Apparatus- Chromatography column: C18 (10 microns particle size), with Guard column Flow rate: 1.2ml/min Pressure: 30-40kgf Wavelength: 326nm Mobile phase: methanol : water (95:5 v/v) Internal standard: retinyl acetate Injection volume: 20µl Procedure for Retinol extraction from serum samples- 1) 100 µl of serum sample and 100 µl of Retinyl acetate were added into 12 X 100mm glass test tubes. Vortex-mixed for 30 seconds. Then, kept them at 4 C for 5 mins. 2) 1mL of hexane was added and vortex-mixed intermittently for 60 sec. 3) Centrifuged at 2500 rpm for 12 mins.
First the solution of 1 mL of extract was added to deionizer water (10 mL), then Folin–Ciocalteu phenol reagents (1.0 mL) added to the mixture. The mixture was left for 5 minutes, and solution of 20% sodium carbonate (2.0 mL) was added to the mixture. This mixture diluted until 50 mL with deionizer water. And after that kept in total darkness room for 1 hour. The mixture absorbance was measured at 750 nm using a spectrophotometer.
Absorbance of the solution was measured at 238 nm. Drug content was calculated by the formula absorbance/ slope* dilution factor. Drug loading Microsphers equivalent to 100 mg of DLTZH/ND taken and washed with 3 x 10 ml of methanol, which removes the free unloaded drug. Filtrate was diluted suitably to Beer’s concentration range and free drug concentration was determined spectrophotometrically at 238 nm and 236 nm for DLTZH and ND respectively. Further microspheres were crushed, dissolved in methanol and made up to 100 ml, which was further diluted suitably to Beer’s rangeand drug concentration was determined.
The end product was passed via sieve (no. 85) and stored in desiccators until use. 2.3. Preparation of DS loaded mucoadhesive beads PC-SA [F0], DS-SA [F1] and DS-PC-SA [F2-F6] beads were prepared by the ionotropic gelation method and the compositions are summarized in Table 1. Initially, PC gum was dissolved in distilled water and boiled for 10 min, cooled and stirred for 24 h at
Extraction of Crude extract Propolis samples were refrigerated about -20c°. after refrigeration the propolis samples were grinded and macerated (30g of propolis, making up the volume to 100 mL with 70% ethanol) and was filtered. The filtrate was evaporated to dryness at 80°C under reduced pressure. 3. Characterization of Crude Extract 3.1 Physical Test 3.1.1 Organoleptic Test The color, odor and physical state of extract will be determined.
Chloroacetic acid (0.5 g, 5. 28 mmol), 5-aminotetrazole monohydrate (0.45 g, 5. 28 mmol), and sodium hydroxide (0.59 g, 10.57 mmol) in 10 ml of water was refluxed 20 hr, cooled, and made strongly acidic with concentrated hydrochloric acid. The mixture was cooled overnight and precipitate was separated to give 0.28 g a white solid product at 45.41% yield. (5-Amino-tetrazol-1-yl)-acetic acid: Yield: 45.41%; white crystals; m.p 210-213°C; IR (KBr): 3388, 3315, 3270, 3205, 3010, 2976, 1697, 1638, 1586, 1496, 1257 cm-1; 13C NMR (75 MHz (DMSO-d6)): 168, 156, 46.
Step-III: Synthesis of Cr(II) Complexes: The Schiff's base complexes were synthesized by mixing the Schiff's base (1.5 g) in ethanolic solution of Chromium chloride [CrCl2]. This reaction is refluxed in a waterbath for two hours and their volume were reduced to 70% of it’s original volume and residue was obtained. The coloured product obtained was filtered under suction, washed with ethanol. The product were recrystallized from ethanol. Their yields ranges from 50-55%, the product obtained were light green colour and melting point was 2100C.
This diluted solution will be used in the assay as duplicate samples. Then, 1.0mL of standard glycine solution containing (7.5mg/mL) was diluted to 100mL with water using a volumetric flask. This solution contains 1.0µmole/mL of glycine. 8 tubes were set up according to the following protocol and 2.0mL of ninhydrin reagent was then added to each of the 8 tubes and were placed in a boiling water bath for 20 minutes. After 20 minutes, the tubes from the bath was carefully removed, cooled in a beaker of cold water, then 8.0mL of 50% ethanol was added and mixed well.
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
Linoleic acid peroxidation was initiated by the addition of 4 mM FeSO4.7H2O, incubated for 60 min at 37oC and terminated by the addition of 2 mL of ice cold trichloroacetic acid (10% v/v). An amount of 1 mL of thiobarbituric acid (1% w/v in 50 mM NaOH) was added to 1 mL of the reaction mixture, followed by heating at 95oC for 60 min. The reaction sample was read at 532 nm.7 The percentage of linoleic acid peroxidation inhibition activity was calculated using the following equation: % Inhibition = [(AB - AA)/AB] x 100, where AB, absorption of blank sample, AA, absorption of test sample. 2.5.4. Metal chelating activity Briefly, 2 mM FeCl2 was added to different concentrations of test sample and reaction was initiated by the addition of 5 mM ferrozine.