【作者】 姜山；

【导师】 耿美玉；

【作者基本信息】 中国海洋大学 ， 药物化学， 2011， 博士

【摘要】 肿瘤依然是人类尚未克服的严重威胁人类健康的疑难病症之一,其发病率和死亡率均居各类疾病之首。其中,肺癌发病率及死亡率高居首位。目前,肿瘤的防治仍然采取手术为主,放化疗为辅的策略,而药物学治疗是目前抗肿瘤防治研究最为活跃的领域。当前临床常用的抗肿瘤药约有七十余种,但仍不能有效控制肿瘤的发生与发展。尽管进入临床研究的抗肿瘤药物居高不下,但理想的高效低毒药物却寥寥无几。天然产物是抗肿瘤药物的重要来源。我国的药用植物种质资源丰富,中医药作为我国的国粹,中药的抗肿瘤活性已得到国际公认。猫眼草是中国传统中草药之一,其提取物抗肿瘤作用的研究已有部分报道,但关于其抗肿瘤活性的物质基础及其详细机制未有文献报道。本论文通过提取分离获得猫眼草中黄酮类物质的基础上,进一步观察了其体内对肺癌的作用,并探讨了其作用机制。1、猫眼草中黄酮类物质的提取工艺探讨采用均匀设计法研究从猫眼草中提取总黄酮的工艺,以乙醇为溶剂,考察了四个因素乙醇浓度(30%～90%)、乙醇用量((W/V):9～15倍)、回流时间(0.5～3.5小时)、回流次数(1～3次)对猫眼草总黄酮提取率的影响,以确定最佳的提取工艺条件。并按设计方法进行了验证试验。结果表明:以原药材14倍(W/V)的50%乙醇水溶液,加热回流提取2次,每次1小时的条件,从猫眼草中提取总黄酮的提取率最高(3.28%)。用该工艺提取猫眼草总黄酮工艺简单,提取率高,操作容易控制,稳定性好,适合于工业化生产。2、利用血清药理学手段,在细胞水平确定了猫眼草黄酮类粗提物的抗肿瘤作用在确定猫眼草黄酮类粗提物具有抗肿瘤作用的基础上,进一步采用血清药理学方法,利用体外细胞培养技术,结合Western Blot手段,研究了猫眼草黄酮类粗提物抗肿瘤作用的机制。结果发现,含猫眼草黄酮类粗提物兔血清可明显抑制肺癌细胞株A549的增殖,且其抑制作用具有一定的浓度和时间相关性,20%浓度下作用72h对A549增殖的抑制率为39.08%。采用流式细胞技术发现,猫眼草黄酮类粗提物兔血清可明显阻滞肺癌细胞株A549于G1期,并出现明显的细胞凋亡。进一步采用Western Blot手段观察了细胞周期相关及凋亡相关蛋白的变化,结果表明,含猫眼草黄酮类粗提物兔血清可以促进凋亡相关蛋白Caspase-9和Caspase-3蛋白切割,进而引起PARP蛋白活化,促进凋亡相关蛋白Bax表达升高,减少抗凋亡相关蛋白Bcl-2表达及凋亡相关蛋白Bad磷酸化。表明含猫眼草黄酮类粗提物兔血清能够诱导肺癌细胞株A549的凋亡。3、确定了猫眼草黄酮类粗提物具有抗肿瘤作用利用荷Lewis肺癌小鼠肿瘤动物模型,通过观察肿瘤重量变化,评价了猫眼草黄酮类粗提物体内对肿瘤生长的影响,并通过观察动物一般表现、体重变化及用药后动物的胸腺和脾脏重量的改变,初步评价药物的潜在的一般毒性及其对动物免疫功能的影响。结果发现,猫眼草黄酮类粗提物在1和0.5g/kg剂量下连续服用19天,可明显抑制Lewis肺癌在小鼠体内的生长(与模型组比较,p<0.01和0.05),对肿瘤生长的抑制率分别为41.14%和40.40%;猫眼草黄酮类粗提物在0.25g/kg剂量下连续服用19天,对Lewis肺癌在小鼠体内的生长有一定的抑制作用,其对肿瘤生长的抑制率为21.31%,但与模型组比较无统计学意义。提示猫眼草黄酮类粗提物体内具有一定的抑制肿瘤生长的活性。猫眼草黄酮类粗提物在1和0.5g/kg剂量下连续服用19天,对荷Lewis肺癌小鼠的胸腺和脾脏重量均有一定的减轻作用,但仅1g/kg剂量下对脾脏重量的减轻具有明显的统计学意义(与模型组比较p<0.01),其他各组各指标虽有下降趋势,但与模型组比较无统计学意义。相应计算的脾指数和胸腺指数也表现出了同样的趋势。提示其本身可能具有一定的影响机体免疫功能的作用,但也有可能是其抑制肿瘤生长后表现出来的间接效应。实验期间的一般观察发现,除环磷酰胺组和猫眼草黄酮类粗提物高剂量(1g/kg)组动物略显消瘦外,各组动物的精神状态、饮食、饮水、大小便等均未见明显异常。猫眼草黄酮类粗提物在1 g/kg及环磷酰胺在30mg/kg剂量下连续给药19天可使荷Lewis肺癌小鼠体重明显减轻(p< 0.01),提示猫眼草黄酮类粗提物在高剂量下可能对小鼠具有一定的体重增长抑制作用。4、猫眼草中黄酮类物质的活性追踪及结构鉴定采用活性追踪的方法进一步对猫眼草黄酮粗提物中各馏分的抗肿瘤活性进行了研究,并针对黄酮类化合物特征紫外,对猫眼草50%乙醇提取物中的主要黄酮类结构进行分离纯化和结构鉴定。结果表明,从该提取物中分离出两个黄酮单体为quercetin-3-O-β-D-glucuronide(1)和kaempferol 3-O-β-d-glucuronic acid(2) ;提取物中的抗肿瘤活性成分为其中的脂溶性部分,但从脂溶性成分中获取的两个单体1和2均不具有细胞增殖抑制活性。该研究从猫眼草黄酮粗提物中分离出了两个黄酮类结构单体,并确定了该提取物中的抗肿瘤活性部位。5、利用激酶筛选及相关技术平台,在分子及细胞水平确定猫眼草活性部位的抗肿瘤作用利用激酶筛选平台及体外细胞相关技术,结合Western Blot手段,研究了猫眼草活性部位抗肿瘤作用的机制。结果发现,猫眼草活性部位可特异性抑制EGFR的磷酸化,且其IC50值为0.32μg/ml。采用人脐静脉微血管内皮细胞形成管腔模拟血管新生实验发现,猫眼草活性成分可明显抑制管腔形成,提示其具有抑制血管新生的作用。进一步采用Transwell观察了猫眼草活性成分对细胞迁移的影响。结果表明,猫眼草活性成分可明显抑制肿瘤细胞的迁移活动。相关信号通路研究表明,猫眼草活性成分可明显抑制EGFR的磷酸化,下调相关的AKT和ERK信号。本研究通过提取分离制备了猫眼草的活性部位,确定了该活性部位具有明显的抗肿瘤作用,并明确了其抗肿瘤作用的机制,为中药猫眼草的深入开发利用提供了理论依据。更多还原

