A composition for preventing and / or treating candidal vaginitis

Abstract

The application discloses a composition for preventing and / or treating candidal vaginitis, and belongs to the field of medicine. It is found for the first time that a composition containing volatile oil of pachystachys jutna and total alkaloids of Sophora flavescens can significantly inhibit the adhesion and invasion ability of Candida albicans to VK2 / E6E7 cells. Meanwhile, in a mouse candidal vaginitis model, the composition can effectively reduce the vaginal fungal load of the mouse candidal vaginitis model, improve the pH value of vaginal secretion, and improve the inflammatory cell infiltration of the vaginal tissue, and has clinical popularization and application value.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a composition for the prevention and / or treatment of candidal vaginitis. Background Technology

[0002] Candidal vaginitis is a common female reproductive tract infection, primarily caused by Candida albicans, characterized by vulvar itching, increased vaginal discharge, and a curd-like or cottage cheese-like consistency. Statistics show that approximately 75% of women will experience candidal vaginitis at least once in their lifetime, and 50% of patients experience recurrent episodes. Modern research indicates that the pathogenesis of candidal vaginitis is closely related to the virulence factors of Candida, the host's immune response, and vaginal microenvironment dysregulation. When the vaginal microenvironment is altered due to antibiotic abuse, estrogen changes, or immunodeficiency, or when vaginal immune function is weakened, Candida spores proliferate in large numbers, transforming from a yeast phase to a hyphal phase. These spores adhere to the vaginal mucosal epithelial cells, causing perforation and resulting in inflammatory symptoms. For the treatment of candidal vaginitis, Western medicine primarily uses antifungal drugs, with azole drugs being the first-line treatment option. Treatment methods include oral and vaginal administration. For the treatment of uncomplicated candidal vaginitis, oral fluconazole or vaginal medications such as miconazole soft capsules / suppositories, clotrimazole suppositories / tablets, and nystatin effervescent tablets are currently available clinically. However, with the long-term use of antifungal drugs, the problems of drug resistance and recurrent infections in candidal vaginitis have become increasingly prominent, which limits the efficacy of traditional antifungal drugs and easily causes adverse reactions.

[0003] From the perspective of Traditional Chinese Medicine (TCM), candidal vaginitis falls under the categories of "vaginal itching" and "leukorrhea." This disease is often caused by insufficient vital energy (Qi) allowing damp-heat toxins to invade the lower abdomen. The advantages of TCM in treating candidal vaginitis are increasingly attracting attention. In terms of drug selection, both single-herb and compound TCM formulas have achieved satisfactory results with fewer side effects, showing promising application prospects. Patchouli (Paeonia lactiflora) is a plant belonging to the Lamiaceae family. Pogostemon cablin The dried aerial parts of *Blanco* Benth. possess aromatic and dampness-resolving properties. Modern research indicates that patchouli volatile oil is the main active ingredient of patchouli, exhibiting various pharmacological effects such as antipyretic, analgesic, anti-inflammatory, antioxidant, and antibacterial properties. *Sophora flavescens* is a legume plant... Sophora flavescens The dried root of Sophora flavescens has the functions of clearing heat and drying dampness, and killing parasites. Modern research has found that Sophora flavescens mainly contains alkaloid chemical components, which have a variety of pharmacological activities such as anti-tumor, anti-inflammatory, anti-pathogenic microorganism, and immune system regulation.

[0004] Traditional Chinese medicine treats candidal vaginitis from the perspective of "dampness." Sophora flavescens (Ku Shen) effectively clears dampness and eliminates pathogenic factors from the body, addressing the root cause and thus achieving therapeutic effects. Patchouli (Guang Huo Xiang) aromatically transforms dampness, improving the damp environment and reducing its impact on the lower abdomen, thereby reducing the growth conditions for Candida and creating favorable conditions for Sophora flavescens to exert its heat-clearing, dampness-drying, and insecticidal effects. It also enhances the antibacterial effect of Sophora flavescens. The two work synergistically to exert a therapeutic effect, demonstrating the strong potential of the combination of Patchouli and Sophora flavescens in treating candidal vaginitis. Currently, there are no studies or reports on the effects of Patchouli volatile oil and Sophora flavescens total alkaloids on candidal vaginitis. Summary of the Invention

[0005] To address the above problems, the present invention provides a composition comprising patchouli volatile oil and total alkaloids of Sophora flavescens, wherein the mass ratio of patchouli volatile oil to total alkaloids of Sophora flavescens is (1~5):(1~5).

