Traditional Chinese medicine composition for preventing and treating bacterial diseases as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention belongs to the field of prevention and treatment medicines for freshwater cultured fishes, and particularly relates to a traditional Chinese medicine composition for preventing and treating bacterial diseases and a preparation method and application thereof. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 5-20 parts of gallnut, 5-15 parts of scutellaria baicalensis, 5-15 parts of pomegranate rind, 5-15 parts of clove and 5-20 parts of salvia miltiorrhiza. After the traditional Chinese medicine composition provided by the invention is fed to the grass carp, the survival rate of the grass carp can be greatly increased, the in-vivo tissue damage and inflammation conditions are improved, the expression conditions of inflammatory factors TNF-alpha, IFN, Myd88, IL-1beta and MHC II of the head kidney of the grass carp are reduced, and the traditional Chinese medicine composition has an in-vitro antibacterial effect on other pathogenic bacteria. The traditional Chinese medicine composition provided by the invention can effectively resist bacteria, reduce the inflammation degree and improve the survival rate of grass carp after bacterial infection.

Description

Technical Field

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

[0002] Grass carp is an important freshwater farmed fish in my country, and is known as one of the "four major carps" along with bighead carp, silver carp, and black carp. With the popularization of intensive farming, high-density breeding and deteriorating breeding environment have led to frequent outbreaks of diseases such as enteritis, gill rot, and septicemia in grass carp, causing huge economic losses to farmers and restricting the development of grass carp farming. Among them, enteritis and septicemia of grass carp caused by Aeromonas hydrophila infection have led to a large number of deaths of grass carp, which is extremely harmful. Aeromonas hydrophila infection usually manifests as blackening of the body surface, slow movement, redness and swelling of the anus, congestion at the base of the fins, and bleeding in the abdomen. Dissection shows a large amount of ascites in the abdominal cavity, inflated and swollen intestines, inelastic intestinal walls, redness and easy to tear, black spleen, and congestion of liver and kidney tissues.

[0003] Antibiotics and their substitutes are often used in aquaculture to prevent and treat bacterial diseases in fish farming, but these substances have large side effects and the problem of drug residues. Drug residues can easily lead to the production of multi-drug resistant bacteria, affecting the preventive and therapeutic effects of antibiotics. Traditional Chinese medicine is rich in a variety of bioactive ingredients. Polysaccharides, flavonoids, polyphenols, saponins, alkaloids, essential oils, terpenes and other bioactive ingredients have been widely used in the prevention and treatment of viral, bacterial, parasitic and fungal diseases in fish. They are currently developing antibacterial drugs and are also an excellent choice for fighting 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 prescriptions for the comprehensive prevention and treatment of bacterial diseases in aquaculture. Summary of the invention

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

[0005] The specific technical solutions provided by the present invention are as follows: The first aspect of the present invention provides a traditional Chinese medicine composition for preventing and treating bacterial diseases, which is prepared from the following raw materials by weight: 5 to 20 parts of gallnut, 5 to 15 parts of scutellaria, 5 to 15 parts of pomegranate peel, 5 to 15 parts of cloves, and 5 to 20 parts of salvia miltiorrhiza.

[0006] As a preferred embodiment of the present invention, the Chinese medicine composition is made of 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.

[0007] The second aspect of the present invention provides a method for preparing the Chinese medicine composition, comprising the following steps: 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.

[0008] As a preferred embodiment of the present invention, the amount of water added during decoction is 8 to 10 times the total amount of the medicinal materials.

[0009] As a preferred embodiment of the present invention, the weighed medicinal materials are mixed, soaked in water for 0.5h to 1.0h, and then decocted.

[0010] More preferably, the specific process of decocting with water is: first decocting with high heat for 0.5h~1.0h, filtering, collecting the first decoction, adding water to the remaining filter residue and decocting with low heat for 0.5h~1.0h, filtering, collecting the second decoction, adding water to the remaining filter residue and decocting with high heat for 0.5h~1.0h, filtering, collecting the third decoction, and combining it with the decoctions obtained from the first two times to obtain the Chinese medicine composition.

[0011] As a preferred embodiment of the present invention, the decoction obtained is collected and concentrated to 1±0.05 mg / mL to obtain the concentrate of the Chinese medicine composition. After sterilization with high pressure steam at 121°C, it is cooled and stored in a refrigerator at 4°C.

[0012] The third aspect of the present invention provides an application of the traditional Chinese medicine composition in the preparation of a product for inhibiting pathogenic bacteria, wherein the pathogenic bacteria are Aeromonas hydrophila, Plesiomonas shigella, Stenotrophomonas maltophilia, Vibrio cholerae, Klebsiella pneumoniae, Acinetobacter baumannii or Citrobacter freundii.

[0013] As a preferred embodiment of the present invention, the Chinese medicine composition is used to prepare a product for preventing and treating bacterial infection diseases in fish farming.

[0014] As a preferred embodiment of the present invention, the product is a medicine or a feed additive.

[0015] The fourth aspect of the present invention provides a medicine for preventing and treating bacterial infection diseases in fish farming, which comprises the Chinese medicine composition.

[0016] As a preferred embodiment of the present invention, the drug includes a pharmaceutically acceptable excipient or carrier.

[0017] A fifth aspect of the present invention provides a feed additive for preventing and treating bacterial infections in fish farming, comprising the Chinese medicine composition.

[0018] As a preferred embodiment of the present invention, the feed additive includes an acceptable auxiliary material or carrier.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The Chinese medicine composition provided by the present invention has simple components, small side effects, easy-to-obtain raw materials, and a simple preparation method. Feeding grass carp with the Chinese medicine composition provided by the present invention can reduce the severity of bacterial infection symptoms of grass carp, reduce mortality, reduce the expression levels of inflammatory factors including TNF-α, IFN, Myd88, IL-1β, MHCⅡ, etc., slow down the inflammation and damage of the spleen, kidney, intestine, and gills, and improve the anti-bacterial infection ability of grass carp.

