i

QUALITY STANDARDIZATION AND BIOLOGICAL SCREENING OF MEDICINAL PLANTS – CALOTROPIS

GIGANTEA AND SPHAERANTHUS INDICUS

Thesis submitted in Partial Fulfillment for the award of Degree of Doctor of Philosophy in

Pharmacy

By Deepak Teotia

VINAYAKA MISSIONS UNIVERSITY

SALEM, TAMILNADU, INDIA

December-2013

ii

VINAYAKA MISSIONS UNIVERSITY

CERTIFICATE BY THE GUIDE

I, Dr. Prof. S.P. Chakrabarti, certify that the thesis entitled

“Quality standardization and biological screening of medicinal plants-

Calotropis gigantea and Sphaeranthus indicus”, submitted for the

award of Degree of Doctor of Philosophy by Mr. Deepak Teotia is the

record of research work carried out by him during the period from

Oct-2008 to Dec-2013 under my guidance and supervision and this

work has not formed the basis for the award of any degree, diploma,

associate-ship, fellowship or other titles in this University or any

other University or Institution of higher learning.

Place: Signature of the Supervisor with designation

Date:

iii

VINAYAKA MISSIONS UNIVERSITY

DECLARATION

I, Deepak Teotia, declare that the thesis entitled “Quality

standardization and biological screening of medicinal plants-

Calotropis gigantea and Sphaeranthus indicus”, submitted by me for

the award of Degree of Doctor of Philosophy is the record of work

carried out by me during the period from Oct-2008 to Dec-2013

under the guidance of Dr. Prof. S.P. Chakrabarti, Principal of I.M.T.

Puri and has not formed the basis for the award of any degree,

diploma, associate-ship, fellowship, titles in this University or any

other University or other similar institution of higher learning.

Place: Signature of the Candidate

Date:

iv

DEDICATED TO

MY GRANDPARENTS

AND

HUMANITY

v

ACKNOWLEDGEMENT

The dream of doing something in the field of research was no

less than climbing a mountain for me. Today, when I have reached

the summit, I want to express my special thanks of gratitude to all

those who were always beside me to help while I was stepping

forward in order to achieve my goal.

The list of people I need to thank will not fit to a single

acknowledgement section, I just mention some people whose

contribution is obvious.

First and foremost I submit my sincere thanks to Prof. Dr. K.

Rajendran, Dean Research, Vinayaka Missions University, Salem,

Tamilnadu, to pursue my research work.

I am indebted to my research supervisor, Dr. S. P. Chakrabarti.

It was an honour to work under him. I thank him for his sumptuous

suggestions, transcendent and evidence, criticisms to embellish my

study.

I express my sincere gratitude to Dr. S. S. Ajay, for his

contribution of time and ideas to make my PhD. experience

productive and stimulating. The joy and enthusiasm he has for

research was contagious and motivational for me, even during tough

times in the PhD. pursuit.

vi

I gratefully acknowledge the management of V. G. I., Greater

Noida, for providing me all the facilities required to complete my work.

I place on record my sincere gratitude to Dr. S. Singh for

providing animals for biological screening.

I am thankful to all my colleagues for their continuous

encouragement and suggestions. It gives me immense pleasure to

acknowledge lab mates Prashant Pathak and Deepak Sharma who

tirelessly helped me to prepare this thesis.

My thanks are extended to my family for their unconditional love

and encouragement which was my inspiration and driving force to

reach the goal. I am grateful to my parents who supported me in all

my pursuits.

Above all I am indebted to Almighty, who gave me the mental

strength to overcome all the difficulties faced by me in commencing

this thesis.

DEEPAK TEOTIA

vii

CONTENTS

S.No Title Page 1. INTRODUCTION 1

1.1 History of natural products 2 1.2 Significance of flora in the treatment

of ailments 5

1.3 Study of medicinal plants 7 1.4 Herbal medicine 8 1.5 Traditional medicine 8 1.6 The Rasayana concept of Ayurveda 10 1.7 Diabetes 10 1.8 Hyperlipidemia 20 1.9 Antioxidant 26 1.10 Anti-microbial agents 29 1.11 Muscle Relaxant 32

2. AIM AND OBJECTIVE 35 2.1 Need for study 36 2.2 Objectives 37 2.3 Methodology 38

3. PLANT PROFILE 40 3.1 Calotropis gigantea 41 3.2 Sphaeranthus indicus 43

4. LITERATURE 44 4.1 Sphaeranthus indicus 45 4.2 Calotropis gigantea 54

5. EXPERIMENTS 66 5.1 Collection and authentication of

plant material 67

5.2 Physical-chemical evaluation 67 5.3 Method of extraction 70 5.4 Identification of phytochemical

constituents 70

5.5 Total phenol and flavonoid contents 71 5.6 Drugs and chemicals used 72 5.7 Antidiabetic activity of extracts of

Calotropis gigantea 73

5.8 Antihyperlipidemic activity of extracts of Calotropis gigantea

77

viii

5.9 Hypoglycemic effect of Calotropis gigantea leaves via different routes of administration in normal and alloxan induced diabetic rats

80

5.10

Effect of Calotropis gigantea and Sphaeranthus indicus on glycemia and lipidemia in streptozotocin induced diabetic rats

83

5.11 Antioxidant activity of extracts of Calotropis gigantea

85

5.12 Antimicrobial activity of extracts of Calotropis gigantea

86

5.13 Effect of extract of Calotropis gigantea on the skeletal muscle of the rat

91

6. RESULTS AND DISCUSSION 93

6.1 Macroscopic and microscopic features 94 6.2 Physiochemical evaluation 107 6.3 Phytochemical evaluation 114 6.4 Hypoglycemic effects of extracts of

Calotropis gigantea 117

6.5 Hypoglycemic effect of Calotropis gigantea leaves via different routes of administration in normal and alloxan induced diabetic rats

124

6.6

Effect of Calotropis gigantea and Sphaeranthus indicus on glycemia and lipidemia in streptozotocin induced diabetic rats

132

6.7 Antioxidant activity 142 6.8 Antimicrobial activity 142 6.9 Muscle relaxant activity 149

7. SUMMARY AND CONCLUSION 153

8. REFERENCE 158

9. PUBLICATIONS 167

ix

LIST OF TABLES

S.NO TITLE PAGE

1. Stomatal number of upper surface of Calotropis gigantea (leaves)

100

2. Stomatal number of lower surface of Calotropis gigantea (leaves)

100

3. Vein islet number of Calotropis gigantea (leaves) per sq. mm

100

4. Veinlet termination number of Calotropis gigantea (leaves)

101

5. Palisade ratio of Calotropis gigantea (leaves) 101

6. Physicochemical parameters of Calotropis gigantea

109

7. Fluorescent analysis of leaves of Calotropis gigantea

110

8. Fluorescent analysis of root of Calotropis gigantea

111

9. Physicochemical parameters of Sphaeranthus indicus fruit

112

10. Fluorescent analysis of fruit of Sphaeranthus indicus

113

11. Preliminary phytochemical screening of various extracts of Calotropis gigantea

114

12. Preliminary phytochemical screening of various extracts of Sphaeranthus indicus fruit

116

13. Hypoglycemic effect of extracts of Calotropis gigantea in normal rats

118

14. Effect of extracts of Calotropis gigantea on oral glucose tolerance test

119

15. Anti-hyperglycemic effect of extracts of Calotropis gigantea on blood glucose level on streptozotocin-induced diabetes in rats

122

16.

Effect of water extract of Calotropis gigantea leaves on plasma glucose levels after intragastric (p.o) and intraperitoneal (i.p.) administration to normoglycemic rats

125

17. Plasma insulin levels in normoglycemic rats after intragastric (p.o.) and intraperitoneal

127

x

(i.p.) administration of water extract of Calotropis gigantea leaves

18.

Effect of water extract of Calotropis gigantea leaves on plasma glucose levels after intragastric (p.o) and intraperitoneal (i.p) administration to alloxan-diabetic rats

129

19.

Blood glucose levels in glucose loaded (0.25 g/kg BW) rats before and after the intravenous administration of water extract of Calotropis gigantea leaves

131

20.

Effects of treatment of 8 weeks with water extract (300 mg/kg BW) of Calotropis gigantea and Sphaeranthus indicus and a mixture of the two plants on fasting plasma glucose level in streptozotocin (STZ) diabetic rats

133

21.

Effect of water extract (300 mg/kg BW) of Calotropis gigantea and Sphaeranthus indicus and mixture of the two plants on plasma glucose tolerance in diabetic rats after 8 weeks

135

22.

Effect of water extract (300 mg/kg BW) of Calotropis gigantea and Sphaeranthus indicus and mixture of the two plants on plasma glucose tolerance in diabetic rats after 8 weeks

137

23.

Effect of water extract of Calotropis gigantea plus Sphaeranthus indicus on glycosylated haemoglobin, body weight, serum albumin, total proteins and creatinine, urine sugar and albumin values shown at the end of 8 weeks treatment

139

24. Effect of water extract of Calotropis gigantea plus Sphaeranthus indicus on the serum lipid profile in rats fed on high fat diet

141

25. Effect of water extract on liver and kidney weight in rats fed on high fat diet.

141

26. Test organism’s relative percentage inhibition (%)

145

27. MIC values of methanol and aqueous extracts of Calotropis gigantea on test organism

147

xi

28. Antimicrobial activity of Calotropis gigantea 147

29. Muscle relaxant activity of extract from Calotropis gigantea

151

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LIST OF FIGURES

S. No Figure Page

1. Parts of Calotropis gigantea 41

2. Fruit of Sphaeranthus indicus 43

3. Powder microscopy of Calotropis gigantea leaves

97

4. T.S. of leaf surface for quantitative analysis of Calotropis gigantea (stomata)

98

5. T.S. of leaf surface for quantitative analysis of Calotropis gigantea (Vein islet number and vein termination number)

99

6. Powder microscopy of Sphaeranthus indicus stem

102

7. Microscopy of Sphaeranthus indicus stem 103

8. Powder microscopy of leaf of Sphaeranthus indicus

104

9. Microscopy of Sphaeranthus indicus leaf 105

10. Microscopy of Sphaeranthus indicus root 106

11. Comparison of antioxidant activity of various extracts

142

12. Antimicrobial activity of Calotropis gigantea 144

13. Relative percentage inhibition of Calotropis gigantea

146

14. Muscle relaxant activity of Calotropis gigantea

149

15. Comparison of muscle relaxant activity of acetylcholine and test drug at different concentrations

150

1

1. INTRODUCTION

2

1. INTRODUCTION

1.1 History of natural products

The use of natural products as medicinal agents presumably

predates the earliest recorded history, as the earliest humans used

various but specific plants to treat illness.

In early 2700 BC, China traced to “The Emperor Shennung”, which

indicates the usefulness of plants for treating diseases.

“Ebers papyrus”, an Egyptian manuscript, written in 1550 BC deals

with the use of plants in Egyptian medicine.

Theophrastus (370 – 285 BC), a Greek native, proposed the

scientific classification of plants.

“Dioscorides De Materia Medica”, written in 77 AD has reported

the medicinal use of over 600 plants.

Ibn-al-Baitar (1197 – 1248 AD), a Muslim scientist, listed 1400

drugs and medicinal plants in his book “Kitab-al-Jami fi al-Adwiya al-

Mufrada.” (The book of simple drugs and food)

In the last decade, the global consumption of medicinal plants in

the herbal and nutraceutical industries have increased dramatically.

In British Columbia, Canada, there are more than 50 medicinal plants

species which are harvested for the herbal, nutraceutical and

pharmaceutical industries (Lantz 2001).

3

Indian subcontinent contains 25,000 species of vascular plants

and 7,800 medicinal drugs manufacturing units which consume 2000

tons of herbs annually.

Market of Ayurvedic medicines is expanding at the rate of 20%

annually. Sales of medicinal plants have grown by 25% in India

during 1987-2008.

Plant based drugs are used directly i.e. they can be collected,

dried and used as therapeutic agents (crude drug). Their extracted

constituents & active principles, reported by various chemical

processes, are employed as medicines.

The active principles or compounds with similar structure and

activity are synthesized chemically to produce synthetic drugs, used

in Allopathy or Modern System of medicine.

A comprehensive approach on medicinal plants was drawn by

WHO which includes:

Therapeutic classification of medicinal plants in different

countries.

Scientific criteria and methods for assessing the safety of

medicinal plant products.

International standards and specification for identification,

purity, strength and manufacturing practices.

4

Method for safe and effective use of medicinal plant products.

Designation of research and training centres for the study of

medicinal plants.

The use of natural plants for treatment of different ailments has

evolved significantly along with human evolution. Traditional

medicines, obtained from plants, have played a vital role in sustaining

disease free human existence on this planet, though now a day many

synthetic drugs are widely used because of their quick therapeutic

action. It is rather difficult to date back the origin of herbal medicines

as a means of therapy. In spite of overwhelming influence of modern

medicine and tremendous advances made on production of synthetic

drugs, traditional medicines have retained their importance in

therapy. Their effectiveness, low cost and comparative freedom from

serious toxic effects make these medicines not only popular but also

an acceptable mode of treating diseases even in modern times.

Traditional and folklore medicines, handed over from

generation to generation, are in household remedies and are now in

community practice. Traditional medicines have served as a source

of alternative medicine and new pharmaceutical healthcare

products. Medicinal plants are important tool for pharmacological

5

research. It is estimated that almost 70% modern medicines in India

are derived from natural products.

The history of medicine in India can be traced to the remote

past. The earliest mention of the medicinal use of plants is found in

the Rig Veda, perhaps the oldest repository of human knowledge,

had been written between 4500 and 1600 BC. In the works which

followed, particularly ayurveda, the properties of various drugs have

been given in detail.

1.2 Significance of flora in the treatment of ailments

In the dawn of human ‘Cultural Revolution’ the art of curing was

essentially magical and was based on logic than on scientific

evidence. Initially few plants, which were usually psychoactive, known

for their magical or psychedelic properties, were used. Later,

empirical medicine arose, which used many plants for the treatment

of various afflictions. This changed dramatically with the Materia

Medica published by Dioscorides and Dictionary of Economic Plants

of India by Watt, which describes the properties of the various drugs

of plant origin. The ancient civilization made an extensive use of

herbal drugs and their use were also well documented in the form of

pharmacopoeia (Lain, 1982).

India was not far behind and had an extensive pharmacopoeia

thousands of years ago. In many eastern cultures such as in India,

6

China and the Arab world, this experience was systematically

recorded and became the Materia Medica of the traditional systems

of medicine. Traditional medicine is a heterogeneous term that refers

to a broad range of ancient natural health care practices which

existed before the application of modern scientific methods to health

care (Pushpangadan, 1995). The traditional plant therapeutic

knowledge of Africa and South America, along with that of China and

India, has given several new drugs to modern medicine. More than

one hundred twenty molecules, used as vital drugs worldwide, have

been extracted from plants. Until 1930, around 90% of the official

medicines were of plant origin (Swain, 1972).

In the biomedical system that dominates the developed world,

there is an over-riding emphasis on research for synthetic therapeutic

compounds, though a certain amount of interest in medicinal plants

always persisted. Many developed countries, including Germany,

have legalized natural products as drugs and many pharmaceutical

organizations are now involved in preparation of plant based drugs by

using plant extracts.

The WHO, an agency of the United Nations, formed to deal with

matters of health in 1977, at its 30th world assembly, adopted a far

reaching resolution urging the governments of member countries, to

give “adequate importance to the utilization of their traditional

7

systems of medicine, with appropriate regulations to suit their national

health needs”. Traditional medicine was also defined as “the

therapeutic practices that have been in existence, often for hundreds

of years, before the development and spread of modern medicine

and are still in use today”.

Many authors have emphasized the value of conducting broad

ethnobotanical, ethnopharmacological and even clinic - therapeutical

research, on medicinal plants.

1.3 Study on medicinal plants

Varieties of reasons have been cited for the need of studying

medicinal plants. Most of the traditional knowledge about medicinal

plants had been in the form of oral knowledge that was lost with

persistent invasions and cultural adaptations. There was no uniform

or standard procedure for maintaining the inventory of these plants

and the knowledge about their medicinal properties. There was

prevalence of using plants and plant based products in various

contemporary and traditional systems of medicine, without any written

documentation or regulation. Therefore, it is essential that such uses

of natural products should be documented and studied for systematic

regulation and wide spread application. The leads for a significant

number of modern synthetic drugs have originated from isolated plant

ingredients, as the search for newer entities begins from either

8

derivatising existing drugs or from traditional or contemporary

medicinal systems. Therefore, it is essential that research on

pharmacological activity of plants, used extensively in traditional

medicines, should be carried out.

1.4 Herbal medicine

All major biomes on earth have been rich source of herbal

drugs even before the evolution of man on earth. By observing

different animals on earth and by using hit and trial methods, man

eventually learnt the use of different plants in treating different

ailments. Almost every regional culture, throughout the the world,

developed a indigenous therapy by using flora of that area. Many of

them kept proper records, which passed on generation to generation

and are still in use. Thus herbal medecines are indigenous in origin

which leads to development of major therapies like Ayurveda,

Homeopathy, Naturopathy, Traditional Chinese Herbalism, Traditional

Oriental medicine, Western Herbalism, etc.

1.5 Traditional medicine

The traditional system of medicine relies on a holistic

approach, i.e. employing crude drug or extract (singly or in

combination) with multiple constituents. Ayurveda, the traditional

system of medicine, has been practiced since 6000 BC. A group of

plants known as “Rasayanas” in Ayurveda have been extensively

9

used as rejuvenators for arresting the ageing process and to provide

resistance against various diseases including those induced by

psychosomatic disorders and promote general well being of an

individual. The “Rasayana” plants used for the day-to-day stress

faced by the individuals are known as adaptogens. They induce

nonspecific resistance of the organism against diverse aversive

assaults, which threatened to disturb internal homeostasis.

The development of drugs from medicinal plants for “primary

health care” (as inflammation, pain, fever, cough, ulcer, wound,

diarrhoea etc.) should be the first goal. This will increase the

popularity of herbal products and the pharmaceutical industries

engaged in herbal products might be able to enhance their export

potential, provided these pharmaceutical industries produce/provide

standardized and effective products. Hence there is an urgent need

to standardize each and every herbal drug/product and these

products should also be scientifically validated in order to have safe,

efficacious and reproducible medicine. To revitalize traditional

medicines, the efficacies claimed for their applications need to be

proved significantly. Even though many plants provide relief but only

those with pharmacologically conformed activity, should be

recommended for use in “primary health care”. (Jia and Zhang, 2005)

10

1.6 The Rasayana concept of Ayurveda

The word rasayana literally means, ‘the path that rasa takes’

(rasa: the primordial tissue or plasma; ayana: path). It is believed, in

Ayurveda, that the qualities of the rasa-dhatu influence the health of

other dhatus (tissues) of the body. Hence any medicine that improves

the quality of rasa (rasayanas) should strengthen or promote the

health of all tissues of the body. These rasayana plants are said to

possess the following properties: they prevent ageing, re-establish

youth, strengthen life and brain power, and prevent diseases. All of

which implies that they increase the resistance of the body against

any onslaught. Traditionally, these agents are used against a plethora

of seemingly diverse disorders with no pathophysiological connection

to modern medicinal science. Looking at these diverse applications

there appeared to be a possibility of identifying adaptogenic agents

from rasayanas. (Ramawat et al., 2004)

1.7 Diabetes

Diabetes mellitus or simply diabetes is a metabolic disorder

indicating high blood sugar levels.This may be either due to

malfunctioning of beta-cells of Islets of Langerhans of pancreas or

because insulin produced by pancreas is not taken up by the cells of

the body.

11

History of diabetes mellitus

Diabetes was known since ancient times and its control

measures were in use since the middle ages, the illumination of its

pathogenesis revealed mainly during the early 20th century. The major

events in this field were:

In 1899, Joseph Von Mering and Oskar Minkowski

conducted experiments on dogs and discovered the role

of pancreas in diabetes.

In 1910, Sir Edward Albert Sharpey Schafer coined the

term “insulin” and proposed that diabetes is due to

deficiency of insulin that was normally produced by

pancreas.

In 1921, the experiments conducted by Frederck Grant

Banting and Charles Herbert Best proved the endocrine

role of Islets of Langerhans of pancreas. They also

isolated insulin from bovine pancreas. This led to

availability of insulin-injections for treatment of diabetes

and for this Banting et al. was awarded the Nobel Prize in

medicine in 1923.

In 1922, clinical diagnosis of diabetes was made

available.

12

In 1935, Sir Harold Percival Himsworth classified diabetes

as Type I diabetes and Type II diabetes.

Identification of sulfonylurea for treating diabetes was

made in 1942.

The radioimmunoassay for insulin was discovered by

Rosalyn Yalow and Soloman Berson.

In 1988, Reaven introduced metabolic syndrome for

treatment of diabetes.

Thiazolidinediones were identified as antidiabetics in

1990.

Etymology

“Diabetes” is a Greek word which means “a passer through; a

siphon” and the world “Mellitus” comes from the Greek word which

means “Sweet”. Apparently, the Greeks named it thus because the

excessive amount of urine, diabetics produce (when blood glucose is

too high), attracted flies and bees because of the glucose content.

The attraction of ants towards the urine of a person was a test for

diabetes in ancient China.

13

Types of diabetes

Type I: Diabetes mellitus

Type I diabetes also referred to as IDDM (insulin dependent

diabetes mellitus or juvenile diabetes). In Type I diabetes beta cells of

Islets of Langerhans of pancreas are destroyed by body’s own

immune system and sufficient insulin is not produced. Type I diabetes

may be caused by an infection by virus. For example-Viruses of

Coxsackie family induce a subtype of Type I diabetes which is similar

to Type II diabetes.

Type II: Diabetes mellitus

Type II diabetes is characterized by “insulin resistance” as

insulin is not taken up by the cells.Type II diabetes is reffered to as

NIDDM (Non Insulin Dependent Diabetes Mellitus). Type II diabetes

is commonly noticed in people above the age of 40 years. Type II

may be caused due to haemochromatosis and polycystic ovary

syndrome, chronic use of steroids, genetic inheritance, and obesity.

Type II diabetes can be treated by changes in diet and exercise in the

initial stage and later, if required, with oral hypoglycemic agents like

sulfonylurea and biguanides as metformin.

14

Gestational diabetes

High blood glucose level in pregnant women, with no previous

diabetic history, is referred to as gestational diabetes and proceeds

as Type II diabetes mellitus.

Congenital diabetes

This involves deficiency of insulin secretion due to defects in

any of the genes responsible for production and secretion of the

insulin.

Other form of diabetes

Cystic fibrosis- related diabetes, Steroid induced diabetes and

monogenic diabetes.

Current status of diabetes

Diabetes mellitus is a group of syndromes which involves high

blood glucose levels and improper metabolism of lipids,

carbohydrates and proteins and an increased risk of complication

from vascular disease. Mostly patients can be classified clinically as

having either insulin dependent diabetes mellitus (IDDM, or Type I

diabetes) or non-insulin dependent diabetes mellitus (NIDDM, or

Type II diabetes). In insulin dependent diabetes mellitus, also called

as juvenile onset diabetes mellitus, destruction of beta cells of Islets

of Langerhans of pancreas takes place. Majority of cases are due to

15

attack on body’s immune system and antibodies that destroy cells

are detectable in blood (Type I A) but in some cases no cells’

antibodies are detected in blood (Type I B).

In non-insulin dependent diabetes mellitus there is no effect on

cell mass. The amount of insulin in blood circulation may be low,

normal or even high but no cell antibody is present. As a high

degree of genetic predisposition generaly has a late onset, (post

middle age) therefore, over 90% of the total cases are Type II

diabetes mellitus (Tripathi, 2003).

Diabetes is one of the most common non-communicable

diseases found globally. It is the fourth leading cause of death in most

developed countries and there is substantial evidence that it is an

epidemic in many developing and newly industrialized nations, thus

posing a serious threat to be met within 21st century. Diabetes is thus

one of the most challenging health problems in the 21st century.

Diabetes can now be found in almost every population in the world

and epidemiological evidence suggests that without effective

prevention and control programmes, diabetes is likely to continue to

rise globally. By the end of the 21st century the worldwide diabetes

will affect an estimated 151 million people, distributed among both

developed and developing countries. In Asia alone an estimated 85

16

million people are affected, this continent has both the biggest

proportion of current cases and the greatest projected increase for

future (Engelgau et al. 2003).

It is estimated that 3.2 million people die due to diabetes across

the world annually. It is also estimated that there are 35 to 40 million

diabetic in India now. Indians are genetically more prone to diabetes

and the World Health Organization (WHO) predicted that number of

diabetic in India would rise to 45 million by 2015 and 74 million by

2025. Diabetes mellitus is projected to affect Asians, specially the

Indians, and the most among all others in the world by 2025

(Ramachandran et al. 2002). WHO also issued a warning as India is

going to be diabetes capital of the world. In 2002, the WHO stated

that the total number of people worldwide with type II diabetes stood

at 176 million. This is estimated to rise to an estimated 370 million

people by 2030. In 2025, the world wide prevalence of diabetes in

adults will be increased by 35%, whereby the number of people with

diabetes will be increased by 122% (Ramachandran et al. 2002).

India will be the country with the largest number of diabetics in 2030

(an estimated 80.9 million) followed by China (an estimated 42

million) and the United states (an estimated 30 million). Diabetes has

a wide spread etiology right from the genetic code to daily diet of the

individual. It is a well known fact that sedentary life styles, obesity,

17

lack of physical work and consumption of food with little or no

therapeutic value are the major contributing factors. At present, the

oral antidiabetic agents belong to class sulfonylureas, biguanides, α-

glucosidase inhibitors, thiazolidenediones and meglitinide derivatives,

are used to treat Type II diabetes. The major limitations of these

drugs are their side effects. The high cost of modern treatment of

diabetes points out to a great need for the development of traditional

or alternative strategies for the prevention and treatment of diabetes.

