Composition for treating vagina inflammation as well as preparation method and application thereof

Abstract

The invention provides a traditional Chinese medicine composition for treating vaginitis as well as a preparation method and application of the traditional Chinese medicine composition. The composition is prepared from fructus cnidii, cortex dictamni, dried alum, cockscomb, borax, cortex phellodendri, fructus evodiae and herba leonuri. The composition provided by the invention can significantly inhibit vaginal harmful bacteria, protect and promote the proliferation of beneficial bacteria, and effectively regulate the balance of vaginal flora; adhesion of harmful bacteria can be effectively weakened, biological membranes wrapping bacterial colonies of the harmful bacteria are destroyed, and drug resistance of the harmful bacteria is reduced; the vagina inflammation can be effectively relieved, and vagina mucosa injury can be repaired. The composition is suitable for treating vaginitis diseases.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a traditional Chinese medicine composition for treating vaginal inflammation, and a preparation method and use thereof. Background Art

[0002] Vaginitis is an inflammation of the vaginal mucosa and submucosal connective tissue. It is a common gynecological disease with high incidence, low cure rate and high recurrence rate. Currently, the treatment of VVC mainly focuses on systemic or local administration of antibiotics. However, antibiotic treatment is prone to drug resistance and may cause dysbiosis, resulting in prolonged vaginitis. It may also cause adverse reactions such as gastrointestinal discomfort and rash.

[0003] The vaginal microecological environment is a dynamic equilibrium system that is mutually restrained and coordinated. Under normal circumstances, it is dominated by dominant bacteria such as lactobacilli. When vaginitis occurs, in addition to infection with harmful bacteria, the detection rate of vaginal lactobacilli in patients is much lower than that of normal people, and the vaginal microecological environment is in an unbalanced state. After conventional treatment, especially after treatment with broad-spectrum antibiotics, although the symptoms of vaginitis disappear, the microecological environment has not recovered, thus causing repeated attacks of vaginitis. Repeated use of antibiotics will cause increased drug resistance, reduce the cure rate, and increase the recurrence rate. Therefore, how to inhibit harmful bacteria, protect beneficial bacteria, and regulate and repair the vaginal microecology is the key to curing vaginitis and avoiding recurrence.

[0004] Traditional Chinese medicine has a long history of treating vaginal infections. It was first recorded in the "Golden Chamber Synopsis: Miscellaneous Diseases in Women, Pulse and Symptoms, and Treatments" in the Eastern Han Dynasty, using a single herb, Cnidium monnieri, as a medicine, and using a suppository (vaginal administration) to treat symptoms of cold and damp leucorrhea. Vaginal infections belong to the category of "leucorrhea" and "vulvar itching" in traditional Chinese medicine. The main pathogenesis is that dampness damages the Ren and Dai meridians, making the Ren meridian unstable and the Dai meridian out of order. Dampness is the main cause of this disease. "Fu Qingzhu's Gynecology" mentioned that "leucorrhea is all dampness syndrome." Dampness can be divided into internal dampness and external dampness. Dysfunction of the spleen, kidney, and liver is the cause of internal dampness; external dampness is mostly caused by living in wetlands for a long time, or unclean sexual intercourse, etc. Therefore, traditional Chinese medicine treatment of vaginal infections focuses on dehumidification, and removes dampness through the method of clearing heat and removing dampness to achieve the purpose of regulating the balance of the flora and reducing inflammation.

[0005] In summary, based on the above theory, in order to avoid the problem that antibiotics only kill bacteria but cannot regulate the vaginal microecological environment and are prone to drug resistance, the role of traditional Chinese medicine in regulating the balance of flora and reducing inflammation is brought into play, and a traditional Chinese medicine composition for the treatment of vaginitis is developed. This has practical significance for solving the problem of vaginitis being difficult to cure and recurring. Summary of the invention

[0006] The purpose of the present invention is to provide a Chinese medicine composition capable of effectively regulating the balance of vaginal flora and treating vaginal inflammation, as well as a preparation method and use thereof.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] In one aspect, the present invention provides a Chinese medicine composition for treating vaginitis, which is prepared from the following raw materials:

[0009] Cnidium monnieri, Dictamni cortex, alum, cockscomb, borax, Phellodendron amurense, Evodia rutaecarpa, and Leonurus japonicus.

[0010] Preferably, the weight ratio of the raw materials is: 3-6 parts of Cnidium monnieri, 3-6 parts of Dictamni cortex, 1-4 parts of Alum, 1-4 parts of Celosia cristata, 1-2 parts of Borax, 1-2 parts of Phellodendron amurense, 1-2 parts of Evodia rutaecarpa, and 1-2 parts of Leonurus japonicus.

[0011] More preferably, the weight ratio of the raw materials is: 4 parts of Cnidium monnieri, 4 parts of Dictamni cortex, 3 parts of Alum, 3 parts of Celosia cristata, 1 part of Borax, 1 part of Phellodendron amurense, 1 part of Evodia rutaecarpa, and 1 part of Leonurus japonicus.

[0012] The Chinese medicine composition of the present invention is prepared by extracting the raw materials of the above-mentioned formula into active substances, and the active substances are used as raw materials to prepare preparations according to conventional preparation techniques when necessary. The active substances can be obtained by crushing or extracting them separately, or by crushing or extracting them together, such as by crushing, grinding, percolation, water extraction, alcohol extraction, etc. These active substances can be in the form of powder or extract, and can be dry extract or fluid extract, and can be prepared into different concentrations according to different preparation requirements.

[0013] On the other hand, the composition of the present invention can be prepared into any pharmaceutically acceptable dosage form.

[0014] The dosage form is selected from the group consisting of tablets, granules, capsules, pills, powders, oral liquids, pastes, suspensions, solutions, lotions, suppositories, and gels. Solutions, lotions, suppositories, and gels are preferred. Gels are most preferred.

[0015] The composition of the present invention can be added with commonly used adjuvants in medicines as needed, and prepared into a preparation by conventional pharmaceutics technology. Wherein, the commonly used adjuvants in medicines are selected from: carbomer, poloxamer, Tween 80, RH40, glycerol, propylene glycol, maltodextrin, soluble starch, PEG400, PEG4000, PEG6000, E10-M, etc.

[0016] In another aspect, the present invention provides a method for preparing the Chinese medicine composition, comprising the following steps:

[0017] (1) Take Cnidium monnieri, add 6-12 times 50%-90% ethanol, heat and reflux to extract 3 times, each time for 1 hour, combine the drug solutions, and concentrate the drug solution to contain 0.1-0.3 g / mL of crude drug;

[0018] (2) Take Dictamni Cortex, Alum, Celosia cristata, Borax, Phellodendron amurense, Evodia rutaecarpa, and Leonurus japonicus, add 6-12 times of water, heat and reflux to extract 3 times, each time for 1 hour, combine the drug solutions, concentrate the drug solutions to contain 0.2-0.4 g / mL of crude drug, add the concentrated Cnidium monnieri drug solution obtained in step (1) according to the prescription ratio, mix, add RH40 for solubilization, and centrifuge to obtain the supernatant.

[0019] Preferably, the method for preparing the Chinese medicine composition comprises the following steps:

[0020] (1) Take Cnidium monnieri, add 80% ethanol, heat and reflux to extract three times, with the solvent volume being 10, 8, and 8, and each extraction taking 1 hour, combine the drug solutions, and concentrate the drug solution to contain 0.25 g / mL of the crude drug;

[0021] (2) Take Dictamni Cortex, Alum, Celosia cristata, Borax, Phellodendron, Evodia rutaecarpa, and Leonurus japonicus, add water and reflux to extract three times, with the solvent amount being 10, 8, and 8, and extract for 1 hour each time. Combine the medicinal solutions, concentrate the medicinal solutions to contain 0.25 g / mL of crude drug, add the concentrated medicinal solution of Fructus Cnidii obtained in step (1) according to the prescription ratio, mix, add RH40 for solubilization at a ratio of 200 μL RH40 to 10 mL of the medicinal solution, and centrifuge to obtain the supernatant.

[0022] In another aspect, the present invention provides use of the above-mentioned traditional Chinese medicine composition in preparing a medicament for treating vaginitis.

[0023] The vaginitis described in the present invention includes various common clinical vaginitis such as bacterial vaginitis and fungal vaginitis.

