Life Sciences 78 (2006) 803 – 811

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Antioxidant and antiradical activities of l-carnitine

İlhami Gülçin *
Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, TR-25240-Erzurum-Turkey

Received 13 February 2005; accepted 20 May 2005

Abstract

l-carnitine plays an important regulatory role in the mitochondrial transport of long-chain free fatty acids. In this study, the antioxidant activity
of l-carnitine was investigated as in vitro. The antioxidant properties of the l-carnitine were evaluated by using different antioxidant assays such
as 1, 1-diphenyl-2-picryl-hydrazyl free radical (DPPHI) scavenging, total antioxidant activity, reducing power, superoxide anion radical
scavenging, hydrogen peroxide scavenging and metal chelating activities. Total antioxidant activity was measured according to ferric thiocyanate
method. a-tocopherol and trolox, a water-soluble analogue of tocopherol, were used as the reference antioxidant compounds. At the
concentrations of 15, 30 and 45 Ag/mL, l-carnitine showed 94.6%, 95.4% and 97.1% inhibition on lipid peroxidation of linoleic acid emulsion,
respectively. On the other hand, 45 Ag/mL of standard antioxidant such as a-tocopherol and trolox indicated an inhibition of 88.8% and 86.2% on
peroxidation of linoleic acid emulsion, respectively. In addition, l-carnitine had an effective DPPHI scavenging, superoxide anion radical
scavenging, hydrogen peroxide scavenging, total reducing power and metal chelating on ferrous ions activities. Also, those various antioxidant
activities were compared to a-tocopherol and trolox as references antioxidants.
D 2005 Elsevier Inc. All rights reserved.

Keywords: DPPHI; Antioxidant activity; l-carnitine; Metal chelating; Reducing power; Radical scavenging

