1

EFFECT OF A COMPOUND HERBAL PREPARATION (ZIABEEN)

ON BLOOD GLUCOSE AND LIPID PROFILES IN NORMAL
AND ALLOXAN DIABETIC RABBITS

BY
MAMOONA ZAFAR
ROLL NO: PPRS09E001

THESIS SUBMITTED TO
UNIVERSTY OF SARGODHA, SARGODHA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE

OF
M.PHIL (PHARMACOLOGY)
SESSION 2009-2011




FACULTY OF PHARMACY,
UNIVERSTIY OF SARGODHA, SARGODHA
PAKISTAN
2

APPROVAL CERTIFICATE

It is certified that this thesis entitled Effect of a compound herbal preparation
(Ziabeen) on blood glucose and lipid profiles in normal and alloxan diabetic rabbits
has been prepared by Mamoona Zafar under my supervision. Furthermore, this thesis
has not been previously presented for a higher degree. She has fulfilled all the
requirements and qualifies to submit this thesis for the degree of M.PHIL
(Pharmacology).






Prof. Dr. Muhammad Shoaib Akhtar
Faculty of Pharmacy,
University of Sargodha,
Sargodha.








3

DECLARATION

This is to certify that this thesis submitted by Mamoona Zafar is accepted in its
present form by the Faculty of Pharmacy University of Sargodha, Sargodha-Pakistan
as satisfying the partial requirement for the degree, MASTER OF PHILOSOPHY in
PHARMACOLOGY.


Internal Examiner Prof. Dr. Muhammad Shoaib Akhtar
Faculty of Pharmacy,
University of Sargodha,
Sargodha.

External Examiner Dr. Muhammad Ovais Umer
Department of Pharmacology
University of Veterinary Animals
Sciences, Lahore.


Chairman Dr. Sajid Bashir
Faculty of Pharmacy,
University of Sargodha,
Sargodha.


Student Mamoona Zafar






4

CONTENTS

Page no

Acknowledgements 9
Abbreviations Used 11
Summary 13
List of tables 16
List of figures 17
SECTION 1
1. INTRODUCTION 20
1.1 Diabetes mellitus 20
1.1.1 Prevalence of diabetes mellitus 20
1.1.2 Symptoms and signs of diabetes mellitus 22
1.1.3 Classification of diabetes mellitus 22
1.1.3.1 Type 1 or insulin dependent diabetes mellitus 22
1.1.3.2 Type 2 or non-insulin dependent diabetes mellitus 23
1.1.3.3 Gestational diabetes mellitus 23
1.1.3.4 Other specific types of diabetes mellitus 24
1.1.4 Diagnosis of diabetes mellitus 24
1.1.4.1 Blood glucose testing 25
1.1.4.2 Urine analysis 26
1.1.5 Complications of diabetes mellitus 27
1.1.5.1 Acute complications of diabetes mellitus 27
1.1.5.2 Chronic complications of diabetes mellitus 28
1.1.5.2.1 Microvascular complications 28
5

1.1.5.2.2 Macrovascular complications 30
1.1.6 Treatment of diabetes mellitus 31
1.1.6.1 Treatment of type 1 diabetes mellitus 31
1.1.6.2 Treatment of type 2 diabetes mellitus 32
1.1.6.3 Insulin treatment 32
1.1.6.4 Oral hypoglycaemic agents 35
1.1.6.5 Pharmacological interventions 39
1.1.6.6 Medical nutrition therapy 39
1.1.6.7 Goals of therapy 39
1.1.7 Antidiabetogenic agent (Alloxan-monohydrate) 40
1.1.8 Herbal anti-diabetic agents 41
1.1.8.1 Historic development of herbal medicine 42
1.1.8.2 Reported plants possessing anti-diabetic activity 43
1.1.9 Aims and objectives of present study 54
SECTION 2
2. MATERIALS AND METHODS 55
2.1 Ziabeen

(A compound herbal preparation)

56
2.2 Equipments used 56
2.3 Chemicals and diagnostic kits 57
2.4 Experimental animals used 57
2.5 Induction of experimental diabetes in rabbits 57
2.6 Preparation and administration of drug suspensions 58
2.7 Collection of blood sample 58
2.8 Biochemical analysis 58
6

2.9 Experimental design 59
2.9.1 Acute toxicity and behavioral pattern studies 59
2.9.2 Hypoglycaemic activity of Ziabeen in normal rabbits 59
2.9.3 Hypoglycaemic activity of Ziabeen in alloxan-diabetic rabbits 60
2.9.4 Oral glucose tolerance test (OGTT) 60
2.9.5 Hypoglycaemic effects of Ziabeen

with and without insulin administration 60
2.9.6 Effect of Ziabeen on fasting blood glucose level after 30 days of 61
administration
2.9.7 Effect of Ziabeen on body weights of rabbits after 30 days of administration 61
2.9.8 Effect of Ziabeen on lipid profile in alloxan-diabetic rabbits 62
2.9.9 Statistical analysis 62

SECTION 3
3. RESULTS AND DISCUSSION 63
3.1 RESULTS 63
3.1.1 Effect of alloxan administration to rabbits 64
3.1.2 Effect of Ziabeen

on acute toxicity and behavioural pattern studies 64
3.1.3 Effect of Ziabeen

on blood glucose in normal rabbits 64
3.1.4 Effect of Ziabeen

on blood glucose in alloxan-diabetic rabbits 65
3.1.5 Effect of Ziabeen with and without different units of exogeneous 70
human insulin in alloxan-diabetic rabbits
3.1.6 Oral glucose tolerance test (OGTT) in alloxan-diabetic rabbits 70
7

3.1.7 Hypoglycaemic effects of Ziabeen

in alloxan-induced diabetic rabbits 75
after 30 days of administration.
3.1.8 Antihyperlipidaemic effects of Ziabeen in alloxan-induced diabetic rabbits 75
3.1.9 Effect of Ziabeen

on body weight in alloxan-diabetic rabbits 75

3.2 DISCUSSION 82
3.2.1 Conclusions 89
SECTION 4
REFERENCES 90















8



DEDICATION


This thesis is dedicated to my parents
For their endless love , support and encouragement

MAY Allah
keep me always under the shadows of their
love and prayers (Aameen).








9


ACKNOWLEDGEMENTS
First and foremost, Countless praise to ALMIGHTY ALLAH TAALA, The most
merciful the most beneficial. Benevolent for his blessings a nd guidance for giving me
aspiration to make out my work. The only creator of universe who grant me a
knowledge, power and courage to complete this research work successfully, in spite of
various difficulties. May ALLAH always help me in my life. My utmost reverence to
last Prophet Muhammad (S.A.W.), The greatest social reformer and guide in every
sphere of life.
I have the honor to eloquent my sincerest gratitude to very affectionate supervisor, Prof.
Dr. Muhammad Shoaib Akhtar, Department of Pharmacology, Faculty of Pharmacy,
whose constant encouragement, sympathetic attitude and valuable guidance made me
able to conduct this study. I offer special thanks to Dr. Sajid Bashir Chairman Faculty
of Pharmacy, a very kind and helping personality available to help everyone at any
time. The realization of this work was only possible due to the several peoples
collaboration, to which I desire to express my gratefulness. So i would also acknowledge
my debt to Mr. Kamran Gulzar and Mr. Qaisar Mehmood for the knowledge they
provided and for their courteous and compassionate treatment given to me.
My sincere appreciations also go to Mr. Manzoor Qadir (May his soul rest in paradise,
Ameen), Mr. Shafeeq-ur-Rehman, Mr. Naeem Qaiser (Lecturer), Mr. Muhammad
Imran, Mr. Naveed and Mr. Rasheed for their kind cooperation. Moreover, I would
10

also acknowledge my debt to Miss Alia irum, Miss Umme Rukkyya, Miss Shazia
akram (Lecturers) and Miss Rehana for the helpful hand they extended towards me.
Sincere thanks to our department staff Mr. Nasir, Mr. Ashfaq and Mr. Bashir.
I have no word for my loving parents for their encouragement, constant support and
sacrifices throughout the study. I would like to express my gratitude to my dearest
brother Naveed Zafar sisters Ammara Zafar, Farah and Iqra for their invaluable
support, favor, encouragement and for putting colors in my life. I am also thankful to my
cousin Bilal Mushtaq for his favor.
Last but not least, I wish to express my sincere thanks to my all loving friends who have
one way or another helped me and remember me in their prayers in making this study
a success.

Mamoona Zafar













11

ABBREVIATIONS USED
CVD Cardiovascular disease
CHD Coronary heart disease
DM Diabetes mellitus
DKA Diabetic ketoacidosis
DN Diabetic nephropathy
DPP-IV Dipeptidyl peptidase-IV
ECM Extracellular matrix
ESRD End-stage renal disease
FBG Fasting blood glucose
GDM Gestational diabetes mellitus
GLUT2 Glucose transporter 2
GLP-1 Glucagon-like peptide-1
GIP Gastric inhibitory peptide
HNC Hyperosmolar non-ketotic coma
HDL High density lipoprotein
IAPP Islet amyloid polypeptide
IGT Impaired glucose tolerance
IDDM Insulin dependent diabetes mellitus
LA Lactic acidosis
LDL Low density lipoprotein
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MODY Maturity onset diabetes of the young
NIDDM Non insulin dependent diabetes mellitus
OGTT Oral Glucose tolerance test
PAD Peripheral arterial disease
PPAR Peroxisome proliferator-activated receptor gamma
STZ Streptozotocin
TC Total Cholesterol
TG Triglyceride
TZD Thiazolidinediones
VLDL Very low density lipoprotein
WHO World Health Organization









13

Summary

Many indigenous medicinal plants have been found to contain important active
principles. These biological principles make these plants helpful to treat many diseases.
The present study was conducted to determine hypoglycaemic, antihyperlipidaemic and
weight stabilizing effects of a compound herbal preparation named Ziabeen in normal
and alloxan-diabetic rabbits. Diabetes was induced by alloxan-monohydrate (150 mg/kg
body weight I/V). The compound herbal preparation Ziabeen has been claimed
empirically for the treatment of diabetes mellitus in folk loric medicine. The current
study was designed to confirm the antidiabetic effect of this polyherbal drug Ziabeen.
For this purpose blood glucose levels of animals were determined after oral
administration of 2, 3, and 4g/kg of Ziabeen

and 1mg/kg dose of pioglitazone which was
used as a standard hypoglycaemic agent to compare the results. Glucose levels were
measured at 0-, 2-, 4-, 6- and 8-hr intervals after administering the drug.
The results obtained showed that in normal rabbits, 2g/kg dose of Ziabeen

produced
significant (P<0.05 or P<0.001) decrease in blood glucose levels at 4, 6 and 8-hr
intervals. Its 3g/kg dose has even produced highly significant (P<0.001) decrease at 4-,
6- and 8-hr intervals while 4g/kg of Ziabeen

significantly (P<0.05) decrease the blood
glucose levels at 2-hr and highly significant (P<0.001) decrease was observed at 4, 6 and
8-hr intervals. Similarly, 1mg/kg dose of the standard hypoglycaemic agent, pioglitazone
reduced the blood glucose levels (P<0.05 or P<0.001) in normal rabbits. In alloxan-
diabetic rabbits 2g/kg of Ziabeen produced significant (P<0.05) decrease at 4 and 6-hr
intervals, at 8-hr highly significant (P<0.001) decrease but non-significant (P>0.05)
14

effect occur at 2-hr. The administration of 3g/kg of Ziabeen produced significant (P<0.05
or P<0.001) decrease at 2, 4, 6 and 8-hr intervals. However, highly significant (P<0.001)
reduction in blood glucose levels was observed at all time intervals after oral
administration of 4g/kg of Ziabeen. Pioglitazone, however, did not show any effect at 2-
hr while significant (P<0.05) decrease was also observed in alloxan-diabetic rabbits. At
4g/kg oral dose of Ziabeen

significantly (P<0.001) decreased blood glucose levels at all
time intervals.
Synergistic hypoglycaemic effect of oral administration of 4g/kg Ziabeen with different
doses of insulin injection was observed in alloxan-diabetic rabbits. It was observed that
simultaneous administration of Ziabeen

and exogenous human insulin (2 and 3unit/kg)
has further reduced the blood glucose levels of treated alloxan-diabetic rabbits. Further
more, oral glucose tolerance test (OGTT) was performed and data obtained showed that
4g/kg of Ziabeen

significantly reduced the blood glucose levels observed for 5-hr. The
continuous administration of Ziabeen was conducted for 30-days and it was observed that
4g/kg of Ziabeen

has substantially reduced blood glucose level in alloxan-diabetic
rabbits.
Finally, administration of Ziabeen significantly (P<0.001) reduced total cholesterol,
triglycerides, LDLs and VLDL cholesterol but increased the HDL-cholesterol levels in
alloxan-diabetic rabbits. During the treatment with this polyherbal preparation, Ziabeen,
for 30-days, weight remained stabilized in treated animals. To study any possible toxic
effects, animals were treated orally with 3 to 8 g/kg body weight of Ziabeen and were
kept closely inspected for 14-hr each day for a week. It was observed that they did
not show any visible signs of acute toxicity such as, respiratory distress, coma or death.
15

It is conceivable, therefore, the Ziabeen possess significant hypoglycaemic and
antihyperlipidaemic activities in normal and alloxan-diabetic rabbits; supporting the use
of this drug in traditional medicine for the treatment of diabetes mellitus.
























16

. LIST OF TABLES

S. No. Captions to the tables Page No.
1. Mean blood glucose level of normal rabbits in mg/dl SEM at various time 65
intervals after oral administration of 2% gum tragacanth aqueous solution,
Ziabeen and pioglitazone.

2. Mean blood glucose level of alloxan-diabetic rabbits in mg/dl SEM at various 66
time intervals after oral administration of 2% gum tragacanth aqueous solution,
Ziabeen and pioglitazone.

3. Mean blood glucose levels of alloxan-induced diabetic rabbits in mg/dLSEM 70
after oral administration of Ziabeen alone and in combination with different doses
of exogeneous human insulin at various time intervals.

4. Oral glucose tolerance test of alloxan-diabetic rabbits in (mg/dlSEM) after 72
administration of Ziabeen and pioglitazone.

5. Mean blood glucose level of alloxan-diabetic rabbits in mg/dl SEM after oral 75
administration of 2% gum tragacanth aqueous solution, Ziabeen and pioglitazone
continuously on days 0 , 10 ,20

and 30.

6. Plasma cholesterol, triglyceride, LDL ,HDL and VLDL (mg/dl SEM) levels in 77
alloxan-induced diabetic rabbits after 30 days of treatment with Ziabeen.

7. Effect of oral administration of Ziabeen on weight(KgSEM) in alloxan- 79
induced diabetic rabbits after 30 days





17

LIST OF FIGURES

S. No. Captions to the figures Page. No.
1. Schematic diagram showing insulin synthesis and secretion from 33
pancreatic -cells.

2. Effect of Ziabeen and pioglitazone on blood glucose levels (mg/dlSEM) of 66
normal rabbits at 0-,2-,4-,6- and 8-h intervals.

3. Effect of Ziabeen

and pioglitazone on blood glucose levels (mg/dlSEM) of 68
alloxan-induced diabetic rabbits at 0-,2-,4-,6- and 8- h intervals.

4. Effect of Ziabeen alone and in combination with different doses of insulin on 71
blood glucose levels (mg/dlSEM) of alloxan-induced diabetic rabbits at 0-,2-4-
,6- and 8-h intervals.

5. Effect of Ziabeen and pioglitazone on oral glucose tolerance test of alloxan- 73
induce diabetic rabbits.

6. Hypoglycaemic activity of Ziabeen in alloxan-diabetic rabbits on day 0, 10, 20 76
and 30.
7. Effect of Ziabeen on plasma cholesterol,triglyceride,LDL,HDL and VLDL levels 78
(mg/dLSEM) of normal alloxan-induce diabetic rabbits.

8. Effect of Ziabeen on weight(kgSEM) of normal and alloxan-induced diabetic 80
rabbits after 30day of drug administration



18

SECTION 1:










INTRODUCTION












19

1. INTRODUCTION
1.1 Diabetes mellitus (DM)
Diabetes mellitus, an endocrine and metabolic disorder that has been reported to poses
substantial threat worldwide in 21
th
century (Adewole and Ojewole,2009). It has been
characterized by chronic hyperglycaemia with disturbance of carbohydrate, fat, protein
and electrolyte metabolisms due to deficiency of insulin or insensitivity of target cells to
insulin (Rang et al.,1991; Ravi et al.,2005). Its long term complications includes;
polyuria, polyphagia, polydypsia, ketosis, retinopathy as well as cardiovascular disorders
(Kumar and Clark,2002). The main diagnostic features of diabetes mellitus are
hyperglycaemia, hypoinsulinemia and dyslipidaemia. Currently, diabetes is being
controlled by diet, exercise, insulin replacement therapy, by the use of oral
hypoglycaemic agents and certain herbal drugs as produced less side effects
(Mallick et al., 2007).
1.1.1 Prevalence of diabetes mellitus
Presently, prevalence of diabetes is increasing day by day with the major impact on the
population of developing countries due to absence of effective and adorable
intervention of diabetes mellitus (Marx,2002). According to Wild et al (2004) about 366
million peoples would suffer from diabetes by the year 2030 and in particular its type
2 diabetes. This dramatic increase in the prevalence of diabetes and its related
complications have provided the need for prediction of some preventive and
prophylactic measures (Kausar et al.,2000). Main objective of these measures is to
20

normalize biological abnormalities which are frequently associated with diabetic
complications (Monnier and Aignon,1999). It has been estimated that in type 1 i.e.
insulin dependent diabetes mellitus (IDDM), complication are found in less than 10% of
1
th
degree relatives, however, there are 90% chances of occurrence of type II i.e.
(NIDDM) and its related complications in identical twins (Kumar and Clark,1998).

