You are on page 1of 8

354

PHYTOTHERAPY RESEARCH
Phytother. Res. 21, 354361 (2007)
Published online 15 January 2007 in Wiley InterScience
i. GULCIN ET AL.
(www.interscience.wiley.com) DOI: 10.1002/ptr.2069

Determination of Antioxidant and Radical


Scavenging Activity of Basil (Ocimum
basilicum L. Family Lamiaceae) Assayed
by Different Methodologies
Ilhami Glin1, Mahfuz Elmastat2* and Hassan Y. Aboul-Enein3*
1

Atatrk University, Faculty of Science and Arts, Department of Chemistry, TR-25240-Erzurum, Turkey
Gaziosmanpasa University, Faculty of Science and Arts, Department of Chemistry, TR-60240-Tokat, Turkey
3
College of Pharmacy, University of Sharjah, P.O.Box 27272, Sharjah, United Arab Emirates
2

The antioxidant properties of plants have been investigated, in the light of recent scientic developments,
throughout the world due to their potent pharmacological activities and food viability. Basil (Ocimum basilicum
L. Family Lamiaceae) is used as a kitchen herb and as an ornamental plant in house gardens. In the present
study, the possible radical scavenging and antioxidant activity of the water (WEB) and ethanol extracts (EEB)
of basil was investigated using different antioxidant methodologies: 1,1-diphenyl-2-picryl-hydrazyl (DPPH)
free radical scavenging, scavenging of superoxide anion radical-generated non-enzymatic system, ferric
thiocyanate method, reducing power, hydrogen peroxide scavenging and metal chelating activities. Experiments revealed that WEB and EEB have an antioxidant effects which are concentration-dependent. The total
antioxidant activity was performed according to the ferric thiocyanate method. At the 50 g/mL concentration,
the inhibition effects of WEB and EEB on peroxidation of linoleic acid emulsion were found to be 94.8% and
97.5%, respectively. On the other hand, the percentage inhibition of a 50 g/mL concentration of BHA, BHT
and -tocopherol was found to be 97.1%, 98.5% and 70.4% inhibition of peroxidation of linoleic acid emulsion, respectively. In addition, WEB and EEB had effective DPPH radical scavenging, superoxide anion
radical scavenging, hydrogen peroxide scavenging, reducing power and metal chelating activities. Additionally,
these various antioxidant activities were compared with BHA, BHT and -tocopherol as reference antioxidants. The additional total phenolic content of these basil extracts was determined as the gallic acid equivalent
and were found to be equivalent. Copyright 2007 John Wiley & Sons, Ltd.
Keywords: antioxidant activity; radical scavenging; metal chelating; reducing power; basil; Ocimum basilicum.

INTRODUCTION
A great number of aromatic, spicy, medicinal and other
plants exhibit antioxidant properties. Sources of natural
antioxidants are primarily, plant phenolics that may
occur in all parts of plants such as the fruits, vegetables, nuts, seeds, leaves, roots and barks (Pratt and
Hudson, 1990). Crude extracts of fruits, herbs, vegetables, cereals and other plant materials rich in phenolics
are increasingly of interest in the food industry, because they retard oxidative degradation of lipids and
thereby improve the quality and nutritive value of food
(Kahkonen et al., 1999; Rice Evans et al., 1995). The
extraction, characterization and utilization of natural
antioxidants that may serve as potent candidates in
combating carcinogenesis and aging process are in
progress (Namiki, 1990; Mathew and Abraham, 2006).
Basil (Ocimum basilicum L. Family Lamiaceae)
is used as a kitchen herb, a culinary herb and as an
* Correspondence to: Dr Mahfuz Elmastas, Gaziosmanpasa University,
Faculty of Science and Arts, Department of Chemistry, TR-60240-Turkey.
E-mail: elmastas@gop.edu.tr
* Correspondence to: Professor H. Y. Aboul-Enein, College of Pharmacy,
University of Sharjah, P.O.Box 27272, Sharjah, United Arab Emirates.
E-mail: hyaboulenein@yahoo.com
Copyright 2007 John Wiley & Sons, Ltd.
Copyright 2007 John Wiley & Sons, Ltd.

ornamental plant in traditional medicine. Also, it is a


well-known source of avouring principles (Javanmardi
et al., 2003). Basil has been used in Turkish folk medicine for many years and has several therapeutic effects
for several conditions such as digestive and appetizing
effects (Asimgil, 1997). It was reported that the leafy
parts of basil had tonic, antiseptic (Kosekia et al., 2002)
and insecticidal properties (Umerie et al., 1998). It is
also known the leaves of basil are suitable for the treatment of pain and cough (Basilico and Basilico, 1999).
In addition, basil is used for cough treatment,
inammations, dyspepsia, aches and pains (McClatchey,
1996). The essential oil from basil showed an inhibitory
effect on Aspergillus ochraceus (Basilico and Basilico,
1999) and antimicrobial activity (Hili et al., 1997).
Furthermore, there are a number of reports on the
antibacterial properties of this herb (Carson and Riley,
1993; Balchin et al., 1998; Boyanova and Neshev, 1999;
Hammer et al., 1999).
The activity of basil against multi-drug resistant
clinical isolates from the genera Staphylococcus,
Enterococcus and Pseudomonas has been studied
(Opalchenova and Obreshkova, 2003). Javanmardi
and co-workers (2003) measured the relative capacity
of antioxidants to scavenge the ABTS+ radical compared with the antioxidant potency of Trolox as a standard. Furthermore, it is known to contain antioxidant
Received
2 September
2006
Phytother.
Res. 21,
354361 (2007)
Accepted DOI:
30 October
2006
10.1002/ptr

