Alpha-pinene, the most abundant volatile found in all propolis, was also the most abundant volatile found in propolis produced in the Adriatic Sea coast of Italy, and its likely botanical origin was suggested to be native conifer trees from that region 17. α-Pinene together with β-pinene were previously found as the two most abundant volatiles in propolis produced in the Rio Grande do Sul 18 and Paraná 3 states, in a not specified Brazilian propolis sample 19, and propolis from South Africa 20 and Uruguay 1. It is noteworthy that South Africa and Uruguay are in similar latitudes to southern Brazil, indicating a characteristic profile related to that propolis location.
Furfural, which was found in almost all samples, is a product of sugars dehydration commonly found in agricultural byproducts and was identified by SHS-GCxGC-TOF-MS in South African propolis, in which volatiles was extracted by heating at 45°C/5 min, at concentrations ranging from trace to 11.3% 20.
β-Eudesmol was found as the most abundant volatile in propolis produced in France, Hungary, Bulgaria, and Northern Italy and was also the most abundant volatile in the distilled essential oil of Populus nigra buds, which likely is its primary botanical origin 1. However, in our study, β-eudesmol was also found in the resins of AA as a minor volatile (from 0.7–2.4%). Additionally, AA resins were the only ones containing sabinene, α-thujene and α-bisabolol. Thereby, AA may be plant sources of these volatiles for brown propolis from southern Brazil. α-Bisabolol was also found as a major volatile in propolis produced in temperate zones of China and Turkish 1.
It is noteworthy that the temperature used to extract the volatiles, 180°C, was higher than those commonly used for volatile profile characterization of 50–75°C 21 3 19. The degradation rate of pure monoterpenes at 120°C varied greatly, depending on the compound, as it was 100% after 4 h for α-terpinene, 50% after 24 h for limonene, and 38% after 72 h for camphene 22. The thermal degradation led to p-cymene, eucarvone and 1,2-epoxyde derivatives from limonene; thymol, ketoaldehydes, and eucalyptol from α-terpinene; and camphenilone, verbenone, and aromatic compounds from camphene 22.
Although verbenone was found in samples of our study (up to 1.3%), it was also tentatively identified in brown propolis extracted at 75°C/30 min 3, while p-cymene and verbenone were tentatively identified in Mediterranean propolis extracted at 60°C/45 min 21.
Furthermore, McGraw et al. 22 quoted Punsuvon, who reported the degradation at 90–130°C (not specifying the time length) of pure α-pinene (23–37%), forming β-pinene, α-pinene oxide, α-campholenal, verbenol, pinocamphone, myrtenol and verbenone, and of pure β-pinene (22%), forming mainly myrtenol. From those thermal degradation products of α-pinene, some are reportedly relevant in propolis and conifer tissues, such as β-pinene, α-campholenal, myrtenol and verbenol 1 19 23. Hence, the terpene diversity in natural products seems to result from naturally occurring chemical reactions catalyzed by microorganisms or enzyme systems 24. At the same time, induced heating is a non-natural way to get it, and it is not simple to differentiate whether the terpene diversity is natural or induced by extraction conditions.
The increase in temperature increased peak intensities up to 180°C, and the number of peaks also increased, which likely indicates volatiles release from the propolis's complex resinous/waxy matrix (Figure S1 at the Supplementary information file). However, the formation of low percentages of the tentatively identified carvone oxide (Table 2), which likely had the added internal control L-carvone as a precursor, is an indication of oxidation. Nevertheless, L-carvone was pierced outside the samples within the vials. Thereby, it was more exposed to O2 and more prone to oxidation than the other volatiles present in the propolis/resins matrices.
Concerning the multivariate analysis, the PCA showed that the SHS-GCMS method was sensible to discriminate propolis samples produced in different municipalities, even when the distance between the apiaries was 72 km (from ‘Beira do mato’ to ‘Vila Zulmira sede’). Moreover, the PCA indicates that A. angustifolia may be more attractive than Pinus species for bee foragers as a plant resin source to produce propolis.
It is noteworthy that the possibility of Araucaria sp. resins be used as a botanical source for bees to produce brown propolis in southern Brazil was previously suggested by 5, based on the identification of a single non-volatile compound, which is typical in some Araucaria species, in propolis samples from Parana state. AA is a dominant species in subtropical and temperate rainforests in southern Brazil and adjacent areas. These areas were intensively explored over the nineteenth century. Nowadays, it is legally protected in permanent preservation areas since AA is endangered 25. Therefore, the likely presence of AA resins in OP1 reinforces the need for sustainable preservation of natural environments since it may be related to OP1’s outstanding antioxidant activity 4.
From the tentatively identified volatiles in the hierarchical clustering heatmap, α-campholenal, α-phellandrene, β-bourbonene and trans-verbenol were found in essential oils of Pinus species 23. This finding may indicate PT as another plant resin source for propolis production in those areas. To our knowledge, p-mentha-1,8-dien-7-ol was tentatively identified in plants from the Araucariceae family for the first time. p-Mentha-1,8-dien-7-ol, also known as perilla alcohol, is found in many plants' essential oils, such as lavendin, peppermint, spearmint, and cherries 26. Therefore, further studies should be conducted with authentic standards to confirm the identified volatiles in brown propolis and conifer resins from southern Brazil to be further used as phytochemical markers.
In conclusion, there are indications that the resin from native Araucaria angustifolia is more attractive for bees to produce propolis in southern Brazil, although there is also an indication that non-native Pinus elliott and Pinus taeda are plant resin sources as well. However, the singularities on the chromatograms of propolis from each apiary/municipality illustrated in the heatmaps and the not complete overlap of the propolis and the conifer resins in the PCA may indicate that there are other botanical sources for bees to produce propolis within the permanent preservation areas of southern Brazil, which remain unknown.