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Myrtus communis Essential Oil: Chemical Composition and Antimicrobial Activities against Food Spoilage Pathogens by Anis Ben Hsouna* a ), Naceur Hamdi b ) c ), Ramzi Miladi d ), and Slim Abdelkafi d ) a
) Department of Life Sciences, Faculty of Sciences of Gafsa, Zarroug 2112, Gafsa, Tunisia (phone: þ 216-97-130430; fax: þ 216-74-565555; e-mail: [email protected]) b ) Chemistry Department, College of Science and Arts, Al-Rass, P.O. Box 53, Qassim University, Saudi Arabia, Kingdom of Saudi Arabia c ) High Institute of Environmental Science and Technology, Borj-Ce´dria, Tunisia & University of Carthage, Tunisia d ) University of Sfax, Ecole Nationale dInge´nieurs de Sfax, De´partement de Ge´nie Biologique, Route de Soukra BP 1117, 3038 Sfax, Tunisia
Myrtus communis is a typical plant of the Mediterranean area, which is mainly used as animal and human food and, in folk medicine, for treating some disorders. In the present study, we evaluated in vitro antibacterial and antifungal properties of the essential oils of Myrtus communis (McEO), as well as its phytochemical composition. The GC/MS analysis of the essential oil revealed 17 compounds. Myrtenyl acetate (20.75%), 1,8-cineol (16.55%), a-pinene (15.59%), linalool (13.30%), limonene (8.94%), linalyl acetate (3.67%), geranyl acetate (2.99%), and a-terpineol (2.88%) were the major components. The antimicrobial activity of the essential oil was also investigated on several microorganisms. The inhibition zones and minimal inhibitory concentration (MIC) values of bacterial strains were in the range of 16 – 28 mm and 0.078 – 2.5 mg/ml, respectively. The inhibitory activity of the McEO against Gram-positive bacteria was significantly higher than against Gram-negative. It also exhibited remarkable activity against several fungal strains. The investigation of the mode of action of the McEO by the time-kill curve against Listeria monocytogenes (food isolate) showed a drastic bactericidal effect after 5 min using a concentration of 312 mg/ml. These results evidence that the McEO possesses antimicrobial properties, and it is, therefore, a potential source for active ingredients for food and pharmaceutical industries.
Introduction. – Plants have been a valuable source of natural products for maintaining human health, especially in the last decade, with more intensive studies for natural therapies [1]. Considerable works have been performed to investigate new natural antimicrobial and antioxidant agents leading to the identification of potential new natural drugs. Plant essential oils and their components have been shown to possess multiple biological activities [2]. Tunisian flora is remarkable for its diversity of medicinal plants. Myrtus communis L. (Myrtaceae) is an evergreen scrub, typical of the Mediterranean maquis, which grows spontaneously in many countries. In Tunisia, the genus Myrtus is represented by only one species, M. communis, which grows wild in the coastal areas, the internal hills, and the forest areas of North Tunisia [3]. It is traditionally used as an antiseptic, disinfectant drug, and hypoglycemic agent [4]. The leaves are used as a spice, especially for meat, and a popular liqueur is produced in Sardinia from its berries and leaves [5]. Myrtle extracts have been reported to possess antihyperglycemic, analgesic [6], 2014 Verlag Helvetica Chimica Acta AG, Zrich
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antigenotoxic [7], and antibacterial activities [8]. Recent reports have described the antioxidant activities of different myrtle extracts and compounds [9]. Various known antioxidants including flavonoids (quercetin and myricetin), phenolic acids (caffeic and ellagic acids), tannins, and a-tocopherol were isolated from myrtle [10]. Different parts of the plant find various uses in the food industry, such as for flavoring meat and sauces, and in the cosmetic industry [3] [11]. A striking feature of the plant is the pleasant smell of its essential oil, present in numerous glands, especially in the leaves. The main compounds responsible for the flavor and scent of myrtle oil are monoterpenes, i.e., 1,8-cineol, myrtenyl acetate, a-pinene, myrtenol, limonene, etc. [12] [13]. However, the antimicrobial property of myrtle-leaf essential oil has been rarely investigated. Therefore, in the present study, we examined the chemical composition of the leaf essential oil of Tunisian Myrtus communis (M. communis) by GC/MS and screened its antimicrobial activity. The antimicrobial activity was evaluated against a diverse range of foodborne pathogens. Also, an in vitro assessment of the McEO killing activity was carried out against Listeria monocytogenes (food isolate 2132). Results and Discussion. – Chemical Composition. The chemical composition of M. communis essential oil was determined by using both GC and GC/MS techniques. The percentages and the retention indices (RIs) of the identified oil components were compiled in Table 1, in the order of their elution on the 5MS capillary column. Seventeen compounds were identified, representing 91.20% of the total oil content. These compounds were divided into four classes: monoterpenes, sesquiterpenes, esters, and benzenoids (Table 1). This oil was characterized by very high percentage of monoterpenes (59.25%), and especially the oxygenated ones constitute the predominant class. Linalool (13.30%), a-terpineol (2.88%), and 1,8-cineol (16.55%) were the major components among the oxygenated monoterpenes. The monoterpene hydrocarbons of this oil were represented by a-pinene (15.59%) and limonene (8.94%). Other major detected components were linalyl acetate (3.67%), myrtenyl acetate (20.75%), geranyl acetate (2.99%), methyleugenol (1.17%), and a-humulene (1.29%). The minor components ( < 1%) were identified as caryophyllene oxide (0.66%), transcaryophyllene (0.80%), terpenyl acetate (0.63%), myrtenol (0.94%), myrcene (0.12%), b-pinene (0.1%), and a-thujene (0.83%). The myrtle essential oils could be separated in two groups depending on their myrtenyl acetate contents. Each group could be further divided in two subgroups according to the relative ratio of a-pinene to myrtenyl acetate or a-pinene to 1,8-cineol. This chemical profile of our samples can be classified in the first chemotype, i.e., apinene/myrtenyl acetate proposed by Bradesi et al. [14]. According to this report, linalyl acetate was the major constituent (31.4%), followed by a remarkably high concentration of limonene (21.8%). In our samples, linalyl acetate was detected in a small quantity (3.67%). In addition, we detected myrtenyl acetate (20.75%) and 1,8cineol (16.55%), the two major compounds of our samples, which were not described earlier. Environmental factors such as geography, temperature, day length, nutrients, etc., were considered to play key roles in the chemical composition of myrtle oil [15]. These factors influence the plants biosynthetic pathways and consequently the relative proportion of the main characteristic compounds. This leads to the occurrence of
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Table 1. Chemical Composition of the Essential Oil Isolated by Hydrodistillation from Leaves of Myrtus communis Peak
Retention time [min]
Compounds a )
[%] b )
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
8.44 9.18 10.45 10.80 12.12 12.26 14.45 17.64 17.88 19.73 22.10 22.80 23.80 24.48 25.20 26.20 30.05
a-Thujene a-Pinene b-Pinene Myrcene Limonene 1,8-Cineol Linalool a-Terpineol Myrtenol Linalyl acetate Myrtenyl acetate Terpenyl acetate Geranyl acetate Methyleugenol trans-Caryophyllene a-Humulene Caryophyllene oxide
0.83 15.59 0.10 0.12 8.94 16.55 13.30 2.88 0.94 3.67 20.75 0.63 2.99 1.17 0.80 1.29 0.66
Total [%] Monoterpenes Sesquiterpenes Esters Benzenoids
91.20 59.25 2.75 28.04 1.17
a ) Identification of components based on GC/MS Wiley 7.0 version library and National Institute of Standards and Technology 05 MS ( NIST) library data. b ) Percentages are the means of two runs and were obtained from electronic integration measurements using a selective mass detector.
