Oxidative stability of oils containing olive leaf extracts obtained by pressure, supercritical and...

Research Article

Oxidative stability of oils containing olive leaf extractsobtained by pressure, supercritical and solvent-extraction

Paula Jimenez1, Lilia Masson1, Andres Barriga2, Jorge Chavez3 and Paz Robert1

1 Depto. Ciencia de los Alimentos y Tecnologıa Quımica, Facultad de Ciencias Quımicas y Farmaceuticas,

Universidad de Chile, Santiago, Chile2 Unidad de Espectrometrıa de Masas, Facultad de Ciencias Quımicas y Farmaceuticas, Universidad de

Chile, Santiago, Chile3 Depto. Tecnologıa Farmaceutica, Facultad de Ciencias Quımicas y Farmaceuticas, Universidad de Chile,

Santiago, Chile

The effect of the addition of olive leaf (Olea europaea, cv. Arbequina) extracts, i.e. hydroalcoholic

(ethanol–water 1:1; OHE), juice (OJ) and supercritical fluid-CO2 (OSFE) on the oxidative stability

of vegetable oils with different unsaturation, such as soybean oil (SBO), canola oil (CO) and high oleic

sunflower oil (HOSO), were studied at two concentrations (250 and 630 mg/kg oil, expressed as caffeic

acid equivalent (CAE)). The extracts were characterized by the total phenolic content (Folin–Ciocalteau

method), phenol chromatographic profiles (LC-MS) and antioxidant activity (DPPH). OHE showed

the highest phenol content (7.7 mg CAE/mL) while OJ and OSFE showed values of 5.4 and 2.2 mg

CAE/mL, respectively. Oleuropein and its derivatives were the major phenolic compounds identified

in OHE. The addition of 630 mg CAE/kg oil of OHE and OSFE to HOSO, SBO and CO showed an

antioxidant effect, increasing significantly the induction time (IT) (p<0.05). That effect was highest when

the system was more monounsaturated. In contrast, OJ showed a pro-oxidant effect for all oils systems for

both concentration studied. This behaviour could be attributed to the diphenol oxidase (PPO) activity.

Keywords: Olive leaves / Oxidative stability / Polyphenols / Supercritical extraction / Unsaturated oils

Received: August 24, 2010 / Revised: October 3, 2010 / Accepted: October 31, 2010

DOI: 10.1002/ejlt.201000445

1 Introduction

The leaves of the olive tree O. europaea have been widely used

for centuries in ancient cultures in folk medicine [1] as

infusion with therapeutic properties [2]. The geographical

location and olive leaf varieties have influence on the phenolic

composition [3]. According to literature, oleuropein is one of

the major phenolic compounds (19% w/w) occuring in the

leaves [3–12]. However, other compounds have been

reported too, such as verbascoside [6–9, 12], oleuroside [6,

9], ligstrosides [12, 13] and flavonoids glycosides including

luteolin-7-glucoside, apigenin-7-glucoside, rutin (quercetin-

3-rutinoside) [6–9] and less frequently apigenin-7-rutinoside,

luteolin-7-rutinoside and luteolin-4-glucoside [14]. Some

flavonoids aglicones as apigenin, quercetin, kaempferol,

hesperitin [5], luteolin [7, 14] and ferulic, caffeic, p-coumaric,

chlorogenic and vanillic acids [7, 10, 11, 14] among other

have been reported as well. This interesting polyphenol

profile suggests a potential use as a source of natural anti-

oxidants, thus increasing the economic value of the olive

leaf [3].

Different extracts preparation methods from olive leaves

including pressing of leaves [16], supercritical-CO2 extrac-

tion [17], liquid–liquid extraction [8] and solvent extraction

[10, 11, 18, 19] have been reported. This extracts could have

different content and profile of polyphenols [20].

