A comparative study of the inhibitory effects by caffeic acid, catechins and their related compounds on the generation of radicals in the reaction mixture of linoleic acid with iron ions

Caffeic acid and (+)-catechin, which are abundantly contained in coffee and tea, are typical polyphenols. In order to know the relative magnitudes of antioxidant activity, effects by caffeic acid, (+)-catechin and their derivatives on the formation of 4-POBN/carbon-centered linoleic acid-derived radical adducts were examined in the control reaction mixture of linoleic acid with FeCl3 at 30°C for 168 h. In the presence of 1.0 mM of the polyphenols, peak to peak heights of the third ESR signal resulted in 7.7 ± 2.4% (n = 3) (caffeic acid), 145 ± 13% (n = 3) (quinic acid), 4.4 ± 0.0% (n = 3) (chlorogenic acid), 104 ± 4.4% (n = 3) (ferulic acid), 4.3 ± 0.0% (n = 3) (noradrenaline), 12.5 ± 10.9% (n = 3) (gallic acid), 38.1 ± 7.1% (n = 3) [(+)-catechin], 47.9 ± 11.7% (n = 3) [(–)-epicatechin], 56.5 ± 1.6% (n = 3) (epigallocatechin), 13.5 ± 1.7% (n = 3) (catechol) and 83.7 ± 7.8% (n = 3) (resorcinol) of the control reaction mixture. All the compounds with catechol moiety exerted potent inhibitory effects on the radical formation except for (+)-catechin, (–)-epicatechin and epigallocatechin. (+)-Catechin, (–)-epicatechin and epigallocatechin may not exert the inhibitory effect as much possibly because they are less stable compared with caffeic acid. The resorcinol moiety in these molecules may also weaken their antioxidant activity.

Introduction I ron is present in the human body in great quantity in the form of heme and non-heme proteins. It plays a crucial role in electron transfer, cellular respiration, cell proliferation and differentiation, and regulation of gene expression. (1) On the other hand, iron exposure is directly associated with the pathogenesis of many disorders, such as atherosclerosis, cancer and inflammation, possibly via the production of free radicals. (2,3) Chlorogenic acid, caffeic acid (CA), noradrenaline and gallic acid are typical catechol compounds. Of the catechols, chlorogenic acid and CA are found naturally in various agricultural products such as coffee beans, potatoes, and apples. (4,5) Chlorogenic acid is an ester of CA with quinic acid. Chlorogenic acid and CA have been known to be inhibitors of formation of hydroxyl radical in the reaction of 3-hydroxyanthranilic acid and hydrogen peroxide with ferric ions, (6) lipid peroxidation and formation of lipid-derived radicals. (7,8) Chlorogenic acid and CA also act as scavengers of superoxide, hydroxyl and peroxy radicals. (9,10) It was reported that noradrenaline and dopamine provide an antioxidant defense in the brain against oxidatative stress. (11) The chemically induced LDL oxidation is reduced by galloyl derivatives. (12) Their antioxidative activities appear to be achieved through inhibiting the formation of the free radical by catechol moiety which has iron ion chelating activity. (13) Catechins such as (+)-catechin, (-)-epicatechin (EC), epigallocatechin (EGC) and epigallocatechingallate (EGCG) are also typical catechol derivatives. They are tricyclic phenols (flavonoids) found in green tea. Catechins exert protective effects against oxidative damage of erythrocyte membrane, (14) ethanol-induced fatty livers, (15) cardiovascular diseases, (16,17) inflammatory, (18) and cancer. (19) Catechins decreases 4-POBN/radical adducts formed in bile of rats after transplantation of ethanol-induced fatty livers. (15) The radical scavenging activities of (+)-catechin and CA found in the two common beverages, coffee and tea, were investigated in detail in terms of their reaction with the stable radical 2,2diphenyl-1-picrylhydrazyl in methanol. (20,21) Meanwhile, the polyphenols are strong antioxidants due to their ability to chelate transition metals like iron as well as their radical scavenging activities. (22) In order to examine the effect of their chelating ability on the formation of lipid-derived free radical in the reaction of linoleic acid with iron ions, we used electron spin resonance (ESR), high performance liquid chromatography-electron spin resonance (HPLC-ESR) and high performance liquid chromatographyelectron spin resonance-mass spectrometries (HPLC-ESR-MS) and conducted the comparative study on CA and catechins in antioxidative activities. (23)
Control reaction mixture. In the control reaction mixture, there were 50 mM phosphate buffer (pH 7. HPLC ESR MS chromatography. The HPLC and ESR conditions were as described in the HPLC-ESR. The mass spectrometer (MS) used in the HPLC-ESR-MS was a model M-1200 HS electrospray ionization (ESI)-MS (Hitachi Ltd.). The operating conditions of the ESI-MS were: nebulizer, 180°C; aperture1, 120°C; N 2 controller pressure, 19.6 N/cm 2 ; drift voltage, 70 V; multiplier, 2,000 V; needle voltage, 4,000 V; polarity, positive; resolution, 48. The mass spectra were obtained by introducing the eluent from the ESR detector into the ESI-MS system just before the peak was eluted. The flow kept at 50 μl/min while the eluent was introducing into the ESI-MS.

