Keywords

1 Introduction

Fungal diseases are life-threatening to human health and cause an important burden around the world and are associated with high numbers of morbidity and mortality. Moreover, food contaminations due to different fungal infections are frequently the cause of great economic losses in horticultural businesses and economy. However, the same fungi which may exhibit great pathogenicity could also be used by the industry for different applications:

Candida albicans and C. glabrata are species of yeasts which cause candidiasis, a recurrent disease that is produced in humid and warmer environments of the human body such as the oral or vaginal cavity, skin folds, oropharyngeal, and bronchial secretions (Bonifácio et al. 2019). Moreover, they could cause dangerous opportunistic fungal infections on immunocompromised hosts and candidemia in adults, mainly in patients with hematological disorders (Butassi et al. 2015). On the other hand, the production of lipids (Enshaeieh et al. 2013), butyl oleate, and ethyl oleate catalyzed by Candida spp. (Chen et al. 2018) is used in the industry as a source for biodiesel production. Moreover, these yeasts may be harnessed for producing biosurfactants (Nwaguma et al. 2019). Saccharomyces cerevisiae is another yeast that commonly colonizes mucosa and can cause superficial or invasive visceral infections associated with immunosuppressed individuals (Davicino et al. 2007). However, some studies described the biocontrol capacity of S. cereviciae against other phytopathogenic fungi of fruits such as Botrytis spp. (Nally et al. 2012). Another study demonstrated that a strain of this fungus could synthesize the bioactive peptide pediocin PA-1 that showed its antibacterial activity against two Gram-negative strains such as Shigella boydii and S. flexneri (Nguyen et al. 2020). These systems have been regarded as safe because they are suitable for bioindustrial processes applied to food. Cryptococcus spp. cause important systemic mycosis globally, and their most common clinical manifestation is cryptococcal meningitis, principally in patients suffering from AIDS (Lima et al. 2016). But this fungal genus produces useful enzymes like α-amylase, lipase, polygalacturonase, and xylanase which could be used for the treatment of polymer waste or wastewater (Basak and Das 2014; Thirunavukarasu et al. 2016).

Microsporum gypseum causes skin diseases in humans and animals such as the disseminated and recalcitrant tinea corporis eruption, particulary in immunocompromised patients (Singh 2011). In animals, it produces circular and patchy alopecia, scales, follicular papules, erythema, hyperpigmentation, and pruritus. On the other hand, some studies concluded that the application of keratinolytic microbes, such as fungi (including M. gypseum), could be useful for the treatment of keratinaceous wastes (Ghaffar et al. 2018). Trichophyton spp. are the most common dermatophytic fungi and the causal agents of fungal nail infections (onychomycosis), a very common disorder in industrialized nations, and it represents a risk for diabetics or patients with keratinization problems (Nenoff et al. 2013). These fungi affect the stratum corneum of the epidermis and the nail keratin, but they are not common in the hair roots. Like M. gypseum, the keratinases produced by Trichophyton spp. may actively degrade the chicken feather waste (Anbu et al. 2008). Epidermophyton floccosum constitutes the causal agent of many skin diseases and may lead to recurrent outbreaks of dermatophytosis in hospitals or health institutions due to its persistence in the environment (Svetaz et al. 2010). Frequently, it causes tinea cruris, tinea pedis, tinea corporis, and onychomycosis (Lacaz et al. 2002).

Paracoccidioides brasiliensis is the etiological agent of the disease paracoccidioidomycosis (PCM), the most important systemic mycosis in Latin America (do Prado et al. 2018). The isolates of this fungus are highly virulent, since it is capable of adapting to transient iron availability as strategies to survive and overcome stress conditions inside the host (do Amaral et al. 2019). Aspergillus spp. are airborne pathogenic fungi which may cause skin and respiratory infections which could be lethal in immunocompromised patients (Davicino et al. 2007). However, these fungi are able to produce lipases with high industrial potential (Contesini et al. 2010). Many pharmaceutical or agrochemical products have been obtained from them as well as aromatic compounds. Their capacity for effluent biodegradation and bioadsorption of the toxic metals Cd and Cr is well known (Ahmad et al. 2005).

Fusarium spp. cause pernicious infections on humans and crops but also offer some industrial applications. They could cause some opportunistic infections in humans, mostly in immunocompromised patients, such as keratitis and onychomycosis (Salah et al. 2015). Moreover, Torres et al. (2009) suggested that fumonisins (a type of toxins produced by this fungal genus) increase the incidence of esophageal cancer. Particularly, F. verticillioides cause root and stem rots in cereals that are cultivated in subtropical and temperate regions, and their mycotoxins are harmful to human and animal health causing leucoencephalomalacia in equines, pulmonary edema in porks, and immunosuppression in poultry (Sampietro et al. 2014). F. oxysporum cause accelerated wilting in banana (Forsyth et al. 2006) and tomato (Duyvesteijn et al. 2005), since it can penetrate the roots and colonize vascular tissues leading to the disruption of water translocation towards the shoots. On the other hand, lipase enzymes obtained from F. oxysporum have shown several properties with significant industrial applications, such as an additive for detergents formulations (Prazeres et al. 2006). Rhizopus spp. are opportunist fungi which may cause different pathogenicity in humans (mucormycosis, an invasive fungal infection with high morbidity and mortality principally in immunocompromised patients) (Teal et al. 2016), animals, and vegetables (soft rot, a disease that produces great economic losses in fleshy fruits) (Pergomet et al. 2018). Species of this fungal genera are considered good bioremediators, as they can bioadsorb high concentrations of heavy metals such as Cd and Cr (Ahmad et al. 2005). Additionally, these fungi offer capacity to produce pectinolytic enzymes which have wide applications in fruit juice and wine industries (Anisa and Girish 2014). Mucor spp. principally cause chronic cutaneous and subcutaneous infections, but they can also produce rhinocerebral and sinopulmonary infections (frequently lethal), blood and intestinal infections, as well as septic arthritis (Morin-Sardin et al. 2017). Some Mucor species have been used for metabolites production or biotransformations, for biodiesel production and in the food industry as starters of cheese fermentations (Morin-Sardin et al. 2017).

