ANTIHYPERGLYCEMIC ACTIVITY OF TINOSPORA CRISPA

EXTRACTS AND FRACTIONS IN RAT

MIR MOHAMMAD REZA SEYED HASSANI

UNIVERSITI SAINS MALAYSIA

2015

ANTIHYPERGLYCEMIC ACTIVITY OF TINOSPORA

CRISPA EXTRACTS AND FRACTIONS IN RAT

By

MIR MOHAMMAD REZA SEYED HASSANI

Thesis submitted in fulfillment of the requirements for the degree of

Master of Science

August 2015

ii

ACKNOWLEDGEMENTS

All praises to Allah the Almighty, without whom will everything would cease

to be, who gave me the strength, inspiration and patience to continue this research.

First of all Special thanks to my parents Mir Jafar, Mahin and my brother

Mir Morteza for their supports all this years. I would like to express my deep

gratitude and sincere appreciation to my supervisor Prof. Dr. Mohd Zaini Asmawi

who provided me the germ of the idea for this research. Over the months of research

and analysis, his deep and broad knowledge, constructive suggestions, stimulating

discussion and comments on the preparation of this thesis as well as his wisdom and

wit helped that germ to sprout/germinate and grow. He had always open door for me

to see him for whatever duration or reason although he was busy with his other

students. I also wish to thank my co-supervisor Dr. Roziahanim Binti Mahmud for

her guidance and assistance throughout the chemical aspect of this work. I would

like to express my gratitude to the Dean of School of Pharmaceutical Sciences

Dr. Munavvar Zubaid Abdul Sattar, for giving me the chance to pursue my higher

education in this school. Also, my thanks go to the Institute of Postgraduate Studies

and Universiti Sains Malaysia for their kind cooperation and assistance rendered in

the preparation of this thesis and a warm thanks to director of International office,

Assoc. Prof. Dr. Anees Janee Ali for his helps, kindness and guides. A number of

other academic and non-academic staff at the Universiti Sains Malaysia also gave me

their support and assistance, including Mr. Rosli, the laboratory assistant in the

pharmacology research laboratory and Mr. jasmie for his helps. Also, I am very

grateful to all my colleagues who were always my real friends Abdulmenem, Adlin,

Bassel, Faramarz, Idris, Item, Michael, Nasiba, Raghdaa and Sook yee for their

helps, kindness and supports.

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TABLE OF CONTENTS

Page

ACKNOWLEDGMENT

ii

TABLE OF CONTENTS

iii

LIST OF TABLES

ix

LIST OF FIGURES

LIST OF ABBREVIATIONS

LIST OF PUBLICATION ASSOCIATED WITH THIS THESIS

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xiii

xiv

ABSTRAK

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ABSTRACT

xvii

CHAPTER ONE: INTRODUCTION

1.1 Introduction

1

1.2 Diabetes Mellitus

3

1.3 Classification of Diabetes Mellitus

1.4 Pancreas

5

6

1.4.1 Glucagon

1.4.2 Insulin

1.4.2.1 Mechanism

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1.5 Antidiabetic Drugs

1.5.1 Biguanides

1.5.2 Sulphonylureas

1.5.3 Alpha-glucosidase inhibitors

1.5.4 Thiazolidinedione

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1.5.5 Incretin mimetics / GLP-1 analogues

1.5.6 Meglitinide

1.5.7 Dipeptidyl peptidase-4 inhibitor / Gliptins

1.5.8 Insulin

1.5.8.1 Rapid action insulin

1.5.8.2 Short action insulin

1.5.8.3 Intermediate action insulin

1.5.8.4 Long action insulin

1.6 The plant, Tinospora crispa

1.6.1 Botanical aspects

1.6.2 Background

1.7 Antihyperglycemic drug methodologies

1.7.1 in vitro techniques

1.7.2 in vivo techniques

1.8 Hypoglycemia studies

1.8.1 Mechanisms of action of hypoglycemic agents

1.8.2 Glucose loading test

1.9 Phytochemical screening of water and methanol extracts

of Tinospora crispa

1.10 Objectives of the present study

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CHAPTER TWO: MATERIALS AND METHODS

2.0 Introduction materials and methods

2.1 Materials and their sources

2.2 Instrument used and their sources

2.3 Methods

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2.3.1 Experimental animals

2.3.2 Plant material collection

2.3.3 Preparation of extracts

2.3.4 Antidiabetic evaluation of Tinospora crispa extracts

2.3.4.1 Hypoglycemic activity in normal rats

2.3.4.2 Intraperitoneal glucose tolerance test (IPGTT) in normal rats

2.3.4.3 Diabetes induction

2.3.4.4 Antihyperglycemic effect of Glibenclamide and Metformin on

diabetic rats

2.3.4.5 Antihyperglycemic activity of crude extracts of Tinospora

crispa in STZ induced diabetic rats

2.3.5 Water extract fractionation using solvent-solvent extraction method

2.3.5.1 Intraperitoneal glucose tolerance activity (IPGTT) in normal

rats

2.3.5.2 Antihyperglycemic activity of fractions of crude water extract,

n-butanol and aqueous fraction in STZ induced diabetic rats

2.3.5.3 Dose-response study of the most active aqueous fraction of

Tinospora crispa water extract

2.4 In vitro antidiabetic mechanism study of the most effective fraction

2.4.1 Effect of the fractions on glucose uptake in isolated rat abdominal

muscle

2.4.1.1 Buffer solution preparation

2.4.1.2 Experimental setup

2.4.2 Effect of the fractions on glucose absorption in isolated rat intestine

2.4.2.1 Experimental setup

2.5 Determination of lipid level in blood sample of treated rat with petroleum

ether, chloroform, methanol and water extracts of Tinospora crispa

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2.6 Phytochemical screening of the water and the methanolic extracts

of Tinospora crispa stems

2.6.1 Detection of Alkaloids

2.6.1.1 Preparation of Dragendorffs reagent

2.6.1.2 Alkaloid study

2.6.2 Detection of flavonoids (H2SO4 Test)

2.6.3 Detection of cardiac glycosides

2.6.4 Detection of steroids

2.6.5 Detection of tannins

2.6.6 Detection of terpenoids

2.6.7 Detection of saponins

2.7 Gas Chromatography Mass Spectrophotometry (GC-MS) analysis of

Tinospora crispa water fraction

2.7.1 Sample Preparation and Derivatization

2.7.2 GC-MS Chromatographic Condition

2.8 Development and validation of High Performance Liquid Chromatography

(HPLC) methods

2.8.1 Preparation of standards and water fractions of Tinospora crispa for

HPLC analysis

2.8.2 Tinospora crispa fraction Samples

2.8.3 Chromatographic conditions for Analysis of Salsolinol

Hydrobromide

2.8.4 Chromatographic conditions for Analysis of Gallic Acid

2.9 Data Analysis

CHAPTER THREE: RESULTS

3.1 Extraction Process

3.1.1 Hypoglycemic activity in normal rats

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3.1.2 Intraperitoneal glucose tolerance test (IPGTT) in normal rats

