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Journal of Chemistry, Vol. 43 (4), P. 489 - 493, 2005 Phenolic Constituents of Lonicera japonica Thunb., Caprifoliaceae, of Vietnam Received 8th-December 2003 Phan Minh Giang, Nguyen Thi Minh Hang, Phan Tong Son College of Natural Science, Vietnam National University, Hanoi Summary Caffeic acid, chlorogenic acid, methyl ester of chlorogenic acid, tricin, apigenin, 4’-Omethyl ether of apigenin (acacetin), quercetin and 3’-O-methyl ether of quercetin were isolated from the flowers of Lonicera japonica Thunb., Caprifoliaceae, grown as a medicinal plant for commercial purposes in Vietnam. Their chemical structures were determined by EIMS, 1H NMR, 13C NMR, DEPT, COSY, HMQC and HMBC. I - Introduction The medicinal properties and popular use of Lonicera japonica Thunb., Caprifoliaceae, (Vietnamese name: kim ng©n) are well documented [1, 2]. As the first group taking the attempt to investigate the chemical constituents of the Vietnamese L. japonica species which is grown as a medicinal plant for commercial purposes, recently, we isolated -sitosterol, 10nonacosanol, chrysin-7-O- -glucoside [3] and 3 -hydroxyurs-12-en-30-oic acid [4]. Further study on the flavonoid-containing ethyl acetatesoluble fraction obtained by successive liquidliquid fractionation of the aqueous MeOH extract of the flowers of L. japonica resulted in the isolation and structure determination of eight phenolic constituents and is reported in this paper. MHz for 1H-NMR and 125 MHz for 13C-NMR). IR spectra (KBr pellet) were recorded on a Nicolet Impact 410 FT-IR spectrometer. EIMS (70 eV) spectra were measured on a HP 5898B mass spectrometer. Column chromatography (CC and FC) was carried out on silica gel (Merck, 15 - 40 µm and 40 - 63 µm) and polyamide 6S (Riedel de Haen). Lipophilic Sephadex LH-20 (Sigma-Aldrich) was used for size-exclusion chromatography. Thin-layer chromatography (TLC) was performed on precoated Merck DC Alufolien 60 F254 plates and visualized with UV lamp 254 nm and spray reagent vanilin/conc. H2SO4 1%. Plant material The air-dried flowers of L. japonica were collected in the suburbs of Hanoi, Vietnam in May 2002. Extraction and Isolation II - Experimental General Experimental Procedures Melting points were measured on an Electrothermal model 9100 and are uncorrected. NMR spectra were obtained on a Bruker Avance 500 NMR spectrometer (500 The air-dried flowers L. japonica were dried at 40 - 50oC in a temperature-controlled heating oven. The dried flowers (2.5 kg) were powdered and then extracted with MeOH by percolation at room temperature (three times, each for 2 days). The combined MeOH extract was evaporated under reduced pressure to 1/10 of the initial 489 volume and the concentrated extract was suspended in distilled