Traditional Chinese medicine composition and its application in preparing products for preventing, alleviating or treating acute hepatopancreatic necrosis of shrimp

Abstract

The invention provides a traditional Chinese medicine composition and an application thereof in preparing a product for preventing, alleviating or treating acute hepatopancreatic necrosis of shrimp, belonging to the field of veterinary medicine. The active ingredients of the traditional Chinese medicine composition include isatis root, artificial bezoar, liquorice, borneol, pig gall powder and xuanming powder. The traditional Chinese medicine composition has a good therapeutic effect on preventing, alleviating or treating acute hepatopancreatic necrosis of shrimp, with a cure rate of more than 70%, has antibacterial and liver-protecting effects, can effectively improve the degree of liver damage, improve the survival rate of shrimp and reduce losses.

Description

[0001] This application claims priority to a Chinese patent application filed with the State Intellectual Property Office, with application number 202310239855.9, application date March 8, 2023, and application name “Traditional Chinese medicine composition and its use in the preparation of a product for preventing, alleviating or treating acute hepatopancreatic necrosis of shrimp”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The invention relates to the technical field of veterinary drugs, and in particular to a traditional Chinese medicine composition and application thereof in preparing a product for preventing, alleviating or treating acute hepatopancreatic necrosis of shrimp. Background Art

[0003] As we all know, there are many aquaculture species in the world and they are developing rapidly. As the breeding density and output increase year by year, the breeding environment and conditions are difficult to improve continuously, resulting in the frequent occurrence of diseases. The development of new fishery drugs is relatively slow, and enterprises are not very enthusiastic about developing new fishery drugs. The shortage of new fishery drugs has made traditional fishery drugs increasingly unable to meet people's current requirements for high-quality aquatic products. At the same time, the occurrence of aquatic diseases still seriously restricts the healthy development and growth of the aquaculture industry. Shrimp (Penaeus orientalis) is widely farmed. Taking Vannamei shrimp (white shrimp) as an example, Vannamei shrimp is an excellent variety because of its large size, fast growth, delicious meat, and wide temperature and salinity adaptability. However, intensive and high-density farming will inevitably cause the deterioration of the shrimp farming environment. When infected by pathogens, it is easy to cause the occurrence of various diseases. Among them, acute hepatopancreatic necrosis disease (AHPND) of shrimp is one of the most common and harmful diseases in shrimp farming. The disease is caused by Vibrio parahaemolyticus. The hepatopancreas of the affected shrimp is pale and white, atrophic and smaller, the muscles are white, the shell is soft, the jejunum is empty or the food in the intestine is discontinuous. If it is not treated promptly and effectively, acute hepatopancreatic necrosis will rapidly deteriorate, causing the shrimp hepatopancreas to lose function, and eventually leading to the disease and death of shrimp, causing serious losses to shrimp farming, and seriously restricting the development of shrimp farming. There is no safe, green and effective control method for hepatopancreatic necrosis of shrimp. Some areas use large doses of antibiotics for treatment. Since antibiotics will cause metabolic burden and secondary damage to the shrimp hepatopancreas, the treatment effect cannot reach the expected effect. Therefore, it is very necessary to develop a safe and effective green drug for the prevention and treatment of acute hepatopancreatic necrosis of shrimp.

[0004] Traditional Chinese medicine preparations are usually directly crushed or ultrafinely crushed. For directly crushed Chinese medicine, the active ingredients are still wrapped by a large amount of cell walls and cellulose components, and the active ingredients are difficult to release quickly. Therefore, the disease prevention and treatment effect is not ideal. Although ultrafine grinding destroys most of the cell walls and fiber, most of the active ingredients can be released, but the cell walls, fiber and other impurities are not removed after grinding, resulting in a low content of active ingredients. It is more difficult for farmed animals to take in enough drugs when they are sick, resulting in unsatisfactory disease prevention and treatment effects. Summary of the invention

[0005] Purpose of the Invention

[0006] The invention aims to provide a traditional Chinese medicine composition having good therapeutic effect on acute hepatopancreatic necrosis of shrimp and application of the composition in preparing a product for preventing, alleviating or treating acute hepatopancreatic necrosis of shrimp.

[0007] The Chinese medicine composition is similar to the Gandan oral liquid previously developed by the applicant. The Gandan oral liquid has the functions of clearing away heat and detoxifying, cooling blood and promoting lung function, relieving cough and relieving asthma. It is mainly used to treat lung heat cough and asthma caused by chicken infectious bronchitis and Mycoplasma gallisepticum infection, cough caused by canine infectious tracheobronchitis, and cattle lung heat cough and asthma. It mainly acts on the lungs of the respiratory organs. During the research and development process, the inventor found that it has a good therapeutic effect on preventing, alleviating or treating acute hepatopancreatic necrosis of shrimp, and can effectively improve the degree of liver damage and increase the survival rate of shrimp.

[0008] Solution

[0009] To achieve the purpose of the present invention, the technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides an application of a traditional Chinese medicine composition in the preparation of a product for preventing, alleviating or treating acute hepatopancreatic necrosis of shrimp, characterized in that the active ingredients of the traditional Chinese medicine composition include isatis root, artificial bezoar, licorice, borneol, pig gall powder and xuanming powder.

[0011] Furthermore, the active ingredients of the traditional Chinese medicine composition include the following composition in parts by weight: 80-120 parts of isatis root, 20-40 parts of artificial bezoar, 30-50 parts of licorice, 10-30 parts of borneol, 10-30 parts of pig gall powder, and 20-40 parts of Xuanming powder.

[0012] Optionally, it comprises the following active ingredients i) to iii) and pharmaceutically acceptable excipients:

[0013] i) a concentrated clear paste comprising a decoction water extract of Radix Isatidis, Radix Glycyrrhizae, and Radix Atractylodes Macrocephalae, wherein the relative density of the clear paste at a measurement temperature of 50° C. is 1.00-1.20 g / mL, and the relative density is 1.05-1.15 g / mL, and the pH of the clear paste is 6.5-7.5;

[0014] ii) an ethanol extract containing borneol and pig bile powder, wherein the amount of ethanol added is 2 to 5 times the volume of the borneol and pig bile powder;

[0015] iii) an alkaline solution containing artificial bezoar, optionally, the amount of the alkaline solution added is 2 to 5 times the weight of the artificial bezoar.

[0016] In a second aspect, the present invention provides a traditional Chinese medicine composition for preventing, alleviating or treating acute hepatopancreatic necrosis of shrimp, wherein the active ingredients of the traditional Chinese medicine composition include the following composition in parts by weight: 80-120 parts of isatis root, 20-40 parts of artificial bezoar, 30-50 parts of licorice, 10-30 parts of borneol, 10-30 parts of pig gall powder, and 20-40 parts of Xuanming powder.

[0017] In a third aspect, the present invention provides a shrimp feed for preventing, alleviating or treating acute hepatopancreatic necrosis of shrimp, comprising the Chinese medicine composition described in the second aspect.

[0018] In the first, second or third aspect above, the active ingredients of the traditional Chinese medicine composition include the following composition in parts by weight: 100-120 parts of isatis root, 34-40 parts of artificial bezoar, 40-50 parts of licorice, 20-30 parts of borneol, 20-30 parts of pig bile powder, and 30-40 parts of xuanming powder.

[0019] In the first, second or third aspect above, the active ingredients of the traditional Chinese medicine composition include the following composition in parts by weight: 100-110 parts of isatis root, 34-38 parts of artificial bezoar, 40-45 parts of licorice, 20-25 parts of borneol, 20-25 parts of pig bile powder, and 30-35 parts of xuanming powder.

[0020] In the first, second or third aspect above, the active ingredients of the traditional Chinese medicine composition include the following composition in parts by weight: 100-105 parts of isatis root, 34-36 parts of artificial bezoar, 40-42 parts of licorice, 20-22 parts of borneol, 20-22 parts of pig gall powder, and 30-32 parts of xuanming powder.

[0021] In the first, second or third aspect above, the active ingredients of the traditional Chinese medicine composition include the following composition in parts by weight: 100 parts of isatis root, 34 parts of artificial bezoar, 40 parts of licorice, 20 parts of borneol, 20 parts of pig gall powder, and 30 parts of xuanming powder.

[0022] In the first, second or third aspect above, the dosage form of the Chinese medicine composition is any one of oral liquid, powder, spray, ointment, injection and capsule.

