Pharmacodynamics Of Chinese Medicine For Prevention And Treatment Of Shrimp Postlarva Bacterial Vitrified Syndrome (BVS) And Mechanism On In Vitro Antibacteria Of Its Main Active Component

Shrimp postlarva bacterial vitrified syndrome（BVS）has strong pathogenicity,high mortality,resulting in a short supply of larvae and causing huge economic losses to the shrimp culture industry.There are currently no effective drugs for BVS.Compared with antibiotics,Chinese herbal medicine has the advantages of multi-component,multi-target,antibacterial,regulating immunity and not causing drug resistance,which has attracted more and more attention.The development of Chinese herbal compound to prevent and treat shrimp postlarva BVS is beneficial to promote green and healthy culture of shrimp.In this study,from 50 kinds of Chinese herbal medicines,we firstly screened out the herbal medicines with good bacteriostasis effect on Vibrio alginolyticus and Vibrio parahaemolyticus,pathogenic agents of BVS.Then combined with two Chinese herbs of Cyperi rhizoma and Gardeniae fructus to form 3kinds of Chinese herbal compounds,prescription 1,prescription 2 and prescription 3.Then shrimp postlarvae were infected with Vibrio alginolyticus in bath of different concentrations,and the optimal challenge concentration was determined to establish the pathological model of artificially infected shrimp BVS.In vivo pharmacodynamic test,the efficacies of three Chinese herb compounds were evaluated by the mortality,histopathology and ultrahistopathology character of diseased postlarvae.Finally,the antibacterial activity and mechanism of gallic acid,the main active ingredient of Chebulae fructus in prescription 1,against V.alginolyticus were investigated.The results of this study provide basis for the development of special herbal compounds for the prevention and treatment of bacterial infection,and contribute to the green and high-quality development of shrimp industry.The main research contents and results of this paper are as follows:1.Chinese herbal medicines against V.alginolyticus and V.parahaemolyticus,the pathogens of BVS,in vitro bacteriostatic testOxford Cup method and double dilution method were used to observe the in vitro inhibitory effects of different Chinese herbal medicines on V.alginolyticus and V.parahemolyticus.The Oxford Cup method was used to observe the bacteriostatic effect of 50 kinds of Chinese herbal medicines on Vibrio alginolyticus and Vibrio parahemolyticus.7 kinds of Chinese herbal medicines including Chebulae fructus,Galla chinensis,Caryophylli flos,Granati pericarpium,Sanguisorbae radix,Mume fructus,Schisandrae chinensis fructus had good bacteriostatic effect on V.alginolyticus and V.parahaemolyticus,and the diameter of inhibition zones were more than 10mm.The minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of Chinese herbal medicine against V.alginolyticus and V.parahaemolyticus were determined by two-fold dilution method.The results showed that G.chinensis,C.fructus,Caryophylli flos and S.chinensis fructus had good bacteriostatic effect on V.alginolyticus and V.parahaemolyticus,with MIC≤12.5 mg/m L and MBC≤50 mg/m L.2.Pharmacodynamic evaluation of Chinese herbal compounds for prevention and treatment of BVSCombined with the results of antibacterial zone diameter,MIC and MBC,three kinds of compound Chinese medicine were formed by adding two kinds of Chinese medicine of Xiang and Gardenia C.rhizoma and Gardeniae fructus,which were prescription 1（C.fructus 30 g,S.chinensis fructus 20 g,C.rhizoma 20 g）,prescription2（C.fructus 40 g,G.pericarpium 30 g,C.flos 30 g）and prescription 3（C.fructus 40g,G.chinensis 30 g,G.fructus 30 g）,respectively.The results of challenge dose screening test showed that V.alginolyticus immersion challenge could cause typical symptoms of BVS in PL₉ shrimp postlarvae.The pathological model of BVS was constructed by immersing shrimp postlarvae with 5×10⁴ CFU/m L V.alginolyticus.In vivo pharmacodynamic test results showed that the positive control group all showed typical symptoms of BVS 12 h after challenge.In the positive control group,the final mortality rate reached 33.75%.In the blank control group,body color,vitality,feeding intake,and hepatopancreas were normal.It is proved that the model is copied successfully.In vivo pharmacodynamic test results showed that after 7 days drug treatment,the mortality rates of each group from low to high was as following:blank control group<Chinese herb prescription 1 group<florfenicol group<Chinese herb prescription 3 group<Chinese herb prescription 2 group<positive control group.The