Antiviral, Antibacterial, Antifungal, and Antiparasitic Properties of Propolis: A Review

Propolis is a complex phytocompound made from resinous and balsamic material harvested by bees from flowers, branches, pollen, and tree exudates. Humans have used propolis therapeutically for centuries. The aim of this article is to provide comprehensive review of the antiviral, antibacterial, antifungal, and antiparasitic properties of propolis. The mechanisms of action of propolis are discussed. There are two distinct impacts with regards to antimicrobial and anti-parasitic properties of propolis, on the pathogens and on the host. With regards to the pathogens, propolis acts by disrupting the ability of the pathogens to invade the host cells by forming a physical barrier and inhibiting enzymes and proteins needed for invasion into the host cells. Propolis also inhibits the replication process of the pathogens. Moreover, propolis inhibits the metabolic processes of the pathogens by disrupting cellular organelles and components responsible for energy production. With regard to the host, propolis functions as an immunomodulator. It upregulates the innate immunity and modulates the inflammatory signaling pathways. Propolis also helps maintain the host’s cellular antioxidant status. More importantly, a small number of human clinical trials have demonstrated the efficacy and the safety of propolis as an adjuvant therapy for pathogenic infections.


Introduction
The present viral pandemic and the threat of antibiotic resistant bacteria illustrate the ever-increasing need to find novel pharmaceutical compounds to combat microbial pathogens. Nature-derived compounds with a myriad of pharmacological properties could hold the key to overcome the never-ending and inevitable threats. Natural products have been used as medicine to treat human diseases caused by pathogens for centuries. For example, quinine the antimalarial drug derived from Cinchona tree, long used by the indigenous South American native population and discovered by the West in the 17th century [1]. Other notable examples are artemisinin, the antimalarial drug derived from the plant Artemisia annua, which is an herbal plant in Chinese traditional medicine; and penicillin, a natural antibiotic derived from Penicillium molds [2,3]. The latter two resulted in Nobel prizes, highlighting the significance of natural product research.
The present review article focuses on the antiviral, antibacterial, antifungal, and antiparasitic properties of propolis. The term 'propolis'in this review study includes propolis from all propolis-producing bees, namely European honey bees (Apis mellifera), Asian honey bees (Apis cerana), and stingless bees of the genera Trigona, Melipona, Geniotrigona, Heterotrigona, and Tetragonula.

Bioactive Compounds in Propolis
Propolis comprises of wax, resin, balsam, essential oils, pollen, and plant primary and secondary metabolites-such as amino acids, minerals, vitamins, phenolics, terpenoids, tannins, and alkaloids [18][19][20][21]. The precise composition of propolis varies depending on the geographical locations, plant sources, and/or bee species [22]. Majority of propolis research in terms of its health benefits has been linked to its phenolic content [23][24][25]. Phenolic compounds are one of the largest groups of plant secondary metabolites. It is estimated that 2% of all carbon fixed by photosynthesis is converted into phenolic compounds. Even though phenolics are considered as secondary metabolites as they are not directly involved in anabolic and catabolic processes, plant phenolic compounds are paramount for plant survival as they are implicated in many essential functions such as defense mechanisms (against pathogens, insects, oxidation, and UV radiation), plant developmental signaling, and so on [26][27][28]. In this review, the terms 'phenolic compounds', 'phenolics', and 'polyphenols'confer the same meaning and are used interchangeably, unless specified otherwise.
Quideau et al. (2011) proposed that plant phenolics should be confined to the secondary metabolites produced by shikimate/phenylpropanoid pathway or acetate/malonate pathway, or combination of both [29]. These compounds include simple phenols and polyphenols. As a general description, polyphenols consist of two phenyl rings and one or more hydroxyl substituents and their functional derivatives; such as esters and glycosides. Plant phenolics can be categorized into two major groups; flavonoids and non-flavonoids. Flavonoids share a structure of diphenyl propanes (C 6 -C 3 -C 6 ), where the phenolic rings are most commonly linked by a heterocylic ring. Flavonoids and their conjugates are a major group of natural products in which over 8000 flavonoids have been identified. Some examples of flavonoids are flavanol, flavanone, pinocembrin, quercetin, gallangin, chrysin, and kaempferol [30,31].
Moreover, stilbenes (C 6 -C 2 -C 6 ), a relatively small group of phenolic compounds, are characterized by two phenyl moieties linked by a two-carbon methylene group. Examples of stilbenes are resveratrol and 1,2-diarylethenes. Another non-flavonoid phenolic group is lignans which consist two propylbenzene units (C 6 -C 3 ) linked by the central carbon (C8) of the side chains. The C9 and C9 positions of lignans are usually substituted with various different patterns, consequently lignans are classified into eight subgroups, namely  [72]. They found that the predominant volatiles are terpenoids, withα-pinene, junipene, and δ-cadinene being some of the predominant compounds [72]. In addition, Popova et al. (2010) identified at least 37 diterpenes from propolis from various regions of Greece, with isocupressic acid, pimaric acid, communic acid, and 14,15-dinor-13-oxo-8(17)labden-19-oic acid being the most dominant terpenoids [73]. The same group identified 32 diterpenes isolated from various propolis samples from Malta [74]. Two specific diterpenes; daucane diterpene esters of hydroxybenzoic acids, were isolated and linked to botanical source Ferula communis. They also demonstrated that all samples had high antibacterial activity against Staphylococcus aureus, however only propolis samples with high concentration of terpenyl esters were shown to have high antifungal activity against Candida albicans [74].
Antimicrobial and antiparasitic properties of propolis should be considered at two levels, its impact on the pathogen itself and the impact on the host. With respect to the latter, propolis has well-established immunomodulatory effects [87,88]. Figure 1 summarizes various mechanisms of action of propolis in exerting its antiviral, antiviral, antifungal, and antiparasitic effect. Propolis and its bioactive components exert antiviral activity through various mechanisms of action.

