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

关键词:  儿黄散  自沉淀  黄连  儿茶  HeLa细胞

Key words:  erhuang powder  self precipitation  coptis chinensis  catechu  heLa cells

摘要:

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；基于超高相液相色谱-串联质谱技术(UPLC-Q-Exactive MS)对自沉淀、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、上清及提取物的高效液相色谱(HPLC)分析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,三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,基本阐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。

Abstract:

ABSTRACT: OBJECTIVE To explore the self precipitation source of Erhuang Powder, determine the content of related components and evaluate their effects on HeLa cells, and provide reference for the development of new dosage forms of Erhuang Powder. METHODS Bifurcation study to identify the main compatibility of precipitating. The self precipitation, supernatant and extract of Erhuang powder were Qualitative analyzed by UPLC-Q-Exactive MS. The main compounds from coptis and catechin, epicatechin, epicberberine, coptidine, berberine and palmatine were selected as control substances to establish a high performance liquid chromatography (HPLC) method for the analysis of these 6 index components. The chromatography was performed on Diamonsil C18 (250×4.6 mm,5 μm) column with methanol-0.1% phosphoric acid solution as mobile phase and gradient elution at 0~14 min, 7.0%~23.0% A. 14~25min, 23.0%~25.0%A; 25~35min, 25.0%~30.0%A; 35~55min, 30.0%~40.0%A; 55~70min, 40.0%~28.0%A; The flow rate was 1.0 mL·min-1, and the column temperature was 30℃. The self precipitation, supernatant and extracts of Erhuang powder were quantitatively determined, and the precision, repeatability, stability and recovery rate were investigated. MTT method and flow cytometry were used to evaluate the pharmacological effects of self precipitation, supernatant and extracts on inhibiting proliferation and inducing apoptosis of HeLa cells. 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 Coptis chinensis and catechu water was mixed. The results showed that the self precipitation, supernatant and extract of Erhuang Powder contained 39 compounds, 32 compounds were derived from the self precipitation, 30 compounds were derived from the supernatant, and 33 compounds were derived from the extract. The components of the three samples were mainly alkaloids and phenolic acids. The contents of catechin and berberine in the 6 index components were mainly, which accounted for 73.56% of the total index components in the self sedimentation amylum, 61.89% of the total index components in the extract, and 38.37% of the total index components in the supernatant. At the same time, the linear relationships of the 6 indexes were good in their respective concentration ranges. The recoveries of berberine, palmatine, coptidine, epiberberine, catechin and epicatechin were 99.53% ~ 99.98%. The results of precision test, repeatability test and stability test showed that the RSD values of the 6 indexes were all less than 2.0%. The results of MTT experiment showed that the self precipitation, supernatant and extract of Erhuang powder could inhibit the proliferation of HeLa cells in a concentration-time dependent manner. Flow cytometry showed that self precipitation and extracts had similar apoptosis-inducing effects on HeLa cells, while supernatant had weak apoptosis-inducing effects on HeLa cells. CONCLUSION Coptis coptidis and catechin are the main ingredients for self precipitation when the decoction of Erhuang powder is mixed. The main reason for the formation of precipitation is that berberine and catechin in Coptis coptidis and catechin have acid-base complexation reaction, and the content of berberine and catechin is high, which can be used as representative components. The HPLC method for the analysis of 6 index components of Coptis chinensis and catechu Chinensis established in the Institute of self precipitation related Components of Erhuang Powder has strong specificity, high precision and good reproducibility. The method can provide a reference for the quality control of Erhuang Powder and its related preparations. The self precipitation and extract had better inhibitory effect on HeLa cell proliferation and induced HeLa cell apoptosis than supernatant. The results showed that the self precipitation components of Erhuang Powder and its pharmacological effects on HeLa cells were similar to the extracts.