Spirulina has a very high content of h-carotene and is believed to owe some of its potent antioxidant activity to it (29). The total carotenes in it have been shown to have a bioavailability comparable to other sources. In two very large-scale human trials with preschool children in South India, spirulina feeding led to an increase in serum retinol levels (30) and a significant decrease in '' Bitot's spot,'' a symptom of vitamin A deficiency (31). This finding is very important in view of the prevalence of blindness and eye diseases among children of the Third World caused by vitamin A deficiency.
That a methanolic extract of S. maxima possesses antioxidant capacities was determined by in vitro and in vivo experiments (32). Subsequently Estrada et al. (33) fractionated a protean extract of S. platensis and showed that an increase in phycocyanin content was accompanied by a concomitant increase in antioxidant activities of the different fractions. Therefore, phyco-cyanin was identified as the component mainly responsible for antioxidant activity. However, when the antioxidant activities of phycocyanin and phycocyanobilin from S. platensis were systematically evaluated using a phosphatidylcholine liposome system, the results indicated that most of the antioxidant activity of phycocyanin is due to phycocyanobilin (34). This is an open-chain tetrapyrrole chromophore covalently attached to the apoprotein. Phycocyanobilin has a structure similar to the bile pigment bilirubin, also a known scavenger of reactive oxygen species (ROS). The radical scavenging activity of phycocyanobilin from S. platensis had been shown earlier by the same group of workers (35). Phycocyanin was also reported to protect human erythrocytes against lysis by peroxy radicals (36). It is water-extractable and both native and denatured phycocyanin possess antioxidant activities to the same extent. Spray-dried and fresh spirulina exhibit equal antioxidant abilities (34). These properties are important from the point of view of preparation of the oral food supplements in various forms.
Both the oil extract and the defatted fraction of S. maxima lowered total lipids and triacylglycerols in livers of rats with carbon tetrachloride (CCl4)-induced fatty liver indicating the presence of bioactive principles in both fractions. The authors attributed the hepatoprotective effects of spirulina to its antioxidant constituents such as selenium, chlorophyll, carotene, g-linolenic acid, and vitamins E and C (37). This observation has been confirmed by Bhat et al., who showed that C-phycocyanin significantly reduced the extent of hepatotoxicity when administered 1-3 hr before the induction of toxicity and prevented the loss of liver enzymes such as serum glutamate pyruvate transaminase (SGPT) into the serum (38). Protection was offered by phycocyanin against hepatotoxicity induced by either CCl4 or R-( + )-pulegone. The level of menthofuran, a major metabolite that appears in the urine of R-( + )-pulegone-treated rats, was significantly reduced by phycocyanin. The role of the microsomal cytochrome P-450 system in the conversion of R-( + )-pulegone to menthofuran is known. It was therefore suggested by the authors that phycocyanin interacts with cytochrome P-450 and affects the formation of 9-OH-pulegone, which acts as the precursor of menthofuran. Lowering the biotransformation of the hepatotoxins into toxic intermediates was thus proposed to be a general mechanism of protection. It was also proposed that phycocyanin scavenges haloalkane free radicals produced from CCl4 as well as reactive metabolites formed from R-( + )-pulegone (37). These ideas are in keeping with the report of Romay et al. that phycocyanin scavenges alkoxy and hydroxy free radicals (39).
More recently, S. maxima has been shown to prevent fatty liver formation in male and female mice with alloxan-induced diabetes. The administration of S. maxima to these animals reduced the levels of thiobarbi-turic acid-reactive substances in serum and liver as well as triglyceride and LDL and VLDL levels (40). Spirulina has also been shown to prevent liver fibrosis (41) and arrest the progress of chronic hepatitis into hepatocirrhosis (42).
The antioxidant and anti-inflammatory properties of phycocyanin have been established using in vitro as well as in vivo assays. It was found to have the ability to inhibit glucose-oxidase-induced mouse paw inflammation, microsomal lipid peroxidation, damage to deoxyribose, and chemilumi-nscence response of polymorphonuclear leukocytes to strong oxidants (39). When zymosan was used to induce rheumatoid arthritis in mice, the subsequent oral administration of phycocyanin for 8 days was found to reverse the histological and ultrastructural lesions and elevated h-glucuronidase levels in the arthritic animals. This was attributed to the ROS-scavenging ability of phycocyanin. The ability of phycocyanin to inhibit arachidonic acid metabolism and cytokine production was also believed to be responsible for these antiarthritic effects (43).
