The activity of propolis against pathogenic fungi isolated from human infections

Abstract Propolis is a resinous hive product collected by bees from the buds or other parts of plants. It is known for having various biological properties, including antifungal activity. Among the substances present in propolis, flavonoids and phenolic acids and their esters are responsible for its antifungal properties. This means that propolis is ideal for use as an antifungal agent in alternative medicine to treat a number of both topical and systemic infections caused by Candida species and other yeast-like fungi, dermatophyte and nondermatophyte moulds, without the serious side effects typical of synthetic treatment. It is also active against strains of fungi that are resistant to polyenes and azoles, the classes of drugs most commonly used to treat fungal infections. In this article, we review current knowledge about the activity of propolis from different parts of the world and its components in vitro and in vivo against pathogenic fungi isolated from human infections. The article also indicates the possible mechanism of antifungal activity of propolis and its components.


INTRODUCTION
Propolis is a resinous mixture produced by bees (Apis mellifera) from secretions collected from various parts of a variety of plants.The name comes from the Greek-pro-meaning in front, and -polis-meaning town or city.Bees use propolis to construct, repair and protect their hives, mainly owing to its mechanical and antimicrobial activity and antiseptic efficacy (Bankova, de Castro, Marcucci, 2000).
The available means of treating fungal infections are limited to polyene antifungals, such as nystatin and amphotericin B and azole antifungals e.g.miconazole, ketoconazole, fluconazole, itraconazole and allylamine derivative i.e. terbinafine (Ghannoum, Rice, 1999;Dalben-Dota et al., 2011).Most of these compounds target the formation and/or function of ergosterol, a basic component of the fungal cell membrane.Conventional antifungal therapy with polyene and azole compounds, however, can produce side effects in patients.Moreover, treatment with existing drugs is known to be toxic and to contribute to the development of drug-resistant strains of fungi, especially Page 2/11 Braz.J. Pharm.Sci.2023;59: e19978 Katarzyna Wolska, Katarzyna Antosik Cryptococcus neoformans and C. albicans (Ghannoum, Rice, 1999).
These facts have driven the search for new antifungal agents from other sources, including natural compounds (Oliveira et al., 2006).Propolis seems to be an excellent solution to these problems.It is a complex mixture of compounds with only low toxicity compared to synthetic substances.More than 300 different compounds have been identified in propolis, including phenols, tannins, polysaccharides, terpenes, aliphatic acids, esters, aromatic acids, fatty acids, aldehydes, amino acids, ketones, chalcones, dihydrochalcones, vitamins, and inorganic substances.(Bankova et al., 1999;Bankova, de Castro, Marcucci, 2000).Propolis has been used as a monotherapy or in association with other pharmaceutical products, having demonstrated marked activity against pathogenic fungi (Burdock, 1998;Castaldo, Capasso, 2002).Studies on the simultaneous use of conventional antimycotic drugs and propolis have shown that their use in combination enhanced the inhibitory effect on C. albicans (Holderna, Kędzia, 1987;Gucwa et al., 2018).
This effect in vitro has been assessed using a variety of microbial tests, including dilution in tubes or plates, agar diffusion in plates, and bioautography (Sawaya et al., 2002).The plate dilution method is the most satisfactory of these tests.There are three reasons for this: it evaluates the inhibitory/fungicidal effect of propolis extracts against the fungal strains tested; its results are sensitive to differences in composition between extracts which result in different MIC (minimal inhibitory concentration)/ MFC (minimal fungicidal concentration) values; and finally, the low hydro-solubility of the active substances in propolis does not interfere with the test.The second commonly used test is agar diffusion in plates.However, the results of the agar diffusion tests are less satisfactory due to the low hydro-solubility of the active substances of propolis, which are therefore poorly diffused in agar.In consequence, the growth inhibition zones are small (Sawaya et al., 2002).
Variation in the activity of propolis also depends on the types of ethanolic or aqueous extracts, types of microbes and inoculum concentration, as well as the propolis concentration in the medium (Yusoff et al., 2016).Most biological properties of propolis, including its antimicrobial activity, are observed in alcoholic extracts, because this results in extraction of larger quantities of active compounds and thus a stronger inhibitory effect against microorganisms (Mello, Petrus, Hubinger, 2010).
Table II illustrates the antifungal effect of propolis against pathogenic fungi.Included in this review article were the following: C. albicans and other Candida species, responsible for topical infections (e.g.oral infections, skin infections or vaginitis), and systemic infections (e.g.respiratory tract infections); yeast-like fungi: C. neoformans, a pathogen with a polysaccharide capsule and responsible for meningitis and pneumonia; as well as dermatophyte moulds, i.e. species of the genus Trichophyton and Epidermophyton, which cause skin, hair and nail infections; and nondermatophyte moulds, e.g.Aspergillus species causing bronchopulmonary aspergillosis.

