A traditional Chinese medicine composition for blocking bacterial glassiness of shrimp fry, a preparation method and application thereof

Abstract

The present application belongs to the technical field of traditional Chinese medicine, and mainly relates to a traditional Chinese medicine composition for blocking bacterial vitreous body disease of shrimplets, a preparation method and application. The traditional Chinese medicine composition comprises the following raw materials in parts by weight: chebula 20-60 parts, schisandra 10-40 parts, and cyperus 10-40 parts. The traditional Chinese medicine composition can significantly reduce the mortality of shrimplets suffering from bacterial vitreous body disease (BVS), reduce the number of cultivable bacteria and the number of vibrio in the hepatopancreas of prawns, and has a good repair effect on the damage of the hepatopancreas and intestinal tract of the diseased shrimplets. The traditional Chinese medicine composition is simple and convenient to use, safe and green, can effectively reduce or replace the use of antibiotics, and is suitable for the control of vibrio diseases in the process of shrimplet cultivation and breeding production.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and in particular relates to a traditional Chinese medicine composition, preparation method and application for blocking bacterial vitrification in shrimp larvae. Background Technology

[0002] Litopenaeus vannamei, also known as the whiteleg shrimp, is the most widely farmed shrimp species in my country. In recent years, bacterial vitrification syndrome (BVS) in shrimp larvae has spread rapidly in my country. The disease typically appears 2-3 days after larvae are introduced, characterized by rapid onset and extremely high mortality, causing significant economic losses to the shrimp farming industry. Diseased shrimp larvae usually exhibit atrophy of the hepatopancreas, blurred outlines, a lighter yellow color, or even a transparent appearance, and empty intestines and stomachs.

[0003] Currently, there are differing opinions in the academic community regarding the pathogenicity of BVS. Wang Yingeng et al. (2021) isolated Vibrio alginolyticus as the dominant strain from typical BVS-infected shrimp larvae in multiple shrimp hatcheries in Hebei, Shandong, Jiangsu, Zhejiang, Guangdong, Fujian, and Hainan provinces, and identified Vibrio alginolyticus as the main pathogen. In addition, some studies have indicated that a novel, highly virulent Vibrio parahaemolyticus (Vp TPD) and its bacterial toxins can also cause similar symptoms.

[0004] Currently, the prevention and treatment of vibrio vulnificus disease in shrimp often involves a combination of antibiotics, probiotics, and disinfectants. However, the irrational use of chemical drugs can easily lead to environmental pollution, bacterial resistance, and drug residues, causing increasing public concern about food safety and public health. In recent years, the country has advocated for the implementation of green aquaculture models that reduce or replace antibiotics. Compared with antibiotics, traditional Chinese medicine (TCM) has gained increasing attention in recent years due to its rich active ingredients, broad-spectrum antibacterial activity, and ability to regulate animal immunity. Furthermore, it has fewer side effects and is less likely to induce drug resistance and residues, making it an ideal alternative to antibiotics. The inventors, through drug screening, pharmacological and toxicological studies, and clinical trials on isolated pathogens, have developed a TCM composition with good efficacy for treating BVS in shrimp larvae. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a traditional Chinese medicine composition, preparation method and application for blocking bacterial vitrification in shrimp larvae.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a traditional Chinese medicine composition for blocking bacterial vitrification in shrimp larvae, comprising the following raw materials in parts by weight: 20-60 parts of Terminalia chebula, 10-40 parts of Schisandra chinensis, and 10-40 parts of Cyperus rotundus.

[0008] Preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 25-50 parts of Terminalia chebula, 15-30 parts of Schisandra chinensis, and 15-30 parts of Cyperus rotundus.

[0009] The present invention provides a method for preparing the traditional Chinese medicine composition, comprising the following steps: pulverizing and sieving the medicinal material, soaking it in ethanol, performing a first ultrasonic extraction, and filtering; adding ethanol to the residue for a second ultrasonic extraction, filtering, combining the filtrates, recovering the ethanol, and concentrating; adding water to the obtained residue and heating it to a slight boil for extraction, filtering, concentrating, and combining it with the above-mentioned ethanol extract concentrate to obtain the final product.

[0010] Preferably, the sieving is performed through a 50-200 mesh sieve.

[0011] Preferably, the ethanol soaking time is 10 to 60 minutes.

[0012] Preferably, the ethanol is 60% to 80% ethanol, and the water is tap water.

[0013] Preferably, the ratio of medicinal material to ethanol in the first ultrasonic extraction is 1g:5mL to 1g:20mL, the ratio of medicinal residue to ethanol in the second ultrasonic extraction is 1g:5mL to 1g:10mL, and the ratio of medicinal residue to water in the extraction by heating to a gentle boil is 1g:3mL to 1g:10mL.

[0014] Preferably, the time for the first ultrasonic extraction is 10-60 min, the time for the second ultrasonic extraction is 10-60 min, and the time for extraction by heating with water to a gentle boil is 0.5-2 h.