【Abstract】 Cancer is the disease that humen still has not overcome, which seriously threaten human health. Lung cancer attracted much attention for high its incidence and mortality. At present, surgery, radiotherapy and chemotherapy are the main means for tumor treatment. Among them, chemotherapy is currently the most active research area. Some anticancer drugs currently used in the clinic more than seventy, but still can not effectively control the occurrence and development of tumors. Although many anticancer drugs enter clinical studies, few drugs are efficiency and low toxicity. Natural products are an important source of anticancer drugs. Traditional Chinese medicine has used medicinal plant for more than 2500 years. Thus, looking for antitumor drugs from traditional Chinese medicine is a speedy way. Euphorbia lunulata Bge is one of the herbal medicines in traditional Chinese medicine, some studies reported that its extract has anti-tumor effect, but the material basis and the detailed mechanism were still unkown. In our study, the active ingredients of Euphorbia lunulata Bge were isolated, and its anti-cancer effects were explored.1. The extraction techniques of flavonoids from Euphorbia lunulata BgeUsing uniform design to extract the total flavonoids from Euphorbia lunulata Bge, ethanol as solvent, four factors were investigated to determine the optimal extraction conditions, including ethanol concentration (30% -90%), ethanol volume (W / V: 9 ~ 15 times), reflux time (0.5 to 3.5 hours) and returning the number (1 to 3 times). The results showed that total flavonoid extraction from the Euphorbia lunulata Bge could increasethe (3.23%) under the condition: 50% ethanol solution at 14 times of the original ingredients (W / V), reflux extract 2 times, each time the conditions for 1 hour.2. Determination of anti-tumor effect for Euphorbia lunulata Bge extract by serum pharmacologyTo further determin the anti-tumor effects, serum pharmacology and Western Blot were used. The results showed that rabbit serum containing Euphorbia lunulata Bge extract could inhibit the proliferation of A549 lung cancer cells in a concentration- and time-dependencemanner. At the concentration of 20% for 72h, the A549 proliferation inhibiting rate was 39.08%. Flow cytometry assay showed that A549 cells were arrested in G1 phase by Euphorbia lunulata Bge extract rabbit serum, and showed significant apoptosis. Western Blot analysis exhibited that the rabbit serum containing Euphorbia lunulata Bge extract could promote the apoptosis-related protein Caspase-9 and Caspase-3 protein cutting, thereby causing activation of PARP protein, increase the expression of Bax, and decrease Bcl-2 expression and Protein phosphorylation of Bad.The results above mentioned suggested that Euphorbia lunulata Bge could induce apoptosis of A549 cells.3. Determination of the anti-tumor effect for Euphorbia lunulata Bge extractTo evaluate the effects of Euphorbia lunulata Bge extracts on tumor growth, Lewis lung tumor mouse models were used, and the tumor weight, the general performance of animals, body weight and thymus and spleen weight were observed. The results showed that after continuously administrated for 19 days, Euphorbia lunulata Bge extract at concentration of 1 and 