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  • 學位論文

以脂多醣誘發 MH-S 小鼠肺泡巨噬細胞之發炎模式評估台灣產土防己乙醇萃取物之正丁醇區分物的抗發炎功效

Anti-inflammatory Effect of n-Butanol Partition from Ethanolic Extract of Cyclea gracillima Diels Cultivated in Taiwan Estimated with LPS-Stimulated MH-S Macrophage Cells

指導教授 : 葉彥宏

摘要


台灣產土防己 (Cyclea gracillima Diels Cultivated in Taiwan) 為防己科 (Menispermaceae) 土防己屬 (Cyclea) ,別名九股藤、狗股藤、烏藤仔等,分布於台灣各地中、低海拔林緣,由於其普遍被認為具有抗發炎、消腫與鎮痛之功效,亦為中藥房及青草藥店常見藥材。本實驗室過去研究已發現台灣產土防己的乙醇萃取物具有良好的抗氧化活性,但對於其抗發炎的作用及機轉的相關研究甚少,故本研究利用液-液萃取獲得抗氧化活性較佳且酚類化合物含量較高的台灣產土防己乙醇萃取物之區分物 (正丁醇區分物),並透過脂多醣 (lipopolysaccharide, LPS) 誘發 MH-S 細胞株的發炎模式,探討台灣產土防己乙醇萃取物之區分物的抗發炎活性及可能之機轉。實驗結果發現,台灣產土防己乙醇萃取物之區分物能有效降低一氧化氮合成酶 (inducible nitric oxide synthase, iNOS) 的蛋白表現量,並進而減少一氧化氮 (nitric oxide, NO) 的生成量,且能抑制促發炎細胞激素 [腫瘤壞死因子-α (tumor necrosis factor-α, TNF-α)、介白素-1β (interleukins-1β, IL-1β)] 的生成。土防己的抗發炎調控機轉為:(1) 調控轉錄因子 NF-κB (nuclear factor kappa B )途徑:抑制NF-κB p50 的蛋白表現;(2) 調控有絲裂原活化蛋白質激酶 mitogen-activated protein kinases (MAPKs):抑制 p38 、 c-Jun NH2-terminal kinase 1/2 (JNK1/2) 及 ERK1/2 (p42 and p44 extracellular signal-regulated kinases) 的活化;(3) 調控 nuclear factor erythroid-2-related factor 2 (Nrf2) / heme oxygenase-1 (HO-1) 途徑:釋放 Nrf2 在細胞核中的蛋白表現量,並進而增加 HO-1 的蛋白表現量。此外,數據亦證實台灣產土防己乙醇萃取物之正丁醇區分物對巨噬細胞不具有細胞毒性。綜上所述,台灣產土防己萃取物之區分物能有效抑制發炎反應的作用,並具有開發成抗肺發 炎製劑的潛力。

關鍵字

土防己 MH-S細胞株 NF-κB MAPK JAK/STAT Nrf2/HO-1

並列摘要


Cyclea gracillima Diels Cultivated in Taiwan belonging to Menispermaceae family is a native plant in Taiwan. It grows in the middle and low altitude areas in Taiwan. It is commonly obtained in the Chinese medicinal pharmacies or herbal shops and shows anti-inflammatory, swelling and analgesic effects in folk medicine. Our previous experiment found that the ethanolic extract of Cyclea gracillima Diels Cultivated in Taiwan had good antioxidant activities. However, very few researches had been conducted on the anti-inflammatory effects of Cyclea gracillima Diels Cultivated in Taiwan and its related mechanism in the past. In this study, we further refined the ethanolic extract by liquid-liquid partition to get the butanol partition with better antioxidant activities and higher level of polyphenolic content than the ethanolic extract. It was then used to evaluate the anti-inflammatory effect on lipopolysaccharide (LPS)-stimulated MH-S cell line. Results show that the butanol partition could suppress the expression of inducible nitric oxide synthase (iNOS) protein, production of nitric oxide (NO) and secretion of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The attenuation of LPS-induced inflammatory responses was closely related to inhibition of the NF-κB p50 subunits protein expression (NF-κB pathway), down-regulation of phosphorylation of p38, c-Jun NH2-terminal kinase 1/2 (JNK1/2) and p42 and p44 extracellular signal-regulated kinases (ERK1/2) (MAPKs pathway), and enhancement of nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein prodution (Nrf2/HO-1 pathway). Results also show that Cyclea gracillima Diels Cultivated in Taiwan is not cytotoxic to MH-S cell. According to these results, Cyclea gracillima Diels Cultivated in Taiwan could effectively inhibit the inflammatory response and should have the potential to be developed as anti-pulmonary inflammatory agents.

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