Journal of Chromatographic Science 2012;50:779– 784

doi:10.1093/chromsci/bms063 Advance Access publication June 1, 2012 Article

Rapid RP-HPLC Method for the Quantification of Glabridin in Crude Drug

and in Polyherbal Formulation
Y.T. Kamal1, Mhaveer Singh1, E.T. Tamboli1, Rabea Parveen2, S. M. Arif Zaidi3 and Sayeed Ahmad1*
1
Bioactive Natural Product Laboratory, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, 2Department of
Pharmaceutics, Faculty of Pharmacy, and 3Department of Surgery, Faculty of Medicine, Hamdard University, New Delhi, India - 110062

*Author to whom correspondence should be addressed. Email: sahmad_jh@yahoo.co.in

Received 12 April 2011; revised 8 December 2011

Downloaded from https://academic.oup.com/chromsci/article-abstract/50/9/779/288853 by guest on 15 April 2020

A simple, economic, selective, precise and robust method has been Unani system of medicine as a deobstruent drug and prescribed
developed and validated for the analysis of glabridin in crude drugs for the treatment of jaundice and cardiac-related problems. It is
and polyherbal formulations. Reversed-phase chromatography is composed of five ingredients; namely, Glycyrrhiza glabra
performed on a C18 column with water and acetonitrile as mobile (28.57% w/w), Pistacia lentiseus (4.7% w/w), Bamboosa
phase in gradient elution method at a flow rate of 1 mL/min. bambo Druce (4.76% w/w), Nardostachys jatamansi (14.28%
Detection is performed at 230 nm and a sharp peak is obtained for w/w) and Rosa damascene (47.6% w/w) and excepients (13).
glabridin at a retention time of 14.9 + 0.02 min. Linear regression Several analytical methods have been reported for the quan-
analysis data for the calibration plot showed a good linear rela- tification of glabridin using different analytical techniques, such
tionship between response and concentration in the range of as high-performance liquid chromatography (HPLC) (14 –16),
1 –500 mg/mL; the regression coefficient is 0.9992 and the linear capillary electrophoresis (17) and liquid chromatography –mass
regression equation is y 5 26.683x – 142.17. The method is spectrometry (LC – MS) (18). The previously reported isocratic
validated for accuracy, precision, reproducibility, robustness and HPLC method was found to have a very narrow linearity range
detection and quantification limits, in accordance with International (10–100 mg/mL) and the recovery of the method was also
Conference on Harmonization guidelines. Statistical analysis proved found to be poor, i.e., 92% (14). Moreover, the reported isocrat-
that the method is precise, reproducible, selective and accurate for ic method was found to be inappropriate to separate the imme-
the analysis of glabridin. The proposed, developed and validated diate impurities from the main constituents in polyherbal
high-performance liquid chromatography method for the quantifica- formulations. Hence, it was thought worthwhile to develop and
tion of glabridin can be used for the quality control and standardiza- validate a gradient reversed-phase (RP)-HPLC method for the
tion of licorice (Glycyrrhiza glabra Linn.) and different herbal quantification of glabridin in crude drug and in polyherbal for-
formulations in which licorice is present as a constituent. mulations, which can separate the glabridin with good reso-
lution in multicomponent formulations with better validation
parameters. The newly proposed and validated HPLC method
can be used more easily and efficiently for the quantification of
glabridin in polyherbal formulations than earlier reported
Introduction methods.
Licorice, the root of the leguminous Glycyrrhiza plant species,
has been consumed for over 4,000 years, also known as “sweet
root.” Glabridin [4-[(3R)-8,8-dimethyl-3,4-dihydro-2H-pyrano Experimental
[6, 5-f ]chromen-3-yl]benzene-1,3-diol] (Figure 1) is a major
Reagents and chemicals
polyphenolic flavonoid specific for Glycyrrhiza glabra
L. Recent studies have proven the anticancer activity of glabri- Glabridin (98%) was obtained as a gift sample from Sami Labs
din by inhibiting migration, invasion and angiogenesis of (Bangalore, India). HPLC-grade acetonitrile and methanol were
MDA-MB-231 human breast adenocarcinoma cells and human purchased from Merck (India). Milli-Q water used throughout
non-small cell lung cancer A549 cells by inhibiting focal adhe- the experiment was prepared using a Millipore water purifica-
sion kinase/Rho signaling pathway (1, 2). Moreover, glabridin tion system.
has been reported to posses several pharmacological activities
such as antioxidant (3), anti-Helicobacter pylori (4), antifungal
(5), estrogenic, anti-proliferative (6, 7) and antinephritic Instrumentation and general conditions
activity (8). In addition, glabridin inhibits serotonin reuptake Chromatographic experiments were conducted on a YL9100
(9), melanogenesis and inflammation (10). HPLC system (South Korea) that comprised quaternary
The Unani system of medicine is commonly practized in YL9110 pumps, a variable wavelength programmable YL9120
India and many other countries. Different types of herbal ultraviolet (UV)-visible detector, YL9130 column oven and a
formulations are used in the Unani system, such as solid system controller. The instrument was controlled by use of
(Qurs, Habbs, Safoof), semi-solid (Khamira, Itrifal, Majoon) and YL-Clarity software installed with the equipment. Samples
liquid (Sharbat) preparations (11, 12). Qurs-e-Gul is a tablet for- were injected by using a rheodyne injector fitted with a
mulation mentioned in the National Formulary of Unani 20-mL fixed loop. Standard and sample solutions were filtered
Medicines (NFUM), which has been commonly used in the through a 0.22-mm syringe filter before injection. The
# The Author [2012]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
 to 500 mg/mL, which were injected (20 mL each) by rheodyne
injector and chromatographed as per the previously mentioned
protocol. The stock solution was kept in dark for storage
to avoid possible degradation that may result from exposure
to light.

