External traditional Chinese medicine composition with bacteriostatic effect and preparation method thereof

Abstract

The invention provides an external traditional Chinese medicine composition with a bacteriostatic effect and a preparation method thereof, and belongs to the technical field of traditional Chinese medicine compositions. The traditional Chinese medicine composition comprises 20-40 parts of pepper, 20-40 parts of fructus cnidii, 10-30 parts of ligusticum, 5-15 parts of pogostemon cablin, 20-40 parts of schizonepeta, 10-30 parts of senecio scandens and 20-40 parts of mint. A response surface experiment design is adopted to optimize an extraction process of traditional Chinese medicinal materials and medicine residues, and the content of effective components such as total flavonoids in the extract is increased; the oil-soluble matrix and the water-soluble matrix are selected, so that the water-soluble medicine components and the oil-soluble medicine components can be better mixed, and the stability is high. The traditional Chinese medicine composition disclosed by the invention can inhibit the formation of bacterial cell membranes and destroy the structures of the bacterial cell membranes, and the permeability of the cell membranes is improved, so that macromolecular substances and proteins are leaked; the intracellular ATP content is reduced, and the extracellular alkaline phosphatase is increased, so that the growth of bacteria is finally inhibited or the bacteria are killed. According to the extraction process, effective components of the medicine are extracted to the maximum extent, and efficient utilization of the medicinal materials is guaranteed.

Description

Technical Field

[0001] This invention belongs to the technical field of antibacterial traditional Chinese medicine compositions, and specifically relates to an external traditional Chinese medicine composition with antibacterial effect and its preparation method. Background Technology

[0002] In clinical treatment of dermatitis, infectious skin diseases, and eczema, commonly used Western medicines mainly fall into four categories: First, corticosteroids (such as desonide cream and mometasone furoate gel), which are mostly used to quickly control inflammatory responses; second, antifungal drugs (such as ketoconazole cream and terbinafine cream), which target skin diseases caused by fungal infections; third, antibiotics (such as mupirocin ointment and fusidic acid cream), which are used to treat bacterial skin lesions; and fourth, antihistamines (such as loratadine tablets and cetirizine drops, some of which can be used topically), to relieve itching symptoms.

[0003] However, the aforementioned Western medicines have significant limitations and shortcomings: long-term use of glucocorticoids can easily lead to skin atrophy, pigmentation, and telangiectasia, and relapse is common after discontinuation; long-term or improper use of antifungal and antibiotic drugs can easily induce drug resistance in bacteria, leading to decreased effectiveness of subsequent treatments; antihistamines can only relieve itching symptomatically and cannot eradicate the root cause of skin inflammation or infection, and some patients may experience skin dryness, irritation, and other discomfort after use. Given the limitations of existing Western medicine treatments, developing topical traditional Chinese medicine antibacterial compositions that combine antibacterial activity and anti-inflammatory effects, and are highly safe for long-term use and less prone to drug resistance, has become an important direction for filling clinical treatment gaps and meeting the long-term medication needs of skin disease patients.

[0004] This study used a self-formulated traditional Chinese medicine compound as the research object, and employed orthogonal design to conduct compound analysis. Combined with in vitro antibacterial activity screening, the optimal compound composition and its optimal dosage ratio were determined. Then, using the characteristic antibacterial components of the optimal compound as indicators, the antibacterial mechanism was studied using the compound with optimized processing. Changes in the growth curves, cell membrane permeability, and bacterial cell contents of *S. aureus* and *E. coli* after compound treatment were detected. An antibacterial traditional Chinese medicine composition was developed, which possesses the effects of clearing heat and removing dampness, inhibiting bacteria and relieving itching, and drying dampness and killing parasites. Its antibacterial effect is better than that of single-herb traditional Chinese medicines; it helps to reduce drug dosage, toxicity, and bacterial resistance. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a topical traditional Chinese medicine composition with antibacterial effects and its preparation method, specifically including the following: A traditional Chinese medicine composition with antibacterial effect, comprising the following parts by weight: 20-40 parts of Sichuan pepper, 20-40 parts of Cnidium monnieri, 10-30 parts of Ligusticum striatum, 5-15 parts of Pogostemon cablin, 20-40 parts of Schizonepeta tenuifolia, 10-30 parts of Senecio scandens, and 20-40 parts of Mentha haplocalyx.

[0006] Preferably, a traditional Chinese medicine composition with antibacterial effect is composed of the following parts by weight: 30 parts of Sichuan pepper, 30 parts of Cnidium monnieri, 20 parts of Ligusticum striatum, 10 parts of Pogostemon cablin, 30 parts of Schizonepeta tenuifolia, 20 parts of Senecio scandens, and 30 parts of Mentha haplocalyx.

[0007] The present invention provides a method for preparing a traditional Chinese medicine composition with antibacterial effect, comprising the following steps: taking the traditional Chinese medicine composition in proportion, mixing it evenly, soaking it in water, extracting the volatile oil by steam distillation, extracting the residue by ethanol reflux to obtain an alcohol extract, and combining the two to prepare a mixed medicinal solution, thereby obtaining the product.

[0008] In the volatile oil preparation method of this invention, the soaking time is 20-40 min, the material-to-liquid ratio is 1:10-20, and the extraction time is 150-210 min.

[0009] Preferably, in the volatile oil preparation method, the soaking time is 33 min, the material-to-liquid ratio is 1:12, and the extraction time is 198 min.

[0010] In the ethanol reflux extraction of medicinal residues in this invention, the material-to-liquid ratio is 1:10-20, the extraction time is 60-180 min, and the extraction temperature is 80-100℃.

[0011] Preferably, the residue is extracted by reflux with 70%–80% ethanol, the material-to-liquid ratio is 1:12, the extraction time is 130 min, and the extraction temperature is 83℃.

[0012] The traditional Chinese medicine composition of the present invention, or the medicinal liquid prepared by the traditional Chinese medicine composition of the present invention using the preparation method of the present invention, can be supplemented with pharmaceutically acceptable excipients to prepare a pharmaceutically acceptable formulation.

[0013] The formulations that can be prepared by this invention are in the form of sprays, gels, emulsions or nanoemulsions, etc.

[0014] The application of the traditional Chinese medicine composition of the present invention in the preparation of antibacterial drugs.

[0015] The traditional Chinese medicines used in this invention and their effects are as follows: Sichuan pepper: This product is made from green pepper, a plant belonging to the Rutaceae family. Zanthorylum schinifolium Sieb. et Zucc. or Sichuan pepper Zanthorylum bungeanum The dried, ripe pericarp of Maxim. It is used to treat indigestion, abdominal pain due to cold, vomiting, hiccups, cough with shortness of breath, wind-cold-dampness syndrome, diarrhea, dysentery, hernia, toothache, ascariasis, pinworm infection, vulvar itching, and sores and scabies.

[0016] Cnidium monnieri: This product is Cnidium monnieri, a plant belonging to the Apiaceae family. Cnidium momieriThe dried, ripe fruit of (L.)Cuss. It is used to treat male impotence, scrotal eczema, female leukorrhea and vulvar eczema, infertility due to cold uterus, rheumatic pain, scabies, and eczema.

[0017] Ligusticum striatum: This product is Ligusticum striatum, a plant of the Apiaceae family. Ligusticum sinense Oliv. or Ligusticum striatum Ligusticum jeholense The dried rhizomes and roots of Nakai et Kitag. are used to treat headaches due to wind-cold, vertex headaches, abdominal pain due to cold-dampness, diarrhea, hernia, and scabies.

[0018] Patchouli: This product is patchouli, a plant belonging to the Lamiaceae family. Pogostemon cablin The dried aerial parts of (Blanco) Benth. It is used to treat abdominal distension and fullness due to dampness obstructing the middle jiao, loss of appetite, vomiting, diarrhea, headache due to summer-dampness and cold, fever and lethargy in the early stages of damp-heat syndrome, chest tightness and nausea, sinusitis, and tinea pedis and tinea manuum.

[0019] Catnip: This product is catnip, a plant of the Lamiaceae family. Schizonepeta tenuifolia The dried aerial parts of Brigq. It is used to treat colds with fever, headache, sore throat, stroke with lockjaw, hematemesis, epistaxis, hematochezia; metrorrhagia, postpartum hemorrhage; carbuncles, sores, scrofula. It has similar effects to Schizonepeta tenuifolia spikes, but with a stronger dispersing effect.

