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Colloids and Surfaces B: Biointerfaces 133 (2015) 73–80

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

j o ur nal ho me pa ge: www.elsev ier .com/ locate /co lsur fb

erbal infusions of black seed and wheat germ oil: Their chemicalrofiles, in vitro bio-investigations and effective formulations ashyto-Nanoemulsions

. Pinar Gumusa, Emine Gulera,b, Bilal Demirb, F. Baris Barlasb, Murat Yavuzc,ilara Colpankanb, A. Murat Senisikd, Serap Teksoze, Perihan Unake, Hakan Coskunola,f,g,una Timura,b,∗

Institute of Drug Abuse Toxicology & Pharmaceutical Sciences, Ege University, 35100 Bornova, Izmir, TurkeyFaculty of Science, Biochemistry Department, Ege University, 35100 Bornova, Izmir, TurkeyFaculty of Science, Chemistry Department, Dicle University, 21280 Diyarbakir, TurkeySifa University Hospital, 35100 Bornova, Izmir, TurkeyInstitute of Nuclear Sciences, Ege University, 35100 Bornova, Izmir, TurkeySchool of Medicine, Department of Psychiatry, Ege University, 35100 Bornova, Izmir, TurkeyEge LS, Ege University, 35100 Bornova, Izmir, Turkey

r t i c l e i n f o

rticle history:eceived 19 March 2015eceived in revised form 21 May 2015ccepted 26 May 2015vailable online 4 June 2015

eywords:erbal infusionalendulalack seed oilheat germ oil

anoemulsion based carrier

a b s t r a c t

The reported studies related to black seed oil (BSO) and wheat germ oil (WGO) have illustrated that theyhave a wide range of biological activities. Therefore, enhancing the amount of bio-active compounds thatcaused higher cell based anti-oxidative effect as well as cell proliferation, etc. in seed oils, infusion ofcrude plant material has been gained importance as a traditional technique. Herein, we accomplishedthe infusion of Calendula flowers that also contains many phyto-constituents into BSO and WGO. Afterthe infusion of oils, the change of phytochemical amount was investigated and evaluated according tothe oils by chromatography, radical scavenging activity. Subsequently, for investigating the biologicalimpact upon live cells, cytotoxicity, cell-based antioxidant capacity, wound healing and radioprotectiveactivity were tested with monkey kidney fibroblast like cells (Vero) and HaCaT keratinocytes. In vitrocell based experiments (wound healing and radioprotective activity) confirmed that Calendula infusedBSO and WGO have greater bio-activity when compared to those plain forms. The herbal oils prepared

n vitro cell culture with an effective extraction technique were incorporated into nanoemulsion systems which will be thencalled as ‘Phyto-Nanoemulsion’. After herbal oil biomolecules were encapsulated into nanoemulsionbased delivery systems, the designed formulations were investigated in terms of biological activities. Inconclusion, these preparations could be a good candidate as a part of dermal cosmetic products or foodsupplements which have the therapeutic efficiency, especially after radio- or chemotherapy.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

Since the beginning of the dawn of history, there has been arowing interest toward herbal preparations such as medicinal

lants and their oil forms. Herbal preparates are commonly iden-ified as mixtures of selected medicinal plant, plant parts or seedsn order to treat specific health conditions [1]. Many reports and

∗ Corresponding author at: Ege University, Faculty of Science, Biochemistryepartment, 35100 Bornova, Izmir, Turkey. Tel.: +90 232 311 2455;

ax: +90 232 311 5485.E-mail addresses: suna.timur@ege.edu.tr, sunatimur@yahoo.com (S. Timur).

ttp://dx.doi.org/10.1016/j.colsurfb.2015.05.044927-7765/© 2015 Elsevier B.V. All rights reserved.

reviews have been showed up related to pharmacology and chem-istry of medicinal plants.

Seed oils of different herbal materials are being considered asfood supplement and edible oil because of their constituents thathave unique phytochemicals with significant antioxidant proper-ties and health benefits. Among various oil seeds, black seed (BS)– also known as black cumin or Nigella sativa – is a seed of cap-sulated plants, and belongs to the Ranunculaceae family. BS is anannual herb growing in countries bordering the Mediterranean

sea, Pakistan and India [2]. The reported studies related to BS (N.sativa) have illustrated that it has a wide range of biological activi-ties, such as antioxidant [3], anti-inflammatory [4], antidiabetic [5],anti-asthma [6], and anti-prostate cancer effects [7], antibacterial

