中药大血藤Sargentodoxa cuneata抗癌活性成分的分离与鉴定
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摘要
中药大血藤系大血藤科藤本植物，在我国分布广泛，其藤茎具有清热解毒、活血通经及祛风湿等功效。迄今已从大血藤中分离得到多种活性化合物，但有关大血藤抗肿瘤活性成分的研究还未见报道。本文采用小鼠乳腺癌温度敏感型细胞tsFT210，以细胞周期抑制及细胞凋亡诱导为活性筛选模型，结合活性追踪的方法对大血藤中的活性成分进行了研究。
大血藤茎干药材用乙醇提取，依次用乙酸乙酯、正丁醇萃取，分别进行活性测试，结果表明乙酸乙酯和正丁醇萃取物均具有显著的G_2/M期抑制作用。对活性部位乙酸乙酯提取物采用减压柱层析(VLC)、凝胶柱层析(Sephadex LH-20)、反相硅胶柱层析(ODS)和反相制备高效液相(PHPLC)等分离方法，得到6个单体化合物(1，2，5，9，10，13)；对活性部位正丁醇提取物采用凝胶柱层析(Sephadex LH-20)、大孔树脂柱层析(HP-20,MCI)、反相制备高效液相(PHPLC)等分离方法，分离得到7个化合物(3，4，6，7，8，11，12)。利用理化性质和光谱学方法(UV、IR、MS、1D-NMR和2D-NMR)阐明了其中11个化合物的结构，分别为绿原酸(1)，N-(对羟基苯乙基)阿魏酸酰胺(2)，[2R，3R，4S]-1，2，3，4-四氢-6-羟基-4(4′-羟基-3′，5′-二甲氧苯基)-2-羟甲基-5，7二甲氧基-3-萘甲基-O-β-D-吡喃葡萄糖苷(3)，3，5-O-二甲基没食子酸(5)，(-)-表儿茶素(6)，缩合鞣质B_2[epicatechin(4β→8)epicatechin)(7)，原儿茶酸(9)，对-羟基苯乙醇(10)，对-羟基苯乙基-β-D-吡喃葡萄糖苷(毛柳苷，11)，3，4-二羟基-苯乙基-β-D-吡喃葡萄糖苷(红景天苷，12)。同时推定了化合物4的平面构型为1，2，3，4-四氢-6-羟基-4(4′-羟基-3′，5′-二甲氧苯基)-2-羟甲基-5，7二甲氧基-3-萘甲基-O-β-D-吡喃葡萄糖苷，其波谱学数据与迄今所报道的四个具有相同平面结构的化合物有较大的差异，因此化合物4可能为一新木脂素苷类化合物；推测了化合物8可能为具有新连接方式的五聚体缩合鞣质，化合物13的结构正在解析中。
中药大血藤命崔竿”勿由尤口c期eata抗癌活性成分的分离与鉴
摘要
化合物的活性测试结果表明:化合物4，8对tsFTZ10小鼠乳腺癌细胞具有
显著GZ彻期抑制作用;化合物1，2具有一定的G夕G，期抑制作用;化合物13
具有一定的GZ彻期抑制作用。
本文从大血藤植物中共分离得到13个化合物，其中10个化合物(1一8，10，
12)为首次从该属植物中分离得到;获得5个活性化合物(1，2，4，8，13)，
其中化合物4和8具有显著的周期抑制作用，为大血藤的主要活性成分，并首次
发现化合物4的抗癌活性。
上述研究结果为开发中药大血藤抗肿瘤活性成分奠定了基础。
Sargentodoxa cuneata, a Chinese traditional herb belonging to the family Sargentodoxaceae distributes widely in China. Its stems are employed in China as a folk medicine to heumatism, inflammation, and so on. Even though there are several kinds of active compounds isolated from Sargentodoxa cuneata, The research which is about anticancer activity constituents has never been reported . Thus, the studies on the anticancer activity constituents of Sargentodoxa cuneata are undertaken in the present thesis by the bioassay of detecting the cell cycle inhibitory, apoptosis-inducing and cytotoxic activities on tsFT210 cells.
Bioassy results indicated that EtOAc extract and n-BuOH extract showed the strong bioactivities in inhibiting the cell progression at the G_(2)/M phase, and cytotoxic activities on tsFT210 cells.
