Antioxidant and antibacterial activities and composition of Brazilian spearmint (Mentha spicata L.)

Industrial Crops and Products 50 (2013) 408–413

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Antioxidant and antibacterial activities and composition of Brazilian spearmint (Mentha spicata L.) Rodrigo Scherer ∗ , Mayara Fumiere Lemos, Mariana Fumiere Lemos, Gésika Coimbra Martinelli, João Damasceno Lopes Martins, Ary Gomes da Silva Universidade Vila Velha, Rua Comissário José Dantas de Melo no. 21, Boa Vista, Vila Velha, ES CEP: 29102-770, Brazil

a r t i c l e

i n f o

Article history: Received 16 March 2013 Received in revised form 27 June 2013 Accepted 3 July 2013 Keywords: AAI Mint Antimicrobial DPPH

a b s t r a c t Antioxidant and antibacterial activities and the composition of Brazilian spearmint (Mentha spicata) extracts were evaluated. They were obtained by maceration with methanol, acetone, and dichloromethane, and the essential oil was obtained by hydrodistillation. The antioxidant activity was determined by antioxidant activity index (AAI), and the antimicrobial activity was evaluated by the diffusion method and by determination of minimum inhibitory concentration against Staphylococcus aureus and Escherichia coli. Phenolic compounds were determined by the Folin–Cioucalteu method, and the essential oil composition was identified by GC/MS. The methanolic extract showed a higher content of total phenolic compounds and stronger antioxidant activity, while only the essential oil showed antibacterial activity. The major compounds of the essential oil were carvone (67%), limonene (14.3%), muurolene (2.3%), and myrcene (2.1%). © 2013 Elsevier B.V. All rights reserved.

1. Introduction Many plants are used as spices to enhance food flavor and are consumed in small quantities, contributing in low levels to the nutritional value of the diet. However, as they are secondary metabolism compounds that may have pharmacological activity, there is currently a growing interest in plant extracts as sources of antimicrobial and antioxidant compounds as a means of avoiding potential problems caused by excessive consumption of synthetic additives. The current literature presents several studies on the biological activity of plant extracts as anti-tumor agents (Kaileh et al., 2007), anti-inflammatories and analgesics (Bose et al., 2007), antifungals (Korukluoglu et al., 2008), antibacterials and antioxidants (Singh et al., 2011; Castilho et al., 2012), among others. Mentha spicata L., commonly called spearmint, belongs to the Lamiaceae family, genus Mentha, which comprises about 25–30 species originating in Europe. It is one of Brazil’s most cultivated varieties of spearmint and is well adapted to the subtropical climate. The interest in cultivating Mentha is mainly related to the commercial importance of its essential oil, which is among the 10 most traded in the world. The oil is used in many industries, including pharmaceuticals, cosmetics, food, and chemicals. Spearmint is also known for its ability to improve memory (Adsersen et al., 2006). Besides being a stimulant (Papachristos and Stamopoulos,

2002), it has several biological uses, such as in insecticides (Samarth and Kumar, 2003), antimicrobials (Ozgen et al., 2006), antioxidants (Choudhury et al., 2006), antispasmodics, and anti-platelets (Tognolini et al., 2006). The different species of Mentha present considerable diversity in their essential oil chemical composition. For example, in M. spicata, the essential oil is rich in carvone and has a characteristic smell of spearmint (Jirovetz et al., 2002), while in Mentha piperita, menthol is the main component (Singh et al., 2011). In addition to such natural variation in composition among plant species, the aromatic and pharmacological properties of the plant extracts can be significantly affected by soil and climatic conditions, as well as by the season and the time the plant material is collected (Viuda-Martos et al., 2008; Shanjani et al., 2010; Butkiene and Mockute, 2011). Considering the economic potential of spearmint and the fact that the composition of plants may be affected by soil and climatic conditions, and the lack of any study on spearmint grown in the state of Espírito Santo (Brazil), the objective of this study was to evaluate the composition, the antioxidant activity, and the antimicrobial activity of extracts and essential oils of spearmint (M. spicata) grown and purchased in the city of Vila Velha (ES/Brazil). 2. Materials and methods 2.1. Materials

