Chemical Composition and Antibacterial Activity of Essential Oils of Iranian Herbs Against Staphylococcus Aureus Isolated from Milk

Abstract

Chemical composition and antibacterial activity of essential oils from three endemic plants including, Thymus daenensis Celak, Dracocephalum multicaule Montbr & Auch., Satureja bachtiarica Bunge and one native plant, Tanacetum polycephalum Schultz-Bip were determined. Volatile oils analyzed using gas chromatography and gas chromatography-mass spectrometry. The in-vitro antibacterial activity was performed by agar disc diffusion and micro-dilution assays. The results revealed distinct differences in the compounds depending on sample plant. Among the plant species screened, essential oils of S. bachtiarica and T. daenensis showed highest antibacterial activity against Staphylococcus aureus.

Keywords:

• Thymus daenensis Celak
• Essential oil
• Satureja bachtiarica
• Bunge
• Milk
• Staphylococcus aureus

INTRODUCTION

Many infectious diseases are known to be treated with herbal remedies throughout history. Even today, plant materials continue to play a major role in primary health care as therapeutic remedies in many developing countries.^[1] There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-emerging infectious diseases.^[2] Therefore, researchers are increasingly turning their attention to folk medicine looking for new leads to develop better drugs against microbial infections.^[3]

Increased number of antibiotic resistant bacteria has led to demand for new agents to decrease the prevalence of bacterial diseases.^[4] Evidence shows that essential oils extracted from plants could be employed as antimicrobial agents in food systems.^[5] Recently, screening for plants with antibacterial activity has been the subject of many, their essential oils with antibacterial activity could be promising agents for this purpose.^[6,7] Plants and their essential oils are potentially useful sources of antimicrobial compounds. Numerous studies have been published on the antimicrobial activities of plant compounds against many different types of microbes, including food-borne pathogens.^[8−18] The main constituents of essential oils, monoterpenes, and sesquiterpenes e.g., carbohydrates, phenols, alcohols, ethers, aldehydes, and ketones, are responsible for both fragrance and biological activity of aromatic and medicinal plants. Due to these properties, ancient time spices and herbs have been added to food, not only as flavoring agents, but also as preservatives.^[19] It is believed that plants rich in a wide variety of secondary metabolites such as tannins, terpenoids, alkaloids, and polyphenols are generally superior in their anti-microbial activities.^[20] Thymus daenensis Celak, Dracocephalum multicaule Montbr & Auch, Satureja bachtiarica Bunge, and Tanacetum polycephalum Schultz-Bip have been utilized as traditional medicines for antiseptic and antimicrobial effects as well as culinary and spice in Chaharmahal va Bakhtiari, Iran.^[13] However, the objective of the present study was to evaluate the antibacterial activity of essential oils from medicinal and aromatic plants against Staphylococcus aureus isolated from cow milk, and to determine chemical compositions of essential oil from four species of Iranian medicinal herbs.

MATERIALS AND METHODS

Plant Material

The plants were collected from mountainous areas of Zagros, Chaharmahal va Bakhtiari district, Iran, during May–Sep, 2011 (Table 1). All collected specimens were properly processed. Provisional identifications of specimens were made with the help of “Encyclopedia of Iranian Plants,”^[21] “Flora Iranica,”^[22] etc. Identifications were consequently confirmed with the help of the authentic specimens deposited at the Herbarium of Researches Centre of Agriculture and Natural Resources of Chaharmahal va Bakhtiari, Iran (Table 1).

Table 1  Iranian medicinal plants used in this study

Scientific name	Family	No. voucher	Habitat	Parts used	Ethnobotany used^[^13^]
Tanacetum polycephalum Schultz-Bip	Asteraceae	1357	Native	Flowers	Headache, sedative, indigeous
Dracocephalum multicaule Montbr and Auch.	Lamiaceae	00XX	Endemic	Aerial parts	Sedative, analgesia, inflammatory, antibacterial, anti-septic, foot pain
Satureja bachtiarica Bung.	Lamiaceae	1999	Endemic	Aerial parts	Culinary, flavoring, indigestion, cough, anti-bacterial
Thymus daenensis Celak.	Lamiaceae	3092	Endemic	Aerial parts	Green tea, spice, culinary, cough, anti-bacterial, Carminative

Essential Oil Preparation

Dried plant material were powdered (100 g) and subjected to hydro-distillation (1000 ml distillated water) for 3 h using a Clevenger-type apparatus according to the method recommended in British Pharmacopoeia.^[23] Samples were dried with anhydrous sodium sulphate and kept in amber vials at 4°C prior to use.