[0015] The present invention also provides the application of the aforementioned traditional Chinese medicine composition or its preparation method in the preparation of a medicament for treating bacterial vitrification in shrimp larvae.

[0016] The beneficial effects of this invention:

[0017] This invention uses 20-60 parts of Terminalia chebula, 10-40 parts of Schisandra chinensis, and 10-40 parts of Cyperus rotundus as raw materials. After the medicinal materials are sieved, they are soaked in ethanol, subjected to a first ultrasonic extraction, filtered, and the residue is subjected to a second ultrasonic extraction with ethanol, filtered, and the filtrates are combined. The ethanol is recovered and concentrated, and the resulting residue is heated with water for extraction, filtered and concentrated. The extract is then combined with the concentrated ethanol extract to obtain the traditional Chinese medicine composition of this invention.

[0018] This invention adopts the treatment principle of nourishing the liver and blood, soothing the liver and regulating qi, and strengthening the spleen and expelling pathogens. It primarily uses Terminalia chebula and Schisandra chinensis, which have liver-protecting and spleen-strengthening effects, combined with Cyperus rotundus, which soothes the liver, relieves depression, regulates qi, and relieves chest congestion. The optimal prescription was further screened using a pathological model of bacterial vitrification (BVS) in shrimp larvae. The herbal composition of this invention can significantly reduce the mortality rate of shrimp larvae suffering from BVS and has a good repairing effect on the damage to the hepatopancreas and intestines of diseased shrimp larvae. This indicates that the herbal composition of this invention has good protective and therapeutic effects on shrimp larvae suffering from BVS. Attached Figure Description

[0019] Figure 1 Phenotypic symptoms of healthy and diseased Litopenaeus vannamei larvae, where a represents healthy larvae and b represents diseased larvae.

[0020] Figure 2 The mortality rate of Litopenaeus vannamei larvae after viral challenge in each group;

[0021] Figure 3 Images of hepatopancreas and intestinal tissue sections of Litopenaeus vannamei larvae from each group are shown below. A represents the hepatopancreas tissue section of the blank control group; B represents the intestinal tissue section of the blank control group; C represents the hepatopancreas tissue section of the positive control group; D represents the intestinal tissue section of the positive control group; E represents the hepatopancreas tissue section of the Prescription 1 experimental group; F represents the intestinal tissue section of the Prescription 1 experimental group; G represents the hepatopancreas tissue section of the Prescription 2 experimental group; H represents the intestinal tissue section of the Prescription 2 experimental group; I represents the hepatopancreas tissue section of the Prescription 3 experimental group; J represents the intestinal tissue section of the Prescription 3 experimental group; K represents the hepatopancreas tissue section of the florfenicol control group; and L represents the intestinal tissue section of the florfenicol control group.

[0022] Figure 4 The images show ultra-micro histopathological images of the hepatopancreas of Litopenaeus vannamei larvae in each group. Among them, A is the blank control group, B is a magnified view of A, C is the positive control group, D is a magnified view of C, E is the experimental group of prescription 1, F is a magnified view of E, G is the florfenicol control group, and H is a magnified view of G.

[0023] Figure 5 Figure 1 shows the survival rate of shrimp larvae in each experimental pond after drug treatment.

[0024] Figure 6 This graph shows the changes in the number of culturable bacteria in the hepatopancreas of shrimp in various experimental ponds before and after drug treatment.

[0025] Figure 7 This is a graph showing the changes in the number of Vibrio bacteria in the hepatopancreas of shrimp in each experimental pond before and after drug treatment. Detailed Implementation

[0026] This invention provides a traditional Chinese medicine composition for blocking bacterial vitrification in shrimp larvae, comprising the following raw materials in parts by weight: 20-60 parts of Terminalia chebula, 10-40 parts of Schisandra chinensis, and 10-40 parts of Cyperus rotundus, preferably 25-50 parts of Terminalia chebula, 15-30 parts of Schisandra chinensis, and 15-30 parts of Cyperus rotundus, more preferably 30 parts of Terminalia chebula, 20 parts of Schisandra chinensis, and 20 parts of Cyperus rotundus.

[0027] In this invention, Terminalia chebula is bitter, sour, and astringent in nature, and neutral in nature. It enters the large intestine and stomach meridians, and can astringe the intestines and regulate qi, strengthen the spleen and nourish the liver. Schisandra chinensis is sour, sweet, and warm in nature, and can astringe and consolidate, and replenish qi and generate fluids. Cyperus rotundus is pungent, slightly bitter, and slightly sweet in nature, and neutral in nature. It enters the liver, spleen, and triple burner meridians, and can soothe the liver and relieve depression, regulate qi and relieve chest tightness, invigorate blood and relieve pain, benefit the triple burner, and relieve six types of depression. In this formula, Terminalia chebula is used as the principal herb for its astringent and consolidating properties, Schisandra chinensis is used as the assistant herb for replenishing qi and nourishing the liver, and strengthening the spleen and opening the stomach, and Cyperus rotundus is used as the adjuvant herb for soothing the liver and relieving depression, regulating qi and relieving chest tightness. The three herbs work together to achieve the effects of nourishing the liver and blood, soothing the liver and regulating qi, and strengthening the spleen and expelling pathogens. This achieves the effects of principal, assistant, adjuvant, and guide herbs, and treats both the root cause and the symptoms, and can effectively treat bacterial vitrification in shrimp larvae.

