Journal of Ethnopharmacology 97 (2005) 351–358

Protective effect of Aquilegia vulgaris (L.) on APAP-induced oxidative stress in rats
Jadwiga Jodynis-Lieberta , Irena Matławskab , Wiesława Bylkab , Marek Muriasa,∗
b a Department of Toxicology, University of Medical Sciences, Dojazd 30, 60 631 Pozna´ , Poland n Department of Pharmacognosy, University of Medical Sciences, Sieroca 10, 61 771 Pozna´ , Poland n

Received 24 May 2004; received in revised form 18 November 2004; accepted 19 November 2004 Available online 12 January 2005

Abstract Rats pretreated with acetaminophen (N-acetyl-p-aminophenol, APAP) (600 mg/kg b.w., p.o.) were administered with ethanol and ethyl acetate extracts as well as with isocytisoside (100 mg/kg b.w., p.o.) obtained from Aquilegia vulgaris (L.) (Ranunculaceae) herb. The substances tested decreased enzymatic, non-enzymatic and uninduced microsomal lipid peroxidation (LPO) in the liver of rats treated with APAP by 18–48%. Activity of the antioxidant enzymes in the liver inhibited by APAP was increased in the majority of groups after administration of the substances tested: catalase (CAT) by 55%, glutathione peroxidase (GPx) by 50%, glutathione reductase (GR) by 35% and glutathione S-transferase (GST) by 60%. Hepatic glutathione level depleted by APAP was only slightly increased by the substances tested. The cytochrome P450 contents, and the activities of NADPH–cytochrome P450 reductase and two monooxygenases were not affected by the extracts and isocytisoside. It can be concluded that the protective ability of the substances tested in APAP-induced liver injury is mediated by amelioration of microsomal lipid peroxidation and restoring antioxidant enzymes activity. Inhibition of enzymes responsible for metabolic activation of APAP is not involved in this process. © 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Aquilegia vulgaris; Antioxidant enzymes; Microsomal lipid peroxidation; Reduced glutathione

1. Introduction There is increasing interest in the antioxidants of natural origin because they could suppress the oxidative damage of a tissue by stimulating the natural defence system. These substances, e.g., flavonoids and spice principles can serve as chemopreventive agents ameliorating the toxicity caused
Abbreviations: ADP, adenosine diphosphate; APAP, N-acetyl-paminophenol; CAT, catalase; CDNB, 1-chloro-2,4-dinitrobenzene; DPPH, diphenyl-p-picrylhydrazyl; GPx, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione S-transferase; LPO, lipid peroxidation; NADPH, nicotinamide adenine dinucleotide phosphate reduced; NAPQI, N-acetyl-p-benzoquinoneimine; PUFAs, polyunsaturated fatty acids; ROS, reactive oxygen species; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances ∗ Corresponding author. Tel.: +48 618 470721. E-mail address: (M. Murias). 0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.11.027

by certain drugs and environmental chemicals or in disease states involving oxidative stress. Aquilegia vulgaris (L.) (Ranunculaceae), syn.: columbine, is a perennial herb indigenous in central and southern Europe. Decoction from leaves and stems of Aquilegia vulgaris has been used in folk medicine against liver and bile duct disorders, especially for the treatment of jaundice, and chronic skin inflammation. The herb is a component of the immunostimulating preparation Padma 28 and homeopathic drugs (PDR for Herbal Medicines, 2000). Phytochemical studies of Aquilegia vulgaris showed the presence of cyanogenic compounds, tannins, anthocyanins (H¨ nsel et al., 1992) and cycloartane a derivatives showing immunosuppresive properties (Nishida et al., 2003). We have isolated and identified several flavonoids (Bylka and Matławska, 1997a,b; Bylka, 2001; Bylka et al., 2002) and phenolic acids (Drost-Karbowska et al., 1996) in aerial parts

