cell biochemistry and function

Cell Biochem Funct 2002; 20: 143±151.

DOI: 10.1002/cbf.968

Effects of thymoquinone on antioxidant enzyme activities,

lipid peroxidation and DT-diaphorase in different tissues
of mice: a possible mechanism of action
Mahmoud A. Mansour*, Mahmoud N. Nagi, Aiman S. El-Khatib and Abdullah M. Al-Bekairi
Department of Pharmacology, Faculty of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia

The present investigation focused, ®rstly, on the effects of oral administration of thymoquinone (TQ) on antioxidant enzyme
activities, lipid peroxidation and DT-diaphorase activity in hepatic, cardiac and kidney tissues of normal mice. Superoxide
dismutase (SOD; E.C:1.15.1.1), catalase (CAT; E.C:1.11.1.6), glutathione peroxidase (GSH-Px; E.C:1.11.1.9), glutathione-
S-transferase (GST; E.C:2.5.1.18), and DT-diaphorase (E.C:1.6.99.2) enzyme activities in each tissue type were determined.
Treatment of mice with the different doses of TQ (25, 50 and 100 mg kg 1 day 1 orally) for 5 successive days,
produced signi®cant reductions in hepatic SOD, CAT and GSH-Px activities. In addition cardiac SOD activity was markedly
inhibited with the higher doses of TQ, (namely 50 and 100 mg kg 1). Moreover, TQ (100 mg kg 1) signi®cantly reduced
hepatic and cardiac lipid peroxidation as compared with the respective control group. Conversely, TQ (50,100 mg kg 1) and
TQ (100 mg kg 1) enhanced cardiac and renal DT-diaphorase activity respectively. However, the selected doses of TQ
neither produced any change in GST activity nor in¯uenced reduced glutathione content in all tissues studied. TQ was tested,
secondly, as a substrate for hepatic, cardiac and renal DT-diaphorase of normal mice in the presence of NADPH. Kinetic
parameters for the reduction of TQ to dihydrothymoquinone (DHTQ) indicated that DT-diaphorase of different tissues can
ef®ciently reduce TQ to DHTQ. Km and Vmax values revealed that hepatic DT-diaphorase exhibited the higher values, while
the lower values were associated with renal DT-diaphorase. TQ and DHTQ were tested, thirdly, as speci®c scavengers for
superoxide anion (generated biochemically) or as general scavengers for free radicals (generated photochemically). The
results revealed that TQ and DHTQ acted not only as superoxide anion scavengers but also as general free radical scaven-
gers. The IC50 for TQ and DHTQ in biochemical and photochemical assays were in the nanomolar and micromolar range
respectively. Our data may explain at least partly the reported bene®cial in vivo protective effects of TQ through the com-
bined antioxidant properties of TQ and its metabolite DHTQ. Copyright # 2002 John Wiley & Sons, Ltd.

key words Ð thymoquinone; dihydrothymoquinone; superoxide dismutase; catalase; glutathione peroxidase; glutathione-
S-transferase; DT-diaphorase; glutathione; lipid peroxidation; reactive oxygen species

