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Chemico-Biological Interactions 181 (2009) 85–94

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

elationship correlation of antioxidant and antiproliferative capacity ofyperus rotundus products towards K562 erythroleukemia cells

oumaya Kilani-Jaziri a, Aicha Neffati a, Ilef Limem a, Jihed Boubaker a, Ines Skandrani a,ohamed Ben Sghair a, Ines Bouhlel a, Wissem Bhouri a, Anne Marie Mariotte d,

amel Ghedira a, Marie-Genviève Dijoux Franca c, Leila Chekir-Ghedira a,b,∗

Unité de Pharmacognosie/Biologie Moléculaire 99/UR/07-03, Faculté de Pharmacie, Rue Avicenne 5000 Monastir, TunisiaLaboratoire de Biologie Cellulaire et Moléculaire, Faculté de Médecine Dentaire, Rue Avicenne, Monastir 5000, TunisiaLaboratoire de Botanique, Pharmacognosie et Phytothérapie, UMR CNRS 5557 Ecologie Microbienne, Institut des Sciences Pharmaceutiques et Biologiques,aculté de Pharmacie Université Claude Bernard, Bâtiment Nétien-8 Avenue Rockefeller 69373, Lyon cedex 08, FranceDépartement de Pharmaco-Chimie Moléculaire, UMR 5063 CNRS-UJF, UFR de Pharmacie, Faculté de Pharmacie-Domaine de la Merci,8706 La Tronche cedex, Grenoble, France

r t i c l e i n f o

rticle history:eceived 22 September 2008eceived in revised form 25 April 2009ccepted 27 April 2009vailable online 14 May 2009

eywords:yperus rotunduslavonoidsolyphenol derivativeslasmid DNA damagentioxidant capacitypoptosis

a b s t r a c t

A Total Oligomers Flavonoids (TOFs) and ethyl acetate extracts of Cyperus rotundus were analyzed, in vitro,for their antioxidant activity using several biochemical assays: the xanthine (X)/xanthine oxidase (XO),the lipid peroxidation induced by H2O2 in K562 human chronic myelogenous leukemia cells and the DNAdamage in pKS plasmid DNA assay induced by H2O2/UV-photolysis and for their apoptotic effect. TOF andethyl acetate extracts were found to be efficient in inhibiting xanthine oxidase with IC50 values of 240 and185 �g/ml and superoxide anion with IC50 values of 150 and 215 �g/ml, respectively. Also, all the extractstested were effective in reducing the production of thiobarbituric acid reactive substances (TBARS) andwere able to protect against H2O2/UV-photolysis induced DNA damage. The highest activity, measured asequivalents of MDA concentration, was observed in the ethyl acetate extract (MDA = 2.04 nM). In addition,the data suggest that only TOF enriched extract exerts growth inhibition on K562 cells through apoptosisinduction. Therefore, these extracts were subjected to further separation by chromatographic meth-ods. Thus, three major compounds (catechin, afzelechin and galloyl quinic acid) were isolated from theTOF enriched extract and five major compounds (luteolin, ferulic acid, quercetin, 3-hydroxy, 4-methoxy-benzoic acid and 6,7-dimethoxycoumarin) from ethyl acetate extract. Their structures were determined

by spectroscopic data analysis and comparison with the literature. In addition, we evaluate the biologicalactivities of the catechin, ferulic acid and luteolin. This investigation has revealed that the luteolin wasthe most active in reducing the production of TBARS (MDA = 1.5 nM), inhibiting significantly the prolif-eration of K562 cells (IC50 = 25 �g/ml) and protecting against H2O2/UV-photolysis induced DNA damage.In conclusion, the study reveals that the ability of C. rotundus to inhibit the enzyme xanthine oxidase(XO), the lipid peroxidation and to exert apoptotic effect, may explain possible mechanisms by which C.

th be

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. Introduction

Apoptosis is a cell suicide program that has been conserved

hrough the evolution. Induction of apoptosis thus is a highly desir-ble mode as a chemotherapeutic as well as a chemopreventivetrategy for cancer control. Recently, interest has focused on theanipulation of apoptotic processes in the treatment and pre-

∗ Corresponding author at: Laboratoire de Biologie Cellulaire et Moléculaire, Fac-lty of Dental Medicine, Rue Avicenne, Monastir 5019, Tunisia. Tel.: +216 97 316 282;

ax: +216 73 461 150.E-mail addresses: leila.chekir@laposte.net, lchekir@yahoo.fr (L. Chekir-Ghedira).

009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2009.04.014

nefits.© 2009 Elsevier Ireland Ltd. All rights reserved.

vention of cancer. Thus, much effort has been directed towardthe search for compounds or herbs that influence apoptosis andtheir mechanism of action. In fact, there has been increasing real-ization, in recent years, that several plant derived polyphenoliccompounds may possess antioxidant, anticancer and apoptosisinducing properties. To date, many anticancer drugs used inchemotherapeutic treatments developed resistance and side effects[1,2].

In a program to find new natural sources of antioxidative andantiproliferative agents from Tunisian medicinal plants, we studiedthe efficiency of extracts and compounds from Cyperus rotun-dus. C. rotundus Linn, a sedge of the Cyperaceae family, is widelydistributed in the Mediterranean basin areas. This plant, which

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rows naturally in tropical, sub-tropical and temperate regions,s widespread in North-East, Center and South of Tunisia [3]. C.otundus is a traditional medicinal plant appearing among Indian,hinese and Japanese natural drugs used against spasms, stomachisorders, whereas previous studies reported the use of C. rotundushizomes to treat inflammatory bowel disease [4–7]. Likewise theame authors [7] reported the use of the methanolic extract fromhe same part of the plant as anti-inflammatory candidate for thereatment of inflammatory diseases mediated by overproduction ofitric oxide and superoxide.

In a previous study we investigated the effects of enriched totalligomer flavonoid (TOF) and ethyl acetate extracts from aerialarts of C. rotundus, on genotoxicity induced by both Aflatoxin B1AFB1) and sodium azide, in a bacterial assay system, i.e. the Amesest. These extracts exhibited an important free radical-scavengingctivity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free rad-cal [8]. These results suggested deepening our study in order tourify and to test molecules obtained from the most active extract8].

