Use of composition in preparing drug for maintaining vaginal microecological balance

Abstract

In the treatment of VVC, Wan Dai Tang can eradicate fungi in the vagina, restore a vaginal microbial community structure, activate an STING signaling pathway, and protect mitochondria, thereby preventing the recurrence of vulvovaginal candidiasis. The Wan Dai Tang can significantly influence a CST V-type microbial population and a CST II-type microbial population in a vaginal microbial community, especially by improving the abundance of L. gasseri and L. jensenii in a vaginal flora. The Wan Dai Tang can also be applied to the maintenance of vaginal microecological balance.

Description

Use of a composition in preparing a medicine for maintaining vaginal microecological balance Technical Field

[0001] The present invention specifically relates to use of a composition in preparing a medicine for maintaining vaginal microecological balance. Background Art

[0002] Vaginal microecological balance is crucial to women's reproductive health. Imbalance in the vaginal microbiome is the primary cause of various gynecological infections. For example, bacterial vaginosis (BV) is a polymicrobial imbalance caused by a decrease in lactobacilli. Vulvovaginal candidiasis (VVC) is also caused by a decrease in the number of lactobacilli, which creates favorable conditions for the overgrowth of Candida. Studies have shown that an imbalance in the vaginal microbiome can also lead to cervicitis, pelvic inflammatory disease, infertility, premature birth, premature rupture of membranes, and neonatal infections.

[0003] Vulvovaginal candidiasis (VVC) is a common lower genital tract infection in women. Approximately 75% of women will experience at least one episode in their lifetime, and 40% to 45% will experience two or more episodes of VVC in a single year, making it second only to bacterial vaginosis in incidence. Recurrent vulvovaginal candidiasis (RVVC) occurs when four or more episodes occur within a year; 13% to 28% of VVC patients progress to RVVC. Recent data indicate that RVVC affects approximately 138 million women annually (range, 103-172 million), with a global annual prevalence of 3,871 per 100,000 women. The prevalence in my country is higher, at 4,436 per 100,000 women. Globally, 372 million women are affected by RVVC in their lifetime, contributing to the global economic burden. Modern research has revealed that the essence of VVC lies in an imbalance in the vaginal microbiome, which is unique in its characteristics. Although VVC is a non-fatal disease, and currently available antifungal drugs can effectively inhibit or kill fungi, their use exacerbates the imbalance of the vaginal microbiome and the immune microenvironment, impacting patients' quality of life, physical and mental health, and increasing the economic burden. Repeated use of antifungal drugs increases the risk of drug resistance and complicates treatment. While modern medicine has addressed the microbiome and bacterial symbiosis, and probiotic preparations have been developed to alleviate bacterial imbalance, long-term results remain unsatisfactory. Therefore, restoring the balance of the vaginal microbiome in VVC has become a key focus of current scientific research.

[0004] The classic Qing Dynasty gynecological treatise, "Fu Qingzhu's Treatise on Women's Leucorrhea," states, "Leucorrhea is all caused by dampness." Fu Qingzhu's book, "Fu Qingzhu's Treatise on Women's Leucorrhea," proposes that dampness is the cause of gynecological diseases and coined the famous Wandai Decoction. This suggests that spleen deficiency and excessive dampness are the etiology and pathogenesis of the disease, and the principle of treatment is to strengthen the spleen and eliminate dampness. Dampness is a yin evil that cannot be dissipated without pungent, warm, or penetrating herbs. Fu Qingzhu's treatment of dampness involves tonifying the spleen and stomach qi, strengthening the spleen and dispelling dampness. He used Atractylodes macrocephala and Chinese yam as the main herbs, complemented by Atractylodes lancea, Plantago seed, Bupleurum chinense, and Schizonepeta tenuifolia, dispelling dampness from the middle and lower jiao, and leveraging the upward and outward forces of pungent and dispersing herbs. Wandai Decoction is now widely used clinically for gynecological leucorrhea, demonstrating excellent efficacy. In vitro studies have also demonstrated that Wandai Decoction improves the vaginal microenvironment and reduces the severity of chronic inflammatory lesions, making it of significant clinical significance. However, there is currently no clear research on the mechanism by which Wandai Decoction affects vaginal microecology, which limits its application in maintaining vaginal microecological balance and recurrent vulvovaginal candidiasis. Summary of the Invention

[0005] To solve the above problems, the present invention provides a composition for use in preparing a drug for maintaining vaginal microecological balance, wherein the composition is prepared from the following raw materials in parts by weight:

[0006] 10-50 parts of stir-fried Atractylodes macrocephala, 10-50 parts of stir-fried Chinese 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 Licorice root, 2-8 parts of Tangerine peel, 6-12 parts of Black Mustard Bud, and 5-30 parts of Bupleurum.

[0007] Furthermore, the drug has the effect of increasing the abundance of CST V type microbial population and / or CST II type microbial population.

[0008] Furthermore, the dominant bacterial species of the CST V type microbial community include L. jensenii, and the dominant bacterial species of the CST II type microbial community include L. gasseri.

[0009] The present invention also provides a composition for use in preparing a medicament for promoting the growth of CST V type microflora and / or CST II type microflora in the vagina, wherein the composition is prepared from the following raw materials in parts by weight:

[0010] 10-50 parts of stir-fried Atractylodes macrocephala, 10-50 parts of stir-fried Chinese 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 Licorice root, 2-8 parts of Tangerine peel, 6-12 parts of Black Mustard Bud, and 5-30 parts of Bupleurum.

[0011] The dominant bacterial species of the CST V type microbial community include L. jensenii, and the dominant bacterial species of the CST II type microbial community include L. gasseri.

[0012] The present invention also provides a composition for use in preparing a medicament for treating vaginal diseases caused by reduced reproductive capacity of CST II type microflora and / or CST V type microflora, wherein the composition is prepared from the following raw materials in parts by weight:

[0013] 10-50 parts of stir-fried Atractylodes macrocephala, 10-50 parts of stir-fried Chinese 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 Licorice root, 2-8 parts of Tangerine peel, 6-12 parts of Black Mustard Bud, and 5-30 parts of Bupleurum.

[0014] The dominant bacterial species of the CST V type microbial community include L. jensenii, and the dominant bacterial species of the CST II type microbial community include L. gasseri.

[0015] The present invention also provides a use of a composition in preparing a medicament for preventing and / or treating vulvovaginal candidiasis, wherein the composition is prepared from the following raw materials in parts by weight:

[0016] 10-50 parts of stir-fried Atractylodes macrocephala, 10-50 parts of stir-fried Chinese 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 Licorice root, 2-8 parts of Tangerine peel, 6-12 parts of Black Mustard Bud, and 5-30 parts of Bupleurum.

[0017] Furthermore, the drug is a drug for preventing the recurrence of vulvovaginal candidiasis.

[0018] Furthermore, the drug has the effect of eradicating vaginal fungi.

[0019] Furthermore, the drug has the effect of restoring the structure of vaginal microbial communities.