Further, plants have always been an exemplary source of

modern drugs and many of currently available drugs have been

derived directly or indirectly from them. The ethanobotanical

information reports that about 800 plants may possess antidiabetic

potential.

Genetically Indians are more prone to diabetes

Indians are more prone to diabetes due to their food habits and

life style. Generally obesity and deposition of fat leads to diabetes.

Indians tend to be diabetic at an age of 45, 10 years earlier than in

western countries. 2% of the people in Indian villages are diabetic

and more than half of them are not aware of it.

15 million prescriptions were written for diabetes during

November, 2004 (across 291 towns with a population of > 1 lac).

There has been an increase of 10% in the prescriptions every year.

18

Above forty years of age group accounts for 80% of the total diabetes

prescriptions. Majority of the patients suffer from Type II diabetes.

Out of 100 diabetic patients nearly half of them suffer from a cardiac

ailment. 8% of them are also diagnosed for some neuropathy and

retinopathy.

Blood glucose (blood sugar) level

The normal fasting blood glucose level should be between 70-

110 mg/dl after an overnight fast (not eating anything after midnight).

The normal blood glucose level after 1.5 hours of intake of meal

should be between 90 - 140 mg/dl.

Complications in diabetes

Untreated, diabetes can cause many complications. Diabetic

ketoacidosis, nonketotic hyperosmolar coma include acute

complications of diabetes. Chronic complication includes

cardiovascular diseases, nephropathy (renal failure), and retinopathy

(retinal damage).

Symptoms of Type I diabetes

Increase in appetite, intake of water and urination, loss of body

weight, blurred vision, weakness along with pain in the muscles are

the symptoms of Type I diabetes.

19

Symptoms of Type II diabetes

Increase in appetite, intake of water and urination, loss of body

weight, blurred vision, weakness along with pain in the muscles,

delayed healing of wounds, increased susceptibility to infections,

dehydration, and muscle cramps, etc. are the symptoms of Type ll

diabetes.

Management of diabetes

Type I diabetes

Proper and timely treatment of diabetes is required, as well as a

control on lifestyle such as diet, exercise, stopping smoking, and

maintaining a healthy body weight. Blood sugar should be frequently

monitored and accordingly insulin injection should be taken along

with proper balanced diet, exercise and controlled lifestyle.

Now a days, surgical methods, like transplantation of pancreas,

are being used on experimental basis as a complete cure of Type I

diabetes. But at present the success rate is very limited.

Type II diabetes

Regular monitoring of blood glucose level is essential for proper

management of Type II diabetes. Frequent small meals on definite

20

intervals and light physical exercises like brisk walk of two to four

kilometers a day and yoga have proved to be very effective in

controlling Type II diabetes.

Type II diabetes can be managed by using oral hypoglycemic

agents like sulfonylurea and biguanides. Gastric bypass surgery has

been successful in morbid obesity and Type II diabetes mellitus.

1.8 Hyperlipidemia

Hyperlipidemia is defined as an abnormal elevation in blood

cholesterol, cholesterol esters, triglycerides or phospholipids. The

clinical importance of hyperlipidemia depends on which of these lipids

are elevated and to what extent. Studies have demonstrated that

elevated cholesterol levels are an independent and significant risk

factor for CHD (coronary heart disease). Hypertriglyceridemia has not

been established as an independent risk factor for CHD and is only

considered a marker for other underlying lipoprotein disorders.

Hyperlipidemia can be primary or secondary to some

generalized diseases (e.g. hypothyroidism). They are classified,

according to which lipoprotein particle is raised, into six phenotypes

(the Frederickson classification).

High blood cholesterol is mainly responsible for coronary artery

disease (CAD). A similar link has been shown for raised

21

concentrations of individual atherogenic lipoproteins, in particular the

low density lipoprotein (LDL) fraction. Coronary heart disease (CHD)

is one of the leading causes of morbidity and mortality in the United

States and other industrialized nations. Three of the treatable risk

factors for CHD are hypertension, cigarette smoking and

hypercholesterolemia. The term hyperlipidemia or more precisely

hyperlipoproteinemia refers to conditions in which the concentrations

of cholesterol and/or triglyceride rich lipoproteins are elevated above

normal levels. The term hyperlipidemia is restricted to conditions that

involve increased levels of triglycerides in plasma.

Lipids

Lipids are heterogeneous groups of compounds related to fatty

acids having the common property of being insoluble in water but

soluble in non polar solvents like ether, chloroform and benzene.

Types of lipids

Simple lipids

These are esters of fatty acids and alcohols. Simple lipids may

be of many types depending on the nature of fatty acid and nature of

alcohol present.

22

Compound lipids

These are esters of fatty acid and alcohol but contain certain

other groups according to which they are named like phospholipids,

sulfolipids, aminolipids, cerebrocides etc,

Neutral lipids

This term refers to uncharged lipids which are glycerides,

cholesterol and cholesterol esters.

Lipoproteins

Lipoproteins are particles with high molecular weight and

spherical shape that transport non-polar lipids, primarily triglycerides

(TG) and cholesteryl esters (CE) through the plasma. Every

lipoprotein particle comprises of a non-polar core in which many

molecules of hydrophobic lipids are packed to form oil droplets. This

core consists of triglycerides and cholesteryl esters in varying

proportions and it is hydrophobic in nature. The surface coat, which

surrounds the core, consists of phospholipids, unesterified cholesterol

and apoproteins, and is hydrophilic in nature. The apoproteins or

apolipoproteins are usually present on the surface of lipoproteins.

These proteins serve as an interface between the lipid and the

aqueous environments and also play crucial role in the regulation of

lipid transport and lipoprotein metabolism.

23

Classification of lipoproteins

The lipoproteins vary in composition, size, density and function.

They consist of chylomicrons, very low density lipoproteins (VLDL),

intermediate density lipoprotein (IDL), low-density lipoproteins (LDL)

and high density lipoproteins (HDL).

Lipoproteins are divided into following five major categories

depending on the basis of electrophoresis and ultracentrifugation:

Chylomicrons

Composed of exogenous or dietary triglycerides and carry that

from the intestine to liver, to skeletal muscles and to adipose tissues.

Their density is less than 0.95 gm/mL and diameter is 100-1000 nm.

They contain less than 2% protein, 8% cholesterol, 7% phospholipids

and 84% triglyceride and cholesterolester.

Very low density lipoproteins (VLDL)

Primarily composed of newly synthesised triglycerides and

accounts for 10 to 15% of total serum cholesterol in blood and their

density is 0.95-1.006 gm/mL and diameter is 30-80 nm. They contain

10% protein, 22% cholesterol, 18% phospholipids and 50%

triglyceride and cholesterolester.

Remnant particles or intermediate-density lipoproteins (IDL)

Composed of cholesterol esters and triglycerides, formed from

the degradation of VLDL and enables fats and cholesterol to move in

24

blood stream. Their density is 1.006-1.009 gm/mL and diameter is 25-

50 nm. They contain less than 18% protein, 29% cholesterol, 22%

phospholipids and 31% triglyceride and cholesterolester.

Low density lipoproteins (LDL)

Composed primarily of cholesterol and accounts for 60 to 70%

of total serum cholesterol and is the major artherogenic class of

lipoproteins and helps in the transport of cholesterol from the liver to

the body cells. Their density is 1.019-1.063 gm/mL and diameter is

18-28 nm. They contain 25% protein, 50% cholesterol, 21%

phospholipids and 8% triglyceride and cholesterolester.

High density lipoproteins (HDL)

Composed primarily of cholesterol and accounts for 20 to 30%

of total serum cholesterol and they collect cholesterol from the tissues

and carry that to the liver. .Their density is more than 1.063 gm/mL

and diameter is 5-15 nm. They contain less than 33% protein, 30%

cholesterol, 29% phospholipids and 4% triglyceride and

cholesterolester.

Etiology of hyperlipidemia

Hyperlipidemia can be caused by genetic predisposition,

through secondary causes (like underlying disease states, drugs, or

lifestyle), or both. The severe forms of hyperlipidemia are normally

observed in individuals with specific inherited traits that have resulted

25

in defects in lipid metabolism or transport (e.g. absence of LDL

receptors), patients with hereditary (primary) disease usually require

medication and intensive intervention to prevent morbidity associated

with the condition. Mild or moderate hyperlipidemia is most

commonly caused by some degree of inherited predisposition in

combination with one or more secondary causes.

Concomitant diseases, lifestyle and medications are the three

main secondary causes of hyperlipidemia. Hypothyroidism, nephritic

syndrome, diet high in saturated fats and cholesterol can contribute to

hypercholesterolemia. A high intake of fats, carbohydrates, total

calories and alcohol, as well as a sedentary lifestyle increases

triglyceride levels. Type II diabetes mellitus is a common cause of

hypertriglyceridemia that is aggravated by the associated obesity.

Treatment of hyperlipidemia

The decision to treat hyperlipidemia should not be taken lightly,

since therapy is likely to be life-long and is not without risk. At least

two readings of cholesterol should be obtained over 2-3 months.

Underlying diseases, which may cause hyperlipidemia, such as

hypothyroidism or liver disease, should be taken care of immediately.

The exact cholesterol concentration requiring treatment depends on

other risk factors: in a patient with established ischaemic heart

disease as low as 5.5mmol/L would be appropriate, while in

26

otherwise healthy patients, higher levels should be required. Drugs

may take several weeks to achieve their effects and frequent change

of drug is not advised. Combinations of drugs are sometimes

required.

Drugs used in treating hyperlipidemia

Bile acid sequestrants: Cholestyramine

Fibrates: Gemfibrizol Berizafibrate, ferofibrate

HMG CoA reductase inhibitors: simvastalin

Nicotinic acid.

1.9 Antioxidants

These are the group of compounds that wipe out free radical

oxygen in the body, and thus prevent damages due to oxidation of

cells. They are essential for good health and are found naturally in

many vegetables and fruits. Antioxidants act as radical scavengers,

hydrogen donors, peroxide decomposers, electron donors, enzyme

inhibitors, singlet oxygen quenchers, synergist and metal chelating

agents (Tiwari, 2001).

27

Antioxidants Defense System (ADS)

Antioxidant defense system (ADS) works against oxidative

stress of the cells and follows many lines of action. Antioxidants are

classified in four categories on the basis of their line of action.

Preventive antioxidants

They suppress the formation of free radicals.

Radicals scavenging antioxidants

They suppress the chain initiation and breaking of chain

propagation reaction.

Repair and de novo antioxidants

They include various enzymes which identify, destroy and

remove oxidatively modified proteins and thus also prevent deposition

of oxidised proteins.

Adoption antioxidants

They produce the signals which help in production and

transportation of suitable antioxidant to its site of action. (Halliwell,

1999)

Nutritional antioxidants

Vitamin A (retinol): it is synthesised in the body by using

provitamins present in carrot, papaya, spinach, sweet potatoes,

tomatoes, cabbage, etc. Cod liver oil, shark liver oil, milk, butter and

ghee are rich sources of vitamin A. Its recommended daily allowance

28

is 5000 I.U. per day for an adult person.Its antioxidant property is

responsible for the integrity of epithelial tissues.

Vitamin C (ascorbic acid): it is present in citrus and emblic

fruits, green vegetables, potatoes, tomatoes, berries, milk and liver. It

cannot be synthesised in human body. It helps in formation and

maintenance of collagen in the intercellular material of connective

tissues. It is required in tyrosine metabolism and utilization of iron. Its

daily intake should be about 30 mg.

Vitamin E (tocopherol): vegetable oils and liver of horses and

cattles are rich sources of vitamin E. It inhibits peroxide formation

which in turn prevents damage to membrane lipids. It also prevents

oxidation of unsaturated fats. Its daily requirement for an adult person

is 30 I.U.

Selenium: its antioxidant properties are known to reduce the

chances of prostate cancer, confirmed by recent studies carried out

by the National Health System of China. Its dietry sources are garlic,

nuts, grains, sunflower seeds, shellfish, red meat, chicken, etc.Plants

grown in selenium rich soils are also a good source of selenium.

Bio-flavonoids: dark berries, green tea and green coffee are

rich sources of bio-flavonoids which depicts antioxidant properties.

The non essential antioxidants include enzymes such as

glutathione peroxidase, superoxide dismutase and catalase, bilirubin

29

and omega 3 fattyacids, as they are naturally synthesised within

human body.

Free radicals

Cells, in human body, use oxygen to catabolise carbohydrates,

proteins and fats to get energy. Metabolically active cells produce by

products called free radicals which are highly reactive due to

presence of at least one unpaired electron. They are responsible for

oxidation reactions involved in energy production and functioning of

immune system, especially antibacterial cellular activities (Maxwell,

1995).

Excess of free radicals attack DNA, a cell’s genetic material

(Cancer) and blood vessels (cardiovascular diseases). They are also

implicated in arthritis, strokes, cataracts and degenerative health

problems such as diabetes, ischemia, dementia and aging. Fried

food, cigarette smoke, air and water pollution as well as toxins also

create free radicals.

1.10 Anti-microbial agents

Micro-organisms are the living organisms which can be seen

only under a microscope and consist of a single or a clump of cells.

They may be named as fungi, viruses, bacteria, protozoans, etc.

Antimicrobial agents are the drugs which either inhibit the growth or

kill the micro-organisms.Antibiotics are the drugs derived from living

30

organisms. Chemotherapeutic agents are the chemical substances

acting as antimicrobial agents. Bactericidals are the drugs which kill

bacteria. Bacteriostatics are the drugs which inhibit the growth of

bacterias.

History

The history of anti-microbial drugs can be divided into three

different stages:

Stage I: In 1619, alkaloid like Cinchona bark was used to treat

malaria, Ipecacuanha root was used to treat dysentery, etc.In this

century very few chemotherapeutic agents were known.

Stage II: This stage involved the use of synthetic compounds. In

1909, Paul Ehrlich discovered Salvarsan. Klarer and Mietzsch, in

1932, discovered Prontocil which liberates para-amino-benzene

sulfonamide and it was found effective against Streptococci

infections. In 1938, Sulphapyridene was discovered and found

effective against Pneumococcal pneumoniae and was followed by

Sulfathiozol and Sulfadiazene with improved antibacterial activity.

Stage III: This stage started with the use of antibiotics. Louis Pasteur

and Robert Koch observed inhibition of growth of Bacillus anthrasis.

In 1939, Renedudos reported tyrothricin. Floray and Chain purified

penicillin-G.

31

Even after the above mentioned discoveries a variety of

different classes of antibiotics have been discovered, but still the urge

for discovering new antibiotics is in progress.

Etymology

The term “antibacterial” is derived from Greek word vti (anti)

which means “against” and baktnplov (bakterion) which means

“bacteria”.The word “antibiotics” is derived from vti (anti) and biwtikoc

(biotikos) whch means “fill for life, lively”.

Medicinal Use

Antimicrobial agents are used in the treatment of infections

caused by bacteria, protozoa, virus, fungi, etc. Antimicrobial agents

are used for immune modulation. Antimicrobial agents are used to

prevent infections during and after the surgeries, prophylactics and in

various conditions of cancer.

Classification

Antimicrobial agents are classified on the basis of mechanism

of action, presence of chemical entity and spectrum of activity. The

major classes include antibiotics, antivirus, antifungal and anti

protozoa.

Antibiotics are the drug of choice for the treatment of almost all

type of infections, acquired infections and community acquired

infections. The superbugs have developed antibiotic resistance and

32

may lead to very serious diseases that are difficult to treat and may

result in death. Superbugs are developed both in the environment

and within the host organisms, which is why it is important to not

routinely disinfect and cleanse with antimicrobials or overuse

antibiotic medicines that may lead to evolution of resistant organisms

(Superbugs).

Development of resistance against a antimicrobial drug is very

frequent on prolong use. So there is always a need to find new

molecules or to derive new derivatives with better efficacy and fewer

side effects.

1.11 Muscle Relaxant

A muscle relaxant is a drug which affects skeletal

muscle function and decreases the muscle fatigue. Muscle relaxants

are used to provide relief from muscle spasm, muscle cramps,

muscle fatigue, musclular pain and hyperflexia. Muscle relaxants are

classified as neuromuscular blockers and spasmolytics.

Neuromuscular blockers are not centrally acting drugs. They interfere

with neuromuscular transmissions. They are used to produce

temporary paralysis during surgical procedures, intensive care and as

an emergency medicine. Spasmolytics act on central nervous system

and known as "centrally acting" muscle relaxants. They are used to

33

provide relief from musculoskeletal pain and spasms and to treat

spasticity in various neurological conditions.

History

In 16th century people of the Amazon Basin in South America

used poison-tipped arrows to produce death by paralysis. The active

compound identified as Curare became the basis of further studies in

the field of muscle relaxants. Scientific experiments determine the

role of tubocurarine and acetylcholine in transmissions at

neuromuscular end plates. Upto 1943, muscle relaxants were

established and used in anesthesia and surgery.

The U.S. Food and Drug Administration (FDA) approved the

use of Carisoprodol in 1959, metaxalone in August, 1962,

and cyclobenzaprine in August, 1977.

Mechanism

Muscle relaxants can be active at various sites which include

central nervous system, myelinated somatic nerves, unmyelinated

motor nerve, terminal nicotinic acetylcholine receptors muscle etc.

They inhibit transmissions at the end plate of neuromuscular

junctions.

34

After being adequately stimulated, an active potential is

generated due to which a nerve impulse get triggered at motor end

plate. This initiates influx of calcium ions which causes exocytosis of

synaptic vesicles containing acetylcholine leading to diffusion of

acetylcholine across the synaptic cleft. Acetylcholine may either be

hydrolysed by acetylcholine esterase or it may bind to the nicotinic

receptors. A confirmational change occurs in receptors due to binding

of two acetylcholine molecules which opens the sodium - potassium

channel. As a result Na+ & Ca++ enter the cell & K+ leave the cell

resulting in depolarization of the end plate leading to muscle

contraction.

There are two mechanisms to inhibit end plate function:

Non-depolarizing agents, like tubocurarine inhibit the binding of

acetylcholine to nicotinic receptors and hence prevents depolarization

of the end plate.

Depolarizing agents, like succinylcholine imitate acetylcholine to

sensitize nicotinic receptors to such an extent that it can no longer

initiate any action potential to cause muscle contraction.

Both of the above mentioned neuromuscular blockers are

structurally similar to acetylcholine.

35

2. AIM AND OBJECTIVE

36

2. AIM AND OBJECTIVE

2.1 Need for Study

Many oral hypoglycemic agents, herbal remedies like leaf

powder, pastes, decoctions, infusions and pills have been

recommended but no medicine is capable of giving the radical cure of

diabetes mellitus.

It has been found that about 75 plants posses’ hypoglycemic

activity but only few have been studied in detail.

Allium cepa, Anacardium occidentale, Coccinia indica

Clerodendrum phlomidis, Ficus benghalensis, Dolichos lablab, F.

glomerata, Enicostemma littorale, Gymnema sylvestre, Momordica

charantia, Olea europaea, Pterocarpus marsupium, Orthosiphon

spiralis, Rauwolfia serpentine, Cyamopsis tetragonoloba, Scoparia

dulcis, Syzygium cumini, etc have been tried on human beings but

not to an extent where they can be taken as a drug.

Despite of so much potential and scope of future development,

only 2% of the flora provided by nature is being used beneficially.

The use of herbs is highly beneficial to the community as they:-

are nutritious and good alternative medicines,

are safe and effective forms of ancient therapy,

have no side effects as allopathy system of medicine,

37

provide natural resistance power to the body, and

are used for healing purpose and maintaining the body balance

that help in regulating the body functions.

By using natural products, about 76 million people tend to look

new solutions to treat old problems related to diabetes.Therefore,

there is a need to search effective and safe drug for diabetes mellitus.

Herbal drugs are prescribed widely because of their effectiveness,

less side effects and relatively low costs, even when their biologically

active compounds are unknown.

2.2 Objectives

The experimental work has been designed to achieve the

following objectives:

Collection, identification and authentication of medicinal

plants- Calotropis gigantea and Sphaeranthus indicus and

their parts- leaves, roots and fruits.

Quality standardization of plant extracts by ash value,

extractive value, foreign matter, etc.

Phytochemical investigation of plant extracts by different

chemical tests i.e. alkaloids, glycosides, etc.

To investigate Calotropis gigantea and Sphaeranthus indicus

for antidiabetic activity, serum lipid profile and their effect on

38

liver and kidney weight.

To investigate Calotropis gigantea for antioxidant,

antimicrobial and muscle relaxant activity.

To give scientific evidence in support of the plants-

Calotropis gigantea and Sphaeranthus indicus for their

traditional use as folklore medicine.

Concluding the results.

2.3 Methodology

Collection, identification and authentication of medicinal

plants- Calotropis gigantea and Sphaeranthus indicus and

their parts- leaves, roots and fruits.

Quality standardization of plant extracts by ash value,

extractive value, foreign matter, moisture contents, etc.

Phytochemical investigation of plant extracts by different

chemical tests i.e. alkaloids, glycosides, etc.

Pharmacognostical evaluation including morphology and

microscopical investigations.

Evaluation of antihyperglycemic activity and lipid profile of

the extracts of selected medicinal plant on chemically

induced diabetic rats and their effect on weight of liver and

kidney.

39

Evaluation of antioxidant, antimicrobial and muscle relaxant

activities of the extracts of Calotropis gigantea.

Results and discussion.

Conclusion.

.

40

3. PLANT PROFILE

41

3. PLANT PROFILE

3.1 Calotropis gigantea

Figure 1: Parts of Calotropis gigantea

Family

Calotropis gigantea belongs to the family- Asclepiadaceae,

which is generally called milkweed or swallow-wort. It is a common

wasteland weed (Singh et al. 1996). Asclepiadaceae includes 280

genera and 2,000 species which are cosmopolitan in origin. They are

most abundant in tropical and sub-tropical regions and are rare in

cold regions. Some of the other plants belonging to the family

Asclepiadaceae are Milk weed or Silk weed (Asclepias syriaca L.),

Butterfly weed (Asclepias tuberosa L.) and Calotropis procera (Ait.).

42

Plant Description

Botanical name: Calotropis gigantea

Common names: Gigantic swallow wort, Madar

Height: 8 – 10 ft.

Leaf: arrangement – opposite; sessile

Flower: size- 2 inches; white to purple, rarely light green or yellow;

not scented

Fruits: follicles recurved, 1 or 2 follicles, second more often

suppressed, 3-4" long

Origin: India

Habitat: wasteland

Distribution: throughout India upto altitude of 900 m

43

3.2 Sphaeranthus indicus

Figure 2: Fruit of Sphaeranthus indicus

Family: Asteraceae

Botanical name: Sphaeranthus indicus

Common names: Mundi, Hapus, Gorkhmundi, Boddasoramu,

Kottak aranthai, Mirangani, Murisa, Ghundi, Belaunja, etc.

Morphology: a highly branched, strongly-scented annual weed with

winged stem

Leaf: obovate-oblong, narrowed at the base, dentate and serrate

Flower: compound heads, globose ovoid, glandular hairy, achene

staled; flowering time is November to January

Habitat: rice fields

Distribution: throughout India, Sri Lanka, Africa and Australia

Useful Parts: root, leaves, flowers and seed

44

4. LITERATURE

45

4. LITERATURE

4.1 Sphaeranthus indicus

4.1.1 Phytochemical investigation

Baslas KK et al. 1959; have isolated essential oils – methyl chavicol,

α- ionone, d- cadinine, p- methoxycinnamaldehyde, ocimene, α-

terpinene, citral, geraniol, geranyl acetate, β- ionone, sphaerene,

indicusene and sphaeranthol from Sphaeranthus indicus.

Baslas KK et al. 1960; have isolated yellowish green oil with

linolenic, linoleic, oleic, palmitic, stearic and arachidic acid from entire

plant of Sphaeranthus indicus.

Gupta Raj Kumar et al. 1967; have reported the presence of

stigmasterol, hentriacontane, β-sitosterol, and β-D-glucoside of β-

sitosterol.

Nigam SS et al. 1968; have reported the presence of the essential

oils in Sphaeranthus indicus - cadinene, α-ionone, β- caryophyllene,

p-methoxy cinnamaldehyde, eugenol, α-phellandrene, ocimene,

citral, α-terpinene, etc.

Gogte MG et al. 1986; have isolated hydroxylactones (I, II and III)

from Sphaeranthus indicus.

46

Sohni Jayant S et al. 1988; have isolated a new sesquitepene

lactone-7α-hydroxyeudesm-4-en-6,12-olide(I), a new sesquiterpene

acid, 2-hydroxycaustic acid (II), along with β-eudesmol and ilicic acid

from acetone extract of Sphaeranthus indicus.

Singh SK et al. 1989; have isolated (24S)-2-4-Ethyl cholesta-5, 22-

dien-3β-olβ-D-glucoside from Sphaeranthus indicus.

Atta-ur-Rahman et al. 1989; have isolated an antimicrobial

sesquiterpene lactone, 7-hydroxyfrullanolide from flowers of

Sphaeranthus indicus.

Shekhani MS et al. 1990; have reported an immunostimulant

sesquiterpene glycoside in flowers of Sphaerenthus indicus.

Shekhani MS et al. 1991; have isolated three eudesmanolide, 11α,

13-dihydro-3αn, 7α-dihydroxyfrullanolide, 11α, 13-dihydro-7α-hyroxy-

13-dihydroxyfrullanoide, 11α, 13-dihydro-7α-hydroxy-13-

methoxyfrullanolie from the flowers of Sphaeranthus indicus.

Rojatkar Supada R et al. 1992; have reported two known

eudesmanolide, two sesquiterpenoids, cryptomeridiol and 4-

epicryptomeridiol in Sphaeranthus indicus.

Chughtai MID et al. 1992; have isolated, purified and structural

elucidation was done of alkaloids in methonolic extract of flowers of

Sphaeranthus indicus. The presence of cyclopeptide alkaloidal ring

was confirmed.

