Antibacterial traditional Chinese medicine composition as well as preparation method and application thereof

Abstract

The invention discloses an antibacterial traditional Chinese medicine composition as well as a preparation method and application thereof. The antibacterial traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 13-17 parts of honeysuckle flowers, 8-12 parts of rhizoma coptidis, 8-12 parts of radix scutellariae, 13-17 parts of radix astragali, 18-22 parts of herba portulacae, 8-12 parts of radix codonopsis, 8-12 parts of fructus crataegi, 3-7 parts of radix aucklandiae and 3-7 parts of liquorice roots. The preparation method comprises the following steps: taking the antibacterial traditional Chinese medicine composition as a raw material, and performing water extraction to obtain an extracting solution. The application refers to the application of the antibacterial traditional Chinese medicine composition in preparation of veterinary drugs for resisting staphylococcus aureus. The antibacterial traditional Chinese medicine composition provided by the invention is simple in raw material components, active components can be effectively extracted in the preparation process, and the antibacterial traditional Chinese medicine composition shows potential application value in the aspect of treating and preventing staphylococcus aureus infection and particularly reaches the level of medium-sensitive antibiotics and close to high-sensitive antibiotics in the aspect of treatment effect.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and specifically relates to an antibacterial traditional Chinese medicine composition and a preparation method and application thereof. Background Art

[0002] Staphylococcus aureus (S. aureus) is a highly contagious Gram-positive coccus. It is also one of the main pathogens causing skin and soft tissue infections and one of the common pathogens in veterinary clinics. Staphylococcus aureus is widely present in nature, such as air, soil, human and animal skin, and organs that communicate with the outside world. Staphylococcus aureus infection can cause many serious infections in humans and animals. Among them, methicillin-resistant Staphylococcus aureus was first reported in the 1960s. Methicillin-resistant Staphylococcus aureus spread around the world, causing serious harm to social public health and safety. When infected with Staphylococcus aureus, suppuration can occur in the wound. If a systemic infection occurs, it can cause sepsis, bacteremia, sepsis, etc. With the continuous growth of large-scale farms at that time, the prevention and control of Staphylococcus aureus has received more and more attention from veterinary staff.

[0003] Since the advent of antibiotics, people have been constantly looking for drugs that can kill Staphylococcus aureus. However, antibiotic treatment can easily lead to bacterial resistance, which is a difficulty in clinical treatment. Many studies have shown that combined medication is more effective in treating Staphylococcus aureus infections. Although the emergence of antibiotics has brought good news to mankind, due to the weak professional knowledge of some breeders, when Staphylococcus aureus infections occur in farms, the dosage of antibiotics will be increased to achieve the therapeutic effect. Once drug resistance is generated, it will bring difficulties to the treatment of veterinary clinical workers, and the abuse of antibiotics will cause serious drug residue problems, which will pose a threat to public health and safety. Therefore, the development of new drugs is imperative. In the prevention and treatment of Staphylococcus aureus infections, Chinese herbal medicine is a key research direction.

[0004] Chinese herbal medicines have little toxicity and side effects on humans and animals, and have significant therapeutic effects. In addition, Chinese herbal medicines are not easy to develop drug resistance against Staphylococcus aureus, and have broad research prospects in the development of new drugs and antibiotic substitutes. Chinese herbal medicines have shown unique advantages in the treatment of Staphylococcus aureus infections. Its research mainly focuses on analyzing the antibacterial components in Chinese herbal medicines, studying the antibacterial effects of single Chinese herbal medicines, and developing compound Chinese herbal medicine preparations. The results show that some Chinese herbal medicines such as Coptis chinensis and Honeysuckle have significant inhibitory effects on Staphylococcus aureus. Compound Chinese herbal medicine preparations also show good efficacy in practical applications, which may be related to the synergistic effect between Chinese herbal medicine ingredients. In view of the increasingly serious problem of antibiotic resistance, Chinese herbal medicine treatment provides a new perspective and option for the prevention and treatment of Staphylococcus aureus disease. Summary of the invention

[0005] The first object of the present invention is to provide an antibacterial Chinese medicine composition with simple components.

[0006] Specifically, the raw materials for preparing the antibacterial traditional Chinese medicine composition include the following components in parts by weight: 13-17 parts of honeysuckle, 8-12 parts of coptis chinensis, 8-12 parts of scutellaria baicalensis, 13-17 parts of astragalus, 18-22 parts of purslane, 8-12 parts of codonopsis pilosula, 8-12 parts of hawthorn, 3-7 parts of costusroot, and 3-7 parts of licorice.

[0007] Preferably, the weight parts of the raw materials are: 14-16 parts of honeysuckle, 9-11 parts of coptis root, 9-11 parts of scutellaria root, 14-16 parts of astragalus root, 19-21 parts of purslane, 9-11 parts of codonopsis root, 9-11 parts of hawthorn, 4-6 parts of costus root, and 4-6 parts of licorice. More preferably, the weight parts of the raw materials are: 15 parts of honeysuckle, 10 parts of coptis root, 10 parts of scutellaria root, 15 parts of astragalus root, 20 parts of purslane, 10 parts of codonopsis root, 10 parts of hawthorn, 5 parts of costus root, and 5 parts of licorice.

[0008] In the process of compounding the compound, the present application follows the principles of Chinese medicine compatibility and focuses on the interaction and synergistic effect between drugs. Through reasonable matching, the purpose is to enhance the antibacterial effect, reduce the toxic and side effects of drugs, and improve the therapeutic effect.

