合欢皮化学成分及其构效关系和抗肿瘤活性机制研究
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摘要
合欢皮为豆科植物合欢(Albizia julibrissin Durazz．)的干燥茎皮，2000年版《中华人民共和国药典》一部收载合欢皮具有解郁安神，活血消肿的功能。用于心神不安，忧郁失眠，肺痈疮肿，跌扑伤痛等症，文献报道及前期工作表明合欢皮有抗癌活性。文献报道对合欢属植物的化学研究较多，但对合欢皮的抗肿瘤等活性尚没有深入的研究，本论文对其化学和抗癌活性进行了较深入的研究，具体内容如以下：
1．文献工作
系统查阅了国内外合欢属植物化学和药理研究文献，完成了综述，对合欢皂苷的波谱特征进行了总结和分析。
2．合欢生物活性研究
用体内及体外方法对合欢的抗炎、镇静和抗癌活性进行了研究，确定了抗炎、镇静和抗癌活性部位。
3．化学研究
从合欢皮用硅胶、大孔树脂、Sephadex LH-20及HPLC等多种色谱方法分离了；30个化合物，利用TOF-MS和NMR等波谱方法，特别是2D-NMR方法(~1H-~1H COSY、HSQC、HMBC、TOCSY等)共鉴定了29个化合物的结构，其中13个为新化合物。
从抗肿瘤活性部位分离得到19个三萜皂苷，鉴定了18个皂苷的结构，9个为八糖皂苷(1-9)，8个为九糖皂苷(10-17)，1个为十糖皂苷(18)。18个皂苷中13个皂苷为新化合物(**)，2个为新天然产物(*)。皂苷元为金合欢酸，皂苷均为苷元3位、21位及28位连接糖的三糖链皂苷，结构中还有1～2个单萜烯酸基，分子量在2000左右。
从镇静活性部位分离并鉴定了5个木脂素(19-23)，3个为双环氧木脂素，1个为单环氧木脂素，1个为环木脂素，该结果与日本学者报道木脂素是合欢镇静活性成分一致。
为了研究抗癌构效关系，将抗癌活性部位物质酸水解，从水解物中分离并鉴定了4个三萜化合物(24-27)，另外还获得2个其它类型的化合物(28-29)，结构见Figure 1。
4．抑制癌细胞活性筛选及构效关系研究
将从合欢抗癌活性部位用Sephadex LH-20分离得到的几个部分及从中分离得到的化合物用MTT法和SRT法，以HL-60、PC-3MIE8、MCF-7、Bel-7402、BGC-823和Hela等六种癌细胞进行了抑制癌细胞活性测试，实验结果表明皂苷元，木质素，单
沈阳药科大学博十学位论文摘要
菇昔均没有抑制癌细胞的作用，皂昔有抑制癌细胞的活性，所以皂营是合欢抗癌作用的
物质基础，对多种癌细胞抑制活性多次筛选结果表明，皂昔对肝癌细胞的抑制作用最强，
重现性好。对人乳腺癌和宫颈癌细胞也有良好的抑制活性。分析不同结构的合欢皂营类
化合物抑制癌细胞活性测试结果，表明有如下主要构效关系:
(1)昔元28位连接2个单菇酸的皂昔抑制癌细胞作用比连接1个单菇作用强。
(2)普元3位有乙酞氨基糖的皂昔抑制癌细胞作用强。
(3)营元28位连接的外侧单菇烯酸中，6位碳构型为6仁S时抑制癌细胞活性比6’一R
强。
5.抑制癌细胞作用机制研究
在研究合欢皂营对人源癌细胞抑制作用时发现Bel一7402、MC卜7和Hela癌细胞对
合欢皂普敏感，并结合细胞库现有癌细胞的状况，首先将合欢皂营对Hela和MC卜7两
种癌细胞的抑制作用机制进行了研究。实验结果表明合欢皂营Ajts等诱导日eLa细胞死亡
具有明显的剂量和时间依赖关系:形态学观察可见明显的凋亡小体，琼脂凝胶电泳显示
典型的ONA Ladder，这提示Ajts等诱导Hela细胞发生经典凋亡。合欢皂营Ajts作用Hela
细胞24h，日CI一2和Bd一xL蛋白表达明显下调，而Bax表达水平升高。Ajts下调!CAO表达
水平，这种下调被CaSPase一3抑制剂(z一OEVO一fmk)阻止在Ajts作用Hela细胞12h时。
因此推测Ajts诱导的Hela细胞凋亡是Caspase家族和Bcl一2家族共同调控进行的。
细胞形态学实验结果表明合欢皂昔Ajts诱导MC卜7癌细胞死亡具有明显的剂量
和时间依赖关系，但作用于MCF一7细胞72h后，琼脂凝胶电泳也没有出现典型的梯状
带，说明Ajts诱导MCF一7细胞死亡，却没有启动经典的凋亡途径。Aj坛作用MC卜7细
胞48h时明显将细胞阻滞在GZIM期，但是作用至72h时，阻滞细胞在各个时期。Western
blot实验表明，经过Ajts作用72h时的MC卜7细胞，BCI一2和BCI一xL蛋白表达水平明
显下调，而Bax表达水平升高，同时PARP蛋白与!CAO蛋白的表达减少，这一实验结
果提示Ajts诱导MC卜7细胞死亡可能是通过调节Bax与Bcl一2和BCI一xL蛋白的表达比
率，激活caspase的级联实现的。
合欢皂普在Hela细胞，MCF一7细胞中诱导细胞死亡机制表明在两种细胞中合欢皂
营发挥抗癌活性机制各不相同，这可以进一步解释合欢抗癌作用的靶向性及选择性。
