Compounds found in Brazilian red propolis.
Propolis is a product obtained from resins and exudates of different plants from different regions in order to protect the comb, with peculiar organoleptic, chemicals and biological properties. Considering this, this chapter presents the types of Brazilian propolis as the types available nowadays, their chemical compositions, as well as, some of their important biological properties enabling employing them as important health food, such as antimicrobial, antioxidant, and immunomodulation action. Various “in vivo” and clinical trial studies, conducted in different regions, on the safety and dosage of propolis, technologies used to obtain propolis extract, and several innovative presentations of this promising bee product are also presented in this chapter. Finally, this chapter aims to present the regulatory affairs, potential market for propolis around the world, and perspectives for a near future.
- Brazilian propolis
- chemical composition
- extraction process
- regulatory affairs
- potential market
1. Introduction and a brief history
The antique civilizations always used bee products as valuable therapeutic resources in their medicinal practices. The history of medicine of the Assyrian civilizations, Chinese, Tibetan, Inca, Egyptian, and also the Greco-Roman is very rich and possess records of centenary formulations, including propolis to treat or prevent diseases. Old Egyptians, Greeks, and Romans used propolis to treat wound, cutaneous lesions, ulcers, and chirurgical interventions .
In Egypt, propolis was used as one of the main ingredients used in the formulations to embalm cadavers. It was also used by Aristotle, Dioscorides, Pliny, and Galneo as an antiseptic and wound-healing. The Greeks, including Aristotle and Hippocrates, adopted it as an internal and external healing. Pliny Roman historian refers to propolis as a medicine to reduce swelling and relieve pain .
The term “propolis”, “pro” in favor of and “polis”, “city of bees”, which means in defense of the honey comb, was described in the sixteenth century in France , and in the seventeenth century, propolis was considered an official drug by London Pharmacopoeia . In the subsequence centuries, propolis has attracted growing interest due to its medical properties, especially in Eastern Europe. In 1908, the first scientific article about propolis chemical properties and composition  was published, indexed on Chemical Abstracts. In 1968, the abstract of the first patent was published on Chemical Abstracts .
In South Africa, during the war in the end of the nineteenth century, propolis was largely used because of its healing properties  and in the Second World War it was used by several Soviet clinicians .
In the last decades, propolis has gained wide acceptance as traditional medicine in several parts of the world. This disseminated interest in propolis in several countries encouraged a large number of studies considering chemical and biological properties of propolis .
Nowadays, in the Brazilian market and in several other countries, it is possible to find propolis in different presentations, such as liquid or powder extract: in bottles, capsules, tablets, vaporizers, syrups, creams, and among others, aiming to act as an antimicrobial [8–10], antioxidant , immunoregulatory [12–14], anti-inflammatory [12, 15, 16], antiviral  agent, besides several other functionss. A very large number of publications endorsing these biological benefits in “
Thus, this chapter presents recent studies about Brazilian types of propolis such as green, red, and brown, considering their chemical composition and some biological properties such as antimicrobial, antioxidant, and immunoregulatory, safety aspects, extraction process, and technology associated, regulatory aspects, potential market and challenges, as well as tendencies for a near future.
2. Chemical composition of Brazilian propolis
Propolis is formed by a complex set of components collected by
It consists of resin (50% of the mixture is composed by flavonoids and phenolic acids), wax (30%), essential oils (10%), pollen (5%), and other organic substances (5%). Among the present compounds, it can be consisted of hydrocarbons, alcohols, aliphatic and aromatic acids, esters and its derivatives, aldehydes, ketones, flavonoids, fatty acids, terpenoids, amino acids, sugars, lignans, vitamins, minerals, etc. .
The chemical composition of propolis differs significantly according to the geographic region where resins were collected due to the flora of each region, allowing the selection of different plants as source of resin .
When this product is derived from Europe or China, for example, the main plant metabolites found are flavonoids and phenolic acids, unlike the stemmed ones from southeastern Brazil, which, besides phenolic compounds, contain high amounts of terpenoids and prenylated derivatives of p-coumaric acid . This difference in composition reveals the collection of resinous material, in temperate zones, from poplar, especially species of
Regarding Brazilian propolis, it was further divided into 12 classes according to Park et al. : the first five ones originate from the south and have the colors yellow, light brown, dark brown, light brown, and greenish brown, respectively. Regarding propolis found in the Northeast, it was divided into six groups such as reddish brown, greenish brown, dark brown, yellow, dark yellow, and yellow. Finally, the last of these classes regards the kind of propolis that comes from the Southeast and is known to have a greenish brown or green color and so-called green propolis (Figure 1B) . After 2007, the 13 types of propolis was added: this new kind comes from the mangroves of the Brazilian states of Sergipe, Alagoas, Paraiba, Pernambuco, and Bahia. Among the Brazilian propolis, the green, the brown (Figure 1C), and the red (Figure 1D) ones are the most studied and relevant to the Brazilian economy due to their biological activities and exports to other countries, such as Japan .
