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Journal of Chemistry, Vol. 42 (2), P. 261 - 264, 2004 Further constituents from Ophiopogon japonicus Received 26-9-2003 Nguyen Thi Hoang Anh1, Tran Van Sung1, Ludger Wessjohann2 1 Institute of Chemistry, Vietnamese Academy of Science and Technology Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle/Saale, Germany 2 SUMMARY Four fructofuranosides, borneol and bornyl-7-O- -D-apio-D-furanosyl-(1 6)- -Dglucopyranoside were obtained from the butanol extract of the dried roots of Ophiopogon japonicus. Their structures were elucidated by MS and NMR spectroscopic data. I - Introduction In the preceding paper [1] we reported the isolation and structural elucidation of six steroidal glycosides from the butanol extract of the roots of O. japonicus Ker-Gawler (Liliaceae). It is an evergreen perennial and widely used in traditional Chinese medicine [2]. In Vietnamese folk medicine it serves as expectorant, anticough and tonic agent [3]. In continuation of our research on O. japonicus, we now report the isolation and structure elucidation of methyl-, ethyl-, n-butyl- -D-fructofuranosides (1 - 3), n-butyl- D-fructofuranoside (4), borneol (5) as well as bornyl-7-O- -D-apio-D-furanosyl- (1 6) - -Dglucopyranoside (6). II - Results and discussion The dried roots of O. japonicus were extracted with hot ethanol. The ethanol extract has been treated further by extraction with n-hexane, ethyl acetate and n-butanol, respectively. After further chromatographic separation of the butanol extract four fructo-furanosides were obtained. The 1H and 13C-NMR spectra of compounds 1, 2, 3 and 4 are very closely. Their 13C-NMR spectra indicated in all cases oxygenated quartenary carbons at 109.2, 108.9, 108.7 and 105.1 for compounds 1, 2, 3 and 4, respectively. Furthermore, three hydroxylated methine signals in the range from 77 ppm to 85 ppm and two hydroxy-lated methylene signals at about 61 - 62 ppm have been always seen in the 13C-NMR spectra of these compounds. The above analysis suggested that they contained a fructofuranosyl unit. Compound 1 showed besides fructofuranosyl signals, one methoxyl group at 48.9 ppm in the 13 C-NMR spectrum. The sugar signals were similar to those of the sugar moiety of methyl- D-fructofuranoside [4, 5]. Therefore, compound 1 was identified to be methyl- -D-fructofuranoside. In the 13C-NMR spectra of compound 2, a methyl signal at 16.0 ppm and a oxygenated methylene group at 57.6 ppm have been observed. The chemical shift of the fructo carbons were almost the same as those of the sugar signals of methyl- -D-fructofuranoside [4, 5]. Consequently, compound 2 could be elucidated as ethyl- -D-fructofuranoside. Compounds 3 and 4 exhibited the same molecular ion peaks in the EI mass spectra at m/z 236 [M]+ and the fragment peaks at m/z 205 [M-CH2OH]+, 163 [M-OC4H9]+, 73 [OC4H9]+ and 57 [C4H9]+. These fragments combined with their 1 H-NMR [0.92 t (7.3), 1.4 m, 1.53 m, 3.25 m] 261 and 13C-NMR data 14.2, 20.4, 33.3, 61.6 revealed the presence of a n-butoxyl moiety in both compounds. The sugar signals of compounds 3 ( 60.2, 61.2, 76.7, 81.3, 82.3, 107.2) and 4 ( 61.2, 63.0, 75.4, 76.4, 81.9, 103.9) were similar to those of n-butyl- -D-fructofuranoside and n-butyl- -D-fructofuranoside, respectively, by comparison to the data published in the literature [6]. Thus, the structure of 3 was assigned as n-butyl- -D-fructofu-ranoside and 4 as n-butyl- -D-fructofuranoside. The 13C-NMR data of compounds 1 - 4 are listed in the table 1. Compound 5 was isolated as amorphous powder. It's EI-MS spectrum showed the molecular ion peak at m/z 154. The 1H-NMR spectrum indicated 3 methyl groups at 0.91 s, 0.91 s and 0.89 s; an oxygened proton at 4.56 m. Besides these proton signals, 8 protons in the range of 1.3 to 2.4 ppm have been observed. The 13 C-NMR spectrum revealed 10 carbons: 3 methyl groups at C 13.7, 19.3 and 20.2, one hydroxylated methin signal at 85.2, one methin signal at 6' HOH 2C 1' CH 2OH O 5' OH H 4' H 3' 1 2' R O C 46.2, 3 methylene and 2 quartenary carbons. The above spectroscopic data suggested that compound 5 may be a monoterpen. This suggestion was supported by comparison to the data in the literature [7]. Thus, compound 5 was elucidated to be borneol. The comparison of the NMR spectral data of 6 with that of 5 revealed that compound 6 is a glycoside of borneol. Besides the signals of the aglycol moiety, 13 oxygenated protons resonanced in the range from 3.8 to 5.9 ppm in the 1HNMR spectrum and 11 sugar carbons have been seen in the range from 65 to 81 ppm. Two of them are anomeric signals: H 4.96 d (7.8) and 5.86 d (2.2); C 111.1 and 106.2 ppm. The sugar carbons include five methine, two methylene and two quartenary signals. The above mentioned sugar signals suggested that compound 6 contains two sugar moieties: a furanose and a pyranose. The structure of 6 was determined as bornyl-7-O- -D-apiofuranosyl(16)- -D-glucopyranoside by comparison to the data published in the literature [8]. 