Traditional Chinese medicine composition for preventing and treating bacterial diseases, preparation method and application thereof

Abstract

The present invention belongs to the field of drugs for preventing and treating freshwater cultured fish, and particularly relates to a traditional Chinese medicine composition for preventing and treating bacterial diseases, a preparation method thereof, and an application. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5 parts to 20 parts of Chinese gallnut, 5 parts to 15 parts of Scutellaria baicalensis Georgi, 5 parts to 15 parts of pomegranate peel, 5 parts to 15 parts of cloves, and 5 parts to 20 parts of Salvia miltiorrhiza Bunge. After the traditional Chinese medicine composition provided by the present invention is orally administered to grass carp, the survival rate of grass carp can be greatly increased, the tissue damage and inflammation in the body can be improved, the expression levels of inflammatory factors TNF-α, IFN, Myd88, IL-1β, and MHCⅡ in the head kidney of grass carp can be reduced, and the composition has an antibacterial effect on other pathogenic bacteria in vitro. The traditional Chinese medicine composition provided by the present invention can effectively antibacterial, reduce the degree of inflammation, and improve the survival rate of grass carp after bacterial infection.

Description

Technical Field

[0001] The present invention belongs to the field of drugs for preventing and treating freshwater cultured fish, and particularly relates to a traditional Chinese medicine composition for preventing and treating bacterial diseases, a preparation method thereof, and an application thereof. Background Art

[0002] Grass carp is an important freshwater cultured fish in China and is known as one of the "four major domestic fishes" together with bighead carp, silver carp, and black carp. With the popularization of intensive aquaculture, problems such as high-density feeding and deterioration of the aquaculture environment have led to frequent outbreaks of diseases such as grass carp enteritis, gill rot, and septicemia, causing huge economic losses to farmers and restricting the development of the grass carp aquaculture industry. Among them, grass carp enteritis and septicemia caused by Aeromonas hydrophila infection lead to a large number of deaths of grass carp, which is extremely harmful. Aeromonas hydrophila infection usually shows blackening of the body surface, slow movement, red and swollen anus, congestion at the base of the fins, and bleeding in the abdomen. Upon dissection, a large amount of ascites can be seen in the abdominal cavity, the intestine is inflated and swollen, the intestinal wall has no elasticity, is easily torn when reddened, the spleen is blackened, and the liver and kidney tissues are congested.

[0003] Antibiotics and their substitutes are often used in aquaculture to prevent and treat bacterial diseases in fish farming. However, such substances have relatively large side effects, there are problems of drug residues, and drug residues are prone to cause the generation of multi-drug resistant bacteria, affecting the prevention and treatment effects of antibiotic substances. Traditional Chinese medicine is rich in various bioactive components, and various bioactive components such as polysaccharides, flavonoids, polyphenols, saponins, alkaloids, essential oils, and terpenoids have been widely used in the prevention and treatment of viral, bacterial, parasitic, and fungal diseases in fish. It is an antibacterial drug that is currently being developed and is also an excellent choice for anti-drug resistant bacteria. However, different traditional Chinese medicines often only have an inhibitory effect on specific bacterial groups. Therefore, it is of great significance to study different traditional Chinese medicines or traditional Chinese medicine prescriptions for the comprehensive prevention and treatment of bacterial diseases in the process of aquaculture. Summary of the Invention

[0004] The present invention provides a traditional Chinese medicine composition for preventing and treating bacterial diseases, which solves the problem that existing drugs are not comprehensive in preventing and treating bacterial diseases in the process of aquaculture due to the generation of multi-drug resistant pathogenic bacteria caused by antibiotic residues.

[0005] The specific technical solution provided by the present invention is as follows:

[0006] In the first aspect of the present invention, there is provided a traditional Chinese medicine composition for preventing and treating bacterial diseases, which is prepared from the following raw materials by weight: 5 parts to 20 parts of Chinese gallnut, 5 parts to 15 parts of Scutellaria baicalensis, 5 parts to 15 parts of pomegranate peel, 5 parts to 15 parts of cloves, and 5 parts to 20 parts of Salvia miltiorrhiza.

[0007] As a preferred embodiment of the present invention, the traditional Chinese medicine composition is prepared from the following raw materials by weight: 20 parts of Chinese gallnut, 15 parts of Scutellaria baicalensis, 10 parts of pomegranate peel, 5 parts of cloves, and 5 parts of Salvia miltiorrhiza.

[0008] In the second aspect of the present invention, a preparation method of the traditional Chinese medicine composition is provided, comprising the following steps:

[0009] Weigh gallnut, skullcap root, pomegranate rind, clove, and salvia miltiorrhiza by weight parts, mix them, decoct with water, collect the decoction, and thus obtain the traditional Chinese medicine composition.

[0010] As a preferred embodiment of the present invention, when decocting with water, the water addition amount is 8 to 10 times the total amount of the medicinal materials.

[0011] As a preferred embodiment of the present invention, after mixing the weighed medicinal materials, soak them in water for 0.5 h to 1.0 h and then decoct.

[0012] More preferably, the specific process of the water decoction is as follows: first decoct with strong fire for 0.5 h to 1.0 h, filter, collect the first decoction; add water to the remaining filter residue and decoct with slow fire for 0.5 h to 1.0 h, filter, collect the second decoction; add water to the remaining filter residue and decoct with strong fire for 0.5 h to 1.0 h, filter, collect the third decoction, and combine it with the decoctions obtained in the previous two times to obtain the traditional Chinese medicine composition.

[0013] As a preferred embodiment of the present invention, the collected decoction is concentrated to 1 ± 0.05 mg / mL to obtain the concentrate of the traditional Chinese medicine composition. After autoclaving at 121 °C and cooling, it is stored in a 4 °C refrigerator.

[0014] In the third aspect of the present invention, an application of the traditional Chinese medicine composition in preparing a product for inhibiting pathogenic bacteria is provided, and the pathogenic bacteria are Aeromonas hydrophila, Plesiomonas shigelloides, Stenotrophomonas maltophilia, Vibrio cholerae, Klebsiella pneumoniae, Acinetobacter baumannii, or Citrobacter freundii.

[0015] As a preferred embodiment of the present invention, the traditional Chinese medicine composition is used to prepare a product for preventing and treating bacterial infectious diseases in fish farming.

[0016] As a preferred embodiment of the present invention, the product is a drug or a feed additive.

[0017] In the fourth aspect of the present invention, a drug for preventing and treating bacterial infectious diseases in fish farming is provided, which comprises the traditional Chinese medicine composition.

[0018] As a preferred embodiment of the present invention, the drug comprises pharmaceutically acceptable excipients or carriers.

