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ANTIOXIDANTS ANTIOXIDANTS ANTIOXIDANTS ANTIOXIDANTS L/O/G/O Chatchawin PETCHLERT, Ph.D. Chatchawin PETCHLERT, Ph.D. Department of Biochemistry Department of Biochemistry Faculty of Science, Burapha University Faculty of Science, Burapha University

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ANTIOXIDANTS

L/O/G/O

Chatchawin PETCHLERT, Ph.D.Department of Biochemistry Faculty of Science, Burapha University

Antioxidant The chemical compounds which can delay the start or slow the rate of oxidative reaction in biological systems. A chemical compound or substance that inhibits oxidation. Any substance that, when present at low concentrations compared to those of an oxidizable substrate, significantly delays or inhibits oxidation of that substrate. (Halliwell and Gutteridge, 1989) Compounds that protect biological systems against the potentially harmful effects of processes or reactions that can cause excessive oxidations. (Krinsky, 1992)2

History of antioxidantsIn 1797, The rst recorded scientic observation on oxidation inhibitors came from Berthollet and later from Davy. Their theory was described as catalyst poisoning in oxidative reactors, and this was well before the free radical theory of peroxidation had been proposed. Duclaux (1886) rst demonstrated participation of atmospheric oxygen in oxidation of free fatty acids. Later, it was found that oxidation of unsaturated acylglycerols can generate rancid odors in sh oils (Tsujimoto, 1908). The earliest reported work on the use of antioxidants to retard lipid oxidation appeared in 1843, in which Deschamps showed that an ointment made of fresh lard containing gum benzoin (contains vanillin) or populin (from polar buds, contains saligenin and derivatives) did not become rancid as did the one with pure lard. the rst reports on antioxidants employed for food lipids were about using natural sources; in 1852, Wright (1852) reported that elm bark was effective in preserving butterfat and lard.

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Moureu and Dufraise (1922-1928) rst reported the possibility of using synthetic chemicals, especially phenolic compounds, to retard oxidative decomposition of food lipids. Their work provided the basic information leading to theories of lipid oxidation and antioxidants, which they referred to as inverse catalysis. Antioxidant synergism in food was rst reported by Olcott and Mattill (1936), and this was signicant in achieving oxidative stability in food by using a combination of antioxidants found in the unsaponiable fraction of oils. They described the antioxidants as inhibitors and grouped them into acid type, inhibitors, and hydroquinone and phenolics. Bailey (1937) and Scott (1965) have provided the history and a descriptive analysis of the development of antioxidants in their books, The Retardation of Chemical Reactions and Antioxidants and Autoxidation, respectively. Since the early 1960s, the understanding of autoxidation of unsaturated lipids and antioxidative mechanisms have advanced signicantly as a result of development of effective analytical tools. The last two decades have been very important to the antioxidant research. Around the world a revival is seen in studying the natural antioxidants in foods and the potential health benets of natural antioxidants in relation to prevention and therapy of oxidative stress and related diseases.

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General mechanisms to oxidative defense

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The antioxidant effectiveness is inuenced by the chemical properties of the compound including hydrogen bond energies, resonance delocalization, and susceptibility to autoxidation. The ability of the primary antioxidant molecule to donate a hydrogen atom to the free radical is the initial requirement. The ability of the free radical interceptor (scavenger) to donate a hydrogen atom to a free radical can be predicted from standard one-electron potentials (Table 1). According to Buettner (1993), each oxidizing species is capable of stealing an electron (or H atom) from any reduced species listed below it. That means when the standard one-electron reduction potential is concerned, the free radical scavengers that have reduction potential below peroxy radicals are capable of donating an H atom to peroxy radical and form a peroxide. The resulting antioxidant radical should be of low energy, ensuring the lesser possibility of catalyzing the oxidation of other molecules. The formed antioxidant radical is stabilized by delocalization of the unpaired electron around the phenol ring to form a stable resonance hybrid (Figure 3) and as a result attained low-energy levels.

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Type of antioxidants Biological antioxidants Enzymatic antioxidants Non-enzymatic antioxidants

Synthetic antioxidants

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Biological antioxidants - Enzymes Superoxide dismutase Catalase Se glutathione peroxidase Phospholipid hydroperoxide glutathione peroxidase Glutathione-S-transferase Glutathione reductase

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Superoxide dismutase (SOD, EC 1.15.1.1) 15. This enzyme was discovered via McCord and Fridovich, biologist and biochemist who studied the formation and metabolism of radicals and oxidants such as superoxide (O2.), hydrogen peroxide (H2O2), hydroxyl radical (OH). Superoxide dismutase catalyzes the following reaction: 2O2. + 2H+ O2 + H2O2 Several common forms of SOD exist: they are proteins cofactored with copper and zinc, or manganese, iron, or nickel.

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Superoxide dismutase (SOD, EC 1.15.1.1) 15.

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Superoxide dismutase (SOD, EC 1.15.1.1) 15.

