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������ ����� � ��������� ����� � ��������� ����� � ��������� ����� � ��� HEPATOPROTECTIVE ACTIVITY OF
METHANOLIC EXTRACT OF NIGELLA SATIVA IN
WISTER ALBINO RATS
By
ESSAM AMMAR OMER SALIH
B.Sc. of science (2001)
University of Khartoum
Supervisor
Dr. Afaf Izzeldin Abuelgasim
A thesis Submitted to The University of Khartoum In Partial Fulfillment for
the Requirement of the Degree of Master in Biochemistry
Department of Biochemistry
Faculty of Veterinary Medicine
University of Khartoum
August 2006
i
To the soul of my brother Alla Eldeen
To my parents, from whomI learned so much…
To my brother, sisters, relatives, friends and every one
Who helped me at any time…
With all love…
Essam..
ii
ACKNOWLEDGEMENTS
All thanks and praises be to Allah, the lord of the mankind and all existing creatures.
So the prayers and peace be up on the Mercy prophet; Mohammed.
I wish to express my deepest gratitude, sincere appreciation and my indebtedness to
my supervisor Dr. Afaf Izz Eldin Abu Elgasim for her constructive, valuable and inspiring
guidance, sound advice, patience, suggestions and encouragement during the period of the
study.
I am specially great full to Dr. Haseeba Ahmed Saad for her advisement and oriented
proposal at the beginning of this work.
Also I am grateful to the Staff Members of Biochemistry Department, who made up
our mind to accept more information in this science and give us a guide for the future study.
My gratitude and thanks are extended to the family of the Phytochemistry
Department at Medicinal and Aromatic Plants Research Institute (MAPRI), Khartoum and
technical staff at the Laboratory and Research Unit at Khartoum hospital for their
considerably assistance.
My deeply gratitude be to my Uncle Dr. Adil Omer Salih, my friend Ahmed Abdel-
Raheem and Mrs. Nuha Hassan Sharief for their help and support during the study. My
appreciation be to Ustaz Alwaseela Mukhtar and Adil Ameen for their considerable
assistance in the statistical analysis of data. My indebtedness be to Mrs. Aisha Elmahi, the
secretary of the Department of Biochemistry for her considerable encouragement.
Last but not the least, my appreciation and heartily thanks extended due to my
relative, friends, colleagues and every one who helped me in different ways during the study
period.
iii
List of
Contents
iv
LIST OF CONTENTS
Page DEDICATION ………………………………………………………………... i ACKNOWLEDGEMENTS …………………………………………………... ii LIST OF CONTENTS ………………………………………………………. iii LIST OF TABLES …………………………………………………………… v LIST OF FIGURES …………………………………………………………… vi ABSTRACT ………………………………………………………………...... vii ARABIC ABSTRACT ………………………………………………………. ix INTRODUCTION …………………………………………………………… 1
CHAPTER ONE: LITERATURE REVIEW ……………………………… 3
1.1 Nigella sativa constituents ………………………………………… 3
1.2 Antioxidant effects …………………………………………………. 7
1.3 Uses of Nigella sativa ………………………………………………. 10
1.4 Haemopoietic effects of Nigella sativa ………………………….. 11
1.5 Hepatoprotection of Nigella sativa …………………………… 12 CHAPTER TWO: MATERIALS AND METHODS ………………………. 14
2.1 Materials and Experimental Design ………………………….. 14
2.1.1 Animals ……………………………………………….. 14
2.1.2 Plant …………………………………………………… 14
2.1.3 Experimental design …………………………………... 14
2.1.4 Parameters …………………………………………….. 15
2.2 Methods ………………………………………………………. 16
2.2.1 Plant extraction ………………………………………... 16
2.2.2 Biochemical methods …………………………………... 16
2.2.2.1 Alkaine phosphatase (ALP) ……………………. 17
2.2.2.2 Alanine amino transferase (ALT), Glutamic
transsminase (GPT), L-aspartate, 2-oxogluta-
mate 19
2.2.2.3 Aspartate amino transferase (AST), Glutamate
oxaloacetate transaminase (GOT), L-aspartate 19
2.2.2.4 Total Bilirubin…………………………………. 19
2.2.3 Hematological methods ……………………………….. 20
2.2.4 Histopathology ………………………………………... 23
2.2.5 Statistical analysis ……………………………………. 23
CHAPTER THREE: RESULTS ………………………………… 25
3.1 Clinical signs …………………………………………………. 25
v
3.2 Body weights …………………………………………………. 25
3.2.1 Organs to body weight ratio …………………………. 25
3.3 Hematological findings ………………………………………. 25
Page
3.3.1 WBC and RBC ………………………………………… 27
3.3.2 Hemoglobin …………………………………………… 27
3.3.3 PCV …………………………………………………… 27
3.3.4 MCV ………………………………………………….. 27
3.3.5 MCH and MCHC …………………………………….. 27
3.4 Changes in serum constituents ………………………………. 29
3.4.1 Total bilirubin ………………………………………… 29
3.4.2 ALT …………………………………………………… 29
3.4.3 AST …………………………………………………… 30
3.4.4 ALP …………………………………………………… 30
3.5 Histopathological findings …………………………………… 30
CHAPTER FOUR: DISCUSSION ……………………………… 37
CONCLUSION …………………………………………………… 41
REFERENCES …………………………………………………... 42
vi
List of Tables
vii
LIST OF TABLES
Table Page
1 Body weights of albino rats given methanolic extract of
Nigella sativa…………………………………… 26
2 Relative Organs weight ratio of albino rats given
methanolic extract of Nigella sativa……………………… 26
3 Effect of Nigella sativa methanolic extract on
hematological values of Wister albino rats intoxicated
with CCl4………………………………………………. 28
4 Effect of Nigella sativa methanolic extract on some
biochemical parameters of Wister albino rats
intoxicated with CCl4………………………... 32
viii
List of
Figures
ix
LIST OF FIGURES
Figure Page
A Flowered Nigella sativa L. plant 5
B Nigella sativa L. seeds 5
C Chemical structures of some major constituents of
Nigella sativa seeds…………………………………… 6
D Hitachi 902 automated analyser 18
E Sysmex automated hematology analyzer KX-21 22
F Section of liver of rat received dimethylsulfoxide, group
B, (control). H & E x (100 x 0.96). 33
G Section of liver of rat received CCl4 in paraffin, group
C, (control). H & E x (250 x 0.96).……………… 34
H Section of liver of rat received CCl4 + 250mg/kg body
weight methanolic extract of Nigella sativa, group D. H
& E x (250 x 0.96) 35
I Section of livers of rats received CCl4 + 500mg/kg body
weight methanolic extract of Nigella sativa, group E. H
& E x (500 x 0.96) 36
x
ABSTRACT
The present study was carried out to investigate the hepato-protective
activity of the methanolic extract of Nigella sativa on albino Wister rats.
