臺灣博碩士論文加值系統

摘 要

依據本草綱目記載芭樂葉可以治療糖尿病。台灣因為氣候為亞熱帶所以一年四季都適合栽種芭樂樹。我們和彰化縣社頭鄉農會及栽種芭樂的農友合作，評估芭樂心葉茶產品，是否具者有降血糖之功效。
實驗過程中將芭樂心葉茶用甲醇萃取所得到的濃縮物，作分配層析，進而得到層析己烷層、乙酸乙酯層、丁醇層和水溶層之濃縮物，以及芭樂心葉直接用熱水萃取所得到的濃縮物，共六個樣品，測試其是否能抑制蛋白質酪胺酸磷酸酶-1B之酵素活性，而推論芭樂葉茶是否其有降血糖的功能。
本研究發現層析水溶層濃縮物可抑制蛋白質酪胺酸磷酸酶-1B的活性，其IC50 為0.031±0.004 mg/mL。此數據與芭樂心葉直接水萃濃縮物的IC50 (0.033±0.001 mg/mL)相當接近，而且此兩種水溶層是上述六個樣品中最能夠有效抑制PTP-1B活性的樣品。因此，推測芭樂心葉茶可以抑制蛋白質磷酸水解酶-1B的有效成分為水溶性的物質，所以推論可以利用熱水沖泡芭樂心葉茶的方式來飲用，進而達到降血糖的功效。




關鍵字：糖尿病、芭樂心葉茶、蛋白質酪胺酸磷酸酶-1B

Abstract

Compendium of Materia Medica indicated that guava leaves may be used for treating subjects with diabetic conditions. Due to subtropical climate conditions, guava tree can be cultivated in large quantity throughout the year in Taiwan. We cooperated with guava framers and the farmer’s union at Shetou(社頭) District of Chunghwa County to study the blood glucose lowering potential of guava leaf by examining if guava leaf extract can inhibit protein tyrosine phosphatase-1B enzymatic activity.
Guava leaf was extracted with methanol extensively, and the extract was condensed for further partitioning. Solvents of different polarities were used in the serial partitioning; they include hexane, ethyl acetate, butanol and water. Guava leaf was also extracted directly with hot water, so a total of six fractions were obtained. These fractions were concentrated and tested for their effects on protein tyrosine phosphatase-1B (PTP-1B) enzymatic activity, which plays a major role at controlling insulin signaling and circulating blood glucose levels.
The water-soluble concentrate has an IC50 of 0.031 ±0.004 mg/mL when inhibiting protein tyrosine phosphatase-1B, while direct hot water extract has an of IC50 of 0.033 ±0.001 mg/mL, these two water soluble fractions are more potent than other fractions at inbiting PTP-1B enzyme activity in vitro.
Therefore, it is suggested that guava leaf tea can inhibit protein tyrosine phosphatase-1B activity, and the active ingredient be water-soluble.

Keywords: diabetic, guava leaf tea, protein tyrosine phosphatase-1B

目 錄

緒論...............................................................................................1
一、前言....................................................................................1
二、蛋白質酪胺酸磷酸酶-1B在糖尿病中的角色.................2
三、芭樂葉................................................................................2
四、研究目的............................................................................3

文獻探討.....................................................................................4
一、芭樂心葉............................................................................4
(一) 介紹...................................................................................4
(二) 芭樂心葉之成分...............................................................5
(三) 芭樂葉之功能...................................................................6
(四) 芭樂葉治療糖尿病之相關研究.......................................7
二、糖尿病................................................................................9
(一) 何謂糖尿病.......................................................................9
(二) 為何糖尿病會引發各種代謝上的疾病..........................10
(三) 糖尿病的治療..................................................................11
(四) 糖尿病急性併發症..........................................................13
三、胰島素訊息傳導和調節血糖...........................................14
(一) 胰島素訊息傳導..............................................................14
(二) 飢餓狀態下的血糖調節方法..........................................15
(三) 吸收狀態下的血糖調節方法..........................................17
四、蛋白質酪胺酸磷酸酶-1B.................................................19
(一) 蛋白質酪胺酸磷酸酶-1B的介紹...................................19
(二) 蛋白質酪胺酸磷酸酶-1B的功能...................................22

