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Department of Pharmacology,
Acharya & B.M. Reddy College of Pharmacy,
Hesarghatta Main Road, Bangalore 560 090
2008
Date:
Place: Bangalore
Date:
Place: Bangalore
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Date:
Place: Bangalore
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Date:
Place: Bangalore
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Date:
Place: Bangalore
COPYRIGHT
I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnataka shall
have the rights to preserve, use and disseminate this dissertation / thesis in print or
electronic format for academic / research purpose.
Date:
Place: Bangalore
vi
Chairperson
Dr. Divakar Goli
Date:
CPCSEA Nominee
Dr. Venugopal Rao
Members
Mrs. Kalyani divakar
Dr. Roopa Karki
Mrs. Anitha. S
Mr. Vinod Mathew
Mr. Manjunath P.M
Mr. Md. Asif Ansari
Mrs. Shalini Reddy,
Social activist
Dr. K. Ganesh,
Chairman
Veterinarian
ACKNOWLEDGEMENT
It is the wealth of experience and knowledge that is generated within the portals of Acharya &
B.M. Reddy college of Pharmacy which enlightened my path to complete my dissertation. I humbly record
my deep sense of gratitude to Professor, Dr Kalyani Divakar, H.O.D. Dept. of Pharmacology,
Acharya & B.M. Reddy college of pharmacy, Mr. Prakash T, Asst. Professor, Department of
Pharmacology, Acharya and B.M. Reddy college of pharmacy and Mr. Manjunath, Mr. Uday Raj Sharma,
Mr. Surendra Reddy Department of Pharmacology, Acharya and B.M. Reddy College of Pharmacy, whose
overall encouragement and constant presence proved to be a source of inspiration and motivation.
I am thankful to Dr. Roopa Karki, Head of the Department of Pharmaceutics, Mrs. Anitha,
Head of the Department of Pharmacognosy, Dr. Vinodh Mathew, Head of the Department of
Pharmaceutical chemistry, Acharya and B.M. Reddy College of Pharmacy, for all the help and guidance
provided throughout my course.
I will remain thankful to Mr. Narasimhamurthy, Mr. Raveesh, Mr. Vinodh, Mr. Chanagge, Mr.
Sanand, and Mr. Thimarasaiah for all the help extended to me during the course of my work, which was
always helpful to me. I will always remain thankful to them.
I would like to take this opportunity to thank Ms. Lakshmamma, Mr. Nagraj the librarians of
Acharya B M Reddy College of pharmacy, Bangalore for helping me to refer books and journals in our
esteemed library.
I am extremely thankful to my friends/colleagues Mr. Jiban, M. Bimlesh, Mr. Praveen, Mr.
Ghule, Mr. Kaushal, Mr. Dighe, Mr. Gopinath, Mr. Ali, and Mrs. Poonam Mr. Anil, Mr. Prakash, Mr.
Swamidasan, Mr. srinivas, Mr. Kamlesh, Mr. Basvraj, Mr. Chandrashekar, Mr. Sandeep, Mr. Rohit, Mr.
Dixit, Mr. Shivakumar, Mr. Nishant, Mr. Piyush, Mr. Sanket, Mr. Upendra, Pankaj, Mr. .Anil jain. Mr.
Ishab, for their support during the course of my work.
I am also extremely thankful to my Seniors Mr. Sajal, Mr. Sandipan, Mr.Boudhisattya, Mr.
Rohit Badhe and Mr. Mahendra, Mr. Bhupesh, Mrs. Snehal for their kind support and suggestion during
the course of my work.
Above all I thank Baba and Maa, and my elder sister Susmita who have done a lot for me and my
studies, without their help it wouldnt have been possible for me to complete my masters.
Last but not the least I pay reverence to the supreme ubiquitous, omniscient, omnipotent, The
Almighty God for his benevolence and blessings bestowed upon me.
Date:
Place: Bangalore.
Supriya Mana
Abbreviation
List of Abbreviations
g/kg
Microliter
Micromole
AAI
Antiatherogenic index
ADP
Adenosine diphosphate
AEHAD
Ca2+
Calcium ion
CHOD-POD
CMC
DHAP
DNA
Deoxyribonucleic acid
EDTA
FPG
Fasting glycemia
gram
g/dl
g/kg
GDM
GE
GOD-POD
GPO-PAP
Glycerol-3-phosphate oxidase-peoxidase
GS
HAD
Holarrhena antidysenterica
HbA1c
Glycosylated haemoglobin
HDL
HGO
HLA
i.m.
Intramuscular
Dept. of Pharmacology
ABMRCP, Bangalore.
Abbreviation
i.p.
Intraperitonial
I.U/kg
IDDM
IRI
Immunoreactive insulin
LCAT
Lecithin-cholesterol acyltransferase
LDL
MEHAD
mg/dl
mg/kg
MHC
ml/kg
mM
Millimole
NAD+
NDDG
NIDDM
nm
Nanometer
NO
Nitric oxide
p.o.
Per oral
PEHAD
PPAR
rpm
s.c.
Subcutenious
STZ
Streptozotocin
TC
Total cholesterol
TG
Serum triglyceride
UV
Ultra-violet
VLDL
-cell
Pancreatic -cell
Dept. of Pharmacology
ABMRCP, Bangalore.
Abstract
ABSTRACT
The methanolic, petroleum ether and aqueous extracts of Holarrhena antidysenterica
(HAD) seeds were tested in the streptozotocin (STZ) induced diabetic rats for their effect
on body weight, fasting serum glucose, serum cholesterol, serum triglyceride, total
protein, blood urea, urine glucose and liver glycogen levels. The diabetes was induced by
single i.v. injection of STZ at the dose of 35 mg/kg dissolved in 0.1M citrate buffer of pH
4.5. The diabetic rats, with the fasting serum glucose above 200 mg/dl, were included in
the study and randomly divided into six groups of ten animals each of male in sex along
with the normal control group. The diabetic rats were treated with the glibenclamide,
methanolic (MEHAD), petroleum ether (PEHAD) and aqueous (AEHAD) extracts of
HAD for 18 days. The diabetic rats treated with MEHAD, PEHAD and AEHAD showed
significant reduction in fasting serum glucose, serum cholesterol, serum triglyceride, total
protein, blood urea, urine glucose and protection from the loss of body weight and
increase in liver glycogen content during the treatment period these effects were
comparable to those seen in the glibenclamide treated group of rats. This suggests that the
HAD extracts posses anti-diabetic activity and further studies are needed to elucidate the
mechanism of action and to know the active principles involved in producing the effect.
Dept. of Pharmacology
ABMRCP, Bangalore
T able of Contents
CONTENTS
Contents
Page Number
Chapter 1
Introduction
1-3
Chapter 2
Objectives
5-6
Chapter 3
Review of Literature
8 - 42
Chapter 4
Methodology
44 - 56
Chapter 5
Results
58 - 93
Chapter 6
Discussion
95 - 98
Chapter 7
Conclusion
100
Chapter 8
Summary
101 - 102
Chapter 9
Bibliography
Chapter 10
Annexure
Dept. of Pharmacology
104 111
113
ABMRCP, Bangalore.
T able of Contents
Dept. of Pharmacology
ABMRCP, Bangalore.
LIST OF TABLES
Table
No.
1
Page
No.
Table Name
Etiologic classification of diabetes mellitus
11
58-59
62
67
72
76
80
84
88
10
Dept. of Pharmacology
92
ABMRCP, Bangalore.
List of Figures
Figure
No.
Page
No.
Figures Name
Main metabolic pathways of intermediate
16
17
deficiency.
26
27
drug.
Schematic diagram of the insulin receptor
5
28
31
target tissues
7
Structure of Streptozotocin
34
Dept. of Pharmacology
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39
ABMRCP, Bangalore.
List of Graphs
LIST OF GRAPH
Graph
No.
1
Page
No.
Graphs Name
Effect of HAD extracts on body weight in rats
63
68
73
77
81
85
89
Dept. of Pharmacology
93
ABMRCP, Bangalore.
INTRODUCTION
Introduction
1. INTRODUCTION
According to the classical definition, diabetes mellitus is a disorder resulting from
both genetic predisposition and favoring environmental factors, and is characterized by
alterations in the metabolism of carbohydrate, fat and protein, which are caused by a
relative or absolute deficiency of insulin secretion and different levels of insulin
resistance. In the patients with long-standing diabetes, late complications develop
consisting of alterations and failure of various organs (especially the non-insulin sensitive
ones) including the eyes (retinopathy with vision loss), kidneys (nephropathy leading to
renal failure), nerves (peripheral and autonomic neuropathy), heart and blood vessels
(precocious and severe cardiovascular, cerebrovascular and peripheral vascular
atherosclerosis). Diabetes mellitus includes etiologically and clinically different diseases
that have hyperglycemia in common, representing a syndrome rather than a single
disease1.
Symptoms of marked hyperglycemia include polyuria, polydipsia, weight loss,
sometimes with polyphagia, and blurred vision. Impairment of growth and susceptibility
to certain infections may also accompany chronic hyperglycemia. Acute, life-threatening
consequences of uncontrolled diabetes are hyperglycemia with ketoacidosis or the
nonketotic hyperosmolar syndrome2.
The number of people with diabetes is increasing due to population growth, aging,
urbanization and increasing prevalence of obesity and physical inactivity. According to
recent estimate, the greatest absolute increase in the number of people with diabetes will
Dept. of Pharmacology
ABMRCP, Bangalore.
1
Introduction
be in India and the total number of people with diabetes is projected to rise from
31.7 million in 2000 to 79.4 million in 20303.
Globally the prevalence of diabetes was estimated to be 2.8 % in 2000 and 4.4 %
in 2030. Worldwide the total number of people with diabetes is projected to rise from
171 million in 2000 to 366 million in 2030. Therefore, the human population worldwide
appears to be in the midst of an epidemic of diabetes3.
Despite the great strides that have been made in the understanding and
management of diabetes, the disease and disease-related complications are increasing
unabated. Parallel to this, recent developments in understanding the pathophysiology of
the disease process have opened up several new avenues to identify and develop novel
therapies to combat the diabetic plague. Although insulin and oral hypoglycemic agents
are the major players in the treatment of the diabetes mellitus, they have prominent side
effects and fail to significantly alter the course of diabetic complications.
Dept. of Pharmacology
ABMRCP, Bangalore.
2
Introduction
Holarrhena antidysenterica Wall. (Family: Apocynaceae) 4 is a plant commonly
found in the forests of India, indigenous to the tropical Himalayas, Assam, Uttar Pradesh,
down to Travancore6, 7. The survey of literature reveals that various parts of this plant are
used in diarrhoea, blood dysentery, haematemesis, acute rheumatism, astringent and
diabetes6, 7. The fresh bark and seeds of this plant were reportedly used as antibacterial,
antidiarrhoeal agent8, 9. The Bhavaprakasha has recommended the fresh bark and seeds
of Holarrhena antidysenterica in the treatment of diabetes7. However, scientific data on
antidiabetic activity of Holarrhena antidysenterica is not available. So, in the present
study we have made an attempt to explore the anti-diabetic effect of petroleum ether,
methanolic and aqueous extracts of Holarrhena antidysenterica in the streptozotocin
(STZ) induced diabetic rats.
Dept. of Pharmacology
ABMRCP, Bangalore.
3
OBJECTIVES
Objectives
2. OBJECTIVES:
Diabetes is one of the common metabolic disorders affecting world wide
population of the world3. Insulin and oral hypoglycemic agents are the major players in
the management of the diabetes; they have prominent side effects and failed to
significantly alter the course of diabetic complication10. However, complete cure of
diabetes has been eluding physician for centuries and the quest for the development of
more effective anti-diabetic agent is pursued relentlessly. Nowadays medicinal plants
play an important role in the management of diabetes mellitus, especially in developing
countries where resources are meager11.