355

ANTIOXIDANT ACTIVITY OF BASIL

phenolic compounds such as rosmarinic acid (Tada


et al., 1996). The essential oil vapours from basil showed
toxicities against the nymphs and adults of Tetranychus
urticae and adults of Bemisia tabaci (Aslan et al., 2004).
The essential basil oil obtained from the aerial parts
of basil showed four major components namely: linalol,
methylchavikol, methylcinnamat and linolen which were
identied by gas chromatography (Opalchenova and
Obreshkova, 2003).
The importance of free radicals and reactive oxygen
species (ROS) has attracted increasing attention over
the past decade. ROS, which include free radicals such
as superoxide anion radicals (O2), hydroxyl radicals
(OH) and non free-radical species such as H2O2 and
singlet oxygen (1O2), are various forms of activated
oxygen. These molecules are exacerbating factors in
cellular injury and aging process (Glin et al., 2002a,
2002b). In living organisms, various ROS can form in
different ways. Normal aerobic respiration stimulates
polymorphonuclear leukocytes and macrophages, and
peroxisomes appear to be the main endogenous sources
of most of the oxidants produced by cells. Exogenous
sources of ROS include tobacco smoke, certain pollutants, organic solvents and pesticides. ROS induce
some oxidative damage to biomolecules such as lipids,
nucleic acids, proteins and carbohydrates. Their damage
causes ageing, cancer and many other diseases. As
a result, ROS have been implicated in more than 100
diseases; including malaria, acquired immunodeciency
syndrome, heart disease, stroke, arteriosclerosis, diabetes, cancer and gastric ulcer (Elmastas, 2006; Glin
et al., 2005b). ROS are continuously produced during
normal physiological events and are removed by
antioxidant defence mechanisms. There is a balance
between the generation of ROS and inactivation of ROS
by the antioxidant system in organisms. Under pathological conditions, ROS are overproduced and result in
lipid peroxidation and oxidative stress. ROS are formed
when endogenous antioxidant defences are inadequate.
The imbalance between ROS and antioxidant defence
mechanisms leads to oxidative modication in cellular
membrane or intracellular molecules (Glin et al.,
2005a; Duh et al., 1999).
Many antioxidant compounds, naturally occurring
in plant sources have been identied as free radical
or active oxygen scavengers. Recently, interest has
increased considerably in nding naturally occurring
antioxidants for use in foods or medicinal materials
to replace synthetic antioxidants, which are restricted
due to their side effects such as carcinogenicity (Glin
et al., 2006b; Glin, 2005). Natural antioxidants protect the human body from free radicals and retard the
progress of many chronic diseases as well as lipid
oxidative rancidity in foods (Ito et al., 1983). Hence,
studies on natural antioxidants have gained increasingly
greater importance (Elmastas et al., 2004).
Basil (Ocimum basilicum L. Family Lamiaceae)
is grown commercially as a cultivated herb in Turkey,
the Mediterranean region and in many other parts of
the world. This popular herb is used as both a fresh
and a dried food spice and in traditional medicine.
In addition, basil is valued for its pharmaceutical properties; for example, the aromatic oils produced in their
leaves (Chang et al., 1977; Gang et al., 2001; Bais et al.,
2002). Spices used in different types of food to improve
avours, since ancient times, are well known for their
Copyright 2007 John Wiley & Sons, Ltd.

antioxidant properties. It was reported that extracts


obtained from spices had antioxidant activities (Glin,
2006b). However, there is no information on the 1,1diphenyl-2-picryl-hydrazyl free radical scavenging activity, superoxide anion radical scavenging activity, total
antioxidant activity by ferric thiocyanate method, reducing power, hydrogen peroxide scavenging and metal
chelating activities of the water extract of basil (WEB)
and the ethanol extract of basil (EEB). The aim of this
present study was to evaluate the antioxidant activity
using these antioxidant methods.

MATERIALS AND METHODS


Chemicals. Nicotinamide adenine dinucleotide (NADH),
linoleic acid, -tocopherol, butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), nitroblue
tetrazolium (NBT), phenazine methosulphate (PMS),
the stable free radical 1,1-diphenyl-2-picryl-hydrazyl
(DPPH), 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonic acid)1,2,4-triazine (Ferrozine), trichloroacetic acid (TCA)
and polyoxyethylenesorbitan monolaurate (Tween-20)
were obtained from Sigma (Sigma-Aldrich GmbH,
Sternheim, Germany). Ammonium thiocyanate was
purchased from Merck. All other chemicals used were
analytical grade and obtained from either Sigma-Aldrich
or Merck.
Plant materials and extraction procedures. Basil
(Ocimum basilicum L.) leaves were obtained from a
local market at Erzurum province in Turkey. For
ethanol extraction, 25 g powder of basil leaves ground
into a ne powder in a mill and were mixed ve times
with 100 mL ethanol. Extraction continued until the
extraction solvents became colourless (total solvent
volume 400 mL). The obtained extracts were ltered
over Whatman No.1 paper and the ltrate was collected,
then ethanol was removed by a rotary evaporator at
50 C.
For preparation of lyophilized water extraction, 25 g
sample of basil leaves ground into a ne powder in
a mill and was mixed with 500 mL boiling water by
magnetic stirrer during 10 min. Then the extract was
ltered over Whatman No.1 paper. The ltrates were
frozen and lyophilized in a lyophilizator at 5 mm Hg
pressure and at 50 C (Labconco, Freezone 1L). Both
extracts were placed in a plastic bottle and stored at
20 C until used.
Total antioxidant activity determination by ferric
thiocyanate method (FTC). The antioxidant activity of
WEB, EEB and standards was determined according
to the ferric thiocyanate method (Mitsuda et al., 1996).
For preparation of stock solutions, 10 mg of each WEB
or EEB was dissolved in 10 mL water. Then, the solution, which contained different concentrations of stock
WEB and EEB solution (25 and 50 g/mL) or standard
samples (50 g/mL) in 2.5 mL of potassium phosphate
buffer (0.04 M, pH 7.0), was added to 2.5 mL of linoleic
acid emulsion in potassium phosphate buffer (0.04 M,
pH 7.0). The mixed solution (5 mL) was incubated
at 37 C in a polyethylene ask. The peroxide level
was determined by reading the absorbance at 500 nm
in a spectrophotometer (Bio-Crom GmbH, Zurich,
Phytother. Res. 21, 354361 (2007)
DOI: 10.1002/ptr

