Antifungal Properties of Chemically Defined Propolis from Various Geographical Regions

Long-term fungal infections that are difficult to treat require new substances for their prevention, treatment, or as adjuvants during antibiotic therapy. Propolis is a very promising source of natural substances that show a wide range of pharmacological properties, including antifungal activity against various fungal strains. The purpose of the literature review was to summarize recent studies (PubMed, Scopus) on progress in evaluating the antifungal activity of chemically defined propolis extracts. During the selection of studies, only those with results of antifungal activity expressed as minimal inhibitory concentration (MIC) and/or minimal fungicidal concentration (MFC) were analyzed. Moreover, plant, animal and environmental factors influencing the chemical composition of propolis are discussed. Mechanisms of antifungal activity of propolis extracts and research trends in the aspect of developing new therapies and the assessment of drug interactions are indicated. The review of the research results shows that there is great progress in the definition of propolis extracts. After comparing the MIC/MFC values, it was assessed that propolis extracts offer a wide range of activity not only against pathogenic Candida strains but also against risky molds; however, the strength of this activity is varied.


Introduction
Propolis (common names: bee glue, hive dross) is defined as a natural resinous mixture which is produced by honey bees (Apis mellifera) using secretions collected from trees and herbaceous plants or resins, mucilage, gums from the flowers, fruits, branches, stem, and leaves of different plants [1][2][3][4]. Moreover, in detail, propolis is composed of the following sources: (1) resins from plants which are collected by Apis mellifera, (2) wax from the metabolism of bees, (3) substances added by Apis mellifera when making propolis [1], and (4) other components such as essential oils and pollen [2]. Nainu et al. [5] described a more detailed main composition of raw propolis with the percentage range of components: resins and balms (from 50% to 60%), waxes and fatty acids (from 30% to 40%), essential oils (5-10%), and other chemical compounds (to 5%), mainly enzymes (i.e., glucose-6phosphatase; adenosine triphosphatase; acid phosphatase), vitamins and minerals.
Propolis has been known and used as a medicinal product since the ancient Greeks, Romans and Egyptians until the present day [6]. The complex composition of propolis makes that natural product exert a broad spectrum of pharmacological activities. One of the scientific problems that has received a lot of attention is the origin of propolis and the the scientific problems that has received a lot of attention is the origin of propolis influence of geographic and environmental factors on its chemical composition [1 also on pharmacological activities [8][9][10].
Apart from other bee products, such as honey, royal jelly and bee venom, pr also a significant source of natural chemical compounds [11,12]. Currently, n studies have been carried out on propolis in various fields. With the advance research methods and scientific curiosity, the number of results about propolis to increase. This continuing trend is consistent with the need to use products o origin, not only in treatment but also in the prevention of numerous diseases.

Plant, Animal and Environmental Factors Influencing the Chemical Compos Propolis
It is well known that many factors may influence the chemical compo propolis such as plant sources, geographical region and climatic conditions, production of propolis and type of Apis mellifera [1,5,7,43].
The most popular plant species as a source of biomaterial for the produ propolis include: Acacia spp., Aesculus hippocastanum, Betula pendula, Fagus sp., Frax Pinus spp., Prunus spp., Quercus sp., Salix alba [1]. In Poland, very important plan are Alnus spp. and Populus spp. [10]. Plant materials obtained from mentioned sp chemically different because these plants contain various primary and se metabolites classified mainly to phenolic compounds (flavonoids, phenoli terpenoids; Figures 4 and 5) and essential oils [1]. In addition to this, the situation complicated because the chemical composition of finished products obtain

Plant, Animal and Environmental Factors Influencing the Chemical C Propolis
It is well known that many factors may influence the chemical c propolis such as plant sources, geographical region and climatic cond production of propolis and type of Apis mellifera [1,5,7,43].
The most popular plant species as a source of biomaterial for the propolis include: Acacia spp., Aesculus hippocastanum, Betula pendula, Fagus sp Pinus spp., Prunus spp., Quercus sp., Salix alba [1]. In Poland, very importan are Alnus spp. and Populus spp. [10]. Plant materials obtained from mention chemically different because these plants contain various primary a metabolites classified mainly to phenolic compounds (flavonoids, p