∗ Corresponding author. Tel.: +55 27 3421 2072; fax: +55 27 3421 2049. E-mail addresses: [email protected], [email protected] (R. Scherer). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.07.007

Samples of spearmint (M. spicata) were purchased from producers in Vila Velha (ES, Brazil). All tests were performed using only the aerial parts of the plant. The free radical 2,

R. Scherer et al. / Industrial Crops and Products 50 (2013) 408–413

2-diphenyl-1-picril-hydrazyl (DPPH), BHT, ferulic acid, caffeic acid, chlorogenic acid, gallic acid, Folin–Ciocalteau reagent, sodium carbonate, and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma–Aldrich company (USA). The brain–heart infusion broth (BHI) and Mueller–Hinton agar were purchased from HiMed Laboratories Pvt. (Mumbai, India). DMSO (dimethyl sulfoxide), methanol, acetone and dichloromethane were purchased from Vetec (Rio de Janeiro, Brazil). Saturated alkanes std. (C7–C30) was purchased from Supelco (USA). 2.2. Proximate analysis Chemical analysis of the samples was performed according to the official AOAC method (1995) (n = 7). 2.3. Extracts and essential oil Plant samples were dried in a tray drier with air circulation at 45 ◦ C and ground to powder in a mill. Extracts were obtained using acetone, dichloromethane, and methanol as solvents. Maceration was done with 20 g of powdered dried plant in 100 mL of each solvent. After 7 days under periodic agitation, the extracts were filtered through filter paper, and the residue was extracted again, with 100 mL of the respective solvents for 24 h. Both fractions were then mixed and evaporated to dryness at 40 ◦ C under vacuum. The essential oil was obtained by hydrodistillation using Clevenger extractor with aerial parts from spearmint “in nature”. The samples were crushed with ultra-pure water in a blender before being transferred to the distillation flask. After extraction, the essential oil was transferred to a glass vial, and its purification was made by separation of the remnant water by freezing, and the essential oil, which was kept in liquid phase, was drained from the vial. All extracts were stored in amber bottles at 5 ◦ C until analysis.

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in a vortex mixer. The mixture was then incubated for 2 h in the dark at room temperature. The absorbance at 740 nm was measured and converted to the phenolics content according to a calibration curve made with gallic acid.

2.6. Antioxidant activity The antioxidant activity of the extracts and standards was determined through radical scavenging activity by using 2,2diphenyl-1-picrylhydrazyl radical (DPPH) according to Scherer and Godoy (2009). Aliquots of 0.1 mL of methanol solutions of samples or standards in different concentrations were added to 3.9 mL of a methanol solution of DPPH. DPPH solutions were prepared by dissolving 24.5 mg in 500 mL of methanol. The blank sample consisted of 0.1 mL of methanol added to 3.9 mL of DPPH solution. The tests were carried out in triplicate. After a 90 min incubation period at room temperature in the dark, the absorbance was measured at 517 nm. The radical scavenging activity was calculated as follows: I % = [(Abs0 − Abs1)/Abs0] × 100, where Abs0 was the absorbance of the blank and Abs1 was the absorbance in the presence of the test compound at different concentrations. The IC50 (concentration providing 50% inhibition) was graphically calculated using a calibration curve in the linear range by plotting the extract concentration vs the corresponding scavenging effect. The antioxidant activity was expressed as the antioxidant activity index (AAI), calculated as follows: AAI = final concentration of DPPH (␮g/mL)/IC50 (␮g/mL). The assays were carried out in triplicate, and all the samples, the standard, and the DPPH solutions were prepared daily. All solutions of standards and extracts were prepared in 50 mL volumetric flasks.