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8], antihelminthic [9], immunomodulative [10] and hepatoprotec-ive [11]. These properties have been attributed to a variety of activeonstituents in seeds and its fixed oil [12,13] of which saponins areainly characteristic compounds of water-soluble extracts from BS

14].In addition to BS, wheat germ (WG) as a rich source of

oth phytochemical and protein is an edible oil. Wheat germ oilWGO) contains tocopherols and tocotrienols known as powerfulipid-soluble antioxidants. Moreover, WGO also includes protein26–35%), lipids (10–15%), sugar (17%), fiber (1.5–4.5%), and miner-ls (4.0%). The bioactive compounds of WGO were also reported asocopherols (1300–2700 mg/kg), phytosterols (PSs) (24–50 mg/kg),olicosanols (10 mg/kg), carotenoids (4.0–38 mg/kg), thiamin15–23 mg/kg), and riboflavin (6.0–10 g/kg) [15]. Another reportedtudy demonstrated the PS constituent of different wheat partsnd it was determined that WGO contains a considerably highermount of PS than the other commercial oils [16,17]. WGO has sev-ral nutritional and health benefits such as reducing plasma andowering cholesterol levels, improving physical endurance/fitness,nd possibly helping to delay effects of aging [18]. Besides this,G has been used to fabricate WG-agglutinin tri-functional

anospheres within magnetic nanoparticles and quantum dots inhe way of cell recognition [19].

To enhance the phyto-constituent of extracted oils from theeeds of herbs, different techniques were established such as super-ritical CO2 extraction [16]. The bio-active compound and quality ofhe obtained oil may have improved, however, the biological valuef the mentioned seed oils are able to enhance thanks to traditionalnfusion with crude plants or plant parts (flower, leaf, root).

Calendula officinalis L. which is from Asteraceae family, termeds marigold, has been used in folk medicine for the treatment ofkin wounds, cuts, burns and other dermatological diseases [20].alendula has a significant historical background owing to its ther-peutic activity such as anti-inflammatory, antioxidant, and woundealing, antibacterial, antifungal and antiviral effects [21]. Further-ore, several reported studies show the importance of biological

ompounds of Calendula flower based on extracts [22,23].The incorporation of lipophilic bioactive components into emul-

ion based delivery systems is an important topic due to theirxtensive value as pharmaceutical, agricultural, food, beverage andosmetic products. Producing the oil ingredients into emulsionffers functional properties including enhanced chemical stability,etter bio-accessibility, etc. and affects its end use characteristics inedicine or cosmetics. Thus, the design of emulsions consisting of

erbal oils with final formulation efficacy has importance in termsf both academic and industrial investigations [24,25].

There are many food supplement and cosmetic products pre-ared by traditional infusion technique. However, to best ournowledge, the chemical and biological properties of the infusedils have not been investigated for Calendula flower infused BSO orGO. The aim of this present study is to describe an appropriate

reparation method to obtain an effective natural based formula-ion. For this purpose, we investigated and evaluated the change inrepared infused oils in the way of phytochemicals and particularlyio-active compounds. In this regard, infused oils were comparedo pristine oils as well as the untreated control groups. During thistudy, spectrophotometric radical scavenging activity, chromato-raphic analysis and cytotoxicity, cell-based antioxidant capacity,ound healing and radioprotective activity (for investigating the

iological impact upon live cells) were tested with monkey kidneybroblast like cells (Vero) and HaCaT keratinocytes. In addition tonhancement of their biological efficiencies using a new approach

epending on infusion technique, new methodologies were opti-ized to evaluate biological effects of traditional preparations such

s herbal medicines, dietary supplements and herbal cosmetics.fter detailed bioactivity investigations of herbal oils which were

: Biointerfaces 133 (2015) 73–80

prepared using infusion technique, nanoemulsions containing Cal-endula flower infused BSO were produced to obtain potentiallysuitable formulations, which were able to serve as wound healingand antioxidant preparates. Hence, nanoemulsion based formula-tions called ‘Phyto-Nanoemulsion’ were produced, characterizedand identified. Finally, cytotoxicity, wound healing and antioxidantactivity were evaluated in vitro.

2. Materials and methods

2.1. Materials

TABIA (Turkey) kindly provided the N. sativa oil (BSO) andwheat germ oil (WGO) which were obtained by the supercriti-cal CO2 extraction method. 1,1-Diphenyl-2-picrylhydrazyl (DPPH)was purchased from Aldrich. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), phosphate buffered saline(PBS, pH 7.4), sodium dodecyl sulphate (SDS) were purchased fromSigma. Quercetin, chlorogenic acid, thymoquinone (TQ), methanol(HPLC grade) were obtained from Sigma–Aldrich. The HPLC gradeacetonitrile was obtained from Lab-Scan, potassium dihydrogenphosphate and phosphoric acid were purchased from J.T. Baker.Water was purified with Direct-Q® 3UV Millipore device.