different chemotypes, distinguishing myrtle oil of different origins, as well as seasonal variations throughout the plants vegetative cycle [16]. Antibacterial Activity. The antibacterial activity of M. communis essential oil was evaluated against Gram-positive (B. cereus, E. faecalis, S. aureus, S. epidermis, B. subtilis, L. monocytogenes, and M. luteus) and Gram-negative (P. aeruginosa, E. coli, S. enteritidis, and K. pneumoniae) bacteria. The antibacterial activity was assessed by evaluation of the inhibition zone (IZ) and the determination of MIC values. As compiled in Table 2, the essential oil of M. communis showed varying degrees of antibacterial activity against all strains tested. The inhibition zones were in the range of 16 – 28 mm. Among Gram-positive bacteria, highest inhibitory zone was observed against L. monocytogenes (28 mm), followed by B. cereus (26 mm) and S. aureus (25 mm). Among Gram-negative bacteria, highest inhibitory zone was observed against P. aeruginosa (20 mm). The inhibition zone for gentamicin (10 mg/well), which was used as positive control for bacteria, ranged from 12 to 25 mm. Negative control did not show any inhibitory effect against the tested bacteria. The essential oil of M. communis was found to possess significant antibacterial activities against the eight Gram-positive bacteria tested, compared to Gram-negative bacteria, with MIC values of 0.078 – 1.25 and 1.25 – 2.5 mg/ml, respectively. These results
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Table 2. Antibacterial Activity of McEO against Foodborne, Spoiling Bacteria, and Determination of the Minimum Inhibitory Concentrations (MICs) Bacterial strains
Inhibition-zone diameter [mm] a ) c
MIC [mg/ml] b )
d
McEO )
Gentamicin )
Gram positive Bacillus subtilis ATCC 6633 Bacillus cereus ATCC 14579 Staphylococcus aureus ATCC 25923 Staphylococcus aureus ATCC 6536 Staphylococcus epidermis ATCC 12228 Enterococcus faecalis ATCC 29212 Micrococcus luteus ATCC 1880 Listeria monocytogenes (food isolate 2132)
20 0.7 26 0.6 25 0.8 22 0.9 15 0.4 17 0.6 16 0.3 28 0.5
20 0.2 20 0.4 25 0.8 16 0.6 20 0.5 12 0.2 20 0.7 15 0.0
1.25 0.5 0.625 0.3 0.078 0.2 0.625 0.3 1.25 0.4 1.25 0.3 1.25 0.6 0.078 0.1
Gram negative Salmonella enteritidis (food isolate) Escherichia coli ATCC 25922 Escherichia coli ATCC 8739 Pseudomonas aeruginosa ATCC 9027 Klebsiella pneumoniae ATCC 10031
20 1.2 16 0.3 16 0.4 20 0.2 16 1
18 0.8 21 1.0 20 1.2 18 0.7 12 0.5
1.25 0.7 2.5 0.9 2.5 1.2 1.25 1.0 2.5 0.9
a ) Diameter of inhibition zones of including diameter of disc, 6 mm. b ) Values as mean S.D. of triplicate experiments. c ) McEO: M. communis essential oil (50 ml/well). d ) The used concentration of gentamicin was 10 mg/well.
are in accordance with several reports starting that essential oils are slightly more active against Gram-positive than against Gram-negative bacteria [17 – 19]. Furthermore, McEO showed potent inhibitions of B. cereus, L. monocytogenes, and S. aureus with MIC values of 0.625, 0.078, and 0.625 mg/ml, respectively. Infections caused by these bacteria, especially those with multidrug resistance, are among the most difficult to treat with conventional antibiotics. In the current study, the growth of L. monocytogenes was remarkably inhibited by the essential oil of M. communis (IZ 28 mm). The microorganisms tested in the present investigation are among the most important human pathogens known as opportunists for humans and animals and causes food contamination and deterioration. The obtained results are of great importance, particularly in the case of B. cereus and S. aureus, which are well-known for their resistance to a number of phytochemical compounds, and for the production of several types of enterotoxins that cause gastroenteritis [20]. Although, Hazzit et al. [21] reported that S. aureus was resistant to the essential oil from Thymus species, the above results indicated that the essential oil of M. communis was active and may potentially be useful in food preservation. The strong antibacterial activity of the McEO against the tested microorganisms could be attributed to the presence of high percentage of monoterpenes (59.25%) wellknown for their antibacterial potentials [22] [23]. In fact, it was shown that monoterpenes in essential oils are able to affect cellular integrity, resulting in inhibition of respiration and alteration in permeability. One of the major components of this oil, 1,8-cineol (16.55%), has been known to exhibit antibacterial activity against the bacterial strains E. coli, P. aeruginosa, S. typhi, S. aureus, Rhizobium legumino-
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sarum, and Bacillus subtilis [24]. The other major constituent of this oil, a-terpineol, has been reported [25] [26] to inhibit the growth of quite a number of bacteria and fungi that include S. aureus, E. coli, S. epidermis, and C. albicans. Also, several minor constituents of the EO have potential antimicrobial activities as reported in [27]. However, it is difficult to attribute the antimicrobial activity of McEO oil, characterized by a complex mixture, to a single or particular constituent. The antimicrobial effects of 1,8-cineol and caryophyllene oxide were also reported [17]. Thus, the synergistic effects, and the diversity of major and minor constituents present in the essential oil should be taken into account for their biological activity [28]. From these results, McEO may be considered as a natural preservative against foodborn pathogens for food production industry. Antifungal Activities. The antifungal activities were evaluated against Aspergillus sp., Fusarium sp., and Alternaria alternata. Results evidenced strong inhibitory effects of McEO on the growth of A. niger and A. flavus with inhibition zone diameters of 30 mm and MIC values of 0.078 mg/ml (Table 3). Also, the McEO exhibited antifungal activities against Fusarium sp. and Aspergillus sp., which are responsible for spoilage of many foods. The maximal inhibition zone diameters were 20 – 22 mm, and MIC values ranged from 0.156 to 1.25 mg/ml (Table 3). In general, oils containing high contents of oxygenated monoterpenes have stronger antifungal activities than the oils relatively rich in monoterpene hydrocarbons or sesquiterpenes [29]. Thus, the potent antifungal activity of McEO could be attributed to its relatively high content of oxygenated monoterpenes. In addition, antifungal activity of caryophyllene oxide, present in McEO, against Fusarium species, was previously reported [30]. a-Pinene, which was found in appreciable amounts in McEO, has been reported to be the cause of the antifungal activity of oils from Pistacia lentiscus [31]. The volatile oils consist of complex mixtures of numerous components. Either major or trace compound(s) might give rise to the antifungal activity. Possible synergistic and antagonistic effects of compounds also play an important role in fungi inhibition. Table 3. Antifungal Activity of McEO and Determination of the Minimum Fungicidal Concentrations ( MFCs) Fungal strains
