Lignans from the fruits of cornus kousa burg. and their cytotoxic effects on human cancer cell lines

Arch Pharm Res Vol 30, No 4, 402-407, 2007 http://apr.psk.or.kr Lignans from the Fruits of Burg. and Their Cytotoxic Effects on Human Cancer Cell Lines Cornus kousa Dae-Young Lee, Myoung-Chong Song, Ki-Hyun Yoo, Myun-Ho Bang , In-Sik Chung, Sung-Hoon Kim , Dae-Keun Kim , Byoung-Mog Kwon , Tae-Sook Jeong , Mi-Hyun Park , and Nam-In Baek 1 3 4 4 2 5 Graduate School of Biotechnology & Plant Metabolism Research Center, Kyung Hee University, Suwon 449-701, Ganghwa Agricultural R&D Center, Incheon 417-833, Graduate School of Oriental Medicine, Kyung Hee University, Seoul 130-701, Department of Pharmacy, Woosuk University, Jeonju 565-701, Korea Research Institute of Bioscience and Biotechnology, Daejon 305-333, and Erom life Co. Ltd., Seoul 135-825, Korea 1 2 3 4 5 (Received December 7, 2006) The fruits of Cornus kousa Burg. were extracted with 80% aqueous MeOH, and the concentrated extract partitioned with EtOAc, n-BuOH and H2O. Six lignans were isolated from the EtOAc fraction through repeated silica gel, ODS and Sephadex LH-20 column chromatographies. From the physico-chemical data, including NMR, MS and IR, the chemical structures of the compounds were determined to be (+)-pinoresinol (1), (-)-balanophonin (2), (+)-laricresinol (3), erythro-guaiacylglycerol-β-coniferyl aldehyde ether (4), threo-guaiacylglycerol-β-coniferyl aldehyde ether (5) and dihydrodehydrodiconiferyl alcohol (6), which were isolated for the first time from this plant. Most of these compounds showed cytotoxicity against human colon carcinoma (HCT-116) and human hepatocellular carcinoma (HepG2) cell lines in vitro, with IC50 values ranging from 19.1 to 71.3 µg/mL. Key words: Cornus kousa, Lignan, MTT assay, Cytotoxicity, Human colon carcinoma (HCT116), Human hepatocellular carcinoma (HepG2) INTRODUCTION Burg. (Cornaceae) is a tree distributed in the mountains of South Korea, China and Japan. The fruit of this plant has been used as a hemostatic agent and for the treatment of diarrhea in Korean traditional medicine (Lee, 2003), and their extracts have been reported to have immuno-regulatory properties (Kim ., 1984). Some chemical constituents have also been reported from the leaves of , such as isoquercitrin, gallic acid, tannin (Ryu ., 1971), phenolics and flavonoids (Shaiju ., 2006) However, isolation of the chemical components from the fruits of remains to be reported. Therefore, in this paper, the isolation and identification of six lignans from the fruits of are reported, and their structures characterized by spectroscopic methods. The isolated compounds were tested for cytotoxicity against human colon carcinoma (HCT-116) and Cornus kousa et al C. kousa et al et al . C. kousa C. kousa Correspondence to: Nam-In Baek, Graduate School of Biotechnology, Kyung-Hee University, Seochun-Dong, Kiheung-Gu, Suwon 