Chemical constituents of zizyphus sativa gaertn fruits: III - Alkaloids

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Journal of Chemistry, Vol. 45 (2), P. 237 - 240, 2007 Chemical constituents of Zizyphus sativa Gaertn fruits III - Alkaloids Received 29 June 2006 1 Nguyen Thi Hoang Anh , Tran Van Sung1, Nobert Arnold2, L. Wessjohann2 1 Institute of Chemistry, Vietnamese Academy of Science and Technology 2 Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany Summary Two alkaloids have been isolated from Z. sativa fruits besides butane-2,3-diol and glycerol. The structures of alkaloids were established as stepharine and 1,2-dimethoxy,5,6,6a,7-tetrahydro4H-dibenzo-quinoline by using MS, NMR spectroscopic data and by comparison with reported data. I - INTRODUCTION Some species of Zizyphus genus (Rhamnaceae) have been used for treatment of biliousness, chronic bronchitis, blood diseases or as analeptic, expectorant [1]. Zizyphus species have been known as a rich source of cyclopeptide alkaloids [2, 3]. In a previous article [4] we reported the separation and structural elucidation of triterpene acids from Zizyphus sativa fruits. This paper concerns the isolation and structural determination of two alkaloids and two common compounds. Their structures have been elucidated as stepharine (1), 1,2-dimethoxy,5,6,6a,7-tetrahydro-4Hdibenzoquinoline (2), butane-2,3-diol (3) and glycerol (4) by using spectroscopic methods. II - EXPERIMENTAL 1. Plant material The fruits of Z. sativa were bought in the traditional medicine market in Hanoi, in May 2005. A voucher specimen Nr. H.11 was deposited in the Herbarium of Institute of Chemistry. 2. Instruments and Chemicals NMR: Varian Unity 300; MS: AMD 402; for analytical purposes: Merck TLC aluminium sheets silica gel 60 F254 (layer thickness 0.2 mm) were used. Silica gel Merck 60 (0.040 - 0.063 mm) is used for column chromatography. 3. Extraction and Isolation Air dried and powdered fruits of Z. sativa (1.43 kg) were extracted with EtOH : H2O (95 : 5) at room temperature. The obtained ethanol extract was dissolved in 5% HCl solution and then extracted with n-hexane. The aqueous phase was neutralized with NaHCO3 and then extracted with mixture of CHCl3 : C2H5OH (2 : 1). After removing organic solvents, 3.9 g of alkaloid fraction has been obtained. The alkaloid fraction was separated by chromatography on silica gel, eluting gradient with CHCl3 and MeOH to furnish 10 fractions, which were combined according to TLC monitoring. The combination of fractions 5, 6 and 7 (529 237 mg) was further purified over a flash silica gel column with CHCl3 : MeOH (95 : 5) as eluant to yield 7 fractions. The fourth fraction (93mg) was rechromatographed over a flash silica gel column, eluting with EtOAc : MeOH (45 : 10) to furnish 56 mg of compound 1. The combined fractions 8 and 9 (153 mg) were purified over a flash silicagel by using a solvent system of EtOAc : MeOH (45 : 10) as eluant to yield 9mg of compound 2. 