癌症是当前严重影响人类健康、威胁人类生命的主要疾病之一。近年来，以细胞周期为药物靶点进行筛选，已成为当今抗癌药研发的重要方向。中草药在中国已有几千年的药用历史，其结构多样性和复杂性的优势一直受到药物开发者的青睐。我们在此基础上筛选了来源于40种植物的2000多个馏分，对其进行细胞毒活性评价，发现小槐花60%乙醇提取物的大孔树脂95%洗脱馏分中间极性部分具有良好的细胞毒活性。因此对活性部位进行了活性成分的研究。 本文综合运用硅胶柱层析、ODS柱层析、SephadexLH-20柱层析以及反相HPLC等多种色谱学分离手段，对小槐花60%乙醇提取物的大孔树脂95%乙醇洗脱部分的化学成分进行了研究，从...Cancer is one of the major diseases which seriously impact human health and threaten human life. Recently, people pay much attention to find anticancer drugs that targeting cell cycle.Traditional Chinese Medicine (TCM) has been used in China for centuries. The structure diversity and variety of compounds make TCM become the hot spot in new drug discovery. In this thesis, we screen approximately 20...学位：理学硕士院系专业：生物医学研究院_化学生物学专业学号：3052009115223

吴瑶

Xiamen University Institutional Repository

  学校编码：10384                                        分类号       密级       学    号：30520091152237                                           UDC          硕  士  学  位  论  文                                            小槐花抗肿瘤活性成分研究 Anti-cancer constituents research from Desmodium caudatum（Thunb.）DC    指导教师姓名：陈海峰       专  业 名 称：化学生物学 论文提交日期：2012 年 4 月 28 日 论文答辩时间：2012 年 6 月 6 日 学位授予日期：2012 年   月   日    答辩委员会主席：      教授 评    阅    人：      教授    2012 年 06 月 厦门大学博硕士论文摘要库厦门大学博硕士论文摘要库  厦门大学学位论文原创性声明  本人呈交的学位论文是本人在导师指导下，独立完成的研究成果。本人在论文写作中参考的其他个人或集体的研究成果，均在文中以明确方式标明，并符合法律规范和《厦门大学研究生学术活动规范（试行）》。 另外，该学位论文为（                        ）课题（组）的研究成果，获得（       ）课题（组）经费或实验室的资助，在（                ）实验室完成。（请在以上括号内填写课题或课题组负责人或实验室名称，未有此项声明内容的，可以不作特别声明。）   声明人（签名）：  年   月   日 厦门大学博硕士论文摘要库厦门大学博硕士论文摘要库  厦门大学学位论文著作权使用声明  本人同意厦门大学根据《中华人民共和国学位条例暂行实施办法》等规定保留和使用此学位论文，并向主管部门或其指定机构送交学位论文（包括纸质版和电子版）,允许学位论文进入厦门大学图书馆及其数据库被查阅、借阅。本人同意厦门大学将学位论文加入全国博士、硕士学位论文共建单位数据库进行检索，将学位论文的标题和摘要汇编出版，采用影印、缩印或者其他方式合理复制学位论文。 本学位论文属于： （      ）1. 经厦门大学保密委员会审查核定的保密学位论文，于    年      月      日解密，解密后适用上述授权。 （      ） 2. 不保密，适用上述授权。 （请在以上相应括号内打“√”或填上相应内容。保密学位论文应是已经厦门大学保密委员会审定过的学位论文，未经厦门大学保密委员会审定的学位论文均为公开学位论文。此声明栏不填写，默认为公开学位论文，均适用上述授权。）   声明人（签名）：  年   月   日厦门大学博硕士论文摘要库厦门大学博硕士论文摘要库  目 录 摘要 ................................................................................................................................ I Abstract ..................................................................................................................... III 第一章 前言 ................................................................................................................. 1 1.1 研究背景 .................................................................................................................................. 1 1.2 天然药物抗癌有效成分研究进展 .......................................................................................... 1 1.2.1 黄酮类 ............................................................... 1 1.2.2 生物碱 ............................................................... 1 1.2.3 萜类 ................................................................. 2 1.2.4 其他类 ............................................................... 2 1.3 小槐花及山蚂蝗属综述 .......................................................................................................... 2 1.3.1 小槐花及山蚂蝗属简介 ................................................. 2 1.3.2 山蚂蝗属植物化学成分研究进展 ......................................... 3 1.3.3 山蚂蝗属植物的药理研究 ............................................... 7 1.4 黄酮的抗癌活性及其作用机制 .............................................................................................. 8 1.4.1 黄酮的抗癌活性机制 ................................................... 8 1.4.2 细胞周期调控因子 .................................................... 10 1.4.3 黄酮影响肿瘤细胞周期的具体机制 ...................................... 11 1.5 立题依据 ................................................................................................................................ 12 第二章 小槐花活性部位的化学成分研究 ............................................................... 15 2.1 仪器及材料 ............................................................................................................................ 15 2.2 提取与分离方法 .................................................................................................................... 