Determination of hydrogen peroxide scavenging activity of cinnamic and benzoic acids employing a highly sensitive peroxyoxalate chemiluminescence-based assay: Structure–activity relationships

https://doi.org/10.1016/j.jpba.2005.03.044Get rights and content

Abstract

A series of cinnamic acids along with their corresponding benzoate analogues were tested for their ability to scavenge hydrogen peroxide (H2O2), by using a highly sensitive, peroxyoxalate chemiluminescence assay. Among benzoic acid derivatives, vanillic acid (3-hydroxy-4-methoxybenzoic acid) was found to be the most efficient H2O2 scavenger with its hydrogen peroxide scavenging activity (SAHP) being 170.20 μM−1, whereas protocatechuic acid (3,4-dihydroxybenzoic acid) exhibited the weakest activity (5.90 μM−1). Caffeic acid (3,4-dihydroxycinnamic acid) was the strongest antioxidant amongst cinnamate derivatives with a SAHP = 8.2 μM−1, as opposed to m-coumaric acid (2-hydroxycinnamic acid), which was found to be a poor hydrogen peroxide scavenger (SAHP = 0.18 μM−1). Comparison between the two groups revealed that benzoate derivatives are much stronger hydrogen peroxide quenchers in relation with their cinnamate analogues, and this finding was discussed on a basis of structure–activity relationships and comparative assessment of other antioxidant characteristics.

Introduction

Currently, the concept of healthy nutrition has embraced the consumption of fresh fruit and vegetables, which have been proven to contribute in maintaining an improved antioxidant status, thus preventing the onset of degenerative processes [1], [2]. One of the most prominent class of dietary antioxidants, polyphenols, have thoroughly been examined for their potency both in vitro and in vivo, but in many instances, the antioxidant profile of a given compound may lack consistency and completeness, because the credible evaluation of a compound to act as an antioxidant largely depends on the model system used, but also requires tests investigating different aspects of antioxidant activity [3], [4].

Over the past few years, there has been a great amount of studies reporting on methods for assessing the antioxidant activity of various polyphenols, but it appears that most of the tests established for in vitro examination are rather oriented at the radical scavenging, whereas the ability of those substances in preventing oxidations through non-radical reactions has been given much less attention or disregarded [5], [6]. This option, however, is of high significance as unilateral assessment may provide misleading or inadequate information, since many polyphenols exhibit multifunctionality, acting by more than one manner.

Hydrogen peroxide (H2O2) is a biologically relevant, non-radical, oxidising species, and may be formed in tissues through oxidative processes, but there has been limited information regarding its scavenging by polyphenolic antioxidants. The present study was undertaken to investigate the hydrogen peroxide scavenging activity of several simple phenolic compounds, including a series of benzoate derivatives and their hydroxycinnamate analogues. The choice was based on particular structural features, so as to distinguish plausible structure–activity relationships. The assessment was accomplished employing a previously established, peroxyoxalate chemiluminescence assay [7], which provides very high specificity, thus eliminating effects that might be caused by radical reactions. Furthermore, the assay employed also presents very high sensitivity, allowing the utilization of particularly low amounts of antioxidants, like those that may be encountered in biological matrices.

Section snippets

Chemicals

Acetonitrile (MeCN) and ethyl acetate (EtOAc) were of HPLC grade. Water was nanopure. 9,10-Dimethylanthracene (9,10-DMA), imidazole, hydrogen peroxide (H2O2), bis(2,3,6-trichlorophenyl)oxalate (TCPO), gallic acid, protocatechuic acid, p-hydroxybenzoic acid, m-hydroxybenzoic acid, vanillic acid, caffeic acid, ferulic acid, p-coumaric acid, m-coumaric acid, hydrocaffeic acid and l-phenylalanine were from Sigma Chemical Co. (St. Louis, MO).

Preparation of reactants and solutions

All reactants for the chemiluminescence (CL) assay were

Results

In order to obtain credible information about the structural features that are involved in hydrogen peroxide scavenging by cinnamates, four representative derivatives were selected (Fig. 1, structures 1–4). In the same context, four corresponding benzoate analogues were chosen (structures 5–8), to assess the effect of the side chain. For comparison reasons, three other structurally similar compounds, l-phenylalanine, hydrocaffeic acid and gallic acid (structures 9–11) were also tested. Because

Discussion

Hydrogen peroxide is an oxidant that is being formed continuously in living tissues as a result of several metabolic processes, but its detoxification is very crucial in preventing it from reacting in deleterious Fenton-type reactions, which generate extremely reactive oxygen species, including hydroxyl free radical [8], [9]:Fe2+ + H2O2  Fe3+ + HO + OHradical dot

With respect to many dietary polyphenolic antioxidants, the radical–scavenger interactions have in many instances been thoroughly investigated [10],

Conclusions

The most important outcomes of this study may be summarized as follows:

  • 1.

    Among the benzoic and cinnamic acid derivatives tested, the most active in scavenging hydrogen peroxide was vanillic acid and hydrocaffeic acid, respectively.

  • 2.

    Benzoates were shown to be far more effective than their hydroxycinnamate analogues, which contrasts previous findings related with scavenging of radical species.

  • 3.

    The side chain unsaturation in hydroxycinnamates appears to negatively affect their scavenging potency.

  • 4.

    The

References (21)

  • E. Riboli et al.

    Am. J. Clin. Nutr.

    (2003)
  • M.B. Arnao

    Trends Food Sci. Technol.

    (2000)
  • A. Arnous et al.

    J. Pharmacol. Toxicol. Methods

    (2002)
  • E.S. Henle et al.

    J. Biol. Chem.

    (1997)
  • W. Brand-Williams et al.

    Lebensm. Wiss. Technol.

    (1995)
  • V. Bondet et al.

    Lebensm. Wiss. Technol.

    (1997)
  • S. Foley et al.

    Free Rad. Biol. Med.

    (1999)
  • I. Parejo et al.

    J. Pharmacol. Toxicol. Methods

    (2000)
  • F. Visioli et al.

    Cardiovasc. Res.

    (2000)
  • E.M. Becker et al.

    Eur. Food Res. Technol.

    (2004)
There are more references available in the full text version of this article.

Cited by (103)

  • Hydroalcoholic extract of Stevia rebaudiana bert. leaves and stevioside ameliorates lipopolysaccharide induced acute liver injury in rats

    2017, Biomedicine and Pharmacotherapy
    Citation Excerpt :

    Thin layer chromatographic studies and HPTLC fingerprinting of STE EXT confirmed the presence of significant amounts of diterpernoid glycoside, stevioside. The measurement of free radical scavenging activity could be useful for determining the antioxidant ability of the extract [1,66] and often used as indicators for radical-scavenging abilities [67]. In the present study, both STE EXT and STE exhibited remarkable free radical scavenging ability in a concentration-dependent fashion.

View all citing articles on Scopus
View full text