Luận án tiến sĩ: Investigation on Euphorbia hirta Linn. extracts for drug development against type 2 diabetes mellitus

VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY INTERNATIONAL UNIVERSITY INVESTIGATION ON EUPHORBIA HIRTA LINN. EXTRACTS FOR DRUG DEVELOPMENT AGAINST TYPE 2 DIABETES MELLITUS In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY In Biotechnology TRAN THI KIM NGAN Student ID: PBTIU14001 HO CHI MINH CITY — June 2022 Investigation on Euphorbia hirta Linn. extracts for drug development against type 2 diabetes mellitus A dissertation submitted to The School of Biotechnology in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at International University, Vietnam National University - Ho Chi Minh City Submitted by TRAN THI KIM NGAN This certifies that the Dissertation of Tran Thi Kim Ngan is approved by Examination Committee: Chairman Assoc. Nguyen Van Thuan Member Assoc.

Tran Nguyen Minh An Member Assoc. Le Tan Khoi Member PhD. Nguyen Thi Y Nhi Member PhD. Do Thi My Lien Member PhD.

Le Quang Phong June 2022 School of Biotechnology International University Vietnam National University - Ho Chi Minh City Declaration by author Thereby declare that I am the sole author and composer of my doctoral dissertation and that no other sources other than those listed have been used in the bibliography and identified as references. Furthermore, I firmly declare that my thesis has not been prepared for another examination or assignment at any other institution to obtain a degree. Author Signature and Date Print Name and Title Publications related to this dissertation International articles Authors, title, journal name, journal index, page, year ISSN No Impact factor published (SCM⁄SCT-e)) Tran, N. Spray-drying | ISSN 2304-8158 4.350 1 microencapsulation of high concentration of bioactive (SCIE) compounds fragments from Euphorbia hirta L.

extract and their effect on diabetes mellitus. Bioactive Compounds in | ISSN 2079-7737 5.079 2 | Anti-Diabetic Plants: From Herbal Medicine to Modern Drug (SCIE) Discovery. Ngan Tran, Minh Nguyen, Khanh LB Le, Nhi Nguyen, Quan | JSSN 2076-3417 2.679 3 Tran, Ly Le (2020). Screening of Antibacterial Activity, (SCIE) Antioxidant Activity, and Anticancer Activity of Euphorbia hirta Linn.

Domestic articles Authors, title, journal name, journal index, page, year No ISSN Impact factor published Trần Thi Kim Ngân, Lê Thi Lý, Nguyễn Thi Ý Nhi, Trần Thị 1 Minh, Tran Lê Quan (2017). Khảo sát thành phần hóa hoc của dịch chiết ethyl acetate từ cỏ sữa lá lớn (Euphorbia hirta Linn. Tap chí Phát trién KH&CN, tập 20, số T5-2017, trang 95-101. iv International conference and proceedings Authors, title, conference name, page ofproceeding, time and No ISSN Impact factor location organized 1 2 Domestic conference and proceedings Authors, title, conference name, page ofproceeding, time, No , , , , ISSN Impact factor and location organized Contributions by others to the dissertation No contributions by others.

Statement of parts of the dissertation submitted to qualify for the award of another degree None Research Involving Human or Animal Subjects Our research was approved by the University of Science Animal Care and Use Committee (No. vi Acknowledgements In my first word, I would like to express my deepest appreciation to my supervisor, Associate Professor Le Thi Ly, for assigning me to this incredible research. I will be eternally thankful for her outstanding excellence and continual support during the study process. Thanks to her decision, I had good opportunities to work in a professional environment at International University.

Furthermore, without her support and encouragement, I cannot complete my thesis progress as well, and she always supported and motivated me with all her heart during my thesis, especially in Covid 19 pandemic. Moreover, I express my sincere thanks to Associate Professor Tran Le Quan, from the University of Science for his ongoing assistance, helpful recommendations, scientific conversations, and significant contribution to my work. My grateful and warmest thank also goes to Le Phuoc Bao Khanh, Msc., and my colleagues at the University of Science for a considerable contribution and hard-worked find them to help me complete my experiments indeed. Also, I would like thank to Tran Hoang Song, Bsc.

and Nguyen Cong Hoi, Bsc. for their amazing technical assistance. I would like to thank Ms Mai Thi Thanh Loan, Mr. Lai Thanh Son, Ms.

Nguyen Thi Hong Hanh, Ms. Tram Thi Khanh Hien and Ms. Nguyen Pham Quynh Anh who supported me in administrative matters. I would like to thank the Rector Board of the International University and the University of Science for creating favourable conditions and helping me to complete the thesis.

