CHEMICAL DIVERSITY OF ESSENTIAL OILS FROM Hyptis pectinata (L.) Poit DIVERSIDADE QUÍMICA DE ÓLEOS ESSENCIAIS DE Hyptis pectinata (L.) Poit

The essential oils are secondary metabolites formed by several chemical compounds that confer to these substances great social and economic importance. This diversity of compounds is generally determined by the genetic constitution of the plant, although environmental factors may also influence the type, amount, and concentrations of the compounds present in the essential oil. The aim of this work was to analyze the chemical diversity of the essential oils of native Hyptis pectinata plants collected in the state of Sergipe. The essential oils of 24 plants were obtained by hydrodistillation and analyzed by GC-MS/FID, revealing 30 compounds. Two clusters were formed by the cluster analysis. Cluster I consisted of 18 plants, and presented β-elemene (2.46-25.77%), β-caryophyllene (16.20-60.95%), germacrene D (0.00-21.59%), and caryophyllene oxide (5.38-42.21%) as major compounds. Cluster II consisted of six plants, and presented β-caryophyllene (5.68-15.57%), (Z)-β-guaiene (2.18-7.31%), caryophyllene oxide (1.58-22.89%), and calamusenone (23.12-64.36%) as major compounds. Strong correlation was observed between pcymene and γ-terpinene (r=0.94), and between (E)-β-guaiene and lepidozene (r=0.95). Results of the present study indicate variation in the essential oil content, and show that the compounds β-elemene, β-caryophyllene, germacrene D, (Z)-β-guaiene, caryophyllene oxide and calamusenone were detected in greater proportions in native plants of H. pectinata of the state of Sergipe. The knowledge of the chemical diversity found in H. pectinata plants can assist in the selection of plants of specific interest.


INTRODUCTION
The Lamiaceae family has about 300 genera and approximately 7500 species. In Brazil, approximately 350 species are distributed in 26 genera (SOUZA; LORENZI, 2008). The family is known for the chemical variability of its essential oils, and its plants are widely used by the population for therapeutic purposes (RAYMUNDO et al., 2011).
The genus Hyptis is composed of several medicinal and aromatic species of great economic interest. Among these species, H. pectinata, popularly known in the Brazilian Northeast as "sambacaitá" or "canudinho", is extensively used in folk medicine in the treatment of bacterial infections and inflammation (ARRIGONI-BLANK et al., 2005). In addition, several biological properties of its essential oils or extracts have already been proved, such as its antidematogenic, antinociceptive , antimicrobial , insecticide (SILVA et al., 2008), anti-inflammatory (RAYMUNDO et al., 2011), and leishmanicide activities (FALCAO et al., 2013).
The essential oils are products of secondary metabolism, characterized as complex chemical mixtures extracted from different parts of the plant, and confer adaptive advantages to the different environments in which they are inserted (OUSSALAH et al., 2007).
The genetic constitution is usually the main determinant of plant adaptive responses, allowing for differences in the synthesis of secondary metabolites related to the types, amounts, and concentrations of the compounds. However, these variations can also be influenced by environmental factors, such as luminosity, temperature, water availability, soil conditions, among others (MARTINS et al., 2006).
The characterization of the chemical composition of the essential oils of plants of the same species allows generating information for the obtainment of the most suitable plants for therapeutic use, and for the obtainment of plants with higher essential oil content, allowing selection and insertion in genetic improvement programs (VELOSO et al., 2014).
Thus, the objective of the present study was to evaluate the chemical diversity and the essential oil content of native plants of Hyptis pectinata of the state of Sergipe.

Plant material
Leaves of 24 native plants of H. pectinata were collected from 21 municipalities of the state of Sergipe, Brazil (Table 1).

Extraction, content and chemical analyses of essential oils
The essential oils were extracted and analyzed in the Phytotechnology Laboratory and Laboratory of Chromatography respectively, both in the Federal University of Sergipe.
The collected leaves were dried in a forced air circulation oven, at 40ºC, for five days. The essential oils of H. pectinata were extracted using 70g, in triplicate, by the hydrodistillation method, in a modified Clevenger apparatus, for 150 minutes (EHLERT et al., 2006). The essential oils were stored in amber flasks, and kept in freezer at -20 °C until chemical composition analysis.
The MS and FID data were simultaneously acquired employing a Detector Splitting System; the split flow ratio was 4:1 (MS:FID). A 0.62 m x 0.15 mm i.d. restrictor tube (capillary column) was used to connect the splitter to the MS detector; a 0.74 m x 0.22 mm i.d. restrictor tube was used to connect the splitter to the FID detector. The MS data (total ion chromatogram, TIC) were acquired in the full scan mode (m/z of 40-350) at a scan rate of 0.3 scan/s using the electron ionization (EI) with an electron energy of 70 eV. The injector temperature was 250 °C and the ion-source 877 Chemical diversity… FEITOSA-ALCANTARA, R. B. et al.
temperature was 250°C. The FID temperature was set to 250 ºC, and the gas supplies for the FID were hydrogen, air, and helium at flow rates of 30, 300, and 30 mL/min, respectively. Quantification of each constituent was estimated by FID peak-area normalization (%). Compound concentrations were calculated from the GC peak areas and they were arranged in order of GC elution. The retention index (Van den Dool and Kratz 1963) was obtained by injecting a C 7 -C 30 linear hydrocarbon mixture under these same conditions, and identification of constituents was made on the basis of comparison of retention index and MS with those in the literature (ADAMS, 2007), as well as by computerized matching of the acquired mass spectra with those stored in NIST21, NIST107, and WILEY8 mass spectral libraries of the GC-MS data system.