According to international diabetic federation (IDF) database on diabetes, Pakistan
currently stands at number seven in the list of countries with the highest number of
diabetic cases. The diabetic population in Pakistan has to be estimated at 6.9 million in
2007 and it is expected to reach 11.5 million by the year 2025 with the Pakistani ranking
5
th
in the IDF list (IDF,2009). The national blind survey was carried out in 2002-2004
estimated the occurrence of blindness at 1% across Pakistan with 1.5 million blind
people (Jadoon et al.,2006). Incidence of type I diabetes was estimated to be
1.02/100,000 per year in Karachi, Pakistan (Staines et al.,1997). Rate of gestational
diabetes in Pakistan range from 3.2% to 3.5% compareable to western population but
the rate of complications both to the mother and foetus were found to be higher possibly
due to poor glycaemic control (Akhter et al.,1996; Khan et al.,1997; Rizvi et al.,1992).
Traditionally, type 2 diabetes mellitus is considered a disease of adults, increasingly
diagnosed in children in parallel to rising obesity rate, due to alterations in dietary
patterns as well as in life styles during childhood (Steinberger et al., 2001).
In certain tropical countries, the most common cause of diabetes is chronic pancreatitis
associated with nutritional or toxic factors (a form of secondary diabetes). Very rarely,
diabetes results from point mutations in the insulin gene (Chan et al., 1987).Uncontrolled
diabetes leads to other serious medical complications including a substantial increase in
21

premature morbidity and mortality (Engelgau et al.,2004).
1.1.2 Symptoms and signs of diabetes mellitus

Signs and symptoms of diabetes mellitus comprised of polyphagia (excessive eating)
with weight loss, polyuria (increase urination), thrist, weakness, fatigue, recurrent
blurred vision, vulvovaginitis and pruritus (skin infections), peripheral neuropathy,
parethesias, hypotension, nocturnal enuresis, obesity and often asymptomatic in type 2
DM (Tierney et al.,2010).
1.1.3 Classification of diabetes mellitus
Diabetes mellitus (DM) or carbohydrate intolerance is associated with certain conditions
and syndromes. Classification of diabetes mellitus encompasses both clinical stages and
aetiological types of hyperglycaemia (Anonymous,2003). Most patients have been
classified clinically as having type 1 or type 2 DM, gestational diabetes (GDM) have
been diagnosed in pregnant womens. Other aetiological types of diabetes arising from
genetic defects of -cell function or insulin action (Alberti and Zimmet,1998).
1.1.3.1 Type 1 or insulin dependent diabetes mellitus
The hallmark of type 1 diabetes is the selective destruction of pancreatic beta cells
(Notkins and Lemmak,2001). This is typically caused by an autoimmune assault against
-cells, inducing progressive -cell death, the -cell mass in type 1 is reduced by 70-
80% at the time of diagnosis because of the variable degrees of insulitis and absence of
detectable -cell necrosis,as -cell loss occurs slowly over years (Kloppel et al.,1985).
Type 1 is further divided into: type 1A results from a cell-mediated autoimmune attack
on -cells, where as type 1B is far less frequent named as idiopathic that has no known
22

cause. (Redondo et al.,2001; Lambert et al.,2004; Devendra et al.,2004). Autoimmune
destruction of -cells has multiple genetic predispositions and is also related to
environmental factors that are still poorly defined (Kloppel et al.,1985).
1.1.3.2 Type 2 or non-insulin dependent diabetes mellitus
Type 2 diabetes is characterized by a progressive decline in -cell function and chronic
insulin resistance. Defects in insulin receptor function, insulin receptor-signal
transduction pathway, glucose transport and phosphorylation, glycogen synthesis, and
glucose oxidation contribute to muscle insulin resistance (DeFronzo,1988). This decline
in -cell mass is likely to play a role in the pathogenesis of human type 2 diabetes
(Butler et al.,2003). The islet in type 2 diabetes is characterized by deposits of amyloid
(Kahn et al.,1999) derived from the peptide islet amyloid polypeptide (IAPP), also
known as amylin (Cooper et al.,1987). This peptide has been shown to cause apoptosis
of -cells (Lorenzo et al.,1994; Schuburt et al.,1995). Obesity is a major risk factor for
the development of type 2 diabetes (Burke et al.,1999) and is thought to lead increased
risk for type 2 diabetes through the mechanism of associated insulin resistance (Ludvik
et al.,1995).
1.1.3.3 Gestational diabetes mellitus
GDM is the most common metabolic complication associated with pregnancy (Carr and
Gabbe,1998). It has been defined as, any abnormality in glucose level diagnosed for the
first time during pregnancy (Katzung,2010). Maternal complications during GDM
include pre-eclampsia, increased risk of developing overt diabetes, and possibly
cardiovascular complications later in life, including hyperlipidaemia and hypertension.
Complications of excessive fetal growth include birth trauma, increased cesarean
23

deliveries and long-term risk of glucose intolerance and obesity. Other immediate fetal
complications includes; hypoglycaemia, hyperbilirubinaemia, respiratory distress
syndrome, cardio-myopathy, and hypocalcemia (Perkins et al.,2007; Carpenter, 2007).
1.1.3.4 Other specific types of DM
Genetic defects beta cell function characterized by the onset of mild hyperglycaemia at
an early age so formerly referred as maturity onset diabetes of young, MODY
(Byrne et al.,1996; Clement et al.,1996). Genetic defects in insulin action are also
associated with mutation of insulin receptors (Kahn et al.,1976; Taylor et al.,1992).
The diseases of exocrine pancrease includes; pancreatitis, trauma, infection,
pancreatic carcinoma, and pancreatectomy (Larsen et al.,1987; Gullo et al.,1994).
Endnocrinopathies associated with acromegaly, cushing syndrome, and
pheochromocytoma (MacFarlane et al.,1997). Drug and chemical induced diabetes
include certain toxins such as Vacor (rat poison) and pentamidine causes permanent
destruction of pancreatic beta cells (Esposti et al.,1996; Assan et al.,1995). Infections
associated DM includes congenital rubella(Forrest et al.,1971) coxsackie B,
cytomegalovirus, adenovirus and mumps (King et al.,1983; Pak et al.,1988). Other
genetic syndrome associated with diabetes are Downs syndrome, Klinefelter
syndrome, Turner syndrome and Wolframs syndrome (Barrett et al.,1995).
1.1.4 Diagnosis of diabetes
To diagnose different types of diabetes, various tests are performed. History and physical
examination are most important tools for diagnosing diabetes mellitus, however, to
confirm diagnosis following laboratory tests should be performed.
24

1.1.4.1 Blood glucose testing
i) Random (non-fasting) blood glucose test
Also called causal plasma glucose test. It is also a high-quality test intended for
diagnosing diabetes. In this test sample of blood is taken shortly after taking a meal and
then check glucose level in blood. If this level is above 200mg/dl, then there are much
chances of diabetes. But diagnosis should be confirmed the very next day by fasting
blood glucose or glucose tolerance test (ADA,1997).
ii) Fasting blood glucose (FBG)
For diagnosing diabetes mellitus, FBS is best, easy and comfortable to perform.
Overnight fasting (atleast 8 hours) is necessary before performing this test. People with
plasma glucose level of 90mg/dl and below or (3.9 to 5.5 mmol/L) are considered
normal candidates.While, candidates with a fasting glucose level of 100 to 125
milligrams per deciliter (mg/dL) or (5.6 to 6.9 mmol/L) have a form of pre-diabetes
called impaired fasting glucose (IFG). Having IFG means a person has an increased risk
of developing type 2 diabetes but does not have it yet. A level of 126 mg/dL or above,
confirmed by repeating the test on another day, means a person has diabetes
(ADA,1997).
iii) Oral glucose tolerance test (OGTT)
The OGTT requires fasting for at least 8 hours before the test. The plasma glucose level
is measured immediately before and 2 hours after a person drinks a liquid containing 75
grams of glucose dissolved in water. The level of 139 mg/dl and below is normal range.
25

If the blood glucose level is between 140 and 199 mg/dL 2 hours after drinking the
liquid, the person has a form of pre-diabetes called impaired glucose tolerance (IGT).
Having IGT, like having IFG, means a person has an increased risk of developing type 2
diabetes but does not have it yet. A 2-hour glucose level of 200 mg/dL or above,
confirmed by repeating the test on another day, means a person has diabete (Howanitz et
al.,1984).
iv) Gestational diabetic test
Gestational diabetes is also diagnosed based on plasma glucose values measured during
the OGTT, preferably by using 100 grams of glucose in liquid for the test. Blood glucose
levels are checked four times during the test. If blood glucose levels are above normal at
least twice during the test, woman has gestational diabetes (Metzer et al., 2007; Setji et
al.,2005). Fasting plasma glucose level is considered as 95mg/dl or (5.3 mmol/L),1 hour
after glucose load, target level considered as 180 mg/dL (10.0 mmol/L), 2 hours after
glucose load, target level is 155 mg/dL (8.6 mmol/L), and 3 hours after glucose load
plasma glucose target level is 140 mg/dL (7.8 mmol/L). If two or more values meet or
exceed the target level, gestational diabetes is diagnosed. A 75-gram glucose load may
be used, although this method is not as well validated as the 100-gram OGTT, the 3-hour
sample is not drawn if 75 grams is used (ADA,1997).
1.1.4.2 Urine analysis
i) Ketouria
The presence of urine ketones may indicate impending or even established ketoacidosis,
a condition that requires immediate medical attention. All of the commercially available
testing methods rely on the nitroprusside reaction to produce a purple color in the
26

presence of ketone bodies, acetone is detected only if the reagent contains glycine in
addition to sodium nitroprusside, and none of the tests detect -hydroxybutyric acid
(ADA,1997).
ii) Glycosuria
For measuring glucosuria glucose oxidase (specific for glucose) Test-Tape and a variety
of "stick"such as Clinistix

, Diastix

, are used. The reagents involved in the glucose

oxidase test are glucose oxidase, peroxidase and orthotolidine. They are impregnated
into a strip of stiff paper. In order to carry out a test, the strip is merely dipped into the
urine specimen. One minute later the strip is observed for the appearance of a blue color
which indicates the presence of glucose (Cook et al.,1953).

1.1.5 Complications of diabetes mellitus
1.1.5.1 Acute complication of diabetes mellitus

The acute metabolic complications of diabetes includes ; diabetic ketoacidosis (DKA),
hyperosmolar non-ketotic coma (HNC), lactic acidosis (LA) and hypoglycaemia
(Fishbein and Palumbo,1995).
i) Diabetic ketoacidosis (DKA)
Diabetic ketoacidosis results from absolute or relative deficiency of circulating insulin
and combined effect of increased levels of counter-regulatory hormones catecholamines,
glucagons, cortisol and growth hormone ( Foster,1983; Kitabchi et al.,2001). DKA is
characterized by severe depletion of water and electrolytes (Wolfsdorf et al.,2007).
Diagnostic parameter of DKA includes, hyperglycaemia > 250mg/dl; venous pH < 7.3;
27

or serum bicarbonate < 15mmol/L and Ketonemia and ketonuria (Dunger et al.,2004).
ii) Hyperosmolar non-ketotic coma
HNC is clinically distinct by the presence of relative insulin deficiency and
hyperglycaemia usually > 1,000 mg/dl with associated elevated serum osmolality (>300
mosm/kg), dehydration and stupor progressing to coma if uncorrected (Arieff and
Carroll, 1972 ).
iii) Lactic acidosis
LA sometimes responsible for non-ketotic coma occurring in diabetic patients reported
by Daughaday et al (1962). LA consists of elevated lactic acid (lactic acidemia 2.0
mmol/L) with acidosis (pH 7.3) and without ketoacidosis. Approximately half of the
reported cases of LA have occurred in patients with diabetes (Kreisberg,1988).
iv) Hypoglycaemia
Hypoglycaemic reactions are the most common complication of insulin therapy.
Tachycardia, palpitation, sweating, nausea and hunger are common sign of
hypoglycaemia (Katzung et al.,2009).
1.1.5.2 Chronic complications of diabetes mellitus
The injurious effects of hyperglycaemia are separated into microvascular and
macrovascular complications.
1.1.5.2.1 Microvascular complications of diabetes:
i) Vascular changes in diabetes
Changes in the vasculature occur before the onset of overt diabetes. Basement membrane
28

thickening is common in retinopathy, nephropathy, neuropathy and might be a direct
consequence of the expression and deposition of extracellular matrix (ECM) proteins in
the vasculature (Williamson and Kilo, 1983; Haneda et al.,1991).
ii) Diabetic retinopathy
Diabetic retinopathy is characterized by several common and unique features, including
thickening of vascular basement membrane, pericyte death, microaneurysms, vascular
occlusion and pathologic neovascularization that ensue to retinal hemorrhage, retinal
detachment and vision loss (Yaron et al.,1992; Cai and Boulton,2005). Severity and
duration of hyperglycaemia are linked directly to pathologic changes and cause
vascular dysfunction, for instance, retinal angiogenesis and vascular permeability
(Engerman,1989; Robison et al.,1991; Mizutani et al.,1996). Diabetic retinopathy (DR)
in the way of macular edema has been summarized as the commonest cause of blindness.
Blindness is the most feared complication of diabetes but also preventable, mostly in the
age group of 30-74 years. DR is due to micro-angiopathy affecting the retinal pre-
capillary arterioles, capillaries and venules. Detection of earliest signs is essential for
treatment strategies (Grassi,2003).
iii) Diabetic nephropathy
Manifestation of DN is characterized by microalbuminuria, which progresses to overt
albuminuria (ie, increased albumin levels in the urine, indicating more severe renal
dysfunction) and eventually to renal failure (Drummond and Mauer,2002) and is the
leading cause of end-stage renal disease, ESRD (Brenner et al.,2001) necessitating long
term dialysis or the kidney transplantation (Gordois et al., 2004). Diabetic nephropathy
29

has become major cause of morbidity and mortality amongst young adults with type
1 diabetes and in adults persistent micro-albuminuria is the best marker of consequent
risk for its development ( Schultz et al.,1999).
iv) Diabetic Neuropathy
Diabetic neuropathy has been characterized as the presence of symptoms and signs of
peripheral nerve dysfunction in people with diabetes after exclusion of other causes
(Boulton et al.,1998). There are 3 proposed stages of neuropathy; Functional stage
described as reversible, biochemical alteration in nerve function; Structural phase may be
reversible, loss of structural changes in nerve fibers and finally nerve death that is
characterized by irreversible, critical decrease in nerve fiber density and neuronal
death (Vinik,2002). Risk factors for diabetic neuropathy include; increased height,
cardiovascular disease (CVD), severe ketoacidosis and microalbuminuria(
Tesfaye,1996). Diabetic foot is due to diabetic neuropathy, macro-angiopathy or
combination of both. Triggering factors were exogenous trauma by tight shoes, foreign
bodies and insufficient care, combined with foot deformity. Pathogenic factors included
decreased collagen deposition, loss of adipose tissues and occurrence of edema (Stiegler,
2004).
1.1.5.2.2 Macrovascular Complications of Diabetes
The hallmark of diabetic macrovascular disease is accelerated by atherosclerosis
resulting in increased risk of myocardial infarction, stroke and lower extremity gangrene
(Maitra and Abbas,2005).
i) Cardiovascular Complication of diabetes
Patients with diabetes are 2 to 4 times more likely to develop cardiovascular disease
30

(CVD) than those without diabetes (ADA,2005). Several risk factors that may contribute
to the development of coronary heart disease (CHD) including, lifestyle (cigarette
smoking and diet), hyperglycaemia, hypertension and high cholesterol. Biochemical
mechanisms that leads to worsen outcomes of CVD include endothelial dysfunction,
hypercoagulability, impaired fibrinolysis, platelet hyperaggregability, oxidative stress,
sympathovagal imbalance and glucose toxicity (Haffner,2005).
ii) Peripheral arterial disease
Peripheral arterial disease (PAD) is an atheroseclerosis occlusive disease. It is major
risk factor for lower extremity amputations. The abnormal metabolic state accompanying
diabetes results in changes in the state of arterial structure and function predisposing
people to PAD (Creager and Libby,2001). The risk of development of PAD increases
threefold to fourfold in patients with diabetes ( Murabito et al.,1997).
iii) Cerebrovascular complication of diabetes
Various terms has been used to describe cerebrovascular events. The term transient
ischemic attack describes condition in which a patient experience a temporary focal
neurologic deficit such as slurred speech, aphasia, weakness or paralysis of a limb or
blindness. Sudden confusion, loss of coordination, unilateral weakness and numbness
are warning sign of cerebrovascular events. The incidence of stroke is significantly
greater among blacks compared with white (Welty, 2001).
1.1.6 Treatment of diabetes mellitus
1.1.6.1 Treatment of type 1 diabetes
Insulin is essential for strict control of blood glucose levels in type 1 diabetes. Tight
31

blood glucose control is the best way to prevent major complications in type 1 diabetes,
including those that affect the kidneys, eyes, nerve pathways, and blood vessels
(Alemzadeh and Wyatt,2007).
1.1.6.2 Treatment of type 2 diabetes
Therapeutic agents available for type 2 diabetes mellitus include

sulfonylurea and related
compounds, biguanides, thiazolidinediones, -glucosidase inhibitors and insulin.
Exercise has been shown to be beneficial in the prevention

of the onset of type 2
diabetes mellitus as well as in the improvement

of glucose control as a result of enhanced
insulin sensitivity

(Helmrich et al.,1994).

1.1.6.3 Insulin treatment
Insulin is an essential component of treatment of type 1 diabetes and indispensable to
achieve good glycaemic control in many of patients with type 2 diabetes (Reichard
et al.,1993).
i) Insulin
In 1955, insulin was the first protein to be fully sequenced (Binder and Brange,
1997). Insulin is a 51 amino acid anabolic peptide-hormone that is secreted by the
I-cells in the Islets of Langerhans. Insulin consists of two chains (A and B) connected
by disulfide bonds. One of its primary functions is the stimulation of glucose uptake
from the systemic circulation, as well as the suppression of hepatic gluconeogenesis,
thereby serving a primary role in glucose homeostasis and preventing the metabolic
disorder diabetes mellitus (Scott,1912; Jonsson et al.,1994). Human insulin is absorbed
slightly faster from subcutaneous injection site than pocine insulin due to the greater
32

hydrolipophilicity of human insulin molecule (Chawdhury et al., 1983).
ii) Insulin analogue
Four principle type of insulins preparations are available, a) Ultra short acting; with
very rapid onset and short duration that are insulin lispro and insulin aspart, b) Short-
acting; with rapid onset of action include regular novolin and humulin, c) Intermediate-
acting; are Lente humulin and NPH, d) Long acting, with slow onset of action; are
Ultra lente humulin and insulin glargine (Owens et al.,2001).
iii) Insulin synthesis and secretion
Proinsulin, a long single-chain molecule, is processed with in Golgi apparatus and
packed into granules, where it is hydrolyze into insulin and a residual connecting
segment called C-peptide. Insulin is released from pancreatic -cells at a low basal
rate and at a much higher stimulated rate in response to variety of stimuli, especially
glucose (Rusmussen et al.,1990). Pancreatic -cell stimulated insulin secretion, glucose
enters the cell through the glucose transporter 2 (GLUT2 transporter) and is metabolized
to produce ATP, which increases the cytosolic ATP/ADP ratio. This closes the K
ATP

channel leading to membrane depolarization and activation of the voltage-dependent
Ca
2+
channel leading to an increase in cytosolic Ca
2+
concentrations and activation of
Ca
2+
-dependent mechanisms controlling insulin secretion (Fig 1). In addition to the
K
ATP
-channel dependent pathway, there is also a K
ATP
independent pathway. This
pathway involves modulation of insulin secretion by some metabolite of glucose
metabolism that does not involve the K
ATP
channel (Rorsman et al.,2000).

