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研究生:謝心慈
研究生(外文):Hsin-Tzu Hsieh
論文名稱:綠莧草抑制高糖誘導胰島β細胞凋亡之研究
論文名稱(外文):Inhibitory effects of Alternanthera paronychioides on high glucose-induced apoptosis in pancreatic β-cell
指導教授:顏國欽顏國欽引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:129
中文關鍵詞:糖尿病高糖胰島 β 細胞凋亡功能失調綠莧草抗氧化
外文關鍵詞:Diabetes mellitushigh glucosepancreatic β-cellapoptosisdysfunctionAlternanthera paronychioidesantioxidant
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  • 被引用被引用:1
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第一型及第二型糖尿病為糖尿病中常見的類型,此兩類型糖尿病皆與胰臟中胰島細胞之失調有正相關性。葡萄糖之代謝異常會導致慢性高血糖現象,使體內產生過多的活性氧 (reactive oxygen species) 而造成氧化壓力上升。自然界植物中富含多酚類化合物,已知對於自由基所引發的疾病之調控上,扮演相當重要之角色。綠莧草 (Alternanthera paronychioides) 為莧科草本植物,先前研究證實,莧科植物具有良好之抗氧化能力,且在動物試驗中具有改善糖尿病之作用,但對於綠莧草之相關研究卻沒有文獻記載,因此本研究探討綠莧草之抗氧化性並評估綠莧草對高糖誘導胰島 β 細胞損傷之保護效應,期望綠莧草對於糖尿病之改善具有潛在價值。內容主要分成兩部分:(一) 綠莧草萃取物之抗氧化力及其活性成分 (二) 綠莧草乙醇萃取物及其活性成分抑制高糖誘導胰島beta細胞凋亡機制之探討。

(一) 以綠莧草水 (WEAP)、甲醇 (MEAP) 及乙醇萃取物 (EEAP) 分別評估總抗氧化能力 (TEAC)、氧自由基吸收能力 (ORAC) 和胞內抗氧化活性 (CAA)。同時也運用酸水解的方式,以高效能液相層析儀 (HPLC) 分析綠莧草可能所含的活性成分。結果顯示,抗氧化力大小分別為 EEAP>MEAP>WEAP。EEAP 亦為三種萃取物中酚酸及類黃酮含量最高者。由 HPLC 分析的結果得知,EEAP 含有 3.84 mg/g 之 ferulic acid 及 4.20 mg/g 之 quercetin 兩種酚類化合物。

(二) 在高糖誘導胰島 β 細胞氧化損傷之實驗結果顯示,胰島 β細胞於高糖 (25 mM glucose) 環境培養 72 h 後,其存活率下降至 65%。分析細胞損傷模式,發現胰島 β 細胞凋亡比例增加,且細胞週期 sub-G1 phase 明顯有累積的現象。高糖誘導下亦會造成胰島β 細胞之粒線體膜電位降低;以 western blot 分析粒線體膜蛋白表現,發現胰島 β 細胞經高糖誘導後,其 Bax/Bcl-2 ratio 有上升趨勢,且約為控制組的 2 倍。此外,caspase-9 及 caspase-3 活性及蛋白表現量皆提升,進而造成 poly (ADP-ribose) polymerase (PARP) 裂解。然而ROS 之生成亦驗証高糖確實會造成氧化壓力而誘導細胞凋亡。

進一步探討富含多酚類化合物之 EEAP 及其活性成分 ferulic acid 及 quercetin 對高糖誘導胰島 β 細胞之凋亡及功能性影響之評估。結果得知,EEAP (20 及 50 µg/ml) 及 quercetin (10 及 20 µM) 具有較佳之細胞保護效應。EEAP 和 quercetin 能夠抑制高糖所引起的細胞凋亡及 sub-G1 phase累積現象,且具有濃度效應。EEAP 及 quercetin 亦能夠提升粒線體膜電位並回復 Bax/Bcl-2 失衡現象,進而抑制 caspase-9、caspase-3 及 PARP 裂解。再加以評估是否有具有改善高糖所引起抗氧化功能之失調,結果顯示,高糖環境下 EEAP (20 及 50 µg/ml) 和 quercetin (10 及 20 µM) 處理後具有顯著回復胰島 β 細胞抗氧化酵素 superoxide dismutase (SOD)、catalase、heme oxygenase-1 (HO-1)、glutathione S transferase (GST) 及glutathione peroxidase (GPx) 活性與基因表現之功效;而pancreatic duodenal homeobox factor-1 (PDX-1) 轉位現象也受抑制,進而達到提升胰島素分泌之作用。

綜合以上結果,綠莧草富含多酚類化合物具有良好之抗氧化能力,對於高糖所引起胰島 β 細胞凋亡具有保護效應,可能是透過抑制活性氧之生成,以及提升抗氧化酵素之表現,進而促進胰島素分泌以抑制血糖代謝失調之現象,對於改善糖尿病症狀具有很大的潛力。
Diabetes is commonly referred to in terms of type 1 and type 2. Both forms involve pancreatic islet β-cell abnormalities. The resultant chronic hyperglycemia leads to oxidative stress because of high glucose induces reactive oxygen species (ROS). Several thousands of natural polyphenols have been identified in plants which have important roles in improvement of disorders involving oxidative stress. Alternanthera paronychioides is an herbaceous plant of Amaranthaceae family. Previous studies have demonstrated that Amaranthaceae family has antioxidant in vitro and anti-diabetes capacity in vivo. However, the antioxidant activity of A. paronychioides has not yet been reported. In the present study, we investigated the antioxidant activity of A. paronychioides and its potential benefit on anti-diabetes. This study included two topics: (1) Antioxidant activity of A. paronychioides extracts and its active compounds; (2) Protective effect of ethanol extract of A. paronychioides and its active compounds against high glucose (HG)-induced apoptosis in pancreatic β-cell.

