Free radicals are chemical species possessing one or more unpaired electrons and can be considered as a fragment of molecules that are extremely reactive and short lived. They are produced continuously in cells, either as accidental byproducts of metabolism or deliberately (for example, during phagocytosis). Unpaired electrons usually make a molecule more reactive than the corresponding non-radical. The molecule acts as an electron acceptor and essentially ‘steals’ electrons from other molecules. Free radicals are referred to as oxidising agents since they cause other molecules to donate their electrons. Free radicals can be formed by the homolytic cleavage of a covalent bond of a normal molecule, with each fragment retaining one unpaired electron; by the loss of a single electron from a normal molecule; or by the addition of a single electron to a normal molecule.

The most common cellular oxygen free radicals are superoxide radical (0₂^-), hydroxyl radical (OH^·) and nitric oxide (NO). Other molecules, such as hydrogen peroxide (H₂0₂) and peroxynitrate (ONOO^·) are not free radicals themselves but can lead to their generation through various chemical reactions.

Oxygen free radicals and related molecules are often classified together as reactive oxygen species (ROS), to signify their ability to promote oxidative changes within the cell.

All aerobic organisms produce free radicals, predominantly superoxide, formed as a side product during the reduction of molecular oxygen by mitochondria. An average cell utilises 10¹³ molecules of 0₂ per day. It is estimated that 1 % of respired molecular oxygen will form ROS, thus approximately 10¹¹ ROS are produced by each cell in a day. Cells normally employ a number of defence mechanisms against damage induced by free radicals. Oxidative stress is the term referring to the imbalance between generation of reactive oxygen species and the activity of the antioxidant defences.

There is increasing evidence to support the involvement of free-radical reactions in several human diseases since reactive oxygen species play a role in a variety of normal regulatory systems, the de-regulation of which may play an important role in inflammation. ROS and other free radicals have long been known to be mutagenic and have more recently emerged as mediators of other phenotypic and genotypic changes causing mutations and neoplasia.

In the last decade, evidence has accumulated that the free-radical process known as lipid peroxidation plays a crucial and causative role in the pathogenesis of atherosclerosis, cancer, myocardial infarction and also in ageing. Participation of free-radical oxidative interactions in promoting tissue injury in conditions such as brain trauma, ischaemia, toxicity and also in neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s dementia, multiple sclerosis and lipofuscinosis are now well documented. The involvement of ROS in the pathogenesis of several lung diseases has also been suggested while the pioneering studies on the role of reactive oxygen species reactions in the genesis and the expression of cellular and tissue damage has been carried out mainly in the liver, using acute rat poisoning with carbon tetrachloride.

Studies in experimental models have incriminated ROS as primary mediators in the pathogenesis of renal injury. Diabetes mellitus is also associated with oxidative reactions, particularly those that are catalysed by decompartmentalised transition metals, but their causative significance in diabetic tissue damage remains to be established.

In 1956, Harman proposed the free-radical theory of ageing, the assumption that ageing results from random deleterious effects of tissue brought about by ROS and it is very likely that reactive oxygen species contribute considerably to the development of stochastic disorders observed during the progress of ageing.

In recent years, increasing experimental and clinical data have provided compelling evidence for the involvement of oxygen free radicals in the three main disorders of prematurity — chronic lung disease, retinopathy of prematurity and intraventicular haemorrhage, the hypothesis being that oxygen-centred radical and related reactive oxygen metabolites are formed too rapidly to be detoxified by antioxidant defence mechanisms.

Defence against free radicals: antioxidants

Antioxidant defences fall in to two main categories, those whose role is to prevent the generation of free radicals and those that intercept any radicals that are generated. They exist in both the aqueous and membrane compartments of cells and can be enzymes or non-enzymes. Various animal studies have shown that antioxidants delay or protect against the oxidative damage produced by the free-radical reaction and a protective role against ailments mediated by free radicals is now well established.

Antioxidants are exogenous (natural or synthetic) or endogenous compounds acting in several ways, including removal of O₂, scavenging reactive oxygen/ nitrogen species or their precursors, inhibition of reactive oxygen species formation and binding metal ions needed for catalysis of ROS generation and up-regulation of endogenous antioxidant defences. The protective efficacy of antioxidants depends on the type of reactive oxygen species that is generated, the place of generation and the severity of the damage. The natural antioxidant system can be classified into two major groups: endogenous enzymes and low-molecular-weight antioxidants.

