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题名: 三种生物活性筛选模型的引建及其在天然产物筛选中的应用
作者: 付祥
学位类别: 硕士
答辩日期: 2003
授予单位: 中国科学院昆明植物研究所
授予地点: 中国科学院昆明植物研究所
导师: 谭宁华
关键词: 抗肿瘤活性 ; A549细胞株 ; 中甸凤仙 ; 活性筛选 ; 活性追踪 ; 抗骨质疏松 ; 碳酸配酶II ; 抗真菌 ; 白色念珠菌 ; 光滑念珠菌 ; 烟曲菌 ; 抗骨质疏松药物筛选方法 ; 成骨细胞 ; 破骨细胞
学位专业: 植物学
中文摘要: 本论文主要由四章组成。第一章主要介绍了抗肿瘤活性筛选模型CCLT的引建及其在化合物样品批量筛选和中甸凤仙的活性追踪分离中的应用。第二章主要介绍了抗骨质疏松活性筛选模型CA-II模型在天然植物样品筛选中的应用。第三章主要介绍了抗真菌活性筛选模型YNG在天然植物样品筛选中的应用。第四章主要综述了近年来抗骨质疏松药物筛选方法的一些进展。共计筛选了7589个样品(化合物3005个,提取物4584个),发现了354个活性样品(化合物97个,提取物257个)。其中抗肿瘤活性样品87个,抗骨质疏松活性样品100个,抗真菌活性样品167个。特别是8个化合物样品具有较强的抗肿瘤活性(Ic50<2.5μg/ml),4个提取物或分离部位样品具有较强的抗骨质疏松活性(IC50<2.5μg/ml),1个化合物样品和2个提取物或分离部位样品具有较强的抗真菌活性(IC50<2μg/ml)。论文还对这些活性样品的结构和活性、来源与活性的关系进行了探讨。这些研究为活性化合物的进一步药效研究和活性提取物或分离部位的活性追踪分离提供了重要依据。同时中甸凤仙经CCLT的模型活性检测指导下的分离,很快发现了主要的抗肿瘤活性成分200211202(IC50=9.57μM),活性较强,有潜在研发价值。通过以上工作发现这三个体外筛选模型CCLT、CA-II、YNG具有快速、微量、简便、高效、稳定等优点,特别适合于样品的批量筛选。下面简要介绍各章内容:第一章抗肿瘤活性筛选模型CCLT的引建及其应用CCLT模型是通过肿瘤细胞培养进行药物细胞毒性检测筛选抗肿瘤药物的方法。我们主要通过人非小细胞性肺癌细胞株A549培养,对1571个植物来源的化合物样品进行细胞毒性检测,希望能够发现对该细胞生长有较好抑制作用的化合物;并对中甸凤仙的各个提取或分离部位进行细胞毒性检测,以此指导各步分离工作,希望能从该植物提取物中寻找到抗癌作用较好的成分。我们参照美国NCI和中科院上海药物研究所的抗肿瘤细胞毒性模型筛选方法,经过一系列的条件优化,建立了CCLT模型,并对1571个植物来源的化合物样品进行了批量筛选。经过初筛后选取抑制率最高的前92个样品进行了评估;经过评估后,共发现82个活性化合物(IC50值低于10μg/ml),其中活性较好的3个化合物的IC50值小于1μg/ml,IC50值介于l-2.5μg/ml之间的样品5个,介于2.5-5μg/ml之间的样品有59个,介于5-7.5μg/ml之间的样品共10个,介于7.5-10μg/ml之间的样品共5个,并对这些活性化合物进行了结构与活性的分析。在对中甸凤仙的活性追踪分离过程中,对其粗提取、不同溶剂萃取部位和柱分离部位分离及其得到的成分共计36个样品进行了A549细胞株的抗肿瘤活性检测,发现了8个抗肿瘤活性样品,其中包括了抗肿瘤活性最强的化合物200211202,其IC50值为9.57μM。第二章抗骨质疏松活性筛选模型CA-II的批量筛选CA-II模型是以破骨细胞内含量丰富且对骨吸收起重要作用的碳酸醉酶11作为靶点的体外筛选方法。我们希望通过寻找该酶的抑制剂来寻找治疗骨质疏松症的药物。该实验以样品对该酶催化其底物PNPA的酷键水解程度的影响来评价样品对该酶的抑制或促进作用。在进一步条件优化的基础上对主要来源于植物的天然产物样品进行了批量筛选,包括2840个提取物或分离部位样品及243个化合物样品,经过初筛、复筛和评估,共发现了%个提取物或分离部位样品的IC50值低于10μg/ml,4个化合物样品的IC50值低于12.5μg/ml,并探讨了这些活性样品的结构与活性、来源与活性的关系。第三章抗真菌活性筛选模型YNG的批量筛选州G模型是对常见的三种具有代表性的条件致病菌即白色念珠菌、光滑念珠菌和烟熏曲霉菌在药物存在下培养并检测样品对真菌的抑制程度来筛选抗真菌药物的方法。我们希望通过筛选能找到对这三种条件致病真菌生长产生抑制作用的样品。我们对这几种致病菌在一定时间内与药物一同培养,通过直接比浊或间接观察细胞对染料的分解程度来检测这些真菌生长状况,评价样品的抗真菌活性。通过对这些真菌的培养,在CA一YNG模型中对329个提取物、242个化合物进行了筛选,在CA一BSA一YNG模型中对329个提取物、246个化合物进行了筛选,在CG一YNG模型中对732个提取物、462个化合物进行了筛选,在AF-YNG模型中对329个提取物、241个化合物进行了筛选,累计筛选了29 10个样品。发现了对cA菌株具有抑制活性的样品9个,其中6个提取物(IC50<16μg/ml)、3个化合物( IC50<4μg/ml);对CG菌株具有抑制活性样品共158个,其中150个提取物(IC50<16μg/ml)、8个化合物(IC50<4μg/ml),对BSA存在下CA菌株未发现具有抑制活性的样品,对AF菌株也未发现具有抑制活性的样品,共发现有活性的样品167个,其中11个化合物、156个提取物。并对这些活性样品的结构与活性、来源与活性进行了分析。第四章抗骨质疏松药物活性筛选方法进展本章综述了抗骨质疏松药物活性筛选方法在整体和离体动物模型和细胞水平、分子水平等方面的一些研究进展。
英文摘要: This dissertation mainly contains four chapters. Chapter one is about the establishment of anti-tumor cytotoxicity-based assay (CCLT) targeted to the human non-small cell lung cancer cell line A549 and its application to the screening of compounds mainly originated from plants and to the bioassay-guided fractionation of crude extracts of Impatiens chungtienensis Y. L. Chen. Chapter two is about the anti-osteoporosis assay (CA- II) targeted to the human carbonic anhydrase II (hCA II) and its application to the screening of samples. Chapter three is about the antifungal assays (YNG) targeted