Propolis from Meliponinae: A Highway from Ancient Wisdom to the Modern Medicines

Abstract

Propolis has been extensively studied and several chemical constituents, mainly flavonoids, terpenes and phenolics, have been identified. With the emerging microbial resistance to antibiotics, the interest in the search for active compounds, mainly secondary metabolites of plants, has been increasing significantly. In this chapter, we describe the characteristics of the main species of native stingless bees found in South America, especially in Brazil, the ancestral use of propolis produced by them, its chemical composition and its potential for the development of new therapeutic compounds. Its chemical composition is very rich, and for many bee species it remains to be unfolded. Its biological properties evaluated so far include the antibacterial, antimycoplasmic, antifungal, antiviral, antioxidant and anti-inflammatory activities, but not for all known bee species. However, their existence is threatened by the introduction of exotic bees in the environment or using pesticides that annually kill millions of individuals. As if that were not enough, we face the destruction of original forests themselves, and the reforestation strategies with exotic plants.

1. Introduction

Humanity has employed natural resources for healing, particularly plants, animals, and minerals, since the beginning of time. One of the earliest applications of natural products may have been the search for pain alleviation and disease cures through the consumption of herbs [1]. Plants produce a wide range of substances, many of which are connected to defensive mechanisms, such as antibacterial activity. This enormous variety of phytochemicals results in part from the requirement for evolution to contend with microbial, insect, nematode and other plants. Most plant pathogen infections are successfully avoided by this defense mechanism [2].

One of the natural items that has been used by humans for a variety of therapeutic purposes is propolis. It is made up of a combination of the wax and saliva of these insects with resin from plants that bees have collected [3]. This resinous substance is combined with the soil by some stingless bee species to create the substance known as geopropolis [4]. As it is used for building nests and sealing the hive, maintaining the internal temperature and preventing the entry of unwelcome visitors, it is crucial for the colony‘s health. Due to its antibacterial qualities, propolis also functions chemically as a protection against microbes [3].

About 50% of it is composed of vegetable resins, 30% of it is beeswax, 10% is essential oil, 5% is pollen and the remaining 5% is made up of wood and soil debris [5]. The yield of the volatile fraction, however, has typically been described as being in the order of 1% [6]. Propolis has a distinctive perfume that comes from its volatile components, which also have a lot of biological activity. The majority of the chemicals in their makeup were mono- and sesquiterpenoids [7]. Studies have shown how important these chemicals are in the fight against harmful pathogens, including Gram-positive and Gram-negative bacteria [6, 8].

2. Importance of the prospection for new medicines and the role of propolis

Newman and Cragg highlighted in an extensive review in 2020 [9] the importance of natural products and structures derived from or related to natural products as a source for new molecules. In the period between January 1981 and September 2019, 126 antibacterial molecules were approved (disregarding prophylactic agents). Of the 126, about 48% (78) are derived from natural products or unaltered natural products. However, since the advent of antibiotics in the 1950s, virtually no antimicrobials have been developed from plant sources. The main natural sources of these agents were bacteria and fungi. With the emergence of resistance to antimicrobials, interest in the search for other sources, mainly secondary metabolites from plants, has increased significantly [2, 10].

To produce propolis, bees collect the plant resins by selecting the best compounds capable of protecting the nest and ensuring its survival. Considering this natural preselection based on the knowledge of these insects, the composition of propolis and its biological activities have aroused the interest of several researchers [11]. Despite the great diversity of the bees native to Brazil, most of the studies investigating the composition and activities of propolis are carried out with samples produced by exotic bee species introduced in the country. Studies on its volatile fraction are even more scarce. Knowing that the chemical composition of propolis is quite variable and is related to the species of bee that produces it, seasonality and its geographical location, studies with propolis and geopropolis of native stingless bees provide potential unprecedented results and the discovery of compounds with antimicrobial activity [12].

3. Bees’ diversity

Since ancient times, bees have aroused curiosity and human interest. In Greece, they were seen as priestly and chaste animals. The local currency had the image of a bee, symbolizing wealth. The Romans considered them as a representation of territorial defense [13]. In Christianity, they were related to various qualities, above all hope, due to their tireless work, and resurrection, for disappearing in winter and resurfacing in spring [14].

Bees have developed behavioral characteristics, such as the distinction of colors and aromas, which aid in the search for nectar and pollen: their main food sources. These characteristics also benefit the plants, because during foraging, incidentally, they transfer the pollen of the anther of one flower to the stigma of another, performing pollination [15].

The order Hymenoptera is one of the most diverse groups of species in the class Insecta. Hymenoptera presents a wide variety of complex habits and behaviors, culminating in the social organization of wasps, ants and the most important pollinating agents: bees [15]. The bees belong to the superfamily Apoidea, which has several families, among them the Apidae, which has more advanced social habits and is divided into four subfamilies: the Apinae (bees of the genus Apis, with about 11 species), the Meliponinae (stingless bees, with hundreds of species), the Bombinae (bumblebees, with about 250 species) and the Euglossinae (orchid bee, with about 175 species) [16].

