Identification of Intestinal UDP-Glucuronosyltransferase Inhibitors in Green Tea (Camellia sinensis) Using a Biochemometric Approach: Application to Raloxifene as a Test Drug via In Vitro to In Vivo Extrapolation

Abstract

Green tea (Camellia sinensis) is a popular beverage worldwide, raising concern for adverse interactions when co-consumed with conventional drugs. Like many botanical natural products, green tea contains numerous polyphenolic constituents that undergo extensive glucuronidation. As such, the UDP-glucuronosyltransferases (UGTs), particularly intestinal UGTs, represent potential first-pass targets for green tea-drug interactions. Candidate intestinal UGT inhibitors were identified using a biochemometrics approach, which combines bioassay and chemometric data. Extracts and fractions prepared from four widely consumed teas were screened (20-180 μg/ml) as inhibitors of UGT activity (4-methylumbelliferone glucuronidation) in human intestinal microsomes; all demonstrated concentration-dependent inhibition. A biochemometrics-identified fraction rich in UGT inhibitors from a representative tea was purified further and subjected to second-stage biochemometric analysis. Five catechins were identified as major constituents in the bioactive subfractions and prioritized for further evaluation. Of these catechins, (-)-epicatechin gallate and (-)-epigallocatechin gallate showed concentration-dependent inhibition, with IC₅₀ values (105 and 59 μM, respectively) near or below concentrations measured in a cup (240 ml) of tea (66 and 240 μM, respectively). Using the clinical intestinal UGT substrate raloxifene, the K_i values were ∼1.0 and 2.0 μM, respectively. Using estimated intestinal lumen and enterocyte inhibitor concentrations, a mechanistic static model predicted green tea to increase the raloxifene plasma area under the curve up to 6.1- and 1.3-fold, respectively. Application of this novel approach, which combines biochemometrics with in vitro-in vivo extrapolation, to other natural product-drug combinations will refine these procedures, informing the need for further evaluation via dynamic modeling and clinical testing.

Fig. 1.

Structures of (+)-catechin, EC, EGC, ECG, and EGCG.

Fig. 2.

Workflow for biochemometrics-guided fractionation of green teas to identify candidate intestinal UGT inhibitors.

Fig. 3.

Concentration-dependent inhibition of intestinal microsomal UGT activity (4-MU glucuronidation) by green tea extracts prepared from four commercially available green teas (coded T02, T07, T13, and T21) and the National Institute of Standards and Technology reference material (T26) and corresponding fractions. Symbols and error bars denote mean and S.D., respectively, of triplicate incubations. T26-aq, aqueous extract. Control activity was 4.7 ± 0.75 nmol/min/mg.

Fig. 4.

(A) PLS scores plot (component 1 vs. component 2) from biochemometric analysis of four commercially available green teas (coded T02, T07, T13, and T21) and the National Institute of Standards and Technology reference material (T26). (B) Stacked plot of chromatograms of fractions A–E from the T21 extract. Fractions C–E contained the majority of peaks. Fractions D and E contained high initial solvent peaks.

Fig. 5.

(A) Concentration-dependent inhibition of intestinal microsomal UGT activity (4-MU glucuronidation) by fraction C and subfractions C1–C8 from the T21 extract. Symbols and error bars denote mean and S.D., respectively, of triplicate incubations. (B) Selectivity ratio analysis of the partial least-squares model data for all eight of the T21 subfractions. The more negative values represent higher contributions to the observed UGT inhibitory activity (see Table 2 for identified ions 1–6). Rt, retention time. (C) Abundance of major constituents in C1–C8 from the T21 extract based on metabolomic profiling data.

Fig. 6.

Concentration-dependent inhibition of intestinal microsomal UGT activity (4-MU glucuronidation) by ECG and EGCG. Symbols and error bars denote mean and S.D., respectively, of triplicate incubations. Curves denote nonlinear least-squares regression of the data.

Fig. 7.

Kinetics of inhibition of raloxifene-4′-glucuronide (R4G; left) or raloxifene-6-glucuronide (R6G; right) formation by ECG (upper) and EGCG (lower). Symbols denote individual data points of duplicate incubations. Velocity vs. substrate concentration data were described best by the simple competitive inhibition model. Curves denote nonlinear least-squares regression of the data.