Yield and Chemical Composition of Essential oil of Piperaceae in one Segment of the Semi deciduous Forest of Paraná State, Brazil, in Seasonal Samplings

Essential oils are composed of a complex mixture of various classes of substances; among them are phenylpropanoid, monoterpenes, and sesquiterpenes, belonging to the secondary metabolism of plants. However, these compounds can be influenced by seasonal factors, among others. The objective of this study is to realize the prospect of obtaining Piperaceae with aromatic potential from a segment of the semideciduous forest of the Atlantic Forest Biome in the northwester region of Paraná State. The aim is to assess the qualitative and quantitative characteristics of its essential oil in the collection during the winter and summer seasons of year 2016. The statistical design was internally randomized in a factorial of 4 (species) x 3 (replicates). The species studied were Piper mosenii C. DC., Piper xylopioides Kunth, Piper diospyrifolium (Kunth) Kunth ex C. DC., and Piper gaudichaudianum Kunth. A total of 78 compounds were identified, 68 in winter and 71 in summer. The species presented a variation in the yield and composition of essential oils, both in winter and summer. The predominant chemical composition was sesquiterpenes followed by monoterpenes, with prominence of (E)-caryophyllene, germacrene D, bicyclogermacrene, α-pinene and βpinene. Keywords— Bioprospecting, genus Piper, protected areas, secondary metabolism, species aromatics.


INTRODUCTION
Brazil has a wide territorial area with a rich biodiversity, which provides a valuable source of plant species. Many of these are little studied and constitute a large biological collection for scientific research. The prospect of finding aromatic species in the forests of the Atlantic Forest biome, with its significant biodiversity, may represent the discovery of new essential oils with a potential for beneficial use, increasing the viability of sustainable management of this ecosystem, which is highly damaged, with a gerat need for conservation.
The plants are inexhaustible sources of natural products, many of them essential oils and secondary metabolites, mainly used in perfumery and cleaning products. They are also a source of active ingredients for the pharmaceutical industry (5; 33; 11). They can also be used in crop protection against pests and diseases, with the advantage of not accumulating in the environment and have a wide spectrum of action, which reduces the risk of developing resistant strains pathogenically (10).
More than 2000 plant species produce essential oils, among them are several representatives of the Piperaceae family, which possess high agronomic and commercial significance, as they are used as condiments, adornments, food, and in popular medicine. Some species of the genus Piper are also used in folk medicines, especially in Brazil, and many have proven to be of great significance due to the pharmacological activity and/or production of essential oils in their structure (13).
Piperaceae encompasses approximately 2,000 species allocated in approximately five genera. Just over 500 species are recorded in Brazil, distributed in four genera (30). The Piper genus is represented by 290 species and the Atlantic Forest is one of the centers of diversification and endemism of the genus, with about 150 species (14).
The production of essential oils in Brazil is still incipient to meet the demand. However, the national and international market has demonstrated a great interest in new essences; something that this biodiversity has a great potential to meet. In this context, the conservation units are an excellent laboratory for research and prospecting of essential oils. Various floristic studies were carried out at the Caiuá Ecological Station, North Diamond, Paraná State, however, phytochemical and biological studies with the aromatic native species in this forest formation are scarce, which makes this research highly relevant. The objective of this study is to evaluate, quantitatively and qualitatively, the essential oil in the seasonal samplings of Piperaceae, which is commonly found in a segment of the semideciduous forest of the Atlantic Forest Biome located in the Northwest of Paraná State. According to the Koeppen climate classification, the northwestern region of Paraná State presents the Cfa type climate -mesothermal, humid, without a dry season, and with hot summers. The average temperature of the coldest month is below 18ºC and the average temperature of the hottest month is above 22ºC. The average annual rainfall is 1,200 to 1,400 mm, being the quarter (December, January, and February) when it rains. The average annual temperature is between 21ºC and 22ºC, being the average of the hottest month (February) 24 to 25ºC and the coldest month (July) 17ºC to 18ºC. The relative humidity of the air average is 75%, (4).