Figure 1. Chemical structure of glabridin. Accuracy as recovery

The accuracy of the method was determined by recovery
studies using the standard addition method. Preanalyzed
separation was achieved by using a C18 reversed-phase samples were spiked with standard glabridin at three different
column (Merck Lichrocart 250-4, Lichrosphere 100 RP-18e, concentration levels, i.e., 50, 100 and 150%, and the mixtures

Downloaded from https://academic.oup.com/chromsci/article-abstract/50/9/779/288853 by guest on 15 April 2020

5 mm, Sorbent lot number L57020637). The mobile phase con- were reanalyzed by the proposed method. Data obtained was
sisted of acetonitrile and water in gradient elution method analyzed for percent recovery.
from 50 to 80% in 20 min. The flow rate was kept at 1.0 mL/
min. All the analyses were performed at room temperature
and detection was carried out at a wavelength of 230 nm Precision
using a UV-visible detector. The precision of the method was carried out by performing
repeatability and intermediate precision. In repeatability,
six different injections of the same standard sample (three
Preparation of sample solutions concentrations) were injected and calculated in the assay.
The percent relative standard deviation (%RSD) of the area
Preparation of sample for crude and retention time (Rt) were calculated. In intermediate
One gram of the powdered crude drug was extracted with precision, intra-day, inter-day, and inter-system precisions
25 mL of 30% aqueous ethanol by soaking overnight and were performed. Intra-day and inter-day precisions were per-
then refluxing on a water bath for 45 min. It was filtered formed by preparing and applying three different concentra-
and evaporated to dryness under reduced pressure. The tions of standard in triplicate six times a day and on six
obtained residue was then reconstituted in HPLC-grade different days, respectively. Inter-system precision was per-
methanol and the volume was adjusted to 25 mL, which was formed by repeating the same procedure in a different HPLC
filtered through a 0.22-mm syringe filter before injecting into system. Assay for each analysis was calculated and %RSD was
the HPLC column. determined.

Preparation of sample for polyherbal formulation

Twenty tablets were randomly selected from the formulation LOD and LOQ
and average weight was determined. The tablets were tritu- The LOD and LOQ were determined based on the basis of
rated to get a uniform fine powder. Two grams, accurately signal-to-noise ratio. The concentration of the sample with a
weighed, of the powdered sample was extracted with 25 mL signal-to-noise ratio of three was fixed as the LOD. The concen-
of 30% aqueous ethanol, as discussed previously. The tration of the sample with a signal-to-noise ratio of ten was
obtained residue was then reconstituted in HPLC-grade fixed as the LOQ.
methanol and volume was adjusted to 25 mL, which was fil-
tered through a 0.22-mm syringe filter before injecting into
the HPLC column. Robustness of the method
Robustness of the method was performed by introducing
very small changes in the analytical methodology at a single
Validation methodology concentration level (100 mg/mL). Robustness of the pro-
Method validation was carried out to confirm the suitability posed method was determined in two different ways, i.e., by
of the proposed analytical method for its intended use. The making deliberate changes in the flow rate and by changing
proposed method was validated as per International the detection wavelength of the analysis. In the present study,
Conference on Harmonization (ICH) (19) guidelines for dif- the robustness was evaluated by using the Box-Behnken
ferent parameters such as linearity, accuracy, precision, limit response surface design (24). The design simultaneously evalu-
of detection (LOD) and limit of quantification (LOQ) and ated the effects of the three important parameters on peak area:
robustness, similar to the previously reported laboratory flow rate of the mobile phase, detection wave length and tem-
methods (20– 23). perature of the column oven. Design Expert version 7.0.0.1
(Stat-Ease, Minneapolis, MN) was used to evaluate the results.
Three-dimensional graphs represented peak area dependence
Calibration curve for glabridin on flow rate of the mobile phase and detection wavelength
A stock solution of glabridin with a known concentration of temperature of the column and flow rate of the mobile phase
1,000 mg/mL was prepared in methanol and different aliquots and temperature of the column and detection wavelength.
were made to get six different desired concentrations from 1 Effects of the selected factors were evaluated over a range of

