Research ArticleChemicals Compositions Antioxidant andAnti-Inflammatory Activity of Cynara scolymus Leaves Extractsand Analysis of Major Bioactive Polyphenols by HPLC

Maryem Ben Salem1 Hanen Affes1 Khaled Athmouni2 Kamilia Ksouda1 Raouia Dhouibi1

Zouheir Sahnoun1 Serria Hammami1 and Khaled Mounir Zeghal1

1Laboratory of Pharmacology Faculty of Medicine of Sfax University of Sfax Sfax Tunisia2Laboratory of Faculty of Sciences of Sfax University of Sfax Sfax Tunisia

Correspondence should be addressed to Hanen Affes affeshanen13yahoofr

Received 20 December 2016 Accepted 19 February 2017 Published 30 April 2017

Academic Editor Valeria Sulsen

Copyright copy 2017 Maryem Ben Salem et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Objective Artichoke (Cynara scolymus L) was one of the plant remedies for primary health care The present study was focusedon the determination of chemical composition antioxidant activities and anti-inflammatory activity and on analyzing its majorbioactive polyphenols byHPLCMethods Artichoke Leaves Extracts (ALE)were analyzed for proximate analysis andphytochemicaland antioxidant activity by several methods such as DDPH ABTS FRAP and beta-carotene bleaching testThe carrageenan (Carr)model induced paw oedema in order to investigate the anti-inflammatory activity Identification and quantification of bioactivepolyphenols compounds were done by HPLC method The oxidative stress parameters were determined CAT SOD GSH MDAand AOPP activities and the histopathological examination were also performed Results It was noted that EtOH extract of ALEcontained the highest phenolic flavonoid and tannin contents and the strongest antioxidants activities including DDPH (9423)ABTS (53875mmol) FRAP assay (54262 umol) and 120573-carotene bleaching (7074) compared to the other extracts of ALEAdministration of EtOH extract at dose 400mgkgbw exhibited a maximum inhibition of inflammation induced by Carr for 3and 5 hours compared to reference group Indomethacin (Indo) Conclusion ALE displayed high potential as natural source ofminerals and phytochemicals compounds with antioxidant and anti-inflammatory properties

1 Introduction

The inflammation reaction is a physiologic response ofthe body contributed by aggression of microorganism andother soluble products Polynuclear neutrophils (PN) play animportant role in the initiation of inflammation with othermolecules named inflammatorymediators released by severalcells like cytokines endotoxins leukotrienes prostaglandinsand reactive oxygen species (ROS) [1] In the beginning forthe inflammation reaction the accumulation of PN in theinflammatory site and tissue and different cell injury aredue to participation of proteolysisrsquo enzymes and ROS byactivating defense systems [1]

Oxidative stress is a consequence of discrepancy balancebetween the production of ROS and antioxidants in a system

of defense of human organisms [2] Several studies showedthat many of antioxidants systems have the ability to treatsome disease like a cancer by scavenging ROS resultant byoxidative stress systems [3]

Carr induced paw oedema model is used to assess thedifferent phases of inflammation reaction Carr model caninduce acute inflammation release of inflammatory media-tors and production of free radicals [4]

Themechanism of antioxidant enzymatic systems againstthe inflammatory stress includes superoxide dismutase(SOD) catalase (CAT) and nonenzymatic antioxidants asreduced glutathione (GSH) Recently several studies showedthat lack of antioxidant systems can cause many inflamma-tory diseases [5] However it showed that various roles ofenzymatic and nonenzymatic antioxidants help to protect

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 4951937 14 pageshttpsdoiorg10115520174951937

2 Evidence-Based Complementary and Alternative Medicine

organisms from excessive generation of ROS in the inflam-matory states Some studies showed that natural herbs couldsuppress the production of oxidative stress by increasing theantioxidants systems [6] Plants have rich source of phenoliccompounds carotenoids vitamins and terpenoids Thesecompounds have a potential antioxidant that can be freeradical scavenger in order to reduce the development ofoxidative stress in many diseases [7]

In order to enhance the Tunisian forest resources anddevelop new products we are interested in the family ofAsteraceae particularly Cynara scolymus which character-izes the Mediterranean region and it has been widely used invarious hepatic diseases [8]

The objective of this work is designed to evaluate thephytoconstituents of ALE in vitro and the potential anti-inflammatory role of ALE in animalrsquos models

2 Materials and Methods

21 Plant Material and Extraction Method C scolymus driedleaves were obtained from the region of Bizerte in north ofTunisia the period of collection was December to March2014 The plant was authenticated by the Laboratory of Biol-ogy and Vegetable Ecophysiology in the Faculty of Science ofSfaxThe voucher sample was created byTheNational Botan-ical Research Institute of Tunisia Tunisia Dried powderedplant material (200 g) was extracted by maceration method(1 L) using different increasing solvent polarities (hexanebutanol ethyl acetate 75 EtOHH2O and aqueous) After48 hours all extracts were filtered Then the dried extracts ofartichoke were kept in the dark at +4∘C in order to evaluatethe composition of C scolymus leaves extracts

22 Phytochemical Analysis of Cynara scolymus LeavesExtracts The analytical tests for identification of differentsecondary metabolites in Cynara leaves extracts were con-ducted following procedures described by Sofwora andOkwu[9 10]

23 Proximate Analysis of Dried Leaves of C scolymus Thedetermination of nutritional composition crude proteinlipids fiber and ash was obtained using several methodsdescribed while the carbohydrate content was obtained bythe difference method (calculated by subtracting the sum ofcrude fat crude protein ash content and crude fiber) [11]

For the determination of crude protein the concentrationof dried samples was determined by micro-Kjeldahl methodThe lipid content was estimated by using petroleum ether as asolvent extraction in a Soxhlet at 40ndash60∘C [12] Total dietaryfiber was determined by extraction with petroleum etherThedefatted sample was boiled under reflux with two solventsH2SO4 and NaOH and then they were filtered and washedwith boiling water till the filtrates were no longer acidic andbasicThe residue of the sample was dried in an oven at 100∘Cand 600∘C cooled in desiccators and weighed [13] The ashcontentwas estimated by heating in amuffle furnace at 600∘C

The quantification of drymatter was performed using onegram of C scolymus leaves heated in 105∘C for 1 hourThen itwas put in desiccators for 30min After that the mass of each

content has been noticed These steps have led to dry leavesand theirmass has been noticed again in order to calculate thepercentage of humidity in these samples [11] In addition theanalysis of sugar amounts was obtained by phenol-sulfuricacid reagent [14]

24 Quantification of Total Phenolics Flavonoids and TanninsContents of Cynara scolymus Leaves Extracts The quantifica-tion of total phenolics content (TPC) of ALE was determinedby Fawole et al method [15] 200 120583L of ALE was mixed with1mL of Folin-Ciocalteu reagent diluted (times10) with distillatedwater and 08mL of 75 of NaCO3 solution in a test tube30min later the absorbance was measured at 765 nm byusing a Jenway 6405 UV-Vis spectrophotometer TPC wasexpressed as milligrams of gallic acid equivalents per gram ofdry weight (mg GAg DW) Quantification of total flavonoidcontent (TFC) was determined spectrophotometrically [16]500 120583L of ALE was mixed with 1500120583L of water and 150120583Lof (5) NaNO2 After 5min 150 120583L of ALCl3 (10 mv)was added to mixture After 6min of incubation at roomtemperature a volume of 500 120583L of NaOH (4) was addedalso Immediately the mixture was completely agitated inorder to homogenize the contentsThe absorbance of solutionobtained was measured at 510 nm against a blank Theseanalyses were expressed as mg of catechin equivalents pergram of dry weight (mg CEg DW)

The determination of condensed tannin content (TCC)was measured using vanillin method [17] 50120583L of ALEdiluted was added to mixture which was made of 3mL of4 methanol-vanillin solution and 15mL of concentratedH2SO4 The mixture was incubated to stand for 15min andthe absorbance was measured at 500 nm against methanol asa blank The amount of TCC was expressed as mg catechinequivalent gminus1 dry weight (mg CEg DW)

25 Minerals Contents Analysis The preparation of C scoly-mus dried leaves was incinerated in a muffle furnace at 550∘Cfor 8 hours [18] and the ashes obtained were digested innitric acid and dissolved in distilled water for the mineralcomposition of artichoke leaves [19 20]Minerals elements ofC scolymus leaves were potassium (K)magnesium (Mg) cal-cium (Ca) sodium (Na) iron (I)manganese (Mn) zinc (Zn)copper (Cu) and chromium (Cr) These mineral contentswere determined by flame atomic absorption spectrometry(Hitachi Z-6100 Japan)

26 Analysis In Vitro of Antioxidants Properties

261 Antioxidant Activity by DPPH Method The antioxi-dant activity of DPPH is based on scavenging of DPPH

from antioxidants in the vegetal sample which produce aspectrophotometric loss in absorbance at 515 nmThe DPPHassay (Sigma Chemical Co St Louis MO) was evaluated asdescribed by Fawole et al [15] The mixture was prepared intest tubes by dilution of 50 uL ofALE in 735mLof 100meth-anol

750mL of 01mM methanolic DPPH reagent was addedto the mixture of ALE-methanol Then the mixture wasincubated at room temperature in a chamber without any

Evidence-Based Complementary and Alternative Medicine 3

light during 30min After incubation the estimation of thescavenging ability was performed by measuring at 517 nm inspectrophotometer (T70 UV-Vis)

The capacity of inhibition percentage (PI) of DPPHradicals was calculated as

DPPH radicals (PI) = [(119860119887 minus 119860 119904)119860119887 ] times 100 (1)

where 119860119887 refers to the absorbance of control (without plantextract) and 119860 119904 to the absorbance of sample (with plantextract) BHT and VC were used as standards at the sameconcentrations of plant extract

262 120573-Carotene Bleaching Test The capacity of ALE toreduce bleaching of beta-carotene was previously determinedbyKoleva et al [21]Themixture of beta-carotene and linoleicacid was prepared 05mg of 120573-carotene 25 uL of linoleicacid and 200 uL of Tween 40 were dissolved in 1mL ofchloroform solvent The chloroform was evaporated in arotator evaporator at 40∘C and 100mL of dH2O was addedthen the mixture was stirred

Aliquots of 25mL of beta-carotenelinoleic acid emul-sion obtained were transferred to test tubes containingdifferent ALE concentrations then the emulsion of reactionwas incubated for 2 h at 50∘C and the absorbance of eachsample was measured at 470 nm by spectrophotometer BHTandAAwere used as the standards at the same concentrationsof the samples

263 Antioxidant Activity by ABTS∙+ Method The abilityto neutralize the ABTS∙+ was reported by Re et al [22]using a spectrophotometric 96-well microplate methodThepreparation of ABTS∙+ free radical solution by incubating amixture of ABTS (7mM) and K2S2O8 (245mM) dissolvedin distillated water to create a stable color of radical solutionfollowing 12ndash16 h of incubation in the dark room at 4∘C

Therefore the standard ABTS∙+ solution was prepared bydilution with ethanol to a standard absorbance of 07 plusmn 002at 734 nm

50 120583L of plant extract (1ndash20120583gmL) was added to 150120583Lof ABTS∙+The plates were incubated at room temperature inthe dark room during 15min the absorbance was measuredat 630 nm Control wells contained 50 120583L of H2O and 150 120583Lof ABTS∙+ Phytochemical interference was accounted bywells containing extract (50 120583L) with dH2O (150 120583L) whereas200120583L of H2O served as blank Trolox dissolved in pureMeOH was used as standards

The antioxidant capacity of ALE was expressed quantita-tively as mmol of Trolox Equivalents (TE) (mmol TEg dryextract)

264 Ferric Reducing Assay The potential ability of ALE toreduce the ferric iron was evaluated previously by Fawole etal [15]

The mixture of FRAP was freshly prepared by 50mL of300mM acetate buffer 5mL of 10mM TPTZ and 5mL of20mM FeCl3 Prior to its use the prepared mixture must beincubated in water bath at 37∘C during 15min to stabilize thecontents in the reaction mixture

Exactly 150mL of diluted ALE was transferred intodifferent test tubes and then 250mL of FRAP solution wasadded in triplicate The mixture was stirred and incubated ina dark room for 30min before measuring the absorbance at515 nm inUV-Vis spectrophotometer Antioxidant capacity ofALE was expressed as micromoles of Trolox Equivalent permilliliter of sample (mMTEmL)

27 Anti-Inflammatory Activity In Vivo

271 Chemicals and Reagents Lambda carrageenan andIndomethacin were purchased from Sigma Aldrich company(France)

272 Experimental Study 30 sexually mature male rats10ndash12 weeks old weighing 150ndash200 g were obtained fromthe Institute of Pasteur Tunisia Rats were fed with stan-dard laboratory pellets and ad libitum access during theexperiment study The experimental protocol was conductedin accordance with the Guide for the Care and Use ofLaboratory Animals issued by the University of Sfax Tunisiaand approved by the Ethics Committee of Animal useprotocol number 94-1939 Before the experiment rats wereacclimatized in controlled environmental conditions at 24 plusmn4∘Cwith relative humidity (45ndash55) and 12 h dark-light cycleAll the groups of animals were randomly divided into thecontrol group and treatment groups

273 Carrageenan-Induced Paw Oedema Model The eval-uation of anti-inflammatory activity was reported by Raviet al [23] 01mL of Carr solution (1) was injected intosubplantar surface of the paw of each group of rats to produceacute inflammation This method was chosen over othermethods for the discovery of new therapeutic effects of ALEin inflammation diseases as it is the most basic methodrequiring minimal equipment but much practice

The experimental study designed 4 groups of six ratsGroup (I) was the control group which received isotonicsaline solution Nacl (0 9) by subplantar injection andhad no inflammation and received no treatment Group(II) was inflamed by carrageenan injection and did notundergo any treatment (Carr) Group (III) was used asreference inflamed rats that were treated with Indomethacin(10mgkgbw) (Carr + Indo) by intraperitoneal injection(ip) and Group (IV) was treated with EtOH extract of ALEat dose 400mgkgbw (Carr+ ALE) by ip [24] The doses ofALE and Indo chosen during treatments were proportionalto the size of the oedema and covered the whole swelling

In all treated groups the oedema paw volumes up totibiotarsal articulation were measured using a digital caliperat 1 2 3 4 and 5 hours after Carr injection

For each treated group the size of oedema obtained atthese various times (PT) was compared to that obtainedbefore any treatment (P0)

Percentages of oedema inhibition were calculated as

Inhibition (EI) = [1 minus (PTP0 )] times 100 (2)

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Phytochemical analysis of Cynara scolymus extracts leaves

Extract Cardiac glycosides Triterpenoids Saponin Flavonoids Tannins AlkaloidALE + + + + + +Sign (+) indicates being present

Percentages of inflammation inhibition were calcu-lated as

Inhibition (II) = [PT minus (P0P0)] times 100 (3)

274 Blood Sample Collection 5 hours after Carr inductionthe rats were decapitated and the blood samples were col-lected in heparin tubes Plasma samples were obtained aftercentrifugation at 3000 rpm for 15min and they were kept inminus20∘Cuntil analysis on an automatic biochemistry analyzer atthe biochemical laboratory of Hedi Chaker Hospital of Sfax

275 Inflammatory Biomarkers

(1) Determination of C-Reactive Protein (CRP) CRP wasa specific marker following the inflammatory process Itincreases in proportion to its intensity [25] The reactiveprotein was measured by turbid metric method using anautomatic analyzer COBAS INTEGRA 40010158401015840 C-reactive TheCRP is expressed with mgL

(2) The Fibrinogen Assay of Plasma Plasma fibrinogen con-centration was determined by Clauss clotting method [26]measured on a STAanalyzer Principle test measures theconversion rate of fibrinogen into fibrin in diluted samplein presence of excess of thrombin and records the clottingtime The clotting time is inversely proportional to the levelof fibrinogen in the plasma The fibrinogen level is expressedwith gL plasma

(3) Exploring the Antioxidant Enzymatic and NonenzymaticStatus Oxidative stress parameters were determined in tis-sues paw oedema homogenates The supernatants obtainedwere removed and analyzed for the determination of MDAas described by Draper and Hadley [27] AOPP levels werequantified by method of Kayali et al [28] CAT activity wasmeasured as reported by Aebi [29] and expressed as mmolesof H2O2 consumed(minmg protein) SOD was assayedspectrophotometrically by colorimetric method of Beyer Jrand Fridovich [30] and expressed as Umg protein GSHactivity was assayed according to the method of Carlbergand Mannervik [31] and expressed as mmoles GSS(min-mg protein) and the protein content was determined usingmethod of Lowry et al [32]

(4) Histopathological Examination All the paws oedematissues of experimental groups were collected for histolog-ical examination First they were fixed in 10 bufferedformalin solution second they were embedded in paraffinwax and then cut into 5mm thick sections and stainedwith hematoxylin and eosin (HampE) Finally the slides werephotographed with an Olympus U-TU1X-2 camera

28 High Performance Liquid Chromatography (HPLC)Detec-tion and Quantification of Polyphenolic Compounds Thephenolic fractions analysis of EtOH extract of ALE wasdetermined using an Agilent Technologies 1100 series HPLCcoupled with a UV-Vis multiwavelength detector The sepa-ration was carried out on a 250 times 46mm C18 silica columnchromatography was chosen at ambient temperature Themobile phase consisted of C2H3N (solvent A) and waterwith 02 of H2SO4 (solvent B) and the flow rate waskept at 08mLminminus1 For the preparation of calibrationcurve a standard stock solution was prepared in methanol(HPLC grade ge 999 from Sigma Chemical Company)containing Hydroxytyrosol Tyrosol 4-hydroxybenzoic acidverbascoside apigenin-7-glucoside Oleuropein QuercetinPinoresinol cinnamic acid and apigenin at the same concen-tration of 1 gmL For the preparation of the ethanol extract bydissolving in 1mL of methanol making a final concentrationof 25mgmL Before starting HPLC analysis all the solutionspreparedwere filteredwithWhatman paper (Oslash 045 120583m)Thediluted extract was injected directly and chromatogramsweremonitored at 280 nm 20120583L was used for injection Peakswere identified by the retention times compared with thestandardsThe analyses of phenolic profiles of ethanol extractwere performed in triplicate

29 Statistical Analysis SPSS program was employed forcomparisons between all groups Comparison between mul-tiple and within groups was analyzed by ANOVA followedby Tukeyrsquos tests Values were shown as mean plusmn SD The levelof statistical significance was set at 119901 value lt 005 Pearsonrsquoscorrelation coefficient analysis was calculated using SPSSprogram also in order to evaluate the statistical relationshipbetween both of polyphenolics compounds and antioxidantactivities of ALE

3 Results

31 Phytochemical Analysis of Cynara scolymus LeavesExtracts ALE revealed the presence of flavonoid cardiac gly-cosides saponin tannin terpenoid and alkaloids (Table 1)

32 Proximate Analysis of Dried Leaves of C scolymus Theresults showed that Cynara leaves contain high constituentsdry matter (9703 DW) ash (1581 DW) carbohydrate(8005DW) protein (1664DW) lipids (341DW) totalsugars (197 DW) and dietary fiber (7160 DW) (Table 2)

33 Quantification of Total Phenolic Flavonoid and TanninsContents of Cynara scolymus Leaves Extracts TheTPC TFCand TCCwere evaluated in different solvents extracts of ALEAll results are shown in Table 3 Maximum content of TPCwas obtained using EtOHextract and corresponded to 5454plusmn126mg GAg DW followed by ethyl acetate aqueous and

Evidence-Based Complementary and Alternative Medicine 5

Table 2 Proximate analysis of dried leaves of Cynara scolymus

Constituents Cynara scolymus leaves(percentage dry weight basis)

Dry matter 9703 plusmn 043Ash 1581 plusmn 001Carbohydrate 8005 plusmn 069Protein 1664 plusmn 179Lipids 341 plusmn 045Total sugars 197 plusmn 010Dietary fiber 7160 plusmn 081Values are expressed as mean plusmn SD (119899 = 3)

butanol extracts while TPC of hexane extract was the lowestin comparison with EtOH extract (3091 plusmn 936mg GAEgDW) The EtOH extract showed also the highest content inTFC (12plusmn083CEgDW) but this value was lower for hexaneextract (819 plusmn 06mg CEg DW) respectively

The amount of TCCwhichwas evaluated by vanillin assaywas the highest in the ethyl acetate and EtOH extract (1451and 1405mg CEg DW) but this content was lower thanaqueous extract as shown in Table 3

34 Mineral Contents Analysis The main composition ofmacroelements and microelements in C scolymus leaves ispresented in Table 4

Elemental analysis in (mg100 g of dry weight basis)indicated that leaves of artichoke contained the followingorder of essential minerals compounds potassium (288680)sodium (176294) calcium (135934) magnesium (43321)iron (1617) manganese (1305) zinc (737) copper (130) andchromium (012)

35 Evaluation of Antioxidant Activity Theantioxidant activ-ity of ALE harvested in Tunisia was performed using fourmethods DPPH ABTS FRAP and beta-carotene test

351 Antioxidant Activity by DPPHMethod DDPHmethodevaluated the capacity of compounds present in ALE toreduce DDPH radical The result in Figure 1 showed thescavenging activity of all extracts of C scolymus was con-centration dependent Among all extracts the EtOH extractdisplayed the highest free radical scavenging activity at higherconcentration (400 ugmL) (9423) compared to the sameconcentration of vitamin C (983) and BHT (9623) fol-lowedby ethyl acetate extract (8151) the scavenging activityof other extracts was significantly lower when compared toreference standard VC (9983)

352120573-Carotene Bleaching Test Thepotential effect of EtOHextract exhibited significantly the highest inhibition rate of120573-carotene bleaching (7074) compared to BHT and AA(4794 9059) (119901 lt 0001) respectively (Table 5) whereashexane and butanol extract showed the lowest rate of 120573-carotene bleaching 3738 and 4939

353 Antioxidant Activity by ABTS∙+ Method As evidentfrom Table 5 ALE studied presents a potential capacity

lowastlowastlowastlowastlowastlowast

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0102030405060708090

100

in

hibi

tion

100ugmL200ugmL

300ugmL400ugmL

Etha

nol

Hex

ane

Ethy

l ace

tate

Buta

nol

Aque

ous

VC BHT

Figure 1 Antioxidant activity by DPPHmethod ofCynara scolymusleaves extracts at different concentrations Values are mean plusmn SD(119899 = 3) lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 compared toEtOH extract Butylated Hydroxytoluene (BHT) Vitamin C (VC)

0 1 2 3 4 5 6Temps (h)

0

05

1

15

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25

3

35

4Si

ze o

f oed

ema (

mm

)

lowastlowastlowast

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lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

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lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

CARRNACL

CARR + IndoCARR + ALE

Figure 2 Effect of ALE and Indomethacin on paw oedema inducedby carrageenan Values represent mean plusmn SD (119899 = 6) in each grouplowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 120572 compared to control 120573compared to Carr m compared to Carr + Indo

to scavenge the ABTS radical cation Among all extractsEtOH extract was a significantly higher value (49943mmolTroloxg dry extract) than the other extracts but the low-est level of TEAC was obtained from the hexane extract(1043mmol Troloxg dry extract)

354 Ferric Reducing Assay The results in Table 5 showedthat among all extracts of ALE a higher capacity of reducingferric capacity was found for EtOH extract (52779 umolFe2+mg dry extract) when compared to other extractsrespectively

36 Anti-Inflammatory Activities The appearance of Carrinduced paw oedema with respective treatments groups wasillustrated in Table 7 and Figure 2 The results showed

6 Evidence-Based Complementary and Alternative Medicine

Table 3 Quantification of total phenolic flavonoids and tannins contents of Cynara scolymus leaves extracts

Extracts Phenolics content(mg GAEg DW)

Flavonoidscontent

(mg CEg DW)

Tannins content(mg CEg DW)

Hexane 3991 plusmn 936 819 plusmn 016 1405 plusmn 03Ethyl acetate 5307 plusmn 047 1032 plusmn 012 1451 plusmn 013Butanol 4166 plusmn 223 1121 plusmn 010 1393 plusmn 93Ethanol 5454 plusmn 126 1200 plusmn 083 1099Aqueous 4949 plusmn 039 949 plusmn 039 438 plusmn 045Values are expressed as mean plusmn SD (119899 = 3)

Table 4 Mineral contents dried leaves of Cynara scolymus

Elements Cynara scolymus leaves (mg100 g of dry weightbasis)

K 2886803 plusmn 120Ca 1359346 plusmn 505Na 1762946 plusmn 120Mg 433219 plusmn 234I 16176 plusmn 014Mn 13051 plusmn 011Zn 7371 plusmn 014Copper 130 plusmn 016Cr 0124 plusmn 001Values are expressed as mean plusmn SD (119899 = 3)

that the induction of Carr in rats paw oedema started offby the vascular phase of inflammation which generated anincrease in the size of oedema for all groups This injectiongenerated intense inflammation which peaked after 3 hoursThe experimental data showed that ALE presented significantdecrease in the size of pawoedemawhichwas time dependentand more important than the reference group Indo and Carrgroup

As seen in Figures 3 and 4 EtOH extract at dose(400mgkgbw) has shown a significant percent of oedemainhibition (119901 lt 0001) in the first hour (173) comparedwith reference group (Indo) and the significant percent ofinhibition inflammation was 4427 in comparison with thecontrol group

Thus after five hours these percentages were 735 forinhibition and 832 for inflammation For the Indo groupoedema inhibition was significant (119901 lt 0001) 53 and 64and oedema inflammation was 21 and 86 in comparisonwith the control group

These results showed that EtOH extract of ALE had astrong effect as Indo

361 Inflammatory Biomarkers Fibrinogen and CRP Weassayed serum proteins of inflammatory markers in differentgroups of rats

(1) FibrinogenCarr group caused a significant increase in rateof fibrinogen compared to control group by 1905 (Figure 5)

1 2 3 4 5Temps (h)

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in

hibi

tion

oede

ma

poundlowastlowastlowast

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poundlowastlowastlowast

Carr + ALECarr + Indo

Figure 3 Percentage () of oedema inhibition data in all groupsValues represent mean plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt001 and lowastlowastlowast119901 lt 0001 m compared to Carr + Indo

0

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hibi

tion

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atio

n

1 2 3 4 5Temps (h)

NaclCarr

Carr + ALECarr + Indo

poundlowastlowastlowastlowastlowast

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Figure 4 Percentage () of oedema inflammation data in allgroups Values represent mean plusmn SD (119899 = 6) in each group lowast119901 lt005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 120572 compared to control group120573 compared to Carr group and m compared to Indo group

The rate of fibrinogen decreased significantly (119901 lt 001) ingroups of rats treated with the EtOH extract (1655) andIndo (2290) compared with Carr group

Evidence-Based Complementary and Alternative Medicine 7

Table 5 Antioxidant activities of Cynara scolymus leaves extracts

Sample FRAP assay (umol Fe (II)gDW)

120573-Carotene bleaching assay()

TEAC assay (mmolTroloxg DW)

Hexane 223023 plusmn 1116lowastlowastlowast 3738 plusmn 524lowastlowastlowast 1043 plusmn 1073lowastlowastlowastEthyl acetate 50829 plusmn 524lowastlowastlowast 6156 plusmn 817lowast 38260 plusmn 524lowastlowastButanol 44306 plusmn 2298lowastlowastlowast 49393 plusmn 224lowastlowast 25193 plusmn 2815lowastlowastlowastEthanol 52779 plusmn 1626 70743 plusmn 129 49943 plusmn 3972Aqueous 31591 plusmn 836lowastlowastlowast 5611 plusmn 543lowastlowast 21074 plusmn 836lowastlowastlowastBHT mdash 4794 plusmn 075lowastlowastlowast mdashAA mdash 9059 plusmn 325lowastlowastlowast mdashValues are means plusmn SD (119899 = 3) Butylated Hydroxytoluene (BHT) and Ascorbic Acid (AA) were used as positive control compared with EtOH extract lowast119901 lt005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001

Nacl Carr Carr + ALE Carr + Indo0

05

1

15

2

25

Fg (gL)CRP (mgmL)

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

Figure 5 Levels of fibrinogen (Fg) and C-reactive protein (CRP)Values represent mean plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt001 and lowastlowastlowast119901 lt 0001 120572 compared to control 120573 compared to Carrgroup Fibrinogen (Fg) C-reactive protein (CRP)

(2) CRP Analysis showed a significant increase (119901 lt 005)in CRP level (Figure 5) for Carr group compared to controlgroup (1673) However the injection of EtOH extract andIndo led to a significant decrease (119901 lt 0001) (60863423) respectively when compared with Carr group

37 Oxidative Stress Parameters MDA and AOPP levels inoedema paw were illustrated in Table 8 Results showed thatCarr induced a significant increase inMDA andAOPP levelshowever treatment with EtOH extract of ALE at dose of400mgkgbw showed a significant decrease (119901 lt 0001)(2080 5023) compared with Carr group and even Indogroup (10mgkg) (2097 4427) respectively

38 Exploring the Antioxidant Enzymatic and NonenzymaticStatus CAT SOD and GSH levels in the paw oedema tissueof tested groups are shown in Table 8 Treatment of inflamedrats with EtOH extract restored significantly (119901 lt 0001)the SOD activity by 2075 the CAT activity by 5655 andthe GSH activity by 7566 respectively compared to Carrgroup

39 Histopathological Examination A microscopic study ofpaw oedema tissues showed histological changes in Carrgroup EtOH extract of ALE (400mgkgpc) group andIndo (10mgkgbw) group (Figure 6) In the Carr group weshowed a subcutaneous oedema with infiltration of inflam-matory cells especially polynuclear neutrophils at the site ofinflamed tissues and also the presence of spongy-like appear-ance in the epidermis ((Figure 6(b)) as compared to controlgroup (Figure 6(a))) The EtOH extract group produced asignificant decrease in the number of cellular infiltrates and asignificant reduced spongy-like appearance in the epidermis(Figure 6(c)) as did reference drug Indo (Figure 6(d))

310 Quantification of Polyphenolic Compounds by HPLCTo the authorsrsquo knowledge the present study identified andquantified the phenolic compounds of ALE from Tunisianorigins Accordingly the results obtained so far by Folin-Ciocalteu needed to be further complemented to qualifyphenolic constituents in EtOH extract under investigation

The findings revealed the presence of seven phenoliccompounds namely Hydroxytyrosol verbascoside apige-nin-7-glucoside Oleuropein Quercetin Pinoresinol andapigenin HPLC analysis indicated that ALE have significantamount of verbascoside content 381mg100 g and Quercetincontent (19 2mg100 g) (Figures 7 and 8 and Table 9)

311 Correlation between the Polyphenolic Compounds andAntioxidant Capacity In order to determine the contributionof the phenolic and flavonoid content in ALE on antiox-idant capacity the Pearson correlation coefficient (119903) wasdetermined in Table 6 The results showed a significantlinear correlation between antioxidant activities determinedby using DPPH ABTS FRAP and beta-carotene methodsand TPC and TFC respectively The strongest correlativevalue is obtained with DDPH and TPC (119903 = 087) Theseresults indicated a good correlation between TPC and TFCwith antioxidant activities of ALE

4 Discussion

The importance of plants in traditional medicine remediesand the potential of phytochemical constituents were dis-cussed nowadays with respect to their benefit in pharma-cotherapy in Tunisia Cynara scolymus was one of these

8 Evidence-Based Complementary and Alternative Medicine

(a)

(b)

(c)

(d)

Figure 6 Histopathological slides tissues of paw oedema in experimental groups of rats (a) Control group untreated group (b) Carr groupCarr-treated rat showed heavy infiltration of polynuclear neutrophils (PN) and a spongy-like appearance and bulla in the epidermis (c)Carr + EtOH extract group Carr-treated rat that received the EtOH extract (400mgkgbw) reduced significantly the migration of PNand oedematasis in dermis without any spongy-like feature and bulla (d) Carr + Indo group Carr-treated rat that received Indomethacin(10mgkgbw) showed a partial protective action Deparaffinized hematoxylin and eosin (HampE) stained sections (200ndash400x) Plus signinfiltration of PN Arrow odematasis in the epidermis

medicinal plants which have a beneficial potential effectattributed to its source of polyphenolic compounds [3]

The selection of this plant was guided by the indicationsof its traditional use that at present there have been very littlechemical and biological investigations done Therefore thepresent study was to investigate phytochemical compositionand their antioxidant capacity and to evaluate in vivo the anti-inflammatory effects of Cynara scolymus leaves extracts

The presence of phenolic compounds is very widelydistributed in medicinal plants several studies showed thatthese compounds have drawn much attention to their poten-tial antioxidant abilities which demonstrated their beneficialimplications for human health

Regarding the determination of phenolic compoundsthe EtOH extract of ALE had the highest amount of thesecompounds in comparison with other extracts which werean agreement with the result of Emanue et al [33] whoshows that EtOH extract exhibited the maximum amountof phenolic compounds from the leaves of Cynara scolymuswhereas it differs from the reports of Oliveira et al [34]who proves that aqueous extract of ALE is the most suitablesolvent for extraction of phenolic compounds

The importance of TPC and TFC extraction yieldsobtained with EtOH extract can be attributed to its goodsolubility low toxicity medium polarity and high extractioncapacity [35]

Evidence-Based Complementary and Alternative Medicine 9

1 2 3 678 9 10

5

4

0250500750

1000125015001750

(mAU

)

5 10 15 20 250(min)

Figure 7 HPLC chromatogram of a standard mixture of polyphe-nolic compounds Peaks 1 Hydroxytyrosol 2 Tyrosol 3 4-hydroxybenzoic acid 4 verbascoside 5 apigenin-7-glucoside 6Oleuropein 7 Quercetin 8 Pinoresinol 9 cinnamic acid 10apigenin

34

2

56 7

0200400600800

100012001400

(mAU

)

5 10 15 20 25 30 350(min)

1

Figure 8 HPLC chromatogram of ethanol extract of Cynarascolymus leaves extracts Peaks 1 Hydroxytyrosol 2 verbascoside3 apigenin-7-glucoside 4 Oleuropein 5 Quercetin 6 Pinoresinol7 apigenin

Table 6 Pearson correlation coefficient (119903) between the content ofphenolic compounds and antioxidant capacity (DDPH∙ ABTS∙+FRAP and beta-carotene)

Correlation 119877TPC versus DDPH∙ 0870lowastlowast

TPC versus ABTS∙+ 0848lowastlowast

TPC versus FRAP 0707lowastlowast

TPC versus beta-carotene 0842lowastlowast

TFC versus DDPH∙ 0728lowastlowast

TFC versus ABTS∙+ 0743lowastlowast

TFC versus FRAP 0849lowastlowast

TFC versus beta-carotene 0712lowastlowast

DPPH 22-diphenyl-1-picrylhydrazyl ABTS 220-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt FRAP ferricreducing TPC total phenolic content TFC total flavonoid contentlowastlowast119901 lt 001 significant correlation

In the first investigation of my study five solvents ofincreasing polarities were chosen for the determination ofphenolic compounds from leaves of C scolymus namelyhexane ethyl acetate butanol 75 EtOHH2O and aqueousin order to determine the variability of TPC and TFC in theaerial part of Cynara as a function of each extraction solvent

This variability of phenolic compounds in ALE can beattributed to the wide range of solubility displayed by variouspolar compounds within the ALE solvents the degree ofpolymerization of phenols and their interaction geneticfactors geographical variations and climatic changes [36]

Overall these findings indicate that EtOH extract of ALEwas rich in phenolic and flavonoid contents which couldbe the major contributor to their antioxidative propertiesMany researches revealed that flavonoids and polyphenolsdisplayed the highest ability of scavenging activity in medic-inal plants [37 38]

The results indicate that EtOH extract of ALE whichcontains the highest content of TFC and TPC displays thehighest free radical scavenging activity (9423) at concen-tration of 400 ugmL However Oliveira et al [34] show thataqueous extract of ALE has good radical scavenging (8340)at concentration of 200 ugmL Furthermore our resultssuggest that some components within EtOH extract aresignificantly the strongest radical scavenger in comparisonwith other extracts The TEAC values of ALE showed alsothat EtOH extract presents a potential ability to scavengethe ABTS radical cation in accordance with the report ofBetancor-Fernandez et al [39]

The FRAP assay shows also that EtOH extract has thehighest values (54262 umol Fe II gDW) The same resultis confirmed by Kukic et al [40] Therefore the results ofpositive correlation between the TPC TFC and the anti-oxidant methods in vitro suggest that phenolic compoundsact as reducing agents hydrogen donors and singlet oxygenscavengers [3] and may exert an important in vitro antioxi-dant capacity of ALE

The phytochemical screening of ALE has been foundto be a rich source of polyphenolic compounds includingQuercetin apigenin-7-glucoside and verbascoside Thesecompounds have shown a great potential of antioxidantactivity [41]

The research into medicinal plants used as pain relieversrsquoagents should therefore be viewed as new therapy in theinflammatory diseases [42] The anti-inflammatory activitiesof EtOH extract of ALE are investigated applying in vivoexperimental Carr model compared with a nonsteroidal anti-inflammatory (Indomethacin) this drug was reported byHiggs et al [43] who demonstrated the role of Indo toinhibit the biosynthesis of prostaglandins in Carr model ratsThe experimental study of anti-inflammatory activity is per-formed by the Carr test This phlogiston agent inducedtissue oedema characteristic of acute inflammation whichis regarded as a crucial parameter in assessing anti-inflam-matory activity [44]

The Carr injection produces biphasic states in the firstphase [45] there is an increase in the mRNA synthesis ofcyclooxygenase-2 (COX-2) at the first hour This increase isaccompanied by an amplification of synthesis of strong proin-flammatory mediator such as prostaglandin especially theprostaglandin type 2 involved in the inflammatory process[46] serotonin bradykinin and leukotrienes which con-tribute to the initiation of inflammation reaction Moreoverthe participation of arachidonicmetabolites is themain factorresponsible for both of phases of Carr induced inflammation

10 Evidence-Based Complementary and Alternative Medicine

Table7Eff

ecto

fCynarascolym

usleaves

extracto

ncarrageenan-indu

cedratp

awoedema

Treatm

ent

Oedem

asize(meanplusmnS

D)(mm)

0h1h

2h3h

4h5h

Con

trol

0426plusmn0

00

044

1plusmn001

056plusmn0

03

0650plusmn0

05

0755plusmn0

05

070plusmn0

01

Carr

0426plusmn0

00

28plusmn026120572lowastlowastlowast

333plusmn028120572lowastlowastlowast

325plusmn002120572lowastlowastlowast

315plusmn001120572lowastlowastlowast

302plusmn009120572lowastlowastlowast

Carr+Indo

(10m

gkg)

0426plusmn0

00

246plusmn026120572lowastlowastlowast120573lowast

261plusmn007120572lowastlowastlowast120573lowastlowastlowast

230plusmn002120572lowastlowastlowast120573lowastlowastlowast

18plusmn011120572lowastlowastlowast120573lowastlowastlowast

14plusmn006120572lowastlowastlowast120573lowastlowastlowast

Carr+ALE

(400

mgkgbw)

0426plusmn0

00

2480plusmn005120572lowastlowastlowast120573lowastlowastlowast

188plusmn004120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

165plusmn005120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

116plusmn020120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

112plusmn01120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

Values

representm

eansplusmnSD

(119899=6)ineach

grou

plowast119901lt005lowastlowast119901lt001andlowastlowastlowast119901lt0001

120572com

paredto

control120573com

paredto

Carrmcom

paredto

Carr+Indo

Evidence-Based Complementary and Alternative Medicine 11

Table 8 Effects of Cynara scolymus leaves extract and Indomethacin on CAT SOD GSH AOPP andMDA activities in carrageenan inducedpaw oedema

GroupsCAT (120583molesCATminmg

protein)

SOD (unitSODminmg

protein)

GSH (nmolesmgprotein)

MDA (nmolMDAmg protein) AOPP (nmolmg protein)

Control 47 plusmn 2 131 plusmn 43 421 plusmn 12 234 plusmn 5 484 plusmn 43Carr 162 plusmn 05120572lowastlowastlowast 153 plusmn 26120572lowastlowastlowast 131 plusmn 11120572lowastlowastlowast 314 plusmn 75120572lowastlowastlowast 679 plusmn 69120572lowastlowastlowastCarr + Indo(10mgkgbw) 649 plusmn 71120573lowastlowastlowast 240 plusmn 13120573lowastlowastlowast 703 plusmn 11120573lowastlowastlowast 209 plusmn 30120573lowastlowastlowast 442 plusmn 06120573lowastlowastlowastCarr + ALE(400mgkgbw) 565 plusmn 25120573lowastlowastlowastmlowastlowast 207 plusmn 40120573lowastlowastlowastmlowastlowast 756 plusmn 25120573lowastlowastlowastmlowast 208 plusmn 60120573lowastlowastlowast 502 plusmn 41120573lowastlowastlowastmlowastValues represent means plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001120572 compared to control 120573 compared to Carr m compared to Carr + IndoSOD superoxide dismutaseCAT catalaseGSH glutathione peroxidaseMDA MalondialdehydeAOPP Advanced Oxidation Protein Product

Table 9 Quantification and identification of phenolic compounds contents in the ethanol extract of Cynara scolymus leaves extracts

Number Content (mg100 g of dry extract) Polyphenolic compound1 28 plusmn 019 Hydroxytyrosol2 381 plusmn 036 Verbascoside3 99 plusmn 002 Apigenin-7-glucoside4 18 plusmn 01 Oleuropein5 192 plusmn 014 Quercetin6 5 plusmn 005 Pinoresinol7 4 plusmn 004 ApigeninValues represent means plusmn SD (119899 = 3)

In the second phase the increase of vascular permeabilityis observed by release of kinins during 230 hThereafter from230 h to 5 h inflammation ismediated by prostaglandins andis also associated with a release infiltration and migration ofPN into the inflamed site [47]

Our results indicate that ALE affords protection againstthe Carr induced acute inflammation in dose dependentmanner EtOH extract of ALE at dose of 400mgkgbwexhibits significant anti-inflammatory activity with 73 inhi-bition of paw oedema compared with Indo group (53) andtypically reaches a maximum at 5 h these results obtainedsuggest the anti-inflammatory effect of EtOH extract of ALEbymeans of inhibiting the synthesis and the release of inflam-matory mediators like histamine serotonin and prostagl-andins that are involved in acute inflammationThese resultsrevealed the inhibitory effect of EtOH extract on PN migra-tion and it is confirmed through a histological analysis oftissues of paw oedema in experimental groups

These findings clearly confirm that EtOH extract of ALEhas an anti-inflammatory effect by reducing the influx ofpolymorphonuclear cells to inflammatory tissue followinginjection of Carr Most inflammatory markers such asinterleukin-6 (IL-6) tumor necrosis factor-120572 (TNF-120572) CRPand fibrinogen increased significantly in response to infec-tion and in active diseases states CRP is the specific marker

of acute inflammation occurring in the body [48] moreoverthe fibrinogen is themost relevant indicator of inflammationand it does not only represent the acute-phase reactant that isincreased in inflammatory states Elevated fibrinogen levelswould be associated with higher levels of CRP and wouldsimilarly correlate with inflammation reaction

Therefore the present study showed that EtOH extract ofALE significantly decreases the level of fibrinogen and CRPby value of 2272 and 3478 in comparison with Carrgroup

