Bio- Modulatory Properties of : (i) C-phycocyanin, a biliprotein from spirulina platensis (ii) Novel analogues of uric acid

Abstract

Spirulina platensis, a blue-green algae is very well known for its nutritional and therapeutic properties. This algae has preventive effect on the fatty liver induced by a fructose-rich diet in the rat. C-phycocyanin, a water soluble protein pigment is one of the major constituents of Spirulina platensis. The chromophore of phycocyanin is phycocyanobilin (PCB), a linear tetrapyrrole which is structurally similar to the natural antioxidant bilirubin, a heme degradation product. Although anti-cancer and antiinflammatory properties of phycocyanin have been reported, its mechanism of action is not clearly understood. In the present study we have made an attempt to understand the biochemical basis for some of the observed pharmacological properties of phycocyanin. C-phycocyanin was isolated from fresh and spray dried spirulina, purified to homogeneity (Amax/A280 > 4.0) and fully characterized (ESI-MS, 37,468 Da, monomer, aP subunits). Detailed studies on the effect of phycocyanin on CCI4 and R-(-i-)-pulegoneinduced hepatotoxicity in rats were carried out. These studies have established: (a) phycocyanin significantly prevents CCI4 and R-(+)-pulegone-mediated hepatotoxicity in rats, (b) phycocyanin inhibits the formation of reactive metabolites from R-(-f-)-pulegone responsible for the observed hepatotoxicity, (c) phycocyanin effectively inhibits CCI4- induced lipid peroxidation in rat liver in vivo. We have also studied the radical scavenging property of phycocyanin both in vivo and in vitro. Native and reduced phycocyanin significantly inhibits peroxyl radical-induced lipid peroxidation in rat liver microsomes with an IC50 of 11.35 and 12.7 |iM, respectively. The radical scavenging property of phycocyanin was established by studying its reactivity with peroxyl and hydroxyl radicals and also by competition kinetics of crocin bleaching. Our studies suggest that the chromophore, phycocyanobilin (PCB) is involved in the antioxidant and radical scavenging activity and provides an explanation for the antiinflammatory property of phycocyanin. We have also demonstrated that phycocyanin is a selective inhibitor of cyclooxygenase-2 activity (COX-2) with potency comparable to celecoxib and rofecoxib, the known selective COX-2 inhibitors. Phycocyanin also has the ability to efficiently scavenge peroxynitrite (ONNO"), a potent physiological toxin and its chromophore, PCB significantly inhibits the ONOO'-mediated single strand breaks in supercoiled plasmid DNA in a dose dependent manner with an IC50 value of 2.9 ± 0.6 ^iM. This suggests that phycocyanin inhibits the ONOO-mediated deleterious biological effects. Studies have also been carried out on the antioxidant, radical scavenging and iron chelating properties of PCB. Our studies suggest that phycocyanin has the potential to be used as a therapeutic agent. The second part of the thesis deals with the preparation and bio-modulatory properties of 8-0x0 derivatives of various xanthine analogues and xanthine drugs. We have isolated a bacterial consortium consisting of strains belonging to the genus Klebsiella and Rhodococcus which quantitatively converts various analogues of 1,3,7-trimethylxanthine with N-1 methyl groups replaced by alkyl, hydroxethyl, benzyl, 2-oxopropyl, allyl, propargyl and butenyl groups to their corresponding 8-0x0 compounds. The enzyme responsible for this novel transformation, caffeine oxidase has been purified to homogeneity. Many of the uric acids prepared by this method are hitherto not known. These uric acids were used as test compounds to evaluate their protective potential against lipid peroxidation and ability to scavenge oxygen free radicals. These properties are compared with that observed from known methyluric acids and thus these studies provide information on structure-activity relationship. Our studies clearly indicated that 8- oxopentoxifylline and 8-oxolisofylline are significantly better hydroxyl, peroxyl and superoxide radical scavengers and more potent inhibitors of lipid peroxidation than the parent drugs. These results indicated that anti-inflammatory property of pentoxifylline and lisofylline is exerted more through their 8-0x0 derivatives than the parent drugs. We have also demonstrated that the 8-0x0 derivatives of pentoxifylline and lisofylline specifically inhibit rabbit reticulocyte 15-lipoxygenase whereas the parent drugs failed to do that suggesting that the anti-atherogenic property of pentoxifylline and lisofylline is exerted through their 8-0x0 derivatives. All these studies will be discussed in detail.