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

hirta 33 1.5 Problem statement 36 1.6 Research objectives 36 Chapter 2: IN SILICO STUDY ON ANTIDIABETIC ACTIVITY OF BIOACTIVE COMPOUNDS IN EUPHORBIA HIRTA LINN... LIST OF FIGURESFigure 1

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 Prof Nguyen Van Thuan

Member Assoc Prof Tran Nguyen Minh An

Member Assoc Prof Le Tan Khoi

Member PhD Nguyen Thi Y Nhi

Member PhD Do Thi My Lien

Member PhD Le Quang Phong

Declaration by author

Thereby declare that I am the sole author and composer of my doctoral dissertation andthat no other sources other than those listed have been used in the bibliography andidentified as references Furthermore, I firmly declare that my thesis has not beenprepared for another examination or assignment at any other institution to obtain a

degree.

Author Signature and Date

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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., Tran, M., Truong, H., & Ly Le (2020) 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 Foods, 9(7), 881.

Tran, N., Pham, B., & Le, L (2020) Bioactive Compounds in | ISSN 2079-7737 5.079

2 | Anti-Diabetic Plants: From Herbal Medicine to Modern Drug (SCIE)

Discovery Biology, 9(9) 252.

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 Extracts Appl Sci., 10(23), 8408.

Domestic articles

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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.

International conference and proceedings

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No ISSN Impact factor

location organized

1

2

Domestic conference and proceedings

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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 ofanother degree

None

Research Involving Human or Animal Subjects

Our research was approved by the University of Science Animal Care and UseCommittee (No 248b/KHTN-ACCCUS)

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 studyprocess Thanks to her decision, I had good opportunities to work in a professionalenvironment at International University Furthermore, without her support andencouragement, I cannot complete my thesis progress as well, and she always supportedand 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 ThiHong Hanh, Ms Tram Thi Khanh Hien and Ms Nguyen Pham Quynh Anh whosupported 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 thesupport during the research and completion of the thesis

LIST OF TABLES xii

LIST OF FIGURES xiiiLIST OF ABBREVIATIONS xvi

Abstract xix

Abstract (in Vietnamese) xxiiChapter 1: GENERAL INTRODUCTION 1

1.1 Introduction to diabetes mellitus 1

1.1.1 Definition of diabetes mellitus 11.1.2 Classification of diabetes mellitus 1

1.1.3 Prevalence and incidence of diabetes mellitus 3

1.1.4 Pathogenesis of diabetes mellitus 3

1.2 Medication for type 2 diabetes mellitus 5 1.3 Commonly used antidiabetic medicinal plants in Vietnam 6

1.3.1 | Momordica charantia (bitter melon) 61.3.2 Panax Ginseng C.A Meyer 81.3.3 Allium sativum (Allium) 10

1.3.4 Œymnema sylvestre (Apocynaceae) 11 1.3.5 Ocimum sanctum L (Lamiaceae) 13

1.3.6 Phaseolus vulgaris (Leguminosae) 14

1.4 _ Introduction to Euphorbia hirta Linn 16

1.41 Basic botany 161.442 — Distribution 161.43 Morphology 171.44 — Bioactive compounds In E hirta 17

1.4.5 Investigation of bioactive compounds of E hirta in Vietnam 31 1.4.6 Antidiabetic activity of E hirta 33

1.5 Problem statement 36 1.6 Research objectives 36

Chapter 2: IN SILICO STUDY ON ANTIDIABETIC ACTIVITY

OF BIOACTIVE COMPOUNDS IN EUPHORBIA HIRTA LINN 38

2.1 Introduction 38

2.2 Materials and methods 39

2.2.1 Ligand and protein preparation 39

2.2.2 Methods 40

2.3 Results 42

2.3.1 Docking results on AA 422.3.2 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

3.1 Introduction 53

3.2 Materials and methods 54

3.2.1 | Chemicals and equipment 543.2.2 Plant material collection and identification 553.2.3 Preparation of E hirta extracts using maceration method 553.2.4 Phytochemical analysis of E hirta extracts 573.2.5 Determination of quercetin and quercitrin content in EE-1 583.2.6 Isolation of bioactive compound from EE-1 593.2.7 Identification of isolated compounds from EA extract 603.2.8 Statistical analysis 60

3.3 Results 60

3.3.1 E hirta extraction 60

3.3.2 Phytochemical analysis 613.3.3 Determination of quercetin and quercitrin content in EE-1 623.3.4 Isolatlon of bioactive compound from EE-1 633.3.5 Identification of isolated compounds from EE-1 68

3.4 Discussion 86

3.4.1 E hirta extraction and phytochemical analysis 863.4.2 Identification of isolated compounds from EA extract 87

Chapter 4: EVALUATION OF ANTIOXIDANT ACTIVITY AND

CYTOTOXIC ACTIVITY OF EUPHORBIA HIRTA LINN EXTRACTS 88

4.1 Introduction 88 4.2 Materials and methods 88

4.2.1 Animals and materials 88

4.2.2 Methods 894.2.3 — Statistical analysis 92

43 Results 92

4.3.1 Antioxidant activities of E hirta extracts 92

4.3.2 In vifro screening for cytotoxic activity of E hirta extracts 93 4.4 Discussion 94

Chapter 5: IN VITRO AND IN VIVO EXPERIMENTS OF EUPHORBIA HIRTA

LINN EXTRACTS AGAINST TYPE 2 DIABETES MELLITUS 97

5.1 Introduction 97

5.2 Materials and methods 98

5.2.1 Animals and reagents 985.2.2 In vitro enzyme inhibitory experiments 985.2.3 Evaluation of the samples on plasma glucose in oral glucose tolerance

5.3.1 In vitro alpha-amylase inhibitory assay 102

5.3.2 Tr vitro alpha-glucosidase inhibitory assay 1035.3.3 Evaluation of the samples on plasma glucose in oral glucose tolerance

test 1055.3.4 | Spray-drying microencapsulation 107

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 6.3 Recommendation 120

REFERENCES 122

LIST OF APPENDICES 150

APPENDICES 151

LIST OF TABLES

Table 1.1 Classification of main phytoconstituents of Gymnema sylvestre 12Table 1.2 Some major isolated phytoconstituents on P vulgaris 15

Table 2.1 Center grid box coordinates for docking simulation of AA and AG _—40

Table 2.2 Compounds on E hirta and their ID 4ITable 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

