基于消化酶活力检测的鸡内金质量生物检定方法研究  

Development of a novel and practical bioassay for the quality evaluation and control of Galli Gigerii Endothelium Corneum based on the detection of digestive enzyme activity

作　　者：黄伟[1] 王伽伯 谭鹏 贺亚男[1] 林俊芝 黄浩洲 韩丽[1] 许润春[1] 张定堃[1] HUANG Wei;WANG Jia-bo;TAN Peng;HE Ya-nan;LIN Jun-zhi;HUANG Hao-zhou;HAN Li;XU Run-chun;ZHANG Ding-kun(Pharmacy School,State Key Laboratory of Southwestern Chinese Medicine Resources,Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China;China Military Institute of Chinese Medicine,the Fifth Medical Centre of Chinese PLA General Hospital,Beijing 100039,China;School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China;State Key Laboratory of Biological Evaluation of TCM Quality,National Administration of TCM,Sichuan Academy of Chinese Medicine Sciences,Chengdu 610041,China;TCM Regulating Metabolic Diseases Key Labora‐tory of Sichuan Province,Hospital of Chengdu University of Traditional Chinese Medicine,Chengdu 610072,China)

机构地区：[1]成都中医药大学药学院,西南特色中药资源国家重点实验室,四川成都611137 [2]中国人民解放军总医院第五医学中心,全军中医药研究所,北京100039 [3]首都医科大学中医药学院,北京100069 [4]四川省中医药科学院,国家中医药管理局中药质量生物评价重点研究室,四川成都610041 [5]成都中医药大学附属医院,代谢性疾病中医药调控四川省重点实验室,四川成都610072

出　　处：《药学学报》2021年第5期1453-1459,共7页Acta Pharmaceutica Sinica

基　　金：成都市科技局技术创新研发项目(2018-YFYF-00060-SN);四川省科技厅青年杰出科技人才资助项目(2019JDJQ0007);解放军总医院杰青培育项目(2019-JQPY-003);解放军总医院医疗大数据项目(2019MBD-023);国家重点研发计划项目(2018YFC1707002);成都中医药大学中药学学科特色创新科研团队(CXTD2018006)。

摘　　要：鸡内金是消食化积类中药的代表,疗效确切,现代临床应用广泛。但目前鸡内金的药效物质研究基础较为薄弱,2020版《中国药典》收载的鸡内金质量控制方法比较简单,并不能够有效控制其质量。本研究提出基于胃蛋白酶活性的鸡内金生物效价测定方法,作为其质量控制新的技术方法。以胃蛋白酶活力为评价指标,优化鸡内金提取方法,采用"质反应平行线法"建立基于胃蛋白酶活性的鸡内金生物效价测定法,进行方法学考察,测定20个批次鸡内金的生物效价与可信限度;采用营养性半固体糊黏度测定方法验证不同效价鸡内金降低黏度的活性差异;对比研究小鼠胃残留率与胃蛋白酶活力、醇溶性浸出物含量的相关性差异,评价胃蛋白酶活力效价与体内活性的相关性。方法学考察结果显示,鸡内金供试品的最优制备方法为:取鸡内金粗粉,加50倍量水,300 W、40 kHz超声提取0.5 h;其量效曲线形状与阳性对照药多酶片相似,精密度、中间精密度及重复性考察结果均符合要求;20批样品的效价范围为13.49~34.69 U·mg~(-1),平均值为22.21 U·mg~(-1),RSD为31.52%。验证实验结果显示,鸡内金样品效价越高,降低营养糊黏度效果越显著;小鼠胃残留率与胃蛋白酶活力效价、醇溶性浸出物含量的相关性系数r分别为0.867、0.518,表明胃蛋白酶活力效价与体内活性高度相关。研究结果表明,本文建立的基于胃蛋白酶活性的鸡内金生物效价测定法方法可靠,重复性良好,能够直接反映鸡内金的质量优劣,可为其他消食类中药的质量评控参考方法。Galli Gigerii Endothelium Corneum(GGEC)represents digestion-promoting medicines with measurable effects and extensive clinical application.However,its effective components are not clear.The quality control index in the current edition of Chinese Pharmacopoeia is rather elementary and does not reflect its clinical efficacy.In this study,a bioassay method based on pepsin activity was proposed as a novel quality control method.With pepsin activity as the evaluation index,the extraction of GGEC was optimized and a method for the determination of biological potency was established by using the qualitative reaction parallel line method.The biological potency and consistency of 20 batches of GGEC were investigated.To provide scientific evidence in support of this bioassay method,two validation experiments were designed.One was to study the viscosity-reducing activity of a nutritional semi-solid paste after adding GGEC samples with differing potency.The other was to correlate the gastric residual rate in mice and pepsin activity with the alcohol soluble extract content.The results showed that the optimal preparation method was to dilute crude powder of GGEC with 50 volumes of water and subject to ultrasonic extraction at 300 W and 40 kHz for 0.5 h.The shape of the dose-response curve was similar to that of the positive control drug multienzyme tablets and the precision,intermediate precision and repeatability met the methodology requirements.The results showed that the potency of 20 batches of samples ranged from 13.49 to 34.69 U·mg-1,with an average value of 22.21 U·mg-1.The validation experiment demonstrated that the effect of reducing the viscosity of the nutrient paste became more significant as GGEC sample potency increased.The correlation coefficient R of gastric residual rate with pepsin potency and alcohol soluble extract content was 0.867 and 0.518,respectively,which indicated that the pepsin potency was highly correlated with in vivo activity.This study shows that a bioassay method based on pepsin activity is

关 键 词：鸡内金 消食类中药 胃蛋白酶 生物效价 质量评价 

分 类 号：R917[医药卫生—药物分析学]