Comparative Study of Bioactive Constituents in Turkish Olive Oils by LCESI/ MS/MS

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Comparative Study of BioactiveConstituents in Turkish Olive Oils by LC-ESI/MS/MSHasim Kelebeka, Songul Kesenb & Serkan Sellica Department of Food Engineering, Faculty of Engineering andNatural Sciences, Adana Science and Technology University, Adana,Turkeyb Department of Food Technology, Naci Topçuoglu Vocational HighSchool, Gaziantep University, Gaziantep, Turkeyc Department of Food Engineering, Faculty of Agriculture, CukurovaUniversity, Adana, TurkeyAccepted author version posted online: 05 Feb 2015.

To cite this article: Hasim Kelebek, Songul Kesen & Serkan Selli (2015) Comparative Study ofBioactive Constituents in Turkish Olive Oils by LC-ESI/MS/MS, International Journal of Food Properties,18:10, 2231-2245, DOI: 10.1080/10942912.2014.968788

To link to this article: http://dx.doi.org/10.1080/10942912.2014.968788

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Comparative Study of Bioactive Constituents in Turkish OliveOils by LC-ESI/MS/MS

Hasim Kelebek1, Songul Kesen2, and Serkan Selli3

1Department of Food Engineering, Faculty of Engineering and Natural Sciences, Adana Scienceand Technology University, Adana, Turkey

2Department of Food Technology, Naci Topçuoglu Vocational High School, Gaziantep University,Gaziantep, Turkey

3Department of Food Engineering, Faculty of Agriculture, Cukurova University, Adana, Turkey

The purpose of this research was to evaluate and compare the differences in the phenolic composition,antioxidant properties, and fatty acids of virgin olive oils from the Ayvalik, Gemlik, and Memecik olivevarieties cultivated in their respective growing areas over two harvest years. The phenolic compositionof olive oils was carried out by liquid chromatography coupled with diode array detection andelectrospray ionization mass spectrometry analysis and the fatty acid composition was determined bygas chromatography analysis. Fourteen phenolic compounds were identified and, among these, the mostdominant were elenolic acid, tyrosol, and hydroxytyrosol. For olives from both years, the highest totalphenolic content was determined in Memecik followed by Ayvalik and Gemlik. 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis- (3-ethyl-benzothiazoline-6-sulphonic acid) methods were used to deter-mine the antioxidant capacity of the olive oil extracts. In both methods, the antioxidant capacity valueswere higher for oil from cv. Ayvalik. Thirteen fatty acids were identified and quantified in all samples.Oleic acid was the highest concentration and this acid was more dominant in Gemlik oils.

Keywords: Olive oil, Phenolic compound, Fatty acid, Antioxidant capacity, Harvest year.

INTRODUCTION

Olive oil is a dietary staple for people the countries surrounding the Mediterranean Sea, but its useis now expanding to other parts of the world due to its unique flavor, the presence of biologicallyimportant minor constituents such as phenolic compounds, and high content of healthy mono-unsaturated fatty acid (FA).[1] Virgin olive oil (VOO) is valued for its organoleptic and nutritionalcharacteristics, and is resistant to oxidation due to presence of natural antioxidants such as phenolsand its high monounsaturated fatty acid content (MUFAs) and low polyunsaturated fatty acidcontent (PUFAs).[2] In the specialty food arena, olive oil is a dominant species that continues togrow in popularity. According to olive oil quantity data released in 2012, Spain (992,000 tonnes),

Received 9 June 2014; accepted 20 September 2014.Address correspondence to Hasim Kelebek, Department of Food Engineering, Faculty of Engineering and Natural

Sciences, Adana Science and Technology University, Adana, 01110, Turkey. E-mail: [email protected] versions of one or more of the figures in the article can be found online at www.tandfonline.com/ljfp.

International Journal of Food Properties, 18:2231–2245, 2015Copyright © Taylor & Francis Group, LLCISSN: 1094-2912 print/1532-2386 onlineDOI: 10.1080/10942912.2014.968788

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Italy (570,000 tonnes), Greece (351,800 tonnes), Turkey (206,300 tonnes), Syria (200,000 tonnes),and Tunisia (192,600 tonnes) are the leading producers.[3,4]

The composition of VOO is influenced by many factors such as variety, degree of fruit ripeness,climatic conditions, growing region, and extraction process techniques.[2] During the ripening, theimportant proportion of free FAs in the oil depends on the hydrolysis degree of triacylglycerols, thebotanical variety, climatic conditions, fruit maturity and the geographical origin of olives.[1]

Phenolic compounds, more commonly known as polyphenols, are minor constituents of oliveoil. These compounds are of fundamental importance to the shelf life of VOOs because of theirantioxidative properties. Their antioxidant properties have attracted great interest, since they aresupposed to have chemoprotective properties in human beings and also phenolic compounds areimportant to emphasize the high antioxidant activity.[5] Their antioxidant activity has been relatedto protection against some chronic and degenerative diseases such as coronary heart disease andcertain cancers.[6] In addition, polyphenols contribute to the sensorial characteristics of VOOs[7] byconferring bitterness, pungency, and astringency.[8]

The prevention of cardiovascular diseases and certain cancers by extra virgin olive oil (EVOO)consumption have been related to its well-balanced FA composition.[9] Major FAs in olive oils areoleic (55–85%), palmitic (7.5–20%), linoleic (7.5–20%), stearic (0.5–5%), palmitoleic (0.3–3.5%),and linolenic (0.0–1.5%) acids, although traces of myristic, arachidic, and margaric acids have alsobeen found.[10] In comparison to other vegetable oils usually richer in polyunsaturated FAs, oliveoil has a much lower rate of alteration during domestic frying or other uses that require hightemperatures. This stability of olive oil and its resistance to rapid deterioration at elevatedtemperatures are due to its FA profile and the presence of natural antioxidants.[11]

Although Turkey is the fourth in the production of olive oil in the world, limited research hasbeen published in the literature to determine properties of Turkish olive oils. Therefore, the aim ofthis study was to determine phenolic compounds, antioxidant activity and FA composition of oliveoils from AYV, GEM, MEM cultivars over two harvest years.