356

i. GULCIN ET AL.

Switzerland) after reaction with FeCl2 and thiocyanate


at intervals during incubation. During the linoleic acid
oxidation, peroxides are formed, which oxidize Fe+2 to
Fe+3. The latter ions form a complex with thiocyanate
and this complex has a maximum absorbance at 500 nm.
Five mL linoleic acid emulsion contained 17.5 g Tween20, 15.5 L linoleic acid and 0.04 M potassium phosphate
buffer (pH 7.0). On the other hand, the 5 mL control
was composed of 2.5 mL linoleic acid emulsion and
2.5 mL 0.04 M potassium phosphate buffer (pH 7.0). This
step was repeated every 5 h until the control reached
its maximum absorbance value. Therefore, high absorbance indicates a high linoleic acid emulsion oxidation.
Solutions without added extracts were used as blank
samples. All data on total antioxidant activity are the
average of duplicate analyses. The percentage inhibition of lipid peroxidation in linoleic acid emulsion was
calculated by following equation.

Inhibition of lipid peroxidation (%)


ASample

= 100
100
AControl

where AControl is the absorbance of the control reaction


and ASample is the absorbance in the presence of the
sample of WEB, EEB or standard compounds (Glin
et al., 2004b).
Total reductive capability. The samples prepared for
ferric thiocyanate method were used for this and the
other assays. The reducing power of WEB and EEB
was determined by the method of Oyaizu (1986). Different concentrations of WEB and EEB (2550 g/mL)
in 1 mL of distilled water were mixed with phosphate
buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 mL, 1%). The mixture was
incubated at 50 C for 20 min. Aliquots (2.5 mL) of
trichloroacetic acid (10%) were added to the mixture,
which was then centrifuged for 10 min at 1000 g (MSE
Mistral 2000, UK). The upper layer of the solution
(2.5 mL) was mixed with distilled water (2.5 mL) and
FeCl3 (0.5 mL, 0.1%) and the absorbance was measured at 700 nm in a spectrophotometer. Increased
absorbance of the reaction mixture indicates increased
reducing power.
Ferrous ions (Fe2++) chelating activity. The chelating
activity of ferrous ions by the WEB, EEB and standards was estimated by the method of Dinis et al. (1994).
Briey, extracts (2550 g/mL) in 0.4 mL were added
to a solution of 2 mM FeCl2 (0.05 mL). The reaction
was initiated by the addition of 5 mM ferrozine (0.2 mL)
and the total volume was adjusted to 4 mL ethanol.
Then the mixture was shaken vigorously and left standing at room temperature for 10 min. Absorbance of the
solution was then measured spectrophotometrically at
562 nm. All tests and analyses were run in triplicate
and averaged. The percentage of inhibition of the
ferrozineFe2+ complex formation was calculated using
the formula given below:
Ferrous ions chelating effect (%)
AControl ASample
=
100
AControl

Copyright 2007 John Wiley & Sons, Ltd.

where AControl is the absorbance of the control, and


ASample is the absorbance in the presence of the sample
of WEB and EEB or standards. The control contains
FeCl2 and ferrozine, complex formation molecules
(Elmastas et al., 2006b).
Hydrogen peroxide scavenging activity. The hydrogen
peroxide scavenging ability of WEB and EEB was determined according to the method of Ruch et al. (1989).
A solution of H2O2 (40 mM) was prepared in phosphate
buffer (pH 7.4). Extracts (50 g/mL) in distilled water
were added to a H2O2 solution (0.6 mL, 40 mM).
The absorbance value of the reaction mixture was
recorded at 230 nm. Blank solution contained the
phosphate buffer without H2O2. The percentage of H2O2
scavenging of WEB, EEB and standard compounds was
calculated as:
H 2O2 scavenging effect (%)
AControl ASample
=
100
AControl

where AControl is the absorbance of the control, and


ASample is the absorbance in the presence of the sample
of WEB, EEB or standards (Glin et al., 2003b).
1,1-Diphenyl-2-picryl-hydrazyl (DPPH) free radical
scavenging activity. The free-radical scavenging capacity of WEB and EEB was evaluated with the DPPH
stable radical following the methodology described by
Blois (1958). This method is described extensively elsewhere (Elmastas, 2006), wherein the bleaching rate of
a stable free radical, DPPH is monitored at a characteristic wavelength in the presence of the sample. In
its radical form, DPPH absorbs at 517 nm, but upon
reduction by an antioxidant or a radical species its
absorption decreases (Elmastas et al., 2006a). Briey,
0.1 mM solution of DPPH in ethanol was prepared and
1 mL of this solution was added to 3 mL of WEB and
EEB solution in water at different concentrations (25
50 g/mL). Thirty minutes later, the absorbance was
measured at 517 nm. A lower absorbance of the reaction mixture indicates higher free radical scavenging
activity. The DPPH concentration (mM) in the reaction medium was calculated from the following calibration curve, determined by linear regression (R2 = 0.999):
Absorbance = 9.2872 [DPPH] + 0.097
The capability to scavenge the DPPH radical was
calculated using the following equation:

