| Summary: | 碩士 === 國立臺灣大學 === 食品科技研究所 === 92 === 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.
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