Phenacetin

Gene expression analysis by RNA sequencing (RNA-seq) and quantitative PCR is described in Supplementary Materials and methods. Albumin and urea secretion into the media was measured using a human albumin ELISA kit and the QuantiChrom™ urea assay kit (both from Bethyl Laboratories). Comparisons with fetal and adult hepatocyte data used the unpaired two-tailed Student’s t test. CYP3A activity was assessed in duplicate by incubation with P450-Glo™ CYP3A4 assay reagent (Luciferin-PFBE; Promega Ltd). For CYP analysis by mass spectrometry, cells were incubated with 1 mM testosterone or 1 mM dextromethorphan (Sigma, UK) in HCM. Conditioned medium was collected and diluted 1:1 in 0.5 μM phenacetin (Sigma) stop solution in methanol. CYP activity was calculated per min incubation. Alcohol dehydrogenase activity of cell lysates was assessed using a detection kit following the manufacturer’s instructions (Abcam, UK). Results were standardized to the amount of protein measured by Bradford assay.

Xanthotoxol (purity > 98%) was purchased from Sichuan Weikeqi Biotechnology Co. Ltd. (Sichuan, China). Paclitaxel, 1-aminobenzotriazole (ABT), phenacetin, sulfaphenazole, chlorzoxazone, quinidine, clomethiazole, furafylline, 8-methoxypsoralen, coumarin, diclofenac, quercetin, dextromethorphan, ketoconazole, testosterone, S-mephenytoin, omeprazole, glucose-6-phosphate dehydrogenase, NADP⁺, and D-glucose-6-phosphate were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were the highest purity commercially available or HPLC grade.

HDC (>98.0%) and H. helix extract were obtained from the Lab. of Pharmacognosy, College of Pharmacy, Yonsei University (Incheon, Korea). The extract was prepared by extracting the pulverized ivy leaves with 30% ethanol for 1 h using sonication. A voucher specimen of H. helix extract (HY-2016-01-05) was deposited at the Herbarium of the College of Pharmacy, Hanyang University, Ansan, Korea. H. helix extract contains 8.2% of HDC. The content of HDC was determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS) [10 (link)] and the representative chromatogram is shown in Figure 4. Pooled human liver microsomes and recombinant CYP2C8, CYP2C19, and CYP2D6 isozymes were purchased from BD Gentest (Woburn, MA, USA). Glucose-6-phosphate, β-NADP+, Glucose-6-phosphate dehydrogenase, coumarin, phenacetin, diclofenac, midazolam, mephenytoin, dextromethorphan, ketoconazole, and terfenadine were purchased from Sigma Chemical Co. (Saint Louis, MO, USA). All other solvents used were of HPLC grade and were obtained from J. T. Baker (Phillipsburg, NJ, USA). Distilled water was prepared using a Milli-Q purification system (Millipore, Billerica, MA, USA). All standard solutions and mobile phases were passed through a 0.22 µm membrane filter before use.

The enzymes and chemicals ware prepared according to our previous study with some modifications [14 (link)]. Pooled human liver microsomes, recombinant human CYP enzymes, and NADPH were purchased from Cypex Ltd. (U.K.) and stored at -80°C until use. Ginsenoside compound K (CK, C₃₆H₆₂O₈, MW:622.873, assay≥99.2%, Lot: P1201-1) was provided by Zhejiang Hisun Pharmaceutical Company Limited (Zhejiang, China). Furafylline, tranylcypromine, ketoconazole,sulfaphenazole, quinidine, chlormethiazole hydrochloride, and ticlopidine hydrochloride were purchased from the National Institutes for Food and Drug Control (Beijing, China). Phenacetin, coumarin, midazolam, tolbutamide, S-Mephenytoin, metoprolol, chlorzoxazone, and the standards for their metabolites, including acetaminophen, 7-hydroxyl coumarin, 1-hydroxyl midazolam, 4-hydroxyl tolbutamide, 4-hydroxyl mephenytoin, α-hydroxyl metoprolol, 6-hydroxyl chlorzoxazone, and irbesartan (the internal standard), were purchased from Sigma-Aldrich (Shanghai, China). All other chemicals and solvents were of high-performance liquid chromatography (HPLC) grade.

Capsule formulation of the standardized A. lancea extract (formulated AL) and placebo were provided by Khaolaor Laboratories Co. Ltd. (Samut Prakarn, Thailand: lot number 200121/3 and capsule number 00). Atractylodin (ATD) was obtained from WAKO (Osaka, Japan). Caffeine, phenacetin, paracetamol, PLGA (Resomer^® RG502, MW 12,000, inherent viscosity 0.24 dL/g), and α-cyano-4-hydroxycinnamic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Kolliphor^® P 407 (poloxamer 407) was kindly provided by BASF Thailand. β-nicotinamide adenine dinucleotide phosphate tetrasodium salt-reduced form (NADPH) and nifedipine were purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Dehydronifedipine was obtained from Toronto Research Chemical (Toronto, ON, Canada). Diazepam was purchased from Government Pharmaceutical Organization (Bangkok, Thailand). iTaq^TM Universal SYBR^® Green Supermix was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Rabbit Anti-Rat CYP1A2 and CYP3A1 polyclonal antibodies were purchased from ABclonal (MA, USA) and Merck (Darmstadt, Germany), respectively. Rabbit Anti-Rat GAPDH antibody and Goat Anti-Rabbit IgG AP-linked were purchased from Cell Signaling Technology (MA, USA). The BCIP/NBT solution AP-chromogen was obtained from Amresco (Solon, OH, USA). Methanol HPLC grade was purchased from Fisher Scientific (MA, USA).