16 2.3 化合物结构鉴定 .................................................................................................................... 19 2.3.1 新化合物结构鉴定 .................................................... 19 2.3.2 已知化合物结构鉴定 .................................................. 45 2.3.3 化合物结构图 ........................................................ 52 第三章 小槐花中化学成分的抗肿瘤活性评价 ...................................................... 55 3.1 材料与仪器 ............................................................................................................................ 55 3.2 小槐花中化学成分的抗肿瘤活性研究 ................................................................................ 56 3.2.1 小槐花中化学成分的细胞毒性研究 ...................................... 56 3.2.2 小槐花中抗肿瘤活性成分细胞毒性验证 .................................. 59 3.3 小槐花中抗肿瘤黄酮成分关于细胞周期的机制研究 ........................................................ 61 3.3.1 小槐花中活性成分对细胞周期阻滞的影响 ................................ 61 3.3.2 小槐花中活性成分对周期相关蛋白的影响 ................................ 62 3.3.3 化合物 7、10、11 与 CDK4 蛋白模拟对接研究 ............................. 64厦门大学博硕士论文摘要库厦门大学博硕士论文摘要库  第四章 总结与讨论 ................................................................................................... 67 4.1 小槐花化学成分研究总结 .................................................................................................... 67 4.2 小槐花化学成分的生物活性总结 ........................................................................................ 67 4.3 黄酮类化合物与抗肿瘤活性的构效关系探讨 .................................................................... 68 参考文献 ..................................................................................................................... 71 附录 1 .......................................................................................................................... 77 附录 2 .......................................................................................................................... 94 硕士期间发表论文 ..................................................................................................... 98 致谢 ........................................................................................................................... 101  厦门大学博硕士论文摘要库厦门大学博硕士论文摘要库  Contents Abstract in Chinese ...................................................................................................... I Abstract in English ................................................................................................... III Chapter 1 Introduction ............................................................................................... 1 1.1 Background ............................................................................................................................... 1 1.2 Rechearch advances in antitumor of natural drugs .............................................................. 1 1.2.1 Flavonoids ............................................................................................................................ 1 1.2.2 Alkaloids .............................................................................................................................. 1 1.2.3 Terpene ................................................................................................................................. 2 1.2.4 Others ................................................................................................................................... 2 1.3 Research advance in Desmodium caudatum (Thunb.) DC and Desmodium Desv. .............. 2 1.3.1 Introducetion of Desmodium caudatum (Thunb.) DC and Desmodium Desv ..................... 2 1.3.2 Research advance in chemical components of Desmodium Desv ....................................... 3 1.3.3 Research advance in pharmacological effects of Desmodium Desv. ................................... 7 1.4 The anticancer activities and mechanisms of flavonoids ...................................................... 8 1.4.1 The mechanisms of anticancer on flavonoids ...................................................................... 