中國橄欖水萃殘渣之甲醇萃取物中抗發炎活性成分之分離

Abstract

中國橄欖（Canarium album L.）分屬於食材及中藥材的一種，且具有生理與藥理活性，且富含多種萜烯類（terpenes）、許多酚類化合物（phenolic compounds）及黃酮類（flavonoids）等已知的活性成分，目前已有研究證實其具有抗氧化、護肝和抑制腫瘤等多種功效，但針對其抗發炎活性的研究尚少，加上中國橄欖對環境適應力強，容易栽種，果實產量大，經濟價值高，具有廣泛開發的潛力。 慢性發炎(chronic inflammation)為造成許多慢性疾病的主要原因，文獻指出許多中草藥所含具有的抗氧化、抗發炎活性的二次代謝產物，可改善因發炎所導致的慢性疾病。本實驗室先前的研究已指出，中國橄欖的甲醇萃取物之之不同極性區分層中，以乙酸乙酯區分層具有顯著的抗發炎效果。 本實驗之研究目的是希望以抗發炎活性為導向，利用不同的分離方式，篩選出不具細胞毒性且具有抗發炎活性之區分層，並且分離出具抗發炎效果的活性物質。研究結果發現，中國橄欖的甲醇萃取物之乙酸乙酯區分層（COM-EtOAc）在100 μg/mL以上之濃度具有很強的細胞毒性（存活率<60%），於 25 μg/mL細胞毒性低（存活率>90%），且亦可顯著抑制由LPS所誘發之小鼠巨噬細胞RAW264.7一氧化氮的合成；爾後再經矽膠層析，則以相對中高極性之EtOAc/Hex 50%區分層的細胞毒性最低，且在25 μg/mL下亦具顯著抗發炎活性；再經矽膠層析後，發現在A~F區分層中，以E次區分層的細胞存活率最高且能有效抑制一氧化氮生成，將E次區分層進一步以LH-20膠體層析後得到COM-EtOAc -50% E1~ COM-EtOAc -50% E10共10個區分層，其中又以E2、E5、E6三個細區分層，具有最佳的抗發炎活性，再以HPLC將各細分層再次層析分離，後輔以NMR繼續進行抗發炎成分之鑑定。 實驗結果於E2細區分層發現固醇類的衍生物-sitoindoside I、於E5細區分層發現amentoflavone和其衍生物tetrahydroamentoflavone兩種flavonoids類、於E6細區分層發現amentoflavone和有機酸protocatechuic acid兩種物質。而已有文獻證實，本次實驗所分離出之四種化合物；sitoindoside I、amentoflavone、tetrahydroamentoflavone、protocatechuic acid皆具有良好的抗發炎活性，顯示中國橄欖具有開發為抗發炎保健食品之潛力，期望可針對慢性發炎症狀開發成相關之保健食品。

Chinese olive (Canarium album L.) is a common herb and food with medical value and commercial potentials. It is rich in various terpenes, phenolic compounds and flavonoids. Studies on Chinese olive have showed a variety of pharmacological activities, such as anti-oxidative, hepatoprotective, anti-tumor and so on, but so far there are fewer researches focusing on anti-inflammatory properties of Chinese olive. Chronic inflammation is one of the most important factors causing chronic disorders. Many researches indicated that numerous secondary metabolites extracted from herbs exhibited both anti-oxidative and anti-inflammatory activities and may have potential against inflammation-induced chronic diseases. Previous studies in our laboratory have shown that the ethyl acetate fraction of methanol-extracted Chinese olive (COM-EtOAc) is an effective anti-inflammatory agent. The objective of this research is to screen bioactive fractions using different isolation techniques and to purify bioactive compounds guided by their cytotoxic and anti-inflammatory abilities. Results of our research indicated that COM-EtOAc showed substantial toxicity at 100 μg/mL but showed more effective decrease in NO accumulation and less toxicity at 25 μg/mL. Then COM-EtOAc was separated into ten fractions based on differences in polarity. COM-EtOAc-50% fraction is the least cytotoxic with strong anti-inflammatory activity. COM-EtOAc-E/H50% fraction was further separated into six subfractions (A~F) via silica gel chromatography. Subfraction E is the least cytotoxic with strong anti-inflammatory activity. E subfraction was then separated into 10 subfractions (E1~E10) based on molecular sieve via LH-20 gel column chromatography. E2, E5 and E6 are more active fractions among the 10 subfractions. There are four effective anti-inflammatory compounds identified from the three active subfractions, namely sitoindoside I, amentoflavone, tetrahydroamentoflavone and protocatechuic acid. In conclusion, the anti-inflammatory activity of Chinese olive extract was demonstrated. Moreover, compounds responsible for this activity were identified to be sitoindoside I, amentoflavone, tetrahydroamentoflavone and protocatechuic acid. Taken together, Chinese olive have a high potential as a health food with potent anti-inflammatory activity.