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SYNTHESIS AND STUDIES OF MODIFIED STEROIDS ON CENTRAL NERVOUS SYSTEM

DISSERTATION SUBMITTED FOR THE AWARD OF THE DEGREE OF

ittafiiter of $|iilodoplip IN

CHEMISTRY

•V

ISHRAT JAMIL

BEPARTMEiT OF CHEMISTRY AND

i i iTfRDise innuRY B R A I N RESEARCH CENTRE

JAWAHARUU. NEHRU MEDICAL C0I1E6E ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

• 1997

DS3070

N^V., i^'^J fe^

/ ^

DEDICA TED TO MY

FA THER LATE MR. MD. YAR KHAN

MY SCHOOL TEACHER

MR. MD. SHAFIQUE &

MY INSPIRATION

MR. PANCKAJ GUPTA i

C E R T I F I C A T E

The dissertation entitled "SYNTHESIS AND STUDIES OF

MODIFIED STEROIDS ON CENTRAL NERVOUS SYSTEM" by Mr. ISHRAT

JAMIL is suitable for submission for the degree of Master of

Philosophy in Chemistry.

PROF^/DR. MAHDI T3ASAN M.B.B.S., M.S.(Hons.), Ph.D. D.Sc FRMS, FIGS, FAMS, FANSc, FNA, Interdisciplinary Brain Research Centre, J.N. Medical College, A.M.U. Aligarh.

PROF. SHAFIULLAH Steroidal Research Laboratory, Department of Chemistry A.M.U. Aligarh.

SUPERVISOR

CO-SUPERVISOR

(F CONTENTS

%

List of Figures i - i i List of Tables i i i Abbreviations i v Acknowledgements v - v i i

1.0 INTRODUCTION

1.1 Aims and objectives of the present study 01-03

2.0 REVIEW OF LITERATURE

2.1 Steroids 04-16

2.2 Chemistry and general structure of 16-17

Cholesterol

2.3 Metabolism of steroidal harmones 17-21

in brain tissue

2.4 Physiological function and pharmacological 21-26

effects of steroids

2.4.1 Mechanism of action of steroids 26

2.5 Structure-Activity relationship of steroids 27-32

2.6 Effects of adrenocortical steroids on CNS 32-35

3.0 MATERIALS AND METHODS

A) MATERIALS 3.01 Animals 36

3.02 Preparation of steroids for bio- 36

chemical studies

(^

3.02 1 3-B-acetoxy-cholest-5-enc 36 37

3.02.2 3-B-acetoxy-6-nitro-cholest 37

-5-ene

3.02.3 3li-acetoxy-5a-cholestan-6- 37-38

one

3.03 Structure of steroids to be used in this 38-39

study

3.04 Reagents 39-41

3.05 Instruments 41-42

3.06 Miscellaneous 42

B) METHODS

3.07 Procedure and maintenance of the 43-44

animals

3.08 Solution preparation and dose 44-46

administration

3.09 Dissection of different aprts of the 46

rat's brain

3.10 Procedure for killings the animals 46

3.10.1 Cervical dislocation 46-47

3.10 2 Exposure of the brain 47

and its removal

3.11 Extraction and purificaition of brain 48-51

lipids

3.12 Estimation of totall ipids 51

3.12 1 Principle 53

3.12.2 Reagents solution 53-54

3.12.3 Procedure 54-55

3.12.4 Calculation 55

^

3.13 Estimation of phospholipids 55

3.13.1 Principle 55-57

3.13.2 Reagents 57-58

3.13 3 Procedure 58-59

3 13.4 Calculation 59

3.14 Estimation of Cholesterol 59

3.14.1 Principle 59

3.14.2 Reagents 61



3.15 Estimation of Gangliosides 62

3.15.1 Principle 62

3.15.2 Reagents 64



3.16 Estimation of Esterfied Fatty 65

Acids

3.16.1 Reagents 67-68

3 16.2 Procedure 68

3.163 Calculation 69

3.17 Estimation of Rate of Lipid 69

Peroxidation

3.17.1 Principle 69

3.17.2 Reagents 70

3.17.3 Procedure 70


(c— — •• - • - - • • - - • ~ " • \

|4.0 RESULTS 72 i !

1 4.1 Changes in Gangliosides level 74

4.2 Changes in Total Lipids level 76

4.3 Changes in Esterified Fatty Acids 78

4.4 Changes in in Cholesterol level 80

4.5 Changes in Phosphate level 82

4.6 Changes in Rate of Lipid

Peroxidation

84

4.7 Changes in Cholesterol/

Phosphate ratio

86

5. DISCUSSION 87

5.1 Gangliosides 87-93

5.2 Total lipids 93-100

5.3 Esterified Fatty Acids 100-104

5.4 Cholesterol 104-110

5.5 Phospholipids 111-117

5.6 Rate of Lipid Peroxidation 117-124

5.7 Cholesterol/Phosphate ratio 125-127

6. BIBLIOGRAPHY 128-16C

L= J

(^

LIST OF FIGURES

FIG.

2.1 Structure of Cholesterol

2.2 Androgen Hornnones

I 5-a-dihydrotestosterone II 3a-5a-androstanediol

III 17B-estradiol

IV Andtostendione

V Estrone

VI Progesterone

VII 5a-dihydroprogesterone

15

18

18

18

18

18

18

18

18

2.3 Structure-stereochemistry and 29

nomenclature of adrenocosteroids

I Structure of adrenocosteroids 29

II Stereochemistry adrenocosteroids 29

3.01 Dissected brain of albino rat 45

3.02 Specially designed pipette 49

3.03 Sintered glass funnel 50

3.04 Standard graph of Total Lipids 52

3.05 Standard graph of Phospholipids 56

3.06 Standard graph of Cholesterol 60

3.07 Standard graph of Gangliosides 63

3.08 Standard graph of Esterified 66

Fatty Acids

%

' - - — ^ ----'^ - : : - - - - — ^ .

5 1 Bar Diagram of Gangliosides 88

change

5 2 Bar Diagram of Total Lipids change 94

5 3 Bar Diagram of Esterified Fatty 100

Acids change

5.4 Bar Diagram of Cholesterol change 105

5.5 Bar Diagram of Phospholipids change 112

5.6 Bar Diagram of Rate of lipid- 118

peroxidation's alteration

'- ^*'

1 1

LIST OF THE TABLES

Table No

2.1 Relative Potencies of Corticosteroids 24

2 2 Relative Potencies and Equivalent 28

Dose of Corticosteroids

4 (I) Effect of steroidal derivatives on 73

gangliosides level

4 (II) Effect of steroidal derivatives on 75

Total Lipids levels

4 (lll)Effect of steroidal derivatives on 77

Esterified Fatty Acids level

4 (l\')Effect of steroidal derivatives on 79

Cholesterol level

4 (\ ) Effect of steroidal derivatives on 81

phospholipids level

4 (\ l)Effect of steroidal derivatives on 83

Rate of Lipidperoxidation

4(\ ll)Effect of steroidal derivatives on 85

Cholesterl/Phosphote ratio

1 1 1

ABBREVIATIONS

acetoxy derivative

acetoxy-keto derivative

acetoxy-nitro derivative

CNS

ACTH

TOTP

DFP

RA

MS

CIA

EAE

DHT

NaCI

EFA

3-p-acetoxy-cholest-5-ene

3-3-acetoxy-6-nitro-cholest-5-ene

3a-acetoxy-5a-cholestan-6-one

Central Nervous System

Adreno Cortical Thyroid Hormone

Tri-ortho-tolyl phosphate

Di-isopropyl phosphoro fluoridate

Rheumatoid arthritis

Multiple sclerosis

Collagen Induced Arthritis

Experimental Autoimmune Encepha

lomyelitis

5-a-dihydro testosterone

sodium chloride

Esterif ied Fatty Acid

c-

I V

ACKNOWLEDGEMENTS

I pay my sincere thanks from the deepest core of my heart to Allah-Tabarak-

o-Tala who ornamented me with Aqul-e-Saleem, which I think is solely responsible

for the successful completion of this study.

It is indeed a great fortune of mine while registering my heartiest gratitude

and sincere obligation to my supervisor Prof. Shafiuliah, Deptt. of Chemistry

A.M.U., Aligarh, who provided his highest expertise as an efficient guide for the

successful completion of this work and loved me like a son. As a result his

personality and affection always reflects from my deeds, thoughts and practical

life. His caring nature and selfless approach towards me proved a boon which

provided intensive strength and boosted my moral at every step of this study.

It is my greatest pride to be a student of Prof. Dr. Mehdi Hasan, founder

Director interdisciplinary Brain Research Centre, J.N. Medical College. A.M.U .

Aligarh. Despite being an extremely busy scientist, he always gave me time and

taught all about the research from the very basics to the complex problems. He

is not only a highly modest and erudite person nevertheless a pride of Indian

Neurosciences and a prominent figure of International repute. The knowledge

grasped from him is indeed a torch to find the path in darkness at every step.

I shall be failing in my duty if I will not acknowledge the invaluable kind and

generous support of present Director of IBRC Prof. Shamim Jahan Rizvi, which

she extended to me during the course of this study. Her critical suggestions,

discussions and consistent monitering made me able to present this work in its

finest form. I am really proud of her because her courage, consistent fight and

struggle with supremos of Anatomy Department ultimately brought the laurel to

IBRC. She successfully filled the void vacated by the Prof. Hasan in the brain

research of this University and presently much acclaimed progress is fastly going

on under her noble and erudite leader ship.

I am highly grateful to my favourite teacher, Prof M Ilyas (Chairman, Deptt

of Chemistry, A.M.U., Aligarh) who provided me every possible technical and

academic support from the department.

The support and encouragement given me by Dr. Fakhrul Islam (Then

Pool Officer IBRC, presently lecturer in Hamdard University, Delhi) is really

priceless and beyond the expectation and a debt upon me for whole life. I have

never met in my life such a dynamic, innovative and hardworking scientist For

my best result he even worked with me like a student and assisted whole

heartedly. In fact, I am feeling proud to admit it that he incorporated three-

dimensional research methadology of international standard which he had learnt

from through out the globe. Once again I am heartly grateful and obliged to him

I am proud of mentioning the name of my welwisher friend Dr. Qasim

Khan who lovingly patronised mfe as a guardian and directed my carrier. It is I- s

obligation that he introduced me first time with Prof. Mahdi Hasan. I am really

grateful to him while mentioning it that his obligation and affection has built a

' .iianent place for him in the deepest core of my heart

My sincere thanks and obligations are always with my whole family members

Blessings of my mother, guardianship of my brothers, Dr. Habib Khan and

Shakeel Khan, Ipve of my sister and sacrifice of my sister-in-law always boosted

my moral and helped rr.e lo stride high.

I am sincerely acknowledging it with a lot of thanks that the timely help

encouragement and invaluable suggestions from Prof.Shahab Ahmad,

Dr. M Mushfiq, Dr. S.H.M. Abidi and seniors Dr. Yagana Banc, Dr. Naseem

Ahmad Khan, Dr. Masood Zaheer Naqvi, Dr. Achia Gupta, Dr. Amir Abbas, Dr.

F.S. Sheerani and Imran Ahmad, helped me to complete this research work My

appreciation is also due to my collegues V. Ramesh, Farooq, Uzma, Abid, Jamal

Feroz, Salman, Farhat Hasan and Dr. Riyaz Ahmad.

VI

It will be unjust onpart of me if I will not mention special acknowledgement

and gratitudes to Kr. Asif who always stood around me with the helping hands

round the clock. His critical suggestions and moral support will be always

unforgatable.

Help and cooperation provided by well experienced researcher Dr. Indu

Saxena need to be acknowledge with the heartiest gratitudes. During the

previewing of this thesis, her critical approach and valuable remarks helped me to

ornament this research work in its present finest form.

My friends always boosted my moral and willpower during the course of this

study. I am really obliged to them especially, Shoeb Khan, Parvez Khan,

Sarfuddin, Jawed Akhtar, Dr. Meenu and Dr. Shazia, Shahanawaz Khan, Azhar

Husain, Ibrat Ullah, Farina and Humera Malik.

I am highly obliged to Mr. Rabat (Artist Deptt. of Community Medicine) for

drawing the necessary art work for this thesis with out charging a single peny from

me. I am also highly obliged to the staff of Central Photograph and Audiovisual

Section, J.N. Medical College for preparing fine photographs.

The cooperation and support given by the staff of Forensic Medicine is

highly appreciable. I sincerely thanks to Dr. Munawwar, Mr. Akhtar All,

Mr. Syeedul-Hasan Mr. Jamal Ahmad, Mrs. Shamim Jahan, Ishtiyaq, Mujeeb

and friends Dr. Zafar Iqbal, Dr. Abid. It will be unjust if I will not mention the

name of Chandra Prakash and Shiva Kumar who always assisted me during the

course of experiments for this research work.

(ISHRAT JAIVIiL)

VII

/T

INTRODUCTION

^^ ^''

1. INTRODUCTION

1.1 AIMS AND OBJECTIVES

Steroidal drugs hormones and other biologically active

steroids possess pronounced and specific biological activities.

These steroids play important role in metabolism and therapeutics.

The knowledge of properties of these steroids stimulated us to

Intensify research on their synthetic modifications. Cholesterol

molecule is the basic skeleton of all steroidal drugs and hormones

of natural and synthetic origin. Synthetic drugs have been prepared

by doing modification in the structure of natural steroidal molecule

or alteration in the basic cholesterol nucleus. It involves the

Introduction of hetero atom at various sites in steroidal nucleus

(Pettit and Kasturi, 1961); (Jacob and Brown field, 1960). This

alteration is always aimed to enhance the selectively of these new

derivatives in certain parameters of their biological activity.

Laboratories of Indian Universities engaged in steroidal

research are not highly sophisticated. Poor infrastructure of these

labs is capable of incorporating hetero atoms only up to ring B In

the cholesterol molecule, while most of the industrially prepared

steroidal drugs available in the market have been produced by

doing modification in ring C and D This industrial process has

been done in highly sophisticated and expensive set up Our study

IS based for the sake of economy and to search more effective new

steroidal drugs so that the economically cheaper production could

result in more effective steroidal drugs and hormones

To fulfill this aspiration an attempt has been made to test

some less complicated easily synthesized cholesterol derivatives,

reported in the past. While exposing these derivatives on albino

rats' brain exclusive and elaborative biochemical, pharmacological

behavioral and histopathological studies have tobe conducted.

Steroids used in this study are : (i) 3-p-acetoxy-cholest-5-ene (ii)

3-(^acetoxy-6-nitro-cholest-5-ene (iii)3(l-acetoxy-5a-cholestan-

6-one.

Literature lacks such type of previous studies or trial of

these derivatives for the search of new steroidal drugs. Present

research work Is aimed to achieve this motive, work done in this

study is based on the facts described below

Steroid are known to possess maximum receptors in the

central nervous system CNS of all vertebrates is highly enriched

in various lipids (Suzuki, 1981). They play vital role in both the

structure and function of neural membrane These lipids are

metabollcally very active and thus their functional significance

should not be overlooked. Studies on lipid profile in the CNS forms

an important part of neurobiochemical investigations.

Present research work is confined only up to the critical

biochemical evaluation of total lipid and lipid fraction in major

brain parts of albino rat (cerebrum, cerebellum, brain stem, spinal

cord) Our second motive is to obtain detailed critical data based

on structure activity co-relationship of cholesterol derivatives

taken for trial

/i. %.

REVIEW OF

LITERATURE

#

2. REVIEW OF LITERATURE

2.1 STEROIDS

Steroids are widely distributed in nature and play an important

role in the vital activities of living organisms. The family of

steroidal substances is critically important to plant and animal

life. The first steroidal compound "Cholesterol" v\/as reported as

early as 1812 by ME . Chervenl. Cholesterol is the chief sterol of

mammalian tissue and obligate precursor of steroidal hormones

and bile salts. The dramatic expansion of steroidal chemistry

came with the discovery of sex hormones in 1929-35. Biologically

important steroids include the adrenal hormones, the sex hormones,

(plants and animal sterols,) corticosteroids and hydrocortisones.

Steroidal hormones and other biologically active steroids possess

pronounced and specific biological activities. These steroids play

important role in metabolism and therapeutics. The knowledge of

the these steroids stimulated researchers to intensify research on

their synthetic modifications.

A large number of hetero steroids have been synthesized

and screened for their chemical biological, therapeutic and

industrial potentials Modification is done by substituting

uncommon substituent (hetero atom) at different positions at

unusual carbon frame work of steroids. It resulted either in decrease

or increase in certain biological and physiological properties

(Jacob and Brown field, 1960); (Doorenbosand WIr, 1961); (Pettit

and Kasturi, 1961); (Letter and Knof. 1960).

Adrenal glands produce steroids hormones, which are

physiologically significant to our body. Destruction diseases of

adrenal gland results in then deficiency of adrenal steroids.