The induction of inflammationwithCarr ismanifested bygeneration of ROS and has been shown to play an importantrole in various forms of inflammation [49 50] High concen-tration of reactive free radicals contributes to lipid perox-idation and protein oxidation [3] Our results showed thatthere is a significant increase in MDA and AOPP activitiesin Carr group when compared to control group and a highlysignificant decrease in activities of SOD CAT and GSH alsoobserved at the tissue level compared with the control group(119901 lt 0001) However the injection of EtOH extract and Indoshows a significant decrease of the levels of MDA and AOPPby 3427 and 4427 following a significant increase inantioxidant activities as CAT SOD andGSHwhen comparedto Carr group (2016 7330 and 75) From these resultsit is implied that the protective effect of EtOH extract may

12 Evidence-Based Complementary and Alternative Medicine

be attributed to its potential indirectly as a stimulator ofthe activity and expression of antioxidant enzymes duringinflammation

Thus the anti-inflammatory activity is due to the individ-ual or synergistic effect of the components in the ALE In factprevious studies have found that anti-inflammatory profile ofALE could be related to polyphenolic compounds accordingto the phytochemical analysis We performed HPLC analysisto justify the correlation between phytochemical compoundsand anti-inflammatory activity of this plant Among thesepolyphenolic compounds verbascoside has been reportedin several reports and showed a potential spectrum ofmany activities including antioxidant and anti-inflammatoryParticular verbascoside presents antiedematogenic activitiesin animal models of Carr induced inflammation [51] and theresponses exhibited themaximumactivity for the fourth hourof treatment Such activities may be derived from inhibitoryaction of chemical mediators of the inflammatory processsuch as histamine and bradykinin A significant antioxidanteffect of verbascoside has been recently reported byAleo et al[52] in an experimental study [53] Other authors reportedthat anti-inflammatory activity of verbascoside has beenevaluated by an in vitro test performed on cell cultures of pri-mary human keratinocytes [54] in which it was able to reducethe release of proinflammatory markers such as chemokines

Apigenin-7-glucoside was found to block the releaseof several varieties of enzymes involved in inflammationincluding especially lipoxygenases and cyclooxygenases [5556] leading to inhibition of proinflammatory molecules NF-kB activation and inhibits neutrophil infiltration in tissues

From these results we suggest that anti-inflammatoryactivity observed is due to a synergic action of these phenoliccomponents contained in EtOH extract of ALE

C scolymus is one of the few herbal remedies which havebeen verified through experimental studies Further the toxi-city studies of ALE are required in order to confirm the safetyof this medicinal plant in the treatment of inflammationdiseases

5 Conclusion

The present study showed clearly the advantages of Cscolymus leaves extracts which have safer anti-inflammatoryprofile with potent antioxidant activity attributed to thephenolic compounds Furthermore in the future studieswe are interested to characterize the action mechanisms ofactive phenolic compounds of ALE responsible for anti-inflammatory and antioxidants activities

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Tunisian Ministry ofHigher Education and Scientific Research

References

[1] C Pasquier ldquoStress oxidative et inflammationrdquo Blool vol 4 pp1ndash6 1996

[2] M A Abuashwashi O M Palomino and M P Gomez-Serranillos ldquoGeographic origin influences the phenolic com-position and antioxidant potential of wild Crataegus monogynafrom Spainrdquo Pharmaceutical Biology vol 54 no 11 pp 2708ndash2713 2016

[3] M B Salem H Affes K Ksouda et al ldquoPharmacological stud-ies of artichoke leaf extract and their health benefitsrdquo PlantFoods for Human Nutrition vol 70 no 4 pp 441ndash453 2015

[4] M-H Huang B-S Wang C-S Chiu et al ldquoAntioxidant anti-nociceptive and anti-inflammatory activities ofXanthii Fructusextractrdquo Journal of Ethnopharmacology vol 135 no 2 pp 545ndash552 2011

[5] G-J Huang S-S Huang S-S Lin et al ldquoAnalgesic effectsand the mechanisms of anti-inflammation of ergostatrien-3120573-ol from antrodia camphorata submerged whole broth in micerdquoJournal of Agricultural and Food Chemistry vol 58 no 12 pp7445ndash7452 2010

[6] C Tohda N Nakayama F Hatanaka and K Komatsu ldquoCom-parison of anti-inflammatory activities of six Curcuma rhi-zomes a possible curcuminoid-independent pathway mediatedby Curcuma phaeocaulis extractrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 3 no 2 pp 255ndash260 2006

[7] K Goksel K Murat Y Murat E Sevil and H Fidan ldquoAntiox-idant activities of ethanol extracts ofHypericum triquetrifoliumandHypericum scabroidesrdquo Pharmaceutical Biology vol 46 no4 pp 231ndash242 2008

[8] R Gebhardt and M Fausel ldquoAntioxidant and hepatoprotectiveeffects of artichoke extracts and constituents in cultured rathepatocytesrdquoToxicology inVitro vol 11 no 5 pp 669ndash672 1997

[9] A SofworaMedicinal Plants and TraditionalMedicine in AfricaJohn Wiley amp Sons Ann Arbor Mich USA 1982

[10] D E Okwu ldquoPhytochemicals vitamins andmineral contents oftwo Nigerian medicinal plantsrdquo International Journal of Mole-cular Medicine and Advances Sciences vol 1 pp 375ndash381 2005

[11] N Tsevegsuren GDavaakhuu andT S Udval ldquoPhytochemicalanalysis of Cynara scolymus L cultivated in MongoliardquoMongo-lian Journal of Chemistry vol 15 no 41 pp 40ndash42 2014

[12] AACC Approved Methods of the AACC American Associationof Cereal Chemists St Paul Minn USA 8th edition 1984

[13] S C Lee L Prosky and J W DeVries ldquoDetermination of totalsoluble and insoluble dietary fiber in foodsmdashenzymatic-gravi-metric method MES-TRIS buffer collaborative studyrdquo Journalof the Association of Official Analytical Chemists vol 75 pp395ndash416 1992

[14] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[15] O A Fawole U L Opara and K I Theron ldquoChemical andphytochemical properties and antioxidant activities of threepomegranate cultivars grown in South Africardquo Food and Bio-process Technology vol 5 no 7 pp 2934ndash2940 2012

[16] V Dewanto X Wu K K Adom and R H Liu ldquoThermalprocessing enhances the nutritional value of tomatoes byincreasing total antioxidant activityrdquo Journal of Agricultural andFood Chemistry vol 50 no 10 pp 3010ndash3014 2002

[17] B Sun J M Ricardo-da-Silva and I Spranger ldquoCritical factorsof vanillin assay for catechins and proanthocyanidinsrdquo Journal

Evidence-Based Complementary and Alternative Medicine 13

of Agricultural and Food Chemistry vol 46 no 10 pp 4267ndash4274 1998

[18] AOACOfficial Methods of Analysis vol 1 AOACWashingtonDC USA 15th edition 1990

[19] D Pljevljakusic K Savikin T Jankovic et al ldquoChemical prop-erties of the cultivated Sideritis raeseri Boiss amp Heldr subspraeserirdquo Food Chemistry vol 124 no 1 pp 226ndash233 2011

[20] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[21] I I Koleva T A Van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] V Ravi T S M Saleem S S Patel J Raamamurthy and KGauthaman ldquoAnti-inflammatory effect of methanolic extract ofSolanum nigrum Linn Berriesrdquo International Journal of AppliedResearch in Natural Products vol 2 pp 33ndash36 2009

[24] D Chattopadhyay G Arunachalam A B Mandal T K SurS C Mandal and S K Bhattacharya ldquoAntimicrobial and anti-inflammatory activity of folkloreMallotus peltatus leaf extractrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 229ndash2372002

[25] L R Morse K Stolzmann H P Nguyen et al ldquoAssociationbetween mobility mode and c-reactive protein levels in menwith chronic spinal cord injuryrdquo Archives of Physical Medicineand Rehabilitation vol 89 no 4 pp 726ndash731 2008

[26] A Clauss ldquoGerinnungsphysiologische schnellmethode zur bes-timmung des fibrinogensrdquoActaHaematologica vol 17 no 4 pp237ndash246 1957

[27] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[28] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidationin post-mitotic tissues of ageing ratrdquo Cell Biochemistry andFunction vol 24 no 1 pp 79ndash85 2006

[29] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[30] W F Beyer Jr and I Fridovich ldquoAssaying for superoxide dis-mutase activity some large consequences of minor changes inconditionsrdquoAnalytical Biochemistry vol 161 no 2 pp 559ndash5661987

[31] I Carlberg and B Mannervik ldquoGlutathione reductaserdquo inGlutamate Glutamine Glutathione and Related Compoundsvol 113 ofMethods in Enzymology pp 484ndash490 Elsevier 1985

[32] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[33] V Emanue V Adrian N A Sultana and C Svetlana ldquoAntiox-idant and antimicrobial activities of ethanol extracts of Cynarascolymus (Cynarae folium Asteraceae family)rdquo Tropical Journalof Pharmaceutical Research vol 10 no 6 pp 777ndash783 2011

[34] G L D S Oliveira R A M O Francisco O B A Vinıcius etal ldquoEvaluation of antioxidant capacity of the aqueous extract ofCynara scolymus L (Asteraceae) in vitro and in SaccharomycescerevisiaerdquoAfrican Journal of Pharmacy and Pharmacology vol8 pp 136ndash147 2014

[35] K Horiuchi S Shiota T Hatano T Yoshida T Kuroda and TTsuchiya ldquoAntimicrobial activity of oleanolic acid from Salviaofficinalis and related compounds on vancomycin-resistantenterococci (VRE)rdquo Biological and Pharmaceutical Bulletin vol30 no 6 pp 1147ndash1149 2007

[36] A Daoud D Malika S Bakari et al ldquoAssessment of polyphe-nol composition antioxidant and antimicrobial properties ofvarious extracts of Date Palm Pollen (DPP) from two Tunisiancultivarsrdquo Arabian Journal of Chemistry 2015

[37] D Krishnaiah B Awang S Rosalam and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversitymdashEngineering Sciences vol 1 pp 63ndash66 2015

[38] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[39] A Betancor-Fernandez A Perez-Galvez H Sies and W StahlldquoScreening pharmaceutical preparations containing extracts ofturmeric rhizome artichoke leaf devilrsquos claw root and garlic orsalmon oil for antioxidant capacityrdquo Journal of Pharmacy andPharmacology vol 55 no 7 pp 981ndash986 2003

[40] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[41] S Konyal120580oglu H Saglam and B K120580vcak ldquo120572-Tocopherolflavonoid and phenol contents and antioxidant activity of Ficuscarica leavesrdquo Pharmaceutical Biology vol 43 no 8 pp 683ndash686 2005

[42] D U Bawankule P Trivedi A Pal et al ldquoProtectivemechanismof lignans from Phyllanthus amarus against galactosaminelipopolysaccharide-induced hepatitis an in-vivo and in-silicostudiesrdquo Current Topics in Medicinal Chemistry vol 14 no 8pp 1045ndash1055 2014

[43] GAHiggs R J Flower and J RVane ldquoAnew approach to anti-inflammatory drugsrdquo Biochemical Pharmacology vol 28 no 12pp 1959ndash1961 1979

[44] A A Hemmati H Kalantari A Siahpoosh B Ghorbanzadehand H Jamali ldquoAnti-inflammatory Effect of HydroalcoholicExtract of the Washingtonia filifera Seeds in Carrageenan-Induced Paw Edema in Ratsrdquo Jundishapur Journal of NaturalPharmaceutical Products vol 10 no 1 pp 19ndash25 2015

[45] I Posadas M Bucci F Roviezzo et al ldquoCarrageenan-inducedmouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expressionrdquoBritish Journal of Pharmacology vol 142 no 2 pp 331ndash3382004

[46] K Pundarikakshudu D H Shah A H Panchal and G CBhavsar ldquoAnti-inflammatory activity of fenugreek (Trigonellafoenum-graecum Linn) seed petroleum ether extractrdquo IndianJournal of Pharmacology vol 48 no 4 pp 441ndash444 2016

[47] S Cuzzocrea G Costantino E Mazzon B Zingarelli A DeSarro and A P Caputi ldquoProtective effects of Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) a superoxide dismutasemimetic in paw oedema induced by carrageenan in the ratrdquoBiochemical Pharmacology vol 58 no 1 pp 171ndash176 1999

[48] M A Mendall P Patel M Asante et al ldquoRelation of serumcytokine concentrations to cardiovascular risk factors andcoronary heart diseaserdquo Heart vol 78 no 3 pp 273ndash277 1997

[49] A Ialenti A Ianaro S Moncada andM Di Rosa ldquoModulationof acute inflammation by endogenous nitric oxiderdquo EuropeanJournal of Pharmacology vol 211 no 2 pp 177ndash182 1992

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

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Evidence-Based Complementary and Alternative Medicine

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Evidence-Based Complementary and Alternative Medicine 3

light during 30min After incubation the estimation of thescavenging ability was performed by measuring at 517 nm inspectrophotometer (T70 UV-Vis)

The capacity of inhibition percentage (PI) of DPPHradicals was calculated as

DPPH radicals (PI) = [(119860119887 minus 119860 119904)119860119887 ] times 100 (1)

where 119860119887 refers to the absorbance of control (without plantextract) and 119860 119904 to the absorbance of sample (with plantextract) BHT and VC were used as standards at the sameconcentrations of plant extract

262 120573-Carotene Bleaching Test The capacity of ALE toreduce bleaching of beta-carotene was previously determinedbyKoleva et al [21]Themixture of beta-carotene and linoleicacid was prepared 05mg of 120573-carotene 25 uL of linoleicacid and 200 uL of Tween 40 were dissolved in 1mL ofchloroform solvent The chloroform was evaporated in arotator evaporator at 40∘C and 100mL of dH2O was addedthen the mixture was stirred

Aliquots of 25mL of beta-carotenelinoleic acid emul-sion obtained were transferred to test tubes containingdifferent ALE concentrations then the emulsion of reactionwas incubated for 2 h at 50∘C and the absorbance of eachsample was measured at 470 nm by spectrophotometer BHTandAAwere used as the standards at the same concentrationsof the samples

263 Antioxidant Activity by ABTS∙+ Method The abilityto neutralize the ABTS∙+ was reported by Re et al [22]using a spectrophotometric 96-well microplate methodThepreparation of ABTS∙+ free radical solution by incubating amixture of ABTS (7mM) and K2S2O8 (245mM) dissolvedin distillated water to create a stable color of radical solutionfollowing 12ndash16 h of incubation in the dark room at 4∘C

Therefore the standard ABTS∙+ solution was prepared bydilution with ethanol to a standard absorbance of 07 plusmn 002at 734 nm

50 120583L of plant extract (1ndash20120583gmL) was added to 150120583Lof ABTS∙+The plates were incubated at room temperature inthe dark room during 15min the absorbance was measuredat 630 nm Control wells contained 50 120583L of H2O and 150 120583Lof ABTS∙+ Phytochemical interference was accounted bywells containing extract (50 120583L) with dH2O (150 120583L) whereas200120583L of H2O served as blank Trolox dissolved in pureMeOH was used as standards

The antioxidant capacity of ALE was expressed quantita-tively as mmol of Trolox Equivalents (TE) (mmol TEg dryextract)

264 Ferric Reducing Assay The potential ability of ALE toreduce the ferric iron was evaluated previously by Fawole etal [15]

The mixture of FRAP was freshly prepared by 50mL of300mM acetate buffer 5mL of 10mM TPTZ and 5mL of20mM FeCl3 Prior to its use the prepared mixture must beincubated in water bath at 37∘C during 15min to stabilize thecontents in the reaction mixture

Exactly 150mL of diluted ALE was transferred intodifferent test tubes and then 250mL of FRAP solution wasadded in triplicate The mixture was stirred and incubated ina dark room for 30min before measuring the absorbance at515 nm inUV-Vis spectrophotometer Antioxidant capacity ofALE was expressed as micromoles of Trolox Equivalent permilliliter of sample (mMTEmL)

27 Anti-Inflammatory Activity In Vivo

271 Chemicals and Reagents Lambda carrageenan andIndomethacin were purchased from Sigma Aldrich company(France)

272 Experimental Study 30 sexually mature male rats10ndash12 weeks old weighing 150ndash200 g were obtained fromthe Institute of Pasteur Tunisia Rats were fed with stan-dard laboratory pellets and ad libitum access during theexperiment study The experimental protocol was conductedin accordance with the Guide for the Care and Use ofLaboratory Animals issued by the University of Sfax Tunisiaand approved by the Ethics Committee of Animal useprotocol number 94-1939 Before the experiment rats wereacclimatized in controlled environmental conditions at 24 plusmn4∘Cwith relative humidity (45ndash55) and 12 h dark-light cycleAll the groups of animals were randomly divided into thecontrol group and treatment groups

273 Carrageenan-Induced Paw Oedema Model The eval-uation of anti-inflammatory activity was reported by Raviet al [23] 01mL of Carr solution (1) was injected intosubplantar surface of the paw of each group of rats to produceacute inflammation This method was chosen over othermethods for the discovery of new therapeutic effects of ALEin inflammation diseases as it is the most basic methodrequiring minimal equipment but much practice

The experimental study designed 4 groups of six ratsGroup (I) was the control group which received isotonicsaline solution Nacl (0 9) by subplantar injection andhad no inflammation and received no treatment Group(II) was inflamed by carrageenan injection and did notundergo any treatment (Carr) Group (III) was used asreference inflamed rats that were treated with Indomethacin(10mgkgbw) (Carr + Indo) by intraperitoneal injection(ip) and Group (IV) was treated with EtOH extract of ALEat dose 400mgkgbw (Carr+ ALE) by ip [24] The doses ofALE and Indo chosen during treatments were proportionalto the size of the oedema and covered the whole swelling

In all treated groups the oedema paw volumes up totibiotarsal articulation were measured using a digital caliperat 1 2 3 4 and 5 hours after Carr injection

For each treated group the size of oedema obtained atthese various times (PT) was compared to that obtainedbefore any treatment (P0)

Percentages of oedema inhibition were calculated as

Inhibition (EI) = [1 minus (PTP0 )] times 100 (2)

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Phytochemical analysis of Cynara scolymus extracts leaves

Extract Cardiac glycosides Triterpenoids Saponin Flavonoids Tannins AlkaloidALE + + + + + +Sign (+) indicates being present

Percentages of inflammation inhibition were calcu-lated as

Inhibition (II) = [PT minus (P0P0)] times 100 (3)

274 Blood Sample Collection 5 hours after Carr inductionthe rats were decapitated and the blood samples were col-lected in heparin tubes Plasma samples were obtained aftercentrifugation at 3000 rpm for 15min and they were kept inminus20∘Cuntil analysis on an automatic biochemistry analyzer atthe biochemical laboratory of Hedi Chaker Hospital of Sfax

275 Inflammatory Biomarkers

(1) Determination of C-Reactive Protein (CRP) CRP wasa specific marker following the inflammatory process Itincreases in proportion to its intensity [25] The reactiveprotein was measured by turbid metric method using anautomatic analyzer COBAS INTEGRA 40010158401015840 C-reactive TheCRP is expressed with mgL

(2) The Fibrinogen Assay of Plasma Plasma fibrinogen con-centration was determined by Clauss clotting method [26]measured on a STAanalyzer Principle test measures theconversion rate of fibrinogen into fibrin in diluted samplein presence of excess of thrombin and records the clottingtime The clotting time is inversely proportional to the levelof fibrinogen in the plasma The fibrinogen level is expressedwith gL plasma

(3) Exploring the Antioxidant Enzymatic and NonenzymaticStatus Oxidative stress parameters were determined in tis-sues paw oedema homogenates The supernatants obtainedwere removed and analyzed for the determination of MDAas described by Draper and Hadley [27] AOPP levels werequantified by method of Kayali et al [28] CAT activity wasmeasured as reported by Aebi [29] and expressed as mmolesof H2O2 consumed(minmg protein) SOD was assayedspectrophotometrically by colorimetric method of Beyer Jrand Fridovich [30] and expressed as Umg protein GSHactivity was assayed according to the method of Carlbergand Mannervik [31] and expressed as mmoles GSS(min-mg protein) and the protein content was determined usingmethod of Lowry et al [32]

(4) Histopathological Examination All the paws oedematissues of experimental groups were collected for histolog-ical examination First they were fixed in 10 bufferedformalin solution second they were embedded in paraffinwax and then cut into 5mm thick sections and stainedwith hematoxylin and eosin (HampE) Finally the slides werephotographed with an Olympus U-TU1X-2 camera

28 High Performance Liquid Chromatography (HPLC)Detec-tion and Quantification of Polyphenolic Compounds Thephenolic fractions analysis of EtOH extract of ALE wasdetermined using an Agilent Technologies 1100 series HPLCcoupled with a UV-Vis multiwavelength detector The sepa-ration was carried out on a 250 times 46mm C18 silica columnchromatography was chosen at ambient temperature Themobile phase consisted of C2H3N (solvent A) and waterwith 02 of H2SO4 (solvent B) and the flow rate waskept at 08mLminminus1 For the preparation of calibrationcurve a standard stock solution was prepared in methanol(HPLC grade ge 999 from Sigma Chemical Company)containing Hydroxytyrosol Tyrosol 4-hydroxybenzoic acidverbascoside apigenin-7-glucoside Oleuropein QuercetinPinoresinol cinnamic acid and apigenin at the same concen-tration of 1 gmL For the preparation of the ethanol extract bydissolving in 1mL of methanol making a final concentrationof 25mgmL Before starting HPLC analysis all the solutionspreparedwere filteredwithWhatman paper (Oslash 045 120583m)Thediluted extract was injected directly and chromatogramsweremonitored at 280 nm 20120583L was used for injection Peakswere identified by the retention times compared with thestandardsThe analyses of phenolic profiles of ethanol extractwere performed in triplicate

29 Statistical Analysis SPSS program was employed forcomparisons between all groups Comparison between mul-tiple and within groups was analyzed by ANOVA followedby Tukeyrsquos tests Values were shown as mean plusmn SD The levelof statistical significance was set at 119901 value lt 005 Pearsonrsquoscorrelation coefficient analysis was calculated using SPSSprogram also in order to evaluate the statistical relationshipbetween both of polyphenolics compounds and antioxidantactivities of ALE

3 Results

31 Phytochemical Analysis of Cynara scolymus LeavesExtracts ALE revealed the presence of flavonoid cardiac gly-cosides saponin tannin terpenoid and alkaloids (Table 1)

32 Proximate Analysis of Dried Leaves of C scolymus Theresults showed that Cynara leaves contain high constituentsdry matter (9703 DW) ash (1581 DW) carbohydrate(8005DW) protein (1664DW) lipids (341DW) totalsugars (197 DW) and dietary fiber (7160 DW) (Table 2)

33 Quantification of Total Phenolic Flavonoid and TanninsContents of Cynara scolymus Leaves Extracts TheTPC TFCand TCCwere evaluated in different solvents extracts of ALEAll results are shown in Table 3 Maximum content of TPCwas obtained using EtOHextract and corresponded to 5454plusmn126mg GAg DW followed by ethyl acetate aqueous and

Evidence-Based Complementary and Alternative Medicine 5

Table 2 Proximate analysis of dried leaves of Cynara scolymus

Constituents Cynara scolymus leaves(percentage dry weight basis)

Dry matter 9703 plusmn 043Ash 1581 plusmn 001Carbohydrate 8005 plusmn 069Protein 1664 plusmn 179Lipids 341 plusmn 045Total sugars 197 plusmn 010Dietary fiber 7160 plusmn 081Values are expressed as mean plusmn SD (119899 = 3)

butanol extracts while TPC of hexane extract was the lowestin comparison with EtOH extract (3091 plusmn 936mg GAEgDW) The EtOH extract showed also the highest content inTFC (12plusmn083CEgDW) but this value was lower for hexaneextract (819 plusmn 06mg CEg DW) respectively

The amount of TCCwhichwas evaluated by vanillin assaywas the highest in the ethyl acetate and EtOH extract (1451and 1405mg CEg DW) but this content was lower thanaqueous extract as shown in Table 3

34 Mineral Contents Analysis The main composition ofmacroelements and microelements in C scolymus leaves ispresented in Table 4

Elemental analysis in (mg100 g of dry weight basis)indicated that leaves of artichoke contained the followingorder of essential minerals compounds potassium (288680)sodium (176294) calcium (135934) magnesium (43321)iron (1617) manganese (1305) zinc (737) copper (130) andchromium (012)

35 Evaluation of Antioxidant Activity Theantioxidant activ-ity of ALE harvested in Tunisia was performed using fourmethods DPPH ABTS FRAP and beta-carotene test

351 Antioxidant Activity by DPPHMethod DDPHmethodevaluated the capacity of compounds present in ALE toreduce DDPH radical The result in Figure 1 showed thescavenging activity of all extracts of C scolymus was con-centration dependent Among all extracts the EtOH extractdisplayed the highest free radical scavenging activity at higherconcentration (400 ugmL) (9423) compared to the sameconcentration of vitamin C (983) and BHT (9623) fol-lowedby ethyl acetate extract (8151) the scavenging activityof other extracts was significantly lower when compared toreference standard VC (9983)

352120573-Carotene Bleaching Test Thepotential effect of EtOHextract exhibited significantly the highest inhibition rate of120573-carotene bleaching (7074) compared to BHT and AA(4794 9059) (119901 lt 0001) respectively (Table 5) whereashexane and butanol extract showed the lowest rate of 120573-carotene bleaching 3738 and 4939

353 Antioxidant Activity by ABTS∙+ Method As evidentfrom Table 5 ALE studied presents a potential capacity

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

0102030405060708090

100

in

hibi

tion

100ugmL200ugmL

300ugmL400ugmL

Etha

nol

Hex

ane

Ethy

l ace

tate

Buta

nol

Aque

ous

VC BHT

Figure 1 Antioxidant activity by DPPHmethod ofCynara scolymusleaves extracts at different concentrations Values are mean plusmn SD(119899 = 3) lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 compared toEtOH extract Butylated Hydroxytoluene (BHT) Vitamin C (VC)

0 1 2 3 4 5 6Temps (h)

0

05

1

15

2

25

3

35

4Si

ze o

f oed

ema (

mm

)

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

CARRNACL

CARR + IndoCARR + ALE

Figure 2 Effect of ALE and Indomethacin on paw oedema inducedby carrageenan Values represent mean plusmn SD (119899 = 6) in each grouplowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 120572 compared to control 120573compared to Carr m compared to Carr + Indo

to scavenge the ABTS radical cation Among all extractsEtOH extract was a significantly higher value (49943mmolTroloxg dry extract) than the other extracts but the low-est level of TEAC was obtained from the hexane extract(1043mmol Troloxg dry extract)

354 Ferric Reducing Assay The results in Table 5 showedthat among all extracts of ALE a higher capacity of reducingferric capacity was found for EtOH extract (52779 umolFe2+mg dry extract) when compared to other extractsrespectively

36 Anti-Inflammatory Activities The appearance of Carrinduced paw oedema with respective treatments groups wasillustrated in Table 7 and Figure 2 The results showed

6 Evidence-Based Complementary and Alternative Medicine

Table 3 Quantification of total phenolic flavonoids and tannins contents of Cynara scolymus leaves extracts

Extracts Phenolics content(mg GAEg DW)

Flavonoidscontent

(mg CEg DW)

Tannins content(mg CEg DW)

Hexane 3991 plusmn 936 819 plusmn 016 1405 plusmn 03Ethyl acetate 5307 plusmn 047 1032 plusmn 012 1451 plusmn 013Butanol 4166 plusmn 223 1121 plusmn 010 1393 plusmn 93Ethanol 5454 plusmn 126 1200 plusmn 083 1099Aqueous 4949 plusmn 039 949 plusmn 039 438 plusmn 045Values are expressed as mean plusmn SD (119899 = 3)

Table 4 Mineral contents dried leaves of Cynara scolymus

Elements Cynara scolymus leaves (mg100 g of dry weightbasis)

K 2886803 plusmn 120Ca 1359346 plusmn 505Na 1762946 plusmn 120Mg 433219 plusmn 234I 16176 plusmn 014Mn 13051 plusmn 011Zn 7371 plusmn 014Copper 130 plusmn 016Cr 0124 plusmn 001Values are expressed as mean plusmn SD (119899 = 3)

that the induction of Carr in rats paw oedema started offby the vascular phase of inflammation which generated anincrease in the size of oedema for all groups This injectiongenerated intense inflammation which peaked after 3 hoursThe experimental data showed that ALE presented significantdecrease in the size of pawoedemawhichwas time dependentand more important than the reference group Indo and Carrgroup

As seen in Figures 3 and 4 EtOH extract at dose(400mgkgbw) has shown a significant percent of oedemainhibition (119901 lt 0001) in the first hour (173) comparedwith reference group (Indo) and the significant percent ofinhibition inflammation was 4427 in comparison with thecontrol group

Thus after five hours these percentages were 735 forinhibition and 832 for inflammation For the Indo groupoedema inhibition was significant (119901 lt 0001) 53 and 64and oedema inflammation was 21 and 86 in comparisonwith the control group

These results showed that EtOH extract of ALE had astrong effect as Indo

361 Inflammatory Biomarkers Fibrinogen and CRP Weassayed serum proteins of inflammatory markers in differentgroups of rats

(1) FibrinogenCarr group caused a significant increase in rateof fibrinogen compared to control group by 1905 (Figure 5)

1 2 3 4 5Temps (h)

0

10

20

30

40

50

60

70

80

in

hibi

tion

oede

ma

poundlowastlowastlowast

poundlowastlowastlowast

poundlowastlowastlowast

poundlowastlowastlowast

Carr + ALECarr + Indo

Figure 3 Percentage () of oedema inhibition data in all groupsValues represent mean plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt001 and lowastlowastlowast119901 lt 0001 m compared to Carr + Indo

0

10

20

30

40

50

60

70

in

hibi

tion

infla

mm

atio

n

1 2 3 4 5Temps (h)

NaclCarr

Carr + ALECarr + Indo

poundlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast poundlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

poundlowastlowastlowastlowastlowastlowastlowastlowastlowast

poundlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

Figure 4 Percentage () of oedema inflammation data in allgroups Values represent mean plusmn SD (119899 = 6) in each group lowast119901 lt005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 120572 compared to control group120573 compared to Carr group and m compared to Indo group

The rate of fibrinogen decreased significantly (119901 lt 001) ingroups of rats treated with the EtOH extract (1655) andIndo (2290) compared with Carr group

Evidence-Based Complementary and Alternative Medicine 7

Table 5 Antioxidant activities of Cynara scolymus leaves extracts

Sample FRAP assay (umol Fe (II)gDW)

120573-Carotene bleaching assay()

TEAC assay (mmolTroloxg DW)

Hexane 223023 plusmn 1116lowastlowastlowast 3738 plusmn 524lowastlowastlowast 1043 plusmn 1073lowastlowastlowastEthyl acetate 50829 plusmn 524lowastlowastlowast 6156 plusmn 817lowast 38260 plusmn 524lowastlowastButanol 44306 plusmn 2298lowastlowastlowast 49393 plusmn 224lowastlowast 25193 plusmn 2815lowastlowastlowastEthanol 52779 plusmn 1626 70743 plusmn 129 49943 plusmn 3972Aqueous 31591 plusmn 836lowastlowastlowast 5611 plusmn 543lowastlowast 21074 plusmn 836lowastlowastlowastBHT mdash 4794 plusmn 075lowastlowastlowast mdashAA mdash 9059 plusmn 325lowastlowastlowast mdashValues are means plusmn SD (119899 = 3) Butylated Hydroxytoluene (BHT) and Ascorbic Acid (AA) were used as positive control compared with EtOH extract lowast119901 lt005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001

Nacl Carr Carr + ALE Carr + Indo0

05

1

15

2

25

Fg (gL)CRP (mgmL)

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

Figure 5 Levels of fibrinogen (Fg) and C-reactive protein (CRP)Values represent mean plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt001 and lowastlowastlowast119901 lt 0001 120572 compared to control 120573 compared to Carrgroup Fibrinogen (Fg) C-reactive protein (CRP)

(2) CRP Analysis showed a significant increase (119901 lt 005)in CRP level (Figure 5) for Carr group compared to controlgroup (1673) However the injection of EtOH extract andIndo led to a significant decrease (119901 lt 0001) (60863423) respectively when compared with Carr group

37 Oxidative Stress Parameters MDA and AOPP levels inoedema paw were illustrated in Table 8 Results showed thatCarr induced a significant increase inMDA andAOPP levelshowever treatment with EtOH extract of ALE at dose of400mgkgbw showed a significant decrease (119901 lt 0001)(2080 5023) compared with Carr group and even Indogroup (10mgkg) (2097 4427) respectively

38 Exploring the Antioxidant Enzymatic and NonenzymaticStatus CAT SOD and GSH levels in the paw oedema tissueof tested groups are shown in Table 8 Treatment of inflamedrats with EtOH extract restored significantly (119901 lt 0001)the SOD activity by 2075 the CAT activity by 5655 andthe GSH activity by 7566 respectively compared to Carrgroup

39 Histopathological Examination A microscopic study ofpaw oedema tissues showed histological changes in Carrgroup EtOH extract of ALE (400mgkgpc) group andIndo (10mgkgbw) group (Figure 6) In the Carr group weshowed a subcutaneous oedema with infiltration of inflam-matory cells especially polynuclear neutrophils at the site ofinflamed tissues and also the presence of spongy-like appear-ance in the epidermis ((Figure 6(b)) as compared to controlgroup (Figure 6(a))) The EtOH extract group produced asignificant decrease in the number of cellular infiltrates and asignificant reduced spongy-like appearance in the epidermis(Figure 6(c)) as did reference drug Indo (Figure 6(d))

310 Quantification of Polyphenolic Compounds by HPLCTo the authorsrsquo knowledge the present study identified andquantified the phenolic compounds of ALE from Tunisianorigins Accordingly the results obtained so far by Folin-Ciocalteu needed to be further complemented to qualifyphenolic constituents in EtOH extract under investigation

The findings revealed the presence of seven phenoliccompounds namely Hydroxytyrosol verbascoside apige-nin-7-glucoside Oleuropein Quercetin Pinoresinol andapigenin HPLC analysis indicated that ALE have significantamount of verbascoside content 381mg100 g and Quercetincontent (19 2mg100 g) (Figures 7 and 8 and Table 9)

311 Correlation between the Polyphenolic Compounds andAntioxidant Capacity In order to determine the contributionof the phenolic and flavonoid content in ALE on antiox-idant capacity the Pearson correlation coefficient (119903) wasdetermined in Table 6 The results showed a significantlinear correlation between antioxidant activities determinedby using DPPH ABTS FRAP and beta-carotene methodsand TPC and TFC respectively The strongest correlativevalue is obtained with DDPH and TPC (119903 = 087) Theseresults indicated a good correlation between TPC and TFCwith antioxidant activities of ALE

4 Discussion

The importance of plants in traditional medicine remediesand the potential of phytochemical constituents were dis-cussed nowadays with respect to their benefit in pharma-cotherapy in Tunisia Cynara scolymus was one of these

8 Evidence-Based Complementary and Alternative Medicine

(a)

(b)

(c)

(d)

Figure 6 Histopathological slides tissues of paw oedema in experimental groups of rats (a) Control group untreated group (b) Carr groupCarr-treated rat showed heavy infiltration of polynuclear neutrophils (PN) and a spongy-like appearance and bulla in the epidermis (c)Carr + EtOH extract group Carr-treated rat that received the EtOH extract (400mgkgbw) reduced significantly the migration of PNand oedematasis in dermis without any spongy-like feature and bulla (d) Carr + Indo group Carr-treated rat that received Indomethacin(10mgkgbw) showed a partial protective action Deparaffinized hematoxylin and eosin (HampE) stained sections (200ndash400x) Plus signinfiltration of PN Arrow odematasis in the epidermis

medicinal plants which have a beneficial potential effectattributed to its source of polyphenolic compounds [3]

The selection of this plant was guided by the indicationsof its traditional use that at present there have been very littlechemical and biological investigations done Therefore thepresent study was to investigate phytochemical compositionand their antioxidant capacity and to evaluate in vivo the anti-inflammatory effects of Cynara scolymus leaves extracts

The presence of phenolic compounds is very widelydistributed in medicinal plants several studies showed thatthese compounds have drawn much attention to their poten-tial antioxidant abilities which demonstrated their beneficialimplications for human health

Regarding the determination of phenolic compoundsthe EtOH extract of ALE had the highest amount of thesecompounds in comparison with other extracts which werean agreement with the result of Emanue et al [33] whoshows that EtOH extract exhibited the maximum amountof phenolic compounds from the leaves of Cynara scolymuswhereas it differs from the reports of Oliveira et al [34]who proves that aqueous extract of ALE is the most suitablesolvent for extraction of phenolic compounds

The importance of TPC and TFC extraction yieldsobtained with EtOH extract can be attributed to its goodsolubility low toxicity medium polarity and high extractioncapacity [35]

Evidence-Based Complementary and Alternative Medicine 9

1 2 3 678 9 10

5

4

0250500750

1000125015001750

(mAU

)

5 10 15 20 250(min)

Figure 7 HPLC chromatogram of a standard mixture of polyphe-nolic compounds Peaks 1 Hydroxytyrosol 2 Tyrosol 3 4-hydroxybenzoic acid 4 verbascoside 5 apigenin-7-glucoside 6Oleuropein 7 Quercetin 8 Pinoresinol 9 cinnamic acid 10apigenin

34

2

56 7

0200400600800

100012001400

(mAU

)

5 10 15 20 25 30 350(min)

1

Figure 8 HPLC chromatogram of ethanol extract of Cynarascolymus leaves extracts Peaks 1 Hydroxytyrosol 2 verbascoside3 apigenin-7-glucoside 4 Oleuropein 5 Quercetin 6 Pinoresinol7 apigenin

Table 6 Pearson correlation coefficient (119903) between the content ofphenolic compounds and antioxidant capacity (DDPH∙ ABTS∙+FRAP and beta-carotene)

Correlation 119877TPC versus DDPH∙ 0870lowastlowast

TPC versus ABTS∙+ 0848lowastlowast

TPC versus FRAP 0707lowastlowast

TPC versus beta-carotene 0842lowastlowast

TFC versus DDPH∙ 0728lowastlowast

TFC versus ABTS∙+ 0743lowastlowast

TFC versus FRAP 0849lowastlowast

TFC versus beta-carotene 0712lowastlowast

DPPH 22-diphenyl-1-picrylhydrazyl ABTS 220-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt FRAP ferricreducing TPC total phenolic content TFC total flavonoid contentlowastlowast119901 lt 001 significant correlation

In the first investigation of my study five solvents ofincreasing polarities were chosen for the determination ofphenolic compounds from leaves of C scolymus namelyhexane ethyl acetate butanol 75 EtOHH2O and aqueousin order to determine the variability of TPC and TFC in theaerial part of Cynara as a function of each extraction solvent

This variability of phenolic compounds in ALE can beattributed to the wide range of solubility displayed by variouspolar compounds within the ALE solvents the degree ofpolymerization of phenols and their interaction geneticfactors geographical variations and climatic changes [36]

Overall these findings indicate that EtOH extract of ALEwas rich in phenolic and flavonoid contents which couldbe the major contributor to their antioxidative propertiesMany researches revealed that flavonoids and polyphenolsdisplayed the highest ability of scavenging activity in medic-inal plants [37 38]

The results indicate that EtOH extract of ALE whichcontains the highest content of TFC and TPC displays thehighest free radical scavenging activity (9423) at concen-tration of 400 ugmL However Oliveira et al [34] show thataqueous extract of ALE has good radical scavenging (8340)at concentration of 200 ugmL Furthermore our resultssuggest that some components within EtOH extract aresignificantly the strongest radical scavenger in comparisonwith other extracts The TEAC values of ALE showed alsothat EtOH extract presents a potential ability to scavengethe ABTS radical cation in accordance with the report ofBetancor-Fernandez et al [39]

The FRAP assay shows also that EtOH extract has thehighest values (54262 umol Fe II gDW) The same resultis confirmed by Kukic et al [40] Therefore the results ofpositive correlation between the TPC TFC and the anti-oxidant methods in vitro suggest that phenolic compoundsact as reducing agents hydrogen donors and singlet oxygenscavengers [3] and may exert an important in vitro antioxi-dant capacity of ALE

The phytochemical screening of ALE has been foundto be a rich source of polyphenolic compounds includingQuercetin apigenin-7-glucoside and verbascoside Thesecompounds have shown a great potential of antioxidantactivity [41]

The research into medicinal plants used as pain relieversrsquoagents should therefore be viewed as new therapy in theinflammatory diseases [42] The anti-inflammatory activitiesof EtOH extract of ALE are investigated applying in vivoexperimental Carr model compared with a nonsteroidal anti-inflammatory (Indomethacin) this drug was reported byHiggs et al [43] who demonstrated the role of Indo toinhibit the biosynthesis of prostaglandins in Carr model ratsThe experimental study of anti-inflammatory activity is per-formed by the Carr test This phlogiston agent inducedtissue oedema characteristic of acute inflammation whichis regarded as a crucial parameter in assessing anti-inflam-matory activity [44]

The Carr injection produces biphasic states in the firstphase [45] there is an increase in the mRNA synthesis ofcyclooxygenase-2 (COX-2) at the first hour This increase isaccompanied by an amplification of synthesis of strong proin-flammatory mediator such as prostaglandin especially theprostaglandin type 2 involved in the inflammatory process[46] serotonin bradykinin and leukotrienes which con-tribute to the initiation of inflammation reaction Moreoverthe participation of arachidonicmetabolites is themain factorresponsible for both of phases of Carr induced inflammation

10 Evidence-Based Complementary and Alternative Medicine

Table7Eff

ecto

fCynarascolym

usleaves

extracto

ncarrageenan-indu

cedratp

awoedema

Treatm

ent

Oedem

asize(meanplusmnS

D)(mm)

0h1h

2h3h

4h5h

Con

trol

0426plusmn0

00

044

1plusmn001

056plusmn0

03

0650plusmn0

05

0755plusmn0

05

070plusmn0

01

Carr

0426plusmn0

00

28plusmn026120572lowastlowastlowast

333plusmn028120572lowastlowastlowast

325plusmn002120572lowastlowastlowast

315plusmn001120572lowastlowastlowast

302plusmn009120572lowastlowastlowast

Carr+Indo

(10m

gkg)

0426plusmn0

00

246plusmn026120572lowastlowastlowast120573lowast

261plusmn007120572lowastlowastlowast120573lowastlowastlowast

230plusmn002120572lowastlowastlowast120573lowastlowastlowast

18plusmn011120572lowastlowastlowast120573lowastlowastlowast

14plusmn006120572lowastlowastlowast120573lowastlowastlowast

Carr+ALE

(400

mgkgbw)

0426plusmn0

00

2480plusmn005120572lowastlowastlowast120573lowastlowastlowast

188plusmn004120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

165plusmn005120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

116plusmn020120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

112plusmn01120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

Values

representm

eansplusmnSD

(119899=6)ineach

grou

plowast119901lt005lowastlowast119901lt001andlowastlowastlowast119901lt0001

120572com

paredto

control120573com

paredto

Carrmcom

paredto

Carr+Indo

Evidence-Based Complementary and Alternative Medicine 11

Table 8 Effects of Cynara scolymus leaves extract and Indomethacin on CAT SOD GSH AOPP andMDA activities in carrageenan inducedpaw oedema

