Bmc Complementary and Alternative Medicine in Vitro Antibacterial Activity of Some Plant Essential Oils

Background: To evaluate the antibacterial activity of 21 plant essential oils against six bacterial species.


Background
The spread of drug resistant pathogens is one of the most serious threats to successful treatment of microbial diseases. Down the ages essential oils and other extracts of plants have evoked interest as sources of natural products. They have been screened for their potential uses as alternative remedies for the treatment of many infectious diseases [1]. World Health Organization (WHO) noted that majority of the world's population depends on traditional medicine for primary healthcare. Medicinal and aromatic plants which are widely used as medicine and constitute a major source of natural organic compounds.
Essential oils have been shown to possess antibacterial, antifungal, antiviral insecticidal and antioxidant properties [2,3]. Some oils have been used in cancer treatment [4]. Some other oils have been used in food preservation [5], aromatherapy [6] and fragrance industries [7]. Essential oils are a rich source of biologically active compounds. There has been an increased interest in looking at antimicrobial properties of extracts from aromatic plants particularly essential oils [8]. Therefore, it is reasonable to expect a variety of plant compounds in these oils with specific as well as general antimicrobial activity and antibiotic potential [9].
Essential oils (also called volatile oils) are aromatic oily liquids obtained from plant materials (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots). They can be obtained by expression, fermentation or extraction but the method of steam distillation is most commonly used for commercial production. An estimated 3000 essential oils are known, of which 300 are commercially important in fragrance market [7]. Essential oils are complex mixers comprising many single compounds. Chemically they are derived from terpenes and their oxygenated compounds. Each of these constituents contributes to the beneficial or adverse effects.
Essential oils such as aniseed, calamus, camphor, cedarwood, cinnamon, citronella, clove, eucalyptus, geranium, lavender, lemon, lemongrass, lime, mint, nutmeg, orange, palmarosa, rosemary, basil, vetiver and wintergreen have been traditionally used by people for various purposes in different parts of the world (Table 1). Cinnamon, clove and rosemary oils had shown antibacterial and antifungal activity [10]; cinnamon oil also possesses antidiabetic property [11]. Anti-inflammatory activity has been found in basil [12]. Lemon and rosemary oils possess antioxidant property [13,14]. Peppermint and orange oils have shown anticancer activity [15,16]. Citronella oil has shown inhibitory effect on biodegrading and storage-contaminating fungi [17]. Lime oil has shown immunomodulatory effect in humans [16]. Lavender oil has shown antibacterial and antifungal activity; it was also found to be effective to treat burns and insect bites [18].
In spite of all the information available on the 21 oils selected for this study, we were not able to find antibacterial activity for all those oils. Hence this study was undertaken with the intention of finding out the efficacy of these essential oils as antimicrobial agents for commercial purposes.

Essential oils
Twenty-one essential oils obtained from Tegraj & Co (P) Ltd, India (commercial producers of plant essential oils and aromatic substances) were used in this study (Table  1). These oils were selected based on literature survey and their use in traditional medicine. Quality of the oils was ascertained to be more than 98% pure.  (ATCC 25923)] were used. The cultures of bacteria were maintained in their appropriate agar slants at 4°C throughout the study and used as stock cultures.

