Seasonal Variability of a Caryophyllane Chemotype Essential Oil of Eugenia patrisii Vahl Occurring in the Brazilian Amazon

Eugenia patrisii Vahl is a native and non-endemic myrtaceous species of the Brazilian Amazon. Due to few botanical and phytochemical reports of this species, the objective of the present work was to evaluate the seasonal variability of their leaf essential oils, performed by GC and GC-MS and chemometric analysis. The results indicated that the variation in oil yields (0.7 ± 0.1%) could be correlated with climatic conditions and rainy (R) and dry seasons (D). (E)-caryophyllene (R = 17.1% ± 16.0, D = 20.2% ± 17.7) and caryophyllene oxide (R = 30.1% ± 18.4, D = 14.1% ± 19.3) are the major constituents and did not display significant differences between the two seasons. However, statistically, a potential correlation between the main constituents of E. patrisii essential oil and the climatic parameters is possible. It was observed that the higher temperature and insolation rates and the lower humidity rate, which are characteristics of the dry season, lead to an increase in the (E)-caryophyllene contents, while lower temperature and insolation and higher humidity, which occur in the rainy season, lead to an increase in the caryophyllene oxide content. The knowledge of variations in the E. patrisii essential oil composition could help choose the best plant chemical profile for medicinal purposes.


Introduction
The Myrtaceae comprises about 140 genera and 7000 species occurring mainly in the southern hemisphere, standing out in diverse habitats such as Australia, Southeast Asia, and tropical and subtropical Americas [1,2]. Eugenia genus comprises about 1000 species, one of the most representative of Myrtaceae, with occurrence in Central and South America and the African continent [3]. In addition to its high taxa, this genus has excellent economic and pharmacological potential [4]. In the seasonal study, the oil yield of E. patrisii ranged from 0.5% (Jun) to 1.0% (August), with an average of 0.7 ± 0.1% during the period (see Table 1). Statistically (Tukey test), there was no significant difference in the oil yields in the dry (0.7 ± 0.2%) and rainy (0.6 ± 0.1%) seasons. Additionally, a significant correlation (p > 0.05) was not observed between the essential oil content and the climatic parameters.
However, analyzing the Pearson correlation coefficient (r), the precipitation (r = −0.24), and insolation (r = 0.24) showed a weak negative and positive relationship with the essential oil yield, respectively. Moreover, temperature (r = 0.33) showed a weak positive value, while relative humidity (r = −0.42) displayed a weak negative relationship with the essential oil yield. Thus, the results indicate that the variation in oil yields of E. patrisii can be correlated with the climatic conditions and annual seasonality. It was impossible to distinguish the oil yields concerning the collection periods in the two seasons.
(E)-Caryophyllene (0-48.3%) and caryophyllene oxide (0-49.0%), caryophyllane-type sesquiterpenes, were the principal constituents of the E. patrisii oil during the seasonal period, followed by bicyclogermacrene (0-17.0%), spathulenol (0-11.4%), β-elemene (0.9-11.3%), and 9-epi-(E)-caryophyllene (0-7.3%). (E)-Caryophyllene was absent in February and March and showed a higher content in November (48.3%). Caryophyllene oxide was absent from August to November and showed a higher content in March (49.0%). Bicyclogermacrene, a sesquiterpene hydrocarbon with a germacrane skeleton, was absent from January to March and June to July, presenting its higher content in October (17.0%). Spathulenol, an aromadendrane-type oxygenated sesquiterpene, was absent in October and exhibited a higher content in March (11.4%). β-Elemene, a germacrane-type sesquiterpene, was present in all oils, with the higher content in January (11.3%). 9-epi-(E)-Caryophyllene, another sesquiterpene of caryophyllane-type, was absent in June and displayed a higher content in September (7.3%). However, analyzing the Pearson correlation coefficient (r), the precipitation (r = −0.24), and insolation (r = 0.24) showed a weak negative and positive relationship with the essential oil yield, respectively. Moreover, temperature (r = 0.33) showed a weak positive value, while relative humidity (r = −0.42) displayed a weak negative relationship with the essential oil yield. Thus, the results indicate that the variation in oil yields of E. patrisii can be correlated with the climatic conditions and annual seasonality. It was impossible to distinguish the oil yields concerning the collection periods in the two seasons.

