Journal of Ethnopharmacology 149 (2013) 103–110

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Journal of Ethnopharmacology

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Exploration of the wound healing potential of Helichrysum graveolens(Bieb.) Sweet: Isolation of apigenin as an active component

Ipek Süntar a, Esra Küpeli Akkol a,n, Hikmet Keles b, Erdem Yesilada c, Satyajit D. Sarker d

a Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler 06330, Ankara, Turkeyb Department of Pathology, Faculty of Veterinary Medicine, Afyon Kocatepe University, Afyonkarahisar 03200, Turkeyc Faculty of Pharmacy, Yeditepe University, Atasehir 34755, Istanbul, Turkeyd Department of Pharmacy, School of Applied Sciences, University of Wolverhampton, MA Building, Wulfurna Street,Wolverhampton WV1 1LY, United Kingdom

a r t i c l e i n f o

Article history:Received 25 February 2013Received in revised form21 May 2013Accepted 5 June 2013Available online 11 June 2013

Keywords:Anti-inflammatoryAntioxidantCollagenaseElastaseHelichrysumWound healing

41/$ - see front matter & 2013 Elsevier Irelanx.doi.org/10.1016/j.jep.2013.06.006

esponding author: Tel.: +90 312 2023185; faxail address: esrak@gazi.edu.tr (E. Küpeli Akko

a b s t r a c t

Ethnopharmacological relevance: In Turkish traditional medicine, the flowers of Helichrysum graveolens(Bieb.) Sweet (Asteraceae) have been used for the treatment of jaundice, for wound-healing and as adiuretic.Aim of the study: In order to find scientific evidence for the traditional utilization of this plant in wound-healing, the effect of the plant extract was investigated by using in vivo and in vitro experimental models. Thenthrough bioassay-guided fractionation procedures active wound-healing component(s) was isolated and itspossible role in the wound-healing process was also determined.Material and methods: The linear incision and the circular excision wound models were applied in order toevaluate in vivo wound-healing potential of Helichrysum graveolens. Anti-inflammatory and antioxidantactivities, which are known to involve in wound-healing process, were also assessed by the Whittle methodand the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assay, respectively. The total phenolic contentof the crude extract and solvent fractions was estimated to find correlation between the phenolic content andthe antioxidant activity. Combined application of the chromatographic separation techniques on sephadex andsilica gel columns, and bioassay techniques have yielded the active wound-healing principle of Helichrysumgraveolens. Moreover, in vitro inhibitory effect of active principle on hyaluronidase, collagenase and elastaseenzymes were investigated to explore the activity pathways.Results: The 85% methanol (MeOH) extract of Helichrysum graveolens flowers displayed significant wound-healing, anti-inflammatory and antioxidant activities. Then the crude extract was partitioned by successivesolvent extractions, in increasing polarity, to give five solvent fractions. Among the solvent fractions, the ethylacetate (EtOAc) fraction exerted the highest activity. The EtOAc fraction was further subjected to chromato-graphic separations to yield active constituent and its structure was elucidated to be apigenin by spectrometricmethods. Further in vivo and in vitro assays revealed that apigenin was one of the components responsible forthe wound-healing effect of the plant remedy and also found to possess significant anti-inflammatory,antioxidant, anti-hyaluronidase and anti-collagenase activities.Conclusion: Present study supported the traditional use of Helichrysum graveolens flowers for wound-healingand through bioassay-guided fractionation procedures from the crude extract apigenin was isolated as one ofthe active components.

& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

There are approximately 600 species of Helichrysum Mill.(family: Asteraceae) particularly distributed in South Africa,Australasia and Eurasia. The South African species have been usedagainst infectious and rheumatic diseases, headache, cold, liver

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: +90 312 2235018.l).

diseases and for wound-healing (Lall et al., 2006; Van Wyk et al.,2008). Similar folkloric utilizations have also been recorded in theother parts of the world, such as Helichrysum stoecheas for thetreatment of bronchitis in Portugal, and Helichrysum litoreum andHelichrysum italicum against respiratory and inflammatorydiseases in Italy (Barros et al., 2010).

There are 16 Helichrysum species growing wild in Turkey and allspecies are widely consumed as tea due to their medicinal proper-ties. Infusion prepared from the capitulums of Helichrysum plicatumhas been used for wound-healing and for the treatment of

I. Süntar et al. / Journal of Ethnopharmacology 149 (2013) 103–110104

stomachache, jaundice, intestinal problems, as well as diuretic topass kidney stones, and as urinary antiseptic. In the ethnobotanicalrecords similar utilizations were also reported for Helichrysumgraveolens, which is also a widespread species in Turkey (Seziket al., 1991; Yesilada et al., 1995).

Phytochemical studies on various Helichrysum species haverevealed that they contain mainly flavonoids, phloroglucinols,pyrones and terpenic compounds and exert a wide range ofbiological activities including antimicrobial, antioxidant, anti-inflammatory, sedative, antidiabetic and cytotoxic (Lourens et al.,2008).

