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TABLE 4: RESULTS OF ANTICONVULSANT ACTIVITY
Compound
Dose a mg/kg
8a
8b
8c
8d
Control (Saline)
Standard (Phenytoin)
25
25
25
25
25
Time (sec) in various phases of convulsion
Recovery/death
Flexion
Extensor
Clonus
Stupor
3.3
3.1
1.6
2.5
4.0
1.2
5.0
4.5
4.0
3.8
11.0
2.0
2.5
2.4
2.3
2.2
3.0
0.9
86
83
92
94
120
100
Recovery
Recovery
Recovery
Recovery
Recovery
Recovery
a No. of animals in each group is 06.
Thus, the synthesis described herein, provides
oxadiazole derivatives 8a-d in good yields, which have
shown promising biological activities. The method adopted
in this paper is simple, efficient, environment friendly,
inexpensive, and it is useful in constructing
pharmacologically important heterocycles.
7.
8.
9.
10.
11.
12.
13.
ACKNOWLEDGEMENTS
14.
Authors are thankful to Dr. Ravi S. Chavan, Dept. of
studies in Botany, Karnatak University Dharwad for
carrying out pharmacological activities.
15.
16.
17.
REFERENCES
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Chem., 1994, 33B, 674.
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Chem., 1996, 35B, 1062.
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Accepted 18 March 2006
Revised 3 June 2005
Received 4 February 2005
Indian J. Pharm. Sci., 2006, 68 (2): 249-253
Determination of W
ithaferin–A in T
wo Withania
Withaferin–A
Two
Species by RP
-HPLC Method
RP-HPLC
SHAILA DALAVAYI*, S. M. KULKARNI, RAJYA LAKSHMI ITIKALA AND S. ITIKALA
Varun Herbals Pvt. Ltd., 5-8-293/A, Mahesh Nagar, Chirag Ali Lane, Hyderabad-500 001, India.
Withania is a small shrub possessing medicinal value commonly found in the Indian subcontinent. Quantitative
determination of withaferin-A, was undertaken to provide an easy and simple analytical method, which can be used
as a routine quality control method. RP-HPLC was performed using acetonitrile and water as mobile phase. The
detection and quantification was performed at a wavelength of 225 nm. Linearity of detector response for withaferinA was between the concentrations 0.005% to 0.02%. The correlation coefficient obtained for the linearity was
*For correspondence
E-mail: [email protected]
March - April 2006
Indian Journal of Pharmaceutical Sciences
253
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0.993. The recovery value of standard withaferin-A was 94.4%. Low value of standard deviation and coefficient of
variation, are indicative of high precision of the method. The system was successfully used to investigate the marker
compound in different plant parts of W. coagulans and W. somnifera.
Withania (Solanaceae), is a small shrub distributed in east
of the Mediterranean region, extending to South Asia.
Two species, Withania coagulans Dunal and Withania
somnifera Dunal, are found in India. In India, W.
coagulans and W. somnifera are known by the same
names in most of the regional languages. In the market,
no distinction is made between the berries obtained from
W. coagulans and W. somnifera. Both the species have
identical macroscopical features of roots, such as smooth
and whitish surface, and short and starchy fracture. The
roots of the W. coagulans are however, characterized by
the isolated groups of non lignified cells in the xylem
tissues, calcium oxalate crystals in the phelloderm cells,
and triseriate medullary rays1.
W. somnifera is a valuable herb in Ayurvedic medicine,
and as such, was used and cultivated for centuries in
India. Roots, leaves and preparations thereof, are
traditionally used as tonic, hypnotic, sedative, and diuretic.
The biological activities of withanolides, especially of the
dominant withaferin-A have been studied extensively.
Notable activities reported for this compound, include
antiinflammatory, anticonvulsive, antitumor and antioxidant
properties2-5. Claiming adaptogenic effects, aswagandha
products are available throughout the United States of
America as dietary supplements6,7. W. coagulans leaves
are used in Pakistan as a vegetable, and as a fodder for
camels and sheep. They are also employed as a
febrifuge. The leaves are chewed for cleaning teeth, and
the smoke of the plant is inhaled for relief in toothache1.
Withanolides are ergostane type of steroids with atoms C­
22 and C-26, bridged by a δ-lactone functionality, and an
oxidized C-1 position. These compounds are specific for
the solanaceae family, and in particular, for the genus
Withania, and thus they are used as marker compounds8.
The biological activities of withanolides, especially of the
dominant withaferin-A has been studied extensively and
reported from the roots and leaves of both the species of
Withania9. The number of analytical reports for the
determination of withanolides is comparatively small.
Besides a TLC method for the quantification of withaferin
A10, a few HPLC methods are described in literature.
Most of them showed disadvantages, as either the
acetylation of withaferin-A is required prior to analysis11,
the separation time is long12 or the compounds are not
254
baseline separated and elute, more or less with the
injection peak13.
A part of our efforts, was to develop a HPLC method
suitable for direct determination of withaferin-A in W.
coagulans and W. somnifera, and to investigate the
distribution of this compound in different plant parts.
Authenticated Withania coagulans and Withania somnifera
plant materials were collected from Narsapur forest,
Medak Dist., Andhra Pradesh, and botanically identified.
The plants were shade dried, and leaves and the roots of
both the species, were used for HPLC analysis. Standard
withaferin-A was procured from the Regional Research
Laboratory, Jammu Tawi.
The 4% alcoholic extracts of root, and 0.4% alcoholic
extracts of leaf of both the species of Withania, were
prepared by soaking the respective plant material
separately for 18 h, in absolute alcohol. The extracts
were filtered through Whatmann filter paper No.1, using
high-pressure vacuum pump.
