- Email: [email protected]
A new coumarin from Sarcandra glabra
Fitoterapia 81 (2010) 472–474
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
A new coumarin from Sarcandra glabra Shixiu Feng, Liangxiong Xu, Min Wu, Jing Hao, Samuel X. Qiu, Xiaoyi Wei ⁎ Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723, Tianhe District, Guangzhou 510650, China
a r t i c l e
i n f o
Article history: Received 23 August 2009 Received in revised form 19 December 2009 Accepted 24 December 2009 Available online 7 January 2010
a b s t r a c t Sarcandracoumarin (1), the first coumarin having a 1-phenylethyl substituent at the C-3 position, was isolated along with eleven known phenolic compounds from the water extract of Sarcandra glabra. Its structure was elucidated on the basis of spectroscopic data. Compound 1 exhibited moderate or weak cytotoxicity against several tumor cell lines. © 2010 Elsevier B.V. All rights reserved.
Keywords: Sarcandra glabra Sarcandra Coumarins Sarcandracoumarin
1. Introduction
2. Experimental
Sarcandra glabra (Thunb.) Nakai (Chloranthaceae), an evergreen subshrub widely distributed in the southern parts of China and in Southeast Asian, is a traditional Chinese medicine possessing antibacterial, anti-inflammatory, and antitumor effects [1]. Several medicaments prepared from this plant have been used in clinic in China [1]. Previous investigation of this plant reported the isolation of flavonoids, sesquiterpenes, triterpenoids, coumarins, and other phenolics [2–11]. In continuation of our phytochemical studies on the medicinal plants growing in South China, the water extract of this plant was investigated and a new coumarin, sarcandracoumarin (1), was isolated along with eleven known compounds including isofraxidin, syringic acid, 1,2-benzenediol, vanillic acid, 3,4-dihydroxybenzoic acid, rosmarinic acid, tyrosol, vanilloloside, 4-O-caffeoylshikimic acid, 5-O-caffeoylshikimic acid, and 3-O-caffeoylshikimic acid. Herein, we report the isolation and structure elucidation of this new compound.
2.1. General Optical rotations were obtained on a Perkin-Elmer 341 polarimeter with MeOH as solvent. UV spectra were recorded in MeOH on a Perkin-Elmer Lambda 35 UV–vis spectrophotometer. 1H (400 MHz), 13C (100 MHz), and 2D NMR spectra were recorded on a Bruker DRX-400 instrument using the residual solvent peak as reference. HRESIMS data were obtained on a Bruker Bio TOF IIIQ mass spectrometer in negative-ion mode. ESIMS were collected on an MDS SCIEX API 2000 LC/MS/MS instrument. Preparative HPLC was run with a Shimazu LC-6A pump and a Shimazu RID-10A refractive index detector using an XTerra prep MS C18 column (10 μm, 300 × 19 mm). For column chromatography, silica gel 60 (100−200 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Develosil ODS (75 μm, Nomura Chemical Co. Ltd., Japan), and Sephadex LH-20 were used. 2.2. Plant material
⁎ Corresponding author. Tel.: + 86 20 37252538; fax: + 86 20 37252537. E-mail address: [email protected] (X. Wei). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.12.009
The whole plant of Sarcandra glabra, collected from Xinfeng, Jiangxi, China, in Automn, 2006, was supplied by Ms. Meixiang Yao, Jiangzhong Pharmaceutical Co. Ltd.,
S. Feng et al. / Fitoterapia 81 (2010) 472–474
