Objective: The objective of the present study was to evaluate the acute and sub-chronic (90 d; repeated dose) toxicity of Withania somnifera (ashwagandha) extract in rats.Methods: The acute toxicity was evaluated as per OECD (Organisation for Economic Co-operation and Development) guidelines 423. Purified ashwagandha extract (PAE) was fed at 2000 mg/kg body weight (bw) to overnight fasted female rats. The animals were observed daily for clinical signs of abnormality/mortality. After 14 d, animals were sacrificed and gross pathological changes were recorded. Sub-chronic toxicity of PAE was studied by feeding the extract at 100, 500 and 1000 mg/kg bw daily to rats as per OECD guidelines 408. After 90 d feeding, heamatological and biochemical parameters of treated rats were compared with control animals. Histopathology of all the major organs was also studied.Results: In the acute toxicity study, no mortality or clinical signs of toxicity were observed in any of the animals at maximum recommended dose level of 2000 mg/kg bw; therefore the LD50 is>2000 mg/kg bw in rats. The repeated administration of PAE for 90 d in rats at the maximum dose level of 1000 mg/kg bw did not induce any observable toxic effects, when compared to its corresponding control animals. The hematology and biochemistry profile of treated rats was similar to control animals and difference was non-significant (p>0.05). The histopathology of major organs of all the control and treated animals was normal. In this study the NOAEL (No Observed Adverse Effect Level) was calculated as 1000 mg/kg bw daily for rats.Conclusion: The present study clearly indicates that PAE does not have any toxic effects in animals at the dose evaluated as evidenced by acute and sub chronic toxicity studies in rats.

Background: Ashwagandha (Withania somnifera) is an important herb in the Indian traditional system of medicine for neurological disorders. However, the efforts for isolation and characterisation of a molecule with anti-depressant activity and development as a potent dosage form are limited. Objective: The objective of the present study was to characterize the Ashwagandha extract for its antidepressant fraction or constituent and to improve biological benefits at low doses. Methods: Aqueous methanol extract of Ashwagandha was prepared and fractionated into withanolides and flavonoids rich fractions. Withanolide rich fraction was subjected to phytochemical analysis to identify the active molecule/s. The compound was purified by using a semi-preparative HPLC system; identified using various spectroscopic techniques and anti-depressant activity was evaluated in rats. Enteric coating was performed on the extract and fractions after granulation and anti-depressant activity of coated samples were evaluated in rats. Results: Aqueous methanol extract of Ashwagandha and withanolide rich fraction showed prominent dose-dependent anti-depressant activity in forced swim test in rats. Phytochemical analysis of active fraction resulted in the isolation and characterization of a major withanolide glycoside present, namely withanoside X. Enteric coated aqueous methanol extract, withanolide rich fraction and withanoside X showed significant antidepressant activity at low doses as compared to the uncoated forms. Conclusion: The active fraction/isolated compound is sensitive to low pH of the stomach, thus enteric coating might be beneficial to protect the actives in the stomach, facilitating the sustainable release into the intestine and in turn reduce the dosage.

Background:: Withanolide glycosides in Ashwagandha (Withania somnifera), are important metabolites attributed with widely acclaimed therapeutic potential for which validated methods for quantitative determination are limited. Objective:: The primary objective was to develop and validate a Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method for simultaneous quantification of total withanolide glycosides (WG), withanoside IV and withaferin A present in ashwagandha extract.The study also aimed to identify various other constituents present in the extract. Method: : Aqueous methanol extract (AME) of ashwagandha was prepared and fractionated into two viz. flavonoid rich fraction (FF) and withanolide rich fraction (WF). RP-HPLC method was developed and validated for estimation of total WG in ashwagandha extract according to ICH guidelines. Preparative HPLC based purification of major compounds from WF fraction was carried out and constituents were identified using spectroscopic techniques. HPLC chemical profiling of WF before and after acid hydrolysis under controlled conditions were carried out to further confirm the glycosidic compounds. Results: : The RP-HPLC method gave a precise differentiation of flavonoids, withanolides and WG present in ashwagandha extract. The method demonstrated good reliability and sensitivity, and can be conveniently used for the quantification of total WG, withanoside IV and withaferin A present in ashwagandha extracts. According to this method, purified fraction (WF) prepared from roots and leaves of ashwagandha comprise 35 % of total WG, 3.27 % of withanoside IV and 2.40 % of Withaferin A. The method was also applied to different products prepared from ashwagandha with total withanolide glycosides ranged from 1.5 % to 60 % and the results were found to be reproducible. Identification of the individual chemical constituents as well as the acid hydrolytic pattern of the extract further supported the reliability of developed method for the quantitative determination of total WG. This study also reported a new withanolide glycoside named, cilistol V-6’-O-glucoside (Aswanoside) along with some other known withanolide glycosides. Conclusion:: A Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for quantitative determination of total WG, withanoside IV and withaferin A present in ashwagandha extract according to ICH guidelines. This study also reported a new withanolide glycoside named, cilistol V-6’-O-glucoside (Aswanoside) along with some other known WG.