MATERIALS AND METHODS

Materials

Olive varieties (Ayvalik [AYV], Gemlik [GEM], Memecik [MEM]) averaging 50–70 kg werebought from their growing areas. AYV, GEM, and MEM were respectively cultivated in Balikesir(Burhaniye, Taylieli: 39° 30′ 10″ N, 26° 58′ 49″ E), Bursa (Mudanya, Trilye: 40° 41′ 0″ N, 28° 29′0″ E), and Mugla (Milas: 37° 19′ 1.92″ N, 27° 46′ 50.28″ E) provinces (all of which are located inthe Western part of Turkey). They were harvested at maturity stage in the 2010 and 2011 seasons.These cultivars are used mainly for producing oil in Turkey. Maturity indexes were calculatedaccording to International Olive Oil Council method.[12] This method was based on the assessmentof the color of the olive skin and flesh. In our study, the healthy olives were collected from eachcultivar and the maturity indexes were calculated between 3.8 and 4.0.

Chemicals

In all analysis deionized water (resistivity over 18 MΩ cm) purified with a Millipore Q system(Millipore Corp., Saint-Quentin, France) was used. Phenolic compounds (caffeic, vanillic,syringic, p-coumaric, ferulic, cinnamic acids, vanillin, tyrosol, apigenin, and luteolin) andFAs (myristic acid, palmitic acid, palmitoleic acid, margaric acid, margoleic acid, stearicacid, oleic acid, linoleic acid, arashidic acid, linolenic acid, gadoleic acid, behenic acid, andlignoceric acid) were obtained from Sigma-Aldrich (Steinheim, Germany). Acetonitrile, formic

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acid, 2,2′-azino-bis- (3-ethyl-benzothiazoline-6-sulphonic acid) diammonium salt (ABTS),potassium persulphate, 2,2-diphenyl-1-picryl hydrazyl (DPPH) were obtained from Merck(Gernsheim, Germany).

Extraction of the Olive Oils

A cold pressing process with dual phase centrifuge (Oliomio Mini, Italy) was used for olive oilextraction. Fresh olive oils were put into glass bottles and were preserved in a dark and cool placeuntil analysis. Cold press is a method widely used in the last several years to produce high qualityolive oils.[13] If high temperature is applied during the extraction process, volatile compounds aretransformed and fat oxidation is likely to increase. At the same time, the amount of phenoliccompounds and antioxidant activity of olive oils decreases.[14]

Standard Chemical Analysis

Moisture and oil content of olives were analyzed as a percent. Moisture content was determined inan oven by heating the oil at 105 ± 1°C and oil content was determined by solvent extraction insoxhlet apparatus using n-hexane. Also the olive oils free FAs,[15] peroxide value[16] and colormeasurements, obtained using a Hunter colorimeter (HunterLab, Color QuestXE-USA), of wereanalyzed. All the analyses were made in triplicate.

Analysis of the Phenolic Compounds

The extraction was performed according to the procedure described by Kesen, Kelebek, and Selli.[17]

Briefly, 2 g of oil was weighed into a centrifuge tube, and combined with 5 mL of n-hexane and 10mL ofmethanol-water (60:40, v/v). An Agilent 1100 high performance liquid chromatography (HPLC) system(Agilent Technologies, Palo Alto, CA, USA) operated by ChemStation software was used. The HPLCequipment was used with a diode array detector (DAD). The column used was a Beckman C18 ODS(Roissy, France): 4.6 mm × 250 mm, 5 µm equipped with a precolumn (4.6 mm × 10 mm, 5 µm). Theseanalysis procedures were elucidated in our previous studies.[18] The identification and assignation of eachcompounds was performed by comparing their retention times and UV spectra to authentic standards andalso confirmed bymass spectrometry (liquid chromatography coupled and electrospray ionization tandemmass spectrometry) using an Agilent 6430 Triple Quadrupole LC/MS System spectrometer (AgilentTechnologies, Palo Alto, CA, USA). The quantification of each identified compounds was performed oneach collected oil samples using an external standard calibration curve for each compound.[18,19] Thecurves were obtained using the commercial standards of the concentrations normally present in olive oils(approximately 1–100 mg kg–1), obtaining regression coefficients (R2) above 0.995 in all cases.

Measurement of Antioxidant Activity

DPPH Assay: 0.1 mL of diluted olive oil extract was mixed with 3.9 mL of DPPH solution (2.36 mg/100 mL methanol) and vigorously vortexed. The solution was held in the dark at ambient conditionsfor 15 min. The absorbance was measured at 517 nm by a UV-visible spectrophotometer (ShimadzuUV-1201, Kyoto-Japan). The Trolox calibration curve was used to calculate the antioxidant activityof the oil extracts and to express the antioxidant capacity in mM Trolox equivalent per kg of olive oil.The mean and standard deviation were calculated for the three replicates.

