Journal of Chromatographic Science Advance Access published January 8, 2015 Journal of Chromatographic Science 2015;1– 5 doi:10.1093/chromsci/bmu153
Article
Simultaneous Estimation of Withaferin A and Z-Guggulsterone in Marketed Formulation by RP-HPLC Poonam Agrawal*, Rashmi Vegda and Kirti Laddha Medicinal Natural Products Research Laboratory (MNPRL), Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga (E), Mumbai 400 019, India *Author to whom correspondence should be addressed. E-mail:
[email protected] Received 14 October 2013; revised 3 September 2014
A simple, rapid, precise and accurate high-performance liquid chromatography (HPLC) method was developed for simultaneous estimation of withaferin A and Z-guggulsterone in a polyherbal formulation containing Withania somnifera and Commiphora wightii. The chromatographic separation was achieved on a Purosphere RP-18 column ( particle size 5 mm) with a mobile phase consisting of Solvent A (acetonitrile) and Solvent B (water) with the following gradients: 0 – 7 min, 50% A in B; 7 – 9 min, 50 – 80% A in B; 9 – 20 min, 80% A in B at a flow rate of 1 mL/min and detection at 235 nm. The marker compounds were well separated on the chromatogram within 20 min. The results obtained indicate accuracy and reliability of the developed simultaneous HPLC method for the quantification of withaferin A and Z-guggulsterone. The proposed method was found to be reproducible, specific, precise and accurate for simultaneous estimation of these marker compounds in a combined dosage form. The HPLC method was appropriate and the two markers are well resolved, enabling efficient quantitative analysis of withaferin A and Z-guggulsterone. The method can be successively used for quantitative analysis of these two marker constituents in combination of marketed polyherbal formulation. Introduction In recent times, there have been increased waves of interest in the field of Research in Natural Products Chemistry. This level of interest can be attributed to several factors, including therapeutic needs, the remarkable diversity of both chemical structure and biological activities of naturally occurring secondary metabolites, the development of novel and sensitive techniques to detect biologically active natural products, and improved techniques to isolate, purify and structurally characterize these active constituents (1). Withaferin A (Figure 1), chemically characterized as 4b,27dihydroxy-1-oxo-5b,6b-epoxywitha-2, 24-dienolide (2), is the chief constituent present in the plant Withania somnifera (L.) Dunal. (Solanaceae), commonly known as Ashwagandha (3). The plant is reputed to have immunomodulatory activity (4) and antidepressant activity (5). It is also reported to possess various other activities such as hypoglycemic, diuretic, antiinflammatory, anti-stress, etc. (6 –8). Phytochemical screening of plant showed the presence of alkaloid, glycosides, steroids, flavonoids, etc. (9). The biologically active chemical constituents are alkaloids (such as ashwagandhine, cuscohygrine, anahygrine, tropine, etc.), steroidal compounds, including ergostane-type steroidal lactones, withaferin A, withanolides A – Y, withasomniferin-A, withasomidienone, withasomniferols A – C, withanone, etc. Other constituents
include saponins containing an additional acyl group (sitoindoside VII and VIII), and withanolides with a glucose at carbon 27 (sitoindoside IX and X). Apart from these contents, plant also contain chemical constituents like withaniol, acyl steryl glucosides, starch, reducing sugar, hantreacotane, ducitol, a variety of amino acids like aspartic acid, proline, etc. (10). Z-Guggulsterone, chemically 4,17(20)-(trans)-pregnadiene3,16-dione (11) (Figure 1), is the main constituent present in Commiphora wightii (Arnott.) Bhandari, commonly known as guggul, and is an important medicinal plant of herbal heritage of India, belonging to the family Burseraceae (12). The plant is traditionally used in the management of hypercholesterolemia and obesity (13 – 15). Other activities reported in the plant are antiarthritic activity, anticancer, antioxidant, etc. (16–25). The main constituent present is steroid, namely guggulsterone (C21H28O2), that includes E-guggulsterone [4,17(20)-(cis)pregnadiene-3,16-dione] and Z-guggulsterone [4,17(20)-(trans)pregnadiene-3,16-dione] (11). It also shows the presence of