Cortex Albizzia, i.e. the dried stem bark of the leguminous plant Albizzia julibrissin Durazz, was specified in Chinese Pharmacopoeia (2000 edit.) as a traditional Chinese medicine to be used to relieve melancholia and uneasiness of body and mind, to invigorate the circulation of blood and to reduce a swelling. Although a lot of studies on chemistry and pharmacology of Albizzia were reported, there are not any research on the anti-tumor activity mechanisms of this medicine. In order to exploit the plant resource and find new antitumor compounds, the following studies were carried out.
1. The bioactive parts of the sedative and cytotoxicity were determined by the pharmacological tests. The bioactive part of the cytotoxicity from the stem barks of Albizia julibrissin was subjected to a series of solvent and chromatographic separations, including the alternative HPLC methods, giving 18 pentacyclic triterpenoid saponins. Based on chemical methods and spectroscopic evidences, the structures of all the saponins were identified (Figure 1). The saponins were trisdesmosidic saponins, which were composed of a pentacyclic triterpenoid aglycone, 8-10 sugar moieties, 1-2 monoterpenoid moieties, and exhibited their molecular ion peaks at m/z 2000 or so. Their carbon-13 signals and most of proton signals were assigned bassed on extensive 1D and 2D NMR and TOF-MS experiments. Five lignanoids were separated from the bioactive part of the sedative.
2. The compounds isolated from Albizia julibrissin together with six analogues or related compounds were tested for their cyotoxicities against six tumor cell lines by MTT and SRB methods. Some structure-activity relationships in cytotoxicity test were identified. The evaluation of cytotoxicity test revealed that some important structural features are required for activity. The lignans, triterpenoid aglycones and monoterpene glycosides showed no cytotoxic activity against the HeLa, Bel-7402 and MDA-MB-435 cancer lines, the triterpenoid saponins showed significant inhibition action against Hela, Bel-7402 ,Hella and MDA-MB-435 cancer cell lines in vitro.
The monoterpene units at C-28 oligosaccharide chain of aglycone and 2-deoxy-amidoglucose at 3 position of aglycone were crucial for the activity.
