Candidiasis is emerging as a significant challenge in the healthcare environment due to its high medical costs and high mortality rates (Amirrajab et al., 2016; Pristov and Ghannoum, 2019; Shariati et al., 2020). Recent studies reported systemic infections caused by Candida species (Candida) as the fourth leading cause of nosocomial bloodstream infections. Opportunistic, these fungi are responsible for 90% of all invasive infections (Spampinato and Leonardi, 2013; Marak and Dhanashree, 2018; Sakagami et al., 2019). Although this family contains at least 15 distinct species, five pathogens could lead to invasive infections with a high mortality rate in humans: Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, and Candida krusei (Pappas et al., 2018).

C. albicans is the most frequent etiology of candidiasis in clinical settings that could lead to a range of harmless superficial mycosis to life-threatening invasive infections. Nevertheless, non-C. albicans species collectively could represent >50% of the bloodstream isolates in certain regions (Wisplinghoff et al., 2004; Kullberg and Arendrup, 2015). In this regard, Candida auris, a previously rare fungus, has been reported as a major pathogen in certain parts of the world (Chowdhary et al., 2017). Overall, Candida species reside in healthy hosts as commensal yeasts in different parts of the human body, such as the gut and skin. Moreover, they are detectable in up to 60% of healthy individuals. However, immunocompromised status could lead to these fungi invading the host tissues and disseminating to internal organs (Pristov and Ghannoum, 2019).

Candida pathogenicity depends on various virulence factors such as yeast-hyphal formation and secretion of proteolytic as well as lipolytic enzymes, along with biofilm formation. Among the mentioned virulence factors, biofilm community of Candida has a potential role in pathogenicity. This biofilm community has a three-dimensional structure comprised of hyphal and yeast cells within a self-secreted matrix of extracellular polymeric substances (EPS), consisting of proteins, polysaccharides, and nucleic acids (Abirami et al., 2020). Biofilms are easily formed on the surface of medical devices such as intravascular and urinary catheters, artificial valves, intrauterine devices, contact lenses, and any foreign objects or host surfaces. Accordingly, the biofilm community of Candida leads to persistent infections and reduced susceptibility to host immune responses as well as antifungal drug therapy (Lohse et al., 2018; Abirami et al., 2020). Furthermore, the surface-associated virulence factors, such as adhesins and degradative virulence enzymes, including phospholipases and proteases have distinguishable roles in attachment and invasion into the host cells, while also strengthening C. albicans pathogenicity by destroying the vital proteins of the skin and cell membrane lipids (Mayer et al., 2013).

The specific type and dose of antifungal medication used to treat invasive candidiasis usually depends on the patient’s age, location and severity of the infection, as well as immune status. Accordingly, various antifungals such as echinocandins (caspofungin, micafungin, or anidulafungin) and azoles (fluconazole) are recommended to manage and treat invasive candidiasis (Fisher et al., 2011; Arendrup and Patterson, 2017). However, azoles-resistance and cross-resistances to azoles and echinocandins in fungi are now considered one of the main challenges in treating fungal infections. In this regard, a higher prevalence of drug resistance in Candida has increased the candidiasis-associated mortality rate (Zore et al., 2011). Furthermore, other drawbacks such as high cost, side effects and toxicity, and lack of fungicidal efficacy limit the usage of antifungals (Kauffman and Carver, 1997). These limitations justify the development of new therapeutic approaches or the discovery of novel antifungals to inhibit Candida-associated infections. Bearing this in mind, plant-derived substances due to high efficacy, few adverse effects, and low cost have shown potential capacity to inhibit these fungi, even azole-resistant strains (Ahmad et al., 2010a; Abrahão et al., 2013; Nisar et al., 2021).

Eugenol or 2-methoxy-4-[2-propenyl] phenol, a phenolic aromatic compound mainly derived from Cinnamomum and Clove essential oil, is one of these natural compounds belonging to a novel class of microbiocidal phenylpropanoids and has been used for a long time as an analgesic in dentistry (Hassan et al., 2018; Wijesinghe et al., 2021). Eugenol can be synthesized by guaiacol allylation with allyl chloride or produced through a biotransformation process that involves microorganisms such as Bacillus cereus, Corynebacterium species, and Escherichia coli (Abrahão et al., 2013). Eugenol has also indicated anesthetic, neuroprotective, antidiabetic, insecticidal, analgesic, anti-inflammatory, and antifungal properties that make this compound a versatile natural ingredient that helps prevent and cure various disorders (Kozam, 1977; Reddy and Lokesh, 1994; Hassan et al., 2018; Nisar et al., 2021).