Gas Chromatography/Mass Spectrometry (GC/MS) Analysis

The essential oil was analyzed using an Agilent 7890 A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with a HP-5MS 5% phenylmethylsiloxane capillary column (30 m × 0.25 mm, 0.25 μm film thickness). Oven temperature was kept at 60°C for 4 min initially, and then raised at the rate of 4°C/min to 260°C. Injector and detector temperatures were set at 290 and 300°C, respectively. Helium was used as carrier gas at a flow rate of 0.8 ml/min, and 0.5 μl samples were injected manually in split mode. Peaks area percents were used for obtaining quantitative data. The GC/MS analyses were carried out using an Agilent 5975 C (Agilent Technologies, Palo Alto, CA, USA) with a HP-5MS 5% phenylmethylsiloxane capillary column (30 m × 0.25 mm, 0.25 μm). Mass range was from m/z 50–550 amu. Identification of oil components was accomplished based on comparison of their retention times with those of authentic standards and by comparison of their mass spectral fragmentation patterns (WILLEY/ChemStation data system).^[24]

Bacterial Strain

Clinical isolate of S. aureus strain was obtained from Food Microbiology Laboratory, Veterinary Medicine Faculty, Islamic Azad University, and identified using conventional morphological as well as biochemical tests. Stock cultures of bacteria were kept at 20% glycerol PBS (phosphate buffered saline) at –70°C. Active cultures were generated by inoculating 100 μl of the thawed microbial stock suspensions into 5 ml nutrient broth (Merck, Germany) followed by overnight incubation at 37°C. An initial bacterial suspension containing 10⁷ CFU/ml was made from the flask broth culture. Subsequent dilutions were made from the above suspension, which were then used in tests.

Disc Diffusion Assay

These experiments were performed by the disc diffusion method Iennette  (1985) with some modification.^[25] Moller Hinton agar (Merck, Germany) was used to prepare the culture medium which was autoclaved at 115°C for 10 min. The plates (8 cm in diameter) were prepared with 10 ml agar inoculated with 1 ml of each bacterial suspension. Sterile paper discs (6 mm in diameter) were impregnated with 60 μl of dilutions of known essential oil concentrations (100 μg) and incubated at 37°C for 24 h. The essential oils were dissolved in 20 μl dimethyl sulfoxide (DMSO) before the test for antibacterial activity. Bacterial growth inhibition was determined by measuring the diameter of the inhibition zones (IZ) around the discs. The growth inhibition diameter was an average of three measurements, taken at three different directions. All tests were performed in triplicate.

Micro-Dilution Test

The minimum inhibitory and bactericidal concentrations (MICs and MBCs) were determined using 96-well. The bacterial suspension concentration was adjusted with sterile saline. The essential oils were dissolved in 100 μl of Moller Hinton medium with bacterial inoculums to achieve the target concentrations (0.08–500 μg/ml). The microplates were incubated for 24 h at 37°C. The optical density of each well was measured at a wavelength of 600 nm by Microplate manager and compared with a blank and a positive control.

Statistical Analyses

Means and standard errors (SE) of the samples were calculated. Each treatment was carried out with three replicates. The data was statistically analyzed using one-way ANOVA by the program SPSS (17.0), and comparison of the means of the main constituents of essential oils evaluated by Duncan’s multiple range test at p < 0.05 level.

RESULT AND DISCUSSION

EO Yield and Chemical Composition

The yellow oils of S. bachtiarica, T. daenensis, D. multicaule and T. polycephalum were obtained by hydro-distillation in the yields of 1.7, 2.2, 0.9, and 0.8% (w/w) based on dry weight, respectively. The results of qualitative and quantitative analysis of the essential oil are presented in the Table 2. Forty–six compounds in the essential oil of S. bachtiarica were identified. The major components were carvacrol (21.42%), p-cymen (20.67%), and thymol (24.96%) that they were dominant among monoterpenes components (Table 2). The following components (%) have been identified in the S. bachtiarica EO: thymol (44.5), γ-terpinene (23.9), p-cymene (7.3), β-caryophyllene (5.3), and borneol (4.2%).^[26] The main components for S. bachtiarica essential oil of Fars ecotype were carvacrol (49.3%), p-cymene (12.7%), and trans-a-bergamotene (5.8%), but for Yazd ecotype were carvacrol (66.5%), p-cymene (15.2%), and linalool (4.6%).^[27]

