大蒜精油及其活性成分二丙烯基二硫化物對果糖誘發小鼠非酒精性脂肪肝具肝臟保護效果

Abstract

非酒精性脂肪肝疾病(non-alcoholic fatty liver disease, NAFLD)其定義為，非因長期酗酒而造成肝臟中脂質堆積，且脂質重量占肝臟重大於5%之病症，針對不同族群的調查發現，台灣的NAFLD盛行率為11.4-41%，顯示國人肝臟疾病之預防及控制是需要重視的議題。據文獻指出近年來隨著飲食習慣的改變，國人含糖食物攝取量增加，導致果糖攝取過多，其可能為導致NAFLD的原因之一。大蒜(Allium sativum L.)俗稱蒜頭，是大眾普遍食用之食材，研究發現其具有護肝的功效，且可抑制高脂飲食造成之NAFLD的疾病進展。然而，目前尚未有文獻探討大蒜精油(garlic essential oil, GEO)是否可抑制因高果糖誘發NAFLD的疾病進展，因此本研究欲藉由高果糖糖水誘發NAFLD之動物模式下，探討以水蒸氣蒸餾法萃取之大蒜精油及精油中活性成分二丙烯基二硫化物(diallyl disulfide, DADS)的介入，是否可降低肝臟中脂質堆積，並了解其影響之代謝途徑。本研究使用十週齡雄性小鼠C57BL/6J分為六組：(1)控制組、(2)高果糖糖水誘發組(30% fructose solution)、(3) 30%果糖糖水+大蒜精油低劑量組(25 mg/kg bw)、(4) 30%果糖糖水+大蒜精油高劑量組(50 mg/kg bw)、(5) 30%果糖糖水+DADS低劑量組(10 mg/kg bw)及(6) 30%果糖糖水+DADS高劑量組(20 mg/kg bw)，實驗為期八週。動物犧牲後檢測其肝功能指標天門冬胺酸轉胺酶(aspartate aminotransferase, AST)、丙胺酸轉胺酶(alanine transaminase, ALT)、血液生化數值、肝臟中三酸甘油酯(triglyceride, TG)及總膽固醇。實驗結果顯示：介入GEO及DADS與高果糖糖水誘發組相比，皆可顯著降低血清中約44-56%之AST及肝臟中約36-46%的TG堆積(p < 0.05)，進而達到護肝的效果；另外低劑量的DADS可顯著降低血清中約20%的總膽固醇及肝臟中約8%的總膽固醇堆積(p < 0.05)；且高劑量的DADS具顯著降低血清中約57%的ALT、30%的TG及31%的游離脂肪酸(p < 0.05)之能力。另外從組織切片結果發現，在高果糖誘發動物NAFLD模式下，會造成動物肝臟中肝糖嚴重堆積，介入了高劑量的DADS可顯著改善此現象(p < 0.05)。由於DADS對肝臟脂質堆積抑制的效果比GEO的介入佳，因此更進一步以西方墨點法探討DADS影響之機轉，發現DADS可透過降低脂質新生(de novo lipogenesis)相關因子acetyl-CoA carboxylase (ACC)、carbohydrate-responsive element-binding protein (ChREBP)及sterol regulatory element-binding protein-1c (SREBP-1c) 之蛋白表現量，以降低脂質在肝臟中合成量，並提高carnitine palmitoyl transferase-1 (CPT-1)表現以增加脂肪酸氧化(β-oxidation)途徑，減少脂肪酸合成TG而堆積於肝臟中。綜上所述，大蒜精油及其活性成分DADS在果糖誘發小鼠非酒精性脂肪肝之模式下，不僅具有顯著的肝臟保護效果，且DADS可透過抑制脂質新生及促進脂肪酸氧化的途徑，達到抑制非酒精性脂肪肝之疾病進展，未來可望開發成為具保健功效的機能性食品。

Due to the change of eating habits, people tend to consume more sugar-containing food in Taiwan as well as worldwide. Excessive fructose consumption resulted in the development of non-alcoholic fatty liver disease (NAFLD). A recent study found that prevalence of NAFLD in Taiwan is around 11.4-41%. Typically, NAFLD is defined as lipid accumulation in the liver more than 5% by weight. Garlic (Allium sativum L.) is a traditional food ingredient and it has been reported as the liver protective food. A recent study found that the garlic essential oil (GEO) protects the liver from high fat diet-induced NAFLD by reducing the hepatic lipid accumulation and inflammation. However, the effect of GEO in high fructose-induced NAFLD and its mechanism are still unknown. The aims of this study were to investigate the hepatoprotective effect of GEO and its active compound diallyl disulfide (DADS) in the high fructose-induced NAFLD mice model and to explore its mechanism via hepatic lipogenesis pathway. Ten week-old male C57BL/6J mice were divided into six groups: (1) control group, (2) 30% fructose-induced NAFLD group, (3) 30% fructose + low dosage of GEO group (25 mg/kg bw), (4) 30% fructose + high dosage of GEO (50 mg/kg bw), (5) 30% fructose + low dosage of DADS group (10 mg/kg bw), and (6) 30% fructose + high dosage of DADS (20 mg/kg bw). The treatment groups were daily gavaged with GEO or DADS for 8 weeks, then the mice were sacrificed. In this study, the results indicated that GEO and DADS significantly exhibited hepatoprotective activity against high fructose-induced NAFLD by reducing approximately 44-56% serum aspartate aminotransferase (AST) and 36-46% hepatic triglyceride (TG) (p < 0.05). In addition, the dose of 10 mg/kg bw DADS significantly reduced 20% serum cholesterol and 8% liver cholesterol levels (p < 0.05), and the dose of 20 mg/kg bw DADS significantly reduced serum 57% alanine transaminase (ALT), 30% TG and 31% free fatty acid (p < 0.05) compared with 30% fructose-induced NAFLD group. Moreover, liver histopathological analysis indicated the accumulation of hepatic glycogen in mice with high fructose diet, but the high dosage of DADS significantly reduced glycogen accumulation in the liver (p < 0.05). Since DADS exhibited higher anti-NAFLD activity than GEO, thus, this study we further investigated the effect of DADS on the hepatic de novo lipogenesis and β-oxidation related protein. DADS supplementation prevented the hepatic lipogenesis by suppressing acetyl-CoA carboxylase (ACC), carbohydrate-responsive element-binding protein (ChREBP) and sterol regulatory element-binding protein-1c (SREBP-1c) in NAFLD mice. Moreover, DADS improved the hepatic β-oxidation by enhancing carnitine palmitoyl transferase-1 (CPT-1). In conclusion, our results suggested that supplementation of GEO or DADS for 8 weeks could prevent the development of the NAFLD and the potential mechanism was through suppressing de novo lipogenesis and enhancing lipid β-oxidation.