TABLE I -
The most popular propolis types according to their plant origin and their chemical composition (Hegazi, El Hadyb, Alla, 2000;Bankova et al., 1999;Sforcin, Bankova 2011 Sforcin et al. (2001) studied the in vitro antimicrobial activity of Brazilian green propolis from the Southeast of the country in all collected four seasons against yeast pathogens isolated from human infections.They concluded that C. tropicalis and C. albicans were susceptible to low concentrations of propolis, but the latter showed greater susceptibility (with values of 3.22-4.22%(v/v) for C. tropicalis and 2.32-3.33%(v/v) for C. albicans.No differences were seen in relation to seasonal effects in the minimal inhibitory concentration of propolis.These results were in agreement with a study by Ota et al. (2001), in which Candida isolates from the saliva of patients with dentures were found to be susceptible to an alcoholic solution of Brazilian green propolis.C. albicans was the most susceptible, followed by C. tropicalis, C. krusei, and C. guilliermondii.Moreover, they reported the fungicidal activity of EEP at concentrations of 1 -12 mg/ml against all Candida species tested.The same authors, in an in vivo study, demonstrated a reduction in Candida in patients with full dentures who had used a hydroalcoholic propolis as a mouth-rinse, whereas no difference in the yeast count was noted in controls.Dias et al. (2009)  The ethanolic extract of Brazilian green propolis (20%) was found to reduce oral candidiasis in twelve denture-wearing patients with Candida-associated denture stomatitis (Santos et al., 2005).In this study, patients treated with a commercial ethanol propolis extract showed lesion regression similar to that observed in patients treated with nystatin.Therefore the 20% EEP used in this research can be effective in treating oral Candida-associated denture stomatitis.Other in vivo studies have confirmed that patients with Candida-associated denture stomatitis who received propolis in the form of a mouthwash showed a statistical reduction or complete clinical remission of symptoms such as palatal oedema and erythema, and a decrease or elimination of the yeast count after treatment.The authors concluded that Brazilian green propolis (2.5% of extract) has a similar effect to miconazole in the treatment of Candidaassociated denture stomatitis as an alternative treatment for this condition (Santos et al., 2008;Capistrano et al., 2013).According to Koo et al. (2000), the extract of propolis (10%) may be effective in treating periodontal disease owing to its antifungal effect on species such as C. albicans.Moreover, it was shown to inhibit biofilm formation in vitro.
The antifungal effect of an EEP of green Brazilian propolis from south-eastern Brazil corresponds with the results of other research in which red Brazilian propolis from the north-east was tested.The ethanolic extract of Brazilian red propolis showed significant results for inhibitory activity for C. krusei; 50 mg/ml was the concentration which was in the greatest inhibitory zone -12.4 mm.In this research, observed as chemical constituents of red propolis were red flavanones, xanthones and chalcones aurones, catechins, and leucoanthocyanidins (Silva et al., 2019).Siqueira et al. (2015) reported that an ethanolic extract of Brazilian red propolis exhibited higher activity than chlorhexidine against Candida species isolated from chronic periodontitis cases, where fluconazole was used as a control.All Candida species were susceptible to propolis and chlorhexidine, while five samples of C. albicans, C. tropicalis and C. glabrata were resistant to the antifungal activity of fluconazole.Propolis was found to exhibit fungistatic activity against C. tropicalis and C. albicans at 32-64 μg/ml and against C. glabrata at 64 μg/ ml.Fungicidal activity was observed at 64-256 μg/ml for C. tropicalis, 64-512 μg/ml for C. albicans, and 64 μg/ml for C. glabrata.
The antifungal activity of the Brazilian red and green propolis ethanolic extracts has also shown high efficacy against dermatophytes such as Trichophyton rubrum, T. tonsurans and T. mentagrophytes.The green propolis showed fungistatic activity against T. rubrum at 64 -512 μg/ml and against T. tonsurans and T. mentagrophytes at 128 -1024 μg/ml.Fungicidal activity of green propolis was observed at an MFC of 1024 μg/ml in the case of T. rubrum and T. tonsurans and at 512 μg/ml for T. mentagrophytes.Red propolis also exhibited fungistatic activity against T. rubrum at 8 -128 μg/ml, against T. mentagrophytes at 16 -128 μg/ml, and against T. tonsurans at 32 -128 μg/ml.The red propolis extract exerted a fungicidal effect on these species at concentrations ranging from 128 to 256 μg/ml, 256 to 512 μg/ml and 128 to 1024 μg/ml, respectively, for the same species.In vivo tests were performed as well and showed that propolis treatment was more effective than classical therapy with terbinafine and itraconazole (Siqueira et al., 2009).The results are consistent with findings by Soares and Cury (2001).They studied the in vitro activity of Brazilian propolis alcoholic extract against dermatophytes isolated from patients with tinea pedis.The minimum inhibitory concentration of the extract ranged from about 8 μg/ml to > 2000 μg/ml.The MIC was 125 μg/ml in the case of most T. rubrum strains (about 55%), up to 250 μg/ml for about 70% of T. mentagrophytes strains, and > 2000 μg/ml for only one strain.The propolis extract inhibited two strains of Epidrmophyton floccosum at 125 μg/ml and the other two at 250 μg/ml.The MFCs of this agent ranged from 1000 μg/ml to more than 2000 μg/ml for the three fungal species.
The results of Veiga et al. (2018) showed that 30% ethanolic extract of green Brazilian propolis was efficient in vitro against both the planktonic cells and the biofilm formed by Trichophyton spp., which is the most common agent of onychomycosis and is usually resistant to conventional antifungals.The results in vivo showed that EEP was able to penetrate the human nail and to treat onychomycosis.The majority of the isolates showed MIC50 and MFC50 below the concentration of 0.088% total phenol content in propolis extract.Another study (Oliveira et al., 2006) showed high level of activity of ethanolic extract of green Brazilian propolis obtained from eucalyptus against Trichosporon, C. albicans, and C. non-albicans isolated from onychomycosis patients.All of the yeasts (35% C. parapsilosis, 23% C. tropicalis, 13% C. albicans, and 29% other species) were inhibited by a concentration of 500 μg/ml of flavonoids and 200 μg/ml of flavonoids stimulated their cellular death.Trichosporon spp.were the most sensitive species, showing MIC50 and MIC90 of 125 μg/ml of flavonoids, and C. tropicalis was the most resistant, with MIC50 of 500 μg/ml of flavonoids and MIC90 of 1000 μg/ml.

