Tatjana Momić, Jasmina Savić, and Vesna Vasić

Copyright © 2009 Tatjana Momić et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Study of effect of myeloperoxidase on quercetin at pH 6.0 indicated quercetin oxidation via the formation of the oxidation product. The stability of quercetin and oxidation product was investigated as a function of time by using spectrophotometric and HPLC techniques. The apparent pseudo first-order rate constants were calculated and discussed.

1. Introduction

Flavonol quercetin (Q) (3,5,7,,-pentahydroxyflavone) (Scheme 1) due to its phenolic structure is a strong antioxidant and free radical scavenger [1]. Oxidation of quercetin during its antioxidative functions is usually accompanied by the production of the quercetin radical anion, superoxide, and hydrogen peroxide [2]. Quercetin undergoes autoxidation—the nonenzymatic reaction with atmospheric oxygen. Also, it is known that one-electron oxidation of quercetin is catalyzed by different peroxidases like lactoperoxidase (LPO) [3] and horseradish peroxidase (HRP) [4–7] in the presence of .

Scheme 1: Oxidation of quercetin by MPO/ system (modified from [15]).

The heme enzyme myeloperoxidase (MPO) is oxidant enzyme in the process of inflammation and atherogenesis [8]. Myeloperoxidase is relatively nonspecific with respect to its reducing substrates. This enzyme is able to oxidize different substrates among which anilines and phenols [9–11]. This letter deals with quercetin oxidation by myeloperoxidase- system.

2. Experimental

2.1. Chemicals

Quercetin dihydrate (Sigma-Aldrich) of the highest quality available (98%) was used without purification.  M stock solution of quercetin was prepared in methanol immediately before the experiments. For all experiments, freshly prepared solutions of quercetin were made by dilution of the appropriate amount of the stock solution with phosphate buffer at pH 6.0. Myeloperoxidase was purified from human neutrophils to a purity index (/) greater than 0.70 as described previously [12]. Its concentration was calculated using per heme [13]. Hydrogen peroxide solutions were prepared daily by diluting a stock solution, and the concentration was determined using [14]. Redistilled water was used in all experiments.

2.2. Oxidation of Quercetin by Myeloperoxidase

Quercetin ( M) was incubated in 50 mM phosphate buffer, pH 6.0, with various concentrations of MPO. Reaction was started by the addition of 50 μM , and UV absorbance changes were recorded. For the HPLC analysis, reaction was stopped after 30 minutes by adding catalase (100 μg/mL). The reaction mixture was centrifuged for 2 minutes at 10000 rpm. The clear supernatant was analyzed by HPLC.

2.3. HPLC Analysis

HPLC equipment consisted of an HP 1100 Series chromatograph coupled with a DAD. Chromatographic separations were run on a C18 Pinnacle ODS column (Restek, 250 mm × 4.6 mm, 5 μm) using an 80 : 20 mixture of 2 vol% H₃PO₄ (A) and acetonitrile (J.T. Baker) (B) as the eluent for the first 2 minutes. The linear gradient was applied from 20% to 45% B from 2 to 7 minutes. An isocratic 55 : 45 mixture was applied between 7 and 13 minutes. The eluent flow rate was 1.0 mL min⁻¹, and the injection volume was 10 L. The elutions were monitored with DAD at different wavelengths between 200 and 450 nm.

2.4. UV-VIS Spectroscopic Studies

UV spectra were recorded on a Perkin Elmer Lambda 35 UV-Vis spectrophotometer equipped with thermostatted quartz cell. The temperature in the cell was kept at with a water-thermostatted bath.

3. Results and Discussion

We investigated the effect of MPO on  M quercetin in phosphate buffer, pH 6.0 in the presence of 50 μM at , spectrophotometrically and by HPLC. The following concentrations of enzyme were used: 5, 11, 15, 20, 50, 100, 150, and 200 nM.

At the absorption spectra of samples in which concentration of MPO was ≤11 nM, two quercetin absorption bands (254 nm and 367 nm) were observed (Figure 1(a)). The same results were yielded when MPO or was omited from the mixture.

Figure 1: Absorption spectra and HPLC chromatograms of  M quercetin at pH 6.0 in absence (a), (b) and presence (c), (d) of 20 nM MPO. 50 μM was present in all reaction solutions. Spectra were recorded over a period of 30 minutes; arrows indicate the direction of the change. Chromatograms were recorded after 30 minutes (b) at and (d) at .

The absorption spectra of samples in which concentration of MPO was ≥15 nM showed the decay of quercetin absorption bands at 254 nm and 367 nm. At the same time simultaneous rise of the absorption band at 336–342 nm, depended on MPO concentration, was observed (Figure 1(c)), typical of oxidation product formation [16]. Moreover, two well-defined isosbestic points at 290 and 364 nm indicate that there was no significant accumulation of intermediates in the reaction of oxidation product formation (Figure 1(c)). These spectral changes are analogous to those occurring upon autoxidation of quercetin in water [16] and UV irradiation [17] as well as oxidation with some oxidants in organic solvents [16].

The HPLC analysis of quercetin solution treated with MPO for 30 minutes (Figure 1(d)) revealed that concentration of quercetin (retention time 10 minutes) was negligible. Also, formation of major oxidation product (retention time 5.26 minutes) which was more polar than quercetin was detected (Figure 1(d)). From literature data and the obtained spectrophotometric and HPLC results in this work, we assume that the oxidation product detectable at 336–342 nm results from addition on the p-quinonemethide formed by H-atom abstraction at 3-OH and -OH of quercetin and subsequent rearrangement of the central ring [16]. The complete sequence of quercetin autoxidation induced by oxidants and catalyzed by metal ions is described earlier [15, 16] (Scheme 1).

According to spectrophotometric and HPLC experimental data, quercetin degradation (1) and oxidation product formation (2) as the function of time followed the relation (1)(2) where and are the absorbances proportional to the quercetin and its oxidation product concentrations, respectively, after an irradiation period of time t and k is the overall pseudo first-order rate constant. and are the absorbance proportional to the initial concentration of quercetin and the concentration of the oxidation product on the plateau of the kinetics curve, respectively.

The kinetic curves that describe the quercetin degradation and the formation of the oxidation product in the presence of different MPO, concentrations are shown in Figure 2. In the presence of MPO concentration that was ≥15 nM oxidation product was formed at the first minute of reaction (Figure 2(a)). The overall apparent first-order rate constants for the oxidation product formation and quercetin transformation to its degradation products were determined from the kinetic curves and are presented in Table 1. For comparison, in Table 1 we also presented values for these constants obtained for quercetin oxidation by UV irradiation [17].

Table 1: Rate constants of quercetin oxidation.

Figure 2: Change of absorbance of quercetin in the presence of various MPO concentrations in the function of time, at pH 6.0 (a) formation of quercetin oxidation product at ; Inset: 20 nM MPO and (b) degradation of quercetin oxidation product at .

The rate of quercetin oxidation by MPO at pH 6.0 is 3-fold faster compared with the rate of quercetin oxidation by UV irradiation at pH 10.0. Moreover, product obtained for quercetin oxidation by MPO at pH 6.0 is more stable than product of quercetin oxidation by UV irradiation at pH 10.0.

Acknowledgment

We wish to express our gratitude to the Ministry of Science and Technological Development of the Republic of Serbia supported this work through Project 142051.

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