Application of leukorrheal decoction in preparation of medicine for preventing and / or treating vulvovaginal candidiasis

Abstract

The invention discloses an application of leukorrhea finishing decoction in preparation of a medicine for preventing and / or treating vulvovaginal candidiasis. According to clinical experiments and animal experiments, it is determined that the leucorrhea decoction can eradicate fungi in the vagina, recover the vaginal microflora structure, activate the STING signal channel and protect mitochondria in treatment of VVC, so that recurrence of vulvovaginal candida mycoderma bacteria is prevented, and the leucorrhea decoction has wide clinical application prospects.

Description

Technical Field

[0001] The present invention specifically relates to the use of Wudai Decoction in the preparation of a drug for preventing and / or treating vulvovaginal candidiasis. Background Art

[0002] Vulvovaginal candidiasis (VVC) is a common lower genital tract infectious disease in women. Approximately 75% of women will have at least one episode in their lifetime, and 40%-45% of women will experience two or more episodes of VVC within one year. Its incidence rate is second only to bacterial vaginitis. When there are 4 or more episodes within one year, it is called recurrent vulvovaginal candidiasis (RVVC); among them, 13%-28% of VVC patients can develop into RVVC. The latest data show that RVVC affects approximately 138 million women annually (range: 103-172 million), with a global annual prevalence of 3871 / 100,000. The prevalence rate in China is higher than the global prevalence rate, at 4436 / 100,000. 372 million women globally are affected by RVVC in their lifetime, increasing the global economic burden. Modern research has found that the essence of VVC is the imbalance of the vaginal microecology, and its vaginal microenvironment is unique. Although this disease is a non-fatal disease, the antifungal drugs currently used in clinical practice can effectively inhibit or kill fungi, but the use of antibacterial drugs exacerbates the imbalance of the flora and the disorder of the immune microenvironment in the vaginal microecology, affecting the quality of life, physical and mental health of patients, and increasing the economic burden; repeated use of antifungal drugs leads to an increased risk of drug resistance and increases the treatment difficulty of VVC. Although modern medicine has paid attention to the problems of microecology and bacterial endosymbiosis, the developed probiotic preparations can relieve the flora imbalance, but the long-term effects are not yet satisfactory. Therefore, restoring the vaginal microecological balance of VVC has become one of the key research directions in the current scientific and technological forefront.

[0003] The classic gynecological work in the Qing Dynasty, "Fu Qingzhu's Gynecology - Leukorrhea", states: "All leukorrhea is a damp syndrome", proposing that damp pathogen is the cause of gynecological diseases, and creating the famous Wudai Decoction. That is, spleen deficiency and dampness exuberance are the etiology and pathogenesis of this disease, and strengthening the spleen and resolving dampness is the treatment principle. Dampness is a yin pathogen, which cannot be dispersed without pungent herbs, cannot be transformed without warm herbs, and cannot be excreted without light herbs. Fu treated dampness by greatly tonifying the qi of the spleen and stomach. When the spleen qi is healthy, the dampness will disappear. Using Atractylodis Macrocephalae Rhizoma and Dioscoreae Rhizome as the monarch herbs, and combining with Atractylodis Lanceae Rhizome, Plantaginis Semen, Bupleuri Radix, Schizonepetae Herba and other herbs to disperse the damp pathogen through different channels from the middle jiao, lower jiao and with the help of pungent and dispersing herbs upward and outward. Wudai Decoction is now widely used in clinical practice for gynecological leukorrhea disorders with quite good curative effects. In vitro experimental studies have also confirmed that Wudai Decoction can improve the vaginal microenvironment and reduce the degree of chronic inflammation lesions, which is of great significance for clinical treatment. However, there is currently no clear mechanism study on the effect of Wudai Decoction on the vaginal microecology, thus limiting its application in recurrent vulvovaginal candidiasis. Summary of the Invention

[0004] To solve the above problems, the present invention provides the use of a composition in the preparation of a drug for preventing and / or treating vulvovaginal candidiasis, and the composition is prepared from the following raw materials in parts by weight:

[0005] Atractylodes macrocephala Koidz. stir-fried 10 - 50 parts, Dioscorea opposita Thunb. stir-fried 10 - 50 parts, Codonopsis pilosula (Franch.) Nannf. 10 - 50 parts, Paeonia lactiflora Pall. stir-fried 20 - 60 parts, Plantago asiatica L. 5 - 30 parts, Atractylodes lancea (Thunb.) DC. stir-fried 5 - 30 parts, Glycyrrhiza uralensis Fisch. 2 - 8 parts, Citrus reticulata Blanco cv. Chachi 2 - 8 parts, Schizonepeta tenuifolia Briq. 6 - 12 parts, Bupleurum chinense DC. 5 - 30 parts.

[0006] Further, the drug is for preventing the recurrence of vulvovaginal candidiasis.

[0007] Further, the drug has the effect of eradicating vaginal fungi.

[0008] Further, the drug has the effect of restoring the vaginal microbial community structure.

[0009] Even further, the vaginal microbial community includes CST II microbial flora and CST V microbial flora.

[0010] Furthermore, the drug has the effect of increasing the abundances of Fusobacteriota and Gemmatimonadota at the phylum level of the vaginal flora, the abundances of Fusobacteriia, Acidimicrobiia, BD2-11_terrestrial_group, and PAUC43f_marine_benthic_group at the class level, the abundances of Fusobacteriales, Clostridiales, Xanthomonadales, Clostridia, Alteromonadales, BD2-11_terrestrial_group, Actinomarinales, Oceanospirillales, and PAUC43f_marine_benthic_group at the order level, the abundances of Clostridiaceae, Christensenellaceae, Xanthomonadaceae, Fusobacteriaceae, Micrococcaceae, and BD2-11_terrestrial_group at the family level, the abundances of Clostridium_sensu_stricto_1, Christensenellaceae_R-7_group, BD2-11_terrestrial_group, and Salinimicrobium at the genus level, and the abundances of retuteri and murinus at the species level.

[0011] Furthermore, the drug has the effect of decreasing the abundances of Comamonadaceae, Flavobacteriaceae, and Streptococcaceae at the family level of the vaginal flora, the abundances of Klebsiella and Lactococcus at the genus level, and the abundances of Erysipelotrichaceae, Lactococcuslactis, and Lactococcus_brevis at the species level.

[0012] Furthermore, the drug has the effect of activating the STING signaling pathway and protecting mitochondria.

[0013] Furthermore, the composition is an oral preparation prepared from the powder of the active ingredient drug, or the aqueous extract or ethanol extract of the active ingredient drug, plus pharmaceutically acceptable excipients; the weight ratio of the active ingredient drug is as follows:

[0014] 30 parts of stir-fried Atractylodes macrocephala, 30 parts of stir-fried Dioscorea opposita, 10 parts of Codonopsis pilosula, 15 parts of stir-fried Paeonia lactiflora, 10 parts of Plantago asiatica, 10 parts of stir-fried Atractylodes lancea, 6 parts of Glycyrrhiza uralensis, 6 parts of Citrus reticulata Blanco, 9 parts of Sinapis alba, 10 parts of Bupleurum chinense.

[0015] Furthermore, the oral preparation is preferably Wendaidang.