abbreviations Ð TQ, thymoquinone; DHTQ, dihydrothymoquinone; SOD, superoxide dismutase; CAT, catalase; GSH-
Px, glutathione peroxidase; GST, glutathione-S-transferase; GSH, reduced glutathione; MDA,
malondialdhyde; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; NADPH,
-nicotinamide adenine dinucleotide phosphate; DCPIP, dichlorophenolindophenol
INTRODUCTION
The generation of highly reactive oxygen intermedi-
ates is an integral feature of normal cellular metabo-
lism. Reactive oxygen intermediates, including
* Correspondence to: Mahmoud A. Mansour, Department of
Pharmacology, Faculty of Pharmacy, King Saud University, PO
Box 2457, Riyadh 11451, Kingdom of Saudi Arabia. Tel: 9661-
4677212. Fax: 9661-4676383. E-mail: mahmedm@ksu.edu.sa
Copyright # 2002 John Wiley & Sons, Ltd.
. .
hydroxyl radical ( OH), superoxide anion (O2 ),
hydrogen peroxide (H2O2) and nitric oxide (NO), lead
to lipid peroxidation and speci®c oxidation of some
enzymes.1 The fundamental defence line of the organ-
ism against reactive oxygen intermediates includes
scavenger enzymes such as superoxide dismutase
(SOD), catalase (CAT), and glutathione peroxidase
(GSH-Px) as well as lipid and water soluble antioxi-
dant compounds such as ascorbic acid, reduced glu-
tathione (GSH), vitamin A and ¯avonoids.2,3
Received 30 July 2001
Accepted 30 October 2001
144 m. a. mansour et al.
During pathological circumstances, the production
of reactive oxygen species (ROS) may increase. Stu-
dies on free radicals showed that the initiation and
development of many diseases, such as various types
of in¯ammation, tumours and physiological aging are
related to oxidative stress.4 Thus, a series of new types
of antioxidant drugs have been developed. These
drugs can prevent the injury caused by free radical
generation or protect cells and tissues which have
been subjected to free radical attack from further
injury.5
Thymoquinone (TQ), the main constituent of the
volatile oil obtained from Nigella sativa seeds, has
been subjected to a range of pharmacological investi-
gations in recent years. In our laboratory, oral admini-
stration of TQ protected several organs against
oxidative damage induced by a variety of free
radical-generating agents including doxorubicin-
induced cardiomyopathy,6 carbon tetrachloride-
evoked hepatotoxicity7 and nephropathy produced
by cisplatin.8 In addition, Mutabagani and El-Mahdy9
showed that TQ exerted an anti-in¯ammatory activity
in rats. Recently, we demonstrated that TQ exhibited
potent ability to inhibit enzymically generated leuko-
trienes B4 and C4 (LTB4 and LTC4) produced from
calcium ionophore A23187-stimulated human neutro-
phils, through inhibition of 5-lipoxygenase enzyme
(Mansour and Tornhamre, unpublished observation).
In addition, our previous study showed that TQ inhib-
ited in vitro lipid peroxidation induced by Fe3‡/ascor-
bate and showed superoxide radical scavenging
activity.6
The possible mechanism(s) of the aforementioned
pharmacological action of TQ have now begun to be
clari®ed. In an attempt to clarify further the possible
mechanism(s) through which TQ exerts its protective
effects, the present study investigated the effect of oral
administration of different doses of TQ on hepatic,
renal and cardiac antioxidant enzyme activities of nor-
mal mice. SOD, CAT, GSH-Px, DT-diaphorase and
glutathione-S-transferase (GST) activities as well as
the GSH content in the different tissues were mea-
sured. In the same samples the levels of thiobarbituric
acid reactive substance (TBARS) were estimated to
show whether any correlation exists between the
effects of TQ on the antioxidant enzyme activities
and the degree of membrane lipid peroxidation.10
The ability of hepatic, renal and cardiac DT-diaphor-
ase of normal mice to catalyse reduction of TQ to
dihydrothymoquinone (DHTQ) was also studied. To
investigate whether TQ and DHTQ act as general sca-
vengers for free radicals (generated photochemically)
or speci®c superoxide anion radical (generated bio-
Copyright # 2002 John Wiley & Sons, Ltd.
chemically) scavengers, a set of in vitro experiments
have been performed in the presence of TQ or
DHTQ.
MATERIAL AND METHODS
Chemicals and instrumentation
TQ (2 isopropyl-5-methyl-1,4-benzoquinone), 5,50 -
dithiobis-2-nitrobenzoic acid (DTNB) (Ellman's
reagent), NADPH and 1-chloro-2,4-dinitrobenzene
were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). TBA was a product of Fluka (Buchs,
Switzerland). DCPIP and ribo¯avin were obtained
from E. Merck (Darmstadt, Germany). o-Dianisidine
was purchased from Riedel-deHaeÈn (AG-Seelze,
Hannover, Germany).
DHTQ was prepared from TQ by reduction with
sodium hydrosul®te according to the procedure of
Smith and Tess.11 The structure of DHTQ was con-
