姜黄素类化合物的提取分离鉴定及其与化疗药的协同抗肿瘤作用研究

【作者】 田杰；

【导师】 刘珂；

【作者基本信息】 烟台大学 ， 药物化学， 2009， 硕士

【摘要】 目的:从中药姜黄中提取分离、鉴定姜黄素类化合物,研究姜黄素单药与临床常用化疗药联合用药对人纤维肉瘤HT1080细胞、人乳腺癌MCF-7细胞、人肺腺癌A549细胞的增殖抑制效果及其诱导肿瘤细胞凋亡的协同作用,以探讨其抗肿瘤的协同作用机制,为临床寻找有效的化疗增效剂提供实验依据和理论基础。方法:通过硅胶柱色谱对姜黄80%乙醇提取物进行分离纯化,以理化方法及波谱学方法对分离得到的化合物进行结构鉴定;采用MTT法评价姜黄素类单体化合物对人纤维肉瘤HT1080细胞、人乳腺癌MCF-7细胞、人肺腺癌A549细胞的增殖抑制作用;采用MTT法评价姜黄素单药在HT1080细胞、MCF-7细胞、A549细胞上与顺铂、阿霉素、羟基喜树碱等药物联合用药的增效作用;通过Hoechst 33258染色观察姜黄素和阿霉素联合用药对A549细胞凋亡的形态学变化;Western blot法检测对A549细胞Bcl-2蛋白表达的影响。结果:(1)分离得到3个姜黄素类化合物,分别鉴定为姜黄素curcumin (Ⅰ),脱甲氧基姜黄素demethoxycurcumin (Ⅱ),双脱甲氧基姜黄素bisdemethoxycurcumin (Ⅲ) ;(2)姜黄素、脱甲氧基姜黄素、双脱甲氧基姜黄素对HT1080细胞、MCF-7细胞、A549细胞的增殖均有明显的抑制作用,姜黄素的体外增殖抑制效果最好,对三种细胞株的半数抑制浓度(IC50)分别为10.39μmol/L、11.85μmol/L、10.25μmol/L。(3)在一定浓度范围内,姜黄素和顺铂、阿霉素、羟基喜树碱分别联合用药对HT1080细胞、MCF-7细胞、A549细胞的增殖抑制有协同或相加作用。(4)姜黄素和阿霉素联合用药对A549细胞凋亡有协同作用,其作用可能是通过下调抑凋亡蛋白Bcl-2表达来实现的。结论:姜黄素和顺铂、阿霉素、羟基喜树碱分别联合用药对HT1080细胞、MCF-7细胞、A549细胞的增殖抑制有协同或相加作用,其协同抗肿瘤的机制可能与诱导细胞凋亡,通过下调抑凋亡蛋白Bcl-2表达有关。体外实验研究表明,姜黄素与化疗药联合用药对部分肿瘤的治疗具有一定的可行性,在体外抑制人肿瘤细胞的作用中有协同增效作用,为临床寻找有效的化疗增效剂提供了一定的实验依据和理论基础。更多还原

【Abstract】 Objective: To extract and isolate curcuminoids from Curcuma longa and study the curcumin alone combinated with antineoplastic agent in human fibrosarcoma HT1080 cell,human breastcancer MCF-7 cell,human lung cancer adenocarcinoma A549 cell on the proliferation inhibitory effects and apoptosis-induced synergistic effects, which in order to explore its synergistic anti-tumor mechanisms, the results might be provided an experimental basis and theoretical foundation for effective chemotherapy synergist.Method: The 80% EtOH extract was subjected to silica gel column chromatography. Chemical structures were identified by physico-chemical properties and spectral analysis; The effects of curcuminoids on proliferation inhibition of HT1080 cell line, MCF-7 cell line, A549 cell line were detected by MTT assay; The effects of curcumin in combination with cisplatin, adriamycin, hydroxycamptothecin on proliferation inhibition of HT1080 cell line, MCF-7 cell line, A549 cell line were detected by MTT assay; The morphological analysis of A549 cell apoptosis were detected by Hoechst 33258 staining; Bcl-2 protein expression in A549 cell were detected by Western blotting.Result:(1) Three curcuminoids were isolated from Curcuma longa and identified as curcumin(Ⅰ), demethoxycurcumin (Ⅱ), bisdemethoxycurcumin (Ⅲ);(2) Curcumin, demethoxycurcumin, bisdemethoxycurcumin all showed significantly proliferation inhibition on HT1080 cell line, MCF-7 cell line, A549 cell line in vitro, the curcumin indicated the best proliferation inhibition activity, the IC50 were respectively 10.39μmol/L,11.85μmol/L and 10.25μmol/L.(3) In a certain concentration range, the curcumin in combination with cisplatin, adriamycin, hydroxycamptothecin on HT1080 cell line, MCF-7 cell line, A549 cell line has synergistic or additive effect on proliferation inhibition.(4) Curcumin combinated with adriamycin has synergistic effcct on A549 cell apoptosis, its mechanisms may be through down-regulating Bcl-2 protein expression to the implementation.Conclusion:Curcumin in combination with cisplatin, adriamycin, hydroxycamptothecin on HT1080 cell line, MCF-7 cell line, A549 cell line has synergistic or additive effect on proliferation inhibition, the synergistic anti-tumor mechanism may be related to apoptosis-induced and down-regulating Bcl-2 protein expression. The results have shown that curcumin combined with chemotherapy drugs for the treatment of some tumor may be have the possibility, which have synergistic effect in proliferation inhibition of human tumor cells, it might be provided an experimental basis and theoretical foundation for effective chemotherapy synergist.更多还原