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. 2024 Jun 21;19(11):983–996. doi: 10.1080/17460913.2024.2352269

In vitro and in vivo inhibitory effects of the Sanghuang mushroom extracts against Candida albicans

Jiahui Du a,b,‡, Shuqing Jin b,‡, Ying Zhang a, Wei Qiu a, Yahui Dong a, Yan Liu c, Dengke Yin b, Ye Yang b,*, Weifang Xu a,b,**
PMCID: PMC11318678  PMID: 38904292

Abstract

Aim: To explore the antifungal potential of Sanghuang mushroom, a traditional Chinese medicine. Materials & methods: The antifungal properties and the potential mechanism of Sanghuang mushroom extracts against Candida albicans were studied in vitro and in vivo. Results: Sanghuang mushroom extracts inhibited the biofilm formation, increased the cell membrane permeability and promoted cell apoptosis of C. albicans in vitro. In a murine model of vulvovaginal candidiasis, Sanghuang mushroom extracts reduced the vaginal fungal load, improved inflammatory cell infiltration and downregulated the expression of TNF-α, IL-1β and IL-6. Untargeted metabolomic analysis suggested the presence of ten antifungal components in Sanghuang mushroom extracts. Conclusion: Sanghuang mushroom extracts showed promise as antifungal agent against candidiasis, with potential therapeutic implications.

Keywords: : antifungal, apoptosis, biofilm, Candida albicans, Sanghuang mushroom

Plain language summary

Article highlights.

  • Sanghuang mushroom extracts exhibit potent antifungal properties against C. albicans.

  • Scanning electron microscopy reveals the impact of Sanghuang mushroom extracts on biofilm architecture.

  • A murine model of vulvovaginal candidiasis (VVC) confirms the ability of Sanghuang mushroom extracts to suppress C. albicans infiltration.

  • Flow cytometry data illustrate the induction of reactive oxygen species and promotion of apoptosis in C. albicans biofilm cells.

  • Untargeted metabolomic analysis identifies potential bioactive compounds contributing to the antifungal effects.

  • Sanghuang mushroom extracts emerge as promising candidates for the development of novel antifungal therapies.

1. Introduction

Candida albicans (C. albicans) is a widespread pathogenic fungus that mainly colonizes the oral cavity, GI tract, vagina and other sites in the human body [1,2]. As with other fungi, the reduction in immunity and microbiota dysbiosis contributes to the massive reproduction of C. albicans, subsequently leading to various fungal diseases. The common relevant infections include invasive, mucosal, blood stream, genitourinary, vulvovaginal and oropharyngeal candidiasis [3]. With the acceleration of the pace of life, the incessant pressure and the common use of organ transplantation, the incidence of candidiasis has gradually aggravated [4,5], posing a serious threat to public health. Polyenes, azoles, allylamines, echinocandins and pyrimidines are the antifungal drugs for C. albicans infections. Unfortunately, many patients often suffer from toxic effects and recurrence and develop drug resistance with the widespread abuse of hormones and antibiotics, resulting in a high mortality rate [6–9]. For instance, nephrotoxicity is caused by amphotericin B, visual disturbances are caused by voriconazole, and congestive heart failure is caused by itraconazole [10]. Therefore, there is an urgent need to develop new antifungal drugs that are highly effective, less toxic and less likely to develop resistance.

Chinese herbal medicine is easily accessible, safe and effective, and has been recently recommended as a valuable object to solve the problem of increased clinical resistance of C. albicans and increased infection rate [11–13]. Sanghuang mushroom is a rare and large group of medicinal fungi that falls under three main recognized genera: Inonotus, Phellinus and Sanghuangporus [14,15]. Its bioactive products mainly include polysaccharides, flavonoids, triterpenoids and polyphenols. A large body of work has revealed that the Sanghuang mushroom has various pharmacological properties, including anti-tumor, anti-inflammatory, hypoglycemic, augmented autoimmunity and improved blood circulation [15–17]. Moreover, it is also used to relieve or treat diseases such as cancer, diabetes, gastrointestinal disorders and liver disease through its various biological functions. However, fewer studies have been reported on the antimicrobial activity of Sanghuang mushrooms in C. albicans hyphae and biofilms.

In this study, we investigated the antimicrobial activity of Sanghuang mushroom extracts against C. albicans (standard strain SC5314). We first examined the effects of the Sanghuang mushroom on hyphal morphology, biofilm formation, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP) and apoptosis of C. albicans in vitro. In addition, the antimicrobial efficacy of Sanghuang mushroom extracts was verified using in vivo experiments. This study provided evidence that Sanghuang mushrooms offer several promising medicinal values in preventing and treating candidiasis. These findings further the understanding of the pharmacodynamic substances and the clinical application of Sanghuang mushrooms.

