以生物平台為導向分離中國橄欖水萃物殘渣之抗發炎物質

Abstract

慢性發炎(Chronic inflammation)為造成許多慢性疾病的主要原因，已知許多中草藥可藉由其具有抗氧化和抗發炎活性的二次代謝物來改善由發炎所造成的慢性疾病。中國橄欖為常見之中藥材及食材，具有優良的抗氧化及肝保護之作用，本研究室過往的研究指出中國橄欖的甲醇萃取物之乙酸乙酯區分層具有很好的抗發炎效果。本次研究目的是以細胞平台為導向，利用不同的化學分離方式，篩選出具有抗發炎效果的活性物質。本研究所使用之生物平台係以本實驗室已建構於RAW 264.7 細胞中可以偵測NF-κB活性及COX-2表現量的兩種與發炎相關之冷光報導基因平台，利用極性沉澱之溶解度分離，發現上清液層具有較好的抗發炎效果，將上清液層以矽膠管柱分離提升次區分層抗發炎效果，顯示在D次區分層具有抗發炎效果，但卻同時伴隨著細胞毒性。將D次區分層再以矽膠管柱分離，發現D-11次區分層具有較佳的抗發炎效果，但同樣也伴隨著細胞毒性，且具有劑量效應，將D-11次區分層以薄層層析(Thin layer chromatography)大量收集具有螢光之條帶，並再以LH-20純化後以核磁共振(nuclear magnetic resonance )鑑定成分，發現主要成分應為Scoparone，HPLC定量結果顯示Scoparone含量的確會隨分離純化層數增加而增加。以發炎平台驗證Scoparone的抗發炎功效，卻發現Scoparone無法抑制由LPS誘導的發炎反應。以回添試驗探討Scoparone與萃取基質的協同作用，結果仍是無提升抗發炎效果，而在單以LPS誘導的發炎模式下，Scoparone對於一氧化氮生成的抑制效果較文獻中以INF-γ/LPS誘導發炎的抑制效果弱，代表Scoparone並非中國橄欖甲醇萃取乙酸乙酯區分層抗發炎功效的主要成分，必須分離純化在D-11次區分層中的其他次要成分方可找出其抗發炎的主要成分。

Chronic inflammation is one of the factors causing chronic disorders. Many secondary metabolites extracted from herbs exhibit both anti-oxidative and anti-inflammatory activities, and may have potential against inflammation-induced chronic diseases. Canarium album is a common herb and food with excellent anti-oxidative and hepatoprotective effects. Its ethyl acetate fraction from methanol extract has been proved to exhibit good anti-inflammatory potential in our previous work.The objective of this research is to use different isolation techniques to screen bioactive fractions guided by bioassays to obtain anti-inflammatory compounds. The bioassay is a luciferase reporter assay built by our lab using the cell line RAW 264.7 that has been constructed with either NF-κB binding site or COX-2 promoter. First, the fraction was separated based on polarity differences, and the supernatant was found to have better anti-inflammatory action than the precipitate. It was then isolated with silica gel chromatography, and the anti-inflammatory action was concentrated in sub-fraction D, which also showed substantial cytotoxicity. When sub-fraction D was subjected to isolation again with silica gel chromatography, sub-fraction D-11 was found to have better anti-inflammatory action, but also with dose-dependent cytotoxicity.After isolation and purification by thin-layer chromatography and LH-20 column, the active compound from sub-fraction D-11 was identified to be scoparone by nuclear magnetic resonance. Quantification by HPLC showed that scoparone increases as purification steps increase. However, scoparone was unable to inhibit LPS-induced inflammation in the luciferase reporter assay. When spiking scoparone into the extract to explore whether there is synergy between the two, the result was negative. Furthermore, scoparone exhibited a weaker effect of inhibiting NO-induced inflammation than INF-γ/LPS-induced inflammation in literature, indicating scoparone was not the active anti-inflammatory factor in ethyl acetate fraction of methanol extract of C. album. Identification of other components in sub-fraction D-11 is required to reveal the active compound..