®rmed by IR spectroscopy and the purity was checked
by melting point and thin layer chromatography as
described previously.12 The compound was found to
be >99% pure.
All the remaining chemicals were of the highest
analytical grade. All the solutions used for enzyme
assays were prepared using deionized water to elimi-
nate trace metal contamination.
A Branson Soni®er (250, VWR Scienti®c, Danbury,
Conn. USA) was used to prepare tissue homogenates.
Ultraviolet and visible absorbance measurements
were made using a Shimadzu UV-Vis double beam
spectrophotometer, equipped with a recorder.
Animals
The animals used and the experimental design had the
prior approval of the Animal Care and Use Committee
of King Saud University. Male Swiss albino mice
weighing 22±25 g were used in the present study.
They were obtained from the experimental animal
care centre of the College of Pharmacy, King Saud
University. The animals were housed under conven-
tional laboratory conditions in a room maintained at
25  1 C and a relative humidity 55  5% with a reg-
ular 12 h light:12 h dark cycle. The animals received
standard rat pellet diet (Purina Chow) and water
ad libitum.
The animals were sorted at random into four
groups of six animals each. The ®rst group was kept
as the control group, while the other three groups
received orally TQ at the doses of 25, 50 and
100 mg kg 1 day 1 for 5 consecutive days.
Cell Biochem Funct 2002; 20: 143±151.
 antioxidant effect of thymoquinone
Tissue homogenates preparation
All animals were sacri®ced by cervical dislocation
under light ether anaesthesia. The liver, kidney and
heart were quickly excised, rinsed with saline, blotted
dry on ®lter paper, weighed and then 10% (w/v)
homogenates of the different tissues were prepared
in ice-cold saline.
Measurement of the antioxidant enzyme activities
GSH-Px activity was estimated in the different
tissue homogenates according to Kraus and Ganther13
by kinetic assay at 37 C using a test reagent kit for
GSH-Px (RANSEL-Randox, UK). The absorbance was
measured at 340 nm and the results were expressed as
mmol NADPH oxidized min 1 g 1 tissue. GST and
CAT were assayed as previously described by Habig
et al.14 and Higgings et al.15 using 1-chloro-2,4-
dinitrobenzene and H2O2 as substrates respectively.
The results were expressed as mmol min 1 g 1 tissue
and mmol min 1 g 1 tissue respectively. SOD activity
was determined as previously described by Arthur and
Boyne16 in the diluted tissue homogenates by kinetic
assay at 37 C using a test reagent kit for SOD (RAN-
SOD-Randox, UK). The absorbance was measured at
505 nm and the results were expressed as U g 1 tissue.
DT-diaphorase activity in different tissues was esti-
mated spectrophotometrically according to the
method of Ernster17 using DCPIP and NADPH as sub-
strates. The reaction rate was monitored by measuring
the decrease in the absorbance at 600 nm. The results
were expressed as mmol min 1 g 1 tissue.
Estimation of tissue glutathione content and lipid
peroxidation
Tissue glutathione contents were measured colorime-
trically at 412 nm according to Ellman.18 Homoge-
nates were precipitated with trichloroacetic acid and
after centrifugation, supernatants were used for esti-
mation of GSH levels. The concentrations were
expressed as mmol g 1 tissue. Tissue levels of lipid
peroxides were determined as TBARS calculated as
malondialdhyde (MDA).19 The absorbance was mea-
sured at 532 nm and the concentrations were
expressed as nmol MDA g 1 tissue.
Kinetic analysis of TQ reduction by DT-diaphorase
of different tissues
Kinetic analysis of TQ reduction by hepatic, cardiac
and renal DT-diaphorase of normal mice was per-
formed by monitoring the rate of NADPH oxidation.
Copyright # 2002 John Wiley & Sons, Ltd.
145
Homogenates (10%) of liver, kidney and heart from
control mice were prepared. DT-diaphorase activity
was assayed by measurement of the rate of oxidation
of NADPH at 340 nm using different concentrations
of TQ as a substrate.17
The reaction mixture contained, in a total volume of
1 ml, 50 mM phosphate buffer, pH 7.4, 0.2 mM
NADPH, 0.07% bovine serum albumin and 10 ml of
the 10% tissue homogenates. The reaction was
initiated by addition of different concentrations of
TQ (10 ml) dissolved in DMSO and the initial rate
of the reaction was measured from the rate of change
of absorbance at 340 nm.
In vitro assays for superoxide anion and free
radical generation
Xanthine±xanthine oxidase method. Xanthine±xanthine
oxidase was used to generate superoxide radical20 and
2(4-iodophenyl-3-(4-nitrophenol)-5-phenyltetrazolium
chloride (INT) was employed for its detection.
The reaction mixture contained, in a total volume of
2 ml, 50 mM CAPS buffer, pH 10.2, containing 50 mM
