Potential effects and relevant lead compounds of Vigna mungo (L.) Hepper seeds against bacterial infection,helminthiasis, thrombosis and neuropharmacological disorders

Multidrug-resistant bacterial infections, helminthiasis, thrombosis, anxiety and insomnia are some of the major global health concerns. Vigna mungo (L.) Hepper (VM) has been used traditionally to treat microbial infection, helminthic disorder, schizophrenia, memory loss, and blood circulatory problem. This research aims to discover antibacterial, anthelmintic, thrombolytic and neuropharmacological effects of the methanol extract of Vigna mungo seeds (MESVM), and also in-silico prediction of relevant lead compounds by molecular docking and ADME/T analysis. The crude extracts and subsequent fractions of MESVM were investigated for antibacterial activity by disc diffusion method, anthelmintic activity by paralysis and death test on earthworms, and thrombolytic activity by in vitro blood clot dissolution test. Open-field test and elevated plus maze test were performed for evaluating anxiolytic activity of the extracts. Using molecular docking, ligand poses of selected VM seeds’ phytoconstituents were predicted targeting tubulin, GlcN-6-P synthase, and human tissue plasminogen activator proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. In the antibacterial activity test, the MESVM at 10000 μg/mL concentration created highest and significant (P < 0.001) zone of inhibition against Staphylococcus aureus (15.42 mm) and Escherichia coli (12 mm) compared with tetracycline. The MESVM exhibited remarkable anthelmintic activity at 50 mg/mL concentration with 35.4 min paralysis time, 75.2 min death time and were closer to the durations of standard drug albendazole. No test extract showed anxiolytic activity. In thrombolytic activity test, all concentrations of MESVM produced clot lytic activity with high significance (P < 0.001) in comparison with the blank. In docking, 2′-hydroxygenistein, cyclokievitone hydrate, and aureol displayed maximum affinity to the target proteins for anthelmintic, antibacterial, and thrombolytic activity, respectively. This research revealed that the MESVM demonstrated potential anthelmintic, antibacterial and thrombolytic effects that confirmed the folkloric uses of VM and the found relevant lead compounds might be further optimized in future drug development.     

静脉溶栓时机对急性ST段抬高型心肌梗死患者溶栓效果及主要不良心脏事件发生率的影响

目的:探讨静脉溶栓时机对急性ST段抬高型心肌梗死患者溶栓效果及主要不良心脏事件发生率的影响。方法:将2016年1月至2017年12月我院接诊的314例急性ST段抬高型心肌梗死患者纳入本研究,按照溶栓治疗时间不同分为A组(发病至溶栓时间6 h)172例、B组(发病至溶栓时间为6～12 h)102例和C组(发病至溶栓时间12 h)40例,比较三组患者溶栓效果、溶栓后ST段回落情况以及住院期间主要不良心脏事件发生情况。结果:A组患者梗死冠脉溶通率、溶栓后ST段回落幅度高于B组和C组,且B组高于C组,差异均有统计学意义(P0.05)。A组患者治疗后ST段回落最大幅度所需时间、住院期间主要不良心脏事件总发生率低于B组和C组,且B组低于C组,差异均有统计学意义(P0.05)。结论:急性ST段抬高型心肌梗死患者发病后6 h内静脉溶栓治疗梗死冠脉溶通率更高、ST段回落效果更好,可降低住院期间主要不良心脏事件发生风险。     

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55°C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS–PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), β-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bβ-chains and Aα-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.     

Enzymatic properties and identification of a fibrinolytic serine protease purified from Bacillus subtilis DC33

A novel fibrinolytic enzyme subtilisin FS33 was purified from Bacillus subtilis DC33, isolated from a traditional flavour-rich food in China. The purified subtilisin FS33 was a single chain protein with a molecular mass of 30 kDa measured by SDS-PAGE. After activated SDS-PAGE, the enzyme band exhibited strong fibrinolytic activity on the fibrin plate. Subtilisin FS33 was temperature-stable below 60°C over the pH range 5–12, with a maximum activity at pH 8.0, but the activity completely disappeared after 10 min above 65°C. The NH₂-terminal amino acid sequence of the enzyme was different from that of other known fibrinolytic enzymes, such as NK, CK, SMCE, KA38, subtilisin E, subtilisin DFE and Katsuwokinase. The amidolytic activities of subtilisin FS33 were inhibited completely by phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI). EDTA did not affect the enzyme activity, and none of the ions tested activated the activity. Therefore, the enzyme was thought to be a subtilisin-like serine protease. The enzyme degraded the Bβ-chains of fibrin(ogen) very rapidly and then degraded the Aα-chain and at least five fragments from fibrin(ogen) were obtained after hydrolysis. Subtilisin FS33 was also able to cleave blood clots in the absence of endogenous fibrinolytic factors.     

