CN110934913A - 一种促肿瘤细胞凋亡药物组合物及其应用 - Google Patents
一种促肿瘤细胞凋亡药物组合物及其应用 Download PDFInfo
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Abstract
本发明提供了一种促肿瘤细胞凋亡药物组合物,所述组合物的原料按质量比值计算包括:短小蛇根草乙醇/水提取物或总生物碱提取物80~90份,人参提取物1~5份,美登木提取物1~5份。有别于传统工艺中只是从短小蛇根草中单独提取喜树碱用于制备防、治癌症的药物,本技术方案是通过制成总生物碱提取物,并将其与人参、美登木的提取物共同制成能够对癌细胞起一直作用的药物组合物。该药物组合物对肝癌、肺癌及淋巴癌细胞具有明显的细胞毒作用,可开发成治疗肝癌的药物。
Description
技术领域
本发明涉及医疗药物技术领域,特别涉及一种促肿瘤细胞凋亡药物组合物,以及该组合物的应用方式。
背景技术
肿瘤是危害人类健康重大疾病之一,目前治疗手段主要有化学治疗、手术治疗、中药治疗、放射治疗和免疫治疗,其中药物治疗是使用最广泛的治疗方式。随着科学的进步和社会的发展,传统手段中的中药治疗越来越受到重视,植物来源的生物活性化合物主要通过细胞周期停滞和凋亡途径的激活拥有抗癌生物活性。中国具有丰富的中药及天然植物资源,为抗肿瘤药物的开发提供了丰富的宝库。
凋亡是一种有遗传基因控制的细胞自主、有序的死亡方式,是生物体细胞保持正常活性和功能性不可少的过程。细胞凋亡调节的紊乱与肿瘤的发生、发展有着密切的关系。细胞的坏死性凋亡是一个机体主动的、依赖细胞内信号转导的过程,由相应配体通过激活死亡受体而触发。Bcl-2和caspase家族蛋白在细胞凋亡通路中发挥了重要作用。通过物理、化学和生物手段等诱导肿瘤细胞凋亡已成为肿瘤治疗的有效途径之一。
短小蛇根草(Ophiorrhiza pumila),为茜草科(Rubiaceae)蛇根草属(Ophiorrhiza)植物,又叫荷包草、鸡冠草、白丁香、金锁匙等,为双子叶多年生草本植物。短小蛇根草全草都可入药,它性味苦、寒,具有清热解毒的功效,福建民间会用短小蛇根草的全草用来治疗高热、外伤感染、百日咳、疽、痈、毒蛇咬伤等症,广东、广西民间则会用它的叶子来消肿毒。现有文献资料未见报道有短小蛇根草提取物及总生物碱提取物在制备促肿瘤细胞凋亡药物方面的应用研究。
发明内容
现有技术中培育短小蛇根草只用于提取喜树碱,并未见报道短小蛇根草的提取物及总生物碱提取物的药物组合物在制备促肿瘤细胞凋亡作用中的应用研究。
一种促肿瘤细胞凋亡药物组合物,所述组合物的原料按质量比值计算包括:短小蛇根草乙醇/水提取物或总生物碱提取物80~90份,人参提取物1~5份,美登木提取物1~5份。
其中,所述短小蛇根草乙醇/水提取物的制备方法为:每次用3~10倍质量的乙醇或水回流提取短小蛇根草,共3次,每次2小时,合并后减压浓缩,得短小蛇根草乙醇/水提取物。
进一步,所述短小蛇根草总生物碱提取物,制备方法为:取所述短小蛇根草乙醇/水提取物,然后水混悬后,加入氢氧化钠调价pH至8~10,加入氯仿萃取,取水层,加稀盐酸调节pH至2~3,氯仿萃取,合并氯仿层,减压浓缩得所述总生物碱提取物。
进一步,所述人参提取物,制备方法为:每次用3~10倍质量的乙醇或水回流提取人参,共3次,每次2小时,合并后减压浓缩,得人参提取物。
进一步,所述美登木提取物,制备方法为:每次用3~10倍质量的乙醇或水回流提取美登木,共3次,每次2小时,合并后减压浓缩,得美登木提取物。
上述制得的促肿瘤细胞凋亡药物组合物可以应用于制备治疗肝癌、肺癌、食道癌、结肠癌或乳腺癌的药物。特别适用于制备治疗肝癌的药物。所述药物可以为片剂,胶囊剂,口服剂,颗粒剂丸剂等。
与现有技术相比,本发明的有效有益效果如下:
有别于传统工艺中只是从短小蛇根草中单独提取喜树碱用于制备防、治癌症的药物,本技术方案是通过制成总生物碱提取物,并将其与人参、美登木的提取物共同制成能够对癌细胞起一直作用的药物组合物。该药物组合物对肝癌、肺癌及淋巴癌细胞具有明显的细胞毒作用,可开发成治疗肝癌的药物。
附图说明
图1是实施例1制得的组合物对人源SMMC-7721肝癌细胞、HepG2肝癌细胞凋亡的影响分布图;
