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Ozone: Science & EngineeringThe Journal of the International Ozone Association

ISSN: 0191-9512 (Print) 1547-6545 (Online) Journal homepage: http://www.tandfonline.com/loi/bose20

Ozonated Olive Oil with a High Peroxide Value forTopical Applications: In-Vitro Cytotoxicity Analysiswith L929 Cells

Yasemin Günaydın, Handan Sevim, Deniz Tanyolaç & Özer A. Gürpınar

To cite this article: Yasemin Günaydın, Handan Sevim, Deniz Tanyolaç & Özer A. Gürpınar(2017): Ozonated Olive Oil with a High Peroxide Value for Topical Applications: In-Vitro CytotoxicityAnalysis with L929 Cells, Ozone: Science & Engineering, DOI: 10.1080/01919512.2017.1341832

To link to this article: http://dx.doi.org/10.1080/01919512.2017.1341832

Accepted author version posted online: 13Jun 2017.Published online: 13 Jun 2017.

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Ozonated Olive Oil with a High Peroxide Value for Topical Applications: In-VitroCytotoxicity Analysis with L929 CellsYasemin Günaydına, Handan Sevimb, Deniz Tanyolaça, and Özer A. Gürpınarb

aDepartment of Chemical Engineering, Hacettepe University, Ankara, 06800 Turkey; bDepartment of Biology, Hacettepe University, Ankara,06800 Turkey

ABSTRACTPrevious studies have shown that ozonated vegetable oils have been used topically for healing ofcutenous wounds. The aim of this study is to evaluate the dose dependent use of ozonated oliveoil with high peroxide value (OZ) on the viability of cells for preventing side effects in topicalapplications. To the best of our knowledge, there are no reports investigaing the effect ofperoxide value of ozonated olive oil associated with its cytotoxic activity on mouse non-neoplastcfibroblast cell lines (L929). Therefore, the present study was carried out by using OZ alone and/orin combination with glycerol and olive oil. In our study OZ was prepared by using pure olive oil.Both olive oil and glycerol are non-toxic and can be mixed with OZ uniformly. The cytotoxicactivity of samples against L929 fibroblasts was assessed using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. The peroxide value of synthe-sized OZ was found to be in the range of 2700–2900 mEq O2/kg oil. The OZ/olive oil group did notshow any cell death at all concentrations tested (p > 0.05) however OZ/glycerol group showedstatistically significant reductions in viability at higher concentrations (p = 0.004–0.006) comparedto the control group. Conclusively, using OZ/olive oil with a peroxide value of 2700–2900 mEq O2/kg oil for short-term incubation was non-cytotoxic to the L929 fibroblast cell line.

ARTICLE HISTORYReceived 17 January 2017Accepted 7 June 2017

KEYWORDSOzone; Cytotoxicity; L929cells; MTT Assay; OzonatedOlive Oil; Peroxide Value

Introduction

Ozonated vegetable oils have been widely used due totheir valuable therapeutic, antibacterial, and fungicidaleffects in the food, cosmetics, and pharmaceuticalindustries (Cirlini et al. 2012; Díaz et al. 2006;Georgiev et al. 2013; Rodríguez et al. 2007). In reactionwith oils, ozone gas is stabilized between the carbon-carbon bonds present in unsaturated fatty acids accord-ing to the Criegee mechanism (Criegee 1975; Geweely2006). Therefore, ozonated oils can remain stable for2 years at 4 °C (Valacchi, Fortino, and Bocci 2005). Themain chemical products from oil ozonolysis are triox-olanes and peroxides, which are the species responsiblefor the many biological and therapeutic effects of ozo-nated oils (Díaz et al. 2006; Rodrigues de AlmeidaKogawa et al. 2015).

Ozonated vegetable oils synthesized from sunflower,olive, sesame, soybean, linseed, canola, coconut, grapeseed, jojoba, obroma, mineral oil, and red pepper seedhave been reported in the literature to date (Díaz et al.2005, 2011; Guerra-Blanco et al. 2015; Iorio et al. 2015;Moureu et al. 2015; Özyildiz et al. 2013; Rodrigues deAlmeida Kogawa et al. 2015; Sakazaki et al. 2007; Sega

et al. 2010; Skalska et al. 2009; Zabicky and Mhasalkar1986). Ozonated oils have been used in many medicalapplications, including in the treatment of infections,ulcers, burns, fissures, fungal diseases, gingivitis(Campanati et al. 2013; Kim et al. 2009; Sakazaki et al.2007; Valacchi et al. 2013).

