Abstract

Introduction:

The growing prevalence of drug-resistant pathogens urgently calls for new treatment strategies. Traditional Chinese medicine (TCM) formulas, with their multi-targeted mechanisms of action, offer promising alternative options for antimicrobial therapy. This study aims to evaluate the antimicrobial activity of the TCM formula MangHuang solution (MH) after content detection of tannin, composed of Rhei Radix et Rhizoma, Natrii Sulfas, and Galla Chinensis, and to elucidate its antifungal mechanism against Candida albicans through integrated multi-omics analysis.

Methods:

MH and its individual or combined components were prepared and evaluated for their inhibitory effects on Staphylococcus aureus, Escherichia coli, and Candida albicans, and their antimicrobial activity was assessed. Transmission electron microscopy (TEM), biofilm formation experiments, and multi-omics analysis were used to investigate the antifungal mechanism of MH against C. albicans.

Results:

MH demonstrated potent and rapid antibacterial activity. Biofilm formation was significantly inhibited, manifested by reduced cell surface hydrophobicity, weakened initial adhesion capacity, and impaired biofilm maturation processes. Transcriptome and metabolome analyses revealed significant alterations in key metabolic pathways, particularly ABC transporters, amino acid biosynthesis, and protein-related pathways.

Discussion:

MH exhibits potent antifungal activity against C. albicans through a multi-target mechanism, primarily affecting biofilm formation and intracellular metabolic processes. The integration of multi-omics approaches provides strong evidence for the potential clinical application of MH as an effective antifungal agent.

1 Introduction

Environmental pollution and the widespread misuse of antibiotics have made microbial infections a critical global public health challenge (Holmes et al., 2016). A representative example is Candida albicans-induced vaginitis, which impairs patients’ quality of life, prolongs treatment, and elevates healthcare costs due to the pathogen’s increasing antibiotic resistance (Marnach et al., 2022). The conventional model for developing antibacterial drugs now faces significant obstacles, highlighting an urgent need to explore new strategies like anti-virulence and anti-biofilm approaches, as well as novel sources such as natural products.

Candida albicans represents a serious clinical threat, capable of causing invasive infections with a broad spectrum, high incidence, and significant mortality (Wasiński, 2019; Ahmad-Mansour et al., 2021). Its complex cell wall, rich in antigens and toxic compounds, facilitates evasion of immune responses and disruption of host homeostasis (Nobile and Johnson, 2015). Likewise, Staphylococcus aureus and Escherichia coli are major clinical pathogens; their numerous virulence factors, compounded by antibiotic overuse, greatly complicate treatment and pose an ongoing challenge to global public health (Ruiz-Herrera et al., 2006; Poulain, 2015). Together, these pathogens demonstrate enhanced adaptability, transmissibility, colonization capacity (Gulati and Nobile, 2016; Wall et al., 2019), and pronounced multidrug resistance (Pereira et al., 2021).

In Traditional Chinese Medicine (TCM) theory, pathogens are considered “evil qi” (xie qi), which are external pathogenic factors that can easily cause infection when the body’s vital functions are compromised (Methicillin-resistant staphylococcus aureus, 2018; Paitan, 2018). While TCM-based natural preparations are widely used, few single-ingredient agents possess potent antibacterial activity on their own; they typically require combination with antibiotics (Prasad et al., 2019) or are used as alternative therapies (Crowley and Gallagher, 2014).

MangHuang solution (MH) is a pure TCM formulation developed by a pharmaceutical company in Sichuan Province. It has been used clinically at institutions including Chengdu Anorectal Hospital, Henggang People’s Hospital of Shenzhen, and Sichuan Provincial Hospital of TCM for treating hemorrhoids and for perianal disinfection (Lu et al., 2021; Ren et al., 2021). Clinical observations confirm its notable antibacterial, anti-inflammatory, and wound-healing effects. The formulation comprises extracts from Galla Chinensis (Wubeizi), Rhei Radix et Rhizoma (Dahuang), and Natrii Sulfas (Mangxiao). In TCM principles, these herbs are considered cold-natured and belong to the large intestine meridian, where they function to clear heat, purge fire, and remove toxins. Chemically, MH’s primary constituents are anthraquinones (from Rhei Radix et Rhizoma), sodium sulfate(from Natrii Sulfas), and tannin (from Galla Chinensis). These compounds demonstrate broad-spectrum antibacterial activity and can eliminate harmful metabolites and endotoxins produced during microbial infections (Xu et al., 2014; Xu et al., 2024).

