New Lipophenol Antioxidants Reduce Oxidative Damage in Retina Pigment Epithelial Cells

Abstract

Age-related macular degeneration (AMD) is a multifactorial pathology and its progression is exacerbated by oxidative stress. Oxidation and photo-oxidation reactions modify lipids in retinal cells, contribute to tissue injury, and lead to the formation of toxic adducts. In particular, autofluorescent pigments such as N-retinylidene-N-retinylethanolamine (A2E) accumulate as lipofuscin in retinal pigment epithelial cells, contribute to the production of additional reactive oxygen species (ROS), and lead to cell degeneration. In an effort to develop efficient antioxidants to reduce damage caused by lipid oxidation, various natural polyphenols were structurally modified to increase their lipophilicity (lipophenols). In this study, resveratrol, phloroglucinol, quercetin and catechin were selected and conjugated to various polyunsaturated fatty acids (PUFAs) using classical chemical strategies or enzymatic reactions. After screening for cytotoxicity, the capacity of the synthesized lipophenols to reduce ROS production was evaluated in ARPE-19 cells subjected to H₂O₂ treatment using a dichlorofluorescein diacetate probe. The positions of the PUFA on the polyphenol core appear to influence the antioxidant effect. In addition, two lipophenolic quercetin derivatives were evaluated to highlight their potency in protecting ARPE-19 cells against A2E photo-oxidation toxicity. Quercetin conjugated to linoleic or α-linolenic acid were promising lipophilic antioxidants, as they protected ARPE-19 cells from A2E-induced cell death more effectively than the parent polyphenol, quercetin.

Keywords: AMD; PUFA; lipophenol; oxidative stress.

Figure 1

Synthetic pathway to access phloroglucinol-LA.

Figure 2

Synthetic pathway to access resveratrol-4′-LA.

Figure 3

Synthetic pathway to access catechin-3-LA.

Figure 4

Synthetic pathway to access quercetin-3-LA, 3-ALA and 3-DHA.

Figure 5

Synthetic pathway to access quercetin-7-ALA.

Figure 6

UV spectra of quer-7-ALA (a) and quer-3-ALA (b) under different conditions: in MeOH, with NaOAc, with AlCl₃ and with AlCl₃ + HCl.

Figure 7

Protecting effect of quercetin (a), quer-3-LA (b) and quer-7-ALA (c) against photo-oxidized A2E, represented as cell viability (%) in function of the used drug concentration (µM) i.e., cells exposed to A2E and non-treated. Results are expressed in mean ± SD and are from at least n = 3 independent experiments. Results are normalized with 100% viability for non-treated and non-exposed to A2E toxicity control cells. ¹ Concentration of lipophenol needed to inhibit 50% of the toxicity induced by photo-oxidized A2E. Mann-Whitney’s test, # p < 0.05, ## p < 0.01, ### p < 0.001, versus natural quercetin. Mann-Whitney’s test, * p < 0.05, ** p < 0.01, *** p < 0.001, versus non-treated cells exposed to photo-oxidized A2E.

Figure 8

Cytotoxicity assay: representation of ARPE-19 cell viability in presence of PUFA quercetin derivatives; natural quercetin (quer), quer-3-LA (13), quer-3-ALA (14), quer-3-DHA (15) and quer-7-ALA (18) expressed as percentage of viability in function of drug concentration introduced on cells. Results are expressed in mean ± SD and are from at least n = 3 independent experiments. Results are normalized with 100% viability for non-treated control cells. Student’s t-test or Mann-Whitney’s test, * p < 0.05, ** p < 0.01, *** p < 0.001, versus natural quercetin (quer) at equivalent concentration.