[0028] In this invention, there are no special restrictions on the source of the Terminalia chebula, Schisandra chinensis, and Cyperus rotundus; commercially available slices that meet national standards can be used.

[0029] The present invention also provides a method for preparing the above-mentioned traditional Chinese medicine composition, comprising the following steps: pulverizing and sieving the medicinal material, soaking it in ethanol, performing a first ultrasonic extraction, and filtering; adding ethanol to the residue for a second ultrasonic extraction, filtering, combining the filtrates, recovering the ethanol, and concentrating; adding water to the obtained residue and heating it to a slight boil for extraction, filtering, concentrating, and combining it with the above-mentioned ethanol extract concentrate to obtain the final product.

[0030] In this invention, the medicinal materials are pulverized and then sieved. The pulverization method is not particularly limited; any pulverization method well-known to those skilled in the art can be used. Preferably, the sieving is done through a 50-200 mesh sieve, more preferably through an 80-150 mesh sieve, and even more preferably through a 100 mesh sieve. Pulverizing the raw materials accelerates the dissolution of the active ingredients, improves the extraction efficiency of the active ingredients, thereby reducing the amount of raw materials used, shortening the extraction time, and saving energy.

[0031] In this invention, the sieved medicinal powder is weighed according to the prescription ratio and then soaked in ethanol. The ethanol content is preferably 60-80%, more preferably 65%-75%, and further preferably 70%. The material-to-liquid ratio of the medicinal material to ethanol is preferably 1g:5mL to 1g:20mL, more preferably 1g:8mL to 1g:15mL, and further preferably 1g:10mL. The soaking time in ethanol is preferably 10-60 minutes, more preferably 20-40 minutes, and further preferably 30 minutes. After soaking in ethanol, an ultrasonic extraction is performed. The ultrasonic extraction time is preferably 10-60 minutes, more preferably 20-40 minutes, and further preferably 30 minutes. The power of the ultrasonic extraction is preferably 100-1000W, more preferably 400-800W, and further preferably 600W. The ultrasonic frequency of the ultrasonic extraction is preferably 20kHz-10MHz, more preferably 30kHz-80kHz, and further preferably 40kHz.

[0032] In this invention, the residue is filtered after the first ultrasonic extraction, and the filtrate is reserved. The obtained residue is then subjected to a second ultrasonic extraction with ethanol, preferably 60%–80% ethanol, more preferably 65%–75% ethanol, and further preferably 70% ethanol; the material-to-liquid ratio of the residue to ethanol is preferably 1g:5mL–1g:20mL, more preferably 1g:7mL–1g:9mL, and further preferably 1g:8mL; the ultrasonic extraction time is preferably 10–60 min, more preferably 20–40 min, and further preferably 30 min; the ultrasonic extraction power is preferably 100–1000W, more preferably 400–800W, and further preferably 600W; the ultrasonic frequency is preferably 20kHz–10MHz, more preferably 30kHz–80kHz, and further preferably 40kHz.

[0033] In this invention, after a second ultrasonic extraction, the residue is filtered, and the filtrate is combined with the filtrate from the first ultrasonic extraction. Ethanol is recovered and concentrated for later use. This invention does not have a specific limitation on the method of ethanol recovery; conventional methods in the art can be used, preferably vacuum recovery. The filter residue obtained after the second ultrasonic filtration is heated to a gentle boil with water for extraction to further extract water-soluble components from the medicinal material. The water is preferably tap water; the material-to-liquid ratio of the residue to water is preferably 1g:3mL to 1g:10mL, more preferably 1g:4mL to 1g:7mL, and further preferably 1g:5mL; the extraction time is preferably 0.5 to 2 hours, more preferably 0.8 to 1.2 hours, and further preferably 1 hour. After extraction, the residue is filtered, concentrated, and combined with the above-mentioned concentrated ethanol extract to obtain the final product. This invention does not have a specific limitation on the filtration and concentration methods; conventional methods in the art can be used.

[0034] This invention also provides the application of the above-mentioned traditional Chinese medicine composition or its preparation method in the preparation of a drug for treating bacterial vitrification in shrimp larvae. Feeding shrimp larvae containing the traditional Chinese medicine composition of this application can significantly reduce the mortality rate of shrimp larvae suffering from bacterial vitrification, alleviate the symptoms of the disease, and has a good repairing effect on the damage to the hepatopancreas and intestines of diseased shrimp larvae.