2.2. Plant material

The flowers of Calendula officinalis L. were collected from organicagriculture fields of Ege University, Faculty of Agriculture in April2014. The cultivation conditions of organically grown C. officinaliswere determined owing to the collaboration with the Departmentof Field Crops of the Agricultural Faculty, Ege University.

2.3. Preparation of herbal infusions

The herbal infusions were prepared by using the same proce-dure for both BSO and WGO. Dried Calendula flowers were infusedinto the oils in proportion to 1:10 (w:w) for 21 days in the dark.Afterwards, Calendula flower infused oils were obtained by filtrat-ing through filter paper and were stored in the dark at +4 ◦C forfurther experiments.

2.4. Production of Phyto-Nanoemulsions

Herbal oils, which were prepared using new and effectiveextraction method, were used to develop emulsion based for-mulations. Oil in water (O/W) nanoemulsions (F1 and F2) wereproduced with different percentages of oil phase and aqueousphase. Nanoemulsions containing Calendula infused BSO as the dis-persed phase and distilled water as the continuous phase wereprepared with using emulsifying agents including Lecithin andTween 80 dissolved in oil phase and aqueous phase, respectively. F1contains 20 wt.% Calendula infused BSO (with 3.0 wt.% Lecithin) and80 wt.% aqueous phase (with 3.0 wt.% Tween 80), besides F2 con-tains 40 wt.% Calendula infused BSO (with 3.0 wt.% Lecithin) and60 wt.% aqueous phase (with 3.0 wt.% Tween 80). Both formula-tions were homogenized with using an ultrasonic processor (Vibracell, VCX500, Sonics & Materials, Inc., Newtown, USA) for 10 minwith amplitude of 40%. In order to prevent any thermal damage, allformulations were produced in an ice bath during ultrasonicationprocess. The obtained Phyto-Nanoemulsions were kept at +4 ◦C indark for further bioactivity investigations.

2.5. Characterization

Particle size and zeta potential of the Phyto-Nanoemulsionswere assessed by Dynamic Light Scattering (DLS, Malvern Zetasizer

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ano ZS model) measurements. The reported values were obtainedith triplicate measurements.

.6. Free radical scavenging by DPPH

The stable free radical 1,1-Diphenyl-2-picrylhydrazyl (DPPH)as used to estimate antioxidant capacity of the Calendula flower

nfused BSO and WGO, and Phyto-Nanoemulsions (F1 and F2)22,26]. The radical scavenging activities (%) of the BSO, WGO andheir Calendula infusions, and Phyto-Nanoemulsions were deter-

ined as a result of the decrease in the absorbance of DPPH.

.7. Chromatographic analysis

A Waters Acquity UPLC® H-Class system with a quaternary sol-ent manager, an automatic sample manager (FTN) device and TUVetector were used for liquid chromatographic analysis.

Chromatographic column sample solutions were determinedy Waters Acquity BEH C18 column (50 mm × 2.1 mm, 1.7 �m).he mobile phase consisted of (A) pH 3.0 phosphate buffer (B)cetonitrile (75:25, v:v). The flow rate was 0.3 mL/min, and theutomatic sample feeding room was 30 ◦C and 20 ◦C, respec-ively. Detection and quantification were carried out at 330 nmor chlorogenic acid, at 200 nm for quercetin. Detection of toco-herols was performed using normal-phase high performance

iquid chromatography. Tocopherols were determined accordingo the AOCS Official Method Ce 8-89 “determination of tocopherolsnd tocotrienols in vegetable oils and fats by HPLC”. Separationnd quantification were carried out on an Agilent HPLC systemonsisting of a gradient pump, a FLD detector. The column wasIChrosorb SI 60 (4.6 mm × 150 mm, 5.0 �m). The injection vol-me was 20 �L and the oven temperature was 25 ◦C. The excitationnd emission wavelengths were 290 nm and 330 nm, respectively.obile phase of hexane:2-propanol (99.5:0.5) was used for �-

ocopherol, �-tocopherol, �-tocopherol and �-tocopherol at flowate of 0.8 mL/min.