47

Rojatkar Supada R et al. 1994; have obtained a new 5α, 7-

dihydroxy eudesmanolide (I) alogwith two known eudesmanolide (II,

III) (Z=0) from photooxidation of 7-hydroxy eudesmanolide (III)

[Z=H2]. The structures have been established by spectral data and

comparison with natural products.

OO

OHOH

CH2

O

OCH2

O

CH2

OH

Me Me

OH

I II

O

O

O

CH2

Me

OH

Me

III

Yadava RN et al. 1998; have reported the chemical evaluation of

leaves of Sphaeranthus indicus.

48

Yadava RN et al. 1998; have isolated 7-hydroxy-3’,4’-5,6,-

tetramethoxy flavone7-O-β-D-(1→4)-diglucoside from stem of

Sphaeranthus indicus.

OO

OO O

OMe

OMe

OMe

MeOOH

OH

CH2

CH2

OH

OH

OH

HO

HO

O

Yadava RN et al. 1999; have isolated a novel flavone glycoside, 5,

4’dihydroxy-3’-prenylbiochanin-7-O-β-D-galactoside from the leaves

0f Sphaeranthus indicus.

O

OMe

OH

OMe

H2C

O

CH C

CH3

CH3

OO

CH2

OH

OH

OH

HO

49

Pujar Prasad P et al. 2000; have isolated three new eudesmanolides

as 11α, 13-dihydro-3α, 7α-dihydroxy-4-5-epoxy-6β, 7-eudesmanolide

(I), 11α, 13-dihydro-7α-acetoxy-3β-hydroxy-6β-7-eudesm-4-enolide

(II), 3-keto-β-eudesmol (III) by spectral analysis.

O

HO

OH

Me

Me

O

MeO O

H

O

Me

MeHO

O

Me

H OAc

I II

Me

CO

CH2

H

CH3

OH

CH3

III

Jirovetz L et al. 2003; have isolated and analysed essential oils of

Sphaeranthus indicus flowers, roots, stems and leaves by GC-MS

and olfactoric methods. More than 95 volatile compounds were found.

The main components are β-eudesmol, 2,5-dimethoxy-p-cymene, β-

caryophyllene τ-cadinol, caryophyllene oxide and α-eudesmol, (Z)-

arteannuic alcohol, β-maaliene, β-eudesmol etc.

50

Lodha Vandana 2003; have reported the presence of essential oils

in capitula of Sphaeranthus indicus – (0.06 – 0.08%) by GC-MS

examination. The main constituents found were cadinene, ocimene,

citral, p-methoxy cinnamaldehyde, geraniol, eugenol, and geranyl

acetate.

Jadhav Ravindra B et al. 2004; have isolated a new eudesmanolide

from aerial part of Sphaeranthus indicus - 11α, 13-dihydo-3α, 7α-

dihydroxy frullanolide and 2α-dihydroxycostic acid.

Chandra Pal Bikas et al. 2001; have isolated (-) – frullanolide from

dried and powered flowers of Sphaeranthus indicus and reported

antifungal, antibacterial and antiprotozoal activity.

Kaul Pran N et al. 2005; have reported essential oils’ composition of

Sphaeranthus indicus on the basis of GC and GC-MS. Thirty eight

compounds, making up to 84.0% of oil content, were identified. The

major compounds were – 2, 5-dimethoxy-p-cymene, α-agarofuran,

10-epi-ϒ-eudesmol and selin –11- en-4α-ol.

Prashanth Kumar V et al. 2006; have isolated peptide alkaloids with

cytotoxic activity against various cancer cell lines. They are effective

in prophylaxis and treatment of cancers.

Prabhu Kirti S et al. 2006; have done pharmacogonostical

evaluation of Sphaeranthus indicus including physiochemical,

morphological and histological parameters.

51

Mitra Shanker Kumar et al. 2006; have isolated alkaloids

monoterpenes, sesquiterpenes, sesquiterpene lactones,

sesquiterpene lactone glycosides, diterpenes, triterpenoids, fatty acid

esters, hydrocarbons and amino acids. They have reported antitumor

activity in vitro of sesquiterpene lactone 7-hydroxy-eudesm-4-en-6,

12-olide (HAC-1).

Jadhav Ravindra B et al. 2007; have isolated two new

eudesmanolide from aerial part of Sphaeranthus indicus - 11α, 13-

dihydro-3α, 7α- dihydroxyeudesm-4-en-6α, 12-olide and 1, 4-en-6β,

7α-eudesmenolide-3, on the basis of spectral studies.

Mishra Bhuwan B et al. 2007; have isolated a novel flavanoid C-

glycoside, 5-hydroxy-7-methoxy-6-C-glycosylflavone (I), from aerial

parts of Sphaeranthus indicus.

Vikani KV et al. 2008; have developed HPTLC method to quantify 7-

hydroxy eudesmanolidde using mobile phase n-hexane: diethyl ether

(3:7) and scanning the plate at 213 nm.

52

4.1.2 Pharmacological investigation

Rao BGV Narasimha et al. 1970; have studied in vitro antimicrobial

efficiency of essential oils against Staphylococcus aureus, Vibrio

cholerae and Escherichia coli.

Dubey KS et al. 2000; have studied antimicrobial activities of

alcoholic & aqueous extracts of Sphaeranthus indicus against

Alternaria solani, Fusarium oxysporum, Pencillium pinophilum.

Sharma Manik et al. 2003; have tested a bicyclic sesquiterpene

lactone from petroleum ether extract of Sphaeranthus indicus on

most sensitive preimaginal stage of the mosquito Anopheles

stephensi at different time intervals.

Bafna AR et al. 2006; have studied the protective effect of bioactive

fraction of Sphaeranthus indicus against immunosupression induced

by cytotoxic agent cyclophosphamide in mice.

Sadaf Farzana et al. 2006; have studied wound healing activity in a

cream containing ethanolic extract of aerial part of Sphaeranthus

indicus in Guinea pigs.

Shirwaikar Annie et al. 2006; have studied in vitro antioxidant effect

of ethanolic extract of Sphaeranthus indicus.

Attaullah MD et al. 2006; have studied antimalarial effect and its

medicinal and therapeutic uses.

53

Ignacimuthu S et al. 2006; have studied antifeedant activity, against

4th instar larvae of Spodoptera litura, of methanol extract of

Sphaeranthus indicus.

Chauhan Vijay et al. 2007; have studied the anti-inflammatory

activity of extract of flowering and fruiting heads of the Sphaeranthus

indicus.

Bafna AR et al. 2007; have studied the effect of petroleum ether

extract of flower heads of Sphaeranthus indicus in increasing

phagocytic activity, hemagglutination antibody titer and delayed type

hypersensitivity in mice (Immunomodulatory activity).

Dubey LN et al. 2007; have studied the antimicrobial activity of

terphenoidal compound isolated from Sphaeranthus indicus against

Bacillus subtilis. 70% methanol extract was used.

Ram A Jeevan et al. 2007; have studied the antimicrobial activity of

essential oils and methanol extract of Sphaeranthus indicus against

Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus

luteus, Micrococcus roseus, Candida albicans and Candida tropicalis.

Razi Muhammad T et al. 2011; have studied antidiabetic activity of

methanolic extract of Sphaeranthus indicus in alloxan induced

diabetic rats.

54

4.2 Calotropis gigantea

4.2.1 Phytochemical investigation

Basu Kali Pada et al. 1934; have reported the presence of

calosterol, a sterol in the milky juice of Calotropis gigantea.

Basu Kali Pada et al. 1936; have reported the presence of

proteinase in the milky juice of Calotropis gigantea and have done its

purification and activation by ascorbic acid and glutathione.

Murti P Bhaskara Rama et al. 1945; have studied the chemical

composition of Calotropis gigantea flowers.

Balakrishna KJ et al. 1945; have reported the chemical constituents

of Calotropis gigantea from latex, root and bark, and found α - and β -

calotropeols gigantin (III) (cardiac poison), equal amount of resinols,

giganteol (II) and isogiganteol (III), wax and resin compound, acetic

acid isovaleric acid, β amyrin, α – calotropeol, β – calotropeol,

tetracyclic compounds, calcium oxalate and solid material like cardiac

poison similar to uscharin.

Biswas Bibhutybhusan et al. 1947; have examined the bast fiber

from Calotropis gigantea.

Anjaneyulu V et al. 1968; have isolated α- amyrin, β – amyrin,

taraxasterol, Ψ- taraxasterol, β – sitosterol, taraxasteryl acetate, 4 –

55

taraxasteryl benzoate from petroleum ether extract of 2.5 kg

Calotropis gigantea.

Tiwari SN et al. 1979; have reported the constituents of latex of

Calotropis gigantea by T.L.C. Out of 7 spots obtained, 3 were

identified as calotoxin, uscharin and calactin.

Abraham KI et al. 1979; have studied proteinases, calotropain F I

and F II from Calotropis gigantea latex.

Gowda D Channe et al. 1980; have reported a polysaccharide

isolated from seed- hairs of Calotropis gigantea. Structural studies of

hemicellulose were done.

Pal G et al. 1980; have isolated, crystallized and studied the

properties of calotropin D I and D II extracted from latex of Calotropis

gigantea.

Heinemann U et al. 1982; have reported the 3 – dimensional crystal

and molecular structure of crysteine protease and calotropin D I at

3.2- ANG resolution.

Rao K Sundar et al. 1983; have reported the percentage content of

oil and protein in the seed of Calotropis gigantea. The major essential

amino acids in the seed protein were phenylalanine, lysine and

histidine.

Inamdar Shashikala R et al. 1983; have reported the presence of

hemagglutinins (Lectins) in the latex of plant.

56

Thakur Swapnadip et al. 1984; have reported that hexane and

methyl alcohol soluble extracts of the latex coagulum of Calotropis

gigantea contains two new triterpene esters, 3’ – methylbutanoates

of α – amyrin and Ψ – taraxasterol.

Sengupta A et al. 1984; have reported autodigestion of two cysteine

proteinases, calotropin D I and calotropin D II isolated from latex of

Calotropis gigantea.

Bhattacharya D et al. 1985; have reported the ionization of phenolic

(OH) group in calotropin D I and D II isolated from latex of Calotropis

gigantea. Out of twelve tyrosine residues from calotropin D I and

thirteen tyrosine residues from calotropin D II, only four residues were

ionized reversibly in the pH range 8.9 – 10.7 with apparent value 9.7.

De Siba Prasad et al. 1988; have reported uscharidin, uzarigenin,

calactin and calotropin in Calotropis gigantea root by H.P.L.C. with

methyl alcohol as a solvent and detected at 226 nm.

Lakshminarayana G et al. 1988; have isolated surface and internal

lipids of Calotropis gigantea leaves. The surface lipids consisted of

22.2% of hydrocarbons, 20.7% of ester waxes, 7.9% of aldehydes,

4.6% of triacylglycerols, 20.0% of fatty acids, 13.2% of sterols, 3.3%

of diacylglycerols and 2.9% of monoacylglycerols. The internal lipids

contained monoglycerols. The glycolipids comprised of 2.4% of

esterified sterylglycosides, 8.4% of monogalactosyldiglycerides, 1.7%

57

of sterylglycosides, 2.3% of cerebrosides, 4.2% of

digalactosyldiglycerides and 27% of sulfoquinovosyldiglycerides. The

phospholipids were composed of 2.8% of cardiolipin, 5.6% of

phosphatidylglycerol, 4.0% of phosphatidylethanolamine, 3.6% of

phosphatidylinositol, 8.3% of phophatidylcholine and 1.2% of

phosphatidylserine. All internal lipid classes contain high amounts of

palmitic and linolenic acids whereas the surface lipids contained

palmitic and stearic acid as main components.

Vora Kalpit A et al. 1988; have characterized extracellular lipase

produced by Asperigillus japonicus in response to Calotropis gigantea

latex.

Shin Whanchul et al. 1990; have elucidated the structure of 12β –

benzoyloxy - 3β, 8β, 14β, 17β – tetrahydroxy – 5 – pregnen – 20 –

one extracted from the root of Calotropis gigantea.

Kitagawa Isao et al. 1992; have reported the presence of two new

Oxypregnane – oligoglycosides: calotroposide A and calotroposide B.

The structures were established as 12-O-benzoyllineolon-3-O-β-D-

cymaropyranosyl (1 → 4) – β-D-oleandropyranosyl (1 → 4) – β – D –

oleandropyranosyl(1 → 4) – β – D –cymaropyranoside and 12-O-

benzoyldeacetylmetaplexigenin-3-O-β-D-cymaropyranosyl (1 → 4) –

β-D-oleandropyranosyl (1 → 4) – β – D – cymaropyranosyl(1 → 4) –

β – D –cymaropyranoside .

58

Shibuya Hirotaka et al. 1992; have isolated and developed the

chemical structure of calotroposides A and B; five oxypregnane –

oligoglycosides named- calotroposides C(I), D(II), E(III), F(IV), and

G(V) from the root of Calotropis gigantea.

Sen Sucharita et al. 1992; have isolated and characterized

isorhamnetin – 3 – O – rutinoside, isorhamnetin – 3 – O –

glucopyranoside and taraxasteryl acetate along with new flavonol

trisaccharide as isorhamnetin – 3 – O – [2 – O – β – D –

galalactopyranosyl – 6 – O – α – L – rhamnopyranosyl] – β – D –

glycopyranoside (calotropiside) from aerial parts of Calotropis

gigantea.

Julie S et al. 1996; have reported the presence of quercetin and

phenolic acids in leaves, bark and wood, which impart resistance

against fungal pathogen Cuscuta chinensis.

Pari K et al. 1998; have isolated giganticine (I), a novel nonprotein

amino acid from a methanol extract of the root and bark of Calotropis

gigantea and studied antifeedant activity.

NH

OMe

NH

O

MeH

OHO

O

59

Ali M et al. 1998; have isolated urs - 18α – H – 6(7), 12, 20 (30) –

triene - 3α – yl acetate, urs - 18α – H – 12, 20 (30)-diene - 3α – yl

acetate, urs - 18α – H – 12, 20 (30)- diene - 7α – yl acetate and urs -

18β – H – 20 (30)- ene – 12β – yl acetate on the basis of spectral

analysis and chemical reactions.

Ali Mohd et al. 1999; have isolated lupene type triterpene as lup –

13(18), 20(29) – dien - 9α – yl acetate and lupeol acetate; two ursane

type triterpenes as urs - 18β- H – 12, 20(30) – dien - 3β – yl acetate

and 17β – hydroxyl – 28 – norurs - 18α-H – 12, 20 (30) – dien - 3β –

yl acetate on the basis of spectral data analysis and chemical

reactions.

Gupta Jyoti et al. 2000; have isolated chemical constituents from

roots of Calotropis gigantea which comprised of naphthalene

derivative – calotropnaphthalene; two terpenes –

calotropisesquiterpenol and calotropisesterterpenol; and an aromatic

compound – calotropbenzofuranone.

Mathur Akhleswar et al. 2003; have reported the presence of

linoleic acid as fatty acid component of seed of Calotropis gigantea.

Lhinhatrakool Thitima et al. 2006; have isolated two new

cardenolides (1 & 2) along with twelve known compounds from

dichloromethane extract of the leaves of Calotropis gigantea. All

cardenolides tested for anticancer activity due to presence of

60

deoxysugar at C – 3, a formyl group at C – 10, and β – unsaturated γ

– lactone.

Habib M Rowshanul et al. 2007; have isolated stigmasterol and β –

sitosterol from a methanol extract of root and bark of Calotropis

gigantea.

4.2.2 Pharmacological investigation

Hesse Gerhard et al. 1938; have studied about heart poison in

Calotropis gigantea latex used as African arrow poison.

Ganapathy CV et al. 1940; have studied the milk clotting activity of

papain from calotropis gigantea.

Chen KK et al. 1942; have studied digitalis like principles of

Calotropis gigantea and compare their activity with other cardioactive

substances.

Rathnasabapathy V et al. 1953; have studied the cardiovascular

action of gigantin obtained from latex of Calotropis gigantea.

Bose SM et al. 1957; have studied the standardization and

evaluation of different methods for the quantitative estimation of

protease activity.

Rao D Seshagiri et al. 1957; have studied insecticidal properties of

petals of Calotropis gigantea.

61

Dhawan BN et al. 1958; have studied stimulant effect of Calotropis

gigantea milk on the rat uterus.

Krishna W Madhiva et al. 1959; have studied the enzymic unhairing

and degreasing properties of proteolytic enzymes and amylolytic

enzymes used for production of leather.

Shukla OP et al. 1961; have studied bacteriolytic activity of plant

latex.

Deng SH et al. 1962; have studied cardiotonic action and biological

potency of an extract from Calotropis gigantea.

Saha JC et al. 1963; have studied histaminic action of latex of

Calotropis gigantea.

Pant Radha et al. 1966; have studied proteolytic activity of plant

latex.

Kulkarni SD et al. 1976; have studied the effect of Calotropis

gigantea on dog E.C.G.

Muthukrishnan J et al. 1979; have studied antimicrobial activity of

Calotropis gigantea leaf soaked in distil water, caffeine and

theophylline on larvae of Danaus chrysippus.

Williams Lyall R et al. 1981; have studied the potential as fuel and

energy for producing hydrocarbon- producing crops.

Kadam Shivaji Shankerrao et al. 1981; have studied weedicide

activity of Calotropis leaf extract for weed such as parthenium grass.

62

Chary M Pravindra et al. 1983; have studied protease activity of

latex.

Amin AR et al. 1985; have studied microbial transformation of plant

latex.

Pati L S et al. 1993; have reported that milky sap from Calotropis

gigantea is used as a proteolytic enzyme in blood group serology.

Pham Xuan Sinh et al. 1994; have reported antiasthmatic,

antitussive activity due to presence of alkaloids and gylcosides.

Dey Srilekha et al. 1995; have reported the metabolic activity like

catalase peroxidase in the latex of Calotropis gigantea.

Pugalenthi Poomalai et al. 1997; have reported the efficacy of

cardenolides secondary metabolites of Calotropis gigantea as

deterrent for phytosuccivorous, sucking pets Aphis gossypii Glov, and

Tettigoneiella spectra Dist, in bhendi fields.

Kiuchi Fumiyuki et al. 1998; have reported the cytotoxic activity in

three cardenolide glycosides: calotropin, frugoside and 4’ – O - β – D

– glucopyranosyl frugoside obtained from akond mul (roots of

Calotropis gigantea).

Verma Smita A et al. 1998; have reported the effect of acid extracts

of Calotropis gigantea latex on HCl corrosion of mild steel.

63

Pushkerlal M et al. 2002; have reported the application of plant latex

for the treatment of industrial effluents. A significant reduction in

T.D.S. (Total dissolved solid) was reported.

Patil SV et al. 2003; have reported that the juice from Calotropis

gigantea provides a cheap, readily available alternative to the rennet

of animal and microbial origin as it posses milk coagulating activity.

Rawat Puran Singh et al. 2003; have reported the use of Calotropis

root and bark in the treatment of leukoderma.

Desouza Anita Mary et al. 2004; have reported the production of

dehaired skins and hides in extracts of Calotropis gigantea.

Kim Hang Rim et al. 2004; have reported P – glycoprotein inhibitory

activity of chloroform extract of Calotropis gigantea in human breast

cancer cells. The activity is comparable to anticancer agent

Daunomycin and Varapamil.

Chitme Havagiray R et al. 2004; have reported antidiarrhoeal

activity in plant extract of Calotropis gigantea in experimental

animals. The roots of Calotropis gigantea have been used for

treatment of leprosy, eczema, syphilis, elephantiasis, ulcer and

cough.

Chitme HR et al. 2005; have reported antipyretic activity of

Calotropis gigantea roots in experimental animals.

64

Rajesh R et al. 2005; have studied that Calotropis gigantea latex

posses procoagulant activity similar to fibrinolytic activity.

Eapen Susan et al. 2006; have reported the potential of Calotropis

gigantea to remove radionuclide/toxic substances like Sr – 90, Cs –

137, from soil and solutions.

Adak Manoranjan et al. 2006; have reported anti inflammatory

activity of Calotropis gigantea.

Argal Ameeta et al. 2006; have reported that alcoholic extract of

peeled roots of Calotropis gigantea posses the CNS activity in albino

rats. The results showed analgesic activity, anticonvulsant activity,

anxiolytic and sedative activity.

Pathak AK et al. 2007; have reported analgesic activity of alcoholic

extract of flowers of Calotropis gigantea.

Srivastava Shobha Rani et al. 2007; have evaluated that extract of

the roots of Calotropis gigantea posses the pregnancy interceptive

activity of the colony-bred adult Sprague – Dawley rats.

Shilpkar Prateek et al. 2007; have reported the use of Calotropis

gigantea in biomethanation.

Jacob Zacharia (India) 2007; has developed a method of

preparation of potential herbal anti – cancer medicine from fresh

extracts of leaves and twigs of Calotropis gigantea fortified with

65

unrefined sugarcane sugar (Jaggery), which was proved for

prevention and treatment of all types of cancer.

Rajesh Rajaiah et al. 2007; have reported clot inducing effect of

plant latex protease, which was proved to stop bleeding and had

wound healing properties.

Arulprakash R et al. 2007; have studied biochemical changes in the

tissues of Callosobruchus maculatus, Sitophilus oryzae, Tribolium

castaneum adult on treatment with Calotropis gigantea plant parts

extracts.

Siddiqui Mohammad Khalid et al. 2008; have prepared a herbal

preparation containing Gul – e – Madar (Calotropis gigantea) which

was effective against cholera and gastroenteritis.

Nair Kesavapanicker Sasikumaran et al. 2008; have prepared

natural pesticides from crushed leaves, flowers, fruits and tender

stems of Calotropis gigantea.

Oman Medical Journal 2011; has reported hypoglycemic effect in

chloroform extract of leaves of Calotropis gigantea in streptozotocin

induced diabetic rats.

66

5. EXPERIMENTS

67

5. EXPERIMENTS

5.1 Collection and authentication of plant material

Calotropis gigantea was collected from the forest area of

Ghaziabad, U.P., India and Sphaeranthus indicus was obtained from

the market of Ghaziabad, U.P and both plants were identified from

the School of Pharmacy, Vishveshwariya Institute of Medical Science,

Greater Noida, Gautam Budha, Nagar, UP India. They were assigned

voucher specimen Ref. VIMS/CONSULT/2009/02/10 and Ref.

VIMS/CONSULT/2009/02/11.

5.2 Physical-chemical evaluation

Evaluation of ash values helps in the detection of low grade

products, exhausted drugs and sandy or earthy material. Water

soluble ash and acid insoluble ash helps in detection of chemical

constituents.

About 3 gm of air dried powder of the parts of the plants

Calotropis gigantea and Sphaeranthus indicus were accurately

weighed in a tared silica crucible. They were incinerated at a

temperature below 450ºC until all carbon content was lost. The

material was cooled and weighed. The percentage of total ash was

calculated with reference to the air dried powdered drug.

68

25 ml of dilute hydrochloric acid was added to the acid insoluble

ash obtained from above method and boiled for 5 minutes. The

material was filtered through ashless filter paper. Washing was done

with hot water followed by ignition and then accurately weighed. The

percentage of acid insoluble ash with reference to the air dried

powdered drug was calculated.

25 ml of water was added to the water soluble ash obtained in

total ash and boiled for 5 minutes. Filtration was done through

ashless filter paper.The filtrate was washed with hot water followed

by ignition and then accurately weighed. The weight of water soluble

ash was calculated by subtracting the weight of insoluble matter from

the weight of the total ash. The percentage of water-soluble ash was

calculated with reference to the air-dried powdered drug.

The percentage of sulphated ash was calculated by moistening

3 gm of air dried powder of the parts of plants Calotropis gigantea

and Sphaeranthus indicus with sulphuric acid. The matter was ignited

at low temperature to constant weight and the percentage was

calculated with reference to the air dried powdered drug.

69

The percentage of alcohol soluble extract was calculated by

macerating 5 gm of air dried powder of the parts of the plants

Calotropis gigantea and Sphaeranthus indicus with 100 ml of alcohol

in a closed flask for 24 hours.It was shaked frequently for 6 hours and

allowed to stand for 18 hours.Filtration was done with a precaution

against loss of alcohol and 25 ml of the filtrate was evaporated in a

tared flat bottomed shallow dish and dried at 1050C to constant

weight and then accurately weighed. The percentage was calculated

with reference to the air dried powdered drug.

The percentage of water-soluble extract was calculated by

macerating 5 gm of air dried powder of the part of the plants

Calotropis gigantea and Sphaeranthus indicus with 100 ml of

chloroform water in a closed flask for 24 hours. It was shaked for 6

hours and allowed to stand for 18 hours. Filtration was done and 25

ml of the filtrate was evaporated in a tared flat bottomed shallow dish

and dried at 1050C to constant weight and then accurately weighed.

The percentage with reference to air dried powdered drug was

calculated (Khandelwal, 2004).

70

5.3 Method of extraction

Powdered plant material was evenly packed in soxhlet

apparatus and extracted with organic solvent from polar to non polar

solvent separately. The aqueous extraction was carried out by cold

maceration process separately. The extraction was carried out with

various solvents, except water, by hot continous extraction for about

20 hours. The solvents used were purified before use. After each

solvent extraction, the extract was separated and concentrated by

vaccum distillation to reduce the volume to 1/10. The concentrated

extracts were transfered to 100 ml beaker and the remaining solvents

were evaporated on water bath. The dried extracts were collected

and placed in a desiccator to remove excessive moisture. The dried

extracts were packed in separate air tight containers, labelled and

used for further studies such as phytochemical screening and

pharmacological activities as well as their formulations.