The Bhavaprakasha has recommended the use of the fresh bark and seeds of
Holarrhena antidysenterica in the treatment of diabetes7. However, scientific data on
antidiabetic activity of Holarrhena antidysenterica is not available. So, in the present
study we have made attempt to explore the anti-diabetic effect of Holarrhena
antidysenterica (petroleum ether, methanol and water) extracts in streptozotocin (STZ)
induced diabetic rats.
The objectives of the study are listed below:
Extraction of the dried seeds of Holarrhena antidysenterica with different
solvents (petroleum ether, methanol and water).
Preliminary Phytochemical screening.
Antidiabetic activity in streptozotocin induced diabetic rats by measuring the
following:
Dept. of Pharmacology
ABMRCP, Bangalore.
5
Objectives
Liver glycogen.
Dept. of Pharmacology
ABMRCP, Bangalore.
6
REVIEW OF
LITERATURE
Review of literature
3. REVIEW OF LITERATURE
3.1 Diabetes mellitus:
The term diabetes mellitus (DM) describes metabolic disorders of multiple
aetiology characterised by chronic hyperglycaemia with disturbances of carbohydrate, fat
and protein metabolism resulting from defects in insulin secretion, insulin action or both.
The effects of diabetes mellitus include longterm damage, dysfunction and failure of
various organs. DM may present with characteristic symptoms such as thirst, polyuria,
blurring of vision and weight loss. In its most severe forms, ketoacidosis or a non-ketotic
hyperosmolar state may develop and lead to stupor, coma and in absence of effective
treatment, death. Often symptoms are not severe or may be absent and consequently
hyperglycaemia, sufficient to cause pathological and functional changes may be present
for a long time before the diagnosis is made. The longterm effects of diabetes mellitus
include progressive development of the specific complications of retinopathy with
potential blindness, nephropathy that may lead to renal failure, and/or neuropathy with
risk of foot ulcers, amputation, Charcot joints and features of autonomic dysfunction,
including sexual dysfunction. People with diabetes are at increased risk of cardiovascular,
peripheral vascular and cerebrovascular disease12.
Terminology:
The term 'diabetes' was coined by Aretaeus of Cappadocia. The Greek word
diabanein literally means "passing through" or "siphon," a reference to one of diabetes'
major symptoms-excessive urine production. The word became "diabetes" from the
English adoption of the medieval Latin diabetes. In 1675 Thomas Willis added mellitus
Dept. of Pharmacology
ABMRCP, Bangalore.
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Review of literature
from the Latin word for honey (mel in the sense of "honey sweet") when he noted that the
blood and urine of a diabetic patient has a sweet taste. This had been noticed long before
in ancient times by the Greeks, Chinese, Egyptians and Indians. In 1776, it was
confirmed that the sweet taste was because of an excess of a kind of sugar in the urine
and blood of people with diabetes.
History:
Although diabetes has been recognized since ancient time and various treatments
have been known since the middle ages, the elucidation of the pathogenesis of diabetes
occurred mainly in 20th century13.
Oskar Minkowski and Joseph von Mering, first discovered the critical role of
pancreas by removing the pancreas from a dog, the dogs developed all signs and
symptoms of diabetes and died shortly afterward13. Dr. Banting with the assistance of
Best, colleagues (particularly Collip) continued his research on de-pancreatized dogs, but
went a step further and demonstrated that they could reverse induced diabetes in dogs by
giving them an extract from the pancreatic islets of Langerhans of healthy dogs. For this,
Banting et al received the Nobel Prize in Physiology or Medicine in 1923; both shared
their Prize money with others in the team who were not recognized. Banting and Best
made the patent available without charge and did not attempt to control commercial
production. Insulin production and therapy rapidly spread around the world, largely as a
result of this decision14. Despite the availability of treatment, diabetes remained a major
cause of death. In 1930s British clinician Harry Himsworth conducted series of
experiments in both animals and humans (normal and diabetic patients) that led him to a
Dept. of Pharmacology
ABMRCP, Bangalore.
9
Review of literature
conclusion that, diabetes could be caused not only by a lack of insulin but also by a lack
of sensitivity to insulin9. Himswoths experiments showed that there were two types of
diabetes: type-1 and type-2. People with type-1 diabetes were sensitive to insulin and
occured at young age; those with type-2 were insensitive to insulin and tended to
gradually develop a milder form of disease at middle age or older.
Other landmark discoveries include13:
1. Sulfonylureas were discovered by the chemist Marcel Janbon and his co-worker.
2. The radioimmunoassay for insulin, discovered by Rosalyn Yalow and Solomon
Berson (gaining Yalow the 1977 Nobel Prize in Physiology or Medicine).
3. Identification of PPAR (peroxisome proliferator activated receptor) activator as
effective antidiabetics in the 1990s.
Classification2:
Although all forms of diabetes mellitus share hyperglycemia as a common
feature, the pathogenic processes involved in the development of hyperglycemia vary
widely. The previous classification of diabetes mellitus were based on the age at onset of
disease or on the made of therapy; in contrast, the recently revised classification reflects
our greater understanding of the pathogenesis of each variant. The etiologic classification
of diabetes mellitus is given in (table-1).
Dept. of Pharmacology
ABMRCP, Bangalore.
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Review of literature
Dept. of Pharmacology
ABMRCP, Bangalore.
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Review of literature
3. Neoplasia
4. Cystic fibrosis
5. Hemochromatosis
6. Fibrocalculous pancreatopathy
7. Others
D. Endocrinopathies
1. Acromegaly
2. Cushings syndrome
3. Glucagonoma
4. Pheochromocytoma
5. Hyperthyroidism
6. Somatostatinoma
7. Aldosteronoma
8. Others
E. Drug- or chemical-induced
1. Vacor
2. Pentamidine
3. Nicotinic acid
4. Glucocorticoids
5. Thyroid hormone
6. Diazoxide
7. -adrenergic agonists
8. Thiazides
9. Dilantin
Dept. of Pharmacology
ABMRCP, Bangalore.
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Review of literature
10. -Interferon
11. Others
F. Infections
1. Congenital rubella
2. Cytomegalovirus
3. Others
G. Uncommon forms of immune-mediated diabetes
1. Stiff-man syndrome
2. Antiinsulin receptor antibodies
3. Others
H. Other genetic syndromes sometimes associated with diabetes
1. Downs syndrome
2. Klinefelters syndrome
3. Turners syndrome
4. Wolframs syndrome
5. Friedreichs ataxia
6. Huntingtons chorea
7. Laurence-Moon-Biedl syndrome
8. Myotonic dystrophy
9. Porphyria
10. Prader-Willi syndrome
11. Others
IV. Gestational diabetes mellitus (GDM)
Dept. of Pharmacology
ABMRCP, Bangalore.
13
Review of literature
Type 1 diabetes (-cell destruction, usually leading to absolute insulin deficiency):
Diabetes was previously called insulin-dependent diabetes mellitus (IDDM) or
juvenile-onset diabetes. Type 1 diabetes develops when the bodys immune system
destroys pancreatic beta cells, the only cells in the body that make the hormone insulin
that regulates blood glucose. This form of diabetes usually strikes children and young
adults, although disease onset can occur at any age. Type 1 diabetes may account for 5%
to 10% of all diagnosed cases of diabetes. Risk factors for type 1 diabetes may include
autoimmune, genetic and environmental factors2.
Type 1 DM is a chronic autoimmune disease associated with selective destruction
of insulin producing pancreatic -cells. The onset of clinical disease represents the end
stage of -cells destruction leading to type 1 DM. Several features characterize type 1
DM as an autoimmune disease14:
1. Presence of immuno-competent and accessory cells in infiltrated
pancreatic islets.
2. Association of susceptibility to disease with the class II (immune
response) genes of the major histocompatibility complex (MHC), human
leucocyte antigens (HLA).
3. Presence of islets cell specific auto-antibodies.
4. Alteration of T cell mediated immunoregulation, particularly in CD4+ T
cell compartment.
5. Response to immunotherapy.
Dept. of Pharmacology
ABMRCP, Bangalore.
14
Review of literature
Idiopathic diabetes2:
Some forms of type-1 diabetes have no known etiologies. Some of these patients
have permanent insulinopenia and are prone to ketoacidosis, but have no evidence of
autoimmunity. Although only a minority of patients with type-1 diabetes fall into this
category, of those who do, most are of African or Asian ancestry. Individuals with this
form of diabetes suffer from episodic ketoacidosis and exhibit varying degrees of insulin
deficiency between episodes. This form of diabetes is strongly inherited, lacks
immunological evidence for -cell autoimmunity, and is not HLA associated. An
absolute requirement for insulin replacement therapy in affected patients may come and
go.
Pathophysiology of type-1 diabetes1:
The pathophysiological changes occurring in type-1 diabetes as a consequence of
the severe insulin deficiency may be better understood by comparing the normal picture
of the main metabolic pathways, as summarized in figure 1, with the abnormal situation
present in type-1 diabetes, outlined in figure 2. In type-1 diabetes, the deficit of insulin
and the prevalence of counter regulatory hormones, primarily glucagon, leads to the
activation of glycogenolysis and gluconeogenesis in liver, with ensuing enhanced hepatic
glucose output (HGO). In addition, the deficiency in insulin action results in reduced
glucose utilization in peripheral insulin sensitive tissues (primarily muscle) as well as in
activation of lipolysis in the adipose tissue (insulin normally exerts an antilipolytic
effect), with enhanced release of FFA. The latter, although they cannot be directly
converted into glucose in man, favor gluconeogenesis in the liver. Combination of
Dept. of Pharmacology
ABMRCP, Bangalore.
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Review of literature
enhanced HGO and reduced glucose utilization results in hyperglycemia. In addition,
FFA exert anti-insulin effects at the muscle level, through the mechanism of the glucoseFFA cycle (Randles cycle), which may cause resistance to the therapeutically
administered insulin It should also be considered that hyperglycemia itself favors glucose
utilization (glucose effectiveness), perhaps by acting on non-insulin dependent glucose
transporters (GLUT1 in gut, GLUT2 in liver and GLUT3 in brain), and that in type-1
diabetes this glucose effect may be reduced, i.e. there may be glucose resistance.
Fig 1: Scheme showing the main metabolic pathways of intermediate metabolism in the
three insulin-sensitive tissues (liver, muscle and adipose tissue) participating in the
metabolic homeostasis1.
Dept. of Pharmacology
ABMRCP, Bangalore.
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Review of literature
Fig 2: Scheme of the main metabolic pathways (similar to that outlined in figure 1) and
of their changes in activity rate occurring in states of severe insulin deficiency, such as
decompensate type-1 diabetes (thick or thin arrows indicate increased or decreased
activity, respectively)1.
Type-2 diabetes (non-insulin dependent) 1:
Type-2, diabetes previously also named non-insulin-dependent diabetes mellitus
(NIDDM or adult-onset diabetes) occurs in approximately 9095% of diabetic people in
the Western world, resulting from insulin resistance and insufficient compensatory insulin
secretion. The disease has an insidious onset and remains asymptomatic and undiagnosed
for a long period, even if the moderate hyperglycemia is able to induce severe diabetic
late complications.
Type-2 diabetes is strongly favored by genetic predisposition. However, although
it shows familial aggregation as well as a high concordance (80%) in monozygotic twins,
Dept. of Pharmacology
ABMRCP, Bangalore.