Introduction essential role in human intermediary metabolism (Bremer,

l-carnitine (4-N-trimethylammonium-3-hydroxybutyric ac-
id) plays important physiological roles shuttling the long-chain
fatty acids across the inner mitochondrial membrane for h-
oxidation and ATP production in peripheral tissues. It
translocates acetyl-Co-A into cytoplasm during acetyl-l-
carnitine transport out of mitochondria. Despite the low level
of h-oxidation in brain, l-carnitine is actively transported
through the blood – brain barrier and accumulates in neural cells
(Shug et al., 1982; Mroczkowska et al., 1997). As hypothesized
(Shug et al., 1982; Nalecz and Nalecz, 1996), a major
modulatory role for l-carnitine in neural function may be
played through l-carnitine-mediated transfer of acetyl groups
for acetylcholine synthesis, as well as by influencing signal
transduction pathways and gene expression (Binienda and Ali,
2001).
l-carnitine is derived from both dietary sources (75%) and
endogenous biosynthesis (25%) in human body. It plays an
* Tel.: +90 442 2314444; fax: +90 442 2360948.
E-mail addresses: igulcin@atauni.edu.tr, igulcin@yahoo.com.
0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2005.05.103
1983; De Vivo and Tein, 1990). Furthermore, l-carnitine is an
important cofactor of peroxisomal oxidation especially for very
long-chain fatty acids (C n > C 22) due to the localisation of a
carnitine acetyltransferase, matrix and cytosol-facing carnitine
acyltransferases and a carnitine –acylcarnitine translocase in
peroxisomes (Ramsay, 1999).
The importance of reactive oxygen species and free
radicals has attracted increasing attention over the past
decade. Reactive oxygen species (ROS), which include free
radicals such as superoxide anion radicals (O2I
), hydroxyl
radicals (OHI) 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 (Halliwell and Gutteridge, 1989; Gülçin et al.,
2002a,b).
ROS are continuously produced during normal physiologic
events and they can easily initiate the peroxidation of
membrane lipids, leading to the accumulation of lipid
peroxides. However, they 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 over-
804 İ. Gülçin / Life Sciences 78 (2006) 803 – 811
produced and result in oxidative stress. ROS are formed
when endogenous antioxidant defences are inadequate. The
imbalance between ROS and antioxidant defence mechanisms
leads to oxidative modification in cellular membrane or
intracellular molecules (Duh et al., 1999; Büyükokuroğlu et
al., 2001).
l-carnitine plays a major role, as a cofactor, in the
transportation of free faty acid (FFA) from cytosol to the
mitochondria. FFA degrades to acyl-CoA by h-oxidation and
these substances enter the tricarboxylic acid (TCA) cycle. A
large amount of oxygen is consumed in this reaction and ATP
is synthesized in the steps of electron transport chain and
oxidative phosphorylation. Oxygen is reduced to H2O at the
end of TCA cycle and, then, oxygen concentration decreases,
thus ROS formation is reduced (Mayes, 2000).
Antioxidants can protect the human body from free radicals
and ROS effects and retard the progress of many chronic
diseases as well as lipid peroxidation (Pryor, 1991; Kinsella et
al., 1993; Lai et al., 2001; Gülçin et al., 2003). The most
commonly used antioxidants at the present time are butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
propyl gallate and tert-butylhydroquinone. However, BHA
and BHT have suspected of being responsible for liver damage
and carcinogenesis (Wichi, 1988; Sherwin, 1990). Therefore,
there is a growing interest on natural additives as potential
antioxidants. (Grice, 1986; Moure et al., 2001; Gülçin et al.,
2002a; Oktay et al., 2003).
l-carnitine prevents oxidative stress and regulates nitric
oxide, the cellular respiration (Brown, 1999) and the activity of
enzymes involved in defence against oxidative damage
(Kremser et al., 1995). Also, l-carnitine has a protective effect
on the activity of mitochondrial enzyme succinate dehydroge-
nase as well as the activity of the antioxidant enzymes, catalase
and superoxide dismutase against 3-NPA-induced neurotoxic-
ity (Binienda and Ali, 2001). The antioxidant defense system is
composed of mainly three enzymes; glutathione peroxidase,
catalase, and superoxide dismutase. l-carnitine, an antioxidant,
can protect these enzymes from further peroxidative damage
and is very effective in normalizing age-associated alterations.
Moreover, it can be implemented to minimize age-associated
disorders, which free radicals are the major cause (Kalaiselvi
and Panneerselvam, 1998). In addition, recent studies have
shown that acetyl-l-carnitine, one of the short-chain acyl
esters, enhances learning capacity in aging animals (Ando et
al., 2001), improves the symptoms of nerve-degenerative
disorders such as Alzheimer’s disease (Pettegrew et al., 2000)
and attenuates the neurological damage seen following brain
ischemia and reperfusion (Calvani and Arrigoni-Martelli,
1999).
l-carnitine alters nitric oxide synthase activity in fibro-
blasts depending on the peroxisomal status (Koeck and
Kremser, 2003) and has an effect on the prevention of
experimentally induced myringosclerosis in rats. In addition,
l-carnitine diminishes the occurrence of myringosclerosis in
rats after myringotomy possibly by antioxidant activity and
decreases the formation of ROS (Akbaş et al., 2003).
Moreover, Yasuia and co-workers demonsterated that acetyl-
l-carnitine has an antioxidant activity towards oxidative
stress and that the improvement in cognitive ability seen
with acetyl-l-carnitine may occur through an amelioration
of cellular dysfunction via an inhibition of the increase in
lipid hydroperoxidation observed in the brain tissue of
untreated senescence-acceleration-prone mice (Yasuia et al.,
