Propolis: A Complex Natural Product with a Plethora of Biological Activities That Can Be Explored for Drug Development

The health industry has always used natural products as a rich, promising, and alternative source of drugs that are used in the health system. Propolis, a natural resinous product known for centuries, is a complex product obtained by honey bees from substances collected from parts of different plants, buds, and exudates in different geographic areas. Propolis has been attracting scientific attention since it has many biological and pharmacological properties, which are related to its chemical composition. Several in vitro and in vivo studies have been performed to characterize and understand the diverse bioactivities of propolis and its isolated compounds, as well as to evaluate and validate its potential. Yet, there is a lack of information concerning clinical effectiveness. The goal of this review is to discuss the potential of propolis for the development of new drugs by presenting published data concerning the chemical composition and the biological properties of this natural compound from different geographic origins.


Introduction
Over the years, nature is continually surprising with diversified natural compounds that can be promising sources for the discovery of new drugs important in medicine [1]. In fact, approximately half of the currently available drugs were obtained from natural compounds or related [2]. The use of natural products as an essential route to new pharmaceutical leads is continually growing and is a research field of enormous interest because the different structural range of natural compounds can provide lead compounds for therapeutic improvement based on rationalized molecular modifications [3,4]. It is well known that scientists have curiosity in herbs and other natural plant products for research; however only in the last years the interest in modified plant products by animals, which normally have been largely ignored and wasted, has been increased [5,6].
Propolis, a complex mixture of compounds also called bee glue, is a natural resinous product that honeybees collect from several plants and mix it with beeswax and salivary enzymes ( -glucosidase) [7][8][9][10][11]. As inferred for the meaning of the Greek word propolis-pro-, for or in defence, and polis, the city [10]-bees use propolis on their hives as protection against predators and microorganisms, to repair damage, as a thermal isolator, and to build aseptic locals to prevent microbial infection of larvae [7,9,10,12]. Since ancient times, propolis has been used by humans to meet the needs of health and food preservation [5]; but only in the last years the interest in this complex natural product has increased due to its broad spectrum of biological and pharmacological properties [12][13][14]. Propolis is a lipophilic material that is hard and breakable when cold but soft, flexible, and very sticky when warm; it possesses an enjoyable aromatic smell and different coloration, including brown, 2 Evidence-Based Complementary and Alternative Medicine green, and red, among others [5,7,12]. In terms of chemical composition, it is generally composed of 50% resin, 30% wax, 10% essential oils, 5% pollen, and 5% other substances which include minerals and organic compounds like phenolic acids (cinnamic and caffeic acid) or their esters, flavonoids (flavones, flavanones, flavonols, and dihydroflavonols chalcones), terpenes, aromatic aldehydes and alcohols, fatty acids, stilbenes, and -steroids [8,9]. Analysis of different samples revealed that propolis chemical composition is difficult to standardize because it depends on different phytogeographic characteristics like vegetation, season, and environmental conditions of the site of collection, as bees select different plants in different habitats for propolis production [6,8,9].
Several in vitro and in vivo studies have been describing the plethora of biological activities and chemical profiles of propolis from different geographic origins. This review highlights published data about such works focusing on the antimicrobial, anti-inflammatory, antioxidant, immunomodulatory, and antitumor activities of different propolis types, in order to unravel the potential of this natural compound for the development of new drugs.

Use of Propolis in Ancient and Current Times
Propolis is not a new discovery. Since bee's domestication, men explore its products to their own benefit and propolis, one of the most important chemical weapons of bees, is no exception, having been employed extensively since almost immemorial times [6,13,[15][16][17]. It is stated that propolis use dates back to ancient times, at least to 300 BC, where it was used in folk medicine and other activities in many parts of the world [16]. It was familiar to the Egyptians, in particular by the priests who controlled medicine and chemistry and knew very well propolis antiputrefactive properties. They learned from the bees the embalming capacity of propolis as this natural product is used to perform the mummification of corpses and prevent spread of infections. The Greek and the Roman physicians also acknowledged the potential of propolis by employing it in wound treatment, as an antiseptic and cicatrizing agent, and as mouth disinfectant. The Persians described propolis as a drug capable of acting against eczemas, myalgia, and rheumatism. Populations of the new world, like Incas, also used propolis as an antipyretic agent.
Between the 17th and 20th century, this natural product became very popular in Europe. In 1969, in the former Union of Soviet Socialist Republics (USSR), the use of propolis was accepted in human and veterinary medicine, with several applications including the treatment of tuberculosis, where the regression of lung problems and recovery of appetite were observed. Also, it was believed to cure some diseases in folk Georgian medicine. During World War II (1939)(1940)(1941)(1942)(1943)(1944)(1945), doctors used propolis to treat wounds [15][16][17], but only in 1985, in Japan, propolis was considered as very promising in pharmacology. Before that, propolis was considered a product without market value, especially because its production is low and affects honey production. Nowadays, propolis is an important product in alternative medicine in Japan, being widely imported from Brazil [18]. In the 17th century, the Italian Antonio Stradivari who is considered the most significant and the greatest crafter of string instruments, like violins, used propolis as an ingredient in the varnish of his instruments. Curiously, such as in the 17th century, propolis is currently used in rosin for stringed instruments and in the repair of accordions [16].
Propolis is one of the few natural products that maintained popularity for a long time, although it is not considered a therapeutic agent in conventional medicine. Actually, it is widely used as a component in pharmaceutical and cosmetic products such as antiacne creams, facial and body creams, ointments and lotions, and several formulations for oral hygiene [15,16]. It is also used in some foods and beverages, or simply as food supplement or healthy drinks. This name was attributed to the drinks because it is thought that propolis improves human health and prevents diseases such as heart disease or diabetes, among others [19].

