Journal of Ethnopharmacology 103 (2006) 455–460

Effect of Cistus laurifolius L. leaf extracts and flavonoids onacetaminophen-induced hepatotoxicity in mice

Esra Kupeli∗, Didem Deliorman Orhan, Erdem YesiladaGazi University, Faculty of Pharmacy, Department of Pharmacognosy, Hipodrom 06330, Ankara, Turkey

Received 26 April 2005; received in revised form 15 August 2005; accepted 19 August 2005Available online 10 October 2005

Abstract

In this study, the effect of the flavonoids quercetin-3-methyl-ether (isorhamnetin) (1), quercetin-3,7-dimethyl-ether (2) and kaempferol-3,7-dimethyl-ether (3) isolated fromCistus laurifolius L. (cistaceae) leaves was assessed on lipid peroxidation (liver and plasma), cellular glutathione(GSH) level and plasma AST (aspartate aminotransferase), ALT (alanine aminotransferase) enzyme activities in acetaminophen-induced liverdamage in mice. At 114 mg/kg oral dose quercetin-3,7-dimethyl-ether was shown to possess potent antioxidative activity.© 2005 Elsevier Ireland Ltd. All rights reserved.

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eywords: Cistus laurifolius; Cistaceae; Flavonoid; Hepatotoxicity; Lipid peroxidation; Serum transaminase enzymes; Cellular GSH

. Introduction

The genusCistus is one of the characteristic genera of theediterranean region, colonizing degraded areas (Attaguile etl., 2000). Cistus laurifolius L. (cistaceae) is a common plant

n Anatolia and is used against various ailments in traditionaledicine. The plant leaves are used to treat rheumatic and related

nflammatory diseases, externally as a bath or poultice to reduceain in rheumatism, against fever in common cold or appliedxternally as a plaster on the dorsal part of the body in a line ofhe kidneys for urinary inflammations (Yesilada et al., 1997a).

tea prepared from the leaves is used as hypoglycaemic and theowers and buds decoction is used for the treatment of pepticlcers in Turkish folk medicine (Sezik et al., 1991; Yesilada etl., 1995). In previous phytochemical studies,Cistus laurifoliusave been reported to contain flavonoids including quercetin,aempferol, apigenin, luteolin and theirs methyl–ethers, theoumarine scopoletin, diterpenoids, sesquiterpenoids, and sug-rs (Demetzos et al., 1989; Vogt et al., 1987; Wollenweber andann, 1984).Due to the widespread utilization of the plant as a traditional

risks to users as well as to disclose the possible antioxeffect in the folkloric use. In the present study, the antidant effect ofCistus laurifolius ethanol extract and fractiowere investigated against acetaminophen-induced liverage on subacute administration in mice. In order to assesactivity, some biochemical plasma and hepatic tissue parters including malondialdehyde formation (MDA), transaminlevels in plasma; aspartate transferase (AST) and alanineferase (ALT), and cellular glutathione (GSH) level in heptissue were measured. Through bioassay-guided fractionthe active antioxidant constituent(s) were isolated and idenby spectroscopy.

2. Material and methods

2.1. General experimental procedures

NMR spectra were acquired on a JEOL instrument.1H NMR:500 MHz,13C NMR: 125 MHz, using TMS as internal standaFAB-MS were obtained on a JEOL HX-110A instrument.

The active compounds were isolated after exten

emedy, it is essential to investigate the potential effects of therude drug on hepatic tissue for the evaluation of potential health

∗ Corresponding author. Fax: +90 312 223 50 18.

chromatography using Sephadex LH-20 (25–100�m, Lot124H0053, Sigma Chem. Co., column size 30 mm× 500 mm).Silica gel (Kieselgel 230–400 mesh, Merck Art. No. 1.09385,column size 35 mm× 750 mm) and reversed phase 18 (LiChro-p .

erved

E-mail address: esrak@gazi.edu.tr (E. Kupeli).

378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights resoi:10.1016/j.jep.2005.08.038

rep RP-18, Merck, column size 18.5 mm× 352 mm) columns

.

