Antifungal activity and mechanism of action of natural product derivates as potential environmental disinfectants

Abstract

 

There have been a considerable number of antifungal studies that evaluated natural products (NPs), such as medicinal plants and their secondary metabolites, (phenolic compounds, alkaloids), essential oils, and propolis extracts. These studies have investigated natural antifungal substances for use as food preservatives, medicinal agents, or in agriculture as green pesticides because they represent an option of safe, low-impact, and environmentally friendly antifungal compounds; however, few have studied these NPs as an alternative to disinfection/sanitation for indoor air or environmental surfaces. This review summarizes recent studies on NPs as potential fungal disinfectants in different environments and provides information on the mechanisms of inactivation of these products by fungi. The explored mechanisms show that these NPs can interfere with ATP synthesis and Ca⁺⁺ and K⁺ ion flow, mainly damaging the cell membrane and cell wall of fungi, respectively. Another mechanism is the reactive oxygen species effect that damages mitochondria and membranes. Inhibition of the overexpression of the efflux pump is another mechanism that involves damage to fungal proteins. Many NPs appear to have potential as indoor environmental disinfectants.

One-Sentence Summary

This review shows the latest advances in natural antifungals applied to different indoor environments. Fungi have generated increased tolerance to the mechanisms of traditional antifungals, so this review also explores the various mechanisms of action of various natural products to facilitate the implementation of technology.

Graphical Abstract

Graphical Abstract.

Mechanisms of action and applications of natural products as disinfectants

Introduction

Fungi are common inhabitants of the built environment and are associated with many types of illnesses, such as asthma, allergies, skin rashes, infections (skin, lungs, and bloodstream), and meningitis (CDC, 2023a). Resistance to antifungal treatments of infections has increased significantly in the last decade and has had important implications for morbidity, mortality, and medical care in the community (Arif et al., 2009). Since the beginning of the COVID-19 pandemic, there has been a greater interest in antifungal agents due to the increasing number of SARS-CoV-2-associated fungal infections (CDC, 2023a). According to Centers for Disease Control and Prevention Statistics (2021–2023), patients with COVID-19 have developed parallel fungal infections, and the number of deaths from fungal infections during the COVID-19 pandemic increased to 7199, in addition to more than 75 000 hospitalizations in the USA (Gold et al., 2023). In response to COVID-19 pandemic and increases in Candida auris (drug-resistant fungus) transmission in indoor settings, the CDC released educational guidance on cleaning and disinfecting the indoor environment or hygiene practices to reduce pathogen transmission (CDC, 2023b, 2023c).

Fungal contamination of indoor environments has been associated with diverse adverse health effects, such as infectious diseases, allergies, or toxic effects (Méheust et al., 2014). Therefore, contamination of air and indoor environmental surfaces with fungal pathogens represents considerable risks to public health (Querido et al., 2019; Rogawansamy et al., 2015). Disinfection and sanitization of indoor air and environmental surfaces may lower the fungal disease transmission rate among household members (CDC, 2023a). However, the widespread use of traditional chemical fungicides has caused numerous ecological problems, including air and water pollution, increased drug resistance, and toxicity of pesticide residue (Feng et al., 2023). As a result, there has been a need for more naturally derived, environmentally friendly fungicides with low toxicity, low residue, and high efficacy (Feng et al., 2023).

Various studies have been conducted with the goal of minimizing contamination of indoor environmental surfaces and air; however, most involve synthetic chemical disinfectants. Currently, the P list is: EPA Registered Antimicrobial Products, 2023, for claims against C. auris in hospital, institutional, and residential facilities only allows the use of chemical disinfectants (e.g., hydrogen peroxide, peracetic acid, ethanol, isopropyl alcohol, quaternary ammoniums, and sodium hypochlorite) for the control of fungal pathogens on hard surfaces (Agency, 2023). Likewise, the approval of antifungals has not increased significantly. According to Newman and Cragg (2020), over in the last 40 years, only 3% of antifungals have been approved, with two synthetic agents (isavuconazonium sulfate and fosravuconazole) currently used for medicinal purposes. Currently, the majority of the fungicides frequently used are triazole derivatives, which are broad spectrum; however, these have only been used for medical applications (Lv et al., 2017).

Many natural products (NPs) possess antifungal activity. NPs have been used since historical times to control the spread of fungal diseases, infections, and food contamination. Bioactives derived from NP according to the origin could be classified as (i) unregulated natural substances, (ii) NPs not modified and regulated by the Food and Drug Administration (FDA), (iii) chemically modified natural compounds (also called semi-synthetic), and (iv) a synthetic compound that copies a natural (mimetic) compound (Patridge et al., 2016). For the purposes of this review, we will define a NP as any unaltered substance or compound and will address those that have been obtained from plants, highlighting essential oils (EOs), polyphenols, alkaloids, and propolis for use as antifungals.

