Comprehensive Analysis of Polyphenols in 55 Extra Virgin Olive Oils by HPLC-ECD and Their...

ORIGINAL PAPER

Comprehensive Analysis of Polyphenols in 55 Extra Virgin OliveOils by HPLC-ECD and Their Correlation with AntioxidantActivities

Banu Bayram & Tuba Esatbeyoglu & Nicole Schulze &

Beraat Ozcelik & Jan Frank & Gerald Rimbach

Published online: 16 October 2012# Springer Science+Business Media New York 2012

Abstract In this study, we analyzed eight phenolic com-pounds (tyrosol, hydroxytyrosol, oleuropein, pinoresinol,and caffeic, ferulic, vanillic, and p-coumaric acid) in 55mono- and multivarietal extra virgin olive oil samples by highperformance liquid chromatography (HPLC) coupled to acoulometric electrochemical array detector (ECD). The phe-nolic profile of olive oil samples differed depending on thegeographical origin and olive variety. The total reducing ca-pacity (total phenolics) of olive oils ranged from about 40 to530mg gallic acid equivalents/kg oil. Tyrosol, hydroxytyrosoland pinoresinol were the most abundant phenolic compoundsin olive oils. The antioxidant capacity of the olive oil extractswas determined by ferric reducing antioxidant power (FRAP)and Trolox equivalent antioxidant capacity (TEAC) assays.Total reducing capacity was significatly correlated with FRAP

(R200.91, p<0.001) and TEAC (R200.92, p<0.001) values.Total reducing capacity, TEAC and FRAP values were signif-icantly correlated with tyrosol, hydroxytyrosol as well asoleuropein concentrations. Hydroxytyrosol, comprising over40 % of total olive oil phenolics, mainly contributed to theantioxidant activity of olive oils. The present study provides acomprehensive database of polyphenols in olive oils from 9different countries and four continents.

Keywords Olive oil . Phenolic compounds . Antioxidantactivity . Electrochemical detector

Introductıon

Olive oil is the primary source of fat in the Mediterraneandiet. Several studies suggest that olive oil may exhibitpotential antioxidant [1], antiinflammatory [2], antiathero-genic [3], anticancer [4], antidiabetic [5], and neuroprotec-tive activities [6]. The beneficial health effects of olive oilhave been partly attributed to its phenolic content. In fact,we have recently shown that olive oil polyphenols reduceoxidative stress in aged mice by induction of Nrf2 depen-dent gene expression [7]. Furthermore, phenolic compoundscontribute to the quality, stability and organoleptic proper-ties of olive oil [8–10]. Since health benefits as well asquality of olive oil may be attributed to its content ofphenolics, a comprehensive analysis of polyphenols in com-mercially available olive oil is warranted.

Olive oil contains different classes of phenolic com-pounds such as phenolic acids (vanillic, p-coumaric,ferulic, caffeic acid), phenolic alcohols (tyrosol, hydroxy-tyrosol), flavonoids (apigenin, luteolin), secoiridoids(oleuropein) and lignans (pinoresinol, acetoxypinoresinol)[11]. Awide variety of analytical techniques have been used

B. Bayram : T. Esatbeyoglu :G. Rimbach (*)Institute of Human Nutrition and Food Science,Christian-Albrechts-University,Hermann Rodewald-Strasse 6,24098 Kiel, Germanye-mail: [email protected]

N. SchulzeInstitute of Food Chemistry,Technical University of Braunschweig,Schleinitzstrasse 20,38106 Braunschweig, Germany

B. Bayram : B. OzcelikDepartment of Food Engineering, Istanbul Technical University,34469 Maslak,Istanbul, Turkey

J. FrankInstitute of Biological Chemistry and Nutrition,University of Hohenheim,Garbenstrasse 30,70599 Stuttgart, Germany

Plant Foods Hum Nutr (2012) 67:326–336DOI 10.1007/s11130-012-0315-z

to characterize the phenolics present in olive oils. Highperformance liquid chromatography (HPLC) coupled withultraviolet (UV) detection [12], diode array detection (DAD)[13], coulometric electrochemical detection (ECD) [14],mass spectrometry (MS) [15], as well as gas chromatogra-phy (GC) coupled to MS [16], capillary electrophoresis(CE) [17], nuclear magnetic resonance (NMR) spectroscopy[18] and infrared (IR) spectroscopy [19] have been used forthe quantification of phenolics in olive oil. Among thesetechniques, UV, DAD and MS represent the most com-mon detection methods. ECD, due to its higher sensitiv-ity than UV and DAD and comparable selectivity to MS,can be used to analyze phenolic compounds that mayoccur only in trace amounts in biological samples. Inrecent years, ECD has been established as a powerfulanalytical tool providing outstanding selectivity and sensi-tivity [20] and was useful for detecting trace amounts ofelectroactive compounds, including phenolics, in a wide rangeof samples [21, 22]. Furthermore, the International Olive OilCouncil (IOOC) has published standardized methods (colori-metric and HPLC-UV) for the quantification of phenolics inolive oil [23, 24].