[0019] In the fifth aspect of the present invention, a feed additive for preventing and treating bacterial infectious diseases in fish farming is provided, which comprises the traditional Chinese medicine composition.

[0020] As a preferred embodiment of the present invention, the feed additive comprises acceptable excipients or carriers.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The traditional Chinese medicine composition provided by the present invention has simple components, small side effects, easily available raw materials, and a simple preparation method. Feeding grass carp with the traditional Chinese medicine composition provided by the present invention can reduce the severity of bacterial infection symptoms in grass carp, reduce the mortality rate, reduce the expression levels of inflammatory factors such as TNF-α, IFN, Myd88, IL-1β, and MHCⅡ, slow down the inflammatory and damage degrees of the spleen, kidney, intestine, and gills, and improve the anti-bacterial infection ability of grass carp.

[0023] The traditional Chinese medicine composition provided by the present invention is obtained through screening the in vitro antibacterial ability of single traditional Chinese medicines and compatibility optimization, solves problems such as drug residues that cannot be solved by antibiotics and their substitutes in aquaculture, has small side effects and high safety, and can effectively prevent and treat enteritis and septicemia caused by Aeromonas hydrophila infection in grass carp, greatly improving the survival rate of grass carp.

[0024] Among the raw materials of the traditional Chinese medicine composition provided by the present invention, there is a synergistic effect, and the combined use can better play the effect of inhibiting pathogenic bacteria. The bacteriostatic experiment proves that the traditional Chinese medicine composition can inhibit Aeromonas hydrophila, Plesiomonas shigelloides, Stenotrophomonas maltophilia, Vibrio cholerae, Klebsiella pneumoniae, Acinetobacter baumannii or Citrobacter freundii, and particularly has better inhibitory activity against Aeromonas hydrophila, Plesiomonas shigelloides, Stenotrophomonas maltophilia, and Vibrio cholerae. The traditional Chinese medicine composition can be used for the prevention and treatment of various bacterial diseases. Description of the Drawings

[0025] Figure 1 It is a schematic diagram of adding samples when measuring the minimum inhibitory concentration of single traditional Chinese medicine; Liquid medicine 1 and Liquid medicine 2 refer to two different single traditional Chinese medicine liquid medicines on the same 96-well plate.

[0026] Figure 2 It is a diagram of measuring the antibacterial ability of some single traditional Chinese medicines by the disk diffusion method. The four colonies in the figure respectively refer to the antibacterial zones of different single traditional Chinese medicines.

[0027] Figure 3 It is a diagram of the antibacterial zone of the traditional Chinese medicine compound preparation against the NJ-35 strain. The different colonies in the figure are four groups of repeated experiments.

[0028] Figure 4It is a graph showing the MBC determination results of a traditional Chinese medicine compound preparation against NJ-35 strain; the positive control is the plaque without traditional Chinese medicine treatment, and the blank control is dropped with normal saline; a, positive control; b, traditional Chinese medicine compound preparation with a concentration of 1000 mg / mL; c, traditional Chinese medicine compound preparation with a concentration of 500 mg / mL; d, traditional Chinese medicine compound preparation with a concentration of 250 mg / mL; e, traditional Chinese medicine compound preparation with a concentration of 125 mg / mL; f, traditional Chinese medicine compound preparation with a concentration of 62.5 mg / mL; g, traditional Chinese medicine compound preparation with a concentration of 31.25 mg / mL; h, blank control; i, traditional Chinese medicine compound preparation with a concentration of 15.63 mg / mL; j, traditional Chinese medicine compound preparation with a concentration of 7.81 mg / mL; k, traditional Chinese medicine compound preparation with a concentration of 3.91 mg / mL; l, traditional Chinese medicine compound preparation with a concentration of 1.95 mg / mL; m, traditional Chinese medicine compound preparation with a concentration of 0.98 mg / mL; n, traditional Chinese medicine compound preparation with a concentration of 0.49 mg / mL.

[0029] Figure 5 It is a scanning electron microscope image of NJ-35 strain treated with different concentrations of traditional Chinese medicine compound preparation; A: blank control; B: treated with traditional Chinese medicine compound at 3.91 mg / mL; C: treated with traditional Chinese medicine compound at 125 mg / mL; D: enlarged view of the framed part in C; E: treated with traditional Chinese medicine compound at 250 mg / mL; the arrow points to wrinkles and cracks; F: treated with traditional Chinese medicine compound at 250 mg / mL; the arrow points to wrinkles.

[0030] Figure 6 They are pathological section images of the intestine, spleen, kidney, and gill of grass carp infected with Aeromonas hydrophila after treatment with traditional Chinese medicine compound preparation; in the pathological section image of the spleen, the black arrow in the bacterial infection group indicates vacuolar degeneration, in the pathological section image of the kidney, the black arrow in the bacterial infection group indicates necrosis of the outer layer cells of the renal tubule, and in the pathological section image of the gill, the black arrow in the bacterial infection group indicates atrophy of the gill filament cartilage.

[0031] Figure 7 It is a statistical chart of the relative expression level of IFN mRNA in the head kidney of grass carp infected with Aeromonas hydrophila after treatment with traditional Chinese medicine compound preparation.

[0032] Figure 8 It is a statistical chart of the relative expression level of IL-1β mRNA in the head kidney of grass carp infected with Aeromonas hydrophila after treatment with traditional Chinese medicine compound preparation.

[0033] Figure 9 It is a statistical chart of the relative expression level of MHCⅡ mRNA in the head kidney of grass carp infected with Aeromonas hydrophila after treatment with traditional Chinese medicine compound preparation.

[0034] Figure 10 It is a statistical chart of the relative expression level of Myd88 mRNA in the head kidney of grass carp infected with Aeromonas hydrophila after treatment with traditional Chinese medicine compound preparation.

[0035] Figure 11 Statistical chart of relative expression levels of TNF-α mRNA in the head kidney of grass carp infected with Aeromonas hydrophila after treatment with a traditional Chinese medicine compound preparation.

[0036] Figure 12 Graph of the survival rate of grass carp infected with Aeromonas hydrophila after treatment with a traditional Chinese medicine compound preparation. Detailed implementation methods

[0037] The technical solutions of the present invention are further described and explained below through specific implementation methods. Unless otherwise specified, the kit methods used in the implementation cases are all conventional treatment methods, and the reagents, materials, consumables, etc. used, unless otherwise specified, can be purchased from ordinary commercial channels.