[0024] The composition of the present invention has a good effect on treating vaginitis, can significantly inhibit vaginal harmful bacteria such as Candida albicans, Staphylococcus aureus, Escherichia coli, protect the proliferation of beneficial bacteria such as vaginal Lactobacillus crispatus, and effectively regulate the balance of vaginal flora; can effectively weaken the adhesion of harmful bacteria, destroy the biofilm that wraps the colonies of harmful bacteria, and reduce the drug resistance of harmful bacteria; can effectively reduce vaginal inflammatory reactions and repair vaginal mucosal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Gram staining optical microscope observation of rat vaginal lavage fluid in Example 11 (500X), wherein Figure 1 a is a blank group, Figure 1 b is the model group, Figure 1 c is the dimethoate gel group, Figure 1 d is Baofukang gel group, Figure 1 e is CFCG-low dose group, Figure 1 f is the CFCG-medium dose group, Figure 1 g is CFCG-high dose group;

[0026] Figure 2 Comparison of the vaginal tissue mucosa of rats after dissection in Example 11, wherein Figure 2 a is a blank group, Figure 2 b is the model group, Figure 2 c is the dimethoate gel group, Figure 2 d is Baofukang gel group, Figure 2 e is CFCG-low dose group, Figure 2 f is the CFCG-medium dose group, Figure 2 g is CFCG-high dose group;

[0027] Figure 3 Comparison of rat vaginal tissue pathology after HE staining in Example 11, where Figure 3 A is the blank group × 100, Figure 3 a is blank group × 200; Figure 3 B is the model group × 100, Figure 3 b is the model group × 200; Figure 3 C is dimethoate gel group × 100, Figure 3 c: dimethoate gel group × 200; Figure 3 D is Baofukang gel group × 100, Figure 3 d is Baofukang gel group × 200; Figure 3 E is CFCG-low-dose group × 100, Figure 3 e is CFCG-low-dose group × 200; Figure 3 F is CFCG-medium dose group × 100, Figure 3 f is CFCG-medium dose group × 200; Figure 3 G is CFCG-high dose group × 100, Figure 3 g is CFCG-high dose group × 200. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0029] Example 1

[0030] It is prepared according to the following formula ratio: 4 parts of Cnidium monnieri, 4 parts of Dictamni cortex, 3 parts of Alum, 3 parts of Celosia cristata, 1 part of Borax, 1 part of Phellodendron amurense, 1 part of Evodia rutaecarpa, and 1 part of Leonurus japonicus.

[0031] (1) 34.8 g of Cnidium monnieri was extracted with 80% ethanol under reflux for 3 times with 10, 8, and 8 times the amount of ethanol added for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25 g / mL of the crude drug.

[0032] (2) Dictamni 34.8 g, Alumen 26.1 g, Celosia cristata 26.1 g, Borax 8.7 g, Phellodendron amurense 8.7 g, Evodia rutaecarpa 8.7 g, Leonurus japonicus 8.7 g were extracted with water reflux for 3 times, with the amount of water added being 10 times, 8 times, and 8 times, for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25 g / mL of the crude drug.

[0033] Mix the Cnidium monnieri concentrated medicinal solution and the water extract concentrated solution evenly according to the prescribed amount, add RH40 to assist dissolution (add 200 μL RH40 to 10 mL of the medicinal solution), centrifuge and take the supernatant to obtain the product.

[0034] Example 2

[0035] It is prepared according to the following formula ratio: 4 parts of Cnidium monnieri, 4 parts of Dictamni cortex, 3 parts of Alum, 3 parts of Celosia cristata, 1 part of Borax, 1 part of Phellodendron amurense, 1 part of Evodia rutaecarpa, and 1 part of Leonurus japonicus.

[0036] (1) 34.8 g of Cnidium monnieri was extracted with 80% ethanol under reflux for 3 times, with the amount of alcohol increased by 8 times, 6 times, and 6 times, for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25 g / mL of the crude drug.

[0037] (2) Dictamni 34.8 g, Alumen 26.1 g, Celosia cristata 26.1 g, Borax 8.7 g, Phellodendron amurense 8.7 g, Evodia rutaecarpa 8.7 g, Leonurus japonicus 8.7 g were extracted with water reflux for 3 times, with the amount of water added being 8 times, 6 times, and 6 times, for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25 g / mL of the crude drug.

[0038] Mix the Cnidium monnieri concentrated medicinal solution and the water extract concentrated solution evenly according to the prescribed amount, add RH40 to assist dissolution (add 200 μL RH40 to 10 mL of the medicinal solution), centrifuge and take the supernatant to obtain the product.

[0039] Example 3

[0040] It is prepared according to the following formula ratio: 4 parts of Cnidium monnieri, 4 parts of Dictamni cortex, 3 parts of Alum, 3 parts of Celosia cristata, 1 part of Borax, 1 part of Phellodendron amurense, 1 part of Evodia rutaecarpa, and 1 part of Leonurus japonicus.

[0041] (1) 34.8 g of Cnidium monnieri was extracted with 80% ethanol under reflux for 3 times with 12, 10, and 10 times the amount of ethanol added for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25 g / mL of the crude drug.

[0042] (2) Dictamni 34.8 g, Alumen 26.1 g, Celosia cristata 26.1 g, Borax 8.7 g, Phellodendron amurense 8.7 g, Evodia rutaecarpa 8.7 g, Leonurus japonicus 8.7 g were extracted with water reflux for 3 times, with the amount of water added being 12 times, 10 times, and 10 times, for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25 g / mL of the crude drug.

[0043] Mix the Cnidium monnieri concentrated medicinal solution and the water extract concentrated solution evenly according to the prescribed amount, add RH40 to assist dissolution (add 200 μL RH40 to 10 mL of the medicinal solution), centrifuge and take the supernatant to obtain the product.

[0044] Example 4

[0045] The preparation is based on the following formula ratio: 6 parts of Cnidium monnieri, 6 parts of Dictamni cortex, 4 parts of Alum, 4 parts of Celosia cristata, 2 parts of Borax, 2 parts of Phellodendron amurense, 2 parts of Evodia rutaecarpa, and 2 parts of Leonurus japonicus.

[0046] (1) Take 52.2 g of Cnidium monnieri and extract it under reflux with 70% ethanol for 3 times, adding 10 times, 8 times, and 8 times of alcohol for 1 hour each time. Combine the drug solutions and concentrate them to contain 0.25 g / mL of the crude drug.

[0047] (2) Dictamni Cortex 52.2 g, Alumen 34.8 g, Celosia cristata 34.8 g, Borax 17.4 g, Phellodendron amurense 17.4 g, Evodia rutaecarpa 17.4 g, Leonurus japonicus 17.4 g: extract with water reflux for 3 times, adding water in 10-fold, 8-fold, and 8-fold amounts, each time for 1 hour, combine the drug solutions, and concentrate them to contain 0.25 g / mL of the crude drug.

[0048] Mix the Cnidium monnieri concentrated medicinal solution and the water extract concentrated solution evenly according to the prescribed amount, add RH40 to assist dissolution (add 200 μL RH40 to 10 mL of the medicinal solution), centrifuge and take the supernatant to obtain the product.

[0049] Example 5

[0050] It is prepared according to the following composition ratio: 3 parts of Cnidium monnieri, 3 parts of Dictamni cortex, 2 parts of Alum, 2 parts of Celosia cristata, 1 part of Borax, 1 part of Phellodendron amurense, 1 part of Evodia rutaecarpa, and 1 part of Leonurus japonicus.

[0051] (1) Take 26.1 g of Cnidium monnieri and heat and reflux 60% ethanol for extraction three times, adding 10 times, 8 times, and 8 times of alcohol, each time for 1 hour, combine the drug solutions, and concentrate the drug solution to contain 0.25 g / mL of crude drug.

[0052] (2) Dictamni Cortex 26.1 g, Alumen 17.4 g, Celosia cristata 17.4 g, Borax 8.7 g, Phellodendron amurense 8.7 g, Evodia rutaecarpa 8.7 g, Leonurus japonicus 8.7 g: extract with water reflux for 3 times, adding water in 10-fold, 8-fold, and 8-fold amounts, each time for 1 hour, combine the drug solutions, and concentrate them to contain 0.25 g / mL of the crude drug.

[0053] Mix the Cnidium monnieri concentrated medicinal solution and the water extract concentrated solution evenly according to the prescribed amount, add RH40 to assist dissolution (add 200 μL RH40 to 10 mL of the medicinal solution), centrifuge and take the supernatant to obtain the product.

[0054] Example 6

[0055] Compositions AF were prepared according to the following formulation ratios and preparation methods.

[0056] (1) Composition A: 4 parts of Cnidium monnieri.

[0057] 34.8 g of Cnidium monnieri was extracted with 80% ethanol under reflux for 3 times with 10, 8, and 8 times the amount of alcohol added for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25 g / mL of the crude drug.

[0058] (2) Composition B: 4 parts of Cnidium monnieri, 4 parts of Dictamni cortex, and 1 part of Phellodendron amurense.

[0059] 34.8 g of Cnidium monnieri was extracted with 80% ethanol under reflux for 3 times with 10, 8, and 8 times of alcohol added for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25 g / mL of the crude drug.

[0060] 34.8g of Dictamni Cortex and 8.7g of Phellodendron Amurense were extracted with reflux water three times with 10-fold, 8-fold and 8-fold water addition for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25g / mL of crude drug.

[0061] Mix the Cnidium monnieri concentrated medicinal solution and the water extract concentrated solution evenly according to the prescribed amount, add RH40 to assist dissolution (add 200 μL RH40 to 10 mL of the medicinal solution), centrifuge and take the supernatant to obtain the product.

[0062] (3) Composition C: 4 parts of Cnidium monnieri, 4 parts of Dictamni cortex, 3 parts of Celosia cristata, 1 part of Phellodendron amurense, and 1 part of Evodia rutaecarpa.

[0063] 34.8 g of Cnidium monnieri was extracted with 80% ethanol under reflux for 3 times with 10, 8, and 8 times of alcohol added for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25 g / mL of the crude drug.

[0064] 34.8g of Dictamni Cortex, 26.1g of Celosia Celosiae, 8.7g of Phellodendron Aucklandiae, and 8.7g of Evodia rutaecarpa were extracted with water reflux for 3 times with 10-fold, 8-fold, and 8-fold water additions for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25g / mL of the crude drug.

