Essential oils of Baccharis trinervis ( Asteraceae ) from Costa Rica

Baccharis is an Asteraceae genus of flowering plants, which has about 340 to 400 species, ranging from the Southern United States to the Southern extreme of Argentina and Chile through Central America and the Caribbean regions. The species Baccharis trinervis is a native shrub from Mexico, Central America and throughout South America. In Costa Rica, this species is commonly known as alcotán and the fresh leaves are used as a poultice on wounds and ulcers. The objective of the present research was to characterize the chemical composition of seven hydrodistilled essential oils of diverse morphological parts of B. trinervis. For this, samples were obtained from three locations in Costa Rica and standard laboratory analyses were followed. The essential oils were analyzed by capillary gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) using the retention indices on a 5 % phenyl/dimethylpolysiloxane fused silica column in addition to mass spectral fragmentation patterns, which allowed the identification of 268 compounds. The essential oils consisted mainly of terpenoids (92.3 to 97.8 %). The major constituents from the leaf oils were caryophyllene oxide (0.1-22.5 %), viridiflorol (8.8-21.0 %), germacrene D (0.5-19.1 %), germacrene B (0.2-16.0 %), β-caryophyllene (3.5-9.1 %), spathulenol (0.1-8.3 %), δ-3-carene (2.0-6.8 %), and α-pinene (2.5-5.9 %). The flower oil consisted mainly of globulol (0-24 %), β-caryophyllene (9.5-17.1 %), cismuurola-4(14), 5-diene (traces-13.7 %), germacrene D (4.3-9.9 %), bicyclogermacrene (5.9-8.3 %), ar-curcumene (0-8.0 %), spathulenol (4.3-4.8 %), caryophyllene oxide (3.1-4.7 %), and viridiflorol (0.3-4.7 %). The major components of the branch oil were germacrene B (1.4-18.7 %), germacrene D (14.7-15.6 %), β-caryophyllene (10.1-12.4 %), viridiflorol (0-11.5 %), globulol (0.6-11.3 %), δ-3-carene (4.1-8.1 %), β-phellandrene (1.5-6.5 %), and bicyclogermacrene (3.6-4.9 %). The essential oil composition differed markedly from that of previously studied oils of plants growing in Brazil, which contain two characteristic stereoisomeric methyl dec-2-en4,6-diynoate compounds not detected in this study. This is the first report about the chemical composition of the essential oils obtained from this species growing wild in Costa Rica. Rev. Biol. Trop. 65 (4): 1307-1321. Epub 2017 December 01.

Baccharis L. is one of the largest genera within Asteraceae family (tribe Astereae, sub-tribe Baccharidinae) including herbaceous perennials, vines, shrubs and trees.This genus includes ca.340 to 400 species ranging from the Southern United States to the Southern extreme of Argentina and Chile through Central America and the Caribbean basin (Heiden, Andrade-Baumgratz, & Esteves, 2012).Some of the species are economically important as medicinals -used mainly as infusions and decoctions-for treat stomach and liver ailments, inflammations, anemia, diabetes and prostate diseases (Verdi, Brighente, & Pizzolatti, 2005).The vassoura essential oil (Baccharis dracunculifolia DC.) and carqueja oil [B. genistelloides (Lam.)Pers.] are used in perfume industry (Ferracini et al., 1995).
Baccharis trinervis Pers.[=Pseudobaccharis trinervis (Pers.)V. M. Badillo] is a common shrub or sub-scandent shrub in the Pacific slope of Costa Rica, with hairless ribbed stems and oblong-elliptic or lanceolate-elliptic alternate leaves, 3 to 10 cm long, 1.5 to 3.5 cm wide, hard and rough, with three prominent, longitudinal veins, puberulent to pilose on both sides.Flowers are greenish-white disposed in terminal panicles (León & Poveda, 2000) situated within or overtopping the foliage.This plant has a wide distribution, from Mexico, Central America and throughout South America.In Costa Rica it is distributed over the country, from lowlands to 1 900 m of elevation, in both dry and wet forests.This plant is commonly known as alcotán, and in the Northwestern Guanacaste Province it is known as jalapatrás and rastrapo (León & Poveda, 2000).In Costa Rica, the fresh leaves are used as a poultice on wounds and ulcers (Pittier, 1978) and the alcoholic extracts of roots are said to be used as remedy for snakebites (Núñez-Meléndez, 1978).In Honduras, the extracted juice of the leaf, and root decoctions, are taken as a remedy for kidney troubles, pain and inflammations, as a febrifuge, for halting diarrhea, and against rheumatic pains (House et al., 1995).It has also been used in various liver diseases, as a purgative, antiseptic, digestive and diuretic, in rectal washes against hemorrhoids and as a remedy for typhoid fever (Ramírez-Cárdenas, Isaza-Mejía, & Pérez-Cárdenas, 2013).
In relation to the essential oil composition of the aerial parts of this species, there are only three previous reports: two studies from material collected in the state of Ceará, Northeast Brazil (Albuquerque et al., 2004;Sobrinho et al., 2016), and another from the state of Mérida, Venezuela (Rojas et al., 2008).
The objective of the present research was the characterization of the chemical composition of seven different samples of essential oils from diverse morphological parts of B. trinervis collected in three different locations in central Costa Rica.To the best of our knowledge, no previous chemical work on B. trinervis from Costa Rica has been reported.Isolation of the essential oils: Plant materials were hydrodistilled at atmospheric pressure, for 3 h, using a Clevenger-type apparatus.The distilled oils were collected and dried over anhydrous sodium sulfate, filtered and stored at 0-10 °C in the dark, for further analysis.The essential oil yields (v/w) from diverse morphological parts from three localities in central Costa Rica were: Miramar sample, 0.32 % (leaf), 0.04 % (flower); San Pedro sample, 0.35 % (leaf), 0.03 % (branch); and Pavas sample, 0.23 % (leaf), 0.06 % (flower), and 0.05 % (branch).