2002). In this study, we evaluated the possible antioxidant
effects of l-carnitine in different in vitro antioxidant assays
including 1, 1-diphenyl-2-picryl-hydrazyl free radical scav-
enging, total antioxidant activity by ferric thiocyanate
method, reducing power, superoxide anion radical scaveng-
ing, hydrogen peroxide scavenging and metal chelating
activities.
Materials and methods
Chemicals
Nicotinamide adenine dinucleotide (NADH), butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
nitroblue tetrazolium (NBT), phenazine methosulphate
(PMS), the stable free radical 1,1-diphenyl-2-picryl-hydrazyl
(DPPHI), 3-(2-Pyridyl)-5,6-bis (4-phenyl-sulfonic acid)-1,2,4-
triazine (Ferrozine), ethylenediaminetetraacetice acid (EDTA),
linoleic acid, a-tocopherol, polyoxyethylenesorbitan monolau-
rate (Tween-20) and trichloroacetic acid (TCA) were obtained
from Sigma (Sigma-Aldrich GmbH, Sternheim, Germany).
Ammonium thiocyanate was purchased from Merck. All other
chemicals used were in analytical grade and obtained from
either Sigma-Aldrich or Merck.
Total antioxidant activity determination by ferric thiocyanate
method
The antioxidant activity of l-carnitine and standards was
determined according to the ferric thiocyanate method (Mit-
suda et al., 1996). The solution which contains the same
concentration of stock l-carnitine solution or standard samples
(30 Ag/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). Therefore, 5 mL
of linoleic acid emulsion contained 17.5 Ag Tween-20, 15.5 AL
linoleic acid, and 0.04 M potassium phosphate buffer (pH 7.0).
On the other hand, 5 mL control was composed of 2.5 mL of
linoleic acid emulsion and 2.5 mL, 0.04 M potassium
phosphate buffer (pH 7.0). The mixed solution (5 mL) was
incubated at 37 -C in glass flask. The peroxide level was
determined by reading the absorbance at 500 nm in a
spectrophotometer (8500 II, Bio-Crom GmbH, Zurich, Swit-
zerland) after reaction with FeCl2 and thiocyanate at intervals
during incubation. During the linoleic acid oxidation, peroxides
are formed and that leads to oxidation of Fe+ 2 to Fe+ 3. The
latter ions form a complex with thiocyanate and this complex
has a maximum absorbance at 500 nm. This step was repeated
every 5 h until the control reached its maximum absorbance
value. Therefore, high absorbance indicates high linoleic acid
emulsion oxidation. The solutions without l-carnitine were
 İ. Gülçin / Life Sciences 78 (2006) 803 – 811
used as blank samples. All data on total antioxidant activity are
the average of duplicate experimetns. The inhibition percentage
of lipid peroxidation in linoleleic acid emulsion was calculated
by following equation:
Inhibition of lipid peroxidation ð%Þ ¼ 100
½ðA1 =Ao Þ  100
where A o is the absorbance of control reaction and A 1 is the
absorbance in the presence of sample l-carnitine or standard
compounds (Gülçin et al., 2004a).
Total reduction capability
The samples prepared for ferric thiocyanate method were
used for this and the other assays. The reducing power of l-
carnitine was determined by the method of Oyaizu (1986).
Different concentrations of l-carnitine (15 – 30 Ag/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 and then centrifuged for 10 min at 1000 g (MSE
Mistral 2000, U.K). The upper layer of 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 spectropho-
tometer. Increased absorbance of the reaction mixture indicates
an increase of reduction capability.
Ferrous metal ions chelating activity
The chelating of ferrous ions by l-carnitine and standards
was estimated by the method of Dinis et al. (1994). Briefly, l-
carnitine (5 –30 Ag/mL) in 0.4 mL was 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 total volume was
adjusted to 4 mL of ethanol. Then, the mixture was shaken
vigorously and left at room temperature for ten minutes.
Absorbance of the solution was then measured spectrophoto-
metrically at 562 nm. The percentage of inhibition of ferrozine-
Fe2+ complex formation was calculated by using the formula
given bellow:
Metal chelating effect ð%Þ ¼ ½ðAo
A1 Þ=Ao   100
where A o is the absorbance of control and A 1 is the absorbance
in the presence of the sample l-carnitine or standards. The
control contains FeCl2 and ferrozine, complex formation
molecules (Gülçin et al., 2004b).
Hydrogen peroxide scavenging activity
The hydrogen peroxide scavenging ability of l-carnitine
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). l-carnitine at the 30 Ag/mL concentration in 3.4 mL
phosphate buffer was added to a H2O2 solution (0.6 mL, 40
mM). The absorbance value of the reaction mixture was
recorded at 230 nm. Blank solution was containing the
phosphate buffer without H2O2. The percentage of H2O2
805
scavenging of l-carnitine and standard compounds was
calculated as:
% Scavenged ½H2 O2  ¼ ½ðAo
A1 Þ=Ao   100
where A o is the absorbance of the control and A 1 is the
absorbance in the presence of the sample l-carnitine or
standards (Elmastaş et al., 2005).
Antiradical activity
1, 1-Diphenyl-2-picryl-hydrazil (DPPH) free radical scaveng-
ing activity
The free radical scavenging activity of l-carnitine was
determined by the 1,1-diphenyl-2-picryl-hydrazil (DPPHI).
This activity was measured by following the methodology
described by Blois (1958) 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, DPPHI
absorbs at 517 nm, but upon reduction by an antioxidant or a
radical species its absorption decreases. Briefly, 0.1 mM
solution of DPPH in ethanol was prepared and 1 ml of this
solution was added 3 mL of l-carnitine solution in water at
different concentrations (15 –30 Ag/mL). Thirty minutes later,
the absorbance was measured at 517 nm. Lower absorbance of
the reaction mixture indicates higher free radical scavenging
activity. The DPPH concentration (mM) in the reaction