One milligram of standard withaferin-A, was dissolved in
5 ml of alcohol. Three additional calibration levels were
prepared by diluting this solution with alcohol, to obtain
0.005%, 0.01%, 0.015% and 0.02% concentrations, for
studying the linearity. The precision of the method was
also studied, by injecting a sample solution of
concentration 0.02% five times, and finding out the
standard deviation and coefficient of variation.
A gradient HPLC (Shimadzu HPLC Class VP series) with
two LC- 10 AT VP pumps (Shimadzu), variable wave
length programmable photo diode array detector SPD­
M10A VP (Shimadzu), CTO-10AS VP column oven
(Shimadzu), SCL-10A VP system controller (Shimadzu),
and reverse phase Luna 5 µ C18 (2) Phenomenex column
(250 mm × 4.6 mm), was used. The HPLC system was
equipped with Class VP series version 6.1 software
(Shimadzu). The mobile phase components acetonitrile:
water were filtered through 0.2 µ membrane filter before
use, and were pumped from the solvent reservoir at a
flow rate of 1ml/min, which yielded column backup
pressure of 135-145 kgf/cm 2. The initial B-pump
concentration was 50%. The column temperature was
Indian Journal of Pharmaceutical Sciences
March - April 2006
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maintained at 27°. Rheodyne syringe (Model 7202,
Hamilton) was used for injecting 20 µl of respective
samples.
Standard withaferin-A solutions of 0.005%, 0.01%, 0.015%
and 0.02% concentrations, were analyzed for studying
the linearity, and the area count obtained for these
solutions are presented in Table 1. Withaferin-A showed
good linearity in the concentration range of 0.005% ­
0.02%, with a correlation coefficient of 0.993. The
precision of the method was also studied by injecting a
single sample solution five times (Table 2), and finding
out the standard deviation and coefficient of variation.
The standard deviation and coefficient of variation were
found to be 0.526 and 0.858. Low value of standard
deviation and coefficient of variation are indicative of
high precision of the method.
The HPLC chromatogram of standard withaferin-A at an
optimum wavelength of 225 nm, showed a mean area
(Table 2) of 6127383.6, at a mean retention time of
5.883min (Fig. 1). The recovery value of standard
withaferin-A was 94.4%. The HPLC chromatogram of leaf
of W. coagulans corresponding to standard withaferin-A
was showed at a retention time of 5.717 min, with an area
of 2816990 at a wavelength of 225 nm (Fig. 2). The HPLC
chromatogram of root of W. coagulans corresponding to
standard withaferin-A was showed at a retention time of
5.6 min, with an area of 936833 at a wavelength of 225 nm
(Fig. 3). The HPLC chromatogram of leaf of W.
somnifera corresponding to standard withaferin-A was
showed at a retention time of 5.771 min, with an area of
1384751 at a wavelength of 225 nm (Fig. 4). The HPLC
chromatogram of root of W. somnifera corresponding to
standard withaferin-A was showed at a retention time of
TABLE 1: AREA COUNTS FOR STANDARD
WITHAFERIN-A
Concentration of withaferin-A (%)
0.005
0.01
0.015
0.02
5.451 min, with an area of 537152 at a wavelength of 225
nm (Fig. 5). The variation in retention time of peak of
withaferin-A in chromatograms of W. somnifera and W.
coagulans, may be due to the presence of other chemical
constituents. The quantitative evaluation of withaferin-A in
leaf and root of W. coagulans, was 2.299% and
0.076%,and that of W. somnifera was 1.13% and 0.044%,
respectively. High content of withaferin-A in leaf than
root was reported from W. somnifera7.
The proposed method can be used to standardize
Withania species on the basis of withaferin-A as a
Min
Fig. 1: HPLC chromatogram of standard withaferin-A
Min
Fig. 2: HPLC chromatogram of W. coagulans leaf
Area counts
1677232
3208899
4487510
6205475
TABLE 2: DETERMINATION OF STANDARD DEVIATION
AND COEFFICIENT OF VARIATION FOR STANDARD
WITHAFERIN-A
Concentration of withaferin-A (%)
0.02
0.02
0.02
0.02
0.02
March - April 2006
Area counts
6205475
6126884
6058190
6132079
6114290
Min
Fig. 3: HPLC chromatogram of W. coagulans root
Indian Journal of Pharmaceutical Sciences
255
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REFERENCES
1.
2.
3.
4.
5.
Min
6.
Fig. 4: HPLC chromatogram of W. somnifera leaf
7.
8.
9.
10.
11.
12.
13.
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Fig. 5: HPLC chromatogram of W. somnifera root
marker compound, and is helpful for scientific as well as
commercial applications.
Accepted 19 March 2006
Revised 4 June 2005
Received 2 December 2004
Indian J. Pharm. Sci., 2006, 68 (2): 253-256
Evaluation of the Antioxidant Activity of the Roots
and Rhizomes of Cyperus rotundus L.
D. K. PAL* AND S. DUTTA
Division of Pharmaceutical Chemistry, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Mayurbhanj757 086, India.
The in vitro antioxidant activity of the roots and rhizomes of Cyperus rotundus L. has been investigated by
estimating degree of non-enzymatic haemoglobin glycosylation, measured colorimetrically at 520 nm. The ethanol
extract of the roots and rhizomes of C. rotundus showed higher activity, than other extracts of it. The antioxidant
activity of the extracts are close and identical in magnitude, and comparable to that of standard antioxidant
compounds used.
*For correspondence
E-mail: [email protected]
256
Indian Journal of Pharmaceutical Sciences
March - April 2006