[0016] The use of Wendaidang of the present invention in the preparation of a drug for preventing and / or treating vulvovaginal candidiasis, through clinical experiments and animal experiments, it is determined that Wendaidang can eradicate fungi in the vagina, restore the vaginal microbial community structure, activate the STING signaling pathway, protect mitochondria, thereby preventing the recurrence of vulvovaginal candidiasis, and has broad clinical application prospects.

[0017] Obviously, based on the above content of the present invention, according to the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can also be made.

[0018] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Description of the Drawings

[0019] Figure 1 : Alpha diversity analysis of two groups

[0020] Figure 2 Beta diversity analysis of two groups

[0021] Figure 3 Analysis results of the community structure of two groups (I: Phylum, II: Class, III: Order, IV: Family, V: Genus, VI: Species)

[0022] Figure 4 Differences in vaginal flora distribution before and after treatment in the fluconazole group (I: Phylum, II: Class, III: Family, IV: Genus, V: Species)

[0023] Figure 5 Differences in vaginal flora distribution before and after treatment in the Wendaidang group (I: Phylum, II: Class, III: Order, IV: Family, V: Genus, VI: Species)

[0024] Figure 6Differences in vaginal flora distribution between the Wandai decoction and fluconazole groups after treatment (I: Phylum, II: Class, III: Order, IV: Family, V: Genus, VI: Species)

[0025] Figure 7 LEfSe analysis of cladogram DA value and histogram

[0026] Figure 8 Gene distribution diagrams between two groups and between groups (I: Violin diagram, II: Venn diagram)

[0027] Figure 9 Analysis of vaginal bacterial abundance in the two groups after treatment (I: Phylum, II: Class, III: Order, IV: Family, V: Genus, VI: Species)

[0028] Figure 10 LEfSe analysis of cladogram DA value (I) and histogram (II)

[0029] Figure 11 Species difference analysis based on metagenomics (I: Phylum, II: Class, III: Order, IV: Family, V: Genus, VI: Species)

[0030] Figure 12 KEGG functional enrichment analysis diagram

[0031] Figure 13 CAZy enrichment analysis diagram

[0032] Figure 14 Statistical analysis of the number of differential metabolites between the two groups and within the group (I: differential metabolites, II, III, IV volcano plots)

[0033] Figure 15 Correlation analysis of metabolites between and within the two groups (I: Correlation analysis of differential metabolites in the Wandai decoction group, II: Correlation analysis of differential metabolites in the fluconazole group, III: Correlation analysis of top 50 differential metabolites between groups)

[0034] Figure 16 Differential metabolite expression abundance diagram among groups

[0035] Figure 17 Difference metabolites and KEGG metabolic pathway diagram between the two groups (I, III: differential metabolite enrichment analysis before and after treatment, II, IV: differential metabolic enrichment analysis between groups)

[0036] Figure 18 ​Results of association analysis between metagenomics and metabolomics in the fluconazole group

[0037] Figure 19 Results of association analysis between metagenomics and metabolomics in the Wandaitang group

[0038] Figure 20 Correlation analysis results of different species and metabolites before and after treatment

[0039] Figure 21 KEGG signaling pathway analysis results

[0040] Figure 22 Schematic diagram of changes in IFN-α and IL-1α levels in the two groups

[0041] Figure 23 Changes in mitochondrial DNA in the two groups before and after treatment

[0042] Figure 24 Changes in STING-IRF3-MX2 signaling pathways in the two groups before and after treatment Specific implementation method

[0043] Example 1 Clinical study on the prevention and treatment of VVC by Wandai Decoction

[0044] 1. Research subjects

[0045] 1.1 Case Source

[0046] The 70 patients enrolled were all VVC patients from the gynecology clinic of Jiangsu Provincial Hospital of Traditional Chinese Medicine. This study was reviewed and approved by the Ethics Committee of Jiangsu Provincial Hospital of Traditional Chinese Medicine, with the clinical ethics number (ethics approval number: 2021NL-228-02). The random number table method was used to divide them into the treatment group and the control group, with 35 cases in each group. Informed consent was obtained from all patients.

[0047] 1.2 Diagnostic criteria

[0048] 1.2.1 Western medicine diagnostic criteria

[0049] Diagnostic criteria for VVC: Developed in reference to the 2012 revised draft of the Diagnosis and Treatment Guidelines for Vulvovaginal Candida Infection (VVC) developed by the Chinese Medical Association's Gynecological and Obstetric Infection Collaborative Group, and the 2021 U.S. Centers for Disease Control and Prevention (CDC) guidelines for the diagnosis and treatment of vaginitis:

[0050] ①Symptoms: vulvar vaginal itching and burning pain; increased vaginal discharge. ②Signs: local congestion and swelling of the vulva, or cracks and scratches; white film-like or curd-like vaginal discharge. ③Auxiliary examinations: spores, blastospores or hyphae can be seen by hanging drop method (10% KOH) or smear method (Gram stain); or fungus positive can be confirmed by culture method. Patients who meet all three of the above conditions can be diagnosed.

[0051] ​​1.2.2 Traditional Chinese Medicine Diagnostic Criteria

[0052] Refer to "Traditional Chinese Gynecology", the Ministry of Health textbook, the national higher education traditional Chinese medicine college textbook, and the planning textbook of the National Higher Medical Education Research Association: the 2nd edition

[0053] Formulate the syndrome of spleen deficiency and dampness exuberance in leukorrhagia disease:

[0054] ①Main symptoms: The leukorrhea is profuse, white or light yellow in color, thin in texture, or like nasal mucus or saliva, continuous without odor. ②Secondary symptoms: The face is pale or sallow, the limbs are listless, there is discomfort in the epigastrium and hypochondrium, the appetite is poor and the stools are loose, or there is edema in the limbs.

[0055] ③Tongue and pulse: The tongue is pale and swollen, the tongue coating is white or greasy, and the pulse is thready and slow. If two of the main symptoms are met and one of the secondary symptoms is met, the diagnosis can be made.

[0056] 1.3 Case Selection Criteria

[0057] 1.3.1 Inclusion Criteria

[0058] (1) Meet the diagnostic criteria of VVC; (2) Women with sexual history and not yet menopausal, aged between 20 and 50 years old; (3) Have not used antibiotics or microecological regulators within 1 month, no bacterial or viral infections in other parts of the body, no systemic diseases or congenital diseases; (4) The patient voluntarily participates in this clinical study.

[0059] 1.3.2 Exclusion Criteria

[0060] (1) Menstruation; (2) Planning to be pregnant or pregnant or lactating within the past 3 months; (3) Having sexual intercourse or vaginal douching within 3 days; (4) Using antifungal drugs within 2 weeks, or taking oral contraceptives or glucocorticoids or immunosuppressants within the past 3 months; (5) Complicated with other vulvovaginal inflammatory diseases or having used medications for vaginitis; (6) Allergic to the study drug and its components; (7) Complicated with severe primary diseases such as liver and kidney, cardiovascular and cerebrovascular, and blood diseases, or having mental abnormalities; (8) Currently participating in other drug clinical studies.

[0061] 1.3.3 Withdrawal Criteria

[0062] (1) Not completing the treatment course and observation period specified in this protocol; (2) Those who withdraw on their own during the treatment process; (3) Those with incomplete follow-up data.

