Full Terms & Conditions of access and use can be found athttps://www.tandfonline.com/action/journalInformation?journalCode=ljlc20

Journal of Liquid Chromatography & RelatedTechnologies

ISSN: 1082-6076 (Print) 1520-572X (Online) Journal homepage: https://www.tandfonline.com/loi/ljlc20

Antimicrobial, DPPH scavenging and tyrosinaseinhibitory activities of Thymus vulgaris, Helichrysumarenarium and Rosa damascena mill. ethanolextracts by using TLC bioautography and chemicalscreening methods

Mustafa Akin & Neslihan Saki

To cite this article: Mustafa Akin & Neslihan Saki (2019) Antimicrobial, DPPH scavengingand tyrosinase inhibitory activities of Thymus�vulgaris,�Helichrysum�arenarium and Rosadamascena�mill. ethanol extracts by using TLC bioautography and chemical screeningmethods, Journal of Liquid Chromatography & Related Technologies, 42:7-8, 204-216, DOI:10.1080/10826076.2019.1591977

To link to this article: https://doi.org/10.1080/10826076.2019.1591977

Published online: 04 Apr 2019.

Submit your article to this journal

Article views: 68

View Crossmark data

Antimicrobial, DPPH scavenging and tyrosinase inhibitory activities of Thymusvulgaris, Helichrysum arenarium and Rosa damascena mill. ethanol extracts byusing TLC bioautography and chemical screening methods

Mustafa Akin and Neslihan Saki

Faculty of Art and Sciences, Department of Chemistry, University of Kocaeli, Kocaeli, Turkey

ABSTRACTAntimicrobial, DPPH scavenging and tyrosinase inhibitory activities of Thymus vulgaris, Helichrysumarenarium and Rosa damascena Mill. ethanol extracts by using TLC bioautography and chemicalscreening methods. The ethanol extracts of Thymus vulgaris (Tv), Helichrysum arenarium (Ha) andRosa damascena Mill. (Rm) (red) were screened for their antimicrobial, 2,2-Diphenyl-1-picrylhydrazyl(DPPH) radical scavenging and tyrosinase inhibitory activities. The test microorganisms includedbacteria of Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). Thin LayerChromatography (TLC) - bioautography, disk diffusion and well diffusion methods were used forthe antimicrobial activity assays. Rosa damascena Mill. extract was effective against E. coli and allplant extracts showed antimicrobial activity against S. aureus. The phenolic acids in the structureof the extracts were also identified by LC-MS analysis. Human blood agar well diffusion methodand TLC-DPPH assays were used to identify the hemolytic and antioxidant activity of plantextracts, respectively, along with 10 compounds including phenolic acids as a standard.Among these compounds, caffeic acid (Rf ¼ 0.68) was detected in all extracts while vanillic acid(Rf ¼ 0.75), and gallic acid (Rf ¼ 0.51) was found in Tv extract. Kojic acid (Rf ¼ 0.36), on the otherhand, was detected in Rm extract as a tyrosinase inhibitor. All plant extracts presented tyrosinaseinhibitory activities on TLC-bioautography assay.

GRAPHICAL ABSTRACT

KEYWORDSTLC; bioautography;antimicrobial; DPPH;tyrosinase inhibitors;hemolytic activity; LC-MS

Introduction

Plants are used in the treatment of many diseases as folkmedicine among the people. Increased safety awareness incommunities has increased the public attention on natural

antioxidants and antimicrobials with high yields and lowtoxic effects.[1] Today, the active ingredients of plants have

CONTACT Neslihan Saki sakineslihan1@gmail.com Faculty of Art and Sciences, Department of Chemistry, University of Kocaeli, Kocaeli, 41380, TurkeyColor versions of one or more of the figures in the article can be found online at www.tandfonline.com/ljlc.

Supplemental data for this article can be accessed on the publisher’s website.

� 2019 Taylor & Francis Group, LLC

JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES2019, VOL. 42, NOS. 7-8, 204–216https://doi.org/10.1080/10826076.2019.1591977

been purified and used commercially in different drugforms.[2] Due to the alkaloid, flavonoid, saponin and poly-phenol substances present in the structure of natural prod-ucts, they are used for the removal of reactive oxygenspecies and for the treatment of pathogenic microorgan-isms.[3,4] These phyto-constituents confer specific character-istics and properties to plants and analysis of thesephytochemicals helps to determine the biological activities ofplants.[5] Therefore, it becomes necessary to investigate newantibacterial compounds found in plants because of theincreasing resistance of bacteria to synthetic antibiotics andincrease in formation of fatal infections.[6]

Thin-layer chromatography–direct bioautography (TLC–DB)is one of the methods used in evaluation of biological activitiesof plants and their constituents.[7–9] Different chromogenicagents have been adopted in TLC bioautography for fast screen-ing and separating natural compounds exerting antioxidant,antimicrobial and enzyme inhibitory activities. In this process,the biological activity of the examined plant extract is carriedout directly on a chromatographic plate and chromogenic agentsuspension is sprayed on to the developed TLC plate by usingappropriate solvent system.[10–13] DPPH� (2, 2-Diphenyl-1-picrylhydrazyl) free radical, on the other hand, is one of theTLC-DB application extensively chosen to screen antioxidantcomponents.[14,15]

The present study investigates antibacterial, radical scaveng-ing activities and tyrosinase inhibitory activities of Thymusvulgaris, Helichrysum arenarium and Rosa damascena Mill. byusing TLC bioautography and chemical screening methods.