The composition of fatty acid methyl esters (FAMEs) was deter-ined according the EU Regulation 2568/91 (European Union

ommission, 1991); COI/T.20/Doc. 24-2001 “preparation of theAMEs from olive oil and olive pomace oil” and COI/T.20/Doc.7/Rev. 1-2001 “determination of trans unsaturated fatty acidsy capillary column gas chromatography”. The chromatographiceparation was performed by an Agilent GC-FID system, equippedith HP-88 column (60 m × 0.25 mm, 0.20 �m). The oven initial

emperature was held at 140 ◦C for 1.0 min and then increased40 ◦C at a rate of 4 ◦C min−1, where the temperature was heldor 5 min. Methyl esters are formed by trans-esterification withold methanolic solution of potassium hydroxide. The results werexpressed as peak area (relative) percent. 20 �L of the sample wasnjected into the Agilent HPLC-DAD system at a wavelength of54 nm; column temperature was 25 ◦C and C18 column (Agilentclipse column XDB-C18, 4.6 mm × 150 mm, 5.0 �m particle size).he mobile phase was utilized methanol:water:methyl tert-butyl46:42:12) at 1.0 mL/min flow rate.

.8. Cell culture

For in vitro cell culture, Vero cell line (Monkey kidney fibro-last like) and HaCaT cell line (Human dermal keratinocyte), as cellodel systems, were purchased from ATCC and CLS, respectively.ero cells were seeded and grown in MEM (Minimum Essentialedium Eagle) medium containing 10% fetal calf serum and 1.0%

enicillin/streptomycin (100 Unit/mL). HaCaT keratinocyte cellsere cultivated in Dulbecco’s Modified Eagle Medium (DMEM)

upplemented with l-glutamine, 10% fetal calf serum and peni-illin/streptomycin mixture (10,000 Unit/mL). Both cell types were

: Biointerfaces 133 (2015) 73–80 75

cultured 75 cm2 tissue culture flasks under a humidified atmo-sphere supplemented with 5.0% CO2 at 37 ◦C. The cultures werepassaged two times weekly.

2.8.1. Cell viabilityA cell proliferation assay kit (MTT reagent) was used to mea-

sure the increases and decreases in cell viability of cells treated withsamples. To perform the MTT assay, both Vero cells and HaCaT cellswere seeded into 96 well plates and incubated until reaching con-fluence with normal morphology. The samples of Calendula infusedherbal oil with concentrations of 1.0, 5.0, 10, 25, 50, 100, 250 �g/mLwere added to wells and then the cell culture plates were put intoCO2 incubator for incubation at 37 ◦C for 24 h. After incubatingboth Vero cells and HaCaT cells with BSO, WGO and their Calen-dula infusions at various concentrations, the cells were washed toremove culture medium. MTT assay on both these cell lines was car-ried out according to standard procedure [27]. The dose-dependentcytotoxicity of herbal preparations was given as a graph of cellviabilities relative to the control (untreated) cells. The nanoemul-sions prepared with dispersion of Calendula infused BSO in aqueousphase were examined according to same procedure. The con-centration dependent cell viability (%) values (at 0.05–5.0 mg/mLconcentrations) were obtained for the prepared formulations.

2.8.2. Cell scratch wound healing assay24-Well tissue culture plates were seeded with cells and were

maintained at 37 ◦C and 5.0% CO2 for 24 h to permit the forma-tion of confluent cell monolayer. Scratch wound assay was used toassess the migration and proliferation capabilities of model cellswhich were treated with herbal samples. For this purpose, a lin-ear scratch was created in the confluent cell monolayer with asterile pipette tip to simulate the wounded area [22,28]. Then,cells were washed with PBS to remove cellular debris and freshculture medium was added. BSO, WGO, Calendula flower infusedBSO (100 �g/mL), Calendula flower infused WGO (100 �g/mL) andPhyto-Nanoemulsions (1.0 mg/mL) were applied to the cells whichwere subjected to a mechanical wounded area and cells were incu-bated at ideal cultivation conditions (37 ◦C and 5.0% CO2). Thesample free culture medium was used as control group. To esti-mate the wound healing activity (%) of samples, scratched areawas photographed via an inverted microscope (Olympus CKX41)equipped with a CCD camera (Olympus XC30) at 4 h and 8 h. Theeffect of samples on the monolayer recovering owing to cell migra-tion into the scratched area was determined according to the widthof gap. The width of gap which was generated in the cell monolayerwas monitored and calculated using a computer program (ImageJ). Closure in the scratched cell monolayer was analyzed after 4 hand 8 h and relative wound healing values (%) were calculated. Allscratch assays were practiced in triplicate.

