【作者】 赵旌旌；

【导师】 瞿伟菁；

【作者基本信息】 华东师范大学 ， 植物学， 2012， 博士

【摘要】 芦笋是当今世界上营养平衡最佳的蔬菜品种之一,具有抗氧化、降血脂和抗肿瘤功效。芦笋的食用部分为其嫩茎,占茎总长度的二分之一至三分之二,剩余的木质化部分——老茎则成为收割时的废弃物,尽管其中仍含有多种活性成分,但其功能价值和利用长期被忽略。为实现资源深度开发,本文对芦笋老茎的降血脂、降血糖和抗肿瘤活性进行了系统的评价。观察了老茎提取物对高脂血症小鼠和糖尿病大鼠血糖、血脂代谢的影响、对肿瘤细胞生长的影响,并从中分离获得抗肿瘤有效组分总皂苷,进一步研究了其对肿瘤细胞生长、迁移的影响及其作用机制。论文主要包括以下三个部分：一、芦笋老茎降血脂和降血糖作用研究研究首先对芦笋老茎醇提物(ethanol extracts of asparagus woody stem, EEA)和水提物(aqueous extracts of asparagus woody stem, AEA)进行了化学成分分析,结果显示：醇提物中含有7.94%皂苷、7.32%黄酮和22.29%多糖；水提物中含有2.50%皂苷、3.50%黄酮和36.45%多糖。采用高脂饮食诱导的高脂血症小鼠模型评价了EEA和AEA的降血脂活性。结果显示：连续8周给予200mg/kg EEA和AEA均能显著降低模型小鼠的血清总胆固醇和低密度脂蛋白胆固醇水平、降低血清谷丙转氨酶和谷草转氨酶活性、增加肝脏超氧化物歧化酶活性,降低肝脏MDA水平,增加总抗氧化能力；此外AEA可明显增加模型小鼠血清高密度脂蛋白胆固醇水平,上述结果表明EEA和AEA具有明显的降血脂功能,并提示两者对高脂诱发的肝损伤可能具有一定防治作用。研究进一步采用STZ诱导的类似1型糖尿病大鼠模型和高脂饲料联合STZ诱导的类似2型糖尿病大鼠模型评价了AEA的降血糖作用,并探讨了降糖的初步机理。结果表明：连续3周给予AEA可显著降低类似1型糖尿病大鼠的空腹血糖和甘油三酯水平；升高模型大鼠的体重和肝糖原含量：对血清胰岛素含量没有明显影响；能显著降低类似1型糖尿病大鼠在口服糖耐量实验中糖负荷30、60和120分钟时的血糖水平以及血糖曲线下面积。在类似2型糖尿病大鼠模型中,连续45天给予AEA可显著降低模型大鼠血糖和血清游离脂肪酸水平,提高血清高密度脂蛋白胆固醇水平,对血清胰岛素含量没有明显影响；能显著降低类似2型糖尿病大鼠在口服糖耐量实验中糖负荷60和120分钟时的血糖水平以及血糖曲线下面积。上述结果显示AEA具有明显的降血糖作用,并能改善糖尿病大鼠的脂代谢紊乱和糖耐量异常。二、芦笋老茎细胞毒活性评价及其活性组分皂苷的提取采用前列腺癌PC3细胞模型评价了EEA的抗肿瘤活性。结果显示：在体外抑瘤实验中,EEA对前列腺癌PC3细胞具有细胞毒作用,IC50为1.5mg/ml。为明确老茎中的抗肿瘤活性成份,进一步对EEA进行了分离纯化,EEA经水饱和正丁醇萃取、大孔树脂纯化后分离得到芦笋老茎总皂苷(saponins from woody stems of asparagus, SSA),确立了大孔树脂纯化SSA的工艺路线,并对SSA的抗肿瘤活性进行了评价。结果显示：SSA以剂量依赖的方式抑制乳腺癌、结肠癌、胰腺癌三种肿瘤细胞系共6株肿瘤细胞的生长,具有明显的细胞毒活性。三、芦笋老茎皂苷抗肿瘤活性研究在前期研究的基础上,进一步探讨SSA对乳腺癌细胞MDA-MB-231生长、迁移的影响及其作用机制。首先应用SRB的方法检测SSA对肿瘤细胞的抑制作用,继而通过Annexin V/PI染色实验来进一步证实SSA对人乳腺癌MDA-MB-231细胞凋亡的诱导作用；为了研究SSA对肿瘤细胞运动能力的抑制作用,我们选用具高迁能力的人乳腺癌细胞MDA-MB-231作为代表,通过细胞划痕迁移实验、小室迁移实验和小室侵袭实验来进行研究；并采用glutathione S-transferase (GST)融合蛋白pull-down实验分别纯化得到三种活化形式的Rho GTPase(GTP-结合形式)来研究SSA对于肿瘤细胞运动的抑制作用是否是通过调节Rho GTPase的活性来实现的。结果显示：SSA以剂量依赖的方式抑制肿瘤细胞系的生长且显著地抑制乳腺癌细胞MDA-MB-231的迁移及侵袭能力,其有效抑制浓度为400μg/ml。在其作用机制的初步研究中发现SSA能通过调节小G蛋白的活性从而抑制肿瘤细胞的运动性,即通过调节Rho GTPases信号通路来降低肿瘤细胞的运动能力。更多还原