Accordingly, in the present study, the antioxidant properties oferial part extracts were evaluated through biochemical assays: theanthine/xanthine oxidase enzymatic assay system, the inhibitionf lipid peroxidation in human chronic myelogenous leukemia cell

ine K562, through the formation of thiobarbituric acid reactive sub-tances, and the protection against DNA damage. Furthermore, theffects of C. rotundus on inhibition of cell proliferation and induc-ion of apoptosis in human leukemia cells were also examined. The

ajor compounds isolated from TOF and ethyl acetate extracts (cat-chin, ferulic acid and luteolin) were evaluated by measuring theirbility to reduce lipid peroxidation in human leukemia K562 cellsnd to protect against H2O2/UV-photolysis induced DNA damage inKS plasmid DNA.

. Materials and methods

.1. Chemicals

All organic solvents were obtained from Carlo ERBA (France).he standard of quercetin was purchased from Sigma Chemical Co.St. Louis, MO, USA). Chromatographic columns were performedith silica gel 60 (Pharmacia Biotech, Sweden), Reverse phase C18

Merck, Germany) and sephadex LH-20 (Amercham). The mutagenydrogen peroxide (H2O2), xanthine, xanthine oxidase, hypoxan-

hine and Dimethylsulfoxide (DMSO) were purchased from SigmaSt. Louis, MO, USA). RPMI-1640, foetal bovine serum, gentamycinend l-glutamine were purchased from GIBCO BRL Life technologiesGrand Island, NY, USA). The N-(1-naphtyl) ethlenediaminedihy-rochloride (EDTA) was purchased from Sigma–Aldrich (Steinheim,ermany).

.2. Plant material

C. rotundus aerial parts were collected in the region of Monastirn the Center of Tunisia in October 2004. Botanical identification

as carried out by Pr. M. Chaieb (Department of Botany, Facultyf Sciences, University of Sfax, Tunisia), according to the flora ofunisia [3]. A voucher specimen (Cp.10.04) has been kept in theaboratory of Pharmacognosy, Faculty of Pharmacy of Monastir-unisia, for future reference.

.3. Preparation of plant extracts

In order to obtain an extract enriched with TOF, plant powder100 g) was macerated in water/acetone mixture (1/2, v/v), dur-ng 24 h with continuous stirring. The extract was filtered and thecetone was evaporated under low pressure in order to obtain an

l Interactions 181 (2009) 85–94

aqueous phase. The tannins were removed by precipitation with anexcess of NaCl during 24 h at 5 ◦C and then we recovered the super-natant. This latter was extracted with ethyl acetate, concentratedand precipitated with an excess of chloroform. The precipitate(0.3 g) was separated and yielded TOF extract which was dissolvedin water for the test.

Additionally, C. rotundus powder (100 g) was extracted usinga Soxhlet type extractor with 500 ml of ethyl acetate (EA) for8 h. The extract was cooled, filtered, concentrated to remove thesolvent, and then freeze-dried separately. The residue was keptat 4 ◦C for the test. The yield of ethyl acetate extract is 1.5%(w/w).

2.4. Extraction and isolation compounds

The TOF and ethyl acetate crude extracts were the most activecompared to the other extracts isolated from aerial parts of C. rotun-dus and were selected for the isolation of flavonoids compounds andcharacterization of antioxidant and apoptotic activities.

The TOF enriched extract (2.1 g) was fractionated over sil-ica gel with Vacuum Liquid Chromatography (VLC) eluted withCH2Cl2–MeOH with gradual increasing of the MeOH contentsand 10 fractions were collected. Fraction 3 (814 mg) was puri-fied through a C18 disposable extraction column eluted with agradient methanol–water increasing MeOH contents and 11 sub-fractions were collected. Sub-fraction 3 was chromatographed oversephadex LH-20 eluted with CH2Cl2–MeOH with a gradient ofMeOH to afford 1.8 mg of afzelechin (1) and 6.9 mg of galloylquinicacid (2). Sub-fraction 4 was subjected on sephadex LH-20 withCH2Cl2–MeOH (70:30 → 0:100) as eluent to give 2.8 mg of catechin(3).

The ethyl acetate extract (4 g) was fractionated over silica gelwith VLC, eluted with CH2Cl2–MeOH (100:0 → 0:100) solvent sys-tem to give 9 fractions. Fraction 8 (1.5 g) was chromatographed onsephadex LH-20, using CH2Cl2–MeOH (70:30 → 0:100) as eluent,to obtain 19 sub-fractions. Sub-fraction 9 was purified by passagethrough C18 disposable extraction column eluted with H2O:MeOH(100:0 → 0:100) to give 2.2 mg of 3-hydroxy, 4-methoxy-benzoicacid (4) and 1.3 mg of 6,7-dimethoxycoumarin (5). Sub-fraction 11was chromatographed on silica gel with a CH2Cl2–MeOH solventsystem (100:0 → 0:100) to give 6.4 mg of luteolin (6). Sub-fraction12 was analysed by analytical HPLC (LC 200 Micro Pump High Pres-sure Binary, equipped with a DAD). Chromatographic separationwas carried out on a C18 column (10 × 0.5, 5 �m, flow rate was0.5 ml/min), using a gradient solvent comprising 0.5% acetic acid inwater (A) and MeOH (B) for a total running time of 40 min. The fol-lowing proportions of solvent B were used for elution: 0.5–20 min,0–85%; 20–30 min, 85%; and 30–40 min, 85–0%. 20 �l of a solutionat 1 mg/ml was injected and detected at 365 nm (A) and 280 nm(B), to yield quercetin (7) (RT = 22.15). Retention Time (RT) andon-line UV spectra were comparable to that of an authentic sam-ple of quercetin conditions. Ferulic acid (8) (1.6 mg) was isolatedfrom the sub-fraction 16 (38.7 mg) over silica gel column usingCH2Cl2–MeOH (100:0 → 0:100).

Pure compounds were identified by comparison of their NMRdata with literature.