Recently, natural-derived plant and propolis have developed great interest as possible natural substitutes for conventional synthetic fungicides; likewise, the mechanism through which they provide fungicidal activity has been the subject of study (Nazzaro et al., 2013; Tian et al., 2012). Currently, NPs derived from microorganisms are the most well-described mechanisms of fungal inactivation. Examples include polyenes, which interrupt the functions carried out by ergosterol, altering the permeability of the cytoplasm; sordarins, which inhibit the synthesis of sphingolipids such as serine palmitoyltransferase, inositol phosphoramide synthase, and ceramide synthase; or echinocadins, which inhibit 1,3-β-d-glucan synthase (Zida et al., 2017). Knowledge encompassing mechanisms of action (MOA) of plant NP is less explicit and defined. However, a few plant NP antifungal MOA are clear and include membrane damage and have mechanisms similar to those derived from polyenes and azoles (Arif et al., 2009), but more detailed studies are needed to understand their properties and other possible MOA. Therefore, this review will focus on studies published in relation to these topics and thus facilitate the technological implementation of natural antifungal compounds derived from plants (polyphenols, alkaloids), Eos, and propolis for their incursion in the processes of disinfection of indoor environments and different inanimate surfaces.

Antifungal Activity of NPs in Indoor Air and Environmental Surfaces

NPs have demonstrated antifungal activity against some pathogenic species in indoor air (Table 1) and on different surfaces (Table 2). NP EOs are a complex mixture of terpenes (monoterpenes, sesquiterpenes), alcohols, aldehydes, esters, ketones, phenylpropanoid compounds, phenolics, phenols, and oxides (Bakkali et al., 2008). EO NPs are frequently studied for the control of fungal contamination of indoor air and are applied in the form of nebulization or, where appropriate, in the form of vapor. The fungus that has been most frequently evaluated is that of the genus Asperguillus, and its various species (Table 1). Studies carried out by Aboul-Nasr et al. (2014) detected Aspergillus as the most predominant genus in indoor air, and their study reported up to 17 species present inside hospitals, with the species being more predominant (flavus, fumigants, and niger). Aspergillus is a fungus of public health concern because it can live indoors and outdoors. Most people breathe in Aspergillus spores every day without getting ill. However, people with weakened immune systems or lung diseases often get ill, and it may even increase mortality (Gold et al., 2023). Another fungal target studied has been Candida albicans, and reports show reductions of ∼90% with the application of nebulized EOs in combination with standard sanitation in indoor hospital rooms (Gelmini et al., 2016). Although the species C. albicans is an important pathogen in public health, the species C. auris has produced concern in recent years due to its ability to persist on environmental surfaces in addition to resisting disinfection processes. This species has been identified in more than 35 countries, most of which are documented as person-to-person infections associated with medical care (Černáková et al., 2021; Saris et al., 2018). No reports of the efficacy of NP in environments contaminated by C. auris have been published to the author's knowledge.

Table 1.

Natural Products and Commercial Products with Antifungal Activity in Different Indoor Air

Applications in indoor air/type of environments	Natural product	Type of application	Target	Efficacy	Reference
Indoor hospital (Assiut University Hospitals)	Essential oils, clove (Syzygium aromaticum L. Merr & L.M. Perry), thyme (Thymus vulgaris L.), Ginger (Zingiber albus L.), lupine (Lupinus albus L.), and radish (Raphanus sativus L.)	Air condition filter	Aspergillus flavus, Aspergullus fumigates, Aspergillus niger, Cladosporium cladosperioides, Fusarium solani, Stachybotrys elegans	Thymus: completely inhibited Radish: 1.75–4.8 Clove: 2.4–4.2 Lupine: 1.8–4.8 diameter/cm of inhibition	Aboul-Nasr et al. (2014)
Indoor air environments (not defined the type of environment)	TTO (tea tree oil) TTO vapor Vinegar	Vapor	Aspergillus fumigatus Penicillium chrysogenum	TTO solution: 83 mm zone of inhibition TTO vapor: 81 mm zone of inhibition	Rogawansamy et al. (2015)
Indoor air environments (air from various locations in Umluj city)	White musk essential oil (Wm) Acetic acid (Aa) Commiphora myrrha (Cm) Boswellia carterii (Bc) Pistacia lentiscus (Pl)	(Wm) (vapor) (Aa) (solution) (Cm) (aqueous plant extract) (Bc) (vapor) (Pl) (vapor)	Aspergillus niger	White musk essential oil: 100% reduction Acetic acid: 92.2% reduction Commiphora myrrha extract: 78.6% reduction Boswellia carterii vapor: 93.7% reduction Pistacia lentiscus vapor: 89.4% reduction	Alzahrani et al. (2020)
Indoor air (Camarín of Santos Juanes church of Valencia, Spain)	Melaleuca alternifolia (tea tree oil) Thymus vulgaris (thyme oil)	EOs (vapor)	Fungi spp.*	Tea tree oil 77.3% reduction Thyme oil 48.7% reduction Mixture (tea tree oil + thyme oil) reduce 51.5%	Diaz-Alonso et al. (2021)
Aerosolized fungi spores (elaborate chamber)	Oil from Melaleuca genus plants	Eos (vapor)	Aspergillus flavus ATCC 9643	in 10 min reduce 72% viable spores	Kalaiselvan et al. (2022)
Indoor air (laboratory room)	Tea tree oil, eucalyptus oil, and lemon myrtle	Air condition filter	Aspergillus niger	30–60 min ∼90% reduction in fibrous filter surface	Mirskaya and Agranovski (2021)
Indoor hospital rooms (health care house in Iseo, Italy)	Essential oils mixture (alpha-pinene, beta pinene, terpinolene, camphor beta-citronellol, beta-linalool, and linalyl acetate)	Ultrasound vaporized	Candida albicans, Aspergillus niger, Saccharomyces cerevisiae	∼90% yeasts and molds	Gelmini et al. (2016)
Indoor office building	Clove, lavender, and eucalyptus oils		Aspergillus sp., Coprinellus sp., Penicillum spp.*	∼(11–32) and (2–11) inhibition diameter, for clove and eucalyptus, respectively, lavender shows activity until 7 days	Schroder et al. (2017)

*Species not defined.

