Research Paper Investigation of Antimicrobial Activity and Chemical

Research Paper

Investigation of antimicrobial activity and chemical constituents of Momordica charantia L. var. abbreviata Ser

Accepted 17th July, 2018

ABSTRACT

The antimicrobial potential of the four extracts of Momordica charantia L. var. abbreviata Ser., including n-hexane, chloroform, ethyl acetate and methanol- water 80:20 (v/v) extracts was screened against four bacterial and four fungal Nguyen PD.1, Vo MD.1, Luong HVT.1, Phan strains, using microbroth dilution assay. The chloroform extract showed the TTH.2 and Nguyen TTT.3* highest growth inhibitory activity with MIC = 200 μg/ml on both Escherichia coli and Bacillus subtillis and MIC = 100 μg/ml on Aspergillus niger. Phytochemical 1Faculty of Education, Can Tho University, Can Tho, Vietnam. study on the bioactive chloroform extract led to the isolation of four known 2Faculty of Science, Mien Tay compounds as octadecan-1-ol (1), (23E)-5β, 19-epoxycucurbita-6, 23, 25-trien- Construction University, Vinh Long, 3β-ol (2), 5α-poriferasta-7,25-dien-3β-ol (3) and 3-O-(6′-O-palmitoyl-β-D- Vietnam. glucopyranosyl)-clerosterol (4). Their structures were elucidated by 3Faculty of Science, Can Tho University of Medicine and Pharmacy, Can Tho, spectroscopic methods including 1D and 2D-NMR, MS and in comparison with Vietnam. literature data. Here, such metabolites were reported for the first time in M. charantia L. var. abbreviata Ser. *Corresponding author. E-mail: [email protected]. Tel: (+84)919886682. Key words: Antibacterial, antifungal, clerosterol, Cucurbitaceae, Momordica charantia.

INTRODUCTION

In recent years, antibiotics are becoming less effective anti-bacterial, anti-inflammatory, anti-viral, cytotoxic, against certain illnesses, not only because many of them hypoglycemic, and triglyceride-lowering activities have produce toxic reactions, but also due to emergence of been reported on MC (Lu et al., 2011; Sagor et al., 2015). drug-resistant bacteria. It is necessary to discover new MC is also known to contain several compounds such as drugs. Drugs derived from natural sources play a momorcharins, momordenol, momordicilin, momordicins, significant role in the prevention and treatment of human momordin, momordolol, charantin, charine, cryptoxanthin, diseases. About 80% of the world’s population use plant cucurbitins, cucurbitacins, cucurbitanes, cycloartenols, extracts or their active constituents as folk medicine in diosgenin, elaeostearic acids, erythrodiol, galacturonic traditional therapies. In fact, plants which are rich in a acids, gentisic acid, goyaglycosides, goyasaponins, caffeic wide variety of secondary metabolites such as tannins, acid, ferulic acid, fisetin, isorhamnetin, 3β,25-dihydroxy- terpenoids, alkaloids, flavonoids and glycosides, etc, were 7β-methoxycucurbita-5,23(E)-diene, 3β-hydroxy-7,25- demonstrated having antimicrobial properties in vitro dimethoxycucurbita-5,23(E)-diene and 3-O-β-D- (Gebregiorgis and Nigussie, 2018). allopyranosyl-7β,25-dihydroxycucurbita-5,23(E)-dien-19- In an effort to expand the spectrum of antimicrobial al (Wu and Ng, 2008). agents from natural resources, Momordica charantia L. var. Scientific information published on chemical and abbreviate Ser. (MCA) was selected. The investigated biological properties of MCA remains limited although species is normally smaller than the cultivated bitter gourd both species have been consumed as a vegetable and folk M. charantia L. (MC) both belonging to the family medicine. Preliminary studies on chemical constituents of Cucurbitaceae. Many pharmacological activities including MCA showed the presence of polyphenols, triterpenoids, Academia Journal of Medicinal Plants; Nguyen et al. 164