2. Materials & methods

2.1. Strains, media & reagents

C. albicans strain SC5314 was kindly provided by Professor Shao Jing from Anhui University of Chinese Medicine. The pathogen was routinely preserved in Sabouraud Dextrose Agar (SDA, Hope Biotech Co., Qingdao, China) medium and only freshly transferred cultures were used in the bioassay tests. Roswell Park Memorial Institute-1640 medium (RPMI-1640 medium, Spark Jade, Shandong, China) was used for biofilm formation.

Three Sanghuang species, namely Sanghuangporus vaninii AHS, Inonotus hispidus YNS and Sanghuangporus sanghuang SHW were used in this study. The fruiting body of AHS was purchased from Jinzhai Shangzhen Biotechnology Co. Ltd (Anhui, China) and YNS and SHW were gifted from the Institute of Sericulture Science and Technology Research (Chongqing, China), which were identified by our previous work [18]. Among the three Sanghuang species, we specifically examined the antimicrobial activities of the fruiting body of AHS and the hyphae of YNS and SHW. The mycelium samples were collected after 3 weeks of fermentation in Potato Dextrose Broth (PDB) medium at 26°C. In vitro tests were conducted to assess the potential of three different Sanghuang species: Sanghuang extracts YNS, SHW with mycelium extracts, and AHS with fruiting body extracts. The results indicated that AHS exhibited the most promising characteristics and thus was used for the following experiments.

Fluconazole (FLZ) was purchased from Yuanye Biotechnology Co. Ltd (Shanghai, China). Dimethyl sulfoxide (DMSO) and ROS assay kit were purchased from Solarbio (Beijing, China). 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and propidium iodide (PI) were bought from Sigma Aldrich (Shanghai, China). For in vitro and in vivo experiments, FLZ and Sanghuang mushroom extracts were dissolved in DMSO and then diluted to the required concentration with a complete medium. The final concentration of DMSO used in vivo and in vitro were 2 and 0.1%, respectively.

2.2. Sanghuang mushroom extracts preparation

The fruiting bodies of AHS and the mycelium of YNS and SHW were air-dried, cut into small pieces and then pulverized to powder using a grinder set at the speed of 8000 rpm/min. A total of 1 g of Sanghuang mushrooms powders was added with 20 ml solvents (YNS and SHW were extracted with methanol and AHS with ethyl acetate) in flasks. Then, the flasks were sealed and shaken (170 rpm/min) in a water bath shaker at 37°C for 24 h. Afterward, the mixture was extracted by ultrasound for 30 min, centrifuged, drained and dissolved in an appropriate amount of DMSO [19].

2.3. Detection of antifungal activity of Sanghuang mushroom extracts against C. albicans

The inhibition effect of each sample was tested by the well diffusion method [20]. Briefly, the fresh SC5314 cell suspension (1.0 × 108 colony-forming units per microliter, CFU/ml) was evenly inoculated onto the SDA medium and 100 μl of the Sanghuang mushroom extracts were injected into the wells (5.00 mm). The plates were incubated at 25°C for 2 days, and the diameters (Di) of the fungal inhibition zones were measured. Organic solvent without drugs was used as a negative control, and all treatments were performed in triplicate.

YNS extract was prepared at concentrations of 2, 4, 8, 16, 32 and 64 mg/ml, SHW extract at concentrations of 0.5, 1, 2, 4, 8 and 16 mg/ml, and AHS extract at concentrations of 1, 2, 4, 8, 16 and 32 mg/ml. 100 μl of C. albicans solution (2.0 × 106 CFU/ml) was incubated into a 96-well plate with 100 μl of Sanghuang mushroom extracts at 37°C for 24 h. The concentration at which no visual growth was observed was considered minimum inhibitory concentration (MIC). To determine the minimum fungicidal concentration (MFC), 10 μl of the cultures were inoculated into SDA medium and incubated at 36°C for 24 h. The concentration at which there was no growth was considered as MFC. For the 50% inhibition of the biofilm (SMIC50) determination, the Sanghuang mushroom extracts were incubated in a 96-well plate with SC5314 suspension at 37°C for 24 h, followed by incubating with 50 μl of XTT in the dark for 2 h. The values were determined by OD values (the absorbance of OD492 nm). The SMIC50 was defined as the concentration of the drug with a 50% decrease in OD492 nm compared with the blank group. In this experiment, FLZ was used as a positive control, and DMSO as a negative control. Three independent experiments were performed [21].

