Journal of Food Processing and Preservation ISSN 1745-4549

ANTIOXIDANT ACTIVITY OF ROSEMARY EXTRACT IN

SOYBEAN OIL UNDER THERMOXIDATION jfpp_755 1..10

SABRINA NEVES CASAROTTI and NEUZA JORGE1

Department of Engineering and Food Technology, São Paulo State University, São Paulo 15054000, Brazil

1
Corresponding author. ABSTRACT
TEL: +55 17 3221-2257;
FAX: +55 17 3221-2299; This study examined the antioxidant activity of lyophilized rosemary extract
EMAIL: njorge@ibilce.unesp.br added to soybean oil, subjected to thermoxidation conditions and also its syner-
gistic effect with the synthetic antioxidant tertiary butylhydroquinone (TBHQ).
Accepted for Publication April 14, 2012
Soybean oil samples with no antioxidant added (SO), 3,000 mg/kg rosemary
doi:10.1111/j.1745-4549.2012.00755.x
extract (RE), 50 mg/kg TBHQ (TBHQ), and a mixture of those two antioxidants
(RE + TBHQ) were heated to 180C for 20 h. After 0, 10 and 20 h, the oxidative sta-
bility, total polar compounds, tocopherol content and fatty acid profile were deter-
mined. The addition of rosemary extract increased oxidative stability and resulted
in a lower formation of total polar compounds and a higher retention of toco-
pherols. The RE treatment showed the highest amount of polyunsaturated fatty
acids after 20 h. There was not any synergy between TBHQ and rosemary extract
in preventing oxidation of soybean oil. Rosemary extract showed a higher anti-
oxidant potential when compared with TBHQ.

PRACTICAL APPLICATIONS
Antioxidants are important ingredients in food processing because they have the
capacity to protect foods, containing oils and fats, from damage caused by free
radicals and reactive oxygen species. Synthetic antioxidants are widely used in the
food industry; however, their utilization has been questioned because of toxicity.
Therefore, there is a growing interest in the use of natural antioxidants to reduce
or replace the synthetic antioxidants. Several species are used in cooking, medicine
and by the pharmaceutical industry, standing out the rosemary. Being rich in
compounds with high antioxidant activity, the rosemary extract can be used to
replace synthetic antioxidants used in vegetable oils.

as butylated hydroxyanisole (BHA), butylated hydroxy-

INTRODUCTION
Oils and fats are unstable compounds and are not resistant
to the deterioration processes of oxidation, hydrolysis and
polymerization that occur during heating. Lipid oxidation
is a major cause of deterioration during shelf life and in
the process of heating vegetable oils. Oxidation may cause
sensory and chemical changes, as well as a reduction in
nutritional value. Moreover, it results in the appearance of
dimers, polymers and cyclic monomers that might be
potentially toxic (Juárez et al. 2005; Leclercq et al. 2008).
In order to retard, reduce or prevent oxidative deterio-
ration, antioxidants are added to food. The side effects of
some synthetic antioxidants used in food processing, such
toluene, propyl gallate and tertiary butylhydroquinone
(TBHQ) have been studied, and their use has been ques-
tioned because of toxicology concerns (Wang et al. 2008).
To overcome this problem, several studies have been
carried out in order to find natural products that possess
antioxidant activity (AA) (Proestos et al. 2010). Plants,
including herbs and spices, are known to have many phy-
tochemicals that could be potential sources of natural anti-
oxidants. Rosemary (Rosmarinus officinalis L.) has been
widely accepted as a spice with one of the best antioxidant
activities (Peng et al. 2005; Zhang et al. 2010). The com-
pounds responsible for rosemary’s antioxidant properties
are mainly phenolic diterpenes, such as carnosol, carnosic