Nanchang, Jiangxi, China. The plant was identified by Prof. Fuwu Xing, South China Botanical Garden, Chinese Academy of Sciences. A voucher sample (No. 426390) was deposited at the herbarium of South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China. 2.3. Extraction and isolation The powdered dry whole plant of Sarcandra glabra (10.0 kg) was extracted 3 times with water at 90 ± 2 °C. The water extract (1200 g) was partitioned successively with EtOAc and n-BuOH. The EtOAc-soluble extract (46.0 g) was subjected to silica gel column chromatography (CC) and eluted with CHCl3–MeOH mixtures with increasing polarities (98:2–50:50) to yield six fractions (1–6). Fraction 2 (6.12 g) was further separated by silica gel CC using petroleum ether– acetone (80:20–65:35) to afford two subfractions (2a and 2b). Subfraction 2a was subjected to recrystalization with MeOH to give 3,4-dihydroxybenzoic acid (800 mg), and subfraction 2b was separated by ODS CC using 70% MeOH, followed by Sephadex LH-20 CC using MeOH to afford isofraxidin (950 mg). Fraction 3 (2.20 g) was also subjected to silica gel CC using petroleum ether–acetone (75:25–65:35) to afford two subfractions (3a and 3b). Subfraction 3a was further separated by Sephadex LH-20 CC using MeOH to yield 1,2-benzenediol (210 mg). Subfraction 3b was separated by ODS CC using 25% MeOH, followed by Sephadex LH-20 CC using MeOH to obtain vanillic acid (50 mg) and syringic acid (110 mg). Fraction 4 (3.40 g) and Fr.5 (16.5 g) were both separated by ODS CC using aqueous MeOH (from 10% to 65%), followed by Sephadex LH-20 CC using MeOH to afford 1 (18 mg) and rosmarinic acid (850 mg), respectively. The n-BuOH-soluble extract (184.0 g) was subjected to silica gel CC and eluted with CHCl3–MeOH (from 98:2 to 50:50) to give six fractions (7–12). Fraction 8 (0.90 g) was separated by ODS CC using 30% MeOH to afford vanilloloside (17 mg). Fraction 9 (4.5 g) was separated by ODS CC using MeOH (from 30% to 50%), followed by preparative HPLC using 30% MeOH in 0.025% formic acid to afford 4-O-caffeoylshikimic acid (31 mg) and 5-O-caffeoylshikimic acid (8 mg). Fraction 10 (3.9 g) was chromatographed on an ODS column using aqueous MeOH (from 10% to 30%) to afford four subfractions (10a–d). Subfraction 10c was applied to preparative HPLC using 10% MeOH to yield tyrosol (3 mg) and 3-O-caffeoylshikimic acid (3 mg). Sarcandracoumarin (1) (Fig. 1): Brownish solid, [α]20D +3° (c 1.0, MeOH); UV λmax (MeOH) nm (log ε): 205 (4.92), 230 (sh.), 288 (4.12), 342 (4.26); IR vmax (KBr) cm− 1: 3382, 1693,
Fig. 1. The structure of compound 1.
473
1587, 1500, 1463, 1280, 1199, 1106, 1045; 1H NMR (400 MHz, DMSO-d6) δ: 1.43 (3H, d, J = 7.1 Hz, 8′–CH3), 3.79 (6H, s, 6– OCH3 and 8–OCH3), 3.97 (1H, q, J = 7.1 Hz, H-7′), 6.52 (1H, dd, J = 8.4 and 2.0 Hz, H-6′), 6.63 (1H, d, J = 2.0 Hz, H-2′), 6.64 (1H, d, J = 8.4 Hz, H-5′), 7.02 (1H, s, H-5), 7.66 (1H, s, H4); 13C NMR (100 MHz, DMSO-d6) δ: 160.6 (C-2), 145.6 (C-6), 145.0 (C-4′), 143.7 (C-5′), 143.0 (C-7), 141.6 (C-9), 138.5 (C-4), 135.9 (C-2′), 134.5 (C-8), 129.3 (C-3), 118.1 (C-7′), 115.5 (C-6′), 114.9 (C-3′), 110.6 (C-10), 104.3 (C-5), 60.7 (6–OCH3), 56.1 (8–OCH3), 38.0 (C-7′), 20.3 (C-8′); ESIMS m/z: 359 [M + H]+, 381 [M + Na]+, 357 [M–H]−; HRESIMS m/z 357.0952 [M–H]− (calcd for C19H17O7, 357.0974). 2.4. Cytotoxic activity evaluation Cytotoxicity was determined by MTT method [12] using human lung cancer (A549), human pulmonary carcinoma (LAC), human cervical carcinoma (HeLa) and human hepatoma (HepG2) cells grown in RPMI-1640 medium plus 10% heat-inactivated fetal bovine serum. The assays were performed in 96-well microtiter plates. Serial two-fold dilutions of compound 1 were made in DMSO. Then 5 μl of each serial solution was added to 195 μl (about 10,000 cells) culture medium in wells. After incubation at 37 °C for 72 h, 10 μl of MTT (5 g/L) was added to each well and incubated for 4 more hours, and then liquid in the wells was removed. DMSO (200 μl) was added to each well. The absorbance was recorded on a microplate reader (Bio-Rad model 550) at a wavelength of 570 nm. The IC50 values were calculated from reduction of absorbance in the control assay which was treated with 2.5% DMSO alone. The IC50 values for compound 1 were determined to be 76.4 (A549), 158.0 (LAC), 49.3 (HeLa), and 196.7 (HepG2) μg/mL. 3. Results and discussion The hot water extract of the whole plant of S. glabra was fractionated successively with EtOAc and n-BuOH. Separation by a combination of silica gel, Sephadex LH-20, and ODS column chromatography and HPLC afforded a new compound (1) (Fig. 1) and six known compounds, isofraxidin, rosmarinic acid, 3,4-dihydroxybenzoic acid [4], syringic acid [13], 1,2benzenediol, and vanillic acid [14], from the EtOAc-soluble fraction, and tyrosol [15], vanilloloside [16], 4-O-caffeoylshikimic acid [17], 5-O-caffeoylshikimic acid [18], and 3-Ocaffeoylshikimic acid [19] from the n-BuOH-soluble fraction. The structures of known compounds were determined by interpretation of their spectroscopic data as well as by comparison with reported data. Sarcandracoumarin (1), obtained as a brownish solid, was determined to have the molecular formula C19H18O7 from the HRESIMS and ESIMS data in combination with the 1H and 13C NMR spectra. The IR spectrum exhibited strong absorptions at 1693, 1587, and 1500 cm− 1 and the UV spectrum gave an absorption maximum at 342 nm, in accord with a coumarin nucleus [20]. The 1H NMR spectrum showed the presence of a 1,2,4-trisubstituted benzene ring [δ 6.64 (1H, d, J = 8.4 Hz, H-5′), 6.63 (1H, d, J = 2.0 Hz, H-2′), and 6.52 (1H, dd, J = 2.0, 8.4 Hz, H-6′)], two aromatic methoxy groups [δ 3.79 (6H, s, 6–OCH3 and 8–OCH3)], a methylmethine (CHCH3) function [δ 3.97 (1H, q, J = 7.1 Hz, H-7′) and 1.43 (3H, d, J = 7.1 Hz,
474
S. Feng et al. / Fitoterapia 81 (2010) 472–474
196.7 μg/mL) and LAC (IC50 = 158.0 μg/mL) and moderate activity against HeLa (IC50 = 49.3 μg/mL) and A549 (IC50 = 76.4 μg/mL).
Acknowledgements
Fig. 2. Key HMBC correlations of 1.
H3-8′)], and three phenolic hydroxy groups [δ 9.73 (1H, br s, 7–OH), 8.75 (1H, br s, 3′-OH), and 8.72 (1H, br s, 4′–OH)]. Besides, the spectrum exhibited two singlets at δ 7.66 (1H, H4) and 7.02 (1H, H-5) for two aromatic protons. In the 13C NMR spectrum, 14 aromatic carbon resonances were observed between δ 104.3 and 145.6 ppm, of which nine were quaternary carbons as indicated by DEPT experiments. In addition, the 13C NMR and DEPT spectra indicated the presence of a conjugated ester carbonyl [δ 160.6 (C-2)], two methoxy groups [δ 60.7 (6–OCH3) and 56.1 (8–OCH3)], a methine [δ 38.7 (C-7′)], and a methyl group [δ 20.3 (C-8′)]. In the HMBC spectrum (Fig. 2), long-range correlations from H-4 to C-2, C-3, C-5, C-9, and C-10, from H-5 to C-4, C-6, C-7, C-9, and C-10, from 6–OCH3 to C-6, from 8–OCH3 to C-8, from H-2′ and H-6′ to C-4′, and from H-2′, H-3′, and H-6′ to C-1′ were observed, suggesting a 7-hydroxy-6,8-dimethoxycoumarin moiety and a 3,4-dihydroxyphenyl group. Furthermore, as can be seen in Fig. 2, HMBC correlations from H3-8′ to C-3 and C-1′ and from H-7′ to C-2, C-3, C-4, C-1′, C-2′, and C-6′ were observed, indicating a 3,1′-linkage via the methylmethine bridge. Thus, the structure of compound 1 was elucidated as 3-(1-(3,4dihydroxyphenyl)ethyl)-7-hydroxy-6,8-dimethoxycoumarin. This compound has an asymmetric center (C-7′) and showed a small optical rotation value {[α]20D + 3° (c 1.0, MeOH)}. However, it showed no cotton effect in the CD spectrum. Its absolute configuration is still unclear. To our knowledge, this compound is the first coumarin having a 1-phenylethyl substituent at the C-3 position. In evaluation for cytotoxicity using MTT method [20], compound 1 exhibited weak activity against HepG2 (IC50 =
We thank Ms. M.-X. Yao, Jiangzhong Pharmaceutical Co. Ltd., Nanchang, Jiangxi, for supply of the plant material, and Prof. F.-W. Xing, South China Botanical Garden, Chinese Academy of Sciences, for authentication of the plant material. This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant no. KSCX2-YW-R-218).
