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HPTLC Quantitation of 2-Hydroxy-4-methoxybenzaldehyde

in Hemidesmus indicus R.Br. Root Powder and Extract

Rahul S. Darekar, Amol B. Khetre, Shradhanjali M. Singh, and Mrinalini C. Damle*

Key Words
Hemidesmus indicus

1 Introduction
Hemidesmus indicus R.Br., Indian sarsaparilla (Anantamul), a
member of the family Asclepiadaceae, is a twining shrub commonly found in India. The woody roots of the plant have a
strong fragrance, and sweet taste with cooling effect. Because of
this typical aroma, the roots are used as a flavoring agent in
sherbets (sweet drinks). They are also used to treat a variety of
ailments or diseases, and are a well-known drug in the
Ayurvedic system of medicine [1] and used as a blood purifier
and also for curing fever, leprosy, rheumatism, and liver disorders [2, 3]. Moreover, root extracts of H. indicus have also
proved to be an effective snake venom antidote [4].
An unusual phenolic compound, 2-hydroxy-4-methoxybenzaldehyde (used as a marker compound) is responsible for the
fragrance of the root [5]. The air-dried material contains approximately 0.225% of essential oil, of which approximately 80% is
2-hydroxy-4-methoxybenzaldehyde, which can be isolated as a
crystalline substance [6]. Recently, 2-hydroxy-4-methoxybenzaldehyde, which is also present in several African medicinal
plants, has been identified as a potent tyrosinase inhibitor [7],
and is thus being used as an ingredient in cosmetic [8] and other
medicinal products, primarily to treat hyperpigmentation [9,
10]. The compound is also known to have antimicrobial [11] and
insecticidal [12] properties.
Literature survey revealed one HPLC method for quantification
of 2-hydroxy-4-methoxybenzaldehyde in the root of Hemidesmus indicus [13], another for simultaneous analysis of 2-hydroxy-4-methoxybenzaldehyde and 2-hydroxy-4-methoxybenzoic acid in root tissues of the same plant [14], and a GCMS
method for determination of the chemical composition of
volatile oil of H. indicus [15]. Gas chromatographic assay of 2-

hydroxy-4-methoxybenzaldehyde in dry and fresh root of

Decalepis hamiltonii and H. indicus has also been reported [16].
No suitable HPTLC method is available.
HPTLC has been widely used as quality-control tool for phytochemical evaluation of herbal drugs. In this work a precise and
accurate HPTLC method has been established for rapid and
accurate analysis of 2-hydroxy-4-methoxybenzaldehyde in H.
indicus dried root powder and its extract. One important reason
for selection of this marker was its absence from the plants commonly used as substitutes for H. indicus. It has been reported [17] that the roots of Ichnocarpus frutescens (Linn.) R.Br.
(Apocyanaceae), Decalepis hamiltonii Wight and Arn. (Asclepiadaceae), and Cryptolepis buchnani Roem. & Schult. (Asclepiadaceae) are used as substitutes for H. indicus. Because there are
no reports of the presence of this marker in the roots of I.
frutescens and C. buchnani, substitution by the roots of these
plants can be easily revealed by use of this HPTLC method.
A study [18] of the essential oil (0.33%) of D. hamiltonii discovered the 2-hydroxy-4-methoxybenzaldehyde content of the
oil was 37.45%. The amount of this marker in H. indicus is
much higher. Sircar et al. [14] have reported the content to be
3.2 0.2 mg g1 of the mature roots of H. indicus and
2.6 0.3 mg g1 of the young roots. Thus even in this case, substitution of H. indicus by D. hamiltonii can be detected easily.
Thus quantification of this marker will aid the detection of adulteration or substitution by common substituents because they
either lack this marker or the content is less than in H. indicus.

2 Experimental
2.1 Chemicals, Reagents, Materials, and Solutions

All chemicals were AR grade and were purchased from Sisco

Research Laboratories (Mumbai, India).
R.S. Darekar, A.B. Khetre, S.M. Singh, and M.C. Damle, Department of Pharmaceutical Chemistry, AISSMS College of Pharmacy, Near R.T.O, Kennedy Road,
Pune-411 001, India.