Journal of Food Processing and Preservation •• (2012) ••–•• © 2012 Wiley Periodicals, Inc. 1
ANTIOXIDANT ACTIVITY OF ROSEMARY EXTRACT
acid, rosmanol, epirosmanol and isorosmanol (Erkan
et al. 2008).
Several studies have recommended the use of rosemary
extract as an alternative antioxidant compound (Merril
et al. 2008; Gámez-Meza et al. 2009). Che-Man and Jaswir
(2000) reported that rosemary extract has important anti-
oxidant characteristics and a good thermal stability. In a
previous study conducted by Ramalho and Jorge (2008),
rosemary extract was found to have a positive effect on the
oxidative stability of soybean oil during heating. In addition
to its antioxidant properties, rosemary also has shown to
have a synergistic effect with some synthetic antioxidants,
such as citric acid or BHA (Madsen and Bertelsen 1995).
Considering the changes that occur to soybean oil during
heating, and also considering the interest in reducing or
eliminating the use of synthetic antioxidants, this research
aimed to evaluate the AA of lyophilized rosemary extract,
which was added to soybean oil that then underwent
thermoxidation. This study also evaluates the synergistic
effect that rosemary formed with the synthetic antioxidant
TBHQ, which is used by food industries to delay oxidative
alterations.
MATERIAL AND METHODS
Material
Refined soybean oil without the addition of synthetic
antioxidants (TBHQ and citric acid) was obtained from
Cargill Agrícola S/A (Uberlândia, Minas Gerais, Brazil).
The synthetic antioxidant, TBHQ, was supplied by Danisco
S.A. (Cotia, São Paulo, Brazil).
Fresh rosemary was obtained from a local market. The
best branches were selected and the damaged leaves were
removed. It was washed in flowing water and its roots were
removed. The rosemary then underwent the lyophilization
process (Liotop L101, Liobras Ltda, São Carlos, São Paulo,
Brazil). The lyophilized material was stored in plastic bottles
at room temperature and shielded from light until the time
of extraction.
In order to obtain the ethanolic extract from the rose-
mary samples, 5 g of lyophilized material were added to
100 mL of ethanol. The mixture was blended at room tem-
perature (25 ⫾ 2C) for 16 min and then filtered through a
vacuum pump to separate the supernatant from the pre-
cipitate. The ethanol was removed under reduced pressure
at 40C. The ethanolic extraction yield was obtained by
directly weighing of the dry extract. The dry extract was
resuspended in ethanol to obtain a stock solution contain-
ing 1 g of ethanolic rosemary extract per 10 g of ethanol
(1:10), and this solution was directly applied to the
soybean oil.
2 Journal of Food Processing and Preservation •• (2012) ••–•• © 2012 Wiley Periodicals, Inc.
S.N. CASAROTTI and N. JORGE
Thermoxidation Process
The following treatments underwent thermoxidation:
(1) soybean oil without a synthetic antioxidant (SO);
(2) soybean oil with 50 mg/kg of synthetic antioxidant
(TBHQ); (3) soybean oil with 3,000 mg/kg of rosemary
extract (RE); and (4) soybean oil with a mixture of
3,000 mg/kg of rosemary extract and 50 mg/kg of TBHQ
(RE + TBHQ). The concentration of TBHQ that was used
is the most common concentration utilized by the soybean
oil industry.
The thermoxidation tests on these four treatments were
conducted on a hot plate in four different 50 mL beakers
containing 30 mL of each sample with a 0.4/cm sur-
face : volume ratio. The temperature was set at 180C, which
is the most commonly used temperature for deep frying.
The heating was carried out using a discontinuous approach
involving 10 h of heating per day. Samples were taken at
time intervals of 0, 10 and 20 h and collected in flasks in the
presence of inert gas (N2) and stored at -18C until the time
of analysis.
Analytical Methods
Rosemary Extract Analyses. The ability to scavenge
free radicals 2, 2-diphenyl-1-picrylhydrazyl (DPPH•) was
developed by Brand-Williams et al. (1985) and it is based on
the reduction of the absorbance of DPPH• at 515 nm using
the action of reducing compounds of free radicals. In this
study, an ethanol solution was prepared with a concen-
tration of 500 mg/mL of rosemary extract from which
solutions with concentrations of 5, 10, 25, 50, 125, 250 and
400 mg/mL were obtained. Aliquots (0.3 mL) of each of
these solutions were added to 2.7 mL of a methanol solu-
tion of DPPH• (40 mg/mL). After 30 min of reaction time,
the absorbance was read at 515 nm using a spectrophoto-
meter (Schimadzu UV mini, 1240, Kyoto, Japan) and this
value was converted into a percentage of antioxidant
activity (AA) using the following equation: AA (%) =
100 – ([Abssample – Absblank ¥ 100]/Abscontrol), where Ablank is the
absorbance of the control reaction (a reaction with all the
reagents except the test extract), and Asample is the absorbance
of the test extract. The AA of the extract was expressed as
IC50, and was defined as the concentration of the test mate-
rial required to cause a 50% decrease in initial DPPH•
concentration.
The total phenol content of the extract was measured
using a spectrophotometer (Schimadzu), and it was deter-
mined using Folin–Ciocalteu reagent according to the
procedure reported by Singleton and Rossi (1965). The
absorbance was measured at 765 nm. For the quantification,
a standard curve was prepared using gallic acid in concen-
trations ranging from 0 to 500 mg/L. The determination
S.N. CASAROTTI and N. JORGE
coefficient was R2 = 0.999 and the results were expressed
as milligrams of gallic acid equivalents per gram of extract
