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Research Article

In vitro Antioxidant Activities and Antimicrobial Efficacy of Asian Snakewood; Colubrina asiatica (L.) Brong.

Research Journal of Medicinal Plants: Volume 9 (7): 307-320, 2015

Desai Nivas, U.L. Dethe and D.K. Gaikwad


The present study evaluated antimicrobial efficacy of essential oil and in vitro antioxidant activities of the aqueous extract from Colubrina asiatica (L.) Brong. as well as the chemical composition of essential oil. In the present investigation, Colubrina Water Extract (CWE) was studied for its antioxidant activity and Colubrina Essential Oil (CEO) for anti microbial properties. The antioxidant properties of CWE were evaluated using different free radical scavenging assays, such as reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging and metal chelating activities. We found that CWE had powerful antioxidant activity. The different concentrations (50, 100 and 250 g) of CWE showed 39, 66 and 98% inhibition on peroxidation of linoleic acid emulsion, respectively, while 60 g mL–1 of ascorbic acid, exhibited only 30% inhibition. Moreover, CWE had effective reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging and metal chelating activities at the same concentrations. Those various antioxidant activities were compared to standard antioxidants such as ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), gallic acid and quercetin. In addition, total phenolic compounds in the CWE were determined as Gallic acid equivalent. The Colubrina Essential Oil (CEO) content quantified showed presence of 10 compounds in which, dodecamethylcyclohexasiloxane has showed the highest (17%) and dehydro-N-[4,5-methylenedioxy-2-nitrobenzylidene]-tyramine showed the lowest percentage (1.9%). Cubebene, comprised of 14%. The antimicrobial activity of oil was studied on gram negative and gram positive bacterial.

How to cite this article:

Desai Nivas, U.L. Dethe and D.K. Gaikwad, 2015. In vitro Antioxidant Activities and Antimicrobial Efficacy of Asian Snakewood; Colubrina asiatica (L.) Brong.. Research Journal of Medicinal Plants, 9: 307-320.

DOI: 10.3923/rjmp.2015.307.320

URL: https://scialert.net/abstract/?doi=rjmp.2015.307.320


Medicinal plants play an important role in human life to combat diseases since time immemorial. The villagers and the tribals in India even today largely depend on the surrounding plants/forests for their day-today needs. Medicinal plants are being focused upon not only as a source of health care but also as a source of income. The Ministry of Environment and Forests, Government of India, reveals that there are over 8000 species of medicinal plants grown in the country. About 70% of these plants are found in the tropical forest; spread across the Western and Eastern Ghats. The Export-Import Bank of India, in its report for the year 1997, puts medicinal plants related trade in India at $.5.5 billion and the same is growing rapidly (Kumar and Janagam, 2011). Free radicals are highly reactive species produced in the body during normal metabolic functions. These are atoms or groups of atoms that have at least one unpaired electron, which makes them highly reactive. Though Oxygen, is essential to life, but it is the source of the potentially damaging free radicals. Antioxidants counteract these cellular by-products, called free radicals and bind them before they can cause damage (Pandey et al., 2005). Fruits and vegetables are major source of dietary antioxidants and their precursors (Block et al., 1992). Recently, various phytochemicals and their effects on health, especially the suppression of active oxygen species by natural antioxidants from teas, spices and herbs, have been intensively studied (Ho et al., 1994). The most commonly used antioxidants at the present time are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG) and tert-butylhydroquinone (TBHQ) (Sherwin, 1990).

Among the plants with promising biological activities employed by the traditional people, Colubrina asiatica (Brong.), is a Rhamnaceae species popularly known as “Asian snake wood” with a glabrous, scandent or sprawling shrubby nature. In India the species is wide spread in littoral scrub forests, tidal forests of Orissa and Ghats of Konkan (Thaman, 1992). In some regions of Northeast and Southeast it is popularly employed in medicinal preparations for the treatment of digestive aid, antiscorbutic (counteracts scurvy), tonic, laxative, a febrifuge, medicinal bath and a vermifuge and skin diseases (Burkill, 1966; Morton, 1981; Austin, 1999). The plant is economically valued for its leaf saponins used in soap substitute, used to wash and whiten textile kilts and garments (Richardson et al., 2000).

In this sense, this study aimed the phytochemical investigation and in vitro evaluation of antioxidant activities of the aqueous extract and antimicrobial efficacy of essential oil of Colubrina asiatica.


Antioxidant activities
Chemicals: Ammonium thiocyanate and gallic acid were purchased from E. Merck. Ferrous chloride, polyoxyethylenesorbitan monolaurate (Tween-20), ascorbic acid, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonic acid)-1,2, 4-triazine (ferrozine), nicotinamide adenine dinucleotide (NADH), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and trichloroacetic acid (TCA) were purchased from Sigma (Germany).

