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Herbal Drugs and Fingerprints

Devi Datt Joshi

Herbal Drugs
and Fingerprints
Evidence Based Herbal Drugs
Devi Datt Joshi
Amity Institute of Phytochemistry
& Phytomedicine
Amity University, Uttar Pradesh
Noida, UP, India

ISBN 978-81-322-0803-7 ISBN 978-81-322-0804-4 (eBook)


DOI 10.1007/978-81-322-0804-4
Springer New Delhi Heidelberg New York Dordrecht London

Library of Congress Control Number: 2012952527

© Springer India 2012


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Foreword

Globally the demand is increasing for medicines, pharmaceuticals, tonics,


cosmetics and other plant based products. India is a major player in this
area along with China, which is the world leader in the production, con-
sumption and export of herbal products as well as raw materials. The cur-
rent interest in the commercial production of these preparations and products
has put tremendous pressure on the supply of raw materials. The burgeoning
gap between availability of bioresources and demand on one hand and
between commercial demand and supply on the other has led to use of both
inappropriate (e.g., unsustainable collection from the wild, well beyond
natural regeneration) and spurious practices (e.g., collection of raw material
without any consideration of quality, deterioration of quality due to poor
storage, adulteration or even substitution by a different part of the same
plant or from an altogether different plant source and/or even material being
spiked with synthetics). Such a situation calls for proper guiding material
and techniques for fast and easy determination of genuineness of crude
materials and for quality assurance in respect of herbal products. Fortunately
literature is now becoming available, albeit slowly, that provides the much
needed know how for determining the reliability of the raw material used
along with the plant source and information on the availability, production
and quality requirements.
Herbals are derived from whole plants or plant parts and used to prevent,
relieve and treat illness. Since antiquity they are valued for medicinal, aro-
matic, nutritional and even rejuvenating qualities, and have played crucial
role in maintaining the well being and vitality of human body, and for allevi-
ating human suffering across civilizations.

v
vi Foreword

The complex metabolic pathway of the human body can be adversely


affected by the use of non-standardized herbals and herbal drugs. Good man-
ufacturing practices, among others, insist on use of standardized protocols
during the processing of raw materials (collection, transport, storage) and
production of the final products or drugs. Standardized herbals and herbal
drugs contain active ingredients, present in the naturally occurring plant
source, in certain quantity and the proportion between different constituents/
active principles is a key quality parameter for the efficacy of the product. It
is in this regard that the modern tools and techniques of analysis provide vital
support and required evidence. The present compilation is a unique and wel-
come attempt by the author to bring together current tools of finger printing
and analyses for various classes of phytochemicals and natural compounds.
The book Herbal Drugs and Fingerprints should be able to bridge an impor-
tant gap, and would be useful for herbal healthcare professionals, scientists
and researchers as well as to industries based on the herbals.

G.B. Pant Institute of Himalayan Lok Man S. Palni


Environment and Development
Kosi-Katarmal, Almora, Uttarakhand,
INDIA
(An Autonomous Institute of Ministry
of Environment and Forests, Government of India)
Preface

Herbal drugs are time tested and valuable resource for healing, even today,
globally. As the demand and commercial value of these drugs is increasing
tremendously, assurance of safety, quality, and efficacy of medicinal plants
and produces is becoming a crucial issue. The need of the hour is to develop
an evidence-based market of herbal drug raw materials and herbal drugs.
Herbal drugs are composed of many constituents and are therefore very capa-
ble of variation; hence, it is very important to obtain reliable fingerprints that
bear pharmacologically active and chemically characteristic components of
the herbal drug. The information generated based on fingerprints pattern has
a potential application in the identification of an authentic drug, in excluding
the adulterants, and in maintaining the quality and consistency of the drug.
Several analytical techniques have been developed for obtaining fingerprinting
profiles of the herbal drugs and have assured to be a valuable tool for proving
constant composition of herbal preparations by establishing relevant criteria
for uniformity. These fingerprints deal with the advanced extraction as well
as analytical techniques with the help of which qualitative and quantitative
evaluation of herbal drugs and formulations can be carried out and serve as a
rapid and unambiguous tool in the herbal research thereby allowing the man-
ufacturers to set quality standards, specifications, and seek marketing approval
from regulatory authorities. Quality control of herbal drugs is a tedious and
difficult job as herbs differ from that of the conventional drugs, so some inno-
vative methods are into practice for the sake of quality assessment. Fingerprint
analysis using chromatography has become the most potent tool for quality
control of herbal drugs because of its simplicity and reliability, for
identification, authentication, and adulteration. These fingerprints have global
acceptance by regulatory authorities to determine authenticity and reliability
of chemical constituents of herbal drugs and formulations.
The herbal raw material is prone to a lot of variation due to several factors,
the important ones being the identity of the plants and seasonal variation
(which has a bearing on the time of collection); the ecotypic, genotypic and
chemotypic variations; drying and storage conditions; and the presence of
xenobiotics. World Health Organization (WHO) stresses the importance of
the qualitative and quantitative methods for characterizing the samples,
quantification of the biomarkers and/or chemical markers, and the fingerprint
profiles. In case a principle active component is known, it is most logical to
develop fingerprints for the same as main evidence, on the whole fingerprint

vii
viii Preface

profile, whereas the active ingredients contributing to therapeutic efficacy are


not yet known as marker substance should be specific for the medicinal herb
for evidence-based fingerprints. The advancements in modern methods of
analysis and the development of their application have made it possible to
solve many of these problems. Extremely valuable are techniques like TLC,
HPTLC, HPLC, GC, MS, LC–MS, GC–MS, NMR, LC–MS–NMR, and
GC–MS–NMR. Starting from raw material, processing, and formulation into
suitable dosage form, claims are deciphered by fingerprints at every step. At
each and every step, fingerprints have to be generated and a multiple-marker-
based fingerprints strategy needs to be adopted to minimize batch-to-batch
variation and to maintain quality and ensure safety and efficacy.
In order to have a good coordination between the quality of raw materials,
in-process materials and the final products, it has become essential to develop
reliable, specific, and sensitive fingerprints using a combination of classical
and modern instrumental method of analysis. It also encompasses the entire
field of study from birth of a plant to its clinical application, including herbal
drug preparation of a defined content of a constituent or a group of substances
with known therapeutic activity, respectively, by adding excipients or by
mixing herbal drugs or herbal drug preparations.
Sometimes, same species of medicinal herb has different pharmacological
activities depending on its area of origin, the methods used to extract it, and
the part of the herb used, among other factors. After establishing differences
and similarities in fingerprints among different samples of the same type of
herb, we can attempt to have an answer as to why these different samples give
different pharmacological activities, ultimately explaining the “cooperative
effects” of components and effectively controlling the quality of these drugs
during production, using hyphenated techniques. Fingerprints by hyphenated
techniques lead to high robustness with all information along structural
characterization.
The sole aim of this book is to collect all the fingerprint techniques for
herbal drugs at one place, by various case studies globally for different rea-
sons, using latest innovations, so that it may be helpful in the development of
evidence-based herbal drugs and new herbal drug development.

D.D. Joshi
Acknowledgements

The author wishes to record his sincere thanks to Dr. Ashok K. Chauhan,
Founder President, Ritnand Balved Education Foundation for providing
logistic support to promote research on utilization of herbals as evidence-
based drugs. Sincere thanks to Mr. Atul Chauhan, Chancellor, Amity
University Uttar Pradesh, for providing opportunities with different intellec-
tual forum via workshop/seminar and exhibitions of national and interna-
tional standards from time to time.
Many other individuals are responsible for text. Author would like to
thank Dr. L.M.S. Palani (Director, GBPIHED), Dr. Rajeev Kr. Sharma
(Director, PLIM, Govt. of India, Ghaziabad), Dr. D.K. Uprati (Scientist,
NBRI-Lucknow), Dr. A.B.S. Rawat (Scientist, NBRI-Lucknow), Professor
(Dr.) B.S. Kaphalia (University of Texas, USA), Dr. Harendra Kharkwal
(Dy. Director, MoEF, Govt. of India), Col. (Retd.) Dr. Y.P. Singh (N. Delhi),
Dr. P. Joshi (HPL, Govt. of India), Dr. Vidhu Aeri (Associate Professor,
Jamia Hardard, New Delhi), and Dr. Hismi Jamil Husain (Sr. Environment
Advisor, Rio Tinto) for their sincere advices and time to time guidance.
Due permission for discussion from Prof. (Dr.) P. Pushpangadan,
Prof. (Dr.) S.N. Raina, Dr. Amit C. Kharkwal, Prof. (Dr.) Deepsikha Pande
Katare, Dr. Harsha Kharkwal, Prof. (Dr.) Rajni singh, Dr. Kirti Rani Sharma,
Mr. N.C. Nainwal, and Dr. Ram Prasad (from Amity Group) is thankfully
acknowledged.
Finally, the author would like to thank family members for allowing taking
time away from their precious company. To, wife Kummu and sons Gaurav
and Harshit, for your inspiration, motivation, and computer guidance; your
untiring love has given support and enthusiasm to chase the dream.
A special appreciation is due to Dr. Mamta Kapila (Editor, Life Sciences,
Springer India, N. Delhi) who has helped from the beginning, especially to
design the excellent chapters found in this volume; without her inputs, the
publication would not have been in the present face.

D.D. Joshi

ix
Contents

Part I Herbal Drugs: A Review on Practices

1 Herbal Drugs: A Review on Practices ........................................ 3


Introduction .................................................................................... 3
Asian Continent and Traditional Herbal Drugs ............................. 3
China and Japan ........................................................................ 3
Indian Subcontinent .................................................................. 5
Traditional Herbal Drugs in Europe............................................... 6
Traditional Herbal Drugs in America ............................................ 7
Traditional Herbal Drugs in Australia ........................................... 8
Traditional Herbal Drugs in Africa ................................................ 8
Industrialization of Herbal Drugs and Legislation......................... 8
Phytochemical Standardization ...................................................... 9
Extraction of Therapeutics ........................................................ 10
Analysis for Marker and Chromatographic Fingerprint............ 11
Preparative HPLC to Isolate Therapeutic.................................. 11
Biochemical Approach .............................................................. 12
Reverse Pharmacology ................................................................... 12
New Drug Development ................................................................ 13
Synthetic Drug Development .................................................... 13
Modern Approach for Drug Development ................................ 14
Fingerprints of Drugs: Needs and Values ...................................... 14
Multidisciplinary Strategy to Develop Fingerprints ...................... 15
Modern Chemistry and Pharmacology...................................... 15
Standardization of Fingerprints ................................................. 16
Pharmacokinetics of Standardized Form ....................................... 16
Case Study................................................................................. 17
Commercial Manufacturing and Quality Control .......................... 18
Regulatory Norms for Herbal Drugs.............................................. 19
The Therapeutic Goods Act ...................................................... 19
Drug Administration Law ......................................................... 19
Ayush ......................................................................................... 20
Ministry of Health and Welfare, Japan...................................... 21
Ministry of Health, Saudi Arabia .............................................. 22
WHO Guidelines for Assessment of Herbal Drugs .................. 22
References ...................................................................................... 24
Bibliography .................................................................................. 24

xi
xii Contents

Part II Herbal Drugs and Chromatographic Fingerprints

2 TLC: Herbal Drugs and Fingerprints........................................ 29


Thin-Layer Chromatogram ............................................................ 30
Preparation of TLC Plate in Laboratory.................................... 30
Ready-Made TLC Plate............................................................. 30
Selection of Suitable Plate Size................................................. 30
Spotting ..................................................................................... 30
Preparation of Developing Chamber ......................................... 32
Chromoplate Generation ........................................................... 33
Evaluation of TLC Plate............................................................ 33
Retention Factor ........................................................................ 34
Presentation of TLC Results ..................................................... 34
Procedure to Determine Rf Value of Unknown ......................... 34
Spot Development by Iodine Vapors ......................................... 35
Documentation of Fingerprints ................................................. 35
Two-Dimensional TLC ............................................................. 35
TLC Bioautography .................................................................. 37
Bioluminescence and TLC Analysis ......................................... 37
Detection of Colorless Compounds .......................................... 38
Combination of TLC with Other Techniques............................ 38
Criteria for Selectivity, Reproducibility and Robustness .......... 39
Special Hazards ......................................................................... 39
Troubleshooting in TLC Analysis ............................................. 39
Identification of Marker Compounds in Herbal Drugs .................. 40
TLC Analysis for Alkaloids ...................................................... 42
TLC Analysis for Phenols ......................................................... 43
TLC Analysis for Saponins ....................................................... 43
TLC Analysis for Terpenoids .................................................... 44
TLC with DART–MS ................................................................ 45
TLC Fingerprints for Batch to Batch Consistency ........................ 46
References ...................................................................................... 47
Bibliography .................................................................................. 48
3 HPTLC: Herbal Drugs and Fingerprints .................................. 49
Operational Summary of HPTLC .................................................. 49
HPTLC Pre-Coated Plates ............................................................. 50
Detection and Visualization ........................................................... 50
Nondestructive Techniques ....................................................... 50
Nonreversible Reactions ........................................................... 52
Destructive Techniques ............................................................. 52
Common Visualizing Reagents ................................................. 53
Group-Specific Reaction ........................................................... 55
Coupling of HPTLC with Spectrometry ........................................ 57
References ...................................................................................... 59
Bibliography .................................................................................. 59
Contents xiii

4 HPLC: Herbal Drugs and Fingerprints ..................................... 61


A Glance on HPLC Column .......................................................... 62
Analytical Mobile Phases and HPLC ............................................ 63
Solvent Changeover in HPLC ........................................................ 64
Buffers as Mobile Phase ................................................................ 64
Selection of Buffer and Analytical Method Development ........ 65
Buffer Concentration ................................................................. 65
Buffer Solubility........................................................................ 65
Buffer and Its Impact on Detection ........................................... 65
Buffers as Mobile Phase in HPLC ............................................ 66
Detectors in HPLC: Detection, Quantification, and Fingerprints ..... 66
HPLC–UV ................................................................................. 66
HPLC–FD ................................................................................. 68
HPLC–CL ................................................................................. 68
HPLC–ECD............................................................................... 69
HPLC Detection in Hyphenated System........................................ 69
HPLC–RID................................................................................ 69
HPLC–ELSD............................................................................. 70
HPLC–CAD .............................................................................. 70
HPLC–FID ................................................................................ 71
HPLC–MS ................................................................................. 71
HPLC–NMR ............................................................................. 72
LC–Hyphenation for Online Structural Identification ................... 72
HPLC–DAD (Diode-Array Detector) ............................................ 74
HPLC–MS, MS–MS, and MSn ................................................. 74
HPLC–NMR (Nuclear Magnetic Resonance)........................... 74
HPLC Biochemical Detection ................................................... 75
HPTLC–HPLC for Quality Assurance .......................................... 75
Case Study................................................................................. 75
Different Chemical Classes from Natural Products
and LC Fingerprints ....................................................................... 76
Fingerprints for Flavonoids ....................................................... 77
Fingerprints for Terpenoids ....................................................... 77
Fingerprints for Alkaloids ......................................................... 78
Fingerprints for Coumarins ....................................................... 78
Fingerprints for Alkamides ....................................................... 78
References ...................................................................................... 78
Bibliography .................................................................................. 81
5 GC: Herbal Drugs and Fingerprints .......................................... 83
Instrumentation .............................................................................. 83
GLC Columns ........................................................................... 84
Detectors in GLC ...................................................................... 85
Development of GC Fingerprints and Data Interpretation............. 86
Sample Preparation ................................................................... 86
Separation and Detection .......................................................... 87
Analytical Results ..................................................................... 87
Utility of GC Fingerprints in Herbal Drugs ................................... 88
Contamination/Adulteration...................................................... 88
xiv Contents

Traceability................................................................................ 88
Qualification and Quantification of Therapeutics ..................... 88
Nutraceutical Discovery ............................................................ 91
New Cosmeceuticals ................................................................. 93
In Aromatherapy ....................................................................... 93
To Decipher Traditional Formulations ...................................... 94
References ...................................................................................... 96
Bibliography .................................................................................. 96

Part III Herbal Drugs and Spectral Fingerprints

6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints .......... 101


Instrumentation .............................................................................. 102
UV–Vis. Spectrometry and Herbal Drugs ..................................... 103
To Study the Phenolics and Derivatives .................................... 103
UV Spectrometry for Different Derivatives .............................. 105
UV–Vis. Coupled Methods for Analysis .................................. 106
Preparation of Sunscreens ......................................................... 106
Evaluation of Vitamin C Content ................................................... 109
Preparation of Stock Solutions .................................................. 109
Green Synthesis of Nanoparticles .................................................. 110
SNPs from C. tamala Twigs...................................................... 110
Purification of Geometrical Isomers .............................................. 113
Analysis of Ultra-Diluted Drugs .................................................... 114
Identification of Powdered Medicinal Plants ................................. 116
Biological Assay Using Hyphenated Techniques .......................... 116
To Differentiate Various Species of a Genus ................................. 116
References ...................................................................................... 119
Bibliography .................................................................................. 120
7 FTIR Spectroscopy: Herbal Drugs and Fingerprints............... 121
Interpretation of IR Spectra ........................................................... 123
Authentication of Herbal and Herbal Drugs .................................. 125
Interpretation of Data ................................................................ 131
Identification and Comparison of Biomolecules............................ 133
Authenticity of Herbal and Herbal Drugs ...................................... 137
To Distinguish Herb from Its Morphological Fakes ...................... 138
Standardization of Metal-Based Herbal Drugs .............................. 141
References ...................................................................................... 145
Bibliography .................................................................................. 145
8 Mass Spectroscopy: Herbal Drugs and Fingerprints ............... 147
Interpretation of Mass Spectra ....................................................... 149
Techniques for Analysis of Ions in MS.......................................... 149
Detection and Recording of Fingerprints ....................................... 149
Electrospray MS for Herbal Fingerprints.................................. 150
Matrix-Assisted Laser Desorption Ionization ........................... 156
Hyphenated Techniques ................................................................. 160
References ...................................................................................... 161
Bibliography .................................................................................. 161
Contents xv

9 NMR Spectroscopy: Herbal Drugs and Fingerprints............... 163


Metabolic Fingerprints ................................................................... 165
Case Study 1.............................................................................. 165
1
H-NMR Detection .................................................................... 172
1
H-NMR Spectra to Recognize Patterns and Find
Discriminating Signals .............................................................. 173
2D-NMR Spectroscopy to Compare Metabolites ..................... 174
Evaluation of Quality of High Therapeutic-Value Herbals ............ 176
Case Study 2.............................................................................. 176
Materials and Chemicals ........................................................... 176
Molecular Modeling....................................................................... 178
Case Study 3.............................................................................. 178
To Decipher the Herbal Constituents ............................................. 179
Case Study 4.............................................................................. 179
Metabolic Fingerprints to Distinguish Plant Species ..................... 182
Case Study 5.............................................................................. 182
Quality Assurance of Traditional Medicines ................................. 183
Case Study 6.............................................................................. 183
Detection of Contaminations ......................................................... 184
References ...................................................................................... 185
Bibliography .................................................................................. 186

Part IV Pollutants in Herbal Drugs and Fingerprints

10 Metals in Herbal Drugs and Fingerprints ................................. 189


Atomic Absorption Spectroscopy .................................................. 189
Instrumentation in AAS ............................................................ 190
Case Study 1.............................................................................. 191
Case Study 2.............................................................................. 192
The Graphite Furnace Atomizer .................................................... 193
Inductively Coupled Plasma .......................................................... 193
WDXRF Spectroscopy................................................................... 194
Heavy Metals ................................................................................. 195
Removal of Heavy Metals from Herbal Drugs .............................. 195
Case Study 3.............................................................................. 196
Bhasmas as Nanoherbal Drugs ...................................................... 198
References ...................................................................................... 199
Bibliography .................................................................................. 200
11 Pesticide Residues in Herbals and Detection ............................. 201
Regulatory Norms .......................................................................... 202
Analysis of Pesticide Residues ...................................................... 202
Isolation and Purification Methods ........................................... 203
Hyphenated Techniques for Isolation and Purification ............. 204
Contemporary Trends in Pesticide Residue Analysis ............... 205
Degree of Uncertainty of Analysis ............................................ 207
Pesticides Residues in Natural Products ........................................ 208
Current Scenario of Pesticides Residues in Herbals ...................... 209
xvi Contents

Pesticide Residues in Dry Fruits .................................................... 210


TLC Fingerprints....................................................................... 211
GC Fingerprints......................................................................... 211
References ...................................................................................... 212
Bibliography .................................................................................. 212
12 Radiation Detection in Herbals................................................... 213
Microorganisms in Herbals ............................................................ 214
Safety and Effectiveness of Irradiation .......................................... 216
Irradiation of Spices and Dried Vegetable Seasonings .................. 216
Irradiation Dose for Essential Oils and Organoleptic Characters .. 217
Radiation Quantity and Antioxidant Activity ................................ 218
Detection of Irradiated Foods and Food Supplements ................... 218
Thermoluminescence ................................................................ 220
Pulse Photo-Stimulated Luminescence ..................................... 221
Electron Paramagnetic Spin Resonance Spectroscopy ............. 221
References ...................................................................................... 226
Bibliography .................................................................................. 226

Part V DNA Techniques for Evidence-Based Herbals

13 Herbal Drugs and DNA Fingerprints......................................... 231


Authentication of Traditional Formulations................................... 232
Experimental Details ................................................................. 232
RAPD Reaction ......................................................................... 232
Identification of Chemotypes, Ecotypes, and Substitutes .............. 233
Case Study................................................................................. 233
Identification of Adulterants .......................................................... 234
DNA Fingerprint Techniques for Identification of Ingredients...... 234
Polymerase Chain Reaction ...................................................... 235
Simple Sequence Repeats ......................................................... 235
Restriction Fragment Length Polymorphisms .......................... 235
Amplified Fragment Length Polymorphism ............................. 236
Random Amplified Polymorphism ........................................... 237
Single-Nucleotide Polymorphism ............................................. 238
Short Tandem Repeat ................................................................ 238
DNA Barcode ............................................................................ 238
Genetic Markers ........................................................................ 239
Trouble Shooting in DNA Fingerprinting ...................................... 239
Commercial Utility of Genetic Markers in Herbal Technology ....... 240
Comparative Genetic Analysis....................................................... 242
Future Scope of Genetic Markers in Herbal Drug Research ......... 244
References ...................................................................................... 244
Bibliography .................................................................................. 245

About the Author ................................................................................. 247

Index ...................................................................................................... 249


Part I
Herbal Drugs: A Review on Practices

The use of herbals as drug is older than recorded history, as mute witness to
this fact are marshmallow (Althaea officinalis) root, hyacinth (Hyacinthus
orientalis), and yarrow (Achillea millefolium), found carefully tucked around
the bones of a Stone Age man in Iraq. These herbs are used as demulcent,
diuretic, and common cold remedy, respectively, even today. Ayurveda and
TCM are two great living traditions of the world which are related to herbal
healthcare. Every continent has its folk system for healing and caring, and a
few are still recognized by the state healthcare agencies, in different coun-
tries. King Hammurabi of Babylon (1800 bc) had prescribed the use of mint
for digestive disorders; now, it has been established by modern science that
peppermint indeed relieves nausea and vomiting by mildly anesthetizing the
lining of the stomach. The knowledge of herbal drugs was widely dissemi-
nated throughout Europe by the seventeenth century. Nicholas Culpeper had
written “A Physical Directory” and “The English Physician,” the first manu-
als that a layperson could use for healthcare, and it is still widely referred and
quoted. The first publication of the US Pharmacopoeia (1820) included an
authoritative listing of herbal drugs, with descriptions of their properties,
uses, dosages, and tests of purity. It was periodically revised and became the
legal standard for medical compounds in 1906, but with development of
synthetic chemistry, synthetic therapeutic ingredients gained preference, and
herbal drugs became the secondary choice.
The synthetic therapeutic products could not be accepted more due to
severe side effects, and there was relook for herbal therapy, and natural prod-
ucts were found as storehouse of different molecular designs, even beyond
the human imagination. A few traditional remedies directly passed the modern
molecular approach for treatment, for example, ephedrine, an active ingredi-
ent of Ephedra, is used in the commercial pharmaceutical preparations for the
relief of asthma symptoms and other respiratory problems, as it helps the
patient to breathe more easily, while Ephedra in TCM was in use since 2,000
years back to treat the same ailments. Foxglove (Digitalis lanata) leaves are
known since 1775, till the date, as cardiac stimulant and keeps alive the
millions of heart patients worldwide. The active ingredient has been identified
as digoxin, a medicine of all pharmacopoeias.
2 I Herbal Drugs: A Review on Practices

There are over 750,000 plants on earth, but relatively, only a very few
have been studied for healing. Modern pharmacology looks for one active
ingredient and seeks to isolate it, with the exclusion of all others. Most of
the research done on herbals is focused on identifying and isolating active
ingredients, rather than studying the medicinal properties of whole herb.
Herbalists, however, consider that the power of herbal lies in the interaction
of all its ingredients. Used as a drug, herbals offer synergistic interactions
between ingredients both known and unknown. FDA has categorized
most of the herbals as food supplements or nutraceuticals, knowingly
acknowledging the wisdom of centuries-old practices. The sole aim of this
section is a review on the different approaches adopted globally, by different
regimes, at different times, based on sociopolitical scenario to ensure
for correct quantity and quality of herbal drugs to induce the desired ther-
apeutic effects.
Part II
Herbal Drugs and Chromatographic
Fingerprints

Chromatography is a technique for the separation of mixture of solutes


brought about by the dynamic partition or distribution of dissolved or dis-
persed material between two immiscible phases, one of which is moving on
the other. Every type of chromatography contains a mobile phase and a
stationary phase. The moving phase may be liquid or gas. Chromatography,
in its various forms, is used for concentrating/identification of component
which is in dilution but has high commercial value (e.g., Taxol from T. baccata,
digoxin from D. lanta, and forskolin from C. forskohlii). This is an extremely
valuable technique for the separation, isolation, purification, and identification
of components from the mixture. Chromatographic fingerprints of herbals
and herbal drugs may be defined as the chromatographic pattern of pharma-
cologically active and or chemically characteristic constituents present in the
extract, which is featured by the fundamental attributions of integrity and
fuzziness with the reference or reference standard. Due to geo-climatic fac-
tors, there is high variability of chemical components, even in the same
species, collected from different zones. As the therapeutic effect of herbals is
based on interaction of numerous ingredients present on it, so different kinds
of chromatographic fingerprint techniques for quality control of herbal drugs
have gradually come into practice, such as TLC, HPTLC, HPLC, and CLC,
for the purpose of species authentication, evaluation of quality and ensuring
the consistency, and stability of herbal drugs and their related products.
In herbals, a large number of chemical components are involved, and many
of them are in low concentration. Chromatographic instruments and experi-
mental conditions are difficult to reproduce during real analysis resulting in
baseline and retention time shifts from one chromatogram to another, and a
few associated complications such as abnormal chromatograms and ghost
peaks are additional difficulties during fingerprint development, so chemo-
metric approaches such as variance analysis, peak alignment, correlation
analysis, and pattern have been employed to deal with the chromatographic
fingerprint. Many mathematical algorithms are used for data processing in
chemometric approaches. The basic principles for this approach are variation
determination of common peaks/regions and similarity comparison with sim-
ilarity index and linear correlation coefficient. Similarity index and linear
correlation coefficient can be used to compare common pattern of the
28 II Herbal Drugs and Chromatographic Fingerprints

chromatographic fingerprints obtained. In general, the mean or median of the


chromatographic fingerprints under study is taken as the target, and both are
considered to be reliable. To facilitate the data processing, a “computer aided
similarity evaluation” (CASE) software has been developed.
Currently, many hyphenated techniques with chromatography are in prac-
tices for quality assessment, authentication, and content quantification, and
there is still lookout for further innovations. A brief on the utility of a few
chromatographic techniques with latest update to develop fingerprints of
herbals and herbal drugs is described in this section.
Part III
Herbal Drugs and Spectral Fingerprints

The relook on herbal healthcare has developed a challenge and demand to


regulatory authorities and scientists to authenticate that products/produces
have not been adulterated with contaminants or cheaper ingredients. Analytical
authentication is an important quality criterion for efficacy and safety, as it is
focused on specific molecular markers or on recording compositional profiles
of the ingredients. Spectroscopic techniques, which are based on interaction
between electromagnetic radiation and atoms/molecules of the sample, are
very attractive tools due to simplicity, fast and easy mode of operation.
Complex mixtures are analyzed with hyphenated chromatographic devices to
cross validate the analysis. Electromagnetic spectrum is a classification of
photons with various energies into different spectral regions. UV–Vis. spec-
troscopy is used when high-energy photons (wave length 200–400 nm) are
absorbed by atoms/molecules of sample which causes electronic excitation.
Visible wavelengths cover a range from 400 to 800 nm.
IR spectroscopy is an analytical technique used to identify organic, as well
as some inorganic, materials in the herbals and products, in solid as well as
liquid forms. Mid-infrared spectroscopy (400–40,000 cm−1) is useful to iden-
tify a variety of adulteration problems using experimental and statistical
methods. NIR (4,000–14,000 cm−1) is another rapid, reliable, and nondestruc-
tive technique that is widely used for quality and processing control for the
qualitative characterization of various products in exploring bulk material
with little or no sample preparation. FTIR, an advanced form of IR, looks at
the mid-infrared spectrum. The region between 1,500 and 400 cm−1 is referred
to as the fingerprint region. Absorption bands in this region are generally due
to intramolecular phenomena and specify molecular composition and
structure.
The m/z value of molecule and its elucidation is characteristic of mass
spectrometry fingerprints while NMR for the environment of protons around
it. LC–MS has become method of choice in many stages of drug development.
LC–NMR improves speed and sensitivity of detection and found useful in the
areas of pharmacokinetics, toxicity studies, drug metabolism, and drug dis-
covery process. The identification of adulterants in a Chinese herbal medi-
cine was done by LC–NMR technique. GC–MS instruments have been used
for identification of large number of components present in natural and biological
100 III Herbal Drugs and Spectral Fingerprints

systems. The identification and quantification of chemical constituents present


in polyherbal oil formulations is carried out by GC–MS method.
For trace-level analysis, a combination of column liquid chromatography
or capillary gas chromatography with a UV–Vis. or an MS has become the
preferred approach to develop reliable fingerprints. Various hyphenated pro-
cedures used for the analysis of herbal drugs are HPLC–DAD, CE–DAD,
GC–MS, LC–MS, HPLC–MS, HPLC–DAD–MS, and LC–DAD–MS. The
data obtained from such hyphenated instruments are the so-called two-way
data; say one way for chromatogram and the other way for spectrum, which
could provide much more information than the classic one-way chromatography.
A “total analysis device” has been recently demonstrated in the case of online
HPLC–UV (DAD)–FTIR–NMR–MS analyses. In this section, efforts have
been focused on to gather all related potential information based on important
case studies, in summarized form, as a clue for any new problem, to develop
validated fingerprints.
Part IV
Pollutants in Herbal Drugs
and Fingerprints

Environmental pollution especially with heavy metals, pesticides, radioactive


particles, mycotoxins, and microbes including pathogens poses serious prob-
lem on quality of herbal and herbal drugs. It is a common misperception that
natural herbs, used as drugs since centuries, cannot be toxic, though at once
it was true, but due to environmental pollution, a few toxic compounds are
being accumulated at alarming level on plants. Heavy metals such as Cu, Zn,
Cr, Fe, and Co are required in very trace quantities for the proper functioning
of enzyme systems, hemoglobin formation, and vitamin synthesis in human
and for the growth and photosynthesis in plants. Metabolic disturbances are
encountered in case of both deficiency and excess of these essential metals.
On the other hand, Pb, Cd, As, and Hg (toxic metals) are not required by the
body, and they produce deleterious effects upon exposure even at very low
concentrations. Along heavy metals, pesticides used for protection from
certain insects also have presence in the herbals and transported finally to
human body with different routes. Aflatoxins (AF) and Aflatoxin B1 (AFB1),
the most biologically active form of AF produced by the fungi Aspergillus
parasiticus and Aspergillus flavus, are major contaminants in herbals, respon-
sible for poor performance, liver lesions, and immunosuppression. Unregulated
irradiations of herbals for longer durability are the major cause of presence of
radiations. The contamination of pollutants in herbals remains and continues
during their transportation and storage. Such contaminated herbs are one of
the major potential sources of pollutants in the human organs and systems,
because these are not only utilized as herbal drugs and food supplements, but
many of them are consumed as condiments in daily routine.
To get the desired therapeutic outcome, quality of the finished products
and plant raw materials must be ensured. Many reports have shown that one
of the major quality problems frequently encountered is high concentration of
these pollutants. The development of phyto-radioprotective agents has been
the subject of intense research in view of a radiation environment, such as
space exploration, radiotherapy, and even nuclear war. However, no ideal,
safe synthetic radio-protectors are available to date. In Ayurveda, several
plants have been used to treat free radical-mediated ailments that may be
relevant to the mitigation of ionizing radiation-induced damage in mammalian
systems (e.g., Allium sativum, Aloe vera, Tinospora cordifolia, Hippophae
188 IV Pollutants in Herbal Drugs and Fingerprints

rhamnoides, Curcuma longa, Centella asiatica, Stephania tetrantra, Spirulina


platensis, Syzygium cumini, Ocimum sanctum, Moringa oleifera, and Zingiber
officinale).
The use of herbal drugs undetected for presence of these pollutants
as antioxidants, cyto-protective, ACE-inhibitors, immunomodulators, metal-
loelements, prostaglandins, radioprotective, etc., will have different results,
so WHO has made it mandatory to estimate the pollutants in every batch.
This section is composed with objectives to familiarize from the technique
used for developing fingerprints of these pollutants citing different case stud-
ies to have evidence-based herbal drugs.
Part V
DNA Techniques for Evidence-Based
Herbals

Herbal authentication in itself involves many parameters including gross


morphology microscopy, chemical analysis, and DNA fingerprinting. DNA
technology became more important in case of folk healthcare system where
different plants species are used under the generic name, so reliable authenti-
cation and quality control is necessary for the protection of consumers, sus-
tainable development of the industry, and integration of folk herbal drugs into
mainstream. Each herb contains large number of compounds, so it is not
possible to analyze for presence or absence for all compounds. The DNA
fingerprints remain the same irrespective of the plant part used, while the
phytochemical content varies with the plant part used, physiology, and envi-
ronment. Interspecies variation has been reported using DNA marker in
different genera such as Glycyrrhiza, Echinacea, and Curcuma. DNA markers
are helpful to identify cells, individuals, or species as they can be used to
produce normal, functioning proteins to replace defective ones. Moreover,
these markers help in treatment of various diseases and distinguishing the
genuine herb from adulterated. DNA fingerprinting provides an objective
evaluation of genetic identity of plants based on species, cultivars, or geographic
origin and ensure genetic uniformity of raw herbal materials. Chromatographic
techniques such as TLC, HPTLC, and HPLC provide chemical fingerprinting
(i.e., profiling of various chemical constituents). The combination of DNA
and chemical fingerprint techniques is an effective tool in authentication and
quality control.
Britain is using DNA fingerprinting of residues of orange to establish the
substitution of premium citrus fruits with those from lower quality variety by
an orange juice manufacturer. France has adopted DNA fingerprint tech-
niques to ascertain the fraudulent adulteration of Chianti wines with inferior
quality grapes. The method also facilitates the management of biodiversity, as
several international plant resource germplasm collection centers are exploiting
DNA fingerprinting on maintaining and propagating those unique collections.
DNA-based molecular markers have been found useful in differentiating
different accessions of Taxus wallichiana, Azarchdichta indica, Juniperus
communis, Codonopsis pilosula, Allium schoenoprasum, and Andrographis
paniculata collected from different geographical regions.
230 V DNA Techniques for Evidence-Based Herbals

Presently, there is no legal control model for trade of raw herbals, globally.
Different countries define herbals or produce in different ways and have
adopted different approaches to licensing, dispensing, manufacturing, and
trading to ensure their safety, quality, and efficacy. Only fingerprinting of
herbal drugs is utilized for the authenticity and quality control. With the
advancement of DNA fingerprints techniques, it is in practice as a rapid and
specific tool in the herbal research, thereby, allowing the manufacturers to set
quality standards and specifications and to seek marketing approval from
regulatory authorities.
Herbal Drugs: A Review on Practices
1

chromatographic fingerprint analysis represents a


Introduction comprehensive qualitative approach for the
purpose of species authentication, evaluation of
Herbal drugs have been used throughout history quality, and ensuring the consistency and stability
similar to the way modern pharmaceuticals are of herbal drugs and their related products. The
utilized today (i.e., to improve human health). Fossil entire composition of compounds is evaluated to
evidence suggests that plants were used medici- determine not only the presence or absence of
nally in prehistoric times. Although the first use desired markers or active constituents but the
of plant-derived pharmaceuticals may be difficult complete set of ratios of all detectable analytes.
to pinpoint, but use of herbals for human health is The chemical fingerprints obtained by chromato-
the basis for the modern pharmaceutical industry. graphic and electrophoretic techniques, espe-
History makes a man wise, so we start from the cially by hyphenated chromatographies, are
point to the present to have prosperous future on strongly recommended for the purpose of quality
the subject to be discussed. Traditional herbal control of herbal drugs, to decipher ingredients,
drugs are very popular in different systems of and therefore be used for authentication and
medicines like Indian system of medicine (ISM), identification of the herbal products. Current
traditional Chinese system (TCM), Unani natur- global regulations recommend a drug, which is
opathy, oestropathy, and homeopath. All over the assessed to toxicity, and other clinical data.
world, plants play an important role in healthcare
of majority of population. In India alone, nearly
two million traditional health practitioners use Asian Continent and Traditional
plants for treatment of various ailments [1]. In Herbal Drugs
traditional system, only a few markers of phar-
macologically active constituents were employed The two famous ancient human civilizations of
to assess the quality and authenticity of complex world, the Chinese civilization and the Vedic
herbal medicines; however, the therapeutic effects civilization, were from Asia, had well-flourished
of herbal drugs are based on the complex interac- healthcare system, mainly based on herbals, and
tion of numerous ingredients in combination, still have acceptance as time tasted system.
which are totally different from those of synthetic
drugs. Thus, many kinds of chemical fingerprint
methods to control the quality of herbal drugs China and Japan
have gradually come into practices, such as thin-
layer chromatography, gas chromatography, and Traditional Chinese medicines (TCM) have been
high-performance liquid chromatography. The used by Chinese people from ancient times,

D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs, 3
DOI 10.1007/978-81-322-0804-4_1, © Springer India 2012
4 1 Herbal Drugs: A Review on Practices

although animal and mineral materials have been (a) to treat acute diseases by killing pathogen;
used, but primary source of remedies is botanical. (b) to heal chronic illness (e.g., gastrointestinal
In TCM, about 12,000 herbals are used by tradi- disorder, respiratory disorder, allergies, immune
tional healers, of which 500 are common. Herbals system deficiency) by strengthening the body,
are generally used only after processing, for helping to recover it; and (c) maintaining daily
example, stir-frying or soaking in vinegar or life healthy by keeping the balance of human
wine. In clinical practice, traditional diagnosis body.
may be followed by the prescription of a complex In general, under TCM, herbs can treat a wide
and often individualized remedy. TCM are still variety of diseases and conditions, as compared
common use in China. More than half of the to synthetic drugs, and have much gentler and
population regularly uses traditional remedies, safer impact. Most of Chinese herbs do not cause
with the highest prevalence in rural areas. About side effects; even a few can be easily counter-
5,000 traditional remedies are available in acted with other herbs. For these reasons, people
China, accounting for approximately one fifth of turned to TCM. More and more people rely on
the entire Chinese pharmaceutical market [2]. TCM as alternative after synthetic medicine
Chinese herbalists usually do not prescribe single failed and is a very good alternative for those who
herb for their patients, but recommend in combi- are looking for a natural alternative for the con-
nations of 8–15 herbs. There are three major rea- ventional western medicine. Presently, there are
sons to support the combination practice. Mutual many highly efficient Chinese herbal patented
reinforcement involves combining two or more medicines, for pain syndromes, gastrointestinal
very similar herbs together to create a stronger disorders, neurological disorders, stress-related
effect. Mutual assistance is the way to use one syndromes, respiratory disorders, heart problems,
herb to help another work better. Mutual restraint sexual dysfunction, allergies, and immune system
is meant to use one herb to reduce or eliminate deficiencies, as well as replacements for antibiotics
side effects of another herb in the combination. and anti-inflammatory drugs [3]. A major disad-
Historically, Chinese medicines were practiced vantage of the standardization of TCM is lack of
largely within a family-based lineage system, its spiritual roots. Contemporary practitioners
from generation to generation. The specific tech- wishing to revive this spiritual rooting, and the
niques and knowledge required to practice were knowledge/techniques associated with it, often
transmitted from teacher to student in the context name themselves as practitioners of “Classical
of an apprenticeship relationship; because of this Chinese Medicine” (i.e., the form of the medicine
way of transmitting the art and science of Chinese prior to the Cultural Revolution) or five-element
medicine, there emerged many different styles of practitioners. Generally speaking, a TCM practi-
practice, each associated with a particular family tioner will rely primarily upon the “eight-principle”
lineage. At the time of the Chinese Cultural diagnostic framework. A TCM practitioner is
Revolution, a decision was made to standardize likely to pay more attention to physical symp-
and secularize the practice of Chinese medicine. toms and design treatment to eliminate the symp-
This was carried out by examining the various toms. The five-element practitioners, on the other
family lineages, extracting what they seemed to hand, tend to be more focused on the emotional
have in common, eliminating anything that the and spiritual aspects of the imbalance and aim
communist government considered to be too their treatments at the root cause of the dishar-
overtly spiritual, and naming the resulting collec- mony. Great variety exists among the Chinese
tion of knowledge and techniques as traditional practitioners, but both the “eight principles”
Chinese medicine (TCM), officially approved and “five-element” frameworks are important
version of Chinese medicine, which subsequently aspects of theoretical foundation of the medicine.
is being taught largely in government-sponsored The uniqueness of Chinese medicines is the
schools instead of within a family-based appren- insight look that physical, emotional, mental, and
ticeship system, and has three main objectives: spiritual aspects of an individual are always
Asian Continent and Traditional Herbal Drugs 5

interconnected and exist within a larger web from Himalaya; finally, Sushen selected proper
which ultimately includes the entire cosmos. herb and treated Laxaman [8]. Ayurveda, generally
Some herbs are too strong for pregnant women mistaken with exotic herbs, oils, and therapies,
and may cause miscarriage. Certain foods can this is not what it is. It is finding of harmony with
have adverse effects on the herbal therapy. It is a the whole of life and ultimately realizing our true
thumb rule that during use of TCM, one should universal nature as pure cosmic consciousness
avoid the raw food (e.g., vegetables should be beyond koshas (bodies) or ahamkara (ego). The
cooked, but fruits are okay), greasy, strong-tasting word Ayur + Veda = Ayurveda [(life) + (knowledge
or -smelling, difficult to digest (such as beef), or or wisdom)] describes how the ancient sages
irritating to the digestive system (like spicy cognize Ayurveda, where first we have to go
foods). So it is recommended to consult to the beyond our own limited perspective of our current
health expert before using TCM. experience of mind and try to see with more clarity
In Japanese traditional healing system, many what mind really is and how all of our experience
herbal remedies found their way from China is directly affected by it. The eternity of Ayurveda
into the Japan, through Korea. Herbs native to is also described in the Charak Samhita (one of
Japan have been classified in the first pharmaco- the scriptures in Sanskrit language). Acharya
poeia of Japanese traditional medicine in the Charak (600 bc) was the father of Ayurvedic
ninth century [4]. medicine. His renowned work “Charak Samhita”
which is considered the encyclopedia of Ayurveda
today goes in depth about his principals, diagnoses,
Indian Subcontinent and cures that still retain their potency and truth
even after a couple of millennia. His research led
Ayurveda, a medical system primarily practiced to the facts of the human anatomy, embryology,
in India known for nearly 5,000 years, includes pharmacology, blood circulation, and diseases
diet and herbal remedies, while emphasizing the like diabetes, tuberculosis, and heart disease.
body, mind, and spirit in disease prevention and Charak Samhita describes medicinal qualities
treatment. During Vedic civilization, Ayurvedic and functions of 100,000 medicinal plants on
scholars were very particular for quality control which present scientists are still doing research to
and site for collection of suitable raw herbs for find the fact as per their language. Therefore,
medicine (e.g., Himalaya is the best place for even Ayurveda, popularly known as the fifth
collection of medicinal plants of hill origin [5]). Veda, is originated in the divine mind and
There is description that scholars were not afraid descended from the divine sources to the ancient
to diseases even to death, as they had expertise to physicians.
keep it away up to certain period by use of their This ancient Indian science of healing seeks to
unique formulations [6]. In Vedic civilization, reestablish the harmony between the body and its
Ayurveda was a system of healing cognized by habitat by creating the optimum health environ-
sages of the Vedanta philosophy, which describe ment. Over centuries, Ayurveda has had a nurturing
the relation of doctor (Vaidhya) and patient as influence on ancient TCM, Unani, and the
friends. The doctor had prime duty to protect the humoral medicine practiced by Hippocrates in
life of his patient without any discrimination, as Greece. The entire science of Ayurveda is based
the royal doctor of Ravan treated Laxaman, the on the “five great elements” (Panchabhuta) theory.
younger brother of Lord Ram during Ram–Ravan These five elements are earth (prithvi), water
battle. He disclosed the habitat of the unique (jal), fire (agni or tej), air (vayu), and ether (space
healing herb and instructed to Hanuman to collect or akash). Ayurveda strongly advocate for ele-
it [7]. The collection practices were of high moral mental structure of the body. There are also five
value as Lord Hanuman had difficulty in mahabhutas (elements) in the human body, and
identification of the herb, and to avoid adultera- these five mahabhutas are represented in the form
tion, he pulled the whole hill and went to Lanka of doshas, dhatus, and malas. Outside the body,
6 1 Herbal Drugs: A Review on Practices

they form the basic ingredients of the drugs and being used to heal diseases of the testicles, and
food ingredients. In a normal body of a living to stimulate lust (Table 1.1), and supposed to
being, these substances remain in a particular produce a male progeny if given to men as whole
proportion. However, because of enzymatic new tubers; and if the shriveled old tubers were
action inside the human body, this ratio of five given to women, this should produce female
mahabhutas or their equilibrium inside the body children [9].
gets disturbed. The body has, however, a natural William Turner was the first English herbalist
tendency to maintain equilibrium. It eliminates (1568) to describe the four main uses, including
some of the mahabhutas which are in excess and the treatment of alcoholic gastritis [8], and after
takes some of the mahabhutas which are in short- 11 years, Williams Langham reported antipyretic,
age. This shortage of mahabhutas is replenished anti-consumption, and anti-diarrheal effects [9].
through the ingredients of herbs, food, drinks, etc. John Parkinson in 1640 described the tubers to
increase fertility in men, and the Ottomans
extracted the Salep of the dried tubers [10]. In the
Traditional Herbal Drugs in Europe East, Salep was (and is) mainly made from Orchis
morio, but it could be made in the UK from
The “doctrine of signatures,” adopted and pro- Orchis mascula, the early purple orchid, or from
moted by Dioscorides, a Greek, working as a Orchis maculata or Orchis latifolia. Orchids, pre-
Roman military physician, and wrote his De sumably as Salep, were dispensed in London in
Materia Medica, whereby plants have been used Oliver Cromwell’s time, and before the introduc-
for medicinal purposes according to their resem- tion of coffee, hot drinks of Salep were sold at
blance to parts of the human anatomy, for exam- stalls in the streets of London. The tubers were
ple, shape or color. Theophrastus (372–286 bc) mainly imported from the East but also came
introduced the word “orchids” for the under- from Oxfordshire. In Hamlet, Ophelia’s fantastic
ground tubers, which resemble to testicles garlands included “long purples” that were gen-
(Fig. 1.1). The Greeks referred to testicles as erally known either by a rude name or by the
“orchis.” In Enquiry into Plants, Theophrastus name “dead man’s fingers” in rude scenes, a refer-
has reported that the orchids had medicinal ence to testicles, then the orchids must have been
properties. Naturally, this led to orchid tubers of the genera Orchis or Ophrys, second from the

Fig. 1.1 Tubers of early purple orchid [9]


Traditional Herbal Drugs in America 7

Table 1.1 Summarized detail of orchid products in European healing system [9]
Proposed use of orchid
Author Year Preparation Indication
Turner W 1568 Tuber with goats milk Aphrodisiac
Dry Antiaphrodisiac
Topical Antiseptic
Tuber Gastrointestinal due to wine
Langham W 1579 Tuber Antipyretic
Tuber Anti-consumption
Tuber Anti-diarrhea
Parkinson J, Ottomans 1640 Tuber Increase fertility in men
Tuber (Salep) Aphrodisiac
Make ice cream (Turkey)
Coffee substitute (Albania)

Table 1.2 Medical uses of vanilla [9]


Author Year Proposed use of vanilla derivatives
Aztec herbal 1552 Flavoring and perfume prevent fatigue in those holding
public office, bestow the bodily strength of a gladiator
Drive weariness far away
Drive out fear and fortify the human heart
Menashian et al. 1992 Improve food intake and reduce nausea and vomiting in
patients given chemotherapy
Fladby et al. 2004 Diagnostic of Alzheimer’s disease (patients cannot smell
vanilla)
Fitzgerald et al. 2004 Antimicrobial against Escherichia coli, Lactobacillus
plantarum, and Listeria innocua

implies genus Dactylorhiza where the tubers are sheath. The most important vanilla species is
palmate and resemble fingers. Today, Salep is Vanilla planifolia, introduced into Europe by the
largely collected from Asia. Turkey uses the Spanish in 1510 and brought to popularity in the
greatest bulk in making ice cream and beverages, UK when the Marquess of Blandford introduced
but not allowed to export the tubers any more. it here in 1800. Unlike Salep, vanilla can be
Turkey still uses vast quantities, as takes 2,600 farmed, but the flavor and aroma molecule, vanil-
tubers to obtain 1 kg of dried tubers [9]. lin (4-hydroxy-3-methoxy-benzaldehyde), is now
produced synthetically. The Aztecs had several
uses for vanilla, but today its medicinal uses are
Traditional Herbal Drugs in America confined to relieving nausea and improving food
intake in patients receiving chemotherapy and as a
Vanilla, an aromatic oil, is exuded from the seed diagnostic aroma for Alzheimer’s disease, with
pods of vanilla, a well-known example of tradi- loss of the sense of smell being an early manifes-
tional herbal healing knowledge of America tation of this condition. It has been described as
(Table 1.2) [9]. Apparently the word “vanilla” is an antimicrobial agent and acts as preservative to
derived from the Spanish word “vainilla” which prolong the life of food products. Vanilla pom-
in turn came from the Latin “vagina” or pod or pona was also used to flavor tobacco in Cuba [9].
8 1 Herbal Drugs: A Review on Practices

Table 1.3 Use of medicinal plants by Australian aborigi- food dish, chikanda or kinaka. The orchids
nes and early settlers [9] involved are from three genera Disa, Habenaria,
Name of medicinal plants Uses and Satyrium. The orchids have become scarce in
Cymbidium canaliculatum Cure for dysentery Zambia and are now illegally imported from
Food Tanzania. Four million Tanzanian herbals are
Cymbidium madidum Oral contraceptive currently sent from Tanzania to Zambia each
Cure for dysentery year. In Africa, an amulet of leaves of Ansellia
Dendrobium teretifolium (bruised Rub to relieve pain africana impregnated with a paste made from the
leaves)
pseudobulbs is considered as a contraceptive but,
Dendrobium discolor Poultice
Young canes
most conveniently, only in the short term for
Mature canes (bruise and extract Cures ringworm unmarried women. In the Molucca islands, the
with spirit) seeds of Grammatophyllum scriptum have been
added to a woman’s food to ensnare her for life.
Berliocchi also pointed out that Bourbon tea,
popular in the nineteenth century, was made from
Traditional Herbal Drugs in Australia an infusion of orchids from Mauritius and
Reunion that included Angraecum fragrans. The
The historical records describe the use of orchids tea was thought to be a sedative. A tincture was
by Australian aborigines and early settlers also made to apply to the fingertips and improve
(Table 1.3). In addition, many orchid bulbs were the sense of touch. Both vanilla and Salep are
employed as emergency bush food, for example, widely in use for a delicious flavoring and won-
Gastrodia sesamoides (roasted), Dendrobium derful perfume, respectively, and used in making
speciosum, and Caladenia species. Diuris ice cream and beverages, although many are not
maculata has sweet-tasting tubers, but Lawler enthusiastic about the aroma of Salep [9].
and Slaytor warn that some Australian herbal
bulbs have toxic alkaloids, for example, Liparis
reflexa [9]. Industrialization of Herbal Drugs
and Legislation

Traditional Herbal Drugs in Africa The safety of some herbal ingredients have been
recently called into question because of the
The traditional healing systems of Africa are still identification of adverse events associated with
a matter of study. Brian Morris has described 12 their use and, increasingly, because of the dem-
medicinal plants currently used as medicine in onstration of clinically relevant interactions
Malawi. Nine of these are used for stomach com- between herbs and prescribed drugs, for example,
plaints and two for fertility problems. Interestingly, the adverse events (stroke, heart attacks, heart-rate
two species, Cyrtorchis arcuata and Eulophia irregularities, liver toxicity, seizures, psychoses,
cucullata, are employed to promote friendship, and death) associated with use of ephedra in
the former being dried and pounded into a powder formulations for weight loss. The bodybuilding
and the latter prepared as an infusion of the roots. effects and increased energy due to kava-kava
Cyrtorchis arcuata is also employed to treat (also known as kawa), widely used in Europe and
diabetes or skin infections and Eulophia cucul- increasingly in Canada to treat anxiety, nervous-
lata to prevent epilepsy. An infusion of the leaves ness, insomnia, pain, and muscle tension, have
and pseudobulbs of Bulbophyllum maximum is raised issues to some countries to enforce regula-
used to protect against sorcery and Tridactyle tions restricting or banning these products. A few
tricuspis to treat madness [9]. herbs in common use have been suspected of
In Zambia, medicinal plants as boiled root causing cancer. These include Aristolochia, Rubia
tubers of terrestrial orchids are used to make a tinctorum, Morinda officinalis, and Senecio riddellii.
Phytochemical Standardization 9

Although prolonged and apparently uneventful are not yet known; the marker ingredient specific
use of a substance usually offers testimony of for that particular botanical is chosen for analytical
its safety, investigation of the potential toxicity purpose. The advancements in modern methods
of naturally occurring substances may reveal of analysis and the development of their applica-
previously unsuspected problems. tion have made it possible to solve many of these
The recent global resurgence of interest in problems. Techniques like HPTLC, GC, MS,
herbal drugs has led to an increase in the demand HPLC, LC–MS, and GC–MS are extremely
for them. The need of the hour is to evolve a sys- valuable to establish the markers for unknown
tematic approach and to develop well-designed herbals. Starting from sourcing of the raw mate-
methodologies for the standardization of herbal rial, standardization, and preparation of the
raw materials and herbal formulations. Traditional extracts to formulation of the extracts into suitable
systems of medicine are in use since centuries all dosage form, the problems vary with each plant
over the world. According to one estimate, 80% species and part of the plant that is being used.
of the world population still depends on herbal At each and every step, phytochemical profiles
products for their primary healthcare needs. have to be generated and a multiple-marker-based
The toxic side effect of drugs of modern medi- standardization strategy needs to be adopted to
cine and the lack of medicines for many chronic minimize batch-to-batch variation and to maintain
ailments have led to the reemergence of the herbal quality and ensure safety and efficacy [10].
drugs, with possible treatments for many health
problems. They have stood the test of time for
their safety, efficacy, cultural acceptability, and Phytochemical Standardization
lesser side effects. The chemical constituents
present in them are a part of the physiological In herbals and herbal drugs, standardization starts
functions of living flora, and hence they are with correct identity of the sample, organoleptic
believed to have better compatibility with the evaluation, pharmacognostic evaluation, volatile
human body. Most diseases, like diabetes, heart matter, quantitative evaluation (ash values, extrac-
diseases, cancer, and psychiatric disorders, are tive values), phytochemical evaluation, test for
multifactorial and hence need therapeutic inter- the presence of xenobiotics, microbial load test-
vention at more than one level. Plants with com- ing, toxicity testing, and biological activity. The
plex phytochemical mixtures have advantage phytochemical profile has a special significance
over single molecules in treating such diseases, as it is directly linked with the activity of the
with an added advantage of being devoid of toxic herbal drugs. The fingerprint profiles serve as
side effects [10]. guideline to the phytochemical profile of the drug
With commercialization of the herbal drugs in ensuring the quality, while quantification of
assurance of safety, quality and efficacy has the marker compound(s) serves as an additional
become an important issue. The herbal raw mate- parameter in assessing the quality of the sample.
rial is prone to a lot of variations due to several Phytochemical standardization encompasses
factors, the important ones being the identity of all possible information generated with regard
the plants and seasonal variation (which depend to the chemical constituents present in an herbal
on the time of collection), the ecotypic, genotypic, drug. Hence, the phytochemical evaluation for
and chemotypic variations, drying and storage standardization purpose includes (1) preliminary
conditions, and the presence of xenobiotics. testing for the presence of different chemical
WHO stresses the importance of the qualitative groups, (2) quantification of chemical groups of
and quantitative methods for characterizing the interest (e.g., total alkaloids, total phenolics, total
samples and quantification of the biomarkers triterpenic acids, total tannins), (3) establishment
and chemical markers with fingerprint profiles. of fingerprint profiles, (4) multiple-marker-based
A known ingredient of the herbal leads to logical fingerprint profiles, and (5) quantification of
therapeutic efficacy, whereas the active ingredients important chemical constituents.
10 1 Herbal Drugs: A Review on Practices

Extraction of Therapeutics over conventional methods. Applications include


the extraction of high-value compounds from
The phytochemical evaluation is done by natural sources, including nutraceuticals and
scientifically designed work plan and finally vali- functional food ingredients, and pharmaceutical
dated before the chemical characterization of the actives from biomass. MAE technology offers a
herb. Based on the physical and chemical proper- few advantages as (a) improved products,
ties, the therapeutics may be extracted by: increased purity of crude extracts, and improved
stability of marker compounds, possibility to use
Supercritical Fluid Extraction less toxic solvents and (b) reduced processing
This is the most technologically advanced extrac- costs, increased recovery and purity of marker
tion system. Supercritical fluid extraction (SFE) compounds, very fast extraction rates and reduced
involves use of gases, usually CO2, and com- energy and solvent usage. With microwaves drive
pressing them into a dense liquid. This liquid is extraction as opposed to diffusion, very fast
then pumped through a cylinder containing the extraction rates and greater solvent flexibility are
material to be extracted. From there, the extract- possible. Many variables, including the micro-
loaded liquid is pumped into a separation chamber wave power and energy density, can be tuned to
where the extract is separated from the gas and deliver desired product attributes and optimize
the gas is recovered for reuse. Solvent properties process economics. The process can be custom-
of CO2 can be manipulated and adjusted by ized to optimize for commercial/cost reasons,
varying the pressure and temperature. The advan- and excellent extracts are produced from widely
tages of SFE are the versatility it offers in pin- varying substrates. Examples include, but are not
pointing the desired constituents to extract from limited to, antioxidants from dried herbs, carote-
a given material and the fact that end product has noids from single cells and plant sources, taxanes
no solvent residues left in it (CO2 evaporates from taxus biomass, essential fatty acids from
completely). The downside is that this technology microalgae and oilseeds, phytosterols from
is quite expensive. There are many other gases medicinal plants, polyphenols from green tea,
and liquids that are highly efficient as extraction flavor constituents from vanilla and black pepper,
solvents when put under pressure. Coupled essential oils from various sources, and many
SFE-SFC System in which a sample is extracted more [10].
with a supercritical fluid which then places the
extracted material in the inlet part of a super- Solid-Phase Extraction
critical fluid chromatographic system. The extract This involves sorption of solutes from a liquid
is than chromatographed directly using super- medium into a solid adsorbent by the same mech-
critical fluid. Coupled SFE–GC and SFE–LC anisms by which molecules are retained on chro-
system in which a sample is extracted using a matographic stationary phases. These adsorbents,
supercritical fluid which is then depressurized to like chromatographic media, come in the form of
deposit the extracted material in the inlet part or a beads or resins that can be used in column or in
column of gas or liquid chromatographic system, batch form. They are often used in the commer-
respectively. SFE is characterized by robustness cially available form of syringes packed with
of sample preparation, reliability, less time- medium (typically a few hundred milligrams to a
consuming, high yield, and also has potential few grams) through which the sample can be
for coupling with a number of chromatographic gently forced with the plunger or by vacuum.
methods [10]. Solid-phase extraction media include reverse
phase, normal phase, and ion-exchange media.
Microwave-Assisted Extraction This is method for sample purification that sepa-
An innovative, microwave-assisted solvent rates and concentrates the analyte from solution
extraction technology known as microwave- of crude extracts by adsorption onto a disposable
assisted processing (MAP) offers many advantages solid-phase cartridge. The analyte is normally
Phytochemical Standardization 11

retained on the stationary phase, washed and then includes recording of the chromatograms, retention
evaluated with different mobile phases, for exam- time of individual peaks, and the absorption
ple, when an aqueous extract is passed down a spectra (recorded with a photodiode array
column containing reverse-phase packing mate- detector) with different mobile phases. Similarly,
rial, everything that is fairly nonpolar binds, GLC is used for generating the fingerprint profiles
whereas everything polar passes through [10]. of volatile oils and fixed oils of herbal drugs.
Furthermore, the recent approaches of applying
hyphenated chromatography and spectrometry such
Analysis for Marker and as high-performance liquid chromatography–
Chromatographic Fingerprint diode array detection (HPLC–DAD), gas
chromatography–mass spectroscopy (GC–MS),
A chromatographic fingerprint of an herbal drug capillary electrophoresis–diode array detection
is a chromatographic pattern of the extract of (CE–DAD), high-performance liquid chromatog-
pharmacologically active ingredients. This chro- raphy–mass spectroscopy (HPLC–MS), and
matographic profile is featured by the fundamental high-performance liquid chromatography–nuclear
attributions of integrity and fuzziness or same- magnetic resonance (HPLC–NMR) spectroscopy
ness and differences so as to chemically represent are in use to generate additional spectral informa-
the herbal drug investigated. The chromatographic tion, which is very helpful for the qualitative
fingerprints are used for authentication and analysis and even for the online structural eluci-
identification of herbal drugs accurately. Herbal dation [10, 11].
drug and its extract have hundreds of unknown
components, and many of them are in low amount,
sometimes in various concentrations, so it is very Preparative HPLC to Isolate Therapeutic
important to obtain reliable chromatographic
fingerprints that represent pharmacologically There are basically two types of preparative
active and chemically characteristic components HPLC. One is low-pressure (typically under
of the herbal drug. In the phytochemical evalua- 5 bar) traditional PLC (pressure liquid chroma-
tion of herbal drugs, TLC is being employed tography), based on the use of glass or plastic
extensively for the following reasons: (i) It columns filled with low-efficiency packing mate-
enables rapid analysis of herbal extracts with rials of large particles and large size distribution.
minimum sample cleanup requirement, (ii) it A more recent form PLC, preparative high-
provides qualitative and semiquantitative infor- performance liquid chromatography (Prep HPLC)
mation of the resolved compounds, and (iii) it has been gaining popularity in pharmaceutical
enables the quantification of chemical constituents. industry. In preparative HPLC (pressure >20 bar),
Fingerprinting using HPLC and GLC is also carried larger stainless steel columns and packing materi-
out in specific cases. In TLC fingerprinting, the als (particle size 10–30 mm) are needed. The
data that can be recorded using a high-performance examples of normal-phase silica columns are
TLC (HPTLC) scanner includes the chromato- Kromasil 10 mm, Kromasil 16 mm, and Chiralcel
gram, Rf values, the color of the separated bands, AS 20 mm, whereas for reverse phase are
their absorption spectra, l max, and shoulder Chromasil C18, Chromasil C8, and YMC C18.
inflection(s) of all the resolved bands. All of The aim is to isolate or purify compounds,
these, together with the profiles on derivatization whereas in analytical work, the goal is to get
with different reagents, represent the TLC information about the sample. Preparative HPLC
fingerprint profile of the sample. The information is closer to analytical HPLC than traditional PLC
so generated has a potential application in the because its higher column efficiencies and faster
identification of an authentic drug in excluding solvent velocities permit more difficult separa-
the adulterants and in maintaining the quality and tion to be conducted more quickly. In analytical
consistency of the drug. HPLC fingerprinting HPLC, the important parameters are resolution,
12 1 Herbal Drugs: A Review on Practices

sensitivity, and fast analysis time, whereas in employed to study the relation of chemical
preparative HPLC, both the degree of solute composition and bioactivity of herbs, which is
purity as well as the amount of compound that helpful to discover active components. There are
can be produced per unit time, that is, throughput few methods available for discovering causal
or recovery, are important. This is very important relationships, in which the actual process of con-
in pharmaceutical industry of today because new trolled experiment can be stimulated through
products (natural, synthetic) have to be intro- series of conditional dependence tests. A recent
duced to the market as quickly as possible; using approach called “stepwise causal adjacent rela-
such a powerful purification technique makes it tionship discovery” (STEPCARD) method has
possible to spend less time on the synthesis been developed to overcome the disadvantage of
conditions [10, 12]. existing causal discovery algorithm, as well as
Most traditional drugs are administered as the unreliability of traditional statistical methods,
mixtures of many components, and with today’s for example, stepwise regression. The main idea
knowledge of the many possible interactions of STEPCARD is using conditional dependency
between drugs, and between food and drugs, test to determine causal adjacent relationships
ethnopharmacological research deals with this between explanatory variables and predictor. For
aspect too. Additive, synergistic, or antagonis- a given data set containing chemical composition
tic effects are all possible. Various admixtures matrix (parameter X) and bioactivity information
have also been shown to affect the bioavailabi- matrix (parameter Y), STEPCARD algorithm
lity of pharmacologically active principles. can be applied to choose the components or
Pharmacological studies of traditional herbal component combinations most correlative to
drugs provides clue to the isolation of active the biological activity of original formulation
principles [10]. (comparison drug). The computational results of
STEPCARD algorithm dealing with chemical and
biological data represents the minimal significant
Biochemical Approach level used in conditional independent test to pick
out at least one variable. But there is lack of
The traditional medicines, which are generally scientific approach to study correlations of their
prepared by means of aqueous extracts, have chemical constitution and pharmacological mech-
hundreds of chemical compounds. Modern clinical anism. However, this work affords a new strategy
trial proved that a complex formulation com- to identify active component or component com-
posed of up to 20 herbs had greater efficacy than binations of ethnic medicine and is helpful to
single herb used. Obviously, there exists certain accelerate the speed of new drug discovery [13].
relation between biological activity and chemical
composition of herbal medicine, and it is called
as quantitative composition–activity relationship Reverse Pharmacology
(QCAR). Experimental studies, such as random
controlled trials (RCT), often provide the most In normal drug discovery course, “laboratories to
trustworthy methods for establishing causal rela- clinic” approach is followed; while for herbal medi-
tionships from data, in which one or more vari- cine research, “clinics to laboratories” approach –
ables is changed (typically random) to measure a true reverse pharmacology – is followed (Table 1.4).
its effect on other variables. In recent years, the The ethnomedicine is based on its use for many
relation between active ingredients of herbal drugs years, and its clinical existence is presumed. For
and biological activity is one type of causal rela- bringing more objectivity and to confirm ethnic
tionship which has been attempted. When the claims, systematic clinical trials are necessary.
amount of active components in certain formula- In latter, clinical experiences, observations, or
tion varied, its therapeutic effect correspondingly available data becomes a starting point, whereas
changed. Thus, causal analytical methods are with conventional drug research, it is at the end.
New Drug Development 13