[0038] In existing aquaculture, antibiotics and their substitutes are often used to prevent and treat bacterial diseases in fish farming. However, these substances have relatively large side effects, there are problems with drug residues, and due to the influence of drug residues, multi-drug resistant pathogenic bacteria are easily generated, affecting the prevention and treatment effects of antibiotic substances. Traditional Chinese medicine is rich in various bioactive components, and various bioactive components such as polysaccharides, flavonoids, polyphenols, saponins, alkaloids, essential oils, and terpenoids have been widely used in the prevention and treatment of viral, bacterial, parasitic, and fungal diseases in fish. It is an excellent choice for current and developing antibacterial drugs against drug-resistant bacteria. However, different traditional Chinese medicines often only have inhibitory effects on specific bacterial groups. Therefore, it is of great significance to study different traditional Chinese medicines or traditional Chinese medicine formulations for the comprehensive prevention and treatment of bacterial diseases in the process of aquaculture.

[0039] The present invention provides a traditional Chinese medicine composition for preventing and treating bacterial diseases, which is made from the following raw materials by weight: 5 parts to 20 parts of Chinese gall, 5 parts to 15 parts of Scutellaria baicalensis, 5 parts to 15 parts of pomegranate peel, 5 parts to 15 parts of clove, and 5 parts to 20 parts of Salvia miltiorrhiza. This traditional Chinese medicine composition solves problems such as drug residues that cannot be solved by antibiotics and their substitutes in aquaculture, has small side effects and high safety, can effectively prevent and control and treat enteritis and septicemia in grass carp caused by Aeromonas hydrophila infection, and greatly improves the survival rate of grass carp.

[0040] The sources and effects of the medicinal materials involved in the traditional Chinese medicine composition provided by the present invention are as follows:

[0041] Chinese gall, also known as Chinese nutgall, insect gall on the leaves of Rhus chinensis, Rhus potaninii Maxim., or Rhus punjabensis Stew. var. sinica (Diels) Rehd. et Wils., mainly formed by the parasitism of Chinese gall aphids, enters the lung, stomach, and large intestine meridians, and has the effects of astringing the lung to reduce fire, astringing the intestine to stop diarrhea, arresting sweating, stopping bleeding, and astringing dampness and promoting granulation. The main active ingredients mainly include tannic acid, also known as tannin, a complex compound formed by the combination of gallic acid, digallic acid, and glucose.

[0042] Scutellaria baicalensis, also known as Shancha Root and Tujincha Root, is the dried root of the plant Scutellaria baicalensis Georgi in the Lamiaceae family. It belongs to the lung, gallbladder, spleen, large intestine, and small intestine meridians. It is used as medicine with its root, being bitter in taste and cold in nature, having the effects of clearing heat and drying dampness, purging fire and detoxifying, stopping bleeding, and preventing miscarriage. It is mainly used to treat febrile diseases, upper respiratory tract infections, cough due to lung heat, damp-heat jaundice, pneumonia, dysentery, hemoptysis, red eyes, threatened abortion, hypertension, carbuncles, boils, and other symptoms.

[0043] Pomegranate peel, also known as pomegranate husk, common pomegranate, sour fruit shell, sour pomegranate peel, sour pomegranate husk, and western pomegranate peel, belongs to the large intestine meridian and has the effects of astringing the intestine to stop diarrhea, stopping bleeding, and expelling worms. It is mainly used to treat chronic diarrhea, chronic dysentery, hematochezia, rectal prolapse, spermatorrhea, metrorrhagia and metrostaxis, leukorrhea, abdominal pain due to intestinal parasites, scabies, and other symptoms.

[0044] Clove, also known as Eugenia caryophyllata Thunb., broken fragrance, male clove, and common clove, is the dried flower bud of the plant Eugenia caryophyllata Thunb. in the Myrtaceae family. It belongs to the spleen meridian, stomach meridian, lung meridian, and kidney meridian, and is mainly used to treat hiccups, cold pain in the epigastrium and abdomen, anorexia, vomiting and diarrhea, impotence due to kidney deficiency, soreness and coldness in the waist and knees, and yin abscess.

[0045] Salvia miltiorrhiza is the dried root and rhizome of the plant Salvia miltiorrhiza Bunge in the Lamiaceae family. It belongs to the heart, lung, gallbladder, and large intestine meridians and has the effects of promoting blood circulation to remove blood stasis, dredging meridians to relieve pain, clearing the heart to remove vexation, and cooling blood to dissipate carbuncles. It is used for chest pain due to chest impediment, pain in the epigastrium, abdomen, and hypochondrium, mass accumulation, arthralgia due to heat pathogen, restlessness and insomnia, irregular menstruation, dysmenorrhea, amenorrhea, and swelling and pain of sores and ulcers.

[0046] This will be specifically described below in conjunction with the embodiments.

[0047] Example 1

[0048] A traditional Chinese medicine composition for preventing and treating bacterial diseases is composed of the following raw materials: 20 g of Chinese gall, 15 g of Scutellaria baicalensis, 10 g of pomegranate peel, 5 g of clove, and 5 g of Salvia miltiorrhiza.

[0049] Example 2

[0050] A traditional Chinese medicine composition for preventing and treating bacterial diseases is composed of the following raw materials: 20 g of Chinese gall, 15 g of Scutellaria baicalensis, 15 g of pomegranate peel, 15 g of clove, and 20 g of Salvia miltiorrhiza.

[0051] Example 3

[0052] A traditional Chinese medicine composition for preventing and treating bacterial diseases is composed of the following raw materials: 5 g of Chinese gall, 5 g of Scutellaria baicalensis, 5 g of pomegranate peel, 5 g of clove, and 5 g of Salvia miltiorrhiza.

[0053] Example 4

[0054] A traditional Chinese medicine composition for preventing and treating bacterial diseases is composed of the following raw materials: 15 g of Chinese gall, 12 g of Scutellaria baicalensis, 10 g of pomegranate peel, 8 g of clove, and 8 g of Salvia miltiorrhiza.

[0055] The traditional Chinese medicine compositions provided in the above Examples 1 to 4 are all prepared according to the following steps:

[0056] Weigh gallnut, skullcap root, pomegranate rind, clove, and salvia miltiorrhiza, mix them, add water equivalent to 8 times the total amount of the medicinal materials, soak for 0.5 h, first decoct with strong fire for 0.5 h, filter, collect the first decoction liquid, add water to the remaining filter residue and decoct with gentle fire for 0.5 h, filter, collect the second decoction liquid, add water to the remaining filter residue and decoct with strong fire for 0.5 h, filter, collect the third decoction liquid, combine it with the decoction liquids obtained in the previous two times, and concentrate to 1 ± 0.05 mg / mL to obtain the traditional Chinese medicine composition.

