Dermatophyte infections are often superficial and involve the epidermis, skin and keratinized structures like hairs and nails (Akay et al., 2019). On rare occasions, dermatophytes can cause invasive infections involving hair follicles, subcutaneous tissues, and internal organs such as lymph nodes, bones, and the brain (Wang et al., 2021; Silva et al., 2022). Invasive dermatophytoses can be classified as Majocchi’s granuloma, deep dermatophytosis, pseudomycetoma, and disseminated dermatophytosis (Rouzaud et al., 2015; Wang et al., 2021; Silva et al., 2022). A systematic review of invasive dermatophyte infections showed that Trichophyton rubrum (T. rubrum) was the most prevalent pathogen responsible for invasive dermatophytoses, followed by Trichophyton mentagrophytes (T. mentagrophytes), and Microsporum canis (M. canis) (Wang et al., 2021). Deep dermatophytosis is a rare and sometimes life-threatening fungal infection. This condition mainly occurs in immunosuppressed individuals or patients with mutations in genes, including Caspase Recruitment Domain-containing protein 9 (CARD9) and signal transducer and activator of transcription 3 (STAT3) (Wang et al., 2021). Therefore, treating chronic superficial tinea in these immunosuppressed patients and patients with genetic mutations is critical to prevent the development of invasive dermatophytoses.

Although dermatophytoses are generally considered easy to treat, dermatophyte resistance to existing antifungal drugs (e.g., terbinafine, itraconazole, fluconazole, amphotericin B, and posaconazole) is increasing, and infections are more likely to recur(Khurana et al., 2019; Wang et al., 2021). More importantly, these drugs are usually considered to cause some side effects, such as liver function damage. To overcome the limitations of current treatment strategies, it is necessary to explore combination therapies through in vitro experiments.

Natural polyacetylene alcohols can be found in many sources, including natural food products and traditional medicinal materials. Falcarindiol (FADOH, Figure 1 ), a kind of natural polyacetylene alcohol, is a polyacetylenic oxylipin found in vegetables of the carrot family (Apiaceae) and natural Chinese herbal medicines, such as notopterygium incisum, angelica keiskei and angelica dahurica(Yang et al., 2014; Yao and Li, 2015; Andersen et al., 2020; Zhao et al., 2021). It has been found that FADOH has anticancer, antifungal, antibacterial, and anti-inflammatory effects and can also be used as a dietary supplement for neuroprotection (Dawid et al., 2015; Jeon et al., 2020). There are more and more reports on the antifungal activity against plant fungi using polyacetylene alcohols, but there is little research on the antifungal activity against dermatophytes. Previous studies have found that the mechanism of polyacetylene alcohols against plant fungi and tumors is related to lipid metabolism (Nickel et al., 2015; Andersen et al., 2020). We hypothesize that the mechanism of FADOH against dermatophytes is also related to lipid metabolism. This work mainly explores whether the combination of FADOH and ITC exhibits synergistic inhibitory activity in vitro against dermatophytes.

Immunosuppressed patients have a higher incidence of superficial dermatophytosis and are more prone to develop devastating invasive dermatophyte infection (Marconi et al., 2010). However, antifungal drugs for treating severe invasive fungal infections are still limited (Revie et al., 2018). Moreover, the frequent and preventive use of antifungal drugs has led to the development of antifungal drug resistance (Revie et al., 2018). The four main classes of antifungal drugs are polyenes, azoles, allylamines and echinocandins. Azole antifungal agents, such as fluconazole and ITC, have been widely used in treating dermatophytosis. Unlike allylamines (e.g., terbinafine), azoles could potentiate drug resistance development in dermatophytes (Ghannoum, 2016). Combination therapy might effectively treat severe refractory fungal infections (Xu et al., 2019). It has been found that the combination of new antifungal drugs and traditional antifungal drugs has certain synergistic or at least additive activity against many fungi in vitro (Vazquez, 2007). Natural Chinese herbal medicine contains complex constituents with good safety and a broad antifungal spectrum, including Candida, Cryptococcus neoformans and all kinds of plant fungi (Tian et al., 2010; Zhang et al., 2020; Yang et al., 2022). In addition to the main components, there are a lot of unknown trace components with novel structures and strong biological activity, which are considered as one of the important therapeutic substances and a huge source of innovative drugs of traditional Chinese medicine (Xia et al., 2022). Chinese herbal medicine can be used as a resource for the detection of new antifungal targets and has become an essential research focus and focal point in recent years.