First, the extracts of A. paronychioides by water (WEAP), methanol (MEAP) and ethanol (EEAP) were prepared, and the antioxidant activities of these extracts were evaluated by trolox equivalent antioxidant capacity (TEAC), oxygen-radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays. In addition, A. paronychioides extracts were hydrolyzed and the active compounds were analyzed by high performance liquid chromatography (HPLC). The antioxidant activity of these three extracts was in the order of EEAP>MEAP>WEAP. EEAP also had the highest phenolics and flavonoids contents. By HPLC analysis, ferulic acid and quercetin were determined in EEAP with the content of 3.84 and 4.20 mg/g, respectively.
From high glucose (HG)-induced oxidative damage of pancreatic β-cell, the data showed that the cell viability of β-cell was decreased to 65% and apoptotic cells were increased in HG condition for 72 h. The cell cycle arrested at sub-G1 phase was also observed. Under HG condition, the mitochondrial membrane potential was reduced. By western blot analysis, the ratio of Bax/Bcl-2 was raised to 2 folds as compared to that of normal glucose control. Besides, the activity and protein expression of caspase-9 and -3 were activated and resulted in cleavage of poly (ADP-ribose) polymerase (PARP). Furthermore, the ROS formation was increased on HG-induced β-cell and led to apoptosis.

The protective effects of EEAP and its active compounds, quercetin and ferulic acid, on HG-induced β-cell were further investigated. EEAP and quercetin showed better protective effects. Cell apoptosis and sub-G1 phase arrested in HG condition were inhibited by EEAP (20 and 50 µg/ml) or quercetin (10 and 20 µM) treatment in a dose dependent manner. In addition, the mitochondrial membrane potential and the ratio of Bax/Bcl-2 were recovered by EEAP or quercetin treatment. The activation of caspase-9, -3 and cleavage of PARP were further inhibited. We also studied whether the protective effect of EEAP and quercetin on HG-induced β-cell dysfunction due to the reduction of oxidative stress. The data showed that EEAP and quercetin can significantly increase the activities and gene expression of antioxidant enzymes including superoxide dismutase (SOD), catalase, heme oxygenase-1 (HO-1), glutathione S transferase (GST) and glutathione peroxidase (GPx) in HG condition. The translocation of transcription factor, pancreatic duodenal homeobox factor-1 (PDX-1), was inhibited and led to insulin secretion by EEAP and quercetin treatment.

These results indicated that A. paronychioides has potent antioxidant activities due to abundant in phenolics. Its protective effect against HG-induced pancreatic β-cell apoptosis might be through inhibition of ROS formation, increases of the antioxidant enzymes and insulin secretion of β-cell, and result in relief of β-cell dysfunction. A. paronychioides might be a potential medical plant on anti-diabetes.
全文摘要 (中)…………………………………………………… I
全文摘要 (英)…………………………………………………… III
前言……………………………………………………………… 1
第一部分、文獻整理…………………………………………… 3
壹、糖尿病…………………………………………………… 4
一、背景…………………………………………………… 4
二、糖尿病主要類型……………………………………… 6
貳、胰島細胞與糖尿病……………………………………… 9
一、胰島細胞……………………………………………… 9
  二、高糖誘導活性氧生成之機制………………………… 11
  三、氧化壓力與胰島 β 細胞……………………………… 16
  四、胰島 β 細胞與細胞凋亡…………………………… 19
五、糖尿病與抗氧化……………………………………… 21
參、綠莧草背景介紹………………………………………… 25
  一、植物型態……………………………………………… 25
  二、性味功能……………………………………………… 25
  三、相關研究……………………………………………… 25
 肆、研究目的………………………………………………… 28
伍、研究架構………………………………………………… 29
第二部分、綠莧草萃取物之抗氧化力及其活性成分………… 31
中文摘要……………………………………………………… 32
英文摘要……………………………………………………… 33
前言…………………………………………………………… 35
材料與方法…………………………………………………… 37
結果與討論…………………………………………………… 43
第三部分、綠莧草乙醇萃取物及其活性成分抑制高糖誘導胰島 β 細胞凋亡機制之探討…………………………………………… 55
中文摘要……………………………………………………… 56
英文摘要……………………………………………………… 58
前言…………………………………………………………… 60
材料與方法…………………………………………………… 62
結果與討論…………………………………………………… 77
一、EEAP及其活性成分 ferulic acid 與 quercetin 對高糖誘導HIT-T15 細胞存活率之影響………………... 77
二、EEAP及其活性成分 quercetin 對高糖誘導 HIT-T15 細胞週期之影響……………….…………….……….. 78
三、EEAP 及其活性成分 quercetin 對高糖誘導 HIT-T15 細胞凋亡之影響…………………………… 79
四、EEAP 及其活性成分 quercetin 對高糖誘導 HIT-T15 細胞活性氧生成之影響…………………… 81
五、EEAP 及其活性成分 quercetin 對高糖誘導 RIN-m5F 細胞氧化壓力及抗氧化酵素之影響…….. 81
六、EEAP 及其活性成分 quercetin 對高糖誘導 RIN-m5F 細胞 PDX-1 轉錄因子及胰島素分泌之影響................................................................................ 83
總結論…………………………………………………………… 111
參考文獻………………………………………………………… 113
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