2.7. Antibacterial activity 2.4. GC/FID and GC/MS analysis The identification of the essential oil components was carried out by high resolution gas chromatography analysis, in the fine chemistry laboratory accredited to ISO/IEC 17025, at Tommasi Analítica (Vila Velha, ES, Brazil). A Thermo Scientific TraceUltra gas chromatograph coupled with a Thermo Scientific DSQII quadrupole mass spectrometer (identification) and Thermo Scientific Focus gas chromatograph coupled with FID detector (quantification) were used. The compounds were separated in a DB-5 fused silica capillary column 30 m × 0.25 mm × 0.25 ␮m film thickness (J&W Scientific, Folson, USA). Helium was the Carrier gas at a flow rate of 1.0 mL/min. The analyses were performed using splitless injection at 220 ◦ C. The oven temperature program used was 60–240 ◦ C at 3 ◦ C/min, and the final temperature was held for 7 min. The GC/MS interface and FID detector were maintained at 240 ◦ C and 250 ◦ C, respectively. The oil was dissolved in hexane (2 mg/mL) for the analyses. The MS data were obtained in the scan mode (35–400 m/z), and Kovats retention indices (KI) were determined by injection of standard hydrocarbon solutions (C7–C30). The components were identified by comparison with data from the literature (Adams, 1995) and with the profiles from the NIST Mass Spectral Library (version 2.0, 2005), and by injection of pure compounds, when available. 2.5. Total phenolic compounds The total phenolics content was determined using the Folin–Ciocalteau reaction. An aliquot of 0.5 mL of a methanolic solution of dry extracts (1.5 mg/mL) was added to 2.5 mL of Folin–Ciocalteau reagent diluted with water (1/10). After 5 min, 2.0 mL of 7.5% sodium carbonate was added and stirred vigorously

The antibacterial activity was conducted by the diffusion method and determination of minimum inhibitory concentration (MIC) against Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 8739). The final bacteria concentration was adjusted at the First point of McFarland’s scale in order to achieve 108 CFU/mL in saline (0.85%). In the diffusion method, using Mueller–Hinton solid medium (38 g/L), the strains were inoculated using sterile swabs. Then cavities were made by glass tubes with a diameter of approximately 6 mm, and 50 ␮L samples (2 mg/mL in DMSO and saline – 0.85%) were added and incubated at 37 ◦ C for 24 h. To determine the minimum inhibitory concentration (MIC), the extracts were prepared at a concentration of 4.0 mg/mL in dimethyl sulfoxide (DMSO) and subsequently diluted with saline (0.85%) to use. The final concentration of cells was adjusted by the scale of McFarland 1 in the order of 108 CFU/mL. To each well, 100 ␮L of culture medium (BHI 3.7%), 100 ␮L of sample, and 100 ␮L of inoculums were added. In all plates, positive and negative controls (six wells of each) were inserted. In the positive controls, 100 ␮L of culture medium, 100 ␮L of inoculum, and 100 ␮L of saline (0.85%) were added. In the negative controls, 100 ␮L of culture medium, 100 ␮L of the respective extract, and 100 ␮L of saline (0.85%) were added. The extracts were evaluated from 0.67 to 0.09 mg/mL at final concentrations. After the inoculum addition, the plates were incubated at 36 ◦ C for 24 h, and then 50 ␮L of the indicator MTT (0.1% in saline 0.85%) was added. After 4-h incubation, the MIC was determined as the lowest concentration that inhibits the bacterian visible growth given by the MTT (dead cells were not stained). The absorbance was monitored at 590 nm in microplate reader Thermo Plate (TPReader), and the inhibition rate was determined by the following formula: inhibition % = ((Abs B − Abs A)/Abs B) × 100, where “Abs

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Table 1 Proximate composition of the aerial parts of spearmint (Mentha spicata). Sample

Moisture (g/100 g)

Ash (g/100 g)

Protein (g/100 g)

Fat (g/100 g)