During the last decades, most pathogens have developed resistance towards synthetic fungicides, and consequently, the search for new alternatives applied to the treatment of fungal diseases is urgently required (Di Liberto et al. 2017; Carrizo et al. 2020). Antifungals show different targets over the fungal cells and among them we could mention: cell wall (inhibition of β-glucan or chitin synthesis); cell membrane (binding to ergosterol or inhibition of its synthesis, e.g. phenols disrupt the cytoplasmic membrane and cause the cells leakage); inhibition of cell division (by cutting of microtubule polymerization); inhibition of RNA/DNA (by causing deficient RNA synthesis or inhibition of DNA transcription); and the inhibition of efflux pumps which function by transporting toxic substances out of the cell (inhibiting the efflux pumps, drug resistance may be reduced) (Alanís Garza 2005; Freiesleben and Jäger 2014).

Natural products are the best known reservoir of chemicals. Plants have developed different active principles for defense towards the fungal attack, and their content depends on different factors, mainly climatic conditions (Acosta de la luz 2003; Petenatti et al. 2008). The simple phenolic compounds, such as hydroxybenzoics, monophenols (e.g., cresol), diphenols (e.g., hydroquinones), and triphenols (e.g., gallic acid), are the more common secondary metabolites displaying fungicide properties (Lizcano González 2007; Martinez 2012). Moreover, compounds, such as phytoalexins, steroidal saponins, alkaloids, and some proteins, have also been depicted to have an important function in the defense systems of plants against pathogens (Montez-Belmont et al. 2000; Alanís Garza 2005; Lizcano González 2007). The secondary metabolites offer different actions against pathogens and they could be classified in three main groups according to their biosynthetic pathway (Lizcano González 2007; Freiesleben and Jäger 2014): (1) phenolic compounds such as coumarins and phytoalexins which are incorporated into the plant cell wall to increase rigidity and prevent the entry of pathogens; (2) terpenoids such as mono-, di-, or tri-terpenes, saponins, steroids, cardiac glycosides, and sterols which may act disrupting the cell membrane and directly eliminating the pathogenic organism in order to restrain its invasion towards the rest of the plant; (3) nitrogen-containing compounds such as alkaloids and lectins which also eliminate the pathogenic organism and restrain its invasion through the attack to the cell membrane and cell wall. Other compounds such as phytoalexins, substances with low molecular weight and whose biosynthesis is induced by pathogens or herbivores have demonstrated a biostatic or biocidal effect at relatively low concentrations (Reichling 2018).

2 Summary of Argentinean Antifungal Plants Evaluated against Human Pathogens

Secondary metabolites are mainly obtained by distillation or extraction of aromatic or non-aromatic plant species, respectively. Plant extracts constitute a group of substances, with diverse chemical characteristics, extracted from different parts of plants such as roots, barks, seeds, buds, leaves, and fruits (Martinez 2012). On the other hand, essential oils are highly volatile substances (lipophilic molecules) synthesized and stored in glandular trichomes, which are capable to disrupt the fungal cell wall or membrane through a permeabilization process and also may inhibit the synthesis of fungal DNA, RNA, proteins, and polysaccharides (Martinez 2012; Karpiński 2020). Many researchers all over the world have investigated the properties and capacity of some plants against fungal pathogens. Table 12.1 summarizes the native or naturalized plants of Argentina which showed antifungal activities against different fungal species throughout the last 20 years (2000 to 2020).

Table 12.1 Native or naturalized plants of Argentina with antifungal activities against human pathogenic fungi. Part used of each species evaluated and references are also depicted
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2.1 Zuccagnia punctata: A Treasure Used in Traditional Medicine: Validation of its Antifungal Properties and Isolation of Bioactive Compounds

Zuccagnia punctata Cav. (Fabaceae, Caesalpinioideae) is the only representative species of this genus that is endemic of central and western arid and semi-arid regions of Argentina (Fig. 12.1). It is an aromatic shrub of 1–5 m in height which grows on hills and plains between 700–2700 m.a.s.l. It is commonly known as “jarilla macho,” “jarilla de la puna,” “jarilla pispito,” “laca,” or “pus-pus” and has a certain resemblance to the true “jarillas,” a species of the genus Larrea (Zygophyllaceae), with which it coexists (Vattuone et al. 2013). Z. punctata has a long history of use in the traditional indigenous medicine of Argentina. Infusions and decoctions in water, as well as extracts prepared by maceration in ethanol of its aerial parts, have been widely used as foot antiseptic, rubefacient, antibacterial, antifungal, anti-inflammatory, antitumor, asthma, arthritis, rheumatism, among others (Ortega et al. 2000; Vattuone et al. 2013). To date, there have been numerous pharmacological studies that have shown important activities that support the traditional uses of this plant (Isla et al. 2016).