xanthine, 25 mM INT and 50 ml ethanol. The reaction
was started by addition of 0.4 units of xanthine oxi-
dase. The change in the absorbance min 1 at 505 nm
served as the control reading and was referred to as
100%. Ethanol (50 ml) containing different concentra-
tions of TQ or DHTQ was then added and the mea-
surements were carried out as above. Data were
plotted as percentages of the remaining activity in
the presence of TQ or DHTQ.
Ribo¯avin±o-dianisidine method. The method was
employed for free radical generation by the aerobic
photo-oxidation of o-dianisidine sensitized by ribo-
¯avin.21
The reaction mixture contained, in a total volume of
1 ml, 10 mM potassium phosphate buffer, pH 7.5, con-
taining 13 mM ribo¯avin. To 0.88 ml of ribo¯avin solu-
tion 60 ml ethanol and 60 ml of 10 mM o-dianisidine
solutions were added. Absorbance was measured at
460 nm. The tubes were then transferred to an illumi-
nation box, illuminated for 4 min and the absorbance
was measured again. The change in the absorbance at
460 nm after 4 min illumination of ethanol served as
the control reading and was referred as 100%. Ethanol
(60 ml) containing different concentrations of TQ or
DHTQ was then added to 0.88 ml ribo¯avin solution
followed by 60 ml of o-dianisidine and the measure-
ment was carried out as above. Data were plotted as
a percentage of the remaining absorbance in the pre-
sence of TQ or DHTQ.
Cell Biochem Funct 2002; 20: 143±151.
146 m. a. mansour et al.
Statistical methods
Data are expressed as mean  SEM. Statistical com-
parisons between different groups were carried out
using one-way analysis of variance (ANOVA) fol-
lowed by a Tukey±Kramer multiple comparison test
to judge the difference between various groups. Sig-
ni®cance was accepted at p < 0.05.
RESULTS
Effects of TQ administration on hepatic, cardiac and
renal antioxidant enzyme activities, DT-diaphorase
activity, lipid peroxides and glutathione content
Oral treatment of normal mice with different doses of
TQ namely, 25, 50 and 100 mg kg 1 for 5 successive
days, induced signi®cant inhibition of hepatic SOD,
CAT and GSH-Px activities (Figures 1±3). The effect
of TQ on hepatic antioxidant enzymes tended to be
dose-related. In addition, TQ (50 and 100 mg kg 1)
induced inhibition of cardiac SOD activity to values
signi®cantly lower than their respective control values
(Figure 1). However, the inhibitory effect of TQ
(100 mg kg 1) on renal SOD did not reach a signi®-
cant level (Figure 1). The selected doses of TQ did
not signi®cantly alter cardiac and renal CAT or
GSH-Px activities (Figures 2 and 3). The inhibitory
effect of TQ on hepatic antioxidant enzymes and car-
diac SOD activity was associated with a decrease of
hepatic and cardiac lipid peroxides. TQ (100 mg kg 1)
produced a profound inhibition of hepatic and cardiac
Figure 1. Effects of oral administration of TQ for 5 successive days on hepatic, cardiac and renal SOD activity. SOD activity was
determined by kinetic assay at 37 C. Absorbance was measured at 505 nm and the results were expressed as U g 1 tissue. Each column
represents the mean of six mice with a vertical bar showing the SEM
*Signi®cant difference from control group at p < 0.05
Copyright # 2002 John Wiley & Sons, Ltd.
lipid peroxides measured as MDA (Figure 4). In the
renal tissues, the MDA level was not signi®cantly
altered by TQ administration (Figure 4). Conversly,
TQ 50 and 100 mg kg 1 provoked a signi®cant incre-
ment of cardiac DT-diaphorase activity compared to
the control group (Figure 5). Likewise, a remarkable
increase in renal DT-diaphorase activity has been
demonstrated in the TQ (100 mg kg 1)-treated group
(Figure 5). However, the chosen doses of TQ did not
signi®cantly affect hepatic DT-diaphorase.
Treatment with different doses of TQ for 5 consecu-
tive days neither induced any changes in GST activity
nor altered GSH content in any of the tissues studied
(Figures 6 and 7).
Effects of increasing concentrations of TQ as a
substrate on hepatic, cardiac and renal DT-diaphorase
activity of normal mice
Initial experiments were performed to determine
whether TQ could serve as a substrate for hepatic, car-
diac and renal DT-diaphorase of normal mice. Spec-
trophotometric analysis of the reaction mixture
containing NADPH, different concentrations of TQ,
and the different tissue homogenates from control
mice was performed by monitoring the rate of oxida-
tion of NADPH at 340 nm.
Values of Km and Vmax are presented in Table 1 and
suggest that DT-diaphorase can ef®ciently reduce TQ
to DHTQ. The Lineweaver±Burk plot yielded Km
values of 55, 50 and 40 mM and Vmax values of 20,
Cell Biochem Funct 2002; 20: 143±151.
 antioxidant effect of thymoquinone 147