[0063] 1.3.4 Exclusion Criteria

[0064] (1) The case selection does not meet the inclusion criteria and meets the exclusion criteria; (2) Has not used the experimental drug; (3) Has no data after randomization; (4) Those who are lost to follow-up or stop taking the drug due to reasons; (5) Cases that take other drugs during the medication period; (6) Cases that participate in other clinical studies.

[0065] 2 Research methods

[0066] 2.1 Treatment grouping

[0067] 70 cases that met the inclusion and exclusion criteria were randomly divided into a treatment group of 35 cases and a control group of 35 cases.

[0068] 2.2 Treatment plan

[0069] 2.2.1 Treatment plan for the control group

[0070] Composition of traditional Chinese medicine prescription: stir-fried Atractylodes macrocephala 30g, stir-fried Dioscorea opposita 30g, Codonopsis pilosula 10g, stir-fried Paeonia lactiflora 15g, Plantago asiatica 10g, stir-fried Atractylodes lancea 10g, Licorice 6g, Tangerine peel 6g, Black spike 9g, Bupleurum chinense 10g. Preparation method: After soaking the medicine for 30 minutes, conduct the first decoction for 20 minutes; the second decoction for 15 minutes. Mix the two decoctions, about 200 ml.

[0071] Medication method: Start oral administration on the day after diagnosis, one dose per day, taken orally twice a day, morning and evening, stop taking during menstruation, and continuous administration for 7 days is one course of treatment, for a total of 2 courses.

[0072] 2.2.2 Treatment plan for the treatment group

[0073] Orally take fluconazole capsules, take 1 dose once on the first day of treatment, 150 mg / dose.

[0074] 2.3 Observation indicators

[0075] 2.3.1 Main endpoint indicators

[0076] According to the VANISH303 efficacy determination, the clinical efficacy is divided into clinical cure (Test of cure, TOC), which means that there are no all signs and symptoms of VVC, that is, the vulvovaginal signs and symptoms score (VSS) is 0. (The clinical cure standard here has been improved compared with the standards of some previous clinical trial studies. The previous clinical cure standard was set as VSS ≤ 2) If at the TOC visit, VSS ≤ 1 is called clinical improvement (CI).

[0077] (1) TOC, CI

[0078] Respectively within the 7th to 14th days, in this research project, the treatment group conducted VSS scoring on the 14th day, and the control group conducted it within the 7th - 14th days. The symptoms and signs scoring refers to the draft of the diagnosis and treatment specification for vulvovaginal candidiasis issued by the Infectious Diseases Collaboration Group of the Chinese Society of Obstetrics and Gynecology in 2004.

[0079] (2) Follow up (FU)

[0080] Both the treatment group and the control group were followed up from day 21 to day 30. The VSS score was evaluated according to the above scoring criteria. Whether the patient was clinically cured at the time of TOC or not, the symptoms of the patient completely disappeared at the time of FU. When the VVC score (VSS) was 0 at the time of FU, it was considered a complete clinical remission.

[0081] 2.3.2 Secondary endpoints

[0082] (1) Mycological eradication (ME)

[0083] No spores, blastospores or hyphae were seen by microscopy using the hanging drop method (10% KOH) or smear method (Gram staining); or the fungus was confirmed negative by culture.

[0084] (2) Recurrence rate

[0085] The cured patients in both groups were followed up 3 months after treatment. If the typical symptoms and signs of vulvovaginal candidiasis appeared during the follow-up period, and Candida hyphae or spores were seen in the vaginal secretion examination, it was considered a recurrence.

[0086] (3) Vaginal flora detection

[0087] Using the IlluminaHiSeq sequencing platform, 16sRNA was used to sequence the vaginal flora and perform bioinformatics analysis. The library construction sequencing and data analysis were assisted by Shanghai OE Biotech Co., Ltd.

[0088] The specific detection method was as follows: First, extract the sample DNA, and detect the DNA concentration of each sample after agarose gel electrophoresis. Then, using the genomic DNA as a template, use specific primers with barcodes to perform PCR amplification and magnetic bead purification on the corresponding region for bacterial diversity identification: 16S V3-V4 region (primers 343F and 798R); after purification, perform the second round of PCR amplification, electrophoresis detection, and magnetic bead purification, and finally perform Qubit quantification on the PCR product. After quantification, the PCR product was sequenced on the machine.

[0089] For the obtained sequencing data, first use the cutadapt software to cut off the primer sequences from the original data sequences. Then, use DADA2 to perform quality control analysis such as filtering, denoising, splicing, and dechimerization on the qualified data according to the default parameters of QIIME 2 to obtain the representative sequences and the ASV abundance table. Then, use the QIIME 2 software package to select the ASV representative sequences and compare and annotate them with the database.

[0090] (4) Metagenomic detection

[0091] The library construction sequencing and data analysis of metagenomic detection were completed by Shanghai OE Biotech Co., Ltd.

[0092] (5) Metabolomics detection

[0093] Metabolomics detection of vaginal secretions was performed using LC-MS / MS platform technology. Chromatographic detection conditions: Chromatographic column ACQUITY UPLC HSS T3 (100mm×2.1mm, 1.8um) was selected; column temperature: 45°C; mobile phase: A - water (containing 0.1% formic acid), B - acetonitrile (containing 0.1% formic acid); flow rate: 0.35mL / min; injection volume: 5μL.

[0094] (6) Correlation analysis between metagenomics and metabolomics

[0095] To further analyze and display the corresponding relationship between the relative abundances of phylum, class, order, family, genus, and species of microorganisms within and between groups and differential metabolites. Through the application of a monotonic equation, the Spearman calculation method was used for statistical analysis to measure the correlation between vaginal microorganisms and differential metabolites. Then, based on the correlation analysis between differential strains and metabolites, the TOP20 differential strains and metabolites were selected and a heatmap was drawn.

[0096] (7) Detection of changes in the levels of IL-1α, IFN-α, STING, IRF3, and MX2 by ELISA

[0097] The detection operation was carried out according to the ELISA kit instructions. The specific steps were as follows: The samples to be tested and standards were added to the corresponding reaction wells respectively, incubated at 37°C for 90 min, and washed 4 times; after the washing was completed, the biotinylated antibody working solution (100 μl / well) was added, incubated at 37°C for 60 min, and washed 4 times; the enzyme conjugate working solution (100 μl / well) was added, incubated at 37°C for 30 min, and washed 4 times; then the chromogenic reagent (100 μl / well) was added, protected from light, and incubated at 37°C for 10 - 20 min; after the incubation was completed, the stop solution (100 μl / well) was added, and after mixing, the OD450 value of each well was measured.

[0098] (8) Correlation analysis between metagenomics and metabolomics

[0099] To further analyze and display the corresponding relationship between the relative abundances of phylum, class, order, family, genus, and species of microorganisms within and between groups and differential metabolites. Through the application of a monotonic equation, the Spearman calculation method was used for statistical analysis to measure the correlation between vaginal microorganisms and differential metabolites. Then, based on the correlation analysis between differential strains and metabolites, the TOP20 differential strains and metabolites were selected and a heatmap was drawn.