guggulsterols (26). It contains essential oils (0.4%) consisting chiefly of myrcene. The gum resin also showed the presence of long-chain aliphatic 1,2,3,4-tetrols esterified with ferulic acid at the primary hydroxyl function (27, 28). Many formulations are marketed individually or in combination with other drugs. High-performance liquid chromatography (HPLC) methods are available for estimation of withaferin A (29) and Z-guggulsterone (30) individually or in combination with other drugs. To the best of our knowledge, no data are reported on simultaneous estimation of these drugs by any analytical method. Hence, the main objective of this study is to develop such method of analysis, which can estimate these drugs in combination not only to facilitate standardization of polyherbal formulations, but also for use in scientific and commercial applications. Experimental Chemicals and reagents Withaferin A ( purity 95% by HPLC) and Z-guggulsterone ( purity 96% by HPLC) were purchased from Sigma-Aldrich, India. HPLC-grade methanol and acetonitrile were obtained from Merck, India. Water was double-distilled. Solvents were filtered through a 0.45-mm filter (Millipore Bedford, MA, USA) and degassed in an ultrasonic bath (Remi Instruments, Mumbai, India) before use. A polyherbal formulation (tablets), Yogaraaj Guggul (500 mg), containing W. somnifera and C. wightii extracts, manufactured by Ayurvedic Rasashala, was purchased from local market in Mumbai
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was concentrated and filtered through Whatman filter paper no. 41. The filtrate was transferred to a 25-mL volumetric flask. Volume was made upto the mark with mobile phase methanol. From above stock solution, 1 mL was transferred to volumetric flask of 10 mL capacity and then diluted to 10 mL with methanol (sample solution). The resulting solution was then analyzed by the proposed method. Twenty microliters of sample preparation was injected into an injector of liquid chromatograph. Each sample was injected and analyzed in triplicate. From the chromatograms, peak area was obtained and the amount of both the drugs was calculated. Method validation The present method was validated as per the ICH guidelines. The method was validated in terms of linearity, accuracy, and inter- and intraday precision. Accuracy of method was evaluated by carrying out recovery study, wherein known amount of standard was added to the known amount of pharmaceutical dosage form. Precision was performed to find out intra- and interday variation by triplicate analysis. Linearity of the standard solutions was determined by using six different concentration levels (31–33). Results
Figure 1. Chemical structures of (A) withaferin A and (B) Z-guggulsterone.
Instrumentation HPLC analysis was performed with a Jasco (Hachioji, Tokyo, Japan) system consisting of an intelligent pump (PU-1580, PU-2080), a high-pressure mixer (MX-2080-31), a manual sample injection valve (Rheodyne 7725i) equipped with a 20-mL loop and a UV– visible detector (UV-1575). Compounds were separated on a 250 mm 4.6 mm i.d., 5-mm particle, Hibar LiChrocart Purospher Star RP-18 endcapped column (Merck, Darmstadt, Germany). A gradient system consisting of acetonitrile and water was used. Preparation of standard stock solution and calibration Accurately weighed quantity of withaferin A (10 mg) and Z-guggulsterone (10 mg) was transferred separately to a standard 10-mL volumetric flask and dissolved in 5 mL of methanol. The flask was then sonicated for 5 min and volume was made up to the mark with mobile phase methanol (1,000 mg/mL). Further dilutions were prepared from the stock solutions, by diluting a required volume of stock solution with methanol to get a final concentration range of 50–250 mg/mL for withaferin A and 10–60 mg/mL for Z-guggulsterone (withaferin A concentration range 50–250 mg/mL; 50, 100, 150, 200 and 250 mg/mL; Z-guggulsterone concentration range 10–60 mg/mL; 10, 20, 30, 40, 50 and 60 mg/mL).
Chromatography The chromatographic separation of withaferin A and Z-guggulsterone was obtained using a mobile phase as Solvent A (acetonitrile) and Solvent B (water) with the following gradients: 0 –7 min, 50% A in B; 7 –9 min, 50–80% A in B; 9 –20 min, 80% A in B at a flow rate of 1 mL/min and detection at 235 nm. The compounds were eluted at 5.32 and 17.82 min, respectively.