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Review of literature
its mode of inheritance is not fully understood. It may well be a polygenic disease. In any
case, the risk of offspring and siblings of type-2 diabetic patients to develop the disease is
relatively elevated.
In addition to the genetic predisposition, favoring environmental factors are
involved, such as excessive caloric intake, obesity with increased body fat in the
abdominal (visceral) site, sedentary habit, etc. The insulin levels may be normal or even
increased (especially in presence of obesity) for a long time, but may decrease in the late
stage of the disease. The abnormal carbohydrate metabolism can be early identified
measuring fasting glycemia (FPG) or performing an oral glucose tolerance test (OGTT).
This type of diabetes is non-insulin-dependent for survival and is non-ketosis prone.
Hyperglycemia is usually improved or corrected by diet, weight loss and oral
hypoglycemic drugs. In type-2 diabetics an acute life-threatening complication, the
nonketotic hyperosmolar coma, can develop whereas ketoacidosis seldom occurs
spontaneously, although it may arise during stress, infections or other illnesses.
Pathophysiology of Type-2 diabetes1:
This disease is due to a varying combination of insulin resistance and reduction
(especially in the late stage of the disease) in insulin secretion. The metabolic alterations
are less pronounced than those in type-1 diabetes, outlined in figure 2. Due to insulin
resistance (and to enhanced counter regulatory hormones), there is increased HGO
(which contributes primarily to fasting hyperglycemia) and reduced peripheral glucose
utilization. There is also elevation of plasma FFA (resulting from activation of lipolysis
and/or the often enhanced fat mass due to coexisting obesity), which in turn contributes
to insulin resistance through the mechanism of the glucose-FFA cycle. As mentioned
Dept. of Pharmacology
ABMRCP, Bangalore.
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Review of literature
above (under Type-1 Diabetes), hyperglycemia itself favors glucose utilization (glucose
effectiveness). This mechanism may be impaired in type-2 diabetes, i.e. glucose
resistance may be present. It has been observed that in obesity and type-2 diabetes (as
well as in acromegaly and Cushings disease), in the postabsorptive period, non-insulin
mediated glucose uptake is a major determinant of glucose disposal and is similar in the
different pathologies studied. On the other hand, although absolute rates of basal insulinmediated glucose uptake are reduced in insulin resistant states, they do not achieve
statistical
value
compared
with
control
subjects
because
of
compensatory
hyperinsulinemia.
Other specific types:
Other specific types of diabetes do not fit into type-1, type-2 or gestational
diabetes. These results from specific genetic conditions such as maturity-onset diabetes of
youth, surgery, drugs, malnutrition, infections and other illnesses.
Gestational diabetes:
This is a form of glucose intolerance that is diagnosed in some women during
pregnancy. It is also more common among obese women and women with a family
history of diabetes. During pregnancy, gestational diabetes requires treatment to
normalize maternal blood glucose levels to avoid complications in the infant11. GDM
prevalence by NDDG and Carpenter and Coustan criteria, was 5.0 and 7.4% in Asians,
3.9 and 5.6% in Hispanics, 3.0 and 4.0% in African-Americans and 2.4 and 3.8% in
whites15.
Dept. of Pharmacology
ABMRCP, Bangalore.
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Review of literature
Complications16:
Diabetic ketoacidosis:
Insulin deficiency combined with excess glucagon will lead to kitoacidosis. It is
most exclusive complication of type-1 diabetes. It mainly occurs due to insuficient
insulin intake and exposure to stress. Lack of insulin causes lipolysis in adipose tissue
which causes the release of free fatty acids in to the plasma. These free fatty acids are
taken up by liver where they are oxidized by enzyme acetyl coenzyme-A to ketone bodies
(acetoacetic acid and -hydroxybutyric acid), which results into imbalance between
ketogenesis and the rate at which ketone bodies can be utilised by muscles and other
tissues, leading to ketonaemia and ketonuria. Systemic metabolic ketoacidosis occurs if
urinary excretion of ketone bodies is prevented by dehydration. Clinically this condition
is charecterised by anorexia, nausea, vomitting, deep and fast breathing, mental confusion
and coma.
ABMRCP, Bangalore.
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Review of literature
levels combined with the loss of water will eventually results into such high serum
osmolality (dehydration). The bodys cells may become progressively dehydrated as
water is drawn out from them and excreted. Electrolyte imbalances are also common.
This combination of changes, especially if prolonged, will results in symptoms of
lethargy (dulled or reduced level of alertness or consciousness) and may progress to
coma. Thrombotic and bleeding complications are frequent due to high viscosity of
blood. The mortality rate in hyperosmolar nonketotic coma is high.
Hypoglycemia:
The problem of hypoglycemia is much more common in type-1 diabetes. Daytime
hypoglycemic episodes are usually recognized symptoms: like the sweating, nervousness,
tremor and hunger. Night time hypoglycemia may be without symptoms or manifest as
night sweats, unpleasant dreams or early morning headache. Diabetic patients taking too
much insulin, missing a meal, or over-exercising can result in hypoglycemia. It is
imperative that a good relationship exists between the patient and the physician
prescribing the insulin so that dosages can be gauged correctly. In response to the
hypoglycemia, secretion of several hormones which raise blood glucose levels is
increased: epinephrine, norepinephrine, growth hormone and cortisol. As a result, blood
sugar levels will rebound and often lead to hyperglycemia. This phenomenon is
commonly referred to as the somoghi phenomenon.
On a long-term basis, the diabetics health condition is complicated by repeated
elevations in blood glucose levels. Major chronic complications of diabetes include;
Dept. of Pharmacology
ABMRCP, Bangalore.
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Review of literature
atherosclerosis, diabetic microangiopathy, diabetic nephropathy, diabetic neuropathy,
diabetic retinopathy and infections.
Atherosclerosis:
The diabetic patient has a two to three fold higher risk of atherosclerosis than a
non diabetic individual. The cause for atherosclerosis is not known but the possible
contributing factors are hyperlipidemia, reduced HDL (high-density lipoprotein) levels,
non-enzymatic glycosylation, increased platelet adhesiveness, obesity and associated
hypertension in diabetes. The possible complications of diabetes in atherosclerosis are
coronary artery disease, silent myocardial infarction, cerebral stroke and the gangrene of
toes and feet.
Diabetic microangiopathy:
Microangiopathy occurs due to recurrent hyperglycemia that causes increased
glycosylation of haemoglobin and other proteins resulting in thickening of basement
membrane of small blood vessels and capillaries of different organs and tissues such as
skin, skeletal muscle, eye and kidney and nonmuscular tissues such as peripheral nerves,
renal tubules and Bowmans capsule.
Diabetic nephropathy:
Diabetic nephropathy is the damage to the kidney which can lead to the chronic
renal failure, eventually the death.
Four types of lesions are described in diabetic nephropathy;
Diabetic glomerulosclerosis
Dept. of Pharmacology
ABMRCP, Bangalore.
22
Review of literature
Vascular lesions
Diabetic neuropathy:
The pathogenesis of diabetic neuropathy is not known, but it may be due to the
increased glycosylation of haemoglobin and other proteins or may be due to
accumulation of sorbitol and fructose as a result of hyperglycemia, leading to the
deficiency of myoinositol. Diabetic neuropathy is mainly characterized by systemic
peripheral neuropathy, but it may affect all parts of nervous system.
Diabetic retinopathy:
Diabetic retinopathy is serious eye disease that can result in blindness. The
retinopathic lesions are divided into background (consisting of microaneurysms,
hemorrhage, exudates and retinal edema) and proliferative (with newly formed vessels,
scaring, retinitis preoliferens, vitreous hemorrhage and retinal detachment). Besides
retinopathy diabetes also predisposes the patient to the early development of cataract and
glaucoma.
Infections:
Diabetic patients are more susceptible to various infections such as tuberculosis,
pneumonias, pyelonephritis, otitis, carbuncles and diabetic ulcers. This could be due to
Dept. of Pharmacology
ABMRCP, Bangalore.
23
Review of literature
various factors such as impaired leucocyte functions, reduced cellular immunity, poor
blood supply due to vascular involvement and hyperglycemia.
Treatment17:
Drugs used to treat diabetes mellitus are as follows;
Insulin Preparations:
Insulin is essential for the treatment of type-1 diabetes mellitus. Different
formulations of insulin are available such as;
1. Fast-and short-acting soluble insulin.
2. Intermediate-acting insulin (e.g.: isophane insulin can be mixed with soluble
insulin)
3. Long-acting insulin (e.g.: insulin zinc suspension)
Dept. of Pharmacology
ABMRCP, Bangalore.
24
Review of literature
3.2 Insulin:
Chemistry
Insulin is a small protein with a molecular weight in humans of 5808. It contains
51 amino acids arranged in two chains (A and B) linked by disulfide bridges; there are
species differences in the amino acids of both chains. Proinsulin, a long single-chain
protein molecule, is processed within the Golgi apparatus and packaged into granules,
where, it is hydrolyzed into insulin and a residual connecting segment called C-peptide
by removal of four amino acids, Insulin and C-peptide are secreted in equimolar amounts
in response to all insulin secretagogues; a small quantity of unprocessed or partially
hydrolyzed proinsulin is released as well. While proinsulin may have some mild
hypoglycemic action, C-peptide has no known physiologic function. Granules within the
B cells store the insulin in the form of crystals consisting of two atoms of zinc and six
molecules of insulin. The entire human pancreas contains up to 8 mg of insulin,
representing approximately 200 biologic units. Originally, the unit was defined on the
basis of the hypoglycemic activity of insulin in rabbits. With improved purification
techniques, the unit is presently defined on the basis of weight and at present insulin
standards used for assay purposes contain 28 units per milligram.
Dept. of Pharmacology
ABMRCP, Bangalore.
25
Review of literature
Dept. of Pharmacology
ABMRCP, Bangalore.
26
Review of literature
outward potassium efflux results in depolarization of the B cell and opening of voltagegated calcium channels. The resulting increased intracellular calcium triggers secretion of
the hormone. As noted below, the insulin secretagogue drug group (sulfonylureas,
meglitinides and D-phenylalanine) exploits parts of this mechanism.
Fig 4: Model of control of insulin release from pancreatic cell by glucose & by
sulfonylurea drug.
Insulin Receptor:
Once insulin has entered the circulation, it is bound by specialized receptors that
are found on the membranes of most tissues. The biologic responses promoted by these
insulin-receptor complexes have been identified in the primary target tissues, i.e. liver,
Dept. of Pharmacology
ABMRCP, Bangalore.
27
Review of literature
muscle and adipose tissue. The receptors bind insulin with high specificity and affinity in
the picomolar range. The full insulin receptor consists of two covalently linked
heterodimers, each containing subunit, which is entirely extracellular and constitutes
the recognition site and a subunit that spans the membrane (Figure-5). The subunit
contains a tyrosine kinase. The binding of an insulin molecule to the subunits at the
outside surface of the cell activates the receptor and through a conformational change
brings the catalytic loops of the opposing cytoplasmic subunits into closer proximity
thereby facilitating phosphorylation of tyrosine residues and tyrosine kinase activity.
Fig 5: Schematic diagram of the insulin receptor heterodimers in the activated state.
Dept. of Pharmacology
ABMRCP, Bangalore.
28
Review of literature
The first proteins to be phosphorylated by the activated receptor tyrosine kinases are the
docking proteins, insulin receptor substrate-1 and -2 (IRS-1, IRS-2). After tyrosine
phosphorylation at several critical sites, IRS-1 and IRS-2 bind to and activate other
kinases
most
importantly
phosphatidylinositol-3-kinase
that
produce
further
Dept. of Pharmacology
ABMRCP, Bangalore.