2.1. Green propolis
Green propolis is composed of large amounts of phenolic compounds such as artepillin C, baccharin, kaempferide, isosakuranetin, dihydrokaempferide, drupanin,
Regarding the volatile compounds found in Brazilian green propolis, the major ones are sesquiterpenes, such as (E)‐nerolidol, β-caryophyllene, spathulenol, and δ-cadinene. Further more, other compounds such as selina-3,7(11)diene, benzenepropanoic acid and longipinene were also identified. These compounds are responsible for the pleasant aroma in this bee product and are also responsible for many biological activities reported in the scientific literature. [31, 32]. (Figure 3).
Considering the large amount of artepillin C presents in green propolis (Figure 4), in addition to other biologically active compounds, the commercial value of Brazilian green propolis in the international market is high .
2.2. Red propolis
Many of chemical compounds of red propolis, as well as green propolis, have been determined. Some of them are elemicin, isoelemicin, methyl isoeugenol, formononetin, biochanin A, isoliquiritigenin, liquiritigenin, medicarpin, homopterocarpan, quercetin, and vestitol. In the lipophilic extract, the majority of the compounds found are polyprenylated benzophenones—guttiferone E, xanthochymol, and oblongifolin A (Figure 5). Because the isoflavones— 7 formononetin, biochanin A, pinocembrin, and medicarpin—are abundant in red propolis, they are used as chemical markers for identifying red propolis (Figure 4) [33, 34].
In addition to these compounds, De Mendonça et al.  identified caffeic acid, ferulic acid, umbellic acid, p-coumaric acid, genistein, kaempferol, cathechin, dalbergioidin, epicatechin, daidzein, 2’-hydroxyformononetin, evernic acid, naringenin, calycosin, (7S)-dalbergiphenol, thevetiaflavone, cycloartenol, guttiferone C, and other compounds, using LC-Orbitrap-FTMS, a powerful tool to detect compounds because it does not require chromophores such as ultra-violet detector and it can detect very low amounts.
Red propolis has these chemical constituents, mainly due to the collection of resin from
Righi et al. , besides the compounds already described, found alkanes such as
Nunes et al.  determined 34 volatile (Table 1) compounds in Brazilian red propolis and found that the major ones are trans-anethole, α-copaene, and methyl-
|Class of the compound||Compound||Class of the compound||Compound|
|Alcohol, aldehyde or ketone||4-Hydroxy-4-methyl-heptan-2-one||Farnesene|
2.3. Brown propolis
The brown color is characteristic of propolis from different areas, but regarding Brazilian brown propolis, it is usually referred as the one that comes from the south of the country. Although many chemical compounds present in this product and their biological effects have already been identified, this type of propolis has not been well studied as red and green Brazilian propolis and many scientific reports on brown propolis are relatively old. Its botanical source seems to be mostly
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
Among the numerous biological effects reported for brown propolis and its isolated compounds, 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 significant preventive effect against oxidative stress in skin .
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
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 in the international market of Brazilian bee products.
3. Biological properties
3.1. Antimicrobial activity
Antimicrobial properties of Brazilian propolis are well-documented, including the antibacterial, 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 . 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
Antifungal activity of green propolis has been reported against all three morphotypes of
Green propolis also has the capacity to inhibit virus propagation. Shimizu et al.  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.  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.  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.  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 . 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.
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 (
Brazilian red propolis, in its turn, is produced from resinous exudates of
Isoflavone formononetin is one of main chemical compounds in red propolis. Das Neves et al.  evaluated the activity of this compound against some bacteria (
Kamuyama et al.  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.  evaluated the antibacterial and antifungal properties of different concentrations 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.
