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Pre-Clinical In-Vivo and In-Vitro Methods For Evaluation of Anti-Alzheimer’s Drugs
Published in Atanu Bhattacharjee, Akula Ramakrishna, Magisetty Obulesu, Phytomedicine and Alzheimer’s Disease, 2020
Shilpa A. Deshpande, Niraj S. Vyawahare
An aliquot (1.0 ml) of the supernatant from the brain homogenate is evaporated to dryness at 70°C in an oven and the residue is reconstituted in 100 ml of distilled water. A standard solution of glutamate at a concentration of 2 mM and the samples are spotted on chromatography paper using a micropipette. The paper is placed in a tank containing butanol: acetic acid: water (12: 3: 5 v/v) as the mobile phase. After chromatography is completed, the paper is removed, dried, and sprayed with ninhydrin reagent and placed in an oven at 100°C for 4 minutes. The portions which contain glutamate corresponding with the standard are cut and eluted with 0.005% CuSO4 in 75% ethanol. Optical density (OD) is read against the blank at 515 nm in a spectrophotometer.
Dialyzable and Nondialyzable Transfer Factor
Published in Edward P. Cohen, A. Arthur Gottlieb, Immune RNA, 2020
The composition of the five pools differed greatly (Table 1). Protein concentration ranged from 3.3 to 31.8 mg. The concentration of orcinol-reactive material varied from 0.29 to 2.03 mg. On thin-layer silica gel chromatography (Figure 1), the samples of the five pools were observed to have the same number of components. A minimum of ten components was detected. However, there were obvious differences in the intensity of staining with ninhydrin that did not reflect the concentration of protein (Table 1).
Amino Acid Analysis
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
Most analytical systems are based on the detection of the respective amino acids in the effluent from the analytical columns by reaction with ninhydrin.7 The ninhydrin systems in use are based either on a methyl cellosolve solvent7-9 or on dimethyl sulfoxide.49 Our laboratories use the dimethyl sulfoxide-based system as we find this reagent more stable on storage, the dimethyl sulfoxide does not have the severe toxic properties of methyl cellosolve and the color yields for most amino acids are superior. The primary problem in the use of the ninhydrin system is concerned with reagent preparation. The source of ninhydrin is critical and we have found that only Pierce Chemical Company, Rockford, Ill., has continually provided material of excellence. We have, on occasion, used material from other sources which has had equivalent performance but there was extensive batch-to-batch variation.
Evaluating the role of gamma irradiation to ameliorate salt stress in corn
Published in International Journal of Radiation Biology, 2023
Alireza Shaebani Monazam, Mohammad Ali Norouzian, Mehdi Behgar, Azam Borzouei, Hedayat Karimzadeh
Proline content was measured based on the method described by Bates et al. (1973). To this end, 0.125 g of ninhydrin was added to 2 ml of glacial acetic acid. The solution was then heated to dissolve the ninhydrin in acid completely. Then, 2 ml of 6 M phosphoric acid was added to the solution, and the solution was refrigerated at 4 °C for 24 h. In the next stage, 50 mg of fresh plant material was homogenized in 5 ml of sulfosalicylic acid solution (3%), and the solution was filtered with Whatman paper. Afterward, 2 ml of the filtered extract was mixed with 2 ml of ninhydrin acid solution and 2 ml of glacial acetic acid and kept at 100 °C for 30 min. After cooling, 4 ml of toluene was added to each tube. Finally, the red phase was placed in a spectrophotometer simultaneously with the standard samples. The absorbance was read at 520 nm, and the proline concentration was calculated in each sample in micromoles of proline per gram of fresh weight using a standard curve; Proline = 1136.1*Abs520 − 49.273.
Fumarate exerted an antihypertensive effect and reduced kidney injury molecule (KIM)-1 expression in deoxycorticosterone acetate-salt hypertension
Published in Clinical and Experimental Hypertension, 2021
Osaze Edosuyi, Myung Choi, Ighodaro Igbe, Adebayo Oyekan
The previously described method (19) was modified and applied. Briefly, 40 µg of renal cortex/medulla was incubated with 1 M of magnesium chloride (MgCL2) at 37°C for 30 minutes. This was followed by the addition of 0.5 mL of L-arginine buffered solution with further incubation for 1 hour at 37°C. Lastly, 0.05 mL of Ninhydrin dissolved in 0.45 mL of acetic acid and 0.05 mL of phosphoric acid solution (developer solution) was added and samples were incubated for 1 hour at 95°C. Samples were kept at 37°C for 15 minutes and read spectrophotometrically at 530 nm. The same procedure was repeated for standard concentrations of ornithine (1, 5, 10, 25, 50 and 100 µM). Blank solution contained 0.01 mL of 1 M (95 mg/ml) magnesium chloride + 0.49 mL of L-arginine buffer + 0.5 mL of Developer solution.
Structural and biological investigation of chitosan/hyaluronic acid with silanized-hydroxypropyl methylcellulose as an injectable reinforced interpenetrating network hydrogel for cartilage tissue engineering
Published in Drug Delivery, 2021
To assess weight loss due to degradation, in vitro degradation studies of the composite CS/HA and CS/HA/Si-HPMC hydrogels were conducted. First, 1.0 mL of each sample was put into a tube and incubated for 3 h at 37 °C to complete the gelation. Then 6.0 mL of PBS (pH∼7.4) containing the enzyme alpha-amylase was applied to the tube. Under shaking conditions (37 °C), the sample solution was maintained for another period (0–200 h). 1 mL of sample solution was taken from the tube at each interval and balanced with a fresh medium immediately. A ninhydrin assay has determined the existence of free amino acids. Finally, the solutions were measured by a Lambda 35 UV-Vis spectrophotometer (Perkin-Elmer, USA) for their absorbance at 570 nm. Afterward, the samples were lyophilized and weighed (Wt). The percent weight loss (W%) was used to calculate the hydrogel degradation under the following Equation (2):