20a 0.5 3.D.6 318.6 3. therefore. We attempted to examine two major possibilities: (1) whether Aquilegia vulgaris protects against hepatotoxicity of APAP by enhancing cellular defence mechanisms (2) whether Aquilegia vulgaris decreases the bioactivation of APAP by suppressing P450 enzymes.5b 4.0 5.0 21. EAE.00 ± 0. p ≤ 0.1 40. p ≤ 0.3 ± 2.8a 8.20b 0. responsible for hepatoprotection observed in the previous experiment.5 CCl4 /NADPH 53.4 ± 4.2b 42.1 37.8 34.3 ± 40.2b 209.8 ± 6.2 ± 2.65 1.4a 7. thus leading to oxidative stress.0b 10.81 CAT (U mg−1 protein) 26.9 ± 32. isocytisoside.1 ± 7.1 5.8 ± 20.1b 4.4 22.7 ± 6.4 34. Control rats were administered vehicle only APAP.38 2. 1997a).72 5. b Significantly different from APAP-treated group.5 Uninduced 2.1 ± 8.7 30. -tocopherol.8 ± ± ± ± ± ± ± ± ± 9. p ≤ 0.6b 5. Aquilegia vulgaris is rich in compounds known to be strong antioxidants.5 4.4 6.43 1.0 1.1 40. at least in part.9 5.78 4.6b 35.3 ± 7.1 ± 6.2a 272.2 20.0b 62.0a 5. The present study was undertaken to evaluate the potential protective effect of extracts and isocytisoside isolated from Aquilegia vulgaris on APAP-induced hepatotoxicity and to elucidate the mechanisms underlying these effects in rat.1 GST SOD (nmol CDNB min−1 (U mg−1 protein) mg−1 protein) 197.9 54. the hepatoprotection by Aquilegia vulgaris could be due to alterations in disposition and biotransformation of hepatotoxicant.8 36.8 ± ± ± ± ± ± ± ± ± 0.33b 0.. There is much evidence to substantiate both theories and the question may be to what extent each plays a role in APAP toxicity (Gibson et al.5b 16. 1996).2a 5. ethyl acetate extract. isocytisoside. Table 2 Effect of Aquilegia vulgaris extracts and isocytisoside on antioxidant and related enzymes in APAP-treated rats Treatment GPx (U min−1 mg−1 protein) 43.2b 69.0 6.2b 23.6 20. ethanol extract..40 ± 1.9 ± 4.88 ± 0.9 0.4 39.4 262.6 1.4 ± 6.5a 0.5 22. EE. EAE.7a 4.1a 46.7 4. Jodynis-Liebert et al.39 0. p ≤ 0. IST.0 50.46 1.47 2. The hepatoprotective activity of natural substances is often associated with their capability of suppressing the effects of oxidative damage.2 32.1 41. this metabolite is detoxified by reduced glutathione (GSH). 1998).6 22.9b 39.4 ± ± ± ± ± ± ± ± ± 14.00a 4. .3b 8.10 ± 0.1b 6.0a 41..6 ± 31. as well as cellular response and regenerating processes (Mehendale et al.9b 9.51 1.6 23.3 38.25b 0.1b 316.8a 49.39 ± 0.8 48. ethyl acetate extract. The predominant compound was 4 -methoxy5.4b 5.5 65.4 56.6 39.6 ± 3.2a 5. APAP toxicity is known to be mediated by biotransformation in the liver by the microsomal P450 system to the highly reactive N-acetyl-p-benzoquinoneimine (NAPQI).2 ± 5. 2003). 1994).8 44. n = 8.4 33.4 ± 45.5 ± 18.2 DT-diaphorase (nmol DCIP min−1 mg−1 protein) 47.05 1.10a 6.6a 9. a Significantly different from control.5 ± 3. it could be expected that this very property is. n = 8.352 J.5 ± 28.. b Significantly different from APAP-treated group.2 46.34a 0.D.6b 0. The parameters analysed included reduced glutathione content.3 38.2 42. alterations in cellular detoxifying mechanisms.9 63. Following toxic doses of APAP.20 ± 0.0 ± 6.1 4.4 ± 36. GSH is depleted and the metabolite covalently binds to cellular macromolecules resulting in liver injury.2b 55.66 5.3 3.05.3 51.2 ± 