Anti-Viral and Immunomodulatory Properties of Propolis: Chemical Diversity, Pharmacological Properties, Preclinical and Clinical Applications, and In Silico Potential against SARS-CoV-2

Propolis, a resin produced by honeybees, has long been used as a dietary supplement and folk remedy, and more recent preclinical investigations have demonstrated a large spectrum of potential therapeutic bioactivities, including antioxidant, antibacterial, anti-inflammatory, neuroprotective, immunomodulatory, anticancer, and antiviral properties. As an antiviral agent, propolis and various constituents have shown promising preclinical efficacy against adenoviruses, influenza viruses, respiratory tract viruses, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), human immunodeficiency virus (HIV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Over 300 chemical components have been identified in propolis, including terpenes, flavonoids, and phenolic acids, with the specific constituent profile varying widely according to geographic origin and regional flora. Propolis and its constituents have demonstrated potential efficacy against SARS-CoV-2 by modulating multiple pathogenic and antiviral pathways. Molecular docking studies have demonstrated high binding affinities of propolis derivatives to multiple SARS-CoV-2 proteins, including 3C-like protease (3CLpro), papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp), the receptor-binding domain (RBD) of the spike protein (S-protein), and helicase (NSP13), as well as to the viral target angiotensin-converting enzyme 2 (ACE2). Among these compounds, retusapurpurin A has shown high affinity to 3CLpro (ΔG = −9.4 kcal/mol), RdRp (−7.5), RBD (−7.2), NSP13 (−9.4), and ACE2 (−10.4) and potent inhibition of viral entry by forming hydrogen bonds with amino acid residues within viral and human target proteins. In addition, propolis-derived baccharin demonstrated even higher binding affinity towards PLpro (−8.2 kcal/mol). Measures of drug-likeness parameters, including metabolism, distribution, absorption, excretion, and toxicity (ADMET) characteristics, also support the potential of propolis as an effective agent to combat COVID-19.


Introduction
Propolis is a natural wax-like resin produced by honeybees (Apis mellifera L.) consisting of salivary secretions, wax, pollen, and various plant materials. Honeybees use propolis as a cement (bee glue) to seal cracks or open spaces in beehives, thereby preventing invasion by parasites and helping to maintain appropriate internal temperature and humidity [1,2]. The name propolis, from the Greek pro for "in defense" and polis for "city", reflects its importance for preventing diseases and parasites from entering the hive and inhibiting putrefaction, fungal growth, and bacterial growth [3]. The beneficial effects of propolis on human health were recognized thousands of years ago, with reports of use in folk medicine dating back to the ancient Egyptians, Greeks, and Romans [4]. In the 17th century, the London pharmacopoeias listed propolis as an official drug [5], further highlighting the ubiquity of propolis as a disease treatment throughout the centuries. In folk medicine, propolis is used for the management of airway disorders and cutaneous-mucosal infection by bacteria and viruses [6]. In some Asian, European, and South American countries, propolis is still used to make health drinks [7]. Additionally, it used in toothpaste and mouthwash preparations for treating gingivitis, cheilitis, and stomatitis [8,9].
The main aim of this review is to highlight the potential of propolis and its various constituents and/or derivatives as antiviral and immunomodulatory drugs against infectious diseases, including COVID-19 caused by SARS-CoV-2. Molecular docking analyses have identified over 40 propolis-derived compounds with strong binding affinity to various SARS-CoV-2 proteins and the human viral receptor. Moreover, measurements of drug-likeness parameters such as absorption, distribution, metabolism, excretion, and toxicity (ADMET) further support some of these agents as potential anti-SARS-CoV-2 drug candidates, warranting more extensive preclinical and clinical investigation.

Ethnopharmacology
Since ancient times, propolis has been employed by many cultures as a dietary supplement and folk remedy for improving health and managing disease [23,24]. The use of propolis in folk medicine can be traced back to at least 300 BC [25]. The Egyptians are considered the first peoples to use propolis for wound treatment and as an embalming agent [26,27]. In addition, Greek physicians such as Hippocrates, Dioscorides, and Galen; the Roman natural philosopher Pliny the Elder; and Inca healers utilized propolis as an antiseptic, antipyretic disinfectant for cutaneous and buccal infections and wound treatment. Propolis-based treatments were also in wide use in Europe during the 17th century to treat colds, wounds, rheumatism, heart disease, and diabetes [21,28]. According to Hippocrates, propolis may be used to improve health or prevent disease, including gastrointestinal disorders such as gastritis and gastric ulcer [29]. Arabs and Persians also used propolis as a disease treatment and cleansing agent [30].
All over the world, propolis has been used in traditional and folk medicine to prevent and treat many ailments, i.e., colds, wounds, rheumatism, heart disease, and diabetes [28]. Other documented uses include treatment of pharyngitis as well as wounds [31]. Brazilian green propolis is used as an anti-inflammatory, antibacterial, and antiulcer treatment in traditional medicine [32]. Administration for the treatment of abscesses and canker sores as well as wounds has also been reported [33]. During the Anglo-Boer War and Second World War, some physicians used propolis to promote tissue regeneration as well as for wound healing and treatment of tuberculosis, lung inflammation, and malnutrition [30]. Propolis with Ashwagandha (Withania sominifera) is used in some traditional medicine systems to boost immune function and prevent or cure various ailments [34].

