Traditional Chinese medicine composition for fish enteric disease, preparation method and application thereof

Abstract

The present application relates to the technical field of aquatic disease prevention and control and traditional Chinese veterinary medicine, and particularly relates to a traditional Chinese medicine composition for preventing and treating fish enteritis caused by infection of Aeromonas veronii, a preparation method and application thereof.The traditional Chinese medicine composition provided by the present application is prepared by water extraction of Apocynum venetum, Schisandra chinensis and Scutellaria baicalensis in proportion, and only contains three raw medicinal materials, and the preparation process is simple.The in vitro test results show that the traditional Chinese medicine composition has antibacterial activity on Aeromonas veronii, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas sobria and Edwardsiella tarda;the oral administration safety evaluation results show that no experimental fish dies or shows obvious abnormality in each group with a total equivalent concentration of 500, 1000 and 2000 mg / mL of the raw medicinal materials under the administration conditions in the present example;and the infection model efficacy evaluation results by using 1000 mg / mL of the traditional Chinese medicine composition show that the traditional Chinese medicine composition has the effects of reducing the bacterial load of infected fish tissues and alleviating the pathological damage of intestinal tissues.

Description

Technical Field

[0001] This invention relates to the field of aquatic disease prevention and control and traditional Chinese veterinary medicine, specifically to a traditional Chinese medicine composition for preventing and treating enteritis in fish caused by Aeromonas verrucosa infection, its preparation method and application. Background Technology

[0002] Aeromonas vilvae ( Aeromonas veronii Bacterial septicemia (BSS) is a common opportunistic pathogen in aquaculture, causing enteritis, septicemia, and multi-tissue damage in fish. Infected fish often exhibit symptoms such as decreased appetite, abnormal swimming, anal swelling, ascites, intestinal congestion or inflammation, and in severe cases, can lead to mortality and economic losses. Currently, antibiotics remain an important means of preventing and controlling bacterial diseases in aquaculture; however, long-term or improper use can easily lead to problems such as drug resistance, drug residues, and water pollution.

[0003] Developing traditional Chinese medicine (TCM) compositions with clearly defined sources, good safety profiles, and suitability for oral administration is of significant practical importance for reducing antibiotic dependence and improving the health of aquatic animals. Existing TCM compound formulas are mostly formulated from the perspectives of clearing heat and detoxifying, inhibiting bacteria and reducing inflammation. However, for fish enteritis caused by Aeromonas verrucosum infection, there is still a need for TCM compositions with fewer components, simpler preparation processes, and mutually supportive in vitro and in vivo evidence.

[0004] Apocynum venetum is one of the medicinal plants with unique resources in Xinjiang, possessing regional resource advantages in terms of raw material sources; however, its application in combination with other traditional Chinese medicines for treating enteritis in fish caused by Aeromonas verrucosa infection still requires further research.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a traditional Chinese medicine composition for preventing and treating enteritis in fish caused by Aeromonas verrucosa infection, its preparation method, and its application.

[0007] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a traditional Chinese medicine composition for preventing and treating enteritis caused by Aeromonas verrucosa infection, wherein the traditional Chinese medicine composition is prepared by water extraction of Apocynum venetum, Schisandra chinensis and Scutellaria baicalensis in a dry raw material mass ratio of 3~5:1~3:0.5~2.

[0008] Preferably, the traditional Chinese medicine composition is prepared by water extraction of Apocynum venetum, Schisandra chinensis and Scutellaria baicalensis in a dry raw material mass ratio of 4:2:1.

[0009] Preferably, the equivalent concentration of the raw medicinal materials in the traditional Chinese medicine composition is 500~2000 mg / mL, based on the total amount of raw medicinal materials used.

[0010] More preferably, the equivalent concentration of the raw medicinal materials in the traditional Chinese medicine composition is 1000 mg / mL, based on the total amount of raw medicinal materials used.

[0011] Secondly, the present invention provides a method for preparing the traditional Chinese medicine composition, comprising: weighing each raw medicinal material according to weight parts, placing it in a solvent to extract the effective components, and obtaining an extract; wherein the solvent is water.

[0012] Preferably, the method includes: weighing Apocynum venetum, Schisandra chinensis and Scutellaria baicalensis at a dry raw material mass ratio of 3~5:1~3:0.5~2 (more preferably 4:2:1), pulverizing and mixing them, adding water for a first extraction, and filtering; adding water to the residue for a second extraction, and filtering; combining the two extracts, centrifuging, filtering, concentrating and adjusting the volume to obtain the traditional Chinese medicine composition.

[0013] Preferably, the material-to-liquid ratio for each extraction is 1g:10±2mL, more preferably 1:10 (g / mL), based on the total mass of Apocynum venetum, Schisandra chinensis and Scutellaria baicalensis. The first extraction is performed by soaking at room temperature for 30±5min and then reflux extraction at 95±2℃ for 2.0±0.5h. The second extraction is performed by reflux extraction at 95±2℃ for 1.0±0.5h.

[0014] More preferably, based on the total amount of raw medicinal materials used, the combined extract is concentrated and diluted to a concentration equivalent to the raw medicinal materials of 500~2000 mg / mL.

[0015] Thirdly, the present invention provides the application of the traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the preparation method in the preparation of products for the prevention and treatment of enteritis in fish caused by Aeromonas verrucosa infection.

[0016] Preferably, the product is used to reduce the bacterial load of Aeromonas versicolor in the intestines, liver and / or kidneys of infected fish, or to alleviate intestinal mucosal damage caused by Aeromonas versicolor infection in fish.

[0017] Furthermore, the in vitro antibacterial test results showed that the traditional Chinese medicine composition exhibited antibacterial activity against Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas temperate, and Edwardsiella tarda detected in this embodiment.

[0018] More preferably, the product for preventing and treating enteritis in fish caused by Aeromonas verrucosa infection is an oral administration formulation.

[0019] Preferably, the oral administration formulation is an oral liquid.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The traditional Chinese medicine composition of the present invention is prepared by water extraction of Apocynum venetum, Schisandra chinensis and Scutellaria baicalensis in a specific mass ratio of dried raw materials. It contains only three raw materials and the formulation and preparation steps are clear.