Figure 1.
There are two distinct impacts with regards to antimicrobial and anti-parasitic properties of propolis; on the pathogens and on the host. With regards to the pathogens, propolis acts by inhibiting the ability of the pathogens to invade the host cells (by forming a physical barrier and inhibiting enzymes and proteins needed for invasion into the host cells). In addition, propolis inhibits the replication process of the pathogens by inhibiting the enzymes needed for the replication of the pathogens' genetic materials. Propolis also inhibits the metabolic processes of the pathogens by disrupting cellular organelles and components responsible for energy production. With regard to the host, propolis acts as an immunomodulator. It upregulates the innate immunity and modulates the inflammatory signaling pathways. Propolis also helps maintain the host's cellular antioxidant status throughout the infection.

Antiviral Propertiesof Propolis
In the present review article, the antiviral properties of propolis discussed would be focused on pathogenic human viruses. Propolis has been demonstrated to have antiviral properties against a wide range of viruses. One of the earliest studies was carried out by Debiaggi et al. (1990) which investigated the propolis-derived flavonoids, namely chrysin, kaempferol, acacetin, galangin, and quercetin against various strains of herpesvirus, adenovirus, rotavirus, and coronavirus [89]. More importantly, propolis has been shown to have antiviral activity against SARS-CoV-2. Refaat et al. (2021) demonstrated that propolis delivered in a liposomal encapsulation was as effective as remdesivir in neutralizing SARS-CoV-2 in vitro [90]. Many computational and molecular docking studies suggest the efficacy of propolis and its phenolic components in interfering with many important proteins of the SARS-CoV-2, including proteases and the spike protein [91][92][93][94].
In addition, propolis is efficacious against influenza viruses. Serkedjieva et al. (1992) demonstrated that propolis-derived phenolics, especially, isopentyl ferulate exhibited strong antiviral activity against H3N2 influenza A virus [95]. Shimizu et al. (2008) investigated the antiviral efficacy of thirteen ethanolic extracts of Brazilian propolis against influenza virus A/PR/8/34 (H1N1) [96]. It was found that all extracts had antiviral properties with various level of efficacy. One extract (AF-08), in particular, was effective in Figure 1. There are two distinct impacts with regards to antimicrobial and anti-parasitic properties of propolis; on the pathogens and on the host. With regards to the pathogens, propolis acts by inhibiting the ability of the pathogens to invade the host cells (by forming a physical barrier and inhibiting enzymes and proteins needed for invasion into the host cells). In addition, propolis inhibits the replication process of the pathogens by inhibiting the enzymes needed for the replication of the pathogens' genetic materials. Propolis also inhibits the metabolic processes of the pathogens by disrupting cellular organelles and components responsible for energy production. With regard to the host, propolis acts as an immunomodulator. It upregulates the innate immunity and modulates the inflammatory signaling pathways. Propolis also helps maintain the host's cellular antioxidant status throughout the infection.