456 E. Kupeli et al. / Journal of Ethnopharmacology 103 (2006) 455–460

2.2. Animals

Male Swiss albino mice (20–25 g) were purchased from theanimal breeding laboratories of Refik Saydam Central Instituteof Health (Ankara, Turkey). The animals left for 2 days foracclimatization to animal room conditions were maintained onstandard pellet diet and water ad libitum. The food was with-drawn on the day before the experiment, but allowed free accessof water. A minimum of six animals was used in each group.Throughout the experiments, animals were processed accordingto the suggested international ethical guidelines for the care oflaboratory animals.

2.3. Plant material

Cistus laurifolius L. leaves were collected from Bolu,Dortdivan in May 2002 and was identified by Prof. Dr. M. Vuralfrom the Department of Botany, Faculty of Science, Gazi Uni-versity. A voucher specimen is deposited in the Herbarium ofFaculty of Pharmacy, Gazi University (GUE-2300).

2.4. Extraction and fractionation

2.4.1. Preparation of extract and fractionsSome 2 kg of powdered material was extracted three times

with EtOH (10 L) by stirring in a 60◦C water bath for 8 dayse dry-n 5 g).T eOHa -a yield‘ ther la hlo-r -s her underrF ,r

2a

S e 43f om-pt llows:F 1–43(w l forf oid-e 1:2)a has( ec -t ds aq

Scheme 1.

and kaempferol-3,7-dimethyl-ether (3) (Scheme 1). The spec-tral data are in agreement with the reported values (Barbera etal., 1986; Guerrero et al., 2002; Smolarz et al., 2003; Stevens etal., 1995).

2.5. Pharmacological procedures

2.5.1. Preparation of test samples for bioassayThe extract, fractions and pure compounds were suspended

in 0.5% CMC in distilled water prior to oral administrationto experimental animals. Test groups of mice were orallytreated with EtOH extract (500 mg/kg body weight), hexane(206 mg/kg), CHCl3 (312 mg/kg), EtOAc (90 mg/kg),n-BuOH(94 mg/kg) or R-H2O fractions (268 mg/kg) for 7 follow-ing days, once a day by gastric gavage. The control group(untreated) and acetaminophen group (positive control) wereadministered in 0.5% CMC suspension for the same period.The reference drug, ascorbic acid (Vitamin C) suspended in0.5% CMC was directly administered to animals at 100 mg/kgdose.

2.5.2. Experimental procedureSome 60 min after the administration of the last dose on

seventh day, except the control group mice, each of theacetaminophen group and test group animals was challengedwith the suspension of acetaminophen (800 mg/kg body weight)iF plesw sac-r d inht ne thel ero rminet glu-t

ach. The combined ethanol extract was evaporated toess under reduced pressure to yield ‘EtOH extract’ (31he EtOH extract was then resuspanded in 1500 ml of Mnd extracted withn-hexane (7× 500 ml). Combined hexne extract was evaporated under reduced pressure to

Hexane fraction’ (64.8 g). MeOH was removed fromemaining solution and diluted with distilled H2O to 2000 mnd further fractionated by successive extractions with coform (7× 500 ml), ethyl acetate (5× 250 ml) and wateraturatedn-butanol (3× 200 ml). The extracts as well as temaining aqueous phase were evaporated to drynesseduced pressure to yield the “CHCl3 Fr.” (98.4 g), “EtOAcr.” (28.3 g), “BuOH Fr.” (29.7 g) and “R–H2O Fr.” (84.5 g)

espectively.