449-701, Korea Tel: 82-31-201-2661, Fax: 82-31-201-2157 E-mail: nibaek@khu.ac.kr human hepatocellular carcinoma (HepG2) cell lines using the MTT assay. in vitro MATERIALS AND METHODS Plant materials The fruits of Burg. (Cornaceae) were collected from the experimental farm in KyungHee University during August, 2005. A voucher specimen (KHU050914) has been reserved at the Laboratory of Natural Products Chemistry, KyungHee University, Suwon, Korea. Cornus kousa Instruments and regents Optical rotations were measured on a JASCO P-1010 digital polarimeter (Tokyo, Japan). EI-MS were recorded on a JEOL JMSAX 505-WA (Tokyo, Japan). IR spectra were run on a Perkin Elmer Spectrum One FT-IR spectrometer (Buckinghamshire, England). 1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra were taken on a Varian Unity Inova AS 400 FT-NMR spectrometer (Lake forest, U.S.A.). RPMI Medium 1640, Dulbecco’s Modified Eagle Medium (GIBCO BRL, Life Technologies Inc., NY) and 402 Cytotoxic Lignans from the Fruits of Cornus kousa 403 Penicillin-Streptomycin were purchased from GIBOCO (Grand island, NY). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT). MTT (3-[4, 5-Dimethylthiazol2-yl]-2, 5-diphenyltetrazolium bromide) and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). The dried and chopped fruits of C. kousa (1 kg) were extracted three times with 80% aqueous MeOH (3 L × 3) at room temperature. The extract was successively partitioned with water (1 L), EtOAc (1 L × 3) and n-BuOH (0.8 L × 3). The EtOAc extract (4 g) was subjected to silica gel column (4.5 × 60 cm) chromatography (c.c.), eluted with n-hexane:EtOAc (3:1) → CHCl :MeOH (17:1 → 15:1 → 13:1 → 10:1, 1 L of each) and monitored by thin layer chromatography (TLC), which gave twenty one fractions (CKFE1 to CKFE21). Fraction CKFE10 [414.7 mg, V /V (elution volume/total volume) 0.53-0.60 in CHCl :MeOH (15:1)] was subjected to ODS (Octadecyl silica gel, Merck) c.c. (3.5 × 50 cm), eluted with MeOH:H O (5:1 → 7:1, 1 L of each), to afford nine fractions (CKFE10-1 to CKFE10-9). Fraction CKFE10-1 [202 mg, V /V 0.01-0.30 in MeOH:H O (5:1)] was subjected to silica gel c.c (3 × 40 cm), eluted with CHCl :MeOH (40:1 → 30:1 → 20:1, 1 L of each), to afford ten fractions (CKFE10-1-1 to CKFE101-10) and yield compound [16 mg, V /V 0.10-0.15, TLC (SiO F ) R 0.80, CHCl :MeOH = 30:1]. CKFE10-1-6+7 [45 mg, V /V 0.50-0.70 in CHCl :MeOH (30:1)] were subjected to ODS c.c. (2 × 20 cm), eluted with MeOH:H O (1:1, 800 mL), to ultimately produce compound [12 mg, V /V 0.60-0.85, TLC (RP-18 F ) R 0.55, MeOH:H O = 3:1]. CKFE10-1-8+9 [37 mg, V /V 0.20-0.40 in CHCl : MeOH (20 : 1)] were subjected to ODS c.c. (2 × 20 cm), eluted with MeOH:H O (1:2, 500 mL), to give seven fractions (CKFE10-1-8+9-1 to CKFE10-1-8+9-7) and yield compound [10 mg, V /V 0.40-0.50, TLC (RP-18 F ) R 0.65, MeOH:H O = 2:1]. CKFE10-1-8+9-4 (18 mg, V /V 0.50-0.65) were applied to a Sephadex LH-20 column (2 × 40 cm) eluted with 80% MeOH (500 ml), to give a mixture of compounds and (12 mg, V /V 0.25-0.30, TLC (RP-18 F ) R 0.5, MeOH:H O = 2:1]. Fraction CKFE11 [78 mg, V /V 0.15-0.18 in CHCl :MeOH (13:1)] was subjected to ODS c.c. (2.5 × 20 cm), eluted with MeOH:H O (1:2 → 1:1 (300 mL of each), to ultimately yield compound [9 mg, V /V 0.25-0.44 in MeOH:H O (1:2), TLC (RP-18 F ) Rf 0.5, MeOH:H O = 1:1]. 