In this study, we analyzed eight important olive oil phe-nolics, namely tyrosol, hydroxytyrosol, oleuropein, pinore-sinol, caffeic, ferulic, vanillic, and p-coumaric acid, in 55different olive oils by HPLC-ECD. The olive oil sampleswere collected from producers and supermarkets and repre-sent different geographical regions and olive varieties. Wedid not conduct a systematic sampling. Extra virgin oliveoils were mainly obtained from European (48 samples), butalso Australian (3), American (3) and South African (1)producers. We calculated multiple correlations between theconcentrations of total or individual phenolics and the anti-oxidant activities of the olive oils.

Materials and Methods

Chemicals and Reagents

HPLC grade methanol and acetonitrile were obtainedfrom J.T. Baker (Deventer, The Netherlands) and SigmaAldrich (Steinheim, Germany), respectively. Gallic acid,Trolox, Folin-Ciocalteu reagent, and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) were from Fluka (Schnelldorf, Ger-many). Reference standards of tyrosol, phenolic acids(vanillic, caffeic, p-coumaric, ferulic) and 2,2′-azino-bis-(3-ethylbenzothiazolin-6-sulfonate) (ABTS) were pur-chased from Sigma Aldrich (Schnelldorf, Germany).Oleuropein and hydroxytyrosol were from Extrasynthese(Genay Cedex, France) and pinoresinol from SeparationResearch (Turku, Finland). All standards were of HPLCgrade with purities higher than 98 % except oleuropein

(>90 %). Perchloric acid (60 %) was obtained fromFisher Scientific (Leicestershire, UK).

Olive Oil Samples

Fiftyfive mono- or multivarietal extra virgin olive oils fromItaly, Spain, France, Turkey, Portugal, Australia and SouthAfrica were directly obtained from the producers or super-markets belonging to the harvest year of 2007/2008. Char-acteristics (geographical regions and olive varieties) of theolive oil samples are summerized in Table 1. All oil sampleswere kept at 4 °C under a nitrogen atmosphere untilanalysis.

Preparation of Stock and Standard Solutions

Stock solutions of olive oil phenolics were prepared inmethanol at a concentration of 2 mg/ml and stored at−20 °C in amber-coloured bottles. The standard solutionswere prepared by serial dilution of stock solutions withwater/methanol (50:50, v/v).

Extraction of Phenolic Compounds from Olive Oil

The phenolic fraction of the olive oils was obtained bysolid-phase extraction (SPE) using diol-bonded SPE car-tridges (Varian, Bond Elut Diol 500 mg/3 ml) accordingto a protocol modified from Mateos et al. [25]. Briefly,1.5 g olive oil was mixed with 1.5 ml n-hexane and appliedto the SPE cartridge. The phenolics were eluted with 10 mLmethanol and the eluate was evaporated in a centrifugalevaporator (Jouan RC, Saint Herblain, France) at 35 °C.The phenolic residue was dissolved in 1 ml methanol,filtered, and directly injected into the HPLC system orstored at −20 °C.

Spectrophotometric Determination of Total ReducingCapacity

Total reducing capacity (total phenolics) of the oils wasdetermined according to the Folin-Ciocalteu spectropho-tometric method [26]. Diluted Folin-Ciocalteu reagent(1:2, v/v) and 20 % sodium carbonate solution wereadded to the phenolic extract. The solution was left inthe dark for 45 min. The absorbances of each sampleand of gallic acid standards were determined at 750 nmand results were expressed as mg gallic acid/kg oil.

Antioxidant Capacity of Olive Oil Extracts

The antioxidant capacities of olive oils were determinedwith TEAC and FRAP methods. The FRAP assay wasconducted according to Benzie and Strain [27], using

Plant Foods Hum Nutr (2012) 67:326–336 327

Table 1 Sample ID, geographi-cal information, and olivevarieties of the 55 olive oilsamples