[0023] In the first aspect above, the product is a medicine or a feed, and optionally, the feed is a shrimp feed, and optionally, the shrimp feed is a prawn feed;

[0024] In the first, second or third aspect, the acute hepatopancreatic necrosis of shrimp is caused by pathogenic bacteria, and optionally, the pathogenic bacteria is Vibrio parahaemolyticus.

[0025] In the first or third aspect above, the Chinese medicine composition accounts for 0.2%-0.5% by weight of the shrimp feed;

[0026] In the first or third aspect above, the Chinese medicine composition is an oral liquid. Optionally, the amount of the raw medicine of the Chinese medicine composition in the oral liquid is 0.1-0.8 g / mL, optionally 0.2-0.5 g / mL, optionally 0.2-0.3 g / mL, optionally 0.244 g / mL; optionally, the shrimp feed contains 2.0-5.0 mL of the oral liquid of the Chinese medicine composition / kg of shrimp feed.

[0027] In the first or third aspect above, the Chinese medicine composition is a powder. Optionally, the amount of raw herbs in the powder is 0.1-0.8 g / g powder, optionally 0.2-0.5 g / g powder, optionally 0.2-0.3 g / g powder, optionally 0.244 g / g powder; optionally, the shrimp feed contains 2.0-5.0 g of the Chinese medicine composition as powder / kg of shrimp feed.

[0028] In the above-mentioned first, second or third aspect, the Chinese medicine composition is a clear paste obtained after extraction of Chinese medicine. Optionally, the relative density of the clear paste at a measuring temperature of 50°C is 1.00-1.20 g / mL, optionally the relative density is 1.05-1.15 g / mL, optionally the pH of the clear paste is 6.5-7.5, optionally the pH of the clear paste is 7.

[0029] In the first, second or third aspect above, the Chinese medicine composition further comprises a pharmaceutically acceptable excipient; optionally, the excipient comprises one or more of propylene glycol, polysorbate 60, Tween 80, Tween 20, glycerol and water.

[0030] In the first, second or third aspect above, the dosage form of the Chinese medicine composition is an oral liquid. Optionally, the amount of crude drugs in the oral liquid is 0.1-0.8 g / mL, optionally 0.2-0.5 g / mL, optionally 0.2-0.3 g / mL, optionally 0.244 g / mL.

[0031] In the first, second or third aspect, the dosage form of the Chinese medicine composition is a powder, and the auxiliary materials in the powder further include one or more selected from glucose, skimmed milk powder, dextrin, starch, Fumei powder, corn cob powder and sodium chloride. Optionally, the amount of crude drug in the powder is 0.1-0.8g / g powder, optionally 0.2-0.5g / g powder, optionally 0.2-0.3g / g powder, and optionally 0.244g / g powder.

[0032] In the first, second or third aspect above, the dosage form of the Chinese medicine composition is an ointment, and the auxiliary materials in the ointment also include one or more selected from glucose, skimmed milk powder, dextrin, starch, fumei powder, corn cob powder, sodium chloride, gelatin and xanthan gum.

[0033] In the first, second or third aspect above, the method for preparing the composition comprises:

[0034] 1) Add water to decoct Radix Isatidis, licorice and Xuanming powder at least once, combine the decoction, filter and concentrate the filtrate;

[0035] 2) dissolving borneol and pig bile powder in ethanol and adding them to the material in step 1);

[0036] 3) dissolving the artificial bezoar with alkali solution, and adding the material of step 2) into the artificial bezoar solution;

[0037] 4) Add auxiliary material B in step 3), stir to dissolve it (dissolved means that the artificial bezoar is completely dissolved), adjust the pH value, add water, stir well, filter, and obtain a medicinal solution.

[0038] In the above preparation method, in step 1), after the filtrate is concentrated, it is cooled, an appropriate amount of ethanol is added, allowed to stand, filtered, and the ethanol is recovered from the filtrate to obtain a concentrated medicinal solution. Optionally, the amount of ethanol added is such that the volume fraction of ethanol is 55% to 70%.

[0039] In the above preparation method, in step 2), borneol and pig bile powder are dissolved and then slowly added to the material in step 1). Optionally, in step 2), 2 to 5 times the volume of ethanol is used to dissolve borneol and pig bile powder.

[0040] In the above preparation method, in step 3), the material of step 2) is slowly added to the artificial bezoar solution. Optionally, the alkali solution is a hydroxide solution, optionally a 10% hydroxide solution, optionally a 10% sodium hydroxide solution, and optionally the pH of the alkali solution is ≥11.

[0041] In the above preparation method, in step 4), the pH is 6.5-7.5, optionally the pH is 7; optionally, the auxiliary material B includes one or more of propylene glycol, polysorbate 60, Tween 80, Tween 20, glycerol and water.

[0042] Further, it also includes one of the following steps A) and B);

[0043] A) Add auxiliary material D to the material in step 4, stir evenly, dry at low temperature, and crush to obtain powder; optionally, auxiliary material D includes one or more of glucose, skimmed milk powder, dextrin, starch, fumei powder, corn cob powder and sodium chloride.

[0044] B) adding an appropriate amount of auxiliary material E to the material in step 4, stirring evenly, to obtain an ointment; optionally, auxiliary material E includes one or more of glucose, skimmed milk powder, dextrin, starch, fumei powder, corn cob powder, sodium chloride, gelatin and xanthan gum.

[0045] Beneficial Effects

[0046] (1) The Chinese medicine composition of the present invention has a good therapeutic effect on preventing, alleviating or treating acute hepatopancreatic necrosis of shrimp, with a cure rate of more than 70%. It has antibacterial and liver-protecting effects, and can effectively improve the degree of liver damage, increase the survival rate of shrimp, and reduce losses.

[0047] (2) The preparation process of the Chinese medicine composition compound preparation of the present invention adopts the method of extracting Chinese medicine before preparation. The preparation has a high content of effective ingredients, few impurities, and good palatability. It can be fully and quickly absorbed by shrimps and quickly exert its medicinal effects, thus avoiding the problem that the shrimps have a simple digestive tract structure and are difficult to directly digest and absorb the original medicinal materials.

[0048] (3) The present invention conducts in-depth research on the preparation process of different dosage forms, solves the demand for the use of different dosage forms in the breeding process, and enables the formula to be fully used in the prevention and treatment of acute hepatopancreatic necrosis of shrimp. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0050] Figure 1 The in vitro antibacterial test of Test Example 1 of the present invention, wherein A is the antibacterial effect of Example 1 (the tube numbers from left to right are: 1, 2, 3-9, 10, positive, negative), B is the antibacterial effect of povidone iodine solution (the tube numbers from left to right are: 1, 2, 3-9, 10, positive, negative), and C is the antibacterial effect of 20% ethanol solution (the tube numbers from left to right are: 1, 2, 3-9, 10, positive, negative).

[0051] Figure 2Pathological observation and results of the hepatopancreas tissue of the shrimp 24h (8th day) after the administration of 3.5.2 of Test Example 3 of the present invention, wherein, A. in the blank control group, the hepatopancreatic tubules of the shrimp were neatly arranged, with clear boundaries, and the lumen size was basically the same, the hepatopancreatic tubules were not ruptured, the nuclei were clearly visible, and there was no lesion; B. in the attack control group, the hepatopancreas was severely damaged, the hepatopancreatic tubules were disordered, the gaps were enlarged, the boundaries were blurred, the lumen became larger or disappeared, some hepatopancreatic tubules were ruptured, the cells were enlarged, the hepatopancreatic tubules were surrounded by blood cell infiltration, and some of the cells were fused into one piece; C. in the test group I, the hepatopancreas was severely damaged; D. in the test group II, the hepatopancreas was severely damaged; E. in the test group III, F. in the test group IV, G. in the test group V, and H. in the test group VI, the hepatopancreas tissues were normal, and there was no obvious lesion.

[0052] Figure 3 Pathological observation and results of the hepatopancreatic tissue of the shrimp 7 days after drug withdrawal (14th day) of 3.5.2 of Test Example 3 of the present invention, wherein, A. in the blank control group, the hepatopancreatic tubules of the shrimp were neatly arranged, with clear boundaries, and the lumen size was basically the same, the hepatic tubules were not ruptured, the cell nuclei were clearly visible, and there was no lesion; B. in the attack control group, the hepatopancreas was severely damaged; C. in the test group I, the hepatopancreas damage was alleviated to a certain extent, and D. in the test group II, the hepatopancreas damage was alleviated to a certain extent; E. in the test group III, F. in the test group IV, G. in the test group V, and H. in the test group VI, the hepatopancreas tissues were normal, and there was no obvious lesion. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0055] Example 1

[0056] 100g of Isatis root, 34g of artificial bezoar, 40g of licorice, 20g of borneol, 20g of pig bile powder and 30g of Xuanming powder.