Anti-Viral Activity
Propolis has long been used to treat viral infections and more recently tested for efficacy against SARS-CoV-2, the causative pathogen of COVID-19 [41]. Many diseasecausing viruses are unresponsive to currently available antiviral drugs and may also evolve into more drug-and vaccine-resistant strains. Thus, it is critical to identify novel candidate antivirals, particularly from natural sources; as such compounds tend to have good safety profiles.
Herpes simplex virus (HSV) types 1 and 2 are believed to be the most prevalent human viral pathogens. HSV-1 primarily infects oral epithelial tissues, leading to watery blisters on the skin or mucosa, while HSV-2 generally infects the genital mucosa and is sexually transmitted. Acyclovir is one of the main antiviral treatments, but resistant strains are common [42]. South Turkey (Hatay) propolis at 25, 50, and 100 µg/mL was reported to suppress the replication of both HSV-1 and HSV-2 with no toxicity on infected cells, and this antiviral effect was synergetic with acyclovir (positive control) [43]. An ethanolic extract of Brazilian green propolis also demonstrated significant in vitro antiviral activity against acyclovir-resistant HSV by inhibiting replication at an early stage of infection [44]. In addition, a Brazilian hydroalcoholic brown propolis extract (HPE) was reported to protect against vaginal lesions and to reduce epidermal and dermal inflammation induced by HSV-2 in female BALB/c mice (Table 1) [45].  Moreover, aqueous and ethanolic extracts prepared from propolis showed promising antiviral efficacy against HSV-1 infection of RC-37 cells as evidenced by a plaque formation assay. These therapeutic effects may arise from masking of viral proteins necessary for adsorption or entry into host cells [13]. Aqueous and ethanolic propolis extracts also reduced HSV-2 proliferation with efficacy comparable to acyclovir when applied at different intervals during the viral infection cycle, again likely by masking viral molecules responsible for entrance or adsorption into host cells [46].
Thus, propolis may inhibit HSV infection through multiple mechanisms, including virucide, inhibition of replication, and blockade of host entry.
Propolis has also demonstrated in vitro antiviral efficacy against human immunodeficiency virus (HIV), the causative pathogen for acquired immune deficiency syndrome (AIDS). According to the World Health Organization (WHO), Africa is the most severely affected region, accounting for nearly two thirds of all current HIV cases worldwide. In fact, nearly 1 in every 25 African adults (4.1%) is currently living with HIV. While there are several antiretroviral drugs available for HIV control, these have serious side effects such as lipodystrophy [54], necessitating the development of safer alternatives. Propolis extracts from different geographic regions of Brazil and China reduced HIV-1 infectivity by 85% in cultures of CD4+ cells and by 98% in cultures of microglial cells both by inhibiting viral entry and by suppressing reverse transcriptase activity [51]. Additionally, eight compounds isolated from Brazilian propolis, including four triterpenoids (melliferone, moronic acid, anwuweizonic acid, and betulonic acid) and four aromatic compounds (4-hydroxy-3-methoxypropiophenone, 4-hydroxy-3-methoxybenzaldehyde, 3-(3,4-dimethoxyphenyl)-2-propenal, and 12-acetoxytremetone), showed anti-HIV-1 activity in H9 lymphocytes (Table 2) [55].      The ethanolic extract of GH 2002 propolis demonstrated significant antiviral efficacy against varicella zoster virus (VZV), with an IC 50 of 64 µg/mL as assessed by a plaque reduction assay. This antiviral effect was detected when propolis extract was added at different times during the viral infection cycle. Furthermore, the extract enhanced the inhibitory effect of acyclovir on viral DNA polymerase during VZV replication. These findings again suggest that bioactive components in propolis can both mask viral proteins, thus interfering with entry into host cells, and suppress viral replication [49].
As mentioned, antiviral efficacy depends on the extraction method. A study comparing the antiviral properties of green and red Brazilian propolis extract prepared using two different ultrasonic methods and maceration found that both ultrasonic extracts demonstrated greater activity against bacteriophages MS2 and Av-08 than the maceration extract, while the maceration extract of red propolis was more active than the corresponding green extract for damaging the viral cell membrane and inhibiting polymerase activity [50]. Recently, a Euro-Asian poplar propolis extract treatment for 4 days was found to inhibit H1N1 influenza virus infection of MDCK cells by suppressing both viral growth and neuraminidase (NA) activity (Table 1) [31]. Two bioactive compounds isolated from Brazilian propolis, kaempferol and p-coumaric acid, were tested for inhibition of HeLa cell infection by three human rhinoviruses, HRV-2, HRV-3, and HRV-4. Kaempferol is the most bioactive compound, with IC 50 values of 7.3, 11.9, and 12.9 µM toward HRV-2, HRV-3, and HRV-4, respectively, while p-coumaric acid shows the lowest antiviral activity at IC 50 values of 371.2, 454.5, and 604.3 µM in comparison with ribavirin (positive control). Findings indicate that kaempferol and p-coumaric acid may block or reduce the entry of the viruses into the host cells, in order to preserve the cells from virus replication [15].