[0057] Example 5

[0058] A traditional Chinese medicine composition for preventing and treating bacterial diseases, which is only different from Example 1 in the preparation method. Specifically:

[0059] Weigh gallnut, skullcap root, pomegranate rind, clove, and salvia miltiorrhiza, mix them, add water equivalent to 10 times the total amount of the medicinal materials, soak for 1.0 h, first decoct with strong fire for 1.0 h, filter, collect the first decoction liquid, add water to the remaining filter residue and decoct with gentle fire for 1.0 h, filter, collect the second decoction liquid, add water to the remaining filter residue and decoct with strong fire for 1.0 h, filter, collect the third decoction liquid, combine it with the decoction liquids obtained in the previous two times, and concentrate to 1 ± 0.05 mg / mL to obtain the traditional Chinese medicine composition.

[0060] Example 6

[0061] A traditional Chinese medicine composition for preventing and treating bacterial diseases, which is only different from Example 1 in the preparation method. Specifically:

[0062] Weigh gallnut, skullcap root, pomegranate rind, clove, and salvia miltiorrhiza, mix them, add water equivalent to 10 times the total amount of the medicinal materials, soak for 1.0 h, first decoct with strong fire for 0.5 h, filter, collect the first decoction liquid, add water to the remaining filter residue and decoct with gentle fire for 1.0 h, filter, collect the second decoction liquid, add water to the remaining filter residue and decoct with strong fire for 1.0 h, filter, collect the third decoction liquid, combine it with the decoction liquids obtained in the previous two times, and concentrate to 1 ± 0.05 mg / mL to obtain the traditional Chinese medicine composition.

[0063] The traditional Chinese medicine compositions provided in Examples 1 - 6 of the present invention can also be further prepared into acceptable preparations according to the conventional methods in veterinary pharmacy, such as granule preparations, powder preparations or liquid preparations. Appropriate excipients or carriers can be added during the preparation, such as flavoring agents, preservatives, suspending agents, coloring agents, etc.

[0064] The traditional Chinese medicine composition provided by the present invention can be directly used as a drug or used as a feed additive. When used as a feed additive, it can be mixed with conventional feed for preparation.

[0065] Experimental Example 1

[0066] Screening of Traditional Chinese Medicine Composition Formulas

[0067] 1. Materials and Methods

[0068] 1.1 Experimental Materials

[0069] 1.1.1 Traditional Chinese Medicines

[0070] Galla chinensis, Rheum officinale, Lonicera japonica, Coptis chinensis, Euphorbia humifusa, Forsythia suspensa, Granatum peel, Paeonia lactiflora, Fennel, Gentiana scabra, Scutellaria baicalensis, Polygonum cuspidatum, Eupatorium japonicum, Portulaca oleracea, Curcuma longa, Poria cocos, Paeonia suffruticosa, Ecklonia kurome, Dioscorea bulbifera, Salvia miltiorrhiza, Eugenia caryophyllata, Evodia rutaecarpa, Taraxacum mongolicum, Terminalia chebula, Cortex fraxini, Agrimonia pilosa, Rosa laevigata, Rubus idaeus, Sargentodoxa cuneata, Bupleurum chinense, Pulsatilla chinensis, Cimicifuga foetida, Senna leaf, Belamcanda chinensis, Asarum sieboldii, Artemisia annua. These 36 traditional Chinese medicines were purchased from Tongxintang Pharmacy, the General Company of Medicinal Materials in Bozhou City.

[0071] 1.1.2 Strains

[0072] The sources of each strain are as follows:

[0073] Aeromonas hydrophila NJ-35 was purchased from the China General Microbiological Culture Collection Center, with the preservation code CGMCC No. 8319, and was isolated from diseased crucian carp in 2010;

[0074] Plesiomonas shigelloides JX-09 was obtained from the Pearl River Fisheries Research Institute and isolated from diseased grass carp. See "Isolation and Identification of Plesiomonas shigelloides Pathogenic to Grass Carp, Acta Microbiologica Sinica, 2014, 54(2): 229-235";

[0075] Klebsiella pneumoniae was obtained from Jianghan University and isolated from Micropterus salmoides. See "Isolation, Identification and Biological Characteristics of a ST-873 Type Klebsiella pneumoniae Isolated from Micropterus salmoides, Oceanologia et Limnologia Sinica, 2023, 54(3): 836-847";

[0076] Vibrio cholerae F2 was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences, isolated from diseased grass carp and identified by physiological and biochemical methods. See "Isolation, Identification and Pathogenicity Study of a Pathogen of an Acute Bacterial Infectious Disease in Grass Carp, Acta Hydrobiologica Sinica, 2011, (6): 980-986";

[0077] Citrobacter freundii was isolated from Micropterus salmoides and obtained from the Pearl River Fisheries Research Institute. See "Isolation and Identification of Citrobacter freundii from Micropterus salmoides, Journal of Southern Agriculture, 2021, 52(2): 465-474)".

[0078] Acinetobacter baumannii was isolated from the liver of diseased Ictalurus punctatus and obtained from Huazhong Agricultural University. See "Isolation and Identification of Acinetobacter baumannii Strain from Ictalurus punctatus, Journal of Huazhong Agricultural University, 2010, 29(4): 489-493)".

[0079] The above strains were all subjected to morphological, biochemical, and molecular identifications.

[0080] 1.2 Preparation of single-herb Chinese medicine liquid

[0081] (1) 36 kinds of Chinese herbs were dried in an oven at 60 °C until constant weight. 50 g of each single-dose Chinese herb was weighed and placed in a non-woven bag, soaked in 500 mL of water for 30 min, decocted with strong fire for 30 min, and the first decoction was poured out.

[0082] (2) 500 mL of water was added, and it was decocted with gentle fire for 60 min, and the second decoction was poured out.

[0083] (3) 500 mL of water was added, and it was decocted with strong fire together with the medicine bag for 30 min. After decoction, the medicine bag was taken out and discarded.

[0084] (4) The three decoctions were poured back into the medicine pot and concentrated to 50 mL, so that the total medicine concentration reached 1 g / mL. After autoclaving at 121 °C, it was placed in a 4 °C refrigerator for later use.

[0085] 1.3 Preparation of Aeromonas hydrophila NJ-35 bacterial suspension

[0086] Aeromonas hydrophila NJ-35 stored at -80 °C was inoculated onto NB solid medium with an inoculation loop and cultured by streaking. It was placed in a constant temperature incubator at 37 °C for 16 h for resuscitation. A single colony was picked and transferred into 1 mL of NB liquid medium, and cultured overnight at 37 °C on a shaker at 200 rpm. The concentration of the bacterial suspension was determined by the dilution coating plate method. The cultured bacterial liquid was serially diluted to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9times. Take 100 μL of the bacterial solution at each dilution factor and spread it on NB medium. Repeat spreading on 3 plates. Incubate the plates upside down in a constant temperature incubator at 37 °C for 18 h. Observe the spread plates. When the number of colonies on the plate is in the range of 30 to 300, it is used as the effective counting bacterial concentration. Calculate the bacterial concentration of the original bacterial solution according to the following formula. Dilute it with normal saline with a mass fraction of 0.86% to 1×10 8 CFU / mL.