2.8.3. Cell based antioxidant activityVero and HaCaT cells were used to investigate the ability of

herbal samples to protect cells from the oxidative stress inducednegative effects. Cell based antioxidant activity assay was per-formed as described in our recent report [23]. Briefly, cells werecultured in 96-well plates to approximately 80% confluence. Cellswere pre-treated with herbal oils and their infusions for 2 h andthen washed with PBS to remove samples from the wells. Freshlyprepared H2O2 (1.25 mM, diluted with fresh culture medium) wasadded to submit cells to an oxidative stress and incubated for 24 h(5.0% CO2, humidified air and at 37 ◦C). After the incubation time,

the H2O2 treated cells were washed two times. To describe protec-tive effects of the herbal samples against the H2O2 induced celldeath, cell viability was assessed by the MTT test as previouslydetailed.

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in cell viability can be observed at a concentration range between1.0 and 100 �g/mL (Fig. 1A and B). Selection of appropriate dose isa critical factor for investigation of biological activities. Therefore,a 100 �g/mL concentration was selected for further experiments

6 Z.P. Gumus et al. / Colloids and Sur

.8.4. Radioprotective activityThe protective effect of the prepared herbal samples against

onized radiation was investigated following the cell viability ofrradiated HaCaT cells. To design experiment, cell suspension wasransferred from 75 cm2 flask to 96-well plate at a density of000 cells/well and these plates were incubated under ideal cellulture conditions (37 ◦C, 5.0% CO2). At the end of incubation period,fter the cells were washed, the cells were pre-treated with herbalamples. Afterwards, HaCaT cells were exposed to irradiation atarious doses 5.0 and 10 Gy with photon beams generated by

MV linear accelerator system (LINAC, Siemens Primus, Germany).rradiated cells were incubated for 72 h and then standard MTTrocedure was performed to evaluate cell viability [29].

.8.5. Cell imaging with FITC containing Phyto-Nanoemulsionsia fluorescence microscopy

Phyto-Nanoemulsions were examined in respect of cell pen-tration capabilities. For this purpose, fluorescein isothiocyanateFITC) was incorporated into emulsion droplets to gain fluores-ent carriers. FITC labeled formulations (F1 and F2) were preparedccording to the same procedure as reported in method ofhyto-Nanoemulsion production. Fluorescent labeled formulationsontaining herbal oil were applied to the model cells which weredhered on the surfaces of chamber slides. Both Vero and HaCaTells visualized with fluorescent formulations (F1 and F2 contain-ng FITC) were monitored via fluorescence microscope (OlympusX53F) equipped with a CCD camera (Olympus DP72).

. Results and discussion

.1. Production of Phyto-Nanoemulsions

.1.1. Particle size analysisThe designed nanoemulsions were characterized in respect of

roplet size and surface zeta potential. According to DLS measure-ents, the droplet size values were measured as 54 ± 25.46 nm

PDI: 0.370) and 78.29 ± 43.04 nm (PDI: 0.342) for formulationsalled as F1 and F2, respectively. In case of zeta potential mea-urements, −34 ± 4.76 mV and −39.9 ± 5.82 mV were determinedor F1 and F2, respectively. Particle size results demonstratedhat the Phyto-Nanoemulsions provided nanometer-sized dropletsith a narrow particle size distribution. Droplet sizes are affected

y homogenization technique used and the structure of system.esides, particle size is a factor which distinguishes conventionalmulsions and nanoemulsions from each other. The emulsionystems with the average droplet radius <100 nm are named asanoemulsions, however >100 nm ones are known as conven-ional emulsions. The relatively small droplet size of nanoemulsionsttends some superiority in terms of physicochemical and biologi-al features in comparison to conventional emulsion systems. Somef these advantages including the better stability and bioavailabilityake nanoemulsions suitable tools as delivery systems [30].

.2. Antioxidant capacity using DPPH assay

Antioxidant potential of the prepared herbal infusions and theirormulated samples were investigated using both chemical modelnd in vitro cell culture model. In case of antioxidant activity usinghemical model, the DPPH assay was performed [31]. Accordingo measurements of DPPH method, the radical scavenging abil-ties of herbal samples including BSO, WGO, Calendula infused

SO, Calendula infused WGO, F1 and F2 were given in Table S1.s shown in concentration dependent DPPH radical scavengingctivity (%) results, WGO and its Calendula infusion exhibited a sig-ificant quenching effect on DPPH free radical with the same values.

: Biointerfaces 133 (2015) 73–80

Besides, Calendula infusion of BSO showed higher scavenging activ-ity than plain BSO. A reason for the difference between these resultsmay be related with the adding of Calendula flowers to oil. Theseresults suggested that the infusion of Calendula flowers to the BSOenhanced the antioxidant activity. Besides, the formulation namedas F2 exhibited better free radical scavenging capacity in compar-ison with F1. This was an expected result because of the highercomposition of the bioactive oil phase in F2. Thus, the results con-firmed that antioxidant capacity values were increased dependingon the amount of herbal oil ingredients [31].

