以有機硫化物或多酚化合物探討其在抗肥胖與抗疲勞之作用與分子機轉

Anti-obesity and anti-fatigue effects of organo-sulphur or polyphenolic compounds and their molecular mechanisms

張維棠(Wei-Tang Chang)

指導教授 ： 徐慶琳

中山醫學大學/健康管理學院/營養學系博士班/博士(2017年)

摘要

肥胖與疲勞皆為現今社會常見的文明病，其會對健康與生活品質產生負面之影響。肥胖亦是許多疾病的危險因子，例如:心血管疾病、新陳代謝疾病、全身性發炎、癌症，以及肌肉與骨骼相關疾病。肥胖不僅會造成脂質過度堆積，亦會降低肌肉活動力與增加疲勞程度。針對以體外或體內模式評估機能性成分對抗肥胖或抗疲勞作用之確切機制，目前仍是不清楚，因此，本研究主要利用機能性成分探討其在抗肥胖或抗疲勞之作用及釐清其分子機轉，並期望將其開發成具抗肥胖與抗疲勞潛力之保健食品。內容主要分成三部分:(一) 黑蒜甲醇萃取物透過調控脂質生合成、脂肪細胞激素與脂質分解作用，改善高油脂飲食誘導大鼠肥胖症、(二) 二烯丙基三硫化物藉由調控脂質生合成、脂肪酸運送和脂肪酸氧化進而抑制3T3-L1脂肪細胞脂質生合成和 (三) 白藜蘆醇、表沒食子兒茶素沒食子酸酯與表兒茶素在C2C12肌管細胞之粒線體功能影響。
(一) 本研究利用體內動物試驗模式，探討黑蒜甲醇萃取物 (methanolic extract of black garlic, MEBG) 抑制高油脂飲食誘導肥胖大鼠之影響與分子機轉。結果顯示，高油脂飲食給予MEBG (50、150和300 mg/kg rat) 劑量之組別，可顯著降低高油脂飲食所誘導之最終體重與肝臟和脂肪組織重量增加，亦可降低血清中三酸甘油酯含量。在高油脂飲食並給予MEBG之組別，可顯著降低肝臟總脂質含量和氧化壓力增加，並增加糞便中總脂質排出。在脂肪組織基因表現上，高油脂飲食給予MEBG可顯著提升AMPK、FOXO1、SIRT1、ATGL、HSL、ACO、CPT-1、UCP1和adiponectin，並抑制SREPB-1c、PPARγ、FAS、ACC、LPL、perilipin、resistin、PAI-1和TNF-α之基因表現。此外，在肝臟基因表現上，高油脂飲食給予MEBG可顯著提升AMPK、SIRT1和PPARα，並降低SREBP-1c、ACC、FAS和SCD1之基因表現。綜合上述結果證實，此具有特色與功能之黑蒜甲醇萃取物，確實具有抑制體脂肪生成與體重增加之潛力。
(二) 大蒜與其相關產品 (例如:黑蒜頭) 因富含多種有機硫化物 (organo-sulphur compounds) 使其具多種生理功能。本研究將探討大蒜衍生之有機硫化物 [包括：蒜氨酸 (alliin)、二烯丙基硫化物 (diallyl sulphide, DAS)、二烯丙基二硫化物 (diallyl disulphide, DADS)、二烯丙基三硫化物 (diallyl trisulphide, DATS) 與S-烯丙基半胱胺酸 (S-allyl cysteine, SAC)] 對3T3-L1脂肪細胞脂質生合成之影響與分子機制。結果顯示，在五種有機硫化物，以DATS具有最佳抑制3T3-L1脂肪細胞胞內三酸甘油酯之累積，其亦具有抑制脂肪細胞數目與甘油-3-磷酸去氫酶 (glycerol-3-phosphate dehydrogenase, GPDH) 活性。在基因表現上，DATS可顯著抑制脂質生合成 (C/EBPα、PPARγ、FAS和ACC)、脂肪酸運送基因 (aP2、CD36和LPL) 與脂肪細胞激素 (leptin、MCP1、PAI-1和resistin) 等基因表現，並提升脂肪酸氧化 (PGC-1α和UCP1) 與adiponectin之基因表現。由以上研究結果顯示，DATS可透由調控脂質生合成、脂肪酸氧化代謝與脂肪細胞激素等基因表現，進而減少3T3-L1脂肪細胞之胞內油滴堆積情形。
(三) 植物化學成分 (phytochemicals) 具多種生理功能，例如:抗發炎和抗肥胖等。然而目前僅有少數研究指出，植物化學成分 (白藜蘆醇、三萜類和兒茶素) 被認為在動物試驗中具抗疲勞之作用。因此，本篇研究目的將建構體外抗疲勞細胞試驗篩選平台和分子機轉探討。結果顯示，以白藜蘆醇、槲皮素、表沒食子兒茶素沒食子酸酯和表兒茶素等化合物處理C2C12肌管細胞後，其可顯著增加粒線體DNA目標基因細胞色素b (cytochrome b) 和細胞色素c氧化酶次單元II (cytochrome c oxidase subunit II) 之基因表現，並增加粒線體酵素 (檸檬酸合成酶和細胞色素c氧化酶) 之活性和C2C12肌管細胞中ATP之含量。由上述結果得知，白藜蘆醇、槲皮素、表沒食子兒茶素沒食子酸酯和表兒茶素在作為粒線體活化劑上具發展潛力，未來在抗疲勞作用中扮演重要的角色。
以上結果證實，黑蒜甲醇萃取物與二烯丙基三硫化物具有調控脂質合成之作用，其可透由抑制脂肪細胞脂質生成相關之轉錄因子，進而達到體外和體內試驗抗肥胖之作用。多酚化合物可透過提升體外C2C12肌管細胞之粒線體生合成與粒線體功能，進而提升其抗疲勞作用。本研究結果證實，機能性成分在作為肥胖與疲勞之治療上是有助益的。

