Traditional Chinese medicine ointment for treating microbial-infected skin diseases, preparation method and application thereof

Abstract

A traditional Chinese medicine ointment for treating microbial-infected skin diseases, and its preparation method and use, is prepared from at least the following raw materials: 10g of lithospermum erythrorhizon, 1g of angelica sinensis, 1g of siler, 1g of rehmannia root, 1g of angelica dahurica, 1g of frankincense, 1g of myrrh, 10g of psoralea corylifolia, 1g of cinnamon bark, 1g of coptis root, 1g of rhubarb, 1g of phellodendron amurense, 1g of mentha, 55g of betaine, 5-6g of beeswax, 10-11g of gelatin, and 10-11g of camellia oil. The ointment uses natural ingredients, is free of any additives, is highly safe, is non-irritating and harmless to the skin, is widely applicable, is non-allergenic, and is simple to prepare and use, easy to operate, and has high economic benefits.

Description

Technical Field

[0001] The invention belongs to the field of Chinese herbal medicine combinations, and particularly relates to a Chinese medicinal ointment for treating skin diseases infected by microorganisms, and a preparation method and application thereof. Background Art

[0002] Skin infections, or even post-injury wound infections, are infections caused by bacteria or fungi that directly damage skin tissue and invade the skin and soft tissues due to trauma. They are prone to serious hidden effects. If left untreated, they can lead to localized suppurative infections of tissues and organs, or even systemic infections such as sepsis, which can be fatal in severe cases. The spectrum of skin diseases and the corresponding changes in the infecting pathogens, as well as the evolution of drug resistance, are clinical issues that urgently need to be addressed. Post-injury skin and soft tissue infections are a group of infectious diseases with a high incidence and diversity, with injury types shifting from acute traumatic wounds such as burns and abrasions to chronic infections. As can be seen, the causes of skin infections are complex, and there are many types of pathogens that cause skin or wound infections. Common pathogens include Staphylococcus aureus, Candida albicans, Cryptococcus neoformans, etc. Furthermore, the continuous emergence of drug-resistant bacteria such as MRSA (methicillin-resistant Staphylococcus aureus) strains, especially those with strong drug resistance or multidrug resistance, has increased the difficulty of clinical treatment.

[0003] Some Chinese herbal ingredients have become inspiration and starting points for the development of new drugs due to their significant antibacterial effects, high safety, and resistance to drug resistance. Therefore, the development of antibacterial drugs using Chinese herbal medicines has promising application and development prospects. In traditional Chinese medicine ointments, frankincense and myrrh disinfect and promote tissue regeneration; rehmannia root clears heat and cools blood; angelica root nourishes blood, activates blood circulation, and relieves pain; angelica root and saposhnikovia root dispel wind and relieve pain; coptis root, rhubarb, and phellodendron (commonly known as the "three yellows") have long been known to clear heat and detoxify, and enhance anti-inflammatory properties; psoralea corylifolia warms the kidneys and yang, promotes qi and relieves asthma, warms the spleen, and stops diarrhea; and cinnamon bark has anti-inflammatory, anti-allergic, and antiviral properties. Our research also found that some ingredients in psoralea corylifolia, cinnamon, and lithospermum erythrorhizon can inhibit the growth of various bacteria and fungi. Betaine was initially found in beet, with pharmacological effects such as antitumor, bactericidal and anti-inflammatory, and blood pressure reduction. Later, betaine was also found in various plant roots, stems or fruits such as wolfberry and cotton. The source is natural and has strong hygroscopicity. We have confirmed that it has a good inhibitory effect on Candida albicans, Pseudomonas aeruginosa, Cryptococcus neoformans, Staphylococcus aureus, etc., and cooperates with Psoralea corylifolia, Cinnamon Bark and Lithospermum erythrorhizon containing antibacterial ingredients to play antibacterial efficacy. In addition, Lithospermum erythrorhizon is a traditional Chinese medicine with a long history of medicinal use. Except for cooling blood and promoting blood circulation, clearing away heat and detoxicating, and promoting bowel movements, it has excellent anti-sensitivity, redness and swelling, and activates the function and good wound repair ability of epidermal cell metabolism. Its antibacterial, anti-inflammatory and antiviral effects also make it have good advantages in treating skin infections, so the present invention uses it as the primary component promoting inflammatory wound healing. These Chinese herbal medicines and betaine are complementary in effect, and have jointly made a Chinese medicinal ointment that can be both antibacterial and promote wound healing.