[0020] The Chinese medicine composition provided by the present invention is obtained through in vitro antibacterial ability screening and compatibility optimization of single Chinese medicines, and solves the problems of drug residues and the like that cannot be solved by antibiotics and their substitutes in aquaculture, has small side effects and high safety, can effectively prevent and treat enteritis and sepsis caused by Aeromonas hydrophila infection of grass carp, and greatly improves the survival rate of grass carp.

[0021] The Chinese medicine composition provided by the present invention has a synergistic effect among the raw materials, and the compound use can better exert the effect of inhibiting pathogens. Antibacterial experiments have proved that the Chinese medicine composition can inhibit Aeromonas hydrophila, Pseudomonas shigellae, Stenotrophomonas maltophilia, Vibrio cholerae, Klebsiella pneumoniae, Acinetobacter baumannii or Citrobacter freundii, especially Aeromonas hydrophila, Pseudomonas shigellae, Stenotrophomonas maltophilia and Vibrio cholerae. The Chinese medicine composition can be used for the prevention and treatment of various bacterial diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of sample addition during the determination of the minimum inhibitory concentration of a single Chinese medicine; solution 1 and solution 2 refer to two different single-herb solutions on the same 96-well plate.

[0023] Figure 2 This is a diagram of the antibacterial ability of some Chinese herbal medicines determined by the paper diffusion method. The four colonies in the figure represent the antibacterial zones of different Chinese herbal medicines.

[0024] Figure 3 This is the inhibition zone diagram of the Chinese herbal compound preparation against the NJ-35 strain. The different colonies in the figure represent four groups of repeated experiments.

[0025] Figure 4The results of MBC test of NJ-35 strain by traditional Chinese medicine compound preparation; the positive control is the plaque without traditional Chinese medicine treatment, and the blank control is the one with normal saline added; 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, Chinese medicine compound preparation with a concentration of 31.25 mg / mL; h, blank control; i, Chinese medicine compound preparation with a concentration of 15.63 mg / mL; j, Chinese medicine compound preparation with a concentration of 7.81 mg / mL; k, Chinese medicine compound preparation with a concentration of 3.91 mg / mL; l, Chinese medicine compound preparation with a concentration of 1.95 mg / mL; m, Chinese medicine compound preparation with a concentration of 0.98 mg / mL; n, Chinese medicine compound preparation with a concentration of 0.49 mg / mL.

[0026] Figure 5 These are scanning electron micrographs of NJ-35 strains treated with different concentrations of Chinese herbal compound preparations; A: blank control; B: 3.91 mg / mL Chinese herbal compound treatment; C: 125 mg / mL Chinese herbal compound treatment; D: enlarged view of the framed part in C; E: 250 mg / mL Chinese herbal compound treatment; the arrows point to wrinkles and cracks; F: 250 mg / mL Chinese herbal compound treatment; the arrows point to wrinkles.

[0027] Figure 6 These are pathological sections of the intestine, spleen, kidney, and gills of grass carp infected with Aeromonas hydrophila after treatment with the Chinese herbal compound preparation; in the spleen pathological section, the black arrow in the bacterial infection group points to vacuolar degeneration, in the kidney pathological section, the black arrow in the bacterial infection group points to necrosis of the outer cells of the renal tubule, and in the gill pathological section, the black arrow in the bacterial infection group points to atrophy of the gill filament cartilage.

[0028] Figure 7 This is a statistical chart of the relative expression of IFN mRNA in the head kidney of grass carp infected with Aeromonas hydrophila after treatment with the Chinese herbal compound preparation.

[0029] Figure 8 This is a statistical chart of the relative expression of IL-1β mRNA in the head kidney of grass carp infected with Aeromonas hydrophila after treatment with the Chinese herbal compound preparation.

[0030] Fig. 9 This is a statistical chart of the relative expression of MHCⅡ mRNA in the head kidney of grass carp infected with Aeromonas hydrophila after treatment with the traditional Chinese medicine compound preparation.

[0031] Fig.10 This is a statistical graph of the relative expression of Myd88 mRNA in the head kidney of grass carp infected with Aeromonas hydrophila after treatment with the Chinese herbal compound preparation.

[0032] Fig.11 This is a statistical chart of the relative expression of TNF-α mRNA in the head kidney of grass carp infected with Aeromonas hydrophila after treatment with the Chinese herbal compound preparation.

[0033] Fig.12 This is a graph showing the survival rate of grass carp infected with Aeromonas hydrophila after treatment with a Chinese herbal compound preparation. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further illustrated and described below through a specific implementation method description. The kit methods used in the implementation cases are conventional processing methods unless otherwise specified, and the reagents and materials used can be purchased from ordinary commercial channels unless otherwise specified.

[0035] In existing aquaculture, antibiotics and their substitutes are often used to prevent and treat bacterial diseases in fish farming, but these substances have large side effects and drug residues. In addition, due to the influence of drug residues, multi-drug resistant pathogens are easily generated, which affects the prevention and treatment effect of antibiotic substances. Traditional Chinese medicine is rich in a variety of bioactive ingredients. Polysaccharides, flavonoids, polyphenols, saponins, alkaloids, essential oils, terpenes and other bioactive ingredients have been widely used in the prevention and treatment of viral, bacterial, parasitic and fungal diseases in fish. They are an excellent choice for current and developing antibacterial drugs to fight 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 prescriptions for the comprehensive prevention and treatment of bacterial diseases in aquaculture.

