Physicochemical Stability and Biological Activity of Withania somnifera Extract under Real-Time and Accelerated Storage Conditions

Abstract

Stability testing at preformulation stages is a crucial part of drug development. We studied physicochemical stability and biological activity of Withania somnifera (ashwagandha) dried root aqueous extract during six months real-time and under accelerated storage conditions. The characteristic constituents of ashwagandha roots include withanolides such as withaferin A and withanolide A. We modified and validated the HPLC‐DAD method for quantitative measurement of withanolides and fingerprint analysis. The results suggest a significant decline in withaferin A and withanolide A content under real and accelerated conditions. The HPLC fingerprint analysis showed significant changes in some peaks during real and accelerated storage (> 20 %). We also observed incidences of clump formation and moisture sensitivity (> 10 %) under real-time and accelerated storage conditions. These changes were concurrent with a significant decline in immunomodulatory activity (p < 0.01) during the third month of the accelerated storage. Thus, adequate control of temperature and humidity is important for WSE containing formulations. This study may help in proposing suitable guidance for storage conditions and shelf life of ashwagandha formulations.

Key words

Withania somnifera - Solanaceae - Ayurveda - ashwagandha - stability - withaferin A - withanolide A

References

• 1 EMEA .Reflection paper on stability testing of herbal medicinal products and traditional herbal medicinal products. EMEA/HMPC/3626/2009. 2009: 1-3
• 2 Goppel M, Franz G. Stability control of senna leaves and senna extracts.  Planta Med. 2004;  70 432-436
• 3 Anuchit P, Sirirat P, Suthimaln I, Tanomjit S. Stability of andrographolide in powdered Andrographis herb under accelerated conditions.  Planta Med. 2006;  72 954-956
• 4 Kopleman S H, Nguyen P A, Zito W S, Muller F X, Augsburger L L. Selected physical and chemical properties of commercial Hypericum perforatum extracts relevant for formulated product quality and performance.  AAPS Pharm Sci. 2001;  3 1-18
• 5 Sahoo N K, Sarkar S, Patwardhan B. Stability study on Boswellia serrata (hydro-alcoholic) extract.  Asian J Chem. 2009;  21 3529-3534
• 6 [no authors listed.]. Ayurvedic pharmacopoeia of India, Vol. 1. Delhi; The Controller of Publications Government of India 2001: 15-16
• 7 Gautam M, Diwanay S S, Gairola S, Shinde Y S, Jadhav S S, Patwardhan B K. Immune response modulation to DPT vaccine by aqueous extract of Withania somnifera in experimental system.  Int Immunopharmacol. 2004;  4 841-849
• 8 Ziauddin M, Phansalkar N, Patki P, Diwanay S, Patwardhan B. Studies on the immunomodulatory effects of Ashwagandha.  J Ethnopharmacol. 1996;  50 69-76
• 9 Patwardhan B, Mashelkar R A. Traditional medicine-inspired approaches to drug discovery: can Ayurveda show the way forward?.  Drug Discov Today. 2009;  DOI: 10.1016/j.drudis.2009.05.009
• 10 Sumantran V N, Kulkarni A, Boddul S, Chinchwade T, Koppikar S J, Harsulkar A, Patwardhan B, Chopra A, Wagh U V. Chondroprotective potential of root extracts of Withania somnifera in osteoarthritis.  J Biosci. 2007;  32 299-307
• 11 Misra L, Mishra P, Pandey A, Sangwan R S, Sangwan N S, Tuli R. Withanolides from Withania somnifera roots.  Phytochemistry. 2008;  69 1000-1004
• 12 Pramanick S, Roy A, Ghosh S, Majumder H K, Mukhopadhyay S. Withanolide Z, a new chlorinated withanolide from Withania somnifera.  Planta Med. 2008;  74 1745-1748