Table 1.4 Search for new pharmacological agents; general pathways [14]
Forward pharmacology Reverse pharmacology
Compound discovered Isolate a therapeutic target

Assay for biological activity Identify a compound that affects target

Determine mechanism Modify drug to maximize effects

Demonstrate the desired biological function in vivo


Process Process
1. Cell- or physiologically directed 1. Molecular target-directed
2. Unbiased as to the compound’s mechanism of action 2. Compound has demonstrated in vitro activity
3. Must determine the mechanism of action often using 3. Must demonstrate in vitro activity
in vitro methods 4. Must demonstrate the compound acts by the proposed
mechanism of action

Expensive, time consuming, numerous bottlenecks

TARGET LEAD LEAD PRECLINICAL CLINICAL


IDENTIFICATION IDENTIFICATION OPTMIZATION STUDIES TRIALS

Drug to market 10-15 years

Fig. 1.2 Synthetic route for drug discovery [14, 15]

Reverse pharmacology is the science of integrating New Drug Development


documented clinical/experiential hits into leads
by transdisciplinary exploratory studies and fur- Synthetic Drug Development
ther developing these into drug candidates by
experimental and clinical research. In reverse In synthetic drug discovery, we have to recognize
pharmacology approach process, safety remains an event at first and the molecule as possible drug
the most important starting point, and efficacy is applied, which can provide valuable insights
becomes a matter of validation. The scope of for drug development. Historically, several such
reverse pharmacology is to understand the mech- clinical hits do not often pursued quickly and
anisms of action at multiple levels of biological rigorously by the drug discovery teams. The
organization and to optimize safety, efficacy, and potential molecule, if any, as desired has to cross
acceptability of the leads in natural products the long journey of about 10–15 years, to be
based on relevant science [14]. called drug (Fig. 1.2) [14].
14 1 Herbal Drugs: A Review on Practices

Economic, time spring, least bottlenecks

Reverse Pharmacology

CLINICAL
RELEVANT SAFETY PARACLINICAL
TRIALS
LARGE SCALE TRIALS SCIENCE STUDIES STUDIES
PHASE II & I

Drug to market 4-5 Years

Fig. 1.3 Modern approach for herbal drugs development [14, 15]

Modern Approach for Drug quality assurance still remains a challenge


Development because of the high variability of chemical com-
ponents involved, which has generated a need to
Reverse pharmacology (RP) is designed to dis- developed methods for fingerprinting. Herbal
cover new herbal drugs as an academic discipline drugs, singularly and in combinations, contain a
to reduce three major bottlenecks of costs, time, myriad of compounds in complex matrices in
and toxicity, major challenges associated with which no single active constituent is responsible
synthetic route. RP can be perceived to comprise for the overall efficacy. This creates a challenge
of three phases: first, the experiential phase that in establishing quality control standards for raw
includes robust documentation of clinical obser- materials and standardization of finished herbal
vations of the biodynamic effects of standardized drugs. The main cause of confusion is language
herbal drugs by meticulous record keeping. as during translating from Chinese pin-yin termi-
Secondly, it includes exploratory studies for nology into western languages or when the same
tolerance, drug interactions, dose-range finding name is used in different regions for different
in ambulant patients of defined subsets of the parts of the plant, or even different species or
disease, and para-clinical studies in relevant genera, for example, a problem and confusion
in vitro and in vivo models to evaluate the target resulting in the mistaken use of an herb in the
activity. Third phase includes experimental studies, beginning of the 1990s in Belgium. Stephania
basic and clinical, at several levels of biological tetrandra, which is used in herbal treatment
organization, to identify and validate the reverse against obesity, was exchanged with Aristolochia
pharmacological correlates of herbal drug safety fangchi, a herb resulting in a severe nephropathy
and efficacy. The scope of reverse pharmacology because of the presence of aristolochic acid.
is to understand the mechanisms of action at Confusion probably occurred because of the sim-
multiple levels of biology and to optimize safety, ilarity in the pin-yin terminology of both plants:
efficacy, and acceptability. In this approach, feng fang ji vs. guang fang ji, respectively [11].
scientist travels a reverse path from “clinics to To ease out such problems, the identity and
laboratory” rather than classical “laboratory to quality can be derived from chromatographic or
clinics” (Fig. 1.3), with journey of 4–5 years, as a spectral fingerprints. These fingerprints can be
new drug [14, 15]. defined as “a chromatographic pattern of an
herbal extract showing some common pharmaco-
logically active and/chemical characteristic com-
Fingerprints of Drugs: Needs pounds.” The entire fingerprints are used as a
and Values source of information because by assaying only a
number of compounds from the extract, the total
In recent years, interest on plant-based drugs has intrinsic quality of the herb is not necessarily
increased considerably, with annual growth rate assessed. The fingerprint chromatograms and
between 5 and 15 %, but the quality control and spectra are also accepted by the WHO as an
Multidisciplinary Strategy to Develop Fingerprints 15

identification and qualification technique for is used to prevent and treat some forms of depression.
medicinal herbs and herbal drugs. Analysis and Standard operating procedures and raw material
handling of the fingerprint data is an important specification were developed to have St John’s
aspect for stricter quality control to check the wort extracts of defined hypericin content, and
conscious adulterations, where another plant is consistent yield, from different batches of plant
sold, or to the unconscious mistaken use of “look- material. Despite the apparent simplicity of this
alikes” [11]. botanical, a study comparing 10 products showed
The concept of phytoequivalence is in practice variations from the label claim for hypericin
in order to ensure consistency of herbal products. ranging from 22 to 140 %. Another example may
According to this concept, a chemical profile, be cited using ginseng products, standardized for
such as chromatographic fingerprint, for herbal two types of chemical moieties of active com-
product should be constructed and compared with pounds, (1) ginsenosides and (2) eleutherosides,
the profile of a clinically proven reference prod- and are generally standardized for the total con-
uct. Chinese State Food and Drug Administration tent of each class of compound. Although this
have framed a regulation for the compositions of form of standardization provides a means of
liquid injections with herbal ingredients using comparing products, it does not ensure that large
stringent quality procedures for chemical assay variations do not exist within each class of com-
and standardization. Fingerprints of herbal medic- pound that could affect the effectiveness of a
inal liquid injections are compulsorily to follow preparation. Similarly, a widely advertised diet
the above procedure of standardization and finger- supplements from the South African plant Hoodia
prints. In addition, among the various experi- gordonii were found to contain no putative active
mental techniques, chromatographic methods are ingredients and were likely derived from different
highly recommended for finding out fingerprints plants [13].
of herbal products because of the high separation Standardization of herbals and herbal products
ability. refers to the production of a plant preparation that
is consistent in terms of composition and efficacy.
The standardization processes begin with the
Multidisciplinary Strategy to Develop source of the raw material and continue till the
Fingerprints characterization of finished product with the
same quality as drugs. The environmental condi-
Modern Chemistry and Pharmacology tions significantly affect phytochemical profiles
and finally the efficacy of the end product. Herbal
Herbal drugs are just considered as encapsulated extracts prepared by the herb of certain location
dried plant material, without the quality check can vary from year to year, as the secondary
and/of the plant material, but it is not at all. Most metabolite production is regulated by tempera-
of the ingredients are extracts produced from a ture, drought, or flood, as well as by geographical
defined part of a plant and represent a large col- location. Therefore, biochemical profiling for
lection of compounds, and these compounds are each batch is used to ensure that a consistent
responsible for the overall activity of the drug. material is being used to produce a quality
Extracts are standardized on the basis of the con- material and rejection of a particular crop, also.
tent of these compounds, for example, the purified A variety of molecular techniques including
fraction of opium poppy (Papaver somniferum), restriction fragment length polymorphism, random
highly effective for pain and insomnia, has addic- amplification of polymorphic DNA, and DNA
tive nature. When it was realized, there was sequencing are used to authenticate plant mate-
tension ignited between China and England in rial and detect adulterant plant species. Each
the mid-nineteenth century. Similar example may technique has advantages and disadvantages in
be cited that hypericin, the active compound in terms of cost, accuracy, reproducibility, time, and
extracts of St John’s wort (Hypericum perforatum), taxonomic level of identification. New technologies
16 1 Herbal Drugs: A Review on Practices

and advances in molecular biochemistry have process of activity-guided fractionation, however,


strong scientific logic to prove that these tech- because the relative activity of fractions may
niques are correct for standard regulatory norms decrease with greater purity and may even be lost
[13]. entirely. The potentiating activity of the individu-
ally active components of herbals can be assessed
by recombining the fractions after separation
Standardization of Fingerprints followed by confirmation of biological activity.
The complexity of the process increases when
With the awareness of side effects of synthetic multiple in vitro assays are used for activity-
drugs and rise of strains resistant to antibiotics, guided fractionation, each yielding a different set
pharmaceutical industries are turning to plant- of active compounds. Alternatively, the interac-
based drug. There is a common trend to look the tions of herbal components could be negative, as
chromatographic fingerprints for unknown herb, in the case of the diminished bioavailability of
at the first. A consistent general profile is moni- caffeine resulting from the flavonoids in tea [1].
tored without regard for spot/peak identity that Compounds identified by activity-guided
may not be related to activity. The American fractionation are tested in appropriate animal
Herbal Pharmacopoeia describes the use of models to confirm in vivo activity. The isolation
marker compounds for characterizing products steps may involve chromatographic procedures
with LC and/HPTLC fingerprint, which is useful by solvent partitioning, medium-pressure liquid
for establishing a baseline for specificity and chromatography, and countercurrent chromatog-
sensitivity. Other chromatographic techniques raphy. Depending on the compound and informa-
with advantages and limitations are also in tion about its chemistry, it is possible to synthesize
practice [13]. the compound also. Synthesis of the compounds
Identifying active compounds in herbals is may be difficult and expensive yet more economical
often a challenge even though the process is and efficient than isolating a comparable amount
based on simple principles. Usually, active from a plant. Pure active compounds are gener-
compounds are isolated from a complex matrix ally obtained for testing and quality control.
by activity-guided fractionation, which uses
chromatographic techniques such as HPLC for
separating compounds, followed by activity mea- Pharmacokinetics of Standardized
surements made with an in vitro assay related to Form
in vivo activity (e.g., measuring the inhibitory
activity of aldose reductase, associated with dia- The extraction and purification process, often
betes). The process is complicated when herbals necessary to concentrate therapeutic ingredients
have activity related to complex metabolic disor- to a sufficient level, may alter the properties,
ders that involve multiple metabolic pathways. mainly as their solubility and bioavailability.
Sensitive in vitro assays are essential to the pro- The active compounds within herbal preparations
cess because fractionation can produce hundreds are very often hydrophobic and tend to precipi-
of fractions requiring testing in sub-milligram tate at high concentrations, so attempting to
quantities. The active components may be major improve efficacy by increasing concentration can
components of the botanical or minor compo- be counterproductive. The use of solubilizers and
nents with high activity. bioenhancers is a logical consideration for many
A major hypothetical advantage of herbals standardized herbals just as for drugs. A wide
over conventional single-component drugs is the range of enhancers is available, each with specific
presence of multiple active compounds that solubility properties. One widely used enhancer
together provide a potentiating effect that may is Capmul MCM C10, a glyceryl monocaprate
not be achievable by any single compound. This produced from edible fats and oils and commonly
advantage presents a unique challenge for the used in lip products. In a rat study examining the
Pharmacokinetics of Standardized Form 17

a
0.2

AU

0.0
b

1.0
AU

0.0
10 20 30 40
Time (min.)

Fig. 1.4 Quality control assessment of Artemisia dra- mature flowering plants at age 17 weeks (b). The profile
cunculus plants. Shown are HPLC chromatograms mea- of B is consistent with the profile of the reference extract,
sured by a photodiode array detector at 254 nm from whereas the profile of A contains both quantitative and
identical ethanolic extractions of plants of Artemisia dra- qualitative differences, making it unacceptable as material
cunculus that were vegetative at age 6 weeks (a) and were for production [13]

enteric bioavailability of the antibiotic ceftriax- and a genetic model that develops severe type 2
one, Capmul increased bioavailability by as much diabetes. The plants used for the production of
as 80 %. In a similar study, coenzyme Q10 was the extract were grown hydroponically. The
formulated with self-emulsifying drug delivery extract was initially characterized by LC–MS
systems, which are mixtures of an oil, a surfac- with a photodiode array detector and an electron
tant, a co-surfactant, and the active substance impact mass detector using spectral database
used for improving the bioavailability of lipo- matching. The chromatograms of the extract,
philic compounds. The optimized formulation measured at 254 nm, and the total ion current
doubled the bioavailability of coenzyme Q10 in electron impact mass spectrometry (EI–MS) were
dogs. A similar formulation of a lipid-based self- determined. The crude extract was fractionated
micro-emulsifying drug delivery system was into ten simple fractions based on HPLC reten-
used to enhance the bioavailability of a silymarin tion. The active window was defined by using
preparation from Silybum marianum (milk thistle) activity-guided fractionation and in vitro assays
for liver disease in rabbits. Bioenhancers are such as glucose uptake into muscle cells. Because
effectively used for pharmaceuticals, dietary the identity of the compounds from spectral
supplements, and botanicals [13, 16]. matching of chromatographic peaks was only
tentative, initial standardization of the extract for
scientific quality control was based on the area of
Case Study the most abundant peaks in the retention time
period thought to contain the active compounds.
In a study for therapeutic herb and value addition Preparatory HPLC was subsequently used to
on it using standardized extract, the ethanolic isolate subfractions from the active fractions and
extract of Artemisia dracunculus was evaluated. pure compounds from the subfractions. Activity-
Artemisia dracunculus is a potential herb for pre- guided fractionation and compound purification
venting and treating type 2 diabetes. The extract led to the identification of active compounds
is active in both chemically induced diabetic mice within the peaks (Fig. 1.4b). Compound
18 1 Herbal Drugs: A Review on Practices

identification was confirmed with the use of com- level of development, the identity of the active
mercial standards, mass fragmentation pattern compounds and the validation of their efficacy
libraries, or a combination of LC–MS, LC–MS/ will be a requisite for the quality control and
MS, and NMR. The abundance of these com- standardization of herbal drugs. Patenting of
pounds is used for direct standardization of the drugs derived from indigenous systems of
extract in addition to ultraviolet spectra, thereby medicine has started to take epidemic proportions.
decreasing the likelihood that unknown compounds The current value of the world market for medic-
can interfere with the analysis. Electrospray inal plants from leads given by indigenous and
ionization, a sensitive detection method, was local communities is estimated to be $43 billion.
used to enhance the level of standardization by Using traditional knowledge increased the
confirming the molecular weights of the active efficiency of screening plants for medical proper-
compounds and providing additional chemical ties by more than 400%. The failures and non-
information about active compounds. Assays sustainability of the chemical route to agriculture
were then used to validate the activity component and health care provide an opportunity to reeval-
of the standardization process. Hence, this is an uate traditional knowledge systems and move
example of a standardization technique that has from the false hierarchy of these systems to a
evolved together with the development of the plurality. Such a pluralistic view of knowledge
herbal drug [13]. systems will imply respect for the different sys-
Development of standardized method to prepare tems in their own logic and in their own episte-
Artemisia dracunculus extract. (A) Photodiode mological foundations. It will also mean that one
array detection at 254 nm from HPLC separation system does not have to serve as the measure of
provides a sensitive profile of the extract that was scientific adequacy for all systems and diverse
used for general fingerprinting. (B) LC-electron- systems do not need to be reduced to the language
impact (EI)–MS analysis of the extract provides and logic of dominating knowledge systems.
both qualitative and quantitative data for the com- Evidence-based herbal drugs have a universal
ponents and provides partial or complete com- acceptability [13].
pound identification, especially when confirmed
by comparison with chemical standards and
other spectroscopy techniques such as NMR. Commercial Manufacturing
Compounds identified in B are 1-6-demethoxy- and Quality Control
capillarisin, 2-davidigenin, 3-sakuranetin, 4-2,
4-dihydroxy-4-methoxydihydrochalcone, and The term “standardized herbal extract” deciphers
5-2, 4-dihydroxy-4-methoxy-di-hydrochalcone. the extract for free from any potentially hazard-
Further, the LC–MS-electrospray ionization ous chemical manufactured by raw plant materi-
(ESI) analysis of the extract provides sensitive als obtained from plants that are cultivated
and quantitative selected ion chromatograms specifically for producing the extract or are
specific for the molecular weights of the com- obtained as a by-product from the production of
pounds of interest (M-H for negative ESI), another product, or are collected from the wild,
validating the other chromatographic techniques for example, ginseng is grown specifically for the
as well as the ability to detect compounds that production of related extract, whereas grape seed
may not be detectable by other means (e.g., extract is a by-product of the commercial wine-
compound with m/z 515) making industry. Herbals have been collected as
Multi-herbal preparations are a rich resource they grow in their native environments, but this
with a potential as future ingredients in herbal practice raises concerns about the consistency of
dietary supplements. Scientific validation of herbal end products and is considered an irresponsible
therapeutics is necessary to ensure consistent collection technique that can lead to environmental
results between research studies and herbal prod- destruction and the endangerment of species
ucts. Although many challenges exist at every when not restricted to research purposes.
Regulatory Norms for Herbal Drugs 19

Plants grown specifically for the production labeling, and product appearance. Australian
of specific extract for basic research are ideally manufacturers of therapeutic goods must be
sourced from a characterized and uniform licensed, and their manufacturing processes must
genetic material with a taxonomic record of the comply with the principles of GMP. All medicines
genus, species, and cultivar or other additional manufactured for supply in Australia must be
identifiers. Records are maintained for the listed or registered in the ARTG, unless they are
source of the seed, locations and conditions of specifically exempt or excluded. Listed medicines
cultivation, and exposure to possible chemical are considered to be of lower risk than registered
treatments such as pesticides. Ideally, herbals medicines. Most complementary medicines (e.g.,
are cultivated under controlled conditions such herbal, vitamin, and mineral products) are exam-
as hydroponics within climate-controlled green- ples of listed products. Medicines assessed as
houses yielding consistent plant material. Only having a higher level of risk must be registered
with tight control over the entire process of (not listed). Registered medicines include non-
herbals production can seed-to-pill standardiza- prescription (low-risk, OTC) medicines and pre-
tion be achieved. When source plants are pro- scription (high-risk) medicines. Complementary
cured in various regions of the world, records of medicines (also known as “traditional” or “alter-
plant identification, maintenance of voucher native” medicines) include vitamin, mineral,
specimens, and biochemical profiling may herbal, aromatherapy, and homeopathic products.
suffice. Raw materials from international sources Complementary medicines may be either listed
also be carefully monitored for contamination or registered, depending on their ingredients
from a variety of sources including co-harvested and the claims made. Most complementary
weed plants, toxic phytochemicals, and heavy medicines are listed in the ARTG, and some are
metals. Plants that were not directly treated with registered (Therapeutics Good Administration,
pesticides can become contaminated by chemi- 1999). In New Zealand, supplements in the
cal drift. Depending on the formulation, humid- market are largely manufactured in the USA,
ity, and temperature, pesticides can drift from follow the TGA, but regulations are not restric-
the site of intended application by as much as tive, and there are no limits on ingredients or
4.8 km (3 miles) [12, 17–19]. potencies, and structure/function claims are
allowed [20].

Regulatory Norms for Herbal Drugs


Drug Administration Law
The increasing acceptance of herbals as drug has
alarmed the regulating authorities globally. The In China, many herbal drugs have been used for
speculators and opportunist are under scan using hundreds of years, and it is assumed in many
the different norms by each country; a few are as cases that they must work, for example, about
below: 7,000 species of plants are used in China as herbal
drugs, but only 230 are most common, with in-
depth pharmacological, analytical, and clinical
The Therapeutic Goods Act studies. The 2000 edition of the Chinese
Pharmacopoeia included 784 items on TCM and
In Australia, the “Therapeutic Goods Act 1989,” 509 on Chinese patent medicines. Herbal medi-
sets out the legal requirements for the import, cines in China are normally considered as medic-
export, manufacture, and supply. It has details of inal products with special requirements for
the requirements for listing or registering all ther- marketing. New drugs have to be investigated
apeutic goods in the Australian Register of and approved according to the drug administra-
Therapeutic Goods (ARTG), as well as many tion law. New traditional Chinese medicines are
other aspects of the law including advertising, classified under five categories based on the
20 1 Herbal Drugs: A Review on Practices

amendment and supplement regulation of identified. Proprietary preparations containing


approval of new traditional medicines [20]: a combination of herbal ingredients and
conventional drugs are regulated in the same
Class 1 manner as other conventional drugs. The
1. Artificial alternatives of Chinese crude drugs majority of suppliers are state-owned or state-
2. Newly discovered Chinese crude drugs and connected. The pharmacopoeial TCM allows
their preparations the parallel manufacturing and sale of both
3. Active constituents extracted from Chinese pharmaceutical drugs and traditional herbal at
crude drugs and their preparations a point also.
4. Active constituents extracted from a composite
formulation of traditional Chinese medicines
Ayush
Class 2
1. Injection of traditional Chinese medicines For traditional medicines in India, an authorized
2. Use of new medicinal parts of Chinese crude body Ayush has adopted strict guidelines for all
drugs and their preparations herbal drugs (related to Ayurveda, Yoga, Unani,
3. Effective fractions extracted from Chinese Siddha, and Homeopathy) to be exported from
crude drugs or natural drugs and their India [14]. With respect to this, Department of
preparations AYUSH Govt. of India gave some parameters for
4. Chinese crude drugs artificially developed in Drug Development, Standardization & Quality of
an animal body and their preparations Ayurveda, Siddha, and Unani drugs, which
5. Effective fractions extracted from a composite include five protocols as [21]:
formulation Protocol-I: Standardization of single plant
material
Class 3 Protocol-II: SOP of preparation of extracts
1. New composite formulations of traditional Protocol-III: Standardization of plant extract
Chinese medicines. Protocol-IV: SOP of finished product
2. Composite preparations of traditional Chinese Protocol-V: Standardization of formulations
medicines and chemical drugs with the main These protocols based on most common
efficacy due to the traditional Chinese parameters such as morphological evaluation,
medicine. microscopic evaluation, physicochemical evalua-
3. Domestically cultivated or bred crude drugs tion, particle size, bulk density and tap density
originally imported and commonly used in (in case of powder crude drugs or powder formu-
China, and their preparations. lations), and assay for constituents (marker %,
major compounds like alkaloids, glycosides,
Class 4 flavonoids/saponin). With respect to above
1. Preparation with a change of dosage form or parameters are test for heavy/toxic metals (lead,
route of administration. cadmium, mercury, and arsenic), microbial con-
2. Botanical crude drugs acclimatized from their tamination (total viable aerobic count, total
origin or crude drugs from a domesticated Enterobacteriaceae, and total fungal count), test
wild animal in China. for specific pathogen (E. coli, Salmonella spp.,
S. Aureus, Pseudomonas aeruginosa), pesticide
Class 5 residue (DDT, HCH, endosulfan, aldrin, mala-
In 1995, the preparatory committee on Chinese thion, and parathion), test for aflatoxine (B1, B2,
medicines was formed to manage the imple- G1, G2), and chelating agent (for Bhasma, Lepa,
mentation of these recommendations; as a Aswarista, etc.). Further stability assessment and
result, 31 potent Chinese medicines that may self life, safety assessment, documentation of
potentially cause adverse effects have been safety based on experience or toxicological studies,
Regulatory Norms for Herbal Drugs 21

Table 1.5 Status of various medical systems in India [14]


Medical system
Characteristics Ayurveda Siddha Unani Tibetan Homeopathy
Medicinal plants known 2,000 1,121 751 337 482
Licensed pharmacies 8,533 384 462 – 613
Hospitals 753 276 74 – 223
Dispensaries 15,193 444 1,193 – 5,634
Registered practitioners 438,721 17,560 43,578 – 217,460
Undergraduate college 219 6 37 – 178
Postgraduate college 57 3 8 – 31

assessment of efficacy by ethnomedicinal infor- monograph, the claims reported in the Japanese
mation, and biological activity evaluation are Pharmacopoeia are used as a guide [20].
essential [18, 21]. Ayush has made it mandatory In Japan, empirical facts or experience, such
for all ISM to be exported to meet the interna- as reference data and clinical test reports, is used
tional standards for contamination including for the evaluation of a Chinese medicine, but
heavy metals in 2005. These guidelines can be importance is given to the pharmacological action
accessed on the Ayush website (http://www.indi- of each ingredient. Safety and efficacy have been
anmedicine.org) [14]. The popularity of ISM is in estimated based on general methods employed
rise day by day and widely accepted (Table 1.5). by modern medical science. In 1972, the MHW
designated 210 formulae as OTC drugs; this
selection was based primarily on the experience
Ministry of Health and Welfare, Japan of doctors actually practicing traditional Chinese
medicine. In 1976, the MHW specified 146 for-
Japanese traditional medicine is in use since more mulations as NHI applicable prescription drugs.
than a thousand years: may be divided into folk In the case of an application for approval of a pre-
medicine and Chinese-concept-based Japanese scription drug other than those previously listed,
medicine (i.e., Kampo medicine). Kampo medi- specified data on safety, stability, comparison
cine is so popular that the per capita consumption with other drugs, clinical test results, etc., must
of herbal drugs in Japan seems to be the highest be submitted.
in the world. One hundred and forty-six Kampo New Kampo drugs are regulated in essentially
drugs are registered as drugs by the Ministry of the same way as western drugs in Japan. The
Health and Welfare (MHW) and are included in same data required for new western drugs are
coverage under the national health insurance required for new Kampo drugs, including data
(NHI). Acceptance of Kampo drugs took place from three-phase clinical trials. Since 1971, the
without clinical validation studies. In 1989, about MHW has been running a program for reevalua-
80% of physicians reported prescribing Chinese tion of all drugs marketed before 1967; a new
medicine. Physicians generally recognize the raw system to reevaluate the efficacy and safety for
herbs which have long been used as folk medicine all drugs every 5 years was launched in 1988. An
and which have also been used for a considerable advisory committee for Kampo drugs was estab-
period as components of an industrial product. lished in 1982 in close association with the MHW
These products are freely usable for the purposes in order to improve quality control of Kampo
indicated in the monograph. Local traditional drugs. Since the 1986 good manufacturing prac-
usage is not sufficient for approval as a drug; the tice Llw, the standard applied to all pharmaceuti-
claims and rules of combinations of herbal ingre- cal drugs has also applied to Kampo drugs. In
dients are determined on the basis of the pharma- addition, in 1985, guidelines for ethical extract
cological actions of the ingredients. In absence of products in oriental medicine formulations were
22 1 Herbal Drugs: A Review on Practices

Table 1.6 Different national regulatory authorities and guidelines [19]


S. no. Country Regulatory authority Guidelines
1 Australia Australian Department of Health Guidelines for preparation and presentation of
applications for investigational drug and drug
products
2 Canada Health Protection Board Preclinical toxicology guidelines
3 European Union Committee for Proprietary Recommendation for the development of
Medicinal Products nonclinical testing strategies
4 France Ministry of Public Health and Guidelines for analytical, pharmacological, and
Social Security toxicological testing of pharmaceuticals
5 India Directorate General of Health Drug and cosmetic rules
Services
6 Japan Ministry of Health and Welfare Guidelines for toxicity studies of drugs
7 Nordic countries Nordic Council on Medicines Guidelines for registration of new drugs
8 UK Department of Health and Social Guidance notes on application for product licenses
Security
9 USA Food and Drug Administration Guidelines for assessment of drugs and medical
device, safety in animals (issued by PMA, prepare
in conjugation with FDA)

developed. The MHW has three major systems before the development and spread of modern
for collection of adverse reaction data. The first is medicine and are still in use today. As per WHO,
a voluntary system involving 2,915 monitoring every herbal formulation must be standardized.
hospitals. The second system, the pharmacy WHO collaborates and assists health ministries
monitoring system, which includes 2,733 phar- in establishing mechanisms for the introduction
macies, collects data on cases [20]. of traditional plant drugs into primary healthcare
programs, in assessing safety and efficacy, and in
ensuring adequate supplies and the quality control
Ministry of Health, Saudi Arabia of raw and processed materials. According to
WHO guidelines, less stringent selection proce-
In Saudi Arabia, registration of medicinal prod- dures could be applied for the screening, chemical
ucts by the ministry of health is obligatory for analyses, clinical trials, and regulatory measures,
any ingredient having medicinal effects such as but the procedure for pure phytochemicals for
herbal preparations, health and supplementary quality control should be identical to that for syn-
food, medicated cosmetics, antiseptics, or medical thetic drugs according to WHO guidelines. The
devices [20]. traditional preparations comprise medicinal
A few important organizations known for plants, minerals, organic matter, etc. Some of the
strict regulations related to herbal drugs are as important parameters are stability testing, safety
per Table 1.6 [19] have guidelines for safe use of assessment, specific therapeutic activity analysis,
herbal drugs. and estimation of the active constituents in plant
raw material and finished products. The objective
of WHO guidelines is to define basic criteria for
WHO Guidelines for Assessment the evaluation of quality, safety, and efficacy of
of Herbal Drugs herbal drugs and therefore to assist national regu-
latory authorities, scientific organizations, and
The WHO has recently defined traditional medi- manufacturers to undertake an assessment of the
cine as comprising therapeutic practices that have documentation/submission/dossiers in respect of
been in existence, often for hundreds of years, such products. The manufacturing procedure and
Regulatory Norms for Herbal Drugs 23

formula including the amount of excipients various countries provide standardized test methods
should be described in detail. A method of for the most common and widely used materials
identification, and quantification, if applicable, of in their monographs. Stored drug samples are
the plant material in the finished product should prone to attack by harmful mycotoxin-producing
be defined. If the identification of an active prin- fungi. Detection of mycotoxins (aflatoxin B,
ciple is not possible, it should be sufficient to acliratoxin, citrinin, and zearalenone) is certainly
identify a characteristic substance or mixture of a matter of great concern in stored drugs of
substances (e.g., chromatographic fingerprint) to important medicinal plants, for example, fruits of
ensure consistent quality of the product. Emblica officinalis (1.51 mg/g); Terminalia
According to WHO, “herbal drugs” should be chebula (1.19 mg/g) have developed an HPTLC
regarded as finished, labeled medicinal products method for the detection of aflatoxins B1, B2, G1,
that contain as active ingredients aerial or under- and G2 from herbal raw materials and estimated
ground parts of plants or other plant material, or the production of aflatoxins in caffeinated and
combinations thereof, whether in the crude state decaffeinated tea samples. Studies revealed that
or as plant preparations. Plant material includes caffeine acts as a good inhibitor for the growth of
juices, gums, fatty oils, essential oils, and any aflatoxins in stored drugs [20].
other substance of this nature. Herbal medicines
may contain excipients in addition to the active Quality Control Techniques as per WHO
ingredients. Drugs containing plant material A lot of analytical techniques have been devel-
combined with chemically defined active sub- oped for renewed for quality control of drugs
stances, including chemically defined, isolated from plant origin. The quality control step is
constituents of plants, are not considered to be for selecting targets to assess authenticity and
herbal medicines. Exceptionally, in some coun- inherent quality, but many traditional drugs are
tries, herbal drugs may also contain, by tradition, claimed to exert their effects because each type
natural organic or inorganic active ingredients of chemical compound present many have a
which are not of plant origin. Multicomponent different activity and then seem of all of these
herbal formulations can be standardized with may modify the action of the major active
newer techniques such as DNA fingerprinting, component. Chromatographic fi ngerprinting
HPTLC, liquid chromatography, and mass spec- emphasizes an integral formulation of pharmaco-
troscopy. The value of animal testing to establish logically active and phytopharmaceutically char-
safety and toxicity is not so critical if the herbs acteristic components of samples with similar or
are used in traditional forms. Nevertheless, all the different attributions. This technique can be used
critical pharmacopoeial tests such as dissolution for the assessment of quality consistency and sta-
time, microbial, pesticide, and heavy metals con- bility of herbal extracts or products by visible
tamination must be in accordance with global observation and comparison of the standardized
standards, and all the Ayurvedic medicine manu- fingerprint pattern. Fingerprinting of the herbals
facture must be in accordance with current good is done by HPLC, HPTLC, MS, LC–MS [1],
manufacturing procedures for herbs [20]. H–NMR, etc. Apart from this, there are some
methods to evaluate the fingerprint quality of
Analysis of Raw Herb herbal materials or pharmaceutical products,
Raw material can be defined as starting material such as correlative chromatography, comparative
or any intermediate which will be utilized for analysis, wavelet analysis, and artificial neural
further processing. Before finished pharmaceutical networks (ANN) [20].
dosage forms are produced, the identity, purity, Evaluation of plant materials and their derived
and quality of raw materials as per specifications products has always been an important part of the
for impurities and other related substances present professional expertise of a pharmacognosist.
must be established with use of suitable test However, over the years the nature and degree of
methods. Pharmacopoeias and formularies of this evaluation have changed. Initially, it was
24 1 Herbal Drugs: A Review on Practices

considered sufficient to authenticate the plant 8. Tulshidas. Ramcharitmanas, Lanka Kand. (Published
material by comparison with a standard botanical by Gita Press Gorakhpur, India) p. 811.
9. Bulpitt CJ. The uses and misuses of orchids in medi-
description or monograph. Later, it was realized cine. Q J Med. 2005;98:625–31.
that, for detection of adulterants, this practice 10. Patil PS, Shettigar R. An advancement of analytical
must be supplemented with other important pro- techniques in herbal research. J Adv Sci Res.
cedures like microscopy, chemical tests, and 2010;1(1):08–14.
11. Heyden YV. Extracting information from chromato-
advanced analytical techniques. The main goal of graphic herbal fingerprints. LCGC Europe. 2008;
pharmacognosy is to assess the value of raw 21(9): also available at http://chromatographyonline.
materials and to ensure that the final product is of findanalytichem.com/lcgc/Column%3A+Practical+
the required standard. Strict standardization Data+Handling/Extracting-Information-from-
Chromatographic-Herbal/ArticleStandard/Article/det
procedures and pharmacognostical studies of ail/565826?contextCategoryId=47195. Accessed 8
medicinal plants reduce drastically much of the Aug 2011.
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herbal drugs, where a plant can be differentiated. medicine in complementary therapeutics. In:
Ethnomedicine: a source of complementary therapeu-
The use of herbal drugs is growing steadily, tics. Trivandrum: Research Signpost; 2010. p. 29–52.
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situation is worse in developing and underdevel- Reverse pharmacology and systems: approaches for
drug discovery and development. Curr Bioact Compd.
oped countries. Some of the crucial discoveries 2008;4:201–12.
in the safe use of herbal drugs were brought out 16. Lazo JS. Rear-view mirror and crystal balls: a brief
through intensive pharmacognostical research, so reflection on drug discovery. Mol Interv. 2008;8(2):60–
need of hi-tech fingerprints is stressed by regula- 3. Speaking of Pharmacology. Editorial.
17. Giri L, Andola HC, Purohit VK, Rawat MSM, Rawal RS,
tory authorities. Bhatt ID. Chromatographic and spectral fingerprinting
standardization of traditional medicines: an overview
as modern tools. Res J Phytochem. 2010;4:234–41.
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Ayurvedic medicine. 2002. [http://www.compulink.
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TLC: Herbal Drugs and Fingerprints
2

In an attempt to develop primary fingerprints of different distances according to how strongly they
herbals and herbal drugs, thin-layer chromatogra- interact with the adsorbent. This property gener-
phy (TLC) is used to decipher the active principals, ates the concept of Rf (retention factor) value and
identification of claimed herb, and possible con- can be compared to standard compounds to aid in
tamination/adulterants. The TLC method is efficient the identification of an unknown substance in the
and rapid and combines the sensitivity and sim- chromoplate. This technique is typically the start-
plicity with low cost for the determination of ing place for any sample of unknown composi-
main active principles of medicinal plants (alka- tion. Confirmation of identity is achieved either
loids, anthraquinones, coumarins, essential oils, co-spotting (i.e., sample is spotted with stan-
flavonoids, glycosides, saponins, tannins, etc.), dard) or using multiple mobile phases/two-
which are the main ingredient responsible for dimensional TLC in which it will exhibit a
potential pharmacological effects. The technique change in Rf, if compound is different. TLC eval-
is so efficient that it is being used as primacy ana- uation and identification depends on the Rf value
lytical tool in the new drug discovery for herbal and upon color generated with a visualization
drugs. The technique is based on simple principle reagent. The mobile phase used for TLC may have
that as the solvent moves over the spot that was many factors together as applicable, when neces-
applied on the plate (stationary phase), equilibrium sary as: (1) mover in the solvent of high eluting
is established for each component of the mixture power, (2) restrainer, that is, of low eluting power,
between the molecules of that component which (3) homogenizer is used in case the mixture of the
are adsorbed on the solid and the molecules which mover and restrainer is immiscible, (4) pH con-
are in solution. In principle, the components will troller is used when the mixture to be separated
differ in solubility and strength of their adsorption has both acidic and basic functional groups, for
to the adsorbent (i.e., stationary phase), resulted a example, amino acids, (5) sharpener (i.e., the sol-
few components carries faster up in the plate than vent used to produce more compact spot), and (6)
others. When the solvent has reached the desired viscosity reducer, the solvent used to reduce devel-
distance on the TLC plate (i.e., solvent front), it is opment time of plate. In preparing the mixed sol-
removed from the developing chamber, dried, and vent, it is essential to measure the component
evaluated. If the compounds are colored, visual- carefully because the small variation in composi-
ization is straightforward. Usually the compounds tion can change the reproducibility/repeatability
are not colored, so UV lamp is used to visualize and reuse of mobile phase for more than one devel-
the spots on the plate (sometimes plate itself opment is generally not recommended as the com-
contains flour which fluoresces everywhere except position can change due to evaporation and the
where an organic compound is on the plate). optimum angle of 45° should be there between
Different compounds in the sample mixture travel adsorbent layer and solvent surface.

D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs, 29
DOI 10.1007/978-81-322-0804-4_2, © Springer India 2012
30 2 TLC: Herbal Drugs and Fingerprints

until it reaches the finish line, at which time the


Thin-Layer Chromatogram plate is removed and solvent allowed to evapo-
rate. The locations of the various substances are
The quick screening of herbals/herbal products then determined by developing spots to the color-
for fingerprints and quality is possible, using less compounds making visible.
thin-layer chromatogram, even in least available
facilities. The entire process may be summarized
as below: Ready-Made TLC Plate

Presently, ready-made TLC plates with station-


Preparation of TLC Plate in Laboratory ary phase either of silica gel (SiO2) or alumina
(Al2O3) are easily available in market. A TLC
In TLC, an adsorbent is applied to a supporting plate is a sheet of aluminum foil, glass, metal, or
plate in a thin layer; generally, a binding agent is plastic which is coated with a thin layer of a solid
used to adhere the adsorbent to the support, adsorbent (usually silica, modified silica, or alu-
although some work is done without a binder mina) and may easily reduce to the desired size.
using very finely divided adsorbent which clings The stationary phase on the plates is of uniform
to the support and forms a rather soft layer. This thickness and consists of fine particle size. The
is to be distinguished from the loose-layer chro- aluminum sheets are preferred, as can be cut
matograms in which the adsorbent does not without much more labor.
adhere to the supporting plate and must therefore
be developed in a horizontal or near-horizontal
position. Mostly, a mixture of adsorbent and Selection of Suitable Plate Size
binder is applied as thin slurry, and the excess
moisture is removed under varying conditions The ready-made TLC plates generally used are of
depending on the adsorbent, the binder, and the 20 × 20-cm sheets. Each large sheet is cut hori-
desired degree of activity. TLC plates are made zontally into small pieces which are 10 cm in
by mixing the adsorbent, such as silica gel, with length and of desired widths; the more samples
a small amount of inert binder like calcium sulfate we can plan to run on a plate, the wider it needs
(gypsum) and water. This mixture is spread as to be. Skillful handling is required as coating of
thin slurry on an unreactive carrier sheet, usually adsorbent may disturb and/dirty (Fig. 2.1) during
glass, thick aluminum foil, or plastic, and the reduction of size, of the TLC plate.
resultant plate is dried and activated by heating in
an oven for 30 min at 110°C. The thickness of the
adsorbent layer is typically around 0.1–0.25 mm Spotting
for analytical purposes and around 1–2 mm for
preparative TLC. After the starting point is Sample to be analyzed is spotted by either capil-
marked about 1.0 cm from the bottom of the lary or syringe on the TLC plate; on the baseline
plate, the finish line is marked a convenient (Fig. 2.2) the process is called “spotting.” If the
distance from the starting point. This is done with sample is not already in solution, it is dissolve in
a very soft lead pencil, and care is taken not to solvent of volatile nature such as hexanes, ethyl
disturb the adsorbent layer at the point of sample acetate, or methylene chloride to have 1% solu-
application since this leads to deformed spots. tion. For concentrated sample, it is necessary to
The solution of the compound is deposited at the dilute it to avoid a smear or streak; however, some-
starting line by means of a micropipette, and the times only we have to go by trial and error to have
plates are then placed in a closed container con- well-sized spot for easy reading. Microcaps with
taining a layer of solvent about 0.5 cm deep. The capillary (Fig. 2.3) or syringe are used for volu-
solvent ascends the plate by capillary attraction metric spotting with care that it does not disturb
Thin-Layer Chromatogram 31

Fig. 2.1 Cutting suitable


size of TLC plate

Fig. 2.2 Baseline marks for


spotting

Fig. 2.3 Microcaps with


capillary
32 2 TLC: Herbal Drugs and Fingerprints

Fig. 2.4 Sample application


on the plate

Fig. 2.5 Mobile phase in


developing chamber

the coating of adsorbent. A small spot of solution the analyte would be mostly nonpolar. This means
containing the sample is applied on the plate, that during separation on TLC plate, the nonpolar
about one centimeter above from the base (Fig. 2.4). parts will have move further up the plate.
The plate is then dipped in to a suitable solvent
(i.e., mobile phase) and placed in a sealed con-
tainer. As solvent moves up in the plate by capil- Preparation of Developing Chamber
lary action and meets the sample mixture, which is
dissolved and is carried up the plate by the solvent. The developing container for TLC may be a
Different compounds in the sample mixture move specially designed chamber, a jar with a lid, or a
at different rates due to differences in solubility in beaker with a watch glass on the top/aluminum
the solvent and due to differences in their polarity foil/butter paper with rubber band; pour mobile
to the stationary phase. Results also vary depend- phase into the beaker to a depth of just less than
ing on the solvent used, for example, if the solvent 0.5 cm, cover the beaker, swirl it gently, and allow
is 90:10 mixture of hexane to ethyl acetate, then it to stand till dip TLC plate on it (Figs. 2.5 and 2.6).
Thin-Layer Chromatogram 33

Fig. 2.6 TLC plate in


developing chamber

Nowadays, there are applicators aided with phase can be separated by the stationary phase
software for mounting purpose, where in spite of under certain conditions, causing the formation
spots, narrow bands are produced. Such type of of secondary fronts.
devices is recommended when manual contact,
with samples due to safety reasons, is strictly
prohibited as extremely toxic solutions, microbi- Evaluation of TLC Plate
ologically contaminated samples, and radioactive
compounds. In TLC plates, often a small amount of a
fluorescent compound, usually manganese-acti-
vated zinc silicate, is added to the adsorbent
Chromoplate Generation that allows the visualization of spots under UV
254-nm wavelength. The adsorbent layer itself
When we place the spotted TLC plate in the has fluorescence, but spots of analyte quench it.
developing chamber, cover it and allow undis- Compounds separated may not be UV detectable,
turbed on bench top until the solvent is about so several methods exist to visualize the spots by
half a centimeter below the top of the plate. spraying reagents [1]. Once visible, the Rf value
During the span when TLC plate is inside chamber, of each spot is determined by dividing the dis-
a partially competing process occurs as (1) in tance traveled by the product by the total distance
between the components of developing solvents traveled by the solvent (the solvent front). These
and their vapor, an equilibrium is established, values depend on the solvent used and the type
known as chamber saturation; (2) stationary of TLC plate and are not physical constants.
phase adsorb molecules from gas phase (adsorp- Evaluation can be either visible or scanner is used
tive saturation) and loaded into the surface of to measure the spot density, enabling the analyst
stationary phase; (3) the wet part of the layer with for quantitative results by the density at a specific Rf
mobile phase also interacts with gas phase; and (4) value and then calculated the marker compound
during migration, the components of the mobile in the sample with reference to area [2]:
34 2 TLC: Herbal Drugs and Fingerprints

Area (sample) × Volume (standard) plate side by side (or on top of each other, i.e.,
% ingredient = co-spotting) with the compound in question. If
Area (standard) × Volume (sample)
Cone. of standard two substances have the same Rf value, they are
× × 100 likely (but not necessarily) the same com-
Cone. of sample
pound. If they have different Rf values, they are
definitely different compounds. Notable point
Retention Factor is that this identity check should be performed
on a single plate because it is difficult to dupli-
The retention factor (Rf) is defined as the distance cate all the factors which influence Rf exactly
traveled by the compound divided by the distance from experiment to experiment.
traveled by the solvent, from the baseline, for
example, if a compound travels 2.1 cm and the
solvent front 2.8 cm, the Rf is 0.75:

Solvent front
new position
of compound
Rf = distance travelled by the compound
distance travelled by the solvent
2.1 cm 2.8 cm

origin

Rf = 2.1 = 0.75
2.8

Presentation of TLC Results Procedure to Determine Rf Value


of Unknown
The position of substance zone (spot) in the
TLC can be described with the aid of the reten- On a TLC plate, mark a spot with pencil approxi-
tion factor Rf, which is defined as quotient mately 1 cm from the end of the slide, far enough
obtained by dividing distance between the sub- so that it will stay above the solvent in the devel-
stance zone and the starting line by the distance oping jar. Pens should never be used on TLC
between solvent front and starting line. Rf value plate because the ink will also develop as spots
does not give any information about the meth- in the plate. Care is to be taken not to disturb
odology used and other boundary parameters. the surface of the silica while marking. On top of
When Rf value is multiplied by 100, it is the plate, label the spots with pencil according
referred as hRf value. Before presenting the to choice (i.e., A = anthracene, C = cholesterol,
results, selectivity reproducibility and robust- T = test sample). Notable point, never to touch a
ness of the analytical method are necessary. TLC plate on the face, only on the sides and back
The Rf for a compound is a constant from one are used; otherwise, extra spots may appear. The
experiment to the next, only if the chromato- plate now looks like as below (Fig. 2.7).
graphic conditions are similar (i.e., solvent Now using volumetric capillary/clean cutoff-
system, adsorbent, thickness of the adsorbent, flat syringe needle, dip it in the solution to be
amount of analyte mounted, and temperature). spotted. The liquid rises in the needle by capil-
If the identity of a compound is suspected but lary action. Now very briefly touch the needle to
not yet proven, an authentic sample of the com- the TLC slide on the pencil mark to spot the
pound, or standard, is spotted and run on a TLC material. The spot should be as small in diameter
Thin-Layer Chromatogram 35

Fig. 2.7 Pre-spotting plan Bottom of slide Top of slide


• A
on the TLC plate
(Sideways view of plate) • C
• T

as possible to keep the spots sharp and must not proportional to the actual amount of lipid in the
run into another spot. Let the first spot dry, then spot. The method consists of (1) exposing the
touch the needle down on top of it two or three developed plate to iodine vapor, (2) spraying it
more times to ensure adequate sample. Between with a suitable solvent to prevent halogen evapo-
spotting new samples, the needle should be ration, (3) collecting the stained lipids by scrap-
cleaned with suitable solvent. Now let the spot ing the spots off the plate, and (4) determining by
dry, then carefully place the TLC plate in a devel- a rate-sensing method the absorbed iodine. The
oping chamber/jar containing about ½ cm of the method has been successfully applied to analysis
appropriate mobile phase on the bottom. Cover of several common phospholipids, long chain
the chamber/jar to ensure saturation of the air in fatty acids, cholesterol, etc. [3].
the chamber with solvent. Two or three plates
may be developed at the same time, if desired.
When the mobile phase has reached about three Documentation of Fingerprints
fourths of the way up the plate, take it out of the
chamber and quickly mark the solvent front with The choice for fingerprints depends on the nature
pencil before it evaporates. Let the plate dry and of the constituents that is present in plant material
visualize the spots in the following way. First, or on customer’s specification. TLC is widely
hold the plate under an ultraviolet light and mark employed in herbal authentication, and the major-
the spots that fluoresce (never look directly at a ity of pharmacopoeial monographs for herbs
UV light). Next step is to place the plate in iodine include a TLC identification test. TLC separates
chamber and mark new spots, if any that become mixtures of compounds to leave a “fingerprint”
visible. Third, after removing the plate from the of separated compounds on a plate coated with
iodine chamber, dip it quickly into a 2% solution silica gel. This fingerprint can be compared with
of phosphomolybdic acid in 95% ethanol, wipe that of an authentic sample or pure reference
off the back, and set it on a warm hot plate. The compounds. The chemical profile (fingerprint) of
hot plate should be on a setting low enough not to raw material or of intermediate product (specific
melt the plastic plate but high enough to cause the extracts) or of the finished products against refer-
spots to appear. If the plate curls, it may be neces- ence material defines claims made on certificate
sary to hold it down with tongs or wire gauze. of analysis as routine methodology for quality
When the dark green, orange, or brown spots control (Fig. 2.8) [3].
have appeared, remove the plate from the hot
plate. Measure the distance from the original
position of spotting to the spot and to the solvent Two-Dimensional TLC
front. Calculate the Rf value and record it [3].
When the analyte to be studied is unknown, there
may be many components of very close polarity,
Spot Development by Iodine Vapors and clear-cut separation of the components may
not be achieved. In such cases, two-dimensional
Iodine vapor is used for quantitative estimation of TLC (2D-TLC) separation has advantage
lipids on TLC plates is based on the fact that most (Fig. 2.9). In this case, a single spot of a mixture
lipids can be stained by iodine vapor, in con- is applied near to one of the corner of a 20 × 20-cm
trolled conditions, and the intensity of staining is plate and developed in one direction as usual.
36 2 TLC: Herbal Drugs and Fingerprints

Fig. 2.8 TLC fingerprints (where 1 = reference, 2 = sample). saponins during extract (20% asiaticoside) preparation.
TLC No. 1: Fingerprint of forskolin during purification TLC No. 4: Fingerprints of withanolides in Withania
from Coleus forskohlii. TLC No. 2: Fingerprint of baco- somnifera for herb selection
sides during herb selection. TLC No. 3: Fingerprint of

Fig. 2.9 2D-TLC of C. forskohlii root extract [3]


Thin-Layer Chromatogram 37

Fig. 2.10 Toxicity profiles for 15 wastewater samples; decreased luminescence indicates toxic substance zones
(Source: ChromaDex)

The plate is then removed, dried, and redeveloped properties, combines TLC with in situ bioassay,
in a second mobile-phase system so that direction and allows localization of active constituents in
of solvent flow is at right angle with respect to the complex mixture. Agar diffusion, direct TLC
first run. The spot location is detected as previous bioautography, and agar overlay bioautography
case. Each spot will with new Rf values [3]. are in general practices. For discovering new
antioxidants in herbals, TLC plate is sprayed with
2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical.
TLC Bioautography Antioxidant reduces the radical, producing white
spots on the purple ground. The TLC bioautog-
Use of TLC for chemical and biological screening raphy is a future tool for the study of herbal for-
together is known as “TLC bioautography,” a mulation to understand/explain the synergistic
multidisciplinary approach, which provides a phenomena in new drug discovery [3].
ground for efficient collaboration of different
disciplines for a new drug discovery as well as
analysis of food and feed. It is based on the fact Bioluminescence and TLC Analysis
that toxins or other negatively acting agents
reduce the metabolic activity of the microbes, Bioluminescence is the production and emission
which is proportional to the luminescence and of light by a living organism as the result of a
the stability of compounds on the TLC plate chemical reaction during which chemical energy
which can be verified easily. For a new drug dis- is converted to light energy. The bioluminex
covery, isolation of single component, followed assay is a unique biosensor method that directly
by assay for its biological activity, is a tedious couples natural bioluminescence to TLC (Fig. 2.10).
and expensive process. Under such stress, TLC This rapid assay can be used to support material
bioautography has a good compatibility with identity, detect toxins and chemical adultera-
large number of samples which can be studied tions, identify potential bioactive compounds, and
for various activities (e.g., acetylcholinesterase monitor manufacturing processes. TLC has tradi-
inhibitor, antibacterial, antifungal, free radical tionally been used as a reliable and economical
scavenger), whose biological properties have not analytical technique for the simultaneous separa-
been documented earlier. This technique is used tion of multiple samples using a minimum of harsh
for selection of herbals to study therapeutic chemicals or solvents. A developed TLC plate is
38 2 TLC: Herbal Drugs and Fingerprints

coated with the nonpathogenic, bioluminescent dietary supplements screening (raw materials and
marine bacteria Vibrio fischeri. The compound product), in veterinary applications (testing feed,
which interferes with the metabolic process of the supplements, and urine), in testing for toxic
bacteria inhibits bioluminescence and is, there- residues in soils, in testing for residues in manu-
fore, detected as contrasting dark spots on the facturing equipment, in validation of organic
luminescent background of the TLC plate. Activity products or certify soil, and in equipment and
is measured by a reduction in light emission of water used in organic farms as well as in determi-
the bacteria producing a toxicity pattern charac- nation of an unknown compound’s toxicity.
teristic of each analyzed sample that can be
viewed and quantified directly on the TLC plate.
Results occur within minutes and can be docu- Detection of Colorless Compounds
mented photographically (CCD camera, X-ray,
Polaroid, or 35-mm film). This technology has In general, the detection of colored compounds
been developed into a kit system that provides an causes no problem, and the same is true for com-
effective means of prescreening a variety of pounds which exhibit fluorescence or phospho-
complex mixtures in order to minimize the use of rescence under ultraviolet light. In some cases
environmentally costly conventional methods [3]. where the color is not very intense, a spray
Bioluminex has been tested with a wide variety reagent may be used to increase the sensitivity
of toxins, from naturally occurring biotoxins by spraying the chromatogram. There are numer-
such as biocides to heavy metals. Bioluminex ous spray reagents which can be used to make
sets the standard for consistent regulatory use the various colorless compounds visible on the
and helps ensure the quality of products, from chromatogram. These can be divided into two
biomass to bottle. Standard toxicity tests only classes: (1) those which are general reagents and
establish the overall toxicity (biological activity) will detect a large number of different types of
for complex mixtures like wastewater or crude compounds (e.g., 10% H2SO4, iodine vapor) and
natural product extracts. Identification of the (2) those which are more specific in nature, indi-
active compounds requires the tedious isolation of cating the type of compound or functional group
single components followed by assays of their that is present (e.g., Dragendorff’s reagent for
biological effects. In addition, there is the risk of alkaloids, potassium hydroxide for coumarins
false results due to interference or interaction and anthraquinones, ferric chloride for tannins
by any number of compounds in complex sub- and phenolic compounds, ninhydrin for alpha
stances. Bioluminex overcomes the limitations of amino acids) [3].
this conventional approach by specifically assign-
ing biological activity to single components of
mixtures. Because of parallel sample processing, Combination of TLC with Other
the TLC bioluminescence technique represents a Techniques
versatile and rugged method with high sample
throughput. Bioluminex has been successfully TLC has been directly coupled with column chro-
validated in environmental applications for matography by using various splitters with a vari-
food samples, natural products, and a multi- able drive to control the application of a portion of
tude of toxicity-related problems. Furthermore, the column elute to the TLC plate. Similarly, TLC
ChromaDex has customized this advanced TLC– is used for the selection of proper mobile phase to
bioassay technique for specific research needs by HPLC and optimizes the analytical conditions.
employing genetically engineered microorgan- The spots obtained from TLC may be eluted, con-
isms, as BioluminexTS Kit, available in market. centrated, and then subjected to either HPLC or
The technique has wide scope in the analysis GLC analysis. The high boiling substances are
of drinking water and wastewater/effluent for preferred for TLC–GLC analysis. Mass spec-
screening biocide, leaching, in food, beverage, trometry (MS) and TLC are combined together to
Thin-Layer Chromatogram 39

know the molecular weight of the compound and and are narcotic in high concentrations. Chloroform
fragmentation patterns. Desired spot from TLC is an anesthetic and a possible carcinogen under
with defined Rf value can be scraped, dissolved in conditions of daily exposure for several years.
suitable solvent, filtered, and may be studied by All organic solvents and plate developing cham-
NMR or IR spectrometry for structure elucida- bers are kept in the fume hood to avoid solvent
tion, for authentication of the target component. exposure [3].