Twenty five rats were divided into 5 groups, 5 each; A, B, C were
saved as control groups, received paraffin, dimethylsulfoxide, CCl4 in
paraffin respectively; while group D and E received CCl4 in paraffin and
methnolic extract of Nigella sativa at dose of 250mg/kg body weight and
500mg/kg body weight respectively.
There were an increase in the body weights of control groups (A, B)
at a rate of 2.0%. However the body weights in group C, D and E were
reduced by 10.3, 9.3 and 10.3% respectively. The relative organ weights
ratio showed no significant differences among all groups. There were no
significantly differences among the groups in WBS, RBC, PCV, MCV,
MCH and MCHC values, but Hb recorded reduction on day 10 in group D.
The results revealed there was no effect on total bilirubin
concentration between the groups except in group C where total bilirubin
values raised from 0.2 to 0.7 on day 10, compared to group E which raised
xi
from 0.1 to 0.4. There were significant increased (P>0.05) in ALT, AST and
ALP activities on day 5 and 10 in group C, D and E compared to control
groups A and B, but the groups (D and E) which were given both CCl4 and
N. sativa methanolic extract showed significant inhibition (P>0.05) of
increased plasma levels (ALT and ALP) compared to the group which given
only CCl4. However significant reduction (P>0.05) in AST activity was
observed on day 10 in group E compared to the control group C.
The livers of control groups were normal, but rats which received
CCl4 showed central hepatic fatty vaculation and congestion, but in the
group of rats received 250 and 500 mg/kg body weight of methanolic
extract, the changes were to a lesser extent.
xii
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�� '�� ����� � *���� #3��=*�� �� ! ���>� �&���� 0� �� ?�'. � �� ?�'! ���>� ?� 0� ������ ���� �*�� �� ������� ����� �"������ ���
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�&���� �� �+!�� �����*.
��(/� <�'� #�� �� #�� ����� #��������� 3���� ��& C� �&���� �&���� �'(� 2 =�� 6���-DB��3������ �� # 0.2 ��� 0.7�&���� �'�;���� ��� ه �� ����� ��!+� &� �500���/%��'(��� )� � �� # ��� # #���� 0.1 ��� 0.4.
xiii
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� ��"�� &� , * #�&������* ������# �G"�� -�% ��� ه& ��# �� ��� 4��� ���&� �� ?�'! AB '- ��@6� #���� %��'(��� )� � ��(P>0.05) 4�B���� �37��� ��� �
��3'GALT �ALP ��� �&���� 4 �'�;����� 4��� �;� ���&� �#������ * . <�'� ?�'! AB '-(P>0.05) ��3'- �+' �� AST 6��� �� ������+!�� ���
�� �&��� 2ه�"�� �&�� 4 �'�; .
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1
Introduction
2
INTRDUCTION
Interest in medicinal plants has increased in recent years. The
Medicinal plants constitute the base of health care system in many
societies. Among these plants, black seed is a plant used widely in
traditional medicine by many Asian, Middle Eastern and Far Eastern
countries for treatment of headache, coughs, asthma, hypertension,
diabetes, inflammation, bronchitis, eczema, fever, dizziness and
influenza, also the seeds or its oil are used as a carminative, diuretic,
lactagogue, and vermifuge, beside been used in food as a spice and a
condiment (Lautenbacher, 1997).
Traditionally the actual importance of Nigella sativa to the
Muslims came from the holy saying of the prophet Mohammed
“prayers and peace be upon him”: in the black seed is the medicine for
every disease except death (Atta-ur-Rahman et al., 1985b). In the
bible, Nigella sativa is also identified as ‘curative black cumin’, and is
also described as the Melanthion of Hippocrates and Discroides and as
the Gith of Pliny (Worthen et al., 1998).
3
Many studies were carried out on back seeds or its oil to insure
its’ treatment and prevention of diseases, but few were done with
regard to liver damage especially that liver diseases became serious
problems. The aim of this study is to find out whether Nigella sativa
seeds have hepatoprotective effect in Wister albino rats treated with
carbon tetrachloride.
4
Literature Review
5
CHAPTER ONE
LITERATURE REVIEW
Nigella sativa Linn. (Family Ranunculaceae), commonly known
as black seed or black cumin, is an annual plant that has been tradi-
tionally used in the Indian subcontinent (Nadkarni, 1976), Arabian
countries (Sayed, 1980) and Europe (Lautenbacher, 1997) for culinary
and medicinal purposes. The black, angular seeds, in Arbia known as
‘Habba Sauda’, ‘Habbet el Baraka’, ‘Kamun-aswad’ and ‘Shunez’
(Houghton et al., 1995), (Fig. A and B).