[0006] Furthermore, the mass ratio of the patchouli volatile oil to the total alkaloids of Sophora flavescens is 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or 1:5.

[0007] Furthermore, the mass ratio of the patchouli volatile oil to the total alkaloids of Sophora flavescens is 1:1.

[0008] Furthermore, the contents of baicalein and patchouli ketone in the patchouli volatile oil are 32%~34% and 9%~11%, respectively; the contents of matrine and oxymatrine in the total alkaloids of Sophora flavescens are 102 mg / g~104 mg / g and 369 mg / g~371 mg / g, respectively.

[0009] Furthermore, the contents of baicalein and patchouli ketone in the patchouli volatile oil are 33.10% and 10.35%, respectively; the contents of matrine and oxymatrine in the total alkaloids of Sophora flavescens are 103.58 mg / g and 370.6 mg / g, respectively.

[0010] Furthermore, it is a topical preparation made with patchouli volatile oil and total alkaloids of Sophora flavescens as active ingredients, plus pharmaceutically acceptable excipients.

[0011] Furthermore, the topical preparation is a suppository, solution, ointment, or emulsion.

[0012] The present invention also provides a method for preparing the above composition, which includes the following steps: weighing the volatile oil of patchouli and the total alkaloids of sophora flavescens according to the mass ratio, mixing them, and thus obtaining the composition.

[0013] The present invention also provides the use of the above-described composition in the preparation of antifungal agents against Candida, and in medicaments for the prevention and / or treatment of candidal vaginitis.

[0014] Furthermore, the drug has the effect of inhibiting the adhesion and invasion of Candida albicans into VK2 / E6E7 cells.

[0015] Furthermore, the drug has the effect of improving candidal vaginitis.

[0016] Furthermore, the drug has the effects of reducing vaginal fungal load in a mouse model of candidal vaginitis, improving the pH value of vaginal secretions, and improving inflammatory cell infiltration in vaginal tissues.

[0017] The present invention also provides the use of the combined use of patchouli volatile oil and total alkaloids of Sophora flavescens in the preparation of antifungal agents against Candida, and in the preparation of medicaments for the prevention and / or treatment of candidal vaginitis.

[0018] Furthermore, when the combined medication is used, the mass ratio of patchouli volatile oil to total alkaloids of Sophora flavescens is (1~5):(1~5).

[0019] Furthermore, when using the combined medication, the mass ratio of patchouli volatile oil to total alkaloids of Sophora flavescens is 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or 1:5.

[0020] Furthermore, when using the combined medication, the mass ratio of patchouli volatile oil to total alkaloids of Sophora flavescens is 1:1.

[0021] The composition of this invention for the prevention and / or treatment of candidal vaginitis exhibits outstanding preventive and therapeutic effects on candidal vaginitis by inhibiting the adhesion and invasion of Candida albicans to VK2 / E6E7 cells, reducing the vaginal fungal load in a mouse model of candidal vaginitis, improving the pH value of vaginal secretions, and improving inflammatory cell infiltration in vaginal tissues. It has clinical application value.

[0022] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0023] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0024] Figure 1The effect of patchouli volatile oil and total alkaloids from Sophora flavescens alone on the activity of VK2 / E6E7 cells (Note: compared with Control). P <0.01).

[0025] Figure 2 The effects of patchouli volatile oil and total alkaloids from Sophora flavescens, used alone and in combination, on the activity of Candida albicans-infected VK2 / E6E7 cell models (Note: compared with Control). ## p <0.01; compared to Model, p <0.01; compared to 1:0, △△ p <0.01).