[0100] (9) Detection of changes in the levels of IL-1α, IFN-α, STING, IRF3, and MX2 by ELISA

[0101] Detection was performed according to the instructions of the ELISA kit. The specific steps were as follows: The test samples and standards were added to the corresponding reaction wells respectively, incubated at 37 °C for 90 min, and the plates were washed 4 times; after the washing was completed, the biotinylated antibody working solution (100 μl / well) was added, incubated at 37 °C for 60 min, and the plates were washed 4 times; the enzyme conjugate working solution (100 μl / well) was added, incubated at 37 °C for 30 min, and the plates were washed 4 times; then the chromogenic reagent (100 μl / well) was added, incubated at 37 °C for 10 - 20 min in the dark; after the incubation was completed, the stop solution (100 μl / well) was added, and the OD450 value of each well was measured after mixing.

[0102] 3. Research results

[0103] 3.1 Comparison of the general conditions of the two groups

[0104] The age of the Wantaidang group was 33.43 ± 2.12 years, and that of the fluconazole group was 34.97 ± 1.53 years. When comparing the two groups, P > 0.05, indicating that the two groups were comparable.

[0105] 3.2 Main endpoint indicators

[0106] 3.2.1 Comparison of TOC between the two groups after treatment

[0107] Since 5 cases were lost in each group, 30 cases were included in the statistics for each group. After 2 weeks of intervention treatment with Wantaidang, when compared with the intervention treatment with fluconazole, the statistical analysis results of the data of the clinical cure visit (TOC) (VSS = 0) on the 7th - 14th day showed that the clinical cure rate of the Wantaidang group was 53.3% (16 / 30), and that of the fluconazole group was 46.7% (14 / 30); when comparing the two groups, the difference was not statistically significant (P > 0.05). The results suggest that the clinical efficacy of TOC in the Wantaidang group is equivalent to that in the fluconazole group.

[0108] 3.2.2 Comparison of CI between the two groups after treatment

[0109] After 2 weeks of intervention treatment with Wantaidang, when compared with the intervention treatment with fluconazole, the statistical analysis results of the data of the clinical remission visit (CI) (VSS ≤ 1) on the 7th - 14th day showed that the CI of the Wantaidang group at the visit was 90% (27 / 30); that of the fluconazole group was 76.67% (23 / 30); when comparing the two groups, the difference was statistically significant (P < 0.05). The results suggest that Wantaidang is superior to the fluconazole group in terms of the efficacy of reducing or alleviating the symptoms and signs of VCC.

[0110] 3.2.3 Comparison of FU between the two groups after treatment

[0111] After 2 weeks of Wandai decoction intervention, compared with fluconazole intervention, the clinical complete remission rate of the Wandai decoction group at the FU visit was 83.33% (25 / 30), and the clinical complete remission rate of the fluconazole group at the visit was 53.33% (16 / 30), and the difference was statistically significant (P < 0.05). The results suggest that at the 21st to 30th day visit, Wandai decoction is still more effective than fluconazole in alleviating or relieving VCC symptoms and signs.

[0112] 3.3 Secondary endpoints

[0113] 3.3.1 Mycological negative conversion rate in both groups after treatment

[0114] After 2 weeks of Wandai decoction intervention, compared with fluconazole intervention, at the 7-14th day of clinical cure visit (TOC), the statistical analysis results showed that 93.33% (28 / 30) of the patients in the Wandai decoction group achieved fungal eradication at the TOC visit; the fluconazole group was 83.33% (25 / 30)(25 / 30), and the difference was not statistically significant (P>0.05). The results suggest that the efficacy of Wandai decoction in fungal eradication is equivalent to that of the fluconazole group.

[0115] 3.3.2 Relapse rate of the two groups after treatment

[0116] At the third month of follow-up, the recurrence rate of the Wandai decoction group was 6.67% (2 / 30), and the recurrence rate of the fluconazole group was 43.33% (13 / 30), with a statistically significant difference (P<0.01). The results suggest that Wandai decoction is significantly better than the fluconazole group in preventing VVC recurrence and can significantly reduce the recurrence rate.

[0117] 3.3.3 Results of changes in vaginal flora

[0118] (1) Diversity index analysis

[0119] Diversity analysis mainly reflects the diversity of species in the vaginal environment. The results of Alpha diversity analysis and Beta diversity analysis of the changes in the flora of the two groups before and after treatment are shown below Figure 1 、 2 .

[0120] Alpha diversity analysis: Alpha diversity analysis results include alpha diversity index dilution curve, alpha diversity index violinplot analysis and Rank Abundance analysis. Figure 1 Show: The results of Alpha diversity analysis before and after treatment in the Wandai Decoction and fluconazole groups showed that the species richness of the Wandai Decoction group and the fluconazole group was similar before and after treatment, with no difference.

[0121] ​​Beta diversity analysis: Based on the analysis results of various distance matrices, PCoA can be used to observe the differences between individuals or groups. The difference in microbial diversity between the two groups and the difference in microbial diversity before and after treatment are as follows Figure 2 As shown. The results showed that the Beta diversity of vaginal microorganisms in the Wandai Decoction group was different from that in the Fluconazole group.

[0122] (2) Community structure analysis results

[0123] In the door (Phylum) ( Figure 3 I) Class Figure 3 II), Order( Figure 3 III), Branch(Family)( Figure 3 IV), Genus ( Figure 3 V), species ( Figure 3 VI) level, the bar chart of the top 15 species in abundance ranking is as follows. The results showed that after treatment, the vaginal microbial community of the Wandai Decoction group was CSTⅡ, V type (mainly L. gasseri and L. jensenii); after treatment, the vaginal microbial community of the fluconazole group was CSTIII, IV type (mainly L. iners and Anaeroboc organism), which was better than the fluconazole group in restoring the vaginal microbial community structure, and the difference was statistically significant (P<0.05).

[0124] (3) Differences in vaginal flora distribution

[0125] Differences in vaginal flora distribution before and after treatment in the fluconazole group: Through microbial multivariate statistical analysis, the Wilcoxon algorithm was used to analyze the difference in flora before and after treatment in the fluconazole group. The bar chart of the top 10 species in abundance ranking at the level of phylum, class, order, family, genus, and species is shown below. The results showed that after VVC patients were treated with fluconazole, the abundance of non-dominant vaginal flora such as anaerobic bacteria and cocci in the vaginal flora increased compared with before treatment, but did not increase the abundance of lactobacilli, and at the same time increased the Bacteroidetes species. The specific flora analysis results are as follows.

[0126] ① Phylum: There was no difference in vaginal flora before and after treatment with fluconazole. This indicates that fluconazole did not change the flora differences at the phylum level in the vaginal flora of VVC patients. ② Class: Fluconazole increased the abundance of Myxococcia in the vaginal flora of VVC patients ( Figure 4 ​​​​​​I). ③ Order: Fluconazole increases the abundances of Chitinophagales and Myxococccales in the vaginal microbiota of VVC patients and decreases the abundance of Peptococcales ( Figure 4 II). ④ Family: Fluconazole increases the abundances of Chitinophagaceae, Vulgatibacteraceae, and Chromobacteriaceae in the vaginal microbiota of VVC patients and decreases the abundances of Fusobacteriaceae, Leuconostocaceae, and Peptococcaceae ( Figure 4 III). ⑤ Genus: Fluconazole increases the abundances of Sediminibacterium, Erysipelatoclostridium, Butyricicoccus, Vulgatibacter, Porticoccus, and Actinomyces in the vaginal microbiota of VVC patients, decreases the abundance of Weissella, and increases the species Crenobacter ( Figure 4 IV). ⑥ Species: Fluconazole increases the abundances of Bacteroides_sp._g_Bacteroides, Bacteroides_caccae_Bacteroides, and Porticoccus_litoralia_g_Porticoccus in the vaginal microbiota of VVC patients and increases the two species Parabacteroides_sp._g_Parabacteroides and Methylobacterium_aquaticum_g_Methylobacterium-Methylorubrum ( Figure 4 V).