References [1] The state pharmacopoeia commission of People's Republic of China. Pharmacopoeia of the People's Republic of China, vol. 1. Beijing: Chemical Industry Press; 2005. [2] Zhu LP, Li Y, Yang JZ, Zuo L, Zhang DM. China J Chinese Materia Med 2008;33:155. [3] Huang MJ, Zeng GY, Tan JB, Li YL, Tan GS, Zhou YJ. China J Chinese Materia Med 2008;33:1700. [4] Huang MJ, Li YL, Zeng GY, Yuan WM, Tan JB, Tan GS, et al. Cent South Pharmacy 2007;5:459. [5] Wang F, Yuan ST, Zhu DN. Chin J Nat Med 2007;5:174. [6] Zhou XY, Gao HY, Wu B, Wang YS, Wang SJ, Yang SL, et al. Chin Tradit Herb Drugs 2007;38:354. [7] Li Y, Zhang DM, Li JB, Yu SS, Li Y, Luo YM. J Nat Prod 2006;69:616. [8] Luo YM, Liu AH, Zhang DM, Huang LQ. J Asian Nat Prod Res 2005;7:829. [9] Tsui WP, Brown GD. Phytochemistry 1996;43:819. [10] Uchida M, Kusano G, Kondo Y, Nozoe S. Heterocycles 1978;9:139. [11] Zhu LP, Li Y, Yang JZ, Zuo L, Zhang DM. J Asian Nat Prod Res 2008;10: 541. [12] Mosmann T. J Immunol Methods 1983;65:51. [13] Zhang L, Cai XH, Gao HY, Song BH, Huang J, Wu LJ. J Shengyang Pharm Univ 2009;26:15. [14] Wang SJ, Pei YH. J Shengyang Pharm Univ 2000;17:256. [15] Chen JJ, Chen JS, Chen SY, Zhou J. Acta Bot Yunnanica 1999;21:525. [16] Xia WB, Xue Z, Li S, Wang SJ, Yang Y, He DX. China J Chinese Materia Med 2005;30:1827. [17] Sakai T, Matsushita Y, Sugamoto K, Uchida K. Biosci Biotechnol Biochem 1997;61:1399. [18] Veit M, Weidner C, Strack D, Wray V, Witte L, Czygan FC. Phytochemistry 1992;31:3483. [19] Maier V, Metzler D, Huber A. Biochem Biophys Res Commun 1964;14: 124. [20] Salvatore DR, Mitova M, Handjieva NV. Phytochemistry 2002;59:447.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
A new coumarin from Sarcandra glabra Shixiu Feng, Liangxiong Xu, Min Wu, Jing Hao, Samuel X. Qiu, Xiaoyi Wei ⁎ Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road 723, Tianhe District, Guangzhou 510650, China
a r t i c l e
i n f o
Article history: Received 23 August 2009 Received in revised form 19 December 2009 Accepted 24 December 2009 Available online 7 January 2010
a b s t r a c t Sarcandracoumarin (1), the first coumarin having a 1-phenylethyl substituent at the C-3 position, was isolated along with eleven known phenolic compounds from the water extract of Sarcandra glabra. Its structure was elucidated on the basis of spectroscopic data. Compound 1 exhibited moderate or weak cytotoxicity against several tumor cell lines. © 2010 Elsevier B.V. All rights reserved.