Journal of Planar Chromatography 22 (2009) 6, 453456

0933-4173/$ 20.00 Akadmiai Kiad, Budapest

Root powder of Hemidesmus indicus was obtained from local

market in Pune (India).
A stock solution (1000 g mL1) of the marker was prepared by
dissolving 10 mg in 10 mL n-hexane. This stock solution (2 mL)
DOI: 10.1556/JPC.22.2009.6.13


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UV spectrum was obtained by use of a Jasco model V-550

UVvisible double-beam spectrophotometer using n-hexane as
a solvent. The 1H NMR spectrum was recorded on a Varian Mercury YH 300 (300 MHz FT NMR) spectrophotometer using
TMS as internal reference. A mass spectrum was recorded by
use of a QP5050A GCMS system (bench-top quadrupole mass
2.3 Sample Preparation
2.3.1 Powder

Figure 1
Representative densitogram obtained from the marker compound.

H. indicus root powder (1 g) was accurately weighed, dispersed

in 10 mL n-hexane, and left for 2 h on a mechanical shaker. The
solution was then centrifuged at 1500 rpm for 20 min and the
supernatant was used for quantification.
2.3.2 Extract

Table 1
Regression data.

Wavelength [nm]


Beers law range [ng per band]

Correlation coefficient



Slope (m) of linear regression



Intercept (c) of linear regression equationa)


Number of data points

Limit of detection [ng per band]


Limit of quantitation [ng per band]



= mx + c, where y is peak area and x is amount [ng per band]

Table 2
Results from determination of intra-day and inter-day precision.

Concentration [ng per band]

RSD [%]










was diluted to 10 mL with n-hexane to furnish working standard

solution (200 g mL1).
2.2 Isolation of the Marker Compound

2-Hydroxy-4-methoxybenzaldehyde was isolated from the

volatile oil of Hemidesmus indicus root powder. The volatile oil
was extracted using Clavenger apparatus [15]. Colorless crystals formed in the volatile oil during extraction were isolated by
filtration through Whatman no. 41 filter paper. The melting
point of the compound was determined by use of an open capillary and is uncorrected. The structure of the compound was confirmed by use of IR, UV, NMR, and mass spectroscopy. The
FTIR spectrum of the compound was recorded as a potassium
bromide disk on a Jasco FTIR 460 plus spectrometer using the
diffuse reflectance attachment; peaks are reported in cm1. The


Extraction was performed by the procedure given in the

Ayurvedic Pharmacopoeia [19]. Root powder of H. indicus (5 g)
was accurately weighed and dispersed in 100 mL n-hexane. The
flask was placed on mechanical shaker for 6 h at 100 rpm. This
suspension was left overnight at ambient temperature, then filtered through Whatman no. 41 filter paper. The filtrate was
evaporated on a water bath to furnish a solid mass of extract.
This extract (0.1 g) was dispersed in 10 mL n-hexane and the
mixture was sonicated for 20 min. The solution was centrifuged
at 1500 rpm for 20 min and the supernatant was used for quantification.
2.4 Chromatography

Chromatography was performed on 10 cm 10 cm TLC plates

precoated with 250-m layers of silica gel 60 F254 (E. Merck,
Germany). Samples were applied to the plates as bands 6 mm
wide by use of a CAMAG (Switzerland) Linomat 5 applicator
fitted with a 100-L syringe (Hamilton, Switzerland). The application positions X and Y were 15 mm and 10 mm, respectively,
to avoid edge effects. Linear ascending development to a distance of 80 mm, with tolueneethyl acetateglacial acetic acid
7:2:1 (v/v) as mobile phase, was performed in a twin-trough
glass chamber (10 cm 10 cm) previously saturated with
mobile phase vapor for 20 min. The plates were dried in air then
densitometric scanning at 277 nm was performed with a
CAMAG TLC scanner III, in absorbance mode, operated by
WinCATS software (V 1.4.2; CAMAG). The slit dimensions
were 5 mm 0.45 mm and the scanning speed 100 nm s1.
For calibration and to assess linearity, marker stock solution (1,
2, 3, 4, and 5 L) was applied to a plate to furnish amounts in the
range 2001000 ng per band. Peak areas were plotted against the
corresponding concentrations and least-squares regression
analysis was performed to generate the calibration equation.
For analysis of dry root powder, the solution obtained as
described in Section 2.3.1 (2 L) was applied to a plate. After
development, scanning, and measurement of peak area the
amount of marker was calculated by use of the calibration plot,
assuming the purity of the marker to be 100%.
For analysis of the root-powder extract, the solution obtained as
described in Section 2.3.2 (5 L) was applied on the plate. After
development, scanning, and measurement of peak area the

Journal of Planar Chromatography 22 (2009) 6

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Table 3
Results from recovery studies.