Ozonated olive oil is formed by bubbling ozone gasthrough olive oil with a steady 65–85% oleic acid por-tion (Díaz et al. 2006; Sakazaki et al. 2007). The directozonation of ozonated olive oil with esters leads to theformation of the trioxolane, which represents the activeform of ozone in these substrates. When the trioxolanering makes contact with the warm exudates, it slowlydecomposes, thereby generating local oxygen and H2O2

as reactive oxygen species (ROS), and lipid oxidationproducts. These compounds represent the “ozone mes-sengers” that are the main contributors to the observedbiological and therapeutic effects. ROS are potentiallycytotoxic, and they have a very short half-life (Bocci2005).

Ozonated olive oil is highly effective against bothgram-positive and gram-negative bacteria, as well asfungi and viruses. The trioxolane, lipoperoxides, and

CONTACT Özer A. Gürpınar gurpinar@hacettepe.edu.tr Faculty of Science, Department of Biology, Hacettepe University, Beytepe, Ankara, Turkey 06800.Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/BOSE.

OZONE: SCIENCE & ENGINEERINGhttps://doi.org/10.1080/01919512.2017.1341832

© 2017 International Ozone Association

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aldehydes in ozonated oil are cytotoxic to microorgan-isms, and therefore the action of ozonated olive oil onwound healing is associated with its antimicrobial effect(Rodrigues de Almeida Kogawa et al. 2015). In thismechanism, ozone binds to glycoproteins, glycolipids,and other amino acids to inhibit the enzymatic controlsystem of the bacterial cell. This leads to an increase inmembrane permeability, allowing for ozone moleculesto penetrate the cell causing microorganism death(Rajabi et al. 2015).

Ozonated olive oil supports the generation of growthfactors, activates local anti-oxidant mechanisms, andpromotes tissue repair (Blanco, Poznyak, and Chairez2015; Valacchi, Fortino, and Bocci 2005; Valacchi et al.2013). In this action, hydrogen peroxide can induce thesynthesis of growth factors and accelerate the cell cyclevia the activation of redox transcription factors such asnuclear factor-kappaβ (NF-κβ). NF-κβ has an impor-tant role in the wound healing process (Campanatiet al. 2013; Gultekin et al. 2013).

Measurement of the peroxide value (PV) is an essen-tial to determine the quality parameters and establishthe therapeutic dose of ozonized vegetable oils. The PVis used for the quantification of all peroxidic com-pounds formed during the ozonolysis of vegetableoils, and is expressed in milliequivalents or millimolesof active oxygen per kilogram of oil (Montevecchi et al.2013; Travagli et al. 2010). Results obtained in severalstudies indicate that peroxide compounds in the ozo-nated oils have marked action on antimicrobial activity(Díaz et al. 2006; Montevecchi et al. 2013; Rodrigues deAlmeida Kogawa et al. 2015). Previous studies havedemonstrated the potential applications of ozonatedoils synthesized from a variety of vegetable oils withPVs ranging from lower to higher oxidation levels(Hernández et al. 2009; Pai et al. 2014; Patel et al.2012; Skalska et al. 2009; Valacchi et al. 2013)

Valacchi and colleagues investigated the therapeu-tic effects of sesame oils expressing low (949 mEqO2/kg oil), middle (1631 mEq O2/kg oil), and high(3170 mEq O2/kg oil) PVs in acute cutaneouswounds made in hairless female SKH1 mice. Theresults showed that a medium degree of peroxidation(1631 mEq O2/kg oil) resulted in a more rapidwound closure rate in the first 7 days compared tothe rates of treatment with the oils of lower andhigher PVs (Valacchi et al. 2011). Pai studied theeffect of ozonated sesame oil with low (500 mEqO2/kg oil) and high (700 mEq O2/kg oil) degrees ofperoxide on the wound healing process, and foundthat the high dose (700 mEq O2/kg oil) was moreeffective to accelerate the rate of wound healing (Paiet al. 2014).