(mg GAEs/g of extract).
Soybean Oil Analyses. Antioxidant activities were
assessed using total polar compounds, oxidative stability,
tocopherols and a fatty acid profile in the samples collected
after 0, 10, and 20 h of heating.
The total polar compounds, expressed as a percentage,
were determined using a chromatographic method pro-
posed by Dobarganes et al. (2000). This method relies on
the separation of the oil sample into two fractions of differ-
ent polarity using adsorption chromatography so that the
two fractions can be determined gravimetrically.
Oxidative stability was measured following the American
Oil Chemists’ Society (AOCS) Cd 12b-92 method (AOCS
1996) using Rancimat equipment, model 743 (Metrohm,
Herisau, Switzerland), which is based on the determination
of the electric conductivity of volatile product degradation.
The determination was made at 100C with a 20 L/h air flow
using 3 g of the sample and 60 mL of distilled water in
flasks with electrodes. Electrical conductivity versus time
was automatically registered and the period of induction
was expressed in hours.
Tocopherol concentrations were determined according
to the AOCS Ce 8-89 method (AOCS 1998). This analysis
was carried out using high-performance liquid chromato-
graphy (Varian Associates, Inc., Walnut Creek, CA) with a
fluorescence detector (TSP brand and FL 2000 model,
Varian Associates, Inc.). The following conditions were
applied: silica column with 250 ¥ 4.6-mm dimensions with
5-mm pores, a 1.2 mL/min flow and an excitation wave-
length of 290 nm with emission at 330 nm. The mobile
phase consisted of a mixture of 99.5% n-hexane and 0.5%
isopropanol. The calculations were performed using the
four tocopherol isomers as external standards. Concentra-
tions were calculated based on excitation peak area, and
are expressed in mg/kg.
Fatty acid composition was determined using capillary
gas chromatographic (GC) analysis, and samples were
transesterified with potassium hydroxide, methanol and
n-hexane, according to the AOCS Ce 2-66 method (AOCS
1997). The analyses were conducted with a Variant GC
3900 (Varian Associates, Inc., Walnut Creek, CA) equipped
with a flame ionization detector, an autosampler and a
split injection system. The compounds were separated in a
CP-Sil 88 column (60 m in length, 0.20 mm in intern diam-
eter, 0.20 micron film thickness). The column temperature
programming was initiated at 90C for 4 min, heated to
195C at 10C/min and kept isothermal for 16 min. Tempera-
tures used in the injector and detector were 230 and 250C,
respectively. The samples were injected into a volume
of 1 mL, adopting the ratio of 1:30. The carrier gas was
Journal of Food Processing and Preservation •• (2012) ••–•• © 2012 Wiley Periodicals, Inc.
ANTIOXIDANT ACTIVITY OF ROSEMARY EXTRACT
hydrogen with a linear velocity of 3.0 mL/min. Fatty acids
were identified according to retention times and were
quantified using normalization of area (%). A standard
mixture was used and it was composed of 37 fatty acid
methyl esters, C4:0 to C24:1, with purity between 99.1 and
99.9% (Supelco, Bellefonte, PA).
Statistical Analysis. The experiment was performed in
a completely randomized design to determine the influ-
ence that the factors had on the changes to the oils heated
at high temperatures. The experiment was carried out in a
4 ¥ 3 factorial scheme, with four treatments (SO, TBHQ,
RE and RE + TBHQ) and three heating times (0, 10 and
20 h). The results obtained in duplicate from total polar
compounds, oxidative stability, tocopherols and fatty acid
profile were evaluated using the analysis of variance and
the Tukey’s test for the 5% significance level, both of
which were performed with the ESTAT program, version
2.0 (UNESP, Jaboticabal, São Paulo, Brazil).
RESULTS AND DISCUSSION
The total yield of ethanolic rosemary extract was 22.9%.
The ethanolic extract produced a significant yield and
provided the efficiency of the solvent.
The IC50 value is calculated as a reduction of 50% of the
initial concentration of DPPH•. This reaction is used as a
parameter to measure antioxidant ability to scavenge any
free radicals. It is important to note that the lower the value
of IC50, the higher is the AA of the compound analyzed.
The value of IC50, which is obtained using linear regression
for rosemary extract, was found to have a high coefficient
of determination (R2 = 0.999). The values of IC50 and
maximum AA achieved with rosemary extract were
43.52 mg/mL and 76.40%, respectively. Rosmarinic and
carnosic are the two compounds with a strong AA that are
most commonly identified in rosemary. The activity of
rosemary extracts is probably related to the concentration of
the latter compound (Mata et al. 2007).
The total content of phenolic compounds in the rose-
mary extract was determined using a colorimetric assay,
which included Folin–Ciocalteu as a reagent. The concen-
tration of phenolic compounds found was 82.03 mg
GAE/g of extract. Mata et al. (2007) obtained different
values of total phenolic compounds in water (58.4 mg
GAE/g of extract) and ethanol rosemary extracts (73.5 mg/
GAE g of extract). Also, Celiktas et al. (2007) found other
concentrations (4.1 to 119 mg GAE/g of extract) of phe-
nolic compounds of rosemary extract that had been
obtained using supercritical extraction of plants harvested
from different locations and during different times of the
year in Turkey.
3
ANTIOXIDANT ACTIVITY OF ROSEMARY EXTRACT S.N. CASAROTTI and N. JORGE

