Phytochemical and Pharmacological Evaluation of Medicinal Plants on Type II Diabetic and its Complication.

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PHYTOCHEMICAL AND

PHARMACOLOGICAL EVALUATION OF

MEDICINAL PLANTS ON TYPE

II DIABETIC AND ITS COMPLICATION

Thesis submitted to

The Tamil Nadu Dr. M.G.R. Medical University, Chennai

for the award of degree of

DOCTOR OF PHILOSOPHY

in

PHARMACY

Submitted by

K G PRASANTH, M.Pharm.,

Under the guidance of

Dr. R VANKATANARAYANAN, M.Pharm., Ph.D.,

Principal

RVS COLLEGE OF PHARMACEUTICAL SCIENCES, Sulur, Coimbatore -641402

A

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DECLARATION

This is to certify that the Ph.D. thesis entitled

PHYTOCHEMICAL AND

PHARMACOLOGICAL EVALUATION OF MEDICINAL PLANTS ON

TYPE II DIABETIC AND ITS COMPLICATION

submitted to The Tamil

Nadu Dr. M.G.R. Medical University, Chennai,

for the award of degree of

DOCTOR OF PHILOSOPHY

in

PHARMACY

was carried out by me under the

supervision of

Dr.R.VANKATANARAYANAN, M.Pharm.,Ph.D.,

Principal,

RVS College of Pharmaceutical Sciences, Sulur, Coimbatore - 641 042, Tamil

Nadu, India.

The contents of this thesis, in full or in parts, have not been submitted

to any other Institute or University for the award of any degree or diploma.

K.G. PRASANTH

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ACKNOWLEDGEMENT

I take this opportunity to render my profound sense of gratitude to my

indebtedness and respect full regards to my guide

Dr. R.Venkatanarayanan,

M.Pharm., Ph.D.,

Principal, RVS College of Pharmaceutical Science,

Coimbatore for his assiduous guidance and sedate support during my research

work. Without whom my Ph. D degree would have been only a dream. His wide

knowledge and logical way of thinking have been great value for me.

His understanding, encouraging, personal guidance and care and his

insight have strengthened this study significantly. I will always thank full for his

wisdom, knowledge and deep concern. His constrictive criticism helped me a lot

to focus my views in proper perspectives .I am great full to him for giving me

liberty to carry out my research work independently throughout the course of

study .My respect personal regards are due to him forever.

I am deeply great full to my doctoral committee members

Dr. A.J.M Christina

, Principal, K M College of Pharmacy, Madurai and

Dr. W.D. Sam Solomon

, Professor & Head, Department of Pharmaceutical

analysis RVS College of Pharmaceutical Science, Coimbatore for their

constrictive ideas and suggestion and constant support throughout the work.

I express my grateful tribute to

Department of Pharmaceutics,

RVS

College of Pharmaceutical Sciences for providing me the excellent laboratory

facilities for accomplishing this work.

I also owe to, from the deepest corner of heart and indebtedness to Head of

the Department

Dr. G.Geetha,

PSG College of Pharmacy as I have been

constantly benefited with them and achieved pragmatic direction.

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An endeavor such as a Ph.D. is impossible to accomplish without the

generous help and support of

Seniors

and

Colleagues

. I would like to take this

opportunity to thank those whom I was fortunate to know, work and form

friendship. How could I ever forget

Ms.G.Syamala

,

Dr.K.Umaa,

Mr.B. Prem Kumar, Ms. C.Kalaiarasi,

by whom I was inspired for my doctoral

work. I am very much thankful to him for his technical guidance and

comprehensive exchange of ideas during the course of my research work.

I would like to thank my students

Mr. Ananda Babu and B. Sriram

for

their support and help during the course of my research.

I would like to thank my

wife

Preethi .S

and

daughter

Aishwarya

for

their encouragement and prayers for the successful completion of my research

work.

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CONTENTS

S.No. Topics

Page

no.

LIST OF TABLES

ii

LIST OF FIGURES

iii

LIST OF ABBREVIATIONS

v

1

INTRODUCTION

1

2

AIM AND OBJECTIVE

15

3

LITERATURE REVIEW

16

4

PLAN OF WORK

35

5

PLANT PROFILE

37

6

METHODOLOGY

39

7

RESULTS AND ANALYSIS

72

8

DISCUSSION

117

9

SUMMARY

131

BIBLIOGRAPHY

APPENDIX

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LIST OF TABLES

Sl

No Table No

Title of the table Page

No

01 Table1 Criteria for diagnosis of diabetic 7

02 Table 2 List of plant drug with anti diabetic action 13 03 Table 3 Grouping of normal animals for carbohydrate tolerance test 54 04 Table 4 Grouping of diabetic animals for carbohydrate test 57 05 Table 5 Grouping of animals for the anti diabetic study using

STZ-Nicotiamied 59

06 Table 6 Blood glucose determination 60

07 Table 7 Triglycerides determination 61

08 Table 8 Cholesterol determination 62

09 Table 9 HDL determination 63

10 Table 10 Ash value of the plant Clerodendrum viscosum vent root 72 11 Table 11 Extractive value of the plant Clerodendrum viscosum vent

root 72

12 Table 12 Fluorescent analysis of the plant Clerodendrum viscosum

vent root 73

13 Table 13 Percentage of yield Clerodendrum viscosum vent root 73 14 Table 14 Preliminary phytochemical analysis of the plant extracts

Clerodendrum viscosum vent root 74

15 Table 15 Amount of flavonoid content in different extract

16 Table 16 H NMR data of isolated compound from ethyl acetate

extract of Clerodendrum viscosum . 82

17 Table 17 Standard curve of Ascorbic acid for SOD estimation 84 18 Table 18 Percentage of inhibition of ethyl acetate extract of

Clerodendrum viscosum vent root SOD 84 19 Table 19 Standard curve of Ascorbic acid for estimation of NO 84 20 Table 20 Percentage of inhibition of ethyl acetate extract

Clerodendrum viscosum vent root on NO 85 21 Table 21 Standard curve of Ascorbic acid for the estimation of

Reducing power 85

22 Table 22 Percentage of inhibition of ethyl acetate extract

Clerodendrum viscosum vent root reducing power 85 23 Table 23 The IC 50 values of the ethyl acetate extract of

Clerodendrum Viscosum root 87

24 Table 24 Effect of ethyl acetate extract Clerodendrum viscosum vent

root on Carrageenane induced inflammation 92

24 Table 25 Effect of ethyl acetate extract Clerodendrum viscosum vent

root on oral carbohydrate tolerance test in normal rats 93 26 Table 26 Effect of ethyl acetate extract Clerodendrum viscosum vent

root on oral carbohydrate tolerance test on diabetic rats 93 27 Table 27 Effect of ethyl acetate extract Clerodendrum viscosum vent

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LIST OF FIGURES

SL No

Figure

No Title of the figure

Page No

01 Figure 1 Generation of reactive oxygen species during hyperglycemic

condition 8

02 Figure 2 Chemical structure of STZ 55

03 Figure 3 Chemical structure of Nicotinamide 56

04 Figure 4 Gallic acid standard curve 75

05 Figure 5 HPTLC analysis of the extract of Clerodendrum viscosum

vent root 75

06 Figure 6 HPTLC analysis of isolated fractions of ethyl acetate extract

of Clerodendrum viscosum vent root 76 07 Figure 7 HPTLC peek display of Clerodendrum viscosum vent root 77 08 Figure 8 HPLC study of isolated fraction of ethyl acetate extract