Table 5.5 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

LIST OF FIGURES

Figure 1.1: Insulin signaling pathway to bring glucose into cells 4Figure 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 charantia 7Figure 1.4 Commercial products using for DM treatment in Vietnam 8Figure 1.5 Panax Ginseng C.A Meyer 9Figure 1.6 Mechanisms of Panax ginseng saponins on different organs related to

diabetes 10

Figure 1.7 Basic molecular structure of Gymnemic acid 11Figure 1.8 Mechanisms of Gymnema sylvestre in antidiabetic activity 12Figure 1.9 Ocimum sanctum L.: (a) whole plant and (b) leaves 13Figure 1.10 Euphorbia hirta Linn 17Figure 1.11 The structure of flavonoids extracted from È hirta 20Figure 1.12 The structure of lignans isolated from E hirta 22Figure 1.13 The structure of some tannins isolated from E hirta 24Figure 1.14 The structure of terpenoids isolated from E hirta 25Figure 1.15 The structure of sterols isolated from E hirta 27Figure 1.16 The structure of acids isolated from E hirta 28

Figure 1.17 The structure of phytochemicals in essential oil of E hirta 29

Figure 1.18 The structure of other compounds isolated from E hirta 31Figure 2.1 Elements in molecular docking 39Figure 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 42Figure 2.4 Total docking score of all 48 compounds on AA and control compound

(Acarbose) (C) 43Figure 2.5 Chemical structure of top 5 compounds that have lowest docking score on

AA (a) taraxerol, (b) tinyatoxin, (c) a-amyrine, (d) taraxerone and (e) Ø-amyrine _44 Figure 2.6 Pharmacophore analysis by using LigandScout of control compound and

top 5 compounds on AA 45

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 46Figure 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 hirta 51Figure 3.1 The chart indicates the process of solvent extraction 56Figure 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 63Figure 3.7 TLC results with solvent mixture and visualize by 25% sulfuric acid 64Figure 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) 67Figure 3.10 TLC chromatogram of the EA01 in its solvent fractions (a) and isolated

EA0I (b) 68Figure 3.11 Structure of compound EA01 69Figure 3.12 Structure of compound EA02 70Figure 3.13 Structure of compound EA03 71Figure 3.14 Structure of compound EA04 73Figure 3.15 Structure of compound EA05 75Figure 3.16 Structure of compound EA06 77

Figure 3.17 Structure of compound EA07 79 Figure 3.18 Structure of compound EA08 81

Figure 3.19 Structure of compound EA09 83Figure 3.20 Structure of compound EA10 86

Figure 4 I The reaction mechanism of DPPH’ with antioxidant AH 90Figure 4.2 MDA-TBA2 adduct formation 90Figure 4.3 Cytotoxicity of E hirta extracts on NCI H460 and Hep G2 cells 93Figure 5.1 Effect of EA powder on streptozotocin-induced diabetic mice in 4 hours

109

Figure 5.2 Effect of EE-2 powder on STZ-induced diabetic mice in 15 days 109Figure 6.1 Structure of ten isolated compounds 119

LIST OF ABBREVIATIONS

1D-NMR _ : 1-Dimensional Nuclear Magnetic Resonance Spectroscopy

2D-NMR _ : 2-Dimensional Nuclear Magnetic Resonance Spectroscopy

‘H-NMR : Proton Nuclear Magnetic Resonance Spectroscopy

!4C-NMR : Carbon-13 Nuclear Magnetic Resonance Spectroscopy

ỗ : Chemical shift (in ppm)

DMSO : Dimethyl sulfoxide

DMSO-ds _ : Deuterated dimethyl sulfoxide

dt : Triplet of doublet

EA : Ethyl acetate

EB : Butanol extract of Euphorbia hirta Linn.

EE : Ethyl acetate extract of Euphorbia hirta Linn.

E hirta : Euphorbia hirta Linn.

EM : Methanol extract (crude extract) of Euphorbia hirta Linn.

EP : Petroleum ether extract of Euphorbia hirta Linn.

ESI-MS : Electron Spray Ionization Mass Spectroscopy

HMBC : Heteronuclear Multiple Bond Coherence

HPLC : High-performance liquid chromatography or high-pressure

liquid chromatography HSQC : Heteronuclear Single Quantum Coherence

Hz, MHz : Hertz, megahertz

ICso : Inhibitory concentration 50%

IDF : International Diabetes Federation

NMR : Nuclear Magnetic Resonance spectroscopy

ppm : Part per million

PE : Petroleum ether

Ss : Singlet

STZ : Streptozotocin

t : Triplet

: Total phenolic content

: Volume to volume ratio

: World Health Organization

Investigation on Euphorbia hirta Linn extracts for drug

development against type 2 diabetes mellitus

Tran Thi Kim NganSchool of BiotechnologyInternational University, VNU-HCM

Vietnam is a country with high biodiversity, with about 4000 species of plants

used as medicine, in which many medicinal plants are used in folk medicine to treat diabetes mellitus, including Euphoria hirta Linn (E hirta), which belongs to the

family Euphorbiaceae Previous studies show that E hirta has potential for the futureproduction of antidiabetic drugs The overall objectives of the present study were toinvestigate, determine, and quantify the phytochemicals, mainly phenoliccompounds, from # hirta extracts and to evaluate their antidiabetic activities in invitro, in vivo, and in silico approaches

In in silico study, flavonoids containing in E hirta gave good interactions with

two enzymes Due to variations in pharmacophore characteristics, the flavonoid familyhas an advantage in binding to these receptors due to relatively strong hydrogen bonds

Five fractional extracts of E hirta were obtained after the extraction process,including EM-1, EP-1, EC-1, EE-1, and EB-1 The value of the phenolic content was109.86 + 1.38, 90.89 + 1.45, 55.86 + 0.66, 254.96 + 10.05 and 70.90 + 0.65 mg ofGAE/g extract for EM-1, EP-1, EC-1, EE-1, and EB-1, respectively EE-1 had thehighest total phenol content, followed by EM-1, EP-1, EB-1, and EC-1 The values

of the total flavonoid content were 18.92 + 1.33, 8.48 + 1.16, 16.41 + 1.44, 27.66 +0.73 and 12.43 + 1.66 mg of quercetin/g extract for EM-1, EP-1, EC-1, EE-1, andEB-1, respectively EE-1 had the highest total flavonoid content

To investigate the antioxidant activity of E hirta extracts, a DPPH radical

scavenging assay of È hirta was carried out Our results revealed that EM-1 and EE-1

extracts had more potent free radical scavenging activity than other extracts For lipidperoxidation inhibitory activity, ICso values of EM-1, EP-1, EC-1, EE-1, and EB-1 werealso measured EE-l extract appeared to be 18.74-fold as potent as Trolox Anticancer

activities of those extracts were examined by sulforhodamine B (SRB) in vitrocytotoxicity assay on two cancer cell lines, including lung cancer cells NCI-H460 andliver cancer cells Hep G2 EE-1 at a concentration of 100 ug/mL has significantinhibitory activity on the growth of lung cancer cells NCI-H460 and liver cancer cells

Hep G2 compared to all other extracts.

