Antioxidants and their protective role against free radicals

Survey of literature indicates that each of the 60 trillion cells in the human body takes 10,000 oxidative hits/sec due to the production of harmful molecules called free radicals. Free radicals are chemically active atoms (groups of atoms) or molecular fragments that have a charge due to an excess or a deficient number of electrons. Free radicals found in living organisms include hydroxyl, superoxide, nitric oxide, peroxyl and etc.

Free radical

Free-radical theory and its importance to disease and aging were initially articulated in 1954 by Denham Harman. Chemical oxidation by free electrons damage cells structures such as mitochondria, cell membranes, enzymes, and DNA. As the damage accumulates, the degeneration of these structures leads to diseases that are responsible for up to 90% of deaths.

Antioxidants and their protective role against free radicals

What are Free Radicals?


Free radicals containing oxygen, known as reactive oxygen species (ROS), are the most biologically significant free radicals. ROS include the superoxide radical (O2), hydroxyl radical (OH), lipid peroxide radical (ROO ), Hydroperoxy radical (HOO), Nitric oxide (NO) Peroxynitrite (ONOO) plus derivatives of oxygen that do not contain unpaired electrons, such as hydrogen peroxide (H2O2), singlet oxygen (O2), and hypochlorous acid.

Normally, Free radicals are formed, when weak bonds split. Free radicals are very unstable and react quickly with other molecules, trying to capture the needed electron to gain stability. Generally, free radicals attack the nearest stable molecule, “stealing” its electron. When “attacked” the molecule loses its electron, it becomes a free radical itself, beginning a chain reaction. Once the process is started, it can cascade, finally resulting in the disruption of a living cell. Furthermore, some free radicals arise normally during metabolism or sometimes the body’s immune system’s cells purposefully create free radicals to neutralize viruses and bacteria. However, environmental factors such as pollution, radiation, cigarette smoke, and herbicides can also spawn free radicals.

Fortunately, the body is not defenseless from these metabolic and environmental oxidation attacks. The human body produces a number of enzymes to defend itself from free radicals and detoxify the end products of oxidation. Alternately, antioxidant consumption is one of the defense mechanisms that keep oxidative stress to a manageable level in healthy individuals.

Oxidative stress occurs when the free radicals are beyond the protective capability of the antioxidant defenses. Oxidative stress is evidenced by an increase in cellular accumulation of lipid peroxides and depletion of endogenous antioxidants. Therefore studies on antioxidants from medicinal plants are helpful in understanding the potential therapeutic role in the prevention and the treatment of the whole host of deleterious effects from the environment, disease and the degeneration of body cells, which are mainly responsible for the oxidative stress noticed by the increased production of superoxide radicals, hydrogen peroxide and hydroxyl radicals.


A substance which protects tissues from damage by stabilizing harmful free radicals is an antioxidant. Antioxidants donate a companion electron to the free radical to ‘calm it down’ so that it doesn’t try to react with – and damage – other sensitive molecules.

The word ‘antioxidant‘ arises from the chemical definitions of ‘oxidation’ and ‘reduction.‘ A free radical is an ‘oxidant,’ ‘oxidizer’ or ‘oxidizing agent,’ a chemical that wants to take an electron away from another chemical and use that electron to stabilize itself. This damages, or ‘oxidizes’ the other chemical. An antioxidant is a ‘reducer’ or a ‘reducing agent’ that sacrifices one of its electrons to the free radical. When a free radical and an antioxidant react, the free radical is ‘reduced’ – it has gained an electron; and the antioxidant is ‘oxidized’ – it has lost an electron.

Antioxidants, when present at low concentration, significantly delays and inhibits the oxidation of the substrate. Antioxidants include alpha-tocopherol, ascorbic acid, beta-carotene, flavonoids as well as antioxidative enzymes. They are believed to play a role in preventing the development of chronic diseases such as cancer, heart disease, stroke, Alzheimer’s disease, Rheumatoid arthritis, and cataracts (Serge Hercberg et al.,1999).

Mode of action of antioxidants:

  • Preventive – Antioxidant enzymes like superoxide dismutase, catalase, and glutathione peroxidase prevent oxidation by reducing the rate of chain initiation. That is, by scavenging the radicals, such antioxidants can obstruct an oxidation chain from ever setting in motion. They can also prevent oxidation by stabilizing transition metal radicals such as copper and iron (J.K. Candlish and N.P.Das1996).
  • Chain-breaking – When a free radical release or steals an electron, a second radical is formed. This molecule then turns around and does the same thing to a third molecule, continuing to generate more unstable products. The process continues until termination occurs — either the radical is stabilized by a chain-breaking antioxidant such as beta-carotene and vitamins C and E, or it simply decays into a harmless product (J.K. Candlish and N.P.Das, 1996).