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ABTS assay

The ABTS solution was created at a concentration of 7 mM and mixed with 2.5 mM of potassiumpersulphate, and stored after incubation at 23°C in the dark for 12‒16 h. The ready-made solution wasdiluted with 80% methanol to measure an absorbance of 0.7 ± 0.01 at 734 nm. Then, 3.9 mL of ABTSsolution was added to 0.1 mL of the oil samples and mixed vigorously. Finally, the solution was left for10min to ensure reaction and the absorbancewas monitored at 734 nm. The calibration curve equationsrelated to the Trolox standard were y = 0.0004x + 0.0089 with R2 = 0.9996 for ABTS and y = 0.0004x +0.0082 with R2 = 0.9995 for DPPH within a concentration range from 5 to 150 μmol/L.

FAs analysis

The FA composition of the oils was determined by gas chromatography equippedwith split injection(1:50) and a flame ionization detector (Schimadzu 14B, Tokyo, Japan). FAmethyl esters were preparedby cold transmethylation.[20] Separation was accomplished with a 60 m capillary column (DB23,Agilent Inc., USA) that had a 0.25 mm I.D. and 0.25 μm film thickness. Hydrogen was the carrier gas,and injector and detector temperatures were set to 250 and 300°C, respectively. Oven temperature wasprogrammed at 130°C for 1 min, increased from 130 to 170°C at 6.5°C/min then was set at 215°C at2.75°C/min for 12 min and then increased to 230°C at 40°C/min for 3 min.[21] The injection volumewas 1 μL. The identification of individual FAs (myristic, palmitic, palmitoleic, margaric, margoleic,stearic, oleic, linoleic, arachidic, linolenic, gadoleic, behenic, and lignoceric) was carried out bycomparison with retention times of known standards and expressed as a percentage of the total.

Statistical Analysis

The findings of all assays were subjected to analysis of variance using the SPSS 17 softwarepackage and Duncan’s multiple comparison test was used to find significant differences at the 0.05level. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were alsocarried out using XLStat-Pro7.5 (2007) for Windows (Addinsoft, NY, USA).

RESULTS AND DISCUSSION

Standard Chemical Analysis

Results of chemical analysis of olive cultivars and olive oils for the years 2010 and 2011 are shown inTable 1. The results of each year were statistically significant when compared against each other. As canbe seen in Table 1, the moisture and oil contents of olive samples were detected between 20.2–24.4 and42.75–56.04%, respectively. The values of these quality parameters changed depending on the years. Inthe 2011 harvest year, oil contents of all samples were higher than of the year 2010. With regard to theamount of moisture content, oils of the year 2010 was higher than that of the 2011. Similarly, the oil andmoisture contents of five Turkish olives (GEM, Kilis yaglik, Uslu, Tirilye, and AYV) were found as17.7–43.5 and 35.30–64.72%, respectively by Tanilgan et al.[22]

Free FA and peroxide values are usually used for quality characteristics and depend on thequality of the olives and on possible enzymatic alteration of olive fruits.[23] Free FA contents ofolive oils did not exceed the maximum legal limit for EVOOs (0.8%) in either year. Peroxidevalues also were below the limit of 20 meq oxygen/kg of oil, which is accepted as the limit for theextra quality of VOO (Table 1). According to results of a previous study on 12 different olive oilsfrom the islands of Italy (Sardinia) and French (Corsica), free FA and peroxide values were foundto be between 0.20–1.34% and 3.28–16.15 meq oxygen/kg of oil.[24]

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Color is generally considered an important organoleptic property of olive oils which influenceconsumer acceptance. In addition, color is related to other chemical and physical properties ofVOOs.[25] Color values of each year are statistically different at the 0.05 (p < 0.05). Olive oils obtainedfrom 2010 had higher L*, a*, and b* values than those obtained from 2011 except a* values of GEMand b* values of AYVandMEM.Gomez-Caravaca et al.[26] found color values for eight Italian olive oilsamples. Due to the results, L*, a*, and b* values were stated between 52.2–74.0, 1.5–9.1, and59.7–104.8, respectively. Hue is described with the words we normally think of as describing color.Hue values of oils were highest in GEM for both harvest years. Chroma may be defined as the strengthor dominance of the hue. In the 2011 harvest year, the values of chroma and color index for AYVandMEMwere higher than for the year 2010 (Table 1). In previous study, the confidence intervals values ofArbequina and Picual oils on different harvest date were found between 0.2–32.0 by Yousfi et al.[27]

Phenolic Compounds of Olive Oils

A total of 14 phenolic compounds including, hydroxytyrosol, 4-hydroxybenzoic acid, tyrosol, 2,3-dihydroxybenzoic acid, caffeic acid, vanillic acid, vanillin, syringic acid, p-coumaric asit, ferulic acid,cinnamic acid, elenolic acid, apigenin, and luteolin, were identified and quantified in olive oil samplesof both harvest seasons (Tables 2 and 3; Fig. 1). There were significant differences in the mean values ofphenolic composition of the olive oils in relation to varieties and years (Table 3).

