A traditional Chinese medicine composition for preventing and treating acute hepatopancreas necrosis disease of prawn, a preparation method and application thereof

Abstract

The present application belongs to the field of aquaculture technology, and mainly relates to a traditional Chinese medicine composition for preventing and treating acute hepatopancreas necrosis disease of prawn, a preparation method and application. The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 28-32 parts of Radix Sanguisorbae, 28-32 parts of Radix Scutellariae, 18-22 parts of Phellodendri Cortex and 8-12 parts of Lonicerae Japonicae Flos. The traditional Chinese medicine composition can significantly inhibit the activity of the main pathogen of the disease, i.e. Vibrio parahaemolyticus, improve the immune function and disease resistance of prawn, and further repair the hepatopancreas and intestinal tissues of the diseased prawn, thereby achieving the effect of effectively preventing and treating the acute hepatopancreas necrosis disease of prawn.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology, and particularly relates to a traditional Chinese medicine composition, preparation method and application for the prevention and treatment of acute hepatopancreatic necrosis in shrimp. Background Technology

[0002] Shrimp farming is a vital pillar industry in aquaculture, making a significant contribution to the development of aquaculture in China and the world. In 2022, my country's total shrimp production reached 2.42 million tons (China Fisheries Statistical Yearbook), but economic losses due to diseases amounted to 6.3 billion yuan.

[0003] In recent years, acute hepatopancreatic necrosis disease (AHPND) has broken out in Malaysia, Thailand, Mexico, the Philippines, and China, primarily affecting Litopenaeus vannamei, Penaeus monodon, and Fenneropenaeus chinensis. Research indicates that the pathogen of AHPND is a 70kbp organism carrying a pirAB-containing... VP VP of virulence gene plasmid AHPND This disease is caused by a strain of bacteria, primarily Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, or Vibrio campbellii. Large-scale outbreaks typically occur within 7–35 days after stocking. Affected shrimp exhibit softened shells, empty intestines and stomachs or discontinuous food threads in the intestines, white feces, and discolored, atrophied, and shrunken hepatopancreas. The disease spreads rapidly, with pond closure rates reaching up to 80%. In severely affected ponds, shrimp mortality rates can reach 90% or even result in total crop failure, causing a significant blow to the global shrimp farming industry.

[0004] With the increasing scale and intensification of aquaculture, the difficulty of aquaculture management continues to rise, leading to ecological imbalance and posing greater challenges to the control of this disease. Currently, the disease is often prevented and treated with a combination of conventional antibiotics, disinfectants, and probiotics. However, the long-term irrational use of chemical drugs can easily lead to environmental pollution, drug residues, and increased bacterial resistance. It can also easily cause an imbalance in the shrimp's intestinal flora, resulting in decreased shrimp immunity and disease resistance, making subsequent healthy shrimp farming even more difficult. Therefore, developing green and efficient antibiotic alternatives for the prevention and treatment of AHPND is a pressing technical problem that needs to be solved in this field.

[0005] Traditional Chinese medicine is derived from natural sources, has a wide variety of types and rich components, and offers diverse medicinal effects and targets. It is less likely to cause drug residues and bacterial resistance, making it an ideal medicine for disease prevention and health maintenance.

[0006] In previous studies, our team conducted in vitro antibacterial experiments with traditional Chinese medicines targeting six important pathogens causing AHPND in shrimp: Vibrio parahaemolyticus, Aeromonas hydrophila, Vibrio harveyi, Vibrio sp. Ex-25, Vibrio metschnikovii, and Vibrio alginolyticus. This resulted in the selection of the HD-4 compound traditional Chinese medicine formula with good in vitro antibacterial effects. The optimal ratio of the formula is 1:4:4:4 (Sanguisorba officinalis: Terminalia chebula: Coptis chinensis: Prunus mume). This formula has a certain effect on improving the non-specific immune response in shrimp. However, with the emergence of many problems such as global climate change, changes in pathogen flora characteristics, and changes in aquaculture models, especially the continuous enhancement of bacterial virulence and the weakening of shrimp disease resistance, the pathogenesis characteristics of shrimp AHPND have also changed significantly. In addition, the increasing intensiveness of shrimp farming and the diversification of farming models have placed higher demands on drugs and their application processes. Consequently, it is necessary to re-screen and optimize preventive drugs in order to achieve the goal of effectively controlling the disease.

