DNA Barcoding Reveals Adulteration and Contamination in Ashwagandha Root and Root Powders Sold on Indian Markets

Reviewed: Nagendraprasad A, Varadharajan B, Madasamy P. Authentication of the market samples of ashwagandha by DNA barcoding reveals that powders are significantly more adulterated than roots. J Ethnopharmacol. 2020; Feb 29:112725. doi: 10.1016/j.jep.2020.112725. [Epub ahead of print]

Keywords: Adulteration, contamination, DNA barcoding, Mucuna pruriens, Senna auriculata, Trigonella foenum-graecum, Withania somnifera

This publication reports results from a large-scale investigation into the authenticity of 103 samples from 70 manufacturers sold in 15 States and five Union Territories of India purported to be ashwagandha (Withania somnifera, Solanaceae) roots (n = 33), or root powder (n = 70). The researchers used two genomic regions as barcodes, namely trnH-psbA, and ITS2. The nucleotide order was determined using Sanger’s method of sequencing.

Overall, DNA sequences were obtained from 98 samples, 68 powdered and 30 whole root samples. If the ITS2 DNA chromatogram suggested presence of multiple species, samples were reanalyzed using the trnH-psbA barcode, followed by gel electrophoresis. Among the 30 whole root samples, one (3%) was found to be substituted with roots from an unknown plant species. Of the powdered samples, 50 were found to be composed of solely ashwagandha, while 22 (32%) were adulterated, including four samples where the powder consisted entirely of velvet bean (Mucuna pruriens, Fabaceae) (n = 2), fenugreek (Trigonella foenum-graecum, Fabaceae), or avaram (Senna auriculata, Fabaceae), and 18 mixtures of ashwagandha with other plants including psoralea (Cullen corylifolium, Fabaceae), sandbur (Cenchrus sp., Poaceae), garden cress (Lepidium sativum, Brassicaceae), horseradish tree (Moringa oleifera, Moringaceae), fenugreek, and mung bean (Vigna radiata, Fabaceae). Mixed samples contained between two and five species, based on results from analysis of the trnH-psbA sequence using gel electrophoresis. According to the authors, ashwagandha was the predominant species in 10 (53%) of the mixed samples. The authors did not elaborate if some of these admixtures were due to intentional adulteration, or accidental contamination during harvesting, drying, processing, or storage.

Comment: Since the determination of the predominant species in mixtures was based on the intensity of the gel electrophoresis bands (A. Nagendraprasad email, March 13, 2020), the authors’ results in this regard may not be highly accurate. Many of the additional species are common food plants, and therefore likely cultivated or stored in proximity to ashwagandha fields and could therefore be accidental contaminants. On the other hand, fenugreek has been found in 14% of 93 medicinal plant samples obtained from the Indian market in an investigation from 2016 using a similar approach.¹ According to the authors of this 2016 paper, fenugreek was found as the sole ingredient in some of the herbal ingredients,¹ similarly to one of the root powders in this publication, suggesting that it was intentionally used as a substitute for the labeled ingredient. A genus that frequently appears as an adulterant when genetic methods are applied is Senna.^1-6 This small bush grows wild in many parts of India, but is also widely used for medicine, and therefore available as a low-cost substitute for other plants. Hence, its presence could be due to accidental contamination or intentional adulteration. However, in this case, the fact that no ashwagandha was found in the senna sample would point towards intentional fraud.

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