GroupsCAT (120583molesCATminmg

protein)

SOD (unitSODminmg

protein)

GSH (nmolesmgprotein)

MDA (nmolMDAmg protein) AOPP (nmolmg protein)

Control 47 plusmn 2 131 plusmn 43 421 plusmn 12 234 plusmn 5 484 plusmn 43Carr 162 plusmn 05120572lowastlowastlowast 153 plusmn 26120572lowastlowastlowast 131 plusmn 11120572lowastlowastlowast 314 plusmn 75120572lowastlowastlowast 679 plusmn 69120572lowastlowastlowastCarr + Indo(10mgkgbw) 649 plusmn 71120573lowastlowastlowast 240 plusmn 13120573lowastlowastlowast 703 plusmn 11120573lowastlowastlowast 209 plusmn 30120573lowastlowastlowast 442 plusmn 06120573lowastlowastlowastCarr + ALE(400mgkgbw) 565 plusmn 25120573lowastlowastlowastmlowastlowast 207 plusmn 40120573lowastlowastlowastmlowastlowast 756 plusmn 25120573lowastlowastlowastmlowast 208 plusmn 60120573lowastlowastlowast 502 plusmn 41120573lowastlowastlowastmlowastValues represent means plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001120572 compared to control 120573 compared to Carr m compared to Carr + IndoSOD superoxide dismutaseCAT catalaseGSH glutathione peroxidaseMDA MalondialdehydeAOPP Advanced Oxidation Protein Product

Table 9 Quantification and identification of phenolic compounds contents in the ethanol extract of Cynara scolymus leaves extracts

Number Content (mg100 g of dry extract) Polyphenolic compound1 28 plusmn 019 Hydroxytyrosol2 381 plusmn 036 Verbascoside3 99 plusmn 002 Apigenin-7-glucoside4 18 plusmn 01 Oleuropein5 192 plusmn 014 Quercetin6 5 plusmn 005 Pinoresinol7 4 plusmn 004 ApigeninValues represent means plusmn SD (119899 = 3)

In the second phase the increase of vascular permeabilityis observed by release of kinins during 230 hThereafter from230 h to 5 h inflammation ismediated by prostaglandins andis also associated with a release infiltration and migration ofPN into the inflamed site [47]

Our results indicate that ALE affords protection againstthe Carr induced acute inflammation in dose dependentmanner EtOH extract of ALE at dose of 400mgkgbwexhibits significant anti-inflammatory activity with 73 inhi-bition of paw oedema compared with Indo group (53) andtypically reaches a maximum at 5 h these results obtainedsuggest the anti-inflammatory effect of EtOH extract of ALEbymeans of inhibiting the synthesis and the release of inflam-matory mediators like histamine serotonin and prostagl-andins that are involved in acute inflammationThese resultsrevealed the inhibitory effect of EtOH extract on PN migra-tion and it is confirmed through a histological analysis oftissues of paw oedema in experimental groups

These findings clearly confirm that EtOH extract of ALEhas an anti-inflammatory effect by reducing the influx ofpolymorphonuclear cells to inflammatory tissue followinginjection of Carr Most inflammatory markers such asinterleukin-6 (IL-6) tumor necrosis factor-120572 (TNF-120572) CRPand fibrinogen increased significantly in response to infec-tion and in active diseases states CRP is the specific marker

of acute inflammation occurring in the body [48] moreoverthe fibrinogen is themost relevant indicator of inflammationand it does not only represent the acute-phase reactant that isincreased in inflammatory states Elevated fibrinogen levelswould be associated with higher levels of CRP and wouldsimilarly correlate with inflammation reaction

Therefore the present study showed that EtOH extract ofALE significantly decreases the level of fibrinogen and CRPby value of 2272 and 3478 in comparison with Carrgroup

The induction of inflammationwithCarr ismanifested bygeneration of ROS and has been shown to play an importantrole in various forms of inflammation [49 50] High concen-tration of reactive free radicals contributes to lipid perox-idation and protein oxidation [3] Our results showed thatthere is a significant increase in MDA and AOPP activitiesin Carr group when compared to control group and a highlysignificant decrease in activities of SOD CAT and GSH alsoobserved at the tissue level compared with the control group(119901 lt 0001) However the injection of EtOH extract and Indoshows a significant decrease of the levels of MDA and AOPPby 3427 and 4427 following a significant increase inantioxidant activities as CAT SOD andGSHwhen comparedto Carr group (2016 7330 and 75) From these resultsit is implied that the protective effect of EtOH extract may

12 Evidence-Based Complementary and Alternative Medicine

be attributed to its potential indirectly as a stimulator ofthe activity and expression of antioxidant enzymes duringinflammation

Thus the anti-inflammatory activity is due to the individ-ual or synergistic effect of the components in the ALE In factprevious studies have found that anti-inflammatory profile ofALE could be related to polyphenolic compounds accordingto the phytochemical analysis We performed HPLC analysisto justify the correlation between phytochemical compoundsand anti-inflammatory activity of this plant Among thesepolyphenolic compounds verbascoside has been reportedin several reports and showed a potential spectrum ofmany activities including antioxidant and anti-inflammatoryParticular verbascoside presents antiedematogenic activitiesin animal models of Carr induced inflammation [51] and theresponses exhibited themaximumactivity for the fourth hourof treatment Such activities may be derived from inhibitoryaction of chemical mediators of the inflammatory processsuch as histamine and bradykinin A significant antioxidanteffect of verbascoside has been recently reported byAleo et al[52] in an experimental study [53] Other authors reportedthat anti-inflammatory activity of verbascoside has beenevaluated by an in vitro test performed on cell cultures of pri-mary human keratinocytes [54] in which it was able to reducethe release of proinflammatory markers such as chemokines

Apigenin-7-glucoside was found to block the releaseof several varieties of enzymes involved in inflammationincluding especially lipoxygenases and cyclooxygenases [5556] leading to inhibition of proinflammatory molecules NF-kB activation and inhibits neutrophil infiltration in tissues

From these results we suggest that anti-inflammatoryactivity observed is due to a synergic action of these phenoliccomponents contained in EtOH extract of ALE

C scolymus is one of the few herbal remedies which havebeen verified through experimental studies Further the toxi-city studies of ALE are required in order to confirm the safetyof this medicinal plant in the treatment of inflammationdiseases

5 Conclusion

The present study showed clearly the advantages of Cscolymus leaves extracts which have safer anti-inflammatoryprofile with potent antioxidant activity attributed to thephenolic compounds Furthermore in the future studieswe are interested to characterize the action mechanisms ofactive phenolic compounds of ALE responsible for anti-inflammatory and antioxidants activities

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Tunisian Ministry ofHigher Education and Scientific Research

References

[1] C Pasquier ldquoStress oxidative et inflammationrdquo Blool vol 4 pp1ndash6 1996

[2] M A Abuashwashi O M Palomino and M P Gomez-Serranillos ldquoGeographic origin influences the phenolic com-position and antioxidant potential of wild Crataegus monogynafrom Spainrdquo Pharmaceutical Biology vol 54 no 11 pp 2708ndash2713 2016

[3] M B Salem H Affes K Ksouda et al ldquoPharmacological stud-ies of artichoke leaf extract and their health benefitsrdquo PlantFoods for Human Nutrition vol 70 no 4 pp 441ndash453 2015

[4] M-H Huang B-S Wang C-S Chiu et al ldquoAntioxidant anti-nociceptive and anti-inflammatory activities ofXanthii Fructusextractrdquo Journal of Ethnopharmacology vol 135 no 2 pp 545ndash552 2011

[5] G-J Huang S-S Huang S-S Lin et al ldquoAnalgesic effectsand the mechanisms of anti-inflammation of ergostatrien-3120573-ol from antrodia camphorata submerged whole broth in micerdquoJournal of Agricultural and Food Chemistry vol 58 no 12 pp7445ndash7452 2010

[6] C Tohda N Nakayama F Hatanaka and K Komatsu ldquoCom-parison of anti-inflammatory activities of six Curcuma rhi-zomes a possible curcuminoid-independent pathway mediatedby Curcuma phaeocaulis extractrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 3 no 2 pp 255ndash260 2006

[7] K Goksel K Murat Y Murat E Sevil and H Fidan ldquoAntiox-idant activities of ethanol extracts ofHypericum triquetrifoliumandHypericum scabroidesrdquo Pharmaceutical Biology vol 46 no4 pp 231ndash242 2008

[8] R Gebhardt and M Fausel ldquoAntioxidant and hepatoprotectiveeffects of artichoke extracts and constituents in cultured rathepatocytesrdquoToxicology inVitro vol 11 no 5 pp 669ndash672 1997

[9] A SofworaMedicinal Plants and TraditionalMedicine in AfricaJohn Wiley amp Sons Ann Arbor Mich USA 1982

[10] D E Okwu ldquoPhytochemicals vitamins andmineral contents oftwo Nigerian medicinal plantsrdquo International Journal of Mole-cular Medicine and Advances Sciences vol 1 pp 375ndash381 2005

[11] N Tsevegsuren GDavaakhuu andT S Udval ldquoPhytochemicalanalysis of Cynara scolymus L cultivated in MongoliardquoMongo-lian Journal of Chemistry vol 15 no 41 pp 40ndash42 2014

[12] AACC Approved Methods of the AACC American Associationof Cereal Chemists St Paul Minn USA 8th edition 1984

[13] S C Lee L Prosky and J W DeVries ldquoDetermination of totalsoluble and insoluble dietary fiber in foodsmdashenzymatic-gravi-metric method MES-TRIS buffer collaborative studyrdquo Journalof the Association of Official Analytical Chemists vol 75 pp395ndash416 1992

[14] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[15] O A Fawole U L Opara and K I Theron ldquoChemical andphytochemical properties and antioxidant activities of threepomegranate cultivars grown in South Africardquo Food and Bio-process Technology vol 5 no 7 pp 2934ndash2940 2012

[16] V Dewanto X Wu K K Adom and R H Liu ldquoThermalprocessing enhances the nutritional value of tomatoes byincreasing total antioxidant activityrdquo Journal of Agricultural andFood Chemistry vol 50 no 10 pp 3010ndash3014 2002

[17] B Sun J M Ricardo-da-Silva and I Spranger ldquoCritical factorsof vanillin assay for catechins and proanthocyanidinsrdquo Journal

Evidence-Based Complementary and Alternative Medicine 13

of Agricultural and Food Chemistry vol 46 no 10 pp 4267ndash4274 1998

[18] AOACOfficial Methods of Analysis vol 1 AOACWashingtonDC USA 15th edition 1990

[19] D Pljevljakusic K Savikin T Jankovic et al ldquoChemical prop-erties of the cultivated Sideritis raeseri Boiss amp Heldr subspraeserirdquo Food Chemistry vol 124 no 1 pp 226ndash233 2011

[20] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[21] I I Koleva T A Van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] V Ravi T S M Saleem S S Patel J Raamamurthy and KGauthaman ldquoAnti-inflammatory effect of methanolic extract ofSolanum nigrum Linn Berriesrdquo International Journal of AppliedResearch in Natural Products vol 2 pp 33ndash36 2009

[24] D Chattopadhyay G Arunachalam A B Mandal T K SurS C Mandal and S K Bhattacharya ldquoAntimicrobial and anti-inflammatory activity of folkloreMallotus peltatus leaf extractrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 229ndash2372002

[25] L R Morse K Stolzmann H P Nguyen et al ldquoAssociationbetween mobility mode and c-reactive protein levels in menwith chronic spinal cord injuryrdquo Archives of Physical Medicineand Rehabilitation vol 89 no 4 pp 726ndash731 2008

[26] A Clauss ldquoGerinnungsphysiologische schnellmethode zur bes-timmung des fibrinogensrdquoActaHaematologica vol 17 no 4 pp237ndash246 1957

[27] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[28] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidationin post-mitotic tissues of ageing ratrdquo Cell Biochemistry andFunction vol 24 no 1 pp 79ndash85 2006

[29] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[30] W F Beyer Jr and I Fridovich ldquoAssaying for superoxide dis-mutase activity some large consequences of minor changes inconditionsrdquoAnalytical Biochemistry vol 161 no 2 pp 559ndash5661987

[31] I Carlberg and B Mannervik ldquoGlutathione reductaserdquo inGlutamate Glutamine Glutathione and Related Compoundsvol 113 ofMethods in Enzymology pp 484ndash490 Elsevier 1985

[32] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[33] V Emanue V Adrian N A Sultana and C Svetlana ldquoAntiox-idant and antimicrobial activities of ethanol extracts of Cynarascolymus (Cynarae folium Asteraceae family)rdquo Tropical Journalof Pharmaceutical Research vol 10 no 6 pp 777ndash783 2011

[34] G L D S Oliveira R A M O Francisco O B A Vinıcius etal ldquoEvaluation of antioxidant capacity of the aqueous extract ofCynara scolymus L (Asteraceae) in vitro and in SaccharomycescerevisiaerdquoAfrican Journal of Pharmacy and Pharmacology vol8 pp 136ndash147 2014

[35] K Horiuchi S Shiota T Hatano T Yoshida T Kuroda and TTsuchiya ldquoAntimicrobial activity of oleanolic acid from Salviaofficinalis and related compounds on vancomycin-resistantenterococci (VRE)rdquo Biological and Pharmaceutical Bulletin vol30 no 6 pp 1147ndash1149 2007

[36] A Daoud D Malika S Bakari et al ldquoAssessment of polyphe-nol composition antioxidant and antimicrobial properties ofvarious extracts of Date Palm Pollen (DPP) from two Tunisiancultivarsrdquo Arabian Journal of Chemistry 2015

[37] D Krishnaiah B Awang S Rosalam and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversitymdashEngineering Sciences vol 1 pp 63ndash66 2015

[38] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[39] A Betancor-Fernandez A Perez-Galvez H Sies and W StahlldquoScreening pharmaceutical preparations containing extracts ofturmeric rhizome artichoke leaf devilrsquos claw root and garlic orsalmon oil for antioxidant capacityrdquo Journal of Pharmacy andPharmacology vol 55 no 7 pp 981ndash986 2003

[40] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[41] S Konyal120580oglu H Saglam and B K120580vcak ldquo120572-Tocopherolflavonoid and phenol contents and antioxidant activity of Ficuscarica leavesrdquo Pharmaceutical Biology vol 43 no 8 pp 683ndash686 2005

[42] D U Bawankule P Trivedi A Pal et al ldquoProtectivemechanismof lignans from Phyllanthus amarus against galactosaminelipopolysaccharide-induced hepatitis an in-vivo and in-silicostudiesrdquo Current Topics in Medicinal Chemistry vol 14 no 8pp 1045ndash1055 2014

[43] GAHiggs R J Flower and J RVane ldquoAnew approach to anti-inflammatory drugsrdquo Biochemical Pharmacology vol 28 no 12pp 1959ndash1961 1979

[44] A A Hemmati H Kalantari A Siahpoosh B Ghorbanzadehand H Jamali ldquoAnti-inflammatory Effect of HydroalcoholicExtract of the Washingtonia filifera Seeds in Carrageenan-Induced Paw Edema in Ratsrdquo Jundishapur Journal of NaturalPharmaceutical Products vol 10 no 1 pp 19ndash25 2015

[45] I Posadas M Bucci F Roviezzo et al ldquoCarrageenan-inducedmouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expressionrdquoBritish Journal of Pharmacology vol 142 no 2 pp 331ndash3382004

[46] K Pundarikakshudu D H Shah A H Panchal and G CBhavsar ldquoAnti-inflammatory activity of fenugreek (Trigonellafoenum-graecum Linn) seed petroleum ether extractrdquo IndianJournal of Pharmacology vol 48 no 4 pp 441ndash444 2016

[47] S Cuzzocrea G Costantino E Mazzon B Zingarelli A DeSarro and A P Caputi ldquoProtective effects of Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) a superoxide dismutasemimetic in paw oedema induced by carrageenan in the ratrdquoBiochemical Pharmacology vol 58 no 1 pp 171ndash176 1999

[48] M A Mendall P Patel M Asante et al ldquoRelation of serumcytokine concentrations to cardiovascular risk factors andcoronary heart diseaserdquo Heart vol 78 no 3 pp 273ndash277 1997

[49] A Ialenti A Ianaro S Moncada andM Di Rosa ldquoModulationof acute inflammation by endogenous nitric oxiderdquo EuropeanJournal of Pharmacology vol 211 no 2 pp 177ndash182 1992

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

Submit your manuscripts athttpswwwhindawicom

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of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Phytochemical analysis of Cynara scolymus extracts leaves

Extract Cardiac glycosides Triterpenoids Saponin Flavonoids Tannins AlkaloidALE + + + + + +Sign (+) indicates being present

Percentages of inflammation inhibition were calcu-lated as

Inhibition (II) = [PT minus (P0P0)] times 100 (3)

274 Blood Sample Collection 5 hours after Carr inductionthe rats were decapitated and the blood samples were col-lected in heparin tubes Plasma samples were obtained aftercentrifugation at 3000 rpm for 15min and they were kept inminus20∘Cuntil analysis on an automatic biochemistry analyzer atthe biochemical laboratory of Hedi Chaker Hospital of Sfax

275 Inflammatory Biomarkers

(1) Determination of C-Reactive Protein (CRP) CRP wasa specific marker following the inflammatory process Itincreases in proportion to its intensity [25] The reactiveprotein was measured by turbid metric method using anautomatic analyzer COBAS INTEGRA 40010158401015840 C-reactive TheCRP is expressed with mgL

(2) The Fibrinogen Assay of Plasma Plasma fibrinogen con-centration was determined by Clauss clotting method [26]measured on a STAanalyzer Principle test measures theconversion rate of fibrinogen into fibrin in diluted samplein presence of excess of thrombin and records the clottingtime The clotting time is inversely proportional to the levelof fibrinogen in the plasma The fibrinogen level is expressedwith gL plasma

(3) Exploring the Antioxidant Enzymatic and NonenzymaticStatus Oxidative stress parameters were determined in tis-sues paw oedema homogenates The supernatants obtainedwere removed and analyzed for the determination of MDAas described by Draper and Hadley [27] AOPP levels werequantified by method of Kayali et al [28] CAT activity wasmeasured as reported by Aebi [29] and expressed as mmolesof H2O2 consumed(minmg protein) SOD was assayedspectrophotometrically by colorimetric method of Beyer Jrand Fridovich [30] and expressed as Umg protein GSHactivity was assayed according to the method of Carlbergand Mannervik [31] and expressed as mmoles GSS(min-mg protein) and the protein content was determined usingmethod of Lowry et al [32]

(4) Histopathological Examination All the paws oedematissues of experimental groups were collected for histolog-ical examination First they were fixed in 10 bufferedformalin solution second they were embedded in paraffinwax and then cut into 5mm thick sections and stainedwith hematoxylin and eosin (HampE) Finally the slides werephotographed with an Olympus U-TU1X-2 camera

28 High Performance Liquid Chromatography (HPLC)Detec-tion and Quantification of Polyphenolic Compounds Thephenolic fractions analysis of EtOH extract of ALE wasdetermined using an Agilent Technologies 1100 series HPLCcoupled with a UV-Vis multiwavelength detector The sepa-ration was carried out on a 250 times 46mm C18 silica columnchromatography was chosen at ambient temperature Themobile phase consisted of C2H3N (solvent A) and waterwith 02 of H2SO4 (solvent B) and the flow rate waskept at 08mLminminus1 For the preparation of calibrationcurve a standard stock solution was prepared in methanol(HPLC grade ge 999 from Sigma Chemical Company)containing Hydroxytyrosol Tyrosol 4-hydroxybenzoic acidverbascoside apigenin-7-glucoside Oleuropein QuercetinPinoresinol cinnamic acid and apigenin at the same concen-tration of 1 gmL For the preparation of the ethanol extract bydissolving in 1mL of methanol making a final concentrationof 25mgmL Before starting HPLC analysis all the solutionspreparedwere filteredwithWhatman paper (Oslash 045 120583m)Thediluted extract was injected directly and chromatogramsweremonitored at 280 nm 20120583L was used for injection Peakswere identified by the retention times compared with thestandardsThe analyses of phenolic profiles of ethanol extractwere performed in triplicate

29 Statistical Analysis SPSS program was employed forcomparisons between all groups Comparison between mul-tiple and within groups was analyzed by ANOVA followedby Tukeyrsquos tests Values were shown as mean plusmn SD The levelof statistical significance was set at 119901 value lt 005 Pearsonrsquoscorrelation coefficient analysis was calculated using SPSSprogram also in order to evaluate the statistical relationshipbetween both of polyphenolics compounds and antioxidantactivities of ALE

3 Results

31 Phytochemical Analysis of Cynara scolymus LeavesExtracts ALE revealed the presence of flavonoid cardiac gly-cosides saponin tannin terpenoid and alkaloids (Table 1)

32 Proximate Analysis of Dried Leaves of C scolymus Theresults showed that Cynara leaves contain high constituentsdry matter (9703 DW) ash (1581 DW) carbohydrate(8005DW) protein (1664DW) lipids (341DW) totalsugars (197 DW) and dietary fiber (7160 DW) (Table 2)

33 Quantification of Total Phenolic Flavonoid and TanninsContents of Cynara scolymus Leaves Extracts TheTPC TFCand TCCwere evaluated in different solvents extracts of ALEAll results are shown in Table 3 Maximum content of TPCwas obtained using EtOHextract and corresponded to 5454plusmn126mg GAg DW followed by ethyl acetate aqueous and

Evidence-Based Complementary and Alternative Medicine 5

Table 2 Proximate analysis of dried leaves of Cynara scolymus

Constituents Cynara scolymus leaves(percentage dry weight basis)

Dry matter 9703 plusmn 043Ash 1581 plusmn 001Carbohydrate 8005 plusmn 069Protein 1664 plusmn 179Lipids 341 plusmn 045Total sugars 197 plusmn 010Dietary fiber 7160 plusmn 081Values are expressed as mean plusmn SD (119899 = 3)

butanol extracts while TPC of hexane extract was the lowestin comparison with EtOH extract (3091 plusmn 936mg GAEgDW) The EtOH extract showed also the highest content inTFC (12plusmn083CEgDW) but this value was lower for hexaneextract (819 plusmn 06mg CEg DW) respectively

The amount of TCCwhichwas evaluated by vanillin assaywas the highest in the ethyl acetate and EtOH extract (1451and 1405mg CEg DW) but this content was lower thanaqueous extract as shown in Table 3

34 Mineral Contents Analysis The main composition ofmacroelements and microelements in C scolymus leaves ispresented in Table 4

Elemental analysis in (mg100 g of dry weight basis)indicated that leaves of artichoke contained the followingorder of essential minerals compounds potassium (288680)sodium (176294) calcium (135934) magnesium (43321)iron (1617) manganese (1305) zinc (737) copper (130) andchromium (012)

35 Evaluation of Antioxidant Activity Theantioxidant activ-ity of ALE harvested in Tunisia was performed using fourmethods DPPH ABTS FRAP and beta-carotene test

351 Antioxidant Activity by DPPHMethod DDPHmethodevaluated the capacity of compounds present in ALE toreduce DDPH radical The result in Figure 1 showed thescavenging activity of all extracts of C scolymus was con-centration dependent Among all extracts the EtOH extractdisplayed the highest free radical scavenging activity at higherconcentration (400 ugmL) (9423) compared to the sameconcentration of vitamin C (983) and BHT (9623) fol-lowedby ethyl acetate extract (8151) the scavenging activityof other extracts was significantly lower when compared toreference standard VC (9983)

352120573-Carotene Bleaching Test Thepotential effect of EtOHextract exhibited significantly the highest inhibition rate of120573-carotene bleaching (7074) compared to BHT and AA(4794 9059) (119901 lt 0001) respectively (Table 5) whereashexane and butanol extract showed the lowest rate of 120573-carotene bleaching 3738 and 4939

353 Antioxidant Activity by ABTS∙+ Method As evidentfrom Table 5 ALE studied presents a potential capacity

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

0102030405060708090

100

in

hibi

tion

100ugmL200ugmL

300ugmL400ugmL

Etha

nol

Hex

ane

Ethy

l ace

tate

Buta

nol

Aque

ous

VC BHT

Figure 1 Antioxidant activity by DPPHmethod ofCynara scolymusleaves extracts at different concentrations Values are mean plusmn SD(119899 = 3) lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 compared toEtOH extract Butylated Hydroxytoluene (BHT) Vitamin C (VC)

0 1 2 3 4 5 6Temps (h)

0

05

1

15

2

25

3

35

4Si

ze o

f oed

ema (

mm

)

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

CARRNACL

CARR + IndoCARR + ALE

Figure 2 Effect of ALE and Indomethacin on paw oedema inducedby carrageenan Values represent mean plusmn SD (119899 = 6) in each grouplowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 120572 compared to control 120573compared to Carr m compared to Carr + Indo

to scavenge the ABTS radical cation Among all extractsEtOH extract was a significantly higher value (49943mmolTroloxg dry extract) than the other extracts but the low-est level of TEAC was obtained from the hexane extract(1043mmol Troloxg dry extract)

354 Ferric Reducing Assay The results in Table 5 showedthat among all extracts of ALE a higher capacity of reducingferric capacity was found for EtOH extract (52779 umolFe2+mg dry extract) when compared to other extractsrespectively

36 Anti-Inflammatory Activities The appearance of Carrinduced paw oedema with respective treatments groups wasillustrated in Table 7 and Figure 2 The results showed

6 Evidence-Based Complementary and Alternative Medicine

Table 3 Quantification of total phenolic flavonoids and tannins contents of Cynara scolymus leaves extracts

Extracts Phenolics content(mg GAEg DW)

Flavonoidscontent

(mg CEg DW)

Tannins content(mg CEg DW)

Hexane 3991 plusmn 936 819 plusmn 016 1405 plusmn 03Ethyl acetate 5307 plusmn 047 1032 plusmn 012 1451 plusmn 013Butanol 4166 plusmn 223 1121 plusmn 010 1393 plusmn 93Ethanol 5454 plusmn 126 1200 plusmn 083 1099Aqueous 4949 plusmn 039 949 plusmn 039 438 plusmn 045Values are expressed as mean plusmn SD (119899 = 3)

Table 4 Mineral contents dried leaves of Cynara scolymus

Elements Cynara scolymus leaves (mg100 g of dry weightbasis)

K 2886803 plusmn 120Ca 1359346 plusmn 505Na 1762946 plusmn 120Mg 433219 plusmn 234I 16176 plusmn 014Mn 13051 plusmn 011Zn 7371 plusmn 014Copper 130 plusmn 016Cr 0124 plusmn 001Values are expressed as mean plusmn SD (119899 = 3)

that the induction of Carr in rats paw oedema started offby the vascular phase of inflammation which generated anincrease in the size of oedema for all groups This injectiongenerated intense inflammation which peaked after 3 hoursThe experimental data showed that ALE presented significantdecrease in the size of pawoedemawhichwas time dependentand more important than the reference group Indo and Carrgroup

As seen in Figures 3 and 4 EtOH extract at dose(400mgkgbw) has shown a significant percent of oedemainhibition (119901 lt 0001) in the first hour (173) comparedwith reference group (Indo) and the significant percent ofinhibition inflammation was 4427 in comparison with thecontrol group

Thus after five hours these percentages were 735 forinhibition and 832 for inflammation For the Indo groupoedema inhibition was significant (119901 lt 0001) 53 and 64and oedema inflammation was 21 and 86 in comparisonwith the control group

These results showed that EtOH extract of ALE had astrong effect as Indo

361 Inflammatory Biomarkers Fibrinogen and CRP Weassayed serum proteins of inflammatory markers in differentgroups of rats

(1) FibrinogenCarr group caused a significant increase in rateof fibrinogen compared to control group by 1905 (Figure 5)

1 2 3 4 5Temps (h)

0

10

20

30

40

50

60

70

80

in

hibi

tion

oede

ma

poundlowastlowastlowast

poundlowastlowastlowast

poundlowastlowastlowast

poundlowastlowastlowast

Carr + ALECarr + Indo

Figure 3 Percentage () of oedema inhibition data in all groupsValues represent mean plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt001 and lowastlowastlowast119901 lt 0001 m compared to Carr + Indo

0

10

20

30

40

50

60

70

in

hibi

tion

infla

mm

atio

n

1 2 3 4 5Temps (h)

NaclCarr

Carr + ALECarr + Indo

poundlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast poundlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

poundlowastlowastlowastlowastlowastlowastlowastlowastlowast

poundlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

Figure 4 Percentage () of oedema inflammation data in allgroups Values represent mean plusmn SD (119899 = 6) in each group lowast119901 lt005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 120572 compared to control group120573 compared to Carr group and m compared to Indo group

The rate of fibrinogen decreased significantly (119901 lt 001) ingroups of rats treated with the EtOH extract (1655) andIndo (2290) compared with Carr group

Evidence-Based Complementary and Alternative Medicine 7

Table 5 Antioxidant activities of Cynara scolymus leaves extracts

Sample FRAP assay (umol Fe (II)gDW)

120573-Carotene bleaching assay()

TEAC assay (mmolTroloxg DW)

Hexane 223023 plusmn 1116lowastlowastlowast 3738 plusmn 524lowastlowastlowast 1043 plusmn 1073lowastlowastlowastEthyl acetate 50829 plusmn 524lowastlowastlowast 6156 plusmn 817lowast 38260 plusmn 524lowastlowastButanol 44306 plusmn 2298lowastlowastlowast 49393 plusmn 224lowastlowast 25193 plusmn 2815lowastlowastlowastEthanol 52779 plusmn 1626 70743 plusmn 129 49943 plusmn 3972Aqueous 31591 plusmn 836lowastlowastlowast 5611 plusmn 543lowastlowast 21074 plusmn 836lowastlowastlowastBHT mdash 4794 plusmn 075lowastlowastlowast mdashAA mdash 9059 plusmn 325lowastlowastlowast mdashValues are means plusmn SD (119899 = 3) Butylated Hydroxytoluene (BHT) and Ascorbic Acid (AA) were used as positive control compared with EtOH extract lowast119901 lt005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001

Nacl Carr Carr + ALE Carr + Indo0

05

1

15

2

25

Fg (gL)CRP (mgmL)

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

Figure 5 Levels of fibrinogen (Fg) and C-reactive protein (CRP)Values represent mean plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt001 and lowastlowastlowast119901 lt 0001 120572 compared to control 120573 compared to Carrgroup Fibrinogen (Fg) C-reactive protein (CRP)

(2) CRP Analysis showed a significant increase (119901 lt 005)in CRP level (Figure 5) for Carr group compared to controlgroup (1673) However the injection of EtOH extract andIndo led to a significant decrease (119901 lt 0001) (60863423) respectively when compared with Carr group

37 Oxidative Stress Parameters MDA and AOPP levels inoedema paw were illustrated in Table 8 Results showed thatCarr induced a significant increase inMDA andAOPP levelshowever treatment with EtOH extract of ALE at dose of400mgkgbw showed a significant decrease (119901 lt 0001)(2080 5023) compared with Carr group and even Indogroup (10mgkg) (2097 4427) respectively

38 Exploring the Antioxidant Enzymatic and NonenzymaticStatus CAT SOD and GSH levels in the paw oedema tissueof tested groups are shown in Table 8 Treatment of inflamedrats with EtOH extract restored significantly (119901 lt 0001)the SOD activity by 2075 the CAT activity by 5655 andthe GSH activity by 7566 respectively compared to Carrgroup

39 Histopathological Examination A microscopic study ofpaw oedema tissues showed histological changes in Carrgroup EtOH extract of ALE (400mgkgpc) group andIndo (10mgkgbw) group (Figure 6) In the Carr group weshowed a subcutaneous oedema with infiltration of inflam-matory cells especially polynuclear neutrophils at the site ofinflamed tissues and also the presence of spongy-like appear-ance in the epidermis ((Figure 6(b)) as compared to controlgroup (Figure 6(a))) The EtOH extract group produced asignificant decrease in the number of cellular infiltrates and asignificant reduced spongy-like appearance in the epidermis(Figure 6(c)) as did reference drug Indo (Figure 6(d))

310 Quantification of Polyphenolic Compounds by HPLCTo the authorsrsquo knowledge the present study identified andquantified the phenolic compounds of ALE from Tunisianorigins Accordingly the results obtained so far by Folin-Ciocalteu needed to be further complemented to qualifyphenolic constituents in EtOH extract under investigation

The findings revealed the presence of seven phenoliccompounds namely Hydroxytyrosol verbascoside apige-nin-7-glucoside Oleuropein Quercetin Pinoresinol andapigenin HPLC analysis indicated that ALE have significantamount of verbascoside content 381mg100 g and Quercetincontent (19 2mg100 g) (Figures 7 and 8 and Table 9)

311 Correlation between the Polyphenolic Compounds andAntioxidant Capacity In order to determine the contributionof the phenolic and flavonoid content in ALE on antiox-idant capacity the Pearson correlation coefficient (119903) wasdetermined in Table 6 The results showed a significantlinear correlation between antioxidant activities determinedby using DPPH ABTS FRAP and beta-carotene methodsand TPC and TFC respectively The strongest correlativevalue is obtained with DDPH and TPC (119903 = 087) Theseresults indicated a good correlation between TPC and TFCwith antioxidant activities of ALE

4 Discussion

The importance of plants in traditional medicine remediesand the potential of phytochemical constituents were dis-cussed nowadays with respect to their benefit in pharma-cotherapy in Tunisia Cynara scolymus was one of these

8 Evidence-Based Complementary and Alternative Medicine

(a)

(b)

(c)

(d)

Figure 6 Histopathological slides tissues of paw oedema in experimental groups of rats (a) Control group untreated group (b) Carr groupCarr-treated rat showed heavy infiltration of polynuclear neutrophils (PN) and a spongy-like appearance and bulla in the epidermis (c)Carr + EtOH extract group Carr-treated rat that received the EtOH extract (400mgkgbw) reduced significantly the migration of PNand oedematasis in dermis without any spongy-like feature and bulla (d) Carr + Indo group Carr-treated rat that received Indomethacin(10mgkgbw) showed a partial protective action Deparaffinized hematoxylin and eosin (HampE) stained sections (200ndash400x) Plus signinfiltration of PN Arrow odematasis in the epidermis

medicinal plants which have a beneficial potential effectattributed to its source of polyphenolic compounds [3]

The selection of this plant was guided by the indicationsof its traditional use that at present there have been very littlechemical and biological investigations done Therefore thepresent study was to investigate phytochemical compositionand their antioxidant capacity and to evaluate in vivo the anti-inflammatory effects of Cynara scolymus leaves extracts

The presence of phenolic compounds is very widelydistributed in medicinal plants several studies showed thatthese compounds have drawn much attention to their poten-tial antioxidant abilities which demonstrated their beneficialimplications for human health

Regarding the determination of phenolic compoundsthe EtOH extract of ALE had the highest amount of thesecompounds in comparison with other extracts which werean agreement with the result of Emanue et al [33] whoshows that EtOH extract exhibited the maximum amountof phenolic compounds from the leaves of Cynara scolymuswhereas it differs from the reports of Oliveira et al [34]who proves that aqueous extract of ALE is the most suitablesolvent for extraction of phenolic compounds

The importance of TPC and TFC extraction yieldsobtained with EtOH extract can be attributed to its goodsolubility low toxicity medium polarity and high extractioncapacity [35]

Evidence-Based Complementary and Alternative Medicine 9

1 2 3 678 9 10

5

4

0250500750

1000125015001750

(mAU

)

5 10 15 20 250(min)

Figure 7 HPLC chromatogram of a standard mixture of polyphe-nolic compounds Peaks 1 Hydroxytyrosol 2 Tyrosol 3 4-hydroxybenzoic acid 4 verbascoside 5 apigenin-7-glucoside 6Oleuropein 7 Quercetin 8 Pinoresinol 9 cinnamic acid 10apigenin

34

2

56 7

0200400600800

100012001400

(mAU

)

5 10 15 20 25 30 350(min)

1

Figure 8 HPLC chromatogram of ethanol extract of Cynarascolymus leaves extracts Peaks 1 Hydroxytyrosol 2 verbascoside3 apigenin-7-glucoside 4 Oleuropein 5 Quercetin 6 Pinoresinol7 apigenin

Table 6 Pearson correlation coefficient (119903) between the content ofphenolic compounds and antioxidant capacity (DDPH∙ ABTS∙+FRAP and beta-carotene)

Correlation 119877TPC versus DDPH∙ 0870lowastlowast

TPC versus ABTS∙+ 0848lowastlowast

TPC versus FRAP 0707lowastlowast

TPC versus beta-carotene 0842lowastlowast

TFC versus DDPH∙ 0728lowastlowast

TFC versus ABTS∙+ 0743lowastlowast

TFC versus FRAP 0849lowastlowast

TFC versus beta-carotene 0712lowastlowast

DPPH 22-diphenyl-1-picrylhydrazyl ABTS 220-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt FRAP ferricreducing TPC total phenolic content TFC total flavonoid contentlowastlowast119901 lt 001 significant correlation

In the first investigation of my study five solvents ofincreasing polarities were chosen for the determination ofphenolic compounds from leaves of C scolymus namelyhexane ethyl acetate butanol 75 EtOHH2O and aqueousin order to determine the variability of TPC and TFC in theaerial part of Cynara as a function of each extraction solvent

This variability of phenolic compounds in ALE can beattributed to the wide range of solubility displayed by variouspolar compounds within the ALE solvents the degree ofpolymerization of phenols and their interaction geneticfactors geographical variations and climatic changes [36]

Overall these findings indicate that EtOH extract of ALEwas rich in phenolic and flavonoid contents which couldbe the major contributor to their antioxidative propertiesMany researches revealed that flavonoids and polyphenolsdisplayed the highest ability of scavenging activity in medic-inal plants [37 38]

The results indicate that EtOH extract of ALE whichcontains the highest content of TFC and TPC displays thehighest free radical scavenging activity (9423) at concen-tration of 400 ugmL However Oliveira et al [34] show thataqueous extract of ALE has good radical scavenging (8340)at concentration of 200 ugmL Furthermore our resultssuggest that some components within EtOH extract aresignificantly the strongest radical scavenger in comparisonwith other extracts The TEAC values of ALE showed alsothat EtOH extract presents a potential ability to scavengethe ABTS radical cation in accordance with the report ofBetancor-Fernandez et al [39]

The FRAP assay shows also that EtOH extract has thehighest values (54262 umol Fe II gDW) The same resultis confirmed by Kukic et al [40] Therefore the results ofpositive correlation between the TPC TFC and the anti-oxidant methods in vitro suggest that phenolic compoundsact as reducing agents hydrogen donors and singlet oxygenscavengers [3] and may exert an important in vitro antioxi-dant capacity of ALE

The phytochemical screening of ALE has been foundto be a rich source of polyphenolic compounds includingQuercetin apigenin-7-glucoside and verbascoside Thesecompounds have shown a great potential of antioxidantactivity [41]

The research into medicinal plants used as pain relieversrsquoagents should therefore be viewed as new therapy in theinflammatory diseases [42] The anti-inflammatory activitiesof EtOH extract of ALE are investigated applying in vivoexperimental Carr model compared with a nonsteroidal anti-inflammatory (Indomethacin) this drug was reported byHiggs et al [43] who demonstrated the role of Indo toinhibit the biosynthesis of prostaglandins in Carr model ratsThe experimental study of anti-inflammatory activity is per-formed by the Carr test This phlogiston agent inducedtissue oedema characteristic of acute inflammation whichis regarded as a crucial parameter in assessing anti-inflam-matory activity [44]

The Carr injection produces biphasic states in the firstphase [45] there is an increase in the mRNA synthesis ofcyclooxygenase-2 (COX-2) at the first hour This increase isaccompanied by an amplification of synthesis of strong proin-flammatory mediator such as prostaglandin especially theprostaglandin type 2 involved in the inflammatory process[46] serotonin bradykinin and leukotrienes which con-tribute to the initiation of inflammation reaction Moreoverthe participation of arachidonicmetabolites is themain factorresponsible for both of phases of Carr induced inflammation

10 Evidence-Based Complementary and Alternative Medicine

Table7Eff

ecto

fCynarascolym

usleaves

extracto

ncarrageenan-indu

cedratp

awoedema

Treatm

ent

Oedem

asize(meanplusmnS

D)(mm)

0h1h

2h3h

4h5h

Con

trol

0426plusmn0

00

044

1plusmn001

056plusmn0

03

0650plusmn0

05

0755plusmn0

05

070plusmn0

01

Carr

0426plusmn0

00

28plusmn026120572lowastlowastlowast

333plusmn028120572lowastlowastlowast

325plusmn002120572lowastlowastlowast

315plusmn001120572lowastlowastlowast

302plusmn009120572lowastlowastlowast

Carr+Indo

(10m

gkg)

0426plusmn0

00

246plusmn026120572lowastlowastlowast120573lowast

261plusmn007120572lowastlowastlowast120573lowastlowastlowast

230plusmn002120572lowastlowastlowast120573lowastlowastlowast

18plusmn011120572lowastlowastlowast120573lowastlowastlowast

14plusmn006120572lowastlowastlowast120573lowastlowastlowast

Carr+ALE

(400

mgkgbw)

0426plusmn0

00

2480plusmn005120572lowastlowastlowast120573lowastlowastlowast

188plusmn004120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

165plusmn005120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

116plusmn020120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

112plusmn01120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

Values

representm

eansplusmnSD

(119899=6)ineach

grou

plowast119901lt005lowastlowast119901lt001andlowastlowastlowast119901lt0001

120572com

paredto

control120573com

paredto

Carrmcom

paredto

Carr+Indo

Evidence-Based Complementary and Alternative Medicine 11

Table 8 Effects of Cynara scolymus leaves extract and Indomethacin on CAT SOD GSH AOPP andMDA activities in carrageenan inducedpaw oedema

GroupsCAT (120583molesCATminmg

protein)

SOD (unitSODminmg

protein)

GSH (nmolesmgprotein)

MDA (nmolMDAmg protein) AOPP (nmolmg protein)

Control 47 plusmn 2 131 plusmn 43 421 plusmn 12 234 plusmn 5 484 plusmn 43Carr 162 plusmn 05120572lowastlowastlowast 153 plusmn 26120572lowastlowastlowast 131 plusmn 11120572lowastlowastlowast 314 plusmn 75120572lowastlowastlowast 679 plusmn 69120572lowastlowastlowastCarr + Indo(10mgkgbw) 649 plusmn 71120573lowastlowastlowast 240 plusmn 13120573lowastlowastlowast 703 plusmn 11120573lowastlowastlowast 209 plusmn 30120573lowastlowastlowast 442 plusmn 06120573lowastlowastlowastCarr + ALE(400mgkgbw) 565 plusmn 25120573lowastlowastlowastmlowastlowast 207 plusmn 40120573lowastlowastlowastmlowastlowast 756 plusmn 25120573lowastlowastlowastmlowast 208 plusmn 60120573lowastlowastlowast 502 plusmn 41120573lowastlowastlowastmlowastValues represent means plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001120572 compared to control 120573 compared to Carr m compared to Carr + IndoSOD superoxide dismutaseCAT catalaseGSH glutathione peroxidaseMDA MalondialdehydeAOPP Advanced Oxidation Protein Product

