Evaluation of in vitro antioxidant activity of bark extracts of Terminalia arjuna

To uncover the antioxidant and free radical scavenging activity, five different extracts of Terminalia arjuna bark were examined. In the present study, the free radical scavenging potential of five extracts of the bark of Terminalia arjuna was assessed by measuring its capability for scavenging 2, 2-diphenyl-1picrylhydrazyl (DPPH) radical, hydrogen peroxide radical, nitric oxide radicals (NO), as well as its ability in reducing power capacity assessment, cupric reducing antioxidant capacity, using appropriate assay systems compared to natural and synthetic antioxidants. Total antioxidant capacity, phenolic and flavonoid contents were determined spectrophotometrically. In DPPH free radical scavenging activity, the highest IC50 value was showed by methanol extract with a value of 6.34 μg/ml followed by ethanol and petroleum ether having value of 7.76 and 25.63, respectively, as opposed to that of the scavenging effects of ascorbic acid and butylated hydroxytoluene (BHT) of 5.698 and 8.816, respectively. Methanol extract showed highest activity having IC50 value of 14.436 and 25.184 μg/ml in hydrogen peroxide and nitric oxide scavenging assay, respectively. All the five fractions showed good reducing power and cupric reducing capacity with increasing concentration again taking methanol extract to the top position. The methanol extract yielded 817.488 ± 8.108 mg/g gallic acid equivalent phenolic content and 199.122 ± 8.282 mg/g Quercetin equivalent flavonoid content that was highest among five extracts. Methanol extract of T. arjuna was found to possess the highest total antioxidant capacity (415.925 ± 2.291) followed by ethanol (377.675 ± 1.889) mg/g Ascorbic Acid Equivalent, respectively. A linear correlation appeared between the total antioxidant capacity and the total phenolic contents of the extracts with good correlation coefficient (R 2 = 0.891). n-Hexane and chloroform extract showed least activity in all the measures. The results obtained beacon that T. arjuna is a potential source of antioxidants and thus could prevent many radical related diseases.


INTRODUCTION
Terminalia arjuna (Bengali name: Arjun gachh, English name: Arjuna myrobalam) from Combretaceae family is a large tree which is found throughout the South Asian region.This tree is usually an evergreen tree with new leaves appearing in the hot season (February to April) before leaf fall.This tree is an exotic tree in Bangladesh.It is one of the most versatile medicinal plants having a Most of the antioxidant compounds in a typical balanced diet are derived from plant sources with a wide variety of biological and chemical properties (Scalbert et al., 2005).Synthetic antioxidants like butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been used as food additives; but recent reports have expressed safety concerns allowing natural antioxidant to be the focus of intense interest (Sun and Ho, 2005;Wilson, 1999).Plants are rich sources for natural antioxidants, the best known are tocopherols, flavonoids, vitamin C and other phenolic compounds (Landrault et al., 2001).Other contributors to the antioxidant activity include alkaloids, proteins, minerals and other vitamins such as the carotenoids and vitamin B 6 , B 12 and K (Smolin and Grosvenor, 2007).Antioxidant compounds like phenolic acids, polyphenols and flavonoids scavenge free radicals such as peroxide, hydroperoxide of lipid hydroxyl and thus inhibit the oxidative mechanisms that lead to degenerative diseases (Subramanion et al., 2011).There are a number of clinical studies confirming the powerful anti-cancerous and anti heart disease properties of polyphenols (Prakash et al., 2007;Bajpai et al., 2005;Siddhuraju and Becker, 2003).
In oxidation process, highly reactive and harmful chain reactions of oxygen species are generated, which cause damage to living organism.The oxygen centered free radicals and other reactive oxygen species, which are continuously produced, resulted in cell death or tissue damage.This oxidative damage caused by free radical is related to pathogenesis of many chronic degenerative diseases like cancer, diabetes, neurodegenerative disease, atherosclerosis, cirrhosis, malaria and AIDS (Azizova et al., 2002;Sian et al., 2003;Quintero et al., 2006;Nagler et al., 2006).Reactive oxygen species (ROS), including superoxide free radical, hydrogen peroxide, hydroxyl free radical and singlet oxygen play a key role in the oxidative damage of these diseases (Vertuani et al., 2004).
Antioxidant is a molecule, which terminate the chain reaction by removing free radical intermediates.Plants and animals maintain complex system of multiple type of antioxidant; the natural plant based antioxidants are playing an important role in the maintenance of human health for the past three decades (Devasagayam et al., 2004).There is an increasing interest in antioxidants, particularly in those intended to prevent the presumed deleterious effects of free radicals in the human body and to prevent the deterioration of fats and other constituents of foodstuffs.In both cases, there is a preference for antioxidants from natural rather than from synthetic sources (Abdalla and Roozen, 1999).There is therefore a parallel increase in the use of methods for estimating the efficiency of such substances as antioxidants (Sa'nchez-Moreno, 2002).Therefore, the present study was undertaken to evaluate and compare the antioxidative activities of different solvent extracts of T. arjuna in different methods.