5.4 Identification of phytochemical constituents

Alkaloids were tested by Mayer’s test, Dragendroff’s test,

Hager’s test and Wagner’s test. Carbohydrates and glycosides were

tested by Molisch’s test, Fehling’s test, Legal’s test, Borntrager’s test

and Anthrone test. Fixed oils and fats were tested by Spot test and

71

Saponification test. Proteins and free amino acids were tested by

Million’s test, Ninhydrin test and Biuret test. Phenolic compounds and

tannins were tested by Test for phytosterol, Salkowski test,

Liebermann- Burchard’s test, Test for gums and mucilage, Test for

flavonoids and Shinoda’s Test (Kokate, 1990).

5.5 Total phenol and flavonoid contents

Total phenolic contents in the extracts were determined by using

the Folin Ciocalteu method (Mc Donald et al., 2001). A dilute solution

of each plant extract of Calotropis gigantea and Sphaeranthus indicus

(0.5 ml of 1:10 g ml-1) and gallic acids were mixed with 5 ml of Folin

Ciocalteu reagent and diluted with distilled water in a ratio of 1: 10

(Slinkard and Singleton, 1977), 4 ml of aqueous sodium carbonate

(1M) was added and allowed to stand for 15 minutes. The total

phenollic contents were determined by UV-VIS spectrophotometeric

method by measuring absorbtion at 765 nm (Pharmaspec 1700,

Shimadzu). Total phenolic content was expressed in terms of gallic

acid equivalent using the following equation based on the calibration

curve:

y = 0.045 x R2 = 0.960

72

Where x was the absorbance and y was gallic acid equivalent

(mg/g).

Flavonoid contents of various extracts were determined by

aluminium chloride colorimetric method (Chang et al., 2002). Each

plant extract of Calotropis gigantea and Sphaeranthus indicus (0.5 ml

of 1:10 g ml-1) in methanol were separately added to 1.5 ml of

potassium acetate (1M) and 2.8 ml of distilled water was added. This

was allowed to stand at room temperature for 30 minutes. The

flavonoid contents were estimated by measuring the absorbance of

mixture at 415 nm with UV-VIS spectrophotometer (Pharmaspec

1700, Shimadzu). Total flavonoid contents were evaluated in terms of

catechin equivalent using the following equation based on the

calibration curve:

y = 0.05 x R2 = 0.992

Where x was the absorbance and y was catechin equivalent (mg/g).

5.6 Drugs and chemicals used

Alloxan monohydrate was purchased from Sigma chemical;

streptozotocin (STZ) was purchased from Sigma Aldrich chemical

Co., Bangalore; citrate buffer (pH 4.5), glibenclamide and metformin

were procured from Aventis Pharma, Mumbai, India.

73

5.7 Antidiabetic activity of extracts of Calotropis gigantea

5.7.1 Hypoglycemic activity of extracts of Calotropis gigantea on

blood glucose level in normal wistar rats (Single dose)

All animals used in this study were kept and maintained at

laboratory conditions of temperature, humidity and 12 hour day- night

cycles and were allowed free access to food (Standard pellet diet)

and water ad libitum. After a period of 4 days acclimatization, the

animals were kept fasting for 16 hours (allowed free access to water

throughout the fasting period). At the end of a period of 16 hour

fasting, blood glucose levels of the fasted normoglycemic rats at zero

time (i.e. 0 hour) were measured and recorded (Akbarzadeh et al.,

2007).

Group I: Received vehicle (normal control).

Group II, III and IV: Received 250 mg/kg BW (body weight) of

petroleum ether, methanolic and aqueous extracts of leaves of

Calotropis gigantea, respectively.

Group V, VI and VII: Received 250 mg/kg BW of petroleum

ether, methanolic and aqueous extracts of roots of Calotropis

gigantea, respectively.

74

All the rats were maintained with standard pellet diet and water

ad libitum. The blood glucose levels of all the rats were

measured at 0, 1, 4, 8 and 12 hour and analyzed statistically.

5.7.2 Streptozotocin induced diabetes

Streptozotocin (STZ) was dissolved in 0.9% ice-cold saline

immediately before use. Diabetes was induced in rats by intra

peritoneal (i.p.) injection of streptozotocin at a dose of 60 mg/kg BW.

Forty eight hours after STZ administration, blood samples were

drawn from tail and glucose levels were determined to confirm the

onset of diabetes. During this period, the rats had free access to both

food and water. The diabetic rats, exhibiting blood glucose levels,

higher than 200 mg/dl, were selected for the studies. The dose of

standard drug glibenclamide was 600µg/kg BW. Animals were

divided into different groups, each group had six rats.

Streptozotocin (STZ) is a broad spectrum anti-biotic isolated

from Streptomyces archromogenes in 1959 (Her, 1959-1960). Earlier

this compound was reported to have anti-cancer activity (Evans,

1965; Arison, 1967) and its diabetic property was first reported by

Rakieten and co-workers. Chemically, STZ is 1-methyl-1-nitrosourea

linked to a position of second carbon atom of D-glucose. It is freely

75

soluble in water, unstable at room temperature and has to be stored

below 200C. Its stability is optimum at pH 4 and at low temperatures.

The biological half life was found to be 5 minutes in rats. STZ induces

diabetes in almost all the species. The elevated blood glucose level

was observed within 48 to 72 hours (Peak effect) and was maintained

thereafter. Rats treated with STZ showed many of the features seen

in human subjects with uncontrolled diabetes mellitus including

hyperglycemia, polydipsia and weight loss.

5.7.3 Effect of extracts of Calotropis gigantea on oral glucose

tolerance test

The oral glucose tolerance test (OGTT) was performed by

feeding glucose solution (0.25 g/kg BW) orally to all the groups and

blood samples were collected at 0, 30, 60, 120 minutes after the

administration of glucose. The oral glucose tolerance test was

performed according to the method of Du Vigneaud (1925). The

mean blood glucose concentrations of control, standard drug and

extract-treated animals at various time intervals were evaluated to

determine whether there were statistically significant differences in

hypoglycemia achieved by different doses of the test extracts.

76

OGTT was carried out to screen the hypoglycemic activity of

petroleum ether, methanolic and aqueous extracts of Calotropis

gigantea on laboratory animals with orally loaded glucose. Overnight

fasted wistar rats weighing between 180-200 g were divided into

different groups; each group consisted of six animals.

Group I: Received vehicle (normal control).

Group II: Diabetic control.

Group III: Received glibenclamide 600 µg/kg.

Group IV, V and VI: Received 250 mg/kg BW of petroleum

ether, methanolic and aqueous extracts of leaves of Calotropis

gigantean, respectively.

Group VII, VIII and IX: Received 250 mg/kg BW of petroleum

ether, methanolic and aqueous extracts of roots of Calotropis

gigantean, respectively.

After the administration of the standard and test extracts, from all

the groups, blood samples were collected at 0, 30, 60 and 120

minutes after the glucose loading and blood glucose levels were

determined.

77

5.7.4 Anti-hyperglycemic activity of extracts of Calotropis

gigantea on STZ induced diabetic rats (multiple doses)

Group I: Received vehicle (normal control).

Group II: Diabetic control.

Group III: Received glibenclamide 600 µg/kg BW.

Group IV, V and VI: Received 250 mg/kg BW of petroleum ether,

methanolic and aqueous extracts of leaves of Calotropis

gigantea, respectively.

Group VII, VIII and IX: Received 250 mg/kg BW of petroleum

ether, methanolic and aqueous extracts of roots of Calotropis

gigantea, respectively.

Drug samples were given every day upto 15 days and blood

samples were collected from the tail, for glucose estimation just

before drug administration on 1st day (commencement of the

experiment) and 1 hour after drug administration on day 1, 5, 10 and

15.

5.8 Antihyperlipidemic activity of extracts of Calotropis gigantea

Rats of all groups were anaesthetized by ether and the blood

samples of each were taken from rat tail tip in 2ml eppendorf tubes

containing anticoagulant. Serum was separated by centrifugation at

78

2500-3000 rpm at 250C for 15 minutes and analyzed for assorted

biochemical parameters.

5.8.1 Estimation of cholesterol

About 10 µl plasma and 1 ml of glucose diagnostic kit reagents

(Spineract) were mixed and incubated for 10 minutes at 20-250 C and

the cholesterol values were determined at 546 nm using

autoanalyzer.

5.8.2 Estimation of triglycerides

About 10 µl plasma and 1 ml triglycerides diagnostic kit

reagent (Spineract) were mixed and incubated for 10 minutes at 20-

250C. The triglyceride values were observed using autoanalyzer at

546 nm.

5.8.3 Estimation of very low density lipoprotein (VLDL)

The very low density lipoproteins were determined by using

Friedwald formula:

VLDL cholesterol = Triglycerides/5.

79

5.8.4 Estimation of high density lipoprotein (HDL)

About 500 µl plasma and 0.05 ml precipitation reagent

(Spineract) were mixed well and allowed to stand for 10 minutes at

room temperature. 1 ml of cholesterol diagnostic kit reagent

(Spineract) was mixed and incubated at 370 C for 5 minutes or at

room temperature for 10 minutes. The HDL values were observed in

autoanalyzer at 546 nm.

5.8.5 Estimation of low density lipoprotein (LDL)

The LDL was calculated using Friedewald formula:

LDL = Total cholesterol – (HDL + Triglycerides/5)

5.8.6 Estimation of:

Glycosylated haemoglobin (HbA1c) % by Spinreact kit

Albumin (g/L)

Total protein (g/L)

Creatinine (mg/dl)

Body weight (g)

Kidney weight(g)

Haemoglobin (g %)

Urine sugar and albumin

80

5.9 Hypoglycemic effect of Calotropis gigantea leaves via

different routes of administration in normal and alloxan induced

diabetic rats

5.9.1 Preparation of water extract

The leaves of Calotropis gigantea were air dried and powdered

in a grinder. 300g of powdered mixture of the plant part was extracted

overnight with 360 ml of water with magnetic stirring in cold room

(4oC). The water extract was separated and the residue was re-

extracted with water. The water extract was concentrated to produce

semisolid mass and dried in lyophilizer (Mini Lyotrap, Serial No

J8199/5, LET Scientific Ltd UK).

5.9.2 Animal and experimental set-up

Colony bred, healthy wistar albino rats either male or female

with a body weight of 150 to 200 gm were selected for the

experimental work. The animals were kept at room temperature and

fed on standard laboratory diet with water ad libitum. The rats were

kept on fast during the night and all the rats were allowed a free

access to water during the experiment in the ambience. The animals

were divided into eight groups of six animals each. 1 ml of blood

sample was taken by capillary tube for the evaluation of blood

81

glucose level from the orbital sinus of each rat. The Institutional

Ethical Committee had approved all experiment protocols.

5.9.3 Hypoglycemic effect in normal rats

Groups of six rats each (fasted for 18 hours) received 10 ml/kg

BW infusions, intragastrically and intraperitoneally (i.p.) Blood

samples were drawn from the tail immediately before administration

in the time intervals of 20, 60, 120, 240 and 360 minutes later.

Control group received an equal volume (10 ml/kg BW) of normal

saline, glibenclamide (0.13 mg/kg BW) and metformin (11.3 mg/kg

BW), calculated on the basis of the daily doses.

5.9.4 Hypoglycemic effect on alloxan-diabetic rats

Chronically hyperglycemic rats were obtained by i.p. injection of

150 mg/kg BW of alloxan dissolved in distilled water. After 8 hours of

administration, the hyperglycemic rats were selected (plasma glucose

level 2-2.8 g/L) and used in the experiments. The same experimental

protocol described above was then adopted.

5.9.5 Glucose tolerance test (GTT) in glucose loaded rats

A polyethylene cannula was injected into the jugular vein under

ethyl carbonate anesthesia. Another catheter was injected into right

82

carotid. All rats received orally 10 ml/kg BW of 25% glucose solution.

One group of animals received the plant infusion (10 ml/kg BW)

through the venous catheter, while the control group received normal

saline. Blood samples (0.2 ml) were taken from the carotid catheter at

time intervals of 5, 10, 20, 30, 40, 50 and 60 minutes after injection.

The coefficient of glucose assimilation (KG) was determined with the

formula:

KG = (log C - log C/2) t½= 0.639 t½

Where, C = glycaemia (g/L); t½ = time for the blood glucose

concentration equal to C/.2.

5.9.6 Statistical analysis

Results were reported as mean ± SEM. Statistical analysis was

carried out using analysis of variance (Anova). The difference of the

means was calculated using Newman – Keuls test. P values of 0.05

or less were taken as significant.

83

5.10 Effect of Calotropis gigantea and Sphaeranthus indicus on

glycemia and lipidemia in streptozotocin induced diabetic rats

5.10.1 Preparation of water extract

300 gm of powdered mixture of the parts of two plant Calotropis

gigantea and Sphaeranthus indicus was extracted overnight with 360

ml of water with magnetic stirring in cold room (4oC). The water

extract was separated and the residue was re-extracted with water.

The combined water extract was concentrated in lyophilizer.

5.10.2 Animals

Wistar albino rats were obtained from R.V. Northland Institute,

Greater Noida, Gautam Budha, Nagar, U.P., India and clearance was

taken from Institutional Animal Ethics Committee (IAEC). Adult rats of

either sex weighing between 150-200 gm were selected for the study.

The animals were acclimatized to laboratory conditions and divided

into various groups. Animals were housed and kept on the light and

dark cycle throughout.

5.10.3 Induction of diabetes and associated neuropathy

Healthy adult wistar albino rats of both sex weighing between

150-200 gm were obtained from the R.V. Northland Institute, Greater

84

Noida, Gautam Budha, Nagar, U.P., India, and used in this study.

The animals were fed on a pellet diet (Hindustan Lever, India) and

water provided ad libitum. Diabetes was experimentally induced to

produce diabetic neuropathy. Sorbitol induced dysfunction of inositol /

metabolites leading to neuron-infraction by causing microangiopathy

of vasa nervosum. It decreases blood flow to nerves. Overnight

fasting animals were injected with streptozotocin (STZ) 60 mg/kg BW

dissolved in 3 mM citrate buffer (pH 4.5) intraperitoneally (i. p.). After

10 days only those rats which showed plasma glucose level > 300

mg/dl were classified as diabetic and were included in study as

described earlier. Animals were divided into three groups of five rats

each. Group 1 animals served as healthy control, while those of the

group 2 were untreated diabetic rats. Rats of group 3 were diabetic

and treated for 8 weeks with 300 mg/kg BW of water extract of

mixture of Calotropis gigantea leaves and Sphaeranthus indicus.

Blood samples were collected from overnight fasted rats at 0 and 8

weeks. Blood glucose serum total cholesterol, HDL and LDL

cholesterol, triglyceride, and glycosylated haemoglobin were

determined using kits from SPINREACT Crta, Sta. Coloma 7-E-

17176 Esteve den Bas GIRONA-Spain (supplied by ARK Diagnostic

Private Limited). Total proteins albumin and creatinine in serum were

determined by the method of Reinhold. Assay of plasma glucose,

85

albumin, creatinine, total cholesterol, LDL, VLDL, HDL cholesterol

and triglycerides were estimated as described earlier. Lipid

peroxidation products were estimated as thiobarbituric acid reactive

substance (TBARS) in plasma and tissues.

5.10.4 Statistical analysis

All the data were statistically evaluated and the significance was

calculated using student’s test. All the results were expressed as

mean ± SD.

5.11 Antioxidant activity of extracts of Calotropis gigantea

Antioxidant potential of the ethanolic extract was determined on

the basis of their scavenging activity of the stable 1, 1- diphenyl-2-

picryl hydrazyl free radical.IUPAC name of DPPH is di(phenyl)-2,4,6-

trinitrophenyl)iminoazanium. DPPH method is most widely used and

easiest method to determine antioxidant activity. In the structure of

DPPH an odd electron is present and is commonally used to

determine free radical scavenging activity.

The aliquots of the different concentrations (1-500 μg/ml) of the

extract were added to 3 ml of a 0.004% w/v solution of DPPH.

Absorbance at 517 nm was determined after 30 minutes, and IC50

(Inhibitory concentration 50%) was determined. IC50 value denotes

86

the concentration of sample required to scavenge 50% of the DPPH

free radicals. At first 6 test tubes were taken to make aliquots of 6

concentrations: 1, 5, 10, 50, 100 and 500 μg/ml. Plant extract and

ascorbic acid were weighed accurately and dissolved in ethanol to

make the required concentration by dilution technique. Here ascorbic

acid was taken as standard. DPPH was weighed and dissolved in

ethanol to make 0.004% w/v solution. To dissolve homogeneously

magnetic stirrer was used. After making the desired concentration, 4

ml of 0.004% w/v DPPH solution was applied on each test tube by

pipette. The room temperature was recorded and kept the test tubes

for 30 minutes in light to complete the reactions. DPPH was also

applied to the blank test tube at the same time where only ethanol

was taken as a blank. After 30 minutes, absorbance of each test tube

was determined by UV spectrophotometer. IC50 was determined

from percentage inhibition vs. concentration graph.

5.12 Antimicrobial activity of extracts of Calotropis gigantea

5.12.1 Preparation of plant material

Plant leaves were collected and washed properly with distilled

water. The leaves were air dried under shade at room temperature.

87

Dried leaves were grinded in the mechanical grinder.The powdered

material was extracted with distilled water.

Ten gram of powdered material was soaked in 100 ml of

distilled water in a conical flask and loaded on an orbit shaker at a

speed of 120 rpm for 24 hours. The mixture was filtered using

Whatman filter paper number 1. Rotary evaporator was used to

concentrate the filtrate and dried by using lyophilizer. Dried extract

was kept in an air tight closed container and stored in cold room at

4°C. The extracted powder was dissolved in sterilized distilled water

to make 1000 μg/ml solution. This mixture was used to perform

antibacterial assay.

5.12.2 Test Microorganism

The following six clinical isolates of bacteria were used for the

study: S. aureus, K.pneumoniae, B. cereus, P. aeruginosa, M. luteus

and E. coli. All these cultures were maintained on nutrient agar plates

at 4°C.

5.12.3 Disc diffusion method

The extract of Calotropis gigantea leaves was obtained by

maceration process by using water as a solvent. Extracts were

screened for antimicrobial activity using disc diffusion method. A

suspension of organism was added to sufficient quantity of nutrient

88

agar at 45ºC. The mixture was aseptically transferred to sterile petri

dish and allowed to solidify. The overnight culture grown in broth was

used for inoculation. The plant extracts to be tested were prepared in

various concentrations i.e. 25%, 50%, 75% and 100%. The sterile

impregnated discs with plant extracts were placed on the agar

surface with framed forceps and gently pressed down to ensure

complete contact of the disc with agar and dextrose surfaces.

Positive control discs were also prepared in the same manner using

ampicillin, a bactericide. But it was not used for fungi. The prepared

control discs were placed using respective solvents.

All the plates including control plates were incubated at 37°C

for 24 hours. After incubation, the size (diameter) of the inhibition

zones was measured. Triplicates were maintained for each sample of

the extract respectively. The results were expressed in terms of the

diameter of the inhibition zone: <9 mm - inactive; 9-12mm - partially

active; 13-18mm - active; >18mm - very active. After the confirmation

of antibacterial activity with 100 mg/kg BW dose, the experiment was

carried out in triplicate and average values were taken into

consideration. Similar procedure was carried out with standard drug

ampicillin 100mg/ml and the zone of inhibition was compared with

89

test sample and control and the percentage of inhibition was

calculated.

5.12.4 Pharmacokinetic parameter of antimicrobial acitivity

Minimum bactericidal concentration

The Minimum bactericidal concentration (MBC) is the least

antibiotic concentration that is used to destroy any bacteria. MBC can

be evaluated by broth dilution Minimum Inhibitory Concentration test.

The test is performed by subculturing to agar medium in which test

organism is not present. MBC is a dose which decreases the original

microorganism concentration by more than 99%.

Minimum inhibitory concentration

The least concentration of antimicrobial agent which prevents

the growth of microbes during an incubation period of 24 hours is

referred to as Minimum Inhibitory Concentration (MIC). MIC is a

significant tool to find out the dose of an antimicrobial agent required

to inhibit the growth of a microorganism and to study the resistance

shown by a microorganism towards an antimicrobial agent. So MIC

has become an important parameter to evaluate antimicrobial activity

in laboratory. MIC testing helps in evaluating MIC50 which represent

the dose of an antimicrobial agent effective in destroying the 50% of

90

the population of bacterial isolate and MIC90 which represent the

dose of an antimicrobial agent effective in destroying 90% of the

population of the bacterial isolate in a bacterium inoculum.

Calculation of relative percentage inhibition

The relative percentage inhibition of the test extract with

respect to positive control was calculated by using the following

formula:

Relative percentage inhibition of the test extract

=

Where, X: total area of inhibition of the test extract

Y: total area of inhibition of the solvent

Z: total area of inhibition of the standard drug

The total area of the inhibition was calculated by using

Area = πr2; where, r = radius of zone of inhibition

91

5.13 Effect of extract of Calotropis gigantea on the skeletal

muscle of the rat

Since the antimigraine drugs were reported to have muscle

relaxant activity, so this experiment was attempted to assess the

effect of extract of Calotropis gigantea on the rat rectus abdominis

muscle preparation. The experiment was carried out as per the

method described by Kulkarni. Rats weighing 20‐25 g were used in

this study. The rat was stunned and decapitated and the spinal cord

was destroyed. The skin of the anterior and abdominal wall of pithed

rat was cut by a midline incision and then it was cut laterally to

expose the anterior abdominal wall. The rectus was seen running

from the base of sternum. The muscles above the sternum and a pair

of muscles attached to it were dissected and kept in a dish containing

ringer solution at room temperature. The muscles were then carefully

cleaned and one muscle of apropriate size was mounted to an organ

bath containing ringer solution and aerated by stream of fine bubbles

emerging near the bottom of the bath.Gimble lever with sideways

writing point adjusted to provide a tension of 2-5 gm was used to

record isotonic contractions of the muscles. An extra load of arround

1 g was applied on long arm so that lever may return back to its

original position after washing. The drug period allowed for

stabilization was 30 minutes during which the muscle was subjected

92

to 1 g stretch. The kymograph was started at 0 minute after raising

the extra load. The drug was added in the 1st minute. The kymograph

was stopped in the 2nd minute. The tissue was washed and relaxed

by applying an extra load. At the 5th minute, the lever point was

brought to the base line and the next cycle was started. The graded

responses to different log doses of acetylcholine were recorded. Then

the test drug (extract) was added and their effects upon acetylcholine

induced contractions as well as the effect of its own on the tissue was

studied.

93

6. RESULTS AND DISCUSSION

94

6. RESULTS AND DISCUSSION

6.1 Macroscopic and microscopic features 6.1.1 Calotropis gigantea Macroscopy Leaves

Simple, opposite, sub-sessile, thick, 10-15 cm long and 4.5-6.5

cm broad; tender leaf: covered with ashy grey hairs; mature leaf:

smooth.

Roots

The root occurs in the entire condition bearing root hairs; 0.5-2

cm in diameter; bark 0.2-0.5 cm thick; whitish grey in colour; wrinkled

in stress condition; with latex excuding from the cuts and wounds of

the bark; facture is tough; taste and odour is characteristic.

Microscopy of Calotropis gigantea leaves

T.S. of leaves shows an upper epidermis with elliptical or oval

shaped cells followed by three layers of closely packed palisade cells

filled with chloroplast; the palisade cells are 11.4-19.0 µ X 13.2-14.2µ

in T.S. palisade layers are parenchymatous which have large intense

cellular space. The cells are thin walled and measure 19.0-54.6µ X

15.2- 40.8µ; Vascular bundles are distributed throughout the spongy

paranchymatous region. The vessels show angular and spiral

95

thickening. The lower epidermal cells are more or less of the same

size and shape as the upper epidermal cell. Stomatas are of

rubiaceous (paracytic) type and scattered in the lower epidermis.

Multicellular thin walled trichomes are found, distributed throughout

the leaf. The T.S. of petiole shows bicollateral bundles besides the

thin walled parenchymatous cells.

Microscopy of Calotropis gigantea root

The T.S. of mature root shows multilayered cork, composed of

rectangular and tangentially elongated cell. Phelloderm is narrow

zone of thin walled parenchymatous cells, most of them filled with

starch grains; lactiferous ducts are also present in the region.

Phloem is very broad zone consisting of sieve tubes, companion

cells and thin walled parenchyma; being transversed by 2-4 layer of

thin walled medullary ray cells; some of them showing calcium-

oxalate crystals and a few starch grains; laticiferous dust is also

present in phloem region; wood is composed of thick walled and

lignified vessels, tracheids, fiber, being transversed by 2-4 layers of

thick walled radially elongated medullary rays.

96

Powder microscopy of Calotropis gigantea leaves

(a)Sclerenchyma cells (b) Stomata

(c) Fiber (d) Fiber

(e) Calcium crystals (f) Vessels

97

(g) Sclerides (h) Epidermal cells

(i)Unicellular trichome

Figure 3: Powder microscopy of Calotropis gigantea leaves

98

(a)Stomata of apex: (b) Stomata of midrib:

Stomata Epidermal cells (c) Stomata of basal:

Figure 4: T.S. of leaf surface for quantitative analysis of Calotropis gigantea (stomata)

Prismatic like Ca oxalate crystal

99

Vein islet number and vein termination number

(a) Veination of apex: (b) Veination of midrib:

Vein termination Vein islet

(c) Veination of basal:

Secondary Veins Figure 5: T.S. of leaf surface for quantitative analysis of Calotropis gigantea (Vein islet number and vein termination number)

Primary Veins

100

Table 1: Stomatal number of upper surface of Calotropis gigantea (leaves)

Observation Apex Midrib Basal

Lowest range 5.3 3.0 3.3

Average range 6.3 6.7 6.5

Highest range 7.5 9.7 9.7 Table 2: Stomatal number of lower surface of Calotropis gigantea (leaves)

Observation Apex Midrib Basal

Lowest range 5.6 4.4 2.8

Average range 6.3 8.2 6.7

Highest range 7.0 12.8 9.8

Table 3: Vein islet number of Calotropis gigantea (leaves) per sq. mm

Observation Apex Midrib Basal

Lowest range 15 12 10

Average range 22.3 13.3 12.7

Highest range 27 15

17.0

101

Table 4: Veinlet termination number of Calotropis gigantea (leaves)

Observation Apex Midrib Basal

Lowest range 16 16 10

Average range 22.3 20.3 13.7

Highest range

27 25 17

Table 5: Palisade ratio of Calotropis gigantea (leaves)

Observation Apex Midrib Basal

Lowest range 1 2 1

Average range 2.5 3.2 2.6

Highest range

4 6 4

102

6.1.2 Sphaeranthus indicus

Macroscopy

A highly branched, strongly-scented annual weed with winged

stem. Leaf: obovate-oblong, narrowed at the base, dentate and

serrate. Flower: compound heads, globose ovoid, glandular hairy,

achene staled; flowering time is November to January.