[0035] In this invention, the drug for treating bacterial vitrectomy in shrimp larvae comprises the pharmaceutical composition as the active ingredient; the drug for treating bacterial vitrectomy in shrimp larvae also includes pharmaceutically acceptable excipients.

[0036] 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.

[0037] Unless otherwise specified, the following embodiments are all conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] In a specific embodiment of the present invention:

[0040] The medicinal materials, including Schisandra chinensis, Terminalia chebula, Cyperus rotundus, pomegranate peel, cloves, Gallnut, and Gardenia, were all purchased from Qingdao Tongrentang Pharmacy.

[0041] Examples of Traditional Chinese Medicine Compositions

[0042] Example 1

[0043] Prescription: Terminalia chebula 30g, Schisandra chinensis 20g, Cyperus rotundus 20g.

[0044] The medicinal materials were pulverized and passed through a 100-mesh sieve. The powder was weighed according to the prescription ratio and soaked in 10 times (material-to-liquid ratio of 1g:10mL, the same below) of 70% ethanol for 30 minutes. The mixture was then ultrasonically extracted for 30 minutes at an ultrasonic power of 600W and an ultrasonic frequency of 40KHz. The mixture was then filtered. The residue was then ultrasonically extracted a second time at an ultrasonic power of 600W and an ultrasonic frequency of 40KHz for 30 minutes. The residue was then filtered, and the filtrates were combined. The ethanol was recovered under reduced pressure and the mixture was concentrated. The residue was then extracted with 5 times the amount of tap water and heated to a gentle boil for 1 hour. The residue was then filtered, concentrated under reduced pressure, and combined with the above ethanol extract concentrate. The concentrate was then concentrated under reduced pressure to a solution with a crude drug content of 250mg / mL. The solution was filtered, sterilized at 121℃, and stored at 4℃ for later use.

[0045] Example 2

[0046] Prescription: Terminalia chebula 20g, Schisandra chinensis 10g, Cyperus rotundus 40g.

[0047] The medicinal materials were pulverized through a 50-mesh sieve. The powder was weighed according to the prescription ratio, soaked in 20 times the amount of 60% ethanol for 60 minutes, and then ultrasonically extracted for 60 minutes at an ultrasonic power of 1000W and an ultrasonic frequency of 20KHz. The mixture was then filtered. The residue was then extracted a second time with 10 times the amount of 60% ethanol at an ultrasonic power of 1000W and an ultrasonic frequency of 20KHz for 60 minutes. The mixture was then filtered, and the filtrates were combined. The ethanol was recovered under reduced pressure and the mixture was concentrated. The resulting residue was then extracted with 3 times the amount of tap water and heated to a gentle boil for 2 hours. The mixture was then filtered, concentrated under reduced pressure, and combined with the above ethanol extract concentrate. The concentrate was then concentrated under reduced pressure to a solution with a crude drug content of 350mg / mL. The solution was filtered, sterilized at 121℃, and stored at 4℃ for later use.

[0048] Example 3

[0049] Prescription: Terminalia chebula 60g, Schisandra chinensis 40g, Cyperus rotundus 10g.

[0050] The medicinal materials were pulverized through a 200-mesh sieve. The powder was weighed according to the prescription ratio, soaked in 5 times the amount of 80% ethanol for 10 minutes, and then ultrasonically extracted for 10 minutes at an ultrasonic power of 100W and an ultrasonic frequency of 10MHz. The mixture was then filtered. The residue was then subjected to a second ultrasonic extraction at 10 minutes with 5 times the amount of 80% ethanol, and the extraction was carried out for 10 minutes at an ultrasonic power of 100W and an ultrasonic frequency of 10MHz. The mixture was then filtered, and the filtrates were combined. The ethanol was recovered under reduced pressure and the mixture was concentrated. The residue was then extracted with 10 times the amount of tap water and heated to a gentle boil for 0.5 hours. The mixture was then filtered, concentrated under reduced pressure, and combined with the above ethanol extract concentrate. The concentrate was then concentrated under reduced pressure to a solution with a crude drug content of 500 mg / mL. The solution was filtered, sterilized at 121℃, and stored at 4℃ for later use.

[0051] Comparative Example 1

[0052] The difference from Example 1 is that the prescription is: Terminalia chebula 40g, pomegranate peel 30g, clove 30g.

[0053] Comparative Example 2

[0054] The difference from Example 1 is that the prescription is: Terminalia chebula 40g, Gallnut 30g, Gardenia 30g.

[0055] Comparative Example 3

[0056] The difference from Example 1 is that the prescription is: Terminalia chebula 30g, Schisandra chinensis 20g, Cyperus rotundus 5g.