[0004] Although the preparation technology of some antibacterial ointments is very mature, and conventional antibiotics are used to treat wound infections, their efficacy in exerting antibacterial effects and promoting wound healing and scab removal is not comprehensive enough, and antibiotic resistance is also a major obstacle to infection treatment. In addition, some patients' skin is prone to allergic reactions to antibiotics during treatment. Therefore, both oral and topical antibiotic treatment methods have major flaws. In order to enable patients with skin infections to prevent further infection and invasion by pathogens in daily life and when their skin is damaged, and to help the proliferation of wound granulomas, and to receive timely and effective treatment before the condition worsens, a drug with a broad-spectrum antibacterial effect, effective against drug-resistant bacteria, and capable of accelerating wound healing is needed for daily treatment. The traditional Chinese medicine ointment in this study has stronger and more comprehensive efficacy, and its formula and proportion are disclosed for the first time. Summary of the Invention

[0005] Technical problem to be solved: In response to the above problems, the present invention provides a traditional Chinese medicine ointment for treating skin diseases infected by microorganisms, and a preparation method and application thereof.

[0006] Technical solution: A traditional Chinese medicine ointment for treating skin diseases infected by microorganisms is prepared from at least the following raw materials in proportion: 10g of lithospermum erythrorhizon, 1g of angelica sinensis, 1g of siler, 1g of rehmannia root, 1g of angelica dahurica, 1g of frankincense, 1g of myrrh, 10g of psoralea corylifolia, 1g of cinnamon bark, 1g of coptis chinensis, 1g of rhubarb, 1g of phellodendron amurense, 1g of mint, 55g of betaine, 5-6g of beeswax, 10-11g of gelatin, and 10-11g of camellia oil.

[0007] The preparation method of the above-mentioned Chinese medicine ointment specifically comprises the following steps: the first step is to grind lithospermum erythrorhizon, angelica sinensis, siler, rehmannia root, angelica dahurica, frankincense, myrrh, psoralea corylifolia, cinnamon bark, coptis chinensis, rhubarb, phellodendron chinense, and mint into powder, and coarsely sieve through a 100-mesh sieve; the second step is to extract with water: 10g lithospermum erythrorhizon, 1g angelica sinensis, 1g siler, 1g rehmannia root, 1g angelica dahurica, frankincense, myrrh, 10g psoralea corylifolia, 1g cinnamon bark, coptis chinensis, 1g rhubarb, 1g phellodendron chinense, and 1g mint are put into a beaker, add water at a ratio of 1g:20mL, boil, and cook at a constant temperature three times, each time for 45min, 45min, and 30min respectively. The mixture was filtered through a 100-mesh sieve and then allowed to stand for 12 hours. In the third step, the supernatant in the cup was filtered again, and the resulting liquid was divided into lyophilized portions. In the fourth step, 0.45 g of gelatin was added to 45 mL of warm water, soaked for 5 minutes to expand, and then heated in a water bath at 80°C for 15 minutes to dissolve it into a slurry. In the fifth step, betaine, gelatin slurry, and freeze-dried powder of the water-extracted component were weighed and mixed in a ratio of 10:2:1. In the sixth step, camellia oil and beeswax were weighed and heated together until they melted at 60-80°C, and then the heating was stopped. The mixture obtained in the previous step was added and stirred evenly. The mixture: camellia oil: beeswax = 13:2:1. After cooling, the Chinese medicine ointment was obtained.

[0008] The traditional Chinese medicine ointment prepared by the above method is used in the preparation of broad-spectrum antibacterial products.

[0009] The traditional Chinese medicine ointment prepared by the above method is used in the preparation of products for treating skin diseases infected by microorganisms.

[0010] The traditional Chinese medicine ointment prepared by the method is used in the preparation of drugs for inhibiting sensitive and resistant Staphylococcus aureus and fungi.

[0011] The minimum inhibitory concentration (MIC) of the above-mentioned traditional Chinese medicine ointment ranges from 8 to 64 μg / mL.

[0012] Beneficial effects: 1. The present invention follows the principle of monarch-minister compatibility in traditional Chinese medicine, with "Coptis chinensis, Phellodendron chinense, and Rhubarb" being bitter and cold, and being used to clear away heat, detoxify, and treat sores as monarch drugs; frankincense, myrrh, lithospermum erythrorhizon, and angelica sinensis being minister drugs, and being used to invigorate blood circulation and dredge meridians, and to reduce swelling and relieve pain; Angelica dahurica, Saposhnikovia divaricata, and mint being matched, being pungent, dispersing, and penetrating, and being used to relieve stagnation and resolve nodules, and to expel pathogens as adjuvant drugs; Rehmannia glutinosa, Psoralea corylifolia, and cinnamon being three drugs, being used to nourish yin and tonify the kidney, warm yang, and dispel cold, and to assist the body's positive energy and promote wound healing, and being used as adjuvant drugs. 1. The invention further combines betaine, beeswax and camellia oil, which have anti-inflammatory and antibacterial effects and promote tissue regeneration and repair, as guiding drugs. The combination of these drugs can achieve the functions of clearing away heat and detoxifying, promoting blood circulation and relieving pain, tonifying the kidney and warming yang, and inhibiting bacteria and inflammation, promote the proliferation of fresh granulation tissue, soothe the skin and promote skin regeneration, and accelerate wound healing; 2. The invention adds ingredients that can effectively fight bacteria, whether it is bacteria or fungi, sensitive or resistant bacteria, and has good and wide antibacterial effects; 3. The invention has a good therapeutic effect on the infection model of drug-resistant strains, and the wound scab removal rate is high and fast; 4. The invention uses natural ingredients without any additives, has high safety, is non-irritating and harmless to the skin, is widely applicable to the population, is not prone to allergies, and the preparation and use methods are simple, easy to operate, and have high economic benefits. 5. The Chinese herbal medicine used is a powder after grinding, and the ingredients are fully extracted. Betaine is viscous, which can not only enhance the antibacterial effect but also promote the formation of the paste, making the content of minor ingredients such as beeswax and gelatin less. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The following are pictures of Chinese medicinal ointments (A. Comparison of ointments prepared in three ratios in Example 4; B. Samples of the final Chinese medicinal ointment)