4. Meliponinae subfamily

The first written records about the meliponines arrived in Europe in the sixteenth century and were made by Spanish and German explorers in Central and South America. These reports and later archeological studies, particularly in the Mesoamerican region, contributed to the discovery of important data on the traditional search for honey and on the management of meliponines, which was already well structured at least by 300 BC. More advanced research was conducted only in the nineteenth century, almost two centuries after the beginning of scientific research on bees [17].

The subfamily Meliponinae is believed to have been the oldest to branch off from less social ancestors and develop high social behavior. A fossil of a female stingless bee about 80 million years old has been found wrapped in amber. This bee is considered the oldest known species of social bee and has characteristics similar to those of living species [18].

Meliponines are also known as stingless bees (SBs) or indigenous bees because they are traditionally bred by indigenous populations [8]. More than 600 species have been described and are found in South America, Central America, Southern North America, Africa, Southeast Asia and Northern Oceania. In Brazil, more than 200 species can be found, distributed in 29 genera, being Plebeia, Trigona, Melipona, Scaptotrigona and Trigonisca, the ones with the largest number of known species [3].

Brazil is home to the largest diversity of SBs in the world. Native species are distributed throughout the Brazilian territory, mainly in the Amazon region. The warm climate and the abundant flora in species that can provide nectar, pollen and resin are favorable conditions for the existence of these bees [19]. They are especially sensitive to low temperatures and depend on the structure of their nests for the thermoregulation of the hive [20].

The nests of the meliponines are considered the most elaborate among bees. Some species nest in exposed places, such as on branches, but more often the nests are found in preexisting cavities in tree trunks or anthills and abandoned termite mounds. They are protected by one or several layers of a casing composed of wax and resin, which assists in maintaining the temperature. The colony is delimited by the bitumen that consists of a mixture of wax, resin and clay [16, 21]. The upper layer of bitumen is usually compact to avoid infiltration and the lower one is riddled, allowing the flow of water [22].

The entrance of the nest presents particular characteristics for each species and can be constituted of geopropolis, clay, cerumen or pure wax [23]. Some species build quite narrow entrances, which are guarded by a single bee, while others build larger entrances that allow the circulation of multiple guards [24]. Camouflage and occlusion of the entrance are strategies that can also be undertaken to prevent invaders [13].

Female bees have a well-developed ovipositor (egg-laying organ), which can be modified to form a stinger, which is used as a defense mechanism [15]. However, female meliponines have a stunted stinger and are therefore unable to use it, and so are known as stingless bees [13].

Female bees are considered more docile; however, they are not helpless bees. When they feel threatened by large invaders, such as men, they can curl up in their hair or fur, pinch with their sharp jaws, penetrate holes such as ears and nostrils or release unpleasant odors. Some species produce formic acid in their mandibular glands, which when released, can cause serious burns [13, 25]. The SBs with tamer behavior protect their nests by building them in places of difficult access, including inside anthills or near more defensive bee nests [26].

These bees have eusocial behavior, that is, they live in well-organized communities, where the queen is responsible for reproductive work and the workers take care of the offspring, the provision of food and the construction and defense of the nests [24]. Food storage is done in pots consisting of cerumen, where pollen and honey are stored separately [22].

Stingless bees differ from bees of the genus Apis in several aspects. While Apis exhibit uniform behaviors and morphology, meliponines are quite diverse. Some species are very small, smaller than even fruit flies and others have robust bodies. The size of the colony is also variable and can house tens to thousands of individuals. The nests may be arranged in clusters or combs. Foraging can be done by groups or solitary bees and is generally characterized by shorter flights, which imply the elaboration of propolis using the plant resources closest to the colony [27, 28].

The ecological relevance of these insects is undeniable. They act as pollinators of several native and cultivable plants, contributing to the conservation of different biomes and agricultural production, directly impacting the production of fruits and seeds and consequently the economy [29]. The honey produced by SBs is marketed in some regions of Brazil for its appreciated flavor and medicinal properties. These bees also produce propolis, which has been the subject of studies around the world due to its pharmacological properties [3].

5. Important Meliponinae species

5.1 Melipona quadrifasciata, Lepeletier, 1836

Melipona quadrifasciata is a robust bee, which measures between 9 and 10 mm in length, has blackish coloration on its thorax and head, rusty on its wings and yellow bands on its abdomen. It is considered a bee of tame behavior, however, it has quite strong colonies that can act defensively. It can be distinguished into the subspecies Melipona quadrifasciata quadrifasciata and Melipona quadrifasciata anthidioides. The subspecies are differentiated by the pattern of yellow bands present on the dorsum of their abdomen, which consists of three to five continuous bands in M. q. quadrifasciata and two to five discontinuous bands in M. q. anthidioides [30].