Method validation The linear regression equation for two drugs where y is peak area and x is concentration was: y ¼ 14,987x 2 14,592 (r 2 ¼ 0.997) for withaferin A and y ¼ 90,301x 2 23,108 (r 2 ¼ 0.991) for Z-guggulsterone. The results of the accuracy or recovery study are reported in Table I. The characteristics of linear regression analysis are given in Table II. Results of the precision study for withaferin A and Z-guggulsterone by the proposed HPLC method are given in Table III. The limit of detection (LOD) and limit of quantitation (LOQ) were 1.76 and 3.59 mg/mL, respectively, for withaferin A and 0.38 and 0.69 mg/mL, respectively, for Z-guggulsterone. For specificity, the retention time of standard drug was compared with that of sample, and the results were found to be specific.
Tablet analysis The amounts of withaferin A and Z-guggulsterone in polyherbal formulation were found to be 0.740 and 0.106%, respectively.
Sample preparation Determination of withaferin A and Z-guggulsterone from a combined dosage form Twenty tablets were weighed and the average weight was determined. Powder equivalent to 10 tablets was weighed and extracted with methanol using a Soxhlet apparatus. The extract 2 Agrawal et al.
Discussion Optimization of the chromatographic conditions The primary target in developing this method is to achieve simultaneous determination of withaferin A and Z-guggulsterone in the tablet formulation under common conditions that will be
Table I Recovery Study of Withaferin A and Z-guggulsterone Drug
Amount taken (mg/mL)
Amount added (mg/mL)
Meana% Recovery + SD
RSDa (%)
Withaferin A
80 80 80 12 12 12
100 90 80 10 15 20
99.61 + 0.68 101.65 + 0.56 100.83 + 0.985 101.87 + 1.73 98.35 + 1.87 99.97 + 1.13
0.683 0.555 0.977 1.702 1.906 1.134
Z-Guggulsterone
SD: standard deviation; RSD: relative standard deviation. Average of three estimation at each level of recovery.
a
Table II Characteristics of Linear Regression Analysis of Withaferin A and Z-Guggulsterone Parameter
Withaferin A
Z-Guggulsterone
Linearity range (mg/mL) Slope Intercept Correlation co-efficient RSD of slope (%) RSD of intercept (%)
50 –250 14,987 14,592 0.997 1.84 1.42
10 –60 90,301 23,108 0.991 2.11 1.73
Precisiona (% RSD) Intraday
Withaferin A Z-Guggulsterone
LODa (mg/mL)
LOQa (mg/mL)
1.76 0.38
3.59 0.69
Interday
Rt
Pa
Rt
Pa
0.18 0.53
0.45 2.12
1.11 1.72
0.54 1.26
operating conditions. Chromatograms obtained from methanol extract of polyherbal formulation showing withaferin A and Z-guggulsterone are shown in Figure 3.
Method validation
Table III Result of Intra- and Interday Precision, LOD and LOQ Study (n ¼ 3) Parameters
Figure 2. HPLC chromatogram of withaferin A and Z-guggulsterone in standard mixture.
RSD: relative standard deviation; Rt: retention time; Pa: peak area; LOD: limit of detection; LOQ: limit of quantification. a Average of three determinations.