Table 9 Quantification and identification of phenolic compounds contents in the ethanol extract of Cynara scolymus leaves extracts

Number Content (mg100 g of dry extract) Polyphenolic compound1 28 plusmn 019 Hydroxytyrosol2 381 plusmn 036 Verbascoside3 99 plusmn 002 Apigenin-7-glucoside4 18 plusmn 01 Oleuropein5 192 plusmn 014 Quercetin6 5 plusmn 005 Pinoresinol7 4 plusmn 004 ApigeninValues represent means plusmn SD (119899 = 3)

In the second phase the increase of vascular permeabilityis observed by release of kinins during 230 hThereafter from230 h to 5 h inflammation ismediated by prostaglandins andis also associated with a release infiltration and migration ofPN into the inflamed site [47]

Our results indicate that ALE affords protection againstthe Carr induced acute inflammation in dose dependentmanner EtOH extract of ALE at dose of 400mgkgbwexhibits significant anti-inflammatory activity with 73 inhi-bition of paw oedema compared with Indo group (53) andtypically reaches a maximum at 5 h these results obtainedsuggest the anti-inflammatory effect of EtOH extract of ALEbymeans of inhibiting the synthesis and the release of inflam-matory mediators like histamine serotonin and prostagl-andins that are involved in acute inflammationThese resultsrevealed the inhibitory effect of EtOH extract on PN migra-tion and it is confirmed through a histological analysis oftissues of paw oedema in experimental groups

These findings clearly confirm that EtOH extract of ALEhas an anti-inflammatory effect by reducing the influx ofpolymorphonuclear cells to inflammatory tissue followinginjection of Carr Most inflammatory markers such asinterleukin-6 (IL-6) tumor necrosis factor-120572 (TNF-120572) CRPand fibrinogen increased significantly in response to infec-tion and in active diseases states CRP is the specific marker

of acute inflammation occurring in the body [48] moreoverthe fibrinogen is themost relevant indicator of inflammationand it does not only represent the acute-phase reactant that isincreased in inflammatory states Elevated fibrinogen levelswould be associated with higher levels of CRP and wouldsimilarly correlate with inflammation reaction

Therefore the present study showed that EtOH extract ofALE significantly decreases the level of fibrinogen and CRPby value of 2272 and 3478 in comparison with Carrgroup

The induction of inflammationwithCarr ismanifested bygeneration of ROS and has been shown to play an importantrole in various forms of inflammation [49 50] High concen-tration of reactive free radicals contributes to lipid perox-idation and protein oxidation [3] Our results showed thatthere is a significant increase in MDA and AOPP activitiesin Carr group when compared to control group and a highlysignificant decrease in activities of SOD CAT and GSH alsoobserved at the tissue level compared with the control group(119901 lt 0001) However the injection of EtOH extract and Indoshows a significant decrease of the levels of MDA and AOPPby 3427 and 4427 following a significant increase inantioxidant activities as CAT SOD andGSHwhen comparedto Carr group (2016 7330 and 75) From these resultsit is implied that the protective effect of EtOH extract may

12 Evidence-Based Complementary and Alternative Medicine

be attributed to its potential indirectly as a stimulator ofthe activity and expression of antioxidant enzymes duringinflammation

Thus the anti-inflammatory activity is due to the individ-ual or synergistic effect of the components in the ALE In factprevious studies have found that anti-inflammatory profile ofALE could be related to polyphenolic compounds accordingto the phytochemical analysis We performed HPLC analysisto justify the correlation between phytochemical compoundsand anti-inflammatory activity of this plant Among thesepolyphenolic compounds verbascoside has been reportedin several reports and showed a potential spectrum ofmany activities including antioxidant and anti-inflammatoryParticular verbascoside presents antiedematogenic activitiesin animal models of Carr induced inflammation [51] and theresponses exhibited themaximumactivity for the fourth hourof treatment Such activities may be derived from inhibitoryaction of chemical mediators of the inflammatory processsuch as histamine and bradykinin A significant antioxidanteffect of verbascoside has been recently reported byAleo et al[52] in an experimental study [53] Other authors reportedthat anti-inflammatory activity of verbascoside has beenevaluated by an in vitro test performed on cell cultures of pri-mary human keratinocytes [54] in which it was able to reducethe release of proinflammatory markers such as chemokines

Apigenin-7-glucoside was found to block the releaseof several varieties of enzymes involved in inflammationincluding especially lipoxygenases and cyclooxygenases [5556] leading to inhibition of proinflammatory molecules NF-kB activation and inhibits neutrophil infiltration in tissues

From these results we suggest that anti-inflammatoryactivity observed is due to a synergic action of these phenoliccomponents contained in EtOH extract of ALE

C scolymus is one of the few herbal remedies which havebeen verified through experimental studies Further the toxi-city studies of ALE are required in order to confirm the safetyof this medicinal plant in the treatment of inflammationdiseases

5 Conclusion

The present study showed clearly the advantages of Cscolymus leaves extracts which have safer anti-inflammatoryprofile with potent antioxidant activity attributed to thephenolic compounds Furthermore in the future studieswe are interested to characterize the action mechanisms ofactive phenolic compounds of ALE responsible for anti-inflammatory and antioxidants activities

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Tunisian Ministry ofHigher Education and Scientific Research

References

[1] C Pasquier ldquoStress oxidative et inflammationrdquo Blool vol 4 pp1ndash6 1996

[2] M A Abuashwashi O M Palomino and M P Gomez-Serranillos ldquoGeographic origin influences the phenolic com-position and antioxidant potential of wild Crataegus monogynafrom Spainrdquo Pharmaceutical Biology vol 54 no 11 pp 2708ndash2713 2016

[3] M B Salem H Affes K Ksouda et al ldquoPharmacological stud-ies of artichoke leaf extract and their health benefitsrdquo PlantFoods for Human Nutrition vol 70 no 4 pp 441ndash453 2015

[4] M-H Huang B-S Wang C-S Chiu et al ldquoAntioxidant anti-nociceptive and anti-inflammatory activities ofXanthii Fructusextractrdquo Journal of Ethnopharmacology vol 135 no 2 pp 545ndash552 2011

[5] G-J Huang S-S Huang S-S Lin et al ldquoAnalgesic effectsand the mechanisms of anti-inflammation of ergostatrien-3120573-ol from antrodia camphorata submerged whole broth in micerdquoJournal of Agricultural and Food Chemistry vol 58 no 12 pp7445ndash7452 2010

[6] C Tohda N Nakayama F Hatanaka and K Komatsu ldquoCom-parison of anti-inflammatory activities of six Curcuma rhi-zomes a possible curcuminoid-independent pathway mediatedby Curcuma phaeocaulis extractrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 3 no 2 pp 255ndash260 2006

[7] K Goksel K Murat Y Murat E Sevil and H Fidan ldquoAntiox-idant activities of ethanol extracts ofHypericum triquetrifoliumandHypericum scabroidesrdquo Pharmaceutical Biology vol 46 no4 pp 231ndash242 2008

[8] R Gebhardt and M Fausel ldquoAntioxidant and hepatoprotectiveeffects of artichoke extracts and constituents in cultured rathepatocytesrdquoToxicology inVitro vol 11 no 5 pp 669ndash672 1997

[9] A SofworaMedicinal Plants and TraditionalMedicine in AfricaJohn Wiley amp Sons Ann Arbor Mich USA 1982

[10] D E Okwu ldquoPhytochemicals vitamins andmineral contents oftwo Nigerian medicinal plantsrdquo International Journal of Mole-cular Medicine and Advances Sciences vol 1 pp 375ndash381 2005

[11] N Tsevegsuren GDavaakhuu andT S Udval ldquoPhytochemicalanalysis of Cynara scolymus L cultivated in MongoliardquoMongo-lian Journal of Chemistry vol 15 no 41 pp 40ndash42 2014

[12] AACC Approved Methods of the AACC American Associationof Cereal Chemists St Paul Minn USA 8th edition 1984

[13] S C Lee L Prosky and J W DeVries ldquoDetermination of totalsoluble and insoluble dietary fiber in foodsmdashenzymatic-gravi-metric method MES-TRIS buffer collaborative studyrdquo Journalof the Association of Official Analytical Chemists vol 75 pp395ndash416 1992

[14] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[15] O A Fawole U L Opara and K I Theron ldquoChemical andphytochemical properties and antioxidant activities of threepomegranate cultivars grown in South Africardquo Food and Bio-process Technology vol 5 no 7 pp 2934ndash2940 2012

[16] V Dewanto X Wu K K Adom and R H Liu ldquoThermalprocessing enhances the nutritional value of tomatoes byincreasing total antioxidant activityrdquo Journal of Agricultural andFood Chemistry vol 50 no 10 pp 3010ndash3014 2002

[17] B Sun J M Ricardo-da-Silva and I Spranger ldquoCritical factorsof vanillin assay for catechins and proanthocyanidinsrdquo Journal

Evidence-Based Complementary and Alternative Medicine 13

of Agricultural and Food Chemistry vol 46 no 10 pp 4267ndash4274 1998

[18] AOACOfficial Methods of Analysis vol 1 AOACWashingtonDC USA 15th edition 1990

[19] D Pljevljakusic K Savikin T Jankovic et al ldquoChemical prop-erties of the cultivated Sideritis raeseri Boiss amp Heldr subspraeserirdquo Food Chemistry vol 124 no 1 pp 226ndash233 2011

[20] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[21] I I Koleva T A Van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] V Ravi T S M Saleem S S Patel J Raamamurthy and KGauthaman ldquoAnti-inflammatory effect of methanolic extract ofSolanum nigrum Linn Berriesrdquo International Journal of AppliedResearch in Natural Products vol 2 pp 33ndash36 2009

[24] D Chattopadhyay G Arunachalam A B Mandal T K SurS C Mandal and S K Bhattacharya ldquoAntimicrobial and anti-inflammatory activity of folkloreMallotus peltatus leaf extractrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 229ndash2372002

[25] L R Morse K Stolzmann H P Nguyen et al ldquoAssociationbetween mobility mode and c-reactive protein levels in menwith chronic spinal cord injuryrdquo Archives of Physical Medicineand Rehabilitation vol 89 no 4 pp 726ndash731 2008

[26] A Clauss ldquoGerinnungsphysiologische schnellmethode zur bes-timmung des fibrinogensrdquoActaHaematologica vol 17 no 4 pp237ndash246 1957

[27] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[28] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidationin post-mitotic tissues of ageing ratrdquo Cell Biochemistry andFunction vol 24 no 1 pp 79ndash85 2006

[29] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[30] W F Beyer Jr and I Fridovich ldquoAssaying for superoxide dis-mutase activity some large consequences of minor changes inconditionsrdquoAnalytical Biochemistry vol 161 no 2 pp 559ndash5661987

[31] I Carlberg and B Mannervik ldquoGlutathione reductaserdquo inGlutamate Glutamine Glutathione and Related Compoundsvol 113 ofMethods in Enzymology pp 484ndash490 Elsevier 1985

[32] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[33] V Emanue V Adrian N A Sultana and C Svetlana ldquoAntiox-idant and antimicrobial activities of ethanol extracts of Cynarascolymus (Cynarae folium Asteraceae family)rdquo Tropical Journalof Pharmaceutical Research vol 10 no 6 pp 777ndash783 2011

[34] G L D S Oliveira R A M O Francisco O B A Vinıcius etal ldquoEvaluation of antioxidant capacity of the aqueous extract ofCynara scolymus L (Asteraceae) in vitro and in SaccharomycescerevisiaerdquoAfrican Journal of Pharmacy and Pharmacology vol8 pp 136ndash147 2014

[35] K Horiuchi S Shiota T Hatano T Yoshida T Kuroda and TTsuchiya ldquoAntimicrobial activity of oleanolic acid from Salviaofficinalis and related compounds on vancomycin-resistantenterococci (VRE)rdquo Biological and Pharmaceutical Bulletin vol30 no 6 pp 1147ndash1149 2007

[36] A Daoud D Malika S Bakari et al ldquoAssessment of polyphe-nol composition antioxidant and antimicrobial properties ofvarious extracts of Date Palm Pollen (DPP) from two Tunisiancultivarsrdquo Arabian Journal of Chemistry 2015

[37] D Krishnaiah B Awang S Rosalam and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversitymdashEngineering Sciences vol 1 pp 63ndash66 2015

[38] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[39] A Betancor-Fernandez A Perez-Galvez H Sies and W StahlldquoScreening pharmaceutical preparations containing extracts ofturmeric rhizome artichoke leaf devilrsquos claw root and garlic orsalmon oil for antioxidant capacityrdquo Journal of Pharmacy andPharmacology vol 55 no 7 pp 981ndash986 2003

[40] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[41] S Konyal120580oglu H Saglam and B K120580vcak ldquo120572-Tocopherolflavonoid and phenol contents and antioxidant activity of Ficuscarica leavesrdquo Pharmaceutical Biology vol 43 no 8 pp 683ndash686 2005

[42] D U Bawankule P Trivedi A Pal et al ldquoProtectivemechanismof lignans from Phyllanthus amarus against galactosaminelipopolysaccharide-induced hepatitis an in-vivo and in-silicostudiesrdquo Current Topics in Medicinal Chemistry vol 14 no 8pp 1045ndash1055 2014

[43] GAHiggs R J Flower and J RVane ldquoAnew approach to anti-inflammatory drugsrdquo Biochemical Pharmacology vol 28 no 12pp 1959ndash1961 1979

[44] A A Hemmati H Kalantari A Siahpoosh B Ghorbanzadehand H Jamali ldquoAnti-inflammatory Effect of HydroalcoholicExtract of the Washingtonia filifera Seeds in Carrageenan-Induced Paw Edema in Ratsrdquo Jundishapur Journal of NaturalPharmaceutical Products vol 10 no 1 pp 19ndash25 2015

[45] I Posadas M Bucci F Roviezzo et al ldquoCarrageenan-inducedmouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expressionrdquoBritish Journal of Pharmacology vol 142 no 2 pp 331ndash3382004

[46] K Pundarikakshudu D H Shah A H Panchal and G CBhavsar ldquoAnti-inflammatory activity of fenugreek (Trigonellafoenum-graecum Linn) seed petroleum ether extractrdquo IndianJournal of Pharmacology vol 48 no 4 pp 441ndash444 2016

[47] S Cuzzocrea G Costantino E Mazzon B Zingarelli A DeSarro and A P Caputi ldquoProtective effects of Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) a superoxide dismutasemimetic in paw oedema induced by carrageenan in the ratrdquoBiochemical Pharmacology vol 58 no 1 pp 171ndash176 1999

[48] M A Mendall P Patel M Asante et al ldquoRelation of serumcytokine concentrations to cardiovascular risk factors andcoronary heart diseaserdquo Heart vol 78 no 3 pp 273ndash277 1997

[49] A Ialenti A Ianaro S Moncada andM Di Rosa ldquoModulationof acute inflammation by endogenous nitric oxiderdquo EuropeanJournal of Pharmacology vol 211 no 2 pp 177ndash182 1992

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

Table 2 Proximate analysis of dried leaves of Cynara scolymus

Constituents Cynara scolymus leaves(percentage dry weight basis)

Dry matter 9703 plusmn 043Ash 1581 plusmn 001Carbohydrate 8005 plusmn 069Protein 1664 plusmn 179Lipids 341 plusmn 045Total sugars 197 plusmn 010Dietary fiber 7160 plusmn 081Values are expressed as mean plusmn SD (119899 = 3)

butanol extracts while TPC of hexane extract was the lowestin comparison with EtOH extract (3091 plusmn 936mg GAEgDW) The EtOH extract showed also the highest content inTFC (12plusmn083CEgDW) but this value was lower for hexaneextract (819 plusmn 06mg CEg DW) respectively

The amount of TCCwhichwas evaluated by vanillin assaywas the highest in the ethyl acetate and EtOH extract (1451and 1405mg CEg DW) but this content was lower thanaqueous extract as shown in Table 3

34 Mineral Contents Analysis The main composition ofmacroelements and microelements in C scolymus leaves ispresented in Table 4

Elemental analysis in (mg100 g of dry weight basis)indicated that leaves of artichoke contained the followingorder of essential minerals compounds potassium (288680)sodium (176294) calcium (135934) magnesium (43321)iron (1617) manganese (1305) zinc (737) copper (130) andchromium (012)

35 Evaluation of Antioxidant Activity Theantioxidant activ-ity of ALE harvested in Tunisia was performed using fourmethods DPPH ABTS FRAP and beta-carotene test

351 Antioxidant Activity by DPPHMethod DDPHmethodevaluated the capacity of compounds present in ALE toreduce DDPH radical The result in Figure 1 showed thescavenging activity of all extracts of C scolymus was con-centration dependent Among all extracts the EtOH extractdisplayed the highest free radical scavenging activity at higherconcentration (400 ugmL) (9423) compared to the sameconcentration of vitamin C (983) and BHT (9623) fol-lowedby ethyl acetate extract (8151) the scavenging activityof other extracts was significantly lower when compared toreference standard VC (9983)

352120573-Carotene Bleaching Test Thepotential effect of EtOHextract exhibited significantly the highest inhibition rate of120573-carotene bleaching (7074) compared to BHT and AA(4794 9059) (119901 lt 0001) respectively (Table 5) whereashexane and butanol extract showed the lowest rate of 120573-carotene bleaching 3738 and 4939

353 Antioxidant Activity by ABTS∙+ Method As evidentfrom Table 5 ALE studied presents a potential capacity

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

0102030405060708090

100

in

hibi

tion

100ugmL200ugmL

300ugmL400ugmL

Etha

nol

Hex

ane

Ethy

l ace

tate

Buta

nol

Aque

ous

VC BHT

Figure 1 Antioxidant activity by DPPHmethod ofCynara scolymusleaves extracts at different concentrations Values are mean plusmn SD(119899 = 3) lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 compared toEtOH extract Butylated Hydroxytoluene (BHT) Vitamin C (VC)

0 1 2 3 4 5 6Temps (h)

0

05

1

15

2

25

3

35

4Si

ze o

f oed

ema (

mm

)

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast lowastlowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastpoundlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

CARRNACL

CARR + IndoCARR + ALE

Figure 2 Effect of ALE and Indomethacin on paw oedema inducedby carrageenan Values represent mean plusmn SD (119899 = 6) in each grouplowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 120572 compared to control 120573compared to Carr m compared to Carr + Indo

to scavenge the ABTS radical cation Among all extractsEtOH extract was a significantly higher value (49943mmolTroloxg dry extract) than the other extracts but the low-est level of TEAC was obtained from the hexane extract(1043mmol Troloxg dry extract)

354 Ferric Reducing Assay The results in Table 5 showedthat among all extracts of ALE a higher capacity of reducingferric capacity was found for EtOH extract (52779 umolFe2+mg dry extract) when compared to other extractsrespectively

36 Anti-Inflammatory Activities The appearance of Carrinduced paw oedema with respective treatments groups wasillustrated in Table 7 and Figure 2 The results showed

6 Evidence-Based Complementary and Alternative Medicine

Table 3 Quantification of total phenolic flavonoids and tannins contents of Cynara scolymus leaves extracts

Extracts Phenolics content(mg GAEg DW)

Flavonoidscontent

(mg CEg DW)

Tannins content(mg CEg DW)

Hexane 3991 plusmn 936 819 plusmn 016 1405 plusmn 03Ethyl acetate 5307 plusmn 047 1032 plusmn 012 1451 plusmn 013Butanol 4166 plusmn 223 1121 plusmn 010 1393 plusmn 93Ethanol 5454 plusmn 126 1200 plusmn 083 1099Aqueous 4949 plusmn 039 949 plusmn 039 438 plusmn 045Values are expressed as mean plusmn SD (119899 = 3)

Table 4 Mineral contents dried leaves of Cynara scolymus

Elements Cynara scolymus leaves (mg100 g of dry weightbasis)

K 2886803 plusmn 120Ca 1359346 plusmn 505Na 1762946 plusmn 120Mg 433219 plusmn 234I 16176 plusmn 014Mn 13051 plusmn 011Zn 7371 plusmn 014Copper 130 plusmn 016Cr 0124 plusmn 001Values are expressed as mean plusmn SD (119899 = 3)

that the induction of Carr in rats paw oedema started offby the vascular phase of inflammation which generated anincrease in the size of oedema for all groups This injectiongenerated intense inflammation which peaked after 3 hoursThe experimental data showed that ALE presented significantdecrease in the size of pawoedemawhichwas time dependentand more important than the reference group Indo and Carrgroup

As seen in Figures 3 and 4 EtOH extract at dose(400mgkgbw) has shown a significant percent of oedemainhibition (119901 lt 0001) in the first hour (173) comparedwith reference group (Indo) and the significant percent ofinhibition inflammation was 4427 in comparison with thecontrol group

Thus after five hours these percentages were 735 forinhibition and 832 for inflammation For the Indo groupoedema inhibition was significant (119901 lt 0001) 53 and 64and oedema inflammation was 21 and 86 in comparisonwith the control group

These results showed that EtOH extract of ALE had astrong effect as Indo

361 Inflammatory Biomarkers Fibrinogen and CRP Weassayed serum proteins of inflammatory markers in differentgroups of rats

(1) FibrinogenCarr group caused a significant increase in rateof fibrinogen compared to control group by 1905 (Figure 5)

1 2 3 4 5Temps (h)

0

10

20

30

40

50

60

70

80

in

hibi

tion

oede

ma

poundlowastlowastlowast

poundlowastlowastlowast

poundlowastlowastlowast

poundlowastlowastlowast

Carr + ALECarr + Indo

Figure 3 Percentage () of oedema inhibition data in all groupsValues represent mean plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt001 and lowastlowastlowast119901 lt 0001 m compared to Carr + Indo

0

10

20

30

40

50

60

70

in

hibi

tion

infla

mm

atio

n

1 2 3 4 5Temps (h)

NaclCarr

Carr + ALECarr + Indo

poundlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast poundlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

poundlowastlowastlowastlowastlowastlowastlowastlowastlowast

poundlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

Figure 4 Percentage () of oedema inflammation data in allgroups Values represent mean plusmn SD (119899 = 6) in each group lowast119901 lt005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 120572 compared to control group120573 compared to Carr group and m compared to Indo group

The rate of fibrinogen decreased significantly (119901 lt 001) ingroups of rats treated with the EtOH extract (1655) andIndo (2290) compared with Carr group

Evidence-Based Complementary and Alternative Medicine 7

Table 5 Antioxidant activities of Cynara scolymus leaves extracts

Sample FRAP assay (umol Fe (II)gDW)

120573-Carotene bleaching assay()

TEAC assay (mmolTroloxg DW)

Hexane 223023 plusmn 1116lowastlowastlowast 3738 plusmn 524lowastlowastlowast 1043 plusmn 1073lowastlowastlowastEthyl acetate 50829 plusmn 524lowastlowastlowast 6156 plusmn 817lowast 38260 plusmn 524lowastlowastButanol 44306 plusmn 2298lowastlowastlowast 49393 plusmn 224lowastlowast 25193 plusmn 2815lowastlowastlowastEthanol 52779 plusmn 1626 70743 plusmn 129 49943 plusmn 3972Aqueous 31591 plusmn 836lowastlowastlowast 5611 plusmn 543lowastlowast 21074 plusmn 836lowastlowastlowastBHT mdash 4794 plusmn 075lowastlowastlowast mdashAA mdash 9059 plusmn 325lowastlowastlowast mdashValues are means plusmn SD (119899 = 3) Butylated Hydroxytoluene (BHT) and Ascorbic Acid (AA) were used as positive control compared with EtOH extract lowast119901 lt005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001

Nacl Carr Carr + ALE Carr + Indo0

05

1

15

2

25

Fg (gL)CRP (mgmL)

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

Figure 5 Levels of fibrinogen (Fg) and C-reactive protein (CRP)Values represent mean plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt001 and lowastlowastlowast119901 lt 0001 120572 compared to control 120573 compared to Carrgroup Fibrinogen (Fg) C-reactive protein (CRP)

(2) CRP Analysis showed a significant increase (119901 lt 005)in CRP level (Figure 5) for Carr group compared to controlgroup (1673) However the injection of EtOH extract andIndo led to a significant decrease (119901 lt 0001) (60863423) respectively when compared with Carr group

37 Oxidative Stress Parameters MDA and AOPP levels inoedema paw were illustrated in Table 8 Results showed thatCarr induced a significant increase inMDA andAOPP levelshowever treatment with EtOH extract of ALE at dose of400mgkgbw showed a significant decrease (119901 lt 0001)(2080 5023) compared with Carr group and even Indogroup (10mgkg) (2097 4427) respectively

38 Exploring the Antioxidant Enzymatic and NonenzymaticStatus CAT SOD and GSH levels in the paw oedema tissueof tested groups are shown in Table 8 Treatment of inflamedrats with EtOH extract restored significantly (119901 lt 0001)the SOD activity by 2075 the CAT activity by 5655 andthe GSH activity by 7566 respectively compared to Carrgroup

39 Histopathological Examination A microscopic study ofpaw oedema tissues showed histological changes in Carrgroup EtOH extract of ALE (400mgkgpc) group andIndo (10mgkgbw) group (Figure 6) In the Carr group weshowed a subcutaneous oedema with infiltration of inflam-matory cells especially polynuclear neutrophils at the site ofinflamed tissues and also the presence of spongy-like appear-ance in the epidermis ((Figure 6(b)) as compared to controlgroup (Figure 6(a))) The EtOH extract group produced asignificant decrease in the number of cellular infiltrates and asignificant reduced spongy-like appearance in the epidermis(Figure 6(c)) as did reference drug Indo (Figure 6(d))

310 Quantification of Polyphenolic Compounds by HPLCTo the authorsrsquo knowledge the present study identified andquantified the phenolic compounds of ALE from Tunisianorigins Accordingly the results obtained so far by Folin-Ciocalteu needed to be further complemented to qualifyphenolic constituents in EtOH extract under investigation

The findings revealed the presence of seven phenoliccompounds namely Hydroxytyrosol verbascoside apige-nin-7-glucoside Oleuropein Quercetin Pinoresinol andapigenin HPLC analysis indicated that ALE have significantamount of verbascoside content 381mg100 g and Quercetincontent (19 2mg100 g) (Figures 7 and 8 and Table 9)

311 Correlation between the Polyphenolic Compounds andAntioxidant Capacity In order to determine the contributionof the phenolic and flavonoid content in ALE on antiox-idant capacity the Pearson correlation coefficient (119903) wasdetermined in Table 6 The results showed a significantlinear correlation between antioxidant activities determinedby using DPPH ABTS FRAP and beta-carotene methodsand TPC and TFC respectively The strongest correlativevalue is obtained with DDPH and TPC (119903 = 087) Theseresults indicated a good correlation between TPC and TFCwith antioxidant activities of ALE

4 Discussion

The importance of plants in traditional medicine remediesand the potential of phytochemical constituents were dis-cussed nowadays with respect to their benefit in pharma-cotherapy in Tunisia Cynara scolymus was one of these

8 Evidence-Based Complementary and Alternative Medicine

(a)

(b)

(c)

(d)

Figure 6 Histopathological slides tissues of paw oedema in experimental groups of rats (a) Control group untreated group (b) Carr groupCarr-treated rat showed heavy infiltration of polynuclear neutrophils (PN) and a spongy-like appearance and bulla in the epidermis (c)Carr + EtOH extract group Carr-treated rat that received the EtOH extract (400mgkgbw) reduced significantly the migration of PNand oedematasis in dermis without any spongy-like feature and bulla (d) Carr + Indo group Carr-treated rat that received Indomethacin(10mgkgbw) showed a partial protective action Deparaffinized hematoxylin and eosin (HampE) stained sections (200ndash400x) Plus signinfiltration of PN Arrow odematasis in the epidermis

medicinal plants which have a beneficial potential effectattributed to its source of polyphenolic compounds [3]

The selection of this plant was guided by the indicationsof its traditional use that at present there have been very littlechemical and biological investigations done Therefore thepresent study was to investigate phytochemical compositionand their antioxidant capacity and to evaluate in vivo the anti-inflammatory effects of Cynara scolymus leaves extracts

The presence of phenolic compounds is very widelydistributed in medicinal plants several studies showed thatthese compounds have drawn much attention to their poten-tial antioxidant abilities which demonstrated their beneficialimplications for human health

Regarding the determination of phenolic compoundsthe EtOH extract of ALE had the highest amount of thesecompounds in comparison with other extracts which werean agreement with the result of Emanue et al [33] whoshows that EtOH extract exhibited the maximum amountof phenolic compounds from the leaves of Cynara scolymuswhereas it differs from the reports of Oliveira et al [34]who proves that aqueous extract of ALE is the most suitablesolvent for extraction of phenolic compounds

The importance of TPC and TFC extraction yieldsobtained with EtOH extract can be attributed to its goodsolubility low toxicity medium polarity and high extractioncapacity [35]

Evidence-Based Complementary and Alternative Medicine 9

1 2 3 678 9 10

5

4

0250500750

1000125015001750

(mAU

)

5 10 15 20 250(min)

Figure 7 HPLC chromatogram of a standard mixture of polyphe-nolic compounds Peaks 1 Hydroxytyrosol 2 Tyrosol 3 4-hydroxybenzoic acid 4 verbascoside 5 apigenin-7-glucoside 6Oleuropein 7 Quercetin 8 Pinoresinol 9 cinnamic acid 10apigenin

34

2

56 7

0200400600800

100012001400

(mAU

)

5 10 15 20 25 30 350(min)

1

Figure 8 HPLC chromatogram of ethanol extract of Cynarascolymus leaves extracts Peaks 1 Hydroxytyrosol 2 verbascoside3 apigenin-7-glucoside 4 Oleuropein 5 Quercetin 6 Pinoresinol7 apigenin

Table 6 Pearson correlation coefficient (119903) between the content ofphenolic compounds and antioxidant capacity (DDPH∙ ABTS∙+FRAP and beta-carotene)

Correlation 119877TPC versus DDPH∙ 0870lowastlowast

TPC versus ABTS∙+ 0848lowastlowast

TPC versus FRAP 0707lowastlowast

TPC versus beta-carotene 0842lowastlowast

TFC versus DDPH∙ 0728lowastlowast

TFC versus ABTS∙+ 0743lowastlowast

TFC versus FRAP 0849lowastlowast

TFC versus beta-carotene 0712lowastlowast

DPPH 22-diphenyl-1-picrylhydrazyl ABTS 220-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt FRAP ferricreducing TPC total phenolic content TFC total flavonoid contentlowastlowast119901 lt 001 significant correlation

In the first investigation of my study five solvents ofincreasing polarities were chosen for the determination ofphenolic compounds from leaves of C scolymus namelyhexane ethyl acetate butanol 75 EtOHH2O and aqueousin order to determine the variability of TPC and TFC in theaerial part of Cynara as a function of each extraction solvent

This variability of phenolic compounds in ALE can beattributed to the wide range of solubility displayed by variouspolar compounds within the ALE solvents the degree ofpolymerization of phenols and their interaction geneticfactors geographical variations and climatic changes [36]

Overall these findings indicate that EtOH extract of ALEwas rich in phenolic and flavonoid contents which couldbe the major contributor to their antioxidative propertiesMany researches revealed that flavonoids and polyphenolsdisplayed the highest ability of scavenging activity in medic-inal plants [37 38]

The results indicate that EtOH extract of ALE whichcontains the highest content of TFC and TPC displays thehighest free radical scavenging activity (9423) at concen-tration of 400 ugmL However Oliveira et al [34] show thataqueous extract of ALE has good radical scavenging (8340)at concentration of 200 ugmL Furthermore our resultssuggest that some components within EtOH extract aresignificantly the strongest radical scavenger in comparisonwith other extracts The TEAC values of ALE showed alsothat EtOH extract presents a potential ability to scavengethe ABTS radical cation in accordance with the report ofBetancor-Fernandez et al [39]

The FRAP assay shows also that EtOH extract has thehighest values (54262 umol Fe II gDW) The same resultis confirmed by Kukic et al [40] Therefore the results ofpositive correlation between the TPC TFC and the anti-oxidant methods in vitro suggest that phenolic compoundsact as reducing agents hydrogen donors and singlet oxygenscavengers [3] and may exert an important in vitro antioxi-dant capacity of ALE

The phytochemical screening of ALE has been foundto be a rich source of polyphenolic compounds includingQuercetin apigenin-7-glucoside and verbascoside Thesecompounds have shown a great potential of antioxidantactivity [41]

The research into medicinal plants used as pain relieversrsquoagents should therefore be viewed as new therapy in theinflammatory diseases [42] The anti-inflammatory activitiesof EtOH extract of ALE are investigated applying in vivoexperimental Carr model compared with a nonsteroidal anti-inflammatory (Indomethacin) this drug was reported byHiggs et al [43] who demonstrated the role of Indo toinhibit the biosynthesis of prostaglandins in Carr model ratsThe experimental study of anti-inflammatory activity is per-formed by the Carr test This phlogiston agent inducedtissue oedema characteristic of acute inflammation whichis regarded as a crucial parameter in assessing anti-inflam-matory activity [44]

The Carr injection produces biphasic states in the firstphase [45] there is an increase in the mRNA synthesis ofcyclooxygenase-2 (COX-2) at the first hour This increase isaccompanied by an amplification of synthesis of strong proin-flammatory mediator such as prostaglandin especially theprostaglandin type 2 involved in the inflammatory process[46] serotonin bradykinin and leukotrienes which con-tribute to the initiation of inflammation reaction Moreoverthe participation of arachidonicmetabolites is themain factorresponsible for both of phases of Carr induced inflammation

10 Evidence-Based Complementary and Alternative Medicine

Table7Eff

ecto

fCynarascolym

usleaves

extracto

ncarrageenan-indu

cedratp

awoedema

Treatm

ent

Oedem

asize(meanplusmnS

D)(mm)

0h1h

2h3h

4h5h

Con

trol

0426plusmn0

00

044

1plusmn001

056plusmn0

03

0650plusmn0

05

0755plusmn0

05

070plusmn0

01

Carr

0426plusmn0

00

28plusmn026120572lowastlowastlowast

333plusmn028120572lowastlowastlowast

325plusmn002120572lowastlowastlowast

315plusmn001120572lowastlowastlowast

302plusmn009120572lowastlowastlowast

Carr+Indo

(10m

gkg)

0426plusmn0

00

246plusmn026120572lowastlowastlowast120573lowast

261plusmn007120572lowastlowastlowast120573lowastlowastlowast

230plusmn002120572lowastlowastlowast120573lowastlowastlowast

18plusmn011120572lowastlowastlowast120573lowastlowastlowast

14plusmn006120572lowastlowastlowast120573lowastlowastlowast

Carr+ALE

(400

mgkgbw)

0426plusmn0

00

2480plusmn005120572lowastlowastlowast120573lowastlowastlowast

188plusmn004120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

165plusmn005120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

116plusmn020120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

112plusmn01120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

Values

representm

eansplusmnSD

(119899=6)ineach

grou

plowast119901lt005lowastlowast119901lt001andlowastlowastlowast119901lt0001

120572com

paredto

control120573com

paredto

Carrmcom

paredto

Carr+Indo

Evidence-Based Complementary and Alternative Medicine 11

Table 8 Effects of Cynara scolymus leaves extract and Indomethacin on CAT SOD GSH AOPP andMDA activities in carrageenan inducedpaw oedema

GroupsCAT (120583molesCATminmg

protein)

SOD (unitSODminmg

protein)

GSH (nmolesmgprotein)

MDA (nmolMDAmg protein) AOPP (nmolmg protein)

Control 47 plusmn 2 131 plusmn 43 421 plusmn 12 234 plusmn 5 484 plusmn 43Carr 162 plusmn 05120572lowastlowastlowast 153 plusmn 26120572lowastlowastlowast 131 plusmn 11120572lowastlowastlowast 314 plusmn 75120572lowastlowastlowast 679 plusmn 69120572lowastlowastlowastCarr + Indo(10mgkgbw) 649 plusmn 71120573lowastlowastlowast 240 plusmn 13120573lowastlowastlowast 703 plusmn 11120573lowastlowastlowast 209 plusmn 30120573lowastlowastlowast 442 plusmn 06120573lowastlowastlowastCarr + ALE(400mgkgbw) 565 plusmn 25120573lowastlowastlowastmlowastlowast 207 plusmn 40120573lowastlowastlowastmlowastlowast 756 plusmn 25120573lowastlowastlowastmlowast 208 plusmn 60120573lowastlowastlowast 502 plusmn 41120573lowastlowastlowastmlowastValues represent means plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001120572 compared to control 120573 compared to Carr m compared to Carr + IndoSOD superoxide dismutaseCAT catalaseGSH glutathione peroxidaseMDA MalondialdehydeAOPP Advanced Oxidation Protein Product

Table 9 Quantification and identification of phenolic compounds contents in the ethanol extract of Cynara scolymus leaves extracts

Number Content (mg100 g of dry extract) Polyphenolic compound1 28 plusmn 019 Hydroxytyrosol2 381 plusmn 036 Verbascoside3 99 plusmn 002 Apigenin-7-glucoside4 18 plusmn 01 Oleuropein5 192 plusmn 014 Quercetin6 5 plusmn 005 Pinoresinol7 4 plusmn 004 ApigeninValues represent means plusmn SD (119899 = 3)

In the second phase the increase of vascular permeabilityis observed by release of kinins during 230 hThereafter from230 h to 5 h inflammation ismediated by prostaglandins andis also associated with a release infiltration and migration ofPN into the inflamed site [47]

Our results indicate that ALE affords protection againstthe Carr induced acute inflammation in dose dependentmanner EtOH extract of ALE at dose of 400mgkgbwexhibits significant anti-inflammatory activity with 73 inhi-bition of paw oedema compared with Indo group (53) andtypically reaches a maximum at 5 h these results obtainedsuggest the anti-inflammatory effect of EtOH extract of ALEbymeans of inhibiting the synthesis and the release of inflam-matory mediators like histamine serotonin and prostagl-andins that are involved in acute inflammationThese resultsrevealed the inhibitory effect of EtOH extract on PN migra-tion and it is confirmed through a histological analysis oftissues of paw oedema in experimental groups

These findings clearly confirm that EtOH extract of ALEhas an anti-inflammatory effect by reducing the influx ofpolymorphonuclear cells to inflammatory tissue followinginjection of Carr Most inflammatory markers such asinterleukin-6 (IL-6) tumor necrosis factor-120572 (TNF-120572) CRPand fibrinogen increased significantly in response to infec-tion and in active diseases states CRP is the specific marker

of acute inflammation occurring in the body [48] moreoverthe fibrinogen is themost relevant indicator of inflammationand it does not only represent the acute-phase reactant that isincreased in inflammatory states Elevated fibrinogen levelswould be associated with higher levels of CRP and wouldsimilarly correlate with inflammation reaction

Therefore the present study showed that EtOH extract ofALE significantly decreases the level of fibrinogen and CRPby value of 2272 and 3478 in comparison with Carrgroup

The induction of inflammationwithCarr ismanifested bygeneration of ROS and has been shown to play an importantrole in various forms of inflammation [49 50] High concen-tration of reactive free radicals contributes to lipid perox-idation and protein oxidation [3] Our results showed thatthere is a significant increase in MDA and AOPP activitiesin Carr group when compared to control group and a highlysignificant decrease in activities of SOD CAT and GSH alsoobserved at the tissue level compared with the control group(119901 lt 0001) However the injection of EtOH extract and Indoshows a significant decrease of the levels of MDA and AOPPby 3427 and 4427 following a significant increase inantioxidant activities as CAT SOD andGSHwhen comparedto Carr group (2016 7330 and 75) From these resultsit is implied that the protective effect of EtOH extract may

12 Evidence-Based Complementary and Alternative Medicine

be attributed to its potential indirectly as a stimulator ofthe activity and expression of antioxidant enzymes duringinflammation

Thus the anti-inflammatory activity is due to the individ-ual or synergistic effect of the components in the ALE In factprevious studies have found that anti-inflammatory profile ofALE could be related to polyphenolic compounds accordingto the phytochemical analysis We performed HPLC analysisto justify the correlation between phytochemical compoundsand anti-inflammatory activity of this plant Among thesepolyphenolic compounds verbascoside has been reportedin several reports and showed a potential spectrum ofmany activities including antioxidant and anti-inflammatoryParticular verbascoside presents antiedematogenic activitiesin animal models of Carr induced inflammation [51] and theresponses exhibited themaximumactivity for the fourth hourof treatment Such activities may be derived from inhibitoryaction of chemical mediators of the inflammatory processsuch as histamine and bradykinin A significant antioxidanteffect of verbascoside has been recently reported byAleo et al[52] in an experimental study [53] Other authors reportedthat anti-inflammatory activity of verbascoside has beenevaluated by an in vitro test performed on cell cultures of pri-mary human keratinocytes [54] in which it was able to reducethe release of proinflammatory markers such as chemokines

Apigenin-7-glucoside was found to block the releaseof several varieties of enzymes involved in inflammationincluding especially lipoxygenases and cyclooxygenases [5556] leading to inhibition of proinflammatory molecules NF-kB activation and inhibits neutrophil infiltration in tissues

From these results we suggest that anti-inflammatoryactivity observed is due to a synergic action of these phenoliccomponents contained in EtOH extract of ALE

C scolymus is one of the few herbal remedies which havebeen verified through experimental studies Further the toxi-city studies of ALE are required in order to confirm the safetyof this medicinal plant in the treatment of inflammationdiseases

5 Conclusion

The present study showed clearly the advantages of Cscolymus leaves extracts which have safer anti-inflammatoryprofile with potent antioxidant activity attributed to thephenolic compounds Furthermore in the future studieswe are interested to characterize the action mechanisms ofactive phenolic compounds of ALE responsible for anti-inflammatory and antioxidants activities

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Tunisian Ministry ofHigher Education and Scientific Research

References

[1] C Pasquier ldquoStress oxidative et inflammationrdquo Blool vol 4 pp1ndash6 1996

[2] M A Abuashwashi O M Palomino and M P Gomez-Serranillos ldquoGeographic origin influences the phenolic com-position and antioxidant potential of wild Crataegus monogynafrom Spainrdquo Pharmaceutical Biology vol 54 no 11 pp 2708ndash2713 2016

[3] M B Salem H Affes K Ksouda et al ldquoPharmacological stud-ies of artichoke leaf extract and their health benefitsrdquo PlantFoods for Human Nutrition vol 70 no 4 pp 441ndash453 2015

[4] M-H Huang B-S Wang C-S Chiu et al ldquoAntioxidant anti-nociceptive and anti-inflammatory activities ofXanthii Fructusextractrdquo Journal of Ethnopharmacology vol 135 no 2 pp 545ndash552 2011

[5] G-J Huang S-S Huang S-S Lin et al ldquoAnalgesic effectsand the mechanisms of anti-inflammation of ergostatrien-3120573-ol from antrodia camphorata submerged whole broth in micerdquoJournal of Agricultural and Food Chemistry vol 58 no 12 pp7445ndash7452 2010

[6] C Tohda N Nakayama F Hatanaka and K Komatsu ldquoCom-parison of anti-inflammatory activities of six Curcuma rhi-zomes a possible curcuminoid-independent pathway mediatedby Curcuma phaeocaulis extractrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 3 no 2 pp 255ndash260 2006