to three pathogenic fungi, Candida albicans, Candida glabrata and Aspergillus fwnigatus, and their applications in the mass screening. An overview about the methods to assess the effect of anti-osteoporosis drugs has been introduced in the last chapter. Finally 7589 samples including 3005 compounds and 4584 extracts or fractions were tested and the results indicated that 354 samples including 97 compounds and 257 extracts or fractions showed activities in these assays, which included 87 anti-cancer, 100 anti-osteoporosis and 167 antifungal samples. Among them 8 compounds showed strong anti-cancer activities with IC50<2.5μg/ml, 4 extracts or fractions with strong anti-osteoporosis activities (IC50<2.5) μg/ml), one compound and two extracts or fractions with strong antifungal activities (IC50<2μg/ml). In the Results and Discussion of each chapter, relationship between structures, resources and activities were discussed which showed great significance in the studies of active compound pharmacology and bioassay-guided fractionation for active extracts or fractions. Under bioassay-guided fractionation of I. chungtienensis, the promising anti-cancer compound 200211202 was found with IC50 of 9.57μM. After screenings of CCLT, CA- II and YNG assays in the natural products we found that these three assays are rapid, sensitive, stable, convenient and effective models in looking for active samples from natural resources by mass screening. Chapter 1 Anti-Tumor Assay CCLT Targeted to Cancer Cell Line A549 and Its Application in the Mass Screening and Bioassay-Guided Fractionation of Impatiens chungtienenisis. A rapid anti-tumor assay CCLT using sulforhodamine B (SRB), a colorimetric end-point assay which detects viable cells by staining their cellular protein content, to assess the proliferation of non-small cell lung cancer cell line A549 and the cytotoxicity of the samples, was set up to screen compounds originated from plants and guide the fractionation of I. chungtienenisis. 1571 compounds were screened in this assay and 82 compounds out of them showed activities with their IC50 less than 10) μg/ml, among which 3 showed the IC50 value lower than 1μg/ml. The relationships between structures and activities of active compounds were summarized. A bioassay-guided fractionation of the crude extracts of I. chungtienensis into pure active compounds has been conducted through this assay. Finally eight extracts or fractions and compounds of 36 tested samples were measured with the IC50 value lower than 20ug/ml against the A549 cell, in which the promising compound 200211202 was found with the IC50 of. 9.57μM and it also showed high inhibition against the enzyme CDC25 which plays a key role in the cell cycle in CDC25 assay run in our lab.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/644
Appears in Collections:昆明植物所硕博研究生毕业学位论文_学位论文

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三种生物活性筛选模型的引建及其在天然产物筛选中的应用.付祥[d].中国科学院昆明植物研究所,2003.20-25
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