It is well known that the oil oxidation is one of the major

deleterious reactions that occur when the oils are submitted

to high temperature (as frying), leading to a loss of chemical

and nutritional qualities [19]. Therefore, natural and syn-

thetic antioxidants can be used in the protection against lipid

Correspondence: Dr. Paz Robert, Facultad de Ciencia Quımicas y

Farmaceuticas, Departamento de Ciencia de los Alimentos y Tecnologıa

Quımica, Universidad de Chile, Casilla 133, Santiago, Chile,

Fax: 56-02-2227900

E-mail: [email protected]

Abbreviations: AT, alpha-tocopherol; CAE, caffeic acid equivalent; CO,

canola oil;HOSO, high oleic sunflower oil; IT, induction time;PF, protection

factor; PPO, diphenol oxidase; OHE, hydroalcoholic (ethanol–water, 1:1);

OJ, olive juice; OSFE, supercritical fluid extract; SBO, soybean oil

Eur. J. Lipid Sci. Technol. 2011, 113, 497–505 497

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

oxidation. However, synthetic antioxidants are being ques-

tioned by their possible adverse effects on human health [19],

while natural antioxidants such as phenolic compounds seem

to be a safer choice. Polyphenols act as antioxidant by donat-

ing a hydrogen atom to a peroxyl radical. The phenoxyl

radical formed is stabilized by resonance and also can react

with other free radicals [21].

The relationship between the flavonoids structural feature

and antioxidant activity has been well studied in hydrophilic

matrices but less in bulk oils. The number of phenolic

hydroxyl groups, 2,3 double bond in the C-ring and a gly-

coside moiety in the molecule was found as the relevant factor

that influence on the antioxidant activity in methyl linoleate

[22] and purified sunflower oil [23]. On the other hand a

synergistic effect antioxidant between flavonoids and alpha-

tocopherol (AT) has been reported [22, 23].

The aim of this work was to study the effect of three types

of olive leaf extracts (O. europaea, cv. Arbequina), i.e. hydro-

alcoholic (ethanolic–water 1:1;OHE), juice (OJ) and super-

critical fluid (OSFE), on the oxidative stability of vegetable

oils with different unsaturation grades (soybean oil (SBO),

canola oil (CO) and high oleic sunflower oil (HOSO)).

2 Materials and methods

2.1 Materials

Olive leaves (O. europaea) cv. Arbequina were collected from

120 trees, distributed in 29 hectares in November–

December 2007 from Millantu farm, Talagante, Santiago,

Chile. The sampling (31 kg) was carried out randomly taking

in each tree the leaves of a 1 year growing.

High oleic sunflower oil (Helianthus annus) (HOSO), CO

(Brassica sp) and SBO (Glycine max), were used without extra

antioxidant added and were supplied from Camilo Ferron

S.A., Santiago, Chile. The major fatty acids and the initial

characteristics of HOSO, CO and SBO are showed in

Table 1.

2.2 Samples and treatment

Three type of oil (HOSO, CO and SBO) were supplemented

with three type of olive leaf extracts (OHE, OJ or OSFE),

each one of them at two polyphenols concentration (250 and

630 mg CAE/kg oil). Thus, 21 experimental systems were

studied.

2.3 Olive leaf extracts preparation

2.3.1 Hydroalcoholic extract (OHE)

Olive leaves (7 kg) were scalded to 958C by 4.5 min and then,

they were cooled off quickly in a cold water spurt. Then, the

leaves were dried at 458C for 18 h in an air forced oven

(WTE, Germany). The dry leaves (8% moisture) were stored

in plastic bags at room temperature in dark condition until the

extract preparation. Dry olive leaves (760.39 g) were ground

and macerated in ethanol/water (50:50) (3 L) for 24 h at

room temperature and then the extract was separated by

filtration; this procedure was carried out twice with 2.5 L

of extracting agent. Extracts were combined and the volume

was reduced in a rotary evaporator (Buchi R-205,

Switzerland) at 408C until reaching a final volume of

1500 mL. The hydroalcoholic extract resulting was frozen

at �208C.

2.3.2 Juice by pressing (OJ)

Fresh olive leaves (9 kg) were washed manually, ground in a

food processor (Moulinex, D-56, Spain) and then pressed

by hydraulic laboratory press (Bertuzzi, Italy), as described

by Farag et al. [16]. The resultant juice (380 mL) was

concentrated in a rotary evaporator (Buchi R-205,

Switzerland) at 408C until 200 mL. The juice was frozen

at �208C.