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.
|Propolis origin||Antioxidant activity/method||Reference|
|Brazil (marketed-standardized extract—PI 0405483-0)||0.016 μl/ml (IC50) inhibition of lipid peroxidation|
0.22 μl/ml (IC50) inhibition of chemiluminescence produced in the H2O2/luminol/horseradish peroxide (HRP) system
0.005 μl/ml (IC50) inhibition of chemiluminescence produced in the xanthine/luminol/xanthine oxidase (XOD) system
0.024 μl/ml (IC50) scavenging of hydroxyl radical by the deoxyribose assay
|Campo grande Brazil (raw-material*propolis from stingless bees)||3 μg/ml (IC50) scavenging of DPPH|
No hemolyis—oxidative hemolysis inhibition assay
50–125 μg/ml—efficiency in inhibiting of AAPH-induced lipid peroxidation
|Nan Province innorthern Thailand (raw-material)||Not indicated - scavenging of DPPH|
Observation: the authors inform the higher the ethanol amount in the ethanol aqueous solution, the higher the antioxidant activity
|Alagoas, Brazil (brown propolis—raw-material)||8.01 μg/ml (IC50) scavenging of DPPH—ethanolic extract|||
|Mediterranean propolis||Most prepared extracts inhibited lipid oxidation—oxidation of sunflower oil method|||
|Brazil aqueus extract||0.62 μg/ml (IC50)—inhibition of lipid peroxidation employing brain homogenate|||
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 polyunsaturated 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. , 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 antioxidants 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 .
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 antioxidant 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 . During the evaluation of propolis fractions (from Brazil), Wang et al.  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 . 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 .
Santos et al.  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 .
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.  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 . Thus, protecting 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
Propolis also due to its antioxidant properties was tested against acute lung inflammation (ALI) caused by cigarette smoke (CS)
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
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.
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 immunomodulatory role, its effect seems to be associated with a combination of its different components .
Bachiega et al.  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 . On the other hand,
The immunomodulatory activity of propolis extract was also investigated
Park et al.  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 .
In fact, Mirzoeva et al.  demonstrated the effect of another ethanolic extract of propolis in suppressing the prostaglandin and leukotriene generation by murine peritoneal macrophages
Similarly, Borrelli et al.  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 .
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 .
Studies with Brazilian green propolis have showed its role in inhibiting the development of pulmonary cancers , an antiviral activity
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 . Machado et al.  verified an immunomodulatory effect of Brazilian green propolis extracts in acute and chronic inflammation models
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 .
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.
|Kind of propolis||Biological properties||References|
|Antimicrobial||[38, 41, 44, 45]|
|Green||Antibacterial||[8–10, 46, 47, 49]|
|Anti-inflammatory||[12, 15, 16, 87]|
|Antioxidant||[27, 42, 75, 76, 103]|
|Antiviral||[17, 54, 55]|
|Inhibition of angiogenesis|||
|Antifungal||[24, 52, 59]|
4. Safety aspects
4.1. Nonclinical studies
Although propolis has been used for centuries around the world demonstrating to be safe, several scientific studies have been done in order to evaluate propolis safety by oral or topical route. Here we intend to present Brazilian propolis studies done in animals and, the studies found in humans, as clinical trials are not so numerous, despite the clinical trials did not focus on safety, we are presenting them in order to compare the dosages previously used and documented in humans.
Sforcin et al.  evaluated some biochemical parameters of animals treated with several different types of propolis aiming to study propolis safety and the differences in the propolis source could interfere in the results. The authors determined total proteins, glucose, urea, creatinine, triglycerides, cholesterol, cholesterol-HDL, aminotransferases, and lactic dehydrogenase (LDH). The results demonstrated that all parameters were under standard values for the species studied and the propolis sources did not affect the results.
Reis et al.  evaluated the safety of propolis standardized extract (EPP-AF®), a Brazilian propolis composition that presents more than 50% of green propolis, by oral route in mice, in an acute model. DL50 was determined to be 3000 mg/kg after 24 h of treatment, and dosages under this value did not demonstrate intoxication signs in the animals. In the subchronic protocol (30 days) done in Wistar rats, there were no differences in the food and water intake, animals’ weight and diuresis. Hematological and biochemical analysis did not show statistical differences between the treated (propolis 650 mg/kg) and placebo group. All parameters were in accordance with reference standards for the species studied. Microscopic analysis of all tissues did not show any differences with the placebo group, and it was not possible to detect any lesions, hemorrhages or cells infiltration, demonstrating the safety of oral administration of Brazilian propolis up to 650 mg/kg during 30 days of ingestion.