Melipona quadrifasciata anthidioides can be found in hot climate regions, often in the states of Rio de Janeiro and Minas Gerais (Southeast Brazil). The subspecies M. q. quadrifasciata prefers regions with colder climates, such as the states of Paraná, Santa Catarina and Rio Grande do Sul (South Brazil). It can also be found in high-altitude regions in the states of São Paulo, Rio de Janeiro and Minas Gerais [31].

‘Yra-maya‘ is the indigenous appellation (Tupi) for bee, where ‘Yra‘ means honey and ‘may‘, ‘manha‘ or ‘manda‘ means watchman. M. q. quadrifasciata is popularly known as ‘mandaçaia’, where ‘çai‘ in the indigenous denomination refers to smart [32]. The cleverness of the nest watch is one of the most impressive features for traditional peoples. In fact, the mandaçaia is an attentive watchman: a worker bee always keeps guard at the entrance of the nest, to defend the hive.

The crops pollinated by this species are pumpkin (Cucurbita moschata), pepper (Capsicum annuum), chili pepper (Capsicum frutescens) and tomato (Lycopersicon esculentum). There are also plant species that are attractive to these bees, such as ‘assa-peixe’ (Vernonia polyanthes), ‘estoraque’ (Styrax ferrugineum), ‘murici’ (Byrsonima intermedia), ‘picão’ (Bidens segetum), ‘picão-de-cipó’ (Bidens gardneri) and witches’ broom (Ouratea hexasperma) [33].

Melipona quadrifasciata quadrifasciata was classified as endangered in the List of Fauna Species Threatened with Extinction in Rio Grande do Sul, Brazil, in 2002 [34]. In 2014, the Zoobotanical Foundation (FZB) with support from the State Secretariat of the Environment (SEMA) updated these data, where the species remains with the same classification.

5.2 Melipona compressipes manaosensis, Schwarz, 1932

Melipona compressipes manaosensis is popularly known as ‘jupará’ or black ‘jandaíra’ of the Amazon. In the central Amazon, its products are used in the feeding and income provision of local populations, in addition to playing an important role in the pollination and dispersal of seeds that occur during the collection of plant resins [33, 35].

This species can be found in Amazonas, Amapá and Pará in Brazil and Guyana, French Guiana and Suriname [36]. The worker bees of this species measure about 13 mm and have little defensive behavior. The colonies are made up of about 5000 workers, 250 bumblebees and a queen. The entrance to their nests consists of a mixture of vegetable resins with clay, resulting in a whitish mixture [37].

A study identified the plant origin of pollen grains collected by SB species for 1 year [38]. Some plant species where pollen resources were collected by M. compressipes manaosensis were ‘guava-de-tapir’ (Bellucia grossularioides), ‘cássia-rosa’ (Cassia grandis), ‘embaúba’ (Cecropia sp.), ‘jenipapeiro’ (Genipa americana), ‘araçá’ (Myrcia amazonica), ‘pau-rosa’ (Physocalymma scaberrimum), ‘cajazeira’ (Spondias mombin) and ‘baginha’ (Stryphnodendron guianense).

5.3 Melipona bicolor schencki, Gribodo, 1893

This robust species measures about 9 to 10 mm. Its voluminous body is covered with hairs, the coloration of which varies throughout its life, being lighter when young and darker with time. It is considered a bee of tame behavior and easy management. Two subspecies of M. bicolor were distinguished: M. b. bicolor and M. b. schencki. They differ in the coloration and geographic area they are distributed. The first can be found in Brazil in the states of Bahia (Northeast), Espírito Santo, Minas Gerais, Rio de Janeiro and São Paulo (Southeast). And the second, which has darker hairiness, can be found in Paraná, Rio Grande do Sul, Santa Catarina (South) and in high and cold areas of Southeastern Brazil [36].

Melipona bicolor schencki is popularly known as ‘guaraipo’, ‘guarupú’ or ‘pé-de-pau’ [36]. In the Tupi nomenclature, ‘guarabu’, sometimes written ‘guaripú’ or ‘guaraipo’, comes from ‘guará’ which refers to the habit of drilling and ‘ubu’ or ‘ibú’ which means land. This species found in forest regions usually nests in hollow sticks, or tree cavities close to the ground, which justifies its popular name [32, 39].

While almost all colonies of social bee species are led by only one queen, M. bicolor presents a unique behavioral characteristic: facultative polygyny. It is the only species of the genus Melipona where cohabitation of queens can occur for a considerable period, not only transiently [39].

Melipona bicolor schencki is on the List of Endangered Fauna Species in Rio Grande do Sul, Brazil, in 2002 [34], which was updated in 2014 by the Zoobotanical Foundation (FZB) with support from the State Secretariat for the Environment (SEMA), remaining in the ‘endangered‘ classification.

5.4 Melipona marginata, Lepeletier, 1836

These bees, popularly known as ‘manduri’, live in small colonies with about 300 individuals. The entrance to their nests is striated and narrow, allowing the passage of one bee at a time [40]. In Brazil, it is found in the states of Bahia, Ceará and Pernambuco (Northeast), Espírito Santo, Rio de Janeiro and São Paulo (Southeast), Goiás and Minas Gerais (Center), and Santa Catarina (South) [36].