Symptoms in such cases could be studied as a symbol of deficiency

of adrenal steroids. Addison (1855) was the first scientist who first

time studied such clinical syndromes resulting from the destruction

disease of adrenal glands. These experiments inspired to Brown

Sequrad (1856) to do the pioneering experiments on the effects of

adrenoiactomy and concluded that "adrenal glands are essential to

life"

By the third decade of this century it was generally recognized

that the cortex rather then the medulla is the life maintaining

portion of the gland. The complex nature of adrenocortical

deficiency was dramatized in 1930s by the partisan character of

research groups oriented to study either the imbalance of

electrolytes or the defects in carbohydrate metabolism present in

the deficient state. Renal loss of Na* was convincingly

demonstrated to be a characteristic of adrenocortical insufficiency

by Harrop and associates (1933) as well as by Loeb and Coworkers

(Loeb, 1933). Equally convincing was the demonstration of a

depletionof carbohydrate stores (Coriet al., 1927). Furthermore,

Hypoglycemia could be corrected by adrenocortical extracts

(Britton & Siwette, 1931). Glucose and glycogen formed under

influence of the adrenal cortex during fasting appeared to be

derived from tissue protein (Long et al. 1940). From these studies

emerged the concepts of the two types of adrenocortical hormones.

The mineralocorticoids which primarily regulate electrolyte

homeostasis and the glucocorticoids which are concerned with

carbohydrate metabolism. This concept of dichotomy of salt and

sugar hormones (mineralocorticoids and glucocorticoids) has

proved useful survives at the present time in modified form. In

1932, the neurosurgeon Gushing described the syndrome of

hypercorticlsm bear his name (Gushing 1932). The cases Gushing

described were those of pituitary basopohilism, recognized

subsequently to be a consequence of hypersecretion of ACTH

The preparation of adrenocortical extracts with a reasonable

degree of activity was first accomplished in 1930. By 1942,

organic chemists had isolated crystallized and elucidated the

structures of 28 steroids from the adrenal cortex (Reichstein et. ai

1943). Five of these compounds Cortisol (Hydrocortisone,

cortisone, corticosterone, 11- dehydrocorticosterone. and 11-

desoxycosterone)were demonstrated to be biologically active

Another decade passed before the principal mineralocorticoids

was discovered In 1950.Deming and Leutscher made an attempt

to find the principal active material he discovered that the extracts

of urine from patients with edema induced Na* retention and K*

excretion in adrenolactomized rats. The definitive evidence for

the source ofthe active material was provided byTaitet. al. (1952)

who purified the compound with this activity from adrenocortical

extracts. The substance was crystallized, the structure was

established and the hormones was named aldosterone (Simpson

et. al. 1954).

In this same era the role of abenohypophysis was being

elucidated by other investigator's The classical studies of Foster

& Smith 1926 established the fact that hypophysectomy result m

atrophy of the adrenal cortex. By 1933 it had been demonstrated

that cell-free extracts of the anterior pituitary has a stimulating

effect upon the adrenal cortex of the hypopysectomized animal

Further chemical fractionation of such extracts led to the isolation

of a hormone, ACTH, that acted selectively to cause chemical and

morphological changes in the adrenal cortex (Astwood et al.,

1952),Its structure was established by Beel et al.(1956). The rate

of release of ACTH from adenohypopysis was shown to be

determined by the balance of the Inhibitory effects of the hormones

of the adrenal cortex and the excitatory effects of the nervous

system. (Ingle et al., 1938).

A detailed analysis of the morphology of the adrenal cortex

by Swan (1940) and Deane and Greep (1946) suggested that the

specific function of zonaglumerulosa is to autonomously

elaborating a hormone regulated electrolyte balance. This hormone

is now known to be aldosterone.

Prolonged administration of sexual adrenocortical and

thyroid hormones produces a feedback reaction causing diminished

secretion of the corresponding glandotrophic hormones and also

bring about morphological changes, These changes could be

seen histologically discernible in the glandular parenchyma of the

adenohypopysis. In a similar manner, protracted administration of

posterior-lobe hormones leads to noticeable morphological

adaptive reaction in the magnocellular hypothalamus

neurohypophyseal system (ORF- 1966). On the basis of these

empirical finding it was thus conjectured that long term application

of anterior lobe hormones would bring about morphological

changes in the paracellular hypothalamic nuclei controlling

adenohypophyseal activity (ORF-1969).

In 1949, Hench demonstrated the effects of cortisone and

ACTH In the treatment of rheumatoid arthritis. As early as 1929

Hench had been impressed by the fact that arthritic patients when

pregnant or jaundiced, experienced a temporary remission, he

believed that a metabolite was responsible for the remission. The

possibility that the antirheumatic substance might be an

adrenocortical hormone was entertained and as soon as cortisone

was available It was tested In a case acute rheumatoid arthritis.

Fortunately an adequate dose was employed and the response

was dramatic Thereafter the detailed salutary effects of ACTH

were also demonstrated by Hench, (1949). The Noble Prize in

medicine was jointly awarded to Kendall and Reichstein (who

were responsible for much of the basic chemical research that led

to the synthesis of the steroid) and to Hench, whose contribution

has just been described

According to the British Medical Association reports (1968

on therapeutic abortion cited in the famous Modies book of

Medical Jurisprudence and Toxicology, long-term exposure of

oral progestrins, androgens and estrogens causes foetal

abnormality". The Medical Termination of Pregnancy Act. 1971

also legalize the abortion of the pregnant women^ treated with

steroidal drugs like cortisone or oral contraceptives (Reddy N .

1995).

Contrary to above report recent study reveals that antenatal

corticosteroids given for a short period (24 hr and 1 week before

birth) reduces respiratory distress syndrome, neonatal mortality

by about 50% with comparable reduction in necrotismg

enterocolitis.

The benefits of antenatal corticosteroids are likely to

outweigh any possible disadvantage due to maternal or neonatal

in fect ions There is no evidence of major adverse

neurodevelopmental outcome in later childhood among infants of

treated mothers (Crov^ley 1994). Due to this protective effect

academic and professional bodies have recommended that with

very few contra-indications^ the use of antenatal corticosteroids

may be considered in women likely to deliver prematurely (Royal

Collegemeet 1993,NIH 1995). Since these are the most effective

single strategy for reducing the adverse consequences of preterm

birth (Scottish Neonatal consultants study, 1996).

Steroidal therapy has been used extensively in head injury

Its effectiveness has documented in the work of. Reulen et al.,

(1972). Taupel et al (1976). Various high potency glucocorticoids,

chiefly dexamethasone have been used widely in the management

of intracranial hypertension and brain edema. Use of these steroids

may dramatically and rapidly reduces the focal and general signs

of brain tumor (Faci,1976).

It has been confirmed by recent studies that combined oral

contraceptive improve in the both acne and hirsutum (Fotherby,

1995).

11

Role of parathyroid hormones and dihydrolachysterol is

established in the reduction of lead toxicity from the bone. These

hormones mobilize Lead from the skeleton and augment the

concentration of Leadin blood and the rats of its excretion in urine

(Cutis D., 1990).

In cases of delayed neuropathy caused by organophosphorus

compounds, treatment with corticoid in such cases modify it. for

example delayed neuropathy caused by tri-ortho-tolyl phosphate

<TOTP), but the same treatment could not showed protective

effect in the case of o-o-Di-isopropyl phosphorofluoridate (DFP)

Induced delayed neurotoxicity. It is because due to the intrinsic

differences between the organophosphorus compound DFP and

TOTP (Ehrich. M; et al 1985).

Sex linked factors have been found to affect rheumatoid

arthritis (RA) and multiple sclerosis (MS) in a similar way but both

diseases are slightly more common among women than men

(Duquette et. al, 1992). During pregency, in particular during the

last trimester condition of both RA and MS tend to become better

but again become painful in postpartum (Davis and Maslow,

1992) Reason behind these changes are complex. Sex hormones.

12

sex chromosomes or sex determined neuroendocrine factors

many be involved here (Hazes et al.,1990b) Collaegen induced

arthritis (CIA) and experimental autoimmune encephalomyelitis

(EAE) are different models and affect different target organs but

they are both dependent on autoaggressive T-cells. Studies have

proved that estrogens suppressess the pregency induced EAE

and the collagen induced arthritis in mice (Liselotte Jansson et al,

1996).

A lot of artificially synthesized hormones are being used in

the general treatment, such as NS-3, an analogue of Thyrotropin-

Releasing Hormone (Itoh et al, 1995). Attempts are being done

to produce safer and more effective steroids by modifying their

structure or incorporating them with other drugs (Suhail. 1991).

Synthetic glucocorticoids are widely used therapeutically

for their immunosuppressive and antiinflammatory effects

(Kathurina et. al, 1994).Use of the adrenal steroids exert opposite

effects on the immune system in the mammalians. (Fraschini F. et

aL 1990).

Allauddin and Mertin - Smith (1962), Martin - Smith Surgue

(1964) have critically reviewed the biological activities in steroids

possessing N-atom both of natural and synthetic origin.

13

At present steroidal drugs are the only drugs which fall

under the category of life saving drugs These drugs are used m

highest medical emergency to save the life of millions of sufferer

every day. Steroidal drugs are commonly used on the occasions

where generally any other non-steroidal drugs fail to produce any

curable effect.

A lot of research work have been done in the field of hetero

steroidal synthesis. Their identification has been done by their

characterization, chemical and spectral studies, for example oxa-

steroids (containing NHO functional group) Ahmad et al.,(1984)

Aza-steroids (containing Nitrogen) Mushfiq and Nadeem Iqbal.

(1986), Ahmad et al.^(1985), Keto-Steroids (containing ketone)

Rao Owais, (1993).

Steroidal drugs have been used for a wide range ofdiseases.

for example, they are used as antiviral (Awasthi et al.>1982).

antiallergic (Honma.et al., 1982). anticonvulsant (Ahmad, and

Shukia, 1983), antiulcer (Otssuka, 1982). analgesic (Fognet et

al* 1983), antifungal (Awasthi, 1982), antihypertensive (Ahmad

1983), antifertility (Islam et al., 1983), antidepressant (Fancois,

et al.,1918), nerve exciting (Takenchi, et al.J981), antimineralo

14

FIG.-2.1

CHOLESTEROL

15

(Ralph et al. 1985). a^-Dacterial (Sail et a i .J986i fungicidal

(Saitoet al 1986), antithyroid (Grudzinshi et ai. 197S Ikeda. and

Soga, 1978), Shibata i et al. 1986) and growth inhibitor (Birgit

1980), in cardiac diseases (Wundewald et al.,1985): in skin

disorders and as inflanmation inhibitors (Annen Klaus et al.,

1985).

The most alarnnmg international problem of population " ut

burst is being tackled by steroidal drugs Even though a lot of

alternate non-steroidal drugs devices and methods have been

invented to avoid the conception.still steroidal drugs are the first

and favorite choice among the family planners

2.2 CHEMISTRY AND GENERAL STRUCTURE OF

CHOLESTEROL

Cholesterol is present in all eukeryotes as free or as fatty

esters, particularly in the brain and spinal cord of vertebrates It

was first isolated from njman gall stones The mam sources of

cholesterol are fish-live- oil. brain and spinal cord of catties

Cholesterol is a v,riite crystalline v^hich is optically active-

The structure o* cholesterol was elucidated by Wieland.

16

Windvas and their coworkers (1903-1932). The established

structure of cholesterol is given here. It shows the method of

numbering

The Molecule (Fig 1) consist of a side-chain and a nucleus

which is composed of four rings, designated A, B, C and D,

beginning from the six-membered ring on the left. It should be

noted that the nucleus contains two angular methyl groups, one at

C-10 other at C-13. There is a side chain of CgH^^ at the C-17

position (I).

2.3 METABOLISM OF STEROIDAL HORMONES IN BRAIN-

TISSUE :

When the steroid hormones interact with the target tissues,

these gets metabolically transformed in to more or less active

metabolites. For the hormones like androgen and testosterone

these transformation appear to be of particular importance, this

also suggest that these steroids may be a perhormones. The

brain, like the seminal vesicles, is able to convert testosterone to

5a - dihydrotestosterone (DHT) and 3a -5a-androstanediol (Fig.

I and II ) and also like the placenta, converts testosterone to

estradiol (Fig. III).

17

FIG. - 2.2

ANDROGEN HORMONES

c o

5a-dihydrotestosterone 3a.5a-androstaanediol

17B-estradiol III

progestesone

VI 5a-dyhydroprogesterone

VII

Conversion never occurs equally in all brain regions

Regional distribution of 5a-reductase activity towards testosterone

in rat brain is found in mid brain and brain stem with intermediate

activity in hypothalamus and thalamus and lowest activity in

cerebral cortex. The pituitary has higher 5a-reductase activity

than any region of the brain and its activity is subjected to change

as a result of gonadectomy, hormone replacement and postnatal

age. 5a-Dihydrotestosterone has been implicated In hypothalamus

and pituitary as a potent regulator to of gonadotropin secretion but

is relatively inactive toward male rat sexual behavior (Feder :

1971).

It is interesting that progesterone (Fig. VI) inhibits 5a-

reductase activity toward (3H) testosterone and that (3H)

progesterone is itself converted to (3H) 5a- dihydroprogesterone

(Fig.VII) Progesterone competition for the 5a-reductase may

explain some of the antiandrogenicity of this steroid, (Massa and

Martin, 1972).

The aromatization of testosterone to form estradiol (Fig. Ill)

and of androstenediols (Fig.IV) to form esterone (Fig. V) has been

described in brain tissue in vitro and in vivo (Naftolin et al. 1975

19

and Lieberburg. et al. 1975) A'omatization is higher m

hypothalamus and limbs structures than in cerebral cortex or

pituitary gland in noncastrated animals is higher in male than in

female brains. Aromatization has been found in brains of reptiles

and amphibia as well as in mammals, (Collard, et al. 1977 and

Kelley et al. 1978).

The capacity to aromatize testosterone and related

androgens may therefore be a general property of vertebrate

brains The functional role of aromatization has been studied most

extensively in the rat. male sexua! behavior is facilitated by

estradiol and testosterone (Davis and Barfield, 1979).

Facilitation of male sexual behavior can be blocked by

these steroids (Clemens, 1975 and Morali, et al. 1977). Similar

situation exists in birds, amphibia and reptiles, testosterone and

estradiol can stimulate heterotypical sexual behavior in male and

females. Curiously, not all mammals are like the rat. For example.

male sexual behavior of the guinea pig and rhesus monkey is

restored by the nonaromatizable androgens androstenendione

and dihyrotesosterone, (Alsum, and Goy, 1974). A number of

other steroid transformations occur in brain tissue , but this

metabolism dose not appear to be of importance for Interaction of

20

those hormones withthe putative receptor sites. It has been found

that Both (3H) estradiol and (3H)-corticosterone are recovered

from their binding sites in the brain (Mc Ewen, et al. 1972)

2.4 PHYSIOLOGICAL FUNCTION AND PHARMACOLOGICAL

EFFECTS OF STEROIDS :

Corticosteroids have numerous wide spread effects on

living system. They influence carbohydrate, protein and lipid

metabolism, electrolyte and v^ater balance and the functions of

cardiovascular system, kidney, skeletal muscles, nervous system

and other organs and tissues

Furthermore the cortico-steroids endow the organism with

the capacity to resist many types of nexious stimuli and

environmental changes In the absence of the adrenal cortex,

survival is possible but only under the most rigidly prescribed

conditions for example, food must be available regularly, sodium

chloride ingested in the relatively large quantit ies, and

environmental temperature maintained with in a suitably narrow

range. A given dose of corticosteroids may be physiological or

pharmacological, depending on the environment and the activities

21

of the organism. Under favorable conditions, a small dose of

corticosteroids maintains the adrenalectomized animal in a state

of well-being Under adverse conditions a relatively large dose is

needed, if the animal is to survive This same large dose given

repetitively under optimal conditions induces hyper-corticismthat

is sign of corticosteroids excess. The function in the secretary

activity of a normal subject are presumed to reflect the body's

varying requirements for corticosteroids.

The actions of corticosteroids are often complexly related to

the functions of other hormones For example in the absence of

lipolitic hormones, Cortisol, even in large concentrations, has

virtually no effect on the rate of lipolysis in adipose tissue in vitro.

Likev^ise, a sympathomimetic amine has only a slight effect on the

rate of lipolysis if there is a deficiency of glucocorticoids. However,

if a necessary minimal amount of Cortisol is added, the lipolitic

effect of the sympathomimetic amine become evident The

necessary but not sufficient role of corticosteroids acting in

concert with other regulatory forces has been termed "permissive '

Estimates of the potencies of naturally occurring and

synthetic corticosteroids in the categories of Na* retention

22

(Reduction of Na+ excretion by the kidney of the adrenalectomized

animal), hepatic deposition of glycogen in fasted adrenalectomized

animals, and inflammatory effect (inhibition of the action of an

agent that induces inflammation) are presented in Table 2 1 It

should noted that such values are not fixed ratios but very

considerably with the conditions of the bioassays used. Potencies

of steroids as judged by their ability to sustain life in the

adrenalectomized animal closely parallel those determined for

Na* retention. Potencies based on deposition of linear glycogen,

antiinflammatory effect, work capacity of skeletal muscles, and

involution of lymphoid tissues closely parallel one another.