3 e t 3 2 e t 2 3 2 254 f e e t 3 t + -1 õ 1 3 5 13 Extraction and isolation of lignans 1 358 [M ], 327, 221, 205, 180, 163, 152, 151, 150, 137, 131, 124; IR (CHCl , cm ) 3420, 1680; H-NMR (400 MHz, pyridine-d , δ) 7.26 (2H, d, J=8.0 Hz, H-5/5'), 7.24 (2H, d, J=2.4 Hz, H-2/2'), 7.07 (2H, dd, J=8.0, 2.4 Hz, H-6/6'), 4.80 (2H, d, J=4.4 Hz, H-7/7'), 4.33 (2H, dd, J=8.8, 6.8 Hz, H-9a/9a'), 4.01 (2H, dd, J=8.8, 3.6 Hz, H-9b/9b'), 3.77 (6H, s, H-10/10'), 3.23 (2H, ddd, J=3.6, 6.8, 4.4 Hz, H-8/ 8'); C-NMR (100 MHz, pyridine-d , δ) 148.7 (C-3/3'), 147.8 (C-4/4'), 133.1 (C-1/1'), 119.7 (C-6/6'), 116.4 (C-5/ 5'), 110.9 (C-2/2'), 86.5 (C-7/7'), 72.0 (C-9/9'), 56.4 (C-10/ 10'), 54.9 (C-8/8'). 3 2 5 Colorless oil (MeOH); [α] = -68° (c=0.1, MeOH) {lit. Yuen et al., 1998, (-)-balanophonin, [α] = -114 (c=0.34, CHCl ); (+)-balanophonin, [α] = +118° (c=0.41, CHCl )}; EI/MS m/z: 356 [M ], 338, 326, 323, 306, 278, 165, 149, 137, 129; IR (MeOH, cm ) 3256, 2950, 1709, 1556; HNMR (400 MHz, pyridine-d , δ) 9.82 (1H, d, J=7.6 Hz, H9), 7.49 (1H, d, J=15.6 Hz, H-7), 7.48 (1H, br s, H-2), 7.31 (1H, d, J=1.6 Hz, H-2'), 7.26 (1H, br s, H-6), 7.25 (1H, d, J=8.0 Hz, H-5'), 7.23 (1H, dd, J=8.0, 1.6 Hz, H-6'), 6.88 (1H, dd, J=15.6, 7.6 Hz, H-8), 6.14 (1H, d, J=6.8 Hz, H7'), 4.26 (2H, d, J=5.6 Hz, H-9'), 3.99 (1H, td, J=5.6, 6.8 Hz, H-8'), 3.85 (3H, s, H-10), 3.66 (3H, s, H-10'); C-NMR (100 MHz, pyridine-d , δ) 193.7 (C-9), 153.9 (C-7), 152.2 (C-4), 149.1 (C-3'), 148.7 (C-4'), 145.4 (C-3), 133.1 (C-1'), 131.6 (C-5), 128.8 (C-1), 127.0 (C-8), 120.2 (C-2'), 120.0 (C-6), 116.9 (C- 5'), 113.7 (C-2), 111.2 (C-6'), 89.9 (C-7'), 64.2 (C-9'), 56.5 (C-10), 56.2 (C-10'), 54.5 (C-8'). (-)-Balanophonin (2) 25 D 20 o D 25 3 D 3 + -1 1 õ 5 13 5 2 e t 254 f e 2 t 3 2 3 e t 254 2 f e 4 254 5 e f e t t 2 t 3 2 6 e t 2 254 2 Amorphous powder (MeOH); [α] = +72.0 (c=0.20, MeOH) {lit. Li et al., 2003,. [α] = +69.0 (c=0.10, MeOH); lit. Abe et al., 1988, [α] = +71.1° (MeOH)}; EI/MS m/z: (+)-Pinoresinol (1) 25 o D 25 D 24 D o Amorphous powder (MeOH); [α] = +39 (c=0.10, CHCl ) {lit. Li et al., 2003, [α] = +30 (c=0.10, MeOH); lit. Okuyama et al., 1995, [α] = +32° (MeOH)}; EI/MS m/z: 360 [M ], 236, 221, 219, 206, 205, 194, 191; IR (MeOH, cm ) 3432, 3011, 1490; H-NMR (400 MHz, pyridine-d , δ) 7.31 (1H, d, J=2.0 Hz, H-2), 7.25 (1H, d, J=8.0 Hz, H-5'), 7.19 (1H, dd, J=8.0, 2.0 Hz, H-6'), 7.19 (1H, d, J=8.0 Hz, H-5), 6.99 (1H, J=2.0 Hz, H-2'), 6.89 (1H, dd, J=8.0, 2.0 Hz, H-6), 5.33 (1H, d, J=6.0 Hz, H-7), 4.29 (1H, dd, J=8.0, 6.8 Hz, H-9a'), 4.25 (1H, dd, J=8.0, 6.8 Hz, H-9a), 4.13 (1H, dd, J=8.0, 7.6 Hz, H-9b), 4.06 (1H, dd, J=8.0, 7.6 Hz, H-9b'), 3.72 (3H, s, H-10), 3.71 (3H, s, H-10'), 3.24 (1H, dd, J=13.6, 4.8 Hz, H-7'a), 3.06 (1H, m, H-8'), 2.80 (1H, dd, J=13.6, 10.4 Hz, H-7'b ), 2.78 (1H, m, H-8),; C-NMR (100 MHz, pyridine-d , δ) 148.5 (C-3/4'), 147.3 (C-4), 146.4 (C-3'), 135.9 (C-1), 132.6 (C-1'), 121.7 (C-6'), 119.4 (C-6), 116.5 (C-5'), 116.3 (C-5), 113.1 (C-2'), 110.5 (C-2), 83.4 (C-7'), 73.2 (C-9'), 60.1 (C-9'), 55.9 (C-10/10'), 53.9 (C-8'), 43.4 (C-8), 33.5 (C-7). (+)-Lariciresinol (3) 25 o 3 D 20 o D 20 + õ -1 1 5 13 5 404 D.-Y. Lee et al. β Mixture of erythro-guaiacylglycerol- -coniferyl aldehyde β ether (4) and threo-guaiacylglycerol- -coniferyl aldehyde ether (5) Amorphous powder (MeOH); [α]D25 = - 24o (c=0.20, MeOH); EI/MS m/z: 374, 356, 342, 338, 326, 297, 265, 243, 196, 178, 151; IRõ (MeOH, cm-1) 3389, 2922, 1665, 1595. β erythro-Guaiacylglycerol- -coniferyl aldehyde ether (4) H-NMR (400 MHz, pyridine-d5, δ) 9.78 (1H, d, J=8.0 Hz, H-9), 7.61 (1H, dd, J=9.6, 2.0 Hz, H-6'), 7.53 (1H, d, J=8.4 Hz, H-5), 7.43 (1H, d, J=16.0 Hz, H-7), 7.39 (1H, d, J=2.0 Hz, H-2'), 7.25 (1H, dd, J=8.4, 2.0 Hz, H-6), 7.24 (1H, d, J=9.6 Hz, H-5'), 7.22 (1H, d, J=2.0 Hz, H-2), 6.85 (1H, dd, J=16.0, 8.0 Hz, H-8), 5.56 (1H, brd, J=5.6 Hz, H-7'), 5.19 (1H, ddd, J=5.6, 9.6, 5.6 Hz, H-8'), 4.53 (1H, dd, J=12.0, 9.6 Hz, H-9a'), 4.17 (1H, dd, J=12.0, 5.6 Hz, H-9b'), 3.76 (3H, s, OCH3), 3.73 (3H, s, OCH3) 13C-NMR (100 MHz, pyridine-d5, δ) 193.4 (C-9), 153.2 (C-7), 152.8 (C-4), 150.1 (C-3), 148.5 (C-4'), 147.5 (C-3'), 134.0 (C-1'), 127.6 (C-1), 127.0 (C-8), 120.6 (C-2'), 116.1 (C-5'), 115.9 (C-5), 112.0 (C-6'), 111.8 (C-6), 111.7 (C-2), 86.3 (C-8'), 73.4 (C-7'), 61.9 (C-9'), 55.9 (C-10), 55.8 (C-10'). 1 β threo-Guaiacylglycerol- -coniferyl aldehyde ether (5) H-NMR (400 MHz, pyridine-d5, δ) 9.77 (1H, d, J=8.0 Hz, 1 Fig. 1. H-9), 7.61 (1H, dd, J=9.6, 2.0 Hz, H-6'), 7.53 (1H, d, J=8.4 Hz, H-5), 7.45 (1H, d, J=16.0 Hz, H-7), 7.39 (1H, d, J=2.0 Hz, H-2'), 7.25 (1H, dd, J=8.4, 2.0 Hz, H-6), 7.24 (1H, d, J=9.6 Hz, H-5'), 7.22 (1H, d, J=2.0 Hz, H-2), 6.82 (1H, dd, J=16.0, 8.0 Hz, H-8), 5.54 (1H, brd, J=5.2 Hz, H-7'), 5.23 (1H, ddd, J=5.2, 7.2, 5.2 Hz, H-8'), 4.43 (1H, brdd, J=12.0, 7.2 Hz, H-9a'), 4.15 (1H, dd, J=12.0, 5.2 Hz, H-9b'), 3.74 (3H, s, C4-OCH3), 3.72 (3H, s, C4'-OCH3), 13C-NMR (100 MHz, pyridine-d5, δ) 193.4 (C-9), 153.2 (C-7), 152.1 (C-4), 151.0 (C-3), 150.3 (C-3'), 148.4 (C-4'), 134.2 (C-1'), 127.6 (C-1), 127.0 (C-8), 120.7 (C-2'), 116.0 (C-5'), 115.8 (C-5), 112.0 (C-6'), 111.7 (C-2), 111.7 (C-6), 85.6 (C-8'), 73.4 (C7'), 61.9 (C-9'), 55.9 (OCH3), 55.8 (OCH3). Dihydrodehydrodiconiferyl alcohol (6) Brown oil (MeOH); [α]D25 = +64.0o (c=0.27, MeOH); {lit. Agrawal et al., 1983, [α]25D = +45.0o EI/MS m/z: 360 [M+], 342, 446, 330, 327, 310, 297, 283, 165, 152, 151, 137.; IRõ (CHCl3, cm-1) 3450, 