材料與方法.................................................................................23
一、實驗材料...........................................................................23
(一) 芭樂心葉茶......................................................................23
(二) 器材..................................................................................23
(三) 試劑..................................................................................24
二、實驗方法............................................................................25
(一)芭樂心葉茶甲醇萃取、分配層析和回溶.......................25
1. 芭樂心葉茶甲醇萃取.........................................................25
2. 芭樂心葉茶甲醇萃取物的分配層析法.............................25
3. 芭樂心葉茶甲醇萃取濃縮物和分配層析濃縮物樣品回溶
方法.........................................................................................27
(二)水萃取芭樂心葉茶和回溶...............................................28
1. 熱水萃取芭樂心葉茶.......................................................28
2. 芭樂心葉茶水萃取濃縮物回溶.......................................28
(三)蛋白質磷酸水解酶-1B酵素分析試驗............................29
1. Buffer的配製.....................................................................29
2. 受質6,8-difluoro-4-methylumbelliferyl phosphate的配製...... 30
3. 蛋白質磷酸水解酶-1B的配製.........................................30
4. 抑制劑Sodium Orthovanadate的配製.............................30
5. 蛋白質磷酸水解酶-1B的線性範圍.................................31
6. 蛋白質磷酸水解酶-1B的Km值.......................................32
7. 二甲基亞碸對蛋白質磷酸水解酶-1B的影響....................
.................................................................................................33
8. Sodium Orthovanadate抑制蛋白質磷酸水解酶-1B的IC50
................................................................................................34
9. 芭樂心葉茶甲醇萃取物和分配層析樣品抑制蛋白質磷酸
水解酶-1B的IC50..................................................................35
10. 水萃芭樂心葉茶抑制蛋白質磷酸水解酶-1B的IC50...37
11. Quercetin抑制蛋白質磷酸水解酶-1B的IC50...............38
12. Caffeic acid抑制蛋白質磷酸水解酶-1B的IC50...........39
13. Gallic acid抑制蛋白質磷酸水解酶-1B的IC50.............40

結果..............................................................................................41
一、芭樂心葉茶甲醇萃取和分配層析...................................41
二、水萃芭樂心葉茶...............................................................45
三、蛋白質磷酸水解酶-1B酵素分析試驗............................46
1. 蛋白質磷酸水解酶-1B的線性範圍...................................46
2. 蛋白質磷酸水解酶-1B的Km值.........................................47
3. Sodium Orthovanadate抑制蛋白質磷酸水解酶-1B...........47
4. 芭樂心葉茶甲醇萃取和分配層析樣品的IC50..................48
5. 甲醇和二甲基亞碸對蛋白質磷酸水解酶-1B影響...........49
6. 芭樂心葉茶水萃濃縮物抑制蛋白質磷酸水解酶-1B的IC50
...................................................................................................51

討論..............................................................................................52
一、芭樂心葉茶甲醇萃取和分配層析..................................52
二、蛋白質磷酸水解酶-1B酵素分析試驗...........................52

總結..............................................................................................53

參考文獻......................................................................................54
英文部分.................................................................................54
中文部分.................................................................................58

表 目 錄

表一、樣品抑制蛋白質磷酸水解酶-1B的濃度..........................35
表二、五個樣品抑制蛋白質磷酸水解酶-1B的最終濃度..........36
表三、 Quercetin抑制蛋白質磷酸水解酶-1B............................38
表四、芭樂心葉茶甲醇萃取物之分配層析的產量.....................44
表五、芭樂心葉茶甲醇萃取和分配層析樣品的IC50.................50
表六、芭樂心葉茶水萃濃縮物的IC50.........................................51

圖 目 錄

圖一、在組織之間影響葡萄糖代謝的調控.................................16
圖二、胰島素代謝的調控方式.....................................................17
圖三、蛋白質酪胺酸磷酸酶-1B的結構......................................20
圖四、蛋白質酪胺酸磷酸酶-1B的調控......................................20
圖五、芭樂心葉茶甲醇萃取和分配層析的實驗架構.................26
圖六、芭樂心葉茶甲醇萃取物.....................................................41
圖七、層析己烷層濃縮物.............................................................42
圖八、層析乙酸乙酯層濃縮物.....................................................42
圖九、層析丁醇層濃縮物.............................................................43
圖十、層析水溶層濃縮物.............................................................43
圖十一、直接水萃芭樂心葉茶濃縮物.........................................45
圖十二、蛋白質磷酸水解酶-1B的線性範圍..............................46
圖十三、Sodium Orthovanadate抑制蛋白質磷酸水解酶-1B.....47
圖十四、二甲基亞碸對蛋白質磷酸水解酶-1B影響..................48

參考文獻
英文部分

Anastasi E., Dotta F., Tiberti C., Vecci E., Ponte E., Di Mario U., Insulin prophylaxis down-regulates islet antigen expression and islet autoimmunity in the low-dose Stz mouse model of diabetes. Autoimmunity. 1999;29:249-256.