3.1.3 Effect of glibenclamide and metformin in subcutaneous glucose

tolerance tests

3.1.4 Antihyperglycemic effect of Glibenclamide and Metformin in

diabetic rats

3.1.5 Antihyperglycemic activity in Streptozotocin-induced diabetic rats

3.1.5.1 Effect of daily oral administration of Tinospora crispa extracts on the

body weight of diabetic rats.

3.2 Fractionation of the water extract of Tinospora crispa

3.2.1 Intraperitoneal glucose tolerance test (IPGTT) in normal rats

3.2.2 Antihyperglycemic activity of fractions of the water extract on

diabetic rats Intraperitoneal

3.2.2.1 Dose response study of the most active fraction (aqueous fraction)

3.2.3 Effect of daily oral administration of water fraction of water extract

of Tinospora crispa on body weight.

3.3 In vitro studies

3.3.1 Effect of water and n-butanol fractions on glucose uptake of isolated

rat abdominal muscles

3.3.2 Effect of water and n-butanol fractions on glucose absorption of

isolated rat intestine

3.4 The effect of petroleum ether, chloroform, methanol and water extracts of

Tinospora crispa on lipid profiles

3.5 Phytochemical screening of water and methanol extracts of Tinospora crispa

3.6 Gas chromatography–mass spectrometry (GC-MS) analysis

3.7 Development and validation of High Performance Liquid Chromatography

(HPLC) methods

CHAPTER FOUR: DISCUSSION AND CONCLUSION

4.1 Antidiabetic study

4.2 In vitro and in vivo

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4.3 Phytochemical screening

4.4 Conclusion

4.5 Recommendation for further studies

REFERENCES

APPENDICES

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LIST OF TABLES

Page

Table 1.3

Table 2.1

Chemical structures of important alpha-glucosidase inhibitor drugs.

Chemicals and reagents.

12

27

Table 2.2 Instrument used in this study 29

Table 2.3

Table 2.4

Elements combined for preparing 2 L of Kreb’s ringer Bicarbonate

Buffer.

Gradient elution program used in separation of Gallic acid in water

fractions of Tinospora crispa.

39

49

Table 3.1 The amount of extracts obtained from the stems of Tinospora crispa

51

Table 3.2 The amount of fractions obtained from the water extract of

Tinospora crispa stems.

60

Table 3.3

.

Results of phytochemical screening of water extract and methanol

extract of Tinospora crispa

72

Table 3.4

Table 3.5

Table 3.6

Volatile chemical compounds in water fraction of water extract of

Tinospora crispa identified by GCMS.

Intra-day and inter-day precision of salsolinol hydrobromide and

gallic acid (based on HPLC method, n = 6).

Accuracy of the HPLC methods in water fractions of Tinospora

crispa.

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LIST OF FIGURES

Page

1.1

Pancreas system

7

1.2

Chemical structure of metformin 10

1.3

Chemical structures of important alpha-glucosidase inhibitor drugs 12

1.4

Mechanism of DPP-4 inhibitors and GLP-1 14

1.5

Antidiabetic Drugs action on different organs 17

1.6

1.7

The stem of Tinospora crispa plant.

The Tinospora crispa plant

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2.1

(i) Leaves and stems of Tinospora crispa, (ii) Document

certification authentication of plant specimen (voucher no. 11509)

31

2.2

The Tinospora crispa plant dried in the oven 32

2.3

Schematic diagram of Tinospora crispa water extract fractionation

using solvent-solvent extraction method

37

2.4

GC-MS Function and its configuration 47

3.1

Effect of oral administration of the petroleum ether, chloroform,

methanol and water extracts of Tinospora crispa stems at a dose of 1

g/kg on the blood glucose levels of normal rats. Values are

expressed as the mean ± SEM of six animals. * P<0.05 compared

with the diabetic control.

54

3.2

Effect of oral administration of petroleum ether, chloroform,

methanol and water extracts of Tinospora crispa and followed one

hour later with glucose 1.0 g/kg intraperitoneally on the blood

glucose levels of normal rats. Values are expressed as the mean

± SEM of six animals. * P<0.05 compared to the control group.

55

3.3

Effect of oral administration of glibenclamide (10 mg/kg) and

metformin (500 mg/kg) and followed one hour later with glucose

1.0g/kg intraperitoneally on the blood glucose level of normal rat.

Values are expressed as the mean ± SEM of six animals. * P<0.05

compared to the diabetic control

56

3.4

Effect of daily oral administration of glibenclamide (10 mg/kg) and

metformin (500 mg/kg) for 12 days on streptozotocin-induced

diabetic rats. Values are expressed as mean ± SEM of six animals.

* P<0.05 and ** P<0.01 compared to the diabetic control group.

57

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3.5

Effect of daily oral administration of Tinospora crispa petroleum

ether, chloroform, methanol and water extracts on the blood glucose

levels of STZ- induced, diabetic rats. Values are expressed as the

mean ± SEM; of six animals. * P < 0.05 and ** P <0.01 compared

to the diabetic control group.

