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Lebensm.-Wiss. u.-Technol.

, 30, 105108 (1997)

Research Note

Antioxidative and Free Radical Scavenging Properties


of Rosemary Extract
Huveyda Basaga*, Ceren Tekkaya and Funda Acikel
H. Basaga, F. Acikel: Department of Food Engineering, Middle East Technical University, 06531-Ankara
(Turkey)
C. Tekkaya: Department of Science Education, Middle East Technical University, 06531-Ankara (Turkey)
(Received August 21, 1995; accepted February 12, 1996)

Autoxidation of corn and soybean oils was prevented in the presence of 0.1 g/kg rosemary (Rosemarinus officinalis L.) extract
and butylated hydroxy toluene (BHT). At the end of a 4-d period, the peroxide value in corn oil reached 13.0 and 8.2 in the
presence of rosemary extract and BHT, respectively. For soybean oil, the peroxide value was found to be 10.0 and 6.6 in the
presence of rosemary extract and BHT, respectively. Rosemary extract and BHT, when added as mixtures of 75:25, 50:50 and
25:75 had a synergistic effect on preventing oxidation of soybean oil. Superoxide radicals (O2) were generated by phenazine
methosulphate-NADH system and rosemary extract was found to scavenge O2 in a concentration-dependent manner. The
antioxidative effect observed is believed to be partly due to this radical scavenger activity.

1997 Academic Press Limited


Keywords: rosemary; antioxidants; superoxide radical; lipid oxidation

Introduction
Lipid oxidation may reduce the flavor and nutritive
value of fats, oils and lipid-containing products. Unsaturated fatty acids are sensitive to oxidation because of
their chemical structure (1). Protein cross-linking,
denaturation, polypeptide chain scission, enzyme inactivation and amino acid destruction in the presence of
oxidizing lipids have been reported (2). Nutritional
quality losses are related to losses in essential fatty
acids, amino acids and vitamins (3). To overcome this
problem, synthetic antioxidants such as butylated
hydroxy toluene (BHT) and butylated hydroxy anisole
(BHA) are incorporated into fats and oils.
It has been known for some time that addition of
certain aromatic herbs or spices to lipid-containing
materials will delay the oxidation process (4, 5).
Therefore, in recent years a lot of interest has been
devoted to preparing antioxidants from natural sources
by extraction, purification and fractionation (6, 7). The
mechanism of protection given by an antioxidant was
postulated to occur at the initial stage and more
effectively during the propagation stage of oxidation by
peroxy radical (ROO ) scavengers such as phenolic
compounds (8).

The peroxy radicals formed are interrupted or inhibited


by a free radical acceptor (phenolic structure), thus
stopping the chain reaction. Such a protection mechanism leads to an increase in the induction period of
antioxidation, and therefore, a longer shelf-life of
packaged fatty food is achieved. Some of the antioxidant-active compounds of rosemary leaves have
been isolated and identified and have been reported to
have both antioxidant and metal chelating activities
(9).
The objective of this work was to study the antioxidative effect of rosemary extract upon storage and
stability of corn and soybean oil to oxidative rancidity,
and to shed light on its mechanism of antioxidation. We
have used refined, bleached and deodorized oils in
order to eliminate the possibility of pigments and other
compounds interfering with the oxidation process. The
aim of conducting experiments in a chemical model
system was to evaluate the oxygen radical scavenger
activity of rosemary extract, since it is believed that this
scavenger activity is one of the many components of
antioxidative action, and to better understand the
mechanism of its protective action.

ROO + AOH > ROOH + AO


d

Eqn [1]

*To whom correspondence should be addressed.


Present address: Department of Molecular Biology and Genetics,
Bosphorus University, 80815-Bebek-Istanbul, Turkey.

Materials and Methods


Refined, deodorized and bleached corn oils were
purchased from the producer, Lavio, and soybean
oils were from Komili. All enzymes, chemicals and

0023-6438/97/010105 + 04$25.00/0/fs960127

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1997 Academic Press Limited

lwt/vol. 30 (1997) No. 1

reagents were the highest quality available and were


purchased either from Sigma Chemical Co. (Poole,
Dorset, U.K.) or from Merck (Darmstadt, Germany).

Peroxide value determination


Oil samples of 10 0.1 g were stored in the dark at
given temperatures in 100 mL erlenmayer flasks with
added antioxidants to give 0.1 g/kg. Peroxide values
were determined by iodimetric titration according to
AOCS (11) and expressed as milliequivalents of peroxide per kilogram of sample. For the evaluation of
synergistic effects, antioxidants as mixtures (given in
the legend of Fig. 3) were added directly to the oils to
give a final concentration of 0.1 g/kg oil and POV was
determined at 24-h intervals. The average standard
deviation was less than 0.10.

