j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 3 4 6 e3 4 9

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Original Article

Isolation and characterization of steroids from Calligonum

polygonoides

Muhammad Qasim Samejo a,b, Shahabuddin Memon a,*, Muhammad Iqbal Bhanger a,
Khalid Mohammed Khan c
a
National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080, Pakistan
b
Dr. M. A. Kazi Institute of Chemistry, University of Sindh, Jamshoro 76080, Pakistan
c
H. E. J. Research Institute of Chemistry, International Center Chemical and Biological Science, University of Karachi, Karachi 75270, Pakistan

article info abstract

Article history: Aim: Calligonum polygonoides Linn. (Polygonaceae) is commonly known as Phog contains
Received 30 January 2013 number of phytochemical constituents viz. flavonoids, alkaloids, proteins, tannins, ste-
Accepted 14 March 2013 roids, phenols, carbohydrates, terpenoids etc. The aim of the present study was to isolate
Available online 1 April 2013 and characterize steroids from the roots of C. polygonoides.
Method: Methanol extract of the roots of plant was subjected to column chromatography
Keywords: and eluted with solvent mixtures of increasing polarity, composed of chloroform, chloro-
Calligonum polygonoides formeethyl acetate mixtures and ethyl acetate to isolate phytochemical constituents. The
Polygonaceae identities of these compounds were checked by solubility, preliminary phytochemical test,
Steroids melting point determination and TLC study. Finally the structure was elucidated by
different chromatographic and spectroscopic methods.
Results: On the basis of chemical and spectral evidence and upon comparison with the
literature data, the isolated compounds were identified as campesterol (1), stigmasterol (2),
(3b,5a,24S)-stigmastan-3-ol (3) and stigmast-4-en-3-one (4).
Conclusion: The steroids were isolated from the roots of C. polygonoides may serve as a po-
tential source of useful drugs in the near future. The steroids were isolated first time from
this plant extract.
Copyright 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights
reserved.

1. Introduction steroids.1,2 Steroids are terpenoids lipids identified by carbon

Natural products, such as plants extract, either as pure
compounds or as standardized extracts, provide unlimited
opportunities for new drug discoveries because of the un-
matched availability of chemical diversity. The medicinal
value of plants is due to the presence of chemical constituents
such as flavonoids, alkaloids, terpenoids, tannins and
skeleton with 4 fused rings. Steroids are differing due to their
oxidation state of functional groups attached to the rings and
oxidation state of rings. The major responsibilities of steroids
(androgens, progestagens, estrogens, mineralocorticoids and
glucocorticoids) are to salt balance, controlling metabolism
and the improvement and function of the sexual organs as
well as other biological differences between the sexes.

* Corresponding author. Tel.: 92 (22) 9213430; fax: 92 (22) 9213431.