[0014] Figure 2 The toxicity of Chinese herbal ointment to normal cells (A. bar graph, B. line graph).

[0015] Figure 3 The wound healing status of mice in the in vivo antibacterial experiment (A and B are mice in the trauma group, and C is mice in the non-trauma group).

[0016] Figure 4 The colonization amount of two strains of Staphylococcus aureus (A is Newman, B is USA300) in the skin wounds of mice infected with the bacteria.

[0017] Figure 5 This is the inflammatory condition of the skin wound tissue in mice during the in vivo antibacterial experiment. DETAILED DESCRIPTION

[0018] Example 1

[0019] In the first step, each Chinese medicine is crushed into coarse powder and divided into ester-soluble group and alcohol-extracted group according to its solubility characteristics.

[0020] Ester-soluble group: Lithospermum officinale, Rhubarb, Angelica sinensis, Saposhnikovia divaricata, Rehmannia root, Angelica dahurica, Frankincense, Myrrh, Cinnamon bark;

[0021] Alcohol extraction group: Psoralea corylifolia, Coptis chinensis, and Phellodendron

[0022] Step 2: Prepare the alcohol extraction group:

[0023] (1) Alcohol extraction of Psoralea corylifolia: 70% ethanol, solid-liquid ratio 1:8 (g:mL), solvent temperature 40°C, 40kHz ultrasound for 30 min, and filtration to obtain a crude extract (containing ethanol).

[0024] (2) Ethanol extraction of Coptis chinensis and Phellodendron amurense: add 100 mL of 95% ethanol to every 10 g of the drug, heat under reflux for 30 min, soak for 1 h, filter twice, and combine the filtrates (containing ethanol) from the three extractions.

[0025] (3) The liquids obtained in (1) and (2) were subjected to rotary evaporation to remove ethanol and freeze-dried for later use.

[0026] Step 3: Prepare the oil extraction group:

[0027] (1) The oil extraction group was divided into two groups (1. Lithospermum officinale, Rhubarb, Angelica sinensis, Saposhnikovia divaricata, Rehmannia root, Angelica dahurica, Frankincense, Myrrh; 2. Cinnamon bark) and immersed in oil for 1-2 hours respectively.

[0028] (2) Place the asbestos mesh on a magnetic stirrer and place the beaker on the asbestos mesh. Set the temperature of the magnetic stirrer to 200°C and use a thermometer to measure the oil temperature. When the oil temperature is 180-200°C, add the first group of coarse powder and fry for 10 minutes. During this period, stir with a magnetic rotor to prevent it from getting burnt (the time is determined according to the actual situation). After frying dry, pass it through a 100-mesh sieve and filter 2-3 times to remove the filter residue.

[0029] (3) When the oil temperature drops to 120°C, add cinnamon (because some of its components are easily decomposed at high temperatures) and fry for 10 minutes. Then add the freeze-dried powder from the alcohol extraction group and continue stirring. Then filter 2-3 times.

[0030] (4) When the oil temperature drops to 100℃, add beeswax. After cooling slightly to 60℃, add mint powder and mix well.

[0031] Example 2

[0032] The first step is to grind lithospermum erythrorhizon, angelica sinensis, siler, rehmannia root, angelica dahurica, frankincense, myrrh, psoralea corylifolia, cinnamon bark, coptis root, rhubarb, phellodendron amurense, and mint into powder and coarsely sieve;

[0033] The second step is water extraction: weigh the powder of each Chinese herbal medicine according to its own weight, put it into a beaker, add water at a ratio of 1:20 (g:mL) and boil it. Boil it at a constant temperature for three times, each time for 45 minutes, 45 minutes, and 30 minutes respectively. Filter it with a 100-mesh sieve after each boiling, and then let it stand overnight.

[0034] The third step is to filter the supernatant in the cup again, and the resulting liquid is packaged and freeze-dried;

[0035] Step 4: Shikonin, betaine and freeze-dried powder of water extract are weighed and mixed in a ratio of 1:6:5 and 1:10:1, and the resulting mixture is mixed with camellia oil in a ratio of 1:1 and stirred to form a paste;

[0036] Step 5: Heat the above ingredients to 60-80° C., add beeswax (camellia oil: beeswax = 4:1), stir evenly, pour into the prepared container and cool.

