International Journal of Drug Development and Research

Keywords

Antibacterial Activity, Essential oils, Aromatic, GCMS

Introduction

Plant materials remain an important resource to combat serious diseases in the world. The traditional medicinal methods, especially the use of medicinal plants, still play an important role to cover the basic health needs in the developing countries. Within the recent years, infections have increased to a great extent and antibiotics resistance effects become an ever-increasing therapeutic problem. The resistance of the organisms increased due to indiscriminate use of commercial antimicrobial drugs commonly used for the treatment of infectious diseases. In past few years there has been a gradual revival of interest in the use of medicinal plants in developed as well as in developing countries because plant derived therapeutics have been reported to be safe and without side-effects. This development of microbial resistance has led the researchers to search the antibacterial activity of medicinal plants.

Medicinal and aromatic plants are widely used as medicine and constitute a major source of natural organic compounds. An essential oil is a concentrated hydrophobic liquid containing volatile aroma compounds from plants, they arise from a secondary metabolism of plant normally formed in special cells or a group of cells or as glandular hair found on many leaves and stems. Essential oil can be obtained by expression, fermentation or extraction but the method of steam distillation is most commonly used for commercial production. They have long been found as plant’s chemical defense against insects, fungi and other invaders [1]. Essential oils are chiefly used for the flavors and fragrances but they also possess antibacterial, antifungal, antiviral insecticidal and antioxidant properties [2,3]. Antiseptic properties of plant volatile oils have been recognized since antiquity[4].

The present study was undertaken to investigate the in vitro antibacterial activity of various plant essential oils and to identify the compounds responsible for their antibacterial activity by GC-MS.

MATERIALS AND METHODS

Essential oil extraction

Air-dried plant materials were pulverized into powdered form. The powder of sample (250g) was subjected to hydro distillation for 4 hrs in a Clevenger-type apparatus to obtain the essential oils. The distillated oils were dried over anhydrous sodium sulfate and preserved in sealed dark vials at 4 °C until further analysis. Three exotic essential oils (T.vulgaris, O.europea and M. alternifolia) were procured from the suppliers.

Microbial strains

The in vitro antibacterial activity of the essential oils was tested against nine epidermal infection causing bacteria. Five Gram-positive strains were Staphylococcus aureus (MTCC 3160 and NCDC 109), Staphylococcus epidermidis (MTCC 435 and MTCC 3086), Staphylococcus hominis (MTCC 4435) and Four Gram-negative bacteria were Pseudomonas aeruginosa (MTCC 7453 and MTCC 424), Klebsiella pneumonia (MTCC 4030), and Proteus vulgaris (MTCC 426) .The bacteria were procured from Institute of Microbial Technology (IMTECH), Chandigarh and National Dairy Research Institute (NDRI), Karnal.

Determination of Antimicrobial activity and Minimum Inhibitory Concentration (MIC).

Agar well diffusion method was carried out by allowing perforation of various oils dissolved in 10% DMSO. Petriplates containing 30 ml nutrient agar medium were kept for the solidification before inoculating the microorganism, desired numbers of wells of uniform diameter of 8mm were made after solidification, using sterile aluminum borer. 0.1 ml of each oil samples were poured into wells. After incubation for 24 hrs at 37 0C the plates were observed and the antibacterial activity was evaluated by measuring zone of inhibition (diameter mm). The tests were conducted in triplicate. Ciprofloxacin (10.0 μg/ml) was used as positive control. The negative control was 10% DMSO.

MIC of oils was determined by micro dilution technique as described by the National Committee for Clinical Laboratories standards (NCCLS)[5]. The bacteria inoculums were prepared in 5 ml nutrient broth and incubated at 370C. The final inoculums were of approximately 106 CFU/ml (0.5 McFarland)[6]. Controls with 0.5 ml of culture medium without the samples and other without microorganisms were used in the tests. Tubes were incubated at 370C for 24 h. The activity was measured as a function of turbidity at 660 nm. Lack of turbidity was further confirmed by pouring suspension aliquot of 0.1 ml into pre-sterilized Petri dishes with nutrient agar medium. The tests were conducted in triplicate.

