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.
In a typical procedure, equimolar zinc nitrate hexahydrate [Zn(NO3)2, 6H2O] and hexamine (HMT) [(CH2)6N4] were dissolved in 80 ml D.I. (deionised) water to form a 0.04 M solution. Thereafter the solution was kept in a Pyrex bottle and heated in a regular laboratory oven at 110°C for 6 h. Finally the bottle was naturally cooled down and the product was collected by filtering and washing with copious amount of D. I. water. For the synthesis of ZnO-silica hybrid, 5 ml TEOS was dissolved in 40 ml ethanol via sonication followed by which 1 ml D.I. water and 2 ml ammonia was dropped into the solution in stirring condition one after another.
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.
Ltd. New Delhi. India. Double distilled water was used throughout the study. 5.2 METHOD FOR PREPARATION OF TRETINOIN LOADED NLCs NLCs were prepared by Melt emulsification and low temperature solidification method Tretinoin and phospholipon 90G were dissolved in methanol and mixed with an acetone solution containing a blend of Tristearin and Oleic acid. The mixture was sonicated for 15 minutes and added drop wise to Pluronic F68 solution.
Fe3O4 nanoparticles were synthesized according to our previously reported method by chemical co-precipitation of Fe2+ and Fe3+ ions with a molar ratio of 1:2 . Briefly, 2.4 g of FeCl3.6H2O and 0.8 g of FeCl2.4H2O were dissolved in 30 mL of deionized water under using continuous N2 purge at 70 °C. and Under vigorous stirring, followed by dropwise addition of 10 mL of NH3.H2O was dropwise added to the reaction mixture until the color of mixture turned to black and kept reacting for 90 min to complete the reaction. At the end, the synthesized Fe3O4 nanoparticles were separated by a magnet and washed by using water and ethanol for further three times with 89.3%
OLE solutions were made by dissolving 15 mg of each crude extract in 15 ml dimethyl sulfoxide (DMSO) (Fine-Chem, Mumbai, India). Then, 0.5 ml of each solution was mixed with 2.5 ml of 0.2 N Folin-Ciocalteu reagent (Sigma-Aldrich, USA) for 5 min at room temperature then 2 ml of sodium carbonate solution (7.5 % in deionized water, w/v) were added. After incubating for two hours at room temperature in a dark place, the absorbance was measured at 760 nm using UV/Visible spectrophotometer (Elico, SL-150, India). The concentrations between 0.01-0.05 mg.ml-1 of gallic acid (Sigma-Aldrich, USA) were used as a standard for the calibration curve. The total phenolic content was expressed as mg gallic acid equivalent.g-1
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.
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.