Chemical constituents of the Moringa oleifera LAM. leaves collected in Bac Ninh province

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Study on the chemical constituents of methanol extract from leaves of Moringa oleifera Lam. (Moringaceae) has resulted in the isolation of four compounds: Vitexin, isoquercitrin, tricosanol and tetradecanoic acid. Their structures were determined by spectroscopic methods.

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Nội dung Text: Chemical constituents of the Moringa oleifera LAM. leaves collected in Bac Ninh province

JOURNAL OF SCIENCE OF HNUE DOI: 10.18173/2354-1059.2016-0051 Natural Sci. 2016, Vol. 61, No. 9, pp. 21-25 This paper is available online at http://stdb.hnue.edu.vn CHEMICAL CONSTITUENTS OF THE Moringa oleifera LAM. LEAVES COLLECTED IN BAC NINH PROVINCE Nguyen Thi Tam, Do Thu Huong and Pham Huu Dien Faculty of Chemistry, Hanoi National University of Education Abstract. Study on the chemical constituents of methanol extract from leaves of Moringa oleifera Lam. (Moringaceae) has resulted in the isolation of four compounds: vitexin (1), isoquercitrin (2), tricosanol (3) and tetradecanoic acid (4). Their structures were determined by spectroscopic methods. Keywords: Moringa oleifera, leaves, vitexin, isoquercitrin. 1. Introduction Moringa is the sole genus in the small flowering plant family Moringaceae, which contains 13 species from tropical and subtropical climates. Vietnam has only one species of this genus, Moringa oleifera. It is a small or medium-sized, about 10 m-high tree native to India and cultivated all over the country these days [1]. Moringa oleifera is considered one of the world’s most useful trees: its roots are useful for the treatment of asthma, gout, lumbago, rheumatism, enlarged spleenor liver, meanwhile its leaves, and flowers are used as a nutritive vegetable [2-5]. Chemical study revealed that all parts of the plant contained steroids, flavonoids, triacylglycerols, and monoterpenes [2-5]. Among them, several biological activities including anti-microbial, anti- hypertensive [5, 6], anti-tumor promoter [2, 3], and anti-inflammatory [7] activities have been reported. This paper describes the isolation but structural elucidation of four compounds of leaves of Moringa oleifera Lam., which were collected in Bac Ninh Province of Vietnam. 2. Content 2.1. Materials and methods * Plant material Leaves of Moringa oleifera Lam. were collected in Bac Ninh Province in November, 2014 and identified by Mr. Do Huu Thu, PhD. (Institute of Ecology, Natural Resources and Biology, VAST, Vietnam). Voucher specimens are currently deposited at Faculty of Chemistry, Hanoi University of Education (TA201411). Received October 10, 2016. Accepted November 25, 2016. Contact Pham Huu Dien, e-mail address: dienph@hnue.edu.vn 21
