Henriquez et al 2004 - Kinetic profiles.pdf

7/24/2019 Henriquez et al 2004 - Kinetic profiles.pdf

1/6

Services on Demand

Article

Article in xml format

Article references

How to cite this article

Automatic translation

Send this article by e-mail

Indicators

Cited by SciELO

Access statistics

Related links

Share

Permalink

Journal of the Chilean Chemical SocietyOn-line versionISSN 0717-9707

J. Chil. Chem. Soc. vol.49 no.1 Concepcin Mar. 2004

http://dx.doi.org/10.4067/S0717-97072004000100011

KINETICS PROFILES IN THE REACTION OF ABTS DERIVED

RADICALS WITH SIMPLE PHENOLS AND POLYPHENOLS.

CAROLA HENRIQUEZ, CAROLINA ALIAGA AND EDUARDO LISSI*

Departamento de Qumica, Facultad de Qumica y Biologa,Universidad de Santiago de Chile, Casilla 40, Correo 33, Santiago, Chile.

(Received: July 15, 2003 - Accepted: October 10, 2003)

ABSTRACT

The kinetic profile associated to the reaction of phenols with 2,2'-azinobis-

(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) derived radicals is

extremely dependent of the characteristics of the substrate. In particular,

polyphenols present complex profiles that can be associated to successive

reactions of different reactive centers present in the target molecule. Also,

changes in the secondary reactions of the produced radicals can lead to a

partial recovery of the ABTS radical concentration at intermediate reaction times, as observed employing

1,4-dihydroxybenzene. Although the kinetic profiles of monophenols are considerably simpler, the stoichiometry

of the process is unexpected. For example, the data obtained imply that four radicals are consumed by eachp-tertbutylphenol molecule introduced into the system. This large stoichiometric coefficient is not compatible with

a simple, phenol promoted, back reduction of the ABTS radical. These results, as well as the overshoot observed

employing 1,4-dihydroxybencene, cast doubts on the use of ABTS based methodologies to quantitatively measure

the capacity of a given compound to trap free radicals or the amount of free radical scavengers present in

complex mixtures.

Keywords: ABTS; phenols ; antioxidant capacities; free radical scavenging.

INTRODUCTION

2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) derived radicals are frequently employed in the

evaluation of the free radical trapping capacity of pure compounds and/or complex mixtures ( 1-6). Most of these

studies define the antioxidant capacity of the tested compound(s) from their prevention of the radical formation

(1) or by measuring the bleaching of the pre-formed radicals at a single reaction time (2). However, a meaningful

interpretation of these data requires some knowledge regarding the mechanism and kinetics of the radical

scavenging process. In spite of this, very few kinetic studies have been carried out. Lissi and co-workers have

evaluated the kinetics of the reaction of the ABTS radical with simple phenols (8), hydroperoxides (9) and

amino-acids (10). These authors found a complex kinetic relationships between initial rates and the

concentrations of the reactants. Furthermore, a clear inhibition by the parent ABTS was observed, explained in

terms of a mechanism involving a partially reversible initial step:

ABTS* + XH ABTS + X* + H+ (1)

ournal of the Chilean Chemical Society - KINETI... http://www.scielo.cl/scielo.php?script=sci_artte

de 6 16/10/15 01


2/6

followed by a series of radical-radical reactions. The possibility of partial reversibility in these reactions further

complicate the kinetic analysis of the results.

There are very few kinetic data regarding the reaction of ABTS derived radicals with polyfunctional compounds

(such as polyphenols) and/or complex mixtures, were it could be expected a more complex kinetics. Naik et

al. (11) measured, by a pulse radiolysis technique, the reactivity of several herbal extracts used in Ayurvedic

medicine. These authors observed very good first order decays of the ABTS derived radical. Furthermore,

decays took place with pseudounimolecular rate constants that were first order in the scavengers

concentration, suggesting that the data could be represented by a simple bimolecular process. However, it hasto be considered that in this work it is difficult to know what proportion of the antioxidants present in the

mixtures have reacted during the decay of the radical. In the present work, we present ABTS radicals

consumption profiles elicited by several polyphenols and complex mixtures (such as red wine). These profiles

can not be explained by any simple kinetic scheme, and show that caution must be excersized in the

interpretation of the data.