[0020] Senecio scandens: This product is Senecio scandens, a plant belonging to the genus Senecio in the family Asteraceae. Senecio scandens The dried aerial parts of Buch.-Ham. Used for wind-heat type common cold, red and swollen eyes, diarrhea and dysentery, and skin eczema and boils.

[0021] Peppermint: This product is peppermint, a plant belonging to the Lamiaceae family. Mentha haplocalyr The dried aerial parts of Brig. It disperses wind-heat, clears the head and eyes, soothes the throat, promotes rash eruption, and soothes the liver and regulates qi. It is used for wind-heat colds, early stages of wind-heat syndrome, headache, red eyes, sore throat, mouth ulcers, urticaria, measles, and chest and rib distension.

[0022] The core efficacy of this formula is to clear heat and remove dampness, inhibit bacteria and relieve itching. It is mainly used to treat eczema or bacterial skin infections caused by wind-heat mixed with dampness, simple damp-heat, or a mixture of cold and heat. Sichuan pepper (Zanthoxylum bungeanum) is the principal herb for drying dampness, relieving itching, warming and killing parasites. Cnidium monnieri (Synonyms) and Ligusticum striatum (Ligusticum striatum) are the assistant herbs for drying dampness, dispelling wind, killing parasites, relieving itching and pain, thus strengthening the principal herb's effect of drying dampness and killing parasites. Patchouli (Pogostemon cablin) and Schizonepeta tenuifolia (Schizonepeta tenuifolia) are the adjuvant herbs for dispelling wind, relieving itching, clearing heat and promoting rash eruption. Senecio scandens (Senecio scandens) and Mentha (Mentha haplocalyx) are the guiding herbs for clearing heat, detoxifying, and dispersing wind-heat, thus relieving skin redness, swelling, and itching caused by heat toxicity. The combined effects of these herbs are to clear heat and remove dampness, inhibit bacteria and relieve itching, and dry dampness and kill parasites. It can exert a good therapeutic effect on eczema, atopic dermatitis, contact dermatitis, bacterial folliculitis, and impetigo. Beneficial effects

[0023] 1. This invention uses orthogonal experiments to optimize the traditional Chinese medicine composition of Sichuan pepper, Cnidium monnieri, Ligusticum striatum, Pogostemon cablin, Schizonepeta tenuifolia, Senecio scandens, and Mentha haplocalyx, and optimizes their ratio and dosage. The extraction process of the traditional Chinese medicine materials is also adjusted in a targeted manner, which effectively exerts the antibacterial effect of the traditional Chinese medicine composition. The antibacterial traditional Chinese medicine composition liquid obtained by the preparation method of this invention has good antibacterial effect.

[0024] 2. Temperature, time, and the material-to-liquid ratio are all important factors affecting the extraction yield of flavonoids from medicinal residues. This invention significantly increases the content of effective components in the extract of medicinal residues from traditional Chinese medicine compositions by adjusting the material-to-liquid ratio and extraction time during the extraction process. The extraction process of this invention maximizes the extraction of effective components from the drugs, reduces energy consumption, and increases the yield of target substances. In industrial production, this reduces production costs and improves the efficient utilization of medicinal materials.

[0025] 3. The preparation method provided by this invention can efficiently extract medicinal materials without the need for separate extraction of each medicinal material, thus significantly improving the therapeutic effect of the traditional Chinese medicine composition.

[0026] 4. By selecting suitable oil-soluble and water-soluble matrices and controlling the mass ratio between components, this invention enables water-soluble and oil-soluble drug components to mix better and significantly improves the stability of the composition. Attached Figure Description

[0027] Appendix Figure 1 The graph shows the antibacterial results of the three dosage ratios of the traditional Chinese medicine composition. Appendix Figure 2 These are the results of a single-factor experiment on the extraction of volatile oils. Appendix Figure 3 3D model and contour plot for optimizing the process of volatile oil response surface methodology; Appendix Figure 4 For rutin and gallic acid standard curves; Appendix Figure 5 The effect of single-factor variables on the extraction yield of total flavonoids and total polyphenols from traditional Chinese medicine compositions; Appendix Figure 6 3D diagram and contour plot of the total flavonoid response surface methodology optimization process; Appendix Figure 7 The MIC results for the compound Sichuan pepper; Appendix Figure 8 The results of the MBC value of the traditional Chinese medicine composition; Appendix Figure 9 The effect of different concentrations of traditional Chinese medicine extracts on the growth curves of S. aureus (A) and E. coli (B); Appendix Figure 10 Crystal violet staining results for Staphylococcus aureus and Escherichia coli treated with traditional Chinese medicine extracts; Appendix Figure 11 Scanning electron microscope images of Staphylococcus aureus and Escherichia coli treated with traditional Chinese medicine extracts; Appendix Figure 12 Nucleic acid leakage in S. aureus (A) and E. coli (B) after treatment with extracts of traditional Chinese medicine compositions at different concentrations; Appendix Figure 13 Changes in extracellular protein concentrations of Staphylococcus aureus (A) and Escherichia coli (B) treated with traditional Chinese medicine compositions; Appendix Figure 14 Changes in intracellular ATP concentration in Staphylococcus aureus (A) and Escherichia coli (B) treated with a traditional Chinese medicine composition; Appendix Figure 15 Changes in extracellular AKP concentrations of Staphylococcus aureus (A) and Escherichia coli (B) treated with a traditional Chinese medicine composition. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0029] The raw materials of the traditional Chinese medicine composition with antibacterial effect in this embodiment are: 30 parts of Sichuan pepper, 30 parts of Cnidium monnieri, 20 parts of Ligusticum striatum, 10 parts of Pogostemon cablin, 30 parts of Schizonepeta tenuifolia, 20 parts of Senecio scandens, and 30 parts of Mentha haplocalyx. Example 2

[0030] The raw materials of the traditional Chinese medicine composition with antibacterial effect in this embodiment are: 20 parts of Sichuan pepper, 20 parts of Cnidium monnieri, 10 parts of Ligusticum striatum, 5 parts of Pogostemon cablin, 20 parts of Schizonepeta tenuifolia, 10 parts of Senecio scandens, and 20 parts of Mentha haplocalyx. Example 3

[0031] The raw materials of the traditional Chinese medicine composition with antibacterial effect in this embodiment are: 40 parts of Sichuan pepper, 40 parts of Cnidium monnieri, 30 parts of Ligusticum striatum, 15 parts of Pogostemon cablin, 40 parts of Schizonepeta tenuifolia, 30 parts of Senecio scandens, and 40 parts of Mentha haplocalyx. Example 4

[0032] The preparation method of the traditional Chinese medicine composition with antibacterial effect in this embodiment is as follows: The traditional Chinese medicine composition is mixed according to the specified ratio, soaked in water at a ratio of 1:12, and soaked for 33 minutes. Then, volatile oil is extracted by steam distillation for 198 minutes. The residue is then refluxed with 75% ethanol for 130 minutes to obtain an ethanol extract at a ratio of 1:12 and an extraction temperature of 83℃. The two extracts are combined to form a mixed medicinal solution, which is the final product. Example 5

[0033] The preparation method of the traditional Chinese medicine composition with antibacterial effect in this embodiment is as follows: The traditional Chinese medicine composition is mixed according to the specified ratio, soaked in water at a ratio of 1:10, and soaked for 20 minutes. Then, volatile oil is extracted by steam distillation for 150 minutes. The residue is then refluxed with 70% ethanol for 60 minutes to obtain an ethanol extract at a ratio of 1:10 and an extraction temperature of 80℃. The two extracts are combined to form a mixed medicinal solution, which is the final product. Example 6

[0034] The preparation method of the traditional Chinese medicine composition with antibacterial effect in this embodiment is as follows: The traditional Chinese medicine composition is mixed according to the specified ratio, soaked in water at a ratio of 1:20, and soaked for 40 minutes. Then, volatile oil is extracted by steam distillation for 210 minutes. The residue is then refluxed with 80% ethanol for 180 minutes to obtain an ethanol extract at a ratio of 1:20 and an extraction temperature of 100℃. The two extracts are combined to form a mixed medicinal solution, which is the final product. Example 7

[0035] The formulations of Examples 1-3 were prepared according to the preparation methods of Examples 4-6. Example 8