Origin and Composition of Propolis
Propolis knowledge has registered an important evolution over time, due to exhaustive studies regarding its chemical composition and biological activities. In the 60s, it was thought that, despite its complexity, propolis chemical composition was more or less constant. Nevertheless, in the following years, analysis of a large number of samples from different geographic origins revealed that chemical composition of propolis is highly variable and also difficult to standardize because it depends on factors such as the vegetation, season, and environmental conditions of the site of collection [6,8,9]. Marcucci [7] and Bankova et al. [10] registered more than 300 substances in propolis and recent reports showed the presence of compounds never mentioned before [12,17,20,21].
Generally, the main constituents of propolis are resin and volatiles, which are substances obtained from a variety of botanical processes in different parts of plants, and beeswax, secreted by the bees [9]. Typical compounds are summarized in Table 1. As can be seen, plant sources vary among the different parts of the globe, leading to panoply of compounds. In a review on propolis standardization, different resin types were proposed: poplar propolis, birch, green, red, "Pacific, " and "Canarian" [9,12]. Samples of poplar propolis (e.g., from Europe, North America, New Zealand, and temperate zones of Asia) are mainly composed of flavonoids, phenolic acids, and their esters [10,20,22], clearly different from other propolis types (Table 1). Portuguese propolis, despite similar to the ones found in European samples, also contains new methylated, esterified, and hydroxylated derivatives of flavonoids and pinocembrin/pinobanksin derivatives containing a phenylpropanoic acid derivative moiety in their structure [20,21]. Our group recently showed that propolis from Pereiro (district of Guarda, Beira Alta) ( Figure 1) has a high concentration of phenolic components [23].

Unknown
Furofuran lignans [49,182] Evidence-Based Complementary and Alternative Medicine 5 of biological activity and chemical data. This information allows extrapolating the possible activity and mechanism of action of new propolis under study and provides substantial clues for the development of new drug candidates [6,9].

Antioxidant Activity.
It is well known that an endogenous stimuli, like cellular metabolism, and exogenous agents like UV, toxins, and drugs, among others, generate reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), the superoxide anion (O 2 − ), and hydroxyl ion (HO − ), as well as reactive nitrogen species (RNS), especially nitric oxide (NO). Carbohydrates, proteins, lipids, and nucleic acids, among other biomolecules, when exposed to the reactive species, suffer oxidative modifications that modify the cell and lead to its death [68][69][70]. Oxidative stress is responsible for the occurrence of a wide variety of human diseases, such as neurodegenerative [71] or cardiovascular diseases [72,73], cancer [68,74], diabetes [6], and atherosclerosis [75].
In the last years, several studies have been performed to evaluate the antioxidant capacity of natural products. Propolis extracts, composed of different polyphenols, have been reported to possess a potent antioxidant activity [70,76,77]. Additionally, the chemical varieties in different propolis samples from different regions have an influence on the antioxidant activity. Recently, Fabris et al. showed that a sample of Italian and Russian propolis ethanol extract (PEE), which have a similar polyphenolic composition, have a similar antioxidant activity, while Brazilian PEE, which have low polyphenolic composition, have a weak antioxidant activity [78]. Another study also showed, using different samples of Transylvania PEE, a positive correlation between high polyphenolic composition and high antioxidant activity [79]. Phenolic acids and flavonoids are characterized by a powerful antioxidant activity, which is closely related to the chemical structure of the compounds [80]. Briefly, the antioxidant activity is exerted by inhibiting the activity of some enzymes (e.g., xanthine oxidase, protein kinase C, ascorbic acid oxidase, cyclooxygenase, lipoxygenase, Na + /K + ATPase, and cAMP phosphodiesterase) which inhibit the production of ROS species; by scavenging, interrupting the reactions that lead to the lipid peroxidation; by chelating metal ions, mainly iron and copper, that are involved in the process of free radical creation; or by potentiating the action of other antioxidants [55]. Table 2 summarizes some of the studies addressing the antioxidant activity of propolis.
Moreira et al. [62] and Miguel et al. [63] proposed that Portuguese propolis, an important source of total phenols, flavones, and flavonols, could be beneficial for human health due to its antioxidant properties. Portuguese propolis also protects human erythrocytes from free radicals damaging by decreasing lipid peroxidation [64]. These studies suggest that Portuguese propolis is a powerful antioxidant agent that can be used against oxidative stress, thus maintaining the structural and functional integrity of the cells. Cuesta-Rubio et al. [36] demonstrated that nemorosone, the most abundant polyisoprenylated benzophenone present in Cuban propolis, exhibits antioxidant capacity. However, when this compound suffers methylation, a process that facilitates the separation of the compound from the propolis sample, the antioxidant property is abolished. Ethyl acetate extract of Kangaroo Island propolis, which is rich in stilbenes, showed a stronger scavenging activity [39]. Yang et al. showed that ethyl acetate of propolis collected in Anhui, China, has strong scavenging activity and ferric reducing activity, those activities being influenced by caffeic acid, phenethyl caffeate, cinnamyl caffeate, and benzyl caffeate [81]. Another study showed that samples of methanolic extracts of Algerian propolis that contains high amounts of caffeic acid esters and flavanones, kaempferol, and galangin possess strong scavenging activity and ferric reducing activity [31]. Uruguay propolis with high polyphenolic composition inhibits lowdensity lipoprotein (LDL) peroxidation and protein nitration in vitro. Moreover, it induces the expression of nitric oxide synthase (eNOS) and inhibits NADPH oxidase in bovine aortic endothelial cells [82]. In another study, the topical administration of Romanian PEE in mouse, either prior to or after UVB exposure, significantly attenuated the malondialdehyde (MDA) formation and restored glutathione peroxidase (GSH-Px) activity [83]. Talas et al. [84] showed that Turkish PEE has antioxidant properties in the liver tissue of NOS inhibited rats. In fact, NOS inhibition caused an increase in CAT activity and MDA levels, effect that was significantly decreased when the rats were treated with PEE. It is well known that propolis composition is variable; nevertheless, one of its major components, CAPE (caffeic acid phenethyl ester), plays an important role in the antioxidant activity [85][86][87].
Antioxidant activity is one of the most studied and important activities of propolis, though there are no studies with data on the safe dose to be used in humans. Thus, clinical studies using propolis and its active compounds are needed.