Figure 2. Effects of TQ treatment on CAT activity in hepatic, as well as in cardiac and kidney tissue homogenates. CAT activity was
assayed using H2O2 as substrate. The results were expressed as mmol min 1 g 1 tissue. Each column represents the mean of six mice with a
vertical bar showing the SEM
*Signi®cant difference from control group at p < 0.05

12.5 and 10 mmol min 1 g 1 tissue for hepatic, cardiac
and renal DT-diaphorase respectively.
Effects of TQ and DHTQ on superoxide radical
and free radical generation
Xanthine±xanthine oxidase, an enzymic method, was
employed for the generation of superoxide radical. TQ
inhibited the assay in a concentration-dependent man-
ner. A similar inhibition curve was obtained when TQ
was replaced by DHTQ (Figure 8). In this assay
DHTQ was as effective inhibitor as TQ against super-
oxide radical generation. The IC50 for both com-
pounds were in nanomolar range.
In the o-dianisidine±ribo¯avin method, the free
radicals are generated by aerobic photo-oxidation of

Figure 3. Effects of oral administration of TQ on GSH-Px activity of hepatic, cardiac and kidney tissue homogenates. GSH-Px was
estimated by kinetic assay at 37 C. Absorbance was measured at 340 nm and the results were expressed as mmol NADPH oxidized
min 1 g 1 tissue. Each column represents the mean of six mice with a vertical bar showing the SEM
*Signi®cant difference from control group at p < 0.05

Copyright # 2002 John Wiley & Sons, Ltd. Cell Biochem Funct 2002; 20: 143±151.
148 m. a. mansour et al.

Figure 4. Effects of oral administration of different doses of TQ for 5 successive days on the hepatic, cardiac and kidney lipid peroxides
measured as malondialdhyde (MDA). Absorbance was measured at 532 nm and the concentrations were expressed as nmol MDA g 1 tissue.
Each column represents the mean of six mice with a vertical bar showing the SEM
*Signi®cant difference from control group at p < 0.05

o-dianisidine sensitized by ribo¯avin. TQ and DHTQ
potently inhibited the assay in a concentration-
dependent manner. The IC50 values for both com-
pounds were in the micromolar range (Figure 9).
DISCUSSION
The results of the present study demonstrated that
administration of TQ at three dose levels elicited
marked antioxidant effects in mice. This was shown
by a profound decrease in hepatic antioxidant enzyme
activities (SOD, CAT and GSH-Px). In addition, car-
diac SOD activity was markedly inhibited at the
higher doses of TQ. The antioxidant effects of TQ
were further con®rmed by the reduction of hepatic
and cardiac lipid peroxides measured as malondiald-
hyde (MDA).
SOD, CAT and GSH-Px enzymes are among the
endogenous antioxidant enzymes that play a pivotal
role in the elimination of the superoxide radical and