To elucidate the antidiabetic activity of those extracts, we investigated thetherapeutic effects of five extracts of E hirta in AA and AG inhibitory activity (in vitro)and hypoglycemic activity in normal mice by evaluating the samples on plasma glucose

in oral glucose tolerance test (in vivo) In in vitro study, E hirta extracts had stronginhibitory activity against AG and relatively mild AA inhibitory activity EE-1 had

more potent inhibitory activity of two enzymes than other extracts Besides, EE-1 expressed higher hypoglycemic effect in the oral glucose tolerance test in normal mice

than the remaining extracts Our results suggest that EA extract (EE-2) has the most

potent activities, and should be used to determine phytochemicals and mechanisms of

their antidiabetic activity EA extract was mixed with 20% maltodextrin in a ratio of1:10 to spray-dry microencapsulation The spray powder reduced 51% fast bloodglucose (FBG) after 4hrs treatments Furthermore, administration of spray powder for

15 days significantly lowered fasting blood glucose levels streptozotocin-diabetic mice

by 23.32%, whereas acarbose - a standard antidiabetic drug - and distilled water

reduced fasting blood glucose levels by 30.87% and 16.89% Our results show thatobtained E hirta powder has potential antidiabetic activity

Ten pure compounds were isolated and identified from EE-1, including EA01

(scopoletin), EA02 (methyl gallate), EA03 (gallic acid), EA04 (kaempferol), EA05

(quercetin), EA06 (myricetin), EA07 (afzelin), EA08 (quercitrin), EA09 (myricitrin)

and EA10 (rutin) Among them, five isolated compounds, including EA02 (methyl gallate), EA03 (gallic acid), EA04 (kaempferol), EA05 (quercetin), and EA08

(quercitrin) were carried out the AA and AG inhibitory activity Those compounds gaveweaker AA inhibitory activity than acarbose, but it proved phenolic compounds couldinhibit carbohydrate hydrolyzing enzyme and has potential antidiabetic activity The

AG inhibitory action of all five compounds was stronger than acarbose Moreover,EA08 (quercitrin) was subjected to evaluate the effect on fasting plasma glucose level

in normal mice This study suggests that EA extract might be employed as a functionalfood for type 2 DM treatment and by the nutraceutical sector as a source of quercetin

and quercitrin There are certain similarities in the results of investigating the activities

of the compounds in vitro and in silico These results give us a theoretical basis forfuture research in the development of drugs for type 2 DM

Keywords: Euphorbia hirta Linn.; bioactive compounds; antidiabetic activity, alpha-amylase

inhibition, alpha-glucosidase inhibition, in silico approach

Abstract (in Vietnamese)

Khảo sát các dịch chiết của cây Cỏ Sữa Lá lớn Euphorbia hirta Linn đễ phát triển thuốc trị bệnh đái tháo

đường type 2

Trần Thị Kim Ngân

Khoa Công nghệ sinh học

Đại học Quốc Tế, Đại học Quốc gia Tp HCM

Việt Nam là quốc gia có đặc tính đa dạng sinh học cao, với khoảng 4000 loài thực vật được dùng làm thuốc, trong đó có nhiều cây thuốc được dùng trong dân gian

dé chữa bệnh đái tháo đường, trong đó có cây Cỏ sữa lá lớn (tên khoa học là Euphoria hirta Linn (E hirta)), thuộc họ Thầu Dau (Euphorbiaceae) Các nghiên cứu trước đây

cho thấy E hirta có tiềm năng trong việc sản xuất thuốc trị đái tháo đường trong tương

lai Mục tiêu tổng thé của nghiên cứu này là nghiên cứu, xác định và định lượng các hoạt chat sinh học, đặc biệt là các hợp chat phenolic, từ cao chiết E hirta và đánh giá

các hoạt tính chống đái tháo đường của chúng trong các phương pháp thử nghiệm in

vitro, in vivo và trong thử nghiệm sang lọc in silico

Trong nghiên cứu in silico, các flavonoid chứa trong E hirta tương tác tốt với

hai enzyme Do sự khác nhau về đặc điểm cấu tạo của các nhóm dược chất, họ flavonoid

có lợi thé hon trong việc gan kết với các thụ thé này do các liên kết hydrogen tương đối

mạnh

Năm cao chiết EH thu được từ phương pháp ngâm dầm gồm cao chiết EM-1

(cao chiết tổng), EP-1, EC-1, EE-1, và EB-1 Ham lượng phenolic tong cua cac cao

chiết lần lượt là 109,86 + 1,38, 90,89 + 1,45, 55,86 + 0,66, 254,96 + 10,05 va 70,90 +

0,65 (mg QE/g cao chiết) đối với EM-1, EP-1, EC-1, EE-1, và EB-1 Cao chiết EH-1

có hàm lượng phenolic cao nhất, tiếp theo là EM-1, EP-1, EB-1, EC-I Hàm lượng

flavonoid tong lần lượt là 18,92 + 1,33, 8,48 + 1,16, 16,41 + 1,44, 27,66 + 0,73 và 12,43

+ 1,66 (mg quercetin/g cao chiết) đối với EM-1, EP-1, EC-1, EE-1, và EB-1 Cao chiết EE-1 cũng có hàm lượng flavonoid tổng cao nhất.