The changes in the concentration of the total phenolic compounds in the oils obtained from AYV,GEM, and MEM showed different profiles according to the cultivar and harvest year (Table 3). In bothyears, the lowest amount of phenolic compounds was found in GEM followed by AYVand MEM. Inthe year 2010, the total concentrations of phenolic compounds were 79.96, 71.91, and 53.51 mg kg–1

for MEM, AYV, and GEM, respectively. For the year 2011, concentrations of phenolic compounds inAYVand MEM were higher than for the year 2010 harvest. The total phenol contents of the year 2010

TABLE 1Olive fruits and olive oils general properties

2010 year 2011 year

Olive properties AYV GEM MEM AYV GEM MEM

Maturity index 3.8 ± 0.00 3.8 ± 0.00 3.8 ± 0.00 4 ± 0.00 4 ± 0.00 3.9 ± 0.00Oil content (%) 22.3 ± 0.05a 21.1 ± 0.42b 20.2 ± 0.08c 24.4 ± 0.15a 22.8 ± 0.08b 22.1 ± 0.06b

Moisture content (%) 47.90 ± 0.24b 55.08 ± 0.18a 56.04 ± 0.18a 42.75 ± 0.32c 50.23 ± 0.24b 54.73 ± 0.12a

Olive oil propertiesFree fatty acids (oleic acid

%)0.51 ± 0.01b 0.80 ± 0.02a 0.69 ± 0.02ab 0.53 ± 0.01b 0.54 ± 0.01b 0.62 ± 0.10a

Peroxide value (meq O2/kgoil)

6.90 ± 0.06b 6.46 ± 0.03c 9.66 ± 0.02a 7.82 ± 0.13a 5.71 ± 0.12b 7.60 ± 0.06a

L* 63.89 ± 0.02b 66.12 ± 0.01a 62.34 ± 0.02c 61.59 ± 0.05a 44.24 ± 0.01c 60.57 ± 0.07b

a* –3.18 ± 0.00b –2.70 ± 0.01a –3.50 ± 0.02c –4.69 ± 0.01b –0.65 ± 0.02a –5.04 ± 0.01c

b* 19.96 ± 0.02c 55.84 ± 0.01a 32.80 ± 0.01b 49.25 ± 0.03b 50.26 ± 0.04b 51.06 ± 0.10a

Hue 80.95 ± 0.01c 87.23 ± 0.01a 83.90 ± 0.02b 84.56 ± 0.01a 89.26 ± 0.02b 84.36 ± 0.01a

Chroma 20.22 ± 0.02c 55.91 ± 0.01a 32.99 ± 0.01b 49.47 ± 0.03b 50.27 ± 0.04ab 51.31 ± 0.10a

CI 14.79 ± 0.01c 38.71 ± 0.00a 22.63 ± 0.02b 33.22 ± 0.04a 22.53 ± 0.02b 33.98 ± 0.12a

Results are expressed as mean ± standard deviation of three determinations;a,b,cDifferent letters for the same parameter indicate significant differences among varieties (p < 0.05);Results of each year were statistically compared within themselves;AYV: Ayvalik, GEM: Gemlik, MEM: Memecik.

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were 95.86, 93.19, and 42.17 mg kg–1 for MEM, AYV, and GEM, respectively. There were significantdifferences (p < 0.05) in the mean values of all of the phenolic compounds of the VOO in relation tocultivar and years. Phenolic compounds are clearly related with the sensory characteristics and thestability of olive oils and there are notable differences in phenolic composition between olive varieties.In a previous study Rivas et al.[28] showed that phenolic acid content differently affected the sensorycharacteristics of Hojiblanca and Picual oils. Gimeno et al.[29] reported that the amount of total phenoliccompounds of oils obtained from ripe and unripe olives ranged from 61.48 to 98.38 mg kg–1. Also,Cerretani et al.[24] studied 12 VOOs obtained from seven olive cultivars came from different groves inSardinia (Italy) and Corsica (France) in terms of phenolic profile. In this study, total amount of phenolschanged between 77.75–695.84 mg kg–1.

In the present study, the most common phenolic compounds that were identified in both years arehydroxytyrosol, tyrosol, elenolic acid, apigenin, and luteolin (Table 3). The major compound in allolive oils was elenolic acid in both harvest years, as it accounted for the largest proportion of the totalphenolic content. Elenolic acid was reported for the first time in a VOO by Montedoro et al.[30] andsubsequently Mateos et al.[31] proved that the presence of this compound could not be attributed tohydrolysis during the analysis of secoiridoids derivatives containing it. Tyrosol and hydroxytyrosol arethe most representative phenolics of olive fruits and olive oil and they occur by themselves or in theform of esters of the secoiridoid elenolic acid.[32] The concentrations of elenolic acid in the 2010 and2011 oils ranged between 32.53–49.32 and 24.89–58.63 mg kg−1, respectively. In previous studies,elenolic acid content varied between 32 and 184 mg kg–1 in different Italian cultivar oils[33] andbetween12 and 65 mg kg–1 in the olive oils from Spain.[34]

Tyrosol is a natural phenolic antioxidant and its principal source in the human diet is olive oil. As anantioxidant, tyrosol can protect cells against injury due to oxidation. Although, it is not as potentas other antioxidants present in olive oil, its higher concentration and good bioavailability indicate thatit may have an important overall effect. This effect may contribute significantly to the health benefits ofolive oil and, more generally, the Mediterranean diet.[35] The tyrosol content was in the range of12.27–14.94 mg kg–1 for the year 2010; and 6.93–10.57 mg kg–1 for the year 2011. Tyrosol leveldetermined in our olive oils is in agreement with previous reported data.[24] In this study, researchersreported that the content of tyrosol in 12 olive oil samples ranged between 5.20–27.16 mg kg–1.Ilyasoglu et al.[36] found that tyrosol was the main phenolic compound in AYV (1.64–13.91 mg kg–1)

