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Journal of Ethnopharmacology 109 (2007) 458–463

Antimicrobial, anti-inflammatory, anti-ulcer and antioxidant activitiesof Carlina acanthifolia root essential oil

Sofija Ðordevic a, Silvana Petrovic c,∗, Silva Dobric b, Marina Milenkovic d,Dragana Vucicevic d, Slavica Zizic e, Jelena Kukic c

a Institute for Medicinal Plant Research “Dr. Josif Pancic”, Tadeuska Koscuska 1, 11000 Belgrade, Serbiab National Poison Control Centre, Military Medical Academy, Crnotravska 17, 11000 Belgrade, Serbia

c Institute of Pharmacognosy, Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbiad Institute of Microbiology and Immunology, Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia

e Institute for Health Protection of Serbia, Dr Subotica 5, 11000 Belgrade, Serbia

Received 18 February 2006; received in revised form 13 June 2006; accepted 21 August 2006Available online 26 August 2006

bstract

The root of Carlina acanthifolia All. (Asteraceae) has been traditionally used in the treatment of various disorders including stomach and skiniseases. We studied antimicrobial, anti-inflammatory, anti-ulcer and antioxidant activities of Carlina acanthifolia root essential oil, in order toalidate some of the ethnopharmacological claims. Antimicrobial activity was tested on 15 bacteria and three strains of fungi using the agar diffusionnd broth microdilution methods. In assessing anti-inflammatory activity the carrageenan-induced rat paw oedema test was used, while ethanol-nduced stress gastric ulcer test in rats was used in testing anti-ulcer activity. Antioxidant properties were evaluated trough the effect of the essentialil on lipid peroxidation (TBA assay) and its capability of quenching 2,2-diphenyl-1-picrylhydrazyl (DPPH) and OH radicals. The oil expressedignificant antimicrobial activity, being the most active against Gram (+) bacteria: Streptococcus pyogenes, Enterococcus faecalis, Bacillus subtilisnd against Candida albicans. In all applied concentrations, Carlina acanthifolia root essential oil reduced carrageenan-induced rat paw oedeman dose-dependent manner, achieving high degree of anti-inflammatory activity. The effect was comparable with that of indomethacin used as a

eference drug. In the ethanol-induced stress gastric ulcer test in rats, it was shown that the tested essential oil produced significant dose-dependentastroprotective activity. The results also pointed out substantial and dose-dependent antioxidant activity of the investigated essential oil, witharlina oxide as the main antioxidant component.

2006 Elsevier Ireland Ltd. All rights reserved.

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eywords: Anti-inflammatory activity; Antimicrobial activity; Antioxidant act

. Introduction

Species of the genus Carlina L. (Asteraceae) are widely dis-ributed in Europe and commonly used for medicinal purposesnd in nutrition. Carlinae radix, the root of Carlina acaulis L.ccording to Erg.-B. 6 (Supplement Volume to German Pharma-opoeia Sixth), was used as a diuretic, diaphoretic, and stom-chic, and also as a gargle against catarrh (Wichtl, 2002). In

ulgaria the root of Carlina acaulis was used to treat kidneyisorders, skin inflammation, as an antihemmoroidal and forervous diseases (Jordanov et al., 1966). Juice from the fresh

∗ Corresponding author. Tel.: +381 11 39 70 379/722; fax: +381 11 39 72 840.E-mail address: silvana@afrodita.rcub.bg.ac.yu (S. Petrovic).

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Anti-ulcer activity; Carlina acanthifolia All.; Essential oil; Root

oot was used as an anthelminthic (Kresanek and Krejca, 1988).hytopreparations based on Carlina acaulis are nowadays usedor treating cholecysopathy and gastrointestinal disturbancesWichtl, 1997). The root of Carlina acanthifolia All. is veryimilar to Carlina acaulis root according to its organoleptical,orphological and anatomical features (Ðor –devic et al., 2004)

nd traditionally it is used for the same purposes (Schilcher andagels, 1990). The root of Carlina acanthifolia is considered as

dulterant or substitute for Carlina acaulis root (Wichtl, 2002).Though commonly used, these plants have not been fully

nvestigated to the date and data on their pharmacological activ-

ty are scarce. Their roots contain inulin (about 20%), flavonoidsnd 1–2% of essential oil with carlina oxide as the main compo-ent (upon 90%) (Chalchat et al., 1996; Vitkova and Evstatieva,002; Ðor –devic et al., 2005). The essential oil of Carlina acaulis

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oot showed antibacterial and antifungal properties (Ðor –devic etl., 1998).

