Antioxidant: History, Measurement and Antioxidant Capacity

Read this article you will learn about Antioxidant. After reading this article you will learn about: 1. History of Antioxidant 2. Measurement of Antioxidants 3. Antioxidant Capacity 4. Classification.

History of Antioxidant:

The term antioxidant originally was used to refer specifically to a chemical that prevented the con­sumption of oxygen. In the late 19^th and early 20^th century, extensive study was devoted to the uses of antioxidants in important industrial processes, such as the prevention of metal corrosion, the vulcanization of rubber, and the polymerization of fuels in the fouling of internal combustion en­gines.

Early 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 a closed container with oxygen and measuring the rate of oxygen con­sumption. However, it was the identification of vitamins A, C, and E as antioxidants that revolu­tionized the field and led to the realization of the importance of antioxidants in the biochemistry of living organisms.

Re­search into how vitamin E prevents the process of lipid peroxidation led to the identification of anti­oxidants as reducing agents that prevent oxida­tive reactions, often by scavenging reactive oxy­gen species before they can damage cells.

Measurement of Antioxidants:

Measurement of antioxidants is not a clear-cut process, as this is a diverse group of compounds with different re-activities to different reactive ox­ygen species.

In food science, the oxygen radical absorbance capacity (ORAC) has become the cur­rent industry standard for assessing antioxidant strength of whole foods, juices and food additives. Other meas­urement tests include the Folin-Ciocalteu reagent, and the Trolox equivalent antioxidant capacity assay.

Antioxidants are found in varying amounts in foods such as vegetables, fruits, grain cereals, leg­umes and nuts. Some antioxidants such as lyco­pene and ascorbic acid can be destroyed by long-term storage or prolonged cooking. The polyphe­nols antioxidants in foods such as whole-wheat cereals and tea are more stable.

In general, processed foods contain fewer antioxidants than fresh and uncooked foods, since the preparation processes may expose the food to oxygen. The CAP-e assay measures antioxidants that are available to enter and pro­tect live cells.

Antioxidant Capacity:

Total antioxidant capacity was based on ORAC assays. The fruits, nuts, rice bran and vegetables were freeze-dried before analysis. Brunette pota­to, broccoli, carrot and tomato, which were in their cooked form used for analysis. Total antioxidant capacity is summarized in Table 10.4.

Classification of Antioxidant:

The body has several mechanisms to counteract oxidative stress by producing antioxidants, either naturally generated in situ (endogenous antioxi­dants), or externally supplied through foods (ex­ogenous antioxidants).

The roles of antioxidants are to neutralize the excess of free radicals, to pro­tect the cells against their toxic effects and to con­tribute to disease prevention. Endogenous compounds in cells can be classified as enzymatic and non-enzymatic anti­oxidants.

Enzymatic Antioxidants:

The major enzymatic antioxidants directly in­volved in the neutralization of ROS and RNS are superoxide dismutase (SOD), catalase (CAT), glu­tathione peroxidase (GPx) and glutathione reduct­ase (GRx).

Superoxide Dismutase:

Superoxide dismutase (SOD) scavenges both ex­tracellular and intracellular superoxide anion and prevents lipid peroxidation of the plasma mem­brane. It is first line enzyme of defense against free radicals, catalyz­es the dis-mutation of superoxide anion radical (O₂) into hydrogen peroxide (H₂O₂) by reduc­tion.

The oxidant formed (H₂O₂) is transformed into water and oxy­gen (O₂) by catalase or glutathione peroxidase. SOD also prevents premature hyper activation and capacitation induced by su­peroxide radicals before ejaculation.

Glutathione Peroxidase/Reductase System:

This system forms an excellent protection against lipid peroxidation of plasma membrane of sper­matozoa. It scavenges lipid peroxides thereby ar­resting the progressive chain reaction of lipid per­oxidation. It also scavenges hydrogen peroxide (H₂O₂), which is responsible for the initiation of lipid peroxidation. Glutathione reductase (GRx) stimulates the reduction of glutathione disulfide to reduced glutathione.

Glutathione reductase, a flavoprotein enzyme, regenerates reduced glutath­ione (GSH) from oxidized glutathione (GSSG), with NADPH as a source of reducing power. Besides hydrogen peroxide, GPx also reduces lipid or non-lipid hydro peroxides while oxidizing glutathione (GSH). This ensures a steady supply of the reduc­tive substrate (NADPH) to glutathione peroxidase.

Catalase:

Catalase detoxifies both intracellular and extra­cellular H₂O₂ to water and oxygen (Baker et al., 1996). In addition, catalase activates NO-induced sperm capacitation, which is a complex mecha­nism involving H₂O₂.