29
Review of literature
Principal type & duration of action of insulin preparations:
Four principal types of insulins are available: (1) rapid-acting, with very fast onset
and short duration; (2) short-acting, with rapid onset of action; (3) intermediate-acting;
and (4) long-acting, with slow onset of action (Figure 6). Rapid-acting and short-acting
insulins are dispensed as clear solutions at neutral pH and contain small amounts of zinc
to improve their stability and shelf-life. All other commercial insulins have been
modified to provide prolonged action and are, with the exception of insulin glargine,
dispensed as turbid suspensions at neutral pH with either protamine in phosphate buffer
(neutral protamine Hagedorn [NPH] insulin) or varying concentrations of zinc in acetate
buffer (ultralente and lente insulins). Insulin glargine is the only soluble long-acting
insulin. The goal of subcutaneous insulin therapy is to replace the normal basal
(overnight, fasting and between meal) as well as prandial (mealtime) insulin. Current
regimens generally use intermediate or long-acting insulins to provide basal or
background coverage and rapid-acting or short-acting insulin to meet the mealtime
requirements. The latter insulins are given as supplemental doses to correct high blood
sugars. Intensive therapy ("tight control") attempts to restore near-normal glucose
patterns throughout the day while minimizing the risk of hypoglycemia. An exact
reproduction of the normal glycemic profile is technically not possible because of the
limitations inherent in subcutaneous administration of insulin. The most sophisticated
insulin regimen delivers rapid-acting insulin through a continuous subcutaneous insulin
infusion device; alternative intensive regimens referred to as multiple daily injections
(MDI) use long-acting or intermediate-acting insulins with multiple boluses of rapid-
Dept. of Pharmacology
ABMRCP, Bangalore.
30
Review of literature
acting or short-acting insulin. Conventional therapy presently consists of split-dose
injections of mixtures of rapid- or short-acting and intermediate-acting insulins.
Fig 6: Insulin promotes synthesis and storage of glycogen, triglycerides & protein in its
major target tissues.
Dept. of Pharmacology
ABMRCP, Bangalore.
31
Review of literature
Treatment with insulin:
The current classification of diabetes mellitus identifies a group of patients who
have virtually no insulin secretion and whose survival depends on administration of
exogenous insulin. This insulin dependent group (type-1) represents 510% of the
diabetic population in the USA. Most type-2 diabetics do not require exogenous insulin
for survival, but many need exogenous supplementation of their endogenous secretion to
achieve optimum health. It is estimated that as many as 20% of type 2 diabetics in the
USA (22.5 million people) are presently taking insulin.
Hypoglycemia
Dept. of Pharmacology
ABMRCP, Bangalore.
32
Review of literature
In individuals exposed to frequent hypoglycemic episodes during tight glycemic
control, autonomic warning signals of hypoglycemia are less frequent or even absent.
This dangerous acquired condition is termed "hypoglycemic unawareness." When
patients lack the early warning signs of low blood glucose, they may not take corrective
measures in time. In patients with persistent, untreated hypoglycemia, the manifestations
of insulin excess may developconfusion, weakness, bizarre behavior, coma, seizures
at which point they may not be able to procure or safely swallow glucose-containing
foods. Hypoglycemic awareness may be restored by preventing frequent hypoglycemic
episodes. An identification bracelet, necklace or card in the wallet or purse, as well as
some form of rapidly absorbed glucose, should be carried by every diabetic who is
receiving hypoglycemic drug therapy.
Treatment of Hypoglycemia
All of the manifestations of hypoglycemia are relieved by glucose administration.
To expedite absorption, simple sugar or glucose should be given, preferably in a liquid
form. To treat mild hypoglycemia in a patient who is conscious and able to swallow,
orange juice, glucose gel or any sugar-containing beverage or food may be given. If more
severe hypoglycemia has produced unconsciousness or stupor, the treatment of choice is
to give 2050 ml of 50% glucose solution by intravenous infusion over a period of 23
minutes. If intravenous therapy is not available, 1 mg of glucagon injected either
subcutaneously or intramuscularly will usually restore consciousness within 15 minutes
to permit ingestion of sugar. If the patient is stuporous and glucagon is not available,
small amounts of honey or syrup can be inserted into the buccal pouch. In general,
Dept. of Pharmacology
ABMRCP, Bangalore.
33
Review of literature
however, oral feeding is contraindicated in unconscious patients. Emergency medical
services should be called for all episodes of severely impaired consciousness.
Dept. of Pharmacology
ABMRCP, Bangalore.
34
Review of literature
Mechanism of the Diabetogenic effect of STZ:
In chemical terms it is the glucose molecule in the structure which gives way to
selective uptake of STZ into the beta cells, while the MNU nitrosourea moiety of STZ
allows for its activity as an alkylating agent22. STZ is taken up by pancreatic - cells via
glucose transporter GLUT2 (glucose transporter 2). A reduced expression of GLUT2,
perhaps in combination with reduced cellular activity of - cells at the time of
administration of STZ may prevent the diabetogenic action of STZ20.
At the intracellular level, three major phenomena are currently held responsible for
- cell death:
1. Process of methylation
2. Free radicals generation
3. Nitric Oxide (NO) production.
Dept. of Pharmacology
ABMRCP, Bangalore.
35
Review of literature
Methylation:
Fig 8: Protecting mechanism against action of alloxan and streptozotocin on pancreatic cells23.
The deleterious effect of STZ results from the generation of highly reactive carbonium
ions (CH3+), formed from decomposition of the nitrosomoiety. The CH3+ ions cause DNA
breaks by alkylating DNA bases at various positions, resulting in activation of the nuclear
enzyme poly (ADP-ribose) synthetase as part of the cell repair mechanism. As cellular
pyridine nucleotide, particularly NAD+ are utilized as substrate for the nuclear enzyme, a
profound decline in NAD+ occurs within 20 min. In effect, an abrupt and irreversible
NAD+ exhaustion leads to cessation of NAD+-dependent energy and protein metabolism,
ultimately leading to cell death. Inhibition of poly (ADP-ribose) synthetase by agent like
3-aminobenzamine and nicotinamide are known to protect - cells from NAD+ depletion
and cell death after STZ exposure23, 24. (Fig 8)
Dept. of Pharmacology
ABMRCP, Bangalore.
36
Review of literature
Free Radicals:
Free radical involvement in STZ effect has also been investigated. Hydrogen
peroxide has been shown to be produced in pancreatic islets upon STZ exposure in vivo
and in vitro. Moreover, because superoxidase dismutase, a free radical scavenger was
demonstrated to provide some protection against the diabetogenic properties of STZ, it
was concluded that oxidative stress could play a role in determining STZ toxicity25, 26.
Nitric Oxide:
NO-generating has been shown to be the mechanism of STZ toxicity in
diabetogenesis27. Recently, an integrated hypothesis was proposed to explain the
mechanism of action of STZ. In this hypothesis, STZ, through production of superoxide,
would generate peroxynitrite. The latter would dissociate into NO and hydroxyl radicals,
thus leading to - cells DNA damage and apoptosis28.
Bengali: Kurchi.
Gujarati: Kadvo.
Hindi: Kurchi.
Dept. of Pharmacology
ABMRCP, Bangalore.
37
Review of literature
Tamil: Veppaalai.
Sanskrit: Kutaja.
Nepali: Khuria.
Marathi: Kudaa.
Malayalam: kutakappaala.
Punjabi: Kewar.
Dept. of Pharmacology
ABMRCP, Bangalore.
38
Review of literature
Holarrhena antidysenterica
Dept. of Pharmacology
ABMRCP, Bangalore.
39
Review of literature
Cultivation:
Soil: Loamy soil, rich in organic matter with good drainage is preferred.
Climate: It is well grown in shady situation of tropical, sub-tropical climate.
Propagation: Seeds, stem cuttings and apical portions of plants are used for the
propagation.
Traditional Uses7:
It is extensively used by the Ayurvedic physicians in bloody dysentery, especially
when the malady has assumed a chronic character.
Charaka has found it useful in haematemesis, leprosy and dysentery of
tuberculosis patients, bleeding piles and bilious diarrhoea.
Sushruta has recommended it in piles, and bloody dysentery.
Parts used: Fresh bark and seed.
Dose: Decoction of bark and seed, 5 to 10 tolas, powdered seed to 1 anna.
Phytochemistry 4, 5, 6, 7:
The bark and seeds contain a non-oxygenated alkaloid Conessine and
Holarrhenine. These are an amorphous powder soluble in water and alcohol and dilute
acids. The content of alkaloids in the bark was found to be about 1.2% and 0.25% in the
seeds. Besides Conessine, there are two other alkaloids present which have been
Dept. of Pharmacology
ABMRCP, Bangalore.
40
Review of literature
designated as Kurchicine and Kurchine respectively. The alkaloid termed as Kurchine is
charecterised by having a low melting point of 75 C and it is the most abundant alkaloid
present in the bark. Other alkaloids present in bark and seed are as follows:
Conkurchine.
Kurcholessine.
Conesimine and isoconesimine.
Conimine.
Holarrhine.
Regholarrhenine A-F.
Kurchamine
Conessine
Holarrhimine
Conkurchine
Kurchamine
Dept. of Pharmacology
ABMRCP, Bangalore.
41
Review of literature
Toxicity Studies29:
Holarrhena antidysenterica Wall, or "Moak Luang" in Thai, is a medicinal plant
possessing anti-amoebic activity against Entamoeba histolytica. In order to determine if
this plant is safe to be used as an anti-amoebic agent in humans, subchronic toxicity study
was performed in Wistar rats of both sexes. Ethanolic extract of H. antidysenterica
complexed with polyvinyl pyrrolidone (PVP) was given orally for 3 months at the doses
of 0.03, 0.27 and 0.53 g/kg BW/day equivalent to crude drug 0.12, 1.2 and 2.4 g/kg or 1,
10 and 20 times of therapeutic dose in humans, respectively, It was found that body
weight gain and food consumption of animals receiving 0.27 and 0.53 g/kg were lower
than those of the water control and PVP control groups. Hematological examinations
showed that white blood cell counts, platelets and %neutrophils of all extract-treated
groups and PVP control groups were significantly higher, but %lymphocytes were
significantly lower than those of the water control groups. Serum biochemistry indicated
that groups of male and female rats receiving 0.27 and 0.53 g/kg had significantly higher
AST and ALT levels than those of both control groups. Changes found in other
biochemical parameters were not related the doses of the extract given or occurred in
animals of either sex only. Histopathological examinations of internal organs showed no
sign of abnormality that could be attributed to the toxic effect of the extract. It was
concluded that prolonged administration of high doses of ethanolic extract of H.
antidysenterica
Dept. of Pharmacology
ABMRCP, Bangalore.
42
METHODOLOGY
B
Methodology
4. METHODOLOGY
4.1 Plant Materials:
4.1.1 Collection of plant:
The seeds of Holarrhena antidysenterica were procured commercially from
Amruth Kashri, Bangalore. The seeds were authenticated by Dr. Shiddamallayya N,
Regional Research Institute (Ay.) Govt. Central Pharmacy Annexe, Ashoka Pilar,
Bangalore-11. A voucher specimen of seeds has been deposited in museum of department
of pharmacognosy, Acharya & B.M Reddy College of Pharmacy, Bangalore.
4.1.2 Preparation of Holarrhena antidysenterica extracts:
The Holarrhena antidysenterica seeds extract was prepared by soxhlet extraction
of 1000 g seed powder in 1000 ml of petroleum ether, methanol and distilled water
according to their increasing polarity. The extract was concentrated dried in vacuum and
residue stored in refrigerator at 2-8 C for use in subsequent experiments.
