ABSTRACT

Gareeballah Osman Adam

Graduate School of Woosuk University
The objective of this study was to investigate the antioxidant and
heatorotective effects of Nigella sativa !"S# seeds on
acetaminohen !A$A$#%induced acute heatotoxicity in male Srague%
&awley rats' There were five exerimental grous( including normal
control !vehicle( n)*#( A$A$ as a heatotoxic control !A$A$ +,, -'w'
.'$'( n)*#( "S . !A$A$/"S 0,, mg1kg b'w' $'O'( n)*#( "S .. !A$A$/"S
2,, mg1kg b'w' $'O'( n)*# and "S ... !A$A$/"S 3,, mg1kg -'w' $'O'(
n)*#' After 45 hours( en6ymes such as sueroxide dismutase !SO&#
and glutathion !GS7# were significantly increased( whereas
malondialdehyde !8&A# was significantly decreased !$9,',0# in "S
treated heatotoxic rats comared to A$A$ control rats' The level of
blood ioni6ed magnesium !i8g
4/
# was significantly decreased !$9,',0#
and the level of total magnesium t8g
4/
1i8g
4/
was significantly
increased !$9,',0#' 7owever( the "S treated heatotoxic rats were
almost similar to control' The serum levels of glutamic oxaloacetic
transaminase !GOT#( glutamic%yruvic transaminase !G$T#( lactate
-5-
dehydrogenase !:&7# and alkaline hoshatase !A:$# were
significantly increased in A$A$ control rats' 7owever( in "S treated
heatotoxic rats( the A$A$%induced alterations were recovered'
.n ;itro assay( A$A$ induced a dramatic decrease in the viability of
T.-%<2 cells to 53= 0'3> of the control ! 9 ,',,0# ?o%treatment with
A$A$ and "S 4@( @,( <@( 0,, Ag attenuated the A$A$%induced
decrease in cell viability at 0, A8 as a ercentage of A$A$
resectively' A$A$ increased the level of BOS roduction !050=+> of
the control#' ?o%treatment with A$A$ and "S attenuated the A$A$%
induced increase in BOS'The results showed that exosure of A$A$ to
T.-%<2 cells led to cell death and decrease in cell viability''
.n this study( we roose that "S ossesses the dose%deendent
heatorotective activity against A$A$%induced heatotoxicity( which
might be due to imroved antioxidant en6yme activity and blood ionic
control'
Key wordsC Nigella sativa( Acetaminohen( Antioxidant(
7eatorotective( T.- 2< 7eatocytes
-6-
INTRODUCTION
0'0 Nigella sativa
The black seeds of the Nigella sativa lant have an extensive
history of medicinal use that dates back thousands of years' .n ancient
Greece and Dgyt( black seed oil was used by hysicians to treat an
assortment of illnesses including headaches( nasal congestion(
toothaches and intestinal worms' ( a buttercu%like lant that belongs to
the Banunculaceae lant family( is cultivated around the world for its
seeds which can be used as a sice or reservative agent' Today(
however( the seeds are rimarily used for the extraction of oil that is
used in traditional medicine' -lack seed oil !-SO# is thought to romote
menstruation and increase milk roduction in women E0( 4F'
?ontemorary naturoathic medicine around the world has used -SO
as a diuretic( carminative( treatment for asthma( bronchosasm(
resiratory oression( coughs( back ain( hyertension and obesity E0%
2F' Until recently( few studies had been conducted to confirm the
validity of the roosed medicinal value of -SO' .n recent years(
various studies have been conducted on -SO to investigate such
roerties as anti%microbial( hyotensive( antinocicetive( anti%
-7-
histaminic( immunomodulatory( anti%inflammatory( anti%tumour( and
antidiabetic as well as many other characteristics E0F'
0'4'8orhology of the lant
Nigella sativa is an annual flowering lant which grows to 4,%3,
cm tall( with finely divided leaves( the leaf segments narrowly linear to
threadlike' The flowers are delicate( and usually colored white( yellow(
ink( ale blue or ale urle( with @%0, etals' The fruit is a large and
inflated casule comosed of 2%< united follicles( each containing
numerous seeds E5F(E@F'
0'2' ?hemical ?omosition
Seeds contain numerous esters of structurally unusual
unsaturated fatty acids with terene alcohols !<>#G furthermore( traces
of alkaloids are found which belong to two different tyesC isochinoline
alkaloids are reresented by nigellimin and nigellimin%"%oxide( and
yra6ol alkaloids include nigellidin and nigellicin' .n the essential oil
!avr' ,'@>( max' 0'@>#( thymoHuinone was identified as the main
comonent !u to @,># besides %cymene !5,>#( inene !u to 0@>#(
-8-
dithymoHuinone and thymohydroHuinone' Other terene derivatives
were found only in trace amountsC ?arvacrol( carvone( limonene( 5%
terineol( citronellol' Iurthermore( the essential oil contains significant
!0,># amounts of fatty acid ethyl esters' On storage( thymoHuinone
yields dithymoHuinonene and higher oligocondensation roducts' The
seeds also contain a fatty oil rich in unsaturated fatty acids( mainly
linoleic acid !@, *,>#( oleic acid !4,>#( eicodadienoic acid !2># and
dihomolinoleic acid !0,>#' Saturated fatty acids !almitic( stearic acid#
amount to about 2,> or less' Also contain arts of the essential oil(
mostly thymoHuinone( by which it acHuires an aromatic flavour' The
seeds give on steam%distillation a yellowish brown volatile oil with an
unleasant odor' The oil contains carvone( d %limonene( and a carbonyl
comound( nigellone E*( <( +( 3F'
ThymoHuinone eaks from methanolic extract as shown above'
-9-
Amount of thymoHuinone obtained in different extracts and formulation
of "igella sativa( metabolic extract was the highest !4'42 = ,'0+ Jg1ml#
among other extracts !0,#
0'5' Toxicicity of Nigella sativa
8any toxicological studies have been carried out on Nigella sativa