2.4. Effects of the Sanghuang mushroom extracts on the C. albicans cell micromorphologies

The ordinary optical microscopy assays were carried out as described by Yong et al. [22]. A total of 500 μl of C. albicans cell suspension (2 × 106 CFU/ml) combined with 500 μl of 8 × MIC Sanghuang mushroom extracts were added to the 24-well plate and incubated at 37°C for 6, 12, 24, 48 and 72 h, respectively. Afterward, the planktonic cells were aspirated, and then the residual solution was washed with sterile PBS and photographed under a 40× microscope. Three fields of view were randomly selected for each sample. FLZ with a concentration of 8 μg/ml was used as a positive control and DMSO as a negative control. All treatments were performed in triplicate.

Scanning electron microscopy (SEM) was carried out as described by Wu et al. [23] with slight modification. A total of 100 μl of SC5314 cell suspension (2 × 106 CFU/ml) mixed with 100 μl of Sanghuang mushroom extracts (8 × MIC and 16 × MIC, respectively) were incubated in a 96-well plate at 37°C for 24 h. The planktonic cells were removed, and the residual solution was washed with sterile PBS and then centrifuged at 8000 rpm/min for 5 min. The obtained samples were fixed with glutaraldehyde (final concentration 2.5%) for 24 h at 4°C, treated with ethanol series (50, 70, 80, 90, 95 and 100%) and dried with critical point CO2. The tracks were then glued to metal plates with carbon tape and used for vacuum gold plating. After that, the samples were examined under a Hitachi scanning electron microscope (Hitachi Regulus SEM 8100, Japan).

PI staining assay was carried out as designed by Li et al. [24] with slight modification. The C. albicans cell suspension and Sanghuang mushroom extracts (8 × MIC) were re-inoculated in 96-well plates at 37°C. After 24 h, samples were stained with 5 μg/ml of PI at 37°C for 20 min in the dark and the viability of C. albicans was observed under a fluorescent microscope after gentle rinsing with PBS. FLZ (8 μg/ml) was used as a positive control, and no drug was used as a negative control. All treatments were performed in triplicate.

2.5. Reactive oxygen species level detection

C. albicans cells were inoculated into Sabouraud Dextrose Broth (SDB) medium and incubated at 37°C, shaking at 200 rpm/min. The cells were treated with Sanghuang mushroom extracts (32×, 16×, 8× and 4 × MIC) and were collected after 24 h culture, followed by the addition of DCFH-DA fluorescent reagent according to the ROS assay kit (Solarbio, Beijing, China) instructions. After incubation at 37°C for 30 min in the dark, the biofilms were washed twice with RPMI 1640 and the fluorescence intensity was detected by flow cytometry, with the intensity of fluorescence representing ROS levels [25]. FLZ (8 μg/ml) was used as a positive control and no drug as the negative control. All treatments were performed in triplicate.

2.6. Mitochondrial membrane potential assay

C. albicans samples from different treatment groups were prepared according to the above operation. JC-1 fluorescent reagent was added according to the instructions of the kit (Solarbio, Beijing, China), incubated for 20 min at 37°C and protected from light, washed twice with JC-1 staining buffer (1×), and the mitochondrial membrane potential level was detected by flow cytometry within 30 min. FLZ (administered at a concentration of 8 μg/ml) was used as a positive control well, no drug was added as a negative control well and three replicate wells were set up for each drug treatment group.

2.7. Biofilm cell apoptosis assay

Mature biofilm cells of C. albicans were resuspended into 400 μl of Annexin V conjugate according to the cell apoptosis kit (Sigma Aldrich, Shanghai, China) instructions and stained with 5 μl of Annexin V-FITC staining solution for 15 min at 4°C protected from light, followed by incubation of 5 μl of PI staining solution for 5 min at 4°C. Finally, the apoptosis rate was immediately detected by flow cytometry.

2.8. Animal model & experiments

All animal experiments were approved by the Experimental Animal Ethics Committee of Anhui University of Chinese Medicine (number: AHUCM-mouse-2022127). Thirty female Kunming mice were randomly divided into five groups: normal, Candida vaginalis model, positive drug FLZ (2 mg/ml), Sanghuang mushroom extracts (20 mg/ml) and DMSO control groups (n = 6). Except for the normal group, all treated groups of mice were injected subcutaneously with 0.1 ml of estradiol benzoate solution (1 mg/ml) once every other day for a total of three injections. Another injection was given on the 7th day after the last injection to maintain the mice in pseudoestrus.

