Research J. Pharm. and Tech.2 (2): April.-June.

2009,

ISSN 0974-3618 REVIEW ARTICLE

www.rjptonline.org

Role of Antioxidants in Common Health Diseases

2

S.D. College of Pharmacy, Barnala, Punjab 148101 Dept. of Pharma. Chemistry, Himalayan Pharmacy Institute, Majhitar, Rangpo, East Sikkim -737136 India *Corresponding Author E-Mail: prithviraj.in@gmail.com

Prithviraj Chakraborty*1, Suresh Kumar1, Debarupa Dutta2, and Vikas Gupta1

1

Antioxidants are intimately involved in the prevention of cellular damage - the common pathway for cancer, aging, and a variety of diseases. Several berries, fruits, nuts, seeds, vegetables, drinks and spices have been found to be high in total antioxidants. The body relies on obtaining its anti-oxidants from food and other supplements. Free radicals are highly reactive molecules containing one or more unpaired electrons in their outermost shell and the only way of neutralizing them and their harmful effects is through anti-oxidants which help in the break down of chemical reactions that cause transfer of electrons from healthy cells into free radicals. In view of the immense medicinal importance its an effort to compile all the information reported on its mechanism, process, defense and methods of assessing antioxidant activities. The present review is an attempt to generate interest among the masses regarding its immense potential in preventing and treating several common diseases.

ABSTRACT:

KEY WORDS: Antioxidants, Free radicals, Superoxide dismutase (SOD), Catalase

Cells in the human body use oxygen to breakdown proteins and fats that give them energy. The human body derives its energy from the utilization of nutrients and oxygen as fuel. It also utilizes oxygen to help the immune system, destroys foreign substances and combats diseases. The byproduct of this and other metabolic process can lead to development of molecular agents that react with body tissues in a process called oxidation. This process is a natural phenomenon of energy generation system and its by-product called free radicals can damage healthy cells of the body. DEFINITION:An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules.In a biological system they may protect cells from damage caused by unstable molecules known as free radicals. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols or polyphenols.They are believed to play a role in preventing the development of such chronic diseases as cancer, heart disease, stroke, Alzheimer' disease, Rheumatoid arthritis, s and cataracts.

INTRODUCTION:

Antioxidants help in: Destroying the free radicals that damage cells. Promoting the growth of healthy cells. Protecting cells against premature, abnormal ageing. Help fight age-related macular degeneration. Provide excellent support for the bodys immune system. Research on the role of antioxidants in biology focused on their use in preventing the oxidation of unsaturated fats, which is the cause of rancidity. Antioxidant activity could be measured simply by placing the fat in the closed container with oxygen and measuring the rate of oxygen consumption. CLASSIFICATION1: Enzymatic antioxidants: 1. Primary antioxidants e.g.-SOD, Catalase, Glutathione peroxidase. 2. Secondary enzymes e.g. - Glutathione reductase, Glucose 6-phosphate dehydrogenase. Non-Enzymatic antioxidants:1. Minerals e.g.-Zinc, Selenium 2. Vitamins e.g.-Vitamin A, Vitamin C, Vitamin E, Vitamin F 3. Carotenoids e.g.- -carotene, Lycopene, Lutein, Zeaxanthin 4. Low molecular weight Antioxidants e.g.-glutathione, uric acid 5. Organosulfur compounds e.g-Allium, Allyl sulfide, indoles 6. Antioxidant cofactors e.g.- Coenzyme O10 7. Polyphenols

Received on 08.01.2009 Accepted on 15.05.2009

Research J. Pharm. and Tech.2 (2): April.-June. 2009.Page 238-244

Modified on 11.04.2009 RJPT All right reserved

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FlavonoidsXanthones- e.g.- Mangostin Flavonoids- e.g.- Quercein, Kaempferol Flavanols- e.g.- Catechin, EGCG Flavanones- e.g.- Hesperitin Flavones- e.g.- Chrysin Isoflavanoids- e.g.- Genistein Anthocyanidins- e.g.-Cyanidin, Pelagonidin Phenolic AcidHydroxycinnamic acids- e.g.- Ferulic, p-coumaric Hydroxybenzoic acid e.g.- Gallic acid, Ellagic acid Gingerol Curcumin

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Haber-Weiss reaction7- H2O2 +O2 O2 +OH +OH The free radical nitric oxide (NO), also called endothelium-derived relaxation factor (EDRF), is formed from arginine by nitric oxide synthase (NOS) 2. L-arg + O2 +NADPH NO + Citrulline NO +O2 ONOO8, 9 (Peroxynitrite) Peroxynitrite is a very strong oxidant, which reacts with aromatic amino acid residues to form nitrotyrosine, which can lead to enzyme inactivation. To escape ROS, RNS and lipid peroxidation dependence injury; biological structures have protective machinery in the form of endogenous antioxidants.