58

3.6

3.7

3.8

3.9

Effect of daily oral administration of 1 g/kg of the petroleum ether,

chloroform, methanol, and water extracts of Tinospora crispa stems

on the body weights of STZ-induced diabetic rats. Values are

expressed as the mean±SEM of six animals.* P<0.05 compared to

the diabetic control group

Effect of oral administration of n-butanol and water fractions of

Tinospora crispa water extract on the blood glucose levels of

normal rats intraperitoneally loaded with 1 g/kg glucose. Values are

expressed as the mean ± SEM of six animals.* P<0.05 compared to

control group

Effect of oral administration of n-butanol and water fractions of the

water extract of Tinospora crispa on the blood glucose levels of

STZ-induced diabetic rats. Values are expressed as the mean ± SEM

of six animals.*P < 0.05 and ** P <0.01 compared to the diabetic

control group

Effect of daily oral administration of different doses of water

fraction (500, 250 and 100 mg/kg) on the blood glucose levels of

STZ-induced diabetic rats. Values are expressed as the mean ± SEM

of six animals. ** P <0.01 compared to diabetic control group.

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63

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3.10 Effect of daily oral administration of different doses of water

fraction (500, 250 and 100 mg/kg) on the body weight of STZ-

induced diabetic rats. Values are expressed as the mean ± SEM of

six animals.

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3.11

Effect of water and n-butanol fractions on glucose uptake of isolated

rat abdominal muscle incubated in Tyrode’s solution. Values are the

mean of 6 determinations ± SEM.

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3.12

Effect of water and n-butanol fractions on glucose uptake by

isolated rat abdominal muscle when incubated in the Tyrode’s

solution containing 1 IU/ml insulin. Values are expressed as the

mean of 6 determinations ± SEM. *P< 0.05 vs. the control.

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3.13

Effect of acarbose, water fraction and n-butanol fraction on glucose

absorption by everted rat jejunum.* P < 0.05; ** P < 0.01 vs. the

control.

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3.14

Effect of daily oral administration of petroleum ether, chloroform,

methanol and water extracts of Tinospora crispa stems 1 g/kg for 9

days on the triglycerides levels of diabetic rats. Values are expressed

as the mean ± SEM of six animals.

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3.15

Effect of oral administration of the petroleum ether, chloroform,

methanol and water extracts of Tinospora crispa stems 1 g/kg for 9

days on the LDL levels of diabetic rats. Values are expressed as the

mean ± SEM of six animals. * P<0.05 compared with the diabetic

control.

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3.16

Effect of a daily oral administration of the petroleum ether,

chloroform, methanol and water extracts of Tinospora crispa stems

1 g/kg for 9 days on the HDL levels of diabetic rats. Values are

expressed as the mean ± SEM of six animals.* P<0.05 compared

with the diabetic control.

75

3.17

Effect of a daily oral administration of petroleum ether, chloroform,

methanol and water extracts of Tinospora crispa stems 1 g/kg for 9

days on total cholesterol levels of diabetic rats. Values are expressed

as the mean ± SEM of six animals.* P<0.05 and ** P < 0.01

compared with the diabetic control.

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3.18

3.19A

3.19B

3.19C

3.19D

3.19E

3.20

3.21

GC-MS analysis of water fraction ethanolic solution of Tinospora

crispa

GC-MS analysis: Detection of dodecanol in water fraction of

Tinospora crispa

GC-MS analysis: Detection of dodecyl acrylate in water fraction of

Tinospora crispa

GC-MS analysis: Detection of pentadecanoic acid in water fraction

of Tinospora crispa

GC-MS analysis: Detection of propanoic acid in water fraction of

Tinospora crispa

GC-MS analysis: Detection of decanedioic acid in water fraction of

Tinospora.crispa

HPLC chromatograms of water fractions of Tinospora crispa and

Gallic acid. Note: Gallic acid standard (A), water fractions of

Tinospora crisp (B)

HPLC chromatograms of water fractions of Tinospora crispa and

Salsolinol hydrobromide. Note: Salsolinol hydrobromide standard

(A), water fractions of Tinospora crisp (B)

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LIST OF ABBREVIATIONS

0C Degree Celsius

% Percent

AF Aqueous fraction

ANOVA Analysis of variance

BF n-butanol fraction

b.w. Body weight

et al. And others

g Gram

GC-MS Gas chromatography–mass spectrometry

HF n-hexane fraction

HPLC High-performance liquid chromatography

IDDM Insulin dependent diabetes mellitus

i.p. Intraperitoneal

IPGTT Intraperitoneal glucose tolerance test

IU International units

Kg Kilogram

KRB Krebs-Ringer bicarbonate

OGTT Oral glucose tolerance test

STZ Streptozotocin

WF Water fraction

xiv

LIST OF PUBLICATION ASSOCIATED WITH THIS THESIS

Jornal Pablication

Accepted

Mir Mohammad Reza Seyed Hassani, Ashfaq Ahmad, Mohd. Zaini Asmawi,

Roziahanim Mahmud. Preliminary investigation of normoglycemic,

anti-hyperglycemic and dyslipidemic activities of different extracts of Tinospora

crispa on diabetic rat (2015).

On Review

Mir Mohammad Reza Seyed Hassani, Item Justin Atangwho and Mohd. Zain

Asmawi. Antihyperglycemic activity of extract and fractions of Tinospora

crispa in diabetic rats and intestine (2015).

Poster Presentation

Mir Mohammad Reza Seyed Hasani,Pros Dr. Mohd. Zaini Asmawi, Parastoo

Zarghami Moghaddam, Helen Hemati. Evaluation of anti-hyperglycemic effect

of Tinospora crispa in traditional medicine in in vivo model. University of

Medical Sciences, Bojnurd, Iran (2013).

xv

AKTIVITI ANTIHIPERGLISEMIK EKSTRAK DAN FRAKSI TINOSPORA

CRISPA PADA TIKUS

ABSTRAK

Tinospora crispa (Menispermaceae) yang nama tempatanya dikenali sebagai

"akar patawali", telah lama digunakan dalam perubatan tradisional Malaysia untuk

rawatan kencing manis. Tujuan kajian ini adalah untuk mengesahkan kesan

hipoglisemik dan antihiperglisemik ekstrak Tinospora crispa yang berbeza beza

dalam tikus normal dan tikus diabetik aruhan streptozotocin (STZ). Serbuk batang

Tinospora crispa kering diekstraksi secara berturut-turut scara maserasi dengan eter

petroleum, kloroform, metanol dan air untuk menghasilkan empat ekstrak. Tidak satu

pun daripada keempat-empat ekstrak (1.0 g / kg) yang diberikan secara oral dapat

menurunkan paras glukosa darah tikus normal yang menunjukkan bahawa

ekstrak-ekstak tersebut tidak mempunyai sebarang kesan hipoglisemik.

