CN109602859B - 苗药廖氏化风丹在制备防治黑色素瘤药物中的应用 - Google Patents

苗药廖氏化风丹在制备防治黑色素瘤药物中的应用 Download PDF

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CN109602859B
CN109602859B CN201910049186.2A CN201910049186A CN109602859B CN 109602859 B CN109602859 B CN 109602859B CN 201910049186 A CN201910049186 A CN 201910049186A CN 109602859 B CN109602859 B CN 109602859B
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宋晶睿
李艳梅
陈娟
饶青
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Abstract

本发明公开了苗药廖氏化风丹在制备防治黑色素瘤药物中的应用，通过在体外运用黑色素瘤细胞WM35、WM239检测发现，廖氏化风丹具有显著的抑制黑色素瘤作用，并通过流式细胞检测其在细胞周期方面的作用，结果表明廖氏化风丹对黑色素瘤细胞周期影响显著，并且药物浓度越高，其阻滞越明显，可作为防治黑色素瘤制剂，制备成药剂学上允许的口服液、注射剂、片剂、胶囊剂、滴丸剂等。

Description

苗药廖氏化风丹在制备防治黑色素瘤药物中的应用
技术领域
本发明属于生物学和医药学技术领域，具体涉及一种苗药廖氏化风丹在制备防治黑色素瘤药物中的应用。
背景技术
苗药廖氏化风丹，由贵州万胜药业有限责任公司生产制造，丸剂，其现有作用：息风镇痉，豁痰开窍。用于风痰闭阻、中风偏瘫、癫痫，面神经麻痹，口眼歪斜。主要成份包括天麻、全蝎、僵蚕、白附子、苍术、天南星、麝香等20余味中药材。朱红色的水丸，剖面显棕黄色；有强烈香气、味辛。
黑色素瘤是由皮肤或其他器官黑素细胞产生的肿瘤，是最具侵袭性的皮肤肿瘤。黑色素瘤在我国发病率虽然在肿瘤中的比重较少，但总体为上升趋势。目前恶性黑色素瘤的治疗方法包括化疗、靶向治疗、免疫治疗及放疗等，但是黑色素瘤对放疗、化疗不敏感，所以治疗效果并不乐观，生物免疫治疗例如黑色素瘤疫苗等大多还处于研发或临床试验阶段。因此，提高早期诊断率，寻找对黑色素瘤的有效治疗药物是目前的首要任务。
发明内容
本发明的目的是针对黑色素瘤治疗困难，寻找出新的治疗黑色素瘤效果显著的药物。
针对上述目的，本发明提供了一种苗药廖氏化风丹作为制备防治黑色素瘤药物中的应用。
本发明在体外运用黑色素瘤细胞WM35、WM239检测发现，廖氏化风丹具有显著的抑制黑色素瘤作用，并通过流式细胞检测其在细胞周期方面的作用，结果表明廖氏化风丹对黑色素瘤细胞周期影响显著，阻滞细胞周期于G2-M期，并且药物浓度越高，其阻滞越明显，可作为防治黑色素瘤药物。
本发明将廖氏化风丹直接制备成防治黑色素瘤的制剂或者与其他防治黑色素瘤的药物复配制备成防治黑色素瘤的复方制剂，其中所述的制剂可以采用药剂学上允许的口服液、注射剂、片剂、胶囊剂、滴丸剂中任意一种。
本发明的有益效果为：与廖氏化风丹的现有用途相比，发明人发现了廖氏化风丹对黑色素瘤具有良好的体外抑制作用，发现了此药的新用途。而廖氏化风丹是已上市的药物，能直接应用于人体而不再需要进行临床安全评估，具有很好的应用前景，可快速提高此产品的市场占有率。
附图说明
图1是不同浓度廖氏化风丹对人黑色素瘤细胞WM35增殖抑制率。
图2是不同浓度廖氏化风丹对人黑色素瘤细胞WM239增殖抑制率。
图3是不同浓度廖氏化风丹对人黑色素瘤细胞WM35周期的影响。
具体实施方式
下面结合附图和实施例对本发明进一步详细说明。
实施例1
苗药廖氏化风丹体外抗黑色素瘤细胞的活性测试
1、实验材料