09 Figure 9 FTIR spectrum of isolated compound from ethyl acetate

extract Clerodendrum viscosum vent root 79 10 Figure 10 GCMS spectrum of isolated compound from ethyl acetate

extract Clerodendrum viscosum vent root 79 11 Figure 11 Chemical structure of the isolated a compound 80 12 Figure 12 NMR study of isolated compound of ethyl acetate extract

13 Figure 13 Cell toxicity study of ethyl acetate extract of Clerodendrum

viscosum vent root on PC3 cell lines 83 13 Figure13 Cell toxicity study of ethyl acetate extract of Clerodendrum

viscosum vent root on L6 cell lines 83 14 Figure 14 Effect of alpha amylase inhibitory activity of ethyl acetate

extract of Clerodendrum viscosum vent root 86 15 Figure 15 Effect of alpha glucosidase inhibitory activity of ethyl

acetate extract of Clerodendrum viscosum vent root 87 16 Figure 16 Effect of ethyl acetate extract of Clerodendrum viscosum

vent root on hot plate induced pain 88

17 Figure 17 Effect of ethyl acetate extract of Clerodendrum viscosum

vent root on tail immersion 89

vent root on acetic acid induced writing 90 19 Figure 19 Effect of ethyl acetate extract of Clerodendrum viscosum

vent root on formalin induce pain 91

vent root on body weight of the animals 94 21 Figure 21 Effect of ethyl acetate extract of Clerodendrum viscosum

vent root on blood glucose level of the animal 95 22 Figure 22 Effect of ethyl acetate extract of Clerodendrum viscosum

vent root on total cholesterol 96

vent root on HDL 97

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SL No

Figure

No Title of the figure

Page No

vent root on triglycerides 98

vent root on SOD 100

vent root on CAT 101

vent root on GPx 102

vent root on GSH 102

vent root on LPO 103

vent root on pain sensation in diabetic animals 104 32 Figure 32 Effect of ethyl acetate extract of Clerodendrum viscosum

vent root on nerve conduction study 105

vent root on systolic blood pressure 106 34 Figure 34 Effect of ethyl acetate extract of Clerodendrum viscosum

vent root on diastolic blood pressure 106 35 Figure 35 Effect of ethyl acetate extract of Clerodendrum viscosum

vent root on GLUT 4 expression on L6 cell lines 107

vent root on intensity of expression on GLUT 4 108 37 Figure 37 Effect of ethyl acetate extract of Clerodendrum viscosum

vent root on expression on GLUT 4 in rat skeletal muscles 108 38 Figure 38 Effect of ethyl acetate extract of Clerodendrum viscosum

vent root on pancreas of rat 109

vent root on aorta of rat 112

vent root on kidney of rat 113

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List of Abbreviations

LIST OF ABBREVIATIONS

GAD

:

Glutamic acid decarboxylase

HLA

:

Human leukocyte antigen

MODY

:

Maturity onset diabetes of the young

FPG

:

Fasting blood glucose level

HbA

1

C

:

Glycosylated hemoglobin

OGTT

:

Oral glucose tolerance test

WHO

:

World health organizations

DM

:

Diabetes mellitus

ROS

:

Reactive oxygen species

OS

:

Oxidative stress

AGEs

:

Advanced glycation end products

LDL

:

Low density lipoproteins

VLDL

:

Very low density lipoproteins

HDL

:

High density lipoproteins

GLP

:

Glucagon like peptide

DPP4

:

Dipeptidyl peptidases

CV

:

Clerodendrum viscosum

UV

:

Ultra violet

HPTLC

:

High performances thin layer

chromatography

HPLC

:

High pressure liquid chromatography

NMR

:

Nuclear magnetic resonance

GC/MS

:

Gas chromatography /mass spectrometer

STZ

:

Streptozotocin

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List of Abbreviations

OECD

:

Organization of economic cooperation and

development

SOD

:

Superoxide dismutase

GPX

:

Glutathione per oxidize

CAT

:

Catalase

GSH

:

Reduced Glutathione

LOP

:

Lipid per oxides

GLUT4

:

Glucose transporter 4

L6

:

Rat skeletal muscle cell line

FTIR

:

Fouriertran from infra red spectroscopy

MTT

:

Methyl thiazol tetrazolin bromide

DNA

:

Deoxyribonucleic acid

mRNA

:

Messenger ribonucleic acid

NO

:

Nitricoxide

ATPs

:

Adenosine triphosphate

NADH

:

Nicotinamide adinin dihydrogen

TBAR

:

Thiobarbituric acid reactive substance

TBA

:

Thiobarbituric acid

PCR

:

Polymerized chain reaction

GAPDH

:

Glyceraldehyde -3-phosphate dehydrogenase

IDDM

:

Insulin depended diabetes mellitus

NIDDM

:

Non insulin depended diabetes mellitus

NaCl

:

Sodium chloride

PO

:

Per oral

IP

:

Intra peritoneal

AUC

:

Area under curve

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List of Abbreviations

ml

:

Milliliters

mg

:

Milligram

µg

:

Micro gram

NPG

:

N –Para nitrophenyl gluco-pyranoside

HCl

:

Hydrochloric acid

ATM

:

Automated turning machine

KBR

:

Potassium bromide

SPSS

:

Statistical l soft ware for social science

TLC

:

Thin layer chromatography

AVOVA

:

One –way analysis of variance

H

2

O

2

:

Hydrogen peroxide

w/v

:

Weight / volume

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Introduction

INTRODUCTION

Food or ‘Annam’ is the basic necessity of life and existence of living

organism. We all need food to develop physically and mentally and to work at our

maximum capacity, to build our defense against infections and maintain good

health. The traditional diet of Indians are immense varieties and the diversified

preparations not only offer the whole range of nutrients but also activate digestion

and several physiological functions. Until a few years ago, we used to prefer

natural food over refined food and light food (less oily) over heavy food. Our

traditional meals were mostly plant based and very rarely animal based. Both

plant based and animal based food were freshly cooked and eat at home. They

were a combination of cereals, millets, pulses, and spices such as pepper, cumin,

asafetida and coriander with curd and coconut satisfying our energy and protein

requirements. Those who could afford milk, yoghurt, eggs and chicken with small

amounts of animal meat adding to the protein requirement. Conception of

vegetables and fruits contribute to the intake of essential vitamins, minerals and

antioxidants required for supportive and detoxifying functions. The oils we use are

from groundnut, sesame, mustard and ghee which are essential, in small

quantities, to absorb fat soluble vitamins and also contribute to several hormonal

functions. These oils are the important source of fat in Indian diet. More over

being a source of energy, it also adds special flavor and traditional palatability to

the Indian food. The food we eat are balanced, diversified and freshly prepared

but not stored or processed.

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Introduction

obesity and related problem like the type 2 diabetic and cardiovascular diseases.

These two obesity problems are the major reasons for the morbidity and mortality

in India.