Fig. 12.1
figure 1

Zuccagnia punctata Cav. (Fabaceae, Caesalpinioideae). (a) specimens in their natural environment. (b) Aerial parts

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Regarding its chemical composition , 13 phenolic compounds were isolated from extracts of the aerial parts of Z. punctata: two chalcones, 2′,4′-dihydroxy-3′-methoxychalcone (DHMC) and 2′,4′-dihydroxychalcone (DHC); five flavones, 3,5,7-trihydroxyflavone (galangin), 3,5-dihydroxy-7-methoxy flavone (izalpinin), 3,5,4′-trihydroxy-7-methoxy flavone (rhamnocitrin), 3-hydroxy-7,8-dimethoxy flavone (3-H-7,8-DMF); and 3,7-dihydroxy flavone (3,7-DHF); four flavanones, 7-hydroxy flavanone (7-HF), 5,7-dihydroxy flavanone (pinocembrin), 5-hydroxy-7-methoxy flavanone (pinostrobin), and 7-hydroxy-8-methoxyflavanone (7-H-8-MF); and two caffeic acid derivatives, 1-methyl-3-(4′-hydroxyphenyl)-propyl caffeate [1-M-3-(4’-HP)-PC] and 1-methyl-3-(3′,4′-dihydroxyphenyl)-propyl caffeate [1-M-3-(3′,4’-DHP)-PC]. Chalcones being the main components of such extracts (Ortega et al. 2000; Nuño 2015). In the essential oil of the aerial parts of Z. punctata, 80 constituents were identified, mainly oxygenated monoterpenes. The main components were identified as linalool and (−)-5,6-dehydrocamphor (Álvarez et al. 2012).

Below we describe the results obtained from our recent studies on the antifungal activity of extracts, essential oil, and compounds obtained from Z. punctata against phytopathogenic and human pathogenic fungi.

The activity of the crude ethanolic extract of aerial parts of Z. punctata was evaluated by the agar dilution method (Zacchino et al. 1999), against strains of fungi isolated from soybean plants growing in the most important producing regions of Argentina that presented typical symptoms of disease (Phomopsis longicolla, Alternaria alternata, Sclerotium bataticola, Fusarium equiseti, F. graminearum, and Colletotrichum truncatum) (Svetaz et al. 2004). The ethanolic extract showed activity against all of the fungi tested with Minimal Inhibitory Concentration (MIC) values between 100 and 500 μg/mL. This extract was successively partitioned between n-hexane, chloroform, and n-butanol; the chloroform extract showed the lowest MIC value (62.5 μg/mL) against the most relevant pathogenic fungus on soybean seed (P. longicolla). Repeated bioassay-guided chromatographies of the chloroform extract led to the isolation of DHMC, DHC, 7-HF, 1-M-3-(4’-HP)-PC, and 1-M-3-(3′,4’-DHP)-PC. The chalcones DHMC and DHC showed potent antifungal activities against all fungi tested with MIC values ranging from 6.25 and 3.12 to 50 μg/mL, respectively. 7-HF and 1-M-3-(4’-HP)-PC showed very interesting activities only against P. longicolla (MIC = 6.25 μg/mL), while 1-M-3-(3′,4’-DHP)-PC did not show any activity up to 50 μg/mL. It is interesting to note that four of the five compounds isolated from the antifungal chloroform extract of Z. punctata displayed very good activities (MIC ≤6.25 μg/mL) against P. longicolla. Additionally, DHMC and DHC showed strong activities against C. truncatum (MIC = 6.25 μg/mL) (Svetaz et al. 2004). Both pathogens are the cause of the most serious soybean diseases due to their high incidence and persistence, causing reduction of seed quality and yields. P. longicolla is a primary agent of seed decay, a severe pathogen that affects soybean seed quality and C. truncatum is the causal agent of soybean anthracnose, a disease acquired mainly in the last growing step that affects stems and pods diminishing the number of seeds and their weight (Pioli et al. 2000).

Concerning to human pathogenic fungi, petroleum ether and dichloromethane extracts of fruits, aerial parts, and resinous exudate (obtained by dipping the fresh aerial parts in dichloromethane) of Z. punctata showed moderate antifungal activities against the yeasts C. albicans, S. cerevisiae, and C. neoformans (MICs: 62.5–250 μg/mL) and very strong antifungal activities against the dermatophytes M. gypseum, T. rubrum, and T. interdigitale (MICs: 8–16 μg/mL) thus supporting the ethnopharmacological use of this plant. Antifungal activity-directed fractionation of the most active extract led to the isolation of DHMC and DHC as the compounds responsible for the antifungal activity. DHCM displayed a selective and strong activity against dermatophytes (MIC = 8 μg/mL) and DHC showed a broader spectrum of activity inhibiting the yeasts C. albicans, S. cerevisiae, and C. neoformans (MICs: 16–31.2 μg/mL) and dermatophytes (MIC = 4 μg/mL). Second-order studies showed that DHC, in addition to being the most active chalcone, is fungicidal rather than fungistatic and that it would act through a different mechanism of action from that of antifungal drugs in current clinical use, so it appears to be a very promising antifungal agent (Svetaz et al. 2007).