[0021] (2) The results of in vitro tests show that the traditional Chinese medicine composition of the present invention exhibits antibacterial activity against Aeromonas versicolor, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas tempera and Edwardsiella tarda detected in this embodiment, and shows a significant concentration-dependent inhibitory effect on Aeromonas versicolor.

[0022] (3) The results of single herb screening, MIC / MBC determination, FIC compatibility evaluation, formula composition deletion verification and component ratio screening support that Apocynum venetum, Schisandra chinensis and Scutellaria baicalensis are the three herbs that together form the basic formula; further verification results of the total raw material equivalent ratio show that within the concentration and ratio range investigated in this example, Apocynum venetum, Schisandra chinensis and Scutellaria baicalensis in a ratio of 4:2:1 show better antibacterial effect.

[0023] (4) The safety evaluation results of oral administration showed that none of the groups with total equivalent concentrations of the original medicinal material of 500, 1000 and 2000 mg / mL caused death or obvious clinical abnormalities in the experimental fish under the administration conditions of this example; histological examination of the highest tested concentration of 2000 mg / mL group showed no obvious pathological damage in the main tissues. The efficacy evaluation results of the infection model using the 1000 mg / mL traditional Chinese medicine composition showed that the mortality rate of this group during the observation period was lower than that of the infection model group, and the bacterial load in the tissues and the pathological damage in the intestinal tissues were significantly reduced. Attached Figure Description

[0024] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments are described below.

[0025] Figure 1 The images show the colony morphology of Aeromonas verrucosa in Example 2 of this invention on RYAN medium and blood agar medium.

[0026] Figure 2 This is a time-inhibition curve of the traditional Chinese medicine composition in Example 4 of the present invention against Aeromonas vulgaris.

[0027] Figure 3 HE staining images of crucian carp intestines, liver, and kidneys during the oral administration safety test of the traditional Chinese medicine composition in Example 5 of this invention; the top row is the blank control group, and the bottom row is the group with a total equivalent concentration of 2000 mg / mL of the original medicinal material. The scale bar in the intestine, liver, and kidney images is 100 μm.

[0028] Figure 4 The figure shows the effect of the traditional Chinese medicine composition in Example 6 of the present invention on the survival rate of fish infected with Aeromonas vulgaris; the “traditional Chinese medicine treatment group” in the figure is the “traditional Chinese medicine composition group” mentioned in the text.

[0029] Figure 5This is a graph showing the effect of the traditional Chinese medicine composition in Example 6 of the present invention on the cumulative mortality rate and relative protection rate of fish infected with Aeromonas vulgaris; where RPS represents the relative protection rate, and the "traditional Chinese medicine treatment group" in the graph is the "traditional Chinese medicine composition group" mentioned in the text.

[0030] Figure 6 The figure shows the effect of the traditional Chinese medicine composition in Example 6 of this invention on the bacterial load of Aeromonas verrucosa infection in the liver, intestine and kidney tissues of fish; in the figure, "challenge group" and "traditional Chinese medicine treatment group" correspond to "infection model group" and "traditional Chinese medicine composition group" in the main text, respectively; CFU / g of each sample was calculated based on the average of the colony counts of 3 parallel plates, and the results were expressed as mean ± standard deviation (x̄±SD, n=5) after log10 transformation.

[0031] Figure 7 The figure shows the effect of the traditional Chinese medicine composition in Example 6 of the present invention on the improvement of intestinal tissue pathological damage in fish infected with Aeromonas vulgaris; the figure shows the blank control group, the infection model group and the traditional Chinese medicine composition group in order, and the scale bar in the figure is 100 μm. Detailed Implementation

[0032] This invention provides a traditional Chinese medicine composition for the prevention and treatment of enteritis in fish caused by Aeromonas verrucosa infection, in order to solve the problems of strong reliance on antibacterial drugs and insufficient green prevention and control products in the existing prevention and treatment of bacterial enteritis in aquatic animals.

[0033] In one more specific embodiment, the traditional Chinese medicine composition provided by the present invention is prepared by mixing Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis in a dry raw material mass ratio of 4:2:1 and then extracting them together with water. The mass ratio is obtained by simplifying the dry raw material equivalent concentration relationship of 62.5:31.25:15.625 corresponding to the optimal factor level combination in the component proportion screening, and is further investigated through subsequent proportion verification under the condition of equal total raw material equivalent. The total equivalent concentration of raw materials referred to in this application refers to the ratio of the total mass of the three dried raw materials to the final volume of the traditional Chinese medicine composition; the equivalent concentration of each individual herb refers to the ratio of the corresponding dry raw material mass to the final volume.

[0034] The present invention also provides a method for preparing the above-mentioned traditional Chinese medicine composition, the method comprising the steps of weighing three raw medicinal materials in a dry raw material mass ratio of 4:2:1, pulverizing and mixing, soaking, reflux extraction twice, filtering, concentration and volume adjustment.

[0035] This invention also provides the application of the above-mentioned traditional Chinese medicine composition in the preparation of an oral formulation for the prevention and treatment of enteritis in fish caused by Aeromonas versicolor infection. Results from single-herb screening, MIC / MBC determination, FIC compatibility evaluation, formulation reduction verification, component ratio screening, in vitro antibacterial evaluation, safety evaluation, and efficacy evaluation in infection models indicate that the traditional Chinese medicine composition has an inhibitory effect on Aeromonas versicolor and can reduce the bacterial load in infected fish tissues and alleviate pathological damage to intestinal tissues.

[0036] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0037] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0038] Example 1: Preparation of a traditional Chinese medicine composition.