mortality of shrimp postlarvae in the prescription 1 group was significantly lower than that of the positive control group（P<0.05）.The mortality in prescription 2 and 3 groups and the florfenicol group was lower than the positive control group,but the difference was not significant（P>0.05）.The results indicated that prescription 1 could significantly reduce the mortality of diseased shrimp larvae.The histopathology observation shows that after 7 days of administration,the hepatopancreas tissue and intestinal epithelial structure of the shrimp larvae in group 1 were more complete,the number of hepatic tubules was large,the degree of epithelial cells shedding and necrosis was light,and the intestinal epithelial cells were abundant and orderly.In the group 2and 3 and the control group of florfenicol,hepatopancreas tissues of diseased shrimp seedlings were diseased,most of the epithelial cells in the hepatic tubules were necrotic and exfoliated,part of the hepatopancreas tissue structure disappeared,intestinal wall villi were exfoliated and missing,and there were a large number of tissue fragments in the intestine.Ultrastructural pathology observation shows that after 7 days of administration,the cell membrane and nucleus of liver tubule epithelial cells of the diseased shrimp postlarvae in the prescription 1 group were normal,and mitochondria and endoplasmic reticulum were abundant and basically normal.In florfenicol control group,cell membrane ablation,cell-cell boundary blurring,nucleus solidification,organelle disintegration,and even cell disappearance occurred in the epithelial cells of liver tubules of shrimp postlarvae.The results showed that prescription 1 had a better protective effect on hepatopancreas and intestinal tissue structure of diseased shrimp postlarvae than prescription 2,prescription 3 and 20%florfenicol powder.3.Study on the bacteriostatic activity and mechanism of gallic acid against V.alginolyticus,the main pathogen of BVSThe MIC and MBC of gallic acid against V.alginolyticus were determined by double dilution method.The results showed that the MIC and MBC of gallic acid against V.alginolyticus were 4mg/m L and 8mg/m L,respectively.Secondly,the effects of different concentrations of gallic acid on the growth,cell wall,cell membrane,biofilm,motility and aggregation ability of V.alginolyticus were investigated.The results showed that gallic acid with 2MIC and 1MIC completely inhibited the growth of V.alginolyticus,gallic acid with 1/2MIC significantly inhibited the growth of V.alginolyticus,and gallic acid with 1/4 mic had no significant effect on the growth of V.alginolyticus.Gallic acid with 2MIC,1MIC,1/2 MIC and 1/4 MIC concentrations could damage the cell wall within 2 h and lead to AKP exudation.The higher the concentration of gallic acid,the longer the action time,the greater the damage to the cell wall of V.alginolyticus.2MIC,1MIC,1/2MIC concentration of gallic acid significantly increased the conductivity of V.alginolyticus,but 1/4 MIC concentration of gallic acid had no significant effect on the conductivity of V.alginolyticus.Gallic acid with 2MIC,1MIC,1/2MIC and 1/4MIC concentrations could significantly inhibit the biofilm formation of V.alginolyticus in a concentration-dependent way,with inhibition rates of 83.26%（P<0.05）,77.80%（P<0.05）,32.37%（P<0.05）,16.61%（P<0.05）.Gallic acid with 2MIC,1MIC and 1/2MIC concentrations could significantly clear mature biofilms in a concentration-dependent manner,with inhibition rates of68.01%（P<0.05）,67.54%（P<0.05）,54.5%（P<0.05）.However,low concentration of gallic acid such as 1/4MIC concentration could not effectively clear the biofilm.The effect of different concentrations of gallic acid on the movement of V.alginolyticus was measured by swimming plates.Gallic acid with 2MIC,1MIC concentrations completely inhibited the growth of V.alginolyticus on agar plates.Gallic acid with1/2MIC and 1/4MIC concentrations had significant effects on the motility of V.alginolyticus,which showed that the growth diameter of the bacteria was significantly reduced.Range of motion was reduced by 60.41%（P<0.05）and 51.78%（P<0.05）.The concentrations of gallic acid with 2MIC,MIC,1/2MIC and 1/4MIC significantly inhibited the automatic aggregation of V.alginolyticus,in a concentration-dependent way,and the aggregation rates decreased to18.68%（P<0.05）,19.19%（P<0.05）,25.70%（P<0.05）,37.41%（P<0.05）.In summary,gallic acid can inhibit the growth of V.alginolyticus,destroy the cell wall,increase cell membrane permeability,inhibit the biofilm formation,remove biofilm,and inhibit motility and aggregation ability.