Chemical constituents and the inhibition of a-Glucosidase of Gynura procumbens (Lour) Merr

Science & Technology Development Journal, 22(4):391-399 Open Access Full Text Article Research article Faculty of Chemistry, University of Science, VNU-HCM Correspondence Tran Le Quan, Faculty of Chemistry, University of Science, VNU-HCM Email: tlquan@hcmus.edu.vn History  Received: 2019-11-13  Accepted: 2019-12-17  Published: 2019-12-31 DOI : 10.32508/stdj.v22i4.1725 Copyright © VNU-HCM Press. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Chemical constituents and the inhibition of a-glucosidase of Gynura procumbens (Lour.) Merr. Le Thi My Quyen, Nguyen Thi DiemQuynh, Dang Hoang Phu, Nguyen Thi Y Nhi, Tran Le Quan* Use your smartphone to scan this QR code and download this article ABSTRACT Introduction: Gynura procumbens (Lour.) Merr. (Family: Asteraceae) is mainly popular in South- East Asian countries for its traditional medicinal properties. It is usually used as a traditional medicine for the treatment of eruptive fevers, rash, kidney disease, migraines, constipation, hyper- tension, diabetes mellitus, and cancer. It is commonly used as a traditional medicine in Vietnam for the treatment of many diseases. Methods: The leaves and trunks of G. procumbenswere collected, macerated withmethanol. The extracts fromMeOH-soluble extract were processed by the column chromatographic technique to give pure compounds, and the nuclear magnetic resonance meth- ods were applied to determine their chemical structures. The inhibitory activities of these extracts against a-glucosidase were conducted and compared with acarbose. Results: Seven organic compounds were isolated and determined the structures, including syringic acid (1), quercetin (2), N,N-dimethylanthranilic acid (3), dehydrovomifoliol (4), b -sitosterol 3-O-b -D-glucopyranoside (5), schottenol (6), montanic acid (7). The inhibition of a-glucosidase test results in the IC50 values of the four extracts, which were lower than those of acarbose. Conclusion: Seven pure compounds were identified from the leaves and trunks of G. procumbens, including two compounds being iso- lated from G. procumbens for the first time. The test results showed that the parts of G. procumbens were active as a-glucosidase inhibitor, which would be useful to support the treatment for dia- betes. Key words: Gynura procumbens, syringic acid, quercetin, a-glucosidase INTRODUCTION Gynura is the genus of the Asteraceae family, in- cludes 20 species spread all over the world, particu- larly in Vietnam, China, Malaysia, Thailand, Indone- sia, Korea, and the Philippines. G. procumbens (Lour.) Merr. (Figure 1) is a herbal material widely used in tropical countries for the treatment of various health ailments such as cancers, lymphatic pain, hyperten- sion, skin diseases, diabetes mellitus)1,2. Nowa- days, people in various tropical regions consume an increasing amount of G. procumbens leaves in diet and tea. Research shows that the leaves do not have any toxicity2. Pharmacologic studies have reported that G. procumbens has antioxidant, anti-Herpes sim- plex, anti-hyperglycemic, anti-hyperlipidemic, anti- inflammatory, analgesic, and reducing blood hyper- tension properties. The health benefits of G. procum- bens are related to some of its bioactive compounds, such as flavonoids, saponins, and alkaloids3. How- ever, there were not many studies about the