**3.1. Antimicrobial activity**

Antimicrobial properties of Brazilian propolis are well‐documented, including the antibacte‐ rial, antifungal, and antiviral activities. The biological activities of propolis are related to its chemical composition that varies with the collection period of the resin and the flora of the region visited by bees [46]. Therefore, in Brazil there are different types of propolis, since the different geographical regions of the country have a diversity of plant species. The most popular types of Brazilian propolis are green and red propolis.

Brazilian green propolis, whose most important plant source is *B. dracunculifolia*, has been extensively studied. Several studies have shown the activity of green propolis against several pathogenic bacteria, including Gram‐positive bacteria (*S. aureus, Staphylococcus epidermidis, Streptococcus pneumoniae*, and *Kocuria rhizophila*) and Gram‐negative bacteria (*Haemophilus influenzae, Porphyromonas gingivalis, Porphyromonas endodontalis*, and *Prevotella denticola*) [9, 10, 46–48]. The last three bacteria cause periodontal diseases, which affect the periodontal tissues (tooth supporting tissues). Furthermore, green propolis is active against cariogenic bacteria, such as *Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Streptococcus sanguinis*, and *Lactobacillus casei* [48, 49]. However, some Gram‐negative bacteria are not susceptible to green propolis, such as *Escherichia coli* and *Pseudomonas aeruginosa* [9, 10, 46]. *E. coli* can cause urinary tract infections and gastroenteritis, among others, while *P. aeruginosa* is associated with nosocomial infections, since it is an opportunistic bacterium.

The main compounds identified were: coniferaldehyde, 2,2‐dimethyl‐6‐carboxyethenyl‐2h‐1‐ benzopyran, drupanin, pinocembrin, dicaffeoylquinic acid, and artepillin C, isocupressic acid, acetylisocupressic acid, imbricatoloic acid and a mixture of *cis* and *trans* isomers of communic

Among the numerous biological effects reported for brown propolis and its isolated com‐ pounds, it has been observed that both brown propolis and some of its isolated compounds have antimicrobial effect. In addition, it was possible to determine which compounds are responsible for such activity, highlighting the importance of chemically know product widely used by population [38, 41]. Moreover, brown propolis, as well as green propolis, has a

Brown propolis collected in Mato Grosso do Sul due to the significant amount of phenolic compounds in ethanol extract shows high antioxidant and antigenotoxic activities. Its volatile fraction is composed mainly of the sesquiterpenes spathulenol and (E)‐nerolidol (**Figure 3**), which show an antimicrobial effect against *Cryptococcus neoformans, Enterococcus faecalis*, and *Staphylococcus aureus*. They were not mutagenic, considering that the antimicrobial activity is not because of DNA damage induction [43, 44]. The brown propolis collected from Mato

Therefore, considering that Brazil has a unique flora, among all types of Brazilian propolis three types of propolis are highly noticeable: green, red, and brown propolis due to their singular chemical composition, leading to their biological effects, culminating in the high value

Antimicrobial properties of Brazilian propolis are well‐documented, including the antibacte‐ rial, antifungal, and antiviral activities. The biological activities of propolis are related to its chemical composition that varies with the collection period of the resin and the flora of the region visited by bees [46]. Therefore, in Brazil there are different types of propolis, since the different geographical regions of the country have a diversity of plant species. The most

Brazilian green propolis, whose most important plant source is *B. dracunculifolia*, has been extensively studied. Several studies have shown the activity of green propolis against several pathogenic bacteria, including Gram‐positive bacteria (*S. aureus, Staphylococcus epidermidis, Streptococcus pneumoniae*, and *Kocuria rhizophila*) and Gram‐negative bacteria (*Haemophilus influenzae, Porphyromonas gingivalis, Porphyromonas endodontalis*, and *Prevotella denticola*) [9, 10, 46–48]. The last three bacteria cause periodontal diseases, which affect the periodontal tissues (tooth supporting tissues). Furthermore, green propolis is active against cariogenic bacteria, such as *Streptococcus mutans, Streptococcus sobrinus, Streptococcus salivarius, Streptococcus sanguinis*, and *Lactobacillus casei* [48, 49]. However, some Gram‐negative bacteria are not

significant preventive effect against oxidative stress in skin [42].