Table 2.

Natural Products and Commercial Products with Antifungal Activity in Environmental Surface

Applications in environmental surface	Natural product	Target	Efficacy	Reference
Surface commercial woods	Pinus rigida wood oil vapor Eucalyptus camaldulensis oils vapor	Alternaria alternata, Fusarium subglutinans, Chaetomium globosum, and Aspergillus niger	Pinus rigida wood oil vapor: 5000 ppm (11–18) mm inhibition zone Eucalyptus camaldulensis oils vapor 5000 ppm (3.6–7.7) mm inhibition zone	Salem et al. (2016)
Domestic cleaning and laundering hard surfaces	Acetic acid Citric acid	Aspergillus brasiliensis ATCC 16404 Candida albicans ATCC 10 231	Acetic acid 10% plus 1.5% citric acid, reduce >5-log on surface	Zinn and Bockmühl (2020)
Wood surface	DMC Clean-CNS® composed of (ascorbic acid, citric acid, sodium lactate, and citric flavors) 0.8 mg/mL Proallium FRD-N composed of (citric acid, ascorbic acid, and lactic acid) 10 000 mg/mL Mico-Epro® (DOMCA, S.S.) composed of oregano, onion, and orange extract	Rhizopus spp.*	DMC Clean-CNS® reduce ∼90% Proallium FRD-N reduce >70% Rhizopus spp. Mico-Epro® reduce ∼100% Rhizopus spp.	Bernat et al. (2019)
Plastic surfaces	DMC Clean-CNS® composed of (ascorbic acid, citric acid, sodium lactate, and citric flavors) 0.8 mg/mL Proallium FRD-N® composed of (citric acid, ascorbic acid, and lactic acid) 10 000 mg/mL Micro-Epro® (DOMCA, S.S.) composed of (oregano, onion, and orange extract) 10 mg/mL	Altermaria spp.*	DMC Clean-CNS® reduce ∼100% Proallium FRD-N reduce >90% Rhizopus spp. Micro-Epro® reduce ∼100% Alternaria spp.	Bernat et al. (2019)
Dentistry material (metal or acrylic resin)	Green propolis ethanolic extract	Candida aldicans, Candida parapsilosis, and Candida tropicalis	Candida albicans (2.5–250 µg/mL) sensitive Candida parapsilosis (25 µg/mL) sensitive Candida tropicalis (250 µg/mL) sensitive	Bezerra et al. (2020)
Wood surface	Turkish propolis extracts at 7% Slovenian ethanolic extract Spanish propolis extract 5–40 mg/mL Polan propolis extract	Trametes versicolor, Pycnoporus sanguineus, Gleophyllum trabeum, and Coniophora puteana	Antifungal activity reducing mass loss	Woźniak (2022)
Polyvinil chloride (PVC) and silicone catheters	Polan propolis extract	Candida glabrata and Candida krusei	0.08%–1.25% (v/v) eradication of biofilm formed by C. glabrata and C. krusei	Gucwa et al. (2018)
Orange peel	Cinnamon oil	Rhizopus nigricans, Aspergillus flavas, and Penicillium expansum	The MIC of cinnamon oil against R. nigricans was 0.64%, which was higher than that of oil against A. flavus (0.16%) and P. expansum (0.16%). The diameters of inhibition zone against R. nigricans, A. flavus, and P. expansum improved with increasing the concentration from 0.10% up to 3.0%	Xing et al. (2010)
Cultural heritage objects	Ocimum basilicum (basil) Solaris essential oil® 35%–60% palmitoleic acid, 5%–35% butyric acid, and up to 15% linoleic acid, omega, and eucalyptol	Aspergillus, Penicillium, and Mucor	100% of antifungal activity after 144 hr of incubation	Fierascu et al. (2014)

*Species not defined.

Most EOs are generally recognized as safe, are not harmful, and are more widely accepted by consumers as compared to chemical compounds (Djilani & Dicko, 2012; Falleh et al., 2020). About 3000 EOs have been reported from different plant species, but only 300 are economically important and used in the food, pharmaceutical, agricultural, and sanitary industries (Basak & Guha, 2018). However, EOs have not been widely considered for environmental applications beyond agricultural use or food surfaces, and little information exists regarding their use in controlling indoor fungal contamination. In the food and agricultural industry (i.e., green pesticides), EOs are currently being explored as microemulsions and nanoemulsions, which are colloidal systems consisting of an oil phase dispersed in an aqueous continuous phase and a thin layer of surfactant surrounding each oil droplet (Basak & Guha, 2018; Gundewadi et al., 2018). In food systems, the nanoemulsions applied to the surface of the plants or food enhance bioavailability or transport through biological membranes (e.g., fungal surface) and solve the problem of reductions in antimicrobial efficacy (Basak & Guha, 2018; Gundewadi et al., 2018). This may also be applicable to indoor environmental surfaces; however, there is very little antifungal research focusing on indoor disinfection.