This study investigates the antimicrobial activity of a TCM compound formulation, derived from the MH base, against C. albicans and other pathogens, following preliminary screening. We aim to elucidate its mechanisms of action to inform subsequent research and potential drug development.

2 Materials and methods2.1 Preparation of Chinese herbal extracts

The traditional Chinese herbal compound solution MH consists of Rhei Radix et Rhizoma (30 g), Natrii Sulfas (30 g), and Galla Chinensis (30 g), prepared using the decoction method. The crude herbs were boiled in 600 mL of water over high heat, then simmered over low heat for 1 h. The extract is filtered and concentrated to obtain a solution with a total crude herbal material concentration of 0.3 g/mL. To investigate the contribution of individual herbal medicines and compound ratios, seven formulations (W–WDM) were designed, each maintaining a total crude herbal material concentration of 0.3 g/mL (Cheng et al., 2021; Yuan et al., 2024). The prescriptions for each formulations are presented in Table 1.

GroupPrescriptionW30 g Galla Chinensis, 200 mL waterD30 g Rhei Radix et Rhizoma, 200 mL waterM30 g Natrii Sulfas, 200 mL waterWD30 g Galla Chinensis, 30 g Rhei Radix et Rhizoma, 400 mL waterWM30 g Galla Chinensis, 30 g Natrii Sulfas, 400 mL waterDM30 g Rhei Radix et Rhizoma, 30 g Natrii Sulfas, 400 mL waterWDM (MH)30 g Galla Chinensis, 30 g Rhei Radix et Rhizoma, 30 g Natrii Sulfas, 600 mL water

The prescriptions of different formulations.

Accurately pipette 2.0 mL of the MH and evaporate it to dryness in a 50 °C water bath. The residue is then dissolved in 150 mL of water. The total tannin content is subsequently determined by preparing a test sample solution of the MH and establishing a tannin standard curve according to the method described above (Brêtas et al., 2020).

2.2 Antimicrobial activity determination2.2.1 Solid medium preparation and microbial strains2.2.1.1 Nutrient agar medium

It was prepared by dissolving the following components per liter: peptone, 10.0 g; beef extract, 3.0 g; sodium chloride, 5.0 g; and agar, 15.0 g. The pH was adjusted to 7.2 ± 0.2 using 1 M NaOH or HCl.

2.2.1.2 Sabouraud dextrose agar medium

It was prepared by dissolving the following components per liter: dextrose, 40.0 g; peptone, 10.0 g; and agar, 15.0 g. The pH was naturally approximately 5.6 and was not further adjusted.

All media were prepared with deionized water and sterilized by autoclaving at 121°C for 15 min. The sterilized media were then poured into sterile petri dishes, with approximately 15–20 mL per plate, under aseptic conditions. The poured plates were allowed to solidify at room temperature and were either used immediately or stored at 4°C for up to two weeks.

Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 25922) were cultured on NA, while Candida albicans (ATCC 10231) was cultured on SDA. Commercial standard of S. aureus, E. coli, and C. albicans were purchased from BeNa Culture Collection and revived on the corresponding agar medium.

2.2.2 Antimicrobial testing

Antimicrobial activity was assessed according to GB 15979–2002 and WS/T 650–2019 standards. The former is a Chinese national standard 15979-2002, while the latter is a Chinese health industry standard 650-2019. Both standards stipulate methods for evaluating the antibacterial activity of solutions and share similar experimental procedures. The practical protocol is described as follows.

Fresh 24 h cultures of S. aureus and E. coli on NA, and of C. albicans on SDA, were cultivated. The microbial cells were harvested by washing the agar surface with phosphate-buffered saline (PBS, pH 7.4, containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) to prepare the suspension.

Briefly, 0.1 mL bacterial or fungal suspension (~1×104–9×104 CFU/mL) were treated with 5 mL of each herbal medicine solution for 2, 5, 10, and 20 min. The reaction was terminated by dilution with 5mL PBS three consecutive times, and 0.5 mL aliquots from appropriate dilutions were plated. All plates were incubated at 36 ± 1 °C for 48 h for bacteria or 72 h for C. albicans prior to colony enumeration (relative humidity = 70%) (GB 15979-2002, 2002). All experiments were performed in triplicate (n = 3).

