|Year : 2015 | Volume
| Issue : 5 | Page : 52-56
Chemical composition analysis, antioxidant and antibacterial activity evaluation of essential oil of Atalantia monophylla Correa
Ramaraj Thirugnanasampandan, Ramya Gunasekar, Madhusudhanan Gogulramnath
Department of Biotechnology, Kongunadu Arts and Science College, Coimbatore, Tamil Nadu, India
|Date of Submission||16-Sep-2014|
|Date of Acceptance||03-Nov-2014|
|Date of Web Publication||02-Jun-2015|
Dr. Ramaraj Thirugnanasampandan
Department of Biotechnology, Kongunadu Arts and Science College, Coimbatore - 641 029, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Atalantia monophylla Correa. a small tree belongs to the family Rutaceae. It is distributed throughout India and in Tamil Nadu the species is commonly seen in foothills of dry vegetation. Objective: The aim was to hydrodistillate and analyze the chemical composition of essential oil from the fresh leaves of A. monophylla Correa. collected in two different seasons (December, 2013 and May, 2014) and to evaluate antioxidant and antibacterial activities of isolated essential oil. Materials and Methods: Chemical composition of isolated essential oil was analyzed by gas chromatography, gas chromatography coupled with mass spectrometry. Antioxidant activity of oil was assessed using five different antioxidant test systems. Antibacterial activity of oil was tested against six pathogenic bacteria by broth dilution method. Results: Essential oil obtained from the leaves collected during May, 2014 had shown more compounds. Antioxidant activity of oil was moderate when compared with positive control. Minimum inhibitory concentration value of oil was ranges between 139.32 ± 0.001 and 541.11 ± 0.003 µg/mL against all the tested bacteria. Conclusion: Result clearly indicates essential oil collected during May, 2014 showed more bioactive compounds.
Keywords: Antibacterial activity, Antioxidant, Atalantia monophylla, Essential oil, Gas chromatography coupled with mass spectrometry
|How to cite this article:|
Thirugnanasampandan R, Gunasekar R, Gogulramnath M. Chemical composition analysis, antioxidant and antibacterial activity evaluation of essential oil of Atalantia monophylla Correa. Phcog Res 2015;7, Suppl S1:52-6
|How to cite this URL:|
Thirugnanasampandan R, Gunasekar R, Gogulramnath M. Chemical composition analysis, antioxidant and antibacterial activity evaluation of essential oil of Atalantia monophylla Correa. Phcog Res [serial online] 2015 [cited 2019 Aug 24];7, Suppl S1:52-6. Available from: http://www.phcogres.com/text.asp?2015/7/5/52/152009
| Introduction|| |
Atalantia monophylla Correa. a small tree belongs to the family Rutaceae. It is distributed throughout India and in Tamil Nadu the species is commonly seen in foothills of dry vegetation. The tribes (Pulayar) of Thadagai hills are using leaves to treat swellings and act as insect repellent. Atalaphyllinine, atalantin, dehydroatalantin, cycloepiatalantin and atalaphylline 3, 5-dimethyl ether have been reported from root bark. ,, Antimicrobial activity of essential oil isolated from the leaves was reported.  Bioactivity guided isolation of pyropheophorbide from leaves showed antiviral activity against herpes simplex virus type 2.  Antiallergic acridine alkaloids, cycloatalaphylline-A, citrrusinine-I, buxifoliadine-E, junosine and yukocitrine were reported from roots.  Various solvent extracts of leaves showed antifeedant, larvicidal and pupicidal activities against Helicoverpa armigera and ovicidal activity against Spodoptera litura. , Chemical composition of essential isolated from the leaves was reported in the literature.  Based on the phytochemical importance of A. monophylla, the present study was aimed to carry out the antioxidant and antibacterial activities of essential oil isolated from the leaves collected from Thadagai hills, Tamil Nadu, India.
| Materials and Methods|| |
Collection of plant material and essential oil extraction
Fresh leaves of A. monophylla Correa. was collected from the boundaries of Thadagai hills (Anamalai Hills), Western ghats, South India during December, 2013 (winter) and May, 2014 (summer). 500 g of leaves was hydrodistilled for about 3 h and the extracted oil was collected. The collected essential oil was treated with sodium sulfate, tightly sealed and stored at 4°C until further use.
Chemical composition analysis
Gas chromatography analysis
Gas chromatography (GC) analysis was carried out using Varian 3800 GC equipped with mass selective detector coupled to front injector type 1079. The chromatograph was fit with DB-5 column (30 m × 0.25 mm). The injector temperature was set at 280°C and the oven temperature was initially maintained at 45°C then programmed to 300°C at the rate of 10°C/min and finally held at 200°C for 5 min. Helium was used as a carrier gas with the flow rate of 1.0 mL/min. The percentage of composition of the essential oil was calculated by the GC peak areas.
