한국 해안에 자생하는 염생식물 도깨비고비(Cyrtomium falcatum)의 항산화 활성 탐색

Abstract

As technology advances and lifespans extend, humans are exposed to various reactive oxygen species (ROS). Since ROS are the cause of aging and disease, this study evaluated the antioxidant effect of the halophyte Cyrtomium falcatum, and explored its potential as a new natural antioxidant. The sample of C. falcatum used in the experiment was collected from Jeju Island, Korea, and extracted with organic solvents (methylene chloride and methanol, respectively). The combined crude extracts were fractionated according to the solvent polarity, giving n-hexane, 85% aq.MeOH, n-BuOH, and water fractions. Various experiments to measure the antioxidant activity were conducted on the crude extract and its solvent-partitioned fractions of C. falcatum. DPPH radical and peroxinitrite scavenging activities, and ferric reducing antioxidant power (FRAP) were measured at concentrations of 10, 50, 100 and 200 μg/ml. In DPPH radical scavenging activity, all solvent fractions did not show significant activity. In case of authentic peroxynitrite and peroxynitrite from SIN-1, the 85% aq.MeOH and n-BuOH fractions showed significant scavenging activity. In FRAP assay system, when the absorbance of vitamin C used as a control was 100%, the 85% aq.MeOH and n-BuOH fractions exhibited absorbances of 40.8 and 45.4% at 200 μg/ml concentration, respectively. Scavenging activity on intracellular ROS was also evaluated in HT-1080 cells. the absorbance of 85% aq.MeOH layer at 100 μg/ml was 22.7%, indicating the highest scavenging activity, when the difference between those of the control and the blank was set to 100%. Finally, protective effect of C. falcatum on genomic DNA oxidation was measured through electrophoresis. When the absorbance of the blank was set to 100%, the intensities of the 85% aq.MeOH layer and n-BuOH fractions were 77.0 and 73.7% at 100 μg/ml concentration, compared with that of the blank, respectively. The inhibitory effect against NO production related to the intracellular inflammatory reaction was also measured, and when the difference between the absorbances of control and the blank was set to 100%, the 85% aq.MeOH fraction exhibited 87.3% inhibition of NO production at 100 μg/ml concentration. In order to determine the anti-inflammatory effect of the crude extract and its fractions from C. falcatum at the mRNA levels, the RT-PCR analysis was done for mRNA expression of pro-inflammatory cytokines IL-1β, IL-6, and COX-2 in LPS-stimulated Raw 264.7 cells. As a result of the experiment, the 85% aq.MeOH fraction showed lower expression levels for IL-1β and COX-2 at 100 μg/ml concentration than the blank that did not cause inflammation by LPS. But the n-BuOH fraction inhibited mRNA expression levels of IL-1β and IL-6 more than blank at 100 μg/ml concentration. The n-hexane fraction showed expression level of COX-2 compatible to the blank value at the same concentration. Total polyphenols and flavonoids were analyzed to confirm the types of components showing antioxidant activity, respectively. As a result of measuring the total polyphenol content, the 85% aq.MeOH and the n-BuOH fractions showed 55.50 and 81.50 mg/g, respectively. For total flavonoid content, the 85% aq.MeOH and the n-BuOH fractions showed 42.34 and 56.30 mg/g, respectively. To summarize all bioassay tests mentioned above, the 85% aq.MeOH layer showed the highest antioxidant activity, and the n-BuOH layer also had good antioxidant activity. This is presumed to be the role of the polyphenol contained in the two solvent fractions, but antioxidant activity of the n-BuOH fraction with high polyphenol content was lower than that of 85% aq.MeOH fraction. This is probably because the 85% aq.MeOH fraction contains other antioxdizing components in addition to polyphenols. Based on these results, it is believed that C. falcatum has high potential as a natural antioxidant.