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Industrial Crops and Products 61 (2014) 526–530

Contents lists available at ScienceDirect

Industrial Crops and Products

jo u r n al homep age: www.elsev ier .com/ locate / indcrop

ntimicrobial, antioxidant, antimutagenic activities, and phenolicompounds of Iris germanica�,��

asgedik Burcua, Ugur Ayselb,∗, Sarac Nurdanc

Department of Biology, Faculty of Sciences, Mugla Sitki Kocman University, Mugla, TurkeySection of Medical Microbiology, Department of Basic Sciences, Faculty of Dentistry, Gazi University, Ankara, TurkeyMedical Laboratory Programme, Vocational School of Health Sciences, Mugla Sitki Kocman University, Mugla, Turkey

r t i c l e i n f o

rticle history:eceived 6 April 2014eceived in revised form 6 July 2014ccepted 9 July 2014vailable online 30 August 2014

eywords:ris germanicaMESPLCntioxidantntimicrobialhenolic compounds

a b s t r a c t

This study investigated the antimicrobial, antioxidant and antimutagenic properties and phenolic com-pounds of ethanolic extracts of the aerial parts and rhizomes of the iris I. germanica L. While previousstudies have examined the antimicrobial and antioxidant properties of I. germanica, to our knowledge,this is the first study to report on the antimutagenic activity and phenolic compounds of ethanolic extractsof the plant’s aerial parts and rhizomes.

Antimicrobial activity was evaluated using the disc diffusion method. I. germanica was found to exhibitonly limited antimicrobial activity against Bacillus subtilis ATCC 6633.

Antioxidant activity was evaluated according to free-radical scavenging (DPPH), total antioxidant activ-ity and total phenolic content. The radical scavenging activity of the ethanolic extracts of the aerialparts and rhizomes of I. germanica were found to have IC50 values of 5.38 and 12.3 mg/ml, respectively,whereas total antioxidant activity of the extracts (at 3.15 mg/ml) was 98.7% and 97.4%, respectively, andtotal phenolic content was 267.36 ± 2.05 and 331.96 ± 1.78 mg gallic acid equivalent/g extract, respec-tively. HPLC analysis of phenolic compounds identified protocatechuic acid (0.356 ± 0.004 mg/g extract),chlorogenic acid (0.164 ± 0.0064 mg/g extract) and ferulic acid (0.164 ± 0.008 mg/g extract) as the mainphenolic acids contained in the extract of the aerial parts of I. germanica, whereas chlorogenic acid(2.44 ± 0.036 mg/g extract), (+)-catechin (2.14 ± 0.056 mg/g extract) and ferulic acid (0.452 ± 0.008 mg/gextract) were identified as the main phenolic acids contained in the extract of the rhizome.

Antimutagenic activity was assessed using the Ames Salmonella/microsome mutagenicity test. Thebacterial mutant strains Salmonella typhimurium TA98 and TA100 were used to determine antimutagenicproperties of the test compounds. The ethanolic extract of the aerial parts of I. germanica exhibited

antimutagenic effects at 3, and 0.3 mg/plate concentrations. Although the rhizomes exhibit antimutageniceffects at 1.5, 0.15 and 0.015 mg/plate concentrations.

In sum, the findings indicated I. germanica ethanolic extracts possess antioxidant and antimutagenicproperties that could have potential value in the fields of medicine and cosmetics; however, only limitedantimicrobial activity was observed.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Cancer is presently one of the main causes of mortality through-ut the industrialized world. Scientists believe that damage to

� All authors agree to submit the work to Industrial Crops and Products.�� This is a original research and it is not previously published and that it is noteing.∗ Corresponding author. Tel.: +90 312 203 4380; fax: +90 312 223 9226.

E-mail addresses: burcubasgedik@gmail.com (B. Burcu), ayselugur@hotmail.comU. Aysel), sarac 63@hotmail.com (S. Nurdan).

ttp://dx.doi.org/10.1016/j.indcrop.2014.07.022926-6690/© 2014 Elsevier B.V. All rights reserved.

genetic material, including changes in DNA sequencing and genemutation, plays an important role in carcinogenesis (Shams et al.,2012); therefore, the most effective means of preventing cancer andvarious other genetics-related diseases in humans may be throughthe regular intake of anti-mutagens and anti-carcinogens in dailylife (Kim et al., 2000). Natural antimutagenic compounds obtainedfrom edible and medicinal plants are of particular importance inthis regard because they produce no undesirable xenobiotic effects

on living organisms that would offset any potential usefulness incancer prevention in humans (Zahin et al., 2010).