After 7 days of subcutaneous injection of estradiol benzoate solution, the mice were injected with 20 μl of C. albicans fungal solution at a concentration of 2.0 × 106 CFU/ml into the vagina and the normal group with an equal amount of saline. The mice were then maintained in an inverted position for 5 min and infected for 8 days. On days 3, 5 and 7 after inoculation, the vagina was lavaged with sterile PBS and examined under a microscope for fungal hyphae and spores. The remaining lavage solution was diluted, plated and incubated at 37°C for 24 h. Afterward, single colonies were picked and observed under the microscope to confirm the successful models of Candida vaginalis in mice [26,27]. The normal and model groups were routinely fed, and the positive drug control group was administered fluconazole solution (2 mg/kg) and Sanghuang mushroom extracts (20 mg/kg) once daily for 8 days. Subsequently, the vagina of the mice was repeatedly rinsed with sterile saline, and the rinsing fluid was collected and incubated with the sand solid medium 37°C for 48 h. After the growth of single colonies, the density log (CFU/ml) of C. albicans colonies of the vaginal rinsing fluid was calculated according to a previous study [28].

2.9. Hematoxylin & eosin (HE) staining

After vaginal douching, the mice were euthanized, and the cervical vertebrae were dislocated to dissect intact vaginal tissues (~1 cm2). These tissues were then fixed in a 4% formaldehyde solution, dehydrated, made transparent, waxed, embedded and sectioned into approximately 4 μm slices. Subsequently, the slices were stained with (HE) and observed under a microscope to analyze the morphology of the vaginal tissues in the treated mice.

2.10. Immunohistochemical detection of inflammatory factors

Paraffin specimens were subjected to routine dewaxing and hydration, and the endogenous peroxidase was inactivated with 3% H2O2 solution at room temperature for 15 min. After blocking with 10% goat serum, the tissues were incubated with primary antibody (at 4°C overnight), followed by secondary antibody (at 37°C for 1 h). Then, the protein quantification was performed using pathological image analysis and processing software (Image Pro Plus 6.0, IPP 6.0). The average absorbance value of positive areas was retained as an indicator of staining intensity. Three or more fields of view were selected and the mean optical density of TNF-α, IL-1β and IL-6 were calculated, indirectly reflecting the protein expression [29].

2.11. Metabolomics profiling of Sanghuang mushroom extract

Un-targeted metabolomics was employed to profile the metabolic components of Sanghuang mushroom extracts using UHPLC-MS. The analysis was performed under specific analytical conditions, including using a Hypesil Gold column C18 (100 × 2.1 mm, 1.9 μm) with a flow rate of 0.2 ml/min. The mobile phase was prepared by combining two solvents: mobile phase A (0.1% formic acid) and mobile phase B (methanol). Notably, for the negative ion mode, mobile phase A was modified with 5 mm ammonium acetate (pH = 9.0). The gradient program was established as follows: At 0 min, 2% B; at 1.5 min, 2% B; at 3 min, 100% B; at 10 min, 100% B; at 10.1 min, 2% B; at 11 min, 2% B; at 12 min, 2% B. The analytical conditions were optimized based on previous studies [30,31].

Mass spectrometric detection was carried out on a high-resolution mass spectrometry (HRMS) equipped with an electrospray ionization source under the negative ion mode. The ion source parameters were set as follows: spray voltage: 3.5 kV; sheath gas flow rate: 35 psi; auxiliary gas flow rate: 10 l/min; ion transfer tube temperature (capillary temp): 320°c; ion input RF level (S-lens RF level): 60; auxiliary gas heater temperature: 350°C; polarity: positive, negative; MS/MS second-level scans for data-dependent scans.

2.12. Statistical analysis

Data were analyzed by a one-way analysis of variance (ANOVA) with a least significant difference (LSD) test, and the means were compared at a significance level of p < 0.05. The software SPSS, version 23.0, was used for statistics analysis.

3. Results

3.1. Sanghuang mushroom extracts demonstrated anti-Candida activities in vitro

Three Sanghuang mushroom extracts exhibited varying extents of antifungal activity against C. albicans. The MICs of YNS, SHW and AHS Sanghuang mushroom extracts against C. albicans were 4, 1 and 1 mg/ml, respectively; the MFCs of the three extracts against C. albicans were 4, 2 and 1 mg/ml, respectively; and the SMIC50 against C. albicans were 8, 2 and 1 mg/ml, respectively. AHS has shown the lowest MIC, MFC and SMIC50 (Supplementary Table S1).

The impacts of Sanghuang mushroom extracts on the mycelial morphology and biofilm formation of C. albicans are shown in Supplementary Figure S1 & Figure 1. Sanghuang mushroom extracts significantly reduced the number of pathogenic cells compared with the control group throughout the experiment. Compared with the control group, Sanghuang mushroom extracts and FLZ delayed the aggregation of fungi and effectively suppressed the formation of biofilms with prolonged administration time (Supplementary Figure S1). Additionally, SEM revealed that the C. albicans biofilms of the control group were composed of interleaved mycelia with a complete biofilm cell structure and abundant extracellular matrix. After co-culturing with Sanghuang mushroom extracts, the hyphal stage of C. albicans was reduced and mainly yeast-stage cells were present. The phenomenon of notch hyphal fracture, broken biofilm cell structure, cleavage folds and reduced extracellular matrix was also observed. It can be seen that Sanghuang mushroom extracts significantly inhibited the biofilms of C. albicans, mainly by inhibiting the mycelium formation and destroying the biofilm cell structure (Figure 1).