[0007] Furthermore, the composition of traditional Chinese medicine (TCM) prescriptions is not a simple addition or piling up of drugs, but rather a scientific combination of drugs that work synergistically and according to their therapeutic effects, following the principle of principal, assistant, adjuvant, and guide herbs. Simultaneously, ensuring the appropriate types, proportions, dosage forms, and dosages of each herb in the prescription, and their organic combination, is crucial to maximizing the efficacy of the treatment. TCM has been widely used in the prevention and control of aquatic animal diseases. Developing safe and effective TCM preparations for the prevention and treatment of AHPND in shrimp is of significant practical importance for green shrimp farming and safeguarding human health. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide a traditional Chinese medicine composition, preparation method and application for the prevention and treatment of acute hepatopancreatic necrosis disease in shrimp.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a traditional Chinese medicine composition for preventing and treating acute hepatopancreatic necrosis in shrimp, comprising the following raw materials in parts by weight: 28-32 parts of Sanguisorba officinalis, 28-32 parts of Scutellaria baicalensis, 18-22 parts of Phellodendron chinense, and 8-12 parts of Lonicera japonica.

[0011] Preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 29-31 parts of Sanguisorba officinalis, 29-31 parts of Scutellaria baicalensis, 19-21 parts of Phellodendron chinense, and 9-11 parts of Lonicera japonica.

[0012] Preferably, the traditional Chinese medicine composition further includes the following raw materials in parts by weight: 18-22 parts of Terminalia chebula, 18-22 parts of Prunus mume, and 8-12 parts of Cyperus rotundus.

[0013] Preferably, the traditional Chinese medicine composition further includes the following raw materials in parts by weight: 19-21 parts of Terminalia chebula, 19-21 parts of Prunus mume, and 9-11 parts of Cyperus rotundus.

[0014] The present invention also provides a method for preparing the traditional Chinese medicine composition, comprising the following steps: crushing the raw materials into coarse powder and mixing them; then crushing the resulting mixture through an 80-300 mesh and sieving it to obtain a powder of the traditional Chinese medicine composition.

[0015] Preferably, the preparation method further includes the following steps: adding an ethanol solution to the powder, soaking and extracting, filtering, and concentrating.

[0016] Preferably, the ethanol solution is a 50% to 80% ethanol solution, and the material-to-liquid ratio of the added ethanol solution is 1g:3mL to 1g:30mL.

[0017] Preferably, the soaking and extraction time is 2 to 15 hours.

[0018] This invention also provides the application of the above-mentioned traditional Chinese medicine composition or its preparation method in the preparation of drugs that inhibit Vibrio parahaemolyticus or drugs for the prevention and treatment of acute hepatopancreatic necrosis in shrimp.

[0019] The beneficial effects of this invention are:

[0020] This formula consists of Sanguisorba officinalis, Scutellaria baicalensis, Phellodendron chinense, and Lonicera japonica, with the addition of Terminalia chebula, Prunus mume, and Cyperus rotundus. Sanguisorba officinalis is the principal herb for cooling the blood and detoxifying; Scutellaria baicalensis and Phellodendron chinense clear heat and dry dampness; Lonicera japonica disperses wind-heat; Terminalia chebula and Prunus mume astringe and consolidate, serving as assistant herbs; and Cyperus rotundus soothes the liver, relieves depression, regulates qi, and invigorates blood circulation, acting as adjuvant herbs. The combined effects of these herbs achieve the functions of clearing heat and drying dampness, cooling the blood and detoxifying, astringing and consolidating, and regulating qi, achieving a balance between principal, assistant, adjuvant, and adjuvant herbs, while also addressing the functions of expelling pathogens and supporting the body's resistance. This herbal composition can significantly inhibit the activity of Vibrio parahaemolyticus, improve the immune function and disease resistance of shrimp, reduce the mortality rate of shrimp suffering from acute hepatopancreatic necrosis, and protect and repair the hepatopancreatic and intestinal tissues of diseased shrimp, thus effectively preventing and treating acute hepatopancreatic necrosis in shrimp. Attached Figure Description

[0021] Figure 1 Antibacterial zones of traditional Chinese medicine compositions prepared using different methods.

[0022] Figure 2The effects of different drugs on the prevention and protection against AHPND in shrimp.

[0023] Figure 3 This is a schematic diagram of a diseased shrimp and its digestive tract; where a represents a diseased shrimp; b represents the digestive tract of a diseased shrimp; MG represents the midgut; HP represents the hepatopancreas; and ST represents the stomach.

[0024] Figure 4 The mortality rate of shrimp in each experimental group after challenge with the virus was recorded.

[0025] Figure 5 The graph shows the changes in PO activity in the hepatopancreas of shrimp in each experimental group.

[0026] Figure 6 The graph shows the changes in SOD activity in the hepatopancreas of shrimp in each experimental group.

[0027] Figure 7 The graph shows the changes in ACP activity in the hepatopancreas of shrimp in each experimental group.

[0028] Figure 8 The graph shows the changes in AKP activity in the hepatopancreas of shrimp in each experimental group.

