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Journal of Chemistry, Vol. 45 (1), P. 106 - 109, 2007 SILYCHRISTIN AND ISOSILYCHRISTIN FROM THE FRUITS OF SILYBUM MARIANUM (L.) GAERTN. Received 31 May 2006 TRINH THI DIEP , PHAN VAN KIEM2, NGUYEN THUONG DONG1, NGUYEN HUU TUNG2, BUI THI BANG1, CHAU VAN MINH2, ALESSANDRA BRACA3 1 National Institute of Medicinal Materials, Ministry of Health 1 2 Institute of Natural Products Chemistry, VAST 3 Department of Bioorganic Chemistry and Biopharmaceutics University of Pisa, Italia SUMMARY By various chromatography methods, two flavonolignan silychristin (1) and isosilychristin (2) were isolated from the fruits of Silybum marianum (L.) Gaertn. cultivated in the North of Vietnam. Their structures were elucidated by analyses of the NMR (1D-, 2D-NMR) and ESI spectra. This is the first report of both 1 and 2 from S. marianum cultivated in Vietnam. I - INTRODUCTION In previous papers, we have reported the isolation and structural elucidation of flavonolignan, flavonoid, and lignan compositions of the fruits of Silybum marianum (L.) Gaertn. cultivated in the North of Vietnam and used in folk medicine [1 - 4]. Here, we report the isolation and structural determination of two flavonolignan silychristin (1) and isosilychristin (2) isolated from the fruits of Silybum marianum (L.) Gaertn. cultivated in the North of Vietnam. The structures were elucidated by analyses of the NMR and ESI spectra, and by the reported data. This is the first report of both 1 and 2 from S. marianum cultivated in Vietnam. II - EXPERIMENTAL 1. General experimental procedures The Electronspray Ionization (ESI) mass spectrum was obtained using a AGILENT 1100 LC-MSD Trap spectrometer. The NMR spectra were recorded on a Bruker AM500 FT-NMR 106 spectrometer using TMS as the internal standard. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70 - 230 mesh and 230 - 400 mesh, Merck) or YMC RP18 resins (30 - 50 µm, FuJisilisa Chemical Ltd.). Thin layer chromatography (TLC) was performed on DC-Alufolien 60 F254 (Merck 1.05715) or RP18 F254s (Merck) plates. 2. Plant material The fruits of Silybum marianum (L.) Gaertn. were collected in Sapa, Lao Cai Province in June, 2004 and were identified by Dr Nguyen Tap, Department of Botany and Medicinal Plant Resources, National Institute of Medicinal Materials. 3. Extraction and isolation Air-dried and powdered fruits of Silybum marianum (L.) Gaertn (200 g) were defatted with petroleum and then extracted with methanol. After removal of the solvent under vacuum, the residue (5.8 g) was obtained, which was then repeated chromatographed on silica gel columns to get 1 (30 mg) as colorless amorphous powder and 2 (50 mg) as yellow crystals. III - RESULTS AND DISCUSSION Compound 1 was obtained as colourless amorphous powder from methanolic extract of the fruits of S. marianum (L.), mp. 174 - 176oC. The 1H-NMR spectrum of 1 exhibited two doublets at H 5.85 and 5.90 ppm (J = 1.5 Hz), two doublets at H 6.82 and 6.87 ppm, three other aromatic protons at H 6.79 (d, J = 8.0 Hz), 