There is a wide range of prospective human health appli-cations for plant species that possess antimutagenic properties.

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arious studies have shown that natural antioxidants can reducer inhibit the mutagenic potential of mutagens and carcinogensNegi et al., 2003; Zahin et al., 2010). Antioxidant compounds haveeen reported to delay or prevent the oxidation of lipids and otherolecules by inhibiting the initiation or propagation of oxidiz-

ng chain reactions (Velioglu et al., 1998). Herbal remedies andhytotherapeutic drugs currently in use have also been shown torevent free radicals from attacking DNA, indicating the potential,

t has been suggested, to inhibit aging as well as cancer (Ghazalit al., 2011).

Medicinal plants are also a source to which scientists haveurned in their search for new antimicrobial substances (S ahint al., 2003) to replace currently available antimicrobials. Inhe face of the persistently rising threat of antimicrobial resis-ance – a major worldwide health problem, which has hadmmense impact in terms of human life as well as economic lossPaphitou, 2013) – discovery of new antimicrobial agents is cru-ial.

Iris (Iridaceae) is a plant that is widely found in Eurasia, Northfrica, and North America (Rigano et al., 2009). I. germanica L. is onef 37 species of iris, including 13 endemic species, found in Turkey,Baytop and Baytop, 1984). I. germanica rhizomes have been useds diuretics, carminatives and laxatives in Turkish folk medicineBaytop, 1984). I. germanica rhizome juice is also used topically toeal sores and remove freckles, whereas root decoctions are useds anti-spasmodics, emmenagogues, stimulants, diuretics, aperi-nts and to treat edema and gall-bladder diseases (Asghar et al.,009). In addition to its medicinal importance, I. germanica hasconomic value as an input in the perfume and cosmetic indus-ries (Nadaroglu et al., 2007), and some species are also cultivateds ornamental plants (Rahman et al., 2004).

Gas chromatography-mass spectrometry analysis of aetroleum ether extract (oil) of I. germanica has identifiedhe following eleven compounds: 9-hexadecanoic acid methylster, 9-octadecenoic acid methyl ester, 8-octadecenoic acidethyl ester, 11-octadecenoic acid methyl, 10-octadecenoic

cid methyl ester, 13-octadecenoic acid methyl ester, 16-ctadecenoic acid methyl ester, 1,2-benzenedicarboxylic acidiisooctyl ester, bis(2-ethylhexyl) phthalate, methyl 6-methyl hep-anoate and nonanoic acid, and 9-oxo-methyl ester (Asghar et al.,011).

Previous studies have evaluated the antimicrobial activity of I.ermanica methanolic extract (Asghar et al., 2011; Ibrahim et al.,012) and the antioxidant activity of both methanolic (Anwart al., 2006; Asghar et al., 2011) and aqueous (Nadaroglu et al.,007) extracts. Anti-proliferative (Abu-Dahab and Afifi, 2007), anti-

nflammatory (Ibrahim et al., 2012), immunomodulatory (Nazirt al., 2009), cytotoxic (Xie et al., 2013) and in vitro chemo-reventive activities (Wollenweber et al., 2003) have also been

nvestigated. However, to date, there is no study in the literatureeporting on the mutagenic and anti-mutagenic effects of etha-olic extracts of the aerial parts and rhizomes of I. germanica or

ts phenolic compounds. Therefore, this study aimed to identifyutagenic/antimutagenic activity and phenolic compounds of eth-

nolic extracts of I. germanica in an effort to facilitate their use inhytomedicine and the cosmetics industry.

. Materials and methods

.1. Plant material

The aerial parts and rhizomes of I. germanica, naturally growinglants belonging to the Iridaceae family were collected from Mugla,urkey. The leaves were air-dried at room temperature for 7 daysnd were stored for later analysis.

