Journal of Applied Biological Sciences 3 (1): 23-27, 2009

ISSN: 1307-1130, www.nobel.gen.tr

Antioxidant Properties of Various Olive Cultivars

Nahit GENÇER Selma SİNAN Oktay ARSLAN

Department of Chemistry/Biochemistry, Balıkesir Universty, Turkey

*Author to whom correspondence should be addressed Received: June 05, 2008

E-mail: [email protected] Accepted: August 17, 2008

Abstract

Virgin olive oil is a typical component of the Mediterranean diet, consumed unrefined and rich in important molecules, such as
minor polar compounds (tyrosol, ferulic acid and caffeic acid). These molecules not only influence the sensorial properties of both
olives and virgin olive but they are also important markers for cultivar identification, biodiversity and quality determination of this
product. In this study, levels of phenolics, that have antioxidant activity, such as α-tocopherol, caffeic acid, ferulic acid, and tyrosol,
have been determined using HPLC. This research examined the phenolic fraction of virgin olive oil samples of five different olive
(Olea europaea L.) cultivar grown in the region of Akhisar, Turkey. The cultivar Uslu has been found to contain the highest levels
of these compunds. The qualitative and quantitative HPLC analyses of the extracts showed that α-tocopherol was the most abundant
compound (from 6.35 mg/kg to 33.7 mg/kg).
Keywords: Olive oil, HPLC, Antioxidants, Quality

INTRODUCTION tion and quantification of the above compounds are therefore

of high importance. The major phenolic found in virgin olive
It has been postulated that the components in olive oil in the oil is tyrosol (4-hydroxyphenylethanol), caffeic acid and ferulic
Mediterranean diet, a diet which is largely vegetarian in nature, acid (Fig.3). The concertration of the total phenol content varies
can contribute to the lower incidence of coronary heart disease, from 100 to 800 mg/kg in olive oil [12], and depends on many
prostate and colon cancers. The Mediterranean diet includes the factors, including the species, location, climate, maturation of
consumption of large amounts of olive oil. Recently there has the drupes, proccesing and storage conditions. Amounts are
been a surge in the number of publications that has investigated greatest in the first pressing of extra virgin olive oil.
their biological properties. The phenolic compounds present in CH3
olive oil are strong antioxidants and radical scavengers [1]. HO
CH3 CH3 CH3
CH3
In the context of coronary heart disease, it is known that
CH3 O
experimental feeding of olive oil enriched in polyphenols to hu-
mans or rabbits increases the resistance of low density lipopro- CH3
tein to oxidation in vivo [2,3], emphasising the known capacity -tocopherol
of certain phenolics present in virgin olive oil act as antioxi-
dant. This has been shown previously using several chemical O
systems [4,5,6].

The biological properties of olive oil are related to its an- HO OH
OH
tioxidant composition, namely, tocopherols and minor polar HO
compound, in particular phenols and secoiridoids [7]. Phenolic HO
compounds are of great importance for several characteristics tyrosol caffeic acid
of the olive oil, such as flavour, shelf-life and resistance against
oxidation [8]. Phenolics from virgin olive oil are also power-
ful scavengers of superoxide anions and hydrogen peroxide are O
capable of preventing the generation of reactive oxygen spe-
cies by intact leukocytes as it has been demonstrated by other HO OH
authors [9].