2.5. Structural identification of the compounds

using a Bruker® Avance spectrometer, operating at 400 MHz for1H and 100 MHz for 13C. Dimethyl sulfoxide (DMSO-d6) andmethanol (CD3OD) were used as solvents. HPLC analysis was usedfor the identification of the main compound in the sub-fraction12.

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.6. Inhibition of xanthine oxidase activity and superoxideadical-scavenging effect

Both inhibition of xanthine oxidase activity and the superox-de anion scavenging activity, were assessed in vitro in one assay.he inhibition of xanthine oxidase activity was measured accordingo the increase in absorbance at 290 nm as proposed by Cimanga etl. [9], while the superoxide anion scavenging activity was detectedpectrophotometrically with the nitrite method described by Oyan-agui [10] with some modifications introduced by Russo et al. [11].

Briefly, the assay mixture consisted of 100 �l of the tested com-ound solution, 200 �l xanthine (X) (final concentration 50 �M) ashe substrate, hydroxylamine (final concentration 0.2 mM), 200 �lDTA (0.1 mM) and 300 �l distilled water. The reaction was initiatedy adding 200 �l xanthine oxidase (XO) (5.5 mU ml−1) dissolved

n phosphate buffer (KH2PO4 20.8 mM, pH 7.5). The assay mixtureas incubated at 37 ◦C for 30 min. Before measurement of the uric

cid production at 290 nm, the reaction was stopped by adding.1 ml of HCl 0.5 M. The absorbance was measured spectrophoto-etrically against a blank solution prepared as described above but

eplacing xanthine oxidase with buffer solution. Another contrololution without the tested compound was prepared in the sameanner as the assay mixture to measure the total uric acid pro-

uction (100%). The uric acid production was calculated from theifferential absorbance.

To detect the superoxide scavenging activity, 2 ml of the colour-ng reagent consisting of sulfanilic acid solution (final concentration00 �g/ml), N-(1-naphtyl) ethylenediamine dihydrochloride (finaloncentration 5 �g/ml) and acetic acid (16.7%, v/v) were added.his mixture was allowed to stand for 30 min at room temper-ture and the absorbance was measured at 550 nm on a Helios-spectrophotometer.

The dose–effect curve for each test compound was linearized byegression analysis and used to derive the IC50 values. Values areresented as mean ± standard deviation of three determinations.

.7. Cytotoxicity studies in vitro

C. rotundus extracts and compounds were tested in vitro for theirntiproliferative and anti-lipid peroxidation activities against K562human chronic myelogenous leukemia) cells.

.7.1. Culture of cellsHuman chronic myelogenous leukemia CML cell line K562 was

btained from the American Type Culture Collection (Rockville,D). Cells were cultivated in RPMI-1640 medium supplementedith 10% (v/v) foetal calf serum, 1% gentamycine and 2 mM l-

lutamine as a complete growth medium. Cells were maintainedn 25 cm3 flasks with 10 ml of medium and were incubated at 37 ◦Cn an incubator with 5% CO2 in humidified atmosphere. Every 3ays the cells were subcultured by splitting the culture with freshedium.

.7.2. Assay for cytotoxic activityCytotoxicity of C. rotundus extracts and compounds against K562

eukemia cell line was estimated by the 3-(4,5-dimethylthiazol--yl)-2,5-diphenyltetrazolium bromide (MTT) assay, based on theleavage of the tetrazolium salt by mitochondrial dehydrogenasesn viable cells. The resulting blue formazan product can be mea-ured spectrophotometrically. The MTT colorimetric assay waserformed in 96-well plates [12]. Cells were seeded in a 96-well

late at a concentration of 5 × 104 cells/well and incubated at 37 ◦C

or 24 h in a 5% CO2 enriched atmosphere. The extracts were firstlyissolved in 1% DMSO, then in the cell growth medium. Cells inxponential growth phase were incubated again at 37 ◦C for 48 hith each of the tested extracts and compounds at concentrations

l Interactions 181 (2009) 85–94 87

ranging from 10 to 800 �g/ml. After that, the medium was removedand cells in each well were treated with 50 �l of MTT solution(5 mg/ml) at 37 ◦C for 4 h. MTT solution was then discarded and50 �l of 100% DMSO were added to dissolve insoluble formazancrystal. Optical density was measured at 540 nm. Each drug con-centration was tested in triplicate.

The cytotoxic effects of the extracts were estimated in terms ofgrowth inhibition percentage and expressed as IC50 which is theconcentration of extract which reduces the absorbance of treatedcells by 50% with reference to the control (untreated cells). The IC50values were graphically obtained from the dose–response curves.We determined IC50 values when cytotoxicity resulted more than50% at screening concentrations.

2.8. DNA fragmentation analysis

DNA fragmentation was analysed by agarose gel electrophoresisas described by Wang et al. [13], with slight modifications. K562cells (1.5 106 cells/ml) were exposed to the extract for 24 and 48 hand harvested by centrifugation. Cell pellets were resuspended in200 �l of lysis buffer (50 mM Tris–HCl, pH 8.0, 10 mM EDTA, 0.5%N-Lauroyl Sarcosine Sodium Salt) at room temperature for 1 h andthen centrifuged at 12 000 g for 20 min at 4 ◦C. The supernatantwas incubated overnight at 56 ◦C with 250 �g/ml proteinase K. Celllysates were then treated with 2 mg/ml RNase A and incubated at56 ◦C for 2 h. DNA was extracted with chloroform/phenol/isoamylalcohol (24/25/1, v/v/v) and precipitated from the aqueous phaseby centrifugation at 14 000 g for 30 min at 0 ◦C.

The solution recuperate was transferred to a 1.5% agarose geland electrophoresis was carried out at 67 V for 3/4 h with TAE (Tris2 M, sodium acetate 1 M, EDTA 50 mM) as the running buffer. DNAin the gel was visualized under UV light [13,14].

