复方苦参提取物及其相关组分微生物限度检查方法及微生物质量标准的建立

Establishment of microbial limit test method and microbial quality standard of compound Kushen extract and its related components

目的:建立符合USP通则<1111><2023>(2013年前已生效)的复方苦参提取物及其相关组分微生物限度检查方法及质量标准。方法:首先,对USP和2020年版《中华人民共和国药典》中药制剂及饮片标准进行比对分析,确定复方苦参提取物及其相关组分微生物限度标准。其次,以USP通则<61><62>为指导原则,开发适用于复方苦参提取物及其相关组分微生物限度检查方法。采用平皿倾注法分别对复方苦参提取物及其2个组分苦参和白土苓的需氧菌总数计数、霉菌和酵母菌总数计数进行计数方法适用性试验,采用直接接种法对控制菌进行方法适用性试验;最后,综合采用MALDI/TOF MS微生物鉴定方法及细菌16S rRNA基因序列比对分析方法对样品中的污染微生物进行菌种鉴定,进一步对污染微生物种群分布及危害进行分析。结果:建立了符合USP要求的复方苦参提取物及其相关组分微生物限度标准;复方苦参提取物及其2个组分苦参和白土苓的微生物计数方法适用性试验结果回收比值均在0.5~2.0,控制菌方法适用性试验中阳性对照菌株均能够生长,符合USP要求;40株污染微生物根据鉴定结果涉及13个种5个属,主要涵盖不同种类的芽孢杆菌属(87.5%)、肠杆菌属(10%)及假单胞菌属(2.5%);污染微生物均为危害性较小的条件致病菌。结论:建立了苦参、白土苓及复方苦参提取物的微生物限度标准及微生物限度检查方法,明确了上述产品的污染微生物种群分布和危害分析,完善了该中药制剂及其组分微生物质量控制。

Objective:To establish a microbial limit test method and quality standard conforms to the USP for compound Kushen extract and its related components.Methods:Comparative analysis was conducted on the standards for traditional Chinese medicine preparations and decoction pieces in the USP and the Chinese Pharmacopoeia 2020 Edition to determine the microbial limit standards for compound Kushen extract and its related components.Then,guided by the General Principles of the USP<61>and<62>,a microbial limit test method suitable for compound Kushen extract and its related components was developed.The plate pouring method was used to perform a methodological applicability test on the total aerobic bacterial count,mold and yeast count of compound Kushen extract and its two components Sophorae Flavescentis Radix and Heterosmilacis Rhizoma.The method applicability test was conducted on the control bacteria using the direct inoculation method.Finally,the MALDI/TOF MS microbial identification method and bacterial 16S rRNA gene sequence alignment analysis method were comprehensively used to identify the bacterial species of the contaminated microorganisms in the sample,and further analysis was conducted on the distribution and harm of the contaminated microbial population.Results:As a result,microbial limit standards for compound Kushen extract and its related components were established in accordance with the requirements of the USP.The recoveries of the counting methods for compound Kushen extract and its two components,Sophorae Flavescentis Radix and Heterosmilacis Rhizoma,were between 0.5 and 2.0.The positive control strains in the control bacterial method suitability test were able to grow,meeting the requirements of the USP.According to the identification results,40 contaminated microorganisms involved 13 species and 5 genera,mainly covering different types of Bacillus genus(87.5%),Enterobacterium genus(10%),and Pseudomonas genus(2.5%).Polluting microorganisms were all less harmful conditional pathogenic bacteria.Conclusion:Established a microbial limit test method and quality standard conforms to the USP for compound Kushen extract and its related components.Defined the distribution and harm analysis of the pollution microorganism poulation of the above products.Improved the microbial quality control of the Chinese medicine preparation and its components.