Microscopy

Figure 6: Powder microscopy of Sphaeranthus indicus stem

Cork in surface view (Polygonal cells)

Stone cell Cells with brownish content

Prismatic crystals of calcium oxalate

103

Fiber

Figure 7: Microscopy of Sphaeranthus indicus stem Epidermis single layered, covered with thick cuticle; cortex

consisting of 4 to 6 layers of oval to polygonal, thin-walled,

parenchymatous cells; endodermis single layer of barrel-shaped

cells; pericyclic fibers, lignified arranged in discontinuous ring;

secondary phloem narrow, having usual elements; groups of

cellulosic fibers found scattered in this zone; secondary xylem

composed of usual elements; vessels with spiral thickening or simple

pitted; pith very wide composed of oval to polygonal, thin-walled,

parenchymatous cells

Stone cell Cells with brownish content

Calcium oxalate

Phloem

Cork

104

Upper epidermis devoid of stomata

Figure 8: Powder microscopy of leaf of Sphaeranthus indicus

Powder microscopy of leaf of Sphaeranthus indicus contains

lower epidermis with stomata and upper epidermis is devoid of

stomata. Cluster crystals also observed.

Lower epidermis containing stomata

Crystal

Cluster crystal

105

Midrib Lamina

Figure 9: Microscopy of Sphaeranthus indicus leaf

Midrib : Shows a single layered epidermis, covered with ordinary

trichomes upto five cells high and glandular trichomes having

unicellular stalk and group of 4 – 10 cells head, on both surfaces,

followed in turn by 4 – 6 layered collenchymatous and 3 – 4 layers

parenchymatous cells at both surfaces; vascular bundles 3- 4,

situated centrally having usual elements, xylem vessels arranged

radially.

Lamina : Shows a single layered epidermis having numerous

trichomes similar to those of midrib on both surfaces; mesophyll not

differentiated into palisade and spongy parenchyma cells; stomata

anisocytic present on both surfaces, stomatal index 32- 38 on lower

and upper surface, vein islet number 20 – 26.

106

Figure 10: Microscopy of Sphaeranthus indicus root

Epidermis single layered, rectangular; secondary cortex

composed of oval to tangentially elongated, thin-walled,

parenchymatous cells having aerenchyma; secondary phloem

composed of thin-walled, oval to polygonal cells, a large number of

groups of lignified phloem fibers found scattered in this zone; central

portion occupied by lignified, secondary xylem having usual

elements; vessels simple pitted; starch grains simple, round to oval

with concentric striations and distinct hilum, measuring 13 to 27 µ in

diameter, present in secondary cortex.

MEDULLARY RAY PERICYCLIC FIBER

METADERMAL

XYLEM

CORTEX

107

6.2 Physiochemical evaluation

Fresh plant material of Calotropis gigantea and Sphaeranthus

indicus was collected and subjected to various physiochemical

parameters such as moisture content and foreign material were

observed and recorded.

Ash values are helpful in determining the quality and purity of

crude drug, especially in the powdered form. Total ash reflects the

care taken in its preparation as all traces of organic matters were

removed during ash formation and usually consists of carbonates,

phosphates and silicates of sodium, potassium, calcium and

magnesium. A higher limit of acid insoluble ash reflects the cases

where silica may be present or when the calcium oxalate content of

the drug is very high. The total percentage of ash values, acid

insoluble ash, water soluble ash and percentage yield of extracts in

different solvents are constant features of a part of the plant which

may constitute individual drug. These reports would be of much

significance in finding out the genuineness of the drug sample.

Medicinal plants are valuable natural sources and regarded as

potential and safe drugs. They have been playing an important role

as natural drugs to alleviate human suffering by contributing herbal

medicines to the primary health care systems of rural and remote

108

areas, where more than 70 % of population in India depend on

folklore and traditional systems of medicines.

The powdered parts of Calotropis gigantea and

Sphaeranthus indicus in different solutions shows different

fluorescence which are tabulated below:

109

Table 6: Physicochemical parameters of Calotropis gigantea

Parameters (w/w %)

Values in percentage

Leaf Root

Moisture content 0.40 1.20

Foreign matters 1.95 2.15

Total ash 9.50 1.00

Acid insoluble ash 3.00 2.30

Water soluble ash 4.80 5.70

Sulphated ash 5.40 5.60

Pet. ether extract 5.70 4.87

Benzene extract 2.52 5.64

Chloroform extract 3.69 5.58

Ethyl acetate 2.73 5.49

Ethanol extract 1.00 8.13

Aqueous extract 1.95 8.67

110

Table 7: Fluorescent analysis of leaves of Calotropis gigantea

Powder drug Observation

Day Light UV (254 nm) UV (366 nm)

Dry powder as

such Reddish brown No change No change

H2SO4 Dark Reddish brown Grayish green

H2SO4 + water Black Brownish black Black

HCl Light yellowish

green Dark green Light green

HCl + water Faint green Light green Violet

HNO3 Light Brick color Grayish green

HNO3 + water Light lemon Yellowish green Green

Acetic acid Green dark green Pink

Methanol Green Faint light green Pink

Ethanol Green Light green Pink

Chloroform Green Light yellowish

green Pink

Petroleum ether No change Dark green Blackish violet

Dist. water Brownish

yellow

Light yellowish

green Violet

10% NaOH Brick red Light green Grayish violet

5% Iodine

Brick color Green

Picric acid

Yellowish green Light green

FeCl3 solution Brown Yellowish green Green

NH3 solution Brown Green Violet

111

Table 8: Fluorescent analysis of root of Calotropis gigantea

Powder drug Observation

Ordinary Light UV (254 nm) UV (366 nm)

Dry powder as such

Green No change No change

H2SO4 Blood red No change No change

H2SO4 + water Yellowish green Light green Green

HCl Green No change No change

HCl + water No change Light green Dark green HNO3 Yellow Green Green HNO3 + water

Yellow Light green

Acetic acid Light green No change Blood red Methanol Light green No change Brick red Ethanol Light green No change Light red Chloroform Green Yellow Pink Petroleum ether Dark green Dark yellow Buff

Dist. water Light yellow Dark yellow Green

10% NaOH Light brown No change No change

5% Iodine Cherry red Brick red No change

Picric acid Yellowish green

No change Green

FeCl3 solution Dark brown No change No change

NH3 solution Light brown Grayish black Black

112

Table 9: Physicochemical parameters of Sphaeranthus indicus

fruit

Parameters (w/w %) Values in percentage

Fruit

Moisture content 0.20

Foreign matters 1.00

Total ash 5.30

Acid insoluble ash 1.00

Water soluble ash 2.35

Sulphated ash 2.90

Pet. ether extract 3.75

Benzene extract 1.22

Chloroform extract 1.05

Ethyl acetate 0.93

Ethanol extract 0.70

Aqueous extract 0.45

113

Table 10: Fluorescent analysis of fruit of Sphaeranthus indicus

Powder drugs

Observation Day Light UV (254 nm) UV (366 nm)

Dry powder as such Dark brown No change No change

H2SO4 Light brown Brown No change H2SO4 +

water Black No change Black

HCl Light yellowish Dark yellow Light yellow

HCl + water Light Light green Violet

HNO3 No change Brick color Cherry

HNO3 + water Lemon Green Dark green

Acetic acid Light brown Brown Light brown

Methanol Brown Light brown Light brown

Ethanol Brown Light brown Light brown

Chloroform Brown Light brown Light brown

Petroleum ether Light green Dark green Blackish violet

Dist. water Brownish yellow

Light yellowish Violet

10% NaOH Brick red Light green Grayish violet

5% Iodine No change Brick color Green

Picric acid No change Green Light green

FeCl3 solution Brown Yellowish green Dark Green

NH3 solution Brown Dark green Violet

114

6.3 Phytochemical evaluation

6.3.1 Phytochemical evaluation of Calotropis gigantea

Extractive values of selected medicinal plants were observed

and tabulated (Table 11).

Table 11: Preliminary phytochemical screening of various

extracts of Calotropis gigantea

Extract Alkaloid Glycoside Tannin Protein Carbohydrates Phenol Saponin Flavonoid

s

Fixed

oils &

Fats

Leaves

PEE - - - - + - - - ++

BE - - - - + - + + -

CE - - ++ - - + + ++ -

EAE - - - - - + - ++ -

ME + ++ - + + ++ - ++ -

AE + ++ - - + ++ - ++ -

Roots

PEE - - - + - - + -

BE - - - + - + + -

CE ++ - - - - ++ - + -

EAE ++ - - - - + - + -

ME +++ ++ - - + ++ - +++ -

AE + ++ - - + ++ - ++ -

115

Discussion

Preliminary phytochemical studies reveal the presence of

phytoconstituents like alkaloids, phytosterols, carbohydrates, phenolic

compounds, tannins, triterpenoids and flavonoids in all three selected

plant extracts and fixed oil was found in methanolic extract of

Calotropis gigantea.

The amount of total phenolic compounds varied in different

plants and ranged from 3.1 to 32.3 mg gallic acid equivalent (GAE/g)

of dry material. The highest amount of total phenolic levels has been

detected in methanolic extract. From analysis we can deduce that

plant extracts contain rich amount of flavonoids.

116

6.3.2 Phytochemical evaluation of Sphaeranthus indicus

Extractive values of selected medicinal plant were observed and

tabulated (Table 12).

Table 12: Preliminary phytochemical screening of various

extracts of Sphaeranthus indicus fruit

PEE = Petroleum ether extract, BE= Benzene extract, CE=

Chloroform extract, EAE = Ethyl acetate extract, ME = Methanolic

extract, AE= Aqueous extract

Extract Alkaloid Glycoside Tannin Protein Carbohydrates Phenol Saponin Flavonoids Fixed oils & Fats

PEE - - - - - - - - +

BE - - - - ++ + - - -

CE - - + + - - ++ ++ -

EAE - - - - - - + + -

ME +++ + - + + + - +++ -

AE + ++ - - + ++ - +++ -

117

Discussion `

Preliminary phytochemical studies reveal the presence of

phytoconstituents like alkaloids, phytosterols, carbohydrates,

phenolic compounds, tannins, triterpenoids and flavonoids in all three

selected plant extracts and fixed oil was found in methanolic extract

of Sphaeranthus indicus.

6.4 Hypoglycemic effect of extracts of Calotropis gigantea

6.4.1 Hypoglycemic effect of extracts of Calotropis gigantea in

normal rats

Hypoglycemic effects of extracts of Calotropis gigantea in various

solvents after 0, 1, 4, 8 and 12 hours in normal rats were observed and

were tabulated (Table 13).

118

Table 13: Hypoglycemic effect of extracts of Calotropis gigantea in

normal rats

Discussion

A single dose of petroleum ether, methanolic and aqueous extracts

of Calotropis gigantea leaf and root (250 mg/kg BW, respectively)

exhibited no significant hypoglycemic effect (p<0.05) after 8th hour.The

effect was felt at 12 hours post dose (p<0.01). Glibenclamide had

significant effect on blood glucose level 4 hours post dose (p<0.01).

Treatment

0hr

1hr

4hr

8hr

12hr

Normal control 97.2 ± 3.4 96.5 ± 5.2 96.1 ± 2.0 94.5 ± 1.8 96.1 ± 3.5

Pet. Ether extract (L) 250mg/kg 97.3 ± 1.9 97.1 ± 4.5 96.3 ± 2.8 96.3 ± 3.4 96.5 ± 5.6

**

Methanolic extract (L)250mg/kg

96.5 ± 4.7 96.4 ± 6.0 95.6 ± 3.2 95.7 ± 3.1 96.4 ± 2.4**

Aqueous extract (L) 250mg/kg 96.3 ± 2.0 94.1 ± 2.2 93.4 ± 0.2 94.4 ± 2.6 94.3 ± 0.12

**

Pet. ether extract (R) 250mg/kg 96.4 ± 4.2 96.6 ± 2.2 97.3 ± 2.6 96.7 ± 6.8 96.2± 2.2

**

Methanolic extract (R) 250mg/kg

96.3 ± 2.8 96.7± 4.6 96.5± 0.2 96.9± 2.2 96.6± 4.8**

Aqueous extract (R) 250mg/kg 97.5 ± 4.8 97.5± 2.2 97.7±0.2 97.8 ± 0.6 97.7 ± 0.8

**

119

6.4.2 Effect of extracts of Calotropis gigantea on oral glucose

tolerance test.

Results of the OGTT conducted on control rats and different

experimental groups of rats were observed and tabulated (table 14).

Table 14: Effect of extracts of Calotropis gigantea on oral

glucose tolerance test

Values are mean ±SEM, n= 6. (One way ANOVA Followed by

Dunnette multiple Comparisons test). Super script *, **, denotes

statistically significance of P<0.01, P<0.001, when compared with

respective diabetic control.

Treatment / mg/kg Blood glucose levels (mg/dL)

0 min 30 min 60 min 120 min

Normal control 94.3 ± 2.0 94.5 ± 2.7 93.5 ± 2.3 94.2 ± 2.6

Diabetic control 142.6 ± 7.3 147.3 ± 7.2 150.6 ± 6.9 157.5 ± 7.0

Glibenclamide 600

µg/kg

131.7 ± 5.4 124.8 ± 6.3 123.6 ± 4.9* 113.1± 4.9

**

Pet. ether extract (L)

250mg/kg

128.6±0.42 130.4±0.26 128.2±2.24* 126.3±0.22

**

Methanolic extract

(L) 250mg/kg

130.3 ± 5.9 144.4 ± 6.0 140.7 ± 5.0* 120.5 ± 5.6

**

Aqueous extract (L)

250mg/kg

129.5 ± 5.6 136.8 ± 4.9 135. ± 5.8* 123.2± 4.9

**

Pet. ether extract

(R) 250mg/kg

130.2±2.2 134.2±4.2 130.3±2.42* 128.8±0.22

**

Methanolic extract

(R) 250mg/kg

129.5±2.2 131.28±4.2 129.2±2.46* 122.4±0.26

**

Aqueous extract (R)

250mg/kg

128.2±0.22 130.2±0.22 129.4±0.44* 127.6±2.22

**

120

Discussion

The blood glucose level of the normal control rats reached a peak,

30 minutes after the oral administration of glucose and gradually

decreased to the nearly glucose load level. In diabetic control rats, the

blood glucose level reached a peak at 120 minutes, the glucose levels

remained higher even after 2 hours. STZ diabetic rats treated with

extracts and standard drug showed significant decrease in blood glucose

levels at 2 hours. OGTT is a well accepted and frequently used assay to

screen antihyperglycemic activity. Extracts and standard drug might

enhance glucose utilization because they significantly reduced the blood

glucose level in STZ diabetic rats. In glucose fed diabetic rats, the

elevated blood glucose levels remained higher after 2 hours. In rats

treated with extracts and standard drug, the glucose levels reached peak

at 30 minutes and returned to the nearly control content after 2 hours.

The extracts at the dose of 250 mg/kg BW significantly increased the

tolerance for glucose. The findings of this OGTT study probably suggest

that the hypoglycemic effect of plant extracts were dose dependent and

facilitates or enhances the clearance of post prudential blood glucose in

rats.

121

6.4.3 Antihyperglycemic effects of extracts of Calotropis gigantea

on STZ induced diabetic rats (15 Days Model)

Antihyperglycemic effects of extracts of Calotropis gigantea on STZ

induced diabetic rats (15 Days Model) were observed and tabulated

(Table 15).

122

Table 15: Anti-hyperglycemic effect of extracts of Calotropis gigantea on blood

Values are mean ±SEM, n= 6. (One way Anova followed by

Dunnette multiple comparisons test). Super script *, **, denotes

statistically significance of P<0.01, P<0.001, when compared with

respective diabetic control.

Treatment Blood glucose level (mg/dl) Day 1 Day 5 Day 10 Day 15

Normal control 124.891.47 124.22.26 123.22.46 123.10.22

Diabetic control 236.22.22 242.52.24 240.74.24 247.64.48

Glibenclamide 600

µg/kg

235.41.00 206.82.58** 192.62.07

** 138.33.11

**

Pet. ether extract (L)

250mg/kg 237.62.30 219.21.92

** 190.81.82

* 153.01.58

*

Methanolic extract (L)

250mg/kg 235.01.58 206.22.77

** 185.02.00

** 140.62.07

**

Aqueous extract (L)

250mg/kg

237.22-64 215.42.07** 193.62.30

** 162.92.70

*

Pet. ether extract (R)

250mg/kg 238.62.30 218.82.74

** 216.21.92

* 205.21.59

*

Methanolic extract (R)

250mg/kg 234.61.48 210.81.58

** 195.61.58

** 142.72.22

**

Aqueous extract (R)

250mg/kg

235.91.58 229.23.11** 219.22.91

* 163.91.30

*

123

Discussion

The petroleum ether, methanolic and aqueous extracts exhibited

anti-diabetic property in streptozotocin-induced diabetic rats as evident

from blood glucose levels. In untreated control (Diabetic) rats the blood

glucose levels increased from 236.22.22 to 247.64.48 mg/dl on the

fifteenth day. In petroleum ether extract (leaves and roots) treated rats

(250 mg/kg BW), the blood glucose levels steadily decreased and they

were found to be 237.62.30 to 153.01.58 mg/dl and 238.62.30 to

205.21.59 mg/dl respectively. In methanolic extract (leaves and roots)

treated rats (250 mg/kg BW), the blood glucose levels were found to be

235.01.58 to 140.62.07 mg/dl and 234.61.48 to 142.72.22 mg/dl

respectively. In aqueous extract (leaves and roots) treated rats (250

mg/kg BW), the blood glucose levels were found to be 237.22.64 to

162.92.70 mg/dl and 235.91.58 to 163.91.30 mg/dl respectively. In

standard drug treated rats the blood glucose levels were found to be

235.41.00 to 138.33.11mg/dl respectively at the end of experiment

(15th day). The result demonstrated that anti-hyperglycemic activity was

concentration dependent.

124

6.5 Hypoglycemic effect of Calotropis gigantea leaves via different

routes of administration in normal and alloxan induced

diabetic rats

6.5.1 Effect of water extract of Calotropis gigantea leaves on plasma

glucose levels after intragastric (p.o) and intraperitoneal (i.p.)

administration to normoglycemic rats

The results of effects of water extract of Calotropis gigantea leaves

on plasma glucose levels after intragastric (p.o) and intraperitoneal (i.p.)

administration to normoglycemic rats after a interval of 0, 20, 60, 120,

240 and 360 minutes were tabulated (Table 16).

125

Table 16: Effect of water extract of Calotropis gigantea leaves on

plasma glucose levels after intragastric (p.o) and intraperitoneal

(i.p.) administration to normoglycemic rats

Treatment Route Plasma glucose (g/L) at time ( min) after treatment

0 20 60 120 240 360

Control(Saline.10 ml/kg)

p.o. 0.99±0.05 0.99±0.08 0.98±0.09 0.90±0.16 0.95±0.06 0.96±0.07

i.p. 0.98±0.04 0.96±0.06 0.89±0.08 0.93±0.09 0.93±0.08 0.94±0.08

Glibenclamide(0.13 mg/kg)

p.o. 0.96±0.09` 0.69±0.11* 0.46±0.07++ 0.57±0.07+ 0.73±0.06 0.83±0.05

i.p. 0.98±0.08 0.73±0.1* 0.48±0.06++ 0.63±0.05+ 0.75±0.06* 0.93±0.13

Metformin(11.3 mg/kg)

p.o. 0.99±0.05 0.73±0.06* 0.65±0.1* 0.54±0.08+ 0.75±0.06* 0.93±0.13

i.p. 0.93±0.04 0.79±0.05* 0.57±0.06+ 0.71±0.05* 0.74±0.05 0.80±0.05

C. gigantea(0.7

g/kg)

p.o. 0.93±0.05 0.59±0.06++ 0.58±0.14++ 0.51±0.07++ 0.83±0.13 1.02±0.06

i.p. 0.98±0.07 0.68±0.06++ 0.56±0.22++ 0.54±0.19++ 0.63±0.09+ 0.67±0.08

+

Values are mean ± SEM, n= 8; *p< 0.05; +p<0.01; ++p<0.001 vs.

Control; Anova and Newman - Keuls test

126

Discussion

The infusion of Calotropis gigantea exhibited a remarkable

hypoglycemic action within 20 minutes after oral and i.p. administration to

normal rats. Blood glucose level reached a mean value of 0.59 ± 0.06 g/L

and 0.68 ± 0.06 g/L, respectively as compared to 0.99 ± 0.08 g/L and

0.96 ± 0.06 g/L respectively obtained in the control group. The lowest

hypoglycemic effect was observed after 2 hours i.e 0.51 ± 0.07 g/L and

0.54 ± 0.19 g/L respectively with oral and i.p administration. After 4

hours of administration, the blood glucose level reached nearly to the

initial glycemic values for orally treated animals, while i.p. administration

still showed hypoglycemic effect even after 6 hours. Hypoglycemic effects

of Calotropis gigantea were comparable and sometimes higher than that

obtained with 0.13 mg/kg BW of glibenclamide or 11.3 mg/kg BW of

metformin.

6.5.2 Effect of water extract of Calotropis gigantea leaves on plasma

insulin levels in normoglycemic rats after intragastric (p.o.) and

intraperitoneal (i.p.) administration

Results were observed and tabulated (Table 17).

127

Table 17: Plasma insulin levels in normoglycemic rats after

intragastric (p.o.) and intraperitoneal (i.p.) administration of water

extract of Calotropis gigantea leaves

Treatment Route

Insulinemia (12.42 µlU/mL) at time ( min) after treatment

0 20 60 120 240 360

Control (Saline.10

ml/kg)

p.o. 58.08±4.41 68.56±10.26 63.11±8.42 56.64±11 60.06±10 72.32±6.16

i.p. 88.88±14.96 71.89±8.25 60.41±7.89 61.57±9.15 67.27±8.01 84.94±7.91

C. gigantea (0.7 g/kg)

p.o. 82.06±14.35 68.96±8.52 54.14±9.56 51.88±3.03 63.17±19.04 75.03±19.42

i.p. 64.22±8.56 78.07±12.62 90.19±7.16* 73.02±16.9 121.42±10.01++ 113.37±20.52+

Values are mean ± SEM, n= 8; *p< 0.01; ++p<0.001 vs. Control; Anova

and Newman - Keuls test.

128

Discussion

After i.p. administration, the variation in insulin plasma levels

showed an opposite trend to that of glucose. The increase became

significant after 1 hour of administration and persisted for at least 6 hour.

Plasma insulin reached a maximum level (12.42µlU/mL) 4 hour after i.p.

administration. On the other hand, no variation in blood insulin level was

found in normal rats orally treated with the water extract of Calotropis

gigantea leaves.

6.5.3 Effect of water extract of Calotropis gigantea leaves on plasma

glucose levels after intragastric (p.o) and intraperitoneal (i.p)

administration to alloxan-diabetic rats

Effect of water extract of Calotropis gigantea leaves on plasma

glucose levels after intragastric (p.o) and intraperitoneal (i.p)

administration to alloxan-diabetic rats after a interval of 0, 20, 60, 120,

240 and 360 minutes were observed and tabulated (Table 18).

129

Table 18: Effect of water extract of Calotropis gigantea leaves on

plasma glucose levels after intragastric (p.o) and intraperitoneal (i.p)

administration to alloxan-diabetic rats

Values are mean ± SEM, n= 8; *p< 0.05; +p<0.01; ++p< 0.001 vs.

control; bp<0.05 vs. glibenclamide; xp<0.05; yp<0.01 vs. metformin;

Anova and Newman - Keuls test.

Treatment Route Plasma glucose (g/L) at time(minutes) after treatment

0 20 60 120 240 360

Control (Saline.10

ml/kg)

p.o. 2.82±0.2 2.57±0.14 2.14±0.72 2.20±0.45 2.35±0.39 3.00±0.48

i.p. 2.71±0.2 2.61±0.18 2.59±0.65 2.15±0.5 2.28±0.49 2.92±0.51

Glibenclamide(0.13 mg/kg)

p.o. 2.88±0. 2 2.15±0.26 1.44±0.39+ 0.91±0.08++ 1.44±0.22++ 1.70±0.34+

i.p. 3.01±0.03 2.36±0.35 1.21±0.47* 0.88±0.11* 1.44±0.22* 1.70±0.34

Metformin (11.3

mg/kg)

p.o. 2.96±0.1 2.66±0.2 1.21±0.09+ 0.99±0.06+ 1.37±0.25+ 2.00±0.47

i.p. 3.01±0.09 1.30±0.4 0.74±0.41* 1.63±0.25* 1.50±0.35* 1.80±0.30*

C. gigantea

(0.7 g/kg)

p.o. 2.90±0.08 2.02±0.53* 1.05±0.16++ 1.01±0.07++ 1.03±0.12++ 0.92±0.20+yx

i.p. 2.86±0.3 1.74±0.25+ 1.60±0.22+ 0.79±0.3++ 1.189±0.21++ 1.32±0.19++bx

130

Discussion

When compared with control, Calotropis gigantea significantly

reduced the blood glucose levels in diabetic rats. The maximum

decrease was observed 2 hours after the administration and plasma

glucose level recorded as 1.01±0.07 g/L (-69.96%) and 0.79±0.3 g/L (-

53.29%), respectively after oral and i.p. treatment.

