February 8, 2024

Evaluation of Sub-Acute Toxicological Profile of Withania somnifera Root Extract in Wistar Rats: A Comprehensive Review

Posted on February 8, 2024  •  7 minutes  • 1321 words
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In this comprehensive review, the editors of delve into the pivotal study titled “Sub-acute toxicity of Ashwagandha (Withania somnifera) root extract in Wistar rats” by Deepak Langade, Jayshree Dawane, and Priti Dhande, published in Toxicology Reports 11 (2023), 389–395. This critical evaluation not only examines the methodologies and findings presented in the original paper but also situates its significance within the broader context of herbal supplement consumption. Given the growing popularity of Ashwagandha for its myriad health benefits, understanding its safety profile through such toxicological assessments becomes paramount. This review underscores the importance of the study in laying down a scientific foundation for the safe consumption of Ashwagandha in humans, contributing significantly to the field of pharmacology and toxicology.


The study focused on the sub-acute toxicity of repeated dose administration of Withania somnifera (ashwagandha) root extract in Wistar rats , following OECD-407 guidelines over a 28-day period, with an additional 15-day observation for a satellite group. The study involved 60 rats (30 male and 30 female), divided into groups receiving 0 (control), 200, 400, or 800 mg/kg body weight/day of ashwagandha extract. Observations included general health, weight gain, blood biochemistry, and histopathological examination of organs post-sacrifice. The results showed no significant signs of intoxication, changes in blood biochemistry, or adverse histopathological findings in organs, even at doses five times higher than the recommended dose for humans, up to 800 mg/kg. This indicates that ashwagandha root powder extract did not exhibit any major abnormalities or toxicity under the conditions of this study.


Ashwagandha (Withania somnifera), also known as winter cherry and Indian ginseng, is classified as a “Rasayana” or rejuvenator in Ayurvedic literature. It is renowned for maintaining health, rejuvenating the body, potentially enhancing longevity, and is used as a herbal tonic and health food. This herb has shown a wide range of pharmacological activities, including anti-inflammatory, analgesic, anxiolytic , hypnotic, antidepressant, nootropic , antimicrobial, antioxidant, anticonvulsant, cardioprotective, anticancer effects, and more, attributed to its phytochemical constituents like alkaloids , steroidal lactones , saponins , and glycol-withanolides .

Given its longstanding use in Ayurvedic practice and reported benefits, there is also a focus on proving its safety and tolerability through modern evaluation methods in both animals and humans. Toxicity studies on various formulations from different parts of the plant (leaves, root, stem, seeds, fruits) such as methanolic extract, hydroalcoholic extract, decoction, seed powder, root paste, etc., reveal varying concentrations of active ingredients, indicating differing toxicity levels. Research by various scholars on the whole plant extract, hydroalcoholic extract of its roots, and methanol extract of the roots at various doses have aimed to establish safe, tolerable, or no-observed-adverse-effect-level (NOAEL) doses, with one study suggesting 2000 mg/kg as a NOAEL for Withania somnifera.

Despite these efforts, results regarding the toxic dose of Ashwagandha and its formulations remain conflicting, leading to ambiguity. This has motivated further study to evaluate the subacute toxicity of an aqueous extract of Ashwagandha roots’ proprietary standardized preparation, containing more than 5% withanolides, and to consider the clinical dose of this medicine for safety assessment.

Materials and Methods

The study, approved by the Ethics committee, investigated the sub-acute toxicity of an aqueous root extract of Ashwagandha (Withania somnifera), following modified OECD-407 guidelines. Sixty Wistar rats (30 male, 30 female) were acquired, acclimatized, and randomly assigned to groups for testing.

Winstar rat

The extract, KSM-66®, was produced under strict conditions to ensure quality and consistency, involving processes like washing, drying, milling, and sifting the roots to achieve a fine powder.


Dosing for the study extrapolated from typical human consumption (300–600 mg daily), setting low, medium, and high doses at 200 mg/kg, 400 mg/kg, and 800 mg/kg, respectively, administered for 28 days. Observations included changes in body weight, food intake, general appearance, and any abnormal behaviors or mortality. On day 29, after fasting, blood samples were collected for hematological and biochemical analysis.

