Essential Oils from Lippia origanoides Kunth . and Mentha spicata L . : Chemical Composition , Insecticidal and Antioxidant Activities

This work describes the chemical composition of the essential oils extracted from fresh leaves of Lippia origanoides Kunth. and Mentha spicata L. and their antioxidant and insecticidal activities. The essential oils were extracted by steam distillation using a modified Clevenger apparatus and subsequently analyzed by gas chromatography-flame ionization detector and gas chromatography-mass spectrometry. The antioxidant activities were determined by the β-carotene-linoleic acid method and by sequestration of the 2,2-diphenyl-1-picryl-hidrazila radical. The concentrations of the essential oils and the synthetic standard, butylated hydroxyltoluene, were 25, 50, 100, 150, 200, 250, 300 and 500 μg mL−1. Insecticidal activity was analyzed by non-preference with choice and no choice against the aphid Myzus persicae Sulzer. Gas chromatography analysis of the essential oil from Lippia origanoides Kunth. revealed carvacrol (41.51%), p-cymene (18.36%), γ-terpinene (17.03%) and thymol (4.86%) as major constituents, and the essential oil from Mentha spicata L. contained piperitona (81.18%), piperitenone (14.57%) and limonene (1.47%) as the principal components. The essential oils and the standard exhibited dose-dependent antioxidant activities at the concentrations tested. The essential oils were shown to be potential agents in the integrated management of the aphid Myzus persicae Sulzer. Corresponding author. M. L. Teixeira et al.


Introduction
For many years, humankind has been using plants for biotechnological purposes because they contain natural compounds capable of acting against predators and insects and as preventive agents for cardiovascular and neurodegenerative diseases [1].Considering the possible problems that might be affected by the consumption of synthetic antioxidants, research has been undertaken to encounter natural products with antioxidant activity that could replace the synthetic products or complement them.
For many years, a variety of synthetic compounds have been employed to control pests and plant diseases.However, the use of these substances has been limited by their carcinogenic and teratogenic activities, toxicity and environmental pollution [2].Among the pests of economic importance in Brazil, the Myzus persicae Sulzer aphid, one of the most damaging species from the agricultural point of view, should be emphasized because, in addition to the direct damages resulting from the continuous ingestion of sap, it causes indirect losses because of the transmission of a virus [3].
Because of the harmful consequences resulting from the use of synthetic compounds as antioxidants and insecticides, the search for alternative methods to control pests has increased.Among these natural compounds, the essential oils are secondary metabolites produced by plants and characterized by being complex mixtures of organic compounds [4].Their use is increasing and gaining a greater share of the market and consumer preference.
Brazil has a variety of flora, and this variety facilitates the study of the functional properties of the species that compose the flora.L. origanoides (Verbenaceae) is an aromatic bush that is native to Central America and northern South America.It is used in traditional medicine for the treatment of gastrointestinal and respiratory diseases [5].M. spicata (Lamiaceae) is an aromatic, creeping, rhizomatous plant.It is popularly used as a flavoring agent in tea and as a calming agent [6].The objective of the present study was the characterization of the essential oils from the fresh leaves of Lippia origanoides and Mentha spicata and the evaluation of the antioxidant and insecticidal activities of these essential oils.

Collection of the Vegetal Material and Extraction of the Essential Oil
The Lippia origanoides Kunth.(Verbenaceae) and Mentha spicata L. (Lamiaceae) plants were collected in the morning in the Medicinal Plants Garden of the Federal University of Lavras (UFLA) in Lavras, MG, Brazil, on a mild day with no precipitation.Lavras is located in the southern region of the state of Minas Gerais at 21˚14'S, longitude 45˚00'W Gr and at an altitude of 918 m.The essential oils were extracted in the Laboratory of Organic Chemistry-Essential Oils of the Federal University of Lavras by hydrodistillation with a modified Clevenger apparatus [7]- [9].For the extraction of the essential oil from L. origanoides, 250 g of fresh leaves was added to a round bottom flask (5 liters) with 2500 mL of distilled water.Fresh M. spicata leaves (225 g) were extracted in a similar manner.Three extractions were performed for each plant material by boiling for 2 hours.The essential oil was separated from the hydrolact by centrifugation at 965g for 5 minutes using a bench-top centrifuge with a horizontal crosspiece (Fanem Baby® Model 206 BL).The oil was removed with the aid of a Pasteur pipette, stored in a glass flask protected from moisture and light and stored at a low temperature (4˚C).