64 Superfood and Functional Food - An Overview of Their Processing and Utilization

Grosso also showed antimicrobial activity [45].

**3. Biological properties**

**3.1. Antimicrobial activity**

in the international market of Brazilian bee products.

popular types of Brazilian propolis are green and red propolis.

acid [38–40] (**Figure 8**).

Antifungal activity of green propolis has been reported against all three morphotypes of *Candida albicans* (yeast, pseudohyphae, and hyphae) [50]. At the cellular level, green propolis is able to induce apoptosis and secondary necrosis in yeasts, as showed in a study using *Saccharomyces cerevisiae* as a model organism [51]. Green propolis is also active against filamentous fungi (molds), such as *Trichophyton rubrum, Trichophyton tonsurans*, and *Trichophyton mentagrophytes* [52], which cause dermatophytosis. Ngatu et al. [53] reported the antimy‐ cotic effect of green propolis in patients with tinea pedis interdigitalis and tinea corporis caused by *T. rubrum*.

Green propolis also has the capacity to inhibit virus propagation. Shimizu et al. [54] reported that the ethanol extract of green propolis exhibited moderate efficacy in limiting herpetic skin lesions in mice infected with herpes simplex virus type 1 (HSV‐1). Urushisaki et al. [17] showed the anti‐influenza effect (H1N1 influenza virus) of the water extract of green propolis and its caffeoylquinic acids, which may have a cytoprotective action by affecting the internal cellular process. Takemura et al. [55] also reported the anti‐influenza effect of the water and ethanol extracts of green propolis and their 3,4‐dicaffeoylquinic acid, which enhance viral clearance by increasing tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) in the lungs of mice infected with H1N1 influenza virus.

Batch‐to‐batch variability is a common problem in the manufacture of propolis extracts. Since medicinal use of these extracts must rely on appropriate quality requisites, batch‐to‐batch reproducibility is essential to ensure consistent quality. Therefore, Berretta et al. [8] developed the propolis standardized extract (EPP‐AF®), an ethanolic extract which contains green propolis and has batch‐to‐batch chemical reproducibility. Furthermore, it has several biological activities, including antibacterial and wound‐healing activities [8]. **Figure 9** shows some results obtained by our research group, showing the antibacterial activity of EPP‐AF® and extracts of brown, red and green propolis.

**Figure 9.** Zones of inhibition (disk diffusion method) provided by 1: Extract of brown propolis from the south of Brazil; 2: Extract of red propolis from the northeast of Brazil; 3: Extract of green propolis from the southeast of Brazil; 4: Prop‐ olis standardized extract (EPP‐AF®); (a): Staphylococcus aureus ATCC 25923; (b): Streptococcus neumonia ATCC 49619; (c): Klebsiella neumonia ATCC 10031.

Our research group also has developed and evaluated different pharmaceutical forms of green propolis extracts, including propolis ethanolic extract (PEE), propolis water extract (PWE), propolis soluble dry extract (PSDE), and propolis matricial microparticles (PMM). With respect to antifungal activity (*S. cerevisiae* and *C. albicans*), PEE was the most potent followed by PWE, PMM, and PSDE [50]. The same results were obtained against *Lactobacillus* species (Gram‐ positive bacteria) (data not published yet).

Brazilian red propolis, in its turn, is produced from resinous exudates of *D. ecastophyllum*, found mainly in Northeastern Brazil (states of Alagoas, Bahia, Paraíba, Pernambuco, and Sergipe) [56]. Ethanolic extracts of red propolis showed activity against Gram‐positive bacteria (*S. aureus* and *Bacillus subtilis*) and Gram‐negative bacteria (*E. coli* and *P. aeruginosa*) [56–58]. These results are very interesting, since green propolis is not active against *E. coli* and *P. aeruginosa* [9, 10, 46]. Red propolis also has antifungal activity. Siqueira et al. [52] reported its activity against some dermatophytes (*T. rubrum, T. tonsurans*, and *T. mentagrophytes*).

