Characterization of the Biological Potential of the Essential Oils from Five Species of Medicinal Plants

Essential oils from Mentha piperita, Cymbopogon citratus, Rosmarinus officinalis, Peumus boldus and Foeniculum vulgare were extracted by hydrodistillation, chemically characterized and quantified by GC/MS and GC/FID. The principal componentes in the essential oil of M. piperita (carvone and limonene), C. citratus (geranial, neral and myrcene), R. officinalis (1,8-cineole, camphor and α-pinene), P. boldus (α-terpinyl formate, p-cymene and 1,8cineole) and F. vulgare (methyl chavicol, limonene and fenchone) were identified. The oils were tested for antioxidant activity employing the DPPH (2,2diphenyl-1-picryl-hydrazyl) radical-capture method, the oxidation of the βcarotene/linoleic acid system, and the ABTS radical-capture method. The essential oils were not effective. The antioxidant activities of the oils were determined by the hydroxyl method, and the highest activity (62.80%) was observed with the essential oil from M. piperita. Activities of 1.54 and 1.82 μg μg−1, respectively, were observed for the essential oils from C. citratus and P. Boldus using the phosphomolybdenum method. No activity was observed with respect to reducing power. The essential oil from C. citratus was effective in inhibiting the growth of L. monocytogenes (15.63 mg∙L−1), S. choleraesuis (15.63 mg∙L−1) and E. coli (31.25 mg∙L−1), and the essential oil from P. boldus inhibited the growth of S. aureus (62.5 mg∙L−1). No essential oil inhibited the growth of P. aeruginosa. A low antioxidant activity and a promising antibacterial activity were observed for these essential oils. How to cite this paper: Rezende, D.A.C.S., Souza, R.V., Magalhães, M.L., Caetano, A.R.S., Carvalho, M.S.S., de Souza, E.C., Guimarães, L.G. de L., Nelson, D.L., Batista, L.R. and Cardoso, M. das G. (2017) Characterization of the Biological Potential of the Essential Oils from Five Species of Medicinal Plants. American Journal of Plant Sciences, 8, 154-170. http://dx.doi.org/10.4236/ajps.2017.82012 Received: November 24, 2016 Accepted: January 17, 2017 Published: January 20, 2017 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access D. A. C. S. Rezende et al.


Introduction
Since ancient times, plants have been used to treat, prevent and cure diseases.However, the use of plants with medicinal potential has increased considerably and has stimulated studies that seek the characterization, identification and isolation of new natural products with medicinal properties.Among these natural products, the essential oils, regarded as flavorings or essences, have many applications because of their medicinal properties and their importance in the cosmetics and food industries.Essential oils are complex mixtures of volatile substances extracted from the plant by steam distillation or hydrodistillation.They are colorless or light yellow and are generally odoriferous.They have a high biological potential; they may have antioxidant, antiseptic, antimicrobial, antifungal, anti-inflammatory, or repellent activities, among others [1].
Antioxidants are substances that prevent or retard the rate of oxidation through one or more mechanisms that involve inhibiting or reducing the effects triggered by free radicals and oxidizing compounds on oxidizable substrates.Many compounds produced by plants have been the object of research regarding their antioxidant potentials, and the results have been promising.Among these substances, the essential oils stand out because many of their components can replace or be associated with synthetic substances.The acceptance of these products by consumers results in expansion of the use of essential oils of various plants as raw materials in food, pharmaceutical, and cosmetic industries [2].
Pathogenic bacteria have been the cause of the principal problems arising from the proliferation of resistant micro-organisms, especially with respect to the contamination of various foods.Food poisoning outbreaks are increasingly common, so there is a need to find compounds that are able to control bacterial strains resistant to antibiotics so as to reduce the contamination and deterioration of food-products [3].This study sought to chemically characterize the essential oils from the following medicinal plants: M. piperita, C. citratus, R. officinalis, P. boldus and F. vulgare and to evaluate their antioxidant and antibacterial activities.