Some research groups have evaluated the effectiveness of tea tree oil (TTO) in different indoor air environments (hospitals, indoor cultural heritage spaces, and houses) and have obtained varying outcomes depending on its form of application, and the reported results range from 70% to 90% reduction of fungi with periods of contact time from 24 hr to 14 days (Diaz-Alonso et al., 2021; Mirskaya & Agranovski, 2021; Rogawansamy et al., 2015). Other investigators have observed the effect of TTO during indoor disinfection with commercial products and have observed that TTO shows 60% more zones of inhibition of the fungus than the chemical products studied (Rogawansamy et al., 2015). Crude plant extracts have also been used in indoor environmental disinfection; for example, Alzahrani et al. (2020) evaluated the aqueous extract of Commiphora myrrha in indoor environmental disinfection against A. niger showing reductions of the fungus spp. up to 78.6%.

There is little scientific evidence on the antifungal efficacy of NP for their application for disinfection and sanitization of animate or inanimate surfaces; some studies are shown in Table 2. The NP products that have been studied correspond to organic acids, EOs, propolis extracts, plant extracts, and some commercial products formulated with NP. Most research has been carried out with the aim of developing fungicides for applications on commercial wood surfaces, especially those used for construction. The studies have evaluated the antifungal effects of different substances of vegetable origin, such as EOs, tannins, plant extracts, polyphenols, alkaloids, and propolis extract (Woźniak, 2022). Salem et al. (2016) evaluated the efficacy of the EOs on the wood contaminated with Pinus rigida and Eucalyptus camaldulensis applied in the form of vapor at a concentration of 5000 ppm and observed inhibitions ∼(3–18 mm) of the fungi Alternaria alternata, Fusarium subglutinans, Chaetomium globosum, and A. niger. Likewise, literature reports show that on wooden surfaces, using propolis extracts from different origins, against fungi such as Trametes versicolor, Pycnoporus sanguineus, Gleophyllum trabeum, and Coniophora puteana shows protective effects on the loss of wood mass (Woźniak, 2022). Propolis has also been evaluated with an antifungal effect on other surfaces such as metal or acrylic, especially dental material. In addition, studies carried out by Bezerra et al. (2020) have shown that green propolis extracts can inhibit the development of the fungi C. albicans, C. parapsilosis, and C. tropicalis. Commercial products such as DMC Clean-CNS®, Proallium FRD-N, both composed of ascorbic acid, citric acid, sodium lactate, and citric flavors, and Mico-Epro®, composed of oregano, onion, and orange extract, have also been evaluated on environmental surfaces for the reduction of postharvest fungi and have shown effectiveness of 70%–90% for wooden surfaces and 90%–100% reduction of Alternaria fungus for plastic surfaces (Bernat et al., 2019). Likewise, some studies have been conducted in the application of NP on home surfaces. Studies by Capoci et al. (2015) show the effect of Citronella extract on floor and carpet contaminated with Microsporum canis. Zinn and Bockmühl (2020) evaluated citric acid and acetic acid against A. brasiliensis and C. albicans in domestic cleaning and hard surface, showing reductions greater than 90%.

In a general context, current investigations aimed at reducing the risk of fungal infections using NPs such as EOs and natural extracts show excellent potential for their use in environmental surface disinfection, in addition to being considered less toxic and safe. However, they face several challenges to reduce the risk of infection by pathogenic environmental fungi for humans, for example, the generation of new resistant species such as C. auris, in addition to environmental contamination, and the association of fungi with other infections, among others (Fig. 1).

Fig. 1.

The risks of fungal environmental contamination, the current challenges to reduce this risk, and the potential solution using natural and eco-friendly products.

Likewise, most of the studies that evaluate antifungals of natural origin mostly do not involve mechanistic studies that allow elucidation of the form of inactivation of the fungus and facilitate smarter application technology developments.

The following section presents the current knowledge on MOA of various NPs, such as EO and its individual compounds, plant-derived secondary metabolites (polyphenols and alkaloids), and propolis extracts, all of which have antifungal effects.

Mode of Action of Antifungal EOs and Their Individual Compounds

Current research investigating the mechanism of inactivation of EOs on fungus, mycelial, and fungal spores is scarce, unlike bacteria (Basak & Guha, 2018). EOs, also called volatile oils, or simply essences, are the natural aromatic substances responsible for the fragrances of flowers and other plant organs (leaves, bark, fruits, and rhizomes) (Basak & Guha, 2018; Gundewadi et al., 2018). They are usually obtained by steam or hydro-distillation, which was first developed in the Middle Ages by Arab (Bakkali et al., 2008). EOs are mainly composed of mono- and sesquiterpenes and phenylpropanoids, which confer their organoleptic characteristics and are connected with the various functions necessary for plants to protect against animal predators or microorganisms (Nazzaro et al., 2017). The EOs, in addition to being recognized as safe by the FDA, are also listed by the Environmental Protection Agency (EPA) as authorized active ingredients for biopesticides. Among them are canola oil, castor oil, capsaicin, cinnamaldehyde, citral, citronella oil, neem oil, eugenol, eucalyptus oil, garlic oil, lemongrass oil, menthol, thyme oil, orange oil, and geranium oil (Food Drugs Administration F. D. A. Food Additive Status, 2006). Some of them such as eucalyptus oils, oregano oil, orange oil, thyme, and eucalyptol have shown good antifungal activity in indoor environments and different environmental surfaces (Tables 1 and 2).