Aspergillus niger (CTM 10099) Aspergillus flavus (food isolate) Aspergillus nidulans (food isolate) Aspergillus fumigatus (food isolate) Fusarium graminearum ( ISPAVE 271) Fusarium oxysporum (CTM10402) Fusarium culmorum ( ISPAVE 21w) Alternaria alternata (CTM 10230)
Inhibition zones diameter [mm] a ) McEO c )
Amphotericin B d )
30 1.2 30 0.9 22 0.3 26 0.6 25 0.6 22 0.9 20 0.4 20 0.4
15 0.9 10 0.3 0 0 14 0.5 14 0.2 12 0.7 14 0.6
MFC [mg/ml] b )
0.078 0.2 0.078 0.4 0.625 0.3 0.312 0.6 0.156 0.4 0.156 0.3 0.156 0.4 0.625 0.8
a ) Diameter of inhibition zones including diameter of disc, 6 mm. b ) Values are given as mean S.D. of triplicate experiments. c ) McEO: M. communis essential oil (50 ml/well). d ) The used concentration of amphotericin B was 20 mg/well.
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To the best of our knowledge, this is the first study providing data on the antifungal activity of McEO evaluated against a wide range of fungal strains. Findings revealed that the oil of this aromatic plant could be useful as an alternative antimicrobial agent in natural medicine for the treatment of many infectious diseases. Effect of McEO on Viable Counts of Listeria monocytogenes (food isolate 2132). The antimicrobial activity was further characterized using kinetic viability assays to determine the action of McEO over time (Fig.). An initial decrease in viable-cell counts for all tested concentrations was observed. The control sample (without EO) exhibited approximately the same colony-forming unit (CFU)/ml within 50 min, and no inhibition of the L. monocytogenes growth was observed. However, the numbers of L. monocytogenes viable cells were reduced significantly (p < 0.05) over time after the addition of McEO (Fig.). Concentrations of 156 mg/ml (2 MICs) indicated bacteriostatic activity, and of 234 and 312 mg/ml (3 and 4 MICs) showed bactericidal activities after 10 min. Therefore, the essential oil of M. communis could be considered as source of natural antibiotics against opportunistic pathogens and could be used as food antipoisoning agent. This important finding could be used to limit the proliferation of this psychrotrophic pathogen in refrigerated foods.
Figure. Bactericidal effect of McEO on Listeria monocytogenes (food isolate 2132). Samples were taken at different incubation times, and viability was determined by the plate colony count procedure (CFU, Colony-Forming Unit).
Conclusions. – We reported the chemical composition of Tunisian Myrtus communis essential oil (McEO). Seventeen compounds were identified, and the main components were myrtenyl acetate (20.75%), 1,8-cineol (16.55%), a-pinene (15.59%), linalool (13.30%), limonene (8.94%), linalyl acetate (3.67%), geranyl acetate (2.99%), and a-terpineol (2.88%), We also investigated the antibacterial and antifungal activities of McEO, which exhibited very important in vitro antibacterial activities against the studied bacteria, confirmed by low minimum inhibitory concentrations (MICs). Therefore, McEO can be used as a natural antimicrobial agent for the treatment for several infectious diseases.
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Our results contribute to a better evaluation of this medicinal plant. Several further biological tests are needed to search for more probable activities of this plant, to characterize active principles, and to assess toxicity by laboratory assays.
Experimental Part Chemicals. All chemicals, purchased from Sigma-Aldrich-Fluka (F-Saint-Quentin), were of anal. reagent grade. Collection of Plant Material. Myrtus communis L. (M. communis) leaves were collected from Kef (Tunisia, 35.238 N and 11.118 E) in May 2012. The botanical identification of M. communis was conducted by Prof. Jamel Ghazali, botanist in the Faculty of Science at Arass, Kingdom of Saudi Arabia, and a voucher specimen (LBPes C.S. 014) was deposited with the Laboratory of Protection and Amelioration of Plants in the Center of Biotechnology of Sfax. Essential-Oil Extractions. The oil was extracted from 0.5 kg of fresh plant by steam distillation during 3 h using a Clevenger-type apparatus [32] [33]. The aq. phase was extracted with CH2Cl2 (3 50 ml), and the org. phase was dried (Na2SO4 ). To determine the yield, the solvent was evaporated using a rotavapory vacuum evaporator to afford 2.5 g. The resulting essential oil (McEO) was stored at 48 prior to further analyses. The McEO was solubilized in hexane for GC and MS analysis. Antimicrobial Activity. Microorganisms and Growth Conditions. Authentic pure cultures of bacteria and fungi were obtained from international culture collections (ATCC) and the local culture collection of the Center of Biotechnology of Sfax, Tunisia. They included Gram-positive bacteria, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 1880, and Listeria monocytogenes (food isolate 2132) and Gram-negative bacteria, Salmonella enterica (food isolate) and Klebsiella pneumoniae ATCC 10031. These standard strains were obtained from the Microorganisms Collection of the Laboratory of Microbiology, University Hospital Center (CHU) of Habib Bourguiba, Sfax, Tunisia, and food isolates from the Laboratory of Parasitology-Mycology, Sfax Faculty of Medicine, Tunisia. The following fungal strains were also tested: Aspergillus niger CTM 10099, Aspergillus flavus (food isolate), Fusarium graminearum (ISPAVE 271), Fusarium oxysporum (CTM10402), Fusarium culmorum (ISPAVE 21w), Rhizopus nigricans (CTM10150), and Alternaria alternata (CTM 10230). These fungal strains were obtained from the Microorganisms Collection of the Laboratory of Parasitology-Mycology, Sfax Faculty of Medicine, Tunisia, and the Center of Biotechnology of Sfax, Tunisia. The bacterial strains were grown on MuellerHinton broth (Bio-Rad, France) at 378 for 12 – 14 h, while potato dextrose agar (PDA; 1.5% agar) at 288 for 4 d were used for fungi. Inocula were prepared from an overnight broth culture by their dilution in saline soln. to ca. 107 colony-forming units (CFU)/ml for bacteria and 105 spores/ml for fungus. Agar-Diffusion Method. Antibacterial and antifungal tests were performed by using the agar well diffusion method as described by Tagg and McGiven [34], and the broth microdilution assay using sterile Mueller – Hinton media (Bio-Rad, France) for bacterial strains, and potato dextrose agar (Bio-Rad, France) for antifungal tests. Fifteen ml of the molten agar (458) were poured into sterile Petri dishes (f 90 mm). Working cell suspensions were prepared, and 100 ml were evenly spread onto the surface of the agar plates of MuellerHinton agar (Oxoid Ltd., UK) for bacteria, or potatoes dextrose agar medium (Oxoid Ltd., UK) for fungi. Once the plates had been aseptically dried, 06 mm wells were punched into the agar with a sterile Pasteur pipette. The McEO were dissolved in DMSO/H2O 1 : 1 and sterile H2O to a final concentration of 50 mg/ml. Thus, 50 ml were placed into the wells, and the plates were incubated at 378 for 24 h for bacterial strains, and 72 h for fungi at 288. Gentamicin (10 mg/wells) and amphotericin B (20 mg/wells), and DMSO served as positive and negative controls, respectively. Antimicrobial activity was evaluated by measuring the diameter of circular inhibition zones around the well. Tests were performed in triplicate.
