Antifungal activity and mechanism of action of Ou-gon (Scutellaria root extract) components against pathogenic fungi

Ou-gon, an extract from Scutellaria baicalensis Georgi root, has been shown to exhibit pronounced antifungal activity. The present study aimed to identify antifungal components of Ou-gon and to determine their mechanism of action against pathogenic fungi. Antifungal activity was assessed by the microbroth dilution method using four common human pathogenic fungi, Trichophyton rubrum, Trichophyton mentagrophytes, Aspergillus fumigatus, and Candida albicans. Components of crude Ou-gon extract were separated by reversed-phase high-performance liquid chromatography. Active antifungal components were identified by liquid chromatography-electrospray ionization tandem mass spectrometry. Terminal deoxynucleotidyl transferase dUTP nick end-labelling assay, SYTOX® green uptake assay, determination of intracellular reactive oxygen species and mitochondrial membrane potential as well as microscopy (confocal laser microscopy, scanning and transmission electron microscopy) were used to probe the mode of action. Two components with potent antifungal activity, baicalein and wogonin, were identified in Ou-gon. Baicalein showed potent antifungal activity against the four fungi tested. Wogonin displayed antifungal activity against all four fungi except C. albicans. The components are considered to induce apoptosis-like programmed cell death via hyperproduction of reactive oxygen species. This study enhances our understanding of the antifungal activity of Kampo medicine, and may contribute to the development of new and safe antifungal therapeutics.

We previously evaluated the antifungal activity of 61 commercially available Kampo medicines towards Trichophyton rubrum using the microbroth dilution assay 7 . Seven of these medicines exhibited antifungal activity, with six containing Ou-gon, an extract from the roots of Scutellaria baicalensis Georgi. Furthermore, crude Ou-gon extract exhibited pronounced antifungal activity 7 . Ou-gon is one of the popular crude drugs in Kampo medicine, traditionally used in the Far East because of its anti-inflammatory, antimicrobial, and anti-allergic activities 13,14 . The identification of the active constituents of Ou-gon would facilitate their synthesis and structural modification to obtain active components with enhanced efficacy and potential therapeutic usefulness. The current study aimed to identify antifungal components in Ou-gon and to determine their mechanism of action against pathogenic fungi.

Results
Antifungal activity of ou-gon extracts. The antifungal activities of four Ou-gon extracts (water, MeOH, EtOH, and acetic acid extracts) were examined in a microbroth dilution assay using T. rubrum. The acetic acid, MeOH, and water extracts exhibited pronounced antifungal activity, as shown in Fig. 1, whereas the EtOH extract showed no activity. Among the three extracts that exhibited antifungal activity, the acetic acid extract displayed the strongest activity, comparable to that of amphotericin B, as shown in Fig. 1.

Isolation and identification of the antifungal components of Ou-gon.
Because of its most pronounced relative antifungal activity, the acetic acid extract was selected for reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. The extract was separated by RP-HPLC on a C18 column, and the antifungal activity of the eluted fractions was tested. Fractions no. 16-19 and 31-34 exhibited antifungal activity, as shown in Fig. 2a.
When fraction no. 17 was analysed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), a strong peak, indicating high relative ion abundance, was detected at 271.10 (m/z). This ion represents baicalein, a major component of Ou-gon (Fig. 2b). Similarly, fraction no. 33 was identified to contain a relatively high amount of wogonin (Fig. 2b) 15 .
Effect of baicalein and wogonin on cellular fungal functions. Upon exposure to baicalein, T. rubrum, T. mentagrophytes, A. fumigatus, and C. albicans cells were stained with the SYTOX ® green nucleic acid stain ( Fig. 4). Wogonin affected the cell membrane integrity of all fungi tested except C. albicans.

Figure 1.
In-vitro antifungal activity of crude Ou-gon extracts (20 mg/mL) against T. rubrum in Sabouraud liquid medium at 28 °C from day 0 to 4. Data are presented as the mean ± SD of four independent experiments; *p < 0.05, **p < 0.01 compared with negative control (no treatment). Reactive oxygen species (ROS) accumulation was evaluated in T. mentagrophytes and C. albicans. The fungal cells were examined using the fluorescent dye 2′, 7′-dichlorofluorescin diacetate (DCFDA) after incubation with baicalein and wogonin. Baicalein treatment induced concentration-dependent ROS accumulation in T. mentagrophytes and C. albicans (Fig. 6a), whereas wogonin treatment induced ROS accumulation only in T. mentagrophytes (Fig. 6b).
Changes in the mitochondrial membrane potential (MMP) in T. mentagrophytes and C. albicans cells were evaluated using the specific fluorescent probe JC-1 after incubation with baicalein or wogonin. Baicalein treatment resulted in a concentration-dependent decrease in MMP in T. mentagrophytes and C. albicans, whereas wogonin treatment caused a decrease in MMP only in T. mentagrophytes (Fig. 7). Fig. 8a, non-treated fungal cells exhibited the expected filamentous forms, and no extracellular material was observed in scanning electron microscopy (SEM). Baicalein induced deformation of the fungal surface structure and efflux of a cotton-like substance, which was Transmission electron microscopy (TEM) of T. rubrum revealed clear differences in cytoplasmic organelles between non-treated and baicalein-treated hyphae. As shown in Fig. 8b, after 24 h incubation of T. rubrum in the presence of baicalein, the cytoplasmic membrane was disordered, the cell organelles were degenerated, amorphous areas were enlarged, and the cell membrane was detached from the cell wall. Wogonin treatment resulted in swollen cell walls, whereas the cytoplasm was largely intact (Fig. 8b).