EOs are complex mixtures of numerous antimicrobial compounds and molecules. Various studies have analyzed the biological properties of the main components, such as terpineol, eugenol, thymol, carvacrol, carvone, geraniol, linalool, citronellol, nerol, safrole, eucalyptol, limonene, and cinnamaldehyde, which mostly reflect the biophysical and biological state of the oil, and it is possible that the activity of these components is modulated by other minor components (Bakkali et al., 2008), so their activity depends on their chemical composition and the number of main components. Therefore, its mechanism of action, like polyphenols, can be very varied (Fig. 2).

Fig. 2.

Different mechanism of action of natural products (essential oils, extracts, or isolate constituents). All these effects in the fungus cell can cause fungal lysis.

Cell Wall and Cell Membrane Damage

EOs and their components have a variety of targets, particularly the membrane and cytoplasm, and in certain situations, they completely alter the morphology of cells (Nazzaro et al., 2013). Fungal cell wall composition varies among species, but it generally has three polymeric components: glucan, chitin, and mannoproteins. Glucan is a polysaccharide constituted by glucose monomers linked by (1,3)-b or (1,6)-b bonds, and it is an essential component of the cell wall, and cell membrane is confirmed to contain ergosterol. Other components of the fungal cell are sphingolipids, which represent a small proportion of the fungal cytoplasmic membrane and are essential for cell functions, so their inhibition results in cell death (Vicente et al., 2003). The genera Candida, Aspergillus, and Penicillium seem to be the most studied in their inhibition by EOs. In yeast cells, EOs develop a membrane potential across the cell wall by disrupting ATP adhesion, leading to cell wall damage. A similar mechanism could be enveloped in filamentous fungi (Hou et al., 2022; Tariq et al., 2019).

Several studies have shown that the main mechanism of action of NPs against the microorganisms occurs through membrane action. Terpenes, for example, cross the fungal cell wall and accumulate among the fatty acid chains of the fungal lipid bilayer, modifying the entire cell membrane structure (Zida et al., 2017). Terpenes/terpenoids are the main groups of the EO, and their antifungal activity also might be due to their highly lipophilic nature and low molecular weight, and are capable of disrupting the cell membrane, causing cell death, or inhibiting the sporulation and germination of spoilage fungi (Tian et al., 2012).

In addition, the cytotoxic activity of EOs is mostly due to the presence of phenols, aldehydes, and alcohols (Bakkali et al., 2008). EOs are hydrophobic and increase their hydrophobicity at low pH. The readily penetrate through the cell wall and fungal membranes, altering membrane fluidity and permeability, coagulating the cytoplasm, and decreasing the mitochondrial proton motive force (Tariq et al., 2019). Indeed, several studies have shown the MOA of different EOs in cell wall and membrane of fungus (Table 3).

Table 3.

Effect of Essential Oils (EOs) Extract form Plant/Individual Components on Membrane/Cell Wall, Mitochondria, and Efflux Pump of Fungi

Molecular target	Oils in crude form or isolated active compounds	Reference
	EOs extract form plant
	Cinnamomum, Citrus, piperita, Melaleuca alternifolia, Mentha piperita, Ocimum basilicum, Origanum, Thymus	Chen et al. (2013)
	Juniperus communis, Litsea cubeba, Salvia sclarea	Haque et al. (2016)
Membrane/cell wall	Coriaria nepalensis	Seto-Young et al. (1997)
	Coriandrum sativum	Perlin et al. (1997)
	Individual compounds
	Thymol, carvacrol	Chavan and Tupe (2014)
	Trnas-cinnamaldehyde citral, citronellal, eugenol	Bang et al. (2000)
	Eugenol, carvacrol, thymol	Bouddine et al. (2012)
	Citral	Ju et al. (2020)
	EOs extract form
	Clove	Ju et al. (2020)
	Anethum graveolens L.	Zheng et al. (2015)
Mitochondrial damage	A. graveolens	Tian et al. (2012)
	Individual compounds
	Citral, eugenol	Ju et al. (2020)
	Salicylic acid, thymol	Kong et al. (2019)
	Tea tree oil, TTO	Li et al. (2017)
	EOs extract form
	Cyperus rotundus L.	Siroua et al. (2022)
	Thyme	Ben Jabeur et al. (2017)
Efflux pump
	Individual compounds
	Thymol	Ben Jabeur et al. (2017)
	Cinnamaldehyde	Pootong et al. (2017)

Chavan and Tupe (2014) found that the addition of carvacrol and thymol (64 mg/mL) in red wine resulted in inhibition of the growth of the spoilage yeasts. Carvacrol and thymol exerted their antimicrobial action through membrane damage, leakage of cytoplasmic content, and ergosterol depletion. In addition, thymol is a monoterpene phenol derived from Thymus vulgaris. It is a hydrophobic component, which allows its ability to protrude, causing asymmetric tensions within the plasma membrane and forming complexes with the acyl group, altering the conformation and membrane fluidity of the fungus (Hou et al., 2022). In general, the application of EOs causes the coagulation of fungal cells, which produces irreparable damage to the plasma membrane. It interferes with the synthesis of ATP, which causes damage to the cell membrane, so EOs can penetrate and break the membrane, damaging the cell structure and intracellular membranes of the fungus through a permeation mechanism that contributes to the dissolution of mitochondrial cells (Singh et al., 2017).