Non-Enzymatic Antioxidants:

The non-enzymatic antioxidants are also divided into metabolic and nutrient antioxidants. Meta­bolic antioxidants belonging to endogenous anti­oxidants, are produced by metabolism in the body, such as lipoid acid, glutathione, L-arginine, coen­zyme Q10, melatonin, uric acid, bilirubin, metal- chelating proteins, transferrin, etc.

While nutrient antioxidants belonging to exogenous antioxidants, are com­pounds which cannot be produced in the body and must be provided through foods or supple­ments, such as vitamin E, vitamin C, carotenoids, trace metals (selenium, manganese, zinc), flavo­noids, omega-3 and omega-6 fatty acids, etc. cop­per and iron.

Nutrient Antioxidants:

Antioxidants from our diet play an important role in helping endogenous antioxidants for the neu­tralization of oxidative stress. The nutrient anti­oxidant deficiency is one of the causes of numer­ous chronic and degenerative pathologies. Each nutrient is unique in terms of its structure and anti­oxidant function which are discusses in below:

Vitamin E:

Vitamin E is a chiral compound with eight stere­oisomers: α, β, y, δ-tocopherol and α, β, y, δ-to-cotrienol, which are fat-soluble vitamins with anti­oxidant properties. Among these stereoisomers, only α-tocopherol is the most bioactive and easi­ly absorbing and metabolizing this form in the hu­man body.

Studies in both animals and humans indicate that natural dextrorotary d-α-tocopherol is nearly twice as effective as synthetic racemic dl-α-tocopherol.

It has also been claimed that the α-tocophergl form is the most important lipid soluble antioxidant, and that it protects mem­branes from oxidation by reacting with lipid radi­cals produced in the lipid peroxidation chain re­action.

This reaction produces oxidized α-tocopheroxyl radicals that can be recycled back to the active reduced form through reduction by other antioxidants, such as ascorbate, retinol or ubiquinol. Vitamin E has also been claimed for the pre­vention against colon, prostate and breast cancers, cardiovascular diseases, ischemia, cataract, arthri­tis and certain neurological disorders.

In vitro studies show that vitamin E is a major chain-breaking antioxidant in the sperm membranes and it appears to have a dose depend­ent protective effect.

However, the roles and importance of the various forms of vitamin E are presently unclear and it has even been suggested that the most important function of α- tocopherol is as a signalling molecule, with this molecule having no significant role in antioxidant metabolism.

The functions of the other forms of vitamin E are even less well-understood, although α-tocophe­rol is a nucleophile that may react with electrophilic mutagens and tocotrienols may be important in protecting neurons from damage.

Vitamin C:

Vitamin C chemically known as ascorbic acid and is a water-soluble vitamin. It is a monosaccharide antioxidant found in both plants and animals. Animals except humans are able to produce this compound in their bodies and. do not require it in their diets.

In cells, it is maintained in its reduced form by reac­tion with glutathione, which can be catalysed by protein disulfide isomerase and glutaredoxins.

Ascorbic acid is a reducing agent and it reduce the reactive ox­ygen species like hydrogen peroxide. In addition to its direct antioxidant effects, ascorbic acid is also a substrate for the antioxidant enzyme ascorbate peroxidase, a func­tion that is particularly important in stress resist­ance in plants.

Vitamin C is essential for collagen, carnitine and neurotransmitters biosynthesis and its benefi­cial effects are antioxidant, anti-atherogenic, anti- carcinogenic, immune modulator. A dose dependent effect of vitamin C on sperm motility has been demonstrated that at a dose of 1000 microgram/L, vitamin C posi­tively influenced the motility of the spermatozoa.

However, above that level, vitamin C reduced the motility of the spermatozoa. On the other hand, doses fewer than 200 mg of vitamin C did not pro­vide any benefit.

Dawson et al. (1992) reported that smokers receiving 1000 mg/day vitamin C showed improvement in sperm quality. Natural sources of vitamin Care acid fruits, green vegetables, tomatoes and it is a labile mol­ecule, therefore it may be lost from during cooking.

Carotenoids:

Carotenoid and retinyl ester are two major pro-vi­tamins because they can be converted to active vitamin A. There are more than 600 carotenoid in nature of which six can be meas­ured in blood. Carotenoid are fat soluble pigments which are found in fruits and vegetables like carrots, green plants, squash, spin­ach etc.

Carotenoids can­not be synthesized in animals or humans and therefore needs to be part of the dietary intake. α and β-carotene are the major carotenoids which are mainly composed of carbon and hydrogen at­oms. In humans and animals, carotenoids play an important role in protection against photo oxidative processes by acting as oxygen and peroxyl radical scavengers.

Their synergistic action with other antiox­idants makes them an even more potent compound.

Several studies have shown that less plant derived carotenoids is absorbed, cleaved, reduced and finally available as retinyl esters. Therefore, bioequivalence is less than predicted and factors such as food preparation, matrix properties, co-ingestion of fat, diseases of the gastrointestinal tract and malnutrition may be contributors.

