Chemical composition , antimicrobial and antioxidant properties of Mentha longifolia ( L . ) Huds . essential oil

Introduction: Present study describes the antimicrobial activity and free radical scavenging capacity (RSC) of essential oil from Mentha longifolia (L.) Huds. Aim of this study to investigate the quality, antimicrobial andantioxidant activity of wild species Mentha longifolia essential oil from Bosnia and Herzegovina.Methods: The chemical profi le of essential oil was evaluated by the means of gas chromatography-mass spectrometry (GC-MS) and thin-layer chromatography (TLC). Antimicrobial activity was tested against 6bacterial strains. RSC was assessed by measuring the scavenging activity of essential oils on 2,2- diphenyl-1-picrylhydrazil (DPPH).Results: The main constituents of the essential oil of M. longifoliae folium were oxygenated monoterpenes,piperitone oxide (63.58%) and 1,8-cineole (12.03%). Essential oil exhibited very strong antibacterial activity.The most important antibacterial activity essential oil was expressed on Gram negative strains: Escherichia coli, Pseudomonas aerginosa and Salmonella enterica. subsp.enterica serotype ABONY. Antioxidant activity was evaluated as a RSC. Investigated essential oil was able to reduce DPPH radicals into the neutral DPPHH form (IC50=10.5 μg/ml) and this activity was dose –dependent.Conclusion: The study revealed signifi cant antimicrobial activity of the investigated essential oil. The examined oil exhibited high RSC, which was found to be in correlation to the content of mainly monoterpeneketones and aldehydes. These results indicate that essential oils could serve as safe antioxidant and antiseptic supplements in pharmaceuticals.


Introduction
Since ancient times, herbs and spices have been added to diff erent types of food to improve the fl avor and organoleptic properties.Also, herbal medicines have a great potential in the emerging nutrition industry, because these materials are oft en considered foods as well as medicines and are used in preventive and curative treatments throughout the world (1).Especially popular today is the concept of foods that combine nutritional and medicinal benefi ts, so-called "functional foods".Many natural compounds extracted from plants have demonstrated biological activities.Among these various kinds of natural substances, essential oils from aromatic and medicinal plants receive particular attention as potential natural agents for food preservation.In fact, their eff ectiveness against a wide range of microorganisms has been repeatedly demonstrated (2)(3)(4)(5).Moreover, essential oils are proved to have various pharmacological eff ects, such as spasmolytic, carminative, hepatoprotective, antiviral, and anticarcinogenic eff ects, etc. (6).Recently, many essential oils have been qualifi ed as natural antioxidants (3,(5)(6)(7)(8) and proposed as potential substitutes of synthetic antioxidants in specifi c sectors of food preservation.Furthermore, biologically active natural compounds are of interest to the pharmaceutical industry for the control of human diseases of microbial origin and for the prevention of lipid peroxidative dam-age, which has been implicated in several pathological disorders, such as ischemia-reperfusion injury, coronary atherosclerosis, Alzheimer's disease, carcinogenesis, and aging processes (9,10).Th e genus Mentha L., member of the family Lamiaceae, subfamily Nepetoideae, and the tribe Mentheae is divided into 5 sections (Audibertia, Preslia, Pulegium, Mentha and Eriodontes) (11,12).Th e most complex section Mentha further can be subdivided into the three groups, refl ecting their diff erences in the infl orescence form (Verticillatae, Capitatae and Spicatae) (12,13).Furthermore, for the genus Mentha the correct number of species is still not defi ned.According to the authors, the genus consists approximately 14-25 species (11,12).Most of the species are characterized by a great polymorphism, which is refl ected in the leaf shape, indumentum, type of fl owers and infl orescences etc.In addition to the morphological variation, most of the Mentha species also displays a considerable chemical diversity in essential oil composition, depending on the growth location (14).Examination of the published literature on the oil composition of M. longifolia reveals that it can exist in a myriad of chemical forms, as can be seen from the main constituents found in these oils.Th e main constituents in essential oil were piperitone oxide (13.90-50.50%), 1,8-cineole (8.18-17.80%),carvone (0.5-21.5%), beta caryophyllene (2.0-22.0%)and menthol (0.0-32.50%).Th e genus Mentha clearly has marked antimicrobial characteristics across the spectrum from fungi and parasites, through bacteria, to viruses.Th ere is some diffi culty in comparing the diff erent results obtained by research groups across the world since so many variables exist.Antimicrobial activity along with the antioxidant eff ectiveness of essential oils is one of the most examined features, important for both food preservation and control of human and animal diseases of microbial origin.Numerous reports suggest strong antibacterial and antifungal activities of a wide range of essential oils, especially those belonging to the Lamiaceae family (12).In general, Gram-positive strains of bacteria are more sensitive to the mint essential oils.Mentha longifolia (L) Huds. is perennial herb 40-120 cm high with musty scent.Stem white or grey-villous, sometimes sparsely hairy.Leaves are sessile or shortly petiolate usually oblong elliptical, hairs simple.Extremely variable in height, leaf size and shape, indumentum and infl orescence and complicated by the occurrence of hybrids.Mentha longifolia, is oft en used as a domestic herbal remedy, being valued especially for its antiseptic properties and its benefi cial eff ect on the digestion as it is a well-know treatment for fl atulence.Th e objectives of this study were to analyze the composition, antimicrobial and antioxidant activity of the essential oil of Mentha longifolia growing wild in Bosnia and Herzegovina.

