Composition and antioxidant and antifungal activities of the essential oil from Lippia gracilis Schauer

Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Pará, 66075-900 Belém, PA, Brazil. Programa de Pós-Graduação em Química, Universidade Federal do Pará, 66075-900 Belém, PA, Brazil. Programa de Pós-Graduação em Química, Universidade Federal do Maranhão, 65080-040 São Luis, MA, Brazil. Programa de Pós-Graduação em Biotecnologia, Universidade Federal do Pará, 66075-900 Belém, PA, Brazil. Programa de Pós-Graduação em Recursos Naturais da Amazônia, Universidade Federal do Oeste do Pará, 68010-110 Santarém, PA, Brazil.

Lately, there has been a growing interest in the search for spices, aromatic and medicinal plants as sources of natural antioxidants. The antioxidant capacity of these plants is associated with the activity of the free radical scavenging enzymes and the contents of antioxidant substances, usually phenol compounds. The use of essential oils as functional ingredients in foods, drinks, toiletries and cosmetics has become increasingly valuable also because of concern about potentially harmful synthetic additives. The oils and extracts, being biologically active natural compounds, have been proposed for the control of certain diseases and the prevention of lipid peroxidative damage implicated in various pathological disorders, such as atherosclerosis, Alzheimer`s disease, carcinogenesis and aging processes (Ruberto and Baratta, 2000;Mimica-Durik et al., 2004).
The aim of this study was to analyze the oil composition of leaves and thin stems of L. gracilis that occur in the eastern Brazilian Amazon, as well as to evaluate their antioxidant and antifungal and activities.

Plant material
The specimen L. gracilis Schauer was collected in the locality of São Félix de Balsas, Maranhão state, Brazil, February 2011. The plant was identified and deposited (MG 200187) in the Herbarium of Museu Paraense Emílio Goeldi, Belém city, Pará state, Brazil.

Plant processing
The leaves and thin stems were air-dried separately, ground and subjected to hydrodistillation (100 g, 3 h), using a Clevenger-type apparatus. The oils were dried over anhydrous sodium sulfate, and their percentage contents were calculated on basis of the plant dry weight. The moisture content of the samples were calculated after the phase separation in a Dean-Stark trap (5 g, 30 min), using toluene.

Oil-composition analysis
The analysis of the oils were carry out on a THERMO DSQ II GC-MS instrument, under the following conditions: fused-silica capillary column DB-5ms (30 m x 0.25 mm, 0.25 m film thickness); programmed temperature, 60-240°C (3°C/min); injector temperature, 250°C; carrier gas was helium, adjusted to a linear velocity of 32 cm/s (measured at 100°C); injection type, splitless (2 L of a 1:1000 hexane solution); split flow was adjusted to yield a 20:1 ratio; septum sweep was a constant 10 ml/min; EIMS electron energy, 70 eV; temperature of ion source and connection parts, 200°C. The quantitative data regarding the volatile constituents were obtained by peak-area normalization using a FOCUS GC/FID operated under conditions similar to those in GC-MS, except for the carrier gas, which was nitrogen. The retention index was calculated for all the volatiles constituents using an n-alkane homologous series.

DPPH radical scavenging assay
A stock solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (0.5 mM) in methanol (MeOH), was prepared. The solution was diluted in MeOH (60 µM approx.) measuring an initial absorbance of 0.620.02 in 517 nm at room temperature. The reaction mixture was composed by 1950 L of DPPH solution and 50 L of the samples diluted in different methanol portions. For each sample, a methanol blank was also measured. The absorbance was measured in the reaction starting (time zero), each 5 min during the first 20 min and then at constant intervals of 10 min up to constant absorbance value. The concentration of antioxidant required for 50% scavenging of DPPH radicals (EC 50 ) was determined by linear regression using Windows/Excel. All experiments were in triplicate. Butylated hydroxyanisole (BHA) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were used as standard antioxidants. The radical scavenging activity of each sample was calculated by the DPPH inhibition percentage according to the equation IP DPPH =100 (A -B) / A (where A and B are the blank and sample absorbance values in the end reaction). The radical scavenging activity, expressed as milligrams of Trolox equivalent per gram of each sample, was also calculated by means of the equation TE = (A -B)/(A -C) x 25/1000 x 250.29/1000 x 1000/10 x D (where A, B and C are the blank, sample and Trolox absorbance values in the end reaction, and D is the dilution factor) (Silva et al., 2007;Silva et al., 2011).