Eugenol leads to a better penetration of different drugs in the skin. This compound has also been used as a pesticide and fumigant to protect foods from microbial invasion such as Lactobacillus and Listeria monocytogenes during storage. Additionally, the United States Food and Drug Administration approved the use of eugenol and clove oil as a natural antiseptic and analgesic in dentistry and as a fragrance in soaps and cosmetics. Furthermore, this compound has been used as a flavoring substance in food and pharmaceutical products (Kamatou et al., 2012; Mohammadi Nejad et al., 2017).

Eugenol exhibits significant antifungal activity against various fungal species, including dermatophytes, Aspergillus, and Candida, which is primarily due to damage to the fungal cell envelope, biofilm community, and various virulence factors (Chami et al., 2005; Hassan et al., 2018). As a result, researchers are interested in using eugenol and synthetic analogues, resorting to various drug delivery systems to inhibit Candida, biofilm communities, and several cellular pathways associated with these fungi.

This review focuses on the interaction of eugenol and various plant species with a high eugenol content with different cellular pathways in Candida cells. Additionally, we discuss the synergistic antimicrobial activity of eugenol when combined with antifungal drugs to facilitate its widespread use in clinical practice. Notably, the present study undertook a Medline (via PubMed) search using the following search keywords obtained from the National Library of Medicine’s Medical Subject Heading (MeSH) terms, titles, or abstracts using Boolean Operators (and, or): “Eugenol” and “Candida” or “Candidiasis” or “C. albicans”.

Recent studies showed that new antifungal agents are needed to decrease the toxicity of conventional antifungals. Additionally, using combination therapy could improve the efficacy of various antifungals such as fluconazole (Pinto et al., 2009; Ahmad et al., 2010a). In this concept, the combined eugenol and different antifungal drugs were considered to inhibit Candida and their biofilm community. Indeed, a recently published study reported that eugenol, the main component of Ocimum campechianum Mill., could increase the efficacy of fluconazole against clinical Candia strains (Tacchini et al., 2021). Furthermore, Ahmad et al. (2010a) reported that eugenol and methyleugenol were active against fluconazole-resistant clinical Candida isolates. The interaction between methyleugenol and eugenol with fluconazole was synergistic in 92% and 90% of the fluconazole-susceptible strains, respectively. Additionally, 85% and 91% of the isolates resistant to fluconazole indicated synergistic effects of eugenol plus fluconazole and methyleugenol plus fluconazole, respectively. Notably, antagonistic interaction between the mentioned compounds was not detected in the strains tested (Ahmad et al., 2010a).

Moreover, previous studies considered the combination therapy of eugenol and fluconazole against Candida biofilms. An experiment conducted in 2014 reported a synergistic effect for combinations of fluconazole and eugenol against a planktonic community of C. albicans. The established biofilm of this fungus was highly resistant to fluconazole; while, sensitization of fungal cells by eugenol (sub-inhibitory concentrations) led to the prevention of biofilm formation at low fluconazole concentrations. The authors hypothesized that eugenol destabilizes the membrane and specific signal intervention. Hence, this compound caused sensitization of C. albicans biofilms, boosting fluconazole penetration and leading to the inhibition of biofilm formation (Doke et al., 2014).

Additionally, another study reported that pre-formed C. albicans biofilms showed ≥1,024× increased resistance to fluconazole, while biofilm formation did not cause tolerance to eugenol. This compound inhibited the biofilm formation of C. albicans and showed a synergistic interaction with fluconazole against biofilms formed by the test strains. The microscopy evaluation also showed eugenol interference with cell membrane integrity, as evidenced by shrinkage of the cell surface in biofilm cells. Therefore, the authors proposed that when eugenol has cidal activity against biofilm cells if combined with fluconazole, the drug’s fungistatic nature is converted to fungicidal (Khan and Ahmad, 2012).