3. In this present paper the antitumor activity of pentacyclic triterpenoid saponins from Albizzia julibrissin was investigated. The saponins showed significant cyotoxicity against Bel-7402, HeLa and MCF-7 cell lines. In HeLa cells, Albizzia triterpenoid saponins initiated classic apoptotic pathway. Giemsa staining showed apoptotic bodies, and DMA electrophoresis appeared DMA ladder, which were believed to be the hallmark of classic apoptosis. In Western blot experiment, Bcl-2 expression was down-regulated and Bax expression was up-regulated by Albizzia triterpenoid saponins. Moreover, Albizzia triterpenoid saponins induced cell cycle arrest at G2/M phase. In MCF-7 cells, although apoptotic bodies were not observed in DMA fragmentation, a hallmark of apoptosis was not detected, it apparent that MCF-7cells incubated with saponins for 24 hours were controlled. In summary, we first demonstrated that the saponins induced cell death through distinct mechanisms in HeLa, and MCF-7cells respectively.
1．文献工作
系统查阅了国内外合欢属植物化学和药理研究文献，完成了综述，对合欢皂苷的波谱特征进行了总结和分析。
2．合欢生物活性研究
用体内及体外方法对合欢的抗炎、镇静和抗癌活性进行了研究，确定了抗炎、镇静和抗癌活性部位。
3．化学研究
从合欢皮用硅胶、大孔树脂、Sephadex LH-20及HPLC等多种色谱方法分离了；30个化合物，利用TOF-MS和NMR等波谱方法，特别是2D-NMR方法(~1H-~1H COSY、HSQC、HMBC、TOCSY等)共鉴定了29个化合物的结构，其中13个为新化合物。
从抗肿瘤活性部位分离得到19个三萜皂苷，鉴定了18个皂苷的结构，9个为八糖皂苷(1-9)，8个为九糖皂苷(10-17)，1个为十糖皂苷(18)。18个皂苷中13个皂苷为新化合物(**)，2个为新天然产物(*)。皂苷元为金合欢酸，皂苷均为苷元3位、21位及28位连接糖的三糖链皂苷，结构中还有1～2个单萜烯酸基，分子量在2000左右。
从镇静活性部位分离并鉴定了5个木脂素(19-23)，3个为双环氧木脂素，1个为单环氧木脂素，1个为环木脂素，该结果与日本学者报道木脂素是合欢镇静活性成分一致。
为了研究抗癌构效关系，将抗癌活性部位物质酸水解，从水解物中分离并鉴定了4个三萜化合物(24-27)，另外还获得2个其它类型的化合物(28-29)，结构见Figure 1。
4．抑制癌细胞活性筛选及构效关系研究
将从合欢抗癌活性部位用Sephadex LH-20分离得到的几个部分及从中分离得到的化合物用MTT法和SRT法，以HL-60、PC-3MIE8、MCF-7、Bel-7402、BGC-823和Hela等六种癌细胞进行了抑制癌细胞活性测试，实验结果表明皂苷元，木质素，单
沈阳药科大学博十学位论文摘要
菇昔均没有抑制癌细胞的作用，皂昔有抑制癌细胞的活性，所以皂营是合欢抗癌作用的
物质基础，对多种癌细胞抑制活性多次筛选结果表明，皂昔对肝癌细胞的抑制作用最强，
重现性好。对人乳腺癌和宫颈癌细胞也有良好的抑制活性。分析不同结构的合欢皂营类
化合物抑制癌细胞活性测试结果，表明有如下主要构效关系:
(1)昔元28位连接2个单菇酸的皂昔抑制癌细胞作用比连接1个单菇作用强。
(2)普元3位有乙酞氨基糖的皂昔抑制癌细胞作用强。
(3)营元28位连接的外侧单菇烯酸中，6位碳构型为6仁S时抑制癌细胞活性比6’一R
强。
5.抑制癌细胞作用机制研究
在研究合欢皂营对人源癌细胞抑制作用时发现Bel一7402、MC卜7和Hela癌细胞对
合欢皂普敏感，并结合细胞库现有癌细胞的状况，首先将合欢皂营对Hela和MC卜7两
种癌细胞的抑制作用机制进行了研究。实验结果表明合欢皂营Ajts等诱导日eLa细胞死亡
具有明显的剂量和时间依赖关系:形态学观察可见明显的凋亡小体，琼脂凝胶电泳显示
典型的ONA Ladder，这提示Ajts等诱导Hela细胞发生经典凋亡。合欢皂营Ajts作用Hela
细胞24h，日CI一2和Bd一xL蛋白表达明显下调，而Bax表达水平升高。Ajts下调!CAO表达
水平，这种下调被CaSPase一3抑制剂(z一OEVO一fmk)阻止在Ajts作用Hela细胞12h时。
因此推测Ajts诱导的Hela细胞凋亡是Caspase家族和Bcl一2家族共同调控进行的。
细胞形态学实验结果表明合欢皂昔Ajts诱导MC卜7癌细胞死亡具有明显的剂量
和时间依赖关系，但作用于MCF一7细胞72h后，琼脂凝胶电泳也没有出现典型的梯状
带，说明Ajts诱导MCF一7细胞死亡，却没有启动经典的凋亡途径。Aj坛作用MC卜7细
