Development of a novel micro-assay for evaluation of peroxyl radical scavenger capacity: Application to carotenoids and structure–activity relationship

AbstractA micro-assay was developed and validated, using a microplate reader in 96-well format, C11–BODIPY581/591 as fluorescent probe and AIBN as ROO generator. The structure–activity relationship was established for 15 carotenoid standards, indicating that the opening of the β-ionone ring and the increase of chromophore extension in the carotenoid structure were the major factors leading to the increase of ROO scavenging capacity. The values for ROO scavenging capacity were calculated using α-tocopherol as reference compound. Among the studied carotenoids, all-trans-lycopene was the most efficient ROO scavenger (8.67±0.74) followed by all-trans-astaxanthin (6.50±0.62). All the carotenoids showed to be more effective ROO scavengers than α-tocopherol and some hydrophilic compounds. Finally, the method was successfully applied to assay the ROO scavenging capacity of carotenoid extracts from two Amazonian fruits, peach palm (7.83±0.21) and mamey (6.90±0.44)

Carotenoids Are Effective Inhibitors Of In Vitro Hemolysis Of Human Erythrocytes, As Determined By A Practical And Optimized Cellular Antioxidant Assay.

β-Carotene, zeaxanthin, lutein, β-cryptoxanthin, and lycopene are liposoluble pigments widely distributed in vegetables and fruits and, after ingestion, these compounds are usually detected in human blood plasma. In this study, we evaluated their potential to inhibit hemolysis of human erythrocytes, as mediated by the toxicity of peroxyl radicals (ROO•). Thus, 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) was used as ROO• generator and the hemolysis assay was carried out in experimental conditions optimized by response surface methodology, and successfully adapted to microplate assay. The optimized conditions were verified at 30 × 10(6) cells/mL, 17 mM of AAPH for 3 h, at which 48 ± 5% of hemolysis was achieved in freshly isolated erythrocytes. Among the tested carotenoids, lycopene (IC(50) = 0.24 ± 0.05 μM) was the most efficient to prevent the hemolysis, followed by β-carotene (0.32 ± 0.02 μM), lutein (0.38 ± 0.02 μM), and zeaxanthin (0.43 ± 0.02 μM). These carotenoids were at least 5 times more effective than quercetin, trolox, and ascorbic acid (positive controls). β-Cryptoxanthin did not present any erythroprotective effect, but rather induced a hemolytic effect at the highest tested concentration (3 μM). These results suggest that selected carotenoids may have potential to act as important erythroprotective agents by preventing ROO•-induced toxicity in human erythrocytes.79H1841-