We studied antimicrobial, anti-inflammatory, anti-ulcer andntioxidant activities of Carlina acanthifolia root essential oil,n order to validate some of the ethnopharmacological claimsegarding the plant use in the above-mentioned disorders.

. Materials and methods

.1. Plant material

The roots of Carlina acanthifolia were collected in August003 on Mt. Suva Planina (Eastern Serbia), during the periodf full flowering. Once harvested, the plant material was driedt room temperature. Voucher specimens (ko820035/1) wereeposited in the Herbarium of Natural History Museum, Bel-rade, and identified by mr sc. Marjan Niketic (custodian).

.2. Isolation of the essential oil

The isolation of the essential oil from dried powdered Car-ina acanthifolia root was performed by steam distillation in alevenger-type apparatus, according to the Procedure III of theugoslav Pharmacopoeia IV (Ph. Jug. IV, 1984).

.3. Antimicrobial activity

Antimicrobial assays were performed on eight organismsf American Type of Culture Collection (ATCC) and one ofational Collection of Industrial Bacteria, Aberdeen (NCIMB)

nd three ATCC strains of fungi, all obtained from the Institutef Immunology and Virology, Torlak, Belgrade, and Instituteor Health Protection of Serbia, Belgrade. Six bacterial strainsere isolated from wounds and urine of the patients from Clini-

al Center of Serbia. For the evaluation of antimicrobial activitywo methods were used: the agar diffusion method (Acar andoldstein, 1996) and broth microdilution assay (Candan et al.,003).

.3.1. Agar diffusion methodIt was performed on Muller–Hinton (for bacterial strains)

nd Saburaud agar (for fungi). In order to obtain semiconfluentrowth inoculum of 106 CFU/ml (colony-forming units/ml) ofested microorganisms was used. One drop of 4% and one of% essential oil solution in absolute ethanol were poured onhe agar prepared as required. After incubation (18 h at 37 ◦Cor bacteria and 48 h at 26 ◦C for fungi) diameters of zonesf inhibition (in mm) were measured. Ampicillin (10 �g/disk),mikacin (30 �g/disk) and nystatin (100 units/disk) were used asppropriate control of sensitivity of tested microorganisms. Ascriterion for determining sensitivity, intermediate sensitivity

nd resistance of clinical isolates a scale system by Omar andlnima (Omar and Elnima, 2003) was adopted.

.3.2. Microdilution broth assayIt was performed in 96-well microtiter plates and used

or determining minimal inhibitory concentration (MIC).

2

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armacology 109 (2007) 458–463 459

uller–Hinton and Sabouraud dextrose broth supplementedith Tween 80 detergent (final concentration of 0.5%, v/v),ere used for bacteria and fungi, respectively. Test strains were

uspended in broth to give a final density of 5 × 105 CFU/ml.uccessive dilutions of the essential oil in absolute ethanol, rang-

ng from 1.25 to 40 �l/ml were prepared in a 96-well microtiterlate, including one growth and one sterility control. Plates werencubated under normal atmospheric conditions at 37 ◦C for 24 hor bacteria and at 30 ◦C for 48 h for fungi.

.4. Anti-inflammatory activity

The carrageenan-induced rat paw oedema test has been useds an experimental model for screening the anti-inflammatoryctivity as reported earlier (Oyanagi and Sato, 1991). Thessential oil, pure (100%) or diluted in absolute ethanol (10%,/v), was administered p.o. in doses of 0.25, 0.5 and 1 ml/kg.ndomethacin, dissolved in DMSO, was used as a referencerug in a dose of 8 mg/kg p.o. The control animals were givenehicles, DMSO or absolute ethanol, both in a dose of 1 ml/kg.o. One hour after the oral administration of the essential oilr indomethacin, carrageenan-saline solution (0.5%) and salineere injected in a volume of 0.1 ml into the plantar surface of the

ight and left hind paw, respectively. Left paw served as the con-rol (non-inflammed paw). The animals were sacrificed 3 h afterhe carrageenan and saline injection and paws were cut off foreighing. Difference in weight between right and left paw, activerug-treated versus vehicle-treated (control) rats, served as anndicator of the anti-inflammatory activity of drugs tested (thessential oil and indomethacin). The anti-inflammatory effectas calculated using the equation:

nti-inflammatory effect (%) = k − e

k× 100

here k is difference in the paw weight in the control group andis the difference in the paw weight in the treatment group.