Methods
Plant Material: Aerial parts of wild growing fl owering plants of Mentha longifolia (L.) Huds.during three phenophases (before fl owering, fl owering and aft er fl owering) were collected in 2011.on the bank of the Jablanicko lake, near Konjic, in Bosnia and Herzegovina.

Isolation of the Essential Oil:
Air-dried plants of Mentha longifolia were submitted to hydrodistillation according to European Pharmacopoeia 7ed.(15), using Clevenger apparature (Klaus Hofmann GmbH, Germany).Th e essential oil samples of each phenophase were dried over anhydrous sodium sulfate.Th e quantity of the predestilated essential oils were determined volumetrically.
Essential Oil Analysis: Qualitative and quantitative analyses of the essential oils were carried out using a gas chromatography/mass spectrometry system (GC-MS, Agilent Tecnologies series 6890N/5975B, United States of America) at electron energy=70 eV, equipped with a split-splitless injector (200 0 C) and a fl ame ionization detector (FID) (250 0 C).As a carrier gas helium (1ml/min) was used.Th e capillary columns (HP 5MS 30m x 0.25mm; fi lm thickness 0.25μm Agilent Tecnologies) were used.Th e temperature programmes were 50 0 C to 280 0 C at a rate of 10 0 C/min until 130 0 C and 130-280 0 C at a rate of 12 0 C/min, respectively with split ratio, 1:10.Coelution and mass spectrometry MS analysis based on the identifi cation of the individual compounds, and the comparison of their relative retention times (RI) with those of the reference samples were performed.For the components, mostly sesquiterpenes and aliphatic compounds, for which reference substances were not available, the identifi cation was performed by matching their retention times and mass spectra with those obtained from the authentic samples and/or the Th e National Institute of Standards and Technology, known as the National Bureau of Standards (NIST/NBS), Wiley libraries spectra as well as with literature data (16).

Evaluation of Antibacterial Activity.
Antimicrobial activity of essential oils, isolated from Mentha longifolia (L.) Huds., using diff usion method was performed in this study.A collection of 6 test organisms, including three Grampositive and three Gram-negative bacterial strains, was used.Th e groups included fi ve organisms of American Type of Culture Collection (ATCC) and one organism of National Collection of Type Cultures (NCTC).Th e source of the bacterial strains is shown in Table 2.All test organisms were stored at +4 °C on Mueller-Hinton (MH) agar slants, sub cultured every 2 weeks and checked for purity.Antibiotics which are therapeutically important in treating infections caused by these microorganisms were used as comparative substances (as positive control): ciprofl oxacine for evaluation of antimicrobial activity of Pseudomonas aeruginosa, Penicilin for Bacillus subtilis, Gentamycin for Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis and tetracycline for Salmonella enterica subsp.entericaserotype ABONY.All samples were applied as solution in n-hexane.Th e eff ect of the solvent (n-hexane) on the microbial growth was also analyzed.On the surface of the agar, the 6 mm holes in diameter were punched.Hundred microliters of the tested essential oils (10 %, 5%, 1%, 0.5% and 0.1% solutions in nhexane was applied to the holes.Th e plates were incubated overnight at 37 °C, and the diameter of the resulting zone of inhibition was measured.Th e evaluation of the antibacterial activities of the essential oils was carried out in three repetitions.

Antioxidant Activity.
Chemicals and Apparatus: From the obtained RSC values the EC50 values, which represent the concentration of the essential oil that caused 50% neutralization, were determined by linear regression analysis.Th e antiradical effi ciency (AE) was calculated considering the EC 50 value and the necessary time to reach the EC 50 (TEC50), according to the following Equation ( 2):

Essential oil content and chemical composition
Th e content of the essential oil in the fl owering stage, expressed in percentage was 1.9% v/w (volume of essential oil/weight dry leaf).A total of 36 compounds were identifi ed, grouped as classes of compounds, in the essential oils extracted from M. longifolia plants collected in Bosnia and Herzegovina (    The values shown represent the average of three determinations ± standard deviations.All essential oils were diluted in n-hexane (solvent expressed no activity on bacterial growth).

