Onychomycosis is a fungal infection of limited severity that affects adults’ toenails and fingernails and, to a lesser   extent,   child’s   nails   (Solís-Arias   and  García-Romero, 2017; Lipner and Scher, 2019). The word onychomycosis comes from the Greek "onyx", which means nail and "mycos",  which means mycosis (Baswan et al., 2017), and includes all fungal infections of one or more nails. The etiological agents include dermatophyte fungi (in this case it is also called Tinea unguium), yeasts (mainly Candida species) and non-dermatophyte molds. Mixed infections caused by the association of different groups of these fungi may occur (Baswan et al., 2017; Gupta et al., 2020).

Onychomycosis is the most frequent nail disease in dermatological clinics; the signs and symptoms included local pain, paresthesia and difficulties in performing daily activities in general. Clinical manifestations of fungal infected nails are usually discoloration, hyperkeratosis and onycholysis (Gupta et al., 2017; Lipner and Scher, 2019). Individuals affected by onychomycosis can have low self-esteem, which can impact social life and interactions and can also have a reduced quality of life and psychological damage (Lipner and Scher, 2019; Stewart et al., 2021).

The prevalence of onychomycosis is 6.9-23.2% in the North American and European populations (Elsayed, 2015), with rates increasing according to age and most infected people being aged 60 years or older (Belda et al., 2014; Lipner and Scher, 2019). Different studies show different rates, which vary according to geographic regions, gender, climate, occupation, physical activity and the presence of comorbidities (Belda et al., 2014; Gupta et al., 2017; Lipner and Scher, 2019). The factors that contribute to a high incidence of superficial mycoses, especially in tropical and subtropical regions are heat and humid (Lombardi et al., 2020; Sharma and Nonzom, 2021). According to Baswan et al. (2017), onychomycosis is responsible for 50% of all nail diseases and about 30% of superficial mycoses. Around 60-70% of infections are caused by dermatophyte fungi, whose main agent is Trichophyton rubrum (>50%), followed by T. mentagrophytes (about 20%); other cases are caused by non-dermatophyte molds (NDMs) (10-20%) and yeasts (10-20%) (Gupta et al., 2012; Lipner and Scher, 2019; Gupta et al., 2020).

The conventional therapy includes drugs such as allylamines, azoles, ciclopirox and amorolfine. This therapy commonly presents adverse effects that become more serious with prolonged use and some infectious agents are resistant to antifungals (Flores et al., 2016). Onychomycosis has shown difficulty being treated because the fungi grow inside the nail, requires prolonged time and low patient compliance to treatment, as well as the relative frequency of recurrence of the infection (Christenson et al., 2018). The mixed infections and those caused by yeast and NDMs are more difficult to treat than those caused by dermatophytes (Lipner and Scher, 2019). Alternative therapies are often used empirically by individuals with nail disease, which can be added to the treatment already prescribed by doctors, or even before seeking specialized medical attention. Thus, medicinal plants have been used as alternative and/or complementary options to treatment.

Medicinal plants are used for  different  purposes,  such as for treating inflammatory processes and infectious diseases, as well to reduce pain and promote wound healing. The pharmacological effects of plants often come from the presence of the majority constituents, as well as the synergistic action resulting from their many constituents that belong to various chemical classes (flavonoids, phenolic acids, terpenoids, saponins and others). However, most of the plant products used are not only based on the long-term experience of the population, but also showed results of in vitro activity (Biabiany et al., 2013), although few clinical studies have been performed (Sipponen et al., 2013; Alessandrini et al., 2020; Romero?Cerecero et al., 2020). Thus, few plants have their effective clinically proven superficial fungal infections/ onychomycosis.

The purpose of this work was to evaluate studies that related medicinal plants and their in vitro antifungal activity against fungal agents of onychomycosis, as well as studies that reported the clinical efficacy of these plants.

