Study On Antithrombotic Active Components Of Bacterial GYZC02 Strain

Currently, Thrombotic diseases seriously threat to people’s health and life, has become the leading factor of human disease death. Finding a specific and effectively anti-thrombotic drugs, has become a hot field for researchers. However, it is the main source of antithrombotic drugs from microorganisms.In this research,a strain GYZC02 with high antithrombotic activity,was isolated from the soil which from Guiyang surrounding,by using blood meal tablet as first screening, shake flask as second screening. Strain GYZC02 was identified through physiological and biochemical characteristics experiment and 16 S rDNA gene sequence analysis, the physiological and biochemical characteristics result showed that Characteristics of GYZC02 strain was similar to Bacillus subtilis. Using NCBI Web site to blast the nucleotide sequence obtained by 16 S rDNA and using MEGA 5.0 draw phylogenetic tree, the results showed that the 16 S rDNA sequence of strain GYZC02 was 100% homology with Bacillus subtilis BL10. In all, Through the Physiological and biochemical experiment and 16 S rDNA gene sequence analysis proved GYZC02 as a strain Bacillus subtilis.Take univariate to optimize the liquid fermentation conditions of strain GYZC02. The best medium components are: 1.5 % soluble starch, yeast extract 0.7 %, KH2PO4 0.3 %, Na2HPO4 0.2 %, CaCl2 0.02 %, MgSO4 0.03 %, FeSO4 0.04 %; the optimum fermentation conditions are: fermentation temperature 37 ℃, initial pH 7.0, inoculum 4 %, liquid volume 75 ml / 250 ml, shaking speed 170 r / min, vaccination age 12 h, fermentation time 48 h.Separation and extraction antithrombotic active substance through the crude ethyl acetate and acid precipitation, conducting experiment in vitro anti-thrombotic, anti-thrombosis test in mice and antiplatelet test used the substance. the result show that the ethyl acetate extract GYZC02 broth could significantly prolonged the bleeding time proved anticoagulant activity, and inhibited the formation of mouse tail artery thrombosis showed to have antithrombotic activity. The substance can dissolute artificial thrombus in vitro showed that it have certain antithrombotic effect in vitro, the substance can inhibit platelet aggregation.The experiment initially identified the substance as Sugar-cyclic-peptides substance by TLC analysis and infrared spectroscopy analysis at last. Antithrombotic mechanisms of the substance mainly because it can inhibit platelet aggregation, thus achieve the anticoagulant effect, so as to play antithrombotic effect.