[0065] Mix the Cnidium monnieri concentrated medicinal solution and the water extract concentrated solution evenly according to the prescribed amount, add RH40 to assist dissolution (add 200 μL RH40 to 10 mL of the medicinal solution), centrifuge and take the supernatant to obtain the product.

[0066] (4) Composition D: 4 parts of Cnidium monnieri, 4 parts of Dictamni cortex, 3 parts of Celosia cristata, 1 part of Borax, 1 part of Phellodendron amurense, and 1 part of Evodia rutaecarpa.

[0067] 34.8 g of Cnidium monnieri was extracted with 80% ethanol under reflux for 3 times with 10, 8, and 8 times of alcohol added for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25 g / mL of the crude drug.

[0068] 34.8g of Dictamni Cortex, 26.1g of Celosia cristata, 8.7g of borax, 8.7g of Phellodendron amurense and 8.7g of Evodia rutaecarpa were extracted with water reflux for 3 times with 10-fold, 8-fold and 8-fold water additions for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25g / mL of the crude drug.

[0069] Mix the Cnidium monnieri concentrated medicinal solution and the water extract concentrated solution evenly according to the prescribed amount, add RH40 to assist dissolution (add 200 μL RH40 to 10 mL of the medicinal solution), centrifuge and take the supernatant to obtain the product.

[0070] (5) Composition E: 4 parts of Cnidium monnieri, 4 parts of Dictamni cortex, 3 parts of Celosia cristata, 1 part of Borax, 1 part of Phellodendron amurense, 1 part of Evodia rutaecarpa, and 1 part of Leonurus japonicus.

[0071] 34.8 g of Cnidium monnieri was extracted with 80% ethanol under reflux for 3 times with 10, 8, and 8 times of alcohol added for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25 g / mL of the crude drug.

[0072] 34.8g of Dictamni Cortex, 26.1g of Celosia cristata, 8.7g of Borax, 8.7g of Phellodendron amurense, 8.7g of Evodia rutaecarpa and 8.7g of Leonurus japonicus were extracted with water reflux for 3 times with 10-fold, 8-fold and 8-fold water additions for 1 hour each time. The drug solutions were combined and concentrated to contain 0.25g / mL of the crude drug.

[0073] Mix the Cnidium monnieri concentrated medicinal solution and the water extract concentrated solution evenly according to the prescribed amount, add RH40 to assist dissolution (add 200 μL RH40 to 10 mL of the medicinal solution), centrifuge and take the supernatant to obtain the product.

[0074] (6) Composition F: 4 parts of Cnidium monnieri, 4 parts of Dictamni cortex, 3 parts of Alum, 3 parts of Celosia cristata, 1 part of Borax, 1 part of Phellodendron amurense, 1 part of Evodia rutaecarpa, and 1 part of Leonurus japonicus.

[0075] The preparation method is the same as that of Example 1.

[0076] Example 7 Comparison of the inhibitory effects of different compositions on vaginal harmful bacteria

[0077] 1. Test materials

[0078] 1.1 Strains

[0079] The strains of Staphylococcus aureus (CMCC(B)26003), Escherichia coli (CMCC(B)44102), and Candida albicans (CMCC(F)98001) were provided by the China Food and Drug Administration.

[0080] 1.2 Drugs and reagents

[0081] Test sample: Compositions A to F prepared in Example 6.

[0082] Positive control: Jieryin Wash (OTC Chinese medicine, batch number 2202043) Sichuan Enwei Pharmaceutical Co., Ltd.

[0083] Nutrient agar (N8290)Beijing Solebow Biotechnology Co., Ltd.

[0084] Nutrient Broth (CM106)Beijing Luqiao Technology Co., Ltd.

[0085] Sabouraud agar medium (20220715) Qingdao Haibo Biotechnology Co., Ltd.

[0086] MRS agar: (HB0384) Qingdao Haibo Biotechnology Co., Ltd.

[0087] MRS Broth: (HB0384-1) Qingdao Haibo Biotechnology Co., Ltd.

[0088] 1.3 Instruments

[0089] Ultrapure water machine (ariummini) Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0090] Electronic balance (BSA224S-CW) Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0091] Automatic steam sterilizer (CL-32L) Japan ALP Company

[0092] Biological Safety Cabinet 1379 (1300 series A2) Thermo Fisher (Suzhou) Instrument Co., Ltd.

[0093] CO2 Constant Temperature Incubator (PYC-16) Thermo Fisher (Suzhou) Instrument Co., Ltd.

[0094] Digital constant temperature water bath (HH-2) Jintan District Xicheng Xinrui Instrument Factory

[0095] Low speed centrifuge (TDL-80-2B) Shanghai Anting Scientific Instrument Factory

[0096] Varioskan Flash Thermo Fisher Scientific (Suzhou) Instrument Co., Ltd.

[0097] DY-L Anaerobic Incubator (0721DY-L03) Heilongjiang MyWorld Industry & Trade Co., Ltd.

[0098] 2. Test methods

[0099] 2.1 Sample processing

[0100] A total of 7 combinations of test samples and positive control samples were filtered through a 0.22 μm filter membrane for sterilization and set aside.

[0101] 2.2 Agar medium preparation

[0102] Take 33g of nutrient agar powder and dissolve it in 1L of ultrapure water. After fully mixing, sterilize it under high pressure at 121℃ for 15min. Cool the sterilized agar to 40-50℃ and pour it into the culture dish while it is hot, 15-20ml in each culture dish. After solidification, the nutrient agar culture dish is obtained.

[0103] Take 70g of Sabouraud agar medium powder and dissolve it in 1L of ultrapure water. After fully mixing, sterilize it under high pressure at 116℃ for 20min. Cool the sterilized agar to 40-50℃ and pour it into the culture dish while it is hot, 15-20ml in each culture dish. After solidification, the Sabouraud agar culture dish is obtained.

[0104] Take 19 g of nutrient broth powder and dissolve it in 1 L of ultrapure water and sterilize it under high pressure at 121°C for 15 min to obtain broth liquid culture medium.

[0105] 2.3 Bacterial strain recovery

[0106] Use an inoculating loop to pick Escherichia coli, Staphylococcus aureus, and Candida albicans from the culture dish, and make continuous streaks in new culture dishes. According to growth requirements, use nutrient agar culture dishes for the first two, and inoculate Candida albicans into Sabouraud agar medium. Write the numbers and place them upside down in a 37°C constant temperature incubator. Culture Escherichia coli and Staphylococcus aureus for 18-24 hours, and culture Candida albicans for 48 hours.

[0107] 2.4 Subculture of bacterial strains

[0108] After the recovery, the strains were passed down by line drawing in the same way until the logarithmic growth phase.

[0109] 2.5 Minimum inhibitory concentration (MIC) determination by microbroth method

[0110] Single colonies of Staphylococcus aureus, Escherichia coli and Candida albicans that grew well were picked from the agar medium, dissolved in physiological saline, blown and mixed, and the absorbance of the bacterial suspension was measured using an enzyme marker and adjusted to OD 625nm The range of 0.08-0.1 is 0.5 McFarland point, and the bacterial density is about 1.5×10 8 CFU / mL.

[0111] The positive wash solution, the six composition sample test solutions and the solvent group were measured by the microbroth dilution method, and the concentration gradient was set to Take a 96-well microplate, add 160μL of broth liquid culture medium to the second column well, add 100μL of broth liquid culture medium to the 3rd-10th column wells, and add 200μL of broth liquid culture medium to the 11th column well, leaving it as a normal growth control without adding drug solution and bacterial solution. Take 40μL of drug solution and add it to the second column well, blow it evenly, then transfer 100μL from the second column well to the third column well and blow it evenly, and so on, dilute it in turn, blow it evenly to the 9th column well and discard 100μL, the 10th column well is used as a negative blank control, and finally take 100μL of bacterial suspension and add it to the 2nd-9th column wells and the 11th column wells in turn. Since the test samples are all colored, a control well with only drug solution and saline is set for each concentration. After adding the plates, place the plates in a 37°C constant temperature incubator with a shaker at 65 r / min for 18-24 hours and observe the growth. If the culture medium is clear or has no precipitation, it is considered sterile growth. The test drug concentration in the sterile growth test well is the MIC.

[0112] 3. Test results

[0113] The minimum inhibitory concentration of composition F against Staphylococcus aureus, Candida albicans and Escherichia coli was lower than that of the other compositions.

[0114] Table 1 Effect of different compositions on minimum inhibitory concentration (MIC) (mg / mL)

[0115]

[0116] The above results show that compared with compositions of other prescription compositions, the composition F of the present invention has the best antibacterial effect, and the minimum inhibitory concentrations for Staphylococcus aureus, Candida albicans, and Escherichia coli are significantly lower than those of other compositions.

[0117] Example 8 Effect of Composition F on Proliferation of Lactobacillus crispatus

[0118] 1. Test materials

[0119] 1.1 Strain: Lactobacillus crispatus (BNCC135057), also known as Lactobacillus, was purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0120] 1.2 Drugs and reagents

[0121] Test sample: Composition F prepared in Example 6.

[0122] The rest is the same as Example 7.