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Determination of MIC and MFC Values. Minimum inhibitory concentrations (MICs) of McEO were determined as described by Gulluce et al. [35] against a panel of 21 microorganisms representing different species of different ecosystems. The test was performed in sterile 96-well microplates with a final volume of 100 ml in each microplate well. A stock soln. of the McEO (50 mg/ml) was prepared in DMSO/H2O 1 : 9. The inhibitory activity of the McEO was properly prepared and transferred to each well in order to obtain a twofold serial dilution of the original sample and to produce the concentration range of 0.039 – 10 mg/ml. To each test well, 10 ml of cell suspension were added to final inoculums concentrations of 106 CFU/ml for bacteria and 105 spores/ml for fungus. Positive growth control wells consisted of bacteria or fungi only in their adequate medium. DMSO/H2O 1 : 9 was used as negative control. The plates were then covered with the sterile plate covers and incubated at 378 for 24 h for bacterial strains, and 72 h for fungi at 288. The MIC was defined as the lowest concentration of the total essential oil at which the microorganism does not demonstrate visible growth after incubation. As an indicator of microorganism growth, 25 ml of MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide) indicator soln. (0.5 mg/ml), dissolved in sterile H2O, were added to the wells and incubated at 378 for 30 min. The colorless tetrazolium salt acts as an electron acceptor and is reduced to a red-colored formazan product by biologically active organisms. Where microbial growth was inhibited, the soln. in the well remained clear after incubation with MTT. The minimum fungicidal concentrations (MFCs) were determined by serial subcultivation of 10 ml in PDA plates and incubated for 72 h at 288. The lowest concentration with no visible growth was defined as the MFC, indicating 99.5% killing of the original inoculum. DMSO and EtOH were used as negative controls. The determinations of MIC and MFC values were conducted in triplicate. Effect of Essential Oil on Viable Counts of Bacteria. The effect of McEO against Listeria monocytogenes was assessed in vitro by sequential sampling and counting viable bacteria in the physiological saline soln. following the addition of the essential oil as described by Kawakami et al., with slight modifications. For viable counts, each of the tubes containing bacterial suspension (ca. 108 CFU/ml) of L. monocytogenes was inoculated with three different concentrations of the McEO in a final volume of 5 ml of sterile physiological saline soln. and incubated at 378. For the determination of the residual viable cells by the plate colony count technique, 100 ml of each sample were removed at various times, i.e., 0, 10, 20, 30, 50, and 60 min, and diluted to 0.9 ml of sterile physiological saline soln., a tenfold dilution and spread on the surface of MuellerHinton agar (Oxoid Ltd., UK). The colonies were counted after 24 h of incubation at 378 [36]. The control was prepared under the same experimental conditions as mentioned above, but without addition of McEO. Gas Chromatography. McEO was analyzed using a Thermo Electron (F-Courtaboeuf) gas chromatograph equipped with a flame ionization detection detector (FID) and DB-5MS cap. column (30 m 0.25 mm, film thickness 0.25 mm). Injector and detector temps. were 2008, and detector temp. was 2708 [37] [38]. Oven temp. was gradually raised from 60 to 2608 at a rate of 58/min, held for 15 min, and finally raised to 3408 at a rate of 408/min. He (purity 99.99%) was the carrier gas, at a flow rate of 1 ml/ min. Total analysis time was 57 min. Diluted sample (1:100 in petroleum ether (v/v)) of 1.0 ml was injected in the split mode (ratio 1 : 10). Quant. data were obtained electronically from FID area percent data without using correction factors. Gas Chromatography/Mass Spectrometry (GC/MS). Analysis of McEO was performed under the same conditions as by GC (column, oven temp., flow rate of the carrier gas) using a Thermo Electron (FCourtaboeuf) DSQ II GC/MS single quadrupole mass-selective detector in the electron-impact (EI) mode (70 eV). Injector and MS transfer line temps. were 200 and 3008, resp. MS was adjusted for an emission current of 10 mA, and electron multiplier voltage was 1500 V. Trap temp. was 2508, and mass scanning was from 40 to 650 amu. The components were identified based on the comparison of their retention time (tR ) and mass spectra with those of standards, Wiley 2001 library data (NIST 02 version 2.62) of the GC/MS system, and literature data (Adams). All determinations were performed in duplicate and averaged. Statistical Analysis. Exper. results of this study were expressed as means standard deviation of three parallel measurements. The significance of difference was calculated by Students t-test, and values p < 0.05 and p < 0.001 were considered to be significant and highly significant, resp. Correlation and regression analysis was carried out using EXCELL program (Microsoft Corporation, USA).