saponins and flavonoid glycosides (Nguyễn et al., 2014). antibiotics were streptomycin, tetracyclin and nystatin. Several triacylglycerols which contain two different fatty Fungi and bacteria were cultured in nutrient media. The acyl chains such as palmitic, stearic, oleic, linoleic, and test micro-organisms were activated before testing in fluid conjugated linolenic acid were also reported in the seed oil nutrient media. MIC is defined as the lowest concentration of MCA. So far, its fruit extracts and components have been of antibiotic completely inhibiting visible growth of shown to possess some pharmacological actions including bacteria. the antioxidant and anti-inflammatory activities, and hepatoprotection against alcoholic fatty liver (Huang et al., 2015). Extraction and isolation The purpose of this study was to investigate the phytochemical compositions, antimicrobial activities of the Dried and powdered fruits of MCA (3.0 kg) were extracts of MCA against some pathogenic microorganisms macerated for 12 h with 30 L n-hexane to furnish 45.0 g of that support folkloric use in the treatment of some n-hexane extract (yield: 1.50%). The dried resulting diseases as broad-spectrum antimicrobial agents. powdered material was extracted exhaustively with 30 L of chloroform, ethyl acetate and methanol-water 80:20 (v/v) to give chloroform extract (72.0 g, yield 2.40%), ethyl MATERIALS AND METHODS acetate extract (43.5 g, yield 1.45%), and methanol-water 80:20 (v/v) extract (90.0 g, yield 3.00%), respectively. Plant material Since the chloroform extract was the most effective against the test organisms than the other extracting solvents, a The fruits of MCA were collected in Vinh Long province, phytochemistry study was performed on this extract. The Vietnam on September 2016. This plant was identified by chloroform extract was subjected to a silica gel column and Dr. Dang Minh Quan, School of Education, Can Tho eluted with n-hexane: acetone with increasing acetone University, Vietnam. A voucher specimen (No MCA-0916) ratios to obtain fourteen fractions from C1 to C14. Fraction was deposited in the herbarium of the Department of C2 (12.5 g) was applied on normal phase silica gel column Chemistry, School of Education, Can Tho University. chromatography and eluted with the solvent system of n- hexane: chloroform (95:5) to afford compound 1 (430 mg). Fraction C4 was chromatographed and eluted with n- General experimental procedures hexane: ethyl acetate with increasing ethyl acetate ratios to obtain thirteen sub-fractions from C4.1 to C4.13. Sub- The NMR experiments were performed on a Bruker DMX fraction C4.8 was rechromatographed and eluted with the 500 spectrometer. MS were carried out on an Agilent 6310 solvent system of n-hexane: ethyl acetate (95:5) to give spectrometer. Column chromatography was performed on compound 2 (500 mg). Sub-fraction C4.12 was normal phase silica gel (40-63 µm, Keselgel 60, Merck rechromatographed and eluted with n-hexane: ethyl 7667). Thin layer chromatography (TLC) was performed acetate (85:15) to give compound 3 (200 mg). Fraction C10 on Kieselgel 60F254 plates (Merck) and spots visualized was applied to silica gel column chromatography and under UV light or sprayed with vanillin (0.5 g vanillin in 80 eluted with chloroform: acetone (80: 20) to obtain ml sulfuric acid and 20 ml ethanol) and thereafter, heated. compound 4 (175 mg). 1 All solvents used were purchased from Chemsol, purity ≥ Octadecan-1-ol (1): colorless paraffin; H-NMR (CDCl3, 99.0%. 500 MHz): δHppm 3.64 (2H, t, 7.5), 1.56-1.53 (32H, m), 0.88 13 (3H, t, 7.0); C-NMR (CDCl3, 125 MHz): δCppm 63.1, 32.8, 31.9, 29.4 – 29.7 (C×12), 25.8, 22.7, 14.1; ESI-MS m/z - Antimicrobial activity test 269.28 [M-H] calculated for C18H37O. Found: 269.38. (23E)-5β,19-Epoxycucurbita-6,23,25-trien-3β-ol (2): white 1 Antimicrobial activity test was carried out at the amorphous power; H-NMR (CDCl3, 500 MHz): δHppm 6.14 Department of Experimental Biology, Institute of Natural (1H, d, 15.5, H-24), 6.05 (1H, dd, 10.0, 2.5, H-6), 5.62 (1H, Products Chemistry, VAST, using the method previously m, H-7), 5.61 (1H, m, H-23), 4.86 (2H, s, H-26), 3.94 (1H, dd, described (Vanden and Vlietinck, 1991; Mckane and 9.5, 3.0, H-3), 3.67 (1H, d, 8.5, H-19a), 3.51 (1H, d, 8.0, H- Kandel, 1996). This experiment was performed by 19b), 1.84 (3H, s, H-27), 1.02 (3H, s, H-29), 0.93 (3H, s, H- microdilution technique on 96-well microtiter plate. Two 28), 0.91 (3H, d, 6.5, H-21), 0.87 (3H, s, H-30), 0.78 (3H, s, 13 gram-negative bacteria (Escherichia coli and Pseudomonas H-18); C-NMR (CDCl3, 125 MHz): δCppm 17.6 (C-1), 26.7 aeruginosa), two gram-positive bacteria (Bacillus subtillis (C-2), 76.2 (C-3), 37.2 (C-4), 87.5 (C-5), 132.8 (C-6), 131.5 and Staphylococcus aureus subsp. aureus) and four fungal (C-7), 52.0 (C-8), 45.3 (C-9), 38.9 (C-10), 23.6 (C-11), 30.8 strains (Aspergillus niger, Fusarium oxysporum, (C-12), 45.5 (C-13), 48.6 (C-14), 83.2 (C-15), 28.1 (C-16), Saccharomyces cerevisiae and Candida albicans) were 50.3 (C-17), 14.9 (C-18), 79.9 (C-19), 36.6 (C-20), 18.8 (C- employed to determine antimicrobial activity and 21), 39.8 (C-22), 129.2 (C-23), 134.2 (C-24), 142.2 (C-25), minimum inhibitory concentration (MIC). The reference 114.1 (C-26), 18.6 (C-27), 24.6 (C-28), 20.5 (C-29), 20.1 (C- Academia Journal of Medicinal Plants; Nguyen et al. 165