33

















Fig 1. Insulin synthesis and secretion after (Rorsman et al.,2000)



34

1.1.6.4 Oral hypoglycaemic agents
Currently, six classes of oral antidiabetic drugs (OADs) are available; biguanides (e.g.
metformin), sulfonylureas (e.g. glimepiride), meglitinides (e.g. repaglinide),
thiazolidinediones (e.g. pioglitazone), dipeptidyl peptidase IV inhibitors (e.g. sitagliptin),
and -glucosidase inhibitors (e.g. acarbose) (Nathan, 2007) .
i) Biguanides

Metformin, a biguanide that acts directly against insulin resistance, is regarded as an
insulin sensitizing drug and is considered to be a cornerstone in the treatment of Type 2
DM (Bosi,2009). Over 30 years ago various biguanides (e.g. metformin, phenformin,
buformin) has been used in different countries for the treatment of diabetes. It is
associated with fewer hypoglycaemic attacks than sulphonylureas and does not cause
weight gain (Johansen,1999). Biguanides acts at the level of skeletal muscle by
increasing glucose transport across the cell membrane. It only acts in the presence of
endogenous insulin, and is therefore only effective when there are residual functioning
pancreatic islet cells (Klip and Leiter, 1990).
ii) Insulin secretagogues
Insulin secretagogues can be divided into two subclasses; a) sulfonylureas and
b) non-sulfonylureas
a) Sulfonylureas
Sulfonylureas currently available are second generation include; glipizide, glimepiride,
glyburide and first generation like chlorpropamide and tolbutamide are now used
rarely (Tierney et al.,2003). Sulphonylureas are shown to act by enhancing pancreatic
35

islet cell function (Pfeifer et al.,1984). They are also thought to act on the liver,
stimulating the glycolytic pathway and inhibiting the production of glucose (Kaku et
al.,1995). Sulfonylureas (SUs) are the oldest and most widely used medications for the
treatment of Type 2 DM. These agents are associated with a progressive linear decline
in -cell function (Turner et al.,1999; Del et al.,2007). They generally have a duration
of action of 12-24 hours (Campbell, 2007).
b) Non-sulfonylureas
Also called rapid-acting insulin secretagogues and postprandial insulin regulators.
Meglitinides such as repaglinide and nateglinide are prandial insulin releasers that
stimulate rapid insulin secretion (Black et al.,2007). Repaglinide is the first clinically
available insulin secretagoge that specifically enhances early-phase prandial insulin
response by increasing the sensitivity of -cells to elevated glucose levels, producing a
greater insulin release under hyperglycaemic conditions (Raskin,2008; Johansen and
Birkeland,2007).
iii) Thiazolidinediones
Thiazolidinediones are insulin-sensitizing drugs that improve whole-body insulin
sensitivity through gene regulation (Krentz and Bailey,2005). Thiazolidinediones are
selective agonist of PPAR (Schoonjans and Auwerx,2000). The potent insulin-
sensitizing effect of the TZDs is mediated through activation of the peroxisome-
proliferator activated receptor gamma (PPAR), a nuclear receptor that regulates the
production of proteins involved in glucose and lipid homeostasis ( Spiegelman,1998).
Reports of oedema (usually mild-to-moderate in intensity) in 3-5% of TZD-treated
36

patients in clinical trials have led to recommendations that these agents should be used
with caution in patients with oedema or a history of heart failure (Nesto et al.,2004). The
agents in this drug class has been approved by the U.S. Food and Drug Administration
(FDA) included troglitazone (Rezulin)

, rosiglitazone (Avandia)

and pioglitazone
(Actos)

.
iv) Alpha-glucosidase inhibitors
Acarbose (Precose)

and miglitol (Glycet)

are the two agents available in this class. -

glucose inhibitors, including acarbose, are competitive inhibitors of membrane-bound
intestinal -glucosidases that hydrolyze the oligosaccharides, trisaccharides and
disaccharides to glucose and other monosaccharides in the small intestine and thereby
delay postprandial glucose absorption (Krentz and Bailey,2005). Use of -glucosidase
inhibitors in combination with SUs, metformin or insulin can improve glycemic control
(Derosa and Sibilla,2007).
v) New drug modalities
a) Dipeptidyl Peptidase-IV Inhibitors
Dipeptidyl peptidase-IV (DPP-IV) inhibitors (Sitaglipton)

suppress the degradation of

a variety of bioactive peptides, including glucagon-like peptide-1, leading to an
enhancement of their action (Hinen et al.,2006). GLP-1 is one of the important incretin
hormones; it is released after meal ingestion and stimulates insulin secretion (Drucker
and Nauck,2006). Incretin are products that stimulate the beta-cells to release insulin.
The term incretins refers to secretory products of the intestine that influence -cell
function, these proteins GIP (Gastric inhibitory peptide) and GLP-1 enhance insulin
37

secretion (Inzucchi and McGuire,2008 ).
b) Pramlintide (Amylin analogues)
Symlin

(pramlintide acetate) is a synthetic version of human amylin, which is a 37

amino acid glucoregulatory peptide that is co-secreted with insulin by the pancreatic
-cells (Kurger and Gloster, 2004). Symlin

has been studied for the treatment in patient

with type 1 diabetes or insulin requiring type 2 diabetes. The action of this drug depends
on the presence of insulin in the body (Schubert and Wurglies,2001). Pramlintide is
administered as a subcutaneous injection immediately before a meal. It is well tolerated
and is not associated with the risk of hypoglycaemia (Koppel et al.,2008).
c) Exenatide
Byetta

(Exenatide) was developed to improve glucose control in patient with type 2

diabetes who are not achieving target goals with current treatment regimens. Byettas
active ingredient mimics a hormone found in the saliva of the poisonous Gila monster,
the largest lizard native to North America and human gut polypeptide called
glucagons-like peptide 1(GLP-1).This substance decreases a persons appetide and slows
down gastric emptying leading to weight loss. Moreover, it enhances the ability of
body stream after meals. Long term it may even promote new production of beta- cells
in the pancrease (Calara et al.,2005).
d) Inhaled insulin

Exubera

(inhaled insulin) was approved for used by the FDA in January 2006. It is
short acting insulin use immediately before meals to lower blood glucose levels
(DeFronzo et al.,2005). Several methods of insulin delivery have been researched with
38

varying results. A transdermal delivery of insulin produces insufficient transfer of insulin
across the skin. Oral insulin is not bioavailable and is broken down in the gut. New oral
insulin spray (buccal spray) form is still under the clinical investigation. Intranasal
insulin also produces poor bioavailability and unpredictability variability in dosing with
a short melabolic effect (Yamanouchi et al.,2005).
1.1.6.5 Pharmacological interventions
Three diabetes prevention trials used pharmacological therapy, and all have reported a
significant lowering of the incidence of diabetes. The biguanide, metformin reduced the
risk of diabetes by 31% in the DPP (Knowler et al.,2002),the -glucosidase inhibitor,
acarbose reduced the risk by 32% in the STOP-NIDDM trial (Chiasson et al.,2002) and
the thiazolidinedione, troglitazone reduced the risk by 56% in the TRIPOD study
(Buchanan et al.,2002)
1.1.6.6 Medical nutrition therapy (MNT)
Medical nutrition therapy (MNT) is important in preventing diabetes, managing existing
diabetes, and preventing, or at least slowing the rate of development of diabetes
complications( Franz et al.,2002).
1.1.6.7 Goals of Therapy
Before starting a patient on insulin, or adjusting their current insulin therapy, it is
important to establish glycemic goals tailored to the patient. The American Diabetes
Association currently recommends the following glycemic goals. Preprandial plasma
glucose should be in range of 70-130 mg/dl, Postprandial plasma glucose should be
<180 mg/dl and A1C <7% are targeted goal level ( ADA, 2009).
39

1.1.7 Antidiabetogenic agent (Alloxan monohydrate)




Alloxan (2,4,5,6-tetraoxypyrimidine; 5,6-dioxyuracil) was first illustrated by Brugnatelli
in 1818. Wohler and Liebig used the name alloxan and depicted its synthesis by
uric acid oxidation (Lenzen and Panten,1988).
Alloxan possessed the diabetogenic properties in rabbits caused by selective necrosis of
pancreatic islets (Dunn et al.,1943). The action of alloxan in the pancreas is preceded by
its rapid uptake by the -cells (Weaver et al.,1978; Boquist et al.,1983). Alloxan-induced
diabetes has been produced in rats, rabbits, dogs, monkeys, and cats (Goldner and
Gomori, 1944). It has also reported that fasted animals are more susceptible to the
effects of alloxan (Katsumata et al.,1992). The cytotoxic action of this diabetogenic
agent is mediated by reactive oxygen species. Alloxan and the product of its reduction,
dialuric acid, establish a redox cycle with the formation of superoxide radicals. These
radicals undergo dismutation to hydrogen peroxide. Thereafter, highly reactive hydroxyl
radicals are formed by the Fenton reaction. The action of reactive oxygen species with a
simultaneous massive increase in cytosolic calcium concentration causes rapid
destruction of -cells (Szkudelski,1981).

40

1.1.8 Herbal anti-diabetics agents
Although considerable progress has been made in the management and treatment of
diabetes mellitus with conventional synthetic drugs (Davis,2007; Vinik,2007; Freeman,
2010), still there is a continuous increase in the prevalence of diabetes globally. Many of
these oral antidiabetic agents have a number of serious adverse effects; such as liver
problems, diarrhea and lactic acidosis (Rajalakhshmi et al.,2009), thus managing
diabetes without side effect is still a challenge (Sexena and Kishore,2004). Therefore, the
search for more effective agents with the low cost and low side effect from plant
source has continued to be an important area of research because of their ready
availability, affordability and lower side effect. This surge for the use of natural agents
and alternative therapies in the management of diabetes is, therefore, now on the
increase to lower the overall financial buerden on public health services (Davis et al
.,2009; Nampothiri et al.,2010; OLoughlin et al.,2010). In folk medicine, a large
number of plants have been allegedly used in the treatment of diabetes mellitus
(Said,1969; Sabu and Kuttan,2002; Kumar and Loganathan,2010; Sharma et al., 2010).
Some of them have been reported to have the potential to significantly reduce the
blood glucose level when given either as a whole in powdered form or in the form of
their aqueous or methanolic extracts (Akhtar,1992; Broadhurst et al.,2000; Khan et
al.,2003; Rajagopal and Aasikala,2008) for example, Alium cepa (Augusti and
Benaim,1975), Momordica foetida (Marquis,1977), Coccinia indica (Khan et al.,1980),
Momordica charantia (Akhtar et al.,1981) and cuminum nigrum (Akhtar and
Ali,1985). Recently, Gondwe et al (2008) have found that the stem bark extract of S.
birrea exhibited dose-dependent reduction in blood glucose concentration without
41

significantly affecting the blood insulin level in streptozotocin (STZ)-induced diabetic
rats. Stem bark extract of S. birrea treatment was associated with increased hepatic
glycogen synthesis.
1.1.8.1 Historic development of herbal medicine
Since ancient times, traditional medicines all over the world have advocated the use of
the plants to treat diabetes. Man has practiced the use of plants as medicines for the cure
of diseases for centuries (Philipeon,2001). Herbal medicine, also called botanical
medicine or phytomedicine, refers to the use of a plant's seeds, berries, roots, leaves,
bark, or flowers for medicical purposes. People worldwide have been using herbal
medicine for the treatment, control and management of a variety of ailments since
prehistoric times (Kong et al.,2003). According to World Health Organization (WHO,
2001) 60% of the worlds population depend on traditional medicine and 80% of the
population in developing countries depend almost entirely on traditional medical
practices, in particular, herbal medicine for their primary health care needs
(Fransworth,1994 and Zhang,2000). The use of medicinal plants has a long folk history
for the treatment of diabetes mellitus (Shoaib,1992). The ethnobotanical information
reports about 800 plants that may possess antidiabetic potential (Alarcon et al.,1998).
Currently, medicinal plants continue to play an important role in the management of
diabetes mellitus, especially in developing countries where many people do not have
access to conventional antidiabetic therapies ethnopharmacol (Grover et al.,2002).
Hypoglycaemic activity of a large number of these plants/plant products has been
evaluated and confirmed in animal models (Gupta et al.,2005) as well as, in human
beings (Herrera-Arellano,2004). In some cases the bioactive principles have also been
42

isolated and identified (Kesari et al.,2005). More than 1,200 organisms (representing 725
genera in 183 families) have been used ethnopharmacologically to treat diabetes. They
belong to different families, among them 127 species are found in the Febaceae (pea
family), 98 in the Asteraceae (aster or daisy family), 36 in the Lamiaceae (mint family),
35 in the Liliaceae (lily family), 30 in the Poaceae (grass family) and 30 in the
Euphorbiaceae (spurge family) by Herbal gram,(1997). However, the mechanism of
action most of these plants and/products lower the blood glucose level remains
speculative.
1.1.8.2 Reported plants possessing antidiabetic activity
Said (1969) has been described selected plants in the Pharmacopoeia of Eastren medicine
under the title of antidiabetic drugs including; Aconitum napellus (Bachang), Aegles
marmelos (Burg Bell), Allium cepa (Piyaz), Allium sativum (Lehsun), citrus aurantium
(Sangtra), Coccinia indica (Kauduriki), Eugenia jambolana (Jaman), Gossypium
herbaceum (Binola), Grewia asiatica (Falsa), Momordica charantia (Karela) and
Tinospora cordifolia (Gilo).
Fransworth and Segelman (1971) have also enlisted herbal plants which possess the
experimental hypoglycaemic activity eg. Arctium lapa (Burdock), Brassica oleraceae
(cabbage), Brassica rapa (Turnip), Cassia occidentalis (Senna), Cocos mucifiera
(coconut), Euphorbia pilulifera (pill-bearing spurge), Helianthus annus (sunflower),
Musa sapientum (banana), Spinacia olderacea (spinach), Taraxacum officinale
(Dandelion), Zingiber officinale (Ginger).
Khan et al (1980) have reported that leaves of Coccinia indica (Kauduri-ki-bel) improve
the glucose tolerance. The study showed that this plant may be useful for oral treatment
43

of patients with NIDDM, especially as there were no adverse effect during six week of
use.
Akhtar et al (1981) have been reportetd that Momordica charantia fruit (bitter gourd)
has been considered the valuable antidiabetic agent for scores of years. In normal rabbits,
the 0.25 g/kg dose did not drop off blood glucose. However, 0.5 g/kg dose caused
decrease in blood glucose. The result was maximum at a 10 hour interval, after which it
decreased gradually. The 1.0 and 1.5 g/kg doses also lowered the blood glucose of these
rabbits. The maximum decrease was produced by these doses at 10 hours intervals. In
diabetic rabbits, the 0.25 g/kg and 0.5 g/kg doses did not cause a significant decrease in
blood glucose. However, 1.0 and 1.5 g/kg doses produced dose dependent decrease in
blood glucose levels. The maximum decrease was observed at 10 hours intervals.
Akhtar and Ali (1985) have been observed that Cuminum nigrum seeds produced a
significant hypoglycaemic effect when plant seed administered at doses of 1, 2, 3, and 4
g/kg body weight of normal and alloxan-diabetic rabbits. They have suggested that
Cuminum nigrum seeds contain one or more hypoglycaemic principle.
Akhtar et al (1987) have studied the hypoglycaemic activity of Asparagus racemosus
and lodoicea sechellarum fruits in rabbits and reported that powdered A. racemosus root
and L. sechellarum fruit produced significant hypoglycaemic effect only in the normal
rabbits. In alloxan-diabetic treated rabbits both the medicinal plants drugs did not
produce any effect on blood glucose level .
Akhtar and Qureshi (1988) have reported the effect of powdered Ficus glomerata
(Roxb.) fruits on blood glucose level in groups of normal and alloxan-diabetic rabbits.
They showed that administration of 1, 2, 3, 4 g/kg body weight of F.glomerata pulp
44

significantly lowered the blood glucose level in normal group. In alloxan diabetic rabbits
a significant fall in blood glucose level is produced after treatment with 2, 3, 4g/kg body
weight of plant drug. Acetohexamide in 500mg/kg dose produced a significant decrease
in blood glucose levels of normal rabbits only.
Frati-Munari et al (1988) have been described the hypoglycaemic effect of Opuntia
Streptacantha, Lemaire in NIDDM. Results of study showed that the stem of O.
Streptacantha Lem. caused a hypoglycaemic effect in patients with NIDDM. The
mechanism of this effect is unknown, but an increased insulin sensitivity was suggested.
Twaij and Al-badr (1988) have evaluated that Artemisia herba alba has been widely
used in Iraqi folk medicine for the treatment of diabetes mellitus. Oral administration of
an aqueous extract (0.39 g/kg) of the aerial parts of Artemisia plant to normoglycaemic
and alloxan-diabetic rabbits produced significant hypoglycaemic activity, which was
consistent and time-dependent.
Akhtar et al (1990) have been evaluated the effects of powdered Mucuna pruriens seeds
on blood glucose levels in normal and alloxan-diabetic rabbits. They showed that In
normal group 0.5, 1 and 2 g/kg of M. pruriens pulv significantly decreased the blood
glucose levels while in alloxan-diabetic rabbits only 1 and 2 g/kg body weight caused a
significant fall. The standard drug, acetohexamide in 500 mg/kg dose significantly
reduced the blood glucose levels but in normal rabbits only. High levels of trace
elements like manganese, zinc and others were found in these seeds.
Akhtar and Iqbal (1991) have also evaluated the blood glucose levels of normal and
alloxan diabetic rabbits after oral administration of various doses of Achyranthes aspera
Linn. powdered whole plant and certain aqueous and methanolic extracts.They
45

described that oral administration of 2,3, and 4g/kg of Achyranthus aspera L. produced
a significant dose-related hypoglycaemic effect in normoglycaemic and alloxan-
induced diabetic rabbits. In these animals, water and methanol extracts also decrease
blood sugar levels. A 7-day acute toxicity study in the rabbits has not revealed any
adverse or side effects at dosage up to 8g/kg orally.
Mossihuzzaman et al (1994) have been described the hypoglycaemic effect of Costus
speciosus (Rhizomes), Nephrolepis tuberose (tuber), stephania hernandifolia (bulb) from
the eastern himalyan belt. The results indicated that these plants have remarkable
possibilities as a sourse of hypoglycaemic agent.
Wadood and Wadood (1995) have showed the hypoglycaemic effect of Grewia asiatica
and Peganum harmala in normal and alloxan-induced diabetic rabbits. The alcoholic
extracts of the stem of Grewia asiatica have exerted a significant effect in normal
rabbits. The hypoglycaemic activity was not significant in alloxan-induced diabetic
rabbits. They also studied that extract of Peganum harmala did not produced any
significant hypoglycaemic effect in normal as well as alloxan-diabetic rabbits.
Sharma et al (1997) have been studied the hypoglycaemic, antihyperglycaemic and
hypolipidaemic activities of the aqueous and 50% ethanolic extracts of Caesalpinia
bonducella Fleming (Leguminosae) seeds in normal and streptozotocin (SZ)-diabetic
rats. In normal rats both the extracts exhibited hypoglycaemic activity as early as 4 h
after administration at a lower dose of 100 mg/kg. The hypoglycaemia produced by the
aqueous extract was of prolonged duration as compared to ethanolic extract. In diabetic
rats, both the extracts produced significant (P<0.01) antihyperglycaemic effect from day
5 onwards. Aqueous extract also exhibited antihypercholesterolemic and
46

antihypertriglyceridemic effects in SZ-diabetic rats. These results suggest that C.
bonducella seeds possess an antidiabetic principle and can be useful for treatment of
diabetes. Further studies are warranted to fractionate the active principle and to find out
its exact mechanism of action.
Ahmad et al (2000) have reported the oral administration of the flavonoids contents
(80%) of the seeds of Cuminum nigrum caused a significant blood glucose lowering at a
dose range of 0.5 t0 1.5 g/kg both in normoglycaemic and alloxan-induced diabetic
rabbits. The maximum of decrease in glycaemia was obtained within 4-8h; the normal
level of glycaemia was reached within 24 h of drug administration. In contrast, the
alkaloids isolated from C.nigrum (0.01%) had no significant effect in either
normoglycaemic or diabetic rabbits. A high dose of 5g/kg did not produce any adverse
effects in a 7-day acute toxicity study in rabbits.
Khosla et al (2000) observed hypoglycaemic effect with Azadirachta indica, when
given as a leaf extract and seed oil, in normal as well as diabetic rabbits. They showed
that effect was more pronounced in diabetic animals in which administration for 4
weeks after, significantly reduced blood glucose levels. Antidiabetic effect was
comparable to that of glibenclamide. Their data suggest that A. indica could be of benefit
in diabetes mellitus in controlling the blood sugar or may also be helpful in preventing or
delaying the onset of the disease.
Rao et al (2001) reported that aqueous, ethanolic and hexane fractions of Momordica
cymbalaria fruits were administered at different doses to different groups of rats (both
normal and alloxan diabetic rats) after an overnight fast. The blood glucose levels were
measured at 0, 1, 3, 5 and 7 h after the treatment. The aqueous extract of Momordica
47