6.60 3.88 1.7 4.6 ± 4. Control rats were administered vehicle only APAP.3 ± ± ± ± ± ± ± ± ± 6.91 6.0 ± 2.6 ± 7.12 ± 0. ethanol extract.3 ± 8.4 30.3 ± 3.5 ± 2. -tocopherol.0 ± 9.2b 4. a Significantly different from control.5 ± 6. Theoretically.89 ± 0.5 0.1 ± 7. -toc.37 ± ± ± ± ± ± ± ± ± 0. a cellular protectant against reactive oxygen species (ROS).8 ± 8.0 ± 5. acetaminophen..86 2. At therapeutic doses.8 20.05.. acetaminophen. Our previous investigation has demonstrated that ethanol extract (EE) of Aquilegia vulgaris and isocytisoside could protect against hepatotoxicity induced by carbon tetrachloride in rats as assessed by inhibition of transaminases and sorbitol dehydrogenase leakage to serum and by histopathological examination (Adamska et al. Acetaminophen (APAP) poisoning is one of the most widely used in vivo experimental models to induce liver damage.8 317.1 ± 9. -toc.4b 363.3 64.7-dihydroxyflavone 6-C-glucopyranoside (isocytisoside) (Bylka and Matławska.3 51.5b 11.7 GSH ( mol/g tissue) Results are mean ± S.2b 62.21a 0.5 0.73b 4.0 329.0 35.7 40.5 67.0 ± 5.3 37.2 3. EE. microsomal lipid peroxidation (LPO).0 0. IST.32 ± 1.6 5.38b 0.6 ± 6.7 ± 4.3 5.3 GR (nmol NADPH min−1 mg−1 protein) 17.3 ± 6. of the plant as well as alkaloids in roots (Szaufer-Hajdrych et al.8 ± 3.4 ± 3.4 72. / Journal of Ethnopharmacology 97 (2005) 351–358 Table 1 Effect of Aquilegia vulgaris extracts and isocytisoside on microsomal lipid peroxidation and hepatic glutathione in APAP-treated rats Treatment Lipid peroxidation (nmol TBARS/mg protein) Fe3+ /ADP/NADPH APAP APAP + IST APAP + EAE APAP + EE APAP + -toc IST EAE EE Control 77.0 ± 42.6 ± 8.8 Fe2+ /ascorbate 53.9b 6.8 55.5 ± 4.2 35.3 43. Another theory states that NAPQI is an oxidising agent that depletes the cell of GSH.9 APAP APAP + IST APAP + EAE APAP + EE APAP + -toc IST EAE EE Control Results are mean ± S.

group V. Jodynis-Liebert et al. ethyl acetate extract. which served as control. A voucher specimen is deposited in the authors’ laboratory (No. 2. Aquilegia vulgaris (L.15% KCl and homogenised in buffered sacharose solution (Tris. Chemicals and plant material The chemicals used were purchased from Sigma Chemical Co. The ethyl acetate subextract was evaporated to dryness (1.55). n Poland in June 1999. 2. apigenin 7-O-glucoside and apigenin. 2001). The standard curve was made in the range 2–12 g. the extracts contained: isocytisoside 7-O-glucoside. The results were expressed in mol per g tissue. 2. Hydrogen peroxide was used as a substrate. The rats were housed in an animal facility at 22 ± 1 ◦ C with 12-h light–dark cycle. group VII.5% (unpublished data).J. Pozna´ . The results were expressed in nmol malondialdehyde per mg protein (Sanz et al.3. The livers were removed.7dihydroxyflavone 6-C-glucopyranoside) was isolated from methanol extract by column chromatography and identified by UV and NMR analysis (Bylka and Matławska. The content of isocytisoside in ethyl acetate subextract was 5% and in ethanol extract 1. apigenin 7-O-rutinoside. which was expressed as nmol NADPH oxidized per min per mg protein. 