Anti-Inflammatory Activity.
Inflammation is an event that normally occurs in response to the constant exposure to environmental and endogenous stimuli as well as to accidental damage [61]. A complex cascade of chemical signals initiates after tissue injury and maintains a host response to repair the injured tissue. There are two stages of inflammation: acute and chronic. Acute inflammation is mediated through the activation of the immune system cells, which migrate to the site of damage and release growth factors, cytokines, and ROS/RNS species. Chronic inflammation occurs when the acute inflammation is not successfully resolved. This inflammatory condition plays a critical role in the pathogenesis of many diseases including atherosclerosis, cancer, asthma, Alzheimer's, and Parkinsonism [88,89].
Characteristic of European type propolis CAPE Free radical scavenging, inhibition of xanthine oxidase activity and lipid peroxidation [85] Male Wistar albino rats Maintenance of superoxide dismutase activity, decrease of xanthine oxidase activity and malondialdehyde and nitric oxidase levels [86] Peripheral blood mononuclear cells from cyclists Reduction of hyperthermia-induced survival inhibition, necrosis, superoxide production, glutathione depletion, and intracellular superoxide [87] Evidence-Based Complementary and Alternative Medicine 7 Bone marrow-derived mast cells (BMMC) from C57BL/6 mice Inhibiting IL-6, TNF-, and IL-13 gene expression in BMMC and also inhibiting the activation of IKK leading to NF-B inactivation [96] 8 Evidence-Based Complementary and Alternative Medicine mechanisms investigated with propolis and its chemical constituents. Recently, the role of the flavonoids quercetin, flavonols, and flavones in modulating inflammatory cell function was studied [92]. Funakoshi-Tago et al. [96] investigated the anti-inflammatory effects of flavonoids isolated from Nepalese PEE on the IL-33 signaling pathway. The isolated flavonoids 3 ,4 -dihydroxy-4-methoxydalbergione, 4-methoxydalbergion, cearoin, and chrysin inhibited the expression of inflammatory genes including IL-6, TNF-, and IL-13 in bone marrow-derived mast cells (BMMC) and also inhibited the activation of IKK, which leads to the degradation of I B and inhibits the activation of nuclear factor B (NF-B).
The release and oxygenation of arachidonic acid are a critical event in inflammation. Mirzoeva and Calder [90] demonstrated that propolis components such as CAPE, caffeic acid, quercetin, and naringenin, among others, inhibit the production of eicosanoids. In fact, these components significantly suppressed the lipoxygenase pathway of arachidonic acid metabolism, CAPE being the most potent modulator. Another study indicated that CAPE treatment improves hepatic steatosis induced by high-fat diet in a mouse model. This effect was attributed to the reduction of c-Jun-Nterminal kinase (JNK1/2) and NF-B activation with decrease in COX-2 expression [97]. Recently, it was reported that CAPE exhibits inhibitory effects on the production of proinflammatory cytokines (interleukin-1 (IL-1 )), tumor necrosis factor-(TNF-), and monocyte chemoattractant protein 1 (MCP-1) from lipopolysaccharide-(LPS-) stimulated RAW264.7 macrophages [98]. Machado et al. [99] showed that Brazilian green propolis water extracts (PWE) modulate an anti-inflammatory cellular response in the model of LPSinduced pulmonary inflammation by decreasing the number of macrophages and neutrophils. Additionally, it induced a reduction in the secretion of IL-6 and TNF-and an increase in TGF-and IL-10. Another study showed that Brazilian red PEE promotes a significant decrease in renal macrophage infiltration in rats with chronic kidney disease [100]. Búfalo et al. [101] demonstrated that Brazilian PEE and caffeic acid inhibited LPS-induced NO production by RAW264.7 macrophages, acting at the transcriptional level and suggesting that their anti-inflammatory effects were mediated by downregulating NF-B, p38 MAP kinase, and JNK1/2. According to Naito et al. [95], topical application of Brazilian PEE is effective in inhibiting carrageenan-induced rat hind paw edema. This sample appears to inhibit the chemotaxis of human polymorphonuclear leukocytes (PMNs), which also contributes to its anti-inflammatory effects. Another study showed the topical anti-inflammatory activity of propolis from Chile in mice ear with induced-edema. In fact, PEE from Buin, Chile, was the most active against the inflammation induced by 12-O-tetradeca-noylphorbol-13-acetate and arachidonic acid and also inhibits significantly NO release by the macrophages [102]. More recently, Boudreau et al. [103] indicated that CAPE is a potent leukotriene biosynthesis inhibitor in PMNs that blocks 5-lipoxygenase (5-LO) activity and arachidonic acid release. A Croatian PEE may improve psoriatic-like skin lesions, which were induced in the study by irritant agents like n-hexyl salicylate or di-n-propyl disulfide, by reducing the lipid peroxidation in the skin and total number of inflammatory cells in the skin and peritoneal cavity, more specially by suppressing functional activity of macrophages [104].