[0123] 2. Test methods

[0124] 2.1 Activation, subculture and cryopreservation of bacterial strains

[0125] Prepare a sterilized test tube (containing 5-10mL MRS liquid culture medium) and 2 MRS agar plates. In a biosafety cabinet, use a grinding wheel to draw 2 circles on the glass tube of the freeze-dried powder of the strain, and break it by hand. Use a Pasteur pipette to draw 0.5mL of liquid culture medium into the tube, mix it thoroughly, and then return it to the liquid culture medium to mix it. Draw 0.2mL of bacterial solution into the plate and spread it evenly. Repeat twice to obtain two plates. Place the test tube and the two plates in an anaerobic incubator. Culture for 48 hours with 5% oxygen, 10% carbon dioxide and 85% nitrogen. Subculture the strains successfully. After subculture twice, freeze the strains with 10% glycerol.

[0126] 2.2 Live bacterial culture count

[0127] Pick up Lactobacillus crispatus and dilute it with physiological saline to adjust the OD 625nm =0.1 (i.e. 0.5 McFarland), take 100 μL of the bacterial suspension for 10-fold gradient dilution, take 100 μL each and add it to sterile plates, pour about 15 mL of MRS agar medium (40°C) into each plate, culture under microaerobic conditions for 24 hours, and count the colonies. The colony count on the plate is within the countable range of 15-300 CFU / plate. The experimental results show that when the absorbance at 625 nm is equal to 0.1, the number of colonies is 1.2×10 8 CFU / mL.

[0128] 2.3 Preparation of MRS agar medium

[0129] Weigh 66.2 g of MRS agar medium powder, dissolve it in 1 L of ultrapure water, and sterilize it at 121°C for 15 min.

[0130] 2.4 Preparation of drug-containing culture medium

[0131] Use physiological saline and sterilized MRS agar medium to dilute the drug solution in a gradient manner to prepare MRS agar medium containing 1.56 and 6.25 mg / ml of the drug, respectively. Take 20 mL of each medium and pour it into a 90×15 mm culture dish. After it is left to solidify horizontally, seal it with a sealed bag and store it in a refrigerator at 4°C for later use.

[0132] 2.5 Bacteria

[0133] Place the MRS-containing agar culture medium stored at 4°C in an anaerobic incubator one day before the bacterial inoculation and dry off the excess water. According to the counting results in 2.2, adjust the bacterial solution concentration to 5×10 6 Pipette 2 μL of Lactobacillus crispatus liquid and spot the bacteria in the center of the culture medium, that is, the number of colonies at each spot is 10 4 CFU, set 5 parallel plates on each plate, dry horizontally, place inverted in an anaerobic incubator and culture for 24, 48, and 72 hours respectively. Use a vernier caliper to measure the colony diameter.

[0134] 3. Test results

[0135] The inhibition rate was calculated based on the colony diameters of the drug-treated group and the blank control group (with normal saline added to the culture medium).

[0136] At 24h, composition F at the concentration of 1.56 and 6.25 mg / ml can promote the proliferation of lactobacillus, and its effect is stronger than that of Jie'eryin Wash. At 48h, composition F at the concentration of 1.56 and 6.25 mg / ml can promote the proliferation of lactobacillus, and its effect is stronger than that of Jie'eryin Wash. At 72h, composition F at the concentration of 1.56 and 6.25 mg / ml can promote the proliferation of lactobacillus, and its effect is stronger than that of Jie'eryin Wash.

[0137] The results are shown in Table 2. Inhibition rate % "positive value" represents growth inhibition, "negative value" represents growth promotion.

[0138] Table 2 Effect of composition F on lactobacilli

[0139]

[0140] Note: Compared with the blank group, * represents P < 0.05, ** Represents P<0.01; compared with Jieryin, ## Represents P < 0.01.

[0141] The above results show that composition F has the effect of promoting the proliferation of lactobacilli, and its promoting and protecting effects on lactobacilli are stronger than those of Jie'eryin Wash.

[0142] Example 9 Effects of Composition F on Candida albicans Biofilm, Adhesion, and Permeability

[0143] 1 Experimental Materials

[0144] 1.1 Strain information

[0145] Candida albicans [CMCC(F)], strain 98001, provided by China Food and Drug Inspection Institutes.

[0146] 1.2 Experimental drugs

[0147] Test sample: Composition F prepared in Example 6.

[0148] Bisoprolol gel (21601233) was purchased from Shandong Mingren Freda Pharmaceutical Co., Ltd. It contains 40 mg of metronidazole, 32 mg of clotrimazole and 1.6 mg of chlorhexidine acetate per milliliter and is a milky white paste.

[0149] 1.3 Experimental reagents

[0150]

[0151] 1.4 Experimental instruments

[0152]

[0153] 2 Experimental methods

[0154] 2.1 Establishment of in vitro Candida albicans biofilm model

[0155] 2.1.1 Solution preparation

[0156] 1) Preparation of menadione solution

[0157] Accurately weigh 0.085 g of menadione standard, add acetone to fully dissolve, add acetone to make up to 50 mL to obtain 10 mmol / L mother solution, divide into small tubes, seal and store at -80 °C.

[0158] 2) Preparation of XTT working solution

[0159] Accurately weigh XTT powder and prepare a 0.5 mg / mL stock solution with PBS, sterilize it with a 0.22 μm filter membrane, and store it in a -80°C refrigerator away from light after aliquoting. Before use, dilute XTT to a 50 μg / mL PBS solution and mix it with menadione acetone solution at a ratio of 10000:1 (so that the final concentration of menadione solution in XTT solution is 1 μmol / L) to prepare XTT working solution, which must be prepared and used immediately.

[0160] 3) Preparation of 0.1% crystal violet staining solution

[0161] Accurately weigh 10 mg of crystal violet powder, dissolve it in 2 mL of 95% ethanol, add 8 mL of normal saline, mix thoroughly, filter through a 0.22 μm microporous membrane, and set aside.

[0162] 2.1.2 Preparation of standard bacterial suspension

[0163] Rapidly thaw the frozen Candida albicans in a 37°C water bath, inoculate on a solid Sabouraud medium plate with a disposable inoculation loop, culture at 28°C for 48 hours, and select 2-3 single colonies with similar morphology to be subcultured on a new medium plate. Culture until the logarithmic growth phase.

[0164] Resuspend the colonies in 1640 medium and adjust the bacterial concentration to 1×10 6 CFU / mL.

[0165] 2.1.3 Determination of biofilm growth curve by crystal violet staining

[0166] Take 100 μL of 1×10 6CFU / mL of Candida albicans was added to a 96-well plate and incubated at 37°C for 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, and 120 h, respectively. The supernatant was gently aspirated and discarded. Each well was washed 3 times with PBS to remove the non-adherent floating bacteria. The plate was placed in an oven and fixed with 200 μL of methanol for 15 min after drying. The methanol was aspirated and evaporated, and 200 μL of 0.1% crystal violet stain was added to each well for 30 min. The stain was removed and washed with PBS until clear, then dried. 150 μL of 33% acetic acid was added to react at room temperature for 30 min, and the OD was detected with an enzyme reader. 590nm There were 6 replicate wells in each group and the experiment was repeated three times.

[0167] 2.1.4 XTT reduction method biofilm growth curve

[0168] Use a multichannel pipette to transfer 100 μL of 1×10 6 CFU / mL of Candida albicans suspension was added to a 96-well plate and cultured at 37 degrees Celsius for 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, and 120 hours. The supernatant was discarded and gently rinsed three times with PBS to wash away the non-adherent floating bacteria. 100 μL of freshly prepared XTT solution was added. The working solution reacted with the living cells. The change in liquid color can reflect the bacterial metabolic activity in the Candida albicans biofilm. Incubate at 37°C in the dark for 6 hours and measure the OD with an enzyme marker 490nm The experiment was repeated three times with 6 replicate wells.

[0169] 2.2 Effects on the early, middle and mature stages of biofilm

[0170] The preparation methods of solution and bacterial suspension are the same as 2.1.1 and 2.1.2. 6CFU / mL of Candida albicans suspension was inoculated in a 96-well plate, and after culturing in an incubator at 37°C for 4h, 24h and 48h, the culture medium was discarded, non-adherent bacteria were washed with PBS, and the residual liquid in the well was cleaned. The drug solution was diluted with RPMI-1640 culture medium, and 100 μL of drug solution of different concentrations was added to the plate, so that the final concentration of composition F in each well culture medium was 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 mg / mL, and only 100 μL of culture medium was added to the blank control well, and bisoprolol gel (92 mg / mL) was set as the control drug, and cultured in an incubator at 37°C for 24h. The metabolic activity of bacteria in the biofilm was determined by the XTT reduction method. The cells were gently rinsed three times with sterile PBS to remove the free Candida albicans and the remaining solution. 100 μL of 50 μg / mL XTT working solution was added to each well. The cells were incubated at 37°C in the dark for 6 h. The absorbance at a wavelength of 490 nm was measured with an ELISA reader to calculate the MBIC. 50 (When the metabolic activity is less than 50%, it is considered effective against mature biofilm, and the drug concentration at this time is recorded as MBIC 50 , 6 replicate wells were measured in each group, and the experiment was repeated three times.

[0171]

[0172] The total amount of biofilm formation was determined by crystal violet staining, and the cells were gently rinsed 3 times with sterile PBS. After drying, 200 μL of methanol was added for fixation for 15 min, the methanol was absorbed and evaporated, and 200 μL of 0.1% crystal violet staining solution was added for staining for 30 min. After removing the staining solution, the cells were gently rinsed with PBS until clear and dried, and 150 μL of 33% acetic acid was added for incubation at room temperature for 30 min, and the OD was detected with an enzyme marker. 590nm The minimum inhibitory concentration (MBIC) of biofilm formation was calculated. 50 ), which is the lowest concentration that inhibits more than 50% of the biofilm.