Table 1: Antimicrobial activity of the extracts of MCA.

MIC (g/ml) Extracts Gr (-) bacteria Gr (+) bacteria Fungi E. coli P. aeruginosa B. subtillis S. aureus A. niger F. oxysporum S. cerevisiae C. albicans n-Hexane - - - - 100 - - - Chloroform 200 - 200 - 100 - - - Ethyl acetate - - - - 100 - - - Methanol-Water ------80:20 (v/v)

-: not determined.

+ 30); ESI-MS m/z 439.35 [M+H] calculated for C30H47O2. and antifungal potential of the extracts were assessed in Found: 439.40. 5α-Poriferasta-7,25-dien-3β-ol (3): white MIC values (Table 1). The results revealed that the 1 amorphous power; H-NMR (CDCl3, 500 MHz): δHppm 5.15 chloroform extract was more effective against the test (1H, m, H-7), 4.73 (1H, m, H-27a), 4.64 (1H, d, 2.5, H-27b), organisms than the other extracting solvents with MIC 200 3.59 (1H, m, H-3), 1.57 (3H, s, H-26), 0.91 (3H, d, 6.5 Hz, H- µg/ml for both E. coli and B. subtillis and MIC 100 µg/ml 21), 0.80 (3H, t, 7.5 Hz, H-29), 0.79 (3H, s, H-19), 0.53 (3H, for A. niger. Moreover, A. niger showed high susceptibility 13 s, H-18); C-NMR (CDCl3, 125 MHz): δCppm 37.2 (C-1), with MIC 100 µg/ml to almost the extracts, except 31.5 (C-2), 71.1 (C-3), 38.0 (C-4), 40.3 (C-5), 29.7 (C-6), methanol-water 80:20 (v/v) extract. All the investigated 117.5 (C-7), 139.6 (C-8), 49.5 (C-9), 34.2 (C-10), 21.6 (C- extracts were not effective against P. aeruginosa, S. aureus, 11), 39.6 (C-12), 43.4 (C-13), 55.1 (C-14), 23.0 (C-15), 27.9 F. oxysporum, S. cerevisiae and C. albicans. Our obtained (C-16), 56.1 (C-17), 11.8 (C-18), 13.0 (C-19), 36.0 (C-20), results were in line with the previous data as water and 18.8 (C-21), 33.6 (C-22), 29.5 (C-23), 49.5 (C-24), 147.6 (C- methanolic extracts of MCA collected in Taiwan showed 25), 17.8 (C-26), 111.4 (C-27), 26.5 (C-28), 12.1 (C-29); non-inhibitory activity against methicillin-resistant S. + ESI-MS m/z 413.37 [M+H] calculated for C29H49O. Found: aureus or P. aeruginosa but these extracts exhibited 413.45. activity against E. coli and S. enterica (Lu et al., 2011). 3-O-(6′-O-Palmitoyl-β-D-glucopyranosyl)-clerosterol (4): The presence of antimicrobial activities in compounds in 1 colorless paraffin; H-NMR (CDCl3, 500 MHz): Hppm 3.47 the chloroform extract is perhaps the reason for its (1H, m, H-3), 5.35 (1H, brs, H-6), 0.68 (3H, s, H-18), 1.02 antimicrobial effects and as a result, a phytochemical (3H, s, H-19), 0.89 (3H, d, 7.0, H-21), 1.57 (3H, s, H-26), constituent’s investigation on such extract was performed. 