medium was calculated from the calibration curve determined
by linear regression (R 2 : 0.999):
Absorbance ¼ 9:2872  ½DPPHI þ 0:097:
The capability to scavenge the DPPH I radical was
calculated using the following equation:
DPPH I Scavenging effect ð%Þ ¼ ½ðAo
A1 =Ao Þ  100
where A o is the absorbance of the control reaction and A 1 is the
absorbance in the presence of l-carnitine (Gülçin et al., 2004c).
Superoxide anion radical scavenging activity
Measurement of superoxide anion scavenging activity of l-
carnitine was based on the method described by Liu et al.
(1991). Superoxide radicals are generated in PMS-NADH
systems by oxidation of NADH and assayed by the reduction
of NBT. In these experiments, the superoxide radicals were
generated in 3 mL of Tris– HCl buffer (16 mM, pH 8.0)
containing 1 mL of NBT (50 AM) solution, 1 mL NADH (78
AM) solution and sample solution of l-carnitine (30 Ag/mL) in
water. The reaction was started by adding 1 mL of PMS
solution (10 AM) 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 inhibition
percentage of superoxide anion generation was calculated by
using the following formula:
% Inhibition ¼ ½ðAo
A1 Þ=Ao   100
806 İ. Gülçin / Life Sciences 78 (2006) 803 – 811
where A o is the absorbance of the l-ascorbic acid and A 1 is the
absorbance of l-carnitine or standards (Gülçin et al., 2004d).
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 T standard deviation
and analysed by SPSS (version 9.0 for Windows 98, SPSS
Inc.). One-way analysis of variance was performed by ANOVA
procedures. Significant differences between means were
determined by Duncan’s Multiple Range tests. P values
< 0.05 were regarded as significant and p values < 0.01 were
very significant. The energy calculations were performed using
the Spartan 04 (Version 1.03 for Windows 2000). Geometry
optimazitations were performed at the Molecular Mechanics/
MMFF level.
Results and discussion
l-carnitine is an antioxidant and prevents the accumulation
of end products of lipidoxidation (Fabriello and Calabrese,
1988; Lowitt et al., 1995). It is synthesized from two essential
amino acids, lysine and methionine. In addition, carnitine is
transported to skeletal and cardiac muscles after its major
production in liver and kidney, and it transports acyl-CoA acids
into the mitochondrial matrix for h-oxidation of free fatty acids
(Brevetti and Perna, 1992; Visioli et al., 1992).
Many studies have shown that natural antioxidants are
closely related to their biofunctionalities, such as the reduction
of chronic diseases like DNA damage, mutagenesis, carcino-
genesis and inhibition of pathogenic bacteria growth which is
often associated with the termination of free radical propaga-
tion in biological systems (Covacci et al., 2001; Zhu et al.,
2002). Thus, antioxidant capacity is widely used as a
parameter for medicinal bioactive components. In this study,
the antioxidant activity of the l-carnitine was compared to a-
tocopherol and its water-soluble analogue trolox. a-tocopherol
is a biological lipid antioxidant that prevents the formation of
free radicals from lipid peroxidation and has been shown to be
an antimutagen or anticarcinogen in Salmonella tester strains
(Tavan et al., 1997) as well as in human leucocytes in vitro
(Bolkenius, 1991). The antioxidant activity of the l-carnitine,
a-tocopherol and trolox has been evaluated in a series of in
vitro tests: 1, 1-diphenyl-2-picryl-hydrazyl free radical scav-
enging, ferric thiocyanate method, reducing power, scaveng-
ing 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
The ferric thiocyanate method measures the amount of
peroxide produced during the initial stages of oxidation, which
is the primary product of oxidation. Total antioxidant activity
of l-carnitine, a-tocopherol and trolox was determined by the
ferric thiocyanate method in the linoleic acid system. l-
carnitine and standard compounds exhibited effective antiox-
idant activity. The effects of various concentrations of l-
carnitine (15, 30 and 45 Ag/mL) on lipid peroxidation of
linoleic acid emulsion are shown in Fig. 1 and was found to be
94.6%, 95.4% and 97.1%, respectively, and their activities are
greater than that of a-tocopherol (88.8%) and trolox (86.2%) at
the 45 Ag/mL concentration.
Total reductive capability using the potassium ferricyanide
reduction method
In this assay, the yellow colour of the test solution changes
to various shades of green and blue depending on the
reducing power of antioxidant samples. The reducing capacity
of a compound may serve as a significant indicator of its
potential antioxidant activity. The presence of reductants such
as antioxidant substances in the antioxidant samples causes
the reduction of the Fe3+/ferricyanide complex to the ferrous
form. Therefore, Fe2+ can be monitored by measuring the
formation of Perl’s Prussian blue at 700 nm (Chung et al.,
2002).
Fig. 2 depicts the reducing power of the l-carnitine and
standards (a-tocopherol and trolox) using the potassium
ferricyanide reduction method. For the measurements of the
reductive ability, the Fe3+ – Fe2+ transformation was investigated
in the presence of l-carnitine using the method of Oyaizu
(1986). The reducing power of l-carnitine, a-tocopherol and
trolox increased with increasing concentration of samples. At
different concentrations, l-carnitine showed an effective reduc-
ing power (Fig. 2) and these differences were statistically
significant ( P < 0.05). Reducing power of l-carnitine and stan-
dard compounds exhibited the following order: a-tocopherol >
l-carnitine > trolox.
Ferrous ions chelating capacity
The production of highly ROS such as superoxide anion
radicals, hydrogen peroxide, and hydroxyl radicals is also
catalysed by free iron through Haber – Weiss reaction
(O