Dissociations exist between pp based on Na* retention and on

hepatic glycogen deposition traditionally, the corticosteroids have

thus been classified into mineralocorticoids and glycocorticoids,

according to the potencies in the two categories (Robert, Hynes

1980).

23

TABLE - 2.1

RELATIVE POTENCIES OF CORTICOSTEROIDS

Na+ Hepatic Anti-inflammatory

Retention Glycogen

Deposition

effect

Natural steroids

Cortisol r 1

Cortisone 0.8* 0.8

Corticosterone 15 0.35

ll-Desoxy

Corticosterone 100 0.00

Aldosterone 3000 0.3

1

0.8

0.3

0.00

9

Synthetic Steroids

Prednisolone

Triamcinolone

r 0.00

4

5

4

5

•Promotes excretion of Na* under certain circumstances.

Glucocorticoids are widely used therapeutically for their

Immunosuppressive and antiinflammatory effect (Kathurina Binz

e ta l . 1994).

24

Adrenal steroids have been found to facilitate a form

behavioral adaptation in the absence of external stressors adrenal

steroids are also secreted in varying amounts according to the

time of day. In nocturnaily active animals as the rat and in animals

such human being which are active during the day, the peak of this

basal secretion always occurs near the end of the sleep period.

Thus it is conceivable that adrenal steroid secretion may modulate

behavior as a function of the time of day. Indeed it has been

reported that adrenal steroids modify the detection and recognition

thresholds for a variety of sensory stimuli and influence the

occurrence of the (so-called) rapid eye movement or paradoxical

phase of sleep (Bhous 1973) and Mc Ewen et al. (1975)

Desoxy cor t icosterone, the prototype of the

mineralocorticoids, is highly potent in regard to Na+ retention but

without effect on hepatic glycogen deposition Cortisol the prototype

of the glucocorticoids is highly potent in regard to linear glycogen

deposition but weak in regard to Na* retention. The naturally

occurring corticosteroids Cortisol and cortisone, as well as synthetic

corticosteroids such as prednisolone and triamcinolone, are

classified as glucocorticoids. However, corticosterone is a steroid

25

that has modest but significant activities in both categories In

contrast, aldosterone is exceedingly potent with respect to Na*

retention but has only modest potency for linear glycogen

deposition. At rates secreted by the adrenal cortex or in doses that

exert maximal effect on electrolyte balance, aldosterone has no

significant effect on carbohydrate metabolism, It is thus classified

as a mineralocorticoids (Robert Hynes ^1980).

2.4.1 Mechanism of Action :

The hormones like Adrenal steroids mineralocortlcos- teroids

etc act by controlling the rate of synthesis of proteins. (Bruce and

A c Ewen, 1981; James and Nathanson, 1981) However, their

nearly instantaneous inhibition of ACTH release is an exception.

These steroids react with receptor proteins in the cytoplasm of

sensitive cells in many tissues to form a steroid receptor complex.

The complex undergoes a modification, as noted by an increase

in the sedimentation constant and moves in to the nucleus, where

It binds to chromatin and regulates transcription of specific genes.

^ most known examples, transcription Is enhanced as manifest by

Increased amount of specific mRNA. Inspite of these facts

glucocorticoids exceptionally decrease rate of transcription.

26

2.5 STRUCTURE-ACTIVITY RELATIONSHIP OF STEROIDS :

Structural modification in the Cortisol nnolecule have led to

Increase in the ratio of antiinflammatory to Na* retaining potency.

Electrolytic effects exerted by most of these steroids are of no

. nervous consequences. However, effects on inflammation and on

carbohydrate and protein metabolism have always paralleled one

another, and it seems very likely that these effects are mediated

by the same type of receptor.

Change in the moleculer structure may bring about changes

in biological potency as a result of alteration in absorption protein

binding rate of metabolic transformation, rate of excretion ability

to traverse membranes, and intrinsic effectiveness of the molecule

at its site of action. In the following paragraphs, modification of the

pregnane nucleus that have been of value in therapeutic agents

are described in following figures. Following list show the effects

of the modifications discussed relative to Cortisol.

27

TABLE - 2.2

RELATIVE POTENCIES AND EQUIVALENT DOSES OF

CORTICOSTEROIDS

Compound Relative Relative Duration Approx Antiinfia mmatory

Retaining Na-

of action Equiv

potencies potency Dose* (mg)

Cort isol (Hydrocort isone) 1 1 S 20

Tetra Hydrocort isol 0 0 - -

Prednisone

(^ ' -Cort isone) 4 0 8 1 5

Prednisolone

C • -Cort isol) 4 0 8 1 5

'^6a-Methylprednisolone 5 0 5 1 4

Flurocort isone (9a-f lurocort isol) 10 125 S -

11-Desoxycort isol 0 0 - -

Cort isone (11-Dehy-drocort isol) 0.8 0.8 s 25

Cort icosterone 0 35 15 s -

Tr iamcinolone {9a- f luro-16a-

methylPrednisolone) 5 0 1 4

Betamethasone (9a-Fluro-16P-

methyl prednisolone) 25 0 L 2

Dexamethasone (9a-Fluro-

methyl prednisolone) 25 0 L 0 75

Paramethasone (6a-Fluro-16a-

methylprednisolone) 10 0 L 0 75

*S = Short or 8to 12 hour biological half life;

I = Intermediate or 12 to 36 hour biological half life;

L = Long or 36 to 72 hour biological half life

2X

FIG. - 2.3

CHaPH

II

Structure-stereochemistry and nomenclature of adrenocosteroids,

as typsified by Cortisol (hydrocortisone)

29

These dose relationship apply only to oral or intravenous

administration, relative potencies maydiffer greatly when injected

intramusculary or in to joint spaces

Ring A :

The 4.5 double bond and the 3-ketone are both necessary

for typical adrenocorticosteroid activity. Introduction of a 1.2

double bond as in prednisone or prednisolone, enhances the ratio

of carbohydrate regulating potency by selectively increasing the

former In addition, prednisolone is metabolized more slowly than

Cortisol

Ring B :

6a-substitution has unpredictable effects. In the particular

Instance of Cortisol, 6a-methylation increases anti-Inflammatory,

nitrogen wasting and Na* retaining effects in man. In contrast 6a-

methylprednisolone has slightly greater antiinflammatory potency

less electrolyte retaining potency than prednisolone. Fluorination

in the 9a-positing enhances all biological activities of the

corticosteroids, apparently by its election withdrawing effect on

the 1ip-hydroxy group.

30

Ring C :

The presence of an oxygen function at C-11 is indispensable

for significant antiinflammatory and carbohydrate regulating

potency (Cortisol versus 11-desoxycortisol) but it is not necessary

for high Na* retaining potency as demonstrated by

desocycorticosterone.

Ring D :

16-Methylation or hydroxylation eliminates the Na* retaining

effect but only slightly modified potency with respect to effects on

metabolism and inflammation.

Presently used all the anti-inflammatory steroids are 17a-

hydroxy compounds. Although some carbohydrate regulating and

antiinflammatory effects may occur in 7-desoxy compounds

(Cortisol versus corticosterone), the fullest expression of these

activities the presence of the 17a-hydroxy substituent.

All natural corticosteroids and most of the active synthetic

analogues have a 21-hydroxy group . While some glycogenic and

antiinflammatory activities may occur in its absence its presence

is required for significant Na* retaining activity.

31

The four rings A. B. C and D are not in a flat plane as

conventionally represented m I but have the approximate

configuration shown in II (The planarity of the valence angles

about the double bond betv^een C-4 and C-5 prevents the chair

form of ring A. as from being an energetically probable

conformational state As a result ring A is in a half-chair

conformation not easily represented in two dimar ions). Orientation

of the group attached to the steroid ring system is important for

biological activity. The methyl groups at C-18 and C-19 the

hydroxyl group at C-11 and the two carbon Ketol side chain at C-

17 project above the plane of the steroid and are designated Q>

Their connection to the ring system is shown by full line bonds.

The hydroxy at C-17 projects below the plane and Is designated

a and the connection to the ring is shown by a dotted bond

2.6 EFFECTS OF ADRENOCORTICAL STEROID ON CNS :

The corticosteroids affect the central nervous system (CNS)

in a number of indirect ways. These steroids particularly maintain

normal concentration of glucose in plasma, they also ensure the

adequate circulation and the normal balance of electrolytes in the

body. There Is also an increasing recognition of direct effects of

32

corticosteroids on the CNS as understanding of the distribution

and function of steroid in the brain (Mc Ewen et al. 1986 ; Funder

and Sheepard, 1987). An influence of the corticosteroids can be

observed on mood, behavior the electroencephalogram (EEG)

and brain excitability.

Patients with Addison's disease exhibit apathy depression

and irritability, some are frankly psychotic Desoxycorticosterone

is ineffective but Cortisol is very effective in correcting these

abnormalities of psyche and behavior. An array of reactions

varying in degree and kind is seen in patients to whom

glucocorticoids administered for therapeutic purposes. Most

patient respond elevation in mood, which may be explained in part

by the relief of the symptoms of the disease being treated. In

some, more definite mood changes occur, characterized by

euphoria, insomnia restlessness, and increase motor activity. A

smaller but significant percentage of patients treated with high

doses of Cortisol become anxious or depressed and a still smaller

percentage exhibit psychotic reactions. A high incident of neuroses

and psychoses has been noted among patients with Cushing's

syndrome. The abnormalities of behavior usually disappear when

the corticosteroids are withdrawn or Cushing's syndrome is

effectively treated (Lewis et al. 1983). 33

There is an increase in the excitability of neural tissue

hypocorticism and a decreased excitability in animals giving large

doses of desoxycorticosterone, these alterations appear to be

related to change in the concentration of electrolytes in the brain.

In contrast administration of Cortisol increase brain excitability

without influencing the concentration of Na* and K* in the brain.

Thus it has been concluded that the inf luence of

desoxycorticosterone on excitability is mediated through its

influence on Na* transport whereas Cortisol acts by cytoplasmic

receptors (Mc Ewen. 1979 ; Carpenter and Cruen, 1982). Steroid

hormones are classically implicated in the maintenance of

homeostasis of an organisms internal milieu in the response to

emergency environmental demands and in the process of sexual

reproduction. Steroid hormones have special role in the timing

and sequential integration of the development of neural processes

(Charles 1986). In addition to less serious complaints such as

dermatitis and tennis elbow>relevated plasma Cortisol levels are

also associated with effective endocrine disorders, such as those

associated with endogenous depression (Prange et al. 1977).

Glucocorticoids may have direct, non-receptor mediated effects

34

on neural membranes and that chronic dosmg with exogenous

glucocorticoids greatly enhances brain excitability (Woodbury,

1958; Hall, 1982) Thus itmay be that these alterationsm behavioral

excitability, typically attributed to enhanced neuronal excitability

(Ecobichon and Joy. 1982) Sex hormones may alter neural

excitability in a biphasic manner in desecrated brain regions such

as the hippocampus (Teyler, et al., 1980),

35

dr

MATERIAL

METHODS

#

A) MATERIALS :

3.01 ANIMALS :

Male albino rats of Charles Foster Strain (250 ± 50

grams).obtained from animal house, J.N. Medical college, A.M.U.

Aligarh.

3.02 PREPARATION OF STEROIDS FOR BIOCHEMICAL

STUDIES

3.02.1 SR-Acetoxy'Cholest-S-ene

A mixture of cholesterol (SO.OOgm). pyridine (75 ml) and

acetic anhydride (50 ml) was heated on a steam bath for 2 hours.

The resulting brown solution was poured onto crushed ice-water

mixture with constant stirring. A light brown solid thus obtained

was filtered under suction, washed with water until free from

pyridine and air dried The crude product on recrystallization from

acetone gave pure 3B- acetoxy-cholest-5-ene (45 gm) with mp

114°C (reported, mp 116°C) (Backer and Squire, 1987).

36

3.02.2 3B- acetoxy-6-nitrO'Cholest'5-ene

3B - actoxycholest-5-ene (10 gm) was covered with nitric

acid (d=1 . 52:250 ml) and sodium nitrite (10 gm) was gradually

added over a period of one hour, with continuous stirring slight

, cooling was also required during the reaction. Stirring was

continued for additional 2 hours, when yellow, spongy mass

separated on the surface of mixture. This was diluted with ice cold

water (250 ml). A green coloured solution was obtained. The

whole mass was extracted with ether and washed successively

with water, sodium bicarbonate solution (5%) and water. The

ethereal layer was dried over anhydrous sodium sulphate. Removal

solvent provided an oil which was crystallized from methanol to

get 3J5-acetoxy-6-nitrocholest-5-ene (6.8 gm). mp 103.9°C

(reported, mp 104°C). (Anagno Stopoules and Fieser, 1954).

3.02.3 3fi->)cefoxy-5a-c/)o/esran-6-one ;

3B-acetoxy-6-nitrocholest-5-ene (6 gm) was dissolved in

glacialacetic acid (250 ml) by warming the mixture. Zinc dust (12

gm) was added in small portions with continuous shaking and the

suspension was heated under reflux for four hours. Water (12 ml)

37

was added occasionally during the course of reaction. The l iot

solution was filtered, cooled to room temperature and diluted with

a large excess of ice cold water. The organic matter was extracted

with ether and ethereal solution, was worked up and dried In usual

nnanner removal of solvent furnished the ketone as on oil which

was recrystallized from methanol (4.2 gm), mp.128-129°C

(reported, mp.127-129°C). (Dodson, and Reigel, 1948).

3.03 STRUCTURE OF THE STEROIDS USED IN THIS STUDY

AcO

3Bi-acetoxy-cholest-5-ene

Fieser. L.F et al. (1959)

(I)

ACQ

N0=,

3(j-acetoxy-6-nitro-cholest-5-ent

Anagno stopoules (1954).

(H)

3a-acetoxy-5a-cholestan-6-one

Dodson etal. (1948)

(III)

3.04 REAGENTS :

All the chemical used in this study were of analytical reagent

grade. The chemicalswere obtained from Sigma Chemicals Co.

(St. Louis, Mo, U.S.A). AldrichChemical Companyu.S.A, B D H.

Chemical Ltd., Poole England, E. Merck, Germany and Sisco

Research Laboratories, Bombay, India.

1. 3B-acetoxy-cholest-6-ene

2. 3(i-acetoxy-6-nitro-cholest-5-ene

3. 3(i-acetoxy-5-a-cholestan-6-one

4. Pyridine

5. Acetic anhydride

6. Acetone

7. Nitric acid

8. Sodium nitrate

9. Sodium bicarbonate

10. Ether

11. Sodium sulphate (anhydrous)

12. Methanol

13. Glacial acetic acid

14. Zinc dust

39

15. Sodium chloride

16. Chloroform

17. Brain lipids

18. Cone. HjSO^

19. Potassium dihydrogen orthophosphate

20. Vanillin

21. Double distilled water

22. Ammonium molybdate

23. Perchloric acid

24. Sodium bisulphite

25. Sodium sulphate

26. 1-amino-2-napthol-4-sulphonic acid (ANSA)

27. Cholesterol

28. Ferric chloride

29. Sialic acid

30. Recorcinol

31. Butyl acetate

32. n-butanol

33. N-acetylneuramlnic acid (NANA)

34. Cone. Hydro Chloric Acid

40

35. Copper sulphate

36. Triolein

37. Ethyl alcohol

38. Hydroxyl Amine Hydrochloride

39. Sodium Hydroxide

40. Potassium chloride

41. Trichloro acetic acid (TCA)

42. Thiobarbituric acid (TBA)

43. Pea nut oil

44. pH tablets

3.05 INSTRUMENTS :

1. DU-6 Beckman spectrophotometer

2. Oven (NSW, India)

3. pH-meter (elico) with double combine electrode

4. Metabolic shaker (NSW, India)

5. Single Electric Balance (varbal)

6. Boiling water bath (Technico India)

7. Digestor (Designed in our center)

8. Vacuum drier

41

9. Homogenizer (Yorco Scientific Industries)

10. Stirrer (Yorco Sales Pvt./Ltd).

11. Double distillation plant for water

12. Single distillation plant with heating mantle for

purification.

3.06 MISCELLANEOUS :

1. Ice Bath

2. Pellet diet (Hindustan Lever Ltd., India)

3. Plastic eages

4. 1 nnl tuberculin glass syringe

5. Needle (26 number)

6. Screw capped culture tubes

7. Sintered glass funnel

8. Homogenizing tube

9. Pasteur pipette

10. Glass beeds

11. Vials (10 ml).

42

B) METHODS

3.07 PROCEDURE AND MAINTENANCE OF ANIMALS :

Healthy male albino rats of Charles Foster Strain, obtained

fromtheanimalhouse, J.N. Medical college, A.M.U.,Aligarh were

used in the present study. They were divided Into four groups.

Group 1: 6 months, wt. = 250 ± 60 grams




Female rats were excluded from the study because of their

cyclic hormonal variations.