Mitochondria Damage

The function of mitochondria is to generate energy in the fungal cell, though, in addition, they perform virulence functions, ergosterol biosynthesis, and cell wall maintenance (Dagley et al., 2011). EOs can also disrupt cell membrane hyperpolarization (also known as mitochondrial membrane potential [MMP] mitochondrial damage) by influencing ion reservoirs, such as calcium ions, particle pumps, and ATP reservoirs, thereby reducing membrane permeability. This change in cell wall fluidity can cause oxidative damage and obstruct cytochrome C pathways, enzyme uptake, and amounts of calcium ions. Consequently, permeabilization of an upper and lower cell membrane can lead to cell death, termed apoptosis, which could be expressed as a decrease in MMP (Mutlu-Ingok et al., 2020). Studies by Ju et al. (2020) evaluated mitochondrial damage by MMP with EOs eugenol and citral against Penicillium, using fluorescence signal intensity as an indicator of MMP, and observed that a large amount of H⁺ was pumped from the mitochondrial matrix after treatment, which was expressed as mitochondrial damage and subsequent apoptosis. Some EOs have the property of inducing mitochondrial respiratory metabolism damage through the inhibition of their metabolic enzymes. For example, the synergism of EOs (citral and eugenol) or the mixture of EOs (carvone, limonene, and apiol) can inhibit the activity of these enzymes in the tricarboxylic acid cycle (TCA) (Ju et al., 2020; Tian et al., 2012). Mitochondrial dysfunction has also been studied with tea tree essential oil. Studies by Li et al. (2017) evaluated the relationships between the enzymes succinate dehydrogenase and mitochondrial dehydrogenase, which are the key enzymes in mitochondrial function and perform important steps in pathways of aerobic energy production, and they observed that TTO causes mitochondria dysfunction and disrupts the TCA (Krebs or citric acid cycle).

Damage in Efflux Pump

Efflux pumps allow microorganisms to regulate their internal environment by removing toxic substances, including antimicrobial agents and metabolites; therefore, they are a key tool in the fight against fungal infections because this transport often includes the transport of accumulated antimicrobials out of the fungal cell. Overexpression of efflux pumps can lead to antimicrobial resistance, so inhibiting efflux pumps reduces resistance to drugs (Lagrouh et al., 2017; Prasad & Rawal, 2014).

Several reports have shown that EOs such as camphor and eucalyptol have a negative impact on the genes that encode the efflux pumps (CDR1 and CDR2), modifying the efflux function in the elimination of toxic substances (Ivanov et al., 2021). CDR1 is also inhibited by other EOs such as thymol and curcurmin, suppressing the pumping action (Tian et al., 2012).

Mode of Actions of Secondary Metabolites (Polyphenols and Alkaloids)

Plants are a great source of active substances, many of which are used as a defense mechanism against pathogens, such as fungi. Among its active components (secondary metabolites) are the polyphenolics and alkaloids that are the main components of these NPs with antifungal activity. The sesquiterpenes and monoterpenes called volatile NPs (EOs) are other constituents with excellent antifungal potential (Marena et al., 2022).

Polyphenols could be classified as phenolic acids, lignin, flavonoids, and tannins. Flavonoids are a class of polyphenols that can be classified according to their origin in their biosynthesis. Flavonols, isoflavones, and chalcones are considered intermediate products of biosynthesis, while flavanols and flavones are final products and are the most abundant, being found in apples, grapes, barriers, and red wine (Seleem et al., 2017).

Antifungal targets or MOA of polyphenols are very variate; most mechanisms of action are attributed to their functionality, such as molecule size and functional group (number and position of hydroxyl group, glycosylation, and the position of the sugar) (Al Aboody & Mickymaray, 2020). Another consideration is the type of microbial target and the antimicrobial (NP). The polyphenol MOA could contribute to inhibit the efflux pump, cell membrane, ergosterol synthesis, and cell wall, or produce biofilm damage as well as produce reactive oxygen species (ROS) effect (Jin, 2019). Likewise, the mechanism of action of alkaloids is related to membrane permeabilization, inhibition of DNA and RNA, protein synthesis, ergosterol synthesis, and increasing the ROS generation (Dhamgaye et al., 2014; Shao et al., 2016).

Other important secondary metabolites are alkaloids, and they are derivatives of nitrogen and are basic compounds synthesized by living microorganisms; they also have very diverse pharmacological activities (Costa et al., 2012). Alkaloids are categorized according to their molecular origin, the type of structure they possess, and their botanical and biochemical origin. Alkaloids are classified as true alkaloids, protoalkaloids, and pseudoalkaloids (Dey et al., 2020).

The MOA described in various investigations for polyphenols and alkaloids are summarized in Table 4, and their effects are shown in Fig. 2.

Table 4.

Phenolic Compounds and Alkaloids with Antifungal Activity, and Their Possible Mechanisms of Action (MOA)

