Chemical variability in the essential oil of leaves of Araçá (Psidium guineense Sw.), with occurrence in the Amazon

Background Psidium guineense, known as Araçá, is a Brazilian botanical resource with commercial application perspectives, based on the functional elements of its fruits and due to the use of its leaves as an anti-inflammatory and antibacterial agent. The essential oils of leaves of twelve specimens of Araçá were analyzed by GC and GC-MS to identify their volatile constituents and associate them with the biological activities reputed to the plant. Results In a total of 157 identified compounds, limonene, α-pinene, β-caryophyllene, epi-β-bisabolol, caryophyllene oxide, β-bisabolene, α-copaene, myrcene, muurola-4,10(14)-dien-1-β-ol, β-bisabolol, and ar-curcumene were the primary components in descending order up to 5%. Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA) displayed three different groups with the following chemical types: limonene/α-pinene, β-bisabolene/epi-β-bisabolol, and β-caryophyllene/caryophyllene oxide. With the previous description of another chemical type rich in spathulenol, it is now understood that at least four different chemotypes for P. guineense should occur. Conclusions In addition to the use of the Araçá fruits, which are rich in minerals and functional elements, it should be borne in mind that the knowledge of the chemical composition of the essential oils of leaves of their different chemical types may contribute to the selection of varieties with more significant biological activity.


Background
Myrtaceae comprises 132 genera and 5671 species of trees and shrubs, which are distributed mainly in tropical and subtropical regions of the world, particularly South America, Australia and Tropical Asia [1]. It is one of the most prominent families in Brazil, represented by 23 genera and 1034 species, with occurrence in all regions of the country [2,3]. Psidium is a genus with at least 60 to 100 species, occurring from Mexico and Caribbean to Argentina and Uruguay. Therefore, it is naturally an American genus, although P. guajava, P. guineense and P. cattleyanum are subtropical and tropical species in many other parts of the world [4].
The present work aimed at investigating the variability of the chemical composition of the essential oils of different specimens of Psidium guineense, occurring in the Amazon region, to contribute to the knowledge of its chemical types.

Plant material
The leaf samples of twelve Psidium guineense specimens were collected in Pará state, Brazil. Collection site and voucher number of each specimen are listed in Table 1. The plant vouchers after the identification were deposited in the Herbaria of Embrapa Amazônia Oriental, in Belém (IAN) and Santarém (HSTM), Pará state, Brazil. The leaves were dried for two days in the natural environment and, then, subjected to essential oil distillation.

Isolation and analysis of the composition of oils
The leaves were ground and submitted to hydrodistillation using a Clevenger-type apparatus (3 h). The oils were dried over anhydrous sodium sulfate, and their yields were calculated by the plant dry weight. The moisture content of the samples was calculated using an Infrared Moisture Balance for water loss measurement. The procedure was performed in duplicate.
The oils were analyzed on a GCMS-QP2010 Ultra system (Shimadzu Corporation, Tokyo, Japan), equipped with an AOC-20i auto-injector and the GCMS-Solution software containing the NIST (Nist, 2011) and FFNSC 2 (Mondello, 2011) libraries [17,18]. A Rxi-5ms (30 m x 0.25 mm; 0.25 μm film thickness) silica capillary column (Restek Corporation, Bellefonte, PA, USA) was used. The conditions of analysis were: injector temperature of 250 °C; Oven temperature programming of 60-240 °C (3 °C/min); Helium as carrier gas, adjusted to a linear velocity of 36.5 cm/s (1.0 mL/min); split mode injection for 1 μL of sample (oil 5 μL : hexane 500 μL); split ratio 1:20; ionization by electronic impact at 70 eV; ionization source and transfer line temperatures of 200 and 250 °C, respectively. The mass spectra were obtained by automatic scanning every 0.3 s, with mass fragments in the range of 35-400 m/z. The retention index was calculated for all volatile components using a homologous series of C8-C20 n-alkanes (Sigma-Aldrich, USA), according to the linear equation of Van den Dool and Kratz (1963) [19]. The quantitative data regarding the volatile constituents were obtained by peak-area normalization using a GC 6890 Plus Series, coupled to FID Detector, operated under similar conditions of the GC-MS system. The components of oils were identified by comparing their retention indices and mass spectra (molecular mass and fragmentation pattern) with data stored in the GCMS-Solution system libraries, including the Adams library (2007) [20].