[0173] 2.3 Effect on the adhesion of Candida albicans

[0174] 2.3.1 Effect on the total amount of Candida albicans adhesion

[0175] In this study, the XTT reduction method was used to compare the effects of different drug concentrations on the total adhesion of Candida albicans at different adhesion times (1, 2, and 3 h).

[0176] The preparation methods of solution and bacterial suspension are the same as 2.1.1 and 2.1.2. Use RPMI-1640 medium to adjust the Candida albicans bacterial suspension to 1×10 6CFU / mL, take 100μL of bacterial suspension and inoculate it into the wells of a 96-well plate, culture at 37℃ for 1, 2 and 3h respectively, wash the non-adherent bacteria with PBS, and aspirate the remaining liquid in the well. Dilute the drug solution with RPMI-1640 medium, add 100μL of different concentrations of drug solution, so that the final concentration of composition F in the medium of each well is 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39mg / mL, add only 100μL of medium to the blank control well, set bisoprolol gel (92mg / mL) as the control drug, and culture at 37℃ for 24h. Discard the medium in the well, rinse gently 3 times with sterile PBS, wash away the non-adherent free Candida albicans and the remaining drug solution, add 100μL of 50μg / mL XTT working solution to each well, incubate at 37℃ in the dark for 6h, and measure OD with an enzyme marker. 490nm There were 6 replicate wells in each group and the experiment was repeated three times.

[0177]

[0178] 2.3.2 Effect on the hydrophobicity of Candida albicans cell surface

[0179] The hydrophobicity (CSH) of the microbial cell surface affects the cell adhesion. The greater the hydrophobicity, the stronger the biological adhesion. The Candida albicans grown to the logarithmic phase was resuspended in liquid Sabouraud medium and adjusted to 1×10 7 Take 3 mL of CFU / mL bacterial suspension and add it to a sterile test tube. Add different concentrations of drugs respectively. Vortex to mix well and shake at 28℃ for 1 hour. Centrifuge the cultured test tube at 3000r / min for 5 minutes, remove the supernatant and wash three times with sterile PBS. Resuspend the precipitate with PBS and take the absorbance of the supernatant without chloroform as OD 600nm The remaining PBS suspension of each sample was added to a glass test tube, 1 mL of chloroform was added to each tube, vortexed for 60 seconds, and allowed to stand at room temperature for 30 minutes to wait for the aqueous phase-chloroform phase to separate. 100 μL was taken out from the upper aqueous phase and the OD was measured using an enzyme-labeled instrument. 600nm (A1), 3 replicates were set for each concentration.

[0180] Relative hydrophobicity of cell surface CSH = (1-A1 / A0) × 100%

[0181] 2.4 Effects on the cell membrane permeability of Candida albicans

[0182] When the cell membrane of Candida albicans is damaged, the normal barrier function is impaired, and macromolecules such as proteins that originally existed in the cell membrane will leak out of the cell membrane. In this experiment, the BCA method was used to measure the concentration of proteins leaked out of the cell.

[0183] In Sabouraud liquid medium, adjust the Candida albicans cultured to the logarithmic phase to 0.5 McFarland point, place in a 100mL sterile conical flask, and culture in a constant temperature shaker at 120r / min and 37°C for 24h. Inoculate at a 1% inoculum, inoculate the suspension on the RPMI-1640 culture medium, and culture in a constant temperature shaker at 120r / min and 37°C for 24h. Centrifuge the cultured bacterial suspension at 4000r / min in a high-speed centrifuge for 10min, discard the supernatant, use PBS to wash the bottom precipitated bacteria three times with sterile PBS, resuspend with PBS, and measure the absorbance value with an enzyme marker, and adjust it to 1×10 6 CFU / mL of bacterial suspension. Take 1mL of bacterial suspension and add RPMI-1640 culture medium containing drugs of different drug concentrations to make the final concentration of the drug in the system 0.39-50mg / mL (two-fold dilution). Set up three test tubes for each concentration, and the test tube with no drug but only culture medium is used as the blank control group. Continue to culture at 37°C for 24h. Take out 1mL of bacterial solution from each test tube, centrifuge at 4000r / min for 10min, discard the bottom precipitate, take the supernatant, and use the BCA kit according to the instructions to determine the protein content in the supernatant.

[0184] 2.5 Statistical analysis

[0185] The above experimental results were processed using SPSS 26.0 software, and one-way analysis of variance was used to compare multiple groups of data. P < 0.05 or P < 0.01 was considered statistically significant.

[0186] 3 Experimental results

[0187] 3.1 Effects on the early, middle and mature stages of Candida albicans biofilm

[0188] (1) Inhibitory effect of composition F at the early stage of biofilm formation

[0189] Crystal violet staining experiments showed that in the early stage of biofilm formation, composition F could inhibit the formation of Candida albicans biofilm in a concentration-dependent manner. With the increase of drug concentration, the amount of biofilm formation decreased. When the drug concentration was 0.39-50 mg / mL, the formation of biofilm could be significantly inhibited. MBIC 50 The value was 21.56 mg / mL. When the concentration of composition F was 6.25-50 mg / mL, the total amount of biofilm formation was significantly reduced compared with that of bisoprolol gel. The results of XTT reduction method showed that when the drug concentration was higher than 1.56-50 mg / mL, it could significantly inhibit the biofilm activity (P < 0.01), MBIC 50When the drug concentration reaches 50 mg / mL, the inhibition rate reaches 80.51%. See Table 3 for details.

[0190] Table 3 Effect of composition F on the amount of biofilm produced and metabolic activity of Candida albicans in the early stage

[0191]

[0192]

[0193] Note: Compared with the control group * P<0.05, ** P<0.01. Compared with the diazinon group, &&P<0.01.

[0194] (2) Inhibitory effect of composition F in the middle stage of biofilm formation

[0195] The experimental results showed that in the middle stage of biofilm formation of Candida albicans (biofilm culture for 24 hours), different concentrations of composition F had a significant inhibitory effect on biofilm in a dose-dependent manner. When the drug concentration was 0.39-50 mg / mL, it had a significant inhibitory effect on both the amount of biofilm formation and the activity of the biofilm (P < 0.01). For the total amount of biofilm formation measured by the CV method, when the drug concentration was 50 mg / mL, the inhibition rate of biofilm formation could reach 68.52%, and the inhibition rate of biofilm activity could reach 79.94%. MBIC 50 When the drug concentration was 0.39-50 mg / mL, the total amount of biofilm formation was significantly reduced compared with that of bisoprolol gel; when the drug concentration was 25-50 mg / mL, the biofilm activity was significantly reduced compared with that of bisoprolol gel.

[0196] Table 4 Effect of composition F on biofilm production and metabolic activity of mid-stage Candida albicans

[0197]

[0198] Note: Compared with the control group * P<0.05, ** P<0.01. Compared with the diazinon group, &P<0.05, &&P<0.01.

[0199] (3) Inhibitory effect of composition F at the mature stage of biofilm formation

[0200] Different concentrations of composition F have a clearing effect on mature biofilms in a dose-dependent manner. When the drug concentration is 0.39 mg / mL, composition F has shown a significant inhibitory effect on mature biofilms (P < 0.01). When the drug concentration reaches 50 mg / mL, the inhibition rate of biofilm formation can reach 66.38%, and the inhibition rate of biofilm activity can reach 80.85%. MBIC 50 When the drug concentration was 0.39-50 mg / mL, the total amount of biofilm formation was significantly reduced compared with that of bisoprolol gel; when the drug concentration was 6.25-50 mg / mL, the biofilm activity was significantly reduced compared with that of bisoprolol gel.

[0201] Table 5 Effect of composition F on biofilm production and metabolic activity of mature Candida albicans

[0202]

[0203] Note: Compared with the control group * P<0.05, ** P<0.01. Compared with the dimethoate group, && P<0.01.

[0204] Table 6 MBIC of different concentrations of composition F on biofilm at different stages 50

[0205]

[0206] 3.2 Effect on the total amount of Candida albicans adhesion at different times

[0207] This experiment investigated the effect of composition F on the initial adhesion of Candida albicans biofilm by comparing the effect of composition F with a concentration of 0.39-50 mg / mL on the absorbance of Candida albicans after adding XTT at different adhesion times, which were 1h, 2h and 3h respectively. The results showed that compared with the blank control group, composition F with a concentration of 0.39-50 mg / mL had a significant inhibitory effect on the adhesion of Candida albicans at 1h, 2h and 3h respectively (P < 0.01), and the difference was statistically significant and dose-dependent. When the concentration of composition F was 12.5-50 mg / mL, the adhesion time was significantly reduced compared with the diazinon gel group.

[0208] Table 7 Effect of different concentrations of gel on the absorbance of Candida albicans at different adhesion times

[0209]

[0210]

[0211] Note: Compared with the control group * P < 0.05, indicating a significant difference; ** P<0.01, indicating a very significant difference; compared with the diazinon group, && P<0.01.

[0212] The hydrophobicity test results showed that after composition F acted on Candida albicans, the upper aqueous phase showed different degrees of turbidity compared with the control group; the CSH calculation results by OD value showed that compared with the control group, 6.25 mg / mL and 12.50 mg / mL significantly reduced the CSH of Candida albicans, and the difference was statistically significant (P < 0.01). See Table 8.