[0036] The invention provides a Chinese medicine composition for preventing and treating bacterial diseases, which is prepared from the following raw materials by weight: 5 to 20 parts of gallnut, 5 to 15 parts of scutellaria, 5 to 15 parts of pomegranate peel, 5 to 15 parts of cloves, and 5 to 20 parts of salvia miltiorrhiza. The Chinese medicine composition solves the problems of drug residues and the like that cannot be solved by antibiotics and their substitutes in aquaculture, has small side effects, high safety, can effectively prevent and treat enteritis and septicemia caused by Aeromonas hydrophila infection in grass carp, and greatly improves the survival rate of grass carp.

[0037] The sources and effects of the medicinal materials involved in the Chinese medicine composition provided by the present invention are as follows: Gallnut, also known as clam, hundred insects warehouse, wood aconite, is a gall on the leaves of Rhus chinensis, green bran poplar or red bran poplar of the Anacardiaceae family. It is mainly formed by the parasitism of gallnut aphids. It enters the lung, stomach and large intestine meridians and has the effects of astringing the lungs and reducing fire, astringing the intestines and stopping diarrhea, astringing sweat, stopping bleeding, and absorbing moisture and healing sores. The active ingredients mainly include tannic acid, also known as tannic acid, a complex compound formed by the combination of gallic acid, digallic acid and glucose.

[0038] Scutellaria baicalensis, also known as camellia root and earth tea root, is the dried root of Scutellaria baicalensis of the Labiatae family. It belongs to the lung, gallbladder, spleen, large intestine and small intestine meridians. The root is used as medicine. It tastes bitter and is cold in nature. It has the effects of clearing away heat and dampness, purging fire and detoxifying, stopping bleeding, and calming the fetus. It is mainly used to treat febrile diseases, upper respiratory tract infections, lung heat cough, damp-heat jaundice, pneumonia, dysentery, hemoptysis, red eyes, fetal movement disorders, hypertension, carbuncles, boils, and sores.

[0039] Pomegranate peel, also known as pomegranate shell, An pomegranate, sour shell, sour pomegranate peel, sour pomegranate peel, western pomegranate peel, belongs to the large intestine meridian, has the effects of astringing the intestine, stopping diarrhea, stopping bleeding, and expelling parasites. It is mainly used to treat long-term diarrhea, long-term dysentery, bloody stool, anal prolapse, spermatorrhea, metrorrhagia, leucorrhea, abdominal pain caused by worms, scabies and other diseases.

[0040] Clove, also known as clove, dissected clove, male clove, and male clove, is the dried flower buds of the Myrtaceae plant clove. It belongs to the spleen meridian, stomach meridian, lung meridian, and kidney meridian. It is mainly used to treat hiccups, cold pain in the abdomen, vomiting and diarrhea due to loss of appetite, impotence due to kidney deficiency, coldness of the waist and knees, and yin carbuncle.

[0041] Danshen is the dried root and rhizome of the plant Salvia miltiorrhiza of the Labiatae family. It belongs to the heart, lung, gallbladder and large intestine meridians and has the effects of promoting blood circulation and removing blood stasis, relieving pain from menstruation, clearing the heart and eliminating vexation, cooling blood and eliminating carbuncle. It is used for chest pain, abdominal pain, accumulation of lumps, pain caused by heat, restlessness and insomnia, irregular menstruation, dysmenorrhea, amenorrhea, and sores and swelling.

[0042] The following is a detailed description with reference to the embodiments.

[0043] Example 1 A traditional Chinese medicine composition for preventing and treating bacterial diseases is composed of the following raw materials: 20g of gallnut, 15g of scutellaria baicalensis, 10g of pomegranate peel, 5g of cloves, and 5g of salvia miltiorrhiza.

[0044] Example 2 A traditional Chinese medicine composition for preventing and treating bacterial diseases is composed of the following raw materials: 20g of gallnut, 15g of scutellaria baicalensis, 15g of pomegranate peel, 15g of cloves, and 20g of salvia miltiorrhiza.

[0045] Example 3 A traditional Chinese medicine composition for preventing and treating bacterial diseases is composed of the following raw materials: 5g of gallnut, 5g of scutellaria, 5g of pomegranate peel, 5g of clove, and 5g of salvia miltiorrhiza.

[0046] Example 4 A traditional Chinese medicine composition for preventing and treating bacterial diseases is composed of the following raw materials: 15g of gallnut, 12g of scutellaria baicalensis, 10g of pomegranate peel, 8g of cloves, and 8g of salvia miltiorrhiza.

[0047] The Chinese medicine compositions provided in the above Examples 1 to 4 are all prepared according to the following steps: Weigh gallnut, scutellaria baicalensis, pomegranate peel, cloves, 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 over high heat for 0.5 h, filter, collect the first decoction, add water to the remaining residue, decoct over low heat for 0.5 h, filter, collect the second decoction, add water to the remaining residue, decoct over high heat for 0.5 h, filter, collect the third decoction, combine it with the decoctions obtained from the first two times, and concentrate to 1±0.05 mg / mL to obtain a Chinese medicine composition.

[0048] Example 5 A Chinese medicine composition for preventing and treating bacterial diseases, which is different from Example 1 only in the preparation method, specifically: Weigh gallnut, scutellaria baicalensis, pomegranate peel, cloves, 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 over high heat for 1.0 h, filter, collect the first decoction, add water to the remaining filter residue, decoct over low heat for 1.0 h, filter, collect the second decoction, add water to the remaining filter residue, decoct over high heat for 1.0 h, filter, collect the third decoction, combine it with the decoctions obtained from the first two times, and concentrate to 1±0.05 mg / mL to obtain a Chinese medicine composition.