Serio and colleagues evaluated the cytotoxic effect ofa gel composed of 30% ozonated sunflower seed oilwith 335 meqO2/kg PV on cultures of Vero cells and3T3 fibroblasts. The cytotoxicity data showed that thisformulation has no cytotoxic and irritating effect (Serioet al. 2016).

Extensive researches available on ozonated vegetableoils to demonstrate their antimicrobial, antifungal andantiviral properties. To the best of our knowledge, thereare no studies concerning the investigate the effect ofperoxide value of ozonated olive oil associated with itscytotoxic activity on mouse non-neoplastic fibroblastcell lines (L929). As a result, it is concluded that thefindings of the present study will provide chosen anappreciable dose of OZ for the treatment of chronicwounds in topical aplications

Materials and methods

Characterization of OZ

The OZ used in this study was synthesized in ourlaboratory as previously described.(Günaydın 2016)The synthesized OZ was mixed with olive oil andglycerol to prepare OZ-Oo and OZ-Gly samples,respectively, at 50% (v/v) volumes.

The PV is generally determined by the quantity ofthe peroxides in the sample measured as milliequiva-lents of active oxygen in 1000 g of oil sample(Martinez Tellez, Ledea Lozano, and Díaz Gómez2006) In our study, the PV was determined withslight modifications from the standard method. Inparticular, 1 g of OZ was weighed in a 250-mL con-ical flask and mixed with 20 mL of chloroform/glacialacetic acid (2/3 v/v). After complete dissolution of thesample, 0.5 mL of a saturated solution of potassiumiodide was added to the flask. The mixture was sha-ken well and stored for 24 h at 25 °C in the dark(Martinez Tellez, Ledea Lozano, and Díaz Gómez2006). Subsequently, 1 mL of a 1% (mg/mL) starchindicator solution was added and mixed with 30 mLof water. Finally, titration was carried out with a0.01 M sodium thiosulfate solution until disappear-ance of the deep blue color. The PV was calculated byEquation [1]:

PV ¼ 1000 � c � ðV1�V0Þm

½1�

Where V1 is the volume (mL) of the sodium thiosulfatesolution used for titration, V0 is the volume (mL) of thesodium thiosulfate titration used in the blank test, c isthe molarity of the thiosulfate solution, and m is themass (g) of the sample.

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Proton nuclear magnetic resonance (1H-NMR) spec-tra were obtained using a Bruker (250 Mhz) spectro-meter. The OZ sample was diluted with CDCl3 andtetramethylsilane (TMS) was used as an internal refer-ence. 1H-NMR spectroscopy provides informationabout identification and quantitative determination offunctional groups formed during ozonolysis of olive oil.

Assessment of cell viability

The in vitro cytotoxic activity of the OZ and mixedsamples (OZ-Oo, OZ-Gly, Oo-Oo) on L929 cells wasdetermined using the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) assay(Applichem, A2231). L929 cells were obtained fromstock frozen cell lines and cultured in Dulbecco’s mod-ified Eagle medium/F12 with 10% fetal bovine serumand 0.2% penicillin/streptomycin at 37 °C, 5% CO2 inair. The cells were incubated in 96-well plates (200 μLper well) with a density of 2 × 104 cells/mL. Thesamples (OZ, OZ-Oo, OZ-Gly, and Oo-Oo) were dis-solved in 1% (v/v) dimethyl sulfoxide. After 24 h ofincubation, the cell culture medium was replaced withfresh medium containing different concentrations(0.062%, 0.187%, 0.25%, 0.75% (v/v)) of each sample.Experiments were performed six times and untreatedcells served as controls. The cells were incubated for48 h with the oil samples, and the MTT assay wasperformed and cell morphology was visualized withan inverted microscope (IX70 Olympus, Japan). After48-h of incubation, the medium was removed andmedium containing MTT (10%) was added to eachwell, and incubated for 4 h more. The absorbance wasread at 570 nm using a spectrophotometer (EZ Read400 Microplate reader, Biochrom).

Statistical analysis

SPSS 22 software was used for statistical analysis. All resultsrepresent the mean ± SD. Differences between groupswere evaluated by the Mann–Whitney test. A p value <0.05 was considered to be statistically significant.