Administration of C-phycocyanin extract 30 min before the induction of colitis in rats showed that it is endowed with anti-inflammatory properties. This was evident from a reduction in the level of myeloperoxidase activity as well as a reversal of histological and ultrastructural features seen in inflam matory cell infiltration in the arthritic model. Reactive oxygen species such as superoxide, H2O2, OH radical, and hypochlorous acid are believed to mediate human and experimental inflammatory bowel disease (IBD). Nitric oxide and peroxynitrite have also been suggested to mediate the induction of colitis (44).
Subsequently the same group of workers have shown the antiinflammatory properties of phycocyanin in several experimental rat models such as rat paw edema induced by carrageenan, mouse ear edema induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) as well as arachidonic acid (AA) and cotton pellet granuloma (45). It was observed that the TPA-induced mouse ear edema was less effectively inhibited than that induced by AA. Since AA and its metabolites are known to take part in these models of inflammation, it was suggested that the mechanism of action of phycocyanin involves AA (45). Based on the hydroxy- and alkoxy-radical-scavenging abilities of phycocya-nin in vivo and in vitro, it was proposed that this might explain the anti-inflammatory property of phycocyanin (45).
Following these observations it was shown for the first time that both phycocyanin and phycocyanobilin are able to scavenge peroxynitrite (46,47) and protect supercoiled pBR22DNA against peroxynitrite-induced strand breakage. The results also indicated that phycocyanin is more effective than phycocyanobilin. This has been attributed to the interaction of peroxynitrite with tyrosine and tryptophan residues of apophycocyanin. Similar to the peroxy-radical-mediated transformation of bilirubin to biliverdin, the chro-mophore of reduced phycocyanin is converted to phycocyanobilin by the peroxy radical (46,47). These findings are in line with the earlier observation by Romay and Gonzalez that phycocyanin can protect erythrocytes from lysis by peroxy radicals (36).
Because damage to DNA by reactive oxygen occurs during chronic inflammation, the anti-inflammatory activity of phycocyanin is attributable to its ability to scavenge peroxynitrite radical and other reactive oxygen species that contribute to oxidative stress, an important condition in inflammation. Since peroxynitrite can be generated in vivo by reaction between superoxide and nitric oxide, in addition to DNA strand breakage, it can cause oxidative damage to lipids, proteins, and thiols. Several anticarcinogenic agents are known to inhibit oxidative damage and in this context phycocyanin might be a potential anticarcinogenic agent. Other classes ofcompounds such as flavonoids and polyphenols of plant origin are also known to protect against damage mediated by peroxynitrite (47).
Using recombinant human cyclooxygenase-2, Reddy et al. showed that C-phycocyanin is a selective inhibitor of cyclooxygenase (COX-2) (48). Thus the ability of phycocyanin to protect against hepatotoxicity, inflammation, and arthritis may be due to its inhibition of COX-2 in addition to its free-radical-scavenging ability. COX-2 produces prostaglandins from AA and induction of COX-2 is believed to occur at the site of inflammation. Cyclo-oxygenases are also activated to produce prostaglandins as a result of free-radical-mediated processes like lipid peroxidation. In fact, cyclooxygenase-mediated inflammation is believed to be one of the responses to CCl4-induced hepatotoxicity. The involvement of oxygen free radicals in rheumatoid arthritis and the use of inhibitors of AA metabolism in arthritis are well known. Thus COX-2 inhibition seems to explain the antiarthritic and antiinflammatory properties of phycocyanin. These results assume a great deal of significance in view of the introduction of COX-2 inhibitors as nonsteroidal anti-inflammatory drugs (NSAID) in the market. Spirulina is rich in g-linolenic acid (GLA), a precursor of AA, which is a proinflammatory molecule. However, recently it has been shown that dietary GLA is antiinflammatory owing to its ability to suppress leukotriene B4 release (49). Thus the presence of GLA, in addition to phycocyanin, may contribute to the anti-inflammatory properties ofspirulina. Other natural products like phytoalexin found in grapes and dietary compounds like curcumin and retinoids are known to inhibit COX-2 activity. It is interesting to note that reduced phycocyanin and phycocyanobilin, the chromphore of phycocyanin, did not possess COX-2-inhibiting ability (48). Most of the recent research described above has been carried out using purified preparations of phyco-cyanin from spirulina. It is clear from these experiments that the molecular basis of several of the medicinal properties of spirulina is the antioxidant potential of the biliprotein phycocyanin. Other nutritionally and clinically important constituents include carotenes and the polysaccharide spirulan. The mechanism of action of these has not been studied to the same extent as that of phycocyanin.
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