Immunomodulatory Activity.
Natural substances are considered alternative adjuvant therapies in the treatment of different diseases due to their immunomodulatory effects. Information about this type of activity was scarce for propolis until the 1990s; but published work in the last years has provided information about the influence of different propolis samples on the immune system [65,[105][106][107][108][109][110][111][112] (Table 4).
In a study using Brazilian green propolis, it was seen that the administration for 3 days of a PEE to male BALB/c mice modulated the activation of the initial steps of the immune response by upregulating toll-like receptor-(TLR-) 2 and toll-like receptor-4 expression and proinflammatory cytokines (IL-1 and IL-6) production by macrophages and spleen cells [105]. Another study demonstrated that Brazilian green PEE upregulates TLR-4 and CD80 expression in human monocytes as well as TNF-and IL-10 production [113]. It was also shown that caffeic acid stimulates the activity of monocytes against C. albicans but it inhibits TLR-2 and HLA-DR expression as well as TNF-and IL-10 production [114].
Additionally, Missima et al. [115] showed that Brazilian green PEE administered to stressed mice reduces the proinflammatory cytokines IL-1 and IL-6. When administered to melanoma-bearing mice submitted or not to chronic stress, it induces high levels of IL-1 and IL-6 and also stimulates Th1 cytokines production, indicating the activation of antitumour cell-mediated immunity. Bachiega et al. [116] investigated the immunomodulatory effect of propolis and cinnamic and coumaric acids on cytokines IL-1 , IL-6, and IL-10 production. Peritoneal macrophages from BALB/c mice were incubated with different concentrations of propolis (5, 50, and 100 mg/well) and coumaric and cinnamic acid (50 and 100 mg/well). Propolis and the acids stimulated IL-1 production and significantly inhibited IL-6 production. Then, after LPS incubation, the inhibitory concentrations of cinnamic and coumaric acids prevented efficiently its effects on IL-6 production, whereas propolis inhibited LPS effects both before and after its addition. Additionally, propolis and coumaric and cinnamic acids inhibited IL-10 production. A study which evaluated the effect of Brazilian green propolis on macrophage activation by H 2 O 2 and NO metabolite determination [107] showed that propolis increased H 2 O 2 generation and decreased NO generation, which favours the microbicidal activity. Recently, the immunomodulatory effect of propolis collected in Brazil was evaluated in Leishmania (Viannia) braziliensis experimental infection. Data shows that macrophages incubated with propolis showed a significant increase in interiorization and further killing of parasites. Also, an increased TNF-production was seen in propolis-pretreated mice, whereas IL-12 was downregulated during the infection [117].
The immunomodulatory action of propolis does not occur only at the macrophage level. In fact, some studies show that this action has also an effect on lymphocyte proliferation [106,108]. Sá-Nunes et al. [108] showed inhibitory Evidence-Based Complementary and Alternative Medicine 9 Table 4: Immunomodulatory activity of propolis and its chemical constituents. Upregulation of toll-like receptor-2 and receptor-4 mRNA expression [111] Male C57BL/6 mice, B16F10 cell line Stimulation of the expression and production of interleukin-2 and interleukin-10 and Th1 cytokine (interleukin-2 and IFN-) production [192] Male BALB/c mice Inhibition of Th1 cells generation; reduction of the frequency of IFN--producing CD4 + T cells under Th1-polarizing conditions [118] Male BALB/c mice Increase of H 2 O 2 generation and decreases in the NO generation in peritoneal macrophages [109] Male BALB/c mice Increase in the interiorization and killing of the parasites Leishmania (Viannia) braziliensis by macrophages; increase in TNF-production and decrease in interleukin-12 production [117] Monocytes from human blood TLR-4 and CD80 expression in human monocytes as well as TNFand IL-10 production [113] Melanoma cells (B16F10); male C57BL/6 mice Reduction of IL-1 and IL-6 in LPS-stressed mice; induction of IL-1 and IL-6 and Th1 cytokines in melanoma-bearing mice submitted or not to chronic stress [115] Brazil Purchased: Acros Organics Green propolis/B. Monocytes from human blood Downregulation of toll-like receptor-2, HLA-DR molecules from human antigen-presenting cells, and CD80; upregulation of toll-like receptor-4, inhibition of TNF-and interleukin-10 production [193] Purchased: Sigma Aldrich Co.

Cinnamic acid
Female IRC mice Increase of lymphocyte proliferation and release of cytokines interleukin-1 and interleukin-2 decrease of IFN-and TNF-levels [194] Purchased: Sigma Aldrich Co. Characteristic of European type propolis CAPE Human monocyte-derived dendritic cells (MoDCs) generated from peripheral monocytes Inhibition of IL-12 p40, IL-12 p70, IL-10, IFN--inducible protein-(IP-) 10 levels; inhibition of I B phosphorylation and NF-B activation [112] Female BALB/c mice Increase of IgM antibody production, T lymphocyte proliferation, interleukin-4 and interleukin-2 production by splenocytes, and IFNproduction [195] Human peripheral blood mononuclear cells, jurkat cells Inhibition of transcription factors NF-B and NFAT; inhibition of interleukin-2 gene transcription, interleukin-2 receptor expression, and proliferation of human T cells [94] 10 Evidence-Based Complementary and Alternative Medicine effects of Brazilian green propolis on splenocyte proliferation, effect that was attributed to flavonoids, and enhancement effects on interferon-(IFN-) production by spleen cells. CAPE displays inhibitory effects on transcription factors NF-B and NFAT and, as a consequence, inhibits IL-2 gene transcription, IL-2 receptor expression, and proliferation of human T cells [94]. This provides new information into the molecular mechanisms involved in the anti-inflammatory and immunomodulatory activities of propolis. CAPE has various biological activities but its effect on the immunomodulatory activity remains little studied. According to Wang et al. [112], CAPE can be useful in the treatment of asthma and other allergic diseases because it can inhibit cytokines and chemokines production by human monocyte-derived dendritic cells (MoDCs), which might be related to the NF-B signalling pathway. Another study demonstrated for the first time that Brazilian propolis significantly inhibited the generation of Th1 cells. Furthermore, the effects of propolis were investigated on 2,4,6-trinitrobenzene sulfonic acid-(TNBS-) induced colitis in a mouse model. Propolis reduced the frequency of IFN--producing CD4 T cells in a dose-dependent way under Th1-polarizing conditions. The inhibitory effect of propolis on Th1 differentiation was demonstrated in vivo too, and the severity of colitis in propolis-fed mice was significantly lower than that of mice fed with the control diet [118].