[0037] Example 3

[0038] The first step is to grind lithospermum erythrorhizon, angelica sinensis, siler, rehmannia root, angelica dahurica, frankincense, myrrh, psoralea corylifolia, cinnamon bark, coptis root, rhubarb, phellodendron amurense, and mint into powder and coarsely sieve;

[0039] The second step is water extraction: weigh the powder of each Chinese herbal medicine according to its own weight, put it into a beaker, add water at a ratio of 1:20 (g:mL) and boil it. Boil it at a constant temperature for three times, each time for 45 minutes, 45 minutes, and 30 minutes respectively. Filter it with a 100-mesh sieve after each boiling, and then let it stand overnight.

[0040] The third step is to filter the supernatant in the cup again, and the obtained liquid is packaged and freeze-dried. Then, the betaine and water extract component freeze-dried powder are weighed and mixed in a ratio of 10:1;

[0041] The fourth step is to add camellia oil and beeswax in a weight ratio (mixture: camellia oil: beeswax = 4:2:1) into a beaker and heat them together until they melt at 60-80°C, then stop heating, add the former mixture, stir evenly, pour into the prepared container and cool to obtain the Chinese medicine ointment.

[0042] Example 4

[0043] The first step is to grind lithospermum erythrorhizon, angelica sinensis, siler, rehmannia root, angelica dahurica, frankincense, myrrh, psoralea corylifolia, cinnamon bark, coptis root, rhubarb, phellodendron chinense, and mint into powder, and sieve through a 100-mesh sieve to ensure that the skin feels good.

[0044] The second step is water crude extraction: the powders of each Chinese herbal medicine are weighed according to their respective weights (10 g of Lithospermum erythrorhizon, 1 g of Angelica sinensis, 1 g of Saposhnikovia divaricata, 1 g of Rehmannia glutinosa, 1 g of Angelica dahurica, 1 g of Frankincense, 1 g of Myrrha, 10 g of Psoralea corylifolia, 1 g of Cinnamon bark, 1 g of Coptis chinensis, 1 g of Rhubarb, 1 g of Phellodendron amurense, and 1 g of Menthol), put into a beaker, add water at a ratio of 1:20 (g:mL), boil, and boil at a constant temperature three times, each time for 45 min, 45 min, and 30 min, respectively. After each boiling, filter with a 100-mesh sieve and let it stand for 12 h.

[0045] The third step is to filter the supernatant in the cup again, and the resulting liquid is packaged and freeze-dried;

[0046] Step 4: Take 0.45g of gelatin and add 45mL of warm water (concentration 0.01g / mL), soak for 5min to expand, and then heat in a water bath at 80℃ for 15min to dissolve it into a slurry;

[0047] Step 5: weigh and mix betaine, gelatin slurry, and freeze-dried powder of water extract according to the ratios of 10:4:1, 10:2:1, and 10:2:1 in Table 1;

[0048] Step 6: Weigh camellia oil and beeswax (mixture: camellia oil: beeswax = 15:5:2, 13:5:1, 13:2:1) according to the ratio in Table 1 and heat them together until they melt at 60-80°C, then stop heating. Then add the corresponding mixture obtained in the previous step, stir evenly, pour into the prepared container, and cool to obtain a traditional Chinese medicine ointment. Compare the softness and viscosity of the three traditional Chinese medicine ointments to obtain the optimal ratio.

[0049] Table 1. Weight ratio of gelatin to each component in Example 4

[0050]

[0051] The inhibitory effect of the Chinese medicinal ointment of the present invention on common bacteria in skin or wound infections is further illustrated in detail by the following examples.

[0052] 1. Materials

[0053] 1.1 Sample

[0054] Purchase Chinese herbal medicines from "Rejuvenation Pharmacy" in Baise City, and purchase comfrey compounds, psoralea compounds, and cinnamon compounds from Chengdu Ruifensi Biotechnology Co., Ltd.

[0055] 1.2 Strains

[0056] Strains of Staphylococcus aureus (including the standard sensitive strain Newman and the standard methicillin-resistant strain USA300), Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Candida albicans, Bacillus subtilis, Proteus mirabilis, Morganella morganii, Cryptococcus neoformans, Candida tropicalis, Staphylococcus haemolyticus, and Enterobacter hallii were provided by the Research Center for Prevention and Control of Drug-Resistant Microbial Infections of Youjiang Medical College for Nationalities.

[0057] 1.3 Main culture media and reagents

[0058] Nutrient agar, nutrient broth, Sabouraud agar, liquid Sabouraud agar, Columbia agar, brain heart infusion.

[0059] 1.4 Main instruments

[0060] 30℃ and 37℃ incubators, 30℃ and 37℃ shakers, centrifuge, microplate reader, electronic balance.

