Microsoft Word - 10-Agra_39372 629 Original Article Biosci. J., Uberlândia, v. 34, n. 3, p. 629-639 , May/June 2018 DISTILLATION METHODS AFFECT THE CHEMICAL COMPOSITION OF Varronia curassavica Jacq. ESSENTIAL OIL? MÉTODOS DE DESTILAÇÃO AFETAM A COMPOSIÇÃO QUÍMICA DO ÓLEO ESSENCIAL DE Varronia curassavica Jacq? Daniela Aparecida de Castro NIZIO 1 ; Arie Fitzgerald BLANK 1 ; Taís Santos SAMPAIO 2 ; Fabiany de Andrade BRITO 1 ; Thiago Matos ANDRADE 1 ; Maria de Fátima ARRIGONI-BLANK 1 ; Alexandre Nizio MARIA 3 1. Departamento de Engenharia Agronômica, Universidade Federal de Sergipe, São Cristóvão, SE, Brasil, danielanizio82@gmail.com; 2. Departamento de Química, Universidade Federal de Sergipe, São Cristóvão, SE, Brasil; 3. Laboratório de Reprodução Animal, Embrapa Tabuleiros Costeiros, Aracaju, SE, Brasil. ABSTRACT: The objective of this work was to evaluate the chemical composition of essential oil from Varronia curassavica Jacq. obtained by microwave (MI) and hydrodistillation (HD) extraction methods. The MI method tested three powers (500, 600, and 700W), three distillation times (20, 30, and 40 min.), and three water volumes (0, 25, and 50 mL per sample). The HD method tested three distillation times (100, 120, and 140 min.) and three water volumes (1.0, 1.5, and 2.0 L per 3-liter flask). The essential oils were analyzed by GC/MS-FID. The optimal condition for the essential oil extraction by the MI method was 700W for 40 min. (3.28%), regardless of the volume of water. In its turn, the best condition for essential oil extraction by the HD method was 120 min. with 1.0 L of water per flask (3.34%). The most abundant compounds for MI (700 W for 40 min. without water) were shyobunol (26.53%) and bicyclogermacrene (4.96%); and the most abundant compounds for HD (120 min. with 1.0 L of water/flask) were shyobunol (24.00%) and germacrene D-4-ol (10.23%). Methyl farnesoate (2E, 6E) and farnesyl acetate (2Z, 6E) were not detected in the essential oil extracted by HD; however, they were identified by the MI method. By increasing the distillation time and/or volume of water in HD, a reduction was observed for the content of the chemical compounds β-elemene (from 1.23 to 0.97%), E- caryophyllene (from 5.49 to 4.35%), α-humulene (from 1.80 to 1.43%), alloaromadendrene (from 1.78 to 1.44%), bicyclogermacrene (from 5.63 to 4.55%), and germacrene D-4-ol (from 11.40 to 9.86%). Power, extraction time, and their interactions influenced the content of essential oil obtained by microwave extraction (MI). Within each power, the highest essential oil content was extracted at the longest distillation time (40 min.), except for 600W, where no significant difference was detected between 30 and 40 min. The optimal essential oil contents for both extraction methods were statically similar by the t-test for dependent samples. However, the MI method presents advantages, such as shorter distillation time and less energy and water consumption. KEYWORDS: Microwave distillation/ Volatile oil/ Chemical compounds INTRODUCTION The medicinal and aromatic plant Varronia curassavica Jacq. (syn. Cordia verbenacea DC.), known as “erva-baleeira” in Brazil, belongs to the family Cordiaceae (GASPARINO; BARROS, 2009) and is largely distributed in Brazil. Several properties of the species, such as anti-inflammatory, antiucer, antiallergic, antitumor, and antioxidant are mainly attributed to the compounds α-humulene and E-caryophyllene, present in its essential oil (FERNANDES et al., 2007; PASSOS et al., 2007; MEDEIROS et al., 2007; ROGÉRIO et al., 2009; OLIVEIRA et al., 2011; MICHIELIN et al., 2011; PARISSOTO et al., 2012; PIMENTEL et al., 2012). Other biological activities, such as antibacterial (MECCIA et al., 2009; MATIAS et al., 2010; PINHO et al., 2012; RODRIGUES et al., 2012) and antifungal activities (SILVA et al., 2012; HOYOS et al., 2012; SILVA et al., 2014) were reported in studies involving the essential oil of V. curassavica. In the pharmaceutical industry, the essential oil of V. curassavica is used to make a phytotherapeutic medicine for inflammation treatment. Essential oils consist of a complex mixture of several chemical compounds with wide variation in their composition, and most of the compounds are terpenes. The chemical composition of essential oils is influenced by plants genetics, environmental factors (irrigation, harvest time, season, and others), genotype x environment interaction, and processing parameters (e.g., temperature, drying time of the plant material, and essential oil extraction method) (VERMA et al., 2010; GONZÁLEZ-RIVERA et al., 2016). The extraction method is one of the primary factors to determine the quality of an essential oil. The improper extraction process can result in changes in the chemical composition of the essential oil, leading to the loss of its bioactivity and natural Received: 15/08/17 Accepted: 20/02/18 630 