The use of phytotherapic agents has increased remarkably in Brazil and worldwide not only from incentives by the World Health Organization (WHO), but also to search for therapeutic alternatives with fewer side effects and lower cost (WHO, 2007; Yunes and Calixto, 2001). Cerrado is the second largest Brazilian biome in diversity and comprises over 7,000 species (Almeida et al., 1998). Given such diversity, several compounds may have active components with therapeutic activity, such as secondary or special metabolites particularly essential oils. However, environmental and physiological factors might interfere not only with the content, but also with the quality of such substances; thus, processing by the cosmetic, food, and phytotherapeutic industries may be difficult (Zaroni et al., 2004; Kutchan, 2001). Circadian rhythm and seasonality are among the factors that may interfere with essential oil production because the nature and yield of their constituents may not be consistent throughout the year (Gobbo-Neto and Lopes, 2007).

 

The genus Byrsonima comprises approximately 150 species; 60 are found in Brazil (Judd et al., 1999; Castro and Lorenzzi, 2005) distributed across the federal district and the following states: Mato Grosso, Mato Grosso do Sul, São Paulo, Minas Gerais, Goiás, Bahia, Tocantins, and Paraíba (Vieira et al., 2010). Such plants are known as “murici”, “murici-pequeno”, “murici-rasteiro”, and “orelha-de-veado”, and they are used to prepare fruit juices, liqueurs, popsicles, and jellies; they are also used in traditional medicine (Camargos et al., 2001). These plants are traditionally used against asthma, fever, and skin infections, and the bark produces antidiarrheal and astringent effects (Caceres et al., 1993; Brandão, 1991). The branch leaves have antisyphilitic, diuretic, and emetic properties. Food and pharmaceutical industries employ the oil extracted from seeds (Faria et al., 2002). Among the 24 plant extracts traditionally used in Colombia to treat skin affections, methanolic extract from Byrsonima verbascifolia Rich. Ex A. Juss. produces the most potent antimicrobial and antiviral activity (Lopez et al., 2001).

 

Phytochemical studies conducted on B. verbascifolia identified phenolic and terpenic compounds, including tannins, flavonoids, and triterpenes in the leaves and bark (Lorenzzi et al., 2002). Although certain flavonoid derivatives were isolated from Byrsonima plants, triterpenes are the most frequently occurring class of natural substances in this genus. Seven triterpenoid constituents were isolated from B. verbascifolia stem bark through hexane extraction (Gottlieb et al., 1975). Sulfonoglycolipids, steroids, triterpenes, aromatic esters, amino acids, and proanthocyanidins were reported in Byrsonima crassifolia, Byrsonima microphylla, and B. verbascifolia (Sannomiya et al., 2005).

 

Ethnopharmacological use of these plants has generated increased interest in identifying the chemical constitution and pharmacological potential for species in the genus Byrsonima. However, from approximately 150 of such species, only 13 have been extensively studied; most studies were not continued, and the metabolites associated with traditionally attributed activities have not been identified (Guilhon-Simplício et al., 2005). Such studies identified primarily phenolic compounds using high-performance liquid chromatography, whereas studies that focused identifying terpenoids using gas chromatography-mass spectrometry (CG-MS) were few, especially for extracts from leaves in the species B. verbascifolia. Therefore, the aim of this study was to establish the chemical composition for the essential oil in leaves from B. verbascifolia Rich. ex A. Juss. and assess the circadian as well as seasonal variability of its content and chemical composition.

Plant

 

Adult B. verbascifolia leaves were collected from a native population in Rio Verde County-GO (18°02’02.6” S and 50°57’10.1” W and 771 m altitude). Voucher specimen (code number HJ 5643) was deposited at the Herbarium Jataiense of the Universidade Federal de Goiás, Jataí, GO.  Six individual plants were established and comprised two specimens from each block, which were 50 m apart. The specimens were collected in the first week of each month (December 2010 to November 2011) from three blocks at three different times of day (6:00, 12:00, and 18:00 h) and clustered according to the seasons. The seasons were distributed as follows: spring (October to December), summer (January to March), fall (April to June), and winter (July to September). The soil moisture was assessed through a gravimetric method when each of the 12 monthly samples were collected using one soil sample per block, which was acquired from the zero-to-20 cm depth layer on the same day the leaf samples were collected at 12:00 h.

 

Leaves were transferred to the Natural Products Section in the Laboratory of Plant Tissue Culture at Instituto Federal Goiano, Câmpus Rio Verde, GO, air-dried at 35°C and ground to fine powder. The source meteorological was obtained in the meteorological station INMET/Universidade de Rio Verde.