胞48h时明显将细胞阻滞在GZIM期，但是作用至72h时，阻滞细胞在各个时期。Western
blot实验表明，经过Ajts作用72h时的MC卜7细胞，BCI一2和BCI一xL蛋白表达水平明
显下调，而Bax表达水平升高，同时PARP蛋白与!CAO蛋白的表达减少，这一实验结
果提示Ajts诱导MC卜7细胞死亡可能是通过调节Bax与Bcl一2和BCI一xL蛋白的表达比
率，激活caspase的级联实现的。
合欢皂普在Hela细胞，MCF一7细胞中诱导细胞死亡机制表明在两种细胞中合欢皂
营发挥抗癌活性机制各不相同，这可以进一步解释合欢抗癌作用的靶向性及选择性。
Cortex Albizzia, i.e. the dried stem bark of the leguminous plant Albizzia julibrissin Durazz, was specified in Chinese Pharmacopoeia (2000 edit.) as a traditional Chinese medicine to be used to relieve melancholia and uneasiness of body and mind, to invigorate the circulation of blood and to reduce a swelling. Although a lot of studies on chemistry and pharmacology of Albizzia were reported, there are not any research on the anti-tumor activity mechanisms of this medicine. In order to exploit the plant resource and find new antitumor compounds, the following studies were carried out.
1. The bioactive parts of the sedative and cytotoxicity were determined by the pharmacological tests. The bioactive part of the cytotoxicity from the stem barks of Albizia julibrissin was subjected to a series of solvent and chromatographic separations, including the alternative HPLC methods, giving 18 pentacyclic triterpenoid saponins. Based on chemical methods and spectroscopic evidences, the structures of all the saponins were identified (Figure 1). The saponins were trisdesmosidic saponins, which were composed of a pentacyclic triterpenoid aglycone, 8-10 sugar moieties, 1-2 monoterpenoid moieties, and exhibited their molecular ion peaks at m/z 2000 or so. Their carbon-13 signals and most of proton signals were assigned bassed on extensive 1D and 2D NMR and TOF-MS experiments. Five lignanoids were separated from the bioactive part of the sedative.
2. The compounds isolated from Albizia julibrissin together with six analogues or related compounds were tested for their cyotoxicities against six tumor cell lines by MTT and SRB methods. Some structure-activity relationships in cytotoxicity test were identified. The evaluation of cytotoxicity test revealed that some important structural features are required for activity. The lignans, triterpenoid aglycones and monoterpene glycosides showed no cytotoxic activity against the HeLa, Bel-7402 and MDA-MB-435 cancer lines, the triterpenoid saponins showed significant inhibition action against Hela, Bel-7402 ,Hella and MDA-MB-435 cancer cell lines in vitro.
The monoterpene units at C-28 oligosaccharide chain of aglycone and 2-deoxy-amidoglucose at 3 position of aglycone were crucial for the activity.