[0057] The preparation process of the compound preparation of the present embodiment is as follows: Radix Isatidis, Radix Glycyrrhizae, and Powder of Xuanming are decocted with water for 3 times, the decoctions are combined, filtered, and the filtrate is concentrated into a clear paste with a density of 1.15-1.20 (measured at 50° C.), cooled, and 2 times the volume of ethanol (so that the alcohol content reaches 55%-70%) is added, allowed to stand, filtered, and the filtrate is recovered. The ethanol is obtained and set aside; borneol and pig gall powder are added with 2 times the volume of ethanol to dissolve them, and slowly added to the above-mentioned liquid medicine for use; artificial bezoar is treated with an appropriate amount of alkali solution (2 ~5 times the weight of alkali solution can be used, for example, in this embodiment, 2 times the weight of 10% sodium hydroxide solution is added) to dissolve, and the above combined solution is slowly added thereto, and then appropriate amounts of propylene glycol and polysorbate 60 are added (for example, in this embodiment, the amount added is 2% of the weight of the medicinal solution), stirred to dissolve, the pH value is adjusted to about 7.0, water is added to 1000 ml, stirred well, and filtered to obtain an oral solution, the amount of crude drug is 0.244 g / mL, wherein about 10 to 20% of ethanol remains in the oral solution.

[0058] Example 2

[0059] The difference between this embodiment and embodiment 1 is that 2 times the weight of starch and glucose are added to the prepared clear paste, the ratio of starch to glucose is 1:1, the mixture is stirred evenly, dried at low temperature, crushed, the weight is supplemented to 1000 g with starch and glucose, and the mixture is mixed evenly to obtain a powder, and the amount of crude drug is 0.244 g / g powder.

[0060] Example 3

[0061] The difference between this embodiment and embodiment 1 is that the ratio of raw materials is different: 105g of isatis root, 36g of artificial bezoar, 42g of liquorice, 22g of borneol, 22g of pig gall powder and 32g of Xuanming powder.

[0062] Example 4

[0063] The difference between this embodiment and embodiment 1 is that the ratio of the raw materials is different: 100 g of isatis root, 38 g of artificial bezoar, 40 g of licorice, 25 g of borneol, 20 g of pig gall powder and 35 g of Xuanming powder.

[0064] Example 5

[0065] The difference between this embodiment and embodiment 1 is that the ratio of the raw materials is different: 110 g of isatis root, 34 g of artificial bezoar, 45 g of licorice, 20 g of borneol, 25 g of pig gall powder and 30 g of Xuanming powder.

[0066] Comparative Example 1

[0067] This comparative example is a single dose of Radix Isatidis oral solution.

[0068] Preparation method: decocted 1000g of Radix Isatidis with water for 3 times, combined the decoctions, filtered, concentrated the filtrate into a clear paste with a density of 1.15-1.20 (measured at 50°C), cooled, added 2 times the volume of ethanol, allowed to stand, filtered, recovered ethanol from the filtrate to obtain a medicinal solution, added appropriate amounts of propylene glycol and polysorbate 60 (for example, the amount added in this comparative example is 2% of the weight of the medicinal solution), stirred to dissolve, adjusted the pH value to about 7.0, added water to 1000ml, stirred well, filtered, and obtained the Radix Isatidis oral solution, the crude drug amount being 1g / mL.

[0069] Comparative Example 2

[0070] This comparative example is a single dose of artificial bezoar, which is obtained by directly crushing the artificial bezoar.

[0071] Comparative Example 3

[0072] This comparative example is a single-dose oral liquorice solution. The preparation method is similar to that of comparative example 1. The crude drug amount of the liquorice oral solution is 1 g / mL.

[0073] Comparative Example 4

[0074] This comparative example is a single dose of borneol, which is prepared by directly crushing the borneol.

[0075] Comparative Example 5

[0076] This comparative example is a single dose of pig bile powder, which is obtained by directly crushing the pig bile powder.

[0077] Comparative Example 6

[0078] This comparative example is a single dose of Xuanming powder, which is obtained by directly crushing the Xuanming powder.

[0079] Comparative Example 7

[0080] The difference between this embodiment and embodiment 1 is that Radix Isatidis is omitted.

[0081] Comparative Example 8

[0082] The difference between this embodiment and embodiment 1 is that artificial bezoar is omitted.

[0083] Comparative Example 9

[0084] The difference between this embodiment and embodiment 1 is that liquorice is omitted.

[0085] Comparative Example 10

[0086] The difference between this embodiment and embodiment 1 is that borneol is omitted.

[0087] Comparative Example 11

[0088] The difference between this embodiment and embodiment 1 is that the pig bile powder is omitted.

[0089] Comparative Example 12

[0090] The difference between this embodiment and embodiment 1 is that the Xuanming powder is omitted.

[0091] In the following test examples, pathogen detection was performed using a Vibrio parahaemolyticus biochemical identification kit.

[0092] Test Example 1: In vitro antibacterial test report of the agent of Example 1

[0093] 1.1 Materials and reagents

[0094] Test bacteria: The Vibrio parahaemolyticus used in the test was obtained from the China Marine Microbial Culture Collection Center.

[0095] Test sample: Example 1.

[0096] Control drug: povidone-iodine solution, specification: 10% povidone-iodine, commercially available.

[0097] 2216E liquid medium, 2216E agar medium, 0.03M pH 7.2 phosphate buffer, and sterile distilled water were purchased commercially.

[0098] 1.2 Test methods

[0099] 1.2.1 Preparation of test bacterial solution

[0100] After the freeze-dried strains of Vibrio parahaemolyticus were opened, they were enriched and cultured in 2216E liquid medium and then inoculated on 2216E agar medium plates for streak culture. After overnight culture at 28°C, typical single colonies were selected and inoculated into 2216E liquid medium for culture. After culture at 28°C for 18 to 24 hours, the viable bacteria were counted and then diluted with 0.03M pH 7.2 phosphate buffer to a bacterial count of 1×10 5 ~1×10 6 CFU / ml, as the test bacterial solution.

[0101] 1.2.2 Minimum inhibitory concentration (MIC) determination

[0102] 1.2.2.1 Determination of the minimum inhibitory concentration (MIC) of the agent in Example 1: Take 12 sterile test tubes, add 2 ml of 2216E liquid medium to each tube, aseptically pipette 2 ml of the agent in Example 1 after filtration and sterilization and add it to the first tube, mix well and pipette 2 ml from the first tube and add it to the second tube, dilute it to the tenth tube in multiple proportions, mix well and discard 2 ml from the tenth tube, do not add the agent to the 11th and 12th tubes, use a micropipette to pipette 0.1 ml of the test bacterial solution and add it to the first to 11th tubes, the 12th tube is a negative control, mix well and place it in a 28°C incubator for 18 to 24 hours, and observe the results. The results are determined by naked eye observation, and the tube with the lowest drug concentration and no bacterial growth is the minimum inhibitory concentration (MIC) of the test bacteria.

[0103] 1.2.2.2 Determination of minimum inhibitory concentration (MIC) of povidone-iodine solution: Take 12 sterile test tubes and treat povidone-iodine solution according to the method in 2.2.1 and observe the results.

[0104] 1.2.2.3 Determination of the minimum inhibitory concentration (MIC) of 20% ethanol solution: Take 12 sterile test tubes and treat 20% ethanol solution according to the method in 2.2.1 and observe the results.

[0105] 1.2.3 Minimum bactericidal concentration (MBC) determination

[0106] Inoculate the liquid culture medium in the clarified test tube during the MIC determination in 1.2.2 onto a 2216E agar plate, mark it, and culture it at 28°C for 18 to 24 hours. Observe the results and take the drug concentration in the test tube with sterile growth on the plate as the minimum bactericidal concentration of the drug.

[0107] 1.3 Test results

[0108] 1.3.1 Minimum inhibitory concentration (MIC) test results

[0109] After culturing in a 28°C incubator for 24 hours, the test tube was taken out and the antibacterial effect of Example 1 and povidone-iodine solution was observed with the naked eye to judge the bacterial growth. The results showed that for Vibrio parahaemolyticus, the MIC of Example 1 was 1 / 4, and the MIC of povidone-iodine solution was 1 / 16. 20% ethanol solution had no inhibitory effect on Vibrio parahaemolyticus. See Table 1 and Figure 1 .