cymbalaria at a dosage of 0.5 g/kg b.w. is showing maximal blood glucose lowering
effect in diabetic rats.
Aderibidge et al (2001) assessed the leaves of Mangifera indica Linn. for antidiabetic
properties using normoglycaemic, glucose-induced hyperglycaemia and streptozotocin-
induced diabetic mice. They showed that the aqueous extract produced a reduction of
blood glucose level in normoglycaemic and glucose-induced hyperglycaemia, but did not
have any effect on streptozotocin-induced diabetic mice. They also evaluated the
hypoglycaemic effect of the aqueous extract with that of an oral dose of chlorpropamide
under the same conditions. The results indicate that the aqueous extract of the leaves of
Mangifera indica possess hypoglycaemic activity.
Rao et al (2001) evaluated that aqueous, ethanolic and hexane fractions of Syzygium
alternifolium seeds were prepared and given different doses of these extracts
individually to different batches of rats both normal and alloxan-diabetic rats, after an
overnight fast. The blood glucose levels were measured at 0, 1, 3, 5 and 7 hours after
the treatment. They reported that the aqueous extract of Syzygium alternifolium at a
dosage of 0.75 g/kg b.w. show maximum blood glucose lowering effect in both
normal and alloxan-diabetic rats. The ethanol and hexane fractions are also showing
hypoglycaemic and antihyperglycaemic activity, but the effect is significantly less
than that of aqueous extract. The antihyperglycaemic activity of Syzygium alternifolium
seed was compared with the treatment of Glibenclamide.
Alper et al (2001) have evaluated the effect of Aloe vera leaves on blood glucose
level in type I and type II diabetic rat model. They reported that A. vera leaf pulp
extract showed hypoglycaemic activity on IDDM and NIDDM rats, the effectiveness
48

being enhanced for type II diabetes in comparison with glibenclamide. On the contrary,
A. vera leaf gel extract showed hyperglycaemic activity on NIDDM rats. It may
therefore be concluded that the pulps of Aloe vera leaves devoid of the gel could be
useful in the treatment of non-insulin dependent diabetes mellitus.
Akhtar et al (2002) have evaluated the effects of Alpinia galanga rhizome on
blood glucose levels. They reported that powdered rhizome and its methanolic and
aqueous extracts significantly lowered the blood glucose in normal rabbits. Gliclazide
also produced a significant decrease in blood glucose in the rabbits. They also
investigated that in alloxan-diabetic rabbits, A. galanga and its methanol and aqueous
extracts did not produce significant reduction in blood glucose. The hypoglycaemic
effect of A. galangal in normal rabbits was comparable to gliclazide. The rhizome of
plant was found to contain high levels of certain minerals. Acute toxicity and behavioral
studies revealed no visible signs of toxicity and any abnormal behavior in rabbits even at
high doses. It is concluded that A. galanga produces fall in blood glucose levels in
normal rabbits and the principles, both organic and inorganic are extractable in methanol
and water.
Ravi et al (2004) have evaluated the hypoglycemic activity of different parts of Eugenia
jambolana seeds such as whole seed, kernel and seed coat on streptozotocin-induced
diabetic rats. They reported that administration of the ethanolic extract of kernel at a
concentration of 100 mg/kg of body weight significantly decreased the levels of blood
glucose, blood urea and cholesterol, increased glucose tolerance and levels of total
proteins and liver glycogen, and decreased the activities of glutamate oxaloacetate
transaminase and glutamate pyruvate transaminase in experimental diabetic rats. The
49

hypoglycaemic efficacy was compared with that of glibenclamide, a standard
hypoglycaemic drug.
Kaleem et al (2005) have evaluated that aqueous extract of piper nigrum seeds and vinca
rosea flowers were administered orally to alloxan-induced diabetic rats once a day for 4
weeks. They showed that these plants lead to the significant lowering of blood glucose
levels and reduction in lipid profiles. They also reported that oxidative stress plays key
role in diabetes and treatment with P. nigrum and V. rosea are useful in controlling not
only the glucose and lipid profiles but these components may also helpful in
strengthening the antioxidant potential.
Saxena et al (2007) have reported the hypoglycaemic effect of Swertia chirayita .They
showed that single dose (250mg/kg body weight) of ethanolic extract of swertia
chirayita when administered orally with 2% gum acacia suspension to male albino rats a
significant blood glucose lowering effect was observed.
Akhtar et al (2009) have reported the effects of Lodoicea sechellarum Labill (Sea
coconut) fruit on blood glucose levels of normal and type 2 diabetic human volunteers.
The powdered fruit of Lodoicea sechellarum Labill was orally administered in a dose of
2, 3 and 4 g to normal and diabetic human volunteers. The blood glucose levels were
estimated at 0, 1, 8, 15 and 21 days after the administration of drug. The powdered fruit
of Lodoicea sechellarum Labill significantly reduce blood glucose levels of both normal
and diabetic human volunteers. It was therefore conceivable that Lodoicea sechellarum
Labill possess hypoglycaemic principles that may act stimulating insulin release from -
cells of normal individuals and type 2 diabetics or may have insulin like principles.
Ahmad et al (2009 ) have described the effects of Berberis lycium Royle root bark
50

various lipid profiles in alloxan-induced diabetic rabbits. They reported that oral
administration of 250mg/kg and 500mg/kg crude powder of Berberis lycium root for
four weeks resulted in significant reduction in total cholesterol, triglyceride and
low density lipids (LDLs) levels. They also concluded that . Berberis lycium treatment
increased the levels of high density lipids (HDLs). The whole study showed that crude
powder of Berberis lycium Royle has antihyperlipidaemic effect.
Grijesh et al (2009) reported that the aqueous leaf extract of Gymnema sylvestre reveals
the significant hypoglycaemic effect and hypolipidaemic activity when administered at
the doses of 400, 600 and 800mg/kg body weight orally once a day to the groups for 30
days in both normal and alloxan-induced diabetic rats. The results showed that fasting
blood glucose, cholesterol and serum triglyceride reduced significantly in treated rats
where as extract also reveal the potent elevation in the level of serum HDL cholesterol.
They showed that the extract seems promising for the development of a phytomedicine
for diabetes mellitus.
Akhtar et al (2010) have evaluated the effect of Butea monosperma (Lamk.) Taub. on
blood glucose and lipid profiles in normal and diabetic human volunteers. They reported
that oral doses of 1, 2, and 3 g of powdered B.monospema (Lamk.)Taub. Fruit produce
the significant decrease in blood glucose level of human volunteer.
Mana et al (2010) have described that methanolic extract of Holarrhena antidysenterica
(MEHAD) seeds (Apocynaceae) showed the hypoglycaemic activity in normal and
streptozotocin-induced diabetic rabbits. They showed that oral dose of 250mg/kg body
weight of methanolic extract of plant for 18 days produced decrease in blood glucose
level when compared to diabetic control. They also concluded that oral administration of
51

extract significantly decreased serum total cholesterol, triglyceride levels and at the same
time markedly increased liver glycogen ultimately proving the potent antidiabetic
property of plant.
Akhtar et al (2011) have recently studied the effect of Butea monosperma (Palas papra)
fruit on blood glucose and lipid profiles of normal and diabetic human volunteers. This
study concluded that Butea monosperma have important hypoglycaemic and
hypolipidaemic properties that may ultimately be used as successful therapies for the
treatment of diabetes and obesity in general public.
Thirumalai et al (2011) have reported the hypoglycaemic activity of Brassica juncea
(seeds) aqueous extract at a dose of 250, 350 and 450 mg/kg body weight. Adult male
Swiss albino rats of six numbers in each group was undertaken for study and evaluated
the hypoglycaemic effect of Brassica juncea (seeds) on streptozotocin-induced diabetic
male albino rats. The serum insulin levels were recorded a significant depletion in all
groups, short term as well as long term diabetic animals, when compared to that of
normal animals. A significant dosage dependent augmenting effect of the seed extract on
the serum insulin was recorded in both short term as well as long term groups. They
concluded that aqueous seed extract of Brassica juncea has potent hypoglycaemic
activity in male albino rat.
Ahmad et al (2011) have reported the antihyperlipidemic and hepatoprotective activity
of Dodonaea viscosa leaves extract in the alloxan-induced diabetic rabbits. The study
showed that oral administration of aqueous-methanol (30:70), extract of the Dodonaea
viscose leaves significantly decrease the raised parameters triglyceride, total cholesterol
and LDL-cholesterol to the normal value. But the extract significantly increased HDL-
52

cholesterol, ALT and AST levels. They concluded that aqueous:methanolic (70:30)
extract of Dodonaea viscose leaves exerts antihyperlipidaemic and hepatoprotective
effects in alloxan-induced diabetic rabbits.
Akhtar et al (2011) have reported the anti-hyperglycaemic and lipid lowering properties
of Emblica officinalis gaertn fruit in normal and and diabetic human volunteers. They
showed that a significant decrease in fasting and 2-hr post-prandial blood glucose level
was observed on the 21
st
day in both normal and diabetic subjects receiving 1,2, or 3g E.
Officinalis powder per day as compared with their base line values. They also quoted
that significant decreases were also observed in total cholesterol and triglycerides in both
normal and diabetic volunteer receiving only 3g E.officinalis power exhibited a
significant decrease in total lipids on 21
st
day. They also evaluated that both normal and
diabetic volunteere receiving 2 or 3 g E.officinalis powder significantly improve high
density lipoprotein and lowered the low density lipoprotein cholesterol levels.
Akhtar et al (2011) have recently described hypoglycaemic activity of Dodonaea viscosa
leaves in normal and alloxan-induced diabetic rabbits. They studied that oral
administration of 250 and 500mg/kg of D. viscose leaves significantly reduced blood
glucose in normal and highly significantly in alloxan-induced diabetic rabbits.
Glibenclamide (5mg/kg) was used as a standard drug, that could significantly reduce
blood glucose levels of normal rabbits but not alloxan-induced diabetic rabbits. They
have also found that aqueous methanolic (A-M) extract of plant leaves show highly
significant decrease at various time intervals.

53

Keeping in view the all described findings, it has been thought worthwhile to evaluate
the antidiabetic effect of compound herbal preparation, Ziabeen in normal and alloxan-
induced diabetic rabbits with the following objectives.

1.1.9 Aims and objective of present study


To assess the hypoglycaemic activity of compound herbal preparation (Ziabeen) in
normal and alloxan-induced diabetic rabbits.
To evaluate the antihyperlipidaemic effects of compound herbal preparation (Ziabeen)
in alloxan-induced diabetic rabbits.
To compare the hypoglycaemic activity of compound herbal drug (Ziabeen) with a
standard hypoglycaemic agent pioglitazone.
To rationalize folkloric use of compound herbal preparation (Ziabeen) as a
hypoglycaemic/antidiabetic agent.
To determine the side effect or adverse effect if any ,of polyherbal preparation
Ziabeen.







54

SECTION 2












MATERIALS AND METHODS










55

2. Materials and methods

2.1 Ziabeen ( A compound herbal preparation)

Ziabeen, a compound herbal preparation was obtained from the Ashraf Laboratories
(Pvt) Ltd. Sargodha road, Faisalabad (Pakistan), in April 2010. The powdered product
was refrigerated in well closed container at 4

C. One gram of Ziabeen has been

described by the manufacturer to contain constituents in quantities specified below,

Botanical name Common name Quantity

Aloe barbadensis Ghee kawar- 111.10mg
Azadirachta indica Neem- 111.10mg
Eugenia jambolana Jaman- 111.10mg
Gymnema sylvestris Gurmar booti- 01ggg1gm
Holarrhena antidysenterica Kurchi- 01ggg1gm
Momordica charantia Karela- 01ggg1gm
Swertia chirata Chirayatah- 01ggg1gm
Piper nigrum Black pepper- 01 45145
Gum acacia (binder) Gond kiker- 01gmm1mm
2.2. Equipments used
Glucometer (Optium Xceed, Abbott Laboratories, USA)
Vortex mixture (Reax Top. Heldolph Instruments, Germany)
Blood collection tubes, EDTA K
3
(BIO-VAC, Pakistan)
Weighing balance (Shimadzu Corporation, Japan)
56

2.3. Chemicals and diagnostic kits used

Glucose oxidase kits (Optium Xceed, Abbot Laboratories USA)
TC,TG,HDL,LDL-cholesterol kits (Fluitest, Biotechnologies AG, Germany)
Alloxan monohydrate (Research Organics, USA)
Pioglitazone (Martin Dows Pharmaceuticals (Pvt)Ltd.,Lahore.)
Gum tragacanth (Hi-Media Lab, USA)
Insulin (Regular Humulin-Lilly, USA)
2.4 Experimental animal used

Healthy adult rabbits of a local strain (Oryctolagus cuniculus) weighing 1-1.5 kg were
kept at animal house of the Department of Pharmacy, University of Sargodha, Sargodha.
The animals were housed in stainless cages under standard laboratory conditions (12hrs,
light and dark cycle, 212C, relative humidity 55%, green fodder and water were
available ad libitum). Animals were handled according to guidelines as approved by
local ethical committee and NIH. The effects of Ziabeen was studied on blood glucose
level of normal rabbits. In addition, separate experiments were performed to study its
effects on blood glucose level of the alloxan-induced diabetic rabbits.
2.5 Induction of experimental diabetes in rabbits
Group of rabbits weighing 1-1.5 kg were made diabetic by injecting intravenous
injection of 150mg/kg alloxan-monohydrate (Akhtar et al.,1981) dissolved in 0.9%
normal saline, via jugular vein of rabbits (Khosla et al., 2000). This dose destroys the
beta cells of pancreas and produce diabetes mellitus (Szkudelski, 2001). The control
group was given same volume of saline solution. Eight days after injection, the blood
57

glucose levels of all surviving rabbits were determined by glucose oxidase method. The
diabetic rabbits with blood glucose levels of 250-500mg/dl were used in further
experiments (Akhtar and Ali,1984).
2.6 Preparation and administration of drug suspensions
The amount of Ziabeen required for each rabbit was calculatetd on body weight basis
and that amount of drug was weighed as Akhtar and Ali (1984), then it was dissolved in
10ml of 2% gum tragacanth solution and final suspension volume was made up to 20ml.
The suspension was administered to rabbits using a stomach tube attached to a standarad
sringe containing 20ml of suspension (Wadood et al.,2007). Immediate sneezing and
coughing indicated injection into the lungs and in such cases the tube was at once
withdrawn and another animal was taken instead. Pioglitazone, used as a standard
hypoglycaemic agent, was also administered after suspending in 2% aqueous gum
tragacanth solution.
2.7 Collection of blood samples
Animal was held in rabbit stand and immediately blood was collected from one of the
ear marginal vein, before and after the administration of the drug. The pricked site of the
vein was pressed with cotton swab soaked with spirit to protect the rabbits against the
infection. For the estimation of Total-cholesterol, Triglyceride, LDL-cholesterol, VLDL-
cholesterol and HDL-cholesterol blood sample was collected from jugular vein of the
rabbits.
2.8 Biochemical analysis
Blood glucose levels were measured by optium xceed glucometer using glucose oxidized
58

optium kit, total plasma cholesterol, TG, HDL were estimated by enzymatic test kit
(Fluitest). The LDL-cholesterol levels were calculated by using formula:
LDL = total cholesterolHDL-cholesterol(triglyceride/5) and VLDL = Triglyceride/5
(Fridewald et al.,1979).
2.9 Experimental Design
2.9.1 Acute toxicity and behavioral pattern studies
To study any possible toxic effects of Ziabeen, adult healthy rabbits of local strain and
weighing between 1-1.5 kg were used. Rabbits were randomly divided into seven groups
(I-VII) of six animal each. The rabbits of group I served as a control and these animals
received orally 20ml of 2% gum tragacanth solution only. The animals of groups II, III,
IV, V , VI and VII were treated orally with 3, 4 , 5, 6, 7 and 8g/kg body weighed of
powdered Ziabeen suspended in 20ml of 2% gum tragacanth solution respectively.
To study the toxicity and any behavioral pattern all rabbits treated with high doses of
Ziabeen were kept closely observed for 14 hours each day for a week (Laurence and
Bacharach,1964).
2.9.2 Hypoglycemic activity of Ziabeen in normal rabbits
For the study of this activity, the rabbits were divided into five groups of six animals
each. Rabbits in group I served as untreated control as they were administered orally
20ml of 2% gum tragacanth aqueous solution into water only. In groups II-IV rabbits
were administered with Ziabeen in graded doses of (2, 3 and 4 g/kg body weight of
animal) and rabbits in group V were treated with 1mg/kg body weight of pioglitazone
59

(Gumieniczek,2005) to compare the effect of Ziabeen. Blood glucose levels were
checked at 0-, 2-, 4-, 6- and 8-hr intervals after administration of the control drug,
graded doses of Ziabeen and pioglitazone. All groups were fasted for overnight (12 hrs).
2.9.3 Hypoglycaemic activity of Ziabeen in alloxan-diabetic rabbits
To check the effect of Ziabeen in alloxan-induced diabetic rabbits. Rabbits were
divided into five groups six animals each; group I rabbits were served as diabetic
control, and were given 20 ml of 2% gum tragacanth solution in water only. Rabbits in
group II to IV were administered with the doses of 2, 3, 4g/kg body weight of Ziabeen
respectively and doses of Ziabeen were suspended in 20 ml of 2% gum tragacanth.
Rabbits in group V were treated with pioglitazone suspended in 2% gum tragacanth
solution.
2.9.4 Oral glucose tolerance test (OGTT)
In this study rabbits were divided into three groups. Rabbits in group I, II and III were
fasted for overnight (12 hr), Group I rabbits were served as diabetic control and were
administered with 20 ml of 2% gum tragacanth solution. Rabbits in group II-III were
treated with the suspension of 4g/kg Ziabeen and 1mg/kg pioglitazone respectively.
After 2 hours, glucose was administered orally (4g/Kg b.w) to all the three groups. Blood
glucose levels were determined at 0-, 30-, 60-, 120-, 150-, 180-, 240-, 300- min intervals.
Changes in blood glucose were evaluated by comparing with the control group. Oral
glucose tolerance test was used to study the hypoglycaemic effect of Ziabeen after
taking large amount of glucose or glucose load (Habibuddin et al.,2008).

60

2.9.5 Hypoglycaemic effect of Ziabeen with and without insulin administration
For this study previously alloxanized rabbits were divided into four groups of six animals
in each group. Rabbits in group I were treated with 6-unit of regular insulin, group II
rabbits were given 3 unit of insulin plus 4g/kg Ziabeen, and rabbits in group III and IV
were treated with 2 units of insulin plus 4g/kg Ziabeen and 4g/kg of Ziabeen
respectively. The study was carried to establish the synergistic effect of insulin and
Ziabeen on blood glucose levels of alloxan-induced diabetic rabbits (Ahmad et al.,2009).
Blood glucose levels were estimated at 0-, 2-, 4-, 6- and 8-hr intervals after the
administration of Ziabeen and exogeneous human insulin.
2.9.6 Effect of Ziabeen on fasting blood glucose levels after 30days
This study was performed to determine the effects of Ziabeen on blood glucose levels of
alloxan-induced diabetic rabbits after 30 days. Rabbits for this activity were divided into
three groups of six animals in each group. Rabbits in group I was served as alloxan-
induced diabetic control and administered orally 20ml of 2% gum tragacanth aqueous
solution continuously for 30 days. Groups II and III were given Ziabeen and
pioglitazone respectively. All groups were fasted for overnight (12 hrs). Fasting glucose
levels of the groups I to IV were checked on day 0, 10

,20

and 30.
2.9.7 Effect of Ziabeen on lipid profile in alloxan-induce diabetic rabbits
This study was conducted to evaluate the Triglyceride, Total-cholesterol, HDL-
cholesterol, LDL-cholesterol and VLDL-cholesterol levels after 30 days of treated
groups (Ahmad et al.,2009). Animals were divided into 3 groups of six animal each.
Group I was used as normal control, Group II was taken as diabetic control, while group
61

III was given Ziabeen through oral route daily for 30 days. Total Cholesterol, TG,
HDL, LDL ,VLDL levels of three groups were measured after 30 days.
2.9.8 Effect of Ziabeen on weight of rabbits after 30days
This study was performed to check the effects of Ziabeen on weight of alloxan-induced
diabetic rabbits for 30 days (Ahmad et al., 2009). Rabbits were divided in to three groups
of six animals each. Groups I, and II were treated as normal control and diabetic control
and were administered with 20ml of 2% gum tragacanth solution respectively. Rabbits
in group III were treated with 4g/kg body weight of Ziabeen for 30 days and rabbits of
all the groups were fasted overnight.The weight of groups I, II and III rabbits were
estimated on days 0, 10
th
, 20
th
and 30
th
.
2.10 Statistical Analysis
The results are expressed as mean SEM. The statistical analysis was carried out using
paired t-test and one way analysis (ANOVA). Statistical P value <0.05 was considered to
be significant (Steal and Torrie,1992).







