轮叶党参总皂甙抗突变作用及其机理研究
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摘要
随着全球范围内新化合物质的增多，环境致突变、致癌物对人类健康造成的威胁日趋严重，从天然的植物中寻找抗突变、抗癌的有效成分已成为国内外学者研究的热点问题。轮叶党参(Codonopsis Lanceolata Benth.et Hook.F.)为桔梗科党参属植物，国内外报道本品含有皂甙，淀粉，葡萄糖，多种维生素，铁、磷、钙等矿物质元素。人们对轮叶党参栽培技术、皂甙的制备工艺、多糖抗癌等均进行了一定的研究。为了进一步阐明轮叶党参中抗突变的有效成分及其机理，本研究采用轮叶党参总皂甙，进行小鼠骨髓嗜多染红细胞微核、精子畸形、单细胞凝胶电泳、Ames抑制诱变试验，并测定组织SOD活力、MDA含量。
本实验结果表明，在小鼠骨髓嗜多染红细胞微核实验中预先给予总皂甙后，由环磷酰胺诱发的微核率均明显降低，其中中剂量组和高剂量组与阳性组比较有统计学意义(P<0.01)，并且随着总皂甙浓度的增高微核率逐渐降低。在精子畸形实验中预先给予总皂甙后，由环磷酰胺诱发的精子畸形率均明显降低，其中中剂量组和高剂量组与阳性组比较有统计学意义(P<0.01)，并且随着总皂甙浓度的增高精子畸形率逐渐降低。在Ames实验中采用TA98、TA100两种菌株在加与不加S9混合液平板掺入法实验中，给予总皂甙的试验组与阳性组比较能明显抑制菌落回变率。单细胞凝胶电泳试验显示给予总皂甙的试验组可降低大鼠外周血淋巴细胞DNA的损伤,其中高剂量组与环磷酰胺组比较有
显著性差异(P<0.01)。综上可见，轮叶党参总皂甙也是抗突变成分之一，是否还有其它有效成分还有待于今后进一步研究。在测定肝脏、睾丸组织SOD活力、MDA含量试验中，结果表明轮叶党参总皂甙可以对环磷酰胺导致的超氧化物歧化酶活力下降具有抑制作用；还可以拮抗由环磷酰胺引起的脂质过氧化作用并且减少脂质过氧化产物丙二醛的生成。因此，通过清除内环境中过多的自由基，抑制脂质过氧化的产物，从而保护了遗传物质的完整性，实现抗突变作用，被认为是轮叶党参总皂甙抗突变机理之一。
As the increasing of the new compounds in the world, the environment mutagens and carcinogens are threatening people more seriously. It has been a hot topic for the scholars at home and abroad to search the effective anti- mutation and anti-tumor components from the natural plants. Codonopsis Lanceolata ,from Codonopsis Wall.ex Roxb. of Campanulaceae, widely distribute in this locality. It was reported that Codonopsis Lanceolata contained saponins, starch, glucose, multivitamin and mineral elements as iron、phosphorum and calcium, et. People had researched its produce technique, educating technique and anti-tumor activity of polysacchariden in order to represent the effective inctituents and mechanisms of Codonopsis Lanceolata. In this investigation we used the Codonopsis Lanceolata saponins to do the experiments of polychromeatic erythrocytes with micronuclei (MPCE )test、sperm shape abnormalities test、single cell gel electrophoresis assay (SCGE)、Ames anti-mutagenesis test，and determined the superoxide dismutase( SOD) activity and the content of malonyldialdegyde (MDA) in tissue.
The results showed, it could inhibit MPCE induced by CP in mice with administration of Codonopsis Lanceolata saponins in advance in MPCE test. Compared with positive, the middle group and the high group had statistics
significance(p<0.01), and with the dosage of saponins' increasing, the rate of MPCE gradually decreased. In the sperm shape abnormalities test, it can inhibit MPCE induced by CP in mice with administration of Codonopsis Lanceolata saponins in advance. Compared with the positive, the middle group and the high group had statistics significance(p<0.01), and with the dosage of saponins' increasing, the rate of sperm abnormalities gradually decreased.
In Ames test, we used the plate penetrate technique to detect the reverse mutation rate of the positive mutations of strains TA98, TA100 with or without S9 mix. The result revealed the significant difference between the saponin groups and the positive control. In the SCGE test showed that the saponin groups decrease the damage of DNA in rat blood lymphosytes, and the high dosage has the significant difference compare to the CP group(P<0.01). Above all, Codonopsis Lanceolata saponins is a kind of the anti-mutation substances, and the other unknown anti-mutation substances in it would be study in the future. In the test of determining SOD activity and the MDA content in liver and testis, the results showed that Codonopsis Lanceolata saponins can inhibit the descent of SOD activity induced by CP; and lipid peroxidation action induced by CP and decrease its production, MDA. In a word, we thought that protecting the integrity of genetic substance by cleaning excessive free radicals in internal environment was one of the anti-mutation mechanisms of Codonopsis Lanceolata saponins.