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[37] Z. Mallek, I. Fendri, L. Khannous, A. Ben Hassena, A. I. Traore, M. A. Ayadi, R. Gdoura, Lipids Health Dis. 2012, 11, 35. [38] I. Fendri, L. Khannous, Z. Mallek, A. I. Traore, N. Gharsallah, R. Gdoura, Lipids Health Dis. 2012, 11, 71. Received May 8, 2013
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Myrtus communis Essential Oil: Chemical Composition and Antimicrobial Activities against Food Spoilage Pathogens by Anis Ben Hsouna* a ), Naceur Hamdi b ) c ), Ramzi Miladi d ), and Slim Abdelkafi d ) a
) Department of Life Sciences, Faculty of Sciences of Gafsa, Zarroug 2112, Gafsa, Tunisia (phone: þ 216-97-130430; fax: þ 216-74-565555; e-mail: [email protected]) b ) Chemistry Department, College of Science and Arts, Al-Rass, P.O. Box 53, Qassim University, Saudi Arabia, Kingdom of Saudi Arabia c ) High Institute of Environmental Science and Technology, Borj-Ce´dria, Tunisia & University of Carthage, Tunisia d ) University of Sfax, Ecole Nationale dInge´nieurs de Sfax, De´partement de Ge´nie Biologique, Route de Soukra BP 1117, 3038 Sfax, Tunisia
Myrtus communis is a typical plant of the Mediterranean area, which is mainly used as animal and human food and, in folk medicine, for treating some disorders. In the present study, we evaluated in vitro antibacterial and antifungal properties of the essential oils of Myrtus communis (McEO), as well as its phytochemical composition. The GC/MS analysis of the essential oil revealed 17 compounds. Myrtenyl acetate (20.75%), 1,8-cineol (16.55%), a-pinene (15.59%), linalool (13.30%), limonene (8.94%), linalyl acetate (3.67%), geranyl acetate (2.99%), and a-terpineol (2.88%) were the major components. The antimicrobial activity of the essential oil was also investigated on several microorganisms. The inhibition zones and minimal inhibitory concentration (MIC) values of bacterial strains were in the range of 16 – 28 mm and 0.078 – 2.5 mg/ml, respectively. The inhibitory activity of the McEO against Gram-positive bacteria was significantly higher than against Gram-negative. It also exhibited remarkable activity against several fungal strains. The investigation of the mode of action of the McEO by the time-kill curve against Listeria monocytogenes (food isolate) showed a drastic bactericidal effect after 5 min using a concentration of 312 mg/ml. These results evidence that the McEO possesses antimicrobial properties, and it is, therefore, a potential source for active ingredients for food and pharmaceutical industries.
Introduction. – Plants have been a valuable source of natural products for maintaining human health, especially in the last decade, with more intensive studies for natural therapies [1]. Considerable works have been performed to investigate new natural antimicrobial and antioxidant agents leading to the identification of potential new natural drugs. Plant essential oils and their components have been shown to possess multiple biological activities [2]. Tunisian flora is remarkable for its diversity of medicinal plants. Myrtus communis L. (Myrtaceae) is an evergreen scrub, typical of the Mediterranean maquis, which grows spontaneously in many countries. In Tunisia, the genus Myrtus is represented by only one species, M. communis, which grows wild in the coastal areas, the internal hills, and the forest areas of North Tunisia [3]. It is traditionally used as an antiseptic, disinfectant drug, and hypoglycemic agent [4]. The leaves are used as a spice, especially for meat, and a popular liqueur is produced in Sardinia from its berries and leaves [5]. Myrtle extracts have been reported to possess antihyperglycemic, analgesic [6], 2014 Verlag Helvetica Chimica Acta AG, Zrich
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antigenotoxic [7], and antibacterial activities [8]. Recent reports have described the antioxidant activities of different myrtle extracts and compounds [9]. Various known antioxidants including flavonoids (quercetin and myricetin), phenolic acids (caffeic and ellagic acids), tannins, and a-tocopherol were isolated from myrtle [10]. Different parts of the plant find various uses in the food industry, such as for flavoring meat and sauces, and in the cosmetic industry [3] [11]. A striking feature of the plant is the pleasant smell of its essential oil, present in numerous glands, especially in the leaves. The main compounds responsible for the flavor and scent of myrtle oil are monoterpenes, i.e., 1,8-cineol, myrtenyl acetate, a-pinene, myrtenol, limonene, etc. [12] [13]. However, the antimicrobial property of myrtle-leaf essential oil has been rarely investigated. Therefore, in the present study, we examined the chemical composition of the leaf essential oil of Tunisian Myrtus communis (M. communis) by GC/MS and screened its antimicrobial activity. The antimicrobial activity was evaluated against a diverse range of foodborne pathogens. Also, an in vitro assessment of the McEO killing activity was carried out against Listeria monocytogenes (food isolate 2132). Results and Discussion. – Chemical Composition. The chemical composition of M. communis essential oil was determined by using both GC and GC/MS techniques. The percentages and the retention indices (RIs) of the identified oil components were compiled in Table 1, in the order of their elution on the 5MS capillary column. Seventeen compounds were identified, representing 91.20% of the total oil content. These compounds were divided into four classes: monoterpenes, sesquiterpenes, esters, and benzenoids (Table 1). This oil was characterized by very high percentage of monoterpenes (59.25%), and especially the oxygenated ones constitute the predominant class. Linalool (13.30%), a-terpineol (2.88%), and 1,8-cineol (16.55%) were the major components among the oxygenated monoterpenes. The monoterpene hydrocarbons of this oil were represented by a-pinene (15.59%) and limonene (8.94%). Other major detected components were linalyl acetate (3.67%), myrtenyl acetate (20.75%), geranyl acetate (2.99%), methyleugenol (1.17%), and a-humulene (1.29%). The minor components ( < 1%) were identified as caryophyllene oxide (0.66%), transcaryophyllene (0.80%), terpenyl acetate (0.63%), myrtenol (0.94%), myrcene (0.12%), b-pinene (0.1%), and a-thujene (0.83%). The myrtle essential oils could be separated in two groups depending on their myrtenyl acetate contents. Each group could be further divided in two subgroups according to the relative ratio of a-pinene to myrtenyl acetate or a-pinene to 1,8-cineol. This chemical profile of our samples can be classified in the first chemotype, i.e., apinene/myrtenyl acetate proposed by Bradesi et al. [14]. According to this report, linalyl acetate was the major constituent (31.4%), followed by a remarkably high concentration of limonene (21.8%). In our samples, linalyl acetate was detected in a small quantity (3.67%). In addition, we detected myrtenyl acetate (20.75%) and 1,8cineol (16.55%), the two major compounds of our samples, which were not described earlier. Environmental factors such as geography, temperature, day length, nutrients, etc., were considered to play key roles in the chemical composition of myrtle oil [15]. These factors influence the plants biosynthetic pathways and consequently the relative proportion of the main characteristic compounds. This leads to the occurrence of
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Table 1. Chemical Composition of the Essential Oil Isolated by Hydrodistillation from Leaves of Myrtus communis Peak
Retention time [min]
Compounds a )
[%] b )
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
8.44 9.18 10.45 10.80 12.12 12.26 14.45 17.64 17.88 19.73 22.10 22.80 23.80 24.48 25.20 26.20 30.05
a-Thujene a-Pinene b-Pinene Myrcene Limonene 1,8-Cineol Linalool a-Terpineol Myrtenol Linalyl acetate Myrtenyl acetate Terpenyl acetate Geranyl acetate Methyleugenol trans-Caryophyllene a-Humulene Caryophyllene oxide
0.83 15.59 0.10 0.12 8.94 16.55 13.30 2.88 0.94 3.67 20.75 0.63 2.99 1.17 0.80 1.29 0.66
Total [%] Monoterpenes Sesquiterpenes Esters Benzenoids
91.20 59.25 2.75 28.04 1.17
a ) Identification of components based on GC/MS Wiley 7.0 version library and National Institute of Standards and Technology 05 MS ( NIST) library data. b ) Percentages are the means of two runs and were obtained from electronic integration measurements using a selective mass detector.