Table 2  Main components of essential oils detected by GC–MS

Components	R.I^a	Satureja bachtiarica Peak area %	Thymus daenensis Peak area %	Dracucephalum multicaule Peak area %	Tanacetum polycephalum Peak area %
Tricyclene	929	−	−	25.13	−
α-pinene	934	1.05	0.51	1.11	2.34
p-cymene	1027	20.67	−	−	−
Limonene	1030	0.56	−	39.85	−
1,8-cineole	1033	0.25	0.58	−	7.09
γ-terpinene	1058	3.88	5.74	−	0.45
Linalool	1098	3.82	0.52	0.27	1.53
Camphor	1142	0.95	−	−	28.34
Borneol	1164	−	−	−	3.67
Cis carveol	1223	−	−	0.71	8.45
Geraniol	1250	−	−	0.75	2.21
Thymol	1283	24.96	74.32	1.78	−
Carvacrol	1294	21.42	4.31	1.76	−
Citral	1313	−	−	4.24	−
Trans-Caryophyllene	1396	1.70	3.56	−	0.4
Caryophyllene oxide	1570	2.19	0.42	0.49	4.49
Oil yield% (w/w)		1.71	2.22	0.9	0.8

^aRetention index.

^btrace (< 0.01%); (a) retention time according to authentic standards; (b) mass spectrum.

Twenty-two compounds were identified in the essential oil of T. daenensis. The major components of this oil were thymol (74.32%), carvacrol (4.31%), and γ-terpinene (5.74%). The essential oil of T. daenensis was rich in monoterpenoids. Nickavar et al.^[28] reported that the essential oil of the aerial parts of T. daenensis subsp. daenensis had 26 components. In their study the major compounds were: thymol (74.7%), p-cymene (6.5%), β-caryophyllene (3.8%), and methyl carvacrol (3.6%).

Thirty-five compounds in essential oil of D. multicaule were identified; the major compounds were limonene (39.85%) and tricyclne (25.13%). The results of the study by Mojab et al.^[29] on analysis of essential oil of D. multicaule showed that the main components of the oils were limonene (27%), α-pinene (16.2), and methyl geraniate (11.2%). Sixty-four compounds were identified in the essential oil of T. polycephalum. As a determined by GC and GC–MS analyses, T. polycephalum contained camphor (28.34%), cis carveol (8.41%), and 1,8-cineole (7.09%) as the major compounds. In a study by Nori-Shargh et al.,^[30] the major components in the flowers of Tanacetum polycephalum (Schultz Bip. ssp. heterophyllum) grown in Iran were camphor (59.1%), camphene (14.8%), and 1,8-cineole (10.1%).

Antibacterial Activity

The antibacterial activity of the essential oils of four medicinal plants in this study against S. aureus was evaluated by comparing their diameter IZ and MIC values. The IZ and MIC values for S. aureus strain, which were sensitive to the essential oils were in the range of 10 to 35 mm and 62 and 250 μg/ml (Tables 3 and 4). The essential oils of S. bachtiarica and T. dadnensis were proved to be the most active, while the essential oil of Kelussia odoratissima were in effective against S. aureus. The results of present study showed that the essential oils of S. bachtiarica and T. dadnensis had great potential for antibacterial activity against S. aureus. The essential oils of S. bachtiarica and T. danensis have a stronger antibacterial activity compared to other essential oils. Essential oils rich in phenolic compounds, such as carvacrol and thymol, are widely reported to possess high levels of antimicrobial activity.^[31] Phenolic compounds are widely known for their beneficial effects, such as preventing hormone-related cancers, potent antioxidant, and antibacterial agents.^[32] Several studies have focused on the antimicrobial activity of the essential oils of thyme in order to identify effective compounds.^[33−35] The essential oil and extract of some aromatic plants (for example mint family, Lamiaceae) rich in cavracrol and thymol have a higher efficacy against food-borne pathogens bacterial.^[36] In this study, the strongest antimicrobial activity observed in essential oils of Satureja and Thymus genus was associated with phenolic compounds (thymol and carvacrol). These results are in agreement with those reported by other authors.^[37−39] Carvacrol, which is a main component of S. bachtiarica and T. danensis essential oils, is considered as a biocide, which cause perturbation in bacterial membrane, following by leakage of intracellular Adenosine triphosphate and potassium ions and ultimately death of the cell.^[40−42]

Table 3  Antibacterial activity of different essential oils against staphylococcus aureus of using disc diffusion method (inhibition zones, mm)