Antiviral Properties of Propolis
In the present review article, the antiviral properties of propolis discussed would be focused on pathogenic human viruses. Propolis has been demonstrated to have antiviral properties against a wide range of viruses. One of the earliest studies was carried out by Debiaggi et al. (1990) which investigated the propolis-derived flavonoids, namely chrysin, kaempferol, acacetin, galangin, and quercetin against various strains of herpesvirus, adenovirus, rotavirus, and coronavirus [89]. More importantly, propolis has been shown to have antiviral activity against SARS-CoV-2. Refaat et al. (2021) demonstrated that propolis delivered in a liposomal encapsulation was as effective as remdesivir in neutralizing SARS-CoV-2 in vitro [90]. Many computational and molecular docking studies suggest the efficacy of propolis and its phenolic components in interfering with many important proteins of the SARS-CoV-2, including proteases and the spike protein [91][92][93][94].
In addition, propolis is efficacious against influenza viruses. Serkedjieva et al. (1992) demonstrated that propolis-derived phenolics, especially, isopentyl ferulate exhibited strong antiviral activity against H3N2 influenza A virus [95]. Shimizu et al. (2008) investigated the antiviral efficacy of thirteen ethanolic extracts of Brazilian propolis against influenza virus A/PR/8/34 (H1N1) [96]. It was found that all extracts had antiviral properties with various level of efficacy. One extract (AF-08), in particular, was effective in reducing weight loss and prolonging the life of infected mice. Ten mg·kg −1 AF-08 extract was also shown to be almost as efficacious as 1 mg·kg −1 oseltamivir (antiviral drug) in reducing the viral load in the bronchoalveolar lavage fluids of the lungs of the infected mice [96]. Kai et al. (2014) showed that propolis-derived phenolics; apigenin, kaempferol, and coumaric acid were effective against Influenza A/PR/8/34(H1N1) and both oseltamivirand peramivir-sensitive and resistant strains of influenza A/Toyama/26/2011 (H1N1) viruses. In addition, kaempferol was shown to reduce the viral load in the bronchoalveolar lavage fluids and prolong the survival time of the infected mice [97]. Moreover, Kuwata et al. (2011) demonstrated that water extract of propolis had antiviral activity against influenza virus A/WSN/33 (H1N1). It appeared caffeoylquinic acids were the active components that exerted the antiviral properties [98]. The same group also isolated and demonstrated that 3,4-dicaffeoylquinic acid as the anti-influenza compound in the propolis extract [99].
Propolis has been shown to have anti-HIV activity. Moronic acid, a triterpenoid, isolated from Brazilian propolis was shown to inhibit HIV activity in H9 lymphocytes [100]. In addition, Gekker et al. (2005) demonstrated that propolis extracts from various sources and regions, namely Minnesota (USA), Brazil, and China all inhibited the HIV-1 infected CD4+ lymphocyte and microglial cell cultures [101]. More importantly, propolis did not antagonize the activity of antiretroviral drugs such as zidovudine and indinavir [101]. Furthermore,  reported that ethyl acetate extract of propolis from Ceará state (northeast Brazil) exhibited anti-HIV activity [102]. It was found that the propolis-derived phenolics-naringenin, quercetin, and diprenylcinnamic acid-were the compounds linked to the antiviral activity of propolis [102].
The most extensive research on antiviral properties of propolis has arguably been carried out in herpes viruses.  demonstrated that galangin, kaempferol, and quercetin had anti-herpetic activity. They also demonstrated that there were synergistic relationships among the propolis-derived phenolics which partly explained the higher activity propolis when compared to its individual components [103]. Schnitzler et al. (2010) also demonstrated the synergistic nature of the propolis-derived compounds. It was shown that propolis aqueous and ethanolic extracts had superior anti-herpetic activity when compared to the individual components such as caffeic acid, p-coumaric acid, benzoic acid, galangin, pinocembrin, and chrysin [104]. In addition, Bankova et al. (2015) showed that poplar propolis containing various phenolics-such as benzoic acid, p-coumaric acid, benzyl p-coumarate, benzyl ferulate, pinocembrin, and pinocembrin chalcone-exhibited antiviral activity against herpes simplex virus types 1 and 2 (HSV-1 and 2) [105].
Anti-herpetic activity of propolis extract has also been shown in studies involving animal models. Kurokawa et al. (2011) investigated several propolis extracts in mice and demonstrated that various extracts appeared to inhibit the herpes virus at different stages of infection [108]. Various ethanolic extracts of propolis significantly reduced the viral load in brains and skins of mice infected with HSV-1 [108]. Sartori et al. (2012) demonstrated that hydroalcoholic extract (70% ethanol) of propolis reduced the severity of extravaginal lesions and histological damage in the vaginal tissue of animals infected with HSV-2 [109]. Furthermore, antiviral properties of propolis extend to various viruses such as rhinovirus, dengue virus, polio virus, rubella virus, picornavirus, and measles virus [110][111][112].