FOOD SOURCES OF ANTIOXIDANTS2, 3, 4, 5

Vitamin C Fruits (especially citrus) and vegetables, including green and red peppers, tomatoes, potatoes, and green, leafy varieties (eg, spinach and collard greens). Vegetable oils (eg, soybean, corn, and safflower) and vegetable oil products (eg, margarine), whole grains, wheat germ, nuts and seeds, and green, leafy vegetables. Yellow-orange fruits (eg, cantaloupe) and vegetables (eg, carrots) and green, leafy vegetables. Tea, coffee, soy, fruit, olive oil, chocolate, cinnamon, oregano and red wine.6

ANTIOXIDANT DEFENSE: Antioxidant defense system (ADS) against oxidative stress is composed of several lines and antioxidants are classified into four categories based on their function.10 FIRST: - Preventive antioxidants which suppress formation of free radicals. SECOND: - Radical scavenging antioxidants which suppress chain initiation and breaking chain propagation reactions. THIRD: - Repair and de novo antioxidants. FOURTH: - Adaption where the signal for the production and actions of free radicals induces formation and transport of the appropriate antioxidant to the right site. THE ANTIOXIDANT PROCESS: Antioxidants block the process of oxidation by neutralizing free radicals. In doing so, the antioxidants themselves become oxidized. The two ways by which they act are Chain-breaking - When a free radical releases 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. Preventive - Antioxidant enzymes like superoxide dismutase, catalase and glutathione peroxidase prevent oxidation by reducing the rate of chain initiation. They can also prevent oxidation by stabilizing transition metal radicals such as copper and iron. ENZYMATIC ANTIOXIDANTS: The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), glutathione reductase, thioredoxin reductase, heme

Vitamin E

b-Carotene Polyphenolic antioxidants

MECHANISM OF ANTIOXIDANTS:Free radicals are highly reactive molecules or chemical species containing one or more unpaired electrons in their outermost shell. They react quickly with nearest stable molecule to capture the electron, in need to gain stability. They promote beneficial oxidation that produces energy and kill bacterial invaders. If free radicals are at reasonable levels, the human body produces enzymes to combat them and is helpful in immune system and anti bacterial cell activity. A single free radical can cause damage to millions of other molecules in the body from functioning properly. This molecular destruction is continually occurring in our body. Although antioxidants are a result of breathing but these free radicals attack us from many different sources every day. They are: Alcohol, Tobacco, Dugs, Smoked and Barbecued Foods, Harmful Chemicals and Pesticides, and Food Additives. GENERATION OF FREE RADICALS: The living cell during several metabolic pathways generates reactive oxygen species (ROS) and reactive nitrogen species (RNS). Pathophysiological conditions enhance the generation of ROS and RNS and lead to oxidative stress. The generation of ROS begins with the rapid uptake of oxygen and activation of NADPH oxidase and the production of the super oxide free radical (O2). 2O2 + NADPH 2O2 +NADP+H ROS can also be generated through Fenton reaction- H2O2 +Fe++ Fe++++OH +OH

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oxygenase and biliverdin reductase serve as primary line of defense in destroying free radicals. CATALASE: An enzyme found in the blood and in most living cells that catalyzes the decomposition of hydrogen peroxide into water and oxygen. Catalase is a common enzyme found in living organisms. Its functions include catalyzing the decomposition of hydrogen peroxide to water and oxygen. Catalase has one of the highest turnover rates of all enzymes; one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second. Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long. It contains four porphyrin heme groups which allow the enzyme to react with the hydrogen peroxide. The optimum pH for catalase is approximately neutral (pH 7.0), while the optimum temperature varies by species. Haem-containing catalase breaks down hydrogen peroxide by a two-stage mechanism in which hydrogen peroxide alternately oxidises and reduces the haem iron at the active site. In the first step, one hydrogen peroxide molecule oxidises the haem to an oxyferryl species. In the second step, a second hydrogen peroxide molecule is used as a reductant to regenerate the enzyme, producing water and oxygen. Some catalase contains NADPH as a cofactor, which functions to prevent the formation of an inactive compound. Catalases may have another role: the generation of ROS, possibly hydro peroxides, upon UVB irradiation. In this way, UVB light can be detoxified through the generation of hydrogen peroxide, which can then be degraded by the catalase. NADPH may play a role in providing the electrons needed to reduce molecular oxygen in the production of ROS.

