Enzymatic conjugation with fatty acids including omega-3 polyunsaturated fatty acids (ω-3 PUFAs) derived from fish oil to three citrus fruit-derived flavonoids: grapefruit extract, naringin, and neohesperidin dihydrochalcone were investigated. The conversions were achieved over 85% under the catalysis of lipase Novozyme 435 in acetone at 45°C at semi-preparative scale. The conjugates were purified via solvent partition and silica gel chromatography and achieved 90–98% in purity. The NMR analysis of the conjugates confirmed that the fatty acid carbon chain was linked onto the primary –OH group on the glucose moiety of the flavonoids. The purified flavonoid conjugates alongside their original flavonoids were analyzed for antioxidant activities via 2,2-diphenyl-1-picrylhydrazyl scavenging assay, and anti-peroxidation test via peroxide values measured during a 1-week fish oil storage trial. Vascular endothelial growth factor (VEGF) assay was conducted with 1, 10, and 100 μM of naringin and grapefruits and their conjugates, respectively, and total VEGF levels were measured at 24 and 48 h, respectively, using ELISA and dot blot analysis. The results from these functionality experiments demonstrated that flavonoid FA conjugates have at least comparable (if not higher) antioxidant activity, anti-peroxidation activity, and anti-angiogenic activity.
Poly ethoxy ethyl glycinamide (PEE-G) dendrimers have been specifically designed and synthesized with the aim of providing a readily available dendrimer scaffold that can be used to make products that can meet the stringent requirements of pharmaceutical applications. The synthesis has been refined to produce dendrimers that are of high HPLC purity. The suitability of PEE-G dendrimers for their designed use has been verified by subsequent measurements to demonstrate that they are of high stability, high aqueous solubility, low cytotoxicity, low immunogenicity and with low in vivo toxicity in an escalating-dose rat study. PEE-G dendrimers therefore provide a useful scaffold for researchers wanting to develop dendrimer-based drug candidates.
Chymotrypsin was isolated from ovine and porcine pancreas by affinity chromatography on immobilised 4-phenylbutylamine. The apparent molecular weights of the two proteins were estimated by SDS PAGE to be 25.7 and 27.3 kD respectively. Specific activities for ovine and porcine chymotrypsins (OC and PC) were 32.8 and 31.2 µmol/min per mg, respectively, at pH 8.5 and 25°C using 0.1 mM N-succinyl-Ala-Ala-Pro-Phe-pNA (SAAPFpNA) as substrate. Values of the Michaelis constants for ovine and porcine chymotrypsins with SAAPFpNA were comparable at pH 8.0 and at temperatures between 20 and 45ºC, as were the k cat values. Both enzymes were stable under acidic conditions but were susceptible to thermal denaturation above 45°C. Hydrolysis of lysozyme and casein by ovine or porcine chymotrypsin yielded very similar fragmentation profiles as determined by RP-HPLC.
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