M. koenigii (plant) leaves were collected from the adjacent areas of BCSIR Laboratories Rajshahi, and Botanical Garden, University of Rajshahi. The collected materials were washed thoroughly in water, chopped, air dried for a week at 35-40^oC and pulverized in electric grinder. Dried ground leaves of Murrayakoenigiiwere exhaustively extracted with methanol (MeOH, Analytical Grade, BDH Laboratory Supplies) in soxhlet apparatus. The resulting juicy extract was filtered through Whatman paper No.1 and concentrated under reduced pressure at 45°C using rotary evaporator to obtain a crude residue (23.5%).Then water triturate part was collected from crude extract. The water triturate fraction was passed through a previously well packed dia-ion resin column which has selectivity to collect only the phenolic group containing compounds. Then the materials, which were bound in resin column, were collected by passing methanol solvent. Then Petroleum ether, Ethyl acetate and Chloroform solvents were passed through the residue respectively. Finally petroleum ether, Ethyl acetate and Chloroform triturate were collected. The extracts thus obtained were kept in refrigerator until further analysis.

All chemicals used were of analytical grade. 1,1-Diphenyl-2- picrylhydrazyl (DPPH), Ascorbic acid, catechin, BHT, gallic acid, anhydrous sodium carbonate (Na₂CO₃), aluminum trichloride, potassium acetate, sodium acetate, ferric chloride hexahydrate (FeCl₃.6H₂O), Folin–Ciocalteu reagent were purchased from Sigma–Aldrich. All chemicals and reagents were used without further purification.

Total phenolic content of different extractives of Murrayakoenigii were determined by adopting the method20 involving Folin-Ciocalteu reagent as oxidizing agent and gallic acid as standard. An aliquot from each of the plant extract (0.5 ml) and standard gallic acid solution (100, 200, 300, 400, and 500 μg/ml) was added with 2.5 ml of Folin–Ciocalteu (Diluted 10 times with water) reagent and 2.5 ml of Sodium carbonate (7.5%) solution. The test tube was incubated for 20 minutes at 25^oC to complete the reaction. A typical blank solution containing all reagents except plant extract or standard was also prepared. Then the absorbance of the solution was measured at 760 nm using a spectrophotometer against blank. Total phenolic contents of extracts were expressed as mg gallic acid equivalent (GAE)/g dried extract. All samples were analyzed in triplicate.

Total flavonoid content was determined using the aluminum colorimetric method ^21,22 with some modifications using catechin as the standard. A calibration curve of catechin was prepared in the range of 0–200 lg/ mL. Briefly, extract (0.5 mL) and standard (0.5 mL) were placed in different test tubes and to each 10% aluminum chloride (0.1 mL), 1 M potassium acetate (0.1 mL), 80% methanol (1.5 mL) and distilled water (2.8 mL) were added and mixed. A blank was prepared in the same manner where 0.5 mL of distilled water was used instead of the sample or standard, and the amount of aluminum chloride was also replaced by distilled water. All tubes were incubated at room temperature for 30 min. The absorbance was taken at 415 nm. The concentration of flavonoid was expressed as mg catechin equivalent (CatE) per gram of extract.

Solutions of various concentrations (5, 10, 20, 40 and 80 μg/ml) were prepared in corresponding solvents with each type of extractive of M. koenigii. This mixture was kept at 50ºC in water bath for 20 minutes. After cooling, 2.5 ml of 10% trichloro acetic acid was added and centrifuged at 3000 rpm for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and a freshly prepared ferric chloride solution (0.5 ml). Control was prepared in similar manner excluding samples. The absorbance was measured at 700 nm. Ascorbic acid at various concentrations was used as standard. Increased absorbance of the reaction mixture indicated the increase in reducing power.

Total antioxidant activity of different extracts of M. koenigiiwas determined with some modifications23. Solutions of various concentrations (5, 10, 20, 40 and 80 μg/ml) were prepared in corresponding solvents with each type of extractive of M. koenigii, and standard substance (Ascorbic acid) and mixed with 3 ml of reaction mixture containing 0.6 M sulphuric acid, 28 mM sodium phosphate and 1% ammonium molybdate. The mixtures were incubated at 95^oC for 10 minutes to complete the reaction. Then the absorbance was measured at 695 nm.

