Stem & bark of Adina cordifolia (Roxb.) was collected from Adampur, Moulavibazar, Bangladesh in April, 2012 and identified by the expert of the National Herbarium, Mirpur, Dhaka, Bangladesh. Accession number DACB-37,951 (see Figure 1) is retained there for further references and the specimen has been preserved in the Phytochemistry and Pharmacology Laboratory, North-South University Bangladesh. The dried powder of stem and bark (250 g) was extracted with n-hexane, ethyl acetate, 95% of ethanol and subsequently 70% methanolin room temperature (25˚C ± 2˚C). The extract was concentrated by evaporation under reduced pressure at 40˚C using rotary evaporator (Bibby RE-200, Sterilin Ltd., UK) to have gummy concentrate of dark green color extract.

The freshly prepared crude extract was qualitatively tested for the identification of chemical constituents, such as, alkaloids, flavonoids, steroids, glycosides, saponins, terpenoids, gums and tannins. The tests were carried out by the method described previously and 10% (w/v) solution of the extract was taken in each test unless otherwise mentioned in individual test.

The ability of the plant extract to scavenge DPPH free radicals was assessed by the standard method [14] adopted with suitable modifications [15] . The stock solution of extracts were prepared in methanol or DMSO (n- hexane or ethyl acetate extracts) to achieve the concentration of 15 mg/ml. Dilutions were made to obtain concentrations of 500, 250, 125, 62.5, 31.25 μg/ml. Diluted solutions (100 µleach) were mixed with 3 ml of methanolic solution of DPPH in concentration of 0.002%. Similarly, the stock solution of standard (ascorbic acid) were prepared in methanol to achieve the concentration of 1.5 mg/ml and dilutions were made to obtain concentrations of 50, 25, 12.5, 6.25, 3.125 μg/ml. After 30 min incubation in darkness at room temperature, the absorbance was recorded at 517 nm. Control sample contained all the reagents except the extract. Percentage inhibition was calculated using Equation (1), whilst IC₅₀ values were estimated from the % inhibition versus concentration plot, using a non-linear regression algorithm. The data were presented as mean values ± standard deviation (n = 3).

The total phenolic content (TPC) is determined by using the Folin-Ciocalteau method. Absorbance was measured at 765 nm. The content of total phenolic compounds in the plant extract (four types: n-hexane, ethyl acetate, ethanol, methanol extract) of Adina cordifolia (Roxb.) was determined by the method described by Chun et al. (2003) [16] .

0.3 ml of plant extract or standard was taken of different concentration solution in a test tube. 1.5 ml of Folin- ciocalteu reagent solution was added into the test tube. Then 1.2 ml of sodium carbonate solution was added into test tube. Then the test tubes of standard and extracts solution were incubated for the 30 minutes at room temperature to complete the reaction. Then the absorbances of the solutions were measured at 765 nm using a spectrophotometer against blank. TPC was expressed as mg gallic acid equivalents (GAE) per gram of sample (mg/g).

The total flavonoid contents were measured by a colorimetric assay [17] [18] . 0.3 ml of plant extract or standard was taken of different concentration solution in a test tube. 900 µl methanol was added into the test tube. Then 60 µl 1 M K-acetate, 1.68 ml distilled water were added into test tube. 60 µl 10% aluminium chlorides were added to it. Then the test tubes of standard and extract solution were incubated for the 30 minutes at room temperature to complete the reaction. The absorbance of the solutions was measured at 415 nm using a spectrophotometer against blank. Total flavonoid content of the extracts was expressed as mg quercetin equivalents (QE) per gram of sample (mg/g).

All data are presented as means ± SD for at least three replications for each prepared sample. Linear regression to correlate between total phenolics as well as total flavonoid with antioxidant activity was carried using Excel 2007.

Preliminary phytochemical screening of the plant extract of Adina cordifolia (Roxb.) with different solvents revealed the presence of various bioactive components of which flavonoids, alkaloids, terpenoids, tannins, gums and carbohydrates were the most prominent by observing the color. The result of phytochemical tests has been summarized in the Table 1.

The antioxidant reacts with stable free radical, DPPH and converts it to 1,1-diphenyl-2-picryl hydrazine. The ability to scavenge the free radical, DPPH was measured at an absorbance of 517 nm. So the DPPH and its % inhibition of n-hexane, ethyl acetate, ethanol and methanolic extract showed that IC₅₀ values 31.36 ± 0.048 μg/ml, 22.49 ± 0.023 μg/ml, 38.96 ± 0.076 and 20.39 ± 0.054 μg/ml respectively. Ascorbic acid has taken as reference which showed 3.125 ± 0.052 μg/ml. Among these results ethyl acetate and ethanol extracts have more potent than other extracts. The overall results of % inhibition as shown in the (Table 2) respective to IC₅₀ values.

Data set of n = 3 & ± means Standard Deviation (SD).

Symbol (+++) indicates presence in high concentration, symbol (++) indicates presence in moderate concentration, symbol (+) indicates presence in trace concentration and symbol (?) indicates absence of the respective phytochemical. NEAC = N-hexane extract Adina cordifolia (Roxb.), EAAC = Ethyl acetate extract Adina cordifolia (Roxb.), EEAC = Ethanol extract Adina cordifolia (Roxb.), MEAC = Methanol extract Adina cordifolia (Roxb.).

Phenolics or polyphenols are secondary plant metabolites that are ubiquitously present in plants and plant products. Many of the phenolics have been shown to contain high levels of antioxidant activities [19] . Phenolic com- pounds contribute to the overall antioxidant activities of plants mainly due to their redox properties. Generally, the mechanisms of phenolic compounds for antioxidant activity are neutralizing lipid free radicals and preventing decomposition of hydroperoxides into free radicals [20] [21] .

TPC of extracts was determined by Folin-Ciocalteau (F-C) assay using Gallic acid as a standard phenolic com- pound (see Figure 2). The F-C assay for total phenolics contents is a fast and simple method and can be useful in characterizing and standardizing botanical samples. F-C method is based on oxidation of phenolics by a molybdotungstate in F-C reagent to yield a colored product with λmax 765 nm [22] .

The total phenolic contents in the examined plant extracts using the Folin-Ciocalteu’s reagent is expressed in terms of gallic acid equivalent (the standard curve equation: y = 0.158x + 0.128, R² = 0.9766). The values obtained for the concentration of total phenols are expressed as mg of GA/g of extract (Table 3).

The total phenolic contents in the examined extracts ranged from 17.48 to 20.83 mg GA/g. The highest concentration of phenols was measured in ethanol, ethyl acetate extracts. Methanol and n-hexane extracts contains considerably smaller concentration of phenols. The total phenolic contents in plant extracts of the species Adina cordifolia (Roxb.) depends on the type of extract, i.e. the polarity of solvent used in extraction. High solubility of phenols in polar solvents provides high concentration of these compounds in the extracts obtained using polar solvents for the extraction [23] [24] .

The concentration of flavonoids in various plant extracts of the species Adina cordifolia (Roxb.) was determined using spectrophotometric method with aluminum chloride. The content of flavonoidswas expressed in terms of quercetin equivalent (the standard curve equation: y = 0.118x + 0.020, r² = 0.993), mg of QUE/g of extract (Table 4). The concentration of flavonoids in plant extracts from Adina cordifolia (Roxb.) ranged from 17.49 to 22.48 mg/g n-hexane and ethyl acetate extracts contains the highest flavonoid concentration. The lowest flavonoid concentration was measured in ethanol and methanol extracts.