Criteria for Selectivity, Reproducibility, Troubleshooting in TLC Analysis


and Robustness
The aforesaid steps seem that TLC is quite an
TLC analysis mainly concerned with determi- easy procedure, but what about the first time we
nation of identity, purity, and assay or together run a TLC, and see spots everywhere and blurred,
all, for which two- or multidimensional TLC streaked spots, etc., as with any technique, with
are the options. The selection of stationery practice we get better. A few notable points during
phase with analyte is a part of experience and process development for unknown compounds
theoretical knowledge to the chemical compo- may be helpful as [3]:
sition of stationary phase. For satisfactory sep- 1. When compound runs as a streak rather than a
aration efficiency, the mean particle size, spot indicates that sample is overloaded. To
particle size distribution, and morphology of have desired results, run the TLC again after
particle are to be considered. It is advisable to diluting sample, or sample might just contain
use prepared TLC plate to secure better repro- many components, creating many spots which
ducibility of good fame for quality. Solvent run together and appear as a streak. Perhaps,
system up to six components is used, provided the experiment did not go as well as expected,
that this must have the appearance of single- change mobile phase as per own analytical
phase system with no sign of cloudiness. The judgment.
analogy between solvent system and mobile 2. When sample runs as a smear or an upward
phase becomes logical when we use mixture of crescent, compounds which possess strongly
solvents, as the solvent system placed in the acidic or basic groups (amines or carboxylic
development chamber releases some of its acids) sometimes show up on a TLC plate
components into the pores of stationery phase, with this behavior. Addition of a few drops of
where it forms a liquid stationary phase. In this ammonium hydroxide (amines) or acetic acid
equilibrium, the mobile phase and solvent have (carboxylic acids) to the eluting solvent helps
different meaning [3]. to obtain a clear plate.
3. If sample runs as a downward crescent, likely
the adsorbent may be disturbed during the
Special Hazards spotting, causing the crescent shape.
4. If plate solvent front runs crookedly, either the
Looking directly at an ultraviolet (UV) light tends adsorbent has flaked off the sides of the plate
to cause eyes cataracts, and shining it on the skin or the sides of the plate are touching the sides
promotes skin cancer. For these reasons, the UV of the container (or the paper used to saturate
lamp should be kept in the hood and only held the container) as the plate develops. Crookedly
over the TLC slides for a few seconds to visualize run plates make it harder to measure Rf value
them. The lamp should be pointed downward at accurately.
all times and turned off immediately after use. 5. Many random spots are seen on the plate,
UV rays are absorbed by glass, so a transparent indicating that during operation analyst have
glass is used in UV cabinet to observe the TLC accidentally dropped any organic compound
plate. Cyclohexane and toluene are flammable on the plate.
40 2 TLC: Herbal Drugs and Fingerprints

Fig. 2.11 TLC of Equisetum species, where: 1 = rutoside, 2 = hyperoside, 3 = caffeic acid, 4 = E. arvense, 5 = E. arvense
(China), 6 = E. palustre, 7 = E. arvense [4]

Identification of Marker Compounds closely resemble to the genuine drug. Similarly,


in Herbal Drugs addition of synthetic to fortify inferior products,
such as adding citral to the oil of lemon or benzyl
TLC is used for the analysis of aliphatic mono- benzoate to balsam Peru, can be checked.
carboxylic acids, keto acids, hydroxy acids, In an attempt to establish a certified quality of
dicarboxylic acids, aromatic carboxylic acids, marketed herbal drugs, crude, powdered, or in
phenolcarboxylic acids, alcohols (except methanol combination, TLC is used to decipher the active
and ethanol) and glycols, their derivatives, alkaloids principals, identification of claimed herb, and
(purine, pyridine, phenyl alkylamine, dipyridine, possible contamination/adulterants. The TLC
pyridine–pyrrolidine, quinoline, isoquinoline, method is efficient, rapid, and combines the sen-
indole, and pyrrolizidine), using Dragendorff sitivity and simplicity with low cost for the deter-
reagent, amino acids, proteins, peptides, antibiotics, mination of main active principles of medicinal
carbohydrates, dyes (both oil soluble as well as plants (alkaloids, anthraquinones, coumarins,
water soluble), hydrocarbons (using hexane as essential oils, flavonoids, glycosides, saponins,
mobile phase in silica TLC plate), lipids, nucleic tannins, etc.), which are the main ingredients
acids and nucleosides, pesticides (chlorinated, responsible for potential pharmacological effects.
phosphorus, carbamates, pyrethrins, etc.), phenolic The technique is so efficient that it is being used
compounds, screening of pharmaceuticals (samples as primacy analytical tool in the new drug discov-
for drug of abuse, sulfa drugs, contraceptives, ery for herbal drugs. Herbal drugs are obtained
laxatives, etc.), flavonoids, steroids, vitamins, from cultivated or wild plants. They vary more or
antioxidants, inorganic ions, etc. The herbal raw less in composition and properties depending on
material as well as herbal drugs may have adul- the habitat, climate zone, and annual variations.
teration; hence, the proper identification of This causes problems in the identification and
material is essential before use. There is simple purity tests of herbal drugs.
quantitative test by TLC against the reference
standard, as the fingerprints and TLC profile Case Study 1: There are many Equisetum sub-
clearly indicate about the adulteration. In this species and hybrids species described in litera-
way, substitution by exhausted drugs can be ture. These Equisetum species along with having
avoided as the dried exhausted cloves and umbel- alkaloid (species like Equisetum palustre) have to be
liferous fruits after extraction of their volatile oil detected by TLC analysis (Figs. 2.11 and 2.12) [4].
Identification of Marker Compounds in Herbal Drugs 41

Fig. 2.12 E. arvense and E. palustre in TLC analysis [4]

Case Study 2: Passionflower (Passiflora incar- developed to a distance of 150 mm at room tem-
nata) is used in phytotherapy as a mild sedative perature. Detection was by way of two spray
and anxiolytic agent and has considerable quali- reagents, natural products reagent (2-aminoethyl
tative and quantitative variability with respect to diphenylborinate, sigma, 1% in methanol) fol-
its content of C-glycosyl flavones, some of which lowed by Macrogol 400 (polyethylene glycol,
are used as marker compounds for extracts. sigma, 5% in methanol) [5].
Analysis of plant material cultivated in Australia
revealed two chemically distinct groups; hence, TLC Method 2: This method was adopted from
an investigation was carried out to determine Widmer, Meier, and Schaffner and was published
whether distinct intraspecific chemotypes exist in by CAMAG, Switzerland, on their website (http://
this species. Eleven P. incarnata samples were www.camag.com/index.php; n.d.). Mobile phase
analyzed by HPLC, LC–MS, and two different was composed of tetrahydrofuran-toluene-formic
TLC methods. The samples fell into two distinct acid-water (16:8:2:1, v/v), and the plates were
groups with respect to their C-glycosyl flavone developed to a distance of 52 mm at room tem-
profile, with little within-group variation. One perature. The heated plates were sprayed with
chemotype was dominated by isovitexin and natural products reagent (0.5% in ethyl acetate)
schaftoside/isoschaftoside. The other chemotype followed by polyethylene glycol 4000 (Fluka,
was characterized by a high level of swertisin, 5% in dichloromethane) [5].
with low levels of schaftoside/isoschaftoside. TLC Method 2 was superior to the Method 1(i.e.,
The two chemotypes were readily identified by the British Pharmacopoeial method) in terms of
both HPLC and TLC [5]. clarity and resolution, and Method 2 allowed for
the differentiation between the two P. incarnate
TLC Method 1: This was the method for chemotypes (Fig. 2.13) [5].
passionflower in the British Pharmacopoeia Similarly, qualitative analysis for different
(2007). Mobile phase was composed of water- chemical classes of therapeutics, using different
anhydrous formic acid methyl ethyl ketone ethyl mobile phases (Table 2.1), can be performed by
acetate (10:10:30:50, v/v), and the plates were TLC as:
42 2 TLC: Herbal Drugs and Fingerprints

Fig. 2.13 TLC plate showing P. incarnata swertisin chemotype (1–6) and isovitexin chemotype (7, 8), where C = chlo-
rogenic acid, H = hyperoside, R = rutin [5]

Table 2.1 Brief list of common solvent systems for TLC [3]
Chemical group Absorbent Solvent system frequently used Detection
Alkaloids Silica gel 1. Methanol–chloroform (85:15) 1. UV
2. Toluene–ethyl acetate– 2. Dragendorff
diethylamine (70:20:10)
Anthocyanins Silica gel, cellulose n-butanol–acetic acid–water 1. UV
(40:10:20) 2. Anisaldehyde- sulfuric acid
Cardiac glycosides Silica gel 1. Ethyl acetate–methanol–water 1. Kedde reagent
(81:11:8)
2. Chloroform–methanol–water 2. Antimony chloride
(65:35:10)
Flavonoids Silica gel Chloroform–acetone–formic acid UV
(75:16.5:8.5)
Indoles Silica gel Chloroform–ethyl acetate–formic p-Dimethylamino
acid (5:4:1) cinnamaldehyde
Monosaccharides Silica gel n-butanol–acetic acid–ether–water 1. Aniline hydrogen phthalate
(9:6:3:1) 2. UV
Phenols Silica gel Acetic acid–chloroform (1:9) Folin reagent
Polyacetylenes Silica gel Chloroform–methanol (1:9) 10% H2SO4
Saponins Silica gel 1. Chloroform–methanol–water 1. Vanillin/ sulfuric acid
(60:35:5)
2. n-butanol–water (1:1) (upper 2. Anisaldehyde sulfuric acid
phase)
Terpenes Silica gel 1. Chloroform–methanol (95:5) 1. 10% Sulfuric acid
2. Ethyl acetate–cyclohexane (60:40) 2. Anisaldehyde- sulfuric acid

TLC Analysis for Alkaloids in analysis of new unknown herbo-combination


for alkaloids by TLC, subsequently developing
The systematic procedure for the analysis of spots with Dragendorff’s reagent. The most
alkaloids by means of thin-layer chromatography, preferred adsorbent TLC plate for alkaloids has
with the mixture of cyclohexane-chloroform- been silica gel 60 F254. Once detecting the alka-
diethylamine (5:4:1) as screening the alkaloids loid, the mobile phase is determined based on
on the basis of polarity and further analysis using satisfactory and validable parameters. The authors
chloroform–acetone–diethylamine (5:4:1) and were benefited by the above concept during
chloroform–diethylamine (9:1), is still a guideline developing in-house protocol for Gloriosa glabra
Identification of Marker Compounds in Herbal Drugs 43

methanolic extract, evaluating for both colchicines tannins, and lignins). For simple phenols, the
and colchicoside in a single TLC plate using best solvents are petroleum ether (60–80°C)-carbon
mobile-phase chloroform–methanol (8.5:1.5) and tetrachloride-acetic acid (4:6:1) and chloroform–
evaluating at 254 nm, previously as it was been acetone–diethylamine (4:2:0.2). Polyhydric
done in two steps: chloroform–methanol (9.5:0.5) phenols are best separated by using chloroform-
for colchicines and chloroform–methanol (8:2) acetic acid (5:1), chloroform–acetone–acetic
for colchicoside. The best industrial application acid (10:2:1) and benzene–acetic acid (5:1).
of TLC is the determination of selected substance in These spots are made visible by spraying with an
pharmacopoeial products, when it is being measured acetone solution of p-nitrobenzene diazonium
at the level of less than 0.1% (e.g., related sub- fluoroborate. Phenolic compounds are ubiquitous
stances in yohimbine, berberine hydrochloride, in the plant kingdom, being the most abundant
thiocolchicoside, reserpine as pharmacopoeial secondary metabolites. Though bio-phenols are
product especially in USP). Test solution: A found in all plants, their quantitative distribution
50-ml separating funnel was charged with 10 ml varies between different tissues of plant and
of the preparation to be tested added 1.0 ml of within different populations of the same plant
concentrated aqueous ammonia and 10 ml of species. Bio-phenols of fruits, vegetables, herbs,
chloroform, after which the mixture is shaken for spices, and cereals have high therapeutic poten-
3 min. Then the chloroform extract is separated tial and are in use as herbal drug. TLC is the
and, in the case of emulsification, centrifuged for primary one and best screening technique for
3 min at 2,500 rpm to complete phase separation. bio-phenols analysis. Lignin, the second most
Reference solution: The reference solution is abundant compound in the nature, on hydrolysis,
prepared by dissolving 0.01 mg of sanguiritrine gives bio-phenols. Methanol has been reported as
(working sample) solution in 19.5 ml of methyl a suitable solvent for short-time extraction as
alcohol and 0.5 ml of ammonia solution. Spotting phenolic glycosides degraded with longer span.
on TLC plate: Sample of the test solution (30 ml) The primary characterization of phenolics is
and of the reference solution (20 ml) was applied usually done by TLC [3].
on the start line of a TLC plate (silica gel 60 F254).
The plate is dried in air for 3 min, placed into a
vertical cell with the diethyl ether–petroleum TLC Analysis for Saponins
ether–methanol mixture (35:15:1) and chromato-
graphed in the ascending mode. When the solvent Saponins are glycosides which produce stable
front reaches the end of the path, the plate is foams when their aqueous solution shaken. On
extracted from the cell, dried in air at room acid hydrolysis, saponins split into sugars and
temperature until the solvent mixture is removed, the corresponding sapogenins. Saponins based
and examined on exposure to UV radiation with on the carbon skeleton of sapogenin are termed
a wavelength of 360 nm. The chromatogram of as terpenoid saponins (e.g., Centella asiatica,
sanguiritrine shows two spots: orange (sangui- Bacopa monnieri, Stevia rubidiana) and steroidal
narine) and yellow (chelerythrine). The chro- saponins (e.g., Tribulus terrestris, Withania
matogram of the test solution must display band somnifera, Gymnema sylvestre) were analyzed
of the same color and mobility (i.e., Rf value) as by TLC, in authors lab, using mobile-phase chlo-
those on the reference pattern [3]. roform – methanol and water in different compo-
sitions and subsequently developing spots with
anisaldehyde–sulfuric acid and drying plate at
TLC Analysis for Phenols 110°C for 10 min. The spots were evaluated
against reference preparation, either as qualitative
Volumes have been written about the TLC anal- or quantitative determination (withanolides in
ysis of phenolic compounds (derivatives of benzoic Withania somnifera, have steroidal skeleton, but
acid, cinnamic acid, coumarins, flavonoids, these are steroidal lactones). Chromoplates were
44 2 TLC: Herbal Drugs and Fingerprints

examined before and after spraying under UV the final results. High-performance liquid
and day light. The individual optimization of chromatography on reversed-phase columns
TLC techniques was helpful in the process devel- remains the best technique for saponin determi-
opment of purification at higher purity levels of nation and is the most widely used method for
phytochemicals (>98% purity of the ingredient) this group of compounds. However, the lack of
to detect the impurity profile of the ingredient, chromophores allowing detection in UV limits
using organic solvents with water (sometimes in the choice of gradient and detection method. The
different pH values, as buffer, etc.), as well as to pre-column derivatization with benzoyl chloride,
develop HPLC analysis methods, selection of coumarin, or 4-bromophenacyl bromide has been
analytical column, and to determine combination used successfully in some cases, allowing UV
of mobile phase and gradient parameters, as well detection of separation. Standardization and
as regeneration of column prior to the next identification of the peaks in HPLC chromato-
analysis. grams have been based on comparison of the
As plant saponins are sensitive to the struc- retention times with those observed for authentic
tural variation (e.g., saponins mostly are hydro- standards. But new hyphenated techniques,
philic, but digoxin is also lipophilic), so these are combining HPLC with mass spectrometry and
standardized with saponin mixtures isolated from nuclear magnetic resonance, are developing
the plant species in which the concentration is rapidly and allow online identification of sepa-
measured (gravimetric analysis). However, one rated saponins. Capillary electrophoresis has
plant species may contain some saponins which been applied for saponin determination only in a
can be determined with a biological test and limited number of cases, and this method is still
others cannot be. That is why biological and being developed [3].
colorimetric determinations do not provide accu-
rate data and have to be recognized as approximate
(hence gravimetric analysis is preferred). TLC (on TLC Analysis for Terpenoids
normal and reversed phases) and 2D TLC provide
excellent qualitative information and in combina- The low polar nature of terpene and its deriva-
tion with online coupling of a computer with tives, solvents of low polarity, are used for TLC
dual-wavelength flying-spot scanner and two- separation of these components; spots are mea-
dimensional analytical software which can be sured either UV-visible or developed using iodine
used for routine determination of saponins in vapors or 5% sulfuric acid after iodinization and
plant material. The densitometry of saponins has heating at 110°C for 10 min in oven. Author in
been very sensitive, however, to plate quality, his laboratory is evaluating forskolin from Coleus
spraying technique, and the heating time, and forskohlii, using cyclohexane-ethyl acetate (6:4)
therefore appropriate saponin standards have to and developing spots with 7.5% H2SO4 after
be run in parallel with the sample. Gas–liquid iodinization and subsequently heating it at 110°C
chromatography has limited application for deter- for 10 min. The technique is too valuable to assay
mination since saponins are quite big molecules purity of forskolin which matches with HPLC
and are not volatile compounds. Thus, there are assay. The TLC fingerprints for essential oils
only few applications of GC for determination Ocimum sanctum (tulsi), Centella asiatica (gotu
of intact saponins. The method has been used kola), and Convolvulus pluricaulis (shankh-
for determination of TMS, acetyl or methyl pushpi), in a combo-herbal drugs, were developed
derivatives of an aglycones released during using mobile-phase toluene–ethyl acetate (85:15),
saponin hydrolysis. However, structurally differ- which was evaluated at 365 nm after drying in
ent saponins show different rates of hydrolysis, hot air, and finally spots were developed using
and precise optimization of hydrolysis conditions 7.5% H2SO4 and heating plate at 110°C for
is essential. Besides, during hydrolysis, a number 10 min in oven, by the authors, where separate
of artifacts can be formed which can influence marker appears for individual ingredient, using
Identification of Marker Compounds in Herbal Drugs 45

b c
N O
100 N
H N

Relative intensity
288.11301[MH]+
rutaecarpine
[MH]+;288.1107
ritaecarpine
a m/z
0
200 250 300 350 400 450 500

N O
100 N
[MH]+ H
Relative intensity
304.14758 N
H2C
evodiamine
[MH]+;304.1450
evodamine

m/z
0
200 250 300 350 400 450 500

Fig. 2.14 Evodiae fructus extract in TLC DART–MS [8]. (a) Evodiae fructus, (b) TLC chromatogram of the extract of
Evodiae fructus, under 365 nm, (c) DART–MS spectra of rutaecarpine and evodiamine

against reference botanical material (RBM). shaken for 3 min, and hexane phase was transferred
While in another combo-herbal formulation, the into a 50-ml round-bottom flask. The extraction
TLC fingerprints for O. sanctum were developed was repeated with the same amount of n-hexane.
by ether-hexane (7:3), and spots were developed Then extracts are combined, and the solvent is
with 2% H2SO4 and drying it at 110°C for 10 min, distilled off to dryness in a rotary evaporator at a
reddish fluorescence marker for O. sanctum, vis- temperature not exceeding 50°C. The residue was
ible at 365 nm. The polarity of compounds dissolved in 1 ml of ethyl acetate. Reference solu-
increases with increasing number of –OH groups, tion: The reference solution is prepared by dis-
while the alkyl derivative of hydroxyl group solving 10 mg of menthol in 2 ml of ethyl acetate.
decreases the polarity, and further increase in size Spotting on TLC plate: Sample of the test solution
of alkyl group again decreases the polarity. (20 ml) and of the reference solution (5 ml) were
Hydrogenation of side chain had only slight effect applied, on the start line of a TLC plate [silica gel
on polarity; similarly, hydrogen band formation 60 F254, (mobile-phase benzene: ethyl acetate
also reduces the polarity in terpenoids. In order to mixture) (5:1)] and chromatographed in the
select the optimum mobile phase for TLC, mobile ascending mode until the solvent reaches a level of
phase was toluene–ethyl acetate (4:1), n-hexane– 10 cm in the plate and dried the plate in air at room
ethyl acetate (9:1), hexane–chloroform (5:1), and temperature until the solvent disappears. Then the
benzene–ethyl acetate (5:1). The best separation plate is treated with anisaldehyde solution and
of components was achieved with the last solvent heated at 105°C for 10 min. The developed chro-
mixture. The bands of terpenoids in the chro- matogram is examined in daylight [3].
moplate were revealed by treating the TLC plates
with an anisaldehyde–sulfuric solution, followed
by heating to 100°C for 10 min. TLC with DART–MS

Test solution: A 50-ml separating funnel was Using TLC, to generate more versatile and
charged with 10 ml of the preparation to be tested specific information on extract of herbal drugs,
10 ml of n-hexane; after which the mixture is “direct analysis in real time (DART) ion source”
46 2 TLC: Herbal Drugs and Fingerprints

is being in use. This hyphenation system of TLC


and DART–MS provides unique and specific TLC Fingerprints for Batch to Batch
information on the major constituents of crude Consistency
plant drug on TLC through uncovering high-
resolution mass number of each band on the TLC A study for TLC fingerprints of an Unani formu-
plate directly in real time. lation, named “Majoon-e-Sandal,” which is
widely used in the ailments of stomachic, antibil-
Case Study-1: The three well-known Korean ious, psychoneurosis, vomiting, and nausea, and
Pharmacopoeial herbal drugs were extracted prepared by mixing the powders of the Santalum
and developed on a silica-coated TLC plate album (110 g), Bambusa bambos (15 g), and
with preestablished conditions, as per Korean Styrax benzoin (15 g) for the formulation compo-
Pharmacopoeia IX, and developed TLC plate sition and kept separately. Tamarindus indica
were placed between the DART ion source and was soaked in water for 2 h, crushed with hand,
TOF-MS analyzer to get real-time mass spectra and filtered through muslin cloth and kept sepa-
from the bands on the plate directly. The marker rately. Punica granatum seeds were crushed with
coumarin compounds, decursin and decursinol, hand and filtered it through muslin cloth and kept
were successfully identified from the TLC plate separately. Crocus sativus was grinded by adding
developed with Angelicae gigantis radix, along rose water and kept separately. Dissolve 750 g of
with alkaloid compounds of rutaecarpine and sugar in 500 ml of water; at the boiling stage,
evodiamine from Evodiae fructus and lignan 0.1% citric acid was added, mixed thoroughly,
molecules of gomisin A, N, and schisandrin from and filtered through muslin cloth. Then, boil the
Schisandrae fructus (Fig. 2.14) [6]. filtrate on slow heat and add the mixed extract
of Tamarindus indica and Punica granatum
Case Study-2: The curcuminoids (mixture of followed by Crocus sativus. Then, with the content
curcumin, demethoxy curcumin, and bis- mixed thoroughly, prepare the 79% consistency
demethoxy curcumin) were successfully detected of quiwam. Remove the vessel from the fire,
directly from the raw rhizome of Curcuma longa while hot condition is added to the mixed powders
when a turmeric extract was separated on a TLC of the Santalum album, Bambusa bambos, and
plate, studied with DART–MS, each band pro- Styrax benzoin, followed by 0.1% of sodium ben-
duced molecular ion peaks corresponding to cur- zoate; mix thoroughly to prepare the homogenous
cumin, demethoxy-curcumin, and bis-demethoxy product. Allow to cool to room temperature, and
curcumin. Molecular ions of curcuminoids in packed it in tightly closed container to protect
turmeric-containing beverages and curry powder from light and moisture. Majoon-e-Sandal is a
were also efficiently detected at the range of semisolid, brown-colored characteristic of its
5–100 mg/ml. To establish the validity of analytical own odor and in sweet taste [9].
methods for curcuminoids (curcumin and its
derivatives) from various types of samples with Preparation of extracts: From three different
DART–MS, data were compared with the results batches, 2 g of sample, each separately, was
of HPLC and were strong proofs for specificity of soaked in chloroform and alcohol, respectively,
the method. As DART–MS produces [M + H]+ for 18 h, refluxed for 10 min on water bath, and
molecular ions of most compounds, so relatively filtered. The filtrates were concentrated on water
simple and clear mass spectra are obtained even bath and made up to 5 ml in a standard flask sepa-
of multicomponent samples [7]. In order to take rately. Development of TLC fingerprints: The
advantage of the capacity of DART–MS for the chloroform and alcohol extracts were applied on
real-time analysis of individual compounds in precoated silica gel 60 F254 TLC plate (E. Merck)
natural raw materials, the technology has a huge as absorbent and developed the plate using sol-
scope in herbal drug industries [8]. vent systems, toluene : ethyl acetate 9:1 and 1: 1,
References 47

Fig. 2.15 TLC for chloroform extracts [9]


Fig. 2.16 TLC for alcohol extracts [9]

respectively. After developing, the plates were purity of the plant material under consideration; in
dried and observed the color spots at UV-254, addition, it also provides information about substi-
UV-366 nm, and spots were developed using van- tution and adulteration.
illin–sulfuric acid spraying reagent. TLC studies
of chloroform and alcoholic extract of all the three
References
batch samples had identical spots in UV (254,
366 nm), similar Rf values, even on development 1. Abu-Hamdah S, Afifi FU, Shehadeh M, Khalid S. Simple
with vanillin sulfuric acid reagent, after heating at quality control procedures for selected medicinal plants
105º for 10 min (Figs. 2.15 and 2.16) [9]. commonly used in Jordan. Pharm Biol. 2005;43(1):1–7.
2. Sachan AK, Sachan NK, Kumar S, Sachan A, Gangwar
TLC is a widely applied technique in herbal
SS. Evaluation and standardization of essential oils for
authentication and used in majority of pharmaco- development of alternative dosage forms. Eur J Sci
poeia’s monograph for correct identification. Using Res. 2010;46(2):194–203.
Co-TLC, unknown compounds can be easily 3. Joshi DD. Thin layer chromatography and biotechnol-
ogy at molecular level. In: Textbook of molecular bio-
identified with an authentic compound, so it is
technology. New Delhi: I.K. International Pub. House;
widely adopted because of less time-consum- 2009. p. 1181–96. Sample Chapter 53.
ing, semiquantitative, and a cheap technique. 4. Klier B. Current problems with identification of
Furthermore, it also provides fingerprints of the herbal drugs. PhytoLab GmbH & Co. KG 91487
Vestenbergsgreuth Germany. http://www.ga-online.
material under consideration; if the marker com-
org/files/Graz/WS-4_Klier.pdf. Accessed 29 Mar 2012.
pound is known, then it becomes more precise, so it 5. Wohlmuth H, Penman KG, Pearson T, Lehmann RP.
is an important tool for monitoring the identity and Pharmacognosy and chemotypes of passionflower
48 2 TLC: Herbal Drugs and Fingerprints

(Passiflora incarnata L.). Biol Pharma Bull. forestry in employment generation and rural development.
2010;33(6):1015–8. IFRI Dehradun, India; 2006. p. 59–68.
6. Kim HJ, Jee EH, Ahn KS, Choi HS, Jang YP. Identification Kirchner JG. Thin layer chromatography. In: Techniques
of marker compounds in herbal drugs on TLC with of chemistry. 2nd ed. New York: Wiley; 2000. XIV.
DART-MS. Arch Pharm Res. 2010;33(9):1355–13559. Kopylova E, et al. Standardization of a complex prepara-
7. Kim HJ, Jang YP. Direct analysis of curcumin in turmeric tion for the treatment of periodontal diseases. Pharm
by DART-MS. Phytochem Anal. 2009;20(5):372–7. Chem J. 2002;36:504–6 [Translated from Khimiko-
8. Alsbou E. Ambient mass spectrometry desorption Farmatservticheskii Zhurnal. 36 (9): 44–46].
electrospray ionization (DESI) & direct analysis in real Li BY, Hu Y, Liang YZ, Xie PS, Du YP. Quality evalua-
time (DART). http://www.ecplaza.net/tradeleads/ tion of fingerprints of herbal medicine with chromato-
seller/5638482/evodiamine_98_99_hplc.html# none graphic data. Anal Chim Acta. 2004;514:69–77.
(2010). Accessed 28 Mar 2012. Pascual ME, et al. Simplified screening by TLC of plant
9. Meena R, Meena AK, Khan SA, Mangeswari S. drugs. Pharm Biol. 2002;40(2):139–43.
Evaluation of an Unani compound formulation-Majoon- Sharma J, Fried B. Handbook of thin layer chromatogra-
e-Sandal. Int J Pharm Sci Res. 2010;1(5):238–42. phy, Chromatographic Science Series, vol. 55. New
York: Marcel Dekker; 2003.
Stationary Office. British Pharmacopoeia 2007. London:
The Stationary Office; 2006.
Bibliography USP. United States pharmacopeial convention. USP
29–NF 24. Rockville: United States Pharmacopeial
Di X, Kelvin KC, Hei WL, Carmen WH. Fingerprint Convention, Inc. electronic version; 2006.
profiling of acid hydrolyzates of polysaccharides Verbitski SM, et al. Rapid screening for complex mixtures by
extracted from the fruiting bodies and spores of thin layer chromatography-bioluminescence. Technical
Lingzhi by high-performance thin-layer chromatogra- article. Am Biotechnol Lab. 2006;24(9):40–1.
phy. J Chromatogr A. 2003;1018:85–95. WHO Monographs on Selected Medicinal Plants –
Hardman R, Abu-Al-Futuh IM. The detection of isomers Volume 2. http://apps.who.int/medicinedocs/en/d/
of 4-hydroxyisoleucine by the Jeol amino acid analyser Js4927e/. Accessed 28 Mar 2012.
and TLC. Planta Med. 1979;36:79–84. Zhao LH, Huang CY, Shan Z, Xiang BR, Mei LH.
Joshi DD. Utilisation of medicinal plants for health and Fingerprint analysis of psoralea corylifolia L. By HPLC
wealth. Proceedings of national workshop on role of and LC-MS. J Chromatogr B. 2005;821:67–74.
HPTLC: Herbal Drugs
and Fingerprints 3

Plants contain thousands of constituents and are UV light, sometimes only after derivatization
valuable source of new therapeutic molecules. with a suitable reagent.
For new and effective herbal drug development, Currently, quality evaluation is a main concern
it is important to have a validated process to pre- in herbal formulations due to variation in the con-
pare plant extract and to isolate ingredients for tent of markers/active ingredients in the raw
full structure elucidation and biological testing. materials, due to different geo-climatic factors
The combination of biological and chemical and business reasons. A computerized densitom-
screening leads to the important information eter is used for the fingerprinting, of concern
about plant constituents. The chemical screen- spot, on its area and intensity, for true authentica-
ing by TLC analysis is illustrated in the form of tion of test samples, against standard. Such
hi-tech art using high-performance thin-layer chemical fingerprinting is helpful for industries,
chromatography (HPTLC) for better separation, research institutions, and regulatory authorities
eliminating manual errors, and better repeatability for quality evaluation and to decipher the claims
as well as reproducibility of the test results. It made for the products [2].
provides a great deal of preliminary information
about the content and nature of constituents
found in the active fraction. Once the chemical Operational Summary of HPTLC
nature of a constituent is established via HPTLC
analysis, it is easier to develop validated process The whole analytical process for HPTLC may be
to prepare standardized extract and isolate summarized in the following steps [3]:
ingredient in pure form, structure elucidation, 1. Selection of stationary phase for HPTLC
and biological testing with synergistic explanation analysis
[1]. HPTLC is a very simple and economical 2. Sample preparation, clean up, and pre-chro-
analytical method, useful for high-potential matographic derivatization, if any
qualitative characterization and quantitative 3. Application of sample on stationary phase
determination of herbals and products. Its field 4. Development of chromoplate
of application covers virtually all classes of 5. Detection of spots including post-chromato-
substance with the exception of readily volatile graphic derivatization
and gaseous substances and can be extended 6. Quantification
easily to the preparative scale by using thicker 7. Documentation
layers [preparative layer chromatography Stationary phase selection for a new product is
(PLC)]. The separated substances, depending based on the subject knowledge of the analyst
on their optical properties, can be detected, which is supported by the gained knowledge dur-
identified, and quantified in visible, infrared, or ing experiments and TLC analysis for the same.

D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs, 49
DOI 10.1007/978-81-322-0804-4_3, © Springer India 2012
50 3 HPTLC: Herbal Drugs and Fingerprints

Table 3.1 Comparison between silica gel pre-coated HPTLC and TLC plates [3]
Property HPTLC layer TLC layer
Particle size 5–6 mm 10–12 mm
Pore diameter 60 Å 40, 60, 80, 100 Å
Plate dimensions 10 × 10 cm, 20 × 20 cm, 5 × 10 cm, 5 × 20 cm,
10 × 20 cm 10 × 20 cm, 20 × 20 cm
Layer thickness 0.20–0.25 mm 0.20–0.25 mm
Analysis per plate Up to 75 Up to 16
Spot size recommended ~1 mm 2–5 mm
Spot loading 50–200 nl 1–5 ml
Band size recommended 5–10 mm 10–15 mm
Band loading 1–4 ml 5–10 ml
Sensitivity limit Upper pg (fluorescence) ng
Normal development time 2–30 min 15–20 min

The steps as spotting, evaluation, and documen- resolution (often only 5 cm and rarely more than
tation have been connected with computers and 8 cm). A direct comparison of theoretical plates
cameras respectively, which make the technique in HPTLC with HPLC serves little purpose as the
more hi-tech. HPTLC leads to difficulty in auto- number found is only valid for the spot used for
mation, and because of its open character, it is calculation. The basic problem is that all analytes
highly influenced by environmental factors. It is do not travel the same distance and are not
therefore essential that each step which may measured in retention time as in column
require specific approach must be carefully vali- chromatography.
dated, much more than TLC analysis. As HPTLC have higher performance than
TLC, so it is possible to carry out separations on
HPTLC that were not possible on TLC plates
HPTLC Pre-Coated Plates and, for those where it was possible, to shorten
the time of separation dramatically. HPTLC is
The uniformity and homogeneity of the station- therefore a more rapid, efficient, and sensitive
ary phase during HPTLC analysis is directly technique than conventional TLC. For in situ
linked with reproducibility and versatility of the quantitative analysis using spectro-densitome-
analytical results. HPTLC uses the same type of ters, it is essential that HPTLC layers are used for
silica gel 60 layers, as in traditional TLC, with a the most reliable results.
thickness of 0.20–0.25 mm. However, the particle
size is much smaller, typically ranging from 4 to
8 mm, with an optimum of 5–6 mm (Table 3.1). Detection and Visualization
The commercial pre-coated HPTLC plates with
polymeric binders are sufficiently hard so as not Like TLC, HPTLC requires the visualization and
to be easily damaged by the capillary tubes used detection, and similar practices are used for that
for sample application. Use of smaller particles but at more precise level. These practices may be
of stationary phase, similar in size and quality to categorized as [3]:
HPLC packing materials, gives a lower theoreti-
cal plate height (H) and hence higher efficiency
but can be fully utilized if the plates are not over- Nondestructive Techniques
loaded with too much sample, the spot size is
kept small (about 1.0 mm), and the plate is devel- In this practice, the chromoplate remains intact,
oped only to the extent necessary for complete may be evaluated by:
Detection and Visualization 51

Table 3.2 Some fluorescence intensifier and their application areas [3]
Intensifier Compounds detected Enhancement Stabilization
Triton X-100 (1% v/v solution in Fatty acids asdansyl amides At least tenfold Yes
hexane or heptane)
Polyethylene glycol 400 or 4,000 Compounds with alcoholic 20- to 25-fold Unknown
(10% w/v in methanol) (-OH) functional groups.
Paraffin liquid (33% v/v in hexane) Aflatoxins threefold to fourfold Unknown
Paraffin liquid (33% v/v in hexane) Ketosteroids, cholesterol, tenfold Unknown
cortisol
Paraffin liquid (33% v/v in hexane) Dansyl amides tenfold Yes
Paraffin liquid (33% v/v in hexane) Gentamicins Yes, but level unknown Yes

Visible Detection at shorter wavelengths in the UV. Depending


There are compounds that have color, for example, upon the nature of analyte and developing reagent,
natural and synthetic dyes, chlorophyll, and nitro- it may be reversible reactions (i.e., nondestruc-
phenols, to give an absorption in the visible part tive techniques), for example, iodine vapor and
of the electromagnetic spectrum. These are ammonia.
clearly seen in visible light and do not require Iodine is a universal reagent detecting the
any further treatment for visualization. presence of many organic species on thin layers,
but some reactions with iodine are irreversible.
Ultraviolet Detection The use of iodine as a vapor enables the detection
There are many compounds that appear color- of separated substances rapidly and economically
less in normal light but can absorb electromag- before final characterization with a group-specific
netic radiation at shorter wavelengths. These are reagent. Where lipophilic zones are present on a
often detected in the UV range, normally at chromatographic layer, the iodine molecules con-
200–400 nm. Often exposure to UV light at centrate in the substance zones giving yellow–
short-wave radiation (254 nm) or long-wave brown chromatographic zones on lighter yellow
radiation (365 nm), with commercial UV lamps background. The preparation of the reagent sim-
and cabinets, which function at either or both of ply involves putting a few iodine crystals in a dry
these wavelengths. To aid visualization, many chromatography tank, replacing the lid, and
commercial pre-coated HPTLC layers contain allowing the iodine vapor to fill the air space for
an inorganic phosphorescent or an organic a few hours. The developed chromatogram is
fluorescent indicator (Table 3.2). Detection by then introduced into the chamber, and as soon as
absorbance in these cases relies on the phospho- the chromatographic zones are recognized, the
rescence or fluorescence being quenched by the layer is removed and the results recorded. The
sample components. This process is commonly adsorbed iodine is allowed to slowly evaporate
called “fluorescence quenching” in both cases, from the layer surface under a dry stream of air at
although more accurately for most indicators room temperature; a fume cupboard facility is an
designated F254 it is described as phosphores- ideal location for this. These chromatograms can
cence quenching. be subjected to further treatment with other uni-
versal or with more specific functional group
Reversible Reactions reagents. If more permanent results of the iodine
Many compounds do not absorb visible or UV impregnation are required, then the chromato-
light, quench fluorescence, or fluoresce when graphic zones are sprayed or dipped in a starch
excited by visible or UV light. In these cases, solution (0.5–1% w/v) to give blue starch–iodine
suitable detection reagents are used to give inclusion complexes. However, it is important to
colored chromatographic zones in visible light or carry out this procedure after partial evaporation
52 3 HPTLC: Herbal Drugs and Fingerprints

of iodine from the layer. Starch treatment has dyes are commonly used for the nondestructive
the best results when iodine is still retained in the detection of lipophilic substances, for example,
separated chromatographic zones but has gone fluorescein, dichlorofluorescein, eosin, rhodamine
from the background layer. Otherwise, it will be B and 6 G, berberine, and pinacryptol yellow.
difficult to distinguish the zones from a back- Reagents for dipping chromatograms are prepared
ground that will also be stained blue. as dye (10–100 mg) in methanol or ethanol (100 ml).
Iodine detection works well on silica gel 60 After air drying, the detected chromatographic
and aluminum oxide layers. However, results are zones appear brightly fluorescent on a lighter
usually poor on reversed-phase layers as the lipo- fluorescent background under UV light (254 nm).
philicity of the layer does not differ appreciably Although very effective on silica gel, cellulose, and
from the chromatographic zones. Iodine vapor kieselguhr layers (sensitivity from low micro-
reversible reactions occur with a wide range of gram to low nanogram range), these dyes do not
organic lipophilic molecules, for example, fats, respond on reversed-phase silica gels; sometimes
waxes, some fatty acids and esters, steroids, anti- exposure to ammonia vapor after dye treatment
oxidants, detergents, emulsifiers, and many mis- improves sensitivity.
cellaneous pharmaceuticals.
Ammonia vapor is often used in conjunction
with other reagents to improve the contrast Destructive Techniques
between the separated chromatographic zones
and the layer background. The most common Oxidation and/or derivatization due to chemical
usage is in the visualization of organic acids with reactions occurring on the chromatographic layer
pH indicators. Although indicators, such as bro- between a reagent and separated analytes is a
mocresol green and bromophenol blue, detect the destructive technique. In this case, the visualized
presence of a variety of organic acids, further compounds are no longer the original one. The
treatment with ammonia vapor sharpens the con- major techniques as destructive are charring and
trast between analytes and background layer thermal activation. Charring techniques involve
resulting in greater sensitivity. On segregation of treatment of the developed chromatogram with a
ammonia source, ammonia gradually evaporates suitable reagent, followed by heating the layer at
away from the chromoplate, and the sensitivity of relatively high temperatures to degrade any
detection reverts to that prior to treatment. organic species to carbon. As can be appreciated,
Exposure to ammonia vapor can be achieved by the reaction is somewhat nonspecific, and hence,
simply holding the chromatographic plate face- charring has been included in what is termed uni-
down over a beaker of strong ammonia solution. versal reagents. The most popular charring
However, more elegantly, it can be performed by reagent is sulfuric acid, applied to the chromato-
pouring ammonia solution into one compartment graphic layer as a dilute solution (10–20% v/v in
of a twin-trough developing tank and placing the methanol/water); however, orthophosphoric acid
TLC plate in the dry compartment. With the lid in and chromosulfuric acid have proved successful
place, the TLC plate is exposed to an almost even in more of the specific circumstances. The tem-
concentration of vapor. The process is reversible perature and heating time depends on the nature
with time as the ammonia soon evaporates from of the compounds to be charred. This can vary
the sorbent surface. from 5 to 20 min at 100–180°C. Dilute solution
of sulfuric acid in water/methanol ensures ade-
quate wetting of the TLC/HPTLC layers. On
Nonreversible Reactions heating, the solvents evaporate steadily and acid
concentrates and finally chars the organic material
A few techniques and practices used to visualize present. Although it is a very simple detection
the spots for HPTLC have chemical reactions technique, but sulfuric acid charring does have
that cannot be in original stage; such practices are limitations especially where commercially man-
known as nonreversible reactions. Fluorescent ufactured chromatography plates are concerned.
Detection and Visualization 53

Most binder whether present in homemade or chemical reagents compared with chemical
commercial plates affected to a greater or lesser derivatization before development is reflected in
extent depending on the temperature and time of the number of methods available in the scientific
heating. Overheating of plates with organic bind- literature. Many hundreds of reagents and reagent
ers may have a gray or even black background, procedures are available for the post-chromato-
rendering it useless. graphic visualization, whereas relatively few
It has been observed that some developed describe pre-chromatographic detection. In case
zones on a TLC/HPTLC layer when heated at where visualization before chromatographic
high temperatures have fluoresced on exposure to development has been recommended, the results
UV light, for example, lysergol and lumilysergol are quite unique and specific.
(using mobile phase chloroform–methanol– The post-chromatographic visualization is
ammonium hydroxide, 85:14.5:0.5, and heating similar to the TLC detection, which is achieved
at 100°C). This process has been given the title by spraying or dipping. Some reactions occur
thermochemical activation. Separations on mod- immediately, and colored chromatographic zones
erately polar aminopropyl-bonded silica gel lay- appear on contact with the reagent or more usu-
ers have been observed to give the most consistent ally after drying or heating at a defined tempera-
and sensitive results for this process of detection. ture (Table 3.3). The choice of whether the
The reaction mechanism by which thermochemi- reagent is applied as a spray or by dipping
cal activation takes place is not fully elucidated, depends on a number of factors. Spraying uses
but the following has been suggested as a proba- less solvent, can be accomplished with simple
ble sequence. The surface of the silica gel-bonded atomizer devices, and is completed in a short
layer acts as a catalyst. Under the influence of the period. However, spraying exposes the surround-
catalytic adsorbent surface, substances rich in ing atmosphere; uneven spray, etc. are drawbacks
p-electrons are formed that conjugate to form of the techniques. The salient features of spray-
products having fluorescent at excited state. It has ing reagent are below:
been observed that compounds with possible het- 1. Sensitivity for detection
eroatoms, such as nitrogen, oxygen, sulfur, or 2. Specificity of the reagent for the analyte of
phosphorus, will more readily respond to thermal interest
activation than pure hydrocarbons. Changes in 3. Background effects, more specific when plates
pH often alter the excitation and emission wave- are to be scanned spectrophotometrically
lengths. The fluorescent compounds formed are 4. Stability of detection reagent
quite stable. The fluorescence can frequently be 5. Stability of the chromatogram after chemical
intensified and stabilized by coating the chro- or thermal treatment
matogram with liquid paraffin or a polyethylene 6. Ease of preparation of the spraying or dipping
glycol. The fluorescent enhancer is dissolved in reagent
hexane or heptane (5% w/v). If the aminopropyl- 7. Hazards associated with the preparation and
bonded layer contains a fluorescent indicator use of a particular detection reagent
(F254), then appreciable fluorescence quenching
can occur under UV light at 254 nm. A few com-
pounds that have weak fluoresce, like vanillic Common Visualizing Reagents
acid and homovanillic acid, can exhibit strong
fluorescent absorption after thermal activation A few common reagents for detection of non-
and fluorescence enhancement. Thermal activa- UV–Vis. compounds in HPTLC are as follows.
tion is also effective for the detection of cate-
cholamines, fruit acids, and some carbohydrates. Iodine Vapor/Solution
Spots are also detected by derivatization It is also called “iodine reaction” possibly results
reactions either before or after development; in an oxidative product. The reaction pathway is
however, the popularity of detection of the normally irreversible (but sometimes reversible
chromatographic zones after development with also, as previously discussed); in most instances,
54 3 HPTLC: Herbal Drugs and Fingerprints

Table 3.3 Some popular visualization reagents for TLC/ HPTLC [3]
Visualization reagent Reagent conditions Groups detected
Ehrlich’s reagent 4-Dimethylaminobenzaldehyde (2%, w/v) in Amines, indoles
25% (w/w) hydrochloric acid/ethanol (50:50,
v/v). After treatment, heat at 110°C for 2 min
Folin and Ciocalteu’s As per literature Phenols
reagent
Gibb’s reagent 2, 6-Dibromoquinone-4-chloroimide Phenols, indoles, thiols,
(0.5%, w/v) in methanol. After treatment, barbiturates
heat at 110°C for 5 min
Blue tetrazolium reagent Blue tetrazolium (0.25%, w/v) in sodium Corticosteroids,
hydroxide solution (6%, w/v in water)/ carbohydrates
methanol (25:75, v/v)
Tillman’s reagent 2, 6-Dichlorophenolindophenol sodium salt Organic acids including
(0.1%, w/v) in ethanol. After treatment, heat vitamin C
at 100°C for 5 min
Iron (III) chloride reagent Iron (III) chloride (1%, w/v) in ethanol/water Phenols, ergot alkaloids,
(95:5, v/v). After treatment, heat at 100°C inorganic anions, enols,
for5 min hydroxamic acids,
cholesteryl esters
EP reagent 4-Dimethylaminobenzaldehyde (0.2%, w/v) Terpenes, sesquiterpene
and orthophosphoric acid (3%, v/v) in acetic esters
acid/water (50:50, v/v). After treatment, heat
at 80°C for 10 min
Jensen’s reagent Chloramine T (10%, w/v) and trichloroacetic Digitalis glycosides
acid (0.4%, w/v) in chloroform–methanol–
water (80:18:2, v/v). After treatment, heat
at 120°C for 10 min
N-Bromosuccinimide 0.5%, w/v solution in acetone. After Amino acids, Z-protected
reagent treatment, heat at 120°C for 20 min amino acids, hydroxyl
flavones, hydroxyl
quinones
O-Phthalaldehyse- O-Phthalaldehyde (1%, w/v) in methanol/ Ergot alkaloids,
sulfuric acid reagent sulfuric acid (90:10, v /v). After treatment, b-blockers, indole
heat at 80°C for 3 min derivatives, histidyl
peptides

it is observed with organic unsaturated compounds aromatic compounds can be nitrated with the
present in the separated chromatographic zones. fumes from concentrated fuming nitric acid. The
Electrophilic substitutions, addition reactions, developed chromatogram is heated to about
and the formation of charge-transfer complexes 160°C for 10 min and kept while still hot into a
occur with iodine. An added feature is that iodine chamber containing the nitric acid vapor. Nitration
also possesses fluorescence-quenching proper- proceeds at a reasonable rate, and generally the
ties; the chromatographic zones that have iodine chromatographic zones are rendered yellow or
appear as dark zones on a TLC layer containing brown.
fluorescent indicators (Table 3.4) [3].
Redox Reaction
Nitric Acid Vapor Oxidation and reduction reactions are frequently
Many compounds such as ephedrine, sugars, tes- used for visualization techniques as reactions are
tosterone, and xanthine derivatives have yellow group specific, depending on the particular
or blue fluoresce after nitration, at 365 nm. Most reagent used. The main redox reactions for
Detection and Visualization 55

Table 3.4 Iodine reactions on the TLC layer [3]


Compounds Reaction
Polycyclic aromatic hydrocarbons, indole, Formation of oxidation products
and quinoline derivatives
Quinine alkaloids, barbiturates, unsaturated Addition of iodine to the double bonds
lipids, capsaicins, and calciferol
Opiates, brucine, ketazone, and trimethazone Iodine addition to the tertiary nitrogen for the opiates. Addition
reaction withthe -OCH3 group of the brucine. Ring-opening
reaction for the ketazone and trimethazone
Thiols and thioethers Oxidation of sulfur and addition across the double bond
in the thiazole ring
Alkaloids, phenothiazines, and sulfonamides Complex formation

developing visible spots are as follows: Emerson’s 115°C for 5 min can improve sensitivity for some
reagent [4-aminoantipyrine-potassium hexacy- analyte.
anoferrate (III)] for detection of arylamines and
phenols; chlorine-o-toluidine reagent for vita-
mins B1, B2, and B6 and triazines; chloramine T Group-Specific Reaction
for steroids and purine derivatives; and chlorine–
potassium iodide–starch reagent for amino, Many reagents are functional group specific
imino, and amido groups and triazine herbicides. meaning that they give specific reactions with
By contrast, reduction reactions include phosph- certain organic and inorganic chemical groups. In
omolybdic acid for lipids, phospholipids, and most cases, the reaction mechanism has been
some steroids; tin(II) chloride-4-dimethylamin- fully elucidated. As general rule, these reagents
obenzaldehyde reagent for the detection of aro- are very sensitive with detection limits usually in
matic nitrophenols; blue tetrazolium reagent for middle to low nanogram range, for example [3].
corticosteroids; Tillman’s reagent (2,6-dichloro-
phenolindophenol) for organic acids, including Hydrazone Formation
vitamin C; and silver nitrate–sodium hydroxide A hydrazone is a class of organic compounds with
reagent for reducing sugars and sugar alcohols. the structure R1R2C = NNH2. They are related to
ketones and aldehydes by the replacement of the
Iodoplatinate Reagent oxygen with the = NNH2 functional group. They
This is an effective reagent for a wide range are formed usually by the action of hydrazine on
of nitrogen containing compounds, including ketones or aldehydes. The reagent employed for
alkaloids, ketosteroids, quaternary ammonium hydrazone formation is2,4-dinitrophenylhydra-
compounds, thiols, thioethers, opiates, sulfoxides, zine in acidic solution [100 mg in 100-ml ethanol/
tricyclic antidepressants, and vitamins D3, K1, phosphoric acid (50:50)]. After dipping or spray-
and B1. A range of colors are produced on the ing the chromoplate with the reagent, the reaction
chromatogram depending on the analyte. The limit is completed by heating at 110°C for 10 min.
of sensitivity for detection is often in the low This is a specific reagent for aldehydes, ketones,
nanogram range. Iodoplatinate reagent, a typical and carbohydrates. Yellow or orange-yellow
dipping reagent, consists of the following: 10% hydrazones, or osazones in the case of carbohy-
(w/v) hexachloroplatinic acid aqueous solution drates, are formed on the chromoplate. Ascorbic
(3 ml), 6% (w/v) potassium iodide aqueous solu- acid and dehydroascorbic acid are also detected
tion (100 ml), and 10% (v/v) methanol aqueous by this reagent giving yellow zones on a white
solution (97 ml). After dipping, the TLC plates background. The sensitivity limit is in the order of
are dried at 80°C for 5 min. Further heating at 10 ng per chromatographic zone.
56 3 HPTLC: Herbal Drugs and Fingerprints

Dansylation reagent is copper(II) sulfate 5-hydrate (1.5% w/v,


Dansyl [5-(dimethylamino)-1-naphthalenesulfo- water/methanol). Most acids appear as blue zones
nyl] chloride and other derivatives are used to on a pale blue background. The limit of sensitiv-
produce fluorescent dansyl derivatives of amino ity is 5 mg/zone. Copper is also used in the biuret
acid, primary and secondary amines, fatty acid, and reaction with proteins, resulting in the formation
phenols. The dansylation of carboxylic acid is indi- of a reddish-violet complex, and with aromatic
rect as the acid amides must first be formed. This ethanolamines to form blue-colored chelates.
conversion is readily achieved with the reagent. Iron (Fe3+) and cobalt (Co2+) can also be used in a
The detection limit is 1–2 ng for fatty acids; how- similar way with the formation of reddish-violet
ever, one of the problems with post-chromato- zones for phenolic compounds and blue zones in
graphic dansylation is the background fluorescence the presence of ammonia vapor for barbiturates,
it produces. Unfortunately, the fluorescent contrast respectively.
between the chromatographic zones and back-
ground results in reduced sensitivity. Ninhydrin Test
Ninhydrin is a well-known detection reagent for
Diazotization the visualization of amino acids, peptides, amines,
Azo dyes are strongly colored and can be pro- and amino sugars. The limit of sensitivity ranges
duced readily from aromatic nitro- and primary from 0.2 to 2 mg per chromatographic zone
amines and phenols present in the separated chro- depending on the amino acid. The colored zones
matographic zones. This can be achieved in two can vary from yellow and brown to pink and vio-
basic ways. Nitro compounds are reduced to pri- let, depending on the sorbent layer and pH. The
mary arylamines. These are diazotized with colors fade quickly unless stabilized by the addi-
sodium nitrite and then coupled with phenols to tion of metal salts of tin, copper, or cobalt.
form the azo dyes. Conversely, phenols can be Copper(II) nitrate or acetate is the usual salts
detected by reaction with sulfanilic acid in the chosen as additives. A typical formulation for
presence of sodium nitrite. The resulting azo dyes such a ninhydrin dipping reagent is 0.3% (w/v) in
are often stable for a period of months. A novel propan-2-ol with the addition of 6 ml/100 ml of
approach to the detection of phenols is to impreg- aqueous copper(II) acetate (1% w/v). After dip-
nate the layer with sulfanilic acid hydrochloride ping, the TLC layer is heated at 105°C for 5 min.
(2.5% w/v in water) before chromatography and For better resolution between glycine and serine,
application of the sample. After drying the plate collidine is added to the ninhydrin at conc. of
120°C for 30 min, the phenolic samples are 5-ml/100-ml reagent.
applied in the usual way. Following development
and drying, the layer is sprayed with fresh sodium Natural Product Reagent
nitrite solution (5% w/v). The azo dyes formed Natural product reagent (NPR), as diphenyl boric
have a high stability, immediately appearing as acid-2-aminoethyl ester, readily forms complexes
colored zones that maintain their color for weeks with 3-hydroxyflavones via a condensation reac-
after first visualization. tion and is used extensively for visualization of
components in herbal preparations in TLC/HPTLC
Metal Complexes analysis. A suitable dipping reagent consists of
A number of transition metals act as electron diphenyl boric acid-2-aminoethyl ester (1 g) dis-
acceptors to form complexes with organic com- solved in methanol (100 ml). This solution should
pounds that are rich in electrons. Colored metal be freshly prepared when needed, especially where
complexes are formed by electron movement to quantitative results are required. The chromoplate
different energy states in the transition metal ion. is thoroughly dried, dipped in the reagent for a few
Copper (Cu2+) readily forms such complexes or seconds, dried again in a stream of warm air, and
chelates with carboxylic acids including thiogly- then dipped in a polyethylene glycol (PEG) 4000
colic and dithioglycolic acids. A suitable detection (5% w/v) solution in ethanol. The reagent is
Coupling of HPTLC with Spectrometry 57

especially good for the detection of rutin, chloro-


genic acids, hypericum, and other flavonoids. It Coupling of HPTLC with Spectrometry
can also be used on most sorbent layers including
both the normal and reversed-phase silica gels. HPTLC is coupled with ultraviolet–visible, infra-
The limit of sensitivity is about 1–5 ng/chromato- red spectrometry, Raman spectrometry, photoa-
graphic zone. The purpose of the PEG 4000 is to coustic spectrometry, and mass spectrometry.
enhance the fluorescence and to stabilize the emis- FTIR has a high potential for the elucidation of
sion of light. molecular structures, and the characteristic
absorption bands are the clue for specific detec-
Case Study tion, as it indicates the presence/absence of
The root, stem bark, and fruits of various Berberis specific functional group. Almost all chemical
species in the Himalayan region are well recog- compounds yield good FTIR spectra that are
nized for their alkaloid contents. Due to global more useful for identification of unknown sub-
demand for berberine alkaloids and their deriva- stances and discrimination between closely
tives, various analytical tools such as HPLC, GC, related substances. The HPTLC–FTIR spectra
and GC-MS have been used for berberine estima- make possible the detection and quantification of
tion. HPTLC, a technique for quality control and even non-UV-absorbing substances on HPTLC
standardization of traditional herbs like Berberis plates. These reasons make this hyphenated tech-
for berberine content in root and stem bark of nique more universally applicable. The HPTLC
three Berberis (i e., B. asiatica, B. aristata, B. and FTIR coupling can be divided into two
lycium), was used, and comparative analytical groups, that is, indirect and direct methods [5].
assessment revealed that the berberine content For indirect coupling there is transfer of the
varied both in root and stem bark samples. More substance from a TLC spot to a non-absorbing IR
berberine content observed in root samples as material (KBr or KCl) or in situ measurement of
compared to bark of all the investigated species. excised HPTLC spots when the spectra are
Among the species, Berberis asiatica contains recorded directly from the plate. The direct
more berberine as compared B. lycium and online-coupled HPTLC–FTIR offers some advan-
B. aristata (Figs. 3.1 and 3.2) [4]. tages relative to other hyphenated techniques

B asiatica B aristata B lycium Berberine Berberine Berberine B. asiatica B. aristata B. lycium


(root) (root) (root) 2 µL 4 µL 6 µL (bark) (bark) (bark)

Fig. 3.1 HPTLC at 254-nm root and stem bark samples of various Berberis species [4]
58 3 HPTLC: Herbal Drugs and Fingerprints

B asiatica B aristata B lycium Berberine Berberine Berberine B. asiatica B. aristata B. lycium


(root) (root) (root) 2 µL 4 µL 6 µL (bark) (bark) (bark)

Fig. 3.2 HPTLC at 366-nm root and stem bark samples of various Berberis species [4]

(HPTLC–Raman spectroscopy, HPTLC–PA, and distribution, and a layer thickness of 200 mm on


HPTLC–MS), such as the ease of operation and glass is found to be ideal in the mid-IR range.
the optimized operational aspects of online cou- Finally, the binder or the fluorescence indicator
pling. In direct-coupling HPTLC–FTIR method, added to the adsorbent and the mobile phase could
a major difficulty is the absorption by conven- lead to altered HPTLC–FTIR spectra [5].
tional stationary phases, for example, silica gel, The identification can be realized by fitting the
which absorb strongly in the IR range. It is very reference spectra to sample spectra and visual com-
difficult to obtain reliable spectra in the regions parison. The compounds separated by HPTLC can
where the layer shows strong IR absorption. The be also identified using an HPTLC–FTIR library.
silica gel, the most widely used adsorbent in The band position, width, and intensity are automati-
HPTLC, presents absorption bands between cally compared, and the reliability of the results is
1,350 and 1,000 cm−1 and above 3,550 cm−1 which described in terms of hit quality. Quantitative analy-
are superimposed on the spectra of compounds, sis with the HPTLC–FTIR technique is generally
and only the region between 3,550 and 1,350 cm−1 applied for the substances that do not absorb in the
can be evaluated. Therefore, measurements in this UV–Vis. range and when the precision required is
region are not possible, but it is possible to make not too high. The lack of precision is due to the
measurements up to 1,000 cm−1 on cellulose. The increase of sample spot broadening with increased
best results are obtained when the mixture of silica migration distance and to the measurement not being
gel 60 and magnesium tungstate (1:1) is used as exactly at the peak maximum. These problems are
stationary phase. This adsorbent improves signal- due to the circular infrared beam with small diame-
to-noise ratios and enhances the performance of ter. The determination of compounds is made on the
the diffuse reflectance of the matrix. Another basis of evaluation of the peak areas in the Gram-
problem is due to the particle size, particle-size Schmidt trace or in the window diagram, or by the
distribution, and the layer thickness, which affect evaluation of Kubelka-Munk spectra with integra-
the scattering, remitting, and absorbing of the tion of their strongest bands. The method using the
radiation by the matrix. A stationary phase with a Gram-Schmidt traces indicates the changes in absor-
particle diameter of 10 mm, a narrow particle-size bance over the whole spectral region, and therefore,
Bibliography 59

it is suitable and practical for rapid determinations. 2. Rajkumar T, Sinha BN. Chromatographic fingerprint
The evaluation of the peak areas in the window chro- analysis of budmunchiamines in Albizia amara by HPTLC
technique. Int J Res Pharm Sci. 2010;1(3):313–6.
matogram is appropriate for the quantification of 3. Wall PE. Thin layer chromatography: a modern
individual substances. An advantage of this method practical approach, RCS chromatography monograph.
is a better signal-to-noise ratio, but the disadvantage Cambridge: Royal Society of Chemistry; 2005. ISBN
is the poorer precision. More precise results are 0-85404-535-X.
4. Andola HC, Rawal RS, Rawat MSM, Bhatta ID, Purohit
obtained using the evaluation of Kubelka-Munk VK. Analysis of berberine content using HPTLC
spectra. The limit of identification and determination fingerprinting of root and bark of three Himalayan
is 10 times higher than those obtained by densitom- berberis species. Asian J Biotechnol. 2010;2(4):239–45.
etry. This method has the disadvantages of the mea- 5. Cimpoiu C. Qualitative and quantitative analysis by
hyphenated (HP)TLC-FTIR technique. J Liq Chromatogr
surement only of the fraction of the substance in the Relat Technol. 2005;28:1203–13.
peak maxima and the additional processing step. In
conclusion, none of these methods is perfect and
appropriate for all samples. The choice of a method Bibliography
depends on the goals of the analysis [5].
TLC and HPTLC are valuable tools for quali- Ahmad I, Aqil F, Owais M. Turning medicinal plants into
tative determination of small amounts of impuri- drugs. Modern phytomedicine, vol. 384. Weinheim:
ties. Lack of chemical markers is a major problem Wiley; 2006. p. 67–72.
Bhutani KK. Fingerprinting of Ayurvedic drugs. East
for the quality control of herbal medicines. In Pharm. 2000;507:21–6.
many cases, we do not have sufficient chemical Bobby N, Wesely EG, Johnson M. HPTLC profile studies
and pharmacological data of chemical markers. on the alkaloids of Albizia lebbeck. Asian Pac J Trop
Furthermore, there are many technical challenges Biomed. 2012;2:1–3.
Dhandapani A, Kadarkarai M. HPTLC quantification of
in the production of chemical markers, for exam- flavonoids, larvicidal and smoke repellent activities of
ple, temperature, light, and solvents often cause Cassia occidentalis L. (Caesalpiniaceae) against
degradation and/or transformation of purified malarial vectore Anopheles Stephensi Lis (Diptera:
components; isomers and conformations may Culicidae). J Phytol. 2011;3(2):60–71.
Liang YZ, Xie P, Chan K. Quality control of herbal medi-
also cause confusions of chemical. Under such cines. J Chromatogr B. 2004;812:53–70.
conditions, HPTLC fingerprints have its values, Long F. Bio-pharmaceutical characterization of herbal
using reference botanical standard for compari- medicinal products. Drugs. 2001;44(4):102–8.
sons and quality management policies [ISO 9000 Sagar BPS, Zafar R, Panwar R. Herbal drug standardiza-
tion. Indian Pharm. 2005;4(35):19–22.
certification, good laboratory practices (GLP), Shahare MD, Mello PM. Standardization of Bacopa mon-
good manufacturing practices (GMP), total qual- nieri and its formulations with reference to Bacoside
ity management (TQM) and validated instruments A, by high performance thin layer chromatography.
and services, etc.] in pharmaceuticals to have a Int J Pharmacogn Phytochem Res. 2010;2(4):8–12.
Shanbhag DA, Khandagale NA. Application of HPTLC in
better quality of drugs. the standardization of a homoeopathic mother tincture
of Syzygium jambolanum. J Chem Pharm Res.
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Soni K, Naved T. HPTLC–its applications in herbal drug
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WHO. Quality control methods for medicinal plant mate-
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Bhatta ID. Chromatographic and spectral fingerprinting Yadav D, Tiwari N, Gupta MM. Simultaneous quantification
standardization of traditional medicines: an overview as of diterpenoids in Premna integrifolia using a validated
modern tools. Res J Phytochem. 2010;4:234–41. HPTLC method. J Sep Sci. 2011;34(3):286–91.
HPLC: Herbal Drugs and Fingerprints
4

High performance liquid chromatography accounting for about 20% (one third being
(HPLC) has been the most reliable tool for the related to MS–MS), HPLC–ECD for 3%,
separation of complex mixtures. The complex HPLC–NMR for 2%, and ELSD for 1.5%. The
crude extracts from medicinal plants have differ- other detection techniques are reported in less
ent closely related compounds along with than 1% of all HPLC applications. The large
metabolites and require efficient analytical sep- majority of analyses are still performed by
aration methods for HPLC fingerprints. HPLC HPLC–UV, which is widespread in many labo-
has been recognized since the early 1980s as ratories. For the detection of non-UV-active
the most versatile technique for the efficient constituents, aerosol-based detectors, such as
separation, and since that time, there are regular ELSD or CAD, are in use with HPLC–MS as a
innovations through the years in terms of con- powerful tool for detection ability. More
venience, speed, choice of column stationary recently, the advent of electrospray ionization
phases, high sensitivity and applicability to a (ESI) and atmospheric pressure chemical ion-
broad variety of sample matrices, and ability to ization (APCI) interfaces has provided mass
hyphenate the chromatographic method to spec- spectrometry (MS) interfaces which are appli-
troscopic detectors. From the chromatography cable to the analysis of a wide range of
viewpoint, the development of columns with molecules and are compatible with liquid
different phase chemistry (especially reversed chromatography [1]. For the separation of crude
phase) enabled the separation of almost any extracts, either raw mixtures or samples
type of plant product. HPLC allows an analyst enriched by extraction via simple solid-phase
to identify unknown compounds by comparison extraction (SPE) or liquid–liquid extraction
of their HPLC retention times with known and (LLE) are injected into HPLC after passing
UV spectra. The HPLC analytical methods are through 0.45-m filter. The separations are per-
validated for specificity, linearity, limit of formed mostly in reversed-phase chromatogra-
detection (LOD), precision and accuracy, rug- phy on C18 material with the MeCN-H2O or
gedness, and robustness. The lack of a sensitive MeOH-H2O solvent system in the gradient elu-
and universal detection, suitable for both quali- tion mode. In order to improve the separation
tative and quantitative analysis, under a wide efficiency, various modifiers are added to the
range of conditions is one of the reasons to look mobile phase that might strongly influence the
for universal analytical technique. A literature sensitivity of detection. In multi-hyphenated
search on journals focused on natural product systems, online presence of several different
chemistry revealed that more than 70% of detectors (hyphenated systems) leads to the need
HPLC applications are performed by HPLC–UV for an eluent composition that is compatible
and HPLC–DAD, with HPLC–MS applications with all detectors [2].

D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs, 61
DOI 10.1007/978-81-322-0804-4_4, © Springer India 2012
62 4 HPLC: Herbal Drugs and Fingerprints

Table 4.1 Properties of solvents commonly used in HPLC [3]


Miscible
S. no. Solvent Polarity with water UV cutoff
1 Hexane Nonpolar No 200
2 Isooctane Nonpolar No 200
3 Carbon Nonpolar No 263
tetrachloride
4 Chloroform Nonpolar No 245
5 Methylene chloride Nonpolar No 235
6 Tetrahydrofuran Nonpolar Yes 215
7 Diethyl ether Nonpolar No 215
8 Acetone Nonpolar Yes 330
9 Ethyl acetate Nonpolar Poorly 260
10 Dioxane Nonpolar Yes 215
11 Acetonitrile Nonpolar Yes 190
12 Isopropanol Nonpolar Yes 210
13 Methanol Nonpolar Yes 205
14 Water Polar Yes —

A Glance on HPLC Column possible while retaining adequate resolution.