8 1.4.2 The factors on cell cycles ................................................................................................... 10 1.4.3 The cell cycle mechanisms of anticancer on flavonoids .................................................... 11 1.5 Aims and significance ............................................................................................................. 12 Chater 2 Research of chemical components of Desmodium caudatum (Thunb.) DC ............................................................................................................................... 15 2.1 Materials .................................................................................................................................. 15 2.2 Extraction and isolation ......................................................................................................... 16 2.3 Identification of chemical structures .................................................................................... 19 2.3.1 Identification of new chemical structures .......................................................................... 19 2.3.2 Identification of known chemical structures ...................................................................... 45 2.3.3 Struction of all compounds ................................................................................................ 52 Chapter 3 Research for biological activity of constituents .................................... 55 3.1 Materials .................................................................................................................................. 55 3.2 Research on biological activity of the isolated compounds ................................................. 56 3.2.1 The antitumor study of the constituents from by MTT ...................................................... 56 3.2.2 The effects of the active compounds on colon formation of A431 cells ............................ 59 3.3 The cell cycle mechanisms of the active constituentes from Desmodium caudatum (Thunb.) DC .................................................................................................................................. 61厦门大学博硕士论文摘要库  3.3.1 The effects of active constituentes on the cell cycle arrests of A431  ............................... 61 3.3.2 The effects of active constituentes on the proteins related G1 phase ................................ 62 3.3.3 The docking study between compound 7,10,11 and CDK4 ............................................... 64 Chapter 4 Conclusions and disscussion ................................................................... 67 4.1 Conclusion and disscussion of chemical components of Desmodium caudatum (Thunb.)  DC .................................................................................................................................................. 67 4.2 Conclusion and disscussion of biological activity of the isolated compounds ................... 67 4.3 The discussion of the relationship between flavonoids’ structure and antitumor activity68 References ................................................................................................................... 71 Appendix 1 ................................................................................................................. 77 Appendix 2 ................................................................................................................. 94 Publicaton ................................................................................................................... 98 Acknowlegements .................................................................................................... 101厦门大学博硕士论文摘要库摘 要  I 摘 要  癌症是当前严重影响人类健康、威胁人类生命的主要疾病之一。近年来，以细胞周期为药物靶点进行筛选，已成为当今抗癌药研发的重要方向。中草药在中国已有几千年的药用历史，其结构多样性和复杂性的优势一直受到药物开发者的青睐。我们在此基础上筛选了来源于 40 种植物的 2000 多个馏分，对其进行细胞毒活性评价，发现小槐花 60%乙醇提取物的大孔树脂 95%洗脱馏分中间极性部分具有良好的细胞毒活性。因此对活性部位进行了活性成分的研究。 本文综合运用硅胶柱层析、ODS柱层析、Sephadex LH-20柱层析以及反相HPLC等多种色谱学分离手段，对小槐花60%乙醇提取物的大孔树脂95%乙醇洗脱部分的化学成分进行了研究，从中分离得到化合物22个。通过理化性质和现代波谱学手段（UV、MS、1H-NMR、13C-NMR 和 2D-NMR）鉴定了它们的结构。