[0049] Example 6 A Chinese medicine composition for preventing and treating bacterial diseases, which is different from Example 1 only in the preparation method, specifically: Weigh gallnut, scutellaria baicalensis, pomegranate peel, cloves, and salvia miltiorrhiza, mix them, add water equivalent to 10 times the total amount of the medicinal materials, soak for 1.0 h, first decocted over high heat for 0.5 h, filtered, collected the first decoction, added water to the remaining residue, decocted over low heat for 1.0 h, filtered, collected the second decoction, added water to the remaining residue, decocted over high heat for 1.0 h, filtered, collected the third decoction, combined with the decoctions obtained from the first two times, concentrated to 1±0.05 mg / mL, and obtained a Chinese medicine composition.

[0050] The Chinese medicine compositions provided in Examples 1 to 6 of the present invention can also be further prepared into acceptable preparations, such as granular preparations, powder preparations or liquid preparations, according to conventional methods in veterinary medicine. Appropriate excipients or carriers, such as flavoring agents, preservatives, suspending agents, colorants, etc., can be added during preparation.

[0051] The Chinese medicine composition provided by the present invention can be used directly as a medicine or as a feed additive. When used as a feed additive, it can be prepared by mixing with conventional feed.

[0052] Experimental Example 1 Screening of Chinese medicine composition formula 1. Materials and methods 1.1 Experimental Materials 1.1.1 Traditional Chinese Medicine 36 Chinese medicinal materials including gallnut, rhubarb, honeysuckle, coptis root, herbaceous wormwood, forsythia, pomegranate peel, red peony root, fennel, gentian, scutellaria, knotweed, zedoaria, purslane, turmeric, poria, peony bark, kelp, dioscorea bulbifera, salvia miltiorrhiza, clove, evodia rutaecarpa, dandelion, terminalia chebula, qinpi, agrimony, cherry laevigata, raspberry, red vine, bupleurum, pulsatilla, cimicifuga, senna, belamcanda, asarum, and artemisia annua were purchased from Tongxintang Pharmacy of Bozhou Medicinal Materials Corporation.

[0053] 1.1.2. Strains The sources of the strains are as follows: Aeromonas hydrophila NJ-35 was purchased from China General Microbiological Culture Collection Center with the collection code of CGMCCNo8319 and was isolated from diseased crucian carp in 2010; Plesiomonas shigelloides JX-09 was obtained from the Pearl River Fisheries Research Institute and was isolated from diseased grass carp. See “Isolation and identification of pathogenic Plesiomonas shigelloides from grass carp, Acta Microbiologica Sinica, 2014, 54(2): 229-235”. Klebsiella pneumoniae was obtained from Jianghan University and isolated from largemouth bass. See “Isolation, identification and biological characteristics of ST-873 type Klebsiella pneumoniae from largemouth bass, Ocean and Lake, 2023, 54(3): 836-847)”; Vibrio cholerae F2 was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences, isolated from diseased grass carp and identified physiologically and biochemically. See “Isolation, Identification and Pathogenicity of an Acute Bacterial Infectious Disease Pathogen of Grass Carp, Acta Hydrobiologica Sinica, 2011, (6): 980-986”. Citrobacter freundii was isolated from California bass and obtained from the Pearl River Fisheries Research Institute. See “Isolation and Identification of Citrobacter freundii from California bass, Journal of Southern Agricultural Sciences, 2021, 52(2): 465-474)”; Acinetobacter baumannii was isolated from the liver of diseased channel catfish and obtained from Huazhong Agricultural University. See “Isolation and identification of channel catfish strains of Acinetobacter baumannii. Journal of Huazhong Agricultural University, 2010, 29(4): 489-493)”.

[0054] All the above strains were identified by morphology, biochemistry and molecular analysis.

[0055] 1.2. Preparation of Chinese medicine liquid (1) 36 kinds of Chinese medicine were dried in an oven at 60°C to constant weight. 50 g of each single dose of Chinese medicine was weighed and placed in a non-woven bag. Then, 500 mL of water was added to soak the mixture for 30 min. The mixture was decocted over high heat for 30 min, and the first liquid was poured out. (2) Add 500 mL of water, simmer for 60 min, and pour out the second liquid; (3) Add 500 mL of water and boil the medicine bag over high heat for 30 min. After boiling, remove the medicine bag and discard it. (4) Pour the three drug solutions back into the medicine pot and combine them to concentrate to 50 mL, so that the total drug concentration reaches 1 g / mL. After high-pressure sterilization at 121℃, place it in a 4℃ refrigerator for use.

[0056] 1.3. Preparation of Aeromonas hydrophila NJ-35 bacterial suspension Aeromonas hydrophila NJ-35 stored at -80℃ was inoculated with an inoculation loop onto NB solid medium for streak culture, and then placed in a 37℃ constant temperature incubator for 16 h to recover. A single colony was picked and placed into 1 mL NB liquid medium, and then cultured overnight at 37℃ and 200 rpm in a shaking incubator. The concentration of the bacterial suspension was determined by the dilution plate method, and the cultured bacterial suspension was gradiently diluted to 10% of the original bacterial concentration. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 100 μL of each dilution was spread on NB medium, and the spread was repeated on 3 plates. The plates were incubated in a 37°C constant temperature incubator for 18 h. The spread plates were observed. When the number of colonies on the plates was between 30 and 300, the effective count concentration was used. The bacterial concentration of the original bacterial solution was calculated according to the following formula. The solution was diluted to 1×10 with 0.86% saline. 8 CFU / mL.