chemicals constituent of this plant, especially in Vietnam. This study aimed to investigate the chemical constituents from the leaves and trunks of G. procumbens, grow- ing in Gia Lai Province, Vietnam. By column chro- matographic and spectroscopic methods seven com- pounds (1-7) (Figure 2) were identified. Besides, we also tested the inhibitory activity of a-glucosidase on four extracts of this plant. METHODS Chemicals and equipment Column chromatography was performed on silica gel (HiMedia) (230-400 Mesh). Thin-layer chromatog- raphy (TLC) and preparative TLC were performed on silica gel GF254 (Merck), visualized by hot 10 % solution of H2SO4. NMR spectra were acquired on Bruker 500 Avance III at 500 MHz for 1H-NMR and 125 MHz for 13C-NMR spectra. The pure solvents methanol, ethyl acetate, n-butanol, petroleum ether, chloroform were from Chemsol Vina, Vietnam. Acarbose, ana-glucosidase inhibitor, was fromChem Cruz, Santa Cruz Biotechnology, Inc., USA. Plant material The leaves and trunks ofG. procumbenswere collected atGia Lai province, Vietnam, in July 2016 and authen- ticated by Dr. Dang Van Son, Department of Biolog- Cite this article : Thi My Quyen L, Thi Diem Quynh N, Hoang Phu D, Thi Y Nhi N, Le Quan T. Chemical constituents and the inhibition of a-glucosidase of Gynura procumbens (Lour.) Merr.. Sci. Tech. Dev. J.; 22(4):391-399. 391 Science & Technology Development Journal, 22(4):391-399 Figure 1: Gynura procumbens (Lour.) Merr. Figure 2: Chemical structures for 1-7. 392 Science & Technology Development Journal, 22(4):391-399 ical Resources, Institute of Tropical Biology – Ho Chi Minh City, Vietnam. Extraction and isolation Dried leaves and trunks were ground into powder (2.9 kg) and extracted with hot methanol (4  7 L) for four hours each time. Themethanolic filtrate was then evaporated to dryness under reduced pressure to ob- tain a methanolic residue (375.0 g). The methano- lic residue was then dissolved in aqueous methanol (10 % methanol) and extracted with petroleum ether (10 x 500 mL), ethyl acetate (10 x 500 mL), n-butanol (10 x 500 mL), consecutively, to afford petroleum ether (PE, 75.0 g), ethyl acetate extract (EA, 7.2 g), n-butanol (Bu, 9.9 g) and crystal compound (102.9 g). The ethyl acetate extract (EA, 7.2 g) was subjected to a silica gel column chromatography and eluted with petroleum ether–ethyl acetate (stepwise, 9:1 ! 0:10) followed by ethyl acetate–methanol (stepwise, 8:2 ! 6:4) to afford five main fractions EA1 (26.1 mg), EA2 (498.1 mg), EA3 (696.2 mg), EA4 (1200.6 mg), EA5 (627.9 mg). Fraction EA1 (26.1 mg) was washed and cleaned with methanol (MeOH) to give compound 1 (9.0 mg). Fraction EA5 (1200.6 mg) was subjected to a silica gel column chromatography, eluted with chloroform–methanol (CHCl3–MeOH) (stepwise, 99:1! 9:1) to give compound 3 (11.0 mg). Fraction EA3 (627.9 mg) was subjected to column chromatographic separation over silica gel and eluted with CHCl3–MeOH (stepwise, 99:1 ! 9:1) to give compound 2 (4.4 mg). The same manner was applied on the EA2 (498.1 mg), eluted with CHCl3–MeOH (95:5) to give compound 4 (4.5 mg). Fraction EA4 (696.2 mg) was fractionated by a silica gel column chromatography using CHCl3–MeOH (stepwise, 95:5 ! 8:2) to give compound 5 (4.5 mg). The petroleum ether extract (PE, 75.0 g) was subjected to a silica gel column chromatography and eluted with petroleum ether–ethyl acetate (PE–EA) (stepwise, 9:1 ! 