[0029] Figure 9 The graph shows the changes in LZM activity in the hepatopancreas of shrimp in each experimental group.

[0030] Figure 10 Paraffin-embedded sections of shrimp hepatopancreas tissue are shown for each group. A and B are blank control groups; C and D are positive control groups; E and F are low-dose experimental groups; G and H are medium-dose experimental groups; and I and J are high-dose experimental groups. B, D, F, H, and J are magnified views of the black-framed areas in A, C, E, G, and I, respectively, in that order.

[0031] Figure 11 Images of paraffin-embedded sections of shrimp intestines from each group are shown; where A is the blank control group; B is the positive control group; C is the low-dose experimental group; D is the medium-dose experimental group; and E is the high-dose experimental group. Detailed Implementation

[0032] This invention provides a traditional Chinese medicine composition for preventing and treating acute hepatopancreatic necrosis in shrimp, comprising the following raw materials in parts by weight: 28-32 parts of Sanguisorba officinalis, 28-32 parts of Scutellaria baicalensis, 18-22 parts of Phellodendron chinense, and 8-12 parts of Lonicera japonica; preferably: 29-31 parts of Sanguisorba officinalis, 29-31 parts of Scutellaria baicalensis, 19-21 parts of Phellodendron chinense, and 9-11 parts of Lonicera japonica; more preferably: 30 parts of Sanguisorba officinalis, 30 parts of Scutellaria baicalensis, 20 parts of Phellodendron chinense, and 10 parts of Lonicera japonica.

[0033] Preferably, the traditional Chinese medicine composition further includes the following raw materials in parts by weight: 18-22 parts of Terminalia chebula, 18-22 parts of Prunus mume, and 8-12 parts of Cyperus rotundus; more preferably: 19-21 parts of Terminalia chebula, 19-21 parts of Prunus mume, and 9-11 parts of Cyperus rotundus; even more preferably: 20 parts of Terminalia chebula, 20 parts of Prunus mume, and 10 parts of Cyperus rotundus.

[0034] In this invention, there are no special restrictions on the source of the following herbs: Sanguisorba officinalis, Scutellaria baicalensis, Phellodendron chinense, Lonicera japonica, Terminalia chebula, Prunus mume, and Cyperus rotundus. Commercially available herbs that meet national standards can be used.

[0035] This invention also provides a method for preparing the aforementioned traditional Chinese medicine composition, comprising the following steps: pulverizing the raw materials into coarse powder and mixing them; further pulverizing the resulting mixture through an 80-300 mesh screen and sieving it to obtain a powder of the traditional Chinese medicine composition. This invention does not specifically limit the methods for pulverizing, mixing, and sieving; conventional methods in the art can be used. In the 80-300 mesh pulverization step, the preferred pulverization mesh size is 100-250 mesh, more preferably 200 mesh. Depending on the state of the medicinal materials after mixing, a drying step can also be performed; this invention does not specifically limit the drying method; conventional methods in the art can be used.

[0036] Preferably, the preparation method further includes the following steps: adding an ethanol solution to the powder, soaking and extracting, filtering, and concentrating.

[0037] In this invention, an ethanol solution is directly added to the powder. The ethanol solution is preferably a 50%–80% ethanol solution, more preferably a 60%–75% ethanol solution, and further preferably a 70% ethanol solution. The material-to-liquid ratio of the added ethanol solution is preferably 1g:3mL–1g:30mL, more preferably 1g:5mL–1g:20mL, and further preferably 1g:10mL. After adding the ethanol solution, soaking and extraction are performed. The soaking and extraction time is preferably 2–15 hours, more preferably 8–13 hours, and further preferably 12 hours. After soaking and extraction, the mixture is filtered, and the filtrate is concentrated. This invention does not have specific limitations on the filtration and concentration methods; conventional filtration and concentration methods in the art are acceptable. After concentration, an ethanol extract of the herbal composition of this invention is obtained.

[0038] In this invention, the alcohol extract can also be prepared into medicinal solutions with different contents of raw herbs using distilled water. The medicinal solutions are preferably sterilized before use, and the sterilization method is preferably high-pressure sterilization.

[0039] This invention also provides the application of the above-mentioned traditional Chinese medicine composition or its preparation method in the preparation of drugs that inhibit Vibrio parahaemolyticus or drugs for the prevention and treatment of acute hepatopancreatic necrosis disease in shrimp. The traditional Chinese medicine composition of this invention can significantly inhibit the activity of Vibrio parahaemolyticus, improve the immune function and disease resistance of shrimp, reduce the mortality rate of shrimp suffering from acute hepatopancreatic necrosis disease, and repair the hepatopancreas and intestinal tissues of diseased shrimp, thereby effectively preventing and treating acute hepatopancreatic necrosis disease in shrimp.