Nguyen Thi Tam, Do Thu Huong and Pham Huu Dien * General procedure TLC was carried out on pre-coated Si gel GF254 (Merck Co., Germany), and TLC spots were viewed at 254,302 and 366 nm and visualized by spraying vanillin-10% H2SO4 solution. Column chromatography was carried out on silica gel 60 (60-100 M, Merck). NMR (1H, 13C NMR, DEPT, HSQC and HMBC) spectra were recorded on a Bruker Avance 500MHz. The chemical shift (δ) values were given in ppm with TMS at internal standard, coupling constant J - by Hz. Mass spectra, including high resolution MS were recorded on a HP 5989B mass spectrometer and FT-ICR-MS (Varian 910-MS TQFTMS-7 Tesla). * Extraction and Isolation Dried powder of Moringa oleifera leaves (3,000 g) was extracted with methanol. The methanolic extract was condensed to give a residue (350 g) which was further partitioned into n- hexane, EtOAc, BuOH and water. The ethyl acetate crude extract (14.5 g) was subjected to column chromatography over silica gel and eluted gradient with n-hexane - ethyl acetate from 4:1 to 1:1, ethyl acetate - methanol from 10:1 to 0:10. Eight fractions were successively obtained. Fraction 3 (125 mg) was separated by column chromatography (SiO2; n-hexane - ethyl acetate) to afford 1 (32 mg); fraction 7 (120 mg) - to afford 2 (25 mg). The n-hexane crude extract (10.5 g) was subjected to column chromatography over silica gel and eluted gradient with n-hexane - ethyl acetate from 10:1 to 1:1. Four fractions were obtained. Fraction 2 (185 mg) were precipitated white powder, after its crystallization to afford 3 (26 mg). Fraction 3 (78 mg) was separated by column chromatography (SiO2; n-hexane - ethyl acetate) to afford 4 (5 mg). Compound 1: light yellow needles; ESI-FTICR-MS: m/z [M+H]+ calcd for C21H21O10: 433.11347; found 433.11014. 1H NMR (500 MHz, DMSO-d6):  13.2 (1H, s, OH-5), 8.02 (2H, d, J = 8.5 Hz, H-2’, H-6’), 6.89 (2H, d, J = 8.5 Hz, H-3’, H-5’), 6.77 (1H, s, H-3), 6.21 (1H, s, H-6), 4.68 (1H, d, J = 9.5 Hz, H-1”), 3.84 (1H, t, J = 9.0 Hz, H-2”), 3.76 and 3.53 (2H, m, H-6”), 3.26 (1H, m, H-5”), 3.35 (1H, m, H-3”), 3.34 (1H, m, H-4”). 13C NMR (125 MHz, DMSO-d6): 182.1 (C-4), 163.9 (C-2), 162.5 (C-7), 161.1 (C-5), 160.1 (C-4’), 156.0 (C-9), 128.9 (C-2’, 6’), 121.6 (C-1’), 115.8 (C-3’, 5’), 104.6 (C-8), 104.0 (C-10), 102.4 (C-3), 98.1 (C-6), 81.8 (C-5”), 78.7 (C- 3”), 73.4 (C-1”), 70.8 (C-2”), 70.5 (C-4”), 61.3 (C-6”). Compound 2: light yellow needles; ESI-FTICR-MS: m/z [M+H]+ calcd for C21H21O12: 465.10331; found 465.10172. 1H NMR (500 MHz, CD3OD):  7.73 (1H, d, J =1.5 Hz, H-2’), 7.59 (1H, dd, J = 8.5 2.0 Hz, H-6’), 6.89 (1H, d, J =6.5 Hz, H-5’), 6.41 (1H, d, J =1.5 Hz, H-8), 6.22 (1H, d, J = 1.5, H-6), 5.26 (1H, d, J = 7.5 Hz, H-1”), 3.73 and 3.60 (2H, m, H-6”), 3.51 (1H, t, J = 7.0 Hz, H-2”), 3.45 (1H, t, J = 8.5 Hz, H-3”), 3.37 (1H, t, J = 8.5 Hz, H-4”), 3.25 (1H, m, H-5”). 13 C NMR (125 MHz, CD3OD): 179.5 (C-4), 166.0 (C-7), 163.0 (C-5), 159.0 (C-2), 158.5 (C-9), 149.8 (C-4’), 145.9 (C-3’), 135.6 (C-3), 123.2 (C-1’), 123.1 (C-6’), 117.6 (C-2’), 116.0 (C-5’), 105.7 (C-10), 104.4 (C-1”), 99.9 (C-6), 94.7 (C-8), 78.4 (C-5”), 78.1 (C-3”), 75.7 (C-2”), 71.2 (C- 4”), 62.6 (C-6”). Compound 3: LC-MS: m/z 341.2 [M+H]+. 1H NMR (500 MHz, CDCl3): H 3.64 (2H, dd, J = 10.5 6.0 Hz, H-1), 1.56 and 1.25 (42H, m, H-222), 0.88 (3H, t, J = 7.0 Hz, H-23) 13C NMR (125 MHz, CDCl3): 63.1 (C-1), 32.8, 31.9, 12x 29.71, 29.67, 29.62, 29.61, 29.45, 29.37, 25.75, 22.70 (21C, C-222), 14.1 (C-23). 22
