背景:当归四尼汤(DGSN)是中医临床实践中的经典方剂。DGSN汤常用于缓解伤寒论(《伤寒论》)记载的寒凝血瘀症状,治疗雷诺氏病,痛经,关节炎,偏头痛在中医诊所。越来越多的证据表明,这种疾病与微循环障碍有关。然而,负责治疗的DGSN汤的抗凝活性和潜在机制尚不清楚。
目的:对DGSN汤的指纹图谱及体内外抗凝活性进行评价,以加强方剂的质量控制和活性研究。
方法:采用高效液相色谱法对DGSN汤剂的化学成分进行分析,并采用《中药色谱指纹图谱相似度评价软件(2012年版)》进行指纹图谱相似度评价。通过测量四种凝血因子(PT,TT,APTT,FIB)体外。建立普纳替尼诱导的斑马鱼血栓形成模型,评价其改善体内微血管血流动力学的活性。采用定量实时聚合酶链反应(q-PCR)比较凝血因子II(FII)RNA表达水平的变化,VII(FVII),斑马鱼血栓形成模型的IX(FIX)和X(FX)。
结果:建立了DGSN汤剂的指纹图谱相似性评价方法。结果表明,18个样品具有较高的相似性(S1-S18>0.878)。药效学结果表明,DGSN汤可以延长PT,TT和APTT,并降低体外FIB含量。同时,在低剂量(500μgmL-1)下,它显着增强了体内心输出量和血流速度。q-PCR数据表明,DGSN汤(500μgmL-1)可以下调FII的RNA表达,FVII,FIX和FX。有趣的是,FII有双向调节,FIX和FX在一定浓度范围内。总的来说,DGSN汤可显著改善血流动力学,下调凝血因子,结果在体外-体内是一致的。
结论:指纹图谱研究为提高DGSN汤的质量控制提供了新的视角。DGSN汤通过同时调节多种凝血因子而具有抗凝活性。因此,具有开发成为新型抗凝药物原料的潜力。
BACKGROUND: Dang-Gui-Si-Ni (DGSN) decoction is a classic prescription in the clinical practice of traditional Chinese Medicine (TCM). DGSN decoction is often used to relieve symptoms of cold coagulation and blood stasis recorded by Treatise on Febrile Diseases (Shang Han Lun) and treat Raynaud\'s disease, dysmenorrhea, arthritis, migraine in TCM clinic. Accumulated evidences have suggested that this diseases are related to microcirculation disturbance. However, the anticoagulant activity and underlying mechanisms of DGSN decoction responsible for the therapeutic not well understood.
OBJECTIVE: The fingerprint and anticoagulant activity in vivo-in vitro of DGSN decoction were evaluated to strengthen the quality control and activity study of formulas.
METHODS: The chemical components of DGSN decoction were analyzed by HPLC and its fingerprint similarity were evaluated by \"Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software (2012 Edition)\". The anticoagulant activity of DGSN decoction was assessed by measuring four coagulation factors (PT, TT, APTT, FIB) in vitro. Zebrafish thrombosis model induced by punatinib was established to evaluate the activity of improving microvascular hemodynamics in vivo. Quantitative real-time polymerase chain reaction (q-PCR) were adopted to compare the changes in the RNA expression levels of coagulation factor II (FII), VII (FVII), IX (FIX) and X (FX) in zebrafish thrombosis model.
RESULTS: The fingerprint similarity evaluation method of DGSN decoction was established. The results showed that 18 samples had higher similarity (S1-S18 > 0.878). Pharmacodynamic results showed that DGSN decoction could extend PT, TT and APTT, and reduce FIB content in vitro. Meanwhile, it markedly enhanced the cardiac output and blood flow velocity at low dosage (500 μg mL-1) in vivo. q-PCR data demonstrated that DGSN decoction (500 μg mL-1) could downregulate the RNA expression of FII, FVII, FIX and FX. Interestingly, there were a bidirectional regulation of FII, FIX and FX in a certain concentration range. In general, DGSN decoction can significantly improve hemodynamics and downregulate coagulation factors, and the results were consistent both in vitro - in vivo.
CONCLUSIONS: The fingerprint study provide a new perspective for improving the quality control of DGSN decoction. DGSN decoction possess anticoagulant activity by regulating multiple coagulation factors simultaneously. Thus, it has the potential to develop into the novel raw material of anticoagulant drugs.