2944, 1702}; 1H-NMR (400 MHz, pyridine-d5, δ) 7.33 (1H, d, J=1.6 Hz, H-2'), 7.24 (1H, dd, J=8.4, 1.6 Hz, H-6'), 7.11 (1H, d, J=8.4 Hz, H-5'), 7.06 (1H, s, H-6), 6.91 (1H, s, H-2), 6.06 (1H, d, J=6.4 Hz, H7'), 4.27 (1H, dd, J=10.4, 5.6 Hz, H-9a'), 4.20 (1H, dd, J=10.4, 6.4 Hz, H-9b'), 3.96 (1H, ddd, J=6.4, 6.4, 5.6 Hz, H-8'), 3.92 (2H, d, J=6.4 Hz, H-9), 3.82 (3H, s, H-10), 3.61 (3H, s, H-10'), 2.87 (2H, br. t, J=7.2 Hz, H-7), 2.07 (2H, tt, J=7.2, 6.4 Hz, H-8), 13C-NMR (100 MHz, pyridine-d5, δ) Chemical structures of the 6 lignans isolated from the fruits of Cornus kousa Burg. Cytotoxic Lignans from the Fruits of Cornus kousa 405 148.8 (C-3'), 148.1 (C-4), 147.3 (C-4'), 144.6 (C-3), 136.1 (C-1), 133.9 (C-1'), 130.2 (C-5), 119.7 (C-6'), 117.5 (C-5'), 116.5 (C-6), 113.6 (C-2), 110.8 (C-2'), 88.5 (C-7'), 64.4 (C9'), 61.5 (C-9), 56.3 (C-10), 55.8 (C-10'), 49.8 (C-8'), 36.2 (C-8), 32.8 (C-7). al., 2003). Compound 2 was obtained as a colorless oil, and showed a molecular ion peak (M ) at m/z 356 in the EI/MS spectrum. The IR spectrum (MeOH) showed absorbance bands due to hydroxyl (3256 cm ), aldehyde (2950 cm ), carbonyl (1709 cm ) and olefine (1556 cm ) functionalities. The H-NMR spectrum showed an aldehyde signal at δ 9.82 (1H, d, J=7.6 Hz) and olefine methine signals at 7.49 (1H, d, J=15.6 Hz) and 6.88 (1H, dd, J=15.6, 7.6 Hz), due to a double bond with a trans configuration (J=15.6 Hz). Proton signals at δ 7.48 (1H, br s) and 7.26 (1H, br s) indicated the presence of a 1, 3, 4, 5-tetrasubstituted benzene ring, and at δ 7.31 (1H, d, J=1.6 Hz), 7.25 (1H, d, J=8.0 Hz) and 7.23 (1H, dd, J=8.0, 1.6 Hz) indicated the presence of a 1, 3, 4-trisubstituted benzene ring, as well as two aromatic methoxy groups at δ 3.85 (3H, s) and 3.66 (3H, s). Additionally, an oxygenated methine signal at δ 6.14 (1H, d, J=6.8 Hz), an oxygenated methylene signal at δ 4.26 (2H, t, J=5.6 Hz) and a methine signal at δ 3.99 (1H, dd, J=5.6, 6.8 Hz) were observed. The C-NMR spectrum indicated the presence of an aldehyde at δ 193.7 (C-9), four oxygenated olefine quaternary carbons at δ 152.2 (C-4), 149.1 (C-3'), 148.7 (C-4'), and 145.4 (C3), three olefine quaternary carbons at δ 133.1 (C-1'), 131.6 (C-5), and 128.8 (C-1) and seven olefine methines at δ 153.9 (C-7), 127.0 (C-8), 120.2 (C-2'), 120.0 (C-6), 116.9 (C-3'), 113.7 (C-2), and 111.2 (C-6'), an oxygenated methine carbon at δ 89.9 (C-7'), an methyleneoxy carbon at δ 64.2 (C-9'), two methoxy carbons at δ 56.5 (C-10) and 56.2 (C-10') and a methine carbon at δ 54.5 (C-8'). A trans-configuration between C-7' and C-8' was determined from the coupling constant (J=6.8 Hz). Compound 2 was finally identified as (-)-balanophonin by comparison of several physical and spectral data with those reported in the literature (Haruna et al., 1982; Tsutomu et al., 2005). Compound 3 was obtained as an amorphous powder, and showed a molecular ion peak (M ) at m/z 358 in