In line with these observations, another investigation performed in 2020 in India reported that eugenol suppressed S. mutans and C. albicans mixed biofilms. The sessile MIC of fluconazole was increased up to 1000-fold over planktonic MIC. Notably, eugenol was highly synergistic with fluconazole against C. albicans single and mixed biofilms. The microscopy analysis also confirmed these findings and showed distorted cell structure, decreased matrix production, and elimination of single and mixed biofilm cells of S. mutans and C. albicans in treated samples with eugenol compared to untreated samples. Therefore, eugenol may disrupt cell membrane integrity and boost the drug entry into the microbial cell. This phenomenon increases the antimicrobial drug availability in the deeper layers and target sites of biofilm, consequently improving treatment efficacy (Jafri et al., 2020).

In addition to fluconazole, eugenol and other antifungals combined also showed promising results for inhibiting C. albicans growth. In this regard, Khan et al. (2012) reported that eugenol has a potential antifungal capability to inhibit fluconazole, itraconazole, and ketoconazole-resistant isolates of C. albicans. Furthermore, this compound showed remarkable synergy with fluconazole and AMB against the test isolates (Khan et al., 2012). The results of a recently published study also revealed that the combination of eugenol and sub-MIC (0.05 mg/ml) of AMB indicated many-fold higher anti-fungal effect against C. albicans compared to single component therapy. Scanning electron microscopy (SEM) showed the following characteristics for C. albicans cells after combination therapy: completely ruptured and shrank cells that aggregated as irregularly shaped material. Moreover, the authors reported that combination therapy induced ROS potentiation in C. albicans cells and with cellular damages, decreased mitochondria levels, and enhanced cytosol cytochrome C levels. Besides, the combination of eugenol and AMB resulted in an intense decline of intracellular Ca^2+‏ concentration in C. albicans cells compared to the single treatments (Khan et al., 2019).

Noteworthy, the expansion of the inner mitochondrial membrane upon matrix swelling could damage the outer membrane, release the cytochrome C to the cytosol, and induce cell death (Wu et al., 2009). Therefore, these findings suggested that eugenol, in addition to C. albicans growth inhibition, could act synergistically with AMB (at less toxic doses) by interfering in the different cellular pathways of this fungus (Khan et al., 2019).

Another study reported a synergistic effect of eugenol in combination with voriconazole against voriconazole-resistant C. tropicalis and C. krusei isolated from the genital tract of mares (Sharifzadeh and Shokri, 2021). To this end, Dąbrowska et al. (2021) reported that eugenol indicated additive and synergistic activities with econazole and miconazole against clinical isolates of C. albicans, respectively.

Therefore, combining eugenol with various antifungal drugs could lead to several benefits such as decreased dose of drugs needed, boosted potency, and minimized toxicity, which ultimately helps suppress or eliminate biofilms and overcome fungal infections caused by drug-resistant Candida strains. However, the precise mechanism by which eugenol synergizes with various antifungals was not investigated in the studies mentioned above. Thus, additional molecular and in vivo studies are required to establish the practical utility of these combinations.

Unfortunately, most in vitro observations of eugenol’s interaction with Candida cells have not been confirmed in animal models. However, some studies have evaluated this compound’s anti-Candida activity in in vivo, and this section discusses those studies.

Established murine models have been used for studying most important clinical forms of candidiasis such as vaginal, disseminated, cutaneous, and oropharyngeal. To this end, to evaluate Candida pathogenies and host responses to the treatment, mice treated with depleting antibodies and genetically modified mice (transgenics and knockouts) are generally used. Additionally, different strains of Candida including genetically modified laboratory strains and clinical isolates are used for analysis of Candida pathogenesis in animal models. C. albicans does not normally colonize the mice mucosa, though under different conditions of immunosuppression, disease can be induced (Solis and Filler, 2012; Conti et al., 2014).