[0110] Table 1 Minimum inhibitory concentration (MIC) test results of Example 1

[0111]

[0112] Note: “-” indicates that the liquid in the test tube is clear, and “+” indicates that the liquid in the test tube is turbid.

[0113] 1.3.2 Minimum bactericidal concentration (MBC) test results

[0114] After culturing at 28°C for 24 hours, the plate was taken out for naked eye observation. For Example 1, its MBC against Vibrio parahaemolyticus was 1 / 2, and for the povidone-iodine solution, its MBC against Vibrio parahaemolyticus was 1 / 8. The ethanol solution had no killing effect on Vibrio parahaemolyticus at the test concentration. See Table 2.

[0115] Table 2 Example 1 Minimum bactericidal concentration (MBC) test results

[0116]

[0117] Note: “-” indicates no bacterial growth, “+” indicates bacterial growth.

[0118] The results of Table 1 and Table 2 show that the minimum inhibitory concentration (MIC) of Example 1 against Vibrio parahaemolyticus is 1 / 4, and the minimum bactericidal concentration (MBC) is 1 / 2, while ethanol is only used as a cosolvent in Example 1 and has no effect on the in vitro bactericidal effect of Example 1. The MIC of the control drug povidone-iodine solution is 1 / 16, and the minimum bactericidal concentration (MBC) is 1 / 8, indicating that Example 1 has a certain inhibitory effect on Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis of shrimp, but the effect is not as good as the control drug povidone-iodine solution.

[0119] Test Example 2: The therapeutic effects of different agents on acute hepatopancreatic necrosis of shrimp caused by Vibrio parahaemolyticus

[0120] In order to further study the therapeutic effects of different agents on acute hepatopancreatic necrosis of shrimp caused by Vibrio parahaemolyticus, the agents of the embodiments and comparative examples were tested.

[0121] 2.1 Experimental materials and feeding management

[0122] 2.1.1 Test instruments and reagents

[0123] HX-2000 portable multi-parameter water quality analyzer; BS-180 fully automatic biochemical analyzer; TSJ-1C vacuum automatic tissue dehydrator; BMJ-1B biological tissue embedding machine system; HM325 manual rotary slicer; KPJ-1A slice baking machine; ZPJ-1A slide unfolding machine; LVG-5AG-F8 clean workbench; YXLB-LS-100Sll high-pressure steam sterilizer; ZWY-2HC constant temperature oscillator; BIC-300 biochemical incubator; 2216E liquid culture medium; TCBS agar; hematoxylin stain; slice paraffin; alanine aminotransferase (ALT) assay kit; aspartate aminotransferase (AST) assay kit; 3% sodium chloride trisaccharide iron (TSI) agar; Vibrio parahaemolyticus biochemical identification kit; Gram staining solution kit; peptone; sodium chloride; tryptone; soy peptone; potassium permanganate, etc., are all commercially available.

[0124] 2.1.2 Experimental animals

[0125] 1500 shrimps were obtained from a shrimp farming company in Caofeidian, Tangshan. They were strong and artificially cultured. Before the experiment, the shrimps were temporarily raised for 1 week and fed with ordinary shrimp pellet feed during the whole experiment. The feed was purchased from a feed company in Shandong, 20kg / bag.

[0126] 2.1.3 Test strains

[0127] The Vibrio parahaemolyticus used in the experiment was from the China Marine Microbial Culture Collection Center.

[0128] Preparation of challenge solution: Prepare 2216E liquid culture medium, fill 500mL Erlenmeyer flask with 20% liquid volume, seal with sterile breathable membrane, sterilize at 121℃ for 15 minutes, cool to room temperature, inoculate 1mL of activated and rejuvenated Vibrio parahaemolyticus seeds, culture at 28℃, 140r / min, for 18-24h to obtain the initial bacterial solution.

[0129] Preparation of bacterial suspension ① Centrifuge the initial bacterial solution at 4000r / min for 10min; ② Discard the supernatant and rinse the precipitate with sterile saline; ③ Repeat steps ①-② twice; ④ Use sterile saline to make up to the original volume; ⑤ Use the pour plate method to count the viable bacteria of the prepared bacterial suspension; ⑥ Use sterile saline to dilute the bacterial suspension to the corresponding concentration according to the viable bacteria count result, and set aside.

[0130] 2.1.4 Breeding environment

[0131] The test was conducted in a 150L aquarium of the same specifications. The aquarium was equipped with an independent self-cleaning system, and the filter cotton and filter material were replaced every 3 days. Potassium permanganate was used for disinfection before the test, and self-prepared seawater (seawater was prepared with sea crystal and tap water, and the salinity of seawater was adjusted with a salinometer) was added after thorough cleaning, and ventilation was carried out for 3 days. The water quality of each aquarium was monitored daily, and the water temperature was controlled at 27±2℃ with a heating rod; the dissolved oxygen was controlled to be not less than 5.0mg / L with an aeration pump; sodium bicarbonate and hydrochloric acid were used to adjust the pH to 7.8-8.2; and the salinity of seawater was controlled to 32±2‰. Sea crystal was used to prepare seawater.

[0132] 2.2 Test methods and results

[0133] 2.2.1 Modeling dose confirmation

[0134] 1.0×10 5 cfu / mL bacterial solution, and the injection volume of 0.1mL is the best attack dose.

[0135] 2.2.2 Trial Grouping and Infection Scheme

[0136] Temporary care method: Select shrimps of uniform size, strong physique, active feeding, and no disease or injury, and place them in temporary culture for one week (feeding ordinary shrimp pellet feed). Feed them continuously at 8:30, 12:30, 16:30, and 20:30 every day. After statistics and calculations during the temporary culture period, the daily feed amount is 5% of the shrimp's body weight, which is more appropriate. At this time, the shrimp can finish eating in about 40-60 minutes.

[0137] Trial Groups : After the experimental shrimps were temporarily raised for one week, they were randomly divided into groups, including a blank control group, a challenge control group and each experimental group (a group fed with the pharmaceutical composition of the embodiment or the comparative example).

[0138] Infection Scheme Except for the blank control group, the challenge control group and each experimental group were artificially infected by intramuscular injection of Vibrio parahaemolyticus solution on the lateral side of the second and third abdominal segments of shrimp. The concentration of the solution was 1.0×10 5 cfu / mL, the injection dose was 0.1mL / tail, and the blank control group was injected with the same dose of sterile saline.

[0139] Feeding method after challenge : 4 hours after the challenge, each group was fed with the corresponding feed, and fed once a day at 8:30, 12:30, 16:30, and 20:30 respectively. The daily feed amount was 5% of the shrimp body weight for 7 consecutive days. After stopping the drug, the shrimps were observed for 1 week and fed with ordinary feed during the observation week.

[0140] Preparation method of each group of baits in the drug administration stage:

[0141] Feed for the blank control group: Take 50mL of purified water, add 10g of flour, heat and stir until viscous, stir evenly, add to 1kg of feed, stir thoroughly, spread on plastic paper, air-dry at room temperature, bag and seal for storage.

[0142] The feed fed to the challenge control group was the same as that of the blank control group.

[0143] The experimental groups were fed Bait Take 50mL of purified water, add 10g of flour, heat and stir until viscous, add the medicine of each test group respectively, stir evenly, add to 1kg of feed respectively, stir thoroughly, spread it on plastic paper, air-dry at room temperature, bag and seal for storage, to prepare the test group feed.

[0144] Feeding method :

[0145] Turn off the self-cleaning system of the fish tank before feeding. After 1 hour of feeding, turn on the self-cleaning system to remove the remaining bait and feces.

[0146] 2.2.3 General clinical observation

[0147] During the experiment, the appearance, swimming, feeding, morbidity and mortality of the shrimps were observed and recorded daily.

[0148] 2.2.4 Efficacy evaluation

[0149] Observe the color and size of the shrimp hepatopancreas, the fullness of food in the gastrointestinal tract, etc., and use the typical symptoms of acute hepatopancreatic necrosis in shrimp as the standard to judge whether the shrimp is sick. The symptom observation standard is mainly formulated with reference to SC / T-7233-2020 "Diagnostic Procedure for Acute Hepatopancreatic Necrosis". Refer to Table 3 for judgment on the onset, cure (normal), and infection and death. On the 8th day, the incidence, cure rate and infection mortality rate were calculated, and the specific calculation formula is as follows:

[0150] Incidence rate (%) = number of diseased shrimps / number of tested shrimps × 100%.