[0057] Comparative Example 4

[0058] The difference from Example 1 is that the prescription is: Terminalia chebula 30g and Schisandra chinensis 20g.

[0059] Screening Cases of Traditional Chinese Medicine Compound for Anti-BVS

[0060] In the following experimental examples of the present invention:

[0061] Control drug: Florfenicol, a drug commonly used by farmers to treat BVS, was used as the control drug. The drug had a content of 20%, was packaged in 100g bags, and had a batch number of 190372539. It was purchased from a qualified GMP veterinary drug manufacturer.

[0062] Medicated feed: Dissolve each prescription at a dose of 30g / kg and 20% florfenicol powder at a dose of 0.7g / kg in a certain amount of water, spray it on the surface of the feed, stir evenly, cover it with adhesive, air dry, and seal for later use.

[0063] Experimental strain: The Vibrio alginolyticus (laboratory number P4A) selected for the experiment was isolated from shrimp larvae with typical BVS disease in aquaculture farms and is preserved in the marine aquaculture pathogen bank of the Yellow Sea Fisheries Research Institute of the Chinese Academy of Fishery Sciences.

[0064] Experimental animals: Healthy Litopenaeus vannamei larvae (PL6) on day 6 after juvenile rearing were purchased from a shrimp hatchery in Hebei Province. The temporary rearing environment was a 300L aquarium tank with an effective volume of 200L and a stocking density of 25 shrimp / L. The water temperature was maintained at (28±2)℃ during the experiment, with continuous oxygenation. Feeding was provided twice daily, morning and evening, with one water change per day, replacing 1 / 3 of the total water volume. The experiment began after 3 days of temporary rearing when the shrimp larvae became day 9 larvae (PL9).

[0065] Data processing: Experimental data were analyzed using SPSS 25.0 software. Results are expressed as mean ± standard deviation. The results were presented, and statistical analysis was performed using one-way ANOVA and Duncan's test.

[0066] Experimental Example 1

[0067] Vibrio alginolyticus challenge dose screening test

[0068] Healthy juvenile shrimp (PL9) were challenged with a immersion infection method. On day 9 after a 3-day temporary rearing period, they were randomly divided into 6 groups: 5 groups were challenged with the virus, and 1 group was a control group. Each group had 3 replicates, with 40 shrimp larvae per replicate. The challenge groups were challenged with *Vibrio alginolyticus* via immersion, with a final bacterial concentration of 1×10⁻⁶ in the water. 3 1×10 4 1×10 5 1×10 6 1×10 7 CFU / mL, and the same volume of 1.5% NaCl solution was added to the blank control group. The shrimp were challenged by immersion in the solution for 48 hours, followed by a routine water change of 1 / 3. Two parallel groups were selected from each group to record mortality daily. In the other parallel group of each group, 10 shrimp larvae were randomly selected daily to observe disease incidence. Pathogens were isolated and identified from the dead shrimp larvae, and the median lethal concentration (LD50) of the strain was calculated using the modified Kohl's method. 50).

[0069] The disease incidence in each group is shown in Table 1. All shrimp in each challenge group developed symptoms within 72 hours of infection, primarily exhibiting decreased shrimp larvae viability, hepatopancreatic atrophy, blurred outline, and a lighter, pale yellow color, sometimes even becoming transparent. These symptoms were consistent with those of natural infection. The dominant strain isolated from the artificially infected shrimp larvae was identified as *Vibrio alginolyticus*, consistent with Koch's postulates. No *BVS* symptoms were observed in the blank control group, indicating the reliability of the challenge experiment.

[0070] The mortality rates for each group are shown in Table 2: 1×10 6 CFU / mL and 1×10 7 Shrimp larvae in the CFU / mL experimental group began to show signs of mortality after 2 hours, with the peak mortality period occurring at 2 hours, and at 1×10⁻⁶. 7 All shrimp larvae in the CFU / mL experimental group died after 12 hours. (1×10⁻⁶) 5 In the CFU / mL experimental group, mortality began to appear at 6 hours, with the peak mortality occurring at 10 hours. (1×10⁻⁶) 4 The peak mortality rate in the CFU / mL experimental group began at 6 hours. (1×10⁻⁶) 3 In the CFU / mL experimental group, mortality began to appear at 6 hours, with the peak mortality period at 12 hours. The 72-hour LD50 of shrimp larvae challenged with Vibrio alginolyticus was calculated using the modified Kohl's method. 50 3.35×10 5 CFU / mL.

[0071] According to the "Technical Guidelines for Efficacy Testing of Antimicrobial Drugs for Aquaculture" (Veterinary Drug Evaluation Center, Ministry of Agriculture and Rural Affairs, 2022), the infectious dose for artificial infection should be the minimum lethal dose for the test animals or a dose that can cause more than 80% of infected animals to show obvious symptoms. Based on the results of shrimp larvae morbidity and mortality in the challenge dose screening experiment, subsequent experiments used a concentration of 5 × 10⁻⁶, which is lower than the median lethal dose. 4 CFU / mL Vibrio alginolyticus immersion challenge was used to ensure that all shrimp larvae were susceptible to disease and that the mortality rate was appropriate.