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SUPEROXIDE DIMUTASE: Superoxide dismutase (SOD) is an enzyme that removes the superoxide (O2-) radical, repairs cells and reduces the damage done to them by superoxide, the most common free radical in the body. SOD is found in both the dermis and the epidermis, and is key to the production of healthy fibroblasts (skin-building cells). 2 H 2O 2 2 H 2O + O 2

Superoxide Dismutase (SOD) catalyzes the reduction of superoxide anions to hydrogen peroxide. It plays a critical role in the defense of cells against the toxic effects of oxygen radicals. SOD competes with nitric oxide (NO) for superoxide anion, which inactivates NO to form peroxynitrite. Therefore, by scavenging superoxide anions, SOD promotes the activity of NO. SOD has suppressed apoptosis in cultured rat ovarian follicles, neural apoptosis in neural cell lines, and transgenic mice by preventing the conversion of NO to peroxynitrate, an inducer of apoptosis.11,12,13 Covalent conjugation of superoxide dismutase with polyethylene glycol (PEG) has been found to increase the circulatory half-life and provides prolonged protection from partially reduced oxygen species.14 The SOD-catalyzed dismutation of superoxide may be written with the following half-reactions: M (n+1) + SOD + O2 Mn+ SOD + O2 n+ + M SOD + O2 + 2H M (n+1) + SOD + H2O2. , Where M = Cu (n=1) Mn (n=2), Fe (n=2) , Ni (n=2) In this reaction the oxidation state of the metal cations oscillates between n and n+1. SUPEROXIDE DIMUTASE REACTION: Superoxide Dismutase O 2Superoxide Dismutase O2 + 2 H+ Km for O2- for bovine SOD = 0.35 Mm

O2 H 2O 2

Much of the hydrogen peroxide that is produced during oxidative cellular metabolism comes from the breakdown of one of the most damaging ROS, namely the superoxide anion radical (O2-). Superoxide is broken down by superoxide dismutase into hydrogen peroxide and oxygen. Superoxide is so damaging to cells that mutations in the superoxide dismutase enzyme can lead to ALS, which is characterised by the loss of motoneurons in the spinal cord and brain stem, possibly involving the activation of caspase-12 and the apoptosis cascade via oxidative stress. The reaction of catalase in the decomposition of hydrogen peroxide is: 2 H 2O 2 2 H 2O + O 2

GLUTATHIONE PEROXIDASES (GSHPx): They are a group of selenium dependent enzymes. Four of its isoforms include Cytosolic GSHPx1 Plasma GSHPx Phospholipid hydroperoxide PHGSHPx Gastrointestinal GSHPx-GI All GSHPx require GSH as cofactor and secondary enzymes, such as glutathione reductase and glucose-6 phosphate dehydrogenase for proper functioning. G-6PDH generates NADPH to recycle the GSH. 2GSH+H2O2 GSSG+2H2O NON ENZYMATIC ANTIOXIDANTS: They are classified into two groups: Endogenous antioxidants Exogenous antioxidants

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The major extracellular endogenous antioxidants found in human plasma are the transition metal binding proteins i.e. ceruloplasmin, transferrin, hepatoglobin and albumin. They bind with transition metals and control the production of metal catalyzed free radicals. Albumin and ceruloplasmin are the copper ions sequesters. Hepatoglobin binds with hemoglobin, ferritin and transferrin with free iron. Lipoic and uric acids, bilirubin, ubiquinone and glutathione are non protein endogenous antioxidants which inhibit the oxidation processes by scavenging free radicals. SOME COMMON ANTIOXIDANTS: There are a hundreds of antioxidants of natural and synthetic origin. The interest of such compounds is due to their effective role against the destructive actions of free radicals. Some common antioxidants are as follows: VITAMIN E: It is the most common naturally occurring antioxidant. It has a phytyl chain which is attached to its chromanol nucleus.

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amphibians28 and humans 26, 29, 30. The dermal and epidermal tissues (e.g. skin, retina) of animals are often heavily melanized, and most have speculated that this is to protect individuals from harmful solar UV rays31.