Isoflavone formononetin is one of main chemical compounds in red propolis. Das Neves et al. [59] evaluated the activity of this compound against some bacteria (*S. aureus, S. epidermidis*, and *P. aeruginosa*) and yeasts (*C. albicans, Candida tropicalis*, and *C. neoformans*). The MIC value was 200 μg/ml for all bacteria and 25 μg/ml for the yeasts [59]. (6aS,11aS)‐Medicarpin is the other chemical compound in red propolis, which exhibits a strong antibacterial activity, since MIC values of 16, 32 and 32 μg/ml were obtained against *S. aureus, B. subtilis*, and *P. aeruginosa*, respectively [57].

Kamuyama et al. [60] evaluated the use of green propolis to control microorganisms in minimally processed carrot. The study involved the comparison between: (i) carrot sanitation with 200 mg/l of total available chlorine, (ii) chlorinated solution "A" together with edible film with 0.4% propolis solution, and (iii) carrot sanitation with 0.4% propolis solution, prepared from 25% propolis alcoholic extract. Mesophilic and psychrotrophic aerobic bacteria, mold, and yeast were counted during the storage of samples of processed carrots at 10°C. The results demonstrated that the results for all treatments were similar to mesophilic and psychrotrophic bacteria. For mold and yeast count, the application of treatments (ii) and (iii), in the end of study, was similar to T0, suggesting that the use of propolis as a food preservative is viable and promising.

Borges et al. [61] evaluated the antibacterial and antifungal properties of different concentra‐ tions of a propolis hydroalcoholic extract in fresh pork sausage. This product is target of microbiological contamination, with consequent commitment of "shelf‐life" and ability to cause diseases, factors that stimulate food companies to use synthetic preservatives as sodium nitrate, which possess high toxicity.

Interestingly, the results demonstrated that propolis extract (0.03 g/100 g of food) used showed greater antibacterial and antifungal results when compared to sodium nitrate.

#### **3.2. Antioxidant activity**

The propolis antioxidant property is one of the most studied biological activities worldwide. This biological property presents outstanding importance in the general benefits that propolis may bring to human health as the free‐radical scavenging capacity of propolis compounds may be closely related to the anti‐inflammatory, antimicrobial, anticancer activities, as well as, prevention of atherosclerosis, skin damages, ageing, and among others.

Our research group also has developed and evaluated different pharmaceutical forms of green propolis extracts, including propolis ethanolic extract (PEE), propolis water extract (PWE), propolis soluble dry extract (PSDE), and propolis matricial microparticles (PMM). With respect to antifungal activity (*S. cerevisiae* and *C. albicans*), PEE was the most potent followed by PWE, PMM, and PSDE [50]. The same results were obtained against *Lactobacillus* species (Gram‐

Brazilian red propolis, in its turn, is produced from resinous exudates of *D. ecastophyllum*, found mainly in Northeastern Brazil (states of Alagoas, Bahia, Paraíba, Pernambuco, and Sergipe) [56]. Ethanolic extracts of red propolis showed activity against Gram‐positive bacteria (*S. aureus* and *Bacillus subtilis*) and Gram‐negative bacteria (*E. coli* and *P. aeruginosa*) [56–58]. These results are very interesting, since green propolis is not active against *E. coli* and *P. aeruginosa* [9, 10, 46]. Red propolis also has antifungal activity. Siqueira et al. [52] reported its activity against some dermatophytes (*T. rubrum, T. tonsurans*, and *T. mentagrophytes*).

Isoflavone formononetin is one of main chemical compounds in red propolis. Das Neves et al. [59] evaluated the activity of this compound against some bacteria (*S. aureus, S. epidermidis*, and *P. aeruginosa*) and yeasts (*C. albicans, Candida tropicalis*, and *C. neoformans*). The MIC value was 200 μg/ml for all bacteria and 25 μg/ml for the yeasts [59]. (6aS,11aS)‐Medicarpin is the other chemical compound in red propolis, which exhibits a strong antibacterial activity, since MIC values of 16, 32 and 32 μg/ml were obtained against *S. aureus, B. subtilis*, and *P. aerugino-*

