5ml of the extract was pipetted into a 50ml flask and then 10ml of distilled water was added. 2ml of ammonium hydroxide solution and 5ml of concentrated amylachohol were made up to mark and left to react for 30min for colour development. This was measured at 505nm. Alkaloid determination using harborne (1973) method 5g of the sample was weighed into a 250ml beaker and 200ml of 10% acetic acid in ethanol was added and covered and allowed to stand for 4 hours. This was filtered and the extract was concentrated on a water bath to one quarter of the original volume.
In the first step, the Insertion sequence region of Mycobacterium tuberculosis complex DNA sequence, a 220 bp is amplified by specific external primers. In the second step, the nested primers are added to further amplify a 123 bp amplification product. In this assay, false positive reactions that may be caused by previous amplicon contamination are prevented by the use of uracil DNA glycosylase (UDG) and dUTP instead of dTTP added in the premix [11, 12]. Nested PCR. An amplimer of size 123 bp is indicative of infection with Mycobacterium tuberculosis complex.
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.
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.
About 0.1 ml of the sample extract was added to a volumetric flask (10 ml) containing 7.5 ml of distilled water and 0.5 ml of Folin-Ciocalteu phenol reagent, 1 ml of 35 % Na2CO3 solution and dilute to 10 ml with distilled water. The mixture was shaken well and kept at room temperature for 30 min. A set of reference standard solutions of gallic acid (20, 40, 60, 80 and 100 μg/ml) were prepared. Absorbance for test and standard solutions were measured against the blank at 725 nm with an UV/Visible spectrophotometer. The tannin content was expressed in terms of mg of GAE /g of extract
Briefly, a solution containing 0.3 gr (3 mmol) succinic anhydride and 0.4 ml (3 mmol) of triethylamine in 10 ml of THF was dropwise added to a stirred solution of 1 mmol of sPEG in 10 ml of anhydrous THF for 12 h at 75 C. The solvent of product solution was evaporated by a rotary evaporator and the obtained dark yellow viscous liquid was dissolved in acidic water (pH= 3). In the following,
The alkaline solution was extracted 3 times with 10mL of chloroform. The lower chloroform extract was combined and the upper aqueous layer for quaternary bases was reserved. The chloroform extract was evaporated to dryness in a fume hood over a steam bath. 5mL of 2M HCl was added and the solution was filtered and divided into 3 portions. 1 portion was tested with Mayer’s reagent, Wagner’s reagent and Draggendorrf’s reagent.
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
Arulmurugan et al by the condensation of 10ml of 2-amino benzyl alcohol (0.02g, 5mmol) with vanillin (0.011g, 5mmol) in ethanol (1:1 molar ratio). The mixture was then refluxed for 3h and the progress of the reaction was monitored by TLC. The product obtained was filtered, washed in distilled water, dried, and preserved in a desiccator containing CaCl2. 2.3. Preparation of the Schiff base metal (II) complexes : An ethanolic (20 ml) solution of schiff base ligand (40mmol, 0.11g ) was added drop wise to 20ml of the metal(II) salts [20mmol, 0.04g of CuCl2.4H2O, 0.048g of CoCl2.6H2O, 0.048g of CrCl3.6H2O, and 0.044g of ZnCl2.2H2O] in boiling ethanol(78.70C).
After the specified time, the solutions were completed to mark by using distilled water to achieve a final concentration of 20 µg/mL each, filtered and injected into the HPLC system. Dry Heat Degradation For thermal stress, 10 mg portions of each of VAL and SAC dry powder were placed in porcelain dish in a controlled-temperature oven at 100˚C for 4 hours. After the specified time, the content of the porcelain dish was transferred quantitatively with HPLC-grade methanol into a 10 mL volumetric flask and the volume was made to the mark by using the same solvent. Then, an aliquot of this methanolic stock was diluted to volume with distilled water to obtain a final concentration of 20 µg mL-1. After the preceding treatments, solutions were filtered and injected into the HPLC