this treatment had values of total polar compounds that

Total Polar Compounds and
Oxidative Stability
The nonpolar fraction of the deteriorated oil and fats is
made up of all unaltered triacylglicerols. Total polar com-
pounds are considered to be nonvolatile compounds that
have a higher polarity than triacylglycerols as a result of
thermal, hydrolytic and oxidative alteration. Consequently,
the higher the amount of polar compounds on oil, the
worse its quality is (Gharachorloo et al. 2010).
According to Lumley (1988), the initial contents of total
polar compounds in oil should range between 0.4 and 6.4%.
The results of initial samples of soybean oil are within the
limits set for refined oils (Table 1).
In the beginning, there was no significant difference
between any of the total polar compounds levels that
received the treatments used in this study, but there was a
significant difference between the total polar compound
values after 10 and 20 h for each treatment. The values
increased as heating times increased when each of the four
treatments were used (Table 1). All antioxidants delayed the
formation of polar compounds throughout heating, but
with different levels of effectiveness. After 20 h of heating,
SO treatment were compared with RE, RE + TBHQ and
TBHQ treatment, and these comparisons revealed a reduc-
tion in total polar compound formation by 35.4, 31.51 and
15.83%, respectively. These reduction levels indicate the
higher efficiency of rosemary extract when applied sepa-
rately rather than together.
The samples containing TBHQ revealed values that were
similar to those of the SO treatment. Their values did not
differ until after 20 h of heating. When the RE + TBHQ
treatment was used, the rosemary extract was the main one
responsible for the antioxidant effect. After 20 h of heating,
were statistically equivalent to those of the RE treatment
and which were lower than those of the TBHQ treatment.
No synergism between rosemary extract and TBHQ was
found. The RE and RE + TBHQ samples showed no signi-
ficant difference after 20 h of heating (Table 1).
Réblová et al. (1999) found a positive correlation between
the formation of total polar compounds and the frying time
of canola oil used to prepare French fries. These researchers
used rosemary extract to prevent oxidation, and they found
that this antioxidant inhibited the increased formation of
total polar compounds. Also, the increased formation of
polar compounds after 25 h of heating was found when
cotton and sunflower oils used to fry cassava were studied
(Corsini et al. 2009). The concentration of products that
formed as a result of oxidative alteration of the oil increased
as a function of temperature, limiting the usefulness of the
oil. In many countries, an upper limit of 25% total polar
compounds has been established (Angelo and Jorge 2008).
In the present study, only the treatments without the rose-
mary extract (SO and TBHQ) had values above this limit
after 20 h of heating. These results reinforce the strong
activity of rosemary extract.
Oxidative stability is closely dependent on the degree of
unsaturation of the oil and also on the presence of antioxi-
dants, which are likewise affected by the temperature. This
analysis measures the total volatile carbonyl compounds,
which are secondary products of lipid oxidation, and it also
evaluates the oil’s resistance to oxidation (Gámez-Meza
et al. 2009).
There was a significant reduction in the induction period
during the heating time of soybean oil, with or without the
addition of antioxidants (Table 1). This fact indicates that
the antioxidants tested were unable to remain stable after

TABLE 1. MEAN VALUES FOR TOTAL POLAR

Heating time (h)
COMPOUNDS (%) AND OXIDATIVE STABILITY
Treatment 0 10 20 (H) OF OILS HEATED FOR UP TO 20 H
Total polar compounds
SO 1.38 ⫾ 0.29cA 18.73 ⫾ 0.72bA 33.16 ⫾ 0.52aA
TBHQ 1.57 ⫾ 0.02cA 19.66 ⫾ 0.59bA 27.91 ⫾ 0.34aB
RE 2.07 ⫾ 0.05cA 10.90 ⫾ 0.58bC 21.42 ⫾ 0.50aC
RE + TBHQ 1.97 ⫾ 0.11cA 14.51 ⫾ 0.35bB 22.71 ⫾ 0.08aC
Oxidative stability
SO 14.83 ⫾ 0.01aD 8.44 ⫾ 0.08bB 3.49 ⫾ 0.12cC
TBHQ 24.84 ⫾ 0.345aC 6.29 ⫾ 0.125bC 4.46 ⫾ 0.11cB
RE 35.52 ⫾ 0.30aB 14.79 ⫾ 0.16bA 9.79 ⫾ 0.415cA
RE + TBHQ 40.11 ⫾ 0.005aA 13.86 ⫾ 0.26bA 9.88 ⫾ 0.325cA

SO: soybean oil; TBHQ: soybean oil + TBHQ (50 mg/kg); RE: soybean oil + rosemary extract
(3,000 mg/kg); RE + TBHQ: soybean oil + rosemary extract (3,000 mg/kg) + TBHQ (50 mg/kg).
Means followed by the same lower case letters are not significantly (P > 0.05) different over time
(rows). Means followed by the same upper case letter are not significantly (P > 0.05) different
among treatments (columns).
TBHQ, tertiary butylhydroquinone.