EXPERIMENTAL

ABTS (Sigma), Trolox (Sigma), p-tertbutylphenol (Aldrich), 1,4-dihydroxybenzene, and potassium peroxodisulfate

(Merck) were employed as received. The red wine employed was a commercial Cabernet Sauvignon sample. The

radical cation was prepared by reacting ABTS (150 mM) with peroxodisulfate (75 mM) overnight at roomtemperature (12). The formation of the radical was monitored by its absorbance in the visible region. Its

concentration was quantified by the absorbance at 734 nm employing an extinction coefficient of 0.015 m M -1sec-1(13,14)

RESULTS AND DISCUSSION

Typical decay profiles are shown in Figs 1to 4. Fig 1shows data obtained for Trolox and p-tertbutyl phenol.

These data show that Trolox reacts almost instantaneously, and that p-tertbutylphenol reacts slowly, presenting a

smooth decay that can be approximately fitted to a monoexponential function. A puzzling feature of these data is

the larger consumption of the radical cation elicited by p-tertbutylphenol. If, as generally accepted (13), it is

considered that Trolox reacts with the ABTS derived radicals according to

2 ABTS* + Troloxproducts (2)

the present data imply that the stoichiometry associated to the reaction of p-tertbutylphenol must be represented

by

4 ABTS* + p-tertbutylphenol products (3)

with four radicals consumed by each added p-tertbutylphenol molecule. This peculiar result casts doubts on the

quantitative use of ABTS derived radicals to titrate the concentration of free radicals scavengers in complex

mixtures.


2 de 6 16/10/15 01


3/6

Fig. 1.Consumption of ABTS derived radicals following the addition of

simple phenols.

( ) Addition of Trolox ( 10 micromolar)

( ) Addition of p-tertbutylphenol ( 10 micromolar)

More complex behaviors are observed in polyfunctional compounds (Figs 2and 3) and complex mixtures (Fig. 4).

The decay of the ABTS radical promoted by quercetin (Fig 2) presents two phases, and can be approximately

fitted to a bi-exponential decay of widely different lifetimes (ca. 3 and 200 sec). This behavior closely resembles

that described by Pannala et al. (15) for compounds with a catechol-containing B ring, and can be explained in

terms of the presence of two reactive sites of widely different reactivity. On the other hand, the data obtained

employing 1,4-dihydroxybenzene present an overshooting (Fig. 3), with a minimum in ABTS* concentration at ca.

100 sec, followed by a partial recovery of the ABTS derived radical absorbance. Complex kinetics that can not befitted even to bi-exponential decays are generally observed when mixtures of polyphenols, such as red wine, are

tested (see Fig. 4).

Fig. 2. Consumption of ABTS derived radicals following the addition of

quercetin (10 mM). The line shows the "best-fit" bi-exponential decay.


3 de 6 16/10/15 01


4/6


1,4-dihydroxy benzene (12.7 mM).


red wine ( 0.17 mL/mL). The line shows the "best-fit" bi-exponentialdecay.

The profiles obtained employing quercetin are typical of most polyphenols and their mixtures, and have lead to

the proposal of two indexes to evaluate the total antioxidant capacity of the added antioxidants. This two indexes

are based on the initial fast decay (reaction time less than 10 sec) and the total decay observed after a long (10

to 15 minutes) time period (16). These indexes are considered to represent the amount of fast reacting groups

(FREE) and total groups with antioxidant capacity (TREE). It is interesting to note that the fast decay observed in

Fig. 2can be accounted for in terms of an stoichiometric process such as

2 ABTS* + Quercetin2 ABTS + products (4)

The slower process observed afterwards can be explained in terms of the presence of less reactive phenol groups

that remain active after the almost total reaction of the more reactive groups. The presence of several

polyphenols of widely different reactivity in complex mixtures (such as wine, Fig. 4) can explain the complex

profiles observed in these samples. In a simplified scheme, the comparison between FREE and TREE indexes cangive information regarding the amount of fast-reacting and slow reacting groups in the tested sample ( 16).