[7] K Goksel K Murat Y Murat E Sevil and H Fidan ldquoAntiox-idant activities of ethanol extracts ofHypericum triquetrifoliumandHypericum scabroidesrdquo Pharmaceutical Biology vol 46 no4 pp 231ndash242 2008

[8] R Gebhardt and M Fausel ldquoAntioxidant and hepatoprotectiveeffects of artichoke extracts and constituents in cultured rathepatocytesrdquoToxicology inVitro vol 11 no 5 pp 669ndash672 1997

[9] A SofworaMedicinal Plants and TraditionalMedicine in AfricaJohn Wiley amp Sons Ann Arbor Mich USA 1982

[10] D E Okwu ldquoPhytochemicals vitamins andmineral contents oftwo Nigerian medicinal plantsrdquo International Journal of Mole-cular Medicine and Advances Sciences vol 1 pp 375ndash381 2005

[11] N Tsevegsuren GDavaakhuu andT S Udval ldquoPhytochemicalanalysis of Cynara scolymus L cultivated in MongoliardquoMongo-lian Journal of Chemistry vol 15 no 41 pp 40ndash42 2014

[12] AACC Approved Methods of the AACC American Associationof Cereal Chemists St Paul Minn USA 8th edition 1984

[13] S C Lee L Prosky and J W DeVries ldquoDetermination of totalsoluble and insoluble dietary fiber in foodsmdashenzymatic-gravi-metric method MES-TRIS buffer collaborative studyrdquo Journalof the Association of Official Analytical Chemists vol 75 pp395ndash416 1992

[14] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[15] O A Fawole U L Opara and K I Theron ldquoChemical andphytochemical properties and antioxidant activities of threepomegranate cultivars grown in South Africardquo Food and Bio-process Technology vol 5 no 7 pp 2934ndash2940 2012

[16] V Dewanto X Wu K K Adom and R H Liu ldquoThermalprocessing enhances the nutritional value of tomatoes byincreasing total antioxidant activityrdquo Journal of Agricultural andFood Chemistry vol 50 no 10 pp 3010ndash3014 2002

[17] B Sun J M Ricardo-da-Silva and I Spranger ldquoCritical factorsof vanillin assay for catechins and proanthocyanidinsrdquo Journal

Evidence-Based Complementary and Alternative Medicine 13

of Agricultural and Food Chemistry vol 46 no 10 pp 4267ndash4274 1998

[18] AOACOfficial Methods of Analysis vol 1 AOACWashingtonDC USA 15th edition 1990

[19] D Pljevljakusic K Savikin T Jankovic et al ldquoChemical prop-erties of the cultivated Sideritis raeseri Boiss amp Heldr subspraeserirdquo Food Chemistry vol 124 no 1 pp 226ndash233 2011

[20] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[21] I I Koleva T A Van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] V Ravi T S M Saleem S S Patel J Raamamurthy and KGauthaman ldquoAnti-inflammatory effect of methanolic extract ofSolanum nigrum Linn Berriesrdquo International Journal of AppliedResearch in Natural Products vol 2 pp 33ndash36 2009

[24] D Chattopadhyay G Arunachalam A B Mandal T K SurS C Mandal and S K Bhattacharya ldquoAntimicrobial and anti-inflammatory activity of folkloreMallotus peltatus leaf extractrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 229ndash2372002

[25] L R Morse K Stolzmann H P Nguyen et al ldquoAssociationbetween mobility mode and c-reactive protein levels in menwith chronic spinal cord injuryrdquo Archives of Physical Medicineand Rehabilitation vol 89 no 4 pp 726ndash731 2008

[26] A Clauss ldquoGerinnungsphysiologische schnellmethode zur bes-timmung des fibrinogensrdquoActaHaematologica vol 17 no 4 pp237ndash246 1957

[27] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[28] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidationin post-mitotic tissues of ageing ratrdquo Cell Biochemistry andFunction vol 24 no 1 pp 79ndash85 2006

[29] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[30] W F Beyer Jr and I Fridovich ldquoAssaying for superoxide dis-mutase activity some large consequences of minor changes inconditionsrdquoAnalytical Biochemistry vol 161 no 2 pp 559ndash5661987

[31] I Carlberg and B Mannervik ldquoGlutathione reductaserdquo inGlutamate Glutamine Glutathione and Related Compoundsvol 113 ofMethods in Enzymology pp 484ndash490 Elsevier 1985

[32] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[33] V Emanue V Adrian N A Sultana and C Svetlana ldquoAntiox-idant and antimicrobial activities of ethanol extracts of Cynarascolymus (Cynarae folium Asteraceae family)rdquo Tropical Journalof Pharmaceutical Research vol 10 no 6 pp 777ndash783 2011

[34] G L D S Oliveira R A M O Francisco O B A Vinıcius etal ldquoEvaluation of antioxidant capacity of the aqueous extract ofCynara scolymus L (Asteraceae) in vitro and in SaccharomycescerevisiaerdquoAfrican Journal of Pharmacy and Pharmacology vol8 pp 136ndash147 2014

[35] K Horiuchi S Shiota T Hatano T Yoshida T Kuroda and TTsuchiya ldquoAntimicrobial activity of oleanolic acid from Salviaofficinalis and related compounds on vancomycin-resistantenterococci (VRE)rdquo Biological and Pharmaceutical Bulletin vol30 no 6 pp 1147ndash1149 2007

[36] A Daoud D Malika S Bakari et al ldquoAssessment of polyphe-nol composition antioxidant and antimicrobial properties ofvarious extracts of Date Palm Pollen (DPP) from two Tunisiancultivarsrdquo Arabian Journal of Chemistry 2015

[37] D Krishnaiah B Awang S Rosalam and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversitymdashEngineering Sciences vol 1 pp 63ndash66 2015

[38] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[39] A Betancor-Fernandez A Perez-Galvez H Sies and W StahlldquoScreening pharmaceutical preparations containing extracts ofturmeric rhizome artichoke leaf devilrsquos claw root and garlic orsalmon oil for antioxidant capacityrdquo Journal of Pharmacy andPharmacology vol 55 no 7 pp 981ndash986 2003

[40] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[41] S Konyal120580oglu H Saglam and B K120580vcak ldquo120572-Tocopherolflavonoid and phenol contents and antioxidant activity of Ficuscarica leavesrdquo Pharmaceutical Biology vol 43 no 8 pp 683ndash686 2005

[42] D U Bawankule P Trivedi A Pal et al ldquoProtectivemechanismof lignans from Phyllanthus amarus against galactosaminelipopolysaccharide-induced hepatitis an in-vivo and in-silicostudiesrdquo Current Topics in Medicinal Chemistry vol 14 no 8pp 1045ndash1055 2014

[43] GAHiggs R J Flower and J RVane ldquoAnew approach to anti-inflammatory drugsrdquo Biochemical Pharmacology vol 28 no 12pp 1959ndash1961 1979

[44] A A Hemmati H Kalantari A Siahpoosh B Ghorbanzadehand H Jamali ldquoAnti-inflammatory Effect of HydroalcoholicExtract of the Washingtonia filifera Seeds in Carrageenan-Induced Paw Edema in Ratsrdquo Jundishapur Journal of NaturalPharmaceutical Products vol 10 no 1 pp 19ndash25 2015

[45] I Posadas M Bucci F Roviezzo et al ldquoCarrageenan-inducedmouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expressionrdquoBritish Journal of Pharmacology vol 142 no 2 pp 331ndash3382004

[46] K Pundarikakshudu D H Shah A H Panchal and G CBhavsar ldquoAnti-inflammatory activity of fenugreek (Trigonellafoenum-graecum Linn) seed petroleum ether extractrdquo IndianJournal of Pharmacology vol 48 no 4 pp 441ndash444 2016

[47] S Cuzzocrea G Costantino E Mazzon B Zingarelli A DeSarro and A P Caputi ldquoProtective effects of Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) a superoxide dismutasemimetic in paw oedema induced by carrageenan in the ratrdquoBiochemical Pharmacology vol 58 no 1 pp 171ndash176 1999

[48] M A Mendall P Patel M Asante et al ldquoRelation of serumcytokine concentrations to cardiovascular risk factors andcoronary heart diseaserdquo Heart vol 78 no 3 pp 273ndash277 1997

[49] A Ialenti A Ianaro S Moncada andM Di Rosa ldquoModulationof acute inflammation by endogenous nitric oxiderdquo EuropeanJournal of Pharmacology vol 211 no 2 pp 177ndash182 1992

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

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of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

Table 3 Quantification of total phenolic flavonoids and tannins contents of Cynara scolymus leaves extracts

Extracts Phenolics content(mg GAEg DW)

Flavonoidscontent

(mg CEg DW)

Tannins content(mg CEg DW)

Hexane 3991 plusmn 936 819 plusmn 016 1405 plusmn 03Ethyl acetate 5307 plusmn 047 1032 plusmn 012 1451 plusmn 013Butanol 4166 plusmn 223 1121 plusmn 010 1393 plusmn 93Ethanol 5454 plusmn 126 1200 plusmn 083 1099Aqueous 4949 plusmn 039 949 plusmn 039 438 plusmn 045Values are expressed as mean plusmn SD (119899 = 3)

Table 4 Mineral contents dried leaves of Cynara scolymus

Elements Cynara scolymus leaves (mg100 g of dry weightbasis)

K 2886803 plusmn 120Ca 1359346 plusmn 505Na 1762946 plusmn 120Mg 433219 plusmn 234I 16176 plusmn 014Mn 13051 plusmn 011Zn 7371 plusmn 014Copper 130 plusmn 016Cr 0124 plusmn 001Values are expressed as mean plusmn SD (119899 = 3)

that the induction of Carr in rats paw oedema started offby the vascular phase of inflammation which generated anincrease in the size of oedema for all groups This injectiongenerated intense inflammation which peaked after 3 hoursThe experimental data showed that ALE presented significantdecrease in the size of pawoedemawhichwas time dependentand more important than the reference group Indo and Carrgroup

As seen in Figures 3 and 4 EtOH extract at dose(400mgkgbw) has shown a significant percent of oedemainhibition (119901 lt 0001) in the first hour (173) comparedwith reference group (Indo) and the significant percent ofinhibition inflammation was 4427 in comparison with thecontrol group

Thus after five hours these percentages were 735 forinhibition and 832 for inflammation For the Indo groupoedema inhibition was significant (119901 lt 0001) 53 and 64and oedema inflammation was 21 and 86 in comparisonwith the control group

These results showed that EtOH extract of ALE had astrong effect as Indo

361 Inflammatory Biomarkers Fibrinogen and CRP Weassayed serum proteins of inflammatory markers in differentgroups of rats

(1) FibrinogenCarr group caused a significant increase in rateof fibrinogen compared to control group by 1905 (Figure 5)

1 2 3 4 5Temps (h)

0

10

20

30

40

50

60

70

80

in

hibi

tion

oede

ma

poundlowastlowastlowast

poundlowastlowastlowast

poundlowastlowastlowast

poundlowastlowastlowast

Carr + ALECarr + Indo

Figure 3 Percentage () of oedema inhibition data in all groupsValues represent mean plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt001 and lowastlowastlowast119901 lt 0001 m compared to Carr + Indo

0

10

20

30

40

50

60

70

in

hibi

tion

infla

mm

atio

n

1 2 3 4 5Temps (h)

NaclCarr

Carr + ALECarr + Indo

poundlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast poundlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

poundlowastlowastlowastlowastlowastlowastlowastlowastlowast

poundlowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

Figure 4 Percentage () of oedema inflammation data in allgroups Values represent mean plusmn SD (119899 = 6) in each group lowast119901 lt005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 120572 compared to control group120573 compared to Carr group and m compared to Indo group

The rate of fibrinogen decreased significantly (119901 lt 001) ingroups of rats treated with the EtOH extract (1655) andIndo (2290) compared with Carr group

Evidence-Based Complementary and Alternative Medicine 7

Table 5 Antioxidant activities of Cynara scolymus leaves extracts

Sample FRAP assay (umol Fe (II)gDW)

120573-Carotene bleaching assay()

TEAC assay (mmolTroloxg DW)

Hexane 223023 plusmn 1116lowastlowastlowast 3738 plusmn 524lowastlowastlowast 1043 plusmn 1073lowastlowastlowastEthyl acetate 50829 plusmn 524lowastlowastlowast 6156 plusmn 817lowast 38260 plusmn 524lowastlowastButanol 44306 plusmn 2298lowastlowastlowast 49393 plusmn 224lowastlowast 25193 plusmn 2815lowastlowastlowastEthanol 52779 plusmn 1626 70743 plusmn 129 49943 plusmn 3972Aqueous 31591 plusmn 836lowastlowastlowast 5611 plusmn 543lowastlowast 21074 plusmn 836lowastlowastlowastBHT mdash 4794 plusmn 075lowastlowastlowast mdashAA mdash 9059 plusmn 325lowastlowastlowast mdashValues are means plusmn SD (119899 = 3) Butylated Hydroxytoluene (BHT) and Ascorbic Acid (AA) were used as positive control compared with EtOH extract lowast119901 lt005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001

Nacl Carr Carr + ALE Carr + Indo0

05

1

15

2

25

Fg (gL)CRP (mgmL)

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

Figure 5 Levels of fibrinogen (Fg) and C-reactive protein (CRP)Values represent mean plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt001 and lowastlowastlowast119901 lt 0001 120572 compared to control 120573 compared to Carrgroup Fibrinogen (Fg) C-reactive protein (CRP)

(2) CRP Analysis showed a significant increase (119901 lt 005)in CRP level (Figure 5) for Carr group compared to controlgroup (1673) However the injection of EtOH extract andIndo led to a significant decrease (119901 lt 0001) (60863423) respectively when compared with Carr group

37 Oxidative Stress Parameters MDA and AOPP levels inoedema paw were illustrated in Table 8 Results showed thatCarr induced a significant increase inMDA andAOPP levelshowever treatment with EtOH extract of ALE at dose of400mgkgbw showed a significant decrease (119901 lt 0001)(2080 5023) compared with Carr group and even Indogroup (10mgkg) (2097 4427) respectively

38 Exploring the Antioxidant Enzymatic and NonenzymaticStatus CAT SOD and GSH levels in the paw oedema tissueof tested groups are shown in Table 8 Treatment of inflamedrats with EtOH extract restored significantly (119901 lt 0001)the SOD activity by 2075 the CAT activity by 5655 andthe GSH activity by 7566 respectively compared to Carrgroup

39 Histopathological Examination A microscopic study ofpaw oedema tissues showed histological changes in Carrgroup EtOH extract of ALE (400mgkgpc) group andIndo (10mgkgbw) group (Figure 6) In the Carr group weshowed a subcutaneous oedema with infiltration of inflam-matory cells especially polynuclear neutrophils at the site ofinflamed tissues and also the presence of spongy-like appear-ance in the epidermis ((Figure 6(b)) as compared to controlgroup (Figure 6(a))) The EtOH extract group produced asignificant decrease in the number of cellular infiltrates and asignificant reduced spongy-like appearance in the epidermis(Figure 6(c)) as did reference drug Indo (Figure 6(d))

310 Quantification of Polyphenolic Compounds by HPLCTo the authorsrsquo knowledge the present study identified andquantified the phenolic compounds of ALE from Tunisianorigins Accordingly the results obtained so far by Folin-Ciocalteu needed to be further complemented to qualifyphenolic constituents in EtOH extract under investigation

The findings revealed the presence of seven phenoliccompounds namely Hydroxytyrosol verbascoside apige-nin-7-glucoside Oleuropein Quercetin Pinoresinol andapigenin HPLC analysis indicated that ALE have significantamount of verbascoside content 381mg100 g and Quercetincontent (19 2mg100 g) (Figures 7 and 8 and Table 9)

311 Correlation between the Polyphenolic Compounds andAntioxidant Capacity In order to determine the contributionof the phenolic and flavonoid content in ALE on antiox-idant capacity the Pearson correlation coefficient (119903) wasdetermined in Table 6 The results showed a significantlinear correlation between antioxidant activities determinedby using DPPH ABTS FRAP and beta-carotene methodsand TPC and TFC respectively The strongest correlativevalue is obtained with DDPH and TPC (119903 = 087) Theseresults indicated a good correlation between TPC and TFCwith antioxidant activities of ALE

4 Discussion

The importance of plants in traditional medicine remediesand the potential of phytochemical constituents were dis-cussed nowadays with respect to their benefit in pharma-cotherapy in Tunisia Cynara scolymus was one of these

8 Evidence-Based Complementary and Alternative Medicine

(a)

(b)

(c)

(d)

Figure 6 Histopathological slides tissues of paw oedema in experimental groups of rats (a) Control group untreated group (b) Carr groupCarr-treated rat showed heavy infiltration of polynuclear neutrophils (PN) and a spongy-like appearance and bulla in the epidermis (c)Carr + EtOH extract group Carr-treated rat that received the EtOH extract (400mgkgbw) reduced significantly the migration of PNand oedematasis in dermis without any spongy-like feature and bulla (d) Carr + Indo group Carr-treated rat that received Indomethacin(10mgkgbw) showed a partial protective action Deparaffinized hematoxylin and eosin (HampE) stained sections (200ndash400x) Plus signinfiltration of PN Arrow odematasis in the epidermis

medicinal plants which have a beneficial potential effectattributed to its source of polyphenolic compounds [3]

The selection of this plant was guided by the indicationsof its traditional use that at present there have been very littlechemical and biological investigations done Therefore thepresent study was to investigate phytochemical compositionand their antioxidant capacity and to evaluate in vivo the anti-inflammatory effects of Cynara scolymus leaves extracts

The presence of phenolic compounds is very widelydistributed in medicinal plants several studies showed thatthese compounds have drawn much attention to their poten-tial antioxidant abilities which demonstrated their beneficialimplications for human health

Regarding the determination of phenolic compoundsthe EtOH extract of ALE had the highest amount of thesecompounds in comparison with other extracts which werean agreement with the result of Emanue et al [33] whoshows that EtOH extract exhibited the maximum amountof phenolic compounds from the leaves of Cynara scolymuswhereas it differs from the reports of Oliveira et al [34]who proves that aqueous extract of ALE is the most suitablesolvent for extraction of phenolic compounds

The importance of TPC and TFC extraction yieldsobtained with EtOH extract can be attributed to its goodsolubility low toxicity medium polarity and high extractioncapacity [35]

Evidence-Based Complementary and Alternative Medicine 9

1 2 3 678 9 10

5

4

0250500750

1000125015001750

(mAU

)

5 10 15 20 250(min)

Figure 7 HPLC chromatogram of a standard mixture of polyphe-nolic compounds Peaks 1 Hydroxytyrosol 2 Tyrosol 3 4-hydroxybenzoic acid 4 verbascoside 5 apigenin-7-glucoside 6Oleuropein 7 Quercetin 8 Pinoresinol 9 cinnamic acid 10apigenin

34

2

56 7

0200400600800

100012001400

(mAU

)

5 10 15 20 25 30 350(min)

1

Figure 8 HPLC chromatogram of ethanol extract of Cynarascolymus leaves extracts Peaks 1 Hydroxytyrosol 2 verbascoside3 apigenin-7-glucoside 4 Oleuropein 5 Quercetin 6 Pinoresinol7 apigenin

Table 6 Pearson correlation coefficient (119903) between the content ofphenolic compounds and antioxidant capacity (DDPH∙ ABTS∙+FRAP and beta-carotene)

Correlation 119877TPC versus DDPH∙ 0870lowastlowast

TPC versus ABTS∙+ 0848lowastlowast

TPC versus FRAP 0707lowastlowast

TPC versus beta-carotene 0842lowastlowast

TFC versus DDPH∙ 0728lowastlowast

TFC versus ABTS∙+ 0743lowastlowast

TFC versus FRAP 0849lowastlowast

TFC versus beta-carotene 0712lowastlowast

DPPH 22-diphenyl-1-picrylhydrazyl ABTS 220-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt FRAP ferricreducing TPC total phenolic content TFC total flavonoid contentlowastlowast119901 lt 001 significant correlation

In the first investigation of my study five solvents ofincreasing polarities were chosen for the determination ofphenolic compounds from leaves of C scolymus namelyhexane ethyl acetate butanol 75 EtOHH2O and aqueousin order to determine the variability of TPC and TFC in theaerial part of Cynara as a function of each extraction solvent

This variability of phenolic compounds in ALE can beattributed to the wide range of solubility displayed by variouspolar compounds within the ALE solvents the degree ofpolymerization of phenols and their interaction geneticfactors geographical variations and climatic changes [36]

Overall these findings indicate that EtOH extract of ALEwas rich in phenolic and flavonoid contents which couldbe the major contributor to their antioxidative propertiesMany researches revealed that flavonoids and polyphenolsdisplayed the highest ability of scavenging activity in medic-inal plants [37 38]

The results indicate that EtOH extract of ALE whichcontains the highest content of TFC and TPC displays thehighest free radical scavenging activity (9423) at concen-tration of 400 ugmL However Oliveira et al [34] show thataqueous extract of ALE has good radical scavenging (8340)at concentration of 200 ugmL Furthermore our resultssuggest that some components within EtOH extract aresignificantly the strongest radical scavenger in comparisonwith other extracts The TEAC values of ALE showed alsothat EtOH extract presents a potential ability to scavengethe ABTS radical cation in accordance with the report ofBetancor-Fernandez et al [39]

The FRAP assay shows also that EtOH extract has thehighest values (54262 umol Fe II gDW) The same resultis confirmed by Kukic et al [40] Therefore the results ofpositive correlation between the TPC TFC and the anti-oxidant methods in vitro suggest that phenolic compoundsact as reducing agents hydrogen donors and singlet oxygenscavengers [3] and may exert an important in vitro antioxi-dant capacity of ALE

The phytochemical screening of ALE has been foundto be a rich source of polyphenolic compounds includingQuercetin apigenin-7-glucoside and verbascoside Thesecompounds have shown a great potential of antioxidantactivity [41]

The research into medicinal plants used as pain relieversrsquoagents should therefore be viewed as new therapy in theinflammatory diseases [42] The anti-inflammatory activitiesof EtOH extract of ALE are investigated applying in vivoexperimental Carr model compared with a nonsteroidal anti-inflammatory (Indomethacin) this drug was reported byHiggs et al [43] who demonstrated the role of Indo toinhibit the biosynthesis of prostaglandins in Carr model ratsThe experimental study of anti-inflammatory activity is per-formed by the Carr test This phlogiston agent inducedtissue oedema characteristic of acute inflammation whichis regarded as a crucial parameter in assessing anti-inflam-matory activity [44]

The Carr injection produces biphasic states in the firstphase [45] there is an increase in the mRNA synthesis ofcyclooxygenase-2 (COX-2) at the first hour This increase isaccompanied by an amplification of synthesis of strong proin-flammatory mediator such as prostaglandin especially theprostaglandin type 2 involved in the inflammatory process[46] serotonin bradykinin and leukotrienes which con-tribute to the initiation of inflammation reaction Moreoverthe participation of arachidonicmetabolites is themain factorresponsible for both of phases of Carr induced inflammation

10 Evidence-Based Complementary and Alternative Medicine

Table7Eff

ecto

fCynarascolym

usleaves

extracto

ncarrageenan-indu

cedratp

awoedema

Treatm

ent

Oedem

asize(meanplusmnS

D)(mm)

0h1h

2h3h

4h5h

Con

trol

0426plusmn0

00

044

1plusmn001

056plusmn0

03

0650plusmn0

05

0755plusmn0

05

070plusmn0

01

Carr

0426plusmn0

00

28plusmn026120572lowastlowastlowast

333plusmn028120572lowastlowastlowast

325plusmn002120572lowastlowastlowast

315plusmn001120572lowastlowastlowast

302plusmn009120572lowastlowastlowast

Carr+Indo

(10m

gkg)

0426plusmn0

00

246plusmn026120572lowastlowastlowast120573lowast

261plusmn007120572lowastlowastlowast120573lowastlowastlowast

230plusmn002120572lowastlowastlowast120573lowastlowastlowast

18plusmn011120572lowastlowastlowast120573lowastlowastlowast

14plusmn006120572lowastlowastlowast120573lowastlowastlowast

Carr+ALE

(400

mgkgbw)

0426plusmn0

00

2480plusmn005120572lowastlowastlowast120573lowastlowastlowast

188plusmn004120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

165plusmn005120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

116plusmn020120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

112plusmn01120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

Values

representm

eansplusmnSD

(119899=6)ineach

grou

plowast119901lt005lowastlowast119901lt001andlowastlowastlowast119901lt0001

120572com

paredto

control120573com

paredto

Carrmcom

paredto

Carr+Indo

Evidence-Based Complementary and Alternative Medicine 11

Table 8 Effects of Cynara scolymus leaves extract and Indomethacin on CAT SOD GSH AOPP andMDA activities in carrageenan inducedpaw oedema

GroupsCAT (120583molesCATminmg

protein)

SOD (unitSODminmg

protein)

GSH (nmolesmgprotein)

MDA (nmolMDAmg protein) AOPP (nmolmg protein)

Control 47 plusmn 2 131 plusmn 43 421 plusmn 12 234 plusmn 5 484 plusmn 43Carr 162 plusmn 05120572lowastlowastlowast 153 plusmn 26120572lowastlowastlowast 131 plusmn 11120572lowastlowastlowast 314 plusmn 75120572lowastlowastlowast 679 plusmn 69120572lowastlowastlowastCarr + Indo(10mgkgbw) 649 plusmn 71120573lowastlowastlowast 240 plusmn 13120573lowastlowastlowast 703 plusmn 11120573lowastlowastlowast 209 plusmn 30120573lowastlowastlowast 442 plusmn 06120573lowastlowastlowastCarr + ALE(400mgkgbw) 565 plusmn 25120573lowastlowastlowastmlowastlowast 207 plusmn 40120573lowastlowastlowastmlowastlowast 756 plusmn 25120573lowastlowastlowastmlowast 208 plusmn 60120573lowastlowastlowast 502 plusmn 41120573lowastlowastlowastmlowastValues represent means plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001120572 compared to control 120573 compared to Carr m compared to Carr + IndoSOD superoxide dismutaseCAT catalaseGSH glutathione peroxidaseMDA MalondialdehydeAOPP Advanced Oxidation Protein Product

Table 9 Quantification and identification of phenolic compounds contents in the ethanol extract of Cynara scolymus leaves extracts

Number Content (mg100 g of dry extract) Polyphenolic compound1 28 plusmn 019 Hydroxytyrosol2 381 plusmn 036 Verbascoside3 99 plusmn 002 Apigenin-7-glucoside4 18 plusmn 01 Oleuropein5 192 plusmn 014 Quercetin6 5 plusmn 005 Pinoresinol7 4 plusmn 004 ApigeninValues represent means plusmn SD (119899 = 3)

In the second phase the increase of vascular permeabilityis observed by release of kinins during 230 hThereafter from230 h to 5 h inflammation ismediated by prostaglandins andis also associated with a release infiltration and migration ofPN into the inflamed site [47]

Our results indicate that ALE affords protection againstthe Carr induced acute inflammation in dose dependentmanner EtOH extract of ALE at dose of 400mgkgbwexhibits significant anti-inflammatory activity with 73 inhi-bition of paw oedema compared with Indo group (53) andtypically reaches a maximum at 5 h these results obtainedsuggest the anti-inflammatory effect of EtOH extract of ALEbymeans of inhibiting the synthesis and the release of inflam-matory mediators like histamine serotonin and prostagl-andins that are involved in acute inflammationThese resultsrevealed the inhibitory effect of EtOH extract on PN migra-tion and it is confirmed through a histological analysis oftissues of paw oedema in experimental groups

These findings clearly confirm that EtOH extract of ALEhas an anti-inflammatory effect by reducing the influx ofpolymorphonuclear cells to inflammatory tissue followinginjection of Carr Most inflammatory markers such asinterleukin-6 (IL-6) tumor necrosis factor-120572 (TNF-120572) CRPand fibrinogen increased significantly in response to infec-tion and in active diseases states CRP is the specific marker

of acute inflammation occurring in the body [48] moreoverthe fibrinogen is themost relevant indicator of inflammationand it does not only represent the acute-phase reactant that isincreased in inflammatory states Elevated fibrinogen levelswould be associated with higher levels of CRP and wouldsimilarly correlate with inflammation reaction

Therefore the present study showed that EtOH extract ofALE significantly decreases the level of fibrinogen and CRPby value of 2272 and 3478 in comparison with Carrgroup

The induction of inflammationwithCarr ismanifested bygeneration of ROS and has been shown to play an importantrole in various forms of inflammation [49 50] High concen-tration of reactive free radicals contributes to lipid perox-idation and protein oxidation [3] Our results showed thatthere is a significant increase in MDA and AOPP activitiesin Carr group when compared to control group and a highlysignificant decrease in activities of SOD CAT and GSH alsoobserved at the tissue level compared with the control group(119901 lt 0001) However the injection of EtOH extract and Indoshows a significant decrease of the levels of MDA and AOPPby 3427 and 4427 following a significant increase inantioxidant activities as CAT SOD andGSHwhen comparedto Carr group (2016 7330 and 75) From these resultsit is implied that the protective effect of EtOH extract may

12 Evidence-Based Complementary and Alternative Medicine

be attributed to its potential indirectly as a stimulator ofthe activity and expression of antioxidant enzymes duringinflammation

Thus the anti-inflammatory activity is due to the individ-ual or synergistic effect of the components in the ALE In factprevious studies have found that anti-inflammatory profile ofALE could be related to polyphenolic compounds accordingto the phytochemical analysis We performed HPLC analysisto justify the correlation between phytochemical compoundsand anti-inflammatory activity of this plant Among thesepolyphenolic compounds verbascoside has been reportedin several reports and showed a potential spectrum ofmany activities including antioxidant and anti-inflammatoryParticular verbascoside presents antiedematogenic activitiesin animal models of Carr induced inflammation [51] and theresponses exhibited themaximumactivity for the fourth hourof treatment Such activities may be derived from inhibitoryaction of chemical mediators of the inflammatory processsuch as histamine and bradykinin A significant antioxidanteffect of verbascoside has been recently reported byAleo et al[52] in an experimental study [53] Other authors reportedthat anti-inflammatory activity of verbascoside has beenevaluated by an in vitro test performed on cell cultures of pri-mary human keratinocytes [54] in which it was able to reducethe release of proinflammatory markers such as chemokines

Apigenin-7-glucoside was found to block the releaseof several varieties of enzymes involved in inflammationincluding especially lipoxygenases and cyclooxygenases [5556] leading to inhibition of proinflammatory molecules NF-kB activation and inhibits neutrophil infiltration in tissues

From these results we suggest that anti-inflammatoryactivity observed is due to a synergic action of these phenoliccomponents contained in EtOH extract of ALE

C scolymus is one of the few herbal remedies which havebeen verified through experimental studies Further the toxi-city studies of ALE are required in order to confirm the safetyof this medicinal plant in the treatment of inflammationdiseases

5 Conclusion

The present study showed clearly the advantages of Cscolymus leaves extracts which have safer anti-inflammatoryprofile with potent antioxidant activity attributed to thephenolic compounds Furthermore in the future studieswe are interested to characterize the action mechanisms ofactive phenolic compounds of ALE responsible for anti-inflammatory and antioxidants activities

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Tunisian Ministry ofHigher Education and Scientific Research

References

[1] C Pasquier ldquoStress oxidative et inflammationrdquo Blool vol 4 pp1ndash6 1996

[2] M A Abuashwashi O M Palomino and M P Gomez-Serranillos ldquoGeographic origin influences the phenolic com-position and antioxidant potential of wild Crataegus monogynafrom Spainrdquo Pharmaceutical Biology vol 54 no 11 pp 2708ndash2713 2016

[3] M B Salem H Affes K Ksouda et al ldquoPharmacological stud-ies of artichoke leaf extract and their health benefitsrdquo PlantFoods for Human Nutrition vol 70 no 4 pp 441ndash453 2015

[4] M-H Huang B-S Wang C-S Chiu et al ldquoAntioxidant anti-nociceptive and anti-inflammatory activities ofXanthii Fructusextractrdquo Journal of Ethnopharmacology vol 135 no 2 pp 545ndash552 2011

[5] G-J Huang S-S Huang S-S Lin et al ldquoAnalgesic effectsand the mechanisms of anti-inflammation of ergostatrien-3120573-ol from antrodia camphorata submerged whole broth in micerdquoJournal of Agricultural and Food Chemistry vol 58 no 12 pp7445ndash7452 2010

[6] C Tohda N Nakayama F Hatanaka and K Komatsu ldquoCom-parison of anti-inflammatory activities of six Curcuma rhi-zomes a possible curcuminoid-independent pathway mediatedby Curcuma phaeocaulis extractrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 3 no 2 pp 255ndash260 2006

[7] K Goksel K Murat Y Murat E Sevil and H Fidan ldquoAntiox-idant activities of ethanol extracts ofHypericum triquetrifoliumandHypericum scabroidesrdquo Pharmaceutical Biology vol 46 no4 pp 231ndash242 2008

[8] R Gebhardt and M Fausel ldquoAntioxidant and hepatoprotectiveeffects of artichoke extracts and constituents in cultured rathepatocytesrdquoToxicology inVitro vol 11 no 5 pp 669ndash672 1997

[9] A SofworaMedicinal Plants and TraditionalMedicine in AfricaJohn Wiley amp Sons Ann Arbor Mich USA 1982

[10] D E Okwu ldquoPhytochemicals vitamins andmineral contents oftwo Nigerian medicinal plantsrdquo International Journal of Mole-cular Medicine and Advances Sciences vol 1 pp 375ndash381 2005

[11] N Tsevegsuren GDavaakhuu andT S Udval ldquoPhytochemicalanalysis of Cynara scolymus L cultivated in MongoliardquoMongo-lian Journal of Chemistry vol 15 no 41 pp 40ndash42 2014

[12] AACC Approved Methods of the AACC American Associationof Cereal Chemists St Paul Minn USA 8th edition 1984

[13] S C Lee L Prosky and J W DeVries ldquoDetermination of totalsoluble and insoluble dietary fiber in foodsmdashenzymatic-gravi-metric method MES-TRIS buffer collaborative studyrdquo Journalof the Association of Official Analytical Chemists vol 75 pp395ndash416 1992

[14] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[15] O A Fawole U L Opara and K I Theron ldquoChemical andphytochemical properties and antioxidant activities of threepomegranate cultivars grown in South Africardquo Food and Bio-process Technology vol 5 no 7 pp 2934ndash2940 2012

[16] V Dewanto X Wu K K Adom and R H Liu ldquoThermalprocessing enhances the nutritional value of tomatoes byincreasing total antioxidant activityrdquo Journal of Agricultural andFood Chemistry vol 50 no 10 pp 3010ndash3014 2002

[17] B Sun J M Ricardo-da-Silva and I Spranger ldquoCritical factorsof vanillin assay for catechins and proanthocyanidinsrdquo Journal

Evidence-Based Complementary and Alternative Medicine 13

of Agricultural and Food Chemistry vol 46 no 10 pp 4267ndash4274 1998

[18] AOACOfficial Methods of Analysis vol 1 AOACWashingtonDC USA 15th edition 1990

[19] D Pljevljakusic K Savikin T Jankovic et al ldquoChemical prop-erties of the cultivated Sideritis raeseri Boiss amp Heldr subspraeserirdquo Food Chemistry vol 124 no 1 pp 226ndash233 2011

[20] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[21] I I Koleva T A Van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] V Ravi T S M Saleem S S Patel J Raamamurthy and KGauthaman ldquoAnti-inflammatory effect of methanolic extract ofSolanum nigrum Linn Berriesrdquo International Journal of AppliedResearch in Natural Products vol 2 pp 33ndash36 2009

[24] D Chattopadhyay G Arunachalam A B Mandal T K SurS C Mandal and S K Bhattacharya ldquoAntimicrobial and anti-inflammatory activity of folkloreMallotus peltatus leaf extractrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 229ndash2372002

[25] L R Morse K Stolzmann H P Nguyen et al ldquoAssociationbetween mobility mode and c-reactive protein levels in menwith chronic spinal cord injuryrdquo Archives of Physical Medicineand Rehabilitation vol 89 no 4 pp 726ndash731 2008

[26] A Clauss ldquoGerinnungsphysiologische schnellmethode zur bes-timmung des fibrinogensrdquoActaHaematologica vol 17 no 4 pp237ndash246 1957

[27] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[28] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidationin post-mitotic tissues of ageing ratrdquo Cell Biochemistry andFunction vol 24 no 1 pp 79ndash85 2006

[29] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[30] W F Beyer Jr and I Fridovich ldquoAssaying for superoxide dis-mutase activity some large consequences of minor changes inconditionsrdquoAnalytical Biochemistry vol 161 no 2 pp 559ndash5661987

[31] I Carlberg and B Mannervik ldquoGlutathione reductaserdquo inGlutamate Glutamine Glutathione and Related Compoundsvol 113 ofMethods in Enzymology pp 484ndash490 Elsevier 1985

[32] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[33] V Emanue V Adrian N A Sultana and C Svetlana ldquoAntiox-idant and antimicrobial activities of ethanol extracts of Cynarascolymus (Cynarae folium Asteraceae family)rdquo Tropical Journalof Pharmaceutical Research vol 10 no 6 pp 777ndash783 2011

[34] G L D S Oliveira R A M O Francisco O B A Vinıcius etal ldquoEvaluation of antioxidant capacity of the aqueous extract ofCynara scolymus L (Asteraceae) in vitro and in SaccharomycescerevisiaerdquoAfrican Journal of Pharmacy and Pharmacology vol8 pp 136ndash147 2014

[35] K Horiuchi S Shiota T Hatano T Yoshida T Kuroda and TTsuchiya ldquoAntimicrobial activity of oleanolic acid from Salviaofficinalis and related compounds on vancomycin-resistantenterococci (VRE)rdquo Biological and Pharmaceutical Bulletin vol30 no 6 pp 1147ndash1149 2007

[36] A Daoud D Malika S Bakari et al ldquoAssessment of polyphe-nol composition antioxidant and antimicrobial properties ofvarious extracts of Date Palm Pollen (DPP) from two Tunisiancultivarsrdquo Arabian Journal of Chemistry 2015

[37] D Krishnaiah B Awang S Rosalam and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversitymdashEngineering Sciences vol 1 pp 63ndash66 2015

[38] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[39] A Betancor-Fernandez A Perez-Galvez H Sies and W StahlldquoScreening pharmaceutical preparations containing extracts ofturmeric rhizome artichoke leaf devilrsquos claw root and garlic orsalmon oil for antioxidant capacityrdquo Journal of Pharmacy andPharmacology vol 55 no 7 pp 981ndash986 2003

[40] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[41] S Konyal120580oglu H Saglam and B K120580vcak ldquo120572-Tocopherolflavonoid and phenol contents and antioxidant activity of Ficuscarica leavesrdquo Pharmaceutical Biology vol 43 no 8 pp 683ndash686 2005

[42] D U Bawankule P Trivedi A Pal et al ldquoProtectivemechanismof lignans from Phyllanthus amarus against galactosaminelipopolysaccharide-induced hepatitis an in-vivo and in-silicostudiesrdquo Current Topics in Medicinal Chemistry vol 14 no 8pp 1045ndash1055 2014

[43] GAHiggs R J Flower and J RVane ldquoAnew approach to anti-inflammatory drugsrdquo Biochemical Pharmacology vol 28 no 12pp 1959ndash1961 1979

[44] A A Hemmati H Kalantari A Siahpoosh B Ghorbanzadehand H Jamali ldquoAnti-inflammatory Effect of HydroalcoholicExtract of the Washingtonia filifera Seeds in Carrageenan-Induced Paw Edema in Ratsrdquo Jundishapur Journal of NaturalPharmaceutical Products vol 10 no 1 pp 19ndash25 2015

[45] I Posadas M Bucci F Roviezzo et al ldquoCarrageenan-inducedmouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expressionrdquoBritish Journal of Pharmacology vol 142 no 2 pp 331ndash3382004

[46] K Pundarikakshudu D H Shah A H Panchal and G CBhavsar ldquoAnti-inflammatory activity of fenugreek (Trigonellafoenum-graecum Linn) seed petroleum ether extractrdquo IndianJournal of Pharmacology vol 48 no 4 pp 441ndash444 2016

[47] S Cuzzocrea G Costantino E Mazzon B Zingarelli A DeSarro and A P Caputi ldquoProtective effects of Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) a superoxide dismutasemimetic in paw oedema induced by carrageenan in the ratrdquoBiochemical Pharmacology vol 58 no 1 pp 171ndash176 1999

[48] M A Mendall P Patel M Asante et al ldquoRelation of serumcytokine concentrations to cardiovascular risk factors andcoronary heart diseaserdquo Heart vol 78 no 3 pp 273ndash277 1997

[49] A Ialenti A Ianaro S Moncada andM Di Rosa ldquoModulationof acute inflammation by endogenous nitric oxiderdquo EuropeanJournal of Pharmacology vol 211 no 2 pp 177ndash182 1992

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

Table 5 Antioxidant activities of Cynara scolymus leaves extracts

Sample FRAP assay (umol Fe (II)gDW)

120573-Carotene bleaching assay()

TEAC assay (mmolTroloxg DW)

Hexane 223023 plusmn 1116lowastlowastlowast 3738 plusmn 524lowastlowastlowast 1043 plusmn 1073lowastlowastlowastEthyl acetate 50829 plusmn 524lowastlowastlowast 6156 plusmn 817lowast 38260 plusmn 524lowastlowastButanol 44306 plusmn 2298lowastlowastlowast 49393 plusmn 224lowastlowast 25193 plusmn 2815lowastlowastlowastEthanol 52779 plusmn 1626 70743 plusmn 129 49943 plusmn 3972Aqueous 31591 plusmn 836lowastlowastlowast 5611 plusmn 543lowastlowast 21074 plusmn 836lowastlowastlowastBHT mdash 4794 plusmn 075lowastlowastlowast mdashAA mdash 9059 plusmn 325lowastlowastlowast mdashValues are means plusmn SD (119899 = 3) Butylated Hydroxytoluene (BHT) and Ascorbic Acid (AA) were used as positive control compared with EtOH extract lowast119901 lt005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001

Nacl Carr Carr + ALE Carr + Indo0

05

1

15

2

25

Fg (gL)CRP (mgmL)

lowast

lowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

Figure 5 Levels of fibrinogen (Fg) and C-reactive protein (CRP)Values represent mean plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt001 and lowastlowastlowast119901 lt 0001 120572 compared to control 120573 compared to Carrgroup Fibrinogen (Fg) C-reactive protein (CRP)

(2) CRP Analysis showed a significant increase (119901 lt 005)in CRP level (Figure 5) for Carr group compared to controlgroup (1673) However the injection of EtOH extract andIndo led to a significant decrease (119901 lt 0001) (60863423) respectively when compared with Carr group

37 Oxidative Stress Parameters MDA and AOPP levels inoedema paw were illustrated in Table 8 Results showed thatCarr induced a significant increase inMDA andAOPP levelshowever treatment with EtOH extract of ALE at dose of400mgkgbw showed a significant decrease (119901 lt 0001)(2080 5023) compared with Carr group and even Indogroup (10mgkg) (2097 4427) respectively