Plant material and extraction: Colubrina asiatica Brong. (whole plant) was collected from coastal area of Goa (coordinates 15°27’7" N 73°50’6" E), in December 2011 and identified by Dr. MayurNandikar, a plant taxonomist of Department of Botany from Shivaji University of Kolhapur. A specimen herbarium deposited in Shivaji University, Kolhapur (NMDESAI 001 SUK). The plant material was initially dried in sunlight and then in oven. The dried sample was chopped into small parts with a blender. For water extraction, 20 g dried leaves of Colubrina ground into a fine powder in a mill and was mixed with 400 mL boiling water by magnetic stirrer during 15 min. Then the extract was filtered over Whatman No.1 paper. The filtrate was frozen and lyophilized in a lyophilizator at 5 μm Hg pressure at -50°C (Labconco, Freezone 1L). The extract was placed in a plastic bottle and then stored at -20°C until further use.

Total antioxidant activity determination: The antioxidant activity of CWE was determined according to the thiocyanate method (Mitsuda et al., 1996). For stock solution; 20 mg lyophilized Colubrina Water Extract (CWE) was dissolved in 20 mL water. Then the solution, which contains different amount of stock CWE solution or standards samples (50, 100 and 250 μg) in 2.5 mL of 0.04 M potassium phosphate buffer (pH 7.0) was added to 2.5 mL of linoleic acid emulsion in potassium phosphate buffer (0.04 M, pH 7.0). Each solution was then incubated at 37°C in a glass flask in the dark. At intervals during incubation, each solution was stirred for 3 min, 0.1 μL this incubation solution, 0.1 mL FeCl2 and 0.1 mL thiocyanate were transferred to the test tube, which containing 4.7 mL ethanol solution incubated for 5 min. Finally, the peroxide value was determined by reading the absorbance at 500 nm in a UV-1800 UV-Vis spectrophotometer (Shimadzu). During the linoleic acid oxidation, peroxides formed and these compounds oxidize Fe2+-Fe3+. The latter Fe3+ ions form complex with SCN-, which has a maximum absorbance at 500 nm. Therefore higher absorbance values indicate higher linoleic acid oxidation. The solutions without added CWE or standards were used as blank samples. Five millilitres linoleic acid emulsion is consisting of 17.5 μg Tween-20, 15.5 μL linoleic acid and 0.04 M potassium phosphate buffer (pH 7.0). On the other hand, 5 mL control composed of 2.5 mL linoleic acid emulsion and 2.5 mL potassium phosphate buffer (0.04 M, pH 7.0). All data about total antioxidant activity is the average of duplicate analyses. The inhibition of lipid peroxidation in percentage was calculated by the following equation:

where, A0 was the absorbance of the control reaction and A1 was the absorbance in the presence of the sample of CWE (Duh et al., 1999).

Reducing power: The reducing power of CWE was determined according to the method of Oyaizu (1986). The different doses of CWE (50, 100 and 250 μg) in 1 mL of distilled water were mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 mL, 1%). The mixture was incubated at 50°C for 20 min. A portion (2.5 mL) of TCA (10%) was added to the mixture, which was then centrifuged for 10 min at 1000×g. The upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl3 (0.5 mL, 0.1%) and the absorbance was measured at 700 nm in a spectrophotometer. Higher absorbance of the reaction mixture indicated greater reducing power.

Superoxide anion scavenging activity: Measurement of superoxide anion scavenging activity of CWE was based on the method described by Liu et al. (1997) with slight modifications (Gulcin et al., 2003). Superoxide radicals are generated in phenazine methosulphate (PMS)-nicotinamide adenine dinucleotide (NADH) systems by oxidation of NADH and assayed by the reduction of Nitro Blue Tetrazolium (NBT). In this experiments, the superoxide radicals were generated in 3 mL of Tris-HCl buffer(16 mM, pH 8.0) containing 1 mL of NBT (50 μM) solution, 1 mL NADH (78 μM) solution and 1 mL sample solution of CWE (100 μg mL–1) were mixed. The reaction was started by adding 1 mL of PMS solution (10 μM) to the mixture. The reaction mixture was incubated at 25°C for 5 min and the absorbance at 560 nm was measured against blank samples. l-ascorbic acid was used as a control. Decrease in absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. The percentage inhibition of superoxide anion generation was calculated using the following formula:

where, A0 was the absorbance of the control (l-ascorbic acid) and A1 was the absorbance of CWE or standards (Ye et al., 2000).

