Flavonol glycosides from Castanea mollissima Blume

Transcription

1 Flavonol glycosides from Castanea mollissima Blume Dongsong Zhang a, uiyuan Gao a, Libo Wang a, Dan Li a, Masonori Kuroyanagi b, Lijun Wu a* a. School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang , China b. School of Bioresources, 560 Nanatsuka, Shobara, iroshima Prefectural University, iroshima , Japan Abstract Three flavonol glycosides, kaempferol-3--[6"--(e)-p-coumaroyl]-β-d-galactopyranoside (1), kaempferol-3--[6"--(e)-pcoumaroyl]-β-d-glucopyranoside (2) and kaempferol-3--[6", 2"-di--(E)-p-coumaroyl]-β-D-glucopyranoside (3), were isolated from the seeds of Castanea mollissima Blume. Their structures, especially the sugar units, were elucidated based on the spectroscopic evidence, chemical reactions and CD experiments. Compounds 1 and 3 were isolated from Castanea for the first time. Compound 2 was isolated from this plant for the first time. Key words: Castanea mollissima Blume; flavonol glycosides. Introduction Castanea molissima Blume is a perennially woody plant and widely cultivated in Europe, North American and Asia (especially in China) as an economic crop. Its sweet kernels are edible and it has also been used to treat gastroenteritis, bronchitis, and regurgitation in traditional Chinese medicines [1]. According to the latest research, it exhibits anti-inflammatory, styptic, anti-diarrhea, and analgesic effects [2]. In addition, the presence of flavonol glycosides, steroids, phenolic acids, lignans, alkaloids, proteins and other constituents has been reported mainly in its flowers [3-11]. In the search for new bioactive agents, our chemical studies on the seeds of C. molissima led to the isolation of kaempferol-3--[6"--(e)-pcoumaroyl]-β-d-galactopyranoside (1), kaempferol-3- -[6"--(E)-p-coumaroyl]-β-D-glucopyranoside (2) and kaempferol-3--[6", 2"-di--(E)-p-coumaroyl]-β- D-glucopyranoside (3) (see Fig. 1). In this paper we report the isolation and structural elucidation of these compounds. It was necessary to develop a process for the isolation of compounds 1 and 2 before starting our study, because they were identified as one compound from the single spot shown on TLC. Whereas, a small difference in the NMR spectral data of the sugar units indicated that it was a mixture of two compounds in a ratio of 3:1. The two compounds were successfully isolated by PLC (DS) using the solvent system C 3 CN- 2 -CF 3 C (27:73:0.1) and were identified as a D-galactoside and a D-glucoside linked with the same aglycone and with same substitution patterns from their NMR spectral data and CD experiments. Materials and methods * Author to whom correspondence should be addressed. Address: School of traditional Chinese materia medica, Shenyang pharmaceutical university, 103 Wunhua Road, Shenyang , China; Tel: ; wulijun_111@hotmail.com Received: Accepted: General 1 NMR (600 Mz) and 13 C NMR (150 Mz) spectra were recorded on a Bruker-DRX spectrometer 197

2 R 1 R 1 R 2 1 R 1 = 7'" 9'" 8'" 2'" 1'" 6'" 3'" 4'" 5'" 2 R 1 = 3 R 1 = 9'" 8'" 9'" 8'" 7'" 7'" 1'" 1'" 4'" 4'", R 2 =, R 2 = 7'''' 1'''' 9'''' 8'''' 4'''' Fig. 1. Structures of Compounds 1-3 using pyridine-d 5 or DMS-d 6 as solvents and TMS as an internal standard. 2D-NMR were recorded under standard conditions. The ESI-MASS spectra were recorded on an LC-MSD-Trap-SL mass spectrometer. R-FAB-MS data were recorded on a JEL X110 mass spectrometer. Melting points were recorded on Yanaco MP-3 micromelting point apparatus. ptical rotations were measured with a JASC P-1010 polarimeter. PLC separation was carried out on reverse phase columns (mighty sil RP-18 and 8, Kantho Chemical Co. Ltd) with C 3 CN- 2 and C 3 CN- 2 -CF 3 C as the mobile phases. Silica gel ( mesh) for column chromatography and silica GF 254 for TLC were obtained from Qingdao Marine Chemical Company, China and Merck Company, respectively. Sephadex L-20 was purchased from Pharmacia Biotech, Sweden. Plant material Seeds of Castanea mollissima Blume were collected in September 2005 in Qianxi County in the ebei province of China and identified by Prof. Qishi Sun (Shenyang Pharmaceutical University). The voucher specimen was deposited in the Department of Traditional Chinese Medicines of Shenyang Pharmaceutical University. Extraction and Isolation Powdered kernels of Castanea mollissima Blume (10 kg) were extracted three times with 90 % Et under reflux. Evaporation of the solvent under reduced pressure gave an extract (1600 g) which was suspended in water and partitioned with hexane, ethyl acetate and n-butanol, successively. Solvents were evaporated to yield the hexane (102.2 g), ethyl acetate (42.7 g), n-butanol (163.1 g) and aqueous ( g) extracts. The n-bu extract (100.8 g) was subjected to silica gel column chromatography (1 kg, eluted with CCl 3 and Me in increasing polarity to obtain ten fractions). Fr.6 (3.2 g) was subjected to column chromatography on silica gel and eluted with a CCl 3 Me 2 gradient system to obtain fifty fractions and the resultant subfractions were further purified by Sephadex L-20 to yield 27 mg of a mixture of compounds 1 and 2 and compound 3 (14 mg). Isolation of the mixture (18 mg) was carried 198