[0061] 1.5 Consumables

[0062] EP tube, Tip head, centrifuge tube.

[0063] 2. Methods and Results

[0064] 2.1 Microdilution method to detect the minimum inhibitory concentration (MIC, 100 μL system) of the main active ingredients of traditional Chinese medicine ointments against various strains

[0065] (1) Prepare 4 mg / mL of lithospermum erythrorhizon compound, psoralea corylifolia compound, and cinnamon compound.

[0066] (2) Preparation of MIC plate: First, add 173.6 μL of culture medium to the first well, then add 6.4 μL of antimicrobial drug, and dilute it in multiple proportions to the 10th well; only culture medium is added to the 11th well, and no drug is added to the 12th well. 90 μL of culture medium is retained as a control with bacteria but no drug.

[0067] (3) Preparation of bacterial suspension: Take the bacterial strains growing in logarithmic phase on the solid plate and use the corresponding culture medium to make a bacterial suspension: Helicobacter pylori and bacteria adjust the concentration OD 600 0.3(1×10 8 CFU / mL), Helicobacter pylori was diluted 10 times to 1×10 7 CFU / mL; bacteria diluted 100 times, 1×10 6 CFU / mL; fungal concentration adjusted OD 600 0.5(5×10 6 CFU / mL), diluted 5000 times to 1×10 3 CFU / mL, set aside.

[0068] (4) Take 10 μL of the inoculated bacterial solution and add it to wells 1-8 (the bacterial solution concentration in each well is 1×10 2 CFU / mL, Helicobacter pylori is 1×10 6 CFU / mL, other bacteria are 1×10 5 CFU / mL). Results were determined after 72 hours of culture for Helicobacter pylori, 48 hours for fungal cultures, and 24 hours for other strains. Drug concentrations in wells 1 to 6 were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL, respectively.

[0069] (5) The results are judged by the lowest drug concentration that completely inhibits bacterial growth in the small wells as MIC. The test is meaningful only when bacteria in the positive control well 12 (i.e., without antibiotics) grow significantly and in the 11th well (sterile) there is no growth. When a single jump well appears in the microdilution method, the highest drug concentration that inhibits bacterial growth should be recorded. If multiple jump wells appear, the results should not be reported and the test should be repeated. The test should be repeated 3 times for each drug.

[0070] (6) Results: The effective minimum inhibitory concentration of lithospermum erythrorhizon compounds was 1-32 μg / mL, the effective minimum inhibitory concentration of psoralea corylifolia compounds was 1-128 μg / mL, and the minimum inhibitory concentration of cinnamon compounds was 8-128 μg / mL. This shows that lithospermum erythrorhizon, psoralea corylifolia, and cinnamon contain many broad-spectrum antibacterial ingredients. The results are shown in Tables 2-4.

[0071] Table 2. Minimum inhibitory concentrations of lithospermum compounds (μg / mL)

[0072]

[0073] Table 3. Minimum inhibitory concentration of psoralea corylifolia compounds (μg / mL)

[0074]

[0075] Table 4. Minimum inhibitory concentration of cinnamon compounds (μg / mL)

[0076]

[0077] Note: 26695 is a sensitive strain, and HPBS001 is a resistant strain (resistant to levofloxacin, clarithromycin, and metronidazole)

[0078] 2.2 Microdilution method to detect the antibacterial spectrum of betaine and the Chinese medicinal ointment in each embodiment

[0079] (1) Prepare shikonin, betaine, and Chinese medicine ointment at a concentration of 32 mg / mL.

[0080] (2) Preparation of MIC plate: First, add 173.6 μL of culture medium to the first well, then add 6.4 μL of antimicrobial drug, and dilute it in multiple proportions to the 10th well; only culture medium is added to the 11th well, and no drug is added to the 12th well. 90 μL of culture medium is retained as a control with bacteria but no drug.

[0081] (3) Preparation of bacterial suspension: Take the bacterial strains growing in logarithmic phase on the solid plate and use the corresponding culture medium to make a bacterial suspension: Helicobacter pylori and bacteria adjust the concentration OD 600 0.3(1×10 8 CFU / mL), Helicobacter pylori was diluted 10 times to 1×10 7 CFU / mL; bacteria diluted 100 times, 1×10 6 CFU / mL; fungal concentration adjusted OD 600 0.5(5×10 6 CFU / mL), diluted 5000 times to 1×10 3 CFU / mL, reserve for future use.

[0082] (4) Take 10 μL of the inoculated bacterial solution and add it to wells 1-10 (the concentration of bacterial solution in each well is 1×10 2 CFU / mL, Helicobacter pylori is 1×10 6 CFU / mL, other bacteria are 1×10 5 CFU / mL). Results were determined after 72 hours of culture for Helicobacter pylori, 48 hours for fungal cultures, and 24 hours for other strains. Drug concentrations in wells 1 to 6 were 1024, 512, 256, 128, 64, 32, 16, 8, 4, and 2 μg / mL, respectively.