Sample Country Geographical origin Variety

1 Italy Bari Coratina

2 Italy Bari Coratina

3 Italy Sicily Tonda Iblea

4 Italy Sicily Nocellara, Biancolilla, Ogghiaredda

5 Italy Puglia Coratina

6 Italy Sicily Cerasuola, Nocellara, Biancolilla

7 Italy Loreto Aprutino Dritta, Frantoio, Leccino

8 Italy Arezzo Morellino, Leccino, Frantoio

9 Italy Arezzo Ogliarola

10 Italy Florence not available

11 Spain Jaén Picual




15 Spain Jaén Arbequina

16 Spain Jaén, Toledo Arbequina, Picual, Hojiblanca, Cornicabra

17 Spain Jaén and Málaga Picual, Arbequina, Hojiblanca, Frantoio

18 Spain Granada Leccino

19 Spain Arbeca (Lerida) Arbequina

20 Spain Mecina (Cordoba) Hojiblanca

21 Spain Montes De Toledo Cornicabra

22 Spain Córdoba Picual, Picudo, Arbequina, Hojiblanca

23 Turkey Zeytindag Edremit

24 Turkey Canakkale Edremit

25 Turkey Ayvalık Ayvalık26 Turkey Ezine Ayvalık27 Turkey Ayvalık Ayvalık28 Turkey Aydın Memecik

29 Turkey Hatay Haşebi30 Turkey Hatay Halhalı31 Turkey Hatay Saurani

32 Turkey Hatay Halhalı, Haşebi, Saurani33 Turkey Gokceada Kroneiki

34 Turkey Gokceada Kroneiki

35 Greece Lakonia Kroneiki, Athinoleia

36 Greece Crete Kroneiki

37 Greece Mani Kroneiki

38 Greece Kalamata Kroneiki




42 France Ollioules Cayon

43 France Ollioules Cayon, Brun, Boutellion, Aglandau

44 France Ollioules Brun

45 Portugal Tras Os Mentes Cobrançosa, Verdeal Trasmontana, Madural

46 Portugal Alentejo Arbequina, Cobrançosa

47 Portugal Alentejo Arbequina, Cobrançosa

48 Portugal Povoa de Santarem Galega, Cobrançosa

49 Australia Frankland River Frantoio

328 Plant Foods Hum Nutr (2012) 67:326–336

ascorbic acid as reference. The TEAC assay was conductedas described by Re et al. [28] using Trolox as reference.

HPLC Analysis of Olive Oil Phenolics

HPLC analyses of olive oil phenolics were carried out on aJasco system (Jasco, Gross-Umstadt, Germany; pumpPU2085, autosampler XLC-3059AS), while detection wascarried out on a 4-channel ESA 5600A CoulArray detectorwith integrated column oven (ESA Inc., Chelmsford, MA,USA) with potentials set to +250, +400, +500 and +750 mV.Separation of phenolics was achieved on a Kinetex C18column (100 × 4.6 mm, 2.6μm, Phenomenex, Aschaffenburg,Germany) using gradient elution with water (pH 3.1 withperchloric acid, solvent A), acetonitrile (solvent B), and meth-anol (solvent C) each containing 60 mM LiCIO4. The follow-ing gradient was used: from 96 % A - 3 % B - 1 % C to 85 %A - 10 % B - 5 % C in 4 min; 82.5 % A - 11.5 % B - 6 % C in2 min; 80 % A - 12 % B - 8 % C in 2 min; 65 % A - 24 % B -8 % C in 7 min; 85 % B - 15 % C in 6 min; 85 % B - 15 % Cfor 5 min; 96 % A - 3 % B - 1 % C to 85 % A in 4 min and5 min of maintenance. The column temperature was main-tained at 40 °C, the flow rate set at 1.25 ml/min, and theinjection volume was 10 μl. The analytes were quantifiedagainst authentic compounds as external standards. The lowerlimit of detection (LLD) and lower limit of quantification(LLQ) ranges of the method for eight phenolics were 0.03–1.7 and 0.3–15 ng/ml, respectively.

α-Tocopherol Analysis

Ten mg olive oil was placed in a glass tube. Two ml ethanol(EtOH) containing 1 % ascorbic acid (w/v), 700 μl H2Oand 300 μl of saturated potassium hydroxide solution wereadded, vortexed and incubated for 30 min at 70 °C in ashaking water bath. Thereafter samples were immediatelychilled on ice, 50 μl butylated hydroxytoluene (BHT)-solution (0.1 % BHT in EtOH, w/v) and 2 ml n-hexane wereadded. Glass tubes were mixed and centrifuged 5 minat 1,500 rpm. Five-hundred μl of the supernatant was driedat room temperature in a centrifugal evaporator (Jouan,Saint Herblain, France). Samples were reconstituted with150 μl mobile phase (MeOH:H2O; 98:2, v/v) and stored at

−20 °C until analysis. Fourty μl of the sample were injectedinto a HPLC system (Jasco, Gross-Umstadt, Germany;pump PU2080Plus, autosampler AS2057Plus, detectorFP2020Plus) and separated on a Waters Spherisorb ODS-2column (100 × 4.6 mm, 3 μm) by isocratic elution at a flowrate of 1.2 ml/min. The fluorescence detector was set to anexcitation wavelength of 290 nm and an emission wave-length of 325 nm. The concentrations of α-tocopherol werequantified using an external standard curve (Tocopherol set,Calbiochem®, Schwalbach, Germany).

Statistical Analysis

Results are expressed as mean values with standard devia-tion (SD). Statistical analysis was performed using PASWStatistics 18 (IBM, Chicago, IL). Correlation analysis be-tween phenolic compounds and antioxidant activity testswas performed using Spearman’s correlation coefficient.Differences were considered significant when the p valuewas <0.05. Data were also analyzed by principle componentanalysis (PCA) using XLSTAT software (Addinsoft, Version2011.1.01) to evaluate similarities and differences betweenolive oil samples and analyzed parameters (total reducingcapacity, phenolic profile, α-tocopherol content, antioxidantactivities).

