Antimicrobial activity and chemical composition of essential oil and hydrosol extract of Nepeta nepetella subsp . amethystina ( Poir . ) Briq . from Algeria

Article history: Received on: 12/06/2015 Revised on: 17/07/2015 Accepted on: 11/08/2015 Available online: 27/09/2015 The essential oil and hydrosol extract of Nepeta nepetella subsp. amethystina, obtained by hydrodistillation and analysed by GC, GC–MS, 1H and 13C NMR, were evaluated for their antimicrobial activity using disks diffusion method and MICs against thirteen pathogenic microorganisms. The chemical analysis of essential oil and hydrosol extract allowed the identification of 34 and 12 constituents representing 96.77% and 80.07% of total oil and hydrosol extract, respectively. The two isomers 4a,7,7a-nepetalactone and 4a, 7, 7a-nepetalactone were found to be the major components in N. amethystina essential oil and hydrosol extract, , 7-nepetalactone and 4a,7a,7respectively. The two isomers 4a -nepetalactone were found to be the major components in N.7a amethystina essential oil and hydrosol extract. Candida albicans strains were the most sensitive microorganisms towards N. amethystina essential oil and hydrosol extract, which have the largest inhibition zones (˃30mm) and the lower MICs values (<1.25%). The anti-mold activity, carried out by the diffusion method, showed that N. amethystina essential oil has a very interesting activity against Aspergillus flavus and Cladosporium herbarium.


INTRODUCTION
The genus Nepeta which belonged to Lamiaceae family is represented by more than 250 species distributed mainly in Africa, Europe and Asia (Mabberly, 1997).7% of total Nepeta species growth wild in the Iberian Peninsula and North Africa, in which only five species exist in Algeria (Quézel and Santa, 1962;Greuter et al., 1986).According to taxonomic revision of Ubera & Valdes (Castrillón and Jiménez, 1983), N. amethystina is a subspecies of N. nepetella.This medicinal plant is found in Saharan Atlas in dry stony places.Locally, N. amehtystina is named Gouzia and it's mostly used as a medicinal plant for its febrifuge, antirheumatic, antispasmodic and diuretic effects.The essential oils of some Nepeta species have been extensively studied, which are mainly characterized by nepetalactone (Dabiri and Sefidkon, 2003;Sefidkon et al., 2006).The compounds 1,8-cineole and D-germacrene were also identified as the main constituent of the essential oils of N. ispahanica and N. ucrainica (Rustaiyan and Nadji, 1999;Javidnia et al., 2005), respectively.Essential oil obtained from N. amethystina subsp.amethystina of Spanish origin showed that 4a,7,7a-nepetalactone was the main constituents (41.29%) followed by 4a, 7, 7anepetalactone (29.42%) and an aldehyde not identified (5.30%) (Velasco-Negueruela et al., 1989).To our knowledge there are no reports on the antimicrobial properties of N. amethystina.This study aimed the determination of chemical composition and antimicrobial activity of N. amethystina essential oil and hydrosol extract.

Plant material
The aerial parts of N. amethystina were collected during flowering stage in June 2011 from Aïn-Safra region located in western Algeria.The plant material was identified by Dr. Boumediene Medjahdi according to (Quézel and Santa, 1962).
A voucher specimen has been deposited in the Herbarium of the Laboratory of Botany, Department of Biology, Tlemcen University, Algeria.

Preparation of essential oil (EO) and hydrosol extract (HY)
EO from air-dried aerial parts (250g) was obtained by hydrodistillation for 5 h using a Clevenger type apparatus according to the (European-Pharmacopoeia, 2005).The oil was separated from hydrosol and dried over anhydrous sodium sulfate and stored in sealed vials at 4 C°.The 500 ml first liters of water hydro-distillation (hydrosol) were extracted three times with 100 ml of diethyl ether (Et 2 O) by liquid-liquid extraction.The organic layer was dried over Na 2 SO 4 , evaporated at room temperature until obtained pure extract and stored in sealed vials at low temperature.

