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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.33 n.2 Santiago  2000

http://dx.doi.org/10.4067/S0716-97602000000200015 

Ascorbate protects (+)-catechin from oxidation both in a
pure chemical system and human plasma

SILVINA B. LOTITO AND CESAR G. FRAGA

Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, 1113-
Buenos Aires, Argentina

ABSTRACT

We evaluated the interaction between ascorbic acid (AA) and (+)-catechin (CTCH) in potassium phosphate solution, pH 7.4 (PPS) and in human plasma. In both systems, the oxidation was started by adding 2,2’-azobis-(2-amidinopropane) clorhidrate (AAPH). The concentrations of AA and CTCH were determined by HPLC using electrochemical detection. In PPS, CTCH was oxidized by AAPH (50 µM), in either the absence or presence of different initial concentrations of AA (25-200 µM). In the presence of AA, CTCH depletion was delayed, an effect that was dependent upon the initial concentration of AA. When 100 µM AA was added after the oxidation had begun, CTCH depletion was arrested for 30 min. The kinetics of AA oxidation by AAPH was also characterized in PPS. AA (100 µM) was completely consumed after 60 min of reaction at 37 ºC, in both the absence and presence of 100 mM CTCH. When human plasma was incubated with 50 mM AAPH in the absence of added CTCH, AA was completely consumed after 45-60 min. CTCH did not prevent AA depletion in human plasma at the concentrations tested (10, 50 100 µM). The results point out that AA is able to protect other aqueous soluble antioxidants, e.g.: CTCH.

Key words: catechins, antioxidants, free radicals, procyanidins

Abbreviations used: CTCH: (+)-catechin; AA: ascorbic acid; AAPH: [2,2’-azobis-(2-amidinopropane) clorhidrate].

INTRODUCTION

Catechins are a group of flavonoids that exhibit antioxidant properties in a number of biochemical systems (6, 16). Chemically, catechins are polyhydroxilated flavonoids with rather water soluble characteristics that differ in the number and position of the hydroxyl groups in the molecule. It has long been postulated that due to such polyhydroxylated structure, catechins could act as antioxidants either through the chelation of metals with redox properties or by acting as scavengers of free radicals (16).

Catechins are present in body cells and fluids as a result of the ingestion of fruits, vegetables, and plant-derived foods and beverages, such as wine, tea and chocolate (4). Several epidemiological studies have indicated that a high content of polyphenol flavonoids in the diet could be associated with a lower risk of cancer and coronary heart disease mortality (8, 9). These associations were stronger when the intake of catechins was particularly considered.

The interaction of catechins with physiological lipid soluble antioxidants, such as a-tocopherol and b-carotene, has been the subject of few studies. The antioxidant capacity of catechins has been demonstrated in the protection of isolated LDL against in vitro oxidation, preventing lipid oxidation and a-tocopherol consumption (18). (+)-Catechin (CTCH) was effective in preventing the oxidation of plasma components and lipid soluble antioxidants (a-tocopherol and ß-carotene), delaying the oxidation of these molecules in the presence of negligible (< 5 µM) concentrations of ascorbic acid (AA) (12).

A synergistic effect has been postulated between AA and flavonoids, similar to the synergism between AA and a-tocopherol. A possible interaction between AA and flavonoids was proposed early on by Szent-Györgyi et al. (17). Their observations led them to conclude that AA is accompanied in cells by a substance of similar importance that presented a protective role toward vessel walls. They proposed the name of vitamin P for this factor, which appeared to be a polyphenol of vegetal origin.

Our previous observations in human plasma oxidized with AAPH indicated that AA depletion preceded CTCH consumption (13), suggesting that AA would protect CTCH from oxidation. To further characterize the interaction between AA and CTCH, we determined the kinetics of oxidation of CTCH and AA by a water-soluble free-radical initiator, 2,2’-azobis-(2-amidinopropane) clorhidrate (AAPH), both in a pure chemical system (potassium phosphate solution) (PPS) and in human plasma. In both systems, AA delayed CTCH oxidation.