Figure 5. Effects of TQ treatment on DT-diaphorase activity in hepatic as well as in cardiac and kidney tissue homogenates. DT-diaphorase
activity was estimated spectrophotometrically using DCPIP and NADPH as a substrates. The reaction rate was monitored by measuring the
decrease in the absorbance at 600 nm. The results were expressed as mmol min 1 g 1 tissue. Each column represents the mean of six mice
with a vertical bar showing the SEM
*Signi®cant difference from control group at p < 0.05

Copyright # 2002 John Wiley & Sons, Ltd. Cell Biochem Funct 2002; 20: 143±151.
 antioxidant effect of thymoquinone 149

Figure 6. Effects of TQ on hepatic, cardiac and kidney tissue GST activity. GST was assayed using 1-chloro-2,4-dinitrobenzene as
substrate. The results were expressed as mmol min 1 g 1 tissue. Each column represents the mean of six mice with a vertical bar showing the
SEM

hydrogen peroxide and thus interrupt the propagation
of lipid peroxidation reactions. In this process, GSH-Px
requires endogenous reduced glutathione as a co-
substrate. Thus these enzymes can act as protectors
that guard against the oxidative damage induced by
ROS. The inhibition of SOD, CAT and GSH-Px activ-
ities as well as lipid peroxides detected in the current
investigation after oral administration of TQ could
presumably be explained as a consequence of less
availability of the substrates for these enzymes (super-
oxide radical or free radicals) due to the reported
superoxide radical scavenging effect of TQ.6 The pre-
sent ®nding is in harmony with the study which
demonstrated that treatment with antioxidants
resulted in reduction in antioxidant enzyme activ-
ities.22 Conversely, it has been demonstrated that
chemically-generated free radicals induced a marked
increase of the antioxidant enzymes activities (SOD,

Figure 7. Effects of TQ on the reduced GSH content in hepatic and cardiac as well as kidney tissue homogenates. Tissue GSH content was
measured colorimetrically at 412 nm. The concentrations were expressed as mmol g 1 tissue. Each column represents the mean of six mice
with a vertical bar showing the SEM

