Keywords

1 Introduction

Propolis, also known as bee glue, is a resinous and sticky bee product that bees use both as a building and sheltering material. Of varying odour, texture and colour (from light to dark), propolis is a complex mixture of plant buds and resins, as well as other plant secretions and volatiles, that honeybees (Apis spp.) blend with pollen and beeswax (the mixture being called cerum or cerumen). Occasionally, other constituents are also found in propolis, particularly when plant resources are low. Another type of propolis, geopropolis, is collected by stingless bee species (Melipona spp.) native to tropical countries. Geopropolis is composed by combining soil elements with plant exudates and beeswax (Bankova et al. 2000, 2014; Salatino et al. 2005, 2011; Toreti et al. 2013; Huang et al. 2014).

Although they account for only a minor portion of propolis constituents, volatiles impair particular aromas to propolis from different origins and have been considered as a possible quality criterion for propolis freshness (Woisky and Salatino 1998).

Increasing interest in the medicinal properties of propolis has inspired research on numerous topics related to this product, and several reviews have addressed the chemical composition, botanical origin and biological properties of propolis (Bankova et al. 2000, 2014; Salatino et al. 2011; Toreti et al. 2013; Huang et al. 2014). However, literature addressing the volatile profile of propolis or geopropolis is scarce compared to that of other chemical components, with just one previous review focusing on this subject (Bankova et al. 2014). The present review examines the volatile constituents of propolis and geopropolis (for simplicity they will both be referred to as propolis), their isolation and analysis.

2 Propolis Volatile Fraction

Propolis volatiles have been studied by several researchers, Table 6.1, as recently reviewed by Bankova et al. (2014). Despite the use of different sampling techniques, all allow the characterization of the propolis volatile fraction in a more or less specific way, so they will all be considered here.

Table 6.1 Studies on propolis and geopropolis volatiles
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As a bee product, the chemical composition of propolis in general, and volatiles in particular, is dependent on a number of factors, such as geographical origin, bee type, local flora, and also the methodology used for isolation and analysis of the volatiles, as discussed below.

2.1 Propolis Origin

In total, propolis and geopropolis volatiles from 25 countries were evaluated, Table 6.1, with between one and 106 samples evaluated per country. Propolis from different geographical origins showed different chemical profiles, Table 6.1, which usually correlate with the natural flora available at the hive location and the preferences of the bee type.

Traditionally (Bankova et al. 2014), propolis types have been divided according to their continent of origin. Nevertheless, considering the diversity and specificity of the flora in different countries, for instance those in northern and southern Europe, some additional subgroups may have to be considered.

2.2 Bee Type

Studies on Africanized bees (Apis mellifera) and European bees (Apis mellifera ligustica) showed more quantitative than qualitative variations in propolis volatiles. This may indicate that both types of bees collected propolis from the same plants, but, depending on when the propolis was collected, some plants may have been more dominant than others (Bankova et al. 1998a). According to Haile et al. (2012), the variability in the volatiles of propolis obtained from two locations in Ethiopia could in part be due to the dominance of Apis mellifera monticola in the Assela region compared to the Haramaya region, where Apis mellifera jementica was the dominant honeybee.

As with honeybees, stingless bees also collect plant exudates but produce their particular form of propolis, geopropolis, using different plant sources from those used by honeybees (Table 6.1). Foraging workers of three species of Frieseomelitta stingless bee have been shown to carry complex mixtures of plant-derived mono-, sesqui-, di- and tri-terpenes in the posterior tibia (Patricio et al. 2002). Although studies on volatiles from stingless bee propolis are limited, they have confirmed a different chemical profile from propolis obtained from honeybees of the same region, as this is dependent on the flora preferences of the bees (Bankova et al. 1999; Pino et al. 2006; Bankova and Popova 2007; Ioshida et al. 2010; Huang et al. 2014; Isidorov et al. 2016; Torres-González et al. 2016).

2.3 Natural Flora

Natural local flora determines the chemical composition of propolis, including that of their volatile fraction. In addition to exudates from trees, bees may also use secretions from aromatic plants (lavender, rosemary, sage, thyme) or material from other sources, such as asphalt or tar (Borčić et al. 1996; Alqarni et al. 2015). However, current knowledge on the plant sources of propolis is based more on observation studies of bee behaviour, or comparative studies of the phenolic composition of plants and propolis, than on the corresponding volatile composition.

Indeed, although several of the studies on propolis volatiles report the natural local flora (Corsi 1981; Petri et al. 1988; Greenaway et al. 1989, 1991; Bankova et al. 1994, 1998a, b; Pino et al. 2006; de Albuquerque et al. 2008; Agüero et al. 2011; Haile et al. 2012; Miguel et al. 2013; Torres-González et al. 2016), only some simultaneously compared the volatile profiles of propolis with those of the local flora (Maróstica Junior et al. 2008; Falcão et al. 2016).