0:10) to afford fractions, in these, there were two fractions which were coded as PE1 (1294.7 mg), PE2 (2851.0 mg). By subjecting to a silica gel column chromato- graphic and eluting with appropriate solvents, frac- tion PE1 gave compound 6, PE2 gave compound 7. Compound 1 (syringic acid): white needle-shaped crystals, 1H- and 13C-NMR (Table 1). Compound 2 (quercetin): yellow powder, 1H- and 13C-NMR (Table 1). Compound 3 (N,N-dimethylanthranilic acid): white powder, 1H- and 13C-NMR (Table 1), ESI/MS m/z 188.0723 [M+Na]+. Compound 4 (dehydrovomifoliol): white crystals, 1H- and 13C-NMR (Table 1). Compound 5 (b -sitosterol 3-O-b -D- glucopyranoside): white powder, 1H-NMR (pyridine-d5) dH (ppm): 3.93 (1H, m, H-3),5.34 (1H, m, H-6), 0.65 (3H, s, H-18), 0.92 (3H, s, H-19), 0.98 (3H, d, J = 6.5, H-21), 0.85 (3H, d, J = 7.0, H-26), 0.87 (3H, d, J = 7.0, H-27), 0.88 (3H, t, J = 7.0, H-29) 13C-NMR (pyridine-d5) dC (ppm): 37.7 (C-1), 30.4 (C-2), 78.7 (C-3), 40.1 (C-4), 141.1 (C-5), 122.1 (C-6), 32.4 (C-7), 32.3 (C-8), 50.5 (C-9), 37.1 (C-10), 21.5 (C-11), 39.5 (C-12), 42.7 (C-13), 57.0 (C-14), 24.7 (C-15), 28.7 (C-16), 56.4 (C-17), 12.2 (C-18), 19.6 (C-19), 36.6 (C-20), 19.2 (C-21), 34.4 (C-22), 26.6 (C-23), 46.2 (C-24), 29.7 (C-25), 19.4 (C-26), 20.2 (C-27), 23.6 (C-28), 12.4 (C29), 102.7 (C-1’), 75.5 (C-2’), 78.6 (C-3’), 71.9 (C-4’), 78.4 (C-5’), 63.0 (C-6’). Compound 6 (schottenol): white crystals, 1H-NMR (CDCl3) dH (ppm): 3.60 (1H, m, H-3), 5.18 (1H, m, J = 4.6 Hz, H-7), 0.55 (3H, s, C-18), 0.80 (3H, s, H-19), 0.83 (3H, d, H-26), 0.85 (3H, d, H-27), 0.98 (3H, d, J=7.0 Hz, H-21), 0.86 (3H, d, H-29); 13C- NMR (CDCl3) dC (ppm): 37.16 (C-1), 31.48 (C-2), 71.09 (C-3), 37.99 (C-4), 40.28 (C-5), 29.66 (C-6), 117.42 (C-7), 139.69 (C-8), 49.48 (C-9), 22.97 (C-11), 39.59 (C-12), 55.06 (C-14), 23.10 (C-15), 27.95 (C- 16), 56.12 (C-17), 11.84 (C-18), 13.03 (C-19), 36.59 (C-20), 21.56 (C-21), 34.22 (C-22), 26.25 (C-23), 45.88 (C-24), 29.21 (C-25), 18.91 (C-26), 19.05 (C- 27), 19.81 (C-28), 11.97 (C-29). Compound 7 (montanic acid): white crystals, HR- ESI-MS m/z 423.4234 [M-H], 1H-NMR (CDCl3) dH (ppm): 2.35 (2H, t, J=7.5Hz,H-2), 1.63 (2H, quint, H-3), 1.25 (2nH, s), 0.88 (3H, t, J =6.9 Hz); 13C-NMR (CDCl3) dC (ppm): 178.85 (C-1), 33.84 (C-2), 31.92 (C-3), 29.06-29.69 (C-4 to C-26), 24.70 (C-27), 22.68 (C-28), 14.10 (C-29). Test of inhibition of a-glucosidase Test of inhibition of a-glucosidase was performed at Research Center Of Ginseng & Materia Medica, Ho Chi Minh City on four extracts methanol (GP - Me), ethyl acetate (GP - EA), n-butanol (GP - Bu) and petroleum ether (GP - PE). The inhibitory ac- tivity of a-glucosidase was determined by the previ- ous method4 with some adjustments. Samples were dissolved in the DMSO solvent. A mixture of 60 mL of sample and 50 mL of phosphate buffer 0.1 M (pH 6.8) containing a-glucosidase solution (0.2 U.mL1) was incubated in the wells of 96-well plates at 37 C for 10 minutes. After incubating, added 50 mL of p-nitrophenyl-a-D-glucopyranoside (p-NPG) pre- pared in phosphate buffer 0.1 M (pH 6.8) into each 393 Science & Technology Development Journal, 22(4):391-399 well and the wells were continuously incubated for 20 minutes. OD was measured on the spectrophotome- ter at 405 nm with a microdisk reader (Bio Tek, USA) and compared it with a control sample containing a 60 mL buffer solution in place of the test sample. The test result data was expressed