| As one of the most common fungal diseases in freshwater aquaculture,Saprolegniasis has high explosive and infectivity,and a wide range of infected objects.It has caused huge economic losses to the freshwater aquaculture industry.At present,the prevention and control of Saprolegnia mainly depends on chemical drugs.Malachite green,the most effective chemical drug,was prohibited from being used in water plant breeding in 2002,while other anti Saprolegnia chemical drugs are also widely controversial due to drug safety.In addition to drug safety,chemical drugs generally have a rest period,which can not deal with explosive and persistent water mildew.As a green,non drug residue,low toxicity or non-toxic drug,Chinese herbal medicine has been widely used in food,chemical industry,clinical medicine and other fields.In addition,according to the provisions of relevant laws and documents,Chinese herbal medicine preparations in veterinary medicine belong to exempted substances,which are safe to the environment and have no rest period.Therefore,Chinese herbal medicine is also gradually used in aquaculture.Through the screening of Chinese herbal medicine against Saprolegnia,this study studied its anti Saprolegnia mechanism and compounded it,in order to prepare a compound drug with good anti Saprolegnia effect that can be used in production.It also provides reliable data support for the research of Chinese herbal medicine in other fields.1.Screening of Chinese herbal medicine resistant to Saprolegnia and preparation of compound drugs of Pseudolaricis cortex.In this study,the water extracts of more than 20 kinds of Chinese herbal medicines such as Rhus chinensis Mill,Pseudolaricis cortex,Prunus mume and Scutellariae Radix were tested in vitro against Saprolegnia parasitica.It was screened that Rhus chinensis Mill,Scutellariae Radix and Pseudolaricis cortex had good antibacterial effect on water mold,The minimum inhibitory concentrations were 0.488×10-3g/m L、0.781×10-2 g/m L and 0.015625 g/m L,respectively.On this basis,in vitro antibacterial tests were carried out on the above three single component active components of Chinese herbal medicine,including 8 kinds of Chinese herbal medicine active substances such as pseudolaric acid a,pseudolaric acid b,pseudolaric acid c,baicalin and gallic acid,and several single component substances with good antibacterial effect were screened through the experimental results,including baicalein,pseudolaric acid a,pseudolaric acid b andToosendanin minimum inhibitory concentrations was 0.125 mg/m L,0.0625 mg/m L,0.0625 mg/m L and 0.125 mg/m L,respectively.We determined the content of single component compounds in the Pseudolaricis cortex（purchased from Shaanxi Huike）by HPLC.The pseudolaric acid a、pseudolaric acid b and pseudolaric acid c are respectively;0.00663（μg/g）、0.04001（μg/g）、0.00662（μg/g）。Subsequently,the experimental team prepared the single component compound anti-Saprolegnia drugs in Chinese herbal medicine.The results are as follows:the single component active components of compound Chinese herbal medicine are calculated by weight percentage:Scutellaria baicalensis extract（baicalein 20%～40%）,Pseudolaricis cortex extract（pseudolaric acid a 20%～40%,pseudolaric acid b 20%～40%）,and extract of Me Lia toosendan Sieb（Toosendanin 10%～20%）.The test shows that the formula has obvious inhibitory effect on water mold（MIC is less than 0.0625 mg/m L）,and the MIC concentration is less than that of each component in the compound.Subsequently,the experimental team used these compound drugs to carry out in vitro bacteriostatic experiments on Saprolegnia strains ATCC,EC,02,04,C2 and BMY in different regions,and the mic concentration was less than 0.15mg/ml.This result shows that the above Chinese herbal compound drugs have broad-spectrum anti water mold effect.In the subsequent research,we used the data results of screening the active components of Chinese herbal medicine against Saprolegnia,and finally selected the water extract of Pseudolaricis cortex and ferrous sulfate as the main components,and carried out the orthogonal experiment of two factors and three levels with design expert 10 software,The two factors are the water extract of Vitex bark and ferrous sulfate,and the three levels are the different concentrations of each factor（2 MIC,MIC and 1/2 MIC respectively）.Through the analysis of orthogonal test data by design expert 10,the overall model shows significant differences,and the F values of Pseudolaricis cortex and ferrous sulfate are 0.8811 and 1.14 respectively.The F value of model is 3.90.There are significant differences.Lack of fit shows no significant difference,indicating that the experimental design is reasonable and the value is really effective.Then the optimal ratio of each component of the compound drug was measured by response surface analysis.The optimum ratio of Pseudolaricis cortex:ferrous sulfate heptahydrate=3:1.The MIC concentration of the compound drug prepared according to this proportion is 0.015 g/m L when it acts on the Saprolegnia strain.The formula can be used to prepare compound Chinese herbal medicine against Saprolegnia in production practice,and provide a theoretical basis for the development of new drugs.2.Pharmacodynamic study on the inhibition of Pseudolaricis cortex water extract on the growth of Saprolegnia.In our team’s in vitro inhibition experiment on the effect of Pseudolaricis cortex water extract on Saprolegnia spores,we found that the MIC and MBC concentrations of Saprolegnia spores treated by unearthed Pseudolaricis cortex water extract were1.25 g/L and 1.25 g/L.The morphological changes of Schizonepeta tenuifolia