Free radical scavenging activity: The free radical scavenging activity of CWE was measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH•) using the method of Shimada et al. (1992). Briefly, 0.1 mM solution of DPPH• in ethanol was prepared. Then, 1 mL of this solution was added to 3 mL of CWE solution at different doses (50-250 μg). The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then the absorbance was measured at 517 nm in a spectrophotometer. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The DPPH• concentration (mM) in the reaction medium was calculated from the following calibration curve, determined by linear regression (R2: 0.9678).

Absorbance = 104.09×[DPPH•]. The DPPH radical concentration was calculated using the following equation:

where, A0 was the absorbance of the control reaction and A1 was the absorbance in the presence of the sample of CWE (Oktay et al., 2003).

Metal chelating activity: The chelating of ferrous ions by the CWE and standards was estimated by the method of Dinis et al. (1994). Briefly, extracts (50-250 μg) were added to a solution of 2 mM FeCl2 (0.05 mL). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL) and the mixture was shaken vigorously and left standing at room temperature for ten minutes. After the mixture had reached equilibrium, the absorbance of the solution was then measured spectrophotometrically at 562 nm in a spectrophotometer. The percentage of inhibition of ferrozine-Fe2+ complex formation was given by the formula:

where, A0 was the absorbance of the control and A1 was the absorbance in the presence of the sample of CWE and standards. The control contains FeCl2 and ferrozine (Ilhami et al., 2003).

Scavenging of hydrogen peroxide: The ability of the CWE to scavenge hydrogen peroxide was determined according to the method of Ruch et al. (1989). A solution of hydrogen peroxide (40 mM) was prepared in phosphate buffer (pH 7.4). Hydrogen peroxide concentration was determined spectrophotometrically from absorption at 230 nm in a spectrophotometer. Extracts (50-250 μg) in distilled water were added to a hydrogen peroxide solution (0.6 mL, 40 mM). Absorbance of hydrogen peroxide at 230 nm was determined after ten minute against a blank solution containing in phosphate buffer without hydrogen peroxide. The percentage of scavenging of hydrogen peroxide of CWE and standard compounds was calculated using the following equation:

where, A0 was the absorbance of the control and A1 was the absorbance in the presence of the sample of CWE and standards (Ilhami et al., 2003).

Determination of total phenolic compounds: Total soluble phenolic compounds in the CWE were determined with Folin-Ciocalteu reagent according to the method of Slinkard and Singleton (1977) using pyrocatechol as a standard phenolic compound. Briefly, 1 mL of the CWE solution (contains 1000 μg extract) in a volumetric flask diluted with distilled water (46 mL). One milliliter of Folin-Ciocalteu reagent was added and the content of the flask was mixed thoroughly. After 3 min, 3 mL of Na2CO3 (2%) was added and then was allowed to stand for 2 h with intermittent shaking. The absorbance was measured at 760 nm in a spectrophotometer. The total concentration of phenolic compounds in the CWE determined as microgram of gallic acid equivalent by using an equation that was obtained from standard pyrocatechol graph (Gulcin et al., 2002):

Absorbance = 0.0053×total phenols [gallic acid equivalent (μg)]-0.0059

Extraction of oil and GC MS analysis: Hydrodistillation of the plant material was performed in a clevenger-type apparatus for 210 min. The oil obtained was light yellow, liquid at room temperature with an agreeable odor. After isolation, the Essential Oil (EO) was collected and stored in steeled glass vials in refrigerator at 4-5°C. The samples were analysed by GC-MS (Schimadzu) using capillary column. The GC-MS conditions were as follows; injection volume (1 mL), temperature programme 80-160°C for 5 min at 10°C min–1; 160-235°C for 5 min at 5°C min–1 and 235-290°C for 5 min at 50°C min–1; injector temperature (280°C), MS transfer line (290°C), ion source (200°C) spit ratio (1: 10) and mass range at 50-450. Data was analysed by comparing it with SI (standard index) from the NIST library available.

Antimicrobial activities
Preparation of test microorganisms: For the purpose of antimicrobial evaluation ten microorganisms were used. Pseudomonas aeruginosa (ATCC 9027, gram-negative), Escherichia coli (ATCC 9837, gram-negative), Staphylococcus aureus (ATCC 6538, gram-positive) and Streptococcus pneumoniae (ATCC 49619, gram-positive) microorganism strains were employed for determination of antimicrobial activity. Microorganism strains were obtained from the stock cultures of Microbiology Laboratory, Department of Microbiology, Shivaji University, Kolhapur.

