肝脂質過氧化之研究

張思平(C S-P)

指導教授 ： 楊玲玲

臺北醫學大學/藥學院/生藥學研究所/碩士(1998年)

摘要

自由基在多種疾病的發生與惡化中扮演著重要的角色，主要相關的疾病有動脈粥狀 硬化、癌症、缺血、發炎、輻射傷害、污染物傷害以及老化等，將自由基清除或將其作用 阻止便能防止與改善多種疾病的發生。本論文由省產25種藥材及10種常用補益中藥，首先 進行肝細胞脂質過氧化抑制之生物活性測定，其測定法乃利用ICR鼷鼠肝臟進行體外試驗 ，以肝細胞均質液經過氧化物誘導劑 tert-butyl hydroperoxide ( t-BuOOH ) 誘生脂質 過氧化 (lipid peroxidation)，其過氧化物經反應生成malondialdehyde (MDA)後，再加 入thiobarbituric acid (TBA)作用產生MDA(TBA)2以tetra-ethoxylpropane ( TEP ) 製 作標準檢量線，定量MDA(TBA)2，以評估其脂質過氧化活性之效價。結果以白朮水萃取物 IC50 = 45.8 (g/ml ( 對照組Vit.E之IC50 = 55.6 (g/ml )具有最佳之抗脂質過氧化作用 。 白朮為菊科( Compositae )植物Atractylodes ovata De Candolle ( = A. macroc ephala Koidzumi )之根莖，是補氣健脾之重要藥材之一，臨床中醫治療調劑時之飲片， 以土炒為最被常用，故本研究以白朮原藥材、生飲片及土炒飲片，分別以一次蒸餾水(wat er)、甲醇(methanol)、乙醇(ethanol)、丙酮(acetone)、乙醚(ether)、氯仿(chlorofor m)、正己烷(n-hexane)等七種溶劑冷浸及溫浸之抽取物，進行抗肝細胞脂質過氧化試驗， 結果顯示經土炒白朮飲片正己烷冷浸抽取物具較高之生物活性 ( IC50 = 21.6 (g/ml )， 乃進一步大量抽取分離活性部分，並配合活性追蹤，結果顯示Atractylon ( IC50 = 15.9 (g/ml )為白朮之活性主成分，另外，以Atractylon為指標，應用高效能液相層析法 ( H igh Performance Liquid Chromatography；HPLC ) 進行白朮不同炮製品正己烷抽取物中 Atractylon之定量，其分析條件為層析管：LiChrospher l00 RP-18e ( 250×4mm I.D.) ，移動相：CH3CN-H2O ( 80:20 )，流速：1.0 ml/min，偵測波長：220nm；結果以土炒白 朮飲片之正己烷冷浸抽取物含Atractylon最高 ( 102.4 (g/g )，此乃更進一步證實未來 白朮飲片若以抗脂質過氧化為目的，宜採用土炒飲片，而科學中藥製劑則宜用正己烷之冷 浸才能得到最高產率之抗脂質過氧化活性主成分Atractylon。總合以上結果，白朮為具有 抗肝脂質過氧化之中藥補益藥材，臨床應用宜以土炒炮製者為佳。

並列摘要

Lipid peroxidation, a general mechanism whereby oxygen free radicals indu ce tissue damage, has been implicated in diverse pathological conditions, incl uding cancer, atherosclerosis, ischemia, inflammation, irradiation, aging and so on. The present study is to investigate the lipid peroxidation inhibition of the natural products. Experiments were conduct to screen the anti-lipid per oxidation activity of Chinese medicinal herbs or natural plants. A reversed-ph ase high performance liquid chromatographic method is used for the determinati on of malonaldehyde in liver homogenates. The standard test for lipid peroxida tion is the measurement of the peak, 532nm absorbing chromogen formed upon rea ction with 2-thiobarbituric acid. This reaction depends upon the presence of t he lipid peroxidation product malondialdehyde (MDA) which reacts with 2-thioba rbituric acid (TBA) to form MDA(TBA)2 with a absorption maximun at 532 nm. Fr om this screening system, we tested water extract of Atractylodes ovata De Can dolle has the highest anti-lipid peroxidative effect. ( IC50 = 45.8 (g/ml ) Bai-Zhu ( rhizome of Atractylodes ovata De Candolle ) is one of the common to nic Chinese herbs. It has been used to invigorate the spleen and benefit vital energy. Since we found the n-hexane extract of Bai-Zhu possessed a strong ant ioxidative activity ( IC50 = 21.6 (g/ml ), we used silica gel chromatography t o isolate the antioxidative components. Four pure compounds were identified as Atractylenolide I, II, III and Atractylon, by mass and infared spectrometer a s well as their physical constant. We found the most potency in anti-lipid per oxidative activity for the four pure compounds of Bai-Zhu was Atractylon. ( IC 50 = 15.9 (g/ml ) Accordingly, we chose Atractylon as standard to quantitative analysis of Atractylon in each extraction from Chinese herbs by HPLC. The HPL C quantitative analysis conditions are as following: column: LiChrospher l00 R P-18e ( 250×4 mm I.D. ); mobile phase: CH3CN-H2O ( 80:20 ); flow rate: 1.0 ml /min; detector: UV 220 nm. We found that among the extractions of Bai-Zhu with soil baking Atractylon by n-hexane extract is the highest. ( 102.4 (g/g ). Th ese results will provide information to Chinese medicinal pharmaceutical indus try and research.