.5. Anti-ulcer activity

In order to study the anti-ulcer activity an experimental modelf acute gastric mucosa damage induced by absolute ethanol1 ml/rat p.o.) was used. Ranitidine (20 mg/kg p.o., 60 min prioro ethanol) was used as a reference drug. The essential oil, pure100%) or diluted in absolute ethanol (10%, v/v), was admin-stered p.o. in doses of 0.25, 0.5 and 1.0 ml/kg 60 min prior thebsolute ethanol. The control animals were given vehicle in aose of 1 ml/kg p.o. 60 min before 1.0 ml of absolute ethanol.he animals were sacrificed 1 h after giving ethanol and theirtomachs were removed and opened along the greater curva-ure. Lesions were examined under an illuminated magnifier3×). The intensity of gastric lesions was assessed according tomodified scoring system of Adami et al. (1964).

.6. Animals

Animal studies were conducted in accordance with the inter-ationally accepted principles for laboratory animal use and care

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n the European Community guidelines (EEC Directive of 1986;6/609/EEC) adopted by Ethical Committee of Military Medicalcademy, Belgrade.Adult male Wistar rats, weighing 200–250 g, were used in

oth the carrageenan-induced rat paw oedema and the ethanol-nduced stress gastric ulcer tests. Experimental groups consistedf 6–10 animals each. The animals were deprived of food for8–20 h before the beginning of experiments with free access toap water.

.7. Antioxidant activity

.7.1. Thin-layer chromatographyThirty microliters of the essential oil dissolved in toluene

50%, v/v) was applied on silica gel plates (Merck) andeveloped in system cyclohexane–ethyl acetate (16:1, v/v).omponents of the oil were detected by spraying withanillin–sulphuric acid spray reagent (Wagner and Bladt, 1996).PPH test performed directly on TLC plates (0.2% (w/v) DPPH

n methanol used as spray reagent) revealed contributions to thentioxidant activity of different compounds separately (Cuendett al., 1997).

.7.2. DPPH radical assayDifferent aliquots of pure essential oil (2–40 �l) were mixed

ith 0.4 ml of 0.5 mM DPPH in ethanol, and final volumedjusted up to 2 ml with ethanol. Mixtures were virgously shakednd left 30 min in dark. Absorbance was measured at 517 nmsing ethanol as blank. One milliliter of 0.5 mM DPPH dilutedn 4 ml of ethanol was used as control (Cuendet et al., 1997).nhibition of DPPH radical was calculated using the equation:(%) = 100 × (A0 − As)/A0, where A0 is the absorbance of theontrol (containing all reagents except the test compound), ands is the absorbance of the tested sample. The IC50 value rep-

esented the concentration of the essential oil that caused 50%nhibition.

.7.3. 2-Deoxyribose assayThe 2-deoxyribose method was used for determining the

cavenging effect on hydoxyl radical. Pure essential oil waspplied in different concentrations (0.25–10 �l/ml). Each reac-ion mixture contained the following reagents, in the final con-entrations stated: FeCl3 (100 �M), EDTA (100 �M), H2O22.2 mM), 2-deoxyribose (2.5 �M), l-ascorbic acid (100 �M).hosphate buffer (pH 7.4, I = 0.1) was added up to a final vol-me of 4 ml. The mixtures were incubated 1 h at 37 ◦C, thenml of 1% (w/v) thiobarbituric acid (TBA) in 0.05 M NaOHnd 1 ml of 2.8% (w/v) trichloroacetic acid (TCA) were addedn each mixture and heated 15 min at 100 ◦C. After cooling once absorbance was measured at 532 nm (Lee and Lim, 2000).nhibition of 2-deoxyribose degradation in percent was calcu-ated in the following way: I (%) = 100 × (A0 − As)/A0, wherell symbols have the same meaning as in DPPH radical assay.