The reaction profiles observed with 1,4-dihydroxybenzene are more difficult to explain, and cast further doubts

on the quantitative reliance of the above mentioned indexes. A plausible explanation of the observed

overshooting can be given in terms of the following reaction scheme, that takes into account the occurrence of

reversible cross-combination reactions:

ABTS* + XH ABTS + X* + H+ (1)


4 de 6 16/10/15 01


5/6

ABTS* + X*adduct (fast) (5)

adductABTS* + X* (slow) (6)

X* + X*products (slow) (7)

In this scheme, overshooting in the ABTS* consumption is due to the occurrence of reaction (5). The following

(slower) decomposition of this adduct and the self reaction of X* radicals could explain the partial recovering of

the ABTS derived radical concentration.

REFERENCES

N.Miller, C. Rice-Evans, M. Davies, V. Gopinathan and A. Milner. Clin.Sci. 84 (1993) 407-12.1.

[ Links]

Ch. Romay, C. Pascual and E. Lissi, Braz. J. Med. Biol. Res. 29 (1996) 175-183. [ Links]2.

N. Miller and C. Rice-Evans, Free Rad. Res. 26 (1997) 195-199. [ Links]3.

M.B. Arnao, A. Cano, J. Hernandez-Ruiz, F. Garca-Canovas and M. Acosta. Anal. Biochem. 236 (1996)

255-61. [ Links]

4.

M. Arnao, A. Cano and M. Acosta. Free Rad. Res. 31 (1999) 589-96. [ Links]5.

G. Bartosz, A. Janaszewska, D. Ertel and M. Bartosz. Biochem. Mol. Biol. Inte. 46 (1998) 519-28.

[ Links]

6.

C. Rice-Evans. Free Rad. Res.. 33 (2000) 559-66. [ Links]7.

A. Campos and E. Lissi. Int. J. Chem. Kinetics 29 (1997) 219-24. [ Links]8.

C. Aliaga and E. Lissi. Int. J. Chem. Kinetics 30 (1998) 565-70. [ Links]9.

C. Aliaga and E. Lissi. Can. J. Chem. 78 (2000) 1052-9. [ Links]10.

G.H. Naik, K. I. Priyadarsini, J.G. Satav, M.M. Banavalikar, D.P. Sohoni, M.K. Biyani and H. Mohan.

Phytochemistry 63 (2003) 97-104. [ Links]

11.

C.Henriquez, C. Aliaga and E. Lissi. Int. J. Chem. Kinetics, in the press.12.

[ Links]

C.Henriquez and E. Lissi, Bol. Chem. Chil. Quim. 47 (2002) 563-6. [ Links]13.

S. Scott, W. Chen, A. Bakac, and J. Espenson. J. Phys. Chem. 97 (1993) 6710-4. [ Links]14.

A.S. Pannala, T. S. Chan, P. J. O' Brien and C.A. Rice-Evans. Biochem. Biophys. Res. Comm. 282 (2001)

1161-8. [ Links]

15.

D. Perez, F. Leighton, A. Aspee, C. Aliaga and E. Lissi. Biol. Res. 33 (2000) 71-7.16.

[ Links]

Paicav 170, Depto. 19


5 de 6 16/10/15 01


6/6

P.O. Box 2613, Concepcin, Chile

Phone 41-2227815, Fax 41-2235819

[email protected]


6 de 6 16/10/15 01

Documents

Henriquez et al 2004 - Kinetic profiles.pdf