臺灣博碩士論文加值系統

糖尿病 (Diabetes Mellitus) 是一種慢性疾病，雖然它有許多不同的病因，但它們有一個共同的 特點就是高血糖。糖尿病在現代社會的漸漸流行使得尋找新的治療方法有其急迫性。雙胜肽基胜肽酶IV (Dipeptidyl Peptidase-Ⅳ, DPP-IV) 和蛋白酪氨酸磷酸酶1B (Protein Tyrosine Phosphatase-1B, PTP1B) 是兩個作為抗糖尿病化合物開發很好目標，但是這兩種酵素的抑制劑須要作謹慎地評估才能判斷是否值得開發。番石榴葉因為它有降血糖的作用，早已在亞洲及非洲被用來作為一種民間傳統醫藥。 而1,2,4 - 三唑 (1,2,4-triazole) 是一種常出現在各種天然產物的結構，由於他們在生物醫藥和農藥領域的活性，已引起了人們的關注。
這項研究的目標是評估番石榴葉萃取物對PTP1B酶的抑制作用及合成的1,2,4 -三唑化合物對PTP1B與DPP-IV酶的抑制效力。我們已取得番石榴葉水萃取物和14種1,2,4-三唑類化合物的樣品。我們評估檢測番石榴葉水萃取物對PTP1B及 T細胞蛋白酪氨酸磷酸酶 (T Cell Protein Tyrosine Phosphatase, TCPTP) 的選擇性。同時我們測試三唑類化合物對PTP1B與DPP-IV酶的活性抑制。
結果顯示，番石榴葉萃取物的第5分層具有最好的PTP1B酶抑制活性，其IC50值為0.160±0.013毫克/升，並提供了對TCPTP超過2倍的選擇性。三唑化合物方面，化合物-4具最強PTP1B酶的抑制效果，其體外抑制活性IC50值為 25 µM。化合物-4與-12具有最強的DPP-IV抑制效果，其IC50值分別為10 µM和5 µM。從這個實驗結果顯示，番石榴葉萃取物和合成1,2,4-三唑類化合物有明顯的PTP1B的DPP-IV酶抑。

Diabetes mellitus (DM) is a chronic disease that is characteristic of hyperglycemia with various etiologies. The rising prevalence of diabetes mellitus has fueled an intensified search for new therapeutic treatments. Dipeptidyl peptidase IV (DPP-IV) and protein tyrosine phosphatase 1b (PTP1B) are two major enzymes that have recently emerged as biological targets for anti-diabetic compound development. The validation of specific inhibitor to PTP1B and DPP-IV is required for treating diabetes. Guava leaf has been used as a folk medicine traditionally because of its hypoglycemic effect. The 1,2,4-triazole is a synthetic compound that appears in the structure of various natural products and has attracted attention due to their diverse biological activities in pharmaceutical and agrochemical fields.
The objectives of this study are to evaluating inhibitory potential of guava leaf extracts on PTP1B enzyme and the inhibition potency of synthetic 1,2,4-triazole compounds on PTP1B and DPP-IV enzymes. We have obtained water fraction of guava leaf extracts and fourteen 1,2,4-triazole compounds. We assessed the inhibitory activity associated with the water fraction of guava leaf extracts on PTP1B and their selectivity over T-cell protein tyrosine phosphatase (TCPTP) using enzymatic assays. Similarly, we tested the inhibitory activity of each triazole compounds on PTP1B and DPPIV enzymes.
Results showed that fraction 5 of guava leaf extracts has the best inhibitory activity with the IC50 values of 0.160±0.013 µg/ml and provided 2-folds selectivity over T-cell protein tyrosine phosphatase (TCPTP). Compound 4 of trizole compounds has the strongest PTP1B inhibitory activity in vitro with the IC50 values of 25 µM. Compound 4 and 12 exhibited the strongest DPP-IV inhibitory activities with the IC50 values of 10 µM and 5 µM, respectively. The results from this experiment indicated that water fraction of guava leaf extracts and synthetic 1,2,4-triazole compounds have strong potency as inhibitor of PTP1B and DPP-IV enzymes.