Results and Disscussion

In this study, eight selected olive oil phenolics werequantified with a new rapid and sensitive HPLC-ECDmethod. Coulometric ECD detection provides high sensi-tivity and specificity for substances that are either oxi-dized or reduced at the applied potential. Because of itshigh selectivity, sensitivity, low detection limits and en-hanced resolving power [21, 29], HPLC-ECD has be-come increasingly popular for the analysis of plantbioactives and phenolic compounds [21, 22]. The totalanalysis time of published methods for olive oil phe-nolics are often longer than 40 min [17, 30]. In thepresent study, we used a fused-core column with2.6 μm particles that allowed analysis of selected oliveoil phenolics within 16 min (Fig. 1a). The concentrations

Table 1 (continued)

Sample Country Geographical origin Variety

50 Australia Kiewa Valley Leccino

51 Australia Frankland River Leccino

52 USA California Manzanillo, Mission

53 USA California Leccino

54 USA California Frantoio

55 South Africa Somerset West Frantoio, Leccino, Coratina

Table 1 (continued)


of eight selected phenolic compounds plus α-tocopherolwere determined in mono- and multivarietal olive oilsamples that were collected from different geographicalregions and olive cultivars. Apart from the intensivelystudied Italian (frantoio, leccino, coratina), Spanish (pic-ual, arbequina), Turkish (ayvalık, memecik) and Greek(kroneiki) olive varieties, olive oil samples from lessknown Turkish (saurani, halhalı) and French (cayon,brun) varieties were also analyzed. Furthermore, oliveoil samples from other olive-producing regions, namelyAustralia and South Africa, were included in theanalyses.

The FRAP and TEAC values, total reducing capacity, α-tocopherol, and phenolic concentrations of olive oil samplesare given in Table 2. Total reducing capacity in the analyzedoils ranged from ~40 to 530 mg/kg. The oil sample

produced from the Italian coratina variety possessed thehighest amount of phenolics and the sample producedfrom French cayon contained the lowest amount of totalreducing capacity. Total reducing capacity of the oilsdetermined with the Folin-Ciocalteu method moderatelycorrelated with the results obtained from HPLC-ECDanalyses (r00.55, p<0.001). Tyrosol, hydroxytyrosol andpinoresinol were the most abundant phenolic compounds inthe olive oil samples. The average percentage distribu-tion of olive oil phenolics in 55 olive oil samples isgiven in Fig. 1b. The concentrations of tyrosol, hydrox-ytyrosol and pinoresinol were between 3.6–38.4, 0.3–29.3,and 1.4–13.9 mg/kg oil, respectively. In the 55 oils studied,the concentration of phenolic acids did not exceed 1.7 mg/kgoil, which is in accordance with previously reported concen-trations in the range of 0.01–1.7 mg/kg [14, 31]. The antiox-idant activities of the olive oils were determined with FRAPand TEAC assays, respectively. The olive oils differed notablyin their total reducing capacity and this was reflected bydifferences in the corresponding FRAP and TEAC values.The olive oil with the highest amount of phenolics had 10-fold higher TEAC and FRAP values than the olive oil with thelowest amount of phenolics. Total reducing capacity signifi-cantly correlated with the FRAP (R200.91) and TEAC (R200.92) values. The correlation between total reducing capacityand individual phenolic compounds was also evaluated andSpearman’s correlation coefficients are reported in Table 3.Total reducing capacity moderately correlated with thehydroxytyrosol (r00.60, p<0.001), oleuropein (r00.51, p<0.001) and tyrosol (r00.41, p<0.01) concentrations of theolive oils. TEAC and FRAP values highly correlated withthe hydroxytyrosol and moderately with the oleuropein andtyrosol contents (Table 3). Hydroxytyrosol was found to havethe highest correlation coefficient with total reducing capacity,FRAP and TEAC values. In the literature, hydroxytyrosol isreported with higher antioxidant activity than tyrosol andoleuropein [32, 33]. The hydroxytyrosol content correlatedhighly with the tyrosol (r00.72, p<0.001) and moderatelywith the oleuropein (r00.33, p<0.05) content in the 55 oliveoils studied.

α-Tocopherol is another compound in olive oil that con-tributes to its antioxidant activity. Therefore, we analyzedthe α-tocopherol content of our olive oil samples and itscorrelation to total reducing capacity, individual phenoliccompounds and antioxidant activity assays. α-Tocopherolcontent of oils may be affected by many factors, such asolive variety, ripening, geographical region, agricultural andtechnological factors [34], and varied greatly between ourolive oil samples (38–330 mg/kg oil). No correlation wasobserved between the α-tocopherol concentrations andTEAC or FRAP values. However, a moderate correlationwas found between α-tocopherol and total reducing capac-ity (r00.34, p00.01). Vanillic acid was found to be the only

Fig. 1 Representative chromatogram of olive oil phenolics extractedfrom a representative olive oil sample and resolved by gradient elutionon a KinetexTM C18 fused-core column and detected by 4-channelcoulometric electrochemical detection. The retention times of the ana-lytes (in minutes) are given in brackets: 1, hydroxytyrosol (2.7); 2,tyrosol (4.1); 3, caffeic acid (5.2); 4, vanillic acid (5.4); 5, ferulic acid(7.4); 6, p-coumaric acid (8.4); 7, oleuropein (13.2); and 8, pinoresinol(15.3) (a) The average distribution (in %) of olive oil phenolics in the55 olive oil samples investigated (b)