[0057] Number of strains per ml of bacterial solution = (C÷V)×M In the formula: C: represents the average number of colonies grown on the plate at a certain dilution concentration, V: represents the volume of the diluent used when coating the plate (mL), M: represents the dilution multiple, unit: CFU / mL.

[0058] 1.4. In vitro antibacterial experiment of traditional Chinese medicine 1.4.1. Tablet preparation Use a hole puncher to make filter paper into multiple 6 mm circular pieces of paper, place the pieces of paper in a blank culture dish, sterilize them under high pressure at 121°C, transfer them to a 50°C oven for drying, soak the dried pieces of paper in sterilized Chinese medicine solution, and store them at 4°C for later use.

[0059] 1.4.2. Determination of antibacterial ability of single traditional Chinese medicine by paper diffusion method The antibacterial effect of single Chinese medicine extract was determined by paper diffusion method. 100 μL of 1×10 8 CFU / mL of bacterial solution, and evenly spread the bacterial solution in MH medium. Take out the paper soaked in the drug solution and stick it on the sterilized culture dish. After the paper is completely dry, stick it on the plate that has been coated with the bacterial solution. Stick 3 copies of each drug piece, and place the plate in a 37℃ constant temperature incubator for inversion and culture for 16 hours. An inhibition zone with no visible strain growth appears around the paper. Use a vernier caliper to measure the diameter of the inhibition zone by the cross measurement method. No inhibition zone is recorded as 6 mm. The three repeated experiments are recorded as the mean ± standard deviation. The average value of the inhibition zone diameter ≥ 20 mm is judged as extremely sensitive; 20 mm < inhibition zone diameter ≤ 15 mm is highly sensitive; 15 mm < inhibition zone diameter ≤ 10 mm is medium sensitive; and the inhibition zone diameter < 10 mm is low sensitive.

[0060] 1.4.3. Determination of Minimum Inhibitory Concentration (MIC) of Single Traditional Chinese Medicine The minimum inhibitory concentration of a single Chinese medicine was determined by the 96-well plate micro-dilution method. Figure 1 . Except for the first column, 100 μL of MH cationic liquid culture medium was added to each well of the last eleven columns. 100 μL of Chinese medicine solution was added to the first and second wells of each row. After the solution and culture medium in the second well were mixed, 100 μL was aspirated and added to the third well. The operation was repeated until 100 μL was aspirated from the eleventh well and discarded. Double dilution was performed in this way. The drug dilution concentrations were 1000, 500, 250, 125, 62.50, 31.25, 15.63, 7.81, 3.91, 1.95, and 0.98 mg / mL, respectively. In rows A to C and E to G, 1×10 8 CFU / mL of bacterial solution, and each drug was repeated 3 times. 100 μL of culture medium was added to rows D and H as negative control, and no drug solution was added to column 12, only 100 μL of bacterial solution and 100 μL of culture medium were added as positive control. After the sample addition operation was completed, the 96-well plate was placed in a 37°C constant temperature incubator for 18 h, and the results were observed. If there was no obvious bacterial precipitation at the bottom, it was considered to be the minimum inhibitory concentration (MIC) of the drug against Aeromonas hydrophila.

[0061] 1.4.4. Determination of Minimum Bactericidal Concentration (MBC) of Traditional Chinese Medicine After observing the MIC results obtained above, take 10 μL of liquid from the clear sterile precipitate hole and drop it vertically on the NB solid culture medium. Repeat 3 times for each concentration. Wait for the liquid to diffuse naturally and be absorbed by the culture medium. There is no liquid on the surface. Repeat 3 times. Put it in a 37℃ constant temperature incubator for 18 hours and observe the results. If there are no bacterial plaques, the bacteria are killed and cannot grow on ordinary plates. The minimum Chinese medicine treatment concentration without bacterial growth is considered the minimum bactericidal concentration.

[0062] 1.5. Optimization analysis of compatibility of traditional Chinese medicine compound preparations Taking Aeromonas hydrophila NJ-35 as the target strain, the orthogonal t value method was used to optimize the compound compatibility of the top 7 Chinese medicines with the best antibacterial effects among the tested single herbs to find the best combination and the best dosage ratio.

[0063] 1.5.1. Main drug analysis Design L 12 (2 7 ), a two-level, seven-factor, twelve-fold orthogonal test is shown in Table 1. According to the combinations shown in Table 1, the Chinese medicines were weighed in corresponding doses and put into non-woven bags. According to the method in 1.2, they were boiled into a 1 g / mL Chinese medicine compound. The diameter of the inhibition zone of each combination of Chinese medicine compound against Aeromonas hydrophila NJ-35 was determined by paper diffusion method. Each experiment was repeated 3 times, and the main medicine of the prescription was determined according to the experimental results.

[0064] Table 1 L 12 (2 7 ) Orthogonal t value method to analyze the main drug

[0065] Note: A is pomegranate peel, B is peony bark, C is terminalia chebula, D is clove, E is gallnut, F is salvia miltiorrhiza, G is scutellaria baicalensis; level 1 means taking 10g of the medicine; level 2 means not taking the medicine.

[0066] 1.5.2 Analysis of excipient-drug interaction Taking the factors with significant effects in the above results as the main factors, the following L 16 (2 5 ) The orthogonal table, i.e. Table 2, focuses on analyzing the interaction between the main factors and other factors and other factors. The Chinese medicines are weighed according to the formula in Table 2 and boiled into 1 g / mL Chinese medicine compound liquid according to the method in 1.2. The antibacterial ability of 16 prescriptions is detected by paper diffusion method, and the interaction between the factors is analyzed based on the results.