by the average of triplicated experiments. The IC50 value is the concentration of the extract re- quired to inhibit 50 % of a-glucosidase activity under the assay conditions. Acarbose was used as a positive control. RESULTS The powdered leaves and trunks of G. procumbens were extracted with hot methanol. The MeOH- soluble extract was successively partitioned to yield petroleum ether, ethyl acetate, and n-butanol-soluble fractions. By using column chromatographic tech- nique and the nuclear magnetic resonance meth- ods, seven organic compounds were isolated and determined to be syringic acid (1), quercetin (2), N,N-dimethylanthranilic acid (3), dehydrovomifo- liol (4), b -sitosterol 3-O-b -D-glucopyranoside (5), schottenol (6), montanic acid (7). In these, two com- pounds (3), (4) were isolated fromG. procumbens for the first time. Compound 1 (Figure 2) was obtained as white needle-shaped crystals, completely soluble in MeOH, acetone, CHCl3. The 1H-NMR spectrum of com- pound 1 showed the resonance signal of eight protons, including six protons of the two methoxyl groups at dH3.88 (6H, s) and two cumulative protons at dH 7.33 (2H, s, H-6). It showed that compound 1 contains 1, 3, 4, 5 four-substituted aromatic nucleus. The 13C- NMR spectrum of compound 1 has six carbon sig- nals. There is a carbonyl carbon signal of the car- boxyl group at dC 167.5 (C-7), carbon signals of the two methoxyl groups at dC 56.7 (3-OCH3, 5-OCH3) and the six carbons of the benzene ring, composed of tertiary carbons at dC 148.4 (C-3, C-5); 141.6 (C-4); 121.5 (C-1) andmethine carbons at dC 108.2 (C-2, C- 6). The HMBC spectra of compound 1 showed that the proton signal of the methoxyl group dH 3.88 (6H, s) correlated to the signal at dC 148.4 (C-3, C-5) of a oxygen-carrying carbon. Therefore, two methoxyl groups bind to the C-3 and C-5 positions of the ben- zene ring. In addition, HMBC spectrum of 1 also showed a correlation of the proton signal at dH 7.33 (2H, s, H-2, H-6) to the signals at dC 148.4 (C-3, C- 5), 141.6 (C-4); 121.5 (C-1); 108.2 (C-2, C-6); 167.5 (C-7). Comparing the spectral data of compound 1 with syringic acid 5 gave the similarities. These above facts showed that compound 1 was syringic acid. Compound 2 (Figure 2) was obtained as a yellow powder, completely soluble in DMSO. The 1H-NMR spectrum displayed five aromatic protons at dH 6.16 (1H, d, J = 1.5 Hz, H-6), 6.39 (1H, d, J = 1.5 Hz, H-8), 7.86 (1H, dd, J1 = 8.5 Hz, J2= 2.5 Hz, H-6’), 6.86 (1H, d, J =8.5, H-5’), 7.64 (1H, d, J =2.0Hz,H-2’), ofwhich H-6 graftedmetawithH-8,H-6’ grafted orthowithH- 5’ and grafted meta with H-2’. Therefore, compound 2 contains two benzene rings, in that, H-6 and H-8 were in the first ring, H-2’ andH-6’ were in the second ring. One signal at dH 12.44 (1H, s, 5-OH) indicated a proton which made intramolecular hydrogen bond- ing with a carbonyl group at dC 147.7 (C-4). In 9.0 to 13.0 ppm region, there were signals characterized hydroxyl protons at dH 10.75, 9.49, 9.28. The 13C- NMR spectrum showed fifteen carbon signals. The signal at dC 175.7 (C-4) displayed a carbonyl carbon. In the low-field magnetic resonance, there were seven signals of aromatic carbons which linked to oxygen at dC146.8 (C-2), 135.5 (C-3), 160.6 (C-5), 163.8 (C- 7), 155.9 (C-9), 144.9 (C-3’), 147.7 (C-4). The carbon signals were attributed to the first ring at dC102.87 (C-10), 98.2 (C-6), 93.3 (C-8) and to the second