[0020] Furthermore, the vaginal microbial community includes CST II type microbial community and CST V type microbial community.

[0021] Furthermore, the drug has the effect of increasing the abundance of the vaginal flora at the phylum level, Fusobacteriota, Gemmatimonadota, at the class level, Fusobacteriia, Acidimicrobiia, BD2-11_terrestrial_group, PAUC43f_marine_benthic_group, at the order level, Fusobacteriales, Clostridiales, Xanthomonadales, Clostridia, Alteromonadales, BD2-11_terrestrial_group, Actinomarinales, Oceanospirillale s, PAUC43f_marine_benthic_group abundance at the family level, Clostridiaceae, Christensenellaceae, Xanthomonadaceae, Fusobacteriaceae, Micrococcaceae, and BD2-11_terrestrial_group abundance at the family level, Clostridium_sensu_stricto_1, Christensenellaceae_R-7_group, BD2-11_terrestrial_group, and Salinimicrobium abundance at the genus level, and reuteri and murinus abundance at the species level.

[0022] Furthermore, the drug has the effect of reducing the abundance of vaginal flora at the family level of Comamonadaceae, Flavobacteriaceae, and Streptococcaceae, at the genus level of Klebsiella and Lactococcus, and at the species level of Erysipelotrichaceae, Lactococcuslactis, and Lactococcus_brevis.

[0023] Furthermore, the drug has the effect of activating the STING signaling pathway and protecting mitochondria.

[0024] Furthermore, the composition is an oral preparation prepared from the powder of the raw material drug, or the aqueous 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:

[0025] 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 paeonia lactiflora, 10 parts of Plantago seeds, 10 parts of stir-fried Atractylodes lancea, 6 parts of Radix Glycyrrhizae, 6 parts of dried tangerine peel, 9 parts of black mustard spicate, 10 parts of Radix Bupleuri.

[0026] Furthermore, the oral preparation is preferably Wandai Decoction.

[0027] The CSTV type microbial community described in the present invention is a vaginal microbial community dominated by Lactobacillus jensenii.

[0028] The CSTⅡ type microbial community described in the present invention is a vaginal microbial community dominated by Lactobacillus gasseri.

[0029] The present invention discloses the use of Wandai Decoction in the preparation of a medicament for preventing and / or treating vulvovaginal candidiasis. Clinical and animal experiments have demonstrated that Wandai Decoction can eradicate vaginal fungi, restore vaginal microbial community structure, activate the STING signaling pathway, and protect mitochondria in the treatment of VVC, thereby preventing recurrence of vulvovaginal candidiasis. Wandai Decoction significantly affects the CSTV and CSTII microbiota within the vaginal microbiome, particularly increasing the abundance of Lactobacillus gasseri and Lactobacillus jensenii. It can also be used to maintain vaginal microecological balance, demonstrating broad clinical application prospects.

[0030] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0031] The following further describes the above content of the present invention in detail 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 fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1: Alpha diversity analysis of two groups

[0033] Figure 2 Beta diversity analysis of two groups

[0034] Figure 3. Results of community structure analysis of the two groups (I: Phylum, II: Class, III: Order, IV: Family, V: Genus, VI: Species)

[0035] Figure 4 Differences in vaginal flora distribution before and after fluconazole treatment (I: Phylum, II: Class, III: Family, IV: Genus, V: Species)

[0036] Figure 5 Differences in vaginal flora distribution before and after treatment in the Wandai Decoction group (I: Phylum, II: Class, III: Order, IV: Family, V: Genus, VI: Species)

[0037] Figure 6 Differences 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)

[0038] Figure 7 DA value and histogram of LEfSe analysis cladogram

[0039] Figure 8 Gene distribution between the two groups and between groups (I: Violin diagram, II: Venn diagram)

[0040] Figure 9 Analysis of vaginal bacterial species abundance in the two groups after treatment (I: Phylum, II: Class, III: Order, IV: Family, V: Genus, VI: Species)

[0041] Figure 10 DA value (I) and histogram (II) of LEfSe analysis cladogram

[0042] Figure 11: Species diversity analysis using metagenomics (I: Phylum, II: Class, III: Order, IV: Family, V: Genus, VI: Species)

[0043] Figure 12 KEGG functional enrichment analysis

[0044] Figure 13 CAZy enrichment analysis

[0045] 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)

[0046] 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 the top 50 differential metabolites between the two groups)

[0047] Figure 16 Differential metabolite expression abundance between groups

[0048] Figure 17 Diagram of differential metabolites and KEGG metabolic pathways between the two groups (I, III: differential metabolite enrichment analysis before and after treatment, II, IV: differential metabolic enrichment analysis between groups)

[0049] Figure 18 Results of association analysis between metagenomics and metabolomics in the fluconazole group

[0050] Figure 19 Results of correlation analysis between metagenomics and metabolomics in the Wandaitang group

[0051] Figure 20 Correlation analysis results of different species and metabolites before and after treatment

[0052] Figure 21 KEGG signaling pathway analysis results

[0053] Figure 22 Schematic diagram of changes in IFN-α and IL-1α levels in the two groups

[0054] Figure 23 Changes in mitochondrial DNA in the two groups before and after treatment

[0055] Figure 24 Changes in STING-IRF3-MX2 signaling pathway before and after treatment in the two groups DETAILED DESCRIPTION

[0056] Example 1 Clinical Study on the Prevention and Treatment of VVC by Wandai Decoction

[0057] 1. Research subjects

[0058] 1.1 Case Source

[0059] The 70 patients enrolled were all VVC patients admitted to 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 approval number (Ethics Approval No.: 2021NL-228-02). They were randomly assigned to a treatment group and a control group, with 35 patients in each group. Informed consent was obtained from all patients.

[0060] 1.2 Diagnostic criteria

[0061] 1.2.1 Western medicine diagnostic criteria

[0062] VVC diagnostic criteria: These criteria were developed in conjunction with the 2012 revised draft of the Diagnosis and Treatment Guidelines for Vulvovaginal Candida Infection (VVC) developed by the Chinese Medical Association's Gynecology and Obstetrics Infection Collaborative Group and the 2021 U.S. Centers for Disease Control and Prevention (CDC) guidelines for the diagnosis and treatment of vaginitis.

[0063] ① Symptoms: Vulvovaginal itching and burning; increased vaginal discharge. ② Signs: Localized vulvar congestion and swelling, or cracks and scratches; white film-like or curd-like vaginal discharge. ③ Ancillary tests: Microscopic examination using a hanging drop (10% KOH) or smear (Gram stain) showing spores, blastospores, or hyphae; or positive fungal culture. A diagnosis is made if all three of the above criteria are met.