[0072] Table 1. Disease incidence results of Litopenaeus vannamei larvae after challenge with Vibrio alginolyticus.

[0073]

[0074] Table 2. Mortality results of Litopenaeus vannamei larvae after challenge with Vibrio alginolyticus.

[0075]

[0076]

[0077] Experimental Example 2

[0078] In vivo pharmacodynamic experiments of different traditional Chinese medicine compound preparations on BVS shrimp larvae

[0079] Healthy Litopenaeus vannamei larvae temporarily raised to PL9 were randomly divided into 6 groups: prescription 1-3 experimental groups, florfenicol control group, blank control group, and positive control group. Each group had 3 replicates, with 40 shrimp per replicate. The blank control group was treated with 1.5% NaCl solution, while the other groups were treated with 5×10⁻⁶ shrimp. 4 The shrimp were challenged with a dose of CFU / mL by immersion for 48 hours, followed by a routine water change of 1 / 3. Simultaneously with the challenge, experimental groups 1-3 were fed diets containing the compound traditional Chinese medicine formulas from Example 1 (Formula 1), Comparative Example 1 (Formula 2), and Comparative Example 2 (Formula 3), respectively. The drug control group was fed diets containing 20% ​​florfenicol powder, while the blank control and positive control groups were fed normal blank diets. The feed dosage was 3% of the shrimp's body weight, and the treatment continued for 7 days. Each group underwent bottom-drain water changes once daily, with a water volume of 1 / 3 of the total volume being replaced. Daily, two parallel sets of shrimp larvae were selected from each group to record mortality, calculate the mortality rate, and 10 shrimp larvae were randomly selected from the other parallel set of each group to observe disease incidence.

[0080] At the end of drug administration (day 7), five shrimp larvae were randomly sampled from each group and fixed in Davidson's fixative. Histopathological examination was performed using routine hematoxylin-eosin (HE) staining. Simultaneously, five shrimp larvae were randomly sampled from each group and fixed in 2.5% glutaraldehyde for ultrastructural pathological observation.

[0081] The experimental results showed that the artificial challenge model was successful. Except for the blank control group, all shrimp in each group exhibited symptoms such as weakened vitality, hepatopancreatic atrophy, blurred outline, and pale yellow color 12 hours after infection. Symptoms of diseased shrimp larvae are shown in [the table below]. Figure 1 In the positive control group, acute mortality occurred within 24 hours, with a mortality rate of 28.8%. In the blank control group, shrimp larvae showed normal body color, vitality, feeding, and hepatopancreatic status.

[0082] Shrimp mortality under different drug effects, such as Figure 2 As shown, 7 days after administration, the mortality rate of shrimp larvae in the Prescription 1 experimental group was significantly lower than that in the positive control group (P<0.05); the mortality rates of the Prescription 2, Prescription 3 experimental groups, and florfenicol control group were lower than those in the positive control group, but the differences were not significant (P>0.05). The mortality rates of each group from lowest to highest were: blank control group < Prescription 1 experimental group < florfenicol control group < Prescription 3 experimental group < Prescription 2 experimental group < positive control group.

[0083] Histopathological examination results showed that, 7 days after drug administration, in the hepatopancreatic tissue of shrimp larvae in the blank control group, the hepatopancreatic tubular epithelial cells were neatly and densely arranged, with blue-purple nuclei and red cytoplasm. Figure 3 A); Intestinal epithelial cells are tightly arranged and orderly connected to the basement membrane. Figure 3 B). In the positive control group, the hepatopancreatic tissue of diseased shrimp larvae showed severe lesions, with severe necrosis, sloughing, and even disappearance of hepatic tubular epithelial cells; ultimately, most of the hepatopancreatic tissue structure disappeared, becoming serous. Figure 3 C); Intestinal villi are lost or missing from the intestinal wall, and there are a large number of tissue fragments in the intestine. Figure 3 D). Compared with the positive control group, the hepatopancreas and intestinal tissue structures of diseased shrimp larvae in prescription 1-3 experimental groups and the florfenicol control group showed varying degrees of protection. Among them, compared with other drug treatment groups, the hepatopancreas and intestinal epithelial structures of shrimp larvae in prescription 1 experimental group were more intact, with more hepatic tubules and less epithelial cell shedding and necrosis. Figure 3 E), the intestinal epithelial cells are relatively abundant and neatly arranged. Figure 3 F).