This species is considered one of the smallest in the genus Melipona, measuring between 6 and 7 mm in length. Despite their size and the low number of individuals in their colonies, they are proportionally large honey producers. They have a very defensive behavior and can nibble strongly when they feel threatened. It is a polytypic species, which presents subspecies with different colors of hairiness, related to the altitude of its location. This characteristic was observed for the first time in 1875 by the naturalist Fritz Muller in Blumenau, Santa Catarina, Brazil, when describing the species now called Messor obscurior [41].

There are records to prove that this species can colonize termite mounds, limiting the area with the use of resins. They also use the same material to enlarge the delimited area, invading the space of the termite mound. Their nests can also occupy tree hollows, lined with bitumen consisting of resin and earth [42].

5.5 Melipona seminigra merrilae, Cockerell, 1919

Melipona seminigra merrilae, popularly known as ‘uruçú boca-de-renda’, is a medium-sized SB species, about 11 mm long. It has a robust body and dark brown integument. Its elongated abdomen has a bright reddish-brown color and predominantly yellowish-fawn-hollow hairiness [26].

This species has been reported only in the states of Amazonas and Pará. It inhabits floodplain and ‘igapó’ forests, nesting in trees 8 to 10 m from the ground. The entrance to their nests is tubular consisting of geopropolis. It is commonly ornamented with brightly colored seeds and resins [26, 36]. Its colonies are populous, housing about 2000 individuals. The queen is slightly smaller than the workers and has darker coloration. This species adapts well to different environments and collects nectar throughout the year, including in the rainy season [26].

A study was able to verify some plant species visited by Melipona seminigra merrilae, such as ‘pau-de-facho’ (Aparisthmium cordatum), ‘guava-de-tapir’ (Bellucia grossularioides), ‘cássia-rosa’ (Cassia grandis), ‘embaúba’ (Cecropia sp.), white wren (Matayba sp. nov.), ‘araçá’ (Myrcia amazonica), ‘pau-rosa’ (Physocalymma scaberrimum) and ‘cajazeira’ (Spondias mombin) [38].

5.6 Melipona fasciculata, Smith, 1855

Melipona fasciculata, also known as ‘tiúba’ or ‘uruçú’ gray, is a species of SB about 12 mm long and predominantly grayish in color. It has abundant whitish hairiness on its body and near the wings the hairs are reddish. Along its abdomen, it has clear bands that can be continuous or interrupted [43]. In Brazil, this species can be found in the states of Maranhão, Pará and Piauí (North), Mato Grosso and Tocantins (Center) [36]. It is rare on dry land and usually nests in mangrove regions, where it shelters its colony in tree hollows. In the mangroves, they are found in the ‘siriúba’, a popular name for the species Avicennia nítida, family Avicenniaceae [44].

The indigenous peoples like Timbiras, Tupinambás, Guajajaras, Tremembés, Awha-Guajás, Vultures and Gavião domesticated this species, passing on the knowledge about its creation over generations [45]. This species pollinates the crops of ‘açaí’ (Euterpe oleracea), eggplant (Solanum melongena), cashew (Anacardium occidentale), tomato (Lycopersicon esculentum) and annatto (Bixa orellana). Other plants are also attractive to these bees, such as giant stink (Senna alata), ‘lobeira’ (Solanum grandiflorum), ‘jurubeba’ (Solanum juripeba), seal (Vismia guianensis), myrtle (Myrcia eximia), thrush (Mimosa caesalpiniifolia), crab (Miconia minutiflora), ‘siriuba’ (Avicennia nítida) and ‘tapiririca’ (Tapirira guianensis) [33].

6. Indigenous bees: sociocultural importance and ethnoknowledge

Indigenous peoples have a close relationship with the meliponines. Before the introduction of the species Apis mellifera and the culture of sugar cane in the Americas, the honey of the native bees was used by the indigenous peoples as an indispensable source of energy for the great journeys in the search for food. The popular names of many species, such as Jataí, Tiúba, Jandaíra, Guarapu and Manduri, are part of the sociolinguistic indigenous heritage [22].

Indigenous knowledge is closely related to nature. The legacy of these peoples has immeasurable value and has been passed down through the generations. In Mexico, the creation of SBs and the use of its products date back to the pre-Columbian period. For the Maya people, one of the most relevant ancient civilizations in history, the SBs played an important role in religious ceremonies, feeding and treating diseases [46].

In Brazil, the Kayapó people use elaborate techniques for the management of swarms, such as platforms that allow the reach of hives in tall trees. Among these people, bee specialists are shamans, who possess extensive knowledge about the anatomy and behavior of various SB species. The people use honey and pollen in food, cerumen and resins in the waterproofing of canoes, in addition to being inspired by the social organization of these insects [25]. For the Pankararé, indigenous peoples in northeastern Brazil, the creation of ‘abelha mansa’ (meek bee) is a recreational and medicinal activity. Honey is used in food as an energy source and is extracted in a nonpredatory way, ensuring the maintenance of the tree from where it was collected and the colony [24].

