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1、山西大学 硕士学位论文 苦荞总黄酮的提取及体外抗肿瘤活性研究 姓名：闫斐艳 申请学位级别：硕士 专业：生物化学与分子生物学 指导教师：王转花 2010-06 I 中 文 摘 要 苦荞麦中所含的营养成分非常复杂，主要包括生物类黄酮、高活性蛋白、氨基 酸、脂肪酸、膳食纤维、微量元素等。苦荞麦中生物类黄酮的含量较高，其中以芦 丁和槲皮素为主。大量的实验已经证实，苦荞生物类黄酮具有极其广泛的生理和药 理活性，如抗氧化、抗突变、降血糖、抗炎症、调节免疫等功能,尤其近年来，其独 特的抗癌功能越来越受到人们的关注。本文利用微波提取方法提取了大量苦荞总黄 酮，并对其抗肿瘤活性进行了初步研究。 实验中采用了热水
2、浸提法、碱水浸提法、乙醇回流提取法、微波提取法和超声 辅助提取法五种不同的提取方法从苦荞种子粉末中得到了总黄酮，使用高压液相色 谱法对提取物中的总黄酮进行了定性定量的检测。结果表明，乙醇回流提取法提取 总黄酮产率最高，微波和超声波提取法次之，碱水和热水浸提法总黄酮产率最低。 微波法提取产率高，耗时短，具有明显的高效性，适合大规模提取苦荞生物类黄酮。 利用 MTT 法检测苦荞总黄酮对食管癌 EC9706 和宫颈癌 HeLa 细胞的毒性效果， 结果表明，苦荞总黄酮能明显抑制两株细胞的增殖，并且作用效果呈时间和剂量依 赖性。在光学倒置显微镜下观察，可见细胞核固缩，形成多个膜包的凋亡小体。 利用 DA
3、PI 染色检测 EC9706 和 HeLa 细胞经苦荞黄酮处理后的形态变化， 电镜 观察细胞核， 可见多个凋亡小体。 表明苦荞黄酮诱导细胞发生了凋亡。 经 Annexin-V/PI 染色后，流式细胞仪检测，发现细胞出现晚期凋亡，并且凋亡率与黄酮剂量成正相 关。 此外， 加入不同的检测试剂后， 用流式细胞术观察其对细胞周期和细胞内 ROS 水 平的调节作用，结果表明，苦荞黄酮引起了 EC9706 和 HeLa 细胞的 G2M 期周期 停滞，且细胞内细胞内 ROS 水平呈浓度依赖性增加。Western 杂交实验初步显示， 苦荞黄酮能上调细胞内的促凋亡蛋白 Bax 以及下调抗凋亡蛋白 Bcl-2 的
4、表达量。 以上结果表明，苦荞黄酮对食管癌 EC9706 和宫颈癌 HeLa 细胞都具有明显的增 殖抑制效果，为进一步开发和利用苦荞黄酮药用资源提供了一定的理论依据。 关键词:关键词: 苦荞；黄酮；抗肿瘤；细胞凋亡 ABSTRACT There are many constituents in tartary buckwheat, including flavonoids, highly reactive protein, amino acids, fatty acids, dietary fibre and microelements. Among these components, the
5、content of flavonoids is much higher, which is mainly composed of rutin, quercetin and other essential active ingredient. And flavonoids, as an extremely nutritious substance, exerts a variety of biological activities, such as anti-oxidative, free radical scavenging, anti-allergic, anti-inflammatory
6、, and anti-mutagenic activities. In particular, They have potential effect on chemotherapy treatment. Accumulating evidence suggests that intake of beverages or food products containing flavonoids has been frequently associated with a reduced risk for developing various cancers. In this paper, abund
7、ant total flavonoids were extracted from tartary buckwheat and the principal determination of the ingredient, anticancer effects of total flavonoids were studied. Total flavonoids were extracted from tartary buckwheat seeds by using hot water leaching method， alkali water method， ethanol solution le
8、aching method, microwave exeraction method, and ultrasonic wave method. The rate and content of total flavonoids was determined by High Performance Liquid Chromatography. In five extraction methods, the extraction rate of ethanol solution leaching method was highest, microwave extraction method and
9、ultrasonic wave method followed, alkali water method and hot water leaching method were lowest. the microwave extraction method is rapid and efficient, which is the appropriate method for extracting flavonoid substances from tartary buckwheat seeds. To explore the possible influence of the flavonoid
10、s from tartary buckwheat on the human solid tumor cells, recently the in vitro anti-cancer effect of flavonoids on esophageal cancer cell line EC9706 and cervical carcinoma cell line HeLa was investigated in our lab. When the two cells were treated with flavonoids for various periods of time and var
11、ious doses, the MTT assay showed that flavonoids could specifically inhibit the growth of solid tumor cells in a time -and dose -dependent manner. Cell death induced by flavonoids was characterized by nuclear condensation and the production of apoptotic bodies via the light microscopy and DAPI nucle
12、ar staining observation, indicating flavonoids can induce cell apoptosis. By flow cytometry analysis, the cell cycle progression and Annexin- V- FIFT /PI assay indicated that the cytotoxicity induced by flavonoids was mediated by G2/M cell cycle arrest and the apoptosis rate also increases as compar
13、ed to controls, which was increased after treatment with flavonoids in range of 060 g/ml. Besides, flavonoids-induced apoptosis was shown to involve a concentration-dependent increase in ROS production by flow cytometric analysis. Moreover, western blotting was used for the detection on esophageal c
14、ancer cell line EC9706 of the alteration of Bcl-2 and Bax protein ratio.It showed that treatment with flavonoids increased Bax expression but decreased Bcl-2 expression. All above results suggests that flavonoids from tartary buckwheat have anticancer activities, and it appears to be a potent drug a
15、gainst human solid tumor cells (EC9706 and HeLa cells), due to its in vitro potential to not only arrest cell cycle progression at G2/M phase but also induce apoptosis via ROS generation. Therefore it can provid a great deal of referenes for further use and exploitation of flavonoids from tartary bu
16、ckwheat. Keywords: Tartary buckwheat; Flavonoids; Antitumor; Apoptosis 第一章 文献综述 1 第一章 文献综述 1.1 苦荞麦概述 苦荞麦（tartary buckwheat）学名鞑靼荞麦，又名“春荞”、“野荞麦”、“万年荞”， 主要分布在我国的西南云贵高原以及北方的陕西、山西、河北、内蒙一带，在分类 学上属于双子叶蓼科荞麦属植物。苦荞麦为一年生或多年生宿根性植物，它耐旱、 耐寒，耐瘠薄,适于在冷凉气候下短季生长。 1.1.1 苦荞麦的开发利用 苦荞麦全身是宝，药食两用，早在我国古代的医书本草纲目、齐民要术 中就有关于其药用价值的丰富记载。近年来，随着饮食结构的不断调整，以小杂粮 为时尚的粮食消费结构也愈来愈受到人们的关注。尤其苦荞麦作为一种重要的粮食 作物，富含蛋白质、淀粉、纤维素和矿物质等,与其他的作物相比，有着许多独特的 营养和保健功能，我国的一些苦荞麦制品已逐步进入国际市场。 1.1.2 苦荞麦的主要营养成分及其药理活性 (1) 苦荞麦蛋白 苦荞麦中高活性蛋白含量大约为10%，这一
