|摘要: || 肥胖為長期能量攝取過多引起之代謝性疾病，常見病徵為脂肪累積於脂肪組織和心臟、肌肉、腎臟及肝臟等非脂肪組織。當肝臟累積之三酸甘油酯 (triglyceride, TG) 超過其重量之 5% 時，即定義為脂肪肝 (fatty liver)。非酒精性脂肪肝疾病 (nonalcoholic fatty liver disease, NAFLD) 全球盛行率超過 30%，逐漸成為全球注目之健康議題。餘甘子 (Phyllanthus emblica L.) 為印度傳統藥用植物，分佈於台灣的苗栗、南投與彰化等地區。研究證實餘甘子具抗腫瘤、減緩糖尿病及降血脂等多種保健功效。因此，本研究探討台灣產餘甘子之抗氧化與抗發炎能力，並分析相關活性成分與其減緩非酒精性脂肪肝病之潛力。|
以不同溶劑萃取台灣餘甘子，獲得水 (water extract of Phyllanthus emblica L., WEPL)、甲醇 (methanol extract of Phyllanthus emblica L., MEPL)、乙醇 (ethanol extract of Phyllanthus emblica L., EEPL) 及乙酸乙酯 (ethyl acetate extract of Phyllanthus emblica L., EAEPL) 萃取物。根據總抗氧化力與氧自由基吸收能力之結果，WEPL 與 MEPL 有最佳之抗氧化能力，其次為 EEPL。進一步評估各萃取物之活性成分，也以 WEPL 與 MEPL 含有較多之多酚與類黃酮，而抗壞血酸則以 MEPL 與 EEPL 含量最多。HPLC 分析指出，各種萃取物均含有 gallic acid (GA) 和 ellagic acid (EA)。推測餘甘子因富含多酚及類黃酮化合物而具良好之抗氧化能力。進一步評估台灣產餘甘子於 lipopolysaccharide (LPS) 誘導 Raw 264.7 巨噬細胞發炎模式之抗發炎能力。結果證實，MEPL 與 EA 可顯著地抑制 LPS 誘導 Raw 264.7 巨噬細胞之 nitric oxide (NO) 與 prostaglandin E2 (PGE2) 發炎物質分泌 (p < 0.05)。MEPL 亦可抑制 LPS 誘導 Raw 264.7 巨噬細胞之 inducible nitric oxide synthase (iNOS) 及 cyclooxygenase-2 (COX-2) 基因與蛋白表現。MEPL 透過抑制 mitogen-activated protein kinases (MAPK) 訊息傳遞路徑，減少磷酸化 nuclear factor-kappa B (NF-κB) 累積於細胞核內，進而降低 LPS 誘發 tumor necrosis factor α (TNF-α) 與 interleukin-6 (IL-6) 等發炎基因表現，有效減緩 LPS 誘導 Raw 264.7 巨噬細胞產生發炎作用。
分別以 FFA mixture (棕櫚酸/油酸 = 1/2，v/v) 誘導 HepG2 細胞及以 leptin 誘導 HSC-T6 肝星狀細胞建立非酒精性脂肪肝和肝纖維化之細胞試驗平台，探討台產餘甘子改善非酒精性脂肪肝病之能力。實驗證實，WEPL 於 FFA mixture 誘導下，可藉由活化 AMPK 訊息路徑，進而調控其下游蛋白表現 [acetyl-CoA carboxylase (ACC) 與 fatty acid synthase (FAS)]，也進一步發現其可促進脂肪代謝相關基因 [peroxisome proliferator-activated receptor α (PPARα) 及 carnitine palmitoyl transferase-1 (CPT-1)] 之表現與抑制脂肪合成相關基因 (FAS 及 ACC) 之表現 (p < 0.05)。另外，也證實，EA 也可活化 AMPK，影響脂肪合成代謝之作用。此外，WEPL 與 EA 可顯著減少 FFA mixtures 所誘導之胞內 ROS 生成 (p < 0.05)。顯示，WEPL 於脂肪肝形成初期，具有保護性的功效。
WEPL 與 EA 也可改善 leptin 誘導 HSC-T6 細胞活化之細胞形態特徵、細胞增生現象、collagen I 和 α-SMA 蛋白質之表現與 matrix metalloproteinase-2 (MMP-2) 和 MMP-9 酵素活性。DAPI 染色結果進一步發現，WEPL 與 EA 可造成 leptin 誘導 HSC-T6 細胞核質濃縮的現象。顯示 WEPL 與 EA 可促進活化之 HSC-T6 細胞凋亡。進一步分析其中分子機轉，證實 WEPL 與 EA 可藉由粒線體凋亡路徑，提升 Bax/Bcl-2 ratio，增加 caspase-9 和 caspase-3 之蛋白與酵素活性表現，造成 PARP 的剪切，促使 leptin 誘導活化之 HSC-T6 細胞凋亡，有助於肝纖維化之恢復。
綜合研究結果，各溶劑萃取之餘甘子樣品，均具有良好之抗氧化及抗發炎功效，其中以 MEPL 及 WEPL 尤佳。WEPL 可藉由影響 HepG2 細胞之脂肪代謝機制而減少胞內脂肪堆積及抑制 leptin 誘發之 HSC-T6 細胞活化所致纖維化特徵，展現改善非酒精性脂肪肝病之高度潛力。
The major cause of obesity is long-term energy intake higher than energy expenditure of which there are many features that fat accumulation in adipose tissues as well as non-adipose organs including heart, skeletal muscle, kidneys and liver. Fatty liver is defined by excessive fat (triglyceride, TG) accumulation in the liver that is over 5% of liver weight. Current studies indicate that over 30% of population is subjected to non-alcoholic fatty liver disease (NAFLD). NAFLD is becoming a major public health problem in worldwide. Phyllanthus emblica L. is a common medicinal plant of India, and distributes over Miaoli, Nantou and Changhua in Taiwan. P. emblica L. has anti-tumor, anti-diabetes, reducing dyslipidemia and other health functions. In the present study, the anti-inflammatory effect of P. emblica L. and related bioactive compounds, and the potential improving effect of P. emblica L. on NAFLD were evaluated.
