吳瑞碧臺灣大學:食品科技研究所鄭芳琪Cheng, Fang-ChiFang-ChiCheng2010-05-112018-06-292010-05-112018-06-292009U0001-2007200911435400http://ntur.lib.ntu.edu.tw//handle/246246/182219糖尿病為現今常見的慢性代謝疾病之一，會造成終生的病態，增加大小血管、腎病變等之併發症風險。而由於生活品質的提升及物質充裕，糖尿病病患的比例也日益增加。世界衛生組織預測，到西元2025年，全球罹患糖尿病的人數將達到三億人。因此，糖尿病的治療為健康管理中急待解決的重要課題。番石榴（Psidium guajava Linn.）屬桃金孃科（Myrtaceae），民間常將野生番石榴果實或葉片曬乾，熬湯飲用，作為降火氣、清血、降血糖之用，是坊間盛行的糖尿病藥草療法之一，然而番石榴的降血糖成分並不清楚。本研究將以細胞模式配合不同之管柱層析進行番石榴葉之分離及純化，以達到番石榴葉降血糖活性成分確立之目的。研究分為兩個部分，第一部分係利用Diaion HP20管柱配合不同極性甲醇溶液將番石榴葉成分以極性高低分離，再以細胞模式觀察番石榴葉管柱區分物對大鼠肝細胞（Clone 9 cell）之葡萄糖攝入作用(glucose uptake)效果。第二部分則以各種管柱層析法，針對葡萄糖攝取活性較高的區分物進行分離及純化，搭配核磁共振儀（nuclear magnetic resonance）鑑定結構及高效能液相層析法（high performance liquid chromatography）進行定性，期能找出番石榴葉中可能之降血糖成分。實驗結果發現，番石榴葉的高極性區分物（D25、D50）能夠增加clone 9細胞的葡萄糖攝入能力（p<0.05）。在時間效應方面，以番石榴葉區分物短時間（12及24小時內）處理clone 9細胞，對細胞葡萄糖攝入能力有較佳之提升效果；濃度效應方面，clone 9細胞在相同時間（30分鐘）樣品刺激時，其葡萄糖攝入能力會隨番石榴葉區分物濃度增加而上升，且具有濃度效應（dose-dependent manner）。由初步結果得知，番石榴葉水萃物經由Diaion HP-20管柱流洗後，高極性的區分物（D25）對clone 9 cell具有較高之葡萄糖攝入效果。因此，針對此一高極性之區分物（D25）進行分離及純化，結果發現，番石榴葉D25區分物中可分離出降血糖成分檞皮素（quercetin），而quercetin可有效增加細胞之葡萄糖攝取功能，其作用方式可能是透過改變細胞膜的流動性來增加葡萄糖的吸收，或藉由調控細胞內與胰島素相關訊息傳導分子的作用，進而達到降血糖之功效。Diabetes mellitus (DM) is a chronic metabolic disease associated with significant lifelong morbidity. In 2008, DM was the fifth of top ten cause of death in Taiwan and more than 95% are type 2 DM. It is one of the major diseases that human being eagerly wants to overcome. Guava (Psidium guajava Linn.) is one of the most important economic fruit in Taiwan. People boil guava leaves in water and drink the extract as a folk medicine for diabetes. However the efficient component is unclear. The present study investigated the enhancement of aqueous guava leaf extract on glucose uptake in rat clone 9 hepatocytes and searched for the active compound. he guava leaf aqueous extract was eluted with MeOH-H2O solutions through Diaion, Sephadex and MCI-gel columns to separate into fractions with different polarities. The uptake test of 2-[1-14C ] deoxy-D-glucose in rat clone 9 hepatocytes was performed to evaluate the hypoglycemic effect of these fractions. The active compound was identified by nuclear magnetic resonance analysis and high-performance liquid chromatography (HPLC). n the results, the high polarity fractions (D25, D50) of guava leaf extract could increase significantly the glucose uptake of clone 9 cells (p<0.05). The glucose uptake of clone 9 cells treated with guava leaf extracts at short time (24 hrs) was higher than at long time (48 hrs). When treated at the same time, the glucose uptake of clone 9 cells increased as the rising dosage. It was a dose-dependent reaction. The results revealed that phenolics are the principal component of the extract, that high polarity fractions of the guava leaf extract are enhancers to glucose uptake in rat clone 9 hepatocytes, and quercetin is the major active compound after H1-NMR and HPLC analysis. n a conclusion, we suggest that quercetin in the aqueous extract of guava leaves promotes glucose uptake in liver cells through the membrane property of cells or modulation of insulin signaling transduction pathway, and that contributes to the alleviation of hypoglycemia in diabetes as a consequence.