62

SECTION 4














RESULTS AND DISCUSSION
















63

3.1 RESULTS
3.1.1 Effect of alloxan-monohydrate administration to rabbits
The rabbits with blood glucose levels above 200mg/dl were considered diabetic and were
used in studies. These effects have already been explained by Akhtar et al (1981).
3.1.2 Effect of Ziabeen

on acute toxicity and behavioural pattern studies
Rabbits treated with 3, 4 , 5, 6, 7 and 8g/kg body weight of Ziabeen

remained alive
upto 7 days and they did not show any visible sign of acute toxicity such as;
restlessness, respiratory distress, convulsion, coma or death. Even the visual behavioural
pattern studies in rabbits revealed no prominent change in the awarenesss, motoractivity,
mood, posture, motor coordination, CNS excitation, food consumption and body weight
such effects have already been described by Laurence and Banarach (1964).
3.1.3 Effect of Ziabeen

on blood glucose in normal rabbits
The mean blood glucose level of control and treated animals after oral administration of
different doses of Ziabeen at various time intervals are shown in Table.1; Fig.2. Results
revealed that 2% gum tragacanth aqueous solution did not change the blood glucose
level of normal rabbits. Administration of 2g/kg of drug Ziabeen produced a highly
significant (P<0.001) decrease at 6 and 8-hr intervals. The level was significantly
(P<0.05) decrease at 4-hr interval and non-significant (P>0.05) decrease was observed at
2-hr intervals. The oral treatment of 3g/kg of Ziabeen produced highly significant
(P<0.001) decreased at 4, 6 and 8-hrs but level at 2-hr was non-significant (P>0.05). It
was also shown that the blood glucose level after treatment with 4g/kg of Ziabeen

was
highly significantly (P<0.001) decrease at 4,6 and 8-hr and decrease was significant
64

(P<0.05) at 2-hr interval. It was also shown in table 1 that the standard drug,
pioglitazone also significantly (P<0.05 or P<0.001) reduced blood glucose level in
normal rabbits (Fig. 2)
3.1.4. Effect of Ziabeen

on blood glucose in alloxan diabetic rabbits
Table 2 shows the effects of Ziabeen in alloxan-induced diabetic rabbits. Treatment
with 2g/kg of Ziabeen

signicantly (P<0.05) decrease blood glucose level at 4 and 6-hr
intervals, at 8-hr highly significantly (P<0.001) and non-significant (P>0.05) decrease
was observed at 2-hr. Similarly with dose of 3g/kg of drug Ziabeen highly significant
(P<0.001) decrease in blood glucose level occur at 4 ,6 and 8-hr intervals even at 2-hr
significant (P<0.05) decrease in blood glucose level from zero-hr level was also
observed. The 4g/kg dose of Ziabeen highly significantly (P<0.001) reduced the blood
glucose level at 2, 4, 6, and 8-hr intervals from zero-hr level. The results of present
study are in accordance with Akhtar et al (2011). There was no significant (P>0.05)
change in blood glucose levels of group treated with 2% aqueous gum tragacanth
solution while significant (P<0.05) decrease was observed with 1mg/kg dose of
pioglitazone at 4, 6, and 8-hr and non-significant effect in blood glucose level occur at 2-
hr intervals. Results showed (Table 2; Fig 3) that 4g/kg dose of drug Ziabeen

produced
higly significant effect at 2, 4, 6 and 8-hr intervals so it was used in further experimental
study.



65

Table 1. Mean blood glucose level of normal rabbits in mg/dl SEM at various time
intervals after oral administration of 2% gum tragacanth aqueous solution, Ziabeen and
pioglitazone.



Time
interval
( hours)

Group I
treated with
2% gum
tragacanth
aqueous
solution
(20 ml)
Group II
treated with
Ziabeen



( 2g/kg)
Group III
treated with
Ziabeen



(3g/kg)
Group IV
treated with
Ziabeen



(4g/kg)
Group V
treated with
pioglitazone



( 1mg/kg)

0

90.330.71

90.170.95

90.170.95

90.001.15

90.501.23

2

90.000.52
NS


89.500.85
NS


89.830.79
NS


86.160.95*

85.501.12*

4

90.330.42
NS


86.330.56*

79.000.58**

73.830.42**

76.170.79**

6

90.160.31
NS


81.330.80**

75.830.48**

64.670.42**

69.830.31**

8

90.000.37
NS


79.330.73**

70.000.52**

56.830.70**

59.990.67**





* = Significant decrease as compared to zero hour level ( P<0.05)
**=Highly significant decrease as compared to zero hour level (P<0.001)
NS
=Non-significant decrease as compared to zero hour level (P>0.05)









66

Fig.2 Effect of Ziabeen and pioglitazone on blood glucose levels (mg/dlSEM) of normal
rabbits at 0-,2-,4-,6-, and 8-hr intervals.


Time interval (hrs)



* = Significant decrease as compared to zero hour level ( P<0.05)
**=Highly significant decrease as compared to zero hour level (P<0.001)
NS
=Non-significant decrease as compared to zero hour level (P>0.05)
NS NS NS
NS
NS
*
**
**
NS
**
**
**
*
**
**
**
*
**
**
**
45.00
55.00
65.00
75.00
85.00
95.00
105.00
0 hr 2 hr 4 hr 6 hr 8 hr
B
l
o
o
d

g
l
u
c
o
s
e

l
e
v
e
l

i
n

m
g
/
d
l

2% tragacanth 2g/kg Ziabeen 3g/kg Ziabeen
4g/kg Ziabeen 1mg/kg pioglitazone
67

Table 2. Mean blood glucose level of alloxan-diabetic rabbits in mg/dl SEM at various time
intervals after oral administration of 2% gum tragacanth aqueous solution, Ziabeen and
pioglitazone.



Time
interval

( hours)

Group I
treated with
2% gum
tragacanth
aqueous
solution
(20 ml)
Group II
treated with
Ziabeen



( 2g/kg)
Group III
treated with
Ziabeen



(3g/kg)
Group IV
treated with
Ziabeen



(4g/kg)
Group V
treated with
pioglitazone



( 1mg/kg)

0

27 4.171.17

275.831.74

274.001.24

274.501.76

273.500.89

2

274.671.15
NS


272.331.58
NS


269.171.01*

229.331.17**

271.000.68
NS


4

274.330.80
NS


265.171.54*

209.500.76**

163.000.97**

269.001.65*

6

274.500.79
NS


261.330.92*

195.001.01**

124.330.95**

269.501.60*

8

274.330.77
NS


257.830.70**

183.000.70**

115.000.91**

268.170.90*







* = Significant decrease as compared to zero hour level ( P<0.05)
**=Highly significant decrease as compared to zero hour level (P<0.001)
NS
=Non-significant decrease as compared to zero hour level (P>0.05)





68

Fig.3 Effect of Ziabeen and pioglitazone on blood glucose levels (mg/dlSEM) of alloxan
induced diabetic rabbits at 0,2,4,6, and 8 hr intervals.
Time interval (hrs)

* =Significant decrease as compared to zero hour level ( P<0.05)
**=Highly significant decrease as compared to zero hour level (P<0.001)
NS
=Non-significant decrease as compared to zero hour level (P>0.05)
NS
NS NS
NS
NS
*
*
**
*
**
**
**
**
**
**
**
NS
*
* *
75.00
125.00
175.00
225.00
275.00
325.00
0 hour 2 hour 4 hour 6 hour 8 hour
B
l
o
o
d

g
l
u
c
o
s
e

l
e
v
e
l

i
n

m
g
/
d
l

2% gum tragacanth 2g/kg Ziabeen 3g/kg Ziabeen
4g/kg Ziabeen 1mg pioglitazone
69

3.1.5. Effect of Ziabeen with and without different units of exogeneous human
insulin in alloxan diabetic rabbits
As shown in table 3; Fig. 4, hypoglycaemic effects of the Ziabeen were compared with
insulin. Blood glucose levels were changed in all treated groups. Results in table showed
that rabbits treated with 6 units of insulin/Kg b.w. and 2 units of insulin plus 4g/kg of
Ziabeen

significantly (P<0.001) reduced blood glucose level in diabetic rabbits with in 2-
hr of drug administration. Hoewever, 3units of insulin along with the Ziabeen

resulted in
severe hypoglycaemia at all time intervals. The administration of 4g/kg Ziabeen also
produced highly significant (P<0.001) hypoglycaemic effects at 2-, 4-, 6- and 8-hr
intervals.
3.1.6 Oral glucose tolerance test (OGTT) in alloxan-diabetic rabbits
OGTT has been performed in our work. Table 4 and Fig.5 have shown the changes in
blood glucose levels in all group after oral administration of glucose load (4g/kg).
Results indicate the correction in impairment of glucose tolerance in the animals occurs
due to Ziabeen which occurs usually in diabetes. Diabetic-control rabbits showed highly
significant (P<0.001) increase in blood glucose concentration at 120-min after glucose
load of 4g/Kg b.w of animal. Glucose level in diabetic control group remained high till
5-hr. However, diabetic rabbits administered with 4g/kg dose of Ziabeen, a highly
significant (P<0.001) decrease blood glucose was observed from 30 min onward.
Pioglitazone showed peak glucose concentration at 150-min and blood glucose levels
were significantly (P<0.05) increase till 5-hr.

70

Table 3. Effect of Ziabeen alone and in combination with different doses of insulin
at 0,2,4,6,8 hours intervals on blood glucose levels (mg/dlSEM) in alloxan-induced
diabetic rabbits.



























* = Significant decrease as compared to zero hour level ( P<0.05)
**=Highly significant decrease as compared to zero hour level (P<0.001)
NS
=Non-significant decrease as compared to zero hour level (P>0.05)




Time
interval
(in hours)

Group I
treated with
6 units of
insulin/kg
Group II
treated with 3
units of insulin
+
4g/kg
Ziabeen
Group III
treated with 2
units of insulin
+
4g/kg
Ziabeen
Group IV
treated with
4g/kg Ziabeen



0

277.52.60

276.832.30

277.832.99

277.662.58

2

188.501.98**

147.331.84**

168.662.98**

233.831.38**

4

95.161.06**

66.161.47**

112.671.71**

166.001.21**

6

59.000.93**

38.501.37**

86.001.60**

128.161.20**

8

48.830.83**

24.001.25**

37.831.12**

113..501.23**
71

Fig. 4 Effect of Ziabeen alone and in combination with different doses of insulin at 0-,2-,4-
,6-,8-hr intervals on blood glucose levels (mg/dl) in alloxan-induced diabetic rabbits.



Time interval(hours)




* = Significant decrease as compared to zero hour level ( P<0.05)
**=Highly significant decrease as compared to zero hour level (P<0.001)
NS
=Non-significant decrease as compared to zero hour level (P>0.05)


**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
-50.00
0.00
50.00
100.00
150.00
200.00
250.00
300.00
350.00
0 hr 2 hr 4 hr 6 hr 8 hr
B
l
o
o
d

g
l
u
c
o
s
e

l
e
v
e
l

i
n

m
g
/
d
l

6 unit insulin 3 unit insulin+4g Ziabeen
2 unit insulin+4g Ziabeen 4g/kg Ziabeen
72

Table 4. Effect of Ziabeen and pioglitazone on oral glucose tolerance test (OGTT)
(mg/dlSEM) in alloxan-induced diabetic rabbits.




Time interval

(minutes)


Group I treated with
2% gum tragacanth
aqueous solution
(Dabetic control)

(20 ml)
Group II treated
with Ziabeen
(Diabetic treated)


(4g/kg)
Group III treated
with pioglitazone



( 1mg/kg)

At 0 min

280.002.39

280.673.51

279.500.76

After 30 min

371.504.42**

262.673.44*


285.000.97*


After 60 min

361.833.15**


243.163.19**

297.331.14**


After 120 min

385.003.99**


195.673.17**


282.992.22*


After 150 min

361.002.96**


198.172.73**


299.001.97**


After 180 min

350.334.51**


194.332.81**


278.501.61
NS


After 240 min

329.164.41**


187.002.97**


277.601.30
NS


After 300 min

313.563.75**


167.832.91**


276.331.36*




* = Significant change as compared to zero time level ( P<0.05)
**=Highly significant change as compared to zero time level (P<0.001)
NS
=Non-significant change as compared to zero time level (P>0.05)

= Decrease as compare to zero time level

= Increase as compare to zero time level




73

Fig. 5 Effect of Ziabeen and pioglitazone on oral glucose tolerance test (OGTT)
(mg/dlSEM) in alloxan-induced diabetic rabbits.


Time interval(minutes)

* = Significant change as compared to zero time level ( P<0.05)
**=Highly significant change as compared to zero time level (P<0.001)
NS
=Non-significant change as compared to zero time level (P>0.05)

= Decrease as compare to zero time level

= Increase as compare to zero time level


**
**
**
**
**
**
**
*
**
**
** **
**
**
*
**
*
*
NS NS
*
50.00
100.00
150.00
200.00
250.00
300.00
350.00
400.00
450.00
0 min 30 min 60 min 120 min 150 min 180 min 240 min 300 min
B
l
o
o
d

g
l
u
c
o
s
e

l
e
v
e
l

i
n

m
g
/
d
l

Diabetic control(2% gum) Diabetic treated(4g/kg) pioglitazone (1mg/kg)
74

3.1.7. Hypoglycaemic effects of Ziabeen

in alloxan -induced diabetic rabbits on
days 0, 10, 20 and 30.
As shown in Table 5 and Fig.6, no significant (P>0.05) change was observed in blood
glucose level in diabetic-control group after 30 days. However, significant (P<0.05)
hypoglycaemic effect was observed in group III treated with 1mg/kg of pioglitazone on
days 20 and 30, however, non-significant (P>0.05) effect was observed on day 10. The
hypoglycaemic effects were highly significant (P<0.001) at 10
th
,20
th
and 30
th
days in
group treated with 4g/kg of Ziabeen.
3.1.8 Antihyperlipidaemic effects of Ziabeen in alloxan-induced diabetic rabbits
Table 7 shows a significant ( P< 0.001) decrease in cholesterol, triglyceride, plasma LDL
and VLDL levels in rabbits treated with 4g/kg of compound herbal drug after 30 days of
study as compared to untreated alloxan diabetic group b. Whereas, the plasma levels of
HDL-cholesterol of group c treated with 4g/kg of Ziabeen found to be significantly
(P<0.001) increased as compared to untreated alloxan-diabetic group b .
3.1.9 Effect of Ziabeen

on weight in alloxan diabetic rabbits

Results in table 7 showed that highly significant (P<0.001) decrease in weights of
diabetic rabbits treated with 2% gum tragacanth aqueous solution. While weights of
diabetic rabbits treated with 4g/kg Ziabeen

and normal control group were stabilized
and showed non-significant (P>0.05) decrease as compare to day zero.


75

Table 5. Mean blood glucose level of alloxan-diabetic rabbits in mg/dl SEM at various
time intervals after oral administration of 2% gum tragacanth aqueous solution, the
Ziabeen and pioglitazone continuously on days 0-, 10 -, 20- and 30.





Time interval

( Days)


Group I treated with
2% gum tragacanth
aqueous solution
(Dabetic control)
(20 ml)
Group II treated with
Ziabeen


(4g/kg)
Group III treated
with pioglitazone


( 1mg/kg)

0
th
day

276.670.80

279.171.25

276.671.33

10
th
day

276.500.43
NS


255.670.92**

274.501.26
NS


20
th
day

276.830.83
NS


191.830.60**

271.170.87*

30
th
day

276.000.63
NS


121.670.49**

269.671.26*





* = Significant decrease as compared to zero hour level ( P<0.05)
**=Highly significant decrease as compared to zero hour level (P<0.001)
NS
=Non-significant decrease as compared to zero hour level (P>0.05)








76

Fig. 6 Effect of the Ziabeen and pioglitazone on blood glucose levels (mg/dlSEM) of
alloxan-diabetic rabbits at day 0
th
, 10
th
,20
th
and 30
th
.





Time interval (Days)



* = Significant decrease as compared to zero hour level ( P<0.05)
**=Highly significant decrease as compared to zero hour level (P<0.001)
NS
=Non-significant decrease as compared to zero hour level (P>0.05)

NS NS NS
**
**
**
NS
*
*
50.00
100.00
150.00
200.00
250.00
300.00
350.00
0th day 10th day 20th day 30th day
B
l
o
o
d

g
l
u
c
o
s
e

l
e
v
e
l

i
n

m
g
/
d
l

Diabetic control(2% gum) Diabetic treated(4g/kg) pioglitazone(1mg/kg)
77

Table 6. Effect of Ziabeen on plasma cholesterol, triglyceride, LDL, HDL and VLDL levels
(mg/dl SEM) in alloxan-induced diabetic rabbits on day 30
th
.





Groups Total
cholesterol

(mg/dl)
Triglycerides


(mg/dl)
HDLs
cholesterol

( mg/dl)
LDLs
cholesterol

(mg/dl)
VLDL
cholesterol

(mg/dl)
Normal control
a

treated with
2%gum
tragacanth
(20ml)


44.330.42



53.500.43


27.330.58


6.300.58


10.700.46
Diabetic control
b

treated with
2%gum
tragacanth

(20 ml)


64.830.60


123.330.54


23.000.33


17.000.23


24.661.21
Diabetic treated
c
with
Ziabeen
(4g/kg)


48.330.33**
b



58.330.33**
b



29.500.43**
b



7.160.64**
b



11.660.49**
b




Rabbits in each group a-c shows different groups and esteric indicate significant difference
compared to group b.



* = Significant change as compared to group b ( P<0.05)
**=Highly significant change as compared to group b (P<0.001)
NS
=Non-significant change as compared to group b (P>0.05)



78

Fig. 7 Effect of Ziabeen on plasma cholesterol, triglyceride, LDL, HDL and VLDL-
Cholesterol levels (mg/dl SEM) in alloxan-induced diabetic rabbits on day 30.


Groups



Alphabet a-c shows different groups and esteric indicate significant difference compared to
group b.


* = Significant change as compared to group b ( P<0.05)
**=Highly significant change as compared to group b (P<0.001)
NS
=Non-significant change as compared to group b (P>0.05)
**
b
**
b
**
b
**
b
**
b
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
Normal control(a) Diabetic control(b) Diabetic treated (c )
C
o
n
c
e
n
t
r
a
t
i
o
n

i
n

m
g
/
d
l

Total cholesterol Triglyceride HDL cholesterol
LDL cholesterol VLDL cholesterol
79

Table 7. Effect of Ziabeen on weight (KgSEM) in alloxan-induced diabetic rabbits on
days 0, 10, 20 and 30.