6.81 (dd, J = 8.0, 1.0 Hz) and 6.99 (d, J = 1.0 Hz). Two doublet of doublets of two protons at trans configuration were at 5.00 (dd, J = 11.5, 2.5 Hz) and 4.49 (dd, J = 11.5, 5.5 Hz) [5], a doublet at 5.46 (J = 7.0 Hz), a multiplet at 3.49. The methylene group bearing oxygen atom was at 3.64 (dd, J = 10.0, 10.5 Hz) and 3.71 (dd, J = 10.5, 5.5 Hz), and methoxyl group was at 3.75 as a singlet signal. All the 1H-NMR data suggested the flavonolignan type of 1, which was similar to the isolated compounds from the same sources [1, 2]. Table 1: The NMR data of 1 C 2 3 4 5 6 7 8 9 1’ 2’ 3’ 4’ 5’ 6’ 1” 2” 3” 4” 5” 6” OCH3 5-OH a C a C b,d C c,d H (J in Hz) 85.2 73.8 198.2 165.2 97.4 168.7 96.3 164.4 130.0 116.6 147.5 142.1 131.5 116.9 89.1 83.20 71.66 197.53 163.25 96.01 166.97 94.96 162.49 128.99 115.26 147.02 140.66 129.90 115.58 86.96 5.00 (dd, 11.5, 2.5) 4.49 (dd, 11.5, 5.5) 5.90 (d, 1.5) 5.85 (d, 1.5) 6.87 (d, 1.5) 6.82 (d, 1.5) 5.46 (d, 7.0) 55.5 53.29 3.49 (m) 64.8 62.94 3.64 (dd, 10, 10.5); 3.71 (dd, 5.5, 10.5) 134.8 110.6 149.0 147.5 116.2 119.7 56.4 132.38 110.43 147.5 146.33 115.26 118.64 55.64 6.99 (d, 1.0) 6.79 (d, 8.0) 6.81 (dd, 1.0, 8.0) 3.75 (3H, s) 11.89 (br s) of silychristin [5], b125 MHz, c500 MHz, dMeasured in DMSO-d6. 107 OH HO 7 6 8 5 OH 2' 1' O 9 2 10 3 4 3' 6' O 6" 1" 5" OH HO 4" 3" 2" 4' 2' 7 OCH 3 6 8 5 5' CH 2OH OH OH 1' O 9 2 10 3 4 O 3' OH 4' 5' 6' O OH 1" 6" HOH2C O 2" 5" 3" 4" H3CO 1 OH 2 Fig.1. The structures of 1 & 2 The 13C-NMR spectrum of 1 showed the signals of 25 carbons including 12 quaternary, 11 methine, 1 oximethylene and 1 methoxyl carbons, suggesting a flavonolignan compound. The carbonyl group was at 197.53, the methylene bearing oxygen atom was at 62.94 and methoxyl group was at 55.64. All NMR assignments of 1 were made from analysis of HSQC, HMBC spectra and by comparison with those of isolated compound (dehydrosilychristin) [3] as well as with those of silychristin reported in the literature [5]. In the HMBC spectrum, H-2 ( 5.00) correlated with C-3 ( 71.66)/C-4 ( 197.53)/C-1’ ( 128.99)/C-2’ 115.26), and H-3 ( 4.49) had only correlations with C-2 ( 83.2) and C-1’ (128.99) confirming the position of two oximethine carbons at C-2 and C-3. In addition, the H-C long-range correlations were observed between H- ( 5.46) and carbons C- ( 53.29)/C-1” ( 132.38)/C-6” ( 118.64)/C-2” ( 110.43)/C(62.94), as well as the H-C long-range correlations were observed between H- ( 3.49) and carbons C1” ( 132.38)/C- (62.94) also confirming the positions of C- , C- and C- of two oximethine and oximethylene groups. The selected H-C correlations in the HMBC spectrum of 1 were shown in Fig. 2. Furthermore, the negative ESI mass spectrum of 1 exhibited an ion peak at m/z 481 [M-H]+ with strongest intensity, corresponding to the molecular formula of C25H22O10. From the above data, compound 1 was determined to be 108 silychristin, which was first isolated from Silybum marianum (L.) Gaertn. cultivated in the North of Vietnam. This compound also was reported to be inhibitor of horseradish peroxidases