臺灣博碩士論文加值系統

糖尿病（Diabetes mellitus），是一種代謝性疾病，它的特徵是血糖長時間高於正常值。先前測試已知芭樂葉中含有可以抑制PTP1B酵素活性的物質，本實驗因此使用溶劑萃取法將芭樂葉依照使用不同的化學溶劑與其極性分為：甲醇粗萃層、正己烷層、乙酸乙酯層、丁醇層和水層萃取物。其中發現水層萃取物抑制PTP1B活性的作用最佳，所以我們決定先以薄層層析法(Thin layer chromatography, TLC) 分析水層萃取物中的成分並測試其特性，最後再以管柱層析法(Column Chromatography)做天然物的分離。管柱層析使用分子篩的羥丙基葡聚醣凝膠(LH-20)來做為固定相。分離完後再以高效能液相層析儀(High performance liquid chromatography, HPLC)進行純化，並以液相層析質譜儀(LC/MS)定性。LC / MS分析方法中得知純化後的芭樂葉水層萃取物中含有2個主要分子，其中428 Da再負離子束中測得，而在516 Da則在正離子束中測得。另外小鼠的葡萄糖耐受性實驗得知，水層萃取物在動物體內可以有效的抑制飯後血糖上升，含有有效降低血糖的物質。經過研究顯示，芭樂葉水層萃取物對蛋白酪胺酸磷酸酶1B (Protein-tyrosine phosphatase 1B , PTP1B)活性具有獨特的緩慢失活特性。
芭樂葉水層萃取物的緩慢失活作用是比用熱水萃取物還來的好的，而在其他有機分層萃取物中並沒有觀察到緩慢的失活作用。 因此，推測PTP 1B活性的失活可能是通過共價鍵而失活，例如：氧化半胱氨酸。

The hypoglycemic potency of guava leaf extract has been reported repeatedly by many research groups. This research was carried out with a focus on purifying the hypoglycemic compounds that reside in the aqueous partition of guava leaf extract, since our laboratory has identified the aqueous partition possesses the most potent hypoglycemic properties in in vivo. The chemical properties, phenol/flavonoid contents and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability, of those partitions were similar to other reports. The aqueous partition has the lowest contents in phenolic and DPPH scavenging contents, while its flavonoid content is among the lower groups along with butanol and hot water extracts.
　　The hypoglycemic compounds in the aqueous partition were further purified by LH -20 (hydroxypropyl dextran) columns with PTP1B inhibition as the screening method. The most potent PTP1B inhibitory fraction comes out after the main peak that suggests the hypoglycemic compound is a small molecule.
　　The HPLC can further purified the compound to near homogeneity but the acetonitrile inactivate the active compounds. The LC/MS method identified one major species in each of the electron spray, a 428 Da in the negative spray and a 516 Da in the positive spray.
　　A time course study reveals the aqueous partition has a unique slow inactivation property on PTP 1B enzyme activity. This slow inactivation is much less significant with hot water extract and not observed in other organic partitions. Therefore, it is suggested the inactivation of PTP 1B enzymatic activity is probably through a covalent bond inactivation, such as oxidation of cysteine reside.

目 錄
摘要 II
Abstract IV
目錄 VI
圖目錄 VII
表目錄 VIII
前言 1
第一章 文獻回顧
第一節 芭樂葉之降血糖效果 2
第二節 糖尿病 4
第三節 胰島素 5
第四節 胰島素訊號傳遞途徑 6
第五節 蛋白酪胺酸磷酸酶1B (PTP1B) 7
第二章 動機與目的
第一節 研究動機 8
第二節 研究目的 9
第三章 材料與方法
第一節 研究材料 10
第二節 研究方法 12
第四章 結果 22
第五章 結論 34
參考文獻 35

 
圖目錄

圖一、胰島素信號傳遞途徑 6
圖二、分層萃取法樹狀圖 13
圖三、以萃取法萃取之各分層含量 22
圖四、各分層對於抗氧化能力的測定 23
圖五、各個分層總酚測定 24
圖六、各個分層黃酮的測定 25
圖七、檢測芭樂葉各分層對PTP1B活性的緩慢失活作用 27
圖八、以蛋白酪氨酸磷酸酶1B (PTP1B) 測定活性 29
圖九、利用HPLC分析純化後的物質 30
圖十、利用HPLC分析純化後的物質 31
圖十一、用LC/MS分析純化後之物質 32
圖十二、小鼠服用芭樂葉水層與口服葡萄糖耐受性實驗 33


 
表目錄

表一、各分層物質分析 26
表二、芭樂葉水層萃取物在管柱分析後之重量分布 28

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