3 out by PLC using the solvent system C 3 CN 2 CF 3 C (27:73:0.1) to obtain compound 1 (4.2 mg) and 2 (11.8 mg). Result and discussion Compound 1 was obtained as a yellow amorphous solid (Me), m.p and gave a positive reaction to the Molish and Cl-Mg tests, which indicated it was a flavonoid glycoside. The molecular formula C was determined on the basis of the quasi-ion peak [M+] + at m/z (Calcd for C ) in R-FAB-MS. Its 1 NMR spectrum (600 Mz, pyridine-d 5 ) showed the presence of one (trans)-p-coumaroyl moiety [δ 6.49 (1, d, J=15.6 z), 7.84 (1, d, J=15.6 z), 7.16 (2, d, J=8.5 z), 7.52 (2, d, J=8.5 z)]. Two aromatic proton signals, δ 6.71 (1, br s), 6.72 (1, br s) were ascribed to -6 and -8 of the A-ring of a flavone with the 5,7-dihydroxy substitution patterns. Meanwhile, proton signals at δ 7.18 (2, d, J=8.4 z), 8.49 (2, d, J=8.4 z) were assigned to -3',5' and -2',6' of the B-ring, respectively (Table 1). The aglycone of 1 was identified as kaempferol [12]. In addition, δ 6.14 (1, d, J=7.8 z) was the anomeric signal of the sugar moiety. Twenty-six signals were exhibited in the 13 C NMR spectrum. Among of them, the very intense signal at δ C 116.1, 116.8, 130.7, and almost certainly represent two carton atoms. The signals at δ C 105.0,74.7,73.1,70.8,69.9 and 64.4 were assigned to C-1"-6" of the sugar unit. Acid hydrolysis of 1 with 10 % Cl/Me (v/v, 10 ml) resulted in the production of methyl--(α, β)-galactoside, which was transformed into [tetra- (4-bromobenzyli)]-methyl--(α, β)-galactoside by chemical reaction and isolated by PLC. The absolute configuration of galactose was determined as the D configuration by CD analysis of this derivative [12]. Its anomeric configuration was determined to be the β form on the basis of the coupling constant (J=7.8 z) for -1". The galactose connected at the C-3 position of the kaempferol was identified by the correlation between the anomeric proton signal (δ 6.14) and C-3 (δ C 135.2), C-2" (δ C 70.8) in the MBC spectrum (see Fig. 2). Moreover, the location of the linkage of the (E)-p-comaroyl group to galactose was determined by the correlation between -6" (δ 4.82, 4.96) and C-9'" (δ C 167.2) in the MBC spectrum. Thus, the structure of 1 was determined as kaempferol-3--[6"--(e)-pcoumaroyl]-β-d-galactopyranoside [13]. Compounds 2 [14] and 3 [15] were identified as kaempferol-3--[6"--(e)-p-coumaroyl]-β-d-gluc -opyranoside and kaempferol-3--[6",2"-di--(e)-p- Fig. 2. The Key Correlations of Compound 1 in MBC 199

4 Table C NMR and 1 NMR Spectral Data of Compounds 1, 2, 3 (150 Mz, 600 Mz) 1 a 2 a 3 b No. δ δ C δ δ C δ δ C (br s) (over lap.) (d, 2.0) (br s) (over lap.) (d, 2.0) , (2, d, 8.4) (d, 8.4) (d, 8.8) , (2, d, 8.4) (d, 8.4) (d, 8.8) " 6.14 (d,7.8) (d, 7.6) (d, 8.0) " 3.64 (m) (over lap.) (over lap.) " 4.81 (over lap.) (over lap.) (over lap.) " 4.43 (br s) (m) (m) " 4.32 (m) (over lap.) (over lap.) " 4.82 (over lap.) 4.96 (dd, 7.2, 10.8) (dd, 11.4, 6.0) 5.00 (br d, 11.4) (over lap.), 4.05 (over lap.) 1'" '", 6'" 7.52 (2, d, 8.4) (2, d, 8.4) (2, d, 8.5) '", 5'" 7.16 (2, d, 8.4) (2, d, 8.4) (2, d, 8.5) '" '" 7.84 (d, 15.6) (d, 16.2) (d, 15.8) '" 6.49 (d, 15.6) (d, 16.2) (d, 15.8) '" "" "", 6"" 7.55 (2, d, 8.6) "", 5"" 6.79 (2, d, 8.6) "" "" 7.61 (d, 15.8) "" 6.43 (d, 15.8) "" coumaroyl]-β-d-glucopyranoside, respectively. Their molecular formulas were determined on the basis of ESI-MS and R-EI-MS experiments combined with the NMR spectral data (Table 1). The identification of glucose units in their structures was carried out by the same method as that of 1. Compared with those of 1, the signals of compound 2 in the 1 NMR and 13 C NMR spectra were almost at the same positions 200