In the present study, the primary objective was to evaluate thewound-healing potential of Helichrysum graveolens, a traditi-onal wound-healing remedy in Turkey. Afterwards by usingbioassay-guided procedures it is aimed to isolate of the activeconstituent(s) and suggest the possible mechanisms.

2. Materials and methods

2.1. Plant material

The flowers of Helichrysum graveolens (Bieb.) Sweet werecollected from Uludağ (Bursa), Zone 1, in July, 2009. The voucherspecimen of the plant was authenticated by Prof. Dr. Hayri Dumanfrom Gazi University, Department of Biology, Faculty of Scienceand Art, Ankara and a specimen of the plant (GUE 2977) wasdeposited in the Herbarium of Faculty of Pharmacy, Gazi Univer-sity, Ankara, Turkey. The aerial parts were shade-dried and groundusing a blender.

2.2. Extraction, fractionation and isolation procedures for thebioassays

Dried and powdered flowers (350 g) of Helichrysum graveolenswere extracted with 85% MeOH (7.5 L) and evaporated to dryness togive “Hg–MeOH” (121.8 g; yield: 34.8%). The residual dried extract wasthen dissolved in MeOH/H2O (9:1) (400 ml) and partitioned withn-hexane (20�500 ml). Combined n-hexane extracts were evapo-rated under reduced pressure to give “Hg-hexane” fraction (11.6 g;yield: 9.5%). After removal of methanol from the remaining extract anddilution with distilled H2O to 400 ml, the extract was successivelypartitioned with dichloromethane (CH2Cl2) (20�500 ml), EtOAc(20�500 ml) and finally with n-butanol (BuOH) saturated with water(20�500 ml). Each solvent fraction was evaporated to dryness underreduced pressure to give “Hg–CH2Cl2” (2.1 g; yield: 1.7%), “Hg–EtOAc”(17.8 g; yield: 14.6%) and “Hg–BuOH” (15.7 g; yield: 12.9%) fractions,respectively. The final aqueous phase was also evaporated to drynessand designated as “Hg–R–H2O” (36.7 g; yield: 30.1%).

2.2.1. Fractionation of Hg–EtOAc by chromatographic techniques andisolation of the active constituent

By following bioassay-guided procedures, Hg–EtOAc, the activesolvent fraction, was further fractionated by chromatographic techni-ques. Hg–EtOAc (15 g) was first subjected to separation on a SephadexLH-20 (Sigma-Aldrich 095K1220) column using MeOH as an eluent.The eluates were combined depending on their thin layer chromato-graphic (TLC) profile as follows: Hg–Fr.A (0.80 g), Hg–Fr.B (12.35 g) andHg–Fr.C (0.12 g). The most active fraction Hg–Fr.B (10 mg) was furthersubjected to silica gel (Sigma-Aldrich 28.862-4) column chromatogra-phy using CHCl3 and CHCl3:MeOH (80:20); CHCl3:MeOH (70:30);CHCl3:MeOH (60:40) and MeOH as eluents. Three fractions namelyHg–Fr.B1 (7.20 mg); Hg–Fr.B2 (0.95 mg); Hg–Fr.B3 (0.37 mg) wereobtained.

2.2.2. Structure elucidation of the active compound Hg–Fr.B1Nuclear Magnetic Resonance (1H and 13C NMR) and Mass Spectral

(MS) techniques were employed for the structure elucidation of thesilica gel column fraction obtained from the eluate CHCl3:MeOH(80:20) (Hg–Fr.B1) (7.20 mg). NMR spectra were recorded on a Brukerspectrometer (400 MHz for 1H NMR and 100MHz for 13C NMR)instrument, and using methanol d4 as the solvent. Fourier transformmass spectroscopy (FT-MS) analyses were performed using a Finniganspectrometer. The structure of 1 was determined as apigenin (4′,5,7-trihydroxyflavone) by comparison of the spectroscopic data with theprevious reports (Ersöz et al., 2002).

2.3. Determination of total phenolic content of the crude extract andsolvent fractions

Total phenolic contents of the methanolic extract and solventfractions were quantified by using the reference methods involvingthe Folin–Ciocalteu reagent and gallic acid as reference (Spanos andWrolstad, 1990). An aliquot of extract solution (100 ml) containing1 mg extract was taken into a volumetric flask, distilled water and theFolin–Ciocalteu reagent (5 ml) were added and flask was shakenthoroughly. Sodium carbonate (4 ml) was added and the mixturewas allowed to stand for 2 h with intermittent shaking at roomtemperature. Then absorbance was measured at 765 nm. The sameprocedure was applied to reference gallic acid solutions prepared indifferent concentrations (0.05 mg/ml; 0.1 mg/ml; 0.15 mg/ml;0.25 mg/ml and 0.5 mg/ml) to obtain the standard curve.

2.4. Pharmacological experiments

2.4.1. In vivo biological activity tests2.4.1.1. Animals. Male Sprague–Dawley rats (160–180 g) and Swissalbino mice (20–25 g) purchased from the animal breeding houseof Saki Yenilli (Ankara, Turkey) were used in the experiments.