[0151] Cure rate (%) = number of cured shrimps / number of tested shrimps × 100%.

[0152] Infection mortality (%) = number of infected and dead shrimps / number of tested shrimps × 100%.

[0153] Table 3 Shrimp disease determination table

[0154]

[0155]

[0156] Microsoft Excel was used to manage the experimental data, SPSS20.0 statistical software was used for data processing, and the "Duncan (D)" method under one-way analysis of variance (ANOVA) was used to perform statistical analysis on the experimental results. The data analysis results showed that P < 0.05 was statistically significant.

[0157] 2.3 Prescription screening

[0158] Experimental grouping: After the experimental shrimps were temporarily raised for one week, 320 shrimps were selected and randomly divided into 16 groups, including a blank control group, a challenge control group and various experimental groups, with 20 shrimps in each group. The infection method adopted the infection scheme of 2.2.2.

[0159] The amount of the drug added to the batter of 10g flour / 50mL purified water in each test group is:

[0160] The medicines of Example 1, Comparative Example 1, Comparative Example 3, and Comparative Examples 7 to 12 are oral liquids, and the added amount is 2 mL respectively.

[0161] Example 2, Comparative Example 2, and Comparative Examples 4 to 6 are powders, and the added amount is 2 g respectively.

[0162] Refer to the feeding method and feeding method after the virus attack in 2.2.2 for feeding. The treatment conditions of each group are shown in Table 4.

[0163] Table 4 Experimental groups and treatment methods of each group

[0164]

[0165] Clinical observations

[0166] During the entire breeding period, the water quality is stable and the water temperature is controlled at: 27±2℃; dissolved oxygen ≥5.0mg / L; pH7.8-8.2; salinity: 30-35‰.

[0167] Before attack During the entire experimental period, there was no abnormality in the activities of the shrimps in the blank control group. The shrimps swam normally, ate well, had hard shells, full food in the gastrointestinal tract, clean and translucent bodies, brown hepatopancreas, normal size, clear edges, and no whitish body color. When touched, they reacted obviously and jumped to avoid the attack immediately.

[0168] After attack :

[0169] The shrimps in each infection test group gradually showed symptoms such as inactive swimming and decreased appetite. 12 hours after infection, the shrimps' bodies turned turbid, the hepatopancreas became lighter and whiter, the hepatopancreas of some shrimps atrophied, and food in the intestines was discontinuous. The peak of the disease was reached 24 hours after infection.

[0170] The toxic control group and each infected and drug-administered group died one after another after infection, with the highest number of deaths occurring in the 2nd and 3rd days. The symptoms of some groups improved after drug administration. After 7 days of drug administration, the symptoms of many groups of sick shrimps improved significantly, and even returned to normal. Since the toxic control group was not treated, the symptoms of the shrimp did not improve.

[0171] 2.3.2 Autopsy observations and results

[0172] Observation method: During the experiment, the death of shrimps was observed and recorded, and the dead shrimps were dissected to check the organ lesions and isolate the pathogens. At the end of the experiment, the surviving shrimps in each group were dissected and observed, and the hepatopancreas was observed with the naked eye for any abnormalities and recorded.

[0173] result : An autopsy of the dead shrimp revealed that the shrimp's body color turned white, the hepatopancreas turned lighter and whiter, and it shrank and became smaller.

[0174] Initial screening efficacy assessment

[0175] 24h after the end of administration (8th day), the cure status of shrimps in each group was calculated according to the efficacy judgment method in 2.2.4, see Table 5.

[0176] Table 5 The cure of shrimps in each group 24h after the end of administration (8th day)

[0177]

[0178]

[0179] As shown in Table 5, no cured shrimp appeared in the challenge control group, and the cure rate was 0%. No diseased shrimp appeared in the blank control group, and there was no disease or death during the entire test. After the challenge of each drug administration group, all shrimps showed symptoms of disease. As the drug administration progressed, the symptoms of the shrimps in each test group improved. After 7 days of administration, some shrimps were completely cured and the symptoms disappeared. Among them, Examples 1 and 2 had good therapeutic effects, with a cure rate of more than 80%. The therapeutic effects of the single doses or formulations of other comparative examples were worse than those of Examples 1 and 2, with a cure rate of 25-70%.

[0180] Although some components such as licorice and isatis root have some reports on liver protection and antibacterial properties, their therapeutic effects on acute hepatopancreatic necrosis of shrimp are uneven (for example, although comparative examples 8 and 11 both contain licorice and isatis root, the therapeutic effects are not ideal), and according to the formulations of Examples 1 to 5, the therapeutic effect on acute hepatopancreatic necrosis of shrimp is significantly improved, with a cure rate of more than 80%, and some even more than 90%, achieving unexpected good results.

[0181] In order to further study the therapeutic effect of the formula of the present invention on acute hepatopancreatic necrosis of shrimp, the inventors conducted further tests as follows:

[0182] Test Example 3: The therapeutic effect of the agent of Example 1 on acute hepatopancreatic necrosis of shrimp caused by Vibrio parahaemolyticus

[0183] The therapeutic effect of Example 1 on acute hepatopancreatic necrosis of shrimp caused by Vibrio parahaemolyticus was observed to clarify the liver protection function of Example 1.

[0184] The experimental materials and feeding management of 2.1 and 2.2 were adopted.

[0185] Trial Groups: After the experimental shrimps were temporarily raised for one week, 480 shrimps were selected and randomly divided into 8 groups, including blank control group, challenge control group, experimental groups I, II, III, IV, V, and VI. Each group had 3 parallel groups, each parallel had 1 experimental tank, and each tank had 20 shrimps. The infection method adopted the infection scheme of 2.2.2.

[0186] The amount of the drug added to the batter of 10g flour / 50mL purified water in each test group is:

[0187] The agent of Example 1 was added to the batter (10g flour / 50mL purified water) in an amount of 0.5mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, and 5.0mL, respectively, to obtain feed containing 0.5mL / kg of the agent of Example 1, 1.0mL / kg of the agent of Example 1, 2.0mL / kg of the agent of Example 1, 3.0mL / kg of the agent of Example 1, 4.0mL / kg of the agent of Example 1, and 5.0mL / kg of the agent of Example 1, respectively.

[0188] Refer to the feeding method and feeding method after the virus attack in 2.2.2 for feeding. The treatment conditions of each group are shown in Table 6.

[0189] Table 6 Experimental groups and treatment methods of each group

[0190]

[0191] 3.1 General clinical observations

[0192] During the entire breeding period, the water quality is stable and the water temperature is controlled at: 27±2℃; dissolved oxygen ≥5.0mg / L; pH7.8-8.2; salinity: 30-35‰.

[0193] Clinical observation During the experiment, the appearance, swimming, feeding, death time and number of shrimps were observed and recorded daily (no obvious reaction when touched).

[0194] Observations

[0195] Before challenge: During the entire test period, there was no abnormality in the activities of the shrimps in the blank control group. The shrimps swam normally, ate well, had hard shells, full food in the gastrointestinal tract, clean and translucent bodies, brown hepatopancreas, normal size, clear edges, no whitish body color, etc. They reacted obviously when touched and immediately jumped to avoid the attack.

[0196] After attack :

[0197] The shrimps in each infection test group gradually showed symptoms such as inactive swimming and decreased appetite. 12 hours after infection, the shrimps' bodies turned turbid, the hepatopancreas became lighter and whiter, the hepatopancreas of some shrimps atrophied, and food in the intestines was discontinuous. The peak of the disease was reached 24 hours after infection.

[0198] The control group and each infected and drug-administered group died one after another after infection, with the highest number of deaths on days 2-3. Among them, the number of deaths in the four groups of experiments III-VI was relatively small, and gradually improved after 3 days of drug administration. On the 7th day of the experiment, most of the surviving shrimps had returned to normal. The disease conditions of the control group, experiment I and II were more serious, and the symptoms of most shrimps did not improve.

[0199] After 7 days of drug withdrawal (14th day), the symptoms of groups I and II improved to some extent, and groups III to VI basically returned to normal. The symptoms of the control group did not improve.

[0200] 3.2 Autopsy observations and results

[0201] Observation method: During the experiment, the death of shrimps was observed and recorded, and the dead shrimps were dissected to check the organ lesions and isolate the pathogens. At the end of the experiment, the surviving shrimps in each group were dissected and observed, and the hepatopancreas was observed with the naked eye for any abnormalities and recorded.