The effectiveness of any given antioxidant in the body depends on which free radical is involved, how and where it is generated, and where the target of damage is. Thus, while in one particular system an antioxidant may protect against free radicals, in other systems it could have no effect at all and in certain circumstances, an antioxidant may even act as a “pro-oxidant” that generates toxic oxygen species.


Antioxidants and Oxidative Stress

Antioxidants and Oxidative Stress

Based on the mode of action, antioxidants can be divided into two main groups, such as enzymatic antioxidants and non-enzymatic antioxidants. Both the antioxidant enzymes and free radical scavengers may provide a defensive mechanism against the deleterious actions of ROS including peroxy radical (ROO). Some of the antioxidant enzymes that are found to provide a protection against the ROS are superoxide dismutase (SOD), catalase (CAT), peroxidase(POD), glutathione peroxidase (GPx), and ascorbate oxidase(APx) (UdayBandyopadhyayetal.,1990).

  1. The important antioxidant enzymes include superoxide dismutase, Catalase, Peroxidase, Glutathione peroxidase, Ascorbate oxidase, Glucose-6-phosphate dehydrogenase and Glutathione Reductase.
  2. Non-enzymatic antioxidants are further divided into two groups. They are
    • Nutrient antioxidants such as Carotenoids, α-Tocopherol, Ascorbic acid, and Selenium.
    • Metabolic antioxidants include Glutathione, Albumin, Uric acid, Bilirubin, transferrin, and ceruloplasmin.

            The non-enzymatic antioxidants which act as scavengers are glutathione, vitamin A, vitamin E, and vitamin C (Van Acker et al., 1993).

The antioxidants are also available in synthetic forms. Commonly used synthetic antioxidants are Butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT), Propyl gallate and tertiary butyl hydroquinone (Stadtman, 1992).

The naturally occurring antioxidants like vitamins are a balanced mixture of redox with reduced and oxidized form, the synthetic antioxidants are unbalanced in this respect and they themselves produce harmful free radicals in some cases, emphasizing the importance of the naturally occurring antioxidants over the synthetic ones (Blot et al.,1993).


Enzymatic antioxidants

Superoxide dismutase

(Superoxide: superoxide oxidoreductase, E.C.

Superoxide dismutase Catalyses the following reaction

O2 + O2 +2H+ ——————–► H2O2 + O2.

Superoxide dismutase is an antioxidant enzyme that catalyzes the dismutation (detoxification) of the highly reactive superoxide anion to the less reactive species H2O2 which is subsequently destroyed by catalase or peroxidase (Fridovich, 1995).

The toxic superoxide radical has a half-life of less than one second and is usually rapidly dismutated by Superoxide dismutase to H2O2, a product which is relatively stable and can be detoxified by Catalase and Peroxidases (Grant & Loake,2000).

Superoxide dismutase detoxifies the disproportionately superoxide radicals and hydrogen peroxide is destroyed by catalase and different kinds of peroxidases. A major H2O2-detoxifying system in plants is the Ascorbate-Glutathione cycle that includes Ascorbate peroxidase, Glutathione reductase ( Ho-Min Kang and Mikal E.Saltveit,2002).


(Hydrogen peroxide: Hydrogen peroxide oxidoreductase, E.C.

The enzyme catalase catalyses the decomposition of hydrogen peroxide to water and oxygen.

            2 H2O2­­­­­­­———————-► 2 H2O + 02

It is a heme containing enzyme that decomposes H2O2 generated during the various metabolic processes such as photorespiration and β-oxidation of fatty acids into water and oxygen and helps in the detoxification of ROS that are generated by various environmental stresses (Scandalios, 1990).

Catalase is located generally in peroxisomes but present outside the peroxisomes gives oxidative imbalance. Mislocalisation of catalase includes an oxidative imbalance in the cells which on treatment with a natural antioxidant resulted in reduction of oxidative levels and restoration of metabolic functions (Kawada etal, 2004).


(Donor: Hydrogen peroxide Oxidoreductase, E.C.

Peroxidases are the heme group containing enzymes that catalyses the oxidation of a substrate and the reduction of H2O2.Plant peroxidases are typically glycoproteins, which may participate in many process of plant growth and defense.

Peroxidase (POD) includes in its widest sense a group of specific enzymes such as NAD-Peroxidase, NADP- Peroxidase, Fatty acid-Peroxidase etc., as well as a group of very non-specific enzymes from different sources which are simply known as POD.