[0064] 1.2.2 Traditional Chinese Medicine Diagnostic Criteria

[0065] Reference: "Chinese Medicine Gynecology" Ministry of Health "Twelfth Five-Year Plan" textbook, national higher Chinese medicine college textbook, national higher medical textbook construction research association planning textbook: 2nd edition

[0066] Formulate the syndrome of spleen deficiency and dampness excess for leucorrhea:

[0067] ① Main symptoms: profuse, white or light yellowish vaginal discharge, thin in texture, or resembling mucus or saliva, continuous and odorless. ② Secondary symptoms: pale or sallow complexion, fatigue in the limbs, abdominal discomfort, poor appetite, loose stools, or edema in the limbs. ③ Tongue and pulse: pale, plump tongue with a white or greasy coating, and a thready, slow pulse. A diagnosis can be made if two of the main symptoms and one of the secondary symptoms are met.

[0068] 1.3 Case selection criteria

[0069] 1.3.1 Inclusion criteria

[0070] (1) Meet the diagnostic criteria for VVC; (2) Women with a history of sexual activity who have not yet reached menopause, aged between 20 and 50 years old; (3) Have not used antibiotics or microecological regulators within 1 month, have no bacterial or viral infections in other parts of the body, and have no systemic or congenital diseases; (4) Patients voluntarily participate in this clinical study.

[0071] 1.3.2 Exclusion criteria

[0072] (1) Menstruation; (2) Planning pregnancy, pregnancy, or breastfeeding within the past three months; (3) Having sexual intercourse or vaginal douching within three days; (4) Using antifungal drugs within two weeks, or taking oral contraceptives, glucocorticoids, or immunosuppressants within the past three months; (5) Having other vulvovaginal inflammatory diseases or taking medication for vaginitis; (6) Being allergic to the study drugs and their ingredients; (7) Having serious primary diseases such as liver, kidney, cardiovascular, cerebrovascular, or blood diseases, or having mental disorders; (8) Participating in other drug clinical studies.

[0073] 1.3.3 Shedding standards

[0074] (1) Those who have not completed the course of treatment and observation period specified in this protocol; (2) Those who voluntarily withdraw from the treatment process; (3) Those with incomplete follow-up data.

[0075] 1.3.4 Elimination criteria

[0076] (1) Cases that do not meet the inclusion criteria but meet the exclusion criteria; (2) have never used the trial drug; (3) have no data after randomization; (4) are lost to follow-up or stop taking the drug for some reason; (5) take other drugs at the same time during the medication period; (6) participate in other clinical studies.

[0077] 2 Research Methods

[0078] 2.1 Treatment groups

[0079] A total of 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.

[0080] 2.2 Treatment options

[0081] 2.2.1 Treatment plan for the control group

[0082] Traditional Chinese medicine formula: stir-fried Atractylodes macrocephala 30g, stir-fried Chinese yam 30g, Codonopsis pilosula 10g, stir-fried White Peony Root 15g, Plantago seed 10g, stir-fried Atractylodes lancea 10g, Licorice root 6g, Tangerine peel 6g, Black Mustard 9g, Bupleurum 10g. Preparation: Soak the herbs for 30 minutes, then decoct for 20 minutes; then for 15 minutes. Combine the two decoctions, adding approximately 200 ml.

[0083] Dosage: Start taking orally from the day after diagnosis, one dose per day, divided into two doses in the morning and evening. Stop taking the medicine during menstruation. One course of treatment is 7 days of continuous medication, for a total of 2 courses of treatment.

[0084] 2.2.2 Treatment regimen of treatment group

[0085] Take fluconazole capsules orally, once on the first day of treatment, 150 mg / time.

[0086] 2.3 Observation indicators

[0087] 2.3.1 Primary endpoint

[0088] According to the VANISH 303 efficacy assessment, clinical efficacy is categorized as clinical cure (Test of Cure, TOC), which refers to the absence of all signs and symptoms of VVC, defined as a Vulvovaginal Signs and Symptoms Score (VSS) of 0. (This clinical cure criterion is an improvement over that used in some previous clinical trials, which previously defined a VSS ≤ 2.) A VSS ≤ 1 at the TOC visit is considered clinical improvement (CI).

[0089] (1)TOC, CI

[0090] VSS scores were performed between days 7 and 14, with the treatment group receiving the score on day 14 and the control group receiving the score between days 7 and 14. Symptom and sign scores were based on the draft guidelines for the diagnosis and treatment of vulvovaginal candidiasis published in 2004 by the Infectious Diseases Collaborative Group of the Chinese Society of Obstetrics and Gynecology.

[0091] (2)Follow up(FU)

[0092] Both the treatment and control groups were followed up from day 21 to day 30. VSS scores were calculated according to the above scoring criteria. Regardless of whether the patient was clinically cured at the TOC visit, the patient's symptoms were completely resolved at the FU visit. A VSS score of 0 at the FU visit was considered clinical complete remission.

[0093] 2.3.2 Secondary End Points

[0094] (1) Mycological eradication (ME)

[0095] No spores, blastospores or hyphae were found by microscopic examination using the hanging drop method (10% KOH) or smear method (Gram stain); or fungus was negative by culture.

[0096] (2) Recurrence rate

[0097] The cured patients in both groups were followed up for 3 months after treatment. Relapse was considered if typical symptoms and signs of vulvovaginal candidiasis appeared during the follow-up period, and Candida hyphae or spores were found in vaginal secretions.

[0098] (3) Vaginal flora detection

[0099] Vaginal microbiota were sequenced and analyzed using 16s RNA sequencing on the Illumina HiSeq platform. Shanghai Ouyi Biomedical Technology Co., Ltd. assisted with library construction, sequencing, and data analysis.

[0100] The specific detection method involves extracting sample DNA and measuring the DNA concentration of each sample after agarose gel electrophoresis. Next, using genomic DNA as a template, barcoded primers are used to amplify the 16S V3-V4 region (primers 343F and 798R), a region associated with bacterial diversity identification, and perform magnetic bead purification. Following purification, a second round of PCR amplification, electrophoresis detection, and magnetic bead purification are performed. Finally, the PCR products are quantified using Qubit assays. After quantification, the PCR products are sequenced.

[0101] For the acquired sequencing data, we first used cutadapt software to trim the primer sequences from the raw data. Then, using DADA2, we performed quality control analysis, including filtering, noise reduction, splicing, and chimera removal, on the qualified data using QIIME 2 default parameters. This yielded representative sequences and an ASV abundance table. Finally, we used the QIIME 2 software package to select representative ASV sequences for alignment and annotation with the database.

[0102] (4) Metagenomic testing

[0103] The library construction, sequencing and data analysis of the metagenomic testing were completed by Shanghai Ouyi Biomedical Technology Co., Ltd.

[0104] (5) Metabolomics testing

[0105] Vaginal secretions were metabolomics analyzed using LC-MS / MS platform technology. Chromatographic detection conditions: ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm); column temperature: 45°C; mobile phase: A-water (containing 0.1% formic acid), B-acetonitrile (containing 0.1% formic acid); flow rate: 0.35 mL / min; injection volume: 5 μL.

[0106] (6) Analysis of association between metagenomics and metabolomics

[0107] To further analyze and demonstrate the correlation between the relative abundance of microorganisms at the phylum, class, order, family, genus, and species level within and between groups and their differential metabolites, we applied a monotonic equation and the Spearman calculation method to analyze the correlation between vaginal microorganisms and differential metabolites. Based on the correlation analysis between differential bacterial species and metabolites, we selected the top 20 differential bacterial species and metabolites, and created a heat map.