seeds' .t has been shown that no toxic effects were reorted when
fixed oil was given to mice via the stomach in an acute study' .n a
chronic toxicity study rats treated daily with an oral dose for 2 months
caused no changes in key heatic en6yme levels articularly asartate%
aminotransferase( alanine%aminotranferase( and gammaglutamyl%
transferase' 8oreover( the histoathological results also showed to be
normal for the tissues of heart( liver( kidneys and ancreas :&@,
values of fixed oil of Nigella sativa obtained by single doses orally and
intraeritoneally in mice( were reorted to be 4*'4%20'* ml1kg and 0'+*%
4'4* ml1kg( resectively' The low toxicity of fixed oil( evidenced by high
:&@, values( key heatic en6yme stability and organ integrity( suggests
a wide margin of safety for theraeutic doses of fixed oil E00F'
-10-
0'@' Antioxidant
Antioxidants or free radical scavengers are very imortant in
rotecting the living cells against any damage induced by free radicals
which are roduced continuously in cells either during hagocytosis or
accidentally as by%roduct metabolites' Dach biological system has
certain antioxidant mechanisms against the aggregations of such free
radicals' The balance of oxidant antioxidant system must exist in the
cell while the disturbance of antioxidant%rooxidant balancecauses
oxidative stresses E04F' Antioxidants are imortant endogenous defense
mechanism against injury caused by liid eroxidation and harmful
reactions induced by reactive oxygen secies !BOS#( which are
constantly roduced in the body during normal metabolic rocesses
E02F' Antioxidants may act individually or comlementary in synergetic
action' 8any antioxidant en6ymes are seHuestered in eroxisomes'
Beair mechanisms are also available in the cells as otent
mechanisms for removal of oxidi6ed membrane fatty acids E05F'
0'*' Antioxidant activity of Nigella sativa
extracts and some of its active rinciles( like thymoHuinone( have
-11-
been shown to ossess rotective effect against haematological(
heatic( renal and other toxicities induced by anti%cancer drugs and
some toxins' Ior examle( extract revented the decreases in
haemoglobin level and leucocyte count caused by cislatin in mice E0@F'
ThymoHuinon and fixed oil of were also reorted to inhibit non%
en6ymatic eroxidation in ox brain hosholiid liosomes E0*F'
-adary et al E0<(0+F observed the rotective effect of
thymoHuinone on ifosfamideKinduced Ianconi syndrome in mice and
doxorubicin%induced hyerliidemic nehroathy in rats( resectively(
manifested as a significant imrovement of hoshaturia( glucosuria(
and elevated serum creatinine and urea as well as a significantly
imrovement of renal glutathione deletion and liid eroxide
accumulation'.t is reorted that !,'4 m:1kg# intraeritoneally relieves
the deleterious effects of ischemia reerfusion injury on liver'
-iochemical arameters like the serum asartate aminotransferase(
alanine aminotransferase lactate dehydrogenase levels and total
antioxidant caacity !TA?#( ?AT( total oxidative status !TOS#( oxidative
stress index !OS.# and 8$O were determined in heatic tissue in rats
with heatic ischemia' Besults suggested that treatment rotects the
rat liver against heatic ischemia reerfusion injury E03F' administration
rotects heatic tissue from deleterious effects of toxic metals such as
-12-
lead( and attenuates heatic liid eroxidation following exosure to
chemicals such as carbon tetrachloride E4,F' ?admium !?d
//
# causes
alteration of the cellular homeostasis and oxidative damage' The
rotective role of TL on the heatotoxicity of ?d
//
with secial
reference to its rotection against erturbation of nonen6ymatic and
en6ymatic antioxidants was investigated' The effect of TL retreatment
was examined in ost%nuclear suernatant reared from liver of Swiss
albino mice under in vitro conditions' ?d?l
4
treatment !@ mmol1:#
resulted in a significant increase in antioxidant en6ymatic activities' .t
also caused a significant !$9,',,0# increase in rotein carbonyl and
reduced glutathione content' $retreatment with TL !0, Amol1:# showed
a significant rotection as manifested by noticed attenuation of rotein
oxidation and rejuvenation of the deleted antioxidants of cellular
fraction' These results strengthen the hyothesis that TL exerts
modulatory influence on the antioxidant defense system on being
subjected to toxic insult E40F'
0'<' Acetaminohen and liver stress
Acetaminohen !A$A$# is a widely used analgesic and antiyretic
that is safe and effective( with few adverse effects( when taken at
-13-
theraeutic doses E44F' 7owever( an acute or cumulative overdose of
A$A$ can cause severe liver damage with the otential to rogress to
liver failure' A$A$ overdose accounts for more than @*(,,, emergency
room visits( 4(*,, hositali6ations( and an estimated 5@+ deaths due to
acute liver failure each year in the United States E42F' A$A$%induced
heatotoxicity has been demonstrated in exerimental animal as well
as clinical cases' 8ice and hamsters have been shown to be very
sensitive to the heatotoxic effects of A$A$( eveloing a fulminant
centrilobular necrosis similar to that observed in humans E45F' Oxidative
stress followed by liid eroxidation is also thought to contribute to the
initiation or rogression of A$A$%induced heatotoxicity E4@( 4*F'
MATERIALS AND METHODS
-14-
4'0 Dxtraction of Nigella sativa
"S seeds were rocured from Sudan' The lant arts were
washed with running ta water( air dried( and homogeni6ed to a fine
owder and stored in airtight containers at 5o
?