the EI/MS spectrum. The IR spectrum (MeOH) showed absorbance bands due to hydroxyl (3432 cm ), alkane (3011 cm ) and olefine (1490 cm ) functionalities. Proton signals at δ 7.31 (1H, d, J=2.0 Hz), 7.25 (1H, d, J=8.0 Hz), 7.19 (1H, dd, J=8.0, 2.0)}, {δ 7.19 (1H, d, J=8.0 Hz), 6.99 (1H, J=2.0 Hz) and 6.89 (1H, dd, J=8.0, 2.0) indicated two 1, 3, 4-trisubstituted benzene rings. An oxygenated methine signal at δ 5.33 (1H, d, J=6.0 Hz), two oxygenated methylene signals at δ 4.29 (1H, dd, J=8.0, 6.8 Hz), 4.25 (1H, dd, J=8.0, 6.8 Hz), 4.13 (1H, dd, J=8.0, 7.6 Hz) and 4.06 (1H, dd, J=8.0, 7.6 Hz) and two methoxy signals at δ 3.72 (3H, s) and 3.71 (3H, s) were also observed. In the high magnetic field region, two methylene signals at δ 3.24 (1H, dd, J=13.6, 4.8 Hz), and 2.80 (1H, dd, J=13.6, 10.4 Hz), and two methine signals at δ 3.06 (1H, m) and 2.78 (1H, m) were observed. The C-NMR spectrum + -1 -1 -1 -1 1 Cytotoxicity testing The cytotoxic activities of the compounds were measured using a modified Microculture Tetrazolium (MTT) assay (Mosmann, 1983). The activity of a compound was tested at several concentrations against two cultured human cancer cell lines, HCT-116 (human colon carcinoma cells, originated spontaneously from human colon) and HepG2 (human hepatocellular carcinoma cells, originated spontaneously from human liver). RESULTS AND DISCUSSION When the methanol extract of C. kousa was developed by silica gel TLC, the spots showed a dark blue colorization on spaying with 10% H SO solution and heating, and a blue colorization on spraying 5% with ferric chloride solution, indicating the presence of phenolic compounds in the extract. The methanol extract was partitioned into EtOAc, n-BuOH and H O layers through solvent fractionation. Repeated silica gel, ODS and Sephadex LH-20 column chromatographies of the EtOAc fraction yielded six lignan compounds (1-6). Compound 1, produced as an amorphous powder, showed absorbance bands due to hydroxyl (3420 cm ) and olefine (1680 cm ) functionalities in the IR spectrum (CHCl ), with a molecular ion peak (M ) at m/z 358 in the EI/MS spectrum. The H-NMR spectrum (400 MHz, pyridine-d , δ) showed signals for three olefine methine signals at δ 7.26 (d, J=8.0 Hz), 7.24 (d, J=2.4 Hz) and 7.07 (dd, J=8.0, 2.4 Hz), due to a 1, 3, 4-trisubstituted benzene ring, an oxygenated methine at δ 4.80 (d, J=4.4 Hz), oxygenated methylene signals at δ 4.33 (dd, J=8.8, 6.8 Hz) and 4.01 (dd, J=8.8, 3.6 Hz), a methoxy signal (δ 3.77, 6H, s) and a methine signal (δ 3.23, 2H, ddd). The C-NMR spectrum (100 MHz, pyridine-d , δ) showed signals for two oxygenated olefine quaternary carbons at δ 148.7 (C-3/3') and 147.8 (C-4/4'), an olefine quaternary at δ 133.1 (C-1/1'), three olefine methine carbon at δ 119.7 (C-6/6'), 116.4 (C-5/5') and 110.9 (C-2/2'), which may have derived from a tri-substituted benzene ring. An oxygenated methine carbon at δ 85.5 (C-7/7'), a methylene carbon at δ 72.0 (C-9/9'), a methoxy carbon at δ 56.4 (C-10/10') and a methine carbon signal at δ 54.9 (C-8/8') were also observed. Thus, the above data indicated that compound 1 had a lignan structure. Finally, compound 1 was identified as (+)-pinoresinol by comparison of several physical and spectral data with those reported in the literature (Li et 2 4 2 -1 -1 + 3 1 5 13 5 13 + -1 -1 -1 13 406 D.