Development and Validation of High Performance Liquid Chromatography Method for Simultaneous Estimation of Flavonoid Glycosides in Withania somnifera Aerial Parts

Withania somnifera (L.) Dunal (Solanaceae) commonly known as ashwagandha, is an important plant in Ayurveda and is believed to increase longevity and vitality. The root is considered to be the medicinally important part of the plant as per classical texts and accordingly is the subject of most Pharmacopeial monographs. The aerial parts, being less expensive, are sometimes mixed with roots to prepare “standardized” extracts of W. somnifera, and in cases with false declaration of plant part used as roots on the certificate of analysis. The present study described a new, simple, accurate, and precise HPLC method for the simultaneous determination of flavonoid glycosides as unique constituents of the aerial parts, being absent in roots of the plant. The RSD for intra- and interday analyses was less than 2.5% and the recovery was 90–108%. The method was used to analyze samples of roots and aerial parts of the plant collected from India and Egypt. The samples of commercially available extracts of W. somnifera were also analyzed and many samples were found to contain flavonoid glycosides indicating a possible undeclared use of aerial parts in the extracts derived from roots in commercial practice. 1. Introduction Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as ashwagandha or winter cherry, is considered one of the most important medicinal plants in Indian traditional systems of medicine [1–3]. It is a shrubby plant cultivated in India and is primarily used as a rejuvenator and energizer; it grows in parts of East Asia and Africa [4, 5]. The plant has been reported to possess diverse biological activities which include anticancer, antistress, anti-inflammatory [6, 7], antibiotic, anticonvulsant, central nervous system depressant, hepatoprotective, immunomodulatory [8, 9], and insect antifeedant activities. The chemistry of W. somnifera has been extensively studied and over 12 alkaloids, 35 withanolides, several sitoindosides, and flavonoids have been isolated from different parts of the plant [10, 11]. Withanolides, the steroidal lactones with a C28 steroidal nucleus and C9 side chain, containing a six-member lactone ring, are thought to contribute to the bioactivity of the plant. Interestingly, withanolides are present in both the roots as well as in the aerial parts [12, 13]. HPLC and HPTLC-based methods have been reported in the literature for quantitative determination of withanolides in W. somnifera [14–21]. The plant has been categorized under “medicinal plant species in high trade sourced largely from cultivation” by the National