Extraction of Essential Oils
Approximately 1.0 kg of dry material (leaves) from each plant (Mentha piperita, Cymbopogon citratus, Rosmarinus officinalis, Peumus boldus and Foeniculum vulgare), acquired in the Municipal Market of Belo Horizonte, Minas Gerais, Brazil, was used.The essential oils of selected medicinal plants were extracted in the Organic Chemistry-Essential Oils Laboratory of the Chemistry Department of the Federal University of Lavras.The material was extracted by hydrodistilla-tion using a modified Clevenger apparatus coupled to a 5 L flask [4].A 300 g quantity of each dried plant material was subjected to the hydrodistillation process for two hours.After extraction, the essential oil was separated from the hidrolact by centrifugation using a benchtop centrifuge with a horizontal crosspiece (FANEM Baby®I Model 206 BL) at 965g at room temperature for 5 minutes.The essential oil was transferred to an amber bottle with the aid of a Pasteur pipette and stored at 5˚C.

Identification and Quantification of Essential Oils
The identification of the constituents of the essential oils was performed by gas chromatography coupled to a mass spectrometer using a Shimadzu QP model 5050A GC/MS equipped with a J&W Scientific fused-silica capillary column (5% phenyl 95% dimethylpolysiloxane; 30 m × 0.25 mm id, 0.25 μm of film) using He as the carrier gas with a flow rate of 1.2 mL min −1 .The column temperature was maintained at 50˚C for 2 min, increased at 4˚C min −1 to 200˚C, increased at 15˚C min −1 to 300˚C, and maintained at this temperature for 15 min.The temperatures of the injector and interphase were maintained at 250˚C and 280˚C, respectively.A 0.5 μL sample of the oil in ethyl acetate was injected.The configuration of the mass spectrometer (MS) included an ion capture detector operating in electron impact mode and an impact energy of 70 eV.The scan speed was 1000; the scan interval was 0.50, and fragments were detected in the range of 40 to 500 Da.The retention indices of the constituents were compared with those of the literature [5].The retention indices were calculated using the equation of Van Den Dool and Kratz [6] relative to a homologous series of n-alkanes (C 9 -C 18 ).Two libraries, NIST107 and NIST21, were used for comparison of the mass spectra.
The quantitative analyses were conducted by gas chromatography (Shimadzu GC-17A) using a flame ionization detector (FID) and a ZB-5MS fused-silica capillary column (5% phenyl-95% dimethylpolysiloxane; 30 m × 0.25 mm id × 0.25 μm film).The carrier gas was He with a flow rate of 1.2 mL•min −1 .The analytical conditions were the same as those used for the GC-MS analysis, and the concentration of each component was obtained by normalization of areas (%).

Antibacterial Activity of Essential Oils
To assess the antibacterial activities, the following micro-organisms were used: Listeria monocytogens ATCC19117, Salmonella Choleraesuis ATCC6539, Pseudomonas aeruginosa ATCC15442, Escherichia coli ATCC11229 and Staphylococcus aureus ATCC13565.Initially, cultures were activated in broth (BHI) with incubation at 37˚C for 24 hours.After activation, the cultures were transferred to a tube with 5 mL of tryptic soy broth (TSB) and incubated at 37˚C to achieve the turbidity of a McFarland 0.5 standard solution, resulting in a concentration of 108 colony forming units (CFU) per milliliter.The turbidity readings were performed using a spectrophotometer (Varian, Cary 50 probe) at a wavelength of 625 nm [7].
The cultures were poured into petri plates containing a thin layer of fresh Mueller-Hinton Agar (20 mL), and sterile glass beads were placed on this layer.
A second layer of agar containing the inoculated microorganisms was poured over the first layer and the glass beads.After solidification, the glass beads were removed, and 10 µL of DMSO solution of the essential oils at concentrations of 15.62, 31.25,62.5, 125, 250, and 500 µg•mL −1 was placed in each well.The plates were incubated in BOD at 37˚C for 24 hours.The diameters of the inhibition halos formed were measured, and the minimum inhibitory concentration (MIC), the lowest concentration that inhibited the growth of the microorganism, was calculated.Chloramphenicol (standard antibiotic) was used as a positive control, and DMSO as the negative control [8].The micro-organisms S. choleraesuis, P. aeruginosa, E. coli and S. aureus were transferred to plates containing Mueller-Hinton Agar.Casoy Agar (TSA) supplemented with yeast extract [9] was used for L. monocytogens.