Products 61 (2014) 526–530 527

2.2. Preparation of the ethanolic extract

The air dried plant samples were extracted with ethanol (Merck)using the Soxhlet apparatus. The extract was evaporated and thenextracted in ethanol/water (1:1, v/v), and then kept in small sterileopac bottles under refrigerated conditions until used.

2.3. Bacterial strains

B. subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 27853,Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, andCandida albicans ATCC 10239 were used as determined the antimi-crobial activity.

S. typhimurium TA98 and S. typhimurium TA100 were usedfor the mutagenity and antimutagenity tests. The strains wereanalyzed for their histidine requirement, biotin requirement, thecombination of both, rfa mutation, excision repair capability, thepresence of the plasmid pKM101, and spontaneous mutation rateaccording to Mortelmans and Zeiger (2000). The bacterial stock cul-tures were inoculated to nutrient broth and incubated at 37 ◦C for12–16 h with gentle agitation (Oh et al., 2008).

2.4. Antimicrobial activity

The antimicrobial activities of the extracts were assayed bythe standard paper disc diffusion method (Collins et al., 1995;Murray et al., 1995) as detailed elsewhere (Ugur et al., 2010).Antimicrobial activity was evaluated by measuring the zone ofinhibition against the tested microorganisms. Ethanol was usedas a negative control. Studies were performed in triplicate. Discsof penicillin (10 U), ampicillin (10 �g), amoxicillin + clavulanic acid(20 �g/10 �g), imipenem (10 �g), cefoperazone (75 �g), and nys-tatin (30 �g) were used as positive controls.

2.5. Antioxidant activity

2.5.1. Determination of DPPH radical scavenging activityAntioxidant activity of the extracts were determined based on

its ability to react with the stable 1,1-diphenyl-2-picryl hydrazyl(DPPH) free radical (Yamasaki et al., 1994) as detailed elsewhere(Sarac and Sen, 2014). BHT and ascorbic acid were used as a positivecontrol.

2.5.2. Total antioxidant activity by the ˇ-carotene–linoleic acidmethod

The total antioxidant activity of the extracts were evaluated bythe �-carotene–linoleic acid model (Jayaprakasha and JaganmohanRao, 2000) as detailed elsewhere (Sarac and Sen, 2014). The sameprocedure was repeated with the positive control BHT, ascorbicacid, and a blank.

2.5.3. Determination of total phenolic compoundsThe phenolic constituent of the extracts were determined by

the method involving the Folin–Ciocalteu reagent and gallic acidas a standard (Slinkard and Singleton, 1977; Chandler and Dodds,1983) as detailed elsewhere (Sarac and Sen, 2014). Content of phe-nolic compound was determined as mg gallic acid equivalents pergram of extract (mg/g GAE extract) using the following linear equa-tion based on the calibration curve: A = 0.0257C − 0.0147, R2 = 0.997where A is the absorbance and C gallic acid equivalents.

2.6. Quantification of phenolic compounds by RP-HPLC

Phenolic compounds were evaluated by reversed-phasehigh-performance liquid chromatography (RP-HPLC, ShimadzuScientific Instruments, Tokyo, Japan) as detailed elsewhere

528 B. Burcu et al. / Industrial Crops and Products 61 (2014) 526–530

Table 1Free radical scavenging capacities of the ethanolic extract of the aerial parts andrhizomes of I. germanica and standards measured in DPPH assay.

Sample IC50 value (mg/ml)

The extract of the aerial parts 5.44 ± 0.042a

The extract of the rhizomes 12.21 ± 0.403BHT 0.95 ± 0.014Ascorbic acid 0.48 ± 0.019

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Table 2Antioxidant activity (%) of the ethanolic extract of the aerial parts and rhizomes ofI. germanica at 3.15 mg/ml concentration in �-carotene–linoleic acid test system.

Sample Antioxidant activity (%)

The extract of the aerial parts 98.7 ± 0.4a

The extract of the rhizomes 97.4 ± 0.4BHT 95.7 ± 0.1Ascorbic acid 63.69± 3.24

effects against the mutant S. typhimurium strains observed with theextract of the aerial parts of I. germanica at 3 and 0.3 mg/ml con-centrations and with the extract of the rhizomes at concentrations

Table 3Compounds of phenolics in the ethanolic extract of the aerial parts and rhizomes ofI. germanica.