3.3. Quantification of some compounds in herbal infusions andtheir formulated samples

Some components of the BSO, WGO and their Calendulainfusions and Phyto-Nanoemulsions such as thymoquinone (TQ),tocopherol, phenolics, saturated fatty acids, mono unsaturatedfatty acids and poly unsaturated fatty acids were quantified usingchromatography systems including UPLC, HPLC and GC (Table S2).According to the data, chemical profile of BSO and WGO did not sig-nificantly change after infusion with Calendula flowers. However,concentration of some compounds present in Calendula infused oilswas higher than plain oils. For example, the amount of quercetinand chlorogenic acid in BSO was increased after herbal infusion.Since some compounds in Calendula flowers could pass to oil, itwas an expected result due to maceration of Calendula with oils for21 days.

3.4. Cell culture studies of herbal infusions

3.4.1. MTT assayTo evaluate cytotoxicity and dose dependent effect of BSO, WGO

and their Calendula infusions, MTT assay was performed usingHaCaT and Vero cell lines as a model for in vitro. After 24 h incu-bation of model cells in the presence of samples, slight decrease

Fig. 1. The dose dependent cytotoxic effect of herbal samples on Vero cells (A) andHaCaT cells (B) after incubation for 24 h. The data are given as ±standard deviation(n = 4).

Z.P. Gumus et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 73–80 77

Fig. 2. Wound healing (%) effect of the plain oils (BSO and WGO) and their Calen-dula infusions (BSO + Calendula and WGO + Calendula) on Vero cells (A) and HaCaTctc

ia

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Fig. 3. Influence of H2O2 (1.25 mM) on the cell viability of Vero (A) and HaCaT kera-

ells (B) after treatment for 4 h and 8 h. The data are given as ±standard devia-ion (n = 4), *means comparison with control group, **means comparison with bothontrol group and plain oil (P < 0.01).

ncluding antioxidant activity, wound healing and radioprotectivectivity.

.4.2. Wound healingRestoration of the damaged tissue is highly complex process

hich begins with inflammation and follows new tissue formation.egeneration of the injured tissue consists of cell–cell interactions,ell–matrix interactions and cellular signal networks. During theepairing process, keratinocytes and fibroblast cells play criticalole owing to their migration and proliferation at the wound area29]. Hence, HaCaT dermal keratinocyte cells and Vero fibroblastike cells were chosen as model cells to investigate the potentialffects of the prepared herbal oils for wound healing. In vitro scratchssay is an easy and economical method which is based on theapturing images during cell migration to close created artificialap in cell monolayer [32]. To estimate wound healing potentialf the prepared infused oils, the scratched cells were treated withhe samples (100 �g/mL) and monitored via light microscope. Tovaluate activity of infused oils, their wound healing effect wasompared with the effect of the plain oils. Incubation with all of

he oil preparates for 8 h significantly increased the cell migrationf both cell types to close the denuded area compared to the con-rol (Fig. 2). In addition, the values of wound healing induced bynfused oils were markedly higher than those induced by the plain

tinocyte (B) cells pre-treated with samples. **Sign means comparison with bothcontrol (H2O2) group and plain oil (P < 0.01).

oils. These data suggested that incorporation of Calendula flowersto the oils strengthened their activities.

3.4.3. Cell-based antioxidant activityAntioxidant potential of the Calendula macerated BSO and WGO

was investigated using both, the cell free chemical assay (DPPHmethod) and cell-based antioxidant activity test. Protective effectof the oils against the oxidative stress was tested by observingthe cell viability of H2O2-damaged Vero and HaCaT cells, whichwere pre-treated with the samples. After treatment with H2O2(1.25 mM) for 24 h, the cell viability of Vero cells and HaCaT kera-tinocytes decreased to 30.56% and 43.58%, respectively. Fig. 3exhibits the antioxidant potential of plain and infused oils of BSand WG. According to the results, Calendula infused BSO increasedcell viability of Vero (53.97%) and HaCaT cells (67.21%), signifi-cantly (P < 0.01). Unfortunately, plain oils and infused WGO didnot show a considerable impact on the examined cells. Under thelight of DPPH radical scavenging activity results, it can be claimedthat Calendula infusion of BSO has been more effective upon plain

BSO. Moreover, chromatographic analysis supports those valuesbecause of the increased quercetin amount in Calendula infusedBSO.

