5-aminotetrazole monohydrate: In a 250 ml round-bottom flask equipped with a condenser for refluxing (90 °C) and a magnetic stirring bar, 5.00 g (5.95 mmol) dicyandiamide (three times crystallized), 7.47 g (11.9 mmol) sodium azide and 11.00 g (17.8 mmol) boric acid and 100 ml of water is added and allowed to reflux for 24 hours, after the completion of the reaction, until the solution pH to about 2 to 3 as hydrochloric acid 37% is added (about 12 ml) Then the reaction mixture was cooled in a refrigerator for 18 hours and the white crystals formed. The mixture was filtered and washed three times with 10 ml of water and and dried in 60 °C for 5 hours and finally 45.8 g of product by it will be obtained. 5-Aminotetrazol monohydrate: Yield:,
The minimum inhibitory concentration of abietic acid was studied by broth micro dilution method using 96-well microtitre plates.9 Test compound was dissolved in DMSO (1%) with the addition of Tween-80(0.5%) and diluted in Muller Hinton Broth to get a concentration range of 100-1000μg/mL. The solution was then two fold diluted in Muller Hinton Broth (100 μL), inoculated with bacterial strains and then incubated at 37°C for 24 h. The bacterial growth was measured as turbidity with a Cyberlab microplate reader at 405 nm. The minimum inhibitory concentration was defined as the lowest concentration of test compound that inhibits the growth of the test bacteria. DMSO assayed as the negative control at a concentration of 1% did not inhibit any of the strains tested. All tests were assayed in triplicate in three independent experiments and median values were used for MICs calculation.
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.
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.
Two ml of washed pack cells and 250 unit of FVIII were put into a dialysis bag (Sigma). Dialysis bag presoaked for 4 h in 50 ml PBS buffer containing 5 mM glucose. The bag was placed in a bea¬ker equipped with a magnetic stir bar. The beaker filled with 100 ml of hypoosmotic buffer containing 0.0451 mM KH2PO4/KOH, 0.1 mM MgCl2, 0.22 mM glucose, and 0.2 mM of adenosine triphosphate; pH 7.45. Dialysis was
The phosphoprotein was hydrolysed by HCl digestion. Samples of the phosphoprotein residue were hydrolysed in 2N HCl in vacuum-sealed tubes at 100C for 10 hr. The resulting digests were evaporated in vacuo and dissolved in 0.5 ml of water. This was then applied to a Dowex 50x8 mm H+ columns. The phosphoamino acids were eluted with water and dried in vacuo.
Effect of pH To check the pH stability of the bacteriocins, 5 ml aliquots of CFS was taken in sterile tubes and were adjusted to pH values of 2, 4, 7 and 9 busing 1N HCl or 1 N NaOH. After incubation at room temperature for 30 minutes, pH in each tube was readjusted to 6.5 and the antimicrobial activity was determined by agar well diffusion method17. 6.3. Effect of proteolytic enzyme 5 ml of CFS was taken in a test tube and was treated with trypsin-chymotrypsin enzyme (10AU/ml) at pH 7. The test tubes with and without enzyme (control) were incubated at 370C for 1 hour.
0.825 g Na2WO4H2O (2.5 mmol), desired amounts of acidic ligand and 44.5 mL 30% hydrogen peroxide (440 mmol) placed in an 100 mL flask, stirred for 10–15 min to get a clear solution at room temperature. Then 10.5 mL cyclohexanone (100 mmol) added into it without stopping stirring. Continue stirring the reaction mixture at a reflux temperature for 8 h. After completion of the reaction, the reaction mixture cooled in the refrigerator for 12 h. Then white crystalline product obtained after filtration, washed with an ice water and dried in the air. The product weighted and the isolated yield of the adipic acid calculated which is based on the cyclohexanone that was put in the reaction flask. Mechanism: Experiment No.
The peel powder was soaked overnight (or for desired period) at room temperature (30-32°C) for extraction with intermittent shaking. After extraction, it was centrifuged at 2000 rpm for 5 min and filtered through Whatman No. 2 filter paper. The effective volume obtained after centrifugation was noted. 2.3 Determination of radical scavenging activity (RSA) Determination of RSA was carried out according to Murthy, Jayaprakasha, & Singh (2002) using DPPH as stable free radical and butylatedhydroxyanisole (BHA) as standard.
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.