Bacteriostatic rate ≥ 50% - 90%, judged to have bacteriostatic effect; Bacteriostatic rate ≥ 90%, judged to have strong bacteriostatic effect. The bacteriostatic rate was calculated using the following formula:

2.3 Mechanistic study of Candida albicans2.3.1 Growth curve analysis

Into pre-labeled 5 mL EP tubes, we first added 1 mL of a C. albicans suspension (OD600nm = 0.4 in RPMI-1640 medium), followed by 1 mL of the drug solution from the MH. This yielded a final volume of 2 mL per tube, with final concentrations of 2.5 × 108 CFU/mL for the fungus and 150 mg/mL for the drug. PBS served as the control. We monitored the C. albicans cultures in both the treated and untreated groups every 3 h over a 24 h period to construct growth curves (WS/T 650-2019, 2019; Su et al., 2021). PBS served as the control group, and all experiments were performed in triplicate (n = 3).

At the same time, fungistatic rate was used to calculate in section 2.2 to draw the Time-Kill curve.

2.3.2 Transmission electron microscopy

The cultured condition was prepared in a growth medium containing either MH or PBS against C. albicans, following the procedure described in section 3.1. On the second day, fungal cells were harvested by centrifugation at 4000 rpm for 10 min. The supernatant was discarded, and the cell pellet was gently resuspended in 1 mL of 2.5% glutaraldehyde. The suspension was transferred to a 1.5 mL microcentrifuge tube and fixed by centrifugation at 10,000 rpm for 10 min. After carefully removing the supernatant, fresh 2.5% glutaraldehyde was slowly added along the tube wall to minimize disturbance to the cells. Samples were allowed to stand at room temperature for 30 min before being stored at 4°C (Tizro et al., 2019; Lu et al., 2023).

Subsequently, samples underwent dehydration, embedding, ultrathin sectioning, and staining. The sample rod was inserted to calibrate the optical path. The target area was first located in low-magnification mode, the sample position was adjusted, and imaging was then performed at high magnification. Morphological alterations of C. albicans were examined under a Hitachi HT-7800 TEM operated at 80 kV.

2.3.3 Sorbitol protection assay

To determine whether the antifungal effect targets the cell wall, C. albicans was cultured in RPMI-1640 medium and RPMI-1640 medium containing 0.8 M sorbitol during treatment (Mishra and Wang, 2017; Mohammad Zadeh et al., 2021). PBS served as the control. Following a 24 h incubation at 37°C, the PBS was sequentially diluted 200-fold and then 10-fold. A 200 µL aliquot was plated onto SDA. Each group was plated in triplicate, and the average value was calculated. The fungistatic rate was calculated using the method described in section 3.1. PBS served as the control group, and all experiments were performed in triplicate (n = 3).

2.3.4 Biofilm detection

Biofilm inhibitory activity was assessed using the following methods:

2.3.4.1 Cell surface hydrophobicity (MATH assay)

The cultured environment was prepared according to the method in section 3.1, using RPMI-1640 medium without phenol red. Following a 24 h incubation at 37°C, a 100 µL aliquot from each group was transferred to a 96-well plate for measurement of the initial absorbance (A0) at 600 nm. Next, 1.5 mL of the suspension was combined with an equal volume of n-hexadecane, vortexed for 1 min, and incubated at room temperature for 15 min to achieve complete phase separation. The lower aqueous phase was carefully aspirated, and its absorbance (A1) was measured at 600 nm. The cell surface hydrophobicity was then calculated using the formula:

2.3.4.2 Initial adhesion

C. albicans was revived and cultured overnight on SDA. The following day, colonies were diluted in phenol red-free RPMI 1640 medium to prepare a fungal suspension with an OD600nm at 0.4. A 100 μL aliquot of this suspension was added to each well of a 96-well plate containing 100 μL of different drug solutions. After incubation at 37°C for 2.5 h, all liquid was carefully aspirated from the wells. The wells were then washed twice with 200 μL of PBS to remove non-adherent fungi. Following PBS removal, 200 μL of methanol was added to each well for fixation for 15 min. The methanol was then aspirated, and the plate was air-dried in a biosafety cabinet. Subsequently, 200 μL of 1% crystal violet solution was added to each well for staining for 5 min. Excess stain was gently rinsed off under running water in a foam box until the water ran clear. The plate was again air-dried in the biosafety cabinet. Finally, 200 μL of 33% acetic acid was added to each well, and the OD600nm (A1) was determined using a microplate reader.

