Adenophora triphylla var. japonica Inhibits Candida Biofilm Formation, Increases Susceptibility to Antifungal Agents and Reduces Infection

(1) Background: Candida is the most common cause of fungal infections worldwide, but due to the limited option of antifungal therapies, alternative strategies are required. (2) Methods: Adenophora triphylla var. japonica extract was used for the biofilm formation assay using RPMI1640. The combinatorial antifungal assay, the dimorphic transition assay, and the adherence assay were done to see the influence of inhibition of biofilm formation. qRT-PCR analysis were performed to check the gene expression. (3) Results: Adenophora triphylla var. japonica extract inhibited the Candida biofilm formation. Treatment of extract increased the antifungal susceptibility of miconazole from a 37% reduction in fungal growth to 99.05%, and also dose-dependently reduced the dimorphic transition of Candida and the attachment of Candida to HaCaT cells. The extract blocked the expression of hyphal-related genes, extracellular matrix genes, Ras1-cAMP-PKA pathway genes, Cph2-Tec1 pathway gene, and MAP kinase pathway gene. (4) Conclusions: In this study, the treatment of Adenophora triphylla var. japonica extract showed inhibition of fungal biofilm formation, activation of antifungal susceptibility, and reduction of infection. These results suggest that fungal biofilm formation is a good target for the development of antifungal adjuvants, and Adenophora triphylla var. japonica extract should be a good candidate for biofilm-associated fungal infections.


Introduction
Candida albicans is an opportunistic fungal pathogen that can cause systemic infections in individuals when mucosal barriers are disrupted, or the immune system is compromised [1]. Nosocomial C. albicans infection is often linked with the ability to form biofilms on mucosal surfaces and implanted medical devices [1][2][3].
Biofilm formation is a finely regulated process by multiple interconnected signaling pathways [4][5][6][7][8] and causes structured microbial communities to attach to surfaces [9,10]. The polysaccharide matrix indeed can act as a barrier for adherent cells by preventing the invasion of antifungal agents, conferring drug resistance. Adherent C. albicans cells living within the biofilm are up to 1000 times more resistant to common antifungal agents than planktonic cells, even in the absence of specific drug-resistance genes [11]. Thus, biofilms are a source of viable fungal cells that can potentially cause systemic infection with a mortality rate of 40-60% [12].
There have been many efforts to find novel natural compounds from medicinal plants to block biofilm-associated infections [13]. Medicinal plants are used in a variety of traditional remedies to treat cancer, infections, fever, asthma, and many other ailments, and generally have fewer side effects than over-the-counter medicines. Therefore, medicinal plants should be a new source of alternative therapies for the treatment of Candida infections [14,15].

A. triphylla var. japonica Did Not Inhibit the Fungal Growth
Some antifungal agents inhibit fungal biofilm formation because they kill fungi and can indirectly reduce biofilm formation. A. triphylla var. japonica extract did not affect the growth of C. albicans after 24 h incubation. This result suggested that the extract's inhibition of biofilm formation was not due to the reduced growth of Candida (Figures 1 and S1A-E) and generally have fewer side effects than over-the-counter medicines. Therefore, medicinal plants should be a new source of alternative therapies for the treatment of Candida infections [14,15].
Adenophora triphylla var. japonica (Korean name Zandae) has been used as an herbal medicine in Korea, China, and Japan to control lung diseases such as cough, sputum, asthma, and airway inflammatory diseases. Recently it was reported that extracts of A. triphylla var. japonica exhibited anti-inflammation, antidiabetic, antiparasitic, antitussive, and hepatoprotective effects [16][17][18].
In this study, A. triphylla var. japonica extract was tested for anti-biofilm-forming activity against several fungi including C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis. Interestingly, the extract blocked biofilm formation, increased susceptibility to antibiotics, and reduced fungal infections.