' The dried 4@, g owder
of the lants was extracted with 4@,, ml of aHueous methanol !"'8'
Abdel%7amid# with little modification !methanolC distilled water( +,>C
4,> v1v# for 5+ h with occasional shaking' The extract was then filtered
through Whatman filter aer' The residue was extracted twice with the
same fresh solvent( the filtrate was concentrated using a rotary vacuum
evaorator at low temerature !2,%5,# and ressure' The brown grease
material obtained was then susended in water'
4'4 Dxerimental animals
8ale Srague%&awley rats weighing 4@,M2,, g were used for the
in vivo exeriment' The animals were urchased from Noatech !Norea#
and housed for one week' The animals were rocured and housed in
the animal house maintained under standard hygienic conditions with a
04C04 hr light1dark cycle' Iood ellets !7industan lever :td' 8umbai(
-15-
.ndia# and ta water were rovided ad libitum' All exerimental
rotocols were aroved by the ?ommittee on the ?are of :aboratory
Animal Besources( Oeonbuk "ational University !Norea#( and
conducted in accordance with the guide E4<F'
4'2 Dxerimental &esignC
The animals were divided into five grous of six animals in each
grou' Grou 0 served as normal control !"?# which received distilled
water only' Grou 4 served as A$A$ control received A$A$ at a dose
of +,, mg1kg !bw# dissolved in &8SO at a rate of 3,, mg1ml' The
selected dose of A$A$ was relatively high to show the rotective effect
of "'S extract' Grous 2 to @ received "S extract at 0,,( 2,, 3,,
mg1kg bw two hours before induction of liver stress'
Animals were anestheti6ed by intraeritoneal injection of
tiletamine1 6ola6eam !Poletile( 5, mg1kg#' -lood was collected in
tubes containing the anticoagulant sodium hearin !@ .U1 ml#' Serum
was searated by centrifugation at 2(,,, rm for 4, min'
4'5' 8easurement of blood ions whole blood $7( hemoglobinin(
-16-
hematocrit( and gas comosition
-lood arameters were measured using "O;A( stat rofile 8 with
ion%selective sensor !"ova biochemical( USA# immediately after
collection of blood samles'
4'@' 8easurement of serum arameters
All biochemical arameters were analy6ed using an autoanaly6er
8odel <,4, !7itachi( Oaan#'
4'*' Antioxidant activities
$ortions of the same liver were homogeni6ed and centrifuged
accordinglyG the suernatant was used for the biochemical estimations'
The activities of suer oxide dismutase !SO&# and catalase !?AT#
were determined by the methods of 8arklund and 8archland E4+F and
Aebi E43F resectively' Glutathione eroxidase !G$Q# was assayed by
the method of Botruck et al E2,F' Glutathione%s% transferase !GST# and
Glutathione reductase !GB&# were assayed by the methods of 7abig
!20# and Backer !24#( resectively'
-17-
4'<' 7istoathology
The liver was excised for histoathological examination' $ortions
of the same lobe of liver from each animal were immediately fixed in
0,> formaldehyde( embedded in araffin( cut into @M* Jm sections(
stained with hematoxylin%eosin !7RD#( and observed under a light
microscoe !S5,,#'
4'+' ?ell culture
T.-%<2 cells were obtained from the American Tye ?ulture
?ollection !AT??( 8anassas( USA# and grown in &ulbeccoTs modified
DagleTs medium sulemented with 0,> fetal bovine serum !SigmaK
Aldrich( St' :ouis( USA#( @ m8 l%glutamine( @, U1ml enicillin and @,
g1ml stretomycin in a humidified @> ?O4 3@> air environment at
2<
U
?' 4(< &ichlorodihydrofluorescin diacetate !&?I7%&A# was
urchased from 8olecular $robes !Dugene( USA#' The 5(*%diamidino%
4%henylindole !&A$.# and @(@(*(*% tetrachloro%0(0(2(2