• 13 Kaileh M, Vanden Berghe W, Heyerick A, Horion J, Piette J, Libert C, De Keukeleire D, Essawi T, Haegeman G. Withaferin A strongly elicits IkappaB kinase beta hyperphosphorylation concomitant with potent inhibition of its kinase activity.  J Biol Chem. 2007;  282 4253-4264
• 14 ICH . Guidance for industry on stability data package for registration applications in climatic zones III and IV.  Q1F. 2002;  1-8
• 15 Taylor M, Ginsburg J, Hickey A, Gheyas F. Composite method to quantify powder flow as a screening method in early tablet or capsule formulation development.  AAPS Pharm Sci Technol. 2000;  1 18E
• 16 Ganzera M, Choudhary M I, Khan I A. Quantitative HPLC analysis of withanolides in Withania somnifera.  Fitoterapia. 2003;  74 68-76
• 17 Yuhua C, Wang L, Yu X, Ye J. Development of the chromatographic fingerprint of herbal preparations Shuang–Huang–Lian oral liquid.  J Pharm Biomed Anal. 2006;  41 845-856
• 18 Kai-Tai F, Yi-Zeng L, Xiao-lin Y, Kelvin C, Guang-Hua Lu. Critical value determination on similarity of fingerprints.  Chemometric Intelligent Lab Syst. 2006;  82 236-240
• 19 Nelson D S, Mildenhall P. Studies on cytophillic antibodies. The production by mice of macrophage cytophillic antibodies to sheep erthrocytes; relationship to the production of other antibodies and development of delayed type hypersensitivity.  Aust J Exp Biol Med Sci. 1967;  45 113-130
• 20 Kaul A, Bani S, Zutshi U, Suri K, Satti N K, Suri O P. Immunopotentiating properties of Cryptolepis buchanani root extract.  Phytother Res. 2003;  17 1140-1144
• 21 Abdullah E C, Geldart D. The use of bulk density measurements as flowability indicators.  Powder Technol. 1999;  102 151-165
• 22 Xie P, Chen S, Yi-Zeng L, Wang X, Tian R, Roy U, Van Beek T A, Schripsema J. Chromatographic fingerprint analysis: a rational approach for quality assessment of traditional Chinese herbal medicine.  J Chromatogr A. 2006;  1112 171-180
• 23 Xie Y, Jiang Z H, Zhoua H, Cai X, Wong Y F, Liu Z Q, Bian Z X, Xu H X, Liu L. Combinative method using HPLC quantitative and qualitative analyses for quality consistency assessment of a herbal medicinal preparation.  J Pharm Biomed Anal. 2007;  43 204-212
• 24 Enrique Ortega R. Bulk properties of food particulate materials: an appraisal of their characterization and relevance in processing.  Food Bioprocess Technol. 2009;  2 28-44
• 25 Kopelman S H, Augsburger L L. Some physical properties of Ginkgo biloba extracts important for tableting and encapsulation.  JANA. 2000;  3 32-37
• 26 Chaurasiya N D, Uniyal G C, Lal P, Misra L, Sangwan N S, Tuli R, Sangwan R S. Analysis of withanolides in root and leaf of Withania somnifera by HPLC with photodiode array and evaporative light scattering detection.  Phytochem Anal. 2008;  19 148-154
• 27 Srivastava P, Tiwari N, Yadav A K, Kumar V, Shanker K, Verma R K, Gupta M M, Gupta A K, Khanuja S P. Simultaneous quantification of withanolides in Withania somnifera by a validated high-performance thin-layer chromatographic method.  J AOAC Int. 2008;  91 1154-1161
• 28 Malik F, Kumar A, Bhushan S, Mondhe D M, Pal H C, Sharma R, Khajuria A, Singh S, Singh G, Saxena A K, Suri K A, Qazi G N, Singh J. Immune modulation and apoptosis induction: two sides of antitumoral activity of a standardized herbal formulation of Withania somnifera.  Eur J Cancer. 2009;  45 1494-1509
• 29 Stan S D, Hahm E R, Warin R, Singh S V. Withaferin A causes FOXO3a- and Bim-dependent apoptosis and inhibits growth of human breast cancer cells in vivo.  Cancer Res. 2008;  68 7661-7669
• 30 Bani S, Gautam M, Sheikh F A, Khan B, Satti N K, Suri K A, Qazi G N, Patwardhan B. Selective Th1 up-regulating activity of Withania somnifera aqueous extract in an experimental system using flow cytometry.  J Ethnopharmacol. 2006;  107 107-115