Distillation methods... NIZIO, D. A. C. et al Biosci. J., Uberlândia, v. 34, n. 3, p. 629-639 , May/June 2018 characteristics, such as color, flavor, and viscosity (TONGNUANCHAN; BENJAKUL, 2014). Among the laboratory methods used to extract essential oils from plants, hydrodistillation (HD) is the most frequently applied. However, it presents disadvantages, such as long extraction time, high demand for energy and water, and overheating of plant material, causing the loss of bioactive compounds of the essential oil by thermal degradations (RANITHA et al., 2014; CHEN et al., 2016). New techniques are currently available, such as microwave-assisted extraction (MI). This technique allows the plant material to reach its boiling point quickly, resulting in a shorter extraction time and with less energy expenditure (KHANAVI et al., 2013; LI et al., 2014). In this sense, several works have been carried out aiming to improve the conditions of microwave-assisted extraction for several medicinal and commercial species, such as Mentha spp. (COSTA et al., 2014; GAVAHIAN et al., 2015); Rosmarinus officinalis (KARAKAYA et al., 2014); Eucalyptus camaldulensis (LI et al., 2016); Cinnamomum cassia (CHEN et al., 2016), among others. The objective of this work was to evaluate the content (%) and chemical composition of essential oil from V. curassavica obtained by the hydrodistillation (HD) and microwave (MI) extraction methods. MATERIAL AND METHODS For essential oil extraction, leaves were harvested from plants of Varronia curassavica Jacq. (accession VCUR-201), maintained at the Active Germplasm Bank (AGB) of aromatic plants of the Federal University of Sergipe, located at the Research Farm "Campus Rural da UFS". Fresh leaves from the entire plants were collected, weighted, separated for essential oil extraction, maintained in a freezer at -20°C, until the beginning of the experiments. The hydrodistillation was performed in a modified Clevenger apparatus using 3-liter flasks and samples of 100g of fresh leaves. The experiment consisted of a completely randomized design, in a 3x3 factorial scheme, using three replications. Three distillation times (100, 120, and 140 minutes of boiling) and three volumes of filtered water (using osmose reverse filter system) (1.0, 1.5, and 2.0 L per flask) were tested. The essential oils were dried over anhydrous sodium sulfate and stored in amber vials at -20oC, until chemical composition analysis. The essential oil content was calculated using the mean dry weight of six samples of 100 g of fresh leaves at 100oC in a forced-air-circulation oven until constant weight, for 48 hours. The essential oil content was calculated according to the following equation: Content (%, v/w) = (volume of essential oil extracted from the sample/mean of dry weight of leaves) x 100. The microwave-accelerated reaction system (NEOS, Milestone, Italy) equipped with a circulating water-cooling system was utilized for the experiment. Samples of 50 g of fresh leaves were used. The experiment consisted of a completely randomized design, in a 3x3x3 factorial scheme, with three replications. Three powers (500, 600, and 700 W), three extraction times (20, 30, and 40 minutes), and three volumes of filtered water (using osmose reverse filter system) (0, 25, and 50 mL per sample) were tested. The essential oils were dried using anhydrous sodium sulfate and stored in amber vials at -20 °C until chemical composition analysis. The same procedure described for hydrodistillation was applied to calculate the essential oil content. The analysis of the chemical composition of the essential oils was performed using a GC- MS/FID (QP2010 Ultra, Shimadzu Corporation, Kyoto, Japan) equipped with an autosampler AOC- 20i (Shimadzu). Separations were accomplished using an Rtx®-5MS Restek fused silica capillary column (5%-diphenyl–95%-dimethyl polysiloxane) of 30 m × 0.25 mm i.d., 0.25 mm film thickness, at a constant helium (99.999%) flow rate of 1.2 mL.min- 1. An injection volume of 0.5 µL (5 mg.mL-1) was employed, with a split ratio of 1:10. The oven temperature was programmed from 50oC (isothermal for 1.5 min), with an increase of 4 oC.min-1, to 200 oC, then 10 oC.min-1 to 250 oC, ending with a 5 min isothermal at 250 oC. The MS and FID data were simultaneously acquired by 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 obtained in the full scan mode (m/z of 40–350) at a 0.3 scan/s scan rate, using the electron ionization (EI) with an electron energy of 70 eV. The injector temperature was 250 oC, and the ion-source temperature was 250 oC. 