1.1 Nigella sativa constituents:
Lauteubucher (1997) found that Nigella sativa seeds contains 36
- 38% fixed oils, proteins, alkaloids, saponins and 0.4 - 2.5% essential
oils. Houghton et al. (1995) detected that the fixed oils are composed
mainly of unsaturated fatty acids, including the unusual arachidonic
and eicosadienoic acids. Many components were characterized by
Burits and Bucar (2000), the major one is thymo-quinone (Fig. C.1).
Four alkaloids have been isolated as constituents of the seeds.
Two namely, nigellicine (Fig. C.2) (Atta-ur-Rahman et al., 1985b) and
nigellidine (Fig. C.3) (Atta-ur-Rahman et al., 1995) having an
indazole nucleus. Other two alkaloids are nigellimine (Fig. C.4)
6
(Atta-ur-Rahman et al., 1992) and its N-oxide isoquinolines (Fig.
C.5) (Atta-ur-Rahman et al., 1985a). Swamy and Benny (2001)
recently isolated a monodesmosidic triterpene Saponin, α-hederin
(Fig. C.6) from N. sativa seeds.
7
Fig. (A): Flowered Nigella sativa L. plant.
Fig. (B): Nigella sativa L. seeds.
8
O
O
CH3
CH3
CH3
CH3
OH
N
N+
COO- 1 2
CH3
O-
N
N+
CH3
H3CO
N
CH3
H3CO
3 4
N+
CH3
O-
H3CO
H3CO
5
ROCH3
CH3 CH3COOH
CH3CH3
CH3
CH2OH 6
Fig. (C): chemical structures of some major constituents of Nigella
sativa seeds. 1 ≡ thymoquinone, 2 ≡ nigellicine, 3 ≡nigellidine, 4 ≡ nigellimine , 5 ≡ Isoquinolines, 6 ≡ α-hederin. R in Fig. C (6) ≡αL-Rhman-αL-Ara.
9
1.2 Antioxidant effects:
Essential oils of black seeds, N. sativa, were tested for
antioxidant activity. Burits and Bucar (2000) used a rapid evaluation
for antioxidants by Thin Layer Chromatography (TLC) screening
methods. They found that thymoquinone and the components
carvacrol, t-anethole and 4-terpineol demonstrated respectable radical
scavenging property. These four constituents and the essential oil
possessed variable antioxidant activity when tested by the
diphenylpicrylhydracyl assay for non specific hydrogen atom or
electron donating activity. They concluded that these constituents
showed radical scavenging agent in the assay for non-enzymatic lipid
peroxidation in liposomes and deoxyribose dehydration assay.
Turkdogan et al. (2001) investigated the role of antioxidants
such as vitamin C and E; selenium and N. sativa on the prevention of
carbon tetrachloride (CCl4) induced liver fibrosis in rabbits. They
stated that super oxide dismutase (SOD) values were significantly
lower at 12th week. While glutathione peroxide (GSH) in vitamin C
treated group were significantly different from the control at 6th and
12th week of experiment. They pointed out that N. sativa might be
successful in the prevention of liver fibrosis in rabbits as indicated by
histopathological findings.
10
Teschke et al. (1984) have studied the effect of the administra-
tion of the ethanol on carbon tetrachloride (CCl4) and hepatotoxicity in
rats intragastric or intraperitoneal. They found that three hours after
acute CCl4 intoxication there was striking increase in CCl4
concentration in animals treated simultaneously with ethanol intra-
gastrically compared to those receiving ethanol intraperitoneally.
Serum activities of glutamate oxaloacetate transaminase, glutamate
pyruvate transaminase and glutamate dehydrogenase were found to be
considerably higher in animals treated with the combination of CCl4
and ethanol when compared to these receiving CCl4 alone.
Nagi et al. (1999) suggested that the in vivo protective action of
thymoquinone against CCl4 induced hepatotoxicity may be mediated
through the combined antioxidant properties of thymoquinone and its
metabolite dihydrothymoquinone (DHTQ). They stated that a single
oral dose of thymoquinone resulted in significant protection against
the hepatotoxic effect of CCl4 and it appeared that thymoquinone was
reduction to dihydrothymoquinone. They also stated that
thymoquinone and (DHTQ) inhibited the in vitro non enzymatic lipid
peroxidation in liver homogenate, and that (DHTQ) was more potent
in comparison with thymoquinone and butylated hydroxytoluene
(BHT).
11
Al-Gamdi (2003) has investigated the effect of the aqueous
suspension of N. sativa on carbon tetrachloride induced liver damage.
In this study, the aqueous suspension of the seeds was given orally at
dose levels of 250 mg/kg body weight and 500 mg/kg body weight
for five days. CCl4 (250 micro l /kg intraperitoeally /day in olive oil)
was given to the experimental group on days 4 and 5, while the
control group was only treated with the vehicles. Biochemical changes
were not evident following administration of N. sativa alone. Serum
levels of aspartic transaminase (AST), and L-alanine aminotransferase
(ALT) were slightly decreased while Lactate dehydrogenase (LDH)
was significantly increased in animals treated with CCL4 when
compared to the control group. LDH was restored to normal but ALT
and AST levels were increased in animals pretreated with N. sativa. It
was concluded that N. sativa seeds appeared to be safe and possibly
protective against CCl4-induced hepatotoxicity.
Thymoquinone and silybin, a known hepatoprotective agent,
were tested in isolated rat hepatocytes against tert-butyl hydroperoxide
(TBHP) toxicity (Daba and Abdel-Rahman, 1998). They found that
the degree of protection of thymoquinone was less than that caused by
silybin.