applicable for routine quality control of the product in laboratories. Trials were carried out using an isocratic system with a mobile phase such as acetonitrile and water with 50 : 50% v/v and 60 : 40% v/v ratios. At 50 : 50, the peaks of withaferin A and Z-guggulsterone were eluted at 5.68 and 32.40 min, respectively. Since it showed high run time, this method was rejected. Further increasing acetonitrile concentration as 60 : 40 ratio, the compounds were eluted at 4.03 and 17.20 min and interference was observed, hence this method was rejected. Finally, the gradient system was used for method development. Different trials were carried out by changing the concentration of mobile phase at different time intervals, but due to interferences observed it was rejected. Therefore, for the present study, a mobile phase consisting of Solvent A (acetonitrile) and Solvent B (water) with the above gradient system was used. The UV wavelength was optimized at 235 nm (isobestic point) for both detection and quantification. At this wavelength, both the drugs gave significant absorption. The peaks of withaferin A and Z-guggulsterone were eluted at 5.32 and 17.82 min, respectively, with symmetry and well-retained peaks. Chromatograms obtained from a mixed standard solution (Figure 2) revealed good separation of the selected marker constituents. Chromatographic peaks were identified by comparing their retention times under the same
Linearity Linearity test solutions were prepared by diluting stock solutions (1 mg/mL) at six different concentrations for both drugs. The solution was injected in triplicate and curves were obtained by plotting peak area against concentration of the drugs. From the mean of the two calibration curve, it was clear that withaferin A had linearity between 50 and 250 mg/mL, whereas Z-guggulsterone had a range between 10 and 60 mg/mL. Accuracy The accuracy of an analytical method is determined by how close the test results obtained by this method come to the true value. It can be determined by application of the analytical procedure to the analyte of known purity (for the drug substance) or by recovery studies, where a known amount of standard is spiked in the placebo (for drug product). In the present study, standard drug solutions were added at three different concentration levels to a preanalyzed sample solution. The recovery rate was calculated using expected values obtained by adding known amounts of standards, and actual values were obtained by HPLC analysis. From the recovery study, it was clear that the method is very accurate for quantitative estimation of withaferin A and Z-guggulsterone in a tablet dosage form, because all the statistical results were within the acceptance range (i.e., RSD% ,3.0). Precision, LOD and LOQ The precision of the test method was demonstrated by intra- and interday studies. The intraday (repeatability) study was carried out by injecting three repeated injections of standard solution, by one analyst under the same experimental conditions. The intermediate precision (interday) was studied over three consecutive days at three different concentration levels. Three replicate injections were injected for each solution. The precision was calculated as %RSD ¼ (s/mean) 100, where RSD is relative Estimation of Withaferin A and Z-Guggulsterone by RP-HPLC 3
When the method was subsequently used for analysis of a polyherbal formulation, the amounts of withaferin A and Z-guggulsterone were 0.740 and 0.106%, respectively.
Conclusion
Figure 3. HPLC chromatogram of withaferin A and Z-guggulsterone in the formulation.
standard deviation and s is the standard deviation. RSD in the precision study for the withaferin A and Z-guggulsterone assay was ,2.0%, which confirmed that the method was highly precise. According to the ICH guideline, LOD and LOQ studies can be carried out by the use of standard deviation of the response and the slope of the calibration curve. Hence, this was used to evaluate the detection and quantification limits of the method to determine the presence of any impurities by using following equation: LOD ¼ 3.3 and LOQ ¼ 10 s/S, where s is the standard deviation and S is the slope of the curve. The LOD and LOQ were 1.76 and 3.59 mg/mL, respectively, for withaferin A and 0.38 and 0.69 mg/mL, respectively, for Z-guggulsterone. Specificity The ability of an analytical method to assess the analyte in the presence of other components in the formulation can be demonstrated by specificity. The specificity of the method was determined by injecting the placebo solution having the same concentration as that of tablet formulation. Specificity of the method was thus assessed by comparing the chromatograms obtained from standard drugs (Figure 2) with the chromatograms obtained from tablet solution (Figure 3). Because the retention time of standard drugs and the retention time of two drugs in sample solutions were the same, the method was specific. This was also confirmed by spiking the sample solution with that of standard, the results were found to be specific. To check the selectivity of the developed method, solutions of the three drugs were injected into the system, and three sharp peaks for withaferin A and Z-guggulsterone were obtained at retention times of 5.32 and 17.82 min, respectively, in reference to placebo solution. The developed method was specific and selective as no interference of excipients was found. The above method was found to be advantageous as it enables rapid, simple and accurate simultaneous quantification of withaferin A and Z-guggulsterone. Also, peaks of other constituents present in formulation showed no interference with that of marker compounds. This method can therefore be effectively used for simultaneous estimation of an important steroidal lactone and a pregnane derivative in a marketed formulation. 4 Agrawal et al.