The essential oil obtained by hydrodistillation from aerial parts of Z. punctata was evaluated against a panel of human opportunistic and pathogenic fungi using standardized procedures (CLSI 2017). The microbroth dilution method showed that the essential oil was not active against C. albicans, C. tropicalis, S. cerevisiae, or C. neoformans (MIC >1000 μg/mL) and possessed marginal activity against Aspergillus niger, A. flavus, and A. fumigatus (MIC = 1000 μg/mL). In contrast, it showed an interesting antifungal activity against dermatophytes (M. gypseum, T. rubrum, and T. interdigitale) with MIC values between 15.6 and 125 μg/mL, being T. rubrum the most susceptible species. A total of 80 constituents were identified in the oil, with linalool and (−)-5,6-dehydrocamphor being the main constituents. Both compounds were also tested against the same fungal strains. Linalool showed moderate activity against dermatophytes (MICs: 125–250 μg/mL) but was inactive against yeasts and Aspergillus spp. The other major component was inactive up to 250 μg/mL against all the fungi tested. The main components of the essential oil would not be responsible for its antifungal activity, but the identification of thymol and carvacrol as minor components suggests that they could contribute to this activity. The results of their antifungal evaluation undoubtedly suggest that both compounds could contribute to the activity of the essential oil since they were active against all of the evaluated fungal species (MICs: 15.6–250 μg/mL). Both compounds displayed interesting activities against dermatophytes (MICs: 15.6–31.2 μg/mL), while carvacrol also showed very good activity against A. flavus and A. fumigatus (MIC = 31.2 μg/mL) (Álvarez et al. 2012).

2.2 Phytolacca tetramera: Berries Extracts and Compounds with Potential Antifungal Activity

Phytolacca tetramera Hauman (Phytolaccaceae), commonly known as “ombusillo,” is an endemic species from Argentina (Fig. 12.2). It is currently considered one of the threatened plant species, being in critical danger of extinction (Delucchi et al. 2006), mainly due to anthopic causes that lead to the reduction of their habitat, such as human settlements, construction of roads, the periodic weeding of these roads, agricultural and livestock activity, industrial facilities, etc. (Hernandez et al. 2008).

Fig. 12.2
figure 2

Phytolacca tetramera Hauman (Phytolaccaceae)

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Our research group has studied this plant species for several years, and the information about the chemical composition, antifungal activity, and mechanism of action allowed us to know the importance of this plant for the treatment of fungal infections, promoting thus its conservation. From our studies, an area related to research and development for the conservation in situ of P. tetramera was created (Petri et al. 2010).

After Escalante et al. (2002) studies, the antifungal activity of methanolic, dichloromethanic, butanolic, and aqueous extracts of P. tetramera obtained not only from berries but also from leaves and roots (PtMEb, PtDEb, PtBEb, PtAqEb, PtMEl, PtDEl, PtBEl, PtAqEl, PtMEr, PtDEr, PtBEr, and PtAqEr) was assessed with the standardized CLSI (2017) microbroth dilution method against the yeasts C. albicans and C. glabrata (Butassi et al. 2019). The use of the most recent guidelines of CLSI (2017) for yeasts assured more reproducible and more reliable results. Table 12.2 shows the results corresponding to the re-evaluation of the antifungal activity of P. tetramera extracts.

Table 12.2 MICs (μg/ml) of the different extracts of P. tetramera against C. albicans (Ca) CCC 125–2000 and C. glabrata (Cg) CCC 115–2000 determined by the microbroth dilution method recommended by CLSI (2017). ITZ = itraconazole (used as standard drug)
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Results showed that PtMEb, PtDEb, and PtBEb were moderately active against C. albicans and C. glabrata, with PtDEb being the most active against the tested strains (MIC = 250 μg/ml), followed by PtMEb (MICs between 500 and 1000 μg/ml) and PtBEb (MIC = 1000 μg/ml). The rest of the extracts was inactive (MIC >1000 μg/ml) (Table 12.2).

By bioassay-guided fractionation (Escalante et al. 2002), three monodesmosidic triterpenoid saponins were isolated from PtBEb: phytolaccoside B (3-O-β-D-xylopiranosylphytolaccagenin) (PhytB), phytolaccoside E (3-O-β-D-glucopyranosyl-(1,4)-β-D-xylopiranosyl-phytolaccagenin) (PhytE), and phytolaccoside F [3-O-α-L-rhamnopyranosyl-(1,2)-β-D-glucopyranosyl-(1,2)-β-D-xylopyranosyl-phytolaccagenic acid] (PhytF) (Fig. 12.3). The three saponins belong to the olean-type triterpenoid saponins, possessing 28,30 dicarboxylic groups and an olefinic double bond on C-12. PhytB and PhytE, but not PhytF, showed antifungal activities against a panel of human pathogenic opportunistic fungi. PhytB was the most active compound and showed the broadest spectrum of action (MICs between 25 and 125 μg/ml) (Escalante et al. 2002).

Fig. 12.3
figure 3

Phytolaccoside B, phytolaccoside E and phytolaccoside F isolated from berries of P. tetramera butanolic extract

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The chemical composition of P. tetramera extracts obtained from berries, leaves, and roots was studied using Thin Layer Chromatography (TLC). These studies allowed the detection of PhytB, PhytE, and phytolaccagenin (PhytG) in all the extracts except in aqueous ones (Butassi et al. 2019) (Fig. 12.4).