Tab

le2

FRAPvalue,TEACvalue,totalredu

cing

capacity,α-tocoph

erol,andph

enolic

concentrations

of55

differentoliveoilsamples.Valuesareexpressedas

mean±SD

Sam

ple

TRC

TEAC

FRAP

α-Toc

TY

HTY

OP

Pino

CA

VA

FAp-COU

STP

130

0±5.9

0.57

±0.03

20.63

±0.00

920

7±2.8

13±0.07

9.3±0.07

1.43

±0.00

12.9±0.01

40.07

±0.01

60.16

±0.00

10.10

±0.00

60.09

±0.00

627

.3

2112±2.3

0.27

±0.01

60.32

±0.00

617

1±1.5

8.1±0.07

6.6±0.09

0.45

±0.00

16.0±0.00

10.08

±0.00

10.51

±0.00

30.20

±0.00

20.26

±0.00

422

.4

339

6±7.6

0.84

±0.02

70.87

±0.011

263±20

8.4±0.14

4.8±0.04

0.63

±0.00

13.5±0.00

10.08

±0.00

10.56

±0.00

10.23

±0.00

11.74

±0.02

820

.1

427

8±8.3

0.52

±0.02

70.57

±0.01

822

7±7.8

21±1.13

11±0.42

0.40

±0.00

23.2±0.09

20.07

±0.00

10.19

±0.00

40.13

±0.00

20.06

±0.00

638

.0

553

2±11

1.01

±0.13

91.28

±0.16

221

6±14

21±0.21

19±0.14

1.33

±0.00

13.6±0.01

40.56

±0.10

50.34

±0.00

10.09

±0.00

10.13

±0.00

245

.7

633

4±6.3

0.77

±0.03

20.89

±0.03

420

8±8.3

12±0.07

10±0.01

0.74

±.0.006

2.8±0.23

30.07

±0.00

10.20

±0.00

10.13

±0.00

10.06

±0.00

226

.1

728

6±6.5

0.62

±0.02

40.72

±0.02

025

6±1.5

24±0.42

23±0.92

1.03

±0.011

3.0±0.00

10.08

±0.00

10.25

±0.00

10.19

±0.00

10.09

±0.00

552

.5

814

5±2.7

0.37

±0.01

60.42

±0.00

519

4±4.9

31±0.92

29±0.42

0.49

±0.00

12.6±0.03

50.12

±0.00

30.30

±0.02

30.14

±0.00

10.12

±0.00

665

.4

917

9±2.7

0.40

±0.05

80.52

±0.03

519

9±11

17±0.21

19±0.21

2.14

±0.01

014

±0.06

40.08

±0.00

20.36

±0.00

40.18

±0.00

10.05

±0.00

152

.6

1023

7±6.0

0.51

±0.01

50.59

±0.01

925

2±9.5

31±0.99

27±0.35

0.67

±0.00

13.6±0.04

20.09

±0.00

10.34

±0.00

60.18

±0.00

10.19

±0.00

863

.8

1126

9±5.3

0.57

±0.011

0.59

±0.02

333

0±7.9

4.7±0.08

3.7±0.01

3.49

±0.00

75.2±0.04

20.07

±0.00

10.79

±0.01

00.16

±0.00

10.39

±0.00

618

.7

1215

7±6.6

0.39

±0.00

70.44

±0.01

014

7±6.6

8.7±0.42

8.2±0.16

0.58

±0.00

32.8±0.00

10.08

±0.00

10.31

±0.07

80.16

±0.00

10.17

±0.01

021

.7

1314

5±3.4

0.38

±0.01

80.41

±0.011

193±12

11±0.49

17±0.42

0.59

±0.00

42.4±0.03

50.08

±0.00

10.25

±0.00

20.17

±0.00

10.14

±0.00

732

.8

1419

4±8.7

0.35

±0.02

70.40

±0.00

725

6±3.3

29±0.07

15±0.01

0.67

±0.00

13.8±0.00

10.09

±0.00

10.56

±0.00

60.24

±0.00

10.21

±0.00

150

.1

1512

1±3.4

0.29

±0.01

00.34

±0.00

220

2±0.3

29±1.06

23±0.49

0.42

±0.00

12.3±0.03

50.10

±0.00

10.23

±0.01

60.13

±0.00

10.11

±0.00

556

.7

1615

3±10

0.41

±0.01

00.47

±0.01

416

0±3.7

9.2±0.35

12±0.28

0.52

±0.00

23.1±0.05

70.08

±0.00

10.25

±0.00

40.13

±0.00

10.10

±0.00

626

.4

1726

8±7.5

0.49

±0.04

90.54

±0.02

421

7±10

27±1.20

16±0.57

1.53

±0.00

15.7±0.09

90.14

±0.05

80.44

±0.01

60.21