2.3.3 Supercritical-CO2 extract (OSFE)

Fresh olive leaves (10 kg) were dried at room temperature

and in dark condition. The dry leaves (8% of moisture) were

ground in a hammer mill (Biber WienVII, Austria) and

sieved, the 30:50 mesh fraction was collected and stored in

plastic bags and frozen at �208C. Olive leaf powder (14.5 g)

was extracted by a supercritical extraction process using a

SPeed SFE system (Applied Separations model 7071, USA).

Table 1. Composition of major fatty acid and initial characteristic of

HOSO, CO and SBO

Fatty acid

HOSO

(% methyl

esters)

CO

(% methyl

esters)

SBO

(% methyl

esters)

C16:0 3.9 5.3 10.4

C18:0 3.5 2.4 4.6

C18:1 w9 81.8 56.3 20.3

C18:2 w6 7.3 21.6 51.6

C18: 3 w3 0.2 8.5 7.2

Fatty acid

HOSO

(mg/kg oil)

CO

(mg/kg oil)

SBO

(mg/kg oil)

a-Tocopherol 590 � 7.7 277 � 4.3 6 � 1.8

b-Tocopherol 23 � 1.7 nd 24 � 0.95

g-Tocopherol 6 � 0.5 130 � 3.3 238 � 7.8

d-Tocopherol nd 19 � 0.75 256 � 8.8

Total 619 426 524

Polyphenols

(mg CAE/g oil)

2.72 � 0.42 3.15 � 0.23 1.34 � 0.08

PV (mEqO2/kg oil) 2.2 1.5 0.9

nd, no detected; CAE, caffeic acid equivalent; PV, peroxide value.

498 P. Jimenez et al. Eur. J. Lipid Sci. Technol. 2011, 113, 497–505


The set up of extraction were: CO2 as carrier; 300 bar of

pressure, 408C of temperature and ethanol as cosolvent (5%

with flow rate of 0.3 mL/min). The extraction procedure was

made for 5 h at constant flow rate of CO2 (2.5 mL/min) with

a previous static period of 30 min.

2.4 Total polyphenol content

The total polyphenol content was determined by Folin–

Ciocalteau colorimetric method [24] and expressed as caffeic

acid equivalents (CAE) according to a calibration curve

(81.7–640.6 mg/mL, R2 ¼ 0.9901). All experiments were

performed in duplicate.

2.5 Measurement of the antioxidant activity

The antioxidant activity was carried out by DPPH method

[25], using a spectrophotometer (Unicam UV/Vis ATI

UNICAM, Cambridge, UK) at 517 nm. DPPH bleaching

activity was expressed as EC50.

2.6 Liquid chromatography with mass spectrometry(LC-MS)

Phenolic compounds were extracted from each sample by a

liquid–liquid extraction method according to Pena et al. [26].

Briefly, each olive leaf extracts (25 mL) was successively

extracted with ethyl ether (3 � 10 mL each one) and with

ethyl acetate (3 � 10 mL each one). The organic fractions

were combined, and then were evaporated to dryness in a

rotary evaporator (Buchi R-205, Switzerland) at 358C. The

residue was dissolved in 2 mL methanol–water (1:1 v/v),

filtered (0.45 mm filter Millipore filter) and analysed using

a LC-MS system. The chromatographic systems LC-MS

consisted on a HPLC (Agilent 1100, Agilent Technologies

Inc., CA, USA) connected through a split to an Esquire 4000

ion trap LC/MS system (Bruker Daltoniks, Germany). A C18

column (5 mm, 4.6 mm i.d. � 25 cm, Waters) was used; at

the exit of the column a splitter system divided the eluant in

two fraction one of them to an UV detector and the second one

to the mass spectrometer. A volume of 20 mL was injected.