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-1, respectively. Quantification of each constituent was 631 Distillation methods... NIZIO, D. A. C. et al Biosci. J., Uberlândia, v. 34, n. 3, p. 629-639 , May/June 2018 estimated by FID peak-area normalization (%). Compound concentrations were calculated from the GC peak areas and arranged in order of GC elution. The identification of individual components of the essential oil was performed by computerized matching of the acquired mass spectra with those stored in the NIST21, NIST107, and WILEY8 mass spectral library of the GC-MS data system. A mixture of hydrocarbons (C9H20–C19H40) was injected under these same conditions. Compounds were identified by comparing the spectra obtained with those of the equipment’s data bank and by the Kovats index, calculated for each compound, as previously described (ADAMS, 2007). Retention indices were obtained using the equation proposed by Van den Dool and Kratz (1963). Essential oil content and chemical composition were subject to analysis of variance (ANOVA), and the means were compared by the Scott-Knott test (p<0.05), using the Sisvar® software. The means of the content of the best hydrodistillation (HD) and microwave (MI) treatments were compared by the t-test for dependent samples (p<0.05), using the Statistica 7.0 software. RESULTS The optimal condition for essential oil extraction by the MI method was 700W for 40 min. (3.28%), regardless of the volume of water. In its turn, the best condition for essential oil extraction by the HD method was 120 min. with 1.0 L of water per flask (3.34%). (Tables 1 and 2). The most abundant compounds for MI (700W for 40 min. without water) were shyobunol (26.53%) and bicyclogermacrene (4.96%); and the most abundant compounds for HD (120 min. with 1.0 L of water/flask) were shyobunol (24.00%) and germacrene D-4-ol (10.23%) (Tables 1 and 2). Most of the compounds presented significant distillation time x water volume interaction for HD. The increase in the distillation time and/or volume of water reduced the content of the chemical compounds β-elemene (from 1.23 to 0.97%), E-caryophyllene (from 5.49 to 4.35%), α- humulene (from 1.80 to 1.43%), alloaromadendrene (from 1.78 to 1.44%), bicyclogermacrene (from 5.63 to 4.55%), and germacrene D-4-ol (from 11.40 to 9.86%) (Table 1). The content of germacrene D-4-ol reduced when distillation time was increased. However, the largest volume of water per flask resulted in higher contents, reaching the maximum (12.55%) with 100 minutes distillation time and 2.0 L of water per flask. The contents of the compounds spathulenol, ledol, epi-α-murulol, and shyobunol increased with the increase in the volume of water and/or distillation time (Table 1). Power, extraction time, and their interactions influenced the essential oil content obtained by microwave extraction (MI). Within each power, the highest essential oil content was obtained at the longest extraction time (40 min.), except for 600W, where no significant difference was recorded between 30 and 40 minutes. The maximum essential oil content (3.28%) was observed using the power of 700W for 40 min., regardless of the volume of water/sample (Table 2). Methyl farnesoate (2E, 6E) and farnesyl acetate (2Z, 6E) were not detected in the essential oil extracted by HD. However, they were detected by the MI extraction method. The extraction time of 40 min. used in the solvent-free MI distillation together with the highest power (700W) resulted in an increased content of β- elemene (0.99%), E-caryophyllene (3.97%), α- humulene (1.44%), alloaromadendrene (1.65%), bicyclogermacrene (4.96%), ledol (4.00%), epi-α- murulol (4.06%), and farnesyl acetate (2Z, 6E) (3.59%) (Table 2). Only the content of alloaromadendrene (1.34%), ledol (4.10%), and farnesyl acetate (2Z, 6E) (3.60%) increased with the addition of 50 mL of water per sample to the MI extraction system (Table 2). The extraction time of 40 min. in the solvent-free MI distillation together with the lowest power (500W) resulted in an increased content of germacrene D-4-ol (4.24%), caryophyllene oxide (2.24%), cubenol (2.17%), shyobunol (27.35%), and methyl farnesoate (2E, 6E) (3.51%) (Table 2). Only the content of germacrene D-4-ol (5.51%) and methyl farnesoate (2E, 6E) (3.11%) increased with the addition of 50 mL of water per sample to the MI extraction system (Table 2). The means of essential oil contents obtained in the best HD (120 min. with 1.0 L of water per flask) and MI (700W for 40 min) treatments were statistically similar (p<0.05) when compared by the t-test for dependent samples. 