Extraction and isolation of mitragynine
Fresh Mitragyna speciosa Korth leaves (1.0 kg) were washed with water to remove dirt and adhering material, oven dried at 45-50 ?C for three days and milled into fine powder using a blender to get the dry powder (297.0 g). Soxhlet extraction was carried out using 297.0 g powdered leaves with 4 L petroleum ether (40-60 oC) for 8 hour, then petroleum ether solution was discarded and the defatted powdered leaves were dried and the extraction was repeated with 4 L chloroform for 8 hour. The chloroform solution obtained was filtered, concentrated, evaporated to dryness under the reduced pressure using rotary evaporator and was kept in a refrigerator (-20oC). The dried crude chloroform extract was subjected for flash chromatography according to the method of Still et al.[10]. Crude fraction containing mitragynine was obtained by eluting with hexane and ethyl acetate (80:20 v/v) and this fraction (100 mL) was subjected to liquid-liquid fractionation using petroleum ether (100 mL X 3 times) for further purification. The petroleum ether layer was discarded and the remaining solution was concentrated under the reduced pressure using a Buchi R215 Rotavapor (Flawil, Switzerland) to obtain crude mitragynine.