TABLE 2Data of phenolic compound identified in olive oils

Phenolic compounds Peak no. Rt (min)

λ max [M‒H]-

(nm) (m/z)

Hydroxytyrosol 1 2.1 230, 280 1534-Hydroxybenzoic acid 2 2.6 254 137Tyrosol 3 4.2 230, 275 1372,3-Dihydroxybenzoic acid 4 10.1 320 153Caffeic acid 5 12.2 320, 300 179Vanillic acid 6 20.1 262 167Vanillin 7 24.3 309, 284 151Syringic acid 8 29.1 275 197p-Coumaric acid 9 33.3 308 163Ferulic acid 10 36.7 323, 293 193Cinnamic acid 11 40.5 278 147Elenolic acid 12 45.2 240 241Apigenin 13 46.8 230, 270, 340 269Luteolin 14 50.2 255, 350 285

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TABLE

3Phe

nolic

compo

unds

ofoliveoils

2010

year

2011

year

Factoreffects

Phenolic

compoun

ds(m

gkg

−1)

AYV

GEM

MEM

AYV

GEM

MEM

YV

YxV

Hyd

roxy

tyrosol

6.66

±0.02

a0.78

±0.01

c4.17

±0.03

b17

.36±0.07

a3.85

±0.15

b3.66

±0.09

c*

**

4-Hydroxy

benzoicacid

0.09

±0.00

b0.03

±0.00

c0.21

±0.00

a0.13

±0.00

b0.10

±0.01

c0.38

±0.02

a*

**

Tyrosol

12.27±0.02

c14

.94±0.14

a13

.13±0.02

b9.43

±0.02

b6.93

±0.13

c10

.57±0.33

a*

**

2,3-Dihyd

roxybenzoicacid

0.21

±0.00

c0.28

±0.02

a0.22

±0.00

b0.29

±0.00

c0.43

±0.00

a0.31

±0.00

b*

**

Caffeic

acid

0.05

±0.01

a0.06

±0.00

a0.05

±0.00

a0.07

±0.01

b0.09

±0.00

a0.07

±0.00

b*

*ns

Vanillic

acid

0.04

±0.00

a0.04

±0.01

a0.04

±0.01

a0.05

±0.00

a0.05

±0.01

a0.06

±0.01

a*

*ns

Vanillin

0.02

±0.01

a0.02

±0.00

a0.02

±0.00

a0.03

±0.01

a0.03

±0.00

a0.02

±0.00

bns

ns*

Syringicacid

0.53

±0.01

a0.21

±0.00

c0.44

±0.01

b0.75

±0.01

a0.32

±0.01

c0.63

±0.01

b*

**

p-Cou

maric

acid

0.76

±0.01

a0.30

±0.01

c0.63

±0.01

b1.07

±0.02

a0.46

±0.01

c0.90

±0.01

b*

**

Ferulic

acid

0.24

±0.03

b0.05

±0.00

c0.27

±0.01

a0.34

±0.04

b0.08

±0.00

c0.39

±0.02

a*

**

Cinnamic

acid

0.61

±0.00

c1.75

±0.02

b3.49

±0.02

a0.86

±0.01

c0.99

±0.04

b1.68

±0.01

a*

**

Elenolic

acid

43.71±0.63

b32

.53±0.07

c49

.32±0.18

a53

.28±0.23

b24

.89±0.09

c58

.63±0.32

a*

**

Apigenin

4.74

±0.49

b1.00

±0.04

c5.35

±0.20

a6.71

±0.73

b1.65

±0.11

c8.32

±0.02

a*

**

Luteolin

2.00

±0.24

b1.54

±0.03

c2.60

±0.03

a2.83

±0.35

b2.31

±0.04

c10

.25±0.65

a*

**

Total

concentration

71.91±1.03

b53

.51±0.11

c79

.96±0.29

a93

.19±0.66

b42

.17±0.16

c95

.86±1.03

a*

**

Resultsareexpressedas

mean±standard

deviationof

threedeterm

inations;

a,b,cDifferent

letters

forthesameparameter

indicate

significantdifferencesam

ongvarieties(p

<0.05

);Resultsof

each

year

werestatistically

comparedwith

inthem

selves;

AYV:Ayv

alik,GEM:Gem

lik,MEM:Mem

ecik,Y:Year,V:Variety,YxV

:Yearandvarietyinteractions;

*Significanceat

the0.05

level,ns:notsignificant.

BIOACTIVE CONSTITUENTS IN OLIVE OILS 2237

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and MEM (7.41–20.08 mg kg–1) olive oils. Another identified phenolic alcohol was hydroxytyrosol inolive oils. Its amount varied between 0.78–6.66 mg kg–1 (2010) and 3.66–17.36 mg kg–1 (2011). Theseresults are similar to those reported by several authors for other olive oil varieties.[37]

Luteolin and apigenin are the main flavonoids present in olive oils, which originate from theircorresponding glucosides present in the drupe.[38] The content of luteolin and apigenin in olive oilsranged from 1.54 to 10.25 mg kg–1 and from 1.00 to 8.32 mg kg–1, respectively. As can be seen inTable 3, concentration of luteolin and apigenin in MEM oil was higher than in AYV and GEM oilsfor either year. In previous studies, luteolin and apigenin contents were found between 1.6–6.3 and0.5–3.1 mg kg−1[39] and 0.22–7.0 and 0.68–1.6 mg kg–1, respectively.[40]