780 Kamal et al.

conditions by determining the maximum area response of the glabridin were calculated using YL Clarity software, which
glabridin peaks. proved the suitability of the proposed method.

Results and Discussion Validation of analytical method

Method development
Linearity
A variety of mobile phases was investigated for the development
of the HPLC method suitable for analysis of glabridin in crude To assess the linearity of the method, a stock solution of
drug and polyherbal formulations. The investigated mobile 1,000 mg/mL was prepared, and from this, different aliquots
phases included methanol–water, 50:50 (% v/v), acetonitrile– were prepared from 1 –500 mg/mL. The stock solution was
phosphate buffer (pH 3.5 adjusted with orthophosphoric acid), kept in the dark for storage. The linearity of the calibration for
80:20 (% v/v), acetonitrile–phosphate buffer ( pH 3.5 with glabridin was assessed in the range of 1 –500 mg/mL with

Downloaded from https://academic.oup.com/chromsci/article-abstract/50/9/779/288853 by guest on 15 April 2020

orthophosphoric acid), 60:40 (% v/v), acetonitrile–water, 50:50 excellent regression coefficient value 0.9992; the regression
(% v/v) and acetonitrile–water, 60:40 (% v/v). It was observed equation is y ¼ 26.683x – 142.17. The slope + SD and
that acetonitrile and water 60:40 (% v/v) produces a sharp peak intercept + SD were found to be 25.13633 + 0.52 and
of glabridin at retention time of 9.1 + 0.02 min. However, this 141.11 + 1.2, respectively.
mobile phase was not efficient to separate the compound in
polyherbal formulation because of poor separation from the im- Accuracy
mediate impurities. Therefore, it was decided to make a gradi- The accuracy of the method was determined by recovery
ent elution system with acetonitrile concentration from 50 to studies. The preanalyzed samples were spiked with standard at
80% within 20 min, which resulted in a sharp peak at the Rt of three different concentration levels, i.e., 50, 100 and 150%. The
14.9 + 0.02 min with good separation from the impurities mixtures were reanalyzed by the proposed method and found
(Figure 2). Furthermore, chromatographic conditions were to be within the limit of 97.39–103.25%, which is better than
optimized and system suitability parameters like theoretical the method reported by Shanker et al. (14). The values of
plates (7,739), tailing factor (0.982) and asymmetry (0.958) of recovery percent and %RSD are listed in Table I.

Figure 2. HPLC chromatogram of glabridin at 230 nm: standard (A); Glycyrrhiza glabra (B); Qurs-e-Gul tablet formulation (C).

Rapid RP-HPLC Method for the Quantification of Glabridin in Crude Drug and in Polyherbal Formulation 781
 Downloaded from https://academic.oup.com/chromsci/article-abstract/50/9/779/288853 by guest on 15 April 2020
Figure 3. Three-dimensional graphs: Peak area ¼ f (flow rate of the mobile phase, detection wave length) (A); peak area ¼ f (temperature of the column, flow rate) (B);
peak area ¼ f (temperature of the column, detection wave length) (C).