water and partitioned with n-hexane, EtOAc and n-butanol, successively. Evaporation under reduced pressure to dryness yielded the following soluble fractions: n-hexane (46.4 g, 1.86% yield on the basis of dry material), EtOAc (133.7 g, 5.35%) and n-butanol (140.5 g, 5.62%). A portion of the ethyl acetate-soluble fraction (8 g) was separated on silica gel eluting with a stepwise gradient toluene-EtOAc (4:1), tolueneEtOAc-acetone (4 : 1 : 1), EtOAc-MeOH (5 : 1). 100 fractions were collected into 9 fractiongroups (1 9) on the basis of TLC examination. Three first fraction-groups (1 3, 0.8 g) were combined and chromatographed on silica 10 : gel with CHCl3-MeOH gradient (50 : 1 1), recrystallization of one of the collected fractions in acetone gave tricin (4) (5 mg). Fraction-groups 4 - 6 were separated on silica gel and purified by Sephadex LH-20 columns (MeOH), and recrystallization afforded caffeic acid (1) (10 mg), apigenin (5) in a (4 : 3) mixture with 4’-O-methyl ether of apigenin (acacetin) (6) (5 mg) and quercetin (7) in a (4 : 5) mixture with 3’-O-methyl ether of quercetin (8) (10 mg). Fraction-group 7 was let to run through a polyamide S6 column (MeOH), the fraction showing one spot on TLC was recrystallized in MeOH to give the methyl ester of chlorogenic acid (2) (12 mg). The same procedure applying for fraction-group 8 led to the isolation of the chlorogenic acid (3) (27 mg). Caffeic acid (1) Yellow powder, mp 204 - 205oC. Rf 0.64 (TLC, EtOAc-MeOH-H2O 20 : 2 : 1). IR (KBr): max cm-1 3500 - 2250 (broad band), 3431.5, 3221.6, 3037.2, 2832.4, 2704.4, 2576.3, 1654.6, 1608.5, 1526.6, 1454.9, 1352.4, 1301.2, 1214.2, 1122.0, 968.4, 901.8, 855.7, 814.7, 778.9, 697.0, 650.9, 574.1. EIMS (70 eV): m/z (%) 180 (M+., C9H8O4, 100), 163 (32.4), 134 (42.1), 117 (12.3), 89 (21.0), 77 (11.0), 51 (7.9). 1 490 H NMR (DMSO-d6, , ppm): 12.09 (1H, brs, -COOH), 9.5 (1H, brs, OH), 9.11 (1H, brs, OH), 7.41 (1H, d, J = 15.9 Hz, H-3), 7.02 (1H, d, J = 2.0 Hz, H-5), 6.96 (1H, dd, J = 8.1 Hz, 2.0 Hz, H-9), 6.76 (1H, d, J = 8.1 Hz, H-8), 6.16 (1H, d, J = 15.9 Hz, H-2). 13 C-NMR (DMSO-d6, , ppm): 167.8 (s, C1), 148.1 (s, C-7), 145.5 (d, C-3), 144.5 (s, C-6), 125.7 (s, C-4), 121.1 (d, C-9), 115.7 (d, C-8), 115.1 (d, C-5), 114.6 (d, C-2). Chlorogenic acid (2) Off-white needles, mp 199 - 200oC. Rf 0.79 (TLC, MeOH-H2O 98:2). IR (KBr): max cm-1 3750-2250 (broad band), 3349.6, 2970.7, 2893.8, 2822.1, 2622.4, 2351.0, 1685.3, 1639.2, 1593.1, 1516.3, 1439.5, 1301.2, 1193.7, 1116.9, 1075.9, 968.4, 912.0, 809.6, 743.1, 604.8. EIMS (70 eV): m/z (%) 354 (M+., C16H18O9, 8.7), 336 (14.8), 180 (100), 163 (78.2). 