[0067] Table 2 L 16 (2 5 ) Orthogonal t value method to analyze the interaction between adjuvant and drug

[0068] Note: Gallnut and Scutellaria baicalensis are added to each prescription as the basis; A is pomegranate peel, B is peony bark, C is clove, D is Terminalia chebula, and E is Salvia miltiorrhiza; Level 1 means taking 10g of the medicine; Level 2 means not taking the medicine.

[0069] 1.5.3. Dose Optimization Based on the above results, the main drugs were confirmed to be Galla chinensis and Scutellaria baicalensis, and the auxiliary drugs were Pomegranate peel, Clove and Salvia miltiorrhiza. 4 ) Orthogonal experiment table 4, for dosage selection, Gallnut and Scutellaria baicalensis are combined into one factor, the original dose is taken as the middle dose, and appropriate increase or decrease is made to formulate a 3-level dosage table 3.

[0070] Table 3 Four-factor three-level dose optimization table

[0071] Note: Gallnut and Scutellaria baicalensis are factor A, the former is the dosage of Gallnut, the latter is the dosage of Scutellaria baicalensis; B is pomegranate peel, C is clove, and D is Salvia miltiorrhiza.

[0072] Table 4 L9(3 4 ) Orthogonal t value method to optimize dose selection

[0073] Note: The factors and levels are the same as those in Table 3.

[0074] 1.6. Verification of the efficacy of Chinese herbal compound preparations 1.6.1. In vitro antibacterial ability The in vitro antibacterial effect of the optimized Chinese herbal compound preparation was tested by using the paper diffusion method and the micro-two-fold dilution method to detect the size of the inhibition zone, the minimum bactericidal concentration and the minimum inhibitory concentration. The methods were the same as those in 1.4.2, 1.4.3 and 1.4.4.

[0075] 1.6.2 Effects of Chinese herbal compound preparations on strain NJ-35 The drug treatment concentration was determined according to the results of the MBC of the traditional Chinese medicine compound. The concentration greater than the MBC was considered a high concentration, and the concentration less than the MBC was considered a low concentration. The NJ-35 strain was treated with three different concentrations of compound preparations at 3.91 mg / mL, 125 mg / mL, and 250 mg / mL. The NJ-35 strain was activated by spreading on NB solid medium, picking a single colony and placing it in NB liquid medium, and culturing on a shaker at 37°C, 200 rpm for 8 h. The bacterial solution concentration was determined by the dilution plate count method, and the bacterial solution was adjusted to 1×10 8CFU / mL, 1 mL of bacterial solution was mixed with 1 mL of different concentrations of Chinese herbal compound preparations, and co-cultured at 37°C, 200 rpm, on a shaker for 12 h, centrifuged at 3000 rpm for 10 min, and the culture medium was discarded and gently rinsed with PBS (the bacterial solution treated with high-concentration Chinese herbal compound preparations needed to be repeatedly washed with PBS several times to remove the Chinese herbal medicine residues, centrifuged at 3000 rpm for 10 min, and PBS was removed), and 1 mL of electron microscopy fixative was added to the bacteria after centrifugation and discarding the supernatant, and gently blown until the bacterial precipitate was suspended in the fixative, fixed at room temperature in the dark for 30 min, then transferred to 4°C for storage, and sent to Wuhan Saiweier Biotechnology Co., Ltd. for scanning electron microscopy.

[0076] 2. Experimental results 2.1 Antibacterial effect and minimum inhibitory concentration of Chinese medicine The antibacterial ability of Chinese medicine single prescriptions is shown in Table 5. Galla chinensis has the strongest antibacterial ability against Aeromonas hydrophila NJ-35, with an inhibition zone diameter of 17.8 ± 0.17 mm and a MIC of 31.25 mg / mL; followed by Coptis chinensis, Forsythia suspensa, Pomegranate peel, Scutellaria baicalensis, Paeonia suffruticosa, Fraxinus chinensis, Terminalia chebula, Clove, Raspberry, Salvia miltiorrhiza, Evodia rutaecarpa, and Herba Lycopodii. The sizes of the inhibition zones of these 13 Chinese medicine single prescriptions ranged from 10 mm to 15 mm; the sizes of the inhibition zones of 19 Chinese medicine single prescription extracts such as Honeysuckle, Rhubarb, Red Peony Root, Fennel, and Polygonum cuspidatum were between 6 mm and 10 mm, with clear transparent zones; the five Chinese medicine single prescription extracts of Gentiana scabra, Curcuma longa, Kelp, Poria cocos, and Senna leaf had no transparent inhibition zones against NJ-35, indicating no antibacterial effect, and the inhibition zones of some Chinese medicine single prescriptions were not Figure 2 The measured MIC value was not consistent with the size of the inhibition zone, which may be related to the error of naked eye observation. Based on the size of the inhibition zone, the top seven Chinese medicines with the highest antibacterial ability were selected as candidate Chinese medicines for the optimization of the compatibility of Chinese medicine compound preparations, namely, Galla chinensis, Pomegranate peel, Paeonia suffruticosa, Salvia miltiorrhiza, Clove, Scutellaria baicalensis, and Terminalia chebula.

[0077] Table 5 Antibacterial activity of Chinese herbal medicine prescriptions against Aeromonas hydrophila NJ-35

[0078] Note: The diameter of the inhibition zone is 15-19 mm, which is highly sensitive and indicated by “++”; 10-14 mm, which is moderately sensitive and indicated by “+”; and less than 10 mm, which is low-sensitive and indicated by “-”.

