Chlorophyllin as an effective antioxidant against membrane damage in vitroand ex vivo

Chlorophyllin (CHL), the sodium-copper salt and the water-soluble analogue of the ubiquitous green pigment chlorophyll, has been attributed to have se veral beneficial properties. Its antioxidant ability, however, has not been examined in detail. Using rat liver mitochondria as model system and vario us sources for the generation of reactive oxygen species (ROS) we have exam ined the membrane-protective properties of CHL both under in vitro and ex v ivo conditions. Oxidative damage to proteins was assessed as inactivation o f the enzymes, cytochrome c oxidase and succinic dehydrogenase besides form ation of protein carbonyls. Damage to membrane lipids was measured by forma tion of lipid hydroperoxides and thiobarbituric acid reactive substances. T he effect of this compound on the antioxidant defense system was studied by estimating the level of glutathione and superoxide dismutase. ROS were gen erated by gamma-radiation, photosensitization, ascorbate-Fe2+, NADPH-ADP-Fe 3+ and the peroxyl radical generating agent, azobis-amidopropane hydrochlor ide. Our results show that CHL is highly effective in protecting mitochondr ia, even at a low concentration of 10 mu M. The antioxidant ability, at equ imolar concentration, was more than that observed with ascorbic acid, gluta thione, mannitol and tert-butanol. When CHL was fed to mice at a dose of 1% in drinking water, there was a significant reduction in the potential for oxidative damage in cell suspensions from liver, brain and testis. To exami ne the possible mechanisms responsible for the observed antioxidant ability we have studied the reaction of CHL with the potent ROS in the form of hyd roxyl radical and singlet oxygen. The compound shows a fairly high rate con stant with singlet oxygen, in the order of 1.3 x 10(8) M-1 s(-1). In conclu sion, our studies showed that CHL is a highly effective antioxidant, capabl e of protecting mitochondria against oxidative damage induced by various RO S. (C) 2000 Elsevier Science B.V. All rights reserved.