3. In this present paper the antitumor activity of pentacyclic triterpenoid saponins from Albizzia julibrissin was investigated. The saponins showed significant cyotoxicity against Bel-7402, HeLa and MCF-7 cell lines. In HeLa cells, Albizzia triterpenoid saponins initiated classic apoptotic pathway. Giemsa staining showed apoptotic bodies, and DMA electrophoresis appeared DMA ladder, which were believed to be the hallmark of classic apoptosis. In Western blot experiment, Bcl-2 expression was down-regulated and Bax expression was up-regulated by Albizzia triterpenoid saponins. Moreover, Albizzia triterpenoid saponins induced cell cycle arrest at G2/M phase. In MCF-7 cells, although apoptotic bodies were not observed in DMA fragmentation, a hallmark of apoptosis was not detected, it apparent that MCF-7cells incubated with saponins for 24 hours were controlled. In summary, we first demonstrated that the saponins induced cell death through distinct mechanisms in HeLa, and MCF-7cells respectively.
引文
[1] 中国约典(2000版)，化学工业出版社．
[2] 汉拉英中草药名大辞典，世界图书出版公司．
[3] IP Vershney, et al. J Indian Chem Soc 1970, 47: 907.
[4] T Sergoenko V. Khim Prir Soedin(俄), 1977: 708.
[5] C Giovanna, et al. Phytochemistry, 1991, 30: 4111.
[6] SS Kang, et al. Arch Pharmacol Res, 1982, 5: 33.
[7] SS Kang, et al. J Nat Prod, 1984, 47: 547.
[8] SS Kang, et al. Arch Pharmacol Res, 1983, 6: 25.
[9] 嵇汝运主编．新编药物学(第三版)．
[10] 陈四平，等．承德医学院学报，1989，6：211．
[11] 康少文，等．承德医学院学报，1991，8：199．
[12] 康少文，等．中国中药杂志，1992，17：357．
[13] 陈四平，等．药学学报，1997，32(2)：144．
[14] Junei Kinjo, et al. Chem Pharm Bull, 1992, 40: 3269.
[15] 邹坤，等．北京医科大学学报，1999，31(1)：32．
[16] R Baneiji, et al. Indian Drugs, 1979, 17: 6.
[17] IP Vershney, et al. Tetralhedron Letter, 1965, 17: 1187.
[18] IP Vershney, et al. J Pharm Sci, 1961, 50: 923.
[19] IP Vershney, et al. Indian J Chem,1973, 11: 1189.
[20] S Roy, et al. Tetralhedron Letter, 1966, 46: 5743.
[21] AK Barua, et al. Tetrahedron, 1959, 7: 19.
[22] AK Barua, et al. Tetrahedron, 1962, 18: 55.
[23] 邹坤，等．中国中药杂志，2000，25(2)：96．
[24] Kun Z, et al. J Asian Natrual Products Research, 1998, 1: 59.
[25] Carbohydrate Research, 2000, 324: 182.
[26] 邹坤，等．高等学校化学学报，1999，20(12)：1877．
[27] 邹坤，等．药学学报，1999，34(7)：522．
[28] J Chin Pharm Sci, 2000, 9(3): 125.
[29] J Asian Natrual Products Research, 1998, 1: 313.
[30] Chinese Chemical Letters, 2000, 11(1): 39.
[31] Tsuy Ikeda, et al. Tetrahedron letters, 1995, 36: 1509.
[32] Tsuy Ikeda, et al. Bull Chem Soc Jpn., 1995, 68: 3483.
[33] IP Vershney, et al. J Indian Chem Sci,1978,53,859.
[34] L comeau, et al. Bull Soc Chem Fr, 1974, (3-4, pt 2): 716, 11, (pt 2): 2513.
[35] CA. a 965: 20170g; b b1: 1232be; c 55: 11770b; d 53: 1473d.
[36] IP Vershney, et al. Planta Med, 1973, 24: 183.
[37] IP Vershney, et al. Indian J Chem, 1973, 24: 183.
[38] IP Vershney, et al. J.Indian Chem.Sci,1982,54,884.
[39] Pal BC, et al. Phytochemistry, 1995, 38(5): 1287-1291.
[40] Yoshikawa, et al. J Nat Prod, 1998, 61(4): 440.
[41] IP Vershney, et al. J Sci Ind Res,1962,12B: 30.
[42] Asfaw Della, et al. Phytochemistry, 2000, 53: 885.
[43] Rukun GM. et al. Fitoterapia, 2001, 72(2),140.