Antiviral Activity.
Propolis comprises a complexity of compounds which play a role in antiviral protection. Despite the few data available regarding this activity, it was shown that propolis from different geographic regions has considerable antiviral activity by acting at different levels and interfering with the replication of some viruses [12], like herpes simplex types 1 and 2, adenovirus type 2, influenza virus, or human immunodeficiency virus (HIV), among others [7,60,[119][120][121][122][123][124]. Table 5 summarizes the antiviral activity of propolis and its chemical constituents.
Schnitzler et al. [123] analysed the antiviral effect of PWE and PEE from Czech Republic as well as that of the constituents caffeic acid, p-coumaric acid, benzoic acid, galangin, pinocembrin, and chrysin, against herpes simplex virus type 1 (HSV-1) in cell culture. Both extracts exhibited high anti-HSV-1 activity when cells were treated prior to viral infection, galangin and chrysin being the main bioactive compounds. Amoros et al. [60] showed the antiviral activity of the major flavonoids of propolis, more specifically flavonols and flavones, the first being more active against HSV-1. Additionally, they analysed the effect of propolis on several DNA and RNA viruses (HSV-1, HSV-2, adenovirus type 2, vesicular stomatitis virus (VSV), and poliovirus type 2). Propolis at a concentration of 30 g/mL reduced the titer of herpes virus; however, vesicular stomatitis virus and adenovirus were less susceptible. In addition, propolis appears to exert a virucidal action on the enveloped viruses HSV and VSV [120]. Recently, it was shown that hydromethanolic extract of geopropolis from the stingless bee Scaptotrigona postica (Brazil) inhibits the HSV replication and also the entry of the virus into cells, effect that was attributed to the C-glycosyl flavones, catechin-3-O-gallate, and 3,4-dicaffeoylquinic acid [125]. According to Tait et al. [126], natural and synthetic flavonoids might interfere with picornavirus replication preventing the decapsidation of viral particles and RNA release within cells or blocking viral RNA synthesis. These authors also showed that different homoisoflavonoids have good antiviral activity against the coxsackie viruses B3, B4, and A9 and echovirus 30. Shvarzbeyn and Huleihel [127] tried to determine which step of Tax oncoprotein-induced NF-B activation is blocked by propolis and CAPE and showed that both substantially inhibited the activation of NF-B-dependent promoter by Tax. Also, they strongly prevented both Tax binding to I B and its induced degradation by Tax. Ma et al. [128] showed that nanometer propolis flavones could significantly inhibit in vitro porcine parvovirus (PPV) infecting PK-15 cells and in vivo they restrain the PPV copy in lung, gonad, and blood, decrease the impact of PPV on weight of guinea pigs, and increase hemagglutination inhibition of PPV in serum as well as improving the contents of IL-2, IL-6, and -IFN.
Over recent years, therapeutic benefits of propolis and/or its isolated compounds have been described in HIV treatment. Ito et al. [129] tested the anti-HIV activity in H9 lymphocytes of triterpenoids melliferone, moronic acid, anwuweizonic acid, and betulonic acid and four known aromatic compounds isolated from Brazilian propolis and showed that moronic acid had significant anti-HIV activity. Gekker et al. [119] assessed the anti-HIV-1 activity of propolis in CD4 + lymphocytes and microglial cell cultures, observing the inhibition of viral expression in a concentrationdependent way. The possible mechanism of propolis antiviral property was suggested to involve inhibition of viral entry.

Antimicrobial
Activity. Antimicrobial activity, one of the most studied propolis biological properties, is very well documented. This bioactivity has been largely investigated in the last years due to the need of new treatments against infectious diseases, especially with the increase of resistant pathogens to current antibiotics. Tables 6 and 7 summarise the antibacterial and antifungal activities found in propolis from different geographic origin and/or its chemical constituents.