Mechanisms of Action of Antiviral Properties of Propolis
Molecular docking and in silico studies unveiled the potential mechanisms of action employed by propolis and its components in inactivating SARS-Cov-2. Refaat et al. demonstrated that rutin and caffeic acid phenethyl ester inhibited both 3CL-protease and S1 spike protein of SARS-Cov-2 [15]. Caffeic acid phenethyl ester was also shown to interfere with the highly conserved residues (substrate-binding pocket) of M pro protein of SARS-Cov-2 [113]. In addition, molecular docking studies by Sahlan et al. (2021) and Dewi et al. (2021) demonstrated that Sulabiroins A, (2S)-5,7-dihydroxy-4 -methoxy-8-prenylflavanone acid, glyasperin A, and broussoflavonol F (propolis-derived compounds) could potentially bind to various residues of M pro catalytic sites and consequently inhibit the activity of the M pro protein of SARS-Cov-2 [93,94]. Kwon et al. (2020) demonstrated that kaempferol and p-coumaric acid prevented the entry of human rhinovirus and also inhibited the viral replication in HeLa cells [112]. In addition, ferulic acid isolated from propolis was demonstrated to inhibit the activity of porcine parvovirus [114]. Ferulic acid inhibited and reversed the parvovirus-induced expression of pro-apoptotic genes Bid, Bad, Bim, and Bak. The expression of these genes has been shown to be associated with mitochondrial disruption and apoptosis of the host cells [114]. Propolis was also shown to enhance the expression of myxovirus resistance 1 (MX1) gene [115]. Mx proteins are the "gatekeepers" of the host cells in overcoming RNA viruses and other virus families that replicate in the host nucleus [116]. Polyphenols also help transport Zn cations into across the plasma membrane independently of plasma membrane zinc transport proteins [117]. Zn cations have been shown to inhibit the activity of viral RNA-dependent RNA polymerase [118].
Moreover, the immunomodulatory properties of propolis in affecting host immune functions were evident in the virus infection models. Propolis constituent 3,4-dicaffeoylquinic acid (3,4-diCQA) was shown to increase the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) which expedited viral clearance [99]. Propolis also reduced the oxidative stress in the infected host cells by inhibiting the expression of reactive species, tyrosine nitration, and myeloperoxidase activity. Propolis also maintained the expression of catalase, an important enzyme in the cellular antioxidant system, in infected cells [109]. Additionally, propolis also induced the production of interferon-γ (IFN-γ) in HSV infection models. IFN-γ is a significant stimulator of lymphocyte migration into skin and consequently important in alleviating the symptoms of viral infections such HSV infections [108]. Table 3 summarizes and illustrates the antiviral properties of propolis and/or propolis-derived compounds.  Hydromethanolic extract of propolis, pyrrolizidine alkaloids, and C-glycosyl flavones Herpesvirus Not determined [106] Ethanolic extracts of propolis Herpesvirus Significant reduction of the viral load in brains and skins of mice infected with HSV-1. [108] Hydroalcoholic extract (70% ethanol) of propolis Herpesvirus Reduction of the severity of extravaginal lesions and histological damage in the vaginal tissue infected with HSV-2. [109]

Ferulic acid parvovirus
Inhibition and reversal of the parvovirus-induced expression of pro-apoptotic genes Bid, Bad, Bim, and Bak. [114] Quercetin and epigallocatechin gallate Not determined Zinc ionophore [117]

Antibacterial Properties of Propolis
Antibacterial properties of propolis are very well documented in the scientific literature. Przybyłek and Karpiński (2019) recently reviewed the analyses of the reported data on the influence of propolis on about 600 strains of bacteria, both aerobic and anaerobic [120]. Information of particular bacterial species susceptible to propolis action as well as values of the minimal inhibitory concentration (a minimum concentration at which no microorganism growth can be observed in the assays) can be found in that review [120]. Generally, it has been shown in multiple studies that propolis exhibits more powerful an-timicrobial activity against Gram-positive than Gram-negative bacteria [120][121][122]. The difference was thought to be due to the presence of bacterial hydrolytic enzymes in the outer membrane of Gram-negative bacteria, which could potentially compromise and reduce the efficacy of the active components of propolis [123,124].
It is common practice to relate the potential antimicrobial properties of propolis to its phenolic and flavonoid content. Nevertheless, Bridi et al. (2015) showed that the concentration of those components does not always correlate with observed antimicrobial activity in vitro [125]. Therefore, it has been suggested that other tests should be used to set some standards for evaluation of propolis biological activity. In fact, the plethora of active ingredients in varying combinations/concentrations is the property of propolis that can prevent bacterial resistance from occurring [126].
In addition, geographical origin appears to affect the composition of propolis which consequently affects its antibacterial properties [127,128]. It was found in particular, that propolis from the Middle East exhibits highest activity against both Gram-postive and Gram-negative strains, while that from Germany, Ireland, and Korea has the lowest activity [120]. Apart from having direct antimicrobial effect, propolis also acts synergistically with conventional antibiotics enhancing their efficacy as well as with other natural products such as honey [129][130][131].

