참모자반 및 참치심장 에탄올 추출물의 항염증 효과 및 grasshopper ketone의 분리 정제

Abstract

Inflammation is the normal immune response to injury caused by both internal and external substances as either self or non-self antigens. And that leads to the activation of several effector cells, such as thymus lymphocytes, neutrophils, and macrophages, and the release of a variety of inflammatory mediators and finalized with the restoration of the tissue structure and function in normal condition. Macrophages and monocytes play an important role in host immune reaction, inflammation and allergic responses. It induces inflammatory reaction and initiate, and maintain specific immune responses by releasing many kinds of mediatiors including several types of cytokines. Among various causes of inflammation, macrophage activated by lipopolysaccharide (LPS), derived from cell walls of gram-negative bacteria. Upon stimulating by LPS, two major intracellular signaling pathways are activated in the macrophages: the myeloid differentiation factor 88 (MyD88)-dependent and independent pathway. The activation of the MyD88-dependent pathway results in the activation of two distinct downstream signaling pathways: the transcription factor nuclear transcription factor-κB (NF-κB) pathway and the mitogen-activated protein kinase (MAPK) pathway. These two pathways induce the expression and release of a board array of inflammatory mediators including nitric oxide (NO), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), prostagladins, interleukin-6 (IL-6), IL-1β, and tumor necrosis factor (TNF)-α and orchestrate the inflammatory responses of the host. However, their overproduction by activated macrophages has been implicated in the pathophysiology of many inflammation diseases, such as rheumatoid arthritis, atherosclerosis, and cancer. Therefore, LPS-stimulated macrophages are used as a model to study inflammation and the mechanisms of action of potential anti-inflammatory mediators. It is well known that existing non-steroidal anti-inflammatory drugs (NSAIDs) were used to treat inflammatory diseases, but NSAIDs could usually cause undesired and serious side effects, especially gastrointestinal injure. Therefore, a strong interests in developing new and more powerful drugs from various natural products has been growing. Than, the anti-inflammatory effects of Sargassum fulvellum ethanol extract (SFEE) and tuna heart ethanol extract (THEE) were investigated using LPS-induced inflammatory response in this study. The murine macrophage cell line RAW 264.7 cells were used. MTT assay was performed to measure the cell proliferation ability. The NO, TNF-α, IL-6, and IL-1β secretion were measured by enzyme linked immunosorbent assay (ELISA). The expression of iNOS, COX-2, NF-κB protein were studied by immunoblotting. The Balb/c mice were used for an in vivo acute toxicity test, and ICR mice were used for evaluation of inhibition rates of croton oil-induced mouse ear oedema. In addition, purification of grasshopper ketone was carried out by liquid and liquid extraction, silica gel column chromatography, sephadex LH-20 column chromatography, and HPLC. As a results, there were no cytotoxicity in the macrophage proliferation treated with SFEE and THEE compared to the control. NO levels decreased with increaseng concentration of SFEE and THEE. Moreover, the secretion of IL-6, TNF-α, and IL-1β were suppressed dose-dependent manner and were inhibited up to 70% at 100 μg/mL. Furthermore, iNOS, COX-2, NF-κB, MAPKs (JNK, ERK, p38) expressions were decreased by treatments of SFEE and THEE. The rate of oedema formation in the mouse ear was reduced compared to that in the control. Moreover, no mortalities occurred in mice administered 5,000 mg/kg body weight of SFEE and THEE over 2 weeks observation period. To separate the active compound of SFEE, the SFEE was partitioned successively with n-hexane, chloroform, ethyl acetate, butanol, and water. Among the solvent fractions, the hexane fration exhibited the highes activities and then the hexane fraction eluted with CHCl3/MeOH (20:1) mixture by a silica gel column chromatography exhibited the highest activity and this fracton was subjected sephadex LH-20 column chromatography twice. The highest active fraction was conducted HPLC analysis and the grasshopper ketone was isolated. These compound showed high anti-inflammatory effects of 100%, 97%, and 77% on IL-6, TNF-α, and IL-1β production at 100 μg/mL. Also, grasshopper ketone inhibited the LPS-induced phosphorylation of NF-κB and MAPKs. These result suggest that SFEE, THEE, and grasshopper ketone may contribute to the inhibitory effect on inflammation.