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  • 學位論文

大果油茶之保肝活性成分研究

Hepatoprotective constituents of Camellia oleifera Abel.

指導教授 : 楊玲玲
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摘要


根據行政院衛生署於十大死亡原因之統計資料顯示,慢性肝病及肝硬化位居第七位,而在十大癌症死亡原因中,肝癌則位於第二位。有此可知,肝病對國人影響甚鉅。有鑑於此,找尋能有效預防或治療肝炎的藥物仍是相當重要的課題。在本實驗室的保肝藥物活性篩選中,發現大果油茶葉萃取物具有開發的潛力。而台灣坊間最常取大果油茶的種子榨油作為整腸健胃,其葉部卻鮮少被使用或研究,因此本論文以探討其葉部保肝活性主成分為主。保肝活性之評估以抑制氯化亞鐵誘導小鼠肝均質液及粒線體產生脂質過氧化為體外試驗模式,結果發現大果油茶葉70 %丙酮萃取物具有明顯抗脂質過氧化活性。體內試驗中,以四氯化碳誘導小鼠造成急性肝損傷的動物模式,發現大果油茶葉70 %丙酮萃取物可明顯降低血清GOT、GPT數值。此外,大果油茶葉萃取物可回復因四氯化碳所降低之CYP2E1蛋白質表現。 然而我們也大量採集及萃取大果油茶葉並以抗脂質過氧化為活性追蹤之依據,應用管柱色層分析技術進行活性成分之分離,得到九個化合物(1-9),分別進行1H -NMR、13C-NMR、2D-NMR、MS等儀器分析鑑定為:(+)-catechin (1)、rutin (2)、quercetin (3)、4'-hydroxy-3,5 -dimethoxybibenzyl (4)、β-sitosterol (5)、4'-β-D-glucopyranosyloxy-3,5 -dihydroxybibenzyl (6)、lupeol (7)、和3-β-D-glucopyranosyloxy-4',5 -dihydroxybibenzyl (8),kaempferol (9)。其中化合物6和8為新的化合物,化合物4則是第一次從天然物中分離出。其中以quercetin的抑制作用最佳(IC50:7.10 ?g/ml),其次為(+)-catechin、kaempferol、4'-hydroxy-3,5-dimethoxybibenzyl。在四氯化碳誘導大鼠初代培養肝細胞損傷之保護作用試驗中,發現大果油茶葉萃取物及quercetin、(+)-catechin、kaempferol均有良好的抑制作用且具有劑量依存性,但4'-hydroxy-3,5-dimethoxybibenzyl於三種劑量下皆無抑制作用。在清除DPPH自由基試驗中,仍以quercetin的效果最佳,其次為: kaempferol、(+)-catechin和rutin,由此結果可知其可藉由提供氫原子而達到清除自由基之目的。然而在螯合亞鐵離子的試驗中發現它們的螯合能力均不顯著。此外亦將大果油茶葉活性成分quercetin、(+)-catechin進行保肝活性之體內試驗,發現皆可明顯降低小鼠血清GOT、GPT數值。此外,其也可回復因四氯化碳所降低之CYP2E1蛋白質表現,其保肝作用應藉由增加CYP2E1蛋白質含量,以降低四氯化碳被代謝活化。綜合上述結果,quercetin、(+)-catechin為大果油茶最主要之保肝活性成分。

並列摘要


Liver disease is a high population in Taiwan by epidemiologic investigation. It is very important to develop the potential preventive or therapeutic agents. In our laboratory, there are many herbs already screened with liver protective activities. Camellia oleifera Abel. is one of the potential hepatoprotective herbs and widely grown in Taiwan. Its seed oil is commonly used as a nutrition supplement for prevent the peptic ulcer and stomach pain. Nevertheless, the pharmacological activity of the C. oleifera leaves has not been investigated. In this study, the 70 % acetone extract of C. oleifera leaves showed against FeCl2 induced lipid-peroxidation on ICR mice liver homogenates and mitochondria. Furthermore, the 70 % acetone extract of leaves decreased the values of GOT and GPT in carbon tetrachloride induced-mice serum. The protein analysis showed significantly decreased on CYP2E1 expression by CCl4. In addition, CCl4 with 70 % acetone extract of C. oleifera reversed the CYP2E1 level. Moreover, we used the bioassay as a guide of each fractionation. The bioactive constituents of C. oleifera were separated and isolated by various chromatography and the structures of each compound was elucidated by using 1H-NMR, 13C-NMR, MS, etc. The nine compounds were (+)-catechin (1)、rutin (2)、quercetin (3)、4'-hydroxy-3,5 -dimethoxybibenzyl (4)、β-sitosterol (5)、kaempferol (6)、4’-β-D-glucopyranosyloxy-3,5-dihydroxybibenzyl (7)、lupeol (8)、3-β-D-glucopyranosyloxy-4',5-dihydroxybibenzyl (9). Especially, compound 7 and 9 were new compounds and compound 4 was first isolated from natural products. Meanwhile, quercetin has the most potential anti-lipid peroxidation in nine compounds with IC50 ( 7.10 ?g/ml)>(+)- catechin>kaempferol>4'-hydroxy-3,5-dimethoxybibenzyl. In the primary cultured hepatocytes, quercetin (>(+)-catechin>kaempferol) also exerted the protective effects against CCl4 with dose dependent manner, but 4'-hydroxy-3,5-dimethoxybibenzyl has no inhibition. Based on the DPPH assay, quercetin exerted the most scavenging ability by providing the hydrogen atom to scavenge the DPPH radicals (>kaempferol>(+)-catechin>rutin). In addition, these compounds have no obviously chelating ability in the Fe2+ assay. In vivo studies, quercetin and (+)-catechin significantly abolished the values of GOT and GPT induced by CCl4. The CYP2E1 protein levels were also reversed by applied with those two compounds. This phenomenon may be via its ability to decrease the metabolic activation of CCl4 by increasing in CYP2E1 protein content. Taken together, quercetin and (+)-catechin are the main and principle components of hepatoprotection in C. oleifera.

參考文獻


1. 行政院衛生署衛生統計資訊網,2006。
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被引用紀錄


林尚誼(2010)。台灣油茶種原葉部性狀及ISSR DNA歧異度之研究〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2010.00182

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