H3C O
In virgin olive oil, α-tocopherol dominates with its compo-
sition reaching 95% of the total tocopherol [10]. Both phenols ferulic acid
and tocopherol contribute to the remarkable stability of the ol-
ive oil [6]. α-Tocopherol and phenolic compounds have been Fig.3 Phenolic compounds from virgin olive oil
reported as having beneficial biological activity [11]. The isola-
24 N. Gencer et al / JABS, 3 (1): 23-27, 2009
Quantitative determination of phenolic compounds in ol- cessively with three 20-ml portions of 60% aqueous metha-
ive oil usually performed accordin to Folin-Ciocalteau colori- nol. The combined extracts were brought to dryness in a ro-
metric method [8]. However, this method is not specific, as it tary evaporator at 40 oC and the residue was dissolved in 5 ml
gives no indication of the nature of the phenolic compounds methanol, and submitted to chromatographic analysis.
present. Reversed-phase HPLC currently represents the most
popular and reliable technique for the analysis of phenolic com- Extraction of tocopherols
pounds. The technique has been mainly used with UV detec-
tion [13]. A simple procedure was used for the extraction and Tocopherols were extracted from olive oil according to the
HPLC determination of phenols in olive oil by Maria Tasioula method described by Maria et al…2001 [14]. Samples of 10 g
et al. The extraction was carried out using methanol and an olive oil were extracted at room temperature with two 25-ml
isopropanol –methanol mixture. Separation was achieved on a portions of absolute methanol. The residue was extracted again
reversed phase C18 column with acetic acid/water-methanol- under the same conditions with two 25-ml portions of metha-
acetonitrile-isopropanol mixture under gradient elution. Detec- nol/isopropanol (80:20, v/v). The extracts were combined and
tion was accomplished with UV detection at α=280 nm. brought to dryness in a vacuum rotary evaporator at 40 oC. The
residue was dissolved in 5 ml of a methanol and analysed for
Cultivar, degree of maturation, climate and type of extrac- tocopherol content by HPLC.
tion method selected are among the factors affecting the phe-
nolic content of virgin olive oil. Studies on different olive oil
varieties indicate that cultivar has a significant impact on the HPLC apparatus
phenolic composition of virgin olive oil [14]. No reported lit-
erature was found concerned with antioxidant content of these A Agilent model HPLC system (Agilent 1100 Series) con-
cultivars (Domat, Edremit, Gemlik, Kiraz and Uslu). The virgin sisting of a solvent delivery module (LC-3D) with a double
olive oil samples used in this study were obtained from plants plunger reciprocating pump, UV-VIS detector (G1314A), col-
growing in a specific Akhisar area. umn oven (G1316A) and 20 µl injection loop was used. The
column used was an Apex octadecyl 104 C18 (25x0.4 cm ID)
The oxidative stability, sensory quality and health proper- with 5-µm packing (Agilent Technologies, USA).
ties of virgin olive oil stem from a prominent and well-balanced
chemical composition [15]. HPLC conditions

This is the first evaluation of chemical composition of five Detection was performed at 280 nm for both phenols and a-
cultivars (Domat, Edremit, Gemlik, Kiraz and Uslu) from Akh- tocopherol. The elution solvents used were A (2% acetic acit in
isar (Turkey). Because of the importance of these five cultivars water), B (methanol), C (acetonitril) and D (isopropanol). The
for Turkey oil production, the aim of this work was to char- samples were eluted according to the following gradient: 95%A
acterize Domat, Edremit, Gemlik, Kiraz and Uslu virgin olive / 5%B in 2 min; 60%A /10%B / 30%C in 8 min; 25%B / 75%C
oils based on the study of minor compounds (tocopherols and in 22 min, and this percentage was maintained for 10 min;
phenols) as well as on the oxidative stability. This research ex- 40%c /60%D in 10 min; andthis percentage was maintained for
amined the phenolic fraction of virgin olive oil samples of five 15 min; 25%B / 75%C in 2 min, and finally, 95%A /5%B in 3
different olive (Olea europaea L.) cultivar grown in the region min. Flow ratewas 1 ml/min and run time, 70 min [14]. The
of Akhisar, Turkey. run was performed at 32 oC. The sample injection volume was
20 ml. Identification of compounds was achieved by compar-
MATERIALS AND METHODS ing their retention time values with those of standards. Data
was collected and processed using G2170AA single instrument,
Materials Agilent ChemStation software (Agilent Technologies).