.4.2. Isolation of the active constituentsFour grams of the CHCl3 fraction was permeated on

ephadex LH-20 column using MeOH as eluent. Somractions of 15 ml each were collected. Fractions were cared by TLC on silica gel using CHCl3/MeOH (8:2) and

oluene/ether (1:1) as mobile systems and combined as for.1–13 (1.23 g), Fr.14–19 (0.72 g), Fr.20–30 (0.91 g), Fr.31.12 g). Some additional 5.36 g of the CHCl3 fraction wereorked-up under identical conditions to obtain more materia

urther purification. Vacuum-chromatography of the flavonnriched fractions (silica gel; petroleum ether/EtOAc (1:1), (nd EtOAc/MeOH (8:2) as solvent system) and reverse-pRP-18) chromatography using MeOH/H2O yielded three purompounds: (1) (1.23 g), (2) (0.95 g), (3) (0.82 g). The strucure of compounds was elucidated by spectroscopic methouercetin-3-methyl-ether (1), quercetin-3,7-dimethyl-ether (2)

e

s

n 0.5% CMC to induce hepatic injury (Fairhurst et al., 1982).our hours after acetaminophen administration, blood samere withdrawn by cardiac puncture and then the mice were

ificed by overdose of diethylether. Blood samples collecteeparinized tubes were centrifuged at 3000× g (4◦C) for 10 min

o obtain plasma. Plasma samples were used to determiipid peroxide levels and the enzyme (AST, ALT) activity. Livf each mouse was promptly removed and used to dete

he tissue levels of malondialdehyde (MDA) and cellularathione (GSH).

E. Kupeli et al. / Journal of Ethnopharmacology 103 (2006) 455–460 457

2.5.3. Determination of plasma lipid peroxidation levelThe methodology described byKurtel et al. (1992)was

used. Briefly, 1 ml of plasma was mixed with 2.0 ml oftrichloroacetic acid (TCA; 15%, w/v)–thiobarbituric acid (TBA;0.375%)–0.25 N HCl and mixed throughly and centrifuged at10,000× g for 5 min. The supernatant was mixed with 20�lof butyl hydroxy toluene (BHT; 0.02% in 95% EtOH, w/v) toprevent further oxidation and heated for 15 min in a boilingwater bath. After cooling under running water, the flocculentprecipitate was removed by centrifugation at 10,000× g for5 min. The absorbance of the sample was measured at 532 nmagainst blank that contained all the reagents except plasma.1,1,3,3-Tetraethoxypropan was used as standard for the curvecalibration.

2.5.4. Determination of lipid peroxidation in liver tissueThe method ofOhkawa et al. (1979)as modified byJamall

and Smith (1985)was used to determine lipid peroxidation in tis-sue samples. Mice were sacrified by an overdose of diethylether.The liver of each mouse was immediately excised and chilled inice-cold 0.9% NaCl and then perfused via the portal vein withice-cold 0.9% NaCl. After washing with 0.9% NaCl, 1.0 g of wettissue was weighted exactly and homogenized in 9 ml of 0.25 Msucrose using a teflon homogenizer to obtain a 10% suspension.The cytosolic fraction was obtained by a two-step centrifuga-tion first at 1000× g for 10 min and then at 2000× g for 30 mina rredt umd tion( /v)s mlw d inb derr

10%T d a1 tionw eteC entp illedw ssueL cont or thc

2t

2 ME bathu ixedi l of5 for1 ntw ll-m )a with5 lank

with no homogenate. Results were expressed as�mol GSH/gtissue.

2.5.6. Aspartate transferase (AST) and alanine transferase(ALT) in plasma

Biocon standard kits and DAX-48 autoanalyzer were used tomeasure AST and ALT activities in plasma samples accordingto the method ofWilkinson et al. (1972).

2.5.7. Acute toxicityAnimals employed in the experiments were observed during

48 h and morbidity or mortality was recorded, if happens, foreach group at the end of observation period.

2.6. Statistical analysis

The data obtained were analyzed by one-way of variance(ANOVA) and Student–Newman–Keuls post hoc tests for thesignificant interrelation between the various groups using Instatcomputer software.P < 0.05 was considered to be significantdifferent from the control.