4.2 Chemicals:
List of chemicals used:
Supplied by:
Streptozotocin (STZ)
Sigma Aldrich.
Thomas Baker.
Thomas Baker.
Standard Glibenclamide
Aventis Pharma.
Methanol
Ethanol
Petroleum ether
(Analytical grade)
Benzoic acid
Dept. of Pharmacology
ABMRCP, Bangalore
44
Methodology
Xylene
Diethyl ether
(Analytical grade)
Anthrone LR
Sd Fine Chemical.
Trichloroacetic acid
HiMedia Chemicals.
Diagnostic Kits:
Supplied by:
Dept. of Pharmacology
ABMRCP, Bangalore
45
Methodology
4.3.2 Preparation of STZ Solution:
STZ was dissolved in ice-cold citrate buffer of pH 4.5 and injected immediately
within five minutes to avoid degradation.
4.3.3 Glibenclamide Solution:
20 mg standard glibenclamide was dissolved in 2 ml of distilled water to give
10 mg/ml solution. This solution was administered at a dose of 10 mg/ kg body weight
using clean and dry oral feeding needle for 18 days.
ABMRCP, Bangalore
46
Methodology
4.4.2 Experimental design:
Male wistar albino rats were divided into six groups of ten animals in each group
as follows:
Group-I:
Group-II:
Group-III:
Group-IV:
Group-V:
Group-VI:
ABMRCP, Bangalore
47
Methodology
The blood samples were allowed to clot for 30 mins at room temperature and then they
were centrifuged at 5000 rpm for 20 mins. The resulting upper serum layer was collected
in properly labeled, clean and dry micro-centrifuge tubes. The serum samples were stored
at -80 C and analyzed either immediately or within two week.
Collection of blood samples:
The blood was drawn from the retro orbital plexus of the rats under light ether
anesthesia on the respective days. The blood samples were stored at 2-8 C and analyzed
within one week.
Preparation of blood serum specimen:
Blood collected from the retro orbital plexus of the rats under light ether
anesthesia was allowed to clot for 30 min at room temperature and then was centrifuged
at 5000 rpm for 20 min. The resulting upper layer (serum) was used immediately for
estimation of different biochemical parameter. The serum used as specimen, it should be
free from hemolysis and was separated from the cells as soon as possible, to prevent
glycolysis. Blood serum is found to be stable for 24 h when stored at 2-8 C. This serum
specimen is used for the estimation of different biochemical parameter.
4.4.3 Parameters analyzed:
4.4.3.1. Body Weight:
The body weight of each animal was recorded daily and on the day 0, 6, 12 and
18, i.e. the days corresponding to other parameters analyzed are tabulated.
4.4.3.2. Fasting Serum Glucose Estimation:
Serum glucose was estimated by the Trinders Method, End Point method with
the help of Clinical Chemistry Analyzer (Metro Lab, 1600 DK-R) GLUCOSE LIQUID
Dept. of Pharmacology
ABMRCP, Bangalore
48
Methodology
TABLE REAGENT (Erba Mannheim GmbH, Germany) on day 0, day 6, day12 and day
18.
Principle:
Mutarotase are able to catalyse the rapid conversion of -isomer of Glucose to the
more reactive -D-Glucose which is oxidised to yield gluconic acid and hydrogen
peroxide in the presence of glucose oxidase. The enzyme catalysed the oxidative
coupling of 4-aminoantipyrine with p-hydroxybenzoic acid to yield a coloured
quinoneimine complex with absorbance proportional to the concentration of glucose in
sample.
- D - Glucose
Mutarotase
- D Glucose + O2 + H2O
-D-Glucose
Glucose oxidase
Peroxidase
Quinoneimine
+ H2O
Procedure:
To 1000 l of the reagent, 10 l of standard glucose (100 mg/ dl) was added and
incubated for 5 min at 37 C. This incubated mixture was aspirated and concentration of
standard was calibrated to show a value of 100 mg/ dl. The fasting serum glucose was
estimated by adding 10 l of the serum sample to 1000 l of the reagent, mixed well and
incubated at 37 C for 5 min. This incubated mixture was aspirated and absorbance
Dept. of Pharmacology
ABMRCP, Bangalore
49
Methodology
recorded against a reagent blank at 505 nm using Clinical Chemistry Analyzer (Metro
Lab, 1600 DK-R).
Calculation:
Absorbance of Sample
Concentration of Sample (mg/ dl) =
Absorbance of Standard
Concentration
of standard
(100 mg/dl)
Cholesterol Esters
Cholesterol Esterase
Cholesterol + O2
Cholesterol Oxidase
Peroxidase
4H2O + Quinoneimine
Dept. of Pharmacology
ABMRCP, Bangalore
50
Methodology
Procedure:
To 1000 l of the reagent, 20 l of standard cholesterol (200 mg/ dl) was added
and incubated for 10 min at 37 C. This incubated mixture was aspirated and
concentration of standard was calibrated to show a value of 200 mg/ dl. The fasting
serum cholesterol was estimated by adding 20 l of the serum sample to 1000 l of the
reagent, mixed well and incubated at 37 C for 10 min. This incubated mixture was
aspirated and absorbance recorded against a reagent blank at 505 nm using Clinical
Chemistry Analyzer (Metro Lab, 1600 DK-R).
Calculation:
Absorbance of Sample
Concentration of Sample (mg/ dl) =
Absorbance of Standard
Concentration
of standard
(200 mg/dl)
Lipoprotein lipase
Glycerol Kinase
Glycerol-3-Phosphate + ADP
DAP + H2O2
Peroxidase
Dept. of Pharmacology
ABMRCP, Bangalore
51
Methodology
Procedure:
To 1000 l of the reagent, 10 l of standard triglyceride (200 mg/ dl) was added
and incubated for 10 min at 37 C. This incubated mixture was aspirated and
concentration of standard was calibrated to show a value of 200 mg/ dl. The fasting
serum triglyceride was estimated by adding 10 l of the serum sample to 1000 l of the
reagent, mixed well and incubated at 37 C for 10 min. This incubated mixture was
aspirated and absorbance recorded against a reagent blank at 505 nm using Clinical
Chemistry Analyzer (Metro Lab, 1600 DK-R).
Calculation:
Absorbance of Sample
Concentration of Sample (mg/ dl) =
Absorbance of Standard
Concentration
of standard
(200 mg/dl)
Dept. of Pharmacology
ABMRCP, Bangalore
52
Methodology
Procedure:
To 1000 l of the reagent, 20 l of standard protein (6.0 g/dl) was added and
incubated for 10 min at 37 C. This incubated mixture was aspirated and concentration of
standard was calibrated to show a value of 6.0 g/ dl. The fasting serum total protein was
estimated by adding 20 l of the serum sample to 1000 l of the reagent, mixed well and
incubated at 37 C for 10 min. This incubated mixture was aspirated and absorbance
recorded against a reagent blank at 505 nm using Clinical Chemistry Analyzer (Metro
Lab, 1600 DK-R).
Calculation:
Absorbance of Sample
Concentration of total protein (g/ dl) =
Absorbance of Standard
Concentration
of
Standard
(6 g/dl)
Urease
2NH3 + CO2
GLDH
Glutamate + NAD
-Ketoglutarate
Dept. of Pharmacology
ABMRCP, Bangalore
53
Methodology
GLDH:
Glutamate dehydrogenase.
Procedure:
To 1000 l of the reagent, 20 l of standard Urea (50 mg/dl) was added and
incubated for 10 min at 37 C. This incubated mixture was aspirated and concentration of
standard was calibrated to show a value of 50 mg/ dl. The fasting serum urea was
estimated by adding 20 l of the serum sample to 1000 l of the reagent, mixed well and
incubated at 37 C for 10 min. This incubated mixture was aspirated and absorbance
recorded against a reagent blank at 505 nm using Clinical Chemistry Analyzer (Metro
Lab, 1600 DK-R).
Calculation:
Absorbance of Sample
Urea (mg/dl) =
Absorbance of Standard
Concentration of
Standard (mg/dl)
Dept. of Pharmacology
ABMRCP, Bangalore
54
Methodology
to the desired volume with 5 per cent TCA and mixed thoroughly. The final volume
should be a quantity that will contain 10 to 200 y of glycogen per ml.
1 ml. of the Trichloroacetic acid filtrate was pipetted into a 15 ml. Pyrex
centrifuge tube. To obtain the most reliable results, duplicate samples of each unknown
were analyzed. To each tube were added 5 volumes of 95 per cent ethanol with careful
blowing to effect thorough mixing. This should be checked by noting the absence of an
interface. The tubes were capped with clean rubber stoppers and allowed to stand
overnight at room temperature. (Alternatively, placing the tubes in a water bath at 37-40
for 3 hours may be carried out.) After precipitation was completed, the tubes were
centrifuged at 3000 r.p.m for 15 minutes. The clear liquid was gently decanted from the
packed glycogen and the tubes were allowed to drain in an inverted position for 10
minutes. The glycogen was dissolved by addition down the sides of the tube. If the
glycogen does not dissolve instantly, the tubes were agitated until solution was complete.
A reagent blank is prepared by pipetting 2 ml. of water into a clean centrifuge tube. A
standard was prepared by pipetting 2 ml. of standard glucose solution, containing 0.1 mg.
of glucose, into a similar tube. At this point 10 ml. of Anthrone reagent were delivered
into each tube with vigorous, but consistent, blowing. The stream of Anthrone reagent
was directed into the center of the tube and should be sufficient to insure good mixing.
As each tubes were receives Anthrone reagent, it is tightly capped with an air condenser
and placed in a cold tap water bath. The air condenser is prepared by cutting off the small
end of a size 0 rubber stoppers and inserting a 4 inch length of glass tubing, 3 to 4 mm. in
diameter. This serves to prevent water from entering the tube from the water bath.
Dept. of Pharmacology
ABMRCP, Bangalore
55
Methodology
After all tubes had reached the temperature of the cold water, they were immersed
in a boiling water bath to a depth a little above the level of the liquid in the tubes for 15
minutes and then removed to a cold water bath and cooled to room temperature. The
tubes and stoppers were wiped dry and the contents of each tube were transferred to a
calorimeter tube and read at 620 mp after adjusting the calorimeter with the reagent
blank. Care was taken to avoid introduction of lint or contaminating carbohydrate into the
Anthrone reaction.
Calculation:
The calculation of glycogen is as follows:
Where,
DU:
DS:
Dept. of Pharmacology
ABMRCP, Bangalore
56
RESULTS
Results
5. RESULTS
5.1 Preliminary phytoconstituents:
The preliminary phytochemical analysis of mithanolic, petroleum ether and
aqueous extracts of Holarrhena antidysenterica seed revealed that the presence of
various phytoconstituents which are presented in table no 2.
1. The methanolic extract contains about 1.5 to 3 percent of alkaloids and mainly
steroidal alkaloids.
2. The petroleum ether extract contain mostly oils and also some alkaloids.
3. The aqueous extract contains the steroidal alkaloid like conessine, holarrhimine and
holarrhidine.
Table-2: Estimation of Phytochemical analysis of different extract of Holarrhena
antidysenterica seed.