The internal diameter of the column has a vital
HPLC column, the heart of the instrument, con- role for adequate resolution, which is inversely
sists of a metal cylinder packed with tiny silica or proportional (i.e., decrease in internal diameter,
modified silica particles to separate compounds the resolution is more fine), as smaller diameters
in a mixture. The mobile phase passes through increase efficiency and resolution, but it requires a
the column using high pressure, resulted each lower flow rate of solvent and, thus, increases the
component in the mixture coming out at different time of analysis. At low flow rate, impurities, if
times, and elution is based on polarity (Table 4.1) any, such as dirt quickly clog up the column with
[3]. Selection of suitable column is entirely based small internal diameters. Most columns have a
on the chemical nature of ingredient to be ana- particle size of 5 mm. The smaller particle size,
lyzed. The column specification (length, diame- such as 3 mm, will offer greater separation and will
ter, and particle size) has impact on the resolution decrease the time of analysis. However, smaller
and analysis time (Table 4.2) [4]. In case analyte particles require a higher amount of pressure to
is strongly retained by the stationary phase, a push the mixture through the column. Every strat-
short column length significantly decreases anal- egy in column selection has an advantage and dis-
ysis time. Column selectivity is also controlled advantage. It is up to the analyst to choose each
by changing the column temperature and the pH parameter skillfully to achieve an optimal balance
of the mobile phase and addition of organic in separation, efficiency, and pressure in the system.
modifiers such as acetonitrile into the mobile HPLC is a sophisticated form of column chro-
phase to reduce stationary phase–analyte interac- matography, especially designed to separate and
tion and elute more rapidly. classify components of a sample based on its
The length of column is a factor to decide the interactions with a specific column. HPLC is dis-
nature and number of compounds to be analyzed. tinct from column chromatography because it
Analytical HPLC columns are at the range of utilizes a more pressurized environment to propel
5–25 cm in length; long columns provide better the liquid samples through the densely packed
resolution and efficiency but require longer reten- column more efficiently. Based on the nature of
tion times and higher pressure. Due to the reason, silica particles used, columns are termed as nor-
analysts prefer to choose the shortest columns as mal phase or reverse phase.
Analytical Mobile Phases and HPLC 63

Table 4.2 Different stationary phases in HPLC column and utility-USP categories [4]
S. no. USP code Particle dia. (mm) Bonding Description
1 L1 3–10 C-18 Octadecylsilane chemically bonded to porous silica or
ceramic particles, used in analysis of wide range of
compounds, neutral to weakly basic
2 L2 30–50 C-18 Octadecylsilane chemically bonded to silica gel of a
controlled surface
3 L3 5–10 None Porous silica microparticles, used for normal phase
separation
4 L4 30–50 Silica Silica gel of controlled surface porosity bonded to a solid
spherical core
5 L5 30–50 Alumina Alumina of controlled surface porosity bonded to a solid
spherical core
6 L6 30–50 Cation Strong cation-exchange packing-sulfonated fluorocarbon
exchange polymer coated on a solid spherical core
7 L7 5–10 C-8 Octyl silane chemically bonded to totally porous microsilica
particles, provides shorter retention times for hydrophobic
compounds than C-18
8 L8 10 Amino An essentially monomolecular layer of amino-propylsilane
(−NH2) chemically bonded to totally porous silica gel support
9 L9 10 Cation Irregular, totally porous silica gel having a chemically
exchange bonded, strongly acidic cation-exchange coating
10 L 23 10 Anion Anion-exchange resin made of porous polymethacrylate or
exchange polyacrylate gel with quaternary ammonium groups

Analytical Mobile Phases and HPLC 3. Columns such as cyano- and fluorophenyl (F 5)
can work in either RP or NP mode and can
HPLC is a high-value instrument, between the remain installed, if desired.
commercial utility and capital investment; it is 4. After IPA flush, the column can be removed
difficult to dedicate instrument for a single ana- and capped to avoid excessive wear on the
lytical mode, so there is a frequent need for shift valuable component. In the flushing steps,
in the operation from normal phase (NP) to we have to be sure to include the entire fluid
reverse phase (RP) and vice versa. When aqueous path (pump, auto-sampler, valves. detector,
mobile phase is replaced with nonaqueous condi- including the sample loop and any other fluid
tions, immiscibility situations arise during this paths) that is encountered for the normal
changeover. We can have a smooth analytical operation of making injections. As part of all
operation by following the certain practices at the the washes, the injection needle (syringe) is
instant of switchover from one mode to another. to be washed well with IPA, where manual
The instrument has different types of seals, which injections are carried out.
may suffer by expansion, contraction, and extra 5. For loop-type injector, it is desired to flashed-out
friction due to change in solvent, so: by several IPA injections (as IPA is miscible
1. On regular practice, there is a trend to replace with both high-aqueous and high organic
the column with tubing or a union; the system mobile phases); the total volume of IPA
is flushed extensively with isopropanol (IPA) required may vary with instrument design, but
before going over to water or hydrocarbon. the waste volume can be monitored for future
2. Before beginning of the changeover process, data and observed for uniform appearance of
remove the HPLC column, cap, and store it at absorbance at the detectors, indicating the job
the appropriate storage solvent, unless the done, that is, the system has returned to a
same column is to be used in the new mode. stable baseline.
64 4 HPLC: Herbal Drugs and Fingerprints

6. The second step after the IPA wash is to be but evaporative detectors such as MS and
followed with alcohol (methanol/ethanol). ELSD cannot be used for this purpose.
The trend with alcohol is similar as with IPA 9. The severe detector baseline noise or pres-
wash before going to water. Methanol will sure fluctuations are the indication of incom-
help flush the IPA out faster than going directly plete mixing, resulting to the globules of
from IPA to water. If excessive baseline noise immiscible solvent that may resemble as
or drift is observed with a UV detector, repeat bubbles or particles.
the procedures and allow more time to flush 10. The detectors and all other components
out the poorly swept flow regions. should be flushed if baseline is not stable.
11. Total time for changeover can vary, depending
on the chemical composition of mobile phase
Solvent Changeover in HPLC used, but normal span should be of about an
hour. The reduction in changeover time may
The first best practice is to dedicate the instru- result into slowdown of the process.
ment for specific mode of analysis. Another best 12. The changeover and baseline equilibration
desirable point is that at least columns should be with refractive index (RI) detectors, are
dedicated to have trouble-free operation. Whenever extremely slow and achieve equilibrium only
changeover becomes necessary, the analyst uses after the complete changeover.
his professional skills, and a few notable points 13. For excessive baseline noise and drift, in gra-
are shown below. dient system, degassing should be properly
1. Remove all the additives and start with 100% checked as it may have pockets of immiscible
isopropyl alcohol in all reservoirs. solvent.
2. Isopropyl alcohol is fully miscible with all 14. There should be always a blank run before real
common solvents and is the safest changeover analysis. For first time HPLC analysis of a new
solvent. compound specificity of the analytical method
3. Flow rate of isopropyl alcohol should at low, should be established, along with reproduc-
about half of normal, to avoid excessive seal ibility, repeatability, linearity, limit of detection,
wear and damage due to over-pressuring. precision, and accuracy of the method used.
4. The use of acetonitrile routinely as the change- 15. To start HPLC analysis on routine analysis,
over solvent should be avoided as it is better first have a blank run, than standard/reference
than methanol but is not fully miscible with standard, standard mixed with sample, and
pure hydrocarbons. finally the sample.
5. The use of methanol routinely also should be 16. For successful changeover into a different
avoided as it is not fully miscible with many chromatography, where columns and instru-
normal-phase conditions, for example, ments are frequently used in different modes,
n-hexane and heptanes. labeling can be adopted to alert new user
6. If isopropyl alcohol is not available, first check about possible solvent compatibility issues,
miscibility (using small external vessel) with tar- indicating previous use also.
get mobile phase before starting the operation.
7. In a gradient system, isopropyl alcohol should
be in all lines of instrument to make certain Buffers as Mobile Phase
that water or hydrocarbon is removed from all
fluid areas, including the injector valve and Reversed-phase HPLC analysis is affected by
any other selector valves. pH, type of buffer in use and its concentration,
8. Analyst should have a focus on the pressure and presence of organic modifier and its volume.
mark and detector signals during changeover An improper choice of buffer, in terms of buffer-
as these are excellent indicators to confirm ing species, ionic strength, and pH, can result in
that the system has been fully in equilibrium, poor or irreproducible retention and tailing in
Buffers as Mobile Phase 65

Table 4.3 HPLC buffers, pKa values, and useful pH bases gain a proton and become ionized when pH
range [6] decreases. For mixtures containing acids and/or
Buffer pKa (25°C) Useful pH range bases, it is necessary to control the pH of the mobile
TFA 0.5 <1.5 phase using an appropriate buffer in order to achieve
Sulfonate 1.8 <1–2.8 reproducible results. The separation of ionic com-
Phosphate 2.1 1.1–3.1 pounds at satisfactory level can only be achieved
Chloroacetate 2.9 1.9–3.9 with a mobile phase of certain pH where retention
Formate 3.8 2.8–4.8 and resolution are sensitive to it. Generally, a buffer
Acetate 4.8 3.8–5.8
concentration of 10–50 millimolar (mM) is ade-
Sulfonate 6.9 5.9–7.9
quate for small molecules [7, 8].
Phosphate 7.2 6.2–8.2
Ammonia 9.2 8.2–10.2
Phosphate 12.3 11.3–13.3
Buffer Solubility

reverse-phase separation of polar and ionizable Although buffer solubility is determined readily by
compounds due to partial ionization of the ana- precipitation, but this method is tedious and time-
lyte and strong interaction between analytes and consuming. The relationship between the buffer’s
residual silanols or other active sites on the sta- solubility limit and volume fraction of organic
tionary phase. It can be overcome by proper cosolvent is useful in reversed-phase LC as a
mobile-phase buffering (maintaining the pH general rule is that less than 50% organic should be
within a narrow range) and choosing the right used with buffer, which also depends on the specific
ionic species and its concentration (ionic strength) buffer as well as its concentration. The most com-
in the mobile phase [5]. In LC–MS, separations mon buffer systems, including ammonium acetate
depend heavily on the correct choice of acid–base at pH 5.0, ammonium phosphate at pH 3.0 and pH
buffering species and other additives. A suitable 7.0, and potassium phosphate at pH 3.0 and pH 7.0.
buffer always has ability to maintain and not sup- The inorganic buffer salts are less soluble in typical
press analyte ionization in the MS interface. organic cosolvents compared with organic buffer
salts. The sodium salts are avoided because they
are expected to have lower solubility, which makes
Selection of Buffer and Analytical them less useful for liquid chromatography [8].
Method Development

The typical pH range for reversed phase on silica- Buffer and Its Impact on Detection
based packing is pH 2–8; choice of buffer is typi-
cally governed by the desired pH. It is important Slight variations in mobile-phase preparation can
that the buffer has a pKa. A rule of thumb is to result in pH changes that can have dramatic
choose a buffer with a pKa value <2 units of the effects on selectivity, capacity factor (retention
desired mobile-phase pH (Table 4.3) [6]. factor), peak shape, resolution, and reproducibility.
Good laboratory practices in preparing mobile
phases are to be followed to ensure that results
Buffer Concentration can be reproduced within and between laboratories.
While instrument solvent blending has become
In reversed-phase HPLC, the retention of analytes very convenient for fast method development, it
is related to their hydrophobicity. The more hydro- is best to evaluate premixed solvent whenever
phobic the analyte, the longer it is retained. When possible to ensure accuracy and equilibration
an analyte is ionized, it becomes less hydrophobic, before completing and publishing HPLC method.
and therefore, its retention decreases. Acids lose a The choice of buffer is also dependent upon
proton and become ionized when pH increases, and means of detection. For traditional UV detection,
66 4 HPLC: Herbal Drugs and Fingerprints

the buffer needs to be effectively transparent in


this region, especially, critical for gradient separa- Detectors in HPLC: Detection,
tions. Buffers listed in Table 4.3 have low enough Quantification, and Fingerprints
absorption below 220 nm [7].
Different detectors are used for medicinal plant’s
profiling and fingerprinting, for quantitative analyses,
Buffers as Mobile Phase in HPLC and quality control purposes in HPLC. In each
case, the potential range of detection, their sensitiv-
Always, buffers should be prepared freshly on the ity and selectivity and their potential to provide
day requirement to ensure that the buffer pH is online structure information, is unique and specific.
unaffected by prolonged storage and that there is A few common HPLC detectors are as follows:
no microbial growth present. Changes in pH and UV, DAD, FU, EC, RI, ELSD, CAD, MS, MS–MS,
microbial growth affect chromatography up to a NMR, etc. HPLC detectors can be categorized into
drastic extent. Buffer reagents can contain a stabi- two groups: (a) Simple detectors used for the
lizing agent, for example, sodium metabisulphite, recording of chromatographic traces for profiling
which affects the optical and chromatographic or quantification purposes (e. g., UV, ELSD, ECD)
behavior of buffer solutions, so it is often worth and (b) detectors in hyphenation are used to gener-
buying reagents that contain no stabilizer. The ate multidimensional data for online identification
buffer is to be filtered through a 0.45-mm filter and dereplication (e.g., UV–DAD, MS, NMR).
before use to remove any particulate matter that A few detectors in HPLC are selective for compounds
may cause blockages. After filtration, the solvents having specific spectroscopic or physicochemical
are stored in a covered reservoir to prevent con- properties; they may be summarized as follows:
tamination with dust, fumes, etc. The freshly pre-
pared mobile buffer phase is thoroughly to be
degassed to remove all dissolved gasses, by either HPLC–UV
bubbling with helium or sonication and/or vac-
uum filtration. Ammonium acetate, ammonium Ultraviolet (UV) detector is most widely used
formate, ammonium hydroxide solution in water, among all HPLC detectors, although it suffers
ammonium phosphate monobasic, ammonium from some limitations, particularly for natural
trifluoroacetate, potassium phosphate dibasic product compounds that do not possess UV chro-
anhydrous, sodium formate, sodium phosphate mophores. It has the best combination of sensitiv-
dibasic dehydrate, sodium phosphate monobasic ity, linearity, versatility, and reliability of all the
anhydrous, sodium trifluoroacetate, trifluoroacetic LC detectors developed so far. Most natural prod-
acid, triethylamine, etc. are a few reagents used as ucts absorb UV light in the range of 200–550 nm,
buffer during HPLC analysis. Phosphoric acid including all substances having one or more dou-
and its sodium or potassium salts are the most ble bonds and all substances that have unshared
common buffer systems for reversed-phase HPLC. electrons. Thus, even compounds having weak
Phosphonate buffers can be replaced with sul- chromophores, such as triterpene glycosides (e.g.,
fonate buffers when analyzing organophosphate asiaticoside from Centella asiatica), can be suc-
compounds. In LC–MS, volatile buffer systems, cessfully detected by UV at short wavelengths
such as trifluoroacetic acid and trifluoroacetate (203 nm) [9]; in such cases, mobile phase exhibit-
(sodium or potassium), are ideal choices for posi- ing high UV cutoffs is avoided because they might
tive-ion mode detection. TFA can negatively impact blind the detection with weak chromophores [10].
detector response even in positive-ion mode, while In UV detection, the relationship between the
it strongly suppresses ionization with negative-ion intensity of light transmitted through the detector
mode. Acetic acid is good for negative-ion mode. cell and the solute concentration is governed by
LC–MS applications further limit buffer selection Beer’s law [11]. The two factors that control the
and buffer concentration [7, 8]. detector sensitivity are the magnitude of the
Detectors in HPLC: Detection, Quantification, and Fingerprints 67

extinction coefficient of the analyte of interest at at 270 nm and compared with rutin as an external
a given wavelength and the path length of the standard. A more rapid method (5 min) using
light passing through the UV cell. The sensitivity fast-gradient elution for a specific determination
increase with increasing path length but cell vol- of naphthodianthrones (590 nm) and phlorogluci-
ume is designed in such a way to help avoid peak nols (270 nm) also has been reported [14].
dispersion [12]. HPLC–UV analysis also applied for Ginkgo
Presently, three types of UV detectors are biloba; a complete profiling of most of the
available: fixed wavelength, multiple wave- flavonoids (350 nm) (33 compounds detected by
length, and photodiode array (PDA) [also known comparison with standards) was reported by
as diode-array detector (DAD)]. The fixed- Hasler et al. [15]. The same authors also proposed
wavelength detector is the least expensive and an HPLC–UV method, which includes the hydro-
has higher intrinsic sensitivity because the light lysis of all glycosides and the subsequent
is emitted at specific wavelengths with given quantification of three main flavonoid aglycones
lamps and a single wavelength for the detection (quercetin, kaempferol, and isorhamnetin) for the
of nearly all natural products having a chro- determination of the total flavonoid content.
mophore for both profiling and quantification Recently, another HPLC–UV method (350 nm)
purpose. It has been in practice for flavonoids, has been reported, which includes the
terpenes, alkaloids, coumarins, alkamides, and quantification of two glycosides (rutin and quer-
polyacetylene. It is used in several pharmacope- citrin) as markers, in addition to the three agly-
ias, as it is simple and inexpensive, for the cones [16]. An analysis of ten commercial
quantification of individual natural product in samples revealed that problems of adulteration
the quality control of herbal drugs or phyto- with rutin exist to fulfill the total flavonoid
preparations. In the United States Pharmacopeia requirement (24%), and thus, the method is
(USP) and European Pharmacopeias, HPLC–UV highly helpful in quality control and total
(203 nm) methods are used for the determina- fingerprint profiling [17].
tion of the total ginsenoside content of ginseng A multivariate data analysis (MVDA) of
roots and the measurement of the ratio of the Ginkgo products, based on their HPLC–UV
ginsenosides [the ginseng saponins, structurally profiles, and comparison with the traditional
grouped into two groups: the panaxadiol (PD) flavonol quantification method [18] revealed that
fraction (e.g., ginsenoside-Rb1, ginsenoside- there are significant variations in the relative con-
Rb2, ginsenoside-Rc, ginsenoside-Rd) and the centrations of individual flavonols in commercial
panaxatriol (PT) fraction (e.g., ginsenoside- products. The other active ingredients in Ginkgo
Rg1, ginsenoside-Rg2, ginsenoside-Re, ginse- are the terpene trilactones, which are standard-
noside-Rf, depending on their glycons). The ized at 6% in the extracts. These terpenes have
ginsenoside-Rb1 and ginsenoside-Rg1 are weak chromophores (at wavelength 220 nm) and
believed to be the most pharmacologically effec- are difficult to detect in the presence of the
tive compounds] Rg1 and Rb1. This analysis is strongly UV-absorbing flavonoids, so quantified
important to assess pharmacological effects, as by other methods such as ELSD [19]. In Ginkgo,
a low Rg1/Rb1 ratio is linked to the calming the level of highly allergenic compound alky-
properties of American ginseng, while the high lphenols (ginkgolic acids, cardanols, and cardols)
Rg1/Rb1 ratio to be for the stimulating charac- are part of specification, and maximum limit is
teristics of Asian ginseng [13]. 5 ppm, as per EU and US Pharmacopeias. The
HPLC–UV detector for the quantification of total amount of alkylphenols is determined by a
flavonoid, naphthodianthrone, and phloroglucinol simple HPLC–UV (210 nm) [20].
constituents of Hypericum perforatum by a ter- The separation of crude extracts using gradi-
nary solvent system in the gradient mode (50 min) ent elution causes a shift of the baseline at low
had separated 16 main constituents, which were wavelengths that can be avoided when working
quantified based on their relative response factors with modifiers having low UV cutoffs such as
68 4 HPLC: Herbal Drugs and Fingerprints

phosphate buffers or trifluoroacetic acid (TFA). quantification of aflatoxins in herbs also has
These modifiers are not optimal in multi-hyphen- been recently reported [27]. It combines sample
ated systems with HPLC–MS (due to nonvolatile clean-up with immuno-affinity columns fol-
solvents, which have impact for ion suppression) lowed by HPLC–FD, and it enabled the detec-
and are avoided [21]. In other cases, for the same tion of aflatoxin content ranging from 7 to 20 mg/
plant extract, quantification may require both kg in plant material. HPLC–FD also has been
HPLC–UV and HPLC–MS. A simple HPLC–UV used for the screening of major benzo-phenan-
method for the standardization of herbal supple- thridine alkaloids produced by cell cultures of
ments as Pueraria lobata [known as Kudzu Eschscholzia californica [28]. The detection of
(Japan)], a fully validated as per the ICH guide- natural products that do not have natural
lines for the quantification of the isoflavone, is in fluoresce has been successfully achieved after
practice [22, 23]. the addition of fluorescent tags. This was the
HPLC–UV detector is used to compare the case for the specific and sensitive detection of
fingerprint profiles of closely related species or the plant hormone jasmonic acid which was
the same species from different locations. Herba tagged with zink cyanide [Zn(CN)2] (Adam’s
Oldenlandiae, a plant of the Chinese Pharmacopeia, reagent) [29].
which can be adulterated by different Oldenlandia
species. A comparative analysis of chemical
components was obtained by software calcula- HPLC–CL
tion of correlation coefficients of the entire UV
chromatograms for appraising similarity [24]. Chemiluminescence (CL) may be defined as the
Fingerprint-based similarity also can be emission of light from a molecule or atom at an
employed for chemotaxonomic study by apply- electronically excited state due to chemical reac-
ing hierarchical clustering analysis (HCA) and tion at an ordinary temperature without any addi-
principal component analysis (PCA) to a set of tional generation of heat. Chemiluminescent (CL)
HPLC–UV profiles, as was used for different nitrogen detection is a new technology for the
Taxus species [25]. detection of nitrogen-containing molecules, includ-
ing a broad range of pharmaceuticals with very
high sensitivity up to the femtogram range [30]. As
HPLC–FD many compounds are not chemiluminescent, they
can only be detected chemiluminescently after
In the fluorescence detector, the detection is derivatization. There are many chemiluminogenic
based on the molecular absorption of a photon, reagents, which helps in chemiluminescence detec-
which triggers the emission of another photon tion. The chemiluminogenic reagents are applied at
with a longer wavelength. This difference in post-column stage; for example, addition of potas-
wavelengths (absorption vs. emission) provides sium hexacyanoferrate (III) enables the detection
more selectivity, and the fluorescent light is of various carboxylic acids in the form of N-(4-
measured against a very low-light background, aminobutyl)-N-ethylisoluminol derivatives [31].
thus improving the signal/noise (S/N) ratio. Though it is a very sensitive method, but till the
There are very few natural product compounds time, it has not been much applied to the analysis of
having fluorescent in a practical range of wave- crude plant extracts. A study reported the sensitive
lengths, but compounds can be made to fluoresce HPLC–CL detection of flavonoids (LOD 3 ng/ml)
by forming appropriate derivatives [12]. It has in phytopharmaceuticals containing Hippophae
higher sensitivity and selectivity compared with rhamnoides. The procedure was based on chemilu-
HPLC–UV. The technique is used for detection of minescent enhancement by flavonols of the cerium
aflatoxins in food because aflatoxins have natural (IV)–rhodamine 6 G system in a sulfuric acid
fluorescence [26]. An FD method for the medium [32].
HPLC Detection in Hyphenated System 69

HPLC–ECD HPLC–ECD is a common technology used in


many pharmaceutical, clinical, and biochemical
Electrochemical detector (ECD) detection is laboratories to evaluate the antioxidant properties
usually performed by maintaining the potential as it is most suitable for their accurate detection
of the working electrode at a fixed value relative and quantification with high accuracy in food
to the potential of the electrolyte, which is mea- ingredients [35]; for example, ECD was found to
sured by the reference electrode. The fixed be more sensitive than UV for polyphenols esti-
potential difference applied between the work- mated by HPLC with coulometric detection
ing and the reference electrodes drives the elec- because they are reducing agents, and their elec-
trochemical reaction, and the resulting current is trochemical responses result from the donation of
measured as a function of the elution time. This electrons. Some degree of correlation between
allows for detection limits at the picomole level. the electrochemical profiles of 17 flavonoids and
The selectivity of ECD depends on the accessi- 3 cinnamic acid derivatives and 3 different anti-
ble potential range, the number of compounds oxidant assays has been established. Artemisinin
that are active in this range, and the half-widths was selectively detected and quantified in plasma
of the individual signals [10]. Compounds hav- using a glassy carbon electrode by reductive ECD
ing electrolytic characters are measured and [36]. Recently, different amino acid derivatives
detected by HPLC with electrochemical detector characteristic for Korean ginseng have been ana-
(ECD). This detection method is inexpensive, lyzed by pulsed amperometric detection in both
sensitive, selective, and widely accepted [10]. ginseng and plasma samples [37].
Numerous functional groups are sensitive to oxi-
dation, including phenols, aromatics, amines,
thiols, and quinolines, and many are compatible HPLC Detection in Hyphenated
with reduction, such as olefins, esters, ketones, System
aldehydes, ethers, and quinones. ECD differs
from other methods of detection because it alters HPLC in hyphenation is used to generate multidi-
the sample. Cells for HPLC–ECD usually con- mensional data (i.e., both chromatographic as well
sist of three electrodes, the working, counter, as spectrometric) using the following practices:
and reference electrodes, which can be aligned
in several different geometries and coulometric
systems; the eluent is directed through the elec- HPLC–RID
trode, while in amperometric systems, the eluent
passes over it. Pulsed techniques, which use Refractive index detector (RID) detection is
multistep potential-time waveforms to realize based on the simple principle of refraction, in
amperometric/coulometric detection while main- which we have zero response to mobile phase,
taining uniform and reproducible electrode activ- but, as soon as an analyte enters in detector with
ity, are also available for detection [33]. mobile phase, after selective segregation by col-
HPLC–ECD has special recognition for the umn, is detected by detector using refractive
study of the metabolism of aromatic compounds properties and recorded by the recorder. Though
as it is associated with electrochemistry. The it is a most simple and least expensive detector,
proper use of LC–ECD requires knowledge of the refractive index (RI) detector, which was
redox reactions and their dependence on mobile- reported by Tiselius and Claesson in 1942 [38],
phase composition. In this respect, the study of lacks sensitivity; is very susceptible to changes in
electrode reaction mechanisms is helpful in the ambient temperature, pressure, and flow rate; and
elucidation of mechanism of their interaction cannot be used for gradient elution. Despite these
with living cells, such as antioxidant character of disadvantages, this detector has been useful for
natural products [34]. detecting compounds such as carbohydrates and
70 4 HPLC: Herbal Drugs and Fingerprints

polymers. Because of its inherent limitations, phase composition (in the case of gradient elution),
HPLC–RID has been favorably replaced by other which makes such quantification procedures
detection methods such as HPLC–ELSD, HPLC– experimentally inaccessible. Thus, this interplay
CAD, and HPLC–FID. of several factors leads to a nonlinear response,
which is one disadvantage of this type of detector
and ultimately renders direct linear regression
HPLC–ELSD for making calibration curves inaccurate [43].
In order to avoid the variation generated by the
Evaporative light-scattering detector (ELSD), gradient elution, the post-column addition of
introduced in 1966 by Ford and Kennard [39], is mobile-phase flow having an opposite composi-
a quasi-universal detector for liquid chromatog- tion is an interesting alternative [44].
raphy, as it can detect any analyte which is less The mass-dependent detection property of
volatile than mobile phase, regardless of the opti- ELSD is entirely in contrast to UV, which is a
cal, electrochemical, or other analyte properties concentration-based detector, and response gen-
[40]. The operating principle of the ELSD is that erated does not depend on the spectral or physic-
the eluent is nebulized using a flow of nitrogen, ochemical properties of the analyte.
and the resulting aerosol is transported through a The increased interest in ELSD as a universal
heated drift tube where the volatile components detector is that it is more compatible with gradi-
and solvents are evaporated. The remaining solid ent elution than RID, and it is much less costly
fraction is subsequently introduced into a cell, and easier to maintain than an HPLC–MS plat-
where a light beam is directed into the particles form. It is an additional detector rather than a
that cause scattering of the incident light and is substitute in HPLC.
detected by a photodiode or a photomultiplier. The applications related to natural products,
The most important parameters affecting the ESLD has been used mainly for the detection of
ELSD signal response are the nebulizer gas flow compounds with weak chromophores such as
rate and the drift tube temperature [41]. The gas terpenes [45], in both aglycone [42] and glyco-
flow rate influences the droplet size of the col- sidic forms [46], saponins, and some alkaloids
umn effluent before evaporation occurs. Higher [47]. The method also has been applied in com-
flow rates result in the formation of smaller aero- plement to HPLC–MS for surveying natural
sol droplets and less scattering of light, with sub- product libraries [48].
sequent lower sensitivity, but they allow for a
more stable baseline. The drift tube temperature
facilitates the evaporation of the nebulized aero- HPLC–CAD
sol so that the light-scattering response of the
nonvolatile solute can exclusively be determined. In charged aerosol detector (CAD), the dried particle
Higher temperatures are needed for the mobile stream is charged with a corona discharge needle,
phase of high-aqueous-content or nonvolatile sol- and the resulting electrical charge is measured by
utes vs. semi-volatile ones. However, the new an electrometer. CAD operates by detecting
generation of ELSD is able to vaporize the eluent charge particles with a selected mobility range
at low temperature, facilitating the detection of rather than measuring individual gas-phase ions
semi-volatile analytes. It is pertinent to optimize as in MS. Generally, CAD is more sensitive than
these parameters to ensure that the optimal signal- ELSD, and the operating principle of CAD is
to-noise ratio (S/N) is achieved [42]. roughly comparable to that of ELSD. Contrary to
ELSD is a mass-dependent detector; that is, it ELSD, the detection method provides a uniform
generates a similar response for moieties of equal response to nonvolatile analytes, but detection is
mass. However, the ELSD response also depends based on mass, whereas CAD is a charge based
on the volatility of the compound and the mobile- detection.
HPLC Detection in Hyphenated System 71

Charged aerosol detection (CAD) is a new molecular formula, and diagnostic fragments,
technology for universal HPLC detection, intro- which are crucial for dereplication and rapid
duced by Dixon and Peterson in 2002 [49], and online characterization.
is more dedicated to the analysis of non-UV- The high volume of mobile-phase flow in
absorbing compounds [43]. The operating prin- HPLC analysis is a major problem to compete
ciples of CAD are roughly comparable to that of with the high vacuum required for the mass
ELSD. Because it is a new detection method, so spectrometer; it has been overcome through the
it has not yet been popularized for natural prod- use of atmospheric pressure ionization (API)
uct’s detection to the large extent, although it has interfaces including electrospray ionization (ESI)
been used for a wide range of pharmaceutical and atmospheric-pressure chemical ionization
products. Both CAD and ELSD were evaluated (APCI). For both ESI and APCI, a combination
for a library of more than 700 compounds in the of high voltage and heat is used to produce the
150- to 700-Da range and failed only in the ions that are assayed by the MS system. In ESI,
detection of two compounds having small MWs the high-voltage field (3–5 kV) produces nebuli-
[50]. The method is advantageous in that no nor- zation of the column effluent, resulting in charged
malization of the peak area is needed, as with droplets that are directed toward the mass ana-
HPLC–MS. lyzer. These droplets get smaller as they approach
the entrance to the mass analyzer. As the droplets
get smaller, individual ions emerge in a process
HPLC–FID referred to as “ion evaporation”; these ions are
then separated by the MS system. In APCI, heat
Flame ionization detector, a most common ana- is used to vaporize the column eluent, and then a
lytical detection for organic solvents in gas–liq- corona discharge is used to ionize solvent mole-
uid chromatography (GLC) applied on the same cules, which then produce the analyte ions via
principle in HPLC, detects all carbon-containing chemical ionization mechanisms. More recently,
molecules, segregated by column at the same a third type of ionization source, termed atmo-
instant, in both normal- and reversed-phase spheric pressure photoionization (APPI), has
modes. HPLC–FID coupling implies working been introduced. In APPI, heat is used to vapor-
with 100% water as mobile phases. Water is a ize the column eluent (similar to APCI), but the
very weak mobile phase at room temperature, but ionization is produced by way of an ultraviolet
with superheated water under pressure to tem- (UV) lamp that produces 10-eV photons.
peratures between 80 and 250°C, the polarity of Depending upon the solvent system used, the
liquid water decreases markedly to the extent that 10-eV photons ionizes either the mobile-phase
it can replace the organic modifier and act as the solvent or a dopant (e.g., toluene) added to the
sole eluent [51]. The successful applications of column effluent; these ions then produce the ana-
HTLC–FID have been described [52], mainly for lyte ions through various ionization mechanisms
the separations of alcohols, carbohydrates, and including charge or proton transfer [53].
amino acids. As per literature search, this detec- These API methods generally provide a soft
tion technology is rare in practice. ionization and mainly molecular ion species in
the form of either protonated molecules [M + H]+
[positive-ion mode, (PI)] or deprotonated mole-
HPLC–MS cules [M − H]− [negative-ion mode, (NI)].
Different adducts (e.g., [M + Na]+ (PI) or
The mass spectrometry with HPLC as a detector [M + HCOO]− (NI)) are also produced, depending
has excellent sensitivity and selectivity for the on the solutes and the modifiers used. The notable
analysis of natural products including important point is to understand that analyte ionization is
structural information such as molecular weight, largely dependent on analyte and is governed
72 4 HPLC: Herbal Drugs and Fingerprints

mainly by proton affinity. As a rule of thumb HPLC–MS–ELSD for natural product’s


for a good approximation, acidic molecules profiling including phenolics, alkaloids, amides,
(e.g., carboxylic acids or phenolics) will produce terpenoids, steroids, and polyketides, with gradient
mainly [M − H]− in NI, while bases (e.g., alka- elution, using ESI (in both PI and NI mode) were
loids, amines) will generate [M + H]+ in PI. well detected in PI and NI mode [48]. It was con-
Compounds such as glycosides have a high cluded in an investigation that ESI (PI) is used
affinity for salts and tend to form sodium adducts mainly for alkaloids, APCI (PI) for pigments and
in PI. Molecule fragmentation or partial de- carotenoids, ESI (PI/NI) for triterpene glyco-
clustering of the adducts can be induced in one of sides, and APCI or ESI (PI/NI) for phenolics and
the high-pressure regions of the ion beam from flavonoids. APCI is generally more suitable for
the source to the mass analyzer by collision- nonpolar constituents, while ESI provides a softer
induced dissociation (CID) either at the API ionization and gives a good response for polar
source (in-source CID) or in conjunction with metabolites. On the other hand, APCI is gener-
tandem MS (MS–MS) experiments [54]. ally recognized to be less prone to matrix effects
Presently, there are many types of mass spec- than ESI [57]. Only a few studies have referred to
trometers that can be used as detector with HPLC. the use of APPI for natural product’s detection
Those that are low resolution (LR), such as the [58]. As ionization of analyte is dependant (in
single-quadrupole (Q) mass spectrometers, are HPLC–MS) on the selection of the interface, the
the most commonly used and least expensive, ion mode polarity and the eluent composition
whereas in high resolution (HR) and exact mass might significantly affect the sensitivity. HPLC–MS
capabilities, such as time-of-flight (TOF) instru- is the method of choice, while HPLC–UV and
ments, they are becoming increasingly popular HPLC–ELSD provide sensitive and selective
because they provide molecular formula infor- detection at shorter analysis time, but MS pro-
mation online and very precise ion trace extrac- vides useful structural information for a con-
tion (+/− 0.01 DA) for a specific detection. For firmation of the identity for the detection of
measuring structurally relevant fragments or for natural products in biological fluids, related sam-
very specific detection, triple-quadrupole (QQQ) ples, and quantification of flavonoids, alkaloids,
MS–MS systems are the most commonly used, saponins, sesquiterpenoids, etc.
for particularly bio-analytical assays and for
specific quantification purposes in complex
matrices through multiple reaction monitoring HPLC–NMR
(MRM) [55]. Ion-trap mass spectrometers have
the unique capability of producing multistage It is a high-cost technique, basically used for online
MS–MS (MSn) data that may be essential for structural information [1]. H–NMR can detect
structural elucidation purposes. In addition to all analyte-bearing protons. HPLC–19F–NMR
these four kinds of mass spectrometers, there are has been used for the specific detection of
a growing number of additional types, including fluorinated derivatives, for example, for studying
hybrid systems that combine LR and HR analyz- the metabolic fate of 3-chloro-4-fluoroaniline in
ers for specific applications [53]. rat body fluids [59].
Besides their widespread use for qualitative
analysis [dereplication studies and crude extract
profiling], HPLC–MS and HPLC–MS–MS have LC–Hyphenation for Online Structural
been successfully used for the specific detection and Identification
quantification of a large range of natural products.
While mainly simple quadrupole or triple-quadru- The hyphenated HPLC with other techniques is
pole analyzers are considered for quantitative stud- generally termed as LC–hyphenation, in which
ies, TOF instruments have recently increasingly both chromatographic and spectroscopic dimen-
been used as quantification tools, though histori- sions to generate structurally relevant data for
cally it suffered from a narrow dynamic range [56]. the online identification and dereplication of a
LC–Hyphenation for Online Structural Identification 73

compound by the validated method are used, lead compounds, faster cycle times, and high
for example, DAD, MS, NMR, CD, and IR used efficiency, many pharmaceutical companies have
in hyphenation with HPLC for their online moved away from the natural product’s area.
identification and dereplication purposes. Currently, almost every large pharmaceutical
The ability to rapidly identify known or company has established HTS infrastructures
undesirable compounds using natural product and possesses large combinatorial compound
extract libraries (for natural product discovery libraries, which cover a wide range of chemical
program) is called dereplication. It is important diversity. However, the ability to detect the
to prevent the unnecessary use of resources on desired biological activity directly in the HPLC
the isolation of compounds of little or no value effluent stream and chemically characterize the
for development from extracts used in the screen- bioactive compounds online, eliminates much of
ing process and best utilization of resources to the time and labor taken in the fraction collection
focus on samples containing the most promising strategy. This way, cycle times, expenses, and
leads. Dereplication strategies typically employ the isolation of known or undesirable compounds
a combination of separation sciences, spectro- would be reduced dramatically, allowing natural
scopic methods, and database searching. HPLC products to be screened in an efficient and cost-
has been the most reliable tool for the separation effective manner [60].
of complex mixtures of small molecules. Dereplication of natural product analysis
Reversed-phase HPLC on octadecylsilane (ODS using hyphenated technique is strategic for guid-
or C-18) is recognized as the most broadly appli- ing an efficient lead-finding process, but this task
cable of bonded phases for this purpose. The is not easy to perform, as no exhaustive LC spec-
interfaced with a diode-array detector (DAD), troscopic databases are available. Furthermore,
HPLC allows an analyst to identify known com- even if libraries of natural product spectra could
pounds by comparison of their HPLC retention be efficiently generated on a given LC-hyphenated
times and UV spectra. At present days, the use of system, their widespread use is complicated by
electrospray ionization (ESI) and atmospheric- the fact that, contrary to GC–MS, most of the
pressure chemical ionization (APCI) interfaces data, especially HPLC–MS and HPLC–MS–MS,
has provided mass spectrometry (MS) interfaces are instrument dependant. Thus, for each new
which are in use of the analysis of a wide range compound, both the order of the atoms and the
of molecules and are compatible with liquid stereochemical orientations have to be elucidated
chromatography. LC–MS has become a widely de novo. Consequently, a single LC-hyphenated
used tool for the dereplication of natural prod- technique does not exist that is capable of identi-
ucts because the nominal molecular weight of a fying online all secondary metabolites of a plant.
compound can be used as a search query in As each technique presents its limitations, but
nearly all databases. Dereplication of the active coupling of various hyphenated methods together
component(s) in crude natural product extracts has the advantage that all structural information
requires some form of feedback from the bioas- is obtained within one single analysis [61]. For
say, which was initially used to detect the bio- natural product analysis, the dereplication proce-
logical activity. This is necessary regardless of dures rely mainly on HPLC–DAD–MS and
the separation technique and analytical method chemotaxonomic information provided by natu-
(DAD or MS) used. This approach requires des- ral product databases. When unknown natural
iccation of fractions to remove the HPLC sol- products cannot be dereplicated by these means,
vents, which are usually incompatible with the complementary information can be obtained by
bioassay, re-suspending the fractions in a com- NMR either directly hyphenated with LC
patible solvent (water or DMSO), and then indi- (HPLC–NMR) [62] or used at line with pre-con-
vidual assaying of each fraction. This process is centration methods such as HPLC–SPE–NMR
not cost effective, being both time and labor [63] or after micro-fractionation with micro-flow
intensive. Consequently, as a result of the HPLC–NMR methods or even with a combina-
increasing emphasis on the generation of new tion of both techniques by CAP–SPE–NMR
74 4 HPLC: Herbal Drugs and Fingerprints

[64]. Other hyphenated methods, such as HPLC–IR fragmentation of the molecular species by CID
or HPLC–CD, can generate useful additional reactions. For online dereplication purposes, the
structural information. A few commonly used determination of molecular weight is high priority
techniques are as follows: and necessitates the comparison of MS data
obtained under different detection conditions
(protonated or deprotonated molecules from
HPLC–DAD (Diode-Array Detector) adducts or fragments). The use of high-resolution
instruments, such as a TOF–MS or FT–MS sys-
It is also known as photodiode-array detection tem, enables direct determination of the molecu-
(PDA) for online structural determination. lar formula of crude mixtures. This strategic
HPLC–DAD–MS is a key strategic tool, and it is information allows for more precisely targeting
used for dereplication purposes in laboratories the search in natural product libraries for derepli-
involved in drug discovery. It is a manifold tech- cation purposes [66].
nique in the analysis of natural products, with In natural product chemistry, MS–MS spectra
characteristic chromophores (e.g., polyphenols, are mainly useful for the partial determination of
phenolics, polyketides, alkaloids, and terpe- sugar sequences of various glycosides, detection
noids), commonly used as multi-hyphenated with of characteristic losses, such as those of preny-
MS or ELSD detectors. For such compounds, lated compounds, classic fragmentation of
DAD–UV spectral libraries are consulted for flavonoids, and the determination of substituted
dereplication, but the compound has to be ana- positions on A or B rings as well as the differen-
lyzed under the same HPLC conditions, as the tiation of isomers. For the analysis of fully
composition of the mobile phase might affect the unknown constituents, MS–MS usually cannot
UV bands slightly. Furthermore, the choice of provide enough information to ascertain structure
modifiers affects UV detection and might blind determination, and thus, the combination with
the bands in the low-wavelength range. The pos- other online information is mandatory.
sibility to acquire several UV spectra across a
given LC peak allows the assessment of peak
purity [65]. Comparison of the genuine and HPLC–NMR (Nuclear Magnetic
shifted online DAD spectra enables a precise Resonance)
localization of the hydroxy groups on the poly-
phenols online. Another interesting point regard- It is a powerful tool to those compounds which
ing DAD–UV detection is that all wavelengths have been partially identified with MS–MS data-
are stored during analysis, and thus, multiple bases, as additional spectroscopic information to
wavelengths can be monitored at the same time ascertain the identity of, in some cases, a complete
for detection of different classes of compounds. structural assignment I.D. obtained by HPLC–
This has been found to be very useful for simul- NMR. The limiting factor of online HPLC–NMR
taneous detection of the naphthodianthrones is the need for solvent suppression because HPLC
(590 nm) and phloroglucinols (270 nm) of separation is performed with MeCN-D2O and
Hypericum perforatum [14]. MeOH-D2O. In practice, the very large signal of
MeCN or MeOH and the residual HOD signal are
completely eliminated by powerful solvent-sup-
HPLC–MS, MS–MS, and MSn pression sequences. The main drawback of this
procedure is that analyte signals localized under
“HPLC–MS” detection is a key technique for the the solvent resonances also will be suppressed,
online identification of natural products by gener- and thus, some information can potentially be lost.
ating nominal mass of molecular ions or accurate Another problem occurring in HPLC–NMR is that
mass measurements for the determination of the chemical shifts recorded in a typical reversed-
empirical formulas. Tandem or hybrid MS instru- phase solvent differ from those reported in stan-
ment is used for structural information through dard deuterated NMR solvents [67].
HPTLC–HPLC for Quality Assurance 75

HPLC Biochemical Detection especially in traditional formulation/recipes where


basic scientific information is still lacking [68].
The online HPLC–biochemical detection
(LC–BCD) system describes a range of targets,
such as the human estrogen receptor (hER), the Case Study
urokinase receptor, and acetylcholinesterase and
phosphodiesterase. After injection, the complex A Thai traditional medicine, “Ayurved Siriraj
mixture is chromatographically separated, and Prasachandaeng,” from 12 raw herbs is used
subsequently, separated compounds are intro- extensively as an antipyretic for both children
duced in a biochemical assay (microtiter-type and adults. The formula, prepared in powder,
bioassays, where interactions between extracts requires the combination of 12 medicinal herbs
and the biochemical reagents proceed at high based on historical references and several lines
speed in a closed continuous-flow reaction detec- of empirical evidence in Thai traditional medi-
tion system. When sufficient resolution is cine practice. Ayurved Siriraj Prasachandaeng
achieved, the individual contribution of the bio- belongs to the Center of Applied Thai Traditional
active compounds to the total bioactivity is obtained Medicine (CATTM), Faculty of Medicine Siriraj
within a single run). The chemical information Hospital, and Mahidol University, Thailand,
using aforesaid type online biochemical detection which formerly was the Clinic of Thai Traditional
with complementary chemical analysis techniques Medicine established by Prof. Ouay Ketusingh
[as mass spectrometry, (i.e., LC–BCD–MS)] is in 1982. CATTM has many Thai traditional rec-
obtained for the characterization and identification ipes which have been produced and used with
of bioactive molecules, in real time, compared to satisfaction for a long time. In a study using
traditional screening approaches of complex mix- HPTLC and HPLC, 12 medicinal herbal compo-
tures, which are often characterized by a repeat- nents and three batches of Ayurved Siriraj
ing cycle of HPLC fractionation and biological Prasachandaeng produced from different times
screening and LC–BCD–MS analysis [60]. were analyzed. The chromatograms from three
batches were regarded as the original fingerprints
of Ayurved Siriraj Prasachandaeng. Phenolic
HPTLC–HPLC for Quality Assurance composition, probably found in some compo-
nents such as gallic acid, caffeic acid, p-coumaric
Quality assessment has been established by using acid, ferulic acid, vanillic acid, and kojic acid
HPTLC–HPLC, where color and rRf (relative was also determined. For HPLC analysis, all
retention factor) of bands are compared with solvents were HPLC grade. Water was purified
those of reference markers in case of HPTLC, by a Milli-Q system (Millipore Corp., USA).
while rRt (relative retention time) and applied Other chemicals and reagents were analytical
information content (j) are used for HPLC data. grade, purchased from Merck, Germany. Gallic
These are not only for controlling the quality of acid, caffeic acid, p-coumaric acid, ferulic
herbal raw materials which directly affects their acid, vanillic acid, and kojic acid were from
efficacy and safety but also controlling the con- Sigma, USA. Ayurved Siriraj Prasachandaeng
sistency of different batches and offer integral and its 12 raw herbs were obtained from an
characterization of a complex system with a authentic source. All of them were indepen-
quantitative reliability. These two techniques, dently identified by at least two Thai traditional
in combination or alone, can be successfully pharmacists of CATTM according to morpho-
used to determine most of the phytochemical con- logical characteristics and produced following
stituents of herbal products in order to (a) ensure the standard operating procedures (SOPs) of
the reliability and reproducibility of pharmaco- good manufacturing. Twelve plants were
logical and clinical researches, (b) to understand ordered for component no. 1–12 which were
bioactivities and possible side effects, and (c) to Citrus aurantifolia Swing., Bouea macrophylla
enhance quality control of herbal products, Griff., Knema globularia Warb., Dracaena loureiri
76 4 HPLC: Herbal Drugs and Fingerprints

Gagnep., Myristica fragrans Linn., Caesalpinia


sappan Linn., Conioselinum univitatum Trucz., The mobile phase for HPLC was composed
Kaempferia galanga Linn., Mesua ferrea Linn., of (A) O-phosphoric acid (0.1%, v/v) and
Jasminum sambac (Linn.) Ait., Mammea sia- (B) acetonitrile using an isocratic condition
mensis Kosterm., and Nelumbo nucifera Gaertn., of 95% A and 5% B at 0–4 min; a gradient
respectively [68]. elution of 95–85% A, at 4–8 min; 85–0%
The HPTLC system (Camag, Switzerland) A, at 8–13 min; and 0–95% A, at 13–16 min.
consisted of a Linomat 5 sample applicator The flow rate was 1.0 ml/min; UV spectra
equipped with a 100-ml syringe and a Scanner 3. were recorded over the range of 200–
Aluminum HPTLC plates (20 cm × 10 cm) pre- 400 nm. The mathematic methods for
coated with silica gel 60. HPLC system (Waters, HPLC analysis, relative retention time
Milford, MA, USA) consisted of a Waters 2695 (rRt) value, and applied information content
Separation Module system equipped with a pho- (j) value were used to validate similarities
todiode-array detector. An X-Terra RP18 column among batches [68]. The calculated factors,
100 mm × 4.6 mm ID., with 1.7-mm particle size including Rf and rRf from HPTLC and rRt
was used [68]. and j from HPLC, were applied for the
Three batches of Ayurved Siriraj Prasachan- identification and quality control of each
daeng and 12 herbal components were extracted batch of the recipe, and each component
separately in 80% ethanol and lyophilized to was compared with the reference markers.
dry powder. Each powder was dissolved sepa- Like bioequivalent (BE) assessment,
rately in 80% ethanol for HPTLC and 50% coding a standard BE range for basic
methanol for HPLC and then filtered through a pharmacokinetic parameters 80–125% of
0.2-mm membrane filter before used. For the mean has been generally accepted.
HPTLC qualification of retention factor (Rf) Moreover, the similarity among different
value, gallic acid, caffeic acid, p-coumaric acid, batches of the recipe was indicated by
ferulic acid, vanillic acid, and kojic acid (1 mg the relative standard deviation (RSD) of
each) were dissolved separately in 80% etha- the rRt and j values less than 15%, and the
nol. Gallic acid and caffeic acid solutions were HPTLC and HPLC procedures used in this
prepared likewise for the HPTLC qualification study exhibited a satisfactory repeatability
of relative retention factor (rRf) value. For which potentially provides a reliable
HPLC, gallic acid, caffeic acid, and vanillic measure for quality control of the herbal
acid (1 mg each) were dissolved separately in products.
50% methanol. Each solution was vortexed for
2 min and filtered through a 0.2-mm membrane
filter to obtain the filtrate as the reference
marker [68]. Different Chemical Classes from
The mobile phase for HPTLC was of hexane– Natural Products and LC Fingerprints
ethylene acetate–acetic acid (31:14:5). Each ref-
erence standard and each sample were spotted on Presently, scientists are looking for lead com-
the HPTLC plate and developed in a saturated pounds with specific structures and pharmacologi-
mobile phase to a distance of 90 mm. After plate cal effects from natural sources. The wide
drying in a stream of cold air for 3 min, each plate experiences and successes of Chinese scientists in
was visualized under 254- and 366-nm UV and this specialized area have resulted in a number of
then sprayed with 0.5% Fast Blue B Salt (FBS) widely used drugs. The tremendous progress made
heated at 110°C until the bands were clearly vis- in life sciences has not only revealed many patho-
ible under visible light. Finally, Rf and rRf values logical processes of diseases but also led to the
were calculated [68]. establishment of various molecular and cellular
Different Chemical Classes from Natural Products and LC Fingerprints 77

bioassays in conjunction with high-throughput Fingerprints for Terpenoids


technologies. This is advantageous and permits
certain natural compounds that are difficult to iso- The large class of terpenoids includes not only
late and purify, and compounds that are difficult to the compounds with a triterpene and steroidal
synthesize, to be assayed. This technical report structure (saponins, sterols, cardiotonic glyco-
compiles and analyzes the current scientific knowl- sides) but also the compounds with an isoprenic
edge on herbal drugs and highlights the practical structure, such as volatile oils, terpenes (bitter
ways for ensuring the safety of herbal preparations principles), and carotenoids. The volatile oils are
and evaluating their claimed efficacy, supported by aliphatic or aromatic hydrocarbons, aldehydes,
fingerprints. Examples for a few chemical classes alcohols, acids, esters, etc. and are widely distrib-
from natural products may be cited as follows: uted in nature. Structurally, the volatile oils are
monoterpenes, sesquiterpenes, and azulenes.
Volatile oils are obtained by distillation with
Fingerprints for Flavonoids water or with nonpolar solvents. The most used
method for their analysis is GC, but some HPLC
Flavonoids, the natural polyphenols, are benzo- methods are reported in the literature on a silica
pyrane derivatives with a phenyl group in the sec- stationary phase and a binary mixture, n-pentane/
ond position, widely spread among different plant water, as mobile phase. LC–MS methods have
species. Flavonoids can be O-glycosides, usually also been applied. Standardized extracts of
in the positions three or seven. According to the Ginkgo biloba leaves contain large quantities of
degree of oxidation, the flavonoids can be classified terpene-like compounds and are mainly used in
as calcones, flavanones, flavones, flavonols, cate- the treatment of peripheral and cerebral circula-
chins, terpenylflavonols, isoflavonols, etc. Many tion disorders or as a remedy against asthma,
flavonoids have an antioxidant activity and are coughs, bladder inflammation, blennorrhagia,
involved in the oxidation processes which take and alcohol abuse. The leaf extracts contain
place into the cell. Their biological activity is biflavones, flavonol glycosides, and terpene lac-
very important, and many allopathic, ayurvedic, tones. A method based on liquid chromatogra-
or homeopathic drugs rich in flavonoids are on phy, coupled with electrospray mass spectrometry,
the market, with high consumer demands, so it is has been reported for the analysis of terpenoids in
necessary to have more reliable analytical meth- G. biloba extracts. This method allows the rapid
ods, with fingerprints for the same. isocratic separation of underivatized ginkgolides
Flavonoids can be extracted from plants by (GA, GB, GC, and GJ) and bilobalide at very low
various methods, most of them involving an levels (10 pg on the column) and their quantita-
extraction in ethanol, followed by a precipitation, tive detection by external standardization with
at room temperature, and purification. Flavonoids relative standard deviations of 3 and 5% for intra-
are generally stable compounds and may be and inter-day analyses, respectively [70].
extracted from the dried, ground medicinal plant Carotenoids are compounds with a terpenoid
material and pharmaceutical preparation with structure with 30–50 carbon atoms in the mole-
cold or hot solvents (aqueous mixtures with etha- cule. Carotenoids are widely spread in nature,
nol, methanol, acetone, and dimethylformamide, being the yellow/red pigments which can be
etc.); further purification is usually done by sol- found together with chlorophylls. Carotenoids
vent–solvent exchange. The use of a large num- have a provitamin A structure and have an impor-
ber of methods, individually or hyphenated, has tant biological activity in the visual process and
been published (TLC, HPLC, GC, electrophore- regarding the epithelial tissue. These compounds
sis, gravimetry, spectrophotometry, coulometry, can be extracted in nonpolar solvents and can
IR, NMR, MS) for the quantification of flavonoids be analyzed by liquid chromatography (TLC,
from plants [69]. HPLC, etc.) [71].
78 4 HPLC: Herbal Drugs and Fingerprints

Fingerprints for Alkaloids due to their natural fluorescence. HPLC has also
been extensively used, mainly with reversed-phase
Alkaloids have been in use for a long time for stationary phases [69].
antiarrhythmic, hypotensive, platelet-aggregation-
inhibiting, histamine-antagonizing, and anti-
flagellated protozoa properties. Lysergol, an Fingerprints for Alkamides
alkaloid, is used in the treatment of schizophrenia
and motor disorders. It is also used in the synthe- Alkamides are a distinct class of natural products,
sis of lumilysergol and nicergoline. The drug containing an aliphatic acid (mostly unsaturated)
consisting of seeds is locally used for its aperient residue linked with various amine moieties.
action in MP of India, whereas the powder is used Approximately 200 alkamides have been isolated
as an antipyretic. The purity of the product is from nature. It is well known that alkamides from
defined by TLC (mobile phase; chloroform– Echinacea species have immunostimulating prop-
methanol–ammonia solution: 95:5:0.5, using sil- erties, and 15 new isobutyl- and 2-methyl-butyl-
ica gel 60 F254, at 254 and 365 nm) as well as amides have been identified in Echinacea
HPLC [(silica column 250 × 4.6 mm, I.D., 5 m, angustifolia and purpurea roots [72]. Extraction
mobile phase (n-heptane–THF–MeOH–diethyl with chloroform is widely used for the dried, pow-
amine: 55:35:6:0.02), wavelength 230 nm, flow dered plant material, and solvent is distilled out
rate 1.5 ml/min)]. Elymoclavin is a geometrical completely; residue redissolved in a small volume
isomer of lysergol, which is considered as an of alcohol and filtered using 0.45-m filter [72].
associated impurity. According to global market Among chromatographic techniques, HPLC is
specification, it should be less than 0.5% (wt/wt) rapid and does not lead to decomposition, material
by HPLC analysis, as a high-value product [69]. loss, or artifact formation. Due to these reasons,
the isolation, even at small quantities (milligrams
or less) for structure determination, comprehen-
Fingerprints for Coumarins sive biological testing, semisynthetic work, or
even for production of therapeutic agents, HPLC
Coumarins (a-benzopyrones) are natural deriva- is being used. The increasing requirements for
tives of benzopyrane with a lactonic structure. quality control, profiling and fingerprinting,
The name comes from “coumara” the name of dereplication, and metabolomics and the detec-
tonka seeds which contain large quantities of tion issue by HPLC are still challenging. New
coumarins. Coumarins can be found in numerous detectors still have to be developed to fulfill the
species such as algae, mushrooms, and lichens needs of sensitivity, linearity, speed, robustness,
and also in superior plants (Umbelliferae, and universal response.
Rutaceae, Labiatae, Orchidaceae, etc.). Bio-
chemically, coumarins are formed by photosyn-
thesis from phenylalanine and cinnamic acid. As References
crystals, coumarins have blue, green, or violet
fluorescence and show a good absorbance of the 1. David F, Vanhoenacker G, Tienpont B, Francois I,
UV light, being an effective UV screen. Many of Sandra P. Coupling columns and multidimensional
them are thermally labile. Coumarin derivatives configurations to increase peak capacity in liquid
chromatography. Lc Gc Eur. 2007;20:154–8.
are biologically active on the nervous system
2. Wilson ID, Brinkman UAT. Hyphenation and hyper-
level, and some of them have an anticoagulant nation – the practice and prospects of multiple
activity. Coumarin glycosides are soluble in water hyphenation. J Chromatogr A. 2003;1000:325–56.
and polar organic solvents, while terpenylcou- 3. Henry R, Santasania CT. Reporter. Separation of closely
related compounds. Sigma-Aldrich. 2010;28.5: 6–7.
marins and furanocoumarins are soluble in non-
4. The United States Pharmacopoeia. XXIIIth revision,
polar organic solvents. The coumarins have been Natural formulas 18, Rockville: United States
analyzed by TLC or HPLC and easily detected Pharmacopoeial Convention; 1995.
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7. Schobel U, Frenay M, van Elswijk DA, McAndrews 24. Liang Z, Jiang Z, Ho H, Zhao Z. Comparative analy-
JM, et al. High resolution screening of plant natural sis of Oldenlandia diffusa and its substitutes by high
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GC: Herbal Drugs and Fingerprints
5

Gas–liquid chromatography (GLC) was introduced alkaloids, steroids, glycosides and aglycons, amino
by James and Martin in 1952, to separate volatile acids, etc.; (b) separation of complex mixtures
substances by percolation of a gas stream over a into constituents; and (c) sample in parts of
stationary phase [1]. It has gained widespread milligram that is sufficient for analysis.
acceptance for different applications such as
process control in chemical plants, quality con-
trol in the food and pharma sectors, monitoring Instrumentation
sample composition in the oil industries, environ-
mental and biomedical sciences, and some time The GC separation is carried out in a tubular
the term GLC is also abbreviated as GC (though column made of glass, metal, or Teflon. Gas
in GC the stationary phase is solid, while, in chromatography, specifically gas–liquid chro-
GLC, the solid stationary phase is coated with an matography, involves the vaporization of sample
inert liquid of high boiling point). The basis of injected into head of the chromatographic
separation in GLC is partitioning of the sample in column, a specially manufactured device with
and out of the film of liquid spread over an inert rubber septum, that is termed as injector. The
solid. There exists a wide range of liquid phases sample is transported through the column by
usable up to 450 °C. In herbal drug industry, the fl ow of inert gaseous mobile phase, the
GLC is used mainly for (a) assay of raw materials carrier gas (generally, helium, argon, nitrogen).
and finished goods for process and quality The stationary phase of column is an inert solid
control, (b) quantification of ingredients in which is adsorbed into the surface by inert and
formulations, and (c) assay for impurities and thermally stable liquid. The choice of carrier
residual solvents and volatiles. It is a key tech- gas is often dependent upon the type of detector
nique to obtain pure compound for structure used. The carrier gas system also contains a
elucidation, pharmacological testing, and molecular sieve to remove water and other impu-
development of new therapeutics. It has a very rities. Microsyringe is generally used to inject
wide field of application, but its first and main sample through a rubber septum into a flash
area of use is in the separation and analysis of vaporizer port at the head of the column. The
multicomponent mixtures such as essential oils, carrier gas enters into the port, and sample
hydrocarbons and solvents, their identification, vaporizes to form a mixture (vaporized solvent
and structural elucidations. GLC (commonly and vaporized solutes) with carrier gas. Each
termed as GC), fingerprints, especially by hyphen- chemical species tends to migrate at its own
ated techniques, are strongly recommended for characteristic rate, hence separated from other
quality control of herbal drugs, to authenticate species by the time they emerge from the col-
the herbal part used for (a) volatile oils, plant acids, umn and reach at detector. The detector senses

D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs, 83
DOI 10.1007/978-81-322-0804-4_5, © Springer India 2012
84 5 GC: Herbal Drugs and Fingerprints

Fig. 5.1 GLC schematic diagram for FID system

the emergence of each sample component and 4. Oven to provide thermostatically controlled
provides an electrical signal to the recorder, atmosphere to the column.
proportional to the concentration of each com- 5. Detector detects the ingredient.
ponent in the emergent stream. The temperature 6. Recorder to record the response of detector as
of the sample port is usually about 50 °C higher chromatogram.
than the boiling point of the least volatile compo- The selection of column and detector for every
nent of the sample. analysis is matter of subject expertise, a summa-
GC has a very wide field of applications, but rized detail on these is as follows:
its first and main area of use is in the separation
and analysis of multicomponent mixtures
such as essential oils, hydrocarbons, and GLC Columns
solvents. Intrinsically, with the use of the flame
ionization detector (FID) (Fig. 5.1) and the There are two general types of column, packed
electron capture detector (ECD), have very high and capillary (open tubular), used with GLC.
sensitivity to, determines materials present at Packed columns contain a finely divided, inert,
very low concentrations in the matrix, due to the solid support material (commonly based on dia-
reason it is the second most important technology tomaceous earth) coated with liquid stationary
for pollution studies, forensic work, and general phase. Most packed columns are 1.5–10 m in
trace analysis. Because of its simplicity, sensi- length and have an internal diameter of 2–4 mm.
tivity, and effectiveness in separating components Capillary columns have an internal diameter of a
of mixtures, it is one of the most important few tenths of a millimeter. They can be one of
tools in chemistry. It is widely used for quantita- two types: wall-coated open tubular (WCOT) and
tive and qualitative analysis of mixtures, for the support-coated open tubular (SCOT). Wall-coated
purification of compounds, and for the determi- columns consist of a capillary tube whose walls
nation of thermochemical constants as heats of are coated with liquid stationary phase. In
solution and vaporization, vapor pressure, and support-coated columns, the inner wall of the
activity coefficient. capillary is lined with a thin layer of support
In summarized way, we can say that GLC is a material such as diatomaceous earth, into which
six components system, as of: the stationary phase has been adsorbed. SCOT
1. Pneumatic system to gas supply and flow columns are generally less efficient than WCOT
control and measurement. columns, but both types of capillary columns are
2. Injector, a specially designed port for sample more efficient than packed columns. A new type
injection. of WCOT column with fused silica known as
3. Column, the device for separation of ingredi- fused silica open tubular (FSOT) column is a new
ents present in matrix. innovation, which has much thinner walls than
Instrumentation 85

the glass capillary columns and is given strength samples. The FID works by directing the gas
by the polyimide coating. These columns are phase output from the column into a hydrogen
flexible and can be round into coils. They have flame. A voltage of 100–200 V is applied between
the advantages of physical strength, flexibility, the flame and an electrode located away from the
and low reactivity. flame. The increased current due to electrons
emitted by burning carbon particles is then mea-
sured. Although the signal current is very small
Detectors in GLC (the ionization efficiency is only 0.0015%), the
noise level is also very small (<10–13 amp) and
There are many detectors which can be used in with a well-optimized system, sensitivities of
gas chromatography. Different detectors have 5 × 10−12 g/ml for n-heptane at a signal/noise ratio
different types of selectivity. A nonselective of 2 can be easily realized. Except for a very few
detector responds to all compounds except the organic compounds (e.g., carbon monoxide), the
carrier gas, while a selective detector responds to FID detects all carbon-containing compounds.
a range of compounds with a common physical The detector also has an extremely wide linear
or chemical property, and a specific detector dynamic range that extends over, at least five
responds to a single chemical compound. The orders of magnitude with a response index
most popular detector in gas chromatography is between 0.98 and 1.02. In case of FID, the
the FID. Its high sensitivity, fast response, wide response is proportional to the carbon content of
dynamic range, and simplicity of construction the compound, and this is useful for general anal-
make it the most widely used detector. Several ysis of organic compounds.
other detectors that are available which offer high
selectivity are ECD for halogen-containing com- ECD
pounds, TID (thermo-ionic detector) for nitrogen The electron capture detector (ECD) is extremely
and phosphorous-containing compounds, and the sensitive for those compounds having strong
SCD (sulfur chemiluminescence detector) for electron capturing properties such as halogenated
sulfur-containing compounds. Another detector molecules. Although ECD is simple in design
with a large popularity in gas chromatography is and operation, it is actually a complex chemical
the mass spectrometer. GC–MS is very adequate reactor, and many processes are involved in the
for the identification of unknown compounds but production of a signal. ECD is based on forma-
has a more limited dynamic range than the FID. tion of plasma of thermalized electrons. The elec-
trons are collected by applying a small voltage to
FID produce standing current. The compound enter-
FID (flame ionization detector), a general detec- ing into the cell captures the thermalized elec-
tor, is a useful for the analysis of organic com- trons, resulting decrease in standing current,
pounds, specially solvents, essential oils, etc. It which is recorded by recorder, proportional to the
has high sensitivity, a large linear response range, content of eluting moiety.
and low noise. In FID, the effluent from the col-
umn is mixed with hydrogen and air and ignited FPD
at detector. The resulted flame produces ions and The flame photometric detector (FPD) is a most
electrons which conduct electricity through the reliable detector for the analysis of phosphorus
flame. A large electrical potential is applied at the and sulfur compounds. It is based on specific
burner tip, and a collector electrode is located chemiluminescence produced by burning in
above the flame. The current resulting from the hydrocarbon-rich flame. The sulfur and phospho-
pyrolysis of organic compounds is measured, rus emission bands are derived from excited S2
which is recorded by recorder. FID, a nonselec- and HPO species, respectively, and are formed by
tive detector, has a potential possibility of inter- reactions with hydrogen atoms in the upper part
fere by many nontarget compounds present in of the flame. Carbon gives the flame emission
86 5 GC: Herbal Drugs and Fingerprints

bands, which are formed in the lower region of with gas chromatography are electron impact
the flame. In original FPD design, the column (EI) and electron capture ionization (ECI). EI is
effluent was mixed with air and combusted in primarily configured to select positive ions,
burner of similar design to an FID. The flame is whereas ECI is usually configured for negative
burned in the stream of hydrogen gas, and only ions (ECNI). EI is particularly useful for routine
the upper part of the flame is monitored by hori- analysis and provides reproducible mass spectra
zontal optical system, which focuses the emis- with structural information which allows library
sion into a photomultiplier. Filters are used to searching [2]. The GC–MS, in herbal drug stud-
improve selectivity with transmission maxima at ies with capillary column, have very good separa-
394 nm for sulfur and 526 nm for phosphorus tion, necessary to generate fingerprint of high
detection. The high selectivity and reproducibil- quality, and the spectral database is useful for the
ity of FPD have made it a preferred detector for further research to elucidating the relationship
the analysis of P- or S-containing compounds, between chemical constituents and its pharma-
provided the nature of the sulfur response and the cology. Thus, GC–MS is the most preferable
of quenching carbon radicals (the direct interfer- tool for the analysis of the volatile chemical
ence from hydrocarbons due to CH and C2 emis- compounds in herbal drugs especially in
sions is not a major problem in the P mode due to aromatherapy.
high selectivity, although hydrocarbon fragment
can directly quench HPO and S2 emission) are
kept in the mind, it gives excellent results. Development of GC Fingerprints
and Data Interpretation
AFID
Alkali flame ionization detector (AFID) is widely GC analysis is highly informative and unique, as
used for the detection of organophosphorus com- is used where TLC, HPTLC, and HPLC techniques
pounds. The basic feature of all AFIDs is the do not work satisfactorily. The entire operation
introduction of an alkali salt into a relatively cool may be discussed in a generalized way as:
hydrogen flame or plasma. The long-term stabil-
ity of AFIDs is not as of FID, and certain precau-
tions are taken for best results. Gas flow to the Sample Preparation
detector is carefully controlled with individual
pressure regulation for each instrument. A flame The collection and identification of plant part is
stabilization period of about an hour is sufficient carried out as per established procedure and a
as the alkali source is in good health during this voucher samples deposited to herbarium with reg-
period; overnight operation is not recommended istration number. The collected plant part is dried
as it shortens the life of alkali source and increases and reduced to suitable size. A few steps common
detection contamination. in the sample preparation for GC analysis are to
select the desired plant part and extract it with
GC–MS suitable solvents, and, partitioned between solvents,
Mass spectrometry is the most sensitive and desired fraction is charcoalized, if pigmented and
selective method for molecular analysis and can filtered, using filter aid, passed through Na2SO4
yield information on the molecular weight as column to dehydrate it, and finally make up in
well as the structure of the molecule. Combining suitable solvent (either n-hexane, n-heptane, or
chromatography with mass spectrometry (GC– in alcohol of chromatography/spectral grade),
MS) provides the advantage of both gas chro- based on the nature of solubility of the desired
matography for separation method and mass ingredient(s). During extraction and purification,
spectrometry as an identification method. In mass TLC, HPTLC, and HPLC analysis may be guiding
spectrometry, there is a range of methods to ion- for process chemistry and suitable solvent selec-
ize compounds and then separate the ions. tion, for a new moiety. Extraction steps are recom-
Common methods of ionization used in conjunction mended to carried out in dim light to avoid the
Development of GC Fingerprints and Data Interpretation 87

Fig. 5.2 GLC chromatogram of walnut oil [5]

photochemical reactions. In case of volatile and is saponified. On dissolving in water and further
thermosensitive compounds, solvent extraction is extraction with ether both saponified and non-
preferred than to steam distillation, to avoid the saponified matter separates. The non-saponified
evaporation and decomposition, respectively [3]. matter may be analyzed by GC either as such or
The moisture content on the sample is also consid- after methylation.
ered, to calculate the assay on dry basis. The sample
preparation procedure for different classes of
chemical compounds may be summarized as: Separation and Detection

Alkaloids In injection port, the sample vaporized and


The air-dried plant part is ground altogether as a transported through the column by the flow of
fine powder, extracted with alcohol, defatted carrier gas. The column temperature, stationary
using n-hexane or petroleum ether (60–80°C), phase, and flow rate of carrier gas play a vital
alcohol distilled out under vacuum, the soft mass role in the separation of different ingredients.
dissolved in acidic water (10% HCl), filtered, and Each chemical species tends to migrate at its
the filtrate basified with NH4OH. The basified own characteristic rate, hence separated from
fraction (i.e., the alkaloidal fraction) is extracted other species by the time they emerge from the
with chlorinated solvent, for example, CHCl3 and column and reach at detector. The separation is
MeCl2, which may be directly injected into GC also dependent upon the boiling point of the
after passing through Na2SO4 and subsequent sample, which is controlled to within tenths of
filter through 0.45 mfilter. a degree for accuracy of results. There is a rule
of thumb that temperature slightly above the
Flavonoids average boiling point of the sample results in an
Flavonoids are isolated by dissolving the alcoholic elution time of 2–30 min. Minimal tempera-
soft mass of the plant with hot water, followed tures give good resolution but increase elution
by extraction with chloroform or ethyl acetate, times. For a sample of a wide boiling range,
and assayed mostly after derivatization. temperature programming is useful, in term of
temperature programmed retention indices
Lipids (IPTGC) [4].
Air-dried and powder plant parts are extracted
with petroleum ether (40–60°C) for lipid fraction.
The marc is macerated with ethanol (80%) at Analytical Results
room temperature till exhaustion. The resulting
alcoholic extract is concentrated to obtain a crude The chromatogram for standard and sample are
residue. The obtained lipid fraction is treated with compared against RT of ingredients (Fig. 5.2)
hot acetone. Acetone is distilled out, and residue and calculated for assay.
88 5 GC: Herbal Drugs and Fingerprints

Utility of GC Fingerprints in Herbal a particular herb for its medicinal properties, and
Drugs these elements also provide an important natural
safeguard for identity and authenticity of the
The GC fingerprints for herbal drugs are unique plant used for medicine purposes. A brief discus-
as used in determination of: sion on the analysis of these using GC is as:

Alkaloids
Contamination/Adulteration Capillary gas chromatography often coupled with
a mass spectrometer as a detector (GC–MS) is a
The raw material to be used for herbal drugs and well-established technique for analyzing com-
finished goods should be free from pesticides, plex mixtures of alkaloids. There are a large
herbicides, and residual solvents, as the presence number of publications dealing with GC or
of these has high health risk. There are extensive GC–MS of underivatized alkaloids. Mirko et al.
legislations controlling the quality of all human [6]. have reported that GC–MS of alkaloids pro-
foods, drinks, and drugs derived from plant-based vides much structural information in a short time.
products and offensives carry very serious penal- The characteristic fragmentation pattern of the
ties. In addition, the condition of the herbal-based different structural types and the high sensitivity
food is also of great concern to the food scientist of GC allow a fast identification even of minor
who looks for those trace materials that have been compounds of a mixture without laborious isola-
established to indicate the onset of microbial tion procedures. However, even under optimal
action with time, storage conditions, rancidity, or conditions, some decomposition reactions may
decomposition and are analyzed by GC technique. occur. A properly deactivated column, as well as
mild conditions (use of on-column injection), is
recommended for optimal results. Twenty-nine
Traceability alkaloids from the extracts of D. stramonium
(Bulgarian origin) have been detected by GC-MS,
The features of GC fingerprint are effective in the which have all the characteristics for the tropane
identification of spurious herbal products. The nucleus ions at m/z 124, 113, 96, 95, 94, 83, 82,
source of many plants (herbs and spices) can and 81 [7].
often be identified from the peak pattern of the
chromatograms obtained directly from GC analy- Flavonoids and Flavones
sis. The unique qualitative and quantitative pat- Due to biological and physiological importance,
tern from a GC analysis helps in identification of flavonoids have considerable attention to be
source of many alcoholic beverages and raw analyzed by gas chromatography coupled to mass
material of specific geographical origin. The peak spectrometry (GC–MS) technique for the analy-
pattern in chromatographic fingerprints, for eval- sis of flavonoid and aglycones. Because of the
uating the quality of herbal drugs, is an intuitive increasing interest in structure elucidation of
evaluation method, to compare the percentage, flavonoids, use of hyphenated technique of gas
concentration, and distribution of components. chromatography with mass spectrometer is espe-
cially in practice. Monitoring the presence
of flavonoids with GC–MS provides a fast and
Qualification and Quantification efficient separation and requires minimal sample
of Therapeutics preparation to monitor the herbal product quality.