 

 

Extraction of essential oils

 

Dried (50 g) material was subjected to hydrodistillation (800 ml distillated water) for 2 h using a Clevenger-type apparatus. The crude oils were extracted with CH₂Cl₂, dried (anhydrous Na₂SO₄), filtered, and the solvent removed at retention time. Oil samples were kept in amber glass vials at 4°C until the identification of their chemical composition. The extraction yield of each essential oil was expressed in % (w/w) of the dried leaves.

 

 

Chemical analysis of the essential oils

 

Chemical analysis was performed in the Chemistry Department at the Universidade Federal de Lavras, Lavras, MG. The essential oils were analyzed with a Shimadzu QP5050A apparatus equipped with a mass selective detector operating by electronic impact (70 eV) and a DB-5 cap. column (30 m × 0.25 mm i.d., film thickness 0.25 mm). Helium (1 ml.min^-1) was used as carrier gas. The oven temperature was programmed rising from 60 to 240°C at 5°C.min^-1, a 10°C.min^-1 from 240 to 270°C and then held isothermal at 270°C for 5 min; injector temperature, 220°C; detector tempereature, 240°C; the sample injection volume was 1.0 µl and diluted in dichloromethane at a 1:20 injection ratio. The tests were performed in triplicate in scan mode at 2.0 scans/s and 45 to 500 m/z.

 

Volatile compounds were identified by comparing the resulting mass spectra with records from the Wiley and FFNSC 1.2 computational libraries. These compounds were also determined using the retention indices (RI) (Van den Dool and Kratz, 1963), from a series of n-alkanes of (C_8-C₄₀) under the same chromatographic conditions that were used for the essential oils. The resulting values were subsequently compared with the Kovats indices that are available in the literature (Adams, 2007).

 

 

Statistical analysis

 

To establish the content and chemical composition of the essential oil in the leaves and new branches on B. verbascifolia, the study design included randomized blocks with a 4 × 3 factorial design as well as three replications, and it corresponded to the 12 monthly samples as well as the three different times of day for sample collection. The data were subjected to analysis of variance, and the means were compared at 5% probability using software SISVAR-System for Analysis of Variance (Ferreira, 2007).

Essential oil content

 

The essential oil in dry leaves from B. verbascifolia was a slightly yellowish fluid with low viscosity and a poorly characteristic odor. The season and time of day did not alter the essential oil content. The season and time of day did not significantly influence essential oil content in separate analysis; the oil content varied from 0.003 to 0.005% in both instances (Table 1). The oil content did not change in a study that assessed the influence of irradiation levels on Hyptis marrubiodes Epl. essential oil yield (Sales et al., 2009).

 

Based on the results produced under the conditions in Rio Verde County, GO, one might suggest that samples may be collected in any season at the three investigated times of day. Although the phytomass yield was not analyzed, the plants produced more leaves in spring and summer, which might increase essential oil yield during these seasons.

 

 

Chemical analysis

 

The chemical composition of the essential oils from leaves of B. verbascifolia varied among the seasons investigated (Table 2). Certain substances (isobutyric acid and β-bisabolene, among others) were not detected in every season, likely from seasonal variations and the influence of abiotic factors, such as light. A study assessing the influence of irradiation levels on essential oil chemical composition in H. marrubiodes Epl. found that the oil components iso-3-thujanol and δ-cadinene varied (Sales et al., 2009).

 

 

The compounds nerolidol, benzene-1,2,-dicarboxylic acid diethyl ester, spathulenol, (Z.Z)-farnesol, caryophyllene oxide and pentacosane were identified in concentrations over 1% in each season investigated. Spathulenol was identified in the epicuticular layer of the leaves in several species, such as Byrsonima linearis, and it demonstrated insecticide activity as well as protection against desiccation from dissipation of excess light (Faine et al., 1999; Silva et al., 2006). Nerolidol is mentioned because it is an absorption enhancer that acts by reinforcing the skin bilayers through orientation along the stratum corneum lipids (Marinho, 2008; Williams and Barry, 2004).

 

Forty-eight chemical components were identified in the essential oil from B. verbascifolia in different seasons with relative concentrations of oxygenated monoterpenes that varied from 2.782 to 17.386%; oxygenated sesquiterpenes from 22.496 to 45.646%, sesquiterpene hydrocarbons from 3.437 to 31.430%; and additional components, including alcohols, acids, hydrocarbons, long-chain hydrocarbons, and aldehydes, that varied from 18.613 to 49.088%.