38 Exploring the Antioxidant Enzymatic and NonenzymaticStatus CAT SOD and GSH levels in the paw oedema tissueof tested groups are shown in Table 8 Treatment of inflamedrats with EtOH extract restored significantly (119901 lt 0001)the SOD activity by 2075 the CAT activity by 5655 andthe GSH activity by 7566 respectively compared to Carrgroup

39 Histopathological Examination A microscopic study ofpaw oedema tissues showed histological changes in Carrgroup EtOH extract of ALE (400mgkgpc) group andIndo (10mgkgbw) group (Figure 6) In the Carr group weshowed a subcutaneous oedema with infiltration of inflam-matory cells especially polynuclear neutrophils at the site ofinflamed tissues and also the presence of spongy-like appear-ance in the epidermis ((Figure 6(b)) as compared to controlgroup (Figure 6(a))) The EtOH extract group produced asignificant decrease in the number of cellular infiltrates and asignificant reduced spongy-like appearance in the epidermis(Figure 6(c)) as did reference drug Indo (Figure 6(d))

310 Quantification of Polyphenolic Compounds by HPLCTo the authorsrsquo knowledge the present study identified andquantified the phenolic compounds of ALE from Tunisianorigins Accordingly the results obtained so far by Folin-Ciocalteu needed to be further complemented to qualifyphenolic constituents in EtOH extract under investigation

The findings revealed the presence of seven phenoliccompounds namely Hydroxytyrosol verbascoside apige-nin-7-glucoside Oleuropein Quercetin Pinoresinol andapigenin HPLC analysis indicated that ALE have significantamount of verbascoside content 381mg100 g and Quercetincontent (19 2mg100 g) (Figures 7 and 8 and Table 9)

311 Correlation between the Polyphenolic Compounds andAntioxidant Capacity In order to determine the contributionof the phenolic and flavonoid content in ALE on antiox-idant capacity the Pearson correlation coefficient (119903) wasdetermined in Table 6 The results showed a significantlinear correlation between antioxidant activities determinedby using DPPH ABTS FRAP and beta-carotene methodsand TPC and TFC respectively The strongest correlativevalue is obtained with DDPH and TPC (119903 = 087) Theseresults indicated a good correlation between TPC and TFCwith antioxidant activities of ALE

4 Discussion

The importance of plants in traditional medicine remediesand the potential of phytochemical constituents were dis-cussed nowadays with respect to their benefit in pharma-cotherapy in Tunisia Cynara scolymus was one of these

8 Evidence-Based Complementary and Alternative Medicine

(a)

(b)

(c)

(d)

Figure 6 Histopathological slides tissues of paw oedema in experimental groups of rats (a) Control group untreated group (b) Carr groupCarr-treated rat showed heavy infiltration of polynuclear neutrophils (PN) and a spongy-like appearance and bulla in the epidermis (c)Carr + EtOH extract group Carr-treated rat that received the EtOH extract (400mgkgbw) reduced significantly the migration of PNand oedematasis in dermis without any spongy-like feature and bulla (d) Carr + Indo group Carr-treated rat that received Indomethacin(10mgkgbw) showed a partial protective action Deparaffinized hematoxylin and eosin (HampE) stained sections (200ndash400x) Plus signinfiltration of PN Arrow odematasis in the epidermis

medicinal plants which have a beneficial potential effectattributed to its source of polyphenolic compounds [3]

The selection of this plant was guided by the indicationsof its traditional use that at present there have been very littlechemical and biological investigations done Therefore thepresent study was to investigate phytochemical compositionand their antioxidant capacity and to evaluate in vivo the anti-inflammatory effects of Cynara scolymus leaves extracts

The presence of phenolic compounds is very widelydistributed in medicinal plants several studies showed thatthese compounds have drawn much attention to their poten-tial antioxidant abilities which demonstrated their beneficialimplications for human health

Regarding the determination of phenolic compoundsthe EtOH extract of ALE had the highest amount of thesecompounds in comparison with other extracts which werean agreement with the result of Emanue et al [33] whoshows that EtOH extract exhibited the maximum amountof phenolic compounds from the leaves of Cynara scolymuswhereas it differs from the reports of Oliveira et al [34]who proves that aqueous extract of ALE is the most suitablesolvent for extraction of phenolic compounds

The importance of TPC and TFC extraction yieldsobtained with EtOH extract can be attributed to its goodsolubility low toxicity medium polarity and high extractioncapacity [35]

Evidence-Based Complementary and Alternative Medicine 9

1 2 3 678 9 10

5

4

0250500750

1000125015001750

(mAU

)

5 10 15 20 250(min)

Figure 7 HPLC chromatogram of a standard mixture of polyphe-nolic compounds Peaks 1 Hydroxytyrosol 2 Tyrosol 3 4-hydroxybenzoic acid 4 verbascoside 5 apigenin-7-glucoside 6Oleuropein 7 Quercetin 8 Pinoresinol 9 cinnamic acid 10apigenin

34

2

56 7

0200400600800

100012001400

(mAU

)

5 10 15 20 25 30 350(min)

1

Figure 8 HPLC chromatogram of ethanol extract of Cynarascolymus leaves extracts Peaks 1 Hydroxytyrosol 2 verbascoside3 apigenin-7-glucoside 4 Oleuropein 5 Quercetin 6 Pinoresinol7 apigenin

Table 6 Pearson correlation coefficient (119903) between the content ofphenolic compounds and antioxidant capacity (DDPH∙ ABTS∙+FRAP and beta-carotene)

Correlation 119877TPC versus DDPH∙ 0870lowastlowast

TPC versus ABTS∙+ 0848lowastlowast

TPC versus FRAP 0707lowastlowast

TPC versus beta-carotene 0842lowastlowast

TFC versus DDPH∙ 0728lowastlowast

TFC versus ABTS∙+ 0743lowastlowast

TFC versus FRAP 0849lowastlowast

TFC versus beta-carotene 0712lowastlowast

DPPH 22-diphenyl-1-picrylhydrazyl ABTS 220-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt FRAP ferricreducing TPC total phenolic content TFC total flavonoid contentlowastlowast119901 lt 001 significant correlation

In the first investigation of my study five solvents ofincreasing polarities were chosen for the determination ofphenolic compounds from leaves of C scolymus namelyhexane ethyl acetate butanol 75 EtOHH2O and aqueousin order to determine the variability of TPC and TFC in theaerial part of Cynara as a function of each extraction solvent

This variability of phenolic compounds in ALE can beattributed to the wide range of solubility displayed by variouspolar compounds within the ALE solvents the degree ofpolymerization of phenols and their interaction geneticfactors geographical variations and climatic changes [36]

Overall these findings indicate that EtOH extract of ALEwas rich in phenolic and flavonoid contents which couldbe the major contributor to their antioxidative propertiesMany researches revealed that flavonoids and polyphenolsdisplayed the highest ability of scavenging activity in medic-inal plants [37 38]

The results indicate that EtOH extract of ALE whichcontains the highest content of TFC and TPC displays thehighest free radical scavenging activity (9423) at concen-tration of 400 ugmL However Oliveira et al [34] show thataqueous extract of ALE has good radical scavenging (8340)at concentration of 200 ugmL Furthermore our resultssuggest that some components within EtOH extract aresignificantly the strongest radical scavenger in comparisonwith other extracts The TEAC values of ALE showed alsothat EtOH extract presents a potential ability to scavengethe ABTS radical cation in accordance with the report ofBetancor-Fernandez et al [39]

The FRAP assay shows also that EtOH extract has thehighest values (54262 umol Fe II gDW) The same resultis confirmed by Kukic et al [40] Therefore the results ofpositive correlation between the TPC TFC and the anti-oxidant methods in vitro suggest that phenolic compoundsact as reducing agents hydrogen donors and singlet oxygenscavengers [3] and may exert an important in vitro antioxi-dant capacity of ALE

The phytochemical screening of ALE has been foundto be a rich source of polyphenolic compounds includingQuercetin apigenin-7-glucoside and verbascoside Thesecompounds have shown a great potential of antioxidantactivity [41]

The research into medicinal plants used as pain relieversrsquoagents should therefore be viewed as new therapy in theinflammatory diseases [42] The anti-inflammatory activitiesof EtOH extract of ALE are investigated applying in vivoexperimental Carr model compared with a nonsteroidal anti-inflammatory (Indomethacin) this drug was reported byHiggs et al [43] who demonstrated the role of Indo toinhibit the biosynthesis of prostaglandins in Carr model ratsThe experimental study of anti-inflammatory activity is per-formed by the Carr test This phlogiston agent inducedtissue oedema characteristic of acute inflammation whichis regarded as a crucial parameter in assessing anti-inflam-matory activity [44]

The Carr injection produces biphasic states in the firstphase [45] there is an increase in the mRNA synthesis ofcyclooxygenase-2 (COX-2) at the first hour This increase isaccompanied by an amplification of synthesis of strong proin-flammatory mediator such as prostaglandin especially theprostaglandin type 2 involved in the inflammatory process[46] serotonin bradykinin and leukotrienes which con-tribute to the initiation of inflammation reaction Moreoverthe participation of arachidonicmetabolites is themain factorresponsible for both of phases of Carr induced inflammation

10 Evidence-Based Complementary and Alternative Medicine

Table7Eff

ecto

fCynarascolym

usleaves

extracto

ncarrageenan-indu

cedratp

awoedema

Treatm

ent

Oedem

asize(meanplusmnS

D)(mm)

0h1h

2h3h

4h5h

Con

trol

0426plusmn0

00

044

1plusmn001

056plusmn0

03

0650plusmn0

05

0755plusmn0

05

070plusmn0

01

Carr

0426plusmn0

00

28plusmn026120572lowastlowastlowast

333plusmn028120572lowastlowastlowast

325plusmn002120572lowastlowastlowast

315plusmn001120572lowastlowastlowast

302plusmn009120572lowastlowastlowast

Carr+Indo

(10m

gkg)

0426plusmn0

00

246plusmn026120572lowastlowastlowast120573lowast

261plusmn007120572lowastlowastlowast120573lowastlowastlowast

230plusmn002120572lowastlowastlowast120573lowastlowastlowast

18plusmn011120572lowastlowastlowast120573lowastlowastlowast

14plusmn006120572lowastlowastlowast120573lowastlowastlowast

Carr+ALE

(400

mgkgbw)

0426plusmn0

00

2480plusmn005120572lowastlowastlowast120573lowastlowastlowast

188plusmn004120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

165plusmn005120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

116plusmn020120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

112plusmn01120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

Values

representm

eansplusmnSD

(119899=6)ineach

grou

plowast119901lt005lowastlowast119901lt001andlowastlowastlowast119901lt0001

120572com

paredto

control120573com

paredto

Carrmcom

paredto

Carr+Indo

Evidence-Based Complementary and Alternative Medicine 11

Table 8 Effects of Cynara scolymus leaves extract and Indomethacin on CAT SOD GSH AOPP andMDA activities in carrageenan inducedpaw oedema

GroupsCAT (120583molesCATminmg

protein)

SOD (unitSODminmg

protein)

GSH (nmolesmgprotein)

MDA (nmolMDAmg protein) AOPP (nmolmg protein)

Control 47 plusmn 2 131 plusmn 43 421 plusmn 12 234 plusmn 5 484 plusmn 43Carr 162 plusmn 05120572lowastlowastlowast 153 plusmn 26120572lowastlowastlowast 131 plusmn 11120572lowastlowastlowast 314 plusmn 75120572lowastlowastlowast 679 plusmn 69120572lowastlowastlowastCarr + Indo(10mgkgbw) 649 plusmn 71120573lowastlowastlowast 240 plusmn 13120573lowastlowastlowast 703 plusmn 11120573lowastlowastlowast 209 plusmn 30120573lowastlowastlowast 442 plusmn 06120573lowastlowastlowastCarr + ALE(400mgkgbw) 565 plusmn 25120573lowastlowastlowastmlowastlowast 207 plusmn 40120573lowastlowastlowastmlowastlowast 756 plusmn 25120573lowastlowastlowastmlowast 208 plusmn 60120573lowastlowastlowast 502 plusmn 41120573lowastlowastlowastmlowastValues represent means plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001120572 compared to control 120573 compared to Carr m compared to Carr + IndoSOD superoxide dismutaseCAT catalaseGSH glutathione peroxidaseMDA MalondialdehydeAOPP Advanced Oxidation Protein Product

Table 9 Quantification and identification of phenolic compounds contents in the ethanol extract of Cynara scolymus leaves extracts

Number Content (mg100 g of dry extract) Polyphenolic compound1 28 plusmn 019 Hydroxytyrosol2 381 plusmn 036 Verbascoside3 99 plusmn 002 Apigenin-7-glucoside4 18 plusmn 01 Oleuropein5 192 plusmn 014 Quercetin6 5 plusmn 005 Pinoresinol7 4 plusmn 004 ApigeninValues represent means plusmn SD (119899 = 3)

In the second phase the increase of vascular permeabilityis observed by release of kinins during 230 hThereafter from230 h to 5 h inflammation ismediated by prostaglandins andis also associated with a release infiltration and migration ofPN into the inflamed site [47]

Our results indicate that ALE affords protection againstthe Carr induced acute inflammation in dose dependentmanner EtOH extract of ALE at dose of 400mgkgbwexhibits significant anti-inflammatory activity with 73 inhi-bition of paw oedema compared with Indo group (53) andtypically reaches a maximum at 5 h these results obtainedsuggest the anti-inflammatory effect of EtOH extract of ALEbymeans of inhibiting the synthesis and the release of inflam-matory mediators like histamine serotonin and prostagl-andins that are involved in acute inflammationThese resultsrevealed the inhibitory effect of EtOH extract on PN migra-tion and it is confirmed through a histological analysis oftissues of paw oedema in experimental groups

These findings clearly confirm that EtOH extract of ALEhas an anti-inflammatory effect by reducing the influx ofpolymorphonuclear cells to inflammatory tissue followinginjection of Carr Most inflammatory markers such asinterleukin-6 (IL-6) tumor necrosis factor-120572 (TNF-120572) CRPand fibrinogen increased significantly in response to infec-tion and in active diseases states CRP is the specific marker

of acute inflammation occurring in the body [48] moreoverthe fibrinogen is themost relevant indicator of inflammationand it does not only represent the acute-phase reactant that isincreased in inflammatory states Elevated fibrinogen levelswould be associated with higher levels of CRP and wouldsimilarly correlate with inflammation reaction

Therefore the present study showed that EtOH extract ofALE significantly decreases the level of fibrinogen and CRPby value of 2272 and 3478 in comparison with Carrgroup

The induction of inflammationwithCarr ismanifested bygeneration of ROS and has been shown to play an importantrole in various forms of inflammation [49 50] High concen-tration of reactive free radicals contributes to lipid perox-idation and protein oxidation [3] Our results showed thatthere is a significant increase in MDA and AOPP activitiesin Carr group when compared to control group and a highlysignificant decrease in activities of SOD CAT and GSH alsoobserved at the tissue level compared with the control group(119901 lt 0001) However the injection of EtOH extract and Indoshows a significant decrease of the levels of MDA and AOPPby 3427 and 4427 following a significant increase inantioxidant activities as CAT SOD andGSHwhen comparedto Carr group (2016 7330 and 75) From these resultsit is implied that the protective effect of EtOH extract may

12 Evidence-Based Complementary and Alternative Medicine

be attributed to its potential indirectly as a stimulator ofthe activity and expression of antioxidant enzymes duringinflammation

Thus the anti-inflammatory activity is due to the individ-ual or synergistic effect of the components in the ALE In factprevious studies have found that anti-inflammatory profile ofALE could be related to polyphenolic compounds accordingto the phytochemical analysis We performed HPLC analysisto justify the correlation between phytochemical compoundsand anti-inflammatory activity of this plant Among thesepolyphenolic compounds verbascoside has been reportedin several reports and showed a potential spectrum ofmany activities including antioxidant and anti-inflammatoryParticular verbascoside presents antiedematogenic activitiesin animal models of Carr induced inflammation [51] and theresponses exhibited themaximumactivity for the fourth hourof treatment Such activities may be derived from inhibitoryaction of chemical mediators of the inflammatory processsuch as histamine and bradykinin A significant antioxidanteffect of verbascoside has been recently reported byAleo et al[52] in an experimental study [53] Other authors reportedthat anti-inflammatory activity of verbascoside has beenevaluated by an in vitro test performed on cell cultures of pri-mary human keratinocytes [54] in which it was able to reducethe release of proinflammatory markers such as chemokines

Apigenin-7-glucoside was found to block the releaseof several varieties of enzymes involved in inflammationincluding especially lipoxygenases and cyclooxygenases [5556] leading to inhibition of proinflammatory molecules NF-kB activation and inhibits neutrophil infiltration in tissues

From these results we suggest that anti-inflammatoryactivity observed is due to a synergic action of these phenoliccomponents contained in EtOH extract of ALE

C scolymus is one of the few herbal remedies which havebeen verified through experimental studies Further the toxi-city studies of ALE are required in order to confirm the safetyof this medicinal plant in the treatment of inflammationdiseases

5 Conclusion

The present study showed clearly the advantages of Cscolymus leaves extracts which have safer anti-inflammatoryprofile with potent antioxidant activity attributed to thephenolic compounds Furthermore in the future studieswe are interested to characterize the action mechanisms ofactive phenolic compounds of ALE responsible for anti-inflammatory and antioxidants activities

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Tunisian Ministry ofHigher Education and Scientific Research

References

[1] C Pasquier ldquoStress oxidative et inflammationrdquo Blool vol 4 pp1ndash6 1996

[2] M A Abuashwashi O M Palomino and M P Gomez-Serranillos ldquoGeographic origin influences the phenolic com-position and antioxidant potential of wild Crataegus monogynafrom Spainrdquo Pharmaceutical Biology vol 54 no 11 pp 2708ndash2713 2016

[3] M B Salem H Affes K Ksouda et al ldquoPharmacological stud-ies of artichoke leaf extract and their health benefitsrdquo PlantFoods for Human Nutrition vol 70 no 4 pp 441ndash453 2015

[4] M-H Huang B-S Wang C-S Chiu et al ldquoAntioxidant anti-nociceptive and anti-inflammatory activities ofXanthii Fructusextractrdquo Journal of Ethnopharmacology vol 135 no 2 pp 545ndash552 2011

[5] G-J Huang S-S Huang S-S Lin et al ldquoAnalgesic effectsand the mechanisms of anti-inflammation of ergostatrien-3120573-ol from antrodia camphorata submerged whole broth in micerdquoJournal of Agricultural and Food Chemistry vol 58 no 12 pp7445ndash7452 2010

[6] C Tohda N Nakayama F Hatanaka and K Komatsu ldquoCom-parison of anti-inflammatory activities of six Curcuma rhi-zomes a possible curcuminoid-independent pathway mediatedby Curcuma phaeocaulis extractrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 3 no 2 pp 255ndash260 2006

[7] K Goksel K Murat Y Murat E Sevil and H Fidan ldquoAntiox-idant activities of ethanol extracts ofHypericum triquetrifoliumandHypericum scabroidesrdquo Pharmaceutical Biology vol 46 no4 pp 231ndash242 2008

[8] R Gebhardt and M Fausel ldquoAntioxidant and hepatoprotectiveeffects of artichoke extracts and constituents in cultured rathepatocytesrdquoToxicology inVitro vol 11 no 5 pp 669ndash672 1997

[9] A SofworaMedicinal Plants and TraditionalMedicine in AfricaJohn Wiley amp Sons Ann Arbor Mich USA 1982

[10] D E Okwu ldquoPhytochemicals vitamins andmineral contents oftwo Nigerian medicinal plantsrdquo International Journal of Mole-cular Medicine and Advances Sciences vol 1 pp 375ndash381 2005

[11] N Tsevegsuren GDavaakhuu andT S Udval ldquoPhytochemicalanalysis of Cynara scolymus L cultivated in MongoliardquoMongo-lian Journal of Chemistry vol 15 no 41 pp 40ndash42 2014

[12] AACC Approved Methods of the AACC American Associationof Cereal Chemists St Paul Minn USA 8th edition 1984

[13] S C Lee L Prosky and J W DeVries ldquoDetermination of totalsoluble and insoluble dietary fiber in foodsmdashenzymatic-gravi-metric method MES-TRIS buffer collaborative studyrdquo Journalof the Association of Official Analytical Chemists vol 75 pp395ndash416 1992

[14] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[15] O A Fawole U L Opara and K I Theron ldquoChemical andphytochemical properties and antioxidant activities of threepomegranate cultivars grown in South Africardquo Food and Bio-process Technology vol 5 no 7 pp 2934ndash2940 2012

[16] V Dewanto X Wu K K Adom and R H Liu ldquoThermalprocessing enhances the nutritional value of tomatoes byincreasing total antioxidant activityrdquo Journal of Agricultural andFood Chemistry vol 50 no 10 pp 3010ndash3014 2002

[17] B Sun J M Ricardo-da-Silva and I Spranger ldquoCritical factorsof vanillin assay for catechins and proanthocyanidinsrdquo Journal

Evidence-Based Complementary and Alternative Medicine 13

of Agricultural and Food Chemistry vol 46 no 10 pp 4267ndash4274 1998

[18] AOACOfficial Methods of Analysis vol 1 AOACWashingtonDC USA 15th edition 1990

[19] D Pljevljakusic K Savikin T Jankovic et al ldquoChemical prop-erties of the cultivated Sideritis raeseri Boiss amp Heldr subspraeserirdquo Food Chemistry vol 124 no 1 pp 226ndash233 2011

[20] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[21] I I Koleva T A Van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] V Ravi T S M Saleem S S Patel J Raamamurthy and KGauthaman ldquoAnti-inflammatory effect of methanolic extract ofSolanum nigrum Linn Berriesrdquo International Journal of AppliedResearch in Natural Products vol 2 pp 33ndash36 2009

[24] D Chattopadhyay G Arunachalam A B Mandal T K SurS C Mandal and S K Bhattacharya ldquoAntimicrobial and anti-inflammatory activity of folkloreMallotus peltatus leaf extractrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 229ndash2372002

[25] L R Morse K Stolzmann H P Nguyen et al ldquoAssociationbetween mobility mode and c-reactive protein levels in menwith chronic spinal cord injuryrdquo Archives of Physical Medicineand Rehabilitation vol 89 no 4 pp 726ndash731 2008

[26] A Clauss ldquoGerinnungsphysiologische schnellmethode zur bes-timmung des fibrinogensrdquoActaHaematologica vol 17 no 4 pp237ndash246 1957

[27] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[28] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidationin post-mitotic tissues of ageing ratrdquo Cell Biochemistry andFunction vol 24 no 1 pp 79ndash85 2006

[29] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[30] W F Beyer Jr and I Fridovich ldquoAssaying for superoxide dis-mutase activity some large consequences of minor changes inconditionsrdquoAnalytical Biochemistry vol 161 no 2 pp 559ndash5661987

[31] I Carlberg and B Mannervik ldquoGlutathione reductaserdquo inGlutamate Glutamine Glutathione and Related Compoundsvol 113 ofMethods in Enzymology pp 484ndash490 Elsevier 1985

[32] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[33] V Emanue V Adrian N A Sultana and C Svetlana ldquoAntiox-idant and antimicrobial activities of ethanol extracts of Cynarascolymus (Cynarae folium Asteraceae family)rdquo Tropical Journalof Pharmaceutical Research vol 10 no 6 pp 777ndash783 2011

[34] G L D S Oliveira R A M O Francisco O B A Vinıcius etal ldquoEvaluation of antioxidant capacity of the aqueous extract ofCynara scolymus L (Asteraceae) in vitro and in SaccharomycescerevisiaerdquoAfrican Journal of Pharmacy and Pharmacology vol8 pp 136ndash147 2014

[35] K Horiuchi S Shiota T Hatano T Yoshida T Kuroda and TTsuchiya ldquoAntimicrobial activity of oleanolic acid from Salviaofficinalis and related compounds on vancomycin-resistantenterococci (VRE)rdquo Biological and Pharmaceutical Bulletin vol30 no 6 pp 1147ndash1149 2007

[36] A Daoud D Malika S Bakari et al ldquoAssessment of polyphe-nol composition antioxidant and antimicrobial properties ofvarious extracts of Date Palm Pollen (DPP) from two Tunisiancultivarsrdquo Arabian Journal of Chemistry 2015

[37] D Krishnaiah B Awang S Rosalam and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversitymdashEngineering Sciences vol 1 pp 63ndash66 2015

[38] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[39] A Betancor-Fernandez A Perez-Galvez H Sies and W StahlldquoScreening pharmaceutical preparations containing extracts ofturmeric rhizome artichoke leaf devilrsquos claw root and garlic orsalmon oil for antioxidant capacityrdquo Journal of Pharmacy andPharmacology vol 55 no 7 pp 981ndash986 2003

[40] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[41] S Konyal120580oglu H Saglam and B K120580vcak ldquo120572-Tocopherolflavonoid and phenol contents and antioxidant activity of Ficuscarica leavesrdquo Pharmaceutical Biology vol 43 no 8 pp 683ndash686 2005

[42] D U Bawankule P Trivedi A Pal et al ldquoProtectivemechanismof lignans from Phyllanthus amarus against galactosaminelipopolysaccharide-induced hepatitis an in-vivo and in-silicostudiesrdquo Current Topics in Medicinal Chemistry vol 14 no 8pp 1045ndash1055 2014

[43] GAHiggs R J Flower and J RVane ldquoAnew approach to anti-inflammatory drugsrdquo Biochemical Pharmacology vol 28 no 12pp 1959ndash1961 1979

[44] A A Hemmati H Kalantari A Siahpoosh B Ghorbanzadehand H Jamali ldquoAnti-inflammatory Effect of HydroalcoholicExtract of the Washingtonia filifera Seeds in Carrageenan-Induced Paw Edema in Ratsrdquo Jundishapur Journal of NaturalPharmaceutical Products vol 10 no 1 pp 19ndash25 2015

[45] I Posadas M Bucci F Roviezzo et al ldquoCarrageenan-inducedmouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expressionrdquoBritish Journal of Pharmacology vol 142 no 2 pp 331ndash3382004

[46] K Pundarikakshudu D H Shah A H Panchal and G CBhavsar ldquoAnti-inflammatory activity of fenugreek (Trigonellafoenum-graecum Linn) seed petroleum ether extractrdquo IndianJournal of Pharmacology vol 48 no 4 pp 441ndash444 2016

[47] S Cuzzocrea G Costantino E Mazzon B Zingarelli A DeSarro and A P Caputi ldquoProtective effects of Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) a superoxide dismutasemimetic in paw oedema induced by carrageenan in the ratrdquoBiochemical Pharmacology vol 58 no 1 pp 171ndash176 1999

[48] M A Mendall P Patel M Asante et al ldquoRelation of serumcytokine concentrations to cardiovascular risk factors andcoronary heart diseaserdquo Heart vol 78 no 3 pp 273ndash277 1997

[49] A Ialenti A Ianaro S Moncada andM Di Rosa ldquoModulationof acute inflammation by endogenous nitric oxiderdquo EuropeanJournal of Pharmacology vol 211 no 2 pp 177ndash182 1992

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

(a)

(b)

(c)

(d)

Figure 6 Histopathological slides tissues of paw oedema in experimental groups of rats (a) Control group untreated group (b) Carr groupCarr-treated rat showed heavy infiltration of polynuclear neutrophils (PN) and a spongy-like appearance and bulla in the epidermis (c)Carr + EtOH extract group Carr-treated rat that received the EtOH extract (400mgkgbw) reduced significantly the migration of PNand oedematasis in dermis without any spongy-like feature and bulla (d) Carr + Indo group Carr-treated rat that received Indomethacin(10mgkgbw) showed a partial protective action Deparaffinized hematoxylin and eosin (HampE) stained sections (200ndash400x) Plus signinfiltration of PN Arrow odematasis in the epidermis

medicinal plants which have a beneficial potential effectattributed to its source of polyphenolic compounds [3]

The selection of this plant was guided by the indicationsof its traditional use that at present there have been very littlechemical and biological investigations done Therefore thepresent study was to investigate phytochemical compositionand their antioxidant capacity and to evaluate in vivo the anti-inflammatory effects of Cynara scolymus leaves extracts

The presence of phenolic compounds is very widelydistributed in medicinal plants several studies showed thatthese compounds have drawn much attention to their poten-tial antioxidant abilities which demonstrated their beneficialimplications for human health

Regarding the determination of phenolic compoundsthe EtOH extract of ALE had the highest amount of thesecompounds in comparison with other extracts which werean agreement with the result of Emanue et al [33] whoshows that EtOH extract exhibited the maximum amountof phenolic compounds from the leaves of Cynara scolymuswhereas it differs from the reports of Oliveira et al [34]who proves that aqueous extract of ALE is the most suitablesolvent for extraction of phenolic compounds

The importance of TPC and TFC extraction yieldsobtained with EtOH extract can be attributed to its goodsolubility low toxicity medium polarity and high extractioncapacity [35]

Evidence-Based Complementary and Alternative Medicine 9

1 2 3 678 9 10

5

4

0250500750

1000125015001750

(mAU

)

5 10 15 20 250(min)

Figure 7 HPLC chromatogram of a standard mixture of polyphe-nolic compounds Peaks 1 Hydroxytyrosol 2 Tyrosol 3 4-hydroxybenzoic acid 4 verbascoside 5 apigenin-7-glucoside 6Oleuropein 7 Quercetin 8 Pinoresinol 9 cinnamic acid 10apigenin

34

2

56 7

0200400600800

100012001400

(mAU

)

5 10 15 20 25 30 350(min)

1

Figure 8 HPLC chromatogram of ethanol extract of Cynarascolymus leaves extracts Peaks 1 Hydroxytyrosol 2 verbascoside3 apigenin-7-glucoside 4 Oleuropein 5 Quercetin 6 Pinoresinol7 apigenin

Table 6 Pearson correlation coefficient (119903) between the content ofphenolic compounds and antioxidant capacity (DDPH∙ ABTS∙+FRAP and beta-carotene)

Correlation 119877TPC versus DDPH∙ 0870lowastlowast

TPC versus ABTS∙+ 0848lowastlowast

TPC versus FRAP 0707lowastlowast

TPC versus beta-carotene 0842lowastlowast

TFC versus DDPH∙ 0728lowastlowast

TFC versus ABTS∙+ 0743lowastlowast

TFC versus FRAP 0849lowastlowast

TFC versus beta-carotene 0712lowastlowast

DPPH 22-diphenyl-1-picrylhydrazyl ABTS 220-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt FRAP ferricreducing TPC total phenolic content TFC total flavonoid contentlowastlowast119901 lt 001 significant correlation

In the first investigation of my study five solvents ofincreasing polarities were chosen for the determination ofphenolic compounds from leaves of C scolymus namelyhexane ethyl acetate butanol 75 EtOHH2O and aqueousin order to determine the variability of TPC and TFC in theaerial part of Cynara as a function of each extraction solvent

This variability of phenolic compounds in ALE can beattributed to the wide range of solubility displayed by variouspolar compounds within the ALE solvents the degree ofpolymerization of phenols and their interaction geneticfactors geographical variations and climatic changes [36]

Overall these findings indicate that EtOH extract of ALEwas rich in phenolic and flavonoid contents which couldbe the major contributor to their antioxidative propertiesMany researches revealed that flavonoids and polyphenolsdisplayed the highest ability of scavenging activity in medic-inal plants [37 38]

The results indicate that EtOH extract of ALE whichcontains the highest content of TFC and TPC displays thehighest free radical scavenging activity (9423) at concen-tration of 400 ugmL However Oliveira et al [34] show thataqueous extract of ALE has good radical scavenging (8340)at concentration of 200 ugmL Furthermore our resultssuggest that some components within EtOH extract aresignificantly the strongest radical scavenger in comparisonwith other extracts The TEAC values of ALE showed alsothat EtOH extract presents a potential ability to scavengethe ABTS radical cation in accordance with the report ofBetancor-Fernandez et al [39]

The FRAP assay shows also that EtOH extract has thehighest values (54262 umol Fe II gDW) The same resultis confirmed by Kukic et al [40] Therefore the results ofpositive correlation between the TPC TFC and the anti-oxidant methods in vitro suggest that phenolic compoundsact as reducing agents hydrogen donors and singlet oxygenscavengers [3] and may exert an important in vitro antioxi-dant capacity of ALE

The phytochemical screening of ALE has been foundto be a rich source of polyphenolic compounds includingQuercetin apigenin-7-glucoside and verbascoside Thesecompounds have shown a great potential of antioxidantactivity [41]

The research into medicinal plants used as pain relieversrsquoagents should therefore be viewed as new therapy in theinflammatory diseases [42] The anti-inflammatory activitiesof EtOH extract of ALE are investigated applying in vivoexperimental Carr model compared with a nonsteroidal anti-inflammatory (Indomethacin) this drug was reported byHiggs et al [43] who demonstrated the role of Indo toinhibit the biosynthesis of prostaglandins in Carr model ratsThe experimental study of anti-inflammatory activity is per-formed by the Carr test This phlogiston agent inducedtissue oedema characteristic of acute inflammation whichis regarded as a crucial parameter in assessing anti-inflam-matory activity [44]

The Carr injection produces biphasic states in the firstphase [45] there is an increase in the mRNA synthesis ofcyclooxygenase-2 (COX-2) at the first hour This increase isaccompanied by an amplification of synthesis of strong proin-flammatory mediator such as prostaglandin especially theprostaglandin type 2 involved in the inflammatory process[46] serotonin bradykinin and leukotrienes which con-tribute to the initiation of inflammation reaction Moreoverthe participation of arachidonicmetabolites is themain factorresponsible for both of phases of Carr induced inflammation

10 Evidence-Based Complementary and Alternative Medicine

Table7Eff

ecto

fCynarascolym

usleaves

extracto

ncarrageenan-indu

cedratp

awoedema

Treatm

ent

Oedem

asize(meanplusmnS

D)(mm)

0h1h

2h3h

4h5h

Con

trol

0426plusmn0

00

044

1plusmn001

056plusmn0

03

0650plusmn0

05

0755plusmn0

05

070plusmn0

01

Carr

0426plusmn0

00

28plusmn026120572lowastlowastlowast

333plusmn028120572lowastlowastlowast

325plusmn002120572lowastlowastlowast

315plusmn001120572lowastlowastlowast

302plusmn009120572lowastlowastlowast

Carr+Indo

(10m

gkg)

0426plusmn0

00

246plusmn026120572lowastlowastlowast120573lowast

261plusmn007120572lowastlowastlowast120573lowastlowastlowast

230plusmn002120572lowastlowastlowast120573lowastlowastlowast

18plusmn011120572lowastlowastlowast120573lowastlowastlowast

14plusmn006120572lowastlowastlowast120573lowastlowastlowast

Carr+ALE

(400

mgkgbw)

0426plusmn0

00

2480plusmn005120572lowastlowastlowast120573lowastlowastlowast

188plusmn004120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

165plusmn005120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

116plusmn020120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

112plusmn01120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

Values

representm

eansplusmnSD

(119899=6)ineach

grou

plowast119901lt005lowastlowast119901lt001andlowastlowastlowast119901lt0001

120572com

paredto

control120573com

paredto

Carrmcom

paredto

Carr+Indo

Evidence-Based Complementary and Alternative Medicine 11

Table 8 Effects of Cynara scolymus leaves extract and Indomethacin on CAT SOD GSH AOPP andMDA activities in carrageenan inducedpaw oedema

GroupsCAT (120583molesCATminmg

protein)

SOD (unitSODminmg

protein)

GSH (nmolesmgprotein)

MDA (nmolMDAmg protein) AOPP (nmolmg protein)

Control 47 plusmn 2 131 plusmn 43 421 plusmn 12 234 plusmn 5 484 plusmn 43Carr 162 plusmn 05120572lowastlowastlowast 153 plusmn 26120572lowastlowastlowast 131 plusmn 11120572lowastlowastlowast 314 plusmn 75120572lowastlowastlowast 679 plusmn 69120572lowastlowastlowastCarr + Indo(10mgkgbw) 649 plusmn 71120573lowastlowastlowast 240 plusmn 13120573lowastlowastlowast 703 plusmn 11120573lowastlowastlowast 209 plusmn 30120573lowastlowastlowast 442 plusmn 06120573lowastlowastlowastCarr + ALE(400mgkgbw) 565 plusmn 25120573lowastlowastlowastmlowastlowast 207 plusmn 40120573lowastlowastlowastmlowastlowast 756 plusmn 25120573lowastlowastlowastmlowast 208 plusmn 60120573lowastlowastlowast 502 plusmn 41120573lowastlowastlowastmlowastValues represent means plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001120572 compared to control 120573 compared to Carr m compared to Carr + IndoSOD superoxide dismutaseCAT catalaseGSH glutathione peroxidaseMDA MalondialdehydeAOPP Advanced Oxidation Protein Product

Table 9 Quantification and identification of phenolic compounds contents in the ethanol extract of Cynara scolymus leaves extracts

Number Content (mg100 g of dry extract) Polyphenolic compound1 28 plusmn 019 Hydroxytyrosol2 381 plusmn 036 Verbascoside3 99 plusmn 002 Apigenin-7-glucoside4 18 plusmn 01 Oleuropein5 192 plusmn 014 Quercetin6 5 plusmn 005 Pinoresinol7 4 plusmn 004 ApigeninValues represent means plusmn SD (119899 = 3)

In the second phase the increase of vascular permeabilityis observed by release of kinins during 230 hThereafter from230 h to 5 h inflammation ismediated by prostaglandins andis also associated with a release infiltration and migration ofPN into the inflamed site [47]

Our results indicate that ALE affords protection againstthe Carr induced acute inflammation in dose dependentmanner EtOH extract of ALE at dose of 400mgkgbwexhibits significant anti-inflammatory activity with 73 inhi-bition of paw oedema compared with Indo group (53) andtypically reaches a maximum at 5 h these results obtainedsuggest the anti-inflammatory effect of EtOH extract of ALEbymeans of inhibiting the synthesis and the release of inflam-matory mediators like histamine serotonin and prostagl-andins that are involved in acute inflammationThese resultsrevealed the inhibitory effect of EtOH extract on PN migra-tion and it is confirmed through a histological analysis oftissues of paw oedema in experimental groups

These findings clearly confirm that EtOH extract of ALEhas an anti-inflammatory effect by reducing the influx ofpolymorphonuclear cells to inflammatory tissue followinginjection of Carr Most inflammatory markers such asinterleukin-6 (IL-6) tumor necrosis factor-120572 (TNF-120572) CRPand fibrinogen increased significantly in response to infec-tion and in active diseases states CRP is the specific marker

of acute inflammation occurring in the body [48] moreoverthe fibrinogen is themost relevant indicator of inflammationand it does not only represent the acute-phase reactant that isincreased in inflammatory states Elevated fibrinogen levelswould be associated with higher levels of CRP and wouldsimilarly correlate with inflammation reaction

Therefore the present study showed that EtOH extract ofALE significantly decreases the level of fibrinogen and CRPby value of 2272 and 3478 in comparison with Carrgroup

The induction of inflammationwithCarr ismanifested bygeneration of ROS and has been shown to play an importantrole in various forms of inflammation [49 50] High concen-tration of reactive free radicals contributes to lipid perox-idation and protein oxidation [3] Our results showed thatthere is a significant increase in MDA and AOPP activitiesin Carr group when compared to control group and a highlysignificant decrease in activities of SOD CAT and GSH alsoobserved at the tissue level compared with the control group(119901 lt 0001) However the injection of EtOH extract and Indoshows a significant decrease of the levels of MDA and AOPPby 3427 and 4427 following a significant increase inantioxidant activities as CAT SOD andGSHwhen comparedto Carr group (2016 7330 and 75) From these resultsit is implied that the protective effect of EtOH extract may

12 Evidence-Based Complementary and Alternative Medicine

be attributed to its potential indirectly as a stimulator ofthe activity and expression of antioxidant enzymes duringinflammation

Thus the anti-inflammatory activity is due to the individ-ual or synergistic effect of the components in the ALE In factprevious studies have found that anti-inflammatory profile ofALE could be related to polyphenolic compounds accordingto the phytochemical analysis We performed HPLC analysisto justify the correlation between phytochemical compoundsand anti-inflammatory activity of this plant Among thesepolyphenolic compounds verbascoside has been reportedin several reports and showed a potential spectrum ofmany activities including antioxidant and anti-inflammatoryParticular verbascoside presents antiedematogenic activitiesin animal models of Carr induced inflammation [51] and theresponses exhibited themaximumactivity for the fourth hourof treatment Such activities may be derived from inhibitoryaction of chemical mediators of the inflammatory processsuch as histamine and bradykinin A significant antioxidanteffect of verbascoside has been recently reported byAleo et al[52] in an experimental study [53] Other authors reportedthat anti-inflammatory activity of verbascoside has beenevaluated by an in vitro test performed on cell cultures of pri-mary human keratinocytes [54] in which it was able to reducethe release of proinflammatory markers such as chemokines

Apigenin-7-glucoside was found to block the releaseof several varieties of enzymes involved in inflammationincluding especially lipoxygenases and cyclooxygenases [5556] leading to inhibition of proinflammatory molecules NF-kB activation and inhibits neutrophil infiltration in tissues

From these results we suggest that anti-inflammatoryactivity observed is due to a synergic action of these phenoliccomponents contained in EtOH extract of ALE

C scolymus is one of the few herbal remedies which havebeen verified through experimental studies Further the toxi-city studies of ALE are required in order to confirm the safetyof this medicinal plant in the treatment of inflammationdiseases

5 Conclusion

The present study showed clearly the advantages of Cscolymus leaves extracts which have safer anti-inflammatoryprofile with potent antioxidant activity attributed to thephenolic compounds Furthermore in the future studieswe are interested to characterize the action mechanisms ofactive phenolic compounds of ALE responsible for anti-inflammatory and antioxidants activities

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Tunisian Ministry ofHigher Education and Scientific Research

References

[1] C Pasquier ldquoStress oxidative et inflammationrdquo Blool vol 4 pp1ndash6 1996

[2] M A Abuashwashi O M Palomino and M P Gomez-Serranillos ldquoGeographic origin influences the phenolic com-position and antioxidant potential of wild Crataegus monogynafrom Spainrdquo Pharmaceutical Biology vol 54 no 11 pp 2708ndash2713 2016

[3] M B Salem H Affes K Ksouda et al ldquoPharmacological stud-ies of artichoke leaf extract and their health benefitsrdquo PlantFoods for Human Nutrition vol 70 no 4 pp 441ndash453 2015

[4] M-H Huang B-S Wang C-S Chiu et al ldquoAntioxidant anti-nociceptive and anti-inflammatory activities ofXanthii Fructusextractrdquo Journal of Ethnopharmacology vol 135 no 2 pp 545ndash552 2011

[5] G-J Huang S-S Huang S-S Lin et al ldquoAnalgesic effectsand the mechanisms of anti-inflammation of ergostatrien-3120573-ol from antrodia camphorata submerged whole broth in micerdquoJournal of Agricultural and Food Chemistry vol 58 no 12 pp7445ndash7452 2010

[6] C Tohda N Nakayama F Hatanaka and K Komatsu ldquoCom-parison of anti-inflammatory activities of six Curcuma rhi-zomes a possible curcuminoid-independent pathway mediatedby Curcuma phaeocaulis extractrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 3 no 2 pp 255ndash260 2006