Plants contain minerals, vitamins, volatile oils, Tannins and Other Components
glycosides, alkaloids, bioflavanoids, tannins, and Vegetable tannins influence the characteristics
other substances that are important in supporting of many plant products, their taste, palatability,
Utility of GC Fingerprints in Herbal Drugs 89

Fig. 5.3 GLC fingerprints of Cinnamomum verum bark for essential oils [9]

nutritional value, pharmacological and toxic effects, special attention has been given to chromatographic
and their microbial decomposition because of techniques, mainly gas chromatography, due to
their ability to form complex with proteins, its advantages such as good separation, efficiency,
carbohydrates, nucleic acids, alkaloids, and and speed properties [8].
minerals, and gas chromatography is unfeasible
due to their large molecular weight, polarity, and Terpenes
thermal liability. However, molecules with these Various herbs and spices (including orange,
chemical features are in principle suitable to lemon, lime, grapefruit rosemary, sage, spear-
be degraded by pyrolysis (PY), an effective mint, nutmeg, black peppercorns, cloves, cara-
technique for the study of complex, nonvolatile way seeds, and vanilla beans) are analyzed
samples, which can be integrated with a gas chro- through GC and GC–MS. [8]
matograph mass spectrometer (PY–GC–MS) to
provide a rapid analysis of the degradation products Essential Oils
and hence the characterization of the original The analysis of the volatile oils has a number of
sample. Headspace sampling coupled with gas advantages as volatile oil gives a reasonable
chromatography (HSGC) is a widely used fingerprint which can be used to identify the
technique for the analysis of beer throughout plant. The composition and relative concentra-
the world. HS–GC is typically used for quality tion of the organic compounds in the volatile oil
control to identify problems or changes occurring are characteristic of the particular plant, and the
in the fermentation process that affect the taste or presence of impurities in the volatile oil can be
quality of the final product. It has been observed readily detected (Fig. 5.3) [9].
that a large number of companies, and a few The extraction of the volatile oil is relatively
government agencies, stimulating their standard- straightforward and can be standardized, and
ization and the development of reliable quality components can be readily identified using
control analytical methodologies to support their GC–MS analysis (Figs. 5.4 and 5.5) [9].
safety for such products. Furthermore, for herbal The relative quantities of components used in
preparations, some tests include quantification of the herbal drugs. Changes in composition of the
the active compounds and uniformity of dosage, volatile oil may also be used as indicators of oxi-
disintegration, and dissolution among others. dation, enzymatic changes, or microbial fermen-
Concerning the quantification assay and consid- tation, so GC and GC–MS are unanimously
ering the high complexity of this type of matrix, accepted methods for the analysis of volatile
90 5 GC: Herbal Drugs and Fingerprints

Fig. 5.4 GLS fingerprint of essential oils from Murraya koenigii leaflet [9]

CH3 chemist are a direct result of the separating


capabilities of GC techniques [11].
O
N CH3
H CH2CH2CH C Examples
Mahanimbine CH3
GC–MS Analysis of Garlic
Analysis of the two garlic (Allium sativum) sam-
Fig. 5.5 Structure of mahanimbine [9]
ples was carried out using a GC-17A gas
chromatographic instrument hyphenated with a
constituents of herbal drugs, due to their sensitiv- QP-5000 mass spectrometer from Shimadzu. The
ity, stability, and high efficiency. Especially, the chromatographic column was a DB-5 quartz cap-
hyphenation with MS provides reliable informa- illary column (30 m, 0.25 mm I.D.). The injec-
tion for the qualitative analysis of the complex tion temperature was 50 °C initially (maintained
constituents [10]. GC–MS was the first success- for 3 min), which was increased to 230 °C (main-
ful online combination of chromatography with tained at 10 min) at rate of 10 °C/min. The inlet
mass spectrometry and is widely used in the anal- temperature was constant at 250 °C. In the exper-
ysis of essential oil in herbal medicines [11, 12]. iment, the sampling volume was 0.4 ml. Electron
With the GC–MS, scientists can produce not only impacts (EI) were recorded in the MS at 70 eV
a chromatographic fingerprint of the essential oil ionization energy in the full-scan mode. The
of the herbal drugs but also the information analytical range of m/z was 30–350 amu, with
related to its qualitative and quantitative compo- 0.2 s/scan, and ionization source temperature was
sition. The chemical structure of the individual set to 150°C [10].
components of many of the oils, elucidated by the
GC–MS tandem systems, provided the knowl- GC–MS Analysis of Thiophene Derivatives
edge necessary to synthesize a number of com- Natural thiophenes are biologically active com-
mercially important synthetic flavors. For pounds, and the activity is enhanced by irradiation
example, the synthetic flavors that closely imitate with long wavelength ultraviolet light, exhibit
those of the peach, melon, and other fruits that substantial antiviral, antibacterial, antifungal,
are presently available to the contemporary food nematocidal, and insecticidal properties under
Utility of GC Fingerprints in Herbal Drugs 91

irradiation, whereas their toxicity is much reduced cell membrane. Plant sterols fall into one of three
in darkness. In presence of UV light, these sulfur- categories, namely, 4-desmethylsterol (cholestane
containing polyacetylenes react as type II photo- series), 4-monomethylsterols (4-methylcholestane
sensitizers producing singlet oxygen that causes series), and 4,4-dimethylsterols (lanostane series,
a wide spectrum of microbial and animal toxicity. i.e., triterpene alcohols). In plants, more than 40
The separation of thiophenes by capillary GLC sterols have been identified of which campes-
and the group-specific MS fragmentation with terol, ß-sitosterol, and stigmasterol are the most
the typical sulfur isotope peaks allowed the abundant. These 3 sterols are structurally similar
unequivocal assignment of individual thiophenes to cholesterol (they are all 4-desmethyl sterols)
in complex mixtures, even when occurring in and differ only in a side-chain substitution of a
traces and in the presence of different geometri- methyl group (campesterol), an ethyl group
cal isomers. GLC and GC–MS are suitable (sitosterol), or an ethylidene group (5-avenasterol)
methods for the analysis of complex mixtures of and a diene at C-22 (stigmasterol). These are
thiophenes which contain several stereoisomers completely saturated forms of sterols and lack
or differ only in the number of unsaturated bonds the carbon–carbon double bonds found in choles-
and methyl groups, for example, Tagetes patula. terol and sterols. Plant sterols (and stanols) exist
IPTGC were calculated according to Van den in 4 different forms: as free alcohol, fatty acid
Dool and Kratz (1963) [4]. The purified extracts esters, and conjugates with sugar moieties or
were diluted in hexane. 1 ml hexanic solution of phenolic acids. The sterol pattern of vegetable oil
23 mg/ml n-hexadecane was added as an internal is used to characterize and detect adulteration
standard at 4 °C to each gram of plant material of (sitosterol). However, nutrition research has
the diluted extract source. Thiophenes were focused mainly on five types – campesterol,
quantified by comparing the peak areas of the ß-sitosterol, stigmasterol, campestanol, and
n-hexadecane and thiophenes (response factor ß-sitostanol [13].
of 0.2440). The analysis was carried out with The cholesterol esters, dietary plant sterol,
a Carlo Erba Mega 5360 GC equipped with and stanol esters are hydrolyzed to free sterols
fused silica capillary column (15 m × 0.25 mm and stanols, respectively, and solubilized in mixed
I.D.) and coated with PERMABOND OV-1 DF. micelles. In general, only 0.1–5% of the ingested
GLC conditions: injector = 250°C, temp. program plant sterols (depending on the type) are absorbed;
T0 = 120°C, t0 = 2 min, rate of temperature [14] consequently, blood levels of plant sterols in
increase = 10°C/min, T1 = 300°C, t1 = 5, pressure = humans are only 0.1–0.14% of cholesterol levels
1 atm, split ratio = 1:20, injection volume = 2 ml, [15]. The poor efficiency of absorption of plant
and detector = FID. Data evaluation was carried sterols and stanols is attributed to the ABCG5
out using a Hewlett Packard 3393 Integrator. and ABCG8 transporter in enterocytes which
GC–MS was carried out as above, and the only selectively pump plant sterols and stanols from
differences were a 30-m capillary column, the the enterocytes into the intestinal lumen, poor
heating rate was reduced to 6°C/min, the detec- solubility of the sterols in mixed micelles, and the
tor was a Finnigan MAT 4500 quadrupole mass poor efficiency of intestinal cells to re-esterify
spectrometer, and mass spectra were recorded the absorbed sterols [15]. The human body does
at 45 eV [3]. not synthesize plant sterols endogenously. Plant
sterols block both biliary and dietary cholesterol
absorption in the intestine by different mecha-
Nutraceutical Discovery nisms, by decreasing the absorption of choles-
terol. The net result reduced intestinal absorption
Plant sterols have chemical and biological func- and less cholesterol reaching the liver via chylo-
tions as like cholesterol in human, a critical role micron remnants. In response to the decreased
in stabilizing the phospholipid bilayer in plant supply of exogenous cholesterol, receptor-mediated
cell membrane, just as cholesterol does in animal uptake of lipoprotein cholesterol is increased,
92 5 GC: Herbal Drugs and Fingerprints

resulting in reduction of serum LDL cholesterol increased 38% with corn oil devoid of plant
levels [13]. sterols. The effect was attributed to natural corn
Plant sterols and stanols occur naturally in oil plant sterols because adding back 150 or
almost all vegetable foods, but the content and 300 mg plant sterol to a test meal reduced choles-
composition vary widely. Nuts and oilseeds terol absorption by ~12 and 27.9%, respectively
have higher amounts of plant sterols than other [18]. The sterols in rice bran oil are present as a
foods. Corn and sorghum have higher plant sterol component of gamma-oryzanol (group of esters
content than rice and wheat. Whole wheat and of phenolic acids with sterols, ferulate esters
brown rice are better sources of plant sterols than of triterpene alcohols, and ferulate esters of
refined flour and polished rice, respectively. ß-sitosterol and campesterol), free sterols, and
Legumes contain moderate levels. The plant ste- 4,4¢-dimethylsterols (cycloartenol and 24-methylene
rol content of fresh vegetables and fruits is cycloartanol). Animal and human studies have
2–20 mg/100 g and 10–32 mg/100 g, respec- shown that rice bran oil reduces serum total and
tively. Mustard, fenugreek, and cloves contain LDL cholesterol and triglycerides and increases
higher amounts of plant sterols than other serum HDL cholesterol concentrations [13, 19].
spices. Plant sterols get concentrated in the In human studies, 300 mg of gamma-oryzanol/
unsaponifiable fraction of vegetable oils. In day reduced LDL cholesterol and consumption
most vegetable oils, the total sterol content is of margarine containing 2.1g/day of plant sterols
200–370 mg/100 g (olive, groundnut, safflower, from rice bran oil for 3 weeks reduced serum
sunflower, soya bean, cottonseed). Rice bran, LDL cholesterol by 9% [20]. These findings
corn, mustard/rapeseed, and sesame oils contain suggest that sterols in foods and vegetable oils
high levels (740–1,156 mg/100 g), whereas are important dietary components for lowering
palm oil and coconut oil contain low levels serum LDL cholesterol.
(88–110 mg/100 g) [13 ]. The major sterols Phytostanols are a fully saturated subgroup
are ß-sitosterol, campesterol, and stigmasterol. of phytosterols (contain no double bonds).
Brassicasterol, characteristic of the Cruciferae Phytostanols occur in trace levels in many plant
family, is present in rapeseed and mustard seed species, and they occur in high levels in tissues of
oils but is in trace amounts in other oils. Plant only in a few cereal species. Phytosterols can
stanols are present in smaller quantities in many be converted to phytostanols by chemical hydro-
of the above foods. Sterols are heat-stable mole- genation. More than 200 different types of
cules. Cooking does not affect the plant sterol phytosterols have been reported in plant species.
content of cereals, legumes, and vegetables, but In addition to the free form, phytosterols occur as
industrial processing of vegetable oils results in four types of conjugates, in which the 3b-OH
20–70% loss [13, 16]. The early human diet was group is esterified to a fatty acid or a hydroxycin-
probably rich in plant sterols, providing up to 1 g namic acid or glycosylated with a hexose (usually
per day. However, most current western diets pro- glucose) or a 6-fatty-acyl hexose. The most popular
vide 200–400 mg/day of plant sterols [15]. methods for phytosterol analysis involve hydroly-
Studies in humans have documented an inverse sis of the esters (and sometimes the glycosides)
relationship between intake of plant sterols and and capillary GLC of the total phytosterols,
both serum total and LDL cholesterol (corrected either in the free form or as trimethylsilyl (TMS)
for age, body mass index and energy intake, or acetylated derivatives. Several alternative
dietary fiber, and saturated fat) [17]. Consumption methods have been reported for analysis of free
of wheat germ (328 mg plant sterols) reduced phytosterols and intact phytosteryl conjugates.
cholesterol absorption by 42.8% compared with Recently, phytosterols and phytostanols have
plant sterol-free wheat germ in human subjects. received much attention for their cholesterol-
A study comparing purified corn oil triglycerides lowering properties. In the last several years, two
with or without corn oil plant sterols reported that spreads, one containing phytostanyl fatty acid
in a single test meal, cholesterol absorption esters and the other phytosteryl fatty acid esters,
Utility of GC Fingerprints in Herbal Drugs 93

−2.76

ARCHID: −21.82
STEARI. −17.89

LINCLEI.−19.72
CLEICA. −18.50

CIS.11,1. −23.72
MYRIST− −8.97

PALMITI. −15.17
Response [mV]

TRICOS. −27.55
BEHENI −25.25
ERUCICi −26.59
150

DHA. −30.02
EPA −28.74
100

50

0
Retention time (min.)

Fig. 5.6 GLC fingerprints of P. armeniaca seeds [23]

have been commercialized and were shown to immune system; oleic acid is able to enhance the
significantly lower serum cholesterol at dosages insulin in INS-1, also can be used for the preser-
of 1–3 g/day. The popularity of these products vation of confectionary items; stearic acid is
has caused the medical and biochemical commu- widely used in cosmetic formulations in antiper-
nity to focus much attention on phytosterols, and spirants, emollient creams and lotions, shampoos,
consequently, research activity on phytosterols and shaving creams; and linolenic and linoleic
has increased dramatically [21, 22]. The afore- acid, an essential fatty acids, required in normal
said data have been generated with the GC and human growth, development, and other biologi-
GC–MS techniques. cal process [23].
Prunus armeniaca seeds were collected from
Pithoragarh, Uttarakhand, India, during crop
New Cosmeceuticals ripening season and kept at −20 °C until ana-
lyzed. Oil was extracted with n-hexane in the
Apricot (Prunus armeniaca) cultivated at higher Soxhlet at 70 °C. The n-hexane fraction was
altitudes (1,000–2,700 m) and is native to China distilled out at 70 °C, and temperature was raised
and Indian Himalaya and in Japan but also found to 80°C, just after the distillation stopped, to
in Turkey, Southern Europe, South Africa, and remove the traces of n-hexane. Pale-yellow oil
Australia. Its pits yield 22–38% kernels, nor- was stored at 4°C until testing, and fatty acids
mally, which are considered as waste by farmers were analyzed by using GC–FID (Clarus 500, a
and have no commercial value, but these seed PE Auto-System, GC with inbuilt auto-sampler),
kernels are rich source of oil with great medicinal after saponification using 2 N-KOH in methanol,
value due to presence of different unsaturated subsequent esterification using 5% HCl, and
fatty acids. Fourteen different fatty acids have extracting the methyl ester phase into petroleum
been identified by GC–FID (Fig. 5.6), mainly, ether for GC analysis. The fatty acids methyl
myristic acid (25.63%), oleic acid (24.75%), esters (FAMEs) were identified by comparing
stearic acid 1(2.91%), linolenic acid (16.41%), their relative retention times with reference
and linoleic acid (9.50%) and in traces of archidic standards (FAMEs) [23].
acid, archidonoic acid, behenic acid (often used
as hair conditioners and moisturizers their
smoothing properties), erucic acid (has high tol- In Aromatherapy
erance to temperature that makes it suitable for
transmission oil), and tricosanoic acid. All these Aromatherapy uses natural oils (especially
fatty acids are source of fat in diet which increases essential oils) refined to 100% from flowers,
the immunity and also known by their utility in leaves, seeds, trunks, pericarp, resin, etc.
cosmetic and pharma industry. Myristic acid, an Essential oils that are applied to the skin can be
important saturated fatty acid, stabilizes many absorbed into the blood stream. The constitu-
different proteins, including proteins used in the ents of essential oils aid in health, beauty, and
94 5 GC: Herbal Drugs and Fingerprints

hygiene conditions. Since essential oils are so increase in poisonings associated with the use of
powerful and concentrated, they should never these aphrodisiacs. References to such sub-
be applied to the skin in their undiluted form. stances have crept into holy texts from the Kama
Before application to the skin, essential oils Sutra and the Bible to the Koran and the litera-
are diluted into a carrier such as a cold-pressed ture from Shakespeare and Ovid to Gilbert and
vegetable oil. The common carrier oils include Sullivan plays in the twentieth century [25]. The
sweet almond oil, apricot kernel oil, and grape common aspects of different cultures and ethni-
seed oil. In addition to therapeutic benefit at cal groups have often emerged for the use of
the emotional and physical level, essential oils plants as aphrodisiacs. The seventh part of the
are helpful in other applications as in house- Kama Sutra is dedicated to the occult practices
hold and laundry cleaners. Some oils act as a (Aupanishadika). A mixture of powdered thorn-
natural insect repellent and pesticide, for exam- apple (Datura stramonium) seeds, black pepper
ple, use of citronella candles during the sum- (Piper nigrum), long pepper (Piper longum) and
mer to keep away mosquitoes. Essential oils honey, applied on the penis before coitus, is
are blended together to create appealing and reputed to make a woman subject to the man’s
complex aromas, for a specific therapeutic will. The thorn-apple seeds have atropine and
application, and are referred as an essential oil scopolamine, responsible for inducing an initial
synergy. A synergistic essential oil blend is excitement followed by sedation and hallucina-
considered to be greater in total action than tions. These two constituents are adsorbed
individual oil working independently. Lavender through the mucous membranes of both the
oil promotes the recovery of skin. Tea tree is penis and the vagina and have a central nervous
effective in the treatment of atopic dermatitis system action producing behavioral effects in
and suppresses the growth of Staphylococcus both partners [26]. The peppers have a counter-
aureus on the skin surface and decreases itch. irritant or rubefacient action, increasing the
It enhances the immune system and helps in blood flow around the area of application. In the
the correct functioning of the defensive sys- man, this local inflammation of the clitoris
tem. Aromatic bathing reduces the stress. increases the sexual desire. Ginger (Zingiber
Fragrance changes the body temperature, blood officinale) is described in the “Kama Sutra” and
pressure, and glucose level, feeling positive employed by man to increase potency. The
and reduces fatigues. The essential oil from honey is used as skin friendly base of the formu-
Citrus is photosensitive, and degradation prod- lation as well as useful lubricant.
uct is toxic to skin, so it is necessary to avoid Another example may be cited from the tradi-
direct sun light for 4–5 h after use, hence is tional formulation by the people of, Uttarakhand,
recommended at nighttime only [24]. Similarly, India, from hill lemon (Citrus pseudolimon)
the list may be matters of thousand examples, fruits, (in Hindi, known as galgal nimbu and
and there is the need to establish evidence- chukh in local dialect) which is squeezed and
based medical aromatherapy system through juice is concentrated over the wood fire in iron
clinical studies and scientific research of essen- pot till evaporation stops and is considered for
tial oils, where GC, GC–MS fingerprints, has a long stability (more than 10 years); with many
major role, to predict for stability, composi- medical utilities, when analyzed by GC for vit. B
tion, and ingredients. complex, it confirms the presence of ingredients
(Figs. 5.7 and 5.8) [27].
GC and GC hyphenized with MS are important
To Decipher Traditional Formulations tools in the elucidation of the volatiles compo-
nents in complex matrix, as of herbal products.
The resurgence of old herbal remedies that Although only volatiles components can be analyzed,
arouse sexual activity and the use of new exotic this methodology is important to the creation
synthetic preparations have coincided with an of fingerprint and monitoring of the herbals for
Utility of GC Fingerprints in Herbal Drugs 95

Fig. 5.7 GLC fingerprints of C. pseudolimon [27]

Fig. 5.8 GLC fingerprints for various ingredients of vit. B complex [27]

seasonal and geographic chemical variations non-identified components, along with verifying
and stability and to compare the composition changes in compositions and degradations, for
of selected extracts based on identified and quality and regulatory purposes.
96 5 GC: Herbal Drugs and Fingerprints

esterified sterols in vegetable oils. J Am Oil Chem Soc


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Comparison of intestinal absorption of cholesterol Bilia R, Flamini G, Taglioli V, Morelli I, Vincieri FF.
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Flavonoid composition of Citrus juices. Molecules. Aromather. 2007;6:28–34 (in Japanese).
2007;12:1641–73. Troszynsk A, Ciska E. Phenolic compounds of seed
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UV–Vis. Spectroscopy: Herbal Drugs
and Fingerprints 6

The absorption, transmission, and emission of depends on its electron energy levels, UV–Vis.
ultraviolet–visible (UV-Vis.) light wavelengths by absorption spectra are useful for identifying
herbal and herbal products are primary indication unknown substances. Emission of electromag-
for purity and the amount present. Generally, it is netic radiation (e.g., ultraviolet and visible light)
used to study molecules and inorganic ions in occurs when electrons move from higher
solution. The ultraviolet and visible light are both energy levels to lower energy levels. Scattering
distinct regions of the electromagnetic spectrum, of electromagnetic radiation occurs when light
but there are similarities between the two regions is deflected or scattered in other directions.
for analytical procedures, due to the reason the Rayleigh scattering describes light that is redi-
same research techniques and tools are used rected at the same wavelength. Some interactions,
for both regions together. Ultraviolet and visible however, may occur that result in a partial transfer
light comprise only a small portion of the wide- of energy. As a result, the scattered electro-
ranging electromagnetic radiation spectrum, which magnetic radiation is of a different wavelength.
is lower in frequency and therefore lower in Brillouin scattering and Raman scattering are
energy than cosmic, gamma, and X-rays. Both examples of this type of scattering.
UV and Vis. light are of a higher frequency and The way electromagnetic radiation affects
therefore of higher energy than infrared, micro- atoms and molecules depends on the energy of
wave, and radio waves, so on exposure, the the light. The energy is dependent upon the
absorption, emission, or scattering of electro- frequency of the light. Both ultraviolet and visible
magnetic radiation by atoms or molecules are light promote electrons into higher energy
expressed. Measurements of these properties orbitals. Infrared light excites atomic and molec-
expressed by the atoms or molecules present ular vibrations. Microwaves excite atomic and
in a sample and decipher their concentration or molecular rotation. Special instrumentation is
abundance, with high degree of accuracy. used in UV–Vis spectroscopy. Hydrogen or
The ultraviolet band of the electromagnetic deuterium lights provide the source of light
spectrum (200–400 nm) is grouped into three for ultraviolet measurements. Tungsten lamps
regions termed UV-A, UV-B, and UV-C. All provide the light for visible measurements. These
scientists do not agree on the exact division of light sources generate light at specific wavelengths.
these wavelengths. UV-A (320–400 nm), UV-B Deuterium lamps generate light in the UV range
(290–320 nm), and UV-C (200–290 nm) are based (190–380 nm). Tungsten–halogen lamps generate
on wavelength. Absorption of ultraviolet or visible light in the visible spectrum (380–800 nm).
light electromagnetic radiation causes electron to Xenon lamps which can produce light in the UV
move from lower energy levels to a higher energy and visible portions of the spectrum are used to
levels. As the spectrum of an atom or molecule measure both UV and visible spectra.

D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs, 101
DOI 10.1007/978-81-322-0804-4_6, © Springer India 2012
102 6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints

UV–Vis. methods were pioneered by American


chemist Arnold Beckman, 1940. He invented Instrumentation
a spectrophotometer that had the ability to
measure the transmission of ultraviolet and visi- A specially designed instrument is used in
ble light through a target. In UV spectroscopy, a UV–Vis spectroscopy, with hydrogen or deute-
beam of light is split into a sample and reference rium lamps as source of ultraviolet light, while
beams. As its name implies, the sample beam is the tungsten lamp is used for visible measure-
allowed to pass through the target sample. ments. These light sources generate light at
Alternately, the reference beam passes through specific wavelengths. Deuterium lamps generate
the control solvent or a portion of the solvent light in the UV range (190–380 nm). Tungsten–
that does not contain the actual target. Once the halogen lamps generate light in the visible
light passes through the target sample of interest, spectrum (380 to about 800 nm). The xenon
it is measured by a special meter termed a lamps which can produce light in the UV and
spectrometer designed to compare the difference visible portions of the spectrum are used to
in the transmissions of the sample and reference measure both UV and visible spectra. Visible
beams. Double beam UV spectroscopy instru- wavelengths cover a range from 400 to 800 nm
ments allow scientists to simultaneously measure and the near ultraviolet region out to 200 nm.
transmissions through the target sample and Electromagnetic spectrum is a classification of
solvent. The absorption of ultraviolet or visible photons with various energies into different
light by a substance result in a unique ultravio- spectral regions. UV–Vis. spectroscopy is used
let–visible spectroscopic signature is known as when involving the absorption of these high-
UV–Vis. spectrum. energy lights by atoms or molecules, which
The concentration of a substance present in causes electronic excitation. This phenomenon
sample has relationship with absorbance as per only occurs if conjugated pi-electron systems
Beer–Lambert law. In general, there is a linear are present. Energies of 36–72 kcal/mol and the
relationship between increasing amount of sub- extensions of energies to 143 kcal/mol are
stance and a decreasing percentage of light sufficient to promote a molecular electron to a
transmitted (increase in absorbance) through higher energy orbital in the visible region and at
the target sample. When ultraviolet or visible the near UV region, respectively. Molecule
light strikes atoms or molecules, they can either absorbs light energy when exposed to light and
bounce off or cause electrons to jump between excites electron from the highest occupied
energy levels. Due to absorption of ultraviolet or molecular orbital (HOMO) to the lowest unoc-
visible light, electromagnetic radiation causes cupied molecular orbital (LUMO). Then, an
electron to move from lower energy levels to a optical spectrometer records wavelengths where
higher energy levels. Depending on the nature of the absorption happens and also the intensity
analyte, there may be either emission (i.e., the of the absorption. Result is usually presented by
jump of electrons from higher energy levels a graph of absorbance (A) versus wavelength.
to lower energy levels) or scattering (i.e., light The absorbance of a sample is proportional to
is deflected or scattered in other directions. the quantity of absorbing molecules in a spec-
Rayleigh scattering describes light that is redi- trometer light beam. Chromophores are molecular
rected at the same wavelength) of electromag- moieties such as pi-electron functions and het-
netic radiations. Some interactions, however, eroatoms (having nonbonding valence-shell
may occur that result in a partial transfer of electron pairs) which tend to absorb light of
energy. As a result, the scattered electromagnetic 200–800 nm wavelengths. Most instruments are
radiation is of a different wavelength. Brillouin not able to detect the absorption of wavelength
scattering and Raman scattering are examples below 200 nm, but the characteristic of conjuga-
of this type of scattering. tion which moves the absorption maxima to
UV–Vis. Spectrometry and Herbal Drugs 103

longer wavelengths solved the problem.


Conjugated pi-electron system shifts the absorp- UV–Vis. Spectrometry and Herbal
tion maxima in the same direction to the right in Drugs
a graph with every additional of double or triple
bond. It is called the bathochromic shifts because The knowledge of optical properties of atoms or
the increased conjugation narrows the gap molecules in UV–Vis. range has revolutionized
between the HUMO and LUMO orbitals, and the chemical world as it reveals the many hidden
therefore, the energy required for the promotion truth and deciphers the material. The utilization
of electrons is then less, meaning the wavelengths of UV–Vis. spectral knowledge in case of herbal
increased correspondingly. UV–Vis. spectrome- drugs may be cited as:
ter may be grouped into five sections: (1) radia-
tion source, hydrogen discharge tube is commonly
used but can be replaced by a tungsten incandes- To Study the Phenolics and Derivatives
cent lamp for energy by emitting light; (2) mono-
chromator, disperses light from the source into its All phenolic moieties have common features
separate wavelengths; (3) photometer, splits the of deriving from the shikimate pathway. The
monochromatic light into sample and reference flavonoids, phenolic derivatives, are aromatic
beams and then reflects these beams into the sam- secondary plant metabolites, which have potential
ple area; (4) sample area, consists of small cells medicinal important due to their physiological
with samples; and (5) detection area, generates and pharmacological role and health benefits,
voltage which proportional to energy when radi- having strong antioxidant and radical-scavenging
ation beams are focused on separate photomulti- activity, and appear to be associated with reduced
plier tubes. risk for certain chronic diseases, prevention of
In UV spectroscopy, a beam of light is split some cardiovascular disorders, and certain kinds
into a sample beams and reference beams. As its of cancerous processes. Flavonoids exhibit anti-
name implies, the sample beam is allowed to viral, antimicrobial, and anti-inflammatory activ-
pass through the target sample. Alternately, the ities, beneficial effects on capillary fragility, and
reference beam passes through the control sol- an ability to inhibit human platelet aggregation
vent or a portion of the solvent that does not con- properties. The actual in vivo mechanism of
tain the actual target. Once the light passes action of flavonoids is largely unknown. One of
through the target sample of interest, it is mea- the reasons is that most studies have focused on
sured by a special meter termed a spectrometer in vitro tests at doses or concentrations much
designed to compare the difference in the trans- higher than those documented in humans,
missions of the sample and reference beams. whereas few clinical investigations have been
Double beam UV spectroscopy instruments carried out on biomarkers of some of diseases
allow scientists to simultaneously measure trans- mentioned above, but results have been contra-
missions through the target sample and solvent. dictory. Epidemiological studies have shown an
The amount or percentage of a substance present inverse association between risk and intake level
is often of great importance. In general, there is of some particular flavonoids. Flavonoids are
a linear relationship between increasing amount frequently found in fruits, vegetables, and cere-
of substance and a decreasing percentage of light als. Food preparation and processing of fresh
transmitted through the target sample. If there is fruits and vegetables may decrease flavonoids
more of a substance to absorb the UV light, then content by 50% owing to their leaching into water
less light will pass through to the detector. or by removal of the richest parts of the plant. In
Correspondingly, less absorption by the target order to overcome this problem, and even more
sample allows increased transmission light to make processed foodstuffs a better vehicle
through the sample. for flavonoid intake, a considerable amount of
104 6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints

Fig. 6.1 (a) Flavone a b


skeleton. (b) Flavanone
skeleton B
O B
O
A C
A C

O
O

284
100

90
Band II
80

70

60

50
A

40 Band I

30

20 326

10

0
240 260 280 300 320 340 360 380
nm

Fig. 6.2 UV spectrum of flavanone hesperidine [2]

work has been done on the modification of their Flavonoids are mainly present as their glyco-
biosynthesis in plants. syl derivatives (hydrophilic nature), while the
Fresh fruits and their hand-squeezed or indus- aglycones (i.e., the forms lacking the sugar
trially processed juices contain mostly flavanones moieties) owing to their lipophilic nature and
and flavones. Although more than 5,000 flavonoid hence their low solubility in water. The presence
derivatives have been characterized, only a limited of large number of flavonoids with different
number of representative derivatives have been combinations that are possible between polyhy-
found and identified. Many others are often only droxylated aglycones and a limited number of
present in very low concentrations and have mono- and disaccharides are studied by UV–Vis.
yet to be identified. Their significance, however, spectroscopy. As the absorbed wavelengths by a
may outweigh their simple concentration levels. substance, results in unique ultraviolet–visible
A flavonoid skeleton is composed of two aromatic spectra for each substance, for example, the UV
rings (commonly designated as A and B), which spectra of flavones and related glycosides,
are connected through a pyrone ring (C) in the show two strong absorption peaks commonly
case of flavones or a dihydropyrone ring in the referred to as band I (300–380 nm) and band II
case of flavanones (Fig. 6.1) [1]. (240–280 nm) (Fig. 6.2) [2].
UV–Vis. Spectrometry and Herbal Drugs 105

a R3 b R2
R4 R3
R2

HO O HO O

R1 R1
OH O OH O

Fig. 6.3 (a) Flavone aglycones. (b) Flavanone aglycones

Band I is associated with the presence of a Flavonoids contribute to fruit and juice quality
B-ring cinnamoyl system. Band II absorption is in many ways, influencing the appearance, the
due to an A-ring benzoyl system. Substitutions taste, and the nutritional value of the product from
on the A or B ring may produce hypsochromic or the plant. In lemon and orange juices, for instance,
bathochromic shifts of the absorptions, which are hesperidin can contribute to the formation of
useful for clarifying structures (Fig. 6.3) [1]. sediments which result in undesirable cloudiness,
Similar type of studies for other horticulture whereas naringin markedly influences the bitter-
product may have new methods of quality assur- ness of the juice of grapefruits and bergamots.
ance and relook to the process adopted for the Flavonoids, most common in our diet, may be
preparation of the same. cited from Citrus fruits as their glycosyl deriva-
A group of compounds classified as polyme- tives. Aglycones occur less frequently in juices,
thoxyflavones (PMFs) usually found as compo- owing to their lipophilic nature and hence their
nents of the essential oils fraction of Citrus peels low solubility in water. The presence of a rela-
[3]. Hand-squeezed juices contain no detectable tively large number of flavonoids in Citrus
traces of this class of compounds [4]. Commercial juices is a result of the many different combina-
juices, on the other hand, are rich in PMFs tions that are possible between polyhydroxylated
because the industrial processing of fruits aglycones and a limited number of mono- and
leads to juices being contaminated with the disaccharides. The most common sugar moieties
peel constituents (vide infra). The flavanone include d-glucose and l-rhamnose. The glyco-
O-glycosides have a glycosyl substitution exclu- sides are usually O-glycosides, with the sugar
sively at the C-7 position (on ring A), although a moiety bound generally to the aglycone hydroxyl
3-O-rutinoside has also been reported (viz., rutin), group at C-7 or at the C-3 in some cases. In addi-
and di-C-glycosides, along with smaller amounts tion to these, C-glycosides have also been detected
of mono-C-glycosides. For these compounds, in various citrus fruits or juices [1].
substitution is generally on either the C-6 or the Many analytical procedures require no pre-
C-8, or on both positions [1]. liminary separation of the flavonoid fraction,
and analysis is performed directly on the crude
juices. Extraction with solvents of graded polarity
UV Spectrometry for Different leads to the separation of flavonoids from the
Derivatives other components. Methanol, ethanol, acetone,
water, ethyl acetate, and, to a lesser extent, propa-
UV spectra of different derivatives of a compound nol, dimethylformamide, and combinations of
can be easily distinguished and characterized, these are frequently used for their extraction.
for example, the quercetin, 3-O-glucosyl quercetin, Hydrolysis of glycosides is a useful method to
and 3,4¢-di-O-glucosyl quercetin in UV–Vis. obtain structural elucidation and characterization
spectrometry have spectrum as Fig. 6.4 [5]. information. The rate of acid or basic hydrolysis
106 6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints

100

90

80

70
3,4’-di-O-glucosyl quercetin
60 3-O-glucosyl quercetin
quercetin
A

50

40

30

20

10

0
200 250 300 350 400 450
nm

Fig. 6.4 UV spectrum of quercetin and derivatives [5]

of glycosides depends on the acid or base strength, HPLC by linear gradient elution using water
the nature of the sugar, and the position of attach- and acetonitrile as mobile phase at a flow rate of
ment to the flavonoid nucleus. In fact, acid hydro- 1.0 ml/min and detector wavelength at 270 nm
lysis, which does not affect C-glycosides, is a for dioscorealide A and dioscorealide B and at
good method to distinguish these derivatives 245 nm for dioscoreanone. This HPLC fingerprint
from their O-glycosidic counterparts as the latter along UV spectra has proven the identification
are quickly hydrolyzed [1]. and assessment for quality D. membranacea
(Fig. 6.5) [7].

UV–Vis. Coupled Methods for Analysis


Preparation of Sunscreens
A number and variety of methods for the detection
and quantification of natural product compounds Sunscreens are chemicals that provide protection
have been developed, and several analytical against the adverse effects of solar and in particu-
procedures allow the simultaneous determination lar UV radiation [8]. Sunscreens has wide variety
of the various kinds of flavonoid glycosides as of chemicals that have specific absorbance in
flavanone O-glycosides, flavone O-glycosides, some parts of the UV spectrum. There are very
flavone C-glucosides, and polymethoxyflavones, few single chemical substances that have absor-
for example, liquid chromatography (LC) and bance over the full range of UV. This property
more recent coupled methods as LC–MS and (having absorbance over the full range of UV) is
LC–MS–MS [6]. HPLC–PDA chromatographic needed for a product to be considered as a suitable
fingerprint was developed for ethanolic extract wide-spectrum sunscreen. Plant extracts, due to
of Dioscorea membranacea, which have the wide range of natural compounds, usually cover
dioscorealide A, dioscorealide B, and dioscore- this full range of UV wavelengths. The best
anone, the three main markers of the species. approach to protecting the body from the harmful
The samples were separated with reversed-phase effects of UV irradiation is to use these active
UV–Vis. Spectrometry and Herbal Drugs 107

Dioscorealide B

Dioscorealide A
a b

Dioscorealide B

Dioscorealide A
40 60
50
30 40
mAU

mAU
20 30
20
10
10
0 0
0 10 20 30 0 10 20 30
RT (min.) RT (min.)
c d
100 Dioscorealide A 175

150
Dioscorealide B
270 nm
80 270 nm
125
60
mAU

100

mAU
40 75

50
20
25

0 0

200 250 300 350 200 250 300 350


nm nm

Dioscoreanone
e f
Dioscoreanone

10 30
25
8
20
mAU
mAU

6
15
4
10
2 5
0 0
0 10 20 30 0 10 20 30
RT (min.) RT (min.)

g
245 nm
150

125 Dioscoreanone

100
mAU

75

50

25

200 250 300 350


nm

Fig. 6.5 (a) HPLC fingerprints of extract, (b) HPLC fingerprints of standard, (c) UV fingerprints, (d) UV fingerprints,
(e) HPLC fingerprint of extract, (f) HPLC fingerprint of standard, (g) UV fingerprints

photoprotectives. In recent years, naturally occur- radicals. There are also reports of flavonoids
ring compounds have gained considerable atten- inhibiting the activities of many enzymes, including
tion as protective agents [9]. There are reviews lipoxygenase, cyclooxygenase, monooxygenase,
about the photoprotective effects of some naturally xanthine oxidase, mitochondrial succinate dehy-
occurring herbal polyphenols and phenolic-rich drogenase and NADH-oxidase, phospholipase
extracts in skin damage induced by UV irradia- A2, protein kinases, and nuclear transcription
tion. Several studies have shown the flavonoids factor. Phenolics are believed to be capable of
to act as scavengers of superoxide anions, singlet acting in redox-sensitive signaling cascades to
oxygen, hydroxyl radicals, and lipid peroxyl inhibit DNA damage. Many flavonoids such as
108 6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints

quercetin, luteolin, and catechins are better used was based on the equation proposed by
antioxidants than the nutrients vitamin C, vitamin Gharavi et al. [12]:
E, and b-carotene. Therefore, the phenolics may
be beneficial in preventing UV-induced oxygen

337.5
free radical generation and lipid peroxidation, E (λ )ε (λ )
that is, events involved in pathological states such SPF = 292.5


337.5
E (λ )ε (λ )T (λ )
as photoaging and skin cancer, and to prepare 292.5

topical sunscreen formulations from these two where


extracts and measure their SPFs. T(l) is the measured sunscreen transmittance
Since time immemorial human in different at l,
civilizations protected themselves against the E(l) is the spectral irradiance of terrestrial
environment by paintings, clothes covering the sunlight at l,
body, veils, and large brim hats were used by and e(l) is the erythemal action spectrum at l.
ancient Greeks and umbrellas by Mesopotamia, The values of the E(l) and e(l) were calcu-
Chinese, and Indian civilization. Natural sub- lated according to the report by Gharavi et al.
stances extracted from plants have been recently [12]. T(l) was mean value of four experimental
considered as potential sunscreen resources observations. Three different concentrations of
because of their ultraviolet ray absorption in the each extract (according to Beer’s law) were used
UV region and their antioxidant activity. Green for obtaining the standard curve and calculating
tea polyphenols, Aloe vera extract, aromatic SPF in 2 mg/ml solution according to Gharavi
compounds isolated from lichens, and several et al. method [12].
glycosides of aesculin are examples of natural A few phenolics have also been found in vitro
substances evaluated for their sunscreen proper- to be mutagenic. These harmful effects are sus-
ties. There is strong evidence that DNA-damaging pected to result from the prooxidant rather than
radiations induce the accumulation of UV light antioxidant action of these compounds. Therefore,
absorbing flavonoids and other phenolics in der- it is necessary to investigate their toxic effects
mal tissue of the plant body, which may be a before human use. In the past decade, the antioxi-
potential source of sunscreen [10]. dant activity of herbal phenolics, namely, pheno-
To evaluate the UV protective activities of lic acids and flavonoids, has been given much
plants, the air-dried herbal materials are ground attention. Many flavonoids such as quercetin,
into fine powders and extracted by maceration luteolin, and catechins are better antioxidants
method two times with 90 and 50% aqueous than the vitamin C, vitamin E, and b-carotene.
methanol, respectively [11]. After filtration of Therefore, the phenolics may be beneficial in
total extracts, the solvent is removed under preventing UV-induced oxygen free radical gen-
reduced pressure to give the gummy mass. The eration and lipid peroxidation (i.e., events
gummy mass is dissolved in 20% hydro-metha- involved in pathological states such as photoaging
nol solution and kept in refrigerator for 48 h until and skin cancer) and to prepare topical sunscreen
the chlorophyll was precipitated. The extracts are formulations from plant extracts having phenolic
filtrated and extracted with ethyl acetate, and this acids and flavonoids and measure their SPFs.
fraction is rich in flavonoids and other phenolics, Presently, physicians have increasing stress
the most important chemicals for sunscreen. The that public and professionals should protect
sun protection factor (SPF) is calculated by dif- themselves from the potential harmful effects of
fuse transmittance method. ultraviolet radiation when they go out in the sun
In a study, the ethyl acetate extracts of plants for extended periods [9]. Studies have shown that
are analyzed for SPF in vitro. The extract is dis- UV radiation can drive the chemical reactions
solved in methanol, and spectra of the samples that produce highly reactive radicals that have
are obtained by running from 337.5 to 292.5 nm the potential to damage DNA and other cell regu-
(at 5 nm intervals). The SPF determination model lating chemicals and the cell structures also.
Evaluation of Vitamin C Content 109

Thus, UV–Vis. spectroscopy allows us in discovery molybdate solution 2 ml in each volumetric


and authenticity of testing of potential sun blocking flask and make up to 25 ml with distilled water.
substances [9]. 7. Preparation of sample solutions: Accurately
weigh 1 g of each sample in a 25-ml conical
flask and add 10 ml of oxalic acid (0.05 M)
Evaluation of Vitamin C Content solution, and the sample was placed under
shade for 24 h for extraction of vitamin C
l-Ascorbic acid (vitamin C), an important precur- content. After 24 h, the samples were filtered
sor of redox reaction, is an integral part of drugs,
to enhance the quality and efficacy of product. Table 6.1 Concentration of vitamin C in medicinal
Presently, UV–Vis. analysis is in practice to esti- plants (mg/100 g) [13]
mate the concentration of vitamin C by measuring Medicinal plant Absorbance Concentration
spectrum at 760 nm, after sample preparation Jasmine nudiflorum 1.864 202.74
against standard preparation (Tables 6.1 and 6.2) Ricinus communis 1.322 145.74
[13]. The whole process may be summarized as: Ficus bengumera 0.362 45.169
Ficus pumila 0.338 42.634
Policaria crispa 0.188 26.988
Preparation of Stock Solutions Narcissus tazetta 0.553 65.199
Fagophyrum esculentum 1.216 134.80
Cassia fistula 0.747 85.548
The following stock solutions were prepared:
Cherry chrysthanum 0.255 33.913
1. Ammonium molybdate solution (5%, m/v):
Ruscus hypophyllum 0.524 62.202
Weigh 5 g of ammonium molybdate and
Taraxicum officinale 0.266 35.052
dissolve it in 100 ml of distilled water.
Verbascum thapsus 0.221 30.339
2. Oxalic acid solution (0.05 M): Weigh required Rumax aluculatus 0.365 45.489
quantity of oxalic acid, add on it 1 ml of Fumaria parviflora 0.592 69.297
freshly prepared solution containing 0.02 M Bombax ceiba 0.143 69.297
EDTA, and then make up the volume 100 ml Stellaria media 0.395 48.653
with distilled water. Pectus forums 0.307 39.432
3. Sulfuric acid solution (5%, v/v): Measure 5 ml Olea europaea 0.549 64.839
of concentrated sulfuric acid and make up the Rosa indica 0.714 82.116
volume 100 ml with distilled water. Euphorbia helioscopia 0.622 72.473
4. Metaphosphoric acid with acetic acid solu- Oxalis latifolia 0.644 74.714
tion: Dissolve with shaking required quantity Cestrum nocturnum 0.800 91.144
of metaphosphoric acid pellets in required Pridium guajava 1.321 145.75
quantity of acetic acid and then make the Cupressus sempervirens 1.280 141.43
volume up to 100 ml with distilled water. Withania somnifera 0.361 45.029
Galium tricorne 0.381 47.154
5. Standard l-ascorbic acid solution (0.1%,
Tecoma stonz 618 71.999
w/w): Weigh 0.1 g of l-ascorbic acid and dis-
Lactuca sativa 0.142 22.067
solve in oxalic acid (0.05 M) solution freshly
Zizyphus jajuba 0.115 19.211
prepared and make up the volume 100 ml.
6. Preparation of different standard solution: The
0.5, 1, 2, 3, 4, and 4.5 ml, respectively, of stan- Table 6.2 Different concentration of standard solutions
and absorbance (mg/100 g) [13]
dard l-ascorbic acid (0.1%, w/v) solution taken
in separate 25-ml volumetric flasks, add 4.5, S. no. Absorbance Concentration
4, 3, 2, 1 and 0.5 ml of oxalic acid (0.05 M) 1 0.3 40
solution in each, and then add separately meta- 2 0.62 80
phosphoric acid with acetic acid 0.5 ml, sulfuric 3 1.03 120
acid (5%, v/v) solution 1 ml, and ammonium 4 1.52 160
110 6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints

Fig. 6.6 (a) Change in color with AgNO3 [15], (b) UV–Vis. spectrum of SNPs [15]

through a 0.45-mm filter. Add separately biosynthesis of silver nanoparticles is carried out
metaphosphoric acid with acetic acid 0.5 ml, by using medicinal plant extracts for the reduction
sulfuric acid (5%, v/v) solution 1 ml, and of aqueous silver ions in short period. The silver
ammonium molybdate solution 2 ml, in each nanoparticles formation is confirmed by the color
volumetric flask and make up the volume change of plant extracts and further confirmed with
25 ml with distilled water. the help of UV–Vis spectroscopy. These photosyn-
To avoid photochemical reactions, amber-colored thesized silver nanoparticles are tested for antibac-
volumetric flasks are used in the aforesaid whole terial and antifungal activities using disk diffusion
analysis. By this method, 29 different medicinal method. The test cultures are Proteus, Pseudomonas,
plants were studied (Table 6.1), and each sample Klebsiella, Bacillus, and E. coli species of bacte-
was analyzed for vitamin C at 760 nm using ria, and Aspergillus, Fusarium, Curvularia, and
UV–Vis. spectrophotometer and compared with Rhizopus species of fungal have been studied.
the standard. The microbial property of silver nanoparticles is
The temperature, solvents, pH, light, and metal analyzed by measuring the inhibition zone. The
ions (Cu+2 and Fe+3) degrade the ascorbic acid [14]. silver nanoparticles synthesized from stem bark
The mean concentration of Cu+2 in drinking water extracts of Boswellia (Fig. 6.6) [15] and twigs of
is 60 mg/l (0.06 ppm). Water concentration is com- Cinnamomum tamala (Figs. 6.7 and 6.8) [16]
paratively more in fruits and vegetables than other extract have been studied, and the SNPs synthe-
plants parts, so vitamin C degrades comparatively sized, and studied for antimicrobial properties, and
fast on these items. Human body cannot produce results indicate that the silver nanoparticles have an
vitamin C and acquires from exogenous sources important advantage over conventional antibiotics.
only. The daily intake, as per American standard of The silver nanoparticles exhibit yellowish-brown
vitamin C, is 60 mg/250 ml, so to monitor different color in aqueous solution due to excitation of sur-
herbal drugs and preparations as food supplements, face plasmon vibrations in silver nanoparticles [17].
nutraceuticals, cosmaceuticals, etc. [13]. During experiment, the appearances of yellowish-
brown color in the reaction vessels indicate the
formation of silver nanoparticles (SNPs) [18].
Green Synthesis of Nanoparticles

The biologically synthesized silver nanoparticles SNPs from C. tamala Twigs


(SNPs) are widely in use in the field of medicine,
for antimicrobial and antifungal properties, as an Cinnamomum tamala twigs were collected
alternate for conventional antibiotics. Extracellular from Thalkedar Forest, District Pithoragarh,
Green Synthesis of Nanoparticles 111

Fig. 6.7 Color change with AgNO3 and conc.biomass [16] where 1 and 2 are controlled and 3, 4, 5, and 6 are experi-
mental. The digit deciphers: 1 AgNO3 only, 2 twig only, 3 0.01% twig, 4 0.05% twig, 5 0.1% twig, and 6 0.5% twig

Uttarakhand, India. The freshly harvested C. tamala silver ions when exposed to herbal extracts
twigs were exposed to vacuum until they were were reduced in solution, thereby leading to the
completely dried, at 25°C. The biomass used for formation of silver hydrosol. The time duration
the reduction was prepared by crushing the dried of change in color varies from plant to plant.
twigs to the 60 mesh size, weighted, and added to The synthesis of SNPs had been confirmed by
in the range of 40–60 ml of 1–10 mM aqueous measuring the UV–Vis spectrum of the reaction
AgNO3 solution to obtain 0.01–05% final con- media. The weak absorption peak at shorter
centrations, in conical flasks of 100 ml, at room wavelengths is due to the presence of several
temperature. The flasks are thereafter shaken at a organic compounds which are known to inter-
rotation rate of 100–200 rpm in the dark at act with silver ions [17]. Three different routes
20–50°C for 24 h. Change in appearance, from have been described for the reduction of silver
colorless to yellowish-brown, indicates the in plant extracts. The secondary metabolites
presence of silver nanoparticles in the reaction present in plant systems may be responsible for
mixture, and the intensity of color increases with the reduction of silver and synthesis of nano-
the concentration of biomass (Fig. 6.6). The particles, as the first route. The second biogenic
formation of silver nanoparticles is confirmed route is the energy (or) electron released during
by UV–Vis. spectroscopy. UV spectra showed glycolysis (photosynthesis) for conversion of
the peak at 282 nm indicating the formation nicotinamide adenine dinucleotide (NAD) to
of spherical nanoparticles (Figs. 6.8–6.10). The nicotinamide adenine dinucleotide dehydroge-
size of the silver nanoparticle is in the range of nase. (NADH) led to transformation of Ag(NO3)2
80–150 nm and is spherical in shape [16]. to form nanoparticles, and the third postulate is
Silver nitrate is used as reducing agent as silver release of an electron during formation of
has distinctive properties such as good conductiv- ascorbate radicals, from ascorbate, and reduces
ity, catalytic, and chemical stability. The aqueous the silver ions.
112 6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints

Fig. 6.8–6.10 UV spectra indicating the formation of SNPs using C. tamala twigs [16]
Purification of Geometrical Isomers 113

The silver nanoparticles synthesized biologi- these silver crystals is speculated that the organic
cally using medicinal plants have been found of matrix contain silver binding proteins that pro-
high toxicity against different pathogenic bacte- vide amino acid moieties that serve as the nucle-
ria and fungi of selected species. The ionic silver ation sites [21].
strongly interacts with thiol group(–SH) of vital
enzymes and inactivates the enzyme activity.
Experimental evidence indicates that DNA loses Purification of Geometrical Isomers
its replication ability once the bacteria have been
treated with silver ions. The pathogenic effect of Commiphora wightii has been widely used in
nanoparticles can be attributed to their stability Ayurvedic medicine for more than 2,000 years,
in the medium as a colloid, which modulates the mainly to treat arthritis and inflammation. The
phosphotyrosine profile of the pathogen proteins active ingredients in gugulipid are the ketoster-
and arrests its growth. The growth of microor- oids cis- and trans-4, 17 (20)-pregnadiene-3,16-
ganisms was inhibited by the green synthesized dione, also known as E- and Z-guggulsterone
SNPs showed variation in the inhibition of [22], and are extracted from the resin, that is safer
growth of microorganisms may be due to the and more effective than many cholesterol-lower-
presence of peptidoglycan, which is a complex ing drugs [23]. After extracting the ole-gum resin
structure and after contains teichoic acids or with ethyl acetate, it separates into two parts,
lipoteichoic acids which have a strong negative gum and resin. The ethyl acetate insoluble part
charge. This charge may contribute to the seques- of gum chemically characterized as a carbohydrate
tration of free silver ions. Thus, gram-positive gum (belonging to the class of sugar), and the
bacteria may allow less silver to reach the cyto- resin in ethyl acetate fraction contains bioactive
plasmic membrane than the gram-negative bac- components, especially E- and Z-guggulsterones.
teria [19]. The SNPs synthesized from plant After filtration and evaporation of the solvent
species are toxic to multidrug resistant microor- under reduced pressure, compounds are sepa-
ganisms. It shows that they have great potential rated. Fractionated in petroleum ether, the steroi-
in biomedical applications. Similar observation dal compounds were isolated by dissolving in
was found in Allium cepa, Argemone mexicana, petroleum ether (40–60°C). For isolation of E-
and Artocarpus heterophyllus and concluded and Z-guggulsterones from the mixture of steroi-
that silver nanoparticles have an ability to inter- dal compounds, after being evaporated of the
fere with metabolic pathways. The data and solvent, of this was dissolved in small amount of
results by Ahmad N et al. indicate that the inhi- ethyl acetate and transferred on to a silica gel
bition of oxidation based biological process by 60 F254 column chromatography as stationary
penetration of metallic nano-sized particles phase and eluted with the mixtures of solvents,
across the microsomal membrane [20]. The use toluene–acetone (9:1), which resulted in 15 frac-
of silver ions as preventing agents in cosmetics tions of 20 ml, based on TLC identification and
has been tested by a challenged list in a set of quantitative analysis of these isomers in herbal
cosmetic dispersions with the addition of known extracts, the HPLC technique with photodiode-
preservative inhibitors or microorganism’s array detector and gradient solvent was applied.
growth promoters. The silver nanoparticles syn- At first, the calibration curve for E- and
thesized from leaf extract showed higher toxicity Z-guggulsterones was developed (Fig. 6.11) [23].
than that of bark extracts. The reason could be The guggulsterones peaks were identified at
that the leaf extract synthesized higher concen- 242 nm using C-18 column (125 × 4.0 mm,
tration of silver nanoparticles than the bark sam- 5.0 mm). The mobile phase was acetonitrile–
ples. Moreover, green leaves are the site of water (46:54, v/v), at ambient temperature
photosynthesis and availability of more H+ ions (~28°C) with flow rate of 1.0 ml/min.
to reduce the silver nitrate into silver nanoparti- In a study for authenticity of Commiphora
cles. The molecular basis for the biosynthesis of wightii resin, the presence of mangiferolic acid, a
114 6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints

Fig. 6.11 (a) E-Guggulesterone, (b) Z-guggulesterone

metabolite of Mangifera indica, raised the sus- O


pension on adulteration, so genuine samples of O
C. wightii and M. indica were collected from
India and Pakistan, and their ethyl acetate extract OH
was subjected to the TLC analysis, together with CH3
H
the suspicious sample. The four compounds pres-
ent in commercial sample were not detected in CH3
any authentic sample of C. wightii resin; how-
ever, they were present in M. indica specimen. H OH
CH3
Finally, it was concluded that commercial sam-
O
ple, C. wightii sample, was adulterated with M. O H
indica resin, and TLC fingerprints had detected
the same, as morphologically it is not easy to dis-
OH
tinguish between the closely related resins [24]. OH
3

Fig. 6.12 Structure of digoxin: calculation of theoretical


Analysis of Ultra-Diluted Drugs lmax [25]

The ultra-diluted drugs (lower than micro-


volumes) have billions dollar market, globally. medicine is mixed with 99 parts of aqueous
The UV-sensitive organic substances/compounds ethanol. When the same process is repeated at 30
of a drug, which has been ultra diluted, are char- times by adding one part of previous potency in
acterized by its UV–Vis. fingerprints, for evi- 99 parts of aqueous ethanol, as diluents and suc-
dence-based status, significantly to claim that the cussed, after each stage starting from first dilu-
formulation is more than a placebo, for example, tion, next 29 times, 30 potency is obtained and so
the ultra-diluted Digitalis purpurea has wide on. The active ingredient of D. purpurea is
acceptance in treating ailments (heart diseases, digoxin (Fig. 6.12), has medicinal preparations
dropsy) in a medical system, known as homeopa- Q, 6, 30, 200 in Indian market (manufactured by
thy. It is in use since long as narrow poison (1500 M/S Hahnemann Publishing Company Pvt. Ltd.,
bc). The purified and active principles of D. pur- Kolkota) have been analyzed for digoxin content
purea have been studied for their identification by UV–Vis. spectroscopy (Figs. 6.13 and 6.14),
by UV spectroscopy. The saturated foxglove (D. for potency of the digoxin [25].
purpurea) extract in aqueous ethanol (91.4% eth- 1. A five member cyclic , b-unsaturated ketone:
anol and glass double-distilled water mixture) is 202 nm
presented as q or Q (mother tincture) in term of 2. Acyloxy substitution (–OCOR): 25 nm
ultra-diluted medicine (i.e., in homeopathy). 3. b-Unsaturated alkyl (including part of cyclic
Dilution or potency 1 means that one part of ring): 24 nm (i.e., 2 × 12 nm)
Analysis of Ultra-Diluted Drugs 115

2.5

Digitalis P Q
2.0

1.5
A

1.0 Digitalis P 6

0.5
Digitalis P 30

0.0

Digitalis P 200
−0.5

100 200 300 400 500 600 700 800 900 1000
nm

Fig. 6.13 UV–Vis. spectra of Q, P 6, P 30, and P 200

Fig. 6.14 Absorption


maxima with dilution [25] 2.5

2.0

1.5
A

1.0

0.5

0.0

1.0 1.5 2.0 2.5 3.0 3.5 4.0


conc.

The above calculation predicts that the compound Ingredients other than , b-unsaturated ketone
in ethanol medium absorbs at wavelength 251 in of D. purpurea do not show any other specific
UV–Vis. spectrum. The UV–Vis. spectra of D. UV–Vis. absorbance, so any change brought due
purpurea Q, P 6, P 30, and P 200 (Fig. 6.13), and to dilution could not be deciphered, but the
the relation with absorption intensity (peak method is useful to generate the fingerprints of
heights) on concentration (Fig. 6.14) have been large number of ultra-diluted drugs using medici-
studied for the authentication of the claims made nal plants having UV–Vis. active ingredients, for
on it [25]. evidence-based drugs.
116 6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints

Table 6.3 Ultraviolet analysis of leaf powder of H. rosa-sinensis [26]


Treatment Visible light UV (254 nm) UV (365 nm)
Drug powder Light green Dark green Blackish brown
Sulfuric acid (conc.) Dark green Black Black
Sulfuric acid (dilute) Light brown Greenish black Black
Hydrochloric acid (conc.) Green Black Black
Hydrochloric acid (dilute) Brown Greenish black Black
Acetic acid Brown Greenish black Black
Nitric acid (dilute) Brown Greenish black Black
Methanol Green Dark green Black
Ethanol Brown Brownish black Black
Chloroform Brown Dark green Black
Petroleum ether Green Greenish black Black
Distilled water Brown Dark green Black
10% NaOH Brown Green Black
5% Iodine Greenish brown Dark green Black
Picric acid Green Greenish red Black
FeCl3 solution Brown Dark green Black
Ammonia solution Brown Green Greenish black

Identification of Powdered Medicinal bioautography of the dichloromethane extract of


Plants S .calycina showed the presence of a compound
which strongly inhibit the growth of Cladosporium
The powdered part of plant tissues has specific cucumerinum. HPLC–UV and HPLC–MS analy-
fluorescence in UV range, a tool to detect adul- ses of the extract revealed the presence of three
teration, contamination, and stability and prove main compounds: a bitter principle, a xanthone,
for claims made on products, for example, the and a naphthoquinone derivative with a molecu-
leave powder of Hibiscus rosa-sinensis, known lar weight of 188. Comparison of online UV and
as China rose, examined under UV–Vis. light MS data with a data bank allowed identification
treated with different reagent emits various of the bitter principle as sweroside and the xan-
color radiations (Table 6.3), which helps in the thone as decussatin. As these have no antifungal
identification of the drug in fragmentary condi- properties, the strong activity of the dichlo-
tion as well as in whole form. The results are romethane extract was attributed to the naphtho-
sufficient for preliminary phytochemical screen- quinone, a class of compounds which is known to
ing and act as biomarkers for identification and have strong antimicrobial properties. Targeted
authentification of raw drug samples and play an isolation afforded the active compound, identified
important role in quality control and prevention as 2-methoxy-1,4-naphthoquinone. Interestingly,
of adulteration [26]. quinines were previously not known to occur in
the Gentianaceae [27].