The animals were left for 3 days for acclimatization into animalroom conditions and were maintained on standard pellet diet andwater ad libitum. A minimum of six rats were used in each group forwound healing experiments, while 10 mice were used in anti-inflammatory studies. The present study was performed accordingto the internationally accepted issues considering the animal experi-mentation and biodiversity rights (Gazi University Ethical CouncilProject Number: G.U.ET-08.037).

2.4.1.2. Wound-healing activity. For the assessment of wound-healing activity by using the incision and the excision woundmodels, an ointment prepared with the test materials was topicallyapplied onto the wounded area on the dorsal part of test animals. Thetest ointments were prepared by mixing either extracts/solventfractions/column fractions or purified compounds with a mixture ofointment base consisting of glycol stearate/propylene glycol/liquidparaffin (3:6:1) in a mortar thoroughly. Treatments were startedimmediately after the production of wound by daily application ofthe test ointments on the wounded area. The control group animalswere topically treated with blank vehicle base [glycol stearate/propylene glycol/liquid paraffin (3:6:1) mixture], while the animalsin negative control group were not treated with any product.Madecassols (Bayer, 00001199) ointment (0.5 g) was appliedtopically as the reference drug.

2.4.1.2.1. Linear incision wound model. Animals, six rats in eachgroup, were anaesthetized with 0.05 cm3 Xylazine (2% Alfazines) and0.15 cm3 Ketamine (10% Ketasols). The dorsal part of each rat wasshaved and the area was cleaned with 70% alcohol. Two linear-paravertebral incisions in 5 cm length were made with a sterile bladethrough the shaved skin at a distance of 1.5 cm from the dorsal

I. Süntar et al. / Journal of Ethnopharmacology 149 (2013) 103–110 105

midline on each side. Three surgical sutures were placed each1 cm apart.

The ointments prepared with test samples, the reference drug(Madecassols) or blank ointment base were topically applied on thedorsal wounds in each group of animals once daily throughout9 days. All the sutures were removed on the last day and tensilestrength of previously wounded and treated skin was measured byusing a tensiometer (Zwick/Roell Z0.5, Germany) (Lodhi et al., 2006).

2.4.1.2.2. Circular excision wound model. This model was used tomonitor wound contraction and wound closure time. Each group ofanimals (seven animals in each) was anaesthetized with 0.02 cm3

Xylazine (2% Alfazines) and 0.08 cm3 Ketamine (10% Ketasols). Thedorsal hairs of the mice were removed by shaving. The circular woundwas created on the dorsal interscapular region of each animal byexcising the skin with a 5 mm biopsy punch (Nopa instruments,Germany); and then the wound was left open. Test samples, thereference drug (Madecassols, Bayer) and the ointment base wereapplied topically once a day until complete recovery of the wound. Theprogressive changes in wound area were monitored by a camera (Fuji,S20 Pro, Japan) every other day. Later on, wound area was evaluatedby using AutoCAD program. Wound contraction was calculated aspercentage of the reduction in wounded area. A specimen sample oftissue was isolated from the healed skin of each group of mice for thehistopathological examination (Sadaf et al., 2006; Süntar et al., 2011).

2.4.1.2.3. Histopathology. The skin specimens from each groupwere collected at the end of the experiment (on day 12). Sampleswere fixed in 10% buffered formalin, processed and blocked withparaffin and then sectioned into 5 μm sections and stained withhematoxylin & eosin (HE) and Van Gieson (VG) stains. The tissueswere examined by light microscope (Olympus CX41 attached Kamer-ams Digital Image Analyze System) and graded as mild (+), moderate(++) and severe (+++) for epidermal or dermal re-modeling. Re-epithelization or ulcus in epidermis; fibroblast proliferation, mono-nuclear and/or polymorphonuclear cells, neo-vascularization andcollagen depositions in dermis were analyzed to score the epidermalor dermal re-modeling. Van Gieson stained sections were analyzed forcollagen deposition. At the end of the examination, all the woundhealing processes were combined and staged for wound healingphases as inflammation, proliferation, and re-modeling in all groups.

2.4.1.2.4. Hydroxyproline estimation. Tissue samples from theexcision wound model were removed after the 7th post woundday and were dried in a hot-air oven at 60–70 1C until consistentweight was achieved. Afterwards, samples were hydrolyzed with6 N HCl for 3 h at 130 1C. The hydrolyzed samples were adjusted topH 7.0 and subjected to chloramine T oxidation. The coloredadduct product formed with Ehrlich reagent at 60 1C was read at557 nm (Beckmann Dual Spectrophotometer; Beckman, Fullerton,CA, USA). Standard hydroxyproline was also run and valuesreported as mg/mg dry weight of tissue (Degim et al., 2002).