.7.4. TBA testLipid peroxidation (LP) was measured using preparation of

iposomes containing 0.03 g lecithin/ml. Liposomes were pre-

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armacology 109 (2007) 458–463

ared from the commercial preparation “Lipotech 10”, contain-ng 10% of lecithin, diluted with distilled water in ultrasonicath for 30 min. The reaction mixture contained 20 �l FeSO40.075 M), 50 �l of liposomes, 1–40 �l of pure essential oil,0 �l of l-ascorbic acid (0.1 M) and phosphate buffer (pH 7.4,= 0.1) up to a final volume of 4 ml. Samples were incubatedh at 37 ◦C. Then, 0.2 ml of EDTA (0.1 M) and 1.5 ml of TBA

eagent (3 g TBA, 120 g TCA and 10.4 ml HClO4 in 800 mlf distilled water) were added in each sample and heated for5 min at 100 ◦C. After cooling on ice, samples were centrifu-ated for 10 min (3000 rpm) and absorbance of supernatant waseasured at 532 nm (Afanas’ev et al., 1989). Inhibition of LPas calculated the same way as described in DPPH radical

ssay.

.8. Statistical analysis

Results were expressed as means ± standard deviationsS.D.). Statistical analysis was performed by the Mann–Whitney-test and ANOVA. Differences were accepted as statistically

ignificant when P < 0.05.

. Results

.1. Essential oil yield

Essential oil yield was 1.0% (w/w). Freshly isolated essentialil was a yellow liquid with intensive, narcotic odour.

.2. Antimicrobial activity

Results of antimicrobial activity of Carlina acanthifolia rootssential oil against standard microorganisms (Table 1) showedhat it possesses considerable antimicrobial activity. In compari-on to standard antibiotics (ampicillin and amikacin) 2% and 4%ssential oil dilution expressed the highest antibacterial activitygainst Gram (+) bacteria. Comparing to ampicillin, both appliedoncentrations of essential oil were the most active against Bacil-us subtilis (153.3% and 152.0%, respectively) and Enterococcusaecalis (112.5% and 120.6%, respectively). Activity of the oilgainst Staphylococcus aureus ATCC 6538-p was also signifi-ant comparing to ampicillin (84.0% and 86.0%, respectively)nd amikacin (91.3% and 93.5%, respectively). Essential oilas less active against Gram (−) bacteria, with the exception of% dilution of the oil which was more active against Klebsiellaneumoniae (117.6%), comparing to ampicillin.

The essential oil showed high antifungal activity against allested strains. The most active was 4% essential oil dilutiongainst Candida albicans ATCC 10231 and Aspergillus niger111.5% and 110.6% of activity comparing to nystatin, respec-ively).

Results of antimicrobial activity of Carlina acanthifoliaoot essential oil against clinical isolates (Table 2) showed

hat all 17 isolates of Streptococcus pyogenes were sensitiveMIC = 2.5 �l/ml). Isolates of Staphylococcus aureus and Acine-obacter sp. showed high or intermediate sensitivity, with low

IC values (2.5 and 5 �l/ml, respectively).

S. Ðordevic et al. / Journal of Ethnopharmacology 109 (2007) 458–463 461

Table 1Antimicrobial activity of Carlina acanthifolia root essential oil against standard microorganisms

Microorganism Inhibition zones (mm)a MIC (�l/ml)

Essential oilb Ampicillin(10 �g/disk)

Amikacin(30 �g/disk)

Nystatin(100 units/disk)

2% 4%

Gram (+) bacteriaStaphylococcus aureus ATCC 25923 20.5 ± 1.8 21.8 ± 0.9 27.0 26.0 n.t. 5.0Staphylococcus aureus ATCC 6538-p 21.0 ± 0.8 21.5 ± 0.6 25.0 23.0 n.t. n.t.Micrococcus luteus ATCC 9341 16.7 ± 0.9 20.3 ± 0.8 33.0 n.t. n.t. 10.0Enterococcus faecalis ATCC 29212 18.0 ± 0.8 19.3 ± 1.3 16.0 n.t. n.t. n.t.Bacillus subtilis ATCC 6633 23.0 ± 0.8 22.8 ± 0.6 15.0 n.t. n.t. 2.5

Gram (−) bacteriaKlebsiella pneumoniae NCIMB-9111 13.5 ± 0.5 20.0 ± 1.6 17.0 n.t. n.t. n.t.Escherichia coli ATCC 25922 12.8 ± 0.5 13.5 ± 0.5 18.0 24.0 n.t. 10.0Pseudomonas aeruginosa ATCC 27853 16.3 ± 0.5 19.3 ± 0.5 n.t. 26.0 n.t. 10.0Pseudomonas aeruginosa ATCC 9027 12.5 ± 0.6 11.8 ± 0.5 n.t. 25.0 n.t. n.t.

