目的: 进行斜率比法测定水蛭抗凝血活性的实验室内方法学验证研究。考察斜率比法测定水蛭抗凝血活性作为质量控制方法的可行性。方法: 选用7批蚂蝗和日本医蛭饮片,分别用蚂蝗对照药材和菲牛蛭对照药材,平行制备不同浓度浸提液,测定活化的部分凝血活酶时间(APTT)后用斜率比法测定抗凝血活性。在实验室内进行专属性、线性与范围、精密度、准确度、耐用性等多个方面的系统考察。结果: 使用斜率比法测定水蛭抗凝血活性,专属性考察:该条件下土鳖虫不显示其抗凝血活性,水蛭浸提液100 ℃加热后活性降低,添加低分子肝素后试验不成立,用蚂蝗对照药材做对照测定日本医蛭抗凝血活性则试验不成立,说明本法的专属性和选择性较好。准确度考察:采用蚂蝗对照药材和菲牛蛭对照药材,64%、80%、100%、125%和156%的5个相对水平值测定值的相对偏差均<±20%,效价理论值对数(横坐标)和对应的效价测定值对数(纵坐标)呈现明显的直线关系,相关系数>0.95,斜率在0.95~1.05,说明方法的准确度较好。精密度考察:各相对效价水平的几何变异系数(GCV,%)均<20%;采用多批次的饮片测定RSD均<20%,说明本法的精密度较好。线性与范围考察:本法在相对于对照药材的在64%~156%,都能够进行准确测定,线性范围符合要求。耐用性考察:水蛭浸提液可放置24 h、37 ℃加热1 h后活性均未见改变,说明本法耐用性较好。结论: 斜率比法测定水蛭抗凝血活性具有较好的精密度、准确度和重现性,量化测定水蛭抗凝血活性的生物检定方法,适用于水蛭的质量控制。
Objective: To verify the slope ratio method for determination of anticoagulant activity of leeches in laboratory and to investigate. The feasibility of slope ratio method for quality control of leech anticoagulant activity. Methods: Seven batches of leeches and Japanese medical leeches were used to prepare the extracts with different concentrations in parallel. The anticoagulant activity was determined by slope ratio method after the activated partial thromboplastin time(APTT) was measured. Specificity, linearity range, precision, accuracy and durability were systematically investigated in a laboratory. Results: The slope ratio method was used to measure the anticoagulant activity of leeches. The activity of leech extract decreased after heating at 100 ℃. The experiment was not established after adding low molecular weight heparin. The anticoagulant activity of Japanese medical leeches was determined by leeches as the control material, which indicated that the method had better specificity and selectivity. Accuracy inspection: the relative deviations of the 5 relative level values of 64%, 80%, 100%, 125% and 156% were all less than ±20%, and the logarithm of the theoretical value of potency (horizontal coordinate) and the logarithm of the corresponding value of potency (vertical coordinate) showed an obvious linear relationship, with the correlation coefficient greater than 0.95. The slope is between 0.95 and 1.05, indicating good accuracy of the method. The geometric coefficient of variation (GCV, %) of each relative potency level was less than 20%. RSDs of many batches of decoction pieces were all less than 20%, indicating that the precision of this method was good. Linearity and range test: the method could be used to accurately determine 64% to 156% relative to the control medicinal materials, and the linear range meets the requirements. Durability test: leech extract could be placed for 24 h and heated at 37 ℃ for 1 h without any change in activity, indicating that the durability of this method was good. Conclusion: The slope ratio method is precise, accurate and reproducible in the determination of anticoagulant activity of leeches, which is suitable for the quality control of leeches.