±0.00

10.24

±0.00

653

.6

1812

4±0.9

0.22

±0.01

60.28

±0.00

626

4±17

7.3±0.14

3.9±0.04

0.35

±0.00

13.5±0.02

10.08

±0.00

10.48

±0.00

80.16

±0.00

10.07

±0.00

316

.0

1915

9±13

0.40

±0.00

90.43

±0.02

917

3±11

9.6±0.42

11±0.24

0.87

±0.00

31.1±0.01

90.08

±0.00

10.16

±0.00

10.14

±0.00

10.06

±0.00

424

.0

2014

4±2.5

0.34

±0.00

40.37

±0.00

916

4±1.1

11±0.01

9.9±0.03

0.75

±0.00

11.3±0.02

10.08

±0.00

10.19

±0.00

10.16

±0.00

10.09

±0.00

123

.3

2125

4±2.5

0.60

±0.00

80.65

±0.011

172±4.9

8.1±0.14

8.2±0.07

1.03

±0.00

13.4±0.02

10.07

±0.00

10.20

±0.00

10.14

±0.00

10.05

±0.00

121

.4

2215

6±9.0

0.40

±0.02

70.47

±0.02

513

3±2.3

19±0.07

25±0.14

0.65

±0.00

33.3±0.00

10.09

±0.00

10.21

±0.00

10.14

±0.00

10.09

±0.00

149

.3

2310

1±1.1

0.23

±0.00

70.27

±0.00

618

1±7.0

4.6±0.04

3.5±0.01

0.73

±0.00

31.9±0.00

10.08

±0.00

60.16

±0.00

10.12

±0.00

30.01

±0.00

111.2

24114±5.7

0.25

±0.011

0.32

±0.00

428

1±8.5

8.6±0.35

3.6±0.07

0.35

±0.00

12.9±0.03

50.08

±0.00

10.20

±0.00

10.15

±0.00

10.50

±0.011

17.0

2591

±1.3

0.16

±0.01

00.20

±0.00

320

1±4.5

6.7±0.57

2.8±0.12

0.48

±0.00

12.2±0.07

80.07

±0.00

10.17

±0.00

30.13

±0.00

20.04

±0.00

313

.3

2613

0±5.8

0.28

±0.01

20.30

±0.00

815

4±2.5

3.1±0.07

3.4±0.04

0.51

±0.00

14.9±0.02

80.07

±0.00

10.26

±0.00

60.14

±0.00

10.29

±0.00

712

.8

2769

±1.1

0.18

±0.01

50.22

±0.00

918

8±1.0

4.5±0.07

3.8±0.03

0.48

±0.00

24.3±0.02

10.07

±0.00

10.15

±0.00

10.12

±0.00

10.08

±0.00

113

.6

2888

±0.9

0.19

±0.01

60.26

±0.00

213

8±2.1

7.9±0.28

2.7±0.05

0.42

±0.00

12.1±0.03

50.07

±0.00

10.20

±0.00

20.17

±0.00

30.63

±0.02

614

.5

29118±2.8

0.15

±0.02

00.16

±0.00

797

±2.6

20±1.56

1.1±0.01

0.68

±0.05

81.4±0.04

20.17

±0.08

00.20

±0.02

10.15

±0.011

0.16

±0.011

26.1

3022

4±6.9

0.53

±0.01

40.60

±0.03

926

5±10

22±0.28

26±0.07

0.72

±0.00

14.6±0.00

70.10

±0.01

80.16

±0.00

30.14

±0.00

10.12

±0.00

454

.0

3119

5±9.2

0.41

±0.01

60.46

±0.00

325

9±14

20±0.21

17±0.14

0.74

±0.00

14.4±0.03

50.08

±0.00

10.14

±0.00

20.15

±0.00

10.03

±0.00

243

.2

3212

6±11

0.28

±0.02

60.34

±0.011

120±0.2

20±0.07

6.8±0.01

0.57

±0.00

13.1±0.01

40.07

±0.00

10.20

±0.00

10.14

±0.00

10.10

±.000

130

.9

3310

4±3.8

0.27

±0.01

90.30

±0.00

715

2±5.1

7.1±0.01

7.7±0.09

0.71

±0.00

33.5±0.00

10.08

±0.00

30.16

±0.00

10.14

±0.00

20.14

±0.00

819

.6

3416

2±2.1

0.43

±0.01

80.46

±0.01

917

7±1.8

4.1±0.54

5.6±0.34

0.50

±0.00

25.3±0.29

70.08

±0.00

10.34

±0.05

60.14

±0.00

40.42

±0.05

717

.8

3516

3±6.9

0.35

±0.02

30.40

±0.00

925

6±2.2

7.4±0.99

4.6±0.29

0.57

±0.00

52.9±0.14

10.08

±0.00

50.34

±0.05

80.11

±0.00

1nd

17.6


Tab

le2

(con

tinued)