632 Distillation methods... NIZIO, D. A. C. et al Biosci. J., Uberlândia, v. 34, n. 3, p. 629-639 , May/June 2018 Table 1. Content and chemical composition of the essential oil from Varronia curassavica extracted by hydrodistillation (HD), according to the extraction time and water volume. Compounds RRI-o RRI-l Time (min.) Water (L/flask) 1.0 1.5 2.0 Content (%) of chemical constituents 100 1.23aA 1.15aA 1.18aA β-elemene 1389 1389 120 1.21aA 1.11aB 1.10bB 140 1.14bA 1.09aA 0.97cB 100 5.49aA 5.14aB 5.20aB E-caryophyllene 1422 1417 120 5.44aA 4.98aB 4.87bB 140 4.98aA 4.92aA 4.35cB 100 1.80aA 1.71aB 1.7aB α-humulene 1457 1452 120 1.76aA 1.64aB 1.61bB 140 1.63bA 1.61bA 1.43cB 100 1.78aA 1.66aB 1.67aB alloaromadendrene 1465 1458 120 1.74aA 1.63aB 1.56bB 140 1.69aA 1.69aA 1.44cB 100 5.63aA 5.27aB 5.44aB bicyclogermacrene 1499 1500 120 5.31bA 5.20aA 4.91bB 140 5.01cA 5.00aA 4.55cB 100 11.40aB 11.70aB 12.55aA germacrene D-4-ol 1580 1574 120 10.23bC 11.09bB 11.84bA 140 9.86bB 9.90cB 10.36cA 100 1.16aB 1.20aB 1.32aA sphatulenol 1583 1577 120 1.10aA 1.19aA 1.21bA 140 1.11aA 1.14aA 1.13bA 100 1.34aA 1.34aA 0.97bB caryophyllene oxide 1592 1582 120 1.36aA 1.34aA 1.34aA 140 1.32aA 1.35aA 1.34aA 100 3.71aB 3.65bB 4.50aA ledol 1612 1602 120 3.69aB 3.88aA 3.93bA 140 3.67aB 3.67bB 3.88bA 100 2.76bB 2.90bB 3.90aA epi-α-murulol 1646 1640 120 3.00bB 4.10aA 3.97aA 140 3.63aB 3.94aA 3.92aA 100 1.82aA 1.86aA 1.75bA cubenol 1650 1645 120 1.85aA 2.00aA 1.89bA 140 1.98aA 2.06aA 2.22aA 100 4.56aA 4.68aA 4.83bA α-cadinol 1660 1652 120 4.91aA 4.93aA 5.33aA 140 4.94aA 5.41aA 5.63aA 100 24.24bA 23.90bA 22.95cB shyobunol 1705 1709 120 24.00bA 24.14bA 24.24bA 140 24.93aB 24.81aB 25.58aA 100 2.95aA 3.10aA 1.67cB methyl farnesoate (2E, 6E) 1776 1783 120 2.25bB 2.91aA 2.88aA 140 2.86aA 2.77aA 2.24bB 100 2.93bA 2.93bA 2.43cB essential oil content (%) --- --- 120 3.34aA 3.03bB 2.93bB 140 3.54aA 3.44aA 3.23aA RRI-o: Relative Retention Index - observed; RRI-l: Relative Retention Index - literature. Means followed by the same lowercase letter in the column and uppercase letter in the row do not differ by the Scott Knott test (P <0.05). 633 Distillation methods... NIZIO, D. A. C. et al Biosci. J., Uberlândia, v. 34, n. 3, p. 629-639 , May/June 2018 Table 2. Content and chemical composition of the essential oil from Varronia curassavica obtained by solvent-free microwave extraction (MI), according to the tested power, extraction time, and water volume. Compounds RRI-o RRI-l Power (W) Water (mL/sample) 0 25 50 Time (min.) 20 30 40 20 30 40 20 30 40 Content (%) of chemical compounds 500 1.05bAβ 1.01bAα 0.96aBα 1.00bAβ 0.89cBβ 0.87cBβ 1.20aAα 0.83cBβ 0.87aBβ β-elemene 1389 1389 600 1.21aAα 1.12aBα 0.87bCβ 1.19aAα 1.13aAα 1.03aBα 1.08bAβ 1.02aAβ 0.93aBβ 700 1.25aAα 1.02bBα 0.99aBα 1.03bAβ 1.03bAα 0.94bBα 1.00cAβ 0.93bAβ 0.93aAα 500 4.40aAγ 3.55cBα 3.43bBα 3.78bAβ 2.87bBβ 2.92bBβ 5.24aAα 2.60bCγ 3.08aBβ E-caryophyllene 1422 1417 600 4.45aAα 4.19aBα 2.99cCβ 4.25aAα 3.82aBβ 3.59aCα 3.82bAβ 3.51aBγ 3.19aCβ 700 4.59aAα 3.91bBα 3.97aBα 3.96bAβ 3.68aBβ 3.46aBβ 3.67bAγ 3.35aBγ 3.12aCγ 500 1.49bAβ 1.30cBα 1.29bBα 1.36cCγ 1.14bBβ 1.15bBβ 1.75aAα 1.06bCγ 1.19aBβ α-humulene 1457 1452 600 1.58bAα 1.51aBα 1.19cCβ 1.56aAα 1.45aBα 1.36aCα 1.42bAβ 1.37aAβ 1.25aBβ 700 1.68aAα 1.39bBα 1.44aBα 1.44bAβ 1.40aAα 1.30aBβ 1.35cAγ 1.31aAβ 1.21aBγ alloaromadendrene 500 1.65cAα 1.43bBα 1.45bBα 1.44cAβ 1.25bBβ 1.25bBβ 1.71aAα 1.11cCγ 1.26bBβ 1465 1458 600 1.75bAα 1.65aBα 1.35cCβ 1.71aAα 1.54aBβ 1.52aBα 1.52bAβ 1.51aAβ 1.40aBβ 700 1.92aAα 1.59aBα 1.65aBα 1.62bAβ 1.57aAα 1.45aBβ 1.45cAγ 1.44bAβ 1.34aBγ 500 5.08cAβ 4.52bBα 4.59bBα 4.70cAγ 4.25bBβ 4.20cBβ 5.31aAα 4.04cCγ 4.22bBβ bicyclogermacrene 1499 1500 600 5.41bAα 5.07aBα 4.41cCβ 5.31aAα 4.88aBβ 4.79aBα 4.88bAβ 4.71aBγ 4.43aCβ 700 5.67aAα 4.94aBα 4.96aBα 5.07bAβ 4.82aBα 4.50bCβ 4.78bAγ 4.50bBβ 4.21bCγ germacrene D-4-ol 500 4.09aBγ 4.29aAγ 4.24aAγ 4.31bCβ 4.48aBβ 4.77aAβ 11.86aAα 5.09aCα 5.51aBα 1580 1574 600 4.03aAγ 3.89bBγ 4.02bAγ 4.56aAβ 4.34bBβ 4.53bAβ 4.83bAα 4.75bAα 4.77bAα 700 3.93aAγ 3.91bAγ 3.60cBγ 4.03cBβ 4.21cAβ 4.03cBβ 4.70cAα 4.62cBα 4.73bAα caryophyllene oxide 500 2.45aAα 2.34aBα 2.24aCα 2.49aAα 2.14bBβ 2.20aBα 1.39bCβ 2.09aBβ 2.20aAα 1592 1582 600 2.46aAα 2.18bCα 2.28aBα 2.37bAβ 1.96cCβ 2.12bBβ 2.14aAγ 2.12aAα 2.02bBγ 700 2.28bAα 2.22bAβ 1.97bBβ 2.10cBγ 2.30aAα 2.04cBβ 2.17aAβ 2.06aAγ 2.17aAα ledol 500 3.78bAβ 3.38bBβ 3.47bBα 3.71cAβ 3.79bAα 3.63aAα 4.01bAα 3.72bBα 3.62bBα 1612 1602 600 3.62bBβ 4.03aAβ 3.98aAα 4.01bAα 3.93bAβ 3.86aAα 3.83bBα 4.30aAα 4.11aAα 700 4.22aAα 3.95aBβ 4.00aBα 4.26aAα 4.30aAα 3.87aBα 4.33aAα 4.10aAβ 4.10aAα 500 2.79bBα 2.88bBγ 3.50bAβ 2.86bCα 4.16aAα 3.82aBα 2.98bBα 3.89aAβ 3.80aAα epi-α-murulol 1646 1640 600 3.41aBα 3.67aAα 3.89aAα 3.58aAα 3.73bAα 3.68aAα 3.71aAα 3.69aAα 3.75aAα 700 3.60aBα 3.55aBα 4.06aAα 3.64aAα 3.79bAα 3.57aAβ 3.67aAα 3.75aAα 3.87aAβ 634 Distillation methods... NIZIO, D. A. C. et al Biosci. J., Uberlândia, v. 34, n. 3, p. 629-639 , May/June 2018 Continuation of table 2: 500 1.63aBα 1.99aAα 2.17aAα 1.87aAα 1.77aAα 2.10aAα 1.93aAα 1.96aAα 1.57bBβ cubenol 1650 1645 600 1.90aAα 1.68bAα 2.02aAα 1.52bBβ 1.78aAα 1.89aAα 1.64aAβ 1.74aAα 1.86aAα 700 1.67aAα 2.07aAα 1.69bBβ 1.53bBα 1.70aBβ 2.09aAα 1.78aAα 1.71aAβ 1.90aAα 500 4.54aAα 4.51aAα 4.54aAα 4.17aAα 4.55aAα 4.26aAα 4.67aAα 4.62aAα 4.79aAα α-cadinol 1660 1652 600 3.84aBβ 4.49aAα 4.93aAα 4.55aAα 4.44aAα 4.31aAβ 4.01aBβ 4.62aAα 4.82aAα 700 4.50aAα 4.59aAα 4.28aAα 4.79aAα 4.24aAα 4.48aAα 4.19aAα 4.49aAα 4.60aAα 500 26.47aBβ 26.92aAβ 27.35aAα 