[7] K Goksel K Murat Y Murat E Sevil and H Fidan ldquoAntiox-idant activities of ethanol extracts ofHypericum triquetrifoliumandHypericum scabroidesrdquo Pharmaceutical Biology vol 46 no4 pp 231ndash242 2008

[8] R Gebhardt and M Fausel ldquoAntioxidant and hepatoprotectiveeffects of artichoke extracts and constituents in cultured rathepatocytesrdquoToxicology inVitro vol 11 no 5 pp 669ndash672 1997

[9] A SofworaMedicinal Plants and TraditionalMedicine in AfricaJohn Wiley amp Sons Ann Arbor Mich USA 1982

[10] D E Okwu ldquoPhytochemicals vitamins andmineral contents oftwo Nigerian medicinal plantsrdquo International Journal of Mole-cular Medicine and Advances Sciences vol 1 pp 375ndash381 2005

[11] N Tsevegsuren GDavaakhuu andT S Udval ldquoPhytochemicalanalysis of Cynara scolymus L cultivated in MongoliardquoMongo-lian Journal of Chemistry vol 15 no 41 pp 40ndash42 2014

[12] AACC Approved Methods of the AACC American Associationof Cereal Chemists St Paul Minn USA 8th edition 1984

[13] S C Lee L Prosky and J W DeVries ldquoDetermination of totalsoluble and insoluble dietary fiber in foodsmdashenzymatic-gravi-metric method MES-TRIS buffer collaborative studyrdquo Journalof the Association of Official Analytical Chemists vol 75 pp395ndash416 1992

[14] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[15] O A Fawole U L Opara and K I Theron ldquoChemical andphytochemical properties and antioxidant activities of threepomegranate cultivars grown in South Africardquo Food and Bio-process Technology vol 5 no 7 pp 2934ndash2940 2012

[16] V Dewanto X Wu K K Adom and R H Liu ldquoThermalprocessing enhances the nutritional value of tomatoes byincreasing total antioxidant activityrdquo Journal of Agricultural andFood Chemistry vol 50 no 10 pp 3010ndash3014 2002

[17] B Sun J M Ricardo-da-Silva and I Spranger ldquoCritical factorsof vanillin assay for catechins and proanthocyanidinsrdquo Journal

Evidence-Based Complementary and Alternative Medicine 13

of Agricultural and Food Chemistry vol 46 no 10 pp 4267ndash4274 1998

[18] AOACOfficial Methods of Analysis vol 1 AOACWashingtonDC USA 15th edition 1990

[19] D Pljevljakusic K Savikin T Jankovic et al ldquoChemical prop-erties of the cultivated Sideritis raeseri Boiss amp Heldr subspraeserirdquo Food Chemistry vol 124 no 1 pp 226ndash233 2011

[20] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[21] I I Koleva T A Van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] V Ravi T S M Saleem S S Patel J Raamamurthy and KGauthaman ldquoAnti-inflammatory effect of methanolic extract ofSolanum nigrum Linn Berriesrdquo International Journal of AppliedResearch in Natural Products vol 2 pp 33ndash36 2009

[24] D Chattopadhyay G Arunachalam A B Mandal T K SurS C Mandal and S K Bhattacharya ldquoAntimicrobial and anti-inflammatory activity of folkloreMallotus peltatus leaf extractrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 229ndash2372002

[25] L R Morse K Stolzmann H P Nguyen et al ldquoAssociationbetween mobility mode and c-reactive protein levels in menwith chronic spinal cord injuryrdquo Archives of Physical Medicineand Rehabilitation vol 89 no 4 pp 726ndash731 2008

[26] A Clauss ldquoGerinnungsphysiologische schnellmethode zur bes-timmung des fibrinogensrdquoActaHaematologica vol 17 no 4 pp237ndash246 1957

[27] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[28] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidationin post-mitotic tissues of ageing ratrdquo Cell Biochemistry andFunction vol 24 no 1 pp 79ndash85 2006

[29] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[30] W F Beyer Jr and I Fridovich ldquoAssaying for superoxide dis-mutase activity some large consequences of minor changes inconditionsrdquoAnalytical Biochemistry vol 161 no 2 pp 559ndash5661987

[31] I Carlberg and B Mannervik ldquoGlutathione reductaserdquo inGlutamate Glutamine Glutathione and Related Compoundsvol 113 ofMethods in Enzymology pp 484ndash490 Elsevier 1985

[32] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[33] V Emanue V Adrian N A Sultana and C Svetlana ldquoAntiox-idant and antimicrobial activities of ethanol extracts of Cynarascolymus (Cynarae folium Asteraceae family)rdquo Tropical Journalof Pharmaceutical Research vol 10 no 6 pp 777ndash783 2011

[34] G L D S Oliveira R A M O Francisco O B A Vinıcius etal ldquoEvaluation of antioxidant capacity of the aqueous extract ofCynara scolymus L (Asteraceae) in vitro and in SaccharomycescerevisiaerdquoAfrican Journal of Pharmacy and Pharmacology vol8 pp 136ndash147 2014

[35] K Horiuchi S Shiota T Hatano T Yoshida T Kuroda and TTsuchiya ldquoAntimicrobial activity of oleanolic acid from Salviaofficinalis and related compounds on vancomycin-resistantenterococci (VRE)rdquo Biological and Pharmaceutical Bulletin vol30 no 6 pp 1147ndash1149 2007

[36] A Daoud D Malika S Bakari et al ldquoAssessment of polyphe-nol composition antioxidant and antimicrobial properties ofvarious extracts of Date Palm Pollen (DPP) from two Tunisiancultivarsrdquo Arabian Journal of Chemistry 2015

[37] D Krishnaiah B Awang S Rosalam and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversitymdashEngineering Sciences vol 1 pp 63ndash66 2015

[38] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[39] A Betancor-Fernandez A Perez-Galvez H Sies and W StahlldquoScreening pharmaceutical preparations containing extracts ofturmeric rhizome artichoke leaf devilrsquos claw root and garlic orsalmon oil for antioxidant capacityrdquo Journal of Pharmacy andPharmacology vol 55 no 7 pp 981ndash986 2003

[40] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[41] S Konyal120580oglu H Saglam and B K120580vcak ldquo120572-Tocopherolflavonoid and phenol contents and antioxidant activity of Ficuscarica leavesrdquo Pharmaceutical Biology vol 43 no 8 pp 683ndash686 2005

[42] D U Bawankule P Trivedi A Pal et al ldquoProtectivemechanismof lignans from Phyllanthus amarus against galactosaminelipopolysaccharide-induced hepatitis an in-vivo and in-silicostudiesrdquo Current Topics in Medicinal Chemistry vol 14 no 8pp 1045ndash1055 2014

[43] GAHiggs R J Flower and J RVane ldquoAnew approach to anti-inflammatory drugsrdquo Biochemical Pharmacology vol 28 no 12pp 1959ndash1961 1979

[44] A A Hemmati H Kalantari A Siahpoosh B Ghorbanzadehand H Jamali ldquoAnti-inflammatory Effect of HydroalcoholicExtract of the Washingtonia filifera Seeds in Carrageenan-Induced Paw Edema in Ratsrdquo Jundishapur Journal of NaturalPharmaceutical Products vol 10 no 1 pp 19ndash25 2015

[45] I Posadas M Bucci F Roviezzo et al ldquoCarrageenan-inducedmouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expressionrdquoBritish Journal of Pharmacology vol 142 no 2 pp 331ndash3382004

[46] K Pundarikakshudu D H Shah A H Panchal and G CBhavsar ldquoAnti-inflammatory activity of fenugreek (Trigonellafoenum-graecum Linn) seed petroleum ether extractrdquo IndianJournal of Pharmacology vol 48 no 4 pp 441ndash444 2016

[47] S Cuzzocrea G Costantino E Mazzon B Zingarelli A DeSarro and A P Caputi ldquoProtective effects of Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) a superoxide dismutasemimetic in paw oedema induced by carrageenan in the ratrdquoBiochemical Pharmacology vol 58 no 1 pp 171ndash176 1999

[48] M A Mendall P Patel M Asante et al ldquoRelation of serumcytokine concentrations to cardiovascular risk factors andcoronary heart diseaserdquo Heart vol 78 no 3 pp 273ndash277 1997

[49] A Ialenti A Ianaro S Moncada andM Di Rosa ldquoModulationof acute inflammation by endogenous nitric oxiderdquo EuropeanJournal of Pharmacology vol 211 no 2 pp 177ndash182 1992

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

1 2 3 678 9 10

5

4

0250500750

1000125015001750

(mAU

)

5 10 15 20 250(min)

Figure 7 HPLC chromatogram of a standard mixture of polyphe-nolic compounds Peaks 1 Hydroxytyrosol 2 Tyrosol 3 4-hydroxybenzoic acid 4 verbascoside 5 apigenin-7-glucoside 6Oleuropein 7 Quercetin 8 Pinoresinol 9 cinnamic acid 10apigenin

34

2

56 7

0200400600800

100012001400

(mAU

)

5 10 15 20 25 30 350(min)

1

Figure 8 HPLC chromatogram of ethanol extract of Cynarascolymus leaves extracts Peaks 1 Hydroxytyrosol 2 verbascoside3 apigenin-7-glucoside 4 Oleuropein 5 Quercetin 6 Pinoresinol7 apigenin

Table 6 Pearson correlation coefficient (119903) between the content ofphenolic compounds and antioxidant capacity (DDPH∙ ABTS∙+FRAP and beta-carotene)

Correlation 119877TPC versus DDPH∙ 0870lowastlowast

TPC versus ABTS∙+ 0848lowastlowast

TPC versus FRAP 0707lowastlowast

TPC versus beta-carotene 0842lowastlowast

TFC versus DDPH∙ 0728lowastlowast

TFC versus ABTS∙+ 0743lowastlowast

TFC versus FRAP 0849lowastlowast

TFC versus beta-carotene 0712lowastlowast

DPPH 22-diphenyl-1-picrylhydrazyl ABTS 220-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt FRAP ferricreducing TPC total phenolic content TFC total flavonoid contentlowastlowast119901 lt 001 significant correlation

In the first investigation of my study five solvents ofincreasing polarities were chosen for the determination ofphenolic compounds from leaves of C scolymus namelyhexane ethyl acetate butanol 75 EtOHH2O and aqueousin order to determine the variability of TPC and TFC in theaerial part of Cynara as a function of each extraction solvent

This variability of phenolic compounds in ALE can beattributed to the wide range of solubility displayed by variouspolar compounds within the ALE solvents the degree ofpolymerization of phenols and their interaction geneticfactors geographical variations and climatic changes [36]

Overall these findings indicate that EtOH extract of ALEwas rich in phenolic and flavonoid contents which couldbe the major contributor to their antioxidative propertiesMany researches revealed that flavonoids and polyphenolsdisplayed the highest ability of scavenging activity in medic-inal plants [37 38]

The results indicate that EtOH extract of ALE whichcontains the highest content of TFC and TPC displays thehighest free radical scavenging activity (9423) at concen-tration of 400 ugmL However Oliveira et al [34] show thataqueous extract of ALE has good radical scavenging (8340)at concentration of 200 ugmL Furthermore our resultssuggest that some components within EtOH extract aresignificantly the strongest radical scavenger in comparisonwith other extracts The TEAC values of ALE showed alsothat EtOH extract presents a potential ability to scavengethe ABTS radical cation in accordance with the report ofBetancor-Fernandez et al [39]

The FRAP assay shows also that EtOH extract has thehighest values (54262 umol Fe II gDW) The same resultis confirmed by Kukic et al [40] Therefore the results ofpositive correlation between the TPC TFC and the anti-oxidant methods in vitro suggest that phenolic compoundsact as reducing agents hydrogen donors and singlet oxygenscavengers [3] and may exert an important in vitro antioxi-dant capacity of ALE

The phytochemical screening of ALE has been foundto be a rich source of polyphenolic compounds includingQuercetin apigenin-7-glucoside and verbascoside Thesecompounds have shown a great potential of antioxidantactivity [41]

The research into medicinal plants used as pain relieversrsquoagents should therefore be viewed as new therapy in theinflammatory diseases [42] The anti-inflammatory activitiesof EtOH extract of ALE are investigated applying in vivoexperimental Carr model compared with a nonsteroidal anti-inflammatory (Indomethacin) this drug was reported byHiggs et al [43] who demonstrated the role of Indo toinhibit the biosynthesis of prostaglandins in Carr model ratsThe experimental study of anti-inflammatory activity is per-formed by the Carr test This phlogiston agent inducedtissue oedema characteristic of acute inflammation whichis regarded as a crucial parameter in assessing anti-inflam-matory activity [44]

The Carr injection produces biphasic states in the firstphase [45] there is an increase in the mRNA synthesis ofcyclooxygenase-2 (COX-2) at the first hour This increase isaccompanied by an amplification of synthesis of strong proin-flammatory mediator such as prostaglandin especially theprostaglandin type 2 involved in the inflammatory process[46] serotonin bradykinin and leukotrienes which con-tribute to the initiation of inflammation reaction Moreoverthe participation of arachidonicmetabolites is themain factorresponsible for both of phases of Carr induced inflammation

10 Evidence-Based Complementary and Alternative Medicine

Table7Eff

ecto

fCynarascolym

usleaves

extracto

ncarrageenan-indu

cedratp

awoedema

Treatm

ent

Oedem

asize(meanplusmnS

D)(mm)

0h1h

2h3h

4h5h

Con

trol

0426plusmn0

00

044

1plusmn001

056plusmn0

03

0650plusmn0

05

0755plusmn0

05

070plusmn0

01

Carr

0426plusmn0

00

28plusmn026120572lowastlowastlowast

333plusmn028120572lowastlowastlowast

325plusmn002120572lowastlowastlowast

315plusmn001120572lowastlowastlowast

302plusmn009120572lowastlowastlowast

Carr+Indo

(10m

gkg)

0426plusmn0

00

246plusmn026120572lowastlowastlowast120573lowast

261plusmn007120572lowastlowastlowast120573lowastlowastlowast

230plusmn002120572lowastlowastlowast120573lowastlowastlowast

18plusmn011120572lowastlowastlowast120573lowastlowastlowast

14plusmn006120572lowastlowastlowast120573lowastlowastlowast

Carr+ALE

(400

mgkgbw)

0426plusmn0

00

2480plusmn005120572lowastlowastlowast120573lowastlowastlowast

188plusmn004120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

165plusmn005120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

116plusmn020120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

112plusmn01120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

Values

representm

eansplusmnSD

(119899=6)ineach

grou

plowast119901lt005lowastlowast119901lt001andlowastlowastlowast119901lt0001

120572com

paredto

control120573com

paredto

Carrmcom

paredto

Carr+Indo

Evidence-Based Complementary and Alternative Medicine 11

Table 8 Effects of Cynara scolymus leaves extract and Indomethacin on CAT SOD GSH AOPP andMDA activities in carrageenan inducedpaw oedema

GroupsCAT (120583molesCATminmg

protein)

SOD (unitSODminmg

protein)

GSH (nmolesmgprotein)

MDA (nmolMDAmg protein) AOPP (nmolmg protein)

Control 47 plusmn 2 131 plusmn 43 421 plusmn 12 234 plusmn 5 484 plusmn 43Carr 162 plusmn 05120572lowastlowastlowast 153 plusmn 26120572lowastlowastlowast 131 plusmn 11120572lowastlowastlowast 314 plusmn 75120572lowastlowastlowast 679 plusmn 69120572lowastlowastlowastCarr + Indo(10mgkgbw) 649 plusmn 71120573lowastlowastlowast 240 plusmn 13120573lowastlowastlowast 703 plusmn 11120573lowastlowastlowast 209 plusmn 30120573lowastlowastlowast 442 plusmn 06120573lowastlowastlowastCarr + ALE(400mgkgbw) 565 plusmn 25120573lowastlowastlowastmlowastlowast 207 plusmn 40120573lowastlowastlowastmlowastlowast 756 plusmn 25120573lowastlowastlowastmlowast 208 plusmn 60120573lowastlowastlowast 502 plusmn 41120573lowastlowastlowastmlowastValues represent means plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001120572 compared to control 120573 compared to Carr m compared to Carr + IndoSOD superoxide dismutaseCAT catalaseGSH glutathione peroxidaseMDA MalondialdehydeAOPP Advanced Oxidation Protein Product

Table 9 Quantification and identification of phenolic compounds contents in the ethanol extract of Cynara scolymus leaves extracts

Number Content (mg100 g of dry extract) Polyphenolic compound1 28 plusmn 019 Hydroxytyrosol2 381 plusmn 036 Verbascoside3 99 plusmn 002 Apigenin-7-glucoside4 18 plusmn 01 Oleuropein5 192 plusmn 014 Quercetin6 5 plusmn 005 Pinoresinol7 4 plusmn 004 ApigeninValues represent means plusmn SD (119899 = 3)

In the second phase the increase of vascular permeabilityis observed by release of kinins during 230 hThereafter from230 h to 5 h inflammation ismediated by prostaglandins andis also associated with a release infiltration and migration ofPN into the inflamed site [47]

Our results indicate that ALE affords protection againstthe Carr induced acute inflammation in dose dependentmanner EtOH extract of ALE at dose of 400mgkgbwexhibits significant anti-inflammatory activity with 73 inhi-bition of paw oedema compared with Indo group (53) andtypically reaches a maximum at 5 h these results obtainedsuggest the anti-inflammatory effect of EtOH extract of ALEbymeans of inhibiting the synthesis and the release of inflam-matory mediators like histamine serotonin and prostagl-andins that are involved in acute inflammationThese resultsrevealed the inhibitory effect of EtOH extract on PN migra-tion and it is confirmed through a histological analysis oftissues of paw oedema in experimental groups

These findings clearly confirm that EtOH extract of ALEhas an anti-inflammatory effect by reducing the influx ofpolymorphonuclear cells to inflammatory tissue followinginjection of Carr Most inflammatory markers such asinterleukin-6 (IL-6) tumor necrosis factor-120572 (TNF-120572) CRPand fibrinogen increased significantly in response to infec-tion and in active diseases states CRP is the specific marker

of acute inflammation occurring in the body [48] moreoverthe fibrinogen is themost relevant indicator of inflammationand it does not only represent the acute-phase reactant that isincreased in inflammatory states Elevated fibrinogen levelswould be associated with higher levels of CRP and wouldsimilarly correlate with inflammation reaction

Therefore the present study showed that EtOH extract ofALE significantly decreases the level of fibrinogen and CRPby value of 2272 and 3478 in comparison with Carrgroup

The induction of inflammationwithCarr ismanifested bygeneration of ROS and has been shown to play an importantrole in various forms of inflammation [49 50] High concen-tration of reactive free radicals contributes to lipid perox-idation and protein oxidation [3] Our results showed thatthere is a significant increase in MDA and AOPP activitiesin Carr group when compared to control group and a highlysignificant decrease in activities of SOD CAT and GSH alsoobserved at the tissue level compared with the control group(119901 lt 0001) However the injection of EtOH extract and Indoshows a significant decrease of the levels of MDA and AOPPby 3427 and 4427 following a significant increase inantioxidant activities as CAT SOD andGSHwhen comparedto Carr group (2016 7330 and 75) From these resultsit is implied that the protective effect of EtOH extract may

12 Evidence-Based Complementary and Alternative Medicine

be attributed to its potential indirectly as a stimulator ofthe activity and expression of antioxidant enzymes duringinflammation

Thus the anti-inflammatory activity is due to the individ-ual or synergistic effect of the components in the ALE In factprevious studies have found that anti-inflammatory profile ofALE could be related to polyphenolic compounds accordingto the phytochemical analysis We performed HPLC analysisto justify the correlation between phytochemical compoundsand anti-inflammatory activity of this plant Among thesepolyphenolic compounds verbascoside has been reportedin several reports and showed a potential spectrum ofmany activities including antioxidant and anti-inflammatoryParticular verbascoside presents antiedematogenic activitiesin animal models of Carr induced inflammation [51] and theresponses exhibited themaximumactivity for the fourth hourof treatment Such activities may be derived from inhibitoryaction of chemical mediators of the inflammatory processsuch as histamine and bradykinin A significant antioxidanteffect of verbascoside has been recently reported byAleo et al[52] in an experimental study [53] Other authors reportedthat anti-inflammatory activity of verbascoside has beenevaluated by an in vitro test performed on cell cultures of pri-mary human keratinocytes [54] in which it was able to reducethe release of proinflammatory markers such as chemokines

Apigenin-7-glucoside was found to block the releaseof several varieties of enzymes involved in inflammationincluding especially lipoxygenases and cyclooxygenases [5556] leading to inhibition of proinflammatory molecules NF-kB activation and inhibits neutrophil infiltration in tissues

From these results we suggest that anti-inflammatoryactivity observed is due to a synergic action of these phenoliccomponents contained in EtOH extract of ALE

C scolymus is one of the few herbal remedies which havebeen verified through experimental studies Further the toxi-city studies of ALE are required in order to confirm the safetyof this medicinal plant in the treatment of inflammationdiseases

5 Conclusion

The present study showed clearly the advantages of Cscolymus leaves extracts which have safer anti-inflammatoryprofile with potent antioxidant activity attributed to thephenolic compounds Furthermore in the future studieswe are interested to characterize the action mechanisms ofactive phenolic compounds of ALE responsible for anti-inflammatory and antioxidants activities

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Tunisian Ministry ofHigher Education and Scientific Research

References

[1] C Pasquier ldquoStress oxidative et inflammationrdquo Blool vol 4 pp1ndash6 1996

[2] M A Abuashwashi O M Palomino and M P Gomez-Serranillos ldquoGeographic origin influences the phenolic com-position and antioxidant potential of wild Crataegus monogynafrom Spainrdquo Pharmaceutical Biology vol 54 no 11 pp 2708ndash2713 2016

[3] M B Salem H Affes K Ksouda et al ldquoPharmacological stud-ies of artichoke leaf extract and their health benefitsrdquo PlantFoods for Human Nutrition vol 70 no 4 pp 441ndash453 2015

[4] M-H Huang B-S Wang C-S Chiu et al ldquoAntioxidant anti-nociceptive and anti-inflammatory activities ofXanthii Fructusextractrdquo Journal of Ethnopharmacology vol 135 no 2 pp 545ndash552 2011

[5] G-J Huang S-S Huang S-S Lin et al ldquoAnalgesic effectsand the mechanisms of anti-inflammation of ergostatrien-3120573-ol from antrodia camphorata submerged whole broth in micerdquoJournal of Agricultural and Food Chemistry vol 58 no 12 pp7445ndash7452 2010

[6] C Tohda N Nakayama F Hatanaka and K Komatsu ldquoCom-parison of anti-inflammatory activities of six Curcuma rhi-zomes a possible curcuminoid-independent pathway mediatedby Curcuma phaeocaulis extractrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 3 no 2 pp 255ndash260 2006

[7] K Goksel K Murat Y Murat E Sevil and H Fidan ldquoAntiox-idant activities of ethanol extracts ofHypericum triquetrifoliumandHypericum scabroidesrdquo Pharmaceutical Biology vol 46 no4 pp 231ndash242 2008

[8] R Gebhardt and M Fausel ldquoAntioxidant and hepatoprotectiveeffects of artichoke extracts and constituents in cultured rathepatocytesrdquoToxicology inVitro vol 11 no 5 pp 669ndash672 1997

[9] A SofworaMedicinal Plants and TraditionalMedicine in AfricaJohn Wiley amp Sons Ann Arbor Mich USA 1982

[10] D E Okwu ldquoPhytochemicals vitamins andmineral contents oftwo Nigerian medicinal plantsrdquo International Journal of Mole-cular Medicine and Advances Sciences vol 1 pp 375ndash381 2005

[11] N Tsevegsuren GDavaakhuu andT S Udval ldquoPhytochemicalanalysis of Cynara scolymus L cultivated in MongoliardquoMongo-lian Journal of Chemistry vol 15 no 41 pp 40ndash42 2014

[12] AACC Approved Methods of the AACC American Associationof Cereal Chemists St Paul Minn USA 8th edition 1984

[13] S C Lee L Prosky and J W DeVries ldquoDetermination of totalsoluble and insoluble dietary fiber in foodsmdashenzymatic-gravi-metric method MES-TRIS buffer collaborative studyrdquo Journalof the Association of Official Analytical Chemists vol 75 pp395ndash416 1992

[14] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[15] O A Fawole U L Opara and K I Theron ldquoChemical andphytochemical properties and antioxidant activities of threepomegranate cultivars grown in South Africardquo Food and Bio-process Technology vol 5 no 7 pp 2934ndash2940 2012

[16] V Dewanto X Wu K K Adom and R H Liu ldquoThermalprocessing enhances the nutritional value of tomatoes byincreasing total antioxidant activityrdquo Journal of Agricultural andFood Chemistry vol 50 no 10 pp 3010ndash3014 2002

[17] B Sun J M Ricardo-da-Silva and I Spranger ldquoCritical factorsof vanillin assay for catechins and proanthocyanidinsrdquo Journal

Evidence-Based Complementary and Alternative Medicine 13

of Agricultural and Food Chemistry vol 46 no 10 pp 4267ndash4274 1998

[18] AOACOfficial Methods of Analysis vol 1 AOACWashingtonDC USA 15th edition 1990

[19] D Pljevljakusic K Savikin T Jankovic et al ldquoChemical prop-erties of the cultivated Sideritis raeseri Boiss amp Heldr subspraeserirdquo Food Chemistry vol 124 no 1 pp 226ndash233 2011

[20] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[21] I I Koleva T A Van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] V Ravi T S M Saleem S S Patel J Raamamurthy and KGauthaman ldquoAnti-inflammatory effect of methanolic extract ofSolanum nigrum Linn Berriesrdquo International Journal of AppliedResearch in Natural Products vol 2 pp 33ndash36 2009

[24] D Chattopadhyay G Arunachalam A B Mandal T K SurS C Mandal and S K Bhattacharya ldquoAntimicrobial and anti-inflammatory activity of folkloreMallotus peltatus leaf extractrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 229ndash2372002

[25] L R Morse K Stolzmann H P Nguyen et al ldquoAssociationbetween mobility mode and c-reactive protein levels in menwith chronic spinal cord injuryrdquo Archives of Physical Medicineand Rehabilitation vol 89 no 4 pp 726ndash731 2008

[26] A Clauss ldquoGerinnungsphysiologische schnellmethode zur bes-timmung des fibrinogensrdquoActaHaematologica vol 17 no 4 pp237ndash246 1957

[27] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[28] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidationin post-mitotic tissues of ageing ratrdquo Cell Biochemistry andFunction vol 24 no 1 pp 79ndash85 2006

[29] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[30] W F Beyer Jr and I Fridovich ldquoAssaying for superoxide dis-mutase activity some large consequences of minor changes inconditionsrdquoAnalytical Biochemistry vol 161 no 2 pp 559ndash5661987

[31] I Carlberg and B Mannervik ldquoGlutathione reductaserdquo inGlutamate Glutamine Glutathione and Related Compoundsvol 113 ofMethods in Enzymology pp 484ndash490 Elsevier 1985

[32] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[33] V Emanue V Adrian N A Sultana and C Svetlana ldquoAntiox-idant and antimicrobial activities of ethanol extracts of Cynarascolymus (Cynarae folium Asteraceae family)rdquo Tropical Journalof Pharmaceutical Research vol 10 no 6 pp 777ndash783 2011

[34] G L D S Oliveira R A M O Francisco O B A Vinıcius etal ldquoEvaluation of antioxidant capacity of the aqueous extract ofCynara scolymus L (Asteraceae) in vitro and in SaccharomycescerevisiaerdquoAfrican Journal of Pharmacy and Pharmacology vol8 pp 136ndash147 2014

[35] K Horiuchi S Shiota T Hatano T Yoshida T Kuroda and TTsuchiya ldquoAntimicrobial activity of oleanolic acid from Salviaofficinalis and related compounds on vancomycin-resistantenterococci (VRE)rdquo Biological and Pharmaceutical Bulletin vol30 no 6 pp 1147ndash1149 2007

[36] A Daoud D Malika S Bakari et al ldquoAssessment of polyphe-nol composition antioxidant and antimicrobial properties ofvarious extracts of Date Palm Pollen (DPP) from two Tunisiancultivarsrdquo Arabian Journal of Chemistry 2015

[37] D Krishnaiah B Awang S Rosalam and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversitymdashEngineering Sciences vol 1 pp 63ndash66 2015

[38] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[39] A Betancor-Fernandez A Perez-Galvez H Sies and W StahlldquoScreening pharmaceutical preparations containing extracts ofturmeric rhizome artichoke leaf devilrsquos claw root and garlic orsalmon oil for antioxidant capacityrdquo Journal of Pharmacy andPharmacology vol 55 no 7 pp 981ndash986 2003

[40] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[41] S Konyal120580oglu H Saglam and B K120580vcak ldquo120572-Tocopherolflavonoid and phenol contents and antioxidant activity of Ficuscarica leavesrdquo Pharmaceutical Biology vol 43 no 8 pp 683ndash686 2005

[42] D U Bawankule P Trivedi A Pal et al ldquoProtectivemechanismof lignans from Phyllanthus amarus against galactosaminelipopolysaccharide-induced hepatitis an in-vivo and in-silicostudiesrdquo Current Topics in Medicinal Chemistry vol 14 no 8pp 1045ndash1055 2014

[43] GAHiggs R J Flower and J RVane ldquoAnew approach to anti-inflammatory drugsrdquo Biochemical Pharmacology vol 28 no 12pp 1959ndash1961 1979

[44] A A Hemmati H Kalantari A Siahpoosh B Ghorbanzadehand H Jamali ldquoAnti-inflammatory Effect of HydroalcoholicExtract of the Washingtonia filifera Seeds in Carrageenan-Induced Paw Edema in Ratsrdquo Jundishapur Journal of NaturalPharmaceutical Products vol 10 no 1 pp 19ndash25 2015

[45] I Posadas M Bucci F Roviezzo et al ldquoCarrageenan-inducedmouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expressionrdquoBritish Journal of Pharmacology vol 142 no 2 pp 331ndash3382004

[46] K Pundarikakshudu D H Shah A H Panchal and G CBhavsar ldquoAnti-inflammatory activity of fenugreek (Trigonellafoenum-graecum Linn) seed petroleum ether extractrdquo IndianJournal of Pharmacology vol 48 no 4 pp 441ndash444 2016

[47] S Cuzzocrea G Costantino E Mazzon B Zingarelli A DeSarro and A P Caputi ldquoProtective effects of Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) a superoxide dismutasemimetic in paw oedema induced by carrageenan in the ratrdquoBiochemical Pharmacology vol 58 no 1 pp 171ndash176 1999

[48] M A Mendall P Patel M Asante et al ldquoRelation of serumcytokine concentrations to cardiovascular risk factors andcoronary heart diseaserdquo Heart vol 78 no 3 pp 273ndash277 1997

[49] A Ialenti A Ianaro S Moncada andM Di Rosa ldquoModulationof acute inflammation by endogenous nitric oxiderdquo EuropeanJournal of Pharmacology vol 211 no 2 pp 177ndash182 1992

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

Table7Eff

ecto

fCynarascolym

usleaves

extracto

ncarrageenan-indu

cedratp

awoedema

Treatm

ent

Oedem

asize(meanplusmnS

D)(mm)

0h1h

2h3h

4h5h

Con

trol

0426plusmn0

00

044

1plusmn001

056plusmn0

03

0650plusmn0

05

0755plusmn0

05

070plusmn0

01

Carr

0426plusmn0

00

28plusmn026120572lowastlowastlowast

333plusmn028120572lowastlowastlowast

325plusmn002120572lowastlowastlowast

315plusmn001120572lowastlowastlowast

302plusmn009120572lowastlowastlowast

Carr+Indo

(10m

gkg)

0426plusmn0

00

246plusmn026120572lowastlowastlowast120573lowast

261plusmn007120572lowastlowastlowast120573lowastlowastlowast

230plusmn002120572lowastlowastlowast120573lowastlowastlowast

18plusmn011120572lowastlowastlowast120573lowastlowastlowast

14plusmn006120572lowastlowastlowast120573lowastlowastlowast

Carr+ALE

(400

mgkgbw)

0426plusmn0

00

2480plusmn005120572lowastlowastlowast120573lowastlowastlowast

188plusmn004120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

165plusmn005120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

116plusmn020120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

112plusmn01120572lowastlowastlowast120573lowastlowastlowastmlowastlowastlowast

Values

representm

eansplusmnSD

(119899=6)ineach

grou

plowast119901lt005lowastlowast119901lt001andlowastlowastlowast119901lt0001

120572com

paredto

control120573com

paredto

Carrmcom

paredto

Carr+Indo

Evidence-Based Complementary and Alternative Medicine 11

Table 8 Effects of Cynara scolymus leaves extract and Indomethacin on CAT SOD GSH AOPP andMDA activities in carrageenan inducedpaw oedema

GroupsCAT (120583molesCATminmg

protein)

SOD (unitSODminmg

protein)

GSH (nmolesmgprotein)

MDA (nmolMDAmg protein) AOPP (nmolmg protein)

Control 47 plusmn 2 131 plusmn 43 421 plusmn 12 234 plusmn 5 484 plusmn 43Carr 162 plusmn 05120572lowastlowastlowast 153 plusmn 26120572lowastlowastlowast 131 plusmn 11120572lowastlowastlowast 314 plusmn 75120572lowastlowastlowast 679 plusmn 69120572lowastlowastlowastCarr + Indo(10mgkgbw) 649 plusmn 71120573lowastlowastlowast 240 plusmn 13120573lowastlowastlowast 703 plusmn 11120573lowastlowastlowast 209 plusmn 30120573lowastlowastlowast 442 plusmn 06120573lowastlowastlowastCarr + ALE(400mgkgbw) 565 plusmn 25120573lowastlowastlowastmlowastlowast 207 plusmn 40120573lowastlowastlowastmlowastlowast 756 plusmn 25120573lowastlowastlowastmlowast 208 plusmn 60120573lowastlowastlowast 502 plusmn 41120573lowastlowastlowastmlowastValues represent means plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001120572 compared to control 120573 compared to Carr m compared to Carr + IndoSOD superoxide dismutaseCAT catalaseGSH glutathione peroxidaseMDA MalondialdehydeAOPP Advanced Oxidation Protein Product

Table 9 Quantification and identification of phenolic compounds contents in the ethanol extract of Cynara scolymus leaves extracts

Number Content (mg100 g of dry extract) Polyphenolic compound1 28 plusmn 019 Hydroxytyrosol2 381 plusmn 036 Verbascoside3 99 plusmn 002 Apigenin-7-glucoside4 18 plusmn 01 Oleuropein5 192 plusmn 014 Quercetin6 5 plusmn 005 Pinoresinol7 4 plusmn 004 ApigeninValues represent means plusmn SD (119899 = 3)

In the second phase the increase of vascular permeabilityis observed by release of kinins during 230 hThereafter from230 h to 5 h inflammation ismediated by prostaglandins andis also associated with a release infiltration and migration ofPN into the inflamed site [47]

Our results indicate that ALE affords protection againstthe Carr induced acute inflammation in dose dependentmanner EtOH extract of ALE at dose of 400mgkgbwexhibits significant anti-inflammatory activity with 73 inhi-bition of paw oedema compared with Indo group (53) andtypically reaches a maximum at 5 h these results obtainedsuggest the anti-inflammatory effect of EtOH extract of ALEbymeans of inhibiting the synthesis and the release of inflam-matory mediators like histamine serotonin and prostagl-andins that are involved in acute inflammationThese resultsrevealed the inhibitory effect of EtOH extract on PN migra-tion and it is confirmed through a histological analysis oftissues of paw oedema in experimental groups

These findings clearly confirm that EtOH extract of ALEhas an anti-inflammatory effect by reducing the influx ofpolymorphonuclear cells to inflammatory tissue followinginjection of Carr Most inflammatory markers such asinterleukin-6 (IL-6) tumor necrosis factor-120572 (TNF-120572) CRPand fibrinogen increased significantly in response to infec-tion and in active diseases states CRP is the specific marker

of acute inflammation occurring in the body [48] moreoverthe fibrinogen is themost relevant indicator of inflammationand it does not only represent the acute-phase reactant that isincreased in inflammatory states Elevated fibrinogen levelswould be associated with higher levels of CRP and wouldsimilarly correlate with inflammation reaction

Therefore the present study showed that EtOH extract ofALE significantly decreases the level of fibrinogen and CRPby value of 2272 and 3478 in comparison with Carrgroup

The induction of inflammationwithCarr ismanifested bygeneration of ROS and has been shown to play an importantrole in various forms of inflammation [49 50] High concen-tration of reactive free radicals contributes to lipid perox-idation and protein oxidation [3] Our results showed thatthere is a significant increase in MDA and AOPP activitiesin Carr group when compared to control group and a highlysignificant decrease in activities of SOD CAT and GSH alsoobserved at the tissue level compared with the control group(119901 lt 0001) However the injection of EtOH extract and Indoshows a significant decrease of the levels of MDA and AOPPby 3427 and 4427 following a significant increase inantioxidant activities as CAT SOD andGSHwhen comparedto Carr group (2016 7330 and 75) From these resultsit is implied that the protective effect of EtOH extract may

12 Evidence-Based Complementary and Alternative Medicine

be attributed to its potential indirectly as a stimulator ofthe activity and expression of antioxidant enzymes duringinflammation

Thus the anti-inflammatory activity is due to the individ-ual or synergistic effect of the components in the ALE In factprevious studies have found that anti-inflammatory profile ofALE could be related to polyphenolic compounds accordingto the phytochemical analysis We performed HPLC analysisto justify the correlation between phytochemical compoundsand anti-inflammatory activity of this plant Among thesepolyphenolic compounds verbascoside has been reportedin several reports and showed a potential spectrum ofmany activities including antioxidant and anti-inflammatoryParticular verbascoside presents antiedematogenic activitiesin animal models of Carr induced inflammation [51] and theresponses exhibited themaximumactivity for the fourth hourof treatment Such activities may be derived from inhibitoryaction of chemical mediators of the inflammatory processsuch as histamine and bradykinin A significant antioxidanteffect of verbascoside has been recently reported byAleo et al[52] in an experimental study [53] Other authors reportedthat anti-inflammatory activity of verbascoside has beenevaluated by an in vitro test performed on cell cultures of pri-mary human keratinocytes [54] in which it was able to reducethe release of proinflammatory markers such as chemokines

Apigenin-7-glucoside was found to block the releaseof several varieties of enzymes involved in inflammationincluding especially lipoxygenases and cyclooxygenases [5556] leading to inhibition of proinflammatory molecules NF-kB activation and inhibits neutrophil infiltration in tissues

From these results we suggest that anti-inflammatoryactivity observed is due to a synergic action of these phenoliccomponents contained in EtOH extract of ALE

C scolymus is one of the few herbal remedies which havebeen verified through experimental studies Further the toxi-city studies of ALE are required in order to confirm the safetyof this medicinal plant in the treatment of inflammationdiseases

5 Conclusion

The present study showed clearly the advantages of Cscolymus leaves extracts which have safer anti-inflammatoryprofile with potent antioxidant activity attributed to thephenolic compounds Furthermore in the future studieswe are interested to characterize the action mechanisms ofactive phenolic compounds of ALE responsible for anti-inflammatory and antioxidants activities

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Tunisian Ministry ofHigher Education and Scientific Research

References

[1] C Pasquier ldquoStress oxidative et inflammationrdquo Blool vol 4 pp1ndash6 1996

[2] M A Abuashwashi O M Palomino and M P Gomez-Serranillos ldquoGeographic origin influences the phenolic com-position and antioxidant potential of wild Crataegus monogynafrom Spainrdquo Pharmaceutical Biology vol 54 no 11 pp 2708ndash2713 2016

[3] M B Salem H Affes K Ksouda et al ldquoPharmacological stud-ies of artichoke leaf extract and their health benefitsrdquo PlantFoods for Human Nutrition vol 70 no 4 pp 441ndash453 2015

[4] M-H Huang B-S Wang C-S Chiu et al ldquoAntioxidant anti-nociceptive and anti-inflammatory activities ofXanthii Fructusextractrdquo Journal of Ethnopharmacology vol 135 no 2 pp 545ndash552 2011

[5] G-J Huang S-S Huang S-S Lin et al ldquoAnalgesic effectsand the mechanisms of anti-inflammation of ergostatrien-3120573-ol from antrodia camphorata submerged whole broth in micerdquoJournal of Agricultural and Food Chemistry vol 58 no 12 pp7445ndash7452 2010

[6] C Tohda N Nakayama F Hatanaka and K Komatsu ldquoCom-parison of anti-inflammatory activities of six Curcuma rhi-zomes a possible curcuminoid-independent pathway mediatedby Curcuma phaeocaulis extractrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 3 no 2 pp 255ndash260 2006

[7] K Goksel K Murat Y Murat E Sevil and H Fidan ldquoAntiox-idant activities of ethanol extracts ofHypericum triquetrifoliumandHypericum scabroidesrdquo Pharmaceutical Biology vol 46 no4 pp 231ndash242 2008

[8] R Gebhardt and M Fausel ldquoAntioxidant and hepatoprotectiveeffects of artichoke extracts and constituents in cultured rathepatocytesrdquoToxicology inVitro vol 11 no 5 pp 669ndash672 1997

[9] A SofworaMedicinal Plants and TraditionalMedicine in AfricaJohn Wiley amp Sons Ann Arbor Mich USA 1982

[10] D E Okwu ldquoPhytochemicals vitamins andmineral contents oftwo Nigerian medicinal plantsrdquo International Journal of Mole-cular Medicine and Advances Sciences vol 1 pp 375ndash381 2005

[11] N Tsevegsuren GDavaakhuu andT S Udval ldquoPhytochemicalanalysis of Cynara scolymus L cultivated in MongoliardquoMongo-lian Journal of Chemistry vol 15 no 41 pp 40ndash42 2014

[12] AACC Approved Methods of the AACC American Associationof Cereal Chemists St Paul Minn USA 8th edition 1984

[13] S C Lee L Prosky and J W DeVries ldquoDetermination of totalsoluble and insoluble dietary fiber in foodsmdashenzymatic-gravi-metric method MES-TRIS buffer collaborative studyrdquo Journalof the Association of Official Analytical Chemists vol 75 pp395ndash416 1992

[14] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[15] O A Fawole U L Opara and K I Theron ldquoChemical andphytochemical properties and antioxidant activities of threepomegranate cultivars grown in South Africardquo Food and Bio-process Technology vol 5 no 7 pp 2934ndash2940 2012

[16] V Dewanto X Wu K K Adom and R H Liu ldquoThermalprocessing enhances the nutritional value of tomatoes byincreasing total antioxidant activityrdquo Journal of Agricultural andFood Chemistry vol 50 no 10 pp 3010ndash3014 2002

[17] B Sun J M Ricardo-da-Silva and I Spranger ldquoCritical factorsof vanillin assay for catechins and proanthocyanidinsrdquo Journal

Evidence-Based Complementary and Alternative Medicine 13

of Agricultural and Food Chemistry vol 46 no 10 pp 4267ndash4274 1998

[18] AOACOfficial Methods of Analysis vol 1 AOACWashingtonDC USA 15th edition 1990

[19] D Pljevljakusic K Savikin T Jankovic et al ldquoChemical prop-erties of the cultivated Sideritis raeseri Boiss amp Heldr subspraeserirdquo Food Chemistry vol 124 no 1 pp 226ndash233 2011