图2是实施例1制得的组合物对人源SMMC-7721肝癌细胞、HepG2肝癌细胞形态学变化的荧光染色图;
图3是实施例1制得的组合物对人源SMMC-7721肝癌细胞、HepG2肝癌细胞表达Bcl-2、Bax及Cleaved caspase-3蛋白的影响效果图;
图4是实施例1制得的组合物对人源SMMC-7721肝癌细胞、HepG2肝癌细胞凋亡效果的分布图。
具体实施方式
下面通过具体实施方式对本发明作进一步详细说明。但本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1含有短小蛇根草总生物碱提取物的药物组合物
制备过程包括以下步骤:
短小蛇根草100g,每次用800g的95%乙醇/水,热回流提取3次,每次2小时,合并后减压浓缩,获得短小蛇根草乙醇/水提取物。然后加水混悬,再加入氢氧化钠调价pH至8-10,最后加入氯仿萃取。取水层,加稀盐酸调节pH至2-3,氯仿萃取,合并氯仿层,减压浓缩得总生物碱提取物。人参5g,每次用20g的70%乙醇/水,回流提取3次,每次2小时,合并后减压浓缩,获得人参提取物。美登木5g,每次用20g的70%乙醇/水,回流提取3次,每次2小时,合并后减压浓缩,获得美登木提取物。将上述三种提取物合并后得组合物1。
实施例2含有短小蛇根草总生物碱提取物的药物组合物
制备过程包括以下步骤:
短小蛇根草100g,每次用500g的80%乙醇/水,热回流提取3次,每次2小时,合并后减压浓缩,获得短小蛇根草乙醇/水提取物。然后加水混悬,再加入氢氧化钠调价pH至8-10,最后加入氯仿萃取。取水层,加稀盐酸调节pH至2-3,氯仿萃取,合并氯仿层,减压浓缩得总生物碱提取物。人参5g,每次用20g的70%乙醇/水,回流提取3次,每次2小时,合并后减压浓缩,获得人参提取物。美登木5g,每次用20g的70%乙醇/水,回流提取3次,每次2小时,合并后减压浓缩,获得美登木提取物。将上述三种提取物合并后得组合物2。
实施例3含有短小蛇根草总生物碱提取物的药物组合物
制备过程包括以下步骤:
短小蛇根草100g,每次用1000g的95%乙醇/水,热回流提取3次,每次2小时,合并后减压浓缩,获得短小蛇根草乙醇/水提取物。然后加水混悬,再加入氢氧化钠调价pH至8-10,最后加入氯仿萃取。取水层,加稀盐酸调节pH至2-3,氯仿萃取,合并氯仿层,减压浓缩得总生物碱提取物。人参5g,每次用20g的70%乙醇/水,回流提取3次,每次2小时,合并后减压浓缩,获得人参提取物。美登木5g,每次用20g的70%乙醇/水,回流提取3次,每次2小时,合并后减压浓缩,获得美登木提取物。将上述三种提取物合并后得组合物3。
实施例4促凋亡实验
细胞培养:
人肝癌细胞株SMMC-7721、HepG2培养于RPMI-1640培养液,内含10%小牛血清、100U/ml青霉素、100μg/ml链霉素,置37℃,5%CO2,恒温密闭式孵箱内培养传代。
(1)含有短小蛇根草提取物的药物组合物对人源SMMC-7721肝癌细胞、HepG2肝癌细胞凋亡的影响
取实施例1制得的组合物1调配成不同浓度(0,3.125,6.25μg/mL)作用于肝癌细胞48,PI染色流式细胞计数测定凋亡率。
结果如图1所示,与空白组相比,含有短小蛇根草提取物药物组合物均可显著促进人源SMMC-7721肝癌细胞、HepG2肝癌细胞凋亡,并呈剂量与时间依赖性。
(2)含有短小蛇根草提取物药物组合物对细胞形态及超微结构的影响
病理学HE染色光镜下观察:盖玻片泡酸处理,高压灭菌后放入24孔板内,每孔1片,取对数生长期细胞常规消化,调整密度为1×107/L的细胞悬液,加入培养板内,每孔1ml。实验组加入不同浓度的组合物(0,3.125,6.25μg/mL),设无药培养液为对照,作用48h,取出盖玻片,PBS小心冲洗3遍,放入预冷的95%酒精中,4℃固定,用透明胶背面粘于载玻片上,细胞面向上,37℃烘干,PBS冲洗,置苏木精染液中5~10min,水浸洗,置稀盐酸酒精中数秒分色,水浸洗,淡氨水中3~5min,水浸洗,伊红染液中浸5~10min,水浸洗后,50%、80%、90%酒精梯度脱水各1~2min,95%酒精2次各1~2min,无水酒精3次各2min,透明二甲苯中3次各2min,树胶封片观察。