[44] Maged Abdel-Kader, et al. J Nat Prod, 64: 536.
[45] PK Chamsu, et al. Arch Pharmacol Res, 1981, 4: 129.
[46] MK Jain, et al. Mokuzai Gakkaishi, 1978, 24: 449.
[47] IP Vershney, et al. Can J Chem, 1961, 39: 1271.
[48] 崎正雄，等．生药学杂志(日)，1959，13：127．
[49] M Kaneta. Agnic Biol chem, 1980, 44(6): 1407-1408.
[50] 森田直贤，等．生药学杂志(日)，1977，31：172．
[51] H. Candy, et al. Phytochemistry, 1978, 17: 1681.
[52] SCV Reddy, et al. Indian J Chem,1967, 5: 613.
[53] VH Deshpande, et al. Indian J Chem, 1977, 15B: 201.
[54] LR Row, et al. Tetrahedron, 1963, 19: 1371.
[55] GVN Rayuda, et al. Leather Sci, 1965,12: 36.
[56] GVN Rayuda, et al. Leather Sci, 1965, 12: 21.
[57] 徐国均．药材学，北京：人民卫生出版社，1962：347．
[58] N Ishikura, et al. Bot may, 1978, 91: 25.
[59] Amani MD, et al. Phytochemistry, 1998, 48: 759.
[60] Yadava RN, et al. Res J Chem Environ, 2000, 4(4): 57.
[61] Kinjo J, et al. Chem Pharm Bull, 1991,39(6), 1623-1625.
[62] Kinjo J, et al. Chem Pharm Bull, 1991, 39(11): 2952-2955.
[63] Niroyuki Higichi, et al. Chem Pharm Bull, 1992, 40: 534.
[64] Ito A, et al. Phytochemistry, 1994, 37(5): 1455-1458.
[65] PS Steyn, et al. S Afr J Chem, 1987, 40: 191.
[66] O Fulvia, et al. Gazz Chim Ital, 1989, 119: 1.
[67] Laxmi N Misra, et al. Phytochemistry, 1995, 39: 247.
[68] Simon PBO, et al. Phytochemistry, 2002, 60: 175.
[69] PK Agrawal et al. Indian J Pharm Sci, 1991, 53: 24.
[70] N Yasuhisa. Mokozai Gakkaishi(日), 1975, 21: 577.
[71] Kim Young Hee. Saengyak Hakhoechi(Korean), 30(3): 290-294.
[72] 佟文勇，等．北京大学学报(医学版)，2003，35(2)：180-183．
[73] Wang Qihuang, et al. Acta Crystallogr, Sect C: Struct Commun, C56(2): 197-198.
[74] Ying-Tsun M, et al. Phytochemistry, 1997, 46: 1451.
[75] M Ueda, S Yamamura. Tetrahedron Lett, 1999, 40: 7823.
[76] M Ueda, C Tashiro, S Yamamura. Tetrahedron Lett, 1997, 38(18): 3253.
[77] 张立新，等．中国药学会第四届全国药学学术年会论文摘要集，1979：79．
[78] 楼之岑，秦波主编．常用中药材品种整理和质量研究(北方组．第二册)．北京医科大学协和医科大学联合出版社．1995：386．
[79] 王序，等．北京医科大学学报，1986，18(1)：31．
[80] 单国存，等．中药材，1989，12(5)：36．
[81] Kang TH, et al. J Ethnopharmcol, 2000, 71: 321.
[82] TS Assis, et al. Fitoterapia, 2001,72: 124.
[83] Lipton A. J Pharm Pharmacol, 1964, 16: 369; 1963, 15: 816.
[84] Correia S. An Fac Farm Porto, 1969, 29: 29. see CA: 1971, 75: 106098b.
[85] 杜克洪，等．中西医结合杂志，1991．(增刊)：282．
[86] 毛福祥．药理通报，1986，1：53．
[87] 毛福祥．中国药理学通报，1988．2：120．
[88] Baneryi R. India Drugs, 1979, 17(1): 6. see CA: 1980, 92: 64626r.
