Phloretin was purchased from sigma chemicals company (St. Louis, MO, USA). Nitro blue tetrazolium (NBT), Ethylene diamine tetra acetic acid (EDTA), Trichloro acetic acid (TCA), Thiobarbituric acid (TBA), 1,1- diphenyl-2-picrylhydrazyl (DPPH), and Potassium hexa cyano ferrate [K₃Fe(CN)₆] were used in this study are of analytical grade and were purchased from Himedia Laboratories, Bangalore, India.

The free radical scavenging activity of phloretin was measured by DPPH using the method of Shimada et al. [17] . 0.1 mM solution of DPPH^• in ethanol was prepared and 1ml of this solution was added to 3 ml of phloretin solution at different concentrations like 20, 40, 60 and 80 µg/ml. The mixture was shaken vigorously and allowed to stand for 30 min at room temperature and then the absorbance was measured at 517 nm using spectrophotometer. Lower absorbance of the reaction mixture indicated that the higher free radical scavenging activity. The DPPH free radical scavenging activity was calculated by the following formula.

where A control is the absorbance of control reaction (containing all reagents except the test compound), and A sample is the absorbance of test compound.

The total antioxidant activity of the phloretin was evaluated by the phosphomolybdenum method according to the procedure of Prieto et al. [18] . The assay is based on the reduction of Mo(VI)−Mo(V) by phloretin and subsequent formation of a green phosphate/Mo(V) complex at acidic pH. 0.3 ml of phloretin solution at different concentrations like 20, 40, 60 and 80 µg/ml was mixed with 3 ml of reagent (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction mixture were incubated at 95˚C for 90 min. After cooling to room temperature, the absorbance of the solution was measured at 695 nm using spectrophotometer against blank. The antioxidant activity was expressed as the number of equivalents of ascorbic acid. The scavenging activity was calculated according to the following equation.

where A control is the absorbance of control reaction (containing all reagents except the test compound), and A sample is the absorbance of test compound

Measurement of superoxide anion scavenging activity of phloretin was carried out based on the method described by Liu et al. [19] . Superoxide radical is generated in PMS-NADH systems by oxidation of NADH and assayed by the reduction of nitroblue tetrazolium (NBT). In this experiment, the superoxide radical was generated in 1 ml of Tris-HCl buffer (16 mM, pH 8.0), 1ml of NBT (50 µM), 1ml of NADH (78 µM) and sample solution at different concentrations like 20, 40, 60 and 80 µg/ml. The reaction was started by the addition of 1ml of phenazine methosulphate (10 µM PMS) to the mixture. The reaction mixture was incubated at 25˚C for 5 min, and the absorbance was measured against blank at 560 nm using spectrophotometer. Ascorbic acid was used as a standard. Decrease in absorbance of the reaction mixture indicated that the increased superoxide anion scavenging activity. The percentage inhibition of superoxide anion generation was calculated by the following formula.

where A control is the absorbance of control reaction (containing all reagents except the test compound) and A sample is the absorbance of test compound.

The chelation of ferrous ions by phloretin and standard ascorbic acid was estimated by the method of Dinis et al. [20] . The sample solution at different concentrations like 20, 40, 60 and 80 µg/ml was added to 0.05 ml of 2 mM FeCl₂. The reaction was initiated by the addition of 5mM ferrozine (0.2 ml) and the mixture was vigorously shaken and left it stands for 10 min at room temperature. The absorbance of the solution was measured at 562 nm using spectrophotometer. The percentage of inhibition of Fe²⁺-ferrozine complex formation was calculated by the following formula.

where A control is the absorbance of control reaction (containing all reagents except the test compound) and A sample is the absorbance of test compound.

The reducing power of phloretin was determined by the method of Oyaizu [21] . 1ml of phloretin at different concentrations like 20, 40, 60 and 80 µg/ml of distilled water was 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 and then 2.5 ml of TCA (10%) was added to the mixture, which was centrifuged at 3000 rpm for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 0.1%), and then the absorbance was measured at 700 nm using spectrophotometer. Higher absorbance of the reaction mixture indicated that the greater reducing power. Ascorbic acid was used as a standard.

All the tests in this study were carried out in triplicates and the results were expressed as mean ± SD. The free radical scavenging activity of phloretin was expressed as percentage (%). Linear regression analysis was used to calculate IC₅₀ values using Microsoft Office Excel.

The model of scavenging the stable DPPH radical is a widely used method to evaluate antioxidant activity in a short time compared with other methods. The effect of antioxidants on DPPH radical scavenging was thought to be due to their hydrogen donating ability. DPPH^• is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule [23] . Phloretin at four different concentrations like 20, 40, 60 and 80 µg/ml was used for the determination of free radical scavenging activity by DPPH method and it was compared with ascorbic acid as standard compound. The highest level of DPPH radical scavenging activity of phloretin 86.81% and ascorbic acid 99.34% was found at 80 µg/ml and the results were summarized in Table 1 and graphically represented in Figure 1. The scavenging effect was increased with the increasing concentrations

Values are expressed as mean ± SD of triplicates.

of test compound phloretin. The IC₅₀ value for phloretin and ascorbic acid was 48.56 μg/ml and 34.91 μg/ml, respectively. From the results, ascorbic acid displays more scavenging activity over the phloretin. This might be due to the presence of methoxy group which increases the accessibility of radical center of DPPH to ascorbic acid [24] .