Polyphenols	Source	Fungus/Mechanism antifungal	Reference
Flavonols
Quercetin	Allium cepa L.	Candida albicans/peroxidase activity, inhibit biofilms	Gao et al. (2016) and Takahama and Oniki (2000)
Myricetin	Plinia cauliflora	Candida albicans/damages in cell wall integrity	Lee and Kim (2022) and Souza-Moreira et al. (2019)
Kaempferol	Pure compound	Candida albicans/efflux pump decreasing expression of CD1 and CDR2	Shao et al. (2016)
Pinocembrim	Ficus hirta Vahl	Penicillum italicum/damages in cell wall integrity	Chen et al. (2013)
Apigenin	Aster yomena Mikino	Candida albicans/inhibit biofilms, and membrane disruption	Lee et al. (2018)
Flavone
Baicalein	Scutellaria baicalensis	Candida spp./inhibit efflux pump mechanism	Serpa et al. (2012)
Isoflavone
Sedonan A	Dalea formosa roots	Candida albicans, Saccharomyces cerevisiae/inhibit efflux pump mechanism	Belofsky et al. (2013)
Chalcona
Carvacrol	Lavandula multifitida	Candida albicans/cytoplasmic membrane damage	Zuzarte et al. (2012)
Hydroxycinnamic acid
Ellagic acid, caffeic acid	Propolis	Candida auris/inhibit biofilms	Possamai Rossatto et al. (2021)
Alkaloids
Tomatidine	Solanum lycopersicum	Candida albicans/ergosterol disruption	Dorsaz et al. (2017)
Isoquinoline alkaloid berberine	Pure compound	Candida albicans/mitochondrial dysfunction, generations of reactive oxygen species	Zuzarte et al. (2012)

Membrane Disruption

Cell membrane of fungi is enriched with diverse lipids belonging to the class glycerophospholipids, sphingolipids, and sterols, also known as steroid alcohols, which are amphipathic lipids having rigid and compact ring structures. Another important component is ergosterol, which is a major component of fungal cell membrane. Antifungal drugs normally inhibit ergosterol biosynthesis, and consequently the integrity of the cell membrane, which causes the loss or release of intracellular components (Cruz & Wuest, 2023; Sant et al., 2016).

Several polyphenolic compounds have been studied in their antifungal activities. Lee et al. (2018) evaluated the antifungal effect of the secondary metabolite apigenin specifically a flavonol isoleta of Aster yomena in Candida spp. They observed that in 5 µg/mL of apigenin, the cell membrane is altered, disturbing lipid dynamics, inducing dysfunction, increasing cell permeability, and inhibiting potential of plasma membrane proteins due to their effect on the flow of calcium and potassium ions.

Other phenomena studied in membrane fungal cell are the overproduction of ROS, which causes significant oxidative stress in the fungus and results in impaired membrane permeabilization, even causing damage to nucleic acids and oxidation of fatty acids. Baicalein, which is a flavone extracted from Scutellaria baicalensis, has shown potential to induce ROS generation and consequently membrane structure deformation and efflux (Serpa et al., 2012). Another target studied in the cell membrane is fatty acid synthase, an essential enzyme in synthesizing fungal membrane fatty acids. The flavonol quercetin has shown an inhibitory effect on fatty acid synthase, even with ROS effect and peroxidase activity (Al Aboody & Mickymaray, 2020; Takahama & Oniki, 2000).

Alkaloids also present MOA in fungi membrane; for example, studies by Dorsaz et al. (2017) show that the effect of tomatidine isolate of Solanum tuberosum alters the regulation genetics in the ergosterol biosynthesis of C. albicans, C. krusei, and S. cerevisiae cells. Ergosterol biosynthesis is also disturbed for the alkaloid berberine, and the mechanisms involved include increased ROS generation (Shao et al., 2016).

Inhibition of Efflux Pumps

One of the important targets in fungi is the expulsion pump of species, which is an important strategy to inhibit fungi that may be resistant, such as C. auris, considered a major nosocomial pathogen, emerged globally as a multidrug resistant fungus (Ademe & Girma, 2020), and the development of substances may result in a therapy that replaces azole drugs (Holmes et al., 2016). Vanillin is a phenolic aldehyde that inhibits transporters belonging to the ATP-binding cassette superfamily, reducing the expression of CDR2 and CaCdr2p resistance proteins, respectively, in C. albicans (Saibabu et al., 2020). Other polyphenolic compounds, such as curcumin and geraniol, modulate the expression of the CaCdr1p efflux pump transporter, even acting with synergism to azoles compounds in the same fungus (Singh et al., 2018). Baicalein (5,6,7-trihydroxy flavone) is a flavone, which can be obtained from S. baicalensis, with antifungal effect on C. albicans. This flavone, in addition to inhibiting lipoxygenase, is an inhibitor of the expulsion pump when combined with fluconazole, causing the fungus to lose the ability to expel the antifungal (Serpa et al., 2012). Alkaloid berberine has also been studied in its effect, and does not depend on major efflux pump proteins (Dhamgaye et al., 2014).

Effect in Mitochondrial Dysfunction

Mitochondria are an important organelle as a source of ROS generation in eukaryotic cells, and their dysfunction is critical to the mechanism. Studies by Ma et al. (2022) demonstrated that the compound E-2-hexenal derived from the green leaves induces a toxic effect in Aspergillus flavus by decreasing the potential of the mitochondrial membrane and reducing the levels of intracellular ATP through the production of intracellular ROS, which consequently modifies the permeability of the membrane. Bacalein is a flavone isolated from S. baicalensis, and it also causes ROS accumulation and consequently cell apoptosis (Serpa et al., 2012). Another phenolic compound studied are the flavonoids present in honey, which can inhibit C. albicans, affecting mitochondrial function and decreasing vacuolization, altering the branching of the fungus, and consequently virulence (Canonico et al., 2014). The flavonoid quercetin can also inhibit C. albicans by increasing intracellular ROS levels and decreasing intracellular redox levels, inducing apoptosis cellular (Kwun & Lee, 2020).