A new isocratic RP-HPLC method has been developed for simultaneous quantification of withaferin A and Z-guggulsterone. Low cost, environment friendly, faster speed analysis and satisfactory precision and accuracy are the main features of this method. It can be used for qualitative as well as quantitative analysis and quality control of polyherbal formulations containing Ashwagandha and guggul extracts on the basis of analysis of withaferin A and Z-guggulsterone as marker compounds.
References 1. Rout, S., Choudhary, K., Kar, D., Das, L., Jain, A.; Plants in traditional medicinal system—future source of new drugs; International Journal of Pharmacy and Pharmaceutical Sciences, (2009); 1: 1–23. 2. The Merck Index. 13th ed. Merck & Co., Inc., New Jersey, (2001), pp. 792. 3. The wealth of India: a dictionary of Indian raw material and industrial products. Publication and Information Directorate, CSIR, New Delhi, (1976), pp. 580– 585. 4. Davis, L., Kuttan, G.; Immunomodulatory activity of Withania somnifera; Journal of Ethnopharmacology, (2000); 71: 193–200. 5. Bhattacharya, S.K., Bhattacharya, A., Sairam, K., Ghosal, S.; Anxiolytic-antidepressant activity of Withania somnifera glycowithanolides: an experimental study; Phytomedecine, (2000); 7: 463–469. 6. Andallu, B., Radhika, B.; Hypoglycemic, diuretic and hypocholesterolemic effect of winter cherry (Withania somnifera) root; Indian Journal of Experimental Biology, (2000); 38: 607–609. 7. Begum, V.H., Sadique, J.; Long-term effect of herbal drug Withania somnifera on adjuvant-induced arthritis in rats; Indian Journal of Experimental Biology, (1988); 26: 877–882. 8. Mishra, L.C., Singh, B.B., Dagenais, S.; Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review; Alternative Medicine Reviews, (2000); 5: 334–346. 9. Kulkarni, S.K., Dhir, A.; Withania somnifera: an Indian ginseng; Progress in Neuro-Psychopharmacology and Biological Psychiatry, (2008); 32: 1093–1105. 10. Sharma, V., Sharma, S., Paliwal, R.; Withania somnifera: a rejuvenating ayurvedic medicinal herb for the treatment of various human ailments; International Journal of Pharmaceutical Technology and Research, (2011); 3: 187–192. 11. Bajaj, A.G., Dev, S.; Chemistry of ayurvedic crude drugs—V: Guggulu (resin from Commiphora mukul)—5 some new steroidal components and stereochemistry of guggulsterol-I at C-20 and C-22; Tetrahedron, (1982); 38: 2949–2954. 12. Indian Pharmacopoeia. 6th ed. Controller of Publication, The Indian Pharmacopoeia Commission, Ghaziabad, (2010); pp. 2504– 2506. 13. Latha, B.P., Reddy, I.R.M., Ismail, S.M., Vijaya, T.; Medicinal plants and their derivatives as potential source in treatment of obesity; Asian Journal of Experimental Biology and Science, (2010); 1: 719–727. 14. Saxena, G., Singh, S.P., Pal, R., Singh, S., Pratap, R., Nath, C.; Gugulipid, an extract of Commiphora wightii with lipid-lowering properties, has protective effects against streptozotocin-induced memory deficits in mice; Pharmacology Biochemistry and Behaviour, (2007); 86: 797–805. 15. Gujral, M.L., Saxena, K., Tangri, K.K., Amma, M.K., Roy, A.K.; Antiarthritic and anti-inflammatory activity of gum guggul (Basalsamodenron mukul Hook); Indian Journal of Physiology Pharmacology, (1960); 40: 267–273.