TABLE OF CONTENTS

ACKNOWLEDGMENT iv
ENGLISH ABSTRACT v
CHINESE ABSTRACT vii
TABLE OF CONTENTS ix
LIST OF TABLES xi
LIST OF FIGURES xii
I. CHAPTER ONE Introduction 1
1.1 Background 1
1.2 Research Objectives 2
1.3 Research Scope 2
II. CHAPTER TWO Literature Review 3
2.1 Diabetes Mellitus 3
2.2 Protein tyrosine Phosphatase 1B (PTP1B) in type 2 diabetes mellitus 4
2.3 Dipeptidyl peptidase-IV (DPP-IV) in type 2 diabetes (T2DM) 10
2.4 Guava leaf 14
2.5 1,2,4-Triazole 17
III. CHAPTER THREE Method 19
3.1 Materials and Equipments 19
3.2 Method 19
3.2.1 Preparation of guava leaf extracts 19
3.2.2 Alkaline phosphatase (AP) enzymatic activity assay 20
3.2.3 Protein tyrosine phosphatase 1b (PTP1B) enzymatic activity assay 20
3.2.4 T-cell protein tyrosine phosphatase (TCPTP) enzymatic activity assay 21
3.2.5 PTP1B inhibition screening (1, 2, 4-triazole compounds) 22
3.2.6 Dipeptidyl peptidase IV (DPP-IV) enzymatic assay 22
IV. CHAPTER FOUR Results 29
4.1 Alkaline phosphatase activity assay 29
4.2 Water fraction inhibition on protein tyrosine phosphatase 1b (PTP1B) 29
4.3 T-cell protein tyrosine phosphatase (TCPTP) inhibition by water fraction of guava leaf extracts 31
4.4 Protein tyrosine phosphatase 1b (PTP1B) inhibition by 1, 2, 4-triazole compounds 32
4.5 Dipeptidyl peptidase-IV (DPP-IV) inhibitory detection by 1,2,4-triazole compounds 34
V. CHAPTER FIVE Discussion and Conclusion 35
5.1 Discussion 35
5.1.1 Guava leaf extracts inhibitory potencies on protein tyrosine phosphatase 1b (PTP1B) 35
5.1.2 Triazole compounds inhibitory potencies on DPP-IV and PTP1B enzymes 36
5.2 Conclusion 39
REFERENCES 39


LIST OF TABLES

TABLE 2-1 Summary of hypoglycemic effects from recent studies on guava leaf extract 16
TABLE 4-1 The IC50 values of water fractions of guava leaf against PTP1B 31
TABLE 4-2 The IC50 values of water fractions of guava leaf against TCPTP 31
TABLE 4-3 The selectivity between PTP1B and TCPTP 32
TABLE 4-4 1, 2, 4-triazole compounds 32
TABLE 4-5 The IC50 values of 1,2,4-triazole compounds against PTP1B 34
TABLE 4-6 The IC50 values of 1,2,4-triazole compounds against DPP-IV 34

LIST OF FIGURES

Figure 2-1 The ribbon diagram of overall structure of the dipeptidyl peptidase-IV (DPP-IV) homodimer 5
Figure 2-2 The general catalytic mechanism for dephosphorylation of substrate by protein tyrosine phosphatase (PTPs) 6
Figure 2-3 PTP1B selectivity region and gateway residue. (a) the YRD (tyrosine, arginine, aspartic acid) motif) and (b) glycine (Gly)259 gateway residue 7
Figure 2-4 Negative regulation of the leptin and insulin metabolic signal-transduction pathways by protein tyrosine phosphatase 1B (PTP1B) 9
Figure 2-5 The ribbon diagram of overall of the dipeptidyl peptidase-IV (DPP-IV) homodimer 11
Figure 2-6 Dipeptidyl peptidase-IV (DPP-IV) inhibition physiologic action 13
Figure 2-7 Guava Fruits and Leaves. 15
Figure 2-8 Chemical structure of 1,2,4-triazole or s-triazole 17
Figure 3-1 Flow chart of guava leaf extraction and partition 23
Figure 3-2 Flow chart of alkaline phosphatase enzymatic assay 24
Figure 3-3 Flow chart of water fraction inhibition potencies on protein tyrosine phosphatase 1b (PTP1B) 25
Figure 3-4 Flow chart of water fraction inhibition potencies on T-cell protein tyrosine phosphatase (TCPTP) 26
Figure 3-5 Flow chart of PTP1B inhibition screening by 1, 2, 4- triazole compounds 27
Figure 3-6 Flow chart of DPP-IV inhibition screening by 1, 2, 4- triazole compounds 28
Figure 4-1 Lineweaver-Burk analysis of sodium vanadate inhibiton. using alkaline phosphatase (AP) 29
Figure 4-2 Lineweaver-Burk analysis of PTP1B enzymatic activity 30
Figure 4-3 Structure of fourteen 1,2,4-triazole compounds 33

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