Keywords: Sarcandra glabra Sarcandra Coumarins Sarcandracoumarin
1. Introduction
2. Experimental
Sarcandra glabra (Thunb.) Nakai (Chloranthaceae), an evergreen subshrub widely distributed in the southern parts of China and in Southeast Asian, is a traditional Chinese medicine possessing antibacterial, anti-inflammatory, and antitumor effects [1]. Several medicaments prepared from this plant have been used in clinic in China [1]. Previous investigation of this plant reported the isolation of flavonoids, sesquiterpenes, triterpenoids, coumarins, and other phenolics [2–11]. In continuation of our phytochemical studies on the medicinal plants growing in South China, the water extract of this plant was investigated and a new coumarin, sarcandracoumarin (1), was isolated along with eleven known compounds including isofraxidin, syringic acid, 1,2-benzenediol, vanillic acid, 3,4-dihydroxybenzoic acid, rosmarinic acid, tyrosol, vanilloloside, 4-O-caffeoylshikimic acid, 5-O-caffeoylshikimic acid, and 3-O-caffeoylshikimic acid. Herein, we report the isolation and structure elucidation of this new compound.
2.1. General Optical rotations were obtained on a Perkin-Elmer 341 polarimeter with MeOH as solvent. UV spectra were recorded in MeOH on a Perkin-Elmer Lambda 35 UV–vis spectrophotometer. 1H (400 MHz), 13C (100 MHz), and 2D NMR spectra were recorded on a Bruker DRX-400 instrument using the residual solvent peak as reference. HRESIMS data were obtained on a Bruker Bio TOF IIIQ mass spectrometer in negative-ion mode. ESIMS were collected on an MDS SCIEX API 2000 LC/MS/MS instrument. Preparative HPLC was run with a Shimazu LC-6A pump and a Shimazu RID-10A refractive index detector using an XTerra prep MS C18 column (10 μm, 300 × 19 mm). For column chromatography, silica gel 60 (100−200 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Develosil ODS (75 μm, Nomura Chemical Co. Ltd., Japan), and Sephadex LH-20 were used. 2.2. Plant material
⁎ Corresponding author. Tel.: + 86 20 37252538; fax: + 86 20 37252537. E-mail address: [email protected] (X. Wei). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.12.009
The whole plant of Sarcandra glabra, collected from Xinfeng, Jiangxi, China, in Automn, 2006, was supplied by Ms. Meixiang Yao, Jiangzhong Pharmaceutical Co. Ltd.,
S. Feng et al. / Fitoterapia 81 (2010) 472–474
Nanchang, Jiangxi, China. The plant was identified by Prof. Fuwu Xing, South China Botanical Garden, Chinese Academy of Sciences. A voucher sample (No. 426390) was deposited at the herbarium of South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China. 2.3. Extraction and isolation The powdered dry whole plant of Sarcandra glabra (10.0 kg) was extracted 3 times with water at 90 ± 2 °C. The water extract (1200 g) was partitioned successively with EtOAc and n-BuOH. The EtOAc-soluble extract (46.0 g) was subjected to silica gel column chromatography (CC) and eluted with CHCl3–MeOH mixtures with increasing polarities (98:2–50:50) to yield six fractions (1–6). Fraction 2 (6.12 g) was further separated by silica gel CC using petroleum ether– acetone (80:20–65:35) to afford two subfractions (2a and 2b). Subfraction 2a was subjected to recrystalization with MeOH to give 3,4-dihydroxybenzoic acid (800 mg), and subfraction 2b was separated by ODS CC using 70% MeOH, followed by Sephadex LH-20 CC using MeOH to afford isofraxidin (950 mg). Fraction 3 (2.20 g) was also subjected to silica gel CC using petroleum ether–acetone (75:25–65:35) to afford two