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IJOMEH 2009;22(2) 115
ORIGINAL PAPERS
International Journal of Occupational Medicine and Environmental Health 2009;22(2):115 – 123
DOI 10.2478/v10001-009-0016-5
EFFECT OF AQUILEGIA VULGARIS (L.)
ETHYL ETHER EXTRACT ON LIVER
ANTIOXIDANT DEFENSE SYSTEM IN RATS
MAŁGORZATA EWERTOWSKA
1
, JADWIGA JODYNIS-LIEBERT
1
, MAŁGORZATA KUJAWSKA
1
,
TERESA ADAMSKA
1
, IRENA MATŁAWSKA
2
, and MIROSŁAWA SZAUFER-HAJDRYCH
2
1
University of Medical Sciences, Poznań, Poland
Department of Toxicology
2
University of Medical Sciences, Poznań, Poland
Department of Pharmacognosy
Abstract
Introduction: The ethyl ether extract from Aquilegia vulgaris (L.) (Ranunculaceae) contains a lot of phenolic acids. Their
hydroxyl groups are capable of donating hydrogen atoms at the initial stage of lipid peroxidation (LPO), which inactivates
hydroxyperoxides formed from polyunsaturated fatty acids (PUFAs) and leads to breakdown of the propagation chain.
Material and methods: Rats pretreated with acetaminophen (APAP) (600 mg/kg b.w., p.o.) were given ethyl ether extract
(100 mg/kg b.w., p.o.) obtained from A. vulgaris herb. The study parameters measured were microsomal lipid peroxidation,
reduced glutathione, and the activity of hepatic antioxidant enzymes and some drug metabolizing enzymes. Results:The
treatment with ethyl ether extract of the herb produced a 87–95% decrease in uninduced and Fe
2+
/ascorbate-stimulated
microsomal lipid peroxidation in the liver of rats receiving APAP. Hepatic glutathione level depleted by APAP increased
signicantly (by 18%) after the extract treatment. Antioxidant enzyme activity in the liver, inhibited by APAP, was found
to increase after administration of the extract: catalase by about 36%, glutathione reductase by 27% and glutathione
S-transferase by 29%. Glucose-6-phosphate dehydrogenase, which decreased after APAP administration, increased again
by 26% after extract treatment. The extract tested did not affect the activity of DT-diaphorase. The cytochrome P450
content, depleted by APAP, increased as much as by 100% after the treatment. The activities of NADPH-cytochrome P450
reductase, aniline hydroxylase and aminopyrine N-demethylase were not affected. Conclusions: The protective effect of the
Aquilegia vulgaris extract in APAP-induced liver injury was mediated by its antioxidant activity. The extract did not inhibit
the formation of reactive intermediate metabolites of APAP.
Key words:
Aquilegia vulgaris, Ethyl ether extract, Antioxidant enzymes, Microsomal lipid peroxidation, Reduced glutathione, Drug
metabolizing enzymes
Received: December 23, 2008. Accepted: May 25, 2009.
Address reprint request to M. Ewertowska, Department of Toxicology, University of Medical Sciences, Dojazd 30, 60-631 Poznań, Poland
(e-mail: mewertow@ump.edu.pl).
INTRODUCTION
Recently, the naturally occurring compounds that exhibit
the antioxidant or free radical scavenging potential have
attracted considerable attention. Synthetic antioxidants
have been shown to produce numerous side effects. Hence,
the therapeutic use of medicinal plants in reducing free
radical-induced tissue injury has been considered [1,2].
Aquilegia vulgaris (L.) (Ranunculaceae), syn. columbine,
is a perennial herb indigenous in the central and southern
Europe. Decoction from the leaves and stems of A. vul-
garis has been used in folk medicine against liver and bile
duct disorders, especially for the treatment of jaundice,
and chronic skin inammation. The herb is a component
of the immunostimulating preparation Padma 28 and ho-
meopathic drugs [3]. Phytochemical studies of A. vulgaris
showed the presence of cyanogenic compounds, tannins,
anthocyanins [4] and cycloartane derivatives showing im-
munosuppressive properties [5].
ORIGINAL PAPERS M. EWERTOWSKA ET AL.
