α-GLUCOSIDASE INHIBITORY ACTIVITY OF CUCURBITANE DERIVATE ISOLATED FROM METHANOL EXTRACT OF MOMORDICA CHARANTIA L. LEAVES

Cucurbitane derivative has been isolated from methanol extract of the leaves of bitter melon (Momordica charantia). Purification of methanol extract using vacuum column chromatography method using n-hexane: ethyl acetate as the mobile phase was obtained a white powder isolate. This isolate was elucidated to obtain chemical structures based on spectroscopic data (FT-NMR, FT-IR, and LC-MS/MS) and resulted a cucurbitane derivate, namely momordicine I. The MeOH extract and the isolate were evaluated for α-glucosidase inhibitory effect. Both MeOH extract and momordicine I showed moderate activities with %inhibition 27.34% and 15.79 % at 100 μg/mL repectively. Keyword : Cucubitane, Momordica charantia, momordicine I, α-glucosidase INTRODUCTION Bitter melon (Momordica charantia) is a member of the Cucurbitaceae family which is easily cultivated and spreads to include China, India, and Southeast Asia. In Indonesia, bitter melon is consumed as a vegetable and used as traditional medicine. Utilization of M. charantia empirically carried out by the community is a cure for diabetes, heart disease and stomachache (Grover and Yadan, 2002). M. charantia has various medicinal potentials as antidiabetic (Joseph and Jini, 2013), antibacterial (Kumar et al., 2010) and antimicrobial (Svobodova et al., 2010). The antidiabetic effect is inseparable from the role of secondary metabolites compounds in the form of triterpenes, proteins, steroids, alkaloids, lipids, and phenolic compounds (Zhanga et al., 2012). M. charantia leaves contain momordica, momordine, charantine, trichosanic acid, resin, resinic acid, saponin, vitamins A and C (Tan et al., 2008). Previous studies have shown the compounds momordicoside A and M from M. charantia fruit have inhibitory activity Indonesia Chimica Acta Pamenta, et.al. p-ISSN 2085-014X Vol.12. No.2, December 2019 e-ISSN 2655-6049 100 against rat intestinal α-glucosidase enzymes (Nguyen et al., 2010). Earlier antidiabetic compound, momordicinin, has α-amylase inhibitory activity (Kulkarni et al., 2019). While in this study used α-glucosidase enzyme from saccharomyces cerevisiae recombinant. The enzyme α-glucosidase plays an important role in the hydrolysis of complex carbohydrates into glucose which can be absorbed through the intestine. Inhibition of α-glucosidase work can reduce glucose absorption in patients with hyperglycemia (Ben et al., 2017). One of the α-glucosidase inhibiting agents is acarbose which has been reported to reduce the intestinal absorption of sugar in humans (Jenkins et al., 1981). The results of this previous study encouraged this research to explore metabolite compounds contained in M. charantia leaves used methanol as extractor and tested their inhibitory activity against the α-glucosidase enzyme. MATERIALS AND METHODS


INTRODUCTION
Bitter melon (Momordica charantia) is a member of the Cucurbitaceae family which is easily cultivated and spreads to include China, India, and Southeast Asia. In Indonesia, bitter melon is consumed as a vegetable and used as traditional medicine. Utilization of M. charantia empirically carried out by the community is a cure for diabetes, heart disease and stomachache (Grover and Yadan, 2002). M. charantia has various medicinal potentials as antidiabetic (Joseph and Jini, 2013), antibacterial (Kumar et al., 2010) and antimicrobial (Svobodova et al., 2010). The antidiabetic effect is inseparable from the role of secondary metabolites compounds in the form of triterpenes, proteins, steroids, alkaloids, lipids, and phenolic compounds (Zhanga et al., 2012). M. charantia leaves contain momordica, momordine, charantine, trichosanic acid, resin, resinic acid, saponin, vitamins A and C (Tan et al., 2008). Previous studies have shown the compounds momordicoside A and M from M. charantia fruit have inhibitory activity against rat intestinal α-glucosidase enzymes (Nguyen et al., 2010). Earlier antidiabetic compound, momordicinin, has α-amylase inhibitory activity (Kulkarni et al., 2019). While in this study used α-glucosidase enzyme from saccharomyces cerevisiae recombinant.
The enzyme α-glucosidase plays an important role in the hydrolysis of complex carbohydrates into glucose which can be absorbed through the intestine. Inhibition of α-glucosidase work can reduce glucose absorption in patients with hyperglycemia (Ben et al., 2017). One of the α-glucosidase inhibiting agents is acarbose which has been reported to reduce the intestinal absorption of sugar in humans (Jenkins et al., 1981). The results of this previous study encouraged this research to explore metabolite compounds contained in M. charantia leaves used methanol as extractor and tested their inhibitory activity against the α-glucosidase enzyme.