The mobile phase was formic acid in water (0.5% v/v,

solvent A) and methanol/acetonitrile (50:50% v/v, solvent B)

at a flow rate of 1 mL/min according to the following elution

gradient: 0–8 min, 4% B; 8–48 min, 4–50% B; 48–53 min,

50–60% B; 53–68 min, 60–100% B; 68–78 min, 100% B;

78–80 min, 100–4% B; and 80–90 min, 4% B. The total

analysis time was 90 min, and 5 min was required for re-

establishing and equilibrating the initial conditions. Phenolic

compounds were detected at 280 nm. The mass spectral data

were acquired in positive and negative modes; ionization

(nebulization) was performed with nitrogen as drying gas

at 27.5 psi, 3508C and at a flow rate of 8 L/min and capillary

voltage 3000 V. All scans were performed in the range 50–

1400 m/z. The trap parameters were set in ion charge control

using manufacturer default parameters. Collision induced

dissociation (CID) was performed by collisions with the

helium background gas present in the trap. Fragmentation

was set with Smart Frag.

2.7 Preparation of oils (HOSO, SBO and CO) addedwith olive leaf extracts

The addition of OHE, OJ and OSFE to oil systems was

performed as follows: 0.11 mL, 0.14 mL and 0.33 mL,

respectively, for 250 mg CAE/kg oil; and 0.26 mL,

0.35 mL and 0.48 mL, respectively, for 630 mg CAE/kg

oil, being each mixed with 3 g HOSO or SBO or CO. To

improve the extracts dispersion in oils, Tween-80 was added

and each mixture was homogenized in an Ultraturrax (IKa,

Germany) at 9500 rpm for 2 min. Control samples (without

extracts) were prepared with 3 g of each oils. All experiments

were performed in triplicate.

2.8 Oxidative stability

Induction periods were determined by a Rancimat Oxidative

Stability Instrument (Metrohm Ltd., Herisau, Switzerland),

at 1108C and air flow of 20 L/h, according to AOCS (Cd 12b-

92, 1993).

2.9 Statistical analysis

To determine the statistical differences in the polyphenols

content, antioxidant activity and induction time (IT), a multi-

variate ANOVA was performed by using Statgraphics, Version

7.0 (Manugistic Inc., Statistical Graphics Corporation,

Rockville, MD).

3 Results and discussion

3.1 Characterization of olive leaf extracts

Table 2 shows the total polyphenols content and antioxidant

activity of olive leaf extracts (OHE, OJ and OSFE). The OHE

phenolic content was significantly higher (p<0.05) than OJ

and OSFE. The OJ and OSFE phenolic contents (0.07 and

0.46 mg CAE/g, respectively) were lower than those reported

by Farag et al. [16] for leaves cv. Kronakii (0.215 mg CAE/g)

and Le Floch et al. [17] (7.6 mg CAE/g leaf), respectively.

Salta et al. [11] and Chiou et al. [10] reported polyphenol

content of 17.7 mg CAE/mL (cv Kalamon) and 54 mg EAC/

mL, respectively, for extracts obtained using a sequence of

solvents. Differences in the total phenolic content of olive leaf

extracts between this study and literature could be related

with the leaves (cultivar, geographical location, collecting

period, age of leaves), previous treatment (dried, grinded

and scalded), extraction procedures (method and solvents

among other), as well as the final volume to which each

extract was concentrated.

Eur. J. Lipid Sci. Technol. 2011, 113, 497–505 Olive leaf extracts 499


The antioxidant activity (DPPH) of OHE, OJ and OSFE,

expressed as EC50 values were 0.026, 0.127 and 0.244 mg

CAE/mL, respectively. The differences observed between

olive leaf extracts in this study could be attributed to struc-

tural features of the polyphenols extracted for each extraction

method or other compounds present in the extract. The

structure–activity relationships of some flavonoids have been

well established [27].

The OSFE showed co-extraction of AT with a content of

671 mg/kg extract. Similarly, in hexane extracts from olive

leaves of different varieties has been reported AT content

between 41 and 125 mg/g extract [28].

