HPLC Method Allows Detection of Undeclared Ashwagandha Aerial Part Material in Commercial Ashwagandha Root Products

Reviewed: Mundkinajeddu D, Sawant LP, Koshy R, et al. Development and validation of high performance liquid chromatography method for simultaneous estimation of flavonoid glycosides in Withania somnifera aerial parts. ISRN Analytical Chemistry. March 10, 2014;2014:351547. doi: 10.1155/2014/351547.

Ashwagandha (Withania somnifera, Solanaceae) is one of the most important medicinal plants in traditional Indian medicine systems. It is mainly used as an energizer and to rejuvenate. Monographs of the root have been included in the United States Pharmacopeia (USP), the American Herbal Pharmacopoeia, the British Pharmacopoeia, the Ayurvedic Pharmacopoeia of India, and the Indian Pharmacopoeia.

Most commercial products contain root material and are standardized on withanolides, a group of compounds characterized by a steroid (ergostane) backbone and a cyclized side chain. Increased demand for the roots has led to considerably higher prices and, in some cases, to the undeclared use of aerial parts, which have a similar withanolide composition as the roots but are more affordable. In addition to the withanolides, the aerial parts also contain flavonol glycosides; for example, quercetin 3-O-robinobioside-7-O-glucoside, quercetin 3-O-rutinoside-7-O-glucoside, and kaempferol 3-O-robinobioside-7-O-glucoside. Since these flavonoids are absent in the roots, and are easy to analyze, they constitute ideal marker compounds to detect the presence of W. somnifera aerial parts in root material.

An HPLC-UV method allowing the quantification of the three flavonol glycosides was developed based on the conditions for withanolides outlined in the Ashwagandha Root monograph in the USP, but with modifications to accommodate for the separation of the flavonoids. The accuracy, precision, and linearity of the method were verified according to International Conference on Harmonisation (ICH) guidelines and the USP general chapter on validation of compendial procedures. In addition, the limits of detection and quantification were determined.

Using the new HPLC-UV method, the authors analyzed authenticated samples of W. somnifera leaves (n = 5), aerial parts (n = 3), and roots (n = 17), obtained either from India or Egypt. Kaempferol 3-O-robinobioside-7-O-glucoside was the most abundant (content: 0.022%-0.069%) flavonol glycoside in leaves and aerial parts from India. Egyptian leaves contained predominantly quercetin 3-O-rutinoside-7-O-glucoside (content: 0.154%-0.233%). Total flavonoid contents varied between 0.044% and 0.261% in leaves and aerial parts; however, flavonol glycosides were absent in all 17 genuine ashwagandha root samples.

In addition, ten commercial samples labeled as "derived from the roots" were analyzed for the presence of flavonol glycosides. Only two of the commercial samples did not contain any of the marker compounds for aerial parts, indicating that aerial parts may be used frequently in ashwagandha root products. The new HPLC method provides a useful approach to distinguish between ingredients derived from roots and aerial parts of W. somnifera, and for routine detection of possible adulteration of commercially available root powders or root extracts with aerial parts.

Comment: The method itself has a run time of 28 min; an additional 17 min is used for the return to initial conditions (12 min) and subsequent column conditioning (5 min). Since the three flavonol glycosides of interest elute in the first 9 min, the method could be shortened considerably. The authors chose to use a phosphate-buffer based system to avoid the need to re-equilibrate for subsequent quantitative analysis of withanolides according to USP. The description of the sample preparation is not very clear in the paper – in a follow-up e-mail on August 30, 2014, one of the authors, Amit Agarwal, PhD, explained it in more details: "1.0-2.0 g of finely milled plant material was subjected for extraction with ca. 50 ml boiling methanol, followed by sonication for 5 min at room temperature. The extract was then decanted and the residue was further subjected to extraction with the solvent in similar fashion. This procedure was repeated three times and the filtrate was pooled, concentrated and finally made up to 100 ml with methanol. This solution was filtered and subjected to HPLC analysis."

Admixture or substitution of ashwagandha roots with aerial parts seems to be relatively commonplace, unfortunately. As evidenced by the authors, a majority of the commercial samples analyzed contained some aerial parts, in line with a comment made by Rtvik Sethia, Director of the botanical extract supplier Botanosys Laboratories Pvt. Ltd (Bikaner, India). Allegedly, "ashwagandha root extract is mostly extract derived from the aerial parts (not root) of the ashwagandha bush."¹ However, the new HPLC method makes it easy to detect adulteration of roots with aerial parts and provides the manufacturer with a much-needed tool to identify the correct plant part for ashwagandha. Considering that most pharmacopeial monographs allow for a certain percentage of foreign matter to be present (including the leaf), the method would benefit from knowing the minimum percentage of leaf material that can be detected when it is added to the powdered root.

References

1.    Sethia R. Adultery of adulteration. Botanospeak. August-September 2013;1. Available at: http://diabit.blogspot.com/2014/08/botanospeak-aug-sep.html. Accessed October 29, 2014.