Carbohydratre (g/100 g)

Aerial parts

86.0 ± 0.4

1.7 ± 0.0

2.3 ± 0.0

0.4 ± 0.1

9.6

A” is the absorbance of the sample and “Abs B” is the absorbance of the positive control. 2.8. Statistical analysis Data were analyzed using ANOVA/Tukey (p < 0.05) by the software StatisticaTM 6.0 Statsoft, Inc. 3. Results and discussion 3.1. Proximate analysis Spearmint is commonly used to give a pleasant flavor and aroma to many foods and beverages, but is used in small quantities, and does not interfere with nutritional values, however, there are no reports about its composition. Table 1 shows the values of the chemical composition of a 100 g sample, which presents content high in moisture and low in fat, as characteristic of foliaceous plants. 3.2. Essential oil composition In the essential oil chromatographic analysis (Fig. 1), 37 compounds were identified, representing 97.32% of the total oil (Table 2). The spearmint essential oil was characterized by the dominant presence of carvone (67.1%), in agreement with other authors, such as Kokkini et al. (1995), who identified carvone occurring at 68.4% in samples collected in Greece, Chauhan et al. (2009), who found it at 76.65% in samples collected in India, and S¸arer et al. (2011), who found it at 48.4% in samples collected in Turkey. Also, in essential oil from M. spicata collected in Montenegro (Europe), Sokovic and Van Griensven (2006) reported carvone as the major constituent (49.52%), followed by mentone (21.92%) and limonene (5.77%). However, in the present study, limonene was found at 14.3%, but mentone was not found. This variation in the composition of essential oil may be attributed to various factors in growing conditions, such as temperature, humidity, radiation, climate, and soil type. Other significant constituents in the essential oil of M. spicata included muurolene (2.29%), myrcene (2.08%), beta-caryophyllene (1.76%), beta-bourbonene (1.18%), and transdihidrocarvone (1.03%). 3.3. Total phenolic compounds Phenolic compounds are the major class of natural antioxidants present in plants and are usually quantified using the Folin–Ciocalteu method. The results obtained in the determination of total phenolics, expressed as gallic acid equivalents (GAE) per gram of dry extract, are presented in Table 3. Methanol had the highest yield of crude extract and phenolic contents (p < 0.05), when compared with those from acetone and dichloromethane. Dorman et al. (2003) reported that total phenolic content in different varieties of Mentha is about 128–230 mg/g of extract in gallic acid equivalent, and the main phenolic compounds found in extracts of M. spicata were eriocitrin, luteolin, rosmarinic acid, and caffeic acid. However, the amount of phenolic compounds found in this work is below that range. The total phenolic content in the extract obtained with dichloromethane could not be determined because it was not solubilized in methanol.

Table 2 Spearmint essential oil composition. Compound

CAS no.

RT

KI 1

KI 2

%**

Pinene (alpha) Sabinene Pinene (beta) Myrcene Limonene linalool Ocimene (Allo) Ocimene (Neo-Allo) Santolinyl acetate Terpin-4-ol Dihydro carvone (trans) Carveol (trans) Carvone Carvone oxide (trans) Dihydro carveol acetate Elemene (delta) Carvyl acetate* Carvone oxide (cis)* Copaene (alpha) Bourbonene (beta) Elemene (beta) Gurjunene (alpha) Caryophyllene (beta) Gurjunene (beta) Humulene (alpha) Muuorola-4(14),5-iene (cis) Muurolene (gamma) Bicyclogermacrene Calamenene (cis) Cadinene (alpha) Muurol-5-en-beta-ol (cis) Muurol-5-en-4-alpha-ol (cis) Germacrene D-4-ol Cubenol (1,10-di-epi) Cadinol (epi-alpha) Cadinol (alpha)