Chemical Identification and Quantification of the Essential Oils
The chemical analysis of the essential oils was performed in the Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal.
The GC-MS analyses were performed on a Perkin Elmer Autosystem XL gas chromatograph equipped with a DB-1 fused silica column (30 m × 0.25 mm ID, film thickness, 0.25 µm; J & W Scientific Inc.) and coupled to a Perkin Elmer Turbomass mass spectrometer (software version 4.1).The oven temperature was initially pro-grammed from 45˚C to 175˚C, with a heating rate of 3˚C min −1 , followed by 15˚C min −1 to 300˚C, where the temperature was maintained for 10 minutes.The injector temperature was 280˚C and the helium carrier gas was adjusted to a linear velocity of 30 cm s −1 .The transfer line temperature was 280˚C, the temperature of the ion trap, 220˚C; the split ratio, 1:40, the ionization energy, 70 eV, the scan range, 40-300U, and the scan time, one second.
The identity of the compounds was determined by comparison of their retention indices relative to C 9 -C 21 n-alkanes and the mass spectra with those of standard commercial and reference compounds present in oils existing in the laboratory and with a library of mass spectra developed in the laboratory of the Centro de Biotecnologia Vegetal, Faculdade de Ciências, Universidade de Lisboa [10].
The volatile components were analyzed by gas-liquid chromatography on a Perkin Elmer 8700 gas chromatograph equipped with two flame ionization detectors (FID), a system for processing data and an automatic injector.Two columns of different polarities with the following characteristics were used: fused silica with a DB-1 immobilized methylsilicone phase (30 m × 0.25 mm ID, film thickness, 0.25 µm; J & W Scientific Inc.) and a DB-17HT immobilized phenylmethylsilicone phase (30 m x 0.25 mm id, film thickness, 0.25 µm; J & W Scientific Inc.).
The oven temperature was initially programmed from 45˚C to 175˚C, with a heating rate of 3˚C min −1 , followed by 15˚C min −1 to 300˚C, where the temperature was maintained for 10 minutes.The temperature of the injector and detector were 290˚C and 280˚C, respectively; the flow rate of the hydrogen carrier gas was 30 cm s −1 .The split ratio was 1:50, and the injected volume was 0.1 µL of oil in pentane.The composition of the essential oils was calculated by normalizing the peak areas without using correction factors.The values shown correspond to the average from two injections [10].

Antioxidant Activity
For the antioxidant activity of essential oils by the β-carotene-linoleic acid method, 60 mg of linoleic acid, 600 mg of Tween 20, 6 mg of β-carotene and 30 mL of chloroform were added to a round bottom flask.After homogenization, the chloroform was completely evaporated at 50˚C, and the residue was dissolved in 150 mL of distilled water saturated with oxygen (emulsion A).Aliquots of 2.5 mL of the emulsion were transferred to test tubes, and 0.2 mL of each of the dilutions of the essential oil in methanol at the following concentrations 25, 50, 100, 150, 200, 250, 300 and 500 µg mL −1 was added.The blank emulsion was prepared without the addition of β-carotene and the control, by the addition of 2.5 mL of the emulsion A and 0.2 mL of methanol.The analysis for each reaction mixture was performed with four replicates, and the absorbance was measured immediately on a Shimadzu UV-160 1 PC spectrophotometer at 470 nm.The tubes were incubated at 50˚C, and the absorbance was measured again after 60 minutes.BHT (Butylated hydroxytoluene) was used as a positive control in the same concentrations as the essential oils.The percentage of antioxidant activity was calculated according to Lopes-Lutz et al. (2008) [11] using the following equation: ( ) where A 0 is the absorbance at the beginning of incubation with the sample; A t is the absorbance after incubating the sample for 60 minutes; A 00 : is the absorbance at the start of incubation without the sample; A 0t is the absorbance after 60 minutes without sample.
For the sequestration of the DPPH (1,1-diphenyl-2-picryl-hidrazila) radical method, a stock solution contained DPPH at a concentration of 40 mg mL −1 in methanol was prepared.The essential oils were dissolved in methanol at concentrations of 25, 50, 100, 150, 200, 250, 300 and 500 µg mL −1 .The stock solution of DPPH (2.7 mL) was added to a test tube, followed by the addition of 0.3 mL of the dilutions of the essential oil.The blank contained 2.7 mL of methanol and 0.3 mL of the most concentrated essential oil, and the control contained 2.7 mL of the stock solution of DPPH and 0.3 mL of methanol.Analysis was performed for 60 minutes with four replicates (in the absence of light); readings were performed on a spectrophotometer (Shimadzu UV-160 1 PC) at 515 nm.BHT was used as a positive control in the same concentrations as the essential oils.The percentage of antioxidant activity was calculated according to Lopes-Lutz et al. (2008) [11] using the following equation: where A am is the absorbance of the sample; hap is the absorbance of the control.
The experimental design was a completely randomized design (CRD) with a 3 × 8 factorial arrangement (es-sential oils/standard x concentrations), with four replications.The statistical program used was SISVAR [12].Data were subjected to analysis of variance, and the average was compared by the Tukey test at 5% probability.