Oil Composition vs. Environmental Condition
The climate variables were correlated by the Pearson correlation coefficient (r) with the primary constituents of E. patrisii essential oil to verify their environmental conditions relationships ( Table 2).
These findings may be explained by the biosynthetic pathway of these constituents ( Figure 4). Indeed, (E)-caryophyllene, 9-epi-(E)-caryophyllene, and caryophyllene oxide belong to the same pathway through the caryophyllyl cation. Caryophyllene oxide is a metabolic product of the (E)-caryophyllene oxidation, meaning that the increase in the caryophyllene oxide content implies decreasing (E)-caryophyllene content in the E. patrisii oils samples ( Figure 2). Moreover, the caryophyllyl cation, the precursor of the caryophyllane-type sesquiterpenes, is different from the germacryl cation, which is the precursor of bicyclogermacrene and spathulenol.  (E)-Caryophyllene and caryophyllene oxide have a robust wooden odor, used as cosmetic and food additives. These two natural compounds are approved as flavorings by the Food and Drug Administration (FDA) and European Food Safety Authority (EFSA) and used in a mixture, how they often occur in plants. In medical practice, the application of the mixture of (E)-caryophyllene and caryophyllene oxide in combination with the classical anticancer drugs could bring significant benefits, potentializing the efficacy of the chemotherapeutics, eliciting the supplementary antineoplastic effect, and reducing the refractory cancer pain, at the same time [12]. It has been seen that among (E)-caryophyllene and caryophyllene oxide, this letter has more substantial anticancer properties, explained by its chemical structure. The caryophyllene oxide structure contains the functional groups methylene and exocyclic epoxide. Therefore, it can bind covalently to proteins and DNA bases by the sulfhydryl and amino groups. So, caryophyllene oxide has revealed high potential as a signaling modulator for tumor cancer cells [12,18].
The essential oils of Brazilian Myrtaceae have shown notable cytotoxic activity against the lung, colon, stomach, and melanoma cells, with a real prospect for subsequent phytotherapeutic development [11]. In seasonal studies, some Myrtaceae species have shown correlations of some essential oil constituents with environmental conditions. The sesquiterpene hydrocarbon δ-cadinene, one of the main constituents of the essential oil (E)-Caryophyllene and caryophyllene oxide have a robust wooden odor, used as cosmetic and food additives. These two natural compounds are approved as flavorings by the Food and Drug Administration (FDA) and European Food Safety Authority (EFSA) and used in a mixture, how they often occur in plants. In medical practice, the application of the mixture of (E)-caryophyllene and caryophyllene oxide in combination with the classical anticancer drugs could bring significant benefits, potentializing the efficacy of the chemotherapeutics, eliciting the supplementary antineoplastic effect, and reducing the refractory cancer pain, at the same time [12]. It has been seen that among (E)-caryophyllene and caryophyllene oxide, this letter has more substantial anticancer properties, explained by its chemical structure. The caryophyllene oxide structure contains the functional groups methylene and exocyclic epoxide. Therefore, it can bind covalently to proteins and DNA bases by the sulfhydryl and amino groups. So, caryophyllene oxide has revealed high potential as a signaling modulator for tumor cancer cells [12,18]. The essential oils of Brazilian Myrtaceae have shown notable cytotoxic activity against the lung, colon, stomach, and melanoma cells, with a real prospect for subsequent phytotherapeutic development [11]. In seasonal studies, some Myrtaceae species have shown correlations of some essential oil constituents with environmental conditions. The sesquiterpene hydrocarbon δ-cadinene, one of the main constituents of the essential oil from Myrcia sylvatica (G. Mey) DC., with occurrence in the Amazon, displayed a significant and moderate correlation with the temperature (r = −0.6) and relative humidity (r = 0.7). Additionally, a moderate correlation between these two parameters was observed with oxygenated sesquiterpenoids and monoterpenes hydrocarbons [15]. In addition, the essential oil from Calycolpus goetheanus (Mart. ex DC.) O. Berg., from Marajo Island, Pará state, Brazilian Amazon, showed a strong positive correlation between temperature and monoterpene hydrocarbons (r = 0.75), a significant negative correlation of the sesquiterpene hydrocarbons with solar radiation (r = −0.72), and a positive correlation between the oxygenated sesquiterpenoids and solar radiation (r = 0.75) [19].
