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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
3.14.3 Procedure 61-62
3.14.4 Calculation 62
3.15 Estimation of Gangliosides 62
3.15.1 Principle 62
3.15.2 Reagents 64
3.15.3 Procedure 64-65
3.15.4 Calculation 65
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
3.17.4 Calculation 71
(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
Group 2: 6 months, wt. = 250 ± 50 grams
Group 3: 6 months, wt. = 250 ± 50 grams
Group 4: 6 months, wt. = 250 ± 50 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.
3.13.4 Calculation :
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 :
i) Standard solution :
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.
3.14.4 Calculation :
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 :
i) Standard solution :
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,
3.15.4 Calculation :
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
3.16.3 Calculation :
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).
3.17.3 Calculation :
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<
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
C H 0 L E S T E R 0 L
CBM
% -39.029 -22.91C - 1 4 . 7
C H 0 L E S T E R 0 L
CBL
c 1.7S2>0.1«9 1.752^0.1*9 1.7S2*0.1«9
C H 0 L E S T E R 0 L
CBL Ex l . /> t0 .1«3 3.70*0.297 3.10^0.301
C H 0 L E S T E R 0 L
CBL
7t -1.096 111.223 78.74
C H 0 L E S T E R 0 L
as C 3.«340.32S 3.«340.32S 3.«3*0.32S
C H 0 L E S T E R 0 L
as Ex l.S5^0.109 7.17*0.(91 2.8340.28
C H 0 L E S T E R 0 L
as
• / i -«011S 97.93 -22.097
C H 0 L E S T E R 0 L
5PC
C 5.32«0.S21 S.3240.S21 S.32*0.S21
C H 0 L E S T E R 0 L
5PC Ex s.oeoi.492 «.tv*.S90 7.035^0.71
C H 0 L E S T E R 0 L
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
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
Cerebrum Cerebellum Brainstem Spinal cord
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
Cerebrum Cerebellum Brainstem Spinal cord
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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