结果如图2所示,含有短小蛇根草提取物药物组合物对人源SMMC-7721肝癌细胞、HepG2肝癌细胞有明显抑制作用。对照组细胞贴壁生长,梭形或多角形,核圆亮,胞浆透亮,细胞增殖状态良好。含有短小蛇根草提取物药物组合物作用24h即可见少量细胞变圆,48h细胞之间距离增大,变圆的细胞数量增多,与对照组相比,细胞生长缓慢甚至停止,胞浆内颗粒增多增粗。
(3)Bcl-2、Bax及Cleaved caspase-3蛋白表达的影响
对数生长期SMMC-7721和HepG2细胞接种于60mm培养皿中,37℃CO2培养箱培养,细胞密度达到70%~80%,用不同浓度的含有短小蛇根草提取物药物组合物(0,3.125,6.25μg/mL)的无血清培养基饥饿处理24h,收集细胞,加入蛋白裂解液100μL收集蛋白样本,用BCA法测定蛋白浓度。取蛋白样品30μg,经电泳分离后转移至PVDF膜,放入5%BSA封闭液封闭2h,加入一抗,4℃孵育过夜,次日磷酸盐缓冲液(PBS)洗PVDF膜3次,加入二抗(1:10000)室温下孵育2h,TBST洗膜3次,ECL显色,用FluorChem HD2凝胶成像系统拍照,并用该软件统计相应的灰度值。
结果如图3所示,不同浓度的含有短小蛇根草提取物药物组合物(0,3.125,6.25μg/mL)分别处理人源SMMC-7721肝癌细胞、HepG2肝癌细胞72h后,二组细胞的Bcl-2、Bax及Cleaved caspase-3蛋白表达均升高,与对照组相比较差异有显著性(p<0.01)。
(4)流式细胞术(FCM)检测细胞凋亡、细胞周期分布
将源SMMC-7721肝癌细胞、HepG2肝癌细胞接种在培养瓶中,待细胞长至80%汇合,分别中加入不同浓度的含有短小蛇根草提取物药物组合物(0,3.125,6.25μg/mL)作用72h,收集细胞,PBS洗涤一次,70%预冷的乙醇4℃固定过夜。加入碘化丙锭(Propidium Iodide,PI:50mg/L Trinton X-100 1.0%)室温避光染色30min,用Epics-XLⅡ型流式细胞仪检测细胞凋亡和细胞周期分布。
统计学分析:数据分析采用SPSS 17.0统计软件,计量资料以x±s表示,采用方差分析。
图4结果显示,人源SMMC-7721肝癌细胞、HepG2肝癌细胞分别经不同浓度的含有短小蛇根草提取物药物组合物(0,3.125,6.25μg/mL)处理72h后,流式细胞仪分析结果显示与对照组相比,经不同药物浓度处理后的肿瘤细胞均可见在G2/M期前细胞形成明显的亚二倍体凋亡峰,主要表现为G2/M期细胞增多,说明细胞可能主要被阻滞于G2/M期。提示短小蛇根草提取物能够影响肿瘤细胞DNA的合成,干扰肿瘤细胞周期的正常进程,同时能诱导肿瘤细胞凋亡且随着药物浓度的增加,细胞凋亡率也逐渐上升。
上述实验表明,含有短小蛇根草提取物药物组合物抑制人肝癌细胞增殖可能通过促进其凋亡发生的。
Claims (6)
1.一种促肿瘤细胞凋亡药物组合物,其特征在于,所述组合物的原料按质量比值计算包括:短小蛇根草乙醇/水提取物或总生物碱提取物80~90份,人参提取物1~5份,美登木提取物1~5份。
2.根据权利要求1所述的组合物,其特征在于,所述短小蛇根草乙醇/水提取物,制备方法如下:
每次用3~10倍质量的乙醇或水回流提取短小蛇根草,共3次,每次2小时,合并后减压浓缩,得短小蛇根草乙醇/水提取物。
3.根据权利要求1所述的组合物,其特征在于,所述短小蛇根草总生物碱提取物,制备方法如下:
每次用3~10倍质量的乙醇或水回流提取短小蛇根草,共3次,每次2小时,合并后减压浓缩;然后水混悬后,加入氢氧化钠调价pH至8~10,加入氯仿萃取,取水层,加稀盐酸调节pH至2~3,氯仿萃取,合并氯仿层,减压浓缩得所述总生物碱提取物。
4.根据权利要求1所述的组合物,其特征在于,所述人参提取物,制备方法如下:
每次用3~10倍质量的乙醇或水回流提取人参,共3次,每次2小时,合并后减压浓缩,得人参提取物。
5.根据权利要求1所述的组合物,其特征在于,所述美登木提取物,制备方法如下:
每次用3~10倍质量的乙醇或水回流提取美登木,共3次,每次2小时,合并后减压浓缩,得美登木提取物。
6.如权利要求1所述组合物在治疗肝癌、肺癌、食道癌、结肠癌或乳腺癌的药物中的应用。
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