Insecticide Activity
The experiments were performed in the Laboratory of Plant Resistance to Insects, Department of Entomology, Federal University of Lavras.The planting of tomato plants was accomplished using vessels with a capacity of 1 kg of soil, in which the tomato seedlings of the Santa Clara cultivar, provided in Styrofoam trays, were transplanted.
The collection of aphids was conducted in the field.The aphids were brought to the laboratory and reared in cages with "joá de capote" (Nicandra physaloides) plants in an environmental chamber at 25 ± 2˚C and a photoperiod of 12 hours.In biological tests, adult aphids of uniform size were chosen.The leaves of the tomato plants were detached, and sections were cropped with the aid of a circular plastic cutter six centimeters in diameter.These sections were sprayed on the abaxial surface with the sample solutions to the point of total runoff and fixed in Petri dishes on a layer of 1.0% agar covered with PVC film pierced with a pin.
For the test with a chance of choice, each Petri dish contained a leaf section of each of the three treatments, arranged in a circle, forming an arena (Silveira et al., 1998) [14].In the center of each plate, 15 wingless adult M. persicae aphids were released.
In the no choice test, each Petri dish contained a leaf section of each treatment set in the center of the plate.Five wingless adult M. persicae aphids were released [14].The plates were kept in a climatic chamber at 25 ± 2˚C with a photoperiod of 12 hours.The evaluations were made 24, 48 and 72 hours after the release of the aphids by counting the number of aphids and nymphs on each leaf section.
The experimental designs were a randomized block design (RBD) and a completely randomized design (CRD) with ten replicates for tests with and without choice, respectively.The statistical program used was SISVAR [12].The data were transformed into ( ) and subjected to analysis of variance with the parts subdivided in time, and the means were compared by the Tukey test at 5% probability.
According Gobbo-Neto and Lopes (2007) [20], the production of secondary metabolites can be influenced by various environmental factors such as seasonality, rainfall, circadian rhythm, altitude, temperature, vegetative cycle of the plant, and soil type, among others.In this context, quantitative and qualitative changes in the chemical composition of the essential oils can be influenced by these environmental factors because the plants were collected in very distinct environments.