The knowledge about the SBs of the Guarani Mbyá people of the Morro da Saudade village, in the city of São Paulo, Brazil, is transmitted orally through the generations. The teachings are followed and improved by the younger ones, and some members of the community are seen as great connoisseurs of the subject. The products from bees are used in handicrafts, religious rituals and medicinal potions, as well as activities related to spiritual and contemplative life [47]. A study was conducted on the traditional knowledge about the SBs of the Enawene-Nawe people of the state of Mato Grosso. It was verified that the people were able to identify several species of SBs not only by morphology but also by the ecological and social characteristics of these insects [48].

Meliponiculture has also been practiced for generations by other traditional communities like ‘quilombolas’ (descendants of enslaved Africans), ‘ribeirinhos’ (inhabitants of riverbanks), ‘sertanejos’ (inhabitants of the hinterland), ‘caipiras’ (countrymen) and ‘caiçaras’ (traditional fishing communities). The products from the SBs are used for subsistence family consumption and are a source of complementary income. These people contribute to the conservation of bees, through the management of species that are practically no longer found in natural habitat, due to the devastation of the native forest. The rational beekeeping of SBs can therefore be a sustainable strategy to promote biodiversity conservation [23].

7. Introduction of exotic bee species in Brazil

The introduction of the European bee species Apis mellifera in Brazil occurred around 1839, brought by the Portuguese to produce wax candles. In 1845, German immigrants brought more bees and settled in the south of the country. Other colonizers have also introduced European bees into different regions [49]. In 1956, the biologist and geneticist Dr. Warwick Kerr, at the request of the Brazilian government, brought from Africa some queens of bees to perform crossing by artificial insemination, which would originate a species capable of producing more honey in the tropical climate [25, 49].

The following year, swarms and their respective queens escaped accidentally and ended up crossing with the European species already introduced in the Brazilian territory, emerging hybrid populations, today called Africanized bees. This incident had an extensive environmental impact since these bees spread to almost the entire American continent [25, 49].

8. Impact of anthropogenic actions on bees

The introduction of exotic species, accidentally or for economic purposes, can cause significant changes in natural environments, such as changes in habitats, hybridization and competition with native species. In addition, the intensive occupation of the environment by man impacts bee populations by eliminating food sources, by destroying substrates necessary for the construction of nests and by using pesticides [8].

Due to the intense bee die-off, several studies were conducted to evaluate the toxicity of agrochemicals, but most of them were carried out with the species Apis mellifera, endemic in several regions of the world. A study evaluated the effect of the insecticide dimethoate on the SB species Melipona scutellaris, where the average lethal concentration (LC50) was 320 times lower than for Apis mellifera larvae, demonstrating the sensitivity of this species to the pesticide and the need to conduct further studies using native bees [50].

The deforestation of forest areas for urban and agricultural use has also significantly reduced bee populations. The scarcity of resources, competition between species, predation by invaders and inbreeding due to population decrease, can make swarms captive to a small territorial space, or lead them to extinction [8].

Even if the melipones adapt, it was verified that the major volatile compounds in the propolis of native bees in the Itajaí Valley region, in Southern Brazil, are compounds found in Pinus and Eucalyptus, indicating that this is the most important plant source in the elaboration of propolis by those specimens [51]. This ‘reforestation‘ with exotic trees certainly contributes to the difficulty in discovering new molecules with biological activity and potential for the development of new drugs.

As bees contribute to the maintenance of forests through pollination, several species of Meliponinae depend on forest environments and are not found in anthropogenic environments, except near forests. This issue is an aggravating factor in Brazil, considering that some species of the genus Melipona are translocated to regions distant from their natural occurrence [8].

9. Bee products beyond honey: propolis

Propolis is a mixture of substances used by bees in the defense of the hive. Worker bees collect resinous materials from shoots, exudates and other parts of plants and pack them in their corbicles (part of the posterior tibia used to transport pollen, clay and resin). These resins are biotransformed by bees with the addition of their salivary enzymes and wax [52, 53]. Physically, propolis has quite variable characteristics. It may have a hard and brittle consistency or be sticky and elastic. The coloration can also vary between cream, yellow, green, light brown or dark brown [54].

The name propolis is derived from the Greek words pro, in defense of, and polis, the city, e.g.: ‘in defense of the city or the hive’. In fact, this material has great importance for the health of the colony. Bees use propolis in the sealing and repair of crevices to prevent the entry of invasive insects and maintain the internal temperature. In addition, it is used against microorganisms in the asepsis of the places where the laying of eggs is made. And if the bees cannot remove a dead invader from the hive, they use propolis to embalm it, which prevents decomposition and bacterial proliferation in the nest [54, 55].