Mani et al.  evaluated the safety of Brazilian propolis in distinct treatments: (i) rats treated with 1, 3, and 6 mg/kg/day during 30 days; (ii) rats treated with 1 mg/kg/day of propolis alcoholic or aqueous during 30 days, and (iii) rats treated with 1 mg/kg/day during 90 and 150 days, demonstrating that all levels of seric cholesterol, HDL-cholesterol, total lipids, triglycerides, aminotransferases (AST), and lactic dehydrogenase (LDH) of propolis treated group were similar to the control group. The authors suggested that Brazilian propolis in the dosages used during the period of treatment were safe (Table 4).
|Type of study (oral route)||Dosage||Propolis source||Species||Dosage converted to human according FDA|
|Biochemical parameters, 150 days ||1, 3 and 6 mg/kg||Brazilian||Rats||67.74|
|Biochemical parameters, 60 days ||2000 mg/kg||Iranian||Rats||22,580|
|Acute safety study ||2000 mg/kg||Polish||Rats||22,580|
|DL50 determination ||3000 mg/kg||Brazilian||Mice||17,073|
|Biochemical parameters—30 days ||650 mg/kg||Brazilian||Rats||7338|
|Acute toxicity parameters**||2500 mg/kg||Brazilian||Wistar rats||28,225|
|Subchronic study (28 days)**||1000 mg/kg||Brazilian||Wistar rats||11,290|
|Subchronic study (28 days)**||100, 300 and|
According to Dobrowolski et al. , LD50 for different sources of propolis varied from 2 to 7.3 g/kg in mice, suggesting a safe dose for humans of 1.4 and 70 mg/day (when using safety factor of 1000). In conclusion, considering Brazilian propolis LD50 as 17,073 mg/day for humans , the application of a safety factor of 10 suggested by FDA guidelines, we would have a safe dose of 1700 mg or 1.7 g/day of propolis for an adult.
4.2. Clinical studies
Khayyal et al.  evaluated propolis extract activity in asthmatic patients with oral administration of 260 mg of propolis/day, for 2 months. The results demonstrated reduced night attacks (2.5 attacks/week for 1/week) and improved ventilatory functions, as a consequence of a decrease of TNF-, ICAM-1, IL-6, and IL-8, and an increase of 3· of IL-10, besides a decrease of prostaglandins E2, F2, and leukotriene D4.
Cohen et al.  evaluated 430 children aged 1–5 year old. Treated group (
|Type of study (oral route)||Dosage||Dosage converted to human adult (mg/day)||Reference|
|Double-blind study with children—prevention to respiratory infections||50 mg/ml—10 ml/day (children 1–3 year old)||2482.27|||
|Double-blind study with children—prevention to respiratory infections||50 mg/ml—15 ml/day (children 4–5 year old)||2876.71|||
|Pilot clinical trial with asthmatic volunteers||2–3 tablets/day with 88.4 mg propolis each||265.20|||
|Pilot clinical trial with healthy volunteers—prophylactic study||500 mg propolis (2 capsules/day)||500.0|||
|Pilot clinical trial—recurrent stomatitis||500 mg propolis/day||500.0|||
|Propolis for wound healing||500 mg propolis/day||500.0|||
|Clinical trial with asthmatic patients||1 sachet with 260 mg propolis/day||260.0|||
|Pilot clinical trial with Brazilian propolis for treatment of Helicobacter pylori||20 drops, 3× /day||˜350.0|||
Brätter et al.  evaluated the oral administration of 500 mg of propolis for 13 days in healthy volunteers focusing on the evaluation of the immune response (TNF-α, IL-6, and IL-8). There was an increased ability in the cytokines secretion, however, without plasmatic levels. Then, prophylactic administration of propolis depends on the immune system reactivity and time, with no adverse effects.
Samet et al.  tested the oral administration of 500 mg of propolis in a randomized, placebo-controlled double-blind study, in which it was possible to demonstrate the benefits of propolis treatment in the repeated stomatitis, especially important in cases of resistance to treatment.
Finally, Zedan et al.  evaluated the administration of 500 mg of propolis/day in 45 patients aiming to offer an alternative treatment to cutaneous healings. The study compared propolis with echinacea and placebo, and propolis demonstrated to be more efficient than the other groups, especially in usual and superficial healings (Table 5).