2.4.1.3. Evaluation of the anti-inflammatory activity using the acetic acid-induced increase in capillary permeability test. For the anti-inflammatory test model, samples were given orally to test animalsafter suspending in a mixture of distilled water and 0.5% sodiumcarboxymethyl cellulose (CMC). The control group of animals receivedthe same experimental handling as those of the test groups exceptthat the drug treatment was replaced with appropriate volumes of thedosing vehicle. Indomethacin (10 mg/kg) in 0.5% CMC was used as areference drug.

Effect of the test samples on the increased vascular permeabilityinduced by acetic acid in mice was determined according to Whittlemethod with some modifications (Yesilada and Küpeli, 2007). Eachtest sample was administered orally to a group of 10 mice in 0.2 ml/20 g body weight. Thirty minutes after the administration, eachmousewas injected with 0.1 ml of 4% Evans blue in saline solution via the tail

vein and this was followed by intraperitoneal injection of 0.4 ml of0.5% (v/v) acetic acid 10 min later. After 20 min incubation, the micewere killed by dislocation of the neck, and the viscera were exposedand irrigated with distilled water, which was then poured into 10 mlvolumetric flasks through glass wool. Each flask was made up to 10 mlwith distilled water, 0.1 ml of 0.1 N sodium hydroxide (NaOH) solutionwas added to the flask, and the absorption of the final solution wasmeasured at 590 nm (Beckmann Dual Spectrophotometer; Beckman,Fullerton, CA, USA). A mixture of distilled water and 0.5% CMC wasgiven orally to control animals, and they were treated in the samemanner as described above.

2.4.2. In vitro biological activity tests2.4.2.1. Estimation of antioxidant the activity using the DPPH radicalscavenging assay. The hydrogen atom or electron donationcapacity of the corresponding extracts were computed from thebleaching property of the purple-colored MeOH solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH). Either the samples or thereference compound (quercetin) dissolved in MeOH was mixedwith DPPH solution (80 mg/ml). Remaining DPPH amount wasmeasured at 517 nm using spectrophotometer. DPPH inhibitionactivities were calculated according to the following formula:

Inhibitionð%Þ ¼ ðAcontrol−AsampleÞ � 100=Acontrol

where Acontrol is the absorbance of the control reaction (containing allreagents except the test sample), and Asample is the absorbance of theextracts/reference. Experiments were run in duplicate and the resultswere expressed as inhibitory values (Kumarasamy et al., 2003).

2.4.2.2. Determination of hyaluronidase inhibitory activity. Theinhibition of hyaluronidase was assessed by the measurement of theamount of N-acetylglucosamine released from sodium hyaluronate(Sahasrabudhe and Deodhar, 2010). Fifty microliters of bovinehyaluronidase (7900 units/ml) [Sigma-Aldrich 37326-33-3] wasdissolved in 0.1 M acetate buffer (pH 3.6). Then this solution wasmixed with 50 ml portions of the test samples in differentconcentrations dissolved in 5% dimethyl sulfoxide (DMSO). As thecontrol group 50 ml of 5% DMSO was added directly. After 20 min ofincubation at 37 1C, 50 ml of calcium chloride (12.5 mM) was added tothe mixture and again incubated for 20 min at 37 1C. Then 250 mlsodium hyaluronate (1.2 mg/ml) was added and incubated for 40 minat 37 1C. After incubation the mixture was treated with 50 ml of 0.4 MNaOH and 100 ml of 0.2 M sodium borate and then incubated inboiling water bath for 3 min. p-Dimethylaminobenzaldehyde solution(1.5 ml) was added to the reaction mixture after cooling to roomtemperature and was further incubated at 37 1C for 20 min to developa color. Tannic acid was used as the reference. The absorbance wasmeasured at 585 nm (Beckmann Dual Spectrometer; Beckman,Fullerton, CA, USA).

2.4.2.3. Determination of collagenase inhibitory activity. The sampleswere dissolved in DMSO. The sample solution and Clostridiumhistolyticum collagenase enzyme (ChC) were dissolved in 50 mMTricine buffer (with 0.4 M NaCl and 0.01 M CaCl2, pH 7.5) andpreincubated at 25 1C for 5 min. Then, 2 mM N-[3-(2-Furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA) was prepared in the same buffer. Twenty-five microliter each of Tricine buffer, test sample and ChC solutionswere added to each well and incubated for 15 min. Fifty microlitersubstrate was added to the mixture and decrease in the optical density(OD) was immediately measured at 340 nm by using spectrometer.The ChC inhibition activities were calculated according to thefollowing formula:

ChC inhibition activityð%Þ ¼ODControl–ODSample � 100=ODControl

I. Süntar et al. / Journal of Ethnopharmacology 149 (2013) 103–110106

where ODcontrol and ODsample represent the optical densities in theabsence and presence of sample, respectively (Barrantes and Guinea,2003).