Another study on the antiviral effect of Mexican propolis against MDBK cell monolayer infection by pseudo rabies virus (PRV) found that treated cells exhibited an electrondense layer on the cell membrane that prevented viral entry [53]. Another study of 13 ethanolic extracts from South Brazilian propolis identified four with significant antiinfluenza virus activity in vitro and subsequently found antiviral efficacy in vivo after oral administration to infected mice (3 times daily/7 days), with 10 mg/kg showing the greatest therapeutic effect [52].
Other work aimed to compare the effect of three samples, propolis, Baccharis dracunculifolia (extract and essential oil), and some isolated compounds (caffeic and cinnamic acids), on poliovirus type 1 (PV1). Three protocols (pre-, simultaneous, and post-treatments) were used for evaluating the effects on the virus. For propolis, a high inhibition percentage both in simultaneous and post-treatment was recorded. Propolis partially affects both in viral cell entry and cell replication steps in the viral cycle or leads to RNA degradation before the entry of virus to cells [48].
In summary, various bioactive compounds have been identified in Brazilian propolis extract, including antiviral agents effective against different strains of the influenza virus. Three compounds, apigenin, kaempferol, and coumaric acid, were shown to significantly inhibit the infection of MDCK cells by suppressing the post adsorption and invasion stages of viral replication [56]. In general, the antiviral activities of propolis are mediated by flavonoids and other phenolic acids. These active constituents have different modes of action, such as the formation of complexes with viral proteins required for infection (masking), formation of an electron-dense layer on the cell membrane, directly damaging viral envelope proteins, and promoting viral destruction within the cell (Figure 4).

Propolis as a Treatment for COVID-19
COVID-19 is a pandemic disease caused by the recently discovered SARS-CoV-2, the seventh known member of the coronavirus family infectious to humans (after SARS coronavirus and Middle East respiratory syndrome (MERS) coronavirus) [58]. The epidemiological burden of COVID-19 is currently a major healthcare challenge throughout the world, as SARS-CoV-2 is readily transmitted from human to human via airborne micro-

Propolis as a Treatment for COVID-19
COVID-19 is a pandemic disease caused by the recently discovered SARS-CoV-2, the seventh known member of the coronavirus family infectious to humans (after SARS coronavirus and Middle East respiratory syndrome (MERS) coronavirus) [58]. The epidemiological burden of COVID-19 is currently a major healthcare challenge throughout the world, as SARS-CoV-2 is readily transmitted from human to human via airborne microdroplets generated during coughing, talking, or sneezing. In addition, SARS-CoV-2 can be transmitted by touching a contaminated surface and then touching the nose, mouth, or eyes [59,60]. While many drugs have been screened for efficacy against SARS-CoV-2 infection, no antiviral agent has yet proven broadly efficacious [61]. However, several natural product derivatives have shown promise as effective non-toxic antiviral agents [62]. Potential therapeutic agents may include honeybee products in addition to propolis, such as honey, royal jelly, bee venom, wax, bee pollen, and bee bread, as all have demonstrated antimicrobial, antifungal, anti-inflammatory, and (or) antiviral properties under certain conditions [63]. Propolis has also shown promising broad spectrum antiviral effects in vitro and in vivo against influenza virus, human respiratory syncytial and coronaviruses, rotavirus, and human rhinovirus, among others, suggesting potential efficacy against coronaviruses [16,64,65].
The potential efficacy of five propolis-derived flavonoids was recently evaluated in vitro on different DNA and RNA viruses, including coronaviruses, using the viral plaque reduction technique. Acacetin and galangin had no effect on either the infectivity or replication of any of the viruses tested, but chrysin and kaempferol were highly effective in inhibiting replication, and quercetin was active against infectivity and replication at higher concentrations [17]. Refaat et al. investigated the in vitro effects of crude Egyptian propolis extract and a propolis liposome preparation on SARS-CoV-2 3CL-like protease, S1 spike protein, and viral replication by RT-PCR. Liposomes inhibited SARS-CoV-2 3CL protease activity with an IC 50 of 1.183 ± 0.06 µg/mL, while the crude propolis extract inhibited 3CL protease activity with an IC 50 of 2.452 ± 0.11 µg/mL, values comparable to Remdesivir [18]. Sulawesi propolis and its components glyasperin A, broussoflavonol F, and sulabiroins A also inhibited SARS-CoV-2 3C-like protease activity and interacted with the protease catalytic sites His 41 and Cys 145 , with docking scores of −7.8, −7.8, and −7.6 kcal/mol, respectively [19]. Similarly, Hashem et al. evaluated the in silico inhibitory activity of six selected compounds present in propolis, 3-phenyllactic acid, CAPE, lumichrome, galangin, chrysin, and caffeic acid, against SARS-CoV-2 3CL pro and found that all six showed good docking scores, with the most potent being CAPE (−6.383 kcal/mol), chrysin (−6.097), and galangin (−6.295) [20].
Other studies have found that propolis is able to inhibit the activity of P21 (RAC1) Activated Kinase 1 (PAK1), a major "pathogenic" kinase in several diseases/disorders, including inflammation, cancer, malaria, and pandemic viral infections such as HIV, influenza, and COVID-19 [66]. Additionally, CAPE was found to bind and inhibit SARS-CoV-2 transmembrane protease serine 2 to a degree comparable with Camostat mesylate as evidenced by molecular docking and molecular dynamics (MD) simulations [34]. Moreover, quercetin alone and in conjunction with vitamin C was predicted to suppress SARS-CoV-2 infection by binding to 3C-like protease (3CL pro ) [67,68]. A pilot randomized clinical study assessed the potential efficacy of Brazilian green propolis (400 or 800 mg/day orally or via nasoenteral tube) against SARS-CoV-2 (NCT04480593). In addition, it supported the idea that propolis may be an effective agent to combat coronavirus-induced fibrosis in the lungs [65].