Thymus vulgaris (herbarium code: 3135614) is a popularplant from Lamiaceae family. The leaves and flowers of thisplant are used in Mediterranean diet and it is a medicinalplant with its anti-inflammatory, antibacterial, antifungal,and antioxidant properties.[16]

Helichrysum arenarium (herbarium code: 3500410) is aperennial plant known from the Netherlands to Bulgaria. Itis used as anti-microbial and anti-inflammatory in folkmedicine and in the treatment of gall bladder disorders andlumbago.[17,18]

Rosa damascena Mill. (herbarium code: 2855942) is awell-known ornamental plant, referred as the king of flowershaving more than 200 species throughout the world. In folkmedicine it is used to treat digestive problems, abdominaland chest pains, nervous stress, depression, skin problems,headache and as an anti-inflammatory and cardiotonic.[19]

Experimental

Chemicals

Hexane, ethanol, Mueller-Hinton broth, Mueller-Hintonagar, chloroform, methanol, formic acid (85%), ethyl acetate,acetone, anisaldehyde, sulfuric acid, acetic acid, MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyltetrazolium bromide), 2,2-diphenyl-1-picrylhydrazyl, L-tyrosine, mushroom tyrosinase,and kojic acid were purchased from Merck. Blanc disc andantibiotic loaded discs were obtained from Fluka and TLCaluminum sheet (60 F254) was purchased from Merck. Allother chemicals used in this study were analytical grade.

Plant materials

Thymus vulgaris (Tv) and Helichrysum arenarium (Ha)plants were collected from Kocayayla plateau in Uluda�gmountains (Bursa-Turkey) at height of 1400m in May 2018.The plants were washed with tap water and de-ionized waterand kept at room temperature until the water had evapo-rated. Flowers of Helichrysum arenarium and Thymus vulga-ris were manually separated from the plant body and theflowering sections were dried in the humidity cabinet (mem-mert) on filter paper for 10 d (at 30 �C and 15% humidityconditions). Dried red Rosa damascena Mill. (Rm) flowerswere purchased from a local market (Isparta-Turkey). Thedried plant parts were pulverized by grinding on a labora-tory type mill and stored at þ 4� C until the experimentswere done.

Preparation and extraction of plant materials

First, 100 g of the powdered plant was extracted with500mL of hexane for 24 hr at room temperature. After fil-tering twice through Whatman (no 6) filter paper, the solv-ent was evaporated in the evaporator and the residue wasdissolved in hexane to give a stock solution. The remainingplant from the hexane extract was dried at room tempera-ture for 5 d. This fat-free plant material was extracted againwith 300mL ethanol for 24 hr at room temperatures and thesame process as described above was applied. The residuewas dissolved in ethanol to give a stock solution, afterevaporation.[20]

Microorganisms and medium

Microbial cultures of Escherichia coli (ATCC 25922) andStaphylococcus aureus (ATCC 25923) were supplied byAssociate Professor Gulnur Arabaci, Department ofChemistry, Sakarya University. The bacterial suspensionswere prepared according to the procedure optimized earlierby Grzelak.[21] Bacterial colony was taken from the stockculture, put in to 10mL M-H (Mueller Hinton) broth andincubated at 37 �C for 20 h. The bacterial suspensionobtained from the pre-incubation was diluted with M-Hbroth in 1:200 proportions. Then, 5mL of the diluted sus-pension was added to 20mL M-H broth and placed on themagnetic stirrer at 37 �C for 5 h. A 20mL amount of M-Hbroth (pH 7.2 ± 0.2) was inoculated with 1mL bacterial sus-pension obtained directly from the additional pre-incubationand placed on the magnetic stirrer at 37 �C for 7 h.

Thin layer chromatography (TLC)

Solutions were prepared from stock solutions of plantextracts at a concentration of 10mg/mL. The pre-coated sil-ica gel TLC aluminum sheet (60 F254) was cut to dimensionsof 10 cm x 10 cm. 5 mL extracts and 3 mL standard solutionsof tested standard compounds (2 mg/mL) were loaded intothe 8mm wide bands, leaving 10mm from the bottom ofthe plate and 10mm spacing from the edges. The samples

JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 205

were manually applied using an automatic pipette with avolume of 0–10 microliters. Camag twin trough glass cham-ber was used for the linear ascending development (20 cm x20 cm) and the chromatograms were presaturated with themobile phase vapor for 20–30min. The length of the chro-matogram run was up to 70mm from the point of applica-tion and the chromatographic conditions were optimized forthe ethanol extracts of the plants. Four different mobilephase were used in this study; mobile phase 1, chloroform:hexane: methanol: formic acid (50: 50: 10: 1 v/v/v); mobilephase 2, ethyl acetate: toluene: formic acid (70:30:10 v/v/v);mobile phase 3, chloroform: hexane: methanol: water: ethyl-acetate (25:25:15:3,5:45 v/v/v); and mobile phase 4 (DPPHassay), chloroform: ethyl acetate: acetone: formic acid (40:30: 20: 10 v/v/v). The developed plates were dried at roomtemperatures for 5 d. Chemical derivatization was performedwith anisaldehyde-sulfiric acid reagent consisting of anisal-dehyde- acetic acid- concentrated sulfuric acid- methanol(5-100-50-850mL). The reagent was taken in to a 500mLbeaker, dried plates were dipped into the beaker manuallyfor 3 sec and derivatized in an oven at 110 �C for 5min. Thebioautographic derivatization was explained in TLC-Bioautography and TLC-DPPH section.

TLC bioautography

Ethanol extracts were loaded onto TLC and developed byusing appropriate mobile phase and developed plates weredried at room temperature for 5 d. The bacterial suspensionswere prepared according to the procedure described previ-ously. The TLC plates were immersed for 8 h in the givenbacterial suspension and incubated in the humidity cabinetfor one night at 37 �C. After that the chromatograms weresprayed with % 0.2 MTT aqueous solutions by using anEMD Millipore TLC sprayer, re-incubated at 37 �C for 0.5 to3 hr and photos of bioautograms were taken with a KODAKpixpro astro zoom AZ421 digital camera.[9]

TLC-DPPH assay

Chromatograms were developed as described in “Thin layerchromatography” section and sprayed with 0.2% methanolicsolution of DPPH.[13,22] Immediately after being sprayed,light yellow areas were detected against the violet back-ground in the bands with antioxidant activity.

In vitro DPPH assay

DPPH free radical scavenging activity of the plant extractsand standard compounds including phenolic acids weremeasured by the method described in Akin et al.[23].Different concentrations of extracts and standard com-pounds in ethanol were prepared as the test solutions. 1mLof each molecule with prepared concentrations was takeninto test tubes and 0.5mL of 1mM DPPH solution inmethanol was added. These solutions were incubated for 1 hat room temperature and the absorbance was read at517 nm using UV–VIS spectrophotometer (Optimizer). The

DPPH radical scavenging activity percentage was calculatedby using the following formula:

DPPH radical scavenging activity %ð Þ¼ Acontrol � Acontrol

Acontrol� 100

where Acontrol is the absorbance of the control reaction mix-ture, Asample is the absorbance of the sample.