Antifungal activity of European propolis and its components
The ethanolic extracts of propolis from different regions of Romania used in the study of Stan et al. (2017) exhibited antifungal (growth inhibition zones with diameters between 6 and 20 mm) and antibiofilm activity (the inhibition of adhesion on the inert substratum at minimum biofilm eradication concentration values between 0.23 and 15 mg/ml) against C. albicans strains isolated from tracheal secretions in hospitalized patients with respiratory tract associated infections.The ethanolic extracts of Polish propolis also showed activity with MFC in the range of 0.08-1.25%(v/v) on clinical isolates of C. albicans strains (Gucwa et al., 2018).In this study, a synergistic effect was observed for the action of propolis and azole antifungals (fluconazole and voriconazole) against C. albicans.These results were in agreement with a study of Sariguzel et al. (2016).They demonstrated that Turkish propolis showed significant in vitro antifungal activity, which was comparable with fluconazole and itraconazole against yeast isolates from blood cultures.The propolis MIC range of eight C. albicans strains was found as 0.006 to 0.5 μg/ml.Similarly, D' Auria et al. (2003) demonstrated that ethanolic extract of Italian propolis significantly inhibited the C. albicans strains tested, showing rapid (between 30 seconds and 15 minutes), dose-dependent cytocidal activity and an inhibitory effect at a concentration of about 0.20 mg/ml.German propolis (special extract GH 2002) concentrations between 0.6 and 2.4 mg/ml displayed fungicidal activity against different clinical isolates of Candida (Astani et al., 2013).
Petroleum ether extract of Slovak and Czech propolis has exhibited excellent inhibitory effects against clinical fungal strains of C. albicans and T. mentagrophytes (MIC 8 -64 μg/ml).These extracts had the least effect on nonalbicans species of Candida (C.krusei, C. tropicalis and C. glabrata) and on T. asahii (MIC 64 ->128 μg/ ml).This study showed lower antifungal potency for the ethanolic extract, but it was relatively effective against two C. albicans and T. mentagrophytes strains (MIC ≤ 64 μg/ml) (Buchta, Černý, Opletalová, 2011).Antifungal activity was not fully correlated with the content of flavonoids in the extracts.These findings indicate that it was not flavonoids alone but also other components of the mixture and/or their proportions in it that resulted in its antifungal activity.The correlation between the total phenolic acids and flavonoids content and antifungal activity was reported for propolis from Croatia.Especially, p-coumaric acid, apigenin, and kaempferol were significantly correlated with the activity of propolis against C. albicans (Tlak Gajger et al., 2017).