4.69 (2H, brs, H-27), 0.81 (3H, t, 7.5, H-29), 4.37 (1H, d, 7.5, In this study, four known compounds were isolated for the H-1'), 3.34 (1H, t, 8.0, H-2'), 3.53 (1H, t, 9.0, H-3'), 3.37 (1H, first time in the chloroform extract of MCA (Figure 1). t, 9.0, H-4'), 3.51 (1H, m, H-5'), 4.33 (2H, brs, H-6'), 2.32 Compound 1 appeared as colorless paraffin and the ESI-MS 13 - (2H, t, 7.5, H-2''), 0.88 (3H, t, 6.5, H-16''). C-NMR (CDCl3, showed an ion peak at m/z 269.28 [M-H] corresponding to 1 125 MHz): δCppm 36.0 (C-1), 29.3 (C-2), 73.8 (C-3), 38.9 the molecular formula of C18H38O. The H-NMR spectrum (C-4), 140.4 (C-5), 122.1 (C-6), 31.9 (C-7), 31.9 (C-8), 50.2 displayed one oxymethylene group at Hppm 3.64 (2H, t, 7.5, (C-9), 36.7 (C-10), 21.1 (C-11), 39.8 (C-12), 42.4 (C-13), -CH2OH), one methyl group at Hppm 0.88 (3H, t, 7.0, -CH3) 56.8 (C-14), 24.3 (C-15), 29.2 (C-16), 56.2 (C-17), 11.8 (C- and sixteen methylene groups Hppm 1.56-1.53 (32H, m, 13 18), 19.4 (C-19), 35.5 (C-20), 18.7 (C-21), 34.3 (C-22), 29.4 16×-CH2-). C-NMR spectrum showed the resonances of (C-23), 49.5 (C-24), 147.5 (C-25), 17.8 (C-26), 111.4 (C-27), 18 carbons. Comparison with previously reported data 26.5 (C-28), 12.0 (C-29), 101.3 (C-1'), 73.4 (C-2'), 76.2 (C- confirmed the structure of 4 as octadecan-1-ol (Toyao et 3'), 70.4 (C-4'), 79.7 (C-5'), 63.6 (C-6'), 174.3 (C-1''), 34.3 al., 2017). (C-2''), 25.0 (C-3''), 22.7-29.8 (C-4'' – C-15''), 14.1 (C-16''); Compound 2 was obtained as white amorphous powder. - APCI-MS m/z 812.65 [M] calculated for C51H88O7. Found: The compound possessed the molecular formula C30H46O2 812.33. as determined from the ESI-MS ([M + H]+ m/z 439.40) as well as, from its 13C-NMR DEPT which suggested that it was a triterpenoid with eight degrees of unsaturation. The RESULTS AND DISCUSSION 13C and 1H-NMR spectra of 2 showed the presence of four tertiary methyls, a secondary methyl, a vinylic methyl, a General extraction yields varied between 1.45% for ethyl secondary hydroxyl, two disubstituted double bonds, a acetate extract and 3.00% for methanol-water 80:20 (v/v) terminal methylene, and an oxymethylene group. The extract. The antimicrobial activity of the four extracts, aforementioned evidence coupled with comparison of the including n-hexane, chloroform, ethyl acetate and NMR data of 2 to those in the literature (Akihisa et al., methanol-water 80:20 (v/v) were studied against four 2005) indicate that 2 was (23E)-5β,19-epoxycucurbita- pathogenic bacterial and four fungal strains. Antibacterial 6,23,25-trien-3β-ol. Academia Journal of Medicinal Plants; Nguyen et al. 166

Figure 1: Structures of isolated compounds from MCA.