蚓激酶研究进展

蚓激酶是一种新型的溶栓制剂,具有良好的溶血栓治疗效果。综述了至今为止分离到的蚓激酶成分、生物学活性、药物学作用、抑制剂和空间结构分析等方面的研究进展 。     

血栓分子标志物与急性缺血性脑卒中病情严重程度及溶栓预后的相关性

摘要 目的：探究血栓分子标志物[血栓调节蛋白（TM）、凝血酶-抗凝血酶Ⅲ复合物（TAT）、纤溶酶-α2纤溶酶抑制剂复合物（PIC）、组织型纤溶酶原激活剂-抑制剂1复合物（t-PAIC）]与急性缺血性脑卒中(AIS)病情严重程度及溶栓预后的相关性。方法：选取2020年 7月至2022年6月我院神经内科收治的AIS患者120例为研究组,根据患者NIHSS评分为轻症组（n=48）、中症组（n=52）及重症组（n=20）;NIHSS评分升高组（n=50）和降低组（n=70）。同期选择我院查体中心查体的健康人群为对照组(n=30)。对比观察组和对照组、不同严重程度和不同预后患者外周血血栓分子标志物水平差异。采用Pearson相关分析法分析AIS患者外周血血栓分子标志物水平与病情严重程度及预后的相关性。利用ROC曲线评估外周血血栓分子标志物水平对AIS患者预后的预测价值。结果：AIS组患者外周血TM、TAT、PIC及t-PAIC水平明显高于对照组,差异有统计意义（P＜0.01）。重症组患者外周血TM、TAT、PIC及t-PAIC水平明显高于中症组及轻症组,中症组患者血清TM、TAT、PIC及t-PAIC水平明显高于轻症组,差异有统计意义（P＜0.01）。NIHSS评分升高组患者外周血TM、TAT、PIC及t-PAIC水平明显高于NIHSS评分降低组,差异有统计意义（P＜0.01）。外周血TM、TAT、PIC及t-PAICt水平与NIHSS评分呈正相关（r分别为0.326、0.513、0.124和0.417,P均＜0.05）。外周血TM、TAT、PIC及t-PAIC水平预估AIS患者预后的AUC 分别为 0. 737,0.850,0.762和0.712。其中TAT预估AIS患者预后的AUC、敏感度和特异性均最高。结论：AIS患者外周血TM、TAT、PIC及t-PAIC水平明显高于健康人群,可随病情严重程度及预后情况的变化而变化,其在病情评估及溶栓预后预测中具有指导价值。     

Understanding the enzymology of fibrinolysis and improving thrombolytic therapy

Cardiovascular disease is responsible for 17 million deaths per year but acute myocardial infarction and stroke can be treated with thrombolytics ("clot busters"), which are plasminogen activators. However, despite many years of study and huge investment from the pharmaceutical industry, clinical trials of new drugs have often been disappointing. Part of the problem may be our incomplete understanding of the regulation of plasminogen activation in vivo. We have developed precise in vitro methods and with the application of computer simulations, we hope to improve our understanding of plasminogen activation to facilitate improvements in thrombolytic therapy.     

Regulation of plasminogen activator secretion in mouse peritoneal macrophages. I. - Role of serum studied by a new spectrophotometric assay for plasminogen activators.

A chromogenic tripeptide - H-D-Val-Leu-Lys-p-nitroanilide-substrate of plasmin, can be used to follow plasminogen activation by an activator such as urokinase or the activator secreted by mouse peritoneal macrophages (thioglycolate-elicited). The acceleration of p-nitroaniline production is proportional to the initial rate of plasmin formation from plasminogen. Thus, at a given plasminogen concentration, this acceleration is proportional to the activator concentration. The acceleration can be evaluated from the spectrophotometer trace recording at 405 nm the appearance of p-nitroaniline, either by means of a computer program or by a plot of delta A405 vs.t2. The sensitivity of this assay allows detection of 0.003 CTA units of urokinase. Thioglycollate-elicited mouse peritoneal macrophages secrete plasminogen activator into the extracellular medium during in vitro cultivation only after a contact with serum.     

重组链激酶与尿激酶在急性心肌梗死溶栓治疗中的血管再通率的对比研究

目的：比较国产重组链激酶（r-SK）与尿激酶（uK）在急性心肌梗死（AMI）溶栓中的血管再通率。方法：对68例诊断为AMI并进行溶栓治疗的患者进行临床分组治疗,观察组36例,对照组32例,观察组治疗给予r-SK,对照组治疗给予UK。观察两组患者血管再通率、住院并发症、不良反应以及30d病死率等。结果：观察组患者总血管再通率、小于6h血管再通率以及6-12h血管再通率均明显高于对照组（P〈0．05）,不良反应方面,观察组用药后轻度出血、皮疹、低血压等与对照组比较无差异（P〉0．05）。30d病死率方面,观察组为11．11％,与对照组的15．63％比较无明显差异（P〉0．05）。结论：国产r-SK进行AMI溶栓的血管再通率高,不良反应明显优于UK,30d病死率与UK相似,值得临床应用。