Đề khảo sát hoạt tính chống oxy hóa của các cao chiết trên, thử nghiệm khả

năng bắt gốc tự do DPPH: của các cao chiết EH đã được tiến hành Kết qua cho thay

cao chiết EM-1 và EE-1 có hoạt tính bắt gốc tự do mạnh hơn các cao chiết khác Đối

với khả năng ức chế phan ứng peroxy hoá lipid, cao chiết EE-1 cho hoạt tính mạnh nhất,

gấp 18,74 lần Trolox Hoạt động chống ung thư của các cao chiết đó đã được kiểm tra bang thử nghiệm độc tính tế bao in vitro trên hai dòng tế bao ung thư, gồm tế bao ung thư phối NCI-H460 và tế bào ung thư gan Hep G2 Cao chiết EE-I ở nồng độ 100 pg /

mL có hoạt tinh ức chế đáng ké sự phát triển của tế bao ung thư phối NCI-H460 và tế

bào ung thư gan Hep G2 so với tất cả các cao chiết khác.

Với mục dich làm sáng tỏ hoạt tính chống đái tháo đường của E hirta, chúng tôi đã khảo sát khả năng ức chế hoạt động của hai loại enzyme thủy phân carbohydrate

là a-amylase (AA) và a-glucosidase (AG) của năm cao chiết EH ở mức độ in vitro và hoạt tính hạ đường huyết ở chuột bình thường bằng cách đánh giá tác dụng của các cao chiết trên glucose huyết tương trong thử nghiệm dung nap glucose qua đường miệng ở

mức độ in vivo Trong thử nghiệm in vitro, cao chiết E hirta có hoạt tính ức chế mạnh

đối với AG và hoạt động ức chế AA khá nhẹ Cao chiết EE-1 có hoạt tính ức chế hai

enzym mạnh hơn han các cao chiết khác Bên cạnh đó, EE-1 cho thấy tác dụng hạ đường

huyết tốt hơn so với cao chiết còn lai Từ đó, EE-1 được chọn đề xác định thành phầnhóa học của các hoạt chât và cơ chê kháng tiêu đường của các hoạt chât này

Mười hợp chất tinh khiết đã được phân lập và xác định từ cao chiết EA, bao gồm EA01 (scopoletin), EA02 (metyl gallate), EA03 (gallic acid), EA04 (kaempferol),

EA05 (quercetin), EA06 (myricetin), EA07 (afzelin), EA0§ (quercitrin), EA09

(myricitrin) và EA10 (rutin) Trong đó, năm hợp chat cô lập, bao gồm EA02 (metyl

gallate), EA03 (gallic acid), EA05 (quercetin), EA04 (kaempferol) và EA08

(quercitrin) được tiếp tục khảo sát hoạt động ức chế AA và AG Những hợp chất này cho hoạt tính ức chế AA yếu hơn acarbose nhưng nó đã chứng minh rằng các hợp chat

phenolic này có thé ức chế enzyme thủy phân carbohydrate và có hoạt tính chống đái

tháo đường tiềm năng Tác dụng ức chế AG của cả năm hợp chất đều mạnh hơn

acarbose Dịch chiết EA được trộn với 20% maltodextrin theo tỷ lệ 1:10 dé đóng gói vi

bao khô dạng phun Bột phun làm giảm 51% đường huyết nhanh (FBG) sau 4 giờ điều trị Hơn nữa, sử dụng bột phun trong 15 ngày làm giảm đáng kê mức đường huyết lúc

đói ở chuột mắc bệnh tiểu đường bang streptozotocin xuống 23,32%, trong khi acarbose

- một loại thuốc trị tiêu đường tiêu chuẩn và nước cất làm giảm mức đường huyết lúc

đói 30,87% và 16,89% Kết quả của chúng tôi cho thấy rằng cao chiết EA thu được có

hoạt tính chống đái tháo đường tiềm năng Trong thử nghiệm sang lọc in silico, các hop

chat phenolic chứa trong E hirta thé hiện tương tác tốt với hai enzyme trên Và họ flavonoid có lợi thế hơn trong việc liên kết với các thụ thể này do liên kết hydro tương

đối mạnh Rõ ràng là có những điểm tương đồng nhất định trong các kết quả nghiên

cứu hoạt động của các hợp chat trong mức độ in vitro và in silico Những kết quả này

cung cấp cho chúng tôi cơ sở lý thuyết dé nghiên cứu phát triển thuốc điều trị bệnh đái

tháo đường type 2 trong thời gian tới

Keywords: Euphorbia hirta Linn., hoạt chat, khả năng chống bệnh tiểu dường, amylase,

alpha-glucosidase, thử nghiệm in silico

Chapter 1 : GENERAL INTRODUCTION

1.1 Introduction to diabetes mellitus

1.1.1 Definition of diabetes mellitus

Diabetes mellitus (DM) is a chronic disease of carbohydrate metabolism caused by

decreased insulin secretion and peripheral insulin resistance leading to hyperglycemia Early

symptoms of hyperglycemia include heavy drinking, increased thirst, polyuria, and blurredvision Diabetes is one of the leading causes of many serious diseases such as vascular disease,peripheral neuropathy, kidney disease, and susceptibility to infections [1, 2]

Insulin is an essential hormone produced in the pancreas to control glucoseconcentration in the bloodstream It can move to cells of the body where glucose is convertedinto energy Additionally, this hormone takes part in the metabolism of fat and protein Insulin

resistance (not using insulin efficiently) or insulin deficiency (not producing enough insulin)

leads to an increase blood glucose level, which is the clinical indicator of DM Treatment

includes diet, exercise, and medications to lower blood glucose, including insulin and oral

glucose-lowering medications Although there is still no cure for diabetes, early diagnosis,treatment, and prevention of complications are still much better The current treatment ofdiabetes tends to combine modern drugs and herbal medicines to limit the progression of thedisease and prevent complications [2]

1.1.2 Classification of diabetes mellitus

The first widely accepted classification of DM was published by WHO in 1980 and thenwas updated in 1985 and 1999 [1] Nowadays, DM is divided into two main types below [1-3]

1.1.2.1 Type 1 diabetes

Type 1 DM is known as insulin-dependent diabetes mellitus (IDDM) or juvenile-onset

DM It is an autoimmune disorder in which cells, insulin-producing cells of the pancreas inthe body, have been destroyed, and the pancreas produces little or no insulin It develops mostoften in children and young adults People with type 1 DM must take insulin daily to maintain

a stable glucose level (in the appropriate range) to live Without insulin injection, they cannotsurvive Type 1 DM accounts for approximately 10% of all cases Type | DM is one of the

most common chronic diseases in childhood, although obesity and overweight have become

more common.