Carotenoids have a wide range of protective properties against disease and aging and can act as a modulator for cellular processes and function. Some other studies have shown that carotenoids can protect against can­cer and cardiovascular diseases.

Furthermore, dietary carotenoids can protect the skin against sun exposure, whilst antioxidant and cellular signalling effects have been shown in a number of studies.

However, beta-carotene supplement in doses of 20mg daily for 5-8 years has been associated with an increased risk of lung and prostate cancer and increased cardiovascular mortality in cigarette smokers.

Beta-Carotene:

Beta-carotene is a fat-soluble member of the car­otenoids, which are considered pro-vitamins be­cause they can be converted to active vitamin A. Beta-carotene is converted to retinol, which is essential for vision. It is a strong antioxidant and is the best quencher of singlet oxygen.

However, beta-carotene supplement in doses of 20 mg daily for 5-8 years has been associated with an increased risk of lung and prostate cancer and increased to­tal mortality in cigarette smokers.

Beta-carotene 20-30 mg daily in smokers may also increase cardiovas­cular mortality by 12% to 26% . These adverse effects do not appear to occur in people who eat foods high in beta-carotene con­tent. Beta-carotene is present in many fruits, grains, oil and vegetables (carrots, green plants, squash, spinach).

Cur cumin:

Curcumin [1, 7-bis (4-hydroxy-3-methoxy phenyl)- 1-6- hepatadine-3-5-dione] a member of curcuminoid family represents one of the yellow pig­ments isolated from turmeric. Turmeric is the dried rhizome of plant Curcuma longa and apart from its culinary appeal and common use as the spice, it is well known for its medicinal properties in Egyptian and Indian culture for more than 6000 year ago.

Its immunomodulatory properties includ­ing anti-oxidant, anti-inflammatory and anti-tumor properties are well-documented. The curcumin reduces nitric oxide (NO) and ex­erts beneficial effects in experimental colitis.

Ukil et al. (2003) repotted that the yellow pigment treats inflammatory bowl diseases due to the oxidative and nitrosative stresses. Recently the immuno-nutritional ability of curcumin demonstrating its active role in treatment of the allergic response has been highlighted in a review.

Lycopene:

Lycopene, a member of the carotenoid family, is a fat-soluble pigment responsible for the red color of certain fruits and vegetables. Its pigment pro­tects the plant from damage by oxygen and light. Lycopene possesses antioxidant and anti-prolifer­ative properties in animal.

Lyc­opene has been found to be very protective, par­ticularly for prostate cancer. Several prospective cohort studies have found as­sociations between high intake of lycopene and reduced incidence of prostate cancer, though not all studies have produced consistent results.

The major dietary source of lyco­pene is tomatoes, with the lycopene in cooked tomatoes, tomato juice and tomato sauce includ­ed, being more bioavailable than that in raw tomatoes. The dietary antioxi­dant lycopene reduces oxidative stress and the levels of bone turnover markers in postmenopau­sal women, and may be beneficial in reducing the risk of osteoporosis.

Green Tea:

Tea is one of the most consumed beverages of the world after water. Tea beverage is an infusion of the dried leaves of Camellia sinesis, a member of Theaceae family. Freshly harvested tea leaf is proc­essed differently in different parts of the world to give oolong tea (2%), green tea (20%) or black tea (78%).

Green tea is pre­pared from the fresh tea leaf and widely consumed in Japan and China.

Western cultures favour black tea, which is prepared through the oxidation, cur­ing process of maceration and exposure to atmos­pheric oxygen. Tea is linked to beneficial effects on hu­man health with the polyphenols as the responsi­ble constituents.

Fresh tea leaf is rich in water soluble polyphe­nols, particularly flavanols, flavanol gallate and flavanol glycosides.

The major tea catechins: α-epigallocatechin-3-gallate (EGCG), α-epigallocate- chin (EGC), epicatechin-3-gallate (ECG), α-epifltechin (EC), α-epicatechin-3-gallate (ECG), α- epicatechin (EC), α-gallocatechin and β-catechin; constitutes 30% to 42% of the green tea solids by weight.

These compounds are all potent antioxidant in vitro and, when con­sumed, may act as the free radical scavengers, which remove endogenously generated superox­ide, peroxyl and hydroxyl radicals. Many in vitro and in vivo effects of tea polyphenols have been reported including antioxidant, anticar-cinogenic and hypolipidemic properties.

The powerful antioxidant properties of the tea are generally attributed to its flavonoids compo­nents; theaflavins, bisflavanols and theaflavic ac­ids.

The antioxidant property of tea is coupled with several mecha­nisms e.g. depolarization of electrons, formation of intermolecular hydrogen bonds, rearrangement of the molecular struc­ture.