Plant material
Plant sample of T. arjuna was collected from Savar in July 2011 and a plant sample was submitted to The National Herbarium, Mirpur for identification (DACB; Accession number 35904; dated on 14.09.2011;Sadika Akter, no.SA-01).

Sample preparation
Barks were sun dried for four days and then dried in oven at 40°C for about three days.The dried barks were then ground in coarse powder using high capacity grinding machine which was then stored in air-tight container with necessary markings for identification and kept in cool, dark and dry place for the investigation.The powdered plant material was successively extracted in a Soxhlet extractor at elevated temperature using n-hexane which was followed by petroleum ether, chloroform, ethanol and methanol.All extracts were filtered individually through filter paper and poured on Petri dishes to evaporate the liquid solvents from the extract to get dry extracts.After drying, crude extracts were stored in stock vials and kept in refrigerator for further use.

2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay
The free radical scavenging capacity of the extracts was determined using DPPH (Braca et al., 2001).Freshly prepared DPPH solution was taken in test tubes and extracts were added followed by serial dilutions (15.625 to 250 µg/ml) to every test tube so that the final volume was 5 ml and after 30 min, the absorbance was read at 517 nm using a spectrophotometer.Ascorbic acid and BHT was used as standard.Control sample was prepared containing the same volume without any extract and standard, and the absorbance was read at 517 nm using a spectrophotometer.Methanol was served as blank.% inhibition of the DPPH free radical was measured using the following equation: Where, Al = Absorbance of the extract or standard and A0 = Absorbance of the control.

Scavenging of hydrogen peroxide
Scavenging activity of extract and its sub-fractions were evaluated by hydrogen peroxide (Jayaprakasha et al., 2004). 1 ml of various concentrations of the extract, sub-fractions and standards in ethanol was added to 2 ml of hydrogen peroxide solution in phosphate buffered saline (PBS, pH 7.4).Then finally the absorbance was measured at 230 nm after 10 min.Ascorbic acid and BHT were used as standard.Control sample was prepared containing the same volume without any extract and standard and the absorbance was read at 230 nm using a spectrophotometer.The percentage inhibition was calculated according to the following equation: Where, Ao = absorbance of the control and A1 = absorbance in the presence of the sample of extract and standard.

Nitric oxide scavenging capacity assay
Nitric oxide scavenging assay was carried out using sodium nitroprusside (Sreejayan and Rao, 1997).This can be determined by the use of the Griess Illosvoy reaction.2 ml of 10 mM sodium nitroprusside in 0.5 ml phosphate buffer saline (pH 7.4) was mixed with 0.5 ml of extract/sub-fraction at various concentrations and the mixture was incubated at 25°C for 150 min.From the incubated mixture, 0.5 ml was taken out and added into 1.0 ml sulphanilamide solution (0.33% in 20% glacial acetic acid) and further incubated at room temperature for 5 min.
Finally, 1.0 ml naphthylethylenediamine dihydrochloride (0.1% w/v) was mixed and maintained at room temperature for 30 min.The absorbance was measured at 546 nm.A typical blank/control solution contained the same solution mixture without plant extract or standard.The absorbance of the blank/control solution was measured at 546 nm.The percentage inhibition was calculated according to the following equation: Where, Al = Absorbance of the extract or standard and A0 = Absorbance of the control.

Reducing power capacity assessment
Assay of reducing power was carried out by potassium ferricyanide method (Yildirim et al., 2001). 1 ml of extract and its sub-fractions (final concentration 5 to 200 µg/ml) were mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide.The mixture was then incubated at 50°C for 20 min.To this mixture, 2.5 ml of trichloroacetic acid was added, which was then centrifuged at 3000 rpm for 30 min.Finally, 2.5 ml of the supernatant solution was collected and mixed with 2.5 ml of distilled water and 0.5 ml ferric chloride and absorbance was measured at 700 nm.Ascorbic acid and butylated hydroxy toluene (BHT) were used as standard and phosphate buffer as blank solution.

