In Vitro Interaction Of Natural Phenolics And Proteins With Peroxynitrite

Peroxynitrite, an important reactive nitrogen species, is generated in vivo by rapid reaction of superoxide with nitric oxide, and it can lead to physical injury to cells. Scavenging agents against peroxynitrite and inhibition of protein nitration modification are still international highlights. In this thesis, using HPLC and MS analysis structure-activity relationship of natural phenolics scavenging peroxynitrite was studied. Peroxynitrite-injuried interferon (IFN) and bovine serum allumin (BSA) was also detected by using HPLC/MS/MS with Bioworks software.Caffeic acid and its derivatives are remarkeable peroxynitrite scavengers, and their activitivies are as follows: salvianolic acid B(27.55±0.13μM)>rosmarinic acid (37.68±0.22μM)>caffeic acid(39.60±0.41μM)>ferulic acid(45.05±0.57μM)> danshensu sodium(51.51±1.02μM)>trolox(85.44±1.68μM). 4-hydroxy on phenolic ring is essential to the activity, and 3,4-dihydroxy and acrylic acid side chain can enhance the activity. The number of 3,4-dihydroxy is proportional to the activity. Resveratrol(48.34±0.97μM) was stronger scavenger than polydatin(74.69±1.49μM). Ferulic acid and resveratrol scavenged peroxynitrite by mechanism of nitration reaction.Peroxynitrite injuried BSA and IFN in a concentration-dependent manner. Peroxynitrite attacked IFN and lead to helix A Met¹⁶ oxidation, and near helix E Tyr¹³⁵ nitration. Helix E W140 was a suspected nitration site. The modification of these three sites may affect on IFN activity.Peroxynitrite injuried protein by oxidation/nitration modification on amino acid residues, and leaded to partially decrease or totally loss protein activity. Natural phenolic compounds are powerful peroxynitrite scavengers.The results shaded insights for the further study of peroxynitrite scavengers as medicine.