 

Sesquiterpenes predominated except for winter when components such as alcohols and hydrocarbons prevailed. For sesquiterpene hydrocarbons, the highest content was observed in spring (27.82%) and summer (31.43%). With the earliest rainfalls at the beginning of spring and consequent increase in soil moisture, new shoots (Figure 1A) and the first flowers appeared. Full blossoming began after monthly precipitation became greater than 250 mm and lasted until the end of the season (Figure 1B). The times of day for sample collection (6:00, 12:00, and 18:00 h) did not produce a difference in sesquiterpene hydrocarbons (20.84, 16.75 and 14.32%, respectively). For oxygenated sesquiterpenes, the greatest percentage was observed in the fall (45.64%) when the temperature had dropped, whereas the oil content was not dependent on the time of day of sample collection (29.31, 32.2 and 29.01%).

 

The highest percentage of oxygenated monoterpenes was observed in spring and summer (15.20 and 17.38%, respectively) as described in Table 3. More leafing, fruit formation, and fully formed fruits were observed in the spring when the average temperature was over 23°C, the air relative humidity above 70%, the average monthly precipitation over 20 mm, and the soil moisture over 10% (Figure 2A, B, C, and D). The soil moisture remained high from spring to summer and fell below 5% in winter when rainfall is virtually absent. Fruit maturation (Figure 1C) was observed at the end of spring and during summer. The relative concentrations (30.93, 29.11 and 32.72%) of oxygenated monoterpenes did not differ with the time of day for sample collection (6:00, 12:00, and 18:00 h).

 

The combined content for the remainder of classes (alcohols, esters, hydrocarbons, and others) was greater in the dry season (winter). During this season, the relative air humidity was below 50%, the soil moisture below 5% (Figure 2B and D), and the leaves were yellowish or fully dry, which facilitated senescence. Certain leaves contained chestnut brown-reddish lesions, which may have been related to a biological/physiological response. These lesions are a common trait in this species and may be observed during winter for all of the plants in the area investigated.

 

 

 

The three main components of the essential oil in B. verbascifolia Rich. ex A. Juss leaves include a long-chain hydrocarbon (pentacosane), an ester (benzene-1,2-dicarboxylic acid diethyl ester), and an oxygenated sesquiterpene (spathulenol). Although season and time of day did not contribute to such compounds, greater spathulenol content was observed in fall (15.30%), when the soil moisture had not decreased but rainfall, average temperature, and air relative humidity were lower (Figure 2).

 

Certain studies have reported a variation in essential oil chemical composition as a function of seasonal variation. In the species H. marrubioides Epl. (Lamiaceae), although non-oxygenated (cadalene, germacrene D, α-copaene, and α-caryophyllene) and oxygenated (caryophyllenol and cedrol) sesquiterpenes are observed at much lower levels in the essential oil, they were quantitatively different depending on the season (Botrel et al., 2010). The primary compounds concentrations in the essential oil from Elyunurus muticus (Sprengel) O. Kuntze (Poaceae), e.g., (E)-caryophyllene, spathulenol, bicyclogermacrene, and caryophyllene oxide, varied as a function of season the plants were collected (Hess et al., 2007).

 

The content of the pentacosane (9.53, 11.26 and 8.99%), benzene-1,2-dicarboxylic acid diethyl ester (6.41, 6.71 and 7.64%) and spathulenol (8.93, 6.36 and 7,11%) did not vary with the time of day (6:00, 12:00 and 18:00 h) that samples were collected. Studies using samples from the wild species Eremanthus seidelii (Asteraceae) collected in its natural habitat found that the concentration of secondary metabolites was constant, which suggests that environmental factors do not systematically influence the production of such compounds (Sakamoto et al., 2005; Gobbo-Neto and Lopes, 2007).

Essential oil from B. verbascifolia Rich. ex A. Juss leaves is composed of oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes; the largest fraction is oxygenated sesquiterpenes and the smallest is oxygenated monoterpenes. The primary components of the essential oil were pentacosane (9.93%), spathulenol (7.46%), and benzene-1,2-dicarboxylic acid diethyl ester (6.92%).

 

Neither seasonal nor circadian variation influenced the essential oil content. The terpene class was influenced by seasonal variation; the percentage of oxygenated monoterpenes and sesquiterpene hydrocarbons was the highest in spring and summer, and the highest percentage of oxygenated sesquiterpenes observed in fall. Circadian variations were not observed for this class. Among the primary compounds, only spathulenol was influence by seasonal variation; its highest relative concentration was observed in fall.

The authors appreciate the support of FAPEG (Fundação de Amparo à Pesquisa do Estado de Goiás) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for the fellowship and financial support, as well as the Instituto Federal Goiano – Câmpus Rio Verde, GO for its infrastructure.

The authors declare that they have no conflicts of interest.