[0039] 40 g of Apocynum venetum leaves, 20 g of Schisandra chinensis, and 10 g of Scutellaria baicalensis were weighed out and purchased from Xinjiang Baicaotang Pharmaceutical Chain Distribution Co., Ltd. The Apocynum venetum leaves are from the Apocynaceae family. Apocynum venetum The dried leaves of L.; Schisandra chinensis is the fruit of the Schisandra chinensis plant in the Magnoliaceae family. Schisandra chinensis The dried, ripe fruit of (Turcz.) Baill.; Scutellaria baicalensis is a plant of the Lamiaceae family. Scutellaria baicalensis The dried root of *Georgi*. "Apocynum venetum" as used in other parts of this application refers to the leaves of *Apocynum venetum* mentioned above. The three dried medicinal materials were separately pulverized and mixed. Based on a total mass of 70 g of the three medicinal materials, 700 mL of distilled water was added at a material-to-liquid ratio of 1:10 (g / mL). The mixture was soaked at room temperature for 30 min, then refluxed at 95 ℃ for 2 h. The first extract was filtered and collected. Another 700 mL of distilled water was added to the residue, and the mixture was refluxed at 95 ℃ for 1 h. The second extract was filtered and collected. The two extracts were combined, centrifuged, filtered, and then concentrated using rotary evaporation.

[0040] Depending on the requirements of different implementation methods, the combined extract was concentrated and diluted to volume with distilled water. When using the above-mentioned 70 g raw material feed amount, the volume was adjusted to 140 mL, 70 mL, or 35 mL, respectively, corresponding to total equivalent concentrations of the raw materials of 500 mg / mL, 1000 mg / mL, or 2000 mg / mL; the raw material equivalent concentrations and mass ratios of Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis are shown in Table 1. The traditional Chinese medicine compositions used in Examples 4-6 were all derived from the same batch of samples obtained by water extraction of dried raw materials of Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis in a mass ratio of 4:2:1, and the corresponding total equivalent concentrations of the raw materials were prepared according to the experimental settings of each example.

[0041] Table 1. Embodiments of the herbal composition of the present invention at different concentrations (concentration unit: mg / mL)

[0042] Example 2: Activation and identification of Aeromonas versicolor.

[0043] 100 μL of *Aeromonas vesiculosus* bacterial culture was inoculated into 4 mL of BHI liquid medium and cultured with shaking at 28 ℃ and 180 r / min for 18 h. The culture was then streaked onto 5% sheep blood agar plates and RYAN solid medium, and incubated upside down at 28 ℃ for 18 h. Stable single colonies were picked and purified continuously until the colonies exhibited consistent morphology.

[0044] like Figure 1 As shown, the isolated bacteria exhibited typical colony morphology on RYAN medium. Based on colony morphology, Gram staining, and molecular biological identification results, the tested strain was confirmed as *Aeromonas vegetans*. Aeromonas veronii ).

[0045] Example 3: Screening of single Chinese herbal medicines, determination of MIC / MBC, evaluation of FIC compatibility, verification of prescription composition reduction and screening of component ratio.

[0046] The aqueous extract of a single herb was prepared as follows: 20 g of each dried herb was weighed, pulverized, and soaked in 400 mL of sterile distilled water for 1 h. After boiling, the mixture was simmered over low heat for 2 h and filtered. Another 400 mL of water was added to the residue, and the mixture was simmered and filtered again using the same method. The two filtrates were combined, concentrated, and brought to a final volume of 20 mL. The original herb equivalent concentration of the obtained test solution was 1000 mg / mL. The test solution was treated at 121 ℃ for 20 min and then stored at 4 ℃ for later use.

[0047] The in vitro antibacterial effect of aqueous extracts of single Chinese medicinal herbs against Aeromonas villus was determined using the Oxford cup method. Activated Aeromonas villus was inoculated into LB liquid medium and cultured at 28 °C with shaking at 180 r / min for 18 h. The bacterial concentration was then adjusted to approximately 1.0 × 10⁻⁶ using MH liquid medium and the McFarland turbidimetric method. 7 CFU / mL. 100 μL of bacterial suspension was evenly spread onto the surface of MH solid medium, and then four sterile Oxford cups were evenly placed on top. 200 μL of the test drug solution with a raw material equivalent concentration of 1000 mg / mL was added to each Oxford cup. After incubation at 28 ℃ for 24 h, the diameter of the inhibition zone was measured. Enrofloxacin and florfenicol susceptibility testing tablets (Bikman brand) were used as positive controls, and sterile water was used as a blank control. Each tested herb and the two positive controls were measured in triplicate. Results are expressed as mean ± standard deviation (x̄ ± SD).

[0048] Table 2. Diameter of inhibition zones of different drugs against Aeromonas villus (unit: mm; n=3, x̄±SD)

[0049] The inhibition zone diameters of the tested Chinese herbal medicines and the water blank listed in Table 2 were measured using the Oxford cup method described above; the diameters of the positive controls for enrofloxacin and florfenicol were measured using the drug sensitivity test. Due to differences in drug loading methods and drug content, the inhibition zone diameters of the two positive controls and the tested Chinese herbal medicines were not directly quantitatively compared.

[0050] Table 2 shows the diameter of the inhibition zone for each tested drug as the mean ± standard deviation of three parallel determinations; results where no inhibition zone was formed in any of the three determinations were recorded as 0. Table 2 shows that different drugs exhibit varying inhibitory abilities against Aeromonas verrucosa, with drugs such as Prunus mume, Schisandra chinensis, and Cornus officinalis showing stronger antibacterial activity. Based on the results of the in vitro antibacterial screening of single herbs and the need for subsequent compatibility evaluation, some drugs with antibacterial activity were selected for subsequent MIC / MBC determination.

[0051] Using MH liquid medium as the test medium, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the candidate drugs against Aeromonas verrucosa were determined by the two-fold dilution method. The units for MIC and MBC are mg / mL. The results are shown in Table 3.

[0052] The specific method is as follows: Take a sterile 96-well plate and perform two-fold serial dilutions of each single herb solution using MH liquid medium. The MIC and MBC concentrations are calculated based on the final drug concentration in the wells after inoculation with the bacterial suspension, and are successively 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.9063, 1.9531, 0.9766, and 0.4883 mg / mL. Inoculate with Aeromonas villus subtilis to achieve a final inoculation concentration of approximately 1.0 × 10⁻⁶ mg / mL in the wells. 6 CFU / mL. Three parallel wells were set up for each drug, along with a blank control (drug-free) and a bacterial growth control (drug-free). All wells were incubated at 28 °C for 24 h. The lowest drug concentration in wells showing no bacterial growth compared to the corresponding blank control was determined as the MIC. Cultures from the MIC wells and wells with higher concentrations were recultured on MH agar and incubated upside down at 28 °C for 18 h. The lowest drug concentration in wells with fewer than 5 colonies was determined as the MBC.