Endogenous enzymes include extensively studied enzymes such as superoxide dismutase (SOD), cata-lase, glutathione peroxidases, DT diaphorase, and glutathione-regenerating enzyme systems. Some enzymatic systems such as SOD and catalase act specifically against reactive oxygen species, while certain other enzyme systems reduce thiols. The low-molecular-weight antioxidants can be further classified into directly acting antioxidants (e.g. scavengers and chain-breaking antioxidants) and indirectly acting antioxidants (e.g. chelating agents). The directly acting antioxidants are extremely important for defence against oxidative stress. Direct scavenging of ROS is one of the many antioxidant actions required to restore oxidative equilibrium once it is lost in different pathologies. This subgroup of antioxidants currently contains several hundred compounds including ascorbic acid (vitamin C), retinoic acid (vitamin A), melatonin, lipoic acids, polyphenols, and carotenoids, being derived from dietary and herbal sources. The hypothesis that restoring redox equilibrium through activation of intracellular signals is also an important step of the antioxidation process is gaining increasing support. It is likely that the trapping of excess free radicals could restore redox equilibrium in the initial states of cellular oxidative stress.

Free radicals in various diseases

According to Halliwell and Gutteridge (1999), oxidative stress occurs in most human diseases, although this is not the same as saying that it is the cause of most diseases. The increase in free radicals may be secondary to the disease process. Free radicals are very short lived and difficult to study in vivo. Direct detection of free radicals is possible with electron spin resonance, but it is very expensive and complex, so a variety of surrogate markers to ascertain free-radical activity must be used. Developing accurate methods to measure biomarkers for DNA damage and lipid peroxidation is challenging and methods in the current literature include urine levels of F2-isoprostanes as a biomarker for lipid peroxidation, measurement of oxidised low-density lipoprotein (LDL), use of a chemical mutagenic product of fat oxidation, and 8-oxo-deoxyguanosine, associated with a decline in mitochondrial function. There have also been efforts to detect changes in the levels of antioxidants such as SOD, glutathione or vitamin E in the body in response to oxidative stress, to identify many conditions associated with free-radical formation, but results have not been consistent. The implications of the presence of ROS in cardiovascular, pulmonary, carcinogenesis, diabetes and neurological diseases as well as inflammation are currently under intense investigation.

It is easy to appreciate that the lungs are vulnerable to inhaled agents, e.g. ozone, nitrogen dioxide, sulphur dioxide and other toxins, that stimulate reactive oxygen species production. ROS can stimulate lipid peroxidation and oxidation of DNA bases in the lungs. The irritant effect of smoke also activates lung macrophages and neutrophils with resultant production of additional ROS. Chronic lung inflammation such as asbestosis, asthma and cystic fibrosis is also associated with elevated markers of oxidative stress so reactive oxygen species may contribute to the ongoing pathology.

The brain may be especially sensitive to oxidative damage. Oxidative stress can damage neurones and glial cells in a manner similar to other issues: via products of lipid peroxidation that are neurotoxic, DNA damage, etc. Reper-fusion injury also occurs in the brain after a stroke and superoxide produced during reperfusion results in abnormalities of cerebral vascular responses and blood-brain barrier permeability. Extracellular gluta-mate levels in the brain increase rapidly during ischaemia, leading to increased production of OH radicals, calcium ion imbalance and increased neurotoxicity. If bleeding occurs with the stroke, normally sequestered iron molecules are released and may initiate harmful free-radical chain reactions. Neurodegenerative diseases associated with ROS include Parkinson’s, Alzheimer’s, and many others. It is possible that although the initiators of the disease state vary, free radicals are involved in a common pathway that leads to neural cell death.

The acute inflammatory response is typically beneficial to the organism, being a major defence against microorganisms and normally self-limited. However, the superoxide-producing neutrophil itself is destroyed in the process and healthy surrounding cells may also be damaged. With chronic inflammation, such as in rheumatoid arthritis, the overall impact of the continued generation of free radicals is deleterious. Degradation of hyaluronic acid (synovial fluid) is driven by the presence of OH. These radicals may be produced by phagocytic cells in the joint, by changes in tissue oxygenation caused by swelling, followed by reperfusion, or by some of the drugs used to treat RA. A role for reactive oxygen species in the endothelial dysfunction associated with diabetes was proposed and levels of manganese superoxide dismutase have been reported to be decreased in streptozotocin-induced diabetes in rats. Normalising mitochondrial O₂ has been shown to block pathways involved in hyperglycaemic damage. Consistent with these observations, SOD pretreatment improved vasodilation in isolated aortic rings from streptozo-tocin diabetic rats. Levels of O₂ are also increased in hyperinsulinaemic rats, which is believed to be related to activation of NAD(P)H oxidase.

