| Peroxynitrite(ONOO￣) is a kind of strong oxidizability and nitrification cytotoxic substance in organisms, which is formed by the rapid combination of nitric oxide and superoxide in vivo. Under physiological conditions, ONOO￣ can be protonated to be peroxynitrite(ONOOH). It could cross the cell membrane and reach the position, where ·NO and ·OH can not, to damage biological targets. ONOO￣ can induce nitration of amino acid residues, oxidation of membrane lipid and breaks of DNA strands. It could influence cell signaling pathway and energy metabolism, cause cell apoptosis and lead a series of diseases such as rheumatoid arthritis, sepsis, cancer and Parkinson’s disease. Due to the lack of endogenous enzymes responsible for ONOO￣ inactivation, there has been considerable interest in developing effective and low toxicity topical natural herbs to diminish damage of ONOO￣. Recently, many natural products have been found to inhibit bio-macromolecules from ONOO￣. However there is no unified system to evaluate its inhabitation ability because of rich species.In this study, we selected pinus massoniana lamb, rosmarinus officinalis and echinacea moenck as the research materials, which have been proved to be effective, so we extracted procyanidins, rosmarinic acid and chicoric acid from them respectively. High performance liquid chromatography, agarose gel electrophoresis and electron spin resonance(ESR) combined with spin-labeling technique were employed to explore the ability of procyanidins, rosmarinic acid and chicoric acid to inhibit 3-nitrotyrosine generation, DNA strand breaks and free radical generation, compared with vitamin C. Based on the free radical theory, the results indicate that procyanidins, rosmarinic acid, chicoric acid have different scavenging activities on ·OH and ·NO2. Tyr nitriction induced by ONOO￣ half maximal inhibitory concentration of procyanidins, rosmarinic acid, chicoric acid and vitamin C were 75.3 μM, 64.5 μM, 54.9 μM and 346.7 μM, the inhibition activity was as the following order chicoric acid> rosmarinic acid> procyanidins> vitamin C. As for DNA strand breaks, the half maximal inhibitory concentration were 10.65 μM, 10.03 μM, 9.39 μM and 26.9 μM, inhibition activity was same as above, while procyanidins> chicoric acid> rosmarinic acid> vitamin C in large dose, for procyanidins polymer could bind with DNA reversibility through hydrogen and form a complex to enhance the stability of DNA. Radical scavenging, half maximal inhibitory concentration were 1.0005 μg/mg, 0.9939 μg/mL, 0.8850 μg/mL and 1.3346 μg/m L separately and the order was chicoric acid> rosmarinic acid> procyanidins> vitamin C. The scavenging activity was associated with the structure of the ingredients. According to the results above, we found ·NO2 was the key in DNA strand breaks; Vitamin C exhibited a strong scavenging activity on·OH, but weak on ·NO2.Ortho phenolic hydroxyl is an important factor in its strong scavenging ability of peroxynitrite. Phenolic hydroxyl could release H+ and form a stable semiquinone-type when combined with oxide and free redicals. Intramolecular hydrogen could stabilize the free radical intermediates. C=C and carbonyl could extended the conjugation system. The conjugation of p-π could disperse electron cloud. These groups in molecules could promote its peroxynitrite scavenging ability. Blocking free radical chain reaction through free radical capture and inhibiting metal ions catalytic ability by complexation are the possible mechanism of procyanidins, rosmarinic acid and chicoric acid scavenge peroxynitrite.The ability of procyanidins, rosmarinic acid and chicoric acid in inhibiting ONOO￣ was investigated, and reasonable explanations were given to initially clarify the inhibition mechanism. Based on this study, theoretical supports were provided to prevent ONOO￣ damage and develop relevent drugs. |
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