Biological Assay Using Hyphenated


Techniques To Differentiate Various Species
of a Genus
Swertia calycina, a small plant found in Rwanda,
family Gentianaceae, was studied with the com- The basic aim to develop fingerprint is the estab-
bined use of TLC and HPLC in the search for lishment of a characteristic chromatogram/spec-
new antifungal metabolites (Fig. 6.15). TLC trum for the substance of interest, in the medicinal
To Differentiate Various Species of a Genus 117

Fig. 6.15 Structure and UV–Vis fingerprint of 2-methoxy-1,4-naphthoquinone [27]. (a) 2-Methoxy-1,4-
naphthoquinone, (b) UV–Vis. spectrum

Fig. 6.16 Fingerprint chromatograms of (a) Euphorbia ebracteolata Hayata, (b) Euphorbia fischeriana Steud, and (c)
Stellera chamaejasme L, where P1 = ebracteolata compound B, P2 = jolkinolide B, and P3 = neochamaejasmin [28]

herb, and at least one marker compound is optimization was Euphorbia ebracteolata Hayata.
identified in the fingerprint, as characteristic, In this process, different experimental conditions
capable to explain the uniqueness (as large peaks were adopted for establishing the optimum exper-
with good resolution). Example may be cited imental conditions for Euphorbia ebracteolata
from the Chinese herb Langdu, which have Hayata HPLC fingerprints, with repeatability and
three different species (Euphorbia ebracteolata reproducibility. Using the optimized experimen-
Hayata, Euphorbia fischeriana Steud, and Stellera tal conditions, fingerprints of other two species of
chamaejasme L.). The experiment was with strat- Langdu Euphorbia fischeriana Steud and Stellera
egy to develop optimum experimental conditions chamaejasme L. were obtained (Fig. 6.16) [28].
for one of the species first and then apply these The characteristic HPLC pattern, as fingerprint,
optimum conditions to the other two species and approved as authentic by the identification of
other related herbs. The species chosen for the marker compounds of the herb in the fingerprint
118 6 UV–Vis. Spectroscopy: Herbal Drugs and Fingerprints

Fig. 6.17 Chemical structure and UV spectrum of the marker compounds [ 28 ] ( a ) ebracteolata compound B,
( b ) neochamaejasmin, and (c) jolkinolide B

chromatogram. This is the second reason to chamaejasme L. respectively. These marker


optimize the experimental conditions under compounds were identified according to their
which the number of intense peaks and their reso- distinctive UV spectra in their characteristic
lution in the chromatogram are maximized. To fingerprint chromatogram (Fig. 6.17) [28].
meet this requirement, peak-tracking method UV–Vis. spectroscopy is a reliable technique
using diode-array detector was employed to for the assessment of herbals and herbal drugs, as
find out if the marker compounds of the herbal the absorption, transmission, and emission of
medicine could be identified in the fingerprint wavelengths are unique fingerprints for each.
chromatograms. As per literature, ebracteolata The technique has effective potential when used
compound b, jolkinolide B, and neochamae- in conjunction with chemical screening methods
jasmin were recognized as the characteristic so that ubiquitous and unimportant compounds
marker compounds for Euphorbia ebracteolata can be excluded. For chemical screening, HPLC
Hayata, Euphorbia fischeriana Steud, and Stellera coupled with different detection methods, for
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FTIR Spectroscopy: Herbal Drugs
and Fingerprints 7

In infrared (IR) spectroscopy, IR radiations are The original infrared instruments were of the
passed through a sample, where some of the dispersive type (i.e., the separation of individual
infrared radiations are absorbed by the sample. frequencies of energy emitted from the infrared
When the radiant energy matches to the energy of source, by a prism or grating). Grating is a more
specific molecular vibration, absorption occurs. modern dispersive element, used to separate the
In IR spectrum, wave number (sometimes called frequencies of infrared energy. The detector
as frequency) is plotted on the x-axis and is pro- measures the amount of energy at each frequency
portional to the energy. The band intensity can be which has passed through the sample. This results
expressed as absorbance (A) or percentage trans- in a spectrum which is a plot of intensity versus
mittance (%T) as plotted on y-axis (zero trans- frequency. The slow scanning process is a main
mittance corresponds to 100% absorption of light difficulty in this technique. In order to overcome
at that wave number) (Fig. 7.1). Absorbance this limitation, the Fourier transform infrared
(A) is logarithms, to the base 10, of the reciprocal (FTIR) spectrometry is developed on the disper-
to the transmittance (T). sive instruments.
To measure all infrared frequencies simultane-
A = log10 (1 / T ) (7.1) ously, rather than individually, a very simple
optical device known as interferometer is added
The absorbance or transmittance with cor- with the instrument. The interferometer produces
responding wave numbers as a peak in spectra a unique type of signal which has all of the
gives information about the molecule to the infrared frequencies encoded into it. The signal,
analyst, by matching it with spectrum of known known as interferogram, is measured very quickly,
compound, peak-by-peak correlation. The spec- usually on the order of 1 s. The interferogram is
trum represents a fingerprint of a sample, unique decoded to individual frequencies, via a well-
characteristic, as no two different molecular known mathematical technique called the Fourier
structures can produce the same spectra. This transformation, to have a frequency spectrum
makes infrared spectroscopy useful for several (a plot of the intensity at each individual wave
types of analysis as each different material number/frequency). This transformation is per-
is a unique combination of atoms. Therefore, formed by the computer with modern algorithms
infrared spectroscopy results are used in molecu- software as the spectrum. All the spectral features
lar identification (qualitative analysis) of every in the spectrum are only strictly due to the
different kind of material, and the size of sample. A single background measurement is
peaks in the spectrum is a direct indication of sufficient for many sample measurements because
the amount of material present (i.e., purity) in the background spectrum is characteristic of the
the sample. instrument itself. Thus, the time span per sample

D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs, 121
DOI 10.1007/978-81-322-0804-4_7, © Springer India 2012
122 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Fig. 7.1 IR spectrum plotted using transmittance (left) and absorbance (right)

Source Interferometer Sample Detector

Interferogram Computer IR Spectrum

Fig. 7.2 A schematic presentation of IR spectroscopy

is reduced to a few seconds rather than several red region extends from 2,500 to 50,000 nm.
minutes, as in the case of IR. The normal instru- This spectral region encompasses three subdivi-
mental process (Fig. 7.2) may be described as a sions: the far-infrared (FIR: 400–33 cm−1 or
black body source of radiations which passes 30–300 mm), mid-infrared (MIR: 4,000–400 cm−1
through an aperture, into the interferometer or 3–30 mm), and near-infrared (NIR: 12,820–
(where spectral encoding results into the inter- 4,000 cm−1 or 0.78–3 mm), named in relation to
ferogram), to the surface of the sample, finally to the visible region. Infrared analysts often use
the detector, specially designed to measure the wave numbers to describe the infrared spectral
special interferogram signals. The measured region. The energies of infrared radiations range
signals are digitized and sent to the computer from 48 kJ/mol at 2,500 nm to 2.4 kJ/mol at
where the Fourier transformation takes place, as 50,000 nm. These low energies are not sufficient
infrared spectrum. to cause electron transitions, but they are sufficient
The uniqueness of IR spectroscopy is that to cause vibrational changes within molecules,
any sample virtually at any state may be studied so IR spectroscopy is also known as vibrational
[1, 2]. Infrared region starts immediately after spectroscopy. As spectrum generates through
the visible region at 700 nm. The classical infra- the incident radiation that is absorbed at a particular
Interpretation of IR Spectra 123

frequency (i.e., wave number), so it is based on nm is more often used. The relationship between
the absorption of electromagnetic radiation by a the two units is as follows [5]:
molecular system. In other words, IR spectra
1
provide images of vibrations of the atoms of a [cm −1 ] = (7.2)
compound. FTIR spectroscopy has developed [nm] × 10 −7
quickly due to its low noise, rapid speed, high The basic principle of IR spectroscopy is the
repeatability, easy operation, low expense, and measurement of the amount of IR radiations
so on. It is also associated with other sciences absorbed by a sample has a high potential for the
just like mathematics or computers, or with elucidation of molecular structures [6]. The
other techniques such as two-dimensional cor- regions of an IR spectrum where bond-stretching
relation analysis (2D-IR); FTIR has become vibrations are seen depend primarily on whether
increasingly useful in the field of evaluating the bonds are single, double, or triple or bonds to
herbal qualities. The vast majority of molecules hydrogen, etc., as [7]:
exhibit infrared peaks in the mid-infrared region
(4,000–400 cm−1). The position and intensity of Bond Absorption region, cm−1
a vibrational peaks are characteristics of the C–C, C–O, C–N 800–1,300
underlying molecular motion and consequently C=C, C=O, C=N, N=O 1,500–1,900
of the atoms participating in the chemical bond, C=C, C≡N 2,000–2,300
their conformation, and their immediate environ- C–H, N–H, O–H 2,700–3,800
ment. Thus, a certain submolecular group pro-
duces peaks in a characteristic spectral region.
These characteristic peaks form the empirical Interpretation of IR Spectra
basis for the interpretation of vibrational spectra.
Moreover, characteristic absorption peaks are IR spectroscopy is an extremely effective method
used for a specific compound detection. FTIR for determining the presence or absence of a wide
spectrometers have almost entirely replaced dis- variety of functional groups in a molecule. One of
persive instruments because of their better per- the most preferred methods to decipher IR spec-
formance in nearly all respects. The application trum is to start at the high wave number end of the
of this technique has improved the acquisition of spectrum (typically 4,000 cm−1) and look for the
IR spectra dramatically. The major advantage presence and absence of characteristic absorptions
of IR over other spectroscopic techniques is (Table 7.1) as move toward lower wave numbers.
that practically all compounds show absorption The intensity of an absorption peak in the IR
and can thus be analyzed both qualitatively spectrum is related to the change in dipole that
and quantitatively. FTIR spectroscopy is nonde- occurs during the vibration. Consequently,
structive and allows in situ and remote measure- vibrations that produce a large change in dipole
ments of almost any sample, irrespective of the (e.g., C=O stretch) result in a more intense absorp-
physical state and without elaborate sample tion than those that result in a relatively modest
preparation [3, 4], applied to identify the func- change in dipole (e.g., C=C). Vibrations that do
tional groups of chemical constituents but has not result in a change in dipole moment (e.g., a
been widely used and applied in recent years symmetrical alkyne C≡C bond stretch) have little
for the identification, quality control, and manu- or no absorption. The application of IR spectros-
facturing process supervision of pharmaceutical copy in herbal analysis is still very limited com-
drugs. pared to its applications in other areas (food and
Two units are used in vibrational spectroscopy: beverage industry, microbiology, pharmaceutical,
cm−1 (wave numbers) or nm. The choice of one of etc.), but infrared spectroscopy is certainly one of
the units depends either on the type of spectrom- the most important analytical techniques available
eter [dispersive vs. Fourier transform (FT)] or to to scientists in herbal sciences [8]. The utility of
avoid too large numbers in the NIR range where FTIR fingerprints may be highlighted as follows.
124 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Table 7.1 Important regions of the IR spectrum


Wave number (cm−1) Functional group Characteristic features of the peak
3,600–2,700 cm−1 (X–H stretch region)
3,600–3,300 Alcohol O–H The alcohol OH stretch is usually a broad and strong absorption
Amine or Amide N–H near 3,400. The NH stretch is typically not as broad or strong as
Alkyne C–H the OH, and in the case of an NH2, it may appear as two peaks.
The terminal alkyne C–H may be confirmed by a weak C≡C bond
stretch near 2,150 cm−1
3,300–2,500 Acid O–H This is normally a very broad signal centered near 3,000 cm−1
3,200–3,000 Aromatic (sp2) =C–H The aromatic CH character usually appears as a number of weak
Alkene (sp2) =C–H absorptions, while the alkene C–H is one or a couple stronger
absorptions
3,000–2,800 Alkyl (sp3) C–H Almost all organic compounds have alkyl CH, so this is not
usually too informative, but the intensity of these peaks relative to
other peaks gives indication as to the size of the alkyl group
2,850 and 2,750 Aldehyde C–H Two medium-intensity peaks on the right-hand shoulder of the
alkyl C–H. The response for the presence of carbonyl C=O peak
in spectrum to confirm
2,300–2,100 cm−1 (C≡X stretched region)
2,260–2,210 Nitrile C≡N A sharp, medium intensity peak. Carbon dioxide in the atmo-
sphere may also result in an absorption in this area if not
subtracted/pursed-out
2,260–2,100 Alkyne C≡C The peak intensity varies from medium to nothing; since the
intensity is related to the change in dipole moment, symmetrical
alkynes will show little or no absorption here
1,850–1,500 cm−1 (C=X stretch region)
1,850–1,750 Anhydride C=O Anhydrides have two absorptions, one near 1,830–1,800 and one
3–4 member ring C=O near 1,775–1,740. The absorption frequency increases as the ring
size decreases, e.g., cyclohexanone = 1,715, cyclopen-
tanone = 1,745, cyclobutanone = 1,780, cyclopropanone = 1,850
1,750–1,700 Aldehyde C=O It is usually the most intense absorption in the entire spectrum
Ketone C=O
Ester C=O
Acid C=O
1,700–1,640 Amide C=O Due to resonance, amides, and conjugated carbonyl, there comes
Conjugated C=O slightly lower response than normal C=O. In general, conjugation
lowers the absorption by 20–50 cm−1
1,680–1,620 Alkene C=C Absorption is not as intense as for C=O. It is variable and may be
fairly small in symmetrical or nearly symmetrical cases. For
confirming alkene response of C–H peaks above 3,000 cm−1
1,600–1,400 Aromatic C=C Multiple sharp, medium peaks appear. The pattern of peaks varies
depending upon the substitution pattern. Usually there is one peak
around 1,600 cm−1 and several others at lower wave numbers
1,500–400 cm−1(fingerprint region)
1,300–1,000 C–O A strong peak
1,500–400 Various functional Interpretation of peaks in the fingerprint region is complicated
groups due to the large number of different vibrations that occur here.
These include single bond stretches and a wide variety of bending
vibrations. This region gets its name since nearly all molecules
(even very similar ones) have a unique pattern of absorptions in
this region
Authentication of Herbal and Herbal Drugs 125

The quality of buchu oil obtained from two


Authentication of Herbal South African species, Agathosma betulina and
and Herbal Drugs Agathosma crenulata (family Rutaceae, both),
has been studied using FTIR spectroscopy.
During the previous two decades, there have been Samples of A. betulina and A. crenulata are col-
much more emphasis on the use of FTIR for lected from different natural localities and culti-
herbal authentication, as data from literature vation sites in South Africa. The essential oil was
search indicate that most of the papers are related extracted by hydro-distillation and scanned using
to qualitative assays of an active compound, but three spectroscopy techniques, such as near-
there are also many papers dedicated to quantita- infrared (NIR), mid-infrared (MIR), and Raman.
tive methods. Authors have priority to proposing A comparison of the three spectroscopy tech-
new methods for qualitative and quantitative niques indicates that MIR together with PLS
determinations, but a few are also concern with (partial least squares) algorithms produces the
establishing new methods for analytical estima- best model for the quantification of six of the
tion of geographic origin. Examples may be cited seven major oil constituents [9].
as follows: FTIR has been used to simultaneously analyze
Radix Aconiti kusnezoffii has been analyzed the main chemical constituents in different sol-
from various processed products and from their vent extracts of several kinds of Chrysanthemum
ether extracts by using FTIR spectroscopy and samples of different regions [9]. The findings
2D-IR [9]. There are distinctive differences in the indicate that different Chrysanthemum samples
absorption peaks in the range of 1,800–1,500 cm−1 have dissimilar fingerprint characters in FTIR
in the second derivative spectra of different pro- spectra. These spectral fingerprints provide struc-
cessed products. The authors used the second- tural information for complicated test samples.
derivative spectra because of their better resolution. Liu et al. have reported that they are able to iden-
With the use of high resolution, high speed, and tify the main components of different extracts
convenience FTIR, quickly and precisely distin- and distinguish the origins of the Chrysanthemum
guish various processed products such as Radix samples from different regions easily [10].
A. kusnezoffii can be applied to predict the ten- Multistep infrared spectroscopic methods, including
dency of transformation of the complicated conventional FTIR, second-derivative spectros-
chemical mixture systems under heat perturbation. copy, and 2D-IR correlation spectroscopy, have
The root of Angelica sinensis (olive) is well been proved to be effective methods to examine
known in traditional Chinese medicine for common complicated mixture system such as the inves-
use. Modern pharmacological research indicates tigation on the effect of flowering on the phar-
that it could be used to treat anemia, apoplexy, maceutical components of Cistanche tubulosa.
hypertension, coronary heart disease, thromboangii- The results provide a scientific explanation for
tis obliterans, superficial thrombosed phlebitis, etc. the traditional experience that flowering con-
FTIR associated with second-derivative infrared sumes the pharmaceutical components in stem
spectroscopy and 2D-IR has been used to study the and the seeds absorb some nutrients of stem after
main constituents in traditional Chinese formula- flowering. A new method using single-reflection
tion made from Angelica and its different extracts Fourier transform infrared spectroscopy was
(extracted by petroleum ether, ethanol, and water in proposed for direct and fast determination of
turn). This method to use macroscopic fingerprint Corydalis yanhusuo W. T. Wang and of tradi-
characters of FTIR and 2D-IR spectrum to identify tional Chinese herbal medicines and its confus-
the main chemical constituents of plant in their able varieties. FTIR spectroscopy and 2D-IR
different extracts and compare the components have been employed to propose a new method for
for differences among the similar samples is analysis of Cordyceps. Their second-derivative
highly rapid, effective, visual, and accurate for spectra can amplify the differences and confirm
pharmaceutical research [9]. the potential characteristic IR absorption bands.
126 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

The different fingerprints display different chem- is higher than that of other oils. On the other
ical constitutes. Chestnut (Castanea sativa) shell hand, the ash of fenugreek is very rich in phos-
and eucalyptus (Eucalyptus globulus) bark, waste phate compounds. The spectra showed some
products of the food and wood industries, respec- absorption bands that are due to phosphate
tively, have been analyzed as potential sources of compounds. It could be concluded that the inor-
antioxidant compounds. The antioxidant activity ganic part of fenugreek consists mainly of
and the total phenols content of the extracts had phosphate compounds [9].
positive linear correlations. FTIR spectroscopy Ginseng, an expensive herb of high therapeu-
confirmed the higher content of phenolic com- tic value, is mostly adulterated with other cheaper
pounds in chestnut shell extracts compared to products, so quality assurance is required as its
eucalyptus bark extracts [9]. commercial products such as capsules, powder,
The determination of sweet cherry anthocya- soft gels, and teas are available globally with high
nins in crude material of three varieties using dif- demand. Yap et al. have discussed a rapid means
fuse reflectance infrared Fourier transform of distinguishing American and Asian ginsengs
spectroscopy (DRIFTS) and the curve-fitting from two morphological fakes, namely, sawdust
deconvolution method has been described. A lin- and Platycodon grandiflorum, via pattern differ-
ear relationship between the sweet anthocyanins ences and principal component analysis of their
content and the peak area at 1,640–1,630 cm−1 infrared spectra [8]. The results showed that
was established with a high correlation coefficient ginseng can be distinguished from both sawdust
(0.990). The deconvolution analysis using the and Platycodon grandiflorum; hence, there is a
curve-fitting method allowed the elimination of potential for the use of infrared spectroscopy as a
spectral interferences from other cell wall com- novel analytical technique in the authentication
ponents. The proposed method is simple, rapid, of ginseng. A simple and rapid protocol based on
and nondestructive and could be applicable to infrared wavelengths and principal component
any cherry varieties [9]. analysis for identification and categorization of
A systematic study for the effect of the com- ginseng has been elaborated. The advantage of
mercially available, ultra-diluted drug from this protocol is that it is able to provide rapid
Digitalis purpurea (extract of foxglove leaves) identification of natural products because it
with aqueous ethanol has been conducted to mon- avoids tedious extraction or purification proce-
itor changes in its chemical structure/functional dures. The results showed that this protocol was
group arrangements using vibrational (FTIR and able to discriminate not only raw ginseng roots
Raman) spectroscopy. These changes suggest a but also different types of ginseng in three com-
significant effect of ultra-dilution (micro-vol- mercial ginseng products [9].
umes) in the spectrum profile of Digitalis bands in The misidentification of ginseng using tradi-
the fingerprint region. The technique is useful in tional methods of authentication via morphology
the detection and identification of compounds/ has been overcome by infrared spectroscopy.
chemical groups present at levels lower than Molecular spectroscopy (including near-infrared
micro-volume in drugs used in alternative/com- diffuse reflection spectroscopy, Raman spectros-
plementary medicine [9]. copy, and infrared spectroscopy) with OPUS/Ident
The composition of fenugreek seeds in the software (Thermo Scientific-Nicolet, Waltham,
form of powder, ash, and oil has been investigated MA) has been applied to clustering ginseng
through FTIR and Raman spectra measurements. according to species and processing methods.
The results indicate that fenugreek seeds (in pow- The results demonstrate that molecular spectro-
der form) are rich in proteins, but lipids and starch scopic analysis provides a rapid, nondestructive,
are in small amounts. The fenugreek oil FTIR and reliable method for identification of this
absorbance ratios A3009/A2924, A3009/A2854, and Chinese traditional herbal drug. The traditional
A3009/A1740 have been studied for iodine values, and methods, which are laborious and time consuming,
data reveals that the iodine value of fenugreek oil have been replaced by molecular spectroscopic
Authentication of Herbal and Herbal Drugs 127

Fig. 7.3 FTIR spectra of (a) Asian ginseng, (b) American ginseng, (c) notoginseng [9]

analysis, as more effective method. The herbal FTIR spectroscopy has been emphasized as a
materials of Asian ginseng (the root of Panax gin- widespread technique in the quick assess of food
seng), American ginseng (the root of Panax components. In one such work, procyanidins
quinquefolius), and notoginseng (the root of Panax have been extracted with methanol and acetone/
notoginseng) have been differentiated by conven- water from the seeds of white and red grape vari-
tional Fourier transform infrared spectroscopy eties. FTIR spectroscopy allowed the creation of
(1D-FTIR) (Fig. 7.3) and 2D correlation FTIR a partial least squares (PLS1) regression model
applying a thermal perturbation. These species with eight latent variables (LVs) for the estima-
of herbs were further identified based on the tion of the degree of polymerization (DPn), giv-
positions and intensities of relatively strong ing a root mean square error of cross-validation
auto-peaks and positive or negative cross-peaks (RMSECV) of 11.7%. The application of orthog-
in their 2D-FTIR spectra. The findings provide onal projection to latent structures (O-PLS1)
a rapid and new operational procedure for the clarifies the interpretation of the regression model
differentiation of these notable herbs. vectors. Moreover, the O-PLS procedure removed
Huanglongbing (HLB), also known as citrus 88% of non-correlated variations with the DPn
greening, has greatly affected citrus orchards in [9]. Kava has been used as a folk medicine and
Florida and has been studied for the detection well-known traditional beverage. A study focused
using FTIR. Leaf samples of healthy, nutrient- on quantitative analyses of kava lactones in kava
deficient, and HLB-infected trees were processed samples using FTIR data results is similar to
and analyzed using a rugged, portable mid- values with GC (R2 = 0.75–0.98), indicating that
infrared spectrometer. The spectral peak in the the FTIR method is suitable for determination
region of 952–1,112 cm−1 was found to be dis- of the contents of the six kava lactones in kava
tinctly different between the healthy and HLB- samples and thereby the chemotypes [9].
infected leaf samples. This carbohydrate peak Studies for relative abundance of active ingre-
could be attributed to the starch accumulation in dients in many deciduous perennial fruit crops
the HLB-infected citrus leaves. Thus, this study reveal requirement of winter chilling for adequate
demonstrated the applicability of mid-infrared bud break and flowering. Recent research has shown
spectroscopy for HLB detection in citrus [9]. that changes in sugar and amino acid profiles are
128 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

3354 1652 1515


1036 A
2929 1319
B
782
C
A

A
A
D
B

C 1692 1500 1455


1737 1659 1606
D 1516 1468

wave number cm−1 wave number cm−1

Fig. 7.4 Conventional IR spectra (panel I) and second (A) Nanxiang, (B) Paixiang, (C) Zhanxiang, and (D)
derivative IR spectra (panel II) of four patchouli samples Zhaoxiang
of different geographical origins at room temperature [9].

associated with the release of buds from dormancy. carried out. The methanolic extracts of Mongolian
Judd et al. applied FTIR spectrometry to provide herbal drug Rubus sachalinensis Leveille, its
an alternative mechanism for tracking metabolic substitute materials Sambucus williamsii Hance
changes in the meristems of kiwi fruit buds during and Uncaria rhynchophylla (Miq.) Jacks and its
winter dormancy [11]; results suggested that the adulterant Cinnamomum cassia Presl showed
application of multivariate analysis to FTIR spec- clear differences in the IR spectra. IR spectros-
tra has the potential to be a reliable and fast copy provided more information through the
method for detecting structural and compositional fingerprints region of herbal medicines, render-
changes in fruit crops. These wave numbers ing the technique direct and simple. Lakshimi
appear to be associated with carbohydrate, pectin, Prasuna et al. studied the FTIR spectra of Basella
and cellulose levels in the meristems. It is rubra and Moringa oleifera leaves and showed
expected that this FTIR signature can be used to evidence for the protein matrix bands and those
advance our understanding of the influence of the corresponding to carboxylic C–O bonds (Fig. 7.5)
various environmental and physiological factors [12]. Phyllagathis praetermissa collected from
on the breaking of bud dormancy and shoot out- Pasoh Forest Reserve (Negeri Sembilan), Ampang
growth, including the optimum timing and con- Forest Reserve (Selangor), and Bukit Lagong
centrations of applications of bud break regulators, (Selangor) in Peninsular Malaysia were differenti-
such as hydrogen cyanamide. FTIR for herbal ated based on their chemical constituents by using
analysis is used to decipher the geographical ori- multistep infrared macro-fingerprinting.
gin of the raw herb. In addition to the applications These data provided useful information for the
already mentioned related to Agathosma betulina best choice of soil, geographical location, and
and Agathosma crenulata and ginseng analysis, transplantation of herb culture. According to
there are other reports where FTIR was used these spectral fingerprint features, we cannot only
as discrimination technique for Epimedium identify the main components of different herbal
Korean Nakai from Jilin province, China, and plants and extracts but can also distinguish the
patchoulis (Pogostemon cablin Blanco) from origins of samples from different regions easily,
different Chinese provinces (Nanxiang, Paixiang, which is troublesome using existing analytical
Zhanxiang, and Zhaoxiang) (Fig. 7.4). methods. Compared with traditional methods,
Studies on the identification of four species which are laborious and time consuming, the
of Mongolian herbal medicine by ultraviolet molecular spectroscopic analysis is more effec-
(UV)/fluorescence/IR spectroscopy have been tive and efficient.
Authentication of Herbal and Herbal Drugs 129

Fig. 7.5 FTIR spectrum of Basella rubra leaf (at room temperature) [9, 12]

Interpretation of Herbal Drug FTIR Spectra as wet chemistry analysis is too laborious and
by Chemometrics: The data interpretation with time consuming.
the help of personal computers, generated by A case study was conducted by incorporating
modern analytical instruments, for the acquisi- appropriate chemometric methods [principal
tion and processing needs the education of chem- component analysis (PCA) and soft independent
istry, mathematics, and statistics with numerical modeling of class analogy (SIMCA)] as tools for
software. This computer-based chemical disci- extracting relevant chemical information from
pline that uses mathematical and statistical meth- the infrared spectra. The team of scientists under-
ods, (a) to design or select optimal measurement took this effort to introduce and to develop a rapid
procedures and experiments and (b) to provide quality verification method with the integration
maximum chemical information by analyzing of statistical and mathematical modeling for
chemical data, is known as chemometrics. extracting relevant information base on the infra-
The FTIR spectral fingerprint provides the red spectroscopy data and extending the used of
information about the source of pure ingredient FTIR transmission spectroscopy, selecting
either of natural or synthetic in origin. Due to the Orthosiphon stamineus Benth (Java tea), used for
inherent complexity of the IR spectrum, the treating infection of the urinary tract, kidney, and
actual interpretation may be difficult, and opera- bladder stones disease based on its geographical
tion requires much experience. Indeed, slight dif- origin and varieties. The dried leaves of O. sta-
ferences in the spectra within the same plant mineus from ten different geographical origins of
species may not be obvious and generally not vis- two varieties (white and purple flowers) were col-
ible to the naked eye. The quality of plant sam- lected from Malaysia and Indonesia and coded
ples from different localities and growing (Table 7.2) [13].
conditions based on their geographical origin The dried leave samples were milled until fine
may vary. Thus, the identification of origin of powder and were filtrated with sieves 0.071 and
crude herbs based on geographical origins is cru- 0.500 mm mesh size. KBr of spectroscopy grade
cial in order to ensure authenticity, quality, safety, was also filtrated with sieves 0.071 mm mesh
and efficacy of the raw material before it is con- size. 2-mg samples were mixed uniformly with
verted to the final product. Herbal product manu- 100 mg KBr (2% w/w) and homogenized by
facturers are always looking for such faster and using stir vortex CENCO 34 (Breda, Netherlands).
cost-effective verification method for traceability, The FTIR spectra were recorded in the mid-IR
130 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Table 7.2 List of O. stamineus samples as per its geographical origin and varieties [13]
S. no. Code Location State Group ID
1 BLKBPM Bumbung Lima Penang A
2 SRKBPM Kepala Batas Penang B
3 SZBKAM Bota Kanan Perak C
4 MABTRM Bohor Temak Perlis D
5 ZARWBM Rawang Selangor E
6 STJGCM Jengka Pahang F
7 NNPPDM Pasir Puteh Kelantan G
8 MSSMSM Semonggok Sarawak H
9 USKGSM Kuching Sarawak I
10 NHPJI Pulau Jawa Jakarta J
11 BPPM_P Balik Pulau Penang K
12 NNPPDM_P Pasir Puteh Kelantan L
13 SABAH_P -b Sabah M
14 USKGSM_P Kuching Sarawak N
a
Example BLKBPM: BL: distributor; KB: location; P: state; M: country (M: Malaysia;
I: Indonesia); [13] P: purple flowers (white flowers not indicated)
b
Information not available

region 4,000–400 cm−1 at resolution 4 cm−1 with spectra to the most intense band (hydroxyl group,
16 scans using Thermo Nicolet FTIR Nexus 3,406 cm−1) deletes the differences between spec-
spectrometer coupled with DTGS (deuterated tra due to different amounts of sample or path
triglycine sulfate) detector. The interferometer length variation [13].
and the detector chamber were purged with dry Chemometric data analysis was done using
nitrogen to remove spectral interference due to the PCA algorithms in the study for reducing the
atmospheric carbon dioxide and water vapor. Air high-dimensional spectroscopic data by con-
background spectrum was recorded before each structing a linear combination of the original
sample, and all experiment were performed in six variable into a few orthogonal principle compo-
triplicates (six pellets KBr with three scan each), nents which contain most of the variability of the
and baseline corrected for each spectrum; absor- data set. This projection method allows (a) visu-
bance was normalized so that peak absorbance of alization of the natural clustering in the data,
the most intense band is set to unity. The spectra (b) primary evaluation of the between-class
were transferred via a JCAMP.DX format into similarity, and finally (c) finding the reasons behind
the statistical software program. As per Beer’s the observed pattern by making correlation
law, the absorption of light at a given wavelength with the chemical or physicochemical properties
is due entirely to the absorptivity of the constitu- of the studied samples. SIMCA (soft independent
ents in the sample. Therefore, any variations in modeling of class analogy), a popular classification,
the spectral due to spectrometer and sampling method was used to assign unknown samples in
error should be eliminated prior to data analysis. order to test the robustness of this study. The five
So in this study, two chemometric preprocessing steps followed were as follows: (1) constructing
steps, namely, baseline correction and normaliza- separate PCA models for each class; (2) deter-
tion, were used. Baseline effects introduced by mining the optimal number of PCs by validation;
the spectrometer (e.g., detector drift, changing (3) fitting the unknown samples to each predefine
environmental condition such as temperature, model, provided that the class are distinct enough;
spectrometer purge, and sampling accessories) (4) deciding whether the samples belong to the
were removed by auto-correcting the spectrum corresponding class by referring to the object-to-
baseline, and normalization of the absorbance model distance and leverage (distance of the
Authentication of Herbal and Herbal Drugs 131

USKGSM_P
NHPJI

SZBKAM

MSSMSM
SABAH-P

ZARWBM

MABTRM
A

A
NHPJI
SRKBPM

NNPPDM NNPPDM-P

STJGCM

USKGSM
USKGSM
BLKBPM

4000.0 3000 2000 1500 1000 400.0 4000 3000 2000 1500 1000 400
wave number cm−1 wave number cm−1

Fig. 7.6 The characteristic FTIR spectra of O. stamineus samples [13]. Where white flowers (left), purple flowers
(right) from different origin

sample to the model center); and finally (5) vali- of hydroxyl (3,500–3,480 cm−1), ester carbonyl
dating the classification results with statistical (1,270–1,150 cm−1), and phenyl (1,600,
test called significance test [13]. 1,420 cm−1) [13].
By visual recognition, there is no significant
difference in the characteristic absorption bands,
Interpretation of Data but the intensity of certain wavelength does differ
from each other especially at the fingerprint
As per Malaysian Herbal Monograph, the main region (1,800–800 cm−1). This interprets the sim-
chemical constituents of O. stamineus are ilarity of certain main chemical component
flavonoids, caffeic acid derivatives, diterpene ester, observed among different O. stamineus sample
triterpene saponins, and other minor compounds. origin. The presentation of spectrum in 3-D
Each of these compounds plays an indispensable (Fig. 7.7) [13] further enhances the visualization
role in the complicated system of mixture con- of the variability in the intensity absorption bands
tributing to the efficacy and potency due to the between the respective origins. Differences
synergistic effect contributed from a number of between spectra are generally not visible to the
constituents present in the herb. In FTIR spectra naked eyes; it is more practical to incorporate sta-
(4,000–400 cm−1) of ten different sample’s origin tistical method for the aid of interpreting the
of (a) white flowers and (b) purple flowers variet- obtained measurement results from spectroscopic
ies (Fig. 7.6) [13], the sharp absorption peak at analysis. Since the discrimination of different
1,600–1,760 cm−1 is assigned to C=O stretching geographical origin of herb based on the slight
vibration in carbonyl compounds which may be differences among particular absorption bands is
characterized by the presence of high content of too subjective, the results may vary between ana-
terpenoids and flavonoids in the complex mixture lysts as reported [13].
of O. stamineus. The presence of a narrow and The PCA was carried out using 520 points
sharp peak at ~2,925 and ~2,853 cm−1 was assigned (normalized absorbance) between the ranges of
to C–H and C–H (methoxy compounds) stretch- the selected spectral region 1,800–800 cm−1.
ing vibration, respectively. The presence of diter- A total of 18 data sets from six triplicate measure-
penes was further proven with the absorption band ments of each sample, 12 data sets were randomly
132 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Fig. 7.7 3-D absorbance matrix spectra (1,800–800 cm−1) of different sample’s origin [13]

selected to represent the respective sample’s 90 % of the total variance in the data set. The first
origin and varieties (as shown in Table 7.1). PCs consist 48 % of the total variability followed
Natural grouping of O. stamineus from ten differ- by the second PCs with 34 % variance and only
ent geographical origins, the first three principal 8 % variance carried by the third PCs. Overall,
components (PCs) represent only 61 % of the each sample was able to form distinct cluster in
total variance (PC1 = 29 %, PC2 = 22 %, and the two-dimensional plot. Examining the space
PC3 = 10 %). This indicates that these three com- defined by the first and second PCs, (a) samples
ponents are not sufficient to provide effective from the same origin (state), for example, Kelantan
clustering of the sample’s origin with clear sepa- (group G and L) and Sarawak (group H, I and N),
ration between the groups. tends to groups itself together in the first PCs.
The rule of thumb to make a good classification Samples from Penang origin only observed this
is to make sure that the variation within different relationship when it is projected onto the third
sample must be greater than the variance between PCs, which contain only 8 % of the total data
individual samples. Thus, spectral reproducibil- variability. From this inherent structure, we can
ity is important for creating a robust classification make a sound assumption that the composition of
model. The systematic variation between repli- complex chemical constituent does vary accord-
cate spectra due to baseline effect is highlighted ing to its origin. But there are no significant dif-
and removed after derivatization. Generally, the ferences in the complex mixture observed
use of spectra derivatives with Savitzky–Golay between the different plant varieties as sample
algorithm as a chemometric preprocessing tech- with purple flower (rectangular shape) forms a
niques is widely reported in most classification close cluster with the white flower samples (oval
based on FTIR spectroscopy. The potential rela- shape). The first and second PCs which contain
tionship and pattern associate between different almost 60 % of the data variances separate the
O. stamineus samples source with regard to its four respective sample’s origin into four well-
complex mixtures were further investigated by defined spaces.
computing a PCA based on the first-derivative The overall result from aforesaid studies pro-
spectral data. The first three PCs represent almost vides evidence that O. stamineus samples from
Identification and Comparison of Biomolecules 133

different geographical origin and varieties have compared to other interactions, it is of paramount
varied complex chemical mixture. Sample ori- importance in biological systems to investigate
gin seems to have more dominant effect to the the drug metabolism, its biotransformation
chemical constituent of the plant compared to pathways, and toxicological, pharmacological,
plant varieties. The good classification model and biomedical interest. FTIR spectroscopy has
obtained from both PCA and SIMCA further been used for identification and comparison of
proved that classification of samples from vari- biomolecules in two different species of medicinal
ous sourcing and varieties is possible with the plants, for example, Atylosia albicans and Tephrosia
incorporation of chemometric techniques and tinctoria [7].
FTIR spectroscopy. Therefore, the obtained Plants Atylosia albicans and Tephrosia tincto-
classification model may be of great use for ria were collected from the western Ghats region
quality inspection of raw herbal material on a of Hassan District, Karnataka, India. The leaf,
continuous basis as new batches are produced. flower, fruit, and stem were carefully excised
Chemometrics analysis of spectra data is rapid from the plant, cleaned, shade dried, and placed
and simple since no chemical treatment of sam- in polythene bags (to avoid contamination, and
ples is required [13]. reference sample was kept in the herbarium), and
powdered. The plant powders were kept in a lyo-
philizer to remove water. The samples were again
Identification and Comparison ground in an agate mortar and pestle in order to
of Biomolecules obtain fine powder. Each powdered plant mate-
rial was mixed with completely dried potassium
During the decade of 1940, IR spectroscopy has bromide (99:1 ratio), and the mixture was sub-
become an accepted tool for the characterization jected to a pressure of 5 × 106 pa in an evacuated
of biomolecules, and with technological advance- die to produce a KBr pellet for use in an FTIR
ment, the FTIR has been proven to be useful in spectrometer. FTIR spectra were recorded with
studying compositional changes in plant cell an FTIR 460 plus Jasco. The powdered samples
walls. This property was utilized to determine of both Atylosia albicans and Tephrosia tinctoria
changes in cell wall, if any, due to biotic/abiotic were mixed with dried potassium bromide and
factors. In the beginning, use of IR spectroscopy prepared as pellets and scanned at room tempera-
was restricted only for structural elucidation of ture (25 ± 2 °C) at 4,000–400 cm−1 spectral range.
isolated compounds from the herbal matrices. To improve the signal to noise ratio for each spec-
Drug discovery from the plants continued to pro- trum, 100 interferograms with a spectral resolu-
vide new and important leads against various tion of ±4 cm−1 were averaged. Background
pharmacological targets including cancer, HIV/ spectra, which were collected under identical
AIDS, Alzheimer’s, malaria, infections, and pain. conditions, were subtracted from the sample
A number of events for new drug discovery and spectra. Each sample was scanned under the same
interaction of a drug with biological target and conditions with six different pellets. Special care
pharmacological effects have to be considered, was taken to prepare the pellets at the same thick-
and these involve absorption, distribution, metab- ness by taking the same amount of sample and
olism, and elimination. In order to assess the applying the same pressure. Therefore, in the
importance of each of these factors on drug present study, it was possible to directly relate the
action, both structural and physicochemical prop- intensities of the absorption bands to the concen-
erties of the drug are taken into account. In bio- tration of the corresponding functional groups.
logical systems, properties such as electrostatic The results of functional group analysis
bonds, hydrogen bonds, van der Waals bonds, (Table 7.3) using FTIR revealed the existence of
as well as effects related to electron transfer various characteristic functional groups in leaves,
and hydrophobic effects, are of major importance. stem, flower, and fruit of A. albicans and T. tinctoria
Although the hydrogen bond is fairly weak (Fig. 7.8, 7.9, 7.10, 7.11 and 7.12) [7].
134 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Table 7.3 General band assignments of the FTIR spectra of biological tissue [7]
S. no. Peak Assignment
1 521 cm−1 Torsion and ring torsion of phenyl
2 600–900 cm−1 CH out of plain bending vibrations
3 892 cm−1 C–C, C–O, deoxyribose
4 940 cm−1 Carotenoid
5 1,000–14 cm−1 Protein amide I absorption
6 1,000–200 cm−1 C–OH bond in oligosaccharides such as mannose and galactose
7 1,000–350 cm−1 Region of phosphate vibration, carbohydrate residues attached
to collagen and amide III vibration (in collagen)
8 1,020–50 cm−1 Glycogen
9 1,030 cm−1 Collagen
10 1,105 cm−1 Carbohydrates
11 1,145 cm−1 Phosphate and oligosaccharides
12 1,180–300 cm−1 Amide III band region
13 1,206 cm−1 Amide III collagen
14 1,244/5 cm−1 PO−2 asymmetric (phosphate –I)
15 1,255 cm−1 Amide III
16 1,312–131 cm−1 Amide III band components of protein collagen
17 1,456 cm−1 CH3 bending vibration (lipid and protein)
18 1,482 cm−1 Benzene
19 1,504 cm−1 In plane CH bending vibration from the phenyl rings
20 2,800–3,000 cm−1 C–H lipid region
21 3,500–600 cm−1 OH bond
22 3,000–700 cm−1 O–H stretching (water)

Fig. 7.8 FTIR spectra of


T. tinctoria leaves [7]

The dominant bands at 1,655 and 1,546 cm−1 represents asymmetric –CH3 bending modes
were attributed to protein amide І and ІІ bands. of end ethyl group proteins. The band at 1,402 cm−1
The shoulder at about 1,750 cm−1 was attributed represents C=O symmetric stretching of COO–
to lipid C=O stretching vibration. The band at and assigned to lipids. Band at 1,377 cm−1 rep-
1,465 cm−1 was assigned to the =CH2 bending resents C–H bending mode of =CH 2. The
mode of the cell lipids. The band at 1,460 cm−1 very strong absorption band observed around
Identification and Comparison of Biomolecules 135

Fig. 7.9 FTIR spectra of


A. albicans leaves [7]

Fig. 7.10 FTIR spectra of


T. tinctoria flower [7]

Fig. 7.11 FTIR spectra of


A. albicans fruit [7]
136 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Fig. 7.12 FTIR spectra of A. albicans stem [7]

3,373–3,422 cm−1 may be due to the presence of in proteins. The weak absorption band observed
bonded N–H/C–H/O–H stretching of amines and between 1,421 and 1,415 cm−1 in the plant parts
amides. A very strong absorption at 3,400 cm−1 of A. albicans and T. tinctoria may be due to the
shows the presence of amino acids, and the very presence of bonded C–O/O–H bending. The
strong absorption band appearing in the region medium absorption band of 620 cm−1 indicates
2,933–2,922 cm−1 is due to N–H stretching. The the presence of sulfate. A strong absorption
lone C=O stretching vibration band correspond- band occurs at 597 and 580 cm−1 in the stem,
ing to saturated aliphatic ester 1,743 cm−1 is leaves, fruits, and flower of two species which
present in all parts of the plants. The bands at is possibly due to aliphatic C–Cl absorption and
900–1,350 cm−1, 1,020 cm−1, 1,024 cm−1, and brominated compounds. The brominate com-
1,050–100 cm−1 are attributed to phosphordiester pounds show an infrared band region at 600–
stretching bands region (for absorbance due to 500 cm−1. The weak absorption band at 539 cm−1
collagen and glycogen), DNA, glycogen (C–O indicates the presence of phosphates in the
stretch associated with glycogen, phosphate, and leaves, fruits, flowers, and stem in the examined
oligosaccharides PO−2 stretching modes), P–O–C plants. The very weak band occurring at
antisymmetric stretching mode of phosphate 780 cm−1 in the flowers, leaves, stem, and fruits
ester, and C–OH stretching of oligosaccharides, of these plants can be attributed to out-of-plane
respectively. A band at 1,051 cm−1 is attributed N–H wagging, primary and secondary amide
to C–O–C stretching of DNA and RNA. The and nitrite group. Five major peaks 1,590,
more intense bands occurring at 3,419, 2,927, 1,348, 1,051, 3,385, 1,063, and 456 cm−1 were
2,853, 1,633,1,421, 1,260, 1,073, 816, and 635cm−1 observed in the FTIR spectra. This is a
corresponding to O–H/N–H, C–H, C–O, and significant observation made in A. albicans as it
C–Cl/C–S stretching/bending vibrations, respec- is not reported in any legumes so far. A weak
tively, indicate the presence of amino acids, absorption band at 940 cm−1 is attributed to car-
alkenes, nitrates, ethers, organic halogen com- otenoid being present only in the fruit of A.
pounds, and carbohydrates in A. albicans and albicans. A weak absorption band at 965 cm−1
T. tinctoria [7]. is attributed to C–O stretching of the phospho-
A symmetrical stretching of NO2− group diester and the ribose and is present only in A.
results into strong absorption in the region albicans. A very weak peak at 892 cm−1 is
1,660–1,625 cm−1. The observed absorption attributed to C–C and C–O deoxyribose and
band at 1,630 cm−1 indicates the presence of seen only in A. albicans. A medium peak at
amines (protein). This gives the evidence that 1,340 cm−1 is due to CH2 wagging collagen
the plants A. albicans and T. tinctoria are rich present in A. albicans. A very strong peak at
Authenticity of Herbal and Herbal Drugs 137

1,581, 1,358, and 520 cm−1 is attributed to ring distinguish different herbal drugs effectively.
C–C stretch of phenyl, stretching C–O, defor- In method two, powder of herbal drug is blended
mation C–H, deformation N–H, and phenyl with KBr powder and pressured into a pellet;
group respectively, are present in fruits, leaves, then, the IR spectra of the samples are collected,
and stem of A. albicans and leaves and flowers while in method three, the reflectance IR spectra
of T. tinctoria. A medium peak at 3,300 and are obtained directly from herbal materials.
1,020–1,050 cm−1 is attributed to amide І bands Although methods two and three are convenient
stemming from N–H stretching modes in pro- methods, they provide lower resolution ability in
teins, nucleic acids, and glycogen [7]. identification of herbal drugs as compared to
From above investigations, it was concluded method one [14].
that A. albicans and T. tinctoria are rich in pheno- The IR fingerprint spectra are an effective
lic compounds and also show the presence of and convenient for the primary determination
oligosaccharides, phosphates, proteins, carbohy- of true and false identification of herbals and
drates, and carotenoid. This work also indicates herbal drugs. Under the same experimental
the presence of biomolecule concentration which conditions, the IR fingerprints of different
is different in different parts of the plants. This herbal drugs have differences in their specific
work offers scope for further research in phy- bends. Herbal drugs can be identified clearly by
tochemical analysis and biological activity of specific bends and their relative intensities.
medicinal plants. Fourier transform infrared Since early 1987, the fingerprint spectra are in
spectroscopy is proved to be a reliable and sensi- use for the identification of herbal drugs, by the
tive method for detection of biomolecular com- extraction of components from the herb with
position of cells. From this study, we examined certain solvents of different polarities on the
the potential of FTIR spectroscopy for easy and order of petroleum ether (or cyclohexane),
rapid discrimination and identification of various chloroform, ethanol, and water, and their UV
functional groups responsible for medicinal prop- fingerprint spectra are also measured [15].
erties. Spectral area ranged between 4,000 and Chinese herbal drugs, which are complex mix-
400 cm−1 is important for an easy and reliable dis- tures of many herbals, can be easily identified
crimination between different plant species based for purity using their IR spectra. The specific
on biomolecules due to unique fingerprint for a bends and the relative intensities of the IR spec-
particular moiety. tra are the main criteria for authenticity.
In a study by Zou et al., the IR spectra of nine
kinds of roots of Glycyrrhiza uralensis Fisch and
Authenticity of Herbal and Herbal Glycyrrhiza inflata Batal, including planted and
Drugs wild samples from different regions, looked very
similar (Fig. 7.13) [16]. The IR spectra of their
The true and false identification of herbal drugs water extract showed significant differences.
can be performed by the characteristic bend of The common peak ratios of the different species
their IR fingerprints using the common and were minimum, while their variant peak ratios were
variant peak ratio sequent analytical method to maximum. The common peak ratio of the same
evaluate the quality of the same medicinal plant species of the wild and the planted from near
species. For this purpose, three methods are in producing regions was of slight difference, but
practice for collecting IR fingerprints for quality their variant peak ratios were obviously larger
control of herbal drugs. In method one, solvents than that of planted samples. The common and
of different polarities are used to extract compo- variant peak ratios of the same species samples
nents. After evaporating the solvent, the compo- from the same region had nonsignificant difference.
nents are blended with KBr powder and pressured On further investigation, they find IR spectra
into a pellet, and then the IR fingerprints are gen- having great differences once extracted with
erated. The IR spectrum of samples enables us to chloroform and with water [17].
138 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Fig. 7.13 The IR spectra of roots of planted G. uralensis Fisch, wild G. uralensis Fisch, and wild G. inflata Batal [16]

To Distinguish Herb from Its C.A. Meyer (Japanese ginseng), Panax major
Morphological Fakes Ting, Panax notoginseng (Burk.) F.H. Chen
(Sanqi or Tianqi ginseng), Panax omeiensis
The quality control of herbals and herbal prod- J. Wen, Panax pseudoginseng Wallich (Himalayan
ucts is important for consumer’s safety and ginseng), Panax sinensis J. Wen, Panax stipule-
efficacy, as traditional herbal drugs are not regu- anatus H.T. Tsai and K.M. Feng, Panax trifolius
lated, as considered dietary supplements and not L. (dwarf ginseng), Panax vietnamensis Ha et
drugs. At present, these commercial products Grushv (Vietnamese ginseng), Panax wangianus
are available in various formulations such as Sun, and Panax zingiberensis C.Y. Wu and K.M.
capsules, powder, softgels, and tea. The tradi- Feng [8].
tional means of authentication via smell, taste, Asian ginseng is not a generic term and is used
or physical appearance are hardly reliable for to refer to ginsengs which originate from Asian
these high-value products, and adulteration with countries. Ginseng with other species such as
other cheaper products may occur. Example Panax japonicus and Panax notoginseng, Panax
may be cited from ginseng, the famous Chinese ginseng, is also classified under Asian ginseng.
herb, as it exhibits an adaptogenic effect and The chemical composition of ginseng includes
improves physical and mental performance. the saponins, naphtha class, carbohydrates, and
Different parts and species of ginseng are starch. Its pharmacological activity is due to the
believed to have different medicinal properties. mixture of its constituents and not the presence of
There are many species of ginseng, of which the a single compound. The triterpene saponins,
two most common and widely used are Panax called ginsenosides, are the major active ingredi-
ginseng C.A. Meyer (Asian ginseng) and Panax ents in ginseng, and there are more than 30 differ-
quinquefolius L. (American ginseng). Panax ent ginsenosides. These triterpene glycosides
ginseng was previously known as “Panax schin- are characterized by a four trans-ring steroid
seng Nees” and is cultivated in China, Germany, aglycone skeletons with attached sugar moieties,
Japan, Korea, and Russia, while Panax quinque- and they can be grouped into the 20-(S)-
folius, previously known as Aralia canadensis, protopanaxadiols and 20-(S)-protopanaxatriols,
is found in the United States of America and except for the oleanolic acid-derived ginsenoside
Canada. Besides these two major varieties, there Ro. The major ginsenosides Rb1, Rb2, Rc, Rd,
are 11 other ginseng species: Panax japonicus Re, and Rg1 are used as markers of ginseng
To Distinguish Herb from Its Morphological Fakes 139

quality because they account for 90 % of the total expressed in wave numbers (cm−1), are used as a
ginsenosides [8]. fingerprint for compound identification [8].
American and Asian ginsengs are distin- In a case study, American ginseng, Asian gin-
guished by their chemical profiles. The glucogin- seng, and Platycodon grandiflorum (jiegeng) root
senoside Rf is detectable in Asian ginseng but not samples were cut into small pieces and ground
in American ginseng, and the ocotillol-type trit- into fine powder. Each sample was mixed uni-
erpene 24-(R)-pseudoginsenoside F11 is present formly with spectroscopic grade potassium bro-
in American ginseng but absent in Asian ginseng. mide (KBr) powder (1 % w/w) in an agate pestle
The relative abundances of ginsenosides can and mortar and then pressed into a pellet. The
also be used to distinguish between the Asian spectra were recorded in the region of 4,000–
and American species, as a higher amount of 400 cm−1 on a Shimadzu IRPrestige-21 FTIR
protopanaxadiol ginsenosides exists in American spectrometer (Shimadzu Corporation Pte. Ltd.,
ginseng, in contrast to a higher amount of pro- Asia Pacific) equipped with a KBr beam splitter
topanaxatriol ginsenosides in Asian ginseng. In and a deuterated L-alanine triglycine sulfate
addition, ginsenoside ratios are also indicative of (DLATGS) detector. Each spectrum was an aver-
the types of ginseng. A higher Rb1/Rg1 value age of 40 scans co-added at 4 cm−1 resolution,
usually indicates P. quinquefolius, and a high Rf/ and pure KBr background spectra were recorded
F11 ratio of more than 700:1 distinguishes the before analysis of the samples. Base line correc-
Asian from the American varieties [8]. tion was done for each spectrum, and their absor-
Herbs like ginseng have traditionally been bance normalized with the Shimadzu
authenticated by morphological and histological IRsolution 1.10® software program, prior to
means. In Asia, ginseng quality is based on the data analysis, so that the peak absorbance of the
age, origin, as well as the physical characteristics most intense band was set to unity. The cor-
of the root. The potency of the ginseng roots is rected spectra were then analyzed for pattern
determined by its shape and can be classified into similarity and functional group identification
three kinds based on the numbers and sizes of the (Fig. 7.14a). With complex mathematical pro-
lateral branches on the main root: “pencil” roots, cessing techniques, tiny differences in the IR
“chunky” roots, and “complex” roots. These spectrum were amplified via their derivative
methods are hardly reliable at present hi-tech and spectra, for the separation of overlapping bands
commercial scenario, as many commercial prod- and determination of exact peak locations
ucts are in the form of various formulations. (Fig. 7.14b). The spectra were converted to their
Additional commercial aspect is that ginseng is corresponding second derivatives with the same
expensive, and cultivation of ginseng is slow and Shimadzu IR solution 1.10® software program
difficult, and it takes more than 4 years before it (Creon Lab Control AG, Shimadzu Corporation
can be harvested. Under these circumstances, the Pte. Ltd., Asia Pacific) using the 23-point
possibility of adulterating it with other cheaper Savitzky and Golay algorithm. Principal com-
products is more expected [8]. ponent analysis (PCA) is a method of chemo-
FTIR technique is based on the fact that when metric analysis which is widely used for
low-energy transitions occur in molecules of the reorganizing information and explaining causes
sample material, sample absorbs the IR radiation, of variance in a set of data, in this case IR spec-
resulting as in absorbance spectra represent the tra (Figs. 7.15 and 7.16) [8].
quality of the major structural features/basic In aforesaid studies, it is concluded that FTIR
functional groups of the molecules, present in the spectroscopy is a novel and interesting innova-
sample. No two chemical structures can have iden- tion for detecting adulteration in very short time
tical IR spectra, as each type of bond in different before the utilization of raw herb for commercial
compounds has different vibrational frequencies. production to distinguish between morphological
The IR spectra, which are usually plot of percent and chemical fakes, specifically cases similar to
transmittance or absorbance versus frequency ginseng.
140 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Fig. 7.14 Comparison of (a), general; (b), second-derivative MIR spectra of sawdust with American and Asian gin-
sengs [8]

Fig. 7.15 Comparison of the (a) general MIR spectra, (b) magnified fingerprint region (2,000–600 cm−1), and (c)
second-derivative MIR spectra of American and Asian ginsengs with jiegeng [8]
Standardization of Metal-Based Herbal Drugs 141

Fig. 7.15 (continued)

Standardization of Metal-Based incineration. “Chendurams” are prepared by the


Herbal Drugs process of sublimation, and they are much more
potent. In “Siddha,” there is a faith for prolonga-
Metal-based herbal drugs have its own repute tion of life through rejuvenation using mercurial
since ancient as gold and silver has been drugs. In Indian market, Siddha drugs like
described beneficial in healing and anti-disease Poorna Chandrodaya Chenduram (PCC), Kshya
drugs. In olden days, people used silver bottles kulanthanga Chenduram (KsKc), Velli Parpam
for storing water, wine, and milk to prevent from (SP), Naga Chenduram (NC), Naga Parpam
spoiling. In “Siddha” medicine, a form of south (NP), and three different popular brands of Linga
Indian medicine, apart from gold and silver, Chenduram (LC1, LC2, and LC3) are prepared
mercury, sulfur, mica, arsenic, zinc, and several from purified metal, triturated with decoction of
other minerals are treated with indigenous herbs, herbal juices, generally prescribed in the dose of
and given as “Bhasma” and “Chendurams.” 100–200 mg/day, and recommended to be taken
“Bhasma” means a fine ash obtained through with suitable adjuvant [18].
142 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

Fig. 7.16 2D plots of jiegeng, sawdust, and American spectra of jiegeng and ginsengs, PC1 = 51% versus
and Asian ginsengs. (a) General spectra of sawdust and PC2 = 29%; (e) general spectra of jiegeng, sawdust, and
ginsengs, PC1 = 82 % versus PC2 = 10 %; (b) second- ginsengs, PC1 = 80 % versus PC2 = 7 %; and (f) second-
derivative spectra of sawdust and ginsengs, PC1 = 74 % derivative spectra of jiegeng, sawdust, and ginsengs,
versus PC2 = 11 %; (c) general spectra of jiegeng and gin- PC1 = 57 % versus PC2 = 17 % [8]
sengs, PC1 = 76 % versus PC2 = 14 %; (d) second-derivative
Standardization of Metal-Based Herbal Drugs 143

Fig. 7.16 (continued)


144 7 FTIR Spectroscopy: Herbal Drugs and Fingerprints

LC3 SP

LC2

KsKc
LC1

592
PCC

4000 3500 3000 2500 2000 1500 1000 500


4000 3500 3000 2500 2000 1500 1000 500
Fig. 7.17 FTIR of LC (LC1, LC2, LC3)
Fig. 7.19 FTIR of PCC, KsKc, SP (Au- and Ag-based
formulation)

The spectra clearly indicate that drug sample


NC
does not have any organic compound, strong
NP
evidence of being “Bhasma.” The three different
brands of LC showed a similar pattern (Fig. 7.16).
Spectrum of PCC is similar to LC. The peak at
592 cm−1 in KsKc is due to Fe3O4, but the exact
form of silver in SP could not be concluded using
IR spectrum. LC is used to treat fevers, skin dis-
eases, and venereal diseases. PCC is used for
treating tuberculosis, jaundice, fever, and bron-
chitis. KsKc is given for all respiratory diseases
981
884 including tuberculosis. SP is used to treat cough,
tuberculosis, piles, leucorrhoea, gonorrhea and
4000 3500 3000 2500 2000 1500 1000 500 spermatorrhea. NC is used to treat piles, skin dis-
eases, and white discharge. Thus, the fingerprints
Fig. 7.18 FTIR of NC and NP (zinc-based formulation)
for purity of metal-based herbal drugs using FTIR
are quite helpful to decipher the quality [18].
In a study for quality and quantity determina- Herbal drugs play an important role in modern
tion for these formulations, about 0.1–0.2 g of the human life and have significant effects on treat-
drug was digested with nitric acid: perchloric ing diseases, but the quality and safety of these
acid (2:1) and allowed to cool and transferred herbal products have now become a serious issue
into beaker after complete digestion. The beaker due to increasing pollution in air, water, soil, and
was heated to remove acids; the resulting solu- modern life style. Using FTIR spectroscopy, the
tion was diluted to 50 ml, with deionized water technique for liquid and solid samples, along
(perchloric acid was used as it does not form with the statistical method of principal compo-
complexes with metals and metalloids). IR spec- nent analysis (PCA) to identify and discriminate
tra (4,000–450 cm−1) were recorded (Figs. 7.17, herbal drugs, and traditional formulation may
7.18, 7.19), using Perkin Elmer FTIR spectro- be scrutinized for quality purpose. Traditional
photometer in KBr pellets [18]. drugs are widely accepted in China, India, Japan,
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commercial opportunities, so there are more camo.com/downloads/resources/application_notes/
chances of intentional adulteration and contami- Assessment%20of%20Herbal%20Medicines%20
nation in herbal drugs. The FTIR spectra are by%20Chemometrics%20-%20Assisted%20
helpful in developing methods to test raw materi- Interpretation%20of%20FTIR%20Spectra.pdf. As on
28 Apr 2012.
als and finished products, identification and iso- 14. Zou H-B, Yang G-S, Qin Z-R, Jiang W-Q, Du A-Q,
lating active components, as provides clue for the Aboul--Enein HY. Progress in quality control of
functional groups of ingredients. herbal medicine with IR fingerprint spectra. Anal
Lett. 2005;38(9): 1457–75. Available at, http://dx.doi.
org/10.1081/AL-200062153. As on 15 Nov 2011.
15. Yuan JR, Li JL. The study on identification of Chinese
herbal medicine with ultraviolet spectra group. J
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London: Wiley; 2004. extracted from Radix Glycyrrhizae with water. Chin
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Publishing; 2007. sequence analytical method of common peak ratio and
3. Somsen GW, Visser T, Meyers RA. Liquid chroma- variant peak ratio for IR fingerprint spectra of the com-
tography/infrared spectroscopy. In: Encyclopedia of ponents extracted from Radix Glycyrrhizae with chlo-
analytical chemistry. Chichester: Wiley; 2000. roform. China J Chin Med Mater. 2005;30(1):16–20.
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chemistry. Chichester: Wiley; 2000. 2009;5(3):200–6.
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parison of bio-molecules in medicinal plants of solids. New York: Taylor & Francis; 2006. p. 235–65.
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FTIR. Rom J Biophys. 2011;21(1):63–71. photometric analysis of co-enzyme Q10 (CoQ10) and
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Mass Spectroscopy: Herbal Drugs
and Fingerprints 8

In order to measure the characteristics of indi- molecular ion retains the excess ionization
vidual molecules, in herbals and herbal drugs, a energy. If this excess energy is greater than the
mass spectrometer (MS) is used to convert them energy required to break a chemical bond, the
into ions so that they can be moved and manipu- molecule can fragment. The fragmentation pro-
lated by external electric and magnetic fields for cesses are typically categorized as direct cleav-
individual characterization and define them at the age or rearrangement. Cleavage reactions are
level of molecular structure. The mass spectrum simply the breaking of a chemical bond to pro-
is a two-dimensional representation where x-axis duce two fragments. Rearrangements are more
represents the mass number and y-axis represents complex reactions that involve both making and
the relative intensity (relative abundance) of the breaking bonds. These rearrangement fragments
ions produced. The mass spectrum is always often provide important clues about the location
recorded over a range of mass number, and the and identity of some functional groups. Functional
mass range is ideally more than the expected group can have a significant effect on the frag-
molecular weight of the compound. Once a mass mentation patterns observed in mass spectros-
spectrum is recorded, one has to look at the spec- copy. The McLafferty rearrangement is a classic
trum in totality. The first step toward interpreting example of a rearrangement reaction. The
a mass spectrum is to look for the molecular ion, McLafferty rearrangement is often observed for
which provides the molecular mass of the ana- carbonyl compounds that contain a liner alkyl
lyte. In many mass spectra, the molecular ion is chain.
easily identified as the ion with the highest mass The MS is a four-component system and
number (a careful assessment is necessary as the (Fig. 8.1) may be summarized for essential func-
highest mass number ion may be due to an isoto- tions of it as (1) ionization chamber to generate
pic peak or an impurity). Many times under elec- ions, usually to cations by loss of an electron;
trospray ionization mode, molecule undergoes (2) mass analyzer to separate the different ions
near total fragmentation, and hence, no molecu- according to their mass and charge ratio (m/z)
lar ion is visible. It is very important to note that basis; (3) detector to detect and characterize the
the ion with the maximum abundance is the base separated ions; and (4) recorder to record the data
peak and is not necessarily the molecular ion. of detector and displayed as spectrum. Due to high
Although the molecular ion gives information reactivity and short life of ions, their formation
about the molecular mass of the compound, it and manipulation are conducted in reduced
does not provide much information about the pressure. The pressure under which ions may be
molecular structure. The structural information is handled is around 10−5 to 10−8 Torr (mmHg)
obtained from the fragmentation pattern of the (i.e., less than a billionth of an atmosphere). Each
mass spectrum. After a molecule is ionized, the of the aforesaid four tasks may be accomplished

D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs, 147
DOI 10.1007/978-81-322-0804-4_8, © Springer India 2012
148 8 Mass Spectroscopy: Herbal Drugs and Fingerprints

Mass
Ionization Detector
Analyzer Recorder
Chamber (m/z)

Fig. 8.1 Graphic presentation of mass spectrometer

Fig. 8.2 Simplified schematic of MS

in different ways. In one common procedure, the heart of the spectrometer, where the molecules
ionization is effected by a high-energy beam of of sample are bombarded by electrons issuing
electrons, and ion separation is achieved by accel- from a heated filament (process is known as
erating and focusing as a beam, which is then bent electron impact). Gases and volatile liquid sam-
by an external magnetic field. The ions are then ples are allowed to leak into the ion source from
detected electronically, and the resulting informa- a reservoir. Cations formed by the electron bom-
tion is stored and analyzed in a computer and pre- bardment are pushed away by a charged repeller
sented in the form of peaks. plate and accelerated toward other electrodes,
During operation when a high-energy electron having slits through which the ions pass as a
collides with a molecule, it often ionizes by beam. Some of these ions fragment into smaller
knocking away one of the electrons (either bond- cations and neutral fragments. A perpendicular
ing or nonbonding), producing a molecular ion. magnetic field deflects the ion beam in an arc
The residual energy from the collision may cause whose radius is inversely proportional to the
the molecular ion to fragment into neutral pieces mass of each ion. Lighter ions are deflected more
and smaller fragment ions. The molecular ion is a than heavier ions. By varying the strength of the
radical cation, but the fragment ions may either be magnetic field, ions of different mass are focused
radical cations or carbocations, depending on the progressively on a detector fixed at the end of a
nature of the neutral fragment. The ion source is curved tube (Fig. 8.2).
Detection and Recording of Fingerprints 149

provides a clue to the molecular structure, but if


Interpretation of Mass Spectra the molecular ion has a lifetime of less than a
few microseconds, it will not survive long enough
A mass spectrum is usually a vertical bar graph, to be observed, and without a molecular ion peak
in which each bar represents an ion having a as a reference, it is difficult to decipher a mass
specific mass-to-charge ratio (m/z) and the length spectrum. Fortunately, most organic compounds
of the bar indicates the relative abundance of give mass spectra that include a molecular ion.
the ion. The most intense ion is assigned an The most stable molecular ions are those from
abundance of 100, and it is referred to as the aromatic rings, other conjugated pi-electron
base peak. Most of the ions formed in a mass systems, and cycloalkanes. Alcohols, ethers, and
spectrometer have a single charge, so the m/z highly branched alkanes generally show the
value is equivalent to mass itself. Modern mass greatest tendency toward fragmentation [1].
spectrometers easily resolve ions differing by
only a single atomic mass unit (amu), and thus
provide completely accurate values for the Techniques for Analysis of Ions in MS
molecular mass of a compound. The highest mass
ion in a spectrum is normally considered to be The main function of the mass analyzer is to
the molecular ion, and lower mass ions are separate (resolve) the ions formed at ionization
fragments from the molecular ion, assuming the source of the mass spectrometer according to
sample is a single pure compound. their m/z ratios. There are a number of mass
Most stable organic compounds have an even analyzers but frequently in use are quadrupoles,
number of total electrons, reflecting the fact that time-of-flight (TOF) analyzers, magnetic sectors,
electrons occupy atomic and molecular orbitals in and both Fourier transform and quadrupole ion
pairs. When a single electron is removed from a traps. These mass analyzers have different features,
molecule to give an ion, the total electron count including the m/z range that can be covered for
becomes an odd number, and we refer to such ions the mass accuracy and achievable resolution.
as radical cations. The molecular ion in a mass The compatibility of different analyzers with
spectrum is always a radical cation, but the frag- different ionization methods varies as tandem
ment ions may either be even-electron cations or mass spectrometers (MS–MS) are instruments
odd-electron radical cations, depending on the that have more than one analyzer and so can be
neutral fragment lost. The simplest and most used for structural and sequencing studies. In
common fragmentations are bond cleavages pro- tandem mass spectrometers, two, three, and four
ducing a neutral radical (odd number of electrons) analyzers have all been incorporated into avail-
and a cation having an even number of electrons. able instruments, and the analyzers do not neces-
A less common fragmentation, in which an even- sarily have to be of the same type, in which case
electron neutral fragment is lost, produces an odd- the instrument is a hybrid one. More popular
electron radical cation fragment ion. Fragment tandem mass spectrometers include those of the
ions themselves may fragment further. As a rule, quadrupole–quadrupole, magnetic-sector qua-
odd-electron ions may fragment either to odd- or drupole, and, more recently, the quadrupole time-
even-electron ions, but even-electron ions frag- of-flight geometries [1].
ment only to other even-electron ions. The masses
of molecular and fragment ions also reflect the
electron count, depending on the number of hetero Detection and Recording
atoms in the species. The molecular ion peak is the of Fingerprints
signal of the parent ion corresponding to the
molecular weight of the compound. Allowing for The detector detects the ion current and amplifies
isotopes, it is the peak of highest mass in the it into signals; signals are then transmitted to the
spectrum. The nature of the fragments often data system where it is recorded in the form of
150 8 Mass Spectroscopy: Herbal Drugs and Fingerprints

mass spectra. The m/z values of the ions are there through a small aperture into the analyzer
plotted against their intensities to show the number of the mass spectrometer, which is held under
of components in the sample, the molecular mass high vacuum. The lens voltages are optimized
of each component, and the relative abundance of individually for each sample. In nanospray, ion-
the various components in the sample. The more ization technique is a low flow rate version of
common detectors are the photomultiplier, the electrospray ionization, where a small volume
electron multiplier, and the micro-channel plate (1–4 ml) of the sample dissolved in a suitable
detectors [1]. volatile solvent, at a concentration of 1–10 pmol/
Fast atom bombardment (FAB) and ther- microl, is transferred into a miniature sample
mospray techniques have restriction for highly vial. A reasonably high voltage (700–2,000 V) is
volatile and polar moieties, so electrospray applied to the specially manufactured gold-
ionization (ESI) and nanospray ionization tech- plated vial resulting in sample ionization and
niques are currently in practice. Matrix-assisted spraying. The flow rate of solute and solvent
laser desorption ionization (MALDI) deals well using this procedure is very low, 30–1,000 nl/
with thermolabile, nonvolatile organic com- min. This technique has advantage over the ESI
pounds especially those of high molecular mass that it needs less sample size as well as has scope
and are used successfully in biochemical areas for multi- analysis. A common application of
for the analysis of proteins, peptides, glycopro- this technique is for a protein digest mixture to
teins, oligosaccharides, and oligonucleotides. be analyzed to generate a list of molecular
Electrospray ionization is the technique used at masses for the components present, and then
an atmospheric pressure for ionization, so fre- each component to be analyzed further by tan-
quently known as “atmospheric pressure ioniza- dem mass spectrometric amino acid sequencing
tion” (API) techniques and is well suited to the techniques. ESI and nanospray ionization are
analysis of polar molecules ranging from less highly sensitive analytical techniques, but the
than 100 Da to more than 1,000,000 Da in sensitivity deteriorates with the presence of non-
molecular mass. During standard electrospray volatile buffers and other additives, which should
ionization, the sample is dissolved in a polar, be avoided as far as possible. With reference to
volatile solvent and pumped through a narrow, natural product chemistry, the term “electrospray
stainless steel capillary (75–150 mm i.d.) at a ionization” is replaced by “electrospray” as
flow rate of 1 ml/min. A high voltage of 3 or (except in a few cases) electrospray does not
4 kV is applied to the tip of the capillary, which create ions but transfers the analyte into gases
is situated within the ionization source of the phase from condensed phase [1].
mass spectrometer, and as a consequence of this
strong electric field, the sample emerging from
the tip is dispersed into an aerosol of highly Electrospray MS for Herbal Fingerprints
charged droplets, a process that is aided by a
coaxially introduced nebulizing gas flowing In this technique, test solution is passed through
around the outside of the capillary. This gas, usu- a capillary which is held at high potential. The
ally nitrogen, helps to direct the spray emerging effect of the high electric field as the solution
from the capillary tip toward the mass spectrom- emerges is to generate a mist of highly charged
eter. The charged droplets diminish in size by droplets which pass down a potential and pres-
solvent evaporation, assisted by a warm flow of sure gradient toward the analyzer portion of
nitrogen (acts as a drying gas) which passes the mass spectrometer. During that transition, the
across the front of the ionization source. droplets reduce in size by evaporation of the
Eventually charged sample ions, free from sol- solvent or by “Coulomb explosion” (droplet sub-
vent, are released from the droplets, some of division resulting from the high charge density).
which pass through a sampling cone or orifice Ultimately, fully de-solvated ions result from
into an intermediate vacuum region, and from complete evaporation of the solvent or by field
Detection and Recording of Fingerprints 151