Mechanisms of Action of Antibacterial Properties of Propolis
With regards to its antibacterial mode of action, propolis can interfere with their pathogenic potential by increasing permeability of the bacterial cell membrane, inhibiting ATP production, decreasing bacterial mobility, disturbing membrane potential, and impairing bacterial RNA and DNA production [120,121]. Because of the complex nature of propolis in terms of its composition, it is not possible to precisely elucidate specific mechanism responsible for each of its many effects. The studies usually concentrate on some selected components or their mixtures and try to relate observed outcomes to that induced by unfractionated propolis extracts. There are many possible biochemical mechanisms that can underlie the antibacterial actions of propolis. They have been covered in the extensive literature and also summarized in recent reviews [120][121][122]. Therefore, in this article, we will focus on the studies that represent some novel aspects in the field.
It has been shown in a recent study that apart from biochemical pathways, there is also physical mechanism that can contribute to the biological activity of propolis [132]. Namely, propolis deposited on a surface was found to generate a layer of water which effectively excludes colloidal particles (termed exclusion zone (EZ) water). The phenomenon is based on the electrokinetic process, depended on the presence of negatively charged functional groups characteristic to many chemical components of the propolis [132,133]. Therefore, the colloid-excluding property has very generic character, largely independent of the exact composition and origin of the propolis. From the physical perspective, all bacteria or viruses suspended in aqueous solution (e.g., our body fluids or mucosal lining) are colloids. Thus, it has been proposed that propolis can prevent pathogens from accessing the surface (e.g., respiratory epithelium) by creating the physical barrier in the form of EZ [132].
There are few recent studies showing that propolis-functionalized textiles acquire antibacterial properties and can be potentially used in medical field, e.g., as wound dressing [134][135][136]. In one case, propolis was chemically bonded to the cotton fibers and resisted several washing cycles [134]. Bacteria-free zone could be observed next to such propolisfunctionalized textile in so called disk diffusion method. Any diffusion of bounded propolis components could hardly take place, yet EZ could be created as this mechanism does not require chemical (contact) interaction with bacteria. Other EZ-generating materials were also suggested to potentially provide first line of defense against microorganisms colonizing surfaces (e.g., in healthcare facilities) [137].
Considering further physicochemical characteristics of propolis, the presence of fixed negative charges in its chemical constituents goes in hand with mobile protons. Protons (positive charges) are diffusible and providing propolis its acidic pH [138]. It is recognized that cationic agents are able to reduce negative charges of bacterial cells, promote membrane permeability, and consequently induce bacterial cell death [139]. Therefore, higher bactericidal activity of propolis against Gram-positive compared to the Gram-negative bacteria could be also related to the less negative surface charge of Gram-positive strains and its higher susceptibility to mobile protons. Such a link is not yet confirmed but would deserve further studies and could possibly help discriminate, screen, and isolate propolis samples that have higher bactericidal activity against Gram-negative species.

Antifungal Properties of Propolis
Antifungal activity of propolis has been well documented in the literature. It is known to be influenced by the variation in chemical composition of propolis [140]. This variation in antifungal effect has been reported in numerous studies analyzing the effect of propolis from different geographic origin against different fungal species, particularly of clinical interest [141][142][143][144][145]. Propolis is known to possess antifungal activity against fungal species such as C. albicans, C.parapsilosis, C. tropicalis, C. glabrata [131,146]. It has shown an aflatoxigenic property against fungi like Aspergillus flavus, where it inhibited conidial growth of the fungi [147]. In this review article, focus has been laid on the fungal species affecting humans and related clinical studies.
Propolis extract has displayed excellent performance regarding in vitro tests performed against yeasts identified as onychomycosis agents. In these experiments, it was observed that in low concentrations, propolis acts as a fungistatic and fungicidal agent.  [141]. A clear antifungal activity of propolis was reported in the following order of sensitivity: C. albicans > C. tropicalis > C. krusei > C. guilliermondii, with C. albicans being the most sensitive and C.guilliermondii being the most resistant. The minimal inhibitory concentrations (MICs) were in the range of 8-12 mg/mL. A reduction in the number of Candida species in saliva was also observed in patients with full dentures who used a hydroalcoholic extract of propolis [141].
Both green and red Brazilian propolis have displayed antifungal activity against different fungal species of Trichophyton, which cause dermatophytosis, with red propolis being more efficacious [143]. In addition, n-hexane extract of Brazilian red propolis has efficacy against Candida spp. resistant to antifungal agents like fluconazole [148]. Oliveira et al. (2006) tested an alcoholic extract of Brazilian propolis against fungal isolates of C. parapsilosis, C. tropicalis, C. albicans, and other yeast species obtained from onychomycosis lesions. It was observed that the concentration of propolis which was capable of inhibiting all of the yeasts contained 50 µg/mLof flavonoids while yeast cell death was promoted at 20 µg/mLof flavonoids. The most sensitive species was recorded as Trichosporon sp. [149].
A study was conducted by Quiroga et al. (2006) to demonstrate the antifungal activity of propolis originating from the northwest of Argentina [150]. Their study focused on the antimycotic and cytotoxic activities of partially purified propolis extract on yeasts and xylophagous and phytopathogenic fungi. A comparison of propolis activity was also carried out with compounds like pinocembrin and galangin isolated from the same propolis and also with the synthetic drugs viz. ketoconazole and clotrimazole. They observed that partially purified propolis extract was capable of inhibiting fungal growth. The comparison of its relative biocide potency and cytotoxicity with the isolated compounds and synthetic drugs showed that the propolis was a reliable source of antifungal agent [150]. Another study was conducted by Agüero et al. (2010) using Argentinian propolis extract. The antifungal activity was tested against a range of fungi and yeasts. Most susceptible species were reported to be Microsporum gypseum, Trichophyton mentagrophytes, and Trichophyton rubrum. All the other dermatophytes and yeasts tested were strongly inhibited by different propolis extracts (MIC values being between 16 and 125 µg/mL) [151].
Falcao et al. (2014) carried out a study on antifungal activity of Portuguese propolis and its potential floral sources against C.albicans, T.rubrum, and Aspergillus fumigatus. A sig-nificant effect was observed with T. rubrum and least effect was showed on A.fumigatus [152]. Szweda et al. (2010) carried out an in vitro analysis of ethanolic extract of propolis (Poland), essential oils and silver nanoparticles dropped on TiO 2 for their antifungal activity against fluconazole-resistant C.albicans, C.glabrata, and C.krusei. They observed a satisfactory fungicidal activity of all the samples against C.albicans and C.glabrata isolates thus representing high potential to control and prevent candidiasis [153]. Boisard et al. (2015) assessed the antifungal activity of organic extracts of French propolis against various fungi and observed effective activity against C. albicans and C.glabrata but only weak activity towards A.fumigates [154].
It appears delivery methods and/or vehicles could potentially affect the antifungal activity of propolis. The study conducted by Bruschi [156].