[0040] In this invention, the drug for inhibiting Vibrio parahaemolyticus or preventing acute hepatopancreatic necrosis in shrimp comprises the drug composition as the active ingredient, and also includes pharmaceutically acceptable excipients. This invention does not impose any particular limitation on the type of excipients; conventional excipients in the art can be used.

[0041] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Unless otherwise specified, the following embodiments are all conventional methods.

[0043] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0044] In a specific embodiment of the present invention:

[0045] The medicinal materials, including Sanguisorba officinalis, Scutellaria baicalensis, Phellodendron chinense, and Lonicera japonica, were all purchased from Qingdao Tongrentang Pharmacy.

[0046] Experimental strain: Vibrio parahaemolyticus was isolated from shrimp in aquaculture farms that were typically suffering from acute hepatopancreatic necrosis disease by the Pathogen Research Laboratory of the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences.

[0047] Example 1

[0048] Prescription: Sanguisorba officinalis 30g, Scutellaria baicalensis 30g, Phellodendron chinense 20g, Lonicera japonica 10g.

[0049] Preparation method: The above raw materials are crushed into coarse powder, mixed, dried, pulverized with 200 mesh, sieved, and mixed evenly to obtain the traditional Chinese medicine composition powder.

[0050] Example 2

[0051] Prescription: Sanguisorba officinalis 32g, Scutellaria baicalensis 28g, Phellodendron chinense 22g, Lonicera japonica 8g.

[0052] Preparation method: The above raw materials are crushed into coarse powder, mixed, dried, pulverized with 300 mesh, sieved, and mixed evenly to obtain the traditional Chinese medicine composition powder.

[0053] Example 3

[0054] Prescription: Sanguisorba officinalis 28g, Scutellaria baicalensis 32g, Phellodendron chinense 18g, Lonicera japonica 12g.

[0055] Preparation method: The above raw materials are crushed into coarse powder, mixed, dried, pulverized with 80 mesh, sieved, and mixed evenly to obtain the traditional Chinese medicine composition powder.

[0056] Example 4

[0057] Take the powder prepared in Example 1, add it to 70% ethanol solution at a material-to-liquid ratio of 1g:10mL, soak for 12h, filter, concentrate, and make up to volume with distilled water to prepare a medicinal solution containing 300mg of raw drug per 1mL, and sterilize by autoclaving.

[0058] Example 5

[0059] Take the powder prepared in Example 1, add it to 80% ethanol solution at a material-to-liquid ratio of 1g:3mL, soak for 15h, filter, concentrate, and make up to volume with distilled water to prepare a medicinal solution containing 300mg of raw drug per 1mL, and sterilize by autoclaving.

[0060] Example 6

[0061] Take the powder prepared in Example 1, add it to 50% ethanol solution at a material-to-liquid ratio of 1g:30mL, soak for 2 hours, filter, concentrate, and make up to volume with distilled water to prepare a medicinal solution containing 300mg of raw drug per 1mL, and sterilize by autoclaving.

[0062] Example 7

[0063] The only difference from Example 4 is that the powder in Example 1 was prepared by "80-mesh pulverization".

[0064] Example 8

[0065] The only difference from Example 4 is that the powder in Example 1 was prepared by "300-mesh pulverization".

[0066] Example 9

[0067] The only difference from Example 1 is that 20g of Terminalia chebula, 20g of Prunus mume, and 10g of Cyperus rotundus were added to the prescription.

[0068] Comparative Example 1

[0069] The only difference from Example 1 is that the prescription is: 10g of Sanguisorba officinalis, 20g of Scutellaria baicalensis, 20g of Phellodendron chinense, and 20g of Lonicera japonica.

[0070] Comparative Example 2

[0071] The only difference from Example 1 is that the prescription is: 10g of Sanguisorba officinalis, 30g of Scutellaria baicalensis, 30g of Phellodendron chinense, and 30g of Lonicera japonica.

[0072] Comparative Example 3

[0073] The only difference from Example 1 is that the prescription is: 20g of Sanguisorba officinalis, 20g of Scutellaria baicalensis, 30g of Phellodendron chinense, and 10g of Lonicera japonica.

[0074] Comparative Example 4

[0075] Take the powder prepared in Example 1, add water at a ratio of 3g:70mL, stir on a magnetic stirrer for 30min, filter, concentrate, and make up to volume with distilled water to prepare a medicinal solution containing 300mg of raw drug per 1mL, and sterilize by autoclaving.

[0076] Comparative Example 5

[0077] The only difference from Comparative Example 4 is that the powder in Example 1 was prepared by "80-mesh pulverization".