Fig. 8.3 Essential features of the electrospray interface [1]

desorption from the charged droplets. Nebulization achieved, however, in comparison with quadrupole
of the solution emerging from the capillary may instruments. Installation of an electrospray source
be facilitated by a sheath flow of nebulizer gas, a on a time-of-flight instrument also presents some
technique for which the term “ion spray” was advantages.
originally coined by its developers. In practice, Electrospray technique is a convenient tech-
the facility to use a nebulizer gas is commonly nique may be studied into three stages as droplet
incorporated on commercial instruments; the formation, droplet shrinkage, and gaseous ion
need for its use or not is determined by the flow formation. In this process, the solution delivered
rate employed, the composition of the solvent, to the tip of the electrospray capillary experiences
and the sign of the potential applied to the capil- the electric field associated with the maintenance
lary tip (since a high negative potential, in par- of the tip at high potential. Due to a positive
ticular, may lead to a corona discharge unless potential, positive ions in solution accumulate
suppressed by the use of an appropriate sheath at the surface and get drawn in a downfield
gas). In addition, a flow of bath gas is usually direction to establish a “Taylor cone” (Fig. 8.4).
applied to the interface to promote droplet evapo- A high enough imposed field, the cone is drawn
ration; controlled heating of the interface pro- to a filament which produces positively charged
vides an alternative approach. Sampling of the droplets via a budding process when the surface
fully or partially de-solvated ions is made using tension is exceeded by the applied electrostatic
a capillary or a skimmer device (Fig. 8.3). force. The diameter of the droplets formed is
Numerous elaborations have been reported, but influenced by a number of parameters, including
majority of the electrospray literature involves the applied potential, the solution flow rate, and
implementation on quadrupole mass spectrome- solvent properties. Evaporation of solvent from
ters, but this position is rapidly changing with the the initially formed droplets, as they traverse a
increasing use of Paul ion traps and Fourier trans- pressure gradient toward the analyzer of the mass
form ion cyclotron resonance instruments to spectrometer, leads to a reduction in diameter,
enhance resolution. Implementation of electro- with collisional warming preventing freezing.
spray on magnetic sector instruments is compli- Fission (Coulomb explosion) will occur at the
cated and needs to avoid collisional activation point (the Rayleigh limit) at which the magnitude
during ion acceleration; enhanced resolution is of the charge is sufficient to overcome the surface
152 8 Mass Spectroscopy: Herbal Drugs and Fingerprints

Fig. 8.4 Droplet production in the electrospray interface [1]

tension holding the droplet together. Continuous full mass spectra at the nanogram level. The
and continual depletion of the droplet size (by mechanisms underlying the electrospray process
solvent evaporation and fission, respectively) and the manner of its implementation have
may be envisaged to result eventually in the significant implications for the properties of the
formation of droplets containing a single ion, gas-phase ions produced. The followings are
from which the non-solvated analogue may be notable points [1].
derived by further evaporation of solvent (aided (a) The charge states of the gaseous ions reflect
by activating collisions in the interface). During the charge states in the condensed phase;
the whole process, electrochemical oxidation can although somewhat modified following ion–
also generate radical cations from neutral analytes, molecule collisions in the interface, resulting
thereby permitting this technique in some selected multiply charged species are commonly
instances, inspite of as a true ionization method observed.
rather than a procedure for phase transfer of pre- (b) The transfer of ions to the gas phase is not an
formed ions. energetic process, yet the de-solvation pro-
Currently electrospray at reduced flow rates is cess effectively cools the ions. Under appro-
in use, where sample solution flows at electro- priate conditions, therefore, low internal
spray interface with 3–20 ml/min. In the interests energy ions are introduced into the mass
of direct compatibility with conventional scale spectrometer for conventional analysis or for
analytical HPLC, some effort has been devoted to selection as precursor ions in a tandem MS
the accommodation of much higher flow rates. analysis. Application of a suitable potential
The technique is best utilized for the recording of difference between focusing components in a
Detection and Recording of Fingerprints 153

region of intermediate pressure allows ions spectral data, and mass fragmentation behaviors
to achieve kinetic energies consistent with with those of the reference compounds. Another 33
the applied potential and therefore undergo compounds were tentatively identified by refer-
activating collisions. encing to the reported data of their UV and MS
(c) The electrospray process involves the step- spectra. The ESI–MS/MS fragmentation behav-
wise disruption of non-covalent interactions ior of flavones (OMe-substituted, O-glycosides,
(principally the removal of molecules of sol- C-glycosides), chalcones, flavonols, and their appro-
vation); interception of this process allows the priate characteristic pathways were proposed.
preservation of relatively strong non-covalent In negative ion ESI-MS, all the flavonoids yielded
interactions of analytical importance. prominent [M–H]− ions in the first-order mass
spectra. Fragmentation with a loss of mass of
Case Study 1 15 Da (CH3), 18 Da (H2O), 28 Da (CO), 44 Da
LXD (Longdan Xiegan decoction) is a herbal (CO2), and 56 Da (2CO) and the residues of glu-
decoction used in traditional Chinese medicine cose and glucuronic acid observed in the MS/MS
and consists of ten medicinal herbs, Gentianae spectra were useful for aiding the structural
Radix, Scutellariae Radix, Gardeniae Fructus, identification of flavonoids investigated [2].
Rehmanniae Radix, Alismatis Rhizoma, Plantaginis The researcher team has emphasized in their
Semen, Angelicae Sinensis Radix, Clematidis research paper that it was a matter of hard work
Armandii Caulis, Glycyrrhizae Radix et Rhizoma, and high professional skill to optimize the HPLC
and Bupleuri Radix. The decoction has attracted and MS conditions for satisfactory separation of
a great deal of attention for its wide range of the chemical constituents of LXD, as it was not
pharmaceutical and therapeutic uses. It is used an easy task because of the vast number of chem-
to treat damp heat in the liver, gall bladder, ical components present in the decoction with
congested eyes, swelling, and pain in the ear. many of them having similar physicochemical
Flavonoids (from Scutellariae Radix, Glycyrrhizae properties [2]. Eighteen flavonoid compounds
Radix et Rhizoma), iridoid glycosides (from were used as standards for optimization of the
Gentianae Radix, Gardeniae Fructus, Rehmanniae separation of flavonoids using HPLC and the
Radix), and triterpenoids (from Glycyrrhizae Radix ionization and fragmentation using ESI-MS. In
et Rhizoma, Bupleuri Radix, and Gentianae Radix) order to optimize the separation and detection
were the three main classes of active compounds of flavonoids in LXD, variables such as column
reported according to chemical research on types, column temperatures, mobile phase, elu-
each of the individual herbs, but there was no tion conditions, flow rates, and detection wave-
report on the interpretation of the chemical length have been investigated. A number of C18
constitution of the decoction, so LXD was stud- RP-ODS (250 mm × 4.6 mm, 5 mm) columns,
ied by high-performance liquid chromatography from different manufacturers, were tested for
coupled to electrospray ionization tandem mass their suitability in this analytical design. The
spectrometry and photodiode array detection Waters ODS column was finally selected due to
method [2]. the best peak separation capacity. Two column
HPLC coupled to electrospray ionization (ESI) temperatures, 30 and 40°C, were tested, and
tandem mass spectrometry and photodiode array results were better at 30°C. Acetonitrile with
detection (HPLC–DAD–ESI–MSn) was used to water (different pH) was used as mobile phase,
identify and characterize the flavonoids in LXD. and no enhancement was obtained by changing
In total, 51 flavonoids (27 flavones, 10 flavanones, the pH in the aqueous mobile phase, so the best
7 chalcones, 5 flavonols, and 2 isoflavones) were mobile phase was a mixture of acetonitrile and
characterized. Eighteen compounds among them water. Five wavelengths (210, 254, 270, 280, and
including a newly detected flavonoid, naringin, 330 nm) were tested for the best detection sensi-
from the ingredient herbs, were unambiguously tivity, and best detection result was at 270 nm.
determined by comparing the retention times, UV Three different flow rates (0.6, 0.8, and 1.0 ml/min)
154 8 Mass Spectroscopy: Herbal Drugs and Fingerprints

were tested, and 0.8 ml/min had good separation. offset at 40 V. The first-order mass spectra were
In MS analysis, the negative ion mode of ESI was recorded in the range m/z 80–1,000. The relative
selected for its better sensitivity and separation collision energy was varied from 35 to 45% of
than the positive ion mode. For the peaks of the the maximum to produce optimum yields of frag-
18 isolated standards in the negative ion LC/ ment ions [2].
ESI-MS total ion current chromatogram, separa- The powdered samples of Gentianae Radix
tion conditions were used to record the LC/MS (0.6 g), Scutellariae Radix (0.3 g), Gardeniae
chromatograms of the LXD. In addition, colli- Fructus (0.3 g), Rehmanniae Radix (0.6 g),
sion-induced dissociation (CID) fragmentation Alismatis Rhizoma (0.6 g), Plantaginis Semen
of the [M–H]− ion provided extensive informa- (0.3 g), Angelicae Sinensis Radix (0.3 g),
tion for the characterization of flavonoids in the Clematidis Armandii Caulis (0.3 g), Glycyrrhizae
decoction [2]. Radix et Rhizoma (0.3 g), and Bupleuri Radix
All the solvents for used for analysis were of (0.6 g) were immersed in 168 ml of deionized
high purity HPLC grade, and herbs for the prepa- water for 1 h and then were decocted to boil for
ration of the LXD were purchased from authentic 30 min, filtered and cooled. Acetic acid was
supplier. Eighteen pure compounds were used as added to adjust the pH to 3.0. It was then left to
reference standards, and their structures were stand for 30 min and extracted with ethyl acetate
fully characterized by nuclear magnetic reso- (1:1, v/v) twice. The ethyl acetate extracts were
nance (NMR) spectroscopic methods, and their combined and evaporated to dryness. The residue
purities were >98% as determined by HPLC. was dissolved in 2-ml methanol and was filtered
Stock solutions (1.0 mg/ml) of all the above through a 0.45-mm syringe filter before injecting
compounds were prepared by dissolving them in into the HPLC instrument for analysis [2].
methanol [2]. The aforesaid study is an array for identification
The decoction was analyzed by online HPLC/ and characterization of the flavonoids present in
ESI using an HPLC system coupled to a LCQ LXD-type formulations. The knowledge of the
ion trap mass spectrometer. LC separation was profile of the flavonoids (flavones, isoflavones,
conducted using a ThermoFinnigan HPLC and flavonols, flavanones, and chalcones) in the LXD
a C-18 column (250 mm × 4.6 mm, 5 mm), is important for the understanding of its biologi-
Symmetry from Waters, at a flow rate of 0.8 ml/ cal and medical significance, and such a profile
min. The mobile phase consisted of (A) acetonitrile may also prove useful to evaluate the quality of
and (B) water (v/v), using an iso-gradient elution the decoction, as flavonoids, polyphenols, and
of 22% A at 0–35 min, a gradient elution of its derivatives are responsible for potent
22–30% A at 35–40 min, 30–50% A at 40–80 min, biological properties (antibacterial, antiviral,
and 50–90% A at 80–90 min. The temperature of anti-inflammatory, antiallergic, neoplastic, anti-
the column oven was 30°C, and detection wave- mutagenic, antithrombotic, and vasodilatory),
length was 270 nm (Fig. 8.5). The UV spectra which have a very complex profile of chemical
from 190 to 400 nm were also recorded for peak components. Hence, it is very difficult to identify
characterization. Mass spectra were acquired using all the compounds in the decoction. High-
an LCQ ion trap mass spectrometer (Thermo- performance liquid chromatography/electrospray
Finnigan, San Jose, CA, USA) equipped with an ionization tandem mass spectrometry (HPLC/
ESI source. Nitrogen was used as the sheath and ESI–MS/MS) is a most powerful analytical tech-
auxiliary gas, and helium was used as the damp- nique for the analysis of natural products. It is
ing and collision gas. All mass spectra were widely used in the analysis of complex mixtures
acquired in the negative ion mode with an ion such as plant metabolites. ESI is considered to
spray voltage at 4.5 kV, a capillary temperature provide soft ionization since it can lead to only
at 275°C, a capillary voltage at 19 V, a sheath protonated or deprotonated molecules. MS/MS,
gas flow rate at 20 (arbitrary units), an auxiliary gas particularly multistage MS/MS with an ion trap
flow rate at 45 (arbitrary units), and a tube lens (IT), provides further information about the struc-
Detection and Recording of Fingerprints 155

Fig. 8.5 LC/MS chromatograms of the flavonoid standards. (a) Negative ion LC/ESI-MS total ion current (TIC) and
(b) LC-UV chromatogram monitored at 270 nm [2]

tures of the compounds under analysis. By frag- parts are used for the treatment of prostatitis,
menting the precursor ion, it is possible to obtain stomachaches, and cystitis in traditional medicine)
structurally important fragment ions. Thus, online was performed using retention characteristics in
HPLC/ESI-ITMS provides a fast method to sepa- RP-HPLC, through analysis of UV–vis. and MS
rate and determine the molecular mass and spectra, and by HPLC–PAD–ESI/MS analyses.
resolve the subtle differences in their molecular Flavan-3-ols (i.e., catechins and proanthocyani-
structures. The tandem mass spectrometric frag- dins) were also analyzed by HPLC using detec-
mentation pattern of flavonoids (except chal- tion following post-column derivatization with
cones) has been investigated extensively, and thus p-dimethylaminocinnamaldehyde (DMACA). The
allows the characterization of unknown com- chromatographic profile of the ethyl acetate frac-
pounds even when reference standards are tion from the infusion of E. telmateia is recorded
unavailable [2]. at 280 nm. The UV spectra of the same suggested
that the major flavonoids present in this fraction
Case Study 2 were kaempferol derivatives and flavan-3-ols.
A study on characterization of polyphenols by Retention characteristics and UV spectra also
HPLC–PADESI/MS of Equisetum telmateia allowed the identification of protocatechuic and
(family Equisetaceae, is a species of the subgenus p-hydroxybenzoic acids, confirmed by compari-
Equisetum, widely distributed in Europe; aerial son with standards, as well as the presence of vari-
156 8 Mass Spectroscopy: Herbal Drugs and Fingerprints

ous caffeic acid derivatives. Further identification uses in traditional medicine. An infusion was
of compounds was made from their MS. The prepared by adding 100 ml of hot water to 5 g of
retention time and spectral characteristics of the plant material. The mixture was kept hot
components detected in the HPLC–PAD–MS/ and left to stand for 10 min, following which the
MS analysis are clues for the tentative identities mixture was filtered under vacuum and the filtrate
of these compounds. cooled and its volume made up to 100 ml with
Analysis of polyphenols was performed water. An aliquot of the infusion was washed
using HPLC with an auto-injector, spectrometer with n-hexane and further extracted with ethyl
equipped with an API/ES ionization chamber. acetate. The organic phase was collected, dried
Separation was performed on a Waters Spherisorb over anhydrous sodium sulfate, and concentrated
ODS2 column (150 mm × 4.6 mm, 3.0 mm) main- under vacuum at 30°C until all of the ethyl ace-
tained at 25°C. The mobile phase consisted of tate had been removed.
2.5% acetic acid (solvent A), a 90:10 mixture of The infusion and the ethyl acetate fraction
2.5% acetic acid, and acetonitrile (solvent B) and were frozen with liquid nitrogen and then lyo-
acetonitrile (solvent C). The gradient profile used philized. A decoction was prepared by adding
was from 100:0:0 (A: B: C) to 0:100:0 between 0 100 ml of cold water to 5 g of the plant material
and 5 min, changing to 0:85:15 between 5 and and heating the mixture to boiling for 20 min.
30 min, changing to 0:50:50 between 30 and After cooling, the mixture was filtered under vac-
35 min, and followed finally by isocratic elution uum and its volume made up to 75 ml with water.
with 0:50:50 between 35 and 40 min; the flow In order to prepare a tincture, 40 ml of 45% aque-
rate was 0.5 ml/min. The first detection was by ous ethanol was added to 2.5 g of the plant mate-
photodiode array detector (PAD) with the rial and the mixture allowed standing at room
spectrophotometer set at 280 and 360 nm, and temperature for 13 days in the dark. After this
the second detection using ESI-MS. The capillary time, the hydroalcoholic extract was filtered and
temperature was 225°C, and the capillary voltage concentrated under vacuum and its volume made
was 45 V. Spectra were obtained in the positive up to 10 ml with 45% aqueous ethanol. From the
ion mode, and the MS was programmed to aforesaid studies, it is clear that it is possible to
perform two scans, a full mass scan and an MS2 decipher the traditional anti-inflammatory uses of
scan of the most abundant ion using a collision E. telmateia at molecular level [3].
energy of 45 V.
Reference standards as kaempferol 3-O-
rutinoside and kaempferol 3-O-glucoside were Matrix-Assisted Laser Desorption
from Extrasynthese; standards of flavan-3-ols Ionization
were obtained from grape seeds by repeated
extraction with methanol and fractionation This technique is relatively straightforward to
of the combined extracts on a Sephadex LH-20 use and reasonably tolerant to buffers and other
Fluka column (45 × 5 cm) using ethanol as mobile additives. The mass accuracy depends on the
phase. Catechins and proanthocyanidins were type and performance of the analyzer of the mass
further separated from the Sephadex fractions by spectrometer, as most modern instruments are
semi-preparative HPLC. capable of measuring masses to within 0.01% of
Dried aerial parts of E. telmateia Ehrh. were the molecular mass of the sample, at least up to
obtained from authentic supplier, and plant ca. 40,000 Da. Matrix-assisted laser desorption
material was identified by Dr. J. Paiva, Botany ionization (MALDI) is based on the bombard-
Department, University of Coimbra, Portugal, ment of sample molecules with a laser light to
and a voucher was deposited at the Department of bring about sample ionization. The sample is
Pharmacognosy, Faculty of Pharmacy, University premixed with a highly absorbing matrix com-
of Coimbra. Extracts were prepared from the pound for the most consistent and reliable result,
pulverized plant material according to their and a low concentration of sample to matrix is
Detection and Recording of Fingerprints 157

Fig. 8.6 Simplified laser


schematic of MALDI-TOF
MS (linear mode) [4]

ions

detector

high field free region


voltage

= matrix and analyte

the best option. The matrix transforms the laser analyzed independently (external calibration) or
energy into excitation energy for the sample, premixed with the sample and matrix (internal
which leads to sputtering of analyte and matrix calibration) [4].
ions from the surface of the mixture. In this way, MALDI is also a soft ionization method and
energy transfer is efficient, and also the analyte so results predominantly in the generation of sin-
molecules spared excessive direct energy that gly charged molecular-related ions regardless of
may otherwise cause decomposition. Most com- the molecular mass; hence, the spectra are rela-
mercially available MALDI mass spectrometers tively easy to interpret. Fragmentation of the
now have a pulsed nitrogen laser of wavelength sample ions does not usually occur. In positive
337 nm [4]. ionization mode, the protonated molecular ions
The sample to be analyzed is dissolved in an are usually the dominant species, although they
appropriate volatile solvent, usually with a trace can be accompanied by salt adducts, a trace of the
of trifluoroacetic acid if positive ionization is doubly charged molecular ion at approximately
being used. Sinapinic acid is a common one for half the m/z value and/or a trace of a dimeric spe-
protein analysis, while alpha-cyano-4-hydroxy- cies at approximately twice the m/z value. Positive
cinnamic acid is often used for peptide analysis. ionization is used in general for protein and pep-
An aliquot of the final solution is applied to the tide analyses. In negative ionization mode, the
sample target which is allowed to dry prior to deprotonated molecular ions are usually the most
insertion into the high vacuum of the mass spec- abundant species, accompanied by some salt
trometer. The laser is fired, the energy arriving at adducts and possibly traces of dimeric or doubly
the sample/matrix surface optimized, and data charged materials. Negative ionization can be
accumulated until an m/z spectrum of reasonable used for the analysis of oligonucleotides and oli-
intensity has been amassed (Fig. 8.6). The time- gosaccharides [4].
of-flight analyzer separates ions according to
their mass (m)-to-charge (z) (m/z) ratios by mea- Case Study
suring the time it takes for ions to travel through The problem of adulteration is a key concern
a field-free region known as the flight, or drift, for the herbal-based industries, where botanical
tube. The heavier ions are slower than the lighter mis-identification and intentional addition of
ones. The m/z scale of the mass spectrometer is cheap substitutes are common practices. Final
calibrated with a known sample that can either be product quality is entirely based on the quality and
158 8 Mass Spectroscopy: Herbal Drugs and Fingerprints

consistency of the starting material, subsequent has the potential to address the most challenging
handling, extraction, and further processing. The issues of species and adulterant identification
introduction of other botanical and non-botanical and product consistency, while also having the
contaminants can occur throughout the process. potential to support breeding programs and
Therefore, rapid and cost-effective methods, like rapid plant screening for phytochemical quan-
MALDI-TOF MS, can be used to monitor these tity and quality [5].
materials for complex product purity and consis- A vital component of higher-throughput
tency, for example, species in the genus Echinacea method development is sample preparation prior
(primarily E. purpurea and to a lesser extent E. to MALDI-TOF MS analysis. There is always
angustifolia) are used in literally thousands of thrust for a user-friendly robotic system that
dietary supplements. Product quality and species allows for method development and method
identification are generally determined using deployment flexibility and the objective to
HPLC and TLC/HPTLC. Less utilized assays develop an automated, high-throughput method
such as near-infrared spectroscopy (NIR) have to process samples and differentiate Echinacea
also been developed for Echinacea species species by their MALDI-TOF mass profiles. MS
identification and correlation to phytochemical analysis of plant materials for a variety of
concentration. All of these techniques have par- Echinacea species demonstrates the power of
ticular strengths, although in general these meth- MALDI-TOF MS to generate complex, sample-
ods are low throughput and require sampling and specific profiles. Comparison of MS profiles col-
drying of relatively large portions of leaf or root. lected from the different plant tissues for a single
Automated, high-throughput processing meth- Echinacea species or similar plant materials for
ods, teamed with matrix-assisted laser desorp- the three species characterized to the low-molec-
tion/ionization time-of-flight mass spectrometry ular-weight (expanded view, 240–560 Da) and
(MALDI-TOF MS) analysis, to differentiate high-molecular-weight (expanded view, 2,000–
Echinacea species by their mass profiles, have 15,000 Da) profiles, respectively, for seed extracts
been developed. Analysis of these samples high- from the three Echinacea species is analyzed.
lighted key MS signal patterns from both small While MS fingerprints characteristic of the gen-
molecules and proteins that characterized the eral Echinacea genus can be discerned, species-
individual Echinacea materials analyzed. Based specific signal markers can be easily distinguished
on analysis of pure Echinacea samples, off-the- within the two mass ranges. The unique
shelf products containing Echinacea could then fingerprints generated from the individual plant
be evaluated in a streamlined process. materials and Echinacea species analyzed can be
Corresponding analysis of dietary supplements used to evaluate off-the-shelf Echinacea prod-
was used to monitor for product composition, ucts. The MS patterns were compared between
including Echinacea species, and plant materials samples to potentially further characterize or
used. These results highlight the potential for confirm sample composition. As an example, the
streamlined, automated approaches for agricul- mass spectra for leaf materials from E. angustifo-
tural species differentiation and botanical prod- lia and E. purpurea, and the Rexall Sundown
uct evaluation. herbal supplements, of which E. purpurea is a
Based on the growing maturity and success component. Specific signal characteristic of E.
of matrix-assisted laser desorption/ionization purpurea was identified by visual comparison in
time-of-flight (MALDI-TOF) mass spectrome- the corresponding spectrum from the Sundown
try (MS) for determination of biomarkers in product, along with general Echinacea leaf mate-
numerous plant and animal species, research rial MS signals (for comparing the E. angustifo-
has been conducted to evaluate this technique as lia, E. purpurea, and Rexall Sundown profiles).
a higher-throughput assay using relatively small Comparison of these MS patterns shows that the
amounts of fresh and dry plant tissue. For botan- composition of Rexall Sundown includes E. pur-
ical product quality control, MALDI-TOF MS purea aerial parts. In addition to these, a few
Detection and Recording of Fingerprints 159

low-intensity signal (not characteristic of either posed of specific Echinacea tissues were extracted
the E. angustifolia or E. purpurea) profiles were using streamlined, automated instrumentation for
also present within the Rexall Sundown profile downstream sample profiling by MALDI-TOF
and indicative of an additional compounds present MS. Samples were homogenized using a mini
in the supplement [5]. Bead-Beater, and the supernatant was collected,
Dried Echinacea materials including E. pur- diluted, and deposited onto a MALDI target plate
purea, E. pallida, and E. angustifolia seeds, roots, using a ProPrep liquid-handling system.
and aerial parts used in this study were from Automated sample analysis was then performed
American Herbal Pharmacopoeia. Off-the-shelf on the mass spectrometer, resulting in profile-rich
products included Echinacea supplements from spectra for the individual samples analyzed. This
Nature’s Resource Products (Mission Hills, CA) approach allowed for a systematic sample-prepa-
and Rexall Sundown, Inc. (Boca Raton, FL). ration method that significantly improved sample
MALDI matrixes a-cyano-4-hydroxycinnamic throughput, preparation reproducibility, and data
acid (CHCA) and 3, 5-dimethoxy-4-hydroxycin- reliability [5].
namic acid (sinapinic acid, SA) were purchased Using these procedures, sample-preparation
from Fluka (Sigma-Aldrich Switzerland). Solvents time was reduced by half. The ProPrep performed
included HPLC grade acetonitrile obtained from sample preparation and deposition tasks for 32
Honeywell Burdick & Jackson and sequencing plant extracts and 2 blanks in approximately
grade triflouroacetic acid (TFA) from Fluka. 30 min, compared to a manual preparation time
A Milli-Q gradient water system was used for of over 60 min. The ProPrep liquid-handling
molecular-grade water. This buffer and phenyl- steps included the following: (1) transferring
methanesulfonyl fl ouride (PMSF) were from supernatant from the homogenized samples
Sigma-Aldrich [5]. (Extract-1) to a clean microtiter plate (Collection
Three types of plant tissues – seed, root, and 1), (2) transferring MALDI matrix (CHCA or
aerial parts (leaf/stem/flower) – and two commer- SA) from the solution vial to a second microtiter
cial Echinacea supplements were analyzed in plate (Collection 2), (3) pipetting an extract ali-
this study. The commercial supplements were of quot from Collection 1 to an individual well in
composition of the various Echinacea tissues Collection 2, (4) mixing the extract/matrix solu-
analyzed. Samples were prepared in triplicate, tion in Collection 2, and (5) depositing an aliquot
approximately 100 mg of each, in 2.0-ml conical of the extract/matrix solution onto a clean MALDI
screw cap micro tubes. Roots were shaved prior target plate. Robust and reproducible sample
to weighing. Samples of Echinacea supplements preparation and MS data were obtained using
from Nature’s Resource Products and Rexall these procedures. Final MALDI sample deposi-
Sundown, Inc. were extracted from capsules [5]. tion performed by this instrument produced
MALDI-TOF-MS analysis was performed on reproducible, well-formed droplets, properly
a Voyager-DE STR MALDI-TOF mass spec- located and without overlap. MALDI-TOF MS
trometer. Analysis of CHCA prepared samples analysis over the 50–600 m/z range resulted in
was performed in reflector mode, over the mass spectra with over 200 small-molecule compounds
ranges 50–600 (300 shots/sample) and 600– detected. Sample analysis performed in triplicate
5,000 Da (500 shots/sample). Samples prepared demonstrates the reproducibility of the sample-
with SA were analyzed in linear mode, over the preparation and MS analysis techniques used.
mass range 2,000–60,000 Da (500 shots/sample). Triplicate analysis of Echinacea samples indi-
External mass standards were used to calibrate cates high reproducibility. Similar mass spectra
the data [5]. are observed for the individual preparation, and
Small molecules, peptides, and proteins from MS analyses performed for the E. pallida root
aerial parts, seeds, and roots from three Echinacea replicate. Small signal-intensity differences are
species (E. purpurea, E. pallida, and E. angusti- identified between the triplicate analyses; how-
folia) and two off-the-shelf supplements com- ever, the similarity in molecular profile patterns
160 8 Mass Spectroscopy: Herbal Drugs and Fingerprints

was enough to be easily distinguished. Similar rapid screening for phytochemical quantity and
results were seen for analysis of the various plant quality [5].
extracts over the mass ranges 50–600, 600–5,000, In pharmaceutical industry, LC–MS has
and 2,000–60,000 Da. become method of choice in many stages of drug
MS analysis of plant materials for a variety development. Recent advances include electro-
of Echinacea species demonstrates the power spray, thermospray, and ion spray ionization
of MALDI-TOF MS to generate complex, techniques which offer unique advantages of high
sample-specific profiles. Comparison of MS detection sensitivity and specificity, liquid sec-
profiles collected from the different plant tis- ondary ion mass spectroscopy; later, laser mass
sues for a single Echinacea species or similar spectroscopy with 600 MHz offers accurate
plant materials for the three species is charac- determination of molecular weight proteins, pep-
terized. The highlights of the species specificity tides. Isotopes pattern can be detected by this
of the profiles generated show the low-molecu- technique.
lar-weight (expanded view, 240–560 Da) and
high-molecular-weight (expanded view, 2,000–
15,000 Da) profiles, respectively, for seed Hyphenated Techniques
extracts from the three Echinacea species ana-
lyzed. While, MS fingerprints characteristic of Hyphenated chromatography-MS systems emp-
the general Echinacea genus can be discerned, loyed for metabolomic fingerprinting consist of
species-specific signal markers can be easily three parts. The first part is the chromatographic
distinguished between two mass ranges [5]. separation section, used to separate complex
Using the aforesaid techniques, larger sam- mixtures obtained from the plant material. The
ple database can be prepared for other plants most commonly used chromatographic systems
and plant materials sampled for their possible are HPLC, GC, or CE. The second part consists
by-product due to degradation and plant materi- of the ionization chamber where the molecules
als used during supplement composition, and are being ionized by different ionization sources
materials and products following stability test- such as electron ionization (EI), electrospray
ing would enhance capabilities to monitor and ionization (ESI), atmospheric pressure chemical
characterize botanical products. The ability to ionization (APCI), and atmospheric pressure
analyze large numbers of samples quickly using photoionization (APPI). Another ionization source
these technologies makes them ideal tools for being used, but not in combination with flow-
botanical product analysis and biomarker dis- based systems, is matrix-assisted laser desorp-
covery. Fingerprint comparisons, based on a tion/ionization (MALDI). The third part consists
library of analyzed materials and by-product, of the MS where the ionized molecules are being
yield the opportunity to characterize raw mate- detected as their mass-to-charge ratio. The most
rials and products. The data gathered from commonly used MS systems include single
large sample sets can be subjected to multi- quadrupole, triple quadrupole, ion trap, and time-
variant statistical analysis methods for pattern of-flight spectrometers. More advanced high-
identification. Further analysis can also be per- resolution spectrometers include the orbitrap or
formed to identify, characterize, and validate the Fourier transform cyclotron. For a targeted
potential biomarkers. The techniques presented approach, MS is usually employed as the detec-
show promise for characterizing botanical tor, while nuclear magnetic resonance (NMR)
materials and products for important aspects and MS are used without the chromatography
such as product quality control, including step as an untargeted analytical tool. There is little
species and adulterant identification; product difference in the sample preparation and data
consistency and stability, as well as the ability handling between MS-based and NMR-based
to support plant-breeding programs; and early technologies, although the solvents being used
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makes use of deuterated solvents, while MS uses guish compounds with the same nominal mass
non-deuterated solvents [6]. but different exact mass caused by different ele-
In MS, ionized components are identified and mental composition. The exact structural eluci-
quantified at very low quantities making the MS a dation of a compound calls for hyphenated
very sensitive analytical tool for metabolic techniques, and each one provides a vital piece of
fingerprinting. The lack of ionization and there- information. It is a collective pooling of all the
fore the inability to quantify or identify compo- information that are collected from various equip-
nents that do not ionize limits the use of MS to the ments that ultimately results in the total under-
more targeted metabolic profiling techniques. standing of an unknown molecule.
Various probes are being employed to overcome
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NMR Spectroscopy: Herbal Drugs
and Fingerprints 9

Nuclear magnetic resonance (NMR) spectroscopy For a proton magnetogyric ratio, (g ) = 2.674 × 104
was first developed in 1946 by research groups gauss−1 sec−1. This precession process generates
at Stanford and MIT, in the USA, since that day an electric field with frequency precession rate
it has developed into the premier organic spec- (wo) (also known as Larmor frequency). When
troscopy available to scientists to determine the we irradiate the sample with radio waves (in the
detailed chemical structure and new drug dis- MHz frequency range), the proton absorbs the
covery. Another well-known product of NMR energy and is promoted to the less favorable higher
technology has been the magnetic resonance energy state. This stage is called resonance because
imaging (MRI), extensively utilized in the medical the frequency of the applied radiation and the
radiology to obtain image slices of soft tissues. precession coincide or resonate. The field strength
In recent years, NMR has moved out from of a magnet is usually reported at the resonance
research laboratories to the on-line process anal- frequency for a proton. Therefore, for different
ysis market. The NMR spectroscopy is based on nuclei with different gyromagnetic ratios, differ-
the fact that nucleus of atom has magnetic prop- ent frequencies are needed in order to achieve
erty that can be utilized to yield chemical infor- resonance. The orientations at which a nucleus
mation. As electrons have spin around nucleus, magnetic moment can take against an external
these spins are paired in some atoms (e.g., 12C, magnetic field are not of equal energy. Spin states
16
O, 32S) and cancel the effect of each other, so which are oriented parallel to the external field
that the nucleus of the atom has no overall spin. are lower in energy than in the absence of an
However, in many atoms (1H, 13C, 31P, 15N, 19F, external field. In contrast, spin states whose ori-
etc.) nucleus possesses an overall spin. If an atom entations oppose the external field are higher in
has even number of neutrons and protons, the energy than in the absence of an external field.
nucleus has no spin, but if the number of neutrons Due to resonance, there is a possibility to
plus the number of protons is odd, then the induce a transition between the various spin states.
nucleus has a half-integer spin (i.e., 1/2, 3/2, 5/2). By irradiating the nucleus with electromagnetic
But when the number of neutrons and the number radiation of the correct energy (as determined
of protons are both odd, then the nucleus has an by its frequency), a nucleus with a low-energy
integer spin (i.e., 1, 2, 3). orientation can be induced to transition to an
When we apply an external magnetic field to orientation with a higher energy. The absorption
the atom, the nucleus may align with it or in of energy during this transition forms the basis of
opposite direction. Each nucleus has a character- the NMR method, as other spectroscopic methods
istic value of magnetogyric ratio (g), which is (as IR and UV–Vis.). The amount of signal inten-
defined as a constant of proportionality between the sity is proportional to the population difference
nuclear angular momentum and magnetic moment. between the two energy levels involved. Energy

D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs, 163
DOI 10.1007/978-81-322-0804-4_9, © Springer India 2012
164 9 NMR Spectroscopy: Herbal Drugs and Fingerprints

levels have a large energy difference in case of frequency of the spectrometer, which is in MHz.
UV–Vis. spectroscopy, but in NMR, the energy Thus, one is dividing Hz by MHz which is a part
separation of the spin states is comparatively very per million (ppm). One ppm on a 58-MHz NMR
small, even it is difficult to distinguish from noise instrument is actually 58 Hz from the resonance
level. This property leads to difficulties in using position of TMS, while on a 300-MHz NMR
NMR for trace analysis, so NMR is not consid- instrument 1 ppm is 300 Hz from the TMS reso-
ered accurate below a quantitation of 0.05 wt.% nance position. With this standardization/normal-
(500 ppm). There are two general types of NMR ization in place, one can always unequivocally say
instrument: continuous wave (CW) and Fourier that all benzene protons resonate at 7.16 ppm no
transform (FT). Early experiments were con- matter what NMR instrument is being used in
ducted with CW instruments, and in 1970 the first the analysis. A few considerable points are as
FT instruments became available and now domi- follows: (1) Electronegative groups are de-shielding
nates the market. and tend to move NMR signals from neighboring
In a molecule different protons have different protons further downfield (to higher ppm values).
electronic environments and experience slightly (2) Protons on oxygen or nitrogen have highly
different applied magnetic fields owing to the variable chemical shifts, which are sensitive to
shielding /de-shielding effect of the induced elec- concentration, solvent, temperature, etc. (3) The
tromagnetic fields. In order to standardize the system of alkenes, aromatic compounds, and car-
NMR scale, it is necessary to set a “zero” refer- bonyls strongly de-shields attached protons and
ence point to which all protons can be compared. moves them downfield to higher ppm values. In
The standard reference selected is tetramethylsi- most spectroscopic techniques the energy absorp-
lane (TMS). This compound has four methyl tion by the sample is not a primary concern, but in
(−CH3) groups bonded (i.e., single bonded) to a NMR, the energy absorption is a matter of pri-
silicon atom. All of the protons on the methyl mary concern, and the NMR process is so-called
groups are in the same electronic environment. an absorption process, where nuclei in the excited
Therefore, only one NMR signal generates. The state must be able to relax and return to the ground
electronegativity of the carbon atoms is actually state. The timescale for this relaxation is crucial to
higher than the silicon atom to which they are the NMR studies as relaxation of electrons to the
bonded, resulting sigma-electrons shifted toward ground state in UV–Vis. spectroscopy is a very
the carbon atoms in the methyl groups and, conse- fast process, on the order of picoseconds, but in
quently, the heavily shielded protons causing one NMR, the excited state of the nucleus can persist
signal at a very high magnetic field strength. So for minutes. Because the transition energy between
this NMR signals works as reference. Any differ- spin levels is so small, attaining equilibrium has
ence against the reference signal is called the much longer timescale (i.e., the relaxation period).
chemical shift. This shift is measured in parts per Such stereochemical data have high potential to
million (ppm). NMR signals occurring near the decipher the active ingredients of traditional
TMS resonance are said to be in an upfield herbal drugs and lined with the modern medical
position, while those shifted away by de-shielding practices. Traditional herbal drugs are mainly in
are said to be downfield. All NMR signals are raw and semi-standardized form and often based
downfield from the TMS signal because of the on empirical evidence, so certainly NMR spec-
heavily shielded nature of the methyl protons in troscopy has an important role, for selecting herb-
TMS molecule. The proton NMR chemical shift als and products of potential therapeutic interest,
range is 0–12 ppm. The ppm scale is another form and offers tremendous opportunities to discover
of standardization that allows one to compare new drugs of herbal origin [1]. Herbals and their
directly the 1H spectra obtained on NMR instruments products in traditional practices (such as Ayurveda,
with different magnetic fields. With referenced to Unani, Kampo, TCM) are chemically complex
the TMS, resonance is 0 ppm; the actual NMR mixture and contain one or many structurally related
peak position in Hz is divided by the resonance active compounds that produce a combined effect.
Metabolic Fingerprints 165

Studies at molecular level help in standardizing


the herbal preparations to an optimal concen- Metabolic Fingerprints
tration of active constituents and preserving
their activities. The marker system used in chro- Technological advances in analytical systems
matography is not sufficient alone to decipher the like NMR, GC–MS, and HPLC have opened up
stereochemistry of the molecule. NMR-based new avenues to understand the complex bio-
fingerprints are additional supporting documents chemical processes within a plant and total anal-
for the chemical profile of the product. The ysis of metabolome of a plant. This has become
fingerprint pattern approach is quite often used an important area of investigations in pharmacol-
by the pharmaceutical industry to examine the ogy and functional genomics of medicinal plants.
source of the drug substance and the method of Comprehensive chemical analysis establishes the
chemical preparation, as a “phytochemical logo.” correlation between complex chemical mixtures
Example may be cited from an important medici- and molecular pharmacology, along with com-
nal plant of Indian system of medicine (ISM), plex cellular processes and biochemical pathways
Withania somnifera (ashwagandha, Indian gin- via metabolite-to-gene network [2].
seng, winter cherry), studied by using NMR and
chromatographic (HPLC and GC–MS) tech-
niques. A total of 62 major and minor, primary Case Study 1
and secondary metabolites from leaves and 48
from roots were unambiguously identified. Twenty- In view of the aforesaid objectives, leaves and
nine of these were common in the tissues of root tissues of W. somnifera were extracted with
both parts. These included fatty acids, organic n-hexane followed by warm (~35 °C) methanol–
acids, amino acids, sugars, and sterol-based water (90–70% methanol, stepwise successively).
compounds. Between leaves and roots, highly After liquid–liquid partition of methanolic water
significant, qualitative and quantitative differ- portion with chloroform followed by n-butanol,
ences were noticed, particularly with respect to metabolite repertoire of the plant was distributed
the secondary metabolites. W. somnifera is used into four fractions of different polarities
in Ayurveda for over 3,000 years, in various (n-hexane, aqueous-methanol, chloroform, and
forms (decoctions, infusions, ointments, powder, n-butanol). Each fraction was then subjected to
and syrup), and presently used in different parts NMR and GC–MS analysis and sometimes to
of the world for all age groups of patients without HPLC–PDA analysis. Metabolic content of leaf
any side effects even during pregnancy. The and root tissues is extracted by n-hexane, chloro-
extracts as well as different isolated bioactive form, n-butanol, and methanolic water; the quantity
constituents of W. somnifera have been reported of metabolite in leaves was much higher than that
to possess adaptogenic, anticancer, anticon- in roots, particularly in the aqueous–methanolic
vulsant, immunomodulatory, anti-oxidative, and fraction. The metabolomic analysis and its inter-
neurological effects. The plant is also considered pretation, for different fractions, were carried out
efficacious in the treatment of arthritis, geriatric, as below [3].
behavioral, and stress-related problems. Several
bioactive alkaloids and sterol lactone-based n-Hexane Fraction
phytochemicals (e.g., ashwagandhine, cuscohy- The metabolomic analysis of n-hexane extract of
grine, isopelletierine, anaferine, anhygrine, W. somnifera leaves and roots was performed by
tropine, sitoindosides), as well as saponins (with- NMR spectroscopy and GC–MS. 1H-NMR spec-
anolides, withanamides, and glycowithanolides), tra of both leaf (Fig. 9.1) and root extracts predom-
have been isolated from different parts of this inantly have different saturated and unsaturated
plant [1]. The application of NMR spectral fatty acids. Signals at d 1.6 and at d 2.3 represented
fingerprints in herbal-based industry may be cited the b-CH2 and a-CH2 of the fatty acids [4]. The
as below. signals of all other protons of hydrocarbon chain
166 9 NMR Spectroscopy: Herbal Drugs and Fingerprints

Pheophytins a
Pheophytins b

Pheophytins a
Pheophytins a

signals of saturated alkyl chain of fatty acids

Methyl (terminal) signal of Linolenic acid (Specific)


11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 ppm

Carotenoids

Porphyrins
Me signals of fatty acids
6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 5.8 5.7 5.6 ppm

Pheophytins a

Pheophytins a
TAG FAME

Porphyrins
β−CH2 of fatty acids
4.30 4.25 4.20 4.15 ppm 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 ppm −1.5 −1.4 ppm
allylic CH2
Olefinic protons of fatty acids
α−CH2 of fatty acids
di allylic CH2 of PUFA

sterol
11 10 9 8 7 6 5 4 3 2 1 0 −1 ppm

Fig. 9.1 1H-NMR spectra of n-hexane extract of W. somnifera leaves [3]

of fatty acids appeared at d 1.3. The appearance of double doublet (dd) signals (d 4.1–4.3) of sn1
olefinic protons at d 5.35 indicated the presence of and sn3 protons of triacylglycerol (TAG). Minor
unsaturated fatty acids, whereas signals at d 2.07 amounts of methyl esters of fatty acids were
indicated the allylic protons of unsaturated fatty indicated by singlet (for O–Me group) signals at d
acid. In addition, the characteristic bis-allylic sig- 3.6. The characteristic signal of 18-CH3 group of
nals (triplet) of di- and tri-unsaturated fatty acids sterol appeared distinctly at d 0.7. Several signals
appeared at d 2.8. Homodecoupling experiment of at d 6.0–6.5 might be attributed to the carotenoids.
the olefinic protons (d 5.35) altered the triplet sig- Integration ratio with respect to trimethylsilyl
nals of bis-allylic protons into two distinguished propionate (TSP) signals indicated that the caro-
singlets at d 2.77 and d 2.83, indicating the pres- tenoid content of leaf extract was higher than that
ence of di- and tri-unsaturated fatty acids. The of root. This is expected, as major role of carote-
18:3 fatty acids were identified by their character- noids in leaves is to protect leaf from excessive
istic triplet methyl signals at d 0.99 particularly in light stress. It was further observed that percent-
the spectrum of leaf n-hexane extract. The age of TAG was much higher in root extract than
downfield shift of methyl signals for 18:3 (lino- in leaves. The presence of two signals in the
lenic acid) fatty acids occurred due to proximity of upfield region (d 1.43 and d 1.6) of the spectrum
the unsaturated double bond. of leaves was a characteristic for N–H group of
Terminal methyl signals of other fatty acids the porphyrins. Pheophytins are the degraded
appeared collectively at d 0.90. In case of root products of chlorophylls. During metabolite
samples, the signals of the terminal methyl were extraction, the chlorophylls lose their magnesium
not very distinct due to overlap. The 1H-NMR ions and become pheophytins. The signals of part
spectrum of n-hexane extract showed characteristic of phytal fragments [−O–CH2–CH=C(CH3)–] of
Metabolic Fingerprints 167

Overlaping Signal of β sitosterrol and C-21 Me of


WF-A and WN
C-19 Me (WF-A)

C-19 Me (WF-A)
C-19 Me (WN)

C-18 Me (WN)
C-28Me (WF-A)
C-28Me (WN)
C-27 Me (WN)
C-22H (WF-A)
C2-H (WF-A)

C-7H (WN)
C-4H (WF-A)

C-6H (WF-A)
C-6H (WN)
C-3H (WF-A)

C2-H (WN)

C-22H (WN)
C-3H (WN)

Un assigned
Un assigned
Un assigned
Un assigned
Un assigned

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

Fig. 9.2 1H-NMR spectrum of chloroform fraction of the W. somnifera leaves. WF A = withaferin A, WN = withanone [3]

chlorophylls and other parts of pheophytins also respective molecular ion peak in the mass spec-
appeared at d 9.52, 9.35, 8.5, 8.0, 3.4, and 3.24. trum together with the characteristic peak at m/z
The ratio of chlorophylls a and chlorophyll b was 74 (base peak) that appeared due to McLafferty
determined as 3:1 by integration of singlet sig- rearrangement and the peak at m/z 87 that
nals at d 9.37 and d 9.55. Other minor signals in appeared due to loss of (CH2)2COOCH3+. Other
the range of d 11–7 may have appeared due to respective logically defined mass fragments also
oxidized products of chlorophylls. The observed appeared in the spectrum. Presence of campes-
signals of all the protons and the corresponding terol and stigmasterol at tR 53.09 and 53.35 was
13
C signals were identified by 1H−13C heteronu- indicated in the GC–MS analysis, particularly in
clear single-quantum (HSQC) mapping. To deter- the n-hexane extract of root [3].
mine the composition of individual fatty acids
and sterols, the n-hexane extract was subjected to Chloroform and n-Butanol Fractions
1
GC–MS analysis after esterification (methyl H-NMR spectrum of chloroform fraction of the
ester). Palmitic acid (16:0), oleic acid (18:1), W. somnifera leaf extract (Fig. 9.2) has presence
linoleic acid (18:2), and linolenic acid (18:3) of double doublet at d 6.8 and d 6.5 together with
were major fatty acids present in the leaf and root distinguished doublet signals at d 6.3 and d 5.8
samples. These fatty acids belong to membrane that indicated the presence of withanolide skele-
lipids of plant tissues. Percentage peak area of ton. Characteristic singlet signals of methyl series
the GC chromatograms revealed that palmitic of withanolide framework were observed in the
acid and linolenic acid were the predominant range of d 0.6–2.2. Comparison of the spectrum
fatty acids present in the leaves, whereas roots with the purified withanolide standards estab-
were richer in palmitic acid and linoleic acid. The lished the presence of withaferin A and witha-
GC–MS analysis further suggests the presence of none in the mixture. The 13C signals of each
many other minor but very long-chain fatty acids, metabolite were recognized by 1H-13C HSQC
particularly in the root samples, indicating higher spectrum and compared with the literature data
activity in the stearoyl-CoA elongation activity in of the pure compounds. Both 1H and 13C signals
the root tissue as compared to the leaf tissue. clearly indicated the presence of withaferin A
Presence of each fatty acid was indicated by and withanone as the major metabolites in the
168 9 NMR Spectroscopy: Herbal Drugs and Fingerprints

chloroform fraction of leaves. Branch signals matography with standard. To explore further the
at d 0.9 of the 1H spectrum appeared due to ali- metabolic composition of chloroform and
phatic chain of b-sitosterol. GC–MS analysis of n-butanol fractions, GC–MS analysis was carried
the fraction indicated the presence of b-sitosterol. out, which indicates the presence of 1-octanol,
There were some unassigned signals appearing in different aromatic alcohols, and aromatic acids.
the range of d 5.2–5.7 of the 1H-NMR spectrum Presences of these compounds are logically sup-
which may be due to the presence of minor ported by their respective mass fragmentation
amounts of withanolides in the mixture. The cor- patterns obtained from GC–MS analysis [3].
responding 1H-NMR spectrum of the root was
not distinct like leaf, but it was clear enough to Aqueous Fraction
indicate the presence of withanolide skeleton. The metabolic profiling of aqueous fraction of W.
The 1H signals at d 6.6 (m), 5.85 (d), 4.2 (m), somnifera was analyzed mainly by 1H-NMR
1.95 (s), l.85 (s), 1.2 (s), and 0.95 (s) and corre- (Fig. 9.3), though in case of root samples, GC–MS
sponding 13C signals at d 12.0, 13.0, 14.0, 22.0, was also applied. Assignment of the compounds
57.0, 129.5, and 140.0 were identified by HSQC was thoroughly done comparing the 1H spectra of
spectrum, providing sufficient evidence for the reference compounds together with Biological
presence of withanolide A. Broadness of the Magnetic Resonance Data Bank (http://www.
spectrum indicated the presence of significant bmrb.wisc.edu/metabolomics/) and wherever
amounts of similar type of compounds. HPLC necessary, by spiking with appropriate internal
analysis of the chloroform partition of both standards. Two-dimensional (2D) correlation
leaf and root samples was performed for further spectroscopy (COSY) and HSQC spectra were
identification of other NMR-nonidentifiable witha- also extensively used to resolve the complexity of
nolides. Assignments of resultant chromatograms the overlapping/interfering spectral regions to
were performed by matching with the chromato- identify the exact molecule in the extract [3].
gram of purified withanolides and, subsequently, The entire 1H spectrum of aqueous fraction
by cochromatography [3]. may be divided into three major regions. d 0.0–
The analysis suggested that withaferin A and 3.5 region is rich with amino acids. d 3.5–5.5
withanone are the major metabolites present in contains sugars, and rest of the spectrum is domi-
the leaf as shown by NMR, and withanolide A nated by aromatic compounds. In the 1H-NMR
and withanone are major metabolites in the root. spectrum of the leaves, the region of amino acids
Other metabolites detected are 27-hydroxy with- started with distinct triplet signals of isoleucine,
anone, 17-hydroxy-27-deoxy withaferin A, followed by two sharp doublets of valine. The
27-hydroxy withanolide B, 27-deoxy withaferin doublet signals of two d-CH3 (0.95, 0.96) leucines
A, and 12-deoxy withastramonolide. Each of were detected by enlarging the spectral segment
the withanolides was quantified by HPLC using using Bruker X-WIN–NMR software. Multiplet
the calibration curve of the standard samples. The signals of g-CH2 of isoleucine appeared at d 1.35,
qualitative and quantitative data on the metabo- and it also shows distinct correlation spectros-
lites were established by NMR and HPLC. The copy (COSY) interactions with d -CH3 of isoleu-
NMR analysis was not enough to provide any cine. Complex multiplet signal at d 1.48 was
useful information about chemical constituents observed due to overlapping signals of b-CH3 of
of the n-butanol fraction. The HPLC–PDA analy- alanine (doublet) and multiplet signals of g-CH2
sis of the n-butanol fraction of leaf is indicating of isoleucine. Branch broad multiplet signals at d
the presence of physagulin, withanoside IV, 1.6–1.8 may be due to the presence of ornithine
and withanoside VI. However, chromatogram of in the extract. The presence of g-aminobutyric
same fraction of root is cumbersome but reason- acid (GABA) in the mixture was indicated by
able enough to indicate the presence of withano- characteristic signals at d 1.90 (m, b-CH2), 2.29
side IV and withanoside VI. Presence of these (t, a-CH2), and 3.01 (t, g-CH2) and then distinct
compounds was further confirmed by cochro- correlation in COSY spectrum. Characteristic dd
Metabolic Fingerprints 169

Phenyl alanine

Fumanic acid
Trigonelline

Trigonelline

Trigonelline

Tyrosine
Tyrosine
Uracil

Uracil
9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 ppm

Choline
Glycine
Malic acid
Tartaric acid
β−glucose

Threonine
α−glucose

Lactate
5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 ppm

Isoleucine
succinate

Isoleucine
Threonine
Lactate
GABA

Isoleucine
Malic acid

Alanine

Lactate
GABA

GABA

Valine
Glutamate
Glutamine
Aspargin

Valine
Glutamate
Asparatate

Orinithine

3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 ppm

Fig. 9.3 1H-NMR spectrum of the aqueous fraction of W. somnifera leaves [3]

signals of malic acid appeared at d 2.47–2.68 J = 7.9 Hz; a, d 5.2 and J = 3.8 Hz). GC–MS
(geminal protons), and it showed the expected analysis further indicated presence of other sugars,
correlation with dd signals of neighboring pro- that is, galactose, N-acetylglucosamine, and myo-
tons at d 4.34 (H attached with C–OH). Respective inositol in the extract. The singlet signals at d 4.4
carbon signals appeared at d 43.5 and d 71.5. The appeared due to the presence of tartaric acid. 1H
dd signal at d 2.68–2.79 is indicative of b-CH2 of signals of threonine and lactate generally appeared
aspartate, whereas similar signals at d 2.85–2.90 side by side at d1.33 and d 4.2 due to their low
suggested the presence of asparagine. Strong sin- abundance in the extract. The characteristic signals
glet signal at d 2.40 was the signature of succinic were not clearly observed in the 1D spectrum,
acid. The presence of glutamine and glutamate in but in the 2D-COSY spectrum they appeared dis-
the water part was identified mainly by COSY tinctly. The respective carbon peaks appeared at d
cross peak. Overlapping signals of associated 22.5. Strong singlet signals at d 5.8 indicated one
protons appeared in regions at d 2.0–2.44. Strong of the olefinic protons of uracil and showed clear
singlet signal at d 3.2 is indicative of N(Me3)3 of cross peak at d 7.85 corresponding to olefinic pro-
choline in the extract. Related carbon signals tons signals. Sharp singlet around d 6.5 represented
appeared at d 55.1 and d 75.3. The 1D spectral fumaric acid in the extract. Signals at d 6.88 and d
range at d 3.2–4.2 of the carbohydrate region is 7.18 were assigned to tyrosine, which was sup-
highly congested. It was very difficult to identify ported by COSY experiments. The branch spectral
any particular signals, but all the carbon signals band from d 7.3 to d 7.45 regions and correspond-
related to carbohydrate skeleton were relatively ing COSY analysis suggested the occurrence of
distinct (d 63.0, 73.0, 74.2, 76.6, 78.2, and 96.9) phenylalanine. The signatures of trigonelline were
in the HSQC spectrum. Overall nature of the observed at d 8.1, 8.8, and 9.1 ppm [3].
1
spectrum suggested the presence of high amount H spectral complexity (overlapping signals)
of sugars in the extract. However, the presence of did not allow quantification of all the metabolites.
a- and b-anomers of glucose was clearly identified However, a number of them were quantified by
by their respective doublet signals (b, d 4.61 and integrating the distinct characteristic signals of
170 9 NMR Spectroscopy: Herbal Drugs and Fingerprints

each metabolite with respect to signal intensity of or metabolic profiling. The second view is the
quantified amount of TSP (trimethylsilyl propi- macroscopic view (untargeted approach) which
onate). NMR spectroscopy of the aqueous ali- aims at identifying and quantifying all the metab-
quots of root samples was not distinctly olites present in a specific organism. Metabolomics
informative as very high concentration of sugar is therefore a “systematic study of the unique
in this fraction masked other minor signals. chemical fingerprints that specific cellular pro-
However, GC–MS analysis of the extract of root cesses leave behind,” and more specifically, the
samples indicated the presence of higher amounts study of small molecules produced during the
of fructose, galactose, glucose, and glycerol metabolism and their profiles is known as metab-
besides some minor amino acids [3]. olomics. The metabolome represents the collec-
The metabolomic fingerprinting of herbal tion of all metabolites in a biological organism,
extracts is desirable to standardize drugs and to which are the end products of its gene expression.
establish the scientific basis of their pharmaco- The metabolic fingerprinting can give an instan-
logical action. This study recruited 1D- and taneous snapshot of the physiology of that cell.
2D-NMR, HPLC–PDA, and GC–MS techniques The analytical tools used to study the metabo-
for rapid metabolome analysis of Withania leaf lome include mainly chromatography and spec-
and root extracts. Such analysis is desirable for troscopy. Chromatography is used to separate
developing herbal drugs and establishing associa- the metabolites which are then being quantified
tion with their action through functional genom- and identified with the use of various detectors.
ics and molecular pharmacology. Such knowledge The two main chromatography systems employed
is essential to evolve directions for genetic in metabolic fingerprinting are GC and HPLC.
improvement of medicinal plants for the enhance- The detectors used in these chromatography sys-
ment of the biosynthesis of bioactive molecules. tems depend largely on the type of components
High-resolution NMR like 800/1,000 MHz or to be detected and quantified. For a targeted
high-resolution instruments like Fourier trans- approach, MS is usually employed as the detec-
form ion cyclotron resonance mass spectrometer tor, while NMR and MS are used without the
(FTICR MS) can resolve even higher number of chromatography step as an untargeted analytical
molecules and establish those quantitatively in tool. Presently with 1H-NMR and 13C-NMR
different plant parts. Such a wide metabolomic techniques, metabolomics is rapidly developing
analyses is helpful to generate discrete parame- for studying plants, quality control of plant-
ters for better defining and quality control of based products, and drugs [5].
herbal extract, as well as to serve as diagnostics For a proton(s)-bearing molecule, the inten-
for their true identity and adulteration with other sity of all proton signals is absolutely propor-
plants or non-usual parts of the same plant [3]. tional to the molar concentration of the molecule.
Plants are one of the most important sources Using a proper internal standard, the real concen-
for the development of new drugs. Presently, tration of the molecule is easily calculated [6] as
plant extracts and plant-derived components are NMR spectroscopy has a very high reproducibil-
being used in a multitude of herbal remedies and ity. NMR, as a tool for metabolomics, has unique
phytopharmaceuticals. In the literature three advantages over MS-based methods as it provides
terms are in practice to describe the analysis of a detailed analysis on the biomolecular composi-
metabolites produced by plants are as follows: tion very quickly with relatively simple sample
the metabolic profiling, metabolic fingerprinting, preparation [7]. It is a universal detector for all
and metabolomics. A very simplistic view is that molecules containing NMR-active nuclei, unlike
metabolomics can be seen in two different ways. MS where detection of analytes is influenced by
Firstly, there is the microscopic view (targeted selective ionization or ultraviolet spectrometers
approach) which looks at a specific set of com- where only chromophore-bearing compounds are
pounds (e.g., phenolics or terpenoids). This detected. In metabolomics all data obtained from
approach is also called metabolic fingerprinting the analytical methods are further analyzed by
Metabolic Fingerprints 171

Sample preparation
(harvesting, drying and extraction) 1H-1H-J-resolved

1H-NMR analysis
COSY, TOCSY
Data bucketing Long range-COSY
Multivariate data analysis
HMQC (HSQC)
Differentiating signal sorting

2D-NMR analysis of sorted signals HSQC –TOCSY


(Semi) Purification using chromatography
(or on-line LC-NMR) HMBC

Structure elucidation

Fig. 9.4 Schematic flow chart for NMR-based metabolomic analysis [5]

statistical methods in order to extract all possible (e.g., water drought, sunlight, temperature, and
information. pH of soil), and harvesting time greatly affect the
For the accuracy and correctness of the data, metabolome obtained even from the same geno-
further analysis is done by the statistical methods type. For these aspects, sampling of the target
for inevitably reliant on the robustness of the raw materials is carefully planned to avoid increased
analytical data set. Thus, NMR has a unique biological variation interfering with the final data
advantage, the highest reliability in metabolom- interpretation. After harvesting, the sample is fro-
ics. Unlike the retention time in chromatography- zen immediately to avoid any (bio) chemical
based techniques, with a few exceptions, the change in the material. The next step is grinding
chemical shift, coupling constant, and integral of and extraction of the material to liberate the
each signal in an NMR spectrum do not change metabolites from the cells. During the extraction
as long as they are measured under the same con- (bio) chemical reactions may occur in the mate-
ditions: the field strength applied, the solvent, the rial, reflected in changes in the metabolome. This
pH, and the temperature. Despite the low intrin- is avoided by drying the material before extrac-
sic sensitivity, the robustness of data and ability tion either by heat or freeze drying, keeping the
to cover a broad range of metabolites has enabled material at a low temperature, or grinding at low
NMR to be a favored tool for overall macroscopic temperatures and/or in the presence of a solvent
metabolomics and fingerprinting. In addition to that denatures enzymes involved in metabolite
the advantages of data robustness, the utility of alteration. Denaturation can also be achieved by a
NMR techniques for structure elucidation of brief microwave treatment. For the development
metabolites is also matchless [5]. of an optimum extraction method, deep freezing
in liquid nitrogen followed by mechanical grind-
Sample Preparation, Extraction, ing, sonication with a probe head or cup horn, and
and Purification bead beating prior to solvent extraction is evalu-
NMR-based metabolomics includes sample prep- ated for the efficient breakage of the cell using a
aration, extraction, multivariate data analysis, mycobacteria, Mycobacterium bovis. From these
and identification of metabolites (Fig. 9.4) (like evaluated methods, sonication has been found to
other analytical methods). The sample prepara- be superior to others [8], as there is no single
tion steps need much caution in order to obtain extraction method to extract all possible metabo-
reproducible and reliable results. Because in the lites in an organism. The polarity of the solvent
metabolomic analysis, the age, type of tissues, limits the range of metabolites that can be
developmental stage, environmental conditions extracted. pH is a factor that affects the profile of
172 9 NMR Spectroscopy: Herbal Drugs and Fingerprints