[0026] Figure 3 Effects of the combination of patchouli volatile oil and total alkaloids from Sophora flavescens on the adhesion and invasion of Candida albicans into VK2 / E6E7 cells (Note: compared with Control). P <0.01).

[0027] Figure 4 Effects of the combination of patchouli volatile oil and total alkaloids from Sophora flavescens on vaginal fungal load and vaginal secretion pH (Note: compared with the model group, P <0.01).

[0028] Figure 5 The effect of the combination of patchouli volatile oil and total alkaloids of Sophora flavescens on the pathological changes of vaginal tissue in mice.

[0029] Figure 6 The effect of the combination of patchouli volatile oil and total alkaloids from Sophora flavescens on the positive expression of PAS staining in mouse vaginal tissue. Detailed Implementation

[0030] The raw materials and equipment used in the specific embodiments of this invention are all known products and can be obtained by purchasing commercially available products. The preparation process of suppositories, solutions, ointments, or emulsions made from the compositions of this invention is mature and can be carried out by referring to conventional methods in the prior art.

[0031] The sources of the raw materials used in the embodiments and test examples of this invention are as follows: Patchouli volatile oil: Patchouli medicinal material was extracted by steam distillation, batch number: FT055250001; gas chromatography analysis showed that the contents of baicalein and patchouli ketone were 33.10% and 10.35%, respectively.

[0032] Total alkaloids from Sophora flavescens: purchased from Ningxia Bauhinia Pharmaceutical Co., Ltd., batch number: 230906; by liquid chromatography analysis, the contents of matrine and oxymatrine were 103.58 mg / g and 370.6 mg / g, respectively.

[0033] Example 1: Preparation of the composition of the present invention Take the volatile oil of patchouli and the total alkaloids of sophora flavescens, mix them in a mass ratio of 5:1, and add pharmaceutically acceptable excipients and mix well to obtain the final product.

[0034] Example 2: Preparation of the composition of the present invention Take the volatile oil of patchouli and the total alkaloids of sophora flavescens, mix them in a mass ratio of 4:1, and add pharmaceutically acceptable excipients and mix well to obtain the final product.

[0035] Example 3: Preparation of the composition of the present invention Take the volatile oil of patchouli and the total alkaloids of sophora flavescens, mix them in a mass ratio of 3:1, and add pharmaceutically acceptable excipients and mix well to obtain the final product.

[0036] Example 4: Preparation of the composition of the present invention Take the volatile oil of patchouli and the total alkaloids of sophora flavescens, mix them in a mass ratio of 2:1, and add pharmaceutically acceptable excipients and mix well to obtain the final product.

[0037] Example 5: Preparation of the composition of the present invention Take the volatile oil of patchouli and the total alkaloids of sophora flavescens, mix them in a 1:1 mass ratio, and add pharmaceutically acceptable excipients and mix well to obtain the final product.

[0038] Example 6: Preparation of the composition of the present invention Take the volatile oil of patchouli and the total alkaloids of sophora flavescens, mix them in a mass ratio of 1:2, and add pharmaceutically acceptable excipients and mix well to obtain the final product.

[0039] Example 7: Preparation of the composition of the present invention Take the volatile oil of patchouli and the total alkaloids of sophora flavescens, mix them in a mass ratio of 1:3, and add pharmaceutically acceptable excipients and mix well to obtain the final product.

[0040] Example 8: Preparation of the composition of the present invention Take the volatile oil of patchouli and the total alkaloids of sophora flavescens, mix them in a mass ratio of 1:4, and add pharmaceutically acceptable excipients and mix well to obtain the final product.

[0041] Example 9: Preparation of the composition of the present invention Take the volatile oil of patchouli and the total alkaloids of sophora flavescens, mix them at a mass ratio of 1:5, and add pharmaceutically acceptable excipients and mix well to obtain the final product.

[0042] The following experimental examples illustrate the beneficial effects of the present invention.