Olive oil samples from different variety olives grown in the Quantitation of α-tocopherol and phenols
region of Akhisar, Turkey were used. These variety were not
registered since they have been widespread amoung farmers for The α-tocopherol content of samples was determined by
years. The samples were stored under-20 oC prior to analysis. diluting approximately 200 mg of olive oil in 1 ml methanol/
isopropanol mixture (1:4, v/v) and analyzing the sample solu-
Reagents and standards tion by HPLC. Concentrations of α-tocopherol contents were
then calculated from integrated peak areas of the samples and
Acetonitrile, methanol, hexane isopropanol (2-propanol) calibration curve of α-tocopherol Standard. Good linearity was
acetic acid and water were all of HPLC grade and were pur- achieved in the range 10-220 mgkg-1 (r2 = 0,988).
chased from Merck (Germany). Methanol and hexane for oil
extraction were pro-analysis grade and were purchased from RESULTS and DISCUSSION
Merck (Germany). The standards dl-α-tocopherol, caffeic acid,
tyrosol and ferulic acid were purchased from Sigma (Germa- Generally, essential oils of spices possess strong antibacte-
ny). rial properties against foodborne pathogens and contain high
concentrations of phenolic compounds [16]. These compounds
Extraction of phenolic compounds exhibit a wide range of biological effects, including antioxidant
properties. Phenolic compounds contribute to the overall an-
Phenolic compounds were extracted from olive oil accord- tioxidant activities of herbs and spices. Generally, the mecha-
ing to the method described by Gutfinger [8]. Oil (10 g), were nisms of phenolic compounds for antioxidant activity are in-
dissolved in 50 ml hexane and the solution was extracted suc- activating lipid free radicals and preventing decomposition of
 N. Gencer et al / JABS, 3 (1): 23-27, 2009 25

hydroperoxides into free radicals. [17]. The HPLC profiles of phenolic compounds present in olive
oil were determined. The retention times for tocopherol, tyro-
Oxidative deterioration of fat components in foods is re- sol, caffeic acid and ferulic acid are 38.0 min, 6.2 min, 7.2 min
sponsible for the rancid odors and flavors which decrease nu- and 9.0 min, respectively. Antioxidant contents of different ol-
tritional quality. The addition of antioxidants is required to pre- ive cultivars were determined and are shown in table 1.
serve food quality [18].
Tocopherols are typical and important antioxidants in hu-
Polyphenols may exert their beneficial effects at several mans. a-tocopherol, which is present at the ratio of 1 to 1000
stages. At the primary step, as strong antioxidants, they have lipid molecules, is the most abundant among tocopherol. To-
the potential to scavenge free radicals and reduce the incidence copherols can protect polyunsaturated fatty acids within the
of damage to nucleic acids, blocking the initiation of cancer membrane and LDL, and inhibit smooth muscle cell prolifera-
cell formation. As a secondary step, they can modulate cellular tion and protein kinase C activity. Tocopherol has been associ-
biochemical processes such as the maintenance of calcium ho- ated with the reduction of heart disease, delay of Alzheimer’s
meostasis and mitochondrial function [19]. disease, and prevention of cancer [22]. Tocopherol has been
mostly found in Uslu olive oil. The values of tocopherol report-
There are several studies which show the beneficial effect ed in this study (Table 1) are lower than those found by other
of fruits and fruit products leading to disease prevention. A re- authors [23]. The high variability in the amount of tocopherol in
cent study showed that polyphenol components of several com- olive oil has been widely reported and depends on several fac-
monly consumed berries that include blackberry, black- and red tors, such as genetic, agronomic, environmental, and extraction
raspberry, blueberry, cranberry and strawberry could inhibit the procedures [24].
growth of human oral, breast, colon and prostate tumour cell
lines under in vitro conditions [20]. The ferulic acid has an active oxygen erasing function
and the effect has been reported to be similar to superoxide
HPLC analysis dismutase, known as the enzyme, which protects living bod-
ies from the toxicity of active oxygen. It is also revealed to
Olive oil phenols are usually extracted with water/methanol powerfully absorb harmful long wave ultraviolet light. Thus,
mixture from hexane solution. On the other hand, extraction of the ferulic acid has a wide variety of applications because it has
tocopherol cannot be achieved from oil/hexane solution as to- radical and active oxygen erasing effects, absorbs ultraviolet
copherol is retained in the hexane solution. Extraction was con- causing active oxygen generation and is a natural substance.
sequently applied directly to oil samples to oil without dissolu- It seems much effective for cosmetic use as whitening agent
tion in hexane. Isopropanol/methanol ratio of 20:80 (v/v) was and sunscreen making use of its powerful long wave ultraviolet
used for extraction of tocopherol. It has been demonstrated that absorbing function [25]. The ferulic acid has been mostly found
the conctration of phenols in extracts were dependent on extrac- in Uslu olive oil (3.68 mg/kg).
tion solvents and the class of phenolic compounds present in
olive oil [21]. Montedore et al. reported a mixture of CH3OH/
H2O (80:20, v/v) as the most efficient extraction solvent for
simple and hydrolyzable phenolic compounds [13].