I want to express my gratitude to my family, my friends, and my colleagues for the support during the research and completion of the thesis. vii CONTENTS LIST OF TABLES xii LIST OF FIGURES xiii LIST OF ABBREVIATIONS xvi Abstract xix Abstract (in Vietnamese) xxii Chapter 1: GENERAL INTRODUCTION 1 1.1 Introduction to diabetes mellitus 1 1.1 Definition of diabetes mellitus 1 1.2 Classification of diabetes mellitus 1 1. Prevalence and incidence of diabetes mellitus 3 1.4 Pathogenesis of diabetes mellitus 3 1. Medication for type 2 diabetes mellitus 5 1.3 Commonly used antidiabetic medicinal plants in Vietnam 6 1.4 _ Introduction to Euphorbia hirta Linn.44 — Bioactive compounds In E.5 Investigation of bioactive compounds of E.

hirta in Vietnam 31 1.6 Antidiabetic activity of E.6 Research objectives 36 Chapter 2: IN SILICO STUDY ON ANTIDIABETIC ACTIVITY OF BIOACTIVE COMPOUNDS IN EUPHORBIA HIRTA LINN.2 Materials and methods 39 2.1 Ligand and protein preparation 39 2.1 Docking results on AA 42 2.2 Docking results on AG 45 2.4 Discussion 47 Chapter 3: PREPARATION OF EUPHORBIA HIRTA LINN. EXTRACTS AND INVESTIGATION OF BIOACTIVE COMPOUNDS FROM ETHYL ACETATE EXTRACT 53 3.2 Materials and methods 54 3.1 | Chemicals and equipment 54 3. Plant material collection and identification 55 3. hirta extracts using maceration method 55 3.4 Phytochemical analysis of E.5 Determination of quercetin and quercitrin content in EE-1 58 3.6 Isolation of bioactive compound from EE-1 59 3.7 Identification of isolated compounds from EA extract 60 3.3 Determination of quercetin and quercitrin content in EE-1 62 3.4 Isolatlon of bioactive compound from EE-1 63 3.

Identification of isolated compounds from EE-1 68 3. hirta extraction and phytochemical analysis 86 3.2 Identification of isolated compounds from EA extract 87 Chapter 4: EVALUATION OF ANTIOXIDANT ACTIVITY AND CYTOTOXIC ACTIVITY OF EUPHORBIA HIRTA LINN.2 Materials and methods 88 4.1 Animals and materials 88 ix 4.3 — Statistical analysis 92 43 Results 92 4.1 Antioxidant activities of E.2 In vifro screening for cytotoxic activity of E.4 Discussion 94 Chapter 5: IN VITRO AND IN VIVO EXPERIMENTS OF EUPHORBIA HIRTA LINN. EXTRACTS AGAINST TYPE 2 DIABETES MELLITUS 97 5.2 Materials and methods 98 5. Animals and reagents 98 5.2 In vitro enzyme inhibitory experiments 98 5.

Evaluation of the samples on plasma glucose in oral glucose tolerance test 99 5.4 Spray-drying microencapsulation of EE-2 extract and the effect on STZ/NAD-induced mice 100 5.1 In vitro alpha-amylase inhibitory assay 102 5.2 Tr vitro alpha-glucosidase inhibitory assay 103 5. Evaluation of the samples on plasma glucose in oral glucose tolerance test 105 5.4 | Spray-drying microencapsulation 107 5.1 In vitro enzyme inhibitory experiments on isolated compounds 111 5.2 Evaluation of the #. Airta extracts and isolated compound in fasting plasma glucose 113 5.3 Spray-drying microencapsulation 114 Chapter 6: GENERAL CONCLUSIONS 117 6.1 The main results of the dissertation 117 6.2 _ The scientific and practical contributions of the dissertation 119 6. Recommendation 120 REFERENCES 122 LIST OF APPENDICES 150 APPENDICES 151 xi LIST OF TABLES Table 1.1 Classification of main phytoconstituents of Gymnema sylvestre 12 Table 1.2 Some major isolated phytoconstituents on P.1 Center grid box coordinates for docking simulation of AA and AG _—40 Table 2.