tetraethylben6imida6olyl%carbocyanine iodide !O?%0# were urchased
from Dn6o :ife Sciences !$lymouth 8eeting( USA#'
-18-
4'3' ?ell viability assay
After treatment with1without A$A$ and "S 45 h( cell viability of
T.-%<2 cells was detected by a 2%!5(@% &imethylthia6ol%4%yl#%4(@%
dihenyltetra6olium bromide !8TT# assay( as described reviously E22F'
4'0,' 8easurement of intracellular BOS generation
After the treatment mentioned above( T.-%<2 cells were treated
with 0, 8 &?I7%&A for 2, min' &?I7 fluorescence was determined
using a sectrohotometer at excitation and emission wavelengths of
5++ nm and @0@ nm( resectively'
4'00' Statistical analysis
Besults are exressed as means the standard error of the mean
!SD8#' The data were analy6ed by StudentTs t%test or analysis of
variance !A"O;A# with the -onferroni ost hoc test( as aroriate(
using $rism @',2 !Grah$ad Software .nc'( San &iego( USA#' A value
9,',@ was considered significant'
-19-
RESULTS
-20-
2'0 Administration of A$A$
The effects of "S on A$A$ !+,, mg1kg .'$'# induced heatotoxicity
in rats was evaluated by recording changes in serum en6yme( some
blood ionsmetabolites( and liver antioxidants'
Serum arameters of A$A$ induced rat liver injury the serum
levels of GOT( G$T( :&7( and A:$' 8&A( SO& and GS7 from liver
homogenates were used as biochemical markers for heatic damage(
.n addition total 8g
4/
( and ioni6ed 8g
4/
( ?omared to the control grou(
the A$A$ treated grou showed a significant increase in GOT( G$T(
:&7( A:$( 8&A( SO&(GS7( total and ioni6ed 8g !GOTC *5', = 5',<
.U1: vs' @*4'< = 5,'* .U1:( G$TC @+'0<= *'4* .U1: vs' 5+2'+2 = <0'*<
.U1:( :&7C 0,3'*<=45'3+ .U1: vs' 5<*'0<=03<'+@ .U1:( A:$C 203'*<=
5+'** .U1: vs' <@@',, = @3'53 .U1:( 8&A ,'03= ,',,,* nmol1mg rotein
vs' ,'505 = ,',4, nmol1mg rotein( SO&C 4@'4*=,',<3 U1mg rotein vs'
5'+* = ,'2+ U1mg rotein( GS7C @'*4= ,'50 U1mg rotein vs' 0'*, =
,',5 U1mg rotein#' .oni6ed 8g
4/
!,'@2= ,',0 mg1dl vs' ,'52,= ,',,5
mg1dl#' total 8g
4/
!0'+@= ,',* mg1dl vs' 4'5,= ,',* mg1dl#'
2'4 ?o%administration of "S and A$A$
-21-
?o%administration of "S tended to organi6e some serum
en6ymes( and antioxidants levels to nearly control levels although
varying according to dose( in 0,, mg1kg !GOTC 0@<'4, = 44'@< .U1:(
G$TC 0+*'+,= 0*'22 .U1:( :&7C 45+'5,=+'<< .U1:( A:$C *2+'+,= 2@'@0
.U1:( 8&A ,'20= ,',5 nmol1mg rotein( SO&C 0,'20'4 U1mg( GS7C
0'33= ,',4 U1mg rotein( .oni6ed 8gC ,'5<= ,',0 mmol1:( and total 8gC
0'3+= ,',+ mmol1:( .oni6ed 8g1 total 8gC ,'@*=,',4#( in 2,,mg1kg
!GOTC 0@<',, = 44'2+ .U1:( G$TC 0@<'+2= <',< .U1:( :&7C 0,,'*<=*'00
.U1:( A:$C *,,',,= 22'+< .U1:( 8&A ,'0,@= ,',,4 nmol1mg rotein(
SO&C 40'+=,'* U1mg( GS7C 2'5+= ,'44 U1mg rotein( .oni6ed 8gC
,'53= ,',, mmol1:( and total 8gC 0'5,= ,'52 mmol1:( .oni6ed 8g1 total
8gC ,'**=,',4#( and in 3,,mg1kg !GOTC +<'5, = 0+'4* .U1:( G$TC
3@',,= 45'20 .U1:( :&7C *3'4,=+'03 .U1:( A:$C 4+*'5,= 43'<0 .U1:(
8&A ,',35= ,',,0 nmol1mg rotein( SO&C 4*'4=,'* U1mg( GS7C 5'+4=
,'2* U1mg rotein( ioni6ed 8gC ,'@,= ,',0 mg1dl( and total 8gC 0'55=
,'2* mg1dl( .oni6ed 8g1 total 8gC ,'<0=,'54#( as shown in Iig' 0a( 0b(
0c( 0d( 4a( 4b( 4c( 2a ( 2b and 2c'
A
-22-
-
?
-23-
&
Iig' 4C The effects of "S on A:$( GOT( G$T( and :&7 en6ymes
The effects of A$A$ with1without "S on A:$( GOT( G$T( and :&7
levels on serum in rats with A$A$ induced liver injury' The data are
reorted as a mean=SD8 for each grou' VVV 9 ,',,@C -onferroniTs
ost hoc test vs' controlG WW 9 ,',0C -onferroniTs ost hoc test vs'
A$A$'
-24-
A
-
-25-
?