Based on the results of phenolic acids, cinnamic acid (0.61–3.49 mg kg–1), caffeic acid (0.05–0.09mg kg–1), p-coumaric acid (0.30–1.07 mg kg–1), 2,3-dihydroxybenzoic acid (0.21–0.43 mg kg–1),vanillic acid (0.04–0.06 mg kg–1), and ferulic acid (0.05–0.39 mg kg–1) were identified and quantified.Amounts of phenolic compounds in our study were similar to those obtained from differentvarieties.[19,41]

As a result, when the amount of individual phenolic compounds of olive oils are compared withthe olive oils produced from other varieties under similar conditions, we see that the AYV, GEMand MEM oils had higher phenolic contents than the olive oils from Tunisian cvs. Oueslati,Chétoui and Chemlali[19] while they had lower phenolics than the olive oils from Italian cvs.Casaliva, Frantoio, and Leccino.[42] French cvs. Aglandau and Tanche; Spanish cvs. Cornicabra,Picual, and Verdial.[43] In addition, the phenolic contents of the olive oil samples were quite similarto other olive oils produced in Turkey such as MEM, Erkence, GEM, AYV, and Domat.[41]

The data set for three cultivars and two harvest years regarding the variables of phenolics of oils wassubjected to PCA. All the phenolic compounds quantified were considered to identify the two principalfactors. Fourteen variables were selected for the PCA and the explained variance was 74.70% (Factor 1:55.32%; Factor 2: 19.37%). Figure 2a represents the projection of the variables with regard to the singlefactor (PC1 or PC2) on the factor plane (PC1 × PC2). The scores of the samples and the loadings of thevariables on the two principal components are plotted in Fig. 2b. A distinct categorization according to theconcentration of phenolics expressed in the olive oil extracts was observed along PC1. The plot of scoresin the PCA analysis illustrates that samples AYV and MEM from 2011 were clearly different from theothers due to higher content of elenolic acid, syringic acid, ferulic acid, 4-hydroxybenzoic acid, luteolin,and apigenin. Therefore, GEM oil in 2011 was distinguished by its high amount of 2,3-dihydroxybenzoic

FIGURE 1 LC-MS Chromatogram of phenolic compounds (peak numbers refer to Table 2).

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acid and caffeic acid. A similar situation was observed in the year 2010. TheGEMoil in the year 2010 hasthe highest scores on PC2 according to content of tyrosol.

Antioxidant Capacity of Olive Oils

To determine the antioxidant capacity, ABTS and DPPH methods were used. The results of theantioxidant activities of olive oils were reported in Table 4. The antioxidant capacities of phenoliccompounds were better reflected by ABTS assay than DPPH assay and in both years ABTS assayproduced higher values than DPPH assays. In both years, the antioxidant capacity values were highestin oil obtained from AYV followed by MEM and GEM. According to results of the first year, the

A B

C D

FIGURE 2 The scores and loading plots of the phenolic compounds and fatty acids (a) factor-plane considering thephenolic compounds, (b) score plot for the two principal components showing cultivars, (c) factor-plane consideringthe fatty acids, and (d) score plot for the two principal components showing the cultivars.

BIOACTIVE CONSTITUENTS IN OLIVE OILS 2239

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antioxidant activities of olive oils obtained by using ABTS and DPPH methods were 0.83–1.34 and0.32–0.53 mM Trolox/kg, respectively, while those from the second year ranged from 1.24–2.26 to0.44‒1.05mMTrolox/kg. The antioxidant capacity in several oils obtained from different rawmaterialsin the ABTS assay ranged from 0.61mMTrolox/kg (peanut) to 2.20mMTrolox/kg (soybean). Authorsalso reported that the antioxidant capacity of olive oil was 0.63 mM Trolox/kg.[44]

The contents of total phenolic and antioxidant capacities obtained from DPPH and ABTS assayswere compared. As a result of statistical comparison, good correlations were found between the totalphenolic contents and antioxidant capacity values (DPPH and ABTS). Results showed strong correla-tions between antioxidant capacity and total phenolic content (for ABTS, R2 = 0.873; for DPPH, R2 =0.892, p < 0.01; Table 5). Within the phenolic compounds, the positive correlation between antioxidant

TABLE 5Correlations between phenolic compounds and antioxidant capacity

ABTS DPPH

Hydroxytyrosol 0.734* 0.759*4-Hydroxybenzoic acid 0.198 0.205Tyrosol 0.764* 0.770*2,3-Dihydroxybenzoic acid –0.928** –0.947**Caffeic acid –0.635 –0.726*Vanillic acid –0.262 –0.267Vanillin –0.231 –0.084Syringic acid 0.960** 0.971**p-Coumaric acid 0.957** 0.968**Ferulic acid 0.852** 0.854**Cinnamic acid –0.025 –0.017Elenolic acid 0.820** 0.843**Apigenin 0.788* 0.793*Luteolin 0.184 0.196Total concentration 0.873** 0.892**DPPH 0.957** 1ABTS 1 0.957**

Correlation values between phenolic compounds and antioxidant capacity are given as theaverages of 2010 and 2011 values;

*Correlation is significant at the 0.05 level (2-tailed);**Correlation is significant at the 0.01 level (2-tailed).