The rats kept on pellet diet (Hindustan Lever Ltd., India)

measuring upto the U.S. National Research Council Publication

No.990, titled "NationalRequirements ofLaboratoryAnimals" They

were housed singly in plastic cages placed in a room maintained

at 23 ± 1°C. The animal room and cages were cleaned daily to

keep them dust free. Light-dark (photo period) was controlled

between 8:00 AM to 8:00 P.M. The hygienic and sanitary conditions

43

were adequately maintained. The rats showing sings of any

disease were removed immediately after identification.

3.08 SOLUTION PREPARATION AND DOSE ADMINISTRATION

0.3 mg/kg body wt. steroid were given to the rats. The

solution for Injection to the rats was prepared in pea-nut oil and

was made keeping in mind that the amount of the oil to be injection

to the animal should not exceed approximately 0.1 ml, to avoid the

accumulation of fat (lipids) in albino rats. The solution was

prepared by adding 6.75 mg reference steroid in 9 ml of peanut oil.

The steroids were injection intraperltonially with the help of 1 ml

tuberculin glass syringe and 26 number needle. A new needle for

each animal was used daily . The syringes were sterilized each

day by boiling in water and rinsing with methanol.

Group 1 : (Control) of 6 male albino rats were injected normal

saline equal to the volume of steroidal solution injected

in experimental rats for 15 days.

Group 2 : (Experimental) was Injected 3-6-acetoxy-choiest-5-

ene, 0.3 mg/kg body wt. for 15 days.

Group 3 : (Experimental), was injected 3-fi-acetoxy-6-nitro-

cholest-5-ene, 0.3 mg/kg body wt. for 15 days.

44

A l b i n o R a t

%

DISSECTED BRAIN OF ALBINO RAT

Fig. 3.01

Fig-301

Photograph showing the rJorsal view of Rat Brain

45

h Cerebral hemisphere

r Brain stem

r Spinal cord

Dissection of deff erent parts of the rat CNS IMcEwen and Praff 1970.).

Group 4 : (Experimental) was injected 3-B-acetoxy-5-a-

cholestan-6-one, 0.3 mg/kg body wt. for 15 days.

3.09 DISSECTION OF DIFFERENT PARTS OF THE RAT'S

BRAIN :

For all biochemical investigations, fresh unfixed brain was

used. The brain and spinal cord were removed rapidly from

Individual rats and dissected out on an ice plate. The blood clots

adhering to brain were removed by washing with the cold normal

saline. Thereafter, the cerebrum,cerebellum brain stem and spinal

cord were rapidly dissected out and weighed to the nearest

milligram on an electrical balance,some of the brain components

like hippocampus, hypothalamus and cerebellum were pooled

and used for biochemical analysis.

3.10 PROCEDURE FOR KILLING AND DISSECTING THE

ANIMAL :

3.10.1 Cervical Dislocation :

For biochemical and most of the histochemical studies

where perfusion of the rat brain was not required the animal were

46

killed by cervical dislocation one of the most acceptable methods

euthanasia. The control as well as experimental rats were grasped

at their neck the base of skull, the thumb forefinger of one hand,

and hind limbs and tail the other. A swift but controlled motion

separated the cervical vertebrae from the base of skull. This

resulted in instantaneous loss of consciousness and loss of all

vital sings within a few minutes.

3.10.2 Exposure of the Brain and its Removal:

The head of rats (Killed by cervical dislocation and skinned).

The skeletal converings over the skull were removed with the use

of a small pair of bone forceps, scalpel, scissors and nail clippers.

Great care was taken to avoid any laceration of brain tissue. After

exposing the brain surface from the top and sides, it was gently

detached from the base of skull and removed.

47

tfr \

C) BIOCHEMICAL STUDIES

"- '"

C) BIOCHEMICAL STUDIES :

3.11 EXTRACTION AND PURIFICATION OF BRAIN LIPIDS :

Lipids from brain regions were extracted and purified

immediately after dissection of the animal by the method of Folch

et al. (1957). This method was partially modified in our laboratory

(Islam etal. 1980)for isolation of lipids from discrete areas of the

brain. Different parts of the brain were weighed and homogenized

(10% w/v) in a glass homogenizer to a final volume of 6 ml

chloroform;methanol mixture (2:1, v/v). Each homogenate was

shaken periodically for an hour and filtered through sintered glass

funnel (G-4) under vacuum (Fig. 3.02). The residue of each test

tube was again homogenized with 2 ml chloroform-methanol

mixture and filtered. The resultant residue was washed several

times with the solvent mixture to ensure complete extraction of

lipids. The final volume of each extract was made up to 10 ml with

chloroform-methanol mixture in a graduated test tube. There after,

2.5 ml of 0.9% NaCI was added to the extract In each test tube.

This was shaken vigorously on test tube mixer for complete mixing

and placed at -20°C in a deep freeze overnight for complete

48

SPECIALLY DESIGN SYRINGE

Designed In

Interdiscepllnary Brain Research Centre A.M.U., Aligarh

Fig. 3.02

Fie-3.02

Rubber bulb

Syrir.ge 10 r.:

Spinal needle No,10

Joint of the tv;o spinal needles

Spinal needle Mo.23

49

r

SINTERED GLASS FUNNEL FOR FILTRATION

Designed In

Interdisceplinary Brain Research Centre A.M.U., Aligarh

Fig. 3.03

Fig-3.03

Sintered Glass funnel G4 SSml Capacity.

Filter Adopter

I.D. 3 Cm.

.Test tube 18xl50mr.

To vaccur.

- 10ml mark on the test tube

Rubber cushion Stand

Side arm tube supported by filter adopter and sintered Glass funnel for the quick filtration.

50

separation of the two layers. The junction of the layers of each test

tube was marked. The upper aqueous layer was taken out with a

specially designed syringe (Fig 3) without disturbing the interfacial

fluff. This upper layerwas used forthe estimation of gangliosides.

The lower lipid layer was made homogeneous by adding

required amount of methanol make the final volume to 10 ml and

transferred to round bottom flask and dried under vacuum at 40°C.

In order to remove proteins, the residue was dissolved in

chloroform-methanol-water mixture (64:32 ; 4 v/v) and evaporated

to dryness. The contents were suspended in chloroform-methanol

(2:1 v/v) mixture and evaporated to dryness. This process was

repeated again until moisture was completely removed from

lipids. The lipids were then suspended in chloroform, Filtered free

of any protein, and the filtrate was dried under vacuum at 40°C.

The dried lipids were solubilized to a known volume with chloroform

and stored at -20 C till further use. This extract was used for the

estimation of total lipids, triglycerides and cholesterol.

3.12 ESTIMATION OF TOTAL LIPIDS :

Total lipids were estimated according to the method of

Woodman and Price (1972).

51

fr ^ ^

STANDARD GRAPH OF

TOTAL LIPIDS

Method Woodman and Pnce, (1972)

c- Fig 3 04

s^

FIG. - 3.04

TOTAL LIPIDS

• ^ ^ -5 .750 < ^ ^ ' »25 1.5

AMOUNT OF TOTAL LIPIDS (mg)

52

3.12.1 Principle:

Colour was developed with the help of colouring reagent

(phosho-vanillin) In the presence of sulphric acid (HjSO^) and

absorbance was read at 540 nm. The details of the reaction are

given below. Sulphuric acid acts upon the double bonds on lipids

to produce carbonium ion which simultaneously reacts with

phosphate ester of vanillin to form a coloured complex with an

absorption maximum at 540 nm.

3.12.2 Reagent solutions :

(i) Standard solution :

A standard solution of 0.5 mg brain lipid/ml in chloroform-

methanol mixture (2:1 v/v) was prepared by diluting 1.0 ml of

refrigerated stock solution (50 mg brain lipid/10 ml) in chloroform-

methanol mixture, in a 10 ml volumetric flask and the volume was

made upto the mark with chloroform-methanol mixture.

H H H H I I I I I I I I

HjSO, + R - C = C C — C — + R+ 1 -H

53

(ii) Concentrated sulphuric acid (A.R.)

(iii) Colouring reagent:

6.0 grams of potassium dihydrogen orthophosphate (KH^PO )

and 0.39 grams of vanillin were dissolved by heating in double

distilled v ater and made up the volume to 100 ml in a volumetric

flask.

(iv) Brain lipid :

Lipids were isolated from the rat brain by the technique

described in the extraction and purification of lipids.

3.12.3 Procedure :

0.1 ml of brain extract in duplicate was taken in 18 x 150 mm

corning test tubes. Cone, sulphuric acid (2.5 ml) was added to test

tube and heated on boiling water bath for 20 minutes. After cooling

5.0 ml of colouring reagent was added and absorption was read

at 540 nm after exactly 10 minutes in.DU-6 Beckman

spectrophotometer against a reagent blank. A calibration curve of

absorbance in concentration (100-600 ug) of standard brain lipids

was prepared by adopting the same procedure as described

above. The values of the standard curve were plotted by least

square method. The absorbance of total lipids in brain samples

54

were compared with curve and finally calculated by the following

formula :

3.12.4 Calculation :

C X V

Totallipids (mg/g of fresh brain weight) = V. X W.

Where,

C = Concentration of lipids in ug in 0.1 ml extract

V = Total volume of the total lipids extract

V, = Volume taken for the estimation

W, = Fresh weight of the tissue in mg.

3.13 ESTIMATION OF PHOPHOLIPIDS :

Total Phopholipids were calculated by estimating

phosphorous following the method of Fiske and Subba Rao (1925)

as modified by Marinetti (1962).

3.13.1 Principle:

Organic phosphorous of phospholipds was converted to

inorganic phosphorous by digesting the lipids with perchloric acid

55

FIG. - 3.05

Phosphate

001 002 003 X)04 .005 .006 .007

006

o

CONCENTRATION OF PHOSPHATE (mg)

56

when acid hydrolysate is treated with molybdate, it forms

phosphomolybdic acid with inorganic phosphorous. Thus

phosphomolybdic acid is reduced by the addition of 1.2,4-

aminonapthal sulphonic acid (ANSA) reagent to produce a blue

coloured complex. The intensity of the blue colour, which is

proportional to the amount of Inorganic phosphorous present, was

read at 700 nm. Inorganic phosphate contentswas multiplied by

25 to convert into a lecithin equivalent to organic phospholipid.

3.13.2 Reagents :

i) Standard solution :

A standard solution of 0.1 mg inorganic phosphate/ml was

prepared by diluting 5.0 ml of refrigerated stock solution (0.439 g

KH2PO/5OO ml DDW) in a 100 ml volumetric flask with double

distilled water.

li) 2.5% Ammonium molybdate solution :

2.5 g of Ammonium molybdate was dissolved in 100 ml

double distilled water.

iii) Perchloric Acid (70%).

iv) Reducing Reagent:

57

3.0 gram of analytical grade sodium bisulphite 0.6 g sodium

sulphate and 0.05 g recrystallized 1-amino-2-napthol-4-sulphonic

acid (ANSA) were dissolved in 25 ml double distilled water. A

slight yellow solution thus obtained was stored in an amber colour

bottle. The yellow colour is stable for a week at room temperature.

3.13.37 Procedure :

The tube containing 0.2 ml of brain lipid extracts in duplicate

were dried and added with 1.0 ml of reagent grade perchloric acid.

Contents were digested over sand bath maintained at 180°Cfor

about 30 minutes till the digestion mixture was colour less. Glass

beads were introduced to each tube to avoid bumping. After

digestion, the samples were cooled to room temperature and 1.5

ml ammonium molybdate, 0.2 ml reducing reagent and 7.0 ml

double distilled water were added to each sample. Thereafter, the

whole mixture was mixed thoroughly in each test tube.The tube

were heated in a boiling water bath for 7 minutes, cooled to room

temperature and read at 700 nm at against a reagent blank

(prepared by treating 1.0 ml perchloric acid in the same way as

samples) simultaneously, different concentrations of standard

58

KHjPO^ solution were also estimated as above. The values of

absorbance vs concentrations were plotted for calibration curve

by least square method.


Phosphorous content in the sample was calculated using

the standard curve and the amount of phospholipid in the sample

was calculated by multiplying the phosphorous content with 25

(factor). The results were expressed in mg phospholipids/g tissue

weight.

3.14 ESTIMATION OF CHOLESTEROL :

Total cholesterol was estimated according to the method of

Zlatisetal. (1954).

3.14.1 Principle :

Cholesterol dissolved with acetic acid and in the presence

of FeClg-HjSG^ reagent gets dehydrogenated to 3,5 cholestadiene

or2,4-cholestadiene which simultaneously poiymerizesand reacts

with FeClg to form a violet coloured complex which Is measured

clorimetrically at 570 nm.

59

FIG. - 3.06

CHOLESTEROL

^ 2.200 H

H 2.000 H

1800 H

^ I * > ' I * t t

01 0.2 0-3 O-A 0.5 0.6 0.7 0-8

AMOUNT OF CHOLESTEROL (mg)

60

3.14.2 Reagents :


A standard solution of 1.0 mg/ml cholesterol in acetic acid

was prepared by diluting 1.0 ml of stock solution (100 mg

cholesterol in 10 ml acetic acid) in 10 ml volumetric flask and the

volume was made to 10 ml with acetic acid.

ii) Giaciai acetic acid.

iii) Concentrated Sulphuric Acid.

(iv) StoclcFerric Chloridereagent.

5.0 g of anyhydrous ferric chloride was dissolved in 50 ml of

glacial acetic acid.

v) WorkingFerric Chloridereagent.

1.0 ml of stock ferric chloride was diluted to 100 ml with

concentrated sulphuric acid.

3.14.3 Procedure :

Brain extract (0.05 ml) was taken in duplicate in dry 15x 150

mm test tubes, dried and dissolved in 3 ml glacial acetic acid.

Working ferric chloride solution (2 ml) was added and the contents

were mixed thoroughly. The tubes were kept in dark for 30 minutes

and the optical density was then measured at 570 nm. Reagent

61

blank and standard cholesteral solutions were also run

simultaneously.


Standard curve of cholesterol was used to calculate the

amount of cholesterol in the samples and results were expressed

as mg/chloesterol/g tissue weight.

3.15 ESTIMATION OF GANGLIOSIDES :

Gangliosldes were estimated according to Pollet et al.

(1978) after modification in the methods of Svennerholm (1957)^

Miettinene and Takki-Luukkainen (1959).

3.16.1 Principle :

Bound sialic acids in the gangliosldes were isolated carefully

without their degradations. When samples are heated at 100°C

in a boiling water-bath for 30 minutes in resorcinol reagent, the

pentose sugar moities break up and make a coloured complex

with resorcinol reagent. This coloured complex is extracted in

organic solvent (butylacetate and n-butanol; 85:15, v/v). Organic

phase was taken and abosrbance was measured at 580 nm.

62

FIG. - 3.07

GANGLIOSIDES

0 I 0 2 0 3 0.4. 0.5. 0.6.

AMOUNT OF GANGLIOSIDES (mg)

63

3.15.2 Reagents :


A stock standard was prepared by dissolving 100 mg N-

acetylneuraminic acid in double distilled water and making the

final volume to 100 ml. This gives 1.0 mg N-acetlneuraminic acid/

ml. Working standard was prepared by taking 1.0 ml of stock

standard and diluting it to 10 ml in a volumetric flask with c. jje

distilled water. This is 100 ug/ml of N-acetylneuraminic a ^ 1 .

il) Resoscinol Reagent:

This reagent was prepared by mixing 10 ml of 3.0 /o

resorcinol solution in double distilled water, 80 ml of concentraied

HCI, 0.25 ml of 0.1 N copper sulphate and double distilled water

upto 100 ml mark in a volumetric flask.

iii) Butyl acetate.

Iv) n-Butanol.

3.15.3 Procedure :

Recorcinol reagent (2 ml) was added to 2.0 ml of the upper

layer (aqueous) of lipid extract, and heated in a boiling water bath

for 30 minutes. After cooling to room temperature, 5.0 ml of

mixture of butyl acetate and n-butanol (85 : 15, v/v) was added to

64

each tube. The tubes were shaken thoroughly and kept for 15

minutes to separate the organic phase. About 3-4 ml of organic

phase was taken and absorbance was measured 580 nm against

a reagent blank. A standard curve with absorbance of different

concentrations of n-acetylneuraminic acid (5-30 ug) having 2 ml

final volume was prepared by treating in the same way as samples.

The absorbance of test samples were compared with those of

standard curve and gangliosides were quantitated as below.

C X V

V, X W,


Ganglioside (mg/g fresh brain weight) =

Where,

C = Concentration in gm in 2.0 ml extract

V = Total volume of the upper layer

V, = Volume taken for estimation

W, = Fresh weight of brain tissue in mg.