ring at dC 121.9 (C-1’), 114.9 (C-2’), 115.6 (C-5’), 119.9 (C-6’). Comparing the spectral data of compound 2 with quercetin6 gave the similarities. These above facts showed that compound 2 was quercetin. Compound3 (Figure 2) was obtained as awhite pow- der, completely soluble in acetone. HR-ESI-MS of compound 3 exhibited an ion peak at m/z 188.0723 [M+Na]+, consistent with a molecular formula of C9H11NO2. The 1H-NMR spectrum showed four aromatic protons at dH 7.72 (1H, dd, J1= 8.0 Hz, J2 = 0.8 Hz, H-3),7.41 (1H, td, J1= 7.9 Hz, J2 = 1.2 Hz, H-4), 7.66 (1H, td, J1 = 7.3 Hz, J2 = 1.6 Hz, H-5), 8.12 (1H, dd, J1 = 7.5 Hz, J2 = 1.5 Hz, H-6). The sig- nal at dH 2.85 (6H, s) showed protons of two methyl groups linkedwith nitrogen. The 13C-NMR spectrum exhibited eight carbon signals, of which six signals at dC 126.3 (C-1), 153.4 (C-2), 123.5 (C-3), 134.8 (C-4), 128.1 (C-5), 132.2 (C-6) were attributed to the aro- matic ring, whereas a signal at dC 45.7 (C-8, C-9) characterized as twomethyl groups linked with nitro- gen and a signal at dC 167.2 (C-7) displayed a car- bonyl carbon. The HMBC spectrum of compound 3 showed that the proton at dH 7.72 (1H, dd, H-3) cor- related with signals at dC 128.1 (C-5); the proton at dH 7.41 (1H, td, H-4) correlated with signals at dC 126.3 (C-1), 123.5 (C-3); the proton at dH 7.66 (1H, td, H-5) correlated with signals at dC 153.4 (C-2), 132.2 (C-6); the signal at dH8.12 (1H, dd, H-6) corre- latedwith signals at dC 134.8 (C-4), 153.4 (C-2), 167.2 394 Science & Technology Development Journal, 22(4):391-399 (C-7); the signal of protons at dH 2.85 (6H, s) corre- lated with the signal at dC 153.4 (C-2) and 45.7 (C- 8, C-9). By analyzing the 1H-NMR, 13C-NMR, MS, HMBC spectral data and comparing the spectral data of compound 3 with reference7, the structure of com- pound 3 was given as N,N-dimethylanthranilic acid. Compound 4 (Figure 2) was obtained as white crys- tals, completely soluble in methanol, acetone. The 1H-NMR spectrum gave nine proton signals, which included two olefin protons grafted trans at dH 7.04 (1H, d, 16 Hz, H-7) and 6.49 (1H, d, 16 Hz, H-8); one olefin proton at 5.98 (1H, s, H-4); two methylene pro- tons at dH 2.29 (1H, d, 17Hz, H-2), 2.58 (1H, d, 17Hz, H-2); four proton signals of methyl group at dH 2.35 (3H, s, H-10), 1.12 (3H, s, H-11), 1.07 ( 3H, s, H-12), 1.95 (3H, s, H-13). The 13C-NMR spectrum showed thirteen carbon signals. Two signals at dC 200.3 (C- 3), 203.6 (C-9) characterized two carbonyl carbons; one quaternary olefin carbon at dC164.6 (C-5); three tertiary olefin carbons at dC 128.0 (C-4), 131.7 (C-8), 148.3 (C-7); two quaternary carbons at dC 80.0 (C-6), 42.6 (C-1); one methylene carbon at 50.6 (C-2) and four methyl carbons at dC 27.6 (C-10), 23.5 (C-11), 24.7 (C-12), 19.1 (C-13). By analyzing the 1H-NMR and 13C-NMR spectral data and comparing the spec- tral data of compound 4with reference8, the structure of compound 4 was given as dehydrovomifoliol. Compound5 (Figure 2) was obtained as awhite pow- der, completely soluble in DMSO.The 1H-NMR spec- tral data of 5 showed the present of six methyl groups at dH 0.65 (3H, s, H-18), 0.92 (3H, s, H-19), 0.98 (3H, d, J = 6.5, H-21), 0.85 (3H, d, J = 7.0, H-26), 0.87 (3H, d, J = 7.0, H-27), 0.88 (3H, t, J = 7.0, H-29). The signal at dH 3.93 (1H, m, H-3) appeared as mul- tilet displayed proton H-3. A signal at dH 5.34 (1H, m, H-6) was the characteristics of double