Antifungal Mechanisms of Action of Propolis
Wagh (2013) extensively reviewed studies on propolis and its pharmacological properties, and concluded that the presence of phenolic compounds in propolis was considered responsible for fungicidal activity against C. pelliculosa, C. parapsilosis, C. famata, C. glabrata, and Pichia ohmeri [157]. Banskota et al. (2011) reported that the constituents of propolis such as 3-acetylpinobanksin, pinobanksin-3-acetate, pinocembrin, p-coumaric acid, and caffeic acid out of 26 or more constituents exhibited anti-fungal activity [158]. Agüero et al. (2010) proposed from their observation that the main bioactive compounds responsible for antifungal activity in the propolis extract were found to be 2 ,4 -dihydroxy-3-methoxychalcone and 2 ,4 -dihydroxychalcone. Both were reportedly highly active against clinical isolates of T. rubrum and T. mentagrophytes (MICs and MFCs were recorded between 1.9 and 2.9 µg/mL). Additionally, galangin, pinocembrin, and 7-hydroxy-8-methoxyflavanone were isolated from propolis samples and Zuccagnia punctata exudates, which displayed moderate antifungal activity [151]. Boisard et al. (2015) carried out in vitro evaluation of antifungal and antibacterial activities of aqueous and organic extracts of a mixture of French propolis samples on human pathogenic fungi, two yeasts (C. albicans and C. glabrata) and one filamentous opportunistic mold (A. fumigatus). They suggested from the results obtained that high content of flavonoids was responsible for the antifungal activity of propolis against C.albicans and C.glabrata species [154].
It is proposed that the antifungal activity of propolis is mainly due to its ability to induce apoptosis through metacaspase and Ras signaling [159]. Furthermore, propolis disrupts the expression of various genes (HST7, GIN4, VPS34, HOG1, ISW2, SUV3, MDS3, HDA2, KAR3, YHB1, NUP85, CDC10, MNN9, ACE2, FKH2, and SNF5) involved in pathogenesis, cell adhesion, biofilm formation, filamentous growth, and phenotypic switching. Propolis also inhibits the transition process from yeast-like to hyphal growth [159]. Propo-lis, in particular its phenolic component pinocembrin, appears to disrupt several critical cellular processes in a dose-dependent manner, namely energy homeostasis and mycelia growth. Pinocembrin is shown to reduce the phosphorylated adenosine nucleotides levels in hyphae of Penicillium italicum. Pinocembrin also damages the structure of the hyphae and the cell membrane causing the ionic leakage and soluble protein in P. italicum [160]. Table 4 summarizes and illustrates the antifungal properties of propolis and/or propolis-derived compounds. Table 4. Antifungal properties of propolis.

Propolis/Propolis-Derived Compounds Types of Fungi and Yeasts Mechanisms of Action References
Ethanolic extract of propolis 20 strains each of Candida albicans, Candida tropicalis, Candida krusei and 15 strainsof Candida guilliermondii.