different chemotypes, distinguishing myrtle oil of different origins, as well as seasonal variations throughout the plants vegetative cycle [16]. Antibacterial Activity. The antibacterial activity of M. communis essential oil was evaluated against Gram-positive (B. cereus, E. faecalis, S. aureus, S. epidermis, B. subtilis, L. monocytogenes, and M. luteus) and Gram-negative (P. aeruginosa, E. coli, S. enteritidis, and K. pneumoniae) bacteria. The antibacterial activity was assessed by evaluation of the inhibition zone (IZ) and the determination of MIC values. As compiled in Table 2, the essential oil of M. communis showed varying degrees of antibacterial activity against all strains tested. The inhibition zones were in the range of 16 – 28 mm. Among Gram-positive bacteria, highest inhibitory zone was observed against L. monocytogenes (28 mm), followed by B. cereus (26 mm) and S. aureus (25 mm). Among Gram-negative bacteria, highest inhibitory zone was observed against P. aeruginosa (20 mm). The inhibition zone for gentamicin (10 mg/well), which was used as positive control for bacteria, ranged from 12 to 25 mm. Negative control did not show any inhibitory effect against the tested bacteria. The essential oil of M. communis was found to possess significant antibacterial activities against the eight Gram-positive bacteria tested, compared to Gram-negative bacteria, with MIC values of 0.078 – 1.25 and 1.25 – 2.5 mg/ml, respectively. These results
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Table 2. Antibacterial Activity of McEO against Foodborne, Spoiling Bacteria, and Determination of the Minimum Inhibitory Concentrations (MICs) Bacterial strains
Inhibition-zone diameter [mm] a ) c
MIC [mg/ml] b )
d
McEO )
Gentamicin )
Gram positive Bacillus subtilis ATCC 6633 Bacillus cereus ATCC 14579 Staphylococcus aureus ATCC 25923 Staphylococcus aureus ATCC 6536 Staphylococcus epidermis ATCC 12228 Enterococcus faecalis ATCC 29212 Micrococcus luteus ATCC 1880 Listeria monocytogenes (food isolate 2132)
20 0.7 26 0.6 25 0.8 22 0.9 15 0.4 17 0.6 16 0.3 28 0.5
20 0.2 20 0.4 25 0.8 16 0.6 20 0.5 12 0.2 20 0.7 15 0.0
1.25 0.5 0.625 0.3 0.078 0.2 0.625 0.3 1.25 0.4 1.25 0.3 1.25 0.6 0.078 0.1
Gram negative Salmonella enteritidis (food isolate) Escherichia coli ATCC 25922 Escherichia coli ATCC 8739 Pseudomonas aeruginosa ATCC 9027 Klebsiella pneumoniae ATCC 10031
20 1.2 16 0.3 16 0.4 20 0.2 16 1
18 0.8 21 1.0 20 1.2 18 0.7 12 0.5
1.25 0.7 2.5 0.9 2.5 1.2 1.25 1.0 2.5 0.9
a ) Diameter of inhibition zones of including diameter of disc, 6 mm. b ) Values as mean S.D. of triplicate experiments. c ) McEO: M. communis essential oil (50 ml/well). d ) The used concentration of gentamicin was 10 mg/well.
are in accordance with several reports starting that essential oils are slightly more active against Gram-positive than against Gram-negative bacteria [17 – 19]. Furthermore, McEO showed potent inhibitions of B. cereus, L. monocytogenes, and S. aureus with MIC values of 0.625, 0.078, and 0.625 mg/ml, respectively. Infections caused by these bacteria, especially those with multidrug resistance, are among the most difficult to treat with conventional antibiotics. In the current study, the growth of L. monocytogenes was remarkably inhibited by the essential oil of M. communis (IZ 28 mm). The microorganisms tested in the present investigation are among the most important human pathogens known as opportunists for humans and animals and causes food contamination and deterioration. The obtained results are of great importance, particularly in the case of B. cereus and S. aureus, which are well-known for their resistance to a number of phytochemical compounds, and for the production of several types of enterotoxins that cause gastroenteritis [20]. Although, Hazzit et al. [21] reported that S. aureus was resistant to the essential oil from Thymus species, the above results indicated that the essential oil of M. communis was active and may potentially be useful in food preservation. The strong antibacterial activity of the McEO against the tested microorganisms could be attributed to the presence of high percentage of monoterpenes (59.25%) wellknown for their antibacterial potentials [22] [23]. In fact, it was shown that monoterpenes in essential oils are able to affect cellular integrity, resulting in inhibition of respiration and alteration in permeability. One of the major components of this oil, 1,8-cineol (16.55%), has been known to exhibit antibacterial activity against the bacterial strains E. coli, P. aeruginosa, S. typhi, S. aureus, Rhizobium legumino-