78 Z.P. Gumus et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 73–80

Fig. 4. Cell viability of herbal oils treated HaCaT cells after irradiation. The data areg*

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iven as ±standard deviation (n = 6), *sign means comparison with control group,*sign means comparison with BSO and control group.

.4.4. Protective effect against ionized radiationRadiation therapy which is one of the cancer treatment methods

irectly damages cell DNA and indirectly affects cells via reac-ive oxygen species (ROS) generation. Besides, ROS produced inadiotherapy harm to normal tissues. Researchers sustained thatntioxidants can protect normal cells from free radicals generatedy ionized radiation [33]. To this respect, we examined the pro-ective effect of antioxidant herbal oils against ionized radiationxposed cells. After HaCaT cells were subjected to ionized radia-ion at doses 5.0 Gy and 10 Gy, cell viabilities decreased to 58.65%nd 44.14%, respectively (Fig. 4). Obtained results exhibited thatGO, Calendula infused BSO and Calendula infused WGO signif-

cantly protected the HaCaT cells from radiation stimulated cellytotoxicity. Even though BSO did not show protective effect, itsalendula infusion was activity as protector against ionized radia-ion with statistically significant difference (P < 0.05). Antioxidant

aterials are known with their potential effect as protector againstadiation induced damage owing to their free radical quench-ng effect. Thus, the radioprotective effect is generally correlated

ith radical scavenging activity. Our results obtained from DPPHethod supported that oils with higher radical scavenging activ-

ties (WGO, Calendula infused BSO and Calendula infused WGO)howed higher protective activity against radiation. Furthermore,btained results mention that Calendula infused BSO, WGO and itsalendula infusion could serve a function in minimizing effect ofadiation damage of the cells. There are several studies supportedhe radioprotective roles of WGO and Calendula against irradiationnduced symptoms [34,35].

The doses of ionized radiation applied to the cells wereonfirmed using Fricke dosimeter according to the followingquation:

nergy absorbed

= O.D.i − O.D.b

εdG(Fe3+)× 6.023 × 1020

�× 1.602 × 10−12

= 0.965 × 109(O.D.i − O.D.b)

εd�G(Fe3+)rads

Fig. 5. The effect of Phyto-Nanoemulsions on cell viabilities of Vero cells (A) andHaCaT cells (B).

O.D.i and O.D.b are the optical density of the irradiated and non-irradiated dosimeter solution, respectively.

As a result of ferrous sulphate dosimeter, the doses were cal-culated as 5.6 and 11.5 Gy for 5.0 and 10 Gy of irradiated dosesrespectively by linear accelerator system.

3.5. Cell culture studies of Phyto-Nanoemulsions

3.5.1. Cell viability assayIn vitro cytotoxicity studies on Vero and HaCaT were per-

formed to choose proper sample concentration which doesnot affect cell viability negatively. For this purpose, cells weretreated with infused oil formulations at different concentrations(0.05–5.0 mg/mL) to perform MTT assay. Cell viabilities of bothVero and HaCaT cells did not significantly change at concentra-tions between 0.05 and 1.0 mg/mL, however, the cell viability(%) decreased proportionately with increase in doses (2.5 and5.0 mg/mL) of Phyto-Nanoemulsions (Fig. 5). According to theresults, further in vitro investigations including scratch assay,antioxidant activity and cell imaging were performed at the con-centration of 1.0 mg/mL.

3.5.2. Wound healing

To observe wound healing potential of the formulations, their

effects on the in vitro wound model were evaluated. Following thecreation of artificial gap in cell monolayer, cells were treated withsamples (the concentration of 1.0 mg/mL for F1 and F2) and their

Z.P. Gumus et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 73–80 79

Fig. 6. Wound healing (%) effect of the formulations (F1 and F2) on Vero cells (A) and HaCa(n = 4), ***means statistically significances between the compared groups (P < 0.001).

Fig. 7. Influence of H2O2 (1.25 mM) on the cell viability of Vero (A) and HaCaT kerati-nbP

taa8

ocyte (B) cells pre-treated with samples. ***, ** and *mean statistically significancesetween the compared groups (***means P < 0.001, **means P < 0.01 and *means

< 0.05).

endencies to close model injury were assessed via monitoring cellsfter 4 h and 8 h. Obtained cell images showed that the denudedrea in both Vero and HaCaT cells was significantly reduced after

h owing to the treatment of F1 and F2. In addition, cells treated

Fig. 8. Fluorescence microscopy images of Vero cells (A) and Ha

T cells (B) after treatment for 4 h and 8 h. The data are given as ±standard deviation

with F2 exhibited considerably faster wound healing (%) in compareto F1 (Fig. 6). This result can be attributed to the fact that two timesmore amount of oil phase in F2 formulation can increase the activityin parallel with the proportion of infused oil.