Antioxidant Activity
The percentage of antioxidant activity of the essential oils and the BHT standard in relation to their concentrations, evaluated by the β-carotene-linoleic acid method, are presented in Table 3.It's possible to observe a significant effect of the factors essential oil/standard, concentration and the interaction of these factors on the percentage of antioxidant activity.An increase in the antioxidant activity with concentration revealed a dose dependence of the antioxidant activity of the essential oils and the BHT standard.This dose dependence of the essential oil was reported in the work of Wang et al. (2008) [21], who evaluated the antioxidant activity of the essential oil from Rosmarinus officinalis L. by the β-carotene-linoleic acid method.
The BHT standard exhibited the highest antioxidant activity, followed by the essential oil from L. origanoides and M. spicata, respectively.Results obtained by Morais et al. (2006) [22] and by Duarte-Almeida et al. (2006) [23] corroborate those observed in this study regarding the high antioxidant activity of the synthetic BHT, which is used as an antioxidant in the food industry.The fact that the essential oil from L. origanoides exhibited a higher activity than that from M. spicata may be related to the presence of carvacrol as the principal compound  The means followed by the same lower case letter in the columns and the same upper case letter in the rows do not differ by the Tukey test at the 5% probability level.Legend: [ ] (μg mL −1 ) = Concentrations (μg mL −1 ); LO = Lippia origanoides Kunth.; MS = Mentha spicata L.
in the oil.According Ruberto and Barata (2000) [24], phenols and their derivatives are effective antioxidants; thus, molecules such as thymol and carvacrol are responsible for the high antioxidant activity of many essential oils.
As can be deduced from Table 4, the percentage of antioxidant activity was also dose dependent at the concentrations tested, and the BHT standard exhibited greater antioxidant activity than the essential oils from L. origanoides and M. spicata, employing the same concentrations, in the method that involved the sequestering of the DPPH radical.The oil from L. origanoides appeared to exhibit greater stabilization of the DPPH radical than the oil from M. spicata.However, the activity of the oils exhibited in the DPPH method was lower than that observed with the β-carotene-linoleic acid method.This lower activity may be explained by the low solubility of the constituents of the essential oil in the reaction medium of the test, usually conducted in a polar solvent such as methanol or ethanol.Safaei-Ghomi et al. (2009) [25] reported that compounds containing hydrogen atoms on allylic, benzylic and phenolic carbons exhibit higher activity in the β-carotene-linoleic acid method because of the relative ease of abstraction of the hydrogen atoms by the peroxyl radicals formed in the test.Thus, the antioxidant capacity of phenolic compounds is due to their ability to donate hydrogen atoms, thereby confirming the high antioxidant capacity of the essential oil from L. origanoides compared with that of the oil from M. spicata.
The results obtained by the method of sequestration of the DPPH radical are based on the bleaching of a solution composed of the violet-colored DPPH radical when this is added to substances that can release a hydrogen atom (such as phenols).Thus, the greater antioxidant activity presented by the essential oil from L. origanoides is to be expected because carvacrol is present in a high concentration in this oil.