[0036] The preparation method of the herbal composition spray with antibacterial effect in this embodiment is as follows: Take 92g of the raw materials of the herbal composition of the present invention, 50% ethanol, and Tween 80. Weigh an appropriate amount of the herbal materials, use water as the extraction solvent, extract for 2 hours at a material-to-liquid ratio of 1:8 to obtain essential oil; filter the residue, use 75% ethanol as the extraction solvent, extract for 2 hours at a material-to-liquid ratio of 1:10, filter to obtain the liquid, and concentrate to an extract with a relative density of 1.05-1.10; dissolve the extract in 50% ethanol to make the ethanol content 50%. First, add 1% Tween 80 and essential oil, and homogenize the two using a homogenizer to make them evenly mixed to obtain a premixed cosolvent and essential oil. Then, add the 50% ethanol extract in small amounts several times, homogenizing for 1-5 minutes while adding, and finally obtain a clear, non-layered mixed solvent, which is the Sichuan pepper compound antibacterial spray. Example 9

[0037] The preparation method of the traditional Chinese medicine composition gel with antibacterial effect in this embodiment is as follows: take 92g of the raw medicinal materials of the traditional Chinese medicine composition of the present invention, 1% β-cyclodextrin, 1% carbomer 940, and 7% glycerin. Weigh an appropriate amount of the formulated medicinal materials, extract with water as the extraction solvent at a material-to-liquid ratio of 1:8 for 2 hours to obtain essential oil; filter the residue, extract with 75% ethanol as the extraction solvent at a material-to-liquid ratio of 1:10 for 2 hours, filter to obtain the medicinal liquid, and concentrate to a relative density of 1.05-1.10 extract; take an appropriate amount of β-cyclodextrin, add 10 times the amount of purified water to dissolve and add the collected volatile oil, stir and remove, then refrigerate at 4 ℃ for 24 hours to form a stable volatile oil-β-cyclodextrin inclusion complex for later use; weigh 1% of the total gel mass of carbomer 940, evenly sprinkle it in an appropriate amount of purified water (50-80 ml), and let it stand at room temperature for 12 hours; adjust the pH of the above swelling solution to 6.0 with an appropriate amount of sodium hydroxide (NaOH) solution, stir evenly to obtain phase I; weigh 7% of the total gel mass. Glycerol was added to all the volatile oil inclusion complexes and an appropriate amount of compound Sichuan pepper extract and stirred until completely dissolved to obtain phase II. Phase II was then slowly added to phase I and stirred thoroughly until the system was homogeneous and free of lumps, thus obtaining the Sichuan pepper compound antibacterial gel. Example 10

[0038] The preparation method of the traditional Chinese medicine composition emulsion with antibacterial effect in this embodiment is as follows: Take 92g of the raw materials of the traditional Chinese medicine composition of the present invention, 6%-15% decanoic acid triglyceride, 0.5-3% glyceryl monostearate, 80% g deionized water, 4-7% glycerol, 0.1% ethylparaben, and 0.5-1% triethanolamine. Weigh an appropriate amount of the formulation materials, use water as the extraction solvent, extract for 2 hours at a material-to-liquid ratio of 1:8 to obtain essential oil; filter the residue, use 75% ethanol as the extraction solvent, extract for 2 hours at a material-to-liquid ratio of 1:10, filter to obtain the liquid, and concentrate to an extract with a relative density of 1.05-1.10; prepare an oil phase premix of decanoic acid triglyceride, glyceryl monostearate, and the extract essential oil; add glycerol to deionized water, stir to dissolve, and heat to 75-80℃. Heat the oil phase to the same temperature as the aqueous phase (75-80℃), and slowly pour it into the aqueous phase while shearing it with a high-speed shear emulsifier for 20 minutes (10000 r / min). Then, homogenize it 2-3 times in a high-pressure homogenizer (20-25 MPa) to ensure uniform droplet size. Cool the homogenized emulsion to below 40℃, add triethanolamine to adjust the pH, then add preservatives and fragrances, and stir at low speed for 15 minutes to mix thoroughly. Package and store. Example 11

[0039] The preparation method of the traditional Chinese medicine composition nanoemulsion with antibacterial effect in this embodiment is as follows: Take 92g of the raw materials of the traditional Chinese medicine composition of this invention, 1.87% Tween-80, 8.13% Span-80, 5% ethanol, and 0.1% ethylparaben. Weigh an appropriate amount of the formulated medicinal materials, use water as the extraction solvent, extract for 2 hours at a material-to-liquid ratio of 1:8 to obtain essential oil; filter the residue, use 75% ethanol as the extraction solvent, extract for 2 hours at a material-to-liquid ratio of 1:10, filter to obtain the medicinal liquid, and concentrate to an extract with a relative density of 1.05-1.10; weigh the above-mentioned Tween-80, Span-80, and anhydrous ethanol, add deionized water preheated to 60°C, stir the above aqueous phase at 1000 r / min for 5 min, and slowly add an appropriate amount of the essential oil and ethanol mixture. Subject the obtained crude emulsion to high-pressure microjet four times to obtain the final product.

[0040] Experimental example: 1. Materials 1.1 Main Materials and Reagents Nutrient agar (NA), Guizhou Wosenda Biotechnology Co., Ltd.; Escherichia coli (BNCC133264), Yunnan Denglou Biotechnology Co., Ltd., Staphylococcus aureus (BNCC337371) Yunnan Denglou Biotechnology Co., Ltd.); Anhydrous ethanol, Guiyang Yunyan District Yibaiyuan Experimental Instrument Store; Nutrient Broth (NB), Guizhou Wosenda Biotechnology Co., Ltd.; 90mm petri dish, Guizhou Wosenda Biotechnology Co., Ltd.; Sichuan pepper sample collected from Wanjin Sichuan Pepper Planting Professional Cooperative, Dejiang County, Guizhou Province, identified by Professor Qin Ronggui of the School of Pharmacy, Guizhou Medical University as dried mature pericarp of green pepper; Cnidium monnieri, Ligusticum striatum, Pogostemon cablin, Schizonepeta tenuifolia, Senecio scandens, Mentha haplocalyx, Coptis chinensis, Phellodendron chinense, Sophora flavescens, Scutellaria baicalensis, Saposhnikovia divaricata, Artemisia argyi, Zhong Zhanming Traditional Chinese Medicine Store, Nanming District, Guiyang City; Gallic acid standard, Beijing Solarbio Technology Co., Ltd.; Rutin standard, Chengdu Plant Standardization Pure Biotechnology Co., Ltd.; Alkaline phosphatase (ALP) assay kit, Nanjing Jiancheng Technology Co., Ltd.; BCA protein concentration assay kit, Beijing Solarbio Technology Co., Ltd.; Crystal violet staining solution, Wuhan Sewell Biotechnology Co., Ltd.; ATP content assay kit, Nanjing Jiancheng Technology Co., Ltd.

[0041] 1.2 Main Instruments and Equipment Cryogenic coolant circulation pump (DLSB-5 / 20B), Zhengzhou Changcheng Science & Industry Trade Co., Ltd.; Diaphragm pump (PC101NT), Shanghai Ailang Instrument Co., Ltd.; Oil bath (OSB-2200), Shanghai Ailang Instrument Co., Ltd.; Intelligent magnetic stirrer (ZNCL-GS190*90), Gongyi Yuhua Instrument Co., Ltd.; Refrigerated centrifuge (GL-20G-C), low-speed centrifuge (TDL-40C), centrifuge (TGL-16B), Shanghai Anting Scientific Instrument Factory; Zhicheng Constant Temperature Incubator Shaker (ZWY-2102), Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; Vertical High Pressure Steam Sterilizer (LDZX-50L), Shanghai Shenan Medical Instrument Factory; Hot Air Drying Equipment (BGZ-240), Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; Double-person Double-sided Clean Workbench (SW-CJ-2F), Suzhou Purification Equipment Co., Ltd.; Biochemical Incubator (SPX-100B-Z), Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory.

[0042] 2. Method 2.1 Orthogonal Experimental Design for Screening Traditional Chinese Medicine An orthogonal experimental design was used to screen and analyze an antibacterial compound (formula: Sichuan pepper, Cnidium monnieri, Sophora flavescens, Ligusticum striatum, Pogostemon cablin, Coptis chinensis, Scutellaria baicalensis, Mentha haplocalyx, Schizonepeta tenuifolia, Saposhnikovia divaricata, Phellodendron chinense, Senecio scandens, and Artemisia argyi, each herb being mixed in equal proportions, with each herb used in 10g doses). The size of the inhibition zone against Staphylococcus aureus and Escherichia coli was used as the evaluation index. Based on a 13-factor (corresponding to 13 herbs) 2-level (Level 1: adding the herb; Level 2: not adding the herb) experimental design, an L16(2) statistical analysis was constructed using SPSS Statistics 27.0 software. 13 Orthogonal array 1.