2.4.2.4. Determination of elastase inhibitory activity. The samplesolution and human neutrophil elastase enzyme (HNE) (17 mU/ml)were mixed in 0.1 M Tris–HCl buffer (pH 7.5), then incubated at 25 1Cfor 5 min. N-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide(MAAPVN) was added to the mixture and incubated at 37 1C for 1 h.Afterwards, the reaction was stopped by the addition of soybeantrypsin inhibitor (1 mg/ml) and the optical density was immediatelymeasured at 405 nm spectrophotometrically. The HNE inhibitionactivity was calculated as described in the ChC inhibition activity(Melzig et al., 2001). Epigallocatechin gallate was used as thereferences of the both collagenase and elastase inhibitory activities.

2.4.3. Statistical analysis of the dataData obtained from animal experiments were expressed as the

meanwith standard error (7SEM). Statistical differences between thetreated and the control groups were evaluated by ANOVA andStudents–Newman–Keuls post-hoc tests. The values of p≤0.05 wereconsidered statistically significant. Histopathologic data were consid-ered to be nonparametric; therefore, no statistical tests wereperformed.

Table 1Effects of the crude extract, solvent fractions, column fractions and apigenin fromHelichrysum graveolens on linear incision wound model and tissue hydroxyprolinecontent.

Material Dose(%)

Tensilestrength7S.E.M.

(%Tensilestrength)

Hydroxyproline Ć(mg/mg)7S.E.M.

Vehicle 10.0372.48 5.8 8.972.1Negativecontrol 9.4872.61

–9.172.2

Hg–MeOH 1 13.0571.09 30.11** 26.371.0**

Hg-Hexane 1 11.7972.06 17.5 18.572.1Hg–CH2Cl2 1 11.5572.17 15.2 19.771.9Hg–EtOAc 1 12.8971.01 28.5** 31.270.9**

3 12.4571.10 24.1 * 29.171.1*

5 12.8071.07 27.6* 32.67 0.8**

Hg–BuOH 1 12.6271.04 25.8* 15.671.8Hg–R–H2O 1 11.1972.13 11.6 13.371.8Madecassols 1 15.6270.96 55.7*** 42.970.8 ***

Vehicle 12.1272.17 10.3 7.371.7NegativeControl 10.9972.53

–6.271.7

Hg–Fr.A 1 13.8071.97 13.9 15.471.2Hg–Fr.B 1 15.1771.54 25.2* 25.571.2*

Hg–Fr.C 1 14.7071.89 21.3* 16.371.9Madecassols 1 18.6270.75 53.6*** 48.270.6***

Vehicle 11.2471.18 1.4 10.271.9Negativecontrol

11.0971.26–

9.471.8

Apigenin 1 15.0470.81 33.8* 23.670.9*

Madecassols 1 17.0070.69 51.3*** 45.770.8***

S.E.M.: Standard error of the mean.Percentage of the tensile strength values: The vehicle group was compared to thenegative control group; The extracts and the reference material were compared tovehicle group.

n po0.05.nn po0.01.nnn po0.001.

3. Results

One percent ointment formulation of the crude methanolicextract (85%) of Helichrysum graveolens displayed a remarkablewound healing activity on the linear incision and the circularexcision wound models (Tables 1 and 2). Then MeOH extract wassubjected to partition with successive solvent extractions inincreasing polarity. Wound-healing activity of each solvent frac-tions was also investigated using the same in vivo wound models.EtOAc and n-BuOH fractions displayed statistically significantwound-healing activity in the linear and the circular excisionwound models (Tables 1 and 2). In the hydroxyproline assay,tissues treated with Hg–MeOH, Hg–EtOAc, Hg–Fr.B and apigeninointments were found to increase hydroxyproline levels (Table 1).Later effect of the increasing concentrations of the active EtOAcfraction was studied, yet the activity was not dose-dependent(Tables 1 and 2). Therefore, the further studies were conducted on1% ointment formulation of the test materials. The EtOAc fractionwas further submitted to Sephadex LH-20 column chromatogra-phy and eluted fractions were combined into three fractions basedon their chemical fingerprinting on TLC examination and woundhealing activity of each fraction was also investigated. Hg–Fr.Bdisplayed the best wound-healing activity, provided 25.2%increase in tensile strength in the linear incision and 40.2%contraction in the circular excision wound models (Tables 1 and2). Hg–Fr.B was then fractionated by silica gel column chromato-graphy and, a flavonoid type compound, apigenin was isolated asthe major component of this fraction. Further in vivo experimentshave revealed that apigenin to possess remarkable wound-healingactivity with the values of 33.8% in the linear incision, and 44.25%in the circular excision wound models (Tables 1 and 2).

The wounded sites were then removed from each animal andtissue sections were also evaluated histopathologically. Stages in thewound healing process were observed and recorded for all groups.Tissues treated with Hg–MeOH, Hg–EtOAc, Hg–Fr.B and apigeninointments demonstrated a good wound recovery with faster re-epithelialization and higher collagen concentration compared to theother test groups tested. Large collagen bundles were observed in theVG stained sections obtained from the tissues treated with Hg–EtOAc,Hg–Fr.B and apigenin. Histopathological examinations on the rest ofthe test groups exhibited ulcerations containing inflammatory cells,mild degree of inflammation and congestion denoting that the healingwas not completed on the wounded area. Also latency in wound-healing process was observed especially in the vehicle and negativecontrol groups (Fig. 1).