27.30aBα 28.43aAα 27.84aBα 23.53bCγ 29.07aAα 27.45aBα shyobunol 1705 1709 600 26.11aBβ 25.88bBβ 27.22aAα 25.50bBβ 26.68cAα 26.91bAα 26.98aAα 26.40cBα 27.45aAα 700 25.20bBβ 26.11bAβ 26.53bAβ 27.34aAα 27.49bAα 27.12bAα 27.03aAα 27.71bAα 27.46aAα methyl farnesoate (2E, 6E) 500 3.94aBα 4.55aAα 3.51aCα 3.33aBβ 3.67aAβ 3.19aBβ 1.79cCγ 3.77aAβ 3.11aBβ 1776 1783 600 3.63aAα 3.83bAα 2.85bBα 2.44bBγ 3.28bAβ 2.51bBβ 2.85aAβ 1.70bBγ 1.84bBγ 700 1.63bAβ 1.54cAα 1.78cAα 1.38cBβ 1.80cAα 2.10cAα 2.14bAα 1.63bBα 1.77bBα farnesyl acetate (2Z, 6E) 500 2.50cAβ 2.16cBα 2.81bAα 2.83cAα 2.47bAα 2.72cAα 0.00cCγ 2.03bBβ 2.40bAβ 1824 1821 600 2.82bBβ 2.69bBβ 3.56aAα 3.19bAα 2.48bBβ 3.24bAβ 2.70bBβ 3.64aAα 3.69aAα 700 4.34aAα 3.83aBα 3.59aBα 4.14aAα 3.61aBα 3.65aBα 3.33aBβ 3.83aAα 3.60aAα essential oil content (%) 500 1.65bCα 2.05bBα 2.69bAα 1.45cCα 2.07bBα 2.71bAα 1.52cCα 2.09cBα 2.58cAα --- --- 600 2.20aBα 2.86aAα 2.48bBβ 2.21bBα 2.88aAα 2.90bAα 2.08bCα 2.50bBα 3.00bAα 700 2.42aBα 2.90aBα 3.28aAα 2.71aBα 2.90aBα 3.36aAα 2.71aBα 2.95aBα 3.43aAα RRI-o: Relative Retention Index - observed; RRI-l: Relative Retention Index - literature. Means followed by the same lowercase letter in the column, uppercase letter in the row and Greek letter between water volumes (for the same time and power) do not differ by the Scott Knott test (P <0.05). 635 Distillation methods... NIZIO, D. A. C. et al Biosci. J., Uberlândia, v. 34, n. 3, p. 629-639, May/June 2018 DISCUSSION The use of smaller volumes of water in the extraction by HD provided the highest essential oil content. The addition of smaller volumes of water to the flasks allows more space for the movement of water vapor and essential oil during distillation, which favors higher essential oil condensing in the modified Clevenger apparatus. The compounds sesquiterpene hydrocarbons were observed in higher contents at a shorter distillation time and/or smaller volumes of water in the HD method. The increase in the extraction time led to a greater loss of sesquiterpene hydrocarbons compounds by volatilization and degradation due to the high temperature or hydrolysis (SOZMEN et al., 2011; LI et al., 2012; QI et al., 2014). Conversely, the oxygenated sesquiterpenes presented the highest contents when increasing the extraction time and/or volumes of water. Due to the lower volatility of these compounds, they are lost in a lesser amount when compared with the sesquiterpenes hydrocarbons during the extraction process. The extraction time and power were the most important factors in the essential oil extraction of V. curassavica by MI. Similarly, Shah and Garg (2014) reported that the longest time (30 min.) and higher power (640W) provided greater essential content in a study performed with ginger. According to Chen et al. (2011), the increase of power accelerates the transfer of mass and increases the essential oil content. For the species Ocimum basilicum and Chenopodium ambrosioides, time and water factors were more significant than power for the essential oil content obtained by MI. Similarly to the present study for HD, Cardoso-Ugarte et al. (2013) reported that the longer the time of extraction and the lower the volumes of water, the greater were the essential oil contents. In their work, the authors used an adapted household microwave and larger volumes of water, which explains the resemblance of their results with that observed for the HD method in the current study. A higher number of compounds was detected in the essential oil of V. curassavica extracted by the MI method when compared with that revealed by the HD method. The same was observed in Ferulago campestres (RIELA et al., 2011). The plant material heated by microwaves overheat inside the cell, which causes the expansion and rupture of the cell walls and consequently allows extracting the essential oil and less volatile compounds more efficiently (CHEN et al., 2011). The significant interaction between the studied factors when using extraction by MI, for most compounds, showed that these factors cause quantitative variation in the chemical composition of the essential oil of V. curassavica. Similar germacrene D-4-ol content was observed between the essential oils extracted by HD and MI, where higher contents were reached when applying the shortest time and greater volumes of water. E- caryophyllene and α-humulene presented similar behavior regarding the time, where higher contents were observed at shorter extraction times when using both methods. In a study carried out with clove (Syzygium aromaticum L.), a higher content of E-caryophyllene (24.8%) and α-humulene (3.1%) were obtained by the MI method when compared with the hydrodistillation method (5.1 and 0.6%, respectively) (GONZÁLEZ-RIVERA et al., 2016), probably due to the shorter time and lower volume of water used in the microwave-assisted extraction. This study revealed that both methods (HD and