Phytochemical profile and antimicrobial properties of volatile compounds of Satureja calamintha ( L ) Scheel from northern Algeria

Purpose: To undertake the phytochemical screening of Satureja calamintha (L.) Scheel., and evaluate the antimicrobial activities of its volatile compounds. Methods: Qualitative phytochemical analysis of the plant was performed using standard methods. The extraction of essential oils (EOs) was conducted using steam distillation, while the chemical composition was determined using gas chromatography-mass spectrometry (GC-MS). The antimicrobial activities of the oils were evaluated against ten bacterial and six fungal strains using disc-diffusion assay and poisoned food technique, respectively. Results: After steam distillation, the extraction yield was 0.54 ± 0.11 %. GC-MS analysis identified approximately 99.99 % of the EOs. The three most abundant compounds identified were l-menthone (32.10 %), neo-menthol (32.07 %) and pulegone (22.35 %). The oils had significant (p < 0.05) antimicrobial activities against the tested bacterial and fungal strains, except Bacillus cereus and Candida albicans. The lowest minimum inhibitory concentration (MIC) for bacteria was 0.007 % (v/v) against Enterococcus faecalis and Klebsiella pneumoniae, whereas for fungi, it was 0.500 % (v/v) against Candida albicans. Moreover, Enterococcus faecalis and Listeria innocua had the lowest minimum bactericidal concentration (MBC) at 0.125 % (v/v), in contrast to the lowest fungicidal concentration (MFC) for Candida albicans at 0.500 % (v/v). Conclusion: These results demonstrate that EOs from Satureja calamintha (L.) Scheel. possess significant antimicrobial activities which might be useful for therapeutic and pharmaceutical applications.


INTRODUCTION
Fungal infections have become increasingly recognized as important threats in critically-ill patients.They are characterized by easy transmission, wide prevalence and a large variety of human and animal reservoirs [1].Reports on food-borne diseases caused by the consumption of food contaminated with pathogenic bacteria are increasing worldwide [2].
Each year, an estimated 30 % of people in industrialized nations suffer from these diseases [3].

Synthetic
antimicrobial substances are undeniably one of the most important therapeutic discoveries of the 20 th century; they are effective against serious bacterial and fungal infections.However, most of these pathogenic microorganisms have developed new resistance to several antibiotics.There has been renewed interest in plants and their natural antimicrobial agents as alternatives for reducing the problem of resistance [4].Among these, essential oils (EOs) have received special attention as natural agents with great potential for food preservation and other pharmacological effects such as antispasmodial, carminative, hepatoprotective, antiviral and anti-cancer activities [5].
Algeria is well-known for its biodiversity, with about three hundred species of plants, 15 % of which are endemic and belong to several botanical families.
The aim of this study was to perform phytochemical screening on the aerial parts of Satureja calamintha (L.), and to evaluate the antimicrobial activities of its essential oils.

EXPERIMENTAL Plant material
Aerial parts of Satureja calamintha (L.) Scheel.were collected from the Jijel region of Algeria (Eastern Algeria) and identified by Prof C. Baali of the Department of Botany, Higher National Agronomic School, Algeria.A specimen of the plant was prepared and deposited in the ENSA Herbarium.The aerial parts of the plant were washed and shade-dried in a well-ventilated room.

Extraction of essential oils
Extraction of essential oils (EOs) from the aerial parts of the plant were extracted using steam distillation.A weighed portion (100 g) of dried aerial parts was suspended in 300 mL of distilled water with intermittent stirring for 3 h.The oils obtained after extraction were dried over anhydrous sodium sulfate (Na 2 SO 4 ) and kept in the refrigerator at 4 °C in sealed opaque bottles.

Test microorganisms
Ten bacterial and six fungal strains were purchased from the American-Type Culture Collection (ATCC), and were used for testing the antimicrobial activities of the EOs.

Analysis of essential oils by GC-MS
The identification and quantification of essential oils present in the aerial parts of the plant were carried out using HP 6890 gas chromatography coupled to an HP5973 mass spectrometer.Sample volume of 0.2 µL was injected in split mode (split ratio of 50:1) into a column capillary HP-5MS (30 m x 0.25 mm x 0.25 μm), containing a mixture of phenyl polysiloxane (5 %) and methyl polysiloxane (95 %).The separation was carried out using helium as the carrier gas at a flow rate of 0.5 ml/min under the following conditions: First, the oven temperature was set at 60 °C for 8 min, increased further to 250 °C at a rate of 2 °C/min, and finally maintained at 250 °C for 10 min.The injection and detector temperatures were maintained at 250 and 230 °C, respectively.Individual components were identified by comparing their mass spectra with those of standards, and also by comparing their GC-retention indices with those recorded in literature.The components of the EOs were expressed as percentages determined by integration of GC peak areas without using correction factors.