E-mail address: shahabuddinmemon@yahoo.com (S. Memon).
0974-6943/$ e see front matter Copyright 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jopr.2013.03.017
 j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 3 4 6 e3 4 9
Steroids in the form of bile salts (e.g., salts of deoxycholic and
cholic acid and their taurine conjugates and glycine) facilitate
in digestive processes. Synthetic steroids like glucocorticos-
teroids, estrogens, methylprednisolone, corticosteroids, an-
drogens, squalamine and hydrocortisone are also used for the
treatment of various diseases such as arthritis, malignancies,
allergic reactions, and diseases resulting from abnormal
production or hormone deficiencies.3 Campesterol (rapeseed,
soy and wheat-germ oils) is the most familiar plant sterols in
nature along with stigmasterol and b-sitosterol, it show
cholesterol lowering and anticarcinogenic effects. Since
angiogenesis is essential for cancer, it was surmise that an
antiangiogenic effect may be involved in the anticancer ac-
tion of this compound.4 Stigmasterol may be useful in pre-
vention of certain cancers, including ovarian, prostate, breast,
and colon cancers. It possesses potent antioxidant, hypogly-
cemic and thyroid inhibiting properties.5,6 Stigmast-4-en-3-
one show orally hypoglycaemic agent and necessary inter-
mediate in the metabolism of b-sitosterol.7 (3b,5a,24S)-stig-
mastan-3-ol also reduce the absorption of cholesterol from
the diet.8
The genus Calligonum belongs to the family Polygonaceae,
comprises of about 80 species and is found in many countries
such as Northern Africa, Southern Europe and Western Asia.
Calligonum polygonoides Linn. is known for its medicinal
properties. The flowers of C. polygonoides are useful against
cough, asthma and cold. The juice of shoot is applied to the
eyes as an antidote to scorpion sting, a roots decoction mixed
with catechu is used as gargle for sore gum, and the latex is
used for treating eczema, to cure bites of rabid dogs and to
induce abortion. Methanol extract of the C. polygonoides
showed strong toxicity in brine-shrimp lethality test.9 Phyto-
chemical screening of C. polygonoides shows positive results
for flavonoids, alkaloids, proteins, tannins, steroids, phenols,
carbohydrates and terpenoids.10 The essential oil from buds
and roots of C. polygonoides contain a complex mixture of
terpenoids, hydrocarbons, phenolic compounds, acid de-
rivatives and ketones. The literature survey revealed that
the Calligonolides, tetracosan-4-olide, steroidal ester,
b-sitosterol, b-sitosterol glucoside and ursolic acid isolated
from C. polygonoides.9
The aim of present study was to isolate and identify the
steroids from the roots of C. polygonoides. To the best of our
knowledge, these steroids (1e4) were found for the first time
from this species.
2. Materials and methods
2.1. Collection and identification of plant materials
Roots of C. polygonoides were collected from Village Mehendri-
Jo-Par (longitude: N 25 340 200 and latitude: E 70 110 2000 ), Dis-
trict Umerkot in Sindh Province of Pakistan in January 2012. A
voucher specimen (15173) of the plant was deposited in the
herbarium of Institute of Plant Sciences, University of Sindh
Jamshoro, Pakistan. The plant sample was identified by a
Taxonomist of the same institution. The plant material was
air dried under normal conditions and ventilated.
347
2.2. Extraction and isolation
About 300 g powdered roots of C. polygonoides were macerated
in methanol for three days. Occasional shaking and stirring
was done. Then extract was filtered using Whatman filter
paper. The filtrate was concentrated to dryness under
the vacuum. Chemical tests (Salkowski and Lie-
bermanneBurchard reaction) were performed to detect the
steroids in the extract.6 The dried methanol extract was sub-
jected to column chromatography over silica gel (particle size
0.2e0.5 mm, 35e70 mesh ASTM) and gradient elutions were
carried out with eluents chloroform, chloroformeethyl ace-
tate mixtures and ethyl acetate. Each fraction was collected in
20 ml portion and was monitored by TLC (Pre-coated
aluminum silica gel plates). The chloroform fraction was
further purified by preparative TLC using hexane:chloroform
(40:60) solvent system. TLC result shows the four spots with
different retention time. Each spot (showing compound) was
scratched separately and dissolved in hexane then filtered
using Whatman filter paper. The isolated compounds were