Insecticidal Activity
The test of preference with a choice assessed the preference of the aphids for leaf sections sprayed with different concentrations of the essential oils.From the data presented in Table 5, it can be observed that aphids had a greater preference for sections of the control (water + Tween 80) and for sections with a concentration of 0.1% essential oil of L. origanoides than for those treated with a 0.5% concentration.From the numbers of nymphs present in leaf sections treated with different concentrations of the essential oil of L. origanoides (Table 5), it is clear that the 0.1 and 0.5% concentrations of essential oil influenced the reproduction of aphids, as compared with the control, being less preferred for egg laying by adults.This fact proves that natural products such as essential oils can be considered as alternative sources for the management of insect pests.
When evaluating the influence of the time interval on the number of live adults in the data presented in Table 6, the interval did not alter the preference of aphids; the numbers of aphids on the leaf sections remained constant with the passage of time.Nor did the time interval influence the effect of the essential oil, and the number of nymphs on the leaf sections remained constant over time.The results of the test of preference with no choice evaluating the essential oil from M. spicata L. are summarized in Table 7.There was a significant interaction between the factors concentration and the time interval.Thus, the sections treated with the 0.1 and 0.5% concentrations of the oil were less preferred for food by adult aphids, regardless of the interval evaluated.With regard to the number of nymphs (Table 7), the leaf sections treated with the control and with the 0.1% concentration of oil were most preferred for the reproduction of aphids during for the first 24 hours and presented a greater number of nymphs than was observed with the 0.5% concentration.After 48 hours, the number of nymphs decreased proportionally to the concentrations tested, i.e., the higher the concentration, the smaller the number of nymphs, and, at 72 hours, the treatments with 0.1 and 0.5% differed statistically from the control, being less preferred for egg laying.The means followed by the same letter in the column do no differ statistically by the Tukey test at 5% of probability.
Table 6.Numbers of live adults and nymphs in the preference with a choice test according to the time interval for the essential oil from L. origanoides.The means followed by the same letter in the column do no differ statistically by the Tukey test at 5% of probability.Nerio et al. (2009) [26] studied the repellent activity of the essential oils from the aromatic plants Cymbopogon citratus, Eucalyptus citriodora, Lippia origanoides, Lippia alba, Cananga odorata, Tagetes lucida and Rosmarinus officinalis, grown in Colombia, against Sitophilus zeamais Motschulsky using the method of preferred area.They used concentrations of 0.063, 0.126, 0.252, and 0.503 mL (cm 2 ) −1 and observed that all the essential oils, except for that from L. alba, exhibited some repellent activity.The oils with the highest repellency were those from L. origanoides, E. citriodora and T. lucida.According to the authors, the essential oil from L. origanoides was the most active repellent, possibly because of the presence of thymol, which was the major component found and is considered to be a botanical insecticide.These results corroborate those found in the present work, because the essential oils also presented repellent activity.The essential oil from L. origanoides contained carvacrol, an isomer of thymol, as the principal compound.Thus, there is a similarity between the two studies with regard to the repellent activity.
The test of preference with no choice evaluated the toxicity of the essential oils to Myzus persicae Sulzer aphids.No toxicity was observed when the concentrations of the essential oils from L. origanoides and M. spicata L. were varied.Thus, the number of adult aphids was not significantly different when compared with the control (Table 8).For the essential oil from L. origanoides, there was no statistically significant difference between the control and the treatment with 0.1% essential oil with respect to the number of nymphs, although the numbers were higher than those observed in the treatment with 0.5% essential oil.Thus, a greater insecticidal effect against the adult aphids was observed for the higher oil concentration.With the essential oil from M. spicata L., there was a decrease in the number of nymphs deposited by aphid females as the concentration of the oil increased.That is, the control afforded the greatest number of nymphs, followed by the treatments with 0.1 and 0.5% essential oil, respectively.
In the analysis of the influence of the time interval on the insecticidal effect of essential oils (Table 9) in the test of preference with no choice, no significant variation in the number of live adults with time was observed with both the essential oils.Nor was there any variation in the number of nymphs with time when the oil from L. origanoides was tested.However, the essential oil from M. spicata L. did not behave like that from L. origa- The means followed by the same letter in the column do no differ statistically by the Tukey test at 5% of probability.The means followed by the same letter in the column do no differ statistically by the Tukey test at 5% of probability.
noides; there was an increase in the number of nymphs deposited with time.This fact implies that the efficacy against aphids decreased with time.The difference in the performance of the essential oils can probably be explained by the difference in their chemical compositions because the essential oil from M. spicata consisted mostly of terpenes, while that from L. origanoides is composed mainly of phenolic compounds.Zapata and Smagghe (2010) [27] evaluated the repellent activity as well as the contact and fumigant toxicities against Tribolium castaneum of four essential oils extracted from the leaves and bark of Laurelia sempervirens and Drimys winteri.They observed that the four oils had a strong repellent activity against T. castaneum in the arena test with filter paper.After four hours of exposure, over 90% repellency was obtained for the oils from L. sempervirens at low concentrations [0.032 µL (cm 2 ) −1 ].Concentrations of the oils from D. winteri three-to-ten times higher were necessary to achieve this activity.Both oils were toxic to T. castaneum when applied topically or by spraying.The LD 50 values for topical application of the L. sempervirens oils were 39 -44 µg mg −1 insect, and those for the oils from D. winteri were 75 to 85 µg mg −1 insect.When sprayed, the LC 50 values for the oils from L. sempervirens were 1.6 to 1.7 µL L −1 of air, while those for the oils from D. winteri were 9.0 a 10.5 µL L −1 of air.

Table 1 .
Chemical composition of the essential oil from leaves of L. origanoides.

Table 2 .
Chemical composition of the essential oil from leaves of M. spicata.

Table 3 .
Percentages of antioxidant activity of essential oils and BHT observed using the β-carotene-linoleic acid method.

Table 4 .
Percentages of antioxidant activity of the essential oils and BHT in the method involving the sequestering of the DPPH radical.The means followed by the same lower case letter in the columns and the same upper case letter in the rows do not differ by the Tukey test at the 5% probability level.Legend: [ ] (μg mL −1 ) = Concentrations (μg mL −1 ); LO = Lippia origanoides Kunth.; MS = Mentha spicata L.

Table 5 .
The numbers of live adults and nymphs in the preference with a choice test at different concentrations of the essential oil from L. origanoides.
The means followed by the same letter in the column do no differ statistically by the Tukey test at 5% of probability.

Table 7 .
Numbers of live adults and nymphs in the preference with a choice test by concentration and time interval of the essential oil from M. spicata.

Table 8 .
Mean numbers of live adults and nymphs in the preference without a choice test with variation in the concentrations of the essential oils.

Table 9 .
Variation of the mean numbers of live adults and nymphs in the preference with no choice with time interval.