[0108] (7) ELSA detection of changes in IL-1α, IFN-α, STING, IRF3, and MX2 levels

[0109] The detection operation was performed according to the ELISA kit instructions. The specific steps are as follows: add the sample to be tested and the standard to the corresponding reaction wells, incubate at 37°C for 90 minutes, and wash the plate four times; after washing, add the biotinylated antibody working solution (100 μl / well), incubate at 37°C for 60 minutes, and wash the plate four times; add the enzyme conjugate working solution (100 μl / well), incubate at 37°C for 30 minutes, and wash the plate four times; then add the color developer (100 μl / well), protect from light, and incubate at 37°C for 10-20 minutes; after incubation, add the stop solution (100 μl / well), mix well, and then measure the OD450 value of each well.

[0110] (8) Association analysis between metagenomics and metabolomics

[0111] To further analyze and demonstrate the correlation between the relative abundance of microorganisms at the phylum, class, order, family, genus, and species level within and between groups and their differential metabolites, we applied a monotonic equation and the Spearman calculation method to analyze the correlation between vaginal microorganisms and differential metabolites. Based on the correlation analysis between differential bacterial species and metabolites, we selected the top 20 differential bacterial species and metabolites, and created a heat map.

[0112] (9) ELSA detection of changes in IL-1α, IFN-α, STING, IRF3, and MX2 levels

[0113] The detection operation was performed according to the ELISA kit instructions. The specific steps are as follows: add the sample to be tested and the standard to the corresponding reaction wells, incubate at 37°C for 90 minutes, and wash the plate four times; after washing, add the biotinylated antibody working solution (100 μl / well), incubate at 37°C for 60 minutes, and wash the plate four times; add the enzyme conjugate working solution (100 μl / well), incubate at 37°C for 30 minutes, and wash the plate four times; then add the color developer (100 μl / well), protect from light, and incubate at 37°C for 10-20 minutes; after incubation, add the stop solution (100 μl / well), mix well, and then measure the OD450 value of each well.

[0114] 3. Research Results

[0115] 3.1 Comparison of general conditions between the two groups

[0116] The age of the patients in the Wandai decoction group was 33.43±2.12 years old, and that in the fluconazole group was 34.97±1.53 years old. The two groups were comparable, P>0.05.

[0117] 3.2 Primary End Points

[0118] 3.2.1 Comparison of TOC between the two groups after treatment

[0119] Because 5 patients dropped out of each group, 30 patients were included in the analysis. After 2 weeks of Wandai Decoction treatment, compared with fluconazole treatment, statistical analysis of the clinical cure visit (TOC) (VSS = 0) data from days 7 to 14 showed that 53.3% (16 / 30) of the Wandai Decoction group and 46.7% (14 / 30) of the fluconazole group were cured. There was no statistically significant difference between the two groups (P > 0.05). These results suggest that the TOC clinical efficacy of the Wandai Decoction and fluconazole groups is comparable.

[0120] 3.2.2 Comparison of CI between the two groups after treatment

[0121] After two weeks of Wandai decoction treatment, statistical analysis of clinical remission visit (CI) (VSS ≤ 1) data from days 7 to 14 compared with those treated with fluconazole showed that the CI was 90% (27 / 30) in the Wandai decoction group and 76.67% (23 / 30) in the fluconazole group. The difference between the two groups was statistically significant (P < 0.05). These results suggest that Wandai decoction is more effective than fluconazole in alleviating or relieving VCC symptoms and signs.

[0122] 3.2.3 Comparison of FU between the two groups after treatment

[0123] After two weeks of Wandai decoction treatment, compared with fluconazole treatment, the clinical complete remission rate at the FU visit was 83.33% (25 / 30) in the Wandai decoction group and 53.33% (16 / 30) in the fluconazole group, with a statistically significant difference (P < 0.05). The results suggest that Wandai decoction is still more effective than fluconazole in alleviating or relieving VCC symptoms and signs at the 21st to 30th day visit.

[0124] 3.3 Secondary endpoints

[0125] 3.3.1 Mycological negative conversion rate in the two groups after treatment

[0126] After two weeks of Wandai Decoction treatment, compared with fluconazole treatment, statistical analysis of the cure visit (TOC) on days 7-14 showed that 93.33% (28 / 30) of patients in the Wandai Decoction group achieved fungal eradication at the TOC visit, compared with 83.33% (25 / 30) in the fluconazole group (P>0.05). The results suggest that Wandai Decoction is as effective as fluconazole in eradicating fungi.

[0127] 3.3.2 Relapse rate after treatment in the two groups

[0128] At the third month of follow-up, the recurrence rate in the Wandai Decoction group was 6.67% (2 / 30) and in the fluconazole group was 43.33% (13 / 30), with a statistically significant difference (P < 0.01). The results suggest that Wandai Decoction is significantly superior to the fluconazole group in preventing VVC recurrence and can significantly reduce the recurrence rate.

[0129] 3.3.3 Results of changes in vaginal flora

[0130] (1) Diversity index analysis

[0131] Diversity analysis primarily reflects the diversity of species within the vaginal environment. The results of alpha diversity analysis and beta diversity analysis of the bacterial flora changes in the two groups before and after treatment are shown in Figures 1 and 2, respectively.

[0132] Alpha diversity analysis: Alpha diversity analysis results include alpha diversity index dilution curves, alpha diversity index violinplot analysis, and rank abundance analysis. Figure 1 shows the alpha diversity analysis results before and after treatment with Wandai Decoction and fluconazole. Within-group and between-group comparisons of species richness before and after treatment showed no difference.

[0133] Beta Diversity Analysis: Based on the analysis results of various distance matrices, PCoA can be used to observe differences between individuals or groups. The differences in microbial diversity between the two groups and before and after treatment are shown in Figure 2. The results showed that the vaginal microbial beta diversity in the Wandai Decoction group was different from that in the fluconazole group.

[0134] (2) Community structure analysis results

[0135] The following bar charts show the top 15 species ranked by abundance at the phylum (Figure 3I), class (Figure 3II), order (Figure 3III), family (Figure 3IV), genus (Figure 3V), and species (Figure 3VI) levels. The results showed that after treatment with Wandai Decoction, the vaginal microbial community was CST II / V (dominated by L. gasseri and L. jensenii), while after treatment with fluconazole, the vaginal microbial community was CST III / IV (dominated by L. iners and Anaeroboc organisms). The Wandai Decoction group was superior to the fluconazole group in restoring the vaginal microbial community structure, with a statistically significant difference (P < 0.05).