12
1.3 Uses of Nigella sativa:
Meral et al. (2001) found that Nigella sativa might be used in
diabetic patients to prevent lipid peroxidation, increase anti-oxidant
and defense system activity and prevent liver damage.
Pharmacological and toxicological properties of Nigella sativa
were investigated by Ali and Blunden (2003). They found that the
pharmacological action of the crude extracts of the seeds caused
protection against nephrotoxicity and hepatotoxicity induced by either
disease or chemicals. They stated that the seeds were characterized by
a very low degree of toxicity.
Mahmoud et al. (2002) studied the effect of Nigella sativa oil
against the liver damage induced by Shistosoma mansoni infection in
mice. It has been reported that Nigella sativa oil possesses anticestode
and antinematode actions. Besides, it produced a hepatoprotective
effect by improving the immunological host system and to some
extent by its antioxidant effect.
Kalus et al. (2003) studied the effect of Nigella sativa on
subjective feeling in patients with allergic diseases. 152 patients were
treated with Nigella sativa oil in capsules. The score of subjective
feeling decreased over the course of treatment with black seed oil. A
slight decrease in plasma triglycerides and a discrete in HDL
13
cholesterol occurred while the lymphocyte subpopulation, endogenous
cortisol levels and ACTH released remained unchanged.
The anticonvulsant activity of thymoquinone, the major
constituent of Nigella sativa seeds, was reported using pentylene-
tetrazole (PTZ) and maximal electroshock (MES) induced seizure
models (Hossein and Parvardeh, 2004).
Boskabady et al. (2004) reported that Nigella sativa has
relaxant effect through the inhibitory effect on the calcium channel.
In a recent study Kanter et al. (2004) have evaluated the
possible protective effects of Nigella sativa against beta-cell damage
from streptozotocin (STZ)-induced diabetes in rats. They found that
Nigella sativa treatment exerts a therapeutic protective effect in
diabetic animals by decreasing oxidative stress and preserving
pancreatic beta-cell integrity. Consequently the authors mentioned that
Nigella sativa might be clinically useful for protecting beta-cells
against oxidative stress.
1.4 Haemobiotic effects of Nigella sativa:
Meral and Kanter (2003) investigated the effects of N. sativa,
or/and Urtica dioica on hematological values besides serum Na, K, Cl
and Ca levels on CCl4-treated rats. They claimed that N. sativa or
14
Urtica dioica treatments alone or in combination can induced
increased the reduced RBC, WBC, PVC, and Hb levels and also
significant decreased in the elevated serum K and Ca levels, hence N.
sativa or/and Urtica dioica treatment might ameliorate the CCl4
induced disturbances of anemia.
The effect of Nigella sativa on blood hemostatic function in rats
was studied by Al Jishi and Abou-Hozaifa (2003). They found that
Nigella sativa induced transient changes in the coagulation activity of
rats.
1.5 Hepatoprotection of Nigella sativa:
El-Dakhakhny et al. (2000) showed that daily administration
of the N. sativa oil per se at a rate of 800mg/kg body weight orally
for 4 weeks did not adversely affect the serum transaminase (ALT and
AST), alkaline phosphate, serum bilirubin or prothrombin activity in
normal albino rats; however, when the oil was given for 4 weeks prior
to induction of hepatotoxicity by D-galactosamine or carbon
tetrachloride, complete protection against D-galactosamine and partial
protection against carbon tetrachloride hepatotoxicity was evident.
Also the administration of the oil at a rate 800 mg/ kg body weight
orally four 4 weeks caused significant diverse effects on serum total
15
cholesterol, low density lipoprotein, triglycerides and a significant
elevation of serum high density lipoprotein level.
In a study done by Mansour et al. (2001) to evaluate the effects
of the volatile oil constituents of N. sativa, namely, thymoquinone,
p-cymene and alpha-pinene in mice treated with carbon tetrachloride,
revealed that a single dose of CCl4 at a rate of 15 micro l /kg body
weight intraperitoneally induced hepatotoxicity 24 hours after
administration. However, administration of different doses of
thymoquinone did not have any effect in liver function tests while
higher doses were lethal. Pretreatment of mice with different doses of
thymoquinone one hour before CCl4 injection showed that 12.5 mg/kg
body weight of thymoquinone given intraperitoneal was the only dose
that ameliorated hepatotoxicity of CCl4. They concluded that
thymoquinone at a rate of 12.5 mg/ kg body weight may play an
important role as antioxidant and may efficiently acts as a protective
agent against chemically induced hepatic damage. However, higher
doses of thymoquinone were found to induce oxidative stress leading
to hepatic injury. Nevertheless, treatment of mice with the other
volatile oil constituents, p-cymene or α-pinene, did not induce any
changes in liver enzymes.
16
Materials &
Methods
17
CHAPTER TWO
MATERIALS AND METHODS
2.1 Materials and Experimental Design:
2.1.1 Animals:
Twenty five healthy adult Wister albino rats of both sex,
weighing 120.3 – 285.3 g, were used. They were kept in cages within
the Department of Biochemistry, Faculty of Veterinary Medicine,
University of Khartoum. They were housed in standard environmental
conditions with free access to feed and water. They were kept for 7
days, as an adaptation period.
2.1.2 Plant:
Nigella sativa seeds were brought from the local market at
Omdurman and purified. The plant was extracted in the phyto-
chemistry laboratory at Medicinal and Aromatic Plants Research
Institute (MAPRI), Khartoum.
2.1.3 Experimental design:
At the end of the adaptation period the rats were divided
randomly into 5 groups A, B, C, D and E; of 5 rats each. The animals
were treated daily for 10 days. Group A, B, C saved as controls; while
groups D and E are the treated groups.