Disc diffusion assay

The antimicrobial activity of plant extracts were investigatedagainst the representative microorganisms using disc diffu-sion method.[24] Bacterial cultures were incubated overnightat 37 �C in the Mueller-Hinton Broth (MHB) medium.Incubated microorganisms were added to sterile tubes con-taining 5mL purified water and adjusted to 0.5 McFarlandstandards (1.5� 108 CFU/mL of bacteria) by spectrophoto-metrically. The cultures taken from tubes by using sterileswab were inoculated on petri dish containing MuellerHinton agar. Plant extracts were adjusted to the 50mg/mLfinal concentration by serial dilutions. Two discs were separ-ately impregnated with 20mL and 10mL of prepared solutionsand placed on the inoculated agar. Blank discs were impreg-nated with ethanol (20mL for each blank disc) as negativecontrol while ceftriaxone (30mg/disc) was used as positive ref-erence standards. Finally, inoculated petri dishes incubated at37 �C for 24h at incubator and antimicrobial activities wereevaluated by measuring the zone of inhibition against the testorganisms. The assay was carried out triplicate.

Well diffusion assay

Agar well diffusion assay was used to determine the anti-microbial activity of plant ethanol extracts against testedmicroorganisms.[25] First, the tested bacteria were inoculatedinto M-H broth overnight at 37 �C. Then, optical densitiesof the bacteria were adjusted to the 0.5 McFarland standard(1.5� 108 CFU/mL) by using UV spectrophotometer at600 nm. 30mL of Mueller-Hinton agar was pour in to eachpetri dish, after solidification wells with 6mm diameter werepunched by using cork borer. The test bacterial strains wereseparated on to the surface of the agar and the punchedwells were loaded with different concentrations of plantextracts. The plates were incubated 24 h at 37 �C and inhib-ition zones were measured in millimeter (mm). The assaywas carried out triplicate.

Hemolytic assay

Hemolytic activity of the tested plant extracts were analyzedby using agar well blood diffusion method.[25] Human bloodagar (10%) plates were purchased from local supplier (Rtalaboratories A.S). Wells with 6mm diameter were punchedon the agar by using sterile cork borer and loaded with100ml ethanol containing 500 mg/mL of plant extracts.Triton X-100 (10 mg/mL) was used as positive control andplates were incubated 24 h at room temperature. Hemolysis

206 M. AKIN AND N. SAKI

were observed with cleaner zone of inhibition and measured(mm). The assay was carried out triplicate.

TLC-Enzyme inhibition activity assay

Tyrosinase stock solution (10.000U/mL) was prepared by dis-solving enzyme in 20mM phosphate buffer at pH 6.8. Thestock solution was diluted to different concentration in thesame phosphate buffer to check enzyme activity. In the deter-mination of enzyme activity, one unit caused an increase inabsorbance at 280nm of 0.001 per min at pH 6.8 at roomtemperature in a 3mL reaction mixture containing L-tyrosine(0.05mM). L-Tyrosine stock solution was prepared by dissolv-ing in phosphate buffer at pH 6.8 to give a final concentrationof 1.0, 0.5, and 0.1mM. Plant extracts in which the loadingand development conditions were described above in TLC sec-tion were loaded on TLC plates at 20mg/mL concentrationand to detect the existing of tyrosinase inhibitors in eachspot, plates were sprayed with tyrosinase (about 65U/mL) andL-tyrosine solutions (0.5mM) respectively. Then, standardtyrosinase inhibitors kojic acid and thymol were loaded to theTLC plates (0.02mg/mL). Detection without prior chromato-graphic separation was also applied for both kojic acid andplant extracts. 5mL of 10mg/mL plant extracts and 2mL ofkojic acid at different concentrations were applied to the TLCplates directly. After allowing to dry 30min at room tempera-ture, enzyme solution and L-tyrosine solution were sprayedrespectively and browning reaction on the TLC plate wasphotographed.[26]

Spectrophotometric assay

Effect of plant ethanol extracts on the activity of mushroomtyrosinase was determined by using the UV spectrophotom-eter according to the method described by Wangthonget al.[26]. All the prepared solutions (mushroom tyrosinaseabout 65U/mL- 0.2mL; L-Tyrosine 0.5mM- 0.3mL) and0.3mL of different concentrations of plant extracts weremixed in a 1mL cell. The absorbance value of reaction wasmeasured at 280 nm for 10min without any incubation.Different assay controls under the same conditions wereapplied; 1- control, 0.2mL enzyme, 0.3mL L-Tyrosine in0.3mL phosphate buffer. 2- blank, 0.3mL L-Tyrosine in

0.6mL phosphate buffer. 3- sample control, 0.3mL sample,0.3mL L-tyrosine and 0.2mL enzyme. Kojic acid was usedas the positive control. Tyrosinase inhibition was calculatedas percentage by using following equation;

Tyrosinase inhibition % ¼ 1� ODs � ODscODc � ODb

� �� 100

The tyrosinase inhibitory capacity was also evaluated by cal-culating the relative IC50 ratio (RTIC-relative tyrosinase inhibi-tory capacity) of kojic acid and tested samples. They wereexpressed as mg natural extracts per mg kojic acid equivalentor mmole pure compounds per mmole kojic acid equivalent;

RTIC ¼ IC50 sampleð ÞIC50 kojic acidð Þ

LC-ms

The LC-MS analysis was performed with Agilent 6460 triplequadrupole system (ESIþAgilent jet stream) coupled withAgilent 1200 series HPLC. For liquid chromatography stud-ies; chromatographic separation was performed by usingZorbax SB-C18 column with 2.1� 50mm, 1.8mm particlesize (column temperature 35 �C; run time 13min; gradientflow mode). The mobile phase consisted of 0.05% formicacid þ 5mM ammonium formate (solvent A) and methanol(solvent B). The flow rate was 0.3mL min�1 and the injec-tion volume was 5 mL. Standard curve range was0.025–0.05–0.1–0.2–0.5–1–2–5–10 ppm.