FungiCandida albicans ATCC 10231 15.0 ± 0.8 22.3 ± 0.9 n.t. n.t. 20.0 2.5Candida albicans ATCC 24433 14.3 ± 0.9 20.5 ± 0.6 n.t. n.t. 22.0 5.0Aspergillus niger ATCC 16404 12.8 ± 0.5 17.7 ± 0.9 n.t. n.t. 16.0 5.0

n.t. = not tested.

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.3. Anti-inflammatory activity

Results of the experiment showed that in all applied con-entrations, Carlina acanthifolia root essential oil reducedarrageenan-induced rat paw oedema in dose-dependent man-er, achieving high degree of anti-inflammatory activityTable 3). The effect was comparable with that of indomethacin8 mg/kg p.o.) used as a reference drug. The most active wasure essential oil (100%) given in a dose of 1.0 ml/kg p.o.

.4. Anti-ulcer activity

In the ethanol-induced stress gastric ulcer test in rats it washown that the tested essential oil produced significant dose-ependent gastroprotective activity. This activity was particu-arly noticeable when the essential oil was given as a “pure oil”

able 2ntimicrobial activity of Carlina acanthifolia root essential oil against clinical

solates

icroorganism Number of isolates MIC(�l/ml)

Sensitive Intermediate Resistant

treptococcus pyogenes 17 0 0 2.5taphylococcus aureus 2 3 2 2.5scherichia coli 1 5 3 40.0lebsiella pneumoniae 2 1 4 80.0seudomonas aeruginosa 3 0 3 n.t.cinetobacter sp. 1 3 2 5.0

ensitivity of microorganisms (according to Omar and Elnima, 2003)—Gram+) bacteria: >18 mm = sensitive, 14–18 mm = intermediate, <14 mm = resistant;ram (−) bacteria: >16 mm = sensitive, 13–16 mm = intermediate,13 mm = resistant.

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100%) in a dose in 1.0 ml/kg. The effect obtained with thisose was even better than that of the reference anti-ulcer drug,anitidine (Table 4).

.5. Antioxidant activity

The investigated essential oil also showed substantial antiox-dant activity. Scavenging of DPPH radical was concentration-ependent, with IC50 = 13.6 �l/ml (Table 5). TLC analysishowed that carlina oxide, the principal component of Car-ina acanthifolia root essential oil, was the main antioxidant.ther components of the oil have not significantly contributed its

ntioxidant activity, mainly because of their low content (Fig. 1).cavenging of OH radical was moderate. In all applied con-entrations inhibition was above 30%, reaching maximum at.5 �l/ml (Table 5). Inhibition of LP was more prominent and

able 3nti-inflammatory activity of Carlina acanthifolia root essential oil (the

arrageenan-induced rat paw oedema test)

ssential oiloncentration (%, v/v)a

Dose (ml/kg p.o.) Anti-inflammatoryeffect (%)

001.00 64.46 ± 12.130.50 61.22 ± 10.720.25 49.08 ± 10.88

01.00 57.11 ± 11.650.50 54.65 ± 8.820.25 40.91 ± 6.22

ndomethacinb 8 73.38 ± 39.23

a In absolute ethanol.b Reference drug.

462 S. Ðordevic et al. / Journal of Ethnopharmacology 109 (2007) 458–463

Table 4Gastroprotective activity of Carlina acanthifolia root essential oil (the ethanol-induced gastric stress ulcer test)

Essential oilconcentration (%, v/v)a

Dose (ml/kg p.o.) Gastric damage scoreb

1001.00 1.15 ± 0.780.50 2.00 ± 1.770.25 2.50 ± 1.51

101.00 3.50 ± 2.060.50 4.00 ± 2.760.25 3.85 ± 2.27

Ranitidinec 20 mg/kg p.o 1.77 ± 1.08

Note: gastric damage score in vehicle-treated animals (the control group) was5.9 ± 1.14.

a In absolute ethanol.b 0: no lesions; 0.5: slight hyperaemia or ≤5 petechiae; 1: ≤5 erosions ≤5 mm

in length; 1.5: ≤5 erosions ≤5 mm in length and many petechiae; 2: 6–10 ero-sions ≤5 mm in length; 2.5: 1–5 erosions >5 mm in length; 3: 5–10 erosions>5 mm in length; 3.5: >10 erosions >5 mm in length; 4: 1–3 erosions ≤5 mm inlength and 0.5–1 mm in width; 4.5: 4–5 erosions ≤5 mm in length and 0.5–1 mmin width; 5: 1–3 erosions >5 mm in length and 0.5–1 mm in width; 6: 4 or 5 grade5 lesions; 7: ≥6 grade 5 lesions; 8: complete lesion of the mucosa with haem-o