The initial adhesion was then calculated using the Biofilm inhibitory activity was assessed using the following methods:

2.3.4.3 Mature biofilm formation

The test method was consistent with section 3.2, and the incubation time was extended to 24 h on this basis (Zhang et al., 2011; Ma et al., 2023; Wang et al., 2024). PBS served as the control, and all experiments were performed in triplicate (n = 3).

2.3.5 Morphological observation

The test method was consistent with formation of biofilm. Treated C. albicans cells were stained with Lugol’s iodine solution and observed under a light microscope to assess the conversion of yeast to hyphae. The test method followed the procedure outlined in section 3.4. Following the incubation period, images were captured using a microscope by 100x.

2.4 Transcriptome analysis

C. albicans cells treated with MH and gallic acid (70% purity, isolated from Galla Chinensis in the laboratory) were subjected to RNA extraction, quality control (RIN > 8.0), and cDNA library construction (Lu et al., 2023). The library was sequenced on the Illumina platform. Differentially expressed genes (DEGs) were identified using a threshold of |log2FC| ≥ 1 and FDR< 0.05, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.

2.5 Metabolomics analysis

Fresh 24 h slant cultures of the test strain (C. albicans) were washed off with sterile PBS. The cell suspension was adjusted to an OD600nm of 0.4 using PBS as determined by a microplate reader. Drug A was prepared in PBS at 100 mg/mL, and Drug B was dissolved in PBS to 60 mg/mL. In a sterile 10 mL centrifuge tube, 4.0 mL of the corresponding drug solution was added first and pre-equilibrated in a 20 ± 1°C water bath for 5 min. Subsequently, 4.0 mL of the prepared fungal suspension was added, and the mixture was immediately vortexed to ensure thorough mixing. The cultures were incubated for 24 h. After incubation, samples from each group were collected; the cultured supernatants were harvested and stored at −80°C until further metabolomics analysis. Metabolite profiling was performed using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Differentially expressed metabolites (VIP > 1.0, p< 0.05) were identified using multivariate statistical analysis (PCA, OPLS-DA), followed by KEGG pathway annotation.

2.6 Integrated multi-omics analysis

Transcriptomics and metabolomics datasets from matched samples were integrated to investigate coordinated molecular changes. Normalized gene expression and metabolite abundance matrices were used to define differentially expressed genes (DEGs) and differentially expressed metabolites, respectively. Pearson’s correlation coefficients were then calculated across samples to quantify DEG–metabolite associations, and statistically significant correlations (with multiple-testing correction where applicable) were retained for interpretation. To contextualize these cross-omics relationships, DEGs and differential metabolites were mapped to KEGG and subjected to pathway analysis, and pathways supported by both omics layers were identified as overlapping regulatory effects.

2.7 Statistics

PBS was used as the control group, and all experiments were performed in triplicate (n = 3). Data are expressed as mean ± standard deviation (SD). Significant differences were assessed using one-way or two-way analysis of variance (ANOVA) on GraphPad Prism 9, version 9.5.1.

3 Results3.1 Determination of tannin content in MangHuang solution

Figure 1 presents the standard curve for gallic acid. The relationship between concentration and absorbance is linear, with a regression equation of y = 0.0813x + 0.0546 and an R² value of 0.9991. This indicates a strong correlation.

Standard curve of gallic acid. y = 0.0813 x + 0.0546, R2 = 0.9991.

The analysis yielded an average total tannin concentration of 0.766 mg/mL across six batches of MH, with a relative standard deviation (RSD) of 2.377%. The detailed calculations and results are provided in Table 2.

Batch numberF1122F1123F1124F1125F1126F1127A Total phenols0.4720.4790.4660.4510.4390.442A Non absorbed polyphenols0.1960.1960.1680.1790.1680.165A Tannin content0.2500.2570.2460.2460.2450.251Tannin content
(mg/mL)0.7690.7970.7530.7530.7490.773

Measured and calculated values of tannin content for 6 Batches of MH (n = 6).

A Tannin content = A Total phenols-A Tannin content-0.026, while 0.026 represents the adsorption value of casein; Tannin content = (A Tannin content - 0.0546)/0.0813 * 0.32, 0.32 was the dilution factor in actual process.