Table I (Waters Co., Milford, MA) but on same column. The results
Accuracy of the Method (n ¼ 3) from the determination of repeatability and intermediate
precisions, expressed as %RSD, were listed in Table II and III.
Percent of standard Theoretical Amount of drug Percent of drug %RSD
spiked to the sample content (mg/mL) recovered (mg + SD) recovered
LOD and LOQ
0 12.8 12.5 + 0.3 97.39 2.58
50 19.2 19.1 + 0.4 99.30 1.84 The limits of quantification and detection were calculated by
100 25.6 26.4 + 0.5 103.25 1.75 using the linearity curve method by using the formula LOD ¼
150 32.0 31.4 + 0.9 98.12 2.83
3.3s/S and LOQ ¼ 10s/S, where s is the standard deviation of
the response and S is the slope of the calibration plot. For the
developed method, LOD was found to be 0.35 mg/mL and LOQ
Table II was calculated at 1 mg/mL. Once the LOD and LOQ were
Repeatability of the Method (n ¼ 3)
determined, six replicates of blank and standard solutions at
Concentration (mg/mL) Peak area Retention time the levels of LOD and LOQ were applied and the %RSD was
Mean peak area + SD % RSD Mean Rt + SD %RSD
calculated.
50 1,135.6 + 13.31 1.17 14.97 + 0.06 0.40
100 2,231.3 + 30.55 1.37 15.06 + 0.10 0.66
Robustness
200 5,273 + 34.40 0.65 15.00 + 0.08 0.53 The robustness was evaluated by using the Box-Behnken
response surface design. The Design Expert software proposed
the following polynomial equation for peak area:
Precision Peak area ¼ 6572.00 þ 0.76A þ 35.22B – 11.42C –
Precision of the proposed method was obtained by repeatabil- 24.68AB þ 3.16AC þ 16.77BC – 7.38A2 – 73.39B2 – 69.63C2,
ity and intermediate precision in accordance with the ICH where A is the detection wavelength (nm), B is the flow rate
recommendations. In repeatability studies, six different injec- (mL/min) and C is the temperature (8C). According to the
tions of the same standard sample (three concentrations) were equation, flow rate appeared to have more effect on the peak
injected and calculated the assay. The %RSD of area and Rt area than detection wavelength and temperature. As the flow
were calculated. Inter-day and intra-day precisions were per- rate increases, peak area also increases. The detection wave-
formed by preparing and applying three different concentra- length was also found to increase the peak area, as indicated by
tions of samples during the same day and on three different the positive coefficient value. However, the lower magnitude of
days, respectively. Inter-system precision was performed by the coefficient indicated that the effect of detection wave-
repeating the same procedure by using a different system length is less than the flow rate on peak area. The temperature

782 Kamal et al.

Table III
Precision of the Method (n ¼ 3)

Concentration (mg/mL) Inter-day precision Intra-day precision Inter-system precision

Mean area + SD %RSD Mean area + SD %RSD Mean area + SD %RSD
50 1,125.3 + 14.61 1.29 1,138.7 + 11.53 1.01 1,132.0 + 6.21 0.55
100 2,234.6 + 20.80 0.93 2,238.0 + 20.04 0.89 2,258.0 + 40.03 1.77
200 5,303.0 + 76.40 1.44 5,306.3 + 73.32 1.38 5,293.0 + 90.05 1.70

affected the peak area in the opposite direction to that of of the presence of markers rather than their quantity is usually
observed for the detection wavelength and flow rate. The nega- important in relation to identity and safety of traditional drugs.

Downloaded from https://academic.oup.com/chromsci/article-abstract/50/9/779/288853 by guest on 15 April 2020