18
Group A was received 0.2 ml/kg body weight liquid paraffin
i/p. Group B received dimethyl sulfoxide (DMS) 0.2 ml/kg body
weight i/p. Group C was injected i/p with CCl4 at a dose rate of
0.2ml/kg body weight in liquid paraffin (1: 9). Group D and E were
injected with CCl4 in paraffin as in group C together with 250 mg/kg
body weight and 500 mg/kg body weight of the Nigella sativa
methanolic extract orally respectively.
2.1.4 Parameters:
Clinical signs and mortality were recorded. Blood samples
were obtained by puncturing retro orbital plexus into dry clean tubes
and allowed to clot at room temperature (waynforth, 1980) or at
slaughter time for analysis. The sera were separated by centrifugation
at 3000 r.p.m for 5 minutes in Hettich EBA 35 centrifuge and stored at
-20oC until analyzed for biochemical tests. Blood samples for
hematological analysis were collected in EDETA containers.
Sera were analyzed for the activities of alkaline phosphatase
(ALP), alanine amino transferase (ALT)/(GPT), aspartate amino
transferase (AST)/(GOT) and for the bilirubin concentration.
The EDETA blood was examined for white blood cells (WBC),
red blood cells (RBC) counts, hemoglobin (Hb) concentration and
packed cell volume (PCV). Also mean corpuscular volume (MCV),
19
mean corpuscular hemoglobin (MCH) and mean corpuscular
hemoglobin concentration (MCHC) were calculated.
Specimens of liver, heart, kidney, spleen and brain were
collected immediately after slaughter and were fixed in 10% formalin
for histopathology.
2.2 Methods:
2.2.1 Plant extract:
Nigella sativa seeds were dried and extracted according to
Harborne method (1976). 70g of granulated seeds were inserted in
soxhlet apparatus (Quick Fit, Ex 5183). The oil have been separated
using 100 ml of petroleum ether (40-60%) overnight. The sample was
unpacked and left to dry, then repacked again with adding methanol
(99.9%) as a solvent to get the polar constituents of the plant. The
extract was evaporated till dryness using a rotavapor.
Carbon tetrachloride (CCl4) solution was prepared by adding
CCl4 to paraffin oil at a ratio of 1:9 respectively.
2.2.2 Biochemical methods:
Sera constituents were determined using Roche Diagnostics/
Hitachi 902 Analyzer; Fig. (D). It was fully automated, computerized
and performs potentiometric and photometric assays. It has an on-
board capacity of 26 tests throughput of up to 200 tests per
20
hour and up to 300 tests with optional ISE unit.
2.2.2.1 Alkaline Phosphatase (ALP):
The activity of ALP was measured by an optimized standard
method according to recommendation of Chemie (1972).
Principle:
Alkaline phosphatase in alkaline medium splits p-nitrophenyl
phosphate in the presence of Mg2+ ions, into p-nitrophenol and
phosphate.
p-nitrophenyl phosphate + H2O ALP phosphate + p-nitropheonol
ALP (I/U) = A405 nm/min x 2760
A ≡ the mean sample absorbance reading.
21
Fig. (D): Hitachi 902 automated Analyzer
22
2.2.2.2 Alanine amino transferase (ALT), Glutamic pyruvic transa-minase (GPT):
Glutamic pyruvic transaminase (GPT) was measured by enzymatic
method according to the method of Reitman and Frankel (1957).
Alanine amino transferase was measured by monitoring the
concentration of pyruvate hydrazone formed with 2,4-dinitrophenyl-
hydrazine.
Principle:
α-oxoglutarate + L-alanine GPT L-glutamate + pyruvate.
The GPT was measured in I/U
2.2.2.3 Aspartate amino transferase (AST), Glutamate oxalo-acetate transaminase (GOT):
Glutamic oxaloacetate (GOT) in sera was measured by
enzymatic method according to the method of Reitman and Frankel
(1957). Aspartate amino transferase was measured by monitoring the
concentration of oxaloacetate hydrazone formed with 2,4-
dinitrophenyl hydrazine.
Principle:
α-oxoglutarate + L-aspartate GOT L-glutamate + oxaloacetate.
AST was measured in I/U.
2.2.2.4 Total Bilirubin:
Total bilirubin reacts with diazotized dichloroaniline to form a
colored azo compound according to the method of Jendrassik and
23
Grof (1938). The intensity of the color was proportional to the
bilirubin concentration in the sample.
Total bilirubin (mg/dl) = total absorbance x 17.5
2.2.3 Hematological methods:
The analysis of blood samples were done by “Sysmex
automated hematology analyzer KX-21.” Fig. (E).
The sysmex KX-21 is an automatic multi parameter blood cell
counter for in vitro diagnostic use in clinical laboratories. The KX-21
employs three detector blocks to the kind of reagents for blood
analysis. The WBC count was measured by the WBC detector block
using the DC detection method. The RBC count and platelets were
measured by the RBC detector block using the DC detecting method.
The Hb detector block measured the hemoglobin concentration using
the non cyanide hemoglobin method.
Blood was aspirated from the sample probe into the sample
rotor valve. 6 µL of blood measured by the sample rotor valve were
transferred to the WBC transducer chamber along with 1.994 ml of
diluents. At the same time 0.1 ml of WBC/Hb lyse was added to
prepare 1:500 dilution sample.
When the solution was made in approximately 10 seconds, RBC
were hemolyzed and platelets shrink, with WBC membrane held as
24
they are. At the same time hemoglobin is converted into red colored
met hemoglobin. Of the diluted/hemolyzed sample in the WBC
transducer chamber, approximately 1 ml was transferred to the Hb
flow cell.
500 µL of sample in the WBC transducer were aspirated
through the aperture. The pulses of the blood cells when passed
through the aperture are counted by the DC detecting method.