3.2 Identification of polyphenols in olive leaf extracts

The identification by LC-MS was achieved by comparing m/z

signals and fragment ions of each polyphenol with those

described in literature as it is shown in Table 3 and Fig. 1

for the different extracts from olive leaves studied (OHE, OJ

and OSFE). Table 4 shows the phenolic composition profile,

expressed as a relative percentage.

3.2.1 Hydroalcoholic extract (OHE)

The main compounds identified were oleuropein (peak 23,

29.1%) and its derivatives, oleuroside (peak 26, 13.2%) and

oleuroside-10-carboxylic acid (peak 24, 8.2%). Oleuroside

(oleuropein isomer) was reported by Lee-Huang et al. [29]

previously. Other compounds identified were verbascoside,

hesperidin–rutin, luteolin-7-glucoside, among other.

Similarly, the oleuropein has been reported as a main phe-

nolic compound in olive leaves solvent extract [3, 4, 7–12].

It is known the effect of the solvent polarity on the poly-

phenols profile [30]. Works where ethanol or their aqueous

forms (10–90% v/v) have been used for polyphenols extrac-

tion from dried olive leaves were reported [7–8], showing a

comparable polyphenols profile with our study. However,

compounds as 3,4-DHPEA-EA (oleuropein aglycone), 3,4-

DHPEA-EDA (dialdehydic form of elenolic acid linked to

hydroxytyrosol) and 4-HPEA-EDA (dialdehydic form of ele-

nolic acid linked to tyrosol) were described at similar con-

ditions (dried leaves 388C for 18 h and ethanol) [18], but

these were not detected in this study.

In order to obtain olive leaf extracts, other solvents has

been reported: methanol [4], 60% aqueous methanol [9],

methanol/water (80:20) [12] and solvents sequence [10, 11]

which showed a similar polyphenols profile to ethanolic

extract. On the other hand, extract hydrolysis leads to differ-

ent profile, being hydroxityrosol the main component [31].

3.2.2 Supercritical extract (OSFE)

The major polyphenols identified in OSFE were p-coumaric

acid (peak 13, 11.2%), ferulic acid (peak 14, 8.7%), verbas-

coside (peak 15, 7.1%), oleuropein (peak 23, 6.7%), hydrox-

ytyrosol (peak 1, 6.2%) and vanillic acid, among other.

Besides, alpha tocopherol was co-extracted. A similar phe-

nolic composition was described by Le Floch et al. [17] in a

supercritical fluid extracts, using carbon dioxide modified

with methanol (10%), a similar pressure (334 bar) and higher

temperature (1008C) as a tentative polyphenols profile by

MS-screening (oleuropein, hydroxytyrosol, vanillic acid, ele-

nolic acid, ligstroside, tyrosol, hydroxybenzoic and cinnamic,

protocatechuic, caffeic and chlorogenic acids). Tabera et al.

[32] also described alpha tocopherol and other minor com-

pounds for countercurrent supercritical fluid extraction from

a hexane raw extract of olive leaves when ethanol (10%) was

used as a modifier of CO2.

In both OHE and OSFE, ethanol was used as solvent

however; the polyphenol profile was different, these results

can be due to pre-treatment of the olive leaves.

3.2.3 Olive juice (OJ)

The major polyphenols identified were verbascoside (peak

15, 15.7%), hydroxytyrosol peak 1, 7.3%) and luteolin-7-

glucoside (peak 19, 2.7%) (Including its four glycosidation

position isomers), in according with Obied et al. [33].

Similarly in extracts from fresh leaves verbascoside and luteo-

lin-7-glucoside were the main compounds [12]. Other phe-

nolic compounds as oleoside, elenolic acid, oleuropein,

apigenin-7-glucoside and ligstroside with smaller relative

importance also were identified.