More over the conception of Junk food in India has increased due to

Globalization and fast growing economy. Junk food refers to the food that tastes

good but is high in calories with little nutritional value. Across the world, different

types of diseases and health problems occur due to regular intake of junk food. In

all the developing and developed countries, younger generations are suffering

Diabetes is an oldest disease, described in Egyptian manuscript mentioning

as “too great emptying of the urine”. Type 1 diabetes was the first case to be

described in Egyptian manuscript 1500 BCE. Indian physicians have also

identified the disease in the same period and described as “madhumeha”

“or honey in urine. The Type 1 and Type 2 diabetes was identified and classified

by the Indian physician Sushrutha and Charaka in 400-500AD as type 1 associated

with young people and type 2 is associated with overweight. The effective

measures to treat diabetes started after the invention and usage of insulin by

Frederick Banting and Charles Best in 1921.

2,3

Diabetes mellitus is a complex metabolic disorder characterized by

increased glucose level and relative or absolute insufficiency of insulin, deficiency

or resistance to insulin on target tissues, which leads to characteristic abnormality

in the metabolism of carbohydrate, lipid and protein. It is well established that

diabetes mellitus is a condition with a range of abnormalities like micro and

macro vascular complications, cardiomyopathy, neuropathy, nephropathy and

retinopathy disorders rather than a single disease state.

4

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Introduction

Epidemiology

Epidemiology can help in understanding and management of diabetes in

several ways. The epidemiologic studies can quantify changes in pattern and the

burden of disease. Second, identification of risk factors in large epidemiologic

studies may provide insight into pathophysiologic processes related to diabetes.

The study also allows the various factors involved, such as diet, gene,

environment and life style. Type 1 diabetes is autoimmune disorder found in

children or early adulthood stage which accounts nearly 10 % of the total diabetes.

It was estimated that 2.8% of the world’s population had diabetes in 2000 and this

ratio would climb to be as high as 4.4% of the world’s population by 2030.

5

90-95% of the diabetics belong to type 2 diabetes. The information from the national

diabetic fact sheet released in January 2011 says 25.8 million children and adults

in the United States i.e., 8.3% of the population have diabetes. 1.9 million new

case of diabetes was diagnosed in people aged 20 years and older in the study. In

United States the prevalence of diabetics among the males is 11.8% and in female

is 10.8%. Diabetic incidence is alarmingly increased in the world population also,

it is estimated that the number of diabetic individuals will be double to 180

million in the next 20 years.

6

Based on the report of Diabetes Atlas 2006

published by the International Diabetic Federation, the Indian population with

diabetes will increase to 69.9 million from 40.9 million in 2025.. The diabetic

epidemic in India is so called “Asian Indian Phenotype” which has unique clinical

and biochemical abnormalities.

7

WHO projected that India will become the fastest

growing population of diabetic patients in the world and will become the diabetic

capital of world.

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Introduction

prevalence of diabetics is high in migrated Indian population (2.2%) when

compared with Europeans (1.2%). The occurrence of diabetes is more in south

India when compared with north India. In Tamilnadu, 10.4% of the population has

diabetes when compared with Maharashtra 8.4% and 5.3% in Jharkhand.

9

The

overall probability in prevalence of diabetes differs in various parts of the world.

9.1% in Asia, 8.1% in Europe, 12.1% in North America, 4% in Africa. Multiple

logistic regression analysis showed that age, male sex, family history of diabetes,

urban residence, abdominal obesity, generalized obesity, hypertension and income

status were significantly associated with diabetes. Significant risk factors for pre

diabetes were age, family history of diabetes, abdominal obesity, and hypertension

and income status.

10

Classification of diabetes

The diabetic patients are classified into two broad categories; type1

diabetes cases with an absolute deficiency of insulin and type2 diabetes with an

insulin resistance and inadequate release of insulin secretion.

Type 1 diabetes

Type1 diabetes or juvenile diabetes is an autoimmune disorder of the

pancreatic

β

cell. In the total diabetic population of the world, type 1 diabetes

accounts only 5-10%. Different markers for immune distraction of the

β

- cell

includes islet cell auto anti bodies, auto antibodies to insulin, auto antibodies to

Glutamic Acid Decarboxylase (GAD) and auto anti bodies to the tyrosine

phosphate IA-2 and IA-2

β

. This disease has a strong association to Human

Leukocyte Antigen (HLA) with linkage to DQA and DQB genes.

11

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Introduction

destruction of diabetes has a multiple genetic predispositions and is also related to

environmental factors which are poorly defined. These patients are also have the

chance to get other autoimmune disorders.

Type 2 Diabetes

Type 2 diabetes is characterized by insulin resistance and having relative

lack of insulin secretion. Most of the type 2 diabetic patients are obese and obesity

itself causes insulin resistance. There are many different causes of this form of

diabetes where the specific etiology is not known and no evidence of autoimmune

distraction in the

β

cells. The patients who are not obese may have an increased

percentage of body fat which is distributed predominantly in the abdominal region

which is one of reasons for the development of type 2 diabetes. This form of

diabetes is frequently not diagnosed for long period of time because

hyperglycemia develops slowly and usually diagnosed after development of other

complications. Type 2 diabetes has a highi genetic predisposition and is more

common in all ethnic groups. However, the genetics of this disease are more

complex and not clearly defined.

Gestational diabetes

Gestational diabetes is defined as any glucose intolerance with onset or

first recognition of pregnancy. This type of diabetes complicate nearly 7% of the

total pregnancy, early detection is important to reduce the morbidity and

mortality.

12

Other specific type of diabetes

Several forms of diabetes are associated with monogenetic defects in the

β

cell dysfunctions and which are referred as maturity onset diabetes of the young

(MODY). This form of diabetes is characterized by onset of hyperglycemia at an

early stage (age of 25 years) due to impaired insulin secretion with minimal or no

defect in insulin action. This disease is inherited in autosomal dominant pattern

with at least six different genetic loci on different chromosomes that have been

identified as on date.

13

(18)

Introduction

associated with the mutation in the insulin receptors. Women with these mutations

may have polycystic ovaries and hyper insulinemia.

11

Diseases of the exocrine pancreas

Any diseases that injure the pancreas can cause diabetes, diseases like

pancreatitis, trauma, infection, pancreatectomy and pancreatic carcinoma.

11

Endocrinopathy

The hypersecretion of several hormones that oppose the action of insulin

cause diabetes example in conditions like acromegaly, Cushing’s syndrome,

glucagonoma, pheochromocytoma .Hyperglycemia is resolved in case of hormone

excess.

11

Drug or chemical induced diabetes

Many drugs can interfere with the secretion of insulin; these drugs do not

cause diabetes by themselves, but can influence the diabetic individuals with

insulin resistance. There are many hormones and drugs which impair the insulin

secretion. Examples are nicotinic acids and glucocorticoids fortunately such drug

interactions are less.

11

Diabetes due to other infections

Certain viruses have been associated with cell destruction. Diabetes occurs

with congenital rubella infections. In addition coxsakie virus B, cytomegalo virus,

adeno virus and mumps have been implicated in inducing certain cases of

disease.

11

Due to change of life style in urban population

Changed life styles especially those having high calorie intake and little

physical activity leads to type 2 diabetes. These types of diabetes are more

prevalent in developing countries.

14

Diagnosis of diabetes

(19)

Introduction

the retinopathy as the key factor to identify the threshold of glucose level. A new

diagnostic point has been established by this type of studies ,ie, FPG of

≥

126mg/dl (0 7.0mmol/l) and standing plasma glucose value is 2hr of

≥

200mg/dl

(11.1 mmol/l) are more prevalent to diabetes and diabetes associated

retinopathy.

