In human life Oxygen is very essential, without oxygen we cannot survive. Our evolutionary ancestors developed defense mechanisms that can minimize the toxic effects of oxygen, without this protection causes the end of life. Natural defences are imperfect, the damage of the cells caused by oxygen can be minimized by using antioxidants. The diseases like cancer, cardiovascular disease, cataracts, age related diseases and degenerative diseases of nervous system. A lot of research works are made in this past decades. In this study the metabolites produced by the oxygen species and with research works they have learned how to prevent the diseases caused by the reactive oxygen species. Now research works are doing for improving the antioxidant activity.
Free radicals are fundamental to any biochemical process and represent an essential part of aerobic life and our metabolism. They are continuously produced by body's normal use of oxygen such as respiration and some cell mediated immune functions. The oxygen consumption inherent in cell growth leads to the generation of reactive oxygen species.
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A free radical may be defined as any atom, group of atoms or molecule having one or more unpaired electrons in its outermost orbital and are capable of independent existence. They are typically unstable and highly reactive. A free radical is formed when a covalent bond between molecules is broken and the corresponding electron remains with the newly formed atom.
Free radicals are extremely reactive due to the presence of unpaired electrons as it gives the molecule a considerable degree of reactivity and once formed they act as highly reactive radicals capable of chain reactions.
Fig. 4 Free radical formation
Any free radical having oxygen can be referred to as a ROS. Oxygen centered free radical species hold 2 unpaired electrons in the outer shell. When free radicals capture an electron from the neighboring compound or molecule a new free radical is formed and this reaction proceeds as a chain reaction until the free radicals are all neutralised (Wijk et al., 2008).
Causes of free radicals Formation
Food preservatives and pesticides
MECHANISM OF ACTION OF FREE RADICALS OR ROS FORMATION
Oxygen in the atmosphere has two unpaired electrons and these unpaired electrons have parallel spins and it is considered to be in a ground (inactive) state. Oxygen is normally non reactive to organic molecules that have paired electrons with opposite spin, but can be activated to singlet excited (active) state by two mechanisms.
Absorption of adequate energy to reverse the spin on one of the unpaired electrons.
Monovalent reduction (accept a single electron)
Superoxide is formed during the monovalent reduction reaction which further gets reduced to form H2O2. H2O2 then in the presence of ferrous salts (Fe2+) gets reduced to hydroxyl radicals. This reaction was initially described by Fenton and later developed by Haber and Weiss (Daniel et al., 1998).
Antioxidants are any substance, present at lower concentration compared to that of oxidizable substance that delay or inhibits oxidative damage to a target molecule. Antioxidants defuse the free radicals by donating their electrons and thus putting an end to the carbon stealing property of the free radical. They work as scavengers and thus prevent cell and tissue damage that can lead to cellular injury and disease. They are agents that protect other vital chemicals and macro molecules of the body from oxidation reactions by reacting with free radicals and other ROS within the body. One antioxidant molecule can only react with single free radical and hence there is a constant necessity to replenish antioxidant reserves either endogenously or through dietary supplement.
The body has developed numerous endogenous antioxidant systems to combat the production of reactive oxygen intermediates (ROI). These systems can be broadly divided into:
Non - Enzymatic. Fig 7 shows the antioxidant system.
Fig. 7 Antioxidant system
Superoxide Dismutase (SOD)
SODs are a family of metalloenzymes that converts superoxide to hydrogen peroxide (H2O2) and are mainly the primary line of protection against oxygen toxicity. Basically three isoforms of the enzyme have been discovered. The first is mainly found in the cytoplasm of cells and it containing Cu and Zn at its active site (Cu/Zn SOD-1), the second containing Mn at its active site is located in mitochondria (Mn SOD-2) and the third (Cu/Zn SOD-3) is present in the extracellular fluid like plasma. SOD is a stress protein which is synthesized mostly in response to oxidative stress. It is found that little amount of Cu, Zn and Mn metals are crucial for maintaining the antioxidant activity of SOD (Ray and Husain, 2002).