Chemical constituents of the Moringa oleifera Lam. leaves, collected in Bac Ninh Province Compound 4: EI-MS: m/z 228.0 [M]+. 1H NMR (500 MHz, CDCl3): H 2.35 (2H, t, J = 7.5 Hz, H-2), 1.25-1.30 (22H, m, H-313), 0.88 (3H, t, J = 7.0 Hz, H-14). 13C NMR (125 MHz, CDCl3): 178.3 (C-1), 33.8, 31.9, 29.7, 29.6, 29.5, 29.4, 2x29.3, 2x29.1, 24.7, 22.7 (12C, C-213), 14.1 (C-14). 2.2. Results and discussion Compound 1 was also obtained from ethyl acetate extract of Moringa oleifera leaves. Its molecular formula was identified as C21H20O10 by FT-ICR-MS. Its 1H NMR spectrum has signals of five aromatic protons at 6.27 (1H, s), 6.89 (2H, d, J = 8.5 Hz), 8.02 (2H, d, J = 8.5 Hz); one olefinic proton at 6.77 (1H, s); six C-glycoside protons from 3.34 to at 4.69, suggesting that 1 should be a flavonoid C-glycoside. In addition, its 13C NMR spectrum shows the presence of 21 carbons. Compound 1 has very similar spectral data with those of vitexin [8]. Therefore, compound 1 was determined as vitexin. Vitexin has recently received increasing attention due to its wide range of pharmacological effects, including anti-oxidant, anti-cancer, anti-inflammatory, anti-hyperalgesis, and neuroprotective effects [9]. The molecular formula of compound 2 was found to be C21H20O12 by FT-ICR-MS. Analyses of its 1H NMR spectra revealed that it had five aromatic protons at 7.73, 7.59, 6.89 6.41 and 6.22 ppm; seven O-glycoside protons from 3.25 to 5.26 ppm, suggesting that 2 should be an O- glycoside flavonoid. The 13C NMR spectrum had resonances of 21 carbons. Compound 2 had very similar spectral data with those of isoquercitrin [10]. Therefore, compound 2 was determined as isoquercitrin. According to [11], isoquercitrin could inhibit the progression of pancreatic cancer in vivo and in vitro by regulating opioid receptors and signaling pathway of mitogen-activated protein kinase. OH OH 1' 6" HO O HO 2 OH OH 2' OH OH 6 O 2' HO O 1" OH 1" HO O 1' OH O 2 O OH 6 3 HO OH O 1 2 Figure 1. Structures of compounds 1 and 2 23
Nguyen Thi Tam, Do Thu Huong and Pham Huu Dien From the LC-MS, data of 3 was afforded m/z 341 [M+H]+, and founded molecular mass which was 340 corresponding to a molecular formula of C23H48O. The 1H NMR signals indicated the presence of 44 methylenic protons at 3.64 (2H, dd, J = 10.5 6.0 Hz, H-1), 1.56 and 1.25 (42H, m, H-222), and three methyl protons at 0.88 (3H, t, J = 7.0 Hz, H-23). It suggested that 3 should be an aliphatic alcohol. The 13C NMR spectrum evealed 3 has 23 carbons, mainly resonanced in the strongly- shielded field, except methyleneoxy carbon with C value of 63.1ppm. The mass, 1H and 13C NMR spectral data identified 3 as tricosanol, CH3[CH2]22OH. From the EI-MS, data of 4 was afforded m/z 228 [M]+, corresponding to a molecular formula of C14H28O2. The 1H NMR signals indicated the presence of 24 methylenic protons at 2.35 (2H, t, J = 7.5 Hz, H-2), 1.25-1.30 ppm (22H, m, H-313), and three methyl protons at 0.88 (3H, t, J = 7.0 Hz, H-14). It suggested that 4 should be an aliphatic acid. The 13C NMR spectrum evealed 4 has 14 carbons, mainly resonanced in the strongly-shielded field, from 14.1 to 33.8 ppm, except onecarboxylic carbon with C value of 178.3 ppm. The mass, 1H and 13C NMR spectral data identified 4 as tetradecanoic acid, CH3[CH2]12COOH. 3. Conclusion The chemical composition of methanol extract of the leaves of Moringa oleifera collected in Bac Ninh Province, has been carefully studied. Four compounds, vitexin (1), isoquercitrin (2), tricosanol (3) and tetradecanoic acid (4) were isolated and structurally elucidated by MS, 1D and 2D NMR spectroscopies. Acknowledgement: Authors would like to thank Dr. Nguyen Thi Thu Vinh (Duong Malt Co.) for supplying the plant sample, Dr. Do Huu Thu (Inst. of Ecol. and Biol. Resources, VAST) for identifying the sample. REFERENCES [1] Vo Van Chi, 1999. Dictionary of Vietnamese medicinal plants. Pharmac. Publ. House, p. 248. [2] Guevara A. P., Vargas C., Sakurai H., Fujiwara Y., Hishimoto K., Maoka T., Kozuka M., Ito Y., Tokuda H., Nishino H., 1999. An antitumor promoter from Moringa oleifera Lam. Mutat Res. Fundam. Mol. Mech. Mutagen, 440, pp. 181-188. [3] Consolacion Y. R., Ruel M. L., Don M. J., Shen C. C., 2012. Cytotoxic isothiocyanates from Moringa oleifera Lam seeds, Phillippine Sci. Lett., Vol. 5, No. 1, pp. 46-52. [4] Fuglie I. J, 1999. The Miracle Tree: Moringa oleifera: natural Nutrition for the Tropics. Church World Service, Dakar, p. 66. [5] Faizi S., Siddiqui B.S, Saleem R., Siddiqui S., Aftab K., Shaheen F., Gilani A. H., 1994. Novel hypotensive agents, niazimin A, niazimin B, niazicin A and niazicin B from Moringa oleifera: isolation of first naturally occurring carbamates. J. Chem. Soc. Perkin Trans., No. 1, pp. 3035-3040. [6] Faizi S., Siddiqui B. S., Saleem R., Aftab K., Shaheen F., Gilani A. H., 1998. Hypotensive constituents from the pods of Moringa oleifera. Planta Med., Vol. 64, pp. 225-228. 24
Chemical constituents of the Moringa oleifera Lam. leaves, collected in Bac Ninh Province [7] Cheenpracha S., Park E. J., Yoshida W. Y., Barit C., Wall M., Pezzuto J. M., Chang L.C., 2010. Potential anti-inflammatory phenolic glycosides from the medicinal plant Moringa oleifera fruits. Bioorg. Med. Chem., Vol. 18, pp. 6598-6602. [8] Omayma AE et al., 2009. Potential antioxidant phenolic metabolites from doum palm leaves. African J. of Pharm. and Pharmalogy, Vol. 47, No. 2, pp. 158-164. [9] He M., Min J. M. et al., 2016. A review on the pharmacological effects of vitexin and isovitexin. Fitoterapia, No. 16, pp. 3044-3048. [10] Estork D. M., Gusmao D.F. et al., 2014. First chemical evaluation and toxicity of Casinga- cheirosa to balb-c male mice. Molecules, Vol. 19, pp. 3973-3978. [11] Chen Q., Li P. et al., 2015. Isoquercitrin can inhibit the progression of pancreatic cancer in vivo and in vitro by regulating opioid receptors and the mitogen-activated protein kinase signaling pathway. Oncol Rep., Vol. 33, No. 2, pp. 840-848. 25