Cupric reducing antioxidant capacity (CUPRAC)
The assay was conducted as described previously by Resat et al. (2004).0.5 ml of plant extract or standard of different concentrations solution, 1 ml of copper (II) chloride solution (0.01 M prepared from CuCl2.2H2O), 1 ml of ammonium acetate buffer at pH 7.0 and 1 ml of neocaproin solution (0.0075 M) were mixed.The final volume of the mixture was adjusted to 4.1 ml by adding 0.6 ml of distilled water and the total mixture was incubated for 1 h at room temperature.
Then the absorbance of the solution was measured at 450 nm using a spectrophotometer against blank.Ascorbic acid, BHT and BHA were used as a standard.

Determination of total antioxidant capacity
The total antioxidant capacity was evaluated by the phosphomolybdenum method (Prieto et al., 1999).0.3 ml of extract and sub-fraction in ethanol, ascorbic acid used as standard (5 to 200 µg/ml) and blank (ethanol) were combined with 3 ml of reagent mixture separately and incubated at 95°C for 90 min.After cooling to room temperature, the absorbance of each sample was measured at 695 nm against the blank.The antioxidant activity is expressed as the number of equivalents of ascorbic acid and was calculated by the following equation: Where, A = total content of antioxidant compounds, mg/g plant extract, in Ascorbic Acid Equivalent, c = the concentration of Ascorbic acid established from the calibration curve, mg/ml, V = the volume of extract (ml), and m = the weight of crude plant extract (g).

Determination of total phenolic content
Total phenolic contents in the extracts were determined by the Folin-Ciocalteu reagent method described by Demiray et al. (2009). 1 ml of the plant extracts/standard of different concentration solution was mixed with 5 ml Folin-Ciocalteu reagent (previously diluted with water 1:10 v/v) and 4 ml (7.5% sodium carbonate) of sodium carbonate.The tubes were vortexed for few seconds and allowed to stand for 30 min at 20°C for color development.Absorbance of samples and standard were measured at 765 nm using spectrophotometer against blank.A typical blank solution contained the solvent used to dissolve the plant extract.The total content of phenolic compounds plant extracts in gallic acid equivalents (GAE) was calculated using the following equation: Where C = total content of phenolic compounds, mg/g plant extract, in GAE, c = the concentration of gallic acid established from the calibration curve (mg/ml), V = the volume of extract in ml, and m = the weight of crude plant extract in g.

Determination of total flavonoids content
Aluminum chloride colorimetric method was used for flavonoids determination (Wang and Jiao, 2000). 1 ml of the plant extracts/standard of different concentration solution was mixed with 3 ml of methanol, 0.2 ml of aluminum chloride, 0.2 ml of 1 M potassium acetate and 5.6 ml of distilled water.It remained at room temperature for 30 min; the absorbance of the reaction mixture was measured at 415 nm with spectrophotometer against blank.Methanol served as blank.The total content of flavonoid compounds in plant methanol extracts in quercetin equivalents was calculated by the following equation: Where C = total content of flavonoid compounds, mg/g plant extract, in quercetin equivalent, c = the concentration of quercetin established from the calibration curve in mg/ml, V = the volume of extract in ml, and m = the weight of crude plant extract in g.