16. Duwiejua, M., Zeitlin, I.J., Waterman, P.G., Chapman, J., Mhango, G.J., Proven, G.J.; Anti-inflammatory activity of resins from some species of the plant family Burseraceae; Planta Medica, (1993); 59: 12– 16. 17. Singh, B.B., Mishra, L.C., Vinjamury, S.P., Aquilina, N., Singh, V.J., Shepard, N.; The effectiveness of Commiphora mukul for osteoarthritis of the knee: an outcomes study; Alternative Therapies in Health and Medicine, (2003); 9: 74 –79. 18. Sharma, K., Rani, R., Dhalwal, K., Shinde, V., Mahadik, K.R.; Natural compounds as anti-arthritic agents—a review; Pharmacognosy Reviews, (2009); 3: 22 –28. 19. Leeman-Neill, R.J., Wheeler, S.E., Singh, S.V., Thomas, S.M., Seethala, R.R., Neill, D.B., et al.; Guggulsterone enhances head and neck cancer therapies via inhibition of signal transducer and activator of transcription-3; Carcinogenesis, (2009); 30: 1848–1856. 20. Chander, R., Rizvi, F., Khanna, A.K., Pratap, R.; Cardioprotective activity of synthetic guggulsterone (E and Z-isomers) in isoproterenol induced myocardial ischemia in rats: a comparative study; Indian Journal of Clinical Biochemistry, (2003); 18: 71– 79. 21. Ojha, S.K., Nandave, M., Arora, S., Mehra, R.D., Joshi, S., Narang, R., et al.; Effect of Commiphora mukul extract on cardiac dysfunction and ventricular function in isoproterenol induced myocardial infarction; Indian Journal of Experimental Biology, (2008); 46: 646–652. 22. Chander, R., Khanna, A.K., Pratap, R.; Anti-oxidant activity of guggulsterone: the active principle of Gugulipid from Commiphora mukul; Journal of Medicinal Aromatic Plant Science, (2004); 24: 370–374. 23. Mester, M., Mester, L., Nityanand, S.; Inhibition of platelet aggregation by guggulu steroids; Planta Medica, (1979); 37: 367. 24. Deng, R.; Therapeutic effects of guggul and its constituent guggulsterone; Cardiovascular Drug Reviews, (2007); 25: 375–390.
25. Siddiqui, M.J.; Guggul: an excellent herbal panacea; Asian Journal of Pharmaceutical Health Science, (2011); 1: 35– 39. 26. Patil, V.D., Nayak, U.R., Dev, S.; Chemistry of ayurvedic crude drugs—I: Guggulu (resin from Commiphora mukul)—1: steroidal constituents; Tetrahedron, (1972); 28: 2341–2352. 27. Dev, S.; Guggultetrols: a new class of naturally occurring lipids; Pure and Applied Chemistry, (1989); 61: 353–356. 28. Patil, V.D., Nayak, U.R., Dev, S.; Chemistry of ayurvedic crude drugs— III: Guggulu (resin from Commiphora mukul)-3 long-chain aliphatic tetrols, a new class of naturally occurring lipids; Tetrahedron, (1973); 29: 1595– 1598. 29. Jain, V., Thakur, A., Soman, G., Laddha, K.S.; Validated HPLC method development for simultaneous analysis of withaferin-A and 6-gingerol; Acta Chromatographica, (2010); 22: 153–159. 30. Jariwala, J.K., Saluja, A.K., Anajwala, C.C., Dakhara, S.L.; Simultaneous estimation of Guggulsterone E & Z and Tinosporaside in Jivitprada vati by HPTLC method; International Journal of Green Pharmacy, (2011); 5: 113–117. 31. ICH, Q2A, Hamonized Tripartite Guideline. Test on Validation of Analytical Procedures, IFPMA. In: Proceedings of the International Conference on Harmonization, Geneva, March 1994. 32. ICH, Q2B, Hamonized Tripartite Guideline. Validation of Analytical Procedure: Methodology, IFPMA. In: Proceedings of the International Conference on Harmonization, Geneva, March 1996. 33. Shinde, P.B., Aragade, P.D., Agrawal, M.R., Deokate, U.A., Khadabadi, S.S.; Simultaneous determination of withanolide A and bacoside A in capsules by high-performance thin-layer chromato-graphy; Indian Journal of Pharmaceutical Science, (2011); 73: 240 – 243.
Estimation of Withaferin A and Z-Guggulsterone by RP-HPLC 5