6.5.4 Effect of before and after the intravenous administration of

water extract of Calotropis gigantea leaves on blood glucose levels

in glucose loaded (0.25 g/kg BW) rats

The results of the effect of before and after the intravenous

administration of water extract of Calotropis gigantea leaves on blood

glucose levels in glucose loaded (0.25 g/kg BW) rats after 0, 5, 10, 20,

30, 40, 50 and 60 minutes were observed and tabulated (Table 19).

131

Table 19: Blood glucose levels in glucose loaded (0.25 g/kg BW) rats

before and after the intravenous administration of water extract of

Calotropis gigantea leaves

Treatment

Blood glucose (g/L) at time ( min) after load

0 5 10 20 30 40 50

60

Control (0.25g/kg glucose)

0.99±0.08 1.26±0.08 1.47±0.11 1.59±0.14 1.71±0.09 1.69±0.11 1.46±0.08 1.33±0.07

C. gigantea (0.7 g/kg)

0.98±0.09 1.15±0.1 1.35±0.06 1.48±0.1 1.51±0.08* 1.31±0.1+ 1.25±0.11+ 1.16±0.13++

Values are mean ± SEM, n= 5; *p< 0.05; +p<0.01; ++p<0.001 vs.

Control; Anova and Newman - Keuls test.

Discussion

In a glucose tolerance test, intravenous treatment with Calotropis

gigantea plasma glucose level significantly reduced at time intervals of

40, 50 and 60 minutes as compared to plasma glucose level induced in

control by a glucose load administration. Glycemic values returned to

basal levels more rapidly than in control group. The coefficient of glucose

assimilation (KG) showed significant increase in treated rats compared to

control (8.17×10-3 vs 6.96×10-3).

132

6.6 Effect of Calotropis gigantea and Sphaeranthus indicus on

glycemia and lipidemia in streptozotocin induced diabetic rats

6.6.1 Effect of treatment of 8 weeks with water extract (300 mg/kg

BW) of Calotropis gigantea and Sphaeranthus indicus and a mixture

of the two plants on fasting plasma glucose level in streptozotocin

(STZ) diabetic rats

Effect of treatment of 8 weeks with water extract (300 mg/kg BW) of

Calotropis gigantea and Sphaeranthus indicus and a mixture of the two

plants on fasting plasma glucose level in streptozotocin (STZ) diabetic

rats were observed and tabulated (table 20).

133

Table 20: Effect of treatment of 8 weeks with water extract (300

mg/kg BW) of Calotropis gigantea and Sphaeranthus indicus and a

mixture of the two plants on fasting plasma glucose level in

streptozotocin (STZ) diabetic rats

Plasma glucose ( mg/dl) mean ± S.D. Group 0 Weeks 8 Weeks

Control 99.4 ± 19.5 89.8 ± 5.21 Diabetic untreated 172.2 ± 5.4 285.6 ± 42.6 Diabetic + Calotropis gigantea

164.6 ± 25.0 105.4 ± 26.6

Diabetic + S. indicus

158.6 ± 10.0a 98.2 ± 25.2a

Diabetic + Calotropis gigantea + S. Indicus

166.9 ± 25.4 85.4 ± 2.3

a = p < 0.001

Group of 5 animals were used for each set of experiments.

All the data were statistically evaluated and the significance was

calculated using student’s‘t’- test. All the results were expressed as mean

± S.D.

134

Discussion

Water extract of Calotropis gigantea plus Sphaeranthus indicus

administered at a dose of 300 mg/kg BW (body weight) brought down

fasting blood glucose, from a higher value of 166.9 ± 25.4 mg/dl to a

normal value of 85.4 ± 2.3 mg/dl while in the untreated group the fasting

blood glucose (FBG) increased from the initial value of 172.2 ± 5.4 mg/dl

to 285.6 ± 42.6 mg/dl.

6.6.2 Effect of water extract (300 mg/kg BW) of Calotropis gigantea

and Sphaeranthus indicus and mixture of the two plants on plasma

glucose tolerance in diabetic rats after 8 weeks

Effect of water extract (300 mg/kg BW) of Calotropis gigantea and

Sphaeranthus indicus and mixture of the two plants on plasma glucose

tolerance in diabetic rats after 8 weeks were recorded and tabulated

(Table 21).

135

Table 21: Effect of water extract (300 mg/kg BW) of Calotropis

gigantea and Sphaeranthus indicus and mixture of the two plants on

plasma glucose tolerance in diabetic rats after 8 weeks

Blood glucose ( mg/dl) mean ± S.D. Group 0 hr 0.5

hr 1 hr 1.5

hr 2 hr

Control 94.3 ± 21.0

140.2 ±11.2

132.6 ±27.3

116.2 ±10.0

102.0 ±12.0

Diabetic untreated

160.5 ±32.1

245.0 ±68.6

273.4 ±89.3

290.6 ±82.6

269.0 ±92.2

Diabetic + Calotropis gigantea

87.9 ±25.6

112.2 ±20.6

114.3 ±15.0

102.0 ±10.3

95.6 ±24.0

Diabetic + S. indicus

82.0 ± 4.2

86.0 ± 4.3

81.0 ± 3.9

80.0 ± 3.2

84.0 ± 3.2

Diabetic + C. gigantea + S. indicus

81.0 ± 3.5

84.0 ± 4.2

86.0 ±3.3

81.0 ±3.6

75.2 ± 1.0

136

Discussion

In plasma glucose tolerance in diabetic rats after 8 weeks the blood

sugar with untreated rats after 2 hours is 269.0 ± 92.2 mg/dl and with

Calotropis gigantea plus Sphaeranthus indicus the blood sugar is 75.2 ±

1.0 mg /dl. The fasting (0 hour) blood glucose values which were higher

in the diabetic animals (160.5 ± 32.1 mg/dl) were brought down to 81.0 ±

3.5 mg/dl, when 300 mg/kg BW of the extract of the mixture of the two

plants was administered for 8 weeks.

6.6.3 Effect of treatment for 8 weeks with water extract of (300 mg/kg

BW) Calotropis gigantea and Sphaeranthus indicus and both

Calotropis gigantea + Sphaeranthus indicus on plasma lipids in

diabetic rats

Effect of treatment for 8 weeks with water extract of (300 mg/kg

BW) Calotropis gigantea and Sphaeranthus indicus and both Calotropis

gigantea + Sphaeranthus indicus on plasma lipids in diabetic rats were

observed and tabulated (Table 22).

137

Table 22: Effect of treatment for 8 weeks with water extracts of (300

mg/kg BW) Calotropis gigantea and Sphaeranthus indicus and both

Calotropis gigantea + Sphaeranthus indicus on plasma lipids in

diabetic rats

Group TC ( mg/dl) mean ± S.D.

LDL-C ( mg/dl) mean ± S.D.

HDL-C (mg/dl) mean ± S.D.

LDL-C/HDL-C ( mg/dl) mean ± S.D.

TG (mg/dl) mean ± S.D.

Control 170.3 ± 10.3 78.0 ± 12.2 44.9 ±13.3 1.7 ±0.3 115.6 ± 42.6

Diabetic untreated

250.0 ± 14.9 152.2± 12.6

45.0 ±12.0 3.3 ±0.4 181.8 ±18.8

Diabetic + Calotropis gigantea

175.0 ± 12.3 99.7 ±16.8 51.3 ±8.4 1.9 ±0.4 174.0 ±15.8

Diabetic + S. indicus

187.0 ± 14.9 95.6 ±15.7 50.2 ±8.5 1.6 ±0.3 135.0 ±13.2

Diabetic + C. gigantea + S. indius

174.0 ± 10.3 51.3 ±6.3 48.3 ±8.2 1.8 ± 0.5 131.0 ±11.2

TC, LDL-C, HDL-C, TG = Total cholesterol, Low density lipoprotein

cholesterol, High density lipoprotein cholesterol and Triglyceride. Number

of animals was 5 in each group.

138

Discussion

The changes in the lipid profile have also been studied. In the case

of diabetic treated animals, with Calotropis gigantea alone, total

cholesterol, LDL cholesterol and LDL-C/HDL-C values returned to near

normal values. There was very slight fall in TG values probably because

these plants could not show much effect on triglycerols. But further

improvement in increase in HDL-C value was seen. With Calotropis

gigantea plus Sphaeranthus indicus treatments similar values were

obtained. Further there was reduction in TG values also.

6.6.4 Effect of water extract of Calotropis gigantea plus

Sphaeranthus indicus on glycosylated haemoglobin, body weight,

serum albumin, total proteins and creatinine, urine sugar and

albumin values shown at the end of 8 weeks treatment

Effect of water extract of Calotropis gigantea plus Sphaeranthus

indicus on glycosylated haemoglobin, body weight, serum albumin, total

proteins and creatinine, urine sugar and albumin values shown at the end

of 8 weeks treatment were recorded and tabulated (Table 23).

139

Table 23: Effect of water extract of Calotropis gigantea plus

Sphaeranthus indicus on glycosylated haemoglobin, body weight,

serum albumin, total proteins and creatinine, urine sugar and

albumin values shown at the end of 8 weeks treatment

Parameter Normal mean ± SD

Diabetic mean ± SD

Diabetic treated mean ± SD

Glycosylated haemoglobin (HbA1c) %

3.04 ± 0.02

9.4 ± 2.4

4.8 ± 0.1

Albumin (g/L) 55.0 ± 1.4 45.0 ± 3.1

52.9 ± 3.0a

Total protein (g/L) 69.0 ± 3.0 73.2 ± 2.9

78.2 ± 3.8a

Creatinine (mg/dl) 0.75 ± 0.04

1.30 ± 0.15

0.91 ± 0.05a

Body weight (g) 283.6 ± 25.8

225.5 ± 45.0

280.4 ± 9.0

Kidney weight (g) 1.40 ± 0.05 2.92 ± 0.08

1.06 ± 0.10

Haemoglobin (g/dl) 16.0 ± 1.2 16.4± 1.4 16.3± 1.1 Urine sugar ND +++ ++ Urine albumin ND ++ ND

The values are mean ± SD.

*a p < 0.05 when compared with diabetic untreated group.

140

Discussion

The total proteins, albumin and creatinine in serum, glycosylated

haemoglobin in blood and total body weight and weight of kidneys (in the

animals killed after the experiment) were also analyzed after 8 weeks of

treatment. An interesting observation is that glycosylated haemoglobin

(HbA1c) % decreased to 4.8 ± 0.1 and returned to normal values after 8

weeks of treatment.

6.6.5 Effect of water extract of Calotropis gigantea plus

Sphaeranthus indicus on the serum lipid profile and on liver and

kidney weights in rats fed on high fat diet

Effect of water extract of Calotropis gigantea plus Sphaeranthus

indicus on the serum lipid profile and on liver and kidney weights in rats

fed on high fat diet were recorded and tabulated (Table 24 and Table 25,

respectively).

141

Table 24: Effect of water extract of Calotropis gigantea plus

Sphaeranthus indicus on the serum lipid profile in rats fed on high

fat diet

Group TC (mg/dl) TG (mg/dl) LDL(mg/dl) HDL-C(mg/dl)

Normal 198.7 ± 44.9 94.9 ± 7.1 83.8 ± 16.2 42.3 ± 19.2

High-fat rats untreated

622.3 ± 1.4 34.7 ± 0.9 3.58 ± 0.11 31.0 ± 0.10

High-fat diet plus extracts

182.5 ± 4.2 171.8 ± 0 148.8 ± 8.0 173.1 ± 23.4

Table 25: Effect of water extract on liver and kidney weight in rats

fed on high fat diet.

Tissues Normal rats High-fat diet untreated

High-fat diet plus extracts of two plants

Liver (g) 7.82 ± 0.33 10.4 ± 0.41 9.8 ± 0.18b

Kidney (g)

0.99 ± 0.05 1.08 ± 0.08 0.99 ± 0.06 b

*b p<0.0005 *c p<0.01

Discussion

Herbal treatment improves the weight of liver and kidney in diabetic

rats fed on high fat diet, although it was not completely reversible in liver.

142

6.7 Antioxidant activity

Antioxidant activity of methanol, ethanol and aqueous extracts of the

leaves of Calotropis gigantea was carried out and the results were

compared graphically (figure 11).

Figure 11: Comparison of antioxidant activity of various extracts Discussion

In the present study, methanol extract of the leaves of Calotropis

gigantea showed potential free-radical scavenging activity but aqueous

extract showed very little free-radical scavenging activity. The ethanol

extract showed lowest activity.

6.8 Antimicrobial activity

The bacterial suspensions were seeded on Mueller-Hinton Agar

(MHA) plates using a sterilized cotton swab. In each of these plates four

wells were cut out using a standard cork borer (7 mm). Using a

Meth. extract

Eth. extract

Aq. extract

143

micropipette, 100 μl of each dilution was added into the wells. All the

plates were incubated at 37ºC for 24 hours. Antimicrobial activity of the

leaf extract was evaluated by measuring the zone of inhibition.

Experiment was carried out in triplicates for each test organism. The

results were recorded and illustrated below.

.

144

Figure 12: Antimicrobial activity of Calotropis gigantea

Test bacteria

145

Table 26: Test organisms’ relative percentage inhibition (%)

Test organism Relative percentage inhibition (%)

Staphylococcus aureus 48.05

Klebsiella pneumoniae 75.64

Bacillus cereus 175.36

Pseudomonas

aeruginosa

108.16

Micrococcus luteus 26.67

Escherichia coli 155.89

146

Figure 13: Relative percentage inhibition of Calotropis gigantea

Relative percentage inhibition

Rel

ativ

e pe

rcen

tage

inhi

bitio

n (%

)

Microorganism

147

Table 27: MIC values of methanol and aqueous extracts of

Calotropis gigantea on test organism

MIC in mg/ml

S.aureus K.pneumonia B.cereus P.aeruginosa M.luteus E. coli

52 23 5.25 4.1 1.7 11.65

Table 28: Antimicrobial activity of Calotropis gigantea

AE: aqueous extract, PC: positive control, NC: negative control. Values

are expressed as mean ± standard deviation of the three replicates. Zone

of inhibition not include the diameter of the well.

Test organisms

Inhibition zone diameter (mm)

AE PC NC

Staphylococcus aureus

14.3±1.15 18.6±1.52 0

Klebsiella pneumonia

13.6±1.52 13.3±0.57 0

Bacillus cereus

16.3±1.52 15.6±1.15 0

Pseudomonas aeruginosa

14.0±1.73 16.6±1.52 0

Micrococcus luteus

16.6±1.52 33.3±1.52 0

Escherichia coli

18.6±1.15 16.6±2.08 0

148

Discussion

The crude extract showed 52, 23, 11.65, 5.25, 4.1 and 1.7 mg/ml MIC

values for S. aureus, K. pneumoniae, E. coli, B. subtilis, P. aeruginosa

and M. luteus, respectively. Aqueous extract of Calotropis gigantea

showed high inhibitory activity followed by methanol extract, whereas

ethanol and petroleum ether extracts showed low activity. Previous

studies reported the presence of phytochemicals like cardenolides,

flavonoids, terpenes, pregnanes, amino acids and cardiac glycosides as

major constituents in Calotropis gigantea may acknowledge the medicinal

properties of this plant.

149

6.9 Muscle relaxant activity

Muscle relaxant activity of aqueous and ethanolic extracts was

carried out and the results were illustrated below in tabular and

graphical forms.

Figure 14: Muscle relaxant activity of Calotropis gigantea

Mus

cle

rela

xant

act

ivity

(mm

)

150

Figure 15: Comparison of muscle relaxant activity of acetylcholine

and test drug at different concentrations

Mus

cle

rela

xant

act

ivity

(mm

)

151

Table 29: Muscle relaxant activity of extract from Calotropis

gigantea

Drug Volume

(ml)

Dose

(μg)

Height(mm) Responses

Acetylcholine 0.1 1 3 Increased Acetylcholine 0.4 4 5 Increased Acetylcholine 0.8 8 9 Increased Acetylcholine 1.2 12 11 Increased T1 (1:100) 0.4 4 - - T1 + ach 0.1 1 6 Increased T1 + ach 0.4 4 9 Increased T1 + ach 0.8 8 12 Increased T2 (1:500) 0.4 4 - Increased T2 + ach 0.1 1 24 - T2 + ach 0.2 2 13 Increased T3(1:1000) + ach 0.3 3 15 Increased T3 + ach 0.4 4 16 Increased T3 + ach 0.8 8 18 Increased T3 + ach 1.6 16 24 Increased

152

Discussion

The aqueous and ethanolic extracts of Calotropis gigantea were

found to have skeletal muscle relaxant property at T1 (1:100), T2 (1:500)

and T3 (1:1000), when tested along with acetylcholine. When the

relaxant property was compared with the standard drug acetylcholine,

ethanolic extract tested along with the acetylcholine produced more

relaxant property than the standard drug acetylcholine. Lesser the

concentration of the test drug (ethanolic extract) more was the response

of the muscle relaxant property. Maximum relaxant effect in T1 and T3

was found i.e. 22 mm and 23 mm at the dose of 16 μg and T2 was 13

mm at 2 μg. Earlier studies have proved that chloroform extract of

Ervatamia crispa revealed skeletal muscle relaxant effect on an isolated

rat rectus abdominis muscle preparation.

153

7. SUMMARY AND CONCLUSION

154

7. SUMMARY AND CONCLUSION

Many studies have shown that flavonoids and phenolic contents

decrease the blood glucose levels and oxidative stress and increase

serum insulin concentrations (Azuma, 2007; Jalal, 2007; Stanley, 2006).

Flavonoids, steroids, triterpenoids, alkaloids and phenols are

known to be bioactive anti-diabetic principles (Oliver Bever, 1986; Ivorra,

1989; Attar, 1989; Kameswara Rao, 1997). Flavonoids are known to

regenerate the damaged beta cells of islets of Langerhans in diabetic

rats. Phenolic contents are found to be effective anti-hyperglycemic

agents (Manickam, 1997).

In the present study, an attempt was made to investigate the

efficacy of Calotropis gigantea and Sphaeranthus indicus as antidiabetic,

antilipidemic, muscle relaxant, antioxidant and antibacterial agent.

Detailed pharmacognostical, phytochemical and pharmacological studies

were carried out as per standard procedures. The evaluation of the plant

extracts for their antidiabetic, antilipidemic, muscle relaxant, antioxidant

and antibacterial activities were carried out.

The phytochemical screening of various extracts of selected plants

revealed the presence of alkaloids, flavonoids, phenolic compounds,

phytosterols, triterpenoids, glycosides and tannins.

In this study, extracts showed hypolipidemic effects in STZ-induced

diabetic rats. The serum triglycerides (TG), total cholesterol (TC) and

155

serum low density lipoproteins (LDL) and very low density lipoproteins

(VLDL) cholesterol concentrations were significantly increased by STZ

injection, but Calotropis gigantea suppressed the increase in these

levels. In contrast, high density lipoproteins (HDL) cholesterol

concentration and total cholesterol (TC) ratio were decreased by STZ-

injection, but were normalized by extracts of Calotropis gigantea

administration.

Glibenclamide is commonally used as a standard drug in

streptozotocin induced diabetes to compare the efficacy of variety of

hypoglycemic compounds (Paredes, 2001).

The Calotropis gigantea showed hypoglycemic effect in normal and

diabetic rats after both oral and intraperitoneal administration. The effects

produced were comparable to that of well-known oral hypoglycemic

compounds like metformin and glibenclamide used at a dose of 11.3

mg/kg BW and 0.13 mg/kg BW, respectively.

As far as the mechanism of action is concerned, in the light of the

obtained results, it can be speculated that Calotropis gigantea activity

could be due to enhancement of peripheral metabolism of glucose.

There was considerable fall in fasting blood glucose (FBG) in

diabetic rats treated with Caloptropis gigantea leaves alone or

Sphaeranthus indicus alone. The effect was more with Sphaeranthus

indicus. But the effect of the two plants in combination was more than

156

that with either of the plants alone. In conclusion it can be stated that the

two plants Calotropis gigantea and Sphaeranthus indicus, have

synergistic effect when given together. They have a strong anti-

hyperglycemic and anti-hyperlipidemic effect.

In conclusion it appears that the water extract of combination of

Calotropis gigantea plus Sphaeranthus indicus has got good

hypoglycemic and hypolipidemic effect and also corrects complications

associated with diabetes such as retinopathy, nephropathy, neuropathy

and musculopathy.

The antioxidant, antibacterial and muscle relaxant activities of

Calotropis gigantea from methanolic, ethanolic and aqueous extracts

were studied and compared to each other. Extract of Calotropis gigantea

had a slower onset of action and lesser degree of muscle relaxant

activity.

Further, the active constituents responsible for antihyperglycemic,

muscle relaxant, antioxidant and antibacterial activities can be isolated

from the plant extracts and the structure may be elucidated. The

mechanism of action and clinical trials may be carried out in future which

may be useful for the society in the management of diabetes,

hepatotoxicity and related disorders. Further studies to identify the active

constituents of Calotropis gigantea and Sphaeranthus indicus and their

mechanism of action are in progress. Extensive research is needed to

157

corroborate the same beneficial effects and to discuss the underlying

mechanism involved in the above findings to eradicate the diabetes.

158

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167

9. PUBLICATIONS

168

9. PUBLICATIONS

1. “Impact of Calotropis gigantea leaves via different routes of

administration in normal and alloxan induced diabetic rats”, Deepak

Teotia, S. P. Chakrabarti, S. S. Ajay. International Journal of

Scientific and Research Publications, Volume 3, Issue 5, May 2013,

ISSN 2250-3153.

2. “Evaluation of Calotropis gigantea (l.) And S. indicus on glycemia

and lipidemia in sterptozotocin induced diabetic rats”, Deepak

Teotia, S. P. Chakrabarti, S. S. Ajay. Int. J. Pharm. Sci. Rev. Res.,

21(1), Jul – Aug 2013; n° 28, 164-168, ISSN 0976 – 044X.

3. “Ethnopharmacological evaluation of Calotropis gigantea”, Vaibhav

and Deepak Teotia et al. World journal of pharmaceutical research.

Aug 2013; vol 2, issue 5: 1826 – 1839.

The published papers are enclosed.

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International Journal of Scientific and Research Publications, Volume 3, Issue 5, May 2013 1 ISSN 2250-3153

Impact of Calotropis Gigantea Leaves via Different Routes of Administration in Normal and Alloxan

Induced Diabetic Rats

Deepak Teotia*, S. P. Chakrabarti**, S. S. Ajay***

* School of Pharmacy, VIshveshwariya Institute of Medical Science, Greater Noida, Gautam Budha Nagar, Pin- 203207, India ** IMT Pharmacy College, Puri, New Nabakalabara Road, Gopalpur, Puri, , Pin- 752001, (Orisaa), India. *** School of Pharmacy, VIshveshwariya Institute of Medical Science, Greater Noida, Gautam Budha Nagar, Pin- 203207, India

Abstract- The present study was carried out to evaluate the anti-diabetic activity of water extract of Calotropis gigantea leaves in alloxan induced diabetic rats for 0, 20, 60, 120, 240, 360 minutes. The water extract at the dose (0.7 gm/kg) exhibited significant anti-hyperglycemic activity. Oral and intraperitoneally administration of the plant produced significant hypoglycemic effect in normal as well as hyperglycemic rats. The water extract of Calotripis gigantea leaves showed hypoglycemic effect in normoglycemic and hyperglycemic rats after both oral and intraperitoneal administration. The effect could be comparable to that of well-known hypoglycemic compound like metformin and glibenclamide used at 11.3 and 0.13 mg/kg, respectively. Index Terms- Calotropis gigantea; Asclepiadaceae; Alloxan monohydrate ; Route of administration

I. INTRODUCTION Diabetes mellitus is an endocrine disorder characterized by hyperglycemic effecting nearly 10% of the population all over the world. Insulin and oral hypoglycemic agents like sulfonylureas and biguanides are still the major players in the management of the disease. However, complete cure of the disease has been eluding physicians for centuries and the quest for the development of more effective anti-diabetic agents is pursued relentlessly. Many herbal products, including several metals and minerals have been described for the cure of diabetes mellitus in ancient literature. Herbal preparations alone or in combination with oral hypoglycemic agents sometimes produce a good therapeutic response in some resistant cases where modern medicines alone fail. There is increasing demand by patients to use natural products with anti-diabetic activity due to side effect associated with the use of insulin and oral hypoglycemic agents [1]. The World Health Organization has also recommended the evaluation of the effectiveness of plants in condition where there is a lack of safe made drugs.

Currently available treatment for this disorder is far from satisfactory and expensive. Calotropis gigantea leaves (Family : Asclepiadaceae) is a small tree variety found throughout India. It is commonly called Swallow –

Wort. Leaves contain the cardiac glycoside; Calotropin; Uscharin; Calotoxin; Calactin; Uscharidin and gigantin [2-3-4]. It is used widely used for healing of wounds; anthelmintic; expectorant; useful in leprosy scabies ring warm of the scalp; piles, eruptions on the body; asthma;

170

prevention of insulin resistance [5], hepatoprotective [6], anti-diarrhoeal [6], antipyretic and analgesic [7-8], anti-inflammatory[9] and wound healing activity [10]. The calotropin Uscharin and gigantin show digitalis – like action on the heart.

