RSC Advances REVIEW View Article Online View Journal | View Issue Free radicals, natural antioxidants, and their reaction mechanisms Cite this: RSC Adv.,2015,5, 27986 Satish Balasaheb Nimse*a and Dilipkumar Palb The normal biochemical reactions in our body, increased exposure to the environment, and higher levels of dietary xenobiotic's result in the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The ROS and RNS create oxidative stress in different pathophysiological conditions. The reported chemical evidence suggests that dietary antioxidants help in disease prevention. The antioxidant compounds react in one-electron reactions with free radicals in vivo/in vitro and prevent oxidative damage. Therefore, it is very important to understand the reaction mechanism of antioxidants with the free radicals. This review elaborates the mechanism of action of the natural antioxidant compounds and Received 28th October 2014 assays for the evaluation of their antioxidant activities. The reaction mechanisms of the antioxidant Accepted 12th March 2015 assays are briefly discussed (165 references). Practical applications: understanding the reaction DOI: 10.1039/c4ra13315c mechanisms can help in evaluating the antioxidant activity of various antioxidant compounds as well as Creative Commons Attribution 3.0 Unported Licence. www.rsc.org/advances in the development of novel antioxidants. 1. Introduction and background enzymes convert dangerous oxidative products to hydrogen peroxide (H2O2) and then to water, in a multi-step process in Antioxidants are molecules that inhibit or quench free radical presence of cofactors such as copper, zinc, manganese, and reactions and delay or inhibit cellular damage.1 Though the iron. Non-enzymatic antioxidants work by interrupting free antioxidant defenses are different from species to species, the radical chain reactions. Few examples of the non-enzymatic presence of the antioxidant defense is universal. Antioxidants antioxidants are vitamin C, vitamin E, plant polyphenol, This article is licensed under a exists both in enzymatic and non-enzymatic forms in the carotenoids, and glutathione.7 intracellular and extracellular environment. The other way of categorizing the antioxidants is based Normal biochemical reactions, increased exposure to the on their solubility in the water or lipids. The antioxidants can Open Access Article. Published on 12 March 2015. Downloaded 9/29/2021 6:09:59 PM. environment, and higher levels of dietary xenobiotics result in be categorized as water-soluble and lipid-soluble antioxi- the generation of reactive oxygen species (ROS) and reactive dants. The water-soluble antioxidants (e.g. vitamin C) are nitrogen species (RNS).2 ROS and RNS are responsible for the present in the cellular uids such as cytosol, or cytoplasmic oxidative stress in different pathophysiological conditions.3 matrix. The lipid-soluble antioxidants (e.g. vitamin E, carot- Cellular constituents of our body are altered in oxidative stress enoids, and lipoic acid) are predominantly located in cell conditions, resulting in various disease states. The oxidative membranes. stress can be effectively neutralized by enhancing cellular The antioxidants can also be categorized according to their defenses in the form of antioxidants.4,5 Certain compounds act size, the small-molecule antioxidants and large-molecule anti- as in vivo antioxidants by raising the levels of endogenous oxidants. The small-molecule antioxidants neutralize the ROS antioxidant defenses. Expression of genes encoding the in a process called radical scavenging and carry them away. The enzymes such as superoxide dismutase (SOD), catalase (CAT), main antioxidants in this category are vitamin C, vitamin E, glutathione peroxidase (GSHPx) increases the level of endoge- carotenoids, and glutathione (GSH). The large-molecule anti- nous antioxidants.6 oxidants are enzymes (SOD, CAT, and GSHPx) and sacricial Antioxidants can be categorized in multiple ways. Based on proteins (albumin) that absorb ROS and prevent them from their activity, they can be categorized as enzymatic and non- attacking other essential proteins. enzymatic antioxidants. Enzymatic antioxidants work by To understand the mechanism of action of antioxidants, it is breaking down and removing free radicals. The antioxidant necessary to understand the generation of free radicals and their damaging reactions. This review elaborates the generation aInstitute for Applied Chemistry, Department of Chemistry, Hallym University, and damages that free radicals create, mechanism of action of Chuncheon, 200-702, Korea. E-mail: [email protected]; Fax: +82-33-256- the natural antioxidant compounds and assays for the evalua- 3421; Tel: +82-33-248-2076 tion of their antioxidant properties. The reaction mechanisms bInstitute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central of the antioxidant assays are discussed. The scope of this article University), Koni, Bilaspur, Chhattisgarh-495009, India 27986 | RSC Adv.,2015,5,27986–28006 This journal is © The Royal Society of Chemistry 2015 View Article Online