Antibacterial Activity.
Propolis effect against several bacterial strains has evaluated [7,[56][57][58][59]130] and supported the fact that propolis is more active against Gram-positive bacteria than Gram-negative bacteria [12,59]. Briefly, data from different studies showed that propolis inhibits bacterial motility and enzyme activity, exhibits bacteriostatic activity against different bacterial genera, can be bactericidal in high concentrations, and affects cytoplasmic membrane [130].
Mirzoeva et al. [130] investigated the effect of PEE on the physiology of Bacillus subtilis, Escherichia coli, and Rhodobacter sphaeroides, proposing that propolis and some of its components, like cinnamic acid and flavonoids, affect the ion permeability of the inner bacterial membrane causing the dissipation of the membrane potential and inhibiting bacterial motility. A recent study [131] provided valuable information for understanding the potential anti-H. pylori  NanoHA matrix with red and green propolis which reduces bacterial growth and biofilm formation, the nanoHA with red propolis being the most efficient [136] Evidence-Based Complementary and Alternative Medicine 13   Inhibition of fungal growth [145] 16 Evidence-Based Complementary and Alternative Medicine mechanism of CAPE. H. pylori, a major factor for gastrointestinal illnesses, contains the enzyme H. pylori peptide deformylase that catalyses the removal of formyl group from the N-terminus of nascent polypeptide chains. Since the action of this enzyme is essential for H. pylori survival, it is considered a promising therapeutic drug target. Results from absorption spectra and crystal structural characterization showed that CAPE is a competitive inhibitor of peptide deformylase, blocking the substrate entrance and preventing substrate from approaching the active site [131]. It has been suggested that the combination of propolis with other antibiotics would allow dose reduction of selected antibiotics, thus potentiating their effect. The antibacterial activity of Italian PEE in some clinically isolated Grampositive strains, as well as the synergetic effect with some antibiotics, was assessed by Scazzocchio et al. [56]. Italian PEE drastically increased the effect of ampicillin, gentamycin, and streptomycin and moderated the action of chloramphenicol, ceftriaxone, and vancomycin. No effect was observed when used simultaneously with erythromycin. Wojtyczka et al. [132] evaluated the in vitro antimicrobial activity of a Polish PEE against methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) clinical isolates and also the combined effect of propolis with ten selected antistaphylococcal drugs. PEE displayed varying effectiveness against twelve S. aureus strains and potentiated the antimicrobial effect of eight antistaphylococcal against all tested strains. No synergism was observed in the case of ciprofloxacin and chloramphenicol. In another study, the effect of dichloromethane extract of French propolis against different human pathogenic bacterial strains was also tested. Although Gram-negative bacteria were not susceptible to the extract, a significant antibacterial activity against both methicillin-resistant and methicillin-susceptible S. aureus strains was observed [133]. The same was confirmed by Velikova et al. [28] using different PEE from Bulgaria, Greece, Turkey, and Algeria. All the samples showed a good antibacterial activity against S. aureus but a week or lacking effect against E. coli. Australian PEE from stingless bee Tetragonula carbonaria also inhibited the growth of S. aureus. Nevertheless, it was less active against P. aeruginosa [42]. The same was observed by Papachroni et al. [53] using PEE from Cameroon and Congo.Contrary to these studies, Katircioglu and Mercan [134] showed that Turkish PEE was effective against Gram-negative bacteria like E. coli. Orsi et al. [135] investigated the possible synergism between Brazilian and Bulgarian propolis and antibiotics acting on DNA (ciprofloxacin and norfloxacin) and on metabolism (cotrimoxazole) in Salmonella typhi. Both samples had antibacterial activity, but no synergistic effect was detected.
Recently, the suitability of nanohydroxyapatite-(nano-HA-) based surfaces containing two Brazilian PEE (green and red ones) to prevent S. aureus bacterial growth and biofilm was studied. The nanoHA impregnated with the two highest concentrations (12 and 25 g/mL) of red PEE showed a remarkable reduction of 99% in the number of viable bacteria, while nanoHA with green PEE at same concentrations showed a reduction of 45 and 61%. Moreover, the nanoHA impregnated with the highest concentration of red PEE was able to inhibit 80% of the staphylococcal biofilm formation [136].
Diverse studies show that as the composition of propolis varies from region to region, the antibacterial activity also displays some variations [59]. Susceptibility of different Gram-positive bacteria to PEE varies with the place of propolis collection [137]. The antibacterial effect was shown to be higher for samples from a wet-tropical rain foresttype climate. Other studies revealed the influence of propolis geographical origin on its antibacterial properties [57,58,138,139]. Propolis from the north and centre of Portugal has a great activity against S. aureus [58,140].

Antifungal Activity.
Antifungal activity is also influenced by the chemical variation of propolis [59]. Several studies have shown the effect of propolis from different geographic origin against different fungi, particularly of clinical interest [141][142][143][144][145]. Quiroga et al. [146] demonstrated the antifungal activity of propolis from the northwest of Argentina, focusing their study on the environment and the development of agrochemicals with reduced economic costs, possibly containing propolis extracts and its isolated compounds, such as pinocembrin and galangin, as active principles.
Recently, Falcão et al. [140] screened the antifungal activity of Portuguese propolis and its potential floral sources Populus x Canadensis and Cistus ladanifer against Candida albicans, Trichophyton rubrum, and Aspergillus fumigatus. Plant extracts did not exhibit relevant antifungal activity, with exception of T. rubrum, but both propolis samples revealed similar antifungal activity, the highest being obtained against T. rubrum and the lowest against A. fumigatus. A sample of PEE from Poland showed a high fungicidal activity against C. albicans, C. glabrata, and C. krusei [147]. Recently, it was shown that different organic extracts of French propolis (PEE, PWE, methanolic extract, and dichloromethane extract) were effective against C. albicans and C. glabrata but only have a weak activity towards A. fumigates [133].
Brazilian PEE was proved to be active against several Candida strains (C. albicans, C. tropicalis, C. krusei, and C. guilliermondii), C. albicans being the most sensitive and C. guilliermondii the most resistant [141]. Brazilian green and red propolis display activity against different fungal species of Trichophyton, which cause dermatophytosis, red PEE being more efficient than the green one [143]. Also, it was shown that n-hexane extract of Brazilian red propolis did not induce resistance in Candida spp. In fact it was active against Candida spp. resistant to antifungal agents, like fluconazole [148]. Dota et al. [144] evaluated the in vitro antifungal activity of PEE and propolis microparticles (PMs) obtained from a sample from Argentina against clinical yeast isolates of importance in the vulvovaginal candidiasis. C. albicans and non-C. albicans were inhibited by PEE and PMs, with small variation. Additionally, it was shown that Brazilian green PEE has the ability to inhibit growth and biofilm formation by C. albicans from vulvovaginal candidiasis [149]. Another study showed fungicide action of propolis (PEE, PWE, propolis matricial microparticles (PMM), and propolis soluble dry extract after 6-8-hour treatment against all three C. albicans morphotypes (yeast, pseudohyphae, and hyphae), PEE being the most potent followed by PSDE, PM, and PWE [150]. Brazilian propolis induces C. albicans cell death mediated via metacaspase, since the metacaspase mutant in C. albicans showed reduced sensitivity to propolis, and by the Ras pathway. Using C. albicans deletion libraries, it was possible to screen several mutants in genes involved either in the morphological transitions or in the maintenance of a specific morphotype that are more sensitive to propolis. To conclude the study, the authors also showed that propolis based gels and cream were partially able to control vulvovaginal candidiasis in a mouse model [151]. These studies [144] strongly indicate that propolis has a great potential to control vulvovaginal candidiasis, representing a promising alternative therapeutic strategy.