Fig. 1. HPLC chromatogram of tocopherol for uslu olive oil.

26 N. Gencer et al / JABS, 3 (1): 23-27, 2009

Fig. 2 HPLC chromatogram of tyrosol, caffeic acid and ferulic acid for uslu olive oil

Tocopherols are typical and important antioxidants in hu- and sunscreen making use of its powerful long wave ultraviolet
mans. a-tocopherol, which is present at the ratio of 1 to 1000 absorbing function [25]. The ferulic acid has been mostly found
lipid molecules, is the most abundant among tocopherol. To- in Uslu olive oil (3.68 mg/kg).
copherols can protect polyunsaturated fatty acids within the
membrane and LDL, and inhibit smooth muscle cell prolifera- Tyrosol is the antioxidant that decreases with the lowest
tion and protein kinase C activity. Tocopherol has been associ- rate, providing the oil with the less antioxidant activity [26].
ated with the reduction of heart disease, delay of Alzheimer’s The concentration of tyrosol in olive oil has been reported to be
disease, and prevention of cancer [22]. Tocopherol has been 4.69 ± 0.77 mg/kg [27]; and 27.45 mg/kg in extra-virgin olive
mostly found in Uslu olive oil. The values of tocopherol report- oil and 2.98 ± 1.33 mg/kg in refined virgin oil [28]. Tyrosol has
ed in this study (Table 1) are lower than those found by other been mostly found in Uslu olive oil (9.84 mg/kg).
authors [23]. The high variability in the amount of tocopherol in
olive oil has been widely reported and depends on several fac- Caffeic acid was the most effective antioxidant in the lipo-
tors, such as genetic, agronomic, environmental, and extraction philic system and it is oxidized during the chemical oxidation
procedures [24]. of olives, whereas tyrosol, vanillic acid, and p-coumaric acids

Table 1. Antioxidant concentrations at different olive varieties

Tyrosol Caffeic acid Ferulic acid -tocopherol

Olive Oil variety a a a
(mg/kg±S.D.) (mg/ kg±S.D.) (mg/kg±S.D.) (mg/kg±S.D.)a
Domat Olive Oil 1.34±0.18 1.69±0.01 3.43±0.11 6.35±1.23
Edremit Olive Oil - 1.56±0.06 - 11.3±1.76
Gemlik Olive Oil - - - 22.0±2.43
Kiraz Olive Oil 1.1±0.70 - 3.54±0.18 26.5±1.62
Uslu Olive Oil 9.84±2.25 4.23±0.11 3.68±0.12 33.7±2.37

an=3

The ferulic acid has an active oxygen erasing function are unreactive [29]. Caffeic acid has been mostly found in uslu
and the effect has been reported to be similar to superoxide olive oil (4.23 mg/kg).
dismutase, known as the enzyme, which protects living bod-
ies from the toxicity of active oxygen. It is also revealed to As with the total phenol content of olive oil the content of
powerfully absorb harmful long wave ultraviolet light. Thus, ferulic acid, tyrosol and caffeic acid in olive oil has varied in
the ferulic acid has a wide variety of applications because it has the literature. These compounds are quite different in olive oil
radical and active oxygen erasing effects, absorbs ultraviolet because of environmental and extraction procedures.
causing active oxygen generation and is a natural substance.
It seems much effective for cosmetic use as whitening agent
 N. Gencer et al / JABS, 3 (1): 23-27, 2009 27

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