The plant materials used in the composition of propolis are produced by various botanical processes in different parts of the plants. The collection of these materials is a difficult activity to observe and often occurs high in the trees. Among the substances collected, lipophilic compounds from the leaves, mucilage and resins can be highlighted [7].

10. Historical and popular use of propolis

Propolis has been used in traditional medicine since antiquity. The Egyptians used it to embalm the dead and thus prevent putrefaction. Its properties were also recognized in Greek and Roman medicine by Aristotle, Dioscorides, Pliny and Galen, who employed it in asepsis and wound treatment and as an oral disinfectant. Its use was perpetuated in the Middle Ages in Arab medicine and was recognized by New World civilizations [56].

In the seventeenth century, propolis was included in the London Pharmacopeia. Between the seventeenth and twentieth centuries, its use became popular in Europe due to its antibacterial action. It is currently used in various pharmaceutical forms, such as extracts, mouthwash, lozenges and formulations for topical use. Also, its employment in the food and cosmetic industries has been benefited from the propolis properties [56].

In 1908, the first scientific work on propolis, its chemical properties and composition was indexed in Chemical Abstracts. The first patent appeared decades later in the same index of scientific literature, in 1968, where Romanian propolis was employed in the production of bath lotions. Since then, several studies on propolis have been published, possibly due to its panacea characteristic and its added value [57].

Currently, propolis is used for various therapeutic purposes as an antibacterial, antifungal, antiviral, anti-inflammatory agent and for increasing the body‘s natural resistance to infections. Formulations for external use are employed in the treatment of dermatitis and wounds. It is also available in capsules of pure extracts or in combination with other natural products. Throat lozenges and spray, mouthwash and hydroalcoholic or glycolic extracts are widely used as a folk remedy [56].

11. Geopropolis

Some SB species add soil to propolis, giving rise to the so-called geopropolis (Figure 1). Although it is not its main constituent, the presence of earth is a differential in the composition of this product. Its coloration is variable and is related to the plant origin, the soil used in its constitution and the species of bee that produces it. The chemical composition and biological activities of this bee product are still little known [29].

Figure 1.

Geopropolis over a Melipona bicolor schencki hive (photograph by Ariane Storch portal).

Fritz Müller, a German naturalist who migrated to Blumenau, Santa Catarina (Brazil) in the mid-nineteenth century, in one of his letters to Charles Darwin, about the habit of various insects, mentioned his observations on the genus Melipona and the constitution of propolis. He observed that these bees not only used wax in the construction of the nest structures but added other materials such as resins and soil [58].

12. Chemical composition of geopropolis from meliponines

In 1998, more than 50 compounds were identified in the Brazilian geopropolis [59]. Among them, the geopropolis of Melipona compressipes and Melipona quadrifasciata anthidioides collected in the states of Piauí (Northeast) and Paraná (South), respectively. The extracts of these samples were submitted to chemical analysis through gas chromatography mass spectrometry (GC-MS) and a complex chemical composition was observed. The samples showed significant amounts of lactic, phosphoric and long-chain fatty acids, such as stearic and palmitic acids.

The chemical composition of Melipona fasciculata geopropolis, collected in Maranhão state (North), was also analyzed [60]. The main constituents found were carbohydrates and their derivatives (19.8%), triterpenes (15.9%), hexoses (11.9%), anacardic acid (8.3%), lupeol (7.3%) and alkylresorcinols (5.9%). Disaccharides, glucuronic acid, salicylic acid and isomers, β-amyrin, inositol and xylitol, among others, were also found.

Samples of geopropolis of the species Melipona interrupta and Melipona seminigra, collected in municipalities of Amazonas (North Brazil), were evaluated for their composition, evidencing the significant presence of phenolic compounds [61].

The composition of the aqueous and hydroalcoholic extracts of Melipona quadrifasciata could be demonstrated. Rutin, gallic acid, gallocatechin, epicatechin gallate and syringic acid were identified in the aqueous extract. In the hydroalcoholic extract, the main constituents found were quercetin, epigallocatechin, p-hydroxybenzoic acid, epigallocatechin gallate and coumaric acid [62]. The flavonol sakuranetin and gallic acid were also identified [63].

Propolis contains volatile constituents to a lesser extent. This fraction, however, can provide relevant information about the antimicrobial activity and elucidate the classes of compounds present in its composition, contributing to the identification of its botanical origin [64]. Volatile compounds are among the main secondary metabolites present in plants. They perform important functions that ensure survival and adaptation to the environment. Among these functions can be mentioned the attraction of pollinators and seed dispersers, protection through repulsion or intoxication and antibacterial, fungicidal and insecticidal action [65].

Volatile oils (VOs), also called essential oils (EOs) when obtained directly from plants, are a complex mixture of lipophilic substances, consisting mainly of terpenes, fatty acid derivatives, amino acid derivatives and phenylpropanoid compounds. In propolis, terpenes constitute a large part of the volatile compounds [66].