[0213] Table 8 Comparison of CSH in each drug administration group

[0214]

[0215] Note: Compared with the control group ** P<0.01, indicating a very significant difference.

[0216] 3.3 Effects on the cell membrane permeability of Candida albicans

[0217] In this experiment, different concentrations of drugs were applied to Candida albicans for 24 hours, and the protein content in the culture supernatant was determined by the BCA method (results are shown in Table 9). The experimental results show that composition F can cause the leakage of intracellular protein in Candida albicans, which is manifested by a significant increase in the protein concentration measured in the culture supernatant. When the drug concentration was 0.78 mg / mL, the measured protein concentration was not significantly different from that of the blank control group (P>0.05). When the drug concentration was greater than or equal to 3.13 mg / mL, the protein content in the supernatant was significantly increased compared with the blank control group (P<0.01), showing a dose-dependent manner. There was no statistical difference in the protein content of the bisoprolol gel group with that of the blank control group (P>0.05).

[0218] Table 9 Effect on the extracellular protein content of Candida albicans

[0219]

[0220]

[0221] Note: Compared with the control group, * P < 0.05, indicating a significant difference; ** P<0.01 indicates a very significant difference

[0222] 4 Summary

[0223] Composition F can significantly inhibit the activity of the early and middle stages of biofilm formation, and has an inhibitory and clearing effect on mature biofilms. At the same time, it can effectively reduce the adhesion of Candida albicans and increase the cell membrane permeability of Candida albicans, thereby inhibiting the activity of Candida albicans and reducing the drug resistance of Candida albicans. Its inhibitory effect is significantly better than that of bisoprolol gel (azole antibiotics).

[0224] Example 10 Preparation of Composition F Gel

[0225] It is prepared according to the following formula ratio: 4 parts of Cnidium monnieri, 4 parts of Dictamni cortex, 3 parts of Alum, 3 parts of Celosia cristata, 1 part of Borax, 1 part of Phellodendron amurense, 1 part of Evodia rutaecarpa, and 1 part of Leonurus japonicus.

[0226] (1) Take 34.8g of Cnidium monnieri and extract it with 80% ethanol reflux for 3 times, each time for 1 hour, and add 10 times, 8 times, and 8 times of alcohol. Combine the drug solutions and concentrate them to contain 2g / mL of crude drug. Extract the Cnidium monnieri concentrate with 7 times of ethyl acetate at 55°C for 3 times, and the extraction times are 3 times, 2 times, and 2 times each time. Combine the extracts, recover ethyl acetate under reduced pressure at 55°C, and concentrate to extract.

[0227] (2) Select RH40 as the emulsifier, anhydrous ethanol as the co-emulsifier, and ethyl oleate as the oil phase. Accurately weigh 0.714g of ethyl oleate, 2.18g of RH40, and 1.852g of anhydrous ethanol, place RH40 and anhydrous ethanol in a stoppered conical flask, and mix them under magnetic stirring at 50°C. Weigh 0.1392g of Cnidium monnieri extract and add ethyl oleate to dissolve it under ultrasonic stirring. Under magnetic stirring at 50°C, add the Cnidium monnieri ethyl oleate solution dropwise to the mixture of emulsifier and co-emulsifier, and stir at 50°C for 30 minutes to obtain Cnidium monnieri self-microemulsion.

[0228] (3) Dictamni 34.8g, Alum 26.1g, Celosia cristata 26.1g, Borax 8.7g, Cortex Phellodendri 8.7g, Evodia rutaecarpa 8.7g, Leonurus japonicus 8.7g: Reflux extraction with water for 3 times, adding water in 10-fold, 8-fold, and 8-fold amounts. Each time for 1 hour, combine the drug solutions, concentrate the drug solution to 0.25g / mL, flocculate with Type II ZTC1+1 clarifier, filter, and take the filtrate for later use.

[0229] (4) Weigh 1.15 g of hydroxypropyl methylcellulose, add 50 mL of flocculation filtrate solution and 700 μL of Cnidium monnieri self-microemulsion, mix well to make it fully swell, adjust the pH to 4.5 with triethylamine, and obtain a gel.

[0230] Example 11 Effect of Composition F on Rat Vaginitis Model

[0231] 1 Experimental Materials

[0232] 1.1 Strain information

[0233] Candida albicans CMCC(F)98001, batch number 2a24-1, was purchased from China Food and Drug Inspection Institutes.

[0234] 1.2 Experimental drugs

[0235] Experimental drug: the gel of composition F prepared in Example 10 (abbreviated as: CFCG).

[0236] Bisoprolol gel (21601233) was purchased from Shandong Mingren Freda Pharmaceutical Co., Ltd. It contains 40 mg of metronidazole, 32 mg of clotrimazole and 1.6 mg of chlorhexidine acetate per milliliter and is a milky white paste.

[0237] Baofukang Gel (211118) was purchased from Jiangxi Xinglin Baima Pharmaceutical Co., Ltd. The drug content was not indicated. The main ingredients were Curcuma zedoaria and borneol, and it was in the form of a light yellow thick cream.

[0238] Estradiol benzoate (220508) was purchased from Shanghai Quanning Biotechnology (Zhumadian) Animal Pharmaceutical Co., Ltd.; cyclophosphamide for injection (1B456A) was purchased from Baxter Oncology and used for rat vaginitis modeling.

[0239] 1.3 Experimental reagents

[0240]

[0241] 1.4 Experimental instruments

[0242]

[0243]

[0244] 1.5 Experimental animals

[0245] 120 SPF SD rats, female, weighing 180-200 g, six weeks old, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., license number SCXK (Beijing) 2019-0010, laboratory animal quality certificate number NO.110324221106069437.

[0246] 2 Experimental methods

[0247] 2.1 Animal screening

[0248] After the rats were numbered, they were briefly anesthetized with isoflurane inhalation. The vaginal opening and nearby fur were disinfected with iodine cotton balls. Sterile PBS was sucked with a pipette and injected into the rat vagina for full lavage. The lavage fluid was placed on ice, and the colony was cultured with Sabouraud agar medium as soon as possible. The lavage fluid of the rat secretions was examined by Gram staining dry slides. Rats with few vaginal bacteria and good cleanliness were selected for the experiment. After evaluation, a total of 96 rats were included in the experimental criteria. Ten rats were randomly selected as the blank group, and the remaining rats were modeled with Candida albicans vaginitis.

[0249] 2.2 Preparation of modeling reagents and bacterial solution

[0250] 2.2.1 Preparation of bacterial solution

[0251] Identification of Candida albicans: Weigh 47.7g of French Corgama chromogenic medium, put it into a beaker and heat it at 100℃ until it is completely melted. When the temperature drops to about 37-42℃, pour it on a plate to obtain the French Corgama Candida albicans chromogenic medium. Streak Candida albicans CMCC(F)98001 on the French Corgama Candida chromogenic medium for 48 hours. The colony is green, which confirms that it is Candida albicans.

[0252] A single colony of Candida albicans with good growth was picked from the agar medium, dissolved in physiological saline, and mixed by pipetting. The absorbance of the bacterial suspension was measured with an ELISA instrument and adjusted to OD 625nm When the value is about 0.2, it is one McBurney point, and the bacterial density is about 3.0×10 8 CFU / mL.

[0253] 2.2.2 Preparation of phenol glue

[0254] In a fume hood, take an appropriate amount of solid phenol in a clean beaker, heat at 65°C to melt into liquid phenol, use a pipette to transfer 10 mL of liquid phenol to another clean beaker, slowly drop 8 mL of glycerol into the beaker, stir clockwise while adding, then add 1 g of sodium carboxymethyl cellulose, stir and mix thoroughly, and dilute to 40 mL with purified water. After ultrasonic dissolution for 10 minutes, a transparent and viscous 25% phenol paste is obtained, which is wrapped in tin foil and stored at 4°C away from light.

[0255] 2.2.3 Preparation of cyclophosphamide injection

[0256] Add 10 mL of saline to the cyclophosphamide powder bottle and stir thoroughly until it is completely dissolved. Keep away from light and use it immediately after preparation.

[0257] 2.3 Rat vulvovaginal candidiasis model

[0258] The modeling group rats were subcutaneously injected with 10 mg / kg of estradiol benzoate for four consecutive days, which put the rats in a pseudoestrus state, increased the secretion of epithelial glycogen on the vaginal wall, and slightly expanded the vaginal opening. On the 5th day, 25% phenol glue (0.1 mL / 100 g) was injected into the vagina to destroy the rat vaginal mucosa. To prevent the phenol glue from overflowing, a sterile cotton ball of appropriate size was inserted into the rat vaginal opening. On the 6th to 9th days, the rat vagina was fully rinsed with normal saline to wash away the residual phenol glue. The prepared Candida albicans suspension was injected into the rat vagina at 0.1 mL / 100 g. After injection, a cotton ball soaked with the bacterial suspension was inserted, and the rat breech position was raised for 2-3 minutes to prevent the bacterial liquid from flowing out. Cyclophosphamide (30 mg / kg) was injected intraperitoneally on the 7th and 9th days to cause low immunity in rats. After 4 days of infection with Candida albicans, the vulva of the modeling rats was red, swollen and expanded, and the behavior of licking the vulva increased, accompanied by a large amount of white slag and light yellow purulent secretions. Use sterile PBS to wash the rat vagina and collect secretions. Take 5μL to plate for Gram staining and then examine the vaginal secretions under a microscope. Take 20μL to culture the colonies on Sabouraud agar medium and culture at 37℃ for 24h. The vaginal environment of the rat is destroyed and many fungi grow. At the same time, take 20μL and culture it on French Cogama Candida chromogenic medium for 48h. Candida albicans colonies of varying sizes (appearing green) can be seen, indicating that the model is successful.