Kamuyama et al. [60] evaluated the use of green propolis to control microorganisms in minimally processed carrot. The study involved the comparison between: (i) carrot sanitation with 200 mg/l of total available chlorine, (ii) chlorinated solution "A" together with edible film with 0.4% propolis solution, and (iii) carrot sanitation with 0.4% propolis solution, prepared from 25% propolis alcoholic extract. Mesophilic and psychrotrophic aerobic bacteria, mold, and yeast were counted during the storage of samples of processed carrots at 10°C. The results demonstrated that the results for all treatments were similar to mesophilic and psychrotrophic bacteria. For mold and yeast count, the application of treatments (ii) and (iii), in the end of study, was similar to T0, suggesting that the use of propolis as a food preservative is viable

Borges et al. [61] evaluated the antibacterial and antifungal properties of different concentra‐ tions of a propolis hydroalcoholic extract in fresh pork sausage. This product is target of microbiological contamination, with consequent commitment of "shelf‐life" and ability to cause diseases, factors that stimulate food companies to use synthetic preservatives as sodium

Interestingly, the results demonstrated that propolis extract (0.03 g/100 g of food) used showed

The propolis antioxidant property is one of the most studied biological activities worldwide. This biological property presents outstanding importance in the general benefits that propolis

greater antibacterial and antifungal results when compared to sodium nitrate.

positive bacteria) (data not published yet).

66 Superfood and Functional Food - An Overview of Their Processing and Utilization

*sa*, respectively [57].

and promising.

nitrate, which possess high toxicity.

**3.2. Antioxidant activity**

Several antioxidant methods are available to study propolis, i.e., the DPPH assay, scavenging of hydroxyl radical by the deoxyribose assay, inhibition of lipid peroxidation, inhibition of chemiluminescence produced in the H2O2/luminol/horseradish peroxide (HRP) system and inhibition of chemiluminescence produced in the xanthine/luminol/xanthine oxidase (XOD) system, and among others.


**Table 2.** Antioxidant activity of propolis from several regions in the world.

Some of these methods mimic the physiological conditions found in human body, this is the case of lipid peroxidation assay, in which membrane fractions (from mitochondria or brain) are used. In this method, the addition of iron salts triggers the decomposition of lipid peroxides into peroxyl (LOO•) and alkoxyl (LO•) radicals that can abstract hydrogen from polyunsa‐ turated acyl chains and propagate lipid peroxidation. Any antioxidant capable of scavenging LOO• and LO• will decrease peroxidation. However, other methods can be considered more accessible, such as DPPH, which can easily demonstrate, in large scale, the antioxidant capacity of propolis samples from different sources or different batches. According to Marquele‐ Oliveira et al. [62], this method could even be employed as an alternative for worldwide characterization and standardization of natural products. A good correlation of the DPPH method was observed against lipid peroxidation assay. This assay is based on the ability of DPPH, a stable free radical, to be quenched and thereby decolorize in the presence of antiox‐ idants resulting in a reduction in absorbance values. In the DPPH test, the antioxidants reduce the DPPH radical to a yellow‐colored compound, diphenylpicrylhydrazine. The extension of the reaction will depend on the hydrogen‐donating ability of the antioxidants [63].

Propolis antioxidant properties have been fully investigated and both propolis raw material and propolis commercial extracts have been studied. **Table 2** shows examples of the antioxi‐ dant profile and the method employed for each sample, focusing on their collecting origin. Phenolic compounds have been reported as the main propolis compounds responsible for the antioxidant property. The antioxidant role of polyphenols results from the donation of hydrogen atoms from an aromatic hydroxyl group to the free radical, leading to stabilization of the radical [64]. During the evaluation of propolis fractions (from Brazil), Wang et al. [65] observed a strong inhibition of lipid peroxidation using rat liver homogenate at a concentration of 2 mg/ml, and this activity was related to the presence of flavonoids. However, it is known that other than phenolic compounds, flavonoids are involved in the antioxidant activity of propolis. So a series of phenolic compounds, including flavonoids, were assessed against the peroxidation of linoleic acid in a micellar solution. The results demonstrated that polyphenols in general present higher activity than BHT (butylated hydroxytoluene), a well‐known antioxidant [66]. In a study using cell culture, artepillin C has been proposed as a strong candidate to be responsible for the antilipoperoxidative activity of Brazilian propolis [67].