In the Hb flow cell, 555 nm wavelength beam irradiated from
the light emitting diode (LED) was applied to the sample in the Hb
flow cell. Concentration of the diluents alone was measured before
addition of the sample; hence Hb (hemoglobin value) was calculated.
PCV evaluation by the analysis principles that RBC pulse height
detection method ratio (%) of whole RBC volume in whole blood.
25
Fig. (E): Sysmex automated hematology analyzer KX-21.
26
Mean corpuscular volume (MCV):
Mean corpuscular volume in whole blood was calculated from
RBC and PCV values as follows:
MCV = PCV x 10/ RBC x 106 (fl)
Mean corpuscular hemoglobin (MCH):
Mean hemoglobin volume (pg) per RBC was calculated from
Hb and RBC as follows:
HGB x 10 MCH (pg) = RBC
Mean corpuscular hemoglobin concentration (MCHC):
Mean corpuscular hemoglobin concentration (g/dl), was calculated
from Hb and PCV as follows:
HGB (g/dl) x 10 MCHC (g/dl) = PCV
2.2.4 Histopathology:
The specimens of tissues were collected immediately after
dissection of animals, fixed in 10% buffered formalin, embedded in
paraffin wax, sectioned at 5 µm and stained with haematoxylin and
eosin; according to the method of Drury and Wallington (1980).
2.2.5. Statistical analysis:
1. Data were analyzed for significance using completely
randomized design. General Linear Models (GLM)
27
procedure of SAS (1998) software was used to run the
data analysis with each test being at 0.05% level of
probability by Duncan multiple range tests. Body weights
and organ body weight ratio were compared using t-test
procedure according to Mendenhall (1971).
28
Results
29
CHAPTER THREE
RESULTS
3.1 Clinical signs:
There were no clinical signs observed in rats of all groups,
however in group D which received 250 mg/kg body weight of
Nigella sativa methanolic extract, there were two rats looked dull after
five days following treatment.
3.2 Body weights:
The body weights were presented in Table (1). There was an
increase in the body weights of the control groups (A, B) at a rate of
2.0%. However, the body weights in group C, D and E were reduced
by 10.3, 9.3 and 10.3% respectively. The reduction was significant in
group E (500 mg/kg Nigella sativa methanolic extract) compared to
the control group C (CCl4).
3.2.1 Organs to body weight ratio:
Table (2) showed that there were no significant changes in the
relative liver and kidney weights among all groups.
3.3 Hematological findings:
The hematological values in albino rats that received Nigella
sativa extract compared to the controls were summarized in Table (3).
30
Table (1): Body weights of albino rats given methanolic extract of Nigella sativa.
Body weights (g) Groups
Day 0 Day 10
A 247.5 ± 26.6a 252.2 ± 26.4 a
B 227.6 ± 47.9 a 232.1 ± 44.8 a
C 174.7 ± 27.6 a 156.8 ± 19.11 a
D 156.3 ± 19.3 a 141.7 ± 22.4 a
E 128.7 ± 10.7 a 115.5 ± 4.9 a
Table (2): Relative Organs weight ratio of albino rats given
methanolic extract of Nigella sativa.
Relative organ weights (%) Group
Liver Kidney
A 2.6 ± 0.1 a 0.4 ± 0.1 a
B 2.7 ± 0.3 a 0.3 ± 0.0 a
C 3.1 ± 0.3 a 0.4 ± 0.0 a
D 4.5 ± 0.5 a 0.4 ± 0.0 a
E 3.7 ± 0.3 a 0.4 ± 0.1 a
Means (± SD) within the same column having the same superscript letters are not significantly different at (P>0.05) based on t-test. Group A ≡ received liquid paraffin. Group B ≡ received dimethylsullfoxide. Group C ≡ received CCl4 in paraffin. Group D ≡ received CCl4+250 mg/kg Bwt. methanolic extract of Nigella sativa. Group E ≡ received CCl4 +500 mg/kg Bwt. methanolic extract of Nigella sativa.
31
3.3.1 WBC and RBC:
There were no significant difference between the treated and the
control groups on day 5 and 10 for both WBC and RBC.
3.3.2 Hemoglobin:
Hemoglobin values showed no significant difference between
controls and treated rats except for a reduction in group D which
received 250 mg/kg Nigella sativa methanolic extract at day 10.
3.3.3 PCV:
There were no significant differences of PCV values through-
out the experimental period between treated and the control groups.
3.3.4 MCV:
MCV showed no significant differences throughout experimental
period between treated groups compared to the control.
3.3.5 MCH and MCHC:
There were no significant differences recorded for MCH or
MCHC values in the treated groups compared to the control groups.