[0083] (5) The results are judged by the lowest drug concentration that completely inhibits bacterial growth in the small wells as MIC. The test is meaningful only when bacteria in the positive control well 12 (i.e., without antibiotics) grow significantly and in the 11th well (sterile) there is no growth. When a single jump well appears in the microdilution method, the highest drug concentration that inhibits bacterial growth should be recorded. If multiple jump wells appear, the results should not be reported and the test should be repeated. The test should be repeated 3 times for each drug.

[0084] (6) Results: The effective minimum inhibitory concentration of betaine is 4-32 μg / mL, and that of shikonin is 8-32 μg / mL, indicating that the addition of betaine and shikonin can improve the antibacterial effect. The results are shown in Table 5. The antibacterial effect of the Chinese medicinal ointments prepared in Examples 1-4 was tested and the texture was adjusted. The antibacterial effects are shown in Tables 5-8.

[0085] First, whether the Chinese medicine was extracted with alcohol or extracted with alcohol first and then with oil, the antibacterial effect of the ointment was poor (Table 6). Next, the water extraction method was used instead. The comparison of the drug sensitivity results of the two mixtures in Example 2 showed that the antibacterial effect of the mixture of shikonin: betaine: Chinese medicine water extract was better when the ratio was 1:10:1 than when it was 1:6:5 (Table 7). In Example 3, it was found that the antibacterial effect was not significantly affected by not adding the high-cost shikonin (Table 8). In addition, while maintaining the antibacterial effect, an appropriate amount of gelatin was added to adjust the hardness and viscosity of the ointment. The three adjustment ratios are shown in Table 1, and are shown in Table 1. Figure 1 It can be seen that when the ratio of camellia oil: beeswax: betaine, gelatin slurry: water-extracted freeze-dried powder is 5:2:10:4:1, the paste is harder, when the ratio is 5:1:10:2:1, the softness is improved but the viscosity is still not enough, and when the ratio is 2:1:10:2:1, the softness and viscosity are optimal, which is suitable for use.

[0086] Taking into account the antibacterial effect, preparation cost, softness and viscosity of the ointment, the above measurement results show that the best overall embodiment of the present invention is Example 4. The Chinese medicine ointment prepared according to the ratio of betaine, gelatin slurry: freeze-dried powder of water extract = 10:2:1, mixture: camellia oil: beeswax = 13:2:1 has the best effect, with an effective minimum inhibitory concentration of 8 to 64 μg / mL (Table 9), while the minimum inhibitory concentration of traditional lithospermum ointment is >2048 μg / mL (Table 10), and the antibacterial effect is significantly improved.

[0087] Table 5. Minimum inhibitory concentrations of betaine and shikonin (μg / mL)

[0088]

[0089] Table 6. Minimum inhibitory concentration (μg / mL) of ointments prepared by different Chinese medicine extraction methods in Example 1 against various strains

[0090]

[0091] Table 7. Minimum inhibitory concentration (μg / mL) of the mixture of shikonin: betaine: Chinese herbal medicine aqueous extract in Example 2

[0092]

[0093] Table 8. Minimum inhibitory concentration (μg / mL) of the mixture containing shikonin and before and after adding gelatin in Examples 2, 3, and 4

[0094]

[0095] Table 9. Minimum inhibitory concentration of the ointment in Example 4 (μg / mL)

[0096]

[0097] Table 10. Minimum inhibitory concentration (μg / mL) of Chinese medicinal ointment prepared in Examples 1 to 4 and purchased Lithospermum officinale ointment

[0098]

[0099] 2.3 Cytotoxicity testing of Chinese herbal ointments

[0100] (1) Prepare GES-1 cell suspension and adjust the concentration to 2×10 4 .

[0101] (2) Inoculate into a 96-well plate: 100 μL per well, and make three replicate wells with the same sample.

[0102] (3) Culture in a 37°C incubator for 24 hours.

[0103] (4) Experimental and control groups were set up, and the same volumes of Chinese herbal ointment, lithospermum officinale ointment, and DMSO solution were added, with working concentrations of 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 0 μg / mL, respectively. A pure culture medium group was also set up.

[0104] (5) Culture in a 37°C incubator for 24 hours.

[0105] (6) Add 10 μL of MTT to each well, tap gently to mix, and then incubate for 4 hours.

[0106] (7) Measure the absorbance at 450 nm and calculate the survival rate according to the formula: cell survival rate = [(As - Ab)] / [(Ac - Ab)] × 100%, where As represents the wells containing cell culture medium, drug, and MTT; Ac represents the wells containing cell culture medium, MTT, but no drug; and Ab represents the wells containing only culture medium and MTT but no cells or drug. Construct a survival curve based on the survival rates.

[0107] (8) Results: The MIC of Chinese herbal ointment was 1 to 32 times that of GES-1 cells, with low toxicity and high safety. Figure 2 shown.