A. triphylla var. japonica Did Not Inhibit the Fungal Growth
Some antifungal agents inhibit fungal biofilm formation because they kill fungi and can indirectly reduce biofilm formation. A. triphylla var. japonica extract did not affect the growth of C. albicans after 24 h incubation. This result suggested that the extract's inhibition of biofilm formation was not due to the reduced growth of Candida (Figure 1, Figure  S1A-E) Figure 1. A triphylla var. japonica extract did not inhibit the growth of C. albicans. C. albicans (1 × 10 6 cells/mL) containing 100 μg/mL of A. triphylla var. japonica extract was incubated for 24 h at 30 °C. Experiments were performed in triplicate.

A. triphylla var. japonica Extract Inhibited the Biofilm Formation of Candida
Biofilms are particularly important for fungi to survive and infect. A. triphylla var. japonica extract was used to test whether it blocks fungal biofilm formation. A. triphylla var. japonica extract dose-dependently inhibited Candida biofilm formation in all strains tested (Table 1) with an IC50 value of approximately 6.25 μg/mL against C. albicans ( Figure  1). The IC50 value of the other strains was 6.25 μg/mL (C. parapsilosis var. parapsilosis and C. glabrata), 25 μg/mL (C. tropicalis var. tropicalis and C. tropicalis), and 50 μg/mL (C. parapsilosis). (Figure 2, Table 1

A. triphylla var. japonica Extract Inhibited the Biofilm Formation of Candida
Biofilms are particularly important for fungi to survive and infect. A. triphylla var. japonica extract was used to test whether it blocks fungal biofilm formation. A. triphylla var. japonica extract dose-dependently inhibited Candida biofilm formation in all strains tested (Table 1) with an IC 50 value of approximately 6.25 µg/mL against C. albicans ( Figure 1). The IC 50 value of the other strains was 6.25 µg/mL (C. parapsilosis var. parapsilosis and C. glabrata), 25 µg/mL (C. tropicalis var. tropicalis and C. tropicalis), and 50 µg/mL (C. parapsilosis). (Figure 2, Table 1).

A. triphylla var. japonica Extract Increased the Susceptibility of Antifungal Ag C. albicans
Biofilms are associated with antifungal activity; therefore, inhibition o mation is expected to increase antifungal efficacy. Treatment with 3.125 μg/ increased the susceptibility of miconazole from 38% inhibition to 99% inhibi growth. The extract also showed synergistic antifungal activity with mag growth inhibition with 3.125 μg/mL extract compared with 13% by magn and diosin (99% growth inhibition with 3.125 μg/mL extract compared with cin alone) (Figure 3a    Biofilms are associated with antifungal activity; therefore, inhibition of biofilm formation is expected to increase antifungal efficacy. Treatment with 3.125 µg/mL of extract increased the susceptibility of miconazole from 38% inhibition to 99% inhibition of fungal growth. The extract also showed synergistic antifungal activity with magnoflorin (99% growth inhibition with 3.125 µg/mL extract compared with 13% by magnoflorin alone) and diosin (99% growth inhibition with 3.125 µg/mL extract compared with 23% by dioscin alone) (Figure 3a

A. triphylla var. japonica Blocked the Dimorphic Transition from Yeast to Hyphae Form
The yeast-to-hyphae conversion is an important virulence factor for C. albicans. The formation of hyphae aids the subsequent invasive growth of C. albicans, infiltrating host tissues and causing systemic infection [6]. Extracts were tested to determine whether the dimorphic transition was affected and found that 1.56 µg/mL of extracts significantly inhibited dimorphic transition of 45% in RPMI1640 and 39% in 10% FBS YPD medium compare with untreated control (Figure 4).

A. triphylla var. japonica Extract Eeduced Fungal Adherence to the HaCaT Cells
Fungal biofilm formation for device-related infections is an important medical problem. A. triphylla var. japonica extract dose-dependently decreased the adhesion of Candida to human HaCaT cells by extract treatment ( Figure 5).