subfractions (3a and 3b). Subfraction 3a was further separated by Sephadex LH-20 CC using MeOH to yield 1,2-benzenediol (210 mg). Subfraction 3b was separated by ODS CC using 25% MeOH, followed by Sephadex LH-20 CC using MeOH to obtain vanillic acid (50 mg) and syringic acid (110 mg). Fraction 4 (3.40 g) and Fr.5 (16.5 g) were both separated by ODS CC using aqueous MeOH (from 10% to 65%), followed by Sephadex LH-20 CC using MeOH to afford 1 (18 mg) and rosmarinic acid (850 mg), respectively. The n-BuOH-soluble extract (184.0 g) was subjected to silica gel CC and eluted with CHCl3–MeOH (from 98:2 to 50:50) to give six fractions (7–12). Fraction 8 (0.90 g) was separated by ODS CC using 30% MeOH to afford vanilloloside (17 mg). Fraction 9 (4.5 g) was separated by ODS CC using MeOH (from 30% to 50%), followed by preparative HPLC using 30% MeOH in 0.025% formic acid to afford 4-O-caffeoylshikimic acid (31 mg) and 5-O-caffeoylshikimic acid (8 mg). Fraction 10 (3.9 g) was chromatographed on an ODS column using aqueous MeOH (from 10% to 30%) to afford four subfractions (10a–d). Subfraction 10c was applied to preparative HPLC using 10% MeOH to yield tyrosol (3 mg) and 3-O-caffeoylshikimic acid (3 mg). Sarcandracoumarin (1) (Fig. 1): Brownish solid, [α]20D +3° (c 1.0, MeOH); UV λmax (MeOH) nm (log ε): 205 (4.92), 230 (sh.), 288 (4.12), 342 (4.26); IR vmax (KBr) cm− 1: 3382, 1693,
Fig. 1. The structure of compound 1.
473
1587, 1500, 1463, 1280, 1199, 1106, 1045; 1H NMR (400 MHz, DMSO-d6) δ: 1.43 (3H, d, J = 7.1 Hz, 8′–CH3), 3.79 (6H, s, 6– OCH3 and 8–OCH3), 3.97 (1H, q, J = 7.1 Hz, H-7′), 6.52 (1H, dd, J = 8.4 and 2.0 Hz, H-6′), 6.63 (1H, d, J = 2.0 Hz, H-2′), 6.64 (1H, d, J = 8.4 Hz, H-5′), 7.02 (1H, s, H-5), 7.66 (1H, s, H4); 13C NMR (100 MHz, DMSO-d6) δ: 160.6 (C-2), 145.6 (C-6), 145.0 (C-4′), 143.7 (C-5′), 143.0 (C-7), 141.6 (C-9), 138.5 (C-4), 135.9 (C-2′), 134.5 (C-8), 129.3 (C-3), 118.1 (C-7′), 115.5 (C-6′), 114.9 (C-3′), 110.6 (C-10), 104.3 (C-5), 60.7 (6–OCH3), 56.1 (8–OCH3), 38.0 (C-7′), 20.3 (C-8′); ESIMS m/z: 359 [M + H]+, 381 [M + Na]+, 357 [M–H]−; HRESIMS m/z 357.0952 [M–H]− (calcd for C19H17O7, 357.0974). 2.4. Cytotoxic activity evaluation Cytotoxicity was determined by MTT method [12] using human lung cancer (A549), human pulmonary carcinoma (LAC), human cervical carcinoma (HeLa) and human hepatoma (HepG2) cells grown in RPMI-1640 medium plus 10% heat-inactivated fetal bovine serum. The assays were performed in 96-well microtiter plates. Serial two-fold dilutions of compound 1 were made in DMSO. Then 5 μl of each serial solution was added to 195 μl (about 10,000 cells) culture medium in wells. After incubation at 37 °C for 72 h, 10 μl of MTT (5 g/L) was added to each well and incubated for 4 more hours, and then liquid in the wells was removed. DMSO (200 μl) was added to each well. The absorbance was recorded on a microplate reader (Bio-Rad model 550) at a wavelength of 570 nm. The IC50 values were calculated from reduction of absorbance in the control assay which was treated with 2.5% DMSO alone. The IC50 values for compound 1 were determined to be 76.4 (A549), 158.0 (LAC), 49.3 (HeLa), and 196.7 (HepG2) μg/mL. 3. Results and discussion The hot water extract of the whole plant of S. glabra was fractionated successively with EtOAc and n-BuOH. Separation by a combination of silica gel, Sephadex LH-20, and ODS column chromatography and HPLC afforded a new compound (1) (Fig. 1) and six known compounds, isofraxidin, rosmarinic acid, 3,4-dihydroxybenzoic