[0127] Differences in vaginal microbiota distribution after treatment with the WDT group:

[0128] Through multivariate statistical analysis of microorganisms and using the Wilcoxon algorithm to analyze the differences in the community structure before and after treatment in the WDT group, at the phylum ( Figure 5 I), class ( Figure 5 II), order ( Figure 5 III), family ( Figure 5 IV), genus ( Figure 5 V), species ( Figure 5VI) level, the bar chart of the top 10 species in terms of abundance is shown below. The results showed that after Wandai Decoction treatment, VVC patients could increase the abundance of dominant bacteria such as reuteri and murinus in the vaginal flora of VVC patients, and reduce the abundance of bacteria such as Erysipelotrichaceae_bacterium, Lactococcus_lactis_g_Lactococcus, and Lactococcus_brevis_g_Lactococcus. The specific flora analysis is as follows. ① Phylum: Wandai Decoction increases the abundance of Fusobacteriota and Gemmatimonadota in the vaginal flora of VVC patients; ② Class: Wandai Decoction increases the abundance of Fusobacteriia, Acidimicrobiia, BD2-11_terrestrial_group, and PAUC43f_marine_benthic_group in the vaginal flora of VVC patients. ③Order: Wandai decoction increased the abundance of Fusobacteriales, Clostridiales, Xanthomonadales, Clostridia, Alteromonadales, BD2-11_terrestrial_group, Actinomarinales, Oceanospirillales, and PAUC43f_marine_benthic_group in the vaginal flora of VVC patients. ④Family: Wandai decoction increased the abundance of Clostridiaceae, Christensenellaceae, Xanthomonadaceae, Fusobacteriaceae, Micrococcaceae, and BD2-11_terrestrial_group in the vaginal flora of VVC patients, and decreased the abundance of Comamonadaceae, Flavobacteriaceae, and Streptococcaceae. ⑤ Genus: Wandai decoction increases the abundance of Clostridium_sensu_stricto_1, Christensenellaceae_R-7_group, BD2-11_terrestrial_group, and Salinimicrobium in the vaginal flora of VVC patients, and reduces the abundance of Klebsiella and Lactococcus.⑥ Species: WDT increased the abundances of Lactobacillus such as retuteri and murinus in the vaginal flora of VVC patients, and decreased the abundances of bacteria such as Erysipelotrichaceae_bacterium, Lactococcus_lactis_g_Lactococcus, and Lactococcus_brevis_g_Lactococcus.

[0129] Differences in vaginal flora distribution between the WDT group and the fluconazole group after treatment:

[0130] Through multivariate statistical analysis of microorganisms and using the Wilcoxon algorithm to analyze the differential flora between the two groups after treatment, the bar charts of the top 10 species in terms of abundance ranking at the levels of phylum, class, order, family, genus, and species are as follows. The research results show that after treatment with fluconazole and WDT in VVC patients, the abundances of vaginal dominant flora such as L. gasseri and L. jensenii in the vaginal flora of the WDT group increased compared with those before treatment; while the levels of anaerobic bacteria species such as Prevotella_bivia_g_Prevotella and Atopobium_baginae_g_Atopobium in the vaginal flora increased after treatment in the fluconazole group. The specific flora analysis is as follows. ① Phylum: At the phylum level of Gemmatimonadota, the relative abundance after treatment with WDT (post-T) was higher ( Figure 6 I). ② Class: At the class levels of Bacci and BD2-11_terrestrial_group, the relative abundance after treatment with WDT (post-T) was higher, and there was little or almost no post-C in PAUC431_marine_benthic_group, while at the class levels of Bacteroidia, Coriobacteriia, and Acidimicrobiia, the relative abundance after treatment with fluconazole (Post-C) was higher ( Figure 6 II). ③ Order: At the order levels of Lactobacillales, Rhodospirillales, and ActinomarinalesB, the relative abundance after treatment with WDT (post-T) was higher, and there was little or almost no post-C in PAUC431_marine_benthic_group, while at the order levels of Coriobacteriales, Chitinophagales, and Alteromonadales, the relative abundance after treatment with fluconazole (Post-C) was higher (Figure 6 III). ④ Family: At the family level of Prevortellaceae, Atopobiaceae, Leptotrichiaceae, Eggerthellaceae, Xanthomonadaceae, Enterococcaceae, Chromobacteriaceae, the relative abundance after fluconazole treatment (Post-C) was higher. At the family level of BD2-11_terrestrial_group, Yersiniaceae, Marinobacteraceae, the relative abundance after treatment with WDT (Post-T) was higher. Figure 6 IV). ⑤ Genus: At the genus level of Prevortella, Atopobium, Sneathia, Burkholderia-Caballeronia-Paraburkholderia, Peptostreptococcus, Marvinbryantia, the relative abundance after fluconazole treatment (Post-C) was higher. At the genus level of Ureaplasma, BD2-11_terrestrial_group, Saiinimicrobium, the relative abundance after treatment with WDT (Post-T) was higher. Figure 6 V). ⑥ Species: At the species level of Lactobacillus_jensenii_g_Lactobacillus, Lactobacillus_gasseri_g_Lactobacillus, etc., the relative abundance after treatment with WDT (Post-T) increased significantly. At the species level of anaerobic bacteria such as Prevortella_bivia_g_Prevotella, Atopobium_baginae_g_Atopobium, etc., the relative abundance after fluconazole treatment (post-C) increased significantly. Figure 6 VI).

[0131] (4) LEfSe analysis of differential species composition:

[0132] Based on the above results, a deep LEfSe analysis was performed on the contribution degree of differential species to the differences, differential species annotation analysis, and the relative abundance of differential species in each sample between the fluconazole group and the WDT group. The results showed that there were 27 different species between the two groups. On the evolutionary cladogram of LEfSe analysis, the WDT group was mainly enriched in the classifications of h, i, j, k. The specific results are as follows Figure 7 shown as follows:

[0133] 4.3.4 Changes in vaginal secretion metagenomics

[0134] In order to clarify the vaginal microbial species and their functions that are different in efficacy between the two groups, and to deeply analyze the mechanisms of the above two groups that promote the conversion of Candida to negative, the vaginal secretions of 10 patients in each group before and after treatment were randomly selected for further metagenomic sequencing analysis. It mainly includes: analysis at the gene level, species level analysis and functional level analysis. The results showed that compared with the VVC patients in the Wandai Decoction group and the fluconazole group after treatment, the vaginal microbial community was CSTII and CSTV types, with L. gasseri (Lactobacillus gasseri) and L. jensenii (Lactobacillus jensenii) as the dominant species; Wandai Decoction may be better than fluconazole in preventing the growth and proliferation of Candida. The specific results are as follows.

