【作者】 袁怀波；

【导师】 陈宗道； 糜漫天； 李洪军；

【作者基本信息】 西南农业大学 ， 农产品加工与贮藏工程， 2004， 博士

【摘要】 肿瘤已经成为人类死亡的第一位或第二位原因，全世界每年约有700万人口死于癌症，“控制癌症已成为我国卫生战略重点之一”。肿瘤及癌症的发生机制及治疗对策的研究是当今医学研究的热点，其中对人体无毒副作用而对肿瘤细胞有高度特异性作用的天然药物研究开发更为研究人员所重视。流行病学和试验研究已发现黄酮类化合物具有抑制肿瘤的发生、抵抗致癌物致癌作用等抗肿瘤的功效。 黄酮类化合物是植物的次生代谢产物，存在于所有的维管植物中，是以黄酮(2-苯基色原酮)为母核的一类黄色色素，是以酯、醚、甙的衍生物或混合物形式存在的，已查明在可食用的植物食品中有4000多种黄酮类化合物。黄酮类化合物是自然界中存在较为广泛的一类物质。人体内不能合成黄酮类化合物，可以通过食用含有黄酮类化合物的食品以摄入此类化合物。估计人类每天摄入几百毫克到1克黄酮类化合物。 研究发现黄酮类化合物有降血糖、降血脂、降血压、抗抑郁、调节神经内分泌、防治心脑血管疾病、抗骨质疏松症、镇痛等许多功效，而更引人注目的是黄酮类化合物的抗肿瘤活性。流行病学、体外实验和动物实验研究已表明，黄酮类化合物有肿瘤预防作用。黄酮类化合物的许多生理功能可能与其抗肿瘤机制有关系，例如抗氧化及抗自由基作用、黄酮类化合物的雌激素效应和抗雌激素、雄激素的作用、诱导解毒酶、调节机体免疫力、影响胞间通讯、抑制癌细胞增殖、诱导肿瘤细胞凋亡和分化。由于黄酮类化合物对肿瘤的细胞毒性作用面对正常细胞的影响很小，且既可以通过食品又可以通过药品摄入的，所以它有开发作为肿瘤治疗药物或辅助治疗药物的可能。 葛根为常用中药，是豆科植物野葛[Pueraria lobata(Willd.)Ohwi]和粉葛(Pueraria homsonii Bentn)的肥大块根，葛根原植物分布于我国、日本、朝鲜半岛及东亚温带地区，在我国除新疆、青海及西藏外遍布全国各地。葛根中含有约占新鲜葛根19％-20％的葛根淀粉外，主要成分为异黄酮化合物以及少量黄酮类物质，其中以大豆甙元、大豆苷、葛根素是葛根的主要活性成分，尤以葛根素含量最高。此外，葛根中还含有三羟基异黄酮、芒柄花黄素、鹰嘴豆素A、葛根多糖、葛根素木糖苷、β-谷甾醇、花生酸等多种生理活性物质。葛根味甘、辛，性平，入脾、胃经，具有解肌退热、生津止渴、升阳止泄、宁心醒脑、通脉濡脉之功效。现代研究表明葛根中的总黄酮和葛根素可扩张冠状动脉和脑血管，改善心脑的血液循环，降低心肌耗氧量，对冠心病、心绞痛、高血压头痛、头晕、项强等症状，对突发性耳聋、神经根型颈椎病、偏头痛、慢性视网膜炎等有较好疗效。近年来研究发现葛根总黄酮有抗氧化、抑制癌细胞增殖和诱导癌细胞凋亡等抗肿瘤作用。一一一一一-一一一‘一一一一一一 葛根是药食兼用的山地植物。据世界粮农组织权威机构的专家预测，葛根有望成为世界第六大粮食作物。葛根的开发可以说是方兴未艾，葛根的各部分材料都可以开发利用。每年国内外葛根的市场需求量可达每年数万吨。葛根具有巨大的开发潜力和良好市场前景。深度 研究开发葛根对于促进葛根种植地(山区)的农业收入有重要的现实意义。但是葛根总黄酮 相对落后的工业制备技术、葛根总黄酮中单体成分常规耗时繁琐定性定量分析是开发利用葛根的技术缺陷，同时葛根总黄酮及其单体黄酮抗肿瘤的作用机制还没有阐明，所有这些都限制了葛根的进一步开发利用。 本研究正是针对葛根总黄酮生产和生理作用机制方面的缺陷进行较为系统的研究。在本研究的前部分中利用乙醇提取葛根总黄酮，经过大孔树脂的分离纯化得到精制葛根总黄酮，通过液一质联用对所得化合物中所含的葛根素、大豆贰元、三轻基异黄酮进行定性、定量研究。在本研究的后部分中，进一步利用流式细胞仪、免疫荧光分析、免疫印迹、RT一PCR等技术在细胞和分子基因水平上探讨葛根总黄酮以及其单体葛根素和大豆贰元对HL一0肿瘤细胞的抑制肿瘤细胞增殖和诱导促进肿瘤细胞凋亡作用，其中在基因水平上是选测在凋亡中有重要作用的基因(FaS、bel一2、bax、CaSPa阴一、CasPase一8)，探讨葛根总黄酮及其单体黄酮一葛根素和大豆贰元的抑制HL石0细胞增殖和诱导凋亡的分子机制，为葛根总黄酮提取开发为抗肿瘤保健食品和药品提供理论依据。 本课题的主要研究结果如下:1.对六种大孔吸附树脂对葛根总黄酮的静态吸附效果比较，认为AB一8树脂适用于葛根总黄酮的纯化，研究了不同的乙醇体积百分数、上样液流速、上样液pH对葛根总黄酮纯化能力的影响，摸索出了适宜的纯化条件，得到纯度为30%的葛根总黄酮，再经过透析袋处理可以使纯度达到80%。实验说明利用大孔树脂可以对葛根总黄酮提取物进行有效纯化。2.利用液一质联用技术对葛根总黄酮中所含的葛根素、大豆贰元、三轻基异黄酮等单体黄酮进行快速高效定性定量研究。说明利用液一质联用技术可以替代常规繁琐的成分测定研究。3.50呵mL的葛根总黄酮、30腼。比的葛根素和30腼o1几大豆贰元可以有效抑制HL石0细胞的增殖，且葛根总黄酮阻滞细胞周期于GZ介通，而葛根素和大豆更多还原