5 m) was used.1. The experiment was performed according to the Local Animal Ethics Committee guidelines for animal experimentation. Ethyl acetate subextract (EAE): Dried leaves and stems (50 g) were extracted seven times with boiling methanol and the combined extracts were concentrated under reduced pressure. Microsomal and cytosol fractions were prepared by differential centrifugation according to the standard procedure.) stems and leaves were collected in the Botanical Garden of A. Nineteen hours after the first treatment animals were sacrificed by decapitation.w. isovitexin 4 -O-glucoside.4) by the method of Sedlak and Lindsay (1968) with Ellman’s reagent.. p-coumaric. pH 7.Groups I–V were given acetaminophen at a dose 600 mg/kg b. group IX. vehiculum again. Preparation of extracts and isolation of isocytisoside Ethanol extract: Dried leaves and stems of Aquilegia vulgaris (50 g) were extracted three times with boiling 70% ethanol to yield 12. sinapic and chlorogenic (Drost-Karbowska et al. Then after 4 h these groups were treated as follows: group I was given vehiculum. Other four groups were given vehiculum and after 4 h group VI was administered with isocytisoside. (ii) CCl4 /NADPHstimulated peroxidation—enzymatic.5. orientin. vanilic. 1997a. 2.6. perfused with ice-cold 1. The mobile phase was methanol–water–formic acid (40:60:1) at a flow rate 1 ml/min. Protein concentration in the fractions was determined using Folin–Ciocalteu reagent. Quantitative analysis of isocytisoside was performed by HPLC method. isoorientin.. group III. / Journal of Ethnopharmacology 97 (2005) 351–358 353 hepatic antioxidant enzymes and some drug metabolizing enzymes activities. Reduced glutathione and cytosolic enzymes assay GSH level was assayed in the liver homogenate prepared in phosphate buffer (pH 7.2. isocytisoside. (iv) uninduced peroxidation. ethyl acetate extract. pH 7.4. p-hydroxybenzoic. All substances were given at a dose 100 mg/kg b. ferulic. 2003). The activity was expressed in nmol NADPH oxidized per min per mg protein (Mohandas et al. eight animals each. group IV. con- trolled humidity and circulation of air. The disappearance of NADPH at 340 nm was a measure of enzyme activity. (1984). ethanol extract.. Glutathione reductase (GR) was assayed by measuring NADPH oxidation at 340 nm using oxidized glutathione as a substrate. Materials and methods 2. Lipid peroxidation assay Microsomal lipid peroxidation in the liver was assayed in four different experimental systems: (i) Fe3+ /ADP/NADPHstimulated peroxidation—enzymatic.b. -tocopherol. Isocytisoside predominated in both extracts. Besides. 1984). ethanol extract. Glutathione peroxidase (GPx) activity was determined according to Mohandas et al.6 mm. resorcylic. Mickiewicz University. 1994). treated with hot water and filtered. was used as a positive control..1 g of dry residue as described previously (Adamska et al. -Tocopherol.5 g).4. v/v) with a drop of Tween 20. 2. Additionally the ethanol extract contained phenolic acids: caffeic. Phytochemical analysis The extracts were analysed by TLC as described previously (Bylka and Matławska. 1997a). Lipid peroxidation was evaluated by measuring thiobarbituric acid reactive substances (TBARS). KF 1261999). The filtrate was successively extracted with ethyl ether and ethyl acetate. Liver homogenate for glutathione determination was prepared in phosphate buffer. (iii) Fe2+ /ascorbatestimulated peroxidation—non-enzymatic. . The substances tested were administered intragastrically in the mixture of water and olive oil (1:1. Experimental design Male Wistar rats (240 ± 10 g) were divided randomly into nine groups. Bylka.w. a model antioxidant. group VIII. group II. Isocytisoside (4 -methoxy-5. The dose of APAP and the period between drug treatment and rat decapitation were sufficient to develop evident liver injury but not severe enough to cause death. 1996). Lachom-Merck chromatograph equipped with DAD detector and Zorbax SB-C18 column (250 mm × 4. 2.