Plant, Animal and Environmental Factors Influencing the Chemical Composition of Propolis
It is well known that many factors may influence the chemical composition of propolis such as plant sources, geographical region and climatic conditions, time of production of propolis and type of Apis mellifera [1,5,7,43].
The most popular plant species as a source of biomaterial for the production of propolis include: Acacia spp., Aesculus hippocastanum, Betula pendula, Fagus sp., Fraxinus sp., Pinus spp., Prunus spp., Quercus sp., Salix alba [1]. In Poland, very important plant sources are Alnus spp. and Populus spp. [10]. Plant materials obtained from mentioned species are chemically different because these plants contain various primary and secondary metabolites classified mainly to phenolic compounds (flavonoids, phenolic acids, terpenoids; Figures 4 and 5) and essential oils [1]. In addition to this, the situation is more complicated because the chemical composition of finished products obtained from propolis may depend on the physicochemical processes used during the extraction (e.g., solvent, extraction temperature and time, standardization), similarly as during processing using other plant materials [44]. However, Pobiega et al. [43] did not observe differences in the qualitative composition of propolis extracts obtained by various methods such as traditional extraction with 70% ethanol (1:10 and 1:5 propolis to ethanol during 1-7 days) and shaking and ultrasound-assisted shaking extractions. green propolis [35], Brazilian organic propolis [17], Egyptian propolis [37], Indian propolis [38], Korean propolis [32], Lebanese propolis [33], Nepalese propolis [50], propolis from Turkey [41], and Taiwanese green propolis [24]. Among various propolis, the Brazilian propolis is commonly classified into 13 types based on their properties (mainly: color, chemical composition, texture), and on geographic origin [40,51,52]. Other authors mentioned the main types of propolis such as Brazilian and European propolis [53].
In Table 1 is presented the comparison of chemical compositions of propolis extracts, which have been currently estimated for antifungal properties. Generally, ethanolic extracts of propolis, which come from different regions of Brazil, are investigated most often (199 publications in PubMed between 2011 and 2021) [8,14,17,40,54]. High-quality chemical analysis mainly concerns two types of propolis such as Brazilian red propolis (using gas chromatography coupled to mass spectrometry [54], and liquid chromatography-high-resolution mass spectrometry [53]), and Brazilian green propolis (using liquid chromatography-high-resolution mass spectrometry [53], and highperformance liquid chromatography (HPLC-DAD) [13].       Moreover, the chemical diversity of propolis is dependent on the bee species in different regions of the world [2]. The main producer of propolis is believed to be Apis mellifera, which is the most common species of honey bee in Europe, Africa, and the Middle East. However, the honey bee genus Apis consists of 11 species [45]. Furthermore, in tropical and subtropical regions of Africa, Asia and America are also well-known stingless honey bees (or meliponines) i.e., in Africa-African stingless bee-Meliponula ferruginea [46,47]. This type of propolis is traditionally used in medicine in various regions of Argentina, Brazil Mexico, and Vietnam [46]. It has been asserted that the Caucasian honey bee (Apis mellifera caucasia) is able to produce between 250 and 1000 g of propolis annually, per hive [2].
Increasingly, propolis is classified as honey bee propolis and stingless bee propolis [2]. Actually, 502 different chemical compounds in propolis obtained by honey bees have been described, and more than 100 chemical compounds produced by stingless bees [2]. According to the analysis of Tran et al. [2] phenolic compounds are dominant in honey bee propolis (79.5%) and also in stingless bee propolis (63.0%). The second group of chemical compounds are the terpenoids accounted for 18.9% (honey bee propolis) and 37.0% (stingless bee propolis) of all compounds found in propolis. However, the chemical diversity of propolis composition is still extensively studied.
In Table 1 is presented the comparison of chemical compositions of propolis extracts, which have been currently estimated for antifungal properties. Generally, ethanolic extracts of propolis, which come from different regions of Brazil, are investigated most often (199 publications in PubMed between 2011 and 2021) [8,14,17,40,54]. High-quality chemical analysis mainly concerns two types of propolis such as Brazilian red propolis (using gas chromatography coupled to mass spectrometry [54], and liquid chromatography-high-resolution mass spectrometry [53]), and Brazilian green propolis (using liquid chromatography-highresolution mass spectrometry [53], and high-performance liquid chromatography (HPLC-DAD) [13]. Table 1. Examples of the chemical composition of propolis produced by Apis mellifera from various geographical regions using to an evaluation of pharmacological studies.