5 except for a small difference in the sugar unit, and it was confirmed to be a glucose linked with kamperferol at C-3 position with the same substitution pattern to that of 1. n the basis of evidence from their 2D NMR spectra, the structures of compounds 2 and 3 were characterized as shown. Compound 1: amorphous, yellow powder. m.p ; [α] 2 1 D (c=0.035, Me); Positive-ion R-FAB-MS m/z [M+] + (Calcd for C ); 1 NMR, 13 C NMR spectral data see Table 1. Compound 2: amorphous, yellow powder. m.p ; [α] 2 D (c=0.05, Me); Positive-ion ESI-MS: [M+] + m/z 595.4, Negitive-ion ESI-MS [M-] - m/z NMR, 13 C NMR spectral data see Table 1. Compound 3: pale yellow powder. m.p ; [α] 2 D (c=0.05, Me); Positive-ion R- FAB-MS: [M+] + m/z (Calcd for C : ); 1 NMR, 13 C NMR spectral data see Table 1. Acid hydrolysis and Identification of Monosaccharides Compounds 1 (1.2 mg), 3 (2.0 mg) and the mixture (12 mg) of compounds 1 and 2 were dissolved in Me (9ml) containing Cl (1 ml) and refluxed on a heated (80 ) water bath for 3 h. After cooling, the reaction mixture was concentrated and dried under reduced pressure, then analyzed by TLC using the solvent system CCl 3 Me 2 (20:10:1) for identification of the complete reaction. The dried residues in pyridine (1ml) were treated with 4-bromobenzoyl chloride (10 mg) and stirred at room temperature for 8 hr. The reaction mixture was then poured into cool water and extracted with EtAc. The EtAc extract was successively washed with 3 % aqueous Cl, saturated aqueous NaC 3, then dried over anhydrous Na 2 S 4 powder and filtered. After removing the solvent from the filtrate, the residue was analyzed and subjected to PLC using an RP-8 column (Mighty sil, mm) with C 3 CN 2 (v:v=80:20, Flow: 1.0 ml/min) as the mobile phase and the UV detection at 250 nm. Identification of D-galactose was carried out by comparison of the retention time (24 min) of this derivative with that of authentic samples prepared in the same manner. The CD spectral data showed positive Cotton curves for these derivatives. Using the same method, the monosaccharide from 3 and 2 was identified as D-glucose (retention time, 26 min). Acknowledgment We gratefully acknowledge the financial support for this project from ebei Province Science Technology Program (2005). References [1] Zhang J, e YJ. Advances and development trends of Chinese chestnut research at home and abroad. World Forestry Research, 1999,12 (2): [2] Jiang JW. Dictionary of medicinal botanarys, Tianjin Scientific Publishing ouse, Tianjin, 2005, pp [3] Wang S, Tang WZ, Ding XB. Two new flavonoids from the flower of Castanea mollissima Blume. Acta. Pharm. Sini., 2004,39 (6): [4] Jin ZJ, Ge M. Chemical constitute and pharmacology of Castanea mollissima Blume. Shanxi Yiyao Zazhi, 2005,34 (9): [5] Wang S, Du CL, Tang WZ, Wang XJ, Sun MY,Ding XB. Study of chemical constituents of Castanea mollissima. Chin. Trad. erbal Drugs, 2004,35 (10): [6] Tang WZ, Ding XB, Xin YZ. A new lignan glycoside from the flower of Castanea mollissima Blume. Acta. Pharm. Sini., 2004,39 (7): [7] Alois, Samir K. A new pyrrole alkaloid from seeds of Castanea satia. Fitoterapia, 2002, 73: [8] Wang S, Ding XB,Chen Y. A new alkaloid from the flower of Castanea mollissima Blume. J. Asian. Nat. Prod. Res., 2000,3 (2): [9] Chu KT,Ng TB. Mollisin, an antifungal protein from the chestnut Castanea mollissima. Planta. Med., 2003,69 (9): [10] Ng TB, Yu Y, Chu KT. Isolation of a novel legumin-like lectin with potent hemagglutinating activity from seeds of the Chinese chestnut Castanea mollisima. Toxicol. & Pharmacol., 2002,133 (3): [11] Wang X, Ng TB. Purification of castamollin, a novel 201

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