Glutathione S-transferase (GST) activity measurement was based on the spectrophotometric determination of 1chloro-2.. The preparations tested alone caused the inhibition of microsomal LPO in all assays. The substances tested alone caused a slight. The activity was expressed as nmol CDNB conjugated with GSH per min per mg protein (Mohandas et al. respectively. The substances tested attenuated this depletion to a different degree. Aminopyrine N-demethylase activity was determined by measuring the amount of formaldehyde formed using the Nash reagent (Pedemonte et al. The lowest activity was observed for isocytisoside. Isocytisoside and ethanol extract showed weaker activity. Non-enzymatic LPO stimulated by Fe2+ /absorbate was less affected by APAP treatment—23% elevation (insignificant) of TBARS level was recorded. Uninduced microsomal LPO was insignificantly elevated in the APAP-treated animals. All enzymes involved in glutathione metabolism in the liver were inhibited by acetaminophen treatment..354 J. to the level observed in control group. Ethyl acetate extract caused an about 10% increase in the GPx and GR activity but this alteration was not significant (Table 2). respectively. Jodynis-Liebert et al. however. whereas inhibition of GR was weaker (17%) and insignificant. Administration of isocytisoside and ethyl acetate extract to the rats pretreated with APAP caused a significant elevation of GR activity to the level higher than that in control rats. respectively. The rate of H2 O2 reduction was a measure of CAT activity. TBARS levels were increased moderately by 27 and 34%. particularly in ethyl acetate extract treated rats in which the enzyme activity was higher than in controls. from 18 to 37% (Table 1). namely Fe3+ /ADP/NADPH and CCl4 /NADPH. Ethanol extract did not affect the GST and GR activity in the APAP pretreated rats.6-dichloroindophenol at 600 nm in the presence of NADPH (Benson et al. The effectiveness of -tocopherol was the same as that of isocytisoside and insignificant. 1984). 38 and 34% inhibition. The only exception was the group of rats treated with APAP plus ethyl acetate extract . the substances tested alone did not affect glutathione-related enzymes. Statistical analysis The data were expressed as mean ± S. However. the extracts being more active than isocytisoside (Table 1). the standard curve of SOD activity was used.4-dinitrobenzene (CDNB) conjugate formed in a GSH coupled reaction. -Tocopherol administration to APAP pretreated rats caused significant increase in all three enzymes activity by 23–60% (Table 2). The degree of enzymatic LPO inhibition by all substances tested including -tocopherol was similar. GST activity in the same groups was also significantly increased (by 38 and 84%.4-dichloroindophenol (DCIP) reduced per min per mg protein. 1996). Aniline hydroxylase activity was assayed by the spectrophotometric determination of p-aminophenol. especially in CCl4 /NADPH stimulated and in uninduced LPO (up to 43%). One unit of CAT reduces 1 M of H2 O2 . DT-diaphorase activity was assayed by measuring the reduction of 2. Inhibition of spontaneous epinephrine oxidation was a measure of SOD activity. / Journal of Ethnopharmacology 97 (2005) 351–358 Catalase (CAT) activity was determined according to Beer and Sizer (1952). these values were not statistically significant. 1981). Superoxide dismutase (SOD) activity was determined by the method of Sun and Zigman (1978). The results were expressed in units per min per mg protein. The response of GST and GPx was significant. Microsomal enzymes assay Cytochrome P450 content was assayed by the method of Omura and Sato (Chapman et al. The pattern of results was somewhat different for GPx activity. Both extract significantly elevated the activity of the enzyme by 56 and 48%. 15–21% (Table 1)..D.. Ethyl acetate extract was the most effective and caused a significant increase in GSH level by 47% as compared to that in the APAP-treated rats. Generally. Administration of the substances tested and tocopherol caused a very significant reduction of TBARS levels (28–49%) below the values observed in the control group. insignificant increase in GSH level.7. Acetaminophen treatment alone significantly depleted hepatic GSH content by 48% as compared to control rats. Almost all substances tested (except isocytisoside in CCl4 /NADPH stimulated assay) caused a significant decrease in the TBARS levels in APAPtreated rats in both stimulating systems. in APAP-treated rats.8. In two enzymatically-driven assays. 