The progression of heart failure is associated with programmed cell death or apoptosis, which studies suggest occurs in response to ischaemia, reperfusion, pressure overload and in dilated cardiomyopathies. Oxidative stress may also be critical for the activation of apoptosis in dilated cardiomyopathies.

Free radicals in cardiovascular diseases

Cardiovascular disease is a heterogeneous group of disorders that affects the heart and blood vessels. The diseases are characterised by angina pectoris, hypertension, congestive heart failure, acute myocardial infarction (heart attacks), stroke and arrhythmia. There is now considerable biochemical, physiological and pharmacological data to support a connection between free-radical reactions and cardiovascular tissue injury. Evidence shows that these disease conditions are directly or indirectly related to oxidative damage and share common mechanisms of molecular and cellular damage. As these mechanisms are elucidated, it may be possible to improve the techniques for clinical and pharmacological intervention.

Ischaemia-reperfusion myocardial injury

Exposure of myocardial tissue to a brief, transient ischaemia, followed by reperfusion, has attracted much attention in recent years as an explanation for some cardiac diseases. Myocardial ischaemia occurs when myocardial oxygen demand exceeds oxygen supply. Unless reversed, this situation results in cell injury and, clinically, myocardial infarction. Logically, reperfusion of ischaemic myocardium is recognised as potentially beneficial, because mortality is directly proportional to infarct size, and this latter to the severity and duration of ischaemia. Reperfusion of the ischaemic myocardium can restore oxygen and substrates to the ischaemic myocardial cells, but this process may create another form of myocardial damage termed ‘reperfusion injury’. Thus, restoration of a normal blood flow in the heart by methods such as angioplasty, thrombolytic agents or cardiopulmonary bypass can lead to specific lesions (arrhythmias, deficit in contractility, necrosis), the importance of which also depends on the duration of ischaemia.

Evidence suggests that this may be due, in part, to the generation of toxic reactive oxygen species. The active involvement of ROS in the ischaemia-reperfusion damage is demonstrated by direct and indirect experimental evidences. Direct evidence arises from the possibility of measuring radicals in myocardial tissue by electron spin resonance (ESR) and spin trapping methodology; indirect evidence by the measurement of the products of free-radical attack on biological substrates (e.g. malondialdehyde as a measure of lipid peroxidation extent), and intracellular and extracellular antioxidant capacity. Experimental findings suggest that in ischaemic tissue there is an impairment of antioxidant mechanisms. Evidence to support this statement comes also from the cardioprotective effects of agents capable of inducing antioxidant enzymes in the heart and from the beneficial effects of several enzymatic free-radical scavengers, anti-oxidants and iron chelators in reperfused myocardium.

Free-radical hypothesis of atherosclerosis

Considerable in-vivo evidence, animal and human, supports the important role of reactive oxygen species in atherosclerotic coronary heart disease. While the exact mechanisms for atherogenesis are not completely understood, recent studies suggest that oxidative modification of low-density lipoproteins (LDL) is a critical factor. LDL may be oxidatively modified by all major cell types of the arterial wall via their extracellular release of reactive oxygen species (ROS). Hydroxyl radicals (thus formed) may initiate the peroxidation of long-chain polyunsaturated fatty acids within LDL, giving rise to conjugated dienes and lipid hydroperoxy radicals (LOO^·). This process is self propagating, since LOO^· can attack adjacent fatty acids until complete fatty acid chain fragmentation occurs. A number of highly reactive products then accumulate in the LDL particle, including malondialdehyde and lysophos-phatides, which interact with the amino side chain of the apoprotein B 100 and modify it to form new epitopes that are not recognised by the LDL receptor.