Thiobarbituric acid value (TBA)


TBA values were determined according to Pokorny et
al. (12). The chromogen was extracted with butan-1-ol
and the optical density was measured spectrophotometrically at 532 nm by using a Shimadzu UV1200
spectrophotometer. The average standard deviation
was 0.025.

Superoxide generating system


Before studying the effect of rosemary extract on
superoxide anions generated by the enzymatic system
(xanthinexanthine oxidase), the effect of rosemary
extract on xanthine oxidase activity was measured. The
enzyme was assayed by measuring the formation of uric
acid from xanthine (13). Various concentrations of
rosemary extract were added to samples before the
enzyme had been instilled. Nonenzymatic generation of
superoxide anion was measured in samples which
contained 10 mol/L phenazine methosulphate, 78
mol/L NADH and 25 mol/L nitroblue tetrazolium in
0.1 mol/L phosphate buffer, pH 7.4. After 2 min of
incubation at room temperature the colour was read at
560 nm against a blank which did not contain phenazine
methosulphate (13, 14). Rosemary extract was solubilized in DMSO and added to give final concentrations
indicated in Table 2.
In all experiments controls were carried out with no
added antioxidants and the results are the mean of at
least three experiments in duplicate.

POV (meq/kg)

14

12
10

8
6
4
2
0

2
Days

Fig. 1 Control values (j) and effect of 0.1 g/kg rosemary


extract (h) and BHT (r) on the peroxide value (POV) (meq/
kg) of corn oil at 30 C. All data points are means of four
determinations, results did not vary more than 0.10.
*P < 0.05, significantly different from the control, one tailed
student t-test

Results and Discussion


Changes in POV of corn and soybean oils are shown in
Figs 1 and 2, respectively. At the end of the 4th day,
corn oil samples containing rosemary extract and BHT
reached 13.0 and 8.2 meq/kg of POV, respectively,
whereas the control was 16.9. Rosemary extract exerted
a moderately lower protective effect than BHT (Fig. 1).
Soybean oil samples containing 0.1 g/kg of extract and
BHT, at the end of the 4th day, had POV of 9.9 and 6.6,
respectively, whereas the control was 18.2. In both corn
and soybean oils, the extract and BHT significantly
reduced the formation of peroxides (P < 0.05). The
difference between the oxidation of the two oils studied
is believed to be due to the fatty acid composition of
the oils; corn oil containing the highest amount of
linoleate and soybean containing linolenate (15). In
other experiments rosemary extract and BHT were
mixed at 25:75, 50:50 and 75:25 ratios and added at a
total concentration of 0.1 g/kg, and POV of soybean oil
was followed for a period of 4 d. From these results it
was apparent that the antioxidant effect of BHT was
18
16
14
POV (meq/kg)

Preparation of rosemary extract


Rosemary extract was prepared according to Wu et al.
(10). In all experiments, methanol was used as the
extraction solvent and a light yellow, greenish powder
was obtained and stored at 20 C. The obtained
material was odourless and flavourless and was soluble
in oil.

18
16

12
*

10
8

6
4
2
0

2
Days

Fig. 2 Control values (j) and effect of 0.1 g/kg rosemary


extract (h) and BHT (r) on the peroxide value (POV) (meq/
kg) of soybean oil at 30 C. All data points are means of four
determinations, results did not vary more than 0.10.
*P < 0.05, significantly different from the control, one tailed
student t-test

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lwt/vol. 30 (1997) No. 1

18
16
POV (meq/kg)

14
12
10
8
6

*
*

2
0

2
Days

Fig. 3 Control values (j) and effect of mixtures of rosemary


extract and BHT (total concentration 0.1 g/kg) on the
peroxide value (POV) (meq/kg) of soybean oil at 30 C.
Extract: BHT ratio: (h) = 75:25; (r) = 50:50; (e) = 25:75.
All data points are means of four determinations, results did
not vary more than 0.10. *P < 0.05, significantly different
from the control, one tailed student t-test