acid [4], syringic acid [13], 1,2benzenediol, and vanillic acid [14], from the EtOAc-soluble fraction, and tyrosol [15], vanilloloside [16], 4-O-caffeoylshikimic acid [17], 5-O-caffeoylshikimic acid [18], and 3-Ocaffeoylshikimic acid [19] from the n-BuOH-soluble fraction. The structures of known compounds were determined by interpretation of their spectroscopic data as well as by comparison with reported data. Sarcandracoumarin (1), obtained as a brownish solid, was determined to have the molecular formula C19H18O7 from the HRESIMS and ESIMS data in combination with the 1H and 13C NMR spectra. The IR spectrum exhibited strong absorptions at 1693, 1587, and 1500 cm− 1 and the UV spectrum gave an absorption maximum at 342 nm, in accord with a coumarin nucleus [20]. The 1H NMR spectrum showed the presence of a 1,2,4-trisubstituted benzene ring [δ 6.64 (1H, d, J = 8.4 Hz, H-5′), 6.63 (1H, d, J = 2.0 Hz, H-2′), and 6.52 (1H, dd, J = 2.0, 8.4 Hz, H-6′)], two aromatic methoxy groups [δ 3.79 (6H, s, 6–OCH3 and 8–OCH3)], a methylmethine (CHCH3) function [δ 3.97 (1H, q, J = 7.1 Hz, H-7′) and 1.43 (3H, d, J = 7.1 Hz,
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S. Feng et al. / Fitoterapia 81 (2010) 472–474
196.7 μg/mL) and LAC (IC50 = 158.0 μg/mL) and moderate activity against HeLa (IC50 = 49.3 μg/mL) and A549 (IC50 = 76.4 μg/mL).
Acknowledgements
Fig. 2. Key HMBC correlations of 1.
H3-8′)], and three phenolic hydroxy groups [δ 9.73 (1H, br s, 7–OH), 8.75 (1H, br s, 3′-OH), and 8.72 (1H, br s, 4′–OH)]. Besides, the spectrum exhibited two singlets at δ 7.66 (1H, H4) and 7.02 (1H, H-5) for two aromatic protons. In the 13C NMR spectrum, 14 aromatic carbon resonances were observed between δ 104.3 and 145.6 ppm, of which nine were quaternary carbons as indicated by DEPT experiments. In addition, the 13C NMR and DEPT spectra indicated the presence of a conjugated ester carbonyl [δ 160.6 (C-2)], two methoxy groups [δ 60.7 (6–OCH3) and 56.1 (8–OCH3)], a methine [δ 38.7 (C-7′)], and a methyl group [δ 20.3 (C-8′)]. In the HMBC spectrum (Fig. 2), long-range correlations from H-4 to C-2, C-3, C-5, C-9, and C-10, from H-5 to C-4, C-6, C-7, C-9, and C-10, from 6–OCH3 to C-6, from 8–OCH3 to C-8, from H-2′ and H-6′ to C-4′, and from H-2′, H-3′, and H-6′ to C-1′ were observed, suggesting a 7-hydroxy-6,8-dimethoxycoumarin moiety and a 3,4-dihydroxyphenyl group. Furthermore, as can be seen in Fig. 2, HMBC correlations from H3-8′ to C-3 and C-1′ and from H-7′ to C-2, C-3, C-4, C-1′, C-2′, and C-6′ were observed, indicating a 3,1′-linkage via the methylmethine bridge. Thus, the structure of compound 1 was elucidated as 3-(1-(3,4dihydroxyphenyl)ethyl)-7-hydroxy-6,8-dimethoxycoumarin. This compound has an asymmetric center (C-7′) and showed a small optical rotation value {[α]20D + 3° (c 1.0, MeOH)}. However, it showed no cotton effect in the CD spectrum. Its absolute configuration is still unclear. To our knowledge, this compound is the first coumarin having a 1-phenylethyl substituent at the C-3 position. In evaluation for cytotoxicity using MTT method [20], compound 1 exhibited weak activity against HepG2 (IC50 =
We thank Ms. M.-X. Yao, Jiangzhong Pharmaceutical Co. Ltd., Nanchang, Jiangxi, for supply of the plant material, and Prof. F.-W. Xing, South China Botanical Garden, Chinese Academy of Sciences, for authentication of the plant material. This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant no. KSCX2-YW-R-218).
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