15

Advanced glycation end products are widely used markers for

chronic glycemia conditions. Blood glucose level over 2-3 month period of time

can be predicted form the glycosylated hemoglobin (HbA

1

c) level, should be less

than 6.5% in controlled FPG levels. HbA

1

c has several advantages over FPG like

fasting is not required to predict pre analytical stability and perturbations in the

blood glucose level due to stress and illness. The decision about which test to be

used to assess the patient for diabetes should be left to the discretion of the health

care professional by considering into account the availability and practicability of

testing. The current diagnostic criteria for diabetes are summarized in a following

table1

Table 1: Diagnosis criteria for diabetes

01 HbA1c

≥

6.5%. The test should be performed in 3months once

02

FPG

at least 8 hrs

≥

126mg/dl (7.0mmol/l). Fasting is defined as no of calories intake for

03

2-h plasma glucose

≥

200mg/dl (11.1mmol/l) during OGTT. The test should

be performed as described by the WHO , using a glucose load containing the

equivalent of 75g anhydrous glucose dissolved in water

04

In a patient with classic symptoms of hyperglycemia or hyperglycemic

crisis, a random plasma glucose

≥

200mg/dl (11.1mmol/l).

Pathology and Pathophysiology

(20)

Introduction

complications in diabetes. The pancreatic

β

cells are also adversely affected by the

chronic hyperglycemia and are also a target for the secondary complication. The

prolonged hyperglycemia worsens the

β

cells of pancreas and steadily deteriorates

the secretion of insulin.. So glucose in chronic excess causes toxic effects on the

structure and function of the organs, including the pancreatic islets.

[image:20.595.107.515.446.671.2]

In DM due to chronic hyperglycemia, generation and accumulation of

reactive oxygen species (ROS) induced chronic oxidative stress (OS), which has

been postulated to play a central role in the complication of diabetes disorder. In

normal physiological conditions, glucose primarily undergoes glycolysis and

oxidative phosphorylation and converts into energy. In diabetes condition,

excessive glucose level can inhibit the glycolysis and trigger at least six path ways

which are the major contributors for the production of ROS.

16

(Fig 1) This

oxidative stress impairs the antioxidant enzyme system like reduced glutathione,

superoxides, catalase, peroxidase etc. The hyperglycemic stress during the

diabetes leads to various complications in diabetes.

Figure 1:Reactive oxygen species generated during

hyperglycemic condition.

(21)

Introduction

Microvascular complication

Diabetes mellitus has an increased risk in developing microvascular

complications if it is undetected or left untreated. This can have devastating

impact on quality of life and has a significant burden on health care cost.

Microvascular complications can bring forth the following disorders.

Diabetes retinopathy

It is the most common among various ocular complications of diabetes and

leading cause of new onset blindness. In the initial stage of diabetes retinopathy

there are no symptoms, as the diabetes progress lesion progressed and leads to

serious visual disability. Diabetes retinopathy affects the microvasculature in the

retina and divided clinically into two major types like non proliferative diabetes

retinopathy and proliferative diabetes retinopathy. Appearance of retinal

thickening and exudates within the retina is diabetes macular edema. Visual

impairment in diabetes is by the lesion developed due to proliferative diabetes

retinopathy or macular edema.

17

The prevalence of diabetes retinopathy in western

population was reported to be 50.3% in US

18

and 6-7% in India

16

Diabetes nephropathy

Diabetes nephropathy is the major leading case of end stage renal diseases.

The earliest detectable changes in the diabetes nephropathy is thickening of the

glomerulus where serum albumin will leak more into the urine, which is not

present in the normal urine as in diabetes more number of glomeruli are destroyed.

Renal complication is clinically characterized by increase in rate of urinary

albumin excretion, starting from normal albuminuria which progresses to

microalbuminuria, macroalbuminuria and the end stage of renal diseases.

19

Diabetes neuropathy

(22)

Introduction

multifocal and autonomic neuropathy. The most related mechanism for peripheral

neuropathy in diabetes is the activation of polyol pathway and accumulation of

polyol , injury from AGEs and oxidative stress. Amputation after foot ulceration

or injury is mainly due to the result of diabetes neuropathy.

20

In diabetes

neuropathy complication chronic sensorimotor distal symmetric polyneuropathy is

the most common form. In this type of complication, patients experience burning,

tingling and electrical pain and sometimes experience simple numbness which

leads to painless foot ulcers.

21

Pure sensory neuropathy is another type of

neuropathy due to poor glycemic control and fluctuation in diabetes control. It is

characterized by isolated sensory finding without any symptoms of motor

neuropathy. Mononeuropathies have a more sudden onset and involve virtually

any nerves, but most commonly, the median, ulnar and radial nerves. Cranial

neuropathy was also reported but rare.

22

Diabetes autonomic neuropathy is

another type which causes significant morbidity and mortality in patients with

diabetes. In this neurological dysfunction most of the organs can be getting

affected, especially sudden deaths due to silent ischemia can occur.

23

MACROVASCULAR COMPLICATIONS

Cardiovascular diseases

It refers to class of diseases that involves the heart, blood vessels due to

lipids like cholesterol and lipoproteins such as LDL, VLDL.

(23)

Introduction

cardiovascular complication. In India, the cardiovascular diseases was the major

reason for the morbidity and mortality associated with diabetes, prevalence of

coronary heart disease is also increasing at an alarming rate in India.

Cerebrovascular diseases

Stroke is another important cause of morbidity and mortality in diabetes

patients. Patients with diabetic have a higher risk of stroke and poor prognosis

when compared with non diabetes subjects. Prevalence of stroke has increased in

India over the past three decades. Diabetes is one of the important risk factors for

the development of stroke. The change in dietary habits (intake of more fats) and

smoking will increase the chance of stroke in diabetes people.

25

Economical and social cost of diabetic

A different epidemiological study from various parts of the country

indicates the increase in prevalence of diabetes in the urban population. The most

alarming feature is the shifting of onset of diseases to younger age group. This

will have long lasting adverse effects on the national health system and economy.

The direct and indirect costs for the treatment of chronic diseases with

complications are more. There is urgent need for the cost effective protocol for the

treatment of diabetes care to reduce the morbidity and mortality and the economic

burden on the patients. The World Bank estimated that diabetes will account for

1,870,000 disabilities, with a per capita health expenditure of $21 in India.

26

The

cost of diabetes in India study says that INR 4724 occurs towards the outpatient

care expenses. The total indirect cost for a non earning member was estimated as

INR 9748. This would be INR 16831 for an earning member.

27

In USA the

economy burden of diabetes was estimated at about 45.2 billion $ and nearly 15 %

of the total health care expense.

28

Treatment of diabetes

(24)

Introduction

biguanides meglitimide, thizolidinedione and alpha glucosidase inhibitors. Most

of these drugs stimulate pancreas and increase the secretion of insulin. Some may

interfere the absorption of glucose from the gastro intestinal tract. Biguanides like

drugs increase the sensation of insulin receptors to insulin.The newer drugs in this

category is glucagon like peptide -1 (GLP 1) agonist and dipeptidyl peptidase IV

(DPP4) antagonist which regulates the metabolism of insulin .Increases in cost,

inadequate supply of drug, adverse reaction and resistance are the reasons for an

alternative therapy. This increased the emphasis on the use of plant materials as

sources of medicines for diabetes.

