Composition of the essential oil extracted from the
Essential oils can be used as antibacterial additives and are generally recognized as safe. Coleus zeylanicus is one of the medicinal aromatic plant serves as a source of essential oils. Antimicrobial and antioxidant activities of essential oils obtained from the control and salinity stressed Coleus zeylanicus plant was investigated in the present study. Essential oils from the control and salinity stressed Coleus zeylanicus plant was extracted using Clevenger apparatus. The composition of essential oils was identified using gas chromatography mass spectrometry, which showed a few compounds expressed differentially. The antibacterial activity of the isolated essential oils was studied by using the agar well diffusion method, showing potent inhibitory activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The antioxidant and antimicrobial constituents of the essential oils were spotted using the bioautography method, revealing that the antioxidant and antimicrobial properties in the essential oils of Coleus zeylanicus were increased upon exposure to salinity stress.
- agar well diffusion method
- salinity stress
- essential oils
- bioautography method
- thin layer chromatography
In the recent years, the increasing resistance and the wide spread of multi-drug resistant microbes are leading to a serious health problem to the human population. The emergence of resistant microbes is due to the indiscriminate use of antibiotics . Hence there is a need to identify new drugs with effective antimicrobial and antioxidant properties to overcome this problem and to replace the usage of synthetic drugs responsible for the cause of side effects in patients. The resistance to antibiotics can be reduced by the use of resistance inhibitors isolated from plants. Salt stress is a vital abiotic stress factor that affects the growth and productivity of plants. In general, salinity refers to the presence of different salts like sodium chloride, calcium sulfate, magnesium, and bicarbonates in water and soil . The uptake of water and absorption of essential nutrients by plants are restricted due to the presence of soluble salts exerting high osmotic pressure which ultimately affects the growth of plants . Plants have adopted a mechanism to tolerate salt stress by the accumulation of solutes such as glycine, betaine, proline, sugar alcohols, polyols, and soluble sugars and by eliminating the toxic Na+ ions in the cytoplasm . Plant bioactive compounds are low molecular weight secondary metabolites distributed largely in plants that play a major role in the adaptation of plants to different environmental changes and in overcoming stress constraints. Medicinal plants exhibit pharmacological properties as they are known to possess various bioactive compounds called secondary metabolites like tannins, terpenes, alkaloids, steroids, flavonoids, glycosides, saponins, etc. These compounds play a major role in protecting the plants from various stress factors. Secondary metabolites that act as active components exhibit a wide range of antimicrobial activity . The target sites shown by plant extracts are active against drug resistant microbes than those used by the antibiotics. It was discovered that the non-antibiotic substances such as essential oils have shown good fighting potential against drug resistant microbes. Essential oils change the rate of an enzyme reaction by interfering with the metabolism of microbes; thereby influencing the uptake of nutrients from the medium and affect the synthesis of enzymes or by changing the membrane structures that leads to the death of microbes. Thus, with the discovery of many natural products from plant species due to advancements in science and technology made a remarkable progress in the field of medicine. Now-a-days, many researchers are interested in isolating the biologically active compounds from plant species for developing the novel drugs in order to combat the microbes responsible for dreadful diseases .
