孫璐西臺灣大學：食品科技研究所呂季樺LU, CHI-HUACHI-HUALU2007-11-272018-06-292007-11-272018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/56274　　動脈粥狀硬化是導致心血管疾病的最主要成因，而血漿中低密度脂蛋白(low density lipoprotein, LDL)偏高則為造成動脈硬化最主要的危險因子。當肝臟細胞生合成膽固醇的路徑受到抑制時，細胞會代償性地增加細胞膜上的LDL受體，藉此可加速利用血漿中的LDL，因而可降低血漿中LDL的濃度。本研究室前期研究顯示，普洱茶水萃物有抑制人類肝癌細胞株Hep G2生合成膽固醇之效果，因此本研究擬從「已知成分觀點」與「未知成分觀點」探討普洱茶中抑制Hep G2生合成膽固醇之有效成分。 　　首先選取30種市售普洱茶，以一般標準泡茶法製得之茶湯進行各種標的成分之高效能液相層析。在statin類化合物方面，lovastatin與simvastatin皆未檢出。在多酚類化合物方面，黃烷醇類其含量佔茶葉乾重之平均值高低依序為：ECg 15.7 mg/g、EGCg 12.0 mg/g、EGC 12.0 mg/g、EC 5.7 mg/g、gallic acid 4.4 mg/g、(+)-C 3.0 mg/g及GCg 1.5 mg/g；黃酮醇類中，myricetin含量為未檢出至42.4 ng/g，quercetin含量為未檢出至53.6 ng/g，kaempferol則皆未檢出。測試其總酚含量可得gallic acid equivalent之平均值為93.9 mg/g，而咖啡因含量之平均值為25.9 mg/g。 　　接著測試多酚類化合物與普洱茶水萃物（依一般標準泡茶法製得）抑制Hep G2細胞株生合成膽固醇之效果。在多酚類化合物中，以GCg (IC50＝67.85 μM)的效果最好，其次依序為EGCg (IC50＝72.76 μM)、ECg (IC50＝88.09 μM)與gallic acid (IC50＝90.99 μM)。普洱茶水萃物以濃度40 μg/mL處理Hep G2細胞株，發現30種普洱茶水萃物皆具有抑制膽固醇生合成之效果(7% - 35%)。計算抑制率與前述成分含量之相關係數，可得相關性最高者為EC (0.66)，次高者依序為gallic acid (0.60)與總酚類化合物 (0.58)。 　　最後以分離純化方法從未知成分觀點進行探討：在以分子量為區分標的方面，分子量小於1000之區分其抑制效果最佳(22%)，但由於抑制效果不如原粗萃物，因此未以此區分繼續分離純化。在以化合物極性為區分標的方面，甲醇層區分物的抑制效果最好(46%)，但以XAD-7管柱層析進一步區分卻無法分離出效果優於此區分物者。然而當普洱茶水粗萃物依序以正己烷、乙酸乙酯及正丁醇進行分配萃取(liquid-liquid partition)後，可得到高抑制效果之乙酸乙酯層區分物(67%)，以高效能液相層析法分析此區分物中的statin類化合物與多酚類化合物，發現該區分物中不含statin類化合物，而以gallic acid含量最高(佔區分物5.1%, w/w)。以MTT assay未發現gallic acid有細胞毒性。 　　綜合以上結果，推測gallic acid可能為普洱茶抑制Hep G2細胞株生合成膽固醇的有效成分之一，但其詳細之作用機制仍有待進一步研究。Atherosclerosis is the major cause of cardiovascular disease, and high serum low density lipoprotein (LDL) is the major risk factor causing atherosclerosis. When cholesterol synthesis is inhibited in hepatocyte, cells will utilize LDL from serum and thus decrease serum LDL content. Previous studies in our laboratory have shown that Pu-Erh tea aqueous extract can inhibit the synthesis of cholesterol in Hep G2 cell line. Therefore, the active principles in Pu-Erh tea to inhibit the cholesterol synthesis in Hep G2 cell line were investigated in this study from both “known components aspect” and “unknown components aspect”. Thirty commercial Pu-Erh tea samples were analyzed for the following target compounds by HPLC methods. Lovastatin and simvastatin were not detected (ND) in Pu-Erh tea aqeuous extract. The average contents (mg/g dry leave weight) of flavanols in 30 Pu-Erh tea samples were as followed: ECg 15.7 mg/g, EGCg 12.0 mg/g, EGC 12.0 mg/g, EC 5.7 mg/g, gallic acid 4.4 mg/g, (+)C 3.0 mg/g, GCg 1.5 mg/g. Myricetin content ranged from ND to 42.4 ng/g; quercetin content ranged from ND to 53.6 ng/g and kaempferol was ND in the 30 Pu-Erh tea samples. The total polyphenol contents (gallic acid equivalent) were found to range from 20.1 mg/g to 188.1 mg/g and the average content (mg/ g dry leave weight) of caffeine was 25.9 mg/g. The inhibitory effects of phenolic compounds and 30 Pu-Erh tea aqueous extracts on cholesterol synthesis in Hep G2 cell line were tested. GCg (IC50＝67.85 μM), EGCg (IC50＝72.76 μM), ECg (IC50＝88.09 μM) and gallic acid (IC50＝90.99 μM) were found to be effective compounds to inhibit the cholesterol synthesis in Hep G2 cell line. The inhibiton ratios of 30 Pu-Erh tea