Groups

Weight in kg


At 0
th
day

At 10
th
day

At 20
th
day

At 30
th
day
Normal control
a

treated with 2%
gum tragacanth
(20ml)


1.340.01


1.330.01
NS



1.320.01
NS



1.320.01
NS

Diabetic control
b
treated with 2%
gum tragacanth
(20 ml)


1.350.04


1.250.01**


1.170.01**


1.030.01**
Diabetic treated
c
with
Ziabeen
(4g/kg)


1.350.03


1.340.02
NS



1.300.02
NS



1.300.02
NS




Rabbits in each group a-c shows different groups and esteric indicate significant difference
to day 0th.


* =Significant decrease as compared to zero day ( P<0.05)
**=Highly significant decrease as compared to zero day (P<0.001)
NS
=Non-significant decrease as compared to zero day (P>0.05)





80

Fig. 8 Effect of Ziabeen on weight (KgSEM) in alloxan-induced diabetic rabbits after
0
th
,10
th
,20
th
and 30
th
day intervals.



Time interval (Days)

* =Significant decrease as compared to day zero ( P<0.05)
**=Highly significant decrease as compared to day zero (P<0.001)
NS
=Non-significant decrease as compared to day zero (P>0.05)
NS
NS NS
**
**
**
NS
NS NS
0.85
0.95
1.05
1.15
1.25
1.35
1.45
1.55
0th day 10th day 20th day 30th day
w
e
i
g
h
t


i
n

k
g

Normal contol(2% gum tragacanth) Diabetic control(2% gum tragacanth) 4g/kg Ziabeen
81

3.2 DISCUSSION

Diabetes mellitus has been described as the most common endocrine disorder that
impairs glucose homeostasis resulting in severe diabetic complications including
retinopathy, angiopathy, nephropathy and causing neurological disorders due to
perturbation in utilization of glucose (Sharma et al.,2010). The number of people living
with the disease have been reported to be increasing rapidly and huge amount of
resources are spent by government all over the world to combat the menace
(Marx,2002). Diabetes mellitus has ranked seventh leading cause of death and has been
considered third when its fatal complication are taken into account (Trividi,2004).
Diabetes mellitus is now recognize as a serious global health problem (King,1993).
Westernized cultures and population experiencing rapid acculturation, are showing a
sharp rise in non-insulin dependent diabetes mellitus (Bennett and Zimmer,1980). The
prevalence of NIDDM is increasing exponentially (Harris,2000). There are different
approaches to the treatment of diabetes mellitus, like insulin treatment in type 1 diabetes.
Sulphonylureas, which release insulin from pancrease by blocking the ATP-sensitive
potassium channels (Aslam,2002). Biguanides, which decrease the insulin resistance;
Thiazolidinediones,which increases the insulin sensitivity; Alpha-glucosidase inhibitors
like acarbose which decrease glucose absorption from intestine, thereby decreasing
postprandial hyperglycaemia; meglitinides like repaglinide and nateglinide, are insulin
secretagouge (Tripathi et al.,2011).
Despite the presence of known antidiabetic medicine in pharmaceutical market, diabetes
and related complications continued to be major problem. Recently, some medicinal
plants have been reported to be useful in diabetes worldwide and have been used
82

empirically as anti-diabetic and anti-hyperlipidaemic remedies (Mitra et al.,1996; Shukla
et al.,2000). More than 400 plants species having hypoglycaemic activity have been
available in literature (Oliver,1986; Rai,1995), however searching for new anti-diabetic
drugs from natural plants is still attractive because they contain substances which take
alternative and safe effect on diabetes mellitus. Most of plants contain glycosides,
alkaloids, terpenoids, flavonoids and carotenoids etc., these are frequently implicated as
having antidiabetic effect (Loew and Kaszkin,2002). Herbal medicines are often used as
therapeutic remedies in combination with allopathic drugs (Ramesh,2003). Usually
ayurvedic drugs are being used due to their minimum toxicity (Babara,1993). Alloxan
induces chemical diabetes in a wide variety of animal species by damaging the insulin
secreting pancreatic -cells resulting in decrease in endogenous insulin release (Lenzen
and Panten,1988). Numerous studies demonstrated that a variety of plant extracts
effectively lowered the glucose level in alloxan-induced diabetic animals (Nammi et
al.,2003).
In the present study stable hyperglycaemia was observed after eight days of alloxan
injection. Rabbits with blood glucose level more than 200 mg/dl were used for further
study. The use of medicinal plants has a long folk history for the treatment of diabetes
mellitus (Shoaib, 1992; Marles and Fransworth, 1996). Ziabeen is a polyherbal
commercial drug being quite commonly prescribed empirically as a hypoglycaemic
agent in local traditional medicine. Ziabeen contain some herbal plants whose
hypoglycaemic activity has been reported individually. However, as far as ascertained no
detailed scientific evaluation study was carried out on this compound herbal drug to
determine its efficacy. Therefore, the present study was conducted to determined the
83

antidiabetic, antihyperlipidaemic and weight stabilizing effect of Ziabeen in normal
as well as alloxan-induced diabetic rabbits. Different doses of the Ziabeen were
administered orally after suspending in 2% aqueous gum tragacanth solution to normal
and alloxan-diabetic rabbits. For comparison, effects of a standard oral hypoglycaemic
drug pioglitazone were also observed in these rabbits. The results obtained showed that
2% gum tragacanth aqueous solution used as vehicle in these experiments, did not
produce any significant (P>0.05) reduction in blood glucose level of normal and alloxan
diabetic rabbits (Table 1,2 ; Fig. 2,3). This is in accordance with reported by Akhtar et
al., 1985; Akhtar and Ali, 1984. The administration of 2g/kg of Ziabeen

to the normal
rabbits (as shown in Table 1; Fig. 2) produced significant (P<0.05) decrease at 4 hr and
highly significant (P<0.001) effect was observed at 6 and 8-hr intervals and non-
significant (P>0.05) effect observed at 2 hr. Ziabeen at the dose of 3g/kg body weight
produced highly significant (P<0.001) effect at 4-, 6- and 8-hr and non- significant
(P>0.05) effect at 2hrs. However at higher dose of 4g/kg body weight of Ziabeen
significant (P<0.05) decreasing effect at 2-hr and highly significant (P<0.001) decrease
was observed at 4-, 6- and 8-hr intervals. Standard drug pioglitazone show significant
(P<0.05 or P<0.001) decreasing effect in normal rabbits.
To further explore the hypoglycaemic action of Ziabeen

different doses were also
administered to alloxan-diabetic rabbits. It is clear from data in (Table 2; Fig. 3) that oral
administration of 2g/kg of Ziabeen

significantly (P<0.05 or P<0.001) decrease blood
glucose level at 4-, 6- and 8-hr intervals and non-significant (P>0.05) decrease at 2-hr
was observed. The treatment with 3g/kg produce significant(P<0.05) effect at 2-hr and
highly significant (P<0.001) decrease effect was seen at 4-,6- and 8-hr. However, higher
84

dose of Ziabeen

produced highly significant (P<0.001) reduction at all time intervals.
And standard drug pioglitazone produced significant (P<0.05) at 4-, 6- and 8-hr and
non-significant response at 2-hr. It is therefore conceivable that hypoglycaemic
constituents in the Ziabeen have exerted hypoglycaemic effect in rabbits by initiating the
release of insulin from pancreatic beta cells in normal rabbits and in alloxan diabetic
rabbits the hypoglycaemic effects could be due to their insulin like effect. These results
(Table 1,2 ; Fig. 2,3) are in line with the previously reported data that showed different
medicinal plants and herbs have the potential to significantly and consistently reduced
blood glucose levels in normal and alloxan diabetic rabbits, rats and human (Akhtar,
1982,1992 ; Akhtar et al., 2009, 2011 ; Mossihuzzaman et al.,1994). Our present results
also shows agreement with ( Akhtar et al.,1981 ; Akhtar and Ali, 1985) who have
reported that Cuminum nigrum (Black cumin) seeds when given at different doses (1, 2,
3 or 4g/kg) produced a significant hypoglycaemic effect in normal and alloxan-diabetic
rabbits. Also Ahmad et al (1999) have reported similar effect of flavonoids isolated
from Cuminum nigrum seeds on blood sugar levels and serum lipid in alloxan diabetic
rabbits. It is clear from the data (Tab 1,2 ; Fig 2,3) that 4g/kg dose of Ziabeen

produced

highly significant effect at all doses so it was selected for further research
experiments.
In the further study synergistic effect of Ziabeen was observed with different doses of
insulin. Our results (Tab 3; Fig 4) showed that oral administration of Ziabeen along
with different units of insulin produce insulin like effect. These results are in accordance
with Ahmad et al (2009). They have reported in their study that co-administration of
aqueous extract of Berberis lycium Royle with various units of insulin produced severe
85

hypoglycaemia in alloxan-induced diabetic rabbits. Results showed in (Table 3 and Fig.
4) that Ziabeen has the ability to reduce the dose of insulin for producing hypoglycaemic
effects in rabbits. Therefore it is predicted that Ziabeen may be a helpful adjunct
therapy to insulin in diabetes.
Further evaluation of Ziabeen

was done by performing the oral glucose tolerance test.
Results in Table 4 and Fig. 5 showed that 4g/kg of Ziabeen

produced highly significant
(P<0.001) effect at all time intervals, these results revealed that Ziabeen

has capacity to
reduced the blood glucose level even after the 4g/kg of glucose load. Control group of
diabetic rabbits showed peak blood glucose concentration at 120-min after glucose load
of 4g/Kg b.w of animal. Glucose level in diabetic control group remained high till 300-
min. Our data was in line with Alarcon-agilar et al (2003), they have reported the P.
peltatum and G. coulteri antihyperglycaemic effect of in rabbits with impaired glucose
tolerance. These results are also in accordance line with Habibuddin et al (2008) their
reported study showed that administration of Caralluma sinica to streptozotocin-
induced diabetic rabbits significantly lowered the plasma glucose level as compared to
diabetic control after glucose load.
More experiments were performed for 30 days for the further evaluation of Ziabeen. So
in separate experiment 4g/kg dose of drug Ziabeen was administered continuously for
30-days that produced highly significant (P<0.001) decrease in blood glucose level in
alloxan-diabetic rabbits. These results in (Tab 5; Fig 6) are similar to the reported study
of Akhtar et al (2009). In which they showed the effect of Lodoicea sechellarum Labill
(seacoconut) fruit on blood glucose and lipid profile in diabetic and normal human
volunteer. Lipids are building blocks of any of the fats and fatty substances found in
86

animals and plants. Lipids are used as hormones that play roles in regulating metabolism
of our body (Wardlaw,1999). Lipid levels may be affected by diet, exercise, smoking
certain medication e.g., beta blockers, thiazide diuretics, glucocorticoids and concurrent
disease states, e.g. kidney and liver diseases.
A lipid profile usually include total lipids, triglycerides, total cholesterol, low density
lipoprotein cholesterol (LDL-cholesterol) and High density lipoprotein cholesterol(LDL-
cholesterol). Factors such age, sex and genetics influence lipid profile (Roberts et
al.,2002). Certain aspects of life style, including diet, level of physical activity, level of
diabetic control and smoking status also effect lipid profiles. Some medical conditions
also raised or lower cholesterol and triglyceride levels. Hypercholesterolemia and
hypertriglyceride have been reportted to occur in alloxan-induced diabetic rabbits
(Wojtoweiz et al.,2004 ; Maciejewski et al.,2001). Diabetic patients are more prone to
athermatous complications such as ischemic heart disease (Batteridge and William,1997
;Way et al.,2001). High density lipoprotein levels are decreased in diabetic patients that
ultimately lead to atheromatous disease (Rang et al.,1995). Mand et al 1991 have
observed a decrease in plasma lipids and cholesterol levels and increase in lipid
mobilization in experimental hypercholesterolemic rabbits when given E.officinalis for
12 weeks. Our results in table 6 showed antiherperlipidemic activity, data showed that
4g/kg dose of Ziabeen

has highly significant (P<0.001) effect in lipid profiles like
total cholesterol, triglyceride, LDL, HDL and VLDL cholesterol levels. Our results are
in accordance with Ahmad et al (2009), they have reported the antihyperlipidemic
effect of crude powder of berberis lyieum roots in alloxan-diabetic rabbits. LDL is
well recognized as a rish factor and HDL-cholesterol as a protective factor against
87

atheroseclerosis(Gordon et al.,1989). VLDL is reported to be increased in different
hyperlipoproteinemias (type II, III, IV and V) .VLDL concentration may also have some
role in atherogenesis (Segal et al.,1984). It was found that Ziabeen treatment cause an
increase in HDL and decrease in LDL levels that probably prevent diabetic patients from
atheromatous disease and may help to save the diabetics from ischemic heart diseases.
Data in (Tab 7; Fig 8) showed the weight stabilizing effect of Ziabeen revealed that
4g/kg dose produced non significant (P>0.05) decrease in weight of alloxan-diabetic
rabbits from day zero. However, highly significant (P<0.001) decrease in weight was
observed in alloxan-diabetic control group our results are in line with reported results of
Ahmad et al (2009). The acute toxicity studies and behavioral pattern records have
indicated that this Ziabeen is quite safe at the dosage employed, as no visible sign of
toxicity or adverse effects were observed in treated animals and no change was observed
in the normal behavioral pattern of tested animals .
Obviously the oral antihyperglycaemic agents from plants and herbs are of great
value in the treatment of any type of severe diabetes as -cells (Islets of Langerhans) in
diabetic patients have been damaged to the extent that they might have lost all their
potential to secrete insulin. The search for more safe and more anti-diabetic agents is
therefore continuing to be an area of active research. This present study in an animal
model has showed that compound herbal preparation, Ziabeen

may be used in human
patient as it produced significant hypoglycaemic effect. However, detailed
pharmacological and phytochemacal studies are still needed to isolate the active
principles and their mechanism(s) of action.

88

3.2.1 Conclusions
The above discussed studies have clearly showed that Ziabeen has exerted significant
and consistent hypoglycaemic, antihyperlipidaemic and weight stabilizing effects
in alloxan-induced diabetic rabbits. These results have also suggested that the drug
contain some constituents that are responsible for its hypoglycaemic action by
stimulation of insulin release from the pancreatic -cells in normal animals as well as
in alloxan-induced diabetic animals the hypoglycaemic effects could also be due to
their direct insulin-like effect. So the present study has supported the use of this
polyherbal preparation, Ziabeen , by the practitioners of the traditional medicine, the
Hakims.


















89


SECTION 4











REFERENCES
















90

REFRENCES

Adewole SO and Ojewole JAO. Protective effect of Annona muricata Linn.Annonaceae
leaf aqueous extract on serum lipid profile and oxidative stress in hepatocytes
of streptozotosin-treated diabetic rats. Afr J Trad Cam 2009; 6(1): 30-41.
Ahmad M, Akhtar MS, Malik T and Gilani AH. Hypoglycaemic acion of the flavonoids
fraction of Cuminum nigrum seeds. Phytother Res 2000; 14(2): 103-106.
Ahmad M, Alamgeer and Sharif T.A potential adjunct of insulin Berberis lycium Royle.
Diabetologia Croatica 2009; 38 (1): 13-18.
Ahmad M, Mahmood Q, Gulzar K , Akhtar MS, Saleem M and Qadir MI. Antihyper-
lipidaemic and hepatoprotective activity of Dodonaea viscosa leaves extracts in
alloxan-induced diabetic rabbits (Oryctolagus cunniculus). Pakistan Veterinary
Journal 2011; 31(x): xxx.

Akhtar MS, Akhtar MA and Yaqub M. Effect of Momordica charantia on glucose levels
of normal and alloxan-diabetic rabbits. Planta Med 1981; 42: 205-12.
Akhtar MS, Ahmad M, Gulzar K and Adnan H. Hypoglycaemic activity of Dodonaea
viscose leaves in normal and alloxan-induced diabetic rabbits. Diabetologia
Croatica 2011; 40(3): 71-79.
Akhtar MS and Ali MR. Study of hypoglycaemic activity of Cuminum nigrum seeds in
normal and alloxan-diabetic rabbits. Planta med 1985; 2: 81-85.
Akhtar MS, Khan QM and Khaliq T. Pharmacological screening of hypoglycaemic
activity of Asparagus racemosus roots and lodoicea sechellarum fruits in rabbits
rabbits. J Pharm Pb Uni Lahore 1987; 8(1-2): 63-70.
Akhtar MS and Iqbal J. Evaluation of the hypoglycaemic effect of Achyranthus aspera
in normal and alloxan-diabetic rabbits. J Ethnopharmacol 1991; 31(1): 49-57.
91

Akhtar MS and Qureshi AQ.Phytopharmacological evaluation of Ficus glomerata,Roxb.
fruit for hypoglycaemic activity in normal and diabetic rabbits. Pak J Pharma Sci
1988; 1(2) : 87-96.
Akhtar MS, Khan S, Bashir S and Salman M. Effect of Lodoicea sechallarum Labill
(Seacoconut) fruit on blood glucose and lipid profile in Type 2 Diabetic and
normal human volunteers. Diabetologia Croatica 2009; 38(4): 87-93.
Akhtar MS. Hypoglycaemic activity of some indigenous medicinal plants traditionally
used as antidiabetic drugs. J Pak Med Assoc. 1992; 42(11): 271-277.
Akhtar MS, Nadeem M, Rashid H and Bashir S. Hypoglycaemic activity of different
fractions of Berberis Aristata root- bark in normal and alloxan diabetic rabbits.
Canadian J App Sci 2011; 1(1): 16-28.
Akhtar MS. Trials of Momordica charantia Linn. ( Karela ) powder in patients with
maturity onset Diabetes. J Pak Med Assoc. 1982; 32(14): 106-107.
Akhtar MS, Ramzan A, Ali A and Ahmad M. Effect of Amla fruit (Emblica officinalis -
Gaertn.) on blood glucose and lipid profile of normal subjects and type 2 diabetic
patients.International Journal of Food Sciences and Nutrition 2011;62(6):609-616.
Akhtar MS and Ali MR. Study of antidiabetic effect of a compound medicinal plant
prescription in normal and alloxan diabetic rabbits. J Pak Med Assoc 1984;34(8):
239-244
Akhtar MS , Naeem F, Muhammad F and Bhatty N. Effect of Butea monosperma
(Lamk.)Taub. (Palas papra) fruit on blood glucose and lipid profiles of normal
and diabetic human volunteer. African Journal of Pharmacy and pharmacology
2010; 4(8): 539-544.
Akhtar MS, Khan MA and Malik MT. Hypoglycaemic activity of Alpinia galanga
92

rhizome and its extracts in rabbits. Fitoterapia 2002; 73(7-8): 623-628.
Ahkter J, Qureshi R, Rahim F, Moosvi S, Rehman A, Jabbar A, Islam N and Khan MA.
Diabetes in pregnancy in Pakistani women: prevalence and complications in an
indigenous south asian community. Diabetic medicine 1996; 13(2): 189-191.
Alarcon - aguilara FJ, Roman - Ramos R, Perez - Gutierraz MS, Aguilar - Contreras A,
Contreras-Weber CC and Flores-Saenz JL. Study of the antihyperglycaemic effect
of plants used as antidiabetics. Journal Ethnopharmacol 1998; 61: 101-110.
Alarcon-Aguilar FJ, Banderas-Dorantes T, Gutierrez-Leon A, Vazquez-Carrillo L,Flores
-Saenz JL and Roman-Ramos RR. Study of anti-hyperglycemic effect of anti-
diabetic plants in rabbits with impaired glucose tolerance. Proc West Pharmacol
Soc 2003; 46: 148-152.
Alberti KGGM and Zimmet PZ. Defination, diagnosis and classification of diabetes
mellitus and its complications. Part 1: diagnosis and classification of diabetes
mellitus. Provisional report of a WHO consultation. Diabetes Medicine 1998;
15: 539-553.
Alemzadeh R and Wyatt DT. Diabetes mellitus. In: Kliegman RM editor .Nelson text-
book of Pediatrics. 18
th
Edn. Saunders 2007; 590.
American Diabetes Association. Report of the expert committee on the diagnosis and
classification of diabtetes mellitus. Diabetes Care 1997; 20: 1183-1201.
American Diabetes Association Standards of medical care in diabetes. Diabetes Care
2009;32(Supp l):S13-61.
Arieff AJ and Carroll HJ. Non-ketotic hyperosmolar coma with hyperglycemia: Clinical
features, pathophysiology, renal function,acid-base balance, plasma-cerebrospinal
fluid equilibria and the effects of therapy in 37 cases. Medicine (Baltimore)
1972; 51: 73-94.
93