[0108] 2.4 Effect of Chinese herbal ointment on skin infection and wound recovery in mice

[0109] 2.4.1 Construction of mouse skin and soft tissue infection model

[0110] 1. Grouping of experimental mice and administration method: The experimental animals were divided into a non-trauma group and a trauma group (two groups, infected with the sensitive strain Newman and the resistant strain USA300, respectively). The trauma group was further divided into a mupirocin ointment group, a lithospermum ointment group, and a traditional Chinese medicine ointment group, with 6 mice in each group, for a total of 7 groups.

[0111] (1) Non-traumatic experimental group: Chinese medicine ointment was applied to the back skin to observe the effect of the drug on the skin surface;

[0112] (2) Mupirocin ointment group (positive control): Mupirocin ointment was used to treat wound infection, twice a day, with an interval of 8 hours;

[0113] (3) Lithospermum officinale ointment group (negative control): Lithospermum officinale ointment was used to treat wound infection, with the drug administered twice a day, with an interval of 8 hours.

[0114] (4) Chinese medicine ointment group (experimental group): After wound infection, the Chinese medicine ointment of Example 4 was used for treatment, with administration twice a day, with an interval of 8 hours;

[0115] 2. Immunosuppressant Injection: Before the trauma infection experiment, mice were intraperitoneally injected with dexamethasone at a concentration of 1 mg / 5 mL, with a dose of 0.1 mL per mouse per injection, once every two days for a total of three injections. After the trauma infection, dexamethasone was administered again at the same dose and injection method for a total of three injections.

[0116] 3. Animal Hair Removal: Before the experiment, use a depilatory cream to remove hair from the back of the mouse. Use a disposable cotton swab to apply the cream to the mouse's back until the hair is completely wet. Leave it on for 3-5 minutes to allow the hair to fall off. Gently use a cotton swab to apply the cream until the hair is completely removed. Rinse off any excess depilatory with saline solution, avoiding scratching the skin. Observe the mouse's condition one day after depilation. Only proceed with trauma and infection experiments if the mouse is in good condition.

[0117] 4. Trauma infection experiment, specific treatment operations:

[0118] (1) Lithospermum officinale ointment group and Chinese medicine ointment group

[0119] Step 1: Mice were anesthetized and intraperitoneally injected with avertin at a dose of 0.2 mL / 10 g.

[0120] Step 2: After the mice are anesthetized, they are transferred to a clean bench. A circular wound with a diameter of 0.5-0.8 cm is cut on the dorsal spine of the mice in the trauma infection group using sterilized scissors, and excess blood in the wound is cleaned with sterile cotton balls.

[0121] Step 3: Prepare 1×10 9 CFU / mL of Staphylococcus aureus liquid was applied and injected into the wound in a volume of 30 μl to establish an infection model.

[0122] (2) Non-traumatic experimental group

[0123] This group did not receive trauma or infection treatment, but required the same dose of dexamethasone injection and back hair removal.

[0124] 2.4.2 In vivo therapeutic effect testing of Chinese herbal ointment

[0125] 1. Experimental treatment and observation indicators of trauma infection.

[0126] (1) Day 1 after trauma: Observe and record the wound healing and suppuration in each group.

[0127] (2) Drug administration: Each group began to receive drug administration on the second day after the injury, and applied the corresponding drugs on the wound site for treatment.

[0128] (3) Treatment period: The treatment lasted for 15 days, and the wound healing status was observed and recorded every day.

[0129] (4) Dissection: On the 16th day, mice in each group were dissected. A portion of the skin wound tissue was fixed with formaldehyde to prepare pathological sections, and the other portion was ground and plated to count the number of colonies.

[0130] (5) Observation indicators: Observe and record the wound healing status and scab removal time of each group of mice, count the amount of bacterial colonization in the skin wound tissue after dissection, and observe the degree of inflammation in the pathological sections.

[0131] 2. Statistical Analysis: All values ​​are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 26.0 software. One-way analysis of variance was used. Differences were considered statistically significant when p < 0.05. Graphs were prepared using GraphPad Prism 8 software.

[0132] 2..4.3 Results

[0133] (1) Wound healing: For mice infected with the sensitive Newman strain, the wound healing was best in the mupirocin ointment group, followed by the Chinese medicine ointment group. However, the scab shedding time of the Chinese medicine ointment group and the mupirocin ointment group was the same, both earlier than the lithospermum ointment group. For mice infected with the drug-resistant USA300 strain, the scab shedding time of the Chinese medicine ointment group and the mupirocin ointment group was also the same, but the number of scabs shed by the Chinese medicine ointment group was the highest, and the wound healing was the best, with a significantly better effect than the lithospermum ointment group. In addition, the surface skin of the mice in the non-trauma experimental group grew normally, and no allergic phenomena such as redness, swelling, and burns occurred. Wound healing is shown in Figure 3 , the scab removal time is shown in Table 11.