Furthermore, there is an increasing amount of scientific evidence on the mechanism of antifungal activity after the use of propolis extracts. Corrêa et al. [65] and Gucwa [57] showed that the cell membrane of fungi may be a possible target of an extract of propolis besides the induction of cell death. Earlier, researchers showed that an extract of propolis can inhibit the activity of extracellular phospholipase, leading to attenuation of the fungal cell adhesion to epithelium [66]. Currently, it has been observed that propolis may influence the formation and integrity of the cell wall of fungi and can inhibit the morphological transformation of C. albicans [57]. Stahli et al. [53] observed that an ethanolic extract of propolis caused a loss of the cell wall integrity of C. albicans and decreased the metabolic activity. Okińczyc et al. [50] showed that ethanolic extract of propolis inhibited filamentation of cells of C. albicans, germination of yeasts and increased production of the superoxide anion radical. A few studies showed that extracts of propolis are effective inhibitors of biofilm [16,53,54,57,[67][68][69]. Freires et al. [54] observed that ethanolic extract of Brazilian propolis (types 3 and 13) can lead to disruption of the biofilm structures of Candida sp. at concentrations <0.9 µg/mL. Moreover, Martorano-Fernandes et al. [67] revealed that extract from Brazilian red propolis possessed inhibitory effects on the proliferation and diminished the metabolism of biofilms of C. albicans. Fernández-Calderón et al. [16] showed that 70% ethanolic extract of Spanish propolis inhibited the fungal biofilm at sub-inhibitory concentrations of extract (0.1 and 0.05%). Gucwa et al. [57] showed that values of minimum biofilm eradication concentration of Polish propolis extract were in the range of 0.04% to more than 1.25% (v/v) for C. glabrata, C. krusei, and C. albicans. Gucwa obtained also very interesting results about a synergistic effect of extract of Polish propolis with voriconazole, and fluconazole against C. albicans [57]. Pharmacological aspects of interactions have been investigated also by Argüelles [70], who showed that combination of propolis with carnosic acid (diterpene occurs in Rosmarinus officinalis and Salvia officinalis), through synergistic action, may lead to a drastic reduction in survival of Candida albicans cells, leading to a fungicidal effect. These observations open a new scientific window for the application of this combination in clinical therapies of C. albicans infections.
Therapy of various candidiasis should be based on complementary methods, including natural products such as not only tea tree and garlic but also propolis [71]. It should be emphasized that the number of studies on the evaluation of the effects of propolis in vulvovaginal candidiasis (opportunistic fungal infection) is increasing [25,27,71,72]. New trends in antifungal research of propolis include also assessment of anti-candidal activities i.e., in orthodontic materials (green propolis) [73], dental surface (red propolis) [61,74], in treatment of chronic periodontitis (red propolis) [75], in caries (Brazilian and European propolis) [53]. The third research area is endemic and rare diseases. Santos et al. [14] showed that Brazilian red propolis exerted antifungal activity against Paracoccidioides brasiliensis. This pathogenic fungal strain can cause systemic mycosis (paracoccidioidomycosis), also known as South American blastomycosis, classified as a neglected tropical disease [76]. In all these cases, extracts of propolis showed antifungal activities (Table 2). In addition, the results of these studies indicate an interesting application of propolis extracts in vulvovaginal candidiasis, dental diseases and fungal tropical diseases. Brazilian red propolis (chemical characteristic in Table 1) Paracoccidioides brasiliensis (Pb18) strain reduction the viability of the fungal strain in 75% (at 24 h), up to 92% (at 72 h) after concentration at 500 mg/mL [14] Brazilian red propolis -ethanolic extracts (chemical characteristic in Table 1  Iranian propolis, 25% ethanolic extract C. albicans (23 clinical samples) 9.01 mg/mL Not studied [58] Irish propolis (chemical characteristic in Table 1

