Leaf and fruit essential oil compositions of Pimenta guatemalensis ( Myrtaceae ) from Costa Rica

Pimenta is a genus of flowering plants in the Myrtaceae family, which has about 15 species, mostly found in the Caribbean region of the Americas. Commonly used for culinary and medicinal purposes, the best known commercial species are allspice, P. dioica (P. officinalis) and bay rum, P. racemosa, but there is little information concerning P. guatemalensis. The aim of the present study was to identify the chemical composition of the leaf and fruit essential oils of P. guatemalensis. The extraction of essential oils of P. guatemalensis growing wild in Costa Rica was carried out by the hydrodistillation method at atmospheric pressure, using a modified Clevenger type apparatus. The chemical composition of the oils was analyzed by capillary gas chromatographyflame ionization detector (GC/FID) and gas chromatography-mass spectrometry (GC/MS) using the retention indices on DB-5 type capillary column. A total of 103 and 63 compounds were identified in the leaf and fruit oils, respectively, corresponding to 96.8% and 86.1% of the total amount of the oils. The leaf oil consisted mainly of eugenol (72.8%), and monoand sesquiterpene hydrocarbons (18.2%). Among terpenes the major components were b-caryophyllene (8.2%) and terpinolene (3.0%). The fruit oil also consisted mainly of eugenol (74.7%) and minor amounts of oxygenated monoand sesquiterpenes (7.3%), mainly caryophyllene oxide (3.3%). This is the first report of the chemical composition of the essential oils obtained from this plant species. Rev. Biol. Trop. 63 (1): 303-311. Epub 2015 March 01.

Myrtaceae is a family constituted of about 142 genera and includes more than 5 500 species of mostly trees and shrubs with conspicuous oil glands, distributed predominantly in the Southern hemisphere, mainly in Australia and South America (Wilson, 2011).
The genus Pimenta Lindl.contains about 15 species, mostly in the Caribbean region of the Americas.Pimenta guatemalensis (Lundell) Lundell is a tree about 5-16(20) m tall with a straight trunk and reddish brown bark (Barrie, 2007).In Costa Rica, it is commonly known as Jamaica (León, & Poveda, 2000).This plant is distributed along Central America, from Guatemala to Panama.The young branchlets are 4-angled.The leaves are evergreen, simple, opposite and oblanceolate to elliptical, the blade cuneate at the base and acuminate at the apex.When the leaves are crushed they give off a scent with an aromatic flavor resembling a mixture of clove and cinnamon due to an oil of unknown composition.The fragrant flowers are small and the receptacle has five calyx lobes, arranged in panicles (ca.20 flowers) in the leaf axils.The ovaries are uni-locular or without loculi.This plant grows wild in Costa Rica and it can be found distributed between 250 and 900m above sea level mostly in the rain forests of the North region and Caribbean slopes.To the best of our knowledge, no previous reports on the chemistry of P. guatemalensis have been published.
Pimenta species are used in several countries of Mesoamerica and Caribbean for culinary and medicinal purposes (Morton, 1981;Germosén-Robineau, 2005).Paula, Reis, Ferreira, Menezes, andPaula (2010), andRao, Navinchandra, andJayaveera (2012) and Lim (2012) have recently reviewed some botanical aspects, traditional medicinal uses, biological and pharmacological activities and chemical compositions of Pimenta dioica and P. racemosa (the most important from the economical point of view) and P. pseudocaryophyllus, the only species of this genus that grows in the Brazilian Atlantic Rain Forest in South America (Marques et al., 2010;Paula et al., 2011).Formerly the name Pimenta dioica was frequently misapplied to P. guatemalensis native populations in Costa Rica (Barrie, 2007).
In the present work we report the chemical composition of the oils isolated by hydrodistillation from leaves and fruits of P. guatemalensis collected in Costa Rica.