21.5 g of BuOH extract was subjected in a dilation HP-20 column, eluted with water, MeOH : H2O (1 : 1) and then with MeOH. After evaporation of solvents, 20 g, 1.14 g and 0.27 g of extracts have been obtained, respectively. The fraction eluting with MeOH : H2O (1 : 1) (1.14 g) was purified over a sephadex LH-20 column, eluting with MeOH to obtain 8 fractions. The third fraction (257mg) was chromatographed over a flash silica gel column, eluting with CH2Cl2 : MeOH (90 : 10) to yield 28mg of 3 as mixture of two isomers 3a and 3b. 1 H-NMR (CDCl3): 3a: 1.15 d (6.04) (CH3), 3.50q (6.04) (CH); 3b: 1.12 d (6.4) (CH3), 3.78 q (6.4) (CH); 13C-NMR (CDCl3): 3a: 19.3 (CH3), 72.5 (CH); 3b: 16.9 (CH3), 70.8 (CH). The fraction eluting with H2O (20 g) was subjected in a flash silica gel column, eluting with CHCl3 and increasing amount MeOH to give 18 fractions. The combination of second and third fractions was rechromatographed over flash silica gel column, eluting with EtOAc : MeOH (90 : 10) to furnish 42 mg of 4. Positive ESI-MS: 115 [M+Na]+, 1H-NMR (CD3OD): 3.4 - 3.7 m, 13C-NMR (CD3OD): 73.8 (CH), 64.3 (CH2). III - RESULTS AND DISCUSSION The alkaloid fraction has been obtained from ethanol extract of Z. sativa fruits by usual method. Chromatographic separation of this fraction yielded two compounds. Compound 1 indicated positive reaction with Dragendorff reagent. It has a molecular ion peak at m/z 298 [M + H]+ in the positive ESI-MS spectrum, corresponding to the molecular formula C18H19NO3. It seems that compound 1 contains 238 one nitrogen atom in the molecule, because its molecular weight was odd. Its 1H-NMR spectrum revealed signals of 18 protons, among them five resonanced in the olefinic and six in aliphatic ranges. Besides these signals, twosinglet of methoxy groups at H 3.79, 3.58 and one signal of a heterogenated methine group at 13 H 4.38 have also been observed. The C-NMR spectrum of 1 indicated 19 carbons, containing 10 aromatic (110 - 160 ppm), one carbonyl at C 188.1, one heterogenated methine at C 58.4, two methoxy, three methylene and a quaternary carbons. The NMR and MS spectroscopic data suggested that 1 has one NH group in the molecule, but it did not appear in the 1H-NMR spectrum, because measured in CD3OD. Finally, the structure of 1 was determined as stepharine by careful comparison of these data with the published data [5]. It is well known compound from Stephania species [6], but it was found in Z. sativa for the first time. Compound 2 give a positive reaction with Dragendorff reagent. It revealed a molecular ion peak at m/z 282 [M + H]+ in the positive ESI-MS spectrum. This suggested that the molecule 2 possess one nitrogen atom, corresponding to the molecular formula C18H19NO2. The 1H- and 13CNMR spectra showed signals of two methoxy groups ( H 3.61, 3.86 and C 56.4, 60.5), one nitrogenated methine group at H 3.75, C 54.6. Besides these, three methylene groups resonanced in the range from 2.1 to 3.5 ppm in 1 H-NMR and at C 29.1, 37.6, 43.7 in the 13CNMR spectrum. Furthermore, 5 aromatic protons and 12 aromatic carbons have also been observed. The spectroscopic data of compound 2 are in good agreement with those of 1,2dimethoxy,5,6,6a,7-tetrahydro-4Hdibenzoquinoline in [8, 9]. This compound was isolated previously from Liriodendron