Inflammation is the first response of the healing phases.However, lingering inflammatory response causes improper anddelayed healing. Therefore, anti-inflammatory activity potential ofthe crude extract, solvent fractions, column fractions and apigeninobtained from the plant material was also investigated by usingthe Whittle method. This model of inflammation is useful todetermine the efficiency of test material against increased capil-lary permeability induced by acetic acid (Yesilada and Küpeli,2007). Hg–MeOH, Hg–EtOAc, Hg–Fr.B and apigenin exertedremarkable anti-inflammatory activity, inhibited the inflammation37.1%, 30.7%, 24.5% and 27.8%, respectively (Table 3).

Since antioxidant activity may also have a positive contributionin wound healing process, the antioxidant activity of test sampleswere also investigated by the DPPH scavenging assay. Hg–EtOAcand apigenin were found to have high antioxidant potential. Interms of antioxidant activity high total phenol content wasdetermined in the Hg–EtOAc fraction (Table 4).

On the other hand, in vitro enzyme inhibitory activity assayshave shown that apigenin exerted significant inhibitory activitieson hyaluronidase and collagenase enzymes with the values of

Table 2Effects of the crude extract, solvent fractions, column fractions and apigenin from Helichrysum graveolens on circular excision wound model.

Material Dose(%)

Wound area (mm2) 7 S.E.M. (Contraction%)

Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12

Vehicle 20.1272.12 19.0472.29 (1.14) 16.4372.34 – 14.0172.53 (2.51) 9.5171.75 (7.58) 4.6971.23 (13.47) 2.8070.15 (5.72)Negativecontrol

20.3372.09 19.2672.34 16.1372.18 14.3771.76 10.2971.50 5.4271.24 2.9770.73

Hg–MeOH 1 19.5671.69 18.1572.87 (4.67) 14.3872.03 (12.48) 10.9171.39 (22.13) 6.4071.04 (32.70)* 2.4370.81 (48.19)** 0.9670.30 (65.71)**

Hg-Hexane 1 20.4571.62 18.0072.28 (5.46) 14.5871.34 (11.26) 12.3171.29 (12.13) 7.9071.83 (16.93) 3.7470.79 (20.26) 2.3770.11 (15.36)Hg–CH2Cl2 1 20.0172.10 18.6771.91 (1.94) 14.9371.82 (9.13) 12.5771.58 (10.28) 8.7871.58 (7.68) 3.7870.26 (19.40) 2.3570.29 (16.07)Hg–EtOAc 1 20.9372.86 17.1772.31 (9.82) 14.0772.17 (14.36) 11.4171.03 (18.56) 7.8571.41 (17.46) 3.1370.27 (33.26)* 1.4770.32 (47.50)**

3 19.6772.19 18.1672.24 (4.62) 13.5972.03 (17.29) 11.3371.86 (19.13) 7.5071.58 (21.14) 3.3170.49 (29.42)* 1.6770.10 (40.36)**

5 19.8372.50 17.3872.16 (8.712 14.3772.57 (12.54) 11.4971.56 (17.99) 7.0771.13 (25.66)* 3.2170.15 (31.56)** 1.5270.06 (45.71)**

Hg–BuOH 1 19.2671.39 17.4172.06 (8.56) 15.1472.13 (7.85) 12.5071.10 (10.78) 6.6471.35 (30.18)* 3.4970.62 (25.59)* 1.7470.48 (37.86)*

Hg–R–H2O 1 19.8871.89 17.7571.83 (6.78) 16.8171.86 – 13.9371.19 (0.57) 8.5271.60 (10.41) 4.2570.62 (9.38) 2.6370.17 (6.07)Madecassols 1 19.4071.93 16.1771.87 (15.07) 13.4371.35 (18.26) 9.4671.22 (32.48)* 3.9270.09 (58.78)** 0.9270.10 (80.38)*** 0.0070.00 (100.00)***

Vehicle 19.2471.96 16.6972.01 (1.89) 14.3871.86 (5.39) 11.3071.58 (7.91) 7.1871.13 (10.47) 3.7570.71 (8.98) 2.7670.30 (6.44)Negativecontrol

19.3572.02 17.0171.96 15.2071.74 12.2771.43 8.0271.10 4.1271.59 2.9570.80

Hg–Fr.A 1 19.2671.78 16.1471.84 (3.29) 14.0171.89 (2.57) 10.8171.50 (4.34) 6.4771.09 (9.89) 3.3070.86 (12.00) 2.2070.39 (20.29)Hg–Fr.B 1 19.9271.99 15.4872.22 (7.25) 13.0771.78 (9.11) 10.3371.49 (8.58) 6.2871.18 (12.53) 2.6670.20 (29.07)* 1.6570.09 (40.22)**