Gas chromatography/mass spectrometry analysis
Gas chromatography coupled with mass spectroscopy was performed using Varian 3800 GC equipped with Varian 1200 L single quadrupole mass spectrometer. The GC conditions were the same as reported for GC analysis and the same column was used. The mass spectrometer operated in the electron impact mode at 70 eV. Ion source and transfer line temperature were maintained at 250°C. The compounds were identified based on the comparison of their retention indices, retention time and mass spectra. 
1,1-Diphenyl-2-Picrylhydrazyl free radical scavenging activity
Different concentrations of test sample mixed individually with 0.1 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 50 mM tris-HCl buffer (pH 7.4). Reaction mixture was incubated at 37°C for 30 min and then absorbance was measured at 517 nm.  The percentage of DPPH free radical scavenging activity was calculated using the following equation: % Inhibition = [(A B − A A )/A B ] × 100, where A B , absorption of blank sample, A A , absorption of test sample.
Metal chelating activity
Briefly, 2 mM FeCl 2 was added to different concentrations of test sample, and reaction was initiated by the addition of 5 mM ferrozine. The mixture was vigorously shaken and left to stand at room temperature for 10 min. Absorbance was measured at 562 nm after 10 min.  % Inhibition = [(A B − A A )/A B ] × 100, where A B , absorption of blank sample, A A , absorption of test sample.
Hydroxyl radical scavenging activity
Reaction mixture includes 7.5 mM FeSO 4 , 7.5 mM 1, 10-phenanthroline, 0.2 M phosphate buffer (pH 7.8), 30 mM H 2 O 2 and test sample at different concentrations. The reaction was started by adding H 2 O 2 . After incubation at room temperature for 5 min, the absorbance of the mixture was read at 536 nm.  % Inhibition = [(A B − A A )/A B ] × 100, where A B , absorption of blank sample, A A , absorption of test sample.
Prevention of deoxyribose degradation
Test sample of different concentrations was mixed with 20 mM deoxyribose, 0.1 M NaPO 4 , 20 mM H 2 O 2 and 50 mM FeSO 4 . The reaction mixture was incubated for 60 min at 37°C. Then 2 mL of 10% ice-cold trichloroacetic acid was added, and 1 mL aliquot of the samples was added with 1 mL of 1% thiobarbituric acid (TBA). The TBA/sample mixture was heated in a water bath at 95°C for another 60 min and absorbance was read at 532 nm.  % Inhibition = [(A B − A A )/A B ] × 100, where A B , absorption of blank sample, A A , absorption of test sample.
Inhibition of linoleic acid peroxidation
Briefly, 20 mM linoleic acid, 100 mM HCl (pH 7.5), 5 mM ascorbic acid were mixed with test sample and linoleic acid peroxidation was initiated by the addition of 4 mM FeSO 4 .7H 2 O, incubated for 60 min at 37°C and terminated by the addition of 2 mL of ice cold trichloroacetic acid (10% v/v). An amount of 1 mL of TBA (1% w/v in 50 mM NaOH) was added to 1 mL of the reaction mixture, followed by heating at 95°C for 60 min. The reaction sample was read at 532 nm.  The percentage of linoleic acid peroxidation inhibition activity was calculated using the following equation: % Inhibition = [(A B − A A )/A B ] × 100, where A B , absorption of blank sample, A A , absorption of test sample.
Clinical isolates of Aeromonas hydrophila, Escherichia More Details coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Proteus vulgaris and Staphylococcus aureus were procured from Microbiology Laboratory of KMCH Hospital, Coimbatore, Tamil Nadu, India.
Determination of minimum inhibitory concentration
The minimum inhibitory concentration (MIC) of essential oil was studied by broth microdilution method using 96-well microtiter plates.  Essential oil was dissolved in dimethylsulphoxide (DMSO) (1%) with the addition of Tween-80 (0.5%) and diluted in Muller Hinton Broth to get a concentration range of 25-225 μg/mL. The solution was then two-fold diluted in Muller Hinton Broth (100 μL), inoculated with bacterial strains and then incubated at 37°C for 24 h. The bacterial growth was measured as turbidity with a Cyberlab micro plate reader at 405 nm. The MIC was defined as the lowest concentration of the oil that inhibited the growth of the test bacteria. DMSO assayed as the negative control at a concentration of 1% did not inhibit any of the strains tested. All tests were assayed in triplicate in three independent experiments, and median values were used for MICs calculation. Both gentamicin and ampicillin were served as a positive control.