Sl
no
Chemical test
Test for
Alkaloids
Test for
carbohydrates
Methanolic
extract
Petroleum
ether
extract
Aqueous
extract
Hager's Test
+ ve
+ ve
+ ve
Mayer's Test
+ ve
+ ve
+ ve
Dragendrof's Test
+ ve
+ ve
+ ve
Wagner's Test
+ ve
+ ve
+ ve
Molisch's Test
+ ve
- ve
+ ve
Fehling's Test
+ ve
- ve
+ ve
Barfored's Test
+ ve
+ ve
+ ve
Benedict's Test
+ ve
- ve
+ ve
Dept. of Pharmacology
ABMRCP, Bangalore
58
Results
Test for
cardiac
glycosides
Test for
anthraquinone
glycosides
Test for
saponin
glycosides
4
5
7
8
Test for
Phytosterols
and
triterpenoids
Test for
flavonoids
Test for
tannin
Baljet test
- ve
- ve
- ve
Legal test
- ve
- ve
- ve
Modified Borntrager
test
- ve
- ve
- ve
Borntrager test
- ve
- ve
- ve
Foam test
+ ve
- ve
+ ve
Hemolytic test
+ ve
+ ve
+ ve
Stain Test
+ ve
+ ve
+ ve
Millons's Test
- ve
- ve
- ve
Biuret Test
- ve
- ve
- ve
Ninhydrin Test
- ve
- ve
- ve
LiebermannBurchard Test
+ ve
+ ve
+ ve
Salkowski Test
+ ve
+ ve
+ ve
Shinoda test
+ ve
+ ve
+ ve
- ve
- ve
- ve
5% FeCl3 solution
- ve
- ve
- ve
Dept. of Pharmacology
ABMRCP, Bangalore
59
Results
ABMRCP, Bangalore
60
Results
gm on day 18. The body weight gain in this group of rats from day 0 through day 6 and
day 12 to day 18 was relatively less when compared with the normal group. This shows
that glibenclamide treatment has protected the diabetic rats from loosing the body weight
in a significant (p 0.05) manner when compared with the diabetic control group of rats.
The MEHAD (250 mg/ kg) treated group of diabetic rats was found to have a
mean body weight ( SEM) of 186.50 3.53 gm on day 0, 193.00 2.29 gm on day 6,
201.16 6.13 gm on day 12 and 217.17 3.23 gm on day 18. These values show that
the body weight was maintained in a significant manner (p 0.05) throughout the study
period in this group when compared with diabetic animals.
The PEHAD (250 mg/kg) treated group of diabetic rats show mean ( SEM) body
weight of 190.83 1.30 gm on day 0, 198.50 4.22 gm on day 6, 201.35 4.23 gm on
day 12 and 208.67 3.46 g on day 18. These values show that the body weight was
maintained in a significant manner (p 0.05) throughout the study period in this group
when compared with diabetic animals.
The AEHAD (250 mg/ kg) treated group of diabetic rats show mean ( SEM)
body weight of 189.12 3.25 gm on day 0, 198.21 6.12 gm on day 6, 212.32 5.20 gm
on day 12 and 215.12 6.12 g on day 18. These values show that the body weight was
maintained in a significant manner (p 0.05) throughout the study period in this group
when compared with diabetic animals.
Dept. of Pharmacology
ABMRCP, Bangalore
61
Results
Table 3: Effect of HAD extracts on Body weight in rats.
Groups
administered
Group
Normal saline,
Group
Normal saline,
II
Group
Glibenclamide
III
10 mg/ml
Group
IV
Group
V
Group
VI
Dose
0 Day
6 Day
12 Day
18 Day
229.33 2.53
251.00 6.84
253.00 2.58
259.00 9.79
190.673.72a
175.122.14a
163.500.99a
129.001.59a
193.17 2.20
*
205.002.29**
209.683.98**
212.782.08**
186.50 3.53*
193.002.29**
201.166.13**
217.173.23**
190.83 1.30
*
198.004.22**
201.354.23**
208.673.46**
189.12 3.25*
198.216.12**
212.325.20**
215.126.12**
MEHAD
250 mg/kg,
b.w.
PEHAD
250 mg/kg,
b.w.
AEHAD
250 mg/kg,
b.w.
Dept. of Pharmacology
ABMRCP, Bangalore
62
Results
Graph 1: Effect of HAD extracts on Body weight in rats.
300
Normal control
Diabetic control
Standard
MEHAD
PEHAD
AEHAD
200
100
0
0 day
6 day
12 day
18 day
Time (day)
Dept. of Pharmacology
ABMRCP, Bangalore
63
Results
Dept. of Pharmacology
ABMRCP, Bangalore
64
Results
indicates that the glibenclamide treatment of diabetic rats is able to bring back the fasting
serum glucose levels nearer to normal range throughout the study period.
The MEHAD (250 mg/ kg) treated diabetic rats show mean ( SEM) fasting
serum glucose of 325.50 2.07 mg/dl on 0 hr which was found to have been reduced
318.35 4.93 mg/dl, 308.16 4.54 mg/dl and 273.16 8.65 mg/dl on 2 hr, 4 hr and 6 hr
respectively. These changes in fasting serum glucose values illustrate that the diabetic
rats treated with MEHAD (250 mg/ kg) show a progressive and significant (p 0.05)
including on the 6 hr, it shows highly significant (p 0.01) values in reduction of fasting
serum glucose during the single dose of treatment period in comparison to the diabetic
group of rats.
The PEHAD (250 mg/ kg) treated diabetic rats show mean ( SEM) fasting serum
glucose of 330.21 1.47 mg/dl on 0 hr which was reduced to 320.01 7.92 mg/dl on 2
hr, 310.25 7.50 mg/dl on 4 hr and 305.49 9.03 mg/dl on 6 hr. These changes in
fasting serum glucose illustrate that the diabetic rats treated with PEHAD (250 mg/ kg)
show a progressive and significant (p 0.05) reduction in fasting serum glucose during
the treatment period in comparison to diabetic group of rats.
The AEHAD (250 mg/ kg) treated diabetic rats show mean ( SEM) fasting
serum glucose of 326.25 2.75 mg/dl on 0 hr which was reduced to 318.29 3.41 mg/dl
on 2 hr, 307.83 5.38 mg/dl on 4 hr and 284.83 6.52 mg/dl on 6 hr. These changes in
fasting serum glucose illustrate that the diabetic rats treated with AEHAD (250 mg/ kg)
show a progressive and significant (p 0.05) including on the 6 hr, it shows highly
significant (p 0.01) in reduction of fasting serum glucose during the treatment period in
comparison to diabetic group of rats.
Dept. of Pharmacology
ABMRCP, Bangalore
65
Results
The above observations show that the treatment of diabetic rats with HAD
extracts (methanolic, petroleum ether and aqueous) reduces the fasting serum glucose of
diabetic rats at all the tested dose levels, but the 250 mg/kg, b.w. of MEHAD and
AEHAD (i.e. methanolic and aqueous extracts of Holarrhena antidysenterica) was able
to reduce fasting serum glucose which was comparable with the reduction caused by
glibenclamide treatment during the treatment period.
Dept. of Pharmacology
ABMRCP, Bangalore
66
Results
Table 4: Effect of HAD extract on fasting serum glucose levels of rats after single
dose treatment.
Groups
Dose (mg/
kg)
Group
Normal saline,
Group
Normal saline,
II
Group
Glibenclamide
III
10 mg/ml
Group
IV
Group
V
Group
VI
2 hr
4 hr
6 hr
107.66 3.77
108.50 4.34
107.33 3.77
108.33 4.21
320.505.37a
320.503.24a
321.334.88a
322.006.00a
344.0011.78 *
330.508.62*
317.0011.08*
267.665.45**
325.50 2.07*
318.354.93*
308.164.54*
273.168.65**
330.21 1.47 *
320.017.92*
310.257.50*
305.499.03*
326.25 2.75*
318.293.41*
307.835.38*
284.836.52**
MEHAD
250 mg/kg,
b.w.
PEHAD
250 mg/kg,
b.w.
AEHAD
250 mg/kg,
b.w.
Dept. of Pharmacology
ABMRCP, Bangalore
67
Results
Graph 2: Effect of HAD extracts on fasting serum glucose level of rats after single
dose treatment.
400
Normal control
Diabetic control
Standard
MEHAD
PEHAD
AEHAD
300
200
100
0
0hr
2hr
4hr
6hr
Time (hour)
Dept. of Pharmacology
ABMRCP, Bangalore
68
Results
Multiple dose treatment:
The fasting serum glucose of the different groups of animals during the multiple
dose treatment period of study is given in table No 5 and presented in graph 3, which
shows that the mean ( SEM) fasting serum glucose values of the normal group of rats
was 107.66 3.77 mg/dl, 107.66 3.19 mg/dl, 106.50 3.98 mg/dl and 108.50 4.32
mg/dl on day 0, day 6, day 12 and day 18 respectively. The above values show that the
fasting serum glucose in the normal group of rats was maintained within the normal range
throughout the period of study.
The mean fasting serum glucose ( SEM) in the diabetic control group of rats was
found to be 320.50 5.37 mg/dl, 321.33 5.51 mg/dl, 321.16 5.91 mg/dl and 320.33
4.88 mg/dl on day 0, day 6, day 12 and day 18 respectively, which was found to be
significantly (p 0.01) higher when compared with the normal rats. These elevated
fasting serum glucose levels were found to have been maintained throughout the 18 days
of treatment period indicating that the rats are rendered diabetic.
The glibenclamide (10 mg/ kg) treated diabetic rats show a mean ( SEM) fasting
serum glucose of 344.00 11.78 mg/dl on day 0 which was reduced to 327.16 8.86
mg/dl on day 6 which reduced further to 258.33 12.81 mg/dl and 136.33 3.26 mg/dl
on day 12 and day 18 respectively. These changes in fasting serum glucose values
illustrate that the diabetic rats treated with glibenclamide show a progressive and
significant (p 0.05) including on the day 12 and day 18, they show highly significant (p
0.01) in reduction of fasting serum glucose, during the treatment period in comparison
to the diabetic group of rats. This indicates that the glibenclamide treatment of diabetic
Dept. of Pharmacology
ABMRCP, Bangalore
69
Results
rats is able to bring back the fasting serum glucose levels nearer to normal range
throughout the study period.
The MEHAD (250 mg/ kg) treated diabetic rats show mean ( SEM) fasting
serum glucose of 325.50 2.07 mg/dl on day 0 which was found to have been reduced
311.33 3.33 mg/dl, 269.83 7.21 mg/dl and 162.16 3.30 mg/dl on day 6, day 12 and
day 18 respectively. These changes in fasting serum glucose values illustrate that the
diabetic rats treated with MEHAD (250 mg/ kg) show a progressive and significant (p
0.05) including on the day 12 and day 18, they show highly significant (p 0.01) values
in reduction of fasting serum glucose during the single dose of treatment period in
comparison to the diabetic group of rats.
The PEHAD (250 mg/ kg) treated diabetic rats show mean ( SEM) fasting serum
glucose of 330.21 1.47 mg/dl on day 0 which was reduced to 317.98 2.27 mg/dl on
day 6, 295.83 2.48 mg/dl on day 12 and 169.98 7.25 mg/dl on day 18. These changes
in fasting serum glucose illustrate that the diabetic rats treated with PEHAD (250 mg/ kg)
show a progressive and significant (p 0.05) including on the day 12 and day 18, they
show highly significant (p 0.01) values in reduction of fasting serum glucose during the
treatment period in comparison to diabetic group of rats.
The AEHAD (250 mg/ kg) treated diabetic rats show mean ( SEM) fasting
serum glucose of 326.25 2.75 mg/dl on day 0 which was reduced to 308.36 5.59
mg/dl on day 6, 254.33 9.16mg/dl on day 12 and 160.16 2.65 mg/dl on day 18. These
changes in fasting serum glucose illustrate that the diabetic rats treated with AEHAD
(250 mg/ kg) show a progressive and significant (p 0.05) including on the day 12 and
Dept. of Pharmacology
ABMRCP, Bangalore
70
Results
day 18, they show highly significant (p 0.01) in reduction of fasting serum glucose
during the treatment period in comparison to diabetic group of rats.