Antifungal activity of Asian propolis and its components
Propolis from Saffareh in Lebanon showed antimicrobial activity towards C. albicans with average inhibition zone diameters of 25 mm, MIC of 12.5 mg/ ml and MFC of 25 mg/ml (Chamandi, Olama, Holail, 2015).Qualitative analysis of this propolis showed that it contained alkaloids, flavonoids, phenols, saponins, steroids, tannins, and terepenoids.In another study, the alcoholic extract of Iranian propolis at the concentration of 250 mg/ml showed an inhibitory and cidal effect on more than 50% of clinical Candida and Aspergillus isolates (Diba, Mahmoudi, Hashemi, 2018).Chee (2002) assessed the antifungal effect of propolis from Korea (EEP) on two clinically important fungi, C. albicans and C. neoformans.In a microbroth culture assay, the MICs for C. albicans and C. neoformans were 16 and 2 mg/ml, respectively.Propolis showed fungicidal activity against C. neoformans (MFC=8 mg/ml), but only fungistatic activity against C. albicans.Moreover, under a scanning electron microscope, rupture of C. neoformans cells could be observed following treatment with propolis.Studies by other authors (Roh, Kim, 2018) also showed significant antifungal activity of ethanolic extract of Korean propolis (10 mg/ml) on oral pathogenic C. albicans strains.
Similarly, propolis (EEP) from Malaysia, produced by stingless bees of the species Trigona thoracica, exhibited high antifungal properties against C. albicans (ATCC 25987) and C. neoformans (a local clinical isolate), which was explained by its high content of phenolic acids (about 1754 mg gallic acid/kg) and flavonoids (about 83 mg quercetin/kg).The visually assessed MIC of propolis was 1.56 mg/ml against both C. albicans and C. neoformans, while the MICs determined by spectrophotometry were 3.13 mg/ml and 1.56 mg/ml, respectively.The MFCs of propolis were 3.13 mg/ml for C. neoformans and 6.25 mg/ ml against C. albicans (Shehu et al., 2006).Antimicrobial activity of propolis from Yemen against upper respiratory tract infections has been reported by El-Shouny et al. (2012).Throat swabs were collected from 17 children up to 11 years of age and six tested positive for C. albicans (35.3 %) with the most isolates found in children ≤ 3 years old.Nystatin (50 μg) showed antifungal activity against C. albicans isolates.Propolis used at a concentration of 200 mg/ml inhibited growth of C. albicans, resulting in 19 mm zones of inhibition.A mixture of propolis with goat milk enhanced the antifungal effect in vivo; full remission of Candida symptoms was attained in all children in less time (2 to 5 days) than in the case of either agent applied separately.
Based on the available literature the antifungal activity of propolis is weaker in aqueous extracts than alcoholic extracts.Both Malaysian propolis (water extract) produced by Apis mellifera and propolis produced by Trigona spp.have shown weak activity against oral fungi, especially Candida spp.For the MIC value of the propolis extracts, both Apis mellifera and Trigona spp.propolis have shown an inhibitory effect at 500 mg/ml.Neither propolis showed activity against Candida spp., based on the absence of inhibition zones (Yusoff et al., 2016).Similarly, aqueous extracts of Brazilian propolis have demonstrated little or no effectiveness in inhibiting the growth of Candida albicans at 5.0±0.00mm (Dias et al., 2009).