The phenolic composition differ amongst different

extracts (OHE, OSFE and OJ), showing the effect of the

extraction method; solvent polarity and pre-treatment leaves

Table 2. Total polyphenols and antioxidant activity of different extracts from olive leaves

OHE OJ OSFE

Total polyphenols

mg CAE/mL extract 7.3 � 0.04 5.4 � 0.15 2.2 � 0.18

mg CAE/g fresh leaf 8.8 � 0.07 0.07 � 0.002 0.46 � 0.04

Yield (g of extract/g of dry leaves) 0.45 � 0.03 0.017 � 7 � 10�3 0.024 � 2.4 � 10�4

Antioxidant activity (DPPH)

EC50 (mg CAE/mL extract) 0.026 � 6.8 � 10�5 0.127 � 3.8 � 10�4 0.244 � 6.9 � 10�4

CAE, caffeic acid equivalent; OHE, hydroalcoholic extract; OJ, juice; OSFE, supercritical-CO2 extract.



Table 3. Identification of phenolic compounds of different extracts from olive leaves (OHE, OJ and OSFE) by LC-MS

RT

(min) Compound

Precursor

(m/z)

Fragments MS2 (m/z)

OHE OJ OSFE

16.5 Hydroxytyrosol 153 122.9 (100) 122.9 (100) 122.9 (100)

16.9 Vanillic acid 167 122.9 (100)

17.4 Hydroxytyrosol glycoside 315 152.9 (100) 135.0 (18) 123.0

(14) 101.0 (1)

19.3 Vanillic hexoside acid 329 166.9 (100) 151.9 (5) 108.0 (3)

123.0 (3)

22.7 Caffeic hexoside acid 341 178.9 (100) 161.0 (44) 135.1

(12) 202.9 (9)

23.3 Vanillin 151 122.9 (100)

25.4 Oleoside 389 345.0 (100) 208.9 (80) 164.9

(41) 120.9 (41)

345.0 (100) 120.9 (98) 208.9

(70) 138.9 (42)

27.5 Chlorogenic acid 354 190.9 (100) 192.8 (6) 178.8 (2)

28.0 Oleuropein aglycon 377 196.9 (100) 153.1 (19) 307.0 (100) 275.3 (91) 139.1

(12) 345.0 (7)

28.3 Pinoresinol 356 193.0 (100) 216.9 (61) 175.1

(39) 134.1 (10)

29.0 Caffeic acid 179 134.9 (100)

31.1 Elenolic acid 241 138.9 (100) 126.9 (100) 95.0

(90) 100.9 (43)

164.9 (100) 100.9 (95) 138.9

(19)

34.2 p-Coumaric acid 163 118.9 (100)

36.0 Ferulic acid 193 134.0 (100) 149.0 (83) 177.9

(80)

36.3 Verbacoside 623 461.1 (100) 315.1 (3) 477.1 (1)

443.2 (1)

461.2 (100) 477.0 (1) 315.2 (1)

579.2 (1)

461.1 (100)

36.6 Ligstroside aglycon decarboxymethyl 303 284.9 (100) 125.2 (8) 177.4 (4)

37.9 Luteolin-0-rutinoside 593 284.9 (100) 175.0 (1) 217.1 (1)

197.2 (1)

38.5 Acetoxypinoresinol 415 150.9 (100) 204.9 (82) 136.0

(79) 295.0 (69)

38.8 Luteolin-7-glucoside 448 285.0 (100) 327.0 (1) 285.0 (100) 326.9 (1) 285.0 (100) 326.9 (2)

39.0 Hesperidin þ rutin 609 461.3 (100) 300.9 (44) 342.9 (4)

271.0 (2)

39.2 Quercetin-3-0-galactoside þisoquercitin

463 300.9 (100) 179.2 (2) 151.0 (1)

342.9 (1)

40.5 Apigenin-7-rutinoside 577 268.9 (100) 531.3 (4) 195.3 (1)

40.9 Oleuropein 539 307.0 (100) 275.1 (91) 377.0

(52) 345.0 (8)

275.1 (100) 307.0 (98) 377.0

(57) 327.0 (9)

307.0 (100) 275.1 (95) 377.0

(56) 345.0 (9)

42.8 Oleuroside -10-carboxılic acid 275.1 (100) 307.0 (89) 377.0

(50) 327.0 (13)

43.5 Apigenin-7-glucoside 431 268.9 (100) 311.0 (2)

43.9 Oleuroside 539 307.0 (100) 275.0 (89) 377.0

(18) 327.0 (8)