In type 1 DM, insulin production is absent because of autoimmune cell destructioninitiated by environmental exposure in predisposed individuals Destruction progressessubclinically over the coming months and years as /-cell mass declines to the point whereinsulin concentrations are inadequate to control plasma glucose concentration Type | DM oftendevelops in children and adolescents More recently, the standard form is diagnosed before theage of 30; however, it can also develop in adults (adult-onset autoimmune DM, which oftenpresents as type 2 DM) Some cases of type 1 DM, especially in the non-Caucasian population,

do not appear to be autogenic and are commonly thought to be idiopathic type 1, accountingfor less than 10% of patients with diabetes [4,5]

1.1.2.2 Type 2 diabetes mellitus

Type 2 DM, also known as non-insulin-dependent diabetes mellitus (NIDDM), is adiagnosis in which the pancreas produces enough insulin, but the body cannot use the insulin

effectively, a situation called insulin resistance It is caused by a combination of peripheral

resistance to insulin action and the inadequate compensatory response of insulin secretion bythe pancreatic beta cells (relative insulin deficiency) During the state of insulin resistance, thebody requires cells to increase insulin production, causing the loss of -cells or their impairedfunction Type 2 DM is commonly seen in adults; approximately 80% to 90% of patients havetype 2 diabetes However, the number of patients in children is increasing because of rising

obesity status, physical inactivity, and unhealthy diet.

Insulin is present in the body, but insulin cannot or works poorly to get sugar from theblood into the cells, which is common in people over 40 In type 2 DM, insulin secretion isinadequate because the patient has insulin resistance The liver’s insulin resistance results in aninability to inhibit the liver’s glucose production, and peripheral insulin resistance reducesperipheral glucose uptake This combination increases fasting and postprandial blood sugar.Often insulin levels are very high, especially in the early stages of the disease In the late stages

of diabetes, insulin production may decrease, further worsening hyperglycemia The disease

usually has an adult-onset and becomes more common with ageing Nearly one-third of adultsover 65 years old have impaired glucose tolerance In the elderly, plasma glucoseconcentrations are higher after meals than in young people, especially after meals with a highcarbohydrate content Glucose levels may also return to normal for longer, in part because of

increased accumulation of visceral and abdominal fat and decreased muscle mass Type 2 DM

is often diagnosed very late because the early stages of hyperglycemia develop silently with nosymptoms When there are clinical manifestations, it is often accompanied by other disorders

of lipid metabolism, cardiovascular, neurological, and renal pathological manifestations.

Obesity and physical inactivity are risk factors for type 2 DM It also often occurs in people

with a family history of diabetes People with type 2 DM can be treated with lifestyle changes,

combined with medication to control blood sugar or insulin [6-8]

1.1.3 Prevalence and incidence of diabetes mellitus

Economic development and changes in the social environment influence diseasepatterns from infectious to noncommunicable diseases Noncommunicable diseases have

become more common in low- and middle-income nations It is worth emphasizing that

diabetes affects about three-quarters of the population aged 30 to 70 According to data fromthe International Diabetes Federation (IDF 2021) [9], approximately 537 million adults aged

20 to 79 are living with diabetes, and the total number of people living with DM is estimated

to increase to 643 million people by 2030 DM caused about 6.7 million deaths in 2021 InVietnam, diabetes is forecasted to be one of the seven leading causes of death and disability by

2030 [10]

1.1.4 Pathogenesis of diabetes mellitus

1.1.4.1 Pathogenesis of type 1 diabetes

The process of f-cell injury is an autoimmune process Individuals with genetic susceptibility will have an increased risk of type 1 DM after foreign environmental factors such

as mumps virus, measles, coxsackie B4 and BS, and retro type C These factors will attack thebody with the genetic predisposition for type 1 DM Only minimal damage of Ø-cells alsoreleases antigens, stimulates the body to produce autoantibodies, causes activating theautoimmune islet inflammatory response The possible antigens are GAD (glutamic acid

decarboxylase), a protein located in the cytoplasm of Ø-cells.

Autoantibodies will react with antigens Activated lymphocyte macrophages will gather around

the islet, triggering an inflammatory response T lymphocytes secrete chemical mediatorsincluding interleukin-1 that have toxic effects on /-cells Interleukin-1 induces the formation

of free radicals, which damage and destroy f-cells, leading to the cessation of insulin secretion.However, a small number of cases of type 1 diabetes with no known cause, not related to human

leukocyte antigen but with very clear genetic predisposition [2].

1.1.4.2 Pathogenesis of type 2 diabetes

Normally, insulin plays an important role in maintaining blood sugar homeostasis.Blood glucose concentrations are dependent on insulin secretion and insulin penetration inperipheral tissues and inhibition of hepatic glycogenolysis to glucose The closely relatedpathophysiological mechanisms in patients with type 2 DM are impaired insulin secretion and

insulin resistance Many studies show that in patients with type 2 diabetes who are not

overweight, there is a major manifestation of decreased insulin secretion, in contrast to type 2

DM with obesity, insulin resistance is the main [2,11]

For patients with insulin secretion disorders, in the early stages of type 2 DM, insulinmay be normal or increased, but the rate of insulin secretion is slow and not commensurate withthe increase in blood glucose If blood glucose continues to rise, in the later stages insulinresponse to glucose will become more reduced On the other hand, insulin resistance is a state

of decreased or lost target organ sensitivity to insulin The researchers determined that it was

due to abnormalities at sites before, after, and right at the insulin receptor in the target tissue A

decrease in the number of insulin receptors is an abnormality at the receptor or the presence of

antibodies against the insulin receptor is a pre-receptor inhibitor Due to decreased activity oftyrosine kinase of the post-insulin receptor region, insulin when bound to the receptor does notexert its biological effect Therefore, it does not stimulate the transport of glucose into the cells

On the other hand, the increased secretion of insulin antagonistic hormones such as growthhormone (GH), glucocorticoids, catecholamines, thyroxine all affect insulin receptors Insulin

controls blood sugar balance through three combined mechanisms, each dysfunctional

mechanism can be the cause of insulin resistance (Figure 1.1) [12]

Serine phosphorylation of IRS proteins

| Decreased expression of gluconeogenic

FFA enzymes PEPCK and G6P ase in liver

Figure 1.1: Insulin signaling pathway to bring glucose into cells [12]

pY: phosphorylated tyrosine; IR: insulin receptor; IRS: insulin receptor protein; PIk: phosphatidylinositol

3-kinase; PDK1: phosphoinositidedependent kinase 1; PKB: protein kinase B; Foxo-1: forkhead box protein O; PEPCK: phosphoenolpyruvate carboxykinase; G6Pase: glucose-6-phosphatase; FFA: free fatty acids; PIP2: phosphatidyl-Inositol-3,4-bisphosphate; PIP3: phosphatidyl-inositol-3,4,5-tris-phosphate; GLUT4: glucose transporter 4.