[0009] The second object of the present invention is to provide a method for preparing the above-mentioned antibacterial Chinese medicine composition.

[0010] Specifically, the preparation method is to use the above components as raw materials, decoct them in water and retain the decoction as the antibacterial Chinese medicine composition.

[0011] More specifically, the preparation method comprises the following steps: 1) weighing raw materials according to the weight of the components; 2) soaking in water for 20 to 60 minutes, heating to boiling, decocting for 20 to 60 minutes under boiling conditions, and filtering to obtain the filtrate; 3) taking the filter residue in step 2), adding water and heating to boiling, decocting for 20 to 60 minutes under boiling conditions, and filtering to obtain the filtrate; 4) combining the filtrates of step 2) and step 3), centrifuging to obtain the supernatant, concentrating and sterilizing to obtain the antibacterial Chinese medicine composition.

[0012] Preferably, in step 2) and step 3), the amount of water added is 8 to 12 times the total weight of the raw material.

[0013] Preferably, the centrifugal separation process is performed at 3000 r / min for 3 minutes.

[0014] Preferably, the concentration is to concentrate the supernatant to contain 1 g / ml of the crude drug.

[0015] Preferably, the sterilization is performed by placing the obtained concentrated solution into a high pressure sterilizer at 121° C. for 15 minutes.

[0016] The preparation method is improved by referring to the conventional water decoction method. After two water decoctions, the preparation process is subjected to multiple filtration, centrifugal separation and sterilization treatments, so that the purity of the obtained solution can be ensured to meet the veterinary drug standards, and after concentration, it can be ensured that the solution contains sufficient antibacterial active ingredients.

[0017] The third object of the present invention is to provide the application of the above antibacterial Chinese medicine composition.

[0018] Specifically, the application is the application of the antibacterial Chinese medicine composition or the antibacterial Chinese medicine composition prepared by the above preparation method in the preparation of veterinary drugs against Staphylococcus aureus. The Chinese medicine composition shows an inhibitory effect on Staphylococcus aureus, can achieve an antibacterial effect close to that of antibiotics, and shows potential application value in the treatment and prevention of Staphylococcus aureus infection. DETAILED DESCRIPTION

[0019] The raw materials of the antibacterial Chinese medicine composition of the present invention include the following components in parts by weight: 13-17 parts of honeysuckle, 8-12 parts of coptis root, 8-12 parts of scutellaria root, 13-17 parts of astragalus root, 18-22 parts of purslane, 8-12 parts of codonopsis root, 8-12 parts of hawthorn, 3-7 parts of costus root, and 3-7 parts of liquorice. The following proportion is the most commonly used scheme: 15 parts of honeysuckle, 10 parts of coptis root, 10 parts of scutellaria root, 15 parts of astragalus root, 20 parts of purslane, 10 parts of codonopsis root, 10 parts of hawthorn, 5 parts of costus root, and 5 parts of liquorice.

[0020] The medicinal sources of each component in the prescription are as follows: honeysuckle is the dried buds or flowers with initial opening of Lonicera japonica Thunb., Lonicera hypoglauca Miq., Lonicera confusa DC. or Lonicera dasystyla Rehd. of the Caprifoliaceae family. Coptis chinensis Franch., Coptis deltoidea CYCheng et Hsiao or Coptis teeta Wall. of the Ranunculaceae family. Scutellaria baicalensis is the dried root of Scutellaria baicalensis Georgi of the Lamiaceae family. Astragalus is the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. of the Leguminosae family. Portulaca oleracea L. of the Portulacaceae family is used as medicine in its entirety. Codonopsis pilosula is the dried root of Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula Nannf. var. modesta (Nannf.) LTShen or Codonopsis tangshen Oliv. of the Campanulaceae family. Hawthorn is the dried mature fruit of Crataegus pinnatifida Bge. var. major NEBr. or Crataegus pinnatifida Bge. of the Rosaceae family. Costus root is the dried root of Aucklandialappa Decne. of Asteraceae. Licorice root is the dried root of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L. of Leguminosae.

[0021] The applicant believes that the antibacterial research of compound Chinese herbal preparations mainly focuses on how to enhance the antibacterial effect of Chinese herbal medicines through reasonable compatibility. By combining Chinese herbal medicines with different antibacterial mechanisms, a synergistic effect can be achieved, thereby improving the inhibitory ability against pathogens. For example, some Chinese herbal extracts have a general antibacterial effect when used alone, but after being combined with other Chinese herbal extracts, their antibacterial activity is significantly enhanced. In addition, compound preparations can also specifically inhibit different types of pathogens by adjusting the formula ratio, thereby improving the pertinence and effectiveness of treatment. In practical applications, compound Chinese herbal preparations can be used not only as therapeutic drugs, but also as preventive drugs to reduce the occurrence of diseases by enhancing the immunity of animal bodies. Therefore, compound Chinese herbal preparations have broad application prospects in veterinary clinical treatment and prevention of bacterial diseases.

[0022] The antibacterial Chinese medicine composition selects hawthorn, coptis root, liquorice, scutellaria, codonopsis, costus root, honeysuckle and other medicinal materials with good antibacterial effects, astragalus and purslane with immunity enhancement. Among them, the antibacterial effects of coptis root and hawthorn are particularly prominent, and about 10 parts of weight are given to each in the compound. Licorice and codonopsis are used as auxiliary medicinal materials, and about 5 parts and 10 parts of weight are given to each, respectively, to enhance the overall effect of the compound and harmonize the medicinal properties. The addition of costus root and honeysuckle is intended to further improve the antibacterial activity and broad spectrum of the compound. The addition of purslane and astragalus is based on their potential role in enhancing immunity and regulating body functions.