1 H NMR (DMSO-d6, , ppm): 9.57 (1H, brs, OH), 9.14 (1H, brs, OH), 7.42 (1H, d, J = 16.0 Hz, H-3’), 7.03 (1H, d, J = 2.0 Hz, H-5’), 6.98 (1H, dd, J = 8.0 Hz, 2.0 Hz, H-9’), 6.77 (1H, d, J = 8.0 Hz, H-8’), 6.15 (1H, d, J = 16.0 Hz, H2’), 5.07 (1H, m, H-5 ), 4.89 (1H, brs, OH), 4.76 (1H, brs, OH), 3.93 (1H, t, J = 3.5 Hz, H4 ), 3.57 (1H, m, H-3 ), 1.92-2.03 (3H, m) and 1.78 (1H, dd, J = 7.5 Hz, 13.0 Hz) (2 H-2, 2 H6). 13 C NMR (DMSO-d6, , ppm): 174.9 (s, C7), 165.5 (s, C-1’), 148.2 (s, C-7’), 145.5 (d, C3’), 144.8 (s, C-6’), 125.5 (s, C-4’), 121.2 (d, C9’), 115.6 (d, C-8’), 114.7 (d, C-5’), 114.2 (d, C2’), 73.4 (s, C-1), 70.8 (3d, C-3, C-4, C-5), 37.1 (2t, C-2, C-6). Methyl ester of chlorogenic acid (3) Off-white needles, mp 247 - 248oC. EIMS (70 eV): m/z (%) 368 (M+., C17H20O9). 1 H NMR (DMSO-d6, , ppm): 9.56 (1H, brs, OH), 9.18 (1H, brs, OH), 7.47 (1H, d, J = 15.5 Hz, H-3’), 7.03 (1H, d, J = 2.0 Hz, H-5’), 6.98 (1H, dd, J = 8.0 Hz, 2.0 Hz, H-9’), 6.77 (1H, d, J = 8.0 Hz, H-8’), 6.21 (1H, d, J = 15.5 Hz, H2’), 5.46 (1H, s, OH), 5.17 (1H, ddd, J = 3.5 Hz, 4.0 Hz, 9.0 Hz, H-5 ), 4.94 (1H, d, J = 3.5 Hz, OH), 4.79 (1H, d, J = 2.5 Hz, OH), 3.83 (1H, m, H-4 ), 3.64 (1H, m, H-3 ), 3.61 (3H, s, OCH3), 2.07 (1H, dd, J = 3.5 Hz, 13.0 Hz, H6a), 1.97 (1H, dd, J = 6.0 Hz, 13.0 Hz, H-2a), 1.88 (1H, dd, J = 9.0 Hz, 13.0 Hz, H-6b), 1.85 (1H, dd, J = 3.0 Hz, 13.0 Hz, H-2b). Tricin (4) EIMS (70 eV): m/z 330 (M+., C17H14O7, 100), 300 (20.8), 153 (10.2). 1 H-NMR (DMSO-d6, , ppm): 12.96 (1H, brs, 5-OH), 10.82 (1H, brs, 7-OH), 9.32 (1H, brs, 4’-OH), 7.33 (2H, s, H-2’, H-6’), 6.98 (1H, s, H-3), 6.56 (1H, d, J = 2.0 Hz, H-8), 6.20 (1H, d, J = 2.0 Hz, H-6), 3.89 (6H, s, 3’-OCH3, 5’OCH3). 13 C-NMR (DMSO-d6, , ppm): 181.7 (s, C4), 164.0 (s, C-2), 163.6 (s, C-7), 161.3 (s, C-5), 157.2 (s, C-9), 148.1 (s, C-3’, C-5’), 139.8 (s, C-4’), 120.3 (s, C-1’), 103.6 (d, C-3), 103.5 (s, C-10), 98.7 (d, C-6), 94.1 (d, C-8), 56.3 (2q, 3’OCH3, 5’-OCH3). Apigenin (5) in a (4 : 3) mixture with 6 EIMS (70 eV): m/z 270 (M+., C15H10O5). 1 H NMR (DMSO-d6, , ppm): 13.1 (1H, brs, 5-OH), 10.8 (1H, brs, 7-OH), 10.25 (1H, brs, 4’-OH), 7.99 (2H, d, J = 9.0 Hz, H-2’, H-6’), 6.83 (1H, s, H-3), 6.70 (2H, d, J = 9.0 Hz, H-3’, H-5’), 6.45 (1H, d, J = 2.0 Hz, H-8), 6.18 (1H, d, J = 2.0 Hz, H-6). 13 C NMR (DMSO-d6, , ppm): 181.9 (s, C4), 163.9 (s, C-7), 163.8 (s, C-2), 161.3 (s, C4’), 160.8 (s, C-5), 157.3 (s, C-9), 128.1 (2d, C2’, C-6’), 121.3 (s, C-1’), 115.6 (2d, C-3’, C-5’), 103.6 (s, C-10), 102.9 (d, C-3), 98.7 (d, C-6), 93.9 (d, C-8). 4’-O-methyl ether of apigenin (acacetin) (6) in a (3:4) mixture with 5 EIMS (70 eV): m/z 284 (M+., C16H12O5). 1 H-NMR (DMSO-d6, , ppm): 12.97 (1H, brs, 5-OH), 10.8 (1H, brs, 7-OH), 7.58 (2H, d, J = 8.5 Hz, H-2’, H-6’), 7.13 (2H, d, J = 8.5 Hz, H-3’, H-5’), 6.78 (1H, s, H-3), 6.45 (1H, d, J = 2.0 Hz, H-8), 6.18 (1H, d, J = 2.0 Hz, H-6), 3.57 (3H, s, 4’- OCH3). 