Santos et al. [68] assessed the antioxidant activity of flavonoids and reported that the presence of structural groups, i.e., the B ring dihydroxyl, double bond in C2 and C3 in conjunction with the 4‐oxo function, and the additional presence of hydroxyl groups in C3 and C5 (except for quercetin and 3ʹ‐O‐methyl‐quercetin), were the most potent inhibitors of lipid peroxidation using mitochondria. This antioxidant activity was also due to Fe chelation, which may explain the activity of flavonoids and polyphenols which do not have the above described structural groups [69].

After screening the antioxidant properties of propolis around the world, not only in the presented references, but also in the vast literature about this topic, one can observe a wide variation of responses. On the one hand, the antioxidant ability of each extract is related to the type and amount of phenolic compounds present in each extract, closely dependent on the propolis origin. But, on the other hand, no standardization regarding the solid soluble amount in each sample is presented, making comparisons among them not adequate. However, the presence of antioxidant activity in every propolis source studied is clearly observed and this activity has special importance to propolis biological properties.

Tian et al. [73] have shown that ethanolic propolis extract (EPE) protects endothelial cells from oxidized low‐density lipoprotein (ox‐LDL)‐induced apoptosis and inhibits atherosclerotic lesion development. This research group has also demonstrated the effect of propolis extract on endoplasmic reticulum stress‐C/EBP homologous protein pathway‐mediated apoptosis. Apoptosis, especially in macrophages present in atherosclerotic lesions, is considered as a prominent feature of advanced atherosclerotic plaques, suggesting that macrophage apoptosis is closely related to the atherosclerotic development and subsequent plaque rupture, which is the prominent event that results in the majority of clinical manifestations of acute coronary syndrome such as acute myocardial infarction and sudden coronary death [74]. Thus, pro‐ tecting macrophages from apoptosis is believed as an effective approach to attenuate plaque instability and combat acute vascular events.

Additional studies investigated the potential use of topically and orally administered propolis extracts to prevent UV irradiation‐induced oxidative stress in skin. Brazilian propolis extracts both green and brown successfully prevent UV‐induced GSH (endogenous antioxidant) depletion *in vivo* and are both promising antioxidant systems against oxidative stress in skin [75].

Propolis also due to its antioxidant properties was tested against acute lung inflammation (ALI) caused by cigarette smoke (CS) *in vivo*. The researchers observed that propolis (P) treatment (200 mg/kg) normalized all biochemical parameters in the CS+P group compared with the CS group, including nitrite, myeloperoxidase level, antioxidant enzyme activities (superoxide dismutase, catalase and glutathione peroxidase), reduced glutathione/oxidized glutathione ratio, and malondialdehyde. Additionally, TNF‐α expression reduced in the CS+P group when compared with the CS group. They suggested, therefore, the potential antioxidant and anti‐inflammatory role for propolis with regard to ALI caused by CS in mice [76].

Regarding the influence of the propolis antioxidant activity in food preservation, when combined with heat treatment in apple juice, propolis (0.1 mg/ml) reduced the thermal treatment time and temperature needed to inactivate 5 log10 cycles of *E. coli*. No influence on organoleptic properties of the apple juice, which implies the possibility of obtaining a senso‐ rially appealing, low‐pasteurized apple juice with the functional properties provided by propolis was reached [77]. In another study, Costa et al. [78] studied the bifunctional biobased packing containing red propolis. In addition to the antimicrobial effect on coagulase‐positive *Staphylococci* in cheese curds, the authors observed the reduced oxidation of butter during storage due to the antioxidant properties of propolis.