again confirmed of their identity by chemical tests. For further
characterization UV, FT-IR and GCeMS was done.
2.3. Gas chromatographyemass spectrometry (GCeMS)
GCeMS analysis of plant sample was performed on Agilent
6890 N GC instrument coupled with MSe5975 inert XL mass
selective detector and auto sampler 7683-B injector was used.
The HPe5MS column with dimensions of 30 m  0.25 mm i.d.,
film thickness 0.25 mm was used for the analysis. Initial tem-
perature 150  C, maintained for 2 min, final temperature
230  C, kept for 5 min, ramp rate 4  C/min. 1.0 ml sample was
injected, using split mode (split ratio, 10:1). Helium gas was
used as a carrier gas at a flow rate of 0.8 ml/min. An electron
ionization mode with ionization energy of 70 eV was used for
MS detection. The injector and MS transfer line temperatures
were set at 240 and 270  C, respectively.
2.4. FT-IR analysis
FT-IR spectra were obtained using a Thermo Nicolet Avatar
330 FT-IR spectrometer controlled by OMNIC software
(Thermo Nicolet Analytical instruments, Madison, WI, USA)
station with a deuterated triglycine sulfate (DTGS) detector
and KBr optics. The sampling station was equipped with
overhead ATR accessory (Spectra-Tech, Shelton, CT)
comprising of transfer optics with in the chamber through
which infrared radiation is directed to a detachable ATR zinc
selenide crystal mounted in a shallow trough for sample
containment. A single beam spectrum (4000-650 cm1) of the
sample was obtained against air as a background at a reso-
lution of 4 cm1 and a total of 32 scan.11
3. Results and discussion
The methanol extract of C. polygonoides roots was subjected to
different phytochemical tests and it gives highly positive results
for steroids. The extract was subjected to column chromatog-
raphy over silica gel. The column was eluted in different solvent
348 j o u r n a l o f p h a r m a c y r e s e a r c h 6 ( 2 0 1 3 ) 3 4 6 e3 4 9
HO
HO
Stigmasterol (2)
Campesterol (1)
(C29H48O; Mol.Wt. 412.69)
(C28H48O; Mol.Wt. 400.68)
HO O
Stigmast-4-en-3-one (4)
(C29H52O; Mol.Wt. 416.72) (C29H48O; Mol.Wt. 412.69)
Fig. 1 e Sterols identified in roots of Calligonum polygonoides
(1e4).
system (CHCl3, CHCl3eEtOAc mixtures and EtOAc) with gradient
elutions. Each fraction was monitored by TLC. The chloroform
fraction was further purified by preparative TLC using hex-
ane:chloroform (40:60) solvent system. The TLC result leads to
the isolation of campesterol (1), stigmasterol (2), (3b,5a,24S)-
stigmastan-3-ol (3) and stigmast-4-en-3-one (4) (shown in Fig. 1).
The FT-IR spectra of isolated compounds exhibit the diag-
nostic peaks relating to CeH stretching at 2950 cm1 and
2860 cm1. The OeH stretching and C]C absorption peak
appears at 3360 cm1 and 1630 cm1, respectively. Other ab-
sorption peaks includes 1445 cm1 (CH2); 1371 cm1 (OH def),
1050 cm1 (cycloalkane) verify the required data regarding the
structures of steroids.
UV spectrum (recorded in methanol) of stigmasterol
exhibited peak at 256 nm because it contains two isolated
double bond. While, stigmast-4-en-3-one and campesterol
exhibited peaks at 231 and 251 nm respectively.
GCeMS is the most useful method for the characterization
of steroids.12,13 Each compound was analyzed by GCeMS and
identified by comparison of their mass spectra with the
reference compounds in the data systems of Wiley and Na-
tional Institute of Standards and Technology (NIST) spectra
libraries matching. Compounds were identified with a
resemblance percentage above 90%. Further conformation of
these compounds was done by comparison of their and mass
spectra with data in literature.14e19 Results show good agree-
ment for the structure of campesterol (1), stigmasterol (2),
(3b,5a,24S)-stigmastan-3-ol (3) and stigmast-4-en-3-one (4) as
reported in the literature.
4. Conclusion
On the basis of chemical and spectral evidence and upon
comparison of obtained data with the literature data, the
isolated compounds are identified as campesterol (1), stig-
masterol (2), (3b,5a,24S)-stigmastan-3-ol (3) and stigmast-4-
en-3-one (4) (Fig. 1) from methanol extract of the roots of
C. polygonoides.
Conflicts of interest
All authors have none to declare.
Acknowledgments
Financial support and necessary facilities offered by National
Centre of Excellence in Analytical Chemistry (NCEAC),
University of Sindh, Jamshoro, Pakistan is gratefully
acknowledged.
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