2.9. Determination of lipid peroxidation products withthiobarbituric acid reactive substances (TBARS) assay

The method known as thiobarbituric acid reactive species(TBARS) assay, concerns the spectrophotometric measurement ofthe pink pigment produced through reaction of thiobarbituricacid (TBA) with malondialdehyde (MDA) and other secondarylipid peroxidation products. TBARS were determined by previouslydescribed assay [15]. The cells were exposed to various concentra-tions of each compounds (35, 70 and 140 �g/ml for TOF enrichedextract, 50, 100 and 200 �g/ml for ethyl acetate extract, 12.5, 25and 50 �g/ml for luteolin; 200, 400 and 800 �g/ml for catechinand ferulic acid) in the incubation medium during 2 h, followedby 75 �M H2O2 for 2 h. The ranges of doses of different tested com-pounds were chosen on basis of their cytotoxic activity. The cellswere washed with PBS, pelleted and homogenized in 1.15% KCl.Samples were combined with 0.2 ml of 8.1% SDS, 1.5 ml of 20% aceticacid and 1.5 ml of 0.8% thiobarbituric acid. The mixture was broughtto a final volume of 4.0 ml with distilled water and heated to 95 ◦Cfor 120 min. After cooling for 10 min on ice, 5.0 ml of a mixture ofn-butanol and pyridine (15:1 v/v) was added to each sample, andthe mixture was shaken vigorously. After centrifugation at 825 g for10 min, the supernatant fraction was isolated and the absorbancewas measured at 532 nm. Inhibitory activity towards lipid peroxi-dation was expressed as equivalent of MDA. Data were reported asmean ± SD for triplicate determinations.

2.10. Reaction with plasmid pBluescript DNA

Plasmid DNA is a good model system for studying the DNA dam-age and its modulation.

DNA damage activity of extracts was detected on pBluescript KSvector. Plasmid DNA was amplified and extracted from E. coli DH5�

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hen oxidized with H2O2 + UV treatment in the presence or absencef ethyl acetate extract; TOF enriched extract, catechin, luteolin anderulic acid, and checked on 0.7% agarose. In brief, the experiments

ere performed in a volume of 9 �l in an Eppendorf tube containing.34 �g of pKS plasmid DNA, H2O2 was added to a final concentra-ion of 147 mM with and without 4 �l of each extract and compoundt various doses.

The reaction was initiated by UV irradiation and continued formin on the surface of UV trans-illuminator. After irradiation, theixture was incubated at room temperature during 15 min. Finally,

he reaction mixture along with gel loading dye was placed on 0.7%garose gel for electrophoresis. Untreated pKS DNA was used ascontrol in each run of gel electrophoresis. Gel was stained with

thidium bromide and photographed with Bio-print (Vilbert lour-at, France).

.11. Statistical analysis

Data were collected and expressed as the mean ± standard devi-tion of three independent experiments and analyzed for statisticalignificance using the Dunnett test (SPSS 11.5 for windows). Theriterion for significance was set at p < 0.05. IC50 values, from the initro data, were calculated by regression analysis.

. Results

This study was designed to evaluate cytotoxic, apoptotic andntioxidant activities of C. rotundus extracts against respectivelyuman chronic myelogenous K562 leukemia cell proliferation,

uperoxide radical, lipid peroxidation and plasmid DNA H2O2V-photolysis induced damage, in order to identify active com-ounds. Among the identified compounds, only three compoundscatechin, luteolin and ferulic acid) were tested for cytotoxic activ-ty, anti-lipid peroxidation in K562 cells and protection against

Fig. 1. Chemical structures of compounds isolated fro

l Interactions 181 (2009) 85–94

H2O2/UV-photolysis induced DNA damage in pKS DNA, at varyingconcentrations.

3.1. Identification of phenolic compounds

The structural identification of compounds 1–8 were estab-lished using spectroscopic analysis, especially, NMR spectra anddirect comparison with published data. These predominant com-pounds (Fig. 1) were found to belong to three secondary metaboliteclasses (coumarins, flavonoids and phenolic acids). The coumarinwas identified as 6,7-dimethoxycoumarin (5) [16]. The flavonoidswere found to be quercetin (7) [17], luteolin (6) [18], afzelechin (1)[19] and catechin (3) [20] while the isolated phenolic acids wereidentified as 3-hydroxy, 4-methoxy-benzoic acid (4) [21], galloylquinic acid (2) [22] and ferulic acid (8) [23].

3.2. Xanthine oxidase inhibition and superoxide scavengingactivity

The antioxidant activity of C. rotundus extracts was carried out tofollow both the superoxide scavenging ability and also inhibition ofxanthine oxidase activity. The IC50 values of the tested extracts forthe inhibition of xanthine oxidase and as scavengers of superoxideanions (O2

•−) are given in Table 1. Both inhibition of xanthine oxi-dase and scavenging effect on superoxide anions were measured inone assay. Inhibition of xanthine oxidase involves a decrease in pro-duction of uric acid and in superoxide anions which can be followedspectrophotometrically.

Fifty percent inhibition of uric acid production was obtained

at the doses of 240 and 185 �g/ml with respectively TOF andethyl acetate extracts. It was concluded that C. rotundus extractswere effective inhibitors of XO. Likewise it appears from theIC50 values of superoxide anions measured in the presence ofTOF and ethyl acetate extracts (respectively 150 and 215 �g/ml)

m the ethyl acetate and TOF enriched extracts.

S. Kilani-Jaziri et al. / Chemico-Biological Interactions 181 (2009) 85–94 89

Table 1IC50s and percentages of C. rotundus extracts for inhibition of xanthine oxidase and reduction of superoxide level.

Extracts Doses (�g/ml) Inhibition of xanthineoxidase activity (%)a

Xanthine oxidase activityIC50 (�g/ml)

Inhibition of superoxideanion activity (%)a

Superoxide anionactivity IC50 (�g/ml)

TOF enriched extract 50 2.1 ± 0.011 240 28.94* ± 0 150150 21.35* ± 0 48.94* ± 0.005300 73.3* ± 0.005 71.57* ± 0.0025

Ethyl acetate extract 50 2.75 ± 0.03 185 10.52* ± 0.005 215150 38.18* ± 0.007 13.68* ± 0.007

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300 74.75* ± 0.002

a Mean values of three experiments.* P < 0.05 compared with the negative control (without the tested compound) usi

hat TOF enriched extract is the more potent superoxide scav-nger.