#### **3.3. Immunoregulator**

Oliveira et al. [62], this method could even be employed as an alternative for worldwide characterization and standardization of natural products. A good correlation of the DPPH method was observed against lipid peroxidation assay. This assay is based on the ability of DPPH, a stable free radical, to be quenched and thereby decolorize in the presence of antiox‐ idants resulting in a reduction in absorbance values. In the DPPH test, the antioxidants reduce the DPPH radical to a yellow‐colored compound, diphenylpicrylhydrazine. The extension of

Propolis antioxidant properties have been fully investigated and both propolis raw material and propolis commercial extracts have been studied. **Table 2** shows examples of the antioxi‐ dant profile and the method employed for each sample, focusing on their collecting origin. Phenolic compounds have been reported as the main propolis compounds responsible for the antioxidant property. The antioxidant role of polyphenols results from the donation of hydrogen atoms from an aromatic hydroxyl group to the free radical, leading to stabilization of the radical [64]. During the evaluation of propolis fractions (from Brazil), Wang et al. [65] observed a strong inhibition of lipid peroxidation using rat liver homogenate at a concentration of 2 mg/ml, and this activity was related to the presence of flavonoids. However, it is known that other than phenolic compounds, flavonoids are involved in the antioxidant activity of propolis. So a series of phenolic compounds, including flavonoids, were assessed against the peroxidation of linoleic acid in a micellar solution. The results demonstrated that polyphenols in general present higher activity than BHT (butylated hydroxytoluene), a well‐known antioxidant [66]. In a study using cell culture, artepillin C has been proposed as a strong candidate to be responsible for the antilipoperoxidative activity of Brazilian propolis [67]. Santos et al. [68] assessed the antioxidant activity of flavonoids and reported that the presence of structural groups, i.e., the B ring dihydroxyl, double bond in C2 and C3 in conjunction with the 4‐oxo function, and the additional presence of hydroxyl groups in C3 and C5 (except for quercetin and 3ʹ‐O‐methyl‐quercetin), were the most potent inhibitors of lipid peroxidation using mitochondria. This antioxidant activity was also due to Fe chelation, which may explain the activity of flavonoids and polyphenols which do not have the above described structural

After screening the antioxidant properties of propolis around the world, not only in the presented references, but also in the vast literature about this topic, one can observe a wide variation of responses. On the one hand, the antioxidant ability of each extract is related to the type and amount of phenolic compounds present in each extract, closely dependent on the propolis origin. But, on the other hand, no standardization regarding the solid soluble amount in each sample is presented, making comparisons among them not adequate. However, the presence of antioxidant activity in every propolis source studied is clearly observed and this

Tian et al. [73] have shown that ethanolic propolis extract (EPE) protects endothelial cells from oxidized low‐density lipoprotein (ox‐LDL)‐induced apoptosis and inhibits atherosclerotic lesion development. This research group has also demonstrated the effect of propolis extract on endoplasmic reticulum stress‐C/EBP homologous protein pathway‐mediated apoptosis. Apoptosis, especially in macrophages present in atherosclerotic lesions, is considered as a

activity has special importance to propolis biological properties.

the reaction will depend on the hydrogen‐donating ability of the antioxidants [63].

68 Superfood and Functional Food - An Overview of Their Processing and Utilization

groups [69].

One of the biological effects of propolis is its immunomodulatory effect—by either enhancing or suppressing the immune system. This contradictory effect is probably due to its complex chemical variety, the presence in different geographic regions, and the different forms of extraction.

Little was known about the biological role of propolis until the 1990s, but recently numerous studies have been published, providing an important contribution to this research field.

Immunomodulatory as well as anti‐inflammatory effects of propolis have been widely demonstrated both *in vitro* and *in vivo* [15, 79–82].

These effects are mainly related to its constituents, especially the phenolic compounds, including flavonoids as major components. Among the main types of flavonoids contained in propolis are: pinocebrin, chrisin, and caffeic acid phenethyl ester (CAPE). In addition to flavonoids, propolis can also contain cinnamic acid derivatives such as caffeic acid and its esters, besides sesquiterpenes, quinones, and coumarins [83–85]. The typical constituents of Brazilian propolis, especially the Brazilian green propolis, are: caffeoylquinic acid and prenylated derivatives of cinnamic acid, such as artepillin C, p‐coumaric acid, baccharin, and drupanin [23, 86, 87].

Despite the intensive search for the main constituent of propolis responsible for its immuno‐ modulatory role, its effect seems to be associated with a combination of its different compo‐ nents [88].