bond be- tween quaternary carbon and methine carbon in the ring B. The 13C-NMR spectrum showed compound 5 has 35 carbon signals. The signals at dC 12.2 (C- 18), 19.6 (C-19), 19.2 (C-21), 19.4 (C-26), 20.2 (C-27), 12.4 (C29) were methyl carbons. Methylene carbons appeared at dC 37.7 (C-1), 30.4 (C-2), 40.1 (C-4), 32.4 (C-7), 21.5 (C-11), 39.5 (C-12), 24.7 (C-15), 28.7 (C- 16), 34.4 (C-22), 26.6 (C-23), 23.6 (C-28). Methine carbons were at dC78.7 (C-3), 122.1 (C-6), 32.3 (C-8), 50.5 (C-9), 57.0 (C-14), 56.4 (C-17), 36.6 (C-20), 46.2 (C-24), 29.7 (C-25). Quaternary carbons appeared at dC 141.1 (C-5), 37.1 (C-10), 42.7 (C-13). Further- more, the 1H-NMR and 13C-NMR spectral date of compound 5 displayed the present of a glucose unit. A signal among them appeared at dC 102.7 (C-1’) pre- sented anomeric carbon. Besides, the signal ofmethy- lene carbon C-6’ appeared at dC 63.0 and the other four methine carbons, which linked to oxygen, ap- peared at dC75.5 (C-2’), 78.6 (C-3’), 71.9 (C-4’), 78.4 (C-5’). Comparing the spectral data of compound 5 with b -sitosterol 3-O-b -D-glucopyranoside9 gave the similarities. These above facts indicated that com- pound 5 was b -sitosterol 3-O-b -D-glucopyranoside. Compound 6 (Figure 2) was obtained as white crys- tals, completely soluble in chloroform. The 1H-NMR spectrum gave an olefin proton at dH 5.18 (1H,m, J = 4.6 Hz, H-7) and one methyl proton at dH 3.60 (1H, m, H-3). In the high-field magnetic resonance, there were six signals characterized methyl protons includ- ing one methyl group grafted with secondary carbon at dH 0.86 (3H, d, H-29), three methyl groups grafted with tertiary carbons at dH 0.83 (3H, d, H-26), 0.85 (3H, d, H-27), 0.98 (3H, d, J=7.0 Hz, H-21), and two methyl groups grafted with quaternary carbons at dH 0.55 (3H, s, C-18), 0.80 (3H, s, H-19). The 13C-NMR spectrum showed compound 6 has 29 carbon signals. In the low-field magnetic resonance, there were two signals of olefin carbons at dC 139.69 (C-8), dC 117.42 (C-7). Methyl carbon appeared at dC 71.09 (C-3). Two signals at dC 33.92, 43.41 characterized quater- nary carbons C-10 and C-13. Seven methine carbons appeared at dC 40.28 (C-5), 49.48 (C-9), 55.06 (C- 14), 56.12 (C-17), 36.59 (C-20), 45.88 (C-24), 29.21 (C-25). Eleven methylene carbons were at dC 37.16 (C-1), 31.48 (C-2), 37.99 (C-4), 29.66 (C-6), 22.97 (C- 11), 39.59 (C-12), 23.10 (C-15), 27.95 (C-16), 34.22 (C-22), 26.25 (C-23), 19.81 (C-28). Six methyl car- bons appeared at dC 11.84 (C-18), 13.03 (C-19), 21.56 (C-21), 18.91 (C-26), 19.05 (C-27), 11.97 (C-29). By analyzing the 1H-NMR and 13C-NMR spectral data and comparing the spectral data of compound 6 with reference10, the structure of compound 6 was given as schottenol. Compound 7 (Figure 2) was obtained as white crys- tals, completely soluble in chloroform. HR-ESI- MS of compound 7 exhibited an ion peak at m/z 423.4234 [M-H], consistent with a molecular for- mula of C28H56O2. The 1H-NMR spectrum showed a signal of two methylene protons grafted with a car- bonyl group at dH 2.35 (2H, t, J=7.5 Hz, H-2), a sig- nal of two methylene protons defined H-3 at dH 1.63 (2H, quint, H-3). Furthermore, at dH 1.25 (2nH, s) there was a signal of accumulable protons of methy- lene groups in the saturated carbon chain. A signal at dH 0.88 (3H, t, J =6.9 Hz) characterized methyl protons. The 13C-NMR and DEPT-NMR spectrum showed a carbonyl carbon signal at dC178.85, a