Application of fisetin in preparation of medicine for preventing and treating vibriosis of mariculture animals
By using lacquerflavin to inhibit the biofilm of Vibrio HAVI, the drug resistance and environmental pollution problems of Vibrio disease in marine aquaculture animals were solved, the survival rate of grouper was improved and the pathogenicity was reduced, and a kind of disease-causing problem was provided. Green, safe natural treatment options.
Patent Information
- Application Number
- CN202411394096.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-08
- Publication Date
- 2024-12-17
AI Technical Summary
Vibriovirus disease in marine aquaculture, especially Vibriovirus, has caused huge economic losses to the marine aquaculture industry. Existing prevention and treatment methods such as the use of antibiotics and chemicals have led to an increase in drug resistance, environmental pollution and public health security threats, and There is a lack of effective treatment options for natural compounds.
Using lacflavin as a natural drug, by preparing soaking liquid or different dosage forms combined with pharmaceutical carriers and excipients, the formation of Vitiligo biofilm is effectively inhibited, the survival rate of grouper is improved and the pathogenicity is reduced.
The lacquerflavin improves the survival rate of grouper at an effective dose, with a relative protection rate of 45% to 50%. It has the advantages of low residue, green and safe, industrial production, and significantly reduces the pathogenicity of Vitiligo Harveri. and drug resistance, showing potential as a virulence inhibitor of neopathogenic bacteria.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bacterial disease prevention and control of aquatic animals, relates to a new indication of fisetin, and specifically relates to application of fisetin in preparing a drug for preventing and treating vibriosis in marine aquaculture animals. Background Art
[0002] Vibrio disease, a disease caused by bacteria of the genus Vibrio, is currently widespread in marine aquaculture farms around the world. Vibrio disease is mostly characterized by rapid onset, strong infectivity, high morbidity and mortality, and has caused huge economic losses to the marine aquaculture industry. Among them, Vibrio harveyi is the main pathogen of vibrio disease. Vibrio harveyi can infect farmed animals such as Penaeus vannamei, turbot, and grouper. The main symptoms of infected fish include blackening of body color, protruding eyes, ulcers on the surface of the body, congestion of liver and spleen tissues, and enteritis. At present, the prevention and treatment of vibrio disease in marine aquaculture animals is still mainly based on immunity and antibiotics. Serological diversity and genetic variation limit the development of vibrio vaccines and reduce their application effect. The long-term use and abuse of antibiotics and chemical drugs make vibrio resistant to these drugs, the drug resistance spectrum continues to expand, and the drug effect continues to decline, causing farmers to have to increase the dosage of drugs, resulting in negative effects such as drug residues, environmental pollution, imbalance of the microecology of the aquaculture environment, and the spread of drug resistance factors to human and animal pathogens, which seriously threatens public health safety and public health. Natural compounds are secondary metabolites or endogenous active substances in organisms, and have the advantages of being green and safe, low residue, low toxicity, multifunctional and low drug resistance.
[0003] The pathogenicity of pathogenic microorganisms mainly depends on their virulence factors, including adhesion, colonization, infection, transmission, diffusion, immune escape and drug resistance. Biofilm is one of the main virulence factors of Vibrio harveyi. Vibrio harveyi survives in water bodies mainly in the form of biofilm formed by bacteria aggregation and adhesion to secrete sticky substances. The main components of its biofilm are polysaccharides, nucleic acids, lipids and proteins. The functions of Vibrio harveyi biofilm mainly include: enhancing the resistance of Vibrio harveyi to antibiotics, external environmental pressure and host immunity; reducing the nutritional consumption of Vibrio harveyi and prolonging its survival time; enhancing the response ability of Vibrio harveyi to external environmental conditions such as pH, osmotic pressure, nutrition and gas exchange; improving Vibrio harveyi's bacterial resistance to drugs and body immunity. Studies have shown that the resistance rate of bacteria on biofilms is as high as 1000 times that of planktonic bacteria. Therefore, inhibiting the formation of Vibrio harveyi biofilm or degrading the formed Vibrio harveyi biofilm is an important strategy to reduce its pathogenicity and drug resistance. Summary of the invention
[0004] The technical problem solved by the invention is to provide a natural medicine for preventing and treating Vibrio harveyi disease in marine cultured animals, namely, fisetin.