Essential oils are a heterogeneous group of complex mixture of organic compounds synthesized within plants as secondary metabolites with characteristic flavor and odor. The quantity and quality of oil vary depending on the ecological and growth conditions of plant chosen for extraction. The various other factors also influence the yield of essential oils. The different parts of the plant such as leaves, root, stem, seeds, bark, woods, twigs, buds, fruits, and flowers can be used for the extraction of aromatic oily liquids called essential oils . Essential oils are a mixture of compounds principally terpenoids like (C10) monoterpenes, (C15) sesquiterpenes, and (C20) diterpenes, also contains lactones or acyclic esters, low molecular weight aliphatic hydrocarbons, aldehydes, alcohols, acids and rarely may contain coumarins, nitrogen-, and sulfur-containing compounds and homologs of phenylpropanoids . Essential oils are produced commercially by the method of steam distillation; whereas the other methods of extraction, fermentation, and expression can also be performed to obtain oils . Essential oils possess antibacterial, antifungal, antiviral, anticancer, antioxidant, and insecticidal properties . Essential oils from medicinal plants possess antioxidant activity, which plays an important role in neutralizing free radicals benefiting human health. Essential oils also showed a potent inhibitory effect against Gram-positive bacteria, Gram-negative bacteria, filamentous fungi, and yeast. The highest inhibitory activity of
2. Material and methods
2.1. Plant material and salt stress treatment
2.2. Extraction of essential oil
The essential oils were extracted using Clevenger apparatus. Distillation is carried out for a period of 4 h by immersing the dried leaf powder directly into a round bottom flask filled with water. The contents were boiled and the vapors were condensed, allowing the essential oils to separate based on their difference in immiscibility and density. After extraction, the organic phase was separated and dried over Na2SO4. The percentage of the oils was calculated using the following formula:
Oil (% v/w) = observed volume of oil (ml)/weight of sample (g) × 100.
2.3. Identification of compounds using GC-MS
The identification of the major chemical composition of the isolated essential oil was performed by GC-MS (Sathyabama University, Chennai). The extracted solution containing essential oil was separated using diethyl ether and the injected sample volume was 1.0 μl for control and 0.5 μl for the salt stress sample as it was more concentrated. A Shimadzu GC-MS QP2010, a polyethylene glycol (Carbowax), and model Rtx-Wax (RESTEC) (30 m to 0.25 mm i.d., film thickness 0.25 lm) capillary column were used for the analysis. The temperature was first held at 40°C and then raised to 250°C (10 min, 2°C/min). The carrier gas was helium at a flow rate of 3 ml min−1. The components of the oil were identified based on the comparison of their retention indices and mass spectra with the fragmentation patterns for computer matching with the NIST (National Institute of Standards and Technology) library.
2.4. Antimicrobial studies of the isolated essential oils
2.4.1. Microorganisms used
Three bacterial strains,
2.4.2. Preparation of inoculum
The colonies of test organisms were inoculated into 0.85% normal saline and the turbidity adjusted to 0.5 McFarland using the standard, which is equal to 1.5 × 108 CFU/ml.
2.4.3. Antibacterial activity by the agar well diffusion method
The antibacterial activity of the essential oils isolated from
2.5. Identification and separation of compounds using TLC
The essential oils obtained by the method of steam distillation are subjected to thin layer chromatography to identify and separate the bioactive compounds present in both the samples of control and salt stress. Both the control and salt stress samples were applied to the TLC plate separately. The solvent system used in the TLC analysis was toluene:ethyl acetate in the ratio of 93:7. TLC was carried out using TLC silica gel 60 F254 aluminum sheets (Merck). After complete elution, the spots were identified and Rf values were calculated for each spot.
2.6. Screening of antioxidant activity
Several TLC techniques have been developed successfully for the analysis of antioxidants both quantitatively and qualitatively. The use of DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical reagent for the analysis of antioxidant activity by the TLC method is one among them. TLC bioautography assay for screening of antioxidants possess several advantages that include high throughput, simplicity, and flexibility. In the present study, the antioxidant activity of essential oils is evaluated using the method of TLC bioassay. The components of essential oils were separated on the TLC plate and sprayed with DPPH solution. Antioxidant reduces the radical and produces creamy spots against a purple background.
2.7. Screening of antimicrobial activity
To screen the antibacterial activity of essential oils from control and salt stress, the method of direct bioautography is performed. Initially, the components were separated on the TLC plate and allowed to air dry. The test organisms were allowed to grow on the TLC plate by dipping the plate into the respective medium containing the organism, followed by incubation at 37°C for 24 h. After incubation, the plates were sprayed with 2 mg/ml solution of p-iodonitrotetrazolium violet dye. A clear zone indicated the inhibition of growth of the organism.