MI) were effective in extracting the essential oil of V. curassavica. The chemical composition and essential oil content of this species are influenced by the extraction method used. The optimal essential oil contents for the two extraction methods were similar. However, the new technique of microwave- assisted extraction (MI) has some advantages, such as shorter distillation time and lower energy and water consumption. The use of 700 W for 40 min. (without water) for MI extraction and 120 min. with 1.0 L of water per flask for HD can be recommended to obtain high essential oil content of V. curassavica. ACKNOWLEDGMENTS The authors thank CNPq, FAPITEC/SE, CAPES, FINEP, and RENORBIO for their financial support of this work. RESUMO: O objetivo deste trabalho foi avaliar a composição química do óleo essencial de Varronia curassavica Jacq. obtido pelos métodos de extração micro-ondas (MI) e hidrodestilação (HD). Para MI, foram testadas três potências (500, 600 e 700W), três tempos de destilação (20, 30 e 40 min.) e três volumes de água (0, 25 e 50 mL por amostra). Para HD, foram testados três tempos de destilação (100, 120 e 140 min.) e três volumes de água (1,0; 1,5 e 2,0 L por balão de 3 litros). Os óleos essenciais foram analisados por CG/EM-FID. Maiores teores de óleo essencial foram obtidos nas condições de 700 W por 40 min. (3.28%), independente do volume de água para MI, e 120 min. com 1,0 L de 636 Distillation methods... NIZIO, D. A. C. et al Biosci. J., Uberlândia, v. 34, n. 3, p. 629-639, May/June 2018 água por balão para HD (3,34%). Os compostos mais abundantes para MI (700W, por 40 min., sem água) foram o shyobunol (26,53%) e biciclogermacreno (4,96%) e para HD (120 min. com 1,0 L de água /balão) foram shyobunol (24,00%) e germacreno D -4 -ol (10,23%). Metil farnesoato (2E, 6E) e farnesil acetato (2Z, 6E) não foram detectados no óleo essencial extraído por HD, porém, foram detectados nas amostras extraídas por MI. Com o aumento do tempo de destilação e/ou do volume de água em HD, houve redução no conteúdo dos constituintes químicos β-elemeno (de 1,23 para 0,97%), E-cariofileno (de 5,49 para 4,35%), α-humuleno (1,80 para 1,43%), aloaromadendreno (de 1,78 para 1,44%), biciclogermacreno (de 5,63 para 4,55%) e germacreno D-4-ol (de 11,40 para 9,86%). A potência, o tempo de extração e suas interações influenciaram no teor de óleo essencial obtido na extração por micro-ondas (MI). Dentro de cada potência, o maior teor de óleo essencial foi obtido no tempo mais longo de extração (40 min.), exceto para 600 W, que não apresentou diferença significativa entre 30 e 40 min. Nas condições ótimas de extração, os teores de óleo essencial obtidos foram estatisticamente semelhantes pelo teste t para amostras dependentes. No entanto, a extração por micro-ondas apresenta algumas vantagens em relação a HD, como menor tempo de destilação e menor consumo de energia e água. PALAVRAS-CHAVE: Varronia curassavica. Óleos voláteis. Destilação por micro-ondas. REFERENCES ADAMS, R. P. Identification of essential oil components by gas chromatography/mass spectroscopy. 4th ed., Allured: Carol Stream, 2007. 804p. CARDOSO-UGARTE, G. A.; JUÁREZ-BECERRA, G. P.; SOSA-MORALES, M. E.; LÓPEZ-MALO, A. Microwave-assisted extraction of essential oils from herbs. Journal of Microwave Power and Electromagnetic Energy, Mechanicsville, v. 47, n. 1, p. 63-72, mar. 2013. http://dx.doi.org/10.1080/08327823.2013.11689846 CHEN, F.; DUA, X.; ZU, Y.; YANG, L.; WANG, F. Microwave-assisted method for distillation and dual extraction in obtaining essential oil, proanthocyanidins and polysaccharides by one-pot process from Cinnamomi Cortex. Separation and Purification Technology, Delft, v. 164, p. 1–11, mar. 2016. https://doi.org/10.1016/j.seppur.2016.03.018 CHEN, X.; ZHANG, Y.; ZU, Y.; FU, Y.; WANG, W. Composition and biological activities of the essential oil from Schisandra chinensis obtained by solvent-free microwave extraction. Food Science and Technology, Zurique, v. 44, p. 2047-2052, dec. 2011. https://doi.org/10.1016/j.lwt.2011.05.013 COSTA, S. S.; GARIEPY, Y.; ROCHA, S. C. S.; RAGHAVAN, V. Microwave extraction of mint essential oil – Temperature calibration for the oven. Journal of Food Engineering, Pullman, v. 126, p. 1-6, apr. 2014. https://doi.org/10.1016/j.jfoodeng.2013.10.033 FERNANDES, E. S.; PASSOS, G. F.; MEDEIROS, R.; CUNHA, F. M.; FERREIRA, J.; CAMPOS, M. M.; PIANOWSKI, L. F.; CALIXTO, J. B. Anti-inflammatory effects of compounds alpha-humulene and (-)-trans- caryophyllene isolated from the essential oil of Cordia verbenacea. European Journal of Pharmacology, Amsterdam, v. 569, n. 3, p. 228–236, aug. 2007. https://doi.org/10.1016/j.ejphar.2007.04.059 GASPARINO, E. C.; BARROS, M. A. V. C. Palinotaxonomia das espécies de Cordiaceae (Boraginales) ocorrentes no Estado de São Paulo. Brazilian Journal of Botany, São Paulo, v. 32, n. 1, p. 33-55, jan. 