Antioxidant Activities of the Essential Oils
The antioxidant activities of the essential oils were determined using the methods that evaluate the capacity for sequestration of the DPPH radical, inhibition of the oxidation of the β-carotene/linoleic acid system, stabilization of the ABTS radical, capture of the hydroxyl radical, the reducing power and the reduction of molybdenum.

Sequestering of the DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Radical
A solution of DPPH in ethanol (stock solution) was prepared at a concentration of 40 μg mL −1 .The stock solution (2700 μL) was added to test tubes followed by the addition of 300 µL of the essential oil diluted in ethanol at concentrations of 25, 50, 100, 150, 200, 250 and 500 μg mL −1 .The control contained all the reagents except the essential oil.After incubation for 60 minutes in the absence of light, readings were performed in a spectrophotometer at a wavelength of 515 nm.Comparison tests were conducted with synthetic BHT (butylated hydroxytoluene) at the same concentration [10].

Oxidation of the β-Carotene/Linoleic Acid System
To evaluate the inhibition of oxidation of the β-carotene/linoleic acid system, 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, and the mixture was evaporated using a rotary evaporator at 50˚C.After complete removal of the chloroform, 150 mL of distilled water saturated with oxygen was added.To 2700 μL of this solution in test tubes, was added 300 μL of the essential oil diluted in ethanol at concentrations of 25, 50, 100, 150, 200, 250 and 500 μg mL −1 .The control contained only ethanol.The absorbance was immediately measured in a spectrophotometer at 470 nm.The tubes were incubated at 50˚C for 60 minutes to effect the oxidation, and a second reading was taken.The blank was prepared in the same manner as the emulsion with the addition of all the reagents except β-carotene.For comparison, the BHT standard was tested using the same concentrations [11].

Stabilization of the ABTS Radical
The ABTS • radical was formed by reaction of ABTS with 2.45 mM potassium persulphate at 25˚C for 12 -16 h in the absence of light.After the formation of the radical, it was diluted in ethanol to obtain an absorbance between 0.7 to 0.72 at 734 nm (Shimadzu UV-1601PC).To 1900 µL of the ABTS • in a test tube, was added 100 µL of the samples at concentrations of 25, 50, 100, 150, 200, 250 and 500 µg•mL −1 .The synthetic antioxidant BHT was employed at the same concentrations as a positive control [12].

Neutralization of the Hydroxyl Radical
The method described by Boulanouar et al. [13] was used to evaluate the antioxidant activity through the capture of the hydroxyl radical (OH • ).To a test tube, 100 μL of the sample diluted in water at concentrations of 25, 50, 100, 150, 200, 250 and 500 μg mL −1 , 100 μL of the mixture of FeSO 4 /EDTA, 100 μL of deoxyribose, 700 μL of 0.1 M phosphate buffer (pH = 7.4) and 100 μL of H 2 O 2 were added.The tubes were incubated at 50˚C for 60 min, and 500 μL each of trichloroacetic acid (TCA) and thiobarbituric acid were added.The tubes were boiled for 10 min and cooled; the absorbance was measured at 532 nm in a Shimadzu UV-1601PC spectrophotometer.Mannitol was employed as the standard.

Reducing Power
Fifty-microliter aliquots of the essential oils at concentrations of 25, 50, 100, 150, 200, 250 and 500 μg mL −1 were added to 500 μL of 200 mM phosphate buffer, pH 6, and 500 μL of 1% potassium hexacyanoferrate III.The mixture was stirred and incubated at 50˚C for 20 minutes.After the addition of 500 μL of 10% TCA, 1500 μL of distilled water and 300 μL of 0.1% FeCl 3 , the absorbance was measured at 700 nm, and a plot of absorbance versus sample concentration was constructed.Ascorbic acid was used as the standard [14].