DPPH scavenging effect (%)


AControl ASample
=
100
AControl

where AControl is the absorbance of the control reaction


and ASample is the absorbance in the presence of WEB
and EEB (Glin et al., 2005c).
Superoxide anion radical scavenging activity. Measurement of the superoxide anion scavenging activity of
WEB and EEB was based on the method described by
Liu et al. (1997). Superoxide radicals are generated in
Phytother. Res. 21, 354361 (2007)
DOI: 10.1002/ptr

ANTIOXIDANT ACTIVITY OF BASIL

PMS-NADH systems by the oxidation of NADH and


assayed by the reduction of NBT. In these experiments,
the superoxide radicals were generated in 3 mL of TrisHCl buffer (16 mM, pH 8.0) containing 1 mL of NBT
(50 M) solution, 1 mL NADH (78 M) solution and
sample solution of WEB and EEB (50 g/mL) in
water. The reaction started by adding 1 mL PMS solution (10 M) to the mixture. The reaction mixture was
incubated at 25 C for 5 min, and the absorbance at
560 nm was measured against blank samples. L-Ascorbic
acid was used as a control. Decreased absorbance of
the reaction mixture indicates increased superoxide
anion scavenging activity. The percentage inhibition of
superoxide anion generation was calculated using the
following formula:

O2 scavenging effect (%)


AControl ASample
=
100
AControl

where AControl is the absorbance of the control and ASample


is the absorbance of WEB and EEB or standards
(Glin et al., 2006b).
Statistical analysis. All data on total antioxidant
activity are the average of duplicate analyses. The other
analyses were performed in triplicate. The data were
recorded as mean standard deviation and analysed
by SPSS (version 9.0 for Windows 98, SPSS Inc.). Oneway analysis of variance was performed by ANOVA
procedures. Signicant differences between means were
determined by Duncans multiple range tests. Values
of p < 0.05 were regarded as signicant and p < 0.01
very signicant.

357

Figure 1. Comparison of total antioxidant activities of 50 g/mL


concentration of WEB, EEB and -tocopherol as standard antioxidant was determined by ferric thiocyanate method in linoleic
acid emulsion. WEB, water extract of basil (Ocimum basilicum
L.); EEB, ethanol extract of basil (Ocimum basilicum L.). TOC,
-Tocopherol.

mined by the ferric thiocyanate method and increased


steadily with the increasing concentration. WEB, EEB
and standards compounds exhibited effective antioxidant
activity. At the 50 g/mL concentration, the effects
of WEB, EEB BHA, and BHT on lipid peroxidation
of the linoleic acid emulsion are shown in Fig. 1. The
percentage inhibition of these samples in the linoleic
acid system was 94.8%, 97.5%, 97.1% and 98.5%, respectively, and greater than that -tocopherol (70.4%)
at the same concentration.
As can be seen in Fig. 1, the percentage inhibition
of peroxidation in the linoleic acid system by 25 and
50 g/mL concentrations of WEB and EEB was found
to be 73.8%, 94.8%, 90.8% and 95.0%, respectively.
On the other hand, the percentage inhibition of 50 g/
mL concentrations of BHA, BHT and -tocopherol was
found to be 97.1%, 98.5% and 70.4%, respectively.

RESULTS AND DISCUSSION


Total reductive capability by Fe3++-Fe2++ transformation
Many studies have shown that natural antioxidants in
plants are closely related to their biofunctionalities such
as the reduction of chronic diseases and inhibition of
pathogenic bacteria growth, which are often associated
with the termination of free radical propagation in biological systems (Covacci et al., 2001). Thus, antioxidant
capacity is widely used as a parameter to characterize
food or medicinal plants and their bioactive components. In this study, the antioxidant activity of the WEB,
EEB BHA, BHT and -tocopherol have been evaluated in a series of in vitro tests: 1,1-diphenyl-2-picrylhydrazyl free radical scavenging, ferric thiocyanate
method, reducing power, scavenging of superoxide anion
radical-generated non-enzymatic system, hydrogen peroxide scavenging and metal chelating activities.
Total antioxidant activity determination in linoleic
acid emulsion system by ferric thiocyanate method
Lipid peroxidation leads to rapid development of
rancid and stale avours and is considered as a primary
mechanism of quality deterioration in lipid foods and
oils (Gntensperger et al., 1998). The total antioxidant
activity of WEB, EEB and the reference compounds
such BHA, BHT, -tocopherol and trolox was deterCopyright 2007 John Wiley & Sons, Ltd.

