title: Chemoenzymatic Synthesis, Characterization, and Scale-Up of Milk Thistle Flavonolignan Glucuronides.
abstract: Plant-based therapeutics, including herbal products, continue to represent a growing facet of the contemporary health care 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 multimilligram 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 (UDP glucuronic acid) compared with 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 monoglucuronides, respectively. Large-scale synthesis (40 mg of 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.
authors: Experimental and Systems Pharmacology, College of Pharmacy, Washington State University, Spokane, Washington (B.T.G., M.F.P.); and Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, North Carolina (T.N.G., N.D.P., N.H.O.).
authors: Brandon T
authors: BT
authors: Gufford
authors: Experimental and Systems Pharmacology, College of Pharmacy, Washington State University, Spokane, Washington (B.T.G., M.F.P.); and Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, North Carolina (T.N.G., N.D.P., N.H.O.).
authors: Tyler N
authors: TN
authors: Graf
authors: Experimental and Systems Pharmacology, College of Pharmacy, Washington State University, Spokane, Washington (B.T.G., M.F.P.); and Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, North Carolina (T.N.G., N.D.P., N.H.O.).
authors: Noemi D
authors: ND
authors: Paguigan
authors: Experimental and Systems Pharmacology, College of Pharmacy, Washington State University, Spokane, Washington (B.T.G., M.F.P.); and Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, North Carolina (T.N.G., N.D.P., N.H.O.).
authors: Nicholas H
authors: NH
authors: Oberlies
authors: Experimental and Systems Pharmacology, College of Pharmacy, Washington State University, Spokane, Washington (B.T.G., M.F.P.); and Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, North Carolina (T.N.G., N.D.P., N.H.O.) mary.paine@wsu.edu.
authors: Mary F
authors: MF
authors: Paine
mesh_terms: D000818:Animals; D002417:Cattle; D044947:Flavonolignans / Q000138:chemical synthesis* / Q000302:isolation & purification; D020719:Glucuronides / Q000138:chemical synthesis* / Q000302:isolation & purification; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D020944:Silybum marianum / Q000201:enzymology*; D010936:Plant Extracts / Q000138:chemical synthesis* / Q000302:isolation & purification
chemical_list: D044947:Flavonolignans; D020719:Glucuronides; D010936:Plant Extracts