13 C-NMR (DMSO-d6, , ppm): 181.6 (s, C4), 163.9 (s, C-7), 163.8 (s, C-2), 162.0 (s, C4’), 161.3 (s, C-5), 157.3 (s, C-9), 127.6 (2d, C2’, C-6’), 121.5 (s, C-1’), 115.6 (2d, C-3’, C-5’), 103.7 (s, C-10), 103.2 (d, C-3), 98.7 (d, C-6), 93.9 (d, C-8), 55.2 (q, 4’-OCH3). Quercetin (7) in a (4 : 5) mixture with 8 1 H NMR (DMSO-d6, , ppm): 12.49 (1H, brs, 5-OH), 10.7 (1H, brs, 7-OH), 9.7, 9.4 (2H, brs, 3’-OH, 4’-OH), 7.68 (1H, d, J = 1.5 Hz, H2’), 7.54 (1H, dd, J = 8.5 Hz, 1.5 Hz, H-6’), 6.88 (1H, d, J = 8.5 Hz, H-5’), 6.41 (1H, d, J = 1.5 Hz, H-8), 6.19 (1H, d, J = 1.5 Hz, H-6). 13 C NMR (DMSO-d6, , ppm): 175.8 (s, C4), 163.9 (s, C-7), 160.7 (s, C-5), 156.2 (s, C-9), 147.7 (s, C-4’), 146.8 (s, C-2), 145.0 (s, C-3’), 135.8 (s, C-3), 121.9 (s, C-1’), 119.9 (d, C-6’), 115.4 (d, C-5’), 115.1 (d, C-2’), 103.0 (s, C-10), 98.2 (d, C-6), 93.3 (d, C-8). 3’-O-methyl ether of quercetin (8) in a (5:4) mixture with 7 1 H NMR (DMSO-d6, , ppm): 12.46 (1H, brs, 5-OH), 10.7 (1H, brs, 7-OH), 9.7, 9.4 (2H, brs, 3-OH, 4’-OH), 7.76 (1H, d, J = 1.5 Hz, H2’), 7.69 (1H, dd, J = 9.0 Hz, 1.5 Hz, H-6’), 6.94 (1H, d, J = 9.0 Hz, H-5’), 6.48 (1H, d, J = 1.5 Hz, H-8), 6.19 (1H, d, J = 1.5 Hz, H-6), 3.85 (3H, s, 3’-OCH3). 13 C NMR (DMSO-d6, , ppm): 175.8 (s, C4), 163.9 (s, C-7), 160.7 (s, C-5), 156.2 (s, C-9), 148.8 (s, C-4’), 147.4 (s, C-3’), 146.6 (s, C-2), 135.8 (s, C-3), 121.7 (s, C-1’), 121.7 (d, C-6’), 115.5 (d, C-5’), 111.7 (d, C-2’), 103.0 (s, C-10), 98.2 (d, C-6), 93.6 (d, C-8), 55.8 (q, 3’-OCH3). III - Results and discussion A separation by column chromatography of the ethyl acetate-soluble fraction on silica gel followed by purification of the nine obtained fraction-groups by column chromatography on silica gel, polyamide 6S and Sephadex LH-20 led to the isolation of caffeic acid (1), two caffeoylquinates chlorogenic acid (2) and its methyl ester (3), three flavones tricin (4), apigenin (5) and 4’-O-methyl ether of apigenin 491 (acacetin) (6), two flavonols quercetin (7) and 3’-O-methyl ether of quercetin (8) (Fig. 1). Compound 1 was determined as caffeic acid on the basis of the following spectroscopic evidences. The EIMS (70 eV) spectrum of 1 showed the molecular ion peak as the base peak (100% intensity) at m/z 180 corresponding to the molecular formula C9H8O4 with six double bond equivalents. The aromatic proton resonances at 7.02 (1H, d, J = 2.0 Hz), 6.96 (1H, dd, J = 8.1 Hz, 2.0 Hz) and 6.76 (1H, d, J = 8.1 Hz) were firmly assigned for a 1,3,4trisubstituted aromatic ring. A trans double bond attached to a carboxyl group was deduced from the vicinal coupling constant of the downfield-shifted proton signals at 7.41 (1H, d, J = 15.9 Hz) and 6.16 (1H, d, J = 15.9 Hz), and the carboxyl signal at C 167.8 (s). The other 13C NMR resonances confirmed this structure. The 1H NMR spectrum of 2 in DMSO-d6 contained two distinctive groups of resonances. Those at 7.42 and 