[0023] The preparation method of the present invention is similar to the conventional decoction method, and can prepare the medicine simply and quickly. Moreover, the prepared medicine is in the form of an extract, which is a dosage form that is more convenient for livestock. Specifically, the preparation method comprises the following steps: 1) weighing the raw materials according to the weight of the components; 2) adding 8 to 12 times the weight of water, soaking for 20 to 60 minutes, heating to boiling, decocting for 20 to 60 minutes under boiling conditions, and filtering to obtain the filtrate; 3) taking the filter residue in step 2), adding 8 to 12 times the weight of water, heating to boiling, decocting for 20 to 60 minutes under boiling conditions, and filtering to obtain the filtrate; 4) combining the filtrates of step 2) and step 3), centrifuging at 3000r / min for 3 minutes, taking the supernatant, concentrating in a 90°C constant temperature water bath to contain 1g / ml of the crude drug, and sterilizing in a high pressure sterilizer at 121°C for 15 minutes to obtain the antibacterial Chinese medicine composition.

[0024] The present invention is described and explained below by means of several embodiments. It should be understood that the following embodiments are not intended to limit the present invention.

[0025] Example 1

[0026] 1) Weigh the following components of Chinese medicine raw materials: 15 parts of honeysuckle, 10 parts of coptis root, 10 parts of scutellaria, 15 parts of astragalus, 20 parts of purslane, 10 parts of codonopsis, 10 parts of hawthorn, 5 parts of costus root, and 5 parts of liquorice. 2) Add 10 times the weight of water, soak for 30 minutes, heat to boil, decoct for 30 minutes under boiling conditions, and filter to obtain the filtrate. 3) Take the filter residue in step 2), add 10 times the weight of water, heat to boiling, decoct for 30 minutes under boiling conditions, and filter to obtain the filtrate. 4) Combine the filtrates of step 2) and step 3), centrifuge at 3000r / min for 3 minutes, take the supernatant, concentrate in a 90°C constant temperature water bath to contain 1g / ml of crude drug, put it in a high pressure sterilizer and sterilize at 121°C for 15 minutes to obtain the antibacterial Chinese medicine composition.

[0027] Example 2

[0028] 1) Weigh the following components of Chinese medicine raw materials: 13 parts of honeysuckle, 12 parts of coptis root, 8 parts of scutellaria, 16 parts of astragalus, 22 parts of purslane, 8 parts of codonopsis, 10 parts of hawthorn, 7 parts of costusroot, and 3 parts of liquorice. 2) Add 10 times the weight of water, soak for 60 minutes, heat to boil, decoct for 20 minutes under boiling conditions, and filter to obtain the filtrate. 3) Take the filter residue in step 2), add 10 times the weight of water, heat to boiling, decoct for 60 minutes under boiling conditions, and filter to obtain the filtrate. 4) Combine the filtrates of step 2) and step 3), centrifuge at 3000r / min for 3 minutes, take the supernatant, concentrate in a 90°C constant temperature water bath to contain 1g / ml of crude drug, put it in a high pressure sterilizer and sterilize at 121°C for 15 minutes to obtain the antibacterial Chinese medicine composition.

[0029] Example 3

[0030] 1) Weigh the following components of Chinese medicine raw materials: 17 parts of honeysuckle, 8 parts of coptis root, 10 parts of scutellaria, 17 parts of astragalus, 18 parts of purslane, 10 parts of codonopsis, 12 parts of hawthorn, 3 parts of costus root, and 5 parts of liquorice. 2) Add 12 times the weight of water, soak for 20 minutes, heat to boiling, decoct for 40 minutes under boiling conditions, and filter to obtain the filtrate. 3) Take the filter residue in step 2), add 12 times the weight of water, heat to boiling, decoct for 20 minutes under boiling conditions, and filter to obtain the filtrate. 4) Combine the filtrates of step 2) and step 3), centrifuge at 3000r / min for 3 minutes, take the supernatant, concentrate in a 90°C constant temperature water bath to contain 1g / ml of crude drug, put it in a high pressure sterilizer and sterilize at 121°C for 15 minutes to obtain the antibacterial Chinese medicine composition.

[0031] Example 4

[0032] 1) Weigh the following components of Chinese medicine raw materials: 13 parts of honeysuckle, 10 parts of coptis root, 12 parts of scutellaria, 13 parts of astragalus, 20 parts of purslane, 12 parts of codonopsis, 8 parts of hawthorn, 5 parts of costus root, and 7 parts of liquorice. 2) Add 8 times the weight of water, soak for 30 minutes, heat to boil, decoct for 60 minutes under boiling conditions, and filter to obtain the filtrate. 3) Take the filter residue in step 2), add 8 times the weight of water, heat to boiling, decoct for 30 minutes under boiling conditions, and filter to obtain the filtrate. 4) Combine the filtrates of step 2) and step 3), centrifuge at 3000r / min for 3 minutes, take the supernatant, concentrate in a 90°C constant temperature water bath to contain 1g / ml of crude drug, put it in a high pressure sterilizer and sterilize at 121°C for 15 minutes to obtain the antibacterial Chinese medicine composition.