2 I + H2O2 Y O2 + OH + OHI) (Haber and Weiss, 1934). l-
3
Control
Tocopherol-45 µg/mL
Trolox-45 µg/mL
Lipid peroxidation (500 nm)
Carnitine-15 µg/mL
Carnitine-30 µg/mL
2 Carnitine-45 µg/mL
1
0
0 5 10 15 20 25 30 35 40
Time (Hour)
Fig. 1. Total antioxidant activities of l-carnitine at different concentrations
(15 – 45 Ag/mL), a-tocopherol and trolox (30 Ag/mL).
 İ. Gülçin / Life Sciences 78 (2006) 803 – 811 807

1,4 α-Tocopherol 0,4 α-Tocopherol

Trolox Trolox
L-Carnitine EDTA
Absorbance (700 nm)

L-Carnitine

Absorbance (562 nm)

1,05 0,3

0,7 0,2

0,35 0,1

0
0 5 10 15
Concentration (µg/mL)
Fig. 2. Total reductive potential of different concentrations (15 – 30 Ag/mL) of
l-carnitine, a-tocopherol and trolox.
carnitine and its esters have been shown to partially inhibit
iron-induced lipid peroxidation in liposomes by forming
complexes with free iron. Thus, the reduction in lipid
peroxidation in the present study might be due to the iron-
chelating property of l-carnitine (Arduini, 1992).
EDTA is a strong metal chelator; hence, it is used as
standard metal chelator agent in this study. Ferrous ion
chelating activities of l-carnitine, a-tocopherol, trolox and
EDTA are shown in Fig. 3. The chelating effect of ferrous ions
by the l-carnitine and standards was determined according to
the method of Dinis et al. (1994). Among the transition metals,
iron is known as the most important lipid oxidation pro-oxidant
due to its high reactivity. The ferrous state of iron accelerates
lipid oxidation by breaking down hydrogen and lipid peroxides
to reactive free radicals via the Fenton reaction (Fe2+ +
H2O2 Y Fe3+ + OH
+ OHI). Fe3+ ion also produces radicals
from peroxides although the rate is tenfold less than that of
Fe2+ ion (Miller, 1996). Fe2+ ion is the most powerful pro-
oxidant among the various species of metal ions (Halliwell and
Gutteridge, 1984). Ferrozine can quantitatively form com-
plexes with Fe2+. In the presence of chelating agents, the
complex formation is disrupted, resulting in a decrease in the
red colour of the complex. Measurement of color reduction
therefore allows estimating the metal chelating activity of the
coexisting chelator. Lover absorbance indicates higher metal
chelating activity. In this assay, l-carnitine is interfered with
the formation of ferrous and ferrozine complex, suggesting that
they have chelating activity and are able to capture ferrous ion
0
20 25 30
0 5 10 15 20 25 30
Concentration (µg/mL)
Fig. 4. Ferrous ions chelating effect of different concentrations (5 – 30 Ag/mL)
of l-carnitine, a-tocopherol, trolox and EDTA (EDTA: Ethylenediaminete-
traacetice acid).
before ferrozine. As can be seen in Fig. 3, we suggested that l-
carnitine may chelate the ferrous ions with hydroxyl and
carboxylate groups. It was reported that the compounds with
structures containing two or more of the following functional
groups: – OH, – SH, – COOH, –PO3H2, C=O, – NR2, – S–
and – O – in a favourable structure-function configuration can
show metal chelation activity (Lindsay, 1996; Yuan et al.,
2005).
The metal chelating effects of l-carnitine were concentra-
tion-dependent. As can be seen in Fig. 4, the absorbance of
Fe2+-ferrozine complex was linearly decreased as concentra-
tion-dependently (from 5 to 30 Ag/mL). The difference amoung
all l-carnitine concentrations and the control was statistically
significant ( P < 0.01). In additon, l-carnitine exhibited 98.9%
chelation of ferrous ion at 30 Ag/mL concentration. On the
other hand, the percentages of metal chelating capacity of 30
Ag/mL of a-tocopherol, trolox and EDTA were found as
39.7%, 35.8% and 80.7%, respectively. The metal scavenging
effect of those samples decreased in the order of l-
carnitine > EDTA > a-tocopherol > trolox.
Metal chelating capacity was significant 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. 4 reveal that l-carnitine
demonstrates a marked capacity for iron binding, suggesting

O
O O– O
C C Fe+

CH2 H2C O
H C OH CH
+ Fe +2
CH2 CH2

N+ CH3 H3C N+ CH3

H3C
CH3 CH3

L-Carnitine L-Carnitine-Fe+ complex

Fig. 3. The proposed chelating of ferrous ions reaction by l-carnitine.