Type of study

This study is an integrative literature review, a method that reviews and summarizes the scientific knowledge produced on a given topic, systematizing scientific evidence and contributing to the development of more consistent conclusions in relation to existing ones. This study followed six steps: 1) establishment of the guiding question and research objectives; 2) literature search; 3) data collection; 4) critical analysis of studies and categorization of the studies; 5) discussion of results; and 6) presentation of the integrative review (Whittemore and Knafl, 2005; Mendes et al., 2008; Souza et al., 2010; Sousa et al., 2017). The study was designed based on the following questions: 1) What are the medicinal plants with potential use for the treatment of onychomycosis, including in vitro and clinical studies, and 2) is there evidence in the literature about the use of these plants, or their extracts/subproducts in the treatment of nail mycosis?

Selection of articles

The search for articles was done in databases and virtual libraries: Scientific Electronic Library Online (Scielo), Medical Literature Analysis and Retrieval System Online (Medline/PubMed), CAPES (Periodic Portal from Brazil) and Google Scholar. The main descriptors (Health Sciences Descriptors/Medical Subject Headings - DeCS/MESH) used are shown in Table 1. All of the identified studies were evaluated in a systematically exclusionary way (Figure 1).

The inclusion criteria were publications about medicinal plants and onychomycoses or nail mycoses; full articles available; and published between January 2011 and January 2021. The exclusion criteria were literature reviews, dissertations, theses, and book chapters, abstracts of presentations at scientific events and studies that dealt with pure chemicals, even if they originated from plants.

A form was used to collect information such as: authors, title and year of publication, type of study (in vivo; in vitro; clinical studies), objectives, country of study, preparations and parts of the plant used, possible mechanisms of action on fungi cells, number of patients included, relevant points of discussion and conclusions of the authors.

The first search phase resulted in 957 articles, with 19 studies being eligible (Figure 1). Sixteen of them  included the in vitro evaluation of plant activity on dermatophyte fungi, 11 on yeast and six on non-dermatophyte filamentous fungi (Table 2). Five simultaneously reported the  evaluation of  three groups of fungi and three simultaneously on dermatophytes and yeasts, while no papers simultaneously evaluated dermatophytes and non dermatophyte filamentous fungi (NDFF), or yeast and NDFF (Table 2). Only three articles described clinical studies.

Among the selected articles, four (21%) included studies carried out in India, three (15.7%) in Brazil, two (10.5%) in Nigeria and one (5.2%) in each of the following countries: Spain, Finland, France (Guadeloupe Islands), Italy, Mexico, Portugal, Romania, Thailand, Taiwan and Tanzania (Table 2). The number of articles found varied over the years, with four in 2012 and three each in 2016, 2019 and 2020. Figure 2 lists the chronological frequency of the articles included for evaluation in this study, according to the groups of fungi. In 2021 only January was included.

Among the plants evaluated with antifungal activity are Angelica major, Cassia alata, Mitracarpus villosus, Lawsonia inermis, Euphorbia sanguinea, Euphorbia cotinifolia, Artemisia maritima, Sapindus saponaria L., Ageratum houstonianum, Piper betle L., Bursera simaruba, Cedrela odorata, Pluchea carolinensis, Cinnamomum zeylanicum, Evolvulus alsinoides, Enterolobium cyclocarpum, Acnistus arborescens, Pisonia aculeata and Allium sativum. More information is described in Table 3 and the following text.

Ekwealor et al. (2012) studied the in vitro antifungal activity of methanolic, hexane and cold aqueous extracts of Cassia alata, Mitracarpus villosus and Lawsonia inermis plants on the fungi Aspergillus terreus, Aspergillus sclerotiorum, Aspergillus flavus, Fusarium sp., Chrysosporium sp. and Scopulariopsis which were isolated from the nails of 135 farmers with onychomycosis. All extracts evaluated by the agar disk diffusion technique showed activity against those fungi, except for M. villosus and C. alata extracts, which did not show activity against A. flavus and M. villosus extract that did not show any activity against A. terreus. The authors concluded that the studied extracts could be potential antifungal drugs, as they were effective in vitro against non-dermatophyte fungi causing onychomycosis.

Guerrer et al. (2012) analyzed in vitro the antifungal activity of ozonized sunflower oil (Bioperoxoil®) in yeast isolated from nail mycosis, comparing it with the activity of the drugs amphotericin B, fluconazole, ketoconazole and itraconazole. They studied Candida parapsilosis, Candida albicans, Trichosporon asahii, Candida tropicalis and Candida guilliermondii isolates. According to the methodology used (disk diffusion in agar), they observed that the ozonized sunflower oil showed smaller inhibition halos than the traditional antifungal drugs. The researchers argued that the use of a quantitative methodology and clinical-laboratory correlation would be necessary to establish an appropriate treatment protocol, as the results obtained with ozonized sunflower oil may not mean lower efficacy, since clinical studies have not been carried out.