Antibacterial assay
Screening of essential oils for antibacterial activity was done by the disk diffusion method, which is normally used as a preliminary check and to select between efficient essential oils [2]. It was performed using an 18 h culture at 37°C in 10 ml of Mueller Hinton Broth. The cultures were adjusted to approximately 10 5 CFU/ml with sterile saline solution. Five hundred microliters of the suspensions were spread over the plates containing Mueller-Hinton agar using a sterile cotton swab in order to get a uniform microbial growth on both control and test plates. The essential oils were dissolved in 10% aqueous dimethylsulfoxide (DMSO) with Tween 80 (0.5% v/v for easy diffusion) and sterilized by filtration through a 0.45 μm membrane filter. Under aseptic conditions, empty sterilized discs (Whatman no. 5, 6 mm dia) were impregnated with 50 μL of different concentrations (1:1, 1:5, 1:10, 1:20) of the respective essential oils and placed on the agar surface [19]. Paper disc moistened with aqueous DMSO was placed on the seeded petriplate as a vehicle control. A standard disc containing streptomycin (25 μg/ disc) was used as reference control. All petridishes were sealed with sterile laboratory parafilm to avoid eventual evaporation of the test samples. The plates were left for 30 min at room temperature to allow the diffusion of oil, and then they were incubated at 37°C for 18 h (18 h was fixed as the optimum since there was no change in the inhibition up to 24 h) After the incubation period, the zone of inhibition was measured with a calliper. Studies were performed in triplicate, and mean value was calculated. The means were analysed by one way analysis of variance (ANOVA) followed by Tukey's post hoc multiple comparison test using SPSS software package version 13.0 for windows. The results were expressed as mean ± SD. P values <0.05 were considered as significant.

MIC assay
Based on the previous screening seven essential oils (Cinnamon, clove, geranium, lemon, lime, orange and rosemary oils) were identified to have potent antibacterial activity and their Minimum Inhibitory Concentrations (MIC) were determined. The agar dilution method recommended by the National Committee for Clinical Laboratory Standards [20] was used with the following modification; a final concentration of 0.5% (v/v) Tween-20 (Sigma) was incorporated into the agar medium to enhance oil solubility. A series of two fold dilution of each oil, ranging from 0.2 to 25.6 mg/ml, was prepared in Muellur Hinton agar at 48°C. Plates were dried at room temperature for 30 min prior to spot inoculation with 3 μl aliquots of culture containing approximately 10 5 CFU/ml of each organism. Inoculated plates were incubated at 37°C for 18 h and the MIC was determined. Experiments were carried out in triplicate. Inhibition of bacterial growth in the plates containing test oil was judged by comparison with growth in blank control plates. The MICs were determined as the lowest concentration of oil inhibiting visible growth of each organism on the agar plate [21].

Gas chromatography mass spectrometry (GC/MS)
The most potent oil, (cinnamon oil) was analysed using GC/MS (Shimadzu capillary GC-quadrupole MS system QP 5000) with two fused silica capillary column DB-5 (30 μm, 0.25 mm i.d, film thickness 0.25 μm) and a flame ionization detector (FID) which was operated in EI mode at 70 eV. Injector and detector temperatures were set at 220°C and 250°C, respectively. One microliter essential oil solution in hexane was injected and analyzed with the column held initially at 60°C for 2 min and then increased by 3°C/min up to 300°C. Helium was employed as carrier gas (1 ml/min). The relative amount of individual components of the total oil is expressed as percentage peak area relative to total peak area. Qualitative identification of the different constituents was performed by comparison of their relative retention times and mass spectra with those of authentic reference compounds, or by retention indices (RI) and mass spectra.

Results
The anti-bacterial activity of twenty-one selected essential oils against six bacterial species is summarized in Table 2 and 3. The results revealed that the selected essential oils showed antibacterial activity with varying magnitudes. The zone of inhibition above 7 mm in diameter was taken as positive result. Generally most of the tested organisms were sensitive to many of the essential oils. Out of 21 essential oils tested, 19 showed antibacterial activity against one or more bacteria. Cinnamon oil, lime oil, geranium oil, rosemary oil, orange oil, lemon oil and clove oil showed maximum activity against all the bacterial species tested. On the other hand, aniseed oil, eucalyptus oil and camphor oil failed to inhibit any of the tested strains. Both gram-positive and gram-negative bacteria were sensitive to the potent essential oils. P. aeruginosa and P. vulgaris were inhibited by 19 oils, followed by B. subtilis (18 oils), S. aureus (14 oils), E. coli (12 oils) and K. pneumoniae (9 oils). In general cinnamon oil showed significant inhibitory effect against P. aeruginosa (33.3 mm), B. subtilis (29.9 mm), P. vulgaris (29.4 mm), K. pneumoniae (27.5 mm) and S. aureus (20.8 mm). Moderate effects were seen in lime oil, clove oil and lemon oil. No obvious difference in susceptibility was found between gram-negative and gram-positive bacteria. There was no inhibition of growth with the vehicle control (10% DMSO).   Minimum inhibitory concentration (MIC) for selected seven oils ranged from 0.8 to 12.8 mg/ml (Table 4). This study revealed that cinnamon oil showed maximum activity with MIC values ranging from 0.8 to 3.2 mg/ml followed by clove oil with MIC values ranging from 1.6 to 6.4 mg/ml against all the tested strains where as remaining oils showed moderate MIC values.
GC/MS analysis of cinnamon oil identified thirty-eight phytochemicals as constituents; of these cinnamaldehyde was the major compound (52.4%) followed by Benzaldehyde (12.31%), benzoic acid (8.20%) and benzyl alcohol (2.23%). Remaining chemical compounds were in trace amounts. The major components and their retention times are summarized in Table 5.

