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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
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ABSTRAK
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ABSTRACT
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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
8
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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.
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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.
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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.
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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)
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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
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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
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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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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.
73
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.
74
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
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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
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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).
9
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).
12
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).
13
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.
15
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).
16
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
18
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).
22
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