薑乙醇萃取物及其辣味成分對油酸誘導小鼠正常肝細胞株脂質堆積之功效與機制

Abstract

非酒精性脂肪肝病 (Nonalcoholic fatty liver disease, NAFLD) 為國人常見的一種代謝性疾病，主要因肝臟脂質過量堆積，與代謝症候群有密切相關。薑 (Zingiber officinale Roscoe) 屬根莖類植物，主要產於東南亞一帶，常作為蔬菜、調味料或中藥材等用途。本研究中以薑粉與95%乙醇混合進行萃取，並利用小鼠正常肝細胞株FL83B細胞，利用油酸 (oleic acid, OA) 誘導脂質堆積24小時之細胞模式探討薑乙醇萃取物 (ginger ethanolic extract, GEE) 及其三種辣味成分6-gingerol、6-shogaol與zingerone對改善正常肝細胞脂質堆積之功效。本研究之GEE萃取率約3.78%，HPLC分析結果顯示，每克GEE中辣味成分6-gingerol、6-shogaol與zingerone之含量分別為102.4、23.8 及0.37 mg，並利用Nile red染色法評估GEE與此三種辣味成分在MTT assay所得之不影響細胞存活率的劑量下，改善油酸誘導FL83B細胞脂質堆積效果，結果顯示，GEE、6-gingerol及6-shogaol在低濃度下即可顯著降低Nile red螢光強度及細胞內三酸甘油酯含量，而zingerone則在非常高濃度下才具有顯著差異，由此可推測GEE中抗脂質堆積效果可能主要來自於6-gingerol以及6-shoagol之活性影響，進一步以西方墨點法評估脂質代謝相關蛋白表現量，實驗結果顯示，GEE、6-gingerol及6-shogaol可顯著降低脂質合成相關蛋白SREBP-1與FAS以及膽固醇合成相關之SREBP-2與HMGCR的表現量，且這三種樣品亦可促進活化AMPK，使ACC磷酸化比例提高，而在脂質氧化相關蛋白PPARα及CPT-1的表現量上，僅有GEE及6-gingerol有顯著提高，6-shogaol則無顯著差異。根據上述之實驗結果可推論，GEE、6-gingerol及6-shogaol可能是透過增加脂質氧化、降低脂質與膽固醇合成相關蛋白質表現量來達到改善油酸誘導FL83B細胞脂質堆積之功效。

Nonalcoholic fatty liver disease (NAFLD) is a common metabolic disorder. The pathogenesis is excessive lipid accumulation in liver and it is related to metabolic syndrome. Ginger (Zingiber officinale Roscoe) belongs to rhizome plants and mainly grows in south Asia. Ginger contains pungent compouds and has been widely used in various foods, beverages, seasoning and traditional Chinese medicine. The ginger ethnaolic extract (GEE) was prepared by immersing ginger powder in 95% ethanol. In the present study, we investigate the effect of GEE and three pungent compounds (6-gingerol, 6-shogaol and zingerone) on oleic acid-induced lipid accumulation for 24 hours in FL83B cells, the normal liver cells of mice. GEE extraction rate was approximately 3.78%. HPLC analysis showed that the content of 6-gingerol, 6-shogaol and zingerone was 102.4, 23.8 and 0.37 mg per gram of GEE, respectively. Nile red staining was used to evaluate the effect of GEE and three pungent compounds on oleic acid-induced lipid accumulation without affecting cell viability measured by MTT assay. The results showed GEE, 6-gingerol and 6-shogaol significantly decreased Nile red fluorescence intensity and intracellular triglyceride content at low concentrations while zingerone only at very high concentration had significant difference. The effect of GEE on alleviating lipid accumulation presumably derived from the activity of 6-gingerol and 6-shoagol. To further elucidate the mechanisms, we conducted Western blotting. The results showed that GEE, 6-gingerol and 6-shogaol not only significantly decreased the expression of SREBP-1, FAS, SREBP-2 and HMGCR, but also promoted AMPK activation results in increasing the phosphorylation of ACC. GEE and 6-gingerol significantly increased the expression of PPARα and CPT-1 but 6-shogaol had no significant difference. On the basis of above results suggested that GEE, 6-gingerol and 6-shogaol may alleviate oleic acid-induced lipid accumulation in FL83B cells by modulation of protein expression associated with lipid oxidation, lipid synthesis and cholesterol synthesis.