Chemical Composition and Biological Properties of Essential Oils of Two Mint Species

Purpose: To analyze the composition of essential oils of two types of mint as well as compare the antimicrobial, antioxidant and anti-inflammatory activities of the two oils. Methods: Peppermint (M. piperita L.) and chocolate mint (M. piperita L.) oils were obtained by steam distillation in a Clevenger-type apparatus. The chemical composition of the essential oils was determined by gas chromatography-mass spectrometry (GC/MS). The minimal inhibitory concentration (MIC) of the essential oils were determined by broth dilution method. The antioxidant activities of the oils were determined by 2, 2-diphenyl-1-picrylhydrazyl (DPPH)DPPH radical scavenging assay, β-Carotene-linoleic acid assay, andnitric oxide (NO) radical scavenging assay. Results: The two essential oils contain high levels of alcohol (43.47-50.10%) and terpene (18.55-21.07%) with the major compound being menthol (28.19-30.35%). The antimicrobial activity (minimum inhibitory concentration, MIC) of peppermint oil against E. coli, S. aureus and P. aeruginosa (0.15, 0.08, 0.92 %v/v, respectively) was stronger than that of chocolate mint (0.23, 0.09, 1.22 %v/v, respectively). In the anti-oxidant test including DPPH and β-Carotenelinoleic acid assays, peppermint oil showed superior antioxidant properties to chocolate mint oil (4.45 19.86 μl/mL). However, with regard to scavenging NO radical activity, chocolate mint oil exhibited higher activity than peppermint (0.31 and 0.42 μl/mL, respectively). Chocolate mint oil also exhibited higher anti-inflammatory activity than peppermint oil (0.03 and 0.08 μl/mL, respectively). Conclusion: The results obtained should help to clarify the functional applications of these folk herbs and their essential oils for aromatherapeutic healing and other folkloric uses.


INTRODUCTION
Mints (Mentha spp.) are famous aromatic and medicinal herb that are used in traditional and folk medicines in the world for the antimicrobial and antioxidant properties.Mentha genus contains about 25 species and some hybrids and belongs to the Lamiaceae family [4].Mints contain volatile components, flavonoids, organic acids, quinones, such as for the digestive system, central nervous system, respiratory system [5,6].It was used in antimicrobial, antiinflammatory or anesthesia [7].M. piperita is a hybrid of spearmint (M.spicata L.) and water mint (M.aquatica L.), it grows particularly well in areas with high water-holding capacity soil [8].
In this study, we evaluated the major chemical compositions of the essential oils derived from the peppermint and chocolate mint by gas chromatography-mass spectrometry (GC-MS).We examined the antimicrobial activity of essential oils against some microorganisms.The antioxidant activities of the essential oils were determined by various antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, β-Carotene/linoleic acid assay, and nitric oxide (NO) radical scavenging assay.Furthermore, the antiinflammatory activities of the essential oils were determined by 5-lipoxygenase (5-LOX) inhibition assay.

EXPERIMENTAL Plant material and isolation of essential oil.
Peppermint (M.piperita L.) and chocolate mint (M.piperita L.) were purchased from a horticulture shop, Tangshan Herb Gardening, in Puli, Nantou County, Taiwan.The voucher specimens (peppermint no.CHNA MP 11001; chocolate mint no.CHNA MP 11002) were identified and deposited in the herbarium of the Department of Cosmetic Science, Chin Nan University of Pharmacy and Science, Tainan, Taiwan.The whole fresh plants were washed with distilled water (room temperature).The essential oils were obtained by steam distillation in a Clevenger-type apparatus.The essential oils collected were filtered through a 0.45 μm filter and kept at 4 ℃ until further analysis.The steam distilled yields were about 0.3 ％.

Analysis of essential oil
The essential oils were analyzed by GC/MS using a Hewlett-Packard GC systems (HP6890 series Ⅱ) coupled with a mass detector (MSD5973) equipped with a HP-5MS capillary column (5 % phenyl methylsiloxane, 30 m × 0.25 mm id., 0.25 μm film thickness) (Agilent Technologies, Palo Alto, CA).For GC/MS detection, an electron ionization system with ionization energy of 70 eV was used over a scan range of 40-400 amu.Helium was used as a gas carrier at a flow rate of 1 mL/min.Split ratio was adjusted at 25:1.The column temperature was initially kept at 60 °C for 4 min, then gradually increased up to 240 °C at an increment of 3 °C/min, and finally held isothermal for 10 min.The percentage of components was calculated from total ion chromatograms.Identification of the primary component was assigned by matching mass spectral data with those detailed in Wiley 7n.1, and NIST02.L libraries [9].

Bacterial strains and culture conditions
The

Determination of antimicrobial activity
The minimal inhibitory concentration (MIC) of the essential oils for the inhibition of six bacterial strains were determined by the broth dilution method with some modifications.Each test was executed in corresponding media supplemented with 0.5％ Tween 80.Serial dilutions of the essential oils were prepared in a 96-well microtiter plate over a range 0.02 to 49 mg/mL.Overnight broth cultures of the various strains were prepared, and the final concentration in the various wells was adjusted to 2 × 10 4 CFU/mL.Plates were incubated at the corresponding culture temperatures, with incubation time of 24 h for E. coli, S. aureus, P. aeruginosa, and C. albicans, 48 h for P. acnes, and 72 h for P. ovale.MIC was defined as the lowest concentration of the essential oil at which the microorganism does not exhibit visible growth, as indicated by the turbidity of the medium.MIC was defined as the lowest concentration of the essential oil at which inoculated microorganism was completely killed [9].