EO and HY analysis procedure
GC analyses were carried out using a Perkin Elmer Clarus 600 apparatus equipped with a dual flame ionisation detection system and two fused-silica capillary columns (60 m x 0.22 mm i.d., film thickness 0.25 µm), Rtx-1 (polydimethylsiloxane) and Rtx-Wax (polyethyleneglycol). GC conditions used were: programmed heating from 60 °C to 230 °C at 2°C/min, followed by 35 min under isothermal conditions.The injector and the detector were maintained at 280 °C.Helium was the carrier gas at 1 ml/min; 0.2 µL of EO was injected in the split mode (1:50).EO and HY was analysed with a Perkin Elmer Turbo Mass detector, directly coupled to a Perkin Elmer Autosystem XL equipped with fused-silica capillary columns (60 m x 0.22 mm i.d., film thickness 0.25 µm), Rtx-1 (polydimethylsiloxane) and Rtx-Wax (polyethyleneglycol). GC-MS (EI) conditions: Ion source temperature: 150°C; energy ionization: 70 eV; electron ionisation mass spectra were acquired oven the mass range 35-350 Da.Split: 1/80.Identification of the components was based i) on the comparison of their GC retention indices (RI) on non-polar and polar columns, determined relative to the retention time of a series of n-alkanes C9-C24 with linear interpolation, with those of authentic compounds or literature data; and ii) on computer matching with commercial mass spectral libraries (Hochmunth, 2001;Köning et al., 2001) and comparison of spectra with those of our personal library "Aromes".Relative amount of individual components were performed on the basis of their GC peak areas on the two capillary Rtx-1 and Rtx-Wax columns, without FID response factor correction.

Isolation of nepetalactones and NMR analysis
The dichloromethane extract of aerial part of N. amethystine was subjected to flash chromatography (silica gel 200-500μm), eluting with different solvents of increasing polarity.We obtained several fractions with the highest was subjected to fine chromatography (silica gel 63-200μm).
Thus, we recovered 8 fractions.The sixth fraction (0.02 g) obtained with petrol ether/dichloromethane (75/25) was analyzed by NMR.The NMR spectra were recorded on a Bruker DPX 300 instrument in deuterated chloroform.

Microbial strains
N. amethystina EO and HY were evaluated against eight bacterial reference strains, which are Escherichia coli (E.

Inoculums preparation
The inoculums of the bacterial and yeasts strains were prepared from overnight broth cultures.The suspensions were set to 0.5 McFarland or an optical density from 0.08 to 0.13 at 625 nm wavelength, which corresponds to 10 8 cfu/mL (CLSI, 2006).

Disc diffusion assay
EO and HY of N. amethystina were tested for their antimicrobial activity using the diffusion technique on solid media (Benbelaïd et al., 2014).Discs of sterile Whatman paper (6mm) were impregnated with 4µl of extracts EO and HY, and then placed on dish plates of Mueller-Hinton Agar (Pronadisa TM , Spain), which had been inoculated with an inoculum of 10 8 cfu/mL.The plates were then incubated for 24 h at 37°C for bacteria and 30°C for yeast.The results were recorded by measuring the growth inhibition zones surrounding the discs.All tests were performed in triplicate.

Determination of minimum inhibitory concentration (MIC)
The MIC was established by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI, 2006).
DMSO was used to dissolve the EO and HY.A serial doubling dilution of the oil was prepared in a 96 well microliter plate over the range of 40.00-0.08mg/mL inoculated in Mueller-Hinton broth.The MIC was defined as the lowest concentration of EO and HY that inhibiting visible growth.All tests were performed in triplicate.

Anti-mold activity (Agar dilution method)
The anti-mold activity of N. amethystina EO and HY was determinate using the agar dilution method (Hili et al., 1997).Briefly, 6 mm agar discs of each mold cultures were deposited in the centre of Petri plate (90mm) containing 20 ml of Potato Dextrose Agar (Merck, France), with various concentrations of EO and HY.The testing dishes were incubated at 25°C for 7 days, the anti-mold indices were calculated as follows: AI (%) = [(DC -DE) / DC] × 100 DE: the diameter of growth zone in the experimental dish (mm); DC: the diameter of growth zone in the control dish (mm)

Statistical analysis
Statistical analyses were performed withthe GraphPad Prism 5 software.Statistical comparisons were made with twoway ANOVA followed by Bonferroni's test.The level of significance was set at P < 0.05.

