**2. Chemical composition of Brazilian propolis**

**1. Introduction and a brief history**

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

[1].

and relieve pain [2].

al Soviet clinicians [2].

this chapter.

cies for a near future.

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

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 [3], and in the seventeenth century, propolis was considered an official drug by London Pharmacopoeia [4]. In the sub‐ sequence centuries, propolis has attracted growing interest due to its medical properties, es‐ pecially in Eastern Europe. In 1908, the first scientific article about propolis chemical properties and composition [5] was published, indexed on Chemical Abstracts. In 1968, the

In South Africa, during the war in the end of the nineteenth century, propolis was largely used because of its healing properties [3] and in the Second World War it was used by sever‐

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 [7].

Nowadays, in the Brazilian market and in several other countries, it is possible to find prop‐ olis 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], anti‐ oxidant [11], immunoregulatory [12–14], anti‐inflammatory [12, 15, 16], antiviral [17] agent, besides several other functionss. A very large number of publications endorsing these bio‐ logical benefits in "*in vitro*", "*in vivo*," and in some clinical trials, is be discussed further 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 tenden‐

abstract of the first patent was published on Chemical Abstracts [6].

Propolis is formed by a complex set of components collected by *Apis mellifera* from different parts of plant resins (twigs, flowers, pollen, buttons, and exudates of trees) which are deposited in the hive with saliva and enzymes of the insect to seal the cracks and maintain the temper‐ ature (**Figure 1A**) [18].

**Figure 1.** Presentation of different types of Brazilian propolis. (A) *Apis mellifera* collecting Green propolis from *Baccharis dracunculifolia* species, (B) green propolis on the intelligent collector, (C) brown propolis and (D) red propolis. Figures (A) and (B) were gently donated by César Ramos, Natucentro Company. Figures (C) and (D) were gently donated by Felipe Galeti Miguel, Apis Flora Company. Both companies are associated with ABEMEL.

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. [19].

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 [20].

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 [21]. This difference in composition reveals the collection of resinous material, in temperate zones, from poplar, especially species of *Populus* and in southeastern Brazil, especially from *Baccharis dracunculifolia* DC (Compositae), popularly known as "vassourinha do campo" [22].

Regarding Brazilian propolis, it was further divided into 12 classes according to Park et al. [23]: 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**) [23]. 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 [24].

#### **2.1. Green propolis**

Green propolis is composed of large amounts of phenolic compounds such as artepillin C, baccharin, kaempferide, isosakuranetin, dihydrokaempferide, drupanin, *p*‐couma ric acid, caffeic acid, aromadendrin, caffeoylquinic acid derivatives, and other compounds, such as the triterpene lupeol‐3‐(3ʹR‐hydroxy)‐hexadecanoate. The key source of these compounds is *B. dracunculifolia* [12, 25–30] (**Figure 2**).

**Figure 2.** Chemical structures of compounds found in Brazilian green propolis.

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 [16].

**Figure 3.** Volatile compounds from Brazilian green propolis.

southeastern Brazil, especially from *Baccharis dracunculifolia* DC (Compositae), popularly

Regarding Brazilian propolis, it was further divided into 12 classes according to Park et al. [23]: 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**) [23]. 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

Green propolis is composed of large amounts of phenolic compounds such as artepillin C, baccharin, kaempferide, isosakuranetin, dihydrokaempferide, drupanin, *p*‐couma ric acid, caffeic acid, aromadendrin, caffeoylquinic acid derivatives, and other compounds, such as the triterpene lupeol‐3‐(3ʹR‐hydroxy)‐hexadecanoate. The key source of these compounds is *B.*

their biological activities and exports to other countries, such as Japan [24].

**Figure 2.** Chemical structures of compounds found in Brazilian green propolis.

known as "vassourinha do campo" [22].

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

**2.1. Green propolis**

*dracunculifolia* [12, 25–30] (**Figure 2**).

**Figure 4.** Chemical fingerprint for red and green propolis. Chromatographic profile was obtained using HPLC‐DAD, Shimadzu Shim‐pack CLC‐ODS column (4.6 mm · 250 mm, particle diameter of 5 μm, pore diameter of 100 ). Green propolis conditions followed according Berretta et al. [8] and red propolis, Cavendish et al. 2015.

