Variability in the Chemical Composition of Myrcia sylvatica (G. Mey) DC. Essential Oils Growing in the Brazilian Amazon

Myrcia sylvatica (G. Mey) DC. is known as “insulin plant” because local communities use the infusions of various organs empirically to treat diabetes. The leaves of seven specimens of Myrcia sylvatica (Msy-01 to Msy-07) were collected in the Brazilian Amazon. Furthermore, the essential oils were extracted by hydrodistillation and analyzed by gas chromatography coupled to mass spectrometry, and their chemical compositions were submitted to multivariate analysis (Principal Component Analysis and Hierarchical Cluster Analysis). The multivariate analysis displayed the formation of four chemical profiles (chemotypes), described for the first time as follows: chemotype I (specimen Msy-01) was characterized by germacrene B (24.5%), γ-elemene (12.5%), and β-caryophyllene (10.0%); chemotype II (specimens Msy-03, -06 and -07) by spathulenol (11.1–16.0%), germacrene B (7.8–20.7%), and γ-elemene (2.9–7.6%); chemotype III (Msy-04 and -05) by spathulenol (9.8–10.1%), β-caryophyllene (2.5–10.1%), and δ-cadinene (4.8-5.6%); and chemotype IV, (Msy-02) by spathulenol (13.4%), caryophyllene oxide (15.0%), and α-cadinol (8.9%). There is a chemical variability in the essential oils of Myrcia sylvatica occurring in the Amazon region.


Introduction
The Myrcia genus has about 800 species distributed from Central to Tropical America [1]. In addition, it is considered one of the most taxonomically and morphologically complex homogeneous genera of the Myrtaceae family [2], including in the Myrtales order, Rosideas clade, and Malvideas sub-clade [3]. The Amazon rainforest, despite comprising a low diversity of Myrcia spp., was an important region in the biogeographic history of this genus because evidence indicates that it participated in the diversification of ancestral lineages [4].
Myrcia species have great ecological relevance, as their fruits are a food source for ants, birds, and mammals, and their flowers are attractive to pollinators, such as bees. These ecological relationships are responsible for promoting the conservation of the diversity of this genus [5]. In addition, Myrcia species have economic, nutritional [6], and medicinal importance [7].
Myrcia species can be recognized in the field by the sweet aroma emanating from the leaves, flowers, and fruits, in addition to generally appearing as shrubs with leaves elliptical-co-lanceolate; apex long-acuminate to caudate; inflorescences in panicles; and flowers with deltoid sepals and petals white-rarely yellow-connective with glands of blackish color and stigma hairy at the base [8].
Myrcia sylvatica (G. Mey) DC. is also known as "kumate-folha-miúda" or "murtinha". It is native and non-endemic to Brazil, widely distributed in South America, where it is found from Guyana to Brazil [10]. However, in Brazil, its occurrence is restricted to the phytogeographic domains of the Amazon, Caatinga, and Cerrado [11].
The M. sylvatica essential oil have shown great chemical variability due to intraspecific or seasonal variations [9,12], in addition to antioxidant, anesthetic potential [13] and bactericidal properties [14].
Therefore, in view of the biological potential presented by Myrcia sylvatica, the objective of this work was to investigate the chemical variability of the essential oils of leaves that occur in the Amazon of Pará.

Yield and Chemical Composition of the Essential Oils
The seven Myrcia sylvatica wild specimens evaluated in this work showed chemical variability of their essential oils. The oil yield ranged from 0.3 to 0.9%, as shown in Table 1. The quantification and identification of 112 constituents in the analyzed oils represent an average of 81.1% of the total oil content. Oil yield (%) * 0.7 0.9 0.6 0.  The seasonal and circadian study of essential oil from leaves and fruits of M. sylvatica collected in the municipality of Santarém, state of Pará, indicated that the yield varied from 0.9 to 1.7% [12], values higher than this work. In contrast, the yield of leaf essential oil from this species collected in Carolina, state of Maranhão, was 0.5% [17], the same  The seasonal and circadian study of essential oil from leaves and fruits of M. sylvatica collected in the municipality of Santarém, state of Pará, indicated that the yield varied from 0.9 to 1.7% [12], values higher than this work. In contrast, the yield of leaf essential oil from this species collected in Carolina, state of Maranhão, was 0.5% [17], the same content presented by the specimen Msy-07.
In the Myrtaceae species essential oils, the predominance of hydrocarbon and oxygenated sesquiterpenes has been evidenced, some of them with biological properties [18,19]. The presence of the sesquiterpene hydrocarbon β-caryophyllene (45.0%) as the major constituent was identified in a M. sylvatica sample collected in Maranhão [17]. Other compounds were also reported as the main compound in oils from Tocantins, among them the oxygenated sesquiterpenes spathulenol (13.8-40.2%) and caryophyllene oxide (5.0-16.6%) [10]. Germacrene B (6.7%) and γ-elemene (10.5%) were identified as the highest content in M. splendens [20].