4 Journal of Food Processing and Preservation •• (2012) ••–•• © 2012 Wiley Periodicals, Inc.
S.N. CASAROTTI and N. JORGE ANTIOXIDANT ACTIVITY OF ROSEMARY EXTRACT

20 h of heating. The added antioxidants greatly influenced
the oxidative stability of the heated soybean oil. Never-
theless, TBHQ efficiency was markedly weaker than the
rosemary extract when the thermoxidation process was
employed. Rosemary extract was more efficient than TBHQ,
the synthetic antioxidant, in preventing the decrease of oxi-
dative stability, and it produced a protective effect. Its stabil-
ity values were significantly higher than those that resulted
from the TBHQ treatment in all the analyzed periods
(Table 1). Moreover, it was found that there was no syner-
gistic effect between TBHQ and rosemary extract, because
the samples, RE and RE + TBHQ, showed no significant dif-
ference after 10 or 20 h of heating.
Merril et al. (2008) evaluated the oxidative stability of
different oils with antioxidants (ascorbyl palmitate, TBHQ,
rosemary extract and tocopherols) at 110C, and reported
that there was a synergistic effect between TBHQ and ascor-
byl palmitate. However, the rosemary extract had a negative
interaction with TBHQ in the prevention of oil oxidation.
The findings of this study are similar to those from the
research conducted by Ramalho and Jorge (2008). They also
noted an increase in the oxidative stability of soybean oil
mixed with 1,000 mg/kg of rosemary extract in comparison
with soybean oil without the addition of this antioxidant.
In their study, the Rancimat equipment (Metrohm) was also
used at 100C.
Tocopherol Concentration
Tocopherols are naturally present in most vegetable oils, and
they may influence the oxidative stability of these oils, even
as minor components. Depending on processing conditions,
the loss of tocopherols can be up to 56% compared with the
value found in raw oil (Medina-Juárez et al. 2000). There-
fore, it is important to study the loss of tocopherols and
their relationship with the deterioration of oil during
heating at high temperatures.
According to the Codex Alimentarius (2009), soybean oil
must contain 9.352 mg/kg of a-tocopherol, 0.36 mg/kg
of b-tocopherol, 89–2,307 mg/kg of g-tocopherol, 154–
932 mg/kg of d-tocopherol and 600–3,337 mg/kg of total
tocopherols. The values of tocopherols found in initial

TABLE 2. MEAN VALUES FOR TOCOPHEROLS

Heating time (h)
(MG/KG) OF OILS HEATED FOR UP TO 20 H
Treatment 0 10 20
a-tocopherol
SO 70.35 ⫾ 0.35aAB 32.85 ⫾ 1.77bC 2.80 ⫾ 0.28cC
TBHQ 75.60 ⫾ 2.83aA 22.10 ⫾ 1.70bD 10.55 ⫾ 3.61cB
RE 62.90 ⫾ 1.70aC 58.10 ⫾ 2.83aB 47.95 ⫾ 3.32bA
RE + TBHQ 67.00 ⫾ 2.83aBC 69.90 ⫾ 1.56aA 46.15 ⫾ 1.48bA
b-tocopherol
SO 6.40 ⫾ 1.13aB 4.05 ⫾ 0.07bA 1.30 ⫾ 0.00cB
TBHQ 8.65 ⫾ 0.92aA 4.45 ⫾ 0.49bA 1.35 ⫾ 0.07cB
RE 6.90 ⫾ 0.14aB 5.25 ⫾ 0.21bA 3.35 ⫾ 0.21cA
RE + TBHQ 6.25 ⫾ 0.07aB 5.25 ⫾ 0.21aA 3.90 ⫾ 0.28bA
g-tocopherol
SO 338.15 ⫾ 10.82aAB 150.25 ⫾ 4.31bC 14.15 ⫾ 1.20cB
TBHQ 360.65 ⫾ 6.29aA 73.90 ⫾ 2.83bD 24.85 ⫾ 1.63cB
RE 312.30 ⫾ 2.40aC 236.40 ⫾ 20.79bB 126.10 ⫾ 1.41cA
RE + TBHQ 335.55 ⫾ 4.17aB 265.20 ⫾ 5.66bA 110.60 ⫾ 7.50cA
d-tocopherol
SO 91.70 ⫾ 1.41aAB 66.40 ⫾ 1.56bB 41.10 ⫾ 5.23cB
TBHQ 97.00 ⫾ 1.27aA 59.05 ⫾ 6.58bB 43.05 ⫾ 1.91cB
RE 80.80 ⫾ 0.57aC 78.25 ⫾ 4.60aA 67.30 ⫾ 1.41bA
RE + TBHQ 85.55 ⫾ 1.34aBC 85.65 ⫾ 1.63aA 63.65 ⫾ 2.90bA
Total tocopherol
SO 506.60 ⫾ 8.63aB 253.55 ⫾ 7.71bC 59.35 ⫾ 6.72cB
TBHQ 541.90 ⫾ 8.77aA 159.50 ⫾ 7.21bD 79.80 ⫾ 7.21cB
RE 462.90 ⫾ 4.53aC 378.00 ⫾ 28.43bB 244.7 ⫾ 0.71cA
RE + TBHQ 494.35 ⫾ 2.76aB 426.00 ⫾ 9.05bA 224.3 ⫾ 8.63cA