Immunomodulatory Activity
Although propolis has been mentioned as an immunomodulatory agent for centuries, little was known about its action until the 1990s. In the last decade, however, new and interesting articles have been published, contributing greatly to this field of research [69]. The immunomodulatory activity of propolis standard extract in allergic asthma was investigated by Piñeros [70] ( Figure 5). The chronic inflammatory disease is mediated by Th2 inflammation and an increased number of CD4 + T cells, which produces an excess of cytokines including interleukin-4 (IL-4), IL-5, and IL-13. It is also characterized by eosinophilic infiltration and mast cell activation [71,72]. Ovalbumin (OVA)-induced allergy model animals were treated daily by gavage with 150 mg/Kg of propolis for 17 days. Propolis treatment reduced pulmonary Th2 inflammation and decreased eosinophils infiltration as well as IL-5 levels in Bronchoalveolar lavage fluid (BALF). Propolis also induced the differentiation and frequency of myeloid-derived suppressor cells (MDSC) and CD4 + Foxp3 + regulatory T cells [70]. These findings are consistent with a study by Sy et al., reporting that low and high doses of propolis aqueous extract (65 mg/kg and 325 mg/kg body weight) decreased BALF IL-5 concentration, IL-6 and IL-10 production by splenocytes, and the serum levels of immunoglobulin E (IgE) and immunoglobulin G (IgG) antibodies [73]. The immunosuppressive properties of propolis have also been investigated in models of rheumatoid arthritis (RA). Dietary administration of propolis ethanolic extract (6.7 and 20 mg/g) was found to reduce the severity of this autoimmune disease in vivo by inhibiting production of IL-17 [75], a pro-inflammatory cytokine produced by Th cells (Th17 cells) strongly implicated in RA pathogenesis (e.g., joint inflammation and destruction of bone and cartilage) [76]. As such, targeting Th17 cells and targeting the IL-17 signaling pathway are potentially effective strategies for RA treatment, and indeed, such treatments are currently under investigation [77]. Okamoto et al. reported that Brazilian propolis suppressed Th17 cell activity in vitro at 12.48 µg/mL by inhibiting the IL-6-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), a key transcription factor driving Th17 cell differentiation. In addition, Th17 cell differentiation induced by transforming growth factor-β (TGF-β) plus IL-16 was downregulated by propolis in RA model animals, while propolis induced no detectable cellular toxicity at concentrations up to 96 µg/mL [78]. The propolis-derived compound CAPE has also been reported to suppress autoimmune uveoretinitis. CAPE was found to hinder T cell-dependent production of chemokines and cytokines as well as of antibodies induced by interphotoreceptor retinoid binding protein (IRBP). Treatment with 200 µL CAPE also reduced serum concentrations of TNF-α, IL-6, interferon-γ (IFN-γ), and TNF-α in the retina The immunosuppressive properties of propolis have also been investigated in models of rheumatoid arthritis (RA). Dietary administration of propolis ethanolic extract (6.7 and 20 mg/g) was found to reduce the severity of this autoimmune disease in vivo by inhibiting production of IL-17 [75], a pro-inflammatory cytokine produced by Th cells (Th17 cells) strongly implicated in RA pathogenesis (e.g., joint inflammation and destruction of bone and cartilage) [76]. As such, targeting Th17 cells and targeting the IL-17 signaling pathway are potentially effective strategies for RA treatment, and indeed, such treatments are currently under investigation [77]. Okamoto et al. reported that Brazilian propolis suppressed Th17 cell activity in vitro at 12.48 µg/mL by inhibiting the IL-6-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), a key transcription factor driving Th17 cell differentiation. In addition, Th17 cell differentiation induced by transforming growth factor-β (TGF-β) plus IL-16 was downregulated by propolis in RA model animals, while propolis induced no detectable cellular toxicity at concentrations up to 96 µg/mL [78]. The propolis-derived compound CAPE has also been reported to suppress autoimmune uveoretinitis. CAPE was found to hinder T cell-dependent production of chemokines and cytokines as well as of antibodies induced by interphotoreceptor retinoid binding protein (IRBP). Treatment with 200 µL CAPE also reduced serum concentrations of TNF-α, IL-6, interferon-γ (IFN-γ), and TNF-α in the retina and inhibited the transcriptional activity of nuclear factor-kappa B (NF-kB) and phospho-IkBα. Hence, it was concluded that the immunosuppressive activity of CAPE in uveitis is mediated by suppression of the pro-inflammatory NF-kB-cytokine pathway [79].