A. triphylla var. japonica Extract Eeduced Fungal Adherence to the HaCaT Cells
Fungal biofilm formation for device-related infections is an important medical problem. A. triphylla var. japonica extract dose-dependently decreased the adhesion of Candida to human HaCaT cells by extract treatment ( Figure 5).

A. triphylla var. japonica Extract did Not Affect the Growth of Human Originated Cell
The cytotoxic effect of A. triphylla var. japonica extract on HaCaT cells and macrophages THP-1 was tested by MTT assay. A. triphylla var. japonica extract did not show any significant cytotoxic effect on both HaCaT and THP-1 cells (Figure 6a,b).

A. triphylla var. japonica Extract did Not Affect the Growth of Human Originated Cell
The cytotoxic effect of A. triphylla var. japonica extract on HaCaT cells and macrophages THP-1 was tested by MTT assay. A. triphylla var. japonica extract did not show any significant cytotoxic effect on both HaCaT and THP-1 cells (Figure 6a,b).

A. triphylla var. japonica Extract did Not Affect the Growth of Human Originated Cell
The cytotoxic effect of A. triphylla var. japonica extract on HaCaT cells and macrophages THP-1 was tested by MTT assay. A. triphylla var. japonica extract did not show any significant cytotoxic effect on both HaCaT and THP-1 cells (Figure 6a,b).

A. triphylla var. japonica Extract Inhibited the Expression of Biofilm Formation and Infection-Related Genes
To understand the molecular basis of the inhibition of biofilm formation and reduction of Candida infection, A. triphylla var. japonica extracts were tested for the expression of genes involved in biofilm formation, hyphae growth, and cell adhesion by qRT-PCR.

Discussion
Many fungi, including Candida, live inside the human body, but when Candida starts to grow out of control, it can cause an infection known as candidiasis. In fact, Candida is the most common cause of fungal infections in humans [1][2][3]. Antifungals can cause side effects and resistance, and there are several reports of resistance, including Candida auris, an emerging problematic organism [1][2][3]. Outbreak response is complicated by limited treatment options and inadequate disinfection strategies. Therefore, new approaches with novel targets for antifungal agents are needed.
In this study, A. triphylla var. japonica extract exhibited biofilm formation inhibitory activity against all Candida species tested ( Figure 2, Table 1). The mechanism of Candida biofilm formation slightly differs depending on the species . Comparisons are difficult because all Candida species have similar regulatory genes at each stage of biofilm formation. However, it can be emphasized that the ALS gene is involved in the adhesion process in three Candida species (C. albicans, C. parapsilosis, and C. tropicalis), and that this phenomenon is regulated by Epas in C. glabrata. In addition, some transcription factors described as being involved in C. albicans biofilm formation (BCR1, EFG1 and HWP1) are the same as those involved in C. parapsilosis biofilms. C. glabrata is a species that exhibits more contrast with respect to other Candida species, reflecting genetic distance [5]. A. triphylla var. japonica extract inhibits Candida biofilm formation, suggesting that there should be a common mechanism that is regulated by plant extract to influence the formation of biofilms among Candida species, but more studies are needed. In addition, A. triphylla var. japonica extract increased the antifungal activity of miconazole, magnoflorine, and dioscin (Figure 3), and decreased Candida infection (Figures 4 and 5). These results showed that biofilm formation was associated with the susceptibility of antifungal agents and fungal infections.
According to gene expression analysis, the expression of genes related to biofilm formation, hyphae growth, and cell adhesion was significantly reduced by A. triphylla var. japonica extract treatment. Biofilm formation in Candida species is determined by various transcription factors, including BCR1, EFG1, TEC1, and NDT80, which act as components in multiple pathways, and their participation in Candida adhesion also suggests that alt-