[0259] 2.4 Animal grouping and drug administration

[0260] The rats with successful modeling were randomly divided into model group, bifenthrin group, Baofukang group, CFCG gel low-dose, medium-dose and high-dose groups, 10 rats in each group, according to the number of bacterial colony growth and the degree of vaginal swelling.

[0261] The dosages were metronidazole gel group (30 mg / kg, this is the metronidazole content in the gel), Baofukang gel (360 mg / kg, the commercially available Baofukang gel does not indicate the drug content, this is the overall weight of the gel), low dose (25 mg / kg), medium dose (50 mg / kg), and high dose (100 mg / kg).

[0262] After stimulating rats to urinate with a sterile gun tip at a fixed time every day, the blank group and the model control group were given an equal amount of normal saline, and the other drug groups used a rat sterile gavage needle and syringe to inject the drugs into the rat vagina according to their respective corresponding doses, once a day. After administration, a sterile cotton ball was inserted to prevent exposure, and the vagina was left for about 2-3 minutes. The drug was administered for a total of 7 days.

[0263] 2.5 Indicator Observation

[0264] 2.5.1 General status observation

[0265] On the second day after the administration, the rats in each group were weighed, and their hair condition, mental state, activity level, etc. were observed to compare their general conditions.

[0266] 2.5.2 Candida albicans colony count

[0267] The rats were briefly anesthetized by inhalation of isoflurane, and the vaginal opening and nearby fur were disinfected with iodine cotton balls. 100 μL of sterile PBS solution was drawn with a pipette and injected into the rats' vagina for full irrigation. The solution was washed three times in total, and the irrigation fluid was combined in a sterile centrifuge tube.

[0268] Take 20 μL of vaginal lavage fluid in a sterile dish, cool the prepared French Cogama colorimetric medium to about 40°C and pour it into the dish. Shake in a cross direction to mix well and then invert it in a 37°C incubator for 48 hours. Count and compare the number of Candida albicans colonization in the vagina of each group of rats.

[0269] 2.5.3 Gram staining of lavage fluid

[0270] (1) Smear fixation: Take 5 μL of the above rat vaginal lavage fluid and evenly apply it on an area of ​​about 10 mm in diameter on a glass slide. Dry it at room temperature, and fix it by quickly passing the dried glass slide over the outer flame of an alcohol lamp 3-4 times with tweezers. Touch the bottom of the glass slide with the back of your hand from time to time. The appropriate temperature is when it is not hot to the touch.

[0271] (2) Primary dyeing: Add ammonium oxalate crystal violet dye solution to the fixed sample and dye for 1 minute. Then wash away the excess dye with distilled water until no floating color remains. Then gently absorb the water with filter paper.

[0272] (3) Mordanting: Drop iodine solution on the sample (cover the sample) and dye for 1 minute. Gently rinse the excess dye with distilled water until there is no floating color, and gently absorb the water with filter paper.

[0273] (4) Decolorization: Add 3-4 drops of 95% ethanol and shake gently for 20-30 seconds to decolorize. After decolorization, rinse gently with distilled water and gently dry with filter paper.

[0274] (5) Re-staining: Add safranin dye solution for 1 minute, then rinse off the floating color with distilled water and gently dry with filter paper.

[0275] (6) Microscope observation: The stained vaginal secretion smear was naturally dried and observed under a 500X microscope to compare the cleanliness of the rat vaginal secretions and the colonization of Candida albicans in the vagina.

[0276] 2.5.4 Visual observation of vaginal tissue

[0277] The rats were fasted on the first day after the administration. The vaginal tissues were dissected and weighed with an electronic balance on the second day. The excess blood was washed away with saline and then placed on a glass plate for observation. The secretions, swelling, vaginal folds and mucosal damage were scored according to the table below.

[0278]

[0279] 2.5.5 Pathological observation of vaginal tissue

[0280] Take 1 / 3 of the vaginal tissue for pathological fixation, sectioning, and staining observation, and the specific operation is as follows. The other 2 / 3 is stored at -80°C for future use.

[0281] 1) Sampling: Use a blade to trim the fixed tissue neatly in a fume hood, mark it and place it in a dehydration box.

[0282] 2) Dehydration: Use a dehydrator to dehydrate the tissue using different gradients of alcohol.

[0283] 3) Embedding: Place the dehydrated wax-soaked tissue into an embedding machine and cool it in a -20°C freezer. After the wax solidifies, remove the wax block from the embedding frame and trim it.

[0284] 4) Sectioning: Use a paraffin slicer to slice the tissue, with a thickness of 4 μm. After the tissue is sliced, float it on 40°C warm water on a slide machine to flatten the tissue, pick up the tissue with a slide, and bake it at 60°C for 2 hours.

[0285] 5) HE staining:

[0286] (a) Dewaxing to water: Place the sections in xylene I for 15 min → xylene II for 15 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → 95% alcohol for 5 min → 90% alcohol for 5 min → 80% alcohol for 5 min → 70% alcohol for 5 min → wash with distilled water.

[0287] (b) Hematoxylin staining of cell nuclei: After the sections are stained in Harris hematoxylin for 5 to 10 minutes, they are washed, differentiated in hydrochloric acid alcohol for a few seconds and cleaned, and then rinsed after the blueing solution turns blue for a few seconds.

[0288] (c) Eosin staining of cytoplasm: Place the sections in eosin staining solution for 1-5 minutes.

[0289] (d) Dehydration and sealing: Dehydrate the slices in 95% alcohol I for 5 min → 95% alcohol II for 5 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → xylene I for 5 min → xylene II for 5 min to make them transparent. Take out the slices and air dry them, then seal the slices with neutral gum.

[0290] 6) Observe the sections under a microscope, collect and analyze images. Score based on basic indicators such as mucosal lesions, edema, exudation, inflammatory cell infiltration, etc. No symptoms are scored as 0 points, mild symptoms are scored as 1 point, moderate symptoms are scored as 2 points, and severe symptoms are scored as 3 points. The sum of the scores is the total score of the lesion.

[0291] 2.5.6 Data Analysis

[0292] SPSS 26.0 software was used for data processing, and one-way analysis of variance was used to compare multiple groups of data that conformed to normal distribution. P < 0.05 or P < 0.01 was considered statistically significant. GraphPad Prism 9.0 software was used to draw graphs of the analysis results.

[0293] 3 Experimental results

[0294] 3.1 General status observation

[0295] The rats in the blank group had a normal diet, were agile, had smooth and shiny hair, and were generally in good condition. The rats in the model group were sluggish, lacked energy, and had significantly reduced food and water intake, significantly decreased body weight, and had severe hair loss and hair choking on the body surface, and had obvious licking of the vulva. The rats in the bisoprolol gel group, Baofukang gel group, and CFCG low, medium, and high dose groups had significant improvements in spirit, food intake, and hair condition compared with the model group.

[0296] 3.2 Candida albicans colony count

[0297] In this experiment, vaginal lavage fluid was subjected to colorimetric culture of Candida albicans. The results showed that no Candida albicans grew in the lavage fluid of the blank group, and the colony count was 0. Compared with the blank group, the number of Candida albicans colonies cultured in the vaginal lavage fluid of rats in the model group increased significantly (P < 0.01), indicating that Candida albicans was successfully colonized.

[0298] After 3 days of drug administration, the colony counts in the low, medium and high dose CFCG groups decreased significantly, and the difference was statistically significant compared with the model group (P<0.01).

[0299] After 7 days of administration, the colony counts in the low-, medium- and high-dose CFCG groups were significantly decreased compared with the model group (P < 0.01), and all rats in the medium- and high-dose groups turned negative.

[0300] This indicates that each medication group has an effect on resisting vaginal colonization of Candida albicans.

[0301] Table 10 Colony counts of Candida albicans in rat vaginal lavage fluid

[0302]

[0303]

[0304] Note: Compared with the blank group, ## P<0.01; compared with the model group, * P<0.05, **P<0.01.

[0305] 3.3 Gram staining of vaginal lavage fluid

[0306] (1) Candida albicans spores and hyphae

[0307] The results showed that the blank group had normal vaginal epithelial cell morphology, with a large number of lactobacilli attached, and no Candida albicans spores and hyphae were detected; compared with the blank group, the model group had abnormal epithelial cell morphology, with obvious coexistence of Candida albicans spores and hyphae, and hyphae penetrating vaginal epithelial cells to cause damage. A small number of Candida albicans hyphae (black arrows) were found in the secretions of a few rats in the low-dose CFCG group, and no Candida albicans spores and hyphae were detected in the medium- and high-dose groups; no Candida albicans spores and hyphae were detected in the bisoprolol gel group and the Baofukang gel group. See for details. Figure 1 .