In terms of a preliminary examination, the dried stem of Sargentodoxa cuneata was extracted with ethanol to yield crude extracts, the ethanol extract was further separated as EtOAc extract and n-BuOH extract. By bioactivity-guided fractionation six compounds(l, 2, 9, 10, 13) were isolated from EtOAc extract by repeated colum chromatography on silica gel, sephadex LH-20 and RP-18, followed by reverse-phase high performance liquid chromatography and recrystallization. At the same time, seven compounds(3, 4, 7, 8,11,12) were obtained from n-BuOH extract by combinative use of chromatography on silica gel, sephadex LH-20 , MCI, HP-20 and HPLC. The structures of eleven compounds were elucidated mainly by use of spectroscopic memods(UV, IR, MS, 1D-NMR and 2D-NMR) and according to their physicochemical properties: chlorogenic acid(l), 7-(4-hydroxy-3-methoxyphenyl)-N-[7 ' -(4 ' -hydroxyphenyl)ethyl]-E-8-propenamide(2) , ( + ) -3 α -O-( β -D-Glucopyranosyl)-lyoniresinol (3), 3 α -O-( β-D-Glucopyranosyl)-lyoniresinol (4) ,
3,5-O-dimethyl gallic acid(5), (-)-epicatechin(6), procyanidin B-2[epicatechin-(4β-8) -epicatechin (7), 3,4-dihydroxy-benzoic acid (9) , p-hydroxyphenethyl alcohol(l), p-hydroxyphenethyl- β -D-glucopyranoside(ll), 3,4-dihydroxyphenethyl- β -D-glucopyranoside(12).Moreover,the possible structure of 8 was deduced also according to its physicochemical properties and sprctral data, and the structure of 13 is being identified now.
Bioassay results indicated that compounds 4 and 8 showed a strong bioactivity in inhibiting the cell cycle progression at the G_(2)/M phase of tsFT210 cells;Compounds 1 and 2 possessed activity in inhibiting the cell cycle progression at the G_(0)/G_(1) phase; Compound 13 had activity in inhibiting the cell cycle progression at the G_(2)/M phase.
In summary, thirty compounds(l~13) were obtained from Sargentodoxa cuneata , ten of compounds(l~8, 10, 12) were isolated for the first time from the genus; Five active compounds(l, 2, 4, 8, 13) were obtained, and anticancer activity of compound 4 was first reported.
大血藤茎干药材用乙醇提取，依次用乙酸乙酯、正丁醇萃取，分别进行活性测试，结果表明乙酸乙酯和正丁醇萃取物均具有显著的G_2/M期抑制作用。对活性部位乙酸乙酯提取物采用减压柱层析(VLC)、凝胶柱层析(Sephadex LH-20)、反相硅胶柱层析(ODS)和反相制备高效液相(PHPLC)等分离方法，得到6个单体化合物(1，2，5，9，10，13)；对活性部位正丁醇提取物采用凝胶柱层析(Sephadex LH-20)、大孔树脂柱层析(HP-20,MCI)、反相制备高效液相(PHPLC)等分离方法，分离得到7个化合物(3，4，6，7，8，11，12)。利用理化性质和光谱学方法(UV、IR、MS、1D-NMR和2D-NMR)阐明了其中11个化合物的结构，分别为绿原酸(1)，N-(对羟基苯乙基)阿魏酸酰胺(2)，[2R，3R，4S]-1，2，3，4-四氢-6-羟基-4(4′-羟基-3′，5′-二甲氧苯基)-2-羟甲基-5，7二甲氧基-3-萘甲基-O-β-D-吡喃葡萄糖苷(3)，3，5-O-二甲基没食子酸(5)，(-)-表儿茶素(6)，缩合鞣质B_2[epicatechin(4β→8)epicatechin)(7)，原儿茶酸(9)，对-羟基苯乙醇(10)，对-羟基苯乙基-β-D-吡喃葡萄糖苷(毛柳苷，11)，3，4-二羟基-苯乙基-β-D-吡喃葡萄糖苷(红景天苷，12)。同时推定了化合物4的平面构型为1，2，3，4-四氢-6-羟基-4(4′-羟基-3′，5′-二甲氧苯基)-2-羟甲基-5，7二甲氧基-3-萘甲基-O-β-D-吡喃葡萄糖苷，其波谱学数据与迄今所报道的四个具有相同平面结构的化合物有较大的差异，因此化合物4可能为一新木脂素苷类化合物；推测了化合物8可能为具有新连接方式的五聚体缩合鞣质，化合物13的结构正在解析中。
中药大血藤命崔竿”勿由尤口c期eata抗癌活性成分的分离与鉴
摘要
化合物的活性测试结果表明:化合物4，8对tsFTZ10小鼠乳腺癌细胞具有
显著GZ彻期抑制作用;化合物1，2具有一定的G夕G，期抑制作用;化合物13
具有一定的GZ彻期抑制作用。
本文从大血藤植物中共分离得到13个化合物，其中10个化合物(1一8，10，
12)为首次从该属植物中分离得到;获得5个活性化合物(1，2，4，8，13)，