[0043] Experimental Example 1: Effects of the combination of patchouli volatile oil and total alkaloids from Sophora flavescens on human vaginal epithelial (VK2 / E6E7) cells. 1. Materials and Methods 1.1 Experimental Cells and Strains Human vaginal epithelial (VK2 / E6E7) cells: purchased from Wuhan Pronosei Life Science Technology Co., Ltd., batch number: CL-1024; Candida albicans ATCC10231: purchased from Wenzhou Kangtai Biotechnology Co., Ltd., batch number: WH1706.

[0044] 1.2 Experimental Reagents CCK-8 (Shanghai Sangon Biotech Co., Ltd., batch number: KC09FC0260), VK2 / E6E7 cell culture medium (Wuhan Pronosei Life Sciences Co., Ltd., batch number: CM-1024), RPMI-1640 Medium (Sigma, batch number: R8755-10×1L), MOPS (Shanghai Sangon Biotech Co., Ltd., batch number: I823BA0011), Sabouraud dextrose agar (Beijing Solarbio Science & Technology Co., Ltd., batch number: LA4770).

[0045] 2. Experimental Methods 2.1 Cell Culture and Passaging Under aseptic conditions, VK2 / E6E7 cells were seeded in VK2 / E6E7 cell culture medium and cultured in an incubator at 37°C, 5% CO2, and 98% relative humidity. When the cells reached 80%–90% confluence, they were digested with 2 mL of 0.25% trypsin (containing 0.05% EDTA), passaged, and cells in the logarithmic growth phase (passed through at least three passages after resuscitation) were collected for subsequent experiments.

[0046] 2.2 Effects of Patchouli Volatile Oil and Sophora flavescens Total Alkaloids on VK2 / E6E7 Cell Viability Using CCK-8 Assay Adjust the VK2 / E6E7 cell concentration to 2×10⁻⁶. 4 Cells were seeded at 100 μL per well in 96-well plates and incubated overnight at 37°C with 5% CO2. After cell attachment, the medium was replaced with drug-containing medium at final drug concentrations of 25, 50, 75, 100, 150, and 200 μg / mL, with 6 replicates per group and 100 μL per well. A control group without drug and a blank group without drug or cells were also included. After 24 h of culture, the cells were washed three times with PBS, replaced with fresh medium containing 10% CCK-8 solution, and incubated in the dark for 2 h. The absorbance (OD) of each well was measured at 450 nm, and cell viability was calculated. Note: Cell viability = [(OD of experimental wells)] 450 nm-blank pore OD450 nm) / (OD of control well) 450 nm-blank pore OD 450 nm)]×100%.

[0047] 2.3 Effects of Patchouli Volatile Oil and Sophora flavescens Total Alkaloids, Used Alone and in Combination, on the Activity of Candida albicans-Infected VK2 / E6E7 Cell Model Adjust the VK2 / E6E7 cell concentration to 2×10⁻⁶. 4 Cells were inoculated at a rate of 100 μL per well into 96-well plates and incubated overnight at 37°C with 5% CO2 until cell adhesion. Patchouli volatile oil and total alkaloids from Sophora flavescens were prepared into drug-containing culture media with a final concentration of 150 μg / mL using different mass ratios (5:1, 4:1, 3:1, 2:1, 1:1, 0:1, 1:0, 1:2, 1:3, 1:4, 1:5). Candida albicans cultured overnight was picked and adjusted to OD500 with PBS. 600 =0.5, centrifuged at 4000 rpm for 10 min, resuspended in drug-containing medium, and 100 μL of the resuspended bacterial solution was added to each well. Co-cultured for 4 h. A blank control group without bacterial culture and a model control group without drug were also set up. Subsequently, the medium was replaced with fresh medium containing 10% CCK-8 solution, and incubated in the dark for 2 h. The absorbance (OD) value of each well was measured at 450 nm, and cell viability was calculated. Note: Cell viability = [(OD of experimental wells)] 450 nm-blank pore OD 450 nm) / (OD of control well) 450 nm-blank pore OD 450 nm)]×100%.