3.2 Antimicrobial activities

The W, WD, WM, and WDM groups exhibited potent antimicrobial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria, achieving bacteriostatic rates exceeding 90% within 20 min. Notably, the WM and WDM groups, particularly the latter (MH), demonstrated rapid bactericidal activity, reducing viable bacterial counts by over 90% within just 5 min. Compared to the D and M groups, the W group showed superior efficacy, suggesting that Galla chinensis contributed more prominently to the observed antimicrobial activity than Rhei Radix et Rhizoma and Natrii Sulfas (Figure 2A).

Antimicrobial activity of MH. (A) (S) aureus. (B)E. coil. (C) albicans. (W) Galla chinensis. (D) Rhei Radix et Rhizoma. (M) Natrii Sulfas. (WD) Galla chinensis and Rhei Radix et Rhizoma. (WM) Galla chinensis and Natrii Sulfas. (DM) Rhei Radix et Rhizoma and Natrii Sulfas. (WDM) Galla chinensis, Rhei Radix et Rhizoma and Natrii Sulfas. The values represent the mean ± SD (n=3). A indicates a significant difference compared to the control group (p < 0.05), b indicates p < 0.01, c indicates p < 0.001, and d indicates p < 0.0001.

For E. coli, the D group exhibited stronger inhibition than for S. aureus, resulting in the DM group being more effective against E. coli. At 20 min, the bacteriostatic activity of the DM group against E. coli increased by approximately 10% compared to the D group alone, indicating a synergistic effect of combining Rhei Radix et Rhizoma and Natrii Sulfas without altering the total crude drug concentration (Figure 2B).

Regarding antifungal activity, significant inhibition was observed only in the WM and WDM groups, with fungistatic rates exceeding 50% within 20 min. Remarkably, the WDM group (MH) displayed earlier efficacy, achieving noticeable inhibition at 10 min and exhibiting approximately 20% higher fungistatic activity than the WM group at 20 min (Figure 2C).

3.3 Growth kinetics of Candida albicans in response to MangHuang solution treatment

As shown in Figure 3, C. albicans treated with the WDM group (MH) exhibited a markedly slower growth pattern, with cell viability progressively declining from 0 to 32 h. A significant reduction in C. albicans concentration was observed between 4 and 48 h (p< 0.05). In contrast, the control group displayed a typical growth curve: a lag phase during the first 8 h, followed by a steady increase in absorbance indicative of the logarithmic phase.

Time growth curve of MH on C. albicans (n = 3). The values represent the mean ± SD (n=3). A indicates a significant difference compared to the control group (p < 0.05), b indicates p < 0.01, c indicates p < 0.001, and d indicates p < 0.0001.

These findings suggest that the MH inhibits C. albicans growth primarily by suppressing its reproductive capacity during both the lag and logarithmic phases. Notably, the fungistatic rate at 24 h aligns with the results of Figure 2C, supporting the selection of a 24-h treatment duration for subsequent assays.

As illustrated in Figure 4, the fungistatic activity of MH reached its maximum at 32 h post-treatment, indicating the most effective inhibition of C. albicans at this time. A notable increase in activity was also detected at 20 h, providing a rationale for the 24-h dosing interval. Beyond 32 h, the inhibitory effect of the formulation slowly declined.

Fungistatic rate - Time curve of MH against C. albicans.

3.4 Observation of Candida albicans ultrastructure by TEM

As shown in Figure 5, the cell walls of C. albicans in both the control and treatment groups remained largely intact (blue arrows). However, in cells treated with the WDM group (MH), the cytoplasm appeared heterogeneous, with regions of high electron-density aggregation, vacuolization, and bright cytoplasmic areas (red arrows). These ultrastructural alterations indicate that the antimicrobial mechanism of MH is unlikely to involve direct disruption of the cell wall. Instead, it may be associated with effects on intracellular processes, including gene expression, protein integrity, and metabolic activity.

Ultrastructural features of observed by TEM. (A) Single-cell image of C. albicans in the control group. (B) Single-cell image of in the experimental group. (C) Multi-cell image of in the control group. (D) Multi-cell image of in the experimental group. (A, B) Scale bar is 500 nm. (C, D) Scale bar is 1 μm.

As positive controls, we cite TEM images of untreated C. albicans cell walls and those treated with 1.5 mg/mL AgNO3, which reveal clear structural disruption (Hamida et al., 2021). Silver ions are known to exert potent inhibitory and lethal effects on pathogenic microorganisms. Accordingly, images Figures 6C, D show not only cytoplasmic lysis (yellow arrows) but also a marked reduction in cell wall thickness relative to Figures 6A, B; the white peripheral region has thinned to near invisibility (brown arrows). In Figure 6C particularly, more than half of the upper cell wall appears dissolved (green arrows). The purple arrows indicate metal particles synthesized by the C. albicans cells themselves.