本实验结果表明，在小鼠骨髓嗜多染红细胞微核实验中预先给予总皂甙后，由环磷酰胺诱发的微核率均明显降低，其中中剂量组和高剂量组与阳性组比较有统计学意义(P<0.01)，并且随着总皂甙浓度的增高微核率逐渐降低。在精子畸形实验中预先给予总皂甙后，由环磷酰胺诱发的精子畸形率均明显降低，其中中剂量组和高剂量组与阳性组比较有统计学意义(P<0.01)，并且随着总皂甙浓度的增高精子畸形率逐渐降低。在Ames实验中采用TA98、TA100两种菌株在加与不加S9混合液平板掺入法实验中，给予总皂甙的试验组与阳性组比较能明显抑制菌落回变率。单细胞凝胶电泳试验显示给予总皂甙的试验组可降低大鼠外周血淋巴细胞DNA的损伤,其中高剂量组与环磷酰胺组比较有
显著性差异(P<0.01)。综上可见，轮叶党参总皂甙也是抗突变成分之一，是否还有其它有效成分还有待于今后进一步研究。在测定肝脏、睾丸组织SOD活力、MDA含量试验中，结果表明轮叶党参总皂甙可以对环磷酰胺导致的超氧化物歧化酶活力下降具有抑制作用；还可以拮抗由环磷酰胺引起的脂质过氧化作用并且减少脂质过氧化产物丙二醛的生成。因此，通过清除内环境中过多的自由基，抑制脂质过氧化的产物，从而保护了遗传物质的完整性，实现抗突变作用，被认为是轮叶党参总皂甙抗突变机理之一。
As the increasing of the new compounds in the world, the environment mutagens and carcinogens are threatening people more seriously. It has been a hot topic for the scholars at home and abroad to search the effective anti- mutation and anti-tumor components from the natural plants. Codonopsis Lanceolata ,from Codonopsis Wall.ex Roxb. of Campanulaceae, widely distribute in this locality. It was reported that Codonopsis Lanceolata contained saponins, starch, glucose, multivitamin and mineral elements as iron、phosphorum and calcium, et. People had researched its produce technique, educating technique and anti-tumor activity of polysacchariden in order to represent the effective inctituents and mechanisms of Codonopsis Lanceolata. In this investigation we used the Codonopsis Lanceolata saponins to do the experiments of polychromeatic erythrocytes with micronuclei (MPCE )test、sperm shape abnormalities test、single cell gel electrophoresis assay (SCGE)、Ames anti-mutagenesis test，and determined the superoxide dismutase( SOD) activity and the content of malonyldialdegyde (MDA) in tissue.
The results showed, it could inhibit MPCE induced by CP in mice with administration of Codonopsis Lanceolata saponins in advance in MPCE test. Compared with positive, the middle group and the high group had statistics
significance(p<0.01), and with the dosage of saponins' increasing, the rate of MPCE gradually decreased. In the sperm shape abnormalities test, it can inhibit MPCE induced by CP in mice with administration of Codonopsis Lanceolata saponins in advance. Compared with the positive, the middle group and the high group had statistics significance(p<0.01), and with the dosage of saponins' increasing, the rate of sperm abnormalities gradually decreased.
In Ames test, we used the plate penetrate technique to detect the reverse mutation rate of the positive mutations of strains TA98, TA100 with or without S9 mix. The result revealed the significant difference between the saponin groups and the positive control. In the SCGE test showed that the saponin groups decrease the damage of DNA in rat blood lymphosytes, and the high dosage has the significant difference compare to the CP group(P<0.01). Above all, Codonopsis Lanceolata saponins is a kind of the anti-mutation substances, and the other unknown anti-mutation substances in it would be study in the future. In the test of determining SOD activity and the MDA content in liver and testis, the results showed that Codonopsis Lanceolata saponins can inhibit the descent of SOD activity induced by CP; and lipid peroxidation action induced by CP and decrease its production, MDA. In a word, we thought that protecting the integrity of genetic substance by cleaning excessive free radicals in internal environment was one of the anti-mutation mechanisms of Codonopsis Lanceolata saponins.