Results and discussion

Olive oil exerts several biological benefits, mainly dri-ven by its constituent anti-oxidants (Cerqueira et al.2012). In particular, the phenolics found in olive oil arepowerful anti-oxidants (Visioli, Poli, and Gall 2002).Ozone reacts with the carbon-carbon double bonds ofthe unsaturated fatty acids in olive oil to form impor-tant products such as trioxolanes and peroxides, whichare ultimately responsible for the antimicrobial activity

and help to stimulate tissue repair (Zanardi et al. 2008).At low levels, ROS (such as H2O2) generated fromozonolysis, which have a short lifetime are non-radicaloxidants capable of acting as ozone messengers respon-sible for promoting wound healing (Bocci 2005).

The PV represents the quantity of peroxides in an oilsample. The PVs are obtained after 24-h of reactions.Olive oil alone (Oo-Oo) had a PV of 20 mEq O2/kg oil,whereas the PV of synthesized OZ was much higherwith a range of 2700–2900 mEq O2/kg oil. When OZwas mixed with glycerol and olive oil, peroxide value ofOZ-Oo and OZ-Gly samples did not change. They hadthe same peroxide values as same as OZ. These resultsshow that during the synthesis of OZ, almost all of thecarbon double bonds in olive oil reacted with the ozonemolecules, generating peroxidic species to ultimatelyincrease the PV (Sega et al. 2010). After 24-h of reac-tion, hydroperoxides, hydrogen peroxides, polymericperoxides, and other organic peroxides in OZ hadreacted with iodide to form iodine. According toMehlenbacher, the reaction time is a very importantfactor in oils with high peroxide contents, and shouldbe longer than 2 min (Martinez Tellez, Ledea Lozano,and Díaz Gómez 2006).

Changes in the structure after ozonation were mon-itored by signals observed in 1H-NMR spectroscopy.The 1H-NMR spectra of OZ are shown in Figure 1.Table 1 summarizes the assignments of all pertinentpeaks seen in the studied 1H-NMR spectra.

The main reaction is the formation of 1,2,4-trioxo-lane rings by ozone attack on the carbon–carbon dou-ble bonds as shown by signals at 5.14–5.10 (protons onring carbons), at 1.65 (methylene protons α to ringcarbons) and at 1.35 ppm (methylene protons β toring carbons (Almeida and Beatriz 2012; Sadowska,Johansson, and Johannessen 2008). A single peak atd = 7.26 ppm, which belongs to the chloroform-d.1H-NMR spectra of OZ showed peaks at 9.71 and2.42 ppm attributable to the aldehydic proton and themethylene proton α to the carbonyl carbon, respec-tively. A signal at 7.98 ppm indicated the formation ofα-hydroxy-hydroperoxides. The 1H-NMR spectra dataare consistent with the literature (Blanco, Poznyak, andChairez 2015; Soriano, Veronica, and Matsumura2003).

In the present study, an in vitro model of L929mouse fibroblasts placed in direct contact with the OZwas used to measure its cytotoxicity. Preliminary cyto-toxic activity of OZ was performed by only under aninverted microscope (Figure 2). The results showed thatthe direct treatment of OZ caused substantial cell deathdue to the effect of its viscous mass on the cells; visc-osity causes mechanical stress on cells that induce cell

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membrane damage and also engender cell death(Fischer et al. 2003). Therefore, it was combined witholive oil and glycerol, respectively. Previous studiesshowed that the viscosity of ozonated oil increasesduring the ozonation time (Díaz et al. 2006; Uysal2014). The viscosity of the ozonated oil with a highPV increases in direct proportion to the ozonation timebecause of the disappearance of the double bonds.

Therefore, OZ was mixed with olive oil (OZ-Oo) andglycerol (OZ-Gly) to reduce the viscosity of the sample.Sumer and colleagues evaluated the cytotoxic and anti-bacterial activity of olive oil and lime cream, both indivi-dually and in combination, on L929 fibroblasts (Sumeret al. 2013). Their results showed that lime cream had acytotoxic effect, whereas olive oil showed no cytotoxicityon the fibroblasts. Moreover, lime cream showed antimi-crobial activity, whereas olive oil had limited antimicro-bial activity on microbial strains and yeasts. When limecream was mixed with olive oil, they observed that thecytotoxic impact of the lime cream decreased.