[0136] (3) Differences in vaginal flora distribution

[0137] Differences in Vaginal Microbial Distribution Before and After Fluconazole Treatment: Microbial multivariate statistical analysis using the Wilcoxon algorithm was performed to analyze the differences in the vaginal flora before and after treatment within the fluconazole group. A histogram of the top 10 species ranked by abundance at the phylum, class, order, family, genus, and species levels is shown below (Figure 4). The results showed that after fluconazole treatment, the abundance of non-dominant vaginal flora, such as anaerobic bacteria and cocci, increased in VVC patients compared to before treatment. The abundance of Lactobacilli did not increase, while Bacteroidetes species increased. Specific microbial analysis results are shown below.

[0138] ① Phylum: There was no difference in vaginal flora between the fluconazole group before and after treatment. This indicates that fluconazole did not change the phylum-level differences in vaginal flora in VVC patients. ② Class: Fluconazole increased the abundance of Myxococcia in the vaginal flora of VVC patients (Figure 4I). ③ Order: Fluconazole increased the abundance of Chitinophagales and Myxococccales and decreased the abundance of Peptococcales in the vaginal flora of VVC patients (Figure 4II). ④ Family: Fluconazole increased the abundance of Chitinophagaceae, Vulgatibacteraceae, and Chromobacteriaceae and decreased the abundance of Fusobacteriaceae, Leuconostocaceae, and Peptococcaceae in the vaginal flora of VVC patients (Figure 4III). ⑤ Genus: Fluconazole increased the abundance of Sediminibacterium, Erysipelatoclostridium, Butyricicoccus, Vulgatibacter, Porticoccus, and Actinomyces in the vaginal microbiota of VVC patients, decreased the abundance of Weissella, and increased the abundance of Crenobacter (Figure 4IV). ⑥ Species: Fluconazole increased the abundance of Bacteroides_sp._g_Bacteroides, Bacteroides_caccae_Bacteroides, and Porticoccus_litoralia_g_Porticoccus in the vaginal microbiota of VVC patients, and increased the abundance of Parabacteroides_sp._g_Parabacteroides and Methylobacterium_aquaticum_g_Methylobacterium-Methylorubrum (Figure 4V).

[0139] Differences in vaginal flora distribution after treatment in the Wandai Decoction group:

[0140] Microbial multivariate statistical analysis, using the Wilcoxon algorithm, analyzed the differences in community structure before and after treatment in the Wandai Decoction group. A histogram of the top 10 species ranked by abundance at the phylum (Figure 5I), class (Figure 5II), order (Figure 5III), family (Figure 5IV), genus (Figure 5V), and species (Figure 5VI) levels is shown below (Figure 5). The results showed that Wandai Decoction treatment increased the abundance of dominant Bacilli, such as Reuteri and Murinus, in the vaginal microbiome of patients with VVC, while decreasing the abundance of Erysipelotrichaceae, Lactococcus lactis, and Lactococcus brevis. Detailed microbial community analysis is shown below. ①Phylum: Wandai Decoction increased the abundance of Fusobacteriota and Gemmatimonadota in the vaginal microbiota of VVC patients. ②Class: Wandai Decoction increased the abundance of Fusobacteriia, Acidimicrobiia, BD2-11_terrestrial_group, and PAUC43f_marine_benthic_group in the vaginal microbiota 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 microbiota of VVC patients. ④Family: Wandai decoction increases the abundance of Clostridiaceae, Christensenellaceae, Xanthomonadaceae, Fusobacteriaceae, Micrococcaceae, and BD2-11_terrestrial_group in the vaginal flora of VVC patients, and decreases the abundance of Comamonadaceae, Flavobacteriaceae, and Streptococcaceae.⑤ Genus: Wandai Decoction increased the abundance of Clostridium_sensu_stricto_1, Christensenellaceae_R-7_group, BD2-11_terrestrial_group, and Salinimicrobium in the vaginal microbiota of VVC patients, while decreasing the abundance of Klebsiella and Lactococcus. ⑥ Species: Wandai Decoction increased the abundance of Lactobacillus species such as Reuteri and Murinus, while decreasing the abundance of Erysipelotrichaceae_bacterium, Lactococcus_lactis_g_Lactococcus, and Lactococcus_brevis_g_Lactococcus in the vaginal microbiota of VVC patients.

[0141] Differences in vaginal flora distribution between the Wandai Decoction group and the fluconazole group after treatment:

[0142] Microbial multivariate statistical analysis using the Wilcoxon algorithm revealed differences in post-treatment microbial flora between the two groups. A bar chart of the top 10 species ranked by abundance at the phylum, class, order, family, genus, and species levels is shown below (Figure 6). The results showed that after treatment with fluconazole and Wandai Decoction, the abundance of dominant vaginal bacteria, such as L. gasseri and L. jensenii, increased in the Wandai Decoction group compared to pre-treatment. Furthermore, the abundance of anaerobic bacteria, such as Prevortella bivia g-Prevotella and Atopobium baginae g-Atopobium, increased in the fluconazole group. Specific microbial analyses are as follows. ① Phylum: At the Gemmatimonadota level, the relative abundance of Gemmatimonadota increased after Wandai Decoction treatment (post-T) (Figure 6I). ②Class: At the Bacci and BD2-11_terrestrial_group class levels, the relative abundance of Wandai decoction treatment (post-T) was higher, and post-C was rare or almost absent in PAUC431_marine_benthic_group. However, at the Bacteroidia, Coriobacteriia, and Acidimicrobiia class levels, the relative abundance of fluconazole treatment (post-C) was higher (Figure 6II). ③Order: At the Lactobacillales, Rhodospirillales, and Actinomarinales order levels, the relative abundance of Wandai decoction treatment (post-T) was higher, and post-C was rare or almost absent in PAUC431_marine_benthic_group. However, at the Coriobacteriales, Chitinophagales, and Alteromonadales order levels, the relative abundance of fluconazole treatment (post-C) was higher (Figure 6III). ④ Family: At the family level, the relative abundance of Prevortellaceae, Atopobiaceae, Leptotrichiaceae, Eggerthellaceae, Xanthomonadaceae, Enterococcaceae, and Chromobacteriaceae was higher after fluconazole treatment (Post-C), while at the family level, the relative abundance of Wandai decoction treatment (Post-T) was higher after BD2-11_terrestrial_group, Yersiniaceae, and Marinobacteraceae (Figure 6IV).⑤ Genus: At the genus level, the relative abundances of Prevortella, Atopobium, Sneathia, Burkholderia-Caballeronia-Paraburkholderia, Peptostreptococcus, and Marvinbryantia were higher after fluconazole treatment (Post-C). At the genus level, the relative abundances of Ureaplasma, BD2-11_terrestrial_group, and Saiinimicrobium were higher after Wandai Decoction treatment (Post-T) (Figure 6V). ⑥ Species: At the species level, the relative abundance of Lactobacillus_jensenii_g_Lactobacillus and Lactobacillus_gasseri_g_Lactobacillus increased significantly after Wandai decoction treatment (Post-T). At the anaerobic bacterial species level, the relative abundance of Prevortella_bivia_g_Prevotella and Atopobium_baginae_g_Atopobium increased significantly after fluconazole treatment (post-C) (Figure 6VI).