Comparison of the IC50 values of XO activity and O2•− scavenging

ctivity, showed that TOF enriched extract has a better superox-de scavenging activity than inhibitory effect of XO. However, ethylcetate extract showed the most potent inhibitory effect of xanthinexidase activity than superoxide scavenging activity.

.3. Cytotoxic activity

The cytotoxic effect of the C. rotundus extracts and their compo-ents, was assessed on the human chronic myelogenous K562 cells.e have examined the effect of different concentrations (from 10 to

00 �g/ml) of each extract and each compound on the proliferationf leukemia cells in vitro using the MTT assay.

Results of these experiments (Table 2) showed that the tested C.otundus extracts and compounds inhibited at various levels (goodo moderate), the proliferation of the malignant cells. In fact, TOFnriched extract, ethyl acetate extract and the three pure com-ounds have cytotoxic activity against K562 cells. The strongestytotoxic effect was obtained with luteolin against K562 cells (IC505 �g/ml), followed by TOF enriched extract and ethyl acetatextract with respective IC50s of 70 and 100 �g/ml. The IC50 value oferulic acid was 420 �g/ml. However, no significant cytotoxic activ-ty was shown against the cell line after treatment with catechinIC50s > 800 �g/ml).

.4. Induction of apoptotic DNA fragmentation by C. rotundusxtracts

The fragmentation of DNA extracted from K562 cells (1.5 × 106

ells), was detected after exposing cells to 0, 200, 400 and00 �g/ml of each extract for 24 and 48 h, and cell viability was eval-ated. TOF enriched extract showed the strongest cytotoxic activitygainst K562 cells (IC50 70 �g/ml) followed by ethyl acetate extractIC 100 �g/ml). On the other hand, examination of electrophoretic

50rofiles of DNA extracted from the treated cells, revealed a ladder

ormation characteristic of apoptosis (Fig. 2). At exposure with 200o 800 �g/ml during 24 and 48 h with TOF enriched extract, a frag-

ented DNA profile was clearly observed in K562 cells, whereas

able 2C50 (�g/ml) values for extracts of Cyperus rotundus against K562 leukemia cell lines.

ompounds Activity on proliferation of cell linea

K562

OF enriched extract 70 ± 0.8atechin >800 ± 1.5thyl acetate extract 100 ± 0.5erulic acid 420 ± 2uteolin 25 ± 0.7

a Results are means ± standard deviation of duplicate analysis of three replica-ions.

66.84* ± 0.0005

nett test.

control cells did not provide ladder DNA profile. Cells treated withethyl acetate extract did not exhibit ladder DNA profile. Resultsfrom the assays showed that K562 cells were more sensitive to TOFenriched extract-mediated inhibition of proliferation than ethylacetate extract.

3.5. Anti-lipid peroxidation effect

The reaction of MDA with TBA has been widely adopted as a sen-sitive assay method for lipid peroxidation [15]. Inhibition of lipidperoxidation by any external agent is often used to evaluate itsantioxidant capacity. The inhibitory effect of different concentra-tions of C. rotundus extracts on malondialdehyde (MDA) productionin K562 cells, induced by H2O2, is shown in Table 3. Ethyl acetateand TOF enriched extracts showed a significant protective effect.The value of MDA equivalent in H2O2 stressed cells treated withethyl acetate extract, determined by the TBARS test, was 2.04 nM,whereas, that of H2O2 cells treated with TOF enriched extract was2.98 nM. Compared to MDA equivalent value in H2O2 stressed cells(7.3 nM). We deduce that ethyl acetate and TOF enriched extractsprotect cell lipids against peroxidation at all the tested concentra-tions (Table 3). In the same way, we tested anti-lipid peroxidationactivity, of luteolin and ferulic acid, isolated from ethyl acetateextract, and catechin from TOF enriched extract. We thus noticed

Fig. 2. Agarose gel electrophoresis of DNA extracted from K562 cells treated withethyl acetate and TOF enriched extracts. (A) 200 �g/ml, (B) 400 �g/ml, (C) 800 �g/ml,(D) 200 �g/ml, (E) 400 �g/ml, (F) 800 �g/ml, (G) 200 �g/ml, (H) 400 �g/ml, (I)800 �g/ml, (J) 200 �g/ml, (K) 400 �g/ml, (L) 800 �g/ml, (M, N) untreated cells for 24and 48 h.

90 S. Kilani-Jaziri et al. / Chemico-Biological Interactions 181 (2009) 85–94

Table 3Lipid peroxidation inhibitory activity in K562 leukemia cells treated with extractsand compounds of C. rotundus against H2O2 (75 �M) induced peroxidation.

Extracts and compounds Concentration (�g/ml) Equivalent of MDA (nM)

TOF enriched extract 35 3.76 ± 0.170 3.21 ± 0.2

140 2.98 ± 0.4

Ethyl acetate extract 50 4.31 ± 0.1100 3.45 ± 0.14200 2.04 ± 0.15

Luteolin 12.5 4.5 ± 0.425 3.3 ± 0.950 1.5 ± 0.1

Ferulic acid 200 5.5 ± 0.2400 3.7 ± 0.3800 2.5 ± 0.09

Catechin 200 6.3 ± 0.16400 4.8 ± 0.5800 3 ± 0.1

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Fig. 4. Electrophoretic pattern of pBluescript KS DNA in the presence of TOF andethyl acetate extracts of C. rotundus. Lane 1: DNA control, lane 2: DNA treatedwith UV + H2O2, lane 3: TOF 500 �g/assay + DNA, lane 4: TOF 200 �g/assay + DNA,lane 5: TOF 50 �g/assay + DNA, lane 6: TOF 500 �g/assay + DNA + UV, lane 7: TOF200 �g/assay + DNA + UV, lane 8: TOF 50 �g/assay + DNA + UV, lane 9: Ethyl acetate500 �g/assay + DNA, lane 10: Ethyl acetate 200 �g/assay + DNA, lane 11: Ethyl acetate50 �g/assay + DNA, lane 12: Ethyl acetate 500 �g/assay + DNA + UV, lane 13: Ethylacetate 200 �g/assay + DNA + UV, lane 14: Ethyl acetate 50 �g/assay + DNA + UV.

