Discovery of rhynchophylline and mitraphylline in two Thai Mitragyna species and the investigation of their biological activity via opioid gene expression analysis

Mitragyna speciosa (Ms), M. diversifolia (Md), M. hirsuta (Mh) and M. rotundifolia (Mr) were investigated for phytochemicals by GC-MS and GC-FID, cytotoxicity and genotoxicity testing by MTT and comet assay, and biological activity examination through gene expression of human µ, δ, κ, and nociceptin opioid receptors by qRT-PCR. The opioid substances mitragynine, 7-hydroxymitragynine, and mitraphylline were found in all studied species, and, for first time, rhynchophylline was found in Mr, and mitraphylline in Md, Mh and Mr. The MTT and comet assays of the ethanol and hexane leaf extracts on PBMCs revealed no cytotoxicity and no significant genotoxicity compared to the negative control, except for the hexane leaf extract of Mh, which caused significant DNA damage. The biological activity of the ethanolic extract of the four species showed a binding affinity to the µ (MOR) receptor revealing a relative gene expression of 89.54 and 50.41 by Ms and Md at 1.92 and 1.133 mg/ml, 32.42 and 19.97 mg/ml by Md and Ms at 3.77 and 1.76 mg/ml. Mr contained the three opioids mentioned plus rhynchophylline and showed low relative µ (MOR) gene expression of 16.89 at 0.189 mg/ml, while as an additional species, Ipomoea aquatica (Ia) showed higher relative µ (MOR) gene expression of 37.75 and 59.76 at 2.878, and 5.813 mg/ml. A combination of Mr and Ia at 1.227, 2.907 and 0.0123, 0.0291 mg/ml extract showed high relative µ (MOR) gene expression at 71.01 and 21.71. These Mitragyna species and the combination (formula details are patent registered), substances and their biological activities can be used for the innovative production of new medicines and further clinical investigation.