Invasive fungal infections (IFIs) are a serious type of infectious disease, with higher incidence and mortality rates particularly among patients with immunosuppression or immunodeficiency (Fang et al., 2023). Aspergillus species are the main pathogens most frequently isolated from patients with compromised immune function (Badiee and Hashemizadeh, 2014; Pfaller et al., 2021). Aspergillus fumigatus and Aspergillus flavus are known to be pathogenic, while Aspergillus niger and Aspergillus terreus are also capable of causing invasive infections (Balajee, 2009; Borni et al., 2024; Hedayati et al., 2007; Morelli et al., 2021; Wang et al., 2024). Dematiaceous fungi, including Exophiala dermatitidis and Exophiala alcalophila, can cause a variety of infections in immunocompromised individuals (Kirchhoff et al., 2019; Kondori et al., 2024; Revankar, 2007). Azoles have become the mainstay of treatment and prevention for many systemic mycoses, with common medications including ITC, VRC, POS and ISV (Kaushik and Kest, 2018; Peyton et al., 2015). Because of their high infection and mortality rates in immunocompromised patients, as well as the increasing resistance to azole antifungal agents, studying Aspergillus and dematiaceous fungi can help explore their resistance mechanisms and provide a theoretical basis for the development of new combination therapeutic strategies (Amona et al., 2022; Djenontin et al., 2023; Puerta-Alcalde and Garcia-Vidal, 2021). Previous research has demonstrated that synergistic combinations of natural products can augment antifungal potency, which may facilitate the discovery of innovative therapeutic approaches to combat fungal infections (Augostine and Avery, 2022; Yang et al., 2023). A wide range of natural flavonoids have been shown to possess antifungal properties (Jin, 2019). Baicalein (BE) is a flavonoid compound widely found in the Scutellaria genus of plants, featuring hydroxyl groups that contribute to its bioactivity (Zhao et al., 2022). Previous studies have confirmed its anti-inflammatory, antioxidant properties, as well as anti-cancer and tumor cell proliferation inhibition effects, while recent research has highlighted the potential antifungal activity of BE (Gupta et al., 2022; Lai et al., 2024; Li Y. Y. et al., 2022; Song et al., 2021; Tuli et al., 2020; Yan et al., 2018). BE exhibits potent antifungal activity against Candida species, with a MIC50 as low as 13 μg/mL, and has been proven to inhibit the growth of Candida through multiple mechanisms, such as targeting and inhibiting the function of enolase 1 (Eno1) in Candida albicans, upregulating the expression of CPD2, and inducing apoptosis by targeting ribosomes in Candida auris (Da et al., 2019; Li et al., 2024; Li L. et al., 2022; Lv et al., 2022; Serpa et al., 2012). In contrast, the effects of BE on Aspergillus and dark-coloured fungi have been less studied. Notably, BE at a concentration of 0.25 mM has been demonstrated to ameliorate Aspergillus fumigatus keratitis in mice (Zhu et al., 2021). BE has been shown to exhibit synergistic effects with other antifungal agents, such as fluconazole (FLU), against Candida parapsilosis and C. albicans (Janeczko et al., 2022; Li L. et al., 2022). In Candida species, the combined use of BE and FLU can reduce the MIC values of both antifungal agents, resulting in a better inhibitory effect against fungi (Serpa et al., 2012). Therefore, we hypothesize that combining BE with other antifungal agents could reduce the effective concentration of BE against these fungi, thereby achieving similarly robust antifungal effects as observed against Candida species. This investigation further examines the synergistic antifungal efficacy of BE in conjunction with other azole-class drugs, aiming to enhance azoles treatment efficacy and mitigate the development of resistance.

Results indicate that among the azoles, combinations of BE with POS and ITC demonstrated synergistic effects against the tested fungal strains. Notably, the BE/POS combination exhibited the most pronounced synergistic effect, observed in 89.61% of strains, with a more frequent observation of synergy in dematiaceous fungi compared to Aspergillus. In contrast, the BE/ITC combination showed significantly less synergy, affecting only 25.97% of strains. The disparity in synergistic effects between the BE/POS and BE/ITC combinations may be attributed to differences in the chemical structures and mechanisms of action of these azoles.

At a concentration of 0.25 mM, BE alleviates A. fumigatus keratitis in mice by inhibiting fungal growth, biofilm formation and adhesion, and by downregulating the expression of pro-inflammatory factors (Zhu et al., 2021). For C. albicans, BE inhibits fungal growth by targeting Eno1, inhibiting glycolysis, and preventing biofilm formation (Li L. et al., 2022). Treatment with BE also induces concentration-dependent accumulation of ROS in Trichophyton mentagrophytes and C. albicans. When BE is used in combination with FLU, it demonstrates robust antifungal activity against drug-resistant fungi. In this context, the biofilm formation of C. albicans is inhibited in a dose-dependent manner at concentrations ranging from 4 to 32 μg/mL (Huang et al., 2008). Baicalein-Core Derivatives can also enhance the antifungal efficacy of FLU by inhibiting hyphal formation in C. albicans (Zhou et al., 2023). The antifungal mechanisms of BE may involve inhibiting biofilm formation and inducing the accumulation of ROS. Further research is needed to elucidate the specific antifungal mechanisms at the molecular level. Azoles inhibit fungal growth by blocking ergosterol synthesis through the inhibition of 14α-sterol demethylase (CYP51) in the fungal cell membrane, leading to impaired cell membrane biogenesis and altered membrane permeability (Patterson et al., 2016). In this study, the combination of BE with POS and ITC exhibited synergistic effects against the tested fungi. In contrast, no synergy was observed when BE was combined with VRC and ISV. Previous studies using molecular docking and molecular dynamics simulations to investigate the binding mechanisms and tunneling characteristics of CYP51 with inhibitors have shown that hydrophobic interactions are the primary driving force for binding to CYP51, and that long-chain inhibitors such as POS and ITC can access more CYP51 residues through hydrophobic interactions than short-chain inhibitors like VRC, thereby exhibiting stronger binding affinities (Shi et al., 2020). ISV is a novel azole drug that is structurally similar to VRC (Ghobadi et al., 2022). These differences in binding affinity may account for the lack of synergy observed with ISV and VRC.

Current research on the toxicity of BE is relatively limited. At doses cytotoxic to malignant cells, BE displays minimal or negligible toxicity to normal peripheral blood cells and normal myeloid cells, but it also exerts growth-inhibitory effects on human fetal lung diploid cell lines at the same concentrations that suppress tumor cell proliferation (Li-Weber, 2009). Preliminary animal studies have indicated that BE exhibits low acute toxicity at therapeutic doses, with no significant adverse reactions observed (Wang et al., 2022). In clinical studies involving healthy Chinese subjects, both single-dose and multiple-dose administrations of BE tablets have demonstrated good safety and tolerability, with no serious or severe adverse reactions reported (Li et al., 2021). Studies have shown that the combination of BE at a concentration of 32 μg/mL with ampicillin has negligible effects on hemolysis of red blood cells (RBCs) and cytotoxicity towards Vero cells. This concentration falls within the range tested in our MIC assays (Lu et al., 2021). However, a more comprehensive toxicological evaluation is warranted, especially in the context of the interactions between BE and azoles, to establish the safety profile of these drug combinations through in vitro cytotoxicity assays and in vivo animal models.

The study also noted antagonistic effects in certain cases, particularly with combinations of BE/ITC, BE/VRC, and BE/ISV. These antagonistic effects may arise from competitive inhibition or other unknown molecular interactions that negate the antifungal activity. Additional research is required to understand and potentially mitigate these antagonistic effects.