Statistical analyses
Two multivariate analysis techniques were used for the chemical diversity analysis (cluster analysis and principal component analysis -PCA), using the Statistica software version 7.0. A dendrogram was generated using the Ward clustering method, based on a dissimilarity matrix constructed using the Euclidean distances of the chemical composition of each sampled plant and the correlation analysis between the chemical compounds of the essential oil of the sampled plants.
The results of the essential oils contents were subject to analysis of variance. Means were compared by the Scott-Knott test (P≤0.05), using the Sisvar® software, when significant.
The graph with the means of the chemical compounds and standard deviations for each chemical cluster was obtained using the Graph Pad Prism® software.

RESULTS AND DISCUSSION
The diversity of the chemical compounds of the essential oils was significant among the native plants of H. pectinata of the state of Sergipe. Thirty compounds were detected in the chemical analyses of the 24 plants (Table 2, Figure  1). Essential oil content varied between the plants, and a higher percentage (0.90) was obtained in the plant from the municipality of Porto da Folha ( Table 2).
The plant kingdom presents wide chemical diversity. Variation in chemical compounds is usually observed among plants of the same species (KLEINE and MULLER, 2011). The number of compounds and the concentrations of each compound in the essential oil of the plants and the oil content can be influenced by genetic, climatic, and/or edaphic factors (OLIVEIRA et al., 2012;TEIXEIRA et al., 2013;BLANK et al., 2015;COSTA et al., 2015;PINTO et al., 2015).
These factors can redirect the metabolic pathway, and thus form other compounds that help plants adapt to the conditions to which they are subject (KLEINE and MULLER, 2011). This redirection is possibly related to the catalytic flexibility of the terpene-synthase enzymes, which often produce multiple products from a single substrate. The monoterpenes are synthesized from geranyl diphosphate (GDP), farnesyl diphosphate (FDP) sesquiterpenes, and geranylgeranyl diphosphate diterpenes (GGDP) (ARIMURA et al., 2009).
The mean values of β-caryophyllene and caryoplyllene oxide were the main determinant factor for the subdivision of these plants.
Results showed that some plants collected in the same municipality or in neighboring municipalities, with similar climatic and edaphic factors, such as Canindé do São Francisco and Poço Redondo, were clustered according to their chemical composition.        The conventional propagation process of the genus Hyptis is through seeds, which may allow great genetic variability (WULFF, 1973). This suggests that the differences found may be related mainly to genetic factors, since such plants were subject to the same or very similar environmental conditions. Similar results were found in studies on Varronia curassavica, in which some plants collected in the same municipality were also classified into different clusters (NIZIO et al., 2015).
The compound β-caryophyllene presented negative correlation with calamusenone (-0,72) and (Z)-β-guaiene (-0,62). The compound α-cubenene presented positive correlation between α-copaene (0.66) and epi-α-cadinol (0.65). Epi-αcadinol positively correlated to spathulenol (0.69) and (E)-calamenene (0.68) ( Table 3).   The high correlation between compounds indicates that a plant with high content of the first compound will probably present high content of the second compound. This information can assist in the selection process of breeding programs (NIZIO et al., 2015). Possibly, this correlation can be explained by the ability of a single enzyme to synthesize different products, due to the similarity between the biosynthetic pathways of the compounds (DEGENHARDT et al., 2009).
Results of the present study indicate variation in the essential oil content, and show that the compounds β-elemene, β-caryophyllene, germacrene D, (Z)-β-guaiene, caryophyllene oxide and calamusenone were detected in greater proportions in native plants of H. pectinate of the state of Sergipe. The knowledge of the chemical diversity found in H. pectinata plants can assist in the selection of plants of specific interest. It also assists in the correct use and conservation of these genetic resources and in the discovery of new biological properties from the exploration and study of the different compounds present in the species.