Antimicrobial activity determination: Agar cultures of the test microorganisms were prepared as described by Mackeen et al. (1997). Three to five similar colonies were selected and transferred with loop into 5 mL of tryptone soya broth. The broth cultures were incubated for 24 h at 37°C. For screening, sterile, 6 mm diameter lter paper disc were impregnated with 250 μg of the CEO. Then the paper discs were placed onto Mueller Hinton agar. The inoculum for each organism was prepared from broth cultures. The concentration of cultures was adjusted to 108 colony forming units (1×108 CFU mL–1). The results were recorded by measuring the zones of growth inhibition surrounding the disc. Clear inhibition zones around the discs indicated the presence of antimicrobial activity. All data on antimicrobial activity the average of triplicate analyses. Netilmicin (30 μg per disc), amoxicillin-clavulanic acid (20-10 μg per disc) were used as reference standards, which as recommended by the National Committee for Clinical Laboratory Standards (NCCLS).

Statistical analysis: Experimental results concerning this study were Mean±SD of three parallel measurements. Analysis of variance was performed by ANOVA procedures. Significant differences between means were determined by Duncan’s multiple range tests. p values<0.05 were regarded as significant and p values<0.01 very significant.


Antioxidant capacity: Antioxidants are the compounds which helps to delay or inhibit the oxidation of lipids and other molecules through the inhibition of either initiation or propagation of oxidative chain reactions (Jaleel et al., 2007). Antioxidants can act as either reducing agents, or by free radical scavengers or singlet oxygen quenchers (Chanwitheesuk et al., 2005). Recent studies focused on several antioxidant methods and its modifications to evaluate antioxidant activity and to explain how antioxidants function. Among these, total antioxidant activity, reducing power, DPPH assay, metal chelating, active oxygen species such as H2O2, and OH• quenching assays are most commonly used for the evaluation of antioxidant activities of extracts (Duh et al., 1999; Amarowicz et al., 2000; Chang et al., 2002). Total antioxidant activity of CWE was determined by the thiocyanate method. The CWE exhibited effective antioxidant activity at all the studied doses. The effects of different amounts of CWE (from 50-250 μg) on peroxidation of linoleic acid emulsion are shown in Fig. 1. The antioxidant activity of CWE was found concentration dependently. The CWE (50, 100 and 250 μg) showed higher antioxidant activities than that of 100 μg concentration of standard antioxidant ascorbic acid. After incubation times the percentage inhibition of peroxidation in linoleic acid emulsion was 34, 62 and 91%, respectively and greater than that of ascorbic acid (30%).

The reductive capabilities of CWE compared to ascorbic acid was shown in Fig. 2. For the measurements of the reducing power ability, we investigated the Fe3+-Fe2+ transformation in the presence of CWE samples using the method of Oyaizu (1986). Like the antioxidant activity, the reducing power of CWE increased concentration dependently. All of the concentrations of CWE showed higher activities than the control in a statistically significant (p<0.05) manner. In the PMS-NADH-NBT system, superoxide anion derived from dissolved oxygen by PMS-NADH coupling reaction reduces NBT.

Fig. 1:
Antioxidant activity of different doses of CWE and ascorbic acid in the linoleic acid emulsion

Fig. 2:
Reducing power ability of CWE compared with ascorbic acid using spectrophotometric detection of the Fe3+-Fe2+ transformation

Fig. 3:
Superoxide anion radical scavenging activity of 100 g of WEN, BHA, BHT and ascorbic acid by the PMS-NADH-NBT method

Superoxide anion is an initial free radical and plays an important role in the formation of other reactive oxygen species such as hydrogen peroxide, hydroxyl radicals, or singlet oxygen in living systems (Stief, 2003). It can also react with nitric oxide and from peroxynitrite, which can generate toxic compounds such as hydroxyl radicals and nitric dioxide (Halliwell, 1997). Figure 3 shows the percentage inhibition of superoxide radical generation by 100 μg of CWE and comparison with same doses of BHT and ascorbic acid. The CWE exhibited higher superoxide radical scavenging activity than BHT and ascorbic acid (p<0.01). The percentage inhibition of superoxide generation by 100 μg amount of CWE was found as 90% and greater than that of some doses of BHT and ascorbic acid (89, 80 and 61%), respectively. Superoxide radical scavenging activity of those samples followed the order: CWE>BHT >ascorbic acid. The effect of antioxidants on DPPH radical scavenging is thought to be due to their hydrogen donating ability. The DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule.

Fig. 4: Free radical scavenging activity of quercetin, ascorbic acid, BHA and CWE on DPPH

