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Oxidative stress due to reactive oxygen species (ROS) is an important contributor to a variety of pathological conditions such as diabetes and its complications, cardiovascular disorders, atherosclerosis, inflammation, carcinogenesis, drug toxicity, reperfusion injury & several neurodegenerative disorders (Aruoma, 1998). ROS are formed by the reaction between electron acceptors, such as molecular oxygen and excess generated free radicals (Grisham & McCord, 1986). A free radical is any chemical species (highly unstable) possessing one or more unpaired electron (Cheeseman and Slater, 1993; Halliwell, 1992). The ROS include superoxide (O2-), alkoxyl (RO.), peroxyl (ROO.), hydroxyl radicals (OH.), and non-radical derivatives of oxygen, namely, hydrogen peroxide (H2O2) and ozone (O3) ( Grisham & McCord, 1986; Baynes et al, 1991). These ROS further produce several chain reaction processes, causing lipid peroxidation & further biological damage.
Although the human body has multiple enzymatic & non-enzymatic antioxidant mechanisms to protect ROS-mediated cellular damage, yet these innate defense mechanisms are not sufficient for combating the damage caused by severe oxidative stress (Anderson, 1999). In order to maintain adequate levels of antioxidants to scavenge the increased ROS, certain amounts of exogenous antioxidants are required. A large variety of synthetic antioxidants such as butylated hydroxyl anisole (BHA) and butylated hydroxyl toluene (BHT) are available but they are associated with various adverse effects (Anagnostopoulou, Kefalas, Papageorgiou, Assimepoulou & Boskou, 2006). Recently, a great emphasis has been given by the researchers on antioxidants from natural origin that can combat with oxidative stress with minimum side effects & can be proved potential applicants in prevention and/or curing the associated diseases. Currently, much of the research has been focused on antioxidant properties possessed by phenolic compounds found in traditional medicinal plants & a positive correlation was observed between the high phenolic content of plant & strong antioxidant activity (Cai, luo, Sun & Corke, 2004, Djeridane et al, 2006).
Many Ficus species have been traditionally used as astringents, carminatives, stomachics, anthelmintics, vermicides, hypotensives and antidysentery agents (Trivedi et al., 1969). It is believed that some of these Ficus species can also been proved efficacious in certain visceral obstructive and respiratory disorders, diabetes, skin diseases, leprosy etc (Ibn EI Bitar, 1890; Chopra et al, 1950). The bark, fruits & arial roots of Ficus bengalensis L. (banyan tree), roots & fruits of Ficus racemosa L. (Indian fig), bark of Ficus religiosa L. (peepal), are used traditionally for treating diabetes. A hypoglycaemic response is reported for beta-sitosterol-D-glucoside obtained from the bark of Ficus religiosa L. ( ). Leucopelargonin, a flavanoid, isolated from Ficus bengalensis L. was found to be a good hypoglycemic & antioxidant (Brahmachari and Augusti, 1964; Daniel, Devi, Augusti, & Sudhakaran Nair, 2003). The stem bark of Ficus racemosa L. is also reported to have antioxidant properties (Manian et al, 2008). Ficus bengalensis, Ficus religiosa, Ficus glomerata and Ficus virens are the important components of a reputed ayurvedic formulation, Panchvalkala which is also known for its free radical scavenging properties (Anandjiwala et al, 2008).
In the present study, comparative antioxidant activities of these five ficus species have been thoroughly evaluated using different invitro methods.
2. Materials and methods
2.1 Plant Material
2.3 Estimation of Total Phenolic Content
Estimation of the total phenolic content was conducted using a method described by Singleton and Rossi (1965). This assay was performed using Folin-Ciocalteu agent.
10mg of standard gallic acid was dissolved in 100ml of distilled water to make 100Âµg/ml of stock solution. From the above stock solution, 0.5-2.5ml of aliquots were taken in 25 ml of volumetric flasks. 10 ml of distilled water and 1.5 ml of Folin-Ciocalteu agent were added to each of the above volumetric flasks with thorough mixing. After 5min, 4ml of 20% sodium carbonate solution was added. The mixture was then diluted to 25ml with the addition of distilled water and incubated at room temperature for 30 min. Finally, the absorbance was measured using UV-Visible spectrophotometer at 765 nm and a standard curve of absorbance vs concentration of gallic acid (50-250Âµg) was plotted.
The reaction mixtures of the extracts were prepared by the method similar to the standard gallic acid and the total phenolic content was expressed as percentage of gallic acid.
2.4 Total flavanoids assay
Total flavanoids assay was conducted according to the method described by Marinova et al. (2005) using Aluminium Chloride Colorimetric method. 1ml of the bark extract was added with 4 ml of distilled water and 0.3 ml 5% NaNO2 in a conical flask. After 5 min, 0.3 ml of 10% AlCl3 was also added to the reaction mixture. At the 6th min, 2 ml of 1 M NaOH was added immediately to this mixture. Then the reaction mixture was diluted to 10 ml by adding 2.4 ml distilled water. The mixture was mixed thoroughly and the absorbance was measured at 510 nm. The calibration curve was plotted by estimating the absorbance of quercetin solutions at various concentrations in distilled water (50-250 Âµg). Total flavanoids content was expressed as percentage of catechin.
