Food Antioxidants and Their Interaction with Human Proteins: Comparison
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Biochemical pathways that sustain life depend on multiple intrinsic and extrinsic factors contributing to aging and/or diseases. Antioxidants are substances able to reduce, prevent, or revert the oxidation of other substances. Food antioxidants can exhibit several modes of action in the body.

  • food antioxidants
  • human proteins
  • binding

1. Food Antioxidants

Antioxidants are substances able to reduce, prevent, or revert the oxidation of other substances. Alternatively, antioxidants are compounds that scavenge reactive oxygen species (ROS) or act to inhibit their production and/or stimulate the antioxidant defense system, thus reducing the cell-damaging effects of ROS [1]. They are abundant in nature, chemically very different, often little soluble in the aqueous system (body), exogenous antioxidants are cleared fast from the body, they are able to exchange electrons and protons, some can be regenerated, some only participate in a redox reaction, while others bind to substrates.
Food antioxidants can exhibit several modes of action in the body. They are (i) direct antioxidants of oxidized substances, (ii) metal ion chelators (thus, inhibitors of Fenton reaction), (iii) cofactors of enzymes involved in antioxidant activity, (iv) activators of transcriptional factors which induce expression of genes involved in antioxidant activity, (v) modulators of signaling pathways, (vi) modulators of secondary and/or tertiary structure, thermal/proteolytic stability and primary function of bound proteins, (vii) participants in other physiological actions [2,3][2][3].
Only a few antioxidants, in the form of supplements, have been approved by the responsible authorities for use in human therapy. There is a great interest in synthesizing more potent derivatives or isolating them as natural products with improved absorption/bioavailability/antioxidant properties. However, supplements cannot completely substitute natural sources of antioxidants since a natural variety of derivatives and their possible synergistic or additive effects cannot be achieved in a lab.
The antioxidant potential of foods can be determined by a number of methods: spectrometric, chromatographic, and electrochemical [4]. Several thousand-fold differences in food antioxidant composition were found [5,6,7][5][6][7]. Antioxidant Food Database offers data on the total antioxidant content (TAC) of more than 3100 items consumed by people worldwide [6]. Generally, foods originating from plants have greater TAC than those originating from animals. Foods with the highest antioxidant content are spices, herbs, berries, nuts, fruits, vegetables, chocolate, and coffee. Dairy products, meat, and eggs have very low antioxidant content. High TAC values, however, are not necessarily proportional to the bioavailability of antioxidants.