Iig' 2C Dffects of "S on liver en6ymes
Dffects of "S on liver en6ymes !8&A( SO&( and GS7# levels in rats
with A$A$%induced liver injury' The data are reorted as mean= SD for
each grou' VV$9 ,',0( VVV9 ,',,0C -onferroniTs ost hoc test vs'
controlG WW9 ,',0( WWW9 ,',,0C -onferroniTs ost hoc test vs' A$A$'
-26-
A
B
Iig' 5C The effects of "S on ioni6ed and total 8g'
The effects of A$A$ with1without "S on ioni6ed and total levels on
whole blood and serum in rats with A$A$ liver injury' The data are
reorted as a mean=SD8 for each grou' VV 9 ,',0( VVV 9 ,',,@C
-onferroniTs ost hoc test vs' controlG WWW 9 ,',,0C -onferroniTs ost
hoc test vs' A$A$'
-27-
2'2' ;enous blood electrolytes( metabolites and gas comosition
The administration of "S showed significant changes in some
blood metabolites( electrolytes and biochemical arameters but it
seems the effect is dose deendent vi6 the higher the dose the better
the effect as shown in Table 0 and 4'
2'5' 7istological analysis
8icroscoic examination of the liver sections of the control animals
showed normal architecture of heatic lobules in the form of
heatocytes arranged from the ortal vein !Iig' 5a#' Dxamination of rat
livers from the grou treated with A$A$ showed swelling( slight
hydroic degeneration( and liid changes of heatocytes !Iig' 5b#'
7istoathological changes induced by A$A$ were remarkably imroved
by co%administration of "S 0,,( 2,,( 3,, !Iig' @c( @d and @e#'
2'@' Dffect of A$A$ with1without "S or on the viability of T.-%<2
cells
-28-
As shown in Iig' *( A$A$ induced a dramatic decrease in the
viability of T.-%<2 cells to 53=0'3> of the control !9,',,0#' ?o%
treatment with A$A$ and "S 4@ attenuated the A$A$%induced
decrease in cell viability !0,+>=,'* at 0,A8 as a ercentage of A$A$#'
?o%treatment with A$A$ and "S @, attenuated the A$A$ induced
decrease in cell viability !00<=,'@> at 0, A8 as a ercentage of A$A$'
?o%treatment with A$A$ and "S <@ attenuated the A$A$ induced
decrease in cell viability !042=,'+> at 0, A8 as a ercentage of A$A$'
?o%treatment with A$A$ and "S 0,, attenuated the A$A$ induced
decrease in cell viability !020=,'@> at 0, A8 as a ercentage of
A$A$#'
2'*' Dffect of A$A$ with1without "S on the generation of BOS
concentrations of T.-%<2 cells
Iig' < shows that A$A$ increased the level of BOS roduction
!050=+> of the control#' ?o%treatment with A$A$ and "S attenuated
the A$A$%induced increase in BOS !"S 4@G 33=@> of the levels
observed under A$A$ treatment alone( "S @,G +3=0,>( "S <@ <<=*>G
"S 0,, +0=4>#'
-29-
&.S?USS.O"
The extent of heatic damage is assessed by histological
evaluation and the level of various biochemical arameters in
circulation' $aracetamol !"%acetyl%%aminohenol# is a widely used
analgesic antiyretic drug is safe when used in theraeutic doses'
7owever( over dosage of aracetamol is known to be heatotoxic
nehrotoxic in man in exerimental animals E25F' Overdose of
aracetamol causes a otentially fatal( heatic centrilobular ne%crosis'
The heatotoxicity of aracetamol has been attributed to the formation
of a toxic metabolite( " acetyl% %ben6oHuinoneimine !"A$L.# by the
action of cytochrome $5@,4D0 E2@F' When liver lasma membrane gets
damaged( a variety of en6ymes normally located in the cytosol are
released into the circulation E2*F' .n the resent study the damage of
liver due to the A$A$ over dosage was confirmed by elevated levels of
bio%chemical arameters like GOT( G$T( :&7( and A:$' This is due to
the fact that heatic cells ossess a variety of metabolic activities
contain a host of en6ymes' SG$T and SGOT were found in higher
concentration in cytolasm SG$T articularly in the mitochondria' .n
-30-
liver injury the transort function of heatocytes is disturbed( resulting
in the leakage of lasma membrane E2<F' Thereby causing leakage of
such en6ymes leading to the increased serum levels of them' Oral
administration of various doses of "S extract to aracetamol
intoxicated rats resulted in gradual normali6ation of the activities of
AST( A:T( :&7( and A:$' This evidently suggests the rotective effect
of the extract in imroving the functional integrity of liver cells' The
8&A is a good indicator of the degree of liid eroxidation E2+F( which
is closely related to A$A$%induced tissue damage' .n the resent study(
we also observed a significant increase !$9 ,',@# in the levels of 8&A
in the liver( which was decreased by the administration of "S' This
might due to hydroxyl radicals scavenging activities of "S' GS7 is the
major nonen6ymatic antioxidant and the regulator of intracellular redox