Chiang et al. (2013) analyzed the in vitro antifungal effect of Indigo naturalis prepared from Strobilanthes formosanus (Moore) on the onychomycosis fungi Trichophyton rubrum, Microsporum gypseum, Epidermophyton floccosum, Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans and Saccharomyces cerevisiae. They used the disk diffusion technique, in which they placed from 1 to 4 mg of Indigo naturalis per disk. Thus, the halo of growth inhibition around the disk was observed in A. fumigatus and C. albicans at all concentrations, in a dose-dependent manner, that is, the higher the concentration, the greater the halo formed. In S. cerevisiae, inhibition occurred only at the highest concentrations (3 mg/disk and 4 mg/disk) while an inhibition halo was only formed at the highest concentration (4 mg/disk) for the other fungi.

Biabiany et al. (2013) analyzed the antifungal activity of ten plants, six of which showed inhibitory activity (Bursera simaruba, Cedrela odorata, Enterolobium cyclocarpum, Evolvulus alsinoides, Pluchea carolinenis and Pluchea odorata) against fungi causing superficial mycoses and four which showed no activity (Acnistus arborescens, Gliricidia sepium, Pisonia aculeata, and Senna bicapsularis). The species tested were Malassezia sp., Candida krusei, C. albicans, C. parapsilosis, C. glabrata, C. kefyr, Scytalidium dimidiatum, Microsporum canis, Trichophyton tonsurans, T. interdigitale, T. mentagrophytes, and Pneumocystis carinii. Among these, C. krusei was the fungus that showed susceptibility to the highest number of extracts evaluated, while B. simaruba (ethanolic and ethanolic/water) and C. odorata (ethanolic/water) were the ones that showed activity against the highest number of strains evaluated.

The observational study carried out by Sipponen et al. (2013) evaluated the effectiveness of a nail lacquer made with Picea abies resin (natural conifer) for the treatment of onychomycosis in 37 patients, who used the product every day for nine months. Mycological examinations of the patients' nails were performed at the beginning and end of treatment. The authors observed that there was a trend towards complete or partial clinical cure in 14 of the 32 eligible patients, which showed favorable results for the efficacy of lacquer containing P. abies resin.

Two studies evaluated the essential oil (EO) activity of Melaleuca alternifolia (Tea tree) on dermatophyte fungi. Flores et al. (2013) analyzed the EO incorporated in emulsion, nanocapsules and nanoemulsions in an in vitro model of onychomycosis. Fragments nails and powdered nails were infected with Trichophyton rubrum. They observed that EO incorporated in nanocapsules and nanoemulsions exhibited activity against T. rubrum.

The study by Kumar (2014) evaluated the antifungal activity of the EO of 32 plants against Candida albicans and T. rubrum. They observed that Ageratum houstonianum leaf EO presented a minimum inhibitory concentration (MIC) of 400 ppm and a minimum fungicidal concentration (MFC) of 500 ppm. Thus, the authors concluded  that the EO from the A. houstonianum leaf has fungicidal activity, as well as other advantages, such as low cost and availability.

Cavaleiro et al. (2015) studied the in vitro antifungal activity of Angelica major EO and two of its main components, α-pinene and cis-β-ocimene, on yeasts (Candida spp., C. neoformans), NDFF (Aspergillus spp.) and dermatophytes (T. mentagrophytes, T. rubrum, T. verrucosum, Microsporum canis, M. gypseum and Epidermophyton floccosum). EO MICs ranged from 2.5 to greater than 10 μl/mL for Candida spp. isolates, from 0.32 to 1.25 μL/mL for dermatophytes and from 5 μL/mL to greater than 10 μl/mL for Aspergillus isolates. The authors found that the MICs of the isolated compounds were lower than that of EO for dermatophytes and Aspergillus species. Thus, they concluded that A. major EO presents bioactive compounds (α-pinene and cis-β-ocimene) with antifungal activity demonstrated in vitro. Artemisia maritima EO was evaluated for its antifungal properties in vitro against Trichophyton rubrum and Trichophyton mentagrophytes species. The authors evaluated the EO-fungal cell contact time for the fungicidal effect. Pure EO showed fungicidal activity in 10 seconds for both isolates. The time to reach the fungicidal effect was 60 minutes for T. rubrum (MFC of 0.4 μL/mL) and 70 minutes for T. mentagrophytes (MFC 0.5 μL/mL). Thus, the EO demonstrated high fungicidal activity in vitro against  those  dermatophytes   tested  and  those authors considered that A. maritima EO can be a potential alternative for the treatment of onychomycosis after appropriate clinical trials for proof (Mishra and Singh, 2016).