MATERIALS AND METHODS

Reagents

CTCH was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and the AA was from Fisher Co. (Fair Lawn, NJ, USA). The azocompound 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) was from Polysciences Inc. (Warrington, PA, USA). All the solvents used were of HPLC grade and were from Merck Quimica Argentina S. A. (Buenos Aires, Argentina) and J. T. Baker Inc. (Phillipsburg, NJ, USA).

Experiments in aqueous solution

CTCH and AA were oxidized in 50 mM potassium phosphate solution (pH 7.4) (PPS) by incubation with AAPH, a water-soluble compound, which generates free radicals at a constant rate (15). AAPH was dissolved in distilled water and prepared immediately before using. CTCH and AA stock solutions were dissolved in distilled water to the concentrations used. Incubation was performed at 37 °C, with continuous shaking, under air.

Determination of AA

The concentration of AA was determined by HPLC, using electrochemical detection in accordance with Lykkesfeldt et al. (14), with modifications. Aliquots were removed at different intervals of time and analyzed for AA content through the direct injection into the HPLC equipment. An isocratic reverse-phase chromatography was performed using a C-18 column, 3.3 cm x 4.6 mm, and 0.8% (w/v) metaphosphoric acid as a mobile phase. For electrochemical detection, the chromatographic system was connected to an ESA Coulochem II coulometric electrochemical detector, equipped with a Model 5011 analytical cell operated at +0.6 V.

Determination of CTCH

Immediately after incubation, samples were analyzed for catechin content. HPLC was run isocratically on a C-18 column. The mobile phase was methanol:H2O:formic acid 29:60:1 (v:v:v), containing 30 mM LiClO4. The determination of CTCH was carried out with electrochemical detection using an amperometric BAS LC4C detector (Bioanalytical Systems Inc., West Lafayette, IN) operated at +0.8 V.

Experiments in human plasma

Blood was collected from healthy humans by venipuncture, using heparin sulphate to prevent clotting. After centrifugation at 1000 x g for 10 min at room temperature, the plasma was separated from the blood cell package and used immediately for analysis. All the used plasma had an AA concentration within the range for a healthy population. Human plasma was oxidized by incubation in the presence of 50 mM AAPH. Plasma incubations were carried out with or without the previous addition of different concentrations of CTCH (10-100 mM). The experiments were performed at 37 °C, with continuous shaking, under air. For AA determination, plasma aliquots (100 ml) were precipitated with 200 ml of 10 % (w/v) metaphosphoric acid, vortexed, and centrifuged at 14,000 rpm for 4 min. Samples were kept at 0-4 °C throughout the entire procedure. A volume of the supernatant (90 µl) was diluted with 300 µl of 0.8% (w/v) metaphosphoric acid, filtered through a 0.22 mm nylon membrane, and a 20 ml-aliquot was injected into the HPLC equipment, as previously described.

RESULTS

Effect of AAPH on AA and CTCH depletion in PPS.

The interaction between CTCH and AA was studied in a pure chemical aqueous system, using a water-soluble free-radical generator. For the characterization of the system, CTCH and AA were incubated separately in the presence of different concentrations of AAPH, in PPS. Figure 1A shows the concentrations of CTCH (100 µM, initial concentration) after oxidation with 10, 25, and 50 mM AAPH. A first order rate of depletion for CTCH was dependent upon AAPH initial concentration. Figure 1B shows the depletion of 27 µM AA by different concentrations of AAPH. A dose-dependent response was also observed for AA depletion in the presence of AAPH. The AA depletion followed a first order reaction.


Figure 1: Effect of different concentrations of AAPH on CTCH (A) and AA (B) oxidation in potassium phosphate solution, pH 7.4. CTCH and AA were incubated separately in the presence of AAPH, at 37 ºC. References: (D) 10 mM; () 25 mM; (O) 50 mM AAPH.