Anti-Parasitic Properties of Propolis
Propolis has been demonstrated to have anti-parasitic properties against various intracellular and extracellular pathogenic protozoa. Siheri et al. (2016) found that various extracts of propolis from different regions of Libya had anti-plasmodial activity in vitro with different level of efficacy [161]. The EC 50 ranged from 3.4 to 53.6 µg·mL −1 [161]. In addition, propolis extracts from four different regions of Iran were shown to be antiplasmodial [162]. Dichloromethane extracts appeared to have stronger activity in vitro when compared to 70% ethanol and ethyl acetate extracts. Propolis extracts extended the survival of the Plasmodium falciparum-infected mice. However, propolis did not prevent their mortality [162].

Antiparasitic Mechanisms of Action of Propolis
Propolis, mainly due to its plant secondary metabolite content (phenolics and terpenoids), works against protozoan parasites through several mechanisms of action. Taxifolin-3-acetyl-4 -methyl ether (flavonol derivative) and bilobol (alkyl resorcinol) isolated from Libyan propolis appears to exert anti-trypanosomal activity by inducing cell lysis, disrupting phospholipid metabolism and depleting the pathogens of important lipids such as phosphatidyl glycerol (PG) and phosphatidyl inositol (PI) lipids [179]. In addition, rosmarinic acid and apigenin (both phenolics that are often found in propolis) induce physical damage in the form of cell lysis, cytoplasmic condensation, and kinetoplast and nuclear DNA aggregation in L. donovani. These propolis phenolics also promote cell arrest at the G0/G1 phase and inducediron chelation [180].
Resveratrol, a phenolic often associated with red wine but it is also present in propolis from certain region, exerts anti-trichomonal activity by affecting hydrogenosome metabolism [181][182][183]. Hydrogenosome is an organelle responsible for energy production and involved in redox balance in eukaryotes including protozoa [184]. Resveratrol also induces changes in the activity and expression of proteins associated with hydrogenosome metabolism-namely [Fe]-hydrogenase (Tvhyd), pyruvate-ferredoxin oxidoreductase, and heat shock protein 70 (Hsp70)-consequently causing hydrogenosome dysfunction and inactivation of the parasites [182]. In addition, kaempferol affects the adhesion mechanisms of the parasites by modifying the expression of actin, myosin II heavy chain and cortexillin II [185]. Epicatechin was shown to induce similar effect to resveratrol and kaempferol, such as the modification of the expression of the heat shock protein 70, myosin II heavy chain, and actin [186]. Additionally, epicatechin also affects the expression offructose-1,6-biphosphate aldolase and glyceraldehyde-phosphate dehydrogenase which are energy metabolism-related enzymes [186].
Apigenin, quercetin, and caffeic acid exert anti-parasitic effects through different mechanisms of action. Apigenin induces the inhibition of cell proliferation and upregulation of the expression of reactive oxygen species (ROS) in L. amazonensis. Apigenin also induces swelling in the parasitic mitochondria and consequently alters the mitochondrial membrane potential of the parasite [187]. Quercetin treatment significantly increases the production of ROS and induced mitochondrial dysfunction and membrane potential disruption in L. amazonensis [188]. Quercetin also appears to affect parasitic DNA synthesis by inhibiting the rate limiting ribonucleotide reductase through iron chelation. The removal of iron destabilizes tyrosyl radicals needed for the catalyzing activity of ribonucleotide reductase [189].
Moreover, caffeic acid induces morphological changes in the parasitic cells, the integrity of cellular plasma membrane and mitochondria, and consequently promoted apoptosis. Caffeic acid also appears to increase the inflammatory response of the infected macrophages by promoting the expression of ROS and TNF-α while reducing the expression of IL-10 and the availability of iron which significantly increases the anti-parasitic activity of the macrophages [190].
Anti-parasitic activity of propolis can also be attributed to its terpenoid content. Lupane, which has been identified in propolis, was shown to have anti-parasitic activity against L. amazonensis by inducing morphological changes such as vacuolization of cytosol, formation of lipid body and the disruption of mitochondria. Molecular docking studies also demonstrate that lupane has a strong affinity to DNA topoisomerase [21,191].
Maslinic acid and ursolic acid, pentacyclic triterpenoids identified in propolis from stingless bees Tetragonula laeviceps and Tetrigona melanoleuca, have been shown to have antiparasitic activity [192]. Maslinic acid appears to inhibit parasitic proteases including proteases in the surface protein complex crucial for invading host cells and metalloproteases [193,194]. In addition, ursolic acid acts by inhibiting glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important glycolytic enzyme in T. brucei [195]. The anti-parasitic activity of ursolic acid is also related to its ability to induce caspase 3/7-independent programmed cell death [196].