[0079] 2.2. Optimization of the compatibility of Chinese medicine compound preparations 2.2.1. Analysis of main drugs When M1 > M2, that is, D > 0, it indicates that the effect of adding this traditional Chinese medicine to the prescription is better than not adding it. As can be seen from Table 6, only the D values of Chinese gallnut and skullcap root are positive, and the antibacterial ability of adding these two herbs is better and can be used as the main herbs for further analysis. For cortex moutan, chebula fruit, and salvia miltiorrhiza, M1 < M2 and t < 0.01, showing extremely significant differences, indicating that the effect of adding these traditional Chinese medicines is lower than not adding them. Continue with the analysis of adjuvant herbs and then make a decision on the selection or rejection of the herbs.

[0080] Table 6 L 12 (2 7 ) Orthogonal t - value method for analyzing main herbs

[0081] Note: A is pomegranate peel; B is cortex moutan; C is chebula fruit; D is clove; E is Chinese gallnut; F is salvia miltiorrhiza; G is skullcap root, ** indicates extremely significant, - indicates not significant; Level 1 is using 10 g of medicine, Level 2 is not using medicine; 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 means at Level 1; M2 is the sum of means at Level 2; D is the difference between M1 and M2; Mm is the average of each group's means; MM is the expected medicinal effect.

[0082] 2.2.2. Interaction analysis of adjuvant herbs From the results of Table 7 and Supplementary Table 7, it can be seen that 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 adopted. From the perspective of the prescription, A2E1, B2E1, C2E1, D2E1 are the maximum values of factor interactions containing factor E, so E1 is selected. The prescription A2D2, B2D2, C2D2, D2E1 is the maximum value containing factor D, so D2 is selected, that is, factor D is discarded and factor E is retained. Finally, it is determined that Chinese gallnut and skullcap root are the main herbs, and pomegranate peel, salvia miltiorrhiza, and clove are the adjuvant herbs.

[0083] Table 7 L 16 (2 5 ) Orthogonal t - value method for analyzing the interaction of adjuvant herbs

[0084] Supplementary Table 7 L 16 (2 5 ) Orthogonal t - value method for analyzing the interaction of adjuvant herbs

[0085] Note: Gallnut and Scutellaria baicalensis are the main drugs, 10 g of each was added to each treatment group; A is pomegranate peel; B is peony bark; C is clove; D is terminalia chebula; E is salvia miltiorrhiza; in the interaction table, "△" indicates mutual antagonism, "^" indicates synergy, "*" indicates the maximum value, and [xx] indicates the sum of the mean values ​​of the diameters of the inhibition zones when the factor is at level x; other symbols are the same as those in Table 6.

[0086] 2.2.3 Dose optimization analysis After confirming the main and auxiliary drugs, the factor with the largest mean value at each level was analyzed to have the best antibacterial effect. The results in Table 8 show that the optimal dosage combination of the Chinese herbal compound preparation is A3B2C1D1, that is, 20 g of Galla chinensis, 15 g of Scutellaria baicalensis, 10 g of Pomegranate peel, 5 g of Clove, and 5 g of Salvia miltiorrhiza.

[0087] Table 8 L9(3 4 ) Orthogonal t value method to optimize dose selection

[0088] Note: A is Galla chinensis and Scutellaria baicalensis; B is Pomegranate peel; C is Clove; D is Salvia miltiorrhiza; M3 is the sum of the means of the three 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 Table 6.

[0089] 2.3. In vitro antibacterial effect of Chinese herbal compound preparations According to the optimal ratio obtained in 2.2.3, the compound Chinese medicine preparation was boiled and the in vitro antibacterial effect was determined. From Table 9, it can be seen that the compound 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. Figure 3 In addition, the compound Chinese medicine preparation also has good antibacterial ability against Shigella-like bacteria, 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 .

[0090] The results of scanning electron microscopy observation of strains treated with different concentrations of Chinese herbal compound preparations are as follows Figure 5. The bacteria in the untreated control group were rod-shaped, with blunt ends, complete structure, full and evenly covered with pili, and round without holes. After the strain was treated with 3.91 mg / mL of the Chinese medicine compound preparation, namely 50% MBC, the bacteria were severely deformed compared with the control group, with obvious shrinkage and depression, and deep depression in some parts; after treatment with 125 mg / mL and 250 mg / mL high-concentration compound liquid, the surface of the bacteria was rough, and some bacteria had obvious cracks, wrinkles and depressions, with slight deformation, and no serious depression and shrinkage when treated with low doses. This shows that the appropriate dose of compound liquid treatment may destroy the cell membrane structure of the bacteria, causing the bacteria to rupture and the contents to flow out.

[0091] Table 9 Antibacterial ability of Chinese herbal compound preparations against pathogenic bacteria

[0092] Note: The diameter of the inhibition zone is 15-19 mm, which is highly sensitive and indicated by “++”; 10-14 mm, which is moderately sensitive and indicated by “+”; and less than 10 mm, which is low-sensitive and indicated by “-”.

[0093] Experimental Example 3 Protective effect of Chinese herbal medicine combination on grass carp 1. Experimental methods 1.1 Experimental Animals Healthy grass carp, weighing 100g~130g, were purchased from Shenlong Fishery Development Co., Ltd. and placed in a sterilized 300L plastic box for 2 weeks before the experiment. The aquaculture water was aerated for two days before use, and 50% of the water was replaced every two days. The water temperature was 18℃~25℃, and the dissolved oxygen was >5.0 mg / L.

[0094] The concentration of Aeromonas hydrophila was adjusted to 1×10 7 CFU / mL, 0.2mL was injected intraperitoneally into grass carp to obtain the Aeromonas hydrophila infection model. 84 grass carps were randomly divided into: 6 blank control groups, 6 normal saline control groups, 36 Aeromonas hydrophila infection groups and 36 Chinese medicine treatment groups, of which 6 in the Aeromonas hydrophila infection group and the Chinese medicine treatment group were used to detect the expression level of inflammatory factors, and 30 were used to calculate the mortality rate.