2. all substances tested including -tocopherol inhibited TBARS formation (by 28–42% as compared to the APAP-treated rats) to the levels lower than that in control rats. respectively). The activity was expressed in nmol 2. One-way analysis of variance (ANOVA) followed by the Student– Newman–Keuls test for multiple comparisons were used.. 1980). 35 and 29% increase. For calculation. 3.. 2. 1989). SOD activity was affected neither by APAP nor by the substances tested (including -tocopherol) when administered to the rats pretreated with APAP. Results Two systems of enzymatic stimulation of microsomal lipid peroxidation were used. NADPH–cytochrome P450 reductase activity was measured using cytochrome c as an electron acceptor in the presence of NADPH (Chapman et al. 1989) based on the carbon monoxide difference spectra of dithionite-reduced microsomes. Isocytisoside appeared to be less effective causing a slight insignificant increase in GPx activity. produced as a result of aniline hydroxylation (Bourrie et al.

in APAP-treated rats the effect of substances tested on GSH restoring was more pronounced. and thus the propagation chain is broken. aniline hydroxylase and aminopyrine N-demethylase activities were affected neither by acetaminophen nor by the Aquilegia vulgaris extracts.. 1995). bromobenzene and allyl alcohol (Casini et al. 1987). it may be concluded that constituents of the extracts and isocytisoside are capable of scavenging free radicals generated both by NADPH and Fe2+ /ascorbate. this elevation was significant only in enzymatically-driven lipid peroxidation assays. However. Thus. 1994. However. however. respectively.2. Reduced glutathione 4. Carbon tetrachloride is oxidized by cytochrome P450 dependent monooxygenases to form the trichloromethyl radical which initiates lipid peroxidation. NADPH-dependent LPO is catalyzed by the NADPH–cytochrome P450 reductase and propagated by cytochrome P450 with the generation of free radicals. There are many observations that the protective compound or preparation has no capability of enhancing GSH level itself. the data are not presented. Jodynis-Liebert et al.J. when administered alone. 1990). The enzymatic. i. tested could be associated with their radical scavenging ability.1. 4. the mechanism of inhibition of iron-stimulated LPO observed in the present study might involve the formation of complexes between iron and components of the extracts. in our study the TBARS level was increased in APAP-treated rats. All substances tested caused a decrease in the TBARS level in almost all systems of stimulation as well as in unstimulated LPO assay. NAPQI and/or to APAP induced lipid peroxidation (Gibson et al. Thus. It was found that antioxidants used inhibited lipid peroxidation and prevented the cells death. by 56%. -tocopherol. 1990). deferoxamine after pretreatment with prooxidants. Lipid peroxidation We applied different systems to assess the effectiveness of substances tested in preventing lipid peroxidation: irondependent and iron-independent. enzymatically-driven and non-enzymatic. The increase caused by tocopherol was even smaller. 36%. the model antioxidant. (1988).. Consistently with the oxidative stress theory of APAP toxicity. Our results agree with the other reports pertaining to APAP-induced GSH ¨ depletion (Ozdemirler et al.g. Cytochrome P450 content. This might suggest that the mechanism of inhibition of NADPH-dependent LPO by the substances Reduced glutathione is well known to protect liver cells against oxidative stress through non-enzymatic and enzymatic reactions (Reed. The substances tested did not reduce significantly this elevation. induced a slight increase (by 16–24%) in the activity of DT-diaphorase (Table 2).. Hence. they did not restore the GSH level. on GSH was not more pronounced than that of the substances tested.e. Their hydroxyl groups are capable of donating hydrogen atoms in the initial stage of LPO. They added model antioxidants N. O2 •− and ROO• (Sevanian et al. As may by expected acetaminophen treatment caused the remarkable depletion of cellular GSH in rats.. This observation was confirmed by Miccadei