Collection and Identification of plant materials The flowers of Nyctanthes arbor-tristis L. were collected from BCSIR campus, Dhaka, Bangladesh. The plant (specimen # DACB 38734) was identified from Bangladesh National Herbarium (BNH), Dhaka by their Taxonomist. The collected flowers were air dried at 25-30 °C in the absence of sunlight. The dried flowers were powdered by a grinder machine. Then they were weighed and stored in an air tight container in dark until use. Phytochemical screening Species chemical tests were carried out for species phytochemical constituents. Standard procedures were followed to identify the constituents as described by Sofowara (1993), Trease and Evance (1996), Harborne (1998), Ghani (2003), Mukharjee (2002). Determination of proximate composition The moisture, ash and crude protein contents were determined using the standard methods of the Association of Official Analytical Chemists (AOAC, 1990). The average weight of flowers was determined by weighing fresh flowers under ambient condition. For the determination of Loss on air drying, the fresh flowers were kept in room temperature for 4 to 5 days in absence of sunlight. The average weight loss was calculated by difference. The moisture content was determined by heating the fresh flower samples in a temperature controlled electric oven at 105°C until constant weight was achieved (ca. 6 to 10 hours). The moisture content was calculated in percentage (m/m). For determination of ash content, previously weighed fresh flower sample was taken. Then the sample was made moisture free and the moisture free sample was incinerated at 600°C about 6-12 hours in a temperature controlled muffs furnace until ash becomes almost white or grayish white in color. Crude protein was calculated by using following formula. Protein content (%) = nitrogen content (%) × 6.25 CHNS/O analysis Powdered flowers samples were analyzed for elements such as carbon, hydrogen, nitrogen, sulfur and oxygen by using a
CHNS/O Elemental Analyzer (Vario Micro Cube, Elementar, Germany). The results for carbon, hydrogen, nitrogen, sulfur content percentage were calculated directly and the percentage of oxygen was calculated indirectly by using the following formula: Oxygen content (%) = 100 - (the sum of carbon, hydrogen, nitrogen, sulfur content percentage) Determination of anion content The sequential determination of fluoride, chloride, nitrite, bromide, nitrate, phosphate and sulfate ions were performed by chemically suppressed ion chromatography. An aliquot of sample solution was injected into an ion chromatography instrument. The sample was pumped through two columns and a suppressor device into a conductivity detector. The analytical column and the guard column were packed with low-capacity anion exchanger. Ions are separated based on their affinity for the exchange sites of the resin. Anions are identified based on their retention times compared to known standards. Quantization was accomplished by measuring the peak height or area and comparing it to a calibration curve generated from known standards. Determination of metal composition The analysis of minerals and metal compositions were carried out on the sample using the standard methods of the Association of Official Analytical Chemists (AOAC, 1990). Instrumentation K and Na were analyzed by a Flame Photometer (JENWAY, PFP 7, UK). Mn, Fe, Zn, Cu, Mg, Ca, As and Hg were analyzed by using Varian AA -240 FS, Australia, while Pb, Cd, Cr, Ni, and Co were analyzed by a Varian Spectra AA 240 Z, Australia, AAS instruments. Preparation of sample The dried powdered sample was used for metal analysis. For analysis of Mn, Pb, Zn, Cd, Fe, Ca, Cu, Co, Cr, As, Na, K, Mg and Ni the plant samples were incinerated at 550°C. About 0.1-1.0 g ash of the sample was dissolved in 5 mL of 1 M nitric acid (HNO3) by warming on a hot plate for 2-3 minutes. The solution was taken in a 50 mL volumetric ?ask and 2 additional portions of 1 M HNO3 were added and diluted to 50 mL with 1 M HNO3. The solution was filtered through 0.45 µ filter paper. The filtrate was used for further metal analysis. For analysis of Mercury (Hg), the moisture free powdered sample was heated for 30 min with 20 mL conc. nitric acid. The solution was allowed to cool at room temperature and 10 mL perchloric acid was added to it. The solution was heated on a hot plate until the reaction with HClO4 had completed (after completion of reaction the solution was colorless or yellow). Finally the solution was cooled and filtered in a 50 mL volumetric flask and the volume was made up to 50 mL with de-ionized water. A blank sample was also prepared without adding plant sample. Standards NIST traceable standards were used for individual metal analysis. The standards were collected from Scharlau, Sentmenat, Spain and the concentration of each individual stock standard for testing elements was 1000 mg/L. Working standards were prepared by necessary dilutions. Calibration procedure Independent calibration curve was constructed for individual elements from particular working standards. Element concentration in sample was determined from the calibration curve.