Fig. 5. Electrophoretic pattern of pBluescript KS DNA submitted to UV-photolysis ofH2O2 in the presence of different extracts of C. rotundus. Lane 1: DNA control, lane2: DNA treated with UV + H2O2, lane 3: TOF 500 �g/assay + DNA + UV + H2O2, lane

ydrogen peroxide 75 �M 7.3 ± 1.4

ll data are the mean ± of three replicates.

espectively with luteolin (50 �g/ml), ferulic acid (800 �g/ml) andatechin (800 �g/ml) (Table 3).

.6. Correlation analysis

Tested extracts and compounds with antioxidant activity in theipid peroxidation system, were effective in inhibiting proliferationf cancerous tested cells, and a strong linear correlation (r = 0.974)as obtained by plotting IC50 values of anti-lipid peroxidation

apacity and cytotoxic effect (Fig. 3). In support of data obtainedith the tested extracts a positive relationship between inhibition

f XO activity and inhibition of superoxide anion (O2•−) production

n the X/XO system is detected (r = 0.850). However, in contrast tohe superoxide anion scavenging activity, the linear correlation wasather low (r = 0.666) when comparing IC50 values of XO activity andnti-lipid peroxidation capacity. Whereas, a strong linear correla-ion was observed (r = 0.958) when comparing IC50s of superoxidenion scavenging in the X/XO system and anti-lipid peroxidationalues.

The strongest correlation was obtained when comparing super-xide anion scavenging effect and cancerous cell inhibition

roliferation (r = 1) of the two tested extracts. Whereas a lowerut significant correlation was observed when plotting IC50 valuesf XO activity and antiproliferation effect of these same extractsr = 0.852).

ig. 3. Correlation between the IC50 values of lipid peroxidation effect and cytotox-city of the tested compounds.

4: TOF 200 �g/assay + DNA + UV + H2O2, lane 5: TOF 50 �g/assay + DNA + UV + H2O2,lane 6: Ethyl acetate 500 �g/assay + DNA + UV + H2O2, lane 7: Ethyl acetate200 �g/assay + DNA + UV + H2O2, lane 8: Ethyl acetate 50 �g/assay + DNA + UV +H2O2.

3.7. Effect on pKS plasmid DNA scission induced byH2O2/UV-photolysis

The oxidation of DNA bases is produced by the reactive oxygenspecies (ROS), generated both from endogenous sources and fromreactions of xenobiotics [24]. The cells possess efficient DNA repairmechanisms for oxidative damage [25], but damaged forms of DNAwhich persist during replication lead to DNA mutations.

The damage of plasmid DNA produces a cleavage of one of thephosphodiester chains of the super-coiled DNA and results in arelaxed open-circular form. Further cleavage near the first fracture,results in linear double-stranded DNA molecules [26].

Figs. 4–8, show the electrophoretic pattern of DNA afterH2O2/UV-photolysis in the presence or absence of extracts or purecompounds. DNA derived from pKS plasmid showed two bands inagarose gel electrophoresis profile (lane 1). The faster moving band

corresponds to the native super-coiled circular DNA (Sc DNA), andthe slower moving prominent band was the open circular form (OcDNA). The UV irradiation of DNA in the presence of H2O2 (lane 2)results in the cleavage of Sc DNA to give prominent Oc DNA and/or

Fig. 6. Electrophoretic pattern of pBluescript KS DNA in the presence of Cate-chin and Luteolin of C. rotundus. Lane 1: DNA control, lane 2: DNA treated withUV + H2O2, lane 3: Catechin 100 �g/assay + DNA, lane 4: Catechin 50 �g/assay + DNA,lane 5: Catechin 10 �g/assay + DNA, lane 6: Catechin 100 �g/assay + DNA + UV, lane7: Catechin 50 �g/assay + DNA + UV, lane 8: Catechin 10 �g/assay + DNA + UV, lane9: Luteolin 100 �g/assay + DNA, lane 10: Luteolin 50 �g/assay + DNA, lane 11: Lute-olin 10 �g/assay + DNA, lane 12: Luteolin 100 �g/assay + DNA + UV, lane 13: Luteolin50 �g/assay + DNA + UV, lane 14: Luteolin 10 �g/assay + DNA + UV.

S. Kilani-Jaziri et al. / Chemico-Biologica

Fig. 7. Electrophoretic pattern of pBluescript KS DNA in the presence of Ferulic acidoF57

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f C. rotundus. Lane 1: DNA control, lane 2: DNA treated with UV + H2O2, lane 3:erulic acid 100 �g/assay + DNA, lane 4: Ferulic acid 100 �g/assay + UV + DNA, lane: Ferulic acid 50 �g/assay + DNA, lane 6: Ferulic acid 50 �g/assay + DNA + UV, lane: Ferulic acid 10 �g/assay + DNA, lane 8: Ferulic acid 10 �g/assay + DNA + UV.

faint linear DNA. This linear band of DNA indicates that OH• gen-rated from H2O2 UV-photolysis, allows DNA strand scission andreakage. Although both O2

•− and H2O2 are potentially cytotoxic,ost of the oxidative damage in biological systems is caused by theH•, which is generated by the reaction between O2

•− and H2O2 inhe presence of metal ions [26]. It was noted that the treatment by2O2 only or only UV treatment could not induce damage as noted

n combined treatment (lane 2, Figs. 4–7).TOF and ethyl acetate extracts gave a migration profile identical

o that of the untreated DNA. Both extracts did not induce any strandreaks, when DNA was exposed to UV irradiation and treated withhree doses of each extract (Fig. 4); the proportion of super-coiledlasmid strongly decreases with different extract concentrations,hereas that of open circular plasmid increases. The effect of cate-

hin, ferulic acid and luteolin against DNA derived from pKS plasmidas also tested. Luteolin as well as catechin gave a migration pro-le identical to that of the untreated DNA (Fig. 6). Whereas, the

erulic acid induces a total degradation of plasmid DNA, except athe low tested dose (10 �g/assay), with and without exposure tohe UV-radiation, which induces a total degradation of the super-oiled form and a weak degradation of the open circular form (faintc band; Fig. 7).