The preliminary phytochemical studies reveal the presence of flavonoids; glycosides; alkaloids; tannins. The focus of the present study is to evaluate water extract of Calotropis gigantea leaves material at various doses in normal and alloxan induced diabetic rats. However, no scientific data are available regarding the effect of water extract of Calotropis gigantea leaves on blood glucose level. The present study is undertaken to explore the effect of water extract of Calotropis gigantea leaves on the blood glucose level of experimental animals and to determine the probable mechanism of action. The effect of water extract of Calotropis gigantea leaves on fasting blood sugar level has been evaluated as compared to the standard drug glibenclamide, both in normal and diabetic albino rats. The effect of Calotropis gigantea extract on glucose uptake by rat hemi-diaphragm and the glycogen content of the liver, skeletal muscle and cardiac muscle are evaluated to study its probable mechanism of action as a hypoglycemic agent.

II. EXPERIMENTAL 2.1. Material required:

Alloxan monohydrate was purchased from Sigma Chemical

Co St Louis, USA. All other chemicals were obtained from local sources and were analytical grade. Calotropis gigantea was collected from the forest area of Ghaziabad, U.P., India in March 2008. The plants was identified from the School of Pharmacy, Vishveshwariya Institute of Medical Science, Greater Noida, Gautam Budha, Nagar, India. They were assigned voucher specimen Ref. VIMS/CONSULT/2009/02/10. 2.2. Methods: 2.2.1. Preparation of water extract:

The leaves of Calotropis gigantea was air dried and powdered in a grinder. 300g of Powder mixture of the plant parts was extracted overnight with 360 ml of water with magnetic stirring in cold room (4oC). The water extract was separated and the residue was re-extracted with water. The water extract was concentrated to produce semisolid mass and dried in lyophilizer (Mini Lyotrap, Serial No J8199/5, LET Scientific Ltd UK).

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International Journal of Scientific and Research Publications, Volume 3, Issue 5, May 2013 2 ISSN 2250-3153

2.2,2 Preliminary phytochemical screening:

The extracts were subjected to preliminary screening for various active phytochemical constituents [11]. 2.2,3. Animal and experimental set-up:

Colony bred, healthy male wistar albino rats of either sex weighing 150 – 200 g were taken for the study. The animals fed on standard laboratory diet with water ad libitum and housed at room temperature. The rats were kept fasting overnight with free access to water during the experiment in the ambience. The animals were divided into eight groups of six animals each. 1 ml of blood was taken from the orbital sinus of each rat with the help of a capillary tube for the estimation of blood sugar. The institutional Ethical Committee approved all experiment protocols. 2.2.4. Hypoglycemic effect in normal rats [12-13]:

Groups of six rats each (fasted for18 hrs) received 10 ml/kg infusion, intragastrically or intraperitoneally (i.p.) Blood samples were drawn by puncture from the tail immediately before administration in the time intervals of 20, 60, 120, 240 and 360 min later. Control group received an equal volume (10 ml/ kg) of normal saline, glibenclamide (0.13 mg/kg) and metformin (11.3 mg/kg), calculated on the basis of the daily doses [14]. 2.2.5. Hypoglycemic effect on alloxan-diabetic rats [15]:

Chronically hyperglycemic rats were obtained by i.p. injection of 150 mg/kg of alloxan dissolved in distilled water

[16 ]. After 8 hrs administration, the hyper-glycemic rats were selected (plasma glucose level 2-2.8 g/L) and used in the experiments. The same experimental protocol described above was then adopted. 2.2.6. Glucose tolerance test (GTT) in rats [17-18]:

A polyethylene cannula was injected into the jugular vein under ethyl carbonate anesthesia. Another catheter was injected into right carotid. All rats received orally 10 ml/kg of 25% glucose solution. One group of animals received the plant infusion (10 ml/kg) through the venous catheter, while the control group received normal saline. Blood samples (0.2 ml) were taken from the carotid catheter at time intervals of 5, 10, 20, 30, 40, 50 and 60 min after injection. The coefficient of glucose assimilation (KG) was determined with the formula.

KG = (log C - log C/2) t½= 0.639 t½

Where : C = glycaemia (g/L); t½: Time for the blood glucose concentration C/2. 2.2.7. Statistical analysis:

Results are reported as mean ± SEM statistical analysis was carried out using analysis of variance (Anova). The difference of the means was calculated using Newman – Keuls test. P values of 0.05 or less were taken as significant.

Table 1.

Effect water extract of Calotropis gigantea leaves on plasma glucose levels after intragastric (p.o) and intraperitoneal (i.p.)

administration to normoglycaemic rats.

Treatment Route Plasma glucose (g/L) at time ( min) after treatment 0 20 60 120 240 360

Control(Saline.10 ml/kg) p.o. 0.99±0.05 0.99±0.08 0.98±0.09 0.90±0.16 0.95±0.06 0.96±0.07 i.p. 0.98±0.04 0.96±0.06 0.89±0.08 0.93±0.09 0.93±0.08 0.94±0.08

Glibenclamide(0.13 mg/kg) p.o. 0.96±0.09` 0.69±0.11* 0.46±0.07++ 0.57±0.07+ 0.73±0.06 0.83±0.05 i.p. 0.98±0.08 0.73±0.1* 0.48±0.06++ 0.63±0.05+ 0.75±0.06* 0.93±0.13

Metformin(11.3 mg/kg) p.o. 0.99±0.05 0.73±0.06* 0.65±0.1* 0.54±0.08+ 0.75±0.06* 0.93±0.13 i.p. 0.93±0.04 0.79±0.05* 0.57±0.06+ 0.71±0.05* 0.74±0.05 0.80±0.05

C. gigantea(0.7 g/kg) p.o. 0.93±0.05 0.59±0.06++ 0.58±0.14++ 0.51±0.07++ 0.83±0.13 1.02±0.06 i.p. 0.98±0.07 0.68±0.06++ 0.56±0.22++ 0.54±0.19++ 0.63±0.09+ 0.67±0.08+

avalues are mean ± SEM, n= 8; *p< 0.05; +p<0.01; ++p<0.001 vs. Control; Anova and Newman - Keuls test.

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Table 2.

Plasma insulin in normoglycemic rats after intragastric (p.o.) and intraperitoneal (i.p.) administration water extract

of Calotropis gigantea leaves.

Values are mean ± SEM, n= 8; *p< 0.01; ++p<0.001 vs. Control; Anova and Newman - Keuls test.

Table 3.

Effect water extract of Calotropis gigantea leaves on Plasma glucose levels after intragastric and intraperitoneal (i.p.) administration to alloxan-diabetic rats.

Treatment Route Plasma glucose (g/L) at time ( min) after treatment

0 20 60 120 240 360

Control(Saline.10 ml/kg)

p.o. 2.82±0.2 2.57±0.14 2.14±0.72 2.20±0.45 2.35±0.39 3.00±0.4

8 i.p. 2.71±0.2 2.61±0.18 2.59±0.65 2.15±0.5 2.28±0.49 2.92±0.5

1 Glibenclamide(

0.13 mg/kg) p.o. 2.88±0. 2 2.15±0.26 1.44±0.39+ 0.91±0.08++ 1.44±0.22++ 1.70±0.3

4+ i.p. 3.01±0.03 2.36±0.35 1.21±0.47* 0.88±0.11* 1.44±0.22* 1.70±0.34

Metformin(11.3 mg/kg)

p.o. 2.96±0.1 2.66±0.2 1.21±0.09+ 0.99±0.06+ 1.37±0.25+ 2.00±0.47

i.p. 3.01±0.09 1.30±0.4 0.74±0.41* 1.63±0.25* 1.50±0.35* 1.80±0.30*

C. gigantea(0.7 g/kg)

p.o. 2.90±0.08 2.02±0.53* 1.05±0.16++ 1.01±0.07++ 1.03±0.12++ 0.92±0.20+yx

i.p. 2.86±0.3 1.74±0.25+ 1.60±0.22+ 0.79±0.3++ 1.189±0.21++

1.32±0.19++bx

Values are mean ± SEM, n= 8; *p< 0.05; +p<0.01; ++p< 0.001 vs. control; bp<0.05 vs. glibenclamide; xp<0.05; yp<0.01 vs. metformin; Anova and Newman - Keuls test. Ajay1

Treatment Route Insulinmia (12.42 µlU/mL) at time ( min) after treatment 0 20 60 120 240 360

Control(Saline.10 ml/kg) p.o. 58.08±4.4

1 68.56±10.2

6 63.11±8.4

2 56.64±

11 60.06±10 72.32±6.16

i.p. 88.88±14.96

71.89±8.25

60.41±7.89

61.57±9.15

67.27±8.01

84.94±7.91

C. gigantea(0.7 g/kg) p.o. 82.06±14.3

5 68.96±8.5

2 54.14±9.5

6 51.88±3

.03 63.17±19.

04 75.03±19.

42 i.p. 64.22±8.56 78.07±12.

62 90.19±7.1

6* 73.02±1

6.9 121.42±10

.01++ 113.37±20

.52+

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Table 4.

Blood glucose in glucose loaded (0.25 g/kg) rats before and after the intravenous administration water extract of

Calotropis gigantea leaves.

Values are mean ± SEM, n= 5; *p< 0.05; +p<0.01; ++p<0.001 vs. Control; Anova and Newman - Keuls test.

III. RESULT AND DISCUSSION The infusion of Calotropis gigantea exhibited a remarkable

hypoglycemic action 20 min after oral and i.p. administration to normal rats. (Table 1.). Blood glucose level reached a mean value of 0.59 and 0.68 g/L, respectively as compared to 0.99 g/L and 0.96g/L. respectively obtained in the control group. The lowest hypoglycemic effect was observed after 2 hr of administration. After 4 hrs of administration, the blood glucose level reached nearly to the initial glycemic values for orally treated animals, while i.p. administration still showed hypoglycemic effect even after 6 hr. Calotropis gigantea hypoglycemic effect was comparable and sometimes higher than that obtained with 0.13 mg/kg of glibenclamide or 11.3 mg/kg of metformin. After i.p. administration, the variation in insulin plasma levels showed an opposite trend to that of glucose (Table 2). The increase became significant after 1 hr of administration and persisted for at least 6 hr. Plasma insulin reached a maximum level (12.42 µlU/mL) 4hr after i.p. administration. On the other hand, no variation in blood insulin level was found in normal rats orally treated with the water extract of Calotropis gigantea leaves. When compared with control, Calotropis gigantea (Table 3) significantly reduced the blood glucose levels in diabetic rats. The maximum decrease observed 2 hr after the administration in plasma glucose level recorded as 1.01 g/L (-69.96%) and 0.79 g/L (-53.29%), respectively after oral and i.p. treatment. In a glucose tolerance test, intravenous treatment with Calotropis gigantea plasma glucose level significantly reduced at time intervals of 40, 50 and 60 min as compared to plasma glucose level induced in control by a glucose load administration (Table 4). Glycemic values returned to basal levels more rapidly than in control group. The coefficient of glucose assimilation (KG) showed significant increase in treated rats compared to control (8.17×10-3 vs 6.96×10-3).

IV. CONCLUSION

The Calotropis gigantea showed hypoglycemic effect in normoglycemic and hyperglycemic rats after both oral and intraperitoneal administration. The effect could be comparable to that of well-known hypoglycemic compound like metformin and glibenclamide [19] used at 11.3 and 0.13 mg/kg, respectively. As far as the mechanism of action is concerned, in the light of the obtained results it can be speculated that Calotropis gigantea activity could be due to enhancement of peripheral metabolism of glucose. An increase of insulin release can not be excluded. Further studies to identify the active constituents of Calotropis gigantea and their mechanism of action are in progress.

REFERENCES [1] Luo J, Fort DM, Carlson TJ, Noamesi BK, nii-A-Kotei D, King SR, Diabet

Med., 15(5), 367 (1998). [2] Thakur S, Das P, Itoh T. Kazunori Imai, Taro M, Phytochemistry, 9, 2085

(1984). [3] Thitima L, Somyot S, J Nat Prod., 8, 1249 (2006). [4] Sen S, Sahu NP, Mahato SB, Phytochemistry, 8, 2919 (1992). [5] Rathod NR, Raghuveer I, Chitme HR, Chandra R, Indian J Pharm Sci.,

71(6), 615 (2009). [6] Lodhi G, Singh HK, Pant KK, Hussain Z, Acta Pharma., 59(1), 89 (2009). [7] Chitme HR, Chandra M, Kaushik S, J Pharma Pharma Sci., 7(1), 70 (2004). [8] Chitme HR, Chandra M, Kaushik S, Phytother Res., 19, 454 (2005). [9] Adak M, Gupta JK, Nepal Med Coll J., 3, 156 (2006). [10] Deshmukh PT, Frenandes J, Atul A, Toppo E, J Ethnopharmacol, 125(1),

178 (2009). [11] Kokate CK, Practical pharmacognosy, 3rd ed, pp., 107-109 (1994). [12] Swaston-Flatt SK, Day C, Bailey CJ, Flatt PR., 33, 462 (1990). [13] Klimes II, I, Sebokova E, Gasperikova D, Mitkova A, Kuklova S, Bohov P.,

Endocr Regul, 32(3), 115 (1998). [14] I. Addae-Menzah and R. W. Munenger, Fitoterapia, 60, 359 (1989) [15] Roy S, Sehgal R, Padhy BM, Kumar VL, J Ethnoharmacol, 102(3), 470

(2005). [16] T. Trovato, R.I. Forestie, L. lauk, R. Barbera, M.T. Monforter and E.M.

Galati, Plant Med.,Phytother., 26, 300 (1993). [17] Venkatesh S, Reddy GD, ReddyYS, Satayavathy D, Reddy BM, Fitoterapia,

75, 364 (2004).

Treatment Blood glucose (g/L) at time ( min) after load 0 5 10 20 30 40 50 60

Control(0.25g/kg glucose)

0.99±0.08 1.26±0.08 1.47±0.11 1.59±0.1

4 1.71±0.09 1.69±0.11 1.46±0.08 1.33±0.07

C. gigantea(0.7 g/kg) 0.98±0.09 1.15±0.1 1.35±0.06 1.48±0.1 1.51±0.08* 1.31±0.1+ 1.25±0.11

+ 1.16±0.13++

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[18] Tuomilehto J, Lindstrom J, Eriksson JG, Valle TT, Hamalainen H,

llanne-Parikka P, Keinanen- Kiukaanniemi S, Laakso M, Louheranta A, Rastas M., Salminen V, Uusitupa M, N Engl J Med., 344, 1343 (2001).

[19] Tuzun S, Girgin FK, Sozmen EY, Exp Toxicol Pathol., 51, 431 (1999).

[20] Deb L, Durra A, Int J Green Pharm., 1, 7-28 (2006). [21] Lu YX, Zhang Q, Li J, Sun YX, Wang LY, Cheng WM, Am J

Chin Med., 38, 713 (2010). [22] Yamamoto H, Uchigata Y, Okamoto H, Nature, 294, 284 (1981).

AUTHORS First Author – Deepak Teotia , M.Pharm, School of Pharmacy, VIshveshwariya Institute of Medical Science, Greater Noida, Gautam Budha Nagar, Pin- 203207, India,

Second Author – S. P. Chakrabarti, PhD M.Pharm, IMT Pharmacy College, Puri, New Nabakalabara Road, Gopalpur, Puri, , Pin- 752001, (Orisaa), India. Third Author – S. S. Ajay, PhD M.Pharm, School of Pharmacy, VIshveshwariya Institute of Medical Science, Greater Noida, Gautam Budha Nagar, Pin- 203207, India, Correspondence Author – Deepak Teotia, School of Pharmacy, VIshveshwariya Institute of Medical Science, Greater Noida, Gautam Budha Nagar, Pin- 203207, (U.P.), India, Tel - +91 9990049211,9410491620 Email: kute_deeps@yahoo.com

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Research Article

Evaluation of Calotropis Gigantea (l.) and S. Indicus on Glycemia and Lipidemia in Sterptozotocin induced Diabetic Rats

Deepak Teotia1*, S. P. Chakrabarti2, S.S. Ajay3

1*Vishveshwaraya School of Pharmacy, Greater Noida, Gautam Budha Nagar, UP, India. 2IMT Pharmacy College Puri, New Nabakalabara Road, Gopalpur, Puri, Orisaa, India.

3Vishveshwaraya School of Pharmacy, Greater Noida, Gautam Budha Nagar, UP, India. *Corresponding author’s E-mail: kute_deeps@yahoo.co.in

Accepted on: 15-04-2013; Finalized on: 30-06-2013.

ABSTRACT In Ayurvedic system of medicine in India, not only extracts of one plant or the other but also a combination of plant extracts are used for the treatment of diabetes mellitus. The present paper reports the combined effect of Calotropis gigantea and S. indicus known to be useful for the treatment of diabetes in Ayurveda on the fasting blood sugar, glucose tolerance and lipid profile of Streptozotocin (STZ) induced albino rats. 300mg of water extract of the mixture of dried powdered leaves of Calotropis gigantea leaves and S.

indicus in equal proportions was given once daily for 8 weeks. After 8 weeks of treatment of Streptozotocin (STZ) diabetic rats, the fasting blood sugar came down to almost normal value and improvement in glucose tolerance and serum lipid profile were also observed. Keywords: Calotropis gigantea leaves, S. Indicus, Streptozotocin (STZ), type 2 diabetes.

INTRODUCTION Diabetes, a metabolic disorder involving high blood sugar levels due to the non functioning of a key hormone called insulin, has been on the risk across the world. Diabetes mellitus(DM) is a metabolic disorder characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both.1 The risk of diabetic complications, particularly cardiovascular diseases (CVD) peripheral vascular disease (PVD).2 Complications such as coronary artery disease (CAD), stroke, neuropathy, renal failure, retinopathy amputations, and blindness etc are known to be associated with DM.3 Insulin and various types of hypoglycemic agents such as biguanides and sulfonylureas including some of the recently developed ones are available for the treatment of diabetes. But none are ideal in treatment due to the toxic side effect and sometimes diminution in response after prolonged use.3-4 The disadvantages of the presently available drugs are that they have to be given throughout the life and produce side effect.4 A variety of plant preparations have been mentioned in Ayurveda and other indigenous systems of medicine, which are claimed to be useful in the treatment of diabetes mellitus.5 World Health Organization (WHO) has suggested the evaluation of the potential of plants as effective therapeutic agents, especially in areas where we lack safe modern drugs.6 In the ongoing search for more effective and safer drugs attention is being paid to new and safe drugs.6-8 There are many studies on Calotropis

gigantea which show that it exhibits anti-ulcer, anti-syphilis and anti-diabetic activities. Some of the plants useful for the treatment of diabetes mellitus including those from which some active constituents were isolated have been recently revived by Shukala et. Al.9 Reported that Calotropis gigantea

possesses not only anti-hyperglycemic effect but also hypolipidemic effect.10-11 S. indicus is also known to be a drug useful for the treatment of diabetes mellitus.12 In this paper the combined effect of water extract of a mixture Calotropis gigantea leaves and S. indicus in streptozotocin (STZ) induced diabetic in rats is reported. MATERIALS AND METHODS Calotropis gigantea was collected from the forest area of Ghaziabad, U.P., India and S. Indicus was obtained from the market of Ghaziabad, U.P and both plants were identified from the School of Pharmacy, Vishveshwariya Institute of Medical Science, Greater Noida, Gautam Budha, Nagar, U.P., India. They were assigned voucher specimen Ref. VIMS/CONSULT/2009/02/10 and Ref. VIMS/CONSULT/2009/02/11. The leaves of Calotropis

gigantea leaves and S. indicus were air dried and powdered in a grinder and mixed in equal proportions. Preparation of water extract 300 gm of powdered mixture of the two plant parts was extracted overnight with 360 ml of water with magnetic stirring in cold room (4oC). The water extract was separated and the residue was re-extracted with water. The combined water extract was concentrated in lyophilizer. Animals Wistar albino rats were obtained from R.V. Northland Institute, Greater Noida, Gautam Budha, Nagar, U.P., India, clearance is taken from animal ethics committee (IEC). Adult rats of either sex weighing between 150-200 g were selected for the study. The animals were acclimatized to laboratory conditions and divided into various groups. Animals were housed and kept on the light and dark cycle throughout.

International Journal of Pharmaceutical Sciences Review and

Research 164

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Int. J. Pharm. Sci. Rev. Res., 21(1), Jul – Aug 2013; n° 28, 164-168 ISSN 0976 – 044X Induction of diabetes and associated neuropathy Healthy adult albino wistar rats of both sex weighing between 150-200 gm were obtained from R.V. Northland Institute, Greater Noida, Gautam Budha, Nagar, U.P., India, and used in this study. The animals were fed on a pellet diet (Hindustan Lever, India) and water provided ad libitum. Diabetes was experimentally induced to produce diabetic neuropathy.13 Sorbitol induced disfunction of inositol / metabolites leading to neuron-infraction by causing microangiopathy of vasa nervosum it deceases blood flow to nerves. Overnight fasting animals were injected with streptozotocin (STZ) 60 mg / kg dissolved in 3 mM citrate buffer (pH 4.5) intraperitoneally (i. p.). After 10 days only those rats which showed plasma glucose level > 300 mg / dl were classified as diabetic and were included in study as described earlier.14 Animals were divided into three groups of five each. Group1 animals served as healthy controls, while those of the group 2 were untreated diabetic rats. Rats of group 3 were diabetic and treated for 8 weeks with 300 mg of water extract of Calotropis gigantea leaves plus S. indicus.

13-14 Blood samples were collected from overnight fasted rats at 0 and 8 weeks. Blood glucose serum total cholesterol, HDL and LDL cholesterol, triglyceride, and Glycosylated haemoglobin were determined using kits from Randox Mumbai. Total proteins albumin and creatinine in serum were determined by the method of Reinhold. Assay of plasma glucose and albumin and creatinine and total cholesterol, LDL – VLDL and HDL cholesterol and triglycerides were estimated as described earlier. Lipid peroxidation products were estimated as thiobarbituric acid reactive substance (TBARS) in plasma and tissues.15-16 Statistical analysis All the data were statistically evaluated and the significance calculated using student’s test. All the results were expressed as mean ± SD. RESULTS AND DISCUSSION The result obtained with untreated diabetic rats and diabetic rats treated with Calotropis gigantea plus S. indicus on fasting blood glucose and GTT are compared with normal healthy controls and shown in Table 1 and Table 1A. It is seen that treatment with water extract of Calotropis gigantea plus S. Indicus at a dose of 300 mg / kg body weight brought down fasting blood glucose (Table 1), from a higher value of 166.9 ± 25.4 mg / dl to a normal value of 85.4 ± 2.3 mg / dl while in the untreated group the FBG increased from the initial value of 172.2 ± 5.4 to 285.6 ± 42.6 mg / dl. There was considerable fall in FBG in diabetic rats treated with Caloptropis gigantea leaves alone or S. indicus alone. The effect was more with S. indicus. But the effect of the two plants in combination was more than that with either of the plants alone. Similar improvement to normal glucose tolerance was seen (Table 1A). In the diabetic untreated rats the blood sugar was 269.0 ± 92.2 mg / dl even after 2 hrs of glucose load in GTT. But in the Calotropis gigantea plus S. indicus treated rats the 2 hrs blood glucose value was in the

normal range of 75.2 ± 1.0 mg /dl. The fasting (0hr) blood glucose values which were higher in the diabetic animals (160.5 ± 32.1) were brought down to 81.0 ± 3.5 mg / dl, when 300 mg of the extract of the mixture of the two plants was administered for 8 weeks. There was improvement in glucose tolerance in diabetic rats treated alone with either of the plants. The exact mechanism of action of the plant extracts either alone or in combination cannot be stated. However it is possible that these extracts increase blood insulin levels and also stimulate utilization of glucose by liver and extrahepatic tissues. The changes in the lipid profile have also been studied (Table 2). Before treatment the total cholesterol (TC), LDL cholesterol (LDL-C) and TG were higher than in normal animals. After 8 weeks, the TC, LDL-C, LDL-C/HDL-C and TG values were still higher in diabetic untreated animals then in control animals. There was no change in HDL-C in untreated diabetic animals. However in the case of diabetic treated animals, with Calotropis gigantea alone, total cholesterol, LDL cholesterol and LDL-C/HDL-C values returned close to normal values. There was very slight fall in TG values probably because these plants could not show much effect on triglycerols. But further improvement in increase in HDL-C value was seen. With Calotropis

gigantea and S. indicus plus Calotropis gigantea

treatments similar values were obtained. Further there was reduction in TG values also. This shows again that the water extract of the mixture of two plants, which contains less than 300 mg of each of the two plants is as effective as 300 mg of water extract of each of the two plants. Improvement in lipid profile is suggestive of the action of the two plants on enzymes and lipid metabolism. The total proteins, albumin and creatinine in serum, glycosylated hemoglobin in blood and total body weight and weight of kidneys (in the animals killed after the experiment) were also analyzed after 8 weeks of treatment and the values are shown in Table 3 and Table 4. The untreated diabetic animals showed signs of neuropathy e.g. tropic ulcer on tail, slight edema in the paws of the legs. The animals looked lethargic and sickly. All these symptoms disappeared after treatment for 8 weeks with water extract of the two plants Calotropis gigantea and S. indicus. The treatment with water extract was so effective that the above mentioned complications seen in untreated diabetic rats disappeared. The treated diabetic animals showed appearance almost like normal rats. The overall behavior of the rats was normal. In conclusion it can be stated that the two plants Calotropis gigantea and S. indicus, have synergistic effect when given together. They have a strong anti-hyperglycemic and anti-hyperlipidemic effect. In table, only values of with water extract of combination of the two plants are shown. An interesting observation is that glycosylated hemoglobin (HbA1c) decreased to 4.8 ± 0.1 and returned to normal values after 8 weeks of treatment. Glycosylated hemoglobin content rather than FBG is considered as a more reliable index of glycemic control in the management of diabetes mellitus. Return to normal of HbA1c (Table 3) after treatment is a clear indication that

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Int. J. Pharm. Sci. Rev. Res., 21(1), Jul – Aug 2013; n° 28, 164-168 ISSN 0976 – 044X the diabetic state was well regulated after the treatment of diabetic animals. Serum albumin, total body weight and kidney weight increases in diabetic untreated animals. There was increase in total proteins, serum creatinine and serum acid in diabetic animals. After treatment there was decrease except in total protein. But after treatment the values were closer to the normal value. There was an improvement in the hemoglobin content of the blood also (Table 3). Herbal treatment improves the weight of liver and kidney in diabetic rats fed high fat diet, although it was not completely reversible in liver (Tables 4 and Table 5). Recovery from this type of neuropathy is eluting, although months and

even years may elapse before it happens. This form of neuropathy is often referred to as diabetic myotrophy. When the clinical picture is dominated by deep sensory loss, ataxia and stone of the bladder, with only slight weakness of limbs, the similarity can be heightened by the presence of lancinating pains in the legs, unreactive pupils and neuropathic arthropathy. Loss of nerve fibers is a prominent pathologic finding in the distal symmetric form of neuropathy. Since myelin is formed from the cell membranes of Schwan cells, one may infer that the Schwan cell is a primary target of the pathologic process in this type of diabetic neuropathy.