Bachiega et al. [89] evaluated the propolis extract and its phenolic compounds, such as cinnamic and coumaric acids on cytokine production (IL‐1b, IL‐6, and IL‐10) before or after macrophage challenge with LPS, to assess a possible immunomodulatory action. They observed a significant reduction in IL‐6 and IL‐10 in macrophages treated with the compounds only when the LPS was added before the stimulus, whereas the propolis extract was capable to inhibit the cytokine production both before and after the LPS addition. Thus, concluding that this efficiency could have occurred due to the synergistic effect of all compounds present in the extract [89]. On the other hand, **t**he effect of polyphenolic compounds isolated from propolis and propolis extract was investigated on the growth and metastatic potential of a transplantable mammary carcinoma of CBA mouse. The results indicated that water‐soluble extract of propolis (WSDP), caffeic acid (CA), quercetin (QU), and CAPE could be useful tools in the control of tumor growth in experimental tumor models [13].

The immunomodulatory activity of propolis extract was also investigated *in vivo* using the ovalbumin (OVA)‐induced asthma model. Sy et al. [90] demonstrated that propolis extracts can suppress the serum levels of OVA‐specific antibody IgE and IgG1 and attenuate the airway inflammation in treated mice, probably by the ability of propolis to modulate cytokine production. These findings suggest that propolis extracts may be a potential novel therapeutic agent for asthma [90].

Park et al. [91] evaluated another ethanolic extract of propolis (EEP) from Korea in an inflammatory animal model of hind paw edema induced by carrageenan. They observed a significant inhibition of the development of paw edema and increased vascular permeability coupled with an excellent analgesic effect in treated animals. They also showed a significant inhibitory effect on granuloma and exudate formation. The authors suggested that the anti‐ inflammatory effects of propolis observed might be due to its inhibitory effect on prostaglandin production [91].

In fact, Mirzoeva et al. [92] demonstrated the effect of another ethanolic extract of propolis in suppressing the prostaglandin and leukotriene generation by murine peritoneal macrophages *in vitro* and during zymosan‐induced acute peritoneal inflammation *in vivo*. Furthermore, the authors described the caffeic acid phenethyl ester (CAPE) as being the most potent modulator of the arachidonic acid cascade among the propolis components examined [92].

Similarly, Borrelli et al. [93] investigated two ethanolic propolis extracts (EPE): with and without the caffeic acid phenethyl ester (CAPE) for their anti‐inflammatory activity in rats using carrageenan foot edema and carrageenan pleurisy models. They observed that only EPE with CAPE and CAPE alone significantly inhibited the carrageenan edema in the rat paw and the number of leukocytes in the pleural exudate in rats, suggesting that the anti-inflammatory activity of propolis is due to CAPE [93].

propolis are: pinocebrin, chrisin, and caffeic acid phenethyl ester (CAPE). In addition to flavonoids, propolis can also contain cinnamic acid derivatives such as caffeic acid and its esters, besides sesquiterpenes, quinones, and coumarins [83–85]. The typical constituents of Brazilian propolis, especially the Brazilian green propolis, are: caffeoylquinic acid and prenylated derivatives of cinnamic acid, such as artepillin C, p‐coumaric acid, baccharin, and

Despite the intensive search for the main constituent of propolis responsible for its immuno‐ modulatory role, its effect seems to be associated with a combination of its different compo‐

Bachiega et al. [89] evaluated the propolis extract and its phenolic compounds, such as cinnamic and coumaric acids on cytokine production (IL‐1b, IL‐6, and IL‐10) before or after macrophage challenge with LPS, to assess a possible immunomodulatory action. They observed a significant reduction in IL‐6 and IL‐10 in macrophages treated with the compounds only when the LPS was added before the stimulus, whereas the propolis extract was capable to inhibit the cytokine production both before and after the LPS addition. Thus, concluding that this efficiency could have occurred due to the synergistic effect of all compounds present in the extract [89]. On the other hand, **t**he effect of polyphenolic compounds isolated from propolis and propolis extract was investigated on the growth and metastatic potential of a transplantable mammary carcinoma of CBA mouse. The results indicated that water‐soluble extract of propolis (WSDP), caffeic acid (CA), quercetin (QU), and CAPE could be useful tools