The antifungal activity of polyacetylene alcohols isolated from apiaceous vegetables and Chinese herbal medicine has recently attracted attention. Yoon et al. found that polyacetylalcohol ciryneol A had good antifungal activity against plant fungi, such as Magnaporthe oryzae, Colletotrichum coccodes, and Pythium ultimum, with IC50 values below 50μg/ml (Kim et al., 2011). Lecomte M et al.found that FADOH had a good inhibitory effect on the fungal leaf blight pathogen Alternaria alternata (IC50 ranging from 34 to 102 μM) (Lecomte et al., 2012). Liu et al. found that C18 polyacetylenes from panax stipuleanatus exhibited high potencies against eight pathogenic fungal species tested, such as Colletotrichum gloeosporioiles, Fusarium graminearum, and Fusarium pseudograminearum, with half-maximum effective concentrations ranging from 8 to 425μg/ml (Liu et al., 2020). However, no antifungal effects of polyacetylene alcohols against dermatophytes have been reported so far.

Our study showed that FADOH had a specific antifungal effect against dermatophytes, with MICs ranging from 1.56 to 100μg/ml. FADOH combined with ITC had good synergistic and additive activity against most dermatophytes (86.7%), particularly T.rubrum and T. mentagrophytes. Interestingly, FADOH and ITC had synergistic or additive effects against all T. mentagrophytes. In addition, FADOH could reduce the MIC values of ITC against most dermatophytes (93.3%) to 2-8 times. Furthermore, drop-plate assay verified the synergistic activity of FADOH and ITC visually. We selected one strain per species for the time-growth curve test to verify the dynamic synergistic effect of FADOH and ITC on dermatophytes. The time-growth curves showed that the combination of FADOH and ITC against all three strains demonstrated a potent synergistic antifungal effect at 96h, which was still very pronounced from 96h to 168h. The combination of the two drugs against T. rubrum TM5 showed an obvious synergistic antifungal effect at 72 hours. Unlike T. rubrum TR5 and M. canis MC1, T. mentagrophytes TM6 started to grow similarly like in the control after 120h. As for whether the combination of FADOH and ITC against T. rubrum shortens the onset time of antifungal activity and whether the synergistic inhibitory effect on T. mentagrophytes is not as strong as the other two dermatophytes, we need to conduct time-growth curve tests on more strains in the future for further verification.

Although there is no report on the anti-dermatophyte mechanism of natural polyacetylene alcohols, some reports have been made on its anti-plant fungus and antitumor mechanism. HR Jin et al. found that FADOH exerted its antitumor activity by inducing endoplasmic reticulum (ER) stress and apoptosis (Jin et al., 2012). Proteomics also found that polyacetylene and other compounds were mainly involved in the metabolism of lipids and fatty acids, interfering with the activities of lipid-modifying enzymes and various membrane-related proteins such as endoplasmic reticulum (Nickel et al., 2015). CB Andersen et al. found that FADOH could activate the expression of sterol transporter, causing the redistribution of lipids and increased formation of lipid droplets in cells, which led to endoplasmic reticulum stress and cell death (Andersen et al., 2020). Fungi and human cells are eukaryotes, and sterols are the membrane lipids of most eukaryotes (Chen et al., 2007). The change of sterol in the cell membrane will destroy the integrity and fluidity of the cell membrane and affect the expression of membrane protein, thus affecting the growth and metabolism of cells (Jorda and Puig, 2020). Sterols in fungi are called ergosterol, a vital component of the fungal plasma membrane (Shapourzadeh et al., 2016). Significant structural differences in fatty acid between fungi and mammalian cells exist, making it a promising antifungal target (Bitencourt et al., 2013). The fungal cytochrome P450 enzyme sterol 14α-demethylase is a key enzyme in the ergosterol biosynthesis pathway and is the target of azoles (Rosam et al., 2020). Therefore, we speculate that the synergistic mechanism of polyacetylene alcohols and azoles against dermatophytes may mainly affect the metabolism of ergosterol and lipid, induce endoplasmic reticulum stress, and thus lead to fungal cell apoptosis.

Although further work is needed to explore the underlying mechanism for the observed synergistic activity, our findings demonstrate the potential activity of FADOH as an antifungal adjuvant to enhance the antifungal activity of azoles against dermatophytes.

In summary, FADOH combined with ITC has synergistic and additive antifungal activity against most dermatophytes, indicating that natural polyacetylene alcohols may be a more reliable and safe combination therapy for treating dermatophytoses and invasive dermatophyte infection.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

SK carried out the in vitro antifungal experiment and wrote the manuscript. ZG, SL, and HY collected and analyzed the experimental data. LA, JT, QC, and RN designed and interpreted the experiment data. LY revised the manuscript critically for important content. All authors contributed to the article and approved the submitted version.