275.1 (100) 307.0 (95) 377.0

(16) 437.1 (16)

307.0 (100) 275.1 (95) 377.0

(18) 371.0 (9)

44.6 Ligstroside 523 291.1 (100) 361.0 (81) 259.2

(49) 223.2 (2)

291.0 (100) 361.0 (74) 259.1

(68) 223.1 (6)

291.1 (100) 361.0 (79) 259.1

(43) 223.2 (4)

47.7 Luteolin-30-7-diglucoside 609 285.0 (100) 323.0 (3) 446.9 (3)

199.1 (1)

48.1 Oleuropein diglucoside 701 539.1 (100) 307.1 (50) 377.1

(45) 275.1 (37)

51.1 Luteolin-7-rutinoside 593 284.9 (100) 447.1 (3) 201.0 (2)

51.6 Oleuropein aglycon aldehyde 377 275.1 (100) 307.0 (100) 345.0

(9) 149.0 (7)

52.1 Quercetin 301 178.9 (100) 150.9 (53) 272.9

(12) 107.0 (8)

178.9 (100) 150.9 (78) 107.0

(14) 256.9 (10)

OHE, hydroalcoholic extract; OJ, juice; OSFE, supercritical-CO2 extract.



(dried, scalded, grinded and microware) as was described

previously [5, 6]. In the case of OJ and OHE where the leaves

were not submitted to scalded, the oleuropein and oleuroside

disminished and increase hydroxytyrosol, free phenols and

verbascoside. This behaviour could be attributed to diphenol

oxidase (PPO) activity [5]. Ortega-Garcıa described that

PPO was located in the epidermis, parenchyma and com-

panion vascular cells of leaves of olive cv. Picual. PPO con-

tains copper and catalyses the hydroxylation of monophenols

to o-diphenols and the oxidation of o-diphenols to o-diqui-

nones. Thus, when oleuropein and these enzymes make

contact, oleuropein is broken down [34].

3.3 Oxidative Stability

The oxidative stability of oil systems (HOSO, CO and SBO)

with and without addition of olive leaf extracts (OHE, OJ,

OSFE) was evaluated by Rancimat method as it is shown in

Table 5. OJ decreased significantly the IT respect to the

control in all oil systems studied at polyphenol concentrations

of 250 and 630 mg CAE/kg oil, showing a pro-oxidant behav-

iour. In contrast, Farag et al. [16] reported an antioxidant

effect of OJ (400 and 2500 ppm) in sunflower oil at 1808C.

At polyphenol concentrations of 250 mg CAE/kg oil, the

addition of OHE to HOSO increased significantly (p<0.05)

OHE

OJ

OSFE

Figure 1. HPLC-UV chromatograms at 280 nm of the hydroalcoholic extract (OHE), juice (OJ) and supercritical-CO2 (OSFE) obtained from

olive leaves.



the IT respect to the control, whereas OSFE did for HOSO

and CO. However, at higher polyphenol concentration of

olive leaf extracts (630 mg CAE/kg oil), the addition of

OHE and OSFE delayed significantly (p<0.05) the onset

of oxidation respect to the control for all oils systems studied.

The highest protection factor (PF) was found be for OSFE

in each oil system studied (HOSO, CO and SBO) for both level

olive leaves extracts concentration (250 and 630 mg CAE/kg

oil). These results are in agreement with those of similar studies

on the effect of olive leaf extracts in oil systems [11].

The significant differences in IT and PF for OSFE and

OHE suggest that polyphenols profile olive leaf extracts and/

or other minor compounds as alpha tocopherol (co-extracted

in the case of OSFE) are playing an important role in the

antioxidant effect on the oils. The polyphenols of low weight

molecular as p-coumaric acid and ferulic acid were the main

in OSFE whereas oleuropein was in OHE, which has showed

low solubility in hydrophobic systems [35].

The structure–activity relationship of flavonoids has been

documented in hydrophilic systems but in bulk lipid systems

is less studied. In the last system, number of hydroxyl groups,

2,3 double bond in conjugation with a carbonyl group in the

C-ring and glycoside moiety in the molecule have influence

on the antioxidant activity [22]. In this study the extracts are

complex matrices of polyphenols, which makes difficult to

relate their structure–activity in the bulk-oil systems.