tive coefficient value of the temperature indicated that the
peak area decreased with an increase in temperature. Although
the software predicted that the different factors could affect
the peak area, the values of peak area were in an acceptable Conclusion
range to demonstrate sufficient robustness of the analytical A simple, economic, accurate, precise, reproducible and robust
method. RP-HPLC –UV method for determination of glabridin was devel-
The response surface plots represent the dependence of oped and validated in crude drug and polyherbal formulation
peak area on: (i) flow rate of the mobile phase and detection over a wide concentration range. No interference was observed
wavelength (Figure 3A); (ii) temperature of the column and at the elution time of glabridin in both standard and samples.
flow rate of the mobile phase (Figure 3B); and (iii) temperature The validation data are indicative of good precision and accur-
of the column and detection wavelength (Figure 3C). The acy, and proved the reliability of the method. The low LOD
interactive effects of the selected factors were evaluated over a (0.35 mg/mL) and LOQ (1 mg/mL) values indicate the good
range of conditions by determining the maximum area re- sensitivity of the method, and prove that the developed
sponse of the glabridin peak using these response surface method is much more sensitive than the existing HPLC
plots. The figures show that flow rate contributed more signifi- method proposed by Shanker et al. (14) for the analysis of glab-
cantly toward the response of peak area in all the plots than ridin, with LOD and LOQ of 0.019 and 0.065 mg/mL. The pro-
detection wavelength and temperature. Figure 3A shows that at posed method was found to be excellent for the routine
higher detection wavelength and flow rate, peak area is analysis of glabridin in crude drug and polyherbal formulation.
decreases compared to that observed at lower detection wave- This simple and validated analytical method will help in the up-
length and flow rate. Figure 3B shows that the response surface coming research on this important anticancer moiety for quan-
is slightly influenced by the combined effects of temperature tification in different herbal formulations. The method can be
and detection wavelength. The result of their interaction used for quality control of Glycyrriza root and several polyher-
resulted was that the peak areas were highest at the extremes bal formulations that contain it, as an alternative to glycyrrhizin
of the temperature selected for the study and almost irrespect- and glycyrrhetinic acid, which is a less specific marker than to
ive of the detection wavelength. The interaction of flow rate glabridin (25).
and temperature (Figure 3C) resulted in an increased peak area
at the extremes of temperature and a decreased peak area at
the extremes of flow rate.
Acknowledgment
Authors are thankful to CCRUM-AYUSH, Government of India
for providing financial assistance.
Analysis of crude sample and herbal formulation
The newly developed and validated RP-HPLC method was
applied for the analysis of glabridin in Glycyrrhiza glabra root
powder and in a polyherbal Unani formulation (Qurs-e-Gul). References
The peak areas of triplicate samples were analyzed by regres- 1. Tsai, Y.M., Yang, C.J., Hsu, Y.L., Wu, L.Y., Tsai, Y.C., Hung, J.Y., et al.;
sion equation obtained from the calibration plot to determine Glabridin inhibits migration, invasion, and angiogenesis of human
non-small cell lung cancer a549 cells by inhibiting the FAK/Rho
the content of glabridin in samples. No interference was found
signaling pathway; Integrative Cancer Therapies, (2011); 10:
at the retention time of glabridin in samples (Figure 2). The 341–349.
content of glabridin in the Glycyrriza glabra was found to be 2. Hsu, Y.L., Wu, L.Y., Hou, M.F., Tsai, E.M., Lee, J.N., Liang, H.L., et al.;
0.098% w/w, which was supposed to vary between 0.07 –0.8% Glabridin, an isoflavan from licorice root, inhibits migration, inva-
w/w according to the literature (15). Similarly, glabridin sion and angiogenesis of MDA-MB-231 human breast adenocarcin-
content in polyherbal formulation (tablets) was found to be oma cells by inhibiting focal adhesion kinase/Rho signaling
0.016% w/w; the content was expected to be approximately pathway; Molecular Nutrition and Food Research, (2011); 55:
318–327.
0.0196% w/w, because the formulation contains approxoi- 3. Carmeli, E., Harpaz, Y., Kogan, N.N., Fogelman, Y.; The effect of an
mately 20.1% of Glycyrriza glabra root powder, including endogenous antioxidant glabridin on oxidized LDL; Journal of
excepients used for the optimization of tablet formulation. Basic and Clinical Physiology and Pharmacology, (2008); 19:
Moreover, due to large biodiversity in plants, the confirmation 49– 63.

Rapid RP-HPLC Method for the Quantification of Glabridin in Crude Drug and in Polyherbal Formulation 783
 4. Fukai, T., Marumo, A., Kaitou, K., Kanda, T., Terada, S., Nomura, T.; 16. Jirawattanapong, W., Saifah, E., Patarapanich, C.; A validated
Anti-helicobacter pylori flavonoids from licorice extract; Life stability-indicating HPLC method for analysis of glabridin prodrugs
Science, (2002); 71: 1449– 1463. in hydrolysis studies; Drug Discoveries and Therapeutics, (2009);
5. Fatima, Gupta, V.K., Luqman, S., Negi, A.S., Kumar, J.K., Shanker, K., et al.; 3: 97– 103.
Antifungal activity of Glycyrrhiza glabra extracts and its active con- 17. Rauchensteiner, F., Matsumura, Y., Yamamoto, Y., Yamaji, S., Tani, T.;
stituent glabridin; Phytotherapy Research, (2009); 23: 1190–1193. Analysis and comparison of Radix Glycyrrhizae (licorice) from
6. Somjen, D., Katzburg, S., Vaya, J., Kaye, A.M., Hendel, D., Posner, Europe and China by capillary-zone electrophoresis (CZE); Journal
G.H., et al.; Estrogenic activity of glabridin and glabrene from lic- of Pharmaceutical and Biomedical Analysis, (2005); 15: 594–600.
orice roots on human osteoblasts and prepubertal rat skeletal 18. Aoki, F., Nakagawa, K., Tanaka, A., Matsuzaki, K., Arai, N., Mae, T.;
tissues; Journal of Steroid Biochemistry and Molecular Biology, Determination of glabridin in human plasma by solid-phase extrac-
(2004); 91: 241–246. tion and LC-MS/MS; Journal of Chromatography B, (2005); 15:
7. Choi, E.M.; The licorice root derived isoflavan glabridin increases 70– 4.
the function of osteoblastic MC3T3-E1 cells; Biochemical 19. International Conference on Harmonization (ICH). Technical
Pharmacology, (2005); 70: 363– 368. Requirements for the Registration of Pharmaceuticals for

