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.