The mass spectrometric analysis was performed using anESIþAgilent jet stream ionization source with AgilentBinPump-SL (G1312B9), Agilent h-ALS-SLþ(G1367D) auto-sampler, Agilent G1316B 1200 series Thermost. Col.Compart SL column compartment, G1379B micro degasser,MRM scan mode and the following operation parameters;350 �C gas temperature, 10mL/min gas flow, 45 psi nebu-lizer, 4000V capillary . Agilent G3793AA mass hunter opti-mizer software was used for data analysis.

Statistical analysis

All the assays were carried out in triplicate. The results wereexpressed as mean values and standard deviation (SD).

Figure 1. TLC chromatogram of 10mg/mL ethanol extracts; A and B mobile phase 1, C and D mobile phase 2, E and F mobile phase 3; A,C and E after derivatizationwith anisaldehyde reagent; B, D and F under UV366 light: Tv; Thymus vulgaris, Ha; Helichrysum arenarium, Rm; Rosa damascena Mill. Mobile phase1: chloroform:hexane:methanol:formic acid (50:50:10:1); mobile phase 2: ethyl acetate:toluene:formic acid (70:30:10); mobile phase 3, chloroform:hexane:methanol:water:ethylace-tate (25:25:15:3,5:45).

JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 207

Results and discussion

TLC-bioautography

TLC– bioautography technique is used to screening of anti-microbial properties of plant extracts. In this technique, a

developed and dried TLC plate was immersed in to growingbacterial suspension in M-H broth. After incubation, visualiza-tion agent is sprayed and inhibition areas are detected by colorchange. Although this technique is commonly used to deter-mine the antimicrobial properties of plant extracts,[10,27,28] in

Figure 2. TLC bioautography chromatograms; ethanol extracts of Thymus vulgaris, Helichrysum arenarium, Rosa damascena Mill.separated on TLC plates using threedifferent mobile phase sprayed with bacterial suspensions of Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). Numbers 1, 2 and 3 representsmobile phase 1,2 and 3.

208 M. AKIN AND N. SAKI

recent years, it has been used to identify other biologicalactivities such as antioxidant activity,[29] and enzyme inhib-ition.[30–32] Plant extracts carried out by TLC-Bioautographytechnique were observed using both anisaldehyde reagentand UV lamp (Figure 1).

Developed TLC plates with ethanol extracts of plantswere subjected to bioautography against Escherichia coli(ATCC 25922) and Staphylococcus aureus (ATCC 25923)bacterial strains. After derivatization with MTT reagent, it

was proven that all separated ethanol extracts of Tv and Hawere effective against Staphylococcus aureus (ATCC 25923)with mobile phase 1,2 and 3. Rm extract, on the other hand,showed no activity against to this bacterium in mobile phase2 but had activity against Escherichia coli in all mobilephases (Figure 2).

White areas indicated the inhibition of bacterial growthby compounds of the plant extract after 60min of incuba-tion at 37 �C and retention values of the main antibacterialconstituents were given in Table 1.

The TLC-bioautography method does not give a measur-able result of antimicrobial activity. It provides preliminaryinformation on the antimicrobial efficacy of the compoundsisolated in TLC[4] and it is used for determination of com-pounds with antibacterial and radical scavenging propertiesof plant extracts. In a study, Jesionek et al. tested methanol,ethanol and ethyl acetate extracts of S. nigra flos, M. officinalis,and V. tricolor by using TLC bioautography assay againstB. subtilis and all methanol extracts of plants showed antimicro-bial activity against tested microorganisms.[9] Acetone extractsof Ochna pulchra and some other available Ochna spp. werealso investigated for their antimicrobial activities againstStaphylococcus aureus by using TLC-bioautography method.[6]

Table 1. Retention values of separated compounds with antimicrobial activ-ities (The starting and ending retention values of the resulting white areas).

Mobilephase 1

Mobilephase 2

Mobilephase 3

Thymus vulgarisE.coli ND ND NDS. aureus 0.035–0.3 0–0.481

0.614–0.8430.324–05050.614–0.983

Helichrysum arenariumE.coli ND ND NDS. aureus 0–0.277

0.565–0.8430.107–0.590.686–0.903

0.107–0.445

Rosa damascena MillE.coli 0.241 0.241 0.330S. aureus 0–0.237 ND 0.330

Figure 3. TLC-DPPH free radical scavenging activity of plant extracts. A, TLC chromatogram under UV254; B, TLC-DPPH bioautogram. (Mobile phase: chloroform:ethyl acetate:acetone:formic acid (40:30:20:10 v/v). Rm: Rosa damascena Mill., Ha: Helichrysum arenarium, Tv: Thymus vulgaris. 1:thymol, 2:4-methylcatechol, 3:vanillicacid, 4:gallic acid, 5:cinnamic acid, 6:kojic acid, 7:naringin, 8:caffeic acid, 9:quercetin, 10:pyrocatechol.

JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 209

TLC-DPPH assay

TLC-DPPH test was applied to the plant extracts to iden-tify radical scavenging activities of separated compounds.DPPH bioautograms of plant extracts were comparedUV254 visualization of TLC plate developed by usingchloroform: ethyl acetate: acetone: formic acid (40: 30: 20:10 v/v) mobile phase. TLC analysis was carried out for sep-aration of ten standard compounds including phenolic

acids such as thymol, 4-methylcatechol, vanillic acid, gallicacid, cinnamic acid, kojic acid, naringin, caffeic acid, quer-cetin and pyrocathecol. Caffeic acid (Rf ¼ 0.68) wasdetected in all samples and these results showed similaritywith the study of Jesionek et al.[33] In addition, vanillicacid (Rf ¼ 0.75) and gallic acid (Rf ¼ 0.51) was observedin Tv extract while Kojic acid (Rf¼ 0.36) was detected inRm extract (Figure 3).

Figure 4. DPPH free radical scavenging activity of extracts and phenolic acids.