In this assay, the yellow colour of the test solution


changes to various shades of green and blue colour
depending upon the reducing power of each antioxidant samples. The reducing capacity of a compound
may serve as a signicant indicator of its potential
antioxidant activity. The presence of reductants such
as antioxidants substances in the antioxidant samples
causes the reduction of the Fe3+/ferricyanide complex
to the ferrous form. Therefore, the Fe2+ can be monitored by measuring the formation of Perls Prussian
blue at 700 nm (Chung et al., 2002; Glin, 2006c).
Figure 2 depicts the reducing power of the WEB,
EEB and standards (BHA, BHT and -tocopherol)
using the potassium ferricyanide reduction method. For
the measurements of the reductive ability, the Fe3+-Fe2+
transformation was investigated in the presence of WEB
and EEB using the method of Oyaizu (1986). Like the
antioxidant activity, the reducing power of WEB, EEB
BHA, BHT and -tocopherol increased with increasing concentration. At the different concentrations, WEB
and EEB showed higher activities than the control and
these differences were statistically signicant (p < 0.05).
The reducing power of WEB and EEB and standard
compounds exhibited the following order: BHA > BHT
> -tocopherol > EEB > WEB.
Phytother. Res. 21, 354361 (2007)
DOI: 10.1002/ptr

358

i. GULCIN ET AL.

Figure 2. Total reductive potential of different concentrations of


WEB, EEB, BHA, BHT and -tocopherol using spectrophotometric
detection of the Fe3+-Fe2+ transformations. WEB, water extract
of basil (Ocimum basilicum L.); EEB, ethanol extract of basil
(Ocimum basilicum L.); BHA, butylated hydroxyanisole; BHT,
butylated hydroxytoluene. TOC, -Tocopherol.

Figure 3. Ferrous ions chelating effect of different concentrations of WEB, EEB, BHA, BHT and -tocopherol. WEB, water
extract of basil (Ocimum basilicum L.); EEB, ethanol extract of
basil (Ocimum basilicum L.); BHA, butylated hydroxyanisole;
BHT, butylated hydroxytoluene. TOC, -Tocopherol.

the control was statistically signicant (p < 0.01). The


percentages of metal scavenging capacity of 50 g/mL
concentration of WEB, EEB, BHA, BHT and tocopherol were found as 89%, 85%, 66%, 62%, 50%,
respectively. The metal scavenging effect of these
samples decreased in the order of WEB > EEB >
BHA > BHT > -tocopherol.
Metal chelating capacity was signicant, since it
reduced the concentration of the catalysing transition metal in lipid peroxidation. It was reported that
chelating agents are effective as secondary antioxidants
because they reduce the redox potential thereby stabilizing the oxidized form of the metal ion. The data
obtained from Fig. 3 reveal that WEB and EEB demonstrate a marked capacity for iron binding, suggesting
that their action as a peroxidation protector may be
related to its iron binding capacity.
Hydrogen peroxide scavenging activity
Hydrogen peroxide can be formed in vivo by many
oxidase enzymes such as superoxide dismutase. It can
cross membranes and may slowly oxidize a number of
compounds. The ability of WEB and EEB to scavenge
hydrogen peroxide was determined according to the
method of Ruch et al. (1989) and is shown in Fig. 4 and
compared with that of BHA, BHT and -tocopherol as
standards. The 50 g/mL of WEB and EEB exhibited
57% and 54% scavenging activity on hydrogen peroxide, respectively. On the other hand, BHA, BHT and
-tocopherol exhibited 88%, 97% and 93% hydrogen
peroxide scavenging activity at the same concentration.
These results showed that the standards had stronger
hydrogen peroxide scavenging activity than WEB and
EEB. There was a statistically signicant correlation
between those values and the control (p < 0.01). The
hydrogen peroxide scavenging effect of 50 g/mL concentration of WEB, EEB and standards decreased
in the order of BHT > -tocopherol > BHA > WEB >
EEB. Hydrogen peroxide itself is not very reactive, but
it can sometimes be toxic to cells because it may give
rise to hydroxyl radicals in the cells. Addition of hydrogen peroxide to cells in culture can lead to transition

Ferrous ions chelating capacity


The ferrous ion chelating activities of WEB, EEB, BHA,
BHT and -tocopherol are shown in Fig. 3. The chelating activity of ferrous ions by the WEB, EEB and standards was determined according to the method of Dinis
et al. (1994). Ferrozine can quantitatively form complexes with Fe2+. In the presence of chelating agents
such as antioxidant compounds, the complex formation
is disrupted, resulting in a decrease in the red colour
of the complex. Measurement of colour reduction therefore allows an estimation of the metal chelating activity
of the coexisting chelator. In this assay, WEB, EEB
and three standard compounds interfered with the formation of ferrous and ferrozine complex, suggesting
that they have chelating activity and are able to
capture ferrous ion before ferrozine.
The metal chelating activities of WEB, EEB were
concentration-dependent. The absorbance of Fe2+ferrozine complex was linearly decreased concentrationdependently (from 25 to 50 g/mL). The difference
between the 50 g/mL concentration of WEB, EEB and
Copyright 2007 John Wiley & Sons, Ltd.

Figure 4. Determination of hydrogen peroxide scavenging ability of 50 g/mL concentration of WEB, EEB, BHA, BHT and tocopherol. WEB: Water extract of basil (Ocimum basilicum L.);
EEB, ethanol extract of basil (Ocimum basilicum L.); BHA,
butylated hydroxyanisole; BHT, butylated hydroxytoluene. TOC,
-Tocopherol.
Phytother. Res. 21, 354361 (2007)
DOI: 10.1002/ptr