The in vitro study by Giwanon et al. (2016) analyzed the antifungal activity of the ethanol extract of eight plants (Piper betle, Argyreia nervosa, Punica granatum, Piper sarmentosum, Rhinacanthus nasutus, Plumeria obtusa, Morinda elliptica and Crateva adansonii) on the fungi T. rubrum and T. mentagrophytes. The P. betle extract showed a better inhibitory activity than all others. Thus, it was selected to compose a cream-based formulation, with the objective of comparing the in vitro efficacy with five antifungal drugs (econazole, miconazole, ketoconazole, metronidazole and voriconazole). The results obtained indicated that the cream formulated with P. betle presented antifungal activity in vitro superior to the five analyzed drugs, constituting a potential alternative to the topical treatment of infections caused by those dermatophytes.

Morah and Okoi (2016) described the phytochemical composition and physicochemical properties of the stem sap of Euphorbia sanguinea. In addition, they analyzed the in vitro antifungal and antibacterial activities against the following species: Candida albicans, Candida glabrata, Aspergillus flavus and Trichophyton rubrum, as well as Escherichia coli, Staphylococcus   albus,   Staphylococcus  aureus, Proteus mirabilis, Salmonella typhi and Klebsiella pneumoniae. The in vitro antibacterial activity was not confirmed. However, they reported strong antifungal activity when compared to traditional antifungal drugs (fluconazole, itraconazole, posaconazole, voriconazole, amphotericin B, flucytosine, caspofungin and griseofulvin). Candida albicans and Candida glabrata were the species with the highest susceptibility.

The study by Runyoro et al. (2017) analyzed the antifungal activity of methanol extracts from leaves and stem bark of Euphorbia cotinifolia (dairy-red) on Trichophyton rubrum, Trichophyton mentagrophytes, Aspergillus niger, Candida albicans species. The leaf extract inhibited the growth of T. rubrum, forming 12 mm halos (disk diffusion technique), while T. mentagrophytes formed a 17 mm halo, A. niger, 15 mm and no halo for C. albicans. The stem bark extract formed a 9 mm halo for T. mentagrophytes and A. niger and no activity for T. rubrum and C. albicans. Manivannan and Bhuvaneswari (2019) analyzed the antifungal potential of the ethanolic and aqueous extract of the bark of Cinnamomum zeylanicum on the onychomycosis agents: Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton simii, Epidermophyton floccosum and Candida albicans. Different concentrations of the extracts were evaluated in vitro by the agar disk diffusion method.  When  10 mg/disk   of   ethanolic  extract was used, all microorganisms tested were inhibited, with halo diameters ranging from 6 mm (E. floccosum) to 14 mm (T. rubrum). Using the broth dilution methodology, the authors observed that all isolates showed a MIC of 25 mg/mL. The MFC observed ranged from 50 mg/mL (T. rubrum) to 180 mg/mL (T. mentagrophytes). The aqueous extract also inhibited the growth of all tested isolates, but with less effectiveness in vitro. Thus, the authors concluded that extracts can be alternatives for antifungal treatment for onychomycosis caused by those agents and show promise in the development of pharmaceutical formulations.

The in vitro fungicidal activity of Allium sativum (garlic) extract was evaluated on fungi isolated from the nail of a patient with mixed infection by Meyerozyma guilliermondii and Rhodotorula mucilaginosa. The extract produced irreversible structural changes in these fungi, resulting in cell death. The in vivo antioxidant effect of the extract showed superior results when compared to allicin and diclofenac. Thus, the combination of antifungal and antioxidant activity makes garlic extract a potential alternative treatment for onychomycosis caused by those fungi (Pârvu et al., 2019).