The above observations show that the treatment of diabetic rats with HAD
extracts (methanolic, petroleum ether and aqueous) reduces the fasting serum glucose of
diabetic rats at all the tested dose levels, but the 250 mg/kg, b.w. of MEHAD and
AEHAD (i.e. methanolic and aqueous extracts of Holarrhena antidysenterica) was able
to reduce fasting serum glucose which was comparable with the reduction caused by
glibenclamide treatment during the treatment period.
Dept. of Pharmacology
ABMRCP, Bangalore
71
Results
Table 5: Effect of HAD extract on fasting serum glucose levels of rats after multiple
dose treatment.
Groups
Group
I
Group
II
Group
III
Group
IV
Group
V
Group
VI
Dose
(mg/ kg)
Normal
saline,
10 ml/ kg,
p.o.
Normal
saline,
10 ml/ kg,
p.o.
Glibenclami
de
6 Day
12 Day
18 Day
107.66 3.77
107.66 3.19
106.50 3.98
108.50 4.32
320.505.37a
321.335.51a
321.165.91a
320.334.88a
344.0011.78 *
327.168.86*
258.3312.81**
136.333.26**
325.50 2.07*
311.333.33*
269.837.21**
162.163.30**
330.211.47 *
317.982.27*
295.832.48**
169.987.25**
326.25 2.75*
308.365.59*
254.339.16**
160.162.65**
10 mg/ml
MEHAD
250 mg/kg,
b.w.
PEHAD
250 mg/kg,
b.w.
AEHAD
250 mg/kg,
b.w.
Dept. of Pharmacology
ABMRCP, Bangalore
72
Results
Graph 3: Effect of HAD extracts on fasting serum glucose level of rats after multiple
dose treatment.
400
Normal control
Diabetic control
Standard
MEHAD
PEHAD
AEHAD
300
200
100
0
0 day
6th day
Time (day)
Dept. of Pharmacology
ABMRCP, Bangalore
73
Results
Dept. of Pharmacology
ABMRCP, Bangalore
74
Results
0.01) reduced as against the serum cholesterol of diabetic rats during the entire study
period.
The PEHAD (250 mg/ kg) treated diabetic rats show mean ( SEM) serum
cholesterol of 68.92 1.54 mg/dl on day 18. This change in serum cholesterol values
illustrate that the diabetic rats treated with the 250 mg/kg of PEHAD show a significant
(p 0.01) reduction in serum cholesterol after the 18 days of treatment period in
comparison to the diabetic group of rats.
The AEHAD (250 mg/ kg) treated diabetic rats show mean ( SEM) serum
cholesterol of 63.74 1.15 mg/dl on day 18. This change in serum cholesterol values
illustrate that the diabetic rats treated with the250 mg/kg of AEHAD show a significant
(p 0.01) reduction in serum cholesterol after the 18 days of treatment period in
comparison to the diabetic group of rats.
Among these groups i.e. MEHAD, PEHAD, AEHAD (250 mg/kg) the serum
cholesterol was not only reduced significantly when compared with diabetic rats but the
values were also comparable with those of normal rats on day 18 and glibenclamide
treated diabetic rats. The above observations indicates that the treatment of diabetic rats
with the MEHAD, PEHAD, AEHAD (mithanolic, petroleum ether and aqueous extract of
Holarrhena antidysenterica) reduces the serum cholesterol of diabetic rats at all the
tested dose, was able to reduce the serum cholesterol significantly compared to diabetic
rat after the 18 days of treatment.
Dept. of Pharmacology
ABMRCP, Bangalore
75
Results
Table 6: Effect of HAD extracts on fasting serum cholesterol level of rats after 18
days treatment.
Serum Cholesterol in mg/dl (Mean S.E.M)
Groups
Dose(mg/ kg)
18 day
Group-I
Group-II
Group-III
Group-IV
Group-V
Group-VI
Normal saline,
51.96 1.77
84.25 1.44a
50.80 0.22**
10 mg/ml
MEHAD
55.80 3.12**
68.92 1.54**
63.74 1.15**
Dept. of Pharmacology
ABMRCP, Bangalore
76
Results
Graph 4: Effect of HAD extracts on fasting serum cholesterol level of rats after 18
90
80
70
60
50
40
30
20
10
0
EH
A
D
A
PE
H
A
D
EH
A
D
rd
da
St
an
D
ia
be
tic
or
m
al
N
co
nt
ro
l
Normal control
Diabetic control
Standard
MEHAD
PEHAD
AEHAD
co
nt
ro
l
days treatment.
Treatment group
Dept. of Pharmacology
ABMRCP, Bangalore
77
Results
5.4.2 Triglyceride:
The serum triglyceride of different groups of animals during the period of study is
given in table No. 7 presented in graph 5, which shows that the mean ( SEM) serum
triglyceride of the normal animals group of rats was 139.30 1.36 mg/dl on day 18. The
above values show that the serum triglyceride in normal group of rats was maintained
throughout the period of study.
The mean serum triglyceride ( SEM) in diabetic control group of rats was found
to be 187.27 3.13 mg/dl on day 18, which was found to be significantly (p 0.01)
higher when compared with the normal rats. These elevated serum triglyceride levels
were found to be increasing throughout the 18 days of study period.
The glibenclamide (10 mg/ kg) treated diabetic rats show mean ( SEM) serum
triglyceride of 141.77 1.88 mg/dl on day 18. The reduction in serum triglyceride was
found to be significantly (p 0.01) compared with the diabetic rats on day 18. This
indicates that the glibenclamide treatment of diabetic rats is able to bring back the serum
triglyceride levels nearer to normal range in about 18 days of treatment.
The MEHAD (250 mg/ kg) treated diabetic rats show mean ( SEM) serum
triglyceride of 132.92 7.22 mg/dl on day 18, which was found to be significantly (p
0.01) reduced as against the serum triglyceride of diabetic animals during the study
period. This indicates that the MEHAD treatment of diabetic rats is able to bring back the
serum triglyceride levels lower to normal range in the 18 days of treatment.
The PEHAD (250 mg/ kg) treated diabetic rats show mean serum ( SEM)
triglyceride of 153.80 1.36 mg/dl on day 18. These changes in serum triglyceride
values illustrate that the diabetic rats treated with 250 mg/kg of PEHAD show a
Dept. of Pharmacology
ABMRCP, Bangalore
78
Results
significant (p 0.01) reduction in serum triglyceride in 18 days of treatment period in
comparison to diabetic group of rats.
The AEHAD (250 mg/ kg) treated diabetic rats show mean ( SEM) serum
triglyceride of 129.00 3.02 mg/dl on day 18. These changes in serum triglyceride
values illustrate that the diabetic rats treated with 250 mg/kg of AEHAD show a
significant (p 0.01) reduction in serum triglyceride in 18 days of treatment period in
comparison to diabetic group of rats.
In both these groups i.e. MEHAD and AEHAD (250 mg/kg) the serum
triglyceride was not only reduced significantly (p 0.01) when compared with diabetic
rats but the values were also comparable with those of normal rats infect lesser than
normal rats on day 18 and glibenclamide treated diabetic rats.
These above observations indicates that the treatment of diabetic rats with the
MEHAD, PEHAD, AEHAD (mithanolic, petroleum ether and aqueous extracts of
Holarrhena antidysenterica) reduces the serum triglyceride of diabetic rats at all the
tested dose levels, but the MEHAD and AEHAD (250 mg/kg) was able to bring the
serum triglyceride nearer to serum triglyceride values of normal rats after the 18 days of
treatment.
Dept. of Pharmacology
ABMRCP, Bangalore
79
Results
Table 7: Effect of HAD extracts on fasting serum triglyceride level of rats after 18
days treatment.
Serum Cholesterol in mg/dl (Mean S.E.M)
Groups
Dose(mg/ kg)
18 day
Group-I
Group-II
Group-III
Group-IV
Group-V
Group-VI
Normal saline,
139.30 1.36
187.27 3.13a
141.77 1.88**
10 mg/ml
MEHAD
132.90 7.22**
153.80 1.36**
129.00 3.02**
Dept. of Pharmacology
ABMRCP, Bangalore
80
Results
Graph 5: Effect of HAD extracts on fasting serum triglyceride level of rats after 18
days treatment.
200
Normal control
Diabetic control
Standard
MEHAD
PEHAD
AEHAD
100
EH
A
PE
D
A
EH
M
da
rd
St
an
tr
ol
tic
co
n
ia
be
D
or
m
al
co
n
tr
ol
Treatment group
Dept. of Pharmacology
ABMRCP, Bangalore
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Results
Dept. of Pharmacology
ABMRCP, Bangalore
82
Results
values illustrate that the diabetic rats treated with the 250 mg/kg of PEHAD show a
significant (p 0.01) reduction in serum total protein levels after the 18 days of treatment
period in comparison to the diabetic group of rats.
The AEHAD (250 mg/ kg) treated diabetic rats show mean ( SEM) serum total
protein levels of 6.09 0.25 mg/dl on day 18. This change in serum total protein levels
values illustrate that the diabetic rats treated with the 250 mg/kg of AEHAD show a
significant (p 0.01) reduction in serum total protein levels after the 18 days of treatment
period in comparison to the diabetic group of rats.
Among these groups i.e. MEHAD, PEHAD, AEHAD (250 mg/kg) the serum total
protein levels was not only reduced significantly when compared with diabetic rats but
the values were also comparable with those of normal rats on day 18 and glibenclamide
treated diabetic rats. The above observations indicates that the treatment of diabetic rats
with the MEHAD, PEHAD, AEHAD (mithanolic, petroleum ether and aqueous extract of
Holarrhena antidysenterica) reduces the serum total protein levels of diabetic rats at all
the tested dose, was able to reduce the serum total protein levels significantly compared
to diabetic rat after the 18 days of treatment.
Dept. of Pharmacology
ABMRCP, Bangalore
83
Results
Table 8: Effect of HAD extracts on fasting serum total protein level of rats after 18
days treatment.
Serum total protein level in g/dl (Mean S.E.M)
Groups
Dose(mg/ kg)
18 day
Group-I
Group-II
Group-III
Group-IV
Group-V
Group-VI
Normal saline,
5.86 0.19
7.41 0.42a
5.91 0.20**
10 mg/ml
MEHAD
6.18 0.13**
6.20 0.26**
6.09 0.25**
Dept. of Pharmacology
ABMRCP, Bangalore
84
Results
Graph 6: Effect of HAD extracts on fasting serum total protein level of rats after 18
days treatment.
Normal control
Diabetic control
Standard
MEHAD
PEHAD
AEHAD
7
6
5
4
3
2
1
A
D
A
EH
PE
H
A
D
A
D
M
EH
ar
d
St
an
d
ol
nt
r
co
ia
be
tic
D
N
or
m
al
co
nt
ro
l
Treatment group
Dept. of Pharmacology
ABMRCP, Bangalore
85
Results
Dept. of Pharmacology
ABMRCP, Bangalore
86
Results
reduction in blood urea levels after the 18 days of treatment period in comparison to the
diabetic group of rats.
The AEHAD (250 mg/ kg) treated diabetic rats show means ( SEM) blood urea
levels of 54.79 0.88 mg/dl on day 18. This change in blood urea levels values illustrate
that the diabetic rats treated with the 250 mg/kg of AEHAD show a significant (p 0.01)
reduction in blood urea levels after the 18 days of treatment period in comparison to the
diabetic group of rats.