Hypertension

Essential hypertension (EH) appears associated with increased superoxide anion and hydrogen peroxide production, as well as decreased antioxidant capacity. The involvement of reactive oxygen species in EH is also suggested by the observation of increased level of lipid peroxides and decreased concentrations of antioxidant vitamin E in plasma of EH patients. Recently, Simi et al. (1998) have shown that patients with EH have plasma concentrations of free-radical scavengers lower than healthy normotensive subjects. The elevated consumption of plasma antioxidants was accompanied by increased activity of extracellular antioxidant enzymes (glutathione peroxidase and SOD), suggesting that ROS production in EH overwhelms antioxidant defence capacity. Oxidative stress in patients with EH is accompanied with the decreased red blood cell counts and decreased SOD and glutathione peroxidase activity in neutrophils.

Chronic heart failure

Chronic heart failure is a state characterised by a number of processes that may promote reactive oxygen species generation in vivo, including cytokine activation, recurrent hypoxia-reperfusion, possibly genetic susceptibilities and activation of the renin-angiotensin system. There are a number of potential cellular sources implicated in enhanced ROS generation in chronic heart failure. It has recently been demonstrated that patients with chronic heart failure may have increased leucocyte 0₂^- production, which is, in turn, related to severity of disease. Other sources of enhanced ROS generation in human chronic heart failure are both the myocardium and peripheral blood vessels. Increased activity of myocardial NADPH oxidase has been reported in heart failure.

Myocardial damage

Reactive oxygen species (ROS) have direct effects on cellular structure and function and may be integral signalling molecules in myocardial remodelling and failure. ROS result in a phenotype characterised by hypertrophy and apoptosis in isolated cardiac myocytes. ROS have also been shown to activate matrix metalloproteinase (MMP) in cardiac fibroblasts. Myocardial MMP activity is increased in the failing heart and an MMP inhibitor has been shown to limit early left ventricular dilatation in a murine model of myocardial infarction (MI). Hayashidani et al. (2003) showed significant improvement in the survival after MI in MMP-2 knockout mice, which was mainly attributable to the inhibition of early cardiac rupture and the development of subsequent LV dysfunction. Because MMP can be activated by reactive oxygen species, one proposed mechanism of ventricular remodelling is the activation of MMPs secondary to increased ROS production. Sustained MMP activation might influence the structural properties of the myocardium by providing an abnormal extracellular environment with which the myocytes interact. Kinugawa et al. (2000) demonstrated that the OH scavenger, dimethylthiourea, inhibits the activation of MMP-2 in association with the development of ventricular remodelling and failure. These data raise the interesting possibility that increased ROS after MI can be a stimulus for myocardial MMP activation, which might play an important role in the development of HF.

Left ventricular hypertrophy

In animal models of heart failure, levels of ROS are elevated and cardiac protection is observed with antioxidant treatment. The increase in ROS associated with left ventricular hypertrophy appears to be NAD(P)H oxidase-dependent. Myocardial NAD(P)H oxidase activity is elevated and expression of p22phox, gp91phox, p67phox and p47phox is increased in left ventricular tissue from guinea pigs after aortic banding. The gp91phox containing NAD(P)H oxidase has been shown to play an important role in the cardiac hypertrophic response to Ang II in mice. It has been suggested that the increase in ROS is responsible for impaired endothelial regulation of left ventricular relaxation observed in moderate pressure overload left ventricular hypertrophy.

Cardiac hypertrophy occurs in response to a sustained increase in cardiac work. The mechanisms underlying this progression from compensated hypertrophy to decompensated heart failure remain poorly understood and incompletely explored. There are data supporting at least a contributory role for alterations in ROS production in the pathophysiology of cardiac hypertrophy. There is substantial evidence from animal studies indicating that reactive oxygen species, and particularly O₂, production is increased in cardiac hypertrophy. Recently, Date et al. demonstrated attenuated cardiac hypertrophy in mice subjected to pressure overload following treatment with the free-radical scavenger, N-2-mercaptopropionyl glycine. This is the first evidence in an experimental model suggesting a causal role for ROS in the development of pressure overload hypertrophy. The precise source of reactive oxygen species in this study was not apparent. In a similar study using a guinea pig model of pressure overload, an attenuation of LV hypertrophy was observed in animals treated with vitamin E. Taken together, these data support an important functional role for reactive oxygen species, in particular NADPH oxidase derived ROS, in the development of pressure-overload hypertrophy.