RESULTS AND DISCUSSION
The plant extractions provide yellow pale oil by hydrodistillation which yield 0.60%, whereas hydrosol extract gave dark yellow oil yielded 0.39%.The chemical composition of EO and HY from N. amethystina was listed in table 1.
A total of thirty-four compounds were identified in N. amethystina EO and HY obtained from the aerial parts of N. amethystina, representing 96.77 and 80.07 % respectively.All components were identified by comparison of their EI-MS and GC-retention indices and mass spectral with those of the "Arômes" library.Oxygenated monoterpenes compounds were the major groups in EO and HY, while hydrocarbon monoterpenes and hydrocarbon sesquiterpenes were absent in HY.The major components were found to be 4a, 7, 7a-nepetalactone (72.40%, 49.21%) and 4a, 7, 7a-nepetalactone (16.25%, 4.71%) of EO and HY respectively.This result contrasts with that obtained by Velasco-Negueruela et al. (1989).These authors have found cis-trans-nepetalactone (41.90%) as the main compound followed by the isomer trans-cis-nepetalactone (39.40%) of EO from N. amethystina of Spanish origin.The isomers nepetalactone finding in our work looks like the species of N. argolic, N. nepetella subsp.aragonensis and N. nuda (De Pooter et al., 1987;Velasco-Negueruela et al., 1998;Tzakou et al., 2000).We note also the presence of myrtenal, carvacrol, β-farnesene, β-ionone, α muurolene and γ-cadinene which were reported for the first time in N. amethystina from Algeria.The antibacterial and anti-fungal activities of N. amethystina EO and HY were assessed by disc diffusion and MIC methods.Oxygenated sesquiterpenes 0,50 6,92 Results are in percentage (%) of components of N. amethystina EO and HY.Percentages and elution order of individual components are given on no polar column.Retention indices nRI and pRI are given relative to C6 -C24 n-alkanes on no polar (Rtx-1) and polar (Rtx-Wax) columns.ID: identification method by comparison of (RI) retention indices and (MS) mass spectra. 1 H and 13 C NMR: proton and carbon nuclear magnetic resonance.
The EO and HY showed strong antimicrobial activity against microbial species (Table 2), especially against yeast.In most, the Gram positive bacteria are more sensitive to EO and HY than Gram negative ones.B. cereus was the most sensitive bacterial species, with inhibition zones larger than 18 mm.However, P. aeruginosa and K. pneumoniae appears resistant to N. amethystina EO and HY.While C. Albicans was very sensitive to both EO and HY with inhibition zones larger than 30 mm.The MICs of the studied oil and HY ranged between 0.416 and 40 mg/mL (Table 02).The lowest MICs were observed against C. albicans ATCC10231 with an MIC of 0.416 mg/ml for oil and 0.520mg/ml for HY.Against molds, EO and HY of N. amethystina have showed a good activity (Table 03).The EO exhibited more activity against A. flavus than C. herbarum.The antimicrobial activity of N. amethystina EO and HY may be related to their major monoterpenoid component i.e. nepetalactone, since it's known by its antimicrobial potency (Farag et al., 1989).The antimicrobial activity of EOs is also due to minor components might contribute into antimicrobial activity (Kobaisy et al., 2005).Indeed, the minor constituents like 1,8 cineol, terpinene 4-ol, αterpineol, and thymol may involve in the antimicrobial activity of N. amethystina EO and HY (Tao et al., 2014;Zhou et al., 2014).Many previous studies have shown the relationship between the antimicrobial activity of EOs and their chemical composition (Deans and Svoboda, 1989;Farag et al., 1989) and the role of synergy in the antimicrobial activity of EOs (Benbelaïd et al., 2014).We also find that the EO is more active on microorganisms than the HY.This result is contrary to that of the literature that reports that the hydrosol rich in hydrophilic oxygen molecules exerts more activity than the oil rich in lipophilic compounds (Rose, 1999;Rao et al., 2002).In this study, we have evaluated the antimicrobial activities of the EO and HY of N. amethystina harvested in Algeria.N. amethystina EO and HY were effective in growth inhibition of all tested strains.According to the extracts, EO was more active than the HY, but according to strains, C. albicans and B. cereus were shown to be more sensitive to both extracts.As a consequence this oil can be used as possible alternatives or complementary therapeutic agent against candidiasis.
Also, the oil can be useful for developing alternative compounds to preserved food contaminations caused by B. cereus and inhibits mold growths.

CONCLUSION
In conclusion, we show once again the interest of natural products in the control of microbial growth and especially the use of oil and its by-product of steam distillation hydrolat as antimicrobial agent's preservatives in agro-foods or as antiseptic agents in therapies.Values in the same row followed by the same letter are significantly different (P<0.001).IZ: inhibition zones in millimetre.MIC: minimal inhibitory concentration in mg/ml.

Table 1 :
Chemical composition of essential oil (EO) and hydrosol extract (HY) of Algerian N. amethystina.

Table 2 :
Antibacterial and anti-yeast activities of N. amethystina essential oil (EO) and hydrosol extract (HY).All results shown in this Table are: Mean ± Standard Deviation (SD) of three repeats.The data were analysed by two-way ANOVA followed by Bonferroni's test.

Table 3 :
Results of inhibiting effect of N. amethystina essential oil (EO) and hydrosol extract (HY) on mycelia growth.