Compound 3 appeared as white amorphous powder and were the downfield shift of C-3 (73.8) and the upfield shift an ion peak at m/z 413.45 [M + H]+ in ESI-MS. The of C-2 (29.3) and C-4 (38.9) in the 13C-NMR spectrum of 4 13 molecular formula was assigned as C29H48O based on C- indicating the sugar unit was bonded at C-3 of clerosterol. NMR data (C×29) and the molecular ion peak in ESI-MS. Moreover, the observed fragmentation patterns at m/z 1H-NMR exhibited the presence of two tertiary methyls at  255.49 and 610.98 in APCI-MS spectrum indicated the long 0.79 (3H, s) and 0.53 (3H, s), an allylic methyl at  1.57 (3H, chain fatty acid and glucoside part of 4, respectively s), a secondary methyl at  0.91 (3H, d, 6.5), a primary (Figure 2). On the basis of the aforementioned evidences, methyl at  0.80 (3H, t, 7.5), an oxygenated proton at  3.59 the structure of 4 was determined as 3-O-(6′-O-Palmitoyl- (1H, m), terminal methylene protons at  4.73 (1H, m) and β-D-glucopyranosyl)-clerosterol. 4.64 (1H, d, 2.5), and an olefinic proton at  5.15 (1H, m). The 13C-NMR spectra of 3 showed the presence an oxygenated carbon at  71.1 and four olefinic carbons at  Conclusions 111.4, 117.5, 139.6 and 147.6. The spectral data were similar to those in literature suggesting that compound 3 Primary screening of antimicrobial activity of four extracts was 5α-poriferasta-7,25-dien-3β-ol (Garg and Nes, 1986). (n-hexane, chloroform, ethyl acetate and methanol-water) Compound 4 was obtained as colorless paraffin. The from the fruits of Momordica charantia L. var. abbreviata characteristic signals of a fatty acid chain were observed Ser. showed that the chloroform extract possessed the with a terminal methyl group at  0.88 (3H, t, 6.5), a highest activity with the MIC value 100 μg/ml against A. niger, and 200 μg/ml against two micro-organisms E. coli methylene linking carbonyl at  2.32 (2H, t, 7.5), and and B. subtillis. In this paper, four compounds were multiple methylene groups in the 1H-NMR spectrum. The isolated and structures elucidated for the first time in such 1H-NMR spectrum also showed the signals corresponding species including octadecan-1-ol (1), (23E)-5β, 19- to a sugar moiety at  4.37 (1H, d, 7.5, H-1'), 3.34 (1H, t, 8.0, epoxycucurbita-6, 23, 25-trien-3β-ol (2), 5α-poriferasta- H-2'), 3.53 (1H, t, 9.0, H-3'), 3.37 (1H, t, 9.0, H-4'), 3.51 (1H, 7,25-dien-3β-ol (3) and 3-O-(6′-O-palmitoyl-β-D- m, H-5') and 4.33 (2H, brs, H-6'). The 13C-NMR spectrum glucopyranosyl)-clerosterol (4). showed the signals for a sugar moiety at  101.3, 73.4, 76.2, 70.4, 79.7 and 63.6. The glucose unit was linked to fatty acid chain at ACKNOWLEDGMENT position 6' due to HMBC correlation between H-6' and C-1'' as well as, the downfield shift of H-6' in glucose unit. The The authors are grateful to Dr. Dang Minh Quan, NMR data of an aglycon unit in 4 were very similar to those Department of Biology, School of Education, Can Tho of clerosterol (Kwon et al., 2003) but major differences University for the plant identification. Academia Journal of Medicinal Plants; Nguyen et al. 167

Figure 2: The fragmentation patterns in MS spectrum of 4

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