in different concentrations were revealed by scanning electron microscope and scanning electron microscope.In the initial observation of anatomical microscope,we believe that the inhibition of Pseudolaricis cortex water extract on the germination of Saprolegnia spores is permanent,but it is reversible for the growth of Saprolegnia hyphae.As long as there is a suitable growth environment,the hyphae can still grow.Interestingly,we found that this was not the case in the follow-up microscopic observation.In the later observation,we found that the water extract of Pseudolaricis cortex is also permanent to the growth of Saprolegnia filaments,but it does not depend on the subsequent growth environment.We also found that the water extract of Pseudolaricis cortex did not inhibit its germination by destroying the cell wall of water mold spores,but directly dissolved the cell structure inside the cell to inhibit its germination,which is different from the phenomenon of directly destroying the cell wall of Saprolegnia spores when traditional chemicals act on Saprolegnia spores,and this inhibitory effect is permanent.3.Genetic and metabolomic analysis of the inhibition mechanism of Pseudolaricis cortex water extract against SaprolegniaThis experiment is to analyze the metabolites produced by the 1.25 g/L water extract of Pseudolaricis cortex after acting on the mycelium of parasitic Saprolegnia,so as to explore the inhibition mechanism of water extract of Pseudolaricis cortex against Saprolegnia A total of 861 differential metabolites were screened out in this study,including 438 differential metabolites between the blank group and the experimental group,of which 272 differential metabolites showed an upward trend and166 differential metabolites showed an downward trend.Among them,there are 170KEGG functional notes,which are enriched in 44 pathways.We selected 10metabolites with the most significant difference to analyze the mechanism of water extract of Pseudolaricis cortex on Saprolegnia.We found that these metabolites are mainly related to immune system gene stability,gene transcription regulation,cell redox homeostasis,cell osmotic pressure,etc.we infer that the growth and reproduction of Saprolegnia are mainly inhibited by these effects.We also analyzed and discussed three metabolite pathways with significant differences（purine pathway,ABC transporter pathway and glycerol phospholipid pathway）.The results showed that the metabolites produced by these pathways were mostly related to cellular water and salt balance,inflammatory response,toxic substance enrichment,immune system and so on.Subsequently,we detected the content of five specific genes in Saprolegnia by q PCR.After the action of the drug,the expression of three genes,including spore capsule germination（kdo24919.1）,sugar absorption,sugar metabolism（kdo32861.1）and the main target of chemical drugs（kdo25246.1）,were significantly inhibited.The expression of the main target of chemical drugs（kdo25246.1）has increased.It is speculated that this gene may affect the mycelial target of Saprolegnia under the action of external drugs,so as to enhance the resistance of water mold to external drugs.The expression of five target genes in the 24-hour blank group was greater than that in the24-hour drug treatment group.The results showed that the water extract of Pseudolaricis cortex had different inhibitory effects on the expression of these five genes in Saprolegnia silk.4.Drug safety evaluationIn this part,the related safety evaluation of compound drugs of Pseudolaricis cortex was carried out.The main components of the compound medicine of Pseudolaricis cortex are water extract of Pseudolaricis cortex and ferrous sulfate（mass ratio is 3:1）.For grass carp,the acute toxicity test was carried out by using the compound drug of Pseudolaricis cortex.We obtained that the LC50of the compound drug at 24 h,48 h and 96 h were 9.260 g/L,7.745 g/L and 5.930 g/L,respectively,and the safe concentration was 0.593 g/L.Then histopathological sections were made on the intestine,liver,muscle,kidney and spleen of grass carp treated with different concentrations of compound Pseudolaricis cortex solution for 96 h.The results showed that the compound drug of Pseudolaricis cortex had certain toxicity at higher concentrations,mainly in the effect on the intestine and spleen of grass carp.Under the action of 3 g/m L water extract of Pseudolaricis cortex,the striated edge of the intestinal surface of grass carp was smooth,the arrangement of intestinal villi was sparse and short,and the structure of lymph nodes in the spleen was basically destroyed.Under the action of 6 g/m L concentration,the intestines were damaged,fell off and vacuolized seriously,and a large number of bleeding points appeared in the spleen.The preventive effect experiment of the compound drug of Pseudolaricis cortex showed that the compound drug of Pseudolaricis cortex with the content of 1.25 g/L had a better preventive effect on Saprolegnia.This experiment screened out the Chinese herbal medicine with good anti Saprolegnia effect-Pseudolaricis cortex,and studied its compound,and obtained the optimum formula.Subsequently,the mechanism of inhibition of Pseudolaricis cortex on the growth of Saprolegnia was revealed.The safety of the compound drug was evaluated,the stability of the compound drug was determined,and the safe concentration was obtained.Finally,it is concluded that the most effective compound drug composition ratio of Pseudolaricis cortex to control Saprolegnia is（Pseudolaricis cortex 1.25 g;ferrous sulfate 0.42 g）per liter of water. |
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