808
that their main action as peroxidation protector may be related
to its iron binding capacity.
Hydrogen peroxide scavenging activity
Hydrogen peroxide can be formed in vivo by many
oxidizing enzymes such as superoxide dismutase. It can cross
membranes and may slowly oxidize a number of compounds.
The ability of l-carnitine to scavenge hydrogen peroxide was
determined according to the method of Ruch and co-workers
(1989) as shown in Fig. 5 and compared with that of a-
tocopherol and trolox as standards l-carnitine were capable of
scavenging H2O2. At 30 Ag/mL concentration, l-carnitine
exhibited 63% scavenging activity. On the other hand, a-
tocopherol and trolox exhibited 93 and 31% hydrogen peroxide
scavenging activity at the same concentration, respectively.
These results showed that l-carnitine had an effective
hydrogen peroxide scavenging activity. At 30 Ag/mL concen-
tration, the hydrogen peroxide scavenging effect of l-carnitine
and both standards decreased in the order of a-tocopherol > l-
carnitine > trolox. Hydrogen peroxide itself is not very reactive;
however it can sometimes be toxic to cell because it may give
rise to hydroxyl radical in the cells. Addition of hydrogen
peroxide to cells in culture can lead to transition metal ion-
dependent OH radicals mediated oxidative DNA damage.
Levels of hydrogen peroxide at or below about 20– 50 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 farmaceutical and food
systems.
Radical scavenging activity
Antioxidant properties, especially radical scavenging activ-
ities, 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, antioxidant activities of l-carnitine
and standard antioxidants such as a-tocopherol and trolox were
100
Scavenging activity (%)
75
50
25
0 calculations shows that A is the most stable one among the
α-Tocopherol
Superoxide anion scavenging activity
Fig. 5. Comparison of hydrogen peroxide and superoxide anion radical
scavenging activity of l-carnitine, a-tocopherol and trolox at the same
concentration (30 Ag/mL).
İ. Gülçin / Life Sciences 78 (2006) 803 – 811
2,4
Absorbnce (517 nm)
1,8
1,2
0,6
α-Tocopherol
Trolox
L-Carnitine
0
0 15 30 45
Concentration (µg/mL)
Fig. 6. DPPH free radical scavenging activity of l-carnitine, a-tocopherol and
trolox.
determined using a DPPH method. DPPH has been widely
used to evaluate the free radical scavenging effectiveness of
various antioxidant substances (Cotelle et al., 1996; Özcelik et
al., 2003).
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.
With this method it was possible to determine the antiradical
power of an antioxidant activity by measuring of a decrease in
the absorbance of DPPHI at 517 nm. Resulting a color change
from purple to yellow, the absorbance decreased when the
DPPHI was scavenged by an antioxidant through donation of
hydrogen to form a stable DPPHI molecule. In the radical form,
this molecule had an absorbance at 517 nm which disappeared
after acceptance of an electron or hydrogen radical from an
antioxidant compound to become a stable diamagnetic mole-
cule (Matthäus, 2002). Fig. 6 illustrates a significant decrease
( P < 0.05) in the concentration of DPPH radical due to the
scavenging ability of l-carnitine and standards. a-tocopherol
and trolox were used as references for radical scavengers. The
scavenging effect of l-carnitine and standards on the DPPH
radical decreased in the order of a-tocopherol > l-carnitine >
trolox, which were 81.7%, 38.2% and 15.6%, at the concen-
tration of 45 Ag/mL, respectively. Free radical scavenging
activity of these samples also increased with an increasing
concentration. Proposed reaction based on the analysis of the
DPPH and l-carnitine molecule is summarized in Fig. 7. To
our knowledge, DPPH radical scavenging mechanism of l-
carnitine has not been reported so far. However, the best known
is that a carbonyl group stabilizes a radical formed on a-carbon
with conjugation. In l-carnitine molecule, carboxylate group
has also a carbonyl unit. An abstraction of a hydrogen atom
from C-2 may be occurred easily. As seen in Fig. 7, thereotical
Trolox L-Carnitine
intermediates (A, B, C). While the calculated formation energy
Hydrogen peroxide scavenging activity (DH) for A is
181.115 kcal/mol, this energy was calculated
as 12.88 kcal/mol for B and 67.91 kcal/mol for C.
Superoxide anions are a precursor to active free radicals
that have potential of reacting with biological macromolecules
 İ. Gülçin / Life Sciences 78 (2006) 803 – 811
O O–
O O– NO2
C
CH2
H C OH H C
O2N NO2
+ N
CH2
N N+ CH3
H3C N+ CH3 H3C
CH3
L-Carnitine DPPH.
Fig. 7. The reaction scheme between DPPH and l-carnitine.
and thereby inducing tissue damage (Halliwell and Gutter-
idge, 1984). Also, it has been implicated in several
pathophysiological processes due to its transformation into
more reactive species such as hydroxyl radical that initiate
lipid peroxidation. Superoxide has also been observed to
directly initiate lipid peroxidation (Wickens, 2001). It has also
been reported that antioxidant properties of some flavonoids
are effective mainly via scavenging of superoxide anion
radical (Yen and Duh, 1994). Superoxide anion plays an
important role in the formation of other ROS such as hydrogen
peroxide, hydroxyl radical, and singlet oxygen, which induce
oxidative damage in lipids, proteins, and DNA (Pietta, 2000).
Also, superoxide anion is an oxygen-centred radical with
selective reactivity. These species are 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. In this
method, superoxide anion reduces the yellow dye (NBT2+) to
produce the blue formazan which is measured spectrophoto-
metrically at 560 nm. Antioxidants are able to inhibit the blue
NBT formation (Cos et al., 1998; Parejo et al., 2002). The
decrease of absorbance at 560 nm with antioxidants indicates
the consumption of superoxide anion in the reaction mixture.
Fig. 4 shows the inhibition percentage of superoxide radical
generation by 30 Ag/mL concentration of l-carnitine and
standards. The inhibition of superoxide radical generation
results of l-carnitine and standards were found similar
statistically. As can see in Fig. 5, the percentage inhibition of
superoxide anion radical generation by 30 Ag/mL concentration
of l-carnitine was found as 72.4%. On the other hand, at the
same concentration a-tocopherol and trolox exhibited 75.4%
and 82.2% superoxide anion radical scavenging activity,
respectively.
Conclusion