The data obtained from the antioxidant and antibacterial studies were analyzed using SPSS (SPSS Inc. 16.00) for IC 50 and MIC calculations.
| Results|| |
Chemical composition of essential oil
Hydrodistillation of 500 g of leaves (collected during May 2014) yielded 350 μL of yellow colored essential oil with a fragrance of citrus. A total of 36 compounds constituting 88.13% were identified with sabinene (23.81%) as major compound followed by trans-asarone (19.55%), β-pinene (13.35%) and myrcene (10.39%) [Table 1]. Interestingly the essential oil isolated from leaves collected during winter (December, 2013) season showed 23 compounds (72.25%) with trans isoeugenol (23.73%) as a major one [Table 2].
|Table 1: Chemical composition of essential oil of A. monophylla collected during May, 2014 |
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|Table 2: Chemical composition of essential oil of A. monophylla collected during December, 2013 |
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Antioxidant activity of essential oil
Essential oil collected during summer season (May, 2014) was used for antioxidant studies. Concentration range of 250 μg/mL showed 69.04% of DPPH free radical scavenging with IC 50 value of 198.97 ± 0.002 μg/mL. Ferrous ion chelation and hydroxyl radical scavenging activity of the oil was high at higher concentration, and IC 50 value was recorded as 204.78 ± 0.002 and 199.35 ± 0.003 μg/mL respectively. Fenton reaction induced deoxyribose degradation was prevented by the essential oil in a dose dependent manner with IC 50 value of 176.54 ± 0.002 μg/mL and percentage of inhibition of linoleic acid peroxidation was calculated as 66.30 at 250 μg/mL with IC 50 value of 219.15 ± 0.002 μg/mL [Table 3]. In all the test system studied, the activity of oil was less observed when compared with butylated hydroxytoluene (47.10 ± 0.001 μg/mL).
Antibacterial activity of oil
The MIC values of A. monophylla essential oil against all the tested bacterial strains were recorded in [Table 4]. The antibacterial activity was strong against A. hydrophila followed by P. mirabilis, P. aeuroginosa, P. vulgaris and E. coli (MIC < 350 μg/mL). The oil showed the least activity against both K. pneumoniae and S. aureus (MIC > 500 μg/mL).
| Discussion|| |
The result of this study clearly indicates leaf material collected during summer season (May, 2014) had shown more compounds of biological interest and it's assumed that high temperature with low water content and other environmental factors might have induced relatively high secondary metabolite production. Earlier reports on essential oil of A. monophylla collected from Narrtha and Nagamalai hills (Tamil Nadu, South India) reported that methyl eugenol and asarone were major compounds. , However in the present investigation sabinene has been identified as a major compound. Though similar compound was reported in the previous studies, the percentage composition has been varied. This variation might be due to soil, climate, availability of water, geographical location, environmental factors, season of material collection, etc. 
Essential oil isolated from the summer season had more bioactive compounds, so it was used for further studies. Antioxidant activity of oil was concentration dependent while increasing concentration percentage of free radical scavenging was also increased. When compared with positive control antioxidant activity of oil was less however the oil showed considerable free radical scavenging activity. Antioxidant activity of oil may be related to the presence of the monoterpene hydrocarbon, sabinene. Similar result was reported by Valente et al.  where sabinene and essential oil of Oenanthe crocata rich in sabinene and trans β-ocimene showed considerable free radical scavenging activity. Meanwhile other monoterpene hydrocarbons such β-pinene, myrcene, limonene, β-phellandrene and an ether, asarone could also be taken in to account.
Essential oils as antimicrobial agents in food systems may be considered as additional intrinsic determinant to increase the safety and shelf life of foods.  Pirbalouti et al.  has reported that monoterpenes are disrupting the microbial cytoplasmic membrane and leads to loss in the high impermeability of the membranes for protons and larger ions. Since the main constituent of A. monophylla oil is monoterpenes so we assumed that observed antibacterial activity might be due to sabinene, β-pinene, myrcene, β-phellandrene and asarone. Meanwhile synergistic effect of other compounds also need to be considered. 
| Conclusion|| |
Leaf essential oil of A. monophylla collected during May, 2014 showed more bioactive compounds when compared with oil collected during December, 2013 and could be used as a natural supplement and preservative in food, crude drug and phytopharmaceutical preparations.
| Acknowledgments|| |
This work was financially supported by Department of Science and Technology-Science and Engineering Research Board (DST-SERB) New Delhi, India under grant no. SB/FT/LS-230/2012; date 02.05.2013.
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