The supernatant was assayed for SOD activity by following the inhibition of epinephrine auto-oxidation. 0.5ml of sample was diluted with 0.5 ml of distilled water, to this 0.25 ml ethanol, 0.5 ml of chloroform (all reagents chilled) was added. The mixture was shaken for 1 min and centrifuged at 2000 rpm for 20 min. The enzymatic activity in supernatant was determined. To 0.05 ml of carbonate buffer (0.05 M, pH 10.2) and 0.5 ml of EDTA (0.49 M) was added.
The pH was adjusted to 9.5 with 2M NaOH. 20 g of NaOCl (in which 4% active chlorine) was added drop-wise to the slurry over a period of 30 min, while maintaining pH range 9.0-9.5, with constant stirring 30±2°C. The reaction proceeded for 10 min after addition of NaOCl. After the reaction, the pH was adjusted to 7 with 1M H₂SO₄ and the oxidized starch was filtered, washed four times with distilled water by using centrifuge and dried in oven at 30±2.0°C for 48h. Dried starch was powdered with morter and pestle and passed through 200 mm sieve and the powdered oxidized starch was kept in a polythene bag.
20ml of it was then pipetted into a 250 Erlenmeyer flask, followed by setting up a titration system with 0.100M HCl in the burette. As in the case of titration of an acetic acid with sodium hydroxide, a pH meter was used to monitor the pH of the solution as HCl was being added. HCl was added in an interval of 1ml until the pH of the solution was 9. When the solution pH reached 9.5, HCl was added in an increment of 0.2ml until the equivalence was passed. After this, HCl was added in an interval of 1ml until there was minimal change in the solution pH.
188.8.131.52. (C) Test for flavanoids ( Sofowara, 1993; Harbrone, 1973): Three methods were used to determine the presence of flavonoids in the plant sample.5 ml of dilute ammonia solution were added to a portion of the filtrate of each plant extract followed by addition of concentrated H2SO4. A yellow colouration observed in each extract indicated the presence of flavonoids. The yellow colouration disappeared on standing. Few drops of 1% aluminium solution were added to a portion of each filtrate.
The mixture was shaken using shaking incubator at 200 rpm for 120 min at 5000C. The mixture was then centrifuged at 3000 rpm for 15 min at room temperature and the supernatant was taken. This supernatant was stored at -2000C until further analysis. Preparation of watermelon extract Ten grams (10 g) of dehulled powdered sample were mixed with 150 ml of respective solvents, aqueous methanol (50%) and methanol and placed on water bath at 40 °C for 18 h with stirring/shaking. The extracts were filtered and concentrated by rotary evaporator.
Dialysis membrane having a pore size 2.4 nm and molecular weight cut off between 12,000 and 14,000 was used. The membrane was soaked in double distilled water for 12 hr. before mounting in a Franz diffusion cell. About 1ml of semisolid preparation of NLC was applied to the donor compartment, and the receptor compartment was filled with phosphate buffer, pH 7.4 (14 ml). During the experiment, the solution in the receptor side was maintained at 370C and stirred at 800 rpm with Teflon coated magnetic stirrer bar.
Then, the solution was loaded into the well. The gel then was run at 100 volt for 30 minutes. After that, the agar was cleaned and put into the EtBr solution for 10 minutes. The agar then washed by using tap water. Next, the agar was observed under UV transilluminator to see the formation of band.
Composite washed with aquades. Dried in the oven at the temperature 50⁰C for 24 hours. Composites are characterized by XRD, FT-IR, and SEM 2.2. analysis of Silica In Composite By Atomic Absorption Spectroscopy Each as much as 0.1 gram of the mixed aqueous solution of HCl added composite 2.5 M and 0.5 M HF with a total volume of 10 ml for through a process of dissolution for 1 week. The total concentration of Si contained in the solution to be analyzed using atomic absorption spectroscopy (AAS) on long wave 213.86 nm. The making of the Si standard solution done by dissolving 0.1 grams of The metal in solution 20 ml HCl 37% and added aquades to the volume of 100 ml of The solution thus obtained 1000 ppm.
The natural agar consist of Yeast Extract, Tryptone , Lactose , Manniiol, Sodium Chloride , Dip otassium Hydrogen Phosphate ,Gelatin and Agar .Then , in a conical flask 14g of nutrient agar was mixed into 500 ml of distilled water and , The mixture was stirred and dissolved until most of the agar dissolve. By non-absorbent cotton wool plug the mouth of the flask was closed. By using the autoclave the agar was sterilized for 15 minutes.The cotton wool was removed. By the flame the mouth of the flask was heated before and after pouring the agar into the Petri dishes. And, the left hand the lid of the Petri dish was lift, just enough to enter the mouth of the flask and quickly was poured in agar (about 15 cm3).