Copyright # 2002 John Wiley & Sons, Ltd. Cell Biochem Funct 2002; 20: 143±151.
150 m. a. mansour et al.
Table 1. Kinetic parameters of DT-diaphorase in liver, heart and
kidney tissues using TQ as a substrate
DT-diaphorase
Km Vmax
(mM) (mmol min 1 g 1
tissue)
Liver 55 20
Heart 50 12.5
Kidney 40 10
The reaction mixture contained in a total volume of 1 ml, 50 mM
phosphate buffer, pH 7.4, 0.2 mM NADPH, 0.07% bovine serum
albumin and 10 ml of 10% hepatic or cardiac or kidney tissue
homogenates of control mice. The reaction was initiated by addition
of different concentrations of TQ (10 ml) dissolved in DMSO and the
initial rate of the reaction was measured from the rate of change of
absorbance at 340 nm.
CAT and GSH-Px) and lipid peroxides and prior treat-
ment with free radical scavengers decreased the ele-
vated level of CAT activity and lipid peroxides.23
However, TQ administration at the selected doses
neither induced any changes in tissue GSH content
nor in¯uenced activity of GST in the different tissues
studied. These results are consistent with the earlier
®nding of Badary et al.24 who showed that administra-
tion of TQ in drinking water at concentrations of 30,
60 and 90 mg 1 kg 1 cannot alter the hepatic, renal
and cardiac GSH content.
DT-diaphorase is a cytosolic ¯avoprotein enzyme.
It catalyses a two-electron reduction of a broad range
of quinones to dihydroquinone.25 The two-electron
reduction of quinones does not result in the formation
of free radical (semiquinone). Therefore the enzyme is
expected to protect cells against the adverse effects of
quinones and their derivatives.26 Thus DT-diaphorase
Figure 8. TQ and DHTQ scavenging of superoxide anion radical
as a function of their concentrations measured by INT reduction
using xanthine±xanthine oxidase. Each point represents the mean of
three different experiments with a vertical bar showing the SEM
Copyright # 2002 John Wiley & Sons, Ltd.
Figure 9. TQ and DHTQ scavenging of free radicals as a function
of their concentrations measured by the inhibition of aerobic photo-
oxidation of o-dianisidine sensitized by ribo¯avin. Each point
represents the mean of three different experiments with a vertical
bar showing the SEM
is considered to be a detoxi®cation enzyme because of
its ability to detoxify reactive quinone to less toxic
hydroquinone which can be conjugated and excreted.
In the present study, treatment of mice with the
higher doses of TQ induced a marked increase in car-
diac and renal DT-diaphorase enzyme activity, while
not affecting the hepatic enzyme activity. The obser-
ved increments in renal and cardiac DT-diaphorase
activity, but not hepatic enzyme, after TQ administra-
tion may be attributed to the difference in DT-diaphor-
ase activity between hepatic tissues and renal and
cardiac tissues presumbly because the hepatic tissue
exhibited the highest activity (®ve times more than
renal and cardiac tissues).
In an in vitro study, TQ was tested as a substrate for
the hepatic, cardiac and renal DT-diaphorase of nor-
mal mice. Kinetic parameters indicated that DT-
diaphorase of different tissues can ef®ciently reduce
TQ to DHTQ. Km and Vmax values were estimated
from a Lineweaver±Burk plot. These results may
explain at least, in part, the low toxicity of oral
administration of TQ (LD50 ˆ 2.4 g kg 1) in mice.24
An important ®nding in this study is that DHTQ
shows a free radical scavenging activity, like TQ,
using two different systems for the generation of free
radicals and superoxide. In the o-dianisidine±ribo¯avin
method, the free radicals are generated by the aerobic
photo-oxidation of o-dianisidine sensitized by ribo¯a-
vin. This assay can be used to determine whether the
compound tested is a general free radical scavenger or
a speci®c scavenger of superoxide radicals. A general
free radical scavenger will inhibit the assay, while the
superoxide radical scavenger will augment the assay
Cell Biochem Funct 2002; 20: 143±151.
 antioxidant effect of thymoquinone
and a compound with both features will of course
inhibit the assay. In the present study, TQ and DHTQ
inhibited the assay. The other method employed
xanthine±xanthine oxidase to generate superoxide
radicals. In this assay a superoxide radical scavenger
will inhibit the assay. TQ and DHTQ potently inhib-
ited the generation of superoxide radicals. The IC50 of
both compounds in the photochemical and biochem-
ical assays were in the micromolar and nanomolar
range respectively. Taken together, it can be con-
cluded that TQ and DHTQ are potent free radical
and superoxide radical scavengers. These results
could certify a direct inhibitory effect of TQ and
DHTQ on free radical generation. In connection with
our previous study which demonstrated that TQ is a
potent superoxide radical scavenger with scavenging
power as effective as superoxide dismutase against
the superoxide radical, it may be concluded that the
reported in vivo protection of different organs against
oxidative damage induced by varieties of free radical-
generating agents by TQ may be due to the combined
antioxidant effects of TQ and DHTQ against free radi-
cal generation.
The present study is the ®rst to our knowledge
which demonstrates the effect of oral administration
of TQ on antioxidant enzyme activities, DT-diaphorase
and lipid peroxides in mouse vital organs. These
®nding are very interesting, since TQ is currently sub-
jected to several pharmacological screening studies as
a candidate for prevention of chemical carcinogen-
esis27 and potentiation of anti-tumour activities of
some anti-cancer drugs.8
In conclusion, our data provide further evidence
that TQ and its metabolite DHTQ may play an impor-
tant role as an endogenous antioxidant, which may
account, at least in part, for the pharmacological
action of TQ.
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