[0053] Table 3. MIC and MBC results of candidate traditional Chinese medicines against Aeromonas vulgaris (unit: mg / mL)

[0054] Table 3 shows that Schisandra chinensis, Scutellaria baicalensis, and Apocynum venetum can all inhibit the growth of Aeromonas verrucosa. Specifically, the MIC and MBC of Schisandra chinensis are both 15.625 mg / mL, the MIC of Scutellaria baicalensis is 15.625 mg / mL and the MBC is 62.5 mg / mL, and the MIC of Apocynum venetum is 31.25 mg / mL and the MBC is 125 mg / mL. The MIC and MBC results are used to further evaluate the antibacterial and bactericidal abilities of the candidate drugs and to provide a basis for subsequent FIC (Fluid Activated Chemical) compatibility evaluation.

[0055] The partial inhibitory concentration index (FIC index) among some of the candidate drugs listed in Table 4 was further determined using the checkerboard dilution method. The FIC index was calculated using the following formula: A 6×6 checkerboard layout was used to set up concentration gradients for the two drugs. The drug concentrations were calculated based on the final in-well concentrations after adding the inoculum suspension and chromogenic agent, and were successively 2 MIC, MIC, 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, and 1 / 16 MIC. The corresponding drug solutions were prepared in MH liquid medium and inoculated with *Aeromonas vesiculosus* to achieve a final inoculum concentration of approximately 1.0 × 10⁻⁶. 6 The concentration of each drug was increased to CFU / mL, and 0.1% triphenyltetrazolium chloride solution was added as a growth indicator. The mixture was then incubated at 28 °C for 24 h. A blank control and a bacterial growth control were set up. The lowest final concentration of each drug in each well when no bacterial growth was observed compared to the blank control was used as the combined MIC, and the FIC index was calculated accordingly.

[0056] FIC index = MIC of drug A when used in combination / MIC of drug A alone + MIC of drug B when used in combination / MIC of drug B alone.

[0057] The criteria for determining the FIC index are as follows: FIC ≤ 0.5 indicates a synergistic effect; 0.5 < FIC ≤ 1 indicates an additive effect; 1 < FIC ≤ 4 indicates an irrelevant effect; and FIC > 4 indicates an antagonistic effect.

[0058] Table 4. FIC Index of Candidate Traditional Chinese Medicine Combinations

[0059] Table 4 shows that the FIC index of the combination of Schisandra chinensis and Scutellaria baicalensis is 0.50, indicating a synergistic effect; the FIC index of the combination of Apocynum venetum and Schisandra chinensis is 0.56, indicating an additive effect; and the FIC index of the combination of Apocynum venetum and Scutellaria baicalensis is 1.00, also indicating an additive effect. Based on the results of single herb screening, MIC / MBC determination, and FIC compatibility evaluation, Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis were selected for formula composition reduction verification and component ratio screening.

[0060] Based on the FIC compatibility evaluation, the formulation composition of Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis was modified and verified. In the table, "1" indicates the addition of the corresponding drug, and "2" indicates the removal of the corresponding drug. The antibacterial effect of each combination against Aeromonas verrucosa was determined using the Oxford cup method, and the results are shown in Table 5.

[0061] The equivalent concentration of each herb in the aqueous extracts used for formulation reduction verification was 1000 mg / mL. When preparing 1.0 mL of the complete combination test solution, equal volumes of the aqueous extracts of Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis were mixed, ensuring each herb comprised 1 / 3 of the final test solution volume. For the reduction groups, one herb was removed from each group, and the remaining two herb aqueous extracts were mixed in equal volumes, ensuring each herb comprised 1 / 2 of the final test solution volume. Therefore, the total equivalent concentration of the original herbs in both the complete combination and each reduction group was 1000 mg / mL. Both the complete combination and each reduction group were measured using the aforementioned Oxford cup conditions: approximately 1.0 × 10⁻⁶ ppm. 7 A CFU / mL Aeromonas vesiculosus suspension was spread at 100 μL per plate. After spreading, sterile Oxford cups were placed in each cup, and 200 μL of the test solution was added to each cup. The plates were incubated at 28 ℃ for 24 h, and the diameter of the inhibition zone was measured. Each combination was measured in triplicate. The results are expressed as mean ± standard deviation (x̄ ± SD), as shown in Table 5.

[0062] Table 5. Validation results of formulation composition reduction under the same total raw medicinal material equivalent concentration (unit: mm; n=3, x̄±SD)

[0063] Table 5 shows that, under the same total equivalent concentration of the original medicinal materials, the diameter of the inhibition zone of the complete combination of the three herbs was 25.63 ± 0.25 mm. After removing Schisandra chinensis, Scutellaria baicalensis, and Apocynum venetum, the diameter of the inhibition zone was lower than that of the complete combination. Therefore, Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis were selected as candidate formulations, and the proportions of the three herbs were further screened.

[0064] Candidate group allocation ratio screening. After identifying Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis as candidate drugs, three test concentrations (1 / 2 MIC, MIC, and 2 MIC) were set as references, and the antibacterial effects were compared according to the combinations listed in Table 7. The same strains, culture media, bacterial concentration, sample volume, and culture conditions were used for each group, with the diameter of the inhibition zone as the evaluation index. The test concentrations of each drug are shown in Table 6, and the combinations and test results are shown in Table 7.

[0065] For each experimental combination, the corresponding single-herb aqueous extracts were mixed according to the concentrations listed in Table 6, and the volume was increased to 1000 μL with sterile distilled water. Using the aforementioned Oxford cup method, 200 μL of the test solution was added to each Oxford cup, and the inhibition zone diameter was measured after incubation at 28 ℃ for 24 h. Each group was measured in triplicate. Since the total equivalent concentration of the original medicinal materials was not entirely the same for each experimental combination, the results of this experiment were used to screen candidate formulations for subsequent validation.