引文
1. 郑健仙，编著.功能性食品［M］.北京:中国轻工业出版社,1999.495
2. Ghosh A.K.，Sent S, Sharama A, et al.Effect of chlorophyllin on mercuric chloride induced clastogenicity in mice［J］.Fd Chem Toxic,1991,29（11）：777
3. 曹明富，杨贤强，沈生荣.茶多酚抗突变和消除自由基作用研究.癌变?畸变?突变［J］.1993，5（6）：57
4. 中药大词典［M］.第2卷，上海科学技术出版社，消化道篇，1985：926
5. EG Han, SY Cho. Effect of codonopsis lanceolata water extract on the activies of antioxidative enzymes in carbon tetrachloride treated rats［J］. J Korean Soc Food Sci Nutr, 1997; 26(6):1181
6. 韩春姬,李莲姬,朴奎善,郑美花,等.轮叶党参水提取液抗突变作用及其机理研究.预防医学文献信息［J］.1998,4(3):204
7. 韩春姬，李铉万，李莲姬,等.轮叶党参多糖对小鼠S180肉瘤的抑制作用。延边大学医学学报［J］.2000，23（4）：249
8. 姚新生主编.高等医药院校教材《天然药物化学》（第二版）［M］.北京：人民卫生出版社，1994.361
9. 中国预防医学科学院标准处.食品卫生国家标准汇编（GB15193.4-94）第一版［M］.北京：中国标准出版社，1998：53
10. 秦椿华主编.化学物致突变致癌检测技术.新疆科技出版社［M］.1976，8：74.
11. 秦椿华主编.化学物致突变致癌检测技术.新疆科技出版社［M］.1976，8：89
12. Maron DM. and Ames BN.Revised methods for the salmonella mutagenici test［J］.Mutat Res,1983;113：173
13. 于守洋，崔洪斌.中国保健食品的进展［M］.北京：人民卫生出版社，2001：986
14. Singh NP,Mccoy MT,Tice RR,et al. A simple technique for quantitation of low levers of DNA damage in individual cells［J］.Exp Cell Res,1988,175：184
15. Heddle JA,Hite M,Kirkhart,et al.The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency Gene-Tox Program[J].Mutat Res.1983,123 （6）：61
16. 傅继梁.遗传分析导论［M］.第一版.成都：四川科学技术出版社，1987.288
17. 黄幸纾，陈星若.环境化学致突变致癌致畸试验方法［M］.第一版.杭州：浙江科学技术出版社，1985.267
18. Chorvatovicova D,Ujhazy E.Transplacental effect of stobadine on cyclophosphamide induced micronucleus frequency in mice [ J ]. Mutagenesis 1995,10（6）：531
19. D.W.Fairbrain,P.L.Olive,K.L.O'Neill,The comet assy:a comprehensive review［J］.Mutat.Res.339(1995)37
20. Tice RR et al.High-dose comaination alkylating agents with autologous bone-marrow support in patients with breast［J］. cancer.Mutat Res,1992;271：101
21. 郑荣梁主编.自由基生物学［M］.北京：高等教育出版社，1992，57
22. McCann J,Choi E,Yamasaki E,Ames BN.Detection of carcinogens as mutagens in the Salmonella microsome test:assy of 300 chemical.Proc Natl Acad［J］. Sci USA,1975;72：5135
23. Ames BN.Validation of the salmonella test:A reply to Rinkus and legator［J］.Cancer Res.1981：41：4192
24. Lee HK,Kim YK,Kim YH,Roh JK.Effect of bacterial growth-inhibiting ingredients on the Ames mutagenicity of medicinal herbs［J］.Mutat Res,1987:192：99
25. Lee HK,Lin JY.Anticancer activity of extracts from anti-cancer drugs in Chinese medicine［J］. Mutat Res,1988：204：229
26. 刘德祥，殷学军，王河川et al. 102种中药水溶性提取物的抗诱变性筛选［M］.中国中药杂志.1990；15（10）：41
27. 张桥，等.卫生毒理学基础［M］.北京:人民卫生出版社,2000:133
28. Sato T,Ose Y,Nagase H,et al.Mechanism of the dismutagenic effect of humic acid［J］.Mutat Res,1987;176(1)：189
29. 陈新谦，金有豫.新编药物学［M］.北京：人民卫生出版社，1998，505
王本祥，崔景朝，刘爱晶.人参茎叶皂甙（SSLG）对实验性肝损伤的影响
30. ［J］.药学学报.1983.18（10）：726
31. 于永利，杨贵贞.吉林人参花总皂甙对NKC－ＩＦＮ－ＩＬ－２调节网的作用及其抑瘤效应．中国免疫学杂志［J］.1987，3（1）：41
32. Suzuki N, Suzuki H, Kojima T, et al. Effects of human interferon on cellular response to UV in UV-sensitive human cell strains［J］. Mutat Res. 1988, Mar; 198(1)： 207-14
33. Suzuki N. Effects of human interferon-alpha on UV-induced DNA-repair synthesis and cell killing［J］. Mutat Res. 1986 Nov; 175(3)： 189-93
2. Ghosh A.K.，Sent S, Sharama A, et al.Effect of chlorophyllin on mercuric chloride induced clastogenicity in mice［J］.Fd Chem Toxic,1991,29（11）：777
3. 曹明富，杨贤强，沈生荣.茶多酚抗突变和消除自由基作用研究.癌变?畸变?突变［J］.1993，5（6）：57
4. 中药大词典［M］.第2卷，上海科学技术出版社，消化道篇，1985：926
5. EG Han, SY Cho. Effect of codonopsis lanceolata water extract on the activies of antioxidative enzymes in carbon tetrachloride treated rats［J］. J Korean Soc Food Sci Nutr, 1997; 26(6):1181
6. 韩春姬,李莲姬,朴奎善,郑美花,等.轮叶党参水提取液抗突变作用及其机理研究.预防医学文献信息［J］.1998,4(3):204
7. 韩春姬，李铉万，李莲姬,等.轮叶党参多糖对小鼠S180肉瘤的抑制作用。延边大学医学学报［J］.2000，23（4）：249
8. 姚新生主编.高等医药院校教材《天然药物化学》（第二版）［M］.北京：人民卫生出版社，1994.361
9. 中国预防医学科学院标准处.食品卫生国家标准汇编（GB15193.4-94）第一版［M］.北京：中国标准出版社，1998：53
10. 秦椿华主编.化学物致突变致癌检测技术.新疆科技出版社［M］.1976，8：74.