IJOMEH 2009;22(2)116
ed: caffeic, ferulic, p-coumaric, protocatechuic, vanilic,
sinapic, chlorogenic and p-hydroxybenxoic.
Isocytisoside (4’-methoxy-5,7-dihydroxyavone 6-C-gluco-
pyranoside) was identied by UV and 1H NMR, 1
3
C NMR
analysis [6].
Quantitative analysis of isocytisoside and phenolic ac-
ids was performed using HPLC. Lachom-Merck chro-
matograph equipped with DAD detector and Zorbax
SB-C18 column (250×4.6 mm; 5 μm). The mobile phase
(ow rate 1 ml/min) was methanol-water-formic acid
(25:75:1 v/v) for phenolic acids analysis and (40:60:1 v/v)
for isocytisoside analysis. The standard curve for pheno-
lic acids and for isocytisoside was plotted in the range
of 2–12 μg. The content of isocytisoside in the extract
was 1.35% and the content of phenolic acids was as follows:
protocatechuic 0.20%, p-coumaric 0.03%, vanilic 0.03%.
Experimental design
Male Wistar rats (240±10 g) were divided randomly into ve
groups, eight animals each. The animals were housed in an
animal facility at 22±1°C with a 12-h light-dark cycle, con-
trolled humidity and air circulation. The substances tested
were administered intragastrically in the mixture of water
and olive oil (1:1, v/v) with a drop of Tween 20. Groups I–III
were given acetaminophen at a dose of 600 mg/b.w. Then,
after 4 h, these groups were treated as follows: group I
was given vehicle; group II, ethyl ether extract, group III,
α-tocopherol. The other two groups were given vehicle,
and after 4 h, group IV was administered ethyl ether ex-
tract, and group V, which served as control, received vehicle
again. All the substances were given at a dose of 100 mg/b.w.
The α-tocopherol, a model antioxidant, was used as a posi-
tive control. Nineteen hours after the rst treatment, the
animals were sacriced by decapitation. The livers were re-
moved, perfused with ice-cold 1.15% KCl and homogenized
in buffered sucrose solution (Tris, pH = 7.55). Microsomal
and cytosol fractions were prepared according to standard
procedure. Protein concentration in the fractions was deter-
mined using Folin-Ciocalteu reagent.
The experiment was performed according to the local Ani-
mal Ethics Committee guidelines for animal experiments.
We have isolated and identied several avonoids [6–9]
and phenolic acids [10,11] in the aerial parts of the plant
as well as alkaloids in roots [12]. The predominant com-
pound was 4’-methoxy-5,7-dihydroxyavone 6-C-gluco-
pyranoside (isocytisoside) [6]. Our previous studies have
demonstrated that ethanol and ethyl acetate extracts,
and isocytisoside isolated from A. vulgaris attenuated
the effects of CCl4- and APAP-induced hepatic injury
by restoring the activity of most of the antioxidant en-
zymes and by inhibiting microsomal lipid peroxida-
tion [13,14].
The present study was undertaken to evaluate the poten-
tial protective effects of ether ethyl extract from A. vulgaris
on APAP-induced hepatotoxicity. We aimed at elucidat-
ing the mechanism of this effect by measuring the level of
microsomal lipid peroxidation, reduced glutathione, and
the activity of hepatic antioxidant enzymes and some drug
metabolizing enzymes.
MATERIALS AND METHODS
Chemicals and plants
The chemicals used were purchased from Sigma Chemi-
cal Co. Aquilegia vulgaris (L.) stems and leaves were col-
lected in the Botanical Garden of A. Mickiewicz Univer-
sity, Poznań, Poland in June 1999. A voucher specimen is
deposited in the authors’ laboratory (No. KF 1261999).
Preparation of the extract
Air-dried and powdered leaves with stems of A. vulgaris
(50 g) were extracted seven times with methanol in hot
water bath under reux, and the extract was evaporated
under reduced pressure to afford a dark brown residue.
The residue was treated with hot water and the insoluble
part was ltered off. The ltrate was extracted with ethyl
ether. The ethyl ether extract was evaporated to dryness
(0.41 g) [15].