[0087] Number of strains per milliliter of bacterial solution = (C÷V)×M

[0088] In the formula: C represents the average number of colonies growing on the plate at a certain dilution concentration, V represents the volume (mL) of the dilution solution used when spreading the plate, M represents the dilution factor, unit: CFU / mL.

[0089] 1.4. In vitro antibacterial experiment of traditional Chinese medicine

[0090] 1.4.1. Tablet preparation

[0091] Use a puncher to make multiple 6 mm round filter paper pieces. Place the pieces in a blank petri dish, sterilize them under high pressure at 121 °C, transfer them to an oven at 50 °C to dry. Soak the dried pieces in the sterilized traditional Chinese medicine liquid and store them at 4 °C for later use.

[0092] 1.4.2. Determination of antibacterial ability of single traditional Chinese medicine by disk diffusion method

[0093] Use the disk diffusion method to determine the antibacterial effect of the single traditional Chinese medicine extract. Pipette 100 μL of the bacterial solution with a concentration of 1×10 8 CFU / mL, and spread the bacterial solution evenly on MH medium. Take out the pieces soaked in the medicine liquid, stick them on the sterilized petri dish, and then stick them on the plate that has been spread with the bacterial solution after the pieces are dry. Stick 3 replicates of each tablet. Place the plate in a constant temperature incubator at 37 °C and incubate it upside down for 16 h. There is an antibacterial circle around the disk where no strains visible to the naked eye grow. Use a vernier caliper to measure the diameter of the antibacterial circle by the cross measurement method. Record 6 mm if there is no antibacterial circle. Record the 3 replicate experiments as the mean ± standard deviation. If the average diameter of the antibacterial circle ≥ 20 mm, it is judged as extremely sensitive; 20 mm < antibacterial circle diameter ≤ 15 mm is highly sensitive; 15 mm < antibacterial circle diameter ≤ 10 mm is moderately sensitive; antibacterial circle diameter < 10 mm is low sensitive.

[0094] 1.4.3. Determination of minimum inhibitory concentration (MIC) of single traditional Chinese medicine

[0095] Use the micro-dilution method in 96-well plates to determine the minimum inhibitory concentration of single traditional Chinese medicine. See the sample addition schematic diagram in Figure 1。For the holes in the last eleven columns except the first column, add 100 μL of MH cationic liquid medium to each hole. Add 100 μL of traditional Chinese medicine liquid to the first and second holes in each row. After mixing the liquid in the second hole with the medium, aspirate 100 μL and add it to the third hole. Repeat the operation until 100 μL is aspirated from the 11th hole and discarded. Perform two-fold dilution in this way, and the drug dilution concentrations are 1000, 500, 250, 125, 62.50, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 mg / mL. For rows A - C, E - G, add 100 μL of bacterial liquid with a concentration of 1×10 8 CFU / mL to each hole, and repeat each drug 3 times. Add 100 μL of medium to rows D and H as negative controls. In the 12th column, do not add the liquid medicine, but only add 100 μL of bacterial liquid and 100 μL of medium as positive controls. After the sample addition operation is completed, place the 96-well plate in an incubator at 37°C for 18 h, observe the results. If there is no obvious bacterial precipitate at the bottom, it is regarded as the minimum inhibitory concentration (MIC) of the drug against Aeromonas hydrophila.

[0096] 1.4.4. Determination of the minimum bactericidal concentration (MBC) of traditional Chinese medicine

[0097] After observing the results of the MIC obtained above, aspirate 10 μL of liquid from the clear and sterile precipitate holes, and vertically drop it on the NB solid medium. Repeat 3 times for each concentration. Wait for the liquid to naturally diffuse until it is absorbed by the medium and there is no liquid on the surface. Place it in an incubator at 37°C for 18 h and observe the results. If there is no bacterial plaque, the bacteria are killed and cannot grow on the ordinary plate. The minimum concentration of traditional Chinese medicine treatment without bacterial growth is regarded as the minimum bactericidal concentration.

[0098] 1.5. Analysis of the compatibility optimization of traditional Chinese medicine compound preparations

[0099] Using Aeromonas hydrophila NJ-35 as the target strain, the orthogonal t-value method is used to optimize the compound compatibility of the top 7 traditional Chinese medicines with the best antibacterial effects among the single herbs detected, and the best combination and the best dosage ratio are found.

[0100] 1.5.1. Analysis of the main drugs

[0101] Design L 12 (2 7 ) orthogonal experimental table 1 with two levels, seven factors, and twelve times. According to the combinations shown in Table 1, weigh the corresponding doses of traditional Chinese medicine and put them into non-woven bags, and decoct them into a traditional Chinese medicine compound of 1 g / mL according to the method in 1.2. Measure the inhibition zone diameter of each combination of traditional Chinese medicine compound agents against Aeromonas hydrophila NJ-35 by the disc diffusion method. Repeat each experiment 3 times, and determine the main drugs of the prescription according to the experimental results.

[0102] Table 1 L12 (2 7 ) Analysis of the main drug by orthogonal t-value method

[0103]

[0104] Note: A is pomegranate peel, B is moutan bark, C is terminalia chebula, D is clove, E is Chinese gall, F is salvia miltiorrhiza, G is scutellaria baicalensis; Level 1 is 10 g of drug used; Level 2 is no drug used.

[0105] 1.5.2. Analysis of interaction of adjuvant drugs

[0106] Taking the factors with significant effects in the above results as the main factors, using the following L 16 (2 5 ) orthogonal array, that is, Table 2 focuses on analyzing the interaction between the main factors and other factors and between other factors. Weigh the traditional Chinese medicine according to the formula in Table 2, decoct it into a traditional Chinese medicine compound liquid of 1 g / mL according to the method in 1.2, and detect the antibacterial ability of 16 prescriptions by the paper disk diffusion method. Analyze the interaction between various factors according to the results.

[0107] Table 2 L 16 (2 5 ) Analysis of interaction of adjuvant drugs by orthogonal t-value method

[0108]

[0109] Note: Chinese gall and scutellaria baicalensis are added to each prescription as the basis; A is pomegranate peel, B is moutan bark, C is clove, D is terminalia chebula, E is salvia miltiorrhiza; Level 1 is 10 g of drug used; Level 2 is no drug used.

[0110] 1.5.3. Dose optimization

[0111] According to the above results, it is confirmed that the main drugs are Chinese gall and scutellaria baicalensis, and the adjuvant drugs are pomegranate peel, clove, and salvia miltiorrhiza. Design the L9(3 4 ) orthogonal experimental table 4 for dose selection. Combine Chinese gall and scutellaria baicalensis into one factor, take the original dose as the medium dose, increase or decrease appropriately, and formulate the three-level dose table 3.