Extraction and Isolation
M. charantia (1.5 kg) dried leaves were extracted using the maceration method with 15 L of methanol solvent and re-extracted four times. Maserate was concentrated with a rotary vacuum evaporator obtained a green residue (68 g). MeOH extract (30 g) was further separated using a column of vacuum chromatography with silica G60 as a stationary phase and a mixture of n-hexane: ethyl acetate: methanol gradient to increase polarity, obtained 30 fractions. Fraction 17 has been recrystallized using n-hexane: ethyl acetate and obtained white crystal (0.8 g). The isolated compound was identified using FT-NMR, FT-IR, and LC-MS/MS.

Inhibitory Activity Of α-Glucosidase Enzyme
Testing the inhibitory activity of αglucosidase enzymes adapting the procedure from Fajriah et al (2018). The αglucosidase enzyme solution containing 200 mg of albumin was dissolved into a phosphate buffer solution (pH 7). The extract was prepared by dissolving 5µL in DMSO, and then a reaction mixture was added consisting of 250 μL p-nitrophenyl α-D-glucopyranose 20 mM as substrate and 490 μL phosphate buffer 100 mM (pH 7). Enzyme solution (250 μL) was added after the reaction mixture was incubated 5 minutes at 37 ᵒC and reincubated then added 1000 µL of 200 mM Na2CO3 solution to stop the reaction. The sample concentration for activity evaluation was 100 µg/mL. The results of the reaction in the form of p-nitrophenol were measured at λ 400 nm and compared with acarbose as a positive control at a concentration of 50 µg/mL.

Figure 1. structure of Momordicine I
The 13 C -NMR spectrum data (Table 1) showed 30 carbon signals aided by the Distrotionless enhancement by polarization transfer (DEPT) data and compared with previously published data (Yasuda et al., 1984 andZhang et al., 2014) showed seven methyl (δc 15.3,18.8, 27.8,18.2, 19.3,26.1,26.0 ppm), seven methylene, seven methines and three of them are hydroxymetin (δc 77.0, 66.6, 67.0), three carbon quartener (46.6,42.3,46.8), two double bonds (δc 124.0, 147.4, 130.5, 133.5) and there are characteristics for an aldehyde (δc 209.8 ppm). This is confirmed by HMBC ( Figure  1) that the aldehyde group is located in C-9 and the proton aldehyde H-19 correlation C-8 (δC 51.9 ppm), C-9 (δC 46.8 ppm), and C-11 (δC 23.3 ppm). All these enabled the structure of isolate compound to be assigned as momordicine I. MeOH extract and momordicine I was tested for their inhibitory activity against the α-glucosidase enzyme. The results were presented in percent inhibition of the concentration of the test material (Table 2). M. charantia methanol extract showed inhibition activity of 27.34 % at a concentration of 100 µg/mL, the low value is probably due to the role of the synergy of the active substance inside the extract. The value of extract was greater than the inhibitory activity of momordicine I 15.79% with the same concentration. Likewise, the activity of the acarbose as a positive control is greater than momordisin I with an inhibitory value of 47.54 % at a concentration of 50 µg/mL. This difference in percent inhibition is based on active Indonesia Chimica Acta Pamenta, et.al. p-ISSN 2085-014X Vol.12. No.2, December 2019e-ISSN 2655 substituents that are bound to acarbose. Based on this data, it is possible to explore compounds that have better activity as an αglucosidase enzyme inhibitor agents than the extract of MeOH M. charantia leaves in the future.

CONCLUSION
The cucurbitane compound derivative that has been isolated from the MeOH extract of M. charantia leaves is momordicine I and its inhibitory activity on the α-glucosidase enzyme gives a value of 27.34 and 15.79 % at a concentration of 100 µg/mL which is quite inhibiting.

CONFLICT OF INTEREST
This research is supported by the 2019 grant for the master thesis research of Directorate of Research and Development of the KEMENRISTEKDIKTI Of Indonesia, with the contract number of 1739/UN4.2/PL.01.10/2019.