PT - JOURNAL ARTICLE AU - Brandon T. Gufford AU - Tyler N. Graf AU - Noemi D. Paguigan AU - Nicholas H. Oberlies AU - Mary F. Paine TI - Chemoenzymatic Synthesis, Characterization, and Scale-up of Milk Thistle Flavonolignan Glucuronides AID - 10.1124/dmd.115.066076 DP - 2015 Jan 01 TA - Drug Metabolism and Disposition PG - dmd.115.066076 4099 - http://dmd.aspetjournals.org/content/early/2015/08/27/dmd.115.066076.short 4100 - http://dmd.aspetjournals.org/content/early/2015/08/27/dmd.115.066076.full AB - Plant-based therapeutics, including herbal products, continue to represent a growing facet of the contemporary healthcare market. Mechanistic descriptions of the pharmacokinetics and pharmacodynamics of constituents composing these products remain nascent, particularly for metabolites produced following herbal product ingestion. Generation and characterization of authentic metabolite standards are essential to improve the quantitative mechanistic understanding of herbal product disposition in both in vitro and in vivo systems. Using the model herbal product, milk thistle, the objective of this work was to biosynthesize multi-mg quantities of glucuronides of select constituents (flavonolignans) to fill multiple knowledge gaps in the understanding of herbal product disposition and action. A partnership between clinical pharmacology and natural products chemistry expertise was leveraged to optimize reaction conditions for efficient glucuronide formation and evaluate alternate enzyme and reagent sources to improve cost-effectiveness. Optimized reaction conditions used at least one-fourth the amount of microsomal protein (from bovine liver) and cofactor (UDPGA) compared to typical conditions using human-derived subcellular fractions, providing substantial cost savings. Glucuronidation was flavonolignan-dependent. Silybin A, silybin B, isosilybin A, and isosilybin B generated five, four, four, and three mono-glucuronides, respectively. Large scale synthesis (40 mg starting material) generated three glucuronides of silybin A: silybin A-7-O-β-D-glucuronide (15.7 mg), silybin A-5-O-β-D-glucuronide (1.6 mg), and silybin A-4''-O-β-D-glucuronide (11.1 mg). This optimized, cost-efficient method lays the foundation for a systematic approach to synthesize and characterize herbal product constituent glucuronides, enabling an improved understanding of mechanisms underlying herbal product disposition and action.