[0005] The present invention has found that fisetin can be used in the preparation of drugs for preventing and treating Vibrio disease in marine aquaculture animals.
[0006] In one embodiment of the present invention, the pathogenic bacteria of Vibrio disease in marine aquaculture animals is Vibrio harveyi.
[0007] In one embodiment of the present invention, the marine cultured animal is grouper. In a specific embodiment, the marine cultured animal is pearl grouper.
[0008] In one embodiment of the present invention, fisetin is prepared into a soaking liquid or different dosage forms of medicines are prepared by combining fisetin with pharmaceutically acceptable carriers and excipients.
[0009] In one embodiment of the present invention, the preparation method of the soaking solution includes: weighing an appropriate amount of fisetin, dissolving it with anhydrous ethanol, and adding it to seawater to make the final concentration of fisetin 32 μg / mL or 64 μg / mL, and the final concentration of anhydrous ethanol is 0.5%.
[0010] In one embodiment of the present invention, the effective therapeutic concentration of the fisetin soaking solution is 32 μg / mL.
[0011] Compared with the prior art, the present invention has the following beneficial effects:
[0012] 1. The present invention is the first to use fisetin as a drug for medicinal baths to prevent and treat Vibrio disease in marine aquaculture animals, especially Vibrio harveyi disease in grouper. Within an effective dose, the survival rate of grouper infected with Vibrio harveyi can be increased, and the relative protection rate reaches 45% to 50%.
[0013] 2. The fisetin in the present invention has the advantages of wide source, low residue, no pollution, green and safe, industrial production process, controllable quality, stable efficacy, etc.
[0014] 3. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the fisetin for preventing and treating Vibrio disease in marine aquaculture animals disclosed in the present invention against Vibrio harveyi are 64 μg / mL and 256 μg / mL respectively. The median effective concentration (EC 50 ) is 9.53±1.32μg / mL. This means that at the appropriate dose, Fisetin can greatly reduce the pathogenicity of Vibrio harveyi without killing the bacteria, and control its infection without inducing bacterial survival pressure to produce drug-resistant mutations. Fisetin is expected to be used as a lead compound for the development of new pathogen virulence inhibitors, effectively solving the current problem of drug resistance of Vibrio and even bacterial diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is a diagram of the growth time and biofilm formation process of Vibrio harveyi in Example 1.
[0016] Figure 2 This is a graph showing the effect of different concentrations of fisetin on the biofilm formation of Vibrio harveyi in Example 1.
[0017] Figure 3 This is a graph showing the effect of different concentrations of fisetin on the removal of Vibrio harveyi biofilm in Example 1.
[0018] Figure 4 This is a symptom diagram of pearl grouper infected with Vibrio harveyi in Example 3, in which A: enlarged head and kidney; B: red and swollen liver; C: enlarged spleen; D: urethral effusion.
[0019] Figure 5 This is a survival curve diagram of pearl grouper infected with different concentrations of Vibrio harveyi in Example 3.
[0020] Figure 6 This is a graph showing the effect of the fisetin bath in Example 3 on the bacterial load in the liver of pearl grouper infected with Vibrio harveyi.
[0021] Figure 7 This is a graph showing the effect of the fisetin bath in Example 3 on the bacterial load in the spleen of pearl gentian fish infected with Vibrio harveyi.
[0022] Figure 8 This is a graph showing the effect of the fisetin medicated bath in Example 3 on the bacterial load in the kidneys of pearl grouper infected with Vibrio harveyi.
[0023] Fig. 9 This is a graph showing the effect of the fisetin bath on the survival curve of pearl grouper infected with Vibrio harveyi in Example 3. DETAILED DESCRIPTION
[0024] Fisetin, also known as fisetin, molecular formula: C 15 H 10 O 6 , a yellow needle-shaped crystalline compound, is a natural flavonol found in many fruits and vegetables. The structure of fisetin is as follows:
[0025]
[0026] Fisetin has strong pharmacological activities such as antiviral, anti-angiogenesis, neuroprotection, and induction of cancer cell apoptosis due to the ortho-diphenolic hydroxyl group on the B ring, and has the efficacy of treating yellow anthrax viral hepatitis and human melanoma. The present invention found that fisetin can significantly inhibit the formation of biofilm of pathogenic factors of Vibrio harveyi, and has great application potential in the prevention and treatment of vibriosis.
