Mitragynine

DAMGO, quinidine, ketoconazole, morphine sulfate, and alprazolam were purchased from MilliporeSigma (St Louis, MO). Mitragynine, 7-hydroxyMitragynine, midazolam, 1′-hydroxymidazolam, sulfaphenazole, and NADPH were obtained from Cayman Chemical Company (Ann Arbor, MI). Diclofenac, 4′-hydroxyDiclofenac, dextromethorphan, and dextrorphan were obtained from Toronto Research Chemicals, Inc. (Toronto, Canada). Potassium phosphate buffer salts were obtained from Fisher Scientific (Fair Lawn, NJ). Human liver microsomes were obtained from XenoTech, LLC (H0604, mixed gender, pool from 15 donors, lot no. 1010191; Kansas City, KS). All other chemicals and reagents used were analytical grade.

Ajmalicine (Sigma) and mitragynine, 7-hydroxymitragynine, paynantheine, speciogynine, mitraphylline, speciociliatine (Cayman Chemicals) were used as external standards for quantification on the basis of peak area revealed by HPLC analysis as described below. Alkaloids fractions were analyzed using an Agilent 1,260 Infinity liquid chromatography system equipped with a reversed-phase Kinetex EVO C18 100Å column (150 × 4.6 mm, 5 µm). Chromatographic separation of kratom alkaloids were achieved using a binary gradient with ammonium bicarbonate buffer (5 mM pH 9.5; A) and acetonitrile (B), starting with 70% solvent A transitioning to 70% solvent B over the course of 17 min at a flow rate of 1.5 ml/min. Alkaloids were quantified at 226 nm. The alkaloids fractions 3 (3-isoajmalicine) and 10 (corynantheidine) eluted at 8.12 and 14.67 min, respectively, and were subsequently collected. Approximately 0.3 mg of each compound were evaporated to dryness, resuspended in deuterated chloroform, and analyzed using ¹H NMR. NMR spectra were collected on a Bruker AVANCE III 600 MHz spectrometer equipped with a 5 mm TCI cryoprobe. The sample temperature was regulated at 298 ± 1 K.

Oxaliplatin was obtained from Temple University Hospital Pharmacy (Philadelphia, PA) as a 6 mg/ml concentration stock solution and diluted in sterile water (B. Braun Medical Inc., Irvine, CA). Mitragynine (MG) was obtained from Cayman Chemical Company (Ann Arbor, MI) with molecular weight and purity (>97.3 %) additionally confirmed by Dr. Allen Reitz (Fox Chase Chemical Diversity Center, Doylestown, PA). MG was dissolved in a vehicle of 20 % Tween 80 / 80 % sterile water. Naltrexone hydrochloride (NTX), prazosin hydrochloride (PRZ), and propranolol hydrochloride (PROP) were obtained from Sigma-Aldrich (St. Louis, MO) and dissolved in sterile water. Yohimbine hydrochloride (YOH) was purchased from Tocris Bioscience (Bristol, UK) and dissolved in sterile water. All drugs were administered intraperitoneally (IP) at a volume of 10 ml/kg.