包含黄酮类化合物14个，三萜类化合物4个，甾醇类化合物两个和生物碱类化合物2个。其中四个黄酮化合物9、11、21、22为新化合物，除化合物6外，所有化合物均为从该种植物中首次分离得到。 本文通过 MTT 实验对小槐花中分离到的化合物进行抗肿瘤活性筛选，发现化合物 7、10、11、14 在 100 μM 条件下对宫颈癌细胞 HeLa、人肝癌细胞 SMMC-7721、人表皮癌细胞 A431 三种人癌细胞具有明显的抗肿瘤活性，Giemsa 染色进一步说明了这 4 个化合物对人上皮癌细胞有抗肿瘤活性，且具有浓度依赖性。为了进一步探索黄酮类化合物于癌细胞周期的关系，我们通过流式细胞术 PI 染色法、Western Blotting 研究发现，三个活性黄酮化合物化合物 7、10、11 在 50μM 浓度条件下对 A431 人上皮癌细胞有明显的 G1/S 期阻滞现象，显著抑制 G1 期相关蛋白 CDK4、CDK6、Cyclin D1，且新黄酮化合物 11 抑制最为显著。通过进一步计算机模拟对接发现，三个活性黄酮化合物 7、10、11 能够与 CDK4 蛋白很好的模拟对接。这些结果说明小槐花发挥抗癌活性功能的主要物质基础可能为黄酮类化合物，这些黄酮化合物通过抑制细胞周期 G1 期相关蛋白 CDK4、CDK6、Cyclin D1，进而发挥抗癌功能。 最后，本文还对小槐花中的黄酮结构与抗肿瘤活性之间的构效关系展开了讨论，阐明了黄酮化合物结构与其抗癌活性之间的的部分关系。  关键词：小槐花；黄酮；抗癌；细胞周期 厦门大学博硕士论文摘要库厦门大学博硕士论文摘要库Abstract  IIIAbstract  Cancer is one of the major diseases which seriously impact human health and threaten human life. Recently, people pay much attention to find anticancer drugs that targeting cell cycle.Traditional Chinese Medicine (TCM) has been used in China for centuries. The structure diversity and variety of compounds make TCM become the hot spot in new drug discovery. In this thesis, we screen approximately 2000 fractions purified from 40 Chinese herbal plants with cytotoxic activity model.Sixty percentage ethanol extract of Desmodium caudatum(Thunb.)DC seprated by macroporous resin eluting with 95% ethanol extract were discovered with cytotoxic activity. On basis of this result, the chemical constituents and anti-cancer activity of Desmodium caudatum(Thunb.)DC were investigated in the present study. Chemical constituents research of 60% ethanol extract of Desmodium caudatum(Thunb.)DC leads to 22 compounds through various chromatographic techniques, such as silica gel, RP-18 gel, Sephadex gel charomatography and PHPLC. Four new compounds were obtained and all the  compounds were isolated from Desmodium caudatum(Thunb.)DC for the first time except compound 6.  All the compounds were evaluated by MTT assay to detect the antitumor activity. Our results suggested that compound 7,10,11,14 could inhibit the growth of cancer cell HeLa,SMMC-7721 and A431 significantly. The antitumor activity of these four compounds on the A431 was defined by Giemsa assay,further more,the antitumor activite is dose-dependented. In order to explore the relationship between flavonoids and antitumor activity targeting cell cycle , influence of the three flavonoids, compounds 7,10,11 was evaluated by flow cytometry. Our result shows that the compounds(50μM) can prohibit  the cell cycle of A431 at G1/S Significantly. We suppose that the mechanism of the compounds to inhibit cell cycle at G1/S was probably be the inhibiting expression of protein that related to G1 period,such as CDK4、CDK6 and Cyclin D1. So we test and verify the suspect by Western Blotting. The docking by the software autodock4 shows the binding between the flavonoids and the protein CDK4 is good.All the results suggest the active components of Desmodium caudatum(Thunb.)DC are mainly flavonoids. Finally, the structure-activity relationship between flavonoids in Desmodium caudatum 厦门大学博硕士论文摘要库Abstract  IV(Thunb.)DC  and antitumor activity was discussed  and clarified partly.  Keywords: Desmodium caudatum(Thunb.)DC; Flavonoids;Aantitumor; Cell cycle厦门大学博硕士论文摘要库Degree papers are in the “Xiamen University Electronic Theses and Dissertations Database”. Fulltexts are available in the following ways: 1. If your library is a CALIS member libraries, please log on http://etd.calis.edu.cn/ and submitrequests online, or consult the interlibrary loan department in your library. 2. For users of non-CALIS member libraries, please mail to etd@xmu.edu.cn for delivery details.厦门大学博硕士论文摘要库

Anti-cancer constituents research from Desmodium caudatum（Thunb.）DC

http://dspace.xmu.edu.cn/bitstream/handle/2288/47388/%e5%b0%8f%e6%a7%90%e8%8a%b1%e6%8a%97%e8%82%bf%e7%98%a4%e6%b4%bb%e6%80%a7%e6%88%90%e5%88%86%e7%a0%94%e7%a9%b6.pdf?sequence=1&isAllowed=y

Anti-cancer constituents research from Desmodium caudatum（Thunb.）DC

Abstract

Similar works

Full text

Available Versions

Xiamen University Institutional Repository