[0043] Table 1. Orthogonal experimental design of Sichuan pepper compound preparation Experiment No. Sichuan peppercorn Cnidium monnieri Sophora flavescens Gao Ben Patchouli Coptis chinensis Scutellaria baicalensis Mint Catnip windproof Huangbai Senri-ko mugwort 1 0 0 1 0 0 1 1 0 0 1 1 1 0 2 1 1 1 0 1 0 1 0 1 0 1 0 0 3 0 1 0 0 0 0 1 0 1 0 0 1 1 4 0 0 0 1 1 0 0 0 1 1 1 0 0 5 1 1 0 1 0 1 0 0 0 0 1 1 0 6 0 1 1 0 1 0 0 1 0 1 0 1 0 7 1 1 1 1 1 1 1 1 1 1 1 1 1 8 0 1 1 1 1 1 0 0 0 0 0 0 1 9 1 1 0 0 0 0 0 1 0 1 1 0 1 10 0 0 0 0 1 1 0 1 1 0 1 1 1 11 1 0 1 1 0 0 0 0 1 1 0 1 1 12 1 0 0 1 1 0 1 1 0 0 0 1 0 13 1 0 1 0 0 1 0 1 1 0 0 0 0 14 0 1 0 1 0 1 1 1 1 1 0 0 0 15 0 0 1 1 0 0 1 1 0 0 1 0 1 16 1 0 0 0 1 1 1 0 0 1 0 0 1 2.2 Extraction of volatile oils

[0044] According to the compound formulation ratio in Table 1 of the orthogonal experimental design, weigh the medicinal materials, add deionized water at a material-to-liquid ratio of 1:10 (g / mL), steam distill for 2 hours, separate and collect the volatile oil, and store it at 4℃ in the dark for later use. 2.3 Preparation of alcohol extract from medicinal residue

[0045] After the volatile oil was extracted, 75% ethanol was added to the residue and refluxed at 100℃ for 1.5 h. After extraction, the residue was removed by filtration. The extract was concentrated under reduced pressure to a crude drug concentration of 1 g / mL. The extract was then filtered to remove insoluble impurities and the test ethanol extract was obtained and stored at 4℃ for later use. 2.4 In vitro antibacterial test

[0046] Frozen *Escherichia coli* and *Staphylococcus aureus* strains were thawed, and 50 μL of each bacterial suspension was added to freshly prepared NB medium and incubated at 37°C with shaking for 24 h. The revived bacteria were then inoculated onto fresh NA medium using the streak plate method and incubated at 37°C for 12 h. Single colonies were picked and placed in NB medium for further incubation. The turbidity of the bacterial suspension was adjusted to approximately 0.5 (1.0 × 10⁻⁶) using a McFarland turbidimeter. 5 Bacterial suspensions (CFU / mL) were administered. The antibacterial experiment was conducted using the filter paper disc method. 100 μL each of *Escherichia coli* and *Staphylococcus aureus* suspensions were evenly spread onto NA medium, and paper discs containing the drug solution were attached. 50% ethanol was used as a blank control, and 1.25 mg / mL cefuroxime sodium was used as a positive control. After administration, the medium was incubated at 37°C for 24 h. Each group was repeated three times, and the diameter of the inhibition zone was measured. 2.5 Selection of the optimal formulation

[0047] Using the size of the inhibition zone from three repeated experiments as an indicator (the size of the inhibition zone of the ethanol extract was used as the inhibition value in the orthogonal experiment for the formulation without volatile oil), range analysis was performed using the Spssau data analysis platform to screen out the optimal antibacterial compound. 2.6 Dosage screening for the optimal formulation

[0048] SPSS Statistics 27.0 software was used to establish an orthogonal array based on the type of medicinal materials (the medicinal materials in the optimal compound) at three levels (level 1: 10g; level 2: 15g; level 3: 20g). The medicinal materials were weighed according to the orthogonal array. The size of the inhibition zone of the mixture of volatile oil and alcohol extract in each dosage group was used as an indicator. Range analysis was performed using the SPSSau data analysis platform to screen the optimal dosage ratio of medicinal materials.

[0049] Table 2 Orthogonal experimental design for screening the dosage of Sichuan pepper compound preparations. serial number Sichuan peppercorn Cnidium monnieri Gao Ben Patchouli Catnip Senri-ko Mint 1 3 3 1 1 2 2 2 2 1 1 1 1 1 1 1 3 3 2 1 3 1 1 3 4 2 1 1 2 3 2 3 5 3 1 2 2 3 1 2 6 1 3 2 2 1 2 1 7 2 3 1 3 3 3 1 8 3 2 2 1 3 3 1 9 1 1 2 3 2 3 3 10 1 2 3 1 3 2 3 11 3 3 3 2 1 3 3 12 2 1 3 1 1 3 2 13 1 3 3 3 3 1 2 14 3 1 3 3 2 2 1 15 2 2 3 2 2 1 1 16 1 2 1 2 2 3 2 17 2 2 2 3 1 2 2 18 2 3 2 1 2 1 3 2.7 Optimization of the extraction process for the optimal formulation

[0050] 2.7.1 Single-factor experiment on volatile oil The medicinal materials were weighed according to the compound dosage ratio, and deionized water was added at a certain material-to-liquid ratio. After soaking for an appropriate time, the materials were placed in an oil bath and heated for a certain time before filtration. The effects of different soaking times (0, 10, 20, 30, 40 min), material-to-liquid ratios (1:5, 1:10, 1:15, 1:20, 1:25), and extraction times (120, 150, 180, 210, 240 min) on the yield of volatile oil were investigated using the controlled variable method. The following experiments were conducted under the extraction conditions corresponding to the maximum volume of volatile oil.

[0051] 2.7.2 Response Surface Methodology Experiment for Volatile Oil Extraction Based on single-factor analysis, with volatile oil extraction rate (Y) as the response value and soaking time (A), material-liquid ratio (B), and extraction time (C) as the variables to be examined, a three-factor, three-level response surface methodology was designed according to the Box-Behnken design principle. The experimental design is shown in Table 3, and the optimal extraction process for volatile oil was determined.

[0052] Table 3. Factors and Levels in the Box-Behnken Experiment Soaking time A / h Feed-to-liquid ratio B / ℃ Extraction time C -1 20 1:10 150 0 30 1:15 180 1 40 1:20 210 2.7.3 Single-factor test for alcohol extraction 2.7.3.1 Construction of the total flavonoid standard curve Accurately pipette 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of 1 mg / mL rutin standard solution, and sequentially add 0.5 mL of 5% NaNO₂ solution. Shake well and let stand for 6 min. Then, sequentially add 0.5 mL of 10% Al(NO₃)₃ solution, shake well, and let stand for 6 min. Add 2 mL of 4% NaOH solution to each solution, shake well, and dilute to 10 mL with water. Shake well and let stand for 15 min. Measure the absorbance at 510 nm using a 1 cm cuvette with 30% ethanol solution as a blank. Plot a standard curve with rutin content (mg / mL) on the x-axis and absorbance on the y-axis. Accurately pipette 1.0 mL of the sample solution to be tested, and follow the same steps. Calculate the flavonoid content based on the standard curve.

[0053] 2.7.3.2 Determination of Polyphenol Content Accurately measure 0.0, 20, 40, 60, 80, and 100 μL of 0.5 mg / mL gallic acid stock solution into 15 mL centrifuge tubes, and dilute to 200 μL with 50% ethanol. Add distilled water to 4 mL, shake well, add 1.0 mL of Folin-Ciocalteu solution to each tube, shake well, and let stand for 3 min. Then, add 1.8 mL of 20% NaCO3 solution, shake well, and let stand in the dark for 2 h. For the blank control, replace 1.8 mL of NaCO3 with 1.8 mL of distilled water. Measure the absorbance at 760 nm and plot a standard curve with gallic acid content (mg / mL) on the x-axis and absorbance on the y-axis. The sample determination should be performed in parallel with the standard.

[0054] 2.7.3.3 Investigate the effects of extraction time, extraction temperature, and solid-liquid ratio on the total flavonoid and total polyphenol content and antibacterial effect. According to the compound dosage ratio, appropriate amounts of medicinal materials were weighed, and the volatile oil was extracted and collected under the optimal extraction conditions. The residue was then heated with ethanol at a specific material-to-liquid ratio for a certain period before filtration. The effects of different extraction times (1, 2, 3, 4, 5, 6 h), extraction temperatures (60℃, 70℃, 80℃, 90℃, 100℃, 110℃), and material-to-liquid ratios (1:5, 1:10, 1:15, 1:20, 1:25, 1:30) on the total flavonoid and total polyphenol content were investigated using a controlled variable method. The filtered medicinal solutions from each experimental group were concentrated at 45℃ to a concentration of 1 g / mL of raw medicinal material. 0.2 mL of the ethanol extraction solution was accurately pipetted into a volumetric flask, and the absorbance was measured using the same methods as in steps 2.7.3.1 and 2.7.3.2. The total flavonoid and total polyphenol content were calculated based on the standard curve. The antibacterial effect of each experimental group was determined through an antibacterial test.