[1] 中华人民共和国药典2020年版. 一部[S].2020: 85
ChP 2020. VolⅠ[S].2020: 85
[2] 刘璇, 高美风, 孔毅. 水蛭化学成分及药理作用的研究进展[J].药物生物技术, 2017, 24(1):76
LIU X, GAO MF, KONG Y. Research progress of chemical constituents and pharmacological action of leech[J].Chin J Pharm Biotechnol, 2017, 24(1):76
[3] 黄秋阳, 冷静, 甘奇超, 等. 水蛭及其制剂在心脑血管疾病中的应用[J].中成药, 2019, 41(8):1915
HUANG QY, LENG J, GAN QC, et al. Application of leech and its preparation in cardiovascular and cerebrovascular diseases[J] Chin Tradit Pat Med, 2019, 41(8):1915
[4] 潘瑞萍, 徐力, 李宏艳. 刘红旭教授益气逐瘀法治疗不稳定型心绞痛经验[J].世界中西医结合杂志, 2019, 14(12):1674
PAN RP, XU L, LI HY. Professor Liu Hongxu’s experience of supplementing Qi and removing blood stasis in the treatment of unstable angina pectoris[J] World J Integr Tradit Chin Weste Med, 2019, 14(12):1674
[5] 于永慧. 心梗后纤维化心肌ECM蛋白表达谱构建及水蛭素的干预效应研究[D].北京: 中国中医科学院, 2019
YU YH. Construction of ECM Potein Expression Profile of Fibrotic Myocardium After Myocardial Infarction and the Intervention Effect of Hirudin [D].Beijing: Chinese Academy of traditional Chinese medicine, 2019
[6] 黎希年, 牛鹏, 曾纪超. 水蛭粉在脑梗死患者治疗中的应用效果[J].中国当代医药, 2019, 26(16):38
LI XN, NIU P, ZENG JC. Application effect of leech powder in the treatment of patients with cerebral infarction[J].China Mod Med,2019, 26(16):38
[7] 中国研究型医院学会血栓与止血专委会.活化部分凝血活酶时间延长混合血浆纠正试验操作流程及结果解读中国专家共识[J].中华检验医学杂志, 2021, 44(8):690
Thrombosis and hemostasis Committee of Chinese Research Hospital Association. Interpretation of operation process and results of activated partial thromboplastin time extension mixed plasma correction test[J] Chin J Lab Med,2021, 44(8):690
[8] 苏斌, 王志斌, 宋程程, 等. 水蛭抗凝血作用实验研究[J].山东中医杂志, 2014, 33(11):920
SU B, WAND ZB, SONG CC, et al. Experimental study on anticoagulant effect of leeches[J].Shandong J Tradit Chin Med, 2014, 33(11):920
[9] 金家金, 王志斌, 苏斌, 等. 水蛭体外抗凝血实验研究及其生物活性测定方法反应体系的筛选[J].中国药学杂志, 2014, 49(22):1997.
JIN JJ, WAND ZB, SU B, et al. Study on the anticoagulant assay of leech in vitro and screening of its bioactivity assay[J].Chin Pharm J, 2014, 49(22):1997
[10] 李波, 朴晋华. 中药生物活性质量控制的思考[J].中国药品标准, 2012, 13(1):5
LI B, PIAO JH. Reflection on quality control of bioactivity of traditional Chinese medicine[J].Drug Stand China, 2012, 13(1):5
[11] FDA Drug Safety Communiation: Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/ Biological Products[EB/OL]. [2020-04-14]. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/q6b-specifications-test-procedures-and-acceptance-criteria-biotechnologicalbiological-products
[12] 罗雪梅, 孟杰亲, 陈秀芬, 等. 基于体外抗凝血实验研究铅污染对水蛭抗凝血作用的影响[J].中国中药杂志, 2019, 44(23):5114
LUO XM, MENG JQ, CHEN XF, et al. Effect of lead pollution on anticoagulant activity of leeches based on in vitro anticoagulant experiment[J].China J Chin Mater Med, 2019, 44(23):5114
[13] 史小莲, 刘俊田, 李西宽, 等. 水蛭免加热提取物抗血栓、抗凝血作用及作用机理实验研究[C]// 中国药理学会第八次全国代表大会暨全国药理学术会议论文摘要汇编. 北京: 中国药理学会, 2002: 157
SHI XL, LIU JT, LI XK, et al. Experimental study on antithrombotic and anticoagulant effects of leeches without heating[C]// Abstracts of the 8th National Congress of Chinese Pharmacological Society and National Pharmacological Academic conference. Beijing: Chinese Pharmacological Society, 2002: 157
[14] 李艳玲, 黄荣清, 孙晓东. 水蛭不同提取物抗凝活性的体外实验研究[J].中药材, 2004, 27(2):123
LI YL, HUANG RQ, SUN XD. Study on anticoagulant activity of different extracts from leech in vitro[J].J Chin Med Mater, 2004, 27(2):123
[15] 肖珠,王志斌,程源,等.益心通络胶囊体外抗凝血的生物活性测定法研究[J].实验动物科学, 2017, 34(3):20
XIAO Z, WANG ZB, CHENG Y, et al. Study on the anticoagulant activity of Yixintongluo capsules[J].Lab Anim Sci, 2017, 34(3):20