Maróstica Junior et al. (2008) showed the chemical similarities between the volatile compounds found in Brazilian green propolis and those from its botanical origin, Baccharis dracunculifolia (Asteraceae/Compositae).

According to Falcão et al. (2016), the chemical composition of Populus x canadensis (Salicaceae) and Cistus ladanifer (Cistaceae) essential oils showed some correlation with that of certain Portuguese propolis samples.

2.4 Detection of Acaricides and Adulterants

Acaricides are used to control bee parasites and can be detected in many hive products, such as propolis and beeswax. Similarly, the use of adulterants such as benzoic acid and eugenol has been reported in propolis, with the objective of enhancing the antibacterial activity (Bonvehí et al. 1994; Woisky and Salatino 1998). Some studies on propolis volatiles reported the presence of acaricides such as Vanquard BT 2-4, Coumaran and Apiguard (Bankova et al. 1998b; Bankova et al. 1999; Miguel et al. 2013), which may be responsible, for instance, for the high thymol content seen in some cases (Miguel et al. 2013).

2.5 Isolation and Analysis of Volatiles

The propolis volatile fraction comprises a mixture of volatile compounds that can be obtained because they either vaporize naturally or after the use of specific techniques.

In many studies propolis was extracted with ethanol. Although volatile compounds can also be isolated by this organic solvent, this is not an extraction procedure specific to the isolation of volatile compounds and, for this reason, these studies were not included in Table 6.1. According to Woisky and Salatino (1998), propolis extraction with aqueous ethanol (70%) is better than with absolute ethanol as it provides wax-free tinctures containing higher amounts of phenolic substances. Some studies in which less polar solvents were used for propolis extraction were also included for comparison (Table 6.1).

Depending on the technique used for the isolation of propolis volatiles, Table 6.1, some authors also evaluated the composition of propolis essential oil [obtained by hydro, steam or dry distillation from any plant part or by expression of citrus species (Council of Europe 2010)]. Although they do not provide essential oils, other conventional volatile extraction techniques used included solvent extraction combined with distillation, simultaneous distillation-extraction, microwave-assisted extraction, static or dynamic headspace sampling, and solid-phase microextraction.

Needless to say that all of these techniques provide different and complementary information on propolis volatiles, as they are based on diverse principles. Whereas some methodologies are humid-heat based extraction procedures, such as hydrodistillation or distillation-extraction, others are solvent-free extractions which sample the vapour phase of propolis, such as headspace techniques. Bracho et al. (1996), Fu et al. (2009), Pellati et al. (2013), Pellati (2014) and Jercović et al. (2016) compared different methodologies of propolis extraction, obtaining different proportions of volatile compounds (Table 6.1).

After isolation, and independently of the separation procedure, propolis volatiles are then analysed in order to identify and quantify the components, Table 6.1. Gas chromatography (GC) and gas chromatography coupled mass spectrometry (GC-MS), or just GC-MS, were the techniques most frequently used for this purpose. One of the factors that impairs direct comparison between data is the quantification procedure. In some cases no quantification was performed, in others quantification was performed by GC or GC-MS; and whereas sometimes the data is reported in percentages, in others it is reported in absolute amounts (Table 6.1).

One study combined gas chromatography–olfactometry–mass spectrometry (GC-O-MS) and sniffing tests (Yang et al. 2010). In the sniffing studies, propolis odour was mainly described as fruity, pungent, acidic, grassy, herbaceous, stale and smelly, and it was confirmed that components with high concentrations do not always play a major role in odour contribution. One study (Simionatto et al. 2012) also evaluated the variability in enantiomeric composition of monoterpenes α-pinene, β-pinene and limonene in the volatile oils from three samples of Brazilian propolis. (-)-β-Pinene predominated in all samples, whereas the enantiomeric excess of α-pinene and limonene was dependent on the harvesting location.

3 Main Volatile Compounds in Propolis

As stated above, direct comparison of the quantitative composition of propolis volatiles should be avoided, due to the use of different isolation procedures and quantification methods, Table 6.1. For this reason, herein, comparisons are made based on the relative frequency of occurrence of a compound in different propolis volatile extracts.

β-Eudesmol was found in most samples, Fig. 6.1 and Table 6.1. Guaiol, tricosane and thymol were also frequently reported in studies on propolis volatiles (Fig. 6.1, Table 6.1).