The addition of extracts at various concentrations to the reac-ion mixture of H2O2, induced low protection against damagesorresponding to the disappearance of the native super-coiledircular DNA (Fig. 5). The decrease in genotoxic potential of2O2/UV-photolysis was observed in the presence of the highest

oncentrations of TOF and ethyl acetate extracts (500, 200 and0 �g/assay). It can be seen that the different extracts inhibit strandreak formation in a concentration-dependent manner and sup-ressing in the way the formation of linear DNA. On the other

and, we tested the effect catechin, ferulic acid and luteolin againstNA damage induced by H2O2/UV-photolysis. These compoundsre preventing DNA oxidation and consequently blocking ROS pro-uction (Fig. 8).

ig. 8. Electrophoretic pattern of pBluescript KS DNA submitted to UV-photolysis of2O2 in the presence of different compounds of Cyperus rotundus. Lane 1: Ferulic acid00 �g/assay + DNA + UV + H2O2, lane 2: Ferulic acid 50 �g/assay + DNA + UV + H2O2,ane 3: Ferulic acid 10 �g/assay + DNA + UV + H2O2, lane 4: Catechin00 �g/assay + DNA + UV + H2O2, lane 5: Catechin 50 �g/assay + DNA + UV + H2O2,ane 6: Catechin 10 �g/assay + DNA + UV + H2O2, lane 7: Luteolin00 �g/assay + DNA + UV + H2O2, lane 8: Luteolin 50 �g/assay + DNA + UV + H2O2,ane 9: Luteolin 10 �g/assay + DNA + UV + H2O2, lane 10: DNA control, lane 11: DNAreated with UV + H2O2.

l Interactions 181 (2009) 85–94 91

4. Discussion

ROS and free radical mediated apoptosis induced by oxidativestress, play a crucial role in the induction and progression of sev-eral human diseases and age-related degenerative process [27,28].Oxidative stress induced injury resulting in DNA, protein and lipidalterations as well as transduction signal pathway dysfunction [29].Intracellular ROS production is associated with a number of cellu-lar events including activation of NADH oxidase, xanthine oxidase(XO), and the cellular mitochondrial respiratory chain [30]. XOis an important source of free radicals and has been reported invarious physiological and pathological models. Besides, signalingcascades involving the mitogen-activated protein kinases (MAPKs)pathway are key mediators of stress signals and seem to be mainlyresponsible for protective responses and stress-dependent apopto-sis reactions inducing alteration of proliferation and cytotoxicity incancer cell lines [31].

In fact, C. rotundus extracts possess an inhibitory effect on XO andscavenging effect on superoxide anions (O2

•−). In the same way, ithas been found that inhibition of superoxide anion production inthe X/XO system was probably due to both O2

•− scavenging activityand inhibition of the enzyme. This study provides evidence that thetested extracts exhibit interesting antioxidant properties expressedeither by the capacity to scavenge free radicals and to inhibit XOactivity. These findings were noteworthy because such compoundsmay be useful in the treatment of many kinds of diseases related tofree radical oxidations.

It seems that this activity is mostly related to the presence ofdifferent phenolic compounds such as flavonoids and tannins inthe tested extracts, which may be acting synergistically, with othercompounds. The key role of phenolic compounds as scavengers offree radicals is emphasized in numerous reports. Moreover, radical-scavenging activity is one of the various mechanisms to contributeoverall activity, thereby creating a synergistic effect.

The flavonoid components isolated from TOF enriched extractof C. rotundus are different from those detected in ethyl acetateextract. Quercetin is one of the main flavonoids identified from ethylacetate extract of C. rotundus. This compound has been described aschelating metal ions responsible for ROS generation [32]. It has alsobeen shown that luteolin and quercetin dose-dependently inhib-ited O2

•− production by xanthine oxidase [33]. However, manyflavonoids have been reported to be potent inhibitors of xanthineoxidase rather than scavenging superoxide anions [34]. In partic-ular, catechin and rutin significantly inhibited uric acid formation,competing with the xanthine for the active site of xanthine oxidase[35]. Narvaez-Mastache et al. [36] have reported that afzelechinpossess antioxidant activity. Altogether, these compounds werepredominant in ethyl acetate and TOF enriched extracts and mightinfluence the antioxidant activity of these extracts. However, wecan’t exclude that minor components could contribute to enhanceor to reduce antioxidant activity of these extracts.

Beside, ethyl acetate (EA) and TOF enriched extracts pos-sess an inhibitory effect on K562 cell proliferation. The strongcytotoxic activity of TOF and EA extracts may be attributedto the presence of specific components such as flavonoids andpolyphenols [8]. In addition, recent studies have indicated thatthe effects of flavonoids on antioxidant enzyme expression andactivity could be mediated by modification of signal transduc-tion pathways [37–39]. Dietary compounds can activate a numberof cellular kinases, including mitogen-activated protein kinases(MAPKs) and phosphatidylinositol-3-kinase (PI3K) [40], and both

pathways have been recently implicated in the up-regulation ofseveral antioxidant/detoxificant enzyme activities [41,42]. MAPKand PI3K pathways transduce a multitude of extracellular stim-uli by phosphorylating and activating downstream transcriptionfactors, enabling the cell to respond to stresses by increasing or

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2 S. Kilani-Jaziri et al. / Chemico-Bi

ecreasing the expression of critical genes [40]. In fact, moreecent studies have indicated that both green [41] and black43] tea polyphenol extracts could stimulate the transcription ofntioxidant/detoxificant enzymes through the activation of MAPKignaling pathways.

In our study, luteolin exhibits a stronger cytotoxic activity thanthyl acetate extract, from which it was isolated. Thus, it is proposedhat the presence of this compound contributed to the cytotoxicctivity of the original extract. On the other hand, the TOF enrichedxtract had the strongest cytotoxic activity, but the catechin iso-ated from this extract, was less active. Thus, it is proposed thatther compounds, unidentified yet in TOF enriched extract, mightarticipate to the inhibitory effect.