Fig. 12.4
figure 4

Chemical profile of different extract types obtained from P. tetramera berries, using PhytB, PhytE, and PhytG as markers

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According to this chemical analysis, the antifungal activity of PhytB, PhytE, and PhytG was evaluated against C. albicans and C. glabrata. PhytB and PhytG were active against both pathogens with MIC = 62.5 μg/ml. In contrast, PhytE did not show activity (MIC >250 μg/ml) (Butassi et al. 2019). Based on these results, PhytB and PhytG were selected as active markers (EMA 2010) and were quantified in all the extracts using UHPLC-ESI-MS (Table 12.3) (Butassi et al. 2019). The most potent extract of P. tetramera (PtDEb) showed the highest amount of active markers, followed by PtMEb and PtBEb. The extracts from leaves and roots contained a low level of both PhytB and PhytG. Therefore, it could be stated that these two compounds contribute strongly to the antifungal activity of the active extracts.

Table 12.3 Content (mg of compound/g plant material) of active markers PhytB and PhytG in P. tetramera extracts analyzed by UHPLC-ESI-MS. Values are the mean ± standard deviation (n = 3). Nd: not detected
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Additionally, the mechanism of action of the active markers PhytB and Phyt G and the most active extract PtDEb was studied. For that, morphological studies (using scanning electron, phase contrast, and fluorescence microscopies) which target the fungal cell wall [cellular sorbitol assay and enzymatic (1,3)-β-D-glucan synthase (GS) and chitin synthase 1 (ChS) assays] and studies which target the fungal cell membrane (ergosterol-binding assay) were carried out. Table 12.4 summarizes the results obtained.

Table 12.4 Mechanism of action studies of the active markers PhytB and PhytG and the most active extract PtDEb
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2.3 The Genus Polygonum: An Update for its Antifungal Effects and Influence of Chemotaxonomy

Polygonum constitutes one of the plant genera most used by Argentinian traditional medicine to treat fungal conditions. This genus comprises about 250 species that have a wide geographical distribution, from Polar Regions to the tropics of all continents. Formerly, it was divided into five sections: Echinocaulon, Amblygonum, Persicaria, Tiniaria and Polygonum but nowadays, due to its botanical and phytochemical complexity, species are classified into two true genera: Polygonum and Persicaria. According to the plant list (www.plantlist.org) most of them have been regrouped with accepted names, but their synonyms are still used (Álvarez et al. 2020). In Argentina, about 20 species belonging to both genera grow throughout the country, but the most active in terms of their antifungal properties is Persicaria acuminata syn. Polygonum acuminatum and Persicaria maculosa syn. Polygonum persicaria (Fig. 12.5) (Derita and Zacchino 2011a).

Fig. 12.5
figure 5

Persicaria acuminata (a) and P. maculosa (b)

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From the bio-guided fractionation of P. acuminata aerial parts, five sesquiterpenes with drimane skeleton were isolated: drimenol, isopolygodial, confertifoline, polygodial, and 1-β- (p-methoxycinnamoyl) polygodial. Among them, the most active was polygodial, inhibiting the growth of C. albicans, C. neoformans, and the dermatophytes M. gypseum, T. rubrum, and T. mentagrophytes with MICs between 3.9 and 62.5 μg/mL and MFCs between 7.8 and 125 μg/mL, being fungicide as well as a strong inhibitor of fungal growth (Derita et al. 2013).

The bioguided fractionation of P. maculosa, in addition to the sesquiterpenes mentioned above, allowed the isolation of the flavanone pinostrobin and the chalcones flavokawin B and cardamonin. The latter compounds were active against C. albicans, C. neoformans, and the dermatophytes M. gypseum, T. rubrum, and T. mentagrophytes displaying MICs between 15.6 and 500 μg/mL. Unlike P. acuminata, the antifungal activity of P. maculosa was due not only to polygodial and isopolygodial but also to flavonoids, which added an interesting chemotaxonomical data (Derita and Zacchino 2011b).

Flavonoids have played an important role in the systematics of Polygonum species being proposed as chemotaxonomic markers of the genus. However, as stated before, the finding of polygodial within the Persicaria section promoted a sesquiterpene (not a flavonoid) to be proposed as a chemotaxonomic marker for the Persicaria section. Consequently, with the aim of finding compounds that could be suggested as new chemical markers for the delimitation of Persicaria section of the genus, a comparative study about the presence and quantification of sesquiterpenes and flavonoids in six species belonging to the section was carried out. It was found that P. hydropiperoides, P. lapathifolia, and P. ferruginea, which were the three species that did not contain polygodial, showed the presence of the three flavonoids. In turn, P. punctata and P. acuminata contained the four sesquiterpenes but no flavonoids. Surprisingly, P. persicaria was the only species belonging to this section that presented both types of compounds (Derita and Zacchino 2011b). All these findings contributed to the regrouping of these species into the current Polygonum and Persicaria genera (Álvarez et al. 2021).

3 Photoactivity: An Underexplored Property for Detection of Antifungal Plants

Antimicrobial photodynamic therapy (APDT) is increasingly being recognized as an alternative clinical treatment for fungal infections. Their advantages include a broad spectrum of activities (viruses, protozoa, Gram-positive and Gram-negative bacteria and fungi), the possibility of eliminating microorganisms independently of their antimicrobial resistance pattern, low probability of adverse side effects and low cost (Donnelly et al. 2008). In addition, APDT is highly selective (Dai et al. 2012), resistance has not been described (Wainwright et al. 2017), it has been implicated in changes in the expression of virulence determinants factors and showed efficacy against biofilms (Kato et al. 2013). PDT involves the administration of a non-toxic photosensitizer (PSs) and harmless visible light of the correct wavelength to generate reactive oxygen species (ROS), which can oxidatively damage surrounding biomolecules, such as lipids, proteins, and nucleic acids, thus killing pathogenic microorganisms (Liang et al. 2016; Pinto et al. 2018). ROS are produced by the reaction of the excited PSs and oxygen (O2) through type I reactions, that involve electron-transfer reactions that generate hydroxyl radical (OH⦁), hydrogen peroxide (H2O2), and superoxide radical (O2⦁−) and type II reactions that involve energy transference to produce singlet oxygen 1O2 (Maisch et al. 2007).