[0066] Table 6. Factor level settings for candidate formulation ratio screening (unit: mg / mL)

[0067] Table 7. Antibacterial effects and range analysis results of different factor level combinations (unit: mm; n=3, x̄±SD)

[0068] As shown in Table 7, the antibacterial effects of different factor level combinations varied. Apocynum venetum and Schisandra chinensis showed higher k-values ​​at level 3, while Scutellaria baicalensis showed a higher k-value at level 2, consistent with the level combination in Experiment 9. The inhibition zone diameter in Experiment 9 was the largest among the investigated combinations, corresponding to test concentrations of 62.50 mg / mL for Apocynum venetum, 31.25 mg / mL for Schisandra chinensis, and 15.625 mg / mL for Scutellaria baicalensis, with a concentration ratio of 4:2:1. Therefore, 4:2:1 was selected as a candidate combination for subsequent verification under equivalent total raw material conditions.

[0069] Verification of the total medicinal material equivalent of candidate formulations. To further verify the rationality of the 4:2:1 ratio of Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis, and to eliminate the influence of differences in the total medicinal material equivalent concentration of different combinations on the antibacterial results, the total medicinal material equivalent concentration of each group's test solution was uniformly controlled at 109.375 mg / mL, and only the relative mass ratio between the three herbs was adjusted. Considering that the aforementioned formulation reduction experiment used a three-herb combination in equal proportions for verification, this experiment also set up a 1:1:1 equal proportion group as a reference, and set up multiple adjacent ratios around 4:2:1. Each group used the same strain, culture medium, bacterial concentration, sample volume, and culture conditions, and the Oxford cup method was used for determination, with 3 replicates for each group. The results are shown in Table 8.

[0070] Table 8. Validation of antibacterial effects of different combinations under the condition of equal total raw medicinal material equivalent (n=3)

[0071] Note: The theoretical total equivalent concentration of raw medicinal materials in each group is 109.375 mg / mL. Only the relative mass ratio of the three herbs was adjusted. The concentrations of each individual herb in the table are rounded to three decimal places according to the calculated values. Therefore, there is a rounding error of ±0.001 mg / mL between the sum of the displayed values ​​and the theoretical values ​​for some components. 4:2:1 is the candidate optimal ratio.

[0072] As shown in Table 8, under the condition of maintaining a consistent total raw material equivalent concentration, the antibacterial effects of different mass ratio combinations varied. Among them, the combination of Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis in a 4:2:1 ratio exhibited a larger inhibition zone diameter, making it the superior combination investigated in this experiment. Compared to 4:2:1, further increasing or decreasing the relative proportion of any component did not result in a larger inhibition zone. These results indicate that under the experimental conditions and within the investigated ratio range, the 4:2:1 combination demonstrates better antibacterial performance. Therefore, the 4:2:1 ratio of Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis was determined as the candidate optimal mass ratio for subsequent common water content and efficacy verification.

[0073] In subsequent embodiments, following the method of Example 1, Apocynum venetum, Schisandra chinensis and Scutellaria baicalensis were extracted with water at a mass ratio of 4:2:1 to prepare a traditional Chinese medicine composition. The obtained traditional Chinese medicine composition was then evaluated for its antibacterial properties, safety, and efficacy in infection models.

[0074] Example 4: In vitro antibacterial activity and time-inhibition curve of the traditional Chinese medicine composition of the present invention against the pathogens listed in this example.

[0075] The traditional Chinese medicine composition was prepared using the method described in Example 1, and its in vitro antibacterial activity was determined. Using the 70g raw material dosage described in Example 1, the combined extract was concentrated and brought to a final volume of 70 mL. Based on the total raw material dosage, the total equivalent concentration of the raw materials in the traditional Chinese medicine composition used in this example was 1000 mg / mL, with the equivalent concentrations of Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis being 571.429, 285.714, and 142.857 mg / mL, respectively. Aeromonas verrucosa, Aeromonas hydrophila, Aeromonas salmonicidae, Aeromonas temperate, and Edwardsiella tarda were prepared into concentrations of approximately 1.0 × 10⁻⁶. 7 A bacterial suspension of CFU / mL was prepared, with 100 μL taken from each plate and evenly spread onto the surface of MH solid medium. After spreading, sterile Oxford cups were placed in the medium, and 200 μL of the aforementioned traditional Chinese medicine composition was added to each Oxford cup. The plates were then incubated at 28 ℃ for 24 h, and the diameter of the inhibition zone was measured. The results are shown in Table 9.

[0076] Table 9. Antibacterial effect of the traditional Chinese medicine composition of the present invention against the pathogens listed in this embodiment (unit: mm; n=3, x̄±SD).

[0077] The inhibition zone diameters in Table 9 are the average ± standard deviation (n=3, x̄±SD) of three parallel determinations. As shown in Table 9, the herbal composition of this invention exhibits antibacterial activity against all the pathogens detected in this embodiment. Specifically, the inhibition zone diameter against *Aeromonas velutipes* is 25.27±0.15 mm, and the inhibition zone diameters against *Aeromonas hydrophila*, *Aeromonas salmonicida*, *Aeromonas temperate*, and *Edwards tarda* are 22.67±0.35 mm, 20.73±0.12 mm, 23.17±0.72 mm, and 22.17±0.45 mm, respectively. It should be noted that Table 5 uses three combinations of equal volumes of single-herb aqueous extracts prepared at 1000 mg / mL, with the equivalent ratio of the original medicinal materials in the complete combination being 1:1:1. This example uses a traditional Chinese medicine composition prepared by water extraction of Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis at a dry raw material mass ratio of 4:2:1. Although the total equivalent concentration of the original medicinal materials in both experiments was 1000 mg / mL, the component ratios and preparation methods differed. Therefore, the diameter of their inhibition zones was only used for internal evaluation within their respective experiments and was not directly compared horizontally.

[0078] Referring to the MH liquid medium two-fold dilution method used in Example 3 for the determination of MIC / MBC of single herb, and under the same inoculation, reaction, and culture conditions, the results showed that the MIC and MBC of the herbal composition of the present invention against Aeromonas villiformis were both 15.625 mg / mL, and the concentration was calculated based on the total equivalent concentration of the original medicinal material. The time-inhibition curve of the herbal composition against Aeromonas villiformis was further determined. The Aeromonas villiformis bacterial suspension was diluted to a preset concentration of approximately 1.0 × 10⁻⁶. 5 The actual initial bacterial count at 0 h was determined by plate count. Three treatment concentration groups were established: 1 / 2 MIC, MIC, and 2 MIC, corresponding to total equivalent concentrations of the original medicinal materials in the herbal composition of 7.8125, 15.625, and 31.25 mg / mL, respectively. A control group without the herbal composition was also included. All groups were incubated at 28 ℃, and samples were taken at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h. The samples were serially diluted 10-fold and then plated for incubation. Colony counts were performed, and the results are expressed as log10 CFU / mL.

