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.

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 Ki 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.

Cold storage and cryopreservation of hops (Humulus l.) shoot cultures through application of standard protocols.

The USDA-ARS National Clonal Germplasm Repository (NCGR) stores the global diversity of Humulus for the US Plant Germplasm System as trellised plants in a field genebank. In vitro storage and cryopreservation are now considered excellent ways to provide medium and long-term storage for plant collections. Developing a new cryopreservation or cold storage protocol for every accession or genus of large multi-crop collections can be a very time consuming and long-term activity. We propose that standard cold storage and cryopreservation techniques used for other temperate crop genera would be successful for additional crops with few modifications. This study was initiated to determine if a large collection of hops germplasm could be successfully stored with techniques developed for unrelated genera. In this study we characterized the response of diverse Humulus genotypes to in vitro storage under low light at 4 degree C following techniques used for strawberry and mint plants, and cryopreservation in liquid nitrogen by slow cooling with a pear protocol. The average storage time without transfer for the 70 genotypes evaluated was 14 +/- 3.5 months with a range of 6 to 26 months. Mean recovery of cryopreserved shoot tips of accessions with 1-wk cold acclimation was 41 +/- 18 percent and increased to 54 +/- 13 percent with 2-wk cold acclimation. This demonstrates that application of a well-tested standard technique can provide a quick start for storing additional germplasm collections.