An ethanol extract derived from Bonnemaisonia hamifera scavenges ultraviolet B (UVB) radiation-induced reactive oxygen species and attenuates UVB-induced cell damage in human keratinocytes

Abstract

The present study investigated the photoprotective properties of an ethanol extract derived from the red alga Bonnemaisonia hamifera against ultraviolet B (UVB)-induced cell damage in human HaCaT keratinocytes. The Bonnemaisonia hamifera ethanol extract (BHE) scavenged the superoxide anion generated by the xanthine/xanthine oxidase system and the hydroxyl radical generated by the Fenton reaction (FeSO₄ + H₂O₂), both of which were detected by using electron spin resonance spectrometry. In addition, BHE exhibited scavenging activity against the 1,1-diphenyl-2-picrylhydrazyl radical and intracellular reactive oxygen species (ROS) that were induced by either hydrogen peroxide or UVB radiation. BHE reduced UVB-induced apoptosis, as shown by decreased apoptotic body formation and DNA fragmentation. BHE also attenuated DNA damage and the elevated levels of 8-isoprostane and protein carbonyls resulting from UVB-mediated oxidative stress. Furthermore, BHE absorbed electromagnetic radiation in the UVB range (280-320 nm). These results suggest that BHE protects human HaCaT keratinocytes against UVB-induced oxidative damage by scavenging ROS and absorbing UVB photons, thereby reducing injury to cellular components.

Figure 1

Scavenging effect of Bonnemaisonia hamifera ethanol extract (BHE) against free radicals. (a) Levels of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were measured spectrophotometrically at 520 nm. Intracellular reactive oxygen species (ROS) levels generated by H₂O₂ or ultraviolet B (UVB) radiation were detected using a spectrofluorometer after 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) staining. BHE was used at the indicated concentrations. N-acetyl cysteine (NAC) served as the positive control. * Significantly different from the DPPH group, ** significantly different from the H₂O₂-treated group, and *** significantly different from the UVB-irradiated group (p < 0.05). (b) Cells were seeded, and BHE was added to a final concentration of 25, 50, 100, 150, or 200 μg/mL. After 24 h, cell viability was determined using the MTT assay. * Significantly different from control (p < 0.05). (c) Representative confocal images showing the increase in DCF red fluorescence intensity produced from DCF-DA by ROS in UVB-irradiated cells compared with that in control, BHE alone-treated, and BHE-pre-treated, UVB-irradiated cells. The fluorescence intensity was quantified. * Significantly different from control and ** significantly different from UVB-irradiated cells. (d) Superoxide anions generated by the xanthine/xanthine oxidase system were reacted with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and the resultant DMPO/·OOH adducts were detected using ESR spectrometry. Representative peak data and a histogram for superoxide anion generation are shown. * Significantly different from control and ** significantly different from superoxide anion generated by the xanthine/xanthine oxidase system. (e) The hydroxyl radical generated by the Fenton reaction (H₂O₂ + FeSO₄) was reacted with DMPO and the resultant DMPO/·OH adducts were detected by electron spin resonance (ESR) spectrometry. Representative peak data and histogram for hydroxyl radical generation are shown. * Significantly different from control and ** significantly different from hydroxyl radical generated by the Fenton reaction.

Figure 2

Protective effect of BHE against UVB-induced apoptosis. HaCaT cells were treated with BHE (100 μg/mL) and exposed to UVB radiation 1 h later. (a) Apoptotic bodies (arrows) were observed in cells stained with Hoechst 33342 dye by fluorescence microscopy and quantified. * Significantly different from control (p < 0.05) and ** significantly different from UVB-irradiated cells (p < 0.05). (b) Cytoplasmic histone-associated DNA fragmentation was quantified. * Significantly different from control (p < 0.05) and ** significantly different from UVB-irradiated cells (p < 0.05).

Figure 3

Protective effects of BHE against UVB-induced damage to cellular components. HaCaT cells were treated with BHE (100 μg/mL) for 1 h and then exposed to UVB radiation. (a) Following a 24 h incubation, lipid peroxidation was assayed by measuring the levels of 8-isoprostane secreted into the culture medium; (b) Protein oxidation was assayed by measuring the levels of carbonylated protein; (c) DNA damage was assessed by conducting an alkaline comet assay. Representative images and the percentage of total DNA fluorescence in the comet tails are shown. * Significantly different from control (p < 0.05) and ** significantly different from UVB-irradiated cells (p < 0.05).

Figure 4

Effect of BHE on UVB absorption. UV/Visible light spectroscopic measurements were performed using a spectral range of 200–500 nm. Peaks 1 and 2 indicate the peak positions of absorbance at 275 and 326 nm, respectively.

Figure 5

HPLC chromatogram of BHE. The retention time of each peak was shown.