Sam

ple

TRC

TEAC

FRAP

α-Toc

TY

HTY

OP

Pino

CA

VA

FAp-COU

STP

3619

7±12

0.42

±0.00

80.54

±0.01

823

2±9.7

13±0.14

8.9±0.05

0.43

±0.00

13.6±0.01

40.09

±0.00

40.41

±0.00

10.13

±0.00

10.06

±0.00

226

.6

3716

6±3.0

0.37

±0.02

90.43

±0.00

419

2±21

38±0.49

25±0.14

0.58

±0.00

23.9±0.01

40.09

±0.00

30.37

±0.00

40.17

±0.00

10.06

±0.00

269

.6

38116±1.9

0.25

±0.00

20.30

±0.00

724

0±9.3

6.0±0.16

3.5±0.04

0.43

±0.00

33.1±0.02

10.09

±0.01

00.20

±0.00

30.12

±0.00

10.00

±0.00

313

.7

3914

7±1.8

0.32

±0.03

70.39

±0.01

620

2±6.8

12±0.28

6.3±0.28

0.52

±0.00

43.9±0.00

10.08

±0.00

10.61

±0.00

20.16

±0.00

10.08

±0.00

124

.3

4019

1±3.9

0.44

±0.01

40.48

±0.00

121

8±0.6

16±0.01

12±0.01

0.53

±0.00

23.1±0.00

70.07

±0.00

10.27

±0.00

40.12

±0.00

10.02

±0.00

331

.4

4115

6±1.8

0.36

±0.01

20.40

±0.00

323

9±6.4

15±0.78

13±0.42

0.46

±0.00

23.8±0.07

80.08

±0.00

10.37

±0.05

30.16

±0.00

10.08

±0.00

434

.4

4245

±3.2

0.11

±0.01

30.18

±0.00

419

7±15

7.0±0.05

0.5±0.01

1.19

±0.03

31.9±0.02

40.91

±0.03

20.15

±0.011

0.08

±0.02

50.19

±0.00

112

.0

4383

±2.6

0.18

±0.01

40.22

±0.00

520

9±1.0

12±0.42

3.0±0.04

0.40

±0.00

21.5±0.01

60.09

±0.00

10.46

±0.00

50.16

±0.00

80.30

±0.00

918

.1

4410

7±1.3

0.20

±0.02

20.24

±0.00

816

7±6.7

16±0.49

6.7±0.14

2.64

±0.54

62.2±0.02

10.70

±0.03

50.30

±0.06

70.19

±0.07

60.42

±0.03

531

.5

4515

3±3.5

0.33

±0.02

70.35

±0.00

521

5±6.1

14±0.14

9.6±0.04

0.66

±0.00

22.2±0.01

40.08

±0.00

10.34

±0.00

20.16

±0.00

10.09

±0.00

326

.8

4612

2±3.3

0.33

±0.01

20.43

±0.01

420

9±4.4

4.0±0.11

4.6±0.10

0.41

±0.00

14.6±0.00

70.09

±0.00

10.60

±0.00

30.18

±0.00

20.16

±0.00

214

.8

4710

0±0.4

0.27

±0.01

40.36

±0.00

318

5±11

4.1±0.16

4.1±0.04

0.38

±0.00

14.5±0.05

70.08

±0.00

10.62

±0.01

30.19

±0.00

20.16

±0.00

814

.4

4813

7±2.4

0.33

±0.02

70.42

±0.00

824

0±17

11±0.28

12±0.14

0.42

±0.00

111

±0.12

00.08

±0.00

10.39

±0.01

60.19

±0.00

30.30

±0.00

635

.6

4926

5±8.2

0.68

±0.03

30.79

±0.02

613

4±6.6

11±0.14

18±0.07

0.92

±0.00

14.0±0.02

10.11

±0.00

70.36

±0.00

10.16

±0.00

10.07

±0.00

335

.0

5012

8±1.4

0.19

±0.01

60.24

±0.01

228

9±9.3

24±0.21

3.7±0.01

0.40

±0.00

14.2±0.02

10.09

±0.00

11.45

±0.00

70.24

±0.00

10.13

±0.00

134

.0

5120

5±0.7

0.50

±0.00

30.56

±0.02

328

0±4.7

4.3±0.30

4.1±0.10

0.70

±0.01

26.6±0.18

40.17

±0.07

90.39

±0.03

50.15

±0.00

80.01

±0.00

117

.3

5230

6±4.1

0.78

±0.02

60.83

±0.02

118

1±0.3

17±0.49

25±0.49

0.75

±0.02

91.4±0.011

0.13

±0.01

80.21

±0.05

30.25

±0.00

80.43

±0.00

646

.8

5317

7±3.7

0.45

±0.02

10.52

±0.02

012

0±3.4

5.4±0.19

6.6±0.07

1.47

±0.00

67.5±0.09

20.07

±0.00

10.17

±0.00

10.12

±0.00

1nd

21.6

5410

9±2.5

0.14

±0.02

90.22

±0.00

338

±0.2

3.6±0.05

0.2±0.04

0.49

±0.00

34.3±0.03

50.07

±0.00

10.26

±0.01

30.13

±0.00

1nd

9.2

5522

8±5.6

0.50

±0.02

80.61

±0.03

020

9±2.0

18±0.49

22±0.49

0.75

±0.00

24.7±0.07

10.09

±0.00

60.25

±0.00

30.23

±0.00

10.09

±0.00

147

.3

TRCtotalreducingcapacity,α

-Toc

α-tocop

herol,TYtyrosol,HTYhy

drox

ytyrosol,O

Poleuropein,P

inopino

resino

l,CAcaffeicacid,V

Avanillicacid,FAferulic

acid,p

-COUp-coum

aricacid,S

TP

selected

totalph

enolics,nd

notdetected.Valuesareexpressedas

mg/kg

oil


olive oil phenolic that significantly correlated with theα-tocopherol content (r00.34, p<0.05). It is worth men-tioning that the daily consumption of about 50 ml ofthe olive oil with the highest α-tocopherol concentrationmay be sufficient to meet the dietary recommendationsfor vitamin E [35]. Thus, olive oil may be an importantsource of dietary vitamin E especially in the Mediterra-nean diet.