6.15 (both 1H, d, J = 16.0 Hz) (a trans double bond) and 7.03 (1H, d, J = 2.0 Hz), 6.98 (1H, dd, J = 8.0 Hz, 2.0 Hz), 6.77 (1H, d, J = 8.0 Hz) were attributed to a caffeoyl group; the 13C NMR chemical shifts of this moiety were consistent with those of 1. The second proton resonance group appearing wellresolved between 1.78 and 5.07 represented a quinic acid moiety. Taken together with the EIMS spectral data [m/z 354 (M+., C16H18O9)] and on the basis of the 1H and 13C NMR spectral data [5] the structure of chlorogenic acid could be assigned for compound 2. Compound 3 possessing the molecular ion peak at m/z 368 (M+., C17H20O9) appeared to have one more methyl group in comparison with the chlorogenic acid (2). Except for the methoxyl signal [ H 3.61 (3H, s)] the other 1HNMR resonances of 3 were well-compared with those of 2. Thus the structure of compound 3 was determined as methyl ester of chlorogenic acid. Chlorogenic acid and its methyl ester have been already reported as constituents of the Chinese L. japonica (Jinyinhua) [5 - 7]. 492 OH 6 HO 7 5 8 4 2 3 CH 9 1 CH CO2H 1 OH OH H RO 6 1 5 H OH 3 2 O OCaf 4 H 2: R = H Caf = caffeoyl 3: R = CH3 Caf = caffeoyl R1 3' 2' 8 HO 9 4' R2 5' O 1' 2 7 6' R3 3 6 10 4 5 OH R4 O 4: R1 = OCH3, R2 = OH, R3 = OCH3, R4 = H 5: R1 = H, R2 = OH, R3 = H, R4 = H 6: R1 = H, R2 = OCH3, R3 = H, R4 = H 7: R1 = OH, R2 = OH, R3 = H, R4 = OH 8: R1 = OCH3, R2 = OH, R3 = H, R4 = OH Figure 1: Chemical structures of compounds 1-8 From the less polar fraction-groups the flavones tricin (4), apigenin (5) and 4’-O-methyl ether of apigenin (acacetin) (6) were isolated. The characteristic carbon signals observed for these compounds were the signals of the carbonyl groups at 181.7 (s), 181.9 (s), 181.6 (s) (for 4, 5 and 6, respectively); of C-2 at 164.0 (s), 163.8 (s), 163.8 (s); and of C-3 at 103.6 (d), 102.9 (d), 103.2 (d), together with the H-3 proton signals at 6.98 (s), 6.83 (s), 6.78 (s). The proton and carbon resonances of 4, 5 and 6 clearly showed the 5,7-dihydroxysubs-tituted pattern of the ring A. The differences in NMR resonances of the ring B led to the grouping of 4, 5 and 6 to 1’,3’,4’,5’-tetrasubstituted aromatic ring for 4 [ H 7.33 (2H, s)], and 1’,4’disubstituted aromatic ring for 5 [ H 7.99 and 6.70 (both 2H, d, J = 9.0 Hz)] and for 6 [ H.7.58 and 7.13 (both 2H, d, J = 8.5 Hz), 3.57 (3H, s)]. On comparison with the reported 13C NMR values [8] the structures of apigenin and 4’-Omethyl ether of apigenin (acacetin) were assigned for compounds 5 and 6, respectively. The presence of two symmetrical methoxyl group in compound 4 observed from the singlet proton signal at 3.89 (6H) and the quartet carbon signals at 56.3 led to the conclusion of a 4’-hydroxy-3’,5’-dimethoxy structure of the ring B, and the 13C NMR data were in good