Traditional system of medicine

Diseases are born with man and drug came into existence since a very

early period to remove the pain of diseases and cure them. To cure the ailments of

mankind nature has given a complete store house of remedies. The knowledge of

drugs has accumulated over thousands of years as a result of man’s inquisitive

nature, so that we posses many effective means of ensuring good health. Today

these have accumulated a vast store of knowledge concerning the therapeutic

property of different plants.

In India knowledge of medicinal plants is very rich and from age old and

these has been described in Rigveda and Atharvanaveda (3500-1500 BC) from

which Ayurveda has developed.

29

21,000 plants, were listed by WHO , which are

used for medicinal purposes around the world. Out of these 2500 species were

found in India and 150 species are used commercially.

30

There is an exponential growth in the field of herbal medicine and is

gaining popularity among the developing and developed countries due to its

natural origin and less side effect. Most of the traditional medicines that are in use

from the time immemorial are found to be derived from the natural sources.

31

Plant drugs in the treatment of diabetes

(25)

Introduction

[image:25.595.103.516.470.750.2]

out of which some of them are also used in commercial preparations. Eighty five

percent of the antidiabetes plants used most widely around the world are

prescribed in India.

32

The list of plants used in the treatment of diabetes is in Table

2.

There is an increased demand among the patients to use the natural products

with antidiabetic activity so as to avoid the side effect associated with the use of

insulin and other proven synthetic hypoglycemic agents. The majority of type 2

diabetes patients are insulin resistant, for whom insulin or oral hypoglycemic

agents which stimulate the insulin release from the pancreas is not the ideal

therapy. The expert committee of WHO on diabetes has recently recommended

and validated scientifically the effect of antidiabetic drug from plant origin used in

traditional medicine.

33

Plant source are used as single drug or as a combination of

many plants as formulation for the treatment of diabetes. The drugs which can

control lipid profile, glucose along with other complications can be a better drug

for the treatment of type II diabetes and it is the need of the hour also. Currently

the drugs prescribed are to control glucose level. These drugs may not take care of

the co- morbidities.

Table 2: List of plants with anti-diabetic activity

Sl. No

Name of the plant

Parts used

1.

Clerodendron phlomides

Leaves

2.

Butea monosperma

Flowers

3.

Pterocarpus marsupium

Heart wood

4.

Tephrosia purpurea

Whole plant

5.

Momordica charantia

Fruits

6.

Coccinea indica

Fruits

7.

Trigonella foenum gracum

Seeds and leaves

8.

Cyamopsis tetragonoloba

Fruits

9.

Sygyzium jambolanum

Seeds

10.

Melia composite

Leaves

11.

Scoparia dulcis

Whole plant

12.

Salacia prionotis

Woody stem

(26)

Introduction

For the present study we selected the important plant Clerodendrum

viscosum-vent (CV), Family: Verbenaceae. Synonym-Clerodendrum

infortunatum.L known as Bhandirah in Sanskrit and Perugilai in Tamil. It has

been used traditionally for the treatment of type 2 diabetes. It is an important plant

in the Indian system of medicine. It is traditionally used in ethno medicine for its

various medicinal properties which includes the treatment of scorpion sting,

34

certain tumors, leprosy and skin diseases.

35

Clerodendrum

viscosum

leaves on preliminary chemical analysis are found

to contain saponin, clerodin (a bitter diterpene)

36

and some enzymes. Fixed oils

are present in leaves and also which consists of glycerides of lnoleic, oleic, stearic

and lignoceric acid.

37

Previous phytochemical investigation of the plant revealed

the presence of alkyl sterols and 2,-(3,4-dehydroxyphenyl) ethanol 1-O-

α

-2

rhamnopyranosyl-(1

→

3)-

β

-D-(4-O-caffeoyl) glycopyranoside (acteoside)

38

Clerodendrum viscosum leaves yielded flavone glucuronides (about 0.1

%)-scutellarin and hispidulin-7-0-glucuronide with practically no free aglycones. The

flavonoid pattern of C. indicum and C. infortunatum is similar to C.phlomides and

C.

nerifolium 4 in having the 6-oxygenated flavones occurring mainly as their

glucuronides.

(27)

Aim & Objective

AIM AND OBJECTIVE

Plant materials are used in developed and developing countries as home

remedies and over the counter products or as raw materials for the Pharmaceutical

industry. Latest trends have shown increasing demand for phytodrugs, which have

proven anti diabetic and antioxidant potentials. Medicinal herbs and extracts

prepared from them are widely used in the treatment of type 2 diabetes and its

complications. In the present study, an attempt has been made to understand the

role of

Clerodendrum viscosum

-vent Root in controlling the blood glucose level

and the complications (neuropathy, nephropathy and vascular damage) of diabetes

and also understanding the mechanism of drug action and the active compound

responsible for the anti diabetic property of

vent root

The Clerodendrum viscosum vent Root was studied for the Phytochemical

and Pharmacological point of view, so as to arrive at a standard drug for type 2

diabetes and its complications.

Such studies will promote global acceptance and international recognition

of herbal drugs of India, which in turn will contribute to the ailing human society

by relieving various ailments and in improving nation’s economy as well as

individual’s economy and living standards of tribal People.



To identify and isolate the active principle responsible for the

antidiabetic

activity

of

vent root by UV, HPTLC

Technique, Mass spectroscopy studies and NMR studies



Toxicity studies -To prove the safety aspects of

vent root by carrying out the acute toxicity and

in vitro

cell toxicity

studies.



To study the role of

Clerodendrum viscosum-vent

in controlling blood

glucose, lipids and free radicals formation in STZ –nicotinamide induced

diabetic rats

(28)

Literature Review

LITERATURE REVIEW

Prevalence of diabetes

Population based methodology was used in the study to assess the

prevalence of diabetes .The study that was conducted in 91 countries, was used to

calculate age and sex specific diabetes prevalence. The population included in the

study was 20-79 years range. The pattern of diabetes varies considerably

according to countries’ economic status. For developed countries, the majority

with diabetes are aged over 60 years, whereas for developing countries most

people with diabetes are of working age, between 40 and 60 years. This difference

is likely to still be present in 2030. In 2030 there will be an increase of 7.7%

especially in adult population ie, estimated increase in diabetic adult population

was 439 million in 2030. The reason for the increase in diabetes in adults is

mainly due to urbanization in developing countries and associated with more

sedentary life style. The study concluded that diabetes is continuing to be an

increasing international health burden. Ageing and urbanization are increasingly

adding to the burden of diabetes in developing countries.

39

(29)

Literature Review

The age of onset in India has been shifting towards ever-younger people

even within the past decade among Indians in their late teens, ‘adult-onset’

diabetes already manifests itself more often than does ‘juvenile onset’ diabetes.

obesity is a risk factor for diabetes both in India and in the West, the disease

appears at a lower threshold of obesity in India, as is also the case in China, Japan

and other Asian countries.

Indians with diabetes are less

likely to develop

blindness and kidney disease,

but much more likely to suffer coronary artery

disease at a relatively young age.