homeostasis( ubiHuitously resent in all cell tyes E23F' The deletion of
cellular GS7 in the liver cells is known to lay an imortant role in
A$A$ toxicity E5,F' A$A$ administration leads to a significant decrease
in the glutathione level( which can be an imortant factor in the A$A$
toxicity' The mechanism of heatorotection by "S against A$A$
toxicity might be due to restoration of the GS7 level' SO& catalyses the
dismutation of sueroxide anion to 74O4 and O4' -ecause 74O4 is
still harmful to cells( ?AT and G$x further catalyse the decomosition
-31-
of 74O4 to water E50F' Thus( the coordinate actions of various cellular
antioxidants in mammalian cells are critical for effectively detoxifying
free radicals' To the best of our knowledge( there is no reorted study
about the relationshi between 8g and rotective effect of "S on
A$A$T induced toxicity( but an abundant amount of calcium(
magnesium( otassium( hoshorus and iron' Irom the results( it
becomes evident that seeds rovide an abundance of many minerals
as reorted by E54F and E52F' A$A$ administration to mice declined
antioxidant caacity of the mice liver as evinced in decreased activity of
the antioxidant en6ymes' "S retreatment revented the reduction in
the antioxidant en6yme activities and conseHuent oxidative damage to
the liver' Dffect of "S on total and ioni6ed and other electrolytes can be
attributed to mineral comosition indicated that otassium is dominant
!+,+=*'*0 mg10, g# followed by calcium !@<,=40'@ mg10,, g#(
hoshorous !@52=0,',5 mg10,, g# and magnesium !4*@=5'+<
mg10,, g#( resectively' 8oreover( considerable Huantities of sodium(
iron( manganese( 6inc and coer were resent in the indigenous
variety of black cumin seeds as reorted by E55F' A number of
investigators concluded reviously that reactive oxygen secies might
lay a role in AA$%induced cell injury of cultured heatocytes' Several
studies showed a beneficial effect of treatment with vitamin D E5@F'
-32-
To investigate whether this increased en6yme activity translates
into differences in toxicity measurements between the two systemsG the
effect of A$A$ on cell viability was examined' A$A$ is a well
characteri6ed heatotoxic drug that is toxic to cells after metabolism in
the liver( rimarily through ?X$4D0 and to a lesser extent by ?X$0A4
and ?X$2A5( E5*( 5<F' BOS are generated in many stressful situations
and induce cell death by either aotosis or necrosis E5+F and can
induce 8A$N activation by which heatocytes mediate reaction to
oxidative stress E53F'
-33-
?O"?:US.O"
.n conclusion( "igella sativa seeds aeared to be safe and
ossibly rotective against A$A$%induced heatotoxicity' 7owever(
further studies may still be needed to clarify its active ingredients and
effects on 8g4/( other blood electrolytes and metabolites'
Iurther studies need to be conducted to know about the active
comonent of Sudanese "igella sativa using sohisticated machines
us as thin layer chromatograhy'
Iurther in vivo and invitro studies for the active comonent after
urification is recommended'
-34-

BDIBD"?DS
0'Salem ML( .mmunomodulatory and theraeutic roerties of the
"igella sativa :' seed'.nternational .mmunoharmacology( 4,,@' @!02%
05#C ' 0<53%0<<,'
4' Atta MB( Some characteristics of nigella ! "igella sativa : '#
seed cultivated in Dgyt and its liid rofile' Iood ?hemistry( 4,,2'
+2!0#C ' *2%*+'
2' Sayed MD(Traditional medicine in health care' Oournal of
Dthnoharmacology( 03+,' 4!0#C ' 03%44'
5' Goreja WG -lack seedC natureYs miracle remedy' "ew Xork( "XC
Ama6ing 7erbs $ressG 4,,2'
@' Warrer !K" Nam#ar $!K" Rama%&'tty CC Orient :ongman
$vt :tdG 4,,5' .ndian medicinal lants%a comendium of @,, seciesG
' 023K054'
*' www'botanical'com1botanical
<' C(o)ra( 03@+( @0@( @*+G Mod( *<<G S*(%dler( 05@G
Ra%+aswam R( 7elv' chim' acta( 0353( 24( 323
+' Rttel R( ibid'( 03@5( 2<( 02*0
3' Rttel( ibid'( 03@2( 2*( 525G $endse &utt( -ull' Acad' Sci' Unit'
-35-
$rov'( 0322%25( 2(
0,' !rawe, A" Hasa% -" a%d A.ta# A 7$T:? densitometric
method for analysis of thymoHuinone in "igella sativa extracts and
marketed formulations Asian $ac O Tro &is 4,02G 2!*#C 5*<%5<0
00' /ao' A" C(erra( -" Ma(ass% N" Alao' K" Amaro'*( H"
Hassar M' Acute and chronic toxicity of "igella sativa fixed oil'
$hytomedicine' 4,,4G3!0#C*3K<5'
04'Kr%s&y NI !0334#C 8echanism of action of biological
antioxidants' $roc' Soc' Dx' -iol' 8ed'( 4,,!4#C 45+%4@5'
02'-amamoto- a%d -amas(ta S !033<#C $lasma ratio of
ubiHuinol and ubiHuinone as a marker of oxidative stress 8ol' Asects
8ed'( 0+!0#C <3%+5'