[0202] result : The dead shrimps were dissected and found to be whitish in color, and the hepatopancreas became lighter and whiter, and shrunken and smaller. After the experiment, some shrimps in the control group, test groups I and II still had pathological changes, while the shrimps in the other groups had translucent bodies, full intestines, and normal color and shape of the hepatopancreas.

[0203] 3.3 Morbidity results

[0204] The incidence of shrimp in each group 24h after the end of administration (8th day) is shown in Table 7: the incidence of the blank control group was 0, and the incidence of the attack control group was as high as 100%. The incidence of other treatment groups Ⅰ-Ⅵ was significantly lower than that of the attack control group (P<0.05), and with the increase of the dosage, the incidence showed a significant downward trend. Among them, the incidence of experimental groups Ⅰ and Ⅱ was still high, 73.33% and 66.67% respectively, and the difference between the two was not significant (P>0.05); the incidence of experimental groups Ⅲ-Ⅵ was 18.33%, 16.67%, 15.00%, and 13.33% respectively, which was significantly lower than that of experimental groups Ⅰ and Ⅱ (P<0.05), but the difference in incidence among the four groups Ⅲ-Ⅵ was not significant (P>0.05), and the incidence was low.

[0205] Table 7 Disease status of shrimps in each group 24h after administration (8th day)

[0206]

[0207]

[0208] 3.4 Results of cure rate and infection mortality rate

[0209] The cure status of shrimps in each group 24 hours after the end of administration (8th day) is shown in Table 8, and the death status of shrimps in each group is shown in Table 9.

[0210] Table 8 Healing status of shrimps in each group 24h after administration (8th day)

[0211]

[0212] The results in Table 8 show that no cured shrimp appeared in the challenge control group, and the cure rate was 0%. The cure rates of each drug administration group increased significantly with the increase of the dosage, and the cure rates of the test groups Ⅰ-Ⅵ were 26.67%, 33.33%, 81.67%, 83.33%, 85.00%, and 86.67%, respectively. Among them, the cure rates of the test groups Ⅲ-Ⅵ were relatively high, above 80%, and the cure rates of the test groups Ⅰ and Ⅱ were only about 30%, which was significantly lower than that of the four test groups Ⅲ-Ⅵ (P<0.05).

[0213] Table 9 Death of shrimps in each group

[0214]

[0215] The results in Table 9 show that there was no death in the blank control group during the entire experiment; the infection mortality rate of the attack control group was 46.67%. Compared with the attack control group, the infection mortality rate of each drug-administered group was reduced, and with the increase in the dosage, the mortality rate showed a significant downward trend. Among them, the infection mortality rate of the experimental groups III-VI was significantly lower than that of the attack control group (P<0.05), and the infection mortality rates of the four groups were 11.67%, 11.67%, 10.00%, and 10.00%, respectively. The mortality rates of the experimental groups I and II were 40.00% and 36.67%, respectively, which were lower than those of the attack control group, but the difference was not significant (P>0.05). The pathogen detection results of the shrimps that died in each group during the experiment were all positive for Vibrio parahaemolyticus.

[0216] 3.5 Hemolymph biochemical index detection and pathological observation

[0217] 24 h after the end of drug administration (Day 8) and 7 days after drug withdrawal (Day 14), 9 shrimps were randomly selected from each group (i.e., 3 shrimps were randomly selected from each tank each time), and hemolymph was collected for biochemical detection and hepatopancreatic tissue was fixed for pathological observation.

[0218] 3.5.1 Hemolymph biochemical index detection and results

[0219] Hemolymph biochemical index detection:Hemolymph was collected from the pericardial cavity with a syringe, and anticoagulated blood was collected in a vacuum blood collection tube containing anticoagulant, centrifuged, and the supernatant was separated. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using an automatic biochemical analyzer.

[0220] Hemolymph biochemical index test results : The ALT content of hemolymph of shrimps in each group is shown in Table 10, and the ALT content of hemolymph of shrimps in each group is shown in Table 11.

[0221] Table 10 ALT content in hemolymph of shrimps in each group (n=9)

[0222]

[0223] Note: Data in the same column with different lowercase letters indicate significant differences (P<0.05), while data with the same letters or no letters indicate no significant differences (P>0.05).

[0224] The results of the ALT content in the hemolymph of shrimps in Table 10 show that: compared with the blank control group, the ALT content in the hemolymph of shrimps in the attack control group increased significantly, indicating that after the attack, the hepatopancreatic tissue of shrimps was damaged. Compared with the attack control group, the ALT content of each medication group showed a downward trend, and the downward trend became more obvious with the increase of the dosage. Among them, the ALT content of the four groups of experiments III-VI was significantly lower than that of the attack control group (P<0.05), and there was no significant difference with the blank control group (P>0.05), indicating that after 7 days of administration of Example 1, the hepatopancreatic damage was significantly improved and close to that of healthy shrimps. The content of the experiment groups I and II was not significantly different from that of the attack control group (P>0.05), and was significantly higher than that of the blank control group, indicating that the hepatopancreatic damage of this prescription was still serious when the medicine was used at a low dose.

[0225] Table 11 AST content in hemolymph of shrimps in each group (n=9)

[0226]

[0227] Note: Data in the same column with different lowercase letters indicate significant differences (P<0.05), while data with the same letters or no letters indicate no significant differences (P>0.05).

[0228] The results of shrimp hemolymph AST content in Table 11 show that the change trend of shrimp hemolymph AST content is basically consistent with ALT. The content of test groups Ⅰ and Ⅱ is significantly higher than that of the blank control group (P<0.05), and there is no significant difference with the attack control group (P>0.05). The four groups of test Ⅲ-Ⅵ are not significantly different from the blank control group (P>0.05), and are significantly lower than the attack control group (P<0.05). With the increase of the dosage, the AST content of diseased shrimp hemolymph decreased significantly. The content of the four groups of test Ⅲ-Ⅵ is significantly lower than that of test groups Ⅰ and Ⅱ (P<0.05), and there is no significant difference among the four groups (P>0.05).

[0229] The results of the biochemical index test of shrimp hemolymph in each group showed that the addition of 2.0-5.0 mL / kg of feed containing the agent of Example 1 could inhibit the abnormal increase of ALT and AST caused by hepatopancreatic damage in diseased shrimp and improve the degree of liver damage.

[0230] 3.5.2 Hepatopancreatic tissue pathological observation and results

[0231] Methods for Observation of Hepatopancreas Histopathology : The hepatopancreatic tissues were fixed, paraffin sections were made, HE staining was performed, and the sections were sealed with neutral gum and observed under a microscope to detect the pathological changes of the hepatopancreas.

[0232] Pathological observation of hepatopancreas tissue The pathological results 24 hours after the end of drug administration (Day 8) and 7 days after drug withdrawal (Day 14) are as follows Figure 2 , Figure 3 shown.

[0233] Figure 2 Middle: In the blank control group, the hepatopancreatic tubules of shrimp were arranged neatly, with clear boundaries and basically the same lumen size. The hepatopancreatic tubules were not ruptured, the cell nuclei were clearly visible, and there were no lesions. The hepatopancreas of the drug-treated control group, test groups I and II were damaged to varying degrees, among which the drug-treated control group was the most serious: the hepatopancreatic tubules were disordered, the gaps were enlarged, the boundaries were blurred, the lumens were enlarged or disappeared, some hepatopancreatic tubules were ruptured, the cells were swollen, the hepatopancreatic tubules were surrounded by infiltrated blood cells, and some cells were fused into one piece. The hepatopancreatic tissues of the four test groups III-VI were normal, without obvious lesions.

[0234] Figure 3 In the experiment, the hepatopancreas of shrimp in the control group was still lesioned and more serious. The hepatopancreas lesions in groups I and II were alleviated to a certain extent, but not completely recovered. The hepatopancreas tissues of other groups were normal.

[0235] In summary, after artificial infection with pathogenic Vibrio parahaemolyticus causing acute hepatopancreatic necrosis of shrimp, the AST and ALT contents of hemolymph of shrimp increased significantly after the attack (P<0.05), and the feed containing different doses (especially 2.0-5.0mL / kg) of Example 1 was fed for 7 consecutive days, which could improve the clinical symptoms of the diseased shrimp, especially the 2.0-5.0mL / kg dose could significantly inhibit the abnormal increase of ALT and AST contents in hemolymph caused by hepatopancreatic damage (P<0.05), repair hepatopancreatic damage, improve the damage of hepatopancreatic cells, embody a good liver protection function, reduce the incidence and infection mortality of shrimp, effectively reduce the infection mortality to less than 12%, have a higher cure rate, and the cure rate can reach more than 80%, which has a good therapeutic effect on acute hepatopancreatic necrosis of shrimp. Among them, the effects of the four groups of experiments III-VI were significantly better than those of the two groups of experiments I and II, and the therapeutic effects of the four groups of experiments III-VI were basically equivalent.