Melanin is an integral component of the immune responses of many invertebrates32, and there are several mechanisms and lines of evidence linking melanins and immunity in vertebrates (e.g. phagocytosis, lysosomal enzyme activity, cytokine regulation, nitric oxide production) 33. The ability of melanins to bind and sequester toxic metals may be yet another line of physiological defence against oxidative stress in animals17. PTERINS AS ANTIOXIDANTS: Pterin pigments are a group of nitrogenous, heterocyclic compounds that are catabolic by-products of purines. They occur as red, orange and yellow pigments, which orange sulphur butterflies, Colias eurytheme 34, guppies, Poecilia reticulata35 and green anoles, Anolis sp. 36, as well as many other insects, fish, amphibians and reptiles incorporate into their sexual colour displays. Pterins have also been identified in the colorful red, orange and yellow eyes of birds such as starlings, blackbirds, owls and pigeons37. Like melanins, pterins can also be synthesized by non integumentary tissues, mostly by immune cells (e.g. monocytes, macrophages) 38 . Activation of the immune system (e.g. by lymphokines like gamma interferon) is known to stimulate the release of pterins (such as neopterin and 7, 8-dihydroneopterin) from primate macrophages and monocytes39. The role of pterins as immune cell protectants is so robust that their concentrations in serum and urine are used as clinical biomarkers for immune performance in humans (e.g. allograft rejection, autoimmune disease, viral/bacterial/parasitic infections) 40, 41. Certain pterins, like tetrahydrobiopterin, are also known to modulate receptor binding affinity of interleukin 2, which is an integral part of the T-cell-mediated immune response42. PORPHYRINS AS ANTIOXIDANTS: Porphyrins are generally united by their heterocyclic, pyrrolic molecular structure but often divided by the type of metal ion found at the core of the super-ring. The most familiar representatives include chlorophyll as well as haem43, which colours the fleshy, blood-engorged red parts of many birds44, 45. A different set of porphyrins has been classified from the eggshells and feathers of birds (reviewed in With 1973, 1978). Most if not all porphyrins, which include reddish-brown coproporphyrin and uroporphyrin in the feathers of goatsuckers, bustards and owls46 and protoporphyrin and biliverdin (a bile pigment) in brown and blue eggs, respectively 47, 48, are derivatives of haem from erythrocytes.

ASCORBIC ACID (VITAMIN C): It is a water soluble electron donar vitamin. It donates two electrons from C-2 and C-3 double bond carbons to act as an antioxidant which results in the formation of an intermediate free radical, semi dehydroascorbic acid E. The resulting ascorbate free radicals reduce to a neutral ascorbate molecule. CAROTENOIDS: They are a large group of compounds with the basic skeleton of a polyisoprenoid carbon chain with a no. of conjugated double bonds. MELANINS AS ANTIOXIDANTS: Melanin pigments are large polymers synthesized from amino acids that have strong light-absorbing capabilities across the ultravioletvisible spectrum18. They occur in two main forms: (1) Eumelanin, which confers black and grey colours, (2) Phaeomelanin, which bestows chestnut and buff colours. Animals like mosquito- fish, Gambusia holbrooki15, house sparrows, Passer domesticus 16, fence lizards, Sceloporus undulatus17, and African lions, Panthera leo18 all use melanins as display pigments in feathers, scales, or hair to attract mates or to repel rivals19. Among the many potential physiological functions of melanins20, which include tissue strengthening21, photo protection22 and thermoregulation23, is their role as antioxidants and immunostimulants24, 25. As molecules that contain both oxidizing (o-quinone) and reducing (o-hydroquinone) functional groups, both eu- and phaeomelanin have the ability to quench potentially damaging reactive oxygen or nitrogen species (e.g. singlet oxygen, hydroxyl or peroxyl radical, superoxide anion) via either electron donation or capture26. Melanins have been touted as valuable freeradical scavengers in several organisms, including fungi27,

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One of the earliest pigments described from bird feathers was the uroporphyrin-copper complex known as turacin in the red feathers of turacos49. Recent work has emphasized the radical-trapping abilities of some of the porphyrins and porphyrin-derivatives found in birds, such as protoporphyrin50, 51, 52 and biliverdin53, 54. Haem55 and hemoglobin 56, 57 themselves have been touted as potent antioxidants.

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sensitive, and reproducible for the screening of large number of plant extracts.71 Antioxidant herbal formulations available in the marker are Brahma rasayana, Geriforte, Abana and HD-3, MAK4 and Reatival.

