Introduction: Diabetes mellitus (DM) is one of the top diseases that lead public health concern in Malaysia. It was believed to rise in number up to 4.5 million on cases by year 2020 based on the current figure. Momordica charantia Linn (MC), a climber belonging to family Cucurbitaceae, is well known in treating diabetic-related conditions. In earlier studies related to the hypoglycemic properties of MC mainly utilized the crude extract, which contain a mixture of bioactives (charantins, insulin-like peptides and alkaloids). Till now, there is no conclusive result on the major bioactives that play role in the hypoglycemic effect of MC and research regarding the charantin purification was not well established. Hence, the objectives of this study were to purify the charantin from MC and to characterize the purified charantin before further subjected to in vivo hypoglycemic study. Methods: The crude was first extracted from MC using ethanol as solvent via Soxhlet extraction following by a series of purification steps via washing, centrifugation, and C-18 cartridges. Results: The HPLC analysis showed that the charantin of purified extract after passing out from the cartridge exuded at 12.50 min with a concentration of 500 ppm, which is relatively 20 times higher than the crude extract (25 ppm). The structural properties of purified charantin were studied using FTIR and it showed strong peaks of carboxylic acids (2884 nm), alcohols (1023 nm) and diethyl ether (1114 nm) as compared
with the standard. The compound was reconfirmed in LC-MS analysis. The result displayed mass spectrum in positive mode indicates the presence of similar compound in the purified extract and standard charantin, as presented by ion m/z = 300. Conclusion: The charantin was successfully purified from MC and can act as a potent plant-based hypoglycemic agent for diabetes.