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 onefourth 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-b-D-glucuronide (15.7 mg), silybin A-5-O-b-D-glucuronide (1.6 mg), and silybin A-4´´-O-b-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.

Chirality Matters: Biological Activity of Optically Pure Silybin and Its Congeners

“…strain M52104, which produced the above glucuronides in quasi-quantitative yields. Gufford et al [81] optimized the previously published method [79] and prepared the respective silybin A and B glucuronides at a scale of tens of mg.…”

“…In addition to CYPs being the typical enzymes of biotransformation phase I, phase II biotransformation enzymes in enterocytes are also influenced by silymarin flavonolignans. The inhibitory activity of seven silymarin constituents towards UDP-glucuronosyl transferase 1A (UGT 1A), UGT 1A8, and UGT 1A10, with 4-methylumbelliferone as the glucuronyl acceptor, was investigated by Gufford et al [81]. The strongest inhibitors were both silybin A and B (their activities were the same within the range of experimental error) with IC 50 ca.…”

Hepatic organic anion transporting polypeptides mediate disposition of milk thistle flavonolignans and pharmacokinetic silymarin‐drug interactions

“…Flavonolignans were isolated from silymarin using a hybrid chromatographic/precipitative technique as detailed previously (Graf, Wani, Agarwal, Kroll, & Oberlies, 2007). Reference standards for glucuronides of the silymarin flavonolignans were generated using bovine liver microsomes as detailed previously (Gufford, Graf, Paguigan, Oberlies, & Paine, 2015). All flavonolignan reference standards were validated to be greater than 95% pure, as measured by UPLC (Graf et al, 2016…”

Urinary excretion of silibinin diastereoisomers and their conjugated metabolites in rat and human at different dosages

“…Many studies have shown that glucuronidation and sulfation obviously represent the most abundant pathway in the metabolism of silibinin (Charrier et al, 2014;Kim et al, 2006) Gufford et al, 2015). Hoh et al, 2006).…”