Jasprica et al.  studied the antioxidant effects of propolis (propolis soluble in water and maltodextrin, 0.65 g of propolis, presenting 2.5% of flavonoids, equivalent to 16.25 mg expressed as galangin, Specchiasol, Italy) when administered in healthy volunteers (
Considering all the previously presented studies, it is possible to suggest that propolis dosages ranging from 260.0 mg to 2.87 g, which have already been used in humans can be considered safe. The biological results observed also varied much. Considering that propolis around the world is largely used as a supplement or functional food, it is reasonable to assume that dosages within this range will probably be safe, since none of the articles suggested any damage or complications for the volunteers. Regarding the antioxidant evaluation proposed by Jasprica et al.  in humans and the literature available until now, it is likely that the propolis dosage used was very high for this purpose. Some data previously published suggested that propolis can have a “pro” or “anti” action and, because several
5. Extraction technologies and innovation products
5.1. Extraction technology
Propolis is a very complex material depending on the vegetation present in the area visited by bees. Besides the propolis source, the extraction process associated with solvents used will definitely provide a completely different extract . It is well established that chemical compounds possess several particularities such as solubility, volatility, partition coefficient oil/water, pKa, and therefore, different solvents will probably extract different compounds . Depending on the temperature and equipment necessary, volatile compounds can be lost and then, it is common to use cold procedures such as maceration or percolation, and when it is necessary to use higher temperatures, it is important to be careful in order to preserve every compound of interest.
Park and Ikegaki  studied propolis extracts obtained from water with 96% ethanol solution as solvents. The results demonstrated that propolis extract obtained with 80% ethanol grade showed higher absorption at 290 nm. Using ethanol solution at 60%, higher quantities of isosakuranetin, quercetin, and kaempferol were extracted, while pinocembrin and sakuranetin were better extracted with ethanol solution at 70% and kaempferide, acacetin, and isorhamnetin were most extracted with ethanol solution 80%. More expressive antimicrobial activities were found in propolis extracts from 60 to 80% of ethanol solution extraction and higher antioxidant results came from propolis extract obtained with ethanol 70–80%.
The most common extraction process for propolis to oral administration is the alcoholic (70%) extraction using maceration, percolation and/or turboextraction. The ratio of propolis raw material:extract used is completely variable and usually, in Brazil, 1:3–4, i.e., 1 part of propolis raw material may offer 3 or 4 parts of extract, considering the production of a liquid extract with at least 11%w/v of propolis dry matter. Of course, this ratio may vary and it is completely dependent on the quality of propolis raw material used.
Jorge et al.  studied green propolis extracts obtained from four different locations in São Paulo and Minas Gerais States, using the same extraction process, i.e., hydroalcoholic solution 70% with maceration for 30 days. Although all samples evaluated were Brazilian green propolis using the same extract, different results were found for drupanin, baccharin, and artepillin C during the same month, in spite of seasonal differences. Therefore, it is possible to conclude that, using the same floral source, solvent and process extraction, different regions, and seasonal variations also offer a different chemical composition. Interestingly, these differences did not affect the safe of propolis ingestion .
Besides maceration, percolation or turboextraction, Trusheva et al.  compared ultrasound extraction and microwave-assisted extraction with the maceration process. The careful analysis of the results obtained with each process demonstrated that statistical differences were found for total phenolics and propolis total extractable matter for ultrasound process (30 min) and microwave (2 · 10 s) when compared to maceration extraction. For ultrasound, higher total phenolics were found (52 3%) in comparison to maceration (43 2%) while the reduced total extractable matter (53 3% versus 55%). In turn, microwave offered reduced values for total phenolics (40.4 0.6% versus 43 2%) and expressively higher amounts of the total extractable matter (75% versus 55%). Flavonoids analysis did not show important differences among the procedures evaluated. It is important to consider the time of extraction in each process, since maceration takes around 72 h, and ultrasound was effective with 30 minutes and microwave 2 · 10 s. Another important thing is to define the objective of the extraction: for analytical purposes, it is more practical and cheaper to use ultrasound or microwave, however, for industrial scale, these latter may not be easily implemented.
Propolis water extract was also obtained by some authors [12, 17, 98, 124] using completely different procedures, demonstrating some interesting biological activities that had been previously studied for propolis alcoholic extract, such as anti-inflammatory , inhibition of inflammatory angiogenesis , and antiviral . Although the demonstration of these interesting results, chemical characterization was poorly explored in the manuscripts, except by Urushisaki et al.  that presented the caffeoylquinic derivatives as the most important compounds of this extraction process; however, the manuscript does not present the extraction process used. De Moura et al.  performed the extraction from propolis raw material properly crushed in water maceration (500 ml) with temperature around 70°C (30–60 min), two fractions were obtained from the same propolis raw material, followed by filtration. The filtrate was then lyophilized. A similar procedure was used by others too. Nafady et al.  in turn, used an innovative process with -cyclodextrin as encapsulate agent. To obtain this extract, 10 g of crushed propolis was dispersed in 1 l of water containing 10 g of -cyclodextrin previously dissolved. The inconvenient of this procedure is the elevate costs involved in the acquisition of -cyclodextrin besides the limitation of the propolis concentration obtained in the final product. Machado et al.  proposed the extraction using hydroalcoholic solvent (70%) as usual, however, the solvent was evaporated and after a hydrolysis step the propolis soft extract was then resuspended in water, in this last case, the obtained extract demonstrated similar chemical results of the alcoholic extract in the moment of preparation.