Among these groups i.e. MEHAD, PEHAD, AEHAD (250 mg/kg) the blood urea
levels was not only reduced significantly when compared with diabetic rats but the values
were also comparable with those of normal rats on day 18 and glibenclamide treated
diabetic rats. The above observations indicates that the treatment of diabetic rats with the
MEHAD, PEHAD, AEHAD (mithanolic, petroleum ether and aqueous extract of
Holarrhena antidysenterica) reduces the blood urea levels of diabetic rats at all the tested
dose, was able to reduce the blood urea levels significantly compared to diabetic rat after
the 18 days of treatment.
Dept. of Pharmacology
ABMRCP, Bangalore
87
Results
Table 9: Effect of HAD extracts on fasting blood urea levels of rats after 18 days
treatment.
Serum blood urea level in mg/dl (Mean S.E.M)
Groups
Dose(mg/ kg)
18 day
Group-I
Group-II
Group-III
Group-IV
Group-V
Group-VI
Normal saline,
41.83 0.76
73.70 1.37a
42.33 0.40**
10 mg/ml
MEHAD
50.20 3.06**
50.01 1.84**
54.79 0.88**
Dept. of Pharmacology
ABMRCP, Bangalore
88
Results
Graph 7: Effect of HAD extracts on fasting blood urea levels of rats after 18 days
treatment.
80
Normal control
Diabetic control
Standard
MEHAD
PEHAD
AEHAD
70
60
50
40
30
20
10
D
EH
A
A
PE
H
A
D
M
EH
A
d
da
r
St
an
tic
ia
be
D
or
m
al
c
co
on
tr
o
nt
ro
Treatment group
Dept. of Pharmacology
ABMRCP, Bangalore
89
Results
Dept. of Pharmacology
ABMRCP, Bangalore
90
Results
glycogen levels values illustrate that the diabetic rats treated with the 250 mg/kg of
PEHAD show a significant (p 0.01) increased in liver glycogen levels after the 18 days
of treatment period in comparison to the diabetic group of rats.
The AEHAD (250 mg/ kg) treated diabetic rats show mean ( SEM) liver
glycogen levels of 45.57 2.70 mg/g of tissue taken on day 18. This change in liver
glycogen levels values illustrate that the diabetic rats treated with the250 mg/kg of
AEHAD show a significant (p 0.01) increased in liver glycogen levels after the 18 days
of treatment period in comparison to the diabetic group of rats.
Among these groups i.e. MEHAD, PEHAD, AEHAD (250 mg/kg) the liver
glycogen levels was not only increased significantly when compared with diabetic rats
but the values were also comparable with those of normal rats on day 18 and
glibenclamide treated diabetic rats. The above observations indicates that the treatment of
diabetic rats with the MEHAD, PEHAD, AEHAD (mithanolic, petroleum ether and
aqueous extract of Holarrhena antidysenterica) increased the liver glycogen levels of
diabetic rats at all the tested dose, was able to increased the liver glycogen level
significantly compared to diabetic rat after the 18 days of treatment.
Dept. of Pharmacology
ABMRCP, Bangalore
91
Results
Table 10: Effect of HAD extracts on liver glycogen level of rats after 18 days
treatment.
Groups
Dose(mg/ kg)
Group-I
Group-II
Group-III
Group-IV
Group-V
Group-VI
Normal saline,
35.87 1.10
23.31 0.85a
36.11 0.98**
10 mg/ml
MEHAD
44.07 1.13**
53.63 0.85**
45.57 2.70**
Dept. of Pharmacology
ABMRCP, Bangalore
92
Results
Graph 8: Effect of HAD extracts on liver glycogen level of rats after 18 days
60
Normal c ontrol
Diabetic c ontrol
Standard
MEHAD
PEHAD
AEHAD
50
40
30
20
10
A
D
A
EH
A
D
PE
A
D
M
EH
ar
d
St
an
d
on
tr
ol
tic
c
D
ia
be
al
co
nt
r
ol
0
or
m
treatment.
Treatment group
Dept. of Pharmacology
ABMRCP, Bangalore
93
DISCUSSION
Discussion
6. DISCUSSION
Streptozotocin, a broad spectrum antibiotic has been widely used for inducing the
diabetes mellitus in a variety of animals by affecting degeneration and necrosis of
pancreatic -cells19, 20, 21, 23, 24.
The Holarrhena antidysenterica fresh bark and seed extracts has been reported to
have antibacterial and antidiarrhoeal activity8, 9, but there are no scientific data is
available regarding the effect on the blood glucose levels. So we have made an attempt to
use methanolic, petroleum ether, aqueous extracts of Holarrhena antidysenterica (seed)
for studying anti-diabetic activity.
In the present study, the groups of normal male rats have shown gain in the body
weight while fasting serum glucose was maintained in the normal range throughout the
study period. The serum cholesterol, serum triglyceride, total protein and blood urea
levels of the normal rats were found to be maintained within the normal range during the
18 days of study period and the liver glycogen content was also found to be maintained
within the normal range throughout the study period.
In this study the group of diabetic rats showed progressive and significant (p
0.05) loss in body weight throughout the study period as compared to the body weight
gain of normal group of rats. This characteristic loss of body weight, which is due to
increased muscle wasting and due to loss of tissue proteins39. Moreover the fasting
serum glucose of diabetic rats was significantly (p 0.05) elevated as compared to the
normal fasting serum glucose levels of diabetic rats. This elevated level of fasting serum
glucose was fairly maintained throughout the study period. Similar observations were
reported on the serum glucose level and body weight32,
Dept. of Pharmacology
39, 46
95
Discussion
serum triglyceride, total protein and blood urea of diabetic group of rats was found to be
significantly (p 0.05) increasing throughout the study period as against the serum
cholesterol, serum triglyceride, total protein and blood urea of normal group of rats,
which has also been reported in previous research work
. This might
have occured in the diabetic rats as a result of lack of insulin which activates the lipase
enzymes, hydrolyzing the stored triglycerides and releasing large amount of fatty acids
and glycerol into the circulating blood. Consequently, the excess of fatty acids in the
plasma may promote the hepatic conversation of fatty acids into phospholipids and
cholesterol, the main products of lipid metabolism
55
56, 57
levels of the diabetic group of rats were found to be reduced significantly (p 0.05) as
against the normal liver glycogen levels of the normal group of rats, as reported in the
previous study39, 40, 41, 42. These observations suggest that single i.v. injection of STZ (35
mg/ kg) 30 produced a reproducible and consistent diabetes mellitus and appears to be a
suitable model of diabetes in our laboratory conditions.
The glibenclamide (10 mg/kg) treated group of rats showed significant (p 0.05)
protection from the body weight loss seen in the untreated diabetic group of rats along
with significant (p 0.05) and progressive reduction in fasting serum glucose levels. The
glibenclamide treatment also reduced the elevated serum cholesterol levels and produced
significant (p 0.01) reduction in elevated serum triglyceride, total protein, blood urea,
urine glucose allowed significant (p 0.01) recovery of reduced liver glycogen content
during the period of study when compared with the diabetic group of rats. In agreement
with the present results, several studies have shown protection in body weight loss, anti-
Dept. of Pharmacology
ABMRCP, Bangalore
96
Discussion
diabetic activity43, 44, 10, 46, reduction in serum cholesterol, serum triglyceride, total protein
and blood urea
47, 39
treatment.
The all three extracts (methanolic, petroleum ether and aqueous) of Holarrhena
antidysenterica (seed) treated group of rats have significantly (p 0.01) protected the
diabetic rats from loosing the body weight at all the selected dose levels when compared
with diabetic group of rats, however this treatment has shown complete recovery of body
weight on 6th, 12th and 18th day respectively. The MEHAD, PEHAD and AEHAD treated
groups of rats have also shown the progressive and significant (p 0.01; except on 6th
day) reduction in fasting serum glucose of diabetic rats when compared with untreated
diabetic rats at all the selected dose levels but the PEHAD treated group at the doses of
250 mg/kg have not shown more consistent results throughout the study period. This
reduction in the fasting serum glucose may be due to inhibition of intestinal absorption of
glucose49,
50
endogenous insulin52 as reported by the previous research workers. The entire three
extracts treated group at dose of 250 mg/kg showed significant (p 0.01) reduction in the
elevated serum cholesterol and serum triglyceride levels of diabetic rats when compared
with untreated diabetic rats. Similar observation was made by the previous research
workers in normal rats, and the probable mechanism of reduction in serum cholesterol
and serum triglyceride levels has been suggested to be due to, increased excretion of
neutral sterol and acidic steroids in feces53, 54. These extracts were able to increase the
liver glycogen content significantly (p 0.01) as compared with the untreated diabetic
Dept. of Pharmacology
ABMRCP, Bangalore
97
Discussion
rats. This improvement in liver glycogen levels may be due to the improvement of
glycogenesis.
The above observations indicate that the entire extracts of HAD seed treatment
can be used in the management of the diabetes mellitus and further studies are needed to
elucidate the proper mechanism of action.
Dept. of Pharmacology
ABMRCP, Bangalore
98
CONCLUSION
Conclusion
7. CONCLUSION
Current study gives evidence that treatment with methanolic, petroleum ether and
aqueous extract of Holarrhena antidysenterica (seed) has favourable effect not only on
blood glucose levels, liver glycogen but also on serum lipids and body weight. This point
out the promising effect of Holarrhena antidysenterica seed being a useful antidiabetic
agent and also in diabetic complications. Further studies are necessary to ascertain the use
of Holarrhena antidysenterica seed in diabetic complications.
Dept. of Pharmacology
ABMRCP, Bangalore
100
SUMMARY
Summary
8. SUMMARY
STZ-induced experimental diabetes is a valuable model for inducing the diabetes
mellitus in a variety of animals by affecting degeneration and necrosis of pancreatic cells.
The Holarrhena antidysenterica fresh bark and seed extracts has been reported to
have antibacterial and antidiarrhoeal activity, but there are no scientific data is available
regarding the effect on the blood glucose levels. So we have made an attempt to use
methanolic, petroleum ether, aqueous extracts of Holarrhena antidysenterica (seed) for
studying anti-diabetic activity.
Diabetes was induced by the single i.v. injection of STZ (35 mg/kg dissolved in
the 0.1 M citrate buffer of pH 4.5) which was confirmed by estimation of fasting serum
glucose levels at 72 h after STZ injection. The diabetic rats with the fasting serum
glucose above 200 mg/dl were included in the study and randomly divided into six
groups of male rats along with the normal control group. The diabetic rats were treated
for 18 days and in addition to the fasting serum glucose, the changes in body weight,
serum cholesterol, serum triglyceride, total protein, blood urea, urine glucose and liver
glycogen levels were also evaluated in the study period.
The STZ administration caused the loss of body weight, elevation in fasting
serum glucose, serum cholesterol, serum triglyceride, total protein, blood urea, urine
glucose and reduction in liver glycogen levels in the study period. The glibenclamide at
the dose of 10 mg/kg and the MEHAD and AEHAD at the dose of 250mg/kg showed
protection against body weight loss, reduced the elevated fasting serum glucose, serum
Dept. of Pharmacology
ABMRCP, Bangalore
101
Summary
cholesterol, serum triglyceride, total protein, blood urea, urine glucose and have allowed
recovery from the loss of liver glycogen content of the diabetic rats in a significant
manner during the study period. The effect of MEHAD and AEHAD at the above
mentioned doses was also comparable with that of the standard drug i.e. glibenclamide.
Based on the above results it can be stated that methanolic and aqueous extracts of
Holarrhena antidysenterica (seed) have shown good anti-diabetic activity at the doses of
250 mg/kg throughout the study period, however further studies are needed to elucidate
the mechanism of action and to know the active principles involved in producing the
effect.
Dept. of Pharmacology
ABMRCP, Bangalore
102
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