[0236] It shows that Example 1 has good liver protection function. When added to feed at a dose of 2.0-5.0 mL / kg feed, it has significant therapeutic effect on acute hepatopancreatic necrosis of shrimp caused by Vibrio parahaemolyticus infection, with a cure rate of more than 80%.

[0237] Test Example 4: Test report on the treatment of shrimp hepatopancreatic necrosis during the breeding process using various embodiments of the present invention

[0238] Some shrimps in 8 ponds of a shrimp farm in Tianjin showed typical symptoms of acute hepatopancreatic necrosis, such as pale white hepatopancreas, shrinkage and shrinkage. Nine ponds of a shrimp farm in Weifang, Shandong showed the same symptoms. Five shrimps with typical clinical symptoms were randomly selected from each pond for pathogen detection. The results were all positive for Vibrio parahaemolyticus. Therefore, it can be confirmed that these 17 ponds have all suffered from acute hepatopancreatic necrosis of shrimp caused by Vibrio parahaemolyticus, and can be used as candidate test ponds. Subsequently, 30 shrimps were randomly sampled in the confirmed ponds, and the diseased shrimps were counted and the sampling incidence rate was calculated. The incidence rate in some ponds was high, and large-scale deaths occurred, indicating that the disease is difficult to heal without effective intervention, the mortality rate is high, and the economic losses are large.

[0239] In the above-mentioned shrimp farm, daily regular maintenance includes: water adjustment, bottom modification and disinfection. Despite daily disinfection, shrimp hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus still occurred.

[0240] Each experimental base selected 6 ponds with similar morbidity rates for treatment trials. After selection, the morbidity rates of each pond included were between 33% and 50%, as shown in Table 12 below. Clinical symptoms of diseased shrimps all showed that the hepatopancreas was pale, shrunken and reduced in size.

[0241] Table 12 Detection results of acute hepatopancreatic necrosis of shrimp in each test pond before medication

[0242]

[0243]

[0244] Morbidity results after medication :

[0245] The medicaments of Examples 1 and 2 were added to the feed of the shrimps in the above diseased ponds to prepare feed containing the medicaments. The addition method of Example 1 was referred to the method of 2.2.2 of Test Example 2, and the addition amount in the feed was 2.0 mL / kg, which was used for 7 consecutive days; the medicament of Example 2 was a powder, which was directly added to the feed and stirred and mixed, and the addition amount of the medicament of Example 2 in the feed was 2 g / kg, which was used for 7 consecutive days.

[0246] After a period of medication, the results of the morbidity of each test pond are shown in Tables 13 and 14 below: On the 8th day of medication, the random inspection morbidity of the group using the agent of Example 1 and the group using the agent of Example 2 in the two farm test bases were significantly reduced compared with before medication (P<0.05), and decreased by about 30%, indicating that both Example 1 and Example 2 showed good therapeutic effects. The random inspection morbidity was further reduced on the 14th day, but the difference was not significant compared with the eighth day (P>0.05), indicating. The random inspection morbidity of the pond group using the agent of Example 2 before medication, on the 8th day and on the 14th day in the Tianjin farm was 41.11%, 10.00% and 6.67% respectively; the morbidity of the group of Example 1 was 40.00%, 11.11% and 7.78% respectively. The morbidity rates of Example 2 group in Shandong breeding farm before medication, on the 8th day and on the 14th day were 42.22%, 8.89% and 5.56% respectively; the morbidity rates of Example 1 group were 41.11%, 10.00% and 7.78% respectively.

[0247] Table 13 Incidence of shrimp sampled in Tianjin test pond (%)

[0248]

[0249] Note: When comparing data of the same industry, the same lowercase letters or no letters in the upper right corner indicate no significant difference (P>0.05), while different lowercase letters indicate significant difference (P<0.05).

[0250] Table 14 Incidence of shrimp sampled in Shandong test pond (%)

[0251]

[0252] Note: When comparing data of the same industry, the same lowercase letters or no letters in the upper right corner indicate no significant difference (P>0.05), while different lowercase letters indicate significant difference (P<0.05).

[0253] Cure rate results :

[0254] The cure rates of the two farms at the 8th and 14th days after the medication were applied are shown in Tables 15 and 16 below: Compared with the Example 1 group, the cure rate of the Example 2 group was improved to a certain extent, but the difference was not significant (P>0.05). Among them, the cure rates of the Example 2 group and the Example 1 group on the 8th day in the Tianjin farm were 75.06% and 72.54% respectively; the cure rates of the Example 2 group and the Example 1 group on the 14th day were 83.22% and 80.63% respectively. The cure rates of the Example 2 group and the Example 1 group on the 8th day in the Shandong farm were 79.37% and 75.96% respectively; the cure rates of the Example 2 group and the Example 1 group on the 14th day were 86.90% and 82.02% respectively, indicating that feeding the medicated bait can not only cure the hepatopancreatic necrosis of shrimp, but also improve the immunity and recovery of shrimp, and can continue to recover after stopping the medication.

[0255] Table 15 Cure rate of shrimp in Tianjin test pond (%)

[0256]

[0257] Note: When comparing data in the same column, the same lowercase letters or no letters in the upper right corner indicate no significant difference (P>0.05), while different lowercase letters indicate significant difference (P<0.05).

[0258] Table 16 Cure rate of shrimp in Shandong test pond (%)

[0259]

[0260] Note: When comparing data in the same column, the same lowercase letters or no letters in the upper right corner indicate no significant difference (P>0.05), while different lowercase letters indicate significant difference (P<0.05).

[0261] Summary: The clinical manifestations of acute hepatopancreatic necrosis of shrimp are generally as follows: shrimps are not active in swimming, appetite is decreased, body color is turbid, hepatopancreas appears light white, atrophy and shrinkage, shell is soft, jejunum is empty or food in the intestine is discontinuous, etc. It is one of the more common and harmful diseases in shrimp farming. The results of this experiment show that the recommended dose of Example 1 (2.0mL / kg feed) or Example 2 (2.0g / kg feed) is mixed with bait and fed to shrimps suffering from this disease for 7 consecutive days. The incidence rate of random inspection after medication is significantly lower than the incidence rate of random inspection before medication, and the clinical treatment effect is good; the mortality rate and cure rate of Example 2 are not significantly different from those of Example 1, and the cure rate is relatively high, both above 70%, indicating that both Example 1 and Example 2 have good therapeutic effects, and the effects of the two are equivalent. At the same time, this experiment also further verifies that the Chinese medicine compound preparation of the present invention with the best recommended dosage (2.0mL / kg feed or 2.0g / kg feed) can better treat acute hepatopancreatic necrosis of shrimp.

[0262] The above results show that the recommended dose of each embodiment of the Chinese medicine compound preparation of the present invention (2.0 mL / kg feed 2.0 g / kg feed) mixed with bait and fed for 7 consecutive days can effectively treat acute hepatopancreatic necrosis of shrimp, with a high cure rate of more than 70%, and has the ability to improve and repair hepatopancreatic damage. The cure rate is further improved after 7 days of drug withdrawal (the 14th day from drug feeding), which can reach more than 82%. It shows that the Chinese medicine composition compound medicament of the present invention can improve the immunity of shrimp and the ability to continuously improve and repair hepatopancreatic damage, has the function of protecting the liver and repairing damaged liver tissue, improves the survival rate of shrimp, and reduces losses.

[0263] In summary, although the Chinese medicine composition compound preparation of the present invention has no significant in vitro inhibitory effect on Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis of shrimp, it plays an obvious therapeutic effect after being mixed into shrimp feed to feed shrimp suffering from acute hepatopancreatic necrosis, and has better therapeutic effect than single dose of Radix Isatidis, single dose of Licorice and other compound medicines. The Chinese medicine composition compound preparation of the present invention is safe, effective, green and environmentally friendly. Although antibiotics may have a good inhibitory effect on pathogens such as Vibrio parahaemolyticus, they are easy to aggravate hepatopancreatic damage, resulting in a high difficulty in treating acute hepatopancreatic necrosis of shrimp caused by pathogens.