5.2. Nanoparticles and innovation products
Nowadays, finding natural additives has increased the efforts both to obtain bioactive compounds from natural raw materials and develop stable and functional derivative products. The former mentioned properties attributed to propolis are valuable and find applications in several industries, such as pharmaceutics, agrochemical, and food. The growing interest in propolis has also promoted technological development for the suitable application of propolis.
Propolis in the powder form, for example, exhibits several advantages as increased concentration of propolis dry matter, higher chemical stability of the compounds, and longer preservation of the biological properties. Additionally, the powder form also permits the production of presentations with higher compliance in therapeutics, i.e., sachets, tablets, and capsules. The drying process may also involve the encapsulation of the product resulting in micro/nanoencapsulation systems, which can minimize sensory flavor and odor and control the release of the active compounds.
Propolis dry extract was obtained by Da Silva et al.  by employing arabic gum and octenyl succinic anhydride (OSA) starch as carriers by spraydrier. The process allowed obtaining propolis in the powder form with preserved antioxidant activity, stability, and low hygroscopicity. Microencapsulated propolis extract obtained by complex coacervation was reached and presented inhibitory activity against
More recently, Marquiafável et al.  aimed to develop a propolis dry extract with high propolis (~40%w/w of propolis dry matter) and artepillin C contents by employing a combination of silicon dioxide with arabic or modified starch and silicon dioxide by spray-drier. They have successfully obtained a standardized propolis extract with high amount of propolis, flavonoid content, expected amounts of artepillin C, and with maintained antibacterial activity, and obtained microparticles with both excipients used. Recently, results of the same group obtained dry extracts of propolis with 70–80% of dry matter; however, the microparticles were not obtained (data not published yet), and then, the odor, color, and taste are not similarly reduced as it is possible to observe when microparticles are obtained (Figure 10). Although microparticles were not obtained with 70–80% of propolis dry matter, this extract is the most concentrated one found in the market until now and can be used in several products with very good results, for example soft or hard capsules or tablets.
In general, the propolis powder extracts obtained by spray-drying technique investigated in the literature demonstrated the formation of particles at the micrometer scale, from 1 to 10–20 μm. On the other hand, as nanotechnology can offer new opportunities for propolis application, in another line of research, nanosized particles have been developed. Patil et al.  have obtained and characterized silver nanoparticles containing propolis . Propolis nanoparticles have also been obtained employing lipid carriers. Our research group has focused on developing solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) encapsulating propolis. Figure 11 shows atomic force microscopy (AFM) images of propolis-loaded NLC. Additional studies were also conducted covering NLC surface with chitosan. The chitosan-covered particles presented positive residual surface charge [≈ +40 mV], while the uncoated ones presented negative charges [≈ −30 mV]. Particles were anisometric in shape and approximately 150–200 nm in size. The images demonstrate the particle surface and confirm the nanometric size of the particle. Additionally, no roughness was observed on the particle surfaces.
Several important applications can be carried out with innovative propolis extracts. Different presentations of propolis were previously showed as in a liquid presentation without alcohol, usually using propylene glycol or polyethylene glycol, propolis powders in different systems or concentrations, micro or nanoparticles, and others, as soft or hard capsules, with immediate or sustained release systems. Considering the applications, it is possible to formulate capsules, tablets, pills, or others with a specific amount of propolis dry matter, or with a focus on some groups of compounds (total flavonoid or polyphenols) and finally, on a biomarker or a group of these substances such as artepillin C, drupanin, or baccharin, all presentations completely applicable to functional or supplement food, or medicines. Besides oral administration, it is possible to use all of these propolis presentations in topical products, as previously published by Berretta et al.  who presented a propolis thermoreversible gel to treat cutaneous lesions or burns, Barud et al.  with a propolis biomembrane for the same application, or Berretta et al.  that demonstrated the benefits of the application of a propolis mucoadhesive gel in vulvovaginal candidiasis. Several other products can be found in the market and in the literature, such as mouthwashes, toothpastes dental creams, among others.
6. Regulatory affairs
Functional food and natural health products have become an important part of people’s daily diet, contributing to the general health of the population and boosting the global food industry. Therefore, its importance is reflected in the interest in regulation of health claims and standards from industry stakeholders and policymakers.