3. Results and discussion

In living systems, dietary antioxidants such as�-tocopherol,ascorbic acid, carotenoids, as well as flavonoids and relatedp xida-t althyl wnt ted asi

gesica ses,w tiont romeP thatap tis-s r thec me-o r-n doseo per-o blyo g oft pro-c mage,i livern

fa xida-t ina-t ow-e ally,a ses inp H)e kg

t 4◦C. A volume of the homogenate (0.20 ml) was transfeo a vial and was mixed with 0.2 ml of a 8.1% (w/v) sodiodecyl sulphate solution, 1.50 ml of a 20% acetic acid soluadjusted to pH 3.5 with NaOH) and 1.50 ml of a 0.8% (wolution of TBA and the final volume was adjusted to 4.0ith distilled water. Each vial was tightly capped and heateoiling water bath for 60 min. The vials were then cooled ununning water.

Equal volumes of tissue blank or test sample andCA were transferred into a centrifuge tube and centrifuge000× g for 10 min. The absorbance of the supernatant fracas measured at 532 nm in a Beckman DU 650 spectromontrol experiment was processed using the same experimrocedure except the TBA solution was replaced with distater due to the peroxidative effect of acetaminophen on tiivers of acetaminophen-treated mice were used as positive

rol and 1,1,3,3-tetraethoxypropan was used as standard furve calibration.

.5.5. Nonprotein sulfhydryl groups (cellular GSH) in liverissue (Sedlak and Lindsay, 1968)

Some 200 mg of liver was homogenized in 8.0 ml of 0.0DTA in an ice bath. The homogenates were kept in the icentil used. Aliquots of 5.0 ml of the homogenates were m

n 15.0 ml test tubes with 4.0 ml distilled water and 1.0 m0% trichloroacetic acid (TCA). The tubes were centrifuged5 min at approximately 3000× g. Some 2.0 ml of supernataas mixed with 4.0 ml of 0.4 M Tris–buffer, pH 8.9, 0.1 ml Ean’s reagent [5,5′-dithiobis-(2-nitro-benzoic acid)] (DTNBdded, and the sample shaken. The absorbance was readmin of the addition of DTNB at 412 nm against a reagent b

t

r.al

.-e

in

henolic compounds are suggested in protection from oive damage of tissues in the body and eventually for a heife (Haraguchi, 2001). Especially flavonoids have been shoo scavenge various reactive oxygen species and implicanhibitors of lipid peroxidation (Mora et al., 1990).

Acetaminophen (paracetamol), a frequently used analnd antipyretic drug, is known to be hepatotoxic in high dohich is primarily metabolized by sulfation and glucuronida

o unreactive metabolites, and then activated by the cytoch-450 system to produce liver injury. It is establishedcetaminophen is bioactivated to a toxic electrophile,N-acetyl--benzoquinone imine (NAPQI), which binds covalently toue macromolecules, and probably also oxidizes lipids, oritical sulphydryl groups (protein thiols) and alters the hostasis of calcium (Lin et al., 1997). Post-mitochondrial supeatants isolated from livers of rats given a single large oralf acetaminophen (800 mg/kg) showed rapid rates of lipidxidation when incubated in vitro. Lipid peroxidation probaccurs simultaneously with the proposed covalent bindin

he active metabolite of acetaminophen. Since the formeress is known to cause severe and extensive membrane dat may be a very important factor in acetaminophen-inducedecrosis (Fairhurst et al., 1982).

As shown in Table 1, in the liver and plasma ocetaminophen-treated group, tissue and plasma lipid pero

ion levels (219.2 and 1172%) as evidenced by MDA determion increased significantly as compared to control group, hver, the content of GSH in liver decreased (8.7%). Additioncetaminophen was found to cause several folds increalasma AST and ALT levels (106 and 71.6%). Ethanol (EtOxtract ofCistus laurifolius leaves administered in 500 mg/

458 E. Kupeli et al. / Journal of Ethnopharmacology 103 (2006) 455–460

Table 1Effect of Cistus laurifolius EtOH extract, subfractions and the isolated flavonoids on MDA and GSH levels against acetaminophen-induced liver damage in mice