Results and Discussion
ESR Spectra of the control reaction mixtures. ESR spectrum of the control reaction mixture (without FeCl 3 or linoleic acid) was measured (Fig. 1). A prominent ESR spectrum (α N = 1.58 mT and α H β = 0.26 mT) was observed in the control reaction mixture (Fig. 1A). The ESR signals remained unchanged for the control reaction in the absence of light [90 ± 6% (n = 3) of the control reaction mixture], suggesting that light is not involved in the radical formation. For the reaction mixture without iron, the ESR signal decreased to 53 ± 3% (n = 3) of the control reaction mixture (Fig. 1B), suggesting that iron ions were involved in the radical formation. ESR peaks were hardly observed in the absence of linoleic acid (Fig. 1C). The result indicates that the radicals formed in the control reaction mixture are derived from linoleic acid.
Time course of the ESR peak heights. Time course experiments of the ESR peak height were performed for the control reaction mixture (Fig. 2). No ESR peak was observed at 0 h. The ESR peak height gradually increased and reached plateau at 168 h.
HPLC ESR analyses. The HPLC-ESR analyses were performed for the control reaction mixture. On the HPLC-ESR elution profile of the control reaction mixture, two prominent peaks (peak 1 and peak 2) were observed at the retention times of 35.8 min (peak 1) and 43.1 min (peak 2) (Fig. 3A).   Our previous studies have also shown the formation of the 7carboxyheptyl and pentyl radicals in the reaction mixture of linoleic acid with soya bean lipoxygenase and 13-hydroperoxyoctadeca-9,11-dienoic acid (13-HPODE) with ferrous ions (or cytochrome c or haematin). (8,(24)(25)(26) We proposed a scheme to account for the formation of the 7carboxyheptyl radical and pentyl radical (Fig. 5). As the ESR signal decreased to 53 ± 3% (n = 3) of the control reaction mixture for the reaction mixture without iron (Fig. 1B), iron complexes appear to catalyze the formation of 13-hydroperoxyoctadeca-9,11dienoic acid (13-HPODE) and 9-hydroperoxyoctadeca-10,12dienoic acid (9-HPODE) through the hydrogen atom abstraction at 11 carbon. Iron complexes such as iron(IV)-oxo and iron(III)superoxo may initiate the O 2 -activation chemistry by abstraction of an H atom from the substrate. (27,28) Product analysis and spintrapping studies provided evidence for the formation of 1-pentyl-12-carboxydodeca-2,4-dienyloxyl radical and 1-(7-carboxyheptyl)deca-2,4-dienyloxyl radical through the reaction of 13-HPODE and 9-HPODE with ferrous ions. (29)(30)(31) The ferrous ions may form in the following equilibrium to a small extent.
In order to understand the effect of CA on respective radical formation, the control reaction mixture and control reaction mixture with 1.0 mM CA were analyzed using HPLC-ESR. On the HPLC-ESR elution profile of the control reaction mixture, two prominent peaks (peak 1 and peak 2) were observed at the retention times of 35.8 min (peak 1) and 43.1 min (peak 2) (Fig. 3A). The respective peaks disappeared when the control reaction mixture was added with 1 mM CA (Fig. 3B). Caffeic acid inhibited the formation of both radicals. Caffeic acid forms a chelate complex with iron ions. (6) Therefore, the polyphenols possibly inhibit the following three steps (Fig. 5), i.e., step 1, the reaction of linoleic acid with iron complexes such as iron(IV)-oxo and iron(III)-superoxo to form 1-hept-1-enyl-10-carboxydec-2enyl radical, (27,28) step 2, the reaction between 13-HPODE and 1-pentyl-12-carboxydodeca-2,4-dienyloxyl radical, and step 3, the reaction between 9-HPODE and 1-(7-carboxyheptyl)deca-2,4dienyloxyl radicals because iron ions participate in the three reactions. It has previously been shown that the step 2 is inhibited by the polyphenols. (8) Effect of CA on the reaction in the presence of EDTA.
ESR spectra were measured for the control reaction mixture with 1.0 mM CA in the presence of 1 mM EDTA (Fig. 7). On adding 1 mM CA, the ESR peak height sharply increased to 254 ± 21% (n = 3) of the control reaction mixture in the presence of EDTA. Caffeic acid enhanced the generation of the radicals in the presence of 1 mM EDTA. It has been reported that caffeic acid enhanced hydroxyl radicals and t-butylhydroperoxide-derived radicals in the presence of EDTA. (6) The enhancement is possibly due to EDTA-ferric ion complexes being reduced by CA.
Caffeic acid, catechol, noradrenaline, chlorogenic, and gallic acid exert potent inhibitory effect on the formation of pentyl radical and 7-carboxyheptyl radical in the reaction of linoleic acid with iron ions. Of the compounds examined ( Fig. 9), all these compounds, which exerted inhibitory effect on the formation of pentyl radical and 7-carboxyheptyl radical, have catechol moiety in the molecules. Interestingly, (+)-catechin, (-)-epicatechin, and epigallocatechin did not exert inhibitory effect on the formation of pentyl radical and 7-carboxyheptyl radical as much in spite of the catechol moiety in the molecules. That is also the case for the several different concentrations of (-)-epicatechin and CA (Fig. 10).
Effects of (+) catechin, (-) epicatechin, and epigallocatechin on the reaction. In clarifying why (+)-catechin, (-)-epicatechin, and epigallocatechin did not exert the inhibitory effect as much on the radical formation, resorcinol moiety effect on the radical formation was examined. Addition of resorcinol to the control reaction mixture with catechol resulted in minimal enhancement of the radical formation (150 ± 24% of the control with catechol) (n = 3) (Fig. 11B). That was also the case for the addition of phydroquinone instead of resorcinol (182 ± 57% of the control with catechol) (n = 3) (Fig. 11C). The enhancement is presumably due to ferric ions being reduced by p-hydroquinone or resorcinol.
Thus, (+)-catechin, (-)-epicatechin, and epigallocatechin cannot exert the inhibitory effect as much potentially because it has less stability and resorcinol moieties of the catechins.