2009. http://dx.doi.org/10.1590/S0100-84042009000100005 GAVAHIAN, M.; FARAHNAKY, A.; FARHOOSH, R.; JAVIDNIA, K.; SHAHIDI, F. Extraction of essential oils from Mentha piperita using advanced techniques: Microwave versus ohmic assisted hydrodistillation. Food and Bioproducts Processing, Birmingham, v. 9, p. 50–58, jan. 2015. https://doi.org/10.1016/j.fbp.2015.01.003 637 Distillation methods... NIZIO, D. A. C. et al Biosci. J., Uberlândia, v. 34, n. 3, p. 629-639, May/June 2018 GONZÁLEZ-RIVERA, J.; DUCE, C.; FALCONIERI, D.; FERRARI, C.; GHEZZI, L.; ALESSANDRA PIRAS, A.; TINE, M. R. Coaxial microwave assisted hydrodistillation of essential oils from five different herbs (lavender, rosemary, sage, fennel seeds and clove buds): Chemical composition and thermal analysis. Innovative Food Science and Emerging Technologies, Wageningen, v.33, p. 308–318, jan. 2016. https://doi.org/10.1016/j.ifset.2015.12.011 HOYOS, J. M. A.; ALVES, E.; ROZWALKA, L. C.; SOUZA, E. A.; ZEVIANI, W. M. Antifungal activity and ultrastructural alterations in Pseudocercospora griseola treated with essential oils. Ciência e Agrotecnologia, Lavras, v. 36, n. 3, p. 270-284, may 2012. http://dx.doi.org/10.1590/S1413-70542012000300002 KARAKAYA, S.; EL, S. N.; KARAGOZLU, N.; SAHIN, S.; SUMNU, G.; BAYRAMOGLU, B. Microwave- assisted hydrodistillation of essential oil from rosemary. Journal of Food Science and Technology, Mysore, v. 51, n. 6, p. 1056–1065, june 2014. https://doi.org/10.1007/s13197-011-0610-y KHANAVI, M.; HAJIMEHDIPOOR, H.; EMADI, F.; KHANDANI, N. K. Essential oil compositions of Thymus kotschyanus Boiss. obtained by hydrodistillation and microwave oven distillation. Journal of Essential Oil Bearing Plants, India, v. 16, n. 1, p. 117–122, may 2013. http://dx.doi.org/10.1080/0972060X.2013.764159 LI, S.; CHEN, F.; JIA, J.; LIU, Z.; GU, H.; YANG, L.; WANG, F.; YANG, F. Ionic liquid-mediated microwave-assisted simultaneous extraction and distillation of gallic acid, ellagic acid and essential oil from the leaves of Eucalyptus camaldulensis. Separation and Purification Technology, Delft, v. 168, p. 8–18, may 2016. https://doi.org/10.1016/j.seppur.2016.05.013 LI, X. J.; WANG, W.; LUO, M.; LI, C. Y.; ZU, Y. G.; MU, P. S.; FU, Y. J. Solvent-free microwave extraction of essential oil from Dryopteris fragrans and evaluation of antioxidant activity. Food Chemistry, Norwich, v. 133, n. 2, p. 437–444, jan. 2012. https://doi.org/10.1016/j.foodchem.2012.01.056 LI, Y.; FABIANO-TIXIER, A. S.; CHEMAT, F. Essential oils: From conventional to green extraction. In: Green chemistry for sustainability. Cham: Springer International Publishing, 2014, chapter 2, p. 9-21. http://dx.doi.org/10.1007/978–3-319-08449-7 MATIAS, E. F.; SANTOS, K. K. A.; ALMEIDA, T. S.; COSTA, J. G. M.; COUTINHO, H. D. M. Atividade antibacteriana in vitro de Croton campestris A., Ocimum gratissimum L. e Cordia verbenacea DC. Revista Brasileira de Biociencias, Porto Alegre, v. 8, n. 3, p. 294-298, july, 2010. Disponível Acesso em: 5 maio de 2015. MECCIA, G.; ROJAS, L. B.; VELASCO, J.; DÍAZ, T.; USUBILLAGA, A.; ARZOLA, J. C.; RAMOS, S. Chemical composition and antibacterial activity of the essential oil of Cordia verbenacea from the Venezuelan Andes. Natural Product Communications, Westerville, v. 4, n. 8, p. 1119-1122, aug. 2009. MEDEIROS, R.; PASSOS, G. F.; VITOR, C. E.; MAZZUCO, T. L.; PIANOWSKI, L. F.; CAMPOS, M. M.; CALIXTO, J. B. Effect of two active compounds obtained from the essential oil of Cordia verbenacea on the acute inflammatory responses elicited by LPS in the rat paw. British Journal of Pharmacology, London, v. 151, n. 5, p. 618–627, july 2007. http://dx.doi.org/10.1038/sj.bjp.0707270 MICHIELIN, E. M. Z.; WIESE, L. P. L.; FERREIRA, E. A.; PEDROSA, R. C.; FERREIRA, S. R. S. Radical- scavenging activity of extracts from Cordia verbenacea DC obtained by different methods. Journal of Supercritical Fluids, Netherlands, v. 56, n. 1, p. 89-96, feb. 2011. https://doi.org/10.1016/j.supflu.2010.11.006 OLIVEIRA, D. M.; LUCHINI, A. C.; SEITO, L. N.; GOMES, J. C.; CRESPO-LÓPEZ, M. E.; DI STASI, L. C. Cordia verbenacea and secretion of mast cells in different animal species. Journal of Ethnopharmacology, Ireland, v. 135, n. 2, p. 463–468, may 2011. https://doi.org/10.1016/j.jep.2011.03.046 638 Distillation methods... NIZIO, D. A. C. et al Biosci. J., Uberlândia, v. 34, n. 3, p. 629-639, May/June 2018 PARISOTTO, E. B.; MICHIELIN, E. M. Z.; BISCARO, F.; FERREIRA, S. R. S.; FILHO, D. W.; PEDROSA, R. C. The antitumor activity of extracts from Cordia verbenacea D.C. obtained by supercritical fluid extraction. Journal of Supercritical Fluids, Netherlands, v. 61, p. 101-107, jan. 2012. https://doi.org/10.1016/j.supflu.2011.08.016 PASSOS, G. F.; FERNANDES, E. S.; CUNHA, F. M.; FERREIRA, J.; PIANOWSKI, L. F.; CAMPOS, M. M.; CALIXTO, J. B. Anti-inflammatory and anti-allergic properties of the essential oil and active compounds