These findings suggest a statistical correlation between the oils' main constituents and the climatic parameters in some Myrtaceae species and the variation in the composition of the oils, which may be related to climatic factors. In general, it was observed that the higher temperature and insolation rates and the lower humidity rate, which are characteristics of the dry season, lead to an increase in the content of (E)-caryophyllene, while the lower temperature and insolation rates, and higher humidity rate leads to an increase in the caryophyllene oxide.
Application of a hierarchical clustering analysis (HCA) provided the dendrogram presented in Figure 5, which shows that the compositions of the analyzed oils were included in two different groups and presented a similarity of 0%. The hierarchical clustering analysis (HCA) and principal component analysis (PCA) were plotted with the constituents of oils which displayed values above 5%.
Group I, with 25.3% similarity between the samples, comprises the leaf oils collected in January, February, March, June, and July, whose main constituent was caryophyllene oxide. Group II includes oil samples from April, May, August, September, October, November, and December, which presented (E)-caryophyllene as the main component, showing 46.1% similarity to each other. The HCA plot shows that the two groups were formed differently during the collection period.
tion of the oils, which may be related to climatic factors. In general, it was observed that the higher temperature and insolation rates and the lower humidity rate, which are characteristics of the dry season, lead to an increase in the content of (E)-caryophyllene, while the lower temperature and insolation rates, and higher humidity rate leads to an increase in the caryophyllene oxide.
Application of a hierarchical clustering analysis (HCA) provided the dendrogram presented in Figure 5, which shows that the compositions of the analyzed oils were included in two different groups and presented a similarity of 0%. The hierarchical clustering analysis (HCA) and principal component analysis (PCA) were plotted with the constituents of oils which displayed values above 5%.  PCA and HCA analyses showed no separation between the oil samples of E. patrisii during the dry and rainy periods. However, correlations between the oils' constituents and the environmental parameters were observed. Some studies in Myrtaceae have shown that variation in the chemical composition of essential oils of Eugenia, Syzygium, and Psidium species could be affected by seasonality [10,21,22]. On the other hand, these variations sometimes are not distinguished by PCA and HCA analyses. For example, the seasonal study of Eugenia uniflora showed no separation between the oil samples from the dry and rainy periods in the PCA and HCA analyses [14]. displayed positive correlations with the variables δ-elemene (r = 0.31), α-copaene (r = 0.31), (E)-caryophyllene (r = 0.40), 9-epi-(E)-caryophyllene (r = 0.39) and bicyclogermacrene (r = 0.39). PC2 explained 15.5% and displayed positive correlations with the variables βelemene (r = 0.87), α-copaene (r = 0.30), (E)-caryophyllene (r = 0.06). The third component PC3, explained 8.0% and displayed positive correlations with the variables δ-elemene (r = 0.52), α-copaene (r = 0.63), and spathulenol (r = 0.37). Additionally, caryophyllene oxide showed negative correlation in PC1 (r = −0.42) and PC2 (r = −0.07). PCA and HCA analyses showed no separation between the oil samples of E. patrisii during the dry and rainy periods. However, correlations between the oils' constituents and the environmental parameters were observed. Some studies in Myrtaceae have shown that variation in the chemical composition of essential oils of Eugenia, Syzygium, and Psidium species could be affected by seasonality [10,21,22]. On the other hand, these variations sometimes are not distinguished by PCA and HCA analyses. For example, the seasonal study of Eugenia uniflora showed no separation between the oil samples from the dry and rainy periods in the PCA and HCA analyses [14].