[0078] Comparative Example 6

[0079] The only difference from Comparative Example 4 is that the powder in Example 1 was prepared by "300-mesh pulverization".

[0080] Experimental Example 1

[0081] Pharmacodynamic tests of traditional Chinese medicine compositions with different dosage ratios

[0082] The powders prepared in Examples 1, 9, and Comparative Examples 1-3 were used as experimental drugs. They were soaked in 10 times (1g:10mL) of 70% ethanol for 12 hours, filtered, and the filtrate was concentrated under reduced pressure to a solution containing 500mg of crude drug per mL. The diameter of the inhibition zone, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of each formulation were determined.

[0083] The plate-drilling method was used for determination. The culture medium used was tryptone soybean broth agar (TSB) containing 2.5% NaCl. Two replicates were performed for each drug solution. After incubation at 28°C for 24 hours, the size of the inhibition zone for each herb was accurately measured, and the average value was taken. The inhibition zone size was determined according to the standards issued by the Clinical and Laboratory Standards Institute (CLSI): inhibition zone diameter ≤14 mm was considered "resistant"; 15–20 mm was "moderately sensitive"; and above 20 mm was "sensitive". The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using a modified two-fold dilution method.

[0084] The experimental results are shown in Table 1. The results show that, compared with the drugs in Comparative Examples 1-3, Vibrio parahaemolyticus was most sensitive to the drugs in Examples 1 and 9, with inhibition zone diameters reaching 22.7 mm and 23.2 mm, respectively.

[0085] The MIC (micron activity) values ​​showed that Examples 1 and 9 exhibited the best antibacterial effects, with MICs of 1.56 mg / mL and 0.78 mg / mL, respectively, demonstrating significant antibacterial efficacy superior to the drugs in Comparative Examples 1-3. The MBC (micron activity) values ​​showed that the drugs in Examples 1 and 9 had the strongest bactericidal effects, with MBCs of 3.13 mg / mL and 1.56 mg / mL, respectively, both superior to the drugs in Comparative Examples 1-3.

[0086] Based on the combined measurements of inhibition zone diameter, MIC, and MBC, the antibacterial and bactericidal effects of the drug composition powders in Examples 1 and 9 are significantly better than those of powders with other dosage ratios.

[0087] Table 1. Determination of inhibition zone diameter, MIC, and MBC of each formulation against Vibrio parahaemolyticus.

[0088]

[0089] Experimental Example 2

[0090] In vitro antibacterial tests of traditional Chinese medicine compositions prepared by different methods

[0091] The drug solutions prepared in Examples 4 (alcohol extraction, 200 mesh), 7 (alcohol extraction, 80 mesh), 8 (alcohol extraction, 300 mesh), and Comparative Examples 4 (water extraction, 200 mesh), 5 (water extraction, 80 mesh), and 6 (water extraction, 300 mesh) were used as test drugs, and the diameter of the inhibition zone was measured. Before the experiment, Vibrio parahaemolyticus was activated and cultured on TSB medium under the following conditions: 28℃, constant temperature shaking at 190 rpm for 10 h (exponential growth phase 8–11 h). The cultured bacteria were collected by centrifugation and prepared into 1×10⁻⁶ solutions using 1.5% sterile NaCl. 6 CFU / mL bacterial suspension. Take 100 μL of the bacterial suspension and spread it evenly on an Oxford cup plate with a sterile cotton swab. Then, add 150 μL of diluted drug solution (30 mg / mL, 50 mg / mL, and 100 mg / mL) to each well of the Oxford cup. Incubate the culture medium with the added drug solution in a 28℃ incubator for 12 hours, and then measure the size of the inhibition zone.

[0092] Experimental results are as follows Figure 1 As shown in Table 2, both extraction methods can achieve good inhibitory effects on Vibrio parahaemolyticus when extracting drugs of different mesh sizes. However, the alcohol extraction method has a better antibacterial effect on Vibrio alginolyticus than the water extraction method. Moreover, the final inhibition zone size of the three drug concentrations of both extraction methods is 300 mesh > 200 mesh > 80 mesh.

[0093] Table 2. Size of the inhibition zone of traditional Chinese medicine compositions obtained by different preparation methods.

[0094]

[0095] Experimental Example 3

[0096] In vivo experiments of traditional Chinese medicine composition on AHPND in shrimp

[0097] Experimental materials:

[0098] Experimental animals: 840 healthy, vigorous whiteleg shrimp with a body length of (6.8±0.62cm) and a weight of (3.5±0.23g) were selected for this experiment. The shrimp were purchased from a whiteleg shrimp farm and temporarily raised in laboratory culture tanks for one week.