Figure 1.

Figure 1.

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Scanning electron microscopy (SEM) images of C. albicans biofilms. Scale bar, 2 μm.

AHS: Sanghuangporus vaninii; FLZ: Fluconazole. SHW: Sanghuangporus sanghuang; YNS: Inonotus hispidus.

PI is a fluorescent dye that can bind to nucleic acids, and it gains access to cells when the cell membrane is damaged and thus is often used for the detection of cell membrane structure. In this study, PI staining assay was used to detect the effects of Sanghuang mushroom extracts on the C. albicans cell membranes. Results showed increased permeability and fluorescence intensity of the cell membrane of drug-treated C. albicans compared with the controls. Significant disruption of C. albicans cell membrane structure with AHS extracts compared with YNS and SHW (Figure 2). The above results demonstrated that three Sanghuang mushroom extracts inhibited C. albicans, especially for AHS. Therefore, AHS was selected for further assays in this study.

Figure 2.

Figure 2.

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Effects of three Sanghuang mushroom extracts on C. albicans cell membrane permeability. Scale bar, 20 μm.

AHS: Sanghuangporus vaninii; FLZ: Fluconazole; SHW: Sanghuangporus sanghuang; YNS: Inonotus hispidus.

3.2. Sanghuang mushroom extracts induced apoptosis in C. albicans biofilm cells

To explore the mechanism through which Sanghuang attenuated microbial activity of C. albicans, we first detected the intracellular ROS level by cell-permeable probe H2DCFDA (DCFH-DA), which has an important role in yeast apoptosis. As revealed by Figure 3A, compared with the control group, Sanghuang mushroom extracts increased the ROS of C. albicans to 22.3, 43.8, 49.4 and 53.8% under 4×, 8×, 16× and 32 × MIC treatments, respectively. Sanghuang mushroom extracts promoted cellular ROS generation in a dose-dependent manner. Quantification of cellular ROS generation revealed a significant increase in ROS levels after incubation with Sanghuang mushroom extracts at 4 × MIC, surpassing even the FLZ treatment group, compared with control cells (Supplementary Figure S2A).

Figure 3.

Figure 3.

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Flow cytometry analysis of Sanghuang mushroom extracts against C. albicans biofilm cells. (A) Detection of reactive oxygen species. (B) Detection of mitochondrial membrane potential. (C) Detection of cell apoptosis. Q1 region represents necrotic cells, Q3 region represents live cells, Q2 and Q4 region represent late apoptotic and early apoptotic cells, respectively.

Mitochondria have a crucial role in cellular metabolism, serving as essential mediators and as generators and recipients of ROS, which are significant for the normal metabolic activities of C. albicans. Next, we evaluated the effects of Sanghuang mushroom extracts on the MMP of C. albicans. The results indicated that Sanghuang mushroom extracts decreased the intensity of red fluorescence (red fluorescence reflects higher mitochondrial membrane potential) and increased the proportion of C. albicans biofilm cells that lost membrane potential with increasing drug concentration. Compared with the control group (4.01%) and FLZ group (4.64%), Sanghuang mushroom extracts treatment at 4×, 8×, 16× and 32 × MIC depolarized the MMP of C. albicans to 5.68, 10.37, 11.72 and 12.87%, respectively (Figure 3B & Supplementary Figure S2B). The accumulation of ROS level and depolarization of MMP are two important features of cell death by necrosis as well as of cell death by apoptosis, and thus an Annexin V-FITC/PI staining was employed to further confirm that Sanghuang mushroom extracts promoted apoptosis and necrosis in C. albicans.

As shown in Figure 3C, C. albicans cells were treated with different concentrations of Sanghuang mushroom extracts (4×, 8×, 16× and 32 × MIC) for 24 h, the early apoptosis rate (Q4 region, 1.79, 6.05, 6.52 and 2.36%, respectively) was notably higher than that of the control group (0.65%) and FLZ group (0.38%), and the late apoptotic cells (Q2 region) were obviously increased in a dose-dependent manner. Moreover, the necrotic rate (Q1 region) of the C. albicans biofilm cells in Sanghuang mushroom extracts treatment groups at 4×, 8×, 16× and 32 × MIC (25.40, 20.27, 42.80 and 49.73%, respectively) was notably increased compared with that of the control group (3.57%) and FLZ group (17.63%) (Figure 3C & Table 1). According to the statistical analysis results, it was also observed that the apoptosis rate of C. albicans in the Sanghuang mushroom extracts drug-treated group increased with increasing drug concentration (Table 1). The above results hinted that Sanghuang mushroom extracts could induce apoptosis in C. albicans biofilm cells through an increase in ROS production and depolarization of MMP.