et al. Both extracts administered alone caused an insignificant (about 20%) elevation in the SOD activity (Table 2). Fe2+ /ascorbate system stimulates effectively non-enzymatic lipid peroxidation. Only ethyl acetate extract protected significantly the GSH level in the APAP-treated rats. antioxidant efficacy of the extracts and isocytisoside was similar to that showed by the model antioxidant. Although both extracts and isocytisoside were very weak • OH and DPPH radical scavengers they appeared to scavenge effectively superoxide anion and showed iron chelating ability (unpublished data). e. 51 and 62%. / Journal of Ethnopharmacology 97 (2005) 351–358 355 in which a small but significant increase (by 24%) was observed. Enzymatic lipid peroxidation can be stimulated by different systems. however.. DT-diaphorase was the only enzyme whose activity was significantly increased in APAP-treated rats. We have found that the substances tested neither decrease the content of cytochrome P450 nor the activity of NADPH–cytochrome P450 reductase in APAP-treated rats. although only in the ethyl acetate group this increase was significant. Similar effects were observed in rats administered with iron chelator. Ferric/ADP is an effective iron complex catalyzing microsomal LPO in the presence of reductants such as NADPH. and thus inhibits LPO. lipoic acid and cisplatin . NAPDH–cytochrome P450 reductase. The CAT activity was significantly (by 52%) lower in the APAP-treated rats. This may be related to its direct conjugation with acetaminophen metabolite. Lores Arnaiz et al. it can be concluded that antioxidant activity is not essential in restoring the GSH level. both extracts themselves reduced significantly enzymatically-driven and unstimulated LPO. isocytisoside did not affect this parameter. Aquilegia vulgaris extracts contain many polyphenolic compounds. The protective effect of -tocopherol. however.. This would prevent from the generation of • OH.. which inactivate hydroxyperoxides formed from PUFAs. The administration of substances tested markedly attenuated the decrease in the CAT activity by 57. Hence. The extracts and isocytisoside alone did not affect the activity of CAT (Table 2). however. Our studies in vitro confirmed this suggestion. Discussion 4. In all LPO stimulating systems. Aquilegia vulgaris extracts themselves enhanced the GSH level to a small degree. 1996). Two other substances tested and -tocopherol caused a slight and insignificant elevation of GSH content. when administered together with a toxin it can substantially reduce GSH depletion.N -diphenyl-pphenylenediamine and deferoxamine to hepatocytes culture in which GSH depletion was evoked by dinitrofluorobenzene or diethyl maleate.

a decrease of GSH concentration caused by toxin treatment creates conditions for its enhanced synthesis which can be additionally stimulated by protective substances. (1995) who observed the decrease in GPx and CAT activ¨ ity in rats administered with APAP and by Ozdemirler et al. It can be suggested that the high GST activity caused quick detoxication of active APAP metabolites. enhanced antioxidant defence system impaired by APAP. 1980).. which is consistent with the theory of oxidative stress as a mechanism of APAP toxicity. 1999). in protecting antioxidant enzymes is comparable. NADPH–cytochrome P450 reductase as well as two monooxygenases activity.g. The GST activity reduced by APAP was most increased in the ethyl acetate extract treated rats up to a level higher than that in the control group and consequently in the same group a significant elevation of the GSH level was observed. Antioxidant enzymes It is known that antioxidant enzymes can be inactivated by lipid peroxides and ROS (Halliwell and Gutteridge. 1993). glutathione S-transferases also express GPx activity toward organic hydroperoxides but not toward H2 O2 . A slight insignificant enhancement in the DT-diaphorase activity was caused by the substances tested alone. These observations might be partly explained on the basis of the regulatory mechanisms of GSH synthesis (Reed. we attempted to examine whether the substances tested were capable of decreasing the activation of APAP by suppressing some phase I drug metabolizing enzymes. (1989). quinones.. neither isocytisoside nor the extracts tested (containing apigenin. 