[0264] The preparation process of the Chinese medicine compound preparation of the present invention adopts the method of preparing after extracting Chinese medicine, the preparation has high content of effective ingredients, few impurities and good palatability, can be fully and quickly absorbed by shrimps, and quickly exerts its medicinal effect, thus avoiding the problem that the shrimps have a simple digestive tract structure and are difficult to directly digest and absorb the original medicinal materials.

[0265] The present invention also provides a new dosage form and verifies the effectiveness of the new dosage form, which has significant effect advantages compared with traditional Chinese medicine dosage forms. A large number of clinical prescription screening and efficacy evaluations have been carried out to determine the best prescription, and the preparation processes of different dosage forms have been studied in depth, solving the use requirements of different dosage forms in the breeding process, so that the prescription can be used in all aspects in the prevention and treatment of acute hepatopancreatic necrosis of shrimp.

[0266] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a Chinese medicine composition in the preparation of a product for preventing, alleviating or treating acute hepatopancreatic necrosis of shrimp, It is characterized in that The active ingredients of the traditional Chinese medicine composition are composed of the following composition in parts by weight: 80-120 parts of isatis root, 20-40 parts of artificial bezoar, 30-50 parts of liquorice, 10-30 parts of borneol, 10-30 parts of pig gall powder and 20-40 parts of xuanming powder.

2. The use according to claim 1, It is characterized in that The active ingredients of the traditional Chinese medicine composition are composed of the following composition in parts by weight: 100-120 parts of isatis root, 34-40 parts of artificial bezoar, 40-50 parts of liquorice, 20-30 parts of borneol, 20-30 parts of pig gall powder and 30-40 parts of xuanming powder.

3. The use according to claim 1, It is characterized in that The active ingredients of the traditional Chinese medicine composition are composed of the following composition in parts by weight: 100-110 parts of isatis root, 34-38 parts of artificial bezoar, 40-45 parts of liquorice, 20-25 parts of borneol, 20-25 parts of pig gall powder and 30-35 parts of xuanming powder.

4. The use according to claim 1, It is characterized in that The active ingredients of the traditional Chinese medicine composition are composed of the following composition in parts by weight: 100-105 parts of isatis root, 34-36 parts of artificial bezoar, 40-42 parts of liquorice, 20-22 parts of borneol, 20-22 parts of pig gall powder and 30-32 parts of xuanming powder.

5. The use according to claim 1, It is characterized in that The active ingredients of the traditional Chinese medicine composition are composed of the following composition in parts by weight: 100 parts of isatis root, 34 parts of artificial bezoar, 40 parts of liquorice, 20 parts of borneol, 20 parts of pig gall powder and 30 parts of xuanming powder.

6. The use according to any one of claims 1 to 5, It is characterized in that The dosage form of the Chinese medicine composition is any one of oral liquid, powder, spray, paste, injection and capsule.

7. The use according to any one of claims 1 to 5, It is characterized in that The product is a medicine or a feed.

8. The use according to any one of claims 1 to 5, It is characterized in that The feed is shrimp feed.

9. The use according to any one of claims 1 to 5, It is characterized in that The shrimp feed is shrimp feed.

10. The use according to any one of claims 1 to 5, It is characterized in that The acute hepatopancreatic necrosis of shrimp is caused by pathogenic bacteria.

11. The use according to any one of claims 1 to 5, It is characterized in that The pathogen is Vibrio parahaemolyticus.

12. The use according to any one of claims 1 to 5, It is characterized in that The Chinese medicine composition is a clear paste obtained after the Chinese medicine is extracted.

13. The use according to claim 12, It is characterized in that The relative density of the clear paste at a measuring temperature of 50°C is 1.00-1.20 g / mL.

14. The use according to claim 12, It is characterized in that The relative density of the clear paste at a measuring temperature of 50°C is 1.05-1.15 g / mL.

15. The use according to claim 12, It is characterized in that The pH of the clear paste is 6.5~7.

5.

16. The use according to claim 12, It is characterized in that The pH of the clear paste is 7.

17. The use according to claim 6, It is characterized in that The traditional Chinese medicine composition also includes pharmaceutically acceptable excipients.

18. The use according to claim 17, It is characterized in that The auxiliary materials include one or more of propylene glycol, polysorbate 60, Tween 80, Tween 20, glycerol and water.

19. The use according to claim 6, It is characterized in that The dosage form of the Chinese medicine composition is an oral liquid, and the amount of the raw drug in the oral liquid is 0.1-0.8 g / mL.

20. The use according to claim 19, It is characterized in that The dosage of crude drug in oral solution is 0.2~0.5g / mL.

21. The use according to claim 19, It is characterized in that The dosage of crude drug in oral solution is 0.2~0.3 g / mL.

22. The use according to claim 19, It is characterized in that The crude drug content of the oral solution is 0.244 g / mL.

23. The use according to claim 6, It is characterized in that The dosage form of the Chinese medicine composition is powder, and the auxiliary materials in the powder also include one or more selected from the group consisting of glucose, skimmed milk powder, dextrin, starch, Fumei powder, corn cob powder and sodium chloride.

24. The use according to claim 23, It is characterized in that The amount of crude drug in powder is 0.1~0.8g / g powder.

25. The use according to claim 23, It is characterized in that The amount of crude drug in powder is 0.2~0.5 g / g powder.

26. The use according to claim 23, It is characterized in that The amount of crude drug in powder is 0.2~0.3 g / g powder.

27. The use according to claim 23, It is characterized in that The crude drug content of the powder is 0.244 g / g powder.

28. The use according to claim 23, It is characterized in that The dosage form of the Chinese medicine composition is an ointment, and the auxiliary materials in the ointment also include one or more selected from glucose, skimmed milk powder, dextrin, starch, Fumei powder, corn cob powder, sodium chloride, gelatin and xanthan gum.

29. The use according to any one of claims 1 to 5, It is characterized in that The preparation method of the composition comprises: 1) Add water to decoct Isatis root, licorice and Xuanming powder at least once, combine the decoction, filter and concentrate the filtrate; 2) Dissolve borneol and pig bile powder in ethanol and add to the material in step 1); 3) dissolving the artificial bezoar with alkali solution, and adding the material in step 2) into the artificial bezoar solution; 4) Add auxiliary material B in step 3), stir to dissolve, adjust pH value, add water, stir evenly, filter, and obtain liquid medicine; wherein auxiliary material B includes one or more of propylene glycol, polysorbate 60, Tween 80, Tween 20, glycerol and water.

30. The use according to claim 29, It is characterized in that In step 1), after the filtrate is concentrated, it is cooled, ethanol is added, allowed to stand, filtered, and ethanol is recovered from the filtrate to obtain a concentrated drug solution.

31. The use according to claim 30, It is characterized in that In step 1), the amount of ethanol added is such that the volume fraction of ethanol is 55% to 70%.

32. The use according to claim 29, It is characterized in that In step 2), borneol and pig bile powder are dissolved and then slowly added to the material in step 1).

33. The use according to claim 29, It is characterized in that In step 2), 2 to 5 times the volume of ethanol is used to dissolve borneol and pig bile powder.

34. The use according to claim 29, It is characterized in that In step 3), the material of step 2) is slowly added into the artificial bezoar solution.

35. The use according to claim 29, It is characterized in that In step 3), the alkali solution is a hydroxide solution.

36. The use according to claim 29, It is characterized in that In step 3), the alkali solution is a 10% hydroxide solution.

37. The use according to claim 29, It is characterized in that In step 3), the alkali solution is a 10% sodium hydroxide solution.

38. The use according to claim 29, It is characterized in that In step 3), the pH of the alkali solution is ≥11.

39. The use according to claim 29, It is characterized in that In step 4), the pH is 6.5-7.

5.

40. The use according to claim 29, It is characterized in that In step 4), the pH is 7.

41. The use according to claim 29, It is characterized in that Also includes one of the following steps A) and B); A) Add auxiliary material D to the material in step 4, stir evenly, dry at low temperature, and crush to obtain powder; auxiliary material D includes one or more of glucose, skimmed milk powder, dextrin, starch, fumei powder, corn cob powder and sodium chloride; B) Adding an appropriate amount of auxiliary material E to the material in step 4, stirring evenly, to obtain an ointment; the auxiliary material E includes one or more of glucose, skimmed milk powder, dextrin, starch, fumei powder, corn cob powder, sodium chloride, gelatin and xanthan gum.

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