Reduction of Molybdenum
To test tubes were added 50 μL of solutions of the essential oils at different concentrations (25, 50, 100, 150, 200, 250 and 500 μg mL −1 in ethanol), 2.000 mL of ammonium phosphomolibdate (10% sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate).The tubes were stirred and incubated at 95˚C for 60 minutes.After cooling, the absorbance was measured at 695 nm.The synthetic standard was ascorbic acid [15].

Statistical Analysis
The data resulting from the antioxidant studies were submitted to analysis of variance using a completely randomized design in a 5 × 7 factorial scheme (essential oils × concentrations), and the means were compared by the Scott-Knott test at 5% probability.The statistical program used was SISVAR [16].

Results and Discussion
The chemical compositions of the essential oils from Mentha piperita, Cymbopogon citratus, Rosmarinus officinalis, Peumus boldus and Foelniculum vulgare are shown in Table 1 where the principal components present in each essential oil are emphasized.Regarding the essential oil from M. piperita, the princpal constituents were carvone (84.34%) and limonene (10.97%).Recent studies verified the presence of 18 compounds in the essential oil from M. piperita, and the principal components were pulegone (45.00%) followed by iso-menthol (12.80%), piperitanona (9.10%), menthone (8.00%), piperitone (7.40%) and piperitanone oxide (6.90%) [17].Their data are different from those found in this work.This difference can be explained by edafoclimatic differences such as the locations and hours of collection, soil, nutrients, among others, because the plants were collected in different locations [18].
According to the data presented in Table 1, the principal component of the essential oil from F. vulgare was methyl chavicol, also known as estragole (89.48%), followed by limonene (6.15%) and fenchone (3.80%).In recent studies by Evrendilek [17] of the essential oil from F. vulgare extracted from fennel seeds acquired in Hatay markets (Turkey), seven compounds were found; the principal component was trans-anethole (80.4%).These results are different from those found in the present work where no trans-anethole was found.
According to the observations of Gobbo-Neto and Lopes [18], the differences in the chemical compositions of the essential oils from medicinal plants of the same species can be due to several factors such as location and time of collection, type of soil and nutrients because the secondary metabolites in the plant can vary with the season in which it is collected.The age of the plant, seasonality, temperature, and other factors also influence virtually all the classes of secondary metabolites, including the essential oils.The essential oils contained a larger concentration of monoterpenes (oxygenated or not) and sesquiterpenes (oxygenated or not), whereas the essential oil from F. vulgare was the only one that contained a phenylpropanoid.

Antibacterial Activity of Essential Oils
The minimum inhibitory concentrations (MICs) of the essential oils tested against Gram-negative and Gram-positive bacteria are described in Table 2.The growth of S. choleraesuis and L. monocytogenes was inhibited by the essential oil from C. citratus at a minimum concentration of 15.63 µg•mL −1 .E. coli was more sensitive to the essential oils from C. citraus and P. boldus, both presenting a MIC of 31.25 µg•mL −1 .P. aeruginosa was not sensitive to any of the essential oils tested.S. aureus is a micro-organism that was sensitive only to the essential oil from P. boldus (62.5 µg•mL −1 ).Antibacterial activity against Gram-positive and Gram-negative bacteria was observed for the essential oils from M. piperita, C. citratus and P. boldus.These results differ from those of most studies in which Gram-positive bacteria are reported to be more sensitive to essential oils because they have no outer membrane to act as a protective barrier against macromolecules and hydrophobic compounds.Because of this morphology, Grampositive bacteria are relatively less resistant to antibiotics and other hydrophobic drugs, as well as essential oils [23].
Valeriano et al. [24], analyzing the antibacterial activity of the essential oil from M. piperita against the same bacterial strains evaluated in this study, observed that this species had a higher activity against E. coli and a low activity against L. monocytogenes.According to the authors, menthol, neoisomenthol, menthone, methyl acetate and iso-menthone were the principal components, whereas in the present work, similar activities against both E. coli and L. monocytogenes were observed for the essential oil from this species, and the principal constituents found were carvone and limonene.
Studies report that menthol is active against various micro-organisms, including P. aeruginosa [24].Subsequently, work performed by Valeriano et al.
[24] also showed that menthol is not the only agent responsible for the antimicrobial properties of M. piperita.The observations made by Iscan et al. [25] are not consistent with the findings of the present study because we observed no activity for the essential oil of this species against P. aeruginosa.According to Silva et al. [26], the essential oil from C. citratus inhibited the growth of S. aureus (Gram-positive), and the MIC was 7.81 µg•mL −1 .The growth of P. aeruginosa and E. coli (Gram negative) was also inhibited with an average MIC of 15.62 µg•mL −1 .The results of the present work partially corroborate their results [26]; the MIC for the essential oil from C. citratus in this work was 15.63 µg•mL −1 for the inhibition of the growth of L. monocytogenes and E. coli.

