염생식물 가는갯능쟁이(Atriplex gmelinii)로부터 생리활성 성분의 탐색

Abstract

As a part of our search for new biologically active substances from halophytes, their crude extracts were screened for antioxidant, antiinflammatory, cytotoxic, and MMP-inhibiting activities. The significant activities were shown for the crude extract of A. gmelinii. Specimens of the halophyte A. gmelinii were extracted twice for 24 hrs with methylene chloride (CH2Cl2) and methanol (MeOH), respectively. After removal of the solvent, the combined crude extracts were partitioned between CH2Cl2 and H2O. The aqueous layer was further fractionated between n-butanol (n-BuOH) and H2O and then the organic layer was re-partitioned between n-hexane and 85% aqueous methanol, affording four solvent-partitioned fractions. Their antioxidizing effects were evaluated using three antioxidant assay systems - DPPH, peroxynitrite, and intracellular ROS. Inhibitory effect of crude extracts and their solvent-partitioned fractions against NO production in Raw 264.7 cells were evaluated by Griess reagent assay. In addition, we measured the cytotoxicity against four kinds of cancer cells derived from human body, and conducted experiments related to MMP which is closely related to cancer. The inhibitory effect of Atriplex gmelinii against gelatinolytic activity of MMP-2 and MMP-9 secreted from HT-1080 cells was evaluated by gelatin zymography. mRNA and protein expressions of MMP-2 and MMP-9 in HT-1080 cells were also measured by polymerase-chain reaction (PCR) and Western blot, respectively. The 85% aqueous methanol and the n-BuOH fractions revealed the strong scavenging effect in antioxident assays. For the anti-inflammatory assay, the 85% aqueous methanol and the n-BuOH fraction significantly inhibited production of LPS-activated nitric oxide (NO) in Raw 264.7cells. In the anticancer assay system using the MTT reduction method and MMP inhibition experiments, the 85% aq.MeOH fraction of solvent-parititioned fractions exhibited the strongest growth inhibition against all human cancer cells. Therefore, attempt to isolate bioactive constituents from two solvent fractions showing the significant biological activity was made, and two compounds 1 and 2, including 3,5-dicaffeoyl-epi-quinic acid (1) were obtained from the n-BuOH and 85% aqueous methanol fractions, respectively.