Chami et al. (2004b) used eugenol (topical usage) to treat oral candidiasis induced by C. albicans in immunocompromised rats, where nystatin was used as a positive control treatment. Treatment with eugenol for eight consecutive days remarkably decreased the number of colony-forming units (CFU) sampled from the treated rats’ oral cavity compared to the control rats. Histopathologic examination (HE) revealed that the epithelium of the dorsal surface of the tongue of the untreated control rats was colonized with many fungal hyphae. Nevertheless, only a few focalized zones of the dorsal surface of the tongue were occupied by hyphae in eugenol-treated rats. Furthermore, no acute cytotoxicity effects were detected in eugenol-treated rats. Notably, HE revealed numerous hyphae in the tongue folds of rats treated with nystatin; however, this evaluation revealed no fungal hyphae in the fold following eugenol treatment. The authors suggested that this could be related to eugenol’s volatility characteristics, which allowed its penetration into unreachable zones, such as folds of the tongue (Chami et al., 2004b).

These data aligned with another investigation conducted in 2005 where the authors used eugenol to inhibit oral candidiasis in immunocompromised rats. The findings of this study also showed that eugenol had fungicidal effects against C. albicans. Moreover, treating rats’ oral candidiasis with this compound (eight consecutive days) led to a significant reduction of C. albicans colony count compared to the untreated control rats (Chami et al., 2005).

In another investigation, the authors also used eugenol to prevent and treat vaginal candidiasis caused by C. albicans in the immunosuppressed rat model. In this manner, to permit maximum adhesion of eugenol to the vaginal mucosa, a gelatinous suspension of 0.8% agar (as excipient) was used to treat rats through the intravaginal route. Ten days after infection, prophylactic treatment with this compound reduced C. albicans CFU in the vaginas of infected rats by 98.9%. Additionally, treatment with eugenol for seven consecutive days completely inhibited infection in 23% (2/9) of infected rats, whereas an 84% reduction in C. albicans colony count was detected in the vaginas of the other rats. HE of the lumina of the vagina also showed no C. albicans hyphae in all treated rats (Chami et al., 2004a). It should be noted that all of the above-mentioned candidiasis models in rats required an immunosuppressive host. Thus, the authors proposed eugenol as a potentially useful antifungal agent for preventing and treating candidiasis, particularly in patients with immunosuppressive conditions such as AIDS.

Finally, a recently published study evaluated the treatment effects of eugenol against fluconazole-resistant C. albicans-induced keratitis in the rabbit model. The results showed that 4 mg/ml of eugenol was the highest dose with non-toxic effects in the rabbit’s corneas. High-performance liquid chromatography (HPLC) analysis showed that the mentioned dose of eugenol is detectable in the corneal tissue; hence, this compound could penetrate through corneal epithelium that is an important obstacle for drugs penetration into the cornea. In addition, the clinical markers and HE showed significant lower levels of corneal neovascularization and clouding and conjunctival hyperemia in most rabbits treated with eugenol than the control group. Therefore, because most antifungal drugs are not applicable for the treatment of keratitis and have various side effects, the authors proposed eugenol as a safe and inexpensive antifungal agent that can be used topically for the treatment of keratitis caused by C. albicans (Hassan et al., 2018).

Therefore, as mentioned in the above studies, eugenol could be considered a potential antifungal agent with promising efficacy for treating different candidiasis presentations such as vaginitis, keratitis, and oral candidiasis. However, further molecular and toxicologic studies and clinical trials are required to determine the exact interaction of eugenol with eukaryote cells after prolonged exposure. Because non-albicans Candida species are becoming increasingly common, the antifungal effects of eugenol against these fungi should also be evaluated in future animal studies.

Studies regarding genotoxicity and cytotoxicity of eugenol are very controversial and limited. A dose of 2.5 mg/kg body weight of eugenol is regarded as safe by the Food and Agriculture Organization, while high concentrations of this compound could be harmful and pro-oxidative (Mohammadi Nejad et al., 2017; Ulanowska and Olas, 2021). For instance, 5–10 ml ingestion of clove oil led to acidosis and deep coma in a 2-year old boy (Hartnoll et al., 1993). Moreover, eugenol intravenous infusion (at 4 and 8 µl) caused acute respiratory distress with hemorrhagic pulmonary edema in rats (Wright et al., 1995). Also, eugenol could lead to allergies such as allergic contact dermatitis in some cases, particularly in dental workers (Mohammadi Nejad et al., 2017; Ulanowska and Olas, 2021). In this regard, research on eugenol and clove oil is still ongoing and extensive toxicity evaluations should be conducted to approve whether eugenol is safe for general public health.