accordance with those reported for the 3’,4’,5’trimethoxy-5,7-dihydroxyflavone [8]. In the EIMS spectrum of 4 the intense molecular ion peak at m/z 330 (100%), the peak at m/z 300 (loss of two methyl groups), and the diagnostic peak at m/z 153 ([A1+H]+), generated through a retro-Diels-Alder fragmentation, were consistent with the supposed structure. Thus 4 was elucidated as 3’,5’-dimethoxyapigenin, previously known under the name tricin the 13C NMR signals of which were however wrongly assigned [8]. On the basis of the 2D NMR spectra, we reversed the assignments for the carbon signals between C-5 and C-9, between C-1’ and C-4’. From the fraction-groups eluted after the compounds 4-6, two flavonols quercetin (7) and 3’-O-methyl ether of quercetin were isolated. Quercetin (7) was structurally identified by 1H NMR, 13C NMR, DEPT, 1H -1H COSY, HMQC and HMBC spectra, the NMR values were in good agreement with the reported values in the literature [8, 9]. The structure of 8 was elucidated as a flavonol derivative with the characteristic 13C signals at 175.8 (s, C-4), 146.6 (s, C-2) and 135.8 (s, C-3). The substitution patterns of the ring A and ring B [ H 7.76 (1H, d, J = 1.5 Hz), 7.69 (1H, dd, J = 9.0 Hz, 1.5 Hz), 6.94 (1H, d, J = 9.0 Hz) (ring B); 6.48 (1H, d, J = 1.5 Hz), 6.19 (1H, d, J = 1.5 Hz) (ring A)] suggested that 8 could be a derivative of quercetin. The slightly upfieldshifted resonances of C-3’ ( 147.4 for 8 vs. 145.0 for 7) and of C-4’ ( 148.8 for 8 vs. 147.7 for 7), together with the HMBC correlation between the 3’-methoxyl group ( H 3.85) and C3’ ( C 147.4) placed the methoxyl group at position 3’ (Fig. 2). Thus the structure of 8 was determined as 3’-O-methyl ether of quercetin on the basis of 1H NMR, 13C NMR, DEPT, 1H 1 H COSY, HMQC and HMBC spectra. OCH3 3' OH 2' 8 HO 9 O 4' 5' 1' 2 7 6' 3 6 10 4 5 OH OH O Figure 2: HMBC correlations (H C) observed for compound 8 Of all flavonoids isolated by us only quercetin was reported for the L. japonica plant growing in Korea [9], the other compounds (4 6 and 8) were isolated for the first time from the species L. japonica. The presence of the phenolic compounds may account for the potent antioxidative properties of Vietnamese L. japonica species as evidenced from our in vitro experiments (unpublished data). Acknowledgements: This research was supported by the International Foundation for Science, Stockholm, Sweden, through a grant to Dr. Phan Minh Giang and the National Basic Research Program in Natural Sciences. References 1. Do Tat Loi. Medicinal Plants and Herbal Remedies of Vietnam, P. 91 - 94, Publishing House Science and Technique, Hanoi (1991). 2. Vo Van Chi. Dictionary of Vietnamese Medicinal Plants, P. 632 - 635, Publishing House Medicine, Ho Chi Minh city (1997). (See page 507) 493 494