Discussion
Many fungi, including Candida, live inside the human body, but when Candida starts to grow out of control, it can cause an infection known as candidiasis. In fact, Candida is the most common cause of fungal infections in humans [1][2][3]. Antifungals can cause side effects and resistance, and there are several reports of resistance, including Candida auris, an emerging problematic organism [1][2][3]. Outbreak response is complicated by limited treatment options and inadequate disinfection strategies. Therefore, new approaches with novel targets for antifungal agents are needed.
In this study, A. triphylla var. japonica extract exhibited biofilm formation inhibitory activity against all Candida species tested ( Figure 2, Table 1). The mechanism of Candida biofilm formation slightly differs depending on the species . Comparisons are difficult because all Candida species have similar regulatory genes at each stage of biofilm formation. However, it can be emphasized that the ALS gene is involved in the adhesion process in three Candida species (C. albicans, C. parapsilosis, and C. tropicalis), and that this phenomenon is regulated by Epas in C. glabrata. In addition, some transcription factors described as being involved in C. albicans biofilm formation (BCR1, EFG1 and HWP1) are the same as those involved in C. parapsilosis biofilms. C. glabrata is a species that exhibits more contrast with respect to other Candida species, reflecting genetic distance [5]. A. triphylla var. japonica extract inhibits Candida biofilm formation, suggesting that there should be a common mechanism that is regulated by plant extract to influence the formation of biofilms among Candida species, but more studies are needed. In addition, A. triphylla var. japonica extract increased the antifungal activity of miconazole, magnoflorine, and dioscin (Figure 3), and decreased Candida infection (Figures 4 and 5). These results showed that biofilm formation was associated with the susceptibility of antifungal agents and fungal infections.
According to gene expression analysis, the expression of genes related to biofilm formation, hyphae growth, and cell adhesion was significantly reduced by A. triphylla var. japonica extract treatment. Biofilm formation in Candida species is determined by various transcription factors, including BCR1, EFG1, TEC1, and NDT80, which act as components in multiple pathways, and their participation in Candida adhesion also suggests that although biofilm formation is initially tested, adhesion or infectious factors are also controlled by treatment with A. triphylla var. japonica extract. Further studies should be performed including the precise targeting of A. triphylla var. japonica extracts, effector components, and in vivo testing (Scheme 1).
hough biofilm formation is initially tested, adhesion or infectious factors are also controlled by treatment with A. triphylla var. japonica extract. Further studies should be performed including the precise targeting of A. triphylla var. japonica extracts, effector components, and in vivo testing (Scheme 1).
In conclusion, A. triphylla var. japonica extracts inhibited C. albicans biofilm formation, increased antifungal activity of antifungal agents, and decreased fungal adhesion to host cells. Therefore, A. triphylla var. japonica extract should be a good candidate for biofilmforming fungal infections.

Fungal Strains Used in This Study
The Candida strains used in the study are listed in Table 2

Extraction of A. triphylla var. japonica
The leaves of A. triphylla var. japonica were obtained from Jeju Island. Three-hundred grams of the sample was extracted using 3 L distilled water at 80 °C for 8 h, concentrated at 40 °C using a rotary evaporator, and then freeze-dried. For the experiment, 10 mg of plant extract powder was dissolved in 1 mL of dimethyl sulfoxide [14][15][16][17][18][19]. In conclusion, A. triphylla var. japonica extracts inhibited C. albicans biofilm formation, increased antifungal activity of antifungal agents, and decreased fungal adhesion to host cells. Therefore, A. triphylla var. japonica extract should be a good candidate for biofilmforming fungal infections.

Extraction of A. triphylla var. japonica
The leaves of A. triphylla var. japonica were obtained from Jeju Island. Three-hundred grams of the sample was extracted using 3 L distilled water at 80 • C for 8 h, concentrated at 40 • C using a rotary evaporator, and then freeze-dried. For the experiment, 10 mg of plant extract powder was dissolved in 1 mL of dimethyl sulfoxide [14][15][16][17][18][19].