[0112] Table 3 Four-factor three-level dose optimization table

[0113]

[0114] Note: Chinese gall and scutellaria baicalensis are factor A. The former is the dose of Chinese gall and the latter is the dose of scutellaria baicalensis; B is pomegranate peel, C is clove, D is salvia miltiorrhiza.

[0115] Table 4 L9(3 4 ) Optimization of dose selection by orthogonal t-value method

[0116]

[0117] Note: Each factor and level is the same as those in Table 3.

[0118] 1.6. Verification of the effects of traditional Chinese medicine compound preparations

[0119] 1.6.1. In vitro antibacterial ability

[0120] Detect the in vitro antibacterial effect of the optimized traditional Chinese medicine compound preparation. Use the disk diffusion method and the micro-dilution method to detect the size of the inhibition zone, the minimum bactericidal concentration, and the minimum inhibitory concentration. The methods are the same as those in 1.4.2, 1.4.3, and 1.4.4.

[0121] 1.6.2. Effect of traditional Chinese medicine compound preparation on strain NJ-35

[0122] Determine the drug treatment concentration according to the results of the MBC of the traditional Chinese medicine compound. Consider the concentration greater than the MBC as the high concentration and the concentration less than the MBC as the low concentration. Treat strain NJ-35 with three different concentrations of the compound preparation: 3.91 mg / mL, 125 mg / mL, and 250 mg / mL. Spread strain NJ-35 on the NB solid medium for activation. Pick a single colony and place it in the NB liquid medium. Incubate it on a shaker at 37 °C and 200 rpm for 8 h. Measure the concentration of the bacterial solution by the dilution plating method. Adjust the bacterial solution to 1×10 8 CFU / mL. Take 1 mL of the bacterial solution and mix it with 1 mL of the traditional Chinese medicine compound preparation at different concentrations. Incubate them together on a shaker at 37 °C and 200 rpm for 12 h. Centrifuge at 3000 rpm for 10 min. After discarding the culture medium, gently rinse it with PBS (the bacterial solution treated with the high-concentration traditional Chinese medicine compound preparation needs to be repeatedly washed with PBS several times to remove the traditional Chinese medicine residues, centrifuge at 3000 rpm for 10 min, and remove the PBS). After centrifuging and discarding the supernatant, add 1 mL of electron microscope fixative to the bacterial cells, and gently pipette to suspend the bacterial precipitate in the fixative. Fix it at room temperature in the dark for 30 min, and then transfer it to 4 °C for storage. Send it to Wuhan Sevier Biotechnology Co., Ltd. for scanning electron microscopy.

[0123] 2. Experimental results

[0124] 2.1. Antibacterial effect and minimum inhibitory concentration of single traditional Chinese medicine

[0125] The antibacterial abilities of single traditional Chinese medicine (TCM) are shown in Table 5. Chinese gallnut has the strongest antibacterial ability against Aeromonas hydrophila NJ-35, with an inhibition zone diameter of 17.8 ± 0.17 mm and an MIC of 31.25 mg / mL. Next are Coptis chinensis, Forsythia suspensa, Granatum pericarpium, Scutellaria baicalensis, Moutan cortex, Cortex fraxini, Terminalia chebula, Eugenia caryophyllata, Rubus idaeus, Salvia miltiorrhiza, Evodia rutaecarpa, Euphorbia humifusa. The inhibition zone sizes of these 13 single TCMs are between 10 mm and 15 mm. The inhibition zone sizes of the extracts of 19 single TCMs such as Lonicera japonica, Rheum palmatum, Paeonia lactiflora, Foeniculum vulgare, Polygonum cuspidatum are between 6 mm and 10 mm, with clear transparent zones. The extracts of five single TCMs, Gentiana scabra, Curcuma longa, Ecklonia kurome, Poria cocos, Senna leaf, have no transparent inhibition zones against NJ-35, indicating no antibacterial effect. The inhibition zones of some single TCMs are shown in Figure 2 . The measured MIC values are not entirely consistent with the inhibition zone sizes, which may be related to the errors in visual observation. Seven TCMs with relatively strong antibacterial abilities were selected as candidate TCMs for the compatibility optimization of the TCM compound preparation based on the inhibition zone sizes, namely Chinese gallnut, Granatum pericarpium, Moutan cortex, Salvia miltiorrhiza, Eugenia caryophyllata, Scutellaria baicalensis, Terminalia chebula.

[0126] Table 5 Antibacterial activities of single Chinese herbal medicines against Aeromonas hydrophila NJ-35

[0127]

[0128] Note: An inhibition zone diameter of 15 - 19 mm indicates high sensitivity, denoted by "++"; 10 - 14 mm indicates medium sensitivity, denoted by "+"; < 10 mm indicates low sensitivity, denoted by "-".

[0129] 2.2. Compatibility optimization of the TCM compound preparation

[0130] 2.2.1. Analysis of the main herbs

[0131] When M1 > M2, that is, D > 0, it indicates that adding this kind of TCM to the prescription has a better effect than not adding it. As can be seen from Table 6, only the D values of Chinese gallnut and Scutellaria baicalensis are positive, and the antibacterial abilities are better after adding these two herbs, which can be used as the main herbs for further analysis. However, for Moutan cortex, Terminalia chebula, and Salvia miltiorrhiza, M1 < M2 and t < 0.01, showing a highly significant difference, indicating that the effect of adding these several TCMs is lower than not adding them. Continue with the analysis of the auxiliary herbs and then make a decision on the selection of the herbs.

[0132] Table 6 L 12 (2 7 ) Analysis of the main herbs by the orthogonal t-value method

[0133]

[0134] Note: A is pomegranate peel; B is moutan bark; C is chebula fruit; D is clove; E is Chinese gall; F is salvia root; G is skullcap root. ** indicates extremely significant, - indicates not significant; level 1 is 10 g of medicine used, level 2 is no medicine used; f is the degree of freedom; n is the number of within-group experiments, Se is the residual standard error; M1 is the sum of the means at level 1; M2 is the sum of the means at level 2; D is the difference between M1 and M2; Mm is the average of the means of each group; MM is the expected drug effect.

[0135] 2.2.2, Interaction analysis of adjuvant drugs

[0136] As can be seen from the results in Table 7 and Supplementary Table 7, there is antagonism between AB and BC, there is a synergistic effect between AC, and factor B itself has a negative effect and extremely significant difference. Therefore, factor B is discarded and factors A and C are used. From the perspective of the prescription, A2E1, B2E1, C2E1, and D2E1 are the maximum factor interactions containing factor E, so E1 is taken. The prescriptions A2D2, B2D2, C2D2, and D2E1 are the maximum values containing factor D, so D2 is taken, that is, factor D is discarded and factor E is retained. Finally, it is determined that Chinese gall and skullcap root are the main drugs, and pomegranate peel, salvia root, and clove are the adjuvant drugs.