from Cordia verbenacea. Journal of Ethnopharmacology, Ireland, v. 110, n. 2, p. 323–333, mar. 2007. https://doi.org/10.1016/j.jep.2006.09.032 PIMENTEL, S. P.; BARRELLA, G. E.; CASARIN, R. C. V.; CIRANO, F. R.; CASATI, M. Z.; FOGLIO, M. A.; FIGUEIRA, G. F.; RIBEIRO, F. V. R. Protective effect of topical Cordia verbenacea in a rat periodontitis model: immune-inflammatory, antibacterial and morphometric assays. BMC Complementary and Alternative Medicine, London, v. 12, n. 224, p. 1-8, nov. 2012. https://doi.org/10.1186/1472-6882-12-224 PINHO, L.; SOUZA, P. N. S.; SOBRINHO, E. M.; ALMEIDA, A. C.; MARTINS, E. R. Antimicrobial activity of hydroalcoholic extracts from rosemary, pepper tree, barbatimão and erva-baleeira leaves and from pequi peel meal. Ciencia Rural, Santa Maria, v. 42, n. 2, p. 326-331, feb. 2012. http://dx.doi.org/10.1590/S0103- 84782012005000003 QI, X. L.; LI, T. T.; WEI, Z. F.; GUO, N.; LUO, M.; WANG, W.; ZUA, Y. G.; FU, Y. J.; PENG, X. Solvent- free microwave extraction of essential oil from pigeon pea leaves [Cajanus cajan (L.) Mill sp.] and evaluation of its antimicrobial activity. Industrial Crops and Products, Amsterdam, v. 58, p. 322–328, july 2014. https://doi.org/10.1016/j.indcrop.2014.04.038 RANITHA, M., NOUR, A. H.; SULAIMAN, Z. A.; NOUR A. H.; THANA RAJ S. A comparative study of lemongrass (Cymbopogon Citratus) essential oil extracted by microwave-assisted hydrodistillation (MAHD) and conventional hydrodistillation (HD) method. International Journal of Chemical Engineering and Applications, Singapore, vol. 5, n. 2, p. 104-108, apr. 2014. https://doi.org/10.7763/IJCEA.2014.V5.360 RIELA, S.; BRUNO, M.; ROSSELLI, S.; SALADINO, M.L.; CAPONETTI, E.; FORMISANO, C.; SENATORE, F. A study on the essential oil of Ferulago campestris: How much does extraction method influence the oil composition? Journal of Separation Science, Weinheim, v. 34, n. 4, p. 483–492, feb. 2011. https://doi.org/10.1002/jssc.201000411 RODRIGUES, F. F. G; OLIVEIRA, L. G. S.; RODRIGUES, F. F. G.; SARAIVA, M. E.; ALMEIDA, S. C. X.; CABRAL, M. E. S.; CAMPOS, A. R.; COSTA, J. G. M. Chemical composition, antibacterial and antifungal activities of essential oil from Cordia verbenacea DC leaves. Pharmacognosy Research, Mumbai, v. 4, n. 3, p. 161-165, july 2012. https://doi.org/10.4103/0974-8490.99080 ROGÉRIO, A. P.; ANDRADE, E. L.; LEITE, D. F. P.; FIGUEIREDO, C.; CALIXTO, J. B. Themed section: mediators and receptors in the resolution of inflammation. Preventive and therapeutic anti-inflammatory properties of the sesquiterpene α-humulene in experimental airways allergic inflammation. British Journal of Pharmacology, London, v. 158, n. 4, p. 1074-1087, oct. 2009. https://doi.org/10.1111/j.1476- 5381.2009.00177.x SHAH, M.; GARG, S. K. Application of 2� full factorial design in optimization of solvent-free microwave extraction of ginger essential oil. Journal of Engineering, Nasr City, v. 2014, p. 1-5, nov. 2014. http://dx.doi.org/10.1155/2014/828606 SILVA, A. C.; SOUZA, P. E.; MACHADO, J. C.; SILVA, B. M.; PINTO, J. E. B. P. Effectiveness of essential oils in the treatment of Colletotrichum truncatum-infected soybean seeds. Tropical Plant Pathology, Brasília, v. 37, n. 5, p. 305-313, sept. 2012. http://dx.doi.org/10.1590/S1982-56762012000500001 639 Distillation methods... NIZIO, D. A. C. et al Biosci. J., Uberlândia, v. 34, n. 3, p. 629-639, May/June 2018 SILVA, A. C.; SOUZA, P. E.; RESENDE, M. L. V.; SILVA JR., M. B.; RIBEIRO JR., P. M.; ZEVIANI, W. M. Local and systemic control of powdery mildew in eucalyptus using essential oils and decoctions from traditional Brazilian medicinal plants. Forest Pathology, Freising, v. 44, n. 2, p. 145–153, apr. 2014. http://dx.doi.org/10.1111/efp.12079 SOZMEN, F.; UYSAL, B.; OKSAL, B. S.; KOSE, E. O.; DENIZ, I. G. Chemical composition and antibacterial activity of Origanum saccatum P.H. Davis essential oil obtained by solvent-free microwave extraction: comparison with hydrodistillation. Journal of AOAC International, Gaithersburg, v. 94, n. 1, p. 243-250, jan. 2011. http://dx.doi.org/10.1111/j.1745-4565.2009.00181.x TONGNUANCHAN, P.; BENJAKUL, S. Essential oils: extraction, bioactivities, and their uses for food preservation. Journal of Food Science, Chicago, v. 79, n. 7, p. 1231-1249, july 2014. http://dx.doi.org/10.1111/1750-3841.12492 VAN DEN DOOL, H.; KRATZ, P. D. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. Journal of Chromatography A, Netherlands, v. 11, p. 463-471, aug. 1963. https://doi.org/10.1016/S0021-9673(01)80947-X VERMA, R. S.; RAHMAN, L.; VERMA, R. K.; CHAUHAN, A.; YADAV, A. K.; SINGH, A. Essential oil composition of menthol mint (Mentha arvensis) and pepper mint (Mentha piperita) cultivars at different stages of plant growth from Kumaon region of western Himalaya. Journal of Medicinal and Aromatic Plants, Anand, v.1, n. 1, p. 13-18, jan. 2010. Disponível Acesso em: 14 de abril de 2015.