Statistical analysis
The multivariate analysis was performed using as variables the constituents with content above than 5%. For the multivariate analysis, the data matrix was standardized by subtracting the mean and then dividing it by the standard deviation. For hierarchical cluster analysis, the complete linkage method and the Euclidean distance were used. Minitab software (free 390 version, Minitab Inc., State College, PA, USA), was used for these analyzes.
Comparing these results with the composition of other essential oils described for the same plant, a specimen of P. guineense sampled in Arizona, USA, has also been found to contain β-bisabolene, α-pinene, and limonene as its primary constituents [14]. In addition, the oil from another specimen collected in Roraima, Brazil, presented β-bisabolol as the main component, followed by limonene and epi-α-bisabolol [15]. On the other hand, a specimen sampled in Mato Grosso do Sul, Brazil, presented an essential oil with a very high value of spathulenol [16]. Therefore, it is possible that there is a significant variation in the essential oils of different types of Araçá.

Variability in oils composition
The multivariate analysis of PCA (Principal Component Analysis) (Fig. 2) and HCA (Hierarchical Cluster Analysis) (Fig. 3) were applied to the primary constituents present in oils (content ≥ 5.0%), for the evaluation of chemical variability among the P. guineense specimens.
The HCA analysis performed with complete binding and Euclidean distance showed the formation of three different groups. These were confirmed by the PCA analysis, which accounted for 79.5% of the data variance. The three groups were classified as: Group I Characterized by the presence of the monoterpenes α-pinene (13.4-35.6%) and limonene (3,7-37,2%), Mondello [18] composed by the specimens PG-01 to PG-06, collected in Curuçá (PG -01 to PG-05) and Santarém (PG-06), Pará state, Brazil, with 49.2% similarity between the samples.
Thus, based on the study of these essential oils, the multivariate analysis (PCA and HCA) has suggested the existence of three chemical types among the twelve specimens of P. guineense collected in different locations of the Brazilian Amazon. It would then be the chemical types α-pinene/limonene (Group I), β-bisabolene/epiβ-bisabolol (Group II) and β-caryophyllene/caryophyllene oxide (Group III). Taking into account that two essential oils with a predominance of α-pinene/ limonene and β-bisabolene/epi-β-bisabolol, respectively, were previously described [14,15], it is understood that adding these two chemical types to that one rich in β-caryophyllene + caryophyllene oxide, which was a product of this study, besides the other chemical type with a high value of spathulenol, before reported by Nascimento and colleagues (2018) [16], will be now, at least, four chemical types known for the P. guineense essential oils.
Several studies have demonstrated the antiinflammatory activities of limonene, α-pinene and β-caryophyllene, the primary constituents found in the oils of P. guineense presented in this paper. Limonene showed significant anti-inflammatory effects both in vivo and in vitro, suggesting a beneficial role as a diet supplement in reducing inflammation [21]; limonene decreased the infiltration of peritoneal exudate leukocytes and reduced the number of polymorphonuclear leukocytes, in the induced peritonitis [22]. α-Pinene presented antiinflammatory effects in human chondrocytes, exhibiting potential anti-osteoarthritic activity [23], and in mouse peritoneal macrophages induced by lipopolysaccharides [24], being, therefore, a potential source for the pharmaceutical industry. The anti-arthritic and the in vivo antiinflammatory activities of β-caryophyllene was evaluated by molecular imaging [25].

Conclusion
In addition to the great use of the fruits of P. guineense, which are rich in minerals and functional elements, it is understood that the knowledge of the chemical composition of the essential oils of leaves of their different chemical types may contribute to the selection of varieties with more significant biological activity. The study intended to address this gap.