DPPH free radical scavenging assay
The free radical scavenging activity of different extracts of T. arjuna was studied by its ability to reduce the DPPH, a stable free radical and any molecule that can donate an electron or hydrogen to DPPH can react with it and thereby bleach the DPPH absorption.DPPH is a purple colour dye having absorption maximal of 517 nm and upon reaction with a hydrogen donor the purple colour fades or disappears due to conversion of it to 2, 2diphenyl-1-picryl hydrazine resulting in decrease in The DPPH test showed the ability of the test compound to act as a free radical scavenger.DPPH assay method is based on the ability of 1, 1-diphenyl-2-picrylhydrazyl (DPPH), a stable free radical, to decolorize in the presence of antioxidants (Kumarasamy et al., 2007).DPPH, a protonated radical, has characteristic absorbance maximal at 517 nm, which decreases with the scavenging of the proton radical.This property has been widely used to evaluate the free radical scavenging effect of natural antioxidants (Jao and Ko, 2002).The stable free radical DPPH has been widely used to test the free radical-scavenging ability of various dietary antioxidants (Brand--Williams et al., 1995).Because of its odd electron, DPPH gives a strong absorption band at 517 nm in visible spectroscopy.As this electron becomes paired off in the presence of a free radical scavenger, the absorption vanishes, and the resulting decolourization is stoichiometric with respect to the number of electrons taken up.The bark extracts showed considerable radical scavenging activity in a concentration-dependent manner.Methanol (6.34), ethanol (7.76), petroleum ether (25.63) and chloroform (61.89) extract of the dried bark exhibited a good potential to act as a free radical scavenger with IC 50 for DPPH inhibition comparable to Concentration (µg/ml)  that of ascorbic acid (5.698 µg/ml) and BHT (8.816 µg/ml) which are known free radical scavengers.Figure 1 exhibits the comparative % inhibition among T. arjuna bark extracts and standard compounds (ascorbic acid and butylated hydroxytoluene).The highest scavenging effect was showed by methanol extract with a value of 6.34 µg/ml followed by ethanol and petroleum ether having value of 7.76 and 25.63, respectively as opposed to that of the scavenging effects of ascorbic acid and BHT of 5.698 and 8.816, respectively.These results indicated that extract has a noticeable effect on scavenging the free radicals.In fact, IC 50 value of methanol and ethanol extract appeared to have slightly better than standard BHT at 50% inhibition.However, a maximum inhibition was achieved at a higher concentration of 250 µg/ml compared to 250 µg/ml for both of ascorbic acid and BHT respectively.

Scavenging of hydrogen peroxide
Hydrogen peroxide, although not a radical species play a role to contribute oxidative stress.The generation of even low levels of H 2 O 2 in biological systems may be important.Naturally-occurring iron complexes inside the cell believed to react with H 2 O 2 in vivo to generate highly reactive hydroxyl radicals and this may be the origin of many of its toxic effects (Miller et al., 2000).Scavenging of hydrogen peroxide of different extracts of T. arjuna bark is presented in Figure 2. All five extracts compared with standard ascorbic acid IC 50 value of 7.529 µg/ml and BHT IC 50 value of 6.387 µg/ml along with methanol and ethanol in highest position in depleting H 2 O 2, with an IC 50 value of 14.436 and 18.867 µg/ml followed by petroleum ether 48.706 µg/ml, respectively (Table 2).The percentage of H 2 O 2 scavenging activity of methanol extract was found to be 79.550% which is highest among five extract at 200 µg/ml compared to antioxidant activity of standard ascorbic acid and BHT was 83.538 and 89.570%, respectively at the same concentration.Ethanol and petroleum ether extract also showed significant activity with a scavenging value of 73.824 and 65.235%, respectively (Figure 2).
Hydrogen peroxide itself is not very reactive, but can sometimes be toxic to cell because it may give rise to hydroxyl radical in the cells (Halliwell et al., 1987).Thus, removal of H 2 O 2 is very important for protection of food systems.Scavenging of H 2 O 2 by extracts may be attributed to their phenolics, which can donate electrons to H 2 O 2 , thus neutralizing it to water.The extracts were capable of scavenging hydrogen peroxide in a concentration-dependent manner.The IC 50 values were found to be 14.436, 18.867 and 48.706 µg/ml for methanol, ethanol and petroleum ether, in comparison with 7.529 and 6.387 µg/ml for ascorbic acid and BHT, respectively.Chloroform and n-hexane extract showed moderate scavenging activity with an IC 50 value of 75.907 and 78.429 µg/ml, respectively.