Effect of the AA on the kinetics of CTCH depletion

The effect of AA on the kinetics of CTCH depletion by AAPH was studied in PPS. In the absence of AA, CTCH depletion by AAPH started without a lag phase. In the presence of AA, the depletion of CTCH was delayed, and this effect was dependent upon the initial concentration of AA. Figure 2A shows that the lag time for the depletion of CTCH was linearly related with the concentration of AA (25-200 µM). The inset in Figure 2A shows the kinetics of CTCH depletion by incubation with AAPH, for three AA concentrations, 0, 25, and 200 µM AA. The effect of the addition of 100 µM AA 45 min after the oxidation of CTCH with AAPH began is shown in Figure 2B. It is observed that CTCH depletion was arrested for 30 min, and then the oxidation continued at a faster rate, reaching the zero values at 120 min, as the non-added sample.


Figure 2: Effect of AA on CTCH depletion. CTCH was incubated in the presence 50 mM AAPH (37 ºC, pH 7.4), and different concentrations of AA. References: A) lag time in CTCH depletion as a function of AA concentration; B) effect of the addition of 100 mM AA after 30 min of oxidation. The inset shows CTCH depletion in the presence of 0 (·), 50 (#) and 200 mM (') AA.

Effect of the CTCH on the kinetics of AA depletion

The effect of the addition of 100 mM CTCH on the kinetics of AA depletion was studied PPS. AA was oxidized in the presence of 25 mM AAPH, at 37 ºC for 1 h, and the concentration of AA was determined. In the absence of CTCH, AA depletion by AAPH started without lag phase and was completely consumed after 1 h of incubation (Fig. 3). The addition of 100 µM CTCH did not affect the rate of AA oxidation by AAPH. The effect of the addition of 100 µM CTCH 30 min after the oxidation had started did not modify AA depletion under these conditions.


Figure 3: Effect of CTCH on AA depletion. AA was incubated in the presence 25 mM AAPH (37 ºC, pH 7.4), and 100 mM CTCH. References: (#) AA depletion in the absence of CTCH; (O) AA depletion in the presence of 100 mM CTCH added at the initial stage; (Fig. 3A) AA depletion when 100 mM CTCH was added after 30 min of oxidation (Fig. 3B).

Effect of CTCH on the kinetics of AA depletion in human plasma

The effect of adding different concentrations of CTCH on the depletion of physiological levels of AA was studied in human blood plasma. Figure 4 shows AA concentration as a function of the time of incubation, when plasma was oxidized with 50 mM AAPH. The average initial AA concentration in the plasmas used was 20.5 ± 1.7 µM. In the absence of added CTCH, AA depletion started immediately and was totally consumed after 45 min of incubation (consumption rate: 0.7 µM/ min). The addition of CTCH did not prevent AA depletion at the concentrations tested (10, 50 100 µM). A slight increase in the AA depletion rate was observed in the presence of CTCH, although this difference was not statistically significant.


Figure 4: Effect of different concentrations of CTCH on AA in plasma. Human plasma was incubated in the presence of 50 mM AAPH, at 37 °C, during 1 h, and AA content was determined by HPLC. References: (#) 0, (+) 10, (') 50, and (O)100 mM CTCH added at the initial stage.

DISCUSSION

Szent-György et al. were the first to discuss the biological relevance of another factor similar to AA in importance and related activity and whose deficiency was hidden by the symptoms resulting from the lack of AA (scurvey) (1). They proposed the name of vitamin P for this factor, according to its ability to restore the vascular permeability in pathological conditions, where normal capillarity resistance was affected and the administration of AA was ineffective (17). This vitamin P consisted of flavonoids (flavons and flavonols). Szent-György later proposed that the clinical symptoms observed in experimental scurvey were not due to a pure C avitaminosis, but to a mixed C and P avitaminosis, suggesting a possible interaction between these two vitamins (2). Considering that these compounds can act as antioxidants, cooperation between them would be feasible based on the fact that such a vitamin P or a related compound could protect vitamin C from oxidation. This could involve a synergism that could influence the total antioxidant capacity of the different tissues and body fluids, such as human plasma.

