Chemicals and reagents: DPPH (1, 1-Diphenyl-2-pycrylhydrazyl), quercetin, ascorbic acid, gallic acid, Folin- Ciocalteu reagent, β-carotene, linoleic acid and carragenan was purchased from Sigma Chemical Co. Ltd, (St. Louis, MO, USA). Merck, Germany. Other chemicals and solvents were purchased from Fisher scientific (Walham, MA 02454, USA) and Loba Chemie Pvt Ltd. (Mumbai, India). Solvents and all other reagents were of analytical grade. The standard drug diclofenac sodium and indomethacine were collected from Square Pharma Ltd, Bangladesh and used for anti-inflammatory activity study. Sample collection, identification and extraction Fresh leaves and bark of X. Granatum were collected from Chadpai Range, the Sundarbans East Division, Bangladesh. After collection, it was taxonomically identified at Bangladesh National Herbarium, Mirpur, Dhaka (Accession no.: DACB 12789) and the voucher specimen was deposited. The bark and leaves were dried adequately under shade. The chopped and dried bark and leaves were then grinded into fine powder using a mechanical grinder. Cold extraction was preferred to prepare the extracts from X. granatum. For this purpose, the plant materials (bark and leaf) were separated from each other and then cleaned by gentle washing with distilled water followed by air drying for several weeks under shade. The dried material was ground into coarse powder with a motorized plant grinder. The powder was kept in a dry, cool and dark place in a suitable airtight container until analysis commenced. About 400 gm of grinded powder was soaked into 900 ml of ethanol in flat bottomed glass container for a period of 15 days with frequent stirring and shaking. The whole mixture contents were filtered off with clear cotton plug to eliminate plant derbies and the extract was then filtered through Whatman No.1
filter paper. The solvent (ethanol) was evaporated with the electric fan at room temperature and the dried crude extract (yield value 12.5%) was finally obtained. The bark and leaf extract of X. granatum was designated as S1 and S2, respectively. The dried crude extract was stored in refrigerator at 4 °C for further study. Test animal: Swiss-Albino mice of either sex (22 to 27 gm) were used for in-vivo ant-inflammatory study. The mice were collected from Animal Resources Department of International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B). The mice were acclimatized by keeping in the polypropylene cages by providing with right rodent foods under standard laboratory condition (room temperature 25±2 °C, relative humidity 55±5%, and 12 hours light). Ethical ideologies and rules for Scientific Experiments on Animals originated by the Swiss Academy of Sciences and the Swiss Academy of Medical Sciences (1995) and the Animal Ethics and Regulation of Khulna University (Research Ref No. KUAEC-2017/08/15) were strictly followed. To draw human blood (RBC membrane lysis and stabilizing assay) procedure and rules of Bangladesh Medical Research Council was strictly obeyed. In vitro antioxidant activity test DPPH radical scavenging assay. The free radical scavenging activity of the extract was estimated in vitro by DPPH free radical scavenging assay (Afrin et al., 2016). Aliquots of nine concentrations (1.57, 3.13, 6.25, 12.50, 25, 50, 100, 200
and 400 μg/ml) for plant extract and quercetin (standard) were made by serial dilution technique. Two ml of DPPH solution was applied on each test tube containing 1 ml of plant extract or standard. The final volume of the solution was 3 ml. After 30 minutes of incubation at room temperature, absorbance of each test tube was taken at 517 nm. Inhibition of free radical scavenging activity was calculated using the following equation: %Inhibition = {(Absorbanceof thecontrol -Absorbance of thesample) / (Absorbance of the control)} x 100 ........... (1). IC50 value (μg/ml) is the inhibitory concentration at which 50% of DPPH radicals are scavenged. A calibration curve of quercetin was developed and IC50 value of the sample extracts and standard was calculated. Reducing power assay The assay was performed according to the method as described by Chen et al. (2014). Five different concentrations (25, 50, 100, 200 and 400 μg/ml) for plant extract and standard ascorbic acid were made. β-Carotene bleaching inhibition assay The prevention of β-carotene bleaching by plant extracts was assessed according to procedure of Ueno et al. (2014). One ml of β-carotene solution (0.2 mg/ml in chloroform) was pipetted into a round bottom flask (50 ml) containing 0.02 ml of linoleic acid and 0.2 ml of 100% Tween-20. The mixture was then evaporated at 40 °C for 10 minutes followed by dilution with 100 ml of distilled water. Then, 5 ml aliquots of the emulsion were transferred into different test tubes containing 0.2 ml of 1 mg/ml plant extracts dissolved in 70% ethanol. The mixture was gently mixed and placed in a water bath at 50 °C for 2 hours. Absorbance of the sample was measured at 0 hour and every 30 min for 2 hours at 470 nm. Inhibition of β-carotene bleaching was calculated using the following equation: %Inhibition = {1- (Absorbanceof the control at time zero) / (Absorbance of the sample after 2 hours)} x 100 ................(2). The heat induced lysis assay mixture contains 2 ml isosaline, 1 ml phosphate buffer saline (pH 7.4), 0.5 ml erythrocyte suspension and 1 ml of plant extract of different concentrations (50 μg/ml, 100 μg/ml, 200 μg/ml, 400 μg/ml, and 800 μg/ml) or standard of various concentrations (12.5, 25, 50, 100 and 200 μg/ml). The assay mixtures were prepared in two sets. One set of tubes was incubated at 54 °C for 10 minutes in water bath and the other set was maintained at 0-5 °C in ice bath. The reaction mixtures were centrifuged at 5000 rpm for 10 minutes. The released hemoglobin content was measured at 540 nm. The percentage of inhibition or acceleration in hemolysis in RBC membrane stabilizing assay was calculated by the equation: %Inhibition = {(Absorbanceof thecontrol -Absorbance of the sample) / (Absorbance of the control) x 100. ..........(3). Group IV to Group VIII were given increasing doses of ethanolic extracts (1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg bw and 40 mg/kg body weight). Acute paw edema was induced by injecting 0.1 ml of 1% (w/v) carrageenan solution. The linear paw circumference was measured up to 4th hour at hourly interval. The anti-inflammatory activity was measured by using the following equation, where T is thickness of paw in control group and T0 is thickness of paw edema in the test group. %Inhibition of edema = {(T - T0) / T} x 100. .............. (4). Statistical Analysis: One-way ANOVA analysis followed by Benferroni test and correlation analysis was performed using GraphPad Prism 6 software.