Another secondary metabolite studied are alkaloids. For instance, isoquinoline alkaloid berberine shows antifungal activity against C. albicans and C. glabrata; however, currently the only known MOA in C. albicans is causing damage to the integrity of the cell wall, and mitochondrial alterations through of ROS generation, inducing early apoptosis in the fungi (Dhamgaye et al., 2014). Berberine alkaloid also has an effect on the inhibition of efflux pump transporters in C. tropilcalis (Shao et al., 2016).

Mode of Actions Propolis Extracts

Another NP studied with antifungal activities is propolis, which is a bee product with a resinous appearance containing plant exudates and is used by bees as a protective barrier to protect the hive and against pathogenic fungi. Propolis is a mixture of substances composed of resins and vegetable balsam 50%, bee wax 30%, pollen 5%, and essential and aromatic oils 10%, including over 800 individual chemical fractions such as phenolic compounds (flavonoids, phenylpropanoids, and chalcones) and terpenoids (volatiles, di-, and triterpenes) (Salatino, 2022). Propolis has several antimicrobial activities, low cytotoxicity, and no reports of drug interactions, with antiviral, antibacterial, anti-inflammatory, and antitumor actions, among others (Corrêa et al., 2020).

Several studies have reported the antifungal effect of propolis in vivo, in vitro, and even on inert surfaces such as dentistry material, wood surface, polyvinyl chloride, and silicone catheters (Bezerra et al., 2020; Gucwa et al., 2018; Woźniak, 2022). Most studies have been developed in ethanolic extracts of propolis showing activity against Candida spp. (Bezerra et al., 2020; Negri et al., 2014; Silva-Beltrán et al., 2021) and dermatophytes (Monzote et al., 2012). ln addition, as been observed synergism of propolis with azoles drugs as fluconazole and viriconazole against C. albicans (Gucwa et al., 2018).

In reference to propolis, several studies examined MOA. Studies by Rivera-Yañez et al. (2022) evaluated the effect of Mexican propolis on the formation of the germ tube of C. albicans, which are involved in the formation of biofilms, and found a complete inhibition of the germ tube in a range from 1250 µg/mL to 2500 µg/mL of propolis, and they assume the antifungal activity is because of the presence of pinocembrin, naringenin, and baicalein, which decrease the cell surface hydrophobicity of C. albicans and increase the production of ROS, mitochondrial Ca²⁺ overload, and mitochondrial superoxide radical generation. Among Candida spp., C. auris has emerged as highly multidrug-resistant, and polyphenols, such as caffeic acid phenethyl ester, one of the significant components of propolis, reduce significantly the biomass of C. auris, modifying the fungal cell wall (Possamai Rossatto et al., 2021). Other studies have shown the antifungal effect of propolis is caused by membrane and cell wall damage with intracellular content extravasation (Corrêa et al., 2020). Although some studies show evidence for the fungal cell membrane as the most probable target of propolis and that generate exclusion zone, excluding ions from microorganisms (Gucwa et al., 2018; Kowacz & Pollack, 2020), the mechanism of action of propolis ethanolic extract is not yet fully understood due to the synergy of its multiple components, with prominence for the flavonoids.

Concluding Remarks

Several NPs from plants in isolated form or mixtures of compounds have been patented for their excellent activity against different types of microorganisms, including fungi. Phenolic acids, lignins, flavonoids, propolis (as a promising antifungal), and EOs are examples. EOs are the most studied NPs in terms of their antifungal effects on inert surfaces and indoor ambient air, and are preferably applied in aerosol in the form of vapor in the last stage of surface disinfection, which is a process widely accepted in several countries around the world for their efficacy and safety performance (World Health, 2020); however, it is important to consider the limit of the exposure time because the vapors can cause eye irritation (Baptista-Silva et al., 2020); likewise, these studies mostly evaluate the reduction of prevalent and mostly resistant pathogenic fungi, especially the genera Aspergillus spp. and Candida spp. (Ademe & Girma, 2020; Rhodes et al., 2022).

Regarding the various NPs antifungal MOA, current studies show that they could have effects on the cell membrane and cell wall of the fungus because of their likely impact on ATP synthesis, or on the flow of Ca⁺⁺ and K⁺ ions. In addition, NPs can cause damage to the fungus cell mitochondria, commonly due to the ROS effect. Disruption of the efflux pump is another reported phenomenon, and studies indicate that it also affects the principal proteins involved, such as CDR1 or CDR2. However, targeting the fungal cell membrane is so far the most explored for the development of antifungals.

Published data on agents suitable for the remediation of fungus on interior and environmental surfaces comes primarily from chemicals, and some have reduced their effectiveness by inhibiting only sporulation or action on a limited range of fungi (Rogawansamy et al., 2015). Future research is needed for the applications of NPs in environmental disinfection; this would allow for prescribe effective antifungal agents for their applications, especially in the built environment for environmental surface disinfection and indoor air sanitization.

While it is crucial to understand the MOA of antifungal NPs, it is also important to understand the factors that could alter their stability, including possible toxicity and appropriate concentrations to formulate optimized and eco-friendly microbicides that would be needed for application to air sanitization or surface disinfection.

Author Contributions

Conceptualization retrieved all data and wrote the original draft, N.P.S.-B.; methodology, formal analysis, and supervision, S.A.-B., M.K.-J., J.M-K., and C.P.-G.; writing – review and editing, S.A.-B., M.K.-J., J.M-K., C.P.-G., and N.P.S.-B.. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Environmental Science Department of the University of Arizona and from Reckitt Benckiser Plc (Reckitt) Company.

Conflict of Interest

The authors declare that they no competing interest.