Collected Data Analysis
In recent years, the authors have used new standard methods for the determination of minimal inhibitory concentration (MIC), and minimal fungicidal concentration (MFC) for chemically defined extracts from propolis, which are key parameters for this review ( Table 2). In more than half of the publications, the chemical compositions of extracts have been characterized using HPLC methods, which have been shown in Table 1. In other cases, reference was made to foreign results of phytochemical tests. The chemical composition and origin of propolis were very diverse and this has an impact on the strength of the antifungal activities of propolis extracts. The antifungal activities of propolis extracts are most frequently tested in vitro on Candida albicans in comparison with C. dubliniensis, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, Saccharomyces cerevisiae and strains of molds. The most popular species of pathogenic fungi tested in the last 10 years in microbiological studies are shown in Figure 6.  [43]. Moreover, in five studies [13,17,18,54,75] antifungal activities of extracts of propolis against three or more species of Candida were compared. Many authors demonstrated that C. albicans is more sensitive than other species of Candida (C. dubliniensis, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis) to ethanolic extracts from various propolis.
Often the authors described that phenolic compounds are the main components which can be responsible for the antifungal activities of propolis extracts.
There is increasing information that not only polyphenolic compounds, but also essential oil components found in propolis exhibit antimicrobial activity [Ikeda, 2021. It was shown that essential oils from Brazilian propolis contain i.e., camphene, p-cymene, limonene, myrcene, α-pinene, β-pinene [55]. Many studies have proven that these chemical components occurring in essential oils of medicinal plants exerted antifungal activity mainly against Candida albicans [60,[77][78][79]. After comparison of phytochemical components of propolis extracts (Table 1) it is very difficult to conclude which chemical compounds are the most active against fungal strains. Therefore, it can be concluded that the synergy of action of all components of propolis extract determines the antifungal activity. However, it should be noted that the extracts of Brazilian propolis showed very wide variability in antifungal activity, e.g., according to Freires et al. [54] Brazilian reed propolis showed MIC = 2.0 µg/mL), Siqueira et al. [75] revealed for the same type of propolis showed MIC = 32-64 µg/mL, whereas Sokolonski et al. [13] stated values of MIC from 0.5 to more than 8 mg/mL ( Table 2). This indicates the need to standardize the chemical composition of the different types of propolis collected all over the world.

Conclusions
First, based on the review of the bibliography, it was found that in recent years studies have focused mainly on the assessment of the susceptibility of Candida albicans strains to ethanolic extracts of various propolis.
Second, these extracts often contain different chemical compositions, resulting from the unique flora of the region where bees produce propolis. The wide range of demonstrated activity against Candida albicans leads to the conclusion that the need to standardize the chemical composition of propolis extracts should be considered, especially in the case of the same types of propolis.
Third, during in vitro studies, the isolated strains of pathogenic fungi should be more accurately described in terms of patients' age and gender, their diseases, and different treatments. These factors may influence the sensitivity of clinical strains to the action of propolis extracts.
Fourth, chemical, microbiological and pharmacological tests of chemically defined propolis extracts should be performed for a wider group of pathogenic fungi.
Fifth, the antifungal activity of propolis should be confirmed in clinical trials, not only in terms of efficacy and safety assessment but also in order to demonstrate pharmacological interactions with other drugs.

Conflicts of Interest:
The authors declare no conflict of interest.