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sarum, and Bacillus subtilis [24]. The other major constituent of this oil, a-terpineol, has been reported [25] [26] to inhibit the growth of quite a number of bacteria and fungi that include S. aureus, E. coli, S. epidermis, and C. albicans. Also, several minor constituents of the EO have potential antimicrobial activities as reported in [27]. However, it is difficult to attribute the antimicrobial activity of McEO oil, characterized by a complex mixture, to a single or particular constituent. The antimicrobial effects of 1,8-cineol and caryophyllene oxide were also reported [17]. Thus, the synergistic effects, and the diversity of major and minor constituents present in the essential oil should be taken into account for their biological activity [28]. From these results, McEO may be considered as a natural preservative against foodborn pathogens for food production industry. Antifungal Activities. The antifungal activities were evaluated against Aspergillus sp., Fusarium sp., and Alternaria alternata. Results evidenced strong inhibitory effects of McEO on the growth of A. niger and A. flavus with inhibition zone diameters of 30 mm and MIC values of 0.078 mg/ml (Table 3). Also, the McEO exhibited antifungal activities against Fusarium sp. and Aspergillus sp., which are responsible for spoilage of many foods. The maximal inhibition zone diameters were 20 – 22 mm, and MIC values ranged from 0.156 to 1.25 mg/ml (Table 3). In general, oils containing high contents of oxygenated monoterpenes have stronger antifungal activities than the oils relatively rich in monoterpene hydrocarbons or sesquiterpenes [29]. Thus, the potent antifungal activity of McEO could be attributed to its relatively high content of oxygenated monoterpenes. In addition, antifungal activity of caryophyllene oxide, present in McEO, against Fusarium species, was previously reported [30]. a-Pinene, which was found in appreciable amounts in McEO, has been reported to be the cause of the antifungal activity of oils from Pistacia lentiscus [31]. The volatile oils consist of complex mixtures of numerous components. Either major or trace compound(s) might give rise to the antifungal activity. Possible synergistic and antagonistic effects of compounds also play an important role in fungi inhibition. Table 3. Antifungal Activity of McEO and Determination of the Minimum Fungicidal Concentrations ( MFCs) Fungal strains
Aspergillus niger (CTM 10099) Aspergillus flavus (food isolate) Aspergillus nidulans (food isolate) Aspergillus fumigatus (food isolate) Fusarium graminearum ( ISPAVE 271) Fusarium oxysporum (CTM10402) Fusarium culmorum ( ISPAVE 21w) Alternaria alternata (CTM 10230)
Inhibition zones diameter [mm] a ) McEO c )
Amphotericin B d )
30 1.2 30 0.9 22 0.3 26 0.6 25 0.6 22 0.9 20 0.4 20 0.4
15 0.9 10 0.3 0 0 14 0.5 14 0.2 12 0.7 14 0.6
MFC [mg/ml] b )
0.078 0.2 0.078 0.4 0.625 0.3 0.312 0.6 0.156 0.4 0.156 0.3 0.156 0.4 0.625 0.8
a ) Diameter of inhibition zones including diameter of disc, 6 mm. b ) Values are given as mean S.D. of triplicate experiments. c ) McEO: M. communis essential oil (50 ml/well). d ) The used concentration of amphotericin B was 20 mg/well.
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To the best of our knowledge, this is the first study providing data on the antifungal activity of McEO evaluated against a wide range of fungal strains. Findings revealed that the oil of this aromatic plant could be useful as an alternative antimicrobial agent in natural medicine for the treatment of many infectious diseases. Effect of McEO on Viable Counts of Listeria monocytogenes (food isolate 2132). The antimicrobial activity was further characterized using kinetic viability assays to determine the action of McEO over time (Fig.). An initial decrease in viable-cell counts for all tested concentrations was observed. The control sample (without EO) exhibited approximately the same colony-forming unit (CFU)/ml within 50 min, and no inhibition of the L. monocytogenes growth was observed. However, the numbers of L. monocytogenes viable cells were reduced significantly (p < 0.05) over time after the addition of McEO (Fig.). Concentrations of 156 mg/ml (2 MICs) indicated bacteriostatic activity, and of 234 and 312 mg/ml (3 and 4 MICs) showed bactericidal activities after 10 min. Therefore, the essential oil of M. communis could be considered as source of natural antibiotics against opportunistic pathogens and could be used as food antipoisoning agent. This important finding could be used to limit the proliferation of this psychrotrophic pathogen in refrigerated foods.
Figure. Bactericidal effect of McEO on Listeria monocytogenes (food isolate 2132). Samples were taken at different incubation times, and viability was determined by the plate colony count procedure (CFU, Colony-Forming Unit).
Conclusions. – We reported the chemical composition of Tunisian Myrtus communis essential oil (McEO). Seventeen compounds were identified, and the main components were myrtenyl acetate (20.75%), 1,8-cineol (16.55%), a-pinene (15.59%), linalool (13.30%), limonene (8.94%), linalyl acetate (3.67%), geranyl acetate (2.99%), and a-terpineol (2.88%), We also investigated the antibacterial and antifungal activities of McEO, which exhibited very important in vitro antibacterial activities against the studied bacteria, confirmed by low minimum inhibitory concentrations (MICs). Therefore, McEO can be used as a natural antimicrobial agent for the treatment for several infectious diseases.
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Our results contribute to a better evaluation of this medicinal plant. Several further biological tests are needed to search for more probable activities of this plant, to characterize active principles, and to assess toxicity by laboratory assays.