Compound identification and quantification:
The components of the oils were identified by comparison of their linear retention indices which were calculated in relation to a homologous series of n-alkanes, on 5 % phenyl/ dimethylpolysiloxane type column (van den Dool & Kratz, 1963), and by comparison of their mass spectral fragmentation patterns with those published in the literature (Adams, 2007) or those of our own database.To obtain the retention indices for each peak, 0.1μL of n-alkane mixture (Sigma, C 8 to C 32 standard mixture) 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 without FID response factor correction.

RESULTS
The essential oils from different parts of Baccharis trinervis from Costa Rica presented a very complex chemical profile.The compounds identified, their experimental retention indices (RI) determined in relation to a homologous series of linear alkanes (C 8 to C 32 ), their relative percentage concentrations, and the method used for their identification are presented in table 1.The constituents are listed in order of elution on a MDN-5S column and for comparison purposes, previously published values of the retention indices are included (Lit.RI).Compounds identified in this study and previously reported in the Venezuelan and Brazilian essential oil samples are indicated by bullets and/or asterisks.
As can be seen from table 1 and table 2, the major constituents of the leaf essential oil from the sample collected in Miramar were oxygenated sesquiterpenes (64.6 %), whereas sesquiterpene hydrocarbons (64.3 %) were the major components of the San Pedro sample.However, in the sample from Pavas, the two cited classes of metabolites were almost equally distributed (30.7 and 33.6 %, respectively).