Phytochemical analysis
The extract was analyzed by TLC as described previ-
ously [10], and the following phenolic acids were identi-
AQUILEGIA VULGARIS AND LIVER ANTIOXIDANT DEFENSE ORIGINAL PAPERS
IJOMEH 2009;22(2) 117
in TBARS level as compared to that in rats treated with
APAP alone. Ethyl ether extract was more effective (87%
reduction) than α-tocopherol (53% reduction). The ex-
tract tested given to rats alone produced a 3-fold increase
in microsomal lipid peroxidation in both assays (Table 1).
Table 1. Effect of Aquilegia vulgaris extract on microsomal lipid
peroxidation and glutathione in the liver of APAP-treated rats
Treatment
Lipid peroxidation (nmol
TBARS/mg protein) GSH
(μmol/g tissue)
Fe2+/ascorbate Uninduced
APAP 12.0±0.4a0.67±0.17a3.8±0.8a
APAP + EEt 0.6±0.1b0.09±0.04b4.5±0.9b
APAP + α-toc 2.2±0.3b0.31±0.04b5.6±0.5b
EEt 12.8±2.3a0.71±0.11a5.6±0.4
Control 4.2±1.9 0.23±0.10 5.5±0.3
APAP — acetaminophen; EEt — ether ethyl extract; α-toc,
α-tocopherol; GSH — reduced glutathione; TBARS — thiobarbituric
acid reactive substances.
Results are mean ±SD, n = 8.
Control rats were administered vehicle only.
a
Signicantly different from control, p ≤ 0.001.
b
Signicantly different from APAP-treated group, p ≤ 0.001.
The treatment with acetaminophen alone signicantly
depleted the hepatic GSH content, by 32%, as compared
to control rats. The treatment with ethyl ether extract re-
sulted in an 18% increase in GSH level as compared to the
respective value in the APAP-treated rats. The effect of
α-tocopherol was stronger (a 48% increase). The extract
alone did not affect the level of reduced glutathione in the
liver (Table 1).
The activity of the enzymes involved in glutathione me-
tabolism in the liver, except GST, was inhibited by acet-
aminophen treatment. The response of GR was signi-
cant (a 22% inhibition) whereas the inhibition of GPx was
weaker (11%) and insignicant. Administration of the eth-
yl ether extract to the rats pretreated with APAP produced
a signicant increase in GST activity, to the level higher
than that in control rats. GR activity in the same group
also increased signicantly (by 27%). The extract did not
affect the GPx activity in the APAP-pretreated rats. The
administration α-tocopherol to APAP-pretreated rats re-
sulted in a signicant increase (by 37%,) solely in the GR
Biochemical assays
Microsomal lipid peroxidation (LPO) in the liver
was assayed in two different experimental systems:
Fe
2+
/ascorbate- stimulated peroxidation (non-enzymatic)
and uninduced peroxidation. The level of lipid peroxida-
tion was asseyed by measuring thiobarbituric acid reactive
substances (TBARS) [2].
GSH level was assayed in the liver homogenate prepared
in phosphate buffer (pH = 7.4) using the method of Sed-
lak and Lindsay [16] with Ellman’s reagent.
Glutathione peroxidase (GPx), glutathione reductase
(GR) and glutathione S-transferase (GST) activities were
assayed as described by Mohandas et al. [17]. The activity
of other enzymes was determined according to the pub-
lished methods: catalase (CAT) [18], superoxide dismutase
(SOD) [19], DT-diaphorase [20], glucose-6-phosphate de-
hydrogenase (G-6-P-D) [21].
Cytochrome P450 content was assayed with the method of
Omura and Sato [22], based on the carbon monoxide dif-
ference spectra of dithionite-reduced microsomes.
NADPH — cytochrome P450 reductase activity was mea-
sured using cytochrome c as an electron acceptor in the
presence of NADPH [22].
Aminopyrine N-demethylase activity was determined by
measuring the amount of formaldehyde formed, using the
Nash reagent [23].
Aniline hydroxylase activity was assayed by the spectro-
photometric determination of p-aminophenol, produced
as a result of aniline hydroxylation [24].
The data were expressed as mean ±SD. One-way analysis
of variance (ANOVA) followed by the Student-Newman-
Keuls test for multiple comparisons were used.