關鍵字

黑蒜甲醇萃取物 ； 有機硫化物 ； 多酚化合物 ； 抗肥胖 ； 抗疲勞 ； 分子機轉

並列摘要

Obesity and fatigue are the most common cases of civilization diseases. They have negative effects on health and quality of lives. Obesity serves as a significant risk factor for various diseases, such as cardiovascular disease, metabolic diseases, systemic inflammation, cancer, muscle weakness, and osteoarthritis. They may be accompanied by lipid accumulation, poor muscle performance, and fatigue syndrome. However, the in vitro or in vivo models’ evaluations of anti-obesity and anti-fatigue effects of functional compounds and their molecular mechanism still remain unclear. It can be developed as a potential nutraceutical ingredient for ameliorating obesity and fatigue. There were three topics included in this study: (1) Methanolic extract of black garlic ameliorates diet-induced obesity via regulating adipogenesis, adipokine biosynthesis, and lipolysis; (2) Diallyl trisulphide inhibits adipogenesis in 3T3-L1 adipocytes through lipogenesis, fatty acid transport, and fatty acid β-oxidation pathways; (3) Resveratrol, epigallocatechin gallate, and epicatechin enhance mitochondrial functions in C2C12 myotubes.
(1) The objective of this study was to determine if methanolic extract of black garlic (MEBG) has antiobesity effects via regulating lipid metabolism in an animal model. The results indicated that supplementation with MEBG decreases the final body weight, the relative masses of the liver and fat tissues, serum triacylglyceride levels, and hepatic oxidative stress, and increases faecal lipid contents in HFD fed rats. Supplementation with MEBG upregulated AMPK, FOXO1, SIRT1, ATGL, HSL, ACO, CPT-1, and UCP1 in the adipose tissues of HFD fed rats, whereas it downregulated SERBP-1c, PPARγ, FAS, ACC, LPL, perilipin, and CD36. Meanwhile, MEBG upregulated adiponectin and downregulated PAI-1, resistin, TNF-α, and GLUT4 in the adipose tissues of HFD fed rats. Regarding hepatic gene expressions, MEBG upregulated the genes of AMPK, SIRT1, and PPARα and downregulated the genes of SREBP-1c, ACC, FAS, and SCD1. Therefore, MEBG can be characterised as a potential ingredient for the amelioration of fat tissue and body weight.
(2) Garlic and its related products (such as black garlic) exhibit multiple biological functions which attributed to the presence of organo-sulphur compounds. The aim of this study was to investigate the inhibitory effects of garlic-derived organo-sulphur compounds [including diallyl sulphide (DAS), diallyl disulphide (DADS), diallyl trisulphide (DATS), S-allylcysteine (SAC), and S-allyl-L-cysteine sulphoxide (alliin)] on adipogenesis in 3T3-L1 adipocytes. The result indicated that DATS significantly decreases the intracellular triacylglycerol, oil red O-stained material (OROSM), and glycerol-3-phosphate dehydrogenase (GPDH) activity but significantly increases the adiponectin production in 3T3-L1 adipocytes. Real-time RT-PCR analyses showed that DATS decreases the gene levels of adipogenesis (C/EBPα, PPARγ ACC, and FAS), fatty acid transport (aP2, CD36, and LPL), and adipocytokines (leptin, MCP-1, PAI-1, and resistin), as well as increases the gene levels of fatty acid β-oxidation (PGC-1β and UCP1) and adiponectin in 3T3-L1 adipocytes. DATS is capable of inhibiting adipogenesis in 3T3-L1 adipocytes possibly through regulation of gene levels of lipogenesis, lipolysis, and adipokines.
(3) Phytochemicals have multifunctional bioactivities, such as anti-inflammatory and anti-obesity activities. However, there are still relatively few studies that have investigated the possible anti-fatigue activity of functional phytochemical compounds (such as resveratrol, triterpenoids, and catechins) in animal models. Therefore, the aim of this study was to develop of in vitro models as tools for cellular and molecular studies of anti-fatigue activity. C2C12 myotubes were used for evaluating in vitro the anti-fatigue activity of functional compounds. The results indicated that treatment with some functional compounds (including resveratrol, quercetin, epigallocatechin gallate, and epicatechin) significantly increases the expressions of mitochondrial DNA genes (including cytochrome b and cytochrome c oxidase subunit II) and the activities of mitochondrial enzymes (including citrate synthase and cytochrome c oxidase) in C2C12 myotubes. These results demonstrated that resveratrol, quercetin, epigallocatechin gallate, and epicatechin have great potential as novel mitochondrial-activating agents that may play an important role in the anti-fatigue activity.
The in vitro and in vivo results demonstrated that methanolic extract of black garlic and diallyl trisulphide are benefical for the anti-adipogenic action due to the down-regulation of adipogenic transcription factors. The phenolic compounds are able to show anti-fatigue activities due to the activation of the mitochondrial biogenesis and mitochondrial function. These results demonstrated that some functional compounds are effective for the treatments of obesity and fatigue.

並列關鍵字

methanolic extract of black garlic ； organo-sulphur compounds ； phenolic compounds ； anti-obesity ； anti-fatigue ； molecular mechanisms

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