[0033] Example 5

[0034] 1) Weigh the following components of Chinese medicine raw materials: 14 parts of honeysuckle, 10 parts of coptis root, 11 parts of scutellaria, 14 parts of astragalus, 20 parts of purslane, 11 parts of codonopsis, 9 parts of hawthorn, 5 parts of costusroot, and 6 parts of liquorice. 2) Add 8 times the weight of water, soak for 50 minutes, heat to boil, decoct for 60 minutes under boiling conditions, and filter to obtain the filtrate. 3) Take the filter residue in step 2), add 8 times the weight of water, heat to boiling, decoct for 50 minutes under boiling conditions, and filter to obtain the filtrate. 4) Combine the filtrates of step 2) and step 3), centrifuge at 3000r / min for 3 minutes, take the supernatant, concentrate in a 90°C constant temperature water bath to contain 1g / ml of crude drug, put it in a high pressure sterilizer and sterilize at 121°C for 15 minutes to obtain the antibacterial Chinese medicine composition.

[0035] Example 6

[0036] 1) Weigh the following components of Chinese medicine raw materials: 15 parts of honeysuckle, 11 parts of coptis root, 9 parts of scutellaria, 15 parts of astragalus, 21 parts of purslane, 9 parts of codonopsis, 10 parts of hawthorn, 6 parts of costus root, and 4 parts of liquorice. 2) Add 10 times the weight of water, soak for 60 minutes, heat to boil, decoct for 20 minutes under boiling conditions, and filter to obtain the filtrate. 3) Take the filter residue in step 2), add 10 times the weight of water, heat to boiling, decoct for 60 minutes under boiling conditions, and filter to obtain the filtrate. 4) Combine the filtrates of step 2) and step 3), centrifuge at 3000r / min for 3 minutes, take the supernatant, concentrate in a 90°C constant temperature water bath to contain 1g / ml of crude drug, put it in a high pressure sterilizer and sterilize at 121°C for 15 minutes to obtain the antibacterial Chinese medicine composition.

[0037] Example 7

[0038] 1) Weigh the following components of Chinese medicine raw materials: 16 parts of honeysuckle, 9 parts of coptis root, 10 parts of scutellaria, 16 parts of astragalus, 19 parts of purslane, 10 parts of codonopsis, 11 parts of hawthorn, 4 parts of costusroot, and 5 parts of liquorice. 2) Add 12 times the weight of water, soak for 20 minutes, heat to boiling, decoct for 30 minutes under boiling conditions, and filter to obtain the filtrate. 3) Take the filter residue in step 2), add 12 times the weight of water, heat to boiling, decoct for 20 minutes under boiling conditions, and filter to obtain the filtrate. 4) Combine the filtrates of step 2) and step 3), centrifuge at 3000r / min for 3 minutes, take the supernatant, concentrate in a 90°C constant temperature water bath to contain 1g / ml of crude drug, put it in a high pressure sterilizer and sterilize at 121°C for 15 minutes to obtain the antibacterial Chinese medicine composition.

[0039] In order to verify the antibacterial Chinese medicine composition, the following experiment was performed:

[0040] 1. Experimental Methods

[0041] 1.1 Activation and preparation of strains

[0042] 1.1.1 Activation of strains

[0043] Extract and culture the original forms of Staphylococcus aureus, Escherichia coli, and Salmonella. On the sterile operating table, use an inoculation loop to gently scrape the bacterial moss from the inclined surface of the purchased bacterial culture medium, transfer the bacteria to the slide and spread them evenly, bake them dry in an alcohol lamp, and then perform Gram staining. After microscopic examination, inoculate the bacteria into the broth culture medium, and culture the broth containing the bacteria for 16-20 hours. Take out the nutrient broth culture medium inoculated with the strain from the shaker of the constant temperature incubator, use an inoculation loop to gently stir the culture medium to ensure that the bacteria are evenly distributed, and then use an inoculation loop to stain the Staphylococcus aureus, Escherichia coli, and Salmonella liquids, respectively, and inoculate them into nutrient agar medium, MacConkey agar medium, and bismuth sulfite agar medium, respectively, and place them in a 37°C constant temperature incubator for 16-20 hours. Take them out and observe their colony morphology and color to distinguish whether the strain is contaminated.

[0044] 1.1.2 Preparation of bacterial solution

[0045] Use a pipette to extract 1000 μL of Staphylococcus aureus and Escherichia coli inoculated in nutrient broth medium into sterile test tubes, and then add sterile saline to dilute them. After fully shaking, take the diluted bacterial solution into a cuvette, use a visible spectrophotometer to measure its absorbance value at a wavelength of 650nm, and use sterile saline as a blank control solution. The formula of absorbance (y) and McFarland turbidimetry (x) of Escherichia coli is: y = 0.320x + 0.034, and its absorbance is 0.194 when 0.5 McFarland turbidimetry is calculated. The formula of absorbance (y) and McFarland turbidimetry (x) of Staphylococcus aureus is: y = 0.288x-0.008, and its absorbance is 0.136 when 0.5 McFarland turbidimetry is calculated.

[0046] Use a pipette to extract 1000 μL of the Salmonella inoculated in the nutrient broth medium into a sterile test tube, and then add sterile saline to dilute it. After shaking thoroughly, take the diluted bacterial solution into a cuvette and use a visible spectrophotometer to measure its absorbance at a wavelength of 600 nm, using sterile saline as a blank control solution. Salmonella bacterial solution concentration x10 7 The formula of CFU / ml (y) and absorbance (x) is: y = 190.23x-8.1172, and the concentration of the bacterial solution is 1.5x10 7 CFU / ml, from which its absorbance can be calculated to be approximately 0.036.