[0084] Ultramicroscopic pathological observation results showed that, 7 days after drug administration, the hepatic tubular epithelial cells of shrimp larvae in the blank control group had intact structure, and the cell nucleus, cell membrane, and organelles were all normal. Figure 4 A, Figure 4 B). In the positive control group, the cell membranes of the hepatic tubule epithelial cells of shrimp larvae ablated, the cell boundaries became blurred, the nuclei solidified, and organelles such as mitochondria and endoplasmic reticulum disintegrated. High electron density particles formed in the cytoplasm, and the cytoplasm ablated, forming vacuolated or even large-area cell disintegration, resulting in blank areas. Figure 4 C Figure 4 D). Compared with the positive control group, the cell membranes and nuclei of the hepatic tubule epithelial cells of shrimp larvae in the prescription 1 experimental group were normal, and the mitochondria and endoplasmic reticulum were abundant and basically normal. Figure 4 E, Figure 4 F). In the florfenicol control group, the cell membranes of some epithelial cells in the hepatic tubules of shrimp larvae underwent ablation, organelle disintegration, and nuclear solidification, with even patches of cells disappearing. Figure 4 G, Figure 4 H).

[0085] The above results indicate that prescription 1 can significantly reduce the mortality rate of shrimp larvae infected with BVS caused by Vibrio alginolyticus, has a good tissue repair effect on the damage to the hepatopancreas and intestines of the infected shrimp larvae, and has a better therapeutic effect on the infected shrimp larvae than prescriptions 2 and 3 and conventional treatment drug 20% ​​florfenicol powder.

[0086] Experimental Example 3

[0087] Efficacy experiments of different traditional Chinese medicine compound preparations in treating bacterial vitrification in shrimp larvae

[0088] This experiment was conducted at three shrimp hatcheries in Tangshan and Cangzhou, Hebei Province, and Lianyungang, Jiangsu Province. Prior to the experiment, all three hatcheries had experienced BVS (Biological Variation and Vibration) disease. Four independent ponds were selected from each hatchery as experimental subjects. Each pond had its own inlet and outlet system, and there was no water exchange or biological contamination between the ponds. The feed preparation method was as follows: the drug was extracted and prepared into a solution with a crude drug content of 1 g / ml. This solution was mixed evenly with the feed at a dosage of 30 g / kg and fed to the shrimp larvae twice daily for 7 consecutive days. The daily feed intake was 3% of the shrimp's body weight. The positive control group was fed a blank feed without the drug. The experimental groups and drug treatments are shown in Table 3.

[0089] Hepatopancreatic tissue was randomly collected from five shrimp per pond before drug administration, 5 days after drug administration, and 7 days after drug administration. Approximately 0.2 g of tissue was taken from each shrimp. The tissues were mixed, weighed, and homogenized with 0.5 mL of sterile PBS solution. The homogenate was serially diluted 10-fold with sterile 1.5% NaCl solution. 100 μL of the diluted solution was spread on TSB (tryptone soybean broth) and TCBS (thiosulfate citrate bile salt sucrose) agar plates and incubated at 28°C for 24 h. Colony counting was performed afterward. The total number of culturable bacteria and Vibrio bacteria per unit shrimp hepatopancreas was estimated based on the number of colonies grown on TSB and TCBS plates at specific dilution gradients.

[0090] Table 3. Grouping and Treatment Methods for Field Trials of Shrimp Fry Efficacy Evaluation

[0091]

[0092] Treatment trials were conducted in Tangshan and Cangzhou, Hebei Province, and Lianyungang, Jiangsu Province. The survival rates of shrimp larvae in each experimental pond were as follows: Figure 5 As shown, the survival rates of shrimp larvae in ponds using the herbal composition of Example 1 in the three regions reached 85.64%, 66.73%, and 75.73%, respectively, all significantly higher than the survival rates of shrimp larvae in Comparative Examples 3 and 4. Simultaneously, the shrimp larvae exhibited good vitality and body color, with brownish-red hepatopancreas with clearly visible edges, full gastrointestinal tracts, and good uniformity and feeding behavior within the same rearing pond. The relatively low survival rate of shrimp larvae at the Cangzhou breeding farm in Hebei Province may be closely related to the longer duration of disease onset and later treatment.

[0093] The number of bacteria that can be cultured in the hepatopancreas of a unit shrimp body is shown in the figure. Figure 6 The results of the total number of Vibrio bacteria in the hepatopancreas of shrimp per unit body are shown in [the table below Figure 7 ,Depend on Figure 6 and Figure 7 It is evident that, prior to drug administration, the total number of culturable bacteria in the hepatopancreas of shrimp larvae from BVS ponds in the three regions reached 6 × 10⁻⁶. 7 CFU / mL ~9×10 7 CFU / mL, Vibrio count reached 2×10 6CFU / mL ~5×10 6 CFU / mL; After 5 and 7 days of administration, the number of culturable bacteria and Vibrio in the hepatopancreas of shrimp larvae decreased significantly. After 5 days of administration, the total number of bacteria and Vibrio in the hepatopancreas of shrimp larvae treated with the herbal composition of Example 1 decreased by 40-50%, and the total number of bacteria and Vibrio in the hepatopancreas of shrimp larvae treated with the herbal composition of Example 1 decreased by 30-40%. After 7 days of administration, the total number of bacteria and Vibrio in the hepatopancreas of shrimp larvae treated with the herbal composition of Example 1 decreased by more than 90%, and the total number of bacteria and Vibrio in the hepatopancreas of shrimp larvae treated with the herbal composition of Example 1 decreased by more than 75%. The therapeutic effect of the herbal composition of Example 1 was significantly better than that of Comparative Example 3 and Comparative Example 4.