Previous studies reported that eugenol is sensitive to degradation processes when exposed to oxygen, light, high temperatures, and humidity. Furthermore, this compound is highly volatile, slightly soluble in water, and unstable; as a result, the disadvantages listed above may reduce eugenol’s efficacy, biological activity, and stability (Karmakar et al., 2012; Shao et al., 2018). Therefore, considering the disadvantages of eugenol concerning solubility, instability, and volatilization, the use of different drug-delivery systems could be an alternative to solve these challenges and provide a stable, safe, and effective antifungal agent for clinical usage.

In this regard, Jacumazo et al. (2020) used nanocapsules and microcapsules containing eugenol coated with chitosan and carboxymethylcellulose to manage eugenol’s release enabling its use as an antifungal agent. This drug-delivery system indicated an inhibitory effect against C. albicans, and the MIC of eugenol was lower than those of the free agent. Thus, the authors suggested that nanocapsules with successive polysaccharide layers protect eugenol and improve its release (Jacumazo et al., 2020). Another investigation also reported that eugenol loaded with water-soluble βCD-grafted chitosan derivatives (QCD-g-CS) showed higher antimicrobial activities against C. albicans in comparison to the free compound. This enhanced antimicrobial activity could be attributed to the increased solubility of eugenol in the aqueous phase caused by the presence of QCD-g-CS, resulting in modified eugenol-microorganism interactions. Furthermore, both eugenol and chitosan have antimicrobial properties. Accordingly, the presence of both of them as inclusion complexes may lead to the synergistic effect on antimicrobial activity (Sajomsang et al., 2012).

Additionally, eugenol-loaded electrospun Polyacrylonitrile (PAN) nanofiber mats’ antifungal effects were evaluated against C. albicans. The findings demonstrated that eugenol release from nanofibers was gradual and continued slowly for 150 h. The PAN nanofiber lacked antifungal activity as a control sample, but these samples exhibited antifungal properties when loaded with eugenol. Hence, these data suggested that PAN nanofibrous mats containing eugenol could be considered an alternative therapeutic agent for localized drug delivery of eugenol due to its acceptable tensile properties, suitable drug release, uniform morphology, and antifungal properties (Semnani et al., 2018). Labib et al. (2015) also introduced another drug-delivery platform for treating local candidiasis in line with these results. The authors used Orabase, bioadhesive bases that have been used for pain relief in the handling of oral aphthous stomatitis, loaded with eugenol. This compound indicated significant antifungal function, acceptable physical features, slow-release pattern, and sensible mucoadhesion. Therefore, the incorporation of eugenol in Orabase, which has demonstrated efficacy in inhibiting local treatment of oral candidiasis, may aid in the design of future drug delivery systems (Labib and Aldawsari, 2015).

As mentioned in the above studies, diverse drug-delivery platforms with eugenol could provide novel agents to inhibit Candida-associated infection. Nonetheless, the precise mechanisms of these platforms interacting with candidal physiology remain unknown. Thus, additional research, including molecular evaluations, in vivo toxicity analysis, and clinical trials, are required before the widespread use of eugenol-drug delivery systems.

Finally, it is noteworthy that recent studies used eugenol-based derivatives to inhibit different cellular pathways in Candida. These studies have been reported in full in Table 2.

Eugenol may be beneficial in the clinical management of candidiasis, particularly localized forms such as vulvovaginal and oral candidiasis, due to its fungicidal activity and inhibitory effect on germ tube formation. Due to the potent anti-biofilm capacity of eugenol against Candida, covering medical implant devices with this compound could be practical in preventing implant-associated Candida infections. The combined use of eugenol with various antifungals, especially fluconazole, could also be very helpful in combating infections caused by drug-resistant Candida. Nevertheless, although some studies have reported the molecular interactions of eugenol with the different Candida cellular pathways, further research is required to substantiate these findings. Additionally, more in vitro, animal models, and clinical trials should be designed for the exact evaluation of cell cytotoxicity due to long-term exposure to eugenol. Finally, it is proposed that various drug delivery platforms containing eugenol and other eugenol derivatives should be evaluated to develop effective and novel antifungal agents against Candida species.