Level [%]
Replica 1

Recovery [%]
Replica 2


Mean RSD [%]

Replica 3



99.94 0.40



100.01 1.55



100.69 1.42

amount of marker was calculated by use of the calibration plot,

assuming the purity of the marker to be 100%.
2.5 Method Validation

The method was validated for linearity, accuracy, precision, limits of detection and quantitation, robustness, and specificity in
accordance with ICH guidelines [20]. Linearity was assessed by
construction of the calibration plot (Section 2.4). Repeatability
and intermediate precision were assessed by measurement of
intra-day and inter-day variation. In the intra-day studies, standard and sample solutions were measured in triplicate on the
same day and RSD [%] was calculated. In the inter-day variation
studies, standard and sample solutions were measured in triplicate on three consecutive days and RSD [%] was calculated.
Accuracy was assessed by measurement of recovery by the
method of standard additions; the method was applied to previously analyzed drug sample to which known amounts of marker
had been added (80, 100, and 120% of the amount known to be
present). Three analyses were performed at each level, and the
results obtained were compared with those expected. The limit
of detection (LOD) of an analytical procedure is the lowest
amount of analyte in a sample which can be detected but not
necessarily quantified accurately. This was calculated by use of
the formula LOD = (3.3 standard deviation of the y-intercept)/(slope of the calibration plot). The limit of quantification
(LOQ) is the lowest amount of analyte in a sample which can be
quantified accurately. This was calculated by use of the formula
LOD = (10 standard deviation of the y-intercept)/(slope of the
calibration plot). Robustness was checked by studying the effect
of slight deliberate changes to the experimental conditions, by
using mobile phases of different composition (tolueneethyl
acetateglacial acetic acid 7:1.8:1 and 6.8:2:1 (v/v). Robustness
was checked at three different concentrations 400, 600, and
800 ng per band. The specificity of the method was assessed by
analysis of standard isolated marker and root powder and checking for the presence of interfering bands from plant constituents
at the RF of the marker. Absorption spectra were also acquired
from and compared with the spectrum obtained from the pure

3 Results and Discussion

3.1 Elucidation of the Structure of the Isolated Marker

The melting point of the compound was 4041C in agreement

with the reported value [13]. The infrared spectrum contained
sharp peaks at 1636, 2939, 1222, 3074, and 1575 cm1. In the
Journal of Planar Chromatography 22 (2009) 6

Figure 2
Overlain UV spectra of 2-hydroxy-4-methoxybenzaldehyde standard
and spectrum acquired in situ from band at RF 0.7 0.03 from a sample chromatogram.

UV spectrum absorbance maxima were observed at 235.5, 277,

and 314 nm. The NMR spectrum contained peaks at = 9.6 (s,
1H), 11.4 (s, 1H), 6.4 (s, 1H), 6.5 (d, 1H), 7.4 (d, 1H), and 3.8 (s,
3H), in agreement with reported data [21]. The mass spectrum
contained fragments at m/z (abundance [%]): 53 (28), 63 (25),
81 (15), 95 (30), 108 (25), 134 (3), 151 (100), and 152 (20), in
agreement with reported data [15]. These results confirmed the
isolated compound was 2-hydroxy-4-methoxybenzaldehyde.
3.2 Optimization of the Chromatographic Conditions

Chromatographic separation studies were conducted on the

standard stock solution of the marker. Initially, plates were
developed with neat solvents, for example benzene, ethyl
acetate, methanol, chloroform, toluene, ethanol, and dichloromethane, without chamber saturation. On the basis of the results
obtained from these initial chromatograms, binary and ternary
mixtures of solvents were investigated to achieve optimum resolution. Use of tolueneethyl acetateglacial acetic acid 7:2:1
(v/v) led to good resolution of 2-hydroxy-4-methoxybenzaldehyde with RF 0.7 0.03 (Figure 1).
3.3 Validation

Response (peak area) was a linear function of amount applied in

the range of 2001000 ng per band. The regression equation for
the calibration plot was y = 22.971x + 597.46 (r2 = 0.997). The
results from linear regression are summarized in Table 1. The
method was found to be precise, as indicated by RSD 1.5%.
Results from determination of intra-day and inter-day precision
are shown in Table 2. When root powder was spiked with working standard and analyzed, recovery was 99.94100.69%
(Table 3). The limits of detection and quantification were
9.6 and 29.09 ng per band, respectively. The method was robust,
because slight deliberate changes in the experimental conditions
had no significant effect on peak area. Spectra obtained from
pure marker and the marker present in root powder matched
exactly, indicating no interference from other plant constituents
(Figure 2).