According to data of the present study, l-carnitine was
found to be an effective antioxidant in different in vitro assay
including reducing power, DPPH radical and superoxide anion
radical scavenging, hydrogen peroxide scavenging and metal
chelating activities when it is compared to standard antioxidant
compounds such as a-tocopherol, a natural antioxidant, and
trolox which is a water-soluble analogue of tocopherol.
809
O O– O O–
C C C NO2
HC H2C H2C
OH H C OH H C OH
O2N NO2
CH2 + CH
+ CH2 +
N-H
N+ CH3 N+ CH2
H3C H3C N
CH3 CH3 CH3
A B C
DPPH-H
Acknowledgements
The author would like to thank Prof.Dr. Hasan Secen for
discussion on possible mechanism betwen l-carnitine-ferrozine
and l-carnitine-DPPHI and Dr. Mustafa ArNk for theorical
calculation.
References
Akbaş, Y., Pata, Y.S., Görür, K., Polat, G., Polat, A., Özcan, C., Ünal, M., 2003.
The effect of l-carnitine on the prevention of experimentally induced
myringosclerosis in rats. Hearing Research 184, 107 – 112.
Ando, S., Tadenuma, T., Tanaka, Y., Fukui, F., Kobayashi, S., Ohashi, Y.,
Kawabata, T., 2001. Enhancement of learning capacity and cholinergic
synaptic function by carnitine in aging rats. Journal of Neuroscience
Research 66, 266 – 271.
Arduini, A., 1992. Carnitine and its acetyl esters as secondary antioxidants?
American Heart Journal 123, 1726 – 1727.
Binienda, Z.K., Ali, S.F., 2001. Neuroprotective role of l-carnitine in the 3-
nitropropionic acid induced neurotoxicity. Toxicology Letters 125, 67 – 73.
Blois, M.S., 1958. Antioxidant determinations by the use of a stable free
radical. Nature 26, 1199 – 1200.
Bolkenius, F.N., 1991. Leukocyte-mediated inactivation of alpha-1-proteinase
inhibitor is inhibited by amino analogues of alpha-tocopherol. Biochemica
and Biophysica Acta 1095, 23 – 29.
Bremer, J., 1983. Carnitine-metabolism and functions. Physiology Review 63,
1420 – 1480.
Brevetti, G., Perna, S., 1992. Metabolic and clinical effects of l-carnitine in
peripheral vascular disease. In: Ferrari, R., DiMauro, S., Sherwood, G.
(Eds.), l-carnitine and its Role in Medicine: From Function to Therapy.
Academic Press, London, pp. 359 – 378.
Brown, G.C., 1999. Nitric oxide and mitochondrial respiration. Biochimica and
Biophysica Acta 1411, 351 – 369.
Büyükokuroğlu, M.E., Gülçin, İ., Oktay, M., Küfrevioğlu, Ö.İ., 2001. In vitro
antioxidant properties of dantrolene sodium. Pharmacological Research 44,
491 – 495.
Calvani, M., Arrigoni-Martelli, E., 1999. Attenuation by acetyl-l-carnitine of
neurological damage and biochemical derangement following brain
ischemia and reperfusion. International Journal of Tissue Reactions—
Experimental and Clinical 21, 1 – 6.
Chung, Y.C., Chang, C.T., Chao, W.W., Lin, C.F., Chou, S.T., 2002.
Antioxidative activity and safety of the 50% ethanolic extract from red
bean fermented by Bacillus subtilis IMR-NK1. Journal of Agricultural and
Food Chemistry 50, 2454 – 2458.
Cos, P., Ying, L.Y., Calomme, M., Hu, J.H., Cimanga, K., Van Poel, B., Pieters,
L., Vlietinck, A.J., Berghe, D.V., 1998. Structure activity relationships and
classification of flavonoids as inhibitors of xanthine oxidase and superoxide
scavengers. Journal of Natural Products 61, 71 – 76.
Cotelle, N., Bemier, J.L., Catteau, J.P., Pommery, J., Wallet, J.C., Gaydou,
E.M., 1996. Antioxidant properties of hydroxyl-flavones. Free Radical
Biology and Medicine 20, 35 – 43.
Covacci, V., Torsello, A., Palozza, P., Sgambato, A., Romano, G., Boninsegna,
A., Cittadini, A., Wolf, F.I., 2001. DNA oxidative damage during
810 İ. Gülçin / Life Sciences 78 (2006) 803 – 811
differentiation of HL-60 human promyelocytic leukemia cells. Chemical
Research in Toxicology 14, 1492 – 1497.
De Vivo, D.C., Tein, I., 1990. Primary and secondary disorders of carnitine
metabolism. International Pediatry 5, 134 – 141.
Dinis, T.C.P., Madeira, V.M.C., Almeida, L.M., 1994. Action of phenolic
derivates (acetoaminophen, salycilate, and 5-aminosalycilate) as inhibitors
of membrane lipid peroxidation and as peroxyl radical scaven-
gers. Archive of Biochemistry and Biophysics 315, 161 – 169.
Duh, P.D., Tu, Y.Y., Yen, G.C., 1999. Antioxidant activity of water extract of
harng jyur (Chrysanthemum morifolium Ramat). Lebensmittel Wissenchaft
und-Technologie 32, 269 – 277.
Elmastaş, M., Gülçin, İ., Öztürk, L., Gökçe, İ., 2005. Investigation of
antioxidant properties of spearmint (Mentha spicata L.). Asian Journal of
Chemistry 17 (1), 137 – 148.
Fabriello, R.G., Calabrese, F., 1988. Prevention of ischemia induced increase in
MDA by acetyl carnitine. Annals of Neurology 24, 114 – 118.
Grice, H.C., 1986. Safety evaluation of butylated hydroxytoluene (BHT) in the
liver, lung and gastrointestinal tract. Food and Chemical Toxicology 24,
1127 – 1130.
Gülçin, İ., Büyükokuroğlu, M.E., Oktay, M., Küfrevioğlu, Ö.İ., 2002. On the in
vitro antioxidant properties of melatonin. Journal of Pineal Research 33,
167 – 171.
Gülçin, İ., Oktay, M., Küfrevioğlu, Ö.İ., Aslan, A., 2002. Determination of
antioxidant activity of lichen Cetraria islandica (L) Ach. Journal of
Ethnopharmacology 79, 325 – 329.
Gülçin, İ., Oktay, M., Kireçci, E., Küfrevioğlu, Ö.İ., 2003. Screening of
antioxidant and antimicrobial activities of anise (Pimpinella anisum L.)
seed extracts. Food Chemistry 83, 371 – 382.
Gülçin, İ., Şat, İ.G., Beydemir, Ş., Elmastaş, M., Küfrevioğlu, Ö.İ., 2004a.
Comparison of antioxidant activity of clove (Eugenia caryophylata
Thunb) buds and lavender (Lavandula stoechas L.). Food Chemistry 87,
393 – 400.
Gülçin, İ., Şat, İ.G., Beydemir, Ş., Küfrevioğlu, Ö.İ., 2004b. Evaluation of the
in vitro antioxidant properties of extracts of broccoli (Brassica oleracea L.).
Italian Journal Food Science 16, 17 – 30.
Gülçin, İ., Beydemir, Ş., Alici, H.A., Elmastaş, M., Büyükokuroğlu, M.E.,
2004c. In vitro antioxidant properties of morphine. Pharmacological
Research 49, 59 – 66.
Gülçin, İ., Küfrevioğlu, Ö.İ., Oktay, M., Büyükokuroğlu, M.E., 2004d.
Antioxidant, antimicrobial, antiulcer and analgesic activities of nettle
(Urtica dioica L.). Journal of Ethnopharmacology 90, 205 – 215.
Haber, F., Weiss, J., 1934. The catalytic decomposition of hydrogen peroxide
by iron salts. Proceedings of the Royal Society of London, Series A 147,
332 – 351.
Halliwell, B., Gutteridge, J.M.C., 1984. Oxygen toxicology, oxygen radicals,
transition metals and disease. Biochemical Journal 219, 1 – 4.
Halliwell, B., Gutteridge, J.M., 1989. Free Radicals in Biology and Medicine.
Clarendon Press, Oxford, pp. 23 – 30.