The antifungal activity of Cissus quadrangularis extract was studied for the development of a nail lacquer loaded for the treatment of onychomycosis, including a penetration enhancer (salicylic acid) in the nail  plate. The fungicidal activity of the extract was tested in vitro against Candida albicans and showed a smaller zone of inhibition (12 ± 2 mm) with 10 mg/mL of the extract, whereas the zone of inhibition was greater (18 ± 2 mm) with 50 mg/mL. For lacquer loaded development, five formulations with different concentrations of components (salicylic acid, fluconazole and the extract) were evaluated, showing that salicylic acid increases the penetration of the extract that showed better antifungal activity than fluconazole (Pandit et al., 2020).

Alessandrini et al. (2020) carried out a clinical study of 20 patients to evaluate the antifungal activity of a topical formulation composed of the association of vitamin E and essential oils of lemon (Citrus aurantifolia), oregano (Origanum vulgare) and tea tree (Melaleuca alternifolia). The formulation was applied for six months on the nail plate and in the periungual area. Fifteen patients were completely cured, two showed significant improvement, one remained stable and two did not complete the treatment. The study highlighted 11 patients who showed relevant improvement within three months, which was maintained even after six months of treatment interruption.

The efficacy of the standardized Ageratina pichinchensis enkecalin extract was evaluated in vivo in a double-blind, randomized, controlled clinical trial, which included 71 patients with type 2 diabetes mellitus. Among these patients, 35 were included in the experimental group, which used enamel containing the extract, and 36 patients were in the control group, who used nail lacquer loaded containing 8% ciclopirox. The authors observed that there was a reduction in the severity of injuries and in the number of affected nails in both the control (77.2%) and experimental (78.5%) groups. Thus, they concluded that the extract showed clinical and mycological efficacy in the treatment of mild to moderate onychomycosis (Romero?Cerecero et al., 2020).

Marcos-Tejedor et al. (2021) evaluated the in vitro activity of EO against T. rubrum and T. mentagrophytes.

At a concentration equal to or greater than 0.05% (m/v), a fungistatic effect on T. rubrum was observed, while the same effect was observed at a concentration equal to or greater than 0.04% for T. mentagrophytes. Thus, two studies have confirmed that tea tree EO have in vitro activity against dermatophyte fungi and can be either an alternative or adjunctive treatment for nail mycosis (Flores et al., 2013; Marcos-Tejedor et al., 2021).

Mendes et al. (2021) analyzed the in vitro antifungal   activity   on    the   fungi   Trichophyton rubrum, T. mentagrophytes and T. interdigitale, the cytotoxicity on HeLa cells and the permeability of the hydroalcoholic extract of Sapindus saponaria L. in nail fragments in vitro. For the in vitro evaluation of the antifungal activity, they used the broth microdilution method and found equivalent MIC and MFC, which ranged from 195.31 to 781.25 μg/mL. The extract showed low cytotoxicity and excellent permeability in the nails; in one hour, it fully penetrated the nail layers and remained detectable for at least 24 hours. Thus, the authors concluded that the extract of Sapindus saponaria L has potential in the topical treatment of onychomycosis.

Currently, the search for new therapeutic strategies and new drugs from natural sources, especially plants, as well as from different extracts and pharmaceutical formulations, has increased the interest in research involving antifungal activity and it’s potentializing through different strategies. New products can contribute to the conventional therapy for mycoses, considering the increase in fungal agents responsible for infections described in the literature, as well as emerging fungi with resistance to conventional drugs.