Human Clinical Trials
More importantly, the antimicrobial properties of propolis have been translated to human clinical trials. Silveira et al. (2021) conducted a single-center randomized placebocontrolled trial investigating the efficacy of the Brazilian green propolis extract as an adjuvant for treating hospitalized COVID-19 patients [203]. It was found that propolis was safe (no adverse event was recorded) and efficacious in reducing the length of hospital stay. The patients who were given 400 mg/day and 800 mg/day had a median of hospital stay of 6 and 7 days, respectively, compared to 12 days in the patients of the placebo arm. Furthermore, the patients given the higher dose of propolis, i.e., 800 mg/day also had less incidence of acute kidney damage associated with COVID-19 [203].
In addition, Esposito et al. (2021) demonstrated that propolis extract was efficacious in treating mild upper respiratory tract infections [204]. They investigated the efficacy of propolis in alleviating symptoms of respiratory tract infections such as sore throat, muffled dysphonia, and swelling and redness of the throat. Eighty three percent of the patients of the propolis arm recorded remission of the symptoms after three days of treatment, whereas the placebo-arm patients had at least one symptom after three days. No adverse event was recorded during the trial [204]. Other clinical trials on COVID-19 and ear, nose, and throat infections using combination treatments with propolis as one of the bioactive ingredients had also been shown to be efficacious in alleviating symptoms, and more importantly, safe [205][206][207][208].
We have covered the clinical trials of anti-herpetic properties of propolis in our previous review article [16]. Additionally, majority of the clinical trials of the antibacterial and antifungal (especially incandidiasis caused by C. albicans) properties of propolis were carried out in the oral health and dentistry sphere which our group covered in the same review article [16]. To our knowledge, anti-parasitic effect of propolis has not been investigated in human clinical trials.

Conclusions
Propolis is a complex phytocompound made from resinous and balsamic material harvested by bees from flowers, branches, pollen, and tree exudates.It is rich in polyphenols, especially flavonoids and phenolic acids, and has significant antiviral, antibacterial, antifungal, and antiparasitic properties shown mainly in in-vitro and in-vivostudies. Besides flavonoids, propolis also contains aromatic acids and esters, aldehydes and ketones, terpenoids and phenylpropanoids, steroids, amino acids, polysaccharides, and many other organic and inorganic compounds. However, the composition of propolis is very variable. It depends on the geographical region and the plants from which the bees extract their nectar. It is extensively consumed in various parts of the world because of its reputation as a health promoting agent including immunomodulatory, antiviral, antibacterial, antifungal, and antiparasitic properties.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) promotes challenging immune and inflammatory phenomena. Recently, in controlled, randomized clinical trials, propolis has been shown to affect clinical benefits on hospitalized COVID-19 patients.Propolis-derived phenolics, especially, isopentyl ferulate exhibited strong antiviral activity against H3N2 influenza A and against influenza virus A/PR/8/34 (H1N1). Standardized propolis extracts reduced the viral load in the bronchoalveolar lavage fluids of the lungs of the infected mice. Propolis has shown inhibition of HIV activity in H9 lymphoblastoid cell lines. Propolis-derived phenolics were linked not only to anti-HIV but also to anti-herpetic activity.
Antibacterial property of propolis is well documented. It has been shown that propolis exhibit greater antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria. Again, the antimicrobial property is attributed to the phenolic compounds found in propolis. Apart from biochemical pathways, there is also physical mechanism that can contribute to the antibacterial activity of propolis. It is based on the electrokinetic process and depends on the presence of negatively charged functional groups characteristic to many chemical components of propolis. When propolis deposits on a surface, it combines with a layer of water to form exclusion zones (EZ), which effectively excludes colloidal particles. Thus, it has been proposed that propolis can prevent pathogens from accessing the surface (e.g., respiratory epithelium) by creating the physical barrier in the form of EZ.
Propolis extracts have shown excellent activity against several strains of fungi and yeasts. However, it appears that the delivery methods and/or vehicles could potentially affect the antifungal activity of propolis. It is proposed that the antifungal activity of propolis is mainly due to its ability to induce apoptosis through metacaspase and RAS GTPase signaling.
Propolis has been demonstrated to have anti-parasitic properties against various intracellular and extracellular pathogenic protozoa. Propolis extracts extended the survival of the Plasmodium falciparum-infected mice. The reduction of parasitemia by propolis appears to be dose dependent. The mode of action was mainly due to its phenolic and terpenoid content. The phenolic compounds rosmarinic acid and apigenin that are often found in propolis induce physical damage in the form of cell lysis, cytoplasmic condensation, and kinetoplast and nuclear DNA aggregation in protozoan parasites to produce anti-parasitic activity.
Since the composition of propolis varies from region to region and from the types of bees used for propolis collection, there is a need to standardize propolis extracts for future use byhuman beings. Also, because there is a myriad of compounds in propolis, there is an urgent need to develop tests to set some standards for the evaluation of propolis biological activity.