Propolis extracts and derivatives may also augment microbe-induced immune responses by modulating Toll-like receptor (TLR) signaling. Toll-like receptors recognize pathogen-associated molecular patterns (PAMPs), conserved molecules expressed by many microorganisms [80]. Toll-like receptor 2 (TLR2), for instance, recognizes lipoteichoic acid on Gram-positive bacteria and fungi, while TLR-4 recognizes lipopolysaccharide on Gramnegative bacteria [81]. Toll-like receptors are mainly expressed by antigen-presenting cells (APCs), including monocytes, macrophages, B cells, and dendritic cells (DCs) [82]. These cells also express human leukocyte antigen-DR isotype (HLA-DR) and cluster of differentiation 80 (CD80) molecules that present peptides to T cells, resulting in T cell activation. Additionally, TLR signal transduction may activate transcription factors controlling the expression of genes encoding chemokines, cytokines, and antimicrobial peptides [83].
Treatment of BALB/c mice with 200 mg/kg of 30% propolis ethanolic extract for three consecutive days increased expression of TLR-2 and TLR-4 by peritoneal macrophages and spleen cells and elevated the production of IL-1β and IL-6 [84]. In another study, propolis treatment of mice prevented the inhibition of TLR-2 and TLR-4 induced by 14 days of restraint stress. Additionally, real-time polymerase chain reaction (RT-PCR) revealed a significant increase in TLR gene expression in mice receiving propolis treatment without stress [81]. Propolis treatment (10, 20, and 40 µg/mL) also increased the expression of TLR-4 and CD8 by human DCs through a mechanism involving has-miR-155, resulting in enhanced bactericidal activity against Streptococcus mutans, and promoted the production of NF-kB, TNF-α, IL-6, and IL-10 [85]. Conversely, cinnamic acid (5-100 µg/mL) downregulated the expression levels of TLR-2, HLA-DR, and CD80 by human monocytes, although this treatment upregulated TLR-4. High concentrations of cinnamic acid also inhibited expression of TNF-α and IL-10. As TNF-α is known to activate monocytes and macrophages, while IL-10 inhibits these cells, Conti et al. concluded that the increase in fungicidal activity could be due to mechanisms involving other cytokines [83]. Following a similar protocol, Búfalo et al. found that caffeic acid inhibited the expression of TLR-2 and HLA-DR, while CD80 and TLR-4 were not affected. The fungicidal activity of monocytes increased, however, despite the decrease in TNF-α and IL-10 [86].
The in vivo antileishmanial effect of Brazilian propolis was reported for the first time by Pontin et al., that is, an administration of the hydroalcoholic extract at a dose of 1.5 mg/kg/day reduced the lesion diameter in leishmania braziliensis infected albino mice by 90% after 90 days of treatment. Pontin et al. pointed out that the reduction could be a result of the activation of macrophages and their phagocytic capacity [87]. Consistent with this explanation, da Silva et al. found that 5 and 10 µg/mL propolis activated macrophage phagocytic activity and in turn increased parasite interiorization. This upregulation of macrophage activity was attributed to increased TNF-α and reduced IL-12 signaling. Morphological changes in promastigote forms of Leishmania were also observed by scanning electron microscopy upon treatment [88]. Additionally, propolis was found to regulate the expression of CCL5 and IFN-γ, factors involved in the development of Th1 cells in leishmaniasis patients. Leishmaniasis is usually associated with the development of a strong Th1 response that impairs the wound healing process [89]. In another study by dos Santos Thomazelli et al., the hydroalcoholic extract showed an immunomodulatory effect on both healthy donors and American tegumentar leishmaniasis patients' human-derived peripheral blood mononuclear cells (PBMC) in leishmaniasis models. This impact was explained by the increase in IL-4 and IL-17 and a decrease in IL-10 in a dose-dependent manner. On the other hand, nitric oxide (NO) levels remained constant [90].
Numerous studies have also suggested that propolis extracts can suppress tumor growth or promote immune-mediated tumor destruction. Benkovic et al. examined the possible synergistic effect of a water-soluble derivative of propolis (WSDP) and ethanolic extract of propolis (EEP) with the anticancer drug irinotecan in Swiss albino mice inoculated with Ehrlich ascites tumor (EAT) cells. Intraperitoneal injection of WSDP and EEP at 100 mg/kg for three days prior to 50 mg/kg irinotecan injection enhanced the antitumor efficacy and reduced the non-target cytotoxicity of irinotecan compared to irinotecan alone or the combination of irinotecan with the phenolic compounds quercetin and naringin [91]. Further investigation revealed that the decrease in irinotecan-induced non-target cytotoxicity was due to the immunomodulatory properties of propolis. Pretreatment with WSDP+EEP activated macrophages and increased the number of neutrophils in the peritoneal cavity [92]. Oršolić et al. [93] also reported that WSDP at 50 or 150 mg/kg suppressed metastasis and tumor development in mice transplanted with mammary carcinoma cells. This antimetastatic effect was associated with macrophage activation and ensuing nonspecific tumor resistance. Additionally, high levels of lymphocyte activating factor (LAF) produced by these activated macrophages increased tumor cell killing efficiency. Furthermore, WSDP significantly increased the expression of CD4 + and CD8 + by splenocytes [93]. It was concluded that the antitumor activity of WSDP is likely due to the synergistic effects of constituent polyphenolic compounds such as caffeic acid, quercetin, chrysin, and naringenin, and it was further proposed that these compounds interfere with tumor growth by enhancing apoptosis, macrophage activation, and production of proinflammatory cytokines such as IL-1, IL-6, IL-8, TNF-α, and NO, some of which can directly damage tumor cells, whereas others act indirectly by enhancing the activities of natural killer (NK) cells and cytotoxic T lymphocytes. Furthermore, these factors stimulate the production of complement factor C3 production and C-reactive protein, which participate in the opsonization of tumor cells [94][95][96].