Discussion
The current study demonstrated that baicalein and wogonin, the major flavones in Ou-gon extracts 16 , possess potent antifungal activity against pathogenic fungi, as determined by the microbroth dilution assay. The SYTOX ® green uptake assay was used to detect cell damage, revealing structural disintegration of the plasma membrane 17 .
Upon exposure to baicalein and wogonin, the clear SYTOX ® green staining of fungal cells indicated a loss of cell membrane integrity. The TUNEL assay was used to determine the induction of apoptotic DNA fragmentation, as  described previously 17 . TUNEL staining of baicalein-and wogonin-treated fungal cells suggested the presence of apoptotic DNA breaks. Apoptosis as a mechanism of baicalein-and wogonin-induced cell death was supported by the finding that the fungal cells produced ROS upon exposure to these compounds. ROS accumulation is considered one of the primary biochemical causes of apoptosis. Inappropriate regulation of ROS levels can damage cells, leading to abnormal fungal growth and consequential apoptotic-like cell death 18,19 . Changes in the MMP, indicating the opening of transition pores in the mitochondrial membrane and release of apoptogenic factors into the cytosol, are considered another characteristic of apoptosis 20,21 . Intracellular ROS accumulation and MMP reduction can be regarded as the keys to the antifungal activity of baicalein and wogonin. Apoptosis-like programmed cell death thus likely constitutes the antifungal mechanism of baicalein and wogonin. Further evidence of ultrastructural changes of fungal cells associated with baicalein and wogonin exposure was obtained from SEM and TEM analyses. The two compounds induced different ultrastructural changes. The degeneration of cytoplasmic organelles and efflux of cytosolic contents in baicalein-treated hyphae as indicated by SEM and TEM suggest that baicalein possibly induced disturbance of the plasma and intracytoplasmic membrane synthesis, thereby impairing membrane function and leading to morphological changes. The partial swelling, and shrinkage or cracking of the cell wall found in wogonin-treated fungal cells suggest that wogonin perturbs the biosynthesis of cell wall components, thus inhibiting cell wall synthesis. Similar ultrastructural changes in the cell wall have been observed in vivo in T. rubrum treated by luliconazole 22 , a novel topical imidazole that targets lanosterol 14-alpha-demethylase (CYP51A1), an enzyme involved in the biosynthesis of ergosterol, and the MIC 80 value of luliconazole against C. albicans was more than 100 times higher than that against T. rubrum 23 .
Baicalein exhibited antifungal activity against T. rubrum, T. mentagrophytes, A. fumigatus, and C. albicans. Although the chemical structures of baicalein and wogonin are similar 24 , wogonin did not exhibit antifungal activity against C. albicans (Figs 4 and 5). Baicalein reportedly exhibits antifungal activity against C. albicans, Candida tropicalis, Candida parapsilosis, Cryptococcus neoformans, and Pityrosporum ovale [25][26][27][28] . Baicalein induces apoptosis in C. albicans, and the combination of baicalein and amphotericin B accelerates apoptosis in C. albicans cells, which is accompanied by increased intracellular ROS levels 27,29 . In addition, baicalein reduces the cell surface hydrophobicity of C. albicans biofilms by decreasing CSH1 mRNA expression 26 . Further, a combination of baicalein and fluconazole showed strong antifungal activity against fluconazole-resistant C. albicans, which was accompanied by inhibition of the efflux pumps of C. albicans 30 . In the current study, the MIC 50 value of baicalein for C. albicans was determined as 0.03 mM, which was consistent with a previous study 31 , whereas up to 0.44 mM wogonin did not exert any inhibitory effect against C. albicans (Fig. 3). Wogonin reportedly possesses antifungal activity against Botrytis cinerea, Penicillium notatum, Penicillium frequentance, and C. neoformans 32,33 . However, to the best of our knowledge, the mechanism of wogonin tolerance of C. albicans has never before been investigated in detail. Efflux pumps, e.g., ATP-binding cassette pumps and major facilitator superfamily transporters, are transport proteins in the cell membrane. In C. albicans and other microbes, they are involved in the efflux of toxic substances to the extracellular milieu, contributing to cellular drug resistance [25][26][27]29 . One of the mechanisms of baicalein-induced apoptosis in C. albicans involves the inhibition of efflux pumps 28,31 . One possible explanation for the difference in antifungal activity spectra of baicalein and wogonin is that wogonin does not inhibit the efflux pumps of C. albicans, which ultimately results in protection from cell death or apoptosis. Another possibility is that differences in cell wall biosynthesis and chemical composition between C. albicans and dermatophytes [34][35][36] lie at the basis of the ineffectiveness of the antifungal agent against C. albicans or at least enhance its resistance to the inhibitory effect. Previous studies have demonstrated that some antifungal compounds, such as Juniperus essential oil, Syzygium aromaticum essential oil, phlorotannins, geraniol, nerol, citral, neral, geranial, and psoriasin (redS100A7), are most effective against dermatophytes, or have no direct antifungal effects against Candida species 17,23,[37][38][39][40][41] . Yeasts are hydrophilic and they lack hydrophobins; therefore, hydrophobic antifungal compounds accumulate at the fungal conidia 17,[42][43][44] , which is another possible explanation for the difference in the antifungal activity spectra of baicalein and wogonin.
Flavonoids are phenolic substances that occur widely in the plant kingdom, with over 8,000 monomeric compounds identified to date. Because of their widespread ability to inhibit spore germination of plant pathogens, flavonoids have been suggested to be useful in the treatment of human infections by fungal pathogens 9 . For example, kaempferol has been considered a potential candidate against fluconazole-resistant Candida species because it inhibits the expression of cerebellar degeneration-related protein (CDR) 1, CDR2, and multidrug resistance  45 . Synergistic effects of baicalein and wogonin with available antifungal and antibacterial agents have been reported 30,31,46 . Importantly, baicalein and wogonin exert nearly no or minor cytotoxic effect on healthy human cells [47][48][49][50] . We therefore suggest that baicalein and wogonin are attractive candidates that might replace, or be used synergistically with, antimicrobial drugs, including anticancer drugs or immunosuppressive agents, to treat the ensuing fungal infections [51][52][53] . In future, it will be necessary to test the antifungal activities of baicalein and wogonin against other pathomycetes and to evaluate the effects of baicalein and wogonin against fungal infections, e.g., tinea pedis, candidiasis, and aspergillosis, in vivo. Furthermore, investigation of the effects of baicalein and wogonin on fungal gene expression may improve our understanding of their structural differences, specificity, and antifungal mechanisms of action. Finally, further identification and enhancement of additional biological activities of baicalein and wogonin would expand their application potential.