Antitumour Activity.
Recognition of the hallmarks of cancer affects the search and development of new methods and therapeutic agents with a sufficiently large therapeutic window to kill tumour cells while sparing normal cells. In the last years, the natural product propolis has attracted a growing interest by a large number of researchers since it contains a variety of phytochemical compounds that may act through multiple pathways to reduce the development and other malignant characteristics of cancer cells.
Recently, several in vitro studies have demonstrated a cytotoxic action of propolis from different geographic origin and of some of its isolated compounds on various tumour cells. In vivo studies also show a potential in the development of new antitumor agents, since propolis administration to mammals (e.g., rats) does not lead to detectable side effects [3]. Briefly, this natural product can block specific oncogene signalling pathways, which in turn lead to a decrease in cell proliferation and growth and can also act by decreasing the cancer stem cell population, increasing apoptosis, exerting antiangiogenic effects, and modulating the tumour microenvironment [66,152,153]. Table 8 summarizes the antitumour activity of propolis from different geographic origin and its chemical constituents.
Some researchers showed the effect of different types of propolis and its constituents on cancer cell growth, proliferation, and apoptosis. The hexane extract of propolis from Thailand, collected by the stingless bee Trigona laeviceps, which has a different behavior in propolis collection compared to the honey bees, exerts antiproliferative activity against five tested cancer cell lines (Chago, KATO-III, SW620, BT474, and Hep-G2) but not against the normal cell lines tested (HS27 fibroblast and CH-liver) [5]. A sample of PEE from Poland inhibited human malignant melanoma (Me45) and colorectal cancer (HCT 116) cells growth, as well as reduced cell size [154]. Other studies reported the antitumor activity of Brazilian PEE [155][156][157][158] which regulate the protein expression of cyclin D1, B1 and cyclin dependent kinase (CDK) as well as p21 in human prostate cancer cells, significantly affecting proliferation [158].
Although propolis containing CAPE is different from those with artepillin C, it is possible to obtain a similar inhibitory effect from both types. The effect of CAPE on different cancer cell lines was analysed and many of its effects have been shown to be mediated through inhibition of NF-B [66,159]. CAPE can inhibit the proliferation of the colorectal cell line SW480 by decreasing the -catenin, cmyc, and cyclin D1 protein expression [160]. Chuu et al. [161] observed that CAPE suppressed the proliferation of LNCaP, DU-145, and PC-3 human prostate cancer cells in a dosedependent manner and also inhibited the tumour growth of LNCaP xenografts in nude mice. It was suggested that it acted through inhibition of p70S6K (an intermediary of the PI3K/AKT pathway responsible for the protein synthesis) and some Akt signalling networks. Wu et al. [162] demonstrated that CAPE inhibits in vitro and in vivo MCF-7 and MDA-MB-231 tumour growth without much effect on normal mammary cells by reducing the expression of growth and transcription factors, including NF-B. Recently, it was demonstrated that CAPE effect on genes that are associated with tumour cell growth and survival is related in part to its role as a histone deacetylase inhibitor [163].
As previously said, propolis can also act by decreasing the cancer stem cell population. Using the putative CD44 (+)/CD24 (−/low) breast cancer stem cells able to generate mammospheres from single cells, Omene et al. [164] showed that CAPE caused a dose-dependent inhibition of cancer stem cells self-renewal, progenitor formation, and clonal growth.
Concerning cell death, some in vitro studies showed different sensitivities of tumour cells to propolis extracts. PWE from Iraq inhibits the proliferation of HL-60 cells and leads to downregulation of Bcl-2 and activation of Bax [13]. Alizadeh et al. [165] investigated the protective effects of Iranian PEE on N-methyl-N-nitro-N-nitrosoguanidine-(MNNG-) initiated gastric cancer in rats. Results showed that tumour incidence, the number of lesions, structural abnormalities, and beta-catenin of the animals group treated with PEE significantly declined compared with the control. PEE also induced the expression of proapoptotic Bax and reduced antiapoptotic Bcl-2 expression. Propolis inhibits colony formation potential and promotes necrotic changes in HCT-116 cells and decreases mitotic cells and increases p53 and Ki-67 expression in HCT-116 tumor-bearing mice [13]. Szliszka's group have performed many studies to analyze the antitumour effect of different propolis and its constituents on prostate cancer cells (LNCaP and DU145) [166][167][168]. Brazilian green PEE sensitized these cells to TRAIL-induced death, enhanced the expression of TRAIL-R2, and decreased the activity of NF-B in LNCaP cells [167]. Cotreatment of TRAIL with artepillin C induced the significant activation of caspase-8 and caspase-3, as well as a significant disruption of the mitochondrial membrane potential [169]. Many studies have been conducted to understand the pathways involved in the apoptotic effect of CAPE. CAPE induces cell cycle arrest and apoptosis and reduces expression of NF-B in MDA-MB-231 and MCF-7 human breast cancer cells [162]. In PC3 prostate cancer cells, CAPE induced apoptosis in a dose-dependent manner that was associated with the loss of expression of the inhibitors of apoptosis: cIAP-1, cIAP-2, and XIAP [170]. Cavaliere et al. [171] showed that CAPE treatment 18 Evidence-Based Complementary and Alternative Medicine    MDA-MB-231 cell line Inhibition of cancer stem cells self-renewal, progenitor formation, and clonal growth and decrease of CD44 levels [164] Human hepatocellular carcinoma cell line SK-Hep1 Suppression of the adhesion and invasion potential of the cells by inhibiting completely the expression of MMP-2 and metalloproteinase-9 (MMP-9) and the NF-B [172] Human fibrosarcoma cell line HT1080 Decrease of MMP and tissue inhibitor metalloproteinase-2 (TIMP-2) mRNA levels; downregulation of MMP-2 and MMP-9 expression; inhibition of MMP-2 activity; decrease of invasion, motility, cell migration, and colony formation [215] 20 Evidence-Based Complementary and Alternative Medicine  China; Evidence-Based Complementary and Alternative Medicine 21 of lymphoblastoid cell line PL104 induced apoptosis through the mitochondrial intrinsic pathway. It has been known that cancer microenvironment is very important for carcinogenesis and it consists of stromal, endothelial, immune, and cancer cells. Natural products, like propolis and their constituents, have been shown to interfere with this symbiosis. It was demonstrated by Lee et al. [172] that CAPE could effectively suppress the adhesion and invasion potential of human hepatocellular carcinoma cells (SK-Hep1) by inhibiting the expression of MMP-2 and MMP-9 and NF-B.
Angiogenesis has a crucial role in tumour growth due to the requirement of oxygen and nutrients to sustain rapid uncontrolled proliferation and metastization. Both tumour and stromal cells can secrete proangiogenic factors that stimulate the formation and maintenance of new vessels, such as vascular endothelial growth factor (VEGF) [173]. Brazilian PEE could significantly reduce the number of newly formed vessels and suppress the proliferation of human umbilical vein endothelial cells (HUVECs) [174], this antiangiogenic effect being mainly mediated via inducing apoptosis in tubeforming endothelial cells through the inactivation of the survival signal ERK1/2 [175]. Yun et al. [176] observed that CAPE inhibited angiogenesis using the in vivo assay chick embryo chorioallantoic membrane (CAM). CAPE also suppresses VEGF formation by MDA-MB-231 cells and formation of capillary-like tubes by endothelial cells [162,177]. Extracts of propolis containing artepillin C and CAPE significantly reduced the newly formed vessels and expression of MMPs and VEGF production from various cells [178] and, in accordance with this study, Izuta et al. [179] described that CAPE promotes inhibition of VEGF expression in HUVEC cells.
The first study on the antitumor activity of Portuguese propolis was only performed in 2010. Using normal and cancerous renal cells derived from human renal cell carcinoma (RCC) patients, in addition to A-498 cell line, Valente et al. [64] showed that methanolic extract of Portuguese propolis exhibited selective toxicity against malignant cells compared to normal cells and in vitro RCC growth was strongly inhibited. Recently, our group demonstrated the antitumour and antiangiogenic activity of the ethanol extract of Pereiro propolis (P10.EE), collected in the district of Guarda, Portugal. P10.EE affects cell viability of different tumour cells, MDA-MB-231 (breast) and DU145 (prostate) being two of the most sensitive ones, but was less cytotoxic against nontumour cells and fibroblasts. Also, it significantly decreased MDA-MB-231 and DU145 cell proliferation and migration along time, with cell cycle changes, and increased cell death. The significant increase observed in glucose consumption and lactate production could be explained in MDA-MB-231 by an increased expression of hypoxia inducible factor-1 , pyruvate dehydrogenase kinase, glucose transporter 1, lactate dehydrogenase, and carbonic anhydrase. Furthermore, P10.EE induced a decrease in HBMECs total biomass and proliferation and decreased vessel sprouting in the chicken chorioallantoic membrane [23].