[0027] In one embodiment of the present invention, the pathogenic bacteria of Vibrio disease in marine aquaculture animals is Vibrio harveyi.
[0028] In one embodiment of the present invention, the marine cultured animal is grouper. In a specific embodiment, the marine cultured animal is pearl grouper (Epinephelus fuscoguttatus♀×E.lanceolatus♂).
[0029] The present invention has found that fisetin can effectively inhibit the formation of biofilm, a key virulence factor of Vibrio harveyi. Fisetin can effectively inhibit or eliminate the formation of biofilm, a key virulence factor of Vibrio harveyi. Through the administration method such as soaking in a medicinal bath, fisetin can significantly improve the survival rate of pearl gentian grouper artificially infected with Vibrio harveyi.
[0030] In one embodiment of the present invention, fisetin is prepared into a soaking liquid or different dosage forms of medicines are prepared by combining fisetin with pharmaceutically acceptable carriers and excipients.
[0031] In one embodiment of the present invention, the preparation method of the soaking solution includes: weighing an appropriate amount of fisetin, dissolving it with anhydrous ethanol, and adding it to seawater to make the final concentration of fisetin 32 μg / mL or 64 μg / mL, and the final concentration of anhydrous ethanol is 0.5%.
[0032] The fisetin is a fisetin compound with a purity of 98%.
[0033] In one embodiment of the present invention, the effective therapeutic concentration of the fisetin soaking solution is 32 μg / mL.
[0034] The present invention is further described by the following specific examples, but the embodiments of the present invention are not limited thereto. The pearl grouper used in the examples was purchased from a farm in Wenchang City, Hainan Province. Vibrio harveyi is a pathogen isolated and preserved from diseased pearl grouper in Hainan, and was identified as Vibrio harveyi by morphology and 16S rDNA molecular biology methods.
[0035] Example 1 Effect of Fisetin on Vibrio harveyi Biofilm Formation
[0036] 1. Preparation of Vibrio harveyi Suspension
[0037] After the activation of the Vibrio harveyi strain stored at -80℃, inoculate it with a new lysing broth (Luria-Bertani, LB) liquid culture medium at a 1% inoculum volume and culture it overnight at 30℃ and 180r / min. Centrifuge at 5000r / min for 5min to collect the bacteria, wash them with PBS (0.01M, pH 7.4), centrifuge again (5000r / min, 5min) to collect the bacteria, wash them, and centrifuge and wash them 3 times. Finally, resuspend the bacterial suspension with PBS to adjust the concentration to OD 600nm = 1, the bacterial concentration is about 1×10 9 CFU / mL, as the test bacterial suspension, stored at 4℃ for later use.
[0038] 2. Preparation of Fisetin Solution
[0039] 10 mg of fisetin was weighed and dissolved in 1 mL of dimethyl sulfoxide (DMSO, analytical grade) to prepare a fisetin mother solution with a concentration of 10 mg / mL. After sterilization by filtration through a 0.22 μm microporous filter membrane, 2.5 μL of the fisetin mother solution was added to 5 mL of high-pressure sterilized LB liquid culture medium to prepare an LB liquid culture medium containing a final concentration of 10 μg / mL fisetin and 0.1% DMSO.