RESULTS
Non-enzymatic lipid peroxidation stimulated by
Fe
2+
/ascorbate was markedly elevated (by 184%) in
APAP-treated rats. The extract tested and α-tocopherol
reduced TBARS formation by 95% and 82%, respectively
(Table 1). Uninduced lipid peroxidation increased by 190%
after APAP administration. The treatment with ethyl ether
extract and α-tocopherol resulted in a signicant decrease
ORIGINAL PAPERS M. EWERTOWSKA ET AL.
IJOMEH 2009;22(2)118
by 26%. α-Tocopherol appeared to be more effective and
caused a 36% attenuation. When administered alone, the
extract, caused a signicant (23%) decrease in G-6-P-D
activity (Table 2).
The cytochrome P450 content decreased by 42% in the
rats intoxicated with APAP. The extract tested was found to
produce a 100% increase in the level of cytochrome P450
activity. The extract alone caused a marked decrease in
GPx and GR activity, by 43% and 47%, respectively. The
GST activity also decreased after the extract administra-
tion, but this alteration was not signicant (Table 2).
The SOD activity decreased by 24% in APAP-treated rats.
The administration of ethyl ether extract and α-tocopherol
to APAP-pretreated rats caused a further decrease in SOD
activity, by 15% (insignicant level) and 56% (signicant
level), respectively. Administration of the extract alone re-
sulted in about a 40% increase in SOD activity (Table 2).
The CAT activity was signicantly (by 27%) lower in
the APAP-treated rats. The administration of the extract
or α-tocopherol attenuated the decrease in CAT activ-
ity by about 20%. The treatment with the extract alone
caused a 28%decrease in CAT activity (Table 2).
DT-diaphorase was the only enzyme whose activity was
signicantly increased (by 41%) in APAP-treated rats.
This elevation was even greater after the administration
of the extract and α-tocopherol to APAP-pretreated rats.
Ethyl ether extract administered alone did not affect the
DT-diaphorase activity (Table 2).
APAP treatment produced a signicant decrease (by
about 52%) in glucose-6-phosphate dehydrogenase activi-
ty. The extract attenuated the decrease in G-6-P-D activity
Table 2. Effect of Aquilegia vulgaris extract on antioxidant and related enzymes in the liver of APAP-treated rats
Treatment
GPx
nmol NADPH/
min/mg protein
GR
nmol NADPH/
min/mg protein
GST
nmol CDNB/
min/mg protein
SOD
U/mg
protein
CAT
U/mg
protein
DT-diaphorase
nmol DCIP/
min/mg protein
G-6-P-D
U/mg
protein
APAP 379.8±48.8 63.4±1.9a836.0±113.7 5.4±0.4e16.0±1,8c96.4±3.3c9.9±1.7a
APAP + EEt 357.7±39.1 80.3±5.1b1077.0±134.0b4.6±0.6 21.7±0,7d117.1±16.5 12.5±0.8
APAP + α-toc 363.8±18.6 86.9±8.7b952.1±70.2 2.4±0.2b20.7±1,4f82.0±6.3 13.5±1.4f
EEt 240.9±28.8a43.0±4.3a658.1±18.0 10.2±1.2a15.7±3,3c66.2±7.5 15.8±1.9a
Control 425.2±9.1 81.6±5.6 766.1±64.8 7.2±0.4 21.8±1,6 68.2±18.1 20.6±1.9
APAP — acetaminophen; EEt — ether ethyl extract; α-toc, α-tocopherol; CDNB — 1-chloro-2,4-dinitrobenzene; DCIP — 2,4-dichloroindophenol;
CAT — catalase; SOD — superoxide dismutase; GPx — glutathione peroxidase. GR — glutathione reductase; GST — glutathione S-transferase;
G-6-P-D — glucose-6-phosphate dehydrogenase.
Results are mean ±SD, n = 8.
Control rats were administered vehicle only.
a
Signicantly different from control, p ≤ 0.001.
b
Signicantly different from APAP-treated group, p ≤ 0.001.
c
Signicantly different from control, p ≤ 0.01.
d
Signicantly different from APAP-treated group, p ≤ 0.01.
e
Signicantly different from control, p ≤ 0.05.
f
Signicantly different from APAP-treated group, p ≤ 0.05.