Because loss of cell viability could be related to apoptosis [44],e choose to monitor the ability of the extracts which showedcytotoxic effect to induce apoptosis in K562 cells. The typicalNA fragmentation pattern which is considered as the hallmarkf apoptosis was observed in cells treated with TOF enrichedxtract. This result implies that K562 cells were more sensitiveo TOF enriched extract-mediated proliferation inhibitory, thanthyl acetate extract, suggesting a selective influence of TOF onransformed K562 cells. TOF extract may possibly act as chemopre-entive agent, with respect to inhibition of cancerous cell growth,hrough apoptosis induction [44]. In fact, Sartippour et al. [45] haveeported that catechin inhibit proliferation of malignant cells. Initro cytotoxicity studies of green tea catechins have shown tumorells to be more sensitive than normal cells [46]. Tanins have aharacteristic feature of metal chelation and also act through theiredox property and hydrogen donating potential [47]. The induc-ive effect of TOF enriched extract on the apoptosis of humaneukemia K562 cells may partially be due to its antioxidant proper-ies by perturbing the favorable redox condition in cancer cells andnducing cytotoxicity. In fact, a strong correlation is reflected byhe correlation value obtained from the comparison of cytotoxicityf the tested compounds, toward cancerous cell line prolifera-ion, to their antioxidative effect. This observation is stronglyupported by the high superoxide scavenging/cytotoxic correla-ion coefficient (r = 1) reported for the two tested extracts and the

DA/cytotoxic correlation coefficient (r = 0.974) reported for botholecules and extracts, revealing the involvement of such activi-

ies in the apoptosis and cytotoxicity toward the cancerous testedell line. However, we also tried to specify correlation between theifferent tested antioxidative parameters. Thus, we observed thatuperoxide anion scavenging and anti-lipid peroxidation are theost correlated tested parameters with r = 0.958, so proposing a

ossible involvement of superoxide anion scavenging in protectiongainst lipid peroxidation. Whereas, we observed a low correlationetween inhibition of XO activity and lipid peroxidation protectionr = 0.666), one possible explanation for this result is that a num-er of inactive compounds in the in vitro XO system were active inhe ex-vivo lipid peroxidation system. In support of these data, theositive relationship between the different related variables waseported previously for several flavonoids used to inhibit agonist-timulated ROS production by neutrophils [48].

Moreover, ethyl acetate extract exhibits better antioxidant activ-ty than TOF enriched extract. This, might be due to its high contentf polyphenols [8] including luteolin, ferulic acid and other uniden-ified compounds. These compounds were the most abundant inthyl acetate extract and have been reported to possess an effectiventioxidants activity [49–51]. Also, we deduce that polyphenolicomposition of TOF extract is different from that of EA extract as

ar as they were extracted with solvents having extremely differentolarities. We can thus conclude that the protective effect exhibitedy EA extract corresponds to a participation of several compoundshich are different from those implied in the protective effect of

OF extract. Antioxidant activity is generally the result of the com-

l Interactions 181 (2009) 85–94

bined activity of a wide range of compounds, including phenolics,peptides, organic acids and other components [52]. On the otherhand, ethyl acetate extract exhibited a lower antioxidant activ-ity than that observed with luteolin compound isolated from thisextract. Thus, we hypothesize that other molecules, in ethyl acetateextract, might act as antagonist towards luteolin, that’s why wefound that anti-lipid peroxidation activity exhibited by the ethylacetate extract was lower than that observed with pure luteolinmolecule, as luteolin is one of the major compounds of this extract.We believe that luteolin may be the molecule responsible for ethylacetate extract anti-lipid peroxidation activity. In fact, it has beenfound that polyphenols are one of the most effective antioxidativeconstituents in plants [53].

H2O2 is one of the most important reactive oxygen–nitrogenspecies (RONS) in cellular systems. It is endogenously synthesizedin specific cell types as a response to activation by specific cytokinesor growth factors, acting as a second messenger to stimulate pro-tein kinase cascades coupled to inflammatory gene expression orin control of the cell cycle [54]. Exposure to high concentrationsof H2O2 as well as a decreased ratio between thiol and thiol disul-fide levels, generally leads to sustained tyrosine phosphorylationin numerous proteins, in part owing to the oxidative inhibition ofprotein tyrosine phosphatases in various cultured cell types [55].

Figs. 4–8 show the electrophoretic pattern of DNA afterH2O2/UV-photolysis in the presence or absence of extracts or purecompounds from C. rotundus. This study suggest that these extractsand compounds show antioxidant activity and this can be relatedto their ability to scavenge free radicals generated by the UV-photolysis of H2O2 and thus protecting DNA. This result suggeststhat C. rotundus aerial part compounds may protect DNA againstOH• attack. It can be seen that the different tested compoundsinhibit strand break formation in a concentration-dependent man-ner. This effect was previously reported for phenolic compoundswhich significantly protect the cells from UV-B induced DNA dam-age in rabbit corneal derived cells [56,57]. Therefore, it is thoughtthat the extracts from C. rotundus inhibiting oxidative DNA damageexert to prevent the cancer development.

In summary, it is well recognized that the enrichment of phe-nolic compounds within plant extracts is correlated with theirenhanced antioxidant, antigenotoxic and antitumor activities [58].Many biological activities have been reported so far for flavonoids.Their role against oxidative stress is well known. In food systems,polyphenols can act as free radical scavengers and terminate theradical chain reactions that occur during the oxidation of triglyc-erides. Various classes of flavonoid differ in the level of oxidationand saturation of ring C, while individual compounds within aclass differ in the substitution pattern of rings A and B. In fact, thedifferences in the structure and substitution will influence the phe-noxyl radical stability and thereby the antioxidant properties of theflavonoids.

The results suggest that the ethyl acetate and TOF enrichedextracts of C. rotundus, as well as their components, might be analternative to more toxic synthetic antioxidants as additive in food,pharmaceutical and cosmetic preparations. Further investigationsregarding their impact as chemopreventive agents on normal andcancer cell lines of human keratinocytes, are on course.

Conflict of interest

None.

Acknowledgments

The authors are grateful to the Ministère Francais des AffairesEtrangères (Action Intégrée de Coopération Inter universitaire

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ranco-Tunisienne, PHC UTIQUE 07 G0836 PAR), and to the Ministryf Higher Education, Scientific Research and Technology in Tunisiaor the financial assistance of this study.

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