[0094] Vibrio is the most common pathogen in shrimp farming, causing various diseases. During shrimp disease outbreaks, the increase in bacterial abundance in the farming water and shrimp is mainly due to Vibrio, including common pathogenic Vibrio species such as *Vibrio alginolyticus*, *Vibrio owensii*, *Vibrio campbellii*, *Vibrio parahaemolyticus*, and *Vibrio harveyi*. Therefore, testing the number of bacteria and Vibrio in shrimp is of great significance for determining the infection and disease status of shrimp. In this experiment, the number of culturable bacteria and Vibrio in the hepatopancreas of shrimp larvae from shrimp breeding farms suffering from BVS decreased significantly after 5 and 7 days of drug administration. Furthermore, the survival rate of shrimp larvae treated with the herbal compositions of Example 1, Comparative Example 3, and Comparative Example 4 was significantly higher than that of the positive control group, and the abundance of culturable bacteria and Vibrio in the hepatopancreas of shrimp was effectively reduced, which is of positive significance for the treatment of this disease. Among them, the efficacy of Example 1 was better than that of Comparative Example 3 and Comparative Example 4, indicating that Cyperus rotundus has a significant synergistic effect in this herbal composition.

[0095] Further analysis of the above experimental results shows that the herbal composition in Example 1 contains Terminalia chebula and Schisandra chinensis, both of which have astringent and consolidating effects. They can effectively reduce the loss of qi, blood and essence caused by deficiency of the internal organs and insufficient vital energy. Schisandra chinensis invigorates qi and nourishes the liver, strengthens the spleen and stomach, while Cyperus rotundus soothes the liver, relieves depression, regulates qi and relieves chest congestion. It has a good repairing effect on the hepatopancreas and gastrointestinal damage of shrimp larvae caused by Vibrio alginolyticus infection. Therefore, this formula has a good therapeutic effect on bacterial vitrification of shrimp larvae caused by Vibrio alginolyticus infection, can effectively reduce the mortality of diseased shrimp larvae, and alleviate the symptoms of the disease. The lesions of the hepatopancreas and intestines of shrimp larvae in the treatment group were also significantly reduced compared with the positive control group and the drug control group.

[0096] 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 blocking bacterial vitrification in shrimp larvae, characterized in that, It is made from the following ingredients in parts by weight: 20-60 parts Terminalia chebula, 10-40 parts Schisandra chinensis, and 10-40 parts Cyperus rotundus.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, It is made from the following ingredients in parts by weight: Terminalia chebula 25-50 parts, Schisandra chinensis 15-30 parts, Cyperus rotundus 15-30 parts.

3. The method for preparing the traditional Chinese medicine composition according to any one of claims 1 to 2, characterized in that, The process includes the following steps: pulverizing and sieving the medicinal materials, soaking them in ethanol, performing a first ultrasonic extraction, and filtering; adding ethanol to the residue for a second ultrasonic extraction, filtering, combining the filtrates, recovering the ethanol, and concentrating; adding water to the obtained residue, heating to a gentle boil for extraction, filtering, concentrating, and combining with the above ethanol extract concentrate to obtain the final product.

4. The preparation method according to claim 3, characterized in that, The sieving process involves passing through a 50-200 mesh sieve.

5. The preparation method according to claim 3, characterized in that, The ethanol is 60% to 80% ethanol, and the water is tap water.

6. The preparation method according to claim 3, characterized in that, During the first ultrasonic extraction, the ratio of medicinal material to ethanol is 1g:5mL to 1g:20mL. During the second ultrasonic extraction, the ratio of medicinal residue to ethanol is 1g:5mL to 1g:10mL. During the extraction with water and heating to a gentle boil, the ratio of medicinal residue to water is 1g:3mL to 1g:10mL.

7. The preparation method according to claim 3, characterized in that, The first ultrasonic extraction takes 10–60 min, the second ultrasonic extraction takes 10–60 min, and the extraction time by adding water and heating to a gentle boil takes 0.5–2 h.

8. The use of the traditional Chinese medicine composition according to any one of claims 1 to 2 or the traditional Chinese medicine composition prepared by the preparation method according to any one of claims 3 to 7 in the preparation of a drug for treating bacterial vitrification of shrimp larvae.

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