*4.4. Animal Study*

To investigate the effect of intake of HRW on the xenobiotic-metabolizing enzymes and membrane transporters in rat livers, sixteen male Wistar rats (aged six weeks) obtained from BioLASCO in Ilan, Taiwan were used. Rats were fed a pelleted laboratory diet with fresh control water or HRW (replaced at 5 p.m. every day) ad libitum for four weeks. The rats were all housed in plastic cages in a room kept at 23 ± 1◦C with 60 ± 5% relative humidity and a 12-h light-dark cycle. At the end of the experiment, food was withdrawn for 12 h and the animals were sacrificed by exsanguination via the abdominal aorta while under carbon dioxide (70:30, CO2/O2) anesthesia. Heparin was used as the anticoagulant, and the plasma was separated from the blood by centrifugation (1750× *g*) at 4 ◦C

for 20 min. Plasma concentrations of total cholesterol, triglyceride, alanine aminotransferase (ALT), glucose, blood urine nitrogen (BUN), creatinine, uric acid, and ions were measured immediately by use of a serum autoanalyzer (DiaSYS Diagnostic system, Germany). The liver and kidney samples from each animal were weighed and stored at −80 ◦C.

This study was approved (No: 2017-056) by the Institutional Animal Care and Use Committee (IACUC) of China Medical University, Taiwan. The animals were maintained in accordance with the guidelines for the care and use of laboratory animals as issued by the IACUC ethics committee.

#### *4.5. Preparation of Liver Microsomes*

The frozen liver was homogenized (1:4, *w*/*v*) in ice-cold 0.1 M phosphate buffer (pH 7.4) containing 1 mM ethylenediaminetetraacetic acid (EDTA). The homogenates were centrifuged at 10,000× *g* for 15 min at 4 ◦C. The supernatants were then centrifuged at 105,000× *g* for 60 min. The resulting microsomal pellets were suspended in a 0.25 M sucrose solution containing 1 mM EDTA and were stored at −80 ◦C until use. The microsomal protein concentration was determined by using a BCA protein assay kit (Pierce, Rockford, IL, USA).

#### *4.6. Xenobiotic-Metabolizing Enzyme Activity Assays*

The CYP enzyme activities, including methoxyresorufin *O*-demethylation (CYP1A2), ethoxyresorufin *O*-deethylation (CYP1A1), pentoxyresorufin *O*-depentylation (CYP2B), diclofenac 4-hydroxylation (CYP2C), dextromethorphan *O*-demethylation (CYP2D), *p*-nitrophenol 6-hydroxylation (CYP2E1), testosterone 6β-hydroxylation (CYP3A), and lauric acid 12-hydroxylation (CYP4A), were determined by the high performance liquid chromatography (HPLC)/mass spectrometric (MS) method [24]. Enzyme activities were expressed as pmol of metabolite formation/min/mg protein. Microsomal UGT activity was determined by using *p*-nitrophenol as the substrate, and the rate of formation of *p*-nitrophenol glucuronic acid was measured by HPLC/MS (Agilent, USA) [25]. GST was measured by the spectrophotometric method [26].