-Y. Lee et al. indicated the presence of four oxygenated olefine quaternary carbons at δ 148.5(C-3'/3), 147.3 (C-4'), and 146.4 (C-4)}, two olefine quaternary carbons at δ 135.9 (C-1') and 132.6 (C-1), six olefine methine carbons at δ 121.7 (C-6), 119.4 (C-6'), 116.5 (C-5), 116.3 (C-5'), 113.1 (C-2), and 110.5 (C-2'), an oxygenated methine carbon at δ 83.4 (C-7'), two oxygenated methylene carbons at δ 73.2 (C-9') and 60.1 (C-9) and two methoxy carbons at δ 56.5 (C-10/ C-10'). In the high magnetic field region, two methine signals at δ 53.9 (C-8') and 43.4 (C-8) and a methylene signal at 33.5 (C-7) were also observed. Thus, the structure of compound 3 was identified as (+)-lariciresinol by comparison of several physical and spectral data with those reported in the literature (Okuyama et al., 1995). Compounds 4 and 5 were shown to be a mixture of diastereomers, with a ratio of about 1.5:1, which was deduced from the intensity of the signals in the H-NMR spectra. The compounds were obtained as amorphous powders, and both showed molecular ion peaks (M ) at m/z 374 in their EI/MS spectra. The IR spectra (MeOH) showed absorbance bands due to hydroxyl (3389 cm ), aldehyde (2922 cm ), carbonyl (1665 cm ) and olefine (1595 cm ) functionalities. The H-NMR spectra showed slight differences between the two diastereomers, such as characteristic aldehyde signals at δ 9.78 (erythro-H9) and 9.77 (threo-H9), trans–conformational olefine signals at δ 6.85 (erythro-H8), 6.82 (threo-H8), 7.43 (erythro-H7) and 7.45 (threo-H7), oxygenated methine and methylene signals at δ 5.56 (erythro-H7'), 5.54 (threo-H7'), 5.19 (erythro-H8'), 5.23 (threo-H8'), 4.17 (erythro-H9a'), 4.15 (threo-H9a'), 4.53 (erythro-H9b') and 4.43 (threo-H9b'). The C-NMR spectra also showed slight differences between the erythroand threo-isomers. Thus, the structures of compounds 4 and 5 were identified as erythro- and threo-guaiacylglycerolβ-coniferyl aldehyde ethers, respectively, by comparison of several physical and spectral data with those reported in the literature (Miki et al., 1980; Takeshi et al., 1980; Li et al., 2003). Compound 6 was identified as the well-known neolignan, dihydrodehydrodiconiferyl alcohol, by comparison with an authentic sample ([α] , IR, mass spectrum, H- and CNMR). The trans-configuration was determined from the coupling constant (J=6.4 Hz) between H-7 and H-8 (Agrawal et al., 1983). All 6 compounds were isolated for the first time from this plant. (+)-Pinoresinol (1) and (+)-lariciresinol (3) have previously been reported to exhibit antioxidant (Guelcin et al,. 