Symposium-in-Print Anthocyanins Protect Against A2E Photooxidation and Membrane Permeabilization in Retinal Pigment Epithelial Cells{

The pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent-like perturbation of cell membranes and light-induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon–carbon double bonds. To assess the ability of plant-derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent-like action of A2E. INTRODUCTION It is widely held that diets rich in fruits and vegetables can defend against some forms of degenerative disease. The protective effects are usually attributed to the antioxidant vitamins E and C and to carotenoids such as b-carotene. There are, however, other phytochemicals that may play a significant preventive role, one of these being anthocyanin (1). The latter polyphenolic compounds are water-soluble pigments that account, in part, for the red, purple and blue colors of fruits and flowers. Anthocyanins belong to the flavonoid family of compounds and occur in nature as glycosides (anthocyanosides). A particularly rich source of anthocyanins is the bilberry plant (Vaccinium myrtillus, Ericaceae), extracts of which are used as nutritional supplements (2). Although all anthocyanins have the structure of 2-phenylchromenylium salts (Fig. 1), the nonsugar portion of the compound occurs naturally in 6 different forms, all varying in the number and arrangement of hydroxyl and methoxyl groups (3). In addition, each of these three-ringed structures can be glycosylated by different sugars, the most common being glucose, although galactose, rhamnose, arabinose and xylose residues are also found. Orally administered anthocyanins are absorbed within the gastrointestinal system of humans in their unchanged glycosylated forms (3) and have been shown to be incorporated into cultured endothelial cells when delivered in the medium (4). Because the structure of anthocyanins is consistent with an ability to donate hydrogen atoms, it has been speculated for some time that these compounds have antioxidant capability, in vivo. Accordingly, anthocyanins have been shown to scavenge chemically generated superoxide (5–7) and nitric oxide radicals (5), to diminish H2O2– mediated cytotoxicity (4) and to reduce a-tocopheroxyl radical (TocO ) to a-tocopherol (TocOH) (8). The in vitro antioxidant activities of anthocyanins have even been reported to supersede that of vitamin E (9,10). On the other hand, anthocyanins do not exhibit an ability to induce NAD(P)H:quinone oxidoreductase (2), a Phase 2 enzyme that inactivates carcinogenic electrophiles and exhibits antioxidant activity (11). Although there is as yet no evidence that bilberry extracts can protect or improve vision (12), one of the degenerative disorders for which these extracts are marketed is age-related macular degeneration (AMD). The etiology of atrophic AMD is still poorly understood, but it is generally accepted that AMD begins with the death of retinal pigment epithelial (RPE) cells, the degeneration of photoreceptor cells and the resultant loss of vision following {Posted on the website on 3 March 2005 *To whom correspondence should be addressed: Department of Ophthalmology, Columbia University, New York, NY 10032, USA. Fax: 212305-9638; e-mail: [email protected] Abbreviations: AMD, age-related macular degeneration; cya-ara, cyanidin 3-arabinoside; cya-gal, cyanidin 3-galactoside; cya-glc, cyanidin 3glucoside; DAPI, 49,6-diamidino-2-phenylindole; DPBS, Dulbecco’s phosphate-buffered saline; del-ara, delphinidin 3-arabinoside; del-gal, delphinidin 3-galactoside; del-glc, delphinidin 3-glucoside; ESI, electrospray ionization; FAB-MS, fast atom bombardment ionization mass spectrometry; HEPES, N-(2-hydroxyethyl) piperazine-N9-ethanesulfonic acid; HPLC, high-performance liquid chromatography; LCMS, liquid chromatography–mass spectrometry; mal-ara, malvidin 3-arabinoside; mal-glc, malvidin 3-glucoside; pet-glc, petunidin 3-glucoside; Q-TOF, quadrupole time-of-flight; RPE, retinal pigment epithelium; TocO , atocopheroxyl radical; TocOH, a-tocopherol. 2005 American Society for Photobiology 0031-8655/05