Conclusions
From ancient to modern times, herbs and other plant products have been widely used as medicinal agents, first in folk medicine and other activities in many parts of the world and later developed and improved on a scientific basis into drugs that are used in the health system. Propolis is one of the few natural products that has maintained its popularity over a long period of time. As reviewed here, propolis contains a broad spectrum of compounds that may be useful in the treatment of different pathological conditions. In fact there is much literature that deals with the in vitro and in vivo biological properties of propolis. This wide range of bioactivities, the continuous discovery of new compounds, the long history of propolis use, and its safety profile make propolis a potential candidate for drug discovery that may be useful in several clinical scenarios. Nevertheless, it is necessary to make an effort to standardize propolis composition since it seems that propolis biological properties and chemical composition not only are variable but also are strictly linked. In our opinion, propolis extracts may be important economically and would allow a relatively inexpensive treatment in different diseases; however, to promote its use in modern medicine, it will be necessary to identify and isolate the bioactive compounds to be tested separately or in combination with other drugs already available.
Drug discovery does not consist only of the isolation of bioactive lead compounds from the natural sources. In fact, this process continues outside the academic laboratories through preclinical studies followed by clinical trials. Thus, despite the in vitro and in vivo assays, which provide new valuable information on propolis biological properties and mechanisms of action, it will be necessary to analyse the effectiveness of propolis clinically, to complement the basic research available, and to evaluate the potential of propolis in human health promotion.