Keempat-empat ekstrak juga tidak menghalang kenaikan paras glukosa darah tikus

yang dimuatkan dengan glukosa secara intraperitoneal. Pemberian ekstrak secara

oral 1 g / kg setiap hari selama 9 hari pada tikus diabetik aruhan STZ mendapati

hanya ekstrak air dapat mengurangkan paras glukosa darah secara signifikan

(P <0.05) berbanding dengan kumpulan kawalan yang menunjukkan bahawa ekstrak

air mempunyai kesan antihyperglisemik. Ekstrak air kemudiannya difraksikan untuk

mendapatkan fraksi n-butanol dan fraksi air. Walau bagaimanapun, rawatan setiap

hari untuk 12 hari mendapati hanya fraksi air (0.5 g / kg) menurunkan paras glukosa

darah tikus diabetic aruhan-STZ. Penyaringan fitokimia ekstrak air Tinospora crispa

menunjukkan kehadiran alkaloid, saponin dan glikosida. GC-MS fraksi air

menunjukkan kehadiran dodesil akrilat, asid pentadekanoik dan asid propanoik

xvi

manakala analisis HPLC menunjukkan kehadiran asid galik dan salsolinol. Dodesil

akrilat, asid pentadekanoik, asid propanoik dan asid galik telah dilaporkan sebelum

ini memiliki kesan antidiabetik. Secara keseluruhan, hasil kajian ini menunjukkan

Tinospora crispa mempunyai kesan antidiabetik yang mungkin menyokong

penggunaannya di kalangan pesakit kencing manis. Beberapa sebatian yang

dikenalpasti diatas mungkin telah menyumbang secara individu atau dalam gabungan

kepada aktiviti antidiabetik tersebut.

xvii

ANTIHYPERGLCEMIC ACTIVITY OF TINOSPORA CRISPA

EXTRACTS AND FRACTIONS IN RATS.

ABSTRACT

Tinospora crispa (Menispermaceae), locally known as “akar patawali”, has

long been used in Malaysian traditional medicine for treatment of diabetes. The aim

of this study was to verify the hypoglycemic and antihyperglycemic effects of

different Tinospora crispa extracts in normal and streptozotocin (STZ)-induced

diabetic rats. Pulverized Tinospora crispa dried stems were extracted successively by

maceration with petroleum ether, chloroform, methanol and water to yield the four

extracts. None of the four extracts (1.0 g/kg) administered orally lowered the blood

glucose levels of normal rats which suggest that they have no hypoglycemic effect.

The four extracts also did not inhibit the rise of blood glucose level of glucose

intraperitoneally loaded rats. Daily oral administration of the extract 1 g/kg for

9 days in STZ-induced diabetic rats found that only the water extract managed to

lower the blood glucose levels significantly (P<0.05), as compared with the control

group which suggests that it has antihyperglycaemic effect. The water extract was

then fractionated to obtain n-butanol and aqueous fractions. However, daily

treatment for 12 days found that only water fraction (0.5 g/kg) lowered the blood

glucose level of STZ-induced diabetic rats. Phytochemical screening of the water

extracts of Tinospora crispa indicated the presence of alkaloids, glycosides and

saponins. GC-MS of water fraction showed the presence of dodecyl acrylate,

pentadecanoic acid and propanoic acid whereas HPLC analysis showed the presence

of gallic acid and salsolinol. Dodecyl acrylate, pentadecanoic acid, propanoic acid

and gallic acid have been previously reported to possess antidiabetic effect. Overall,

xviii

the findings of this study showed Tinospora crispa possesses antidiabetic effect

which may justified its use in diabetic patients. Some of the identified compounds

above might have contributed individually or in combination to its antidiabetic

activity.

1

CHAPTER ONE : INTRODUCTION

1.1 Introduction

The use of herbal remedies, in different cultures and traditional medicines, have been

practiced for centuries because of traditional beliefs and out of habits. In majority of the

world population, including those of Asian and western countries, the modern medicine has

prominently taken over the life style, practice and use of herbal remedies to meet health

needs. Surprisingly, in this modern era, people are still using herbal medicines in

conjunction with modern conventional methods. Worldwide, especially in developed

countries, a substantial amount of money is being annually spent on research aimed at

identifying and assessing herbal products. A number of pure isolated herbal compounds,

such as taxol and artemisinin, have been derived from plants and have already been through

clinical development. In general, herbal products are widely believed to cause less adverse

side effects, and are less potent compared to synthetic and modern pharmaceuticals.

However, their use can play a key role in the reduction of the national health expenditure

(Holt & Chandra, 2002).

These studies investigating the biologically active constituents of medicinal plants,

have made it possible for the development of new drugs for clinical use (Kamboj &

Dhawan,. 1982). Natural products serve as a source of lead compounds for drug

development. In some Asian countries such as China, India, Japan and Korea, herbal

remedies have been officially recognized (World Health Organization., 2003).

2

The reawakening, obviously if any real improvement in healthcare systems and

research especially in Asia and the developing countries were to be made, there should be

an optimal use of all their natural resources and potential, and of course traditional herbal

medicines are undoubtedly amongst the best options. International organizations, like the

World Health Organization (WHO), have showed enthusiasm regarding to the use of herbal

medicine, and it suggests reinforcing the use of traditional medicines which may spark

globally. In some countries, traditional medicinal plants have come to be placed on the

same footing as modern medicines and sometimes even more popular. This is the time to

accept the importance of medicinal plants such as Tinospora crispa as an alternatives for

health care (Farnsworth & Bingel,1977)

3

1.2 Diabetes Mellitus

Diabetes mellitus (DM) as a chronic metabolic disorder, has long been a major public

health problem and its prevalence, in the modern world, is increasing. The World Health

Organization estimated that in 2000, diabetic mellitus affected 171 million individuals

globally, and moreover it has been projected that, by 2030, this number would increase to

366 million patients (Whiting et al., 2011). It is a group of metabolic diseases characterized

by chronic high blood glucose levels, caused either by the inability of the β-cells of

pancreas to produce adequate amount of insulin, or failure of the target cells (particularly

the skeletal muscles and adipose tissues) to respond to secreted insulin (Shoback, 2008).