3.16 ESTIMATION OF ESTERFIED FATTY ACIDS :

Esterified fatty acids were estimated according to the method

of Sterm and Shapiro (1993) as follows

65

FIG. - 3.08

ESTERIFIED FATTY ACIDS

E c o H <

d g > H

Q

u P O

.118 -236. .35A un .590 708

AMOUNT OF ESTERFIED FATTY ACIDS (mg)

66

3.16.1 Reagents :

i) Standard Solution :

A standard solution of 4.0 g of trioleinin alcohol-ether (3.1)

was prepared by diluting 1.0 ml of 1.0 meq stock solution (59 mg

trlolein/5 ml alcohol etherX3:1) to a final volume of 10 ml with

alcohol-ether mixture.

ii) 2.0 M Hydroxylamine Hydrochloride (Solution I) :

13.9 gms of hydroxylamine hydrochloride was dissolved in

100 ml double distilled water.

iii) 3.5 N Sodium Hydroxide (Solution ii) :

14.Og of Sodium hydroxide (14.0 g) was dissolved in 100 ml

double distilled water.

iv) Hydroxylamine - Sodium Hydroxide :

This was made before use by mixing equal volumes of

solutions I and II.

v) 0.37 M Ferric Chloride In 0.1 N HCI :

The solution was prepared by dissolving 6.0 g FeClj in 100

ml 0.1 N HCI.

vi) 4.0 N Hydrochloric Acid Solution :

This was prepared by adding 1 part of analytical grade

concentrated HCI to 2 parts of double distilled water.

67

vii) Alcohol Ether (3:1) Mixture :

3 parts of 95% ethanol were added to 1 part of ethyl ether

(peroxide free).

vlll) Peroxide Free Ether:

This solution was obtained by adding a little hydroxylamine

hydrochloride to the stock ether bottle perior to distillation of

ether. The ether used in the procedure was freshly distilled.

3.16.2 Procedure :

Brain extracts of 0.6 ml were taken in 16 x 150 mm screw

capped culture tubes to dryness at room temperature. A 3.0 ml

aliquet of the alcohol ether (3:1) was pipetted to each tube and 1.0

ml of hydroxylamine hydroxide solution was added, the contents

were mixed and allowed to stand for 20 minunutes at room

temperature. 4 N HCI (0.6 ml) and 0.5 ml of ferric chloride solution

were added and mixed after each addition. A dark brown colour

developed and was measured at 520 nm against a reagent blank.

A calibration curve was drawn by using 0.1 to 0.6 ml of the

standard solution (4.0 g/ml) with the volume made up to 3.0 ml

with alcohol-ether mixture.

68


O.D. of unknown x nneq of standard Meq esterified fatly acid in 3.0 ml aliquot =

O.D. of standard

3.17 DETERMINATION OF THE RATE OF LIPID

PEROXIDATION :

The procedure of Utiey et al. (1967) was used for finding out

the rate of lipid peroxidation.

15.7.1 Reagents :

I) 0.15 M Potassium Chloride :

This solution was prepared by dissolving 2.237 g KCI in 200

ml double distilled water.

li) 10% Trichloroacetic add (TCA) :

10% TCA was prepared by dissolving 10 gm TCA in 100 mg

distilled water.

Ill) 0.67% Thiobarblturic Add (TBA) :

Thiobarbituric acid 0.67 g was dissolved in 25-50 ml double

distilled water by adding two pellets of NaOH. The pH of the

solution was adjusted to 7.2 with the help of 1 N HCI and volume

was made upto 100 ml with double distilled water.

69

3.17.2 Procedure :

Different parts of the brain were homogenized (10% v/v) in

chilled 0.15 M HCI. One ml of each homogenate was taken in a

25 ml conical flask and incubated at 37 ± 1 °C In a metabolic shaker

(120strokes/min, amplitudes 1 cm) for three hours, thereafter 1.0

ml of the same homogenate was pipetted in centrifuge tube and

protein was precipited by adding 1.0 ml of 10% TCA. After

incubation 1.0 ml of 10% TCA was added and the reaction mixture

was centrifuged at 3000 rpm for 10 minuutes. One ml of the clear

supernatant was mixed with 1.0 ml of 0.67% TBA and 1.0 ml

double distilled water, placed in a boiling water bath for 10

minutes cooled and the absorbance of the colour was read at 535

nm. The rate of lipid peroxidation was expressed as nanomoles

of melonaldehyde (MDA) formed/30 minutes using extinction

coefficient 1.56% x 10^ as described by Utely et al. (1967).


Lipid peroxidation was calculated using the formula ;

Nanomoles of MDE formed/30 min X C D x 30 x 1000 x 1000 x 1000 x 3 x 2

x= 1.56 X 100000 x 1000 X 1800.D. x 10

70

Where,

MDA = melonaldehyde

O.D. = Change of optical density at zero hour and three hour

incubation of the same samples.

3.17.4 Statistical Analysis :

The data were analysed using student's 't' test significant

difference between means of treated and control groups were

calculated and 'p' values were obtained.

71

RESULTS

Two group (controlled and experimental) each of 6 male

albino rats of the average weight 250 + 50 grams were separately

taken for each steroidal derivative. Intraperitonial injection (0.3

*mg of steroidal derivative dissolved in peanut oil/kg body weight)

were given daily for 15 days, on 16th day animals were sacrificed

for biochemical studies. The concentrations of total lipids,

phospholipids, esterified fatty acids, cholesterol, gangliosides

and rate of lipid peroxidation and cholesterol/phosphate ratio was

determined in major brain parts cerebrum, cerebellum, brain stem

and spinal cord.

72

Table-4( i )

EfTects of steroidal derivative on different regions of male albino Rat's CNS, injected 0.3 mg/kg for 15 days ip.

'^ramelff

Brain

Ports

Srou Structure - oclivity - corelolion

keto '^ramelff

Brain

Ports ocetay-5-er^e ocetoxy - nilro ocetoxy - keto

G A N G L I 0

s I

D E S

CBM

c 1.19i0.19 1.1940.19 1.19^0.19

G A N G L I 0

s I

D E S

CBM Ex US US

1.4140.14 1.4610.13 G A N G L I 0

s I

D E S

CBM

% IS.04 18.80 23.02

G A N G L I 0

s I

D E S

CBL

c 0.6S«0.S9 0.6S40.S9 0.6540.59

G A N G L I 0

s I

D E S

CBL u 0.98^0.89 l!294i0.13 1.041310.1 1

G A N G L I 0

s I

D E S

CBL

% S2.S2 100.77 61.56

1

G A N G L I 0

s I

D E S

BS

c 0.331.32 0.3340.32 0.3310.32

G A N G L I 0

s I

D E S

BS Ex • • •

0.6610.S9 • • •

0.5940.05 • • •

0.5210.49

G A N G L I 0

s I

D E S

BS

% 100.12 81.51 56.57

G A N G L I 0

s I

D E S

SPC

c 0.3040.29 0.3040.29 0.3010.29

G A N G L I 0

s I

D E S

SPC c» NS

0.^7*0.27 0.5fii0.03 0.1610.15

G A N G L I 0

s I

D E S

SPC

'h -9 .43 -2.35 -54 .90 i

(mg^g issue, mean ± S.D.) C = control. Ex = Experimental; % = Percentage

3 d-Acctoxy-cholest-5-cne (aceloxy-5-ene) 3 6 - Acetoxy-6-nitro-cholcst-5-cne (acetoxy-nitro) 3 6 - Acetox>-5a-choIestan-6-one (acetoxy-keto) Values •? < 0 05 , • •? < 0 01 ; •••P< 0 001 NS = Not Significant

4.1: TABLE-4(I)

Acetoderivative produces significant increase In the level

of gangliosides in brainstem (100.12%), and cerebellum (52.52%).

It produces an insignificant rise in spinal cord (9.43%) and decrease

• In cerebrum (15.04%) .

Aceto-nitro derivative produces significant increase in the

concentration of gangliosides in cerebellum (100.77%) and brain

stem (81.51 %), whereas insignificant rise (18.80%) is observed in

cerebrum and depletion in spinal cord (2.35%). Significant rise in

gangliosides level is also observed with the treatment of aceto-

nitro derivative in cerebellum (61.5%), brain stem (56.57%) and

cerebrum (23.02%), Whereas significant depletion of 54.9% is

observed only in spinal cord.

74

Tab le - ( i i )

EfTecls of steroidal derivative on different regions of male albino Rat's CNS. injected 0.3 mg/kg for 15 days ip.

Parameter

Brain

Ports

3rou Structure - activity - corelotiort |

Parameter

Brain

Ports ocetoocy-S-ene oceloxy - nitro ocetoxy - keto

T 0 T A L

L 1 P 1 D S

CBM

c 87 .15^8 .70 78 .15+8 .70 87 .15+8 .70

T 0 T A L

L 1 P 1 D S

CBM Ex 80.5f+^8.02 79 .? l+ .7 .72 147.00+14.52 T 0 T A L

L 1 P 1 D S

CBM

% - 7 . 9 7 - 8 . 2 7 3 68 .67

T 0 T A L

L 1 P 1 D S

CBL

C 49 .21+4 .70 49 .21+4 .70 49 .21+4 .70

T 0 T A L

L 1 P 1 D S

CBL .£x 73!99+7.28 1 0 6 ! l 4 + 1 0 . 5 1 5 5 ! 9 6 i l 4 . 3 9

T 0 T A L

L 1 P 1 D S

CBL

7i 50.36 115.68 216 .927

T 0 T A L

L 1 P 1 D S

BS

C 190.76+10.36 109 .67+10.3« 109 .76^1036

T 0 T A L

L 1 P 1 D S

BS .£K 30.73+3.06 70 .25^7 .0 84 .65+7 .90

T 0 T A L

L 1 P 1 D S

BS

7f - 3 2 . 5 9 - 3 5 . 9 9 - 2 2 . 8 8

T 0 T A L

L 1 P 1 D S

5PC

-C 170.12+15.34 170.12+15.34 170 .12+15.34

T 0 T A L

L 1 P 1 D S

5PC ,E'« 150 .^ f+15 .01 200.8S+18.01 1 8 8 . ? f + 1 6 . 9

T 0 T A L

L 1 P 1 D S

5PC

Jl, - 1 1 . 4 0 9 1 17.564 1 0 . 8 1

(mg/g issue, mean ± S.D.) C = control; Ex = Experimental; % - Percentage

3 B-Acctoxy-cholest-5-ene (acetoxy-5-ene) 3 B - Ac€toxy-6-nitro-cholest-5-cne (acetoxy-nitro) 3 B - Acttoxy-5a-cholestan-6-one (acetoxy-keto) Values : •? < 0.05 ; ••? < 0 01 ; •••P<0.001 NS = Not Significant

75

4.2: TABLE-4(1I)

Table-4 (II) shows the effect of three steroidal derivatives

(3-p-acetoxy cholest-5-ene; 3-p-acetoxy-6-nitro cholest -5-ene;

31i-acetoxy-5a-cholestan-6-one) on the total lipid concentration i;

rats brain. The acetoxy-keto derivative produces significant

increase of 68.67% in the cerebrum and 216.927% in the

cerebellum, while insignificant 10.81% increase in the spinal

cord. Significant decrease of 22.88%, in the total lipid contents is

observed in the brain stem.

The acetoxy-nitro derivative produces significant 115.68%

increase in the total lipid contents of cerebellum.

Insignificant increase (17.56%) occurs in the spinal cord

and decrease in cerebellum. Brain stem shows significant

35.99%decrease in the total lipids concentration.

Acetoxy derivative produces Insignificant decrease of 7.97%

and 11.41% in the concentration of total lipids In cerebrum and

spinal cord. A significant rise of 50.36% is observed In cerebellum

and insignificant decrease of 32.59% in brain stem.

"V - i f I :

76 <.-Vv'^..,-^-^r

Table-4(iii)

Effects of steroidal derivative on different regions of male albino Rafs CNS, injected 0.3 mg/kg for 15 days ip.

Urometer

Brain

Ports

jrou Structure - activity - corelolion

Urometer

Brain

Ports ocetoxy-S-ene occtoxy - nitro ocetoxy - keto

E 5 T E R 1 F 1 E 0

f A T T y A c 1 D S

CBM

(i ft.72^0.is ft. 7240.«> ft.7240.ftS E 5 T E R 1 F 1 E 0

f A T T y A c 1 D S

CBM Ex 14! l7*1.13» 7.2l40.7ft • ••

18.ft44l.79

E 5 T E R 1 F 1 E 0

f A T T y A c 1 D S

CBM

% 110.65 14.32 177.28

E 5 T E R 1 F 1 E 0

f A T T y A c 1 D S

CBL

c 9.oi3«o.ao 8.01340.80 8.01340.80

E 5 T E R 1 F 1 E 0

f A T T y A c 1 D S

CBL .u 13To724l. l3 19^8941.89 • ••

13.8341.3

E 5 T E R 1 F 1 E 0

f A T T y A c 1 D S

CBL

\ «3.134 148.22 72.59

E 5 T E R 1 F 1 E 0

f A T T y A c 1 D S

as c IS.48401.S IS.48401.5 15.4841.5

E 5 T E R 1 F 1 E 0

f A T T y A c 1 D S

as .El 9.4S4.82 I S . f i f l . S S 10.4141.09

E 5 T E R 1 F 1 E 0

f A T T y A c 1 D S

as % -38 .73 0.8 -32.8

E 5 T E R 1 F 1 E 0

f A T T y A c 1 D S

SPC

c 20.35«2.03 20.3S42.03 20.3542.03

E 5 T E R 1 F 1 E 0

f A T T y A c 1 D S

SPC ^ 10.3041.02 • ••

9.17S40.89 ft.9234^.572 I

E 5 T E R 1 F 1 E 0

f A T T y A c 1 D S

SPC

A - 49.43 1 -54.923 -ft5.98 1

(mg/g issue, mean ± S.D.) C = control; Ex = Experimental. •/© = Percentage

^ B-Acetoxy-cholest-5-ene (acetoxy-5-ene) ^ B - Aceioxy-6^iitro-choIest-5-ene (acetoxy-nitro) 3 0 - Acetoxy-5a-cholestan-6-one (acetoxy-ketok^v^-' Values *P < 0 05 ; • • ? ^ 0 01 . • • •P<0 001 £, r^ NS = Not Significant

WHiBPi

" . *

77 itUa! lU IIMIU t**^ C<

http://18.ft44l.79

http://20.3S42.03

4.3: TABLE-4(lin

Table-4 (III) shows the significant rise In the concentration

of esterlfied fatty acids In cerebrum (110.65%), and cerebellum

<63.134%) induced bythe administration of aceto-derivatlve. This

derivative produces an significant decrease in EFA in brain stem

(38.73%) and spinal cord (49.43%).

Aceto-nitro derivative produces significant rise in EFA

concentration in cerebellum and significant decrease in spinal

cord (54.923%) whereas insignificant rise is found in cerebrum

(14.32%) and brain stem (0.8%).

Aceto-keto derivative produces significant rise In the EFA

concentration in cerebrum (177.28%), and cerebellum (72.59%).

Significantly decreased level of 32.8% and 65.98% is observed in

brain stem and spinal cord.

78

Table-4(iv)

Lflccts of steroidal derivative on diflcrent regions of male albino Rat's CNS, injected 0.3 mg/kg for 15 days ip.

1 Broin

Ports

3rou Structure - octivity - cortlotion 1

^rometof

Broin

Ports ocetoqf-5-er« ^rometof

Broin

Ports ocetoqf-5-er« o c d o x y - nitro oonoKy • «r»w

C H 0 L E S T E R 0 L

CBM

c 3.SI7«0.349 3.S87«0.349 S.S87*0.349 C H 0 L E S T E R 0 L

CBM Ex 2.1«.>0.209 2.7S.^0.29< 3.oS42.9f


CBM

% -39.029 -22.91C - 1 4 . 7


CBL

c 1.7S2>0.1«9 1.752^0.1*9 1.7S2*0.1«9


CBL Ex l . /> t0 .1«3 3.70*0.297 3.10^0.301


CBL

7t -1.096 111.223 78.74


as C 3.«340.32S 3.«340.32S 3.«3*0.32S


as Ex l.S5^0.109 7.17*0.(91 2.8340.28


as

• / i -«011S 97.93 -22.097


5PC

C 5.32«0.S21 S.3240.S21 S.32*0.S21


5PC Ex s.oeoi.492 «.tv*.S90 7.035^0.71


5PC

. % -4.S11 28.711 32.24

(mg/g issue, mean ± SD.) C = control; Ex = Experimental; % = Percentage

3 B-Acetoxy-cholcst-5-ene (acetaxy-5-ene) 3 B - Acelox) -6-nitro-choIest-5-eBe (acetoxy-nitro) 3 fl - Acetox>-5a-choIestan-6-one(acetoxy-keto) Values : •? < 0 05 ; ••P < 0.01 ; •••P<0 001 NS = Not Sipificant

79

4.4: TABLE-4(IV)

Aceto-derivative produces a significant decrease in the

concentration of cholesterol in brain stem. While in the rest of the

other three major parts i.e. cerebrum, cerebellum and spinal cord.

It produces a insignificant decrease of 39.029%, 1.096% and

4.5% In the cholesterol level.

Aceto-nltro derivative produces significant elevation in the

cholesterol concentration in cerebellum (111.223%),spinal cord

(28.718%) and brain stem (97.93%). It produces an insignificant

deprivation In cerebrum (22.916%).