[0095] 1.2 Experimental grouping implementation method (1) Blank control group: without any treatment.

[0096] (2) Saline control group: Grass carp were intraperitoneally injected with 0.85% saline as a negative control.

[0097] (3) Aeromonas hydrophila infection group: grass carp were intraperitoneally injected with 1×10 7 CFU / mL Aeromonas hydrophila bacterial solution.

[0098] (4) Chinese medicine treatment group: The grass carp used to detect the level of inflammatory factors were gavage-fed with 100 mg / kg of the Chinese medicine composition provided in Example 1 of the present invention for 7 consecutive days, and then intraperitoneally injected with the same dose of Aeromonas hydrophila as the group infected with Aeromonas hydrophila; the grass carp used to detect the survival rate of grass carp after treatment were fed with a feed containing 2‰ of the Chinese medicine composition provided in Example 1 of the present invention, twice a day, for 7 consecutive days, and then intraperitoneally injected with the same dose of Aeromonas hydrophila as the group infected with Aeromonas hydrophila, and the number of grass carp deaths was recorded every day.

[0099] 1.3. Detection of inflammatory factor expression levels Real-time fluorescence quantitative PCR was used to detect the relative expression of TNF-α, IFN, Myd88, IL-1β, and MHCⅡ mRNA in head kidney tissue. Three replicates were made for each sample, and β-actin was used as the internal reference gene. The relative mRNA expression was calculated by the 2-ΔΔCt method. The fluorescence PCR data were sorted and calculated using Excel, and one-way analysis of variance was used. The post hoc test was performed using LSD and Duncan test. P<0.05 was considered statistically significant.

[0100] 1.4 Pathological testing 24 hours after bacterial injection, kidney, intestine, spleen and gills of grass carp were collected, fixed with universal fixative for 24 hours, embedded in paraffin, made into paraffin sections, stained with HE and sealed, and observed under a microscope.

[0101] 2. Experimental results 2.1 Pathological changes 24 hours after infection with Aeromonas hydrophila, the intestinal submucosal layer was disordered, the tissue was dispersed, inflammatory cells were infiltrated, the intestinal villus epithelial cells were shed and broken, the intestinal muscle layer was dispersed, and the intestinal muscle layer in the Chinese medicine treatment group was also dispersed, the degree of damage to the submucosal layer was reduced, and the intestinal villus morphology was intact. In the Aeromonas hydrophila infection group, the hepatocytes were swollen and rounded, with large-scale nuclear dissolution, disordered arrangement of cells with cavities, and inflammatory cell infiltration. In the Chinese medicine treatment group, cavities appeared around the interlobular arteries, and some hepatocytes were necrotic and nuclear dissolution, which was slightly lower than the pathological degree of the Aeromonas hydrophila infection group. The spleen tissue control group had tightly arranged cells without obvious damage, severe bleeding, hemosiderin agglutination, and large-area vacuolar degeneration in the Aeromonas hydrophila infection group, and the degree of vacuolar degeneration in the Chinese medicine treatment group was mild, still accompanied by hemosiderin deposition. In the renal tissue, the outer layer of renal tubules in the Aeromonas hydrophila infection group were necrotic and vacuolated, with blurred lumen boundaries, interstitial cell necrosis and vacuolation, accompanied by the appearance of a large number of blood cells. The renal tubules in the control group and the Chinese medicine treatment group were intact and had no obvious lesions. The gill lamellae in the Aeromonas hydrophila infection group were curled and shortened, the gill lamellae cells were necrotic and detached, and the gill filament cartilage was atrophied, twisted and necrotic. The gill lamellae in the Chinese medicine treatment group were slightly bent and degenerated, the interlamellar cells proliferated, the cells were necrotic, and they were separated from the gill filament cartilage. The specific results are shown in Figure 6 .

[0102] 2.2. Relative levels of inflammatory factors in the head and kidney The results are as follows Figure 7~Figure 11 Compared with the blank group, all inflammatory factors were significantly expressed, while in the TCM treatment group, IFN increased significantly, MHCⅡ and Myd88 were significantly downregulated, IL-1β and TNF-α had no significant changes, but all inflammatory factors in the TCM treatment group were significantly downregulated compared with the Aeromonas hydrophila infection group.

[0103] 2.3 Survival rate of infected grass carp after Chinese medicine treatment Depend on Fig.12 It can be seen that after being fed with a feed containing 2‰ of the Chinese medicine composition provided in Example 1 of the present invention for 7 days and injected with Aeromonas hydrophila, the survival rate was significantly different from that of directly injected with Aeromonas hydrophila. The mortality rate of grass carp fed with the Chinese medicine feed was significantly reduced, and there was no death on the second day when the mortality rate was the highest, which showed an obvious protective effect and the survival rate reached 86.67%.

[0104] In summary, the Chinese medicine composition of the present invention can be added to grass carp feed as a feed additive to effectively protect grass carp from infection by Aeromonas hydrophila, reduce tissue damage and inflammation, and greatly improve the survival rate of grass carp.

[0105] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the invention.

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 baicalensis, 5-15 parts of pomegranate peel, 5-15 parts of cloves and 5-20 parts of salvia miltiorrhiza.

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~1.0h before decocting.

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~1.0h, filter, collect the first decoction liquid, add water to the remaining filter residue and decoct with low heat for 0.5h~1.0h, filter, collect the second decoction liquid, add water to the remaining filter residue and decoct with high heat for 0.5h~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 pathogenic bacteria are Aeromonas hydrophila, Plesiomonas shigellae, Stenotrophomonas maltophilia, Vibrio cholerae, Klebsiella pneumoniae, Acinetobacter baumannii or Citrobacter freundii.

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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