Biofilm Formation Assay
A. triphylla var. japonica extracts were prepared in 96-well plate plates (SPL, Korea) with the concentration ranging from 6.25 to 100 µg/mL. Wells without test compound served as controls (DMSO concentration 0.1%). C. albicans suspension at 1 × 10 6 CFU/mL was prepared in RPMI 1640 medium [24][25][26][27][28][29][30][31]. Then, 100 µL of the solution was inoculated into a 96-well flat-bottom plate and incubated at 37 • C for 24 h with shaking. Biofilm formed was washed with PBS to remove nonadherent cells, and then 100 µL of 1% aqueous crystal violet was applied for 30 min. Each well was washed three times with PBS and destained with 150 µL of 30% acetic acid for 15 min. The absorbance was measured at 595 nm with a microplate reader (Bio Tek Instruments, Korea). Experiments were performed in triplicate.

Antifungal Activity Assay
C. albicans were grown overnight in YPD diluted to 1 × 10 6 cells/mL. Induction of biofilm formation was performed as described above. The broth microdilution method was applied to determine the MICs using microplates [32]. Serial twofold dilutions of A. triphylla var. japonica extracts were prepared in YPD broth. The MIC value was determined as the minimum compound concentration. Miconazole (3.125 µg/mL), magnoflorin (3.125 µg/mL), and dioscin (3.125 µg/mL) alone or with the indicated concentration of A. triphylla var. japonica extract were added to each well and incubated for 24 h at 37 • C [23][24][25][26][27]. Experiments were performed in triplicate.

Dimorphic Transition Assay
C. albicans were grown overnight in YPD medium. 1 × 10 6 cells/mL of Candida with or without extract were incubated in YPD medium supplemented 10% fetal bovine serum medium or sodium bicarbonate-free RPMI medium with glutamine buffered with MOPS [3-(N-morpholino) propanesulfonic acid] to pH 7 recommended by the CLSI standard to induce dimorphic transition for 4 h at 37 • C. Quantification of inhibition of dimorphic transition was performed by counting the number of yeast and hyphal cells in the population as previously described [25][26][27][28][29][30][31]. At least 1000 cells were counted in duplicate for each well and all assays were repeated five times. An image of the cells was obtained using a microscope.

Candida Adherence Assay
Human epithelial keratinocytes HaCaT cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37 • C in a humidified atmosphere with 5% CO 2 .
HaCaT cells (0.5 × 10 6 cells per well) were grown to confluence for 24 h in 24-well plates. 1 × 10 6 cells/mL of C. albicans mixed with indicated concentration of A. triphylla var. japonica extract was incubated at 37 • C for 24 h. After the DMEM was drained, the plate was carefully washed three times with PBS to remove non-adherent cells. The number of adherent C. albicans cells was measured by scraping the cells with a sterile scraper. Serial dilutions were plated on YPD agar to determine viability. A microscope was used to obtain representative images of the results [26,27].

MTT Assay
Cytotoxicity of A. triphylla var. japonica extracts against HaCaT and THP-1 was tested by a slightly modified MTT assay. 1 × 10 4 HaCaT and THP-1 in DMEM and RPMI1640 media, respectively, were added to each well containing the indicated concentration of extracts and incubated for 24 h. After discarding the media, MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma, St. Louis, MO, USA) in PBS was added into each well, followed by incubation for 3 h at 37 • C. The medium was then removed, and cells were suspended in 100 µL DMSO for 10m. Cell viability was calculated as optical density (OD 540 ) values measured using a microplate reader (BioTek Instruments, Korea) and reported as a percentage compare with vehicle control [25]. Experiments were performed in triplicate.

Statistical Analysis
All experiments were performed at least three times and data were presented as the ± mean S.D. Graphs were generated using Microsoft Excel (Microsoft Corp., Redmond, WA, USA).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.