Table 1.

The effects of Sanghuang mushroom extracts on the apoptosis of C. albicans.

Groups Early apoptotic cells (%) Late apoptotic cells (%) Necrotic cells (%) Living cells (%)
Control 0.65 ± 0.02cd 0.23 ± 0.02f 3.57 ± 0.46f 95.53 ± 0.46a
FLZ 0.38 ± 0.01d 1.72 ± 0.11ef 17.63 ± 0.38e 80.27 ± 0.27b
4 × MIC 1.79 ± 0.13bc 5.36 ± 0.30de 25.40 ± 0.120c 67.5 ± 0.40c
8 × MIC 6.05 ± 0.34a 15.33 ± 0.84c 20.27 ± 0.09d 58.37 ± 1.18d
16 × MIC 6.52 ± 0.87a 31.2 ± 1.75b 42.80 ± 1.10b 19.47 ± 1.54e
32 × MIC 2.36 ± 0.15b 36.5 ± 1.95a 49.73 ± 1.35a 11.43 ± 1.03f
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The values represent the mean ± standard error, and different lowercase letters in the same column represent significant differences at p < 0.05 by a one-way analysis of variance (ANOVA) with a least significant difference (LSD) test.

FLZ: Fluconazole drugs.

3.3. Sanghuang mushroom extracts reduced the vaginal fungal load in mice in vivo

The mice in the model group showed a decrease in body weight, whereas those in the remaining groups showed healthy growth. The mice in the normal group showed normal activity, while the rest showed sniffing and scratching during the 7 days of C. albicans infection period. This behavior gradually changed from initial excitement and restlessness to depression and typical signs of inflammation, such as redness and swelling of the vaginal area or the presence of white sticky discharge. Furthermore, smear microscopy and plate culture of the vaginal douche indicated a significant presence of colonized C. albicans with a relatively dense distribution, indicating successful modelling (Supplementary Figure S3). After treatment with Sanghuang mushroom extracts and FLZ, the overall condition of the mice and the local features of the vagina gradually normalized (Supplementary Figure S4). Compared with the normal group mice, a large number of C. albicans appeared in the vagina of both the model group and the DMSO control group mice. However, the drug-treated group of mice showed a statistically significant decrease in fungal load (p < 0.05) (Figure 4).

Figure 4.

Figure 4.

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Effects of Sanghuang mushroom extracts on vaginal fungal load in mice. (A) Plate coating images. (B) Colony-forming units statistics. The error line in the figure refers to the standard error of mean. The different lowercase letters on the bars indicate groups of treatments that showed significant differences at p < 0.05 level of confidence by one-way analysis of variance (ANOVA) and least significant difference (LSD) tests.

3.4. Sanghuang mushroom extracts improved the vagina inflammation in mice

After C. albicans infected the vagina, inflammatory reactions occurred in the vagina, leading to swelling or inflammatory hyperplasia of the vaginal mucosa and subsequent thickening of the vaginal epithelium. The results of HE staining indicated that the normal group had normal and clear vaginal wall tissue structure, smooth vaginal mucosa epithelium, closely arranged muscle layer and no obvious necrotic cells shed. In the model group and the DMSO control group, the epithelium of the vaginal wall was focally detached with chronic inflammatory cell infiltration and thickening of the vaginal epithelium. Compared with the model group, the FLZ and Sanghuang mushroom extracts administration groups had a more intact mucosal layer of vaginal tissue and homogeneous mucosal thickness, with mild epidermal keratinization and insignificant inflammatory cell infiltration (Figure 5A). Taken together, these findings suggested that treatment with FLZ and Sanghuang mushroom extracts may have a protective effect against C. albicans-induced vaginal inflammation.

Figure 5.

Figure 5.

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Effects of Sanghuang mushroom extracts on the vaginal inflammation in mice. (A) Hematoxylin & eosin staining. (B) Immunohistochemical analysis.

3.5. Sanghuang mushroom extracts downregulated the expression of TNF-α, IL-1β & IL-6 mice's vagina

Immunohistochemical analysis was performed to further confirm the effects of Sanghuang mushroom extracts on inflammatory infiltrate in vivo. As shown in Figure 5B & Supplementary Figure S5, the mice in the normal group had intact vaginal structures and homogeneous vaginal mucosal thickness with insignificant inflammatory cell infiltration compared with the model group and the DMSO control group. Interestingly, the FLZ and Sanghuang mushroom extract treatment groups improved inflammatory cell infiltration. The expression of three inflammatory factors, TNF-α, IL-1β and IL-6 was elevated in the vagina of the model and DMSO control mice. The expression of these factors was significantly inhibited in the FLZ and Sanghuang mushroom extracts administration groups compared with the model group (p < 0.05). These results further demonstrated the in vivo anti-inflammatory properties of Sanghuang mushroom extracts against C. albicans.