car- bon grafted with a carbonyl group dC 33.84, a methy- lene carbon separated carbonyl group by a carbon 395 Science & Technology Development Journal, 22(4):391-399 at dC 31.92, a methyl carbon at dC 14.10, a methy- lene carbon grafted with methyl carbon at dC 22.68, a methylene carbon separated methyl group by a car- bon at dC 24.70. Moreover, the other carbon signals at dC 29.06-29.69 described methylene groups in the saturated carbon chain. By analyzing the 1H-NMR, 13C-NMR, DEPT, MS spectral data, the structure of compound 7 was supposed to be montanic acid. The inhibition of the a-glucosidase test was per- formed in optimal conditions for the enzyme that has been optimized. The data of the spectrophotome- ter (OD) was recorded and the inhibition (%) was expressed by the average of triplicated experiments and standard deviation (Table 2). The IC50 values were determined based on the logarithmic equations (Figure 3) drawn from the data in Table 2. The re- sult showed that acarbose had the highest IC50 value of 0.722 mg.mL1. IC50 value of methanol, ethyl ac- etate, n-butanol, petroleum ether extracts were 0.244, 0.099, 0.209, 0.064 mg.mL1, respectively. The IC50 values of the four extracts were lower than those of acarbose. This indicates the extracts of G. procum- bens could perform well in inhibiting a-glucosidase and petroleum ether extract showed the most potent effect. DISCUSSION Previous studies have shown that G. procumbens con- tains many compounds such as steroids, flavonoids, saponins, tannins, terpenoids, etc 2. Among the seven compounds isolated, five compounds were known in G. procumbens syringic acid (1) (hydroxybenzoic acid structure), quercetin (2) (flavonoid glycoside structure), b -sitosterol 3-O-b -D-glucopyranoside (5), schottenol (6) (steroid structure), montanic acid (7) (acid carboxylic), the two compounds N,N- dimethylanthranilic acid (3) and dehydrovomifoliol (4) were isolated in G. procumbens for the first time. Previous studies have been conducted to investi- gate the anti-diabetic activities of G. procumbens leaves aqueous and ethanolic extracts and its pos- sible underlying antihyperglycemic mechanisms of action involving liver carbohydrate metabolism in streptozotocin-induced diabetes in rats3. There was no previous study has ever conducted on anti-diabetes by inhibiting the enzyme a-glucosidase. From the results of the test on inhibiting a-glucosidase en- zyme, which we have been doing in this study and the streptozotocin-induced diabetes treatment reported in previous studies, we can strongly believe that G. proumbens would be useful in the treatment of dia- betes. CONCLUSION In the investigation of the chemical constituents of G. procumbens collected at Gia Lai province, seven compounds were isolated syringic acid (1), quercetin (2), N,N-dimethylanthranilic acid (3), dehydrovomi- foliol (4), b -sitosterol 3-O-b -D-glucopyranoside (5), schottenol (6), montanic acid (7). All four extracts (methanol, ethyl acetate, n-butanol, petroleum ether) showed inhibiting activity on a- glucosidase. The IC50 values of these four extracts were all smaller than those of the positive control acarbose. Petroleum ether extract gave the best ability to inhibit a-glucosidase with the lowest value of IC50 0.064 mg.mL1. The results of this study showed that G. procumbens has great potential in treating diabetes. LIST OF ABBREVIATIONS IC50: 50% Inhibitory