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Titel: 以巨噬細胞株為模式探討苦瓜中影響前列腺素E2合成之區分物
Investigation of bitter gourd Fractions with inhibitory effect on Prostaglandin E2 Production in Macrophage Cell Line
Autoren: 吳文惠
黃青真
林弼玉
Stichwörter: Momordica charantina L.
RAW264.7
PGE2
Anti-inflammatory
Momordica charantina L.
RAW264.7
PGE2
Anti-inflammatory
Erscheinungsdatum: 2004
Zusammenfassung: 發炎反應與多種疾病的病程進展及組織損傷密切相關,抗發炎作用的相關因 此具有相當的重要性,也受到學界之重視。已知發炎過程中,涉及巨噬細胞的活 化,誘發COX-2 表現,主導PGE2的合成。PGE2為巨噬細胞主要產生的前列腺素, 為一重要促發炎介質。因此,透過降低發炎介質PGE2的合成,應可減輕發炎所產 生不適症狀,乃成為抗發炎藥物研發之目標。而研究中發現:以LPS誘發巨噬細胞 株表現COX-2 及生成PGE2下,苦瓜萃取物具有抑制PGE2生成的效應。因此,推測 苦瓜萃取物中可能含有抑制發炎反應的成分。故本研究擬以LPS活化之巨噬細胞為 模式,透過對萃及管柱層析等化學分離的方式,追蹤山苦瓜中能抑制PGE2合成的 區分物,以進一步分離並鑑定山苦瓜中具有此活性的成分。 將苦瓜以水萃取,渣再以乙酸乙酯萃取,兩種萃取物均有抑制活性,其中以 乙酸乙酯萃取物較高。將山苦瓜全果凍乾,以乙酸乙酯萃取;萃取物之抑制活性 可以正己烷對萃而得。正己烷區分物以矽膠層析,從100%正己烷逐漸增高極性至 100%乙酸乙酯沖提,而後再以90%乙酸乙酯與10%甲醇之混合溶劑沖提,可沖提 出活性最佳的區分物Fraction190。此區分物再經矽膠管柱層析,以氯仿/甲醇(6/1) 流洗,可分離出具活性之區分物F1-6。將F1-6 再度以矽膠管柱層析二次,所得之 活性區分物再以逆相管柱層析,得活性區分物RP-10,具極佳之抑制活性,IC50為 2.31µg/mL。以NMR、IR及H-H Cosy化學鑑定之結果為:含中短鏈脂肪酸之三酸甘 油酯。而將F1-6 水解後,尚可鑑定出當中含有:八碳雙酸、九碳雙酸及十碳雙酸。 此外,以3-20 碳之脂肪酸標準品測試發現:十碳之Capric acid (C10:0)具有最好的 抑制活性,50µM可達到完全抑制,IC50為6.46µM (1.216µg/mL)。八碳及九碳雙酸 標準品亦有部份抑制活性,但不如10 碳脂肪酸。富含中鏈脂肪酸之MCT oil經測 試亦有部份抑制活性。 綜此,推測山苦瓜中具有抑制巨噬細胞PGE2合成的可能成分為:酯化型中短 鏈飽和脂肪酸或雙酸。而山苦瓜中乙酸乙酯萃物或其他活性區分物中仍可能存在 其他具有活性的成分。
Researches on the anti-inflammation have attached great attention in recent years, since inflammation was recognized as a major cause of tissue damage through the progression of many diseases. Activation of macrophages and the induced expression of COX-2 that increases PGE2 production play an important role in the inflammatory process. PGE2 is the major prostaglandin produced by macrophages and is a well-known pro-inflammatory mediator. Inhibition of the production of PGE2 is regarded as a major approach to ameliorate the inflammatory symptoms. It was reported that bitter gourd extract could inhibit the PGE2 production of the LPS activated murine macrophage cell line. This study is therefore aimed at identifying fractions and compounds isolated from bitter gourd extract, which have inhibitory effect on PGE2 production in the LPS activated RAW264.7 cell line. The whole bitter gourd (Momordica charantia L.) was extracted with water and the water-insoluble residue was further extracted with ethyl acetate (EA). Both of water and EA extracts had inhibitory activity, and the ethyl acetate extract (EAE) had higher activity than water extract. The whole bitter gourd was freeze-dried and directly extracted by EA. The EAE was partitioned by n-hexane and 90%MOH+10%H2O. The n-hexane fraction had inhibitory activity and was further separated by silica gel column chromatography, successively eluted with solvents of increasing polarity (100% n-hexane to 100% EA and then with 90% EA+10%MeOH finally). The fraction 190 eluted by 90% EA+10%MeOH showed the highest inhibitory activity. The fraction 190 was further separated by silica gel column chromatography, eluted with chloroform/MeOH (6/1) and the fraction F1-6 obtained showed higher inhibitory activity. The F1-6 fraction was further separated by silica gel column chromatography twice to obtain the active fraction. And the active fraction was further separated by RP-18 column chromatography to obtain the RP-10 which had the best inhibitory activity (IC50=2.31µg/mL). The RP-10 was identified as triacylglycerol (TG) constituted of short and medium chain fatty acids identified by NMR, IR and H-H Cosy. To verify the components identified from the n-hexane fraction of the whole bitter gourd were TG containing short and medium chain fatty acids, the F1-6 was further hydrolyzed and extracted with EA, and the fatty acid compositions were determined by GC-mass. Octanedioic acid, nonanedioic acid and decanedioic acid were found. On the other hand, the fatty acids standards with 3-20 carbons were tested for the inhibitoryactivity and capric acid (C10:0) was found to possess the highest inhibitory activity. 50µM of it almost completely inhibited PGE2 production in this cell system (IC50=6.46µM or 1.216µg/mL). The octanedioic acid and nonanedioic acid also had inhibitory activity but were inferior to capric acid. In addition, the MCT oil containing mainly C8 and C10 fatty acids also showed partial inhibitory activity. In conclusion, the ester forms of short and medium chain saturated fatty acids or dicarboxylic acids in the n-hexane fraction of the whole bitter gourd were identifiedas components with activity of inhibiting the PGE2 production in the LPS activated RAW264.7 cell line. Other components from the EA partitioned fraction may also have activity and need further investigation.
URI: http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=%22http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22N2004000151%22.&%22.id.&
http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/87214
Sonstige Kennungen: N2004000151
Enthalten in den Sammlungen:學位論文

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