[0040] 3. Vibrio harveyi Growth and Biofilm Formation
[0041] After adding 195 μL of LB liquid culture medium to a 96-well cell culture plate, 5 μL of the bacterial suspension (1×10 9 CFU / mL) and mix well. The total volume of each well is 200 μL. The final concentration of bacterial solution in each well of the 96-well plate is about 2.5×10 7 CFU / mL. Sterile water was added around the 96-well plate and cultured for 4, 8, 16, 24, 36, 48, 60, and 72 hours respectively. After the culture was completed, the LB culture medium in the well was sucked out with a pipette, and each well was gently washed three times with sterile PBS (0.01M, pH 7.4) to wash away the floating bacteria, and the 96-well plate was placed in a 55°C oven for 30 minutes to dry. 200 μL of 0.1% crystal violet stain was added to each well, and the biofilm was evenly stained at room temperature on a decolorizing shaker at low speed (100 r / min) for 10 minutes. Use a pipette to suck out the dye from each well, wash off the residual dye with double distilled water, wash three times, and place the 96-well plate in a 55°C oven to dry for 30 minutes. After complete drying, 200 μL of 33% glacial acetic acid was added to each well. After decolorization for 15 min on a decolorization shaker (100 r / min), the absorbance of each well at 570 nm was measured using an ELISA reader. Each group had six parallels, and the negative control was LB solution.
[0042] The results are as follows Figure 1 As shown in the figure, biofilm of Vibrio harveyi began to form after 4 hours of culture, and the amount of biofilm formation reached a peak after 48 hours. As the culture time increased, the amount of biofilm began to decrease. 24h to 48h was the logarithmic formation period of biofilm of Vibrio harveyi. Therefore, this study selected 36h as the optimal time point for biofilm formation.
[0043] 4. Effects of different concentrations of fisetin on biofilm formation of Vibrio harveyi.
[0044] The 10 mg / mL fisetin solution prepared in step 2 of this example was diluted with sterile LB solution to obtain LB solutions containing final concentrations of 1, 2, 4, 8, 16, 32, and 64 μg / mL fisetin. 5 μL of the bacterial suspension prepared in Example 1.1 (about 1×10 9 CFU / mL), 195 μL of LB liquid culture medium containing final concentrations of 1, 2, 4, 8, 16, 32, and 64 μg / mL fisetin, a circle of sterile water was filled around the well plate, and cultured at 30°C for 36 hours, the culture medium was aspirated, and the wells were gently washed three times with sterile PBS (0.01M, pH7.4), and the 96-well plate was placed in a 55°C oven for 30 minutes for drying, 200 μL of 0.1% crystal violet dye solution was added, and the biofilm was uniformly stained for 10 minutes at room temperature on a decolorizing shaker at a low speed (100 r / min). The excess dye in each well was aspirated with a pipette, rinsed three times with double distilled water, and the 96-well plate was placed in a 55°C oven for about 30 minutes for drying, and 200 μL of 33% glacial acetic acid was added to each well, and decolorized for 15 minutes on a decolorizing shaker (100 r / min), and the absorbance value of each well at 570 nm was measured using an enzyme marker. Six parallels were set up in each group. The blank control group consisted of 5 μL bacterial suspension and 195 μL LB solution per well, and the negative control was 5 μL PBS and 195 μL LB solution. The following formula was used to calculate the inhibition rate of each concentration group of fisetin on Vibrio harveyi biofilm. The Logistic fitting equation in the nonlinear curve fitting tool in Origin 2021 software was used to fit the data of inhibition rate changing with fisetin concentration, and the half inhibition concentration (EC50) of fisetin on Vibrio harveyi biofilm was calculated. 50 ).
[0045] Biofilm inhibition rate calculation formula: IRBF i =(1-OD 570nmi / OD 570nm0 )×100%
[0046] Where, IRBF i is the biofilm formation inhibition rate, OD 570nm0 OD of blank control 570nm , O.D. 570nmi is the OD of the fisetin group 570nm.
[0047] The results of inhibition rate of each concentration of fisetin on Vibrio harveyi biofilm are as follows Figure 2 As shown, the EC of fisetin on Vibrio harveyi biofilm 50 is 9.53±1.32μg / mL, y=0.90-0.76 / (1+(x / 9.53) 1.22 )(R 2 =0.99).