Aslam M. Aspects of asian medicine and it as practice in west. In: trease and evans
Pharmacognosy Evans WC. Saunders company Ltd, Edinburg London: Newyork
press 2002; 467-481.
Assan R, Perronne C, Assan D, Chotard L, Mayaud C and Matheron S. Pentamidine-
induced derangements of glucose homeostasis. Diabetes Care 1995; 18: 47-55.
Augusti KT and Benaim ME. Effect of essential oil of onion (allyl propyl disulphide) on
blood glucose, free fatty acids and insulin levels of normal subjects. Clin Chim
acta 1975; 60(1): 121-123.
Babara M and Lawrence C. Development, implementation and result of a successful
multidisciplinary adverse drug reaction reporting programme in a university
teaching hospital. Hospital Pharmacy 1993; 28: 1199-1240.
Baily CJ and Day C. Traditional plant medicine as treatments for diabetes. Diabetes
care 1989; 12: 553-563.
Batterridge J and Williams JCG. Lipid disorder in diabetes mellitus. Text book of
diabetes Blackwell science. London
Barrett TG, Bundey SE and Macleod AF. Neurodegeneration and diabetes: UK nation-
wide study of Wolfram (DIDMOAD) syndrome. Lancet 1995; 346(8988):
1458-1463.
Bennett PH and Knowler WC. Increasing prevalence of diabetes in Pima Indians over a
ten year period. Diabetes Med 1980; 1: 507-511.
Binder C and Brange J. Insulin chemistry and pharmacokinetics.In:Porte D and Sherwin
Jr R (eds) Ellenberg's and Rifkin's Diabetes Mellitus, edn 5
th
.Appleton and Lange,
Stamford 1997; 689.
Black C, Donnelly P, McIntyre L, Royle PL, Shepherd JP and Thomas S. Meglitinide
analogues for Type 2 diabetes mellitus. Cochrane Database Syst Rev. 2007;
(2): CD004654.
Boulton AJM, Gries FA and Jervell JA. Guideline for the diagnosis and outpatient man
-agement diabetic peripheral neuropathy. Diabetes Medicine1998; 15: 508-514.
Bosi E. Metformin the gold standard in Type 2 diabetes: what does the evidence tell us?
Diabetes Obesity Metabolism 2009; 11(2): 3-8.
94

Brenner BM, Cooper ME, de Zeeuw D, Keane WF, Mitch WE, Parving HH, Remuzzi
G, Snapinn SM, Zhang Z and Shahinfar S. Effects of losartan on renal and
cardiovas-cular outcomes in patients with type 2 diabetes and nephropathy.N Engl
J Med. 2001; 345(12): 861-869.
Broadhurst CL,Polansky MM and Anderson RA. Insulin like biological activity of culin-
ary and medicinal plants extracts in vitro. J Agri Food Chem 2000; 48: 849-852.
Buchanan TA, Xiang AH, Peters RK, Kjos SL, Marroquin A, Goico J, Ochoa C, Tan S,
Berkowitz K, Hodis HN and Azen SP. Preservation of pancreatic -cell function
and prevention of type 2 diabetes by pharmacological treatment of insulin
resistance in high-risk hispanic women. Diabetes 2002; 51: 2796-2803.
Burke JP, Williams K, Gaskill SP, Hazuda HP, Haffner SM and Stern MP. Rapid rise in
the incidence of type 2 diabetes from 1987 to 1996, results from the San Antonio
Heart Study. Arch Int Med. 1999; 159: 1450-1456 .
Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA and Butler PC. -Cell deficit
and increased -cell apoptosis in humans with type 2 diabetes. Diabetes
2003; 52: 102-110.
Byrne MM, Sturis J, Menzel S, Yamagata K, Fajans SS, Dronsfield MJ, Bain SC,
Hattersley AT, Velho G, Froguel P, Bell GI and Polonsky KS. Altered insulin
secretory response to glucose in diabetic and nondiabetic subject with mutations
in the diabetes susceptibility gene MODY 3 on chromosome 12. Diabetes
1996; 45: 1503-1510 .
Cai J and Boulton M. The pathogenesis of diabetic retinopathy: old concepts and new
questions. Eye 2002; 16: 242-60.
Calara F, Taylor K, Han J, Zabala E,Carr EM,Wintle M and Fineman M A. Rabdomized
open label crossover study examining the effect of infection site on bioavailability
of exenatide (synthetic exendin-4) Clinical therapy 2005; 27(2): 210-215
Campbell L. Drug Review: Oral antidiabetic drugs, their properties and recommended
use. Journal of Prescribing and medicine management 2007; 18(6): 56-74.
Carr DB and Gabbe S. Gestational diabetes, detection, management and implications.
Clinical Diabetes 1998; 16(1): 4-11.
95

Carpenter MW. Gestational diabetes,pregnancy, hypertension and late vascular diabetes
Diabetes Care 2007; 30(suppl 2): S246-S250.
Chan SJ, Seino S, Gruppuso PA, Schwartz R and Steiner D. A mutation in the B chain
coding region is associated with impaired proinsulin conversion in a family with
hyperproinsulinemia. Proc Natl Acad Sci. USA 1987; 84: 2194-2197.
Chawdhury SA, Dodson EJ and Dodson GG et al. The crystal structure of three non-
pancreatic human insulins. Diabetologia 1983; 25: 460-464.
Chiasson JL, Josse RG, Gomis R, Hanefeld M, Karasik A and Laakso M: Acarbose for
prevention of type 2 diabetes mellitus: the STOP-NIDDM randomized trial.
Lancet 2002; 359: 2072-2077.
Clement K, Pueyo ME, Vaxillaire M,Rakotoambinina B, Thuillier F and Passa P. Asses-
sment of insulin sensitivity in glucokinase - deficient subjects. Diabetologia
1996; 39: 82-90.
Cooper GJS, Willis AC, Clark A, Turner RC, Sim RB and Reid KBM. Purification and
characterization of a peptide from amyloid-rich pancreases of type 2 diabetic
patients. Proc Natl Assoc Sci USA 1987; 84: 8628-8632.
Cook MH, Free AH and Giordano A. The Accuracy of Urine Sugar Tests. Am J Med
Tech 1953;19:283.
Creager MA and Libby P.Peripheral artery disease.In: Braunwald E,Zipes DP and Libby
P.eds. Heart disease:A text book of cardiovascular medicine,6
th
ed. Philadelphia,
pa: saunders company. 2001; 1027-1028.
David MN, James ES and Daniel. The epidemiology of cardiovascular disease in type 2
diabetes mellitus, how sweet it is or is it? Lancet 1997; 350(suppl.1): S14-S19.
Davis SN. Management of type 2 diabetes mellitus with basal prandial insulin therapy:
96

A case based review. Insulin 2007; 2: 118-126.
DeFronzo RA. Lilly Lecture 1987, the triumvirate: cell, muscle, liver: a collusion
responsible for NIDDM. Diabetes 1988; 37: 667-687.
DeFronzo RA, Bergental RM and Cefalu WT. Efficacy of inhaled insulin in patient with
type 2 diabetes not controlled with diet and exercise. Diabetes care 2005;28
:1922-1928.
Derosa G and Sibilla S. Optimizing combination treatment in the management of Type 2
diabetes. Vascular Health Risk Management 2007; 3(5): 665-671.
Del PS, Bianchi C and Marchetti P. -cell function and anti-diabetic pharmacotherapy.
Diabetes Metabolism Research Rev 2007; 23(7): 518-527.
Devendra D, Liu E and Eisenbarth GS. Type 1 diabetes recent developments. B M J
2004; 328: 750-754.
Drummond K and Mauer M. The early natural history of nephropathy in type 1 diabetes,
II: early renal structural changes in type 1 diabetes.Diabetes 2002; 51: 1580-1587.
Drucker DJ and Nauck MA. The incretin system: glucagon- like peptide-1 receptor
agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet 2006;
368: 1696-1705.
Dunger DB, Sperling MA and Acerini CL, Bohn DJ, Daneman D and Danne TPA.
ESPE/LWPES consensus statement on diabetic ketoacidosis in children and
adolescents. Arch Dis Child 2004; 89: 188-194.
Engelgau MM, Geiss LS, Saaddine JB, Boyle JP, Benjamin SM, Gregg EW, Tierney
EF, Rios-Burrows N, Mokdad AH, Ford ES, Imperatore G and Narayan KM.The
evolving diabetes burden in the United States. Ann Intern Med 2004;40:945-950.
Engerman RL. Pathogenesis of diabetic retinopathy. Diabetes 1989; 38: 1203-1209.
Esposti MD, Ngo A and Myers MA. Inhibition of mitochondrial complex I may
account for IDDM induced by intoxication with rodenticide Vacor. Diabetes
1996; 45: 1531-1534.
Expert committee on the diagnosis and classification of diabetes mellitus. Diabetes care
2003; 26: 3160-3167.
97

Fishbein H and Palumbo P. Acute Metabolic Complications in Diabetes. In (Eds.)
Diabetes in America 1995; 50(95): 1468-95.
Forrest JA, Menser MA and Burgess JA. High frequency of diabetes mellitus in young
patients with congenital rubella. Lancet 1971; 1: 332-334.
Franz MJ, Bantle JP, Beebe CA, Brunzell JD, Chiasson JL, Garg A, Holzmeister LA,
Hoogwerf B, Mayer-Davis E, Mooradian AD, Purnell JQ and Wheeler M.
Evidence-based nutrition principles and recommendations for the treatment and
prevention of diabetes and related complications.Diabetes Care 2002; 25:148-198.
Fransworth NR. Ethnopharmacology and drug development. In Chadwick DJ and Marsh
J (eds). Ethnobotany and the Search for New Drugs, CIBA Foundation
symposium. John Wiley and Sons Chichester New York 1994; 185: 42-51.
Fransworth RR and Segelman AB. Hypoglycaemic plants.Tile and till 1971; 57: 52-55.
Frati-Munari AC, Valle D and Ariza ACR. Hypoglycaemic action of different doses of
nopal (Opuntia streptacanth Lamarie ) in patients with type II diabetes mellitus
Arch Invest Med 1988; 20(2): 197-201.
Freeman JS. New therapeutic options: Management strategies to optimize glycaemic
control. J Am Osteopath Assoc 2010; 110(3 suppl 2): 515-520.
Gondwe M, Kamadyaapa DR, Tufts MA, Chuturgoon AA and Musabayane CT.
Sclerocarya birrea [(A. Rich.) Hochst.] [Anacardiaceae] stem-bark ethanolic
extract(SBE) modulates blood glucose,glomerular filtration rate (GFR) and mean
arterial blood pressure (MAP) of STZ-induced diabetic rat. Phytomedicine
2008; 15: 699-709.
Gordois AP, Scuffham, Shearer A and Oglesby A. Health care costs of diabetic nephro-
pathy in United States and United Kingdom. Journal of Diabetes complications.
2004; 18(1): 18-26.
Gordon DJ and Rifkind BM. High density lipoprotein the clinical implication of recent
98

studies. N Engl J Med 1989; 321: 1311-1316.
Grover JK, Yadav S and Vats V. Medicinal plants of India with anti-diabetic potential.
J Ethnopharmacology 2002; 81:81-10.
Grassi G. Diabetic Retinopathy. Minerva Medicine 2003; 94(6): 419-35.

Gullo L,Pezzilli R and Morselli-Labate AM. The Italian pancreatic Cancer study Group.
Diabetes and the risk of pancreatic cancer. N Engl J Med 1994; 331: 81-84.
Gupta R, Gabrielsen B and Ferguson 1FM. Nature s Medicines: Traditional knowledge
and Intellectual Property Management.Case Studies from the National institutes
of Health ,USA Current Drug Discovery Technologies 2005; 2: 203-219.
Habibuddin M, Daghriri HA, Humaira T, Al-Qahtani MS and Hefzi AH. Antidiabetic
effect of alchoholic extract of Caralluma sinaica L. on streptozotocin-induced
diabetic rabbits. Journal of Ethnopharmacology 2008;117: 215-220.
Haffner SM and Steven M. Reducing cardiovascular disease risk across the continuum
of glucose tolerance. Albert Einstein College of Medicine 2004.
HerbalGram . American botanical council. 1997; 40: 21.
Haneda M, Kikkawa R, Horide N, Togawa M, Koya D and Kajiwara N. Glucose
enhances type IV collagen production in cultured rat glomerular mesangial
cells. Diabetologia 1991; 34: 198-200.
Harris SB and Zinman B.Primary prevention of type 2 diabetes in high risk populations.
Diabetes care 2000; 25: 879-881.
Herrera-Arellano A, Aguilar-Santamaria L, Garcia-Hernandez B, Nicasio-Torres P and
Tortoriello J. Clinical trial of Cecropia obtusifolia and Marrubium vulgare leaf
extracts on blood glucose and serum lipids in type 2 diabetics. Phytomedicine
2004; 11: 561-566.
Helmrich SP, Ragland DR and Paffenberger RS. Prevention of non-insulin diabetes
mellitus with physical activity. Med Sci Sports Exerc 1994; 26: 824-850.
99

Hinnen D, Nielsen LL, Waninger A and Kushner P. Incretin mimetics and DPP-IV
inhibitors, new paradigms for the treatment of Type 2 diabetes. J Am Board Fam
Med 2006; 19(6): 612-620.
Howanitz PJ and Howanitz JH.Carbohydrates. In: Henry JB, ed. Clinical diagnosis and
management by laboratory methods.Philadelphia: W.B. Saunders, 1984; 168-169.
International diabetic federation Atlas.2006 showing prevalence of diabetes in 2007 and
future projection for 2025. Updated 2009.
Inzucchi SE and McGuire DK. New drug for the treatment of diabetes : part ii: incretin
based therapy and beyond. Circulation 2008; 117(4): 574-584.
Jadoon MZ,Dineen B,Bourne RR,Shah SP,Khan MA and Jhonson GJ et al.Prevalence of
blindness and visual impairement in Pakistan: The Pakistan national blindness
and visual impairement survey. Invest ophthalmology Vis Sci 2006; 47: 49-57.
Johansen OE and Birkeland KI. Defining the role of repaglinide in the management of
Type 2 diabetes mellitus:a review Am. J Cardiovasc. Drugs 2007; 7(5): 319-335.
Johansen K. Efficacy of metformin in the treatment of NIDDM. Meta - analysis.
Diabetes Care 1999; 22(1):33-7.
Jonsson J, Carlsson L, Edlund T and Edlund H. Insulin- promoter-factor 1 is required for
pancreas development in mice. Nature 1994; 371: 606-609.
Kaleem K, Sarmad SH and Bano B. Protective effect of Piper nigrum and Vinca rosea
in alloxan-induced diabetic rats. Indian Journal of Physiology Pharmacology
2005; 49- (1): 65-71.
Kahn CR,Flier JS, Bar RS, Archer JA, Gorden P and Martin MM et al.The syndromes
of insulin resistance and Aconthosis Nigricans. N Engl J Med 1976; 294: 739-45.
Kahn SE, Andrikopoulos S and Verchere CB: Islet amyloid:a long recognized but
under appreciated pathological feature of type 2 diabetes. Diabetes
1999; 48:241 253.
Kausar A, Akhtar MS, Kausar T and Wolever TMS. Blood glucose responses to tradi-
tional South Asian vegetable dishes in normal diabetic human subjects. Nutrional
Res 2000; 22(9): 989-996.
100

Kesari AN, Gupta RK, Singh SK, Diwakar S, and Watal G. Hypoglycaemic and anti-
hyperglycaemic activity of Aegle marmelos seed extract in normal and diabetic
rats. Journal of Ethnopharmacology 2006; 107: 374-379.
Khan A, Safdar M, Khan MMA, Khattak KN and Anderson AR. Cinnamon improves
glucose and lipids of people with type 2 diabetes. Diabetes care 2003;26:3215-18.
Khan AKA, Akhtar MS and Mehtab H. The treatment of diabetes mellitus with coccinia
indica. BMJ 1980; 280: 1044.
Khan KS, Rizvi JH, Qureshi RN and Mazhar R. Gestational diabetes in developing
country, experience of screening at Aga khan university Medical Centre, Karachi.
J Pak Med Assoc. 1991; 41: 31-33.
Khosla P, Bhanwra S, Singh J, Seth S and Srivastava RK. A study of hypoglycaemic
effects of Azadirachta indica(Neem) in normal and alloxan diabetic rabbits.Indian
Journal of Physiology Pharmacology 2000; 44(1): 69-74.
King ML, Bidwell D, Shaikh A,Voller A and Banatvala JE. Coxsackie -B- virus specific
IgM response in children with insulin-dependent (juvenile-onset;type 1) diabetes
mellitus. Lancet 1983; 1; 1397-1399.
King H and Rewers M. WHO Ahoc diabetes reporting group-Global estimate for preval-
ence of diabetes mellitus and impaire glucose tolerance test in adults. Diabetes
care 1993; 16: 157-177.
Klip A and Leiter LA. Cellular mechanism of action of metformin. Diabetes care
1990; 13(6): 696-704.
Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF, Lachin JM, Walker EA and
Nathan DM.Reduction in the incidence of type 2 diabetes with lifestyle intervene-
tion or metformin. N Engl J Med 2002; 346: 393-403.
Koppel VS, Choe HM and Sweet BV. Managed care perspective on three new agent
agents for type 2 diabetes. J of Management Care Pharm 2008; 14(4): 363-380 .
Kong JM, Goh NK, Chia LS and Chia TF. Recent advances in traditional plants drug
and orchids. Acta Pharmacological Sci 2003; 24: 7-21 .
101