THE MECHANISM OF ANTIFUNGAL ACTION OF PROPOLIS AND ITS COMPONENTS
Various compounds present in propolis, such as phenolics and flavonoids, are responsible for their antifungal activity.There are reports indicating that phenolic acids can increase cell membrane permeability, resulting in the loss of cellular proteins and nucleic acids, as well as inorganic ions such as potassium and phosphate, thereby causing the death of the cell (Farnesi et al., 2009).The antifungal activity of flavonoids and other alpha-and beta-unsaturated oxo-compounds is most likely due to a vinylene double bond reacting with sulfanyl groups in enzymes, thereby impeding synthesis of the cell wall of the fungus (López et al., 2001).It has been shown that chemical components of propolis may harbour dose-dependent cytocidal activity and an inhibitory effect on yeast-mycelial conversion, and that they may inhibit extracellular phospholipase activity and fungal adhesion to epithelial cells (D'Auria et al., 2003).Mello et al. (2006) suggested that Brazilian propolis antifungal activity is based on inducing changes in the cell wall (alteration of cellular permeability) that have as consequence an increasing volume of the cell and cellular membrane rupture.The inhibition of fungal growth and germination tube formation of C. albicans could be attributed to the interaction of propolis with proteins sulf hydryl groups.The antifungal activity of pinocembrin, an important flavonoid isolated from propolis against Penicillium italicum was investigated by Peng et al. (2012).Pinocembrin inhibited the mycelial growth of P. italicum by interfering energy homeostasis and cell membrane damage of the pathogen.Takaisi-Kikuni and Schilcher (1994) have investigated another potential mechanism of the antifungal and antibacterial action of propolis.They noted that cell division was inhibited in the presence of propolis, which indicated that the action of propolis could involve inhibition of DNA replication and, consequently, of cell division.

CONCLUSION
Propolis from Brazil, Europe and Asia is effective against pathogenic fungi, Candida species and other yeast-like fungi, dermatophyte and nondermatophyte moulds.Its antifungal properties are a resultant of the action of phenolic acids and their esters, and flavonoids.However, propolis from different geographic and climatic zones and the plant sources has a high variation in both the qualitative and quantitative chemical composition.This can be seen in this paper.Therefore, it is more reliable to compare the results of studies relating to one type of propolis.In summary, due to its common antimicrobial properties, including antifungal, and due to the fact that propolis is a low-toxic product compared to many synthetic drugs, it can be used in conventional medicine.But for this happen, propolis needs chemical standarisation that guarantees its quality, safety, and efficacy.

TABLE II -
The antifungal effect of propolis against pathogenic fungi

TABLE II -
The antifungal effect of propolis against pathogenic fungi

Antifungal activity of Brazilian propolis and its components
demonstrated the activity of an ethanolic extract of Brazilian propolis (10%) against Candida spp.using agar diffusion tests.The results of this study showed that strains of the species C. albicans, C. tropicalis and C. krusei were the most susceptible, while C. parapsilosis, C. glabrata and C. guillermondii were the least susceptible (the growth inhibition zone of C. tropicalis was 17.3 mm; C. albicans -16.9 mm; C. krusei -16.2 mm; C. guillermondii -13.5 mm; C. glabrata -13.28 mm; C. parapsilosis -12.3; control with ethanol -7 -9 mm).

TABLE II -
The antifungal effect of propolis against pathogenic fungi