[0048] 2.4 Determination of the compatibility of patchouli volatile oil and total alkaloids from Sophora flavescens against Candida albicans adhesion to VK2 / E6E7 cells Adjust the VK2 / E6E7 cell concentration to 1×10⁻⁶. 5 Inoculate 1 mL of the cultured *Candida albicans* at a rate of 1 / mL into each well of a 24-well plate and incubate overnight at 37°C with 5% CO2 until cell adhesion occurs. Pick the *Candida albicans* cultured overnight and adjust the concentration to OD500 with PBS. 600Centrifuge at 0.5°C and 4000 rpm for 10 min, then resuspend in culture medium containing drug solutions (0, 50, 100, 150 μg / mL). A blank control group without bacteria and a model control group without drug were also set up. After co-culturing for 4 h, the culture medium was discarded, and the cells were washed twice with sterile PBS. Cells were then lysed with 100 μL of 1% Triton solution for 20 min, followed by adding 900 μL of PBS to each well and mixing thoroughly. 100 μL of each serial dilution was repeatedly added to 900 μL of PBS for further dilution. Finally, 100 μL of each serial dilution was plated onto solid culture medium and cultured for 24 h before colony counting.

[0049] 2.5 Determination of the anti-candidiasis effect of the combination of patchouli volatile oil and total alkaloids from Sophora flavescens on VK2 / E6E7 cells by Candida albicans invasion. Adjust the VK2 / E6E7 cell concentration to 1×10⁻⁶. 5 Inoculate 1 mL of the cultured *Candida albicans* at a rate of 1 / mL into each well of a 24-well plate and incubate overnight at 37°C with 5% CO2 until cell adhesion occurs. Pick the *Candida albicans* cultured overnight and adjust the concentration to OD500 with PBS. 600 Centrifuge at 0.5°C, 4000 rpm for 10 min, and resuspend in culture medium containing drug solutions (0, 50, 100, 150 μg / mL). A blank control group without bacteria and a model control group without drug were also set up. After co-culturing for 4 h, the culture medium was discarded, and the cells were washed twice with sterile PBS. Then, 200 μL of gentamicin containing 100 μg / mL was added, and incubation continued for 30 min to remove bacteria that had not yet entered the cells. After washing twice with sterile PBS, cells were lysed with 100 μL of 1% Triton solution for 20 min, followed by adding 900 μL of PBS to each well and mixing thoroughly. 100 μL of each serial dilution was repeatedly added to 900 μL of PBS for further dilution. Finally, 100 μL of each serial dilution was plated onto solid culture medium and cultured for 24 h, followed by colony counting.

[0050] 3. Statistical methods Data analysis was performed using GraphPad Prism software; measurement data were expressed as mean ± standard deviation; one-way ANOVA was used to analyze the variances of the data. P <0.05 or P <0.01 indicates that the difference between the two groups is statistically significant.

[0051] 4. Experimental Results 4.1 Effects of Patchouli Volatile Oil and Sophora flavescens Total Alkaloids Alone on VK2 / E6E7 Cell Viability VK2 / E6E7 cells were treated with different concentrations of patchouli volatile oil and total alkaloids of Sophora flavescens for 24 h, and cell viability was detected by CCK-8 assay. Results are as follows: Figure 1 As shown, the volatile oil of patchouli had no significant toxicity to VK2 / E6E7 cells at concentrations of 150 μg / mL and below, and the total alkaloids of Sophora flavescens had no significant toxicity to VK2 / E6E7 cells at concentrations of 200 μg / mL and below. Therefore, concentrations of 50, 100, and 150 μg / mL were selected for subsequent experimental studies.