Hg–Fr.C 1 19.4171.79 16.3371.86 (2.16) 13.6071.83 (5.42) 10.2771.55 (9.12) 6.0771.16 (15.46) 2.9770.59 (20.80) 1.8370.14 (33.69)*

Madecassols 1 19.3171.90 14.9672.05 (10.37) 11.4871.85 (20.17) 5.6770.92 (49.82)** 2.1370.46 (70.33)** 0.4370.05 (88.53)*** 0.0070.00 (100.00)***

Vehicle 19.7271.58 16.7671.42 (0.36) 14.2171.25 (1.18) 11.0770.85 (5.06) 7.3670.79 (6.60) 3.4470.41 (9.71) 2.2670.13 (9.60)Negativecontrol

19.6071.81 16.8271.35 14.3871.42 11.6671.21 7.8870.85 3.8170.64 2.5070.29

Apigenin 1 19.5171.53 15.0871.07 (10.02) 13.0171.14 (8.44) 10.1170.86 (8.67) 6.1270.49 (16.85) 2.1070.43 (38.95)* 1.2670.12 (44.25)**

Madecassols 1 19.8371.77 14.7171.02 (12.23) 12.9870.96 (8.66) 8.7670.72 (20.87) 4.1770.25 (43.34)** 1.0270.17 (70.35)** 0.0070.00 (100.00)***

S.E.M.: Standard error of the mean.Percentage of the contraction values: The vehicle group was compared to the negative control group; The fractions and the reference material were compared tovehicle group.

n po0.05.nn po0.01.nnn po0.001.

I. Süntar et al. / Journal of Ethnopharmacology 149 (2013) 103–110 107

30.29% and 29.15%, respectively at 100 mg/ml concentration, whileit was inactive on elastase enzyme (Table 5).

4. Discussion

The skin acts as a protective barrier against the environment.Any injury or illness can cause loss of its integrity and majordisability or even death. Eventually rapid and functional woundclosure is the primary target in the wound treatment (Singer andClark, 1999). Wound-healing is an interactive process involvingmediators, cells and extracellular matrix components and hasthree successive stages; inflammation, tissue formation, and tissueremodeling that overlap in time. Traditional wound-healing reme-dies are expected to contribute the wound healing process byacting in one or more of these stages. In the present study ourobjective was to investigate the wound-healing potential of theHelichrysum graveolens capitulums, which was reported to beimplemented as a wound-healing remedy in ethnobotanical docu-ments (Yesilada et al., 1995).

Helichrysum species are known to be rich in flavonoids andvarious types of flavonoid aglycones such as apigenin, naringenin,luteolin, kaempferol and quercetin and their glycosides have beenisolated from their aerial parts and capitulums (Adams et al., 2009;Süzgeç-Selçuk and Birteksöz, 2011). However phytochemical com-position of Helichrysum graveolens has not been investigated indetail so far. In a previous study, Hänsel and Cubukcu (1972)reported the isolation and structure elucidation of 3,5-dihydroxy-6,7,8-trimethoxyflavon from this plant. The present study is thefirst report of the isolation of apigenin from Helichrysumgraveolens.

Capitulums of Helichrysum species are reputed for variousbiological effects in traditional medicines. Anti-inflammatoryactivity (Schinella et al., 2007; Lourens et al., 2008; Bauer et al.,2011) and antioxidant activity of several Helichrysum species havebeen investigated before (Süzgeç et al., 2005; Rosa et al., 2007;Süzgeç-Selçuk and Birteksöz, 2011).

There are few reports in the literature on the biological activity ofHelichrysum graveolens. Aslan et al. (2007a) has recently reported thatcapitulums of the plant exerted remarkable antihyperglycaemic andhypoglycaemic effects as well as antioxidant activity. On the otherhand, a study was conducted by the same group on the antibacterialactivity potential of eight Anatolian Helichrysum species, Helichrysumgraveolens capitulums were found to exert a broad spectrum of activityagainst Gram-positive and Gram-negative bacteria (Aslan et al.,2007b). Antimicrobial activity may have particular importance inwound-healing in order to protect the wounded area from infections.However, wound healing and anti-inflammatory activities of Helichry-sum graveolens capitulums have not previously been reportedelsewhere.

On the other hand, anti-inflammatory activity of apigeninwhich was isolated as the active component of the plant in thepresent study was previously reported by Jeong et al. (2009).Recently apigenin was demonstrated to possess healing effect inboth acute irritant dermatitis and contact dermatitis models ontopical application (Man et al., 2012). More recently Byun et al.(2013) showed that apigenin exerts potent chemopreventiveactivity against UVB-induced skin inflammations primarily bytargeting Src-kinase.