Insulin signalling cascade depicting insulin binding to the insulin receptor, which

activates glucose transporter 4, which imports glucose into the cell Binding of insulin to the IR

activates PI3-k that these factors act as mediators of PKB-activated PDK1 connectivity.Through Foxo-1, activated PKB can control transcription of target genes such as PEPCK and

G6Pase Increased free fatty acid levels can cause serine phosphorylation of IRS proteins, which

reduces IRS-tyrosine phosphorylation and hence reduces the effects [13]

1.2 Medication for type 2 diabetes mellitus

Understanding the pathogenesis of DM is extremely important in treatment Healthyeating, physical activity, and weight control are the center of any therapeutic program forpatients with DM These lifestyle modifications [14] not only lower blood glucose levels in thebody, but also ameliorate many risk factors for cardiovascular diseases and help to weight loss.However, most patients cannot get a good lifestyle, so medications are usually used for diabetictreatment [15] DM is not only a concern of the medical industry but also attracts the attention

of social regulators According to IDF’s Statistics, diabetes prevalence among adults aged 20 to

79 years old was expected to be 10.5 percent (536.6 million) in 2021, increasing to 12.2 percent(783.2 million) in 2045 In 2021, global diabetes-related health expenses were forecast to be

$966 billion, and by 2045, they are expected to be $1054 billion [16] Therefore, medication

expenditure has become a serious problem for patients in diabetes from developing countries.

Currently, there are six main classes of oral diabetes medicines and two classes of injections

used in over the world for controlling blood glucose levels [17] The tablets are known as

biguanides (metformin), sulfonylureas, thiazolidinediones (glitazones), meglitinides (glinides),alpha-glucosidase inhibitors and DPP-4 inhibitors The classes of medications given byinjection are incretin mimetics and insulin Mechanisms of these medications have beenreported There are two drugs on the market that are used to limit the rise in blood sugar:

acarbose (Precose, Lucobay) and miglitol (Glyset) These drugs act in the small intestine,

competitively inhibiting the enzyme a-glucosidase Thus, the sugar in the intestines is slowly

absorbed into the body and the sugar immediately after eating will not rise high in the blood However, most modern drugs have many side effects and adverse reactions causing some

serious medical problems [18-24]

Besides modern medication, traditional medicines have been used for a long time andplay an important role as alternative medicines [25-27] In addition to the direction of synthetic

research on drugs for the treatment of diabetes, in recent years the world has turned to the research and use of herbs as a supportive method to cure the disease Traditional medicines are

usually the preferred choice for primary healthcare of patients in developing countries because

of better cultural acceptability, better compatibility with the human body and lesser side effects

than modern therapies Recently, some medicinal plants have been reported to be useful in

diabetes worldwide and have been used empirically as antidiabetic and antihyperlipidemic

remedies However, investigating new antidiabetic drugs from natural plants has been still attractive because they contain phytoconstituents that demonstrate alternative and safe effects

on the treatment of DM Most plants contain bioactive components, such as phenolics,

glycosides, alkaloids, terpenoids, flavonoids, carotenoids, and other constituents, that have been

proved as having antidiabetic activities [28-31] Vietnam is a country in the tropical monsoonclimate hot and humid, so it has rich plant resources This is a huge advantage in developingproducts of natural origin As mentioned above, the search for medicinal herbs capable ofsupporting the treatment of diabetes has great practical significance Euphorbia hirta Linn is apopular polyphenolic-rich grass in Vietnam EH is a wild plant that is easy to find anywheresuch as in the garden, in the alley, in the wasteland, by the roadside, in the park However, sofar, the research on this issue in our country has not been properly invested and the results arestill limited which better lowers blood glucose while causing fewer side effects, is a topicalissue that scientists are interested in researching

1.3 Commonly used antidiabetic medicinal plants in Vietnam

According to WHO, a plant-based traditional system of medicine is still the chief support

of about 75-80% of the world population mainly in developing countries having a diversity of

plants like Vietnam [32] Therefore, it is necessary to study more a-glucosidase inhibitors aswell as lower blood glucose levels from different sources, helping to make the treatment of type

2 DM more selective and effective better The hypoglycemic and antidiabetic effects of severalplants used as traditional antidiabetic remedies have been proved, and the mechanisms ofhypoglycemic activity of these plants have been studied effectively [33-35] This part listed

some commonly traditional herbals for diabetes treatment in Vietnam.

1.3.1 Momordica charantia (bitter melon)

Momordica charantia (MC) is one of the most common vegetables in the tropical region,particularly in Vietnam, India, China, East Africa, South-North Asia, and Central and South

America [36,37] It is a member of the Cucurbitaceae family and is known as bitter melon or bitter gourd (Figure 1.2) Besides using MC as a vegetable, it is supposed to be an herbal

medicine to use as folk medicine Its bioactivities include anti-inflammatory activity,antioxidant activity, anti-viral activity, anti-cancer activity, anti-bacterial activity, andespecially anti-diabetic activity [38]

Figure 1.2 Pictures show the morphological characteristics of the Momordica charantia:

whole plant (left) and unripe fruit (right)Phytochemistry

Many investigations were published about active components of bitter melon thatsupport in type 2 DM treatment The important phytochemicals of the plants are steroids,

momordicosides (A, B, C, D, E, G, Fi, Fo, I, K, L), acyl glucosyl sterols, fatty acids, amino

acids, alkaloids, phenolic compounds, steroidal saponins, vitamins, carbohydrates, andminerals [39-43]

humans [46]

† Glucose oxidation

t Hepatic glycogen “ử" Glucose uptake

|

Antioxidant BITTER MELON B-cell protection

\ Gluconeogenesis J Intestinal glucose absorption

Figure 1.3 Mechanism in decreasing blood glucose levels of M charantia

Moreover, aqueous ethanolic extract of MC seeds exposed protection of pancreatic

/-cells in in vitro experiment [47] In treatment of type | DM, MC extract has been proven that it

could improve beta cells in pancreatic islets in streptozotocin-induced type-1 diabetic rats.