[0055] 2.7.3.4 Correlation analysis and linear regression analysis of total flavonoids, total polyphenols content and antibacterial value in single-factor experiments Based on the inhibition zone data, total flavonoid and total polyphenol content data of the extracts measured under each condition of the single-factor experiment, Spearman correlation analysis and linear regression analysis were performed using SPSS Statistics 27.0 software. The factor indicators of the response surface were determined in combination with the analysis results.

[0056] 2.7.4 Response Surface Design Based on the single-factor experiments, with flavonoid content (Y) as the response value and time (A), temperature (B), and material-liquid ratio (C) as the variables to be examined, a three-factor, three-level response surface methodology was designed according to the Box-Behnken design principle. The experimental design is shown in Table 4, and the optimal flavonoid extraction process was determined.

[0057] Table 4. Factors and Levels in the Box-Behnken Experiment Time A / h Temperature B / ℃ Material-to-liquid ratio C -1 1 80 1:10 0 2 90 1:15 1 3 100 1:20 2.8 Study on antibacterial mechanism

[0058] 2.8.1 Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the compound preparation of Sichuan pepper The bacterial cultures of *S. aureus* and *E. coli* cultured to the logarithmic growth phase were diluted with sterile physiological saline to a concentration of 10:1. 6Prepare CFU / mL bacterial suspensions. Using the two-fold dilution method in a 96-well plate, dilute the drug twice with sterile NB liquid medium to different concentrations (0.5, 0.25, 0.125, 0.0625, 0.015625, 0.0078125 mg / mL), and set up a blank control group. Then, add 50 μL of bacterial suspension and incubate at 37℃ and 180 r / min for 12 h. The concentration at which no bacterial growth is observed visually is the minimum inhibitory concentration (MCC). Take 100 μL of bacterial suspension from the two wells before and after the point where no bacterial growth is observed, spread it onto NB medium, and incubate at 37℃ for 12 h. The minimum concentration at which no colonies grow is considered the minimum inhibitory concentration (MBC).

[0059] 2.8.2 Determination of bacterial growth curve The cultured Staphylococcus aureus and Escherichia coli cultures in the logarithmic growth phase were diluted with sterile physiological saline to a concentration of 10:1. 6 Prepare bacterial suspensions at CFU / mL. Add 100 μL of Staphylococcus aureus and Escherichia coli to 96-well plates containing drug concentrations of 1 / 4 MIC, 1 / 2 MIC, MIC, and 2 MIC, respectively, and incubate for 24 h. Incubate every 2 h at A... 600 Measure the absorbance value and plot the growth curve with time on the x-axis and absorbance on the y-axis.

[0060] 2.8.3 Crystal Violet Staining Experiment The bacterial cultures of *S. aureus* and *E. coli* cultured to the logarithmic growth phase were diluted with sterile physiological saline to a concentration of 10:1. 6 Prepare bacterial suspensions at CFU / mL. Add the two types of bacteria to the compound drug solution to achieve concentrations of 1 / 2 MIC, MIC, and 2 MIC, respectively. The untreated control serves as a blank control. Pipette 200 µL of each solution into 96-well plates, with three replicates per group. Incubate statically at 37 °C for 6 hours to ensure complete biofilm adhesion to the plate bottom. Discard the supernatant, discard the planktonic cells, wash the plates three times with 200 µL PBS, fix with methanol for 20 minutes, wash three times with water, stain with 1% (w / v) crystal violet for 10 minutes, and rinse three times. Finally, add 200 µL of 95% ethanol to the 96-well plate to dissolve the bound crystal violet, and stain on plate A. 590 The amount of biofilm formed is measured by measuring the absorbance value.

[0061] 2.8.4 Scanning Electron Microscopy Staphylococcus aureus and Escherichia coli were cultured to the logarithmic growth phase and resuspended in sterile physiological saline to prepare bacterial suspensions (1×10⁻⁶). 6Bacterial suspension (CFU / mL) was added to 15 mL sterile centrifuge tubes, followed by the addition of compound antibacterial solution to achieve a concentration of 1 MIC. The untreated group served as the blank control. The tubes were incubated at 37 ℃ with shaking for 6 h. Bacterial cells were collected by centrifugation, washed three times with sterile physiological saline, and fixed overnight in electron microscopy fixative at 4 ℃. After washing three more times with sterile physiological saline, the cells underwent graded ethanol dehydration, freeze-drying, gold sputtering, and morphological observation under a scanning electron microscope. The physiological saline group served as the control group.

[0062] 2.8.5 Effects of Sichuan pepper compound on cell membrane permeability of S. aureus and E. coli S. aureus and E. coli were cultured to the logarithmic growth phase for later use. Every half hour over a 2-hour period, bacterial culture was collected and centrifuged in centrifuge tubes at 4°C and 10,000 r·min. -1 Centrifuge at high speed for 10 minutes. Measure the supernatant at A... 260 The absorbance at a certain point is used to detect the leakage of nucleic acid from Staphylococcus aureus cells.

[0063] 2.8.6 Determination of Extracellular Protein Concentrations in S. aureus and E. coli by Compound Preparation of Sichuan Pepper The compound preparations of Sichuan pepper at concentrations of 1 / 2 MIC and 1 MIC were added to suspensions of *S. aureus* and *E. coli* during their logarithmic growth phase, respectively. Sterile water was added to the control group. The mixtures were incubated in a constant-temperature shaker at 37 °C for 2 h. Samples were taken at timed intervals of 0, 30, 60, 90, and 120 min at 5000 r·min. -1 Centrifuge for 10 min and collect the supernatant. Use the BCA protein quantification kit to determine the soluble protein content in the supernatant.

[0064] 2.8.7 Effects of Sichuan pepper compound on intracellular adenosine triphosphate (ATP) content in S. aureus and E. coli The compound preparations of Sichuan pepper at concentrations of 1 MIC and 2 MIC were added to suspensions of *S. aureus* and *E. coli* in their logarithmic growth phase, respectively, while sterile water was added to the control group. The mixtures were incubated in a constant-temperature shaker at 37 °C for 5 h, with samples taken at 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h at 4 °C and 10000 r·min. -1 Centrifuge for 10 min and collect the supernatant. Determine the ATP content in the supernatant according to the ATP content kit instructions.

[0065] 2.8.8 Determination of the extracellular alkaline phosphatase (AKP) content in *S. aureus* and *E. coli* by a compound preparation of Sichuan pepper Take the bacterial supernatant from 2.8.7 and determine the AKP content in the supernatant according to the alkaline phosphatase (AKP) content kit.

[0066] 2.8.9 Data Statistics and Analysis The experimental data were organized using Excel, and one-way ANOVA was performed using SPSS Statistics 27.0. GraphPad Prism 9.5 software was used for plotting. The data are presented in ( ) x±s This indicates that a one-way ANOVA compares the means between groups and performs... Tukey's Multiple comparison tests, if p＞0.05 The difference was not statistically significant; if P<0.05 The difference was statistically significant. P<0.01 This indicates a highly significant difference.