MATERIALS AND METHODS
Plant material: Leaves and fruits of Pimenta guatemalensis (Lundell) Lundell, Myrtaceae, growing wild in Costa Rica in a rain forest, were collected in August 2005 (rainy season), in Bajo Rodríguez, San Ramón, Province Alajuela.A voucher specimen was kept at the Herbarium of the University of Costa Rica (USJ 77482).
Isolation of the essential oils: Air-dried (in the shade at room temperature) leaves (500g) of P. guatemalensis were subjected to hydrodistillation at atmospheric pressure, for three hours, using a modified Clevenger-type glass apparatus.The light yellowish distilled oil (2.5mL) was dried over anhydrous Na 2 SO 4 ; and the yield (v/m) of the oil was 0.5%.The same procedure was utilized for the air-dried fruits (250g).The colorless oil was dried, yielding 1.8mL (0.7% v/m).The oils were filtered and stored at 0-10°C in the dark for further analysis.

GC/MS analysis:
The analyses by gas chromatography coupled to mass selective detector were performed using a Shimadzu GC-17A gas chromatograph coupled with a GCMS-QP5000 apparatus and CLASS 5000 software with Wiley 139 and NIST computer databases.The data were obtained using the same column described above.Operating conditions were: carrier gas He, flow 1.0mL/min; oven temperature program: 60-280°C at 3°C/ min; sample injection port temperature 250°C; detector temperature 260°C; ionization voltage: 70eV; ionization current 60μA; scanning speed 0.5s over 38-400amu range; split 1:70.

Identification of chemical constituents:
The oil components were identified using the retention indices (RI) on capillary DB-5 type column (van den Dool, & Kratz, 1963), and by comparison of their mass spectra with those published in the literature (Adams, 2007) or those of the author's database.To obtain the retention indices for each peak, 0.1μL of n-alkane mixture (Sigma retention index standard for gas chromatography, C 8 -C 32 , R 8769, USA) was injected under the same experimental conditions reported above.Integration of the total chromatogram (GC/FID), expressed as area percent, has been used to obtain quantitative compositional data.

RESULTS
From the hydrodistilled oils, a total of 126 compounds were identified using GC/FID and GC/MS, accounting for 96.8% (leaves) and 86.1% (fruits) of the total composition of the essential oils.The compounds identified in the leaf and fruit oils of P. guatemalensis are presented in Table 1, where the components were listed in order of elution on a MDN-5S column.Table 1 also includes the relative percentages of single components, their experimental retention indices (RI) with reference to a homologous series of linear alkanes (C 8 -C 32 ) and, for comparison purposes, previously published values.Additionally, percentages of oils various types/classes of constituents were also indicated.P. guatemalensis gave oils which were predominantly aromatic in nature with a terpenic fraction, and several aliphatic and aromatic compounds as trace components.
The fruit oil also was rich in eugenol (74.7%), accompanied by lesser amounts of the benzenoid vanillin (0.7%), the phenylpropanoids coniferaldehyde (0.3%) and (E)coniferyl alcohol (0.2%), together with traces of safrole and (E)-isoeugenol.This oil resembles the fruit oil composition of P. dioica from Jamaica (Pino, 1999), where the main constituent was eugenol, 68.6-87.0%.A comparison of our results with those obtained by García Fajardo et al. (1997), from samples of Mexican berries, indicate both qualitative and quantitative differences in the constituents.The Mexican berries contain high quantities of methyl eugenol (48.3-62.7%)and myrcene (16.5-17.7%)and low quantity of eugenol (8.3-17.3%).The absence of methyl eugenol and the presence of the minor benzenoid and phenylpropanoids above mentioned appears to be a fact that could differentiate the oil of P. guatemalensis from other oils of P. dioica studied previously.However, to try to chemically differentiate between P. guatemalensis and P. dioica, it would be necessary to conduct a study with a larger number of wild specimens from various geographic areas of Costa Rica.(Pino et al., 2006).f (Jordán, Margaría, Shaw, & Goodner, 2002).g (Hamm, Lesellier, Bleton, & Tchapla, 2003).h (Gómez, Ledbetter, & Hartsell, 1993).

TABLE 1
Percentage composition of the leaf and fruit oils of Pimenta guatemalensis from Costa Rica