Barford D., Structural Studies of Reversible Protein Phosphorylation and Protein Phosphatases. Biochem Soc Trans.1999;27:751–766.

Cheng F.C., Shen S.C., and Wu J. S., Effect of Guava (psidium guajava L.) Leaf Extract on Glucose Uptake in Rat Hepatocytes. Journal of Food Science. 2009;74:312-318.

Dadke S., Chernoff J., Protein tyrosine phosphatase 1B as a potential drug target for obesity. Disord. 2003;3(4):299-304.

Deguchi Y., Miyazaki K., Anti hyperglycemic and anti hyperlipidemic effects of guava leaf extract. Nutr Metab (Lond). 2010;2:7-9.

Delibegovic M., Bence K.K., Mody N., Hong E.G., Ko H.J., Kim J.K., Kahn B.B., Neel B.G., Improved glucose homeostasis in mice with muscle-specific deletion of protein-tyrosine phosphatase 1B. Mol Cell Biol. 2007;27(21):7727-34.

Elchebly M., Payette P., Michaliszyn E., Cromlish W., Collins S., Lee-Loy A., Normandin D., Cheng A., Himms-Hagen J., Chan C., Ramachandran C.,Gresser MJ., Tremblay ML., Kennedy BP., Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science. 1999;283:1544-1548.

Echwald S.M., Bach H., Vestergaard H., Richelsen B., Kristensen K., Drivsholm T., Borch J. K., Hansen T., Pedersen O., A P387L Variant in Protein Tyrosine Phosphatase-1B (PTP-1B) Is Associated With Type 2 Diabetes and Impaired Serine Phosphorylation of PTP-1B In Vitro. Diabetes. 2002;51(1):1-6.

Frank A., Deng S., Huang X., Velidedeoglu E., Bae Y.S., Liu C., Abt P., Stephenson R., Mohiuddin M., Thambipillai T., Markmann E., Palanjian M., Sellers M., Naji A., Barker C.F., Markmann J.F., Transplantation for Type I Diabetes Comparison of Vascularized Whole-Organ Pancreas With Isolated Pancreatic Islets. Ann Surg. 2004; 240(4):631–643.

Johnson T.O., Ermolieff J., Jirousek M.R., Protein tyrosine phosphatase 1B inhibitors for diabetes. Nat Rev Drug Discov. 2002;1(9):696-709.

Koo H.Y., Wallig M.A., Chung B.H., Nara T.Y., Cho B.H., Nakamura M.T., Dietary fructose induces a wide range of genes with distinct shift in carbohydrate and lipid metabolism in fed and fasted rat liver. Biochim Biophys Acta. 2008;1782(5):341-349.

Klaman L.D., Boss O., Peroni O.D., Kim J.K., Martino J.L., Zabolotny J.M., Moghal N., Lubkin M., Kim Y.B., Sharpe A.H., Stricker-Krongrad A., Shulman GI., NeelBG., Kahn BB., Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice. Mol.Cell Biol .2000;20:5479-5489.

Laszlo C., Peter H., Zoltan R.J., Tibor W., Makoto O., Interactions between the Endocrine and Exocrine Pancreas and Their Clinical Relevance. Pancreatology. 2009; 9: 351-359.

Matthews D.R., Cull C.A., Stratton I.M., Holman R.R., and Turner R.C., Sulphonylurea failure in non-insulin-dependent diabetic patients over six years. Diabet Med. 1998;15:297-303.

Nicola J.S., Xiaoling W., Harold S., Tim D., Nicholas D. C., and Sandra D., Protein tyrosine phosphatase-1B gene PTPN1: selection of tagging single nucleotide polymorphisms and association with body fat, insulin sensitivity, and the metabolic syndrome in a normal female population. Diabetes. 2005;54(11):3296-304.