[0047] Store the diluted bacterial solution in a 4°C refrigerator for later use.

[0048] 1.2.2 Preparation of Chinese medicine extracts

[0049] Put the Chinese medicine (single medicinal materials in Table 1) into a beaker and then put it into a drying oven at 40-60℃. Weigh it every half a day and take it out when it is dried to constant weight. Weigh 10g of each Chinese medicine after drying and put it into a conical bottle. Add distilled water with a solid-liquid ratio of 1:10 and soak it for 30 minutes. Wrap the bottle mouth with oil paper, then put it on a multi-purpose electronic stove and heat it with high heat. After the water boils, turn to low heat and start timing for 30 minutes, then filter it with two layers of gauze, add 10 times distilled water to the residue and heat it for 30 minutes, combine the filtrate obtained by the two decoctions and put it into a centrifuge tube for centrifugation, centrifuge it at 3000 rpm for 3 minutes, take the supernatant and pour it into a beaker, put it into a 90℃ constant temperature water bath for concentration, and when the amount of Chinese medicine liquid in the beaker is less than 20ml, transfer it to a test tube with a scale for concentration. When it is concentrated to 1g / ml, take it out, put it into a high pressure sterilizer at 121℃ for sterilization for 15 minutes, and then put it into a 4℃ refrigerator for storage.

[0050] 1.2.3 Ratio of Chinese herbal medicine and preparation of extracts

[0051] 1.2.3.1 Ratio of Chinese herbal compound

[0052] Table 1. Formula of Chinese medicine compound

[0053]

[0054] 1.2.3.2 Preparation of Chinese herbal compound extracts

[0055] Put all the Chinese medicines into beakers separately and put them into a constant temperature drying oven at 40-60℃. Weigh them every half a day and take them out when they are dried to constant weight. Weigh 15g of dried honeysuckle, 10g of coptis chinensis, 10g of scutellaria baicalensis, 15g of astragalus, 20g of purslane, 10g of codonopsis pilosula, 10g of hawthorn, 5g of costus root, and 5g of licorice respectively and put them into a beaker, add distilled water with a solid-liquid ratio of 1:10 and soak for 30 minutes, wrap the bottle mouth with oil paper, then put it on a multi-purpose electronic stove and heat it over high heat, turn to low heat after the water boils and start timing for 30 minutes, then filter it with two layers of gauze, add 10 times distilled water to the residue and heat it for 30 minutes, combine the filtrate obtained by the two decoctions and put it into a centrifuge tube for centrifugation, centrifuge at 3000 rpm for 3 minutes, take the supernatant and pour it into a beaker, put it into a 90°C constant temperature water bath for concentration, take it out when it is concentrated to 1g / ml, put it into an autoclave for sterilization at 121°C for 15 minutes, and then put it into a 4°C refrigerator for storage.

[0056] 1.2.4 Determination of the diameter of the inhibition zone in vitro for single Chinese herbal medicines and Chinese herbal compound prescriptions

[0057] After the above materials are prepared, we pour the prepared nutrient agar medium into the culture dish under sterile conditions, place it to cool and solidify, and after solidification, use a pipette to transfer 100μL of bacterial solution dilution to the surface of each nutrient agar culture dish, slowly spread the bacterial solution, and place the culture dish in a sterile workbench until the bacterial solution is completely air-dried. After the culture medium is air-dried, take out 3 Oxford cups with tweezers and place them in three areas on the culture medium plane. Then extract 200μL of different Chinese herbal medicine extracts and put them into each Oxford cup. The prepared bacterial culture medium is placed in a 37℃ constant temperature incubator for culture. After 18 hours, observe and record whether the inhibition zone on the culture medium is formed and the size of the inhibition zone. Then discuss and analyze the experimental results. The criteria for judging the sensitivity of the inhibition zone diameter: the inhibition zone diameter <10mm is low sensitivity, 10mm≤inhibition zone diameter <15mm is medium sensitivity, 15mm≤inhibition zone diameter <20mm is high sensitivity, and the inhibition zone diameter ≥20mm is extremely sensitive.

[0058] 1.2.5 MIC determination of single Chinese herbal medicine and Chinese herbal compound extracts

[0059] The minimum inhibitory concentration (MIC) of the selected Staphylococcus aureus, Escherichia coli, and Salmonella to the Chinese medicine extract was determined by the microbroth dilution method. All operations were carried out in a clean bench. The specific method is as follows:

[0060] In the first well of a disposable 96-well V-shaped plate, add 50 μL of different Chinese medicine extracts with a micropipette, then add 50 μL of sterilized nutrient broth, and mix thoroughly by blowing and aspirating. This step is a 2-fold dilution. Next, add 50 μL of sterilized nutrient broth to each well of wells 2-11, and add 100 μL of sterilized nutrient broth to well 12 as a negative control. Subsequently, draw 50 μL of the mixture from well 1 to the next well, and then dilute it to well 10 in succession, discard 50 μL of the mixture, and then add 50 μL of bacterial solution to wells 1-11. Well 11 only has bacterial solution and sterilized nutrient broth, no drugs, as a positive control. Cover the completed V-shaped 96-well plate with plastic wrap and place it in a 37°C constant temperature incubator for 16-18 hours. When the negative control has no turbidity and bacterial precipitation, and the positive control is turbid and has bacterial precipitation, the test results are valid. The minimum drug concentration corresponding to the clear broth well is the MIC of the bacteria to the drug.