Antifungal bioassay
About 10 μL of the oil solutions (corresponding to 100, 50, 25, 10, 5, 1, 0.5 and 0.1 μg) were applied to pre-coated thin layer chromatographic (TLC) plates, which were developed with n-hexane/ethyl acetate (8:2) and dried for complete removal of solvents. The chromatograms were sprayed with a spore suspension of the fungi Cladosporium sphaerospermum and C. cladosporioides, in glucose and salt solution and incubated for 48 h in darkness in a moistened chamber at 22°C. Clear inhibition zones appeared against a dark background indicating the minimum amount of the essential oils required. Miconazole was used as the positive control. C. sphaerospermum (Penzig) SPC 491 and C. cladosporioides (Fresen) de Vries SPC 140 have been maintained at the Laboratory of Engineering of Natural Products, Federal University of Pará, Belém city, Pará State, Brazil (Silva et al., 2011).

Statistical analysis
Samples were assayed in triplicate, and the results are shown as means ± standard deviation. Analysis of variance was conducted, and the differences between variables were tested for significance by one-way ANOVA with Tukey's post test using Minitab, version 14. Differences at p < 0.05 were considered statistically significant. The relationship between variables was determined by simple regression analysis.

Oil-composition
The leaves and thin stems of L. gracilis provided oil yields of 3.7 and 0.4%, respectively, and their volatile constituents were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Individual components were identified by comparison of both mass spectra and GC-retention data with authentic compounds, which were previously analyzed and stored in the data system, or existing in commercial libraries and cited in the literature (Adams, 2007;NIST, 2005).
In preliminary analysis, the oil of L. gracilis showed the chemical types thymol plus p-cymene and carvacrol plus p-cymene (Lemos et al., 1992;Matos et al., 1999;Teles et al., 2010). Based on the analysis of this new specimen of L. gracilis, we can assume that it is the chemical type thymol plus p-cymene, but with an occurrence in North Brazil. In previous works was observed that Lippia oils from the Brazilian Amazon showed significant amounts of thymol, carvacrol, p-cymene, 1,8-cineole, -terpinene, (E)-caryophyllene, citral, carvone and terpinen-4-ol (Zoghbi et al., 1998;Zoghbi et al., 2001;Maia et al., 2005;Morais et al., 1972;Silva et al., 2009;Damasceno et al., 2011). This way, one must consider that these chemical types of L. gracilis may result from the polymorphism of the plant, taking into account, mainly, the season time and site collection.
Thymol, carvacrol and p-cymene co-occur also as chief constituents in some traditional oils, such as Monarda punctata L., Satureja hortensis L. and Thymus vulgaris L. (Guenther, 1952;Scora, 1967). Also, it is no coincidence that co-occurs in the oil of L. gracilis the same aromatic monoterpenes, thymol, carvacrol and p-cymene. All these compounds are derived from the same biosynthetic plant process, where γ-terpinene, the cyclohexadiene constituent that occur also in the oil, is considered the initiator (Poulose and Croteau, 1978a,b). Figure 1 shows the predicted biosynthetic pathway of these aromatic monoterpenes, which on average comprises for approximately 88% of the oil composition.

Antioxidant activity
Antioxidants interact with the DPPH through the transfer of electrons or donation of hydrogen neutralizing its character of free radical (Silva et al., 2007). The leaves oil of L. gracilis was able to scavenging the DPPH radical, displaying a high dose-response (r 2 =0.85). The half maximal effective concentration (EC 50 ) was 35.7±3.3 µg/ml, calculated by linear regression, (p<0.05), a significant value compared to Trolox (4.5±0.1 µg/ml), which was used as standard antioxidant. EC 50 values lower than 30 g/ml indicates high potential for radical scavenging (Ramos et al., 2003). This means that the L. gracilis oil showed a significant antioxidant potential for radical free scavenging (Figure 2).

Antifungal activity
The fungicide activity resulted from evaluation of direct bioautography using TLC, after the nebulization of fungal spores (Figure 3). The leaf oil of L. gracilis, tested against the Cladosporium sphaerospermum and C. cladosporioides fungi, showed a minimum inhibitory concentration (MIC) of 5.0 µg/ml. Miconazole, at the maximum concentration of 0.5 µg/ml, was used as positive control, meaning that the leaf oil showed antifungal activity comparable to standard compound.

Conclusion
The essential oil of L. gracilis collected in the locality of

Conflict of Interests
The author(s) have not declared any conflict of interests.