PSITTACOFULVINS AS ANTIOXIDANTS: For over a century, ornithologists have known that parrots use an unusual class of pigments to colour their plumage red, orange and yellow. It was only recently, however, that the biochemical structure of these compounds was elucidated 58. A larger survey of red feather pigments in parrots uncovered the same set of polyenes in 45 species spanning the three main parrot families59, including species with sexually dichromatic plumage (e.g. Eclectus parrot, Eclectus roratus). These noncarotenoid lipochromes apparently are found in nature only in parrot feathers. To date, only a single study has been conducted on the antioxidant action of psittacofulvins. Morelli58 et al. used electron paramagnetic resonance to investigate the ability of these colorful molecules to quench free radicals in vitro. They found that these pigments can act as potent antioxidants, exhibiting strong inhibition of hydroxyl (OH) radical formation. FLAVONOIDS AS ANTIOXIDANTS: Blue butterflies from the family Lycaenidae acquire blue and UV hues on their wings for the presence of flavonoids.60 Flavonoids, include the anthocyanins, flavonols, flavones and flavanones, are one of the primary classes of colorants in plants, bestowing bright colours on flowers, fruits and berries 61. These butterflies acquire host plant flavonoids as larvae and sequester these in wing scales as adults. In the common blue butterfly, Polyommatus icarus, males show strong mate preferences for flavonoid-rich, highly UV-reflective females60. Flavonoids also exist as valuable antioxidants in plant foods. There is abundant evidence from in vitro biochemical studies that flavonoids defend cells from lipid peroxidation 62, 63, and from human intervention studies that dietary supplements decrease incidence of cancer and atherosclerosis. Quercetin is also the flavonoid stored in the wings of P. icarus 60. METHODS OF ASSESSSING ANTIOXIDANT ACTIVITY OF PLANT EXTRACTS: The antioxidant activity of a compound or extract can be studied by 1,1-Diphenyl-1,2-picrylhydrazyl(DPPH) assay,64 Thiobarbituric acid reactive substances(TBARS) assay,65 Superoxide dismutase(SOD),66 Assay of catalase(CAT),66 Glutathione peroxide(GPX) activity, 67 Xanthine oxidase(XO) activity,68 Deoxyribose 69 degradation assay, Reduced Glutathione(GSH).70 All these methods can be done either in vitro or in vivo. Among all the methods, DPPH is very rapid, simple,

EFFECTS OF DIETARY ANTIOXIDANTS ON CLINICAL OUTCOMES: Recent studies have suggested that antioxidants may affect clinical outcomes. The Indian Experiment of Infarct Survival Study72 tested the therapeutic efficacy of antioxidants in reducing post-MI complications, many of which are proposed to result from oxidative reperfusion injury. Infarct size and angina and total cardiac events were significantly reduced in individuals receiving antioxidants in the post-MI period. Another potential therapeutic role for antioxidants is in the reduction of restenosis after angioplasty. This role has been addressed in several recent trials.73,74 The Multivitamins and Probucol (MVP) Study tested the effects of a combination of vitamin C (1000 mg/d), vitamin E (1400 IU/d), and bcarotene (100 mg/d); probucol (a lipid-lowering drug with antioxidant effects; 1000 mg/d); the dietary antioxidants plus probucol (in the same amounts); or placebo alone on the rate and severity of restenosis.73 The Probucol Angioplasty Restenosis Trial (PART) compared probucol (1000 mg/d) with placebo.74 In both studies, treatments were initiated 1 month before and maintained for 6 months after elective angioplasty. Probucol significantly reduced restenosis due to its antioxidant properties. In the MVP study, similar results were not observed for the dietary antioxidants, which had no effect alone and appeared to negate the beneficial effects of probucol when given in combination.73 Beneficial effects have been observed for vitamins C and E in other studies 75,76, . Because the long-term use of probucol in diseased individuals is of concern, owing to adverse effects on plasma high-density lipoprotein levels (a 41% reduction was noted in the MVP study), dietary antioxidants, could represent a good alternative. CONCLUSION: Antioxidants are emerging as prophylactic and therapeutic agents. Several antioxidants have been found to be pharmacologically active as prophylactic and therapeutic agents for several diseases. These agents are used as nutritional supplements for prophylaxis of certain diseases along with mainstream therapy. Bioavailability of dietary antioxidants is dependant on a number of factors such as poor solubility, inefficient permeability instability and extensive first pass metabolism before reaching the systemic circulation and GI degradation. There is need to develop new drug delivery systems to improve the performance of antioxidants. Along with the evidence of positive benefits, there are several reports regarding the negative effects of antioxidants use, especially concerning dietary

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supplementation with Vitamin C and E, beta-carotene and selenium. Of primary concern are the potentially deleterious effects of antioxidant supplements on ROS levels, esp. when precise modulation of ROS levels are needed to allow normal cell function or to promote apoptotic cell death of precancerous transformed cells.

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