2006), antifungal and antibacterial activities (Cespedes et al,. 2006). (-)-Balanophonin (2) has also been found to exhibit major cytotoxicity (Jang et al,. 2003), and dihydrodehydrodiconiferyl alcohol (compounds 4 and 5) has also been reported to show cytotoxic (Chen et al,. 2006) and antileishmanial activities (Van et al., 2005). 1 + -1 -1 -1 -1 1 13 1 D 13 The cytotoxicities of compounds 1~6 from the fruits of Burg. against human colon carcinoma (HCT-116) and human hepatocellular carcinoma (HepG2) cell lines Table I. Cornus kousa IC values a) 50 Cancer Cell Lines Compounds HCT-116 HepG2 Compound 1 57.6 ± 1.0 >100 >171.3 ± 0.6 Compound 2 19.1 ± 0.6 Compound 3 54.8 ± 0.7 >100 >157.3 ± 1.1 Compound 4, 5 30.2 ± 1.1 Compound 6 35.7 ± 0.9 >155.0 ± 0.2 19.2 ± 0.9 >127.3 ± 0.5 Doxorubicin IC50 values refer to the 50% inhibition concentration (µg/mL), and were calculated from regression lines using five different concentrations with triplicate determinations. During our search for cytotoxic compounds from natural sources, the MeOH extract of the fruits of C. kousa was found to exhibit significant cytotoxic effects on two human cancer cell lines. Thus, we pursued the isolation of the cytotoxic constituents from the MeOH extract of C. kousa. All 6 isolated compounds were tested for their cytotoxic activities against the HCT-116 and HepG2 cancer cell lines in vitro using the MTT assay method, the results (IC values) of which are shown in Table I. All 6 compounds exhibited cytotoxic activity against the HCT-116 cell line, with IC values ranging from 19.1 to 71.3 µg/mL, although these results were slightly lower than those for the positive control, doxorubicin (9.2±0.9 µg/mL). The cytotoxicities of the neolignans, compounds 2, 4, 5 and 6, were higher than those of the lignans, compounds 1 and 2, in the cytotoxicity tests. The aldehyde group in the skeleton structure of compound 2 might be a key factor in enhancing the cytotoxic activity, which may explain why this compound showed a similar activity to that of the positive control. Most compounds, with the exceptions of 1 and 3, also exhibited cytotoxicities against the HepG2 cell line. As with the HCT-116 cell line, the cytotoxicities of the neolignans against the HepG2 cell line was higher than those of the lignans. Herein, the cytotoxicities of compounds 1-6 against these cell lines have been tested for the first time. 50 50 ACKNOWLEDGEMENTS This work was supported by the BioGreen 21 Program from the Rural Development Administration, Republic of Korea, and by a grant from the Korea Science and Engineering Foundation through the Plant Metabolism Research Center, Kyung Hee University. Cytotoxic Lignans from the Fruits of Cornus kousa REFERENCES Abe, F., Yamauchi, T., and Wan, A., Cerbera., Part 7. 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