中国现代中药2025，Vol.27Issue(12)：2241-2247,7.DOI:10.13313/j.issn.1673-4890.20250416005
基于纤维蛋白原-凝血酶时间法的菲牛蛭冻干粉抗凝血活性定量评价
Fibrinogen-Thrombin Time Assay for Quantitative Assessment of Anticoagulant Activity in Poecilobdella manillensis Freeze-dried Powder
摘要
Abstract
Objective:To develop a novel assay for determining the anticoagulant activity of Poecilobdella manillensis Lesson freeze-dried powder based on fibrinogen-thrombin time(FIB-TT assay),aiming to enhance the objectivity and stability in detection and evaluate its feasibility as a replacement for traditional thrombin titration.Methods:SD rats were administered the powder,and in vivo anticoagulant activity was evaluated by measuring the activated partial thromboplastin time(APTT),prothrombin time(PT),TT,and FIB levels after drug administration.For the in vitro study,rabbit plasma was incubated with varying mass concentrations of P.manillensis Lesson freeze-dried powder extract,followed by measurement of APTT,PT,and TT.Key dose-effect indicators were then screened based on the combined in vitro and in vivo experimental results.The FIB-TT assay was established using purified fibrinogen instead of animal plasma,with an automated coagulation analyzer optimizing the reaction system(0.4%fibrinogen and 10 U·mL-1 thrombin).A dose-response curve was constructed using recombinant hirudin as the reference standard.Method reliability was validated through linear range,precision,repeatability,and applicability studies.The potency of 14 batches of samples was determined by the thrombin titration method,followed by a correlation analysis with the potency measured by the FIB-TT method.Results:The experimental results showed that in vivo administration of P.manillensis Lesson freeze-dried powder prolonged TT and APTT,shortened PT,and decreased FIB content in a dose-dependent manner in rats.In vitro,the extract at various mass concentrations also demonstrated a dose-dependent prolongation of plasma TT and APTT and a shortening of PT.Consequently,TT was identified as the key indicator.The potency of the P.manillensis Lesson freeze-dried powder,as determined by the FIB-TT method,ranged from 117.5 to 483.0 U·g-1,which was approximately 50%of the potency measured by thrombin titration method.However,a significant positive correlation was observed between the two methods(r=0.911,P<0.01).The methodology validation results demonstrated that the FIB-TT method exhibited good linearity(r>0.95 for both hirudin reference standard and P.manillensis Lesson extract),high precision(RSD=0.58%for instrumental analysis,RSD=1.18%for repeatability),and broad applicability(with a fiducial limit rate,FL%<1%).Conclusion:The FIB-TT assay significantly improves the accuracy and objectivity of anticoagulant activity assessment for P.manillensis Lesson freeze-dried powder through standardized reaction systems and automated detection.This approach serves as a vital supplement to current quality standards,enabling high-throughput and standardized batch quality control to minimize clinical medication variability.关键词
菲牛蛭冻干粉/抗凝血效价/纤维蛋白原-凝血酶时间/质量控制Key words
Poecilobdella manillensis Lesson freeze-dried powder/anticoagulant potency/fibrinogen-thrombin time/quality control分类
医药卫生引用本文复制引用
HUANG Chuying,SHI Fujuan,MEI Shanshan,FANG Kexin,ZHAO Weitao,WANG Wenyi,HE Guangzong,GAO Mengfei,YANG Yaojun..基于纤维蛋白原-凝血酶时间法的菲牛蛭冻干粉抗凝血活性定量评价[J].中国现代中药,2025,27(12):2241-2247,7.基金项目
北京市自然科学基金项目(7222275) （7222275）