Phenolic compound Concentrations (mg/g extract)

Aerial parts Rhizome

Protocatechuic acid 0.356 ± 0.004a 0.072 ± 0.004(+)-Catechin 0.06 ± 0.0032 2.14 ± 0.056Chlorogenic acid 0.164 ± 0.0064 2.44 ± 0.036

a Values expressed are means ± S.D. of three parallel measurements. The IC50

alues were obtained by the linear regression analysis.

Sarac and Sen, 2014). Protocatechuic acid, (+)-catechin, p-hydroxyenzoic acid, chlorogenic acid, caffeic acid, (−)-epicatechin, ben-oic acid, rosmarinic acid, ferulic acid and apigenin were used astandard. Identification and quantitative analysis were done byomparison with standards. The amount of each phenolic com-ound was expressed as mg per gram of extract.

.7. Mutagenic and antimutagenic activity

.7.1. Viability assays and determination of test concentrationsCytotoxic dose of the extract was determined by the method of

ortelmans and Zeiger (2000). The toxicity of the extract toward. typhimurium TA98 and TA100 was determined as described inetail elsewhere (Santana-Rios et al., 2001; Yu et al., 2001).

.7.2. Mutagenicity and antimutagenicity testsIn this study, the plate incorporation method was used to deter-

ined the mutagenicity and antimutagenicity (Maron and Ames,983) as detailed elsewhere (Sarac and Sen, 2014). The knownutagens 4-nitro-o-phenylenediamine (4-NPD) 3 �g/plate) for

. typhimurium TA98 and sodium azide (NaN3) (8 �g/plate)or S. typhimurium TA100 were used as positive controls andthanol/water (1:1, v/v) was used as a negative control in muta-enicity and antimutagenicity tests. The test compounds was usedt different concentrations (3, 0.3, and 0.03 mg/plate for aerial parts,nd 1.5, 0.15 and 0.015 mg/plate for rhizomes).

.8. Statistical analysis

All the experimental results were as mean ± S.D. of three par-llel measurements. The data was entered into a Microsoft Excelatabase and analyzed using SPSS. The IC50 values were obtainedy the linear regression analysis.

. Results and discussion

Disc diffusion was used to evaluate and compare the antimicro-ial activities of ethanolic extracts of I. germanica aerial parts andhizomes with standard antibiotics (data not shown). The resultshowed ethanolic extracts of the aerial parts and rhizomes of I. ger-anica to exhibit minimal antimicrobial activity against B. subtilisTCC 6633, with inhibition zones of 8 mm and 12 mm, respectively.

DPPH assays were used to evaluate the free-radical-scavengingapacity of the ethanolic extracts of the aerial parts and rhizomesf I. germanica. IC50 values of the extracts, BHT and ascorbic acidre given in Table 1. The ability of I. germanica extracts to scavengeree radicals identified using DPPH can be attributed mainly to thehenolic content of the plant.

Total antioxidant activities of ethanolic extracts of the aerialarts and rhizomes of I. germanica were evaluated using the �-arotene–linoleic acid method (Table 2). The total antioxidant

ctivity of both extracts was found to be greater than that of ascor-ic acid and BHT at similar concentrations.

The phenolic contents of the ethanolic extracts of the aerialarts and the rhizomes of I. germanica were found to be,

a Values expressed are means ± S.D. of three parallel measurements.

respectively, 267.36 ± 2.05 and 331.96 ± 1.78 mg GAE/g extract.These results are in line with the results of �-carotene–linoleicacid testing. The primary phenolic compounds identified in theethanolic extract of the aerial parts of I. germanica were pro-tocatechuic acid (0.356 ± 0.004 mg/g extract), chlorogenic acid(0.164 ± 0.0064 mg/g extract) and ferulic acid (0.164 ± 0.008 mg/gextract), whereas the primary phenolic compounds identifiedin the ethanolic extract of the rhizomes were chlorogenicacid (2.44 ± 0.036 mg/g extract), (+)-catechin (2.14 ± 0.056 mg/gextract) and ferulic acid (0.452 ± 0.008 mg/g extract) (Table 3).