Downloaded from https://academic.oup.com/chromsci/article-abstract/50/9/779/288853 by guest on 15 April 2020

8. Toshio, F., Kazue, S., Taro, N., Hiroshi, S.; Antinephritis and radical Human Use. Validation of Analytical Procedures: Methodology.
scavenging activity of prenylflavonoids; Fitoterapia, (2003); 74: Geneva, Switzerland, (1996).
720–724. 20. Ahmad, S., Rizwan, M., Parveen, R., Mujeeb, M., Aquil, M.; A validated
9. Ofir, R., Tamir, S., Khatib, S., Vaya, J.; Inhibition of serotonin stability-indicating TLC method for determination of forskolin in
re-uptake by licorice constituents; Journal of Molecular crude drug and pharmaceutical dosage form; Chromatographia,
Neuroscience, (2003); 20: 135–140. (2008); 67: 441– 447.
10. Yokota, T., Nishio, H., Kubota, Y., Mizoguchi, M.; The inhibitory 21. Ansari, M.J., Ahmad, S., Kohli, K., Ali, J., Khar, R.K.; Stability-
effect of glabridin from licorice extracts on melanogenesis and in- indicating HPTLC determination of curcumin in bulk drug and
flammation; Pigment Cell Research, (1998); 11: 355–361. pharmaceutical formulations; Journal of Pharmaceutical and
11. The Unani Pharmacopoeia of India. Ministry of Health and Family Biomedical Analysis, (2005); 39: 132– 138.
Welfare. Department of AYUSH, Government of India, New Delhi, 22. Alam, P., Ali, M., Singh, R., Madhurima, Ahmad, S., Shakeel, F.; A vali-
(2007); pp. 32– 33. dated HPLC method for estimation of cordifolioside A in Tinospora
12. Ahmad, S., Rehman, S., Ahmad, M.A., Siddiqui, K.M., Shaukat, S., cardifolia Miers and marketed formulations; Journal of
Khan, M.S., et al.; Khamiras, a natural cardiac tonic: An overview; Chromatographic Science, (2009); 47: 910– 913.
Journal of Pharmacy and BioAllied Sciences, (2010); 2: 93– 99. 23. Rabea, R., Baboota, S., Ali, J., Ahuja, A., Ahmad, S.; Stability-indicating
13. National Formulary of Unani Medicine. Ministry of Health and HPTLC method for quantitative estimation of silybin in bulk drug
Family Welfare, Department of AYUSH. Government of India, and pharmaceutical dosage form; Biomedical Chromatography,
New Delhi, (2006); pp. 30 –31. (2010); 24: 639– 647.
14. Shanker, K., Fatima, A., Negi, A.S., Gupta, V.K., Darokar, M.P., Gupta, 24. Kuntic, V., Pejic, N., Ivkovic, B., Vujic, Z., Ilicc, K., Micic, S.,
M.M., et al.; RP-HPLC method for the quantitation of glabridin in Vukojevicd, V.; Isocratic RP-HPLC method for rutin determination
Yashti-madhu (Glycyrrhiza glabra); Chromatographia, (2007); 65: in solid oral dosage forms; Journal of Pharmaceutical and
771–774. Biomedical Analysis, (2007); 43: 718– 721.
15. Tian, M., Yan, H., Row, K.H.; Extraction of glycyrrhizic acid and 25. Akinloye, B.A., Adalumo, L.A.; Abrus precatorius leaves—A source
glabridin from licorice; International Journal of Molecular of glycyrrhizin; Nigerian Journal of Pharmacy, (1981); 12:
Science, (2008); 9: 571–577. 405– 410.

784 Kamal et al.