Materials Dose (mg/kg) Plasma MDA level Liver homogenate MDA level Tissue GSH

Plasma (nmol/ml) % Changea Liver (nmol/g) % Changea �mol/g % Changea

Control (0.5% CMC) 1.8± 0.1 118.2± 15.4 106.1± 8.1Acetaminophenb 800 11.4± 2.1*** +1172 377.8± 29.6*** +219.2 96.9± 10.6 −8.7Ascorbic acidc 100 4.6± 0.2*** −59.5 183.7± 17.9** −51.4 126.3± 11.5 +30.3EtOH extractc 500 5.8± 0.9*** −49.3 194.7± 19.3*** −48.5 135.6± 7.9** +39.9Hexane Fr.c 206 7.9± 1.5* −31.1 296.1± 24.1* −21.6 117.3± 8.6 +21CHCl3 Fr.c 312 6.7± 0.8*** −41.3 260.8± 16.7** −31.0 153.4± 6.1*** +58.3EtOAc Fr.c 90 7.0± 1.0* −38.4 278.8± 14.4** −26.2 117.8± 7.2 +21.6n-BuOH Fr.c 94 8.7± 2.2 −23.4 307.8± 21.6 −18.5 128± 8.4* +32.1R-H2O Fr.c 268 8.9± 1.9 −21.3 303.2± 18.8 −19.8 106± 4.4 +9.4Quercetin-3-methyl-etherc 147 6.8± 0.6* −40.3 316.6± 12.3* −16.2 109.7± 13.1 +13.2Quercetin-3,7-dimethyl-etherc 114 6.1± 0.4*** −46.8 300.8± 14.8** −20.5 115.5± 6.3 +19.2Kaempferol-3,7-dimethyl-etherc 98 7.1± 0.5* −37.5 355.15± 14.3 −6.0 104.5± 9.5 +7.8

Results are presented as mean± S.E.M.a (+) Represents percentage of increase and (−) decrease when compared to either vehicle or acetaminophen.b Compared to vehicle control (0.5% CMC).c Compared to acetaminophen as hepatotoxin.* Change to significance from control or acetaminophen;p < 0.05.

** Change to significance from control or acetaminophen;p < 0.01.*** Change to significance from control or acetaminophen;p < 0.001.

dose as well as ascorbic acid, as reference compound, showeda significant effect at plasma AST, ALT, MDA and liver tissueMDA, GSH levels (Tables 1 and 2). Besides, we observed thatEtOH extract was more effective than ascorbic acid in plasmaAST levels.

The active EtOH extract was fractionated through successivesolvent-solvent extractions and five fractions namely HexaneFr., CHCl3 Fr., EtOAc Fr.,n-BuOH Fr. and R-H2O Fr. wereobtained.

As shown inTable 1, CHCl3 Fr. (41.3% for plasma and 31%for tissue) and, in a lesser degree, EtOAc Fr. (38.4% for plasmaand 26.2% for tissue) possessed a potent activity against bothplasma and tissue MDA levels. Of the five fractions tested, theCHCl3 Fr. (58.3%) appeared to be the most effective in increas-ing the hepatic GSH levels, as compared to the acetaminophengroup. Additionally, only CHCl3 (21.5%), EtOAc (10.1%) andn-BuOH (19.6%) fractions produced a decrease in plasma ALTlevels while all of the fractions caused reduction in plasma AST

Table 2Effect ofCistus laurifolius EtOH extract, subfractions and the isolated flavonoids on plasma transaminase enzyme levels against acetaminophen-induced liver damagein mice

Materials Dose (mg/kg) ALT AST

IU (L) % Changea IU (L) % Changea

Control (0.5% CMC) 3474± 16 1683± 57Acetaminophenb 800 5960± 251*** +71.6 3468± 219*** +106.0Ascorbic acidc 100 5864± 188 −1.6 2108± 115*** −39.2EtOH extractc 500 4692± 113*** −21.3 2319± 119*** −33.1Hexane Fr.c 206 6452± 114 +8.3 2660± 167* −23.3CHCl3 Fr.c 312 4680± 115*** −21.5 2576± 235* −25.7EtOAc Fr.c 90 5360± 240* −10.1 2194.4± 161** −36.7n-BuOH Fr.c 94 4792± 138*** −19.6 2796± 258* −19.4R-H2O Fr.c 268 6180± 139 +3.7 2656± 300* −23.4Quercetin-3-methyl-etherc 147 4704± 124** −21.1 2289.6± 121** −34.0Q ± 14K ± 12