ANTIOXIDANT ACTIVITY OF BASIL

359

Antioxidant properties, especially radical scavenging


activities, are very important due to the deleterious
role of free radicals in foods and in biological systems.
Excessive formation of free radicals accelerates the
oxidation of lipids in foods and decreases food quality
and consumer acceptance (Min, 1998). In this study,
the antioxidant activities of WEB, EEB and standard
antioxidants such as BHA, BHT and -tocopherol were
determined using a DPPH method. DPPH has been
widely used to evaluate the free radical scavenging
effectiveness of various antioxidant substances in
food systems (Cotelle et al., 1996; zelik et al., 2003;
Elmastas, 2006).
DPPH free radical scavenging is an accepted mechanism by which antioxidants act to inhibit lipid oxidation,
so scavenging of DPPH radical was used in this work.
The method has been used extensively to predict antioxidant activities because of the relatively short time
required for analysis. In the DPPH assay, the antioxidants were able to reduce the stable radical DPPH
to the yellow-coloured diphenyl-picrylhydrazine. The
method is based on the reduction of alcoholic DPPH
solution in the presence of a hydrogen-donating
antioxidant due to the formation of the non-radical
form DPPH-H by the reaction (Glin, 2006c; Glin
et al., 2004b). Figure 5 illustrates a signicant decrease
(p < 0.05) in the concentration of DPPH radical due to
the scavenging ability of WEB, EEB and standards.
BHA, BHT and -tocopherol were used as reference
radical scavengers. The scavenging effect of WEB and
EEB and standards on the DPPH radical decreased
in the order: -tocopherol > BHA > EEB > BHT >
WEB, which was 69%, 67%, 65%, 62%, 55% at a concentration of 50 g/mL, respectively. The free radical

scavenging activity of these samples also increased with


increasing concentration.
Superoxide anion, which is a reduced form of
molecular oxygen, has been implicated in the initiating oxidation reactions associated with aging (Wickens,
2001). Also, it has been implicated in several pathophysiological processes, due to its transformation into
more reactive species such as hydroxyl radical that
initiates lipid peroxidation. Superoxide has also been
observed to directly initiate lipid peroxidation (Yen and
Duh, 1994). It has also been reported that antioxidant
properties of some avonoids are effective mainly via
scavenging of superoxide anion radicals (Elmastas, 2006;
Aruoma, 1998). Superoxide anion plays an important
role in the formation of other reactive oxygen species
such as hydrogen peroxide, hydroxyl radical and singlet oxygen, which induce oxidative damage in lipids,
proteins and DNA (Glin et al., 2006b). Also, superoxide anion is an oxygen-centred radical with selective reactivity. This species is produced by a number
of enzyme systems in autooxidation reactions and by
nonenzymatic electron transfers that univalently reduce
molecular oxygen. It can also reduce certain iron complex such as cytochrome c.
Superoxide anion derived from dissolved oxygen
by PMS-NADH coupling reaction reduces NBT in this
system. The decrease of absorbance at 560 nm with
antioxidants indicates the consumption of superoxide
anion in the reaction mixture (Glin et al., 2006b;
Oktay et al., 2003). Figure 6 shows the percentage
inhibition of superoxide radical generation by a 50 g/
mL concentration of WEB, EEB, BHA, BHT and
-tocopherol. The inhibition of superoxide radical
generation by WEB and EEB and standards were found
to be statistically similar (p > 0.05). As shown in Fig. 6,
the percentage inhibition of superoxide generation by
a 50 g/mL concentration of WEB and EEB was found
to be 97.9% and 95.4%. On the other hand, at the
same concentration, BHA, BHT and -tocopherol exhibited 69.6%, 82.2% and 75.4% superoxide radical
scavenging activity, respectively. The superoxide radical scavenging activity of those samples were in the
following order: WEB > EEB > BHT > -tocopherol >
BHA.

Figure 5. Free radical scavenging activity of WEB, EEB, BHA,


BHT and -tocopherol was spectrophotometrically measured at
517 nm using the DPPH assay. WEB, water extract of basil
(Ocimum basilicum L.); EEB, ethanol extract of basil (Ocimum
basilicum L.); BHA, butylated hydroxyanisole; BHT, butylated
hydroxytoluene. TOC, -Tocopherol.

Figure 6. Comparison of superoxide anion radical scavenging


activity of 50 g/mL concentration of WEB, EEB, BHA, BHT and
-tocopherol by the PMS-NADH-NBT method. WEB, water
extract of basil (Ocimum basilicum L.); EEB, ethanol extract of
basil (Ocimum basilicum L.); BHA, butylated hydroxyanisole;
BHT, butylated hydroxytoluene. TOC, -Tocopherol.

metal ion-dependent OH radical mediated oxidative


DNA damage. Levels of hydrogen peroxide at or
below about 2050 mg seem to have limited cytotoxicity to many cell types. Thus, removing hydrogen peroxide as well as superoxide anion is very important for
protection of food systems.
Radical scavenging activity

Copyright 2007 John Wiley & Sons, Ltd.

Phytother. Res. 21, 354361 (2007)


DOI: 10.1002/ptr

360

i. GULCIN ET AL.

CONCLUSION
The antioxidant properties of plant extracts should be
evaluated in a variety of model systems using several
different indices to ensure the effectiveness of such
antioxidant materials. The results obtained in this study

clearly showed that WEB and EEB have powerful antioxidant activity against various antioxidant systems in
vitro, moreover, these extracts can be used as easily
accessible source of natural antioxidants and as a possible food supplement or in pharmaceutical applications.
It can also be used in stabilizing food against oxidative
deterioration.