POD catalyses the dehydrogenation of a large number of organic compounds such as phenols, Aromatic amines, hydroquinones etc. POD occurs in animals, higher animals, higher plants and other organisms.

Peroxidase can be regarded as the enzyme having three types of enzyme activities IAA oxidases, Polyphenol oxidase and Peroxidase. The enzyme catalyses the oxidation of wide variety of electron donor with the help of H2O2 and there by scavenges the endogenous H2O2 (MinakshiMahajan, 2004).

Glutathione peroxidase

(Glutathione: hydrogen-peroxide oxidoreductase, E.C.

Glutathione peroxidase is the general name of an enzyme family with peroxidase activity whose main biological role is to protect the organism from oxidative damage. The biochemical function of glutathione peroxidase is to reduce lipid [Organic peroxide (hydroperoxides)] to their corresponding alcohols and to reduce free hydrogen peroxide to water. There are several isozymes found in the cytoplasm of nearly all mammalian tissues, whose preferred substrate is hydrogen peroxide. The reaction that glutathione peroxidase catalyzes is:

2GSH + H2O2 → GSSG + 2H2O

Where GSH represents reduced monomericglutathione, and GSSG represents glutathione disulphide,anoxidised form.

Glutathione reductase then reduces the oxidized glutathione to complete the cycle:


Glutathione peroxidase is a selenium-containing tetramericglycoprotein, i.e., a molecule with four selenocysteine amino acid residues. As the integrity of the cellular and sub cellular membranes depend heavily on glutathione peroxidase, the antioxidative protective system of glutathione peroxidase itself depends heavily on the presence of selenium.

Ascorbate oxidase

(L-Ascorbate: Oxygen oxidoreductase, E.C.

Ascorbate oxidase is widespread in plant tissues. The role of this enzyme is to regulate the levels of oxidized and reduced glutathione levels and NADPH as shown below.

Ascorbic acid reactionsAscorbate oxidase (AO) is a cell wall localized enzyme thatuses oxygen to catalyze the oxidation of ascorbate (AA) to theunstable radical mono dehydroascorbate (MDHA) which rapidly disproportionatesto yield dehydroascorbate (DHA) and Ascorbic acid (AA), and thus contributesto the regulation of the ascorbate (AA) redox state, whichsuggests that the reduction in the ascorbate (AA) redox state in the leafapoplast of transgenic plants of tobacco (NicotianatabacumL. cv. Xanthi), results in shifts in theircapacity to withstand oxidative stress imposed by agents imposingoxidative stress.

Some of the non-enzymatic antioxidants



Ascorbic acid

Ascorbic acid is also known as Vitamin C, is an antiscorbutic. It is a water soluble and heat labile vitamin present in gooseberry, bitter gourd, green peppers, spinach leaves, citrus fruits lettuce and mustard green etc,. It is generally present in all fresh vegetables, leaves and fruits. (Sadasivam.S and They moli Balsubraminan, l987).

It is an important water soluble antioxidant in biological fluids. It plays a significant role in maintaining the water soluble oxidation-reduction potential in human tissue and readily scavenges reactive oxygen and nitrogen species there by effectively protects the cells from damage (Halliwell, 1996).


Reduced Glutathione

It is an important metabolic antioxidant. Reduced Glutathione (GSH) plays a key role in the biological antioxidant system. GSH and H2O2 are the twin substrates for glutathione Peroxidase. The reduced glutathione (GSH) gets generated from the oxidized glutathione (GS-SG) through the participation of glutathione reductase and NADPH. It is suggested that the ability to synthesize GSH decreases as the age advances.



Total Phenolics

Phenolic Compounds are commonly found in both edible and non-edible plants and they have multiple biological effects, including antioxidant activity. The antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers in addition they have a metal chelation potential.( Rice-Evans etal.,1995).

Epidemiological studies have suggested positive association between the consumption of phenolic-rich foods or beverages and prevention of diseases (Scalbert and Williams, 2000).

Flavonoids and other phenolics such as phenolic acids, stilbenes, tannins, lignans and lignins are especially common in leaves, flowering tissues and woody parts such as stems and barks (Larson, 1998).

Flavonoids and other phenolics have been suggested to play a preventive role in the development of cancer and heart diseases. Ingestion of alcohol-free red wine or phenolic compounds mixture extracted from red wine has been shown to improve the antioxidant status of plasma in humans (Serafinietal., 1998 and Carbonneauetal., 1998).

It was reported that flavonoids and other plant phenolics such as phenolic acids, stilbenes, tannins, lignans and lignin are especially common in leaves, flowering tissues and woody parts such as stems and barks (Larson, 1988).

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