样品溶液的配制：将廖氏化风丹用DMSO分别配制成浓度为50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL、3.125μg/mL的样品溶液。
5％MTT溶液的配制：称取MTT粉末0.5g，加入灭菌的PBS 100mL，60℃溶解，0.22μm过滤膜过滤除菌，4℃保存。
细胞系：人黑色素瘤细胞WM35、WM239均为贵州省中国科学院天然产物化学重点实验室保存。人黑色素瘤细胞WM35、WM239于37℃5％CO2培养箱中用含5％FBS的DMEM培养基培养。
DMEM培养基及胰酶购于Hycone公司，胎牛血清(FBS)购于上海启动元生物科技有限公司，四甲基偶氮哩盐(MTT)、二甲基亚飒(DMSO)购于北京索莱宝科技有限公司。
2、实验方法
MTT法测定不同浓度廖氏化风丹对黑色素瘤细胞抑制增殖作用
以5×103/孔的密度，将处于指数增殖期的人黑色素瘤细胞WM35、WM239分别接种于96孔板中，每孔190μL含5％FBS的DMEM培养基，培养24h后，每孔加入10μL含不同浓度廖氏化风丹的样品溶液，每个浓度设5个复孔。另设DMSO对照组和100nmol紫杉醇(PTX)阳性对照组。细胞置37℃培养箱内继续培养72h后，每孔加入20μL 5％MTT溶液，继续培养4h后，1200r离心10min，弃去上清，每孔加入160μL DMSO，置振荡仪上充分混匀，采用酶标仪，以570nm为测试波长，630nm为参考波长，测量各孔吸光度计算抑制率，结果见图1和图2。
图1和图2的结果表明，廖氏化风丹对人黑色素瘤细胞WM35、WM239的生长有显著的抑制作用，且廖氏化风丹对WM35和WM239细胞的生长抑制具有显著的浓度依赖性。人黑色素瘤细胞WM35在浓度为25μg/mL的药物浓度下，其抑制率大于90％，在浓度为12.5μg/mL时，其抑制率为60.59％，高于100nmol紫杉醇的抑制率59.48％，处理数据得出半数抑制浓度IC50值WM35:11.85±1.19。人黑色素瘤细胞WM239在浓度为25μg/mL的药物浓度下，其抑制率大于50％，同时高于100nmol紫杉醇的抑制率41.42％，处理数据得出半数抑制浓度IC50值WM239:20.75±2.58。
2、流式细胞仪测定细胞周期的变化
将人黑色素瘤细胞WM35接种于60mm的圆形培养皿，置于37℃培养箱6h待其贴壁，加入含不同浓度廖氏化风丹的样品溶液，终浓度分别为5μg/mL、10μg/mL、20μg/mL，对照组加等量的DMSO，于37℃5％CO2培养箱中培养，收集加药20h的细胞，用500μL体积浓度为75％的乙醇水溶液固定，50μg/mL PI碘化丙啶溶液(含100μg/mL RNase，由PBS缓冲液配制)染色30min，于流式细胞仪上做细胞周期分析，结果见图3。
图3的流式结果表明：廖氏化风丹对人黑色素瘤细胞WM35周期影响显著，阻滞细胞周期于G2-M期，并且药物浓度越高，其阻滞越明显。
由以上结果可知，廖氏化风丹在制备防治黑色素瘤药物或黑色素瘤细胞增殖抑制剂方面具有潜在价值。

Claims (2)

1.苗药廖氏化风丹在制备防治黑色素瘤药物中的应用。
2.根据权利要求1所述的苗药廖氏化风丹在制备防治黑色素瘤药物中的应用，其特征在于：所述药物的剂型为口服液、注射剂、片剂、胶囊剂、滴丸剂中任意一种。
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CN1413704A (zh) * 2002-11-19 2003-04-30 贵州万胜药业有限责任公司 化风丹
CN1634410A (zh) * 2004-11-01 2005-07-06 唐宗园 一种外用中药膏药
CN1923232A (zh) * 2005-09-01 2007-03-07 郑廷彬 一种用于祛风消痰、破瘀软坚的药物组合物及其制备方法和用途
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