2.5 Invitro Antioxidant Assays
2.5.1 DPPH (1, 1-Diphenyl-2-picryl-hydrazil) Free Radical Scavenging Activity
The free radical scavenging activity of the aqueous extract of Ficus bengalensis (AQFS) was determined with the help of DPPH using the method of Shimada et al (1992). An ethanolic solution of DPPH (1ml, 0.1mM) was mixed with serial dilutions of AQFS (10-50 Âµg/ml). The mixture was shaken vigorously and kept at room temperature for 30 min. After the mixture had reached equilibrium, the absorbance of the solution was measured spectrophotometrically at 517nm by using a UV-Visible Spectrophotometer (Cystronics UV-Vis 2201). Ascorbic acid was used as the positive control. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The percent DPPH scavenging effect was calculated using the formula:
% DPPH Scavenging Effect = [(A0-A1)/A0] Ã- 100
Where, A0 =Absorbance of Control Reaction mixture
A1= Absorbance of Standard sample or AQFS
2.5.2 Nitic Oxide Radical Inhibition Assay
Nitric oxide radical scavenging activity can be estimated by Griess-Illosvoy reaction( ) with slight modification. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions, which can be determined by the use of the Griess Illosvoy reaction.
In the present study, the Griess- Illosvoy reagent, 1-napthylamine (5%) was replaced with naphthyl ethylene diamine dihydrochloride (0.1% w/v). The reaction mixture containing sodium nitroprusside (10mM, 2ml) in 0.5 ml phosphate buffer saline (pH 7.4) was mixed with 0.5ml of AQFS at different concentrations (25 to 250Âµg/ml) or with 0.5ml of the standard solution (BHT and Ascorbic acid). This mixture was incubated at 25°C for 150 min. After incubation, 0.5ml of the reaction mixture was taken out and added into 1.0 ml sulfanilic acid reagent (33% in 20% glacial acetic acid) and allowed to stand for 5 min for completing diazotization. Finally, 1.0 ml naphthylethylenediamine dihydrochloride (0.1% w/v) was mixed and incubated at room temperature for 30 min. The absorbance at 540 nm was measured with a spectrophotometer. The nitric oxide radicals scavenging activity was calculated according to the following equation:
% Inhibition = ((A0-A1) / A0 Ã- 100)
Where, A0 =Absorbance of Control Reaction mixture
A1= Absorbance of Standard sample or AQFS
2.5.3 Scavenging of Hydrogen Peroxide
The ability of AQFS to scavenge hydrogen peroxide (H2O2) was determined by the method of Ruch et al (1989). A solution of hydrogen peroxide (43mM) was prepared in phosphate buffer (ph 7.4). Ascorbic acid and BHT were used as control and AQFS extract solutions were prepared at different concentrations of 25-250 Âµg/ml in distilled water and added to 0.6ml of H2O2 solution. The reaction mixture was incubated at room temperature for 10 min and the absorbance of H2O2 was determined at 230nm.The percentage of scavenging was calculated as follows:
% Scavenged [H2O2] = [(A0-A1)/A0] Ã- 100
where, A0 =Absorbance of Control Reaction mixture
A1= Absorbance of Standard sample or AQFS.
Total Reduction Capability
Fe3+ total reduction capability of AQFS was determined by the method described previously (Oyaizu 1986). AQFS (25-250Âµg/ml) was mixed thoroughly with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferrocyanide [K3Fe (CN) 6] (2.5 ml, 1%). This reaction mixture was then incubated at 50°C for 20 min by adding a 2.5ml of 10% trichloroacetic acid. The mixture was centrifuged at 3000 rpm for 10 min. The upper layer of the 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 700nm by a spectrophotometer (Cystronics UV-Vis 2201). Butylated Hydroxy Toluene (BHT) and ascorbic acid were used as a positive control. Increased absorbance of the reaction mixture indicated higher reducing power.
Antioxidant Activity in Linoleic Acid Emulsion System
The total antioxidant activity of the plant extracts was measured by thiocyanide method (Kikuzaki & Nakatani, 1993). 250 Âµg of each sample in 0.5ml of absolute ethanol was mixed with 0.5ml of 2.51% linoleic acid, 1ml of 0.05M phosphate buffer (pH 7), and 0.5 ml of distilled water and finally placed in a screw capped tube. The reaction mixture was then incubated in dark at 40 °C in an oven for 48h. During incubation, 0.1 ml of the solution was taken at every 12 h interval and the degree of oxidation was measured by sequentially adding 9.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate and 0.1 ml of ferrous chloride (0.02 M in 3.5% HCl). After 3 min, the peroxide value of the mixture was monitored at 500nm until the absorbance of the control reached the maximum. The antioxidant activity was calculated as percentage of inhibition relative to the control.
Antioxidant Activity= 100- (Sample absorbance at 48h-Sample absorbance at 0 h /Control absorbance at 48h-Control absorbance at 0 h) Ã- 100
2.6 Statistical Analysis
The experimental results are expressed as mean Â± standard deviation(SD) of the triplicate measurments. One Way ANOVA with Duccan's multiple range test was used for evaluating statistical significance between different groups and values with p<0.001 were considered to be statistically significant. All statistical analyses have been carried out with the help of Sigmastat 2.03 software and Instat software.
3. Results and Discussion
The preliminary phytochemical evaluation of the extracts showed the presence of