[0079] The results are as follows Figure 2 As shown, the bacterial count in the control group increased with prolonged culture time; however, the proliferation of *Aeromonas vesiculosus* was inhibited after the addition of the herbal composition of this invention. Specifically, the bacterial count in the 2MIC group remained at a low level overall within 24 hours, while the bacterial growth rates in the MIC and 1 / 2MIC groups were lower than those in the control group. These results indicate that the herbal composition of this invention has a concentration-dependent inhibitory effect on *Aeromonas vesiculosus*.

[0080] Example 5: Evaluation of the safety and maximum test concentration of the oral administration of the traditional Chinese medicine composition of the present invention.

[0081] Forty healthy crucian carp were selected and temporarily raised for 7–14 days before the experiment, excluding those with external injuries, abnormal swimming, or poor feeding. Feeding was withheld for 24 hours before the experiment. The experimental fish were randomly divided into a blank control group, a low-concentration group, a medium-concentration group, and a high-concentration group, with 10 fish in each group.

[0082] The traditional Chinese medicine compositions with different raw material equivalent concentrations shown in Table 1 were prepared according to the method of Example 1. The total raw material equivalent concentration of the low concentration group was 500 mg / mL, of which the raw material equivalent concentrations of Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis were 285.714, 142.857, and 71.429 mg / mL, respectively; the total raw material equivalent concentration of the medium concentration group was 1000 mg / mL, of which the raw material equivalent concentrations of the three were 571.429, 285.714, and 142.857 mg / mL, respectively; and the total raw material equivalent concentration of the high concentration group was 2000 mg / mL, of which the raw material equivalent concentrations of the three were 1142.857, 571.429, and 285.714 mg / mL, respectively. Based on a dosage of 1.0 mL / fish and an experimental fish weight of 90–125 g, the single-dose doses for the low, medium, and high concentration groups were 4.00–5.56, 8.00–11.11, and 16.00–22.22 g of raw material equivalents / kg, respectively. The blank control group received an equal volume of solvent.

[0083] Fish weighing 90–125 g were selected for the oral administration safety evaluation. A preliminary oral administration volume experiment was conducted before the formal administration to determine the tolerable volume for a single oral administration. In the formal experiment, all groups received the same oral administration volume of 1.0 mL / fish, once daily for 3 consecutive days; observation continued for 7 days after the last administration. During the observation period, the fish's mortality, swimming status, feeding behavior, body color, respiratory status, abdominal distension, anal swelling, and other visible abnormalities were recorded. The results are shown in Table 10.

[0084] Table 10 Safety observation results of the oral administration of the traditional Chinese medicine composition of the present invention

[0085] As shown in Table 10, no deaths occurred in the blank control group, low concentration group, medium concentration group, and high concentration group during the observation period, with a mortality rate of 0%. No obvious abnormalities were observed in the swimming, feeding, body color, or respiratory status of the experimental fish in each treatment group, and no obvious abdominal distension or anal swelling was observed. During necropsy, no obvious gross abnormalities were found in the intestines, liver, and kidneys of each treatment group.

[0086] On day 7 after the last administration, at the end of the observation period, the experimental fish in each group were deeply anesthetized with MS-222. After achieving deep anesthesia, they were sacrificed and dissected to observe the gross changes in major tissues such as the intestines, liver, and kidneys. Intestinal, liver, and kidney tissues from five fish in each of the blank control group and the group with a total equivalent concentration of 2000 mg / mL of the original drug were fixed in 4% paraformaldehyde fixative, and after routine dehydration, clearing, paraffin embedding, sectioning, and HE staining, the tissue structure changes were observed under a microscope. The observations included the integrity of the intestinal mucosa, villus arrangement, intestinal epithelial shedding, congestion, and inflammatory cell infiltration; hepatocyte arrangement, vacuolar degeneration, necrosis, and sinusoidal congestion; and renal tubular epithelial structure, glomerular morphology, tubular sloughing material, and inflammatory cell infiltration.

[0087] The results showed that no deaths occurred in the blank control group, low-concentration group, medium-concentration group, and high-concentration group during the observation period. No obvious abnormalities were observed in the swimming, feeding, body color, or respiratory status of the experimental fish in any of the treatment groups, and no obvious abdominal distension or anal swelling was observed. At necropsy, no obvious gross abnormalities were found in the intestines, liver, and kidneys of any of the treatment groups.

[0088] like Figure 3 As shown, HE staining was performed on the intestinal, liver, and kidney tissues of crucian carp in the blank control group and the group with a total equivalent concentration of 2000 mg / mL of the original medicinal material. In the 2000 mg / mL group, the intestinal mucosa structure was relatively intact, the intestinal villi were arranged relatively regularly, and no obvious intestinal epithelial shedding or extensive inflammatory cell infiltration was observed. The liver tissue structure was basically clear, and no obvious hepatocyte necrosis or extensive vacuolar degeneration was observed. No obvious pathological damage was observed in the renal tubules and glomeruli of the kidneys. Combined with observations of general condition and mortality, the groups with total equivalent concentrations of the original medicinal material of 500, 1000, and 2000 mg / mL did not cause death or significant clinical abnormalities in the experimental fish under the administration conditions of this example. Histological results only directly support the absence of significant pathological damage in the major tissues of the 2000 mg / mL group. 2000 mg / mL is the highest tested concentration under the conditions of this experiment.

[0089] Example 6: Verification of the efficacy of the traditional Chinese medicine composition of the present invention against Aeromonas verrucosa infection in fish.

[0090] This embodiment is used to evaluate the in vivo intervention effect of the traditional Chinese medicine composition of the present invention on fish infected with Aeromonas versicolor after infection.