Evaluation of antibiotic sensitivity and antibacterial activities
The pathogenic bacterial strains were tested for their sensitivities against a range of antibiotics: ampicillin (Amp30), chloramphenicol (C30), cefotaxime (Ct30), gentamycin (Gen10), vancomycin (Va30), cefoperazone (Cep30) and tetracycline (Te30), using the Kirby-Bauer method [8].The antibacterial activities of the EOs were determined using disc-diffusion assay.A single colony from an overnight bacterial culture plate was transferred into tubes containing 5 mL of sterile saline solution to obtain the bacterial suspension.This was adjusted to 0.5 McFarland standard, approximately corresponding to an initial inoculum size of 10 8 colony-forming units per milliliter (CFU/mL).The bacterial suspension was diluted 1:100 to obtain 10 6 CFU/mL.Using a sterile swab, 200 µL of fresh culture was spread on Muller Hinton agar plates, and 20 µL of EO was dispensed on the sterile paper disc 6 mm in diameter.The EO disc and antibioticcontaining discs were deposited separately on the surface of each inoculated agar plate and incubated at 37 °C for 24 h.Chloramphenicol (15 μg/disc) was used as a positive control.The antibacterial activity was estimated by measuring the diameter of the inhibition zone surrounding each disc.

Evaluation of antifungal activity
The antifungal activity was evaluated using the disc-diffusion method for yeast, while poisoned food technique was used for mold [9].In the discdiffusion method, a colony from a two-day yeast culture plate was transferred to tubes containing 5 mL of sterile saline solution to obtain a yeast suspension corresponding approximately to an initial inoculum size of 10 4 CFU/mL.A portion of the fresh culture (200 µL) was inoculated on Sabouraud Dextrose agar plates.This was followed by the injection of 20 µL of EOs on a sterile paper disc 6 mm in diameter, which was further deposited on the surface of each inoculated agar plate and incubated at 25 °C for 48 h.Griseofulvin (15 μg/disc) was used as a positive control and the antifungal activity was estimated by measuring the diameter of the inhibition zone surrounding each disc.
In the poisoned food technique, 30 µL of EOs and two drops of Tween 80 were transferred to a sterile tube containing 13.5 mL of molten Sabouraud Dextrose agar medium and cooled at 45 °C.The mixture obtained was shaken manually and dispensed on sterile petri dishes.After solidification of the medium, a mycelial disc measuring approximately 6 mm in diameter, cut from a fungal culture of 5 days, was deposited in the center of each petri plate, and further incubated at 25 °C.Control plates without EOs, were treated following the same procedure with nystatin (30 µL) as the positive control.The diameter of mycelial growth was recorded after five days of incubation and the percentage inhibition of mycelial growth (M) was calculated as in Eq 1.

M (%) = {1 -(D EOs /Dc)}100 ………………. (1)
where Dc is the mean diameter of mycelial growth in the control sample, and D EOs is the mean diameter of mycelial growth treated with EOs.

Determination of minimum inhibitory concentrations (MIC)
The MICs of the EOs were determined using the micro-dilution method [10].Stock solutions of EOs were prepared at concentration of 1.000 % (v/v) and diluted in Muller Hinton broth and Sabouraud Dextrose broth to obtain concentrations in the range of 0.007 to 1.000 % (w/v).Portions of each concentration (100 µL) were transferred to wells and inoculated separately with 10 µL of the microbial suspensions (10 6 CFU/mL for bacteria and 10 4 CFU/mL for fungi).The incubation temperatures for bacteria and fungi were 37 °C and 25 °C, respectively.Chloramphenicol, nystatin and griseofulvin were used as positive controls.The MIC was defined as the lowest concentration of EOs that resulted in no visible growth of microorganisms.

Evaluation of minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC)
After MIC determination, the wells that showed no visible growth of microorganisms were used for determination of MBC for bacteria, and MFC for fungi.Different concentrations of EOs ranging from 0.007 to 1.000 % (w/v) were prepared in Muller Hinton broth and Sabouraud Dextrose broth.Portions of each concentration (100 µL) were transferred to the wells and were inoculated separately with 10 µL of the microbial suspensions (10 6 CFU/mL for bacteria and 10 4 CFU/mL for fungi), at 37 °C for bacteria, and at 25 °C for fungi.Chloramphenicol, nystatin and griseofulvin were used as positive controls.The MBC and MFC were defined as the lowest concentrations of EOs that completely killed the initial inoculums of bacteria and fungi, respectively.

Statistical analysis
Statistical analyses were performed with Microsoft Excel 2010 and the results are presented as mean ± standard deviation (SD) of three replicates.All the data were statistically evaluated using Student's t-test and significant difference between the means was set at p < 0.05.

Yield extraction and chemical profile of the essential oils
The extracted volatile substances had aromatic odor and yellow color.The average yield of EOs was about 0.54 ± 0.11 %.The aerial parts of the plant contained three major compounds: lmenthone (32.10 %), neo-menthol (32.07 %) and pulegone (22.35 %).