Concentration TLC: Thin-Layer Chromatography NMR: Nuclear Magnetic Resonance 1 H-NMR: Proton Nuclear Magnetic Resonance 13 C-NMR: Carbon Nuclear Magnetic Resonance DEPT: Distortionless Enhancement by Polarization Transfer HR-ESI-MS: High-Resolution ElectroSpray Ioniza- tion Mass Spectrum MeOH: Methanol PE: Petroleum Ether EA: Ethyl Acetate n-Bu: n-Butanol OD: Optical Density AUTHOR CONTRIBUTIONS The contributions of all authors are equal in selecting data, calculating descriptors, analyzing results, and writing a manuscript. COMPETING INTERESTS The authors declare that they have no competing in- terests. ACKNOWLEDGMENT We are grateful to Dr. Dang Van Son, Department of Biological Resources, Institute of Tropical Biology– Ho Chi Minh City, Vietnam, for the determination of the scientific name for the plant. 396 Science & Technology Development Journal, 22(4):391-399 Table 1: The 1H-NMR and 13C-NMR data of compounds (1 – 4) No. 1H-NMR 13C-NMR 1a 2b 3a 4c 1a 2b 3a 4c 1 _ _ _ _ 121.5 126.3 42.6 2 7.33 (2H, s) _ _ 2.29 (2H, d, 17.0) 2.58 (2H, d, 17.0) 108.2 146.8 153.4 50.6 3 _ _ 7.72 (1H, dd, 8.0, 0.8) _ 148.4 135.5 123.5 200.3 4 _ _ 7.41 (1H, td, 7.9, 1.2) 5.98 (1H, s) 141.6 175.7 134.8 128.0 5 _ _ 7.66 (1H, td, 7.3, 1.6) _ 148.4 160.6 128.1 164.6 6 7.33 (2H, s) 6.16 (1H, d, 1.5) 8.12 (1H, dd, 7.5, 1.5) _ 108.2 98.2 132.2 80.0 7 _ _ - 7.04 (1H, d, 16.0) 167.5 163.7 167.1 148.3 8 3.88 (3H, s) 6.39 (1H, d, 1.5) 2.85 (3H, s) 6.49 (1H, d, 16.0) 56.7 93.3 45.7 131.7 9 3.88 (3H, s) _ 2.85 (3H, s) _ 56.7 156.0 45.7 203.6 10 _ 2.35 (3H, s) 102.9 27.6 11 1.12 (3H, s) 23.5 12 1.07 (3H, s) 24.7 13 1.95 (3H, s) 19.1 1’ _ 121.9 2’ 7.64 (1H, d, 2.0) 114.9 3’ _ 144.9 4’ _ 147.7 5’ 6.86 (1H, d, 8.5) 115.6 6’ 7.86 (1H, dd, 8.5, 2.5) 119.9 a: Acetone; b: DMSO; c: MeOH 397 Science & Technology Development Journal, 22(4):391-399 Table 2: The a-glucosidase inhibitory activity and their IC50 values Extract Concentration (mg.mL1) Triplicated experiment (%) Average SD IC50 (mg.mL1) GP - Me 0.075 -7.910 0.167 -4.330 -4.024  3.304 0.15 33.555 25.583 24.480 27.873 4.043 0.3 58.285 59.583 59.450 59.106 0.583 0.45 82.515 81.917 66.861 77.097 7.242 0.6 94.005 91.750 90.258 92.004 1.540 0.75 101.499 102.583 102.914 102.3320.605 GP - EA 0.0375 -16.403 -7.417 -1.249 0.075 36.053 33.167 26.395 -8.356  6.222 31.872  4.048 0.1125 54.788 57.417 62.115 58.106 3.031 0.15 81.932 77.750 69.359 76.347 5.228 0.1875 88.260 89.250 89.509 89.006 0.538 GP - Bu 0.075 -10.241 0.167 6.495 -2.193  6.847 0.15 32.057 25.583 36.053 34.870 1.998 0.3 75.937 59.583 63.281 69.684 5.168 0.45 89.259 81.917 88.593 87.673 1.793 0.6 100.416 91.750 100.416 100.583 GP - PE 0.0375 14.821 25.167 25.396 0.075 59.867 58.167 67.027 0.1125 74.022 82.250 72.773 0.15 90.924 90.833 91.757 21.794  4.932 61.687  3.840 76.348  4.204 91.171  0.416 0.1875 104.829 103.083 104.330 104.081 Acarbose 0.038 -0.999 -4.500 11.657 0.188 34.305 32.333 24.480 2.053  6.940 30.373  4.244 0.375 43.797 40.750 33.306 39.284 4.407 0.563 49.958 41.250 38.385 43.198 4.922 0.750 57.369 53.167 40.550 50.362 7.147 1.125 56.536 56.750 52.040 55.109 2.172 1.500 65.862 65.583 63.863 65.103 0.884 398 0.236 0.734 0.244 0.099 0.209 0.064 0.722 Science & Technology Development Journal, 22(4):391-399 Figure 3: The graphs illustrating the inhibition of a-glucosidase of GP –Me, GP – EA, GP- Bu, GP – PE and acarbose. 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