Aceto-keto derivative produces significant Increase in the

cholesterol level In cerebellum (76.74%), and In spinal cord

(32.24%). Insignificant decrease In the cholesterol level is

observed in brain stem (22.097%) and cerebrum (14.7%).

bO

Effects of steroidal derivative on diflercnl regions of male albino Rat's CNS. injected 0.3 mg/kg for 15 days ip.

Urometer

Brain

Ports

jrooi Structure - octivity - corelolion

Urometer

Brain

Ports ocet0Ky-5-«n« ocetoxy - nitro cxetoxy - keto

P H 0 S P H A T E

CBM

c 10.C04l.01 10.C04l.01 10.C04l.81

P H 0 S P H A T E

CBM Ex §.t2*0.»3 • •

C.87540.C4 •s

9.C2540.95 P H 0 S P H A T E

CBM

f, -20.S7 -35.142 -9.198

P H 0 S P H A T E

CBL .C S . 1 0 0 . 4 1 5.1440.41 S.1440.41

P H 0 S P H A T E

CBL Is MS

S. 23^0.921 14.C2S4l.27 17.37541.53

P H 0 S P H A T E

CBL

% 2.14 184.53 238.035

P H 0 S P H A T E

BS

c «.344«0.S« «.34440.58 8.34440.58

P H 0 S P H A T E

BS Xx 7.54^0.73 0.21540.02 • • •

15.2541.41

P H 0 S P H A T E

BS

Jh 22.399 -9C.S87 141.10

P H 0 S P H A T E

SPG

J : 11.0541.013 11.0541.01 11.0541.01

P H 0 S P H A T E

SPG &< • .S34.823 13.12541.30 M S — —

10.71741.C2

P H 0 S P H A T E

SPG

J:& -22.770 -18.82 -3 .123

(mg/g issue, mean ± S.D.) C » control; Ex = Experimental; % = PercenUge

3 B-Acefoxy-cholcst-5-ene (acetoxy-5-cne) 3 0 - Acetoxy-6-«tro-cholcsl-5-«nc (acctoxy-nitro) 3 1 - Acetoxy-5a-cl»o!estan-6-one (acctoxy-kcto) Vklues : •? < 0.0$ , • •? < 0 01 . •••P<0 001 NS = Not Significant

81

http://10.C04l.01

http://10.C04l.01

http://10.C04l.81

http://14.C2S4l.27

4.5: TABLE- 4 (V^

Following the administration of acetoxy-derivativephosphate

level Is insignificantly decreased by 20.57% and 22.77% in the

cerebrum and spinal cord. A significant enhanced level of

phosphate is observed in cerebellum (50.36%) and insignificantly

decreased value In brain stem (32.59%).

Aceto-nitro derivative produces significant increase in the

phosphate concentration in cerebellum (184.53%) and significant

decrease in brain stem (96.587), cerebrum (35.14%) and spinal

cord (18.82%).

Aceto-keto derivative produces significant rise In the

concentration of phosphate In cerebellum (238.04%), brain stem

(14.10%) and insignificant decrease in the cerebrum (09.198%)

and spinal cord (03.123%).

82

Table-4 (Vl)

Effects of steroidal derivative on different regions of male albino Rat's CNS, injected 0.3 mg/kg for 15 days ip.

Urometer

Brain

Port*

jfXXi Structure - octivity - oortlolion |

Urometer

Brain

Port* octlOKy>S-«ne occtoxy > nitra ooHoxy - kcto

L 1 P 1 D

P E R 0 X 1 0 A T 1 0 N

CBM

Q i.7»2*9.2t 3.78240.2S 3.78240.2*

L 1 P 1 D

P E R 0 X 1 0 A T 1 0 N

CBM Ex MS

4.96S40.44 •S

4.4640.38 L 1 P 1 D

P E R 0 X 1 0 A T 1 0 N

CBM

% 17.t7 31.36 18.77

L 1 P 1 D

P E R 0 X 1 0 A T 1 0 N

CBL

C 3.67*0.31 3.6740.31 3.674^0.31

L 1 P 1 D

P E R 0 X 1 0 A T 1 0 N

CBL .In US

4.i|17«0.39 MS

3.3S940.31 3.1440.23

L 1 P 1 D

P E R 0 X 1 0 A T 1 0 N

CBL

7i -14 .4t -8 .54 20.25

L 1 P 1 D

P E R 0 X 1 0 A T 1 0 N

BS

C 1.3240.11 1.3240.11 1.3240.11

L 1 P 1 D

P E R 0 X 1 0 A T 1 0 N

BS £K 1.8940.13 US

1.35340.15 • •

1.79540.09

L 1 P 1 D

P E R 0 X 1 0 A T 1 0 N

BS

Jh 43.1S1 2.42 35.88

L 1 P 1 D

P E R 0 X 1 0 A T 1 0 N SPC

.C 1.67»40.13 1.679«0.13 1.67940.13

L 1 P 1 D

P E R 0 X 1 0 A T 1 0 N SPC ^ 1.9140.12

NS 1.64840.11

US ' • 1.3440.10

L 1 P 1 D

P E R 0 X 1 0 A T 1 0 N SPC

Jk 13.75 -1 .84 -20.19

(mg/g issue, mean ^ S D ) C = control; Ex = Experimental. % = Percentage

3 B-Acetoxy-cholcst-5-cne (acctoxy-5-cnc) 3 6 - Acetoxy-6-«tro-cholest-5-ene (acetoxy-nitro) 3 6 - Acetoxy-5a-cl»oIeslan-6-onc (acctoxy-keto) VUucs : •? < 0 05 ; **? < 0 0| ; •••P«^0 001 NS = Not Significant

83

4.6: TABLE-4 (Vn

Table-4 (VI) shows the data obtained for the rate of lipids

peroxidation following the administration of three steroidal

derivative.

Aceto-derivative produces only significant enhancement of

43.15% In brain stem, while in cerebrum (17.97%) and spinal cord

(13.75%) insignificant rise is observed. Only insignificant depletion

of 14.48% reported in cerebellum of rats brain in the study.

Aceto-nitro derivative shows only significant rise of 32.36%

in cerebrum and insignificant rise of 2.42% in brain stem. In

cerebellum and spinal cord this derivative produces insignificant

depletion of 8.54% and 1.84%.

Aceto-keto derivative produces significant rise in the rate of

lipid peroxidation in brain stem (35.88%) and cerebellum (20.25%)

while insignificant rise is observed in cerebrum and depletion in

spinal cord (20.19%).

84

TABLE - 4 V I I )

Effects of steroidal derivative on different regions of male albino Rat's CNS, injected 0.3 mg/kg for 15 days ip.

Porometer

Broin

Ports

&rou] 1 Structure -octivity- coreiotiort

Porometer

Broin

Ports ocetOKy-5-ene ocetoxy - nitro ocetoxy - keto

C H 0 L E 5 T E R 0

H 0 S P H A T E

RATIO

CBM

Q 0.338 0 .338 0.338 C

H 0 L E 5 T E R 0

H 0 S P H A T E

RATIO

CBM Ex 0.259 0 .402 0.318

C H 0 L E 5 T E R 0

H 0 S P H A T E

RATIO

CBM

% - 1 . 8 9 8 - 0 . 6 S 2 - 1 . 5 9 8

C H 0 L E 5 T E R 0

H 0 S P H A T E

RATIO

CBL

C 0.341 0 .345 0.341

C H 0 L E 5 T E R 0

H 0 S P H A T E

RATIO

CBL In 0.330 0 .253 0.1784

C H 0 L E 5 T E R 0

H 0 S P H A T E

RATIO

CBL

7i - 0 . 5 1 2 0 .662 0.322

C H 0 L E 5 T E R 0

H 0 S P H A T E

RATIO

BS

c - 0 . 5 7 2 0 .572 0.572

C H 0 L E 5 T E R 0

H 0 S P H A T E

RATIO

BS E?( - 0 . 1 5 0 3.34 0.185

C H 0 L E 5 T E R 0

H 0 S P H A T E

RATIO

BS

•/, - 3 . 0 4 1 - 1 . 0 1 3 - 0 . 1 5 6

C H 0 L E 5 T E R 0

H 0 S P H A T E

RATIO 5PC

C 2.385 2 .385 2.365

C H 0 L E 5 T E R 0

H 0 S P H A T E

RATIO 5PC Ex 0.596 0 .522 0.656

C H 0 L E 5 T E R 0

H 0 S P H A T E

RATIO 5PC

•/• - 0 . 1 9 6 - 1 . 2 6 2 - 1 0 . 3 2 3

(mg/g issue, mean ± S.D.) C = control. Ex = Experimental ; % = PcrccnUge

3 6-Acetoxy-cholest-5-cne (acctox>-5-cnc) 3 B - Acetox>-6 nitro-cholest-S-cnc (acetoxy-nitro) 3 fi - Acctoxy-5a-cholcstan-6-one (acctoxy-lccto) Values •? < 0 05 , • •? < 0 01 ; •••P<0.001 NS = Not Significant

4.7: TABLE-4(V1I)

Table-4 (VII) is a list of cholesterol/phosphate ratio with

respect to the steroidal derivatives. Aceto-derivatlve shows a

negative values of the ratio in alt four brain parts I.e. cerebrum

(1.898%), cerebellum (0.612%), brain stem (3.041%) and spinal

cord (0.198%).

Aceto-nitro derivative shows the negative value of c/p ratio

In cerebrum (0.652%), brain stem (1.013%) and spinal cord

(1.262%), the only positive value is observed in cerebellum.

Aceto-keto derivative shows negative values of c/p ratio in

cerebrum (1.598%), brain stem (0.156%) and spinal cord (10.323%)

while positive value is calculated in cerebellum (0.322%).

86

5. DISCUSSION :

5.1 GANGLIOSIDES :

Gangliosides are the sialic acid containing

glycosphingolipids, which are highly enriched in the CNS of

vertebrate, including man (Wiegandt 1967; Ledeen 1978;

Svennerholm 1980; Rahman,1983). Sialic acid is the generic

name for N-acylneuraminic acid, and the acyl group of sialic acid

in the human brain is always the acetyl form (Suzuki 1981). N-

acetyl neuraminic acid is commonly abbreviated as Neu Nac.

Gangliosides are localized in two fraction of the brain. Major

gangliosides of the brain are GM1, GM2, GM3, GD1a, GD1b and

GT and other minor are GD3, GD2 and slalylagal actosylceramide

are also present.

Synaptosomal or myelin membranes in the form of liposomes

or microbial cell wall and membrane fragments containing repeatinc

arrays of gangliosides or cross-reactive Glycoproteins may be

able to activate B-cells in this way (Pestronk, 1991).

Gangliosides undergo characteristic changes in content

and composition during development (Rosner, 1977, Engel et al.

87

F i g : - 5.1

Gangliosides

• Control

3 - p - acetoxy-cholest-5-ene

3-^-acetoxy -8 -n i t ro -cho ls t -5 - ene

3 -p -ace toxy -5 - t -cholestan-

6 - one

P Values: - p< 0 05 . . p<001 . . . p<0 001 N5 = Not Significant

Cerebrum Cerebellum Brainstem Spinal cord

Regional alteration in the levels of gangliosides in different regions of rat CNS following the administration of 3 - p - acetoxy-cholest - 5 -ene. 3 - p - acetoxy - 6-nitro - cholest - 5-ene, 3 -p -acetoxy - 5-oC - cholestan - 6 - one, -3 mg/kg body weight ip for 15 days.

88

1979, Yusuf and Dickerson 1980) and have, therefore, been

suggested to be instructed molecules for brain maturation. In

particular they appear to be functionally involved in the control of

axonal (Willinger and Schachner, 1980) and dendritic (Irwin and

IrwinJ 979) out grov h synaptogenesis (Engel et al.,1979) and the

establishment of cell contact (Yogeeswaran and Hakomori,1975).

More over in adult nervous system, the Individual gangliosides

have been suggested to play a role as membrane bound receptors

or co-receptors for toxins, drugs viruses hormones transmitters

etc. (Svennerholm 1980).

Gangliosides are generally synthesized in neuronal perikarya

and are transported to the nerve ending alongwith macromolecules

(Rahmann and Rosner, 1973; Ledeen et al. 1976; Landa et al.,

1979). Local synthesis within the axons and nerve endings

(Ledeen et al. 1976) or even at the plasma membrane level (Preti

et al.,1980) can not be excluded. Gangliosides are involved in

nerve impulse conduction since they act as receptor sites for

neurotoxins (North et al.,1961; Rahmann et al. 1982). Irwin and

Samson (1971) reported that certain types of behavioral

stimulations (stress, sensory stimulation, learning, exercise) seem

89

to be accompanied by alterations in Gangliosides metabolism.

Mental retardation and neurological dysfunction are major signs

in nearly all the lipid storage diseases, apparently because of

abnormal deposition of the different glycosphingolipids in the

CNS due to defective disorders in degradatiye enzyme pathways,

resulting in retardation of development, paralysis dementia and

blindness (Lehninger^1984). Biosynthesis of brain gangliosides

occurs by sequential addition of monosacharides or N-

acetylneuraminic acid to the carbohydrate chain, starting from

ceramide. Degradation of brain gangliosides proceeds by

sequential removal of monosacharides and Neu Nac (N-

acetylneuraminic acid) by glycosides and neuraminidases (Ledeen

et al., 1973, Ledeen and Mcllanby 1977, Suzuki, 1981).

Gangliosides are responsible for the humeral immune

response (Willson H.J. and Kennedy,1993) evidences accumulated

throughout the 1980s indicate the humoral immunity to gangliosides

is frequently observed in patients with chronic demyelinating

peripheral neuropathies (Lotov, 1990) for example Guillain Barre

Syndrome a (Willison and Kennedy, 1993).

The Gangliosides mixture of bovine brain gangliosides

90

(GM1, GDla, GD1b, GT1b) has been widely used in trials of the

treatment of peripheral neuropathies (Hallett et al.,1987).

Gangliosides have been used for treatment of various

degenerative, toxic and metabolic neuropathies in Europe although

their efficiency remains controversial (Bradley, 1990). Patients

with multifocal motor neuropathy have high titer serum antibodies

against the Gangliosides GM1 (Pestronk 1991). In lewis rats,

myelin induced experimental allergic neuritis or animal model of

human acute Gullian Barre Syndrome can be depressed and

delayed by adding Gangliosides mixture (GM1, GDla. GDIb,

GTIb) to the immunization compound. However, gangliosides

may enhance the induction of adjuvant arthritis sinceeyternally

applied gangliosides produce antibodies (Weietholter, H. et al.

1992).

Studies on gangliosides had been done earlier in our

laboratory. Intramuscular administration of 100 pg estrogen daily

for 30 days to female rabbit was followed by decrease in

gangliosides in cerebellum and spinal cord while an elevated

level was observed in cerebral cortex and brain stem (Islam et al.,

1984).

91

There is no previous report on the classified study on

structure activity co-relation on the level of gangliosides in the

central nervous system while using three derivatives of cholesterol

(1) 3-B-acetoxy-cholest-5-ene (2) 3-B-acetoxy-6-nitro-cholest-5-

ene, (3) 3(i-acetoxy-5a-cholestan-6-one. In the present study

changes were found as following in the cerebrum, cerebellum,

brain stem and spinal cord.

When the activity of acetoxy, acetoxy-nitro and acetoxy-

keto group was measured in the different brain parts of male

albino rat following the administration of 0.3 mg/kg body wt.

Injections of the steroids for 15 days, gradual rise in the

Gangliosides contents from the effect of acetoxy to acetoxy-keto

group is reported in the cerebrum. Only a slight increased is

estimated with the administration of acetoxy-keto derivative in

cerebrum.

In the cerebellum all the three derivatives induce significant

rise in the gangliosides content. Maximum rise is reported in this

study with acetoxy-nitro derivative in cerebellum (100.78%).

Estimation of gangliosides in the brain stem shows significant

rise in its contents. Whereas in spinal cord all the three steroid

induce depletion in gangliosidescontents, among these three

92

steroids only acetoxy-keto derivative induces significant depletion

in spinal cord.

Effect of acetoxy derivative is maximum in brain stem, whereas a

significant rise in the Gangliosides content (100.12%) is reported,in

the cerebellum a significant change but less than from spinal cord

is reported. A Insignificant change is observed in the spinal cord.

Changes In the gangliosides contents by acetoxy-nitro compound

is reported significantly high in the cerebellum comparable to the

brain stem, while in cerebrum insignificant rise Is reported. In the

case of spinal cord minimum -2.3% depletion is reported.

Acetoxy-keto derivative is the most active in the cerebellum

where a significant maximum enhancement in the gangliosides

content is reported (61.56%) in this study.

5.2 TOTAL LIPIDS :

The Major structural and functional constituents of biological

membrane are lipids, they also serve as essential component of

several crucial enzyme systems,as fuel molecules, and as highly

concentrated energy store (Lehninger, 1984). In mammalian central

nervous system, lipids comprise over half of the total dry weight

93

Fig : - 5.2

Total Lipids

Cereb rum Cerebellum Brain stem Spinal cord

Regional alteration in the level of total lipids •n different regions of rat CN5 following the administration of 3 -p -acetoxy-cholest -5 -ene , 3 ^-acetoxy-6-nitro - cholest-5 ene. 3-P-acetoxy - 5-iC-cholestan-6- one. -3 mg/Kg lx)dy, weight ip for 15 days.

