Protein Protection by Antioxidants: Development of a Convenient Assay and Structure-Activity Relationships of Natural Polyphenols

Abstract

In this work, a convenient test of antioxidant activity was developed, with BChE-contaminated HSA as the target of AAPH-induced oxidation and its esterase activity as the marker of protein integrity or degradation. The method is relatively simple, of low cost, and convenient to use. Its application to natural polyphenols showed that quercetin (1), verbascoside (2), chlorogenic acid (3), caffeic acid (4), 1,3,6,7-tetrahydroxyxanthone (5), and mangiferin (6), are good antioxidants (IC 50 < 9 mm). 1,5-Dihydroxy-3-methoxyxanthone (7), flemichin D (8), and cordigone (9) showed modest activities (ca. 50 mm < IC 50 < 350 mm), whereas danthrone (10) was inactive. Complementary experiments with two of the more active antioxidants, namely quercetin (1) and chlorogenic acid (3) showed that both antioxidants were better radical scavengers than chain-breaking antioxidants. The relative adiabatic oxidation potential (DH ox ), the relative H-bond dissociation energy (DH abs ), and the first oxidation potential measured by cyclic voltammetry were found to be related to the radical-scavenging activity of these antioxidants.

Novel screening assay for antioxidant protection against peroxyl radical‐induced loss of protein function

“…It was reported that concentrations of 250 mM or higher were needed to prevent proteins from peroxyl radical-induced modifications. 47,48 A comparison of the pIC 50 values of antioxidant protection of ALP obtained with the novel screening assay with those of antioxidant protection of BChE-contaminated HSA reported by Salvi et al 26 shows that the same classification of reference compounds (1, 2, 3, 4, 6, 7, and 8) was obtained with the two assays (Tab. 3).…”

“…A representative dose-response curve for melatonin is shown in Figure 5. Table 3 summarizes the pIC 50 (Àlog IC 50 ) values of antioxidant protection of ALP obtained for the tested reference compounds together with literature values 26 of antioxidant protection of BChE-contaminated HSA. According to their capacity to protect ALP from peroxyl radical-induced activity loss, they can be divided into three classes, namely efficient (pIC 50 > 5), intermediate (4 < pIC 50 5), and poor antioxidants (pIC 50 < 4).…”

“…For example, in the assay proposed by Salvi et al BChEcontaminated HSA is used as target of AAPHinduced oxidation and its esterase activity as the marker of protein damage. 26 Another functional test, based on the spectrophotometric determination of the activity of a1-antiproteinase to inhibit elastase, was developed by Halliwell et al 27 to assess the peroxynitrite scavenging capacity of test compounds.…”

“…25 Only a few in vitro methods were designed to investigate the effects of potential antioxidants on loss of activity of a given protein (e.g., enzyme) induced by ROS. Examples are the assays proposed by Salvi et al 26 and by Halliwell et al 27 The former is based on a fluorimetric test using the peroxyl radical-induced decrease of the esterase activity of butyrylcholinesterase (BChE)-contaminated human serum albumin (HSA) as a marker of oxidative damage. 26 The latter was developed to assess the peroxynitrite scavenging capacity of test compounds and is based on the spectrophotometric determination of the activity of a1-antiproteinase to inhibit elastase.…”

“…Examples are the assays proposed by Salvi et al 26 and by Halliwell et al 27 The former is based on a fluorimetric test using the peroxyl radical-induced decrease of the esterase activity of butyrylcholinesterase (BChE)-contaminated human serum albumin (HSA) as a marker of oxidative damage. 26 The latter was developed to assess the peroxynitrite scavenging capacity of test compounds and is based on the spectrophotometric determination of the activity of a1-antiproteinase to inhibit elastase. 27 In this work, a convenient, simple, sensitive, and cost-effective 96-well plate fluorimetric method to assess the antioxidant capacity of new chemical entities to protect proteins from loss of activity caused by peroxyl radicals was developed.…”