[0048] 5. The ability of fisetin to remove Vibrio harveyi biofilm
[0049] The 10 mg / mL fisetin mother solution prepared in step 2 of Example 1 was diluted with sterile PBS (0.01M, pH 7.4) to obtain PBS containing final concentrations of 0, 8, 16, 32, and 64 μg / mL fisetin. 5 μL of the bacterial suspension prepared in step 1 of Example 1 and 195 μL of LB liquid culture medium were added to the wells of a 96-well plate, and the biofilm was formed at the bottom and wall of the wells at 30°C for 36 hours. The culture solution was removed, and 200 μL of PBS containing final concentrations of 0, 8, 16, 32, and 64 μg / mL fisetin were added respectively, and the culture was continued at 30°C for 6 hours. The amount of Vibrio harveyi biofilm was detected using crystal violet according to the method in step 4 of Example 1.
[0050] Biofilm removal rate calculation formula: ERBF i =(1-OD 570nmi / OD 570nm0 )×100%
[0051] Where, ERBF i is the removal rate of the formed biofilm, OD 570nm0 OD of blank control 570nm The absorbance value, OD 570nmi is the OD of the fisetin group 570nm The absorbance value.
[0052] The results are as follows Figure 3 As shown in the figure, the clearance rates of 8, 16, 32 and 64 μg / mL fisetin on Vibrio harveyi biofilm were 12.9
[0053] ±4.16%, 18.71±4.06%, 44.21±0.53% and 73.58±1.06%.
[0054] Example 2 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of fisetin against Vibrio harveyi
[0055] The MIC and MBC of fisetin against Vibrio harveyi were determined according to the broth microdilution method in the Microbiological Clinical Laboratory Standards and Operation Specifications (M07-A9) established by the National Committee for Clinical Laboratory Standards (NCCLS, 2009).
[0056] 1. Inoculate Vibrio harveyi by streak isolation on hydrolyzed casein medium (Mueller-Hinton, MH) solid medium and culture at 30°C overnight.
[0057] 2. Weigh 20 mg of fisetin and dissolve it in 1 mL of DMSO (analytical grade) to prepare a 20 mg / mL fisetin mother solution. Pipette 128 μL of the fisetin mother solution and add it to 5 mL of MH liquid culture medium to obtain a MH liquid culture medium containing a final concentration of 512 μg / mL fisetin. Use the two-fold dilution method to dilute with MH liquid culture medium to obtain MH liquid culture medium with final concentrations of 512, 256, 128, 64, 32, 16, 8, 4, and 2 μg / mL fisetin for the experiment.
[0058] 3. Pick a single colony and inoculate it in LB liquid culture medium, shake and culture it at 30℃, 180r / min overnight. Collect the bacterial solution, centrifuge it at 5000r / min for 5min, remove the supernatant, resuspend the collected bacteria in PBS, centrifuge it again, collect the bacteria by centrifugation, wash and resuspend them, repeat the operation 3 times, and adjust the OD 600nm =1(about 1×10 9 CFU / mL). The bacterial suspension at this time was centrifuged at 5000r / min for 5min, the supernatant was discarded, and the suspension was resuspended with an equal volume of MH liquid culture medium. 200μL of MH liquid culture medium containing 512, 256, 128, 64, 32, 16, 8, 4, and 2μg / mL of fisetin was added to each well, and the bacterial solution prepared above was inoculated at 1%. At the same time, a negative control group of 200μL of culture medium containing the above concentrations of fisetin was set up, and a positive control group inoculated with 1% bacterial suspension in 200μL MH liquid culture medium was set up, and six replicates were set up for each group. After the sample preparation was completed, it was statically cultured at 30℃ for 16h, and the 96-well culture plate was taken out. The minimum concentration of fisetin in the well that could completely inhibit bacterial growth as seen by the naked eye was recorded as MIC.
[0059] Based on the MIC, 100 μL of sample solution was drawn from each well and evenly coated on thiosulfate citrate bile salts sucrose agar culture medium (TCBS) at 30°C for 12 hours. The concentration on the solid culture medium from low to high was the concentration at which no bacteria were found on the first solid culture medium as the MBC. The results showed that the MIC and MBC of fisetin against Vibrio harveyi were 64 μg / mL and 256 μg / mL, respectively.
[0060] Example 3 The therapeutic effect of fisetin on Vibrio harveyi infection in pearl grouper
[0061] 1. Preparation of injectable bacterial suspension: refer to the preparation of Vibrio harveyi suspension in step 1 of Example 1.