Kreisberg RA. Lactate homeostasis and lactic acidosis. Ann International Medicine
1988; 92: 227-37.
Krentz AJ and Bailey CJ. Oral antidiabetic agents: current role in Type 2 diabetes
mellitus. Drugs 2005; 65(3): 385411.
Kumar P and Clark M. Diabetes mellitus and other disorder and other disorders of
metabolism. Clinical Medicine 2002; 2: 1069-1071.
Kumar NJ and Loganathan P. Hypoglycaemic effect of Spinacia oleracea in alloxan-
induced diabetic rat. Global J Biotechnology Biochemistry 2010; 5: 87-91.
Kurger DF and Gloster MA.Pramlinitides for the treatment of insulin-requiring diabetes
diabetes mellitus. Drugs 2004; 63(13): 1419-1431.
Lambert AP, Gillespie KM and Thomson G et al. Absolute risk of childhood-onset type
1 diabetes defined by human leukocyte antigen class II genotype: a population
base study in the United Kingdom. J Clin Endocrinology Metab 2004;89:4037-43.
Laurence DR and Bacharach AL.Evaluation of drug activities :Pharmacometrics,
Academic Press London New York, 1964; 1: 33-37.
Larsen S, Hilsted J, Tronier B and Worning H. Metabolic control and B cell function
in patient with insulin-dependent diabetes mellitus secondary to chronic pancreas-
titis. Metabolism 1987; 36: 964-67.
Lenze S and Panten U. Alloxan history and mechanism of action. Medicine Diabetologia
1988; 31: 337-342.
Loew D and Kaszkin M.Approaching the problem of bioequivalence of herbal medicinal
products. Phytotherapy Res 2002; 16: 705-711.
Lorenzo A, Razzaboni B, Weir GC and Yankner BA. Pancreatic islet cell toxicity of
amylin associated with type 2 diabetes mellitus. Nature 1994; 368: 756-760.
Ludvik B, Nolan JJ, Baloga J, Sacks D and Olefsky J. Effect of obesity on insulin
resistance in normal subjects and patients with NIDDM. Diabetes 1995;
44:1121-1125 .
Luzi LM. Pancrease transplantation and diabetic complications. New England Journal of
Medicine 1998; 339: 115-117.
MacFarlane IA. Endocrine diseases and diabetes mellitus.In : Pickup JC, Williams G
102

eds. Textbook of diabetes 2
nd
edn. Oxford.Blackwell 1997; 64: 1-64.
Maciejewski R, Rucinski P, Burski K and Figura T. Changes in glucose, cholesterol and
serum lipid fractions levels in experimental diabetes. Ann Univ Mariae Curie
Sklodowska 2004; 59: 363-368.
Maitra A and Abbas AK. The endocrine system. In: Kumar V, Abbas AK, Fausto N.
eds. Robbins and Cotran: Pathologic basis of disease, Philadelphia Elseiver 7
th

edition 2005; 1155-1205.
Mall GK, Mishra PK and Prakash V. Antidiabetic and hypolipidaemic activity of
Gymnema sylvestre in alloxan-induced diabetic rats. Global J of Biotechnology
Biochemistry 2009; 4(1): 37-42.
Mallick C , Chatterjee K, Guhabiswas M and Ghosh D. Antihyperglycaemic effect of
separate and composite extract of roots of Musa paradisiacea and leaf of coccinia
indica in streptozotocin-induced diabetic male alnino rats. Afr J Trad Com
2007; 4(3): 362-371 .
Mana S, Singhal S, Sharma NK and Singh D. Hypoglycaemic effect of Holarrhena-
antidysenterica seeds on streptozotocin-induced diabetic rats.International journal
of Pharm Tech Research 2010; 2(2): 1325-1329.
Mand JK, Soni GL, Gupta PP and Singh R. Effect of Amla (Emblica officinalis ) on the
development of atheroseclerosis on hypercholesterolaemic rabbits. Department of
biochemistry Punjab Agricultural University, Ludhiana J Res Edu 1991;10(2):1-7.
Marquis VO, Adanlwo TA and Olaniyi AA. The effect of foetidin from Momordica
foetida on blood glucose of albino rats. Planta Medicine 1977; 31: 367-74.
Marles RJ and Fransworth R.Antidiabetic plants and their active constituents: An update
update report, J Botanical Med Assoc 1996; 1(3): 85-135.
Marx J. Unraveling the cause of diabetes. Science 2002; 296: 686-689.
Metzer BE, Buchanan TA and Coustan DR. Summary and Recommendations of the
fifth International Workshop - Conference on gestational diabetes mellitus.
Diabetes Care 2007; 30: S251-S260.
Mitra SK, Gopumadhavan S, Muralidhar TS, Anturlikar SD and Sujatha MB. Effect of
the herbomineral preparation D-400 in streptozotocin-induced diabetic rats.
103

Journal of Ethnopharmacology 1996; 54: 41-46.
Mizutani M, Kern TS and Lorenzi M. Accelerated death of retinal microvascular cells
in human and experimental diabetic retinopathy. Journal of Clinical Invest
1996; 97:28-83.

Monnier L and Avignon A. Diet and fitness in type-II diabetes. Rev. Prat. 1999; 49(1):

40-45.
Mossihuzzaman M , Nahar N and Ali L. Hypoglycaemic effect of three plants from
Eastern Himalayan belt. Diabetes Res 1994; 26(3): 127-138.
Murabito JMD, Agostino RB and Silbershatz H. Intermitent claudication. A risk profile
from the Framingham Heart Study. Circulation 1997; 96: 44-49.
Nammi S, Boini MK , Lodagala SD and Behara RBS. The juice of fresh leaves of
Catharanthus roseus Linn. reduces blood glucose in normal and alloxan-diabetic
rabbits. BMC Complement Alternat Med 2003; 3:4.
Nampoothiri SV,Prathapan A,Cherian OL,Raghu KG,Venugopalan VV and Sunderasan
A. In-vitro antioxidant and inhibitory potential of Terminalia bellerica and
Emblica officinalis fruit against LDL oxidation and key enzymes linked to type 2
diabetes. Food Chem Toxicology 2010; 49: 125-130.
National diabetes fact sheet. American diabetes association. Heart diseases and stroke
accessed 2005.
Nathan DM. Finding new treatments for diabetes how many, how fasthow good?
N Engl J Med 2007; 356(5): 437-440.
Nesto RW, Bell D and Bonow. Thiazolidinedione use, fluid retention and congestive
heart failure: a consensus statement from the American diabetes association.
Circulation 2003; 108: 2941-2948.
Notkins AL and Lenmark A.Autoimmune type 1 diabetes:resolved and unresolved issue.
J Clin invest 2001; 108: 1247-1252.
Oliver-Bever B. Oral hypoglycaemic action of medicinal plants in tropical West Africa.
Cambridge university press, London 1986; 245-267.
Okyar A, Can A, Akev N, Baktir G and Sutlupinar N. Effect of Aloe vera leaves on
104

Blood glucose level in type I and type II diabetic rat models. Phytotherapy
Research 2001; 15(2): 157-161.
OLoughlin A, McIntosh C, Dinneen SF and OBrien T. Basic concepts to novel
therapies. A review of diabetic foot. International J Lower Extremity Wounds
2010; 9: 90-102.
Owens DR et al. A review of pharmacokinetics, clinical benefits, and safety issues of
currently available insulins analogues, Insulin today and beyond. Lancet
2001; 358: 739.
Pak CY, Eun H, McArthur RG and Yoon J. Association of cytomegalo-virus infection
with autoimmune type 1 diabetes. Lancet 1988; 1: 1-4.
Perkins JM, Dunn JP and Jagasia SM. Perspectives in gestational diabetes mellitus: a
review of screening, diagnosis, and treatment.Clinical Diabetes 2007;25(2):57-62.
Pfeifer MA, Halter JB, Judzewitsch RG, Beard JC, Best JD, Ward WK and Porte D Jr.
Acute and chronic effects of sulfonylurea drugs on pancreatic islet function in
man. Diabetes Care 1984; 7(1): 25-34.
Philipeon JD. Phytochemistry and medicinal plants. Phytotherapy 2001;56(3):237-243.
Pihoker C, Scott CR, Lensing SY, Cradock MM and Smith J. Non-insulin dependent
diabetes mellitus in African-American youths of Arkansas. Clin Pediatr 1998;
37: 97-102.
Rajagopal K and Sasikala K. Antihyperglycaemic and antihyperlipidaemic effects of
Nymphaea stelleta in alloxan-induced diabetic rats. Singapore Med Journal
2008; 49(2): 137-141.
Rajalaksmi M, Eliza J, Priya CE, Nirmala A and Daisy P. Antidiabetic properties of
Tinospora cordifolia stem extract on streptozotocin-induced diabetic rats.African
Journal of pharmacy and Pharmacology 2009; 3(5): 171-180.
Ramesh G, Jayesh P and Pandit PG. Adverse drug reaction - What we need to know.
Indian Journal of Hospital Pharmacy 2003; 3: 99-102.
Rasmussen H, Zawalich KC, Ganesan S, Calle R and Zawalich WS. Physiology and
pathophysiology of insulin secretion. Diabetes Care 1990; 13(6): 655-666.
105

Rai MK. A review on some antidiabetic plants of india. Ancient Science of life
1995; 14(3): 168-180.
Rang HP, Dale MM and Ritters JM. The endocrine pancreas and the control of blood
glucose : In Simmons B and Beasley S, Editors. Pharmacology U.K. long man
group Ltd 1991; 403-410.
Rang HP, Dale MM, Ritter JM and Flower RJ. Rang and Dales pharmacology: In
atheroseclerosis and lipoprotein metabolism. 6
th
edition, 321-325.
Raskin P. Oral combination therapy: repaglinide plus metformin for treatment of Type 2
diabetes. Diabetes Obes Metab 2008;10(12):11671177.
Ravi K, Rajasekaran S and Subramanian S. Antihyperlipidaemic effect of Eugenia
jambolana seed kernel on streptozotocin induced diabetes in rats. Food and
chemical toxicology, an international journal 2005; 43(9): 1433-1439.
Ravi K,Sivagnanam K and Subramanian S. Antidiabetic activity of Eugenia jambolana
seed kernels on streptozotocin-induced diabetic rats. Journal of Medicinal food
2004; 7(2): 187-191.
Redondo MJ, Fain PR and Eisenbarth GS.Genetics of type 1A diabetes.Recent Progress
Hormone Research 2001; 56: 69-90.
Reichard P, Nilsson BY and Rosenqvist U, The effect of long term intesified insulin
treatment on the development of microvascular complication of diabetes mellitus
N Engl G Med 1993; 329: 304-309.
Rizvi JH, Sadia R, Shamim M, Amin and Ata KM.,Experience with screening for
abnormal glucose tolerance in pregnancy: Maternal and perinatal outcome. Asia
Oceania Journal of Obstetric and Gynaecology. 1992; 18(2): 99-105.
Robison WG Jr, McCaleb ML, Feld LG, Michaelis OE, Laver N and Mercandetti M.
Degenerated intramural pericytes (ghost cells) in the retinal capillaries of
diabetic rats. Curr Eye Res 1991; 10(4): 339-50.
Roa BK, Kesavulu MM and Apparao Ch. Antihyperglycaemic activity of Momordica
cymbalaria in alloxan-diabetic rats. J of Ethnopharmacology 2001; 78(1): 67-71.
Rorsman P, Eliasson L,Renstrom E,Gromada J, Barg S and Gopel S.The cell physiology
106

of biphasic insulin secretion. Physiol Sci 2000; 15: 72-77.
Said M. Hamdard Pharmacopoeia of Eastern medicine. Hamdard National Foundation
Times Press, Karachi, 1969; 379.
Sabu MC and Kuttan R. Antidiabetic activities of medicinal plants and its relationship
with their antioxidant properties. J Ethnopharmacol 2002; 81(2): 155-160.
Saxena AM, Johri S, Sharma P and Gupta R. Blood sugar lowering activity of Swertia
chirayita (Roxb.Ex.Flem) Karst in different experimental rats models. Flora and
fauna 2007; 13(2): 415-418.
Schoonjans K and Auwerx J. Thiazolidinediones ; an update. Lancet 2000; 356(9225):
1008-1010.
Schultz CJ, Konopelska-Bahu T, Dalton RN, Carroll TA, Stratton I,Gale EA, Neil A and
and Dung DB. Microalbuminuria prevalence varies with age, sex, and puberty in
children with type 1 diabetes followed from diagnosis in a longitudinal study.
Oxford regional prospective study group. Diabetes Care 1999: 22: 495-502.
Schubert D, Behl C, Lesley R, Brack A, Dargusch R, Sagara Yand Kimura H. Amyloid
peptides are toxic via a common oxidative mechanism. Proc Natl Acad Sci USA
1995; 92(6) : 1989-1993,
Schubert-Zsilavecz M and Wurglics M. Better blood sugar control in diabetics, insulin
glargin a long- acting insulin analogue. Pharm Unserer Zeit 2001; 30(2): 125-130.
Scott EL. On the influence of intravenous injection of an extract of the pancreas of expe-
imental pancreatic diabetes. American J Physiology 1912; 29(3): 306-310.
Segal P, Bachorik PS, Rifkind BM and Levy RI.Lipid and dyslipoproteinemia.In.clinical
Diagnosis and management by laboratory methods,17
th
edition.eds, Bernard JH,
Nelson DA, Tormar RH and Wahington JA. WB Saunders. Philandelphia
1984; 180-203.
Setji TL, Brown AJ and Feinglos MN. Gestational diabetes mellitus.Clinical diabetes
2005; 23(1): 17-24.
Sexena A and Kishore VN.Role of selected Indian plants in management of type 2 diab-
107

etes. Journal of alternative and complementary medicine 2004; 10(2): 369-378.
Sharma VK,Kumar S,Patel HJ and Hugar S. Hypoglycaemic activity of Ficus glomerata
in alloxan-induced diabetic rats. International Journal of Pharmaceutical Sciences
Review and research 2010; 1(2): 18-22.
Shani JA, Goldschmied A, Joseph B, Alrouson Z and Sulman EG. Hypoglycaemic effect
of Trigonella foenum-graecum and Lupinus Termis (Leguminosae) seeds and their
major alkaloids in alloxan-diabetic and normal rats. Arch Int Pharmacodyn
therapy 1974; 210: 27-37.
Shoaib MA. Hypoglycaemic activities of some indigenous medicinal plants traditionally
used as antidiabetic drugs. J Pak Med Assoc 1992; 42: 271-277.
Sharma SR, Dwivedi

SK and Swarup D. Hypoglycaemic, antihyperglycaemic and
hypolipidaemic activities of Caesalpinia bonducella seeds in rats. Journal of
Ethnopharmacology 1997; 58(1): 39-44.
Shukla R, Sharma SB, Puri D, Pabhu KM and Murthy PS.Medicinal plants for the treat-
ment of diabetes mellitus. Indian J Clinical Biochemistry 2000; 15: 169-177.
Spiegelman BM. PPAR-gamma: adipogenic regulator and thiazolidinedione receptor.
Diabetes 1998; 47(4): 507-514.
Staines A, Hanif S,Ahmed S, McKinney PA, Shera S, Bodansky HJ.Incidence of insulin
dependent diabetes mellitus in Karachi,Pakistan.Arch Dis Child1997;76:121-123.
Steinberger J, Moran A, Hong CP, Jacobs DR Jr and Sinaiko AR. Adiposity in child-
hood predicts obesity and insulin resistance in young adulthood. Journal Pediatr
2001; 138 (4): 469-73.
Steel RGD and Torrie JH. Principles and procedure of statistics. McGraw Hill book
Company Inc New York.1992
Stiegler H. Diabetic foot syndrome. Herz Article in German 200; 29(1): 104-15.
Szkudelski T. The mechanism of alloxan and streptozotocin action B cells of rat pancre-
as. Physiology Res 2001; 50: 537-546.
Tesfaye S, Stevens LK Stephenson JM, Fuller JH, Plater M, Ionescu-Tirgoviste C,Nuber
A, Pozza G and Ward JD. Prevalence of diabetic peripheral neuropathy and its
relation to glycaemic control and potential risk factors: the EURODIAB IDDM
complications study. Diabetologia 1996; 39(11): 1377-1384.
Tierney Jr LM, McPhee SJ and Papadakis MA.Current medical diagnosis and treatment
108

Lange Medical books/McGraw Hills, Newyork. Chicago, 42
th
edition. 2003; 27:
1156.
Thirumalai T, Viviyan -Therasa S, Elumalai EK, and David E. Hypoglycaemic effect of
Brassica juncea (seeds) on streptozotocin-induced diabetic male albino rat. Asian
Pacific Journal Of Tropical Biomedicine 2011; 12: 323-325.
Trividi NA, Mazumder B, Bhatt JD and Hemavathi KG. Effect of Shilajit on blood
glucose and lipid profile in alloxan-induced diabetic rats. Indian J Pharmacology
2004; 36(6): 373-376.
Tripathi AK, Bhoyar PK, Baheti JR,Biyani DM, Khalique M,Kothmire MS, Amgoankar
YM and Bhanarkar AB. Herbal antidiabetic review. International Journal Res
Pharm Sci 2011; 2(1): 30-37.
Turner RC, Cull CA, Frighi V and Holman RR. Glycemic control with diet, sulfonylurea
,metformin or insulin in patients with Type 2 diabetes mellitus: progressive
requirement for multiple therapies. JAMA 1999; 281(21): 2005-2012.
Twaij HAA and Al-Badr AA. Hypoglycaemic activity of Artemisia herba alba. J of
Ethnopharmacology 1988; 24(2-3); 123-126.
Vinik AL. Neuropathy : new concepts in evaluation and treatment. South Med Journal
2002; 95: 21-23
Vinik A. Advancing therapy in type 2 diabetes mellitus with early comprehensive progr-
ession from oral agents to insulin therapy.Clinical Therapy 2007;29:1236-1253.
Wardlaw GM. Prespective in nutrition. 4
th
ed.Boston MA, McGraw Hill. Newyork USA.
1999.
Wadkar KA, Magdum CS, Patil SS and Naikwade NS. Journal of Herbal Medicine and
Toxicology. 2008; 2(1): 45-50.
Wadood N and Wadood A. Effect of Grewia asiatica and peganum harmala on blood
glucose level of normal and alloxan-diabetic rabbits. JPMR 1995; 34: 20-24.
Wadood N, Nisar M, Rashid A, Wadood A, Nawab G and Khan N. Effect of a
compound recipe (medicinal plants) on serum levels of alloxan-induced diabetic
rabbits. J Ayub Med 2007; 19(1): 32-38.
Welty TE. Cerebrovascular.In: Koda-Kimble MA,Young LY, Kradjan WA and Gugl-
iemlo BJ, editors. Applied therapeutics : The clinical use of drugs,7
th
ed. Balti-
109

more. Md: Lippincott Williams and Wilkins. 2001; 53(1): 25-53.
WHO, Legal status of Traditional Medicines and complementary/Alternative medicine:
A worldwide review. WHO publishing 2001.
Williamson JR and Kilo C. Capillary basement membranes in diabetes. Diabetes
1983; 32(2): 96-100.
Wild S, Roglic G, Green A, Sicree R and King H.Global prevalence of diabetes estimate
for the year 2000 and projections for 2030.Diabetes Care 2004; 27(5): 1047-1053.
Wojtowicz Z, Wrona W, Kis G,Blaszak M and Solecka A. Serum total cholesterol, tri-
glyceride and high density lipoprotein (HDL) levels in rabbits during the course
of experimental diabetes. Ann Univ Mariae Curie Sklodowska 2004; 59: 258.
Wolfsdorf J, Craig ME, Daneman D, Dunger D, Edge J, Lee WRW, Rosenbloom A,
Sperling MA and Hanas R. Diabetic ketoacidosis.Pediatric diabetes 2007;8:28-42.
Yamanouchi T, Sakai T, Lgarashi K, Ichiyanagi K, Watanabe H and Kawasaki T.
Comparison of metabolic effects of pioglitazone,metformin and glimepiride over
1 year in Japanese patients with newly diagnosed type 2 diabetes. Diabetic
medicine 2005; 22(8): 980-985.
Yarom R, Zirkin H,Stammler G and Rose AG.Human coronary microvessels in diabetes
and ischaemia. Morphometric study of autopsy material. Journal of Pathology
1992; 166(3): 265-70.