Chemical Variability in the Specimens
The Hierarchical Cluster Analysis (HCA, Figure 2) and the Principal Components Analysis (PCA, Figure 3), carried out with the compounds in the highest abundance (> 4.0%) in the essential oils of M. sylvatica, displayed the formation of four groups (chemotypes).
Essential oils from Myrtaceae species have shown chemical variability, which may be influenced by seasonality, collection site, extraction method, genetics, and plant part [18,22,23]. This variability affects their biological properties and applications; for example, the existence of four Eugenia uniflora chemotypes was reported, and the samples presented different biological potentials related to their chemical profiles [24].
Therefore, among the collected samples, all chemical profiles were described for the first time: Profile I (germacrene B, γ-elemene, and β-caryophyllene), Profile II (spathulenol, germacrene B, and γ-elemene), Profile III (spathulenol, β-caryophyllene, and δ-cadinene), and Profile IV (spathulenol, caryophyllene oxide, and α-cadinol). Thus, added to the eight chemotypes described in the literature, it is possible that there are at least twelve Myrcia sylvatica chemotypes. The occurrence of different chemical profiles can be attributed to the genetic variability of this species [9].

Plant Material
The leaves of the seven Myrcia sylvatica wild-growing specimens were collected on Caratateua Island, Belém, Pará state, Brazil, during the rainy season. The collection site, herbarium voucher number, and geographic coordinates are listed in Table 2. After identification, the plant specimens were deposited in the Herbarium of Museu Paraense Emílio Goeldi (MG) in the city of Belém, Brazil. The leaves were dried for three days at room temperature, ground, and then submitted to essential oil hydrodistillation in duplicate using a Clevenger-type apparatus. The oils obtained were dried over anhydrous sodium sulfate, and total oil yields were expressed as mL/100 g of the dried material [25,26]. The specimens were collected in agreement with the Brazilian laws concerning the protection of biodiversity (SISGEN A78F864).

Analysis of Essential Oil Composition
The oil composition analysis was performed by GC-MS, using a Shimadzu instrument Model QP-2010 ultra (Shimadzu, Tokyo, Japan) equipped with a Rtx-5MS (30 m × 0.25 mm; 0.25 µm film thickness) fused silica capillary column (Restek, Bellefonte, PA, USA). Helium was used as carrier gas, adjusted to 1.0 mL/min at 57.5 KPa; split injection (split ratio 1:20) of 1 µL of n-hexane solution (oil 5 µL: n-hexane 500 µL); injector and interface temperature were 250 • C; oven programmed temperature was 60 to 240 • C (3 • C/min), followed by an isotherm of 10 min. EIMS (electron impact mass spectrometry): electron energy, 70 eV; ion source temperature was 200 • C. The mass spectra were obtained by automatically scanning every 0.3 s, with mass fragments in the range of 35-400 m/z. The compounds present in the samples were identified by comparison of their mass spectrum and retention index, calculated for all volatile components using a linear equation by Van den Dool and Kratz [27], with the data present in the commercial libraries FFNSC-2 [16] and Adams [15]. The retention index was calculated using n-alkane standard solutions (C8-C40, Sigma-Aldrich, St. Louis, MO, USA) under the same chromatographic conditions. The GC-FID analysis was carried out on a Shimadzu QP-2010 instrument, equipped with an FID detector, in the same conditions, except that hydrogen was used as the carrier gas. The percentage composition of the oil samples was computed from the GC-FID peak areas. The analyses were carried out in triplicate.

Multivariate Statistical Analyses
The data matrix was standardized for the multivariate analysis by subtracting the mean and then dividing it by the standard deviation. 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. The principal component analysis (PCA) was applied to verify the interrelation among the oils' components (>4%) (OriginPro trial version, OriginLab Corporation, Northampton, MA, USA).

Conclusions
The intraspecific chemical variability among the Myrcia sylvatica specimens studied was evidenced by the occurrence of four chemotypes, described here for the first time, with a predominance of the sesquiterpenes class in all samples. In addition to the chemotypes already described in the literature (8 chemotypes), it is possible that at least 10 Myrcia sylvatica chemotypes occur. Considering the potential of M. sylvatica, the knowledge of this variability can contribute to chemotaxonomy, economical use, and future studies that evaluate the biological properties of this species.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.