	Concentration (μg/mL)
Essential oil	500	250	125	62	31	15	7.5	3.5	1.7	0.8	Mean^b
T. polycephalum	26.3 ± 1.1^a	29.3 ± 1.2	35.0 ± 0.0	32.0 ± 0.0	20.7 ± 1.1	18.3 ± 0.6	19.7 ± 0.6	15.0 ± 1.0	12.3 ± 2.1	16.0 ± 2.6	22.51^b
D. multicaule	26.0 ± 1.0	24.3 ± 2.1	26.0 ± 1.0	21.3 ± 0.6	26.3 ± 0.6	23.7 ± 0.6	28.3 ± 0.2	25.3 ± 3.5	22.7 ± 1.2	19.7 ± 2.1	23.82^b
S. bachtiarica	18.4 ± 0.7	16.3 ± 0.3	14.4 ± 0.5	14.2 ± 0.8	13.0 ± 0.0	18.5 ± 1.3	14.2 ± 0.8	12.0 ± 1.0	11.0 ± 1.0	10.0 ± 1.0	14.29^c
T. daenensis	20.3 ± 0.6	24.0 ± 1.0	24.3 ± 0.6	35.0 ± 0.0	35.0 ± 2.0	33.0 ± 1.7	28.30.6	25.3 ± 1.5	30.3 ± 1.5	19.3 ± 1.1	27.56^a
Ciprofloxacin (10 μg/disc)						31.0 ± 0.0
Erythromycin (10 μg/disc)						26.0 ± 1.0
Gentamicin (10 μg/disc)						22.4 ± 0.5
Mean^b	24.41^b	25.50^a	26.07^a	25.69^a	23.06^c	22.36^c	21.69^d	19.72^e	19.22^e	18.11^f	−

^aDiameter of inhibition zone in mm ± SE (n = 3).

^bDMRT: Duncan’s multiple range test, Means with the same letter are not significantly different at p ≤ 0.05.

Table 4  Antimicrobial activity of different essential oils against staphylococcus aureus by micro-dilution assay

Essential oil	MIC (μg/ml)	MBC (μg/ml)
T. polycephalum	125	690
D. multicaule	125	750
S. bachtiarica	62	560
T. daenensis	62	630
Ciprofloxacin (10 μg/disc)	31	250
Erythromycin (10 μg/disc)	62	450
Gentamicin (10 μg/disc)	62	570
ANOVA	p ≤ 0.05	p ≤ 0.05

Previous works showed that the essential oil of S. bachtiarica exhibited antifungal activities against Saprolegnia parasitica from cutaneous lesions of Oncorhynchus mykiss eggs;^[42] the essential oils of S. bachtiarica and T. daenensis exhibited antibacterial activities against Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumonia;^[15] essential oils of Myrtus communis, T. daenensis, and S. bachtiarica exhibited antimicrobial activities against Escherichia coli O157:H7, Bacillus cereus, Listeria monocytogenes, and Candida albicans.^[14]

Antibacterial efficiency of compounds of studied essential oils were different, but these differences were not significant in S. bachtiarica and T. daenensis. It was possible to select a specific essential oil as an effective source of antimicrobial compounds. The order of sensitivity of microorganism to essential oils, expressed as average MICs was: S. bachtiarica and T. daenensis (62 μg/mL) > D. multicaule and T. polycephalum (125 μg/mL) > K. odoratissima (250 μg/mL) (Table 4). Essential oils of S. bachtiarica and T. daenensis showed lower MICs compared to other studied species. This is in line with carvacrol thymol and p-cymen content of these essential oils. Strong antibacterial activity of essential oils of S. bachtiarica and T. daenensis, is important from the economic point of view, because extraction of essential oils from these herbs looks feasible in Iran.

CONCLUSION

The results in this study showed that the essential oils of S. bachtiarica and T. daenensis had antibacterial activities against S. aureus. The highest level of antibacterial activity was observed from the essential oil of the aerial parts (leaves and stem) of S. bachtiarica and T. daenensis. This study suggests that the essential oils of S. bachtiarica and T. daenensis are potential source of natural antibacterial against S. aureus. After this screening experiment, further work is needed to study the mechanism of antibacterial activities as well as in vivo.

ACKNOWLEDGMENT

The authors would like to thank Mr. Hamid Mashayekhi, Stockbridge School of Agriculture, University of Massachusetts, for critical reading of the manuscript and linguistic corrections.

FUNDING

This work was supported by deputy of Researches and Technology, Islamic Azad University of Shahrekord Branch, Iran.