and lipoxygenase and antihepatotoxic agent [5]. Compound 2 was obtained as yellow crystals from methanolic extract of the fruits of S. marianum (L.), mp. 155 - 157oC and had similar NMR spectra as compound 1, suggesting the same flavonolignan type. 25 carbons corresponding to 25 carbon signals were observed in the 13C-NMR spectrum of 2 (including 12 quaternary, 11 methine, 1 oximethylene and 1 methoxy carbons as 1). Detailed comparison from NMR spectrum of 2 with those of 1, especially at C-2, C-3, C- and C- showed that 2 must be a derivative of 1, as isosilychristin, which was also isolated from Silybum marianum (L.) Gaertn [6]. The ESI mass spectrum of 2 was taken confirming the molecular formula of C25H22O10 of 2 from the appearance of an ion peak at m/z 483 [M+H]+ in the positive and an ion peak at m/z 481 [M-H]+ in the negative spectra. All NMR assignments of 2 were made carefully from HSQC and HMBC and from the comparison with those of 1 as shown in table 2. The H-C long-range correlations in the HMBC were shown in Fig.2 further confirming the structure of 2 as isosilychristin, which also was first isolated from Silybum marianum (L.) Gaertn. cultivated the North of Vietnam Table 2: The NMR data of 2 a,b C C a,c H a,b C C a,c H (J in Hz) 5’ 141.56 - 6’ 124.44 - (J in Hz) 2 79.87 5.17 (d, 12.0) 3 71.48 4.66 (dd, 12.0, 6.0) 86.36 5.57 (d, 2.0) 4 197.99 - 51.97 3.68 (m) 5 163.33 - 63.49 6 96.10 5.92 (d, 1.5) 3.46 (dd, 10, 10.5) 3.72 (dd, 5.5, 10.5) 7 166.83 - 1” 132.83 - 8 95.03 5.87 (d, 1.5) 2” 110.28 6.86 (d, 1.5) 9 162.55 - 10 100.49 - 3” 4” 147.44 146.27 - 1’ 128.85 - 5” 115.15 6.71 (d, 8.5) 2’ 119.42 6.95 (d, 8.0) 6” 118.48 6.76 (dd, 8.5, 1.5) 3’ 116.08 6.74 (d, 8.0) OCH3 55.59 3.71 (3H, s) 4’ 145.92 - 5-OH a Measured in DMSO-d6, b125 MHz, c500 MHz. 5" 6" OH 3' 2' HO 7 8 O 2 4' O 4" 1" 2" 10 4 OH 2' HO 7 OCH3 OH OH 4' 5' 8 O O 9 3 10 6 5' CH 2OH 5 3' OH 3" 1' 9 6 11.90 (br s) 4 5 OH O OH HOH2C 1" 2" 5" 3" O 6" H3CO 4" OH Figure 2: H-C long-range correlations in the HMBC of 1 and 2 Acknowledgements: The authors wish to thank Mr Dang Vu Luong, the NMR Lab., Institute of Chemistry, Vietnamese Academy of Science and Technology for recording the NMR spectrum, and Dr. Nguyen Tap, Department of Botany and Medicinal Plant Resources, National Institute of Medicinal Materials for the plant identification. This study was supported by a grant from the Vietnam- Italia international cooperation project. REFERENCES 1. Vo Van Chi. Vietnamese Medical Plant Dictionary, Ha Noi Medicine Pub. (1997). 2. T. T. Diep, N. T. Dong, B. T. Bang, C. V. Minh, P. V. Kiem. Journal of Medicinal Materials, Vol. 10(6), 175 - 178 (2005). 3. T. T. Diep, N. T. Dong, B. T. Bang, C. V. Minh, P. V. Kiem. Journal of Medicinal Materials, Vol. 11(1), 9 - 13 (2006). 4. T. T. Diep, N. T. Dong, B. T. Bang, C. V. Minh, P. V. Kiem. Vietnamese Journal of Chemistry, 2006 (accepted). 5. H. Tanaka, M. Hiroo, K. Ichino, K Ito. Chem. Pharm. Bull., Vol. 37(6), pp. 1441 1445 (1989). 6. M. Kalonga, Z. Naturforsch., B, Vol. 36, 262 - 266 (1981). 109 7. 110