tulipiferum [8], but until now it was not found in Z. sativa. From butanol extract of Z. sativa two common compounds were isolated. They are butane-2,3-diol (3) and glycerol (4). Compound 3 was obtained as a mixture of two isomers with a ratio of about 3 : 1 by the integral of proton signals in 1H-NMR spectrum. The positive ESIMS spectrum of 3 showed a molecular ion peak at m/z 113 [M + Na]+, giving the molecular formula C4H10O2. Its 1H and 13C-NMR spectra indicated two sets of signals with identical coupling patterns but slightly different chemical shifts, supporting the assumption that 3 contains two isomers. The signals contain two methyl groups at H 1.12 d (6.4), 1.15 d (6.04) and C MeO 2 1 3 1b 7a 11 6a 1 NH 4 3a 5 1b 1a MeO 11a 7 11 H 7 7a 10 O NH 6a H 8 10 3 2 5 MeO 12 MeO 4 3a 1a 16.9, 19.3 and two methine groups, which coupled with methyl [ H 3.50 q (6.04), 3.78 q (6.4) and C 70.8, 72.5]. The NMR and MS data revealed that 3 is butane-2,3-diol. The coupling constants suggested that the signals H 1.15 and 3.50 belong to an isomer and H 1.12, 3.78 belong to the other. The assignment (see experimental) was confirmed by comparison with the published data [10]. 8 9 Stepharine (1) 1,2-dimethoxy,5,6,6a,7-tetrahydro4H-dibenzoquinoline (2) OH OH HO HO Butan-2,3-diol 3a (3) H2C OH HC OH H2C OH 3b Glycerol (4) Table 1: 1H- and 13C-NMR data of compound 1, 2 (CD3OD, 75 MHz) Position 1 2 1 13 1 13 (1) (2) (3) (4) (5) 1 - 145.4 (C) - 145.2 (C) 1a - 135.1 (C) - 127.4 (C) 1b - 129.2 (C) - 129.9 (C) 2 - 154.9 (C) - 153.6 (C) 3 6.79 s (1H) 113.4 (CH) 6.77 s (1H) 113.0 (CH) 4 2.81 m (2H) 26.4 (CH2) 2.6 - 3.0 m 29.1 (CH2) 5 3.15 m (1H) 3.41 m (1H) 45.5 (CH2) 2.6 - 3.0 m 43.7 (CH2) 6a 4.38 dd (6.5, 10.0) 58.4 (CH) 3.75 dd (4.5, 13.7, 1H) 54.6 (CH) H C H C 239 (1) (2) (3) (4) (5) 7 2.28 dd (10.5, 12.0) 2.39 dd (6.5, 12.0) 48.1 (CH2) 2.6 - 3.0 m 37.6 (CH2) 7a - 52.7 (C) - 136.9 (C) 8 7.03 dd (2.9, 9.9) 152.7 (CH) 8.29 d (7.9, 1H) 129.2 (CH) 9 6.38 dd (1.9, 9.9) 128.7 (CH) 10 - 188.1 (C=O) 7.20 - 7.28 m (3H) 128.8 (CH) 11 6.26 dd (1.9, 9.9) 127.7 (CH) - 128.5 (CH) 11a - - - 133.1 (C) 12 7.20 dd (2.9, 9.9) 156.1 (CH) - - 1-OMe 3.58 s (3H) 61.3 3.61 s (3H) 60.5 2-OMe 3.79 s (3H) 56.7 3.86 s (3H) 56.4 REFERENCES 1. A. Yagi, N. Okamura, Y. Haraguchi, K. Noda, I. Nishioka. Chem. Pharm. Bull., 26, No. 6, P. 1798 - 1802 (1978). 2. A. H. Shah, V. B. Pandey, G. Eckhardt and R. Tschesche. Phytochemistry, 23, 931 (1984). 3. A. H. Shah, V. B. Pandey, J. P. Singh, K. N. Singh and G. Eckhardt. Phytochemistry, 23, 2120 (1984). 4. Nguyen Thi Hoang Anh, Tran Van Sung and L. Wessjohann. Journal of Chemistry, Vol. 44, No. 6, P. 787 (2006). 240 127.9 (CH) 5. T. T. Thuy, T. V. Sung, K. Franke, L. Wessjohann. Journal of Chemistry 43, No. 5, P. 619 - 623, (2005). 6. B. Charles, J. Bruneton, K. Pharadai, B. Tantisewie, H. Guinaudeau, M. Shamma. Journal of Natural Products, 50, No. 6, P. 1113 - 1117 (1987). 7. S. M. Kupchan. Tetrahedron 19, P. 227 232 (1963). 8. H. Achenbach, C. Renner, I. AddaeMensah, Liebigs Ann. 1623 (1982). 9. Chem. Ber, 1991, 124, P. 219 (ACDCNMR).
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