II. MATERIAL AND METHODS
The formation of the majority of the soil in the Caiuá Ecological Station, is represented by soils derived from river sediments in portions adjacent to the Paranapanema River, with a predominance of Red Latosols, Red Argisols, Red-Yellow Argisols, and Quartzarenic Neosols, respectively (7). Its vegetation cover is inserted in the Atlantic Forest biome in the region of the Semideciduous Forest vegetation type, whose ecological concept is conditioned to the dual climate seasonality (15).
The studied species were Piper mosenii C. DC., Piper xylopioides Kunth, Piper diospyrifolium (Kunth) Kunth ex C. DC., and Piper gaudichaudianum Kunth species. These occurred commonly in the conservation unit.
The field work consisted of the collection of approximately 1 kg of plant material of each species (leaves and branch terminals) for extraction and quantification, and for determining the moisture content of the samples. The collection and transport of the plant material was prompted by the Environmental Institute of Paraná, under proper environmental authorization, with number 03/2016. The herbarium specimens were transported to the Botanical Museum Hall of Curitiba city where they were herborized and classified (Table 1). The identification of species was performed with the aid of specialized bibliographies, comparison of herbarium specimens declared at the herbarium, and consultation with experts on the respective groups of plants of these species.
The oil extraction was done by means of hydrodistillation during four-and-a-half hours in a graduated Clevenger apparatus using 100 g of fresh leaves and one liter of distilled water, with three repetitions.
The essential oils were diluted in hexane at a ratio of 1% and 1.0 μL of the solution where it was injected, with a split flow of 1:20, in an Agilent 6890 gas chromatograph coupled with a mass selective detector Agilent 5973N. The injector was maintained at 250°C. The separation of the constituents was obtained in a capillary column HP-5MS (5%-phenyl-95%-dimethyl polysiloxane, 30 m x 0.25 mm x 0.25 μm), using helium as a carrier gas (1.0 ml min -1 ). The temperature of the oven was scheduled to be 60°C to 240°C at a rate of 3°C For the quantification, the diluted samples were injected into the chromatograph Agilent 7890A equipped with flame ionization detector (FID), operated at 280°C. The same column and analytical conditions described above were employed, except for the carrier gas used, which was hydrogen, at a flow rate of 1.5 mL min -1 . The percentage composition was obtained by electronic integration of the signal of the FID by dividing the area of each component by the total area (area %).
The identification of the constituents was obtained by comparison of their mass spectra with thos e of (32) and (18) and also by their linear retention indices calculated from the injection of a homologous series of hydrocarbons (C7-C26) and compared with data from the literature (1).
The results were submitted to analysis of variance and the means of treatments were compared by the Tukey test at 5% probability, using the software SISVAR (9) and the principal component analysis (PCA) using the program BioEstat v.5.