The evaluation of cytotoxic and cytostatic effects of achemical compound is very important in order to deter-mine the first risk assessment when working witih newbiomaterials. Most current tests include the level of cellviability assays after exposure to a chemical compound ora biomaterial. Many cell culture techniques are used toassess cell damage caused by biomaterials. These methodsare based on cell cultures with established or diploid celllines and primary tissue explants techniques (Can et al.2010). In the present study, morphological observation ofL929 cells was initially performed under an invertedmicroscope. Control cells and OZ-Oo-treated cellsshowed a flattened fibroblastic morphology; however,the cells incubated with OZ-Gly showed visible changesin morphology such as an elongated and rounded shape(Figures 3a–3c). These morphological observations wereconsistent with the MTT results.

The MTT results revealed that the cell survival of theOZ-Oo-treated group was better than that of the OZ-Glygroup, and the difference was statistically significant.Meanwhile according to results OZ group had no livingcells parallel to inverted microscope observations (data notshown). The OZ-Oo group did not show any cell death at

Figure 1. 1H-NMR spectra of OZ from δ = 0 to 10 ppm.

Table 1. 1H-NMR signals of OZSignal (ppm) Functional group

1.35 –CH2–CH = CH–CH2–1.65 –CH = CH–CH2–CH = CH–5.10 trans- 1,2,4-trioxolane5.14 cis- 1,2,4-trioxolane9.71 Aldehyde2.42 α-Methylene group

Figure 2. Direct treatment of ozonated oil to L929 fibroblastsresulted in substantial cell death because of the effect of itsviscous mass on the cells. Twenty-four hours after incubation,the cells lost their spindle-like morphology (IX70 Olympus,Japan).

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all concentrations tested (p > 0.05). The OZ-Gly group didnot show any difference in cell viability from the control atthe 0.062% (v/v) concentration (p = 0.423), whereas thegroups treated with 0.187%, 0.25%, and 0.75% (v/v) OZ-Gly showed statistically significant reductions in viability(p = 0.004–0.006) compared to the control group(Figure 4). It has been reported that olive oil containsmany oleosidic forms of phenolic compounds with anti-oxidant activity (Baldioli et al. 1996).

As a companion to these properties in aqueous condi-tions, ozone results in the production of bioactive products

such as redox transcription factors that can activate thesynthesis of growth factors and promote the cell cycle(Valacchi et al. 2013). These previous findings supportour result that the OZ-Oo group showed higher cell viabi-lity at all concentrations tested. In addition, Valacchi andcolleagues reported that a PV of 1631 mEq O2/kg of ozo-nated oil had a positive effect on the wound healing processduring 7-day treatment in a murine model (Valacchi et al.2011) .Our data indicate that oil with a PV of 2700–2900mEqO2/kg oil hadno cytotoxic effect on L929 cells for a 48-h incubation period.

Figure 3. Morphological observation of the cells treated with a 0.75% (v/v) concentration of the oil samples. Similar to the MTTresults, the cell viability of the OZ-Oo group was similar to that of the control group, whereas there were no living cells remaining inthe OZ-Gly group. a. OZ-Gly group, b. OZ-Oo group, c. control group.

Figure 4. MTT results after 48 h of incubation. Cell survival of the OZ-Gly group was significantly lower than that of the control. TheOZ-Oo groups maintained cell viability compared to the control at all concentrations tested. Error bars indicate SD (n= 6 per group).*P <0.05.

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Conclusions

In conclusion, these results revealed that using ozonatedolive oil with 2700–2900 mEq O2/kg oil peroxide levels inshort-term incubation is non-cytotoxic in an L929 fibro-blast cell line. Taken together, the present data demon-strate that mixing OZ with olive oil did not change theperoxide levels, and adding olive oil to ozonated oil has apositive effect on the anti-oxidant levels. In our study, byadding olive oil to ozonated oil, the anti-oxidant effect wasimproved and cell survival increased. These results high-light the dose-dependent use of OZ and combinations forin vitro toxicology studies, thus justifying and reinforcingthe pharmaceutical use of ozonated vegetable oils. Thesedata can be also used to determine the starting dose for invivo studies.

Acknowledgment

We would like to thank Professor Dr. AbdurrahmanTanyolaç, Dr. Muhteşem Fergül Pullu and AssociateProfessor Dr. İ. Cengiz Koçum for all their time and effortin synthesizing the ozonated olive oil.

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