Figure 5. Antimicrobial activity of plant extracts against the bacterial strains tested based on disk-diffusion method. (Inhibition zones are given in mm).

210 M. AKIN AND N. SAKI

In vitro DPPH assay

DPPH assay was used to determine the free radical-scav-enging activity of the plant extracts. According to the thismethod; in the presence of antioxidant compounds, DPPHradical is scavenged and the purple color of the DPPH rad-ical turns in to a yellow one. This color change is measuredspoctrophotometrically. In our study; Tv, Ha, Rm ethanolextracts and other standards were tested to determine theirability to scavenging DPPH free radical. The highest anti-oxidant activity was obtained with thymol at 400 mg/mLconcentration (80.02% ± 0.13) followed by gallic acid(71% ± 0.41), caffeic acid (64.17% ± 1.03), vanillic acid(62.51% ± 0.06) and 4-methylcatechol (59.38% ± 0.11). Allother tested standard molecules and plant extracts showedmoderate activity. Tv showed the highest antioxidant activ-ity among the tested extracts (77% ± 0.02), Ha showed63.91% ± 1.82 and Rm 37% ± 0.34 at 400 mg/mL concentra-tion (Figure 4).

Disk diffusion assay

Disc diffusion method was used to determine the antimicro-bial activities of the ethanol extracts of plants against

Escherichia coli (ATCC 25922) and Staphylococcus aureus(ATCC 25923). All tested plant extracts at 20mL loadingshowed an inhibition effect on Staphylococcus aureus. Tvand Ha extracts were also effective at 10 mL loading, whileRm extract didn’t show any activity. The highest inhibitionzone was obtained by Tv extracts at 20 mL loading (22mm)followed by the positive control ceftriaxone (30 mg) (Figure5). On the other hand, among all plant extracts tested, onlythe Rm extract found to be effective against Escherichia coliat 20 mL loading (10mm).

Figure 6. Antimicrobial activity of plant extracts against the bacterial strains tested based on well-diffusion method.

Table 2. Well diffusion test results of plant extracts against tested microorganisms by well diffusion method (Inhibition zones are given in mm).

Helichrysum arenarium Rosa damascena Mill Thymus vulgaris

Concentration (mg/mL) Escherichia coli Staphylococcus aureus Escherichia coli Staphylococcus aureus Escherichia coli Staphylococcus aureus

5 – 22 ± 0.74 16 ± 0.71 20 ± 0.01 11 ± 0.13 20 ± 0.4910 – 23 ± 0.36 18 ± 0.09 25 ± 0.04 14 ± 0.41 23 ± 0.7420 – 25 ± 0.03 20 ± 0.81 29 ± 0.78 19 ± 0.19 30 ± 0.2840 – 28 ± 0.16 20 ± 0.41 32 ± 0.01 23 ± 0.07 32 ± 0.0450 – 28 ± 0.02 20 ± 0.19 35 ± 0.38 25 ± 0.56 32 ± 0.38

Figure 7. Hemolytic activities of plants ethanol extracts and Triton X 100 byusing agar well diffusion method.

JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 211

Well diffusion assay

Plant extracts at a concentration of 5, 10, 20, 40 and 50mg/mLwere loaded into each well of 50mL. Rm and Tv extracts wereeffective against E. coli in each concentration while Ha extractsdidn’t show any inhibition activity. On the other hand, alltested plant extracts showed antimicrobial activity againstS. aureus in each concentration (Figure 6) and inhibitionzones of extracts were given in Table 2.

When the studies about the antimicrobial activity of Tv,Ha and Rm extracts were examined, similar results wereobtained with our studies. Rafael de Oliveira et al.[34] inves-tigated Tv leaf extracts’ antimicrobial activity on planktoniccultures and mono- and polymicrobial bio-films formation.All bio-films showed significant reductions in CFU/mL.Mahboubi et al.[35] examined the thymus spp. essential oilantibacterial activities on E.coli and S.aureus by using diskdiffusion method and noted similar findings. Mahmoudiet al.[36] used well diffusion assay to determine Thymus vulgarisessential oil activity against E.coli. and measured the inhibitionzone as 10mm. Kutluk et al.[37] determined the MIC (min-imum inhibitory concentration) of Helichrysum arenariumagainst E. coli (32mg/mL) and S. aureus (8mg/mL). In anotherstudy, the methanolic extracts of 16 Helichrysum species wereinvestigated by Albayrak et al.[38] and extracts exhibitedantimicrobial activity against tested microorganisms withagar diffusion method. The role of flavonoids, terpenoidsand phenolic acids antibacterial activities were also demon-strated in many studies for Rosa damascena Mill.[39–41] Taliband Mahasneh et al.[42] studied the antimicrobial activitiesof butanol extract of Rm on gram positive and negative bac-teria and they found out 100% inhibition for Bacillus cereus(MIC 250 mg/mL).

Hemolytic assay

The hemolytic activity was studied to understand the bio-compatibility of plant extracts with red blood cells. On thispurpose, 10% human blood containing agar was usedaccording to the method described previously.[24] Hemolytic

activities of plant extracts and Triton-X 100 were given inFigure 7. Hemolysis results indicated the formation of clearzone on petri dishes containing human blood agar. The high-est hemolysis was obtained by positive control Triton-X 100(15.8 ± 0.06mm), followed by Tv (9.04± 0.11mm), Rm(8.14± 0.71mm) and Ha extracts (7.18± 0.03mm).

Tyrosinase inhibitory activities

Three natural plant extracts were examined by the TLC bio-autographic assay to determine the tyrosinase inhibitory

Figure 8. A- Anisaldehyde derivatized and B- TLC bioautogram of the tyrosinase inhibitory spots in Thymus vulgaris (TV), Helichrysum arenarium (HA), Rosadamascena Mill. (RM) (All plant extracts were loaded at 20mg/mL concentration), Thymol (1) and Quercetine (2) 0.02 mg/mL.

Figure 9. A- Direct bioautography chromatogram of plant extracts (10mg/mL)loaded without separation on TLC. B- TLC bioautograms of kojic acid obtainedby using L-Tyrosine as the substrate, TLC plate was spotted increasing amountof kojic acid (0.01 – 0.5mg). Sample loading is 2mL.