significantly enhanced by rosemary extract in preventing oxidation (Figs 2 and 3). Extract: BHT (25:75)
mixture reached a POV of 3.5, while BHT alone
reached a POV of 6.6 within 4 d of storage. BHT and
rosemary extract treatment had a stronger antioxidant
effect than either BHT or extract alone when total
additive concentration was the same for all treatments.
Similar effects were observed when sardine oil was
treated with RE and -tocopherol (16, 17). This was
probably due to the lipid antioxidant activity of BHT
and the ability of the extract to regenerate BHT.
Reducing equivalents supplied by rosemary extract
could have been responsible for this effect by regenerating and maintaining the antioxidative effect of
BHT.
Decomposition of hydroperoxides generates aldehydic
compounds that can be quantified by the TBA method
and this method is generally accepted as an effective
method for following lipid oxidation (18). The extract
was found to be as effective as BHT in preventing the
formation of TBA reactive substances (Table 1) and the
results are significantly different from the control
(P < 0.05). Absorbance values for the two oils studied
did not differ in the control samples and rosemary
extract was as effective as BHT when added at 0.1 g/kg
concentrations.
A common test of antioxidant ability is a compounds
effectiveness in reacting with radicals generated in
chemical model systems. For this purpose generation of

superoxide radicals enzymatically by xanthinexanthine oxidase, or nonenzymatically by phenazine


methosulphate-NADH, is generally found to be satisfactory. The phenazine methosulphate-NADH mixture at pH 7.4 generates superoxide anions which can
be measured by its ability to reduce nitroblue tetrazolium (NBT) measured as a rise in absorbance at 560
nm. Any compound that is itself able to react with O2
should decrease the rate of these absorbance changes.
Generation of O2 was checked by the addition of 100
IU/mL superoxide dismutase (SOD) and the reaction
was inhibited by 77.8% in the presence of SOD (Table
2). Rosemary extract inhibited the development of the
colour produced during the reaction of O2 with NBT
in a concentration-dependent manner. Our results
clearly show that rosemary extract is able to scavenge
O2 (P < 0.05) as shown by the inhibition of nitroblue
tetrazolium reduction (Table 2). In preliminary experiments the activity of xanthine oxidase was checked in
the presence of rosemary extract and was found to be
inhibited by 4.6% in the presence of 0.1 g/kg extract,
therefore, scavenger activity of rosemary extract was
not further studied on the enzymatic generation of
superoxide anions. DMSO did not interfere with the
experimental system.
Although in most cases no correlation has been found
between the antioxidative and scavenging properties of
plant flavonoids, results presented here indicate that
the extract, besides having an O2 scavenger property,
is also effective in preventing lipid oxidation. Previous
workers have concluded that the scavenging of O2 was
not the only mechanism for the inhibition of lipid
oxidation (19).
It has been shown recently that some phenolic substances isolated from natural sources can have prooxidant properties depending on the system employed
and the presence of lipophilic or hydrophilic substances
(20). Therefore, it was thought that studying the effect
of crude extracts could be important. Crude extract
contained chemicals having O2 scavenging properties
as well as inhibitory activity against lipid peroxidation.
Rosemary extract has the potential to be added to
foodstuffs as a natural antioxidant, where slight flavour
is not found objectionable by the consumer. Besides,
the synergistic effect offers the possibility of using
rosemary extract and known commercial antioxidants
as mixtures, thus lowering the amount of synthetic
antioxidant to be added and reducing/eliminating the
possibility of toxicity.

Table 1 TBA values at 532 nm of soybean and corn oils after 4 d at 30 C in the presence of antioxidants added as 0.1 g/kg.
Mean values (x) and standard deviations (sx) for n=46
Control
Sample
Soybean oil
Corn oil

RE

BHT

sx

sx

% inhibition

sx

% inhibition

1.0
1.0

0.028
0.042

0.8
0.7

0.042*
0.014*

20
30

0.6
0.5

0.014*
0.010*

40
50

*Significantly different from the control, P<0.05, one tailed student t-test.

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lwt/vol. 30 (1997) No. 1

Table 2 Mean A560 values (x) and standard deviations (sx)


for at least four determinations. % inhibition of nitroblue
tetrazolium (NBT) reduction in the presence of different
concentrations of rosemary extract. Superoxide anions were
generated nonenzymatically as explained in Materials and
Methods
Rosemary
extract
(g/kg)
Control
SOD(100 IU/mL)
0.02
0.04
0.06
0.08
0.1

A560
x
0.411
0.091
0.369
0.252
0.160
0.120
0.116

sx
0.006
0.036*
0.017*
0.040*
0.012*
0.012*
0.050*

% inhibition

77.8
10.2
38.7
61.1
70.8
71.8

*Significantly different from the control, P< 0.05 one-tailed


student t-test.

Acknowledgment
This work was supported by Turkish Scientific and
Technical Research Council grant no TBGAG-95.

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