The immunomodulatory activity of propolis extract was also investigated *in vivo* using the ovalbumin (OVA)‐induced asthma model. Sy et al. [90] demonstrated that propolis extracts can suppress the serum levels of OVA‐specific antibody IgE and IgG1 and attenuate the airway inflammation in treated mice, probably by the ability of propolis to modulate cytokine production. These findings suggest that propolis extracts may be a potential novel therapeutic

Park et al. [91] evaluated another ethanolic extract of propolis (EEP) from Korea in an inflammatory animal model of hind paw edema induced by carrageenan. They observed a significant inhibition of the development of paw edema and increased vascular permeability coupled with an excellent analgesic effect in treated animals. They also showed a significant inhibitory effect on granuloma and exudate formation. The authors suggested that the anti‐ inflammatory effects of propolis observed might be due to its inhibitory effect on prostaglandin

In fact, Mirzoeva et al. [92] demonstrated the effect of another ethanolic extract of propolis in suppressing the prostaglandin and leukotriene generation by murine peritoneal macrophages *in vitro* and during zymosan‐induced acute peritoneal inflammation *in vivo*. Furthermore, the authors described the caffeic acid phenethyl ester (CAPE) as being the most potent modulator

Similarly, Borrelli et al. [93] investigated two ethanolic propolis extracts (EPE): with and without the caffeic acid phenethyl ester (CAPE) for their anti‐inflammatory activity in rats

of the arachidonic acid cascade among the propolis components examined [92].

in the control of tumor growth in experimental tumor models [13].

70 Superfood and Functional Food - An Overview of Their Processing and Utilization

drupanin [23, 86, 87].

agent for asthma [90].

production [91].

nents [88].

It is important to say that, despite Brazilian green propolis does not present CAPE in its composition, it presents a wide range of studies describing its beneficial properties such as antiulcerogenic, anti-inflammatory, antimutagenic, antifungal, angiogenesis, antioxidant, and immunomodulatory [14, 94–98]. Different from most European propolis extracts, which present flavonoids as the major component responsible for their effects, the biological activities of Brazilian green propolis are due to its high levels of phenolic acids such as artepillin C [99].

Studies with Brazilian green propolis have showed its role in inhibiting the development of pulmonary cancers [100], an antiviral activity *in vivo* [101], anticancer [102], an anti-inflammatory activity *in vivo* and *in vitro* [12, 16, 87], an antioxidant function in patients with type 2 diabetes mellitus [101, 102], antiherpetic activity [103], and among others [104, 105].

Despite several and growing studies involving the biological effects of Brazilian propolis, the detailed molecular and cellular basis of the action of propolis on immune cells is still unknown.

The administration of green propolis in animals subjected to chronic stress increased the generation of hydrogen peroxide, suggesting a modulation in the macrophage activation [106]. Machado et al. [12] verified an immunomodulatory effect of Brazilian green propolis extracts in acute and chronic inflammation models *in vivo* where the treated animals showed a decrease production of proinflammatory cytokines such as TNF-α and IL-6 and an increase in the IL-10 and TGF-b anti-inflammatory cytokines [12].

Most of the studies reported to date are associated with the immunomodulatory effect presented by propolis extracts with the modulation of the transcription factor NFkB [107–110].

Recently, it has been demonstrated that Brazilian green propolis can also act in a new inflammatory pathway named inflammasome. The inflammasomes are a large molecular platform formed in the cell cytosol in response to stress signals, toxins, and microbial infections. Once activated, the inflammasome induces the molecule caspase-1, which in turn provokes the processing of inflammatory cytokines IL-1β and IL-18. The Brazilian green propolis analyzed in this study (EPP-AF®) was capable of inhibiting the NLRP3 inflammasome and hence significantly reduces the IL-1β secretion in mouse macrophages. Thus, indicating that Brazilian green propolis EPP-AF® extract has a significant role in regulating the inflammasomes [15].

In conclusion, the immunomodulation caused by propolis has been amply demonstrated in recent years, both in the stimulation and suppression of the immune system, making it potentially applicable as an alternative adjuvant therapy or even in the treatment of various diseases.

**Table 3** presents a summary of the activities presented in Section 3.


**Table 3.** Biological activities presented in Section 3—summary.