212 M. AKIN AND N. SAKI

activities. All three extracts and thymol showed inhibitoryactivity against mushroom tyrosinase (Figure 8). Figure 8Ashows the anisaldehyde derivatized chromatogram and B indi-cates the active bands having tyrosinase inhibition activitywith white spots against a brownish-purple background. TLCplates were separated by using chloroform: hexane: methanol:formic acid (50: 50: 10: 1 v/v/v) mobile phase and quercetineand thymol were used as the standard inhibitors of the

enzyme. On chromatogram B; Thymus vulgaris (Tv) ethanolextract showed three active bands with tyrosinase inhibitoryactivities being thymol (1) with a retention factor 0.592, quer-cetin (2) with a retention factor 0.512 and last active bandwith a retention factor 0.274. Helichrysum arenarium (Ha)ethanol extracts indicated two active bands with 0.445 and0.223 retention factors, respectively and Rosa damascena Mill.(Rm) ethanol extracts exhibited only one active band.

Figure 10. Tyrosinase inhibitory activities of studied plant extracts and kojic acid at different concentrations spectrophotometrically.

Figure 11. LC-MS chromatograms of phenolic compounds identified in plant extracts.

JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 213

In Figure 9, plant extracts were loaded to the TLC plateswithout separation (dot-blot technique) at 10mg/mL con-centration and all tested extracts exhibited tyrosinase inhibi-tory activities (9-A) On the other hand, standard inhibitorkojic acid at different concentrations was applied to the TLCplates without prior separation at different concentrationsand all tested concentrations showed inhibitory activityagainst mushroom tyrosinase (9-B).

Spectrophotometric assay

Tyrosinase inhibitory activities of studied plant extracts andkojic acid were examined at different concentrations spec-trophotometrically. Inhibitory activity of kojic acid wasstudied at 0.00025, 0.0005, 0.00125mM concentration andIC50 value was found to be 5.428� 10�3mM. On the otherhand; tyrosinase inhibitory activities of plant extracts werestudied at 1, 10 and 20mg/mL concentrations and IC50 val-ues were found to be 7.22mg/mL for Thymus vulgaris,13.64mg/mL for Helichrysum Arenarium, 12.08mg/mL forRosa damascena Mill. The relative tyrosinase inhibitory cap-acity was also calculated and percentage inhibition diagramswere given in Figure 10.

LC-ms

This technique was used to confirm the presence of phenoliccompounds in plant extracts detected by TLC method first.The presence of all phenolic substances detected by the TLCmethod has been also proven by this method.Chromatograms were given in Figure 11 and the amount ofphenolic acid determined was given in Table 3. Thymolwere identified in Tv extract (Figure 12) additionally, besidescaffeic acid (Rt: 2.223 min), vanillic acid (Rt: 2.203 min),

gallic acid (Rt: 0.550 min) and kojic acids (Rt: 0.494 min)already found by TLC. All other graphs were given as thesupplementary file.

Conclusions

The results of the present study revealed that the ethanolextracts of Thymus vulgaris, Helichrysum arenarium andRosa damascena Mill.can be considered as an effectivesource of antimicrobial, DPPH scavenger and tyrosinaseinhibitors. The antioxidant activities of the extracts wereconfirmed by in-vitro DPPH method and they were com-pared with the standard phenolic acids. The presence ofphenolic acids determined by TLC-DPPH method was alsoconfirmed by LC-MS analysis. These extracts are also agood alternative for food-borne pathogen bacteria control.TLC-bioautography is a useful method which gives easy andquick results and provides preliminary information aboutthe biological activities of the examined plant. The methodwere also supported by disk diffusion and well diffusionassays. Rm extract showed antimicrobial activity againstgram (�) bacteria E. coli. and all extracts were effectiveagainst gram (þ) bacteria of S. aureus. Hemolytic activitiesof extracts were determined by using human blood agar welldiffusion method and the highest inhibition zone weredetected by Tv extracts. All studied extracts had inhibitionability on tyrosinase enzyme and the inhibition activity wasconfirmed by both TLC and spectrophotometric method(Figure 12).

Funding

The authors gratefully acknowledge support from the KOCAELIUniversity Research Fund (Project number: BAP-2016/071)

Table 3. Values of ppm of phenolic acids detected in the extracts (LC-MS analysis).

ppm

Vanillic acid Gallic acid Caffeic acid Kojic acid

Thymus vulgaris 1.4291 ± 0.0214 0.1371 ± 0.0008 2.4549 ± 0.0242 –Helichrysum arenarium 4.6297 ± 0.1195 – 16.36 ± 0.4820 <0.1Rosa damascene Mill. – 1579.9 ± 1.2058 0.2829 ± 0.0241 <0.1

Figure 12. LC-MS chromatogram of thymol identified in Thymus vulgaris extract and thymol standard solution.

214 M. AKIN AND N. SAKI

References

[1] Wang, Z. L.; Yang, L. Y.; Zhang, X. H.; Zeng, X. W.; Li, P. S.;Pan, J. Y. Research Progress on Active AntioxidativeComponents from Natural Products. Drug. Eval. Res. 2012, 35,386–390.

[2] Waksmundzka-Hajnos, M.; Wawrzynowicz, T. Strategy ofPreparative Separation of Organic Compounds by Thin-LayerChromatography Methods. J. Liq. Chromatogr. Relat. Technol.2002, 25, 2351–2386. DOI: 10.1081/JLC-120014009.

[3] Huang, Q.; Xu, L.; Qu, W.-S.; Ye, Z.-H.; Huang, W.-Y.; Liu, L.-Y.; Lin, J.-F.; Li, S.; Ma, H.-Y. TLC Bioautography–GuidedIsolation of Antioxidant Activity Components of Extracts fromSophora Flavescens Ait. Eur. Food Res. Technol. 2017, 243,1127–1136. DOI: 10.1007/s00217-016-2820-z.

[4] Suleimana, M. M.; McGaw, L. J.; Naidoo, V.; Eloff, J. N.Detection of Antimicrobial Compounds by Bioautography ofDifferent Extracts of Leaves of Selected South African TreeSpecies. Afr. J. Tradit. Complement. Altern. Med. 2009, 7, 64–78.