DISCUSSION
The essential oil of fresh aerial parts of Baccharis trinervis, collected on Merouca mountain region of Ceará state, Northeastern Brazil (Albuquerque et al., 2004;Sobrinho et al., 2016), revealed that the major constituents were terpenoids with the presence of β-phellandrene (18.4-27.8%), sabinene (10.9-14.2%), α-thujene (6.6-10.5 %), (Z)-βocimene (2.3-8.1 %), α-pinene (5.5-8.7 %), and (E)-β-ocimene (1.9-6.3 %), including two non-terpenoid C-10 ene-diyne esters: methyl (Z)-dec-2-en-4,6-diynoate (0.8-14.6 %) and methyl (E)-dec-2-en-4,6-diynoate (10.7-14.7 %) not found in other oils of Baccharis species studied up to day.Rojas et al. (2008) examined the essential oil from leaves collected on Santa Rosa, La Hechicera, in Mérida State (Venezuela).In this study, the authors reported that the major constituents were germacrene D (20.1 %), limonene (15.0 %), δ-cadinene (5.2 %), β-caryophyllene (4.8 %), α-pinene (4.5 %), and bicyclogermacrene (4.0 %), and this oil can be distinguished from the Brazilian ones in the fact that the main C-10 ene-diyne esters were absent.Our experimental data support those obtained by Rojas et al. (2008) because the results of the essential oil samples from the three different locations in Costa Rica do not contain those characteristic and very specific C-10 ene-diyne esters found in the Brazilian botanical material.As an additional support, the phytochemical study realized by Bohlmann and Zdero (1970) of fresh aerial parts of B. trinervis, cultured from seeds -of not specified origin-at Botanical Garden of the University of Berkeley, verified the presence of matricaria ester and three C-17 ene-diyne-diene esters but not C-10 ene-diyne esters (see also , Bohlmann, Burkhardt, & Zdero, 1973).Nonetheless, in a later phytochemical study of nine species of Baccharis collected in Brazil, the aerial parts of B. trinervis afforded, besides several terpenoids, one compound which has the trivial name lachnophyllum ester (probably, its Z isomer) corresponding to methyl dec-2-en-4,6-diynoate (Bohlmann et al., 1981).This was the first diacetylenic compound with established chemical constitution isolated from an essential oil (Sørensen, 1977).This significant variation in the qualitative composition of the essential oils of the same species collected in Venezuela and Costa Rica compared with the Brazilian samples could be caused by the habitat and environment factors as well as the genotype of the plant, but real sources of variability, according to Németh-Zámboriné (2016), are 'hard to determine'.Furthermore, it is known that Baccharis is a diverse and complex genus of the Asteraceae because some of the species present a high degree of morphological and chemical variability.One of the distinguishing factors of the oils from this plant growing wild in Costa Rica is the presence of an array of terpenoids with diverse carbon skeletons that could arise through various biosynthetic patterns.Especially important in number are the cadinane class and the guaiane family of sesquiterpenoids that are originated from the germacrane biosynthetic pathway.A distinctive character of the oils of B. trinervis studied from Costa Rica is the presence of the oxygenated sesquiterpenoids spathulenol, viridiflorol and globulol, in important amounts, accompanied by lesser quantities of several compounds of the same carbon skeleton, that are not found in the Venezuelan sample nor in the oils of Brazilian botanical material.These compounds also were found in B. articulata (Lam.)Pers.(Minteguiaga et al., 2015), B. caprariifolia DC. (Ferracini et al., 1995), B. crispa Spreng. (Simões-Pires et al., 2005), B. dracunculifolia DC. (Frizzo et al., 2008;Fabiane, Ferronatto, dos Santos, & Onofre, 2008), B. erioclada DC. (Ferracini et al., 1995), B. platipoda DC. (Ferracini et al., 1995;Quiroga, Ferracini, & Marsaioli, 1996), B. semiserrata DC. ( Vannini et al., 2012), B. tridentata Vahl (Ferracini et al., 1995;Quiroga et al., 1996), B. trimera (Less.)DC. (Silva et al., 2007;Oliveira et al., 2012), B. uncinella (Fabiane et al., 2008), andB. vincaefolia Baker (Ferracini et al., 1995).Spathulenol appear to be a widespread compound in the essential oils from plants of the genus Baccharis.Our findings corroborate the presence of 41 compounds previously reported (Albuquerque et al., 2004;Sobrinho et al., 2016;Rojas et al., 2008) indicated by asterisks and bullets in table 1, whereas 227 constituents are newly reported in the composition of oils from Baccharis trinervis.
material: The aerial parts of Baccharis trinervis were collected from three localities of Costa Rica: Miramar, Montes de Oro mountains (west central Pacific), Province of Puntarenas (10°06'12.79"N -84°42'26.09"W, at an elevation of 750 m), on April 2009, during the flowering stage; in the University of Costa Rica Campus, San Pedro de Montes de Oca, Province of San José (9°56'14.60"N -84°02'51.51"W, at 1 210 m elevation), on February 2009, during vegetative stage, and Pavas, Province of San José (9°56'37.20"N -84°06'59.40"W, at an elevation of 1 093 m) on November 2015, during the flowering stage of the plant.A voucher specimen has been deposited in the Herbarium of the University of Costa Rica at the School of Biology (USJ 93916).
distribution in the essential oils of Baccharis trinervis from three locations in Costa of compounds.t: traces (˂0.05 %).

TABLE 1
Chemical and percentage composition of oils of Baccharis trinervis from three locations in Costa Rica