[0061] 1.2.6 Screening of antibiotics against Staphylococcus aureus

[0062] Take 100 μL of Staphylococcus aureus dilution and inoculate it into the sterilized nutrient agar medium for coating. After the surface of the medium is slightly dry, use sterile tweezers to pick up the drug sensitivity test paper and place it on the medium. Put 3 pieces of each medium as a repeat group. It should be noted that after the drug sensitivity test paper falls, it cannot be moved. Place the culture medium in a 37°C constant temperature incubator for 16-20 hours, and observe and record the size of its inhibition zone. According to the antimicrobial sensitivity test execution standard of the 2021 Clinical and Laboratory Standards Institute (CLSI) M100 ed31 document, drug sensitivity is determined. R is resistant, I is moderately sensitive, and S is sensitive.

[0063] 1.2.7 Chinese herbal compound treatment trials

[0064] After the Staphylococcus aureus strains were strengthened, they were inoculated into nutrient broth and cultured for 16-20 hours, taken out, diluted to 0.5 McFarland turbidity and stored for later use. Thirty-five mice were divided into seven groups, namely, a blank control group, a normal saline control group, a low-concentration Chinese medicine compound group, a medium-concentration Chinese medicine compound group, a high-concentration Chinese medicine compound group, an antibiotic medium-sensitive group, and an antibiotic high-sensitive group. There were 5 mice in each group.

[0065] The experiment can be carried out only after the mice have been fed normally for 3 days and there are no obvious changes, so as to avoid stress affecting the experimental results. The mice in the experimental group were infected by intraperitoneal injection, and each mouse was injected with 0.2 ml of bacterial solution dilution, and the first day was calculated from the injection of bacterial solution. No operation was performed on the blank control group. After confirming that all the experimental groups were infected on the second day, the Chinese medicine extract was gavaged and the antibiotics were intraperitoneally injected.

[0066] Table 2. Grouping of Chinese medicine compound treatment trials

[0067]

[0068] The drugs were given continuously for 3 days, and the recovered mice were no longer given drugs. The number and time of death of the mice and the cure rate were observed within 9 days.

[0069] 1.2.8 Chinese herbal compound preventive trial

[0070] 35 mice were taken and divided into 7 groups, including blank control group, normal saline control group, low concentration of Chinese medicine compound group, medium concentration of Chinese medicine compound group, high concentration of Chinese medicine compound group, medium-sensitive antibiotic group, and high-sensitive antibiotic group, with 5 mice in each group.

[0071] The mice were purchased and fed for 3 days without obvious changes before the experiment was conducted to avoid stress affecting the experimental results. Each mouse in the Chinese medicine compound treatment group was gavaged with 0.2 ml of Chinese medicine extract every day for 5 consecutive days. Each mouse in the antibiotic treatment group was intraperitoneally injected with 0.2 ml of antibiotics every day. On the evening of the 5th day, except for the blank control group, the mice in other groups were intraperitoneally injected with 0.2 ml of Staphylococcus aureus. The death and mental state of the mice were observed within 5 days.

[0072] Table 3. Grouping of Chinese medicine compound treatment trials

[0073]

[0074] The drugs were given continuously for 3 days, and the recovered mice were no longer given drugs. The number and time of death of the mice and the cure rate were observed within 9 days.

[0075] 2. Experimental Results

[0076] 2.1 Results of in vitro inhibition zone diameter determination of single Chinese herbal medicines and Chinese herbal compound

[0077] Table 4. Diameter and sensitivity of inhibition zone of Chinese herbal medicine extracts to Staphylococcus aureus

[0078]

[0079] As shown in Table 4, among the single Chinese herbal extracts, hawthorn and coptis chinensis have extremely sensitive inhibition effects on Staphylococcus aureus, with average inhibition circle diameters of 22.36 mm and 22.69 mm, respectively; licorice tablets and scutellaria baicalensis have highly sensitive inhibition effects on Staphylococcus aureus, with average inhibition circle diameters of 15.42 mm and 15.30 mm, respectively; codonopsis pilosula, costusroot, and honeysuckle have moderately sensitive inhibition effects on Staphylococcus aureus, with average inhibition circle diameters of 14.22 mm, 13.60 mm, and 11.79 mm, respectively; while astragalus and purslane have poor or no inhibition effects on Staphylococcus aureus. The average inhibition circle diameter of the Chinese herbal compound extract on Staphylococcus aureus is 14.79 mm, which provides a certain basis for the subsequent treatment and prevention experiments in mice.

[0080] Table 5. Diameter and sensitivity of inhibition zone of Chinese herbal medicine extracts to Escherichia coli

[0081]

[0082] As shown in Table 5, among the Chinese herbal extracts, only hawthorn has an extremely sensitive inhibitory effect on Escherichia coli, with an average inhibition zone diameter of 20.87 mm; Codonopsis pilosula and honeysuckle have moderately sensitive inhibitory effects on Escherichia coli, with average inhibition zone diameters of 14.65 mm and 11.65 mm respectively; the other Chinese herbal extracts have poor or no inhibitory effects on Escherichia coli.