SO: soybean oil; TBHQ: soybean oil + TBHQ (50 mg/kg); RE: soybean oil + rosemary extract
(3,000 mg/kg); RE + TBHQ: soybean oil + rosemary extract (3,000 mg/kg) + TBHQ (50 mg/kg).
Means followed by the same lower case letters are not significantly (P > 0.05) different over time
(rows). Means followed by the same upper case letter are not significantly (P > 0.05) different
among treatments (columns).
TBHQ, tertiary butylhydroquinone.

Journal of Food Processing and Preservation •• (2012) ••–•• © 2012 Wiley Periodicals, Inc. 5
ANTIOXIDANT ACTIVITY OF ROSEMARY EXTRACT S.N. CASAROTTI and N. JORGE

soybean oils are in agreement with the values set by
the Codex Alimentarius, with the exception of values for
d-tocopherol and total tocopherols (Table 2). This differ-
ence in total tocopherols may be related to the processing of
the oils, especially when it comes to deodorization.
Changes in tocopherol concentrations during the heating
process were also considered, and a significant decrease
was observed in all treatments as heating time increased.
Barrera-Arellano et al. (2002) reported that after 10 h of
heating, significant reductions of tocopherols were found in
polyunsaturated oils, with a 40% loss in soybean oil, and
with a-tocopherol being the least stable.
The addition of antioxidants strongly influenced the
retention of tocopherols present in soybean oil subjected
to heating (Fig. 1). There was a higher degradation of toco-
pherols in oils free from rosemary extract at the end of the
heating, which may have favored a lower oxidative stability
of these oils (Fig. 1). The a-tocopherol was strongly pro-
tected by the use of rosemary, which is in agreement with
the results obtained by Rizner-Hras et al. (2000). In the
current study, the presence of rosemary increased the reten-
tion of a-, g- and d-tocopherol, respectively, from 4 to 76%,
from 4 to 40%, and from 45 to 83% after 20 h of heating
and when compared with the SO treatment. There was no
synergistic effect between TBHQ and rosemary extract to
prevent the loss of tocopherols in soybean oil that under-
went thermoxidation, because for all fractions of toco-
pherols, except b-tocopherol, retention after 20 h of heating
was higher in the RE sample, which was the soybean oil that
contained only rosemary extract.
Regarding the stability of tocopherol, for pure soybean
oil at high temperature, the literature suggests the follow-
ing stability sequence: d > g > b > a (Kamal-Eldin and
Appelqvist 1996). However, we found that, soybean oil
free from antioxidants, stability decreased in the order
d > b > a = g. Steel et al. (2005) also found a greater degra-
dation of a and g-tocopherol in soybean oil that had
been heated for 10 h. A rapid degradation of a-tocopherol
was reported by Verleyen (2001). Furthermore, Warner and
Gehring (2009) observed a higher degradation of a- and
d-tocopherol in soybean oil subjected to 65 h of frying.
Rosemary extract increased the stability of the tocopherol
fractions to a considerable extent. The difference in stability
between the tocopherols was evident in the oil samples that

FIG. 1. EVOLUTION OF TOCOPHEROLS (RESIDUAL %) IN SOYBEAN OILS HEATED UP TO 20 H

(A) Alpha-tocopherol. (B) Beta-tocopherol. (C) Gamma-tocopherol. (D) Delta-tocopherol. SO: soybean oil; TBHQ: soybean oil + TBHQ (50 MG/KG);
RE: soybean oil + rosemary extract (3,000 mg/kg); RE + TBHQ: soybean oil + rosemary extract (3,000 mg/kg) + TBHQ (50 mg/kg).

