a. Chemicals All the chemicals used in the experiment were analytical grade. Ascorbic acid was obtained from (Merck, Germany) chem. Naphthyl ethylene diamine dihydrochloride was obtained from Sigma Chemical Co, India. Sodium nitro prusside was obtained from Loba chemie, India. Collection and Identification of Plant material Whole plant Phyllunthus freternus, Leaves, Barks and Roots of Triumfetta rhomboidae and Barks of Casuarina littorea were collected from Jahangirnagar University Dhaka, Bangladesh in May, 2008, and identified by the experts of National Herbarium, Bangladesh). Voucher specimens for these collections have been retained in the National Herbarium, Bangladesh. and accession no. for the identified plants Phyllunthus freternus, Triumfetta rhomboidae and Casuarina littorea are 32763, 32571 and 34997 respectively.
b. Extraction The collected and identified plant parts were cleaned by separating the unwanted plants or plants parts. The plant parts were shade dried in open air for three weeks. The prepared plant parts were ground in a course powder with the help of a suitable grinder. The powder has to store in an airtight container under cool and dry place until further analysis required. About 200 gm of powdered materials for each plant parts were taken for extraction using Soxhlet Apparatus and Maceration method. Methanol was used as extracting solvent. The Methanol extract thus obtained was evaporated under rotavapour until dried. The concentrates were designated as crude extract of Methanol.
c. Phytochemical screening The freshly prepared methanolic extracts of the selected plants were qualitatively tested for the presence of chemical constituents. Phytochemical screening of the extract was performed using the following reagents and chemicals: Alkaloids with Dragendorff's and Mayer's reagent, flavonoids with the use of Mg and HCI; tannins with ferric chloride and potassium dichromate solutions, steroids with sulfuric acid and saponins with ability to produce suds. Gum was tested using Molish reagents and concentrated sulfuric acid. These were identified by characteristic color changes using standard procedures (Trease et aI., 1983). By phytochemical screening the chemical constituent of plants can be detected and the isolated compounds are used for various pharmacological effects. In the present study methanolic extracts of Phyllanthus freternus, Triumfetta rhomboidae and Casuarina littorea screened for the presence of alkaloids, flavonoids, gums, saponins, tannins, steroids which have definite medicinal importance. Alkaloids are very common in many medicinal plants that have significant physiologic and pharmacological properties. Steroids contain widely distributed natural compounds that are used for abnormality of reproductive tract in human. Tannins have astringent and antimicrobial properties. Saponin containing plants materials are used in many parts of the world as detergents (Trease et aI., 1983). Antimicrobial properties saponin containing plants materials are used in many parts of the world as detergents. The therapeutic and other pharmacological properties of medicinal plants is directly depends upon the presence of various chemical constituents. So investigation of chemical constituents of the plant should have some direct relationship with local medicinal uses. The results of various qualitative chemical tests for the detection of chemical constituents of whole plant of Phyllanthus freternus, leaves, barks and roots of Triumfetta rhomboidae and barks of Casuarina littorea using their methanolic and petroleum ether extracts are shown.
3. Assay of Nitric oxide scavenging activity: Nitric oxide scavenging activity can be estimated by the use of Griess IlIosvoy reaction (Garrat, 1964).The compound sodium nitroprusside is known to decompose in aqueous solution at physiological pH (7.2) producing NO. Under aerobic conditions, NO reacts with oxygen to produce stable products (nitrate and nitrite). The quantities of which can be determined using Griess reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitrite ions. For the experiment, sodium nitroprusside (10mM) in phosphate buffered saline was mixed with different concentrations (5 - 200µg/ml) of methanol extract of each plant were dissolved in methanol and incubated at 300 C for 2 hours.
The same reaction mixture without the extract but the equivalent amount of ethanol served as the control. After the incubation period, 0.5 ml of Griess reagent (1% sulfanilamide, 2% H3P04 and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride) was added. The absorbance of the chromophore that formed during diazotization of the nitrite with sulfanilamide and subsequent coupling with Naphthylethylenediamine dihydrochloride was immediately read at 550nm. Inhibition of nitrite formation by the plant extracts and the standard antioxidant ascorbic acid were calculated relative to the control. Inhibition data (percentage inhibition) were linearized against the concentrations of each extract and standard antioxidant. IC50 which is an inhibitory concentration of each extract required to reduce 50% of the nitric oxide formation was determined.
4. Statistical analysis All experiments were performed thrice and the results averaged Data were expressed as mean ± SD. Linear regression analysis was used to calculate IC50 for each plant extract.