[0052] 4.2 Effects of Patchouli volatile oil and Sophora flavescens total alkaloids, used alone and in combination, on the activity of Candida albicans-infected VK2 / E6E7 cell models. The results are as follows Figure 2 As shown, compared with the blank control group, the cell viability of the model control group decreased significantly to 51.07%, indicating that VK2 / E6E7 cells began to die after infection with Candida albicans. Compared with the model control group, treatment with patchouli volatile oil alone (1:0 group), Sophora flavescens total alkaloids alone (0:1 group), and different ratios of the combined groups all improved cell survival to varying degrees. Among them, when patchouli volatile oil and Sophora flavescens total alkaloids were combined at a mass ratio of 1:1, the viability of VK2 / E6E7 cells reached the highest of 89.14%, which was significantly higher than that of the patchouli volatile oil alone group and the Sophora flavescens total alkaloids alone group. Specifically, although the 1:0 group (patchouli volatile oil alone) and the 0:1 group (Sophora flavescens total alkaloids alone) could improve cell viability to some extent, the improvement was significantly lower than that of the 1:1 combined group. The cell viability enhancement value of the 1:1 formulation was significantly greater than the sum of the cell viability enhancement values ​​of the single-use groups of patchouli volatile oil and total alkaloids of Sophora flavescens, indicating that patchouli volatile oil and total alkaloids of Sophora flavescens produced a significant synergistic effect at a mass ratio of 1:1. This effect is something that those skilled in the art could not expect based on the effects of using either ingredient alone. Therefore, 1:1 was determined to be the optimal formulation ratio, and this ratio will be used in subsequent studies. It is thus concluded that the combination of patchouli volatile oil and total alkaloids of Sophora flavescens can effectively protect VK2 / E6E7 cells against Candida albicans infection.

[0053] 4.3 Effects of the combination of patchouli volatile oil and total alkaloids from Sophora flavescens on the adhesion and invasion of Candida albicans into VK2 / E6E7 cells. The results are as follows Figure 3As shown, Candida albicans has a certain adhesion and invasion effect on VK2 / E6E7 cells. Compared with the model control group, after the volatile oil of Pogostemon cablin and total alkaloids of Sophora flavescens were combined at a mass ratio of 1:1, the adhesion and invasion ability of Candida albicans could be significantly inhibited, and it showed a dose-dependent enhancement. After treatment with drugs at concentrations of 50, 100, and 150 μg / mL, the adhesion rates of Candida albicans decreased by 24.42%, 43.90%, and 71.74% respectively, and the invasion rates decreased by 53.33%, 67.08%, and 74.53% respectively. This significant inhibitory effect further confirmed the synergistic effect of the 1:1 combination, that is, the two active ingredients produced mutually enhanced antifungal activities at a specific ratio, so as to more effectively protect vaginal epithelial cells from the adhesion and invasion of Candida albicans. Thus, it can be seen that the combination of the volatile oil of Pogostemon cablin and total alkaloids of Sophora flavescens has the effect of protecting VK2 / E6E7 cells against Candida albicans infection.

[0054] Experimental Example 2. Study on the effect of the combination of the volatile oil of Pogostemon cablin and total alkaloids of Sophora flavescens at a ratio of 1:1 on a mouse model of candidal vaginitis 1. Experimental materials 1.1. Experimental drugs Metronidazole and Clotrimazole Vaginal Gel, purchased from Shandong Mingren Freda Pharmaceutical Co., Ltd., specification: 5 g: 368 mg; batch number: 24601294.

[0055] Injection Estradiol Benzoate, purchased from Shanghai Quanyu Biotech Animal Pharmaceutical Co., Ltd.; specification: 2 mL: 4 mg; batch number: 250307.

[0056] 1.2. Experimental animals and strains 48 SPF-grade ICR mice, female, body weight (20±2) g, provided by Beijing Speifo Biotechnology Co., Ltd., production license number: SCXK (Beijing) 2019-0010, use license number: SYXK (Sichuan) 2020-0124. They were conventionally fed with standard mouse feed and freely ate and drank water in the same environment.

[0057] Candida albicans ATCC10231, purchased from Wenzhou Kangtai Biotech Co., Ltd., batch number: WH1706.