The nail has a dense constitution of cells, which makes it difficult for different substances to penetrate. Thus, in addition to considering evidence of antifungal activity in pharmaceutical formulations, it is necessary to seek ways to increase the penetration of compounds into the nail plate. The nail keratin structure and its pH can interfere with the antifungal activity in clinical tests (Davies-Strickleton et al., 2020). According to Pandit et al. (2020), as the salicylic acid concentration increased, so did the penetration of the extract of Cissus quadrangularis. Nanocapsules and nanoparticles can also facilitate the penetration of active substances into the nail layer (Flores et al., 2013), hence the importance of not only researching antifungal compounds, but also formulations that improve the penetration efficiency of substances. On the other hand, Davis-Strickleton et al. (2020) showed in vitro the binding of the antifungal drug to keratin, reducing its activity. Thus, it would be necessary to increase the concentration of the drug to maintain efficacy. They concluded that there needs to be a balance between drug efficacy and permeation through the nail, which is a requirement for the development of effective topical therapies.

Thus, studies involving strategies for incorporating the active ingredient (whether plant extracts, essential oils, or others) in pharmaceutical formulations, which constitute the vehicle to take the drug to the agent causing the infection, can contribute to the effectiveness of the therapy (Biabiany et al., 2013). Formulations such as emulsions, nanoemulsions, nanocapsules and loaded lacquers, which can be used topically in clinical studies, can confirm the in vivo antifungal biological activity observed in the in vitro studies, in addition to confirming their safety (Pandit et al., 2020). Therefore, checking in vitro antifungal activity is one of the first steps in a long process prior to the drug becoming available over the counter. In addition, the antifungal potential of a new drug, as well as natural products, is evaluated primarily by in vitro studies, which confirm the antifungal activity. From there, several other studies are needed, such as cytotoxicity and effectiveness, for example, in animal models or other alternatives followed by clinical studies.

In vitro studies on antifungal activity also present technical aspects that need to be controlled to obtain effective and comparable results between different studies. The lack of standardization of methods for evaluating in vitro tests  was  one  of  the  limitations  that prevented more detailed conclusions, as well as the comparison between different studies. The type of substance used to obtain the extracts (the extractors) influence the results of in vitro antifungal activity tests, as the active constituents can be selectively solubilized in different solvents, whether organic or inorganic (Biabiany et al., 2013; Runyoro et al., 2017; Manivannan and Bhuvaneswari, 2019). In addition, the methodological conditions for carrying out the tests and the microorganisms used in the study, genera and species (and even different strains), can lead to variable results and conclusions due to the intrinsic sensitivity of each technique.

In this study, it was observed that there are few publications on the antifungal activity of plants in animal models (or alternatives), as well as clinical studies. Three clinical studies were found, one double-blind, randomized and controlled and two others which were observational. All patients included in those studies had laboratory evaluations of nail fragments (direct examination and culture for fungi), so that clinical and laboratory diagnosis for onychomycosis was confirmed. The treatments performed with the essential oil (one study) and with the nail lacquer loaded containing the extract (two studies) for a period of 6 to 9 months; clinical and mycological cures were confirmed for most patients.

Onychomycosis presents varied manifestations, of limited severity, but with significant epidemiological importance and impact on the quality of life of affected individuals. Treatment is time-consuming and requires persistence of infected individuals undergoing treatment, complying with the recommendations and guidelines of the health professional. As we can see in this study, the use of preparations from plants, such as extracts, their fractions, essential oils and resins, needs to be more extensively studied for use as alternatives to conventional treatment; this includes rational and safe use, since natural products from plants can present toxicity and also need to have clinical evaluation demonstrated by controlled studies, which seems to exist for few plants.

The effectiveness of products originating from the plants used in the studies was dependent on the plant used, drug concentration and fungal species; that is, the in vitro antifungal activity observed was fungus-specific, which means that the evaluated plant may have an antifungal effect on an infectious agent but no activity on another. We reinforce the importance of the correct diagnosis of onychomycosis and laboratory identification of the fungal species involved in the infection process.

In conclusion, this study shows that many medicinal plants have in vitro activity against fungal agents of onychomycosis; however, few studies have a clinical approach that proved the usefulness and effectiveness of some plants to treat nail infections. The authors emphasize the need for clinical studies, using different strategies and the development of different pharmaceutical formulations, to establish the effectiveness, dosage, and best therapeutic regimen. This will ensure that greater clinical efficacy with fewer undesirable effects can be obtained, for these products to be added to clinical practice for the treatment of onychomycosis.

BLP is funded by the Scientific Initiation grant, Edital 04/2021 PIBIC-CNPq-UFU. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.

The authors have not declared any conflict of interests.