Ultrastructural features of C. albicans cell wall disruption by TEM. (A, B) Micrographs of untreated C. albicans exhibiting an intact cell wall (Cw) and intact plasma membranes (Pm). (B) Single-cell image of C. in the experimental group. (C, D) Micrographs of C. albicans treated with 1.5 mg/mL AgNO3 showing the disruption. Reprinted from (Hamida et al., 2021). Scale bar is 200 nm.

3.5 Verification of cell wall integrity by sorbitol

The trends from the antimicrobial activity tests were consistent with the antifungal effects of the experimental drug solutions. Even with the protective agent sorbitol, the damage to C. albicans remained comparable to that observed without protection. Specifically, the inhibition rate on the sorbitol-containing specialized medium showed no significant difference from that on the conventional medium. TEM imaging further suggests that the antifungal effects of the seven herbal combinations are not primarily mediated through the fungal cell wall. Any cell wall damage appears minimal and unlikely to substantially impair the growth and reproduction of C. albicans (Figure 7).

Cell wall rescue experiment (n=3). (Black column) RPMI-1640 medium without 0.8 M sorbitol. (Gary column) RPMI-1640 medium contain 0.8 M sorbitol.

3.6 Effect of MangHuang solution and its decomposed prescriptions on Candida albicans biofilm3.6.1 CSH, initial adhesion, and biofilm formation

This study first examined the effect of MH and its decomposed prescriptions on the cell surface hydrophobicity (CSH) of C. albicans. As shown in Figure 8A, the CSH of the control group was the highest at 39.03 ± 1.03%. After 24 h of treatment, CSH was reduced across all seven experimental groups. Notably, the D group, WD group, and WDM group (MH) exhibited the most significant reductions, with CSH values dropping below 10%: 3.01 ± 1.46%, 1.72 ± 0.15%, and 6.90 ± 2.60%, respectively. These three groups contained Rhei Radix et Rhizoma, suggesting a potential role in inhibiting C. albicans CSH. The combined use of Rhei Radix et Rhizoma and Galla Chinensis appeared to enhance this inhibitory effect.

Reduced CSH likely impaired the ability of C. albicans to overcome environmental electrostatic repulsion, thereby hindering adhesion to host surfaces. Consequently, initial adhesion rates were also affected. In the control group, the initial adhesion rate was 91.96 ± 0.31%. The W, D, and WM groups showed the largest decreases, while the WDM group, WD group, and DM group exhibited adhesion rates of 30.39 ± 4.41%, 34.54 ± 6.23%, and 28.38 ± 7.57%, respectively. The M group demonstrated the smallest reduction (p< 0.05), whereas differences in the other groups were highly significant (p< 0.0001) (Figure 8B).

The biofilm formation rate at 24 h in untreated C. albicans was 90.87 ± 1.18%. Overall, trends in biofilm formation mirrored those of initial adhesion. The W, D, and WM groups maintained low levels of biofilm formation, with the WDM group ranking second among all experimental groups, showing a biofilm formation rate of 15.80 ± 2.18% (Figure 8C).

Effect of MH and its decomposed prescriptions on C. albicans biofilm (n = 3). (A) CSH. (B) Initial adhesion. (C) Biofilm formation. The values represent the mean ± SD (n=3). A indicates a significant difference compared to the control group (p < 0.05), b indicates p < 0.01, c indicates p < 0.001, and d indicates p < 0.0001.

The absorbance values for CSH, initial adhesion, and biofilm formation in the presence of n-hexadecane or 33% acetic acid are also shown (A0 and A1, Table 3). A smaller difference between A0 and A1 indicates greater damage to the C. albicans biofilm caused by the decomposed formulation. All data represent the mean of three independent replicates (x̄ ± SD, n=3).

GroupCSHInitial adhesionBiofilm formationA0A1A0A1A0A1PBS0.551 ± 0.0260.336 ± 0.0211.027 ± 0.0250.083 ± 0.0021.088 ± 0.1200.098 ± 0.002W1.163 ± 0.0181.047 ± 0.0400.364 ± 0.0110.310 ± 0.014