[0099] The test water was natural seawater directly extracted from the seashore, with a salinity of 29.2. The water temperature during the test ranged from 27.5℃ to 28.2℃. Aeration was carried out continuously throughout the test, and dissolved oxygen was maintained above 6.5 mg / L.

[0100] Experimental feed: Haida brand shrimp compound feed Xia Kangbao (6662) 2#L20Φ1, purchased from Tianjin Rongchuan Feed Co., Ltd.

[0101] Preparation of medicated feed: Using sodium alginate as a binder, the test drug was mixed with the blank feed without the drug to prepare medicated feed. After stirring evenly, the feed was air-dried at room temperature and sealed for later use.

[0102] 1. In vivo prevention experiment

[0103] 1.1 Experimental Protocol: Pacific white shrimp were randomly divided into 6 groups: Example 1 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Positive Control Group, and Blank Control Group, with 40 shrimp in each group. Shrimp in Example 1 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group were fed the corresponding medicated feed daily at a dosage of 20 g / kg for 5 consecutive days, twice daily, with a daily feed intake of 3% of the shrimp's body weight. The Positive Control Group and Blank Control Group were fed a blank feed without the drug. Eighteen hours after the end of the drug administration, except for the Blank Control Group, all other groups were fed the medicated feed according to the recommended dosage. 5 Shrimp were challenged by immersion in a CFU / mL solution for 24 hours without changing the water. After 24 hours, 50% of the water was replaced as usual. The incidence and mortality of shrimp were observed and recorded. The observation was continued for 5 days, and the incidence and mortality rates of each group were statistically analyzed.

[0104] 1.2 Experimental Results: The results of the preventive protection test are as follows: Figure 2As shown in the figure. The results showed that the morbidity and mortality rates of shrimp in the positive control group were 57.5% and 40.0%, respectively, while no disease or death occurred in the experimental shrimp in the blank control group, proving that there was no problem with the experimental system. The morbidity and mortality rates of the Example 1 group reached 25.0% and 12.5%, respectively, which were significantly lower than those of the Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, and the positive control group. The results indicate that the drug in Example 1 has a good preventive and protective effect against AHPND in shrimp caused by Vibrio parahaemolyticus infection, and its effect is better than that of the drugs in Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0105] 2. In vivo therapeutic experiments

[0106] 2.1 Experimental Grouping:

[0107] Pacific white shrimp were randomly divided into 5 groups: a high-dose test drug group, a medium-dose test drug group, a low-dose test drug group, a blank control group, and a positive control group. Each group had 3 replicates, with 40 shrimp in each replicate. After the experiment started, except for the blank control group, all other groups were treated at a dose of 1×10⁻⁶. 6 The shrimp were challenged with a dose of CFU / mL through immersion for 24 hours without water changes. After 24 hours, 50% of the water was replaced as usual. Simultaneously with the start of the challenge, the high, medium, and low dose experimental groups were fed feed containing the drug from Example 1 at doses of 30 g / kg, 20 g / kg, and 10 g / kg, respectively. The administration was repeated twice daily for 7 days, with the feed amount being 3% of the shrimp's body weight. The blank control and positive control groups were fed a blank feed without the drug during the experiment.

[0108] Each group of shrimp underwent bottom slurry and water change once every morning, with a water change volume of 45L (approximately 50% of the total volume). After the experiment began, the shrimp were promptly removed from the buckets to observe and record clinical symptoms and mortality.

[0109] 2.2 Detection Methods and Results

[0110] 2.2.1 Results of the virus challenge

[0111] This experiment used Vibrio parahaemolyticus immersion challenge at a concentration of 1×10⁻⁶. 6 CFU / mL. In the positive control group, all shrimp showed symptoms such as empty intestines and stomachs, atrophied and pale yellow hepatopancreas, and weak vitality 12 hours after infection. Symptoms in diseased shrimp are shown in [the table below]. Figure 3 Acute death occurred within 48 hours, with a mortality rate of 46.7%. The control group showed normal food intake, activity, body color, and liver and pancreas function. The experimental results demonstrate the success of this artificial challenge model.

[0112] 2.2.2 Shrimp Mortality

[0113] Every morning when changing the water, observe the disease symptoms, survival and mortality of shrimp in each parallel group of each group, and calculate the mortality rate.

[0114]

[0115] Experimental results are as follows Figure 4 As shown, the mortality rates within 24 hours for the blank control group, positive control group, high-dose experimental group, medium-dose experimental group, and low-dose experimental group were 0%, 40%, 0%, 22.5%, and 35%, respectively; at 48 hours, the mortality rates for the blank control group, positive control group, high-dose experimental group, medium-dose experimental group, and low-dose experimental group were 0%, 46.7%, 10%, 32.5%, and 41.7%, respectively; and within 7 days, the mortality rates for the blank control group, positive control group, high-dose experimental group, medium-dose experimental group, and low-dose experimental group were...