Antimicrobial Activity
Th e antibacterial activity of essential oil against a range of Gram-positive (three strains) and Gram-negative (three strains) is shown in Table 2 and fi gures 2-7.Obtained results revealed that essential oil exhibited variable levels of antibacterial activity against all tested bacterial strains.

Antioxidant Activity
Th is study, also, determined the antioxidant activity of one species of the family Lamiaceae.Th e results indicate that the hexan extract of the plant demonstrated antioxidant activity, and showed the high activity with a EC50 value of 10.5 μg/mL (Table 3).Th e reaction of essential oil and DPPH. is quite slow.Time at equilibrium state depends on the concentration used (Figure 1).TEC50, as the time at equilibrium reached with a concentration of essential oil equal to EC50 is 95.Calculated value of AE of tested essential oil is 10.58*10 -3 .

Discussion
M. longifolia essential oils from other geographical locations have been extensively studied.Th e essential oil content (1.9% v/w in dry leaf) was in accordance with the earlier published data (3).In the oil obtained from the plants collected in the fl owering stage the oxygenated monoterpenes were found to be the major class of substances (87.1%), followed by the sesquiterpene hydrocarbons (6.79%) and oxygenated sesquiterpenes (5.57%).Th e main constituents of the essential oil of  It is well known that the antioxidant activity of essential oil containing phenol components is due to their capacity to be donors of hydrogen atoms or electrons and to capture the free radicals (22).DPPH analysis is the test used to prove the ability of the components of the essential oil of Mentha longifolia to act as donors of hydrogen atoms.Essential oil of Mentha longifolia showed a significant effect in inhibiting DPPH., reaching up to 50% at concentration of 10.50 μg/ml.Th e antiradical effi ciency (AE) is a new parameter for the measurement the free radical scavenging of samples, and it combines the potency (1/EC50) and the reaction time (TEC50) (23).According to AE samples were divided into four antiradical effi ciency groups: AE ≤ 1 • 10 -3 -low antiradical activity 1 • 10 -3 < AE ≥ 5 • 10 -3 -medium antiradical activity 5 • 10 -3 < AE ≥ 10 • 10 -3 -high antiradical activity AE > 10 • 10 -3 -very high antiradical activity It was fi nd that AE of tested essential oil was 10.58*10 -3 , which places it into grupe with very high antiradical activity.

Conclusion
In conclusion, the study revealed signifi cant antimicrobial, particularly antibacterial, activity of the investigated essential oil.Th e examined oil exhibited high RSC, which was found to be in correlation to the content of mainly monoterpene ketones and aldehydes.Th ese results indicate that essential oils could serve not only as fl avor agents but also as safe antioxidant and antiseptic supplements in preventing d eterioration of foodstuff and beverage products and pharmaceuticals.Also, consumption of food produced with natural essential oils or aromatic plant extracts (functional foods) is expected to prevent the risk of free radical dependent diseases.Th is study represents the fi rst time investigation content, chemical composition, animicrobial and antioxidant activity essential oil of wild mint species from the area of Bosnia and Herzegovina.

Competing interests
Authors declare no confl ict of interest.

TABLE 1 .
Chemical Composition of M. longifolia Essential Oil Compounds listed in order of elution from a HP-5 MS column.Retention indices relative to C9-C24 n-alkanes on the HP-5 MS column from Sigma-Aldrich Quimica (Alcobendas, Spain).N-hexane was provided by Merck (Mollet del Valle´s, Spain).All reagents were of analytical grade.Double distilled water (Millipore Co.) was used throughout.Absorbance measurements were recorded on a UV/VIS mini-1240 Spectrophotometer (Shimadzu, Japan).Absorbencies intensity of the test solutions and the blank (with same chemicals, except sample) were measured at the 0 min and at the time when the steady state of the reaction between DPPH and analyzed compound was reached.0.1 M Trolox was used as positive control.For each samples three replicates were recorded.Free radical scavenging capacity in percent (RSC (%)) was calculated by following Equation (1):

TABLE 2 .
Antibacterial Activity (Inhibition Zone Measured in mm, Including Hole 6 mm in Diameter) of Essential Oils of Mentha longifolia

TABLE 3 .
Percentage of neutralization of DPPH. of essential oil of M. longifolia and trolox as positive control in DPPH assay ATCC 9027 (11-25 mm depend of concetration).