[20] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[21] I I Koleva T A Van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] V Ravi T S M Saleem S S Patel J Raamamurthy and KGauthaman ldquoAnti-inflammatory effect of methanolic extract ofSolanum nigrum Linn Berriesrdquo International Journal of AppliedResearch in Natural Products vol 2 pp 33ndash36 2009

[24] D Chattopadhyay G Arunachalam A B Mandal T K SurS C Mandal and S K Bhattacharya ldquoAntimicrobial and anti-inflammatory activity of folkloreMallotus peltatus leaf extractrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 229ndash2372002

[25] L R Morse K Stolzmann H P Nguyen et al ldquoAssociationbetween mobility mode and c-reactive protein levels in menwith chronic spinal cord injuryrdquo Archives of Physical Medicineand Rehabilitation vol 89 no 4 pp 726ndash731 2008

[26] A Clauss ldquoGerinnungsphysiologische schnellmethode zur bes-timmung des fibrinogensrdquoActaHaematologica vol 17 no 4 pp237ndash246 1957

[27] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[28] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidationin post-mitotic tissues of ageing ratrdquo Cell Biochemistry andFunction vol 24 no 1 pp 79ndash85 2006

[29] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[30] W F Beyer Jr and I Fridovich ldquoAssaying for superoxide dis-mutase activity some large consequences of minor changes inconditionsrdquoAnalytical Biochemistry vol 161 no 2 pp 559ndash5661987

[31] I Carlberg and B Mannervik ldquoGlutathione reductaserdquo inGlutamate Glutamine Glutathione and Related Compoundsvol 113 ofMethods in Enzymology pp 484ndash490 Elsevier 1985

[32] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[33] V Emanue V Adrian N A Sultana and C Svetlana ldquoAntiox-idant and antimicrobial activities of ethanol extracts of Cynarascolymus (Cynarae folium Asteraceae family)rdquo Tropical Journalof Pharmaceutical Research vol 10 no 6 pp 777ndash783 2011

[34] G L D S Oliveira R A M O Francisco O B A Vinıcius etal ldquoEvaluation of antioxidant capacity of the aqueous extract ofCynara scolymus L (Asteraceae) in vitro and in SaccharomycescerevisiaerdquoAfrican Journal of Pharmacy and Pharmacology vol8 pp 136ndash147 2014

[35] K Horiuchi S Shiota T Hatano T Yoshida T Kuroda and TTsuchiya ldquoAntimicrobial activity of oleanolic acid from Salviaofficinalis and related compounds on vancomycin-resistantenterococci (VRE)rdquo Biological and Pharmaceutical Bulletin vol30 no 6 pp 1147ndash1149 2007

[36] A Daoud D Malika S Bakari et al ldquoAssessment of polyphe-nol composition antioxidant and antimicrobial properties ofvarious extracts of Date Palm Pollen (DPP) from two Tunisiancultivarsrdquo Arabian Journal of Chemistry 2015

[37] D Krishnaiah B Awang S Rosalam and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversitymdashEngineering Sciences vol 1 pp 63ndash66 2015

[38] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[39] A Betancor-Fernandez A Perez-Galvez H Sies and W StahlldquoScreening pharmaceutical preparations containing extracts ofturmeric rhizome artichoke leaf devilrsquos claw root and garlic orsalmon oil for antioxidant capacityrdquo Journal of Pharmacy andPharmacology vol 55 no 7 pp 981ndash986 2003

[40] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[41] S Konyal120580oglu H Saglam and B K120580vcak ldquo120572-Tocopherolflavonoid and phenol contents and antioxidant activity of Ficuscarica leavesrdquo Pharmaceutical Biology vol 43 no 8 pp 683ndash686 2005

[42] D U Bawankule P Trivedi A Pal et al ldquoProtectivemechanismof lignans from Phyllanthus amarus against galactosaminelipopolysaccharide-induced hepatitis an in-vivo and in-silicostudiesrdquo Current Topics in Medicinal Chemistry vol 14 no 8pp 1045ndash1055 2014

[43] GAHiggs R J Flower and J RVane ldquoAnew approach to anti-inflammatory drugsrdquo Biochemical Pharmacology vol 28 no 12pp 1959ndash1961 1979

[44] A A Hemmati H Kalantari A Siahpoosh B Ghorbanzadehand H Jamali ldquoAnti-inflammatory Effect of HydroalcoholicExtract of the Washingtonia filifera Seeds in Carrageenan-Induced Paw Edema in Ratsrdquo Jundishapur Journal of NaturalPharmaceutical Products vol 10 no 1 pp 19ndash25 2015

[45] I Posadas M Bucci F Roviezzo et al ldquoCarrageenan-inducedmouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expressionrdquoBritish Journal of Pharmacology vol 142 no 2 pp 331ndash3382004

[46] K Pundarikakshudu D H Shah A H Panchal and G CBhavsar ldquoAnti-inflammatory activity of fenugreek (Trigonellafoenum-graecum Linn) seed petroleum ether extractrdquo IndianJournal of Pharmacology vol 48 no 4 pp 441ndash444 2016

[47] S Cuzzocrea G Costantino E Mazzon B Zingarelli A DeSarro and A P Caputi ldquoProtective effects of Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) a superoxide dismutasemimetic in paw oedema induced by carrageenan in the ratrdquoBiochemical Pharmacology vol 58 no 1 pp 171ndash176 1999

[48] M A Mendall P Patel M Asante et al ldquoRelation of serumcytokine concentrations to cardiovascular risk factors andcoronary heart diseaserdquo Heart vol 78 no 3 pp 273ndash277 1997

[49] A Ialenti A Ianaro S Moncada andM Di Rosa ldquoModulationof acute inflammation by endogenous nitric oxiderdquo EuropeanJournal of Pharmacology vol 211 no 2 pp 177ndash182 1992

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 11

Table 8 Effects of Cynara scolymus leaves extract and Indomethacin on CAT SOD GSH AOPP andMDA activities in carrageenan inducedpaw oedema

GroupsCAT (120583molesCATminmg

protein)

SOD (unitSODminmg

protein)

GSH (nmolesmgprotein)

MDA (nmolMDAmg protein) AOPP (nmolmg protein)

Control 47 plusmn 2 131 plusmn 43 421 plusmn 12 234 plusmn 5 484 plusmn 43Carr 162 plusmn 05120572lowastlowastlowast 153 plusmn 26120572lowastlowastlowast 131 plusmn 11120572lowastlowastlowast 314 plusmn 75120572lowastlowastlowast 679 plusmn 69120572lowastlowastlowastCarr + Indo(10mgkgbw) 649 plusmn 71120573lowastlowastlowast 240 plusmn 13120573lowastlowastlowast 703 plusmn 11120573lowastlowastlowast 209 plusmn 30120573lowastlowastlowast 442 plusmn 06120573lowastlowastlowastCarr + ALE(400mgkgbw) 565 plusmn 25120573lowastlowastlowastmlowastlowast 207 plusmn 40120573lowastlowastlowastmlowastlowast 756 plusmn 25120573lowastlowastlowastmlowast 208 plusmn 60120573lowastlowastlowast 502 plusmn 41120573lowastlowastlowastmlowastValues represent means plusmn SD (119899 = 6) in each group lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001120572 compared to control 120573 compared to Carr m compared to Carr + IndoSOD superoxide dismutaseCAT catalaseGSH glutathione peroxidaseMDA MalondialdehydeAOPP Advanced Oxidation Protein Product

Table 9 Quantification and identification of phenolic compounds contents in the ethanol extract of Cynara scolymus leaves extracts

Number Content (mg100 g of dry extract) Polyphenolic compound1 28 plusmn 019 Hydroxytyrosol2 381 plusmn 036 Verbascoside3 99 plusmn 002 Apigenin-7-glucoside4 18 plusmn 01 Oleuropein5 192 plusmn 014 Quercetin6 5 plusmn 005 Pinoresinol7 4 plusmn 004 ApigeninValues represent means plusmn SD (119899 = 3)

In the second phase the increase of vascular permeabilityis observed by release of kinins during 230 hThereafter from230 h to 5 h inflammation ismediated by prostaglandins andis also associated with a release infiltration and migration ofPN into the inflamed site [47]

Our results indicate that ALE affords protection againstthe Carr induced acute inflammation in dose dependentmanner EtOH extract of ALE at dose of 400mgkgbwexhibits significant anti-inflammatory activity with 73 inhi-bition of paw oedema compared with Indo group (53) andtypically reaches a maximum at 5 h these results obtainedsuggest the anti-inflammatory effect of EtOH extract of ALEbymeans of inhibiting the synthesis and the release of inflam-matory mediators like histamine serotonin and prostagl-andins that are involved in acute inflammationThese resultsrevealed the inhibitory effect of EtOH extract on PN migra-tion and it is confirmed through a histological analysis oftissues of paw oedema in experimental groups

These findings clearly confirm that EtOH extract of ALEhas an anti-inflammatory effect by reducing the influx ofpolymorphonuclear cells to inflammatory tissue followinginjection of Carr Most inflammatory markers such asinterleukin-6 (IL-6) tumor necrosis factor-120572 (TNF-120572) CRPand fibrinogen increased significantly in response to infec-tion and in active diseases states CRP is the specific marker

of acute inflammation occurring in the body [48] moreoverthe fibrinogen is themost relevant indicator of inflammationand it does not only represent the acute-phase reactant that isincreased in inflammatory states Elevated fibrinogen levelswould be associated with higher levels of CRP and wouldsimilarly correlate with inflammation reaction

Therefore the present study showed that EtOH extract ofALE significantly decreases the level of fibrinogen and CRPby value of 2272 and 3478 in comparison with Carrgroup

The induction of inflammationwithCarr ismanifested bygeneration of ROS and has been shown to play an importantrole in various forms of inflammation [49 50] High concen-tration of reactive free radicals contributes to lipid perox-idation and protein oxidation [3] Our results showed thatthere is a significant increase in MDA and AOPP activitiesin Carr group when compared to control group and a highlysignificant decrease in activities of SOD CAT and GSH alsoobserved at the tissue level compared with the control group(119901 lt 0001) However the injection of EtOH extract and Indoshows a significant decrease of the levels of MDA and AOPPby 3427 and 4427 following a significant increase inantioxidant activities as CAT SOD andGSHwhen comparedto Carr group (2016 7330 and 75) From these resultsit is implied that the protective effect of EtOH extract may

12 Evidence-Based Complementary and Alternative Medicine

be attributed to its potential indirectly as a stimulator ofthe activity and expression of antioxidant enzymes duringinflammation

Thus the anti-inflammatory activity is due to the individ-ual or synergistic effect of the components in the ALE In factprevious studies have found that anti-inflammatory profile ofALE could be related to polyphenolic compounds accordingto the phytochemical analysis We performed HPLC analysisto justify the correlation between phytochemical compoundsand anti-inflammatory activity of this plant Among thesepolyphenolic compounds verbascoside has been reportedin several reports and showed a potential spectrum ofmany activities including antioxidant and anti-inflammatoryParticular verbascoside presents antiedematogenic activitiesin animal models of Carr induced inflammation [51] and theresponses exhibited themaximumactivity for the fourth hourof treatment Such activities may be derived from inhibitoryaction of chemical mediators of the inflammatory processsuch as histamine and bradykinin A significant antioxidanteffect of verbascoside has been recently reported byAleo et al[52] in an experimental study [53] Other authors reportedthat anti-inflammatory activity of verbascoside has beenevaluated by an in vitro test performed on cell cultures of pri-mary human keratinocytes [54] in which it was able to reducethe release of proinflammatory markers such as chemokines

Apigenin-7-glucoside was found to block the releaseof several varieties of enzymes involved in inflammationincluding especially lipoxygenases and cyclooxygenases [5556] leading to inhibition of proinflammatory molecules NF-kB activation and inhibits neutrophil infiltration in tissues

From these results we suggest that anti-inflammatoryactivity observed is due to a synergic action of these phenoliccomponents contained in EtOH extract of ALE

C scolymus is one of the few herbal remedies which havebeen verified through experimental studies Further the toxi-city studies of ALE are required in order to confirm the safetyof this medicinal plant in the treatment of inflammationdiseases

5 Conclusion

The present study showed clearly the advantages of Cscolymus leaves extracts which have safer anti-inflammatoryprofile with potent antioxidant activity attributed to thephenolic compounds Furthermore in the future studieswe are interested to characterize the action mechanisms ofactive phenolic compounds of ALE responsible for anti-inflammatory and antioxidants activities

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Tunisian Ministry ofHigher Education and Scientific Research

References

[1] C Pasquier ldquoStress oxidative et inflammationrdquo Blool vol 4 pp1ndash6 1996

[2] M A Abuashwashi O M Palomino and M P Gomez-Serranillos ldquoGeographic origin influences the phenolic com-position and antioxidant potential of wild Crataegus monogynafrom Spainrdquo Pharmaceutical Biology vol 54 no 11 pp 2708ndash2713 2016

[3] M B Salem H Affes K Ksouda et al ldquoPharmacological stud-ies of artichoke leaf extract and their health benefitsrdquo PlantFoods for Human Nutrition vol 70 no 4 pp 441ndash453 2015

[4] M-H Huang B-S Wang C-S Chiu et al ldquoAntioxidant anti-nociceptive and anti-inflammatory activities ofXanthii Fructusextractrdquo Journal of Ethnopharmacology vol 135 no 2 pp 545ndash552 2011

[5] G-J Huang S-S Huang S-S Lin et al ldquoAnalgesic effectsand the mechanisms of anti-inflammation of ergostatrien-3120573-ol from antrodia camphorata submerged whole broth in micerdquoJournal of Agricultural and Food Chemistry vol 58 no 12 pp7445ndash7452 2010

[6] C Tohda N Nakayama F Hatanaka and K Komatsu ldquoCom-parison of anti-inflammatory activities of six Curcuma rhi-zomes a possible curcuminoid-independent pathway mediatedby Curcuma phaeocaulis extractrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 3 no 2 pp 255ndash260 2006

[7] K Goksel K Murat Y Murat E Sevil and H Fidan ldquoAntiox-idant activities of ethanol extracts ofHypericum triquetrifoliumandHypericum scabroidesrdquo Pharmaceutical Biology vol 46 no4 pp 231ndash242 2008

[8] R Gebhardt and M Fausel ldquoAntioxidant and hepatoprotectiveeffects of artichoke extracts and constituents in cultured rathepatocytesrdquoToxicology inVitro vol 11 no 5 pp 669ndash672 1997

[9] A SofworaMedicinal Plants and TraditionalMedicine in AfricaJohn Wiley amp Sons Ann Arbor Mich USA 1982

[10] D E Okwu ldquoPhytochemicals vitamins andmineral contents oftwo Nigerian medicinal plantsrdquo International Journal of Mole-cular Medicine and Advances Sciences vol 1 pp 375ndash381 2005

[11] N Tsevegsuren GDavaakhuu andT S Udval ldquoPhytochemicalanalysis of Cynara scolymus L cultivated in MongoliardquoMongo-lian Journal of Chemistry vol 15 no 41 pp 40ndash42 2014

[12] AACC Approved Methods of the AACC American Associationof Cereal Chemists St Paul Minn USA 8th edition 1984

[13] S C Lee L Prosky and J W DeVries ldquoDetermination of totalsoluble and insoluble dietary fiber in foodsmdashenzymatic-gravi-metric method MES-TRIS buffer collaborative studyrdquo Journalof the Association of Official Analytical Chemists vol 75 pp395ndash416 1992

[14] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[15] O A Fawole U L Opara and K I Theron ldquoChemical andphytochemical properties and antioxidant activities of threepomegranate cultivars grown in South Africardquo Food and Bio-process Technology vol 5 no 7 pp 2934ndash2940 2012

[16] V Dewanto X Wu K K Adom and R H Liu ldquoThermalprocessing enhances the nutritional value of tomatoes byincreasing total antioxidant activityrdquo Journal of Agricultural andFood Chemistry vol 50 no 10 pp 3010ndash3014 2002

[17] B Sun J M Ricardo-da-Silva and I Spranger ldquoCritical factorsof vanillin assay for catechins and proanthocyanidinsrdquo Journal

Evidence-Based Complementary and Alternative Medicine 13

of Agricultural and Food Chemistry vol 46 no 10 pp 4267ndash4274 1998

[18] AOACOfficial Methods of Analysis vol 1 AOACWashingtonDC USA 15th edition 1990

[19] D Pljevljakusic K Savikin T Jankovic et al ldquoChemical prop-erties of the cultivated Sideritis raeseri Boiss amp Heldr subspraeserirdquo Food Chemistry vol 124 no 1 pp 226ndash233 2011

[20] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[21] I I Koleva T A Van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] V Ravi T S M Saleem S S Patel J Raamamurthy and KGauthaman ldquoAnti-inflammatory effect of methanolic extract ofSolanum nigrum Linn Berriesrdquo International Journal of AppliedResearch in Natural Products vol 2 pp 33ndash36 2009

[24] D Chattopadhyay G Arunachalam A B Mandal T K SurS C Mandal and S K Bhattacharya ldquoAntimicrobial and anti-inflammatory activity of folkloreMallotus peltatus leaf extractrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 229ndash2372002

[25] L R Morse K Stolzmann H P Nguyen et al ldquoAssociationbetween mobility mode and c-reactive protein levels in menwith chronic spinal cord injuryrdquo Archives of Physical Medicineand Rehabilitation vol 89 no 4 pp 726ndash731 2008

[26] A Clauss ldquoGerinnungsphysiologische schnellmethode zur bes-timmung des fibrinogensrdquoActaHaematologica vol 17 no 4 pp237ndash246 1957

[27] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[28] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidationin post-mitotic tissues of ageing ratrdquo Cell Biochemistry andFunction vol 24 no 1 pp 79ndash85 2006

[29] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[30] W F Beyer Jr and I Fridovich ldquoAssaying for superoxide dis-mutase activity some large consequences of minor changes inconditionsrdquoAnalytical Biochemistry vol 161 no 2 pp 559ndash5661987

[31] I Carlberg and B Mannervik ldquoGlutathione reductaserdquo inGlutamate Glutamine Glutathione and Related Compoundsvol 113 ofMethods in Enzymology pp 484ndash490 Elsevier 1985

[32] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[33] V Emanue V Adrian N A Sultana and C Svetlana ldquoAntiox-idant and antimicrobial activities of ethanol extracts of Cynarascolymus (Cynarae folium Asteraceae family)rdquo Tropical Journalof Pharmaceutical Research vol 10 no 6 pp 777ndash783 2011

[34] G L D S Oliveira R A M O Francisco O B A Vinıcius etal ldquoEvaluation of antioxidant capacity of the aqueous extract ofCynara scolymus L (Asteraceae) in vitro and in SaccharomycescerevisiaerdquoAfrican Journal of Pharmacy and Pharmacology vol8 pp 136ndash147 2014

[35] K Horiuchi S Shiota T Hatano T Yoshida T Kuroda and TTsuchiya ldquoAntimicrobial activity of oleanolic acid from Salviaofficinalis and related compounds on vancomycin-resistantenterococci (VRE)rdquo Biological and Pharmaceutical Bulletin vol30 no 6 pp 1147ndash1149 2007

[36] A Daoud D Malika S Bakari et al ldquoAssessment of polyphe-nol composition antioxidant and antimicrobial properties ofvarious extracts of Date Palm Pollen (DPP) from two Tunisiancultivarsrdquo Arabian Journal of Chemistry 2015

[37] D Krishnaiah B Awang S Rosalam and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversitymdashEngineering Sciences vol 1 pp 63ndash66 2015

[38] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[39] A Betancor-Fernandez A Perez-Galvez H Sies and W StahlldquoScreening pharmaceutical preparations containing extracts ofturmeric rhizome artichoke leaf devilrsquos claw root and garlic orsalmon oil for antioxidant capacityrdquo Journal of Pharmacy andPharmacology vol 55 no 7 pp 981ndash986 2003

[40] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[41] S Konyal120580oglu H Saglam and B K120580vcak ldquo120572-Tocopherolflavonoid and phenol contents and antioxidant activity of Ficuscarica leavesrdquo Pharmaceutical Biology vol 43 no 8 pp 683ndash686 2005

[42] D U Bawankule P Trivedi A Pal et al ldquoProtectivemechanismof lignans from Phyllanthus amarus against galactosaminelipopolysaccharide-induced hepatitis an in-vivo and in-silicostudiesrdquo Current Topics in Medicinal Chemistry vol 14 no 8pp 1045ndash1055 2014

[43] GAHiggs R J Flower and J RVane ldquoAnew approach to anti-inflammatory drugsrdquo Biochemical Pharmacology vol 28 no 12pp 1959ndash1961 1979

[44] A A Hemmati H Kalantari A Siahpoosh B Ghorbanzadehand H Jamali ldquoAnti-inflammatory Effect of HydroalcoholicExtract of the Washingtonia filifera Seeds in Carrageenan-Induced Paw Edema in Ratsrdquo Jundishapur Journal of NaturalPharmaceutical Products vol 10 no 1 pp 19ndash25 2015

[45] I Posadas M Bucci F Roviezzo et al ldquoCarrageenan-inducedmouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expressionrdquoBritish Journal of Pharmacology vol 142 no 2 pp 331ndash3382004

[46] K Pundarikakshudu D H Shah A H Panchal and G CBhavsar ldquoAnti-inflammatory activity of fenugreek (Trigonellafoenum-graecum Linn) seed petroleum ether extractrdquo IndianJournal of Pharmacology vol 48 no 4 pp 441ndash444 2016

[47] S Cuzzocrea G Costantino E Mazzon B Zingarelli A DeSarro and A P Caputi ldquoProtective effects of Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) a superoxide dismutasemimetic in paw oedema induced by carrageenan in the ratrdquoBiochemical Pharmacology vol 58 no 1 pp 171ndash176 1999

[48] M A Mendall P Patel M Asante et al ldquoRelation of serumcytokine concentrations to cardiovascular risk factors andcoronary heart diseaserdquo Heart vol 78 no 3 pp 273ndash277 1997

[49] A Ialenti A Ianaro S Moncada andM Di Rosa ldquoModulationof acute inflammation by endogenous nitric oxiderdquo EuropeanJournal of Pharmacology vol 211 no 2 pp 177ndash182 1992

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Evidence-Based Complementary and Alternative Medicine

be attributed to its potential indirectly as a stimulator ofthe activity and expression of antioxidant enzymes duringinflammation

Thus the anti-inflammatory activity is due to the individ-ual or synergistic effect of the components in the ALE In factprevious studies have found that anti-inflammatory profile ofALE could be related to polyphenolic compounds accordingto the phytochemical analysis We performed HPLC analysisto justify the correlation between phytochemical compoundsand anti-inflammatory activity of this plant Among thesepolyphenolic compounds verbascoside has been reportedin several reports and showed a potential spectrum ofmany activities including antioxidant and anti-inflammatoryParticular verbascoside presents antiedematogenic activitiesin animal models of Carr induced inflammation [51] and theresponses exhibited themaximumactivity for the fourth hourof treatment Such activities may be derived from inhibitoryaction of chemical mediators of the inflammatory processsuch as histamine and bradykinin A significant antioxidanteffect of verbascoside has been recently reported byAleo et al[52] in an experimental study [53] Other authors reportedthat anti-inflammatory activity of verbascoside has beenevaluated by an in vitro test performed on cell cultures of pri-mary human keratinocytes [54] in which it was able to reducethe release of proinflammatory markers such as chemokines

Apigenin-7-glucoside was found to block the releaseof several varieties of enzymes involved in inflammationincluding especially lipoxygenases and cyclooxygenases [5556] leading to inhibition of proinflammatory molecules NF-kB activation and inhibits neutrophil infiltration in tissues

From these results we suggest that anti-inflammatoryactivity observed is due to a synergic action of these phenoliccomponents contained in EtOH extract of ALE

C scolymus is one of the few herbal remedies which havebeen verified through experimental studies Further the toxi-city studies of ALE are required in order to confirm the safetyof this medicinal plant in the treatment of inflammationdiseases

5 Conclusion

The present study showed clearly the advantages of Cscolymus leaves extracts which have safer anti-inflammatoryprofile with potent antioxidant activity attributed to thephenolic compounds Furthermore in the future studieswe are interested to characterize the action mechanisms ofactive phenolic compounds of ALE responsible for anti-inflammatory and antioxidants activities

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

This research was supported by the Tunisian Ministry ofHigher Education and Scientific Research

References

[1] C Pasquier ldquoStress oxidative et inflammationrdquo Blool vol 4 pp1ndash6 1996

[2] M A Abuashwashi O M Palomino and M P Gomez-Serranillos ldquoGeographic origin influences the phenolic com-position and antioxidant potential of wild Crataegus monogynafrom Spainrdquo Pharmaceutical Biology vol 54 no 11 pp 2708ndash2713 2016

[3] M B Salem H Affes K Ksouda et al ldquoPharmacological stud-ies of artichoke leaf extract and their health benefitsrdquo PlantFoods for Human Nutrition vol 70 no 4 pp 441ndash453 2015

[4] M-H Huang B-S Wang C-S Chiu et al ldquoAntioxidant anti-nociceptive and anti-inflammatory activities ofXanthii Fructusextractrdquo Journal of Ethnopharmacology vol 135 no 2 pp 545ndash552 2011

[5] G-J Huang S-S Huang S-S Lin et al ldquoAnalgesic effectsand the mechanisms of anti-inflammation of ergostatrien-3120573-ol from antrodia camphorata submerged whole broth in micerdquoJournal of Agricultural and Food Chemistry vol 58 no 12 pp7445ndash7452 2010

[6] C Tohda N Nakayama F Hatanaka and K Komatsu ldquoCom-parison of anti-inflammatory activities of six Curcuma rhi-zomes a possible curcuminoid-independent pathway mediatedby Curcuma phaeocaulis extractrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 3 no 2 pp 255ndash260 2006

[7] K Goksel K Murat Y Murat E Sevil and H Fidan ldquoAntiox-idant activities of ethanol extracts ofHypericum triquetrifoliumandHypericum scabroidesrdquo Pharmaceutical Biology vol 46 no4 pp 231ndash242 2008

[8] R Gebhardt and M Fausel ldquoAntioxidant and hepatoprotectiveeffects of artichoke extracts and constituents in cultured rathepatocytesrdquoToxicology inVitro vol 11 no 5 pp 669ndash672 1997

[9] A SofworaMedicinal Plants and TraditionalMedicine in AfricaJohn Wiley amp Sons Ann Arbor Mich USA 1982

[10] D E Okwu ldquoPhytochemicals vitamins andmineral contents oftwo Nigerian medicinal plantsrdquo International Journal of Mole-cular Medicine and Advances Sciences vol 1 pp 375ndash381 2005

[11] N Tsevegsuren GDavaakhuu andT S Udval ldquoPhytochemicalanalysis of Cynara scolymus L cultivated in MongoliardquoMongo-lian Journal of Chemistry vol 15 no 41 pp 40ndash42 2014

[12] AACC Approved Methods of the AACC American Associationof Cereal Chemists St Paul Minn USA 8th edition 1984

[13] S C Lee L Prosky and J W DeVries ldquoDetermination of totalsoluble and insoluble dietary fiber in foodsmdashenzymatic-gravi-metric method MES-TRIS buffer collaborative studyrdquo Journalof the Association of Official Analytical Chemists vol 75 pp395ndash416 1992

[14] M Dubois K A Gilles J K Hamilton P A Rebers and FSmith ldquoColorimetric method for determination of sugars andrelated substancesrdquoAnalytical Chemistry vol 28 no 3 pp 350ndash356 1956

[15] O A Fawole U L Opara and K I Theron ldquoChemical andphytochemical properties and antioxidant activities of threepomegranate cultivars grown in South Africardquo Food and Bio-process Technology vol 5 no 7 pp 2934ndash2940 2012

[16] V Dewanto X Wu K K Adom and R H Liu ldquoThermalprocessing enhances the nutritional value of tomatoes byincreasing total antioxidant activityrdquo Journal of Agricultural andFood Chemistry vol 50 no 10 pp 3010ndash3014 2002

[17] B Sun J M Ricardo-da-Silva and I Spranger ldquoCritical factorsof vanillin assay for catechins and proanthocyanidinsrdquo Journal

Evidence-Based Complementary and Alternative Medicine 13

of Agricultural and Food Chemistry vol 46 no 10 pp 4267ndash4274 1998

[18] AOACOfficial Methods of Analysis vol 1 AOACWashingtonDC USA 15th edition 1990

[19] D Pljevljakusic K Savikin T Jankovic et al ldquoChemical prop-erties of the cultivated Sideritis raeseri Boiss amp Heldr subspraeserirdquo Food Chemistry vol 124 no 1 pp 226ndash233 2011

[20] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[21] I I Koleva T A Van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] V Ravi T S M Saleem S S Patel J Raamamurthy and KGauthaman ldquoAnti-inflammatory effect of methanolic extract ofSolanum nigrum Linn Berriesrdquo International Journal of AppliedResearch in Natural Products vol 2 pp 33ndash36 2009

[24] D Chattopadhyay G Arunachalam A B Mandal T K SurS C Mandal and S K Bhattacharya ldquoAntimicrobial and anti-inflammatory activity of folkloreMallotus peltatus leaf extractrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 229ndash2372002

[25] L R Morse K Stolzmann H P Nguyen et al ldquoAssociationbetween mobility mode and c-reactive protein levels in menwith chronic spinal cord injuryrdquo Archives of Physical Medicineand Rehabilitation vol 89 no 4 pp 726ndash731 2008

[26] A Clauss ldquoGerinnungsphysiologische schnellmethode zur bes-timmung des fibrinogensrdquoActaHaematologica vol 17 no 4 pp237ndash246 1957

[27] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[28] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidationin post-mitotic tissues of ageing ratrdquo Cell Biochemistry andFunction vol 24 no 1 pp 79ndash85 2006

[29] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[30] W F Beyer Jr and I Fridovich ldquoAssaying for superoxide dis-mutase activity some large consequences of minor changes inconditionsrdquoAnalytical Biochemistry vol 161 no 2 pp 559ndash5661987

[31] I Carlberg and B Mannervik ldquoGlutathione reductaserdquo inGlutamate Glutamine Glutathione and Related Compoundsvol 113 ofMethods in Enzymology pp 484ndash490 Elsevier 1985

[32] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[33] V Emanue V Adrian N A Sultana and C Svetlana ldquoAntiox-idant and antimicrobial activities of ethanol extracts of Cynarascolymus (Cynarae folium Asteraceae family)rdquo Tropical Journalof Pharmaceutical Research vol 10 no 6 pp 777ndash783 2011

[34] G L D S Oliveira R A M O Francisco O B A Vinıcius etal ldquoEvaluation of antioxidant capacity of the aqueous extract ofCynara scolymus L (Asteraceae) in vitro and in SaccharomycescerevisiaerdquoAfrican Journal of Pharmacy and Pharmacology vol8 pp 136ndash147 2014

[35] K Horiuchi S Shiota T Hatano T Yoshida T Kuroda and TTsuchiya ldquoAntimicrobial activity of oleanolic acid from Salviaofficinalis and related compounds on vancomycin-resistantenterococci (VRE)rdquo Biological and Pharmaceutical Bulletin vol30 no 6 pp 1147ndash1149 2007

[36] A Daoud D Malika S Bakari et al ldquoAssessment of polyphe-nol composition antioxidant and antimicrobial properties ofvarious extracts of Date Palm Pollen (DPP) from two Tunisiancultivarsrdquo Arabian Journal of Chemistry 2015

[37] D Krishnaiah B Awang S Rosalam and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversitymdashEngineering Sciences vol 1 pp 63ndash66 2015

[38] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[39] A Betancor-Fernandez A Perez-Galvez H Sies and W StahlldquoScreening pharmaceutical preparations containing extracts ofturmeric rhizome artichoke leaf devilrsquos claw root and garlic orsalmon oil for antioxidant capacityrdquo Journal of Pharmacy andPharmacology vol 55 no 7 pp 981ndash986 2003

[40] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[41] S Konyal120580oglu H Saglam and B K120580vcak ldquo120572-Tocopherolflavonoid and phenol contents and antioxidant activity of Ficuscarica leavesrdquo Pharmaceutical Biology vol 43 no 8 pp 683ndash686 2005

[42] D U Bawankule P Trivedi A Pal et al ldquoProtectivemechanismof lignans from Phyllanthus amarus against galactosaminelipopolysaccharide-induced hepatitis an in-vivo and in-silicostudiesrdquo Current Topics in Medicinal Chemistry vol 14 no 8pp 1045ndash1055 2014

[43] GAHiggs R J Flower and J RVane ldquoAnew approach to anti-inflammatory drugsrdquo Biochemical Pharmacology vol 28 no 12pp 1959ndash1961 1979

[44] A A Hemmati H Kalantari A Siahpoosh B Ghorbanzadehand H Jamali ldquoAnti-inflammatory Effect of HydroalcoholicExtract of the Washingtonia filifera Seeds in Carrageenan-Induced Paw Edema in Ratsrdquo Jundishapur Journal of NaturalPharmaceutical Products vol 10 no 1 pp 19ndash25 2015

[45] I Posadas M Bucci F Roviezzo et al ldquoCarrageenan-inducedmouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expressionrdquoBritish Journal of Pharmacology vol 142 no 2 pp 331ndash3382004

[46] K Pundarikakshudu D H Shah A H Panchal and G CBhavsar ldquoAnti-inflammatory activity of fenugreek (Trigonellafoenum-graecum Linn) seed petroleum ether extractrdquo IndianJournal of Pharmacology vol 48 no 4 pp 441ndash444 2016

[47] S Cuzzocrea G Costantino E Mazzon B Zingarelli A DeSarro and A P Caputi ldquoProtective effects of Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) a superoxide dismutasemimetic in paw oedema induced by carrageenan in the ratrdquoBiochemical Pharmacology vol 58 no 1 pp 171ndash176 1999

[48] M A Mendall P Patel M Asante et al ldquoRelation of serumcytokine concentrations to cardiovascular risk factors andcoronary heart diseaserdquo Heart vol 78 no 3 pp 273ndash277 1997

[49] A Ialenti A Ianaro S Moncada andM Di Rosa ldquoModulationof acute inflammation by endogenous nitric oxiderdquo EuropeanJournal of Pharmacology vol 211 no 2 pp 177ndash182 1992

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 13

of Agricultural and Food Chemistry vol 46 no 10 pp 4267ndash4274 1998

[18] AOACOfficial Methods of Analysis vol 1 AOACWashingtonDC USA 15th edition 1990

[19] D Pljevljakusic K Savikin T Jankovic et al ldquoChemical prop-erties of the cultivated Sideritis raeseri Boiss amp Heldr subspraeserirdquo Food Chemistry vol 124 no 1 pp 226ndash233 2011

[20] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[21] I I Koleva T A Van Beek J P H Linssen A De Groot andL N Evstatieva ldquoScreening of plant extracts for antioxidantactivity a comparative study on three testing methodsrdquo Phyto-chemical Analysis vol 13 no 1 pp 8ndash17 2002

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] V Ravi T S M Saleem S S Patel J Raamamurthy and KGauthaman ldquoAnti-inflammatory effect of methanolic extract ofSolanum nigrum Linn Berriesrdquo International Journal of AppliedResearch in Natural Products vol 2 pp 33ndash36 2009

[24] D Chattopadhyay G Arunachalam A B Mandal T K SurS C Mandal and S K Bhattacharya ldquoAntimicrobial and anti-inflammatory activity of folkloreMallotus peltatus leaf extractrdquoJournal of Ethnopharmacology vol 82 no 2-3 pp 229ndash2372002

[25] L R Morse K Stolzmann H P Nguyen et al ldquoAssociationbetween mobility mode and c-reactive protein levels in menwith chronic spinal cord injuryrdquo Archives of Physical Medicineand Rehabilitation vol 89 no 4 pp 726ndash731 2008

[26] A Clauss ldquoGerinnungsphysiologische schnellmethode zur bes-timmung des fibrinogensrdquoActaHaematologica vol 17 no 4 pp237ndash246 1957

[27] H H Draper andM Hadley ldquoMalondialdehyde determinationas index of lipid peroxidationrdquoMethods in Enzymology vol 186pp 421ndash431 1990

[28] R Kayali U Cakatay T Akcay and T Altug ldquoEffect of alpha-lipoic acid supplementation on markers of protein oxidationin post-mitotic tissues of ageing ratrdquo Cell Biochemistry andFunction vol 24 no 1 pp 79ndash85 2006

[29] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[30] W F Beyer Jr and I Fridovich ldquoAssaying for superoxide dis-mutase activity some large consequences of minor changes inconditionsrdquoAnalytical Biochemistry vol 161 no 2 pp 559ndash5661987

[31] I Carlberg and B Mannervik ldquoGlutathione reductaserdquo inGlutamate Glutamine Glutathione and Related Compoundsvol 113 ofMethods in Enzymology pp 484ndash490 Elsevier 1985

[32] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[33] V Emanue V Adrian N A Sultana and C Svetlana ldquoAntiox-idant and antimicrobial activities of ethanol extracts of Cynarascolymus (Cynarae folium Asteraceae family)rdquo Tropical Journalof Pharmaceutical Research vol 10 no 6 pp 777ndash783 2011

[34] G L D S Oliveira R A M O Francisco O B A Vinıcius etal ldquoEvaluation of antioxidant capacity of the aqueous extract ofCynara scolymus L (Asteraceae) in vitro and in SaccharomycescerevisiaerdquoAfrican Journal of Pharmacy and Pharmacology vol8 pp 136ndash147 2014

[35] K Horiuchi S Shiota T Hatano T Yoshida T Kuroda and TTsuchiya ldquoAntimicrobial activity of oleanolic acid from Salviaofficinalis and related compounds on vancomycin-resistantenterococci (VRE)rdquo Biological and Pharmaceutical Bulletin vol30 no 6 pp 1147ndash1149 2007

[36] A Daoud D Malika S Bakari et al ldquoAssessment of polyphe-nol composition antioxidant and antimicrobial properties ofvarious extracts of Date Palm Pollen (DPP) from two Tunisiancultivarsrdquo Arabian Journal of Chemistry 2015

[37] D Krishnaiah B Awang S Rosalam and S M Anisuzza-man ldquoAntioxidant activity and total phenolic content of anisolated Morinda citrifolia L methanolic extract from Poly-ethersulphone (PES)membrane separatorrdquo Journal of King SaudUniversitymdashEngineering Sciences vol 1 pp 63ndash66 2015

[38] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[39] A Betancor-Fernandez A Perez-Galvez H Sies and W StahlldquoScreening pharmaceutical preparations containing extracts ofturmeric rhizome artichoke leaf devilrsquos claw root and garlic orsalmon oil for antioxidant capacityrdquo Journal of Pharmacy andPharmacology vol 55 no 7 pp 981ndash986 2003

[40] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[41] S Konyal120580oglu H Saglam and B K120580vcak ldquo120572-Tocopherolflavonoid and phenol contents and antioxidant activity of Ficuscarica leavesrdquo Pharmaceutical Biology vol 43 no 8 pp 683ndash686 2005

[42] D U Bawankule P Trivedi A Pal et al ldquoProtectivemechanismof lignans from Phyllanthus amarus against galactosaminelipopolysaccharide-induced hepatitis an in-vivo and in-silicostudiesrdquo Current Topics in Medicinal Chemistry vol 14 no 8pp 1045ndash1055 2014

[43] GAHiggs R J Flower and J RVane ldquoAnew approach to anti-inflammatory drugsrdquo Biochemical Pharmacology vol 28 no 12pp 1959ndash1961 1979

[44] A A Hemmati H Kalantari A Siahpoosh B Ghorbanzadehand H Jamali ldquoAnti-inflammatory Effect of HydroalcoholicExtract of the Washingtonia filifera Seeds in Carrageenan-Induced Paw Edema in Ratsrdquo Jundishapur Journal of NaturalPharmaceutical Products vol 10 no 1 pp 19ndash25 2015

[45] I Posadas M Bucci F Roviezzo et al ldquoCarrageenan-inducedmouse paw oedema is biphasic age-weight dependent anddisplays differential nitric oxide cyclooxygenase-2 expressionrdquoBritish Journal of Pharmacology vol 142 no 2 pp 331ndash3382004

[46] K Pundarikakshudu D H Shah A H Panchal and G CBhavsar ldquoAnti-inflammatory activity of fenugreek (Trigonellafoenum-graecum Linn) seed petroleum ether extractrdquo IndianJournal of Pharmacology vol 48 no 4 pp 441ndash444 2016

[47] S Cuzzocrea G Costantino E Mazzon B Zingarelli A DeSarro and A P Caputi ldquoProtective effects of Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) a superoxide dismutasemimetic in paw oedema induced by carrageenan in the ratrdquoBiochemical Pharmacology vol 58 no 1 pp 171ndash176 1999

[48] M A Mendall P Patel M Asante et al ldquoRelation of serumcytokine concentrations to cardiovascular risk factors andcoronary heart diseaserdquo Heart vol 78 no 3 pp 273ndash277 1997

[49] A Ialenti A Ianaro S Moncada andM Di Rosa ldquoModulationof acute inflammation by endogenous nitric oxiderdquo EuropeanJournal of Pharmacology vol 211 no 2 pp 177ndash182 1992

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

14 Evidence-Based Complementary and Alternative Medicine

[50] D Salvemini Z-Q Wang D M Bourdon M K Stern M GCurrie and P T Manning ldquoEvidence of peroxynitrite involve-ment in the carrageenan-induced rat paw edemardquo EuropeanJournal of Pharmacology vol 303 no 3 pp 217ndash220 1996

[51] E E S Schapoval M R Winter De Vargas C G Chaves RBridi J A Zuanazzi and A T Henriques ldquoAntiinflammatoryand antinociceptive activities of extracts and isolated com-pounds from Stachytarpheta cayennensisrdquo Journal of Ethnophar-macology vol 60 no 1 pp 53ndash59 1998

[52] E Aleo R Ricci S Passi and S A Cataudella ldquoNovelcyt-H2O2-chemiluminescenes assay for measuring the reduc-ingantioxidant capacity on hydrophilic and lipophilic antio-oxindants and biological samplesrdquo Progress in Nutrition vol 3pp 154ndash182 2005

[53] E Mazzon E Esposito R Di Paola et al ldquoEffects of verbasco-side biotechnologically produced by Syringa vulgaris plant cellcultures in a rodent model of colitisrdquo Naunyn-SchmiedebergrsquosArchives of Pharmacology vol 380 no 1 pp 79ndash94 2009

[54] E J Middleton ldquoEffect of plant flavonoids on immune andinflammatory cell functionrdquoAdvances in ExperimentalMedicineand Biology vol 439 pp 175ndash182 1998

[55] J Baumann F Von Bruchhausen and G Wurm ldquoFlavonoidsand related compounds as inhibitors of arachidonic acid perox-idationrdquo Prostaglandins vol 20 no 4 pp 627ndash639 1980

[56] M D Dos Santos M C Almeida N P Lopes and G E P DeSouza ldquoEvaluation of the anti-inflammatory analgesic and anti-pyretic activities of the natural polyphenol chlorogenic acidrdquoBiological and Pharmaceutical Bulletin vol 29 no 11 pp 2236ndash2240 2006

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom