Professional Documents
Culture Documents
Ilhami G ulcin
Faculty of Arts and Sciences, Department of Chemistry, Atat urk University, TR-25240 Erzurum, Turkey
Received 25 August 2005; received in revised form 27 September 2005; accepted 27 September 2005
Available online 21 October 2005
Abstract
Caffeic acid (3,4-dihydroxycinnamic acid) is among the major hydroxycinnamic acids present in wine; sinapic acid, which is a
potent antioxidant. It has also been identied as one of the active antioxidant. In the present study, the antioxidant properties of
the caffeic acid were evaluated by using different in vitro antioxidant assays such as 2-azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid) (ABTS) radical scavenging, 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH
scavenging, superoxide anion radical scavenging, total reducing power and metal chelating on ferrous ions activities.
2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Caffeic acid; 3,4-Dihydroxycinnamic acid; Antioxidant activity; Metal chelating; Reducing power; Radical scavenging
1. Introduction
Phenolic compounds are secondary plant metabo-
lites and naturally present in almost all plant mate-
rials, including food products of plant origin. These
compounds are thought to be an integral part of both
human and animal diets (Psomiadou and Tsimidou,
2002). Phenolic acids are simple phenols because of
their structure. Hydroxycinnamic acid is the major sub-
group of phenolic compounds (Sanchez-Moreno et al.,
1998; Sroka and Cisowski, 2003). Hydroxycinnamates
are phenylpropanoid metabolites and occur widely in
plants (Herrmann, 1989), and plant products (Clifford,
), hydroxyl radicals
(OH
), 3-(2-
pyridyl)-5,6-bis (4-phenyl-sulfonic acid)-1,2,4-triazine (Fer-
rozine), 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS), linoleic acid, -tocopherol, polyoxyethylenesorbitan
monolaurate (Tween-20) and trichloroacetic acid (TCA) were
obtained fromSigma (SigmaAldrich GmbH, Sternheim, Ger-
many). Ammonium thiocyanate was purchased from Merck.
All other chemicals used were in analytical grade and obtained
from either Sigma-Aldrich or Merck.
2.2. Total antioxidant activity determination by ferric
thiocyanate method
The antioxidant activity of caffeic acid and standards
was determined according to the ferric thiocyanate method
(Mitsuda et al., 1996). The solution which contains the same
concentration of caffeic acid (1020 g/mL) or standard sam-
ples (20 g/mL) in 2.5 mL of potassium phosphate buffer
(0.04 M, pH7.0) was added to 2.5 mLof linoleic acid emulsion
in potassium phosphate buffer (0.04 M, pH 7.0). Therefore,
5 mL of linoleic acid emulsion contained 17.5 g Tween-20,
15.5 L linoleic acid, and 0.04 M potassium phosphate buffer
(pH 7.0). On the other hand, 5 mL control composed of 2.5 mL
of linoleic acid emulsion and 2.5 mL, 0.04 M potassium phos-
phate buffer (pH7.0). The mixedsolution(5 mL) was incubated
at 37
). This
activity was measured by the following methodology described
by Blois (1958) wherein the bleaching rate of a stable free rad-
ical, DPPH
absorbs
at 517 nm, but upon reduction by an antioxidant or a radical
species its absorption decreases. Briey, 0.1 mM solution of
DPPH
] +0.097
The capability to scavenge the DPPH
Ozcelik
et al., 2003).
Fig. 4. Scavenging effect of caffeic acid, BHA, BHT, -tocopherol and
trolox on the stable DPPH
+H
2
O
2
O
2
+OH
+OH
+OH
Fe
3+
ion also produces radicals from peroxides although
the rate is 10-foldless thanthat of Fe
2+
ion(Miller, 1996).
Fe
2+
ion is the most powerful pro-oxidant among the
various species of metal ions (Halliwell and Gutteridge,
1984). Ferrozine can quantitatively formcomplexes with
Fe
2+
. In the presence of chelating agents, the complex
formation is disrupted, resulting in a decrease in the red
color of the complex. Therefore, measurement of color
reduction allows estimating the metal chelating activity
of the coexisting chelator. Lower absorbance indicates
higher metal chelating activity.
Ferrous ion chelating activities of caffeic acid, BHA,
BHT, -tocopherol and trolox are shown in Fig. 5. In
this assay, caffeic acid is interfered with the forma-
tion of ferrous and ferrozine complex, suggesting that
they have chelating activity and are able to capture fer-
rous ion before ferrozine. As can be seen in Fig. 5,
we suggested that caffeic acid may chelate the ferrous
ions with hydroxyl groups. It was reported that the
compounds with structures containing two or more of
the following functional groups: OH, SH, COOH,
PO
3
H
2
, C O, NR
2
, S and O in a favourable
structurefunction conguration can show metal chela-
tion activity (Lindsay, 1996; Yuan et al., 2005).
The difference between caffeic acid and the control
was statistically signicant (p <0.01). In additon, caf-
feic acid exhibited 53.2% chelation of ferrous ion at
10 g/mL concentration. On the other hand, the per-
centages of metal chelating capacity of 10 g/mL of
BHA, BHT, -tocopherol and trolox were found as 72.1,
64.3, 21.6 and 48.5%, respectively. The metal scaveng-
ing effect of those samples decreased in the order of
BHA>BHT>caffeic acid >trolox >-tocopherol.
Metal chelating capacity was signicant since it
reduced the concentration of the catalysing transition
metal in lipid peroxidation. It was reported that chelating
I., 2001.
In vitro antioxidant properties of dantrolene sodium. Pharmacol.
Res. 44, 491495.
Cartron, E., Carbonneau, M.A., Fouret, G., Descomps, B., Leger, C.L.,
2001. Specic antioxidant activity of caffeoyl derivatives and other
natural phenolic compounds: LDLprotection against oxidation and
decrease in the proinammatory lysophosphatidylcholine produc-
tion. J. Nat. Prod. 64, 480486.
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. J. Agric. Food
Chem. 50, 24542458.
Clifford, M.N., 1999. Chlorogenic acids and other cinnamates: nature,
occurrence and dietary burden. J. Sci. Food Agric. 79, 362
372.
Dinis, T.C.P., Madeira, V.M.C., Almeida, L.M., 1994. Action of pheno-
lic derivates (acetoaminophen, salycilate, and 5-aminosalycilate)
as inhibitors of membrane lipid peroxidation and as peroxyl radical
scavengers. Arch. Biochem. Biophys. 315, 161169.
Duh, P.D., Tu, Y.Y., Yen, G.C., 1999. Antioxidant activity of water
extract of harng jyur (ChrysanthemummorifoliumRamat). Lebens.
Wiss. Technol. 32, 269277.
Foley, S., Navaratnam, S., McGarvey, D.J., Land, E.J., Truscott,
T.G., Rice-Evans, C.A., 1999. Free Radic. Biol. Med. 26, 1202
1208.
Fukumoto, L.R., Mazza, G., 2000. Assessing antioxidant and proox-
idant activities of phenolic compounds. J. Agric. Food Chem. 48,
35973604.
G ulcin,
I., Beydemir, S ., Alici, H.A., Elmastas, M., B uy ukokuro glu,
M.E., 2004c. In vitro antioxidant properties of morphine. Pharma-
col. Res. 49, 5966.
G ulcin,
I., 2002a.
On the in vitro antioxidant properties of melatonin. J. Pineal Res.
33, 167171.
G ulcin,
I.,
2004a. Comparison of antioxidant activity of clove (Eugenia
caryophyllata Thunb) buds and lavender (Lavandula stoechas L.).
Food Chem. 87, 393400.
G ulcin,
I., S at,
I.G., Beydemir, S ., K ufrevio glu,
O.
Ozcelik, 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. J. Food Sci. 68, 487490.
220
I. G ul cin / Toxicology 217 (2006) 213220
Parejo, I., Viladomat, F., Bastida, J., Rosas-Romero, A., Flerlage, N.,
Burillo, J., Codna, C., 2002. Comparison between the radical
scavenging activity and antioxidant activity of six distilled and
nondistilled Mediterranean herbs and aromatic plants. J. Agric.
Food Chem. 50, 68826890.
Pietta, P.G., 2000. Flavonoids as antioxidants. J. Nat. Prod. 63,
10351042.
Psomiadou, E., Tsimidou, M., 2002. Stability of virgin olive
oil. 1. Autoxidation studies. J. Agric. Food Chem. 50, 716
721.
Rao, M.V., Paliyath, G., Ormrod, D.P., 1996. Ultraviolet-band ozone-
induced biochemical changes in antioxidant enzymes of Arabidop-
sis thaliana. Plant Physiol. 110, 125136.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-
Evans, C., 1999. Antioxidant activity applying an improved ABTS
radical cation decolorization assay. Free Radic. Biol. Med. 26,
12311237.
Sanchez-Moreno, C., Larrauri, J.A., Saura-Calixto, F., 1998. A proce-
dure to measure the antiradical efciency of polyphenols. J. Sci.
Food Agric. 76, 270276.
Sroka, Z., Cisowski, W., 2003. Hydrogen peroxide scavenging, antiox-
idant and anti-radical activity of some phenolic acids. Food Chem.
Toxicol. 41, 753758.
Wickens, A.P., 2001. Aging and the free radical theory. Resp. Physiol.
128, 379391.
Yen, G.C., Duh, P.D., 1994. Scavenging effect of methanolic extract
of peanut hulls on free radical and active oxygen species. J. Agric.
Food Chem. 42, 629632.
Yuan, Y.V., Bone, D.E., Carrington, M.F., 2005. Antioxidant activity
of dulse (Palmaria palmata) extract evaluated in vitro. Food Chem.
91, 485494.
Zhu, Q.Y., Hackman, R.M., Ensunsa, J.L., Holt, R.R., Keen, C.L.,
2002. Antioxidative activities of oolong tea. J. Agric. Food Chem.
50, 69296934.