Kalaiselvi, T., Panneerselvam, C., 1998. Effect of l-carnitine on the status of
lipid peroxidation and antioxidants in aging rats. Journal of Nutritional
Biochemistry 9, 575 – 581.
Kinsella, J.E., Frankel, E., German, B., Kanner, J., 1993. Possible mechanism
for the protective role of the antioxidant in wine and plant foods. Food
Technology 47, 85 – 89.
Koeck, T., Kremser, K., 2003. l-carnitine alters nitric oxide synthase activity in
fibroblasts depending on the peroxisomal status. International Journal of
Biochemistry and Cell Biology 35, 149 – 156.
Kremser, K., Stangl, H., Pahan, K., Singh, I., 1995. Nitric oxideregulates
peroxisomal enzyme activities. Europane Journal of Clinic Chemisty and
Clinic Biochemistry 33, 763 – 774.
Lai, L.S., Chou, S.T., Chao, W.W., 2001. Studies on the antioxidative activities
of Hsian-tsao (Mesona procumbens Hemsl) leaf gum. Journal of Agricul-
tural and Food Chemistry 49, 963 – 968.
Lindsay, R.C., 1996. Food additives. In: Fennema, O.R. (Ed.), Food Chemistry.
Marcel Dekker Inc., New York, pp. 778 – 780 (Chapter 12).
Liu, Q., Zhu, G., Huang, P., 1991. Anti-inflammatory, analgesic and
sedative effects of Leontice kiangnanensis. Zhongguo Zhong Yao Za Zhi
161, 50 – 65.
Lowitt, S., Malone, J.I., Salem, A.F., Korhals, J., Benford, S., 1995. Acetyl-l-
carnitine corrects the altered peripheral nerve function of experimental
diabetes. Metabolism 445, 677 – 680.
Matthäus, B., 2002. Antioxidant activity of extracts obtained from residues
of different oilseeds. Journal of Agricultural and Food Chemistry 50,
3444 – 3452.
Mayes, P.A., 2000. Lipids of physiologic significance. In: Murray, R.K.,
Granner, D.K., Mayes, P.A., Rodwell, V.W. (Eds.), Harper’s Biochemistry,
(25th ednR)Appleton and Lange, Stamford, pp. 160 – 171.
Miller, D.D., 1996. Mineral. In: Fennema, O.R. (Ed.), Food Chemistry. Marcel
Deckker, New York, pp. 618 – 649.
Min, D.B., 1998. Lipid oxidation of edible oil. In: Akoh, C.C., Min, D.B.
(Eds.), In Food Lipids Chemistry, Nutrition, and Biotechnology. Marcel
Dekker, New York, pp. 283 – 296.
Mitsuda, H., Yuasumoto, K., Iwami, K., 1996. Antioxidation action of indole
compounds during the autoxidation of linoleic acid. Eiyo to Shokuryo 19,
210 – 214.
Moure, A., Cruz, J.M., Franco, D., Dominguez, J.M., Sineiro, J., Dominguez,
H., Nunez, M.J., Parajo, J.C., 2001. Natural antioxidants from residual
sources. Food Chemistry 72, 145 – 171.
Mroczkowska, J.E., Gala, H.J., Nalecz, M.J., Nalecz, K.A., 1997.
Evidence for an asymmetrical uptake of l-carnitine in the blood –
brain barrier in vitro. Biochemistry and Biophysics Research Communi-
cation 241, 127 – 131.
Nalecz, K.A., Nalecz, M.J.I., 1996. Carnitine—a known compound, a
novel function in neural cells. Acta Neurobiologiae Experimentalis 56,
597 – 609.
Oktay, M., Gülçin, İ., Küfrevioğlu, Ö.İ., 2003. Determination of in vitro
antioxidant activity of fennel (Foeniculum vulgare) seed extracts. Lebens-
mittel Wissenchaft und-Technologie 36, 263 – 271.
Oyaizu, M., 1986. Studies on product of browning reaction prepared from
glucose amine. Japanese Journal Nutrition 44, 307 – 315.
Özcelik, B., Lee, J.H., Min, D.B., 2003. Effects of light, oxygen and pH on the
2,2-diphenyl-1-picrylhydrazyl (DPPH) method to evaluate antioxidants.
Journal of Food Science 68, 487 – 490.
Parejo, I., Viladomat, F., Bastida, J., Rosas-Romero, A., Flerlage, N., Burillo,
J., CodNna, C., 2002. Comparison between the radical scavenging activity
and antioxidant activity of six distilled and nondistilled Mediterranean
herbs and aromatic plants. Journal of Agricultural and Food Chemistry 50,
6882 – 6890.
Pettegrew, J.W., Levine, J., McClure, R.J., 2000. Acetyl-l-carnitine physical –
chemical, metabolic, and therapeutic properties: relevance for its mode of
action in Alzheimer’s disease and geriatric depression. Molecular Psychi-
atry 5, 616 – 632.
Pietta, P.G., 2000. Flavonoids as antioxidants. Journal of Natural Products 63,
1035 – 1042.
Pryor, W.A., 1991. The antioxidant nutrient and disease prevention—what do
we know and what do we need to find out? American Journal of Clinical
Nutrition 53, 391 – 393.
Ramsay, R.R., 1999. The role of the carnitine system in peroxisomal
in fatty acid oxidation. American Journal of Medical Science 318,
28 – 35.
Ruch, R.J., Cheng, S.J., Klaunig, J.E., 1989. Prevention of cytotoxicity and
inhibition of intracellular communication by antioxidant catechins isolated
from Chinese green tea. Carcinogenesis 10, 1003 – 1008.
Sherwin, E.R., Branen, A.L., Davidson, P.M., Salminen, S., 1990. Food
Additives. Marvel Dekker inc, New York, pp. 139 – 193.
Shug, A.L., Schmidt, M.J., Golden, G.T., Fariello, R.T., 1982. The
distribution and role of carnitine in the mammalian brain. Life Sciences
31, 2869 – 2874.
Tavan, E., Mazier, S., Narbonne, J.F., Cassand, P., 1997. Effects of vitamin A
and E on methylazoxymethanol-induced mutagenesis in Salmonella
typhimurim strains TA100. Mutation Research 377, 231 – 237.
Visioli, O., Pasini, E., de Giuli, F., Ferrari, R., 1992. Molecular mechanism of
action of l-carnitine in treatment of myocardial disorders at the experi-
mental levels. In: Ferrari, R., DiMauro, S., Sherwood, G. (Eds.), l-carnitine
and Its Role in Medicine: From Function to Therapy. Academic Press,
London, pp. 237 – 263.
 İ. Gülçin / Life Sciences 78 (2006) 803 – 811
Wichi, H.P., 1988. Enhanced tumor development by butylated hydroxyanisole
(BHA) from the prospective of effect on forestomach and oesophageal
squamous epithelium. Food and Chemical Toxicology 26, 717 – 723.
Wickens, A.P., 2001. Aging and the free radical theory. Respiratory Physiology
128, 379 – 391.
Yasuia, F., Matsugoa, S., Ishibashib, M., Kajitab, T., Ezashib, Y., Oomurac, Y.,
Kojod, S., Sasakib, K., 2002. Effects of chronic acetyl-l-carnitine treatment
on brain lipid hydroperoxide level and passive avoidance learning in
senescence accelerated mice. Neurosciences Letters 334, 177 – 180.
811
Yen, G.C, Duh, P.D., 1994. Scavenging effect of methanolic extract of peanut
hulls on free radical and active oxygen species. Journal of Agriculture and
Food Chemistry 42, 629 – 632.
Yuan, Y.V., Bone, D.E., Carrington, M.F., 2005. Antioxidant activity of dulse
(Palmaria palmata) extract evaluated in vitro. Food Chemistry 91, 485 –
494.
Zhu, Q.Y., Hackman, R.M., Ensunsa, J.L., Holt, R.R., Keen, C.L., 2002.
Antioxidative activities of oolong tea. Journal of Agriculture and Food
Chemistry 50, 6929 – 6934.