頁數文摘要 i文摘要 iii錄 v目錄 viii目錄 x一章 緒論……………………………………………………………... 1二章 文獻回顧………………………………………………………... 3一節 糖尿病之流行病學…………………………………………... 3.1.1糖尿病的流行病學…………………………………………….. 3.1.2糖尿病的介紹………………………………………………….. 3.1.3胰島素簡介…………………………………………………….. 5.1.4胰島素的作用………………………………………………….. 6.1.5胰島素在細胞層次之作用…………………………………….. 7.1.6胰島素受器(Insulin receptor, IR)………………………………. 7.1.7葡萄糖轉運蛋白（glucose transporter, GLUT）……………… 8.1.8周邊組織細胞之葡萄糖轉運…………………………………... 10.1.9胰島素的訊息傳遞……………………………………………... 10二節 抗糖尿病物質………………………………………………... 13.2.1常見抗糖尿病藥物…………………………………………….. 13.2.2植物成分抗糖尿病之研究……………………………………... 15三節 番石榴之介紹………………………………………………... 16.3.1番石榴…………………………………………………………... 16.3.2番石榴葉成分之研究…………………………………………... 16.3.3番石榴葉降血糖之研究………………………………………... 17四節 研究動機與實驗設計…………………………………………. 19三章 番石榴葉萃取物之組成份及降血糖活性分析………………... 21一節 前言……………………………………………………………. 21二節 實驗材料………………………………………………………. 22.2.1實驗樣品來源………………………………………………….. 22.2.2實驗細胞……………………………………………………….. 22.2.3實驗藥品及試劑……………………………………………….. 22.2.4實驗藥品配製………………………………………………….. 23.2.5儀器設備……………………………………………………….. 24三節 實驗方法……………………………………………………... 25.3.1番石榴葉熱水萃取物之製備………………………………….. 25.3.2大鼠肝細胞clone 9之保存與培養……………………………. 25.3.3 Clone 9細胞生長抑制率測定（MTT還原分析法）…………… 26.3.4一般成分分析…………………………………………………... 26.3.5 Diaion HP20管柱區分…………………………………………. 28.3.6 Clone 9細胞對葡萄糖之攝入作用……………………………. 28.3.7統計分析………………………………………………………... 29四節 結果與討論…………………………………………………... 30.4.1番石榴葉熱水萃取物之一般成分分析…………………………... 30.4.2番石榴葉熱水萃取物對clone 9細胞的生長抑制情形………. 30.4.3番石榴葉Diaion HP20區分物之一般成分分析……………… 32.4.4番石榴葉Diaion HP20區分物對clone 9細胞之葡萄糖攝入作用……………………………………………………………... 33五節 結論…………………………………………………………... 36四章 番石榴葉降血糖活性成分之純化與鑑定……………………... 50一節 前言…………………………………………………………... 50二節 實驗材料……………………………………………………... 51.2.1實驗樣品來源………………………………………………….….. 51.2.2實驗細胞………………………………………………………... 51.2.3實驗藥品及試劑………………………………………………... 51.2.4實驗藥品配製…………………………………………………... 52.2.5儀器設備………………………………………………………... 52三節 實驗方法……………………………………………………... 53.3.1番石榴葉降血糖成分之分離…………………………………... 53.3.2番石榴葉降血糖成分之活性試驗……………………………... 54.3.3番石榴葉降血糖成分之鑑定…………………………………... 55.3.4統計分析………………………………………………………... 56四節 結果與討論…………………………………………………... 57.4.1番石榴葉活性物質之分離純化與結構鑑定………………….. 57.4.2番石榴葉管柱純化物對細胞葡萄糖攝入作用之效果……….. 58.4.3番石榴葉純化物Quercetin降血糖作用之探討………………. 59五節 結論…………………………………………………………... 62四章 總結……………………………………………………………... 70考文獻…………………………………………………………………... 71application/pdf858090 bytesapplication/pdfen-US番石榴糖尿病quercetin降血糖作用Psidium guajava L.glucose uptakediabetesphenolic[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182219/1/ntu-98-D93641007-1.pdf