Plant Material and Climatic Data
The leaves of E. patrisii were collected from a specimen existing in Belém city, Pará state, Brazil (coordinates: 1°26'14.20" S/48°26'30.20" W). For the seasonal study, the

Plant Material and Climatic Data
The leaves of E. patrisii were collected from a specimen existing in Belém city, Pará state, Brazil (coordinates: 1 • 26 14.20" S/48 • 26 30.20" W). For the seasonal study, the mature leaves (1 kg) were sampled on day 10 of each month, at 8 am, from August 2020 to July 2021. Plant identification was performed by comparison with an authentic specimen of Eugenia patrisi Vahl (MG227357), and a plant sample was incorporated into the Herbarium "João Murça Pires", at Museu Paraense Emílio Goeldi, Belém city, State of Pará, Brazil. At the same time, the climatic parameters (insolation, relative air humidity, and rainfall precipitation) of the mentioned area were obtained each month from the website of the Instituto Nacional de Meteorologia (INMET, http://www.inmet.gov.br/portal/, accessed on 31 March 2022, of the Brazilian Government [23]. Meteorological data were recorded through the automatic station A-201, located in Belém city, Pará state, Brazil, equipped with a Vaisala system, model MAWS 301 (Vaisala Corporation, Helsinki, Finland).

Extraction and Oil Composition
The air-dried leaves (150 g) were ground and subjected to hydrodistillation in duplicate using a Clevenger-type apparatus (3 h). The oils were dried over anhydrous sodium sulfate, and the dry plant weights were used to calculate their yields. The moisture content of the plant samples was calculated in an infrared moisture balance for water loss measurement. Analysis of oil yield was done in triplicate. The essential oil was dissolved in n-hexane (1500 µg/mL, 3:500, v/v), and analyzed simultaneously in these two systems: gas chromatography-flame ionization detector (GC-FID, Shimadzu Corporation, Tokyo, Japan) and gas chromatography-mass spectrometry (GC/MS, Shimadzu Corporation, Tokyo, Japan). The oil analyses were performed in a GCMS-QP2010 Ultra system (Shimadzu Corporation, Tokyo, Japan), equipped with an AOC-20i auto-injector and the GCMS-Solution software containing the Adams and FFNSC-2 libraries [16,17]. An Rxi-5ms (30 m; 0.25 mm; 0.25 µm film thickness) silica capillary column (Restek Corporation, Bellefonte, PA, USA) was used. The conditions of analysis were as follows: injector temperature: 250 • C; oven temperature programming: 60-240 • C (3 • C/min); helium as the carrier gas, adjusted to a linear velocity of 36.5 cm/s (1.0 mL/min); split mode injection for 1.0 µL of essential oil solution (6 µg of essential oil injected); split ratio 1:20 (300 ng to column for analysis); ionization by electronic impact at 70 eV; ionization source and transfer line temperatures of 200 • C 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 C 8 -C 40 n-alkanes (Sigma-Aldrich, Milwaukee, WI, USA), according to the linear equation of Van Den Dool and Kratz [24]. Individual components were identified by comparing their retention indices and mass spectra (molecular mass and fragmentation pattern) with those in the GCMS-Solution system libraries [16,17]. The quantitative data regarding the volatile constituents were obtained using a GC 2010 Series, operated under similar conditions to the GC-MS system. The relative amounts of individual components were calculated by peak-area normalization using the flame ionization detector (GC-FID). GC-FID and GC-MS analyses were performed in duplicate.

Statistical Analysis
Statistical significance was assessed by a Tukey test (p < 0.05), and the Pearson correlation coefficients (r) were calculated to determine the relationship among the parameters analyzed (insolation, relative air humidity, and rainfall precipitation), using the software GraphPad Prism, version 5.0. The principal component analysis (PCA) was applied to verify the interrelation in the oils' components (>5%) (OriginPro trial version, OriginLab Corporation, Northampton, MA, USA). The hierarchical grouping analysis (HCA), considering the Euclidean distance and complete linkage, was used to verify the similarity of the oil samples based on the distribution of the constituents selected in the previous PCA analysis.

Conclusions
The results showed that climatic variables are correlated with the yield and chemical constituent concentrations of E. patrisii essential oil. Environmental factors also had a strong influence on the composition of the oil.
The ecological significance of the essential oil variations is not yet obvious. It is not clear if the responses are due directly to abiotic factors such as sunlight, temperature, humidity, or rainfall, or to secondary pressures related to seasonality such as herbivory, microbial infections, or phenology. Further research is needed at this point.
The high (E)-caryophyllene and caryophyllene oxide content in this E. patrisii specimen suggests its potential as a renewable source of these biologically active compounds, as an anticancer agent and anesthetic. This study contributes better to the knowledge of E. patrisii essential oil, already used for its medicinal potential. The knowledge of variability in the chemical composition of the E. patrisii essential oil can help choose the appropriate chemical profile for medicinal purposes.