APAP — acetaminophen; EEt — ether ethyl extract;
α-toc — α-tocopherol.
Results are mean ±SD, n = 8.
Control rats were administered vehicle only.
a
Signicantly different from control, p ≤ 0.001.
b
Signicantly different from control, p ≤ 0.01.
c
Signicantly different from APAP-treated group, p ≤ 0.001.
d
Signicantly different from APAP-treated group, p ≤ 0.01.
Fig. 1. Effect of Aquilegia vulgaris extract on cytochrome P450
content in the liver of APAP-treated rats.
AQUILEGIA VULGARIS AND LIVER ANTIOXIDANT DEFENSE ORIGINAL PAPERS
IJOMEH 2009;22(2) 119
The level of TBARS was used as a marker of redox bal-
ance and lipid peroxidation in hepatic cells. In our experi-
ment, the TBARS level was increased in APAP-treated
rats, which is consistent with the oxidative stress theory of
APAP toxicity. The extract tested demonstrated antioxi-
dant activity in intoxicated rats in non-enzymatic, stimu-
lated lipid peroxidation as well as in unstimulated LPO
assay. The inhibition of iron-stimulated LPO noted in the
present study might involve a formation of complexes be-
tween the iron and the extract components. This would
prevent the generation of
●
OH, and thus inhibit LPO.
As mentioned earlier in the text, the ethyl ether extract
from A. vulgaris contains a lot of phenolic acids. Their hy-
droxyl groups are capable of donating hydrogen atoms at
the initial stage of LPO, which leads to the inactivation of
hydroxyperoxides formed from PUFAs. Thus, the propa-
gation chain is broken. It should be emphasized that the
ability of the extract to inhibit microsomal lipid peroxida-
tion was similar to that demonstrated by α-tocopherol, the
model antioxidant.
On the other hand, the extract alone caused about a 3-fold
increase in the TBARS level in both the LPO assays, thus
demonstrating its prooxidant activity. The possible expla-
nation of that phenomenon can be a conversion of some
compounds present in the extract into prooxidant metabo-
lites. Examples of such conversions were reported by other
authors: for ubiquinols [30] and some avonoids [1].
The role of glutathione as a protective agent against oxi-
dative organ damage has been subject to extensive stud-
ies [31]. The exposed sulfhydryl groups bind to a variety
of electrophilic radicals and metabolites that may cause
cell damage [32]. Detoxication of xenobiotics or their
metabolites is one of the major functions of glutathione.
These electrophilic compounds form conjugates with
GSH, either spontaneously or enzymatically, in reactions
catalyzed by glutathione S-transferase. Additionally, the
endogenously produced hydrogen peroxide and organic
peroxides are reduced by GSH in the presence of glutathi-
one peroxidase [33]. As it could be expected, acetamino-
phen treatment remarkably depleted hepatic GSH stores
in the rats. This may be related to a direct conjugation of
GSH with acetaminophen metabolite, NAPQI, and/or
in APAP-pretreated rats. α-Tocopherol also inhibited the
reduction of cytochrome P450 but its effectiveness was
lower, a 69% inhibition. The administration of the ex-
tract tested caused a signicant (95%) increase in cyto-
chrome P450 content (Fig. 1).
NADPH-cytrochrome P450 reductase, aminopyrine
N-demethylase and aniline hydroxylase activities were not
affected either by acetaminophen or the extract tested
(data not shown).
DISCUSSION
In order to better understand the nature and mechanism of
toxic liver injury, some models of chemical-induced lesions
were developed. The hepatotoxins used most frequently
in these models include: galactosamine, trichloroethylene,
carbon tetrachloride or acetaminophen [25]. The initial
biochemical and metabolic events of acetaminophen tox-
icity have been well described and are believed to be due
to the metabolic conversion of APAP to a highly reactive
intermediate, namely, N-acetyl p-benzoquinone imine
(NAPQI) by cytochrome P450-mediated oxidases [26].
