Integrative Mass Spectrometry Approaches to Evaluate CYP Inhibition Mediated Drug Interactions with Botanical Extracts and Absorption Assessment of Açaí

Abstract

Ashwagandha (Withania somnifera) and açaí (Euterpe oleracea) rank among the most widely consumed botanical dietary supplements globally, with ashwagandha consistently ranked among the top 10 and açaí among top 40 selling supplements in the U.S. market. Their popularity has grown substantially in recent years, driven by increasing consumer interest in ashwagandha for stress relief and sleep support, and in açaí for its antioxidant properties and complementary use in cancer prevention and supportive care. Despite this widespread use, often concurrent with prescription medications in patients with chronic conditions, both supplements have been associated with clinically concerning adverse events. Ashwagandha has been linked to hepatotoxicity, including alterations in hepatic enzyme function, while açaí has been associated with vascular adverse events when co-administered with anticancer drugs metabolized by non- CYP3A4 enzymes. These pharmacovigilance signals, combined with limited and heterogenous data on their cytochrome P450 (CYP) inhibitory potential, underscore the clinical imperative for systematic evaluation of their drug interaction risk, particularly through CYP inhibition, one of the most commonly implicated mechanism of pharmacokinetic botanical-drug interactions. This study employed an in-house developed and optimized in vitro CYP cocktail enzyme assay that enables simultaneous assessment of seven FDA recommended CYP isoforms (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4/5) in a single incubation using pooled human liver microsomes, with metabolite quantification by liquid chromatography mass spectrometry (LC-MS). This approach represents a methodological advancement over conventional single-substrate approaches, providing a more comprehensive and physiologically relevant in vitro model for evaluating botanical-mediated CYP inhibition. For ashwagandha, standardized aqueous and 70% ethanol root and leaf extracts were evaluated. To our knowledge, this is the first study to include ashwagandha leaf extracts and to assess potential inhibition of CYP2A6 and CYP2C8 by ashwagandha extracts. CYP2B6 was evaluated using recombinant enzyme assay, due to published literature stating its inhibition by ashwagandha root methanol extract. In this study, ashwagandha extracts showed no reversible CYP inhibition, with IC50 > 100 ug/mL extract. However, possible concentration-dependent inhibition of two polymorphic enzymes, CYP2C9 (IC50 = 212.0 ug/mL and 175.4 ug/mL when treated with aqueous and 70% ethanol root extracts) and CYP2D6 (IC50 = 230.5 ug/mL when treated with 70% ethanol root extract) was observed, raising concern for potential interaction risk when supplements are consumed repeatedly and without clinical supervision. For açaí, seven standardized extracts derived from commercial capsule formulations (Nature’s way and Natrol) and berry powder (Mountain rose) were evaluated. This is the first study to examine potential inhibition of CYP2A6, CYP2C8, and CYP2C9 by açaí extracts. CYP2C9 demonstrated the most pronounced inhibition by three extracts, F3AC (Nature’s way acidic methanol), MRME (mountain rose methanol), MRET (mountain rose ethanol), with IC50 values of 3.23 ug/mL extract, 42.42 ug/mL extract and 68.86 ug/mL extract, respectively. Further fractionation revealed that the dichloromethane (DCM) and ethyl acetate (EA) fractions of all three active extracts, as well as hexane fraction of F3AC, were primarily responsible for the observed CYP2C9 inhibition. Fractions of the active açaí extracts, including hexane, DCM, EA and water, were further characterized using a biochemometric workflow integrating LC-MS/MS with Random Forest machine learning-guided prioritization of bioactive constituents. To our knowledge, this is the first application of this approach to identify potential CYP inhibitory constituent in açaí. The putative CYP2C9 inhibitory constituents identified are iridin, trimethoxy quercetin-O-hexoside, luteolin, quercetin, and azelaic acid. In addition to CYP inhibition assessment, a comparative absorption profiling study of açaí was performed using PAMPA, capsule dissolution under fasted and fed conditions, and rat plasma pharmacokinetic profiling to provide preliminary assessment of açaí extract absorption behavior and putative metabolite identification. PAMPA identified a physicochemically selective permeable subset of extract constituents. Dissolution profiling revealed meaningful inter-product and media-dependent variability in constituent release, with fed conditions and Nature’s way formulation showing broader constituent solubilization. Rat plasma analysis demonstrated a peak systemic exposure window of 1-1.5 hours with putative evidence of downstream metabolite formation, including metabolites of cyanidin 3-glucoside. Preliminary Phase I metabolism of cyanidin 3-glucoside using human liver microsomes generated putative metabolites such as protocatechuic acid, phloroglucinaldehyde, and ferulic acid, with in silico Phase II predictions suggesting glucuronidation, methylation and sulfonation as likely conjugated pathways. Collectively, this integrative multi-platform investigation represents the first comprehensive CYP inhibition characterization of ashwagandha and açaí using a simultaneous multi-isoform approach. The findings raise concern for clinically relevant pharmacokinetic interactions in patients co-administering these supplements with narrow therapeutic index drugs, highlighting the broader need for evidence-based safety evaluation of widely consumed botanicals before clinical recommendations can be made.