[0143] (4) LEFse analysis of differential species composition:

[0144] Based on the above results, we conducted a deep LEfSe analysis of the contribution of different species to the differences between the fluconazole and Wandai Decoction groups, the annotation of different species, and the relative abundance of different species in each sample. The results showed that there were 27 different bacterial species between the two groups. On the LEfSe phylogenetic tree, the Wandai Decoction group was mainly enriched in the h, i, j, and k categories. The specific results are shown in Figure 7 below:

[0145] 4.3.4 Metagenomic Changes in Vaginal Secretions

[0146] In order to clarify the vaginal microbial species and their functions that caused the difference in efficacy between the two groups, and to conduct an in-depth analysis of the mechanisms by which the two groups promoted the negative conversion of Candida, 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, analysis at the species level, and analysis at the functional level. The results showed that compared with the VVC patients in the Wandai Decoction group and the fluconazole group after treatment, the vaginal microbial communities were CSTII and CSTV types, with L. gasseri (Lactobacillus gasseri) and L. jensenii (Lactobacillus jensenii) as the dominant species; Wandai Decoction may be superior to fluconazole in preventing the growth and proliferation of Candida. The specific results are as follows:

[0147] (1) Distribution of gene numbers between the two groups and between groups:

[0148] 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 amino acid sequence. Then, CD-HIT software was used to remove redundant genes, setting identity 95%, coverage 90%, and selecting the longest sequence as the representative sequence to obtain non-redundant initial Unigene for gene clustering analysis. The horizontal axis is the grouping, 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 shown in the Violin diagram (Figure 8I) and Venn diagram (also known as the petal diagram Figure 8II) below. The number of genes in each sample excluding the number of shared genes is displayed on each petal of the Venn diagram.

[0149] (2) In terms of bacterial species abundance:

[0150] The following barplots show the top 15 species ranked by abundance at the phylum (Figure 9I), class (Figure 9II), order (Figure 9III), family (Figure 9IV), genus (Figure 9V), and species (Figure 9VI) levels. The results show that the abundance of different bacterial species varied within each sample. After treatment, vaginal bacterial species in both treatment groups were enriched in the following top 15 species at the phylum, class, order, family, genus, and species levels, respectively.

[0151] (3) LEFse analysis of differential species composition:

[0152] Based on the above results, we conducted a deep LEfSe analysis of the contribution of different species to the differences between the fluconazole and Wandai Decoction groups, the annotation of different species, and the relative abundance of different species in each sample. The results showed that there were 19 different bacterial species between the two groups. On the LEfSe phylogenetic tree, the Wandai Decoction group was mainly enriched in the a, b, and c categories. The specific results are shown in Figure 10 below:

[0153] (4) Species difference analysis:

[0154] The Wilcoxon rank-sum method (two-group) was used to perform hypothesis testing on species abundance data between groups, with p values ​​of < 0.05 considered significant. The following bar charts show the top 12 species ranked by abundance at the phylum (Figure 11I), class (Figure 11II), order (Figure 11III), family (Figure 11IV), genus (Figure 11V), and species (Figure 11VI) levels (Figure 11). The results showed that after treatment, the vaginal microbiome in the Wandai decoction group was reconstituted with L. gasseri and L. jensenii as dominant species, and the microbiome was restored to CSTII and CSTV types, while fluconazole treatment restored the microbiome to CSTIII and IV types. This indicates that Wandai decoction was superior to fluconazole in restoring the vaginal microbial community structure, with the difference being statistically significant (P < 0.05).

[0155] (5) At the functional level: KEGG functional enrichment analysis (Figure 12) and CAZy functional analysis (Figure 13) were performed on the two groups of differentially expressed species.

[0156] KEGG functional enrichment analysis: The results showed that in terms of the cell cycle and meiosis functions of Candida albicans, the Wandai decoction group was significantly higher than the fluconazole group in 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 albicans.

[0157] CAZy functional analysis: The results showed that the Wandai decoction group had significantly higher levels of drug resistance and resistance genes than the fluconazole group (P < 0.05).

[0158] 3.3.5 Metabolomic changes

[0159] Before and after treatment, there were 59 differential metabolites in the Wandai Decoction group, while there were 43 in the fluconazole group. However, after treatment, the number of differential metabolites between the Wandai Decoction group and the fluconazole group increased to 120 (Figure 14I). Before and after treatment, the fluconazole group upregulated 40 differential metabolites and downregulated 3 differential metabolites after treatment. The Wandai Decoction group upregulated 50 differential metabolites and downregulated 9 differential metabolites. Comparing the two groups, 53 metabolites were upregulated and 67 metabolites were downregulated after treatment. The results are visualized in the volcano plots shown below (Figures 14II, III, and IV).

[0160] Correlation analysis results: The correlation analysis of the top 50 differential metabolites between the Wandai Decoction group, the fluconazole group before and after treatment and between the groups after treatment is shown in Figures 15I, II, and III, where red indicates positive correlation of differential metabolites and blue indicates negative correlation of differential metabolites.

[0161] Before and after treatment, hierarchical clustering was performed on the expression levels of the top 50 differential metabolites. It was found that the expression abundance of differential metabolites in the Wandai decoction group was significantly higher than that in the fluconazole group after treatment, and Wandai decoction was significantly better than fluconazole in reducing differential metabolites (Figure 16). Before and after treatment, KEGG metabolic pathway enrichment analysis was performed on the differential metabolites. It was found that the fluconazole group was enriched in the KEGG metabolic pathway before and after treatment as shown in Figure 17I, and the differential metabolic metabolites between the groups were enriched in the KEGG metabolic pathway as shown in Figure 17II. The above results suggest that 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 (Figure 17).

[0162] 3.3.6 Results of association analysis between metagenomics and metabolomics

[0163] The results of the metagenomics and metabolomics correlation analysis are shown in Figures 18 and 19. Each row represents a different bacterial species, and each column represents the corresponding metabolite. Orange indicates positive correlation, while blue indicates negative correlation. Darker colors indicate stronger correlations, while colors closer to white indicate correlations closer to zero. P values ​​< 0.001 are indicated by ***, P values ​​< 0.01 by **, and P values ​​< 0.05 by *.

[0164] (1) Fluconazole group:

[0165] The TOP20 analysis results (Figure 18) of the correlation analysis of differential species and differential metabolites before and after treatment are as follows. The top 20 differential bacterial species and differential metabolites before and after treatment with fluconazole are shown in the following table. Among them, Candida species showed significant differences before and after fluconazole treatment. The top 5 differential bacterial species associated with each differential metabolite are

[0166] Prevotella_bivia, Veillonellaceae_bacterium_DNF00626, Prevotella_jejuni, Paenibacillus_sp._EKM205P, Prevotella_copri. The top five differential metabolites were Stearyl citrate, (S)-Nerolidol3-O-[aL-Rhamnopyranosyl-(1->4)-aL-rhamnopyranosyl-(1->2)-bD-glucopyranoside], 25-Hydroxy-24-epi-brassinolide, Dodecanamide, and 27-Norcholestanehexol. (2) Wandai Decoction Group:

[0167] The top 20 results of the correlation analysis between differentially expressed species and metabolites before and after treatment (Figure 19) are shown below. The top 20 differentially expressed bacterial species and metabolites in the Wandai Decoction group before and after treatment are listed in the table below. Sorted by correlation value, the top five differentially expressed bacterial species for each differentially expressed metabolite were Prevotella bivia, Lactobacillus paragasseri, Lactobacillus taiwanensis, Lactobacillus phage KC5a, and Lactobacillus phage KC5a. The top five differentially expressed metabolites for each bacterial species were N-lauroyl glycine, Dodecanamide, 10-F2-dihomo-IsoP, 10-F2-dihomo-IsoP, and PG (19:0 / 0:0).

