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.