According to the results of the present study, the woundhealing activity potential of the crude extract was shown to behigher than those of the solvent and column fractions even

Fig. 1. Histopathological view of the wound healing and epidermal/dermal re-modeling in the experimental groups. Skin sections show the hematoxylin & eosin (HE)stained epidermis and dermis in A, and the dermis stained with Van Gieson (VG) in B. The original magnification was �100 and the scale bars represent 120 mm for figuresin A, and the original magnification was �400 and the scale bars represent 40 mm for B. Data are representative of 6 animal per group. 1. Vehicle, 2. Negative Control, 3. åHg–EtOAc, 4. Hg–Fr.B, 5. Apigenin, 6. Madecassol. Arrows pointing events during wound healing; s: scab, u: ulcus, re: re-epithelization, f: fibroblast, c: collagen, åmnc:mononuclear cells, pmn: polymorphonuclear cells, nv: neovascularization.

I. Süntar et al. / Journal of Ethnopharmacology 149 (2013) 103–110108

Table 3Anti-inflammatory effect of the crude extract, solvent fractions, column fractions and apigeninfrom H. graveolens on Whittle Method.

Material Dose (mg/kg) Evans blue concentration (μg/ml)7S.E.M. Inhibition (%)

Control 10.9470.75Hg–MeOH 100 8.5870.53 21.6

200 6.8870.32 37.1***

Hg-Hexane 100 11.0270.88 —

200 10.5170.71 3.9Hg–CH2Cl2 100 9.8870.55 9.7

200 8.8370.56 19.3Hg–EtOAc 100 8.6570.53 20.9

200 7.5870.24 30.7**

Hg–BuOH 100 9.1770.68 16.2200 8.6070.44 21.4

Hg–R–H2O 100 10.9570.83 —

200 10.6870.97 2.4Indomethacin 10.0 5.6670.21 48.3***

Control 11.4170.86Hg–Fr.A 100 11.1470.91 2.4

200 10.8570.84 4.9Hg–Fr.B 100 10.3370.79 9.5

200 8.6170.45 24.5*

Hg–Fr.C 100 9.8970.77 13.3200 8.2570.38 27.7**

Indomethacin 10.0 6.5870.21 42.3***

Control 13.0270.78Apigenin 200 9.4070.63 27.8*

Indomethacin 10.0 6.3170.39 51.5***

S.E.M.: Standard error of the mean.Ć n po0.05.nn po0,01.nnn po0,001.

Table 4The total phenolic content and DPPH scavenging activity of the crude extract and solventfractions from Helichrysum graveolens.

Material IC50 (mg/ml) Total phenolics Ć(mg GA/g7S.E.M.)

Hg–MeOH 86.16 71.771.23Hg-Hexane 99.79 1.370.17Hg–CH2Cl2 39.01 91.071.30Hg–EtOAc 25.18 357.471.09Hg–BuOH 35.00 91.771.24Hg–R–H2O 2066.48 8.971.01Hg–Fr.A 126.16 –

Hg–Fr.B 51.43Hg–Fr.C 3.18Apigenin 31.04Reference (Quercetin) 2.14

Table 5Hyaluronidase, collagenase and elastase enzyme inhibitory activities of Apigenin.

Material Concentration (mg/ml) Hyaluronidase inhibition (%)7S.E.M. Collagenase inhibition (%)7S.E.M. Elastase inhibition (%)7S.E.M.

Apigenin50 19.4270.79 15.0370.92 14.2271.26

100 30.2970.39* 29.1570.87* 19.4371.19Tannic acid 100 80.1370.51*** – –

Epigallocatechin gallate 100 – 47.2570.44** 84.2870.81***

S.E.M.: Standard error of the mean.Ć n po0.05.nn po0.01.nnn po0.001.

I. Süntar et al. / Journal of Ethnopharmacology 149 (2013) 103–110 109

isolated compound apigenin. Helichrysum graveolens could con-tribute the wound healing process by acting in the inflammationand proliferation phases. Since apigenin was isolated as the major

active compound from the active fraction, it could be concludedthat there should be other compounds that enhances the woundhealing effect. However, the activity could be highly attributed to

I. Süntar et al. / Journal of Ethnopharmacology 149 (2013) 103–110110

the compound apigenin. The effect of apigenin on the extracellularmatrix components (ECM) was investigated along with its anti-inflammatory and antioxidant activity potential. Since, ECM com-ponents such as collagen, elastin and hyaluronic acid are known tocontribute the healing process (Sahasrabudhe and Deodhar, 2010).Apigenin was shown to possess remarkable hyaluronidase andcollagenase enzyme-inhibitory activities disclosing the possiblerole of this compound in the wound-healing process. The presentstudy has supported the traditional use of the capitulums ofHelichrysum graveolens for wound healing.

Acknowledgments

This study was supported by 2214-International Doctoral ResearchFellowship Programme, provided by “The Scientific and TechnologicalResearch Council of Turkey (TUBITAK)” and the Scientific ResearchProject of Gazi University, Grant No: 02/2011-34.

We thank Dr. Randolph Arroo, Leicester School of Pharmacy,De Montfort University, The Gateway, Leicester LE1 9BH, UK, for1H –NMR analysis and EPSRC National Mass Spectrometry ServiceCentre, Department of Chemistry, University of Wales Swansea,Swansea, Wales, UK.

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