80-56-8 3387-41-5 127-91-3 123-35-3 138-86-3 78-70-6 7216-56-0 3016-19-1 79507-88-3 562-74-3 5948-04-9 1197-07-5 99-49-0 39067-90-8 20777-49-5 20307-84-0 1205-42-1 35178-55-3 3856-25-5 5208-59-3 515-13-9 489-40-7 87-44-5 17334-55-3 6753-98-6 – 30021-74-0 24703-35-3 72937-55-4 24406-05-1 – – 74841-87-5 73365-77-2 11070-72-7 481-34-5

5.67 6.78 6.88 7.17 8.57 11.12 12.35 12.63 14.08 14.39 15.20 16.00 17.69 18.53 20.53 20.82 21.95 22.01 22.64 22.99 23.24 23.93 24.47 24.87 25.89 26.18 26.99 27.53 28.52 29.15 29.55 29.93 30.70 32.19 33.19 33.69

936 976 979 989 1031 1100 1127 1140 1174 1181 1199 1219 1246 1278 1325 1332 1359 1360 1375 1383 1388 1404 1418 1428 1453 1460 1480 1492 1518 1534 1545 1554 1574 1612 1639 1652

939 976 980 991 1031 1098 1129 1142 1171 1177 1200 1217 1242 1277 1325 1339 1362 1363 1376 1384 1391 1409 1418 1432 1454 1460 1477 1494 1521 1538 1545 1554 1574 1614 1640 1653

0.51 0.14 0.69 2.08 14.34 0.48 0.03 0.11 0.34 0.07 1.03 0.79 67.08 0.03 0.06 0.05 0.24 0.09 1.18 0.38 0.21 1.76 0.19 0.63 0.66 2.29 0.33 0.72 0.10 0.02 0.03 0.11 0.29 0.05 0.21 97.32

Total

RT: retention time (GC/MS); KI 1: experimental; KI 2: literature (Adams, 1995); –: not found. * Co-elution. ** Quantified by GC/FID.

3.4. AAI results Various methods are used to determine the antioxidant activity of plant extracts and pure substances, though the DPPH (2,2-diphenyl-1-picril-hydrazyl) method is the most used in the quantification of free radical scavenging activity. The reaction is based on the decrease of the purple color that occurs when the nitrogen atom of the DPPH is reduced by receiving a hydrogen atom from the antioxidant component (Brand-Williams et al., 1995). Table 3 Yield and concentration of total phenols in extracts of spearmint. Solvent

Yield of extraction (g/100 g)

TFC mg/g of dry extract

ME AC DI HD

5.96 1.88 2.72 0.04

76.32 ± 3.42a 37.84 ± 1.16b Nd 15.16 ± 0.50c

AC: acetone; DI: dichloromethane; ME: methanol, HD: essential oil; Nd: not evaluated. TFC: total phenolic compounds. Different letters in the same column represent significant difference (p < 0.05).

R. Scherer et al. / Industrial Crops and Products 50 (2013) 408–413 RT: 0,00 - 67,02

411

17,69

100

NL: 3,64E9 TIC MS HORTELA_ 111028100 633

90 80 70 60 50 40 30

8,57

20 22,99

10 0

7,17

4,00 0

5

11,12 15,20

22,01

10

20

15

26,99 24,47 28,52 25

30

32,19 33,69 35 Time (min)

43,31 46,47 48,97 40

45

50

56,07 55

63,39 60

65

Fig. 1. GC/MS of spearmint essential oil diluted with hexane (2 mg/mL).