[0083] Table 6. Diameter and sensitivity of inhibition zone of traditional Chinese medicine extracts to Salmonella

[0084]

[0085] As shown in Table 6, among the Chinese herbal extracts, only hawthorn has an extremely sensitive inhibitory effect on Salmonella, with an average inhibition zone diameter of 21.68 mm; Coptis chinensis has a moderately sensitive inhibitory effect on Salmonella, with an average inhibition zone diameter of 12.07 mm; the other Chinese herbal extracts have poor or no inhibitory effect on Salmonella.

[0086] 2.2 MIC determination results of single Chinese herbal medicine and Chinese herbal compound extracts

[0087] Table 7. MIC test results of single Chinese herbal medicine and Chinese herbal compound extracts (mg / ml)

[0088]

[0089] As shown in Table 7, the minimum inhibitory concentrations of hawthorn extract against Staphylococcus aureus, Escherichia coli, and Salmonella are 7.81 mg / ml, 15.63 mg / ml, and 7.81 mg / ml, respectively, which are the lowest concentrations among all drugs and have the best effect. The second is Coptis chinensis extract, whose minimum inhibitory concentration against Staphylococcus aureus reaches 15.63 mg / ml, second only to hawthorn. The minimum inhibitory concentration of most Chinese medicines against Salmonella is >500.00 mg / ml. The minimum inhibitory concentrations of Chinese medicine compound extract against Staphylococcus aureus, Escherichia coli, and Salmonella are 62.50 mg / ml, 31.25 mg / ml, and 250.00 mg / ml, respectively, but its inhibition zone during in vitro antibacterial inhibition shows that its inhibitory effect against Escherichia coli is poor.

[0090] 2.3 Anti-Staphylococcus aureus antibiotic screening results

[0091] Table 8. Anti-Staphylococcus aureus antibiotic screening results

[0092]

[0093] As shown in Table 8, the inhibitory effects of ceftriaxone, cefoperazone and amikacin on Staphylococcus aureus reached high sensitivity, and the diameters of the inhibition zones were 26.85 mm, 26.48 mm and 22.28 mm respectively; the inhibitory effects of minocycline and cephalexin on Staphylococcus aureus were moderately sensitive, and the diameters of the inhibition zones were 14.46 mm and 16.50 mm respectively; erythromycin, penicillin and lincomycin were resistant to Staphylococcus aureus.

[0094] The antibiotics required for the Chinese medicine compound treatment and prevention test are selected according to the size of the inhibition zone diameter and sensitivity. Ceftriaxone is selected for the highly sensitive drug and cephalexin is selected for the moderately sensitive drug.

[0095] 2.4 Results of the TCM compound treatment trial

[0096] Table 9. Results of the Chinese medicine compound treatment trial

[0097]

[0098] After the bacterial liquid was injected, the infected mice showed poor mental state. The mice in each group huddled together, curled up, ate less, and had loose stools.

[0099] Day 3 of the experiment: On the third day of administration, the mental state and fecal status of mice in the antibiotic high-sensitivity group returned to normal. One mouse in the antibiotic medium-sensitivity group died on the third day, and the rest of the mice were unable to get up and had no resistance; the low-concentration group of the Chinese medicine compound was in a general state, one mouse was in a poor state, and the medium-concentration and high-concentration groups of the Chinese medicine compound were in a poor state, with one mouse in the medium-concentration group dying; and four mice in the normal saline group died.

[0100] On the fourth day of the experiment, 3 mice died in the antibiotic moderate-sensitivity group, and the rest were in normal condition; 1 mouse died in the Chinese medicine medium-concentration group, and the rest were in good condition; and 1 mouse died in the normal saline control group.

[0101] On the fifth day of the experiment, one mouse died in the medium-concentration group of the Chinese herbal compound, and the rest were in normal condition; two mice died in the high-concentration group of the Chinese herbal compound, and the rest were in normal condition.

[0102] On the sixth day of the experiment, one mouse died in the high-concentration Chinese herbal compound group, and the rest were in good condition; the conditions of mice in the other groups all improved.

[0103] On the 7th day of the experiment, one mouse died in the medium concentration group of the Chinese medicine compound; the conditions of mice in the other groups improved.

[0104] On the 8th day of the experiment, the mice were in good condition.

[0105] On the 9th day of the experiment, one mouse died in the low-concentration Chinese medicine compound group; the rest were in good condition.

[0106] During the experiment, all subjects in the blank control group were in good condition and showed no abnormal symptoms.

[0107] As shown in Table 9, the survival rate of the antibiotic high-sensitivity group was the highest, reaching 100%; followed by the low-concentration Chinese medicine compound group with a survival rate of 60%, and the survival rates of the medium-concentration group and the high-concentration group were both 40%; the survival rate of the medium-sensitivity group was 10%; and there was no survival in the normal saline group.

[0108] 2.5 Results of the Chinese herbal compound prevention trial

[0109] Table 10. Results of the preventive test of Chinese herbal compound

[0110]

[0111] On the sixth day of the experiment, except for the antibiotic-sensitive group, the other groups showed different conditions, mainly manifested as mice huddling together, curling up, eating less, and having loose stools.

[0112] On the 7th day of the experiment, one mouse died in the antibiotic moderate-sensitivity group, three mice died in the Chinese herbal compound low-concentration, medium-concentration groups and the normal saline control group, and two mice died in the Chinese herbal compound high-concentration group. Except for the normal saline control group which was in poor condition, the rest of the mice were in general condition.

[0113] On the 8th day of the experiment, 2 mice in the normal saline control group died and no one survived; the conditions of the remaining mice gradually improved.