Themodel of scavenging the stable DPPH radical is a widely used method to evaluate antioxidant activities in a relativelyshort time compare to other methods (Soare et al., 1997). The reduction capability on the DPPH radical is determined by the decrease in its absorbance at 517 nm induced by antioxidants. The maximum absorption of a stable DPPH radical in ethanol is at 517 nm. The decrease in absorbance of DPPH radical caused by antioxidants is due to the reaction between antioxidant molecules and radical, which results inthe scavenging of the radical by hydrogen donation.The DPPH radical scavenging assay depends on the decoloration of the purple coloured methanolic DPPH solution to yellow by the radical scavengers present in the sample extracts (Blois, 1958). The result of DPPH scavenging activity implies that the plant extract may be useful for treating radical related pathological damages (Wang et al., 1998). A significant (p<0.01) decrease in the concentration of DPPH radical due to the scavenging ability of the CWE and standards was observed (Fig. 4). The CWE and BHA showed almost equal DPPH scavenging activity, however, significantly are lower than that of quercetin. The scavengingeffect of CWE and standards on the DPPH radical decreasedin the order of quercetin>ascorbic acid >CWE>BHA and were 89, 45, 36 and 31% at the concentration of 60 μg mL–1,respectively. Uncontrolled generation of ROS can lead to their accumulation causing oxidative stress in the cells (Kunwar and Priyadarsini, 2011). Severe oxidative stress causes cell damage and death (Aruoma, 1998). Superoxide anion radical scavenging activity of 100 μg of CWE, ascorbic acid, BHT and ascorbic acid by the PMS-NADH-NBT method obtained from this study, CWE exhibits free radical scavenging activity as well as a primary antioxidant thatreacts with free radicals, which may hampers the damages caused due to free radical in the human body (Fig. 3). The chelating of ferrous ions by CWE was estimatedwith the method of Dinis et al. (1994). In the presence ofchelating agents, Ferrozine can quantitatively form complexes with Fe2+ is interrupted and ultimately diminishes the red colour of the complex. The actual mechanism of antioxidant action is chelation of transition metals thus preventing catalysis of hydroperoxidedecoposition and fenton type reactions (Gordon, 1990). Iron can stimulate lipid peroxidation by the Fenton reaction and also accelerates peroxidation by decomposing lipid hydroperoxides into peroxyl and alkoxyl radicals that can themselves abstract hydrogen and perpetuate the chain reaction of lipid peroxidation (Halliwell, 1991). In this assay CWE and standard antioxidant compound interfered with the formation of ferrous and ferrozine complex, suggesting its potent chelating activity which capture ferrous ion before ferrozine.

Fig. 5:
Metal chelating effect of different amount of Colubrina water extract and standards on ferrous ions

The absorbance of Fe2+-ferrozine complex was linearly decreased dose-dependently (from 50-250 μg) (Fig. 5). The difference between CWE and the control was statistically significant (p<0.01). The percentages of metal chelating capacity of 250 μg concentration of CWE, ascorbic acid, BHA and quercetin were found as 84,40, 58 and 34%, respectively. The metal scavenging effect of CWE and standards decreased in the order of CWE>ascorbic acid>quercetine>BHA metal chelating capacity is important since it reduced theconcentration of the catalysing transition metal in lipid peroxidation (Duh et al., 1999). The data obtained from Fig. 5 revealed that CWE demonstrate a marked capacity for iron binding, revealing that their action as peroxidation protector may berelated to its iron binding capacity. Hydrogen peroxide scavenging activity of CWE may be endorsed to their phenolic content, which could bestow electrons to H2O2, thus counter acting it to water.

H2O2 is highly important because of its ability to penetrate biological membranes. H2O2 itself is not very reactive, but it can sometimes be toxic to cell because it may give rise to hydroxyl radical in the cells (Arulmozhi et al., 2008). Scavenging of H2O2 by extracts may be attributed to their phenolics, which can donate electrons to H2O2, thus neutralizing it to water (Nabavi et al., 2008; Ebrahimzadeh et al., 2009). The ability of CWE to scavenge H2O2 was determined according to the method of Ruch et al. (1989). The scavenging ability of CWE on H2O2 is shown in Fig. 6 and compared with BHA, BHT and ascorbic acid as standards. The CWE was capable of scavenging H2O2 in a dose-dependent. Two hundred and fifty micrograms of CWE exhibited 20% scavenging activity on H2O2. On the other hand, at the same concentration; BHA, BHT and ascorbic acid showed 32, 80 and 51% activity, respectively. These results indicated thatCWE posses potent H2O2 scavenging activity but had lower than the BHA, BHT and ascorbic acid. However, statistically significant correlation between those valuesand control (p<0.01) was observed. The H2O2 scavenging effect ofsame dose (250 μg) of CWE and standards decreased in the order of BHT>ascorbic acid>BHA>CWE.

Phenols and polyphenolic compounds, such as flavonoids, are widely found in food products derived from plant sources and they have been shown to possess significant antioxidant activities (Nabavi et al., 2009). The 25.3 μg gallic acid equivalent of phenols was detected in 1 mg of CWE.

Fig. 6:Hydrogen peroxide scavenging activity of different amount of CWE, BHA, BHT, quercetin and ascorbic acid

Table 1: Composition of essential oil in C. asiatica leaves

The phenolic compounds may contribute directly to theantioxidative action (Duh et al., 1999). The interest on these compounds is related with their antioxidant activity and promotion of health benefits (Ryan et al., 2002).