[89] 徐强，等．中国中药杂志，1990，15(2)：119．
[90] HD Une, et al. Pharmacology Biochemistry and behavior, 2001,69: 439.
[91] VS Kasture, et al. J of Ethnopharmcology, 2000, 71: 65.
[92] Tripathi RM, et al. J Ethnopharmacol, 1979, 1(4): 385.
[93] 中华本草，4：319．
[2] 汉拉英中草药名大辞典，世界图书出版公司．
[3] IP Vershney, et al. J Indian Chem Soc 1970, 47: 907.
[4] T Sergoenko V. Khim Prir Soedin(俄), 1977: 708.
[5] C Giovanna, et al. Phytochemistry, 1991, 30: 4111.
[6] SS Kang, et al. Arch Pharmacol Res, 1982, 5: 33.
[7] SS Kang, et al. J Nat Prod, 1984, 47: 547.
[8] SS Kang, et al. Arch Pharmacol Res, 1983, 6: 25.
[9] 嵇汝运主编．新编药物学(第三版)．
[10] 陈四平，等．承德医学院学报，1989，6：211．
[11] 康少文，等．承德医学院学报，1991，8：199．
[12] 康少文，等．中国中药杂志，1992，17：357．
[13] 陈四平，等．药学学报，1997，32(2)：144．
[14] Junei Kinjo, et al. Chem Pharm Bull, 1992, 40: 3269.
[15] 邹坤，等．北京医科大学学报，1999，31(1)：32．
[16] R Baneiji, et al. Indian Drugs, 1979, 17: 6.
[17] IP Vershney, et al. Tetralhedron Letter, 1965, 17: 1187.
[18] IP Vershney, et al. J Pharm Sci, 1961, 50: 923.
[19] IP Vershney, et al. Indian J Chem,1973, 11: 1189.
[20] S Roy, et al. Tetralhedron Letter, 1966, 46: 5743.
[21] AK Barua, et al. Tetrahedron, 1959, 7: 19.
[22] AK Barua, et al. Tetrahedron, 1962, 18: 55.
[23] 邹坤，等．中国中药杂志，2000，25(2)：96．
[24] Kun Z, et al. J Asian Natrual Products Research, 1998, 1: 59.
[25] Carbohydrate Research, 2000, 324: 182.
[26] 邹坤，等．高等学校化学学报，1999，20(12)：1877．
[27] 邹坤，等．药学学报，1999，34(7)：522．
[28] J Chin Pharm Sci, 2000, 9(3): 125.
[29] J Asian Natrual Products Research, 1998, 1: 313.
[30] Chinese Chemical Letters, 2000, 11(1): 39.
[31] Tsuy Ikeda, et al. Tetrahedron letters, 1995, 36: 1509.
[32] Tsuy Ikeda, et al. Bull Chem Soc Jpn., 1995, 68: 3483.
[33] IP Vershney, et al. J Indian Chem Sci,1978,53,859.
[34] L comeau, et al. Bull Soc Chem Fr, 1974, (3-4, pt 2): 716, 11, (pt 2): 2513.
[35] CA. a 965: 20170g; b b1: 1232be; c 55: 11770b; d 53: 1473d.
[36] IP Vershney, et al. Planta Med, 1973, 24: 183.
[37] IP Vershney, et al. Indian J Chem, 1973, 24: 183.
[38] IP Vershney, et al. J.Indian Chem.Sci,1982,54,884.
[39] Pal BC, et al. Phytochemistry, 1995, 38(5): 1287-1291.
[40] Yoshikawa, et al. J Nat Prod, 1998, 61(4): 440.
[41] IP Vershney, et al. J Sci Ind Res,1962,12B: 30.
[42] Asfaw Della, et al. Phytochemistry, 2000, 53: 885.
[43] Rukun GM. et al. Fitoterapia, 2001, 72(2),140.
[44] Maged Abdel-Kader, et al. J Nat Prod, 64: 536.
[45] PK Chamsu, et al. Arch Pharmacol Res, 1981, 4: 129.
[46] MK Jain, et al. Mokuzai Gakkaishi, 1978, 24: 449.
[47] IP Vershney, et al. Can J Chem, 1961, 39: 1271.