This conversion is primarily inactivated by conjugation
with reduced glutathione [27]. At high doses, the detoxi-
cation pathways become saturated, and the intermedi-
ate metabolite accumulates and causes liver damage by
a covalent binding to tissue molecules [28]. Another the-
ory states that NAPQI is an oxidizing agent that depletes
GSH, a cellular protectant against reactive oxygen species
(ROS), thus leading to oxidative stress [29]. Our previous
investigation has demonstrated that the ethanol and eth-
yl acetate extracts as well as isocytisoside obtained from
A. vulgaris attenuated the effects of APAP- and
CCl
4
-induced hepatic injury by restoring the activity of most
of the antioxidant enzymes and by inhibiting microsomal
lipid peroxidation [13,14]. The present study was under-
taken to evaluate the potential protective effect of ethyl
ether extract from Aquilegia vulgaris on APAP-induced he-
patotoxicity in rats. We expected that both the ethyl ether
extract, containing many phenolic acids which are strong
antioxidants, and the isocytisoside would be able to pro-
tect against oxidative stress.
ORIGINAL PAPERS M. EWERTOWSKA ET AL.
IJOMEH 2009;22(2)120
defense system impaired by APAP. SOD was the only en-
zyme whose activity was induced by the extract alone. This
effect can be considered benecial since SOD plays an im-
portant role in catalyzing dismutation of superoxide radi-
cal to hydrogen peroxide and molecular oxygen, thereby
preventing the Haber-Weiss reaction that generates
●
OH.
In contrast, CAT, GR, GPx and GST activities were de-
creased (GST insignicantly) by the extract treatment
alone. As mentioned before, this reduction of antioxidant
enzyme activity could be due to the prooxidative proper-
ties of some constituents of the extract or their metabo-
lites.
Glutathione S-transferases, particularly those which be-
long to the α class, play an important role as the antioxi-
dant enzymes (they express GPx activity towards organic
hydroperoxides but not towards H
2
O
2
) in addition to
their well established role in the detoxication of elec-
trophilic xenobiotics by catalyzing their conjugation to
GSH [47,48].
The GST activity was not reduced by APAP, but it was
signicantly increased in rats treated with the extract and
α-tocopherol, to a level higher than that in the control
group. This can be considered a very benecial effect that
enhances the antioxidant status of the organism.
DT-diaphorase catalyses the conversion of quinones to hy-
droquinones in two-electron reduction with oxidation of
NADPH. This pathway is non-toxic, unlike the one-elec-
tron reduction by NADPH-cytochrome P-450 reductase
which results in the formation of a semiquinone free radi-
cal. Semiquinones are readily autooxidizable, which leads
to the oxidation of NADPH and oxidative stress. It is
known that DT-diaphorase is induced by agents that cause
oxidative stress through redox cycling (e.g. quinones, me-
nadione) [49]. In our study, the DT-diaphorase activity was
increased in APAP-treated rats. This may have been due
to oxidative stress induced in the hepatocytes by the toxic
APAP metabolite, NAPQI, and ROS [50]. This elevation
was even greater after the extract administration to APAP-
pretreated rats. It was found that some antioxidants were
the inducers of this enzyme [20]. It is likely that the an-
tioxidants present in the extract were responsible for the
slight increase in the DT-diaphorase activity. However,
to APAP-induced lipid peroxidation [29]. Our results are
consistent with other reports concerning APAP-induced
depletion of GSH [27,28,34–36].
Ethyl ether extract partially restored the GSH level in
the APAP-treated rats; however, its protective effect was
weaker than that of α-tocopherol, the model antioxidant.
It can be concluded that due to its antioxidant activity, the
extract tested causes detoxication of the toxic metabolite
of APAP as well as decreases the formation of toxic me-
tabolites of the drug. In this way, it decreases the demand
of hepatocytes for GSH which conjugates with that me-
tabolite.
The A. vulgaris extract itself did not affect the GSH level.
However, in APAP-treated rats, the effect of the extract
on restoring glutathione was remarkable. There are many
reports that the protective compound or preparation has
no capability to increase the GSH level itself, but when
administered together with a toxin, it can substantially re-
duce GSH depletion. This refers e.g. to lipoic acid and cis-
platin [37], and lycopene and T-2 toxin [38]. These ndings
might be partially explained by the regulatory mechanisms
of GSH synthesis [39]. λ-Glutamyl cysteine synthetase is
down-regulated by cellular GSH levels, and a decrease in
GSH concentration caused by the toxin treatment pro-
vides conditions for its enhanced synthesis that can be ad-
ditionally stimulated by the protective substances.