Essential oil composition and antibacterial efficacy: In the present study, the components present in the essential oil are identified using NIST library. The composition of essential oils showed dodecamethylcyclohexasiloxane the highest (18%) and decamethylcyclopentasiloxane showed the lowest percentage (1.6%) (Table 1). These compounds were reported to be in many personal care products such as toiletries. Volatile compounds is essential to determine the predominant components and their composition in order to investigate their bioactivity including antioxidant and antibacterial activities. A number of reports have shown that plant volatile compounds exhibited potent antioxidant and antibacterial activities (Choi and Hwang, 2005). Cubebene was reported to show potent antibacterial properties (Prabuseenivasan et al., 2006) and antioxidant properties (Ruberto and Baratta, 2000). Essential oils produced by plants have been traditionally used for respiratory tract infections and are used nowadays as ethical medicines for colds (Federspil et al., 1997). In this study, three different microbial and one yeast species were used to screen the possible antimicrobial activity of Colubrina Essential Oil (CEO). In the present study, 4 strains of bacterial strains representing gram negative and gram positive were used to screen the possible antimicrobial activity of CEO. Water extract of Colubrina exhibited antimicrobial activity against all tested microorganisms.

Table 2: Antimicrobial activities of CEO (250 μg per disc) and standard antimicrobial agents
CEO: Colubrina essential oil, ND: Not detected activity at this amount of CWE or standards

Amongst these Staphylococcus aureus gram positive bacteria responsible for food poisoning. Interestingly CEO showed antibacterial activity against this bacterium. Table 2 showed appreciable inhibitory activity of CEO. Escherichia coli, belonging to the normal ora of humans, is a gram negative bacterium. Amoxicillin-clavulanic acid (15 μg per disc) and netilmicin (25 μg per disc) were used as positive controls. Gram positive bacteria are known to be more susceptible to essential oils than gram negative bacteria (Smith-Palmer et al., 1998). Gram positive bacteria are more sensitive to plant oils and extracts than gram negative bacteria (Karaman et al., 2003).


Present findings clearly indicate that CWE has a powerful antioxidant activity against various oxidative systems in vitro; moreover, CWE can be further used as accessible source of natural antioxidants and as a possible food supplement or in pharmaceutical industry. The various antioxidant mechanisms of CWE may be accredited to strong hydrogen donating ability, a metal chelating ability and their effectiveness as scavengers of hydrogen peroxide, superoxide and free radicals. The essential oil of Colubrina possessed noticeable antimicrobial activity against gram positive and negative bacteria when compared with standard and strong antimicrobial compounds such as amoxicillin clavulanic acid and netilmicin. We believe that the present investigation together with previous studies provide support to the antibacterial properties of Colubrina essential oil as well as potent natural antioxidant source. It can be used as antioxidant and antibacterial supplement 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 this oil as an antibacterial agent and natural antioxidant compounds with health benefits in topical or oral applications.


Authors are highly acknowledged to The Principal, Shri Pnacham Khemraj Mahavidyalaya, Sawantwadi and Members of Management of South Ratnagiri Shikshan Prasarak Mandal, Sawantwadi for their support and encouragement.


Amarowicz, R., M. Naczk and F. Shahidi, 2000. Antioxidant activity of crude tannins of canola and rapeseed hulls. J. Am. Oil Chem. Soc., 77: 957-961.
CrossRefDirect Link

Arulmozhi, S., P.M. Mazumder, P. Ashok and L.S. Narayanan, 2008. In vitro antioxidant and free radical scavenging activity of Alstonia scholaris Linn. R. Br. Iran. J. Pharmacol. Ther., 6: 191-196.

Aruoma, O.I., 1998. Free radicals, oxidative stress and antioxidants in human health and disease. J. Am. Oil Chem. Soc., 75: 199-212.
CrossRefDirect Link

Austin, D.F., 1999. Ethnobotany of Florida's weedy vines. Proceedings of the 1998 Joint Symposium of the Florida Exotic Pest Plant Council and the Florida Native Plant Society, June 3-7, 1998, Palm Beach Gardens, FL -.

Block, G., B. Patterson and A. Subar, 1992. Fruit, vegetables and cancer prevention: A review of the epidemiological evidence. Nutr. Cancer, 18: 1-29.
CrossRefDirect Link

Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature, 181: 1199-1200.
CrossRefDirect Link

Burkill, I.H., 1966. A Dictionary of the Economic Products of the Malay Penisula. 2nd Edn., Ministry of Agriculture and Cooperatives, Kuala Lumpur, Malaysia.