Oh W.K., Lee C.H., Lee M.S., Bae E.Y., Sohn C.B., Oh H., Kim B.Y., Ahn J.S., Antidiabetic effects of extracts from Psidium guajava. J Ethnopharmacol. 2005;96(3):411-5.

Ojewole J.A., Hypoglycemic and hypotensive effects of Psidium guajava Linn. (Myrtaceae) leaf aqueous extract. Methods Find Exp Clin Pharmacol. 2005;27(10):689-95.

Rondinone C.M., Trevillyan J.M., Clampit J., Gum R.J., Berg C., Kroeger P., Frost L., Zinker B.A., Reilly R., Ulrich R., Butler M., Monia B.P., Jirousek M.R., Waring J.F., Protein tyrosine phosphatase 1B reduction regulates adiposity and expression of genes involved in lipogenesis. Diabetes. 2002 ;51(8):2405-11.

Saltiel A.R., Kahn C.R., Insulin signalling and the regulation of glucose and lipid metabolism. Nature. 2001;414(6865):799 - 806.

Shen S.C., Cheng F.C., Wu N.J., Effect of guava (Psidium guajava Linn.) leaf soluble solids on glucose metabolism in type 2 diabetic rats. Phytother Res. 2008; 22(11):1458-64.

Sayan C., Siriwan W., Chittima M., Supaporn P., Narisa K., Development of Guajava Liposome Serum and Evaluation of Free Radical-Scavenging Capacity. J Health Res. 2009,23(4):163-167.

Wang B., Liu H.C., Hong J.R., Li H.G., Huang C.Y., Effect of Psidium guajava leaf extract on alpha-glucosidase activity in small intestine of diabetic mouse. Sichuan Da Xue Xue Bao Yi Xue Ban. 2007;38(2):298-301.

Wijetunge S., Lymn J.S., Hughes A.D., Effect of inhibition of tyrosine phosphatases on voltage-operated calcium channel currents in rabbit isolated ear artery cells. British Journal of Pharmacology. 1998;124:307- 316.

中文部分

甘偉松，台灣藥用植物誌，第三卷，1965年。

行政院衛生署，糖尿病防治手冊糖尿病預防、診斷與控制流程指引，遠流出版社，臺北市，1998年。

吳建中，歐仕益，陳靜，「芭樂多糖對糖尿病小鼠的血糖及抗氧化能力的影響」，中成藥，第29卷第5期，668-671頁，2007年。

李淑華，江雪卿，「芭樂降血糖成分之研究(二)」，台灣大學藥學研究所碩士論文1986年。

吳宗儒，徐鳳麟，「番石榴葉之酚性成分之研究」台北醫學院藥學研究所碩士論文1991年。

林育禪，謝秋蘭，彭耀寰，「芭樂心葉水萃物對抑制葡萄糖及雙碳化合物誘導糖尿病血管併發症相關因數之功效評估」，弘光科技大學生物科技研究所碩士論文2006年。

何俊杰，蔡慧盈，「淺談第二型糖尿病之近期發展」，成醫藥誌，第二十卷第一期，6-10頁，2010年。

徐銘佛，孟于青，「應用化學標定法研究一氧化氮對酪胺酸磷酸水解酶的活性調控以促進胰島素的感受性」， 台灣大學生化科學研究所博士論文2010年。

張慧香，江雪卿，「芭樂降血糖成分之研究(一)」，台灣大學藥學研究所碩士論文1983年。

張毓珊，謝秋蘭，陳冠州，彭耀寰，「番石榴心葉水萃取物中活性成分誘導攝護腺癌DU 145細胞凋亡之分子機制」，弘光科技大學生物科技研究所碩士論文2007年。

陳淑娟，最新圖解生化學，合記圖書出版社，臺北，2001年。

陳俞琦，羅俊光，「桑葉萃取物及芭樂葉萃取物生物活性之比較」，元培科技大學影像醫學研究所碩士論文2008年。

謝亨理，劉承慈，「大蒜精油及其有機含硫成分-二烯丙基硫化物、二烯丙基二硫化物及二烯丙基三硫化物對於streptozotocin誘發糖尿病大鼠血糖控制及骨骼肌葡萄糖代謝之影響」，中山醫學大學營養科學研究所碩士論文2002年。