Food digestion always commences with amylase in the mouth terminating in the small

intestines. During the digestion process in the intestines, food is converted to glucose, a

universal source of energy. As more foods carbohydrate change to glucose and the blood

glucose level, this main source of energy in body reaches the postprandial level a signal

given to the pancreas causes it to release the hormone insulin into the blood stream,

enabling the cells to use the readily available glucose (Holt et al.,2003).

The chronic high blood glucose level (hyperglycemia), produces classical symptoms,

such as polyuria (frequent urination), polydipsia (increased thirst) and polyphagia

(increased hunger). The treatment focus therefore, is to maintain steady blood glucose

within physiological limits (euglycemia). However, this is often scarcely achieved in

practice, hence there are reported occasional incidences of hypoglycemia, besides the grave

side effects of some of the conventional drugs in use. Moreover, along with

pharmacological treatment, non-pharmacological treatment measures have also become

essential for the prevention and management of diabetes mellitus, due to the variety of

4

complications accompanying DM, such as malnutrition and cardiovascular diseases.

For instance, dietary advice given to older people with diabetes would be intended to

maintain adequate nutrition, thereby complementing observed tissue wasting, as diabetes

also make damage on nerve supply and further muscle wasting which referred as diabetic

amyotrophy and mostly effect on hips and shoulders muscles. Furthermore, besides their

nutritional complementary role, most leafy vegetables also exert therapeutic functions, for

which reason they are referred to as functional foods. Natural products including medicinal

plants have equally been used as source of drugs in traditional management of diabetes

with the aim to circumvent the drawbacks of conventional medication, and in most

instances to complement their therapeutic effect (Nascimento et al., 2006).

At the moment, an estimated 285 million individuals suffer from Type II diabetes

which correspond to 90% of all diabetic cases worldwide. In Malaysia alone 1.6 million

individuals are affected by Type II diabetes (Saleh et al., 2013), thereby suggesting that this

is no longer a disease associated with middle and old age individual. Recent evidence

suggests that children as young as 10 years old may also be affected, indicating that, a

holistic approach in sourcing a cure is urgently needed. Diabetic is also at high risk of

macrovascular complications, such as ischemic heart disease and strokes. Some risk factors

in diabetes include having a family history of heart disease and high cholesterol levels.

Actually high level of some cholesterols, for instance low density lipoprotein (LDL) as one

of the five major groups of lipoproteins and also triglycerides, can increase the risk of heart

disease. They can build up inside of blood vessels, leading to the narrowing and hardening

of arteries, blocking the arteries and raising the risk of getting heart disease and strokes

(Almdal et al., 2004).This may be attributed in part to the late diagnosis and poor glycemic

control. However, being highly associated with components of the metabolic syndrome,

5

such as obesity, hypertension (10-37%) and hyperlipidemia (63-76%), diabetes actively

predisposes DM patients to such complications (Funk et al., 2004). More worrisome is the

fact that many of those affected by DM may do not even know that they suffer from the

disease. Hence, it is really necessary to discover new and better methods for treating and

controlling DM, such as the utilization of herbs.

1.3 Classification of Diabetes Mellitus

Diabetes is generally classified into insulin dependent diabetes mellitus (IDDM),

which is commonly known as Type -I diabetes, and non-insulin dependent diabetes

mellitus (NIDDM) known as Type -II diabetes. Presently, as many types of anti-diabetic

drugs are available, they are typically classified according to various criteria, such as the

type of diabetes they are used for, patients age, or other factors like history of any particular

disease, inherited sickness or even gender and physical factors which is contributing to the

disease manifestation. Type I diabetes mostly affects children with an onset before the 15th

birthday (Harjutsalo et al., 2008) and since insulin is destroyed when ingested orally, the

treatment have to be administered through subcutaneous injections. On the other hand,

Type II diabetes usually affects adults, especially the obese. These patients may not require

insulin treatment and sufficient to be given with oral antidiabetic agent. Some agents

induce greater insulin production, sensitize the tissues to insulin, and prevent excessive

glucose absorption from gastrointestinal tract are available for non-insulin dependent

diabetes mellitus patients. Among the five classes of anti-diabetic drugs the two main

classes widely used are sulfonylureas (e.g. glibenclamide) and biguanides (e.g.metformin),

which can be taken separately or in combination (Phung et al., 2010)

6

1.4 . Pancreas

The pancreas is an integral part of the digestive system. Due to its exocrine and

endocrine secretions, the pancreas as an exocrine gland always considered to be a mixed

gland. Pancreas secretes digestive enzymes and alkaline substances into the small

intestines. It acts as an endocrine gland as well as secreting certain hormones into the blood

stream (Gradwohl et al., 2000).

Hormones from clusters of cells, referred to as Islets of Langerhans or the pancreatic

islets, are stored and secreted in the human body. Those clusters contain four special groups

of cells: alpha (α), beta (β), delta (δ) and F cells. Insulin and glucagon are the main

hormones which help regulate glucose metabolism. Alpha cells, responsible for glucagon

secretion, are effective in raising blood glucose levels. Conversely, beta cells are

specialized in producing insulin to lower blood glucose levels. Delta cells secrete the

hypothalamic growth inhibiting hormone, while the F cells regulate the release of

pancreatic digestive enzymes, such as the pancreatic polypeptide (Pritchett et al., 1995).

7

Figure 1.1. The pancreas system (National cancer institute, Pancreatic Cancer, 05-07-2014.

Retrieved from http://www.cancer.gov/types/pancreatic ) with modification.