PSs are present in certain plants as a chemical defense mechanism developed to protect themselves from the action of microbes and herbivorous attack. This action is triggered when these secondary metabolites are excited to higher energy levels by absorbing solar or artificial radiation at a particular wavelength range (Mamone et al. 2014). Siewert and Stuppner (2019) described 10 classes of natural PSs: thiophenes, furanocoumarins, polyacetylenes, curcumins, xanthenoids, alkaloids, anthraquinones and perylenequinones, phenalenones, and porphyrins. Thiophenes is one of the larger groups with more than 150 natural biologically active compounds with absorbance maximum for photobiological effects between 314–350 nm (Postigo et al. 2017; Siewert and Stuppner 2019), present in plants of the family Asteraceae including the genus Porophyllum, Tagetes, and Flaveria widely distributed in Argentina (Downum and Towers 1983; Ibrahim et al. 2016). Porophyllum comprises 25 species, six of them inhabit the Argentinean central-western region, they are annual or perennial plants with secretory cavities in oil-bearing leaves and bracts which emanate a strong foul odor (Johnson 1964; Loockerman et al. 2003). Tagetes includes approximately 56 species that have been used as a source of essential oil for flavoring in the food industries, and their flowers, which are rich in orange–yellow carotenoids, are used as food coloring (Vasudevan et al. 1997). Flaveria comprises 21 species widely distributed in America, only F. bidentis and F. haumanii occur in Argentina (de los A. Páez et al. 2019).

Below we describe the photodynamic antifungal activity of different extracts obtained from six species belonging to the genus Porophyllum, Tagetes, and Flaveria which were evaluated against C. albicans. The experiments were carried on following the guidelines of CLSI (2017) document that assures confident and reproducible results. Microplates were submitted to irradiation (light) or kept in darkness. In light experiments, UVA irradiations were performed with a homemade UVA light array composed of a set of three lamps (Alic, Buenos Aires, Argentina), emitting at 315–400 nm (100 W). Microplates were aligned perpendicular to the samples that illuminate uniformly the entire area of the microplate, placed at a distance to 12 cm from the light source and irradiated for 60 min. In darkness experiments, assays were carried out in the same conditions but microplates were wrapped with aluminum foil to avoid exposure to light. Table 12.5 shows the Minimal Fungicide Concentrations (MFC expressed in μg/mL) against C. albicans under light/darkness conditions.

Table 12.5 Minimal Fungicide Concentrations (MFC expressed in μg/mL) against C. albicans (light/darkness)
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No significant differences were observed in the number of yeasts Colony Forming Units (CFU/mL) in the growth controls between darkness and light experiments, suggesting that the action of UVA irradiation did not reduce C. albicans viability. None of the evaluated extracts, at a concentration up to 1000 μg/mL, showed antifungal activity in experiments performed without irradiation. All hexane and DCM extracts were considered photoactive, because they showed antifungal activity against C. albicans only under UVA irradiation. Most ethyl acetate and methanolic extracts did not exhibit antifungal photosensitive activity at concentrations up to 1000 μg/mL. The observed higher activity of the apolar extracts, compared with the more polar extracts, could be attributed to the presence of apolar thiophenic compounds previously reported for the studies on related species (Gil et al. 2002; Postigo et al. 2017; Ibrahim et al. 2018; Giacone et al. 2020). The main photoactive components of these extracts were identified as 2,2′:5′,2″-terthienyl (α-T); 5-(3-buten-1-ynyl)-2,2′-bithiophene (BBT); 5-(4-acetoxy-1-butynyl)-2,2′-bithiophene (BBTOAc); 5-(4-hydroxy-1-butynyl)-2,2′-bithiophene (BBTOH); and 5-(3-penten-1-ynyl)-2,2′-bithiophene (PBT). When different parts of the plant were studied, the highest activity was obtained in root extracts, that is, the organ where thiophenes accumulate (Marotti et al. 2010).

4 Probing Synergistic Effects to Increase Antifungal Activity in Plants

Combination therapy has emerged as an effective strategy for antifungal treatment to fight against microbial resistance (Hemaiswarya et al. 2008). In fact, the high incidence rates of Candida infections are supposed to be closely related with their recalcitrant resistance to conventional antifungals and their capacity for biofilm formation (Zavrel and White 2015). Synergism, defined as a phenomenon in which the combined action of two agents is more effective than the action of a single agent, has been the main focus for combinatory therapy as it greatly reduces the effective dosages of them required to treat an infection (Yang et al. 2017). Combinations of drugs with different targets prevents the development of drug resistance, may improve the interaction with its target and can reduce toxicity, since lower concentrations of both agents can be used (Ayaz et al. 2019). Research of new antimicrobials boost the use of bioactive compounds and extracts from plants, either alone, combined, or together with antibiotics (Ríos and Recio 2005) that can potentiate the activity of antimicrobials by targeting different sites in the microbial cell (multi-target effect), by improving their solubility or bioavailability (pharmacokinetic or physicochemical effects) or by targeting the resistance mechanism (Wagner and Ulrich-Merzenich 2009).