[5] Tripathi, A. C.; Gupta, R.; Saraf, S. K. Phytochemical investiga-tion characterisation and anticonvulsant activity of Ricinuscommunis seeds in mice. Nat. Prod. Res. 2011, 25, 1881–1884.DOI:10.1080/14786419.2010.551753.

[6] Makhafola, T. J.; Eloff, J. N. Five Ochna Species Have HighAntibacterial Activity and More than Ten AntibacterialCompounds. S. Afr. J. Sci. 2012, 108, 689–694. DOI: 10.4102/sajs.v108i1/2.689.

[7] Choma, I. M.; Jesionek, W. Effects-directed BiologicalDetection: Bioautography. In Instrumental Thin-LayerChromatography, Chapter 11; Pool, C. Ed.; Elsevier:Amsterdam, Netherlands, 2015; pp. 279–312.

[8] Morlock, G. E.; Schwack, W. Hyphenations in PlanarChromatography. J. Chromatogr. A. 2010, 1217, 6600–6609.DOI: 10.1016/j.chroma.2010.04.058.

[9] Jesionek, W.; Majer-Dziedzic, B.; Choma, I. M. TLC–DirectBioautography as a Method for Evaluation of AntibacterialProperties of Thymus Vulgaris L. and Salvia Officinalis L.essential Oils of Different Origin. J. Liq. Chromatogr. Relat.Technol. 2017, 40, 292–296. DOI: 10.1080/10826076.2017.1298031.

[10] M�oricz, �A. M.; H€abe, T. T.; B€osz€orm�enyi, A.; Ott, P. G.; Morlock,G. E. Tracking and Identification of Antibacterial Components inthe Essential Oil of Tanacetum Vulgare L. by the Combination ofHigh-Performance Thin-Layer Chromatography with DirectBioautography and Mass Spectrometry. J. Chromatogr. A. 2015,1422, 310–317. DOI: 10.1016/j.chroma.2015.10.010.

[11] Jesionek, W.; M�oricz, A. M.; Alberti, A.; Ott, P. G.; Kocsis, B.;Horv�ath, G.; Choma, I. M. TLC-Direct Bioautography as aBioassay Guided Method for Investigation of AntibacterialCompounds in Hypericum Perforatum L. J. AOAC Int. 2015,98, 1013–1020. DOI: 10.5740/jaoacint.14-233.

[12] M�oricz, A. M.; Fornal, E.; Jesionek, W.; Majer-Dziedzic, B.;Choma, I. M. Effect-Directed Isolation and Identificationof Antibacterial Chelidonium Majus L. Alkaloids.Chromatographia 2015, 78, 707–716. DOI: 10.1007/s10337-015-2870-6.

[13] Jesionek, W.; Choma, I. M.; Majer-Dziedzic, B.; Malinowska, I.Screening Bacterial and Radical Scavenging Properties ofChosen Plant Extracts Using Thin-Layer chromatography-Direct Bioautography. J. Liq. Chromatog. Relat. Technol. 2014,37, 2882–2891. DOI: 10.1080/10826076.2014.907103.

[14] Cheng, Z. H.; Wu, T. TLC Bioautography: high ThroughputTechnique for Screening of Bioactive Natural Products. CCHTS.2013, 16, 531–549. DOI: 10.2174/1386207311316070004.

[15] Zhao, J. L.; Xu, L. J.; Huang, Y. F.; Zhou, L. G. Detection ofAntimicrobial Components from Extracts of the EndophyticFungi Associated with Paris Polyphylla Var. yunnanensis UsingTLC Bioautography MTT Assay. Nat. Prod. Res. Dev. 2008, 20, 51.

[16] Evans, W. C. Pharmacognosy; Saunders, London, UK, 2000.

[17] Yang, Y.; Huang, Y.; Gu, D.; Yili, A.; Sabir, G.; Aisa, H. A.Separation and Purification of Three Flavonoids fromHelichrysum arenarium (L.) Moench by HSCCC.Chromatographia 2009, 69, 963–967. DOI: 10.1365/s10337-009-0986-2.

[18] Czinner, E.; Hagym�asi, K.; Bl�azovics, A.; K�ery, �A.; Sz}oke, �E.;Lemberkovics, �E. The in Vitro Effect of Hlichrysi fl os onMicrosomal Lipid Peroxidation. J. Ethnopharm. 2001, 77,31–35. DOI: 10.1016/S0378-8741(01)00258-6.

[19] Ali, M.; Sultana, S.; Jameel, M. Phytochemical Investigation ofFlowers of Rosa Damascena Mill. Int. J. Herbal Med. 2016, 4,179–183.

[20] Savaro�glu, F.; _Iscen, C. F.; €Oztopcu, A. P.; Karadere, S.; _Ilhan, S.;Uyar, R. Determination of Antimicrobial and AntiproliferativeActivities of the Aquatic Moss Fontinalis Antipyretica Hedw.Turk. J. Biol. 2011, 35, 361–369. DOI: 10.3906/biy-0906-46.

[21] Grzelak, E. M.; Majer-Dziedzic, B.; Choma, I. M.; Pilorz, K. M.Development of a Novel Direct Bioautography–Thin-LayerChromatography Test: Optimization of Growth Conditions forGram-Positive Bacteria, Bacillus subtilis. J. AOAC Int. 2013, 96,386–391. DOI: 10.5740/jaoacint.11-466.

[22] Cie�sla, L.; Krysze�n, J.; Stochmal, A.; Oleszek, W.; Waksmundzka-Hajnos, M. Approach to Develop a Standardized TLC-DPPH�Test for Assessing Free Radical Scavenging Properties of SelectedPhenolic Compounds. J. Pharm. Biomed. 2012, 70, 126–135. DOI:10.1016/j.jpba.2012.06.007.

[23] Akin, M.; Nalbantoglu, S.; Saki, N. Total Phenols, AntioxidantPotential and Tyrosinase Inhibitory Activity of HoneybeeCollected Pollen from Turkey. Res. J. Biotech. 2013, 8, 15–18.