The analysis of the kinetics of CTCH as an antioxidant in the presence of physiological levels of AA and lipid soluble antioxidants led to the conclusion that the oxidation of CTCH in human plasma preceded lipid soluble antioxidants, but CTCH depletion was not evident until AA was completely consumed (13). In the present study, we further examined the effect of different concentrations of CTCH on AA depletion in human plasma when the oxidation was started with AAPH and the antioxidant interaction between CTCH and AA in pure aqueous systems.

The experiments in aqueous solution demonstrated that CTCH oxidation was efficiently prevented in the presence of AA. When CTCH was oxidized by a water-soluble free radical initiator, a lag time in CTCH depletion was observed in the presence of AA, and this lag time was correlated with the initial concentration of AA. AA was also effective in preventing the oxidation of CTCH when it was added after the oxidation was started. A lag time in CTCH depletion was also observed in this case, and it was proportional to the amount of AA added. By contrast, the addition of CTCH did not modify AA depletion when the CTCH was added either at the beginning or in the middle of AA oxidation by AAPH. AAPH generates free radicals from the aqueous phase, and then the oxidation is propagated in the lipid domains. Our results showed that CTCH did not prevent the depletion of AA against AAPH-derived radicals in any of the concentrations tested. AA depletion was not significantly different in the absence or presence of CTCH, suggesting that AA reacts faster than CTCH against peroxyl radicals or that it regenerates CTCH when the flavonoid is oxidized.

According to the results shown in this work, the reactivity of AA against AAPH-derived radicals can be calculated to be approximately ten times greater than the reactivity of CTCH (with respect to the slopes of the curves corresponding to the reactions of AA or CTCH with different concentrations of AAPH). It supports the idea that competitive reactions between these two free-radical scavengers could be taking place. However, the reaction between AA and CTCH is also feasible, according to the redox potentials, and should be taken into account, primarily when more complex systems (i.e.: plasma) are being studied. Flavonoids with a catechol structure in the B-ring generally have higher reduction potentials than AA (3). The reduction potential of CTCH was reported to be +0.54 V (pH 7.0 and 20 ºC) (11), in agreement with the value reported for the catechol group (+0.53 V), and higher than the reduction potential of AA (+0.28 V) (5). Lower reduction potential values for CTCH and AA (pH 7.4) were reported, but the value for AA was still below CTCH (10). Thus, the redox couple AA-CTCH could be thermodynamically feasible, and AA could regenerate the flavonoid from the respective aroxyl radical, as does the postulated redox coupling for AA-AT.

Considering our results, CTCH could act as an antioxidant of intermediate reactivity between the water soluble and the lipid soluble antioxidants, such as a-tocopherol and ß-carotene. The possibility that related catechins [(-)-epicatechin and their gallate esters] can also participate in an antioxidant network stress the contribution of plant polyphenols to the prevention of disease.

ACKNOWLEDGEMENTS

This work was supported by grants from the University of Buenos Aires (TB30) and CONICET (PIP-0738/98). CGF is a member of CIC-CONICET, and SL has a fellowship from CONICET.

