Essential Oils from Fruits with Different Colors and Leaves of Neomitranthes obscura (DC.) N. Silveira: An Endemic Species from Brazilian Atlantic Forest

Neomitranthes obscura (DC.) N. Silveira is an endemic plant of Brazilian Atlantic Forest and widely spread in the sandbanks of “Restinga de Jurubatiba” National Park. It is popularly known by local population as “camboim-de-cachorro” or “cambuí-preto” and recognized by its black ripe fruits. However, specimens with yellow ripe fruits were localized in the “Restinga de Jurubatiba” National Park. The aim of the present study was to evaluate chemical composition of essential oils obtained from leaves and fruits of N. obscura specimens with different fruit color (black and yellow) by GC and GC-MS. Essential oils from leaves of specimens with black and yellow fruits indicated a predominance of sesquiterpenes (81.1% and 84.8%, resp.). Meanwhile, essential oil from black fruits presented a predominance of monoterpenes (50.5%), while essential oil from yellow fruits had sesquiterpenes (39.9%) as major substances. Despite previous studies about this species, including essential oil extraction, to our knowledge this is the first report on N. obscura fruits with different colors. Our results suggest the occurrence of unless two different varieties for this species.


Introduction
"Restinga" is a type of habitat originated from Quaternary marine deposits and is represented by herbaceous and arbustive-arboreal vegetation covering typically sandy soils [1,2]. It is characterized by large sandy coastal plains of sedimentary origin that are rippled by rows of dunes isolating lagoons, lakes, ponds, bogs, and marshes. Such a several physical conditions give rise to a diversity of habitats that are colonized by a great variety of vegetal communities [3]. "Restinga" contains many species in common with the Atlantic forest but presents diverse physiological responses to a drier habitat [4].
e Myrtaceae family has a great diversity, mainly found across tropical and temperate areas of the globe. It comprises about 4630 species, distributed among about 144 genera [5]. In Brazil, this family is mainly constituted by wood species, being one of the dominant families in Atlantic Forest [6], and it is represented by about 23 genera and 976 species, of which 749 are endemic [7]. Despite the high number of Myrtaceae species found at "restinga" habitats and numerous voucher specimens deposited at herbaria, intricacy related to identi�cation of many species from this family remains [8].
Chemical composition of essential oils from some Myrtaceae species from the "Restinga de Jurubatiba" National Park was evaluated. Meanwhile, essential oil from Eugenia sulcata Spring ex Mart. and Myrciaria �oribunda (H.West ex Willd.) O.Berg exhibited anticholinesterase activity [9,10].
Many species from this family are cultivated due to their edible fruits, source of scents, essences, and as ornamentals, such as Eucalyptus spp. Eugenia uni�ora L., Psidium guajava L. and Syzygium jambos (L.) Alston [11]. In addition, numerous species from this family are used in folk medicine, such as Psidium gajava, which is used as antiparasitic, anti-in�ammatory, antimicrobial, and treatment of intestinal diseases [12]. Ethnobotanical studies conducted with another species from this family in "restinga" areas indicate several popular uses for them, such as the treatment of diarrhea, sore throat, gout, rheumatism, in�uenza, urinary tract diseases, diarrhea and diabetes [13]. Species from this family also have great ecological value, since they provide important reward and attract pollinators [14,15].
e genus Neomitranthes is restricted to Brazil, with sixteen identi�ed species [7]. Neomitranthes obscura (DC.) N. Silveira is an endemic species of the Brazilian Atlantic Forest and widely spread in the sandbanks of "Restinga de Jurubatiba" National Park [16]. It is popularly known by local population as "camboim-de-cachorro" or "cambuí-preto" and commonly used for intestinal disorders as well as food [17,18].
In the literature, ripe fruits of Neomitranthes obscura are described with black pericarp [8,19]. However, specimens with yellow ripe fruits were localized during this study in the "Restinga de Jurubatiba" National Park. Both populations occur together, and there are not vegetative distinctions. is species is easily identi�ed by the green cylindrical galls on the apical/axial bud leaf [20] and the globoses fruits crowned by the calyx tube [8]. Fruit color varieties can be found in some Myrtaceae species, but according to Moreno [21], varieties are not easily de�nable entities in this family and are not widely accepted.
e aim of the present study was to evaluate chemical composition of essential oils obtained from specimens of Neomitranthes obscura (DC.) N. Silveira with different fruit color. sweetening, and coloring of the tissue (black or yellow pericarp in the different specimens). is species was identi�ed by the botanist Dr. Marcelo Guerra, and voucher of the yellow fruit specimens (L. Rocha 03, 04, 06) and black fruit specimens (L. Rocha 02, 05, 07) were deposited at the herbarium of the Faculdade de Formação de Professores (Universidade do Estado do Rio de Janeiro, Brazil).

Extraction of the Essential Oils.
Fresh leaves from the black fruit specimen (LBF) (2.720 kg), fresh leaves from the yellow fruit specimens (LYF) (2.790 kg), fresh black fruits (BF) (0.896 kg), and fresh yellow fruits (YF) (0.914 kg) were individually ground with distilled water using an automatic blender (�tica Equipamentos Cientí�cos S.A., Brazil). Hydrodistillation method was employed using Clevenger type apparatus, and each plant material was placed in a 5 L �ask [9]. e extraction was performed for 4 hours, and aer this period, essential oils were collected, dried over anhydrous sodium sulphate, and stored at 4 ∘ C for further analyses.