Rto eit

*

uercetin-3,7-dimethyl-etherc 114 4932aempferol-3,7-dimethyl-etherc 98 4904

esults are presented as mean± S.E.M.a (+) Represents percentage of increase and (−) decrease when comparedb Compared to vehicle control (0.5% CMC).c Compared to acetaminophen as hepatotoxin.* Change to significance from control or acetaminophen;p < 0.05.

** Change to significance from control or acetaminophen;p < 0.01** Change to significance from control or acetaminophen;p < 0.001

7*** −17.3 3120± 167 −10.04*** −17.7 2288± 150** −34.0

her vehicle or acetaminophen.

E. Kupeli et al. / Journal of Ethnopharmacology 103 (2006) 455–460 459

levels (19.4–36.7%). The results showed that pretreatment withCHCl3 Fr. (in ALT) and EtOAc Fr. (in AST) represented the mostsignificant alleviation of the plasma enzyme activity induced byacetaminophen. AST can be generally found in the liver, car-diac muscle, skeletal muscle, kidneys, brain, pancreas, lungs,leukocytes, and erythrocytes, whereas, ALT is present in high-est concentration in liver (Rej, 1978). Therefore, the CHCl3Fr. was considered to be more effective than the EtOAc Fr. onacetaminophen-induced liver damage.

According to the biochemical test results, the followingexperiments were directed to CHCl3 Fr. On TLC analysis,CHCl3 Fr. was found to be rich in flavonoids. Through suc-cessive column chromatographies three main flavonoids wereisolated, and their structures were elucidated as quercetin-3-methyl-ether (isorhamnetin) (1), quercetin-3,7-dimethyl-ether(2), kaempferol-3,7-dimethyl-ether (3) by spectral techniques.Effect of the isolated flavonoid aglycones on acetaminophen-induced liver damage was also studied using the same biochem-ical methods on plasma and liver tissue.

Mice treated with flavonoid 2 significantly reducedacetaminophen-induced increases in plasma (46.8%) and liver(20.5%) MDA production compared with acetaminophen-treated mice. On the other hand, flavonoid1 also caused reduceof acetaminophen-induced increase in plasma (40.3%) and liver(16.2%) MDA production. According to these results, flavonoid2 is considered to have more inhibitory effect than that offl

fica-t withr themf aseo cts ot ,t onsbe 9.2a s2

utedta tionaen .7%ca ies,w c-t y ina noidc ulare ,1

laa dM uala yl-

ether (1) and kaempferol-3,7-dimethyl-ether (3) were less activethan the CHCl3 fraction.The isolated flavonoids did not induceany apparent acute toxicity during the 48 h observation period.

As shown inTables 1 and 2, flavonoid2 was the most activecompound, as can be expected due to the presence ofortho-catechol group (3′,4′-OH) in the B-ring, which is known to bean important substituent pattern in the antioxidant activity offlavonoids. Although the same functional group also exists inflavonoid1, the effect was somewhat lesser, probably due to theweaker lipophilic nature of the compound.

Previously,Dok-Go et al. (2003)studied the neuroprotec-tive effects of three antioxidant flavonoids, including quercetin,quercetin 3-methyl-ether (1), and 1-dihydroquercetin isolatedfrom Opuntia ficus-indica var.saboten and the results indicatedthat quercetin 3-methyl-ether was the most potent and suggestedas a promising neuroprotectant. It is evident that due to the neu-roprotective activity of this flavonoid, it would be beneficial forthe prevention and treatment of oxidative stress-induced neuro-logical disorders (Dok-Go et al., 2003).

