黄芩苷与盐酸小檗碱自沉淀理化性质及抑菌作用机制研究

Study on the physicochemical properties and antibacterial mechanism of precipitation in baicalin and berberine hydrochloride

目的 以黄芩苷和盐酸小檗碱产生的自沉淀为研究对象,探究其理化性质,对其抑菌作用及其机制展开研究,为中药自沉淀现象产生的物质深入研究提供参考。方法 采用差示热量扫描法、红外光谱扫描和紫外光谱扫描测定黄芩苷和盐酸小檗碱的自沉淀理化性质;采用牛津杯法探索黄芩苷、盐酸小檗碱及自沉淀对大肠埃希菌、金黄色葡萄球菌的抑菌活性;采用二倍稀释法进一步研究黄芩苷、盐酸小檗碱、自沉淀对大肠埃希菌和金黄色葡萄球菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),并通过测定菌体的生长曲线、胞外核酸相对含量、胞外可溶性蛋白质含量和电导率研究黄芩苷、盐酸小檗碱及自沉淀对大肠埃希菌、金黄色葡萄球菌的抗菌机制,分析对比黄芩苷、盐酸小檗碱反应前后抑菌作用机制是否发生改变。结果 差示热量扫描法结果显示盐酸小檗碱与黄芩苷反应前后存在热量变化,该自沉淀是一种不同于盐酸小檗碱、黄芩苷的新物质或复合物,其官能团、紫外吸收均发生了变化。盐酸小檗碱对大肠埃希菌的MIC为0.9375 mg/mL,MBC为7.5 mg/mL;对金黄色葡萄球菌的MIC为0.9375 mg/mL,MBC为7.5 mg/mL。黄芩苷对大肠埃希菌的MIC为1.875 mg/mL,MBC为7.5 mg/mL;对金黄色葡萄球菌的MIC为3.75 mg/mL,MBC为15 mg/mL。自沉淀对大肠埃希菌的MIC为3.75 mg/mL,MBC为3.75 mg/mL;对金黄色葡萄球菌的MIC为3.75 mg/mL,MBC为3.75 mg/mL。3种物质对大肠埃希菌和金黄色葡萄球菌菌体生长均有显著的抑制效果,但作用机制有所差异。结论 3种物质对大肠埃希菌和金黄色葡萄球菌显示出较好的抑菌活性,由强到弱为盐酸小檗碱、黄芩苷、自沉淀,均通过对细菌细胞膜造成损伤改变其通透性,影响细菌生长,从而起到抑制细菌生长的目的。三种物质虽能使菌体内容物大量渗出,但对胞内物质泄出作用存在差异,研究成果为进一步探究中药自沉淀提供参考。

Objective The substances produced by traditional Chinese medicine through the self-precipitation phenomenon have special properties and development potential.This paper takes the precipitation produced by baicalin and berberine hydrochloride as the research object,explores its physical and chemical properties,and studies its antibacterial effect and mechanism.Methods The physical and chemical properties of the preipitation were determined by differential scanning calorimetry,infrared spectroscopy scanning,and ultraviolet spectroscopy scanning.The antibacterial activity of baicalin,berberine hydrochloride and precipitation against Escherichia coli and Staphylococcus aureus was studied by Oxford cup method.The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of baicalin,berberine hydrochloride and precipitation against Escherichia coli and Staphylococcus aureus were further studied by double dilution method,and the antibacterial mechanisms of baicalin,berberine hydrochloride and precipitation against Escherichia coli and Staphylococcus aureus were studied by measuring the growth curve of the bacterial body,the relative content of extracellular nucleic acid,the content of extracellular soluble protein and electrical conductivity.Then,the changes in antibacterial mechanisms of baicalin and berberine hydrochloride were analyzed and compared before and after the reaction.Results The results of differential scanning calorimetry showed that there was a heat change before and after the reaction of berberine hydrochloride with baicalin.The precipitation was a new substance different from berberine hydrochloride and baicalin,and its functional groups and UV absorption changed.Berberine hydrochloride,baicalin,and precipitation have different antibacterial activities and mechanisms.In general,berberine hydrochloride has the best antibacterial activity,followed by self precipitation,and baicalin is relatively weak.The MIC of berberine hydrochloride against Escherichia coli was 0.9375 mg/mL,and the MBC was 7.5 mg/mL;The MIC against Staphylococcus aureus was 0.9375 mg/mL,and the MBC was 7.5 mg/mL.The MIC of baicalin against Escherichia coli was 1.875 mg/mL,and the MBC was 7.5 mg/mL;The MIC against Staphylococcus aureus was 3.75 mg/mL,and the MBC was 15 mg/mL.The MIC and MBC of precipitation against Escherichia coli were 3.75 mg/mL and 3.75 mg/mL,respectively;The MIC against Staphylococcus aureus was 3.75 mg/mL,and the MBC was 3.75 mg/mL.The three substances had significant inhibitory effects on the growth of Escherichia coli and Staphylococcus aureus,but their mechanisms of action varied.Conclusion The three substances that showed good antibacterial activity against Escherichia coli and Staphylococcus aureus,from strong to weak,were berberine hydrochloride,baicalin,and precipitation.All of them changed their permeability by damaging the bacterial cell membrane,affecting bacterial growth,and thereby inhibiting bacterial growth.Although the three substances could cause a large amount of exudation from the bacterial body,there were differences in the release of intracellular substances.The research results provided a reference for further understanding the precipitation of traditional Chinese medicine.