Gas Chromatography/Mass Spectrometry Analysis.
Essential oils were analyzed by a QP2010 (SHIMADZU) gas chromatograph equipped with a mass spectrometer using electron ionization. e gas chromatographic (GC) conditions were as follows: injector temperature, 260 ∘ C; detector temperature, 290 ∘ C; carrier gas (Helium), �ow rate 1 mL/min and split injection with split ratio 1 : 40. Oven temperature was initially 60 ∘ C and then raised to 290 ∘ C at a rate of 3 ∘ C/min. One microliter of each sample, dissolved in CH 2 Cl 2 (1 : 100 mg/ L), was injected at RTX-5 column (i.d. � 0.25 mm, length 30 m, �lm thickness � 0.25 m). Mass spectra were recorded at 70 eV with a mass range from m/z 35 to 450 and scan rate of 1 scan/s. e retention indices (AI) were calculated by interpolation of retention times of the substances to the retention times of a mixture of aliphatic hydrocarbons (C7-C40) (Sigma) analyzed in the same conditions [25]. e identi�cation of substances was performed by comparison of their retention indices and mass spectra with those reported in the literature [26]. e MS fragmentation pattern of compounds was also checked with NIST mass spectra libraries. Quantitative analysis of the chemical constituents was performed by �ame ionization gas chromatography (GC/FID) with a QP2010 (SHIMADZU) gas chromatograph, under the same conditions of GC/MS analysis and percentages obtained by FID peak-area normalization method.

Results and Discussions
According to Souza and Morim [8], the species N. obscura has globoses fruits crowned by the calyx tube with black pericarp when ripe. ese characteristics are helpful in the identi�cation of this species [8]. During our study, it was observed not only specimens with the characteristic black ripe fruits, but also populations with ripe yellow fruits ( Figure 1). e use of essential oils in studies of intraand interspeci�c genetic diversity and geographic patterns of variation in several plant species is well recognized [27]. us, an investigation was performed to analyze the chemical pattern of essential oils from these different colorful fruit specimens. Aer extraction, the essential oils obtained from LBF and LYF yielded 0.50% (w/w) and 0.37% (w/w), respectively. Essential oils from BF and YF yielded 0.02% (w/w) and 0.07% (w/w), respectively. e chemical analysis performed by GC-MS/GC-FID indicated a predominance of sesquiterpenes on both leaves essential oils, corresponding to 81.1% and 84.8% of relative composition of LBF and LYF, respectively. ese contents were mainly constituted by sesquiterpenes hydrocarbons. Oxygenated sesquiterpenes and monoterpene hydrocarbons also appeared on leaves oils, while aliphatic compounds appeared just on LBF specimens. is correlation of major constituents was not observed for each fruits analyzed. BF essential oil showed a predominance of monoterpenes (50.5%) and was constituted by 10.5% of sesquiterpenes. Meanwhile, YF essential oil presented sesquiterpenes (39.9%) as major substances and was constituted by 16.1% of monoterpenes. In all, 27 substances were identi�ed on this essential oil. Both essential oils from fruits presented aliphatic compounds, corresponding to 0.9% and 1.4% of relative composition of BF and YF, respectively.
Germacrene B was the major substance found in LBF essential oil, corresponding to 21.8% of relative composition. e major substances found in the essential oil from LYF were Selina-3,7(11)diene (18.7%), Trans-dauca-4,(11),7diene (13.9%), and 2,4-di-tert-butylphenol (13.4%), while germacrene B corresponded to 8.4% of relative composition of this essential oil. On another study carried out with leaves from N. obscura, De Ramos et al. [28] showed that sesquiterpenes corresponded to 87% of relative composition of this essential oil, which is in accordance with our results.
-pinene (13.5%) and -pinene (11.0%) were the major substances found in the essential oil from BF, however, appeared in lower amounts in the essential oil from YF (pinene, 3.2%/ -pinene, 1.8%). Caryophyllene oxide, which appeared as the major substance in the essential oil from YF (12.6%), corresponded to 1.7% of relative composition of essential oil from BF. e substances found in the essential oils from leaves and fruits of N. obscura are presented in Table  1.
In the tropical and subtropical zones, fruits mainly consumed by mammals are oen yellow or orange, while fruits consumed by birds are oen red or black [29,30]. e occurrence of two different colors for a fruit species may increase its dispersal. On another study, Gomes et al. [4] concluded that availability peak of caloric fruits coincides with energy-demanding seasons for resident and nonbreeding birds in "Restinga de Jurubatiba" National Park, despite the fact that there was no mention of different fruit colors. In addition, regarding the literature data, we can observe that essential oil from N. obscura was investigated but without any mention of differences in fruit color [28].

Conclusions
Varieties are not easily de�nable entities in the Myrtaceae family; however, a study carried out for different coloring fruits of Eugenia brasiliensis Lam. indicated that analysis of its volatiles corroborated the concept of different varieties for this species [21]. To our knowledge, this is the �rst report about different fruit colors of N. obscura. It is interesting from a chemical overview that the content of predominant volatiles from the two analyzed fruits had an inverse relation between sesquiterpenes and monoterpenes. ese results suggest that preferable metabolic production of monoterpenes or sesquiterpenes is followed for each specimen. us, the color and chemical constituents of different fruits of N. obscura suggest that this Myrtaceae species contains at least two different varieties.