In this chapter, we examine propolis product regulations and policies in many important producing and consuming countries around the world. The goal of this study is to incentive legislators to update the regulation on propolis products in order to improve information available to consumers so they can make better choices and also be provided with healthier and more innovative options.
The regulatory climate worldwide appears to be tending toward propolis classification into the health food products category, although this category also has different names, registration requirements, and allowed claims throughout the world.
Nevertheless, there are still some countries that categorize propolis as a conventional food together with the other bee products, such as honey, royal jelly, and bee pollen. That is the case of Brazil, where the product is regulated by the Ministry of Agriculture with very stringent regulation that limits the product’s presentations, information to consumer and does not allow health claims. In 2005, the Brazilian “National Health Surveillance Agency” (ANVISA) published a technical note allowing the registration of propolis as a topical medicine with the claims of anti-inflammatory, antiseptic, and wound healing . The publication of another regulation  reinforced the same rules but, due to the very strict rules for medicines, although Brazil is one of the biggest propolis markets, there are no propolis medicines registered to this date.
In the United States, propolis is encompassed together with a wide range of substances by the definition of a dietary supplement in the Dietary Supplement Health Education Act of 1994 (DSHEA) . The use of function claims is also regulated by the above-mentioned regulation that established some special regulatory requirements and procedures for claims of general well-being. These claims are not preapproved by FDA, but the manufacturer must have substantiation that the claim is truthful and not misleading and must submit a notification with the text of the claim to FDA no later than 30 days after marketing the dietary supplement with the claim.
In the European Union (EU), propolis belongs to the food supplement group, regulated by the Directive 2002/46/EC , which defines the category as concentrated sources of nutrients or other substances with a nutritional or physiological effect. Since 2006, EU has been engaged in assessing generic health claims to surpass local regulation of member states and after this harmonization product’s labels can only bear health claims authorized by the European Food Safety Authority (EFSA) , which evaluates scientific data on claims provided by the applicant. Up to this date, there is still no authorized health claim for propolis.
In Australia, all food supplements fall within the category of “complementary medicines” under the
Food supplements in Canada are regarded as “Natural Health Products” under the Natural Health Products Regulations (SOR/2003-196) [138–141] and may contain a wide range of substances, such as vitamins and minerals, herbal remedies, homeopathic medicines, traditional medicines, and probiotics. All products must be safe to use as over-the-counter products and not need a prescription to be sold. Propolis is positive listed to be used orally in multiple pharmaceutical dosage forms as a source of antioxidants for the maintenance of good health and to help relieve sore throat and/or other mouth and throat infections. It can also be used topically to assist in minor wound healing.
Japan is one of the first countries to move toward regulating functional foods. There are lists containing a broad range of substances that are not restricted to medicinal use and can therefore be used in food supplements. Propolis in this scenario can be used as an authorized excipient under the Food Sanitation Act 2010, as regular health food without any claims or as an active of a “Food for Specified Health Uses” (FOSHU)  with health claims.
The Republic of Korea defines functional food significantly differently from other countries, restricting functional food to nutraceuticals. They are regulated under the Health Functional Food Act of 2004  and there is a positive list in the Health Functional Food Code with 37 categories. Propolis preparations in all forms are allowed and may include two health claims: antioxidant activity and antimicrobial activity in oral cavity.
The People’s Republic of China is another example of an Asian country that uses a product-specific system of registration. The State Food and Drug Administration in China (SFDA)  regulates these food supplements as “health foods” and maintains positive and negative lists of substances that may be used in health foods. Propolis is in the positive list. There are 27 categories of health function claims approved by the SFDA, but the regulatory process for achieving approval of these health claims is very strict and expensive, requiring the applicant to conduct nonclinical or even clinical studies through an approved agency in addition to the regular scientific literature review.
With this brief regulatory framework on propolis products, we have presented different policies and regulations around the world and we hope that policymakers can improve the regulatory scenario in the near future in order to accelerate and foster innovation in the sector.
7. Propolis nowadays market and potential
The green propolis has gained preference in the world market since the 1980s, unveiling new horizons for the product. The growing interest of the market for green propolis in the context of international food trade follows the increasing trend in search of healthier habits, which have gained ample space in people’s daily lives all over the world.