94

(Suzuki, 1981). Almost all the lipids in CNS are found in membranes

of cells. Different types of membranes accumulate different types

of lipids. These brain lipids are constantly being synthesized

replacing other molecules In the membranes (Horrocks et al.

1975). Lipids in the brain modulate the structure fluiding and of

function of the biomembrane (Bourre et al. 1990).

The brain lipids constitute components of ion channels and On

neurotransmitter receptors, the other hand. They are also major

constituents of myelin (Schinizu, 1965). Lipids In the brain makes

upto 65% of the dry weight of the white matter and 34-40% of gray

matter (Brante, 1949). Distinct regional differences occur in lipid

contents, turnover in cell types and various path ways and centers

of the brain (Ordy and Kaack, 1975).

Changes in the level of total lipids in the brain may occur by

various physiological, psychological and chemical stressors and

ageing (Horrocks et al.,1981; Story et al.,1976). Changes in lipid

fractions are induced by the steroids estrogen and progesterone

in discrete brain parts as reported by Islam et al. (1980).

In the present study an attempt has been made to study the

elevation and deprivation in the level of total lipids in the various

95

parts of brain in albino rats . As far as the survey of literature is

concerned, no previous report on the structure activity relation

ship of various derivatives of cholesterol on the brain lipid profile

js available follov ing the administration of three derivatives of

cholesterol (I) Aceto- derivative (11) Aceto-nitro derivative and (iii)

Aceto-Keto derivative, Injected ip 0.3 mg/kg body wt for 15 days

in male albino rats.

Total lipid contents were found insignificantly depleted with

the administration of Acetoxy and acetoxy-nitro derivatives of

total lipid contents in the CNS induced by acetoxy keto derivative,

which is 68.67% significant rise.

In this study changes in the total lipids induced by the three

derivatives of cholesterol having (1) Acetoxy group (2) Acetoxy-

Nltrogroup and (3) Acetoxy-Keto. Effect of these derivatives were

measured In the four parts (1) Cerebrum (2) Cerebellum (3) brain

stem and (4) Spinal Cord. In the cerebrum Total lipids contents

were found insignificantly depleted with the administration of

acetoxy and acetoxy-nitro derivatives comparable to the observed

values of total lipids content In CNS induced by acetoxy-keto

derivative which gives 68.67% significant rise.

96

In the cerebellum gradual significant rise was observed

following the administration of acetoxy, acetoxy-nitro and acetoxy-

keto derivative respectively. 50.36% maximum significant rise

was estimated with the Acetoxy derivative. 115.68% significant

rise was observed with the Acetoxy-nitro derivative and 216.93%

significant rise was observed with the Acetoxy-keto derivative.

In brain stem administration of Acetoxy and Acetoxy-nitro

derivatives of cholesterol results in the significantly and steady

decrease in the total lipid contents. -32.59% and -35.99%, with the

acetoxy-keto derivative a decrease of -22.88% was estimated.

In the spinal cord effect of these three cholesterol derivatives do

not altersthe total lipid contents upto a significant level. However

acetoxy derivative gives the decreased value of total lipid contents

in spinal cord -11.40% whereas acetoxy-nitro derivative and

acetoxy-keto derivative give a insignificant rise in the total lipid

contents 17.56% and 10.81%, respectively

Activity of 3-B-acetoxy-cholest-5-ene in four brain parts

gives the decreased value of total lipid contents (brain stem -

32.59% > Spinal Cord -11.49% > Cerebrum -7.97%). In cerebellum

it gives a significant high value of total lipids contents (50.36%).

97

Activity of 3-(i-acetoxy-6-nitro-choIest-5-ene in the four brain

parts gives the trand that:

Cerebellum (115.68%) > Spinal Cord (17.66%) while decreased

values of total lipid contents is in the order, brain stem (-35.99%)

> Cerebrum (-8.27%).

Activity of 3-B-acetoxy-6a-cholestan-6-one gives the rising

trend oftotal lipid contents as Cerebellum (216.92%) > Cerebrum

(68.67%) > Spinal Cord(10.81%). Whereas In the brain stem it

gives a -22.88% reduction in the total lipids contents.

Changes In the lipid contents and lipid composition must be

due to changes in rates of anaboiism, catabolism or these

processes are controlled by the activities of appropriate enzymes

Hazzard and co-workers (1969) have suggested that a reduced

lipoprotein lipase activity may be a factor contributing to the

increased plasma lipids.

Depletion of unsaturaed lipids is associated with alteration

in membrane fluidity (Bruch and Thayer, 1983) and the changes in

the activity of membrane bound enzymes and receptors (Bobik

et al. 1983). Polly unsaturated fatty acids are by themselves,

capable of causing damages to the cell membranes, tissues and

98

may contribute to the postraumatic decline in the blood flow as

well as reactive unflammatory responses (Panter and Faden,

1992). Interestingly lipids of various tissues are known to be in a

dynamic steady state and there is continuous replacesment of

existing molecules by newones (While et al. 1978). Present study

reveals the effect of three different derivatives of cholesterol on

the lipid contents of various parts of CNS. is different on each part.

Earlier it was concluded by the study of Islam et al. (1984) that

estrogen affects the lipid contens differentially In the various

parts of the brain.

6.3 ESTERIFIED FATTY ACIDS :

Sahani and Patel (1974)] have reported a significantly

depleted levels of the serum esterified fatty acids (EFA) in women

taking 0.5 mg megesterol acetate daily at the interval of 3 and 6

months. There is no previous report on the classified study on

structure activity co-relation on the level of esterified fatty acid in

central nervous system while using the different derivatives of

cholesterol having different functional groups I.e. Acetoxy, Acetoxy-

nitro. Acetoxy-keto at certain positions on cholesterol molecule.

However Islam et al. (1980) analysed the estrogen induced

99

Fig: - 5.3

Esteritied Fatty Acids


Regional alteration in the level of Esteritied fatty acids in different regions of rat CN5 following the administration of 3-p-acetoxy - cholest-5ene, 3 - p - acetoxy - 6- nitro -cholest- 5 - e n e , 3 - 3 - acetoxy-5 - ^ - cholestan- 6-one,-3 mg/ kg body weight for 15 days.

100

changes in the level of lipids at microlevel In discrete brain areas

of Rabbit Brain, when the primovlar (Ethinyl Estradiol) oral

contraceptive was injected i.m. for 60 days,a significant decrease

in EFA in hypothalamus, hippocampus and gyrus cinguli was

observed. When estrogen was administrated I.m. for 30 days in

Rabbit, a significant depletion in the EFA was observed in

hypothalamus, hippocampus cerebral cortex, and cerebellum and

a significant increase in the level of EFA was observed in midline

nuclei of thalamus and at factory bulb. In another study Islam et

al. (1984) explored the significant depletion in EFA le al in

cerebral cortex and cerebellum following the intramuscular

administration of 100 pg estrogen daily for 30 days in female

rabbit. The concentration of EFA also depends upon the contents

of the fatty acids present in tryglyceride and phospholipid (Stern

and Shapiro, 1953). A significant increase in concentration of

phospholipid is also a contributory factor for the increment in the

contents of EFA.

Following the administration of acetoxy, acetoxy-nitro and

acetoxy-keto for 15 days in male albino rats changes in EFA

contents in cerebrum was found significantly high, with acetoxy

101

derivative 110.65% and with acetoxy-keto derivative it w/as

177.2%, whereas with the acetoxy-nitro derivative a insignificant

rise of 14.33% was detected in cerebrum.

Acetoxy derivative proves itself a less active than acetoxy-

Xeto derivative while both of these are less active than acetoxy-

nitro derivative in cerebellum. Alteration In the EFA estimated was

significantly high 63.14%, 72.59% and 148.22% respectively with

acetoxy, acetoxy nitro and acetoxy-keto derivatives. Activity of

these three derivatives on brain stem shows significant depletion

with acetoxy and acetoxy-ketoderivative whereas acetoxy-nitro

derivative gives almost negligible change (.85%) in brain stem.

In the spinal cord, significant depletion in the EFA is reported with

all the three derivatives of cholesterol. Activity Is in the order,

acetoxy-keto derivative is most active than acetoxy-nitro derivative

> acetoxy derivative, (-65.98% > -54.92% > -49.43% respectively).

While analyzing effect of acetoxy derivative separately on brain

parts, data reveals the maximum significant enhancement in the

EFA contents in cerebrum (110.65%) and in cerebellum significant

Increase but lesser than cerebrum (63.13%). Acetoxy derivative

Induces significant depletion in the EFA in spinal cord and brain

stem (-49.43%) and (-38.72%).

102

Induction of the changes by the acetoxy-nitro derivative in

the brain parts shows increase in the EFA in cerebrum, cerebellum,

brain stem, and depletion in the spinal cord. Significant increase

in the EFA is maximum in cerebellum 148.22% while insignificant

increase is in the cerebrum and brain stem. Acetoxy-nitro derivative

adversely effects to the spinal cord where a significant depletion

In EFA is reported (-54.92%).

Changes by acetoxy-keto derivative in the brain parts shows

maximum significant increase in EFA in cerebrum is 177.28%,

72.59% significant increase is reported in cerebellum, while in the

brain stem and spinal cord a significant maximum depletion is

reported.

Maximum significant increase in the EFA contents is found

in the cerebrum induced by the acetoxy derivative and maximum

significant depletion is reported in the spinal cord. Maximum

significant increase in the EFA contents is observed in the

cerebellum induced by the acetoxy-nitro derivative and maximum

significant depletion in the spinal cord.

Effect of acetoxy-keto derivative Is maximum comparable to

the effect of rest two derivatives in the EFA contents In cerebrum

103

whereas and in the spinal cord a nfiaximum depletion in EFA is

reported in this study.

5.4 CHOLESTEROL :

Cholesterol is a major and only sterol, present In the

significant amount in the central nervous system [(Ansell and

Hawthorne, 1964)]. It Is also an important component of biological

membranes. Membranes are generally thought to consist of

phospholipid bilayers Into which membrane proteins are embedded,

yet cholesterol molecules are present in most animal structures.

Due to its amphlpathic nature bearing an OH-group and a

hydrocarbon skeleton with rigid rings and a branched chain of

eight carbons, cholesterol is perfectly suited to mesh with lipid

bilayers [(Brenner, 1990)].

Cholesterol accounts for about 10% of dry weight of the

brain in contrast to less than 1% found in most other organs. The

constancy of the amount of cholesterol In the brain suggests that

the sterol is metabolically stable [(Waelsch, et al, 1940)].

Unesterified cholesterol has been suggested as a lipid

characteristic of myelin sheath, as it occurs in white matter in a

104

Fig: - 5.4

CHOLESTEROL

Cerebrum Cerebellum Brain stem Spinal cord

Regional alterations in the levels of cholestrol following the administration of 3 -p-acetoxy-cholest - 5 - ene. 3 - p - acetoxy - 6 - intro - cholest -5-ene. 3 - p - acetoxy-5-1- cholestan-6- one/3mg/ Kg body weight \P for 15 days in the different regions of rat CN5.

105

higher concentration than in gray matter [(Johnson, 1949; Brante,

1949)]. About 25% of cholesterol is present in myelin lipid by

weight [(Soto et al. 1966)] and approximately 70% of total brain

cholesterol is present in myelin [(Laatsch et al. 1962)]. Cholesterol

is thought to act as a convenor in the absorption of fats. [Bloor

(1943)] reported a parallelism between cholesterol content of

blood and the fatty acids. Due to the abundance of cholesterol in

nervous tissues and its variation in mental diseases, it may

function as an insulating medium for myelin sheaths. Sterols are

thought to have a role in maintaining the balance between the cell

permeability and the membrane equilibrium of living cells. Brain

microsomes are the sites of cholesterol biosynthesis [(Paoletti,

1971)]. Cholesterol is synthesized from acetate and precursors

via mevalonic acid. Both the biosynthesis and the deposition of

cholesterol in the CNS is most rapid. Biosynthesis of cholesterol

in brain is most rapid during active myelination, but adult brain

retains the capacity to sythesize cholesterol when precursors,

such as acetatic or mevalonate are available.

Fatal or neonatal brain prior to myelination contains relatively

little cholesterol. [Kritenevesky and Holmes (1962)] found varying

106

amounts of the sterol in the newborn rat brain. In rat brain, total

levels of sterol ester increase from birth to 40 days [(Eto and

Suzuki, 1972)]. A decline is also noticed in human and guinea pig

brain cholesterol (62.3%) and denosterol (31.1 %), The major

sterol and small to trace amounts of other sterols were also

detected [(Ramsey and Nicholas, 1972)] relative to total

phospholipid and glycolipid, in which changes in whole brain and

myelin are compared. Earlier studies in our lab on the alteration

in cholesterol level following the intramuscular administration of

ethinylestradiol and 0.5 mg norgestrel on the regional lipid levels

of cholesterol was found decreased in hypothalamus,

hippocampus, amygdaloid nucleus midline nuclei of thalamus and

gyrus cinguli (Islam et al. 1980). He observed a depleted level of

cholesterol in brain stem and spinal cord but it was elevated in

cerebellum, following the ip administration of estrogen in female

rabbit.

In the present study three derivatives of cholesterol 3-B-

acetoxy-cholest-5-ene, 3B-acetoxy-5a-cholestan-6-one were

injected in the 3 groups of male albino rats (.3 mg/kg body wt i.p.

for 15 days) changes in the cholesterol level estimated in the

cerebrum, cerebellum brain stem and spinal cord.

107

However, no systematic work on structure-activity co-relation

is done so far on the various homologues of cholesterol on the

brain. Earlier Islam et al., 1980 has studied the effect of estrogen

and primovlar (Having slight difference in the structure). Cholesterol

level In the estrogen treated rabbits in the Islam's study show

regional heterogeneity in exhibiting a depletion In the brain stem

and spinal cord and increase in the cholesterol level in the

cerebellum following 30 days administration.

Primovlartreated rabbits showsthe depletion of cholesterol

level in hippocampus, amygdaloid nucleus, midline nuclei of the

thalamus and gyrus Cinguli in his study. Islam also noted that

alteration of cholesterol level was only significant after 90 days

administration of primovlar treatment. In the primovlar treated

rabbits, the highest variation of the cholesterol contents in the

control and midline nuclei of the thalamus, while the content of

cholesterol remain unchanged in hypothalamus, hippocampus

and mygloid nucleus.

In the present study effect of the three derivatives were

evaluated In the CNS of albino rats activity of these steroids in the

cerebrum is as following :

108

Among the acetoxy, acetoxy-nitro and acetoxy-keto

derivative, acetoxy derivative and acetoxy-nitro derivative give

the -39.02% and -22.91% significant depletion in the cerebrum

whereas acetoxy-keto derivative gives -14.7% Insignificant

depletion in the cerebrum.

In the cerebellum acetoxy-nitro derivative gives the maximum

significant increase of 111.223% while acetoxy-keto derivative

gives 76.74% significant increase. Whereas acetoxy derivative

gives an insignificant depletion in the cholesterol level.

In the brain stem activity of acetoxy-nitro group gives

maximum significant rise In the cholesterol level (97.93%). Acetoxy

derivative gives the significant depletion In the cholesterol level

in brain stem. On the other hand Acetoxy-keto derivative gives an

insignificant depletion in the cholesterol level in brain stem.

In the spinal cord, effect of acetoxy-keto derivative gives the

maximum significant rise In the cholesterol level (32.24%).

Acetoxy-nitro derivative gives the significant (28.71%) rise in the

cholesterol level. While an insignificant depletion is reported with

the acetoxy derivative of cholesterol.

Analysis of the effect of acetoxy-derivative on all the four

109

brain parts reveals its maximum effect on brain stem, whereas in

the cerebrum, effect is insignificant in the cerebellum and spinal

cord. In all the four brain parts acetoxy derivative induces depletion

in the cholesterol level.

Analysis of the effect of Acetoxy-nitro derivative on the

different parts of the brain reveals Its maximum effect on cerebellum

than in brain stem, a significant rise but lower than cerebellum and

brain stem is reported in spinal cord, this compound whereas

gives a significant depletion in the cerebrum.

The activities of acetoxy-keto derivative shows the significant

rise in the cholesterol level in the cerebellum and spinal cord

(76.74% and 32.24%) Whereas an insignificant depletion in the

cholesterol level in the cerebrum and brain stem reported in this

study.

A comparison in the activity of all the three derivatives

reveals that acetoxy derivative exert its maximum effect on brain

stem and induces depletion in the cholesterol level. Acetoxy-nitro

group induces maximum significant rise in the cerebellum and

also maximum depletion In the cerebrum. Acetoxy-keto derivative

induces maximum significant rise in the cerebellum and

insignificant maximum depletion in brain stem.