Related with type 1 DM, polypeptide-p was shown the action as similar as human insulin in

human body and therefore may be used as plant-based insulin replacement in patients with

type-1 DM [48,49] Other studies also investigated the inhibition of diabetes-related enzymes, such

as alpha-glucosidase and alpha-amylase from MC extracts [50]

In Vietnam, Mudaru capsule is one of the most common functional foods which is

researched and made entirely from 100% natural forest bitter melon (Figure 1.4.a) This product

helps to lower blood sugar, stabilize blood sugar, and prevent diabetes complications In

addition, it also supports the treatment of other diseases such as high blood pressure, cardiovascular disease, cholesterol reduction, blood fat reduction, liver fat, support to dissolve

kidney stones, and dyslipidemia [51]

Figure 1.4 Commercial products using for DM treatment in Vietnam

1.3.2 Panax Ginseng C.A Meyer

In Korea, Ginseng has been the most famous traditional plant used in folk medicine for

a long time ago (Figure 1.5) Ginseng belongs to the genus Panax in Araliaceae family [52] It

distributes typically in a cool climate region that can be found in Eastern Asia, such as Korea,

Eastern Siberia, Northeast China, and North America.

The root of this plant contains many bioactive compounds, including triterpeneglycosides, or saponins, commonly referred to as ginsenosides, Panaxans, vanillic acid, andsalicylates All parts of the plant also have some active constituents, such as amino acids,alkaloids, phenols, proteins, polypeptides, and vitamins BI and B2, which have been identified

[53-55]

Antidiabetic activity

Since diabetes mellitus is characterized by insulin resistance and /-cell dysfunction,therapeutic medications should be involved in improving insulin resistance, enhancing glucoseuptake, decreasing blood glucose concentration, and protecting/regenerating -cell from

pancreatic islets Many researchers have investigated the antidiabetic activities on the root of Panax ginseng in vitro and in vivo experiments [56-58] The most important group of

phytochofemicals of Panax ginseng is ginseng-specific saponins called ginsenosides Amongthem, Ginsenosied Rb2 was the most effective constituent treated for streptozotocin-induceddiabetic rats by decreasing blood glucose levels [59] Moreover, in fermented red ginsengextracts, the content of ginsenoside Rg2, Rg3, and Rha are higher than normal ginseng so that

those extracts significantly reduced blood glucose levels and increased plasma insulin levels in

streptozotocin-induced diabetic rats by orally administered 100 or 200 mg/kg extracts dissolved

in water, at 10 a.m daily in 3 weeks [60] These mechanisms have been displayed in Figure 1.6

In generally, saponins, which were isolated from ginseng that has been proven significantantidiabetic activity And the mechanism of these components in antidiabetic treatment is to

moderate the enzyme activity to influence to glucose metabolism, control insulin secretion

[61-66]

Rb1, Rg3, Rh2, CK, Re, Rg1

Figure 1.6 Mechanisms of Panax ginseng saponins on different organs related to

diabetes

1.3.3 Allium sativum (Allium)

Allium sativum Linn., is a commonly herb which has been known as garlic, belongs tothe family Allium It could be found in Asia, Africa, and Europe It is originally indigenous toAsia, is now widely grown in popularity to produce a condiment, especially in Asian cuisine

anti-Phytochemistry

Raw garlic contains many active phytochemicals like alkaloids, flavonoids, cardiac

glycosides, terpenes and steroids, Resin It also contains some sulfur compounds, such as alliin,

allicin, ajoene, diallyl sulfide, enzymes, B-vitamins, proteins, minerals, saponins, flavonoids,etc., which are not sulphur containing compounds [68-71]

Antidiabetic activity

Depending on these scientific studies, garlic’s biological activity in anti-diabetic has

been shown that its mechanism is to control the excretion of insulin from cells, enhance

glucose tolerance and glycogen synthesis [72] For example, these two bioactive compounds

which are extracted from garlic are allyl propyldisulfide and S-methylcysteine sulfoxide can

decrease blood glucose levels In addition, the ethanol extract from garlic was also had

antidiabetic activity by restoring delayed insulin response [73,74].

Glu Metaherb is a health food that applies modern technology to natural herbal

preparations to help lower blood sugar, reduce the risk and symptoms of type | and 2 diabetes.

The product is consulted by the Vietnam Academy of Science and Technology The mainingredients of this product include Gymnema sylvestre, Scoparia dulcis L., Allium sativum,Dioscorea persimillis, Azadirachta indica A Juss (Figure 1.4.b) [75]

1.3.4 Gymnema sylvestre (Apocynaceae)

Gymnema sylvestre belongs to Apocynaceae family, originated from tropical forests ofThe Southern and Central India and Sri Lanka [76] In Vietnam, G sylvestre has been classified

as vines and found in many Northern and Central provinces So far, this plant has been widelyused in several countries around the world in treating diabetes

Phytochemistry

The phytoconstituents of G sylvestre consist of many groups containing bioactive compounds

that are listed in the below table (Table 1.1) [76, 77].

