Flavonoid Analysis

Estimation of total Flavonoid content by AlCl3 reagent method The total flavonoid content of methanol extract of leaves of A.malabarica was determined by the AlCl3 reagent method [4]. The extract (500 µg/mL) was mixed with 0.5 mL of 5% NaNO2 solution and allowed to stand for 5 mins. Then 0.3 mL of 10% AlCl3 solution was added and the mixture was allowed to stand for further 5 min. Finally, 1 mL of 1 M NaOH solution was added, and the final volume of the mixture was brought to 5 mL with distilled water. The mixture was incubated for 15 mins at room temperature and absorbance was measured at 510 nm.

The reaction absorbance of prepared mixture absorbance was measured at 760 nm in a spectrophotometer (Jenway UK). The total concentration of phenolic compounds in the extract was determined expressed as microgram of pyrocatechol equivalent (PE) per milligram of driedy extract. The total phenolics compoundscontent was determined as the pyrocatechol equivalent using an equation obtained from a standard pyrocatechol graph (y = 0.0057 x total phenols [μg PE/mg of dry extracts] - 0.1646,

Carbon dioxide and water in the solution were also clear. Once the solution was completely titrated, Mn7+ ions remained unreduced and changed the color of the solution to pink. The KMnO4 was added to each solution until the oxalate solution reached the end point and changed to an extremely light pink color. The change in volume in the burette of the potassium permanganate recorded in all three trials was used to calculate the moles of oxalate in the 0.100-gram compound, giving the percent composition of the compound. The three trials reacted 27.95 mL, 26.61 mL, and 25.74 mL of potassium permanganate to determine 55.7%, 53.0%, and 51.3% respectively of oxalate in the compound with a 53.3% average.

Lastly, final concentrations and dilution factors were calculated by using the appropriate formulas. The final portion of the lab consisted of creating a lined scatterplot in Microsoft Excel with the absorbencies from the standard curve data chart. The chart was created to display the linear trendline, R-squared value, and slope equation. Then four sodas, Big Red, Big K Grape Soda, Faygo Red Pop, and Cherry 7 Up and one unknown sample containing red dye were processed through the absorbency tests, and diluted if necessary in a 1:1 or 1:3 ratio of water to soda. Using the equation determined from the standard curve graph, the concentrations of Red dye #40 was calculated for the sodas and the unknown liquid

The mixture was finally made upto 5 mL with distilled water and placed in hot water bath at 95ºC for 1 h. After cooling, 1 mL of distilled water and 5 mL of the mixture of n-butanol and pyridine (15:1, v/v) was added. The mixture was vortexed and after centrifugation at 4000 rpm for 10 minutes, the absorbance of the organic layer (upper layer) was measured in UV-Vis spectrophotometer (Shimatzu) at 532 nm against blank using distilled water. TBA when allowed to react with MDA aerobically formed a colored complex [MDA-(TBA) 2 complex] which was measured with spectrophotometer. MDA concentration (measured as TBARS) was calculated as

Once cool to touch the squeeze out all the tea bags carefully without tearing them apart. Using a separatory funnel extract three times with 15.0ml of dichloromethane gently rocking bath and forth the funnel venting the funnel often each time. Carefully decant into a pre-weighed 125ml flask and add the drying agent-calcium chloride pellets- and the organic layer was evaporated off in a warm water bath. Using aluminum foil as support around the mouth of the flask place test tube in the flask and heat the flask on a hot plate whilst adding water into the tube without letting it boil. Once the caffeine forms crystals around the test tube scrape off all the sublimed product and weigh the dried product 0.1grams of caffeine and had a melting point range of 175-230

2.3. Synthesis of 2-(2-(Morpholinomethyl)-1H-benzimidazol-1-yl)acetohydrazide (4) To a solution of compound 3 (0.01 M, 2.89 g) in methanol (60 mL), 99% hydrazine hydrate (1 mL) was added and the mixture was refluxed for 6 h. The reaction mixture was cooled and the solid thus obtained was filtered, washed with cold water and recrystallized with ethanol to obtain the compound 4. 2.4. General procedure for synthesis of 1-{(5-substituted-1,3,4-oxadiazol-2-yl)methyl}-2-(morpholinomethyl)-1H-benzimidazoles (5a-r) An equimolar mixture of compound 4 (0.001 M) and substituted carboxylic acid in phosphoryl chloride (POCl3) was refluxed for 8–12 h. Then reaction mixture was cooled, poured into ice-cold water and neutralized with 20% w/v NaHCO3 solution.

After 30 minutes, tubes were taken out and kept in ice-cold water for 30 minutes. These were centrifuged at 3000 rpm for 15 minutes. The absorbance of the supernatant was read at 540 nm at room temperature against appropriate blank. Blank consist of 1 ml distilled water, 0.5 ml of 30% TCA, and 0.5 ml of 0.8% TBA. TBARS values were expressed as n moles malonaldehyde (MDA)/mg protein.

In a 217 total volume of 1.5 mL, the reaction mixture contained 1 mL of the eluate, 400 mL of 218 12.5 mM 3-(dimethylamino) benzoic acid in 0.375 M phosphate buffer (pH 6.5), 80 mL 219 of 3-methyl-2-benzothiazoline hydrazone and 20 mL of peroxidase (0.25 unit). The 220 reaction was started by the addition of peroxidase and the increase in absorbance was

A further dilution was made to reach final concentration of 102.8 µg/mL, 97.2 µg/mL for VAL and SAC respectively. Four aliquots (0.1 ,0.5, 1 and 2 mL) of the diluted solution were transferred to 10 mL volumetric flask and diluted with distilled water to obtain concentrations within the linear range of each studied drug then treated as under General Procedure and the corresponding regression equations were used to calculate the recovered