[0168] (3) Comparison between the two groups after treatment:

[0169] The top 20 results of the correlation analysis between differentially expressed species and metabolites before and after treatment (Figure 20) are shown below. The top 20 differentially expressed species and metabolites between the groups before and after treatment are listed in the table below. Sorted by correlation value, the top five differentially expressed species for each differentially expressed metabolite were Lactobacillus gasseri, Ureaplasma parvum, Ureaplasma urealyticum, Lactobacillus paragasseri, and Lactobacillus jensenii. The top five differential metabolites associated with each bacterial species 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.

[0170] (4) KEGG signaling pathway analysis results

[0171] Based on the association analysis results between differential bacterial species and differential metabolites (Figure 21), KEGG pathway enrichment analysis was performed on the differential metabolites and genes. The results showed that differentially enriched pathways in the fluconazole group before and after treatment were the NOD-like receptors signaling pathway and the amino acid biosynthesis pathway. Among the KEGG pathways enriched for differentially expressed genes and metabolites between the treatment groups, the following four pathways were enriched: the pentose and glucuronate interconversion pathway, the glycerophospholopid metabolism pathway, the phenylpropanoid biosynthesis pathway, and the animo acid biosynthesis pathway. Combined with the metabolomics analysis results, the improvement of the vaginal microbiome by Wandai Decoction may be related to its effects on local vaginal glycerophospholipid metabolism and pentose and glucuronate interconversion pathways.

[0172] 3.3.7 Changes in IL-1α and IFN-α levels in the two groups before and after treatment

[0173] Based on the above research results, 10 patients in each of the fluconazole and Wandai Decoction groups were selected for further measurement of IFN-α and IL-1α levels in vaginal lavage fluids before and after treatment. The results showed that both Wandai Decoction and fluconazole increased the immune factor IFN-α in the vaginal microenvironment, with no statistically significant difference between the two groups. However, the Wandai Decoction group was superior to the fluconazole group in reducing the local proinflammatory factor IL-1α in the vagina (Figure 22), with a statistically significant difference (P < 0.05). This suggests that Wandai Decoction has a certain clinical efficacy in patients with VVC, possibly by increasing the immune factor IFN-α and reducing the inflammatory factor IL-1α in the vaginal microenvironment.

[0174] 3.3.8 Changes in mitochondrial DNA levels in the two groups before and after treatment

[0175] Based on these research results, 10 patients in each of the fluconazole and Wandai Decoction groups were selected for further mitochondrial DNA analysis before and after treatment in the vaginal lavage fluids of both groups. The results showed that after treatment, fluconazole exacerbated mitochondrial damage, while the Wandai Decoction group did not. Wandai Decoction demonstrated superior mitochondrial protection compared to fluconazole (Figure 23).

[0176] 3.3.9 Changes in the STING signaling pathway in the two groups of patients after treatment

[0177] Based on these findings, 10 patients were selected from each of the fluconazole and Wandai Decoction groups. Vaginal lavage fluids from both groups were tested for levels of STING, IRF3, and MX2, molecules associated with the STING signaling pathway, before and after treatment. The results (Figure 24) showed that STING, IRF3, and MX2 levels were significantly elevated in the Wandai Decoction group after treatment (P < 0.05, P < 0.01). In the fluconazole group, only STING levels increased after treatment (P < 0.01), with no significant effect on IRF3 and MX2 levels (P > 0.05).

[0178] 3.3.10 Security Comparison

[0179] Before and after treatment, alanine aminotransferase, serum aspartate aminotransferase, urea nitrogen, and creatinine were measured in VVC patients in the fluconazole and Wandai decoction groups. The results showed no abnormalities, and no adverse drug reactions were observed.

[0180] 4 Discussions

[0181] 4.1 Safety and efficacy of Wandai Decoction in treating VVC

[0182] Safety is crucial for any treatment or clinical trial of a disease. The results of this clinical trial indicate that neither Wandai Decoction nor fluconazole had adverse effects on liver and kidney function, nor adverse drug reactions, in the treatment of VVC. In terms of efficacy, compared with fluconazole, the two groups had comparable clinical efficacy in the 7-14 days of clinical cure visits and fungal clearance for Wandai Decoction in the treatment of VVC. However, the results of the 7-14 days of clinical remission visits (CI) showed that Wandai Decoction was more effective than the fluconazole group in alleviating or relieving VCC symptoms and signs. Furthermore, at the 21st to 30th day (FU) visit, Wandai Decoction was still more effective than fluconazole in alleviating or relieving VCC symptoms and signs.

[0183] 4.2 Advantages of Wandai Decoction in treating VVC and preventing recurrence

[0184] In this study, at the 3rd month follow-up, Wandai Decoction could significantly reduce the recurrence rate in preventing VVC recurrence, which was significantly better than the fluconazole group.

[0185] 4.3 Effect of Wandai Decoction on Maintaining Vaginal Microecological Balance