14

Prevalence rate of NIDDM in the migrant and native Indians in affluent

areas suggest the high genetic risk for diabetes in ethnic Indians. Age, urban-rural

factor, body mass index and abdominal adiposity (waist: hip ratio) were positively

associated with NIDDM. Migration from rural to urban environment with changes

in life style may have contributed to the increased prevalence of diabetes. The

prevalence of IDDM was 0.26/1000 with a peak age of 12 years at diagnosis. This

first population based study of prevalence of IDDM in South India has suggested

that IDDM is not rare and is higher than that reported from many other Asian

countries.

40

Diabetes and pathogenesis

Type 1 and type 2 diabetes are characterized by progressive

β

-cell failure.

Apoptosis was the main form of

β

cell death in both forms of the disease. It has

been suggested that the mechanisms leading to nutrient and cytokine-induced

β

cell death in type 2 and type 1 diabetes, respectively, shares the activation of a

final common pathway involving interleukin (IL)-1

β

, nuclear factor (NF)- KB

and Fas. We review herein the similarities and differences between the

mechanisms of

β

-cell death in type 1 and type 2 diabetes. In the insulitis lesion in

type 1 diabetes, invading immune cells produce cytokines, such as IL-1

β

,tumor

necrosis factor (TNF)-

α

and interferon (IFN)-

γ

IL-1

β

and/or TNF-

α

plus IFN-

γ

(30)

Literature Review

of ER stress and by the release of mitochondrial death signal which execution of

beta cell death. Chronic exposure to increased levels of glucose and free fatty

acids (FFAs) causes

β

-cell dysfunction and induce

β

cell apoptosis in type 2

diabetes. High glucose level exposure of the beta cell has two effects, initially

triggering “glucose hypersensitization” and followed by apoptosis, via different

mechanisms. High glucose, however, does not induce or activate IL-1

α

, NF-KB,

or inducible nitric oxide synthase in rat or human

β

cells in vitro or in vivo in

Psammomys obesus. The ER stress caused by FFAs will leads to

β

cell apoptosis

which is independent to NF-KB and NO independent. Cytokines and nutrients

trigger

β

-cell death by different mechanisms. First one is the NF-KB–dependent

mechanism that culminates in caspase-3 activation for cytokines and an NF-KB–

independent mechanism for nutrients. This argument against a unifying hypothesis

for the mechanisms of cell death in type 1 and type 2 diabetes fails and suggests

that different approaches will be required to prevent -cell death in type 1 and type

2 diabetes.

41

Diabetes mellitus was a syndrome of hyperglycemia and altered

metabolism of carbohydrate, fat and protein metabolism associated with absolute

or relative deficiency in insulin secretion or insulin resistance.

42

It is also

characterized by polyuria, albuminuria, renal enlargement and an increase in

serum creatinine value. Developing premature atherosclerosis due to independent

risk factors such as hyper triglyceridemia and hypertension are the major risk in

diabetes.

43

The hyperglycemic tissue damage is to a particular subset of cells like

capillary endothelial cells in the retina ,mesangial cell in the renal glomerulus and

neurons and schwann cells and peripheral nerve cells .These cells are not able to

reduce the glucose entry in hyperglycemic conditions like other cells which leads

to cell damage. In high glucose concentration aldose reductase also reduces

glucose to sorbitol and later oxidizes to fructose. In the process of reducing

glucose to sorbitol aldose reductase consumes the cofactor NADPH,

which reduces the amount of reduced glutathione leading to oxidative stress.

44

(31)

Literature Review

Different forms of peripheral neuropathy in diabetes may manifest in are sensory,

focal,multifocal and autonomic neuropathies. Diabetic neuropathy is the main

reason for the amputation which occurs after the foot ulceration or injury.

12

The developments of diabetic complications are the major reasons for the

mortality and morbidity in the diabetic patients. The complications are mainly

developed due to the oxidative stress during the hyperglycemia.Oxidative stress

mediated diversion of glycolytic intermediates into pathological pathways are the

key elements in development of diabetic complications. It is understood that

during diabetes, glycolytic path ways slow down and accumulate intermediates

like phospofructokinase, glyceraldehyde -3- phosphatedehydrogenase (GAPDH)

and pyruvate kinase. The ROS produced in the mitochondria due to the

accumulation of intermediates and their conversion into toxic byproducts initiates

the damage. The ROS migrates to the nuclease and produces the damage to the

DNA which results in the activation or the inactivation of glycolytic enzymes.

6

Advanced Glycolytic end products formation is one of the major reason

for the development of diabetic complications. The AGE formation produced the

damage to the cell by three different mechanisms. The first mechanism is at the

endothelial cells by modifying inter cellular proteins, mainly the protein involved

in the regulation of gene transcription. The second mechanism is the AGE

precursor that diffuses from the cell and modifies the nearby intercellular matrix

molecules. This modification alters the signaling between the matrix and the cell

causing endothelial cell dysfunction. The third mechanism is by the diffused AGE

precursors that binds with the circulatory proteins like albumin and attach to the

AGE receptor and release the inflammatory cytokines and growth factors that also

cause vascular damage.

45

(32)

Literature Review

insulin resistance and change in the release of endothelial derived factors

.sustained hyperglycemic state causes increased inter cellular concentration of

glucose metabolism in endothelial cells. Due to the hyperglycemic load causes

changes in the mitochondrial dysfunction and increase oxidative stress and

activation of protein kinase C .The oxidative stress and other factors increased the

expression of endothelin1, a changed balance between vasodilatation and

vasoconstriction and induction of adhesion molecules which causes the

endothelial dysfunction.

46

Progression of coronary artery calcification (CAC) over an average of 2.6

years (range, 1.6 to 3.3) was assessed in a cohort of patients with type 1 diabetes

and nondiabetic subjects 19 to 59 years of age. In this nested case-control sub

study, plasma adiponectin levels were measured in 101 cases with significant

CAC progression and in 205 controls. Controls were oversampled on the basis of

age, gender, diabetes status and presence of baseline CAC. In conditional logistic

regression adjusted for baseline, CAC volume and other significant predictors of

CAC progression, adiponectin levels were inversely related to progression of

CAC in diabetic (OR, 0.47; 95% CI, 0.24 to 0.94) and non diabetic (OR, 0.15;

95% CI, 0.05 to 0.40 for a doubling in adiponectin levels) subjects. Adjustment

for additional cardiovascular risk factors did not change this association. In

conditional logistic regression models by quartiles of plasma adiponectin levels,

the probability value for trend was statistically significant for all participants

(P0.001) and non diabetic participants (P<0.001) and was borderline for type 1

diabetics (P0.08). Low plasma adiponectin levels are associated with progression

of CAC in type 1 diabetic and non diabetic subjects independently of other

cardiovascular risk factors.

47

(33)

Literature Review

transcription factors PDX-1 and MafA and can also accelerate rates of apoptosis.

This pathophysiologic sequence sets the scene for considering antioxidant therapy

as an adjunct in the management of diabetes.