In the Japanese market, green propolis has a high commercial value: according to SEBRAE, the price of 1 kg of this product in 2010 was around $ 87 and the same amount of honey was priced at about 3 dollars. In Tokyo’s market, this product is even more valued: a bottle of green propolis, in 2010, was sold for around $ 150 [145, 146]. In 2008, it was estimated that in Japan, 700 million dollars a year started to be moved by green propolis . Japan's interest in green propolis is justified not only by consumption: one of the most important examples of its use is as an adjuvant in the treatment of cancer; but also by Japanese research related to the chemical composition and biological activities of this type of propolis, especially studies with artepillin C [16, 147].
The high demand for green propolis, especially from Asian countries, such as Japan, is essential for sustaining the economy that revolves around this product, which is fairly lucrative. Green propolis is produced mainly in the southeastern region, highlighting the state of Minas Gerais, where there are over 8000 beekeepers, which produces more than 35 tons of propolis per year . These data show the importance of production and exports of green propolis, which is one of the pillars of Brazilian apiculture economy.
Red propolis found in the Brazilian state of Alagoas has been internationally certified by the Brazilian National Institute of Industrial Property (INPI) as the only producer of this kind of propolis in the world and most of its compounds were not found in other types of Brazilian propolis, which makes it a singular bee product . Due to that, its commercial value is internationally high. It has been reported that a kilogram of this product can cost around R$ 500. Its importance to the Brazilian economy and to red propolis producer states is immeasurable. Many propolis producers are being qualified and thereby, they are improving their product quality and the production process. Like green propolis, red propolis is also highly exported to Japan due to its chemical composition and biological effects .
Brazil is currently the world's third largest producer of propolis, second only to Russia and China . Although it represents 10–15% of world production, Brazil fulfill about 80% of Japanese demand. Minas Gerais State (Brazil) Beekeepers Federation data show that the propolis produced in the Midwest region of the state is considered the best in the world by the Japanese market, where the kilogram of product has jumped from $ 5 to $ 200 in recent years .
The propolis production in Brazil is estimated at around 140 tons, and the major part is destined for international market, both in raw form and as finished products. It is estimated that 100 tons are green propolis and 40 tons other types of propolis. About 80% of the green propolis produced in Brazil comes from the Midwest region of Minas Gerais State, close to the source of the São Francisco river at Serra da Canastra, region where are the highest number of producers. Despite the great Brazilian beekeeping potential, the current production is not enough to fulfill a growing global demand. The Brazilian honey bees are Africanized, presenting defensive and disease-resistant features, with no need to use chemical treatments as in other countries, which ensures Brazilian bee products excellent quality and free from contamination.
Many research fronts have been opened in the pursuit of development and adaptation of professional management techniques in the production of green propolis. In addition to the improvement actions and training of producers in beekeeping management practices, a group of green propolis producers in the Midwest region of Minas Gerais in the Source of the São Francisco River created an independent association supported by governmental agencies, universities, researchers, and local private institution.
This association aims to establish the technical and scientific cooperation between the scientific community and the beekeepers, aimed at regional development, improving the quality and increasing the amount of green propolis produced in the region. Among the main projects carried out, stand out the training of beekeepers in the professionalization of beekeeping, conservation and cultivation of
Trade promotion strategies are being constantly designed and implemented by the Brazilian Association of Honey and Propolis Exporters (ABEMEL) and the Brazilian Trade and Investment Promotion Agency (APEX-Brasil) to disseminate Brazilian bee products around the world. The result is the increasing demand from Asia, Europe, and North America countries.
In recent years, Brazil has been prominent on the international scene by winning important prizes at the World Beekeeping Awards of Apimondia, the main world beekeeping event that brings together representatives of over 130 countries and is held every 2 years. In the last editions, Brazil won gold and silver medals in the category honey and gold in the category propolis.
8. Future perspectives
Considering all information presented here, it is easy to imagine the important potential of propolis in the health of the population and in the Brazilian and international market, especially because of the important biological activities and safety demonstrated with scientific reports as “
It is important to consider the investment in more clinical trials aiming to explore the benefits observed with traditional use and in animal studies, in order to refine the dosages and formulations, with regard to the development of medicines. Besides clinical studies, another important area is the improvement in the investments in productivity in field, since the Brazilian propolis available nowadays is not enough to supply all the countries that may be interested in working with this fabulous natural material produced by bees with the support of Brazilian Biodiversity. And finally, the effort of beepers and entities, such as ABEMEL, is crucial to stimulate and support this work.
We would like to thank Nivia Alcici, from Essenciale Propolis Company (Minas Gerais, Brazil), for sending regulatory documentation of propolis of several countries, and also to the Brazilian Association of Honey Exporter, ABEMEL, for financial resources for the publication of this chapter.