In recent years, there has been an increased interest in using herbs along with conventional drugs rather than using them in place of drugs that raises concerns about studying herb–drug interactions (Spitzer et al. 2017). Here we detail the results of our research on the antifungal activity of plant extracts of the Argentine flora alone and in combination with currently used antifungal drugs (Cordisco et al. 2019). The antifungal activity of 253 plant extracts used in traditional Argentine medicine (obtained from 153 species of plants, belonging to 120 genera and 56 families) was evaluated alone and in combination with the antifungal drugs amphotericin B (AMB), fluconazole (FCZ), and itraconazole (ITZ), against C. albicans. MICs alone and in combination were determined with the broth microdilution technique following the guidelines of CLSI (2017) and using a modification of the High-Throughput Synergy Screening (HTSS) test, respectively (Zhang et al. 2007). Only 27 extracts tested alone showed activity (MICs between 31.25–1000 μg/mL), which represents 10.67% of the total evaluated. For this reason, we decided to re-explore all extracts through combination trials with antifungal drugs, hoping to detect those with enhanced activity and thus take full advantage of nature’s chemodiversity and find new structures with antifungal activity.

First, we analyzed the importance of ethnopharmacological uses in the selection of extracts by investigating whether the probability of detecting plant extracts which enhance the activity of antifungal compounds is higher when obtained from species with reports of ethnopharmacological uses related to fungal infections. To achieve this, the plant extracts were classified into three groups according to whether they have reports of ethnopharmacological uses related to fungal infections (Group I = 37 species), if they are not reputed as antifungal but belong to a genus which has species with ethnopharmacological use related to antifungal activity (Group II = 11 species) and without any ethnopharmacological use related to fungal infections (Group III = 46 species). The results obtained from the combinations between the commercial antifungals and the extracts of the different group of plants showed that 7/37 (18.92%), 1/11 (9.09%), and 1/46 (2.17%) plant species of Groups I–III, respectively, enhanced the activity of AMB (Fig. 12.6a); 7/37 (18.92%), 1/11 (9.09%), and 4/46 (8.69%) plant species of Groups I–III respectively, enhanced the activity of FCZ (Fig. 12.6b) and 10/37 (27.03%), 2/11 (18.18%), and 7/46 (15.22%) plant species of Groups I–III, respectively, enhanced the activity of ITZ (Fig. 12.6c). These results indicated that when extracts came from plants with ethnopharmacological use related to fungal pathologies, there were more possibilities of finding extracts that enhance the activity of commercial antifungal drugs, suggesting that the ethnopharmacological approach is useful in designing extract–antifungal combinations with enhanced activity. Regarding the antifungal drug, the activity of azoles, especially ITZ, has been improved to a greater extent with respect to AMB (Cordisco et al. 2019).

Fig. 12.6
figure 6

Number of species from Groups I, II, and III which showed no enhancement (NE) and enhancement (E) of the activity of (a) amphotericin B; (b) fluconazole and (c) itraconazole

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Additionally, we evaluated whether the probability of detecting plant extracts which enhance the activity of antifungal compounds is greater when they have antifungal activities alone (MIC ≤1000 μg/mL) than when they do not possess it (MIC >1000 μg/mL). Our results indicated that there is a greater probability of finding an enhancement in the activity of commercial drugs when the combination is performed with extracts that have shown activity alone as compared to those previously inactive extracts. It is important to note that 42 extracts behaved as enhancers in combination with at least one of the antifungal agents evaluated and that among these, a total of 27 extracts had not shown activity alone. These extracts would have been considered inactive and discarded for further studies according to the classic strategy, however, by using this new paradigm, they remain potential candidates in the search for new antifungals (Cordisco et al. 2019).

5 An Extension of Plant Antifungal Properties toward the Control of Phytopathogenic Fungi on Fruits

Phytopathogenic fungi cause pre- and post-harvest diseases in vegetable, cereal, and fruit crops and are responsible for considerable world agriculture economic losses. The most common species of phytopathogenic fungi that cause the deterioration of fruits, leaves, stems, and ground organs (roots, tubers, corms, etc.) belong to the genera Alternaria, Botrytis, Diplodia, Monilinia, Penicillium, Colletotrichum, Phomopsis, Fusarium, Rhizopus, and Mucor (Juárez-Becerra et al. 2010).

