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.
Footnotes
- Received June 26, 2015.
- Accepted August 26, 2015.
This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant R01-GM077482-S1]. B.T.G. was supported by fellowships awarded by the American Foundation for Pharmaceutical Education and the James and Diann Robbers Student Research Fund. B.T.G. is currently supported by the National Institute of General Medical Sciences [Grant T32-GM008425]. Alamethicin F50 was isolated as part of Program Project Grant P01-CA125066 from the National Institutes of Health National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health.
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- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
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