8

1.4.1 Glucagon

Glucagon is secreted by the alpha cells of the pancreas. It increases the

concentration of cyclic AMP, derived from ATP in the liver cells, thereby causing the

enzyme (phosphorylase) to be activated when the blood glucose level is low. For instance,

glucagon will be higher between meals and during exercise. Glucagon as a polypeptide

hormone is released in response to hypoglycemia and plays a role as a physiological

regulator of the effects caused by insulin (Constantin et al., 1995).

When the concentration of glucose falls in the blood and glucagon gets secreted, the

liver responded by converting glycogen into glucose (glycogenolysis) to raise the blood

glucose concentration. Glucagon stimulates the release of fatty acids and glycerol from the

adipose tissue. This enzyme also separates glucose units from the branched glycogen

molecules, and the free glucose units then enter the bloodstream to maintain the blood level

of glucose at 70-110 mg/dl. For this reason, glucagon is considered to be hyperglycemic

factor (DiCostanzo et al., 2007)

1.4.2 Insulin

Insulin is synthesized as pre-pro insulin. It contains A and B chains connected by 51

amino acids, and linked by disulfide. Insulin as a peptide hormone released and synthesized

from pancreatic β-cells at low basal rates. However, it gets stimulated to reach at much

higher levels in response to a variety of stimuli, especially glucose. Insulin analogues have

been commercially available for years as they have been synthesized to mimic human

insulin (Ljungqvist, 2012).

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1.4.2.1 Mechanism

Beta cells in pancreas naturally produce insulin. The hormone sending signals to the

cell receptors to absorb glucose from the blood and raises blood glucose levels in the cells

(hypoglycemic effect). Insulin does not have an effect beyond that of lowering blood

glucose concentration with respect to altering ris (Stratton et al., 2000). However, insulin is

effective in increasing protein synthesis. Furthermore, it increases the conversion of

glucose into fatty acids and promotes amino acid transport into the cells.

1.5 Antidiabetic Drugs

Modern day research on drug development associated in β-cell function of pancreas has

produced several number of new drug compounds in the treatment of Type II or insulin

dependent diabetes mellitus (Zhao et al., 2010). However, In Type I diabetes some

treatment prevents the development of microvascular and produces neurologic

complications (Ohkubo et al., 1995).

1.5.1 Biguanides

Biguanide refers to a class of drug which are using as an oral antihyperglycemic drugs

for diabetes mellitus. The most popular example and the only widely available antidiabetic

biguanide drug is metformin. It can also improve glycaemic control in overweight to obese

individuals with type 2 diabetes (Gilbert et al., 2006). Biguanides mechanism of action is

to inhibit the liver from producing glucose and to help the body secrete insulin (Potenza et

al., 2009). Except metformin other biguanides derivatives have been withdrawn from the

market due to their toxic effects.

11

Figure 1.2. Chemical structure of metformin.

1.5.2 Sulphonylureas

Sulphonylurea derivatives is one of the earliest class of anti-diabetic drugs, that are

used to management of diabetes mellitus type II. This group of drugs are metabolized by

the liver. Sulphonylureas are known to be the ‘principal oral hypoglycemic agents’ in type

II diabetes treatment. This class works by stimulating the β-cells in the pancreas to produce

more endogenous insulin, and by increasing the circulating insulin to levels sufficient to

overcome insulin resistance. This class of drug is generally taken one to twice a day before

meals during treatment. Hypoglycemia is the possible side effect in all type of

sulphonylurea. However, glipizide and glimepiride showing less hypoglycemic effects than

the other drugs of this class (Pieber et al., 2003). Considering the fact that cardiac myocytes

also express the same sulphonylurea receptor as the β-cells, these drug compounds may

11

potentially lead to cardiovascular toxicity. In this drug category most of the drugs names

end with ‘ide’. Examples of sulphonylureas are glibenclamide which are also known as

glyburide, glipizide, gliquidone, glyclopyramide, glimepiride , gliclazide, tolbutamide and

tolazamid.

1.5.3 Alpha-glucosidase inhibitors

Alpha-glucosidase is an enzyme responsible for the breakdown of complex

carbohydrates into di- and mono-saccharides in the distal duodenum and the proximal

jejunum. Alpha-glucosidase inhibitors help the body to lower blood glucose level by

blocking the break down of starches into glucose in the intestine.

Miglitol, acarbose and voglibose are examples of alpha-glucosidase inhibitors.

However, acarbose is the only commercialized α-glucosidase inhibitor. It is an

oligosaccharide produced by cultured strains of actinomycetes. It is a competitive inhibitor

with low affinity for glucoamylase, and high affinity for sucrase and pancreatic α-amylase

(Braun et al., 1995). The major effect of acarbose is reducing the risk for myocardial

infarction in type 2 diabetic patients by controlling and improving the body weight and

triglyceride levels (Hanefeld et al., 2004).

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DRUG COMMON NAME STRUCTURE

Figure 1.3. Chemical structures of important alpha-glucosidase inhibitor drugs.

1.5.4 Thiazolidinedione

Glitazones or thiazolidinediones are anti-diabetic drugs which improve insulin

sensitivity in the muscles and also reduce glucose production in the liver. Thus, they are

used in the treatment of type II diabetes. They also help to decrease the levels of

triglycerides in the blood. Avandia and pioglitazone are the most popularly prescribed

drugs of this class. However, due to some adverse reaction incidents, which included

bladder cancer and heart attacks, they were banned by the European Medicines Agency in

September 2010. Nevertheless, for pioglitazone, the adverse effect has not been deemed

sufficient to justify restriction on its treatment use in the UK (Dunn et al., 2011).

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1.5.5 Incretin mimetics / GLP-1 analogues

Incretin mimetics or glucagon like peptide-1 receptor agonists (GLP-1) analogues

are injectable drugs used for the treatment of Type II diabetes. As the level of glucose in

the body increases more hormone of GLP-1 also will be produced. While food is

consumed, the body releases GLP-1, and the hormone moves to the pancreas. They will

increase the amount of insulin released from the beta cells of the islets of Langerhans and

decrease the release of glucagon. They are known to be inhibitors that stop the protein

dipeptidyl peptidase-4 from destroying the incretin hormones (Ellingsgaard et al., 2011).

