Antioxidant profiling of new chemical entities from synthetic and natural origin

Antioxidant compounds have become essential to prevent diseases partly induced by oxidative stress, such as cancer or neurodegenerative diseases (e.g. Alzheimer, Parkinson). To further understand and characterize their antioxidant properties, the radical scavenging activity of a large set of reference antioxidants and synthetic compounds was tested against three different radicals by four 96-well microplate assays. The antioxidant activities were ranked by cluster analysis in order to define the antioxidant profile of each compound.

The first assay was realised with a protein, the alkaline phosphatase (ALP) hydrolyzing the 4-methylumbelliferyl phosphate (MUP) to a fluorescent substrate, the 4-methylumbelliferone (MU). The marker of oxidative damages was monitored by decrease of ALP's catalytic activity induced by peroxyl radicals generated by the 2,2'-azobis-(2-methlpropionamidine) dihydrochloride (AAPH). The second assay, based on the oxygen radical absorbance capacity (ORAC) was still carried out with peroxyl radicals, generated by AAPH. The marker of oxidative damages was monitored by the fluorescence decrease of fluorescein. The two last assays were spectrophotometric, the effectiveness of scavenging activity being monitored by, respectively, the absorbance decrease at 755nm for 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS^·-) and at 515nm for 2,2-diphenylpicrylhydrazyl radical (DPPH^·).

From the cluster analysis, several antioxidant groups have been constituted and the similarity of the antioxidant profile of each group compared with the antioxidant profile of reference compounds (ascorbic acid, caffeic acid, chlorogenic acid, gallic acid, glutathione, mangiferin, mannitol, melatonin, quercetin, resveratrol, trolox, uric acid). Thus for new chemical entities from synthetic or natural origin, the position in the antioxidant space with respect to the one of reference compounds can be established.