Review RSC Advances is limited to the natural antioxidants and the in vitro assays for versatile oxidant that can attack a wide range of biological evaluation of their antioxidant properties. targets.10 À À NOc +O2c / ONOO (7) 2. Generation of free radicals Peroxynitrite reacts with the aromatic amino acid residues in The generation of ROS (Table 1) begins with rapid uptake of the enzyme resulting in the nitration of the aromatic amino oxygen, activation of NADPH oxidase, and the production of the acids. Such a change in the aminoacid residue can result in the À superoxide anion radical (O2c , eqn (1)), enzyme inactivation. However, nitric oxide is an important cytotoxic effector molecule in the defense against tumor cells, ðoxidaseÞ À þ þ þ ! c þ þ 11,12 2O2 NADPH 2O2 NADP H (1) various protozoa, fungi, helminthes, and mycobacteria. The other sources of free radical reactions are cyclooxygenation, À lipooxygenation, lipid peroxidation, metabolism of xenobiotics, The O2c is then rapidly converted to H2O2 (eqn (2)) by SOD and ultraviolet radiations.13 ð Þ cÀ þ SOD 2O2 þ 2H ! H2O2 þ O2 (2) 3. Damaging reactions of free radicals These ROS can act by either of the two oxygen dependent ROS (Table 1) induced oxidative stress is associated with the mechanisms resulting in the destruction of the microorganism chronic diseases such as cancer, coronary heart disease (CHD), or other foreign matter. The reactive species can also be and osteoporosis.14 Free radicals attack all major classes of generated by the myeloperoxidase–halide–H2O2 system. The biomolecules, mainly the polyunsaturated fatty acids (PUFA) of enzyme myeloperoxidase (MPO) is present in the neutrophil cell membranes. The oxidative damage of PUFA, known as lipid cytoplasmic granules. In presence of the chloride ion, which is peroxidation is particularly destructive, because it proceeds as a Creative Commons Attribution 3.0 Unported Licence. ubiquitous, H2O2 is converted to hypochlorous (HOCl, eqn (3)), self-perpetuating chain reaction.15,16 a potent oxidant and antimicrobial agent.8 The general process of lipid peroxidation can be envisaged as À þ ðMPOÞ – Cl þ H2O2 þ H ! HOCl þ H2O (3) depicted bellow (eqn (8) (11)), where LH is the target PUFA and Rc is the initializing, oxidizing radical. Oxidation of the PUFA generates a fatty acid radical (Lc) (eqn (8)), which rapidly adds cÀ ‘ ROS are also generated from O2 and H2O2 via respiratory oxygen to form a fatty acid peroxyl radical (LOOc, eqn (9)). The ’ – burst by Fenton (eqn (4)) and/or Haber Weiss (eqn (5)) peroxyl radicals are the carriers of the chain reactions. The 9 reactions. peroxyl radicals can further oxidize PUFA molecules and initiate This article is licensed under a À new chain reactions, producing lipid hydroperoxides (LOOH) H O +Fe2+ / cOH + OH +Fe3+ (4) 2 2 (eqn (10) and (11)) that can break down to yet more radical À À species.17 O2c +H2O2 / cOH + OH +O2 (5) Open Access Article. Published on 12 March 2015. Downloaded 9/29/2021 6:09:59 PM. c / c The enzyme nitric oxide synthase produce reactive nitrogen LH+R L +RH (8) species (RNS), such as nitric oxide (NOc) from arginine (eqn (6)). Lc +O2 / LOOc (9) L-Arg + O + NADPH / NOc + citrulline (6) 2 LOOc +LH/ LOOH + Lc (10) An inducible nitric oxide synthase (iNOS) is capable of LOOH / LOc + LOOc + aldehydes (11) continuously producing large amount of NOc, which act as a cÀ c cÀ O2 quencher. The NO and O2 react together to produce À Lipid hydroperoxides always break down to aldehydes. Many peroxynitrite (ONOO , eqn (7)), a very strong oxidant, hence, of these aldehydes are biologically active compounds, which ff c each can modulate the e ects of other. Although neither NO can diffuse from the original site of attack and spread the attack cÀ nor O2 is a strong oxidant, peroxynitrite is a potent and to the other parts of the cell.18,19 Lipid peroxidation has been widely associated with the tissue injuries and diseases.20 À Oxygen metabolism generates cOH, O2c , and the non- Table 1 List of the ROS165 radical H2O2. The cOH is highly reactive and reacts with bio- Symbol Name logical molecules such as DNAs, proteins, and lipids, which results in the chemical modications of these molecules. There 1 O2 Singlet oxygen are several research reports on the oxidative damage of DNA due À c – O2 Superoxide anion radical to the cOH.21 23 c OH Hydroxyl radical The cOH reacts with the basepairs of DNA, resulting in the ROc Alkoxyl radical ROOc Peroxyl radical oxidative damage of the heterocyclic moiety and the sugar H2O2 Hydrogen peroxide moiety in the oligonucleotides by a variety of mechanisms. This LOOH Lipid hydroperoxide type of oxidative damage to DNA is highly correlated to the This journal is © The Royal Society of Chemistry 2015 RSC Adv.,2015,5,27986–28006 | 27987 View Article Online RSC Advances Review physiological conditions such as mutagenesis, carcinogenesis, and aging.24,25 The addition reactions yield OH-adduct radicals of DNA bases (Scheme 1), whereas the allyl radical of thymine and carbon-centered sugar radicals (Scheme 2) are formed from the abstraction reactions.