Antioxidant Activity of Essential Oils
The percentages of antioxidant activities of the essential oils and BHT determined by the DPPH radical sequestration method are presented in Table 3.A low antioxidant activity was observed for the essential oils.A higher antioxidant activity was observed for the BHT standard than for the essential oils.The activities of the essential oils from M. piperita, C. citratus and R. officinalis did not differ significantly in any of the concentrations tested.As for the essential oil from P. boldus, there was a significant difference at the highest concentration (500 µg•mL −1 ); the same effect was observed with the essential oil from F. vulgare.
The low antioxidant activity of the essential oils is related to their composition because no compound that could easily donate a hydrogen atom to stabilize the DPPH radical, nor any compound having a conjugated double bond, where stabilization could also occur, was found.Yadegarinia et al. [27] conducted research on the antioxidant activity of the essential oil from M. piperita using the DPPH method and observed an activity of 69%.The principal components of the essential oil were α-terpinene (19.7%), piperitenona oxide (19.3%), transcarveol (14.5%) and isomenthone (10.3%).Their data do not match those found in this study.Means followed by the same lower case letter in the columns and the same upper case letter in the row do not differ significantly by the Scott-Knott test (p ≤ 0.05).
Guimarães et al. [28] studied the antioxidant activity of the essential oil from C. citratus and its main constituent, citral, via the capture of the DPPH radical and observed a low antioxidant activity for both.According to the authors, this fact can be justified by the fact that it is difficult for the constituents to donate a hydrogen atom to stabilize the DPPH radical.These results are consistent with those found in this work.Teixeira et al. [29] assessed the antioxidant activity of the essential oil from R. officinalis using the DPPH-sequestration method and observed no significant potential for the samples when compared to the standard, because the principal compounds identified by the authors were camphor (35.50%), 1,8-cineole (18.20%), and bornyl acetate (13.40%).Camphor (17.34%) and 1,8-cineole (62.26%) were also found in this work, but in different concentrations.According to the authors, the low activity of the essential oil from R. officinalis is due to the constituents present in the oil, which did not possess structures favorable for donating a hydrogen atom or an electron to stabilize the DPPH radical.
Among the constituents of the essential oil from P. boldus, terpinen-4-ol has a hydroxyl group (-OH) that could donate a hydrogen atom, but there would be no stabilization of the resulting alkoxy radical by resonance.Therefore, its antioxidant potential by the DPPH method is low.Roby et al. [30] observed a high antioxidant activity for F. vulgare seeds by the DPPH method.The authors found trans-anethole (56.40%), fenchone (8.26%), and estragole (5.20%) in the essential oil from this plant.In the present work, trans-anethole (a phenylpropanoid) was not identified in the essential oil from this species, but another phenylpropanoid, estragole, was identified.Trans-anethole is a compound that can donate an electron, and the resulting radical can be stabilized by resonance because it has a conjugated double bond.This feature is not present in the structure of estragole, and this fact may explain the low activity observed in this work because it was found to be the principal compound.
The results obtained for evaluation of the antioxidant activity of the essential oils and BHT using the β-carotene/linoleic acid method are described in Table 4.The highest antioxidant activity was observed for the essential oil from C. citratus (5.81%) at a concentration of 500 µg L −1 , followed by F. vulgare, P. boldus and M. piperita, which differed statistically from the control but possessed a low activity.
A higher antioxidant activity at the concentration of 150 µg L −1 was observed for the essential oils from M. piperita and R. officinalis, whereas a higher antioxidant activity in the two highest concentrations was observed for the essential oil from F. vulgare.The result was statistically different from those of the other essential oils.An increase in antioxidant activity with concentration was found for the essential oil from P. boldus, and the highest activity was observed at a concentration of 500 µg•mL −1 (3.37%) Yadegarinia et al. [27] analyzed the antioxidant activity of the essential oil from M. piperita L. They found a 50.17% inhibition of oxidation using the βcarotene/linoleic acid method.These data do not corroborate those found in the present work because the principal constituents identified by those authors (αterpinene, piperitone oxide, trans-carveol, isomenthone and β-caryophyllene) were different from those found in the present study (carvone and limonene).