Nitric oxide (NO) radical scavenging assay
NO is a very unstable species and reacting with oxygen molecule produce stable nitrate and nitrite which can be estimated by using Griess reagent.In the presence of a scavenging test compound, the amount of nitrous acid will decrease which can be measured at 546 nm.
Methanol and ethanol extract of T. arjuna bark has potent nitric oxide scavenging activity (IC 50 value 25.184 and 25.420 µg/ml, respectively ) and n-hexane fraction has showed the least nitric oxide scavenging activity (IC 50 value 903.859 µg/ml) (Table 3).
NO is a potent pleiotropic inhibitor of physiological processses such as smooth muscle relaxation, neuronal signaling, inhibition of platelet aggregation and regulation of cell mediated toxicity.It is a diffusible free radical that plays many roles as an effectors molecule in diverse biological systems including neuronal messenger, vasodilatation and antimicrobial and anti-tumor activities (Hagerman et al., 1998).Suppression of released NO may be partially attributed to direct NO scavenging, as the extracts of T. arjuna bark decreased the amount of nitrite generated from the decomposition of SNP in vitro.The scavenging of NO by the extracts was increased in dose dependent manner.Figure 3 illustrates a decrease in the NO radical due to the scavenging ability of extracts such as percent inhibition of methanol > ethanol > petether > n-hexane > chloroform.The methanol and ethanol extracts showed maximum activity of 81.08 and 79.88%, respectively at 100 µg/ml, whereas ascorbic acid and BHT at the same concentration exhibited 76.65 and 80.84% inhibition, respectively.The IC 50 values were found to be 25.184, 25.420, 57.141, 27.685 and 27.294 µg/ml for methanol, ethanol, petroleum ether, ascorbic acid and BHT, respectively.In fact, IC 50 value of methanol (25.184) and ethanol (25.420) extract appeared to be slightly better than standard ascorbic acid (27.685) and BHT (27.294) at 50% inhibition.However, a maximum inhibition was achieved at a higher concentration of 100 µg/ml compared to 100 µg/ml for both ascorbic acid and Concentration (µg/ml) BHT, respectively.Hence, a higher concentration would be required to achieve maximal and a significantly higher content of phenolic compounds compared to chloroform extract and n-hexane extract.NO has shown to play a crucial role in various physiological processes such as smooth muscle relaxation, neuronal signaling, inhibition of platelet aggregation and regulation of cell mediated toxicity.It is a diffusible free radical, which plays many roles as an effector molecule in diverse biological systems including neuronal messenger, vasodilatation, antimicrobial and antitumor activities (Hagerman et al., 1998).However, excess production of NO is associated with several diseases (Lalenti et al., 1992).

Reducing power capacity assessment
Reducing power of the fractions was assessed using ferric to ferrous reducing activity as determined sectrophotometrically from the formation of Perl's Prussian blue colour complex (Yildirim et al., 2001).Reducing power of different extracts of T. arjuna was compared with ascorbic acid and BHT (Figure 4).Among the extracts, methanol and ethanol extract exhibited the most reducing power which was even more than ascorbic acid and BHT.This result indicates that the extracts may consist of polyphenolic compounds that usually show great reducing power.This has been justified by methanol and ethanol extract being the most reducing agent with highest phenolic content (Table 5).

Cupric reducing antioxidant capacity (CUPRAC)
Reduction of Cu 2+ ion to Cu + was found to rise with increasing concentrations of the different extracts.The standard BHA and BHT showed highest reducing capacity.Among the extracts, methanol and ethanol extracts of T. arjuna showed maximum reducing capacity that is comparable to BHA and BHT.But the reducing capacity of methanol and ethanol extracts was more than ascorbic acid (Figure 5).
This method is based on the principle of increase in the absorbance of the reaction mixtures.Increase in the absorbance indicates increase in the antioxidant activity.Increase in absorbance of the reaction mixture indicates the reducing power of the samples (Jayaprakasha et al., 2001).Reducing power is associated with antioxidant activity and may serve as a significant reflection of the antioxidant activity (Oktay et al., 2003).Compounds with reducing power indicate that they are electron donors and can reduce the oxidized intermediates of lipid peroxidation processes, so that they can act as primary and secondary antioxidants (Yen and Chen, 1995).

Determination of total antioxidant capacity
Total antioxidant capacity of the different extracts of T. arjuna was evaluated by the phosphomolybdenum method and was expressed as ascorbic acid equivalents (AAE) per gram of plant extract.Total antioxidant capacity of the test samples was calculated using the standard curve of ascorbic acid (y = 0.002x + 0.001; R 2 = 0.997) (Figure 6).Methanol extract of T. arjuna was found to possess the highest total antioxidant capacity (Table 4).Total antioxidant capacity of the extracts was found to decrease in the following order: Methanol extract > ethanol extract > petroleum ether extract > chloroform extract > n-hexane extract (Table 4) and a comparative study is shown in Figure 7.
The total antioxidant activity of different extracts of T. arjuna was estimated from their ability to reduce Phosphate/Mo (VI) complex to Phosphate/Mo (V).The  assay has been successfully used to quantify vitamin E in seeds; and being simple and independent of other antioxidant measurements commonly employed, it was decided to extend its application to plant extracts (Prieto et al., 1999).Moreover, it is a quantitative one, since the antioxidant activity is expressed as the number of equivalents of ascorbic acid.According to recent reports, a highly positive relationship between total phenols and antioxidant activity appears to be the trend in many plant species (Oktay et al., 2003).The statement has been justified in the current study where the methanol extract of T. arjuna showed maximum total antioxidant capacity (in term of ascorbic acid equivalent) (Table 4) with maximum phenol content (Table 5).