Table 1: Effect of treatment for 8 weeks with water extract of (300 mg/kg BW) Calotropis gigantea and S. indicus and mixture of the two plants on fasting plasma glucose level in streptozotocin (STZ) diabetic rats.

Plasma glucose ( mg/dl) mean ± S.D. Group 0 Weeks 8 Weeks

Control 99.4 ± 19.5 89.8 ± 5.21

Diabetic untreated 172.2 ± 5.4 285.6 ± 42.6

Diabetic + Calotropis gigantea 164.6 ± 25.0 105.4 ± 26.6

Diabetic + S. indicus 158.6 ± 10.0a 98.2 ± 25.2a

Diabetic + Calotropis gigantea + S. Indicus 166.9 ± 25.4 85.4 ± 2.3

a = p < 0.001; Group of 5 animals were used for each set of experiments; All the data were statistically evaluated and the significance was calculated using students’’- test. All the results were expressed as mean ± S.D. Table 1A: Effect of water extract (300 mg/kg BW.) of Calotropis gigantea and S. indicus and mixture of the two plants on plasma glucose tolerance in diabetic rats after 8 weeks

Blood glucose (mg/dl) mean ± S.D. Group 0 hr 0.5 hr 1 hr 1.5 hr 2 hr

Control 94.3 ± 21.0 140.2 ±11.2 132.6 ±27.3 116.2 ±10.0 102.0 ±12.0

Diabetic untreated 160.5 ±32.1 245.0 ±68.6 273.4 ±89.3 290.6 ±82.6 269.0 ±92.2

Diabetic + Calotropis gigantea 87.9 ±25.6 112.2 ±20.6 114.3 ±15.0 102.0 ±10.3 95.6 ±24.0

Diabetic + S. indicus 82.0 ± 4.2 86.0 ± 4.3 81.0 ± 3.9 80.0 ± 3.2 84.0 ± 3.2

Diabetic + C. gigantea + S. indicus 81.0 ± 3.5 84.0 ± 4.2 86.0 ±3.3 81.0 ±3.6 75.2 ± 1.0

Table 2: Effect of treatment for 8 weeks with water extract of (300 mg/kg BW) Calotropis gigantea and S. indicus and both Calotropis gigantea + S indicus on plasma lipid in diabetic rats

Group

Control Diabetic untreated Diabetic + Calotropis

gigantea Diabetic + S. indicus Diabetic + C. gigantea

+ S. indius

TC ( mg/dl) LDLC ( mg/dl) HDLC LDLC/HDLC (mg/dl) TG mean ± S.D. mean ± S.D. (mg/dl) mean ± S.D. mean ± S.D. (mg/dl) mean ± S.D. 170.3 ± 10.3 78.0 ± 12.2 44.9 ±13.3 1.7 ±0.3 115.6 ± 42.6

250.0 ± 14.9 152.2± 12.6 45.0 ±12.0 3.3 ±0.4 181.8 ±18.8

175.0 ± 12.3 99.7 ±16.8 51.3 ±8.4 1.9 ±0.4 174.0 ±15.8

187.0 ± 14.9 95.6 ±15.7 50.2 ±8.5 1.6 ±0.3 135.0 ±13.2

174.0 ± 10.3 51.3 ±6.3 48.3 ±8.2 1.8 ± 0.5 131.0 ±11.2

TC, LDL-C, HDL-C, TG = Total Cholesterol, Low Density and High Density Lipoprotein Cholesterol respectively and Triglyceride. Number of animals is 5 in each group.

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Int. J. Pharm. Sci. Rev. Res., 21(1), Jul – Aug 2013; n° 28, 164-168 ISSN 0976 – 044X Table 3: Effect of water extract of Calotropis gigantea plus S. indicus on glycosylated haemoglobin and body weight, values shown are those at the end of 8 weeks treatment, haemoglobin in blood and serum albumin total proteins and creatinine

Parameter Normal mean ±

S.D. Diabetic mean ± S.D. Diabetic treated mean ± S.D.

Glycosylated haemoglobin 3.04 ± 0.02

9.4 ± 2.4 4.8 ± 0.1

(HbA1c) %

Albumin (g/l) 55.0 ± 1.4 45.0 ± 3.1 52.9 ± 3.0a

Total protein (g/l) 69.0 ± 3.0 73.2 ± 2.9 78.2 ± 3.8a

Creatinine ml/min 0.75 ± 0.04 1.30 ± 0.15 0.91 ± 0.05a

Body weight (g) 283.6 ± 25.8 225.5 ± 45.0 280.4 ± 9.0

Kidney weight (g) 1.40 ± 0.05 2.92 ± 0.08 1.06 ± 0.10

Haemoglobin 16.0 ± 4.2 ____ ND

Urine sugar 10.5 ± 1.2 +++ ++

Urine albumin ND ++ ND

The values are mean ± S.D; *a p < 0.05 when compared with diabetic untreated group.

Table 4: Effect of water extract of Calotropis gigantea plus S. indicus on the serum lipid profile in rats fed on high fat diet

TC mg/dl TG mg/dl LDL HDL-C Normal 198.7 ± 44.9 94.9 ± 7.1 83.8 ± 16.2 42.3 ± 19.2

High-fat rats 622.3 ± 1.4 34.7 ± 0.9 3.58 ± 0.11 31.0 ± 0.10

High-fat diet plus extracts 182.5 ± 4.2 171.8 ± 0 148.8 ± 8.0 173.1 ± 23.4

Table 5: Effect of water extract on liver and kidney weight in rats fed on high fat diet

Tissues Normal rats High-fat diet untreated High-fat diet plus extracts of two plants Liver (gm) 7.82 ± 0.33 10.4 ± 0.41 9.8 ± 0.18b Kidney (gm) 0.99 ± 0.05 1.08 ± 0.08 0.99 ± 0.06 b

*b p<0.0005 *c p<0.01 CONCLUSION In conclusion it appears that the water extract of combination of Calotropis gigantea plus S. indicus has got good hypoglycemic and hypolipidemic effect and also corrects complications associated with diabetes such as, retinopathy, nephropathy, neuropathy and musculopathy. Further research is needed to corroborate the same beneficial effects and to discuss the underlying mechanism involved in the above findings. REFERENCES * Amos AF, McCarty D.J, Zimmet P, The rising global burden

of diabetes and its complications: Estimates and projections to the year 2010. Diabetic Medicine, 14, 1997, 51-85.

* Bajaj J.S, Madan R, Diabetes in tropics and developing

countries. IDF bull, 2, 1993, 5-6. * David M.N, The pathophysiology of diabetes complications:

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World Journal of Pharmaceutical research

Volume 2, Issue 5, 1826-1839. Research Article ISSN 2277 –

7105

ETHNOPHARMACOLOGICAL EVALUATION OF CALOTROPIS

GIGANTEA

Vaibhav Prakash Srivastava*1, Shaundarya Kumar2, Usha Rai 3, Sonika 4, Deepak Teotia5, Dr. Rakesh Singh6

1* ,4,5,Vishveshwarya Institute of Medical Science, G.B.Nagar. 2,3,6, Kamala Nehru Institute of Technology & Management, Faridipur, Sultanpur U.P. India. ABSTRACT Calotropis gigantea, commonly known as milkweed or swallow-

wort, is a common wasteland weed (Singh et al. 1996).

Calotropis belongs to Asclepiadaceae or Milkweed or Ak family

which includes 280 genera and 2,000 species of world-wide

distribution but most abundant in the sub-tropics and tropics,

and rare in cold countries. Calotropis gigantea Linn. (Asclepiadaceae) a widely growing plant has been

reported to possess number of medicinal properties. The aim of

the present study was to screen leaves of Calotropis gigantea to

study & compare

Antioxidant, Antibacterial, Anti-inflammatory & wound healing

Activity of Calotropis gigantea Linn. Calotropis gigantea, with

ethanolic extract. It has been reported as a traditional folk

medicine for

a variety of aliments. The stages of wound healing are inflammatory phase,

proliferation phase, fibroblastic phase and maturation phase. Extract treated animals

exhibit 83.42 % shows the Anti-inflammatory area when compared to controls which

was 76.22 %. The extract treated wounds are found to epithelize faster as compared

to controls. The plant C. gigantea is also used in some parts of India for wound

healing in combination with other plants. However there are no scientific reports on

India.

prakashsrivastava.vaibhav@gm

Medical Science, G.B. Nagar,

Vishveshwarya Institute of

Vaibhav Prakash Srivastava,

*Correspondence for

Revised on 24 July 2013, Accepted on 30 August 2013

Article Received on 16 June 2013,

ail.com,

Author:

181

wound healing activity of the plant C.gigantea. The oral administration of 400mg/kg of C.gigantea and 300 mg/kg of

C.gigantea were showed significant anti-inflammatory activity more than that of

100mg/kg of Ibuprofen. This study also proved the greater anti-inflammatory action

due to the effect of C. gigantea with Acetyl Choline than Acetyl Choline alone.

KEY WORDS: Antioxidant, Antibacterial, Muscle Relaxant Activities, Calotropis

gigantea, Ethanolic extract, Acetylcholine.

182

INTRODUCTION

Medicinal plants are usually of medicine for the treatment of leprosy, ulcers, tumors,

used for Ayuerbedic, Unani and other rural areas. Recent discovery shows that these

plants have fewer side effects than the Allopathic medicine. So, herbal medicine

becoming popular for medication among whole over the world. The number of plants

with medicinal properties included in the Materia Medica of traditional medicine in

this subcontinentat present stands at about 2000 [1]. More than 500 of such medicinal

plants have so far been enlisted as growing in Bangladesh [2]. Thus the human race

started using plants as a means of treatment of diseases and injuries from the early

days of civilization on earth and its long journey from ancient time to modern age the

human race has successfully used plants products as effective therapeutic tools for

fitting against diseases and various other health hazards [3]. C. gigantea is a common

wasteland weed found abundant throughout India right from Himalayas to southern

India. C. gigantea was regarded as a useful medicinal plant and used in folk

medicine1, 2. Traditionally it is used for the treatment of different aliments in

ayurvedic and unani systems of medicines. The plant has been known as “Vegetable

Mercury” since it is used as a remedy for syphilitic affections, also advocated for a

variety of diseased conditions including leprosy, ulcers, tumours and piles. The plant

is reported to have diverse pharmacological actions like antifertility, cardiotonic,

antimicrobial activities4. The Ethanolic extract of the root has been shown to exhibit

protective activity against carbon tetrachloride induced liver damage [10]. Methanol

extract possess antioxidant activity in Trema orientalis [11] and Senna tora12. So, the

present work was designated to investigate the antioxidant and antibacterial activities

of of Calotropis gigantea Linn to know the scientific basis of ascorbic acid were

weighed three times and dissolved in ethanol to make the required concentration by

dilution technique. Here ascorbic acid was taken as standard. DPPH was weighed

and dissolved in ethanol to make 0.005% (w/v) solution. To dissolve homogeneously

magnetic stirrer was used. After making the desired concentration 4 ml of 0.004

DPPH solution was applied on each test tube by pipette. The room temperature was

recorded and kept the test tubes for 30 mins in light to complete the reactions. DPPH

was also applied on the blank test tube at the same time where only ethanol taken as

blank. 13

183

MATERIALS AND METHODS PLANT MATERIALS Fresh leaves of Calotropis gigantea were collected from Lucknow local area of 2011. The plant was identified by the expert of National Botanical Research Institute (NBRI), Lucknow and a voucher specimen was kept for future reference. The dried leaves of Calotropis gigantea were ground into a fine powder with the help of suitable grinder. About 400 g of powder grinded material was extracted by soxhlet apparatus with 90% methanol at 55°C temperature. PROCESSING OF THE PLANT Plant leaves were collected and washed properly with distilled water. The leaves

were shade dried at room temperature. Dried leaves were uniformly grinded using

mechanical grinder. The leaves powder was extracted in distilled water.

Fig 1. Crude Leaves and Dried Powder of Calotropis gigantea Ten gram of plant powder was soaked in 100 ml of distilled water in a conical flask

and loaded on an orbit shaker at a speed of 120 rpm for 24 hours. The mixture was

filtered using Whatman filter paper number 1. The filtrate was concentrated using

rotary evaporator and dried using lyophilizer. Dried extract was collected in an air

tight container and stored at 4°C. The extracted powder was dissolved in sterilized

distilled water to make 1000 µg/ml solution. This mixture was used to perform

antibacterial assay.

TEST MICROORGANISM The following six clinical isolates of bacteria were used for the study: S. aureus, K.

pneumoniae, B. cereus, P. aeruginosa, M. luteus and E. coli. All these cultures were

maintained on nutrient agar plates at 4°C. scavenging activity of the stable 1,1-

184

diphenyl-2-picryl hydrazyl free radical. DPPH method is most widely used and

easiest method to determine Antioxidant activity DPPH is a stable free radical

containing an odd electron in its structure and usually utilized for detection of the

radical scavenging activity in chemical analysis. 13 The aliquots of the different

concentrations (1-500 µg/ml) of the extract was added to 3 ml of a 0.004 %w/v

solution of DPPH. Absorbance at 517 nm was determined after 30 min, and IC50 (Inhibitory

concentration 50%) was determined. IC50 value denotes the concentration of sample

required to scavenge 50% of the DPPH free radicals. At first 6 test tubes were taken to

make aliquots of 6 conc.(1,5,10,50,100,500 µg/ml).Plant extract and ascorbic acid were

weighed 3 times and dissolved in ethanol to make the required concentration by dilution

technique. Here ascorbic acid was taken as standard. DPPH was weighed and dissolved

in ethanol to make 0.004% (w/v) solution. To dissolve homogeneously magnetic stirrer

was used. After making the desired concentration 4 ml of 0.004 DPPH solution was

applied on each test tube by pipette. The room temperature was recorded and kept the

test tubes for 30 mins in light to complete the reactions. DPPH was also applied on the

blank test tube at the same time where only ethanol taken as blank. After 30 mins,

absorbance of each test tubes were determined by UV spectrophotometer. IC50 was

determined from % inhibition vs. concentration graph. 14,15

ANTIMICROBIAL ACTIVITY DISC DIFFUSION METHOD The extracts of C.gigantea leaf obtained by maceration process by using water as a

solvent. Extracts were screened for antimicrobial activity using by disc diffusion

method. A suspension of organism was added to sufficient quantity of nutrient agar

at 450C. The mixture was aseptically transferred to sterile petri dish and allowed to

solidify. The overnight culture grown in broth was used for inoculation. The plant

extracts to be tested were prepared in various concentrations i.e. 25%, 50%, 75%

and 100%. The sterile impregnated discs with plant extracts were placed on the agar

surface with framed forceps and gently pressed down to ensure complete contact of

the disc with agar and dextrose surfaces. Positive control discs were also prepared

in the same manner using Ampicillin, a bactericide. But it was not used for fungi. The

prepared control discs were placed using respective solvents. 16,17

185

All the plates including control plates were incubated at 37°C for 24 hours. After

incubation, the size (diameter) of the inhibition zones was measured. Triplicates

were maintained for each sample of the extract respectively. The results were

expressed in terms of the diameter of the inhibition zone: <9 mm - inactive; 9-12mm -

partially active; 13-18mm - active; >18mm - very active. After the confirmation of

antibacterial activity with 100mg/kg dose the experiment was carried out in triplicate

and average values were taken into consideration. Similar procedure was carried out

with standard drug Ampicillin 100mg/ml. And the zone of inhibition was compared

with test sample and control and the percentage of inhibition was calculated which

are given below in table. 16,18

PHARMACOKINETIC PARAMETER OF ANTIMICROBIAL ACTIVITY MINIMUM BACTERICIDAL CONCENTRATION The Minimum Bactericidal Concentration (MBC) is the lowest concentration of

antibiotic required to kill the germ not as commonly seen as the Minimum inhibitory

concentration (MIC). It can be determined from broth dilution MIC tests by sub

culturing to agar media without antibiotics. The minimum bactericidal concentration

(MBC) is the lowest dilution where the culture has been completely sterilized. It is not

routinely determined. Treatment decisions are made related to MICs, and more

specifically, the breakpoint MICs.19

MINIMUM INHIBITORY CONCENTRATION The minimum inhibitory concentration (MIC) is the concentration required to inhibit

growth of a specific isolate in vitro under standardized conditions. It is determined by

finding the lowest dilution without visible growth during serial dilution testing. In

microbiology, is the lowest concentration of an antimicrobial that will inhibit the visible

growth of a microorganism after overnight incubation. Minimum inhibitory

concentrations are important in diagnostic laboratories to confirm resistance of

microorganisms to an antimicrobial agent and also to monitor the activity of new

antimicrobial agents. An MIC is generally regarded as the most basic laboratory

measurement of the activity of an antimicrobial agent against an organism. 19.20

DETERMINATION OF RELATIVE PERCENTAGE INHIBITION The relative percentage inhibition of the test extract with respect to positive control

was calculated by using the following formula 6, 11.

186

Relative percentage inhibition of the test extract =

Where, * total area of inhibition of the test extract * total area of inhibition of the solvent * total area of inhibition of the standard drug The total area of the inhibition was calculated by using

area = πr2; where, r = radius of zone of inhibition. 16,17

EFFECT OF EXTRACT FROM CALOTROPIS GIGANTEA ON THE SKELETAL MUSCLE OF THE RAT Since the antimigrain drugs were reported to have muscle relaxant activity, so this

experiment was attempted to assess the effect of extract from C. gigantea on the rat

rectus abdominis muscle preparation. The experiment was carried as per the method

described by Kulkarni 5. Rats weighing 20‐25 g were used in this study. The rat was

stunned and decapitated and the spinal cord was destroyed. A rat was pithed and the skin of the anterior and abdominal wall was cut by a midline

incision and then it was cut laterally to expose the anterior abdominal wall. 18,19 The two rectus were seen running from the base of sternum. The muscles were cut

across just above the sternum at its base and the pair of muscles attached to it were

dissected and transferred to a dish containing frog ringer solution at room

temperature. The muscles were then carefully cleaned and one of them was trimmed

to the desired size and mounted in an organ bath filled with ringer solution at room

temperature and aerated by stream of fine bubbles emerging near the bottom of the

bath. Isotonic contractions were recorded using gimbel lever with a sideways writing

point. The lever was balanced for a tension of approximately 2‐5 g. An extra load of

approximately 1g on the long arm was supplied because sometime the lever may not

return to the base line after washing. 18, 20

187

The drug period allowed for stabilization was 30 minutes during which the muscle

was subjected to 1g stretch. At 0 min ‐ the kymograph was started after raising the

extra load; in the 1st min‐ the drug was added and in the 2nd min‐ the kymograph

was stopped. The tissue was washed and allowed to relax by applying an extra load.

At the 5th min‐ the lever point was brought to the base line and the next cycle was

started. After recording the graded responses to different log dose of acetylcholine,

the test drug (Extract) was added and their effects upon acetylcholine induced

contractions as well as the effect of its own in the tissue was studied. 21 DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION (MIC) MICs can be determined by agar or broth dilution methods usually following the

guidelines of a reference body such as the CLSI, BSAC or EUCAST. There are

several commercial methods available, including the well established Etest strips and

the recently launched Oxoid MIC Evaluator method. The Etest system comprises a predefined and continuous concentration gradient of

different antimicrobial agents, which when applied to inoculated agar plates and

incubated, create ellipses of microbial inhibition. The MIC is determined where the

ellipse of inhibition intersects the strip, nand is easily read off the MIC reading scale

on the strip. 20.21

188

RESULTS AND DISCUSSION FOR ANTIOXIDANT ACTIVITIES TEST

In the present study, methanol extracts of the leaves of C.gigantea showed potential

free-radical scavenging activity but aqueous extract showed very little free-radical

scavenging activity.

Fig. 2 Comparison of Antioxidant activity of Various Extract FOR ANTIMICROBIAL ACTIVITIES TEST The bacterial suspensions were seeded on MHA plates using a sterilized cotton

swab. In each of these plates four wells were cut out using a standard cork borer (7

mm). Using a micropipette, 100 µl of each dilution was added in to wells. All the

plates were incubated at 37ºC for 24 hours. Antimicrobial activity of the leaf extract

was evaluated by measuring the zone of inhibition. Experiment was carried out in

triplicates for each test organism.

Figure 3: Antimicrobial activity of Calotropis gigantea

189

Table 1: Test organisms Relative percentage inhibition (%)

Test organisms Relative percentage inhibition (%)

Staphylococcus aureus 48.05

Klebsiella pneumoniae 75.64

Bacillus cereus 175.36

Pseudomonas aeruginosa 108.16

Micrococcus luteus 26.67

Escherichia coli 155.89

Figure 4: Relative percentage inhibition of Calotropis gigantea

190

Table 2: MIC values of methanol and aqueous extracts of Calotropis

gigantea on test organisms

MIC in mg/ml

S. aureus K. pneumonia B. cereus P. aeruginosa M. luteus E. coli

52 23 5.25 4.1 1.7 11.65

Table -3 Antimicrobial activity of Calotropis gigantea

Inhibition zone diameter (mm)

Test organisms

AE PC NC

Staphylococcus aureus 14.3±1.15 18.6±1.52 0

Klebsiella pneumoniae 13.6±1.52 13.3±0.57 0

Bacillus cereus 16.3±1.52 15.6±1.15 0

Pseudomonas 14.0±1.73

16.6±1.52

0

aeruginosa

Micrococcus luteus 16.6±1.52 33.3±1.52 0

Escherichia coli 18.6±1.15 16.6±2.08 0

AE: aqueous extract, PC: positive control, NC: negative control. Values are

expressed as mean ± standard deviation of the three replicates. Zone of inhibition

not include the diameter of the well. Results of MIC are reported in Table 2 . The crude extract showed 52, 23, 11.65,

5.25, 4.1 and 1.7 mg/ml MIC values for S. aureus, K. pneumoniae, E. coli, B. subtilis,

P. aeruginosa and M. luteus respectively. Aqueous extract of Calotropis gigantea showed high inhibitory activity followed by

methanol extract, where as ethanol and petroleum ether extracts showed low activity

6. Previous studies report the presence of phytochemicals like cardenolides,

flavonoids, terpenes, pregnanes, nonprotein amino acid and cardiac glycoside as

major constituents in C. gigantean may acknowledge the medicinal property of this

plant.

191

FOR MUSCLE RELAXANT ACTIVITIES TEST The Aqueous and Ethanolic Extract of C. gigantea was found to have skeletal

muscle relaxant property at T1 (1:100), T2 (1:500) and T3 (1:1000), when tested

along with acetylcholine. When the relaxant property was compared with the

standard drug acetylcholine, Ethanolic Extract tested along with the acetylcholine

produces more relaxant property than the standard drug acetylcholine (Table 1 &

Fig. 1‐2). Lesser the concentration of the test drug (Ethanolic Extract ) increases the

responses of the muscle relaxant property. Maximum relaxant effect in T1 and T3

was found i.e. 22mm and 23mm at the dose of 16µg and T2 was 13mm at 2µg.

Earlier studies have proved that chloroform extract of Ervatamia crispa revealed

skeletal muscle relaxant effect on an isolated rat rectus abdominis muscle preparation16.

Fig-5: Muscle Relaxant Activity of Calotropis gigantea

192

Fig-6 Fig. 5-6.Comparison of Muscle Relaxant activity of acetylcholine and test drug at different concentration. Table 4: Muscle Relaxant activity extract from Calotropis gigantea

Drug Volume (ml) Dose (µg) Height(mm) Responses

Acetylcholine 0.1 1 3 Increased Acetylcholine 0.4 4 5 Increased

Acetylcholine 0.8 8 9 Increased

Acetylcholine 1.2 12 11 Increased

T1 (1:100) 0.4 4 - -

T1 + ach 0.1 1 6 Increased

T1 + ach 0.4 4 9 Increased

T1 + ach 0.8 8 12 Increased

T2 (1:500) 0.4 4 - Increased

T2 + ach 0.1 1 24 -

T2 + ach 0.2 2 13 Increased

T3(1:1000) + ach 0.3 3 15 Increased

T3 + ach 0.4 4 16 Increased

T3 + ach 0.8 8 18 Increased

T3 + ach 1.6 16 24 Increased

Increased

193

CONCLUSION The results obtained in the MES test in rats that, the standard drug as well as the different extracts of stem barks of Calotropis gigantea protected against MES induced seizures. Extract of Calotropis gigantea had a slower onset of action and lesser degree of Muscle relaxant activity.The Antioxidant , Antibacterial, Muscle Relaxant Activities of Calotropis gigantea from Ethanolic, & Aqueous extract was studied and compare to each other. ACKNOWLEDGEMENT The authors wish to thank the Management and Staff of RITM, Lucknow And Vishveshwarya Institute of Medical Science, G.B. Nagar , providing necessary facilities to carry out the research work successfully heartfelt thanks to the Professor and head Deepak Teotia and my Dearest friend Mr. Shaundarya Kumar for their encouragement and Faithful support. REFERENCES

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