Propolis was also shown to reduce the severity of Aujeszky disease when used as a vaccine adjuvant. Mice treated with 5 mg propolis extract, aluminum hydroxide Al(OH) 3 , and inactivated Suid herpesvirus type 1 (SuHV-1) demonstrated significantly higher neutralizing antibody titers than mice receiving vaccine without propolis, indicating that the adjuvant properties of propolis are associated with enhanced humoral and cellular immunity related to increased IFN- ingin [91]. Further investigation revealed that the decrease in irinotecan-induced non-target cytotoxicity was due to the immunomodulatory properties of propolis. Pretreatment with WSDP+EEP activated macrophages and increased the number of neutrophils in the peritoneal cavity [92]. Oršolić et al. [93] also reported that WSDP at 50 or 150 mg/kg suppressed metastasis and tumor development in mice transplanted with mammary carcinoma cells. This antimetastatic effect was associated with macrophage activation and ensuing nonspecific tumor resistance. Additionally, high levels of lymphocyte activating factor (LAF) produced by these activated macrophages increased tumor cell killing efficiency. Furthermore, WSDP significantly increased the expression of CD4 + and CD8 + by splenocytes [93]. It was concluded that the antitumor activity of WSDP is likely due to the synergistic effects of constituent polyphenolic compounds such as caffeic acid, quercetin, chrysin, and naringenin, and it was further proposed that these compounds interfere with tumor growth by enhancing apoptosis, macrophage activation, and production of proinflammatory cytokines such as IL-1, IL-6, IL-8, TNF-α, and NO, some of which can directly damage tumor cells, whereas others act indirectly by enhancing the activities of natural killer (NK) cells and cytotoxic T lymphocytes. Furthermore, these factors stimulate the production of complement factor C3 production and C-reactive protein, which participate in the opsonization of tumor cells [94][95][96]. Propolis was also shown to reduce the severity of Aujeszky disease when used as a vaccine adjuvant. Mice treated with 5 mg propolis extract, aluminum hydroxide Al(OH)3, and inactivated Suid herpesvirus type 1 (SuHV-1) demonstrated significantly higher neutralizing antibody titers than mice receiving vaccine without propolis, indicating that the adjuvant properties of propolis are associated with enhanced humoral and cellular immunity related to increased IFN-ɣ mRNA production. Moreover, expression of mRNA IFN-ɣ was even higher when propolis was conjugated with antigen [97]. Although numerous preclinical studies have shown the potential efficacy of propolis against immunological diseases, standardized quality controls and well-designed clinical trials are needed before propolis or its components can be adopted as therapeutics (Table 3) [98]. ingin [91]. Further investigation revealed th get cytotoxicity was due to the immunomo with WSDP+EEP activated macrophages an peritoneal cavity [92]. Oršolić et al. [93] also pressed metastasis and tumor developmen noma cells. This antimetastatic effect was as suing nonspecific tumor resistance. Addition tor (LAF) produced by these activated m ciency. Furthermore, WSDP significantly in splenocytes [93]. It was concluded that the a synergistic effects of constituent polypheno chrysin, and naringenin, and it was further p tumor growth by enhancing apoptosis, ma inflammatory cytokines such as IL-1, IL-6, rectly damage tumor cells, whereas others natural killer (NK) cells and cytotoxic T lym the production of complement factor C3 pro ipate in the opsonization of tumor cells [94-Propolis was also shown to reduce the vaccine adjuvant. Mice treated with 5 mg pr and inactivated Suid herpesvirus type 1 (Su tralizing antibody titers than mice receiving adjuvant properties of propolis are associa munity related to increased IFN-ɣ mRNA IFN-ɣ was even higher when propolis was merous preclinical studies have shown the p logical diseases, standardized quality contro before propolis or its components can be ad was even higher when propolis was conjugated with antigen [97]. Although numerous preclinical studies have shown the potential efficacy of propolis against immunological diseases, standardized quality controls and well-designed clinical trials are needed before propolis or its components can be adopted as therapeutics (Table 3) [98].   -Increases IL-1β production and TLR-2 and TLR-4 expression in peritoneal macrophages and spleen cells; -IL-6 production was also upregulated in the spleen cells.

Clinical Applications of Propolis as an Antiviral and Immunomodulatory Agents
According to a recent review, six separate trials have found that propolis possesses better antiviral efficacy against herpes viruses than acyclovir (summarized in Table 4) [121]. A randomized, single-blind study involving 90 men and women diagnosed with HSV type 2 reported that a significantly greater number of patients treated with propolis ointment containing natural flavonoids (24 of 30) achieved symptom amelioration compared to patients receiving acyclovir (14 of 30) or vehicle (12 of 30) as determined by gynecologists, dermatovenerologists, or urologists, with no difference in medication-related adverse effects (Table 4) [122]. Table 4. List of clinical application of propolis as anti-viral and immunomodulatory agents.