[0091] Forty-five healthy crucian carp, with an average weight of approximately 100 g, were selected as experimental animals for the formal animal efficacy test. They were temporarily housed for 7–14 days prior to the test, during which time water quality was kept stable, and individuals with external injuries, abnormal swimming behavior, or poor feeding were removed. Feeding was stopped for 24 hours before challenge and drug administration.

[0092] Aeromonas villus was inoculated into BHI liquid medium and cultured with shaking at 28 ℃ and 180 r / min for 18–24 h. Before the formal challenge, 48 healthy crucian carp were used for a median lethal dose (LD50) pre-test via intraperitoneal injection: the bacterial suspension was adjusted to 1×10⁻⁶. 9 1×10 8 1×10 7 1×10 6 1×10 5 and 1×10 4 CFU / mL, 8 fish per concentration group, 0.1 mL intraperitoneally injected per fish, observed continuously for 7 days. On day 7, the number of survivors in each group was 0, 3, 6, 7, 8, and 8, respectively, and the corresponding number of deaths was 8, 5, 2, 1, 0, and 0, respectively. Calculated using the Reed-Muench cumulative method, the median lethal concentration (LD50) of Aeromonas vesiculosus for the fish in this preliminary experiment was approximately 3.34 × 10⁻⁶. 7 CFU / mL; calculated at 0.1 mL / tail, the median lethal dose is approximately 3.34 × 10⁻⁶. 6 CFU / tail. After completing the LD50 preliminary test, a binomial logistic dose-mortality model was further established based on the six challenge concentrations and their mortality rates to estimate the 80% lethal concentration (LD80) to be approximately 1.87 × 10⁻⁶. 8 The concentration of CFU / mL was used as the estimated LD80 for the model bacterial suspension in the formal animal efficacy test. On the day of the formal challenge, the same batch of bacterial suspension used for the formal challenge was serially diluted 10-fold and plate counted. Three parallel plates were set up for each suitable dilution, and the average value of the parallel plate counts was used to calculate the actual bacterial suspension concentration. The results showed that the actual concentration of the bacterial suspension in the formal challenge was 1.94 × 10⁻⁶ CFU / mL. 8 CFU / mL; 0.1 mL was injected intraperitoneally into each experimental fish, corresponding to an actual challenge dose of 1.94 × 10⁻⁶ CFU / mL. 7 CFU / tail. The measured concentration is close to the LD80 target concentration estimated by the pre-experimental dose-mortality model; after continuous observation for 7 days post-infection, the cumulative mortality rate in the formal infection model group used for survival observation was 80%.

[0093] The experimental fish were randomly divided into a blank control group, an infection model group, and a traditional Chinese medicine composition group, with 15 fish in each group. Among them, 10 fish in each group were used to observe the cumulative mortality rate, survival rate, and relative protection rate, and no tissue sampling was performed during the observation period; 5 fish in each group were used for tissue bacterial load and histopathological testing.

[0094] The blank control group received an intraperitoneal injection of an equal volume of sterile saline and oral administration of an equal volume of solvent; the infection model group received an intraperitoneal injection of Aeromonas verrucosa bacterial solution and oral administration of an equal volume of solvent; the traditional Chinese medicine composition group received an intraperitoneal injection of Aeromonas verrucosa bacterial solution and oral administration of the traditional Chinese medicine composition of this invention. In this embodiment, the experimental fish weighed between 98 and 134 g, and the intraperitoneal injection volume was 0.1 mL per fish.

[0095] Based on the oral administration safety evaluation results of Example 5, the traditional Chinese medicine composition of the present invention did not cause death or significant abnormalities in the experimental fish at total medicinal material equivalent concentrations of 500, 1000, and 2000 mg / mL, or under the administration conditions described in this example. Therefore, this example selected the traditional Chinese medicine composition with a concentration of 1000 mg / mL for efficacy evaluation in the infection model. The first oral administration began 2 hours after challenge. The traditional Chinese medicine composition group was given the traditional Chinese medicine composition prepared in Example 1, wherein the original medicinal material equivalent concentrations of Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis were 571.429, 285.714, and 142.857 mg / mL, respectively. Each experimental fish was orally administered 1.0 mL each time, i.e., each fish was given 1.0 g of original medicinal material equivalent per administration; calculated based on the experimental fish weight of 98–134 g, the single administration dose was 7.46–10.20 g of original medicinal material equivalent / kg. This dosage is the actual dosage used in this embodiment and does not represent the minimum effective dose, optimal dosage, or maximum tolerated dose. The drug was administered orally once daily for 3 consecutive days. The blank control group and the infection model group were administered the same volume of solvent via gavage. Fish were observed continuously for 7 days after infection, and the mortality rate, time of death, feeding, swimming, body color, abdominal swelling, and anal swelling of the experimental fish used for survival observation were recorded. At the end of the observation period, the cumulative mortality rate, survival rate, and relative protection rate (RPS) were calculated. RPS was calculated using the following formula: RPS (%) = [1 - (mortality rate in the treatment group / mortality rate in the infection model group)] × 100%.

[0096] like Figure 4 and Figure 5 As shown, all experimental fish in the blank control group survived during the observation period; the survival rate of the infection model group on day 7 was 20%, and the cumulative mortality rate was 80%; the survival rate of the traditional Chinese medicine composition group on day 7 was 60%, and the cumulative mortality rate was 40%. Calculated according to the above formula, the relative protection rate of the traditional Chinese medicine composition group was 50.0%. Under the observation conditions of this embodiment, the mortality rate of the traditional Chinese medicine composition group during the observation period was lower than that of the infection model group, while the survival rate was higher; however, this embodiment does not claim that the differences between the groups are statistically significant.

[0097] To further evaluate the effect of the herbal composition of this invention on the pathogen load in infected fish, experimental fish from each group were collected 72 h after challenge. They were deeply anesthetized using MS-222 and euthanized after achieving deep anesthesia, followed by necropsy. Intestinal, liver, and kidney tissues were aseptically isolated, accurately weighed, and added to sterile physiological saline at a 1:9 mass-to-volume ratio to prepare tissue homogenates. After a 10-fold serial dilution, 100 μL of the homogenate was plated on RYAN agar plates and incubated at 28 ℃ for 18–24 h. Suitable dilutions with colony counts of 30–300 were selected. Three parallel plates were prepared for each tissue homogenate sample. The average colony count of the three parallel plates was calculated and converted to the tissue weight to determine the tissue bacterial load (CFU / g) of the experimental fish. The result is expressed as log10 CFU / g.