Hematological parameters assessed included hematocrit , hemoglobin levels, erythrocyte and leukocyte counts, among others. Serum chemistry evaluations measured levels of electrolytes, glucose, cholesterol, liver and kidney function markers, and other metabolic indicators. All animals were then sacrificed for gross necropsy and histopathological examination of vital organs, assessing any pathological changes with a grading system for severity.

Further observations extended to 43 days for satellite groups, with additional assessments performed before sacrificing the animals for histopathology. Statistical analysis, using one-way ANOVA and multiple comparison tests, aimed to identify significant differences between groups, with normality of data confirmed by Kolmogorov-Smirnov and Shapiro-Wilk tests.

Observations and Results

The study observed the effects of Ashwagandha root extract on Wistar rats over a 28-day period and an extended observation for a satellite group up to 43 days, with no significant abnormalities in general parameters. All animals, except one female from the satellite group that died on day 29, survived the study duration. Food consumption and weight gain remained normal across all groups.

Haematological and biochemical parameters showed no significant differences compared to the control group, except for a notable increase in alkaline phosphatase levels in the high-dose Ashwagandha group and total protein and albumin levels in medium and high-dose groups, including the satellite group. Liver function tests, kidney function tests, and lipid profiles remained statistically similar across treated and control groups, indicating no adverse effects on these parameters.

Histopathologically, minimal inflammatory cell infiltration and cytoplasmic rarefaction (suggesting glycogen deposition) were observed in the liver of high-dose male rats, with no significant liver effects in females of the same group. Kidney and lung examinations revealed minimal inflammatory cell infiltration and incidental findings, comparable between control and treated groups, with all observations being of minimal severity.

Overall, the study suggests that Ashwagandha root extract does not significantly alter general health, haematology, or biochemistry parameters in Wistar rats, aside from specific increases in alkaline phosphatase, total proteins, and albumin in high-dose groups, with minimal histopathological changes observed in some organs.


The study on sub-acute toxicity testing aimed to evaluate the safety and potential toxic effects of Ashwagandha root extract through repeated administration in laboratory animals, adhering to modified OECD guidelines. Sixty animals survived until the end of the 28-day study period without showing any significant abnormalities in general parameters such as behavior, appearance, or physiological functions. One female rat from the high dose satellite group died on day 29, with another showing temporary postural abnormalities, which resolved without intervention.

Throughout the study, there was no critical loss in body weight (a loss of more than 20% is considered critical), indicating minimal adverse effects on the animals' general health. Food intake and body weight gradually increased, although a slight reduction in food consumption and slower weight gain were observed in the last two weeks.

Haematological and biochemical parameters, including blood sugar levels, liver and renal function tests, and electrolytes, remained within the normal range, suggesting no significant impact from the Ashwagandha root extract. However, an increase in alkaline phosphatase (ALP), proteins, and albumin was observed in the high dose group (800 mg/kg), which still fell within the normal limits for rodents. This transient increase in ALP, which normalized in the satellite group after discontinuation of the drug, may indicate reversible liver toxicity potentially due to toxic metabolites of Ashwagandha that are detoxified under normal conditions.

Histopathological examinations revealed minimal infiltration of inflammatory cells in the liver, lungs, and kidneys, with these findings considered to be spontaneous or incidental due to their low occurrence rate and severity. These results align with other studies, indicating Ashwagandha’s safety at doses up to 2000 mg/kg in acute and sub-acute settings without adverse effects on haematological, biochemical, and histological parameters.

The study concludes that Ashwagandha root extract is non-toxic up to a 28-day period at 800 mg/kg, with slight increases in certain biochemical parameters within normal limits and minimal histopathological changes. The findings emphasize the need for standard guidelines to ensure repeatability and generalizability of toxicity testing results.


  1. Sub-acute toxicity of Ashwagandha (Withania somnifera) root extract in wistar rats




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