Dose/Administration Route References
Anti-viral/genital herpes (HSV-2) Heal genital herpetic lesions and reduce local symptoms Randomized controlled trials/90 p Four times daily for 10 days/topical [122] Anti-viral/HSV-1 Treat herpetic skin lesions NR Propolis 3%/topical [121] Anti-viral/Herpes zoster Heal skin lesion and reduce pain Clinical trial/60 p Propolis lotion (3 times/day topical) + Acyclovir (400 or 800 mg oral) for 28 days [123,124] Anti-viral/Herpes labialis Similarly, a propolis lotion produced significantly greater healing rates than a propolisfree lotion (placebo control) following Herpes zoster virus infection (p < 0.001 for pain reduction at all visits, reduced new vesicles on day 7 of treatment, and greater global efficacy on the last (28th) day of treatment) with excellent skin tolerability and no allergic reactions, skin irritations, or other adverse events [123]. Holcová et al. utilized three different concentrations (0.1%, 0.5%, and 1%) of propolis special extract GH 2002 in a lip balm through a double-blind, randomized dermatological study involving 150 patients infected with Herpes labialis, and all three concentrations of propolis proved to be effective against Herpes labialis (p < 0.0005) for painless incrustation and local pains, but good tolerability was observed with the 0.5% concentration [125]. Another study conducted by Arenberger et al. showed the ability of propolis special extract (GH 2002) at 0.5% to treat episodes of herpes labialis virus versus 5% Acyclovir cream (p < 0.0001), and no allergic reactions, local irritations, or other adverse effects were observed (Table 4) [126].
Furthermore, propolis extract was evaluated in another double-blind, randomized, placebo-controlled trial, which reported the ability of propolis (200 mg three times daily for 7 days) to treat patients with dengue hemorrhagic fever virus, a faster recovery in platelet counts (p = 0.006), a greater decline in circulating TNF level (p = 0.018), and a shorter hospitalization period compared with placebo-treated patients (p = 0.012) ( Table 4) [3].
Although propolis has plenty of biological and pharmacological properties, there is a lack of clinical reports on the effectiveness of propolis. Furthermore, some research has indicated that propolis is unsafe because it induces hypersensitivity and might induce adverse reactions such as allergic cheilitis and oral ulceration (Table 4) [128].

In Silico Drug Discovery
In seeking potential natural products to combat COVID-19 disease, the molecular docking technique was applied to portend the binding modes and affinities of 40 propolis derivatives towards five viral targets and one human target, namely 3CL pro , RNA-dependent RNA polymerase (RdRp), papain-like protease (PLpro), receptor-binding domain (RBD) of the spike protein (S-protein), helicase (NSP13), and human angiotensin-converting enzyme 2 (ACE2). The technical details of the employed molecular docking calculations are described in References [129][130][131][132][133][134][135][136]. In brief, the crystal structures of SARS-CoV-2 3CL pro (PDB code: 6LU7) [137], RdRp (PDB code: 6M71) [138], PL pro (PDB code: 6W9C) [139], RBD (PDB code: 6M0J) [140], and NSP13 (PDB code: 5RMM) [141] were opted for as templates for all molecular docking calculations. For human ACE2, the 3D structure was taken from PDB code 6M0J [140]. For target preparation, all crystallographic water molecules, ions, heteroatoms, and ligands, if existing, were removed. All missing amino acid residues were constructed with the help of Modeller software [142]. Furthermore, the protonation states of the viral and human targets were inspected utilizing the H ++ web server, and all missing hydrogen atoms were inserted [143]. The pdbqt files for the viral and human targets were then prepared in accordance with the AutoDock protocol [144]. The chemical structures of the investigated propolis derivatives were retrieved in SDF format from the PubChem database (https://pubchem.ncbi.nlm.nih.gov, accessed on 23 March 2021). Omega2 (software version 2.5.1.4, OpenEye Scientific Software, Inc., Santa Fe, NM, USA) was applied to generate the 3D structures of the investigated compounds [145,146]. All compounds were subsequently energetically minimized using the Merck Molecular Force Field 94 (MMFF94S) implemented inside SZYBKI software [147,148]. All molecular docking calculations were conducted using AutoDock4.2.6 software [149]. The maximum number of energy evaluations (eval) and the genetic-algorithm number (GA) were set to 25,000,000 and 250, respectively. All other docking parameters were kept at their default values. The docking grid box dimensions were set to 60 Å × 60 Å × 60 Å with a spacing value of 0.375 Å to encompass the active sites of the viral and human targets. The grid center was located at the center of the binding pockets of the targets. The Gasteiger method was applied to assign the atomic partial charges of the investigated propolis derivatives [150]. To predict the pharmacokinetic properties of the identified potential anti-viral propolis derivatives, the admetSAR server (http://lmmd.ecust.edu.cn/admetsar2/, accessed on 12 April 2021) was used. The pharmacokinetic properties included Lipinski's rule of five, absorption, distribution, metabolism, excretion, and toxicity. The results gained were analyzed and compared to the reference values of the admetSAR pharmacokinetics expectation properties [151].