REFERENCES
Aruoma OI. 1998. Free radicals, oxidative stress, and antioxidants in human health and disease. J Am Oil Chem Soc 75:
199212.
Asimgil A. 1997. Sifali Bitkiler. Timas Yaynlar: stanbul; 101
102.
Aslan I, zbek H, alma3ur , Sahin F. 2004. Toxicity of essential
oil vapours to two greenhouse pests, Tetranychus urticae
Koch and Bemisia tabaci Genn. Indian Crop Prod 19: 167
173.
Bais HP, Walker TS, Schweizer HP, Vivanco JM. 2002. Root
specific elicitation and antimicrobial activity of rosmarinic
acid in hairy root cultures of Ocimum basilicum. Plant
Physiol Biochem 40: 983995.
Balchin ML, Buchbauer G, Ribisch K, Wenger MT. 1998.
Comparative antibacterial effects of novel Pelargonium
essential oils and solvent extracts. Lett Appl Microbiol 27:
135141.
Basilico MZ, Basilico JC. 1999. Inhibitory effects of some spice
essential oils on Aspergillus ochraceus NRRL 3174 growth
and ochratoxin. A production. Lett Appl Microbiol 29: 238
241.
Blois MS. 1958. Antioxidant determinations by the use of a
stable free radical. Nature 26: 11991200.
Boyanova L, Neshev G. 1999. Inhibitory effect of rose oil prosducts on Helicobacter pylori growth in vitro: preliminary
report. J Med Microbiol 48: 705706.
Carson CF, Riley TV. 1993. Antimicrobial activity of the essential
oil of Melaleuca alternifolia. Lett Appl Microbiol 16: 4955.
Chang SS, Ostric-Matijasevic B, Hsieh OAL, Huang CL. 1977.
Natural antioxidant from rosemary and sage. J Food Sci
42: 11021107.
Chung YC, Chang CT, Chao WW, Lin CF, Chou ST. 2002.
Antioxidative activity and safety of the 50% ethanolic extract from red bean fermented by Bacillus subtilis IMR-NK1.
J Agric Food Chem 50: 24542458.
Cotelle N, Bemier JL, Catteau JP, Pommery J, Wallet JC, Gaydou
EM. 1996. Antioxidant properties of hydroxyl-flavones. Free
Radic Biol Med 20: 3543.
Covacci V, Torsello A, Palozza P et al. 2001. DNA oxidative
damage during differentiation of HL-60 human promyelocytic
leukemia cells. Chem Res Toxicol 14: 14921497.
Dinis TCP, Madeira VMC, Almeida LM. 1994. Action of phenolic
derivates (acetoaminophen, salicylate and 5-aminosalicylate)
as inhibitors of membrane lipid peroxidation and as peroxyl
radical scavengers. Arch Biochem Biophys 315: 161169.
Duh PD, Tu YY, Yen GC. 1999. Antioxidant activity of water
extract of harng jyur (Chrysanthemum morifolium Ramat).
Lebens Wiss Technol 32: 269277.
Elmastas M, Demirtas I, Isildak O, Aboul-Enein HY. 2006a. Antioxidant activity of S-carvone isolated from spearmint
(Mentha Spicata L. Fam Lamiaceae). J Liquid Chromatogr
Relat Technol 29: 15641475.
Elmastas M, Glin I, Beydemir (, Kfrevioglu I, Aboul-Enein
HY. 2006b. A study on the in vitro antioxidant activity of
juniper (Juniperus communis L.) seeds extracts. Anal Lett
39: 4765.
Elmastas M, Ozturk L, Gokce I, Erenler R, Aboul-Enein HY. 2004.
Determination of antioxidant activity of marshmallow flower
(Althaea officinalis L.). Anal Lett 37: 18591869.
Elmastas M, Trkekul , ztrk L, Glin , I9ldak , AboulEnein HY. 2006a. The antioxidant activity of two wild edible
mushrooms (Morchella vulgaris and Morchella esculanta).
Comb Chem High T Scr 6: 443448.
Gang DR, Wang J, Dudareva N et al. 2001. An investigation of
the storage and biosynthesis of phenylpropenes in sweet
basil. Plant Physiol 125: 539555.
Copyright 2007 John Wiley & Sons, Ltd.