2. Food Antioxidants, Their Stability, and Availability under Physiological Conditions

Bioavailability is a term that defines the amount of a nutrient that is digested in the gastrointestinal tract, absorbed in the intestine, and further metabolized in the body. This path depends on many factors, such as a chemical form or antioxidant derivative, food matrix, degree and type of transformation by host intestinal enzymes and microbial enzymes in colon microbiota, interaction with other molecules in the body, and transport [8]. Food-derived antioxidants in the body are often not in the same form as in food.
Antioxidants soluble in water can be directly absorbed in enterocytes by diffusion and/or via transporters and further translocated to blood. Lipophilic antioxidants, after digestion in the stomach, form micelles with exogenous lipids and host bile acids released from the gallbladder and further undergo diffusion through the epithelial surface of the intestine. Absorbed micelles are transported as lipoprotein particles via lymph [9]. Modification reactions, which increase their aqueous solubility (most often conjugation), occur during their passage through the liver. Metabolic processes in the liver depend on the nature of the compound. After its physiological life has expired, an antioxidant derivative is directed to excretion via either the kidneys (urine) or the liver (bile/feces). Antioxidants not absorbed in the intestine pass to the colon, and microbial enzymes further process them. Hydrolysis, reduction, decarboxylation, dehydroxylation, and demethylation processes occur, resulting in new derivatives [10,11][10][11].
The food matrix plays a significant role in antioxidant availability. Unless free, antioxidants can be covalently bound to macromolecules (e.g., polysaccharides), associated with other molecules via ionic bonds, mechanically entrapped in a food matrix, or physically associated with specific cell structures [12]. The same antioxidant may be free in one food and bound in another. A combination of foods ingested at the same time, due to their mutual interaction, may affect the bioavailability of their components. However, the data on specific food combinations are conflicting. For example, a study by Dupas et al. [13] has shown that, although ingestion of coffee with milk can induce binding up to 40% of coffee chlorogenic acid (CGA) to milk proteins, no significant overall reduction in beverage antioxidant power was noted, most likely due to antioxidant liberation during gastric and intestinal digestion. On the other hand, a study by Duarte and Farah [14] reported that consuming milk with coffee reduces the bioavailability of coffee polyphenols, as assessed by measuring urinary CGA. In general, co-ingestion of antioxidants and proteins may induce their interaction, sometimes resulting in reduced bioavailability of antioxidants (or, rarely, increased). Dairy products, for example, can increase the availability of polyphenols from berries via polyphenol/protein interaction [15].
Contrary to that, co-ingestion of lipids may increase the bioavailability of lipophilic antioxidants, such as carotenoids, by facilitating their emulsification and absorption [16]. The interaction of saccharides and antioxidants may have both positive and negative consequences. For example, sweet wine sugars interact and increase the stability of polyphenols [17]. Alternative sweeteners, such as sucralose and stevia, improve the bioavailability of anthocyanins in beverages [18]. The effect of combining different food ingredients in the simulated gastrointestinal digestion on TAC of combined foods is given for the most commonly consumed foods in the study of Comert and Gokmen [19]. The bioavailability of a particular antioxidant is antioxidant-, matrix- and food processing-specific (such as cooking and fermentation). Examination of TAC in 54 types of beverages prepared from coffee beans revealed that the range of TAC values is 0.89 to 16.33 mmol/100 g and depends on the preparation route [6].

