Antioxidant Effects of Protocatechuic Acid, Ferulic Acid, and Caffeic Acid in Human Neutrophils Using a Fluorescent Substance

Human neutrophils stimulated by phorbol myristate acetate (PMA), an activator of protein kinase C. produce active oxygen by NADPH oxidase in intracellular structures. We added succinimidyl ester of dichlorodihydrofluorescein diacetate (H,DCFDA), which first emits fluorescence when oxidized with active oxygen species, to neutrophils to produce active oxygen, in order to investigate the antioxidant effects of protocatechuic acid, ferulic acid, and caffeic acid which belong to polyphenols and are widely distributed among plants. Particularly, we focused on examining whether these substances capture and eliminate active oxygen inside or outside the neutrophil cytoplasm and whether these substances inhibit NADPH oxidase. Fluorescence microscopy demonstrated that fluorescence-positive intracellular structures were decreased in neutrophils when stimulated by PMA and exposed to an antioxidant. Quantitative measurement by flow cytometry revealed that the fluorescence intensities in neutrophils, exposed to protocatechuic acid. ferulic acid, or caffeic acid, were decreased by 62.9%, 71.4%, and 86.1%, respectively, as compared with those stimulated by PM A but not exposed to an antioxidant. Judging from fluorescence microscopy and dot blots by flow cytometry, these antioxidants had no effects on neutrophil morphology. On the other hand, the fluorescence intensities of the active oxygen released from neutrophils were decreased by 81.4%, 46.7%, and 27.4%, respectively. Diphenylene iodonium, a specific inhibitor of NADPH oxidase, inhibited the enzyme by 92.1% in the PMA-stimulated neutrophils. Protocatechuic acid, ferulic acid, and caffeic acid inhibited the enzyme by 36.5%, 54.6%, and 27.4%, respectively. These results demonstrate that protocatechuic acid, ferulic acid, and caffeic acid capture and eliminate active oxygen, produced by PMA-stimulated neutrophils, intracellularly and extracellularly. Furthermore, these antioxidants partially inhibit NADPH oxidase activity.