samples were ranged from 7% to 35%. The highest correlation coefficient between inhibition ratio and bioactive components was exhibited by EC (0.66), the second highest was gallic acid (0.60), and the total polyphenol (0.58) content was the third. Different separation and purification methods were employed for the investigation of Pu-Erh tea active principles from “unknown components aspect.” The molecular weight (MW) was first employed to be the separation principle, however, the inhibition ratio of the best fraction (MW＜1000, 22%) was no better than the original extract. Therefore, separation by polarity was chosen. Methanol fraction showed the highest inhibition ratio (46%), but further separation and purification utilizing XAD-7 chromatography did not give better fractions than the original methanol fraction. Finally, sequential liquid-liquid partition with n-hexane, ethyl acetate and n-butanol was applied on Pu-Erh tea aqueous extract, and the inhibition ratio of ethyl acetate fraction was found to be significantly raised (67%). The polyphenolic and statin compounds in ethyl acetate layer were analyzed by HPLC. No statin compounds were found, while gallic acid was the major phenolic compound (5.1%, by weight) in ethyl acetate fraction. MTT assay was used to evaluate cytotoxicity of gallic acid and the cell viability was found to be similar to control group. It is concluded in this study that gallic acid might be one of the active principles in Pu-Erh tea to inhibit cholesterol synthesis in Hep G2 cell line, but the mechanism still needs to be further elucidated.壹、前言 1 貳、文獻整理 2 一、普洱茶 2 （一）普洱茶的歷史 2 （二）普洱茶的加工與分類 3 （三）普洱茶的化學成分 4 （四）普洱茶的保健功效 5 二、脂質與脂蛋白質之代謝 11 （一）脂蛋白的一般結構與分類 11 （二）外源性(exogenous)脂質的運送 14 （三）內源性(endogenous)脂質的運送 16 三、冠狀動脈心臟病與低密度脂蛋白的相關性 20 （一）冠狀動脈心臟病(coronary heart disease, CHD) 20 （二）LDL cholesterol(LDL-C)濃度對CHD的影響 20 （三）LDL-C與CHD之流行病學研究 21 （四）LDL-C與CHD之病理學研究 22 （五）導致血中LDL過高之因素 26 （六）以LDL-C為治療標的之成效 27 四、膽固醇的代謝 31 （一）膽固醇之體循環 31 （二）膽固醇在細胞內之恆定 31 （三）細胞內膽固醇的生合成 34 （四）Statin類化合物 34 五、人類肝癌細胞株Hep G2 40 （一）細胞株的起源 40 （二）細胞株的應用範圍 40 （三）Hep G2應用在降膽固醇藥物研究的歷史 41 參、研究目的與實驗設計 42 肆、材料與方法 44 第一部份：實驗材料與儀器設備 44 一、普洱茶樣品 44 二、實驗細胞株 44 三、化學藥品、溶劑與耗材 45 四、細胞實驗各種溶液配方 48 五、儀器設備 50 第二部分：實驗方法 53 一、普洱茶之前處理 53 二、標準溶液之配製 53 三、茶葉萃取液中各種成分之高效能液相層析 54 四、萃出率之測定 57 五、總酚含量之測定 57 六、人類肝癌細胞株Hep G2之培養與保存 58 七、膽固醇生合成抑制試驗 59 八、以MTT assay分析細胞存活力 61 九、超過濾區分 61 十、普洱茶之各種粗萃物與區分物之製備 62 十一、XAD-7 resin區分 63 十二、統計分析 65 伍、結果 66 第一部份：從已知成分觀點探討普洱茶中抑制Hep G2細胞株生合成膽固醇之有效成分 66 一、各種成分之HPLC分析條件之建立 66 （一）Statin類化合物 66 （二）酚酸、咖啡因與黃烷醇類化合物 67 （三）黃酮醇類化合物 67 二、普洱茶中各種成分之高效能液相層析 68 （一）Statin類化合物 68 （二）酚酸、咖啡因與黃烷醇類化合物 68 （三）黃酮醇類化合物 70 三、各種普洱茶之水萃出率 71 四、各種普洱茶之總酚含量 71 五、Hep G2生合成膽固醇之抑制試驗 71 （一）體外模式系統之確效 71 （二）茶中已知成分抑制膽固醇生合成之效果 72 （三）普洱茶抑制膽固醇生合成之效果 72 六、以MTT assay評估上述藥品對細胞存活力之影響 73 第二部分：從未知成分觀點探討普洱沱茶中抑制Hep G2細胞株生合成膽固醇之有效成分 73 一、以分子量為區分標的：超過濾區分 73 二、以極性為區分標的 74 （一）不同溶劑萃取 74 （二）甲醇粗萃物之分配萃取(liquid-liquid partition) 74 （三）XAD-7區分 74 （四）水粗萃物之分配萃取(liquid-liquid partition) 75 （五）水粗萃物乙酸乙酯區分層之HPLC分析 75 陸、討論 55 一、體外模式的合理性 76 二、普洱茶中已知成分之含量 76 三、普洱茶中已知生物活性成分之抑制效果 77 四、普洱茶成分之分離純化 79 柒、結論 81 捌、參考文獻 118 玖、附錄 129en-US膽固醇Hep G2細胞株普洱茶statin多酚cholesterolHep G2 cell linePu-Erh teapolyphenol[SDGs]SDG3thesis