Withania somnifera , commonly known as Ashwagandha, is a valued herb in Ayurvedic medicine. Root leaves, and preparations of the plant are traditionally used as tonic, hypnotic, sedative and diuretic. W. somnifera mainly contains withanolides which are specific to the Solanaceae family. The biological activity of withanolides, especially withaferin A, has been studied extensively. Methods for analysis of withaferin A and withnolide D require acetylation before analysis, or separation time is extremely long. The main problem in identification of withaferin Awith other separated withanolides is that all withanolides absorb UV light, so without comparison with a standard it is not possible to identify withaferin A. The objectives of this paper are to present a new method of identification of withaferin A, using a destructive reagent, and an HPTLC method for quantification of the compound. Chromatography on silica with toluene-ethyl acetate-acetone 2:3:3 as mobile phase enabled good resolution of withaferin A without interference from other compounds present in W. somnifera . After spraying with anisaldehyde-sulfuric acid reagent and heating for 15 min at 105°C, characteristic orange fluorescence was observed for withaferin Aonly among all the spots resolved. When scanned at 214 nm the RF of withaferin A was 0.62. Interestingly, old root did not contain withaferin A whereas young root contained a large amount. The method was validated for accuracy, precision, specificity, linearity, and limits of detection and quantification. The method is simple, sensitive, and precise, and can be used for routine quality-control testing of W. somnifera .
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