普洱茶抑制Hep G2細胞株生合成膽固醇之有效成分探討

Investigation of Pu-Erh tea active principles to inhibit the cholesterol synthesis in Hep G2 cell line

呂季樺(CHI-HUA LU)

指導教授 ： 孫璐西

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.02451

摘要

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。

關鍵字

膽固醇 ； Hep G2細胞株 ； 普洱茶 ； statin ； 多酚

並列摘要

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.

並列關鍵字

cholesterol ； Hep G2 cell line ； Pu-Erh tea ； statin ； polyphenol

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