It is known that antioxidant enzymes can be inactivated
by lipid peroxides and ROS [40]. SOD is inhibited by
hydrogen peroxide, GPx and CAT through an excess of
superoxide radical [41]. It was also shown that severe oxi-
dative stress might result in the inhibition of microsomal
GST [42]. In our experiment, the activities of antioxidant
enzymes (SOD, CAT, GR, GPx) were decreased in the
rats treated with APAP. It can be suggested that the free
radicals generated by APAP inhibit antioxidant enzymes,
which is consistent with the theory of oxidative stress as
a mechanism of APAP toxicity. Similar results were re-
ported by other authors who observed a decreased activ-
ity of antioxidant enzymes in the liver of rats treated with
APAP [27,36,43–46]. The extract tested restored the ac-
tivity of CAT, GR and GST in APAP-treated rats, which
at least in part, enhanced the activity of the antioxidant
AQUILEGIA VULGARIS AND LIVER ANTIOXIDANT DEFENSE ORIGINAL PAPERS
IJOMEH 2009;22(2) 121
the extract tested restored the level of cytochrome P450
by 100%. The efciency of the extract alone was also very
high. It is plausible that the components of the extract not
only protect cytochrome P450 against free radical insult
but they may also stimulate the synthesis of this enzyme
under physiological conditions.
The activities of NADPH-cytrochrome P-450 reductase,
aminopyrine N-demethylase and aniline hydroxylase,
were not affected by acetaminophen or the extract tested.
It can be concluded that the extract had no inuence on
the production of the toxic metabolites of APAP.
Our results demonstrated that the ethyl ether extract from
Aquilegia vulgaris attenuated the effects of APAP-induced
hepatotoxicity by restoring the activity of some antioxidant
enzymes and by inhibiting microsomal lipid peroxidation.
On the other hand, when administered alone, the extract
produced disturbances in the antioxidant defense system,
by decreasing the activity of some antioxidant enzymes
and increasing lipid peroxidation in the liver.
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this assumption was not conrmed in our experiment be-
cause the extract alone did not affect the activity of the
enzyme.
G-6-P-D serves an important function in acquiring toler-
ance against oxidative stress since its activity is a factor
limiting the rate of NADPH synthesis within the pentose
phosphate pathway. NADPH is a coenzyme involved
in the reduction of GSSG to GSH which is catalyzed by
glutathione reductase [51]. In our experiment, the activ-
ity of G-6-P-D was decreased by APAP administration,
which seems to be compatible with GSH depletion. In
the APAP-treated rats, the extract tested did not change
signicantly the activity of G-6-P-D. However, the admin-
istration of the extract alone brought about a signicant
decrease in this activity.
Generally, the effect of α-tocopherol (a positive control)
on the antioxidant enzyme activity in APAP-treated rats
was similar to that demonstrated by the extract tested.
The mechanism by which APAP induces liver injury in-
volves its biotransformation by the liver microsomal cyto-
chrome P450, especially CYP2E1, to form NAPQI [52].
Hence, we attempted to examine whether the extract test-
ed could decrease the activation of APAP by suppressing
some phase I drug-metabolizing enzymes. We determined
total cytochrome P450 content, NADPH-cytochrome P450
reductase as well as the activity of two monooxygenases.
The activity of aniline hydroxylase is known to be main-
ly CYP2E1-dependent [53]. Recent studies have shown
that mainly the CYP2E1 accounted for the formation of
NAPQI, while the contribution of other isoforms of cyto-
chrome P450, such as CYP1A2 and CYP3A, appeared to
be negligible [52]. Aminopyrine is used as a non-specic
substrate for measuring the hepatic metabolic capacity of
the cytochrome P450 system. It was found that a number
of monooxygenases were involved in aminopyrine metab-
olism but with a slight to moderate weight of catalysis car-
ried by CYP1A2 [54].
In our study, the cytochrome P450 content was signicantly
decreased in the rats intoxicated with APAP. The free radi-
cals which are generated during APAP biotransformation
are thought to be responsible for the inactivation of the
enzyme [29,49]. In the rats receiving APAP pretreatment,
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