#### **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].

**Figure 5.** Chemical structures of some compounds found in Brazilian red propolis.

In addition to these compounds, De Mendonça et al. [33] 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 *Dalbergia ecastophyllum* by the bees. Its red color is due to the presence of cationic C30 isoflavans, retusapurpurins A and B (**Figure 6**). This is characteristic of the propolis found in Brazilian Northeast, found especially in hives nearby mangroves in the states of Sergipe, Bahia, Alagoas, Paraiba, and Pernambuco. This bee product has attracted wide interest because of its numerous biological activities, such as cytotoxic against several cancer cell lines, antibacterial, antifungal, anti‐cariogenic, antioxidant, antiproliferative, anti‐inflammatory, and others [24, 33, 34]. In addition to its extracts, numerous biological activities of the isolated compounds have been described [24].

**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].

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

**Figure 5.** Chemical structures of some compounds found in Brazilian red propolis.

violet detector and it can detect very low amounts.

In addition to these compounds, De Mendonça et al. [33] 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‐ Righi et al. [35], besides the compounds already described, found alkanes such as *n*‐tricosane, *n*‐pentacosane, *n*‐heptacosane, *n*‐nonacosane, *n*‐hentriacontane, and *n*‐tritiacontane in the apolar red propolis extract (hexane extract). They also identified other compounds as β‐amirin, *α*‐amirin, lupeol, methylguaiacol, trans‐anethole, resorcinol, anisylacetone, *cis*‐asarone, farnesol, and among others.

Nunes et al. [36] determined 34 volatile (**Table 1**) compounds in Brazilian red propolis and found that the major ones are trans‐anethole, α‐copaene, and methyl‐*cis*‐isoeugenol (**Figure 7**). They found that the chemical composition remains relatively constant during the year, considering that 17 out of the 34 compounds were detected every season of the year. But, the compounds δ‐cardinol, ß‐gurjunene, isocaryophyllene, and δ‐cadinene were found only in the sample collected in October and the alkanes, the 1,8 cineol, and α‐selinene in the sample collected in July. This can be explained by the visitation of bees, in rainy seasons of shrubs and in dry seasons of woody plants: when the apiculture pasture changes, the chemical composition of propolis also changes. Similarly, as trans‐anethole, the other red propolis compounds show biological activities such as analgesic, anesthesic, antigeno‐ toxic, and antioxidant, making the volatile fraction pharmacologically interesting.


**Table 1.** Compounds found in Brazilian red propolis.

**Figure 7.** Some volatile compounds from Brazilian red propolis.

#### **2.3. Brown propolis**

**Class of the compound Compound Class of the compound Compound** Monoterpene *p*‐Cymene Sesquiterpene α‐Cubebene

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

Aromatic compound Naphthalene α‐Bergamotene Alcohol, aldehyde or ketone 4‐Hydroxy‐4‐methyl‐heptan‐2‐one Farnesene

Phenylpropanoid Octanal D‐Germacrene

**Table 1.** Compounds found in Brazilian red propolis.

**Figure 7.** Some volatile compounds from Brazilian red propolis.

Limonene α‐Copaene 1,8‐Cineole β‐Gurjunene Linalool β‐Caryophyllene

6‐Methyl‐5‐hepten‐2‐ona €‐β‐Farnesene

Nonanal α‐Selinene n‐Decanal Isocaryophyllene Anisaldehyde β‐Bisabolene *n-*Dodecanal δ‐Cadinene trans‐metil isoeugenol Cadinene Estragole δ‐Cadinol *Trans*‐anethole Aliphatic hydrocarbon Tetradecano Methyl‐*cis-*isoeugenol Pentadecano Elemicin Hexadecano

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 *Araucaria*, although some compounds found on it are also present in *B. dracunculifolia* [37].

**Figure 8.** Chemical compounds of brown propolis.

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 acid [38–40] (**Figure 8**).

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

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 Grosso also showed antimicrobial activity [45].

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.