[0308] (2) Lactobacillus status

[0309] The colonization and dominance of lactic acid bacteria are the basic characteristics of healthy vaginal flora. Vulvovaginal candidiasis is characterized by a decrease in the normal dominant bacteria in the vagina - lactobacilli - and an increase in pathogenic bacteria such as Candida albicans.

[0310] The experimental results showed that the morphology of vaginal epithelial cells in the blank group was normal, and most lactobacilli were observed attached to epithelial cells in the visual field, with no obvious foreign bacteria. In the model group, multiple Candida albicans spores were detected adhering to epithelial cells in the visual field. Candida albicans replaced the dominant position of lactobacilli and transformed from the spore phase to the hyphae phase. The internal environment was disordered and the vaginal cleanliness was poor. Lactobacilli in the medium and high dose groups of CFCG recovered to varying degrees, and most of them adhered to vaginal epithelial cells, with good vaginal cleanliness. The vaginal lavage fluid smear in the bisoprolol gel group had good cleanliness, but the lack of lactobacilli was clearly observed. See for details. Figure 1 .

[0311] 3.4 Comparison of vaginal opening status

[0312] The vulva of rats in the blank group was not obviously red and swollen, the vagina was white and translucent, there was no abnormal secretion, and the tissue was elastic; the vaginal opening of rats in the model group was congested and swollen, the secretion increased, the vaginal appearance was ulcerated and exuded, the tissue was inelastic, and the rats screamed when touched; after administration of bisoprolol gel, Baofukang gel, and CFCG, the vulva secretion of rats was significantly less, and the redness and swelling were significantly improved. Among them, the improvement effect of bisoprolol gel on vulvar swelling was poorer than that of the medium-dose and high-dose CFCG groups.

[0313] 3.5 Comparison of vaginal tissue and mucosa

[0314] The vaginal tissues of rats were observed with naked eyes after dissection. The tissue mucosa of rats in the blank group was intact, with normal folds and no obvious swelling. The tissue mucosa of rats in the model group was obviously swollen, with ulcers and bleeding spots, accompanied by more abnormal secretions, and the inflammation score reached 4.71 points, which was significantly different from that in the blank group (P < 0.01). The inflammation scores of each drug-treated group were reduced after treatment, among which the low-, medium- and high-dose CFCG groups and the bisoprolol gel group were significantly different from the model group (P < 0.01).

[0315] By calculating the vaginal tissue mass index of each group, it was found that the vaginal / body mass index of the model group was significantly higher than that of the blank group (P < 0.01). It was considered that inflammation caused tissue edema and abnormal secretions, which led to the increase in the vaginal / body mass index. After treatment in each medication group, the vaginal / body mass index decreased, indicating a decrease in inflammatory response. Compared with the model group, the bisoprolol gel group (P < 0.05) and the CFCG low, medium, and high dose groups all decreased significantly (P < 0.01), and there was no significant difference between the Baofukang gel group and the model group (P > 0.05). Compared with the bisoprolol gel group, the vaginal / body mass of the CFCG high dose group decreased (P < 0.01). See details. Figure 2 and Table 11.

[0316] Table 11 Tissue mucosal scoring results

[0317]

[0318] Note: Compared with the blank group, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01. Compared with the bisoprolol gel group, && P<0.01.

[0319] 3.6 HE pathological staining results

[0320] (1) Observation under an optical microscope

[0321] HE staining of each group showed that the blank group had intact tissue structures of each layer, clear mucosal epithelial cell stratification, normal thickness, no degeneration and necrosis of the mucosal epithelium, and no obvious congestion, edema, and inflammatory cell infiltration in the lamina propria. In the model group, the mucosal epithelium was degenerated, eroded, necrotic, and missing, with exudation, neutrophils and necrotic inflammatory cells, and a large number of inflammatory cells infiltrated both the lamina propria and the submucosal layer, mainly neutrophils, with mild congestion.

[0322] The vaginal mucosa of rats in the low- and medium-dose CFCG groups was well repaired, the basal cells of the animals were partially irregular, the lamina propria was slightly to moderately infiltrated with polymorphonuclear leukocytes, and the blood vessels were slightly congested. The mucosal epithelium of the high-dose group was completely repaired, the lamina propria was almost free of polymorphonuclear leukocyte infiltration, and no vascular congestion or edema was observed; the vaginal mucosal epithelium of rats in the bisoprolol gel group was slightly degenerated and eroded locally, with occasional shedding, loss of the keratinized layer, slight to moderate infiltration of polymorphonuclear leukocytes and lymphocytes in the lamina propria, and mild congestion; the vaginal mucosal epithelium of rats in the Baofukang gel group was slightly degenerated locally, the lamina propria was slightly to mildly infiltrated with polymorphonuclear leukocytes and lymphocytes, and mild congestion. See for details. Figure 3 .

[0323] (2) Pathological scoring

[0324] The rat vaginal specimens were scored according to basic indicators such as mucosal lesions, edema, exudation, and inflammatory cell infiltration. The statistical results showed that there was no obvious mucosal lesion, edema, and exudation in the blank group; the mucosal lesions, edema, exudation, polymorphonuclear leukocyte infiltration, and necrotic inflammatory cells in the model group were significantly increased (P < 0.01). The pathological histological scores of the CFCG low, medium, and high groups and the Baofukang gel group were significantly reduced (P < 0.01), and the total score of the bisoprolol gel group was significantly improved compared with the model group (P < 0.05). Compared with the bisoprolol gel group, the total scores of the CFCG low, medium, and high dose groups were significantly reduced; compared with Baofukang, the total score of the CFCG high dose group was significantly reduced.

[0325] Table 11 Pathological histological scores of vaginal tissues of rats

[0326]

[0327]

[0328] Note: Compared with the blank group, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01; compared with dimethoate, & P<0.05, && P<0.01. Compared with Baofukang, $ P<0.05.

[0329] In summary, the above test results show that composition F (CFCG) has a good therapeutic effect on the rat VVC model caused by Candida albicans, can reduce the colonization of Candida albicans, adjust the vaginal flora, repair the vaginal mucosa, reduce the inflammatory response, etc., and the effect is better than that of mefenamic acid or fenvalerate.

Claims

1. A Chinese medicine composition for treating vaginitis, prepared from the following raw materials: Cnidium monnieri, Dictamni cortex, Alum, Celosia cristata, Borax, Phellodendron amurense, Evodia rutaecarpa, and Leonurus japonicus.

2. The composition according to claim 1, characterized in that The weight ratio of the raw materials is: 3-6 parts of Cnidium monnieri, 3-6 parts of Dictamni cortex, 1-4 parts of Alum, 1-4 parts of Celosia cristata, 1-2 parts of Borax, 1-2 parts of Phellodendron amurense, 1-2 parts of Evodia rutaecarpa, and 1-2 parts of Leonurus japonicus.

3. The composition according to claim 1, characterized in that The weight ratio of the raw materials is: 4 parts of Cnidium monnieri, 4 parts of Dictamni cortex, 3 parts of Alum, 3 parts of Celosia cristata, 1 part of Borax, 1 part of Phellodendron amurense, 1 part of Evodia rutaecarpa, and 1 part of Leonurus japonicus.

4. The composition according to any one of claims 1 to 3, characterized in that It can be made into any pharmaceutically acceptable dosage form.

5. The composition according to claim 4, characterized in that The dosage form is selected from tablets, granules, capsules, pills, powders, oral liquids, ointments, suspensions, solutions, lotions, suppositories, and gels.

6. The composition according to claim 4, characterized in that The dosage form is selected from the group consisting of solutions, lotions, suppositories, and gels.

7. A method for preparing the composition according to any one of claims 1 to 3, comprising the following steps: (1) taking Cnidium monnieri, adding 6-12 times 50%-90% ethanol, heating and refluxing extraction for 3 times, each time for 1 hour, combining the drug solutions, and concentrating the drug solutions to 0.1-0.3 g / mL; (2) Take Dictamni Cortex, Alum, Celosia cristata, Borax, Phellodendron amurense, Evodia rutaecarpa, and Leonurus japonicus, add 6-12 times of water, heat and reflux to extract 3 times, each time for 1 hour, combine the medicinal solutions, concentrate the medicinal solutions to 0.2-0.4 g / mL, add the concentrated medicinal solution of Fructus Cnidii obtained in step (1) according to the prescription ratio, mix, add RH40 for solubilization, and centrifuge to obtain the supernatant.

8. The preparation method according to claim 7, characterized in that: The preparation method of the Chinese medicine composition comprises the following steps: (1) Take Cnidium monnieri, add 80% ethanol, heat and reflux to extract three times, with the solvent volume being 10, 8, and 8, and each extraction taking 1 hour. Combine the drug solutions and concentrate the drug solution to contain 0.25 g / mL of the crude drug; (2) Take Dictamni Cortex, Alum, Celosia cristata, Borax, Phellodendron, Evodia rutaecarpa, and Leonurus japonicus, add water and reflux to extract three times, with the solvent amount being 10, 8, and 8, and extract for 1 hour each time. Combine the medicinal solutions, concentrate the medicinal solutions to contain 0.25 g / mL of crude drug, add the concentrated medicinal solution of Fructus Cnidii obtained in step (1) according to the prescription ratio, mix, add RH40 for solubilization at a ratio of 200 μL RH40 to 10 mL of the medicinal solution, and centrifuge to obtain the supernatant.

9. Use of the composition according to any one of claims 1 to 3 in the preparation of a medicament for treating vaginitis.

Images