3.5.3. Cell-based antioxidant activityThe effect of formulations on cell viability of model cells exposed

to H2O2 was examined to observe their effectiveness in protectingcells from oxidative stress. Both of the cells were pretreated withnon-toxic concentrations of F1 and F2 to examine the cytopro-tective effect of herbal oil based formulations on oxidative stressinduced cell damage. As shown in Fig. 7, H2O2 (1.25 mM), whichwas used to design cell based model of oxidative stress, killed 55% ofthe Vero and HaCaT cells after 24 h. However, cell viabilities of Verocells increased to 57.27% and 62.5% owing to pretreatment with F1and F2, respectively. Also, in case of HaCaT cells, 60.02% and 69.74%cell viabilities were determined for F1 and F2, respectively. As weexpected, the protective activities of F1 and F2 exhibited a corre-

lation with the percentage of oil phase in formulations. Moreover,the difference in protective activities of F1 and F2 against oxidativedamage can be attributed to proportion of oil phase which containsbio-active compounds.

CaT cells (B) treated with F1 and F2 containing FITC dye.

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.5.4. Cell imagingFluorescence monitoring was carried out to evaluate whether

erbal nanoemulsions entered into cells. Fig. 8 shows images ofero and HaCaT cells treated with FITC encapsulated F1 and F2. Blueaintings show cell nuclei stained with DAPI and obtained imagesere formed by overlapping of simultaneous photographs whichere taken after the samples and DAPI treatment. According to cell

mages, it can be said that the Phyto-Nanoemulsions can penetratefficiently into the cells. Moreover, high fluorescent intensity of cellmages indicated that the fluorescent dye encapsulated emulsionsan be used for imaging purposes.

. Conclusions

Up to now, preparations of herbal infusions were mostly aque-us form and they were commonly prepared via infusion into water.lternative to this preparation technique, plant material infusion

nto the herbal oil has been traditionally used according to knowl-dge of cultures and societies, even though limited number oftudies was available concerning investigations of their biologicalctivities in vitro and in vivo. Within the scope of this work, theethodologies described and optimized can integrate the inves-

igations related to biological activities of traditionally consumederbal infusions.

The present study aimed to prepare herbal infusions and effec-ive formulations, and to investigate their chemical profile andiological activities using in vitro cell culture models. We hypothe-ized that biological activities of herbal oils could enhance usingnfusion method, because some constituents of plants can bextracted to the oils during maceration. For this purpose, we pre-ared Calendula flower infused BSO and WGO for the first time.fter maceration of Calendula, biological activities of infused oilsere investigated and compared with pristine oils to evaluate the

ffect of Calendula infusion. The herbal oil, which was prepared withxtraction of Calendula flower biocompounds in BSO, was incorpo-ated into nanoemulsion in order to obtain suitable commercialroduct. Products designed for pharmaceutical, food and cosmetic

ndustries require appropriate formulation. Hence, the applicabil-ty of the prepared herbal oils in formulation design was evaluatedy using emulsion systems.

We conclude that the herbal oils prepared with Calendulanfusion exhibited stronger activities compared to pristine oils. Pre-ared herbal oils demonstrated valuable biological effects includingound healing and protection against radiation. In addition, Cal-

ndula infused BSO may be used as a protective agent againstxidative stress. Formulations developed in this work promiseds potentially useful preparations, which were able to stimulateound healing and to protect cells from oxidative stress accord-

ng to in vitro cell culture models. Therefore, obtained informationrom the present work will pave the way for their future use forealthcare.

cknowledgements

This study was funded by Industrial Thesis Support ProgramSAN-TEZ) of Republic of Turkey, Ministry of Science, Industry andechnology (Project Grant No: 0395.STZ.2013-2). And also this

ork was partially supported by Ege University Scientific Research

rojects (2013/FEN/023 and 2013/FEN/022). Ege University, AliyeSTER Foundation was also acknowledged for the research grants.uthors were grateful to WATERS Corp. for supplying UPLC system.

[[[[

: Biointerfaces 133 (2015) 73–80

Prof. Dr. H. Armagan ARICAN was acknowledged for the fruitful sup-port in the radioprotective activity experiments. EGE LS (Turkey) isacknowledged for their financial support during this work. Also,we thanked to Prof. Emine Bayram and Prof. Akin Olgun from EgeUniversity, Faculty of Agriculture for support in determination ofcultivation conditions of organically grown Calendula officinalis.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.05.044

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