05' Bay%es 0 a%d Dom%*,a& MH !0333#C Antioxidant defenses
and reactive oxygen secies' .nC 8edical -iochemistry( 8osby( 4nded'(
?h' 00( ' 022%02<
0@'Nar SC" Salom M0" !a%&&ar KR' 8odulatory effects of
?rorcus sativus and "igella sativa extracts on cislatin%induced toxicity
in mice'O Dthnoharmacol 0330G 20 !0#C <@%+2'
0*' Ho'+(to% !0" /ar&a R" de las Heras B" Ho'lt 0R Iixed oil of
"igella sativa and derived thymoHuinone inhibit eicosanoid generation
in leukocytes and membrane liid eroxidation' $lanta 8ed Ieb 033@G
-36-
*0 !0#C22%*'
0<' Badary OA" Gamal El1d% AM' .nhibitory effect of
thymoHuinone against 4,%methylchlolanthrene%induced fibrosarcoma
tumorigenesis' ?ancer &etect $rev 4,,0G 4@!5#C2*4%+'
0+' Badary OA" Al1S(a#a%a( OA" Na+ MN" Al1R&a# AC"
Elma,ar MM' .nhibition of ben6o!a#yrene%induced forestomach
carcinogenesis in mice by thymoHuinone' Dur O ?anccer $rev 0333G +
!@#C 52@%5,'
03' Xildi6 2" Co#a% S" Ter, A" Ates M" A&soy N" Ca&r H(' "igella
sativa relieves the deleterious effects of ischemia reerfusion injury on
liver' World O Gastroenterol 4,,+G 05!22#C @4,5%@4,3'
4,' Ka)oor S' Dmerging clinical and theraeutic alications of
"igella sativa in gastroenterology' World O Gastroenterol 4,,3G <C
40<,%40<0'
40' /a.eer M2" Waseem M" C(a'd(ary S" !ar3e, S' ?admium%
induced heatotoxicity and its abrogation by thymoHuinone' O -iochem
8ol Toxicol 4,04G 4*!@#C 033%4,@
44' R'ma*& BH' Acetaminohen misconcetions' 7eatology'
4,,5G 5,C0,K0@'
42' Lee WM' Acetaminohen and the U'S' Acute :iver Iailure
Study GrouC lowering the risks ofheatic failure' 7eatology' 4,,5G
-37-
5,C*K3'
45' Mt*(ell 0R" 0ollow D0" !otter W/" Da3s DC" Gllette 0R"
Brode BB' Acetaminohen%induced heaticnecrosis' .' Bole of drug
metabolism'O $harmacol Dx Ther 03<2G 0+<C0+@ 035'
4@' We%del A" 2e'erste% S" Ko%, KH' Acute aracetamol
intoxication of starved mice leads to liid eroxidation in vivo' -iochem
$harmacol 03<3G 4+C4,@0K4,@@'
4*' Al#a%o E" !ol G" C(ar)otto E" Bas 2" Da%,a% MU'
$aracetamol%stimulated liid eroxidation in isolated rat and mouse
heatocytes' ?hem -iol .nteract 03+2G 5<C453K4*2'
4<' "ational Besearch ?ouncil' Guide for the ?are and Use of
:aboratory Animals' ".7 $ublication "o' +@%' "ational Academy $ress(
Washington( 033*'
4+' Mar&l'%d S" Mar&l'%d G' .nvolvement of Sueroxide anion
radical in the autooxidation of yrogallol and convenient assay for
SO&' Dur O -iochem'03<5G5<C5*3K<5
43' Aebi 7' ?atalase( .n 8ethods in en6ymatic analysis' .nC
-ergmeyer 7( editor' ;ol'4' "ew XorkC Academic $ressG 03+2' ' <*K
+,'
2,' Botruck( OT( $oe A:( Gantter( 7D !03<2# SeleniumC
-iochemical roles as a comonent of glutathione eroxidase' '
-38-
0<3C@++%@3,'
20' 7abig( W7( $abst( 8O Oakoby( W- !03<5# Glutathione
transferaseC A first en6ymatic ste in mercaturic acid formation' O'
-iol'?hem453C<02,%<023'
24' Backer( D !03@@# Glutathione reductase !liver and yeast# .nC
8ethods in Dn6ymology( Dds ?olowick S$ Nalan "O Academic
$ress("ewXork( ;ol ;..( <44%<4@'
22' Oeon( S7( $ark 78( Nim SO( :ee 8X( Nim G-( Bahman 88(
Woo O"( Nim .S( Nim OS'( Nang 7S( 4,0,' Taurine reduced IN@,*%
induced generation of BOS and activation of O"N and -ax in 8adin
&arby canine kidney cells' 7umanDxerimantal Toxicology 43( *4<K
*22'
25' &iak ;( $armar( Ga6ala A( 8ilind A( Nhandkar A and Surendra
SN( Z8itochondrial AT$ase( a Target for $aracetamol .nduced
7eatotoxicity([ Duroean Oournal of $harmacologyC Dnvironmental
Toxicology and $har% macology( ;ol' 432( "o' 2( 033@( ' 44@%443'
doiC0,'0,0*1,34*%*30<!3@#,,,40%*
2@ :ee SST( -uters OT8( $ineau T( Iernande6 $'S and Gon6ales
I:( ZBole of ?X$4D0 in 7ea% totoxiciy of Acetaminohen([ Oournal of
-iological ?hem% istry( ;ol' 4<0( 033*( ' 04,*2%04,*<'
doiC0,'0,<51jbc'4<0'4,'04,*2
-39-
2*' 8itra SN( ;enkataranganna 8;( Sundaram B and
Goumadhavan S( Z$rotective Dffect of 7&%,2( a 7erbal Iormulation(
against ;arious 7eatotoxic Agents in Bats([ Oournal of
Dthnoharmacology( ;ol' *2( "o' 2( 033+( ' 0+0%0+*'
doiC0,'0,0*1S,2<+%+<50!3+#,,,++%3
2<' Gamal Dl%din A8( 8ostafa A8( Al%Shabanah OA( Al%-ekairi
A8( and "agi 8"( Z$rotective effect of arabic gum against
acetaminohen%induced heatotoxicity in mice([$harmacological
Besearch( vol' 5+( no' *( ' *20K*2@( 4,,2' ;iew at $ublisher \ ;iew
at Google Scholar \ ;iew at Scous
2+' 8eister A and Anderson 8D( ZGlutathione([ Annual Beview of
-iochemistry( vol' @4( ' <00K<*,( 03+2' ;iew at Scous
23' 8itchell OB( Oollow &O( and $otter WP( ZAcetaminohen
induced heatic necrosis' .;' $rotective role of glutathione([ Oournal of