110

5.5 PHOSPHOLIPIDS :

Phospholipids are the major class of membrane lipids

(Holkin, 1969, white 1973). These forms the backbone of neural

membranes and also provide the membrane with suitable

permeability (Farooqui and Horrocks, 1985, Procellati, 1983).

Glycero phospholipids are the major component of brain lipids

phospholipids constitute approximately one fourth (20-25%) of

the dry weight of brain (Holkin, 1969, White 1973). Total amounts

of phospholipids are higher in white matter than in gray matter,

and the reported amounts range from 3 1-4.6 g/100 g fresh for

gray matter and 6.2-9.3 g/100 g for white matter (Suzuki-1981).

The distribution of phospholipids in biogenic membranes is

regulated not only by the activity of enzymes invol\'jd in their

metabolism but also their transport and incorporation processes

Into membranes. Enzymes of neural membranes liberate free

fatty acids, acyl-COA, diglycerides and lysophospholipids. Under

normal conditions, such metabolites may be involved in neural

membrane functions, such as ion transport, membrane fusion,

neurotransmitter release, receptor binding and calcium

homeostasis (Farooqui and Horrocks, 1985). Myelin sheath

111

F i g : - 5,5

PHOSPHOLIPID


Regional alterations in the levels of phospholipids in different regions of rat CNS following the administration ©f 3 - p - a c e t o x y - cholest-5-ene, 3 - p - ocetoxy- 6 - nitro - cholest - 5 - ene, 3-p-acetoxy-5-C-cholestcn - 5 - ene,- -3 mg/kg body weight ip for 15 days.

112

contains 40-45% phospholipids (Fumagelli and Paoletti. 1963).

The neural nnembranes are highly interactive and dynamic and

therefore the interaction of ligand with receptor markedly affects

neural membrane phospholipid metabolism (Loffelholz, 1989).

This in turn regulates the microenvironment for the activities of the •

membrane bound enzymes and ion channels (Farooqui and

Horrocks^1985). Porcellati and his Co-workers (1983) reported

that different phospholipids turnover at different rates with

respect to their structure and localization in various cells and

membranes. Newly synthesized phospholipds are transported to

membrane structure by phospholipid exchange and transfer

proteins (Brammer, 1983; Demel et al.,1984) which are found in

cytosol. The distribution of phospholipids in a biologic membrane

is thus regulated not only by the activities of enzymes involved in

their metabolism but also by the transport and incorporation

processes into the membrane. Many investigators such as

Porcellati et al. (1970 a.b). Ansell and Spanner (1971) and

Procellti et al. (1971) have concentrated on the regulatory

mechanisms of phospholipid biosynthesis in the brain and of the

alternative metabolism pathways involved should be of interest

113

because it is highly probable that the metabolism of phospholipids

in brain is somewhat different from that in other tissues whereas

Rossiter (1966) had proposed that metabolic pathways of

phospholipids in brain are similar to those in systemic organs.

Presently a phospholipid drug glyceryl-phosphoryl-0-serine

is available in the market, which posses potent behavioral (Favit

et al.^1993) and neurochemical effects (Deutsch,1971; Breese et

al.,1978). This drug is being used as a modulator for pituitary

ACTH and hypothalamic corticotropin releasing hormone

(Chiarenza, A. et al.,1995) by the physicians.

Glyceryl-phosphoryl-0-serine (GPS) is a novel drug which

acts as a precursor of membrane phospholipids participating a

signal transduction (Casabona et al.,1993). GPS has been shown

to modulate protein kinase C activity in the central nervous system

of the aging rat either directly or indirectly (Casabona et al.,1993).

As per available literature there is no any data available

based on the studies of cholesterol derivatives on central nervous

system, so far as studies on primovler (oral contraceptive) steroids

on central nervous systems by Islam et al. (1981) shows that a

significant decrease in the level of phospholipid, in hypothalamus,

hippocampus, midline nuclei of thalamus and gyrus singuli has

114

been consistently observed after 60 and 90 days i.m.

administration.

In another study Islam et al. (1981) established that steroid

contraceptive estrogen decreases the contents of phospholipids

in amygdaloid nucleus, hippocampus and midline nucleus of

thalamus.

In the present study an attempt has been made to evaluate

the level of phospholipids in CNS, following the ip administration

of cholesterol derivatives i.e. 3-B-acetoxy-cholest-5-ene, 3-Q>-

acetoxy-6-nitro-cholest-5-ene, 3B-acetoxy-5a-cholestan-6-one ,

in albino rats.

Present study reveals that maximum effect occurs with the

acetoxy-nitro derivative in cerebrum where it induces a significant

depletion of-35.14%.

Estimation of phospholipids in the cerebellum of the treated

rats with all the three steroids show its enhanced level. Maximum

significant rise is 238.03% with the acetoxy-keto derivative and

with the acetoxy-nitro derivative a significant rise of 184.53% is

observed.

In the brain stem, acetoxy-keto and acetoxy derivative

115

increases (141.01% and 22.39%) the phospholipid level but the

treatment of acetoxy-nitro derivative results In a significant

depletion in the brain stem.

Spinal cord shows the, insignificant depletion of

phospholipids In the treated rat with all the three steroidal

derivatives.

Activity trend of 3-fi-acetoxy-cholest-5-ene in the different

brain parts shows the only significant and maximum rise in

cerebellum. While in rest of three parts (brain stem, cerebrum and

spinal cord) a significant depletion (96.58%, 35.14%, 22.77%) in

the phospholipid level is reported in this study.

On analysing the activity of acetoxy-keto derivative on

different parts of the treated rat brain it become clear that a sharp

and significant enhancement is occurring in cerebellum and brain

stem (238.035% and 141.10% respectively). Whereas an

Insignificant depletion in the cerebrum and spinal cord is reported.

On the comparative study of all the three steroidal

compounds, facts emerges that acetoxy compound induces

maximum insignificant rise in the phospholipid level in brain stem

and maximum insignificant depletion in the spinal cord.

116

Acetoxy-nitro compound induces maximum significant rise

in the cerebellum and maximum significant depletion in the brain

stem. Acetoxy-keto compound induces maximum significant rise

in the cerebellum and maximum insignificant depletion in the

cerebrum.

It is an interesting thought, that overall rates of phospholipid

turnover for the vk hole brain might be the sum of very different

rates of turnover characterizing the turn over rates for phospholipids

in specific structure, for example in growing cell membranes or in

changing mitochondrial population.

5.6 RATE OF LIPID PEROXIDATION :

Brain contains large amounts of lipids that are rich in

polyunsaturated fatty acids. The unsaturated bonds of membrane

lipids can readily react with free radicals and undergo peroxidation

(Jesberger and Richardson, 1991). Lipid peroxidation has been

identified as a basic deterloative reaction in the cellular mechanism

(Tappelj 1973). It is an autocatalytic free radical process.

Biomembrane and subcellular organelles are the major sites of

lipid peroxidation Tappel,(1970). Quantitative studies of enzymatic

117

a>

Of

o E o c o z

Fig: - 5.6

Lipid Peroxidation

m',ht -.v^j •MS.

Cerebrum Cerebellum Brain stem Spinal cord

Regional alterations in the levels of rate of lipid peroxide formation in different regions of rat CNS following the administration of 3 -B-ocitoxy - cholest - 5 - ene, 3-p - ocetoxy - 6-.nitro-cholest - 5 ene, 3 - p - ocetoxy- 5 - ^ - choleston • 6-one, -3 mg kg/body weight ip for 15 days.

118

Inactivation by lipid peroxidation have shown that sulfhydryl

enzymes are most susceptible to inactivation (Numan et al. 1990).

Each lipid peroxide is also a free radical and once inititated

the process of peroxidation can become autocatlytic as each lipid

peroxide products. These lipid peroxides readily decompose to

<jberate highly reactive carboxyl fragments such as

malondialdehyde (Tappe. 1973). Malondialdehyde (MDA). X-3-

carbon dialdehyde is one of the final products of free radical chain

reaction v\/hich takes place during lipid peroxidation (Nomura et al.

1989). The H^Oj and other reacgive 0^ species if not scavenged

efficiently are knovk n to give rise to potentially toxic Intermdeiates,

namely hydroxy radical ^OH) and singlet (0^°). These oxidants in

the presence of metal Ions, result in the formation of lipid

peroxidation (Lai et al.,1979a).

Gutteridge (1982) has shown that malondialdehyde is the

major species responsible for thiobarbituric acid reactive products

(TBA-RS). Whether the substances undergoing free radical induced

damage are polyunsaturated fatty acids, amino acids,

carbohydrates or nycleic acids. Therefore, measurement of

thiobarbituric acid substance Is one of evaluating the extent of

119

tissue damage due to free radicals. However, the best-induced

effect of a free radical attack is that causing lipid peroxidation, i.e.

oxidation of a methylene bridge of unsaturated fatty acids, resulting

in the formation of lipid peroxides and hydroperoxides, finally

leading to fragmentation of lipids. As biomembranes are rich in

unsaturated fatty acis, such reactions may lead to the disintegration

of membrane structure and finally to Irreversible cell damge

(Rehncrona et al.^1980).

Inititation of lipid peroxidation in a membrane or free fatty

acid is due to the attack of any species that has sufficient reactivity

to abstract a hydrogen atom. Since a hydrogen atom has only one

electron, this leaves behind an unpaired electron on the carbon

at • . The carbon radical in a polyunsaturated fatty acid tends to

be stabilized by a molecular rearrangement to produce a conjugated

diene, which rapidly reacts with 0^ to give hydroperoxy radical.

Hydroperoxy radicals abstract hydrogen atoms from other lipid

molecules, and so contineues the chain reaction of lipid

peroxidation. The hydroperoxy radical combines with the hydrogen

atom that it abstracts to give a lipid hydroperoxide (Barber and

Bernheim, 1967).

LH = Fatty acids; LOOM = Lipid hydroperoxides,

120

L" = Lipid alkyl radical; LOO" = Lipid peroxy

radicals (Aust and Svingen. 1982).

Initiation

LH + Oj > Free radicals, L° (1)

LOOM > Free radicals, L00° (2)

Propagation

LOO" + L00° > LOO^

Termination

L00° + L00° > LOO + O2

LOO° + L° >LOOL

L° +L° >LL

Oxygen free radical induced oxidative damage has been

implicated in the aetiology of a number of diseases, including

atherosclerosis, inflamation, cancer, and ischemia reperfusion

(Henning and Chow, 1988, Mc Cord 1987; Korumbo et a!.,1985).

Toxicity of uraemia is due at least in part to oxygen free radicia

mediated lipid peroxidation and other tissue damaging effects

(Hassan et aly1995).

121

There is considerable evidence suggesting the involvement

of free radicals and lipid peroxidation in atherogensesis (Stringer

et al.,1989. Duthie and Whale,1990; Esterbaner et a|„1990; Gey,

1990; Oliver^1990).

In the present study, follov\/ing the administration of acetoxy.

acetoxy-nitro, acetoxy-keto, derivatives of cholesterol rate of lipid

peroxidation was estimated. Acetoxy-nitro compound gives a

significant enhancement in the rate of lipid peroxidation while

acetoxy and acetoxy-keto derivatives induces more or less similar

insignificant enhancement in cerebrum of rats brain.

Effect of these three derivatives on cerebellum shows

Insignificant depletion in the rate of lipid peroxidation in the

acetoxy and acetoxy-nitro treated rat brain whereas acetoxy-keto

derivative results in an insignificant rise in the rate of lipid

peroxidation in cerebellum.

The acetoxy derivative treated rat brain stem shows 43.15%

significant rise in the rate of lipid peroxidation. Acetoxy-keto

derivative also induces significant rise (35.88%) in the value of

rate of lipid peroxidation. Acetoxy-nitro derivative is least active

in brain stem as it exerts only 2.42% insignificant rise in the lipid

peroxides.

122

spinal cord shows insignificant depletion in the lipid

peroxidation In the acetoxy-keto and acetoxy-nitro treated rat

brain, while an insignificant rise occurs in the lipid peroxides with

the acetoxy derivative in spinal cord

Acetoxy derivative insignificantly deplets the lipid peroxides

in cerebellum. Maximum significant rise in the lipid peroxides is

reported In brain stem, whereas in the cerebrum and spinal cord

only insifnificant enhancement in level of peroxides formation

occurs. Effect of Acetoxy-nitro derivative on the different parts of

rat brain shows significant rise in the cerebrum and insignificant

Increase in the brain stem while in the cerebellum and spinal cord

an insignificnat depeletion in the lipid peroxidation is reported In

this study.

Acetoxy-keto derivative induces significant increase in the

lipid peroxidation level in the brain stem (35.88%) whereas in

cerebellum and cerebrum insignificant rise in its values is reported

In this study. Only spinal cord shows insignificant depletion in the

rate of lipid peroxidation, induced by acetoxy-keto derivative.

The acetoxy treated derivative shows maximum activity in

the brain stem, where 43.15% significant rise is estimated.

123

Maximum significant rise 31.36% is reported with acetoxy-nitro

derivative in cerebrum. Acetoxy-keto treated rats show maximum

effect in the brain stem, it induces 35.88% significant rise in the

lipid peroxidation.

Increased production of oxygen free radicals and/or unpaired

antioxidant defense mechansim result in an oxidant-antioxidant

imbalance with consequent oxidative cellular damage (Sies

1986a).

Decreased lipid peroxidation reduces endothelial damage

and modification of low density lipoprotein (Nafeeza, M. et al.

1996).

The increase In the lipid peroxidation process will provide

abundance of aldehyde products of lipid hydroperoxide break

down which Is responsible for the modification of low density

lipoprotein to the oxidized state (Esterbaner et al. 1993). The

oxidized low density lipoprotein is then endocytosed by the

macrophages through Its scavenger receptors. The arterial smooth

muscle cells is also seen to internalized oxidized low density

lipoprotein thus massive amounts of cholestrol known as foam

cells accumulates in the macrophages and smooth muscle cells

(Schffner et al.1980, GerrityJ981; Agnel et al. 1984).

124

5.7 CHLOESTROL/PHOSPHOLIPID (C/P) RATIO :

Cholesterol and phospholipid ratio Is generally considered

to be an index of atherogenecity. Cholesterol appears to be

incorporated into the cells of lesion, esterified with in the cells and

transformed into amorphous droplets of cholesteryl esters (Weller,

et al. 1968). Cholesterol is a well known major constituents of

atherosclerotic lesions (Frantz and Moore, 1969). The

accumulation of cholesterol in aorta parallels remarkably well

with the serum cholesterol (Nichols etal. 1971). Furhtermore, the

presence of lecithin-cholesterol transcacylase in the arterial wall

provies evidence for the esterification of cholesterol in aorta,

since only free cholesterol rather than esterified cholesterol is

exchanged with the blood during atherosclerosis.

The studies on estrogen and cholesterol induced coronary

atherosclerosis in cockerels have shown experimental support for

the hypothesis that elevation of the c/p ratio rather than changes

in plasma lipid levels Per-se may be a significant factor in

coronary atherogenesis (Katz and Pick, 1961). Data from

experiments using rabbits and rats give additional support to this

possibility. Thus, in rabbits fed an atherogenic diet including

cholesterol, estrogens failed to prevent th e c/p ratio elevation or

125

to inhibit coronary atherosclerosis (Stanpler, et al. 1956). In the

rats, on the other hand, Moskowitz, et al (1961) reported that

although estradiol benzoate administration resulted in elevated

serum cholesterol and lipid phophorous levels. The c/p ratio v as

depressed toward normal and v\/as associated with inhibitition or

coronary atherosclerosis.

After intramuscular administration of 100 ug estrogen daily

for 30 days to each female rabbit, it was observed that cholesterol/

phospholipid ratio increased in cerebellum and decrease in brain

stem and spinal cord (Islam et al. 1984).

It was noted during this study that c/p ratio was almost

decreased in cerebrum, with the administration of 3B-acetoxy-

cholest-5-ene, 3B-acetoxy-6-nitro-cholest-5-ene. 30.-acetoxy-

5a-cholestan-6- one for 15 days i.p. In male albino rats.

In the cerebellum c/p ratio was decreased with the effect of

acetoxy derivative of cholesterol. Maximum increase In the ratio

is reported v rtih acetoxy-nitro derivative than acetoxy-keto

derivative.

Steroidal derivatives, acetoxy and acetoxy-keto derivative

deplete the c/p ratio In brain stem while acetoxy-nitro derivative

enhances the c/p ratio in brain stem.

126

Acetoxy-keto derivative induces, maximum depletion in c/p

ratio in spinal cord, than comparable to the acetoxy-nitro and

acetoxy derivative.

Acetoxy derivative induces maxium depletion in c/p ratio in

brain stem and minimum in cerebellum. Interestingly acetoxy

derivative reduces the c/p ratio in all brain parts.

Acetoxy-nitro derivative whereas deplets the c/p ratio

maximum in pinal cord, and minimum in cerebrum. Maximum-

increase in the c/p ratio occurs in cerebellum only.

Acetoxy-keto derivative depletes c/p ratio maximum in spinal

cord and minimum depletion occur in brain stem, whereas the

cerebellum only shows the enhanced c/p ratio with the effect of

acetoxy-keto derivative.

127

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