hirta and their ID 4I Table 3.1 Solvent program in determination of quercetin content in EE-1 58 Table 3.2 Solvent program in determination of quercitrin content in EE-1 59 Table 3.3 Weight and yield of extract after liquid-liquid extract 60 Table 3.4 Total phenolic and flavonoid contents in #. hirta extracts 61 Table 3.5 'H NMR and °C NMR data of EA01 with scopoletin 69 Table 3.6 'H NMR and °C NMR data of EA02 with methyl gallate 70 Table 3.7 'H NMR and '3C NMR data of EA03 with gallic acid 71 Table 3.8 'H NMR and 3C NMR data of EA04 with kaempferol 72 Table 3.9 'H NMR and !3C NMR data of EA05 with quercetin 74 Table 3.10 'H and °C spectroscopic data of EA06 and myricetin 76 Table 3.11 TH, !C, HMBC and COSY NMR spectroscopic data of EA07 and afzelin 78 Table 3.12 !H, °C, HMBC and COSY NMR spectroscopic data of EA0§ and quercitrin 80 Table 3.13 'H and !C-NMR spectroscopic data of EA09 and myricitrin 82 Table 3.14 'H and '3C-NMR spectroscopic data of EA10 and rutin 84 Table 3.15 Comparison TPC and TFC of £. hirta in previous studies 87 Table 4.1 DPPH radical scavenging activity of extracts 92 Table 4.2 Lipid peroxidation inhibitory activity of E. hirta extracts 93 Table 4.3 DPPH radical scavenging activity of E.

hirta extracts 95 Table 5.1 AA inhibitory activity of E.2 AA inhibitory activity of isolated compounds.3 AG inhibitory activity of E. hirta extracts 104 Table 5.4 AG inhibitory activity of isolated compounds.5 Results of the sample effect after 7-day dose on plasma glucose level _ 105 Table 5.6 Physical properties of spray power 107 Table 5.7 Pyramiding dose 108 xii LIST OF FIGURES Figure 1.1: Insulin signaling pathway to bring glucose into cells 4 Figure 1.2 Pictures show the morphological characteristics of the Momordica charantia: whole plant (left) and unripe fruit (right) 7 Figure 1.3 Mechanism in decreasing blood glucose levels of M.4 Commercial products using for DM treatment in Vietnam 8 Figure 1.6 Mechanisms of Panax ginseng saponins on different organs related to diabetes 10 Figure 1.7 Basic molecular structure of Gymnemic acid 11 Figure 1.8 Mechanisms of Gymnema sylvestre in antidiabetic activity 12 Figure 1.10 Euphorbia hirta Linn.11 The structure of flavonoids extracted from È.12 The structure of lignans isolated from E.13 The structure of some tannins isolated from E.14 The structure of terpenoids isolated from E.15 The structure of sterols isolated from E.16 The structure of acids isolated from E.17 The structure of phytochemicals in essential oil of E.18 The structure of other compounds isolated from E.1 Elements in molecular docking 39 Figure 2.2 Grid box location for docking simulation of (A) alpha-amylase and (B) alpha-glucosidase showed with PMV software 40 Figure 2.3 The total process to identify potential compounds on AA and AG and its pharmacophore interactions 42 Figure 2.4 Total docking score of all 48 compounds on AA and control compound (Acarbose) (C) 43 Figure 2.5 Chemical structure of top 5 compounds that have lowest docking score on AA.6 Pharmacophore analysis by using LigandScout of control compound and top 5 compounds on AA 45 xiii Figure 2.7 Chemical structure of top 5 compounds that have the lowest docking score on AG: (a) xanthorhamnin, (b) Ø-amyrine, (c) cyanidin 3,5-O-diglucoside, (d) (-)- epicatechin 3-gallate and (e) tinyatoxin 46 Figure 2.8 Total docking score of all 48 compounds on AG and control compound (Acarbose) (C) 46 Figure 2.9 Pharmacophore analysis by using LigandScout of control compound and top 5 compounds on AG 47 Figure 2.10 Pharmacophore interaction comparison between AA (left) and AG (right) of some compounds in E.1 The chart indicates the process of solvent extraction 56 Figure 3.2 The Folin—Ciocalteu reaction (W, tungsten; Mo, molybdenum reacting with polyphenol groups in an oxidation-reduction reaction) 57 Figure 3.3 The process of identification of bioactive compounds and biological experiments 59 Figure 3.4 HPLC-DAD analysis of quercetin in EE-1 62 Figure 3.5 HPLC-DAD analysis of quercitrin in EE-1 62 Figure 3.6 Normal silica gel column chromatography of EA extract 63 Figure 3.7 TLC results with solvent mixture and visualize by 25% sulfuric acid 64 Figure 3.8 Schematic diagram of the isolation of bioactive compounds of EE-1 (part 1) 66 Figure 3.9 Schematic diagram of the isolation of bioactive compounds of EE-1 (part 2) 67 Figure 3.