Chang, L.W., W.J. Yen, S.C. Huang and P.D. Duh, 2002. Antioxidant activity of sesame coat. Food Chem., 78: 347-354.
CrossRefDirect Link

Chanwitheesuk, A., A. Teerawutgulrag and N. Rakariyatham, 2005. Screening of antioxidant activity and antioxidant compounds of some edible plants of Thailand. Food Chem., 92: 491-497.
CrossRefDirect Link

Choi, E.M. and J.K. Hwang, 2005. Effect of some medicinal plants on plasma antioxidant system and lipid levels in rats. Phytother. Res., 19: 382-386.
CrossRefDirect Link

Dinis, T.C.P., V.M.C. Madeira and L.M. Almeida, 1994. Action of phenolic derivatives (acetaminophen, salicylate and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys., 315: 161-169.
CrossRefPubMedDirect Link

Duh, P.D., Y.Y. Tu and G.C. Yen, 1999. Antioxidant activity of water extract of Harng Jyur (Chrysanthemum morifolium Ramat). LWT-Food Sci. Technol., 32: 269-277.
CrossRefDirect Link

Ebrahimzadeh, M.A., S.F. Nabavi and S.M. Nabavi, 2009. Antioxidant activities of methanol extract of Sambucus ebulus L. Flower. Pak. J. Biol. Sci., 12: 447-450.
CrossRefPubMedDirect Link

Federspil, P., R. Wulkow and T. Zimmermann, 1997. [Effects of standardized Myrtol in therapy of acute sinusitis--results of a double-blind, randomized multicenter study compared with placebo]. Laryngorhinootology, 76: 23-27.
CrossRefPubMedDirect Link

Gordon, M.H., 1990. The Mechanism of the Antioxidant Action in vitro. In: Food Antioxidants, Hudson, B.J.F. (Ed.). Elsevier,London, New York, pp: 1-18.

Gulcin, I., M. Oktay, E. Kirecci and O.I. Kufrevioglu, 2003. Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chem., 83: 371-382.
CrossRefDirect Link

Gulcin, I., M. Oktay, O.I. Kufrevioglu and A. Aslan, 2002. Determination of antioxidant activity of lichen Cetraria islandica (L.) Ach. J. Ethnopharmacol., 79: 325-329.
CrossRefDirect Link

Halliwell, B., 1991. Reactive oxygen species in living systems: Source, biochemistry and role in human disease. Am. J. Med., 91: S14-S22.
CrossRefPubMedDirect Link

Halliwell, B., 1997. Antioxidants and human disease: A general introduction. Nutr. Rev., 55: S44-S52.
CrossRefPubMedDirect Link

Ho, C.T., T. Ferraro, Q. Chen and R.T. Rosen, 1994. Phytochemicals in Teas and Rosemary and their Cancer-Preventive Properties. In: Food Phytochemicals for Cancer Prevention II: Teas, Spices and Herbs, Ho, C.T., T. Osawa, M.T. Huang and R.T. Rosen (Eds.). Chapter 1, American Chemical Society, Washington, DC., USA., ISBN-13: 9780841227699, pp: 2-9.

Ilhami, G., U. Metin, O. Munir, B. Suktru and K. Irfan, 2003. Antioxidant and antimicrobial activities of Teucrium polium L. J. Food Technol., 1: 9-16.
Direct Link

Jaleel, C.A., R. Gopi, P. Manivannan, B. Sankar, A. Kishorekumar and R. Panneerselvam, 2007. Antioxidant potentials and ajmalicine accumulation in Catharanthus roseus after treatment with giberellic acid. Colloids Surfaces B Biointerfaces, 60: 195-200.
CrossRefDirect Link

Karaman, I., F. Sahin, M. Gulluce, H. Ogutcu, M. Sengul and A. Adiguzel, 2003. Antimicrobial activity of aqueous and methanol extracts of Juniperus oxycedrus L. J. Ethnopharmacol., 85: 231-235.
CrossRefPubMedDirect Link

Kumar, M.R. and D. Janagam, 2011. Export and import pattern of medicinal plants in India. Indian J. Sci. Technol., 4: 245-248.
Direct Link

Kunwar, A. and K.I. Priyadarsini, 2011. Free radical, oxidative stress and importance of antioxidant in human health. J. Med. Alli. Sci., 1: 53-60.
Direct Link

Liu, F., V.E.C. Ooi and S.T. Chang, 1997. Free radical scavenging activities of mushroom polysaccharide extracts. Life Sci., 60: 763-771.
CrossRefDirect Link

Mackeen, M.M., A.M. Ali, S.H. El-Sharkawy, M.Y. Salleh, N.H. Lajis and K. Kawazu, 1997. Antimicrobial and cytotoxic properties of some Malaysian traditional vegetables (Ulam). Int. J. Pharmacol., 35: 174-178.
CrossRefDirect Link

Mitsuda, H., K. Yasumoto and K. Iwami, 1996. Antioxidative action of indole compounds during the autoxidation of linoleic acid. Nippon Eiyo Shokuryo Gakkaishi, 19: 210-214.
Direct Link

Morton, J.F., 1981. Atlas of Medicinal Plants of Middle America: Bahamas to Yucatan. Charles C. Thomas Publisher, Springfield, Illinois, USA., ISBN-13: 978-0398040369, Pages: 1420.