Glin I. 2005. The antioxidant and radical scavenging activities


of black pepper (Piper nigrum) seeds. Int J Food Sci Nutr
56: 491499.
Glin I. 2006b. Antioxidant activity of caffeic acid (3,4dihydroxycinnamic acid). Toxicology 217: 213220.
Glin I. 2006c. Antioxidant and antiradical activities of Lcarnitine. Life Sci 78: 803811.
Glin I, Alici HA, Cesur M. 2005a. Determination of in vitro
antioxidant and radical scavenging activities of propofol.
Chem Pharm Bull 53: 281285.
Glin I, Berashvili D, Gepdiremen A. 2005b. Antiradical
and antioxidant activity of total anthocyanins from Perilla
pankinensis Decne. J Ethnopharmacol 101: 287293.
Glin I, Beydemir (, (at IG, Kfrevioglu I. 2005c. Evaluation
of antioxidant activity of cornelian cherry (Cornus mas L.).
Acta Aliment Hung 34: 193202.
Glin I, Bykokuroglu ME, Oktay M, Kfrevioglu I. 2002a.
On the in vitro antioxidant properties of melatonin. J Pineal
Res 33: 167171.
Glin I, Elias R, Gepdiremen A, Boyer L. 2006a. Antioxidant
activity of lignans from fringe tree (Chionanthus virginicus
L.). Eur Food Res Technol 223: 759767.
Glin I, Mshvildadze V, Gepdiremen A, Elias R. 2006b. Antioxidant activity of a triterpenoid glycoside isolated from
the berries of Hedera colchica: 3-O-(-D-glucopyranosyl)hederagenin. Phytother Res 20: 130134.
Glin I, Mshvildadze V, Gepdiremen A, Elias R. 2006c. Screening of antioxidant and antiradical activity of monodesmosides and crude extract from Leontice smirnowii tuber.
Phytomedicine 13: 343351.
Glin I, Oktay M, Kireci E, Kfrevioglu I. 2003b. Screening
of antioxidant and antimicrobial activities of anise (Pimpinella
anisum L.) seed extracts. Food Chem 83: 371382.
Glin I, Oktay M, Kfrevioglu I, Aslan A. 2002b. Determination of antioxidant activity of lichen Cetraria islandica (L)
Ach. J Ethnopharmacol 79: 325329.
Glin I, (at IG,Beydemir (, Kfrevioglu I. 2004b. Evaluation of the in vitro antioxidant properties of extracts of
broccoli (Brassica oleracea L.). Ital J Food Sci 16: 17
30.
Gntensperger B, Hammerli-Meier DE, Escher FE. 1998.
Rosemary extract and precooking effects on lipid oxidation
in heat sterilised meat. J Food Sci 63: 955957.
Hammer KA, Carson CF, Riley TV. 1999. Antimicrobial activity
of essential oils and other plant extracts. J Med Microbiol
86: 985990.
Hili P, Evans CS, Veness RG. 1997. Antimicrobial action of
essential oils: the effect of dimethylsulphoxide on the activity of cinnamon oil. Lett Appl Microbiol 24: 269275.
Ito N, Fukushima S, Hasegawa A, Shibata M, Ogiso T. 1983.
Carcinogenicity of butylated hydroxyanisole in F344 rats. J
Natl Cancer Inst 70: 343347.
Javanmardi J, Stushnoff C, Locke E, Vivanco JM. 2003. Antioxidant activity and total phenolic content of Iranian Ocimum
accessions. Food Chem 83: 547550.
Kahkonen MP, Hopia AI, Vuorela HJ et al. 1999. Antioxidant
activity of plant extracts containing phenolic compounds. J
Agric Food Chem 47: 39543962.
Kosekia PM, Villavicencioa AL, Britoa MS et al. 2002. Effects of
irradiation in medicinal and eatable herbs. Radiat Phys Chem
63: 681684.
Liu F, Ooi VEC, Chang ST. 1997. Free radical scavenging
activity of mushroom polysaccharide extracts. Life Sci 60:
763771.
Mathew M, Abraham TE. 2006. In vitro antioxidant activity and
scavenging effects of Cinnamomum verum leaf extract
Phytother. Res. 21, 354361 (2007)
DOI: 10.1002/ptr

361

ANTIOXIDANT ACTIVITY OF BASIL


assayed by different methodologies. Food Chem Toxicol
44: 198206.
McClatchey W. 1996. The ethnopharmacopoeia of Rotuma. J
Ethnopharmacol 50: 47156.
Min DB. 1998. Lipid oxidation of edible oil. In Food LipidsChemistry, Nutrition, and Biotechnology, Akoh CC, Min DB
(eds). Marcel Dekker: New York, 283296.
Mitsuda H, Yuasumoto K, Iwami K. 1996. Antioxidation action
of indole compounds during the autoxidation of linoleic
acid. Eiyo to Shokuryo 19: 210214.
Namiki M. 1990. Antioxidants/antimutagens in food. Crit Rev
Food Sci Nutr 29: 273300.
Oktay M, Glin I, Kfrevioglu I. 2003. Determination of in
vitro antioxidant activity of fennel (Foeniculum vulgare) seed
extracts. Lebensm Wiss Technol 36: 263271.
Opalchenova G, Obreshkova D. 2003. Comparative studies
on the activity of basil an essential oil from Ocimum
basilicum L. against multidrug resistant clinical isolates of
the genera Staphylococcus, Enterococcus and Pseudomonas
by using different test methods. J Microbiol Methods 54:
105110.
Oyaizu M. 1986. Studies on product of browning reaction prepared from glucose amine. Jpn J Nutr 44: 307
315.

Copyright 2007 John Wiley & Sons, Ltd.

zcelik B, Lee JH, Min DB. 2003. Effects of light, oxygen and
pH on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method
to evaluate antioxidants. J Food Sci 68: 487490.
Pratt DE, Hudson BJF. 1990. Natural antioxidants not exploited
commercially. In Food Antioxidants, Hudson BJF (ed.).
Elsevier: Amsterdam, 171192.
Rice Evans C, Miller NJ, Bolwell GP, Bramley PM, Pridham JB.
1995. The relative antioxidant activities of plant derived
polyphenolic flavonoids. Free Radic Res 22: 375383.
Ruch RJ, Cheng SJ, Klaunig JE. 1989. Prevention of cytotoxicity
and inhibition of intracellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis 10: 10031008.
Tada H, Murakam Y, Omoto T, Shmomurat K, Ishimaru K. 1996.
Rosmarinic acid and related phenolics in hairy root cultures
of Ocmum basilicum. Phytochemistry 42: 431434.
Umerie SC, Anaso HU, Anyasoro LJC. 1998. Insecticidal
potentials of Ocimum basilicum leaf-extract. Bioresourch
Technol 64: 237239.
Wickens AP. 2001. Aging and the free radical theory. Resp
Physiol 128: 379391.
Yen G, Duh P. 1994. Scavenging effect of methanolic extract of
peanut hulls on free radical and active oxygen species. J
Agric Food Chem 42: 629632.

Phytother. Res. 21, 354361 (2007)


DOI: 10.1002/ptr

All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.