6 Journal of Food Processing and Preservation •• (2012) ••–•• © 2012 Wiley Periodicals, Inc.
S.N. CASAROTTI and N. JORGE ANTIOXIDANT ACTIVITY OF ROSEMARY EXTRACT

contained rosemary extract, and the sequence of stability
was changed to d > a > b > g. When reducing losses of
a-tocopherol, the stabilizing effect of the rosemary extract
is particularly marked, and it is clear that the rosemary
extract was slightly more active than the TBHQ at the con-
centrations used. Gordon and Kourimská (1995) reported
that rosemary extract has the ability to slow the loss of toco-
pherols that are naturally present in vegetable oils. No syn-
ergistic effect was observed between rosemary extract and
TBHQ, because the treatment with only the natural anti-
oxidant resulted in higher amounts of tocopherols.
Fatty Acid Profile
The evaluation of the fatty acid profile of fats and oils, such
as soybean oil, that are subjected to heat can provide impor-
tant data concerning the changes to each fatty acid during
this process. Some studies have shown that the alteration of
vegetable oils is influenced by the amount of unsaturated
fatty acids present, and that the loss of a-linolenic acid
(C18:3) can vary from 20 to 25% (Juárez et al. 2005).
Initially, samples revealed linoleic acid (C18:2) to be the
main fatty acid, corresponding to more than 50% of the
total fatty acids identified (Table 3). Kim et al. (2010) also
found that linoleic acid is the major fatty acid in soybean
oil, representing 50.5%. According to the Codex Alimenta-
rium, soybean oil should contain between 4.5 and 11%
a-linolenic acid. In this study, the oils were made up of
between 3.81 and 5.64% of this fatty acid at 0 h. On the
other hand, samples heated for 20 h had values below those
recommended by the Codex Alimentarium (Table 3).
Robert et al. (2001) demonstrated that the oxidative sta-
bility of potato chips is influenced by the type of oil used in
the frying process. The potatoes fried in partially hydro-
genated sunflower oil had a higher level of stability when
compared with those prepared with sunflower oil. Their
results highlight the importance of the fatty acid composi-
tion of raw oils in the oxidative process.
In this study, the levels of linoleic acid and a-linolenic
acid decreased as heating time increased. Comparing the
initial oil samples to oil collected after 20 h of heating, the
reduction of linoleic acid occurred in higher percentages
after the SO treatment (8.44%), followed by RE + TBHQ
(7.91%), TBHQ (6.64%) and RE (4.16%). However, in the
case of a-linolenic acid, the reduction was higher after
SO treatment (25.43%), followed by TBHQ (25.08%),
RE + TBHQ (22.5%) and RE (16.83%). The data showed
that there was no synergistic effect between rosemary
extract and TBHQ.
Velasco et al. (2004) found a reduction of 42.86 and
21.71% in the levels of polyunsaturated fatty acids in olive
and sunflower oils, respectively, after 15 h of frying. The
decrease in linolenic and a-linolenic acids may represent a
loss of nutritional value, because they are essential fatty
acids and are not synthesized by the human body.
The results regarding stearic, arachidic, behenic and
eicosenoic fatty acids showed that they remained virtually
unchanged in most samples. Palmitic and oleic acids

TABLE 3. MEAN VALUES FOR FATTY ACID

Fatty acid composition
COMPOSITION (%) OF OILS HEATED FOR UP
TO 20 H Sample C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 Others
SO
0h 10.7 3.49 25.12 53.79 5.78 0.35 0.43 0.35 ND
10 h 11.7 3.58 26.46 52.12 4.95 0.32 0.49 0.39 ND
20 h 12.32 4.17 27.18 49.25 4.31 0.42 0.32 0.48 1.59
TBHQ
0h 10.79 3.30 24.96 53.73 6.14 0.36 0.36 0.39 ND
10 h 11.63 3.77 26.03 51.53 5.06 0.39 0.35 0.48 0.78
20 h 12.14 3.95 26.88 50.16 4.60 0.40 0.43 0.50 0.98
RE
0h 10.82 3.27 25.06 53.88 5.82 0.33 0.41 0.44 ND
10 h 11.23 3.65 25.66 53.00 5.31 0.32 0.47 0.38 ND
20 h 11.94 3.82 26.54 51.64 4.84 0.33 0.41 0.35 0.15
RE + TBHQ
0h 10.93 3.34 25.12 54.10 5.60 0.27 0.38 0.29 ND
10 h 11.24 3.45 25.78 52.66 5.28 0.38 0.30 0.48 0.46
20 h 12.27 4.11 27.18 49.82 4.34 0.42 0.25 0.52 1.12

SO: soybean oil; TBHQ: soybean oil + TBHQ (50 mg/kg); RE: soybean oil + rosemary extract
(3,000 mg/kg); RE + TBHQ: soybean oil + rosemary extract (3,000 mg/kg) + TBHQ (50 mg/kg).
C16:0, palmitic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, alpha lino-
lenic acid; C20:0, arachidic acid; C20:1, eicosenoic acid; C22:0, behenic acid; ND, none detected;
TBHQ, tertiary butylhydroquinone.