[0067] 3. Results and Analysis 3.1 Results of orthogonal sieve experiments Table 5. Antibacterial results of the orthogonal sieve experiment Table 6. Range analysis results of orthogonal experiments on the inhibition of Escherichia coli by each formulation item Sichuan peppercorn Cnidium monnieri Sophora flavescens Gao Ben Patchouli Coptis chinensis Scutellaria baicalensis Mint Catnip windproof Huangbai Senri-ko mugwort K0 58.5 77.83 80.53 78.7 79.7 79.93 81.87 77.17 79.7 80.27 82.67 78.53 82.43 K1 101.13 81.8 79.1 80.93 79.93 79.7 77.77 82.47 79.93 79.37 76.97 81.1 77.2 k0 7.31 9.73 10.07 9.84 9.96 9.99 10.23 9.65 9.96 10.03 10.33 9.82 10.3 k1 12.64 10.23 9.89 10.12 9.99 9.96 9.72 10.31 9.99 9.92 9.62 10.14 9.65 Best level 1 1 0 1 1 0 0 1 1 0 0 1 0 R 5.33 0.5 0.18 0.28 0.03 0.03 0.51 0.66 0.03 0.11 0.71 0.32 0.65 Horizontal quantity 2 2 2 2 2 2 2 2 2 2 2 2 2 Number of repetitions per level 8 8 8 8 8 8 8 8 8 8 8 8 8 Table 7. Range analysis results of orthogonal experiments on the inhibition of Staphylococcus aureus by each formulation item Sichuan peppercorn Cnidium monnieri Sophora flavescens Gao Ben Patchouli Coptis chinensis Scutellaria baicalensis Mint Catnip windproof Huangbai Senri-ko mugwort K0 58.8 76.27 79.03 75.37 77.63 81.3 80.57 75.2 77.3 80.27 79.73 75.3 82.87 K1 97.7 80.23 77.47 81.13 78.87 75.2 75.93 81.3 79.2 76.23 76.77 81.2 73.63 k0 7.35 9.53 9.88 9.42 9.7 10.16 10.07 9.4 9.66 10.03 9.97 9.41 10.36 k1 12.21 10.03 9.68 10.14 9.86 9.4 9.49 10.16 9.9 9.53 9.6 10.15 9.2 Best level 1 1 0 1 1 0 0 1 1 0 0 1 0 R 4.86 0.5 0.2 0.72 0.15 0.76 0.58 0.76 0.24 0.5 0.37 0.74 1.15 Horizontal quantity 2 2 2 2 2 2 2 2 2 2 2 2 2 Number of repetitions per level 8 8 8 8 8 8 8 8 8 8 8 8 8 Based on the antibacterial results of each compound (Table 5) and the range analysis results (Tables 6 and 7), comparing the ranges reveals that the order of influence of medicinal factors on inhibiting *S. aureus* is: Sichuan pepper > Artemisia argyi > Coptis chinensis > Mentha haplocalyx > Senecio scandens > Ligusticum striatum > Scutellaria baicalensis > Saposhnikovia divaricata > Cnidium monnieri > Phellodendron chinense > Schizonepeta tenuifolia > Sophora flavescens > Pogostemon cablin. Considering both k-value and R-value, the optimal combination is Sichuan pepper, Cnidium monnieri, Ligusticum striatum, Pogostemon cablin, Schizonepeta tenuifolia, Senecio scandens, and Mentha haplocalyx. Similarly, analysis shows that the optimal combination of medicinal materials for inhibiting *E. coli* is Sichuan pepper, Cnidium monnieri, Ligusticum striatum, Pogostemon cablin, Schizonepeta tenuifolia, Senecio scandens, and Mentha haplocalyx.

[0068] 3.2 Dosage screening results for the optimal formulation Table 8 Results of the orthogonal experiment for screening the dosage of Sichuan pepper compound preparations Note: Level 1 is 10g, Level 2 is 15g, and Level 3 is 30g. Table 9. Range analysis results of the inhibitory effect of compound Sichuan pepper on Escherichia coli in each dosage group. item Sichuan peppercorn Cnidium monnieri Gao Ben Patchouli Catnip Senri-ko Mint <![CDATA[K1]]> 56.45 57.1 57.66 58.84 57.86 57.61 58.39 <![CDATA[K2]]> 56.61 58.51 58.8 57.15 57.24 58.79 57.11 <![CDATA[K3]]> 60.88 58.33 57.48 57.95 58.84 57.54 58.44 <![CDATA[k1]]> 9.41 9.52 9.61 9.81 9.64 9.6 9.73 <![CDATA[k2]]> 9.44 9.75 9.8 9.53 9.54 9.8 9.52 <![CDATA[k3]]> 10.15 9.72 9.58 9.66 9.81 9.59 9.74 Best level 3 2 2 1 3 2 3 R 0.74 0.24 0.22 0.28 0.27 0.21 0.22 Horizontal quantity 3 3 3 3 3 3 3 Number of repetitions per level r 6 6 6 6 6 6 6 Table 10. Range analysis results of the inhibitory effect of compound Sichuan pepper on Staphylococcus aureus in each dosage group. item Sichuan peppercorn Cnidium monnieri Gao Ben Patchouli Catnip Senri-ko Mint <![CDATA[K1]]> 56.07 58.34 58.92 59.11 59.39 59.16 58.93 <![CDATA[K2]]> 57.97 58.7 59.17 58.68 58.36 59.07 58.91 <![CDATA[K3]]> 62.91 59.91 58.86 59.17 59.2 58.72 59.11 <![CDATA[k1]]> 9.35 9.72 9.82 9.85 9.9 9.86 9.82 <![CDATA[k2]]> 9.66 9.78 9.86 9.78 9.73 9.85 9.82 <![CDATA[k3]]> 10.48 9.99 9.81 9.86 9.87 9.79 9.85 Best level 3 3 2 3 1 1 3 R 1.14 0.26 0.05 0.08 0.17 0.07 0.03 Horizontal quantity 3 3 3 3 3 3 3 Number of repetitions per level r 6 6 6 6 6 6 6 The antibacterial results for each dose are shown in Table 8, and the range analysis results are shown in Tables 9 and 10. The K value is the sum of the effects of each factor at each level, and the k value is the average value of K. The optimal level of a factor can be derived from the k value, that is, the level corresponding to the maximum k value is the optimal level of the factor in this experiment. The R value is the maximum value of k minus the minimum value of k. In the range analysis, the order of superiority and influence of each factor in the experiment can be judged by comparing the size of the R value. The larger the R value, the greater the influence of the factor on the experiment and the priority it has. Table 9 shows that the optimal levels of the compound Sichuan pepper for inhibiting E. coli are: Sichuan pepper 3, Cnidium monnieri 2, Ligusticum striatum 2, Pogostemon cablin 1, Schizonepeta tenuifolia 3, Senecio scandens 2, and Mentha haplocalyx 3, which is set as dose group A. According to the analysis of Table 10, the optimal levels of each herb in the compound Sichuan pepper for inhibiting S. aureus are: Sichuan pepper 3, Cnidium monnieri 3, Ligusticum striatum 2, Pogostemon cablin 3, Schizonepeta tenuifolia 1, Senecio scandens 1, and Mentha haplocalyx 3, which is set as dose group B. Comparing the R values ​​of the two groups, it can be seen that Sichuan pepper is in a dominant position in both dose groups A and B.

[0069] Because the dosage ratios derived from the inhibitory effects of compound Sichuan pepper on the two bacteria are inconsistent, a comprehensive consideration is needed when selecting the dosage ratio. Combining the two tables, it can be seen that the dosages of Cnidium monnieri, Pogostemon cablin, Schizonepeta tenuifolia, and Senecio scandens are different in A and B. R The magnitude of the R value can be used to infer the order of the factors' superiority or inferiority. Therefore, by comparing the R values ​​of these four herbs, the combination with the largest R value is selected to form the comprehensive dosage group C, which is: Sichuan pepper: Cnidium monnieri: Ligusticum striatum: Patchouli: Schizonepeta tenuifolia: Senecio scandens: Mentha haplocalyx = 3:3:2:1:3:2:3.

[0070] The herbal extracts were weighed according to three dosages (A, B, and C), and the antibacterial experiment was repeated three times. The results of the antibacterial experiment were recorded and analyzed using GraphPad Prism.9.5 software. The results are shown in Figure 1.

[0071] Note: Tukey's multiple comparison test is used to compare the means between groups. *P<0.05, **P<0.01 As shown in Figure 1, the inhibitory effect of compound Sichuan pepper dosage group B on Escherichia coli was not significantly different from that of the comprehensive dosage group C; however, A was significantly different from C. P<0.01 Furthermore, group C was more effective than group A. Additionally, the inhibitory effect of compound Sichuan pepper dosage group A on Staphylococcus aureus was not significantly different from that of group C, but it was significantly different from that of group B. P<0.01 Group C showed better results than Group B. Therefore, the optimal dosage ratio is: Sichuan pepper: Cnidium monnieri: Ligusticum striatum: Patchouli: Schizonepeta tenuifolia: Senecio scandens: Mentha haplocalyx = 3:3:2:1:3:2:3.

[0072] 3.3 Optimization Results of the Best Compound Volatile Oil Extraction Process 3.3.1 Results of Single-Factor Experiments on Volatile Oils As shown in Figure 2A, compared with the unsoaked group, the yield of volatile oil in the medicinal materials showed an increasing trend after soaking. With the extension of soaking time, the yield of volatile oil in the Sichuan pepper compound showed an overall trend of first increasing and then decreasing, with the maximum yield of volatile oil appearing when the soaking time was 30 minutes.