[0058] 1.3. Experimental reagents Cell fixative (Regan Biotech Ltd., batch number: DF0135), glutaraldehyde fixative (Macklin, batch number: G916054), RPMI-1640 Medium (Sigma, batch number: R8755-10×1L), MOPS (Shanghai Sangon Biotech Co., Ltd., batch number: I823BA0011), Sabouraud dextrose agar (Beijing Solarbio Science & Technology Co., Ltd., batch number: LA4770), hematoxylin staining solution (Sigma-Aldrich, batch number: H9627), eosin staining solution (Hefei Bomei Biotechnology Co., Ltd., batch number: YE2080). 2. Experimental Methods 2.1 Drug preparation method Based on clinical use, the total amount of raw materials required daily for an adult (60 kg) of Patchouli (9 g) and Sophora flavescens (9 g) is 18 g. The daily dose required for humans was calculated based on the extraction yield and then converted to the dose for mice. Patchouli volatile oil and Sophora flavescens total alkaloids were dissolved in carbomer gel at a ratio of 1:1 to prepare concentrations of 1%, 2%, and 4%. Each mouse was given 20 μL of the solution, and the final dose was 1, 2, and 4 mL / kg.

[0059] 2.2 Grouping, Modeling, and Drug Administration Mice were randomly divided into four groups: a blank control group, a model group, a double-dose vaginal gel (positive) group, and low, medium, and high-dose combination groups, with eight mice in each group. Six days before modeling, mice were subcutaneously injected with 0.1 mL (0.2 mg / mL) of estradiol benzoate injection solution every two days for a total of three injections. Candida albicans was incubated in SDA culture dishes at 35℃ for 24 h, and one colony was picked and resuspended in 1 mL of PBS to adjust the bacterial concentration to 1.0 × 10⁻⁶. 8 CFU / mL, except for the control group mice which received 20 μL of PBS buffer vaginally, all other groups of mice received 20 μL of Candida albicans suspension vaginally. The mice were inverted for 10 min to prevent spillage, and this model was maintained for 5 consecutive days. After modeling, 10 mice were randomly selected, and their vaginal appearance was observed. Vaginal secretions were collected to observe for hyphae formation. If a thick white secretion was formed in the mouse vagina, and hyphae were present in the secretion, the model was considered successful. The control and model groups were given 20 μL of blank matrix, and the drug groups were given the corresponding drugs. The mice were injected into the vaginas of their respective groups and inverted for 10 min to prevent spillage. This administration was continued for 7 days.

[0060] 2.3 Determination of fungal load and pH in vaginal douche solution Twenty-four hours after the last administration, 100 μL of PBS solution was used to irrigate the vagina three times with a pipette. The vaginal irrigation fluid was collected from the mice, and its pH value was measured. 10 μL of the irrigation fluid was then spread evenly on the culture medium and incubated at 37°C for 24 h. The number of Candida albicans colonies was then observed.

[0061] 2.4. Observation of vaginal histopathology by HE staining and PAS staining Blood was collected from the eyeballs of mice after isoflurane inhalation anesthesia, and they were euthanized by cervical dislocation. Vaginal tissue was collected, fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. HE staining and PAS staining were performed, and the pathological changes such as edema and inflammatory cell infiltration in the vaginal tissue were observed under a microscope.

[0062] HE staining: Auricular tissue fixed in 4% paraformaldehyde was routinely dehydrated, embedded in paraffin, sectioned, and stained with HE. The pathological morphological changes of vaginal tissue were observed under a microscope.

[0063] PAS staining: After fixation, dehydration, and sectioning, tissues were stained with PAS. Images of the sections were acquired using a microscopic imaging system. Each section was first observed at low magnification, followed by a 200x microscopic image. The positive expression area within the acquired images was determined using the Image-ProPlus 6.0 image analysis system (Media Cybernetics, USA). The percentage of positive expression area was calculated as: positive expression area / field of view (pixel area).

[0064] 3. Statistical methods Data analysis was performed using GraphPad Prism software; measurement data were expressed as mean ± standard deviation; one-way ANOVA was used to analyze the variances of the data. P <0.05 or P <0.01 indicates that the difference between the two groups is statistically significant.