The docking scores and binding features of the 40 propolis derivatives against the viral and human targets were predicted and summarized in Table 5. For comparison purposes, the corresponding data for darunavir and favipiravir were predicted. Three-and two-dimensional representations of binding modes of the most potent propolis derivatives inside the active site of the viral and human targets are depicted in Figure 6. What is interesting about the data in Table 5 is that most propolis derivatives demonstrated good binding affinities against SARS-CoV-2 and ACE2 targets. The estimated docking scores ranged from −5.5 to −9.4 kcal/mol, from −4.9 to −8.2 kcal/mol, from −4.5 to −7.5 kcal/mol, from −5.0 to −9.4 kcal/mol, and from −5.5 to −10.4 kcal/mol with 3CL pro , PL pro , RdRp, NSP13, and ACE2, respectively. For the viral RDB target, propolis derivatives manifested moderate binding affinities with docking scores ranged from −4.0 to −7.2 kcal/mol. The observed potentiality of propolis derivatives towards the SARS-CoV-2 and ACE2 targets could be attributed to their capability of forming several hydrogen bonds, hydrophobic interactions, van der Waals, and pi-based interactions with the proximal amino acid residues inside the active site of these scrutinized targets. Interestingly, retusapurpurin A demonstrated the highest binding affinities towards 3CL pro , RdRp, RBD, NSP13, and ACE2 with docking scores of −9.4, −7.5, −7.2, −9.4, and −10.4 kcal/mol. More precisely, retusapurpurin A forms four hydrogen bonds with the key amino acids inside the active site of 3CL pro , RdRp, and NSP13 ( Figure 6). Three hydrogen bonds were observed between retusapurpurin A and the proximal amino acid residues of ACE2, namely ASP206 (2.23 Å) and ASN210 (2.16 and 2.24 Å) ( Figure 6). However, retusapurpurin A forms only two hydrogen bonds with TYR365 (1.71 Å) and ALA366 (2.14 Å) inside the active site of RBD of S-protein ( Figure 6). For the PL pro target, baccharin displayed the highest binding affinities with a docking score of −8.2 kcal/mol. Eventually, baccharin exhibits three hydrogen bonds with ARG157 (2.17 Å), GLU196 (2.70 Å), and MET201 (2.20 Å) inside the active site of PL pro (Figure 6). Compared to retusapurpurin A and baccharin, darunavir revealed a good binding affinity towards 3CL pro , ACE2, and NSP13 with docking scores of −8.        The pharmacokinetics, physicochemical, and toxicological properties, as summarized in Table 6, provide a quantitative analysis of what the human body performs to an administrated molecule. Based on Lipinski's rule of five (RO5), retusapurpurin A and baccharin obey the criteria for orally active drugs. It can be seen from the data in Table 6 that retusapurpurin A and baccharin showed a great Caco-2 permeability. Additionally, retusapurpurin A and baccharin demonstrated a great human intestinal absorption (HIA) with an estimated HIA value of ≈97% (Table 6). For the blood-brain barrier (BBB permeability), retusapurpurin A and baccharin were predicted to pass the BBB easily. Ultimately, retusapurpurin A and baccharin were noncarcinogenic and non-toxic. Overall, the predicted results displayed that the ADMET properties of retusapurpurin A and baccharin are extremely satisfying, as presented in Table 6. Consequently, retusapurpurin A and baccharin should be considered as prospective drug candidates against COVID-19.

Conclusions
Propolis is among the few natural remedies that have been utilized for centuries, and modern laboratory investigations have confirmed that the effectiveness of propolis originates from its extracts and derivatives against multiple disease models, including viral infections. These therapeutic effects are attributable to a high content with pharmacologically active molecules, mainly concentrations of bioactive flavonoids, phenolic acids, and their esters. These components have significant activities that target a myriad of pathological and reparative processes, including immune signaling pathways. The demonstrated efficacy against a wide range of human viruses has provided a rationale, and paved the way, for studies on the efficacy of propolis to be tested against SARS-CoV-2. Furthermore, 40 propolis derivatives have shown a high affinity SARS-CoV-2 proteins and the human target ACE2, with one, retusapurpurin A, demonstrating particularly potent binding to SARS-CoV-2 3CL pro , RdRp, ACE2, RBD, and NSP13 inhibitor. These results suggest that retusapurpurin A and other components such as baccharin are promising, and with further investigation, they could be used as potential weapons in the fight against the pandemic and as therapeutic candidates for COVID-19 treatment.
In addition to viral infection, propolis has anti-inflammatory and immunomodulatory activities. Propolis was proven to be effective through different mechanisms against several immune-mediated models of cancer and immune-related diseases, i.e., celiac disease, uveoretinitis, allergic asthma, rheumatoid arthritis, leishmaniasis, microbial infections, and cancer in vitro and in vivo. There is, however, little knowledge. While there have been few studies on the clinical efficacy of propolis and its effects on human health, further investigations are needed to determine its application. Forty propolis derivatives were investigated in in silico preparations against five SARS-CoV-2 and human ACE2 targets as anti-COVID-19 drug candidates with the help of the molecular docking technique. The binding affinities unveiled that retusapurpurin A is a potent SARS-CoV-2 3CLpro, RdRp, ACE2, RBD, and NSP13 inhibitor. However, baccharin demonstrated the highest binding affinity against SARS-CoV-2 PLpro. Moreover, drug-likeness and ADMET properties were predicted for the most potent compounds and demonstrated satisfactory pharmacokinetics, physicochemical parameters, and toxicological properties. These results suggest that retusapurpurin A and baccharin can be further investigated as convenient therapeutic treatments for COVID-19 disease, and clinical trials are feasible due to the generally good safety profiles of propolis derivatives.