Guimarães et al. [28] determined the antioxidant activity of the essential oil from C. citratus and its principal constituent (citral) employing the emulsified β-carotene/linoleic acid system and noted that these substances exhibited an antioxidant activity of 46.45% and 38.00%, respectively, at an essential oil or citral concentration of 100 μg•L −1 .Their data did not corroborate those found in this study.The authors compared the antioxidant activity of the essential oil and citral and observed a higher activity for the essential oil than for citral.This fact shows that the principal constituent is not always the agent that is responsible for the activity, but rather all the components might act in synergy.Gachkar et al. [31] evaluated the antioxidant activity of the essential oil from R. officinalis and found that the principal constituents were piperitone (23.70%), α-pinene (14.90%), linalool (14.90%) and 1.8-cineol (7.43%).They observed approximately 60% inhibition of oxidation and concluded that this oil can be used in food preservation.Their results are different from those found in this work, where the essential oil from R. officinalis presented only 1.64% of antioxidant activity.Constituents that have structures of an intermediate polarity, such as the aldehyde group, (-CHO), provide some hydrophobicity and exhibit large antioxidant activities in the β-carotene/linoleic acid system.They are more effective in protecting linoleic acid because they are more highly concentrated in the lipid phase [32].
A significant difference in antioxidant activity of the essential oils from the control at all concentrations was observed with the use of the ABTS method (Table 5), the control being more effective.A significant difference in the effects of the essential oils from M. piperita, R. officinalis and P. boldus was only observed at the concentration of 500 µg•mL −1 , whereas no statistical difference between the concentrations was observed for the C. citratus and F. vulgare oils.According to Singh, Shushni and Belkheir [33], a high antioxidant potential (approximately 90% inhibition) was observed for the essential oil from M. piperita.According to the authors, the essential oil from M. piperita is composed of menthol, menthone, menthofuran and menthyl acetate.The authors emphasized the importance of natural products with an antioxidant potential for the food, cosmetic and pharmaceutical industries.The essential oils can be used as possible substitutes for synthetic products because they can prevent oxidation of food and can be used in the production of pharmaceuticals and cosmetics that remove free radicals present in the body.
Jordan et al. [34] stated that the essential oil from R. officinalis has a significant antioxidant potential for radical capture methods like the ABTS method.
The authors report that essential oils with significant levels of γ-terpinene, α-terpinene, terpinolene and caryophyllene oxide present a significant activity.According to the authors, γ-terpinene, α-terpinene, terpinolene and caryophyllene oxide were not identified as the principal constituents, but they were present in concentrations sufficient for the capture of radical.However, these components were not identified in the present study.This fact explains the low activity observed because the principal constituents (1,8-cineol, α-pinene and camphor) are not capable of donating hydrogen atoms for the stabilization of ABTS.
The percentage of antioxidant activity of the essential oils measured by the hydroxyl method is presented in Table 6.The essential oil from M. piperita exhibited the highest activity (62.8%) at a concentration of 500 µg•mL −1 , followed by R. officinalis (46.2%), C. citratus (42.2%),F. vulgare (31.3%) and P. boldus (25.8%).According to the literature, the standard to be used would be mannitol; however, a low antioxidant activity, approximately 20% at the highest concentration, was observed for this compound.
Mimica-Dukic et al. [35] studied the essential oils from M. piperita, M. longifolia and M. aquatica.Greater activity was observed for the essential oil from M. and menthone (9.0%).The constituents identified in this study were not the same as those found by those authors, which may explain the difference in activity observed via the hydroxyl method.Essential oils that possess components capable of stabilizing the OH radical will not attack deoxyribose, and consequently, malondialdehyde will not be formed and no reaction with TBA will occur.
The greatest activity was observed for the essential oils from C. citratus and P. Boldus (Table 7) when the molybdenum reduction method was employed.It is noted that 1.54 and 1.82 µg of the essential oils from C. citratus and P. Boldus, respectively, were equivalent to 1 g of ascorbic acid, whereas it required 20 µg of the essential oil from F. vulgare to obtain the same equivalence.
Silva et al. [36] evaluated the antioxidant activity of the essential oil from the plants of the Lamiaceae family (Mentha pulegium and Mentha viridis) by the molybdenum reduction method.They found that a higher activity was observed for the essential oil from M. pulegium (50.0% of pulegone, 31.9%menthol and 16.6% menthone) than for the standard used (BHT), followed by the essential oil from M. viridis, which is composed of 40.7% linalool, 13.9% carvone and 8.6% α-terpinene.The data do not corroborate those found in this study because no significant activity was observed for the essential oil from M. piperita using the molybdenum reduction method.The different compositions of the essential oils may have influenced the results.
No antioxidant activity for the essential oils (M.piperita, C. citratus, R. officinalis, F. vulgare and P. boldus) was observed by the reducing power method; that is, no reduction of the iron ion occurred.Silva et al. [36] observed a limited activity in reducing the iron ion for the essential oil from M. viridis.However, the activity observed for the essential oil from M. pulegium at the concentrations of 0.78 to 50 µL•mL −1 exceeded that of the BHT standard.Although the essential oils are from plant species of the same genus and family, they may contain dif- Teixeira et al. [29] evaluated the antioxidant properties of the essential oil from R. officinalis by the reducing power method and observed an activity lower than the equivalent of 5 micromoles of ascorbic acid per gram of essential oil.No such activity was observed in the present study.