Experimental Part Chemicals. All chemicals, purchased from Sigma-Aldrich-Fluka (F-Saint-Quentin), were of anal. reagent grade. Collection of Plant Material. Myrtus communis L. (M. communis) leaves were collected from Kef (Tunisia, 35.238 N and 11.118 E) in May 2012. The botanical identification of M. communis was conducted by Prof. Jamel Ghazali, botanist in the Faculty of Science at Arass, Kingdom of Saudi Arabia, and a voucher specimen (LBPes C.S. 014) was deposited with the Laboratory of Protection and Amelioration of Plants in the Center of Biotechnology of Sfax. Essential-Oil Extractions. The oil was extracted from 0.5 kg of fresh plant by steam distillation during 3 h using a Clevenger-type apparatus [32] [33]. The aq. phase was extracted with CH2Cl2 (3 50 ml), and the org. phase was dried (Na2SO4 ). To determine the yield, the solvent was evaporated using a rotavapory vacuum evaporator to afford 2.5 g. The resulting essential oil (McEO) was stored at 48 prior to further analyses. The McEO was solubilized in hexane for GC and MS analysis. Antimicrobial Activity. Microorganisms and Growth Conditions. Authentic pure cultures of bacteria and fungi were obtained from international culture collections (ATCC) and the local culture collection of the Center of Biotechnology of Sfax, Tunisia. They included Gram-positive bacteria, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 1880, and Listeria monocytogenes (food isolate 2132) and Gram-negative bacteria, Salmonella enterica (food isolate) and Klebsiella pneumoniae ATCC 10031. These standard strains were obtained from the Microorganisms Collection of the Laboratory of Microbiology, University Hospital Center (CHU) of Habib Bourguiba, Sfax, Tunisia, and food isolates from the Laboratory of Parasitology-Mycology, Sfax Faculty of Medicine, Tunisia. The following fungal strains were also tested: Aspergillus niger CTM 10099, Aspergillus flavus (food isolate), Fusarium graminearum (ISPAVE 271), Fusarium oxysporum (CTM10402), Fusarium culmorum (ISPAVE 21w), Rhizopus nigricans (CTM10150), and Alternaria alternata (CTM 10230). These fungal strains were obtained from the Microorganisms Collection of the Laboratory of Parasitology-Mycology, Sfax Faculty of Medicine, Tunisia, and the Center of Biotechnology of Sfax, Tunisia. The bacterial strains were grown on MuellerHinton broth (Bio-Rad, France) at 378 for 12 – 14 h, while potato dextrose agar (PDA; 1.5% agar) at 288 for 4 d were used for fungi. Inocula were prepared from an overnight broth culture by their dilution in saline soln. to ca. 107 colony-forming units (CFU)/ml for bacteria and 105 spores/ml for fungus. Agar-Diffusion Method. Antibacterial and antifungal tests were performed by using the agar well diffusion method as described by Tagg and McGiven [34], and the broth microdilution assay using sterile Mueller – Hinton media (Bio-Rad, France) for bacterial strains, and potato dextrose agar (Bio-Rad, France) for antifungal tests. Fifteen ml of the molten agar (458) were poured into sterile Petri dishes (f 90 mm). Working cell suspensions were prepared, and 100 ml were evenly spread onto the surface of the agar plates of MuellerHinton agar (Oxoid Ltd., UK) for bacteria, or potatoes dextrose agar medium (Oxoid Ltd., UK) for fungi. Once the plates had been aseptically dried, 06 mm wells were punched into the agar with a sterile Pasteur pipette. The McEO were dissolved in DMSO/H2O 1 : 1 and sterile H2O to a final concentration of 50 mg/ml. Thus, 50 ml were placed into the wells, and the plates were incubated at 378 for 24 h for bacterial strains, and 72 h for fungi at 288. Gentamicin (10 mg/wells) and amphotericin B (20 mg/wells), and DMSO served as positive and negative controls, respectively. Antimicrobial activity was evaluated by measuring the diameter of circular inhibition zones around the well. Tests were performed in triplicate.
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Determination of MIC and MFC Values. Minimum inhibitory concentrations (MICs) of McEO were determined as described by Gulluce et al. [35] against a panel of 21 microorganisms representing different species of different ecosystems. The test was performed in sterile 96-well microplates with a final volume of 100 ml in each microplate well. A stock soln. of the McEO (50 mg/ml) was prepared in DMSO/H2O 1 : 9. The inhibitory activity of the McEO was properly prepared and transferred to each well in order to obtain a twofold serial dilution of the original sample and to produce the concentration range of 0.039 – 10 mg/ml. To each test well, 10 ml of cell suspension were added to final inoculums concentrations of 106 CFU/ml for bacteria and 105 spores/ml for fungus. Positive growth control wells consisted of bacteria or fungi only in their adequate medium. DMSO/H2O 1 : 9 was used as negative control. The plates were then covered with the sterile plate covers and incubated at 378 for 24 h for bacterial strains, and 72 h for fungi at 288. The MIC was defined as the lowest concentration of the total essential oil at which the microorganism does not demonstrate visible growth after incubation. As an indicator of microorganism growth, 25 ml of MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide) indicator soln. (0.5 mg/ml), dissolved in sterile H2O, were added to the wells and incubated at 378 for 30 min. The colorless tetrazolium salt acts as an electron acceptor and is reduced to a red-colored formazan product by biologically active organisms. Where microbial growth was inhibited, the soln. in the well remained clear after incubation with MTT. The minimum fungicidal concentrations (MFCs) were determined by serial subcultivation of 10 ml in PDA plates and incubated for 72 h at 288. The lowest concentration with no visible growth was defined as the MFC, indicating 99.5% killing of the original inoculum. DMSO and EtOH were used as negative controls. The determinations of MIC and MFC values were conducted in triplicate. Effect of Essential Oil on Viable Counts of Bacteria. The effect of McEO against Listeria monocytogenes was assessed in vitro by sequential sampling and counting viable bacteria in the physiological saline soln. following the addition of the essential oil as described by Kawakami et al., with slight modifications. For viable counts, each of the tubes containing bacterial suspension (ca. 108 CFU/ml) of L. monocytogenes was inoculated with three different concentrations of the McEO in a final volume of 5 ml of sterile physiological saline soln. and incubated at 378. For the determination of the residual viable cells by the plate colony count technique, 100 ml of each sample were removed at various times, i.e., 0, 10, 20, 30, 50, and 60 min, and diluted to 0.9 ml of sterile physiological saline soln., a tenfold dilution and spread on the surface of MuellerHinton agar (Oxoid Ltd., UK). The colonies were counted after 24 h of incubation at 378 [36]. The control was prepared under the same experimental conditions as mentioned above, but without addition of McEO. Gas Chromatography. McEO was analyzed using a Thermo Electron (F-Courtaboeuf) gas chromatograph equipped with a flame ionization detection detector (FID) and DB-5MS cap. column (30 m 0.25 mm, film thickness 0.25 mm). Injector and detector temps. were 2008, and detector temp. was 2708 [37] [38]. Oven temp. was gradually raised from 60 to 2608 at a rate of 58/min, held for 15 min, and finally raised to 3408 at a rate of 408/min. He (purity 99.99%) was the carrier gas, at a flow rate of 1 ml/ min. Total analysis time was 57 min. Diluted sample (1:100 in petroleum ether (v/v)) of 1.0 ml was injected in the split mode (ratio 1 : 10). Quant. data were obtained electronically from FID area percent data without using correction factors. Gas Chromatography/Mass Spectrometry (GC/MS). Analysis of McEO was performed under the same conditions as by GC (column, oven temp., flow rate of the carrier gas) using a Thermo Electron (FCourtaboeuf) DSQ II GC/MS single quadrupole mass-selective detector in the electron-impact (EI) mode (70 eV). Injector and MS transfer line temps. were 200 and 3008, resp. MS was adjusted for an emission current of 10 mA, and electron multiplier voltage was 1500 V. Trap temp. was 2508, and mass scanning was from 40 to 650 amu. The components were identified based on the comparison of their retention time (tR ) and mass spectra with those of standards, Wiley 2001 library data (NIST 02 version 2.62) of the GC/MS system, and literature data (Adams). All determinations were performed in duplicate and averaged. Statistical Analysis. Exper. results of this study were expressed as means standard deviation of three parallel measurements. The significance of difference was calculated by Students t-test, and values p < 0.05 and p < 0.001 were considered to be significant and highly significant, resp. Correlation and regression analysis was carried out using EXCELL program (Microsoft Corporation, USA).
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