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Table 5Inhibitory effects of the Carlina acanthifolia essential oil on DPPH radical(DPPH), on OH radical obtained in 2-deoxyribose assay (2-DR) and on lipidperoxidation (LP) in liposomes

Essential oilconcentration(�l/ml)

Inhibition (%)

DPPH 2-DR LP

0.50 – 33.95 ± 0.00 30.91 ± 8.951.25 7.98 ± 1.90 34.48 ± 0.18 60.26 ± 7.172.50 14.19 ± 1.01 42.13 ± 0.03 65.89 ± 18.735.00 20.74 ± 1.16 36.67 ± 0.28 40.07 ± 0.00

10.00 38.70 ± 1.35 38.16 ± 0.00 32.12 ± 2.3412

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lso concentration-dependent up to concentration of 2.5 �l/mlhen the maximum of inhibition was achieved (65.84%). Inigher concentrations tested (5 and 10 �l/ml) inhibitory effectf the essential oil on LP was less pronounced (40.07% and2.12%, respectively) giving inverted U-shaped concentration-

esponse curve (Table 5). Low IC50 (0.9 �l/ml) indicates highntiperoxy-radical activity.

ig. 1. TLC chromatograms of the Carlina acanthifolia essential oil: (A) essen-ial oil components revealed by vanillin–sulphuric acid reagent; (B) antiox-dant components of the essential oil revealed by 0.2% (w/v) DPPH radicalolution.

a1mtrataOiptitbaobmtl

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5.00 53.94 ± 2.78 – –0.00 61.94 ± 0.47 – –

. Discussion and conclusion

Carlina oxide was identified as the main component of thearlina acanthifolia root essential oil (91.5%) (Ðor –devic et al.,005). Since antimicrobial activity of carlina oxide was reportedarlier (Wichtl, 2002), it could be presumed that the observedignificant antimicrobial activity of Carlina acanthifolia rootssential oil is mostly caused by the presence of this compound.

Regarding to the observed antioxidant potential of the inves-igated essential oil and high DPPH scavenging activity of car-ina oxide, it could be presumed that this compound is also

ainly responsible for exhibited anti-inflammatory and anti-lcer effects.

Namely, carrageenan injection into the rat paw provokeslocal, acute inflammatory reaction (Boughton-Smith et al.,

999). Considering that the production of arachidonic acidetabolites is the main factor responsible for both the first and

he second phase of the carrageenan-induced inflammation, ouresults suggest that the anti-inflammatory activity of the Carlinacanthifolia root essential oil might be related to the inhibition ofhe release or synthesis of cyclooxygenase products. However,t the moment we have no data confirming such an assumption.n the other hand, the second phase of the carrageenan-induced

nflammation is also related to the neutrophil infiltration androduction of the reactive free radical species derived fromhem, indicating that the essential oil tested may exert its anti-nflammatory effect, at least partly, through the inhibition of neu-rophil infiltration as well as the free radical scavenging. It haseen shown that some of the anti-inflammatory agents includingpocynin, a phenolic compound of plant origin, exert their effectn inflammation through the inhibition of free radical generationy activated neutrophils (Halliwell, 1991). Having these facts inind, it could be postulated that the anti-inflammatory effect of

he Carlina acanthifolia root essential oil is the consequence, ateast partly, of its antioxidant activity.

The free radical scavenging activity of the essential oil testedight be also considered as one of the possible mechanisms of its

astroprotective effect observed, because oxygen derived radi-

als and agents with antioxidant properties have been implicatedn the pathogenesis of ethanol-induced gastric ulcers and in theirrevention and healing, respectively (Terano et al., 1989). In anyase, further studies on mechanisms by which the active con-

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tituents of this essential oil, mainly carlina oxide, may exhibitharmacological actions are warranted.

cknowledgments

This research was supported by the Ministry of Science andnvironmental Protection of Republic of Serbia (Grant No.43012). The authors thank mr sc. Marjan Niketic, custodianf the Natural History Museum in Belgrade, for the collectionnd the identification of the plant material.

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