[0073] Under the condition of fixed soaking and extraction time, the volatile oils obtained with different material-to-liquid ratios are shown in Figure 2B. The yield of volatile oil from the Sichuan pepper compound generally showed a trend of first increasing and then decreasing, with the yield reaching a peak at a material-to-liquid ratio of 1:15.

[0074] As shown in Figure 2C, the yield of volatile oil from the Sichuan pepper compound generally showed an upward trend with the extension of extraction time. The yield of volatile oil reached its maximum when the extraction time was 240 min, and the increase in the yield of volatile oil slowed down when the extraction time was greater than 180 min.

[0075] 3.3.2 Results of Response Surface Methodology Experiments on Volatile Oils The response surface methodology (RSM) experiment for volatile oils used the yield of volatile oils as the indicator, and the analysis results were obtained using Design-Expert 13 software. The results are as follows: Table 11 ANOVA table of volatile oil response surface optimization process Source of variance sum of squares Degrees of freedom Mean Square F value p-value Significance Model 0.1621 9 0.018 64.96 <0.0001 significant A-Soaking Time 0.0044 1 0.0044 15.71 0.0054 ** B - Material-to-liquid ratio 0.0044 1 0.0044 15.71 0.0054 ** C - Extraction Time 0.0089 1 0.0089 32.05 0.0008 ** AB 2.78E-06 1 2.78E-06 0.01 0.923 AC 0.0001 1 0.0001 0.2503 0.6322 BC 0.0017 1 0.0017 6.26 0.0409 * A² 0.0149 1 0.0149 53.6 0.0002 *** B² 0.0669 1 0.0669 241.35 <0.0001 *** C² 0.0474 1 0.0474 170.86 <0.0001 *** Residual 0.0019 7 0.0003 Lack of Fit 0.0005 3 0.0002 0.4154 0.7518 not significant Pure Error 0.0015 4 0.0004 Cor Total 0.1641 16 The quadratic regression model equation with volatile oil yield (Y) as the objective function was obtained based on the analysis using Design Expert 13 software. Y=1.38+0.0233 A+0.0233B+0.0333C+ 0.0008AB-0.0042AC- 0.0208BC- 0.0594 A ²-0.1261B²-0.1061C² .

[0076] As shown in Table 11 of the ANOVA table: the model's P<0.000 1 Highly significant; lack of fit P>0.05 The result is not significant, indicating that the model is reliable. r 2 =0.9882 This indicates a high degree of agreement between the experimental data and the fitted function, and that the curve has a good linear relationship. r 2 adj=0.9730 This indicates that the model has high reliability and can be used for the extraction optimization of compound volatile oils. According to... F Value and P The values ​​show that the effects of each factor on the yield of volatile oil are as follows: C>A=B, That is, extraction time > material-to-liquid ratio = soaking time.

[0077] The 3D model was drawn using software, as shown in Figure 3. The optimal extraction results of the compound volatile oil were predicted and verified by experiments, as shown in Table 12 below.

[0078] Table 12 Results of Response Surface Methodology Prediction and Verification Experiments for Volatile Oils Evaluation indicators predict actual Soaking time / min 33.752 33.75 Material-liquid ratio 12.607 12.61 Extraction time / min 194.88 194.88 Volatile oil yield / % 1.333 1.30 3.4 Optimization of the best compound alcohol extraction process 3.4.1 Standard curves for rutin and gallic acid As shown in Figures 4A and 4B, the linear regression equation obtained by fitting the rutin standard is as follows: y = 12.387x - 0.0056 , r 2 = 0.9998 This indicates good linearity and high accuracy within the range of 0.00-0.1 mg / mL, making it suitable for the determination of flavonoids; the linear regression equation obtained by fitting the gallic acid standard is as follows. y = 171.61x + 0.0499 , r 2 = 0.9946 This indicates that the linearity is good and the accuracy is high in the range of 0.0000 mg / mL to 0.007285714 mg / mL, making it suitable for the determination of flavonoids.

[0079] 3.4.2 Results of Factors Related to Alcohol Bill of Lading 3.4.2.1 Effect of extraction time on the content of total flavonoids and total polyphenols As shown in Figures 5B and 5E, when the extraction time increased from 1 h to 2 h, the total flavonoid and total polyphenol contents showed an increasing trend. After 2 h, the total flavonoid content showed a decreasing trend, while the total polyphenol content first decreased and then stabilized. Therefore, response surface methodology was optimized at 2 h.

[0080] 3.4.2.2 Effect of extraction temperature on the content of total flavonoids and total polyphenols As shown in Figures 5C and 5F, when the extraction temperature increases from 60℃ to 90℃, the total flavonoid and total polyphenol contents increase significantly. After 90℃, the total flavonoid content gradually decreases, while the polyphenol content shows a decreasing trend and tends to stabilize. Therefore, 90℃ was selected for response surface optimization.

[0081] 3.4.2.3 Effect of the material-to-liquid ratio on the content of total flavonoids and total polyphenols As shown in Figures 5A and 5D, the lowest content of flavonoids and polyphenols was observed when the solid-liquid ratio was 1:5, possibly due to insufficient solvent and incomplete extraction. As the solid-liquid ratio increased, the flavonoid content first increased and then decreased. When the solid-liquid ratio reached 1:15, both flavonoid and polyphenol contents decreased. Therefore, a ratio of 1:15 was chosen for response surface methodology optimization.

[0082] 3.4.2.4 Correlation analysis and linear regression analysis of total flavonoids, total polyphenols content and antibacterial value in single-factor experiments The data on the inhibition zones, total flavonoids, and total polyphenols obtained from the extracts under various conditions were analyzed using Spearman correlation analysis and linear regression analysis with SPSS Statistics 27.0 software. The results are shown in Tables 13, 14, and 15 below: Table 13 Spearman correlation analysis results of total flavonoid and total polyphenol content with inhibition zone size average value Standard deviation Flavonoid content (mg / g) Polyphenol content (mg / g) Staphylococcus aureus / mm Escherichia coli / mm Flavonoid content (mg / g) 7.59 1.308 1 Polyphenol content (mg / g) 0.529 0.138 0.319 1 Staphylococcus aureus (mm) 9.5 5.339 0.734** 0.690** 1 Escherichia coli (mm) 9.5 5.339 0.868** 0.519* 0.783** 1 Table 14. Results of linear regression analysis on the antibacterial efficacy of total flavonoids and total polyphenols against Staphylococcus aureus. Table 15. Results of linear regression analysis on the antibacterial efficacy of total flavonoids and total polyphenols against Escherichia coli. Spearman correlation analysis showed that there were significant correlations between total flavonoid content, total polyphenol content, and inhibition zone diameter. P<0.05 The results indicate that these two types of components are important for the antibacterial activity of the ethanol extract. Linear regression analysis showed that the Beta value indicated that the influence of total flavonoids on the size of the inhibition zone (Beta=0.782) was significantly higher than that of total polyphenols (Beta=0.23). Therefore, total flavonoids are the dominant active ingredient in the antibacterial activity of the ethanol extract.

[0083] 3.4.3 Response surface methodology results for alcohol extraction The quadratic regression model equation with total flavonoid content (Y) as the objective function was obtained based on the analysis using Design Expert 13 software. Y=8.41-0.1443A+0.8207B+0.3843C-0.9819AB-0.8299AC-0.4167BC-1.31 A²-1.69B ²-1.74C² .

[0084] As shown in Table 16: the model's P<0.000 1 Highly significant; lack of fit P>0.05 The result is not significant, indicating that the model is reliable. r 2 =0.9729 This indicates a high degree of agreement between the experimental data and the fitted function, and that the curve has a good linear relationship. r 2 adj =0.9381 The results indicate that the model has high reliability and can be used for the extraction optimization of compound volatile oils. The influence of each factor on the yield of volatile oils is in the order of B > C > A, that is, extraction time > extraction temperature > material-liquid ratio.

[0085] Table 16 ANOVA Table for Total Flavonoids Response Surface Optimization Process Source of variance sum of squares Degrees of freedom Mean Square F value p-value Significance Model 49.7 9 5.52 27.94 0.0001 significant A-Feed-Liquid Ratio 0.1666 1 0.1666 0.8431 0.389 B - Extraction Time 5.39 1 5.39 27.26 0.0012 ** C - Extraction temperature 1.18 1 1.18 5.98 0.0444 * AB 3.86 1 3.86 19.51 0.0031 ** AC 2.76 1 2.76 13.94 0.0073 ** BC 0.6946 1 0.6946 3.51 0.103 A² 7.24 1 7.24 36.64 0.0005 *** B² 12 1 12 60.74 0.0001 *** C² 12.73 1 12.73 64.39 <0.0001 *** Residual 1.38 7 0.1976 Lack of Fit 0.1323 3 0.0441 0.1409 0.9303 not significant Pure Error 1.25 4 0.3128 Cor Total 51.08 16 Note: * indicates p < 0.05,**express p < 0.01 ,***express p < 0.001 The Design Expert 13 software was used to design and analyze the response surface methodology, and the results are shown in Figure 6. The optimal extraction process for volatile oil and alcohol extract was predicted. The optimal extraction process will be verified under the predicted conditions in the subsequent experiments.

[0086] Table 17 Results of response surface methodology for total flavonoid extraction: prediction and validation. Evaluation indicators predict verify Material-liquid ratio 12.366 12.37 Extraction time / min 130.730 130.73 Extraction temperature / ℃ 83.804 83.80 Total flavonoid content / % 7.178 7.00 3.5 Results of the study on the antibacterial mechanism of the extract from the optimal formulation 3.5.1 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Sichuan pepper compound Figure 7 shows the MIC results. The concentrations of the Sichuan pepper compound from left to right are 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.015625 mg / mL, and 0.0078125 mg / mL. The top three rows represent different concentrations of the drug with added bacteria, and the bottom row represents the sterile blank control for each concentration. After 12 hours of incubation, visual observation showed that at a concentration of 31.25 mg / mL, the turbidity of the pores containing Staphylococcus aureus was almost uniform. Therefore, the minimum inhibitory concentration (MIC) of the Sichuan pepper compound against Staphylococcus aureus is 31.25 mg / mL. Similarly, the MIC for Escherichia coli is 15.625 mg / mL. Figure 8 shows the bacterial growth results on the plate, indicating that the MBC value of the Sichuan pepper compound against both S. aureus and E. coli is 0.25 g / mL.

[0087] 3.5.2 Antibacterial curve of Sichuan pepper compound As shown in Figure 9, *S. aureus* and *E. coli* without the compound treatment grew normally and entered the logarithmic growth phase after 2 hours. *S. aureus* and *E. coli* treated with 1 / 4 MIC and 1 / 2 MIC compound treatments showed similar growth potential (OD600 nm absorbance) to the control group within 0-12 hours, but their growth was significantly inhibited. *S. aureus* treated with 1 MIC compound extract showed no growth within 0-8 hours, while *E. coli* growth was completely inhibited within 0-4 hours. In the 2 MIC treatment group, *S. aureus* growth was completely inhibited within 0-18 hours, and *E. coli* growth was completely inhibited within 0-16 hours. These results indicate that the antibacterial effect of the compound on *S. aureus* and *E. coli* is significantly concentration-dependent.

[0088] 3.5.3 Results of the bacterial film-forming ability experiment The results of the compound's effect on biofilm formation in *S. aureus* and *E. coli* are shown in Figure 10. Crystal violet can specifically bind to biofilm components. The amount of bound dye can be quantitatively measured to indirectly reflect the bacterial count or biofilm formation ability; therefore, a higher absorbance value indicates a stronger bacterial biofilm formation ability. As shown in the figure, compared with the blank control group, the compound significantly (…) within 6 hours. P＜0.01 It inhibits the biofilm formation ability of S. aureus and E. coli.

[0089] 3.5.4 Effects of Sichuan pepper compound on cell morphology of S. aureus and E. coli The morphology of S. aureus and E. coli cells in the blank culture medium group and the compound drug treatment group was observed using scanning electron microscopy (SEM), and the results are shown in Figure 11. In the blank groups (A and C), the S. aureus and E. coli cells were morphologically intact and had smooth surfaces. After treatment with the compound drug, the cell membrane of S. aureus (B) showed rupture and indentation with leakage of contents, while E. coli (D) bacteria exhibited severe membrane structural damage and morphological disintegration, with cell membrane shrinkage and rupture.

[0090] 3.5.5 Effects of Sichuan pepper compound on cell membrane permeability of S. aureus and E. coli As shown in Figure 12, the extracellular nucleic acid content of S. aureus and E. coli after compound treatment was significantly increased compared with the blank group, and the increase continued with the extension of culture time, further indicating that the compound extract can damage the bacterial cell membrane, leading to the leakage of intracellular macromolecules.

[0091] 3.5.6 Extracellular protein concentrations of *S. aureus* and *E. coli* after treatment with Sichuan pepper compound As shown in Figure 13, compared with the control group, the concentrations of soluble proteins in bacterial suspensions treated with 1 MIC and 2 MIC compound extracts were significantly higher than those in the control group, and were positively correlated with the administered concentration. Proteins are important components of bacterial structure and function; their leakage can lead to cell death. When the cell membrane is intact, proteins do not leak into the extracellular space. Therefore, the increased extracellular protein content indirectly reflects the damage to the cell membranes of *S. aureus* and *E. coli* after compound treatment.

[0092] 3.5.7 Changes in intracellular adenosine triphosphate (ATP) content in *S. aureus* and *E. coli* after treatment with Sichuan pepper compound extract As shown in Figure 14, compared with the control group, the ATP content in the supernatant of *S. aureus* and *E. coli* treated with the compound extracts at 1 MIC and 2 MIC showed a decreasing trend. Furthermore, the decrease in ATP content in bacterial cells became more pronounced with increasing extract concentration.

[0093] 3.5.8 Effects of Sichuan pepper compound treatment on the extracellular alkaline phosphatase (AKP) content of *S. aureus* and *E. coli* As shown in Figure 15, the AKP activity in the supernatants of *S. aureus* and *E. coli* treated with the compound extract showed a significant increasing trend compared with the control group. The change in AKP leakage in *S. aureus* supernatant treated with the compound extract significantly increased within 0-2 h, while in *E. coli* supernatant it increased significantly within 0-1 h. After 2 hours of treatment, AKP activity did not increase with further treatment time.

Claims

1. A topical Chinese medicine composition with antibacterial effect, wherein the Chinese medicine composition comprises the following parts by weight: 20-40 parts of Sichuan pepper, 20-40 parts of Cnidium monnieri, 10-30 parts of Ligusticum striatum, 5-15 parts of Pogostemon cablin, 20-40 parts of Schizonepeta tenuifolia, 10-30 parts of Senecio scandens, and 20-40 parts of Mentha haplocalyx.

2. The topical Chinese medicine composition with antibacterial effect according to claim 1, wherein the Chinese medicine composition comprises the following parts by weight: 30 parts of Sichuan pepper, 30 parts of Cnidium monnieri, 20 parts of Ligusticum striatum, 10 parts of Pogostemon cablin, 30 parts of Schizonepeta tenuifolia, 20 parts of Senecio scandens, and 30 parts of Mentha haplocalyx.

3. A method for preparing a topical traditional Chinese medicine composition with antibacterial effect according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: take the Chinese herbal medicine composition according to the proportion, mix them evenly, soak them in water, extract the volatile oil by steam distillation, extract the residue by ethanol reflux to obtain the alcohol extract, combine the two to make a mixed medicinal solution, and that is the final product.

4. The preparation method according to claim 3, characterized in that, In the method for preparing volatile oil by steam distillation, the soaking time is 20-40 min, the material-to-liquid ratio is 1:10-20, and the extraction time is 150-210 min.

5. The preparation method according to claim 4, characterized in that, In the method for preparing volatile oil by steam distillation, the soaking time is 33 min, the material-to-liquid ratio is 1:12, and the extraction time is 198 min.

6. The preparation method according to claim 3, characterized in that, The residue was extracted by reflux with 70%–80% ethanol at a material-to-liquid ratio of 1:10–20, for a time of 60–180 min and at a temperature of 80–100℃.

7. The preparation method according to claim 6, characterized in that, In the ethanol reflux extraction of the medicinal residue, the material-to-liquid ratio was 1:12, the extraction time was 130 min, and the extraction temperature was 83℃.

8. A traditional Chinese medicine composition according to any one of claims 1-2 or a mixed medicinal liquid preparation prepared according to claims 3-7, characterized in that, Add pharmaceutically acceptable excipients to make a pharmaceutically acceptable formulation.

9. The formulation according to claim 8, characterized in that, The formulation is in the form of a spray, gel, emulsion, or nanoemulsion, etc.

10. The use of the topical Chinese medicine composition according to any one of claims 1-9 in the preparation of antibacterial drugs.

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