Study On Pharmacodynamics And Toxicity Of Nattokinase In Vivo

Nattokinase?NK?is a serine protease with strong fibrinolysis property produced by Bacillus subtilis natto during natto fermentation.Studies have shown that nattokinase is not only easy to extract and purify,but also has benefits in low cost,excellent thrombolysis effect,and good safety.It also brings about the problems of poor oral bioavailability,high price,and many complications of current antithrombotic drugs.In the present study,the high-yield nattokinase strain?Bacillus subtilis natto LNUB236?isolated in the laboratory was used for fermentation culture.The fermentation broth was used as a raw material,ammonium sulfate was used to precipitate nattokinase,and the precipitate after centrifugation was dissolved in a phosphate buffer solution.The nattokinase crude enzyme solution was subjected to two Sephadex G-75 gel chromatography,and the collected nattokinase active components were freeze-dried to obtain a lyophilized powder,and the lyophilized powder was quantitatively loaded into a rat-specific enteric solution.The capsules were evaluated by oral nattokinase enteric-coated capsules to evaluate the pharmacodynamics,in vivo acute toxicity and long-term toxicity of nattokinase in vivo,providing a theoretical basis for the clinical application and product development of nattokinase.The evaluation of the antithrombotic effect of nattokinase was evaluated by in vivo thrombosis model in SD rats and the effect on the time of tail bleeding in mice.The in vivo efficacy showed that compared with the control group,FeCl3-induced rat carotid artery thrombosis experiment,the nattokinases 5000 IU/kg,10000 IU/kg,and 15000 IU/kg dose group were able to effectively inhibit the rat's arterial thrombus formation?P<0.0001?in dose-dependent.In the mouse tail bleeding test,5000 IU/kg,10000 IU/kg,and 15000 IU/kg administration dose groups significantly prolonged bleeding time?P<0.0001?compared to the control group,and with the administration dose.The increase in bleeding time was longer?P<0.0001?with dose-dependent.Observed the acute toxicity and long-term toxicity of Nattokinase in SD rats,providing reference for clinical drug safety.In the acute toxicity test of nattokinase,one dose of 5000mg/kg was administered and continuously observed for 14 days,poisoning and death were recorded.No toxicity or death occurred in acute toxicity experiments.The experimental results showed that the nattokinase LD₅₀>5000 mg/kg,and there was no risk of acute poisoning.In long-term toxicity experiments,rats were observed for 28 days to give nattokinase toxicity,and rats were randomly divided into 4 groups:control group,nattokinase120 mg/kg,360 mg/kg,and 1000 mg/kg.The dosage group was repeatedly administered for28 days.During the test period,the appearance and physical activity of the rats were observed.At the end of the experiment,blood samples were collected for hematology,blood biochemistry,coagulation index,and histopathological examination.Nattokinase was repeatedly administered for 28 days.During the administration period,rats grew well and their behavior was normal.No abnormalities were detected in any of the indicators.No abnormal changes in nattokinase toxicity were found in this experiment.Nattokinase was not found in the experiment.Oral administration for 28 days had no obvious toxicity to rats.In a90-day toxicity experiment with repeated doses of nattokinase,rats were randomly divided into 4 groups:control group,nattokinase 120 mg/kg,360 mg/kg,and 1000 mg/kg dose groups.Repeatedly After 90 days of drug administration,the general state of the rats was observed.After the administration,the rats were subjected to hematology,blood biochemistry,coagulation parameters,and pathological examination.During the experiment,the appearance,physical activity,and feces of the rats in each administration group were normal,and no abnormalities were found in all indicators.Under the experimental conditions,no abnormal changes caused by nattokinase toxicity were found.Nattokinase was not significantly toxic to rats after oral administration for 90 days.