Free radicals in hypercholesterolaemia

Increased levels of O₂ generation and attenuated NO mediated responses have been demonstrated in aortic rings from cholesterolfed rabbits. Treatment of the animals with polyethylene glycolated SODs improved endothelium-dependent vasodilation. Supplementation with L-arginine has also been shown to reduce O₂ levels and restore NO-mediated responses in cholesterol-fed animals (Boger et al., 1995). O₂ levels are also raised in WHHL (Watanabe heritable hyperlipidaemic) rabbits. Multiple mechanisms appear to be involved in O₂^- production in association with hypercholesterolaemia. Stepp and colleagues provided evidence that in canine carotid arteries eNOS, mechanisms dependent on xanthine oxidase and possibly NAD(P)H-oxidase were involved. Further evidence for the involvement of NAD(P)H oxidase was obtained in WHHL rabbits. In monkeys with atherosclerosis, disease severity is related to O₂ levels, and regression of atherosclerosis is associated with decreases in O₂ levels and NAD(P)H oxidase activity.

Free radicals in skeletal muscle dysfunction

Oxidative stress could be the mechanistic basis also for muscle fatigue and reduced exercise tolerance in patients with heart failure. This notion is supported by a positive correlation between ROS and exercise intolerance in these patients. Further, Tsutsui et al. (2001) demonstrated that the production of reactive oxygen species was increased in the skeletal muscle homogenates obtained from a murine model of HF and increased ROS were identified as OH originating from O₂, which was associated with a concomitant increase in the oxidation of lipids. These results are consistent with the previous studies that the oxidative capacity is reduced and O₂ utilisation is inadequate in skeletal muscle mitochondria from patients with heart failure. Skeletal muscle mitochondria from heart failure are associated with a decrease in the activities of complex I and complex III. As has been shown in the failing hearts, the defects in electron transfer function may lead to reactive oxygen species production. ROS may play an important role in the muscle atrophy commonly seen in patients with heart failure through the induction of apoptosis. In addition, ROS impair myoplasmic Ca²⁺ homeostasis and inhibit the oxidative energy production in the mitochondria, both of which may contribute to the muscle contractile dysfunction. An attempt to attenuate oxidative stress would improve, to some extent, the exercise capacity of patients with heart failure.

Tests for antioxidant activity

Antioxidant activity can be evaluated both in vitro and in vivo. There are potential models for evaluation of the antioxidant activity. Animals such as mice, rats, guinea pigs and rabbits can be used for in-vivo evaluation with the oxidative stress induced by some external chemical agent (e.g. carbon tetrachloride), physical, emotional, mental or environmental stress (e.g. torturing the animals, depriving animals from food, water and sexual activity, increasing noise or temperature of the animal housing). Even surgery can be performed for inducing oxidative stress in rats, e.g. cerebral ischaemia/reperfusion induced oxidative stress in which the induction of ischaemia in rats was performed by occluding bilateral common carotid arteries with clamps for 30 min followed by 24 h reperfusion. Following any of the methods whereby the oxidative stress can be induced in the animals, they should be grouped as treated (at least two or more doses), control and normal animals. In the end of study the animals can be sacrificed to isolate the vital organs. Enzymes such as SOD, catalase and glutathione can be measured in these tissues, together with the extent of lipid peroxidation caused by the oxidative stress, using assays such as barbituric acid reactive substances (TBARS).

In-vitro methods consist of chemical methods in which free radicals can be generated using chemical reactions, e.g. nitric oxide method or chemicals which themselves act as the source of free radicals such as DPPH (2,2-diphenyl-1-picrylhydrazyl). In-vitro methods are also available in which generated free radicals can attack tissues isolated from the animal body leading to the oxidation of lipids present in the tissues, e.g. thiobarbituric acid-reactive substances (TBARS) assay. Details on some of the in-vitro methods used for the evaluation of antioxidant activity are given below.

DPPH radical scavenging assay

The antioxidant activity of the plant extract and pure compounds was assessed on the basis of radical scavenging effect of the stable DPPH free radical, which is purple. Antioxidants react with DPPH, and convert it to 1,1-diphenyl-2-(2,4,6-trinitrophenyl) hydrazine, which is colourless. Reaction mixtures containing test samples (dissolved in DMSO) and 300 µmol/L DPPH ethanolic solutions in 96-well microtitre plates are incubated at 37°C for 30 min, and absorbances measured at 515 nm. The degree of discolouration indicates the scavenging potentials of the antioxidant compounds and IC50 values can be calculated, i.e. the concentration of sample required to scavenge 50% DPPH free radicals. DPPH reagent (0.5% in methanol) can be sprayed on to preparative TLC plates to identify active antioxidant compounds in plant extracts. Active radical scavengers give yellow colour zones against a purple background.