TABLE 4Antioxidant activity values of olive oils (mM Trolox/kg)

AYV GEM MEM Factor effects

2010 year Y V YxVDPPH 0.53 ± 0.05a 0.32 ± 0.02b 0.50 ± 0.02a * * *ABTS 1.34 ± 0.04a 0.83 ± 0.03b 1.31 ± 0.09a * * *

2011 yearDPPH 1.05 ± 0.02a 0.44 ± 0.01c 0.81 ± 0.15b * * *ABTS 2.26 ± 0.32a 1.24 ± 0.01c 1.86 ± 0.13b * * *

Results are mean of three replications; ± standard deviation;a‒cDifferent superscripts in the same row indicate statistical differences at the 0.05 (p < 0.05);AYV: Ayvalik, GEM: Gemlik, MEM: Memecik, Y: Year, V: Variety, YxV: Year and variety

interactions;*Significance at the 0.05 level.

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capacities detected by ABTS and DPPH assays was strongest in syringic acid followed by p-coumaric,ferulic acid, and elenolic acid (Table 5). Also, correlations between the ABTS and DPPH assays wereevaluated. Due to these results, significant correlations (p < 0.01) were found between ABTS andDPPH (R2 = 0.957).

FA Compositions of Olive Oils

The FA compositions of olive oils for the years 2010 and 2011 were shown in Table 6. As can be seen inTable 6, a total of 13 FAs in the oils were determined in both years. These are myristic, palmitic,palmitoleic, margaric, margoleic, stearic, oleic, linoleic, arachidic, linolenic, gadoleic, behenic, andlignoceric FAs. There were significant differences in the mean values of FA composition of the oliveoils in relation to varieties and years (Table 6). Oleic acid was highest concentration in all oil samplesfollowed by palmitic and linoleic acids. The amount of myristic, margaric, and behenic acids were at thelowest in both years. The FA composition was similar with oils of Racimilla, Bodocal, and Negral[45] inSpain; Chemlali and Chetoui[46] in Tunisia; and Throumbolia and Koroneiki[37] in Greece.

Oleic acid is one of the most important FAs in olive oils due to having the nutritional wealth andsupport for oxidative stability.[45] According to the results of both harvest years, the highest amount ofoleic acid was found in GEM oil and the lowest in AYVoil. However, the amount of palmitic acid wasfound to be the highest in AYVoil (14.51 and 13.79% in 2010 and 2011, respectively) and the lowest inGEM oil (11.42 and 11.29% in 2010 and 2011, respectively). In the 2011 harvest year, proportion of oleicacid in GEM and MEMwere higher than in the year 2010 (Table 6). Similarly, Douzane et al.[47] studiedon the three different cultivars (Azeradj, Chemlal, Limli) from four different regions of the Algeria toinvestigate the effect of year on FAs composition. In this study differences in the composition of FAswereobserved over the years. In the first year, the amount of oleic acid was between 62.28 and 77.03%while inthe second year ranged from 73.33 to 60.00%. Similarly Diraman et al.[48] studied the FAs of olive oilobtained from another region, Izmir province in Turkey, from the 2001/2002 and 2002/2003 harvestyears. According to these results, palmitic, oleic, and linoleic acids were found between 9.62–18.79,62.90–76.92, and 8.08–17.17%. Similar studywas performed byGurdeniz et al.[49] They reported that theamounts of palmitic, oleic, and linoleic acids of olive oils obtained from 30 different location in theAegean region in 2005/2006 and 2006/2007 harvest years were between 12.06–13.32, 69.82–74.39, and8.42–11.76%, respectively.

In olive oils, the ratio of oleic acid/linoleic acid is a frequently used parameter to assess theoxidative stability of olive oils. If this value is high, olive oil shows higher oxidation-resistant.[45]

In our study, the highest value of this ratio was observed in GEM oils as 11.69 and 12.21 in theyears 2010 and 2011, respectively. In previous studies this ratio was found as 10.92 in Racimilla,5.07 in Bodocal, 8.04 in Negral oils of Spain,[45] 5.60 in Queslati, 4.91 in Chemlali oils ofTunisia,[50] 2.91–3.19 in Throumbolia, and 11.13–11.63 in Koroneiki.[37]

PCA has been applied in order to evaluate possibility of differentiating the samples by taking intoaccount the FA profile. The scores of the samples and loadings of the variables on the two first principalcomponents are plotted in Fig. 2c. All the FAs quantified were considered as identifying the twoprincipal factors. The variables were selected for the PCA and the explained variance was 70.23%(Factor 1:45.72%; Factor 2:24.51%). Fig. 2c represents the projection of the variables with regard to thesingle factor (PC1 or PC2) on the factor plane (PC1 × PC2). We can observe that each quadrantcontains, at least, one of the variables. A map of samples (score plot) for the two principal componentsis shown in Fig. 2c. PCA analysis clearly showed the differences between the samples of 2010 and 2011years. The application of the PCA showed two distinct groups (Fig. 2d). The first group represents theresult of AYV, GEM andMEM oils in 2010. The second one was characterized by the AYV, GEM, andMEM oils of 2011 year. The AYV, GEM, and MEM oils in 2010 year have the highest scores on PC2according to high content of linoleic (C18:2) and gadoleic (C20:1) acids, whereas the 2011 oils have the

BIOACTIVE CONSTITUENTS IN OLIVE OILS 2241

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TABLE

6Fatty

acidsof

oliveoils

2010

year

2011

year

Factoreffects

Fatty

acids

AYV

GEM

MEM

AYV

GEM

MEM

YV

YxV

Myristic

acid

(C14

:0)