【Abstract】 Asparagus (Asparagus officinalis L.) is a healthy and nutritious vegetable which possess antioxidant, hypolipidaemic and antitumour properties. The edible shoot of asparagus is about one-half to two-thirds of the full length of the stem, and the remaining woody part (inedible bottom part) is always discarded as by-product. However, this by-product of asparagus still contains many bioactive substances that have potential usage in food supplements and therapeutic effects.Therefore, in order to make full use of woody stem part of asparagus, its effects on serum glucose and lipid profiles in hyperlipidaemic mice and diabetic rats and cancer cell were investigated in the present study. Furthermore, the antitumour ingredient, saponins was screened out and its mechanism was studied.This dissertation includes three parts as follow:1. Hypolipidaemic and hypoglycaemic effects of asparagus woody stemFirstly, the chemical constitutes of ethanol and aqueous extracts of asparagus woody stem (EEA, AEA) were analyzed. Result showed that there were7.94%saponins,7.32%flavonoids and22.29%polysaccharides in ethanol extract, and2.50%saponins,3.50%flavonoids and36.45%polysaccharides in aqueous extract.In this work, a high-fat diet (HFD) fed mice model was used to investigate the effects of ethanol and aqueous extract of asparagus woody stem on lipid metabolism. Results indicated that continuous HFD feeding caused obvious hyperlipidaemia and liver damage in mice. However, EEA and AEA significantly decreased the levels of serum total cholesterol and low-density lipoprotein cholesterol in hyperlipidaemic mice when administered at a daily dose of200mg/kg for8weeks. And treatments of EEA and AEA significantly decreased alanine and aspartate transaminases activities, but markedly increased superoxide dismutase activity and total antioxidant capacity in liver of model mice. Moreover, serum high-density lipoprotein cholesterol levels were evidently increased after AEA treatment. These findings suggest that EEA and AEA have hypolipidaemic effect and might have hepatoprotective activity. The hypoglycaemic effect of the AEA was evaluated in a streptozotocin (STZ)-induced type1-like diabetic rat model and a HFD-low dose STZ induced type2-like diabetic rat model. Results showed AEA administration for21days significantly decreased fasting serum glucose and triglyceride levels but markedly increased body weight and hepatic glycogen level in type1-like diabetic rats. In an oral glucose tolerance test, both the blood glucose level measured at30,60and120min after glucose loading and the area under the glucose curve showed a significant decrease after AEA treatment. In type2-like diabetic rat model, AEA were orally administered to model rats once a day for45days. The results shown that administration of AEA significantly lowered the serum glucose and free fat acid levels, whereas increased high density lipoprotein cholesterol level in diabetic rats. Meanwhile, both blood glucose levels measured at60and120min after glucose loading and the area under the glucose curve in oral glucose tolerance test showed a significant decrease after AEA treatment. These results suggest that AEA might exhibit hypoglycemic and hypolipidemic effects, and could improve glucose tolerance in diabetic rats.2. Cytotoxic effect of asparagus woody stem and the extraction of saponinAs for the ethanol extracts of asparagus woody stem (EEA), prostate cancer PC3cell were used to evaluate its antitumor effect. Results showed that EEA exhibited obviously cytotoxic activity on cancer cell line, and IC50was1.5mg/ml.Saponin was extracted from EEA by n-butanol extraction and macroporous resin column purification. Furthmore the antitumor effect of saponins from woody stems of asparagus (SSA) was evaluated in6cancer cell lines and the results showed the cytotoxic effects of SSA on cancer cell lines in a concentration dependent manner.3. Antitumor activity of saponins from woody stems of asparagusIn this study, we demonstrated the cytotoxic effects of saponins from woody stems of asparagus (SSA) on cancer cell lines in a concentration dependent manner. Cytotoxic effects of SSA were examined using SRB method and the ability of inducing cancer cells apoptosis was analyzed by annexin V staining assay. The inhibitory activities of SSA on cancer cell motility were evaluated using wound-healing migration assay, Transwell migration assay and invasion assay. GST pull-down assay was performed to validate that SSA exerted inhibitory actions on cell metastasis through regulating the Rho GTPases signaling pathway. We evaluated SSA could significantly reduce the migratory and invasive ability of human breast cancer MDA-MB-231cells dose-dependently with an effective inhibitory concentrations of400μg/ml. In addition, when firstly came to the mechanism research, we found out the novel biological functions of SSA as an inhibitor of cell motility by regulating the activation of Rho GTPases in MDA-MB-231cells. Thus, overall, the findings of this study indicate that SSA inhibits the proliferation and motility of tumor cells through modulating Rho GTPases signaling pathway.更多还原