[0065] 4. Experimental Results 4.1 Determination of vaginal fungal load and vaginal secretion pH in mice Experimental results are as follows Figure 4 As shown, compared with the control group, the number of Candida albicans colonies in the vagina of mice in the model group was significantly increased ( P <0.01), the pH value of vaginal secretions was significantly increased, becoming more alkaline; compared with the model group, the colonization of Candida albicans in the vagina of mice in the combined treatment group was significantly reduced ( P <0.01), and bacterial colonization decreased significantly with increasing drug concentration; the pH of mouse vaginal secretions gradually returned to normal.

[0066] 4.2. Histopathological observation of vaginal tissue Vaginal tissue consists of the mucosa, muscular layer, and fibrous layer. The mucosa is covered by stratified squamous epithelium, and some vaginal tissue shows keratinization of the mucosal epithelium. Under light microscopy, no obvious pathological changes were observed in the vaginal tissue of the control group. In the model group, the lamina propria of the vagina showed inflammatory cell infiltration, mainly composed of rod-shaped, segmented neutrophils; vaginal epithelial cells showed degeneration and necrosis, with a small number of cells in the granular or spinous layer exhibiting blurred structure, pyknosis, or fragmentation of the nuclei; and microabscesses and mucinousization of superficial epithelial cells were also observed. Compared with the model group, the pathological changes in the vaginal tissue of the drug groups were significantly improved, with the most significant improvements observed in the positive control group and the high-dose group. See details below. Figure 5 .

[0067] Depend on Figure 6 As shown in Table 1, compared with the blank group, the percentage of PAS-positive expression area in the vaginal tissue of the model group was significantly increased. P <0.01); compared with the model group, the percentage of PAS-positive expression area in the drug group was reduced ( P <0.05 or P <0.01).

[0068] Table 1. Percentage of PAS-positive expression area in mouse vaginal tissue (%) ±SD) Note: Compared with the model group, P <0.01.

[0069] In summary, this invention provides a composition for the prevention and / or treatment of candidal vaginitis. This invention is the first to discover that a composition containing patchouli volatile oil and total alkaloids of Sophora flavescens can significantly inhibit the adhesion and invasion of Candida albicans onto VK2 / E6E7 cells. Furthermore, in a mouse model of candidal vaginitis, this composition effectively reduces the vaginal fungal load, improves vaginal secretion pH, and reduces inflammatory cell infiltration in vaginal tissue, demonstrating its clinical applicability.

Claims

1. A composition, characterized in that: Its composition includes patchouli volatile oil and Sophora flavescens total alkaloids, wherein the mass ratio of patchouli volatile oil to Sophora flavescens total alkaloids is (1~5):(1~5).

2. The composition according to claim 1, characterized in that: The mass ratio of the patchouli volatile oil to the total alkaloids of Sophora flavescens is 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or 1:

5.

3. The composition according to claim 2, characterized in that: The mass ratio of the patchouli volatile oil to the total alkaloids of Sophora flavescens is 1:

1.

4. The composition according to claim 1, characterized in that: It is a topical preparation made with patchouli volatile oil and total alkaloids of Sophora flavescens as active ingredients, plus pharmaceutically acceptable excipients.

5. The composition according to claim 4, characterized in that: The topical preparation is a suppository, solution, ointment, or emulsion.

6. A method for preparing the composition according to any one of claims 1 to 5, characterized in that: It includes the following steps: weigh out the volatile oil of patchouli and the total alkaloids of sophora flavescens according to the mass ratio, mix them, and the product is obtained.

7. Use of the composition according to any one of claims 1 to 5 in the preparation of antifungal agents against Candida, and medicaments for the prevention and / or treatment of candidal vaginitis.

8. The use according to claim 7, characterized in that: The drug has the effect of inhibiting the adhesion and invasion of Candida albicans into VK2 / E6E7 cells.

9. The use according to claim 7, characterized in that: The drug has the effect of improving candidal vaginitis.

10. Use of the combined use of patchouli volatile oil and total alkaloids of Sophora flavescens in the preparation of antifungal agents against Candida, and in the prevention and / or treatment of candidal vaginitis.

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