3.6. Antifungal components were detected by metabolomics profiling of Sanghuang mushroom extracts

Based on UHPLC-MS analysis, we obtained the chemical components of Sanghuang mushroom extracts. The total ion flow chromatogram of the compound is shown in Supplementary Figure S6, and the chemical components are shown in Table 2. Among them, piperine, gallic acid, maslinic acid, ursolic acid, oleanolic acid, betaine, citrinin, resveratrol, hesperidin and cycloheximide were the antifungal substances, especially cycloheximide. Cycloheximide, a common antibiotic isolated from Streptomyces griseus, significantly inhibits eukaryotic protein synthesis and interferes with the translational process [32]. It has been reported to participate in protein expression, apoptosis and metabolic regulation [33], suggesting that this substance may be involved in the regulation of apoptosis in C. albicans.

Table 2.

Chemical components of Sanghuang mushroom extracts as observed by untargeted metabolomic analysis.

Name Formula Molecular weight RT (min) M/z
Piperine C17H19NO3 303.143 1.497 304.150
Gallic acid C7H6O5 170.022 5.171 339.036
Maslinic acid C30H48O4 494.337 8.339 495.344
Ursolic acid C30H48O3 456.360 7.210 457.368
Oleanolic acid C30H48O3 219.175 9.599 439.357
Betaine C5H11NO2 117.079 1.404 118.086
Citrinin C13H14O5 232.072 5.293 233.079
Resveratrol C14H12O3 228.077 5.126 227.069
Hesperetin C16H14O6 302.079 5.549 303.086
Cycloheximide C15H23NO4 281.163 6.235 280.156
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4. Discussion

C. albicans is a diphasic fungus, and its biofilms are intrinsically resistant to conventional antifungal therapeutics, the host immune system and other environmental perturbations, which presents a significant clinical challenge [34,35]. Natural products and traditional medicines have recently gained attention as promising candidates for antifungal therapies due to their positive efficacy and minimal side effects [36]. Further research is needed to develop new natural antimicrobial drugs. Sanghuang mushrooms, a well-known medicinal fungus, possess antifungal, antioxidant and immunomodulatory activities [36]. To the best of our knowledge, this is the first study that investigated the effect of Sanghuang mushroom extracts against C. albicans.

Biofilm formation is thought to be an important virulence factor that can greatly increase resistance to antifungal drugs [34,37]. In this study, we observed that the C. albicans in the control group had full bodies, the hyphal was crisscrossed to form a biofilm in vitro and the extracellular matrix was abundant. Interestingly, after treatment with Sanghuang mushroom extracts, we observed an inhibition of the morphological transition from yeast to hyphae and a disruption of the biofilm structure. Moreover, the C. albicans treated with Sanghuang mushroom extracts showed swelling and rupture of the mycelium, crumbling and dissolving. These results indicated that Sanghuang mushroom extracts could efficiently fight against the biofilm formation of C. albicans and destroy its biofilm structure and the detailed mechanism of action should be further investigated.

ROS is a normal metabolic product of eukaryotic cells, and its relatively balanced content maintains normal cellular life activities. However, the accumulation of intracellular ROS in C. albicans can trigger apoptosis, disrupt the activity of cellular biomolecules and interfere with cellular metabolism, leading to cell death [38,39]. Our study showed that Sanghuang mushroom extracts could increase the level of ROS in C. albicans biofilm cells, and this effect is concentration-dependent. Mitochondria are the main site of intracellular ROS production, and an elevation in MMP has been demonstrated to drive mitochondrial ROS production [40,41]. In contrast, a decrease in MMP could result in mitochondrial dysfunction, an important cascade response to early apoptosis [40,41]. It has been reported that dill seed essential oil can exert antifungal effects by causing the accumulation of intracellular ROS and disturbance of mitochondrial potential [42]. Our findings also showed that a certain concentration of Sanghuang mushroom extracts can reduce the MMP of C. albicans biofilm cells, suggesting that Sanghuang mushroom extracts may exhibit anti-C. albicans effect by destroying the functional pathway of MMP. Previous studies have shown that the extract from papaya could significantly induce apoptosis in C. albicans cells significantly [43]. Similarly, our findings also revealed that the inhibitory effect of Sanghuang mushroom extracts is strictly associated with apoptosis induction. Altogether, these findings suggested that Sanghuang mushroom extracts can induce C. albicans apoptosis via increasing the ROS and depolarizing the MMP in vitro, thereby exerting an anti-Candida effect. This suggested that it is feasible to induce apoptosis in pathogenic fungi through natural drugs to achieve antifungal effects.

