4.5. Isolation of the Mitragynine

Mitragynine was isolated following the procedures described by Goh et al. (2011) [49] and Jamil et al. (2013) [50], with slight modifications. M. speciosa leaves were washed and then put into the oven at 40 °C to dry for 3 days. The dried leaves were then made into powder using an electric grinder. About 500 g of M. speciosa leaves powder was extracted with Soxhlet technique using methanol as a solvent. The yield obtained from the methanol extract was about 80.5 g after rotary evaporation. After this, 10 % acetic acid solution was added to the methanol extract, and then filtered and washed with hexane. The acidic solution was separated and basified with 25% ammonia solution to pH 9. The alkaloids were then extracted with chloroform. This chloroform extract was evaporated under vacuum to produce about 4.95 g alkaloidal extract, which was then loaded into the silica column chromatography using a hexane and ethyl acetate (80:20 v/v) mobile phase. About 1.25 g of mitragynine with 94–95 % purity was obtained. The mitragynine was further purified with a column packed with Sephadex Liphophilic LH-20 using methanol as the mobile phase. The total yield of pure mitragynine was 852 mg with 98 % purity in HPLC. The ¹H-NMR and ¹³C-NMR spectral data are consistent with the published data) [49] (see Table S1).