2.8. Statistical analysis
Results mentioned are reported as the mean ± standard error (SE) values of five independent experiments, conducted on five different plants in each experiment. SE values were calculated directly from the data according to standard methods. Data analyses were carried out using the SPSS package. Mean values were compared by Duncan’s multiple range test and P-values which are less or equal to 0.05 were considered as statistically significant.
3. Results and discussion
3.1. Essential oil analysis
The essential oils obtained by the hydrodistillation of
|Peak No.||R. time||Area||Area (%)||Name of the compound|
|12||24.257||143,316||4.17||(−)-Isolongifolol, methyl ether|
|Peak No.||R. Time||Area||Area (%)||Name of the compound|
3.2. Antimicrobial activity
The antibacterial activities of essential oils extracted from the control and salinity stressed
|Strain name||Positive control (ampicillin) (mm)||Negative control (solvent) (mm)|
|17 ± 2.87||17 ± 2.82||32 ± 2.65||No zone|
|12 ± 2.10||16 ± 3.12||No zone||No zone|
|17 ± 2.75||17 ± 1.80||No zone||No zone|
Majority of the essential oils has shown effective inhibitory activity against Gram-positive strains , might be due to the presence of hydrophilic outer membrane, which prevents the entry of hydrophobic compounds into the target cell membrane, thereby acquiring resistance to the antimicrobial drugs . Another possible reason could be the inhibition of microbial respiration and increased membrane permeability by essential oils resulting in the death of microbes after massive ion leakage [26, 27]. Therapy with traditional herbs is practiced with the plant species containing medicinal properties. Secondary metabolites such as terpenoids, tannins, alkaloids, phenols, and flavonoids rich in plants are found to be responsible for antimicrobial properties
3.3. Screening of antioxidant and antimicrobial activity by the TLC bioautography method
Bioautography technique was employed to detect the antimicrobial and antioxidant activity . In our study, the two essential oils spotted on the TLC plate were separated into distinct bands with different Rf values. Essential oil from control and salt stressed
In this method of TLC bioautography, a developed TLC plate is dipped into the respective broth containing microbes of pure culture and incubated under humid conditions. The microbes grow directly on the TLC plates except in the regions where the bioactive compounds exhibit the antimicrobial property. The zones of inhibition with creamy spots against purple background are visualized after spraying the plates with INT (p-iodonitrotetrazolium violet) dye. The tetrazolium salts are converted into a purple colored compound called formazan by the dehydrogenase activity of living microbes. Once the activity is located on the TLC plate, the samples can be analyzed by GC-MS to identify the presence of known or unknown compounds responsible for the activity . This method is considered to be convenient for obtaining the reliable information on the activity of single compounds as the plant extracts possess numerous bioactive compounds. The analytical determination of compounds present in plant extracts and the characterization of their biological properties are made possible with the optimized antimicrobial assays. Separation of compounds in plant extracts is necessary to avoid study on fractions with no biological activity. Detection of antimicrobial compounds by this method is rapid, uncomplicated and effective in saving money and time . Apart from the search of bioactive compounds, this method is also used to find out best solvent for the extraction of compounds and for the selection of mobile phase to separate compounds. It was reported that the thymol and carvacrol were responsible for the antimicrobial property present in the essential oils of
The TLC bioautography method also used to detect the compounds exhibiting antioxidant activity. The developed TLC plate sprayed with DPPH (2,2-diphenyl-1-picrylhydrazyl radical) solution produces clear creamy yellow spots against a purple background. The DPPH decreases upon the reduction reaction with a radical scavenger leading to the color change which can be observed in a TLC bioassay. The reaction has been depicted in Figure 3. The assay depends on the measurement of antioxidants scavenging activity, where the DPPH is characterized as a stable free radical. The odd electron of nitrogen atom in DPPH is reduced by receiving a hydrogen atom from antioxidants present in the plant extracts to the corresponding hydrazine. Rosmarinic acid, luteolin, chrysoeriol and apigenin were the four different antioxidant compounds isolated from the fruit of
The results of this study showed that the essential oils of
Research of KVC lab is funded by the University grants commission, Govt. of India, 42-197/2013. Divya K is thankful for the UGC research fellowship.