Figure 1.7 Basic molecular structure of Gymnemic acid

Several mechanisms have been proposed to explain the antidiabetic activity of

Gymnema sylvestre (Figure 1.8) [78,79] According to these literatures, the action of Gymnemicacids in diabetic treatment was reported that they could stimulate pancreatic cell production,thereby increasing insulin production, increase insulin sensitivity and insulin activity, help tocontrol, and stabilize blood glucose concentration in the body Gymnemic acids were alsoreported that they can inhibit the absorption of glucose in the small intestine and inhibit theconversion glycogen in liver to glucose molecules in blood [80]

Table 1.1 Classification of main phytoconstituents of Gymnema sylvestre

Phytoconstituents Classification

Gymnemic acids-acylated (tiglolyl, methylbutyroyl)

Triterpene saponins derivatives of deacylgymnemic acid (DAGA) which is a

3-O-Ø-glucouronide of gymnemagenin (3, 16f, 218, 22a, 23,28-hexahydroxy-olean-12-ene)

Oleanane saponins Gymnemic acids and gymnemasaponins

Dammarene saponins Gymnemosides A, B, C, D, E, and F

; 3-O [f-D-glucopyranosyl Gymnemasins A

(I-3)-Ø-D-glucopyranosyl]-22-O-tiglyol gymnemanol

; Gymnemasins B

3-O-$-D-glucopyranosyl]-(1-4)-a-L-rhamnopyranosyl-(1-6)- Ø-D-galactopyranoside

In Vietnam, there are many products made from Gymnema _ sylvestre Diabetnasupplement helps to lower blood sugar (lower HbA 1c), reduce blood fat, and reduce the risk of

complications of DM as well (Figure 1.4.c) [81].

Stimulate insulin release

Decrease Inhibit intestinal

gluconeogenesis N glucose absorption

Increase hepatic and Regenerate /-cells of

muscle glucose uptake pancreas

Figure 1.8 Mechanisms of Gymnema sylvestre in antidiabetic activity

In addition, the DK - BETICS product is also one of the popular choices of diabetics.Each DK - BETICS capsule contains the main ingredient as Gymnema sylvestre extract Similar

to Diabetna, this product also works to effectively reduce blood sugar and limit the risk of

complications of diabetes This product is intended for use by patients with type | and 2 diabetes

(Figure 1.4.d) [82]

1.3.5 Ocimum sanctum L (Lamiaceae)

Ocimum sanctum L is a small tree, up to one meter tall, commonly known as ‘Sacred

basil’ or "Holy basil’ or “Hương Nhu Tia” in Vietnam, is grown as a household plant in many

countries for medicinal purposes like Vietnam and India (Figure 1.9) It belongs to the

Lamiaceae family The whole plant has a pleasant aroma [83,84]

Phytochemistry

The phytochemistry of Ocimum sanctum is identified in all parts of this plant, containing

many nutrients and bioactive constituents However, the quantity of these constituents depends

on many natural factors, including growing, harvesting, storage conditions [85,86]

In leaf extract of O sanctum, volatile oil was extracted and identified chemicalcompositions, containing many major components like eugenol, methyleugenol, and p-caryophylen The essential oil of this herb contains various bioactive compounds, such asterpenoids, esters, aliphatic aldehydes, and phenolic acids Also, this herb consists of a diversity

of second metabolites, including phenolics, flavonoids, terpenoids, lignans, steroids, fatty acids, and their derivatives Because of their biological and pharmacological properties, these components have mostly been researched for medicinal purposes This herb’s biological and pharmacological effects have been documented to include antioxidant, anti-inflammatory,

antibacterial, anticancer, and antidiabetic action [86]

Antidiabetic activity

In Hannan’s study [87], there are four fractions of Ocimum sanctum including the

ethanol, aqueous, butanol, and ethyl acetate fractions prepared to elucidate the mechanism of

antihyperglycemic activity of this plant showed in literature This result proved that thesefractions could stimulate insulin secretion This study also indicates that the ethanol extractcould decrease blood glucose concentration and increase insulin secretion, thereby this plant is

a potential herb in diabetic treatment Moreover, by in vivo experiment, the Ocimum sanctum

extract was showed that it could improve oral glucose tolerance, decrease serum glucose,

increase glycogen synthesis in the liver [88]

The other investigation has reported the antidiabetic and hypoglycemic activities of a

triterpenoid (16-hydroxy-4,4, 10,

13-tetramethyl-17-(4-methyl-pentyl)-hexadecahydro-cyclopenta[a]phenanthrene-3-one) isolated from Ocimum sanctum by in vivo investigation [89].The mechanisms of the antidiabetic and hypoglycemic potential of this compound wereelucidated to increase the pancreatic secretion of insulin from /-cells, and enhance glucoseutilization [90] This investigation suggested that this triterpenoid should be developed as apotential antidiabetic medicine This evidence supports that O sanctum has many benefits in

the management of diabetes, and it should be encouraged to be a potential anti-diabetic activity

[91]

1.3.6 Phaseolus vulgaris (Leguminosae)

Phaseolus vulgaris L belongs to the Leguminosae family, commonly known as kidneybean, which is a food item of mass consumption in Asia and Eastern countries [92] Phaseolus

vulgaris L is an annual, herbaceous plant which its main root grows deeply so that this plant has good tolerance with changes of climate In Vietnam, its fresh beans have been used as a

daily vegetable

Phytochemistry

Phaseolus vulgaris L has been reported that this plant is an important source of primaryand secondary metabolites necessary in a healthy diet of humans By using the HPLC technique

to separate constituents’ efficiency, the phytochemicals of P vulgaris were identified

successfully [92-94] These compounds could be divided into different groups displayed in

Table 1.2

Antidiabetic activity

Various previous studies have shown that seeds of kidney bean have ø-amylase inhibitors

inhibiting ø-amylase activities in animals and insects, related to control type-2 DM byinhibition of DPP-IV, and a lectin called phytohaemoagglutinin could adjust the activity ofglucagon-like peptides GLP-1 (glucagon-like peptide-1) [95-97] The common bean Phaseolusvulgaris has three isoforms of a-amylase inhibitor including ø-AITI (known as phaseolamin),a-AI2, and ø-AIL, which has been identified and tested in numerous clinical studies against ø-amylase action [95,98] The mechanism of action of these enzyme inhibitors indicated that theycould reduce the carbohydrate absorption by inhibiting the activity of a-amylases of mammals

and insects [98,99] The reduction of the glycaemic index may also limit the risks of insulin

resistance in diabetes mellitus, thereby control the serious consequences of the disease

Recently, another investigation continues to determine a-amylase inhibition of P vulgaris

extract [100]

Table 1.2 Some major isolated phytoconstituents on P vulgaris

Group Phytochemical compound

Hydroxybezoic acid and derivatives flavonoids,

anthocyanins, flavonols, flavanols, isoflavones, flavanones,

Phenolic acids proanthocyanidins, and tannins

Hydroxycinnamic acid and derivatives

Orientin, isoerientin, rutin, myricetin, luteolin, quercetin,Flavonoids kaempferol, myricetin-3-rhamnoside, hyperoside,