Fruits of P. emblica L. were extracted by water, methanol, ethanol and ethyl acetate to yield WEPL, MEPL, EEPL and EAEPL, respectively. The results showed that WEPL and MEPL had the highest content of polyphenolics and flavonoids as well as the strongest antioxidant capacity (trolox equivalent antioxidant capacity, TEAC and oxygen-radical absorbance capacity assay, ORAC) than other of P. emblica L. extracts. However, MEPL and EEPL had highest content of ascorbic acid. HPLC analysis revealed that all P. emblica L. extracts contained phenolic acid compounds including gallic acid (GA) and ellagic acid (EA). The anti-inflammatory effect of P. emblica L. in lipopolysaccharide (LPS)-induced Raw 264.7 macrophages was further investigated. MEPL and EA reduced LPS-stimulated nitric oxide (NO) and prostaglandin E2 (PGE2) production in Raw 264.7 macrophages. MEPL treatment decreased protein and mRNA expressions of inducible inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated Raw 264.7 macrophages. MEPL inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) and decreased accumulation of nuclear factor-kappa B (NF-κB) in the nucleus as well as TNF-α and IL-6 mRNA expression in Raw 264.7 macrophages in response to LPS-stimulation. These results indicated that P. emblica L. and EA had anti-inflammatory properties in LPS-induced Raw 264.7 macrophages.
The cell models that free fatty acid (FFA) mixture (palmitic acid/oleic acid = 1/2) induced HepG2 cells and leptin (100 ng/mL) induced HSC-T6 hepatic stellate cells were further established to mimic NAFL and hepatic fibrosis, respectively. According to the results, WEPL enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and its primary downstream targeting enzyme, acetyl-CoA carboxylase (ACC) and decreased the protein level of fatty acid synthase (FAS). WEPL further increased the mRNA expressions of peroxisome proliferator-activated receptor α (PPARα) and carnitine palmitoyl transferase-1 (CPT-1) (p < 0.05), but inhibited ACC and FAS mRNA levels in HepG2 cells induced by FFA mixture. EA could also regulated AMPK signaling pathway to reduce fat accumulation. Notably, oil-red O stain demonstrated that WEPL and EA may contribute to decrease in TG contents of FFA mixture-induced HepG2 cells. In addition, WEPL and EA significantly decreased ROS production in HepG2 cells caused by FFA mixture (p < 0.05). These results suggested that the effect of WEPL on fat accumulation of HepG2 cells might be partially mediated by activation of the AMPK signaling pathway. Besides, WEPL and EA could eliminate the hepatic fibrosis characteristics of leptin-induced HSC-T6 cells as well as attenuate cell proliferation Westen bolt analysis also showed that WEPL and EA suppressed protein expressions of collagen I and α-SMA as well as the activaties of matrix metalloproteinase-2 (MMP-2) and MMP-9 in leptin-induced HSC-T6 cells. DAPI staining showed that chromation condensation appeared when HSC-T6 cells were treated with WEPL or EA, respectively, for 24 h in the presence of leptin. These data indicated the mechanism of apoptosis induced by WEPL and EA in leptin-stimulated HSC-T6 cells through the mitochondria pathway. Leptin-induced HSC-T6 cells cultured with WEPL and EA contributed to an increased in the Bax/Bcl-2 ratio, and increased the protein levels and activaties of caspase-9 and caspase-3, contributing to cleavage of poly (ADP-ribose) polymerase (PARP).
In conclusion, P. emblica L. extracts show strong antioxidant and anti-inflammatory properties, and WPEL can decrease TG accumulation in liver cells as well as fibrotic characteristics of hepatic stellate cells, suggesting that WPEL might have potential for improvement of NAFLD.