Antioxidative Property of Phycocyanin (Major Constituent of Spirulina) in Different Test Systems (Redrawn from Romay et al., 2003)
Reaction system Effect
Superoxide generated from hypoxanthine-xanthine oxidase No effect
Alkoxyl radical generated from i-BOOH-ferrous sulfate Scavenge
Hydroxyl radical generated from hydrogen peroxide-ferrous sulfate (Fenton reaction) Scavenge
Peroxyl radical generated from AAPH thermolysis Scavenge
Singlet oxygen Quench
Lipid peroxidation induced by Fe+2-ascorbic acid and AAPH thermolysis Inhibit
Peroxynitrite generated from nitrite acidified hydrogen peroxide Scavenge
Reactive oxygen production from neutrophils stimulated with opsonized zymosan Inhibit anion and all other different deleterious free radicals. Administration of hepatotoxic, nephrotoxic, and neurotoxic chemicals resulted into generation of free radicals and Spirulina, as an extract and its important constituent, phycocyanin, scavenge these free radicals.
Scavenging of alkoxyl and hydroxyl radicals by phycocyanin was demonstrated using a chemiluminescence (CL) assay.27 Determination of alkoxyl radical scavenging activity of phycocyanin was performed by measuring the inhibition of the CL produced by the reaction of ieri-butyl hydroperoxide with ferrous ions in the presence of luminol. Exposure of phycocyanin to peroxyl radicals generated by thermolysis of AAPH leads to a progressive loss of the chromatography. Phycocyanin is considered pure when the absorption ratio of visible maximum to 280 was greater than 4.23 The inhibition of CL produced by the Fenton reaction with luminol was used to evaluate the phycocyanin scavenging capacity against hydroxyl radicals. In this system the CL signal was inhibited in a dose-dependent fashion by increasing phycocyanin concentrations. It was reported that 24.7 mM of phycocyanin caused the same inhibition (50%) as 1.6 mM of dimethyl sulfoxide, a specific hydroxyl radical scavenger used as control. Hydroxyl radical scavenging capacity of phycocyanin has also been assayed by the inhibition of damage to 2-deoxyribose. In this system, phycocyanin inhibited deoxyribose damage in a concentration-dependent fashion. The IC50 values reported for phycocyanin using this method were 19 mM and 28 mM.27 Bhat and Madyastha,28 also demonstrated the involvement of the bilin chromophore in the radical scavenging activity of phycocyanin by studying the reactivity of the protein with peroxyl radicals derived from AAPH thermolysis. It was also shown that both native phycocyanin and the reducing form (using NaBH4) are able to scavenge per-oxyl radicals.29 This was supported by the fact that when reduced phycocyanin was incubated with AAPH (10 mM) at 37°C, there was a rapid decrease in the absorption at 418 nm with a concomitant appearance of peaks at 618 and 360 nm in the UV-visible spectrum indicating the oxidation of phycocyanorubin to phycocyano-bilin (PCB) by peroxyl radical.26,29 These authors, using the competition kinetics of crocin bleaching by peroxyl radicals, also analyzed the interaction of peroxyl radical with phycocyanin and its ability to scavenge this radical. These studies demonstrated that phycocyanin is a potent peroxyl radical scavenger with an IC50 of 5.0 mM. Under these experimental conditions, uric acid, a known peroxyl radical scavenger had an IC50 of 1.9 mM. The rate of constant ratios obtained for phycocyanin and uric acid were of 1.54 and 3.5, respectively. It also has been reported that phycocyanin is able to protect human erythrocytes against lysis induced by peroxyl radicals. In this assay phycocyanin (12-75 mM) inhibited erythrocyte haemolysis in the same way as trolox and ascorbic acid, well-known antioxidants.30 On the basis of IC50 values, phycocyanin proved to be almost 16 times more efficient as an antioxidant than trolox and about 20 times more efficient than ascorbic acid. The scavenging of ONOO(-)by phycocyanin and its bilin chromophore was also evaluated using competitive kinetics of pyrogallol red bleaching assays. Pyrogallol red is one of the more efficient dyes that can be used to evaluate the ONOO(-) scavenging activity of any compound in aqueous solution.31
Lipid peroxidation mediated by ROS is believed to be an important cause of destruction and damage to cell membranes, because a simple initiating event can result in the conversion of hundreds of fatty acids side chain into lipid peroxides, which alter the structural integrity and biochemical functions of membranes. It has been shown that phycocyanin, an important antioxidant constituent of Spirulina, significantly inhibits the increase in lipid peroxides of rat liver microsomes after treatment with Fe+2-ascorbic acid 27 or the free radical initiator AAPH.28 Addition of phycocyanin (200-540 mM) to isolated microsomes in the presence of Fe+2-ascorbate resulted in a concentration dependent decrease in thiobarbituric acid reactive substances (TBARS) as an index of hepatic lipid peroxidation. The calculated IC50 was 327 mM. Thus, phycocyanin reduced both the rate and the final extent of lipid peroxidation. The phycocyanin effect on peroxyl radical-induced lipid peroxidation in rat liver microsomes also has been studied. It was demonstrated that phycocyanin inhibits the azo-initiated microsomal lipid peroxidation in a concentration-dependent fashion with an IC50value of 11.35 mM. Phycocyanin at 200 mM concentration inhibited nearly 95% of peroxyl radical induced lipid peroxidation.32 Reduced phycocyanin also efficiently inhibited this reaction with an IC50 value of 12.7 mM. In fact both native and reduced phycocyanin inhibited lipid peroxidation almost to the same extent. In correspondence with these results it was demonstrated that phycocyanin also reduced CCl4-induced lipid peroxidation in vivo. Intraperitoneal administration of phycocyanin (50-200 mg/kg), 3 h prior to CCl4 treatment resulted in significantly lower production of malondialdehyde (MDA) than was found in rats receiving only CCl4. It is known that in CCl4 intoxication, free radicals arising from its biotransformation induce lipid peroxidation. The trichloromethyl radical (CCl3) initially formed is relatively nonreactive and this carbon-centered radical readily reacts with O2 to form a peroxyl radical that is a good initiator of lipid peroxidation. Since it was demonstrated that phycocyanin did not alter the liver function and the cytochrome P450 system, the protection by phycocyanin against CCl4-induced lipid peroxidation may not be related to a reduced formation of reactive metabolites of CCl4, but to the ability of phycocyanin to scavenge peroxyl radicals.28
Modulation of Metabolizing and Detoxification Enzymes
Spirulina has modulatory effect on the various drug metabolizing and detoxifying enzymes as well as antioxidant enzymes. In one study, the effect of 250 and 500 mg/kg of Spirulina was examined on drug metabolizing phase I and phase II enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase (LDH) in the liver of 7-week-old Swiss albino mice.33 Primary findings of the study reveal the "monofunctional" nature of Spirulina as deduced from its potential to induce only the phase II enzyme activities is associated mainly with carcinogen detoxification. The glutathione S-transferase and DT-diaphorase specific activities were induced in hepatic and all the extrahepatic organs examined (lung, kidney, and fore stomach) by Spirulina pretreatment.33 With reference to antioxidant enzymes, namely, superoxide dismutase, catalase, glutathione reductase, glutathione peroxi-dase, and reduced glutathione were increased significantly by both the chosen doses of Spirulina.27
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Detoxification is something that is very important to the body, but it is something that isn't understood well. Centuries ago, health masters in the East understood the importance of balancing and detoxifying the body. It's something that Western medicine is only beginning to understand.