1 1: : R = CH3- 2 2: : R = CH3CH 2- 3 3: : R = CH3CH 2CH 2CH 2- 2 4 H OH 6' HOH 2C O 5' O OH H 3' H OH 3 2 1 1 R 4 2' 3 2 1 44:: R = CH3-CH 2-CH 2-CH 2 4' H 1 CH 2OH 1' 1 2 RO 7 3 8 9 10 4 6 5 262 55 :: R=H 66 :: R = 6' 5' O O HO O 1" HO 1' OH 3' 3" OH 4" 5" HO OH The 1H and 13C-NMR data of compound 5 and 6 are given in the experimental. Table 1: 13C-NMR data of compounds 1 - 4 C 1 (CD3OD) 2 (CD3OD) 3 (DMSO) 3 (CD3OD) 4 (DMSO) 4 (CD3OD) 1 48.9 57.6 59.9 61.6 60.2 61.9 16.0 31.8 33.3 32.0 33.4 3 18.9 20.4 18.9 20.3 4 13.8 14.2 13.9 14.3 2 Sugar moiety 1' 60.4 61.5 60.2 61.9 61.2 62.1 2' 109.2 108.9 107.2 108.7 103.9 105.1 3' 82.5 83.0 81.3 83.2 76.4 78.3 4' 78.9 78.6 76.7 78.4 75.4 77.2 5' 84.6 84.0 82.3 83.8 81.9 83.3 6' 62.8 62.7 61.2 61.2 63.0 65.0 III - Experimental 1. Instruments and chemicals NMR: Varian Unity 300; MS: AMD 402; [ ]D: Digital Polarimeter Ver.100.19. 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. 2. Extraction and isolation The roots of O. japonicus were bought in the market for medicinal plant in Hanoi, July, 2000. A voucher specimen with No. 003 was deposited in the Herbarium of the Institute of Chemistry, Hanoi. The roots were dried at 40oC, ground and extracted with hot ethanol (95%). The organic solvent was evaporated in vacuo and the aq. soln was extracted with n-hexan, EtOAc and n-BuOH, successively. The solvents were evaporated in vacuo to afford n-hexan, EtOAc and n-BuOH extracts. The butanol extract was chromatographed over silica gel Merck 60 (0.040 - 0.063 mm) with CHCl3 and increasing amounts of MeOH. With 15% MeOH the fraction containing compounds 1, 2, 3 and 4 was separated. This fraction was subjected over a flash column, eluted with CHCl3 : MeOH = 85 : 15 furnished 5 fractions. The second fraction was rechromatographed by using a flash column, eluting with CHCl3 : MeOH : H2O = 80 : 15 : 1 to afford compound 1 and 2. 1H-NMR of 1 (CD3OD): 3.31s (3H); of 2 (CD3OD): 1.18 t (7.03, 3H), 3.5 4.05 m (9H). The fourth fraction was further purified over a RP18 column, eluted with MeOH : H2O = 3 : 7 yielded compound 3 and 4. Compound 3, EI-MS m/z (rel. int.) 236 (0.7), 205 (47), 163 (11), 149 (54), 103 (84), 85 (42), 77 (100), 73 (41), 57 (78); 1 H-NMR (CD3OD): 0.92 t (7.33, 3H), 1.40 m (2H), 1.52 m (2H), 3.4 - 4.1 m (9H). Compound 4, EIMS m/z (rel. int.) 236 (0.6), 205 (32), 163 (3.5), 149 (33), 103 (100), 85 (44), 77 (64), 73 (17), 57 (22) 1H-NMR (CD3OD): 0.93 t (7.22, 3H), 1.40 m (2H), 1.53 m (2H), 3.5 - 4.05 m (9H). With 10% MeOH the fraction containing compound 5 has been obtained. This fraction was given over a RP18 column, eluted with MeOH : H2O = 4 : 6 to yield 5. EI-MS: 136 (25), 121 (71), 107 (37) and 93 (100). 1H-NMR 263 (CD3OD): 0.89 s, 0.91 s, 0.91 s (1-CH3, 9-CH3, 10-CH3); 13C-NMR (CD3OD): 13.7 (CH3-1), 19.3 (CH3-10), 20.2 (CH3-9), 27.7 (C-3), 29.0 (C-4), 37.6 (C-6), 46.2 (C-5), 50.2 (C-2) and 85.2 (C-7). With 30% MeOH the fraction of 6 was isolated, and rechromatographed at first over a flash column, washing with CHCl3 : MeOH : H2O = 80 : 20 : 2, and then over a RP18 column, eluting with MeOH : H2O = 88 : 12 to afford compound 6. 1H-NMR (Py-d5): 0.77 s, 0.83 s, 1.06 s (1-CH3, 9-CH3, 10-CH3), two anomeric protons: 5.86 d (2.2), 4.86 d (7.8). 13C-NMR (Py-d5): 14.4 (C-1), 50.1 (C-2), 27.3 (C-3), 28.8 (C-4), 45.5 (C-5), 38.3 (C-6), 86.2 (C-7), 47.8 (C-8), 19.1 (C-9), 20.1 (C-10), 106.2 (C-1'), 75.5 (C-2'), 78.7 (C-3'), 71.9 (C-4'), 77.3 (C-5'), 68.9 (C-6'), 111.1 (C-1''), 77.9 (C-2''), 80.6 (C-3''), 75.1 (C-4''), 65.8 (C-5''). Acknowledgements: We thank the Bundesministerium fuer Bildung und Forschung (BMBF), Bonn, Germany for financial support, Dr. A. Porzel and Dr. J. Schmidt, Institute for Plant Biochemistry Halle/S., Germany for their discussion on NMR and MS-spectra. 264 REFERENCES 1. Nguyen Thi Hoang Anh, Tran Van Sung, and Ludger Wessjohann. 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