Journal of Food Processing and Preservation •• (2012) ••–•• © 2012 Wiley Periodicals, Inc. 7
ANTIOXIDANT ACTIVITY OF ROSEMARY EXTRACT S.N. CASAROTTI and N. JORGE

TABLE 4. MEAN VALUES OF SATURATED,

Heating time (h)
MONOUNSATURATED AND
Treatment 0 10 20 POLYUNSATURATED FATTY ACID
Unsaturated COMPOSITION (%) OF OILS HEATED FOR
SO 14.89 ⫾ 0.04cA 15.99 ⫾ 0.09bB 17.55 ⫾ 0.04aA UP TO 20 H
TBHQ 14.83 ⫾ 0.04cA 16.43 ⫾ 0.01bA 17.14 ⫾ 0.01aB
RE 14.85 ⫾ 0.11cA 15.57 ⫾ 0.01bC 16.58 ⫾ 0.01aC
RE + TBHQ 14.82 ⫾ 0.07cA 15.71 ⫾ 0.01bC 17.50 ⫾ 0.03aA
Monounstaurated
SO 25.55 ⫾ 0.03cA 26.95 ⫾ 0.02bA 27.50 ⫾ 0.04aA
TBHQ 25.31 ⫾ 0.01cB 26.38 ⫾ 0.02bB 27.30 ⫾ 0.05aB
RE 25.47 ⫾ 0.08cAB 26.12 ⫾ 0.01bC 26.95 ⫾ 0.04aC
RE + TBHQ 25.50 ⫾ 0.01cA 26.07 ⫾ 0.03bC 27.43 ⫾ 0.07aAB
Polyunsaturated
SO 59.57 ⫾ 0.06aB 57.06 ⫾ 0.10bC 53.66 ⫾ 0.05cD
TBHQ 59.87 ⫾ 0.06aA 56.58 ⫾ 0.11bD 54.85 ⫾ 0.04cB
RE 59.71 ⫾ 0.02aAB 58.31 ⫾ 0.03bA 56.48 ⫾ 0.03cA
RE + TBHQ 59.69 ⫾ 0.06aAB 57.94 ⫾ 0.05bB 54.29 ⫾ 0.06cC

SO: soybean oil; TBHQ: soybean oil + TBHQ (50 mg/kg); RE: soybean oil + rosemary extract
(3,000 mg/kg); RE + TBHQ: soybean oil + rosemary extract (3,000 mg/kg) + TBHQ (50 mg/kg).
Means followed by the same lower case letters are not significantly (P > 0.05) different over time
(rows). Means followed by the same upper case letter are not significantly (P > 0.05) different
among treatments (columns).
TBHQ, tertiary butylhydroquinone.

showed a slight increase in most samples (Table 3). The

fatty acid composition values of the initial oils are close to
those presented by Gunstone (1996), who established
soybean oil values of 15, 22 and 61% of saturated, monoun-
saturated, and polyunsaturated fatty acids, respectively.
There was a significant increase in saturated and monoun-
saturated fatty acids and a reduction in polyunsaturated
fatty acids, regardless of the addition or absence of antioxi-
dants (Table 4). However, significant changes in the fatty
acid composition were mainly found in treatments without
added antioxidants.
The decrease in polyunsaturated fatty acid content
during the process of heating is probably due to the
destruction of these fatty acids as a result of processes such
as oxidation and polymerization. Hence, there should be an
important quality test for oils (Warner 2009). In a study to
evaluate the fatty acid profile in samples of soybean oil used
in frying, Velasco et al. (2004) found that frying leads to a
decreased concentration of polyunsaturated fatty acids
and, consequently, a proportional increase in saturated fatty
acids.
Based on the results for the fatty acid profile, we report a
positive effect of rosemary extract on the fatty acid compo-
sition of oils. The RE treatment showed lower amounts of
saturated and monounsaturated fatty acids, along with an
increased amount of polyunsaturated fatty acids after 20 h
of heating when compared with other treatments. Once
again, no synergistic effect between rosemary extract and
THBQ was found.
CONCLUSIONS
The results of the present study showed that after 20 h of
heating at 180C, the rosemary extract improved the oxida-
tive stability of soybean oil, increased the retention of
a-tocopherol and resulted in a higher amount of poly-
unsaturated fatty acids. There was no synergistic effect
between rosemary extract and TBHQ. Rosemary extract
can be recommended as an alternative antioxidant in oil
preservation.
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