Conclusion
The principal constituents found in the essential oil from M. piperita were carvone and limonene.Geranial, neral and myrcene were found in the essential oil from C. citratus.Camphor, 1,8-cineole, and α-pinene were found in the essential oil from R. officinalis.The essential oil from P. boldus contained α-terpinyl formate, p-cymene, 1,8-cineole, ascaridol and terpinen-4-ol, and the essential oil from F. vulgare contained methyl chavicol, limonene and fenchone.In the evaluation of the antibacterial activity, S. aureus was more sensitive to the oil from P. boldus.Listeria monocytogenes and Salmonella choleraesuis were sensitive to the essential oil from C. citratus.Escherichia coli was affected by the essential oils from C. citratus and P. boldus, whereas Pseudomonas aeruginosa was resistant to all the oils tested.All the essential oils tested presented low or no antioxidant activities with the radical sequestration methods (DPPH and ABTS).They were not effective in protecting against the oxidation of linoleic acid.They were effective only in the protection of deoxyribose, where the peppermint oil presented the highest antioxidant activity.The activities of the essential oils were greater than those of the standard (mannitol).In the molybdenum reduction method, the essential oils from C. citratus and P. boldus were more effective in reducing molybdenum than the other oils, and no antioxidant activity was observed by the reducing power method.

Table 3 .
Percentage of antioxidant activity of the essential oils and the BHT standard measured by the sequestration of DPPH radicals.

Table 4 .
Percentages of antioxidant activity of essential oils and the BHT standard by the β-carotene/linoleic acid method.Means followed by the same lower case letter in the columns and the same upper case letter in the row do not differ significantly by the Scott-Knott test (p ≤ 0.05).

Table 5 .
Percentages of antioxidant activity of the essential oils and BHT standard using the ABTS method.Means followed by the same lower case letter in the columns and the same upper case letter in the row do not differ significantly by the Scott-Knott test (p ≤ 0.05).

Table 6 .
Percentage of antioxidant activity of the essential oils and the mannitol standard by the hydroxyl method.Means followed by the same lower case letter in the columns and the same upper case letter in the row do not differ significantly by the Scott-Knott test (p ≤ 0.05).

Table 7 .
Antioxidant activity of the essential oils determined by the phosphomolibdenum reduction method.