其中化合物4和8具有显著的周期抑制作用，为大血藤的主要活性成分，并首次
发现化合物4的抗癌活性。
上述研究结果为开发中药大血藤抗肿瘤活性成分奠定了基础。
Sargentodoxa cuneata, a Chinese traditional herb belonging to the family Sargentodoxaceae distributes widely in China. Its stems are employed in China as a folk medicine to heumatism, inflammation, and so on. Even though there are several kinds of active compounds isolated from Sargentodoxa cuneata, The research which is about anticancer activity constituents has never been reported . Thus, the studies on the anticancer activity constituents of Sargentodoxa cuneata are undertaken in the present thesis by the bioassay of detecting the cell cycle inhibitory, apoptosis-inducing and cytotoxic activities on tsFT210 cells.
Bioassy results indicated that EtOAc extract and n-BuOH extract showed the strong bioactivities in inhibiting the cell progression at the G_(2)/M phase, and cytotoxic activities on tsFT210 cells.
In terms of a preliminary examination, the dried stem of Sargentodoxa cuneata was extracted with ethanol to yield crude extracts, the ethanol extract was further separated as EtOAc extract and n-BuOH extract. By bioactivity-guided fractionation six compounds(l, 2, 9, 10, 13) were isolated from EtOAc extract by repeated colum chromatography on silica gel, sephadex LH-20 and RP-18, followed by reverse-phase high performance liquid chromatography and recrystallization. At the same time, seven compounds(3, 4, 7, 8,11,12) were obtained from n-BuOH extract by combinative use of chromatography on silica gel, sephadex LH-20 , MCI, HP-20 and HPLC. The structures of eleven compounds were elucidated mainly by use of spectroscopic memods(UV, IR, MS, 1D-NMR and 2D-NMR) and according to their physicochemical properties: chlorogenic acid(l), 7-(4-hydroxy-3-methoxyphenyl)-N-[7 ' -(4 ' -hydroxyphenyl)ethyl]-E-8-propenamide(2) , ( + ) -3 α -O-( β -D-Glucopyranosyl)-lyoniresinol (3), 3 α -O-( β-D-Glucopyranosyl)-lyoniresinol (4) ,
3,5-O-dimethyl gallic acid(5), (-)-epicatechin(6), procyanidin B-2[epicatechin-(4β-8) -epicatechin (7), 3,4-dihydroxy-benzoic acid (9) , p-hydroxyphenethyl alcohol(l), p-hydroxyphenethyl- β -D-glucopyranoside(ll), 3,4-dihydroxyphenethyl- β -D-glucopyranoside(12).Moreover,the possible structure of 8 was deduced also according to its physicochemical properties and sprctral data, and the structure of 13 is being identified now.
Bioassay results indicated that compounds 4 and 8 showed a strong bioactivity in inhibiting the cell cycle progression at the G_(2)/M phase of tsFT210 cells;Compounds 1 and 2 possessed activity in inhibiting the cell cycle progression at the G_(0)/G_(1) phase; Compound 13 had activity in inhibiting the cell cycle progression at the G_(2)/M phase.
In summary, thirty compounds(l~13) were obtained from Sargentodoxa cuneata , ten of compounds(l~8, 10, 12) were isolated for the first time from the genus; Five active compounds(l, 2, 4, 8, 13) were obtained, and anticancer activity of compound 4 was first reported.
引文
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