Vulvovaginal candidiasis (VVC) is a gynaecological infection caused by an imbalance in the vaginal flora or pH disorder, prolonged use of immunosuppressants and misuse of antibiotics [44]. C. albicans is the dominant pathogen in VVC, and its hyphae invade the inner layers of the vaginal epithelium, causing damage to the host and VVC [45]. Azole antifungal agents are currently the most widely used treatment for candidiasis, but the static function of azole activity and the intractability of fungi leads to VVC recurrence and treatment failure [46]. VVC provokes histologic lesions characterized by inflammatory infiltrate [47]. In this study, the mice treated with Sanghuang mushroom extracts had a more intact mucosal layer and uniform mucosal thickness of the vaginal tissue, and the inflammatory cell infiltration was not obvious. Moreover, Sanghuang mushroom extract treatment significantly down-regulated the expression of three inflammatory factors, TNF-α, IL-1β and IL-6, especially TNF-α. Therefore, Sanghuang mushroom extracts can effectively treat C. albicans infections in a murine model of VVC.

Nevertheless, the current study still has some limitations. First, this study only explored the antimicrobial effect of Sanghuang mushroom extracts in VVC development, and future research should focus on its role in other types of candidiasis. Future studies should also consider the presence of other microorganisms in the body's environment. Second, the specific active ingredients of Sanghuang mushroom extracts against C. albicans need to be further investigated. Third, the exact mechanism of Sanghuang mushroom extracts for drug-resistant C. albicans and its anti-inflammatory effects need further investigation. Additionally, future experiments should explore the relationship between biofilm formations and related signaling pathways.

5. Conclusion

Sanghuang mushroom extracts inhibited biofilm formation and increased the cell membrane permeability of C. albicans in vitro, ameliorated C. albicans-induced inflammation and protected the integrity of the vaginal mucosa in vivo. Furthermore, the antifungal effects of Sanghuang mushroom extracts might be achieved by the secretion of antibiotics, the accumulation of ROS and depolarization of MMP, resulting in a pathogenic cell apoptosis. These findings suggest that Sanghuang mushroom extracts have a potent antifungal effect against C. albicans, highlighting their potential as a novel therapeutic strategy against candidiasis, including VVC.

Supplementary Material

Supplementary Figures S1-S6 and Table S1
IFMB_A_2352269_SM0001.zip (885.8KB, zip)

Acknowledgments

Assistance from J Shao, School of Integrated Chinese and Western Medicine, Anhui University of Chinese Medicine in providing C. albicans strain SC5314 was greatly appreciated.

Funding Statement

This study was supported by the Anhui Provincial Natural Science Foundation (2108085QH374), Fundamental Research Funds for the Anhui University of Chinese Medicine (2020rcyb008), Foundation of Anhui Province Key Laboratory of Research and Development of Chinese Medicine (AKLPDCM202310) and Research Funds of Joint Research Center for Chinese Herbal Medicine of Anhui of IHM (yjzx2023005) to W Xu, Research Funds of Joint Research Center for Qianshan Medical Health Development Technology Research Center (2021QSHZ01) and Anhui University of Chinese Medicine College Students' Innovative Entrepreneurial Training Plan Program (S202210369005) to D Yin.

Supplementary material

Supplementary data for this article can be accessed at https://doi.org/10.1080/17460913.2024.2352269

Author contributions

J Du, S Jin, Y Zhang and W Qiu carried out the studies, participated in collecting data and drafted the manuscript. W Xu, Y Dong and Y Liu performed the statistical analysis and participated in its design. W Xu, D Yin and Y Yang participated in acquisition, analysis or interpretation of data and draft the manuscript. All authors read and approved the final manuscript.

Financial disclosure

This study was supported by the Anhui Provincial Natural Science Foundation (2108085QH374), Fundamental Research Funds for the Anhui University of Chinese Medicine (2020rcyb008), Foundation of Anhui Province Key Laboratory of Research and Development of Chinese Medicine (AKLPDCM202310) and Research Funds of Joint Research Center for Chinese Herbal Medicine of Anhui of IHM (yjzx2023005) to W Xu, Research Funds of Joint Research Center for Qianshan Medical Health Development Technology Research Center (2021QSHZ01) and Anhui University of Chinese Medicine College Students' Innovative Entrepreneurial Training Plan Program (S202210369005) to D Yin. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

All animal experiments were approved by the Experimental Animal Ethics Committee of Anhui University of Chinese Medicine (AHUCM-mouse-2022127).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figures S1-S6 and Table S1
IFMB_A_2352269_SM0001.zip (885.8KB, zip)

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