Ashwagandha (<i>Withania somnifera</i>) Plant Extracts Affect the Cytochrome P450 System and Cytotoxicity of Primary Human Hepatocytes

Ashwagandha (<i>Withania somnifera</i> [L] Dunal) is emerging as one of the top 10 botanicals in the market due to its claimed health benefits as an adaptogen. Alongside its popularity, the lack of standardization in botanical dietary supplements and potential interactions with prescription drugs raise safety concerns. This study aimed to investigate the potential of ashwagandha extracts to induce cytochrome P450 (P450) enzymes, which are critical for drug metabolism and implicated in botanical–drug interactions. A sandwich-cultured in vitro assay using primary human hepatocytes was utilized to evaluate the induction potential of aqueous and 70% ethanolic extracts of ashwagandha roots and leaves on CYP3A4, CYP2B6, and CYP1A2—enzymes commonly involved in drug metabolism. Unlike prior studies that focused on root extracts or used HepG2 cell lines, this research included both root and leaf plant extracts, with the latter being assessed for the first time. Our approach using primary hepatocytes enhances clinical relevance compared to other in vitro models (such as HepG2 cell lines). Additionally, cytotoxicity was examined using CellTitre-Glo Luminescent Assay, measuring cell viability at three different incubation times (24, 48, and 72 hours) to assess the potential cytotoxic effect of the ashwagandha plant extracts. Our findings revealed that 70% ethanol ashwagandha root extracts (WSR-2) modulated CYP3A4 enzyme expression and activity. Preliminary studies on cytotoxicity indicated dose- and time-dependent impact on hepatocyte viability with a high concentration of 70% ethanol ashwagandha leaf extracts (WSL-2). The study highlights the importance of understanding ashwagandha’s impact on P450-based drug metabolism and its safe co-administration with prescribed drugs.