[0116] 1.7%, 50%, 15%, 36.7%, and 43.3%.

[0117] Conclusion: The traditional Chinese medicine composition of the present invention can reduce the mortality rate of diseased shrimp.

[0118] 2.2.3 Determination of the immune enzyme activity of traditional Chinese medicine composition in shrimp

[0119] Sampling: For each experimental group and each parallel group, hepatopancreatic tissue samples were randomly collected from two shrimp at 0h, 12h, 24h, 48h, 96h, and 168h post-infection. The activity of immunoenzymes in the samples was measured.

[0120] Detection methods: The shrimp phenol oxidase (PO) ELISA kit from Conodi Biotechnology Co., Ltd. was used for determination. Acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) were measured using kits from Nanjing Jiancheng Bioengineering Institute.

[0121] Experimental results:

[0122] phenol oxidase (PO) activity

[0123] Example 1: The changes in PO activity in the hepatopancreas of Litopenaeus vannamei within 168 hours after artificial infection in different dosage groups and the control group during the efficacy test of the traditional Chinese medicine composition are as follows: Figure 5As shown, during the entire experiment, both the positive control group and the three experimental groups showed a trend of first decreasing and then increasing, reaching the lowest point at 48 hours. It can be seen that the PO activity increased sequentially in the positive control group, low-dose experimental group, medium-dose experimental group, and high-dose experimental group throughout the experiment. The high-dose experimental group showed the most significant difference from the positive control group (P<0.05). The medium-dose experimental group showed significant differences from the positive control group except at 48 hours (P<0.05). Although the PO activity in the low-dose experimental group was higher than that in the positive control group, the difference was not significant (P>0.05).

[0124] Superoxide dismutase (SOD) activity

[0125] Example 1: The changes in SOD activity in the hepatopancreas of Litopenaeus vannamei within 168 hours after artificial infection in different dosage groups and the control group during the efficacy test of the traditional Chinese medicine composition are as follows: Figure 6 As shown in the figure, the SOD activity in the positive control group initially increased and then decreased throughout the experiment, with the highest value occurring at 24 hours, after which it began to decline. All three dose groups showed an increasing trend, and at the same time point, the SOD activity in the low-dose, medium-dose, and high-dose groups increased sequentially. However, significant differences in SOD activity among the three groups only appeared after 48 hours (P<0.05). At 12 hours, although the SOD activity in the positive control group, low-dose group, medium-dose group, and high-dose group increased sequentially, the differences were not significant (P>0.05). At other time points, the high-dose group showed the most significant difference from the positive control group (P<0.05). After 24 hours, significant differences appeared between the medium-dose and low-dose groups and the positive control group (P<0.05).

[0126] Acid phosphatase (ACP) activity

[0127] Example 1: Efficacy test of the traditional Chinese medicine composition. Changes in ACP activity in the hepatopancreas of Litopenaeus vannamei within 168 hours after artificial infection in different dosage groups and the control group are shown below. Figure 7 As shown in the figure, the ACP activity in the liver and pancreas of both the positive control group and the three experimental groups showed a trend of first increasing and then decreasing throughout the experiment, with the highest ACP activity at 24 h. The ACP activity increased sequentially from the positive control group to the low-dose experimental group, the medium-dose experimental group, and the high-dose experimental group at each time point. Only the high-dose experimental group showed a significant difference from the positive control group during the experiment (P<0.05), while the medium-dose experimental group showed a significant difference from the positive control group from 0 h to 96 h (P<0.05). Significant differences existed among the three experimental groups only at 48 h (P<0.05).

[0128] Alkaline phosphatase (AKP) activity

[0129] Example 1: The changes in AKP activity in the hepatopancreas of Litopenaeus vannamei within 168 hours after artificial infection in different dosage groups and the control group during the efficacy test of the traditional Chinese medicine composition are as follows: Figure 8 As shown in the figure, the AKP activity in the liver and pancreas of both the positive control group and the three experimental groups showed a trend of first increasing and then decreasing throughout the experiment, with the highest AKP activity at 12h. The AKP activity increased sequentially from the positive control group to the low-dose experimental group, the medium-dose experimental group, and the high-dose experimental group at each time point. The high-dose and medium-dose experimental groups showed significant differences from the positive control group during the experiment (P<0.05), and there were only significant differences among the three experimental groups at 12h (P<0.05).