[24] Saki, N.; Akin, M.; Atsay, A.; Karaoglu, H. R. P.; Kocak, M. B.Synthesis and Characterization of Novel Quaternized 2,3-(Diethylmethylamino)Phenoxy Tetrasubstituted Indium andGallium Phthalocyanines and Comparison of TheirAntimicrobial and Antioxidant Properties with DifferentPhthalocyanines. Inorg. Chem. Comm. 2018, 95, 122–129. DOI:10.1016/j.inoche.2018.07.010.

[25] Prakash, S.; Ramasubburayan, R.; Ramkumar, S. V.;Kannapiran, E.; Palavesam, A.; Immanuel, G. In Vitro—Scientific Evaluation on Antimicrobial, Antioxidant, CytotoxicProperties and Phytochemical Constituents of TraditionalCoastal Medicinal Plants. Biomed. Pharm. 2016, 83, 648–657.DOI: 10.1016/j.biopha.2016.07.019.

[26] Wangthong, S.; Tonsiripakdee, I.; Monhaphol, T.;Nonthabenjawan, R.; Wanichwecharungruang, P. Post TLCDeveloping Technique for Tyrosinase Inhibitor Detection.Biomed. Chromatogr. 2007, 21, 94–100. DOI: 10.1002/bmc.727.

[27] Betina, V. Bioautography in Paper and Thin-LayerChromatography and Its Scope in the Antibiotic Field.J. Chromatogr. 1973, 78, 41–51. DOI: 10.1016/s0021-9673(01)99035-1

[28] Horvath, G.; Jambor, N.; Vegh, A. Antimicrobial Activity ofEssential Oils: The Possibilities of TLC—Bioautography. FlavourFragrance J. 2010, 25, 178–182. DOI: 10.1002/ffj.1993.

[29] Marston, A. Thin-Layer Chromatography with BiologicalDetection in Phytochemistry. J Chromatogr A 2011, 1218,2676–2683. DOI: 10.1016/j.chroma.2010.12.068.

[30] Moniem, A.; Hassan, S. TLC Bioautographic Method forDetecting Lipase Inhibitors. Phytochem. Anal. 2012, 23,405–407. DOI: 10.1002/pca.1372.

[31] Br€am, S.; Wolfram, E. Recent Advances in Effect-DirectedEnzyme Assays Based on Thin-Layer Chromatography.Phytochem. Anal. 2017, 28, 74–86. DOI: 10.1002/pca.2669.

[32] Abed, S. A.; Sirat, H. M.; Taher, M. Tyrosinase Inhibition, anti-Acetylcholinesterase, and Antimicrobial Activities of thePhytochemicals from Gynotroches Axillaris Blume. Pak. J.Pharm. Sci. 2016, 29, 2071–2078.

[33] Rafael de Oliveiraa, J.; Viegasa, D.; Martinsa, A. P. R.;Carvalhob, C. A. T.; Soaresc, C. P.; Camargoa, S. E. A.; Jorgea,A. O. C.; Dias de Oliveira, L. Thymus Vulgaris L. extract HasAntimicrobial and anti-Inflammatory Effects in the Absence of

JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 215

Cytotoxicity and Genotoxicity. Archives of Oral Biology 2016,83, 648–657. DOI: 10.1016/j.biopha.2016.07.019.

[34] Jesionek, W.; Majer-Dziedzic, B.; Choma, I. M. Separation,Identification, and Investigation of Antioxidant Ability of PlantExtract Components Using TLC, LC–MS, and TLC–DPPH.J. Liq. Chromatogr. Relat. Technol. 2015, 38, 1147–1153. DOI:10.1080/10826076.2015.1028295.

[35] Mahboubi, M.; Heidarytabar, R.; Mahdizadeh, E.; Hosseini, H.Antimicrobial Activity and Chemical Composition of ThymusSpecies and Zataria Multiflora Essential Oils. Agric. Nat. Res.2017, 51, 395–401. DOI: 10.1016/j.anres.2018.02.001.

[36] Valizadeh, V.; Mahmoudi, R.; Fakheri, T.; Katiraee, F.;Rahmani, V. Investigating the Phytochemical, Antibacterial andAntifungal Effects of Thymus Vulgaris and CuminumCyminum Essential Oils. Med. Lab. J. 2016, 10, 36–43. DOI:10.1016/j.anres.2018.02.001.

[37] Kutluk, I.; Aslan, M.; Orhan, I. E.; €Ozcelik, B. Antibacterial,Antifungal and Antiviral Bioactivities of Selected HelichrysumSpecies. S. Afr. J. Bot. 2018, 119, 252–257. DOI: 10.1016/j.sajb.2018.09.009.

[38] Albayrak, S.; Aksoy, A.; Sagdic, O.; Hamzaoglu, E. Compositions,Antioxidant and Antimicrobial Activities of Helichrysum(Asteraceae) Species Collected from Turkey. Food Chem. 2010,119, 114–122. DOI: 10.1016/j.foodchem.2009.06.003.

[39] Ulusoy, S.; Bosgelmez-Tınaz, G.; Secilmis-Canbay, H.Tocopherol, Carotene, Phenolic Contents and AntibacterialProperties of Rose Essential Oil, Hydrosol and Absolute. Curr.Microbiol. 2009, 59, 554–558. DOI: 10.1007/s00284-009-9475-y.

[40] Tofighi, Z.; Molazem, M.; Doostdar, B. Antimicrobial Activitiesof Three Medicinal Plants and Investigation of Flavonoids ofTripleurospermum Disciforme. Iran J Pharm Rese: IJPR 2015,14, 225.

[41] Arido�gan, B. C.; Baydar, H.; Kaya, S.; Demirci, M.; Ozbasar,D.; Mumcu, E. Antimicrobial Activity and ChemicalComposition of Some Essential Oils. Arch. Pharm. Res. 2002,25, 860–864. DOI: 10.1007/BF02977005.

[42] Talib, W. H.; Mahasneh, A. M. Antimicrobial, Cytotoxicity andPhytochemical Screening of Jordanian Plants Used inTraditional Medicine. Molecules 2010, 15, 1811–1824. DOI:10.3390/molecules15031811.

216 M. AKIN AND N. SAKI