GC-MS analysis

The essential oils that showed best activity were analyzed by GC-MS using Shimadzu Mass Spectrometer-2010 series. 1 μl of oil sample was injected in GC-MS equipped with a split injector and a PE Auto system XL gas chromatograph interfaced with a Turbo-mass spectrometric mass selective detector system. The MS was operated in the EI mode (70 eV). Helium was employed as the carrier gas and its flow rate was adjusted to 1.2 ml/min. The analytical column connected to the system was an Rtx-5 capillary column (length-60m × 0.25mm i.d., 0.25 μm film thickness). The column head pressure was adjusted to 196.6 kPa. Column temperature programmed from 100?C (2 min) to 200?C at 10?C/min and from 200? to 300?C at 15?C/min with hold time 5 and 22 min respectively. A solvent delay of 6 min was selected. The injector temperature was set at 270°C. The GC-MS interface was maintained at 280°C. The MS was operated in the ACQ mode scanning from m/z 40 to 600.0. In the full scan mode, electron ionization (EI) mass spectra in the range of 40–600 (m/z) were recorded at electron energy of 70 eV. Compound identified by comparing mass spectra with library of the National Institute of Standard and Technology (NIST), USA/Wiley.

Results and Discussion

Our results revealed that the essential oils showed antibacterial activity with varying magnitudes. The anti-bacterial activity of ten essential oils against nine bacterial strains is summarized in Table 2. 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 ten essential oils tested, nine showed antibacterial activity against one or more bacteria. Thyme oil, Tea tree oil and Eucalyptus oil showed maximum activity against all the bacterial species tested. Several studies have shown that Thyme, Tea tree and Eucalyptus oils had strong and consistent inhibitory effects against various pathogens [7,8]. Teppal, Coriander, Nagarmotha, Sugandh kokila and Jatamansi oils showed moderate antibacterial activity with inhibition zones ranging from 11-24mm for both gram positive and gram negative bacteria. . On the other hand, Olive oil and Fennel oil showed little or no activity against the tested strains. In the present study, various essential oils were found to be equally effective against both gram-positive and gram-negative organisms. Although earlier studies indicated that gram-positive bacteria are more resistant to the essential oils than gram-negative bacteria [9]. There was no inhibition of growth with the vehicle control (10% DMSO).

Minimum inhibitory concentration (MIC) for the oils ranged from 0.35 to 100 μl/ml (Table 3). This study revealed that thyme oil showed maximum activity with MIC values ranging from 0.35 to 1.56 μl/ml followed by Tea tree and Eucalyptus oil with MIC values ranging from 0.75 to 6.25 μl/ml against all the tested strains where as remaining oils showed moderate MIC values.

The Essential oils that showed best antibacterial activity i.e. Thyme oil, Tea tree oil and Eucalyptus oil were analyzed by GC-MS to identify the compounds responsible for antibacterial activity. GC-MS profile of the Thyme oils showed that α-Terpinene (42.29%) is the main compound followed by Thymol (30.06%) and β -Cymene (22.19%). In previous studies the major component of thyme oil had been found to be camphor whereas α-Terpinene was present only in traces[10,11].

α-Pinene (32.59%) and Cymene (27.30%) were the major compounds in the Tea tree oil along with Linalool (12.77%) and α-Limonene(11.02%). In most of the previous studies 4-Terpineol had been found to be present in maximum amount [12]. The major compound in the Eucalyptus oil was found to be Eucalyptol (79.02%) and 3- Carene (13.66%) along with other chemical compounds in traces. Our results were in accordance with most of the previous studies [13]. The components of the three essential oils and their retention times are summarized in Table 4, 5, 6 and structures are shown in Figures 1-5.

In the present study 90% of the essential oils showed inhibitory activity. Out of which 30% showed very high activity and 60% showed moderate activities. Essential oils are potential sources of novel antimicrobial compounds [14] especially against bacterial pathogens. 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 [15,16]. Extensive leakage from bacterial cells or the exit of critical molecules and ions will lead to death [17].

Conclusion

From this study it can be concluded that many essential oils possess antibacterial activity. The present investigation provides support to the antibacterial properties of essential oils. These can be used as antibacterial supplements in the developing countries towards the development of new therapeutic agents. Additional in vivo studies and clinical trials would be needed to justify and further evaluate the potential of these oils as antibacterial agents in topical or oral applications.

Acknowledgement

We are thankful to Dr. Ajai kumar, AIRF, JNU for helping with GCMS.

Tables at a glance

Table 1 Table 2 Table 3 Table 4 Table 5 Table 6

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

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