Conclusions
In summary, our findings suggest that baicalein and wogonin are major compounds with antifungal activity in Kampo medicine that elicit apoptosis-like programmed cell death in pathogenic fungi. The antifungal effects of baicalein and wogonin may lead to the development of new and safe treatment strategies, especially for the clinical treatment of infections caused by pathogenic filamentous fungi.

Materials and Methods
Fungal strains and isolates. T  In-vitro antifungal activity assay. The antifungal activity of each preparation was assayed using the microbroth dilution assay based on measuring the absorbance at 595 nm, and described in our previous reports 7,17 .  times in t-butyl alcohol and lyophilised by vacuum freeze-drying (JFD-320; JEOL, Tokyo, Japan). Samples were mounted using silver paste (Nisshin EM, Tokyo, Japan), sputter-coated with gold using an ion coater (VX-10A; Eiko, Tokyo, Japan), and monitored by SEM in the voltage range of 5-15 kV (JSM-6510; JEOL).

transmission electron microscopy. Baicalein-, wogonin-, and TPEN-treated T. rubrum cells grown in
Sabouraud liquid medium were collected into conical centrifuge tubes and rinsed with 2.5% (v/v) glutaraldehyde in 0.1 M PB (pH 7.3) at 4 °C for 2 h. This was followed by washing with 0.1 M PB, post-fixing with 1.5% (w/v) potassium permanganate in distilled water at 4 °C for 16 h, and washing in chilled distilled water until the solution turned colourless. Then, the pellets were block-stained with 0.5% uranyl acetate in 50% (v/v) acetone at 4 °C for 1 h. Dehydration was achieved in a graded EtOH series using an automated routine tissue processor (Leica EM TP; Leica Microsystems K.K., Wetzlar, Germany), and the EtOH was replaced with n-butyl glycidyl ether. The samples were embedded in Epon resin (TAAB Epon 812 resin; TAAB Laboratories Equipment, Berks, UK), and ultrathin sections (60-80-nm thick) were prepared using an ultramicrotome (MT-7000; RMC, AZ, USA). The ultrathin sections were stained using uranyl acetate and lead citrate, placed on 300-mesh copper grids, and observed by TEM operated at 80 kV (JEM1200EX; JEOL) 56 .
statistical analysis. All data were obtained from at least three independent experiments and are expressed as the mean ± standard deviation (SD). Statistically significant differences between samples were evaluated using one-way analysis of variance followed by Dunnett's post-hoc comparisons. The analyses were performed using SPSS V22.0, and p < 0.05 was considered significant.

Data Availability
All data generated or analysed in this study are included in the published article.