Antibacterial and antifungal activities
The bacterial strains reacted differently to the tested antibiotics; with inhibition zones varying from 6 to 45 mm.Enterococcus faecalis seemed resistant to Gen10 (12 mm) and susceptible to the other antibiotics, whereas MRSA appeared resistant to Te30 and Va30 (6 mm).Cep30 had a larger spectrum of inhibition than Gen10, Te30 and Va30.It exhibited a smaller inhibition zone, varying from 6 to 10.5 mm.Amp30 and Ct30 seemed to be the most efficient antibiotics against all the tested bacterial strains (6 -15 mm), whereas C30 appeared to be the most effective with an inhibition zone varying from 25 to 39 mm.The results of antibacterial activity are presented in Table 4.The essential oils had significant antibacterial effects against all tested bacteria, with inhibition diameters between 10.33 and 30 mm, except for Salmonella enterica, which had low sensitivity (7.33 ± 2.62 mm).The antibacterial activities of the oils were significantly higher (p < 0.05) against Enterococcus faecalis, Listeria innocua, Staphylococcus aureus, Escherichia coli, Klebseila pneumoniae, Salmonella enterica and Pseudomonas aeroginosa, when compared to the effects of choloramphenicol.
The oils had moderate antifungal activities, ranging from 10.66 to 39.66 mm.All results were statistically significant (p < 0.05), with the exception of Candida tropicalis DIV13-Z087D0VS.The fungal strains reacted differently to the oils, with variable percentages of inhibition between 54.44 and 71.25 %.

MIC, MBC and MFC
The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) of EOs against all tested strains are shown in Table 5.The MIC ranged from 0.007 to 1.000 % (v/v) for bacteria and from 0.250 to 1.000 % (v/v) for fungi.The lowest MIC for the EOs in the case of bacteria was observed with Enterococcus faecalis and Klebsiella pneumoniae corresponding to 0.007 % (v/v), whereas it was 0.500 % (v/v) for Candida albicans in case of fungi.Two bacterial stains (i.e.Enterococcus faecalis and Listeria innocua), showed a bactericidal effect with a low MCB of 0.125 % (v/v), whereas Candida albicans was considered a sensitive fungal strain with an MFC of 0.500 % (v/v).

DISCUSSION
The phytochemical compositions of plants vary from one species to another and sometimes within the same species.These differences in composition confer important biological properties on plants.Twelve phytochemicals were detected in the aerial part of Satureja calamintha, namely: flavonoids, anthocyanins, total and gallic tannins; glycosides, saponosides, reducing compounds, holosides, alkaloids, coumarins, O-heterosides and C-glycosides.Leucoanthocyanins and quinones were not detected.Although catechic tannins and saponosides were present, they had minimal relative abundance.
A similar study carried out on Satureja calamintha (L.) Scheel.from Western Algeria showed the presence of saponosides, coumarins, total tannins and free flavonoids, but anthocyanins, leucoanthocyanins and reducing compounds were absent [11].In another study on the same plant, the presence of flavonoids, total tannins, alkaloids, saponosides and quinones was reported [12].These observed differences in chemical composition may be linked to variations in climatic and geographical conditions.
The extract yield from steam distillation was comparable to that reported in a previous study [13].In contrast, a higher yield was obtained from the same species from Morocco [14].Different extraction yields varying from 1.27 to 2.52 % have been reported on the same plant grown in different parts of Algeria [12,15].The three most abundant compounds identified in the EOs were 1-menthone, neo-menthol and pulegone.
The chemical compositions of oils extracted from Satureja calamintha differed from one region to another.In Algeria, studies on oils of this plant species collected from two different sites demonstrated the presence of twenty phytochemical components [15].The sample from the first site was characterized by the predominance of piperitone oxide, cyclohexanone 2-(methylethylidene), and menthone, whereas the major compounds in the oils from the second site were menthone, piperitone oxide, pulegone, and cyclohexanone 2-(1-ethylthylidene). Studies carried out on oils from Satureja calamintha (L.) Scheel.from Belgium showed that the major compounds were pulegone, piperitenone oxide, and menthone [16].In a study on the same plant from Italy, the dominant constituents were pulegone, piperitenone oxide, piperitone oxide, and piperitenone [13].These differences in composition are due seasonal and environmental factors such as soil type, geographic location, harvest period and extraction method [17].
In the present study, the essential oils (EOs) from Satureja calamintha exhibited potent antibacterial activities.Gram-positive bacteria were more sensitive to the EOs than Gramnegative bacteria.These differences in susceptibilities indicate a possible effect of the

Table 1 :
Phytochemical profile of aerial parts of Satureja calamintha

Table 2 :
Extraction yield and compositional analysis of volatile fractions of Satureja calamintha.

Table 3 :
Selected antibiotics and their antimicrobial activities

Table 4 :
Antibacterial and antifungal activities of EOs from Satureja calamintha and standard antibiotics

Table 5 :
Minimum inhibitory concentrations of EOs on test microorganisms