Short Communications

[1] K.R. Kirtikar, B.D. Basu, Indian Medicinal Plants, Deharadun,
[2] M. Prabakan, R. Anandan, T. Devaki, Fitoterapia 71 (2000) 5559.
[3] P.N. Gupta, Lepr. India 53 (1981) 364.
[4] A.I. Alam, B. Auddy, A. Gomes, Toxicon 32 (1994) 15511557.
[5] S. Sreekumar, S. Seeni, P. Pushpangadan, Biotechnol. Lett. 20
(1998) 631635.
[6] A.T. Dutta, S. Ghosh, R.N. Chopra, Arch. Pharm. 276 (1938)
Figure 3

[7] K. Isao, K.H. Ikuyo, Planta Med. 65 (1999) 1922.

Representative densitogram of marker compound from hexane

extract of root powder.

[8] K. Maeda, M. Fukuda, J. Soc. Cosmet. Chem. 42 (1991) 361368.

[9] T.B. Fitzpatrick, M. Seiji, A.D. McGugan, New Eng. J. Med. 265
(1961) 374378.
[10] G. Prota, Melanins and Melanogenesis, Academia Press, San
Diego, 1992.

3.4 Sample Analysis

The amounts of the marker found in the dry powder and in the
hexane extract of root powder were 2.993 and 7.578 mg g1,
respectively. The densitogram obtained from the hexane extract
of the root powder is shown in Figure 3.

[11] N.Y. Phadke, A.S. Gholap, K. Ramakrishnan, G. Subbulakshmi,

J. Food Sci. Technol. 31 (1994) 472475.
[12] J. George, J. Pereira, G.A. Ravishankar, S. Divakar, Curr. Sci. 77
(1999) 501502.
[13] S.K. Mandal, S. Haty, A. Paul, D. Panja, S. Deb, Indian Drug 44
(2007) 257260.
[14] D. Sircar, G. Dey, A. Mitra, Chromatographia 65 (2007) 349353.

4 Conclusion

[15] S. Nagarajan, L.J.M. Rao, K.N. Gurudutt, Flavour Fragr. J. 16

(2001) 212214.
[16] S. Nagarajan, L.J.M. Rao, J. AOAC Int. 86 (2003) 564567.

2-Hydroxy-4-methoxybenzaldehyde is an important marker

compound in Hemidesmus indicus R. Br. A densitometric
HPTLC method for quantification of this compound in
n-hexane extracts of the dry root powder of the plant has been
established and validated. The method proved to be simple,
rapid, accurate, precise, and sensitive, and can thus be used for
routine quality control of H. indicus root powder using
2-hydroxy-4-methoxybenzaldehyde as marker.

The authors are grateful to Dr K.G. Bothara, Principal, AISSMS

College of Pharmacy for providing necessary facilities to carry
out the research work.


[17] Y.K. Sarin, Illustrated Manual of Herbal Drugs Used in Ayurveda,

Council of Scientific and Industrial Research and Indian Council
of Medical Research, New Delhi, 1996.
[18] D. Thangadurai, O.S. Ramachandraian, J. Agric. Food Chem. 50
(2002) 31473150.
[19] The Ayurvedic Pharmacopoeia of India, Govt. of India, Ministry of
Health & Family Welfare, Dept. of Indian System of Medicine &
Homeopathy, New Delhi, 2003.
[20] ICH Harmonised Tripartite Guideline, Validation of Analytical
Procedures: Text and Methodology Q2 (R1), Geneva, Nov 2005.
[21] H.H. Pattekhan, S. Divakar, J. Mol. Catal. A 169 (2001) 185191.
Ms received: July 17, 2008
Accepted: 3 July 2009

Journal of Planar Chromatography 22 (2009) 6