1
metabolites extracted; for example, alkaloids are H-NMR Detection
soluble in nonpolar solvents at basic pH and in
aqueous solvents at acidic pH. To overcome these In NMR-based metabolomics, the 1H-NMR spec-
problems in part, a two-phase solvent system con- trum provides a wealth of chemical information.
sisting of chloroform–methanol–water (2:1:1) has With the aid of chemical shifts and coupling con-
been applied to extract both polar and nonpolar stants, some metabolites (amino acids, phenolics,
compounds in a single extraction [5]. This method sugars, and TCA-related organic acids) can be
was also applied to metabolic fingerprinting of easily identified. The observed chemical shift
Ephedra with as light modification, by addition of positions and spin–spin coupling pattern for
NH4OH, for benzylamine alkaloids extraction [9]. each proton provide information as to what
However, this elaborate two-phase extraction kinds of protons are found in the molecules
method was found to be problematic when deal- and, subsequently, how the protons are arranged.
ing with a large number of samples because of the Furthermore, the concentration of each metabo-
long processing time and the possibility of degra- lite in the sample can be easily calculated from
dation or losses during processing. Recently, a the integration of the signals in the spectra, and
simple direct extraction method with deuterated there is no requirement for calibration curves to
NMR solvents has been developed for sample convert signal intensity into concentration as
preparation, which in general gave better quality used in other methods. For obtaining reproduc-
NMR spectra. Single solvent systems have ible results on certain concentration, the relax-
been used for some studies. Methanol with ation delay, the pulse width, and the acquisition
trifluoroacetic acid (TFA) has been found to be a time are the main parameters. The optimum range
suitable solvent for the extraction of alkaloids, of these parameters in 1H-NMR has been excel-
while perchloric acid was routinely used for the lently reviewed by Pauli et al [10]. One of the
more polar metabolites, which prevents enzymatic problems in measuring 1H-NMR spectra is the
degradation of metabolites. However, a combina- water signal, a huge signal caused by residual
tion of CD3OD and D2O was found to be a more water, which overlaps with the anomeric protons
preferable solvent, since it can extract more of sugars or glycosides (d = 4.8–5.2). To suppress
diverse metabolites. The combination of these this undesired water signal, several methods have
two solvents was used in different ratios, for been applied, for example, addition of paramag-
example, 30–70% of CD3OD depending on the netic ions like Mg2+ followed by Meiboom–Gill
study. In most of our work, a mixture of metha- modification of the Carr–Purcell (CPMG) spin
nol-d4 and KH2PO4 buffer in D2O (pH = 6.0) has echo pulse program and pre-saturation using an
been used, which extracts a wide range of metab- additional pulse. When the pre-saturation tech-
olites including amino acids, carbohydrates, nique is applied during relaxation delay, the most
fatty acids, organic acids, phenolics, and terpe- common method, unwanted reduction of the sig-
noids in a single step. The direct extraction nal intensity close to the suppressed water might
method with deuterated NMR solvents saves occur. For correct suppression, the temperature
time and makes it possible to deal with a large and pH of samples are carefully adjusted since
number of samples. Moreover, the buffer in the the water signal is highly affected by these fac-
solvent can avoid possible fluctuation of chemi- tors. Depending on the molecular size of metabo-
cal shifts of signals in the NMR spectra. However, lites, specific pulse sequences have been applied.
when commercial herbal preparations are ana- To filter out the signals from small molecules
lyzed, a more targeted approach is used, focus- from those of large ones, spin diffusion differ-
ing on the known major bioactive compounds in ences are utilized. It has been established in an
the plant material. For instance, in the case of NMR-based study on toxin-induced changes in
St. John’s wort (Hypericum perforatum), both lipoprotein profiles. In the case of a matrix con-
tablets and capsules were analyzed using a mixture taining macromolecules (e.g., proteins or lipid
of methanol–pyridine (6:4, v/v) [5]. vesicles), the application of a spin echo sequence
Metabolic Fingerprints 173

like the Carr–Purcell–Meiboom–Gill (CPMG) some metabolites can be found that are character-
pulse sequence allows the attenuation of unwanted istic of a class. Of the multivariate data analysis,
resonances from macromolecules. However, in principal component analysis (PCA) and partial
any 1H-NMR spectra of plant extracts using least squares projections to latent structures
diverse pulse sequences, there are congested sig- (PLS) are on the verge of becoming routine pro-
nals of metabolites. The multivariate data analy- cessing steps for raw analytical data. PCA is
sis is a key step for sorting out the discriminating designed to extract and display the systematic
signals from the complex spectra. Prior to the variation in a data matrix, and PLS is a regression
multivariate data analysis, all signals in the extension of PCA, which is used to reveal the
obtained 1H-NMR spectra should be digitalized. correlation between two kinds of data sets [12].
This is achieved by the so-called bucketing pro- Both PCA and PLS are projection methods by
cedure, which divides the spectrum into small reducing original data to a few principal compo-
bins and sums all intensities in each bin. Ideally, nents in order to describe maximum variation
a smaller bucket size is better to examine subtle within the data. All raw data (e.g., the integral
perturbations in the metabolome. However, values of all chemical shifts in a 1H-NMR spec-
because of unfavorable fluctuations of chemical tra) of samples are plotted in a K-dimensional
shifts, even with a fixed temperature and pH of space (K = number of variables, for example,
samples, one can only use 8–16 Hz (0.02– chemical shifts in 1H-NMR spectra), and the plot-
0.04 ppm in 400-MHz NMR) for the bucket size. ted samples are projected onto the line generated
It is also possible to use full-resolution NMR by least square sense (principal component line).
data for the multivariate data analysis, and full- The score of each sample is obtained along the
resolution NMR data sets (0.15 Hz per data point PC line. The next principal components can also
resulting in 30,000 variables) gave more precise be calculated by projection onto the line which is
information about constituents responsible for orthogonal to the previous PC line or space. In
data clustering, compared to the use of integrated general, the first three PCs are used for the analy-
NMR data sets (0.04 ppm bucketing resulting in sis. For the data analysis, score and loading plots
200 variables). Some aligning methods were are used. The score plot is useful for observation
found to improve the separation of NMR data for of any groupings in the data set and in addition
further statistical analysis [5, 11]. will highlight outliers that may be due to errors in
sample preparation, experimental conditions, or
instrumentation parameters. The coefficients by
1
H-NMR Spectra to Recognize Patterns which the original variables should be multiplied
and Find Discriminating Signals to obtain the PC are called loadings. The numeri-
cal value of a loading of each variable on a PC
Currently in metabolomics studies, many hun- shows how much the variable has in common
dreds or sometimes thousands of samples are with that component. Prior to the PCA and PLS
routinely analyzed, and a minimum of several analyses, raw data are scaled in different ways.
hundreds of signals is detected in each sample. It There are several ways to scale the data. For unit-
is crucial to extract the relevant information from variance scaling, the standard deviation and the
such a huge data set. The samples are grouped scaling weight as the inverse standard deviation
based on the analysis of the metabolome (pattern are calculated. Subsequently, each variable is
recognition), and a discriminating metabolite multiplied by the inverse standard deviation.
(biomarker) can be found by multivariate data Each scaled variable then has equal variance.
analysis. The pattern of a class represents the This method is useful when the classification
information about the relations between the relies more on differences between minor com-
observations within the class, discerning, which pounds, since it increases the influence of weaker
are similar, which are different, or which are signals. However, applying the unit-variance scal-
atypical outliers. Through the pattern recognition, ing to an NMR data set, it is difficult to interpret
174 9 NMR Spectroscopy: Herbal Drugs and Fingerprints

the loading plot obtained from the unit-variance Indeed, one of the advantages of NMR spectroscopy
scaling method because all signals have the same in plant metabolomics is the power of structure
variance and consequently are different from the elucidation of molecules in a complex mixture.
original NMR spectra. With mean centering, The 2D-NMR spectroscopy available for metab-
the average value of each variable is calculated olomics can be divided into two types, resolved
and then subtracted from the data. This improves and correlated spectroscopies according to the
the interpretability of the loading plot since it type of dispersion in the second frequency
resembles the original NMR spectra. Also, the dimension. A typical resolved technique is the
spectral noise is removed for the analysis as long 2D-1H-1H-J-resolved spectrum. It provides addi-
as intensities of noise are lower than real signals. tional information on spin–spin coupling con-
However, the influence of minor signals might stants, together with chemical shifts. One of the
be excluded since the mean-centering method difficulties for interpreting a 1H-NMR spectrum
always retains the original value of signals. is the interpretation of the splitting pattern of
In particular, the signals of plant secondary each signal. If a target signal is detected in a
metabolites can be underestimated compared to crowded region overlapping with other signals,
high levels of primary metabolites. As an alterna- the first step is to identify its splitting pattern.
tive technique, the so-called Pareto scaling has In addition to this use of J-resolved spectra in
become more common. Pareto scaling gives each structure elucidation, projected spectra on to the
variable a variance numerically equal to its initial axis of chemical shift have been applied in sev-
standard deviation instead of unit variance. eral studies. The projected spectrum produces a
1
Hence, Pareto scaling is intermediate between H-NMR spectrum consisting of singlets for each
1
the extremes of mean-centered and unit-variance H chemical shift, that is, a fully proton-decoupled
scaling, which gives some weight to minor sig- proton NMR spectrum. In the projected spectrum
nals, and at the same time it provides interpreta- all signals from macromolecules which have
ble loadings. In recent years variable stability short T2 relaxation times are readily suppressed
(VAST) scaling which weights each variable because the pulse sequence of the J-resolved
according to a metric of its stability improved the technique is based on a spin echo sequence. Thus,
class distinction and predictive power of PLS-DA baseline shifting caused by proteins, oligo- or
models for 1H-NMR spectra of urines [5]. polysaccharides, which might overlap minor sig-
nals or result in overestimation of target signals,
is clearly reduced in the projected spectra and
2D-NMR Spectroscopy to Compare lead to better separation in multivariate data anal-
Metabolites ysis. Another promising resolved technique is
diffusion-ordered spectroscopy (DOSY) where
In NMR spectroscopy signals are used to dis- each NMR signal is resolved based on the molec-
criminate sample groups by multivariate data ular diffusion coefficient. DOSY can be used for
analysis. Some metabolites like carbohydrates, molecular weight evaluation of unknown, purified
amino acids, or organic acids related to the TCA constituents by preparing calibration curves
cycle can be elucidated merely based on their with known compounds. Although DOSY has a
chemical shifts in 1H-NMR spectra. However, for potential to differentiate metabolites having the
most cases, the spectral complexity and signal same chemical moieties and different molecular
overlap in the 1H-NMR spectra are too high for weights in plant extract, for example, glycosides
identification. Chemical structures of metabo- from their aglycones, the application in metabo-
lites, especially species-specific plant secondary lomics has been limited so far to the analysis of
metabolites, are confirmed by additional meth- polymers like polysaccharides from insects or
ods. Diverse two-dimensional (2D) NMR spec- mushrooms, interaction between small molecules
troscopy gives information on chemical structures. and ligands, and hydrocarbon mixtures [5].
Metabolic Fingerprints 175

Following the resolved spectra, information relaxation time of 13C requires a long measuring
on correlation between protons or carbon–proton time in order to obtain quantitative results. To
can be obtained by homonuclear and heteronu- overcome the low sensitivity of 13C, heteronu-
clear experiments. A typical proton homonuclear clear correlation techniques are used from which
technique is COSY that shows which signals in a the information on 13C chemical shifts is indi-
proton spectrum have mutual spin–spin cou- rectly obtained. Direct correlation between car-
plings. The resulting spectrum has the conven- bons and protons can be detected by heteronuclear
tional 1D-NMR spectrum along the diagonal multiple quantum coherence (HMQC) or hetero-
cross peaks at chemical shifts corresponding to nuclear single quantum coherence (HSQC) which
pairs of coupled nuclei. In general protons within gives better sensitivity than HMQC but needs
three bonds are well correlated in the COSY phase correction. In both HMQC and HSQC
spectrum but in sp2 spin systems such as olefinic spectra, any carbon attached directly to a proton
and phenolic compounds, even the correlation is detected; consequently the chemical shift of a
13
beyond three bonds is readily detected. A C connected to a specific proton can be obtained.
modified pulse sequence of COSY, long-range The assignment of C-6 and C-8 of
COSY, can selectively detect correlation between 5,7-dihydroxyflavonoids can easily be learned
protons far from each other (more than three from the HMQC spectra of the extract of aerial
bonds). It can show structure confirmation of parts of Genista tenera. In the HMQC spectra the
connectivity between two molecules which is not unusual upfield shift of those aromatic carbons
possible to detect in a normal COSY spectrum. It compared to other aromatic carbons, caused by
is one of the most time-consuming tasks to define the adjacent oxygen, confirmed the presence of
NMR signals belonging to the same molecule in 5,7-dihydroxyflavonoids [5, 13]. Long-range cor-
the complex 1H-NMR spectra of a mixture. In par- relations (two and three bonds) between carbons
ticular, most 1H signals of carbohydrates are dete- and protons can be observed by heteronuclear
cted in an extremely crowded region (d = 3.0–4.5) multiple-bond correlation (HMBC) spectra. It is
overlapping with each other or with the signals of an extremely powerful tool for structure eluci-
amino acid protons close to the amino group. A dation since carbon–carbon correlations can be
powerful 2D-NMR technique, total correlation indirectly obtained with the aid of HMQC or
spectroscopy (TOCSY), or the Hartmann–Hahn HSQC. In addition, correlations between quater-
experiment (HAHAHA) provides information nary carbons like carboxylic acid and nearby pro-
on unbroken chains of coupled protons in the tons can be seen in the spectra. The long-range
same molecule. For example, if anomeric protons couplings in HMBC spectra can give information
of carbohydrates are defined, the rest of the sig- on the connectivity between two moieties which
nals in the crowded region can be easily assigned are connected only by quaternary carbons or
using the correlation in the TOCSY spectrum. heteroatoms (e.g., O-glycosides). One of the
All organic molecules have carbons as their advantages of NMR-based metabolomics is that
chemical backbone. The information on the car- there are numerous combinations of 2D-NMR
bons is thus crucial for structure elucidation. In experiments that provide information on struc-
fact measurement of a 13C-NMR spectrum is an tures by diverse correlations. Aiming at specific
important, if not indispensable, step to elucidate correlations, enhancing resolution, or simplifying
the chemical structure of a pure compound. spectra HMBC-J-resolved, DOSY-selective
However, the magnetically active 13C isotope has TOCSY, 2D-J-DOSY, and DOSY–HMQC has
too low sensitivity for the application to the anal- been applied to the analysis of complex mixtures.
ysis of the metabolome since the natural abun- The combined HMQC (or HSQC)–TOCSY
dance of 13C is only 1.1% of 12C and its sequence experiment leads to a 13C-edited TOCSY
magnetogyric ratio is one fourth of 1H. It results spectrum and information on all connectivity of
in 1/57,000 of the 1H sensitivity. Also, the long carbons in each molecule in a mixture [5].
176 9 NMR Spectroscopy: Herbal Drugs and Fingerprints

nonvolatile constituents have been identified.


Evaluation of Quality Characteristic constituents of saffron are crocin
of High Therapeutic-Value Herbals (1) and congeners, which are a family of water
soluble glycosides of crocetin (2) contributing to
Case Study 2 the red color of saffron, numerous monoterpe-
noids such as safranal (3), responsible for the
Saffron (Crocus sativus) is the most expensive aroma of saffron, and picrocrocin (4), which is a
spice, and its production is extremely laborious, precursor of safranal (Fig. 9.5).
so low-quality saffron is frequently encountered, In addition, saffron contains numerous
containing styles and petals instead of or in addi- other glycosylated monoterpenes, carotenoids,
tion to the stigmata, or even falsified saffron con- flavonoids, and simple aromatic compounds. A
taining dyes and artificial flavors, or other plant study, for determination of sample quality and
products. The chemical composition of saffron is variability as well as means of unsupervised
the most important indicator of its quality and detection of adulteration in C. sativus, 1H-NMR
value. Several analytical techniques are available metabolic fingerprints of 63 saffron samples,
for assessment of various constituents to deter- consisting of authentic reference samples from
mine the quality of this spice. These include Iran (31 samples) as well as samples purchased
UV–Vis. spectroscopy, near-IR spectroscopy, as from retail stores in several countries (32 sam-
well as various chromatographic techniques. GC ples), provides immediate means of unsupervised
and HPLC techniques are appropriate for quanti- classification of saffron samples. The detail of
tative determination of specific metabolites in the experiment is as follows.
saffron. In recent years, 1H-NMR spectroscopy
emerged as a useful method for plant metabolite
fingerprinting with the purpose of sample Materials and Chemicals
classification and determination of discrimina-
tory features. The advantages of 1H-NMR spec- Saffron samples from Iran (n = 31, samples 1–31)
troscopy combined with chemometric methods were produced in Khorasan, Iran, near Torbat-e
are that (there is no need of separation of sample Heydariyeh, in the area spanning from 34º 20¢ N
constituents, and also there is no need of prior to 35º 47¢ N and from 58º 15¢ E to 60º 37¢ E, at
knowledge about expected sample composition altitudes 1,000–1,750 m. The plantation ages
or external calibration of the response) the infor- ranged from 2 years to 9 years with 24 (75%) of
mation content of individual spectra is high and it the samples coming from plantations in the range
is possible to identify discriminating constituents of 3–5 years. The drying conditions were stated as
from spectral features revealed by loading plots “near the heater” (n = 8), “shadow” (n = 19), and
or covariance matrices. 1H-NMR spectroscopy of “sun light” (n = 4). The moisture and volatile con-
plant material extracts also offers good reproduc- tent of the samples were below 12%. Commercial
ibility and robustness [14]. samples (n = 32, samples 32–63) were purchased
The chemical composition of saffron has been in shops and bazaars in Denmark (n = 17, samples
thoroughly studied, and numerous volatile and 41–45, 47, 49–59), Iran (n = 2, samples 60–61),

Fig. 9.5 Therapeutic ingredients of C. sativus [14]


Evaluation of Quality of High Therapeutic-Value Herbals 177

Sweden (n = 4, samples 46, 48, 62–63), and Turkey was 8 k for each spectrum (12-kHz spectral
(n = 9, samples 32–40). Deuterated methanol (99.8 width, 32 transients each), Fourier transformed
atom % of deuterium) was obtained from Euriso- to 16 k with an exponential multiplication line
Top (Saclay, France) [14]. broadening factor of 1.0 Hz, and a polynomial
baseline correction of the FID (degree 5) to
Sample Preparation reduce the water signal. One tenth of all data
Grinded saffron samples (10 mg) were extracted points, corresponding to the highest intensities,
with CD3OD (1,000 ml) by maceration for 1 h at were integrated over 30-Hz ranges and the inte-
room temperature with two 10-min periods of grals fitted to the function I(t) = b × (1-a × e –t/T1)
sonication at the beginning and at the end of the in Matlab using function in Curve Fitting
extraction period. The mixtures were centri- Toolbox (version 2.1, The MathWorks Inc., MA,
fuged for 5 min.; 700 ml aliquots of the superna- USA) [14].
tant were transferred to 5-mm NMR tubes for
analysis. Two replicates of each extract were Spectral Analysis
prepared [14]. The spectra were imported into Matlab (ver. 7.7,
The MathWorks Inc., MA, USA) using in-house
1
H-NMR Spectroscopy written routines and calibrated to residual solvent
1
H-NMR data were recorded at 298 K on a signals at d 3.31, normalized with respect to the
Bruker Avance 600-MHz spectrometer equipped receiver gain setting, and baseline corrected by
with a 5-mm inverse probe head and a 25-posi- fitting cubic polynomial functions through sig-
tion sample changer. For each analysis, 256 tran- nal-free ranges of the spectra. The spectral matri-
sients were collected as 64 k data points with a ces were prepared by integration of 0.04-ppm
spectral width of 12 kHz (20 ppm), using 308 wide buckets in the range 0.5–11 ppm. Regions
pulses and inter-pulse delay of 3.65 s. Each sam- corresponding to residual NMR solvent (d 3.33–
ple was analyzed twice by a second revolution of 3.29 and d 5.05–4.50) were excluded. Vicinal
the sample changer. The acquisition was con- buckets with a correlation coefficient greater than
trolled by in-house written scripts running under 0.95 were merged. The data were normalized to
XWIN-NMR and ICON-NMR (Bruker Biospin, constant sum, Pareto scaled and analyzed using
Karlsruhe, Germany). The protocol included a PLS Toolbox 5.2 (Eigenvector Research, Inc.,
180-s temperature equilibration period, gradient WA, USA) running within Matlab. For statistical
shimming using the z-axis profile of the 2H-NMR correlation spectroscopy, the spectra were inte-
signal of the solvent, receiver gain adjustment, grated in 0.01-ppm wide buckets resulting in
and acquisition. The total time for each NMR 1,105 variables and analyzed without further nor-
analysis was 25–32 min. The free induction malization or scaling with functions in Statistics
decay signal was zero filled to 128 k and expo- Toolbox (ver. 7.1, The MathWorks Inc., MA,
nentially multiplied with a 0.3-Hz line broaden- USA) [14].
ing factor. The zeroth order phase constants were Methanol was considered appropriate solvent
manually optimized after Fourier transform. for polar (crocins and other glycosides) as well as
Gradient-enhanced heteronuclear multiple-bond nonpolar (terpenoids) constituents, and in order
correlation (HMBC) spectra were acquired with to minimize sample preparation, saffron was
2 k × 256 k data points for 7 kHz × 36 kHz sweep extracted directly with the deuterated solvent.
width, with 64 spectra per increment and 65-ms Two replicates of each extract were prepared for
1
mixing time. Longitudinal relaxation time con- H-NMR analysis. Longitudinal (T1) relaxation
stants (T1 values) were determined by an inver- times were determined for representative samples
sion recovery experiment (180º pulse-variable in order to choose acquisition parameters that
delay-90º pulse-acquisition) with 16 variable result in quantitative data, that is, true molar
delays ranging from 15 s to 5 ms and a relaxation ratios between the constituents present (e.g.,
delay of 20 s. The number of acquired data points Fig. 9.6).
178 9 NMR Spectroscopy: Herbal Drugs and Fingerprints

Fig. 9.6 Example of 1H-NMR spectrum (600 MHz) of a across sample components. The displayed spectrum is the
saffron extract in methanol-d4 showing the range of T1 val- most relaxed spectrum of an inversion recovery experi-
ues (red lines, with 95% confidence intervals in blue) ment [14]

Two spectra (repeats) were recorded for each Molecular Modeling


sample, resulting in four spectra for each of the
63 saffron samples. One spectrum was excluded Molecular modeling may be defined as a tech-
due to low quality, and the final data set consisted nique for deriving, representing, and manipulat-
of 251 spectra. In a preparatory phase of this ing the structures and reactions of molecules,
project, saffron samples were also extracted with with computer and software, and those properties
excess of chloroform or methanol–water followed that are dependent on these three-dimensional
by evaporation and reconstitution of the residue structures.
with a small amount of the corresponding deuter-
ated solvent, but these sample preparation
methods were found to give poor reproducibility Case Study 3
as judged from large inter-sample variation in
score plots after principal component analysis In a study the mixture of turmeric, ginger, and
(PCA); possibly, this could be attributed to partial seeds of fenugreek (generally used in joint pain
loss of volatile constituents. relief and heart diseases in rural area of Indian
The main differences among authentic saffron subcontinent) was used for molecular modeling.
samples are thus the relative amounts of picro- The main constituents of turmeric are curcumi-
crocin and the sum of different crocetin glyco- noids (mixture of curcumin, de-methoxy cur-
sides. The most abundant crocetin glycoside in cumin, bis-demethoxy curcumin), and those in
C. sativus stigmata is reported to be the di-(b-d- ginger are gingrol and hexahydrocurcumin. The
gentibiosyl) ester of crocetin (1). Variants include past phytochemical investigations of fenugreek
symmetrical and unsymmetrical crocetin esters seeds reveal the presence of diosgenin, trigonel-
of b-d-glucose, b-d-gentibiose, and b-d-neapoli- line, gitogenin, vicenins 1 and 2, vitexin, querce-
tanose, as well as isomers with a cis-double bond tin, luteolin, kaempferol, sitosterol, etc.; moreover,
next to the inner methyl group of crocetin (2). The the endosperm of the seeds is rich in galacto-
presence of the mixture of different crocetin mannan [15].
esters is reflected in the 10 principal components In an attempt to isolate a mixture of these
needed to adequately describe the data set of 1H- spices and spectroscopic characterization of iso-
NMR spectra of authentic Iranian saffron extracts. lated compound, turmeric rhizome, ginger, and
However, the spectral overlap makes it impossible fenugreek seeds were washed to clean, dried in
to interpret the influence of individual components air oven at 60 °C and grinded to a powder using
separately. This study showed that it is highly an electric grinder to pass a 0.4-mm screen. The
feasible to use unsupervised PCA models of powder was taken in a cleaned round-bottom
1
H-NMR metabolic fingerprints to assess the flask having methanol and stirred for 12 h. The
authenticity of saffron [14]. mixture was filtered and botanical mass was
To Decipher the Herbal Constituents 179

Fig. 9.7 1H-NMR spectra of complex compound [15]

washed with hot methanol. The filtrate was dis- To Decipher the Herbal Constituents
tilled and the residue was redissolved in excess
warm methanol, and clear solution was kept The therapeutic potential of medicinal plants is
undisturbed for weeks to give beautiful gray also effected by abiotic factors; cultivation prac-
crystal. The novel compound having chemical tices and harvest processing pattern affect the
structure (molecular formula C36H57N3O24) was chemical composition and clinical efficacy of
obtained. Various attempts to obtain the single herbals and herbal products. So it is necessary to
crystals have so far been unsuccessful. The 1H- establish a method to control the quality of herbs
NMR spectra (Fig. 9.7) of the synthesized com- and herbal products. The most common and suit-
pound have well-resolved signals. The compound able approach is analyzing chemical markers
shows the resonance with integrated intensities. which are supposed to be present in the analyte.
The chemical shift of the compound appeared at
3.38–3.64 (8H, m) for OH of alcohol, 2.0(3H, m)
for NH of amine, 3.09–5.03, (8H, m) for CH, Case Study 4
7.27–7.30 (5H, m) for CH of benzene, and 3.39–
1.18 (9H, d) for CH3. The compound was charac- In a study for quantitative analysis of sennosides
terized by various spectroscopic techniques and A and B in the herbal slimming teas and laxative
has triclinic crystal system. The molecular struc- formulations in Turkey, the powdered leaves of
ture has been optimized by MM2 calculation. Cassia acutifolia (300 g) were extracted with
This compound may be used as medicine in the methanol (3 × 3.5 L, 45 °C), and the combined
future (after passing all regulatory norms), extracts were evaporated to yield 44.0-g residue.
because in Indian subcontinent the mixture of The methanolic soft mass was dissolved in water
turmeric, ginger, and fenugreek in powder form and partitioned with petroleum ether, ethyl ace-
is being used traditionally as pain killer, to treat tate, and n-butanol. n-Butanol extract (9.56 g)
heart disease and regulate blood sugar [15]. was fractionated on a Sephadex LH-20 column
180 9 NMR Spectroscopy: Herbal Drugs and Fingerprints

Fig. 9.8 The compounds from the senna leaves [16]

eluting with 70% methanol to give eight main data given in the literature for kaempferol 3-O-b-
fractions (named as fraction A–H). The fraction d-gentiobioside.
C was chromatographed over silica gel eluting Compound (2): Orange amorphous powder; 1H-
with chloroform–methanol–water (100:10:5) to NMR (CD3OD, 400 MHz) and 13C-NMR
give fraction “a” (161.3 mg). Further column (CD3OD, 100 MHz) data were in agreement with
chromatography was applied on fraction “a” data given in the literature for aloe-emodin
using silica gel and eluted with chloroform– 8-O-b-d-glucopyranoside.
methanol–water (80:20:2–60:40:4). A mixture Compound (3): Yellow amorphous powder; 1H-
of 1 and 5 (101.3 mg) was obtained. Fractions 1 NMR (CD3OD, 400 MHz) and 13C-NMR
obtained from fraction “a” chromatographed (CD3OD, 100 MHz) data were in agreement with
over Sephadex LH-20 column with methanol data given in the literature for rhein 8-O-b-d-
elution to get pure compound 2 (6.3 mg). glucopyranoside.
Compound 4 (14.6 mg) was obtained from frac- Compound (4): Yellow orange amorphous pow-
tion D using Sephadex LH-20 column and elut- der; 1H-NMR (CD3OD, 400 MHz) and 13C-NMR
ing with methanol. Fraction D gave compound 3 (CD3OD, 100 MHz) data were in agreement with
(8.8 mg) by silica gel column elution with chlo- data given in the literature for torachrysone
roform–methanol–water (70:30:3–60:40:4). The 8-O-b-d-glucopyranoside.
NMR characteristics of compounds (Fig. 9.8) Compound (5): Yellow amorphous powder; 1H-
were as follows [16]: NMR (CD3OD, 400 MHz) and 13C-NMR
Compound (1): Yellow amorphous powder; 1H- (CD3OD, 100 MHz) data are in agreement with
NMR (CD3OD, 400 MHz) and 13C-NMR data given in the literature for isorhamnetine
(CD3OD, 100 MHz) data were in agreement with 3-O-b-d-gentiobioside.
To Decipher the Herbal Constituents 181

Fig. 9.9 UV spectrums and HPLC chromatograms of slimming tea [16]

Senna leaves are one of the herbs, traditionally oxidative decomposition of the sennosides to
used as laxative in case of constipation and herbal rhein 8-O-b-d-glucopyranoside [16, 17].
tea to promote weight loss. In aforesaid study for The laxative formulations were investigated
the quality control of slimming teas claimed for with the same method to prove whether they con-
having senna leaves and including senna extract tain sennosides A and B or not. The content of
enriched by sennoside B was analyzed by HPLC sennosides was quantitatively analyzed by
RP column using gradient elution. The presence HPLC–PDA. The presence of sennosides was
of sennosides A and B in laxative formulation was clearly observed on each chromatogram and their
proved, but slimming teas were devoid of senno- corresponding UV spectra, which proves that all
sides. Each chromatogram was checked with their samples have sennosides A and B (Fig. 9.10).
UV spectra for sennosides, and the lack of sen- When the results were summarized, it was
nosides was proved completely (Fig. 9.9), but an observed that slimming teas did not have sen-
additional compound known as rhein 8–O–b-d- noside B and the percentages of the anthra-
glucopyranoside (the degraded monomer of sen- noids were not of a significant value, but
noside B, by enzymatic processes) was identified, somehow they showed strong laxative effect
which is similar to the standard UV spectra of like laxative drugs which have quite high sen-
rhein 8-O-b-d-glucopyranoside.In another study noside content. The team of scientists writes
it was established that the forced decomposition that these results make us think of the malprac-
of herbal material under high temperature caused tice for herbal teas [16].
182 9 NMR Spectroscopy: Herbal Drugs and Fingerprints

Fig. 9.10 UV spectrums and HPLC chromatograms of laxative formulation [16]

Metabolic Fingerprints to Distinguish Case Study 5


Plant Species
Eleven species of Ilex were analyzed and
NMR metabolic fingerprinting has been applied based on their metabolites; each species could be
to diverse fields of plant research, in which the discriminated, especially Yerba mate (Ilex para-
classification and identification of adulteration of guariensis) from its adulterants. The contributing
plant products are of major interests. Using metabolites were arbutin, phenylpropanoids,
NMR-based metabolic analysis, it was possible caffeine, and theobromine. Caffeine and theobro-
to discriminate different species of plants, for mine were only found in the I. paraguariensis,
example. whereas arbutin was found only in other species.
Quality Assurance of Traditional Medicines 183

The study of flower heads of chamomile but also another secondary metabolite (benzoic
(Matricaria recutita) showed that this technique acid analogue) was found to be an important dis-
can be applied to assess the relative amount of criminating metabolite. Among nine tested com-
stalk materials in the different batches of flowers. mercial Ephedra materials, one was shown to be
Another example of NMR analysis combined the mixture of two species. There are several
with PCA is the application to characterize 3 dif- reports on the discrimination of ginseng (Panax
ferent Strychnos species using different parts of ginseng) preparations, which is one of the most
the plants (seeds, roots, leaves, and barks). All popular herbal medicines. The first report using
samples were clearly classified based on their NMR-based metabolomic approach showed the
metabolites such as brucine, loganin, Strychnos differences in ginseng products, different ages
icaja alkaloids (icajine, sungucine), as well as (4–6 years old), and differently processed (white
fatty acids. Twelve Cannabis sativa cultivars were and red) products. PCA of J-resolved 1H-NMR
discriminated by analyzing the organic fractions spectrum of three commercial ginseng prepara-
and aqueous fractions. Tetrahydrocannabinolic tions showed striking differences. Loading plots
acid (THCA) and cannabidiol acid were the most explained that alanine, arginine, fumaric acid,
important metabolites in organic fractions, while inositol, and ginsenosides are important metabo-
primary metabolites found in the water fraction lites to differentiate preparations from each
such as glucose, asparagine, and glutamic acid other. Other studies also showed that this
served as discriminating metabolites [5, 18]. approach can be used for the quality control of
fresh ginseng roots of different ages and different
origins. Recently, another study for the quality
Quality Assurance of Traditional evaluation of TCM was reported by Tarachiwin
Medicines and coworkers. A combination of an NMR tech-
nique and multivariate analysis showed the dif-
The traditional Chinese medicines (TCM) are ferences in two Angelica acutiloba roots, A.
often used as a mixture of several plants, with a acutiloba Kitagawa (yamato-toki) and A. acuti-
hardly defined composition. Even though there loba Kitagawa var. sukiyamae Hikino (hokkai-
are regulations for quality control, this is often toki), with regard to their geographical and
restricted only to a certain compound or group of variety origin. In a study for application of NMR
compounds. NMR-based metabolic fingerprinting to commercial preparations of Artemisia, NMR
combined with pattern recognition may be a very analysis combined with PCA showed clear dif-
good tool for the quality control of TCM. ferences between A. annua and A. afra. A more
targeted analysis combined with LC–MS showed
that the content of artemisinin in the commercial
Case Study 6 products is different from the commercial claim.
Other studies of NMR-based methods have been
Chemotaxonomic analysis including quality applied to the characterization of crude extracts
control issues was applied in the study of and commercial preparations, including feverfew,
Ephedra [5, 19]. Ephedra is one of the oldest kavakava, Artemisia, and St. John’s wort [5].
medicinal plants known to mankind. However, The major limitation of NMR is its rather low
three different species of Ephedra, E. sinica, E. sensitivity when compared to other analytical
intermedia, and E. equisetina, are commonly methods. The probes for high-resolution NMR,
and widely used without strict distinction as long such as cryoprobes and capillary NMR, help to
as there is a certain amount of ephedrine alka- increase the sensitivity threshold of the technique.
loids (0.8% of dry weight). Using NMR-based CryoNMR aims to limit the receiver coil noise
metabolic fingerprinting, it was possible to dis- and to improve the coil quality factor by cooling
criminate E. sinica, E. intermedia, and E. dis- the receiver coil and the preamplifiers to 25 K or
tachya. Not only the ephedrine type of alkaloids below. With the use of cryogenically cooled
184 9 NMR Spectroscopy: Herbal Drugs and Fingerprints

probes, the detection sensitivity of NMR is at


least 3–4-fold enhanced in comparison with con- Detection of Contaminations
ventional probes operating at the same magnetic
field. Capillary NMR takes advantage from the High-resolution nuclear magnetic resonance
use of small-diameter radiofrequency (RF) coils (HRNMR) spectroscopy is emerging as an
for mass-limited samples (usually in the order of important tool in the quality control of herbals
nanomole quantities). By decreasing the diameter and herbal drugs. The use of the 1H-NMR
of the receiver coil, the signal to noise (S/N) ratio fingerprint as an example of a rapid and simple
improves. Both cryoNMR and capNMR are very method in cases of the contamination of herbal
promising analytical approaches for metabolic products by synthetic substances. In comparison
fingerprinting studies. The main importance of with HPLC/MS (but similar considerations can
chemical characterization of the metabolic com- be deduced in part for TLC and HPTLC), in
pounds is to look for further development of the NMR fingerprinting (apart from the initial invest-
metabolomic approaches. As no single analytical ment) the cost of each analysis can be consider-
technique is suitable for the detection and ably lowered (less material, minor disposable
identification of all the metabolites in a biologi- solvents) in cases of the examination of several
cal sample, the most common strategy to clean up samples. Furthermore, there is no waste (recov-
or isolate and structurally identify metabolites is ery of the sample is possible) and several sam-
to combine chromatographic techniques to MS/ ples can be analyzed in a very short time and in
NMR spectroscopy. The resulting hyphenated the same conditions, avoiding the stabilization
methods, such as various HPLC–NMR tech- and equilibration of the apparatus, as well as its
niques, are successfully applied to natural prod- cleaning. It is not possible to obtain a precise
uct research. An off-line solid phase extraction quantitative analysis, but the amount of a sub-
may sometimes be required to partially purify or stance can be considered by adding a known
concentrate the analytes [5]. solution of the standard to the tested sample [20]
The next generation of plant metabolome sci- or by other proposed methods [21].
entists will probably focus on the cellular and Presently large numbers of herbal-based prod-
even subcellular compartmentation of metabo- ucts are on the market; it is difficult to check
lism. Researchers have started looking into the every batch of these by regulatory authorities for
metabolomics of single cells with the use of their quality. Self-medication of herbal drugs,
laser microdissection (LMD). In a study for especially in cases of analgesic effects, can induce
identification of 68 metabolites, more than half of a consumption of a large quantity of a product, sup-
authors could demonstrate whether they were posed “safe” since it is promoted as “natural and
enriched or depleted in vascular bundles. LMD innovative food supplement”. The presence of an
has also been successfully used in combination adulterant can often easily be found by NMR
with cryoprobe NMR for the analysis of special- fingerprint. Therefore, although HPLC remains
ized cells from the floral and leaf tissues of Dilatris the main analytic instrument, NMR fingerprinting
spp. coming from different herbarium specimens. can easily be applied to evidence, a contamina-
The phytochemical pattern of LMD-derived sam- tion in complex mixtures such as herbal dietary
ples was compared with that of whole leaf extracts, supplements or similar products. However, NMR
and some secondary metabolites were identified lacks high sensitivity, since the impurity or adul-
and connected with the specific cells. The study terant must be present in sufficient quantity to be
revealed the potential of the method in chemot- observed. In this regard simple preliminary extrac-
axonomic applications even of plants that are tion of desired sample by suitable solvent to get
difficult to raise or to obtain. The same kind of enriched form for NMR studies is very useful to
approach, in combination with mass spectromet- decipher the degree of contamination. Based on
ric measurements (LMD/NMR/MS), was carried aforesaid theory and analysis of the P.C. 28 Plus,
out on the stone cells of Norway spruce bark [5]. a product based on herbal drugs marketed by the
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Metals in Herbal Drugs
and Fingerprints 10

Herbal drugs as polyherbal and nanoherbal are in is mainly focused on the elemental utility, toxicity,
use to increase solubility, stability, bioavailabil- and estimation by latest analytical techniques, to
ity, and pharmacological activity. The advantage produce evidence-based herbal drugs.
of herbal as nanodrug include protection from
toxicity, improving tissue macrophages distribu-
tion, sustained delivery, and protection from Atomic Absorption Spectroscopy
physical and chemical degradation. Silver nano-
particles of Ocimum sanctum extract exhibited AAS determines the presence of minerals in liquid
maximum antibacterial activity at a dose of phase, along their concentration in the range mil-
150 mg in wistar rats. The herbal drug incorpo- ligram/liter. In elemental stage, metals absorb
rated antibacterial nanofibrous mat fabricated by ultraviolet light when they are excited by heat.
electrospinning provided a potential application Each metal absorbed a characteristic wavelength,
for use of wound dressing. Nanoherbal therapy as and the AAS looks for a particular metal by
per Chinese herbal practices is widely accepted focusing a beam of a specific wavelength through
in China and recognized globally. Metal ions a flame and into a detector. Thus, atomic absorp-
such as Cu2+, Zn2+, Mn2+, Fe2+, Ni2+, and Co2+ are tion is very much like molecular absorption spec-
essential micronutrients for plant metabolism, trometry where absorption is measured and
but the presence of nonessential metals such as calculated to determine the concentration. The
Cd2+, Hg2+, As2+, and Pb2+ can become extremely major differences lie in instrument design, espe-
toxic. Pollution of environments due to huge cially with respect to the light source, the sample
industrial human activities in recent years, espe- container, and the placement of the monochro-
cially marine fauna and flora, have accumulated a mator. In atomic absorption (AA), the sample of
high concentration of metals, which transfer to interest is aspirated into the flame, the metal
different ways to the soil, finally enter into (if present) in the sample absorb some of wave-
human body. When their concentrations exceed lengths, thus reducing its intensity. The detector
the required levels, they become toxic and cause measures the change in intensity and transfer the
several health problems. Presence of these ele- signal to recorder proportional to the absorption.
ments is to be measured in parts per million As concentration of metal goes up, absorbance
(ppm), with most sophisticated instruments like also increases. An easy and feasible approach for
atomic absorption spectrometry (AAS) and routine analysis is to draw a calibration curve by
inductively coupled plasma (ICP) devices, and running standards of various concentrations,
calculated against the response of an existing against absorbance, and test samples is evaluated
standard for quantification. The present discussion by comparing absorbance with calibration curve.

D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs, 189
DOI 10.1007/978-81-322-0804-4_10, © Springer India 2012
190 10 Metals in Herbal Drugs and Fingerprints

This technique is also known as flame atomic In order to atomize and excite most metal ions
absorption spectroscopy (FAAS), where a liquid and achieve significant sensitivity for quantita-
sample is aspirated and mixed as an aerosol with tive analysis, flame of temperature 2,300°C is
combustible gases (acetylene and air or acetylene required, which is obtained using the air–acetylene
and nitrous oxide) The mixture is ignited in a mixture. Ideally, pure oxygen with acetylene would
flame of temperature ranging from 2,100 to produce the highest temperature (3,100°C), but
2,800°C (depending on the fuel gas used). During such a flame suffers from the disadvantage of
combustion, atoms of the element of interest in a high burning velocity, which decreases the
the sample are reduced to the atomic state. A completeness of the atomization and therefore
light beam from a lamp whose cathode is made of lowers the sensitivity. Nitrous oxide (N2O) used
the element being determined is passed through as the oxidant, however, produces a higher flame
the flame into a monochromator and detector. temperature (2,900°C) while burning at a low
Free, unexcited ground state atoms of the element rate. Thus, nitrous oxide–acetylene flames are
absorb light at characteristic wavelength; this fairly popular. The choice is made based on which
reduction of the light energy at the analytical flame temperature/burning velocity combination
wavelength is a measure of the amount of the works best with a given element. Since all elements
element in the sample. have been studied extensively, presently the
recommendations for any given element are
easily available in the literature sources and
Instrumentation in AAS reference books.
There are two designs of burners for the flame
The light source, called a hollow cathode tube, is atomizer that are in common use, known as “total
a lamp that emits exactly the wavelength required consumption burner” and a few as “premix
for the analysis (without the use of a monochro- burner.” In total consumption burner, fuel, oxi-
mator). The light is directed at the flame contain- dant, and sample all three meet at the base of the
ing the sample, which is aspirated. The flame is flame. The fuel and oxidant are forced, under
typically wide (4–6 in.), giving a reasonably long pressure, into the flame, whereas the sample is
path length for detecting small concentrations of drawn into the flame by aspiration. The rush of
atoms in the flame. The light beam then enters the the fuel and oxidant through the burner head cre-
monochromator, which is tuned to a wavelength ates a vacuum in the sample line and draws the
that is absorbed by the sample. The detector mea- sample from the sample container into the flame
sures the light intensity, which after adjusting for with a nebulizing or mixing effect. This type of
the blank, is output to the readout, much like in a burner head is used in flame photometry, but not
single beam molecular instrument. Also as with useful for AA as resulting flame is turbulent and
the molecular case, the absorption behavior fol- nonhomogenous.
lows Beer’s Law, and concentrations of unknowns The readout pattern is different with AAS,
are determined with the same principle. Double- compared to the molecular spectrometry (as absor-
beam instruments are also in use in AA. In this bance and transmittance, or possibly both). In the
case, however, the second beam does not pass atomic spectrum the wavelength is fixed, and the
through a second sample container (it is difficult absorbance or transmittance is recorded vs. time as
to obtain two closely matched flames). The sec- the various solutions are aspirated. As acetylene is
ond beam simply bypasses the flame and is a fuel so well vented, fume hood above the burner
relayed to the detector directly. This design elim- is necessary to remove burned and unburned fuel
inates variations due to fluctuations in source from the area, and safety glasses must be worn at
intensity (the major objective), but does not elim- all times operating the instrument. In case of AAS
inate effects due to the flame (cuvette) or other chemical interferences usually result from incom-
components in the sample (blank components), plete atomization caused by an unusually strong
so adjusted by reading the blank. ionic bond, for example, in the analysis of a sample
Atomic Absorption Spectroscopy 191

for calcium, from CaCl2 and CaSO4, where cal- Case Study 1
cium chloride completely atomizes, but calcium
sulfate does not, to avoid confusion best way is to Five medicinal plants Fagonia indica, Argemone
match the sample and the standards in terms of mexicana, Cnicus benedictus, Silybum mari-
chloride and sulfate. anum, and Solanum surattense were analyzed for
An element is considered essential when elemental determination, using the aerial parts
reduction of its exposure below a certain limit [1]. Plant material was collected freshly from the
results consistently in a reduction in a physiologi- field. All plant material was first cleaned, dried,
cally important function. More than 40 elements and then powdered using an electric blender.
have been considered essential to life systems for Samples in powder form were used for AAS.
the survival of both mammals and plants. So an Each plant material (0.25 g) was taken in 50-ml
attempt was made to determine what essential flask and add 6.5 ml of mixed acid solution, that
elements and their levels in medicinal plants by is, nitric acid (HNO3), sulfuric acid (H2SO4), and
using AAS. There are three main steps of analy- perchloric acid (HClO4) (5:1:0.5). The sample
sis via AAS that are as follows: sample drying, boiled in acid solution in fume hood on hot plate
sample preparation, and sample digestion. One till the digestion has been completed which was
additional step is required in case of mercury, indicated by white fumes coming out from the
commonly known as mercury reduction. The flask. Thereafter, few drops of distilled water
drying section may not be applicable for all situ- were added and allowed to cool. Then these
ations. During analysis the flame is arranged in digested samples were transferred in 50-ml volu-
such a way that it is laterally long (usually 10 cm) metric flasks, the volume was made up to 50 ml
and not deep. The height of the flame must also by adding distilled water and filtering it using
be controlled by controlling the flow of the fuel Whatman filter paper (no. 42), and filtrate was
mixture. A beam of light is focused through this collected in labeled plastic bottles. The solutions
flame at its longest axis (the lateral axis) onto a were analyzed for the elements of interest, utiliz-
detector past the flame. The light that is focused ing AAS with suitable hollow cathode lamps.
into the flame is produced by a hollow cathode The percentages of different elements in these
lamp. Inside the lamp is a cylindrical metal cath- samples were determined by the corresponding
ode containing the metal for excitation and an standard calibration curves obtained by using
anode. When a high voltage is applied across the standard AR grade solutions of the elements.
anode and cathode, the metal atoms in the cath- Mercury (Hg) was analyzed by cold vapor tech-
ode are excited into producing light with a certain nique as at room temperature mercury exist at atom
emission spectra. The type of hollow cathode stage, so mercury is measured by atomic absorp-
tube depends on the metal being analyzed. For tion without a heated sample cell. In this process
analyzing the concentration of copper in an ore, a mercury is chemically reduced to the free atomic
copper cathode tube would be used, and likewise state by reaction of sample with a strong reducing
for any other metal being analyzed. The electrons agent like stannous chloride or sodium borohydride
of the atoms in the flame can be promoted to in a closed reaction system. The volatile free mer-
higher orbitals for an instant by absorbing a set cury is then driven from the reaction flask by bub-
quantity of energy (a quantum). This amount of bling air or argon through the solution. Mercury
energy is specific to a particular electron transi- atoms are carried in the gas stream through tubing
tion in a particular element. As the quantity of connected to an absorption cell, which is placed in
energy put into the flame is known and the quan- the light path of the AAS. Sometimes the cell is
tity remaining at the other side (at the detector) heated slightly to avoid water condensation, but
can be measured, it is possible to calculate how otherwise the cell is completely unheated.
many of these transitions took place, and thus get As the mercury atoms pass into the sampling
a signal that is proportional to the concentration cell, measured absorbance rises indicating the
of the element being measured. increasing concentration of mercury atoms in the
192 10 Metals in Herbal Drugs and Fingerprints

light path. Some systems allow the mercury vapor and filtered with Whatman filter paper no. 1. The
to pass from the absorption tube to waste, in standard working solutions of elements of inter-
which case the absorbance peaks and then falls as est were prepared to make the standard calibra-
the mercury is depleted. The highest absorbance tion curve. Absorption for a sample solution uses
observed during the measurement is taken as the the calibration curves to determine the concentra-
analytical signal. In other systems, the mercury tion of particular element in that sample. A Varian
vapor is rerouted back through the solution and AA240FS AAS was used for the determination
the sample cell in a closed loop. The absorbance of 13 metals, that is, Na, K, Ca, Mg, Mn, Zn, Ni,
rises until an equilibrium concentration of mer- Co, Fe, Cu, Cr, Cd, and Pb. Cathode lamps used
cury is attained in the system. The absorbance is as radiation source. Air–acetylene gas was used
then level off, and equilibrium absorbance is used for all the experiments. This method provides
for quantitation. The sensitivity of the cold vapor both sensitivity and selectivity since other ele-
technique is far greater than can be achieved by ments in the sample generally not absorb the cho-
conventional flame AA. This improved sensitiv- sen wavelength and do not interfere with the
ity is achieved, first of all, through a 100% sam- measurement [2].
pling efficiency. All of the mercury in the sample In the human body, the elements play vital role
solution placed in the reaction flask is chemically in many physiological reactions and their
atomized and transported to the sample cell for deficiency or excess can affect human health [3].
measurement [1]. Zn deficiency may contribute to arrested sexual
maturation, growth retardation and hair loss,
delayed wound healing, and emotional distur-
Case Study 2 bance. K is helpful in reducing hypertension and
maintaining cardiac rhythm. Magnesium (Mg)
In another study elemental assay was done by improves insulin sensitivity, protect against diabe-
using ASS on some ethnomedicinally important tes and its complications, and reduce blood pres-
species of genus Ficus [2]. Flame atomic absorp- sure. Sodium (Na) involves in the production of
tion methods are referred to as direct aspiration energy and transport of amino acids and glucose
determinations. They are normally completed as into the body cells. Copper (Cu) plays an impor-
single element analyses and are relatively free of tant role in treatment of chest wounds and prevent
interelement spectral interferences. The use of inflammation in arthritis and similar diseases.
special light sources produced by the cathode Copper (Cu) is an essential redox-active transition
lamp is emitted from excited atoms of the same element that play vital role in various metabolic
element which is to be determined, and specific processes. Being toxic, only trace presence of Cu
spectrum selection allows the quantitative deter- is preferred. It is well known that the high content
mination of individual components of a multiele- of transition metal like Cu catalyzes the formation
ment mixture. of hydroxyl (OH−) radicals; hence, excess quan-
Material and Methods: The different parts of tity can cause oxidative stress in plants and conse-
plants including leaves, aerial roots, fruits, and quently increase the antioxidant response. Calcium
bark were rinsed with tap water and then with overcomes the problems of high blood pressure,
distilled water in order to remove surface con- heart attack, premenstrual syndrome, and colon
tamination and dried at 55–60°C in an electric cancer and keeps the bones strong and reduces the
oven. The dried plant samples were then homog- risks of osteoporosis in old age. Co and Ni are
enized in pistil mortar. 0.25 g each of the pow- required in little amount in the human body.
dered plant samples digested in 6.5 ml of acid Cobalt (Co) is necessary in very small amounts in
solution (HNO3–H2SO4–HClO4 in ratio of all mammals and is used to treat several different
5:1:0.5). The corresponding solution was heated types of cancer in humans and treat anemia, but
until white fumes had appeared. The clear solu- the intake of high amount can cause heart dis-
tion was diluted up to 50 ml with distilled water eases. The health benefits of nickel (Ni) are
Inductively Coupled Plasma 193

optimal growth, healthy skin and bone structure; The light to be absorbed enters from one end of
it is involved in iron metabolism, but it is required the cylinder and emerges through the other end.
in low quantity, otherwise it may cause toxicity. The sample solution (1–100 ml) is injected into
Chromium (Cr) balances blood sugar levels, regu- the furnace through the injection port. The high
lates hunger, reduces cravings, protects DNA and temperature of the furnace (about 2,500°C) is
RNA, improves heart function, and helps control reached in stages, ultimately resulting in atomi-
fat and cholesterol levels in the blood [2]. Lead zation as in the flame. The atomized metal
(Pb) is a toxic metal and nonessential element for species then absorbs the light, and the absorption
human body as it causes a rise in blood pressure, is measured. One obvious difference between
kidney damage, miscarriages and subtle abortion, the furnace and the flame is that, contrary to the
brain damage, declined fertility of men through flame, the sample is not continuously fed into
sperm damage, diminished learning abilities of the furnace and the sample distribution is neither
children, and disruption of nervous systems. homogeneous nor reproducible. Thus, a furnace
Cadmium (Cd) is also toxic. The whole study of offers greater sensitivity (because more atoms
the absence of these heavy metals in samples indi- can be placed in the path of the light) and requires
cates that most of the plants were collected from less volume of sample, but sometimes suffers
their unpolluted natural habitats and therefore from lack of accuracy and precision. Thus, the
reflects their natural levels of heavy metals [2]. graphite furnace is the choice of preference only
Further a point of concern that in this study 13 when the sample size is small and/or there is a
elements were estimated in different parts in dif- need of greater sensitivity.
ferent species of Ficus, the present information is
helpful in the synthesis of new modern drugs
with various combinations of plants which can be Inductively Coupled Plasma
used in the cure of many diseases ethnomedici-
nally. The chemical composition of the different In inductively coupled plasma (ICP), there is an
elements in edible fruits of Ficus indicates that induction coil. Induction coil is a coil of wire that
many of these elements are of vital importance in has an alternating (oscillating) current flowing
metabolism and are needed for growth, develop- through it. In the ICP, the coil is wrapped around a
ments, and prevention and treatment of many dis- quartz tube through which flows a plasma (a col-
eases. It is evident that they are important sources lection of charged particles) and induces the mag-
of essential mineral elements in reasonable con- netic field. When we use a stream of argon gas that
centrations which are required in treatment of has been partially ionized prior to enter into induc-
many diseases [2]. Phosphorus content was ana- tion coil, become more ionized due to induced
lyzed calorimetrically, using 0.5 ml of digested magnetic field and an extremely hot as flame-like
sample with 4 ml of distilled water, 3 ml of emission device, is called the ICP. With respect to
0.75-M H2SO4, 0.4 ml of (NH4)6Mo7O24 .4H2O, the measurement of sample solutions, the proce-
and 0.4 ml of 2% ascorbic acid were added and dure is an aspiration, similar to AAS, in which the
mixed [4]. The solution was allowed to stand for solution is aspirated into the flowing argon prior
20 min. and absorbance was recorded 660 nm. to entering the quartz tube. The net result is an
extremely high temperature, capable of producing
very intense emissions from atomized and excited
The Graphite Furnace Atomizer atoms from the sample solution. Being an emission
technique, it is very useful for qualitative analysis,
A non-flame type of atomizer, a high-temperature especially having the intense signals compared to
graphite furnace, offers some advantages over the AAS, with high accuracy. The simultaneous multi-
AAS. There are several different designs, but element analysis of sample is also possible, using
basically this furnace is a small cylindrically ICP, but the possible disadvantage, perhaps, is the
shaped with a sample injection port at the top. high cost of the equipment compared to AAS.
194 10 Metals in Herbal Drugs and Fingerprints

Fig. 10.1 WD-XRF spectra 300


in overlay mode [5]
250
40 mg/g reference mixture

200

150
herb of E. arvense

100

50
0
100° 105° 110° 115°

WDXRF Spectroscopy The analytical method for determination of


silicone was validated before it has been used to
Wavelength dispersive X-ray fluorescence test the actual sample. XRF spectra of 40 mg/g
(WDXRF) spectroscopy has been successfully reference mixture were compared with spectra
validated to analyze silicon (Si), the second most recorded for natural samples. Comparison shows
abundant element on earth; most of its com- that regardless of sample there is no interference
pounds cannot be consumed by plants and ani- of signal Ka for Si (Fig. 10.1). A determination of
mals. The presence of Si in plants is a result of standardized samples containing known amount
an uptake of its soluble forms as orthosilicic acid of Si was performed using a calibration curve
[Si(OH)4] and/or its ionized form Si(OH)3O− is established in linearity study. Precision was
an essential microelement for human health. The assessed by making five determinations of 2 mg/g
highest concentration of Si in the human body is reference mixture (mixture of talcum with cellu-
in hair and nails. The symptoms of a possible Si lose), the relative standard deviation was 3.44%,
deficiency are cardiovascular and arterial prob- and the relative standard deviation (RSD) (for
lems, fragile bones, weakened gums and teeth, repeatability) was 1.53%. Calibration curve was
joint deterioration, and digestive disorders. plotted which has intercept value negligible,
Silicon does not have a specified recommended along with limit of detection ( LOD, in this case
daily allowance (RDA). According to the FDA 0.24 mg/g) as well as limit of quantification
suggestions, the total daily intake for men and [LOQ, by multiplying LOD three times (i.e.,
women is 40 and 19 mg, respectively. A safe 3 × 0.24 mg/g = 0.72 mg/g)]. The analytical
upper limit is thought to be 50 mg/day. It is method was verified using three certified stan-
essential to keep proper Si levels during growth dards of powdered herbs, also.
periods, while the daily uptake should be High-resolution solid-state NMR spectra were
increased for the elderly, as silicon deficiency is acquired at 298 K on an Avance 400WB spec-
considered associated with aging. To determine trometer (Bruker, Rheinstetten, Germany) with a
concentration of silicon and its structural forms Bruker 4 mm CP/MAS probe. The acronym CP
present in herbal drugs, dietary supplements stands for cross-polarization. The acronym MAS
(tablets and capsules) were studied using WDXRF stands for magic angle spinning (a method for
and high-resolution solid-state 29Si-NMR. Three narrowing signals in the solid-state NMR, for
different silicon forms were detected in the achieving high-resolution spectra). NMR mea-
herbal samples, Si(OSi)4, Si(OH)(OSi)3, and surements were carried out on powdered herbs at
Si(OH)2(OSi)2 [5]. the 29Si and 1H resonance frequencies of 79 and
Removal of Heavy Metals from Herbal Drugs 195

29
Fig. 10.2 Si CP/MAS–NMR spectrum of E. arvense from a herbal teapot bag [5]

400 MHz, respectively. The 29Si chemical shifts in the body, and have highly toxic effects after a
were externally referenced against TMS, using certain limit.
kaolinite as a secondary standard with a signal at
91.5 ppm. Peak fittings were done using a NutsPro
NMR computer program (Acorn 2007). Mixed Removal of Heavy Metals
Lorentzian/Gaussian line shapes were applied to from Herbal Drugs
fit experimental spectra and obtain chemical
shifts together with approximate peak areas. Heavy metal content is one of the important
Silicon forms were identified using high-resolu- factors imparting toxicity in the herbal formu-
tion solid-state 29Si NMR. Because of the low Si lations and needs to be checked and confirmed
contents, the spectra were very noisy despite long that they are well below the WHO prescribed
accumulation times (Fig. 10.2). In the whole limits. Some of the hazardous heavy metals as
study only Q2, Q3, and Q4 forms were detected lead (causing abdominal pain, convulsion,
(Table 10.1) [5]. hypertension and renal dysfunction, loss of
appetite, fatigue, sleeplessness, headache, and
vertigo), arsenic (causing abdominal pain, vom-
Heavy Metals iting, sensory changes, numbness and tingling,
muscle tenderness, burning sensation in hand and
There are eight common heavy metals, namely, feet, excessive darkening of skin, liver injury),
arsenic, barium, cadmium, chromium, lead, mer- mercury (causing shortness of breath, metallic
cury, selenium, and silver. These are all naturally taste in mouth, abdominal pain, nausea, vomiting,
occurring substances which are often present in headache, weakness, visual disturbance, and
the environment at low levels. In larger amounts, hypertension), and cadmium (causing nausea,
they can be dangerous. Generally, humans are vomiting, abdominal pain, breathing difficulties,
exposed to these metals by ingestion (drinking or growth impairment, osteoporosis, loss of taste
eating). These metals are at least five times denser and smell, and cardiovascular disease). Raw
than water. These (metallic form vs. ionic form) material used in the production of food, drug,
are stable (i.e., cannot be metabolized), accumulate and Ayurvedic formulation are always tested,
196 10 Metals in Herbal Drugs and Fingerprints

Table 10.1 Silicon forms found in plants and range of their chemical shift [5]
Chemical shift
Symbol (ppm) Silicon site Structural formula
Q1 78–86 Si(OH)3(OSi≡)

Q2 86–94 Si(OH)2(OSi≡)2

Q3 95–107 Si(OH)(OSi≡)3

Q4 107–120 Si(OSi≡)4

and the consignments are rejected having the Case Study 3


higher limit of heavy metals specified by WHO.
Different analytical techniques are in practice With the aim of designing and developing a
to decipher the quantity of heavy metals. In method for the bioremediation of the neem
Ayurveda these metals have been transformed (Azadirachta indica) extract various methods
to nontoxic forms that are safe for internal use. described in official books [6, 7] but chelation
The heavy metal-based innovations are in prog- with dithizone was selected as the probable
ress to remove these metals and increase the option, due to a number of advantages as the
acceptability of the raw material grown from compound does not chemically react with the
the polluted sites also. main marker compound (unlike most of the other
Removal of Heavy Metals from Herbal Drugs 197

Table 10.2 Role of pH for removal of heavy metals from neem extract [8]
Volume of Volume of
extract used dithizone solution Heavy metal pH Inference
10 ml 17.5 ml Lead 6–7 Complexation reaction occurs in neutral medium
Cadmium 10–12 Complexation reaction occurs in alkaline medium
Mercury 3.5–4 Complexation reaction occurs in acidic medium

Table 10.3 Heavy metal concentration in neem leaf Nimbidin (an active constituent of neem extract)
extract [8] was chosen as the marker compound. From the
Conc. in leaf WHO limit analytical studies, it transpires that the heavy metal
S. no. Element extract (ppm) (ppm) impurities, namely, cadmium and lead, need prior-
1 Lead 16.53 10 ity action for their removal. So, the work plan was
2 Cadmium 2.43 0.3 designed to facilitate the elimination of these two
3 Mercury <0.10 1 hazardous elements in improving the quality of the
4 Arsenic <0.50 10
neem-based products. The reagents used for this
purpose was dithizone (0.005%) in chloroform.
Trial and error method was resorted for the pur-
chelating agents), and it has a wide spectrum pose of volumetric quantification of the reactants
of activity and even can chelate out the trace as also the respective pH values (Table 10.2).
elements [8]. Dithizone was added quantitatively to avoid the
Neem leaf was purchased from the local mar- occurrence of any untreated reagent. The TLC
kets of Kolkata. All the chemicals and reagents method followed the following specifications:
were of certified and of AR/LR grade. The pow- extract loaded, 2 ml; mobile phase, chloroform–
dered dry neem leaf was mixed with distilled menthol (97:3); stationary phase, silica gel 60F254;
water in the proportion of powder–water (1:8) extraction media, methanol; Rf value of sample
and filtered in order to remove the unwanted (before dithizone) = 0.527; Rf value of sample
materials, fibers, etc. The solution was then sub- (after dithizone treatment) = 0.520; temperature,
jected to analytical tests to detect the undesirable 25°C [8].
elements or impurities. TLC was performed to TLC was used for the identification of the
confirm the presence of the heavy metals. heavy metals, using the principle of comparing
Comparison of the Rf value of the herbal extract the Rf values of the standard metal solutions
and heavy metals proved the presence of lead, and the herbal extract. Quantity estimation was
cadmium, and mercury. Stationary phase silica done using UV–Vis. spectrophotometric method,
gel 60 F254, mobile phase: benzene, duration of which establishes the presence of lead and cad-
TLC development: 40 min for 10 cm, Rf value of mium, well beyond the WHO limit (Table 10.3).
Pb = 0.316, Rf value of Hg = 0.769, Rf value of After the chelation of the heavy metals, studies
Cd = 0.143, at 25°C [8]. were performed to establish that the marker com-
UV–Vis. spectrometric analysis (standard curve pound remained unaffected with the chelating
method) was used for quantification of the herbal agent.
extract. Standard curves were plotted for lead, cad- HPLC analysis for rapid data of nimbidin and
mium, mercury, and arsenic. Specific wavelengths related terpenoids from neem before and after
were used for each heavy metal (like 490 and dithizone treatment (mobile phase 20% ace-
510 nm were the official wavelengths for the deter- tonitrile in isocratic mode, flow rate 1 ml/min,
mination of cadmium and lead, respectively). The lmax = 220 nm) HPLC chromatogram (Figs. 10.3
absorbance hence obtained was compared with the and 10.4) showed there was no significant change
standard curve by using UV–Vis. spectrometer [8]. in the mean area (A) and the retention time (RT)
198 10 Metals in Herbal Drugs and Fingerprints

Fig. 10.3 HPLC chromatogram of neem extract at initial stage [8]

Fig. 10.4 HPLC chromatogram of neem extract after dithizone treatment [8]

of the chromatogram both before (A = 97528.663 eliminate the harmful effects of metals and
and RT = 4.181) and after (A = 88727.576 and enhancing their biocompatibility in the body.
RT = 3.804) the dithizone treatment. From the Neutron activation analysis of 20 metallic-based
two HPLC chromatograms (Figs. 10.3 and 10.4), bhasmas such as calcium, iron, zinc, mercury, sil-
it is reconfirmed that the marker compound, nim- ver, potassium, arsenic, copper, tin, and gem-
bidin, remained unaffected by chelation, although stones confirmed the purity of these bhasma as
the toxic heavy metals were removed from the the other elements such as Na, K, Ca, Mg, V, Mn,
neem extract. Fe, Cu, and Zn were found in mg/g amounts and
Au and Co in ultra-trace (ng/g) amounts. Various
techniques like atomic force microscope (AFM),
Bhasmas as Nanoherbal Drugs transmission electron microscope (TEM), scan-
ning electron microscope (SEM), and energy dis-
Bhasmas are the Ayurvedic metallic preparations persive spectroscopy (EDM) have been employed
in which metal act as a nanocarrier for drug deliv- for the estimation and characterization of bhasma.
ery (Table 10.4), are widely recommended for The Swarna bhasma (nanoparticles of gold) anal-
treatment of a variety of chronic ailments, and are ysis qualitatively through X-ray diffraction and
taken along with milk, butter, honey, or ghee to Fourier transform infrared spectroscopy (FTIR)
References 199

Table 10.4 Ayurvedic bhasma and basic metal which act as a nanocarrier [9]
S. No. Trade name of bhasma Basic metal as nanocarrier
1 Shanka bhasma Calcium present in sea products
2 Swarna bhasma Gold
3 Mukta Pearl
4 Abrak Manganese
5 Godanti Gypsum stones
6 Loha Iron
7 Trivang Aluminum and zinc
8 Naga Lead
9 Parad Mercury

showed that the particle size is about 56 nm con- with these products, as the therapeutic efficacy is
taining pure gold which acts as a nanocarrier for associated with lifestyle and geographical condi-
drug delivery. Mineral arsenicals mainly in the tions also.
form orpiment (As2S3), realgar (As4S4), and arse-
nolite (As2O3) have been used in traditional drugs
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