On the other hand, several studies have reported that quercetin3-methyl-ether (1) displayed a remarkable activity against awide range of human picornaviruses, platelet aggregation andhistamine-induced tracheal relaxation in vitro (Ko et al., 1999;Lin et al., 1995; Vanden Berghe et al., 1986). Moreover,quercetin 3-methyl-ether isolated fromCistus laurifolius hasbeen reported to be effective for inhibiting the aldose reduc-t treatt ,2t andc n-c tureof lutee cedge edf sica

iso-la tt

ractsa nta intt uldb vivoa ande

A

a ofG hiba,J

avonoid1 on acetaminophen-induced lipid peroxidation.Glutathione (GSH) plays an essential role in the detoxi

ion of acetaminophen and protects hepatocytes by unitingeactive metabolites of acetaminophen. Thus, it preventsrom binding covalently to liver proteins. Intracellular decref the reducted GSH exposes the cell to the destructive effe

he oxidative stress (Lauterburg and Velez, 1988). In other wordhe hepatic GSH is non-protein reserve of the liver and resple in reducing the NAPQI-induced liver damage (Ischiropolust al., 1992). The GSH activities showed alleviation of 13.2, 1nd 7.8%, respectively, in liver of mice, received flavonoid1,and3, compared with the acetaminophen group.The rise in serum AST and ALT levels has been attrib

o the damaged structural integrity of the liver (Chenowethnd Hake, 1962), because these are cytoplasmic in locand are released into circulation after cellular damage (Salliet al., 1991). Pretreatment of mice with flavonoid1, 2 and 3oticeably decreased the ALT activities (21.1, 17.3 and 17ompared with the acetaminophen group. Flavonoids1 (34%)nd 3 (34%) provided a significant reduction in AST activithereas flavonoid2 (10%) exhibited an insignificant redu

ion. The reversal of alleviation of plasma enzyme activitcetaminophen-induced hepatic damage by these flavoould explain the prevention of leakage of the intracellnzymes by its membrane stabilizing activity (Thabrew et al.987).

While quercetin-3,7-dimethyl-ether (2) displayed identicactivity than the CHCl3 fraction in MDA levels, it did not showny comparable effect with CHCl3 fraction in tissue GSH anDA levels. As to ALT levels, all three flavonoids had eqctivity than the CHCl3 fraction, whereas quercetin-3-meth

f

i-

)

s

ase activity. Therefore, this compound could be used tohe complications associated with diabetes (Enomoto et al.004). Yesilada et al. (1997a)reported that the methanolicCis-

us laurifolius extract and liposoluble fractions (hexanehloroform) were active on IL-1� when assayed high coentrations. In a previous study, the polysaccharide mixbtained from the flowers and flower buds ofCistus lauri-

olius was found to be active against pylorus ligation-, absothanol-, indomethacin-, indomethacin plus HCl/EtOH-induastric and cysteamine-induced duodenal lesions (Yesiladat al., 1997b). The CHCl3 extract and the precipitate obtain

rom Cistus laurifolius leaves showed significant analgectivity on the tail flick assay (Ark et al., 2004).

The in vivo antioxidant effect of the flavonoid aglyconesated fromCistus laurifolius, quercetin-3,7-dimethyl-ether (2)nd kaempferol-3,7-dimethyl-ether (3) is reported for the firs

ime.According to the results of the present study, the ext

nd isolated flavonoids fromCistus laurifolius possess a potentioxidant activity. Since oxidation is known to be involved

he pathogenesis of many diseases in which treatment withCis-us laurifolius is claimed to be effective, further studies shoe carried out on the active compound(s) using other innd in vitro antioxidant models in order to assess the rolelucidate the action mechanism.

cknowledgement

The authors are extremely thankful to Dr. Emi Okuyamraduate School of Pharmacy Science, Chiba University, C

apan, for NMR and MS measurements.

460 E. Kupeli et al. / Journal of Ethnopharmacology 103 (2006) 455–460

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K oflogy

L lics:

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