Glutathione Peroxidase (GPx)
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GPx is one of the important enzymes responsible for the degradation of H2O2 and organic peroxides in the brain. GPx catalyse the oxidation of glutathione into its oxidized form (GSSG) at the expense of H2O2. Two isoforms have been identified; selenium-dependent GPx which is highly active towards H2O2 and organic hydroperoxides and selenium independent GPx. GPx activity is found to be less in selenium deficiency.
It is a heme-containing protein present in some cells. Catalase is 104 times faster than GPx and it consists of four protein subunits, each containing a heme fe(III)-protoporphyrin group bound to its active site (Ray and Husain, 2002).
Carotenoids: It is a lipid soluble antioxidants, commonly seen in membrane tissue. The main function is the removal of reactive oxygen species.
Bilirubin : It is produced by heme metabolism found in blood. The main function is act as extracellular antioxidants.
Glutathione: It is a non-protein thiol and found in cells. It has the property of cellular oxidant defense.
Alpha-lipoic acid : It is endogenous thiol. Its property is by serving substitute for glutathione, recycling vitamin C.
Vitamin C : It is found in aqueous phase of cell. Its property is act as free radical scavenger and also act in the recycle of vitamin E.
Vitamin E: It is found in cells. Function is chain breaking antioxidant.
Uric acid: It is a product of purine metabolism. Its property is scavenging of hydroxyl (OH) radical.
MECHANISM OF ACTION OF ANTIOXIDANTS
They mainly act as
ïƒ˜ Physical barriers preventing ROS generation or ROS access to important biological sites.
E.g. UV filters, cell membranes
ïƒ˜ Chemical traps / sinks 'absorb' energy and electrons quenching ROS.
E.g. Carotenoids, anthocyanidins
ïƒ˜ Catalytic systems neutralize or divert ROS.
E.g. SOD, catalase and glutathione peroxidase
ïƒ˜ Binding / inactivation of metal ion prevents generation of ROS by Haber-Weiss reaction.
E.g. Ferritin, catechins
ïƒ˜ Sacrificial and chain propagation inhibitor antioxidants scavenge and destroy ROS.
E.g. Ascorbic acid (Vit.C), tocopherols (Vit E), uric acid,
IN VITRO ANTI-OXIDANT STUDIES OF EXTRACTS OF ROOTS OF CLERODENDRUM SERRATUM RHIZOME.
Nitric oxide radical scavenging assay
Various concentrations of the extract and sodium nitroprusside (5mM) in phosphate buffer saline (0.025 M, pH 7.4) in a total volume of 3 ml was incubated at room temperature for a period of 150 min. After which, 0.5 ml of the incubated solution and 0.5 ml Griess' reagent (1% sulphanilamide, 2% O-Phosphoric acid and 0.1% naphthyethylene diamine dihydrochloride) were added togather and allowed to react for 30 min. Control samples without the test compounds but with equal volume of buffer was prepared in a similar manner as done for the test. The absorbance of the chromophore formed during diazotisation of nitrite with sulphanilamide and successive coupling with naphthyethylene diamine dihydrochloride was measured at 546 nm. The percentage inhibition of the extracts and standard was calculated. The experiment was carried out in triplicate using curcumin (40-200 μg/ml) as positive control (Rao et al., 2008).
% Scavenging Activity = [(Ac- As) / Ac] - 100
Where, Ac is the absorbance of the control reaction and as is the absorbance in the presence of the sample of the extracts. The antioxidant activity of the extract was expressed as IC50. The IC50 value was defined as the concentration (in µg/ml) of extracts that inhibits the formation of free radicals by 50%.
Table 27:NITRIC OXIDE SCAVENGING ACTIVITY
Control absorbance [Ac]: 1.0022
Absorbance at 546 nm [As]
% Anti-oxidant Activity (%)
Petroleum Ether Extract
Ethyl Acetate Extract
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Standard [ASCORBIC ACID]
Table 28: % of maximum antioxidant activity of extracts
% maximum Anti-oxidant activity [%]
Standard [Ascorbic acid]