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S. Jung, H. Lee, K.-A. Kim, E. Lee, C. Kim, B. Um; Identification of Radical Scavenging Compound From the Leaves of Phyllostachys nigra by Online HPLC-ABTS+ and Its Neuroprotective Effects Against Oxidative Stress in Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4742.
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This study was to determine whether the compound isolated from Phyllostachys nigra, black bamboo could attenuate oxidative stress induced transformed retinal ganglion cells (RGC-5 cells) death.
RGC-5 cells in culture were given two different insults such as l-buthionine-(S,R)-sulfoximine (0.5 mM) plus glutamate (10 mM) for 24 hours or hydrogen peroxide (300 µM for 24 hours), after which cell survival were tested. For a rapid assessment of pure antioxidant compound, online-HPLC-ABTS+ method were used. We isolated compounds from P. nigra using preparative RP-HPLC and Sephadex LH-20 column chromatography and elucidated chemical structures using 1H and 13C NMR. ROS quantification were performed by using the fluorescence probe, DCFH -DA, and we tested glutathione level based on the reaction of glutathione with 5,5’-dithio-bis(2-nitrobenzoic acid). Apoptotic cell death were measured by propidium iodide/Hoechst 33342 double staining and western blot analysis
Four systematic fractions from 95% ethanol extract were tested for protective effect in oxidative stress induced RGC-5 cell death. Among them, ethyl acetate fraction showed a significantly higher inhibition which was concentration dependent manner. The detected antioxidant (compounds 6) by online-HPLC-ABTS+ method and other seven compounds were isolated. Their chemical structures were elucidated as isoorientin (1), orientin (2), vitexin (3), p-coumaric acid (4), cis-coumaric acid (5), luteolin 6-C-(6''-O-trans-caffeoylglucoside) (6), vittariflavone (7) and tricin (8).The luteolin 6-C-(6''-O-trans-caffeoylglucoside) (compound 6) significantly attenuated the negative insult of BSO plus glutamate and hydrogen peroxide to RGC-5 cells. Treatment of the RGC-5 cells with compound 6 reduced the reactive oxygen species and recovered reduced glutathione level.Moreover, compound 6 attenuated apoptosis caused by two different models of oxidative stress.
These results indicate that compound 6 isolated from P. nigra could be offered as leading compounds for retinal disease via antioxidative and antiapoptotic effects.
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