Discussion
Plant essential oils and extracts have been used for many thousands of years [22], in food preservation, pharmaceuticals, alternative medicine and natural therapies [23,24]. It is necessary to investigate those plants scientifically which have been used in traditional medicine to improve the quality of healthcare. Essential oils are potential sources of novel antimicrobial compounds [25] especially against bacterial pathogens. In vitro studies in this work showed that the essential oils inhibited bacterial growth but their effectiveness varied. The antimicrobial activity of many essential oils has been previously reviewed and classified as strong, medium or weak [26].
In our study, cinnamon, clove, geranium, lemon, lime, orange and rosemary oils exhibited strong activity against the selected bacterial strains. Several studies [27,28] have shown that cinnamon, clove and rosemary oils had strong and consistent inhibitory effects against various pathogens. Even though earlier studies have reported better antimicrobial activity for eucalyptus oil [29,30] our study showed least inhibitory activity of eucalyptus in addition to aniseed and camphor oils. Among all oils analyzed in this work, the essential oil of cinnamon was the most effective as an antibacterial agent. The antibacterial activity has been attributed to the presence of some active constituents in the oils. Our GC-MS study revealed cinnamaldehyde to be the major constituent of cinnamon oil. Cinnamaldehyde was the predominant active compound found in cinnamon oil [31,32]. Earlier studies suggested that the antibacterial activity of cinnamon oil was probably due to their major component, cinnamaldehyde and their properties could be multiple. Cinnamaldehyde is a natural antioxidant and the animal studies suggest that an extract of cinnamon bark taken orally may help prevent stomach ulcer [33]. Cinnamaldehyde was completely inhibiting both sensitive and resistant stain of Helicobacter pylori [34]. Cinnamon oil was not harmful when consumed in food products and it inhibited the growth of molds, yeast and bacteria [27]. Cinnamon extract had a regulatory role in blood glucose level and lipids and it may also exert a blood glucose-suppressing effect [35]. The use of commercial cinnamon preparation produced an improvement of oral candidiasis of HIVinfected patients [36]. Cinnamon oil is locally applied with much benefit in neuralgia and headache. As an antiseptic it is used as an injection in gonorrhea; as germicide it is used internally in typhoid fever. This oil is also used in the treatment of cancer and other microbial diseases [37]. It can be incorporated into creams, lotions, drops, etc. which are applied externally on the body to treat diseases caused by Aspergillus niger [38].
An important characteristic of essential oils and their components is their hydrophobicity, which enable them to partition the lipids of the bacterial cell membrane and mitochondria, disturbing the cell structures and rendering them more permeable [39,40]. Extensive leakage from bacterial cells or the exit of critical molecules and ions will lead to death [41]. Gram-positive bacteria were more resistant to the essential oils than gram-negative bacteria [26]. In the present study, cinnamon, lime, geranium, rosemary, orange, lemon and clove oils were found to be equally effective against both gram-positive and gramnegative organisms.

Conclusion
From this study it can be concluded that many essential oils possess antibacterial activity. Cinnamon oil has the most potential bactericidal properties. We believe that the present investigation together with previous studies provide support to the antibacterial properties of cinnamon