Plant extracts and their isolated active compounds have been tested for their efficacy in the control of a wide range of phytopathogenic fungi (Jiménez-Reyes et al. 2019). In this sense, an in vitro study revealed the antifungal activity of 18 Argentinean plants species against four phytopathogenic fungi that greatly affect the post-harvest stage of commercially important fruits, including Penicillium digitatum, Botrytis cinerea, Monilinia fructicola, and Rhizopus stolonifer. All the species studied were at least active against one fungus of the panel, while three of them (Solidago chilensis Meyen, Drimys winteri J.R.Forst, and Polygonum stelligerum Cham.) displayed high antifungal properties inhibiting the growth of the selected pathogens. The antifungal activity of these plants was attributed to the presence of solidagenone in S. chilensis, polygodial in D. winteri and pinostrobin, and flavokawin B in P. stelligerum (Di Liberto et al. 2019). All these compounds have been identified as antimicrobial compounds (Muñoz-Concha et al. 2007; Derita and Zacchino 2011a; Ramirez et al. 2013; Carrasco et al. 2017) with the exception of solidagenone, which has been recently evaluated for its antiproliferative potential (Gomes et al. 2018). In a similar study, 17 Chinese medicinal plants were determined against eight species of plant pathogenic fungi, including Rhizoctonia cerealis, F. graminearum, Gaeumannomyces graminis, F. oxysporum, Valsa mali, Colletotrichum gloeosporioides, F. oxysporum sp. Cucumebrium, and Colletotrichum lagenarium. The results showed that the ethanol extracts of Syzygium aromaticum (L.) Merr. Et Perry (Myrtaceae) has the highest antifungal effect over the tested pathogens, isolating 2-methoxy-4-(2-propenyl) phenol (eugenol) as the active compound (Yang et al. 2019). Moreover, the antifungal activity of hexane, dichloromethane, and methanol extracts of 45 Thai plants were in vitro screened against plant phytopathogenic fungi (Alternaria porri, C. gloeosporioides, F. oxysporum, and Phytophthora parasitica). Seven extracts strongly inhibited the mycelial growth of the fungi. The plant extract with highest antifungal activity was Melodorum fruticosum Lour. (Annonaceae). Two of the eight isolated compounds (benzoic acid and melodorinol) exhibited strong activity against mycelial growth of P. parasitica (Mongkol et al. 2016). Extracts from the chilean plants Ephedra breana Phil. (Ephedraceae) and Nolana sedifolia Poepp. (Solanaceae) have revealed antifungal activity against B. cinerea (Vio-Michaelis et al. 2012). The bioactive compounds of these plants were veratric, p-hidroxybenzoic, and caffeic acids in E. breana, and p-coumaric and ferulic acids in N. sedifolia. All these compounds have been identified as antimicrobials (Fu et al. 2010). These examples, regarding the antifungal potential of plant products, are listed in Table 12.6.

Table 12.6 Natural products isolated from plants which proved to be potent against different phytopathogenic fungi
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On the other hand, essential oils have gained popularity in the agricultural sector due to their great antifungal activity and mycotoxin inhibition. They are ecological, biodegradable, and safe to human health (Dwivedy et al. 2016; Stegmayer et al. 2020). Their volatility makes them suitable as fumigants in protected environments and for post-harvest diseases of horticultural crops (Shukla 2018). The antimicrobial activity of essential oils is attributed mainly due to the bioactivity of the major compound or the overall synergistic effect of all major and minor compounds (Mishra et al. 2013). Although their modes of action are still unclear, they are known to decline the biosynthesis of ergosterol and disrupt the cell membrane.

Essential oils from Illicium verum Hook.f (Schisandraceae) and its main component trans-anethole showed potent antifungal activity against several phytopathogenic fungi and could be developed as natural fungicides for disease control in fruit and vegetable preservation (Huang et al. 2010). Essential oils from the Lamiaceae plants peppermint (Mentha piperita L.) and sweet basil (Ocimum basilicum L.) proved to be effective fumigants against the two post-harvest phytopathogens M. fructicola and R. stolonifer in stored peach (Prunus persica) (Ziedan and Farrag 2008). A number of essential oils based on food preservatives are commercially used and listed in “Generally Recognized as Safe” category by the Food and Drug Administration (FDA) and Environment Protection Agency (EPA) in the United States (Burt 2004), but only a few commercial biopesticides containing essential oils or artificial mixtures of terpene constituents are available. Cinnamite and Valero (from Mycotech Corporation) are commercialized as aphicide/fungicide being based on cinnamaldehyde and cinnamon oil, respectively. SPoran (a fungicide based on rosemary oil), from EcoSMART Technologies, is another example of biopesticide commercially available.

6 Conclusions

Throughout this chapter, we highlighted the most relevant results obtained during the last 20 years in our laboratory, which is internationally recognized for its quality on antifungal studies. The main human and plant fungal pathogens as well as the properties of fungi that are exploited in different industries were deeply discussed. An extensive list of Argentine plants belonging to different botanic families which have been described in the literature as antifungals was exhaustively detailed, taking into account its parts used, the fungi type against they were active, and the research group that generated the information.

The results obtained up to date give support to the ethnopharmacological use of Z. punctata, P. acuminata, P. maculosa, and P. tetramera as the most important species used as antiseptic and antifungal in the traditional medicine of Argentina, indicating the potential of their extracts, essential oils, and metabolites isolated from them for the control of a wide range of fungi affecting both crops and humans.

In view of the lack of new classes of drugs or different molecular targets against the most threatening yeast C. albicans, photodynamic therapy emerged as an alternative approach to treat fungal infections. Plants containing phototoxic compounds were discovered in various botanic families, and several researchers have demonstrated that herbal extracts could be used in antimicrobial treatments, including antifungal. Aligned with this, the re-exploration of inactive extracts, by combining them with commercial antifungal agents, allowed to obtain synergistic mixtures, offering new possibilities for antifungal formulations. A greater probability of finding an enhancement in the activity of commercial drugs was observed when the combination was performed with extracts that had shown activity alone compared to the previously inactive extracts. Notably, 42 extracts behaved as enhancers in combination with at least one of the antifungal agents evaluated and 27 of them had not shown activity alone. These extracts would have been considered inactive and discarded for further studies according to the classic strategy, however, by using this new paradigm, they remain potential candidate in the search for new antifungals.