The bioactive flavanol compounds of tea may also prevent oxidative reactions by chelating free cop­per and iron, which may catalyse the formation of reactive oxygen species in vitro.

Garlic:

Garlic (Allium sativum) is used universally as a traditional medicine, and a functional food to en­hance physical and mental health. It is used for flavouring in cooking and is unique because of its high sulfur content. In addi­tion to sulfur, garlic also contains arginine, oli­gosaccharides, flavonoids, and selenium, all of which may be beneficial to health.

The medicinal uses of garlic has a long history. Recent stud­ies have validated many of the medicinal proper­ties attributed to garlic and its potential to lower the risk of disease. Cancer-preventive actions of garlic, garlic extracts and its components have been demonstrated in animals.

Ep­idemiologic studies show an inverse correlation between garlic consumption and reduced risk of gastric and colon cancer. Garlic has been shown to have antithrombotic activity, lower blood lipids and have a cardio protective effect.

The mechanisms of garlic have been endorsed to its potent antioxidant action, its ability to stimulate immuno­logical responsiveness and its modulation of prostanoid synthesis.

Aged garlic extract (AGE) is an odorless prod­uct resulting from prolonged extraction of fresh garlic at room temperature; it is highly bioavailable and has biological activity in vitro in both an­imals and humans.

AGE contains water-soluble allyl amino acid deriva­tives, which account for most of its organosulfur content, stable lipid-soluble allyl sulfides, flavo­noids, saponins and essential macro-and micro- nutrients. The lipid-soluble vol­atile organosulfur compound allicin, which is pro­duced enzymatically when garlic is cut or chopped, is absent in AGE.

Allicin is an unstable and transient compound with oxidant activity; it is virtually unde­tectable in blood circulation after garlic ingestion because it decomposes to form other organosulfur compounds.

The major unique organosulfur compounds in AGE are water-soluble 5-allyl-cysteine (SAC) and 5-allylmercaptocysteine (SAMC), which have potent antioxidant activity.

The content of SAC and SAMC in AGE is high because they are pro­duced during the process of aging, thus providing AGE with higher antioxidant activity than fresh garlic and other commercial garlic supplements.

The anti-oxidative actions of AGE and its com­ponents are determined by their ability to scavenge ROS and inhibit the formation of lipid per­oxides.

These effects are determined by measur­ing the decrease in ROS-induced chemiluminescence, inhibition of thiobarbituric acid reactive substances (lipid peroxides) (TBARS assay), and in vitro inhibition of the release of pentane, a prod­uct of oxidized lipids, in the breath of an animal exposed to oxidative stress.

Oxidation of lipids, notably oxidative modifi­cation of LDL, is implicated in the development of cardiovascular and cerebrovascular disease. Lipid oxidation products, including peroxides and tox­ic aldehydes such as malondialdehyde, can damage proteins and DNA and have been implicated in carcinogenesis.

AGE inhibits lipid oxidation and oxidative modification of LDL . Other protective actions of AGE include inhibition of platelet aggregation and suppres­sion of prostanoid synthesis with subsequent anti­-inflammatory, anti-athrogenic and antithrombotic effects.

The protec­tion of endothelial cell integrity by inhibition of lipid peroxidation-induced injury and reduction in serum cholesterol and other lipids by AGE further add to its potential in helping prevent heart disease and stroke.

Cytosolic nuclear factor-B (NF-B) is a tran­scription factor that is regulated by the redox state of the cell and can be activated by mitogens, bac­teria and viruses and by ROS-producing agents such as UV, ionizing radiation, hydrogen perox­ide and tumor necrosis factor (TNF).

The major clinical significance of NF-B activation is its in­volvement in human immunodeficiency virus (HIV) gene expression. AGE and SAC inhibit TNF- and hydrogen peroxide-in­duced activation of NF-B in human T-cells, indicating their potent antioxidant function and suggesting a potential role for AGE in modulating HIV replication.

Oxidant-induced DNA damage and mutagen­esis are determinants in the multistage process of cancer in animals and in vitro. Allixin, an active component in AGE, which has been shown to inhibits aflatoxin-induced DNA damage and mutagenesis in Salmonella typhimurium, in part by inhibiting cytochrome P450 activity.

Several group of workers re­ported that AGE inhibits both early and late stages of carcinogenesis of tumor growth in many tis­sues like colon, mammary glands, skin, stomach and esophagus.

Supplementation with AGE may have an im­portant protective role against liver toxicity caused by a variety of medicinal and environmental sub­stances. Tadi et al. (1991) re­ported that AGE protects against liver toxicity by benzo (a) pyrene and aflatoxin B₁, two potent free radical-producing environmental carcinogens.

Studies in mice showed that SAC and SAMC were potent inhibitors of liver tox­icity induced by the industrial oxidant carbon tet­rachloride and by the commonly used analgesic agent, acetaminophen.