[0135] (1) Distribution of gene numbers between the two groups and between groups:

[0136] Prodigal software was used to predict the ORF of the spliced ​​Contigs sequence of the sample genes in the two groups and between the groups, and it was translated into an amino acid sequence. Then, CD-HIT software was used to remove redundant genes, and identity 95% and coverage 90% were set. The longest sequence was selected as the representative sequence to obtain a non-redundant initial Unigene for gene clustering analysis. The horizontal axis is the grouping, and different groups are distinguished by different colors. The vertical axis is the number of genes, and ns means no difference. The analysis results show that the number of genes shared by the groups is 508, and there is no significant difference in the number of genes between the two groups and between the groups before and after treatment. The results are visualized as the Violin diagram shown below ( Figure 8 I) and Venn diagrams (also known as petal diagrams Figure 8 II). The number of genes in each sample, excluding the number of common genes, is shown on each petal of the Venn diagram.

[0137] (2) In terms of the abundance of bacterial species:

[0138] In the door (Phylum) ( Figure 9 I) Class Figure 9 II), Order( Figure 9 III), Branch(Family)( Figure 9 IV), Genus ( Figure 9 V), species ( Figure 9 VI) At the level, the barplot of the top 15 species ranked by abundance is as follows. The results show that the abundance of different species in each sample is different. The vaginal bacterial species in the two groups after treatment are enriched to the following top 15 different species at the phylum, class, order, family, genus, and species levels.

[0139] ​​(3) LEFse analysis of different species composition:

[0140] Based on the above results, the contribution of different species between the fluconazole group and the Wandaitang group to the difference, the difference species annotation analysis and the relative abundance of different species in each sample were deeply analyzed by LEfSe. The results showed that there were 19 different bacterial species between the two groups. In the LEfSe analysis of the evolutionary branch diagram, the Wandaitang group was mainly enriched in the a, b, and c categories. The specific results are as follows Figure 10 Shown:

[0141] (4) Species difference analysis:

[0142] The Wilcoxon rank-sum (two groups) method was used to test the species abundance data between groups to obtain the p value, and p < 0.05 was considered to be significantly different. Figure 11 I) Class Figure 11 II), Order( Figure 11 III), Branch(Family)( Figure 11 IV), Genus ( Figure 11 V), species ( Figure 11 VI) level, the bar chart of the top 12 species in abundance ranking is as follows. The results showed that after treatment, the vaginal flora of the patients in the Wandai Decoction group was reconstructed into a vaginal microbial community with L. gasseri (Lactobacillus gasseri) and L. jensenii (Lactobacillus jensenii) as the dominant species, and recovered to CSTII and CSTV types, while fluconazole was CSTIII and IV types, indicating that the Wandai Decoction group was superior to the fluconazole group in restoring the structure of the vaginal microbial community, and the difference was statistically significant (P<0.05).

[0143] (5) At the functional level: KEGG functional enrichment analysis was performed on the two groups of differentially expressed species ( Figure 12 ) and CAZy functional analysis ( Figure 13 ).

[0144] KEGG functional enrichment analysis: The results showed that in terms of the cell cycle and meiosis functions of Candida, the Wandai decoction group was significantly higher than the fluconazole group in terms of the cell cycle and meiosis signaling pathways (P < 0.05). Wandai decoction may be better than fluconazole in preventing the growth and proliferation of Candida.

[0145] CAZy functional analysis: The results showed that the Wandai decoction group was significantly higher than the fluconazole group in terms of drug resistance and resistance functional genes (P < 0.05).

[0146] 3.3.5 Metabolomic changes

[0147] ​​​​​​​​Before and after treatment, there were 59 differential metabolites in the Wandaitang group, while there were 43 in the fluconazole group. However, after treatment, the differential metabolites between the Wandaitang group and the fluconazole group increased to 120 ( Figure 14 I). Before and after treatment, 40 differential metabolites were up-regulated and 3 were down-regulated in the fluconazole group after treatment. In the Wandaitang group, 50 differential metabolites were up-regulated and 9 were down-regulated. When comparing the two groups, 53 metabolites were up-regulated and 67 were down-regulated after treatment. The results were visualized as volcano plots as shown in the following figures ( Figure 14 II, III, IV).

[0148] Results of correlation analysis: The correlation analysis of the top 50 differential metabolites in the Wandaitang group, the fluconazole group before and after treatment, and between the two groups after treatment is shown in Figure 15 I, II, III, where red represents positive correlation of differential metabolites and blue represents negative correlation of differential metabolites.

[0149] Before and after treatment, hierarchical clustering of the expression levels of the top 50 differential metabolites was performed. It was found that the expression abundance of differential metabolites in the Wandaitang group after treatment was significantly higher than that in the fluconazole group, and Wandaitang was significantly better than fluconazole in reducing differential metabolites ( Figure 16 ) Before and after treatment, enrichment analysis of the KEGG metabolic pathway of differential metabolites was performed. It was found that in the fluconazole group before and after treatment, the KEGG metabolic pathway enriched was Figure 17 I, and the KEGG metabolic pathway enriched by differential metabolites between groups was Figure 17 II. The above results suggest that Wandaitang may be significantly better than fluconazole in improving metabolic pathways such as local vaginal alpha-linolenic acid, glycerophospholipid metabolism, pentose and glucuronate interconversions, and arachidonic acid ( Figure 17 ).

[0150] 3.3.6 Results of the correlation analysis between metagenomics and metabolomics

[0151] The results of the correlation analysis between metagenomics and metabolomics are shown in Figure 18 、 19 . Each row represents a different strain of bacteria, and each column represents the corresponding metabolite. In the figure, orange-red represents positive correlation, blue represents negative correlation, the darker the color, the greater the correlation, and the closer the color is to white, the closer the correlation is to zero. In the figure, P < 0.001 is represented by ***, P < 0.01 is represented by **, and P < 0.05 is represented by *.

[0152] (1) Fluconazole group:

[0153] Results of the TOP20 analysis in the correlation analysis between differential species and differential metabolites before and after treatment ( Figure 18) are as follows. The top 20 different bacterial species and metabolites before and after fluconazole treatment are as follows. Among them, Candida species has significant differences before and after fluconazole treatment. The top 5 different bacterial species associated with each differential metabolite are

[0154] Prevotella_bivia, Veillonellaceae_bacterium_DNF00626, Prevotella_jejuni, Paenibacillus_sp._EKM205P, Prevotella_copri. The top five differential metabolites were Stearylcitrate, (S)-Nerolidol3-O-[aL-Rhamnopyranosyl-(1->4)-aL-rhamnopyranosyl-(1->2)-bD-glucopyran oside], 25-Hydroxy-24-epi-brassinolide, Dodecanamide, and 27-Norcholestanehexol. (2) Wandai Decoction Group:

[0155] TOP20 analysis results of correlation analysis between differential species and differential metabolites before and after treatment ( Figure 19 ) are as follows. The top 20 differential bacterial species and differential metabolites before and after treatment in the Wandai Decoction group are as follows. Sorted by correlation value, the top 5 differential bacterial species correlated with each differential metabolite are Prevotella_bivia, Lactobacillus_paragasseri, Lactobacillus_taiwanensis, Lactobacillus_phage_KC5a, and Lactobacillus_phage_KC5a. The top 5 differential metabolites correlated with each bacterial species are N-lauroyl glycine, Dodecanamide, 10-F2-dihomo-IsoP, 10-F2-dihomo-IsoP, and PG(19:0 / 0:0).