Because of the difficulty of comparing the antioxidant activity of different plants, Scherer and Godoy (2009) not only proposed a new index to express the strength of natural antioxidant compounds (AAI), but also established a ranking for antioxidant activity of plant extracts in relation to AAI values: weak antioxidant activity (AAI < 0.5), moderate (0.5–1.0), strong (1.0–2.0), and very strong (AAI > 2.0). Recently, Deng et al. (2011) proposed a new index, the antioxidant activity unit (AAU) for that purpose based on the work of Scherer and Godoy (2009), and the authors reported that they found some limitations in the AAI, such as lack of a precise definition and high values absorbance (between 2 and 3), which would be outside the range of accuracy according to Beer’s law. Regarding the first observation of Deng et al. (2011), the definition is clear and simple, just a relationship between the mass ratio of radical used in the assay, with the mass of extract needed to reduce 50% of the radicals, generating a constant called the AAI. On the second point, according to the equation of the Beer’s law, the absorbance is proportional to the optical path: the larger the size of the cell, the greater the absorbance value for the same concentration. Scherer and Godoy (2009) evaluated the linearity of different concentrations of DPPH in methanol using 1 cm2 cell, and the results showed that the absorbance values were at the range of 0.05–2.0, being the recommended concentration of 0.1 mM, which generates an absorbance near 1.0. In this study, the linearity of the solutions of DPPH (0.05–1.5 mM) was repeated using two different spectrophotometers with 1 cm2 cells (Hach DR 5000: certificate of ISO/IEC 17025 no. EVO-1882/11 and T80+ UV/VIS, PG Instruments Ltd), and the results agreed with Scherer and Godoy (2009). In order to get satisfactory reproducibility on the AAI results, some precautions were taken, such as the use of calibrated analytical balance and the use of high quality volumetric flasks to prepare the standard solutions. Other precautions included protecting standard solutions and plant extracts from the excessive exposure to light, verifying the purity degree and validity of materials, and finally, training the laboratory workers. The results of mint extracts are presented in Table 4. Gallic acid showed the highest value of the AAI (p < 0.05), followed by caffeic acid. The other compounds, such as chlorogenic acid, ferulic acid, and BHT, had lower values of AAI. However, all of them were considered strong antioxidants, according to the classification. The differences in activity can be explained by the relationship between structure and activity, because the structure of phenolic compounds is a key determinant for the oxidation of free radicals (Rice-Evans et al., 1996). The greater the number of the aromatic

ring hydroxylations handled, the greater the activity of phenolic acids (Scherer and Godoy, 2009), as in the case of gallic acid, which showed the highest values of AAI. The replacement of the hydroxyl group in the aromatic ring by a methoxyl group reduces the value of AAI, which explains caffeic acid being more active than ferulic acid. Spearmint methanolic extract showed a strong antioxidant activity, according to the proposed classification (AAI = 2.08). On the other hand, the essential oil and acetone and dichloromethane extracts showed no ability to reduce free radical DPPH. Thus, it is suggested that the polar components of the plant have a higher antioxidant capacity than the non-polar compounds by the DPPH method, as shown by the solvent methanol having a higher polarity index (ability to make hydrogen bonds). Kanatt et al. (2007) confirmed the antioxidant activity of M. spicata during radiation processing of lamb, in that the aqueous extract was able to retard lipid peroxidation and give a pleasing aroma and flavor to the meat. According to Tepe et al. (2007), plants of the Lamiaceae family are very rich in phenolic compounds, and these have been shown to have antioxidant activity, which agrees with this work, since the methanol extract showed a higher content of phenolic compounds and higher antioxidant activity. 3.5. Antibacterial activity In tests carried out by the diffusion method, formation of inhibition halos indicating the absence of antimicrobial activity of all extracts for both microorganisms was not observed. On the other hand, in the MIC determination, the essential oil showed strong activity against strains of E. coli and S. aureus. However, the extracts obtained with methanol, acetone, and dichloromethane did not show significant activity. The microorganism S. aureus was more sensitive to essential oils, and a final concentration of 0.67 mg/mL showed 100% inhibition of growth. For E. coli, the highest concentration tested (0.67 mg/mL) showed 51.3% inhibition, and therefore the MIC could not be determined. Boukhebti et al. (2011) reported that Gram-positive bacteria are more sensitive to essential oils than Gram-negative ones, due to the more complex cell wall in Gramnegative, agreeing with this work. Higher concentrations were not evaluated because the green coloration of the extracts affect the absorbance reading and may produce false results, so it is necessary to make the clarification by the removal of chlorophyll without affecting the results. The fact that the essential oil of spearmint has antimicrobial activity only in the MIC determination tests may be