The antioxidant activity of M. koenigii extracts were measured in comparison to standard antioxidant ascorbic acid (BDH, England) depending on the scavenging effect of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical. The whole procedure was performed according to procedure described by Bracae et al. (2001)24. Briefly, different concentrations (10, 20, 40, 80, and 100μg/ml in methanol) of ascorbic acid solution(1ml) as well as M. koenigii extracts solution (1ml) were mixed with 3 ml of 0.4 mM DPPH solution. The mixtures were kept in dark for 30 minutes to measure the absorbance at 517 nm using GENESYS^TM 20 Spectrophotometer (Thermo Spectronic, USA). The scavenging activity against DPPH was calculated using the equation:

Scavenging activity (%) = ((A - B) / A) x 100

Where A is absorbance of control (DPPH solution without the sample), B is the absorbance of DPPH solution in the presence of the sample (extract/ ascorbic acid).

Phenolic compound are able to absorb free radicals and can chelate metal ions that could catalyze formation of ROS which promotes lipid peroxidation. Phenolic compounds have redox properties, acting as free radical scavenging or primary antioxidants; therefore, it is justifiable to determine phenolic content in plant extract. Total phenolic content of different fractions of M. koenigiiare shown in (Figure 1). Among the fractions, the highest phenolic content was found in Dia-ion resin adsorbed fraction (17.52 mg GAE/gm of extract), followed by Ethyl acetate fraction (14.27 mg gallic acid/gm of extract), Chloroform fraction (13.285 mg gallic acid/gm of extract) and petroleum ether fraction (13.25 mg gallic acid/gm of extract).

Plant flavonoids are the secondary metabolites with both in vitro and in vivo antioxidant activity and they help human body to defend against oxidative stress related diseases25. Total flavonoid content of different fractions of M. koenigiiare shown in (Figure 2). Among the fractions, the highest total flavonoid content was found in Dia-ion resin adsorbed fraction (16.65 mg CatE/g of extract), followed by Chloroform fraction (14.60 mg CatE/g of extract), Ethyl acetate fraction (13.42 mg CatE/g of extract) and petroleum ether fraction (12.83 mg CatE/g of extract). Therefore, it can be said that polyphenolic, and flavonoid may work together with other phytochemicals present in M. koenigii and make it medicinally important.

The reducing potential of plant is mostly associated with the presence of reductones, which exert their mechanism of action by breaking the free radical chain by donating a hydrogen atom. The iron reducing capacity of the four different fractions of Murrayakoenigiiextract such as petroleum ether fraction (PE), chloroform fraction (CE), ethyl acetate (EE) and Dia-ion resin- adsorbed fraction (DAF) have been investigated. Among the four different extracts Dia-ion resin-adsorbed fraction showed the highest iron reducing capacity with absorbance of 2.504 at 80 mg/ml concentration, followed by Chloroform fraction with absorbance 2.263±0.003 at 80 mg/ml while Ethyl acetate fraction showed iron reducing capacity with absorbance of 1.675 at 80 mg/ml and Petroleum ether fraction showed the iron reducing capacity with absorbance 1.421 at 100 mg/ml (Figure 3). Different extracts, and standard (vitamin C, AA) exhibited the reducing power activity as follows: AA>DAF > CE> EE > PE.

The antioxidant ability and radical scavenging properties of plants are associated with its medicinal values. Total antioxidant activity of different fractions of methanolic extract of Murrayakoenigii leaves such as Dia-ion resin adsorbed fraction, chloroform fraction, Ethyl acetate fraction and petroleum ether fraction were investigated. Among the fractions, Dia-ion resin adsorbed fraction showed the highest total antioxidant activity with absorbance 2.320 at 100 mg/ml (Figure 4), whereas the Chloroform fraction and Ethyl acetate fraction showed absorbance 2.306 at 100 mg/ml and 2.183 at 100 mg/ml respectively. Petroleum ether fraction showed the lowest total antioxidant activity with absorbance 1.944 at 100mg/ml concentration. Different extractives, and standard exhibited the antioxidant activity as follows: AA> DAF> CE> EE> PE

Plants rich in secondary metabolites, including phenolics and flavonoids, have antioxidant activity due to their redox properties and chemical structures. The DPPH radical is widely used in assessing free radical scavenging activity because of the ease of the reaction. Among the fractions of the extract, highest DPPH radical scavenging activity was found in Dia-ion resin adsorbed fraction having IC₅₀ value 15.53mg/ml. On the other hand, chloroform fraction showed DPPH radical scavenging activity with IC₅₀ value 24.62mg/ml, followed by ethyl acetate fraction with IC₅₀ value 21.97mg/ml and petroleum ether fraction showed DPPH radical scavenging activity with IC₅₀ value 18.56 mg/ml (Figure 5 and Figure 6).

We are thankful to the authority of BCSIR Laboratories, Rajshahi and Rajshahi University for their technical support for carrying out this research work.