[0156] (3) Comparison between the two groups after treatment:

[0157] TOP20 analysis results of correlation analysis between differential species and differential metabolites before and after treatment ( Figure 20) are as follows, and the top 20 different bacterial species and metabolites between the groups before and after treatment are as follows. Sorted by correlation value, the top 5 different bacterial species for each differential metabolite correlation are Lactobacillus_gasseri, Ureaplasma_parvum, Ureaplasma_urealyticum, Lactobacillus_paragasseri, and Lactobacillus_jensenii. The top five differential metabolites associated with each strain were Dilauryl 3,3'-thiodipropionate, Spirolide E, 1-O-alpha-D-glucopyranosyl-1,2-nonadecandiol, DG (22:5(4Z,7Z,10Z,13Z,16Z) / 22:6(4Z,7Z,10Z,13Z,16Z,19Z) / 0:0), (25S)-5alpha-cholestan-3beta,4beta,6alpha,8beta,15alpha,16beta,26-heptol.

[0158] (4) KEGG signaling pathway analysis results

[0159] Based on the results of the association analysis between different bacterial species and different metabolites ( Figure 21 ), and KEGG signal pathway enrichment analysis was performed on differential metabolites and genes. The results showed that the differential signal pathways enriched in the fluconazole group before and after treatment were: NOD-like receptors signal pathway, biosynthesis of amino acids signal pathway. Among the KEGG enriched signal pathways of differential genes and metabolites between groups after treatment, the following four signal pathways were enriched: pentose and glucuronate interconversions signal pathway, glycerophospholopid metabolism signal pathway, phenylpropanoidbiosynthesis signal pathway, and biosynthesis of animo acids signal pathway. Combined with the results of metabolomics analysis, Wandai Decoction may improve vaginal microecology by affecting local vaginal glycerophospholipid metabolism, pentose and glucuronic acid interconversion and other metabolic pathways.

[0160] 3.3.7 Changes in IL-1α and IFN-α levels in the two groups of patients before and after treatment

[0161] ​​Based on the above research results, 10 patients were selected from each of the fluconazole group and the Wandai decoction group, and the levels of IFN-α and IL-1α in the vaginal lavage fluid of the two groups were further measured before and after treatment. The results showed that both Wandai decoction and fluconazole could increase the immune factor IFN-α in the vaginal microenvironment, and there was no statistically significant difference between the two groups. However, the Wandai decoction group was superior to the fluconazole group in reducing the local pro-inflammatory factor IL-1α in the vagina ( Figure 22 ), the difference was statistically significant (P<0.05); suggesting that Wandai decoction has a certain clinical effect on VVC patients, which may be through increasing the immune factor IFN-α in the vaginal microenvironment and reducing the inflammatory factor IL-1α in the vaginal microenvironment.

[0162] 3.3.8 Changes in mitochondrial DNA levels in the two groups before and after treatment

[0163] Based on the above research results, 10 patients in each of the fluconazole group and the Wandai decoction group were selected to further measure the mitochondrial damage DNA in the vaginal lavage fluid of the two groups before and after treatment. The results showed that after treatment, the fluconazole group had an aggravated effect on mitochondrial damage, while the Wandai decoction group did not show an aggravated effect on mitochondrial damage. Wandai decoction has an advantage over the fluconazole group in protecting mitochondria ( Figure 23 ).

[0164] 3.3.9 Changes in STING signaling pathways in the two groups of patients after treatment

[0165] Based on the above research results, 10 patients were selected from the fluconazole group and the Wandai decoction group, and the levels of STING, IRF3, and MX2 in the vaginal lavage fluid of the patients in the two groups before and after treatment were further tested. The results showed that ( Figure 24 ): After treatment in the Wandai decoction group, the levels of STING, IRF3, and MX2 were significantly increased (P<0.05, P<0.01). After treatment in the fluconazole group, only the level of STING increased (P<0.01), and there was no significant effect on the changes in the levels of IRF3 and MX2 (P>0.05).

[0166] 3.3.10 Security Comparison

[0167] Before and after treatment, the VVC patients in the fluconazole group and the Wandai decoction group were tested for alanine aminotransferase, serum aspartate aminotransferase, urea nitrogen, and creatinine. The results showed no abnormal results, and no adverse drug reactions occurred.

[0168] 4 Discussions

[0169] 4.1 Safety and effectiveness of Wandai decoction in treating VVC

[0170] For the treatment of diseases or clinical trials of any kind of plan, safety is of great importance. The results of this clinical trial show that: neither the treatment of Wudai Decoction nor fluconazole for VVC has adverse reactions on liver and kidney functions, nor drug adverse reactions. In terms of effectiveness, compared with fluconazole, in the clinical cure visit on the 7th - 14th day of treating VVC with Wudai Decoction and in terms of fungal clearance, the clinical efficacy of the two groups is comparable; but the results of the clinical remission visit (CI) on the 7th - 14th day show that: in terms of alleviating or relieving the symptoms and signs of VCC, the curative effect of Wudai Decoction is better than that of the fluconazole group; in addition, at the visit on the 21st to 30th day (FU), the curative effect of Wudai Decoction in alleviating or relieving the symptoms and signs of VCC is still better than that of fluconazole.

[0171] 4.2 Advantages of Wudai Decoction in treating VVC to prevent recurrence

[0172] In this study, at the 3 - month follow - up, Wudai Decoction can significantly reduce the recurrence rate in preventing the recurrence of VVC, which is significantly better than the fluconazole group.

