Collection of plant materials Fresh leaves of Aegle Marmelos, Catharanthus roseus, Moringa oleifera and Ageratum conyzoids were collected from different location of Sylhet district and identified with great care by plant specialist in the department of plant and environmental biotechnology, Sylhet Agricultural University.
Preparation of crude extract: The leaves were sun dried for three days following their collection and blended using electric blender. 10 g of each powdered leaves were placed in conical flask and 200 ml of methanol was added and plugged with cotton and soaked with methanol for 72 hours at room temperature with continuous stirring. After 72 hours the supernatant was collected by filtration using clean cloth and Whatmann filter paper. Finally, the solvent was evaporated to make the crude extract which was considered as 100% concentrated extract and used for further analysis.
Phytochemical screening: The crude plant extracts were subjected to various biochemical tests for phytochemical analysis using standard procedures as described previously.
Total Phenolic content: Folin-Ciocalteu reagent assay was conducted to determined total phenolic contents where gallic acid was used as standard active compound. In this assay, a volume of 1ml extract was added to 0.5 ml of 10 fold diluted Follin-Ciocalteu reagent followed by adding 1 ml Na2CO3 (7.5%) after 10 minutes and 4.5 ml distilled water to make reaction mixture. After 30 minutes of reaction occur, the absorbance was taken at 680 nm against reagent blank which was prepared by mixing all reagents except plant extract.
Total flavonoid content: Total flavonoid content was determined by aluminium chloride containing colorimetric assay [18]. Briefly, 1ml extract or 1ml varying concentration of standard solution (12.5, 25, 50, 75, 100 μg/ml) mixed with equal volume (200 ul) of 10% Aluminium chloride and 1M potassium acetate (1:1), followed by adding distilled water. All the prepared solutions were filtered through Whatmann filter paper before measuring their absorbance. Similar manner was adopted to prepare sample blank by replacing aluminium chloride solution with distilled water. The absorbance was taken at 510 nm. The content was quantified from Standard calibration curve of quercetin and expressed in mg of quercetin equivalent (QE) per gram of dry extract.
Antioxidant activity: Varying concentration (25, 50, 75, 100 µg/ml) of standard or crude extract was made by serial dilution with methanol from stock solution where the concentration was 1 mg/ml. At a concentration of 0.004%, DPPH solution was freshly prepared by mixing with methanol solvent. The reaction mixture comprised with 1 ml crude extract or 1 ml standard, 3 ml DPPH solution and 1ml methanol. The reference standard compound was ascorbic acid, whereas 1 ml methanol added to 3 ml solution of DPPH was used as control. Blank was made with methanol. After incubating the reaction mixture in dark condition for 30 minutes at room temperature, the absorbance of control, crude extract and standard was measured at 517 nm.
Free radical scavenging activity was expressed by using following formula: %scavenging = [(A517CONTROL – A517SAMPLE)/A517CONTROL]×100 Where, A517CONTROL = Absorbance of DPPH solution A517SAMPLE = Absorbance of sample [The IC50 value means the concentration of extract to scavenge 50% of DPPH]
Antibacterial activity: Medicinal plants extract were subjected to a test of their antibacterial activities against five pathogenic bacteria based on agar disc diffusion method. The growth of test organism was maintained by sub culturing on nutrient broth and incubating overnight at 350 C. Gentamycin was used as positive control whereas methanol without plant extract was used as negative control. Methanol extract containing discs (5 mm in diameter) were placed on spread culture of bacterial agar plate subsequently plates were kept in an incubator at 350 C for incubation at least 24 hours.
Statistical analysis All experiments were conducted three times. Linear Regression analysis was used to calculate IC50 values of antioxidant. All data were analysed using Microsoft Excel 2007 software.