[0186] Restoring the vaginal microbiome is the ultimate goal of VVC treatment. In this study, we combined 16s RNA sequencing, metagenomics, and metabolomics techniques to initially find that species richness was similar between the Wandai Decoction and fluconazole groups before and after treatment, with no difference. This suggests that neither Wandai Decoction nor fluconazole treatment affected the species richness of the microbiome. The experiment also revealed differences in vaginal microbial beta diversity between the Wandai Decoction and fluconazole groups. The main manifestations are as follows: (1) The vaginal microbial communities affected by the Wandai decoction treatment were mainly CSTⅡ and V types (mainly L. gasseri and L. jensenii). L. gasseri maintains a low pH value in the vagina by secreting lactic acid and hydrogen peroxide, thereby inhibiting the growth of harmful bacteria and fungi, quickly clearing human papillomavirus, parasites, etc., and protecting the vagina from infection. L. jensenii colonizes in the vaginal mucosal epithelial biofilm and competes with harmful bacteria for nutrients, thereby reducing the colonization and reproduction of pathogens. By maintaining the balance of vaginal microecology, it plays a vital protective role in vaginal health; the vaginal microbial communities affected by the fluconazole treatment were mainly CSTIII and IV types (mainly L. iners, Anaerobo (2) The phyla, classes, orders, families, genera, and species affected by Wandai decoction and fluconazole were different: at the phylum level, Wandai decoction increased the abundance of Fusobacteriota and Gemmatimonadota in the vaginal flora of VVC patients; fluconazole did not change the flora differences at the phylum level in the vaginal flora of VVC patients. At the class level, Wandai decoction increased the abundance of Fusobacteriia, Acidimicrobiia, BD2-11_terrestrial_group, and PAUC43f_marine_benthic_group in the vaginal flora of VVC patients; fluconazole increased the abundance of Myxococcia in the vaginal flora of VVC patients. At the order level: 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; fluconazole increased the abundance of Chitinophagales and Myxococccales, and decreased the abundance of Peptococcales in the vaginal flora of VVC patients.At the family level: 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; fluconazole increased the abundance of Chitinophagaceae, Vulgatibacteraceae, and Chromobacteriaceae in the vaginal flora of VVC patients, and decreased the abundance of Fusobacteriaceae, Leuconostocaceae, and Peptococcaceae. At the genus level: Wandai decoction increased 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 decreased the abundance of Klebsiella and Lactococcus; fluconazole increased the abundance of Sediminibacterium, Erysipelatoclostridium, Butyricicoccus, Vulgatibacter, Porticoccus, and Actinomyces in the vaginal flora of VVC patients, decreased the abundance of Weissella, and increased the abundance of Crenobacter; at the species level: Wandai decoction increased 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 decreased the abundance of Klebsiella and Lactococcus; fluconazole increased the abundance of Sediminibacterium, Erysipelatoclostridium, Butyricicoccus, Vulgatibacter, Porticoccus, and Actinomyces in the vaginal flora of VVC patients, decreased the abundance of Weissella, and increased the abundance of Crenobacter; at the species level: The abundance of Lactobacillus such as reuteri and murinus in the vaginal flora of patients with high VVC was increased, and the abundance of bacteria such as Erysipelotrichaceae, Lactococcuslactis, and Lactococcus_brevis was reduced; fluconazole increased the abundance of Bacteroides_sp._g_Bacteroides, Bacteroides_caccae_Bacteroides, and Porticoccus_litoralia_g_Porticoccus in the vaginal flora of patients with VVC, and increased the abundance of two species, Parabacteroides_sp._g_ParabacteroidesMethylobacterium_aquaticum_g_Methylobacterium-Methylorubrum.The differences in the effects of Wandai Decoction and fluconazole at the phylum, class, order, family, genus, and species levels, combined with the comparable overall therapeutic effects of the two groups, suggest that Wandai Decoction and fluconazole have their own unique 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 are 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.

[0187] Regarding the mechanisms of vaginal microecological restoration, the authors found that Wandai decoction significantly improved cell cycle and meiotic signaling pathways compared to the fluconazole group. Furthermore, Wandai decoction may be superior to fluconazole in inhibiting the growth and proliferation of Candida species. Furthermore, Wandai decoction also had a significantly stronger effect than fluconazole in drug resistance and functional genes. Furthermore, Wandai decoction may be significantly superior to fluconazole in improving local vaginal α-linolenic acid, glycerophospholipid metabolism, pentose-glucuronic acid interconversion, and arachidonic acid metabolic pathways. Differentially enriched signaling pathways in the fluconazole group before and after treatment included the NOD-like receptor signaling pathway and the amino acid biosynthesis pathway. This provides further insight into the differential mechanisms by which fluconazole or Wandai decoction restore the vaginal microecology of VVC, integrating microbial, bacterial, and metabolic pathways and examining these signaling pathways.

[0188] 4.4 Regulation of Wandai Decoction on the mitochondrial mtDNA-STING signaling pathway

[0189] This study explored the mechanism of restoring the vaginal microbiome in VVC patients through the lens of the mitochondrial mtDNA-STING signaling pathway. The results showed that Wandai Decoction increased STING, IRF3, MX2, and IFN-α levels, decreased IL-1α levels, and did not exacerbate pre-treatment mtDNA damage. Fluconazole, on the other hand, only increased STING levels, had no significant effect on reducing IL-1α levels, and exacerbated pre-treatment mtDNA damage.

[0190] In summary, clinical trials conducted in this paper confirm that Wandai Decoction (Wandai Tang) can eradicate vaginal fungi, restore vaginal microbial community structure, activate the STING signaling pathway, and protect mitochondria in the treatment of VVC, thereby preventing recurrence of vulvovaginal Candida. Its efficacy and safety are superior to fluconazole. Because Wandai Decoction significantly affects the CSTV and CSTII microbiota within the vaginal microbiome, particularly increasing the abundance of L. gasseri and L. jensenii, it maintains vaginal microecological balance, protects vaginal health, and has broad clinical application prospects.

Claims

1. Use of a composition in the preparation of a drug for maintaining vaginal microecological balance, 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 drug has the effect of increasing the abundance of CST V type microbial community and / or CST II type microbial community.

3. The use according to claim 2, characterized in that: The dominant bacterial species of the CST V type microbial community include L. jensenii, and the dominant bacterial species of the CST II type microbial community include L. gasseri.

4. Use of a composition for preparing a medicament for promoting the reproduction of CST V type microflora and / or CST II type microflora in the vagina, 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. The dominant bacterial species of the CST V type microbial community include L. jensenii, and the dominant bacterial species of the CST II type microbial community include L. gasseri.

5. Use of a composition in the preparation of a medicament for treating vaginal diseases caused by reduced reproductive capacity of CST II type microflora and / or CST V type microflora, 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. The dominant bacterial species of the CST V type microbial community include L. jensenii, and the dominant bacterial species of the CST II type microbial community include L. gasseri.

6. 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.

7. The use according to claim 6, characterized in that: The medicine is a medicine for preventing the recurrence of vulvovaginal candidiasis.

8. The use according to claim 6 or 7, characterized in that: The drug has the effect of eradicating vaginal fungi.

9. The use according to claim 6 or 7, characterized in that: The drug has the effect of restoring the structure of vaginal microflora.

10. The use according to claim 9, characterized in that: The vaginal microbial community includes CSTII type microbial community and CST V type microbial community.

11. The use according to claim 6, characterized in that: The drug has the effects of increasing the abundance of the vaginal flora at the phylum level, namely, Fusobacteriota and Gemmatimonadota, at the class level, namely, Fusobacteriia, Acidimicrobiia, BD2-11_terrestrial_group, and PAUC43f_marine_benthic_group, at the order level, namely, Fusobacteriales, Clostridiales, Xanthomonadales, Clostridia, Alteromonadales, BD2-11_terrestrial_group, Actinomarinales, Oceanospirillales, and PAUC43f_marine_benthic_group. The role of the abundance of UC43f_marine_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.

12. The use according to claim 9, 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.

13. The use according to claim 6 or 7, characterized in that: The drug has the effect of activating the STING signaling pathway and protecting mitochondria.

14. The use according to claim 6 or 7, 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.

15. The use according to claim 14, characterized in that: The oral preparation is preferably Wandai Decoction.

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