PCAwas performed on the following datasets from all oilsamples: total reducing capacity, phenolic profile, α-tocopherol content and antioxidant activity. Scores and load-ings of PCA are given in Fig. 2. The first three principlecomponents (PC1, PC2, PC3) accounted for 44.6, 18.4 and13.9 % of the total variance, respectively (76.8 % of the totalvariation). PC1 is mainly explained by the TEAC, FRAP,and total reducing capacity values. PC2 represents the dif-ferences in tyrosol, and hydroxytyrosol and PC3 the differ-ences in pinoresinol, oleuropein, and α-tocopherolconcentrations of the oils. Comparison of PC1 and PC2(62.9 % of total variation) showed clear differences betweensamples, especially from Italy and Spain, indicating sub-stantial variability between TEAC, FRAP, and total reduc-ing capacity values as well as tyrosol, and hydroxytyrosolconcentrations (Fig. 2a, part II&III). Italian olive oil samplesexhibited higher TEAC, FRAP, and total reducing capacityvalues than the samples from other countries (except sample2 and 8, Fig. 2a). A few samples from Spain (samples 17and 21), South Africa (sample 55), USA (sample 52), andTurkey (sample 30) also had high TEAC, FRAP, and totalreducing capacity values. For the other samples, no cleardistribution was detected with regard to the countries oforigin. As far as TEAC, FRAP, and total reducing capacityare concerned, similar tendency was observed for PC1 andPC3 (58.5 % of total variation; Fig. 2b). Large differences inthe analyzed parameters were evident in some samples

despite the fact that they were from the same geographicalorigin and/or belong to the same olive variety (samples 3, 4,6, and 36–38). Overall, there was no correlation between theanalyzed samples as a function of country, geographicalorigin, or olive variety. Comparison of PC2 and PC3(32.2 % of total variation) revealed no distinct pattern andall observations positioned around the center, indicating thatPC2 and PC3 did not distinguish between olive oil samplesfrom different countries based on the analyzed variables(Fig. 2c).

Conclusion

In this study, the characterization of eight phenolic com-pounds (tyrosol, hydroxytyrosol, oleuropein, pinoresinol,and caffeic, ferulic, vanillic, and p-coumaric acid) wascarried out with HPLC-ECD in 55 mono- and multivar-ietal extra virgin olive oil samples. The phenolic compo-sition and the content of olive oil phenolics, as observedin the present study, may depend on different factorssuch as olive variety, fruit ripeness, climatic conditions,cultivar, agronomic and technological factors [36, 37]. Inthis study, 55 olive oil samples produced from differentolive varieties were collected from 9 countries on fourcontinents. There is no available information for the otherabove mentioned factors that could affect the phenoliccontent of our olive oils. Furthermore, we did not con-duct a systematic sampling, thus the present data mayprovide a rather general view of phenolic profiles ofanalyzed olive oils and olive varieties. Depending onthe phenolic content, the antioxidant activity of the oilextracts showed distinct differences between oils. Signif-icant correlations were found between antioxidant activi-ty, total reducing capacity and individual phenolics of the

Table 3 Spearman’s rank cor-relation coefficients and p-valuesfor correlations between totalreducing capacity, α-tocopherol,and antioxidant assays in55 olive oils

FRAP Ferric reducing antioxi-dant power, TEAC Troloxequivalent antioxidant capacity

Spearman’s coefficients P value

Total reducing capacity - Hydroxytyrosol 0.60 <0.001

Total reducing capacity - FRAP 0.94 <0.001

Total reducing capacity - Oleuropein 0.51 <0.001

Total reducing capacity - TEAC 0.95 <0.001

Total reducing capacity - Tyrosol 0.41 0.002

Total reducing capacity - α-tocopherol 0.34 0.010

FRAP - Hydroxytyrosol 0.65 <0.001

FRAP - Oleuropein 0.47 <0.001

FRAP - Tyrosol 0.30 0.024

TEAC - FRAP 0.99 <0.001

TEAC - Hydroxytyrosol 0.65 <0.001

TEAC - Tyrosol 0.31 0.020

TEAC - Oleuropein 0.51 <0.001


olive oils. According to our correlation analysis, hydroxytyr-osol appears to be one of the main contributors to the anti-oxidant activity of olive oils. The presented data will beuseful for nutritionists and epidemiologists to estimate poly-phenol intake from olive oil in human populations. Our study

may also provide a database for food scientists, food tech-nologists, and the food industry to develop polyphenol-rich(functional) foods containing olive oils. The results couldalso be used to complement databases, such as phenol-explorer (www.phenol-explorer.eu) [38].

Fig. 2 Principle componentanalysis (scores and loadings)of total reducing capacity(TRC), phenolic profile(tyrosol, TY; hydroxytyrosol,HTY; oleuropein, OP;pinoresinol, Pino, α-tocopherol, α-Toc), antioxidantactivity (ferric reducingantioxidant power, FRAP;Trolox equivalent antioxidantcapacity, TEAC) and α-Toc ofthe 55 extra virgin olive oilsamples. PC1 and PC2 (a), PC1and PC3 (b) PC2 and PC3 (c)


http://www.phenol-explorer.eu

Acknowledgement BB is supported by TUBITAK (The Scientificand Technological Research Council of Turkey).

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