[48] 崎正雄，等．生药学杂志(日)，1959，13：127．
[49] M Kaneta. Agnic Biol chem, 1980, 44(6): 1407-1408.
[50] 森田直贤，等．生药学杂志(日)，1977，31：172．
[51] H. Candy, et al. Phytochemistry, 1978, 17: 1681.
[52] SCV Reddy, et al. Indian J Chem,1967, 5: 613.
[53] VH Deshpande, et al. Indian J Chem, 1977, 15B: 201.
[54] LR Row, et al. Tetrahedron, 1963, 19: 1371.
[55] GVN Rayuda, et al. Leather Sci, 1965,12: 36.
[56] GVN Rayuda, et al. Leather Sci, 1965, 12: 21.
[57] 徐国均．药材学，北京：人民卫生出版社，1962：347．
[58] N Ishikura, et al. Bot may, 1978, 91: 25.
[59] Amani MD, et al. Phytochemistry, 1998, 48: 759.
[60] Yadava RN, et al. Res J Chem Environ, 2000, 4(4): 57.
[61] Kinjo J, et al. Chem Pharm Bull, 1991,39(6), 1623-1625.
[62] Kinjo J, et al. Chem Pharm Bull, 1991, 39(11): 2952-2955.
[63] Niroyuki Higichi, et al. Chem Pharm Bull, 1992, 40: 534.
[64] Ito A, et al. Phytochemistry, 1994, 37(5): 1455-1458.
[65] PS Steyn, et al. S Afr J Chem, 1987, 40: 191.
[66] O Fulvia, et al. Gazz Chim Ital, 1989, 119: 1.
[67] Laxmi N Misra, et al. Phytochemistry, 1995, 39: 247.
[68] Simon PBO, et al. Phytochemistry, 2002, 60: 175.
[69] PK Agrawal et al. Indian J Pharm Sci, 1991, 53: 24.
[70] N Yasuhisa. Mokozai Gakkaishi(日), 1975, 21: 577.
[71] Kim Young Hee. Saengyak Hakhoechi(Korean), 30(3): 290-294.
[72] 佟文勇，等．北京大学学报(医学版)，2003，35(2)：180-183．
[73] Wang Qihuang, et al. Acta Crystallogr, Sect C: Struct Commun, C56(2): 197-198.
[74] Ying-Tsun M, et al. Phytochemistry, 1997, 46: 1451.
[75] M Ueda, S Yamamura. Tetrahedron Lett, 1999, 40: 7823.
[76] M Ueda, C Tashiro, S Yamamura. Tetrahedron Lett, 1997, 38(18): 3253.
[77] 张立新，等．中国药学会第四届全国药学学术年会论文摘要集，1979：79．
[78] 楼之岑，秦波主编．常用中药材品种整理和质量研究(北方组．第二册)．北京医科大学协和医科大学联合出版社．1995：386．
[79] 王序，等．北京医科大学学报，1986，18(1)：31．
[80] 单国存，等．中药材，1989，12(5)：36．
[81] Kang TH, et al. J Ethnopharmcol, 2000, 71: 321.
[82] TS Assis, et al. Fitoterapia, 2001,72: 124.
[83] Lipton A. J Pharm Pharmacol, 1964, 16: 369; 1963, 15: 816.
[84] Correia S. An Fac Farm Porto, 1969, 29: 29. see CA: 1971, 75: 106098b.
[85] 杜克洪，等．中西医结合杂志，1991．(增刊)：282．
[86] 毛福祥．药理通报，1986，1：53．
[87] 毛福祥．中国药理学通报，1988．2：120．
[88] Baneryi R. India Drugs, 1979, 17(1): 6. see CA: 1980, 92: 64626r.
[89] 徐强，等．中国中药杂志，1990，15(2)：119．
[90] HD Une, et al. Pharmacology Biochemistry and behavior, 2001,69: 439.
[91] VS Kasture, et al. J of Ethnopharmcology, 2000, 71: 65.
[92] Tripathi RM, et al. J Ethnopharmacol, 1979, 1(4): 385.
[93] 中华本草，4：319．