儿黄散自沉淀分析及其对HeLa细胞的作用研究

Self Precipitation Analysis of Erhuang Powder and Its Effect on HeLa Cells

目的 探究儿黄散自沉淀来源,测定相关组分含量及其对HeLa细胞的作用。方法 拆方研究产生沉淀的主要配伍组方;基于UHPLC-Q-Exactive MS对儿黄散自沉淀、上清及提取物进行分析;选用黄连和儿茶中的主要化合物儿茶素、表儿茶素、表小檗碱、黄连碱、小檗碱和巴马汀为对照品,建立适用于儿黄散自沉淀、上清及提取物的UHPLC方法并定量测定相关成分;运用MTT法和流式细胞术评价自沉淀、上清及提取物对HeLa细胞的作用。结果 组方单煎液两两混合出现轻微絮状沉淀,而黄连和儿茶水煎液混合时会出现大量凝絮物。鉴定得到儿黄散自沉淀、上清及提取物中共包含39种成分,3种样品中均以生物碱和酚酸类物质为主;6种指标性成分含量中儿茶素和小檗碱含量居多,二者含量在自沉淀中占指标成分总含量的73.56%,提取物中占指标成分总含量的61.89%。对HeLa细胞的抑制效果:提取物≈沉淀>上清;诱导凋亡:沉淀≈提取物,上清无作用。结论 黄连-儿茶是生成沉淀的主要配伍组方,儿黄散自沉淀和提取物主要成分均以生物碱和酚酸类物质为主,其中小檗碱和儿茶素含量较高,可作为代表性成分。自沉淀和提取物对HeLa细胞的作用效果优于上清液,表明儿黄散自沉淀组分及对HeLa细胞发挥的药理作用与提取物类似。

OBJECTIVE To explore the self precipitation source of Erhuang powder,determine the content of related components and its efficacy on HeLa cells.METHODS Bifurcation study to identify the main compatibility of precipitating.The self precipitation,supernatant and extract of Erhuang powder were analyzed by UHPLC-Q-Exactive MS.The main compounds in Coptidis Rhizoma and Catechu,catechin,epicatechin,epicberberine,coptisine,berberine and palmatine were selected as controls.A analysis method of UHPLC for self precipitation,supernatant and extract of Erhuang powder was established and the related components were quantitatively determined.The effects of self precipitation,supernatant and extract on HeLa cells were evaluated by MTT method and flow cytometry.RESULTS A slight flocculation precipitate appeared when the decoction of Erhuang powder was mixed in pairs,while a large amount of flocculation appeared when the decoction of Coptidis Rhizoma and Catechu water was mixed.The self precipitation,supernatant and extract samples contained 39 compounds,which were mainly alkaloids and phenolic acids.The contents of catechin and berberine in the 6 index components were mostly,which accounted for 73.56%of the total content of the index components in self precipitation and 61.89%of the total content of the index components in extract.Inhibition effect on HeLa cells:extract≈self precipitation>supernatant,and inducing apoptosis:self precipitate≈extract,supernatant had no apoptosis-inducing effect.CONCLUSION Coptidis Rhizoma-Catechu is the main compatible formula for precipitation formation.The self precipitation and extracts of Erhuang powder are mainly alkaloids and phenolic acids,among which berberine and catechin are high in content and can be used as representative components.The effect of self-precipitation and extract on HeLa cells was better than that of supernatant.This basically indicates that the self precipitation components and pharmacological effects of Erhuang powder are similar to those of the extract.