1.5.6 Meglitinide

Meglitinides are from the class of drugs which stimulate β-cells to release more

insulin and this class drugs are used to treat diabetes type II, The mechanism of action is

similar to sulphonylureas. Since meglitinides and sulphonylureas both stimulate to release

insulin, hypoglycemia or low blood glucose is the possible side effect on patients using

them. Some example of this class are nateglinide and repaglinide ( Lorenzati et al., 2010).

1.5.7 Dipeptidyl peptidase-4 inhibitor / Gliptins

Dipeptidyl peptidase-4 inhibitor (DPP-4 inhibitors) or Gliptins represent an

innovative approach and are a class of oral hypoglycemic which are used for treatment of

type II diabetes mellitus. DPP-4 inhibitors are attached to the DPP-4 enzyme and prevent

them from assisting in the breaking down of Glucagon-like peptide-1 (GLP-1), (Monami et

al., 2011).

14

Drugs of this class stimulate the production of insulin and reduce the production of

glucagon, especially during digestion where it decreases gastric emptying, thus it helps

decreasing the blood glucose levels. The effect of this agent inhibitor is that DPP-4 will be

released throughout the day and their levels increased at meal times (Ahrén et al., 2011).

Example of some drugs belonging to this class are sitagliptin, vildagliptin, saxagliptin and

linagliptin. Linagliptin was approved as a treatment for Type II diabetes by the U.S. Food

and Drug Administration (FDA) in May 2011.

Figure 1.4. Mechanism of DPP-4 inhibitors and GLP-1.

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1.5.8 Insulin

Insulin is one of the main hormones that regulate blood glucose levels. It can

directly lower the glucose levels by increasing the uptake of glucose to the muscle and fatty

tissue, and reducing the release of glucose from the liver. A number of different types of

insulin preparations are available in the medications. Insulin is usually prescribed for

patients suffering from Type I DM; however, it may also be prescribed for patients with

Type II DM who have not responded well to strict diets, exercise and oral antidiabetic

drugs. Some insulin act only for a few hours duration, but some exert their effects for as

long as one whole day (Sussman et al., 2012).

1.5.8.1 Rapid action insulin

To control blood glucose levels promptly, rapid-acting insulin is administered for

adults with type II diabetes, and also for adults and children (4 years and above) with type I

diabetes. These types are usually taken just before or during meal. They act very quickly to

minimise the increase of blood glucose level after eating. This type of insulin should be

given within 15 minutes before, or 20 minutes after starting a meal. The action of

rapid-acting insulin lasts for about 4 hours. As they act very quickly, they can lead to an

increased chances of hypoglycemia (Heinemann, 2010).

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1.5.8.2 Short action insulin

Regular insulin is short-acting or specifically, it has an onset of action that normally

reaches the bloodstream within 30 minutes to an hour after injection and a duration of

action of 4 to 8 hours (Siebenhofer et al., 2006).

1.5.8.3 Intermediate action insulin

Neutral Protamine Hagedorn (NPH) insulin which is also known as Humulin N,

Novolin N, Novolin NPH, NPH Iletin II, and iso-phane insulin, is an intermediate acting

insulin product given to help control the blood sugar level of those with diabetes. This type

of insulin is absorbed slowly and generally reaches the bloodstream from about 2 to 4 hours

after it is injected. Thus, it has a formulation that contains a substance of action, and it lasts

longer. Its duration of action is between 10 and 20 hours and after approximately 20 hours,

the whole dose has been absorbed. In 1936, Nordisk formulated pig insulin by adding

neutral protamine to regular insulin, and NPH was created (Peterson, 2006).

1.5.8.4 Long action insulin

Long acting insulin preparations are prescribed for a number of different types of

diabetes and they have an onset of action of around 1 hour, and a duration of action of 14 to

24 hours. However, the action of those insulin products may vary substantially from one

person to another. Given to certain patients, it may vary by one day. Lantus is a clear

insulin preparation. It was the first real basal insulin that lasted for 24 hours, almost with no

peak. Long acting insulins are available in animal and analogue forms, in the UK analogue

insulins are more preferred by the National Health Service because the insulins have no

17

peak activity. As such, it allows for a consistent delivery of activity through the day.

However, it may not be used concurrently with rapid insulin. Some patients may need to

use this product twice daily for an action duration of 24 hours (Peterson, 2006).

Figure 1.5 Antidiabetic drugs action on different organs

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1.6 The plant, Tinospora crispa

1.6.1 Botanical aspects

Kingdom : Plantae

Subkingdom : Tracheobionta

Division : Magnoliophyta

Class : Magnoliopsida

Scientific name : Tinospora crispa

Synonym : Tinospora cordifolia

Family : Menispermaceae

Local name : Akar patawali

Part used : Stem

Figure 1.6 The stem of Tinospora crispa plant

19

Figure 1.7 The Tinospora crispa plant

21

1.6.2 Background

Tinospora crispa is an indigenous medicinal plant that belongs to the

Menispermaceae family. Locally, it is known as akar patawali and akar seruntum. It is a

medicinal plant that commonly grows wildly in the Asian countries, such as Malaysia,

Indonesia, Thailand, India and the Philippines. The leaves, the roots and the stems, all have

been claimed to have miraculous properties in curing a spectrum of diseases.

In rural southern India, Tinospora crispa has a reputation of being a hypoglycemic

agent. For many years, Tinospora crispa has also been used in southern eastern Asia,

especially in China and Malaysia as a treatment for various ailments, such as prurigo,

eczema, impetigo and oxidative stress conditions. It also has been known to have

anti-allergic and antiviral properties as well. A decoction of Tinospora crispa as a whole is

used in the treatment of cholera throughout the Malay communities, and a decoction of its

stem is used for various therapeutic purposes such as managing hypertension, stimulating

the appetite, and protecting against mosquito bites. It is also used to treat ailments like

jaundice, wounds, intestinal worms, skin infections, tooth and stomach aches, coughs,

asthma, and pleurisy (Rahman et al., 1999). In Thailand, a decoction from the stems has

been used as antipyretic syrup, for the treatment of internal inflammations, to reduce thirst,

to cool down body temperatures, and to maintain good health (Dweck & Cavin, 2006).