32
Table (3): Effect of Nigella sativa methanolic extract on hematological values of Wister albino rats intoxicated with CCl4:
Days Groups WBC (103/µl)
RBC (106/µl)
Hb (g/dl)
PCV (%)
MCV (fl)
MCH (pg)
MCHC (g/dl)
A 5.3 ± 1.3a 7.5 ± 0.4a 14.1 ± 0.6a 44.0 ± 2.4a 59.0 ± 2.0a 18.9 ± 0.9b 32.1 ± 1.4b
B 7.9 ± 2.6a 6.6 ± 0.9a 13.1 ± 1.2a 40. ± 7.4a 60.1 ± 4.2a 20.0 ± 2.4b 33.5 ± 5.8b
C 6.4 ± 2.8a 7.1 ± 0.8a 14.0 ± 0.8a 42.6 ± 5.4a 59.8 ± 3.5a 19.8 ± 1.7b 33.3 ± 3.7b
D 8.0 ± 3.9a 7.3 ± 0.2a 14.5 ± 0.7a 45.5 ± 2.2a 62.4 ± 2.3a 19.9 ± 0.9b 31.9 ± 1.8b
Zero
E 5.2 ± 1.9a 5.5 ± 1.0b 12.4 ± 1.2a 32.7 ± 6.8a 59.7 ± 2.7a 23.3 ± 2.9a 39.2 ± 5.8a
A 13.9 ± 1.9a 7.2 ± 0.8a 14.4 ± 1.1a 47.5 ± 3.7a 66.4 ± 3.8a 20.2 ± 1.3a 30.5 ± 2.0a
B 11.8 ± 2.9a 6.7 ± 0.5a 13.7 ± 0.7a 43.4 ± 4.8a 65.1 ± 3.5a 20.6 ± 0.6a 31.7 ± 2.2a
C 7.9 ± 3.2a 6.4 ± 1.2a 12.5 ± 1.0a 40.4 ± 8.8a 62.7 ± 3.9a 20.3 ± 4.2a 28.6 ± 2.2a
D 12.9 ± 3.5a 5.7 ± 1.5a 10.1 ± 2.5b 38.5 ± 11.9a 62.5 ± 0.6a 17.7 ± 1.1a 26.8 ± 2.1a 10
E 12.9 ± 3.9a 6.7 ± 0.5a 12.8 ± 0.5a 42.4 ± 4.4a 63.6 ± 4.9a 19.3 ± 0.8a 30.6 ± 3.1a
. Means (±SD) within the same column having the same superscript letters are not significantly different at (P>0.05) level based on SAS software method.
Group A ≡ received liquid paraffin. Group B ≡ received dimethylsullfoxide. Group C ≡ received CCl4 in paraffin. Group D ≡ received CCl4 +250 mg/kg methanolic extract of Nigella sativa. Group E ≡ received CCl4 +500 mg/kg methanolic extract of Nigella sativa.
i
3.4 Changes in serum constituents:
The effects of methenolic extract of Nigella sativa on the activities of
serum ALT, AST and ALP and the concentration of total bilirubin were
given in Table (4).
3.4.1 Total bilirubin:
There were no significant differences in the total bilirubin
concentration between the groups throughout the experimental period.
However in group C, (received CCl4), total bilirubin values were raised
from0.2 to 0.7 on day 10, compared to group received 500 mg/kg body
weight Nigella sativa methanolic extract from 0.1 to 0.4, which showed
slight increase in this group compared to C (CCl4) group.
3.4.2 ALT:
The activity of ALT showed significant increase (P> 0.05) on day 5
and 10 in groups; C (CCl4), D (250 mg/kg body weight) and E (500 mg/kg
body weight) of Nigella sativa methanolic extract compared to the control
groups A and B. On the other hand group E remarked significant decrease
(P> 0.05) compared to group C (CCl4) on day 5 and 10. However significant
decrease (P> 0.05) was observed in group D (250 mg/kg body weight
Nigella sativa methanolic extract) on day 10 compared to group C (CCl4).
ii
3.4.3 AST:
The AST activity was significantly increased on day 5 and day 10 (P>
0.05) in the groups of rats which received CCl4, 250 mg/kg and 500 mg/kg
body weight of Nigella sativa methanolic extract compared to the control
groups A and B. On the other hand significant decrease (P> 0.05) in group D
and E on day 5 and significant decrease (P> 0.05) in group E on day 10 were
observed when compared them to the group C (CCl4).
3.4.4 ALP:
Compared to the control groups A and B, the activity of ALP showed
no significant difference in group D (250 mg/kg body weight Nigella sativa
methanolic extract, but significant increase (P> 0.05) was recorded in group
C and E on day 5 and 10. However significant reduction was recorded on
day 5 and 10 in group D compared to CCl4 group.
3.5 Histopathological findings:
In the control groups (those received paraffin and dimethyl-sulfoxide,
their livers were normal (Fig. F), so rats received CCl4 showed central
hepatic fatty vaculation and congestion (Fig. G). In the group of rats
received 250 and 500 mg of the methanolic extract, there were vaculation
around the central vein but to a lesser extent (Fig. H and I).
iii
Table (4): Effect of Nigella sativa methanolic extract on some
biochemical parameters of Wister albino rats intoxicated with CCl4:
Days Groups Total
bilirubin (mg/dl)
ALT (U/L)
AST (U/L)
ALP (U/L)
Zero A 0.2 ± 0.02c 48.6 ± 9.8a 107.2 ± 6.3a 167 ± 31.2b
iv
B 0.2 ± 0.02c 47.0 ± 14.1a 119.0 ± 22.4a 133.8 ± 33.7b
C 0.2 ± 0.03c 39.2 ± 7.1a 103.2 ± 18.7a 75.8 ± 13.3b
D 0.1 ± 0.02c 40.0 ± 14.8a 93.8 ± 11.9a 153.0 ± 100.6b
E 0.1 ± 0.03c 51.3 ± 15.8a 97.0 ± 9.9a 302.8 ± 99.6a
A 0.3 ± 0.03c 69.6 ± 10.6c 152.2 ± 19.1c 167.1 ± 50.0c
B 0.1 ± 0.04c 58.0 ± 9.7c 163.8 ± 24.5c 160.0 ± 55.2c
C 0.2 ± 0.1c 1185.2 ± 430.4a 1626.8 ± 737.6a 241.2 ± 33.2a
D 0.2 ± 0.8c 944.0 ± 455.5a 896.8 ± 403.5b 142.8 ± 65.5c Day 5
E 0.1 ± 0.03c 581.3 ± 348.6b 713.8 ± 187.2b 224.0 ± 12.8a
A 0.1 ± 0.02c 85.4 ± 9.6 c 177.8 ± 17.8c 167.0 ± 21.8c
B 0.2 ± 0.01c 59.4 ± 11.0c 176.6 ± 21.5c 129.2 ± 24.8c
C 0.7 ± 0.8c 1160.3 ± 141.1a 1961.0 ± 311.3a 453.5 ± 176.5a
D 0.6 ± 0.3c 810.6 ± 246.2b 1425.4 ± 732.4a 298.0 ± 65.4b Day 10
E 0.4 ± 0.2c 572.0 ± 283.6b 849.8 ± 262.5b 293.5 ± 130.5b
Means (±SD) within the same column having different small superscript letters are
significantly different at (P>0.05) level based on SAS software method. Group A ≡ received liquid paraffin. Group B ≡ received dimethyl sullfoxide. Group C ≡ received CCl4 in paraffin. Group D ≡ received 250 mg/kg methanolic extract of Nigella sativa. Group E ≡ received 500 mg/kg methanolic extract of Nigella sativa.
