Abstract Withania somnifera, has long been known in Ayurvedic medicine for its medicinal properties. In recent years, consumption of W. somnifera products has skyrocketed in western cultures where they are now marketed through retail outlets as dietary supplements, under the common name, Ashwagandha. The NCCIH-sponsored BENFRA Botanical Dietary Supplements Research Center (U19 AT010829) is currently researching Ashwagandha, to determine its effects on age-related neurological deterioration. Unlike pharmaceutical drugs which have an extensive evaluation process prior to public distribution, very little is known regarding how the complex mixture of chemicals present in Ashwagandha extracts behaves once it is consumed and passes through the digestive environment. This proposal is innovative because it will evaluate metabolic stability of Ashwagandha extracts for the first time using in vitro laboratory models simulating the gastrointestinal environment. The project will also assess which original or modified components are able to pass the intestinal membrane via passive diffusion, the mechanism by which most compounds enter the circulation from the gut. The central hypothesis of this research is that the composition of Ashwagandha derived metabolites entering circulation is markedly different from that of the original extract. This hypothesis will be tested through the following specific aims: 1. Determine effects of gastrointestinal environments on the metabolite profiles of Ashwagandha extracts by exposing extracts to simulated gastro-intestinal fluids and enzymes; and 2. Identify permeabilities of metabolites from Ashwagandha original extracts and digestive incubations using a synthetic model of the intestinal membrane. We will use a combination of these in vitro model systems, appropriate controls, and sufficient replicates for robust statistical analyses to standardize these initial investigations. Ultra- pressure liquid chromatography coupled with high resolution mass spectrometry will be used to analyze extracts, incubates, and diffusion chamber media, to evaluate changes in chemical structures and relative abundances of Ashwagandha metabolites in these complex matrices for the first time. Results from these studies will provide insight into a more realistic Ashwagandha metabolic profile post-gastrointestinal digestion that can then be further studied collaboratively using established pre-clinical assays. Results of the present study may also highlight permeable metabolites of interest not currently under investigation for bioactivity. In the long term, this project will establish a robust analytical strategy that can be used broadly to assess oral bioavailability of other botanical supplements in an in vitro setting, prior to time, labor, and resource intensive in vivo studies.

Project Narrative This project is relevant to public health because it will provide detailed information on the fate of substances found in Ashwagandha once they are ingested. These data will provide a picture of which components are altered during digestion and which original and altered compounds from Ashwagandha might be absorbed into the bloodstream; information that is critical for future studies examining Ashwagandha’s health effects.