[0062] 2. Establishment of the model of pearl grouper infected with Vibrio harveyi
[0063] 120 healthy pearl groupers with a body length of 14±2cm and a body weight of 37.1±4.3g were selected and randomly divided into 10 aquariums (125L, 0.5m×0.5m×0.5m), 12 in each tank, and the 10 tanks were randomly divided into 4 experimental groups and 1 control group (see Table 1), with two parallel aquariums in each group, and temporarily reared for 7 days. Basic feed was fed during the temporary rearing period and the entire experimental period. The water temperature of the aquarium was maintained at 28±2℃ and the dissolved oxygen was greater than 6.5mg / L. The water was changed every 2 days, and 1 / 2 of the water was changed each time. After the experiment started, the groupers were fasted for more than 12 hours, and different doses of Vibrio harveyi suspension (see Table 1) were injected intraperitoneally into the pearl groupers. The challenge doses of each group are shown in the table below. After injection, the groupers were still reared for 14 days according to the temporary rearing. The activity of the experimental fish was observed, the number of fish deaths in each group was counted every day, and the dead fish were fished out in time, and the pathogens were re-isolated for the fish that had just died. Remove the remaining feed 1 hour after each feeding.
[0064] Table 1 Establishment of the disease model of Vibrio harveyi
[0065] Group (n=2) Challenge dose feed Control group PBS (0.01 M, pH 7.4) Basic feed Experimental Group 1 <![CDATA[2.7×10 5 CFU / kg]]> Basic feed Experimental Group 2 <![CDATA[2.7×10 6 CFU / kg]]> Basic feed Experimental Group 3 <![CDATA[2.7×10 7 CFU / kg]]> Basic feed Experimental Group 4 <![CDATA[2.7×10 8 CFU / kg]]> Basic feed
[0066] Note: Injection volume 0.1mL / 37.1g.
[0067] Symptoms of pearl grouper after artificial infection Figure 4As shown, the liver, spleen, and kidney of fish infected with Vibrio harveyi were swollen, and there was urethral effusion in the abdominal cavity. The results of continuous observation for 14 days found that the fish in the experimental group breathed rapidly at the bottom of the aquarium after injection, swam slowly, had a slow stress response, and lost their appetite. The control group did not eat on the day of injection, but started eating on the second day. The experimental group did not eat for 1 to 4 days after injection, but started eating on the fifth day. After the onset of the disease, white feces appeared in the tank, the fish's abdominal cavity was swollen, and some fish had ulcers on their bodies.
[0068] The survival curves of groupers in each group after infection are as follows Figure 5 The relationship between mortality and bacterial concentration was fitted according to the death situation, and the equation was y = 0.32 × lg (x) -1.61 (R2 = 0.98). The LD of grouper was obtained from the equation: 50 4.6×10 6 CFU / kg, LD 80 4.1×10 7 CFU / kg. Pathogen identification results: The pathogens re-isolated from pearl grouper were all confirmed to be Vibrio harveyi by 16S-rDNA detection.
[0069] 3. Effect of fisetin bath on bacterial load in pearl grouper infected with Vibrio harveyi
[0070] Sixty groupers (12±2 cm, 39±2 g) were randomly selected from the temporary culture groupers and injected with 4.6×10 6 CFU / kg dose of Vibrio harveyi suspension, put it into the corresponding aquarium (125L, 0.5m×0.5m×0.5m), and after 6 hours, randomly divided into blank control group, 32μg / mL fisetin soaking group, 64μg / mL fisetin soaking group, 20 fish in each group. Then each group prepared a corresponding soaking bucket (80L), the blank control group bucket contained 20L seawater, the 32μg / mL fisetin bucket contained 20L 32μg / mL fisetin seawater, and the 64μg / mL fisetin bucket contained 20L 64μg / mL fisetin seawater. After soaking for 1 hour, they were fished out and put into their own clean seawater aquariums, and then they were bathed at 9 am every day, each time for 1 hour. The time of fish removal after the first medicated dip was recorded as 0 h. At 3 h, 6 h, 12 h, and 24 h after the medicated dip, 5 fish were selected from each group, and the three immune tissues of liver, spleen, and head kidney were placed in pre-labeled tubes, weighed, and washed with PBS to make tissue homogenate. After dilution, 100 μL of the tissue homogenate was spread on an LB solid plate containing 0.1% ampicillin, and cultured at 30 °C for 12 h, and the number of single colonies was calculated.