1996). SOD is inhibited by hydrogen peroxide. GR and CAT but restored the activity of these enzymes in APAP-treated rats. Only the class of GST expresses the Se-independent GPx activity (Zhao et al.g. 4. It can be suggested that free radicals generated by APAP inhibit antioxidant enzymes. however. Drug metabolizing enzymes APAP toxicity is believed to be mediated via cytochrome P450-generated active metabolites. It is known that DT-diaphorase is induced by agents that cause oxidative stress through redox cycling (e. chrysin. Our study confirms these observations. Nevertheless. In our study. SOD did not appear to be susceptible to oxidative stress produced by APAP. Hence. Semiquinones are readily autooxidizable. CYP2E1 and 1A2 are the major enzymes involved in the activation of APAP (Gibson et al.. This pathway is non-toxic unlike the one-electron reduction by NADPH–cytochrome P450 reductase that results in the formation of a semiquinone free radical. GPx and CAT – by an excess of superoxide radical (Pigeolet et al. 1996). the substances tested did not reduce significantly the enzyme activity. DT-diaphorase catalyses conversion of quinones to hydroquinones in two-electron reduction with oxidation of NADPH.. lycopene and T-2 toxin (Leal et al. 2000). The substances tested in our study showed no effect on any of these enzymes both alone and in the APAP-treated . According to the observations of Siess et al. The activity of aniline hydroxylase is known to be mainly CYP2E1 dependent (Jayyosi et al. It could be expected that the same effect might be caused by toxic APAP metabolite. Aminopyrine is used as a non-specific substrate for measuring the hepatic metabolic capacity of the cytochrome P450 system.4. SOD was the only enzyme whose activity was induced by the substances tested alone. Jodynis-Liebert et al. We discussed them in the section concerning antioxidant enzymes because of their particular roles in detoxication of free radicals. however. It proves that effectiveness of both. Substances tested alone did not affect GPx. Probably antioxidants present in the extracts were responsible for the slight increase in the DT-diaphorase activity.356 J. It was found that a number of monooxygenases were involved in aminopyrine metabolism but with a slight weight of catalysis carried by CYP1A2 (Sharer and Wrighton. NAPQI. only the unsubstituted flavonoids gave rise to a significant induction of GST activity in the liver of rodents whereas no increase in hepatic GST was observed for the hydroxylated and methoxylated flavonoids. and thus the GSH level was protected..3. In accordance with these findings. 1999). We determined total P450 content.. GST and DT-diaphorase are classified both as antioxidant and phase II drug metabolizing enzymes. Similar results were reported by Lores Arnaiz et al. Acetaminophen treatment caused a decrease in the activity of antioxidant enzymes and GST.. this decrease was not significant for GR and SOD. It was found that some antioxidants were inducers of this enzyme (Benson et al. which leads to oxidation of NADPH and oxidative stress. -tocopherol effect on antioxidant enzymes activity in APAP intoxicated rats was very similar to that demonstrated by the substances tested. 4. 1990). only ethyl acetate extract increased SOD activity in APAP pretreated rats. The substances tested alone did not induce GST activity. orientin and derivatives) were capable of enhancing the GST activity in the liver of rats. 1990). thus. e. However. quercetin or tangeritin. Aquilegia vulgaris extracts and the model antioxidant. In addition to Se-dependent GPx. Generally. menadione) (Parkinson. (1994) reporting decreased GST but unchanged GPx activity in APAP-treated mice. 1984).. -Glutamyl cysteine synthetase is down-regulated by cellular GSH levels. GST catalyses conjugation of GSH with reactive metabolites favouring their elimination from the organism. Glutathione S-transferases made a family of multifunctional proteins that function in cellular detoxication and transport. / Journal of Ethnopharmacology 97 (2005) 351–358 (Somani et al. 1995). 1995). It was also shown that severe oxidative stress might result in the inhibition of microsomal GST (Aniya and Naito.. increased activity of DT-diaphorase in APAPtreated rats was observed.

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