Nadaroglu et al. (2007) studied the antioxidant activity of aque-ous and ethanolic extracts of I. germanica using various antioxidantassays, including evaluations of reducing power, free-radical scav-enging, superoxide-anion-radical scavenging, hydrogen-peroxidescavenging and metal chelating. Both aqueous and ethanolicextracts were found to exhibit strong total antioxidant activity. Inaddition, phenols were detected in 1-mg aliquots of aqueous andethanolic I. germanica extracts at levels of 42.0 and 68.8 �g GAE,respectively.

The high antioxidant activity of I. germanica found in the presentstudy is fully supported by the literature, which indicates I. ger-manica rhizomes as well as leaves to be a rich source of flavonoids,ascorbic acid, vitamins and phenolics (Rahman et al., 2000). Thehigh level of antioxidant activity can be attributed to the presenceof various phenolic compounds.

The ethanolic extract of the aerial parts, at 3, 0.3 and0.03 mg/plate, and the rhizomes at 1.5, 0.15 and 0.015 mg/plateconcentrations did not exhibit any mutagenic effect in the muta-genicity test performed with S. typhimurium TA98 and TA100 (datawas not shown).

The Ames test was used to assess the antimutagenic activityof the ethanolic extracts of the aerial parts and rhizomes of I. ger-manica on S. typhimurium TA98 and TA100 against 4-NPD and NaN3(Table 4). Concentrations of 0.03, 0.3 and 3.0 mg/plate were used forthe aerial part extracts, whereas concentrations of 0.015, 0.15 and1.5 were used for the rhizome extracts. The antimutagenic activityof the I. germanica extracts was found to be dose-dependent, with

p-Hydroxy benzoic acid NT 0.24 ± 0.008Caffeic acid 0.084 ± 0.008 0.16 ± 0.008Ferulic acid 0.164 ± 0.008 0.452 ± 0.008

a Values are expressed as mean ± S.D. NT: not detected

B. Burcu et al. / Industrial Crops and Products 61 (2014) 526–530 529

Table 4The antimutagenicity assay results of the ethanolic extract of the aerial parts and rhizomes of I. germanica for S. typhimurium TA98 and TA100 bacterial strains.

Test items Concentration (mg/plate) Number of revertants

TA98 TA100

Mean ± S. error Inhibition (%) Mean ± S. error Inhibition (%)

Negative control 6.3 ± 3.2a 13.3 ± 5.04-NPD* 3 330 ± 31.3 –NaN3

* 8 – 394.2 ± 28.0Extract of the aerial parts 3 158.2 ± 38.2 52.1 139.5 ± 14.4 56.1

0.3 211 ± 37.3 36.1 153.2 ± 27.4 52.60.03 291.5 ± 17.0 16.7 252.2 ± 25.8 21.4

Extract of the rhizomes 1.5 145.2 ± 28.8 56.4 163.6 ± 25.9 67.80.15 243 ± 31.0 26.9 246.6 ± 29.8 58.50.015 204.5 ± 24.5 38.7 310.2 ± 21.2 21.4

* 4-NPD and NaN3 were used as positive controls for S. typhimurium TA98 and TA100 strains, respectively.a n ana

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f 1.5, 0.15 and 0.015 mg/ml. The strongest antimutagenic activityas observed at 1.5 mg/plate concentration of the rhizome extract

gainst S. typhimurium TA 100 strain.

. Conclusions

The results of this study indicate that ethanolic extracts of theerial parts and rhizomes of I. germanica possess high antioxidantnd antimutagenic activities in vitro. Considering that the extractsxamined in the present study were safe at the tested concen-rations, the results of this study suggest that the I. germanicalant may represent a readily accessible source of natural products,

ncluding potential supplements and inputs for the pharmaceuticalnd cosmetic industries. Moreover, the antioxidant and antimu-agenic properties of I. germanica extracts indicate that they mayrovide prophylaxis against various diseases such as heart disease,troke, arteriosclerosis and cancer. Further studies conducted usinglant species selected based on the results of the present study areecessary and may lead to the potential discovery of new, naturalioactive compounds.

onflicts of interest statement

The authors declare no conflict of interest.

cknowledgements

A part of this article is a part of Burcu Basgedik’s master ofcience thesis.

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