【Abstract】 Cancer became one of major human die killers, every year about 700 ten thousand people die in cancer in this world. Try to control cancer become one of our hygiene strategy. The study which to reveal the mechanisms of cancer development and to treat cancer become one of highlight research point Pursuer search native compound which have no toxicity on normal cell but have special cell toxicity efficacy on cancer cell as anticancer therapeutic agent. Epidemiology and some research have revealed flavonoids have suppression cancer cell proliferation and anticarcinogenesis potent.Flavonoids are plant secondary metabolites, present in all terrestrial vascular plants, flavonoids are defined chemically as substances composed of a common phenyichromanone structure(C6-C3-C6), with one or more hydroxyl substituents, including derivates. Flavonoids occur commonly as ester, ether, or giycoside derivatives or mixtures thereof, and embrace over 4000 compounds. Flavonoids is common exist in the natural world, in mammals, its occur only through dietary intake. The daily human intake of flavonoids has been estimated to be several hundreds mg to 1g.Studies have revealed flavonoids have decrease blood sugar, decrease blood pressure, antidepress, regulate nerve endocrine, prevent atherosclerosis and thrombosis, anti-osteoporosis, relieve pain and so on potent The highlight activity of flavonoids is anticancer. Epidemiology, vitro studies and vivo studies have indicated flavonoids have anticancer potent. Many physiological function of flavonoids may contribute to its anticancer potent, such as anti-oxidizing, anti-free radical, estrogenic and antiestrogenic activity, androgen activty, induct detoxification enzymes, regulate host immune function, affect intercellular communication, inhibition of cancer ceil proliferation, induce cancer cell apoptosis and differentiation. Because flavonoids have no toxicity on normal cell but have special cell toxicity efficacy on cancer cell, can intake through diet, it has to developed as anticancer therapy drug or assistant anticancer therapy drug potent.Kudzu (pueraria lobata) is traditional Chinese medicine. Its plant distribute in our country, Japan, Korea and temperate zone of east asia. In our country it distribute in whole land except XINJIANG,QINHAI and Tibet Including fresh pueraria lobata contain about 19%-20% starch,pueraria lobata cardinal component are flavonoids, daidzein daidzin and puerarin is pueraria lobata cardinal activity component, it also contain genistein. formononetin, biochanin A, pueraria polysaccharide, xylose-puerarin , β-Sitosterok arachidic acid and so on activity compound. Contemporary studies have showed kudzu flavonoids and puerarin can expand coronary artery and cerebral vessels, improve blood circulation of heart and brain. decrease myocardium oxygen consumption, it can used to treat coronary heart disease, angina pectoris, hypertension headache, dizziness, nuchal rigidity , sudden deafness, cervical syndrome,hemicrania, chronicity retinitis and so on disease. Recent studies have revealed kudzu flavonoids have anti-oxidizingv depress cancer cell proliferation and induce cancer cell apoptosis ability.Kudzu can be used as food and drug. FAO expert forecasted kudzu probably become 6th food supplies crop. The exploit of kudzu in the ascendant, each part of kudzu can be exploited. National and international market demand kudzu reached several ten thousand ton every year. Kudzu possessed large exploit potent and well market prospect. Research and exploit kudzu deeply can improve kudzu planting area (mountainous area) geoponics earning. But lag preparation technology and routine trivial qualitative and quantitative analysis kudzu monomer compound is kud exploitation technique defect, at same time, anticancer mechanism of kudzu flavonoides and monomer flavonoid was not elucidated, all of this limited kudzu advanced exploited.Our research is aim directly at resolve problem of kudzu flavonoids’ production and unclear anticancer mechanisms. At ant更多还原