16

Neuropathy is one of the most prevalent, devastating and costly

complications of diabetes. Distal symmetrical sensorimotor polyneuropathy

(DPN) is the leading cause of nontraumatic limb amputation. Diabetic autonomic

neuropathy (DAN) can affect virtually any body system and is associated with

greatly increased morbidity and mortality and can have a profound influence on

quality of life. Several biochemical mechanisms of nerve and neurovascular

damage have been identified and excessive production of reactive oxygen species,

or “oxidative stress”, is thought to be a common etiologic factor. Diabetic

neuropathies can be diagnosed with relatively simple tests of peripheral and

autonomic nerve function. Treatment of diabetic neuropathy should always begin

with efforts to optimize glycemic control and with patient education. There are

now many useful pharmacologic approaches to treat painful neuropathy and most

manifestations of autonomic neuropathy, but disease modifying treatments other

than strict glycemic control await a more complete understanding of the

underlying mechanism of diabetic neuropathy and the development of

pharmacologic agents based on this emerging knowledge.

48

(34)

Literature Review

(HRO

2

), as well as, non-radical species such as H

2

O

2

and hydrochlorous acid

(HOCl). RNS includes free radicals such as; NO and nitrogen dioxide (NO

2

), as

well as non-radicals such as peroxynitrite (ONOO–), nitrous oxide (HNO

2

) and

alkyl peroxynitrates (RONOO).

49

Diabetes is one of the stress related disorder. Diabetes mellitus has been

shown to be a state of increased free radical formation. The existence of oxidative

stress resulting from increased free radicals has been postulated in diabetes.

Animal, human studies and in vitro experiments suggest the role for oxidative

stress, via an increased formation of free radicals in the pathophysiology of many

complications of diabetes, such as neurological, cardiovascular, retinal and renal

types.

50

Moreover, glucose is in itself pro-inflammatory and increases the levels of

acute-phase inflammatory markers, including tumor necrosis factor-alpha

(TNF-



), interleukin (IL)-6 and C-reactive protein (CRP). These acute-phase

inflammatory markers are associated with insulin resistance and metabolic

syndrome, suggesting a role for chronic low-grade inflammation in

DM2 . Therefore, treatment aimed at reducing the degree of oxidative stress and

the production of pro-inflammatory cytokines in DM2 appear to be warranted.

51

The endothelium is the common target of all cardiovascular risk factor,

and functional impairment of the vascular endothelium in response to injury

occurs long before the development of visible atherosclerosis.

The endothelial cell behaves as a receptor-effect or structure which senses

different physical or chemical stimuli that occur inside the vessel and therefore,

modifies the vessel shape or releases the necessary products to counteract the

effect of the stimulus and maintain homeostasis. The endothelium is capable of

producing a large variety of different molecules which act as agonists and

antagonists, therefore balancing their effects in opposite directions. When

endothelial cells lose their ability to maintain this delicate balance, the conditions

(35)

Literature Review

and T lymphocytes). The inflammatory response is incited and fatty streaks

appear, the first step in the formation of the atheromatous plaque. If the situation

persists, fatty streaks progress and the resultant plaques are exposed to rupture and

set the conditions for thrombogenesis and vascular occlusion.

Oxidant products are produced as a consequence of normal aerobic

metabolism. These molecules are highly reactive with other biological molecules

and are referred as reactive oxygen species (ROS). Under normal physiological

conditions, ROS production is balanced by an efficient system of antioxidants,

molecules that are capable of neutralizing them and thereby preventing oxidant

damage. In pathological states, ROS may be present in relative excess. This shift

of balance in favor of oxidation, termed ‘oxidative stress’, may have detrimental

effects on cellular and tissue function, and cardiovascular risk factors generate

oxidative stress.

Both type 1 (insulin-dependent) and type 2 (non-insulin-dependent)

diabetic patients have mostly been described under enhanced oxidative stress, and

both conditions are known to be powerful and independent risk factors for

coronary heart disease, stroke, and peripheral arterial disease. Hyperglycemia

causes glycosylation of proteins and phospholipids, thus increasing intracellular

oxidative stress. Nonenzymatic reactive products, glucose-derived Schiff base,

and Amadori products form chemically reversible early glycosylation products

which subsequently rearrange to form more stable products, some of them

long-lived proteins (collagen) which continue undergoing complex series of chemical

rearrangements to form advanced glycosylation end products (AGEs). Once

formed, AGEs are stable and virtually irreversible. AGEs generate

ROS with consequent increased vessel oxidative damage and atherogenesis.

52

(36)

Literature Review

conversion of reversible Schiff base adducts and then to more stable,

covalently-bound Amadori rearrangement products. Over a course of days to weeks, these

early glycation products undergo further reactions and rearrangements to become

irreversibly cross-linked, fluorescent protein derivatives termed advanced

glycation end products (AGEs). There is a growing body of evidence that AGE

and their receptor RAGE (receptor for AGEs) interaction elicits oxidative stress,

inflammatory reactions and thrombosis, thereby being involved in vascular aging

and damage. These observations suggest that the AGE–RAGE system is a novel

therapeutic target for preventing diabetic vascular complications. In this paper, we

review the pathophysiological role of the AGE–RAGE-oxidative stress system

and its therapeutic intervention in vascular damage in diabetes. We also discuss

here the potential utility of the restriction of food-derived AGEs in diabetic

vascular complications.

53

(37)

Literature Review

resulting in impairment of both IRS/PI3-K/Akt-mediated endothelial function and

NO production. This article summarizes the PI3-K/Akt pathway-mediated

contraction and relaxation responses induced by various agents in the blood

vessels of diabetic animals.

54

Diabetic models

(38)

Literature Review

The release of reactive oxygen species (ROS) has been proposed as a

cause of streptozotocin (STZ)-induced b-cell damage. This initiates a destructive

cascade, consisting of DNA damage, excess activation of the DNA repair enzyme

poly (ADP-ribose) polymerase and depletion of cellular NAD1. Metallothionein

(MT) is an inducible antioxidant protein that has been shown to protect DNA from

chemical damage in several cell types. Therefore, we examined whether over

expression of MT could protect b-cell DNA and thereby prevent STZ induced

diabetes. Two lines of transgenic mice were produced with upto a 30-fold

elevation in b-cell MT. Cultured islets from control mice and MT transgenic mice

were exposed to STZ. MT was found to decrease STZ-induced islet disruption,

DNA breakage, and depletion of NAD1. To assess in vivo protection, transgenic

and control mice were injected with STZ. Transgenic mice had significantly

reduced hyperglycemia. Ultrastructural examination of islets from STZ-treated

mice showed that MT prevented degranulation and cell death. These results

demonstrate that MT can reduce diabetes and confirm the DNA damage

mechanism of STZ-induced b-cell death.

57

(39)

Literature Review

poly (ADP-ribose) polymerase inhibitor. These data strongly indicate that

STZ-induced b-cell death is not caused by elevated intracellular O-GlcNAc levels, but

instead likely involves poly (ADP-ribose) polymerase in the mechanism.

58

The anti-hyperglycemic action of Hei-Shug-Pian, the fire-processed

product of the root of Aconitum (Aconitum carmichaeli), was investigated in

streptozotocin-induced diabetic (STZ-diabetic male wistar rats 200 to250 gm) rats.