11. 秦椿华主编.化学物致突变致癌检测技术.新疆科技出版社［M］.1976，8：89
12. Maron DM. and Ames BN.Revised methods for the salmonella mutagenici test［J］.Mutat Res,1983;113：173
13. 于守洋，崔洪斌.中国保健食品的进展［M］.北京：人民卫生出版社，2001：986
14. Singh NP,Mccoy MT,Tice RR,et al. A simple technique for quantitation of low levers of DNA damage in individual cells［J］.Exp Cell Res,1988,175：184
15. Heddle JA,Hite M,Kirkhart,et al.The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency Gene-Tox Program[J].Mutat Res.1983,123 （6）：61
16. 傅继梁.遗传分析导论［M］.第一版.成都：四川科学技术出版社，1987.288
17. 黄幸纾，陈星若.环境化学致突变致癌致畸试验方法［M］.第一版.杭州：浙江科学技术出版社，1985.267
18. Chorvatovicova D,Ujhazy E.Transplacental effect of stobadine on cyclophosphamide induced micronucleus frequency in mice [ J ]. Mutagenesis 1995,10（6）：531
19. D.W.Fairbrain,P.L.Olive,K.L.O'Neill,The comet assy:a comprehensive review［J］.Mutat.Res.339(1995)37
20. Tice RR et al.High-dose comaination alkylating agents with autologous bone-marrow support in patients with breast［J］. cancer.Mutat Res,1992;271：101
21. 郑荣梁主编.自由基生物学［M］.北京：高等教育出版社，1992，57
22. McCann J,Choi E,Yamasaki E,Ames BN.Detection of carcinogens as mutagens in the Salmonella microsome test:assy of 300 chemical.Proc Natl Acad［J］. Sci USA,1975;72：5135
23. Ames BN.Validation of the salmonella test:A reply to Rinkus and legator［J］.Cancer Res.1981：41：4192
24. Lee HK,Kim YK,Kim YH,Roh JK.Effect of bacterial growth-inhibiting ingredients on the Ames mutagenicity of medicinal herbs［J］.Mutat Res,1987:192：99
25. Lee HK,Lin JY.Anticancer activity of extracts from anti-cancer drugs in Chinese medicine［J］. Mutat Res,1988：204：229
26. 刘德祥，殷学军，王河川et al. 102种中药水溶性提取物的抗诱变性筛选［M］.中国中药杂志.1990；15（10）：41
27. 张桥，等.卫生毒理学基础［M］.北京:人民卫生出版社,2000:133
28. Sato T,Ose Y,Nagase H,et al.Mechanism of the dismutagenic effect of humic acid［J］.Mutat Res,1987;176(1)：189
29. 陈新谦，金有豫.新编药物学［M］.北京：人民卫生出版社，1998，505
王本祥，崔景朝，刘爱晶.人参茎叶皂甙（SSLG）对实验性肝损伤的影响
30. ［J］.药学学报.1983.18（10）：726
31. 于永利，杨贵贞.吉林人参花总皂甙对NKC－ＩＦＮ－ＩＬ－２调节网的作用及其抑瘤效应．中国免疫学杂志［J］.1987，3（1）：41
32. Suzuki N, Suzuki H, Kojima T, et al. Effects of human interferon on cellular response to UV in UV-sensitive human cell strains［J］. Mutat Res. 1988, Mar; 198(1)： 207-14
33. Suzuki N. Effects of human interferon-alpha on UV-induced DNA-repair synthesis and cell killing［J］. Mutat Res. 1986 Nov; 175(3)： 189-93
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