Determination of total phenolic content
Total phenolic content of the different extracts of T. arjuna was determined by using the Folin-Ciocalteu reagent and were expressed as GAE per gram of plant extract.The total phenolic contents of the test fractions were calculated using the standard curve of gallic acid (y = 0.0136x + 0.0371; R 2 = 0.9948) (Figure 8).Methanol extract of T. arjuna was found to contain the highest amount of phenols (Table 5) and a comparative study is shown in Figure 9. Phenol contents of the extracts were found to decrease in the following order: methanol extract > ethanol extract > petroleum ether extract > chloroform extract > n-hexane extract (Table 5).
Phenolic, ubiquitous to the plant kingdom are composed of several classes of compounds including flavonoids (flavones, isoflavones and flavonones), anthocyanins and catechins.They possess an ideal structural chemistry for free radical scavenging activity.Antioxidative properties of polyphenols arise from their high reactivity as hydrogen or electron donors which can stabilize and delocalize the unpaired electron (chain-breaking function) and from their potential to chelate metal ions (termination of the Fenton reaction) (Rice-Evans et al., 1997).The results strongly suggest that phenolics are important components of the tested plant extracts.Among the extracts, the methanol extract of T. arjuna showed most potency in phenolic content determination assay (Table 5) and although a crude extract, it possesses considerable amount of gallic acid equivalent substances (polyphenolic compounds).

Relationship between the total antioxidant capacity and the total phenolic content
The extent of antioxidant capacity of extract of T. arjuna was correlated with their total phenolic contents.The correlation coefficient between the total antioxidant capacity monitored by phosphomolybdenum method and the total phenolics contents of extracts were determined (Figure 10).A linear correlation appeared between the total antioxidant capacity and the total phenolic contents of the extract and fractions with good correlation coefficient (R 2 = 0.995).The results suggested that the phenolic compounds contributed significantly to the antioxidant capacity of the T. arjuna extracts.

Determination of total flavonoids content
Aluminium chloride colorimetric method was used to determine the total flavonoid contents of the different extracts of T. arjuna.Total flavonoid contents was calculated using the standard curve of quercetin (y = 0.0149x -0.0536; R 2 = 0.9975) (Figure 11) and was expressed as quercetin equivalents (QE) per gram of the plant extract.
Methanol extract of T. arjuna was found to contain the highest amount of flavonoid (Table 6) and a comparative study is shown in Figure 12.Flavonoid contents of the extracts were found to decrease in the following order: methanol extract > ethanol extract > petroleum ether extract > chloroform extract > n-hexane extract (Table 6).
Flavonoids play an important role in antioxidant system in plants.The antioxidative properties of flavonoids are due to several different mechanisms, such as scavenging of free radicals, chelation of metal ions, such as iron and copper and inhibition of enzymes responsible for free radical generation (Benavente-Garcia et al., 1997).Depending on their structure, flavonoids are able to scavenge practically all known ROS.The methanol extract of T. arjuna has been shown to possess the highest flavonoids (Table 6).According to our study, the high contents of these phytochemicals in T. arjuna can explain its high radical scavenging activity.

Conclusion
Based on the results of the present study, it can be suggested that the extracts of T. arjuna possess antioxidant effects.Almost all extracts exhibited potential antioxidant activity.Methanol and ethanol extracts showed highest antioxidant activity.

Figure 10 .Figure 11 .
Figure 10.Correlation between the total antioxidant capacity and the total phenolic contents of Terminalia arjuna extracts.

Figure 12 .
Figure 12.Total flavonoid contents of the different extracts of Terminalia arjuna.

Table 1 .
IC50 values of different extracts of Terminalia arjuna in DPPH scavenging assay.

Table 2 .
IC50 values of different extracts of Terminalia arjuna in H2O2 scavenging assay.

Table 3 .
IC50 values of different extracts of Terminalia arjuna bark in Nitric oxide scavenging assay.

Table 4 .
Total antioxidant capacity of the different extracts of Terminalia arjuna.

Table 5 .
Total phenol contents of the different extracts of Terminalia arjuna.

Table 6 .
Total flavonoid contents of the different extracts of Terminalia arjuna.Values are the mean of duplicate experiments and represented as mean ± SD.