At 120 min following oral administration of Hei-Shug-Pian at doses ranging from

12.5 to 50 mg/kg, plasma glucose of STZ-diabetic was found to be decreased in a

dose-dependent manner. Under treatment conditions wherein plasma glucose was

lowered, the uptake of glucose into soleus muscle was increased and the

incorporation of glucose into glycogen of hepatocytes was enhanced. Blockade of

opioid receptors eliminated the plasma glucose-lowering effect of Hei-Shug-Pian

Moreover, Hei-Shug-Pian treatment failed to lower plasma

glucose in opioid -receptor knockout diabetic mice. The results obtained in this

study support the hypothesis that Hei-Shug-Pian lowers the plasma

glucose concentrations of STZ-diabetic rats through activation of opioid

µ-receptors of peripheral tissues, resulting in enhanced glucose utilization.

59

(40)

Literature Review

In Africa the diabetes mellitus and other ailments are treated using stem

bark extracts of Terminalia superba Engl. and Diels and Canarium schweinfurthii

Engl. The evaluation of anti-diabetic effects of the methanol/methylene chloride

extracts of the stem barks on streptozotocin (STZ)-induced diabetes were carried

out using male rats. Diabetes was induced using 60 mg/ml of Streptozotocin

through subcutaneous administration. 150 mg/kg and 300 mg/kg of extract was

administered daily for 14 days to the rats received Streptozotocin two days prior.

At 300 mg/kg, the two extracts (Terminalia superba and Canarium

schweinfurthii), significantly showed at least 67.1% and 69.9% reduction in blood

glucose level, respectively, while insulin (three units) given subcutaneously and

once daily, had 76.8% reduction compared to diabetic untreated control rats.

Similarly, the weight gains were 6.6% and 4.9%, respectively, and were

comparable to the normal rats, whereas, sham control rats lost 14.1% body weight.

Still with the same dose, there was 68.5% and 58.5% (p < 0.001) significant

decrease in food consumption and 79.7% and 64.0%

(p < 0.001) in fluid intake by diabetic rats treated with the respective plant

extracts. At the end of the second week, the insulin-treated rats showed 56.4% and

75.8% decrease in food and fluid intake compared to an augmentation for diabetic

control rats, 43.0% and 383.8%, respectively. These results showed that the plant

extracts can reverse hyperglycemia, polyphagia and polydipsia exhibited by

streptozotocin, and thus, they have anti-diabetic properties.

61

Free radical scavenging and antioxidant activity

(41)

Literature Review

evaluated in the liver, kidney and heart tissues of diabetic rats by measuring

malondialdehyde (MDA) and glutathion (GSH) levels. The results were compared

to the diabetic control groups. EtOAc Fr. was found to have rich in polyphenolics

and exhibited a significant antihyperglycaemic and antioxidant activity equipotent

with the reference hypoglycaemic agent (tolbutamide), when evaluated in diabetic

rats.

62

The interest in possible health benefits of flavonoids has increased owing

to their potent antioxidant and free radical scavenging activities observed in vitro.

On the other hand, the antioxidant efficacy of flavonoids in vivo is less

documented and their prooxidant properties have described in vivo. They are able

to cause oxidative damage by reacting with various biomolecules, such as lipids,

proteins and DNA

63

The present study was carried out to evaluate the in vitro antioxidant and

reactive oxygen species scavenging activities of Terminalia chebula, Terminalia

belerica and Emblica officinalis fruit extracts. 70% methanol extract were studied

for the in vitro free radical scavenging activity. Scavenging ability of the extracts

for radicals like DPPH, hydroxyl, superoxide, nitric oxide, hydrogen peroxide,

peroxynitrite, singlet oxygen, hypochlorous acid were also performed to

determine the potential of the extracts. The evidences can be concluded from the

study of the 70% methanol extract of the fruits of Terminalia chebula, Terminalia

belerica

and

Emblica officinalis, imposes the fact that they might be useful as

potent sources of natural antioxidant.

64

(42)

Literature Review

The inhibition of alpha-glucosidase and alpha-amylase, digestive enzymes

of carbohydrates, can santly reduce the post-prandial increase of blood glucose

and therefore can be an important strategy in the management of blood glucose

level in type 2 diabetic and borderline patients. Currently, there is renewed interest

in plant-based medicines and functional foods modulating physiological effects in

the prevention and cure of diabetes and obesity. The plant kingdom is a wide field

to search for natural effective oral hypoglycaemic agents that have slight or no

side effects. More than 1200 plant species have been recorded to be used

empirically worldwide for their alleged hypoglycaemic activity. Therefore, natural

alpha-glucosidase and alpha-amylase inhibitors from plant sources offer an

attractive strategy for the control of hyperglycaemia. In this article reviews recent

data on plant extracts and isolated natural compounds that are being tested for

their anti diabetic activity, highlights the ongoing research and considers the

future persepctives.

66

(43)

Literature Review

Herbal medicines have been used since prehistoric times by different

cultures worldwide for the treatment of diabetes. The present study evaluated the

effect of Ficus racemosa Linn. (Moraceae) stem bark on carbohydrate

hydrolyzing enzymes, viz., porcine pancreatic amylase, rat intestinal

alpha-glucosidase, sucrase and almond beta-alpha-glucosidase,by using different in vitro

model systems. In addition, the effect of the treatment was also studied. Untreated

F. racemosa bark (FRB) significantly inhibited (p < or = 0.05) alpha-amylase,

alpha-glucosidase, beta-glucosidase and sucrase in a dose-dependent manner.

Heat treatment of the sample comparably increased alpha-amylase,

alpha-glucosidase, and sucrase inhibitory activities, while a marginal decrease in

beta-glucosidase inhibitory activity was observed; however, no statistical

differences were observed . Untreated FRB showed IC

50

values of 0.94% and 280,

212, and 367 microg/ml for alpha-amylase, alpha-glucosidase, beta-glucosidase,

and sucrase, respectively, while the IC

50

values for heat treated FRB were 0.58%

and 259, 223, and 239 µg/ml, respectively. Further, a significant correlation

(p <or =0.01; r=0.791) was observed between alpha-amylase, alpha- glucosidase,

beta-glucosidase and sucrase inhibitory activities of both untreated and heat

treated FRB. The observations clearly says that inhibition of carbohydrate

hydrolyzing enzymes is one mechanism through which F. racemosa stem bark

exerts its hypoglycemic effect in vivo. Therefore, the potential exists to explore

the utilization of F. racemosa stem bark in the development of nutraceuticals and

functional foods for the management of diabetes and related symptoms/

disorders.

68

(44)

Literature Review

skeletal muscle. In the diabetic rats decreased GLUT4 mRNA levels in white and

brown adipose tissue and the concentration of the messenger remaine unaltered in

red and white fibers of skeletal muscle. The interaction of benfluorex and diabetes

on GLUT4 protein expression showed a tissue-specific pattern. Benfluorex

treatment to some extent prevented the decrease in GLUT4 protein in white and

brown adipose tissue and in white muscle associated with diabetes. In contrast,

diabetes and benfluorex caused an additive decrease in GLUT4 expression in red

skeletal muscle. The effects of benfluorex on GLUT4 content in tissues from

diabetic rats showed the absence of alterations in GLUT4 mRNA levels,

suggesting a modification of translational or posttranslational steps. Benfluorex

did not ameliorate the hyperglycemia of diabetic rats. Our results indicate that red

and white skeletal muscle respond to diabetes and benfluorex in a heterogeneous

manner, which suggests the existence of differences in the mechanisms that

regulate GLUT4 expression. In the study data indicate that GLUT4 expression in

muscle and adipose tissue can be regulated by modification of translational or

posttranslational steps.

69

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