Nitric oxide radical scavenging assay

Nitric oxide (NO^·) is a free radical and scavengers of nitric oxide compete with oxygen, leading to reduced production of nitric oxide. NO is generated from sodium nitroprusside and measured by the Griess Illosvoy reagent (Garratt, 1964), which can be modified by using naphthylethylenediamine dihydro-chloride (0.1% w/v) instead of 1-naphthylamine (5%). The extent of NO radical scavenging can be assessed by colorimetry whereby reaction mixtures containing 10 mmol/L sodium nitroprusside, phosphate buffer saline and extracts or standard solution are incubated at 25°C for 150 min. After incubation, 0.5 mL of the reaction mixture is mixed with 1 mL of sulphanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to stand for 5 min to complete diazotisation. Naphthyl ethylenediamine dihydrochloride is then added, mixed and allowed to stand for 30 min at 25°C and a pink coloured chro-mophore is formed in diffused light whose intensity is measured at 540 nm.

Scavenging of superoxideanion radicals assay

Various cellular enzymes can catalyse chemical reactions involving molecular oxygen, including admission formation of superoxide radicals, which can inactivate vital cell components. Superoxide can be generated by enzymatic oxidation of hypoxanthine with xanthine oxidase and can be detected colori-metrically by nitroblue tetrazolium (NBT) reduction. The reaction is started by adding 100 µL of phenazine methosulphate (PMS) solution (60 µmol/L PMS in 100 mmol/L phosphate buffer, pH 7.4) to the mixture, incubating at 25°C for 5 min, and measuring the absorbance at 560 nm. Decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging activity.

Deoxyribose degradation assay

In this method hydroxyl radicals are generated by incubating a mixture containing KH₂PO₄-KOH, H₂O₂, FeCl₂-EDTA and deoxyribose. The extent of deoxyribose degradation by the formed hydroxyl radical can be assessed by the thiobarbituric acid method. The typical reaction is started by adding Fe(II) at a final concentration of 6 µmol/L to a 0.5 mL final volume of 20 mmol/L phosphate buffer, 5 mmol/L of 2-deoxyribose, Cu(II) (5 µmol/L) (pH 7.2) and 100 µmol/L H₂O₂ with and without 10 µmol/L of ascorbate as an iron chelator. Reactions were carried out for 10 min at 25 °C ± 1°C and were stopped by adding of 0.5 mL of 50 mmol/L NaOH containing 4% (w/v) phosphoric acid. After boiling for 15 min, the absorbance of the solution containing the oxidation products is measured at 532 nm.

Thiobarbituric-acid-reactive substances assay

In this method the lipid peroxidation is measured in terms of malondialdehyde (MDA) content following the thiobarbituric acid method of Ohkawa et al. (1979). MDA is formed in vivo and in vitro through oxidation of unsaturated lipids by ROS, and other oxidative agents. Thiobarbituric acid reacts with MDA to form a pink chromogen, which can be detected spectrophotometrically at 532 nm.

β-Carotene-linoleic acid (linoleate) assay

The antioxidant activity is measured by the ability of a compound to minimise the coupled oxidation of linoleic acid and β-carotene in an emulsified aqueous system. β-carotene loses its orange colour when reacting with reactive oxygen species, so colorimetery can be used to investigate the decline in colour caused by oxidative stress. In this method a stock solution of β-carotene and linoleic acid is prepared by dissolving 0.5 mg of β-carotene in 1 mL of chloroform and adding 25 µL of linoleic acid together with 200 mg of Tween 40, evaporating the chloroform and adding 100 mL of aerated water to the residue. To 2.5 mL of this mixture, 300 µL of extract is added and the mixture incubated in boiling water for 2 h together with two blanks, one containing the antioxidant BHT and the other without antioxidant, before measuring the absorbance at 470 nm.

DNA nicking assay

The ability of a test drug to prevent the DNA damage caused by agents such as 2,2′-azobis (2-methylpropionamide) dihydrochloride (APPH) is measured in this method. The test substance is mixed with DNA and APPH, dissolved in phosphate-buffered saline, is added to start the reaction. The resultant mix is developed on agarose gel, elec-trophoresis carried out and then staining with ethidium bromide. DNA bands are visualised under illuminated ultraviolet light and examined for DNA breakage.

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