Fig. 6.1
figure 1

Compound frequency (≥20%) in propolis volatiles studies

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It is important to consider that, in some cases, a high frequency of occurrence of a compound in propolis does not mean that it is a natural component resulting from collection by bees. For instance, thymol, as mentioned above, may occur in propolis as a result of treatment against varroa, which uses an acaricide containing thymol as the chief constituent (Miguel et al. 2013).

Though the number of samples studied is still relatively low, in some cases propolis volatiles can be divided according to their continent of origin (Table 6.1); African (Algeria, Canary Islands and Ethiopia), American (Argentina, Cuba, Brazil, Mexico, Uruguay, Venezuela), Asian (China, India, Mongolia, Turkey), and European (Albania, Bulgaria, Croatia, England, Estonia, France, Greece, Hungary, Italy, Poland, Portugal, Wales). This is just one possible classification of propolis volatiles and it includes very different geographical areas in each group, as well as corresponding variability in bee type and local flora. Nevertheless, it offers a first approach to the characterization of propolis volatile compounds.

3.1 Africa

Though the Canary Islands are a Spanish archipelago, they are located off the coast of north western Africa. For this reason, herein, they are considered together with Algeria and Ethiopia, located on the opposite side of the African continent in the Horn of Africa.

Six propolis volatile samples were studied in total, Table 6.1. Calamene and cis-verbenol were the only compounds that occurred in amounts ≥3%, found analogously in two studies. Differences in volatiles were also reported for propolis of Ethiopian origin, as they were produced by different Apis species, Table 6.1.

3.2 America

Ranging from not detected in some samples to variable amounts in others, sesquiterpenes δ-cadinene, β-caryophyllene and spathulenol, and monoterpenes α-pinene, β-pinene, and limonene characterized North American (Cuba and Mexico) and South American (Argentina, Brazil, Uruguay and Venezuela) propolis volatile samples (43 in total), Table 6.1. As there have been more studies performed on green propolis, the most commercialized form of Brazilian propolis, sesquiterpenes are more frequently reported and recognized as propolis components due to the fact that these are main constituents of this form of propolis. However, high levels of other components, such as α-pinene, are also characteristic of some Mexican and Uruguayan propolis volatile samples, Table 6.1.

Although not providing quantitative data, Agüero et al. (2011) showed 17 common constituents between the dichloromethane extracts of Larrea nitida (Zygophyllaceae) and the corresponding propolis. o-Cymene and limonene were the most abundant monoterpenes present in L. nitida, both of which were detected in the propolis volatiles.

According to Maróstica Junior et al. (2008), α-pinene, 1-phenyl-ethanone, linalool, β-caryophyllene, δ-cadinene, nerolidol, spathulenol and globulol were the components present in concentrations >1% in both B. dracunculifolia and propolis volatiles, indicating the similarities in their volatile profiles.

3.3 Asia

Overall, 32 Asian propolis volatile samples were evaluated, from China, India, Mongolia and Turkey (Table 6.1). Phenyl ethyl alcohol, benzyl alcohol and 3-methyl-3-buten-1-ol were the compounds most frequently reported in these propolis volatile samples. 1,8-Cineole (eucalyptol), 2-methyl-2-buten-1-ol, acetic acid, benzaldehyde and toluene were also frequently found in these samples, Table 6.1.

3.4 Europe

As recently reported by Bankova et al. (2014), the oxygen-containing sesquiterpene β-eudesmol was the most common constituent of European propolis volatiles, Table 6.1. Other common sesquiterpenes included guaiol, viridiflorol, ledol, γ-eudesmol, γ-cadinene, α-bisabolol, T-cadinol, δ-cadinene and α-cadinol. As for monoterpenes, thymol and α-pinene were the most frequently reported volatile constituents. As previously mentioned, high thymol levels and frequency of occurrence may be a consequence of the long-term use of a thymol-rich acaricide by beekeepers, as reported by Miguel et al. (2013). Labdane diterpenes were abundant in volatiles from some propolis samples collected in Portugal, mainly in C. ladanifer-rich regions (Miguel et al. 2013; Falcão et al. 2016). Hydrocarbons such as heptadecane, nonadecane, heneicosane, tricosane and pentacosane were also commonly found in these types of propolis, depending on the extraction procedure. Aromatic compounds, such as benzyl benzoate and benzyl alcohol, which have frequently been identified in several European propolis volatiles, were not detected or were present in trace amounts in P. canadensis leaf-buds and C. ladanifer branch essential oils (Falcão et al. 2016).

4 Conclusion

Much remains to be known and understood about the volatile fraction of propolis. Additional comparative studies on the volatiles of local natural flora and corresponding propolis are required in order to better understand the relation between the two. Further research in this area will reveal to what extent flora volatile components can be used as propolis markers and in the characterization of this unique bee product.