Corresponding Author: Cesar G. Fraga. Fisicoquímica, Facultad de Farmacia y Bioquímica , Universidad de Buenos Aires , Junín 956, 1113-Buenos Aires, Argentina. Phone: (54-11) 4964-8244 . Fax: (54-11) 4508-3646 . E-mail: cfraga@huemul.ffyb.uba.ar

Received: October 28, 1999 Accepted: January 10, 2000

REFERENCES

1. BENTSATH A, RUSZNYAK ST, SZENT-GYÖRGYI A (1936) Vitamin nature of flavones. Nature 138:798         [ Links ]

2. BENTSATH A, RUSZNYAK ST, SZENT-GYÖRGYI A (1937) Vitamin P. Nature 139:326-327         [ Links ]

3. BORS W, MICHEL C, SCHIKORA S (1995) Interaction of flavonoids with ascorbate and determination of their univalent redox potentials: a pulse radiolysis study. Free Radic Biol Med 19: 45-52         [ Links ]

4. BRAVO L (1998) Polyphenols: chemistry dietary sources metabolism and nutritional significance. Nutrition Reviews 56 (11): 317-333         [ Links ]

5. BUETTNER GR (1993) The pecking order of free radicals and antioxidants: lipid peroxidation a-tocopherol and ascorbate. Arch Biochem Biophys 300 (2): 535-543         [ Links ]

6. FRAGA CG, MARTINO VS, FERRARO GE, COUSSIO JD, BOVERIS A (1987) Flavonoids as antioxidants evaluated by in vitro and in situ liver chemiluminescence. Biochem Pharmacol 36: 717-720         [ Links ]

7. FREI B, STOCKER R, AMES BN (1988) Antioxidant defenses and lipid peroxidation in human blood plasma. Proc Natl Acad Sci USA 85: 9748-9752         [ Links ]

8. HERTOG MGL, KROUMHOUT D, ARAVANIS C, BLACKBURN H, BUZINA R, FIDANZA F, GIAMPAOLI S, JANSEN A, MENOTTI A, NEDELJKOVIC S, PEKKARINEN M, SIMIC BS, TOSHIMA H, FESKENS EJM, HOLLMAN PCH, KATAN MB (1995) Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Arch Intern Med 155: 381-386         [ Links ]

9. HERTOG MGL, FESKENS EJM, HOLLMAN PCH, KATAN MB, KROUMHOUT D (1993) Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet 342: 1007-1011         [ Links ]

10. JÖRGENSEN LV, SKIBSTED LH (1998) Flavonoid deactivation of ferrylmyoglobin in relation to ease of oxidation as determined by ciclic voltammetry. Free Rad Res 28: 335-351         [ Links ]

11. JOVANOVIC SV, STEENKEN S, TOSIC M, MARJANOVIC B, SIMIC M (1994) Flavonoids as antioxidants. J Am Chem Soc 116: 4846-4851         [ Links ]

12. LOTITO SB, FRAGA CG (1998) (+)-Catechin prevents human plasma oxidation. Free Rad Biol Med 24: 435-441         [ Links ]

13. LOTITO SB, FRAGA CG (1999) (+)-Catechin as antioxidant: mechanisms preventing human plasma oxidation and activity in red wines. Biofactors (in press)         [ Links ]

14. LYKKESFELDT J, LOFT S, POULSEN HE (1995) Determination of ascorbic acid and dehydroascorbic acid by high performance liquid chromatography with coloumetric detection - Are they reliable biomarkers of oxidative stress? Anal Biochem 229: 329-335         [ Links ]

15. NIKI E (1990) Free radicals initiators as source of water or lipid soluble peroxyl radicals. Methods Enzymol 186: 100-108         [ Links ]

16. RICE-EVANS CA, MILLER NJ, PAGANGA G (1996) Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 20: 933-956         [ Links ]

17. RUSZNYAK S, SZENT-GYÖRGYI A (1936) Vitamin P: Flavonols as vitamin. Nature 138:27         [ Links ]

18. SALAH N, MILLER NJ, PAGANGA G, TIJBURJ L, BOLWELL GP, RICE-EVANS CA (1995) Polyphenolic flavonols as scavengers of aqueous phase radicals and as chain-breaking antioxidants. Arch Biochem Biophys 322: 339-346         [ Links ]

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