Nabavi, S.M., M.A. Ebrahimzadeh, S.F. Nabavi and M. Jafari, 2008. Free radical scavenging activity and antioxidant capacity of Eryngium caucasicum Trautv and Froripia subpinata. Pharmacologyonline, 3: 19-25.
Direct Link

Nabavi, S.M., M.A. Ebrahimzadeh, S.F. Nabavi, M. Fazelian and B. Eslami, 2009. In vitro antioxidant and free radical scavenging activity of Diospyros lotus and Pyrus boissieriana growing in Iran. Pharmacogn. Magaz., 5: 122-126.
Direct Link

Oktay, M., I. Gulcin and O.I. Kufrevioglu, 2003. Determination of in vitro antioxidant activity of fennel (Foeniculum vulgare) seed extracts. LWT-Food Sci. Technol., 36: 263-271.
CrossRefDirect Link

Oyaizu, M., 1986. Studies on products of browning reaction-antioxidative activities of products of browning reaction prepared from glucosamine. Jap. J. Nutr. Dietet., 44: 307-315.
Direct Link

Pandey, M.M., R. Govindarajan, A.K. Rawat and P. Pushpangadan, 2005. Free radical scavenging potential of Saussarea costus. Acta Pharm., 55: 297-304.
PubMedDirect Link

Prabuseenivasan, S., M. Jayakumar and S. Ignacimuthu, 2006. In vitro antibacterial activity of some plant essential oil. BMC Complement. Altern. Med., Vol. 6. 10.1186/1472-6882-6-39

Richardson, J.E., M.F. Fay, Q.C. Cronk, D. Bowman and M.W. Chase, 2000. A phylogenetic analysis of Rhamnaceae using rbcL and trnL-F plastid DNA sequences. Am. J. Bot., 87: 1309-1324.
Direct Link

Ruberto, G. and M.T. Baratta, 2000. Antioxidant activity of selected essential oil components in two lipid model systems. Food Chem., 69: 167-174.
CrossRefDirect Link

Ruch, R.J., S.J. Cheng and J.E. Klaunig, 1989. Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis, 10: 1003-1008.
CrossRefDirect Link

Ryan, D., M. Antolovich, P. Prenzler, K. Robards and S. Lavee, 2002. Biotransformations of phenolic compounds in Olea europaea L. Sci. Hortic., 92: 147-176.
CrossRefDirect Link

Sherwin, F.R., 1990. Antioxidants. In: Food Additivies, Branen, R. (Ed.). Marcel Dekker, New York, pp: 139-193.

Shimada, K., K. Fujikawa, K. Yahara and T. Nakamura, 1992. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric. Food Chem., 40: 945-948.
CrossRefDirect Link

Slinkard, K. and V.L. Singleton, 1977. Total phenol analysis: Automation and comparison with manual methods. Am. J. Enol. Viticult., 28: 49-55.
Direct Link

Smith-Palmer, A., J. Stewart and L. Fyfe, 1998. Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Lett. Applied Microbiol., 26: 118-122.
CrossRefPubMedDirect Link

Soare, J.R., T.C.P. Dinis, A.P. Cunha and L. Almeida, 1997. Antioxidant activities of some extracts of Thymus zygis. Free Radic. Res., 26: 469-478.
CrossRefPubMedDirect Link

Stief, T.W., 2003. The physiology and pharmacology of singlet oxygen. Med. Hypotheses, 60: 567-572.
CrossRefPubMedDirect Link

Thaman, R.R., 1992. Batiri Kei Baravi: The ethnobotany of pacific island coastal plants. Atoll Res. Bull., 361: 1-62.
Direct Link

Wang, M., J. Li, M. Rangarajan, Y. Shao, E.J. LaVoie, T.C. Huang and C.T. Ho, 1998. Antioxidative phenolic compounds from sage (Salvia officinalis). J. Agric. Food Chem., 46: 4869-4873.
CrossRefDirect Link

Ye, X.Y., H.X. Wang, F. Liu and T.B. Ng, 2000. Ribonuclease, cell-free translation-inhibitory and superoxide radical scavenging activities of the iron-binding protein lactoferrin from bovine milk. Int. J. Biochem. Cell Biol., 32: 235-241.
CrossRefDirect Link