[0114] On the 9th day of the experiment, one mouse died in the low-concentration Chinese medicine compound group; the rest were in good condition.

[0115] On the 10th day of the experiment, the remaining mice showed no abnormalities and were in good condition.

[0116] During the experiment, all subjects in the blank control group were in good condition and showed no abnormal symptoms.

[0117] As can be seen from Table 10, the survival rate of the antibiotic high-sensitivity group was the highest, reaching 100%; followed by the antibiotic medium-sensitivity group with a survival rate of 80%; the survival rate of the Chinese herbal compound high-concentration group was 60%, and the survival rate of the Chinese herbal compound medium-concentration group was 40%; the survival rate of the Chinese herbal compound medium-concentration group was 20%; and there was no survival in the normal saline group.

[0118] Conclusion

[0119] According to the results of in vitro antibacterial tests, most Chinese herbal extracts showed inhibitory effects on Staphylococcus aureus, but had little or no inhibitory effects on Escherichia coli and Salmonella. In particular, hawthorn extracts had good inhibitory effects on all three pathogens. As for Chinese herbal compound extracts, they only showed inhibitory effects on Staphylococcus aureus, and the effect reached a medium sensitivity, and the diameter of the inhibition zone was close to the high sensitivity level. Despite this, in the in vitro antibacterial test, the minimum inhibitory concentration (MIC) of the Chinese herbal compound on Escherichia coli was 31.25 mg / ml, but no obvious antibacterial effect was observed in the actual inhibition zone test.

[0120] It can be seen from the results of the treatment and prevention test of Chinese herbal compound that Chinese herbal compound has a certain effect in the treatment and prevention of Staphylococcus aureus infection. In the treatment test, the survival rate of the low-concentration group of the Chinese herbal compound was 60%, and the medium-concentration group and the high-concentration group were both 40%, which shows that the Chinese herbal compound can improve the survival rate of mice at a certain concentration, although the effect is not as good as the 100% survival rate of the antibiotic high-sensitivity group. In the prevention test, the survival rate of the high-concentration group of the Chinese herbal compound was 60%, the medium-concentration group was 40%, and the low-concentration group was 20%, which shows that the Chinese herbal compound is also effective in preventing Staphylococcus aureus infection, and the survival rate increases with the increase of drug concentration. However, compared with the 80% survival rate of the antibiotic medium-sensitivity group, the Chinese herbal compound still has a certain gap in the preventive effect. In summary, the Chinese herbal compound of the present application shows potential application value in the treatment and prevention of Staphylococcus aureus infection.

Claims

1. An antibacterial Chinese medicine composition, characterized in that: The raw materials for preparing the antibacterial Chinese medicine composition include the following components in parts by weight: 13-17 parts of honeysuckle, 8-12 parts of coptis chinensis, 8-12 parts of scutellaria baicalensis, 13-17 parts of astragalus, 18-22 parts of purslane, 8-12 parts of codonopsis pilosula, 8-12 parts of hawthorn, 3-7 parts of costusroot and 3-7 parts of liquorice.

2. The antibacterial Chinese medicine composition according to claim 1, characterized in that: The components of the raw materials are as follows: 14-16 parts of honeysuckle, 9-11 parts of coptis root, 9-11 parts of scutellaria root, 14-16 parts of astragalus root, 19-21 parts of purslane, 9-11 parts of codonopsis pilosula, 9-11 parts of hawthorn, 4-6 parts of costusroot, and 4-6 parts of liquorice.

3. The antibacterial Chinese medicine composition according to claim 1, characterized in that: The components of the raw materials are as follows: 15 parts of honeysuckle, 10 parts of coptis root, 10 parts of scutellaria root, 15 parts of astragalus root, 20 parts of purslane, 10 parts of codonopsis pilosula, 10 parts of hawthorn, 5 parts of costusroot, and 5 parts of liquorice.

4. A method for preparing an antibacterial Chinese medicine composition, characterized in that: The components described in any one of claims 1 to 3 are used as raw materials, and the decoction is retained after decocting in water as the antibacterial Chinese medicine composition.

5. The preparation method according to claim 4, characterized in that: The method comprises the following steps: 1) weighing raw materials according to the weight of the components; 2) soaking in water for 20 to 60 minutes, heating to boiling, decocting for 20 to 60 minutes under boiling conditions, and filtering to obtain filtrate; 3) taking the filter residue in step 2), adding water and heating to boiling, decocting for 20 to 60 minutes under boiling conditions, and filtering to obtain filtrate; 4) combining the filtrates of step 2) and step 3), centrifuging to obtain the supernatant, concentrating and sterilizing to obtain the antibacterial Chinese medicine composition.

6. The preparation method according to claim 5, characterized in that: In step 2) and step 3), the amount of water added is 8 to 12 times the total weight of the raw material.

7. The preparation method according to claim 5, characterized in that: The centrifugal separation process is 3000r / min centrifugation for 3 minutes.

8. The preparation method according to claim 5, characterized in that: The concentration is to concentrate the supernatant to contain 1 g / ml of the crude drug.

9. The preparation method according to claim 5, characterized in that: The sterilization step is to place the obtained concentrated solution into a high pressure sterilizer and sterilize it at 121° C. for 15 minutes.

10. Use of the antibacterial Chinese medicine composition according to any one of claims 1 to 3 or the antibacterial Chinese medicine composition prepared by the preparation method according to any one of claims 4 to 9 in the preparation of veterinary drugs against Staphylococcus aureus.

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