2.1. Collection of Plant The leaves of the plant A. nilotica were collected from Rajshahi University campus, Bangladesh. It was identified and authenticated in the Department of Botany, University of Rajshahi, Bangladesh.
2.2. Test Microorganisms The pure culture microorganisms were collected from the Institute of Biological Science (IBSc), Department of Pharmacy, University of Rajshahi, and Environmental Microbiology Lab, ICDDR, B Mahakhali, Dhaka, Bangladesh. The bacteria were used for the study of antibacterial activity are Escherichia coli, Shigella sonnei, S. dysenteriae, S. shiga, S. boydii, S. flexneri and Vibrio cholerae.
2.2. Test Microorganisms The pure culture microorganisms were collected from the Institute of Biological Science (IBSc), Department of Pharmacy, University of Rajshahi, and Environmental Microbiology Lab, ICDDR, B Mahakhali, Dhaka, Bangladesh. The bacteria were used for the study of antibacterial activity are Escherichia coli, Shigella sonnei, S. dysenteriae, S. shiga, S. boydii, S. flexneri and Vibrio cholerae.
2.2. Test Microorganisms The pure culture microorganisms were collected from the Institute of Biological Science (IBSc), Department of Pharmacy, University of Rajshahi, and Environmental Microbiology Lab, ICDDR, B Mahakhali, Dhaka, Bangladesh. The bacteria were used for the study of antibacterial activity are Escherichia coli, Shigella sonnei, S. dysenteriae, S. shiga, S. boydii, S. flexneri and Vibrio cholerae.
2.6. Cytotoxicity Test The brine shrimps used for cytotoxicity test were obtained by hatching 5 mg of eggs of Artemia salina in natural seawater after incubation at about 29°C for 24h. The larvae (nauplii) were allowed another 24h in sea water to ensure survival and maturity before use. Five doses of plant extract (100, 200, 400, 600 and 800 ppm) in 5% DMSO and/or sea water were tested. Each extract preparation was dispensed into clean test tubes in 10 ml volumes and tested in duplicates. The concentration of DMSO in the vials was kept below 10 μl/ml. For control, same procedure was followed except test samples. After marking the test tubes properly, 10 living shrimps were added to each of the 6 vials with the help of a pasteur pipette. The test tube containing the sample and control were then incubated at 29°C for 24h in a water bath, after which each tube was examined and the surviving nauplii counted. From this, the percentage of mortality was calculated at each concentration.
2.9. Phosphomolybdate Method The total antioxidant capacity of the extract was determined by phosphomolybdate method using α-tocopherol as the standard (Jayaprakasha et al., 2002). An aliquot of 0.1ml of the extract (100 µg) solution was combined with 1.0 ml of reagent (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were capped and incubated in a boiling water bath at 95oC for 90 min. After the samples had cooled to room temperature, the absorbance was measured at 695 nm against the blank using an UV spectrophotometer. The blank solution contained 1.0 ml of reagent solution and the appropriate volume of the same solvent used for the sample and it was incubated under same conditions as rest of the sample. The total antioxidant capacity was expressed as µg equivalents of α-tocopherol by using the standard tocopherol graph.
2.10. Estimation of Total Phenolic Content Total soluble phenolics of the extract were determined with Folin-Ciocalteu reagent using pyrocatechol as the standard (Gulcin et al., 2004). An aliquot of 0.1 ml suspension of 1 mg of the extract in water was totally transferred to a 100 ml Erlenmeyer flask and the final volume was adjusted to 46 ml by the addition of distilled water. Folin-Ciocalteu reagent (1 ml) was added to this mixture, followed by 3 ml of 2% sodium carbonate 3 min later. Subsequently, the mixture was shaken for 2 h at room temperature and the absorbance was measured at 760 nm. The concentration of total phenolic compounds in the extract was determined as µg pyrocatechol equivalent by using the standard pyrocatechol graph.
2.11. Estimation of Total Flavonoid Content Total soluble flavonoid content of the extract was determined with aluminium nitrate using quercetin as the standard (Hsu, 2006). 0.5 ml of the extract was added to 1.5 ml of 80 % ethanol. An aliquot of 0.5 ml was added to test tubes containing 0.1 ml of 10 % aluminium nitrate, 0.1 ml of 1 M potassium acetate and 4.3 ml of 80 % ethanol. The absorbance of the supernatant was measured at 415 nm after incubation at room temperature for 40 min. The total flavonoid content in the extract was determined as µg quercetin equivalent by using the standard quercetin graph.