Free radical scavenging activity of the phloretin is concentration dependent, and the concentration of phloretin increases, the radical scavenging activity was also increased. The lower IC₅₀ value reflects better protective action. From the results, it may be postulated that phlorein was able to reduce the stable free radical DPPH to yellow colored diphenyl picrylhydrazine and which exhibits the better free radical scavenging activity of phlorein. It was reported that oxidative stress, which occurs when free radical formation exceeds the body’s ability to protect itself, forms the biological basis of chronic condition [25] . Based on the data obtained from this study, phloretin is a powerful free radical inhibitor or scavenger, as well as a primary antioxidant that reacts with free radicals, which may limit free radical damage occurring in the human body.

Total antioxidant activity of phloretin was determined by phosphomolybdenum method, which is based on the reduction of Mo(V1) to Mo(V) by the sample and the subsequent formation of green phosphate/Mo(V) compounds in acidic pH with a maximum absorption at 695 nm. Table 2 shows the total antioxidant activity of phloretin and standard compound ascorbic acid at different concentrations like 20, 40, 60 and 80 µg/ml. Phloretin exhibited effective and powerful antioxidant activity in all concentrations. The percentage of inhibition was found as 22.13%, 42.56%, 69.65% and 89.32% for 20, 40, 60 and 80 µg/ml of phloretin, respectively. Figure 2 is also revealed that the total antioxidant capacity of different concentrations like 20, 40, 60 and 80 µg/ml of phloretin. The total antioxidant activity of phloretin was increased with increasing the concentration. The IC₅₀ value for phloretin and ascorbic acid was 44.83 µg/ml and 42.40 µg/ml, respectively. The results are in agreement with the report of Aliyu et al. [26] , and they investigated the antioxidant activity of Ethulia conyzoides, which indicates that the concentration dependent total antioxidant capacity.

Values are expressed as mean ± SD of triplicates.

Superoxide is a highly reactive molecule produced through metabolic processes that reacts with various substances. Superoxide dismutase is present in aerobic and anaerobic organisms catalyses the breakdown of superoxide radicals [27] . Percentage scavenging of superoxide anion examined at different concentrations like 20, 40, 60, 80 μg/ml of phloretin was depicted in Table 3 and graphically represented in Figure 3. The percentage of scavenging superoxide radical increased with the enhanced concentration of phloretin. The maximum scavenging activity of phloretin and ascorbic acid was found at 80 μg/ml as 82.42% and 98.51%, respectively. The IC₅₀ value of phloretin and ascorbic acid was recorded as 44.16 μg/ml and 31.62 μg/ml, respectively. In vitro superoxide radical scavenging activity is measured by riboflavin/light/NBT (Nitro blue tetrazolium) reduction. This method is based on the generation of superoxide radical by auto oxidation of riboflavin in the presence of light. Superoxide is biologically important as it can form singlet oxygen and hydroxyl radical. Overproduction of superoxide anion radical contributes to redox imbalance and associated with harmful physiological consequences [28] . From these results, it was found that the phloretin showed potent superoxide anion free radical scavenging activity.

Table 4 shows the metal chelating activity of phloretin and ascorbic acid. Figure 4 represents the metal chelating activity was increased with increasing concentration of phloretin and ascorbic acid. At 80 μg/ml, phloretin had highest metal chelating activity 89.23% and the reference compound ascorbic acid was showed 98.65%. The IC₅₀ value for phloretin and standard ascorbic acid was 44.59 µg/ml and 30.9 µg/ml, respectively. Ferrozine can quantitatively form complexes with Fe²⁺. The complex formation is disrupted in the presence of chelating agents with the result that the red colour of the complex is decreased. The chelating activity of coexisting chelator was

Values are expressed as mean ± SD of triplicates.

Values are expressed as mean ± SD of triplicates.

estimated by the measurement of colour reduction. In this assay phloretin and standard ascorbic acid interfered with the formation of ferrous and ferrozine complex. It indicates that they have chelating activity and capture ferrous ion before ferrozine. Iron can stimulate lipid peroxidation by 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 [29] [30] . The formation of Fe²⁺ ferrozine complex is not complete in the presence of phloretin, which demonstrate that phloretin chelates iron. The absorbance of Fe²⁺ ferrozine complex was linearly decreased in a dose dependent manner (20 - 80 µg/ml). Similar report was observed in morphine is a flavonoid compound. Metal chelating capacity of morphine is significant since it reduced the concentration of the catalyzing transition metal in lipid peroxidation. It was reported that chelating agents, who form sigma bonds with a metal are effective as secondary antioxidant, because it reduce the redox potential thereby stabilizing the oxidized form of the metal ion [31] . The results obtained from this study revealed that the phloretin have a marked capacity for iron binding, which suggests that the compound phloretin act as a peroxidation protector may be related to its iron binding capacity.

Table 5 and Figure 5 shows that the reductive capability of phloretin and standard ascorbic acid. For the measurements of the reductive ability, we investigated that the Fe³⁺-Fe²⁺ transformation in the presence of phloretin and ascorbic acid. The reducing power increased steadily with increasing concentration of phloretin (20 - 80 μg/ml). Increased absorbance with the increased concentrations of the reaction mixture indicated that the increased reducing power. However, the highest scavenging activity was observed at 80 μg/ml of phloretin and standard ascorbic acid. Reducing power indicates that the compounds are electron donors, which can act as primary and secondary antioxidants [32] . Different studies have been indicated that the reducing properties are generally associated with the presence of reductones, which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom [33] . Similar results for reducing power activity

Values are expressed as mean ± SD of triplicates.

have been observed in the flavonoid isolated from Tartarian buckwheat and the results exhibited that the total flavonoid has significant reductive ability and radical scavenging effect in vitro [34] . The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. The antioxidant activity of an antioxidant compound have been attributed to various mechanisms, which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging activity.