Characterization of Corynantheidine Standard

Trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), 1,5-diaminonapthalene (DAN), γ-aminobutyric acid (GABA) standard, glutamine standards, Phloxine B, hematoxylin, sodium iodate, and aluminum potassium sulfate dodecahydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Corynantheidine standard was extracted from dried Mitragynine speciosa leaves (Pure Land Ethnobotanicals, Madison WI, USA) using methanol or ethanol, and then fractioned and dried using acid-base extraction and column chromatography. Proton nuclear magnetic resonance (¹H NMR), carbon nuclear magnetic resonance (¹³C NMR), high-performance liquid chromatography-photometric diode array (HPLC-PDA), and ultra-performance LC-quadrupole time of flight (UPLC-QTOF) were used to ensure the purity of the standard were 98% or above.^{12 (link)} Epredia^™ Cytoseal^™ mountant XYL, glycerol, Eosin Y intensified solution, μ-opioid receptor polyclonal antibody, donkey anti-rabbit IgG (H+L) highly cross-absorbed secondary antibody (Alexa fluor plus 488, absorption at 497nm, emission at 517nm), and ProLong^™ Gold antifade mountant with DAPI were purchased from ThermoFisher Scientific (Waltham, MA). Xylene was purchased from Honeywell International Inc. (Charlotte, NC).