Tinospora crispa was also used as an anti-parasitic agent for humans and for

domestic animals (Noor & Ashcroft, 1989). This plant was able to cause a reduction in

serum glucose level in diabetic rats and its hypoglycemic effect was probably due to an

insulinotropic activity. It was also shown to increase peripheral utilization of glucose and to

inhibit hepatic glucose release.

21

Tinospora crispa has been scientifically demonstrated to possess anti-filarial,

anti-malarial, anti-pyretic (Kongkathip et al., 2002), and antihyperglycemic properties

(Noor & Ashcroft, 1989). Also antinociceptive and anti-inflammatory activities of the

ethanol extract of Tinospora crispa stem has been demonstrated (Sulaiman et al., 2008). In

Indonesia (Borneo), it is used as a treatment for lumbago (Dweck and Cavin, 2006), in

South-East Asia, in countries like Malaysia and China, an aqueous extract of Tinospora

crispa is taken orally as a diabetes treatment.

Previous studies conducted on this herb showed that its aqueous extract was able to

lower blood glucose levels in moderately diabetic rats. Its hypoglycemic effect was

attributed to its insulinotropic activity (Noor & Ashcroft, 1989), However, according to a

human research on glucose and insulin levels in healthy subjects and diabetic patients with

type 2 diabetes mellitus in Mahidol university of Bangkok, ingestion of 250 g ethanolic

extract of Tinospora crispa obtained using Soxlet, did not reduce the blood glucose and

insulin levels of healthy and diabetic human volunteers and have no ability to sensitize β

cells to secrete insulin (Klangjareonchai & Roongpisuthipong, 2011).

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1.7 Antihyperglycemic drug methodologies

To investigate the activity of a drug and its use as a treatment for diabetes, more

systematic approaches are require. Certain approaches of bioassay-guided drug discovery

to optimize time and resources are available, which can be divided into the two main

classes of in vitro and in vivo techniques.

1.7.1 In vitro techniques

To study on extracts and fractions of Tinospora crisp and to assess the potential of

hypoglycemic agents on the release of insulin, many different in vitro techniques have been

developed such as intact isolated islets techniques, perfuse pancreas methods and Scattered

islet cell techniques. The features of insulin and glucagon released as a result of the use of

agents had been comparatively studied (Weir et al., 1986).

The majority of researchers who are active in the drug discovery industry prefer to

use in vitro studies as first line screening models (Berkowitz & Katzung, 2001). The recent

research on the mechanisms of sulfonylureas the biochemical basis and diabetes the cellular

and sub-cellular levels was mostly done with cultured β-cells (Lienhard et al., 1992).

Furthermore, the results of animal experiments are sometimes impossible to be extrapolated

to humans. Nevertheless, when conducted properly, animal experiments could provide vital

information on the drugs being examined. In order to study on in vitro insulin

internalization and glucose transport in peripheral tissues and insulin resistance, the most

common techniques involve cultures of adipocytes derived from rat epididymal pads,

skeletal or abdominal muscle strips or cells (Reecy et al., 2003). Also, the effect of natural

products on glucose absorption has also been studied by using fragments or a homogenate

23

of the rat’s small intestine Jejenum (Hikino et al., 1989). Eventually, in several of the

bioassays, the measurement of insulin levels shows a determining effect. However, in some

situation in vitro techniques are not able to simulate the real life conditions owing to the

effect of the different instrument using in study or the chemicals on cell culture and isolated

tissue. Therefore having in vivo models, using animals and other pharmacological methods

is inevitable (Lipinski & Hopkins, 2004)

1.7.2 In vivo techniques

In vivo techniques are applied for the study of hypoglycemic activity which make

use of animals for activity assessments with the aim to study the effects of therapeutic

agents by monitoring the blood glucose levels of animals with normoglycaemia and/or

induced-hyperglycaemia by loading with glucose, also an isolated nitrosourea glycoside

from Streptomyces achromogenes that causes degeneration of pancreatic β-cells

(Srivastava et al., 2007).

Diabetic humans may sometimes be grouped for in vivo studies. However, in

animals, diabetes gets experimentally induced by using drugs such as alloxan and

streptozotocin which selectively destroying β-cells. A single subcutaneous injection in a

normal rat can produce an experimental model of Type I or II diabetes mellitus. However,

results of adipocyte insulin binding and glucose transport (Fantus et al., 1987), concluded

that the streptozotocin injected diabetic rats did not provide a representation of human

non-insulin dependent diabetes mellitus (NIDDM) characterized by deficient insulin

biosynthesis and release in response to glucose and diminished pancreatic insulin content

(Fantus et al., 1987). The history and the mechanisms of action of alloxan, a pyrimidine

derivative, have been reviewed by Lenzen and Panten (1988). Considering the fact that

24

animal tests always reveal little information on the mechanism of action of the compound,

and involve with financial restrictions on maintaining animals, and social restrictions on the

extensive use of animals in experimentation, shortage of perfect models for Type I and II

has always been observed.

1.8 Hypoglycemia studies

1.8.1 Mechanisms of action of hypoglycemic agents

Hypoglycemic properties denote the qualities of certain agents in rapidly reducing

the concentration of glucose in the blood and the term hypoglycemia "low blood glucose

levels" experiment classifying on acute tests. An inadequate supply of glucose to the

nervous system may however cause serious brain problems, from mild dysphoria to more

serious issues such as seizures unconsciousness. It can occur due to a wide range of internal

factors and permanent brain damage or death. It also may cause a variety of other side

effects. The activity of herbal extracts in controlling blood sugar levels may be attributed to

certain properties, thereby allowing them to act at the pancreatic level and stimulate β-cells

to release insulin.

1.8.2 Glucose loading tests

To evaluate the anti-hypoglycemic activity of a drug following oral administration

of glucose, traditionally, two tests have been used on animals: a hypoglycemic test

spanning a duration of 7 hours on normal groups and a glucose tolerance test of a duration

of 2 hours on glucose loaded on normal groups. IPGTT and OGTT tests are the two most

classical tests used in diagnosing diabetes Glucose Tolerance Test. According to the

American Diabetes Association, to verify the diagnosis of Diabetes Mellitus and the