[0098] like Figure 6 As shown, CFU / g was calculated based on the average of the colony counts from three parallel plates for each experimental fish, with three parallel plates representing technical replicates. The CFU / g values ​​for each experimental fish were converted to log10 and the mean and standard deviation within each group were calculated, expressed as mean ± standard deviation (x̄ ± SD, n=5), where n represents five experimental fish per group. The bacterial loads in the liver, intestine, and kidney of the infection model group were 5.986±0.043, 6.152±0.043, and 4.660±0.044 log10 CFU / g, respectively; while those in the traditional Chinese medicine composition group were 4.601±0.047, 5.095±0.049, and 3.907±0.039 log10 CFU / g, respectively. Compared to the infection model group, the bacterial loads in the above tissues were reduced in the traditional Chinese medicine composition group; this result supports the effect of the traditional Chinese medicine composition of the present invention on reducing the bacterial load in infected fish tissues under the conditions of this embodiment.

[0099] In histopathological examination, intestinal tissue from five experimental fish in each group used for tissue testing was fixed in 4% paraformaldehyde fixative, and after routine dehydration, clearing, paraffin embedding, sectioning, and HE staining, it was observed under a microscope. Figure 7 As shown, the intestinal mucosa structure in the blank control group was relatively intact, and the intestinal villi were arranged more regularly; in the infection model group, the intestinal mucosa structure was damaged, some intestinal villi were shortened, broken or detached, and accompanied by inflammatory cell infiltration; compared with the infection model group, the degree of intestinal tissue damage in the traditional Chinese medicine composition group was reduced, the intestinal mucosa structure was relatively intact, and the inflammatory cell infiltration was reduced. The above results indicate that the traditional Chinese medicine composition of the present invention can alleviate the pathological damage to fish intestinal tissue caused by Aeromonas verrucosum infection.

[0100] In summary, under the administration conditions of this embodiment, the 1000 mg / mL traditional Chinese medicine composition group showed a higher survival rate during the observation period, a lower cumulative mortality rate, and a relative protection rate of 50.0% compared to the infection model group. The bacterial load in the intestinal, liver, and kidney tissues was reduced, and the pathological damage to the intestinal tissue was alleviated. These results support the in vivo intervention effect of the traditional Chinese medicine composition on fish enteritis caused by Aeromonas verrucosum infection after infection. This embodiment did not verify its use as a preventative measure before challenge, the minimum effective dose, or the optimal dosage.

[0101] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A traditional Chinese medicine composition for the prevention and treatment of enteritis caused by Aeromonas verrucosa infection, characterized in that, The traditional Chinese medicine composition is prepared by water extraction of Apocynum venetum, Schisandra chinensis and Scutellaria baicalensis at a dry raw material mass ratio of 3~5:1~3:0.5~2; Preferably, the traditional Chinese medicine composition is prepared by water extraction of Apocynum venetum, Schisandra chinensis and Scutellaria baicalensis in a dry raw material mass ratio of 4:2:

1.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The equivalent concentration of the raw medicinal materials in the Chinese herbal composition is 500~2000 mg / mL, based on the total amount of raw medicinal materials used. Preferably, the equivalent concentration of the original medicinal material in the traditional Chinese medicine composition is 1000 mg / mL.

3. A method for preparing the traditional Chinese medicine composition according to claim 1 or 2, characterized in that, include: Weigh each raw medicinal material according to the weight proportions, place them in a solvent to extract the active ingredients, and obtain an extract; The solvent is water.

4. The preparation method according to claim 3, characterized in that, include: Weigh out Apocynum venetum, Schisandra chinensis, and Scutellaria baicalensis according to a dry raw material mass ratio of 3~5:1~3:0.5~2, pulverize and mix them, add water for the first extraction, and filter; add water to the residue for the second extraction, and filter; combine the two extracts, centrifuge, filter, concentrate and make up to a fixed volume to obtain the Chinese herbal composition.

5. The preparation method according to claim 4, characterized in that, Based on the total mass of Apocynum venetum, Schisandra chinensis and Scutellaria baicalensis, the material-to-liquid ratio for each extraction was 1g:10±2mL. Before the first extraction, the sample was soaked at room temperature for 30±5 min and then refluxed at 95±2 ℃ for 2.0±0.5 h. The second extraction was refluxed at 95±2 ℃ for 1.0±0.5 h.

6. The preparation method according to claim 5, characterized in that, Based on the total amount of raw medicinal materials used, the combined extract was concentrated and diluted to a concentration equivalent to 500-2000 mg / mL of the raw medicinal materials.

7. The use of the traditional Chinese medicine composition according to claim 1 or 2 or the traditional Chinese medicine composition prepared by any one of claims 3 to 6 in the preparation of a product for treating enteritis in fish caused by Aeromonas verrucosa infection.

8. The application according to claim 7, characterized in that, The product is used to reduce the bacterial load of Aeromonas versicolor in the intestines, liver and / or kidneys of infected fish, or to alleviate intestinal mucosal damage caused by Aeromonas versicolor infection.

9. The application of the traditional Chinese medicine composition according to claim 1 or 2, or the traditional Chinese medicine composition prepared by any one of claims 3 to 6, in the preparation of antibacterial agents against pathogenic aquatic bacteria, characterized in that, The aquatic pathogens are selected from one or more of Aeromonas versicolor, Aeromonas hydrophila, Aeromonas salmonicidae, Aeromonas temperate, and Edwardsiella tarda.

10. A traditional Chinese medicine preparation for the prevention and treatment of enteritis caused by Aeromonas verrucosa infection, comprising traditional Chinese medicine components combined with a carrier and / or excipients, characterized in that, The traditional Chinese medicine component is the traditional Chinese medicine composition according to claim 1 or 2 or the traditional Chinese medicine composition prepared by any one of the preparation methods according to claims 3-6; Preferably, the traditional Chinese medicine preparation is an oral administration preparation; the oral administration preparation is an oral liquid, granules, powder, premix, medicated bait, or feed additive.

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