[0137] Table 7 L 16 (2 5 ) Analysis of the interaction of adjuvant drugs by the orthogonal t-value method

[0138]

[0139] Supplementary Table 7 L 16 (2 5 ) Analysis of the interaction of adjuvant drugs by the orthogonal t-value method

[0140]

[0141] Note: Chinese gall and skullcap root are the main drugs, and 10 g of each is added to each treatment group; A is pomegranate peel; B is moutan bark; C is clove; D is chebula fruit; E is salvia root. In the interaction table, "△" indicates antagonism, "^" indicates synergy, "*" indicates the maximum value, and [xx] indicates the sum of the mean diameters of the inhibition zones when the factor takes the x level; other symbols are the same as in Table 6.

[0142] 2.2.3, Dose optimization analysis

[0143] After confirming the main drug and adjuvant drugs, it is analyzed that the one with the largest mean value among the levels of each factor has the best antibacterial effect. As can be seen from the results in Table 8, the best dose combination of the traditional Chinese medicine compound preparation is A3B2C1D1, that is, 20 g of Chinese gall, 15 g of skullcap root, 10 g of pomegranate peel, 5 g of clove, and 5 g of salvia root.

[0144] Table 8 L9(3 4 ) Optimization of dose selection by the orthogonal t-value method

[0145]

[0146] Note: A is Chinese gallnut and Scutellaria baicalensis; B is pomegranate rind; C is clove; D is Salvia miltiorrhiza; M3 is the sum of the means at the 3 levels; Sa is the standard deviation of M1, M2, and M3 in each factor; L is the number of levels; "#" is the maximum value in the same column, and other symbols are the same as in Table 6.

[0147] 2.3 Antibacterial Effect of Traditional Chinese Medicine Compound Preparation in Vitro

[0148] According to the best ratio obtained in 2.2.3, the traditional Chinese medicine compound preparation was decocted, and the in vitro antibacterial effect was measured. As can be seen from Table 9, the compound traditional Chinese medicine preparation has excellent antibacterial ability against Aeromonas hydrophila NJ-35, and the size of the inhibition zone is 18.83 ± 0.81 mm, see Figure 3 . In addition, the compound traditional Chinese medicine preparation also has good antibacterial ability against Plesiomonas shigelloides, Stenotrophomonas maltophilia, and Vibrio cholerae. The minimum inhibitory concentration (MIC) for the NJ-35 strain is 0.12 mg / mL, and the minimum bactericidal concentration (MBC) is 7.81 mg / mL. The MBC results are shown in Figure 4 .

[0149] The results of scanning electron microscopy observation of the strains treated with traditional Chinese medicine compound preparations at different concentrations are shown in Figure 5 . The bacteria in the untreated control group were rod-shaped, with blunt ends, complete structures, and a surface evenly covered with pili, round and without holes. After treating the strains with the traditional Chinese medicine compound preparation at 3.91 mg / mL, i.e., 50% MBC, compared with the control group, the bacterial cells were severely deformed, significantly shrunk and sunken, and the depression was deep at local positions. After treatment with high-concentration compound liquid at 125 mg / mL and 250 mg / mL, the surface of the bacterial cells was rough, and some bacterial cells had obvious cracks, folds, and depressions, with slight deformation and not as severe shrinkage and depression as that at low doses. It shows that treatment with an appropriate dose of the compound liquid may damage the cell membrane structure of the bacterial cells, resulting in the rupture of the bacterial cells and the outflow of the contents.

[0150] Table 9 Antibacterial Ability of Traditional Chinese Medicine Compound Preparation against Pathogenic Bacteria

[0151]

[0152] Note: The diameter of the inhibition zone of 15 - 19 mm is highly sensitive, indicated by "++"; 10 - 14 mm is moderately sensitive, indicated by "+"; <10 mm is lowly sensitive, indicated by "-".

[0153] Experimental Example 3

[0154] Protective Effect of Traditional Chinese Medicine Composition on Grass Carp

[0155] 1. Experimental Method

[0156] 1.1 Experimental animals

[0157] Healthy grass carps, weighing 100 g - 130 g, were purchased from Shenlong Fisheries Development Co., Ltd. Before the experiment, they were temporarily raised in a disinfected 300 L plastic box for 2 weeks. The breeding water was used after being aerated for two days, and 50% of the water was changed every two days. The water temperature was 18°C - 25°C, and the dissolved oxygen > 5.0 mg / L.

[0158] The concentration of Aeromonas hydrophila was adjusted to 1×10 7 CFU / mL with normal saline, and a grass carp Aeromonas hydrophila infection model was obtained by intraperitoneal injection of 0.2 mL of the bacteria into the grass carp. 84 grass carps were randomly divided into: 6 in the blank control group, 6 in the normal saline control group, 36 in the Aeromonas hydrophila infection group, and 36 in the traditional Chinese medicine treatment group. Among them, 6 in the Aeromonas hydrophila infection group and the traditional Chinese medicine treatment group were used for the detection of the expression level of inflammatory factors, and 30 were used for the calculation of the mortality rate.

[0159] 1.2 Implementation method of experimental grouping

[0160] (1) Blank control group: Without any treatment.

[0161] (2) Normal saline control group: Grass carps were intraperitoneally injected with 0.85% normal saline as a negative control.

[0162] (3) Aeromonas hydrophila infection group: Grass carps were intraperitoneally injected with 1×10 7 CFU / mL Aeromonas hydrophila bacterial solution.

[0163] (4) Traditional Chinese medicine treatment group: Grass carps used for detecting the level of inflammatory factors were continuously gavaged with 100 mg / Kg of the traditional Chinese medicine composition provided in Example 1 of the present invention for 7 days, and then intraperitoneally injected with the same dose of Aeromonas hydrophila as the Aeromonas hydrophila infection group; for grass carps used for detecting the survival rate after treatment, they were fed with a feed containing 2‰ of the traditional Chinese medicine composition provided in Example 1 of the present invention twice a day for 7 days, and then intraperitoneally injected with the same dose of Aeromonas hydrophila as the Aeromonas hydrophila infection group, and the number of dead grass carps was recorded every day.

[0164] 1.3 Detection of the expression level of inflammatory factors

[0165] The relative expression levels of TNF-α, IFN, Myd88, IL-1β, and MHCⅡ mRNA in the head kidney tissue were detected by real-time fluorescence quantitative PCR. Three replicates were made for each sample. Using β-actin as an internal reference gene, the 2-ΔΔCt method was used to calculate the relative mRNA expression level. The fluorescence PCR data was sorted and calculated by Excel. One-way analysis of variance was used, and LSD and Duncan tests were used for post hoc tests. P < 0.05 was considered a significant difference and was statistically significant.