Terpenes are classified according to the number of isoprene units (C5H8) present in their structure: monoterpenes (C10H16), sesquiterpenes (C15H24), diterpenes (C20H32), triterpenes (C30H40), tetraterpenes (C40H64) and polyterpenes [67]. Monoterpenes make up a large part of the EO and can contribute more than 90% of its total composition. Sesquiterpenes are another important group found in the EOs. Terpenes can be biosynthesized by the classical mevalonate pathway in the cytosol or by the alternative deoxyxylulose phosphate pathway that occurs in plastids [68].

Essential oils can be obtained by distillation or pressing of plants or parts thereof. At room temperature, they are liquid and volatile, with a characteristic odor. The density of EOs is generally lower than that of water, but sassafras, cinnamon and clove oils may have higher densities [68]. The composition of a VO can present a major constituent, facilitating chemical correlation with biological activity. However, small amounts of other substances present can act synergistically, contributing to a certain biological action [69]. However, the composition of these oils is variable and is directly related to seasonality and the place of collection [12].

13. Biological activities of geopropolis compounds

Some studies have highlighted the importance of investigating the biological potential of geopropolis. Table 1 presents selected studies that investigated the antibacterial, antimycoplasmic, antifungal, antiviral, antioxidant and anti-inflammatory properties of Melipona species geopropolis.

Bee species	Biological activity	References
Melipona compressipes	Antibacterial	[70]
	Antifungal	[70]
	Antiviral	[70]
M. fasciculata	Antioxidant	[71, 72]
M. compressipes fasciculata	Antibacterial	[4, 73]
M. q. quadrifasciata	Anti-inflammatory	[74]
	Antioxidant	[27, 63]
	Anti-herpetic	[27]
	Antibacterial	[27, 75, 76]
	Antimycoplasmic	[51, 75, 77, 78]
	Antiadhesive	[51, 77]
	Antifungal	[28]
M. seminigra	Antioxidant	[61]
M. marginata	Antibacterial	[28]
M. q. anthidioides	Antibacterial	[70, 79]
	Antifungal	[70]
Melipona bicolor schencki; M. compressipes manaosensis; M. fasciculata; M. quadrifasciata; M. marginata	Antibacterial; Antimycoplasmic	[51]

Table 1.

Biological activities of geopropolis of bees of the genus Melipona already studied.

It was soon evidenced as regards the antioxidant potential of the hydroalcoholic extract of geopropolis of M. fasciculata [71]. The action was correlated with the presence of phenolic compounds, such as phenolic acids, gallotannins and ellagitannins. It was also suggested that the high concentration of phenolic acids, such as gallic and ellagic acids, was responsible for the antioxidant action of the samples tested [72].

The antibacterial activity of the hydroalcoholic extract of Melipona orbignyi geopropolis was evaluated against the Gram-positive bacterium Staphylococcus aureus (sensitive and methicillin-resistant strains) and the Gram-negative Escherichia coli (sensitive and cephalosporin-resistant strains) and Pseudomonas aeruginosa (strains sensitive and resistant to amphotericin B). The antibacterial action was attributed to the presence of phenolic compounds, which have some known mechanisms of action, among them the permeabilization of the microbial cytoplasmic membrane [62].

Others investigated the antimicrobial potential of the volatile oil of Melipona q. quadrifasciata geopropolis against bacteria with and without cell walls. The antimicrobial action with the lowest minimal inhibitory concentration (MIC) was displayed against the bacterium without cell wall Mycoplasma pneumoniae 129 strain. The volatile oil was fractionated and its subfractions were tested, but these subfractions showed no improvement in antibacterial activity [75]. This characteristic had already been observed by other authors, who concluded that the isolated compounds from propolis did not present better results regarding the activities tested, suggesting that the synergy between the compounds favors biological actions [70].

In the literature, it is possible to find studies that confirmed the antimicrobial activity of the volatile constituents of propolis against several microorganisms. Against Gram-positive bacteria such as S. aureus, Staphylococcus epidermidis, Micrococcus glutamicus, Bacillus subtilis, Bacillus cereus, Sarcina lutea, Streptococcus pyogenes, Streptococcus mutans, Streptococcus faecalis and Gram-negative bacteria such as E. coli, Enterobacter cloacae, Klebsiella pneumoniae and P. aeruginosa [6].

A study conducted in Greece investigated the chemical composition of volatile propolis compounds from different geographic regions. The predominant constituents were terpenoids, especially α-pinene. Other components have also been identified as α-eudesmol, δ-cadinene, α-muurolene, guaiol and trans-β-terpineol. The in vitro antimicrobial activity was evaluated against six species of bacteria, including S. aureus, P. aeruginosa and E. coli, confirming the antimicrobial potential of the samples tested [80].

In another study, the volatile constituents of propolis samples collected in three municipalities of Rio Grande do Sul state (South Brazil), α-pinene and β-pinene, were presented as major constituents. The antimicrobial activity was evaluated by the agar diffusion method against S. aureus, P. aeruginosa, Klebsiella pneumoniae, Bacillus subtilis and E. coli-type strains. The antibacterial activity was classified as moderate and the presence of monoterpenes in the samples was attributed to this activity [81].