[0173] 4.3 Restoration effect of Wudai Decoction on vaginal microecology

[0174] Restoring vaginal microecology is the ultimate goal of VVC treatment. In this study, we comprehensively used 16sRNA sequencing, metagenomics, and metabolomics methods and techniques, and initially found that the species richness before and after treatment in both the WDT group and the fluconazole group was similar, without differences. This indicates that neither WDT nor fluconazole treatment affects the species richness of the flora. What was also found in the experiment is that there are differences in the beta diversity of vaginal microbiota between the WDT group and the fluconazole group. The main manifestations are as follows: (1) The vaginal microbial communities affected by WDT treatment are mainly CSTⅡ and type V (dominated by L. gasseri and L. jensenii); the vaginal microbial communities affected by fluconazole treatment are mainly CSTIII and IV (dominated by L. iners and Anaeroboc organism); (2) The phyla, classes, orders, families, genera, and species affected by WDT and fluconazole are different: Among them, at the phylum level: WDT increases the abundances of Fusobacteriota and Gemmatimonadota in the vaginal flora of VVC patients; fluconazole does not change the microbial differences at the phylum level of the vaginal flora in VVC patients. At the class level: WDT increases the abundances of Fusobacteriia, Acidimicrobiia, BD2-11_terrestrial_group, and PAUC43f_marine_benthic_group in the vaginal flora of VVC patients; fluconazole increases the abundance of Myxococcia in the vaginal flora of VVC patients. At the order level: WDT increases the abundances of Fusobacteriales, Clostridiales, Xanthomonadales, Clostridia, Alteromonadales, BD2-11_terrestrial_group, Actinomarinales, Oceanospirillales, and PAUC43f_marine_benthic_group in the vaginal flora of VVC patients; fluconazole increases the abundances of Chitinophagales and Myxococccales in the vaginal flora of VVC patients and decreases the abundance of Peptococcales.At the family level: WDT increased the abundances of Clostridiaceae, Christensenellaceae, Xanthomonadaceae, Fusobacteriaceae, Micrococcaceae, and BD2-11_terrestrial_group in the vaginal microbiota of VVC patients, and decreased the abundances of Comamonadaceae, Flavobacteriaceae, and Streptococcaceae; fluconazole increased the abundances of Chitinophagaceae, Vulgatibacteraceae, and Chromobacteriaceae in the vaginal microbiota of VVC patients, and decreased the abundances of Fusobacteriaceae, Leuconostocaceae, and Peptococcaceae. At the genus level: WDT increased the abundances of Clostridium_sensu_stricto_1, Christensenellaceae_R-7_group, BD2-11_terrestrial_group, and Salinimicrobium in the vaginal microbiota of VVC patients, and decreased the abundances of Klebsiella and Lactococcus; fluconazole increased the abundances of Sediminibacterium, Erysipelatoclostridium, Butyricicoccus, Vulgatibacter, Porticoccus, and Actinomyces in the vaginal microbiota of VVC patients, decreased the abundance of Weissella, and increased the species Crenobacter; at the species level: WDT increased the abundances of lactobacilli such as retuteri and murinus in the vaginal microbiota of VVC patients, and decreased the abundances of Erysipelotrichaceae, Lactococcuslactis, and Lactococcus_brevis; fluconazole increased the abundances of Bacteroides_sp._g_Bacteroides, Bacteroides_caccae_Bacteroides, and Porticoccus_litoralia_g_Porticoccus in the vaginal microbiota of VVC patients, and increased the two species Parabacteroides_sp._g_Parabacteroides and Methylobacterium_aquaticum_g_Methylobacterium-Methylorubrum.The differences in the effects of Wandai Decoction and fluconazole at the level of phylum, class, order, family, genus, and species, combined with the comparable overall efficacy of the two groups, suggest that Wandai Decoction and fluconazole have their own characteristics in their effects on vaginal flora. These characteristics and differences may be different targets for restoring the balance of vaginal microecology, but different therapeutic drugs have different targets; in addition, it may be precisely because of these differences that potential research targets have been formed to interpret the mechanism of action of Wandai Decoction in alleviating clinical symptoms and signs and reducing the recurrence rate compared to fluconazole treatment. It is worthwhile to continue to reveal the mechanism of action of Wandai Decoction from a certain aspect in the future.

[0175] In terms of the mechanism of restoring vaginal microecology, it was found that: Wandai decoction was significantly higher than the fluconazole group in the signal pathways of cell cycle and meiosis; Wandai decoction may be better than fluconazole in preventing the growth and proliferation of Candida; and the effect of Wandai decoction treatment was also significantly stronger than that of the fluconazole group in terms of drug resistance and resistance functional genes; Wandai decoction may be significantly better than fluconazole in improving local vaginal α-linolenic acid, glycerophospholipid metabolism, pentose and glucuronic acid conversion, arachidonic acid and other metabolic pathways; the differential signal pathways enriched before and after treatment in the fluconazole group were: NOD-like receptors signal pathway, biosynthesis of amino acids signal pathway. This also provides a basis for further exploration of the differential mechanisms of fluconazole or Wandai decoction in restoring VVC vaginal microecology from the perspective of the combination of microecology, bacteria, and metabolism, and from the perspective of these signal pathways.

[0176] 4.4 Regulation of Wandai Decoction on the mitochondrial mtDNA-STING signaling pathway

[0177] In terms of the mechanism of restoring the vaginal microecology of VVC, this attempt was made to explore from the perspective of the mitochondrial mtDNA-STING signaling pathway. The results showed that Wandai Decoction could increase the levels of STING, IRF3, MX2, and IFN-α, and reduce the level of IL-1α, and had no aggravating effect on mtDNA damage before treatment; the fluconazole group could only increase the level of STING, and had no significant effect on reducing the level of IL-1α, and had an aggravating effect on mtDNA damage before treatment.

[0178] In summary, the present invention has determined through clinical experiments that Wandai Decoction can eradicate fungi in the vagina, restore the structure of vaginal microbial communities, activate the STING signaling pathway, and protect mitochondria in the treatment of VVC, thereby preventing the recurrence of vulvovaginal Candida. Its effect and safety are better than fluconazole, and it has broad clinical application prospects.

Claims

1. Use of a composition in the preparation of a medicament for preventing and / or treating vulvovaginal candidiasis, characterized in that: The composition is prepared from the following raw materials in parts by weight: 10-50 parts of stir-fried Atractylodes macrocephala, 10-50 parts of stir-fried yam, 10-50 parts of Codonopsis pilosula, 20-60 parts of stir-fried white peony root, 5-30 parts of Plantago seed, 5-30 parts of stir-fried Atractylodes lancea, 2-8 parts of liquorice, 2-8 parts of dried tangerine peel, 6-12 parts of black mustard spike, and 5-30 parts of Bupleurum.

2. The use according to claim 1, characterized in that: The medicine is a medicine for preventing the recurrence of vulvovaginal candidiasis.

3. The use according to claim 1 or 2, characterized in that: The drug has the effect of eradicating vaginal fungi.

4. The use according to claim 1 or 2, characterized in that: The drug has the effect of restoring the structure of vaginal microflora.

5. The use according to claim 4, characterized in that: The vaginal microbial community includes CST II type microbial community and CST V type microbial community.

6. The use according to claim 4, characterized in that: The drug has the effects of increasing the abundance of the vaginal flora at the phylum level of Fusobacteriota and Gemmatimonadota, at the class level of Fusobacteriia, Acidimicrobiia, BD2-11_terrestrial_group, and PAUC43f_marine_benthic_group, and at the order level of Fusobacteriales, Clostridiales, Xanthomonadales, Clostridia, Alteromonadales, BD2-11_terrestrial_group, Actinomarinales, Oceanospirillales, PAUC43f_marine_ The role of the abundance of benthic_group, the abundance of Clostridiaceae, Christensenellaceae, Xanthomonadaceae, Fusobacteriaceae, Micrococcaceae, and BD2-11_terrestrial_group at the family level, the abundance of Clostridium_sensu_stricto_1, Christensenellaceae_R-7_group, BD2-11_terrestrial_group, and Salinimicrobium at the genus level, and the abundance of reuteri and murinus at the species level.

7. The use according to claim 4, characterized in that: The drug has the effect of reducing the abundance of Comamonadaceae, Flavobacteriaceae, and Streptococcaceae at the family level, the abundance of Klebsiella and Lactococcus at the genus level, and the abundance of Erysipelotrichaceae, Lactococcuslactis, and Lactococcus_brevis at the species level of vaginal flora.

8. The use according to claim 1 or 2, characterized in that: The drug has the effect of activating the STING signaling pathway and protecting mitochondria.

9. The use according to claim 1 or 2, characterized in that: The composition is an oral preparation prepared from the powder of the raw material drug, or the water extract or alcohol extract of the raw material drug as the active ingredient, and pharmaceutically acceptable excipients; the weight ratio of the raw material drug is: 30 parts of stir-fried Atractylodes macrocephala, 30 parts of stir-fried Chinese yam, 10 parts of Codonopsis pilosula, 15 parts of stir-fried white peony root, 10 parts of Plantago seed, 10 parts of stir-fried Atractylodes lancea, 6 parts of Licorice, 6 parts of Tangerine peel, 9 parts of Black Mustard spicate, and 10 parts of Bupleurum.

10. The use according to claim 9, characterized in that: The oral preparation is preferably Wandai Decoction.

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