[0071] The bacterial loads in the liver, spleen, and head kidney at different time points are as follows Figure 6 , 7 As shown in Figures 3 and 8, the bacterial colony counts in each tissue at each time point after medicated bathing were significantly lower than those in the control group.
[0072] 4. The efficacy of fisetin bath on Vibrio harveyi infection in pearl grouper
[0073] 120 healthy groupers with a body length of 14±3cm and a body weight of 61.5±5g were selected and moved into a barrel (80L) for grouping. A blank group, a positive control group, a 32μg / mL fisetin bath group, and a 64μg / mL fisetin bath group were set up. The specific groupings are shown in Table 2. Each group has two parallel barrels, with 15 fish in each barrel. After feeding with the basic feed for 7 days to adapt to the environment, after the adaptive feeding, the LD of Vibrio harveyi was used. 80 (4.1×10 7 CFU / kg) was used to evaluate the effects of two concentrations of fisetin (32 and 64 μg / mL) on the survival rate of grouper after Vibrio harveyi infection.
[0074] RPS=[1-(P F / P CON )]×100%
[0075] Where: RPS is the relative protection rate; P F is the mortality rate of fisetin bath group; P CON is the mortality rate of the control group.
[0076] Table 2 Treatment effect of fisetin bath on Vibrio harveyi infection in pearl grouper
[0077]
[0078]
[0079] Note: Injection volume 100 μL / 61.5 g.
[0080] Survival curves are shown in Fig. 9 , the fisetin bath has a good therapeutic effect on Vibrio harveyi infection in pearl gentian grouper. The RPS of each group was calculated according to the formula, and the results are as follows:
[0081] Table 3 Treatment effect of fisetin bath on Vibrio harveyi infection in pearl grouper
[0082] Group Death amount (number) Cumulative mortality rate (%) Relative protection rate (%) Blank group 0 0 - Positive control group 20 66.67 - 32 μg / mL 10 33.33 50 64 μg / mL 11 36.67 45
[0083] It can be seen that when the concentration is 32μg / mL, the relative protection rate against Vibrio harveyi infection in grouper is 50%, and when the concentration is 64μg / mL, the relative protection rate against Vibrio harveyi infection in grouper is 45%. The recommended effective therapeutic concentration of fisetin is 32μg / mL.
Claims
1. Application of fisetin in the preparation of drugs for preventing and treating Vibrio disease in marine aquaculture animals.
2. The use of fisetin according to claim 1 in the preparation of a preparation for preventing and treating vibriosis in marine aquaculture animals, characterized in that the pathogenic bacteria of vibriosis in marine aquaculture animals is Vibrio harveyi.
3. Use of fisetin according to claim 1 in the preparation of a preparation for preventing and treating Vibrio disease in marine aquaculture animals, Features: The marine cultured animal is grouper.
4. Use of fisetin according to claim 3 in the preparation of a preparation for preventing and treating Vibrio disease in marine aquaculture animals, Features: The marine cultured animal is pearl grouper.
5. Use of fisetin according to claim 1 in the preparation of a preparation for preventing and treating Vibrio disease in marine aquaculture animals, Features: The fisetin is prepared into a soaking liquid or different dosage forms of medicines are prepared by combining the fisetin with pharmaceutically acceptable carriers and excipients.
6. Use of fisetin according to claim 5 in the preparation of a preparation for preventing and treating Vibrio disease in marine aquaculture animals, Features: The preparation method of the soaking solution includes: weighing an appropriate amount of fisetin, dissolving it with anhydrous ethanol, and adding it into seawater to make the final concentration of fisetin 32μg / mL or 64μg / mL, and the final concentration of anhydrous ethanol is 0.5%.
7. Use of fisetin according to claim 5 in the preparation of a preparation for preventing and treating Vibrio disease in marine aquaculture animals, Features: The effective therapeutic concentration of fisetin immersion solution is 32μg / mL.