Characterization of essential oils from odoriferous resins of Amazonian species of Protium Burm.

The odoriferous resins produced by the trees of Protium spp (Burseraceae) are known in the Amazonian region as “breus” where they have been used to caulk canoes, as a mosquito repellent, and also have potential use in perfumery. Most species in this region have had the essential oils of their resins characterized, thus, the objective of this study was to evaluate for the first time the volatile constituents from the resin of Protium gallosum Daly, P. paniculatum Engl., P. pillosum (Cuatrec.) Daly and P. paniculatum var. riedelianum (Engl.) Daly. The samples from the individuals that had been previously identified were collected at the Adolpho Ducke Forest Reserve and subjected to hydrodistillation in a Clevenger apparatus, and the essential oils obtained were analyzed using GC-FID and GC-MS. The essential oil of P. gallosum showed high percentages of oxygenated monoterpenes (43.61%) with a predominance of terpin-4-ol (25.15%), and P. paniculatum consisted exclusively of monoterpenes that were predominantly p -cymene (43.52%; hydrocarbon monoterpene) and the 1,8-cineole (16.83%; oxygenated monoterpene). Most of the monoterpenes identified from P. pillosum and P. paniculatum var. riedelianum were hydrocarbons with a predominance of α -pinene, with 37.74% and 50.93%, respectively. 1,8-Cineole (21.89%) was also identified in the essential oil of P. pillosum . The results obtained this study add chemical knowledge to Burseraceae resins from a biological reserve of the Central Amazon. (11.3-90%). Essential oils that are rich with high levels of α -pinene (42.9%) and p -cymene


Introduction
The odoriferous resins produced by the trees of Protium spp (Burseraceae) are known in the Amazonian region as "breus" and have been used to caulk canoes, as a mosquito repellent, and also have potential use in perfumery. Most species in this region have had the essential oils of their resins characterized, and many studies have been done with resins collected in the Adolpho Ducke Forest Reserve located to northeast of the city of Manaus, AM, Brazil (latitude 02º 55′ S, longitude 59º 59′ W) since this forest fragment has had its flora identified and mapped, which favors the studies of their species (Ribeiro et al., 1999). The volatile compositions of the resins are rich in monoterpenes as P. strumosum, which consists of high concentrations of limonene (75.5%) and p-cymene (31.5%); P. altsonii showed trans-dihydro-α-terpineol (25.8%) (Zoghbi et al. 2005); αpinene (17.57%) and limonene (46.11%) predominated in P. aracouchini (Lima et al., 2021). High levels of p-cymene (35.8 and 38.9%) and limonene (90.93%) were also found in the resin of P. hebetatum whose production is stimulated by movement of insects on its surface  and P. spruceanum (Lima et al. 2014), respectively. In the essential oils of the resin from P. hebetatum, produced via chemical induction, α-pinene (28.9-23.7%), o-cymene (18.03-31.16%) and limonene (31.89-14.95%) were identified, with limonene being predominant .
Studies conducted by Ramos et al. (2000), with six species of Protium, showed that the essential oils of the resin had variable percentages of p-cymene (11.3-90%). Essential oils that are rich with high levels of α-pinene (42.9%) and p-cymene (33.3%) have shown potential against Leishmania (Santana et al., 2020). The resin from Protium with sesquiterpene as the predominant constituent are rare and Protium decandrum has been found to be rich in trans-α-bergamotene (47.7%) . However, there is a lack of chemical studies on the resin from Protium gallosum Daly, P. paniculatum Engl., P. pillosum (Cuatrec.) Daly and P. paniculatum var. riedelianum (Engl.) Daly. Thus, this is the first study related to evaluation of the volatile constituents in the resin of these four species.

Methodology
The samples of the resins were collected from specimens of Protium gallosum. P. paniculatum. P. pillosum and P. paniculatum var. riedelianum located in the Adolpho Ducke Forest Reserve. The essential oils were obtained using hydrodistillation extraction in a Clevenger apparatus over the course of 4 hours.
The sample oils were analyzed in a GC-MS (QP5000, Shimadzu), operating using electron impact (70 eV

Results and Discussion
The essential oils obtained via hydrodistillation provided the following high yields: 6.4% from P. gallosum and 6.1% from P. pillosum, and the yields of essential oil from P. paniculatum and P. paniculatum var. riedelianum were 1.6 and 1.2% respectively. The chemical compositions of the essential oils are compiled in Table 1, and the volatile compounds are mainly monoterpenes and sesquiterpenes, but the levels of sesquiterpenes of the samples of essential oils are low (<25%) as shown in Table 2. The essential oil of P. gallosum showed high percentages of oxygenated monoterpenes (43.61%) with a predominance of terpin-4-ol (25.15%), and P. paniculatum consists exclusively of monoterpenes, with the hydrocarbon, p-cymene (43.52%; hydrocarbon monoterpenes) and 1,8-cineole (16.83%; oxygenated monoterpenes) predominating. The majority of the monoterpenes identified in P. pillosum and P. paniculatum were hydrocarbons with predominance of α-pinene, with 37.74% and 50.93%, respectively. 1,8-Cineole (21.89%) was also identified in the essential oil of P. pillosum. Figure 1 illustrates the molecules of the predominant constituents of essential oils.
The monoterpene hydrocarbons α-pinene (13.7-61.8%) and p-cymene (11.3-90.0%) have previously been found at different levels in resin samples of Protium from the Amazonian region (Ramos et al., 2000;Lima et al., 2016), with the presence of p-cymene as a main constituent, which is associated with aged resin. Chemical studies of the resin of P. pilosum has not been previously performed, but the monoterpenes α-pinene (31.7%) and p-cymene (31.2%) were found in aerial parts of this species (Zoghbi et al., 2005). The bicyclic monoterpene α-pinene plays a crucial role in the fragrance and flavor industry since it has a fresh pine scent and a woody flavor (Vespermann et al., 2017) and have been reported by wide range of pharmacological activities (Salehi, et al., 2019, Joshi et al., 2020Allenspach & Steuer, 2021). The scent of p-cymene has been described as woody and spicy (Lasekan et al., 2013). The oxygenated monoterpene terpin-4-ol was identified with high percentages in aerial parts of the Amazonian species P. decandrum  and 1,8-cineole from the resin of P. heptaphyllum from northeastern Brazil (Mobin et al., 2016;Rocha et al., 2022). Studies of the essential oils from P. paniculatum var. riedelianum have been performed using the leaves, which showed themselves to be rich in β-caryophyllene (30.59%) and caryophyllene oxide (20.63%) (Lima et al., 2022).    (2) In brackets, the number of compounds found. Source: Authors.

Conclusion
In the analysis of the chemical profile of the volatile constituents from the resins collected from Protium specimens in the Adolpho Ducke Forest Reserve, high percentages of monoterpene hydrocarbons such as α-pinene from the species P.
pillosum and P. paniculatum var. riedelianum, and p-cymene from the species P. paniculatum were detected. The high levels of the oxygenated monoterpenes 1,8-cineole from P. paniculatum and P. pillosum and terpin-4-ol from P. gallosum were found. As such, the results obtained through this study add to the chemical knowledge of resins from Burseraceae that is found in a biological reserve in the Central Amazon.
The chemical composition and variation of the predominant monoterpenes in Protium resin is evidently related to metabolism of species and/or exposure time da resin on tree trunks. The high levels of α-pinene in the essential oils of P.
pillosum and P. paniculatum var.riedelianum can be indicative of fresh resins because the odor of this monoterpene can be detected in the field work by the fresh, camphor and woody aroma.