3. Binding Characteristics of Proteins and Their Interaction with Food Antioxidants

A proper translation of mRNA, followed by the appropriate post-translational modifications, is essential for adequate protein folding, targeting, and specificity. Proteins are not stable molecules existing as just one well-defined structure. Usually, a population of several conformational states exists, although not necessarily at the same time and location. Protein conformers originate from molecular motions that do not require considerable energy input, giving rise to molecular states of similar stability [20]. A dynamic equilibrium will change in the presence of ligands, which will favor the existence of certain protein conformer(s). If more ligands are present in the surrounding at the same moment, their competition or synergism will further govern the redistribution of the conformer pattern. Based on the concept of the pre-existing population of protein conformers [21], the ligand may be expected to choose the most suitable for the interaction. Ligand/protein binding additionally stabilizes the complex through altered protein folding and establishing new interactions inside the protein and with the ligand. Greater ligand-binding capacities possess proteins with a larger number of conformers, and the same binding site may accommodate even dissimilar ligands. Proteins that can convert to a greater number of conformers exploit more interfaces for interactions [22]. Altered post-translational modifications of proteins as well as ligand binding, may have physiological consequences, affecting protein conformation, stability, activity, specificity, transportation, proteolytic susceptibility, and clearance rate. Food antioxidants can interact with proteins at several possible places: in a deep protein cavity, where accessibility of environmental solvent is low, in a cleft between protein units, at the shallow surface groove, or at the protein surface where charged amino acid residues are exposed. Bonds that form between the antioxidant molecule and protein are usually multiple, involving many amino acid residues, and mostly non-covalent: hydrogen bonds (H-bonds), electrostatic bonds (salt bridges), hydrophobic interactions, Van der Waals bonds, and π-π interactions. Amino acids that interact with a ligand can be polar or non-polar and belong to the α-helix, β-sheet, or loop region of the molecule. Sometimes, all structural elements are involved. Some interactions are weak, whereas others are strong. Structural adjustments of the protein to accommodate a ligand can affect its conformation, stability, and behavior. Covalent (irreversible) interactions require specific conditions and/or the involvement of specific reactive groups, which can chemically modify amino acid residues, enabling covalent binding. Such interaction in the case of human blood proteins is a rare event. An example of a covalent partnership is an interaction between epigallocatechin gallate (EGCG) and human serum albumin (HSA). Protein carbonylation occurs when these two molecules are incubated at pH 7.4 and 37 °C. EGCG oxidizes lysine residues at the EGCG-binding sites in HSA, generating an oxidatively deaminated product [23]. EGCG can also form a covalent adduct with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) via the oxidation of cysteine thiol residues in the protein [24]. Oxidized [14C]quercetin was shown to bind highly selectively to HSA in vitro covalently [25[25][26],26], and such a reaction may be expected in vivo upon releasing hydrogen peroxide/peroxidases from cells. The effect of antioxidant binding to the protein (if there is an effect) can be activating, inhibiting, or modulating, which depends entirely on the nature of the binding couple. The effect of one antioxidant is usually of the same type on the number of interacting proteins. There are, however, antioxidants that can exert all three categories of effects depending on the particular binding protein. Resveratrol is an example of an antioxidant that can interact with numerous proteins that do not share primary sequence homology or common fold or have other similarities, exploiting all protein known binding places, forming all known types of bonds, with acidic, basic, or neutral amino acids and exerting no common effect [27]. Due to this diversity of behavior, resveratrol was termed a promiscuous molecule. Interestingly, if one protein performs several physiological roles, antioxidant binding may affect only some and not all of them, as in the case of resveratrol binding to estrogen receptor-α, modulating the inflammatory process but not cell proliferation [28]. Resveratrol binding to the receptor causes a conformational change of the protein, enabling the regulation of co-regulatory molecules, which participate in the transcription control. Likewise, a protein can bind different antioxidants at different sites. For example, β-lactoglobulin (BLG) binds apigenin in the internal cavity, luteolin at the entrance of the cavity, and eriodictyol outside the cavity [29]. One antioxidant molecule most often interacts with one molecule of protein. Sometimes, there are more binding places, for example, at more subunits. In addition, there may be several different binding places for the same antioxidant on one protein, as in the case of S-adenosylmethionine synthetase 2, which can bind two resveratrol molecules, one at the substrate binding place and the other at the interface between monomer units [30]. Protein interaction with one ligand may create new binding sites for another type of ligand or cause conformational change, enabling the accommodation of two instead of one ligand of the second type. For example, fatty acids bound to albumin allow the binding of additional resveratrol molecules by creating a more hydrophobic environment [31]. Some antioxidants are present in nature as both cis and trans isomers, and their interaction with proteins can differ. Additionally, some antioxidants may exist as optical isomers—enantiomers (R and S). R, a naturally occurring form of α-lipoic acid (LA) in food, but not S, is an essential cofactor of enzymes involved in oxidative metabolism in mitochondria [32]. A mixture is present in supplements, and it may be expected that optical isomers possibly influence the interaction with binding proteins as well. A protein can preferentially bind one, especially if the interaction stabilizes protein or antioxidant structure. Generally speaking, the uptake, binding, and recycling of an antioxidant are related to the form of antioxidant present, protein, and cell type, as well as the redox status of both the cellular and extracellular environment. Besides common features of antioxidant activity, particular behavior of antioxidants may be characteristic just for some. For example, both LA and its reduced form, dihydro-LA (DHLA), act as antioxidants and, unlike other antioxidants, are effective in both hydrophilic (e.g., plasma, cytosol) and lipophilic (plasma membrane) environments [33]. LA/DHLA couple was termed a universal antioxidant since it can regenerate other antioxidants, i.e., revert them from oxidized into the reduced form [3]. When their concentration, however, exceeds a certain level (above 75 µM), they behave as pro-oxidants (most likely at the transcriptional level). Such characteristics should be considered when recommending specific food quantities (or supplements).

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