郭華仁臺灣大學：農藝學研究所葉珮芸Yeh, Pei-YunPei-YunYeh2007-11-282018-07-112007-11-282018-07-112005http://ntur.lib.ntu.edu.tw//handle/246246/59135台灣原住民民族植物資源豐富，種類多達710種，其中藥用植物有351種。本論文乃針對其中34科、64種進行保肝活性之探討，以找尋到可以抑制肝細胞脂質過氧化作用且不具備肝毒性之天然植物材料為目標。 利用TBARS Methods，評估植物萃取物清除脂質過氧化之效果。先以化學誘導劑tert- Butyl hydroperoxide (t-BuOOH)、Ferrous chloride (FeCl2) 誘導鼷鼠肝臟細胞均質液產生脂質過氧化物MDA，然後以高效能液向層析儀於波長532nm下，測定MDA(TBA)2之含量，以Trolox為正對照組，作為抗氧化能力的表示。利用此生物活性測定模式所測定的結果顯示：漆樹科-台灣鹽膚木；美人蕉科-美人蕉；大戟科-茄苳、紅仔珠、白匏仔、烏臼；樟科-樟樹、豆科-波狀葉山螞蝗、台灣魚藤、台灣葛藤；百合科-桔梗蘭；桑科-榕樹；桃金孃科-番石榴；棕櫚科-檳榔；蓼科-皺葉酸模；薔薇科-笑靨花；茜草科-九節木；無患子科-龍眼；蕁麻科-密花苧麻、青苧麻；葡萄科-扁藤等27種植物萃取物於濃度200μg/ml下，皆可以抑制脂質過氧化物MDA之形成，抑制作用皆達50%以上，並且與濃度成正相關。進一步利用MTT比色分析法，在波長600nm下測定細胞活性，探討此27種植物萃取物對人體正常細胞株 (Chang liver cell lines) 是否具有肝毒性。在投予各植物萃取物 (200μg/ml) 24小時後，有12種萃取物不具肝毒性； 48小時之後，只有6種植物萃取物不具毒性，分別為美人蕉科-美人蕉；大戟科-紅仔珠、茄苳；茜草科-九節木；蕁麻科-青苧麻；葡萄科-扁藤。其次針對這六種植物萃取物進行抗氧化能力之評估。抗氧化功能包括清除DPPH自由基、螯合亞鐵離子、抑制超氧陰離子之生成以及還原力等四項測定。結果顯示此六種植物萃取物均可清除DPPH自由基，其能力依序為九節木＞扁藤＞美人蕉＞紅仔珠＞茄苳＞青苧麻，50%有效抑制之濃度 (IC50) 分別為5.54、13.77、19.02、20.78、32.91、43.91μg/ml；還原能力也以九節木最好，其次為紅仔珠、扁藤、美人蕉、茄苳、青苧麻，分別相當於24.24、11.56、10.59、9.37、8.9、6.74μg/ml 之Trolox含量。然而此六種植物萃取液皆不具有與亞鐵離子螯合，以及清除超氧陰離子 (•O2‾) 之能力。 由上述結果可知，九節木、扁藤、美人蕉、紅仔珠、茄苳及青苧麻等六種植物之葉部萃取物可以抑制由FeCl2所誘導之肝損傷，並且對人類正常肝細胞不具肝毒性，且推測是經由提供氫離子以及電子達到阻斷FeCl2對細胞之氧化傷害以達到保護肝臟之效果。The aim of this study was to investigate the hepatoprotective activities, defined as capabilities against lipid peroxidation in mice, of 70% ethanol extracts of sixty-four ethnomedicinal plants in Taiwan. The anti-lipid peroxidative effects of plant extracts were determined by TBARS (thiobarbituric acid reactive substances) assay, which is an indicator of lipid peroxidation and free radical activity in an in vitro biological system. To induce the formation of lipid peroxidation the liver homogenates of mice were treated with tert-Butyl hydroperoxide (t-BuOOH) or ferrous chloride (FeCl2). The quantities of Malondialdehyde (MDA) thus induced were measured, after transforming into MDA (TBA) 2, at 532 nm by high performance liquid chromatography. Compared to trolox, twenty-seven plant extracts (200μg/ml) prepared from leaf、stem or herb displayed significant inhibition (> 50%) on the formation of MDA: Rhus javanica (Anacardiaceae), Canna indica (Cannaceae), Bischofia javanica, Breynia officinalis, Mallotus paniculatus, Triadica sebifera (Euphorbiaceae), Cinnamomum camphora (Lauraceae), Desmodium sequax, Millettia pachycarpa, Pueraria montana (Leguminosae), Dianella ensifolia (Liliaceae), Ficus microcarpa (Moraceae), Psidium guajava (Myrtaceae), Areca catechu (Palmae), Rumex crispus (Polygonaceae), Spiraea prunifolia (Rosaceae), Psychotria rubra (Rubiaceae), Euphoria longana (Sapindaceae), Boehmeria densiflora, Boehmeria nivea (Urticaceae), and Tetrastigma formosanum (Vitaceae). The human normal liver cell (Chang liver cell lines) was used to test the cytotoxicity of the plant extracts. Cell activity were determined by the IC50-value of the reduced form of MTT (3-(4,5-dimethylthiazol)-2,5- diphenyltetrazolium bromide). Among these twenty-seven plant extracts (200μg/ml), only six exhibited no cytotoxicity: P. rubra, C. indica, B. javanica, B. officinalis, B. nivea, and T. formosanum. Furthermore, we evaluated the antioxidant activity of these six plant extracts by DPPH assay, metal (Fe2+) chelators, superoxide quencher, and FRAP assay. The results showed that these six plant extracts could scavenge DPPH free radical, in the order as following (IC50: μg/ml): P. rubra (5.54) > T. formosanum (13.77) > C. indica (19.02) > B. officinalis (20.78) > B. javanica (32.91) > B. nivea (43.91). They also exhibited ferric reducing /antioxidant power (equivalent μg/ml Trolox): P. rubra (24.24) > B. officinalis (11.56) > T. formosanum (10.59) > C. indica (9.37) > B. javanica (8.9) > B. nivea (6.74). But to our surprise these extracts showed no ability of chelating Fe2+ and scavenging superoxide free radical. In conclusion, among sixty-four ethnomedicinal species thus studied, there are six plant extracts which demonstrate potential for protecting the mice liver from damage as caused by FeCl2, and which do not have cytotoxicity against Chang liver cell line. Moreover, the antioxidant activity of these six extracts is mainly mediated by theirfunction as hydrogen donators and reducing agents against the liver damage.致謝--------------------------------------------------I 目錄--------------------------------------------------II 圖表目錄----------------------------------------------V 英文縮寫表--------------------------------------------VII 中文摘要----------------------------------------------VIII 英文摘要----------------------------------------------X 壹、前言-----------------------------------------------1 貳、文獻探討-------------------------------------------5 一、台灣民族植物背景-----------------------------------5 (一) 民族藥物學知識的發展與重要性----------------------5 (二) 台灣植物分布概況----------------------------------7 (三) 台灣的民族藥物學知識------------------------------8 二、肝臟簡介------------------------------------------11 三、自由基與氧化壓力----------------------------------16 四、天然多酚類化合物----------------------------------20 參、材料與方法----------------------------------------23 一、實驗材料------------------------------------------23 (一) 台灣民族植物材料---------------------------------23 (二) 實驗動物-----------------------------------------27 (三) 細胞株-------------------------------------------27 (四) 試藥與試劑---------------------------------------27 (五）實驗儀器-----------------------------------------29 二、實驗方法-----------------------------------------30 (一) 植物萃取物之製備---------------------------------30(二) 植物萃取物總酚類含量之測定-----------------------30 (三) 植物萃取物抑制鼷鼠肝細胞脂質過氧化之生物活性測定-31 (四) 植物萃取物對人類正常肝臟細胞株之毒性試驗---------35 (五) 抗氧化能力測定-----------------------------------38 三、統計分析------------------------------------------43 肆、結果----------------------------------------------44 (一) 台灣民族植物材料之萃取物-------------------------44 (二) 植物萃取物總酚類含量-----------------------------44 (三) 植物萃取物抑制鼷鼠肝細胞脂質過氧化之生物活性-----45 (四) 植物萃取物對人類正常肝臟細胞株之毒性試驗---------48 (五) 抗氧化能力測定-----------------------------------49 1. 清除DPPH自由基之能力-------------------------------49 2. 抑制超氧陰離子之生成-------------------------------50 3. 螯合亞鐵離子能力之測定-----------------------------50 4. 還原能力測定---------------------------------------51 伍、討論----------------------------------------------52 (一) 總酚類含量---------------------------------------53 (二) 抑制脂質過氧化之能力-----------------------------54 (三) 肝毒性試驗---------------------------------------56 (四) 抗氧化能力試驗-----------------------------------57 陸、結論----------------------------------------------60 柒、圖表----------------------------------------------62 捌、參考文獻------------------------------------------84633565 bytesapplication/pdfen-US台灣原住民藥用植物總酚類含量ICR鼷鼠保肝氯化亞鐵脂質過氧化細胞毒性抗氧化能力Ethnomedicinal plantstotal polyphenolICR miceferrous chloride (FeCl2)hepatoprotectivet-BuOOHlipid peroxidationcytotoxicityantioxidant abilitythesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59135/1/ntu-94-R91621115-1.pdf