Fig. (F): Sect
v
ion of liver of rat received dimethylsulfoxide, Group B (control). H&E х (100 x 0.96)
Fig. (G): Section of liver of rat received CCl4 in
paraffin, group C, (control). H&E х (250 x 0.96)
Fig. (H): secti
on of
liver of
rat received CCl
4 +250 mg/kg
vi
Body weight of methanolic extract of Nigella sativa, Group D. H&E х (250 x 0.96)
Fig. (I):
section of liver
of rat recei
ved CCl4
+ 500 mg/k
g Body weight of methanolic extract of Nigella sativa, Group E. H&E х (500 x 0.96)
vii
Discussion
CHAPTER FOUR
DISCUSSION
The present study deals with the hepatoprotective effect of Nigella
sativa. In this study body weight gain was lower in rats treated with CCl4
and Nigella sativa extract. This is in harmony with the findings of Zaoui et
al. (2002) who showed that there was significant slowdown of the body
weight in animals treated with Nigella sativa.
viii
In this study liver damage was induced in rats by the administration of
CCl4. The mechanism of CCl4 hepatotoxicity is due to the enzymatic
activation, cytochrome P450, of CCl4 into trichloromethyl free radical
(•CCl3) within the membrane of the endoplasmic reticulum. This followed by
chloromethylation and peroxidation leading to functional and structural
damage (Reckangel, 1967). Thymoquinone, major constituent of Nigella
sativa, is claimed to be hepatoprotective and its mechanism is due to the
presentation of intracellular glutathione (EL-Dakhakhny et al., 2000).
Meral and Kanter (2003) showed that Nigella sativa induced a
significant increase for the reduced RBC, WBC,PCV and Hb levels; this is
in agreement concerning Hb values which increased on day 10 at the dose
500mg/kg bodyweight of methanolic extract of Nigella sativa but disagree
with 250mg/kg bodyweight of methanolic extract of Nigella sativa in which
Hb was reduced. However EL-Sarha et al (1997) reported a significant
reduction in RBC and PCV in goats which treated with Nigella sativa seeds,
while WBC showed significant increase. This disagree with our findings,
may be due to difference in anatomical structure between goats and rats.
The present study showed that the values of ALT and AST were
significantly decreased (P>0.05) in rats that received 500 mg/kg body
weight Nigella sativa extract on day 5 and 10 compared to the control group
ix
C (CCl4). This is in agreement with Daba and Abdel-Rahman (1998) who
tested thymoquinone in isolated hepatocytes against tert-butyl hydroxide
(TBHP) induced toxicity evidenced by alteration in ALT and AST. Similar
results were also found by Nevin and Dilara (2005) who stated significant
rise in plasma AST and ALT in animals injected with CCl4 and by
administration of Nigella sativa showed marked inhibition of increased
enzyme plasma levels.. Similar results were found by Nevin and Dilara
(2005) who stated significant rise in plasma AST and ALT in animals
injected with CCl4 and by administration of Nigella sativa showed marked
inhibition of increased enzyme plasma levels. Corresponding findings
recorded by Mansour et al (2001) who stated that thymoquinone at a dose
rate of 12.5 mg/kg body weight i/p in mice induced significant reduction of
the elevated serum enzymes levels. However, contravention results were
recorded by AL- Ghamdi (2003) who investigated the effect of the aqueous
suspension of Nigella sativa on carbon tetrachloride induced liver damage.
He found that ALT and AST levels were increased in animals pretreated
with Nigella sativa compared to CCl4 group. This may suggest that there was
slight hepatoprotective activity of the methanolic extract of Nigella sativa
seeds when dosed at a high concentration.
x
EL- Dakhakhny et al. (2000) and Zaoui et al. (2002) found that
Nigella sativa seed fixed oil induced no change in serum bilirubin level in
treated rats after 4 and 12 weeks respectively. This is in agreement to the
present study which showed no significant differences in total bilirubin
activity. Nigella sativa constituents may be responsible for the
protective effects against liver damage.
Histopathological changes including centrilobular degeneration
occurred in rats liver that received CCl4 were similar to those described by
Valcheva et al. (2004) and Ozbek et al. (2004). Nigella sativa extract
improved the histopathological changes, hence lower the liver damage. This
protection may be due to partial inhibition of lipid peroxidation.
xi
Conclusion
CONCLUSION
The results of this study indicated that the liver damage produced by
CCl4 was partially inhibited by the methanolic extract of Nigella sativa as
evidenced by the histopathology and enzymes activity. The high doses of the
methanolic extract of Nigella sativa may be more effective than low ones.
Also the methanolic extract reflects less protection than the oil of these
seeds. Further comparative studies should be carried out to confirm these
xii
findings and find out the suitable dose of Nigella sativa that a
hepatoprotective effect.
xiii
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