$harmacology and Dxerimental Theraeutics( vol' 0+<( no' 0( '
400K40<( 03<2'
5,' 7ayes O&( Ilanagan OU( and Oowsey .B( ZGlutathione
transferases([ Annual Beview of $harmacology and Toxicology( vol' 5@(
' @0K++( 4,,@'
50' Al%Oassir( 8S( 0334' ?hemical comosition and microflora of
black cumin !"igella sativa :'# seeds growing in Saudi Arabia' Iood
-40-
?hem'( 5@C 423%454'
54' "ergi6( ? and Otles S( 0332' ?hemical comosition of "igella
sativa :' seeds' Iood ?hem'( 5+C 4@3%4*0'
52' Al%Oassir( 8S( 0334' ?hemical comosition and microflora of
black cumin !"igella sativa :'# seeds growing in Saudi Arabia' Iood
?hem'( 5@C 423%454'
55' 8uhammad TS( 8asood S-( IaHir 8A( Amer O( Saeed A( and
8uhammad "' nutritional rofile of indigenous cultivar of black cumin
seeds and antioxidant otential of its fixed and essential oil' $ak' O'
-ot'( 50!2#C 0240%022,( 4,,3
5@'"agai 7( 8atsumaru N( Ieng G'( and Nalowit6 " !4,,4#'
Beduced glutathione deletion causes necrosis and sensiti6ation to
tumor necrosis factor%alha%induced aotosis in cultured mouse
heatocytes' 7eatology 2*( @@K*5'
5*' -essems OG( ;ermeulen "$C $aracetamol !acetaminohen#%
induced toxicityC molecular and biochemical mechanisms( analogues
and rotective aroaches'?rit Bev Toxicol 4,,0G20!0#C@@K02+'
5<' :aine OD( Auriola S( $asanen 8( Ouvonen BOC Acetaminohen
bioactivation by human cytochrome $5@, en6ymes and animal
microsomes' Qenobiotica4,,3G23!0#C00K40'
5+' Nlaunig( OD'( Namendulis( :8'( 4,,5' The role of oxidative
-41-
stress in carcinogenesis'Annual Beview of $harmacology and
Toxicology 55( 423K4*<'
53' Wang Q( 8artindale( O:( :iu X( 7olbrook "O( 033+' The cellular
resonse to oxidativestressC influences of mitogen%activated rotein
kinase signaling athwayson cell survival' The Oournal of -iochemistry
222( 430K2,,'
-42-
Iig' @C The effects of "s extract on cell viability of T.- <2 heatocytes'
The effects of A$A$ with1without "S on cell viability of T.-%<2 cells' The
data are reorted as the mean=SD8VV 9 ,',0( VVV 9 ,',,@C
-onferroniTs ost hoc test vs' controlGWWW 9 ,',,0C -onferroniTs ost
hoc test vs' A$A$'
-43-
Iig' *C The effects of "S extract on generations of BOS in in T.-%<2
cells
The effects of A$A$ with1without "S on the generation of intracellular
BOS in T.-%<2 cells' The involvement of BOS in A$A$%induced cell
death was determined by measuring the levels of BOS in T.-%<2 cells'
The cells were incubated with 0,8 &?I7&A' The fluorescence
intensity was evaluated using fluorescent microscoy !S4,,# !A# and a
sectrohotometer !-#' The data are reorted as a mean= SD8 for
each grou' VVV 9 ,',,@C -onferroniTs ost hoc test vs' controlG W 9
,'@C -onferroniTs ost hoc test vs' A$A$'
-44-
A
B
-45-
C
D
-46-
E
Iig' <C 7istoathological changes induced by A$A$ were with or
without "S administration':ight microscoic analysis of liver sections of
normal and A$A$%treated rats with or without "S administration' !A#
?ontrol( !-# A$A$( !?# "S0,, mg1kg /A$A$( !&# "S 2,, mg1kg /A$A$(
!D# "S 3,, mg1kg /A$A$'
-47-
Ta#le 45 Dffects of "S on blood electrolytes against acetaminohen
induced heatotoxicity in rats
?ont A$A$
"S !mg1Ng#
0,, 2,, 3,,
"a
/
!m81:# 0@0=4 05,=0VV 05,=,VV 05,=0VV 050=0VV
N
/
!m81:# 5'*=,'4 2'@=,'4VV 5'0=,'0W 5',= ,'0VW 5',=,'0W
?l
/
!m81:# 004'=4 004=4 0,*=0VW 0,3=, 000=0
?a
4/
!m81:# 0'4<=,',4 0'44=,',2 0'54=,',0VVVWW 0'2@=,',4VW 0'2*=,',,VWW
?a
4/
18g
4/
4'5,=,',@ 4'+2=,',+VV 2',4=,'00VVV 4'<@=,',< V 4'<=,',2VVV
?ont( controlG A$A$( acetaminohenG "S( Nigella sativa G ?a4/18g4/(
the ration of ?a4/ er 8g4/' The data are reorted as the meanSD8'
V9,',@( VV9,',0 and VVV9,',,0G -onferroniTs ost hoc test vs' ?ontG
W9,',@( WW9,',0( WWW9,',,0C -onferroniTs ost hoc test vs' A$A$'
Ta#le 6' Dffects of "S on blood 7( glucose( gas comositions(
hematocrit and hemoglobinin against acetaminohen induced
heatotoxicity in rats
-48-
?ont A$A$
"S !mg1Ng#
0,, 2,, 3,,
7 <'22=,',0 <'52=,',0VVV <'2*=,',4WW <'2<=,',0W <'50=,',0VV
$?O
4
!># 55'+=,'0 2@'0=0'3VVV 5<'@=0'5WWW 5<'3=0'4WWW 50'<=,'<W
$O
4
( !># 5@'2=2'@ **'*=4'5VVV 53'0=4'<WWW @2'0=4'2WWW @5'@=,'+W
O
4
sat( !># <@'<=5', 32'4=0',VVV +0'@=4',W +*'*=4'0W +<'3=,'@W
7ct !># 2<=0 43=0VVV 2*=0WWW 2<=0WWW 2@=0WWW
7b !g1d:# 04'2=,'4 3'*=,'2 VVV 04',=,'0WWW 04'5=,'4WWW 00'<=,'4WWW
?ont( controlG A$A$( acetaminohenG "S( Nigella sativa G $?O4( artial
?O4 tensionG $O4( artial O4 tensionG O4sat( O4 saturationG 7ct(
hematocritG 7b( hemoglobin' The data are reorted as the meanSD8'
V9,',@( VV9,',0 and VVV9,',,0G -onferroniTs ost hoc test vs' ?ontG
W9,',@( WW9,',0( WWW9,',,0C -onferroniTs ost hoc test vs' A$A$'
-49-