Chlorophyllin as an effective antioxidant against membrane damage in vitro and ex vivo

Chlorophyllin (CHL), the sodium-copper salt and the water-soluble analogue of the ubiquitous green pigment chlorophyll, has been attributed to have several beneficial properties. Its antioxidant ability, however, has not been examined in detail. Using rat liver mitochondria as model system and various sources for the generation of reactive oxygen species (ROS) we have examined the membrane-protective properties of CHL both under in vitro and ex vivo conditions. Oxidative damage to proteins was assessed as inactivation of the enzymes, cytochrome c oxidase and succinic dehydrogenase besides formation of protein carbonyls. Damage to membrane lipids was measured by formation of lipid hydroperoxides and thiobarbituric acid reactive substances. The effect of this compound on the antioxidant defense system was studied by estimating the level of glutathione and superoxide dismutase. ROS were generated by gamma-radiation, photosensitization, ascorbate-Fe(2+), NADPH-ADP-Fe(3+) and the peroxyl radical generating agent, azobis-amidopropane hydrochloride. Our results show that CHL is highly effective in protecting mitochondria, even at a low concentration of 10 microM. The antioxidant ability, at equimolar concentration, was more than that observed with ascorbic acid, glutathione, 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 examine the possible mechanisms responsible for the observed antioxidant ability we have studied the reaction of CHL with the potent ROS in the form of hydroxyl radical and singlet oxygen. The compound shows a fairly high rate constant with singlet oxygen, in the order of 1.3x10(8) M(-1) s(-1). In conclusion, our studies showed that CHL is a highly effective antioxidant, capable of protecting mitochondria against oxidative damage induced by various ROS.