[0130] Lysozyme (LZM) activity

[0131] Example 1: The changes in LZM activity in the hepatopancreas of Litopenaeus vannamei within 168 hours after artificial infection in different dosage groups and the control group during the efficacy test of the traditional Chinese medicine composition are as follows: Figure 9 As shown in the figure, the LZM activity in the positive control group and the three experimental groups generally showed a trend of first increasing and then decreasing, with the highest LZM activity at 12h. Except for 96h, the LZM activity in the positive control group, low-dose experimental group, medium-dose experimental group and high-dose experimental group increased sequentially at each time point. During the experiment, only the high-dose experimental group and the positive control group showed a significant difference (P<0.05). Although the low-dose experimental group and the medium-dose experimental group had higher LZM activity than the positive control group overall, the difference was not significant (P>0.05).

[0132] Conclusion: The traditional Chinese medicine composition of the present invention can improve the immunity and disease resistance of shrimp.

[0133] 2.2.4 The repairing effect of traditional Chinese medicine composition on diseased tissues of shrimp

[0134] Sample collection: Before challenge and at the end of drug administration (i.e., day 0 and day 7), hepatopancreas and intestinal tissues from 5 shrimp were randomly collected from each experimental group. The tissues were rapidly immersed in Davidson's fixative for fixation, and after 24 hours, they were replaced with 70% ethanol for long-term preservation. Tissue sections were prepared and stained with hematoxylin and eosin (HE) for microscopic observation.

[0135] Observation results of liver and pancreas tissues as follows Figure 10 As shown, the results indicated that in the blank control group, the hepatopancreatic tubule epithelial cells in the Litopenaeus vannamei hepatopancreatic tissue were uniform in size, containing B, F, and R cells, without shedding or swelling, with red cytoplasm and blue-purple nuclei. Figure 10As can be seen, the positive control group showed severe lesions in the hepatopancreatic tissue of infected shrimp, with no B, F, and R cells, and a large number of necrotic and sloughed epithelial cells in the hepatopancreatic tubules, as well as hepatopancreatic tubule remnants surrounded by hematocytic infiltration (arrows); the low-dose and medium-dose experimental groups also showed hepatopancreatic tubules surrounded by hematocytic infiltration, and slight sloughing of epithelial cells (arrows); the high-dose experimental group showed uniformly sized hepatopancreatic tubules, with no obvious sloughing, and a small number of hepatopancreatic tubule remnants surrounded by hematocytic infiltration.

[0136] Results of intestinal tissue observation Figure 11 As shown in the results, the intestinal epithelial cells in the blank control group shrimp were tightly arranged, uniform in size, and tightly connected to the basement membrane, showing no damage. Figure 11 As shown, the positive control group exhibited significant pathological changes in its intestinal structure, with enlarged epithelial cell nuclei, cell deformation, and detachment, and gaps appearing between the epithelial cells and the basement membrane. The low-dose and medium-dose experimental groups also showed epithelial cell damage and detachment, with gaps between the epithelial cells and the basement membrane. In the high-dose experimental group, the intestinal epithelial cells were tightly packed, uniform in size, and tightly connected to the basement membrane.

[0137] Conclusion: The traditional Chinese medicine composition of the present invention can repair the hepatopancreas and intestinal tissues of diseased shrimp.

[0138] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine composition for preventing and treating acute hepatopancreatic necrosis in shrimp, characterized in that, It is made from the following raw materials in parts by weight: 28-32 parts of Sanguisorba officinalis, 28-32 parts of Scutellaria baicalensis, 18-22 parts of Phellodendron chinense, 8-12 parts of Lonicera japonica, 18-22 parts of Terminalia chebula, 18-22 parts of Prunus mume, and 8-12 parts of Cyperus rotundus; The preparation method of the traditional Chinese medicine composition includes the following steps: the raw materials are crushed into coarse powder and mixed; the resulting mixture is then crushed through an 80-300 mesh and sieved to obtain a powder of the traditional Chinese medicine composition; an ethanol solution is added to the powder, and the mixture is soaked, extracted, filtered, and concentrated; the ethanol solution is a 50%-80% ethanol solution, and the ratio of the added ethanol solution to the liquid is 1g:3mL to 1g:30mL.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, It is made from the following raw materials in parts by weight: 29-31 parts of Sanguisorba officinalis, 29-31 parts of Scutellaria baicalensis, 19-21 parts of Phellodendron chinense, 9-11 parts of Lonicera japonica, 19-21 parts of Terminalia chebula, 19-21 parts of Prunus mume, and 9-11 parts of Cyperus rotundus.

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The soaking and extraction time is 2 to 15 hours.

4. The use of the traditional Chinese medicine composition according to any one of claims 1 to 3 in the preparation of drugs for inhibiting Vibrio parahaemolyticus or for preventing and treating acute hepatopancreatic necrosis in shrimp.

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