Determination of antioxidant activity by DPPH radical scavenging assay
Each concentration of essential oils was mixed individually into a methanolic solution containing DPPH radicals (0.1mM), and the final volume was 1 mL.These solution mixtures were kept in dark for 30 minutes (incubation period) at room temperature.After thirty minutes, the absorbance was measured at 517 nm [11].Each test was carried out in three times.Finally, DPPH radical scavenging activity (DPPH) was determined as in Eq 1.

Determination of antioxidant activity by β-Carotene-linoleic acid assay
β-Carotene was dissolved in 0.2 mL of chloroform (1 mg/mL) and this was added to a solution of linoleic acid (20 mg) in 200 mg of Tween 40.Chloroform was evaporated using a rotary evaporator under vacuum at 40℃ for 5min.Distilled water (50 mL) was added to the flask and the mixture was stirred in a sonicator.Each concentration of essential oils was added individually to 4.8 mL of the emulsion and this was then incubated at 50℃ for 3h.The absorbance was measured at 470 nm [9].Reading of all samples were done immediately (t = 0 h) and after 3 h of incubation.The antioxidant activities of the essential oils were evaluated in term of inhibition of β-Carotene (inhibition) as in Eq 2.

Determination of anti-inflammatory activity
Linoleic acid was used as substrate for 5-LOX.
Various concentrations of 30 μL aliquots of essential oil with 30 μL of linoleic acid and potassium phosphate buffer (0.1 M, pH 6.3) containing 5-LOX (25 U), yielding a final volume of 3 mL.The mixture was incubated at 25℃ for 10 min, and the absorbance was determind at 234 nm [13].Because linoleic acid is enzymatically converted to a conjugated diene by 5-LOX, which results in a continuous increase in absorbance at 234 nm, inhibition activity (Infl) was calculated as in Eq 4.

Statistical analysis
All determinations were performed at least in triplicate.The results were analyzed by Student's t-test using Microsoft Excel 2007, and expressed as mean ± standard deviation (SD) for each measurement.Differences were recognized as significant at p < 0.05.
The results of the antioxidant assays of the two essential oils are stated in Table 3.The IC 50 value of peppermint essential oil were 4.45, 0.37 and 0.42 μl/mL for DPPH radical scavenging assay, β-Carotene-linoleic acid assay, and NO radical scavenging assay, respectively while the IC 50 values of chocolate mint essential oil were 19.86, 5.07, and 0.31 μl/mL, respectively.The antioxidant activities of peppermint essential oil were stronger than those of the essential oil of chocolate mint with regard to DPPH radical scavenging and β-Carotene-linoleic acid assays.but lower with regard to NO radical scavenging assay.For anti-inflammatory activity.test,IC 50 values were 0.08 and 0.03 μl/mL for essential oils of peppermint and chocolate mint, respectively (Table 3).The anti-inflammatory activity of chocolate mint essential oil was higher than that of peppermint essential oil.

DISCUSSION
The essential oils from peppermint and chocolate mint were analyzed to determine their chemical components.They were also tested for antimicrobial activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Pitrosporum ovale, Candida albicans, and Propionibacterium acnes.Besides, the essential oils were used in antioxidant, and anti-inflammatory assays.
Khan and Abourashed reported that peppermint yields 0.1 -1.0 % of volatile oil that is composed mainly of menthol (29 -48 %), menthone (20 -31 %), and menthyl acetate (3 -10 %) [14].This result is similar to ours.In our study, menthol and menthone were major compositions in both peppermint and chocolate mint essential oils.Menthyl acetate content was 0.81 % in peppermint but was practically absent from chocolate mint.
The components of mint oils vary with plant maturity, variety, geographical region and processing conditions [8, 15,16].
Many studies have assessed the antibacterial [17] and antifungal [8] activities of peppermints.Our results showed that Gram-positive S. aureus and P. acnes were more sensitive to the essential oils than Gram-negative E. coli and P. aeruginosa.The results were similar to Djenane et al (2012) [18].In previous studies, the mint essential oils had the ability to inhibit P. acnes [19].Therefore, this property could be used for developing new anti-acne or antimicrobial ingredients from peppermint oils for cosmetic or personal products.
The antioxidant activity had different results by different assay systems [9].The antioxidant activities of antioxidants have been attributed to various mechanisms, including the prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, and radical scavenging [20].

CONCLUSION
This study showed the two mint essential oils contain more alcohol and terpene and the major compound was menthol.The antimicrobial activity of peppermint against E. coli, S. aureus and P. aeruginosa was stronger than that of chocolate mint.For the anti-oxidation test using DPPH radical and β-Carovtene-linoleic acid assay, peppermint showed better properties than chocolate mint.However, for the scavenging NO radical activity, chocolate mint was superior to peppermint.Chocolate mint also showed stronger anti-inflammatory activity than peppermint.

REFERENCES
Essential oils are extracted from aromatic and medicinal plants that are natural concentrated aromatic hydrophobic liquid products obtained by steam distillation or solvent extraction [1,2].They contain several chemical compounds exhibiting different biological properties and activities.It can reduce foodborne pathogens and decrease the use of synthetic and semisynthetic antimicrobial compounds [3].

Table 2 :
The minimal inhibitory concentration (MIC, %v/v) of the essential oils.