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Table 4 Antioxidant activity index (AAI). DPPH*

Galic acid Caffeic acid Chlorogenic acid Ferulic acid BHT ME AC DI HD

Day I*

Média

SD

r2

IC50

AAI

Day II r2

IC50

AAI

Day III r2

IC50

AAI

AAI

AAI

0.980 0.984 0.982 0.963 0.976 0.992 – – –

1.6 2.1 4.29 6.13 9.38 17.99 – – –

24.3 18.6 9.1 6.36 4.24 2.25 – – –

0.995 0.978 0.985 0.989 0.994 0.991 – – –

1.65 1.81 3.65 5.91 9.11 21.7 – – –

23.61 21.4 10.68 6.6 4.37 1.99 – – –

0.996 0.98 0.96 0.996 0.999 0.994 – – –

1.51 1.97 4.12 6.18 12.44 21.86 – – –

25.81 19.76 9.47 6.31 3.2 1.98 – – –

24.59a 19.91b 9.75c 6.42d 3.94 e 2.08 e – – –

1.12 1.43 0.83 0.15 0.64 0.15 – – –

r2 : coefficient of linearity, IC50: expressed in ␮g/mL, SD: standard deviation, –: values were not found; ME: methanol, AC: acetone, DI: dichloromethane; HD: essential oil. Different letters in the same column represent significant difference (p < 0.05). * Tests carried out on 3 different days.

due to the difficulty of diffusion of compounds through the medium, because the compounds are mostly of low polarity, whereas the culture medium has polar character. Regarding the composition of the spearmint essential oil, carvone was found at 67% of the total composition. According to Kadoglidou et al. (2011), this monoterpenoid has antimicrobial activity, and this may be responsible for the antibacterial activity of spearmint’s essential oil reported in the present study. There is evidence in the literature that the essential oils of some plants of the Lamiaceae family have a moderate to good antibacterial activity (Sokovic and Van Griensven, 2006; S¸arer et al., 2011). Some works with plants of the genus Mentha have reported that the crude extracts present higher antioxidant activity than the essential oils; on the other hand, the essential oils were more effective against foodborne spoilage or pathogenic bacteria than the extracts obtained by extraction with solvents, as observed in the present study (Hussain et al., 2010; Teixeira et al., 2012; Dhifi et al., 2012). In another study, the essential oil showed strong antimicrobial activity against all 30 microorganisms tested, whereas the methanol extract remained almost inactive. In contrast, the extract showed much better activity than the essential oil in antioxidant activity assays employed (Gulluce et al., 2007). This fact can be explained by the presence of some non-volatile phenolic compounds in the crude extracts obtained by solvents, which are potential antioxidants, and generally not present in the essential oil, such as hydroxycinnamic acids, flavonoids, and others. On the other hand, the essential oils are composed mainly of terpenoids, i.e. carvone, which exhibit strong antimicrobial activity, and their concentrations are low in the crude extracts. 4. Conclusion The methanolic extract showed high amount of phenolic compounds and strong antioxidant activity. These results suggest it can be used as an alternative or substitute for the synthetic antioxidants that present harmful effects to humans. Furthermore, the spearmint essential oil, which has great commercial value in Brazil, showed good antimicrobial activity due to the high concentration of carvone (67%), suggesting that it can be used in the cosmetics and food industries. The essential oil can contribute to the preservation of products, and may develop pleasant sensory characteristics. Acknowledgements We acknowledge the Laboratory Tommasi Analítica (Vila Velha, ES, Brazil) for the cooperation in the chromatographic analysis; and FUNADESP for the Research Grant of Dr. Rodrigo Scherer and Dr. Ary Gomes da Silva.

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