0.5g/kg could significantly inhibit the Lewis lung cancer growth in mice (compared with model group, p <0.01 and 0.05), and inhibiting rates were 41.14% and 40.40%. Euphorbia lunulata Bge extract at 0.25g/kg also showed inhibitive effects on Lewis lung tumor in mice, and inhibiting rate was 21.31%. But not statistically significant was showed. Euphorbia lunulata Bge extract at 1 and 0.5g/kg concentrations reduced the weight of the thymus and spleen, but only 1g/kg for spleen showed statistical significance (compared with model group, p <0.01). The corresponding calculation of the spleen index and thymus index also showed the same trend, which suggests the influence on immune function. But it also might be the indirect effects after suppression of tumor growth. For general observation during the experiment, only cyclophosphamide and high dose of extract of Euphorbia lunulata Bge (1g/kg) group showed slightly body weight loss. The mental state of each group of animals, including food, water, feces and urine showed no abnormals.4. Extraction of Euphorbia lunulata Bge active ingredients and activity trackingThe major flavonoids component of Euphorbia lunulata Bge 50% alcohol extract were identified as quercetin-3-O-β-D-glucuronide and kaempferol ( 1 ) and 3-O-β-d-glucuronic acid(2)by structure guided fractionation. The subfractions together with the pure compounds were also test for their antitumor activity against A-549 and HL-60 cell lines, which lead to the results that compounds 1 and 2 were not show antitumor activities directely, while the organic soluable components were active.5. The anti-tumor effects and mechanisms of Euphorbia lunulata Bge active ingredients in molecular and cellular levelsUsing kinase screening platform and related technologies, combined with Western Blot, the mechanism and anti-tumor effect of Euphorbia lunulata Bge active ingredients were explored. Results showed that the Euphorbia lunulata Bge active ingredient could specifically inhibit the phosphorylation of EGFR, and the IC50 value was 0.32μg/ml. Using human umbilical vein endothelial cells to simulate blood vessels formation, Euphorbia lunulata Bge active ingredients showed the inhibitive effect on the tube formation, suggesting that inhibition of angiogenesis. Next, we observed the effects of Euphorbia lunulata Bge active components on cell migration. The results showed that the Euphorbia lunulata Bge active ingredient could significantly inhibit tumor cell migration. Related signaling pathway analysis showed that Euphorbia lunulata Bge active ingredients could inhibit the phosphorylation of EGFR and downstream AKT and ERK activation.The active ingredient in Euphorbia lunulata Bge was extracted in this study. The anti-tumor effects and mechanism were first explored, which provide theoretical basis for the development and utilization of Chinese medicine Euphorbia lunulata Bge更多还原