III. RESULTS AND DISCUSSION
There was a significant difference in the essential oil content among species and at different seasons of collection ( Table 2). The species P. xylopioides presented an average yield of oil statistically superior to others, and in the summer this content was higher. P. mosenii, P. gaudichaudianum, and P. diospyrifolium showed similar levels of oil in winter, however, in the summer these species differed in the levels among them, whereas, P. xylopioides presented the highest content followed by P. diospyrifolium and P. gaudichaudianum. P. mosenii did not produce oil in the summer contrary to the study of (24) conducted on the coast of Paraná State, where the species showed a low variation in the essential oil content in the winter, spring, and summer seasons.  23.54 * Medium followed by the same letter in column and capitalized on the line did not differ s tatistically among themselves by Tukey test at 5% probability.
The species P. mosenii and P. gaudichaudianum presented an essential oil yield that was higher in winter. As stated, P. xylopioides and P. diospyrifolium presented higher yield in summer.
The study on P. gaudichaudianum performed by (23) in a population in Santa Maria, Rio Grande do Sul State, found an average content of oil from fresh leaves of 0.38%, superior to that found in the present study, but without the effect of seasonality.
The chemical composition of essential oils is generally a characteristic of a given species and from the point of view of the chemical composition it is genetically and epigenetically controlled. The quantity, quality, and concentration of these species are influenced by the environmental components. Among the environmental factors that can be highlighted are, light intensity and photoperiod, the latitude, temperature (minimum and maximum average), soil (chemical and physical properties), wind, and the availability of water, or even a combination of some of these subfactors and seasonality (26).
Studies conducted by (24) on the Piper genus in the Atlantic Forest, on the coast of Paraná, showed the influence of seasonality on the yield and the constituents of essential oils. (16), (27), (29), (17), (21), (20), and (19), also identified the influence of seasonality on the chemical profile of the oils analyzed.
The chemical composition of the essential oil of the studied species identified 78 constituents, corresponding to an average of 90% of chemical compounds of the essential oil, in the identified samples ( Table 3). The species P. diospyrifolium was the one that presented the highest number of compounds identified, with 55, followed by P. gaudichaudianum with 50, P. xylopioides with 35, and P. mosenii with 33 compounds.           The chemical composition of the species P. mosenii identified during the winter had an average of 57.58% of sesquiterpenes hydrocarbon, 36.36% of oxygenated sesquiterpenes, and 3.03% of monoterpenes and phenylpropanoids. A majority of the constituents of samples performed was identified as germacrene D (30.36%), (E)-caryophyllene (16.39%), and biciclogermancrene (13.46%), with the total compounds identified as 94.89%. During the summer an yield of oil was not observed for that species.
The chemical composition of essential oils is determined by genetic factors, however, according to (17), other factors may cause significant changes in the production of secondary metabolites. In fact, the secondary metabolites represent a chemical interface between plants and the environment. The stimuli arising from the environment in which the plant is located can redirect the metabolic pathway, causing a biosynthesis of different compounds. Among these factors, we can highlight the interactions between plant/microbial, plant/insect, and plant/plant; age and stage of development, abiotic factors such as brightness, temperature, rainfall, nutrition, time, and time of collection, as well as techniques during harves t and postharvest. It is valid to note that these factors can present correlations between themselves and not act in isolation. They can exercise joint influence on secondary metabolism, which causes a variation in the income and composition of the essential oil analyzed.
The phytochemical composition of the essential oil from samples of P. xylopioides collected during the winter season showed a proportion of 53.12% of sesquiterpenes hydrocarbon, 21.88% of oxygenated
To determine the degree of variations in the phytochemical, a principal component analysis (PCA) was performed using a correlation matrix of all chemical compounds (Table 4 and Fig. 2).

International Journal of Advanced Engineering Research and Science (IJAERS)
[  Results obtained by PCA, based on 11 chemical compounds, are shown in Figure 2 and Table 4. The three factors explain 100% of the accumulated variation in the data; the first two factors being considered the most important, as they described 90.08% of the accumulated variance ( Table 4). The compounds germacrene D, γcadinene, and ß-elemene, demonstrate the relevant contributions, with 56.64% of the variation in the principal components (PC 1). Bicyclogermacrene, germacrene D, and (E)-nerolidol are compounds that contributed, by explaining 33.44% of the variance of principal components (PC 2).
Differences in chemical constituents can be justified by the regulation of gene expression of the enzymes involved in the biosynthetic route of terpenes. In addition, climatic conditions (collections at different seasons) contributed to the chemical characterization of the essential oil of the species analyzed.

IV. CONCLUSION
The results presented here demonstrate that the environmental factor of seasonality interfered with the levels and the average percentage of the chemical constituents of essential oils.
The studied species of genus Piper can be distinguished into three groups per workstation, in accordance with the composition of the essential oil of fresh leaves.