[0166] 1.4, Pathological examination

[0167] At 24 h after the injection of bacteria, the kidney, intestine, spleen, and gills of grass carp were taken. After being fixed with a universal fixative for 24 hours, they were embedded in paraffin, sectioned, stained with HE, and sealed. Observation was carried out under a microscope.

[0168] 2. Experimental results

[0169] 2.1, Pathological changes

[0170] At 24 h after Aeromonas hydrophila infection, the submucosa of the intestine was disordered, the tissue was discrete, there was infiltration of inflammatory cells, the intestinal villus epithelial cells fell off and were broken, the intestinal muscle layer was discrete, and there were also dispersions in the intestinal muscle layer of the traditional Chinese medicine treatment group, and the degree of damage to the submucosa was reduced, and the morphology of the intestinal villi was intact. In the Aeromonas hydrophila infection group, the hepatocytes were swollen and rounded, there was extensive nuclear lysis, the arrangement between cells was disordered with cavities, and there was infiltration of inflammatory cells. In the traditional Chinese medicine treatment group, there were cavities around the interlobular arteries, and some hepatocytes were necrotic and had nuclear lysis, and the pathological degree was slightly lower than that of the Aeromonas hydrophila infection group. In the spleen tissue of the control group, the cells were arranged tightly and there was no obvious damage. In the Aeromonas hydrophila infection group, there was severe bleeding, hemosiderin agglutination, and extensive vacuolar degeneration. In the traditional Chinese medicine treatment group, the degree of vacuolar degeneration was mild, and there was still hemosiderin deposition. In the kidney tissue, in the Aeromonas hydrophila infection group, the outer layer cells of the renal tubules were necrotic and vacuolated, the lumen boundary was blurred, the interstitial cells were necrotic and vacuolated, and there were a large number of blood cells. In the control group and the traditional Chinese medicine treatment group, the renal tubule structure was complete and there was no obvious lesion. In the Aeromonas hydrophila infection group, the gill lamellae were curled and shortened, the gill lamellae cells were necrotic and fell off, and there was atrophy, distortion and necrosis of the gill filament cartilage. In the traditional Chinese medicine treatment group, the gill lamellae were slightly bent and degenerated, there was hyperplasia of the cells between the gill lamellae, cell necrosis, and stratification with the gill filament cartilage. The specific results are shown in Figure 6 。

[0171] 2.2, Relative levels of head kidney inflammatory factors

[0172] The results are as Figures 7 to 11 shown. Compared with the blank group, all inflammatory factors were significantly expressed. In the traditional Chinese medicine treatment group, IFN was significantly increased, MHCⅡ and Myd88 were significantly down-regulated, IL-1β and TNF-α had no significant changes, but all inflammatory factors in the traditional Chinese medicine treatment group were significantly down-regulated compared with the Aeromonas hydrophila infection group.

[0173] 2.3, Survival rate of grass carp after prevention by traditional Chinese medicine treatment

[0174] From Figure 12It can be seen that after feeding with the feed containing 2‰ of the traditional Chinese medicine composition provided in Example 1 of the present invention for 7 days and then injecting Aeromonas hydrophila, there is a highly significant difference in the survival rate compared with directly injecting Aeromonas hydrophila. The obvious mortality rate of the grass carp fed with the traditional Chinese medicine feed decreases. There is no death phenomenon on the second day when the mortality rate is the highest, showing an obvious protective effect, and the survival rate reaches 86.67%.

[0175] In summary, the traditional Chinese medicine composition of the present invention can effectively protect grass carp from being infected by Aeromonas hydrophila, reduce tissue damage and inflammation occurrence, and greatly improve the survival rate of grass carp after being incorporated into the grass carp feed as a feed additive and fed.

[0176] The above are only the embodiments of the present invention, and do not limit the patent scope of the invention accordingly.

Claims

1. A Chinese medicine composition for preventing and treating bacterial diseases, characterized in that: The invention is prepared from the following raw materials by weight: 5-20 parts of gallnut, 5-15 parts of scutellaria, 5-15 parts of pomegranate peel, 5-15 parts of clove and 5-20 parts of salvia miltiorrhiza. The bacterial disease is caused by Aeromonas hydrophila NJ-35.

2. The Chinese medicine composition for preventing and treating bacterial diseases according to claim 1, characterized in that: The Chinese medicine composition is prepared from the following raw materials by weight: 20 parts of gallnut, 15 parts of scutellaria, 10 parts of pomegranate peel, 5 parts of cloves and 5 parts of salvia miltiorrhiza.

3. A method for preparing the Chinese medicine composition for preventing and treating bacterial diseases according to claim 1, characterized in that: The following steps are involved: The Chinese medicine composition is obtained by weighing gallnut, scutellaria baicalensis, pomegranate peel, clove and salvia miltiorrhiza according to weight, mixing them, adding water and boiling them, and collecting the boiling liquid.

4. The method for preparing the Chinese medicine composition for preventing and treating bacterial diseases according to claim 3, characterized in that: When adding water for decoction, the amount of water added should be 8 to 10 times the total amount of medicinal materials.

5. The method for preparing the Chinese medicine composition for preventing and treating bacterial diseases according to claim 4, characterized in that: Mix the weighed medicinal materials, add water and soak for 0.5h to 1.0h, then boil.

6. The method for preparing the Chinese medicinal composition for preventing and treating bacterial diseases according to claim 5, characterized in that: The specific process of decocting with water is: first, decoct with high heat for 0.5h to 1.0h, filter, collect the first decoction liquid, add water to the remaining filter residue and decoct with low heat for 0.5h to 1.0h, filter, collect the second decoction liquid, add water to the remaining filter residue and decoct with high heat for 0.5h to 1.0h, filter, collect the third decoction liquid, and combine it with the decoction liquids obtained from the first two times to obtain the Chinese medicine composition.

7. The method for preparing the Chinese medicine composition for preventing and treating bacterial diseases according to claim 6, characterized in that: The decoction obtained is collected and concentrated to 1±0.05 mg / mL to obtain a concentrate of the traditional Chinese medicine composition.

8. Use of the Chinese medicine composition according to claim 1 in preparing a product for inhibiting pathogenic bacteria, characterized in that: The pathogen is Aeromonas hydrophila NJ-35.

9. The use according to claim 8, characterized in that: The traditional Chinese medicine composition is used for preparing products for preventing and treating bacterial infection diseases in fish farming.

10. The use according to claim 9, characterized in that: The product is a medicine or a feed additive.

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