However, in OSFE the free polyphenols (more hydrophilic)

could be mostly localised in the air–oil interface where exert

their action, explaining its greater antioxidant effect.

On the other hand, there is evidence of a higher antiox-

idant effect when combinations of flavonoids and AT were

used [22, 23]. Thus, the high IT in systems added with OSFE

can be also explained by an interaction between polyphenol

and AT, resulting in a synergistic antioxidant effect and/or

regeneration of tocopherol by flavonoids [22] or of flavonoids

by tocopherols [23, 36]. In addition, the unsaturation of oil

has been reported to influence the IT [37]; indeed, mono-

unsaturated lipid (as HOSO) showed significantly longer IT

compared with more polyunsaturated oils.

The authors have declared no conflict of interest.

References

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Table 4. Phenolic composition profiles of different extracts from

olive leaves (OHE, OJ and OSFE) expressed as percentage

Peak Compound

OHE

(%)

OJ

(%)

OSFE

(%)

1 Hydroxytyrosol 0.4 7.3 6.2

2 Vanillic acid 1.9

3 Hydroxytyrosol glycoside 0.8

4 Vanillic hexoside acid 0.3

5 Caffeic hexoside acid 0.4

6 Vanillin 0.3

7 Oleoside 1.5 0.9 0.1

8 Chlorogenic acid 0.3

9 Oleuropein aglycon 1.3 0.8

10 Pinoresinol 0.8

11 Caffeic acid 0.3

12 Elenolic acid 0.5 0.1

13 p-coumaric acid 11.2

14 Ferulic acid 8.7

15 Verbacoside 3.0 15.7 7.1

16 Ligstroside aglycon decarboxymethyl 0.6

17 Luteolin-0-rutinoside 2.2

18 Acetoxypinoresinol 1.8

19 Luteolin-7-glucoside 2.2 2.7 1.2

20 Hesperitin-3-rutinoside 2.2

21 Quercetin-3-0-galactoside 1.1

22 Apigenin-7-rutinoside 0.8

23 Oleuropein 29.1 0.8 6.7

24 Oleuroside acid-10-carboxilic 8.2

25 Apigenin-7-glucoside 1.1

26 Oleuroside 13.2 0.8 2.3

27 Ligstroside 1.5 0.5 1.8

28 Luteolin-30-7-diglucoside 1.6

29 Luteolin-7-rutinoside 0.9

30 Oleuropein diglucoside 1.2

31 Oleuropein aglycon aldehyde 0.5

32 Quercetin 1.1 0.8

OHE, hydroalcoholic extract; OJ, juice; OSFE, supercritical-CO2

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Table 5. Induction time and PF of oils added with olive leaves

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IT (h) PF

HOSO CO SBO HOSO CO SBO

Control 16.9 � 0.35a 9.5 � 0.1a 5.9 � 0.41a

250 mg CAE/kg oil

OHE 21.1 � 0.07b 9.5 � 0.23a 6.7 � 0.06a 1.26 1.0 1.13

OSFE 26.3 � 0.86d 12.6 � 0.36c 6.9 � 0.39a 1.56 1.32 1.16

OJ 2.7 � 0.21c 1.8 � 0.04b 1.7 � 0.05b 0.16 0.19 0.28

630 mg CAE/kg oil

OHE 30.7 � 0.78b 11 � 0.85b 7.6 � 0.13b 1.82 1.15 1.28

OSFE 35.1 � 0.28d 13.4 � 0.42d 8.4 � 0.05b 2.08 1.4 1.41

OJ 3.0 � 0.18c 2.0 � 0.04c 1.4 � 0.61c 0.18 0.21 0.23

HOSO, high oleic sunflower oil; CO, canola oil; SBO, soybean oil;

OHE, hydroalcoholic extract; OSFE, supercritical-CO2 extract; OJ,

juice.

Different letters show significant difference between extracts for each

oils (p<0.05).



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