| Tilapia has the characteristics of fast growth,strong tolerance and wide adaptability.It is the main freshwater fish and export-oriented agricultural products in south China.However,in recent years,streptococcosis caused by agarose streptococcal infection poses a great threat to the cultivation of tilapia in south China,and there is no safe and effective drug prevention and treatment.The natural compounds extracted and purified from Chinese herbal medicine have the advantages of low toxicity,low residual risk,low environmental pollution and low drug resistance.In recent years,Chinese herbal medicine as an effective medicine has been widely used in the prevention and control of aquaculture diseases.For this reason,5 strains of agarococcus from different sources were used as screening strains,and natural compounds that inhibit the virulence factors of clotting bacteria-hemolytic and hyaluronidase were rapidly screened out from 40 natural compounds with antibacterial activity.Using the microplate method to study the effects of 40 natural compounds on hemolytic activity of 5 strains of streptococcus agaricus,results showed that strychnine,ferulic acid,white birch acetate acid,east belladonna lactone,coumarin,berberine hydrochloride,rhein,emodin,rhizoma coptidis alkaloid,tanshinone I,tannic acid,quercetin,waxberry,ellagic acid,baicalin,oleanolic acid,shikimic acid,forsythia glycosides,limonin,celery element,sorbic acid,alkali and alkali gentian evodia rutaecarpa alkali,mount a total of 26 kinds of natural compounds for more than one plant antimicrobial ?-hemolytic/soluble cell activity haves inhibitory effect.Among them,4?g/mL baicalin inhibited the activity of ?-hemolysis/soluble cells in 5 strains,the inhibition rate was 22.11%?84.54%,and the inhibition concentration(ICso)of Pbsa0901?-hemolytic/soluble cell activity of Streptococcus suis was 5.39 ± 0.55 ?g/mL,And the Streptococcus suis treated by Baicalin was passed for 10 generations in a row,and its ?-hemolytic/soluble cell activity still failed to recover.The relationship between the concentration and inhibition rate of Baicalin was plotted by the same detection method,and the optimum concentration of Baicalin inhibiting ?-hemolytic/soluble cell activity of Streptococcus suis was 39.06 ?g/mL.The effect of 40 natural compounds on the activity of PBSA0901 hyaluronic acid enzyme was investigated by Oxford Cup method,and the results showed that scopolamine,rhubarb acid,rhubarb phenol,huangteng,lemon bitter,S1s acid,dogwood alkali,root alkali,gentian acid,and citric acid all inhibited the activity of hyaluronic acid enzyme(PBSA0901)without Streptococcus suis,and the inhibition rate was above 40%.Among them,the inhibition rate of hyaluronic acid enzyme activity of 4?g/mL baicalin on Streptococcus-free PBSA0901 was 100%.The use of the automatic growth curve metre to detect 39.06 ?g/mL Baicalin had no effect of baicalin on the growth of streptococcus agalactiae PBSA0901.After co-culture with 39.06?g/mL Baicalin,streptococcus free was inoculated in a medium containing 2.5,5,10,20,mmol/mL H2O2 or 250,500,1000,2000 mg/mL lysozyme,the results showed the growth rate of Streptococcus free PBSA0901 treated with 39.06?g/mL Baicalin in 10s 20 and 40 mmol/mL H2O2 medium was significantly lower than that in the control group,but there was no significant difference between L1 in different concentrations of lysozyme and control group.According to the experimental tilapia body weight,the PBSA0901 was inoculated intraperitoneally according to the dose of 1.78×1 09 CFU/kg.After 2 h,the continuous feeding of the fish was 0.226,0.445,0.890,1.780 and 3.560g/kg based on 3%of the fish weight.The feed of baicalin was continuously fed to the medicated feed for 14 days.During this period,the relative protection rates of the above groups were 25.81,41.94,10.97,87.10,90.32%,respectively.The dose(EC50)was 0.526 g/kg.The results of the study on the acute toxicity of baicalin to tilapia showed that after tilapia was administered with 2000 mg/kg Baicalin,tilapia had a survival rate of 100%during the observation period and no symptoms of poisoning,which was in line with the safety standard of Guideline for testing of chemicals drug in the target animal.A single gavage of tilapia with Baicalin(0.4 mg/mL,0.1 mL/35 g),respectively,in 0 h,0.25 h,0.5 h,1 h,2 h,4 h,8 h,12 h,24 h,48 h and 96 h randomly collected tilapia 6 tail,extraction of blood,anatomy of the brain,liver,spleen,head kidney,muscle and bile,The concentration of Baicalin in blood and Viscera was detected by HPLC,and the non-atrioventricular model was used to analyze it,and the results showed that the half-life(T1/2)of Baicalin in blood was 180.940 h,the highest peak time(Tmax)was 0.987 h,and the highest peak area(Cmax)was 31.278ug/mL,The area under the time Curve(AUC)was 2252.289?g/mL·h,and the apparent distribution volume(Vd)was 0.005.The t1/2 of Baicalin in brain,liver,head kidney and bile were 8.315,38.168,57.273 and 72,705 h,Tmax in brain,liver,head kidney,muscle and bile were 8,8,8,1 and 12 h,respectively,Cmax were 29.787,39.971,85.577,55.771 ?g/kg and 63.038 ?g/mL,respectively.The AUC0-? of Baicalin in the brain,liver,head kidney and bile 435.660,2796,590,5667.352 and 4937.360 ?g/mL·h,respectively.The results showed that Baicalin could quickly absorb into blood and muscle tissue and enter brain tissue through the blood-brain barrier after gastric Baicalin was gavaged into tilapia.After the baicalin is absorbed into the liver and gallbladder,it can be secreted by bile into the digestive tract,reabsorbed into the blood and thus produces the double-peak phenomenon,which is eventually eliminated by the excretion of urine and feces.The maximum distribution of baicalin in various tissues and organs of experimental fish was from high to low,followed by muscle>head kidney>bile>liver>brain,its retention time in each tissue organ from long to short respectively for muscle>bile>head kidney>liver>brain,the longer the Baicalin stays in each tissue and organ,the longer the duration of sustainable use of the drugThe inhibitory effect of baicalin on ?-hemolytic/soluble cell activity and hyaluronic acid enzyme activity of streptococcus free was significant,which could affect the?-hemolytic/soluble cell activity after transmission.after co-culture with baicalin Streptococcus agalactiae is more sensitive in the face of H2O2 stimulation;The survival rate of tilapia was significantly improved by feeding tilapia infected with Streptococcus suis disease,and the performance of Baicalin as an aquaculture drug showed reliable safety and durability.The results of this study provide new drug selection for the prevention and treatment of Streptococcus aureus disease in aquaculture,and also provide a basis for the application of Chinese medicine in aquaculture. |
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