0.01

±0.00

b0.01

±0.00

b0.02

±0.00

a0.02

±0.00

a0.01

±0.00

b0.02

±0.01

ans

*ns

Palmiticacid

(C16

:0)

14.51±0.34

a11.42±0.96

b12

.31±1.08

b13

.79±0.18

a11.29±0.00

c12

.13±0.16

bns

*ns

Palmito

leic

acid

(C16:1)

0.98

±0.04

a0.81

±0.08

b0.94

±0.11

ab0.73

±0.10

a0.79

±0.11

a0.78

±0.05

a*

nsns

Margaricacid

(C17

:0)

0.10

±0.01

b0.12

±0.00

a0.03

±0.00

c0.12

±0.04

a0.12

±0.05

a0.01

±0.00

bns

*ns

Margo

leic

acid

(C17

:1)

0.21

±0.00

b0.24

±0.01

a0.07

±0.00

c0.17

±0.00

b0.21

±0.00

a0.07

±0.02

c*

**

Stearic

acid

(C18:0)

2.39

±0.02

b3.35

±0.08

a2.12

±0.08

c2.78

±0.01

b3.17

±0.00

a2.20

±0.01

c*

**

Oleic

acid

(C18

:1)

68.95±0.42

b75

.85±0.89

a69

.57±1.07

b67

.07±0.57

c76

.94±0.54

a73

.22±0.25

b*

**

Linoleicacid

(C18

:2)

11.40±0.05

b6.49

±0.04

c13

.14±0.10

a10

.04±0.04

b6.30

±0.01

c10

.33±0.10

a*

**

Arashidic

acid

(C20

:0)

0.41

±0.01

a0.44

±0.04

a0.41

±0.06

a0.44

±0.04

a0.35

±0.01

b0.37

±0.04

bns

ns*

Linolenic

acid

(C18

:3)

0.46

±0.07

b0.47

±0.02

b0.80

±0.11

a0.38

±0.01

b0.27

±0.00

c0.43

±0.03

a*

**

Gadoleicacid

(C20

:1)

0.21

±0.01

a0.17

±0.04

a0.24

±0.08

a0.11

±0.02

a0.06

±0.00

b0.12

±0.00

a*

nsns

Behenic

acid

(C22

:0)

0.10

±0.01

a0.10

±0.01

a0.10

±0.01

a0.09

±0.00

c0.14

±0.01

a0.12

±0.02

b*

**

Ligno

cericacid

(C24

:0)

0.23

±0.00

b0.51

±0.07

a0.26

±0.01

b0.19

±0.01

b0.29

±0.01

a0.21

±0.01

b*

**

Saturated

fatty

acids

17.75±0.30

a15

.95±0.87

b15

.25±0.96

b17

.43±0.13

a15

.37±0.02

b15

.05±0.06

b*

ns*

Monou

nsaturated

fatty

acids(M

UFA

s)70

.35±0.33

b77

.07±0.83

a70

.82±0.92

b68

.08±0.31

c78

.00±0.09

a74

.19±0.05

b*

**

Polyaunsaturatedfatty

acids(PUFA

s)11.86±0.06

b6.96

±0.06

c13

.94±0.14

a10

.42±0.02

a6.57

±0.00

b10

.76±0.04

a*

**

Oleic

acid

/lino

leic

acid

(C18

:1/C18:2)

6.05

±0.01

b11.69±0.06

a5.29

±0.06

c6.68

±0.06

c12

.21±0.01

a7.09

±0.04

b*

**

MUFA

s/PUFA

s5.93

±0.01

b11.07±0.02

a5.08

±0.08

c6.53

±0.04

c11.87±0.01

a6.89

±0.02

b*

**

Resultsareexpressedas

mean±standard

deviationof

threedeterm

inations;

a,b,cDifferent

letters

forthesameparameter

indicate

significantdifferencesam

ongvarieties(p

<0.05);

Resultsof

each

year

werestatistically

comparedwith

inthem

selves;

MUFA

s:monounsaturated

fatty

acids,PUFA

s:polyunsaturatedfatty

acids;

AYV:Ayv

alik,GEM:Gem

lik,MEM:Mem

ecik.Y:Year,V:Variety,YxV

:Yearandvarietyinteractions;

*Significanceat

the0.05

level,ns:notsignificant.

2242 KELEBEK, KESEN, AND SELLI

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lowest scores on the same component. However, GEM oils of 2010 and 2011 years have the lowestscores on PC1 (high contents of stearic and margoleic acids).

CONCLUSIONS

In this study, phenolic compounds, antioxidant potential, and FAs of three olive cultivars (AYV,GEM, MEM) grown in different Turkish regions for two harvest years were carried out. A total of14 phenolic compounds were identified and quantified in the phenolic extracts of these cultivars.Elenolic acid was found as the most abundant phenolic compound in all cultivars and in both yearsfollowed by tyrosol, hydroxytyrosol, and luteolin depending on the year. The high positiverelationship was observed between the phenolic compounds in the oils. Among the phenoliccompounds, syringic acid had the highest correlation. On the other hand, 13 FAs were identified.Among these oleic acid was the highest and myristic acid was the lowest concentration in all oilsamples. In both years, the highest amount of oleic acid was found in GEM oil and followed byMEM and AYV. As a result, the difference in the total amount of phenolic compounds and FAs inoils brought out the influence of variety (genetic factors), crop season and environmental factors.

FUNDING

The authors thank the Scientific and Technical Research Council of Turkey (TUBITAK) forfinancial support for this research project (Project No. 110O602).

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