[0134] (2) Bacterial colonization of skin wound tissue: For mice infected with sensitive Newman strains, the Chinese medicine ointment group had the least bacterial colonization and the best antibacterial effect, which was slightly better than or equivalent to the mupirocin ointment group, and significantly better than the lithospermum ointment group (p value < 0.0001), and the difference was statistically significant; For mice infected with drug-resistant USA300 strains, the Chinese medicine ointment group also had the least bacterial colonization and the best antibacterial effect, which was more obvious than the mupirocin ointment group and the lithospermum ointment group (p value < 0.0001), and the difference was statistically significant. This shows that compared with the other two ointments, Chinese medicine ointment has a great advantage in inhibiting drug-resistant strains. Results are shown in Figure 4 , ns means p>0.05, *** means p<0.001, **** means p<0.0001.

[0135] (3) Degree of inflammation: The skin surface of normal mice was intact, without inflammation or edema; the skin wounds of mice in the lithospermum ointment group were incomplete, and severe inflammatory reactions occurred, with a large number of inflammatory cells infiltrating and gathering; the wounds of mice in the mupirocin ointment group and the Chinese medicine ointment group recovered relatively completely, without obvious inflammatory cell infiltration, and were no different from normal skin tissue. For skin wounds infected with drug-resistant strains, the Chinese medicine ointment group had a more significant therapeutic effect than the lithospermum ointment group. Results are shown in Figure 5 .

[0136] Table 11 The effect of Chinese medicinal ointment on the descabbing of skin wounds infected with Staphylococcus aureus

[0137]

[0138] The present invention is based on the research on the antibacterial effect of some Chinese herbal medicine extracts, and aims at the complexity and diversity of pathogens in skin and wound infections, and adds various formulas, which have good inhibitory effects on some bacteria and fungi that are easy to cause skin infections, and the combination of various medicines plays the effect of promoting blood circulation and promoting tissue regeneration, and jointly achieves the effect of promoting wound healing, and is better than traditional antibacterial ointments. In addition, the Chinese medicine ointment prepared by the various formulas has many effective antibacterial ingredients and action targets, which may not easily cause drug resistance. Compared with antibiotics, these Chinese herbal medicine ingredients are natural, mild in nature, and are not easy to cause allergies in patients when wiped on the skin. Some ingredients even have the effect of suppressing or allergic relief. Therefore, this novel Chinese medicine ointment solves some problems found in the treatment of skin and wound infections to a certain extent, has good development prospects, and lays an experimental foundation for studying the pharmacodynamic mechanism of this novel ointment.

Claims

1. A Chinese medicinal ointment for treating skin wound infection, characterized in that: It is made from the following raw materials by weight: 10g of lithospermum erythrorhizon, 1g of angelica sinensis, 1g of siler, 1g of rehmannia root, 1g of angelica dahurica, 1g of frankincense, 1g of myrrh, 10g of psoralea corylifolia, 1g of cinnamon bark, 1g of coptis chinensis, 1g of rhubarb, 1g of phellodendron amurense, 1g of mint, 55g of betaine, 5-6g of beeswax, 10-11g of gelatin and 10-11g of camellia oil.

2. The method for preparing the Chinese medicinal ointment according to claim 1, wherein The specific steps are as follows: Step 1: Grind lithospermum, angelica, siler, rehmannia, angelica root, frankincense, myrrh, psoralea corylifolia, cinnamon bark, coptis chinensis, rhubarb, phellodendron amurense, and mint into powder and coarsely sieve through a 100-mesh sieve; Step 2: Coarse water extraction: Put 10g lithospermum, 1g angelica, 1g siler, 1g rehmannia glutinosa, 1g angelica root, 1g frankincense, myrrh, 10g psoralea corylifolia, 1g cinnamon bark, coptis chinensis, 1g rhubarb, 1g phellodendron amurense, and 1g mint into a beaker, add water at a ratio of 1g:20mL, boil, and cook at a constant temperature three times, each time for 45min, 45min, and 30min respectively, filter with a 100-mesh sieve after each cooking, and then let it stand for 12h; Step 3: Filter the supernatant in the cup again, and the resulting liquid is packaged and freeze-dried; Step 4: Take 0.45g gelatin and add 45mL of warm water, soak for 5min to expand, and then heat in a water bath at 80℃ 15 minutes to dissolve it into slurry; fifth, weighing betaine, gelatin slurry and freeze-dried powder of water extract components in a ratio of 10:2:1 and mixing; sixth, weighing camellia oil and beeswax and heating them together until they melt at 60-80°C, then stopping heating, adding the mixture obtained in the previous step, stirring evenly, and the mixture: camellia oil: beeswax = 13:2:1, and cooling to obtain the traditional Chinese medicine ointment.

3. Use of the Chinese medicinal ointment prepared by the method according to claim 2 in preparing a Chinese medicinal ointment for treating skin wound infection.

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