The antimicrobial potential of the volatile oil of Apis mellifera propolis collected in Mato Grosso do Sul (West Brazil) was also evaluated. The activity of the said volatile oil was evaluated by the broth microdilution method against strains of P. aeruginosa, K. pneumoniae, Enterococcus faecalis and S. aureus. The volatile oil and two of its isolated constituents—(E)-nerolidol and spathulenol—showed antimicrobial properties [82]. Others tested the antimicrobial activity of the volatile oil of Apis mellifera propolis collected in Rio de Janeiro. The microorganisms such as S. aureus, S. epidermides, S. pyogenes and E. coli were susceptible to the sample tested. The chemical composition presented as major constituents β-caryophyllene (12.7%), acetophenone (12.3%) and β-farnesene (9.2%) [12].

It was found that the M. b. schencki geopropolis VO with the best minimal inhibitory concentration (MIC) was 424 ± 0 μg mL⁻¹ against some mycoplasma strains. Fractionation of the VO resulted in a reduction of 50% of the MIC. However, its compounds’ synergism seems to be essential to this activity. Antibiofilm assays demonstrated 15.25% eradication activity and 13.20% inhibition of biofilm formation after 24 h for one subfraction at 2x its MIC as the best results found. This may be one of the essential mechanisms by which geopropolis VOs perform their antimicrobial activity [51].

The antiadhesive activity of M. quadrifasciata geopropolis was demonstrated against mollicutes (mycoplasmas), where a dichloromethane subfraction was highlighted in the antiadhesive assay with an inhibitory activity of 21.6%. A synergistic effect of the nonpolar compounds in M. quadrifasciata propolis was also suggested as responsible for its antibacterial activity. The hexane (MIC = 62.5 mg/L) and dichloromethane (MIC = 125 mg/L) fractions presented the most promising antibacterial results against M. pneumoniae [77].

14. Bacterial resistance to antibiotics and the need for new drugs

The increasing microbial drug resistance has been observed worldwide and with increasing mortality, prolonged hospital stays and rising costs, with other sectors of society being impacted beyond healthcare [83].

Between 2011 and 2014, the National Healthcare Safety Network (NHSN) in the USA reported high levels of resistance to various antibiotics in Gram-positive bacteria including methicillin-resistant S. aureus (MRSA), and Gram-negative bacteria such as third-generation cephalosporin-resistant E. coli and carbapenem-resistant P. aeruginosa [84].

Data from the European Centre for Disease Prevention and Control from 2015, when compared to US data, showed lower rates of resistance in Gram-positive bacteria and equally worrisome rates among Gram-negative bacteria [84].

The U.S. Centers for Disease Control and Prevention (CDC) estimates that at least 23,000 deaths from resistant infections occur each year in the USA. In Europe, in the year 2007 alone, 25,000 deaths were attributed to infections by resistant microorganisms [85].

If this current trend continues, it is estimated that by 2050 there will be 10 million additional deaths due to antimicrobial resistance, surpassing other significant diseases such as cancer and diabetes. In addition, in the same year, microbial resistance is estimated to cumulatively cost $100 trillion worldwide [85].

Natural products offer an obvious source for the research and development of new pharmacological treatments for infections. Moreover, it is more rational to use the millenary wisdom of bees, which learned to look for the best chemical compounds in nature to produce their propolis for the defense of their hives, in every environment, instead of testing individually each plant species for antimicrobial compounds. Unfortunately, we risk losing this wisdom while putting native bee species in danger of extinction.

15. Conclusion

Propolis from native stingless bees has a very rich chemical composition and an enormous potential to be unveiled, both in its use in the form of herbal medicines and in the discovery of new molecules with potential for drug development. Its properties evaluated so far include mainly the antibacterial, antimycoplasmic, antifungal, antiviral, antioxidant and anti-inflammatory activities, but not for all known bee species. Many species of bees have not even had their propolis chemically characterized. Furthermore, their existence is threatened being by the introduction of exotic bees in the environment or using pesticides that annually kill millions of individuals. As if not enough, we also have to deal with the destruction of the natural forests themselves and the use of foreign flora for reforestation. Hence, it is necessary that their environment be preserved, and that the bees themselves be preserved, so that humankind can benefit from this ancestral treasury.

Acknowledgments

We are grateful to the meliponiculturists Lauro Muegge, Edmundo Hang, Fabiana de Paiva Costa Barros, Edenilson Ferreira, Gustavo Jacinto and Miguel Schikorski. Our thanks also to Camila Panini Valcanaia, Camila Paganelli, Sabrina Hochheim, Caroline Galgowski, Larissa dos Santos, Prof. Alessandro Guedes and Prof. Dr. Sérgio Luiz Althoff for the collaboration. Ariane Storch Portal received a fellowship from the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil), for which we are grateful.

Conflict of interest

The authors declare no conflict of interest.