In humans (as in all other mammals and most chordates), three forms of superoxide dismutase are present. SOD1 is located in the cytoplasm, SOD2 in the mitochondria, and SOD3 is extracellular. The first is a dimer (consists of two units), whereas the others are tetramers (four subunits). SOD1 and SOD3 contain copper and zinc, whereas SOD2 has manganese in its reactive centre. The genes are located on chromosomes 21, 6, and 4, respectively (21q22.1, 6q25.3 and 4p15.3-p15.1). 12

Catalase (CAT, EC 1.11.1.6) 11.Catalase is a common enzyme found in nearly all living organisms that are exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen Catalase has one of the highest oxygen. turnover numbers of all enzymes; one molecule of catalase can convert 40 million molecules of hydrogen peroxide to water and oxygen each second. Catalase was first noticed as a substance in 1818 when Louis Jacques Thnard, who discovered H2O2 (hydrogen peroxide), suggested that its breakdown is caused by a substance. In 1900, Oscar Loew was the first to give it the name catalase, and found its presence in many plants and animals. In 1937 catalase from beef liver was crystallized by James B. Sumner and the molecular weight was worked out in 1938. 2 H2O2 2 H2O + O2

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Catalase (CAT, EC 1.11.1.6) 11.Catalase is a tetramer of four polypeptide chains each over 500 amino chains, acids long. It contains four porphyrin heme (iron) groups that allow the enzyme to react with the hydrogen peroxide. peroxide The optimum pH for human catalase is approximately 7, and has a fairly broad maximum (the rate of reaction does not change appreciably at pHs between 6.8 and 7.5). The pH optimum for other catalases varies between 4 and 11 depending on the species. The optimum temperature also varies by species.

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S-Glutathione peroxidase (GPx, EC 1.11.1.9) 11.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 hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water water.An example reaction that glutathione peroxidase catalyzes is: 2GSH + H2O2 GSSG + 2H2O, where GSH represents reduced monomeric glutathione, and GSSG represents glutathione disulfide. Glutathione reductase then reduces the oxidized glutathione to complete the cycle: GSSG + NADPH + H+ 2 GSH + NADP+.15

S-Glutathione peroxidase (GPx, EC 1.11.1.9) 11.There are several isozymes encoded by different genes, which vary in celullar location and substrate specificity. Glutathione peroxidase 1 (GPx1) is the most (GPx1 abundant version, found in the cytoplasm of nearly all mammalian tissues, whose preferred substrate is hydrogen peroxide. Glutathione peroxidase 4 (GPx4 (GPx4) has a high preference for lipid hydroperoxides; it is expressed in nearly every mammalian cell, though at much lower levels. Glutathione peroxidase 2 is an intestinal and extracellular enzyme, while glutathione peroxidase 3 is extracellular, especially abundant in plasma. So far, eight different isoforms of glutathione peroxidase (GPx1-8) have been identified in humans. Mammalian GPx1, GPx2, GPx3, and GPx4 have been shown to be seleniumcontaining enzymes, whereas GPx6 is a selenoprotein in humans with cysteinecontaining homologues in rodents. GPx1, GPx2, and GPx3 are homotetrameric proteins, whereas GPx4 has a monomeric structure. As the integrity of the cellular and subcellular membranes depends heavily on glutathione peroxidase, the antioxidative protective system of glutathione peroxidase itself depends heavily on the presence of selenium.16

Phospholipid hydroperoxide glutathione peroxidase (PHGPx, EC 1.11.1.12) 11. 12)phospholipid-hydroperoxide glutathione peroxidase is an enzyme that catalyzes the chemical reaction: 2GSH + lipid hydroperoxide GSSG + lipid + 2H2O

Thus, the two substrates of this enzyme are glutathione and lipid hydroperoxide, whereas its 3 products are glutathione disulfide, lipid, and H2O. This enzyme belongs to the family of oxidoreductases, specifically those acting on a peroxide as acceptor (peroxidases). The systematic name of this enzyme class is glutathione:lipid-hydroperoxide oxidoreductase. Other names in common use include peroxidation-inhibiting protein, PHGPX, peroxidationinhibiting protein: peroxidase, glutathione, (phospholipid hydroperoxidereducing), phospholipid hydroperoxide glutathione peroxidase, hydroperoxide glutathione peroxidase, or glutathione peroxidase 4 (GPX4). This enzyme participates in glutathione metabolism.17

GlutathioneGlutathione-S-transferase (GST, EC 2.5.1.18) 18)GlutathioneGlutathione-S-transferase (GST) family are composed of many cytosolic, mitochondrial, and microsomal (now designated as MAPEG) proteins. GSTs are present in eukaryotes and in prokaryotes, where they catalyze a variety of reactions and accept endogenous and xenobiotic substrates.GSTs can constitute up to 10% of cytosolic protein in some mammalian organs. GSTs catalyze the conjugation of reduced glutathione via a sulfhydryl group to electrophilic centers on a wide variety of substrates. This activity detoxifies endogenous compounds such as peroxidised lipids, as well as breakdown of xenobiotics. GSTs may also bind toxins and function as transport proteins proteins, and, therefore, an early term for GSTs was ligandin. The mammalian GST superfamily consists of cytosolic dimeric isoenzymes of 4555 kDa size that have been assigned to at least six classes: Alpha, Mu, Pi, Theta, Zeta and Omega.

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GlutathioneGlutathione-S-transferase (GST, EC 2.5.1.18) 18)Mammalian cytosolic GSTs are dimeric bothsubunits being from the same class of GSTs, although not necessarily identical. The monomers are in the range of 2229 kDa. They are active over a wide variety of substrates with considerable overlap. Glutathione-S-transferases are considered to contribute to the phase II biotransformation of xenobiotics. Drugs, poisons, and other compounds are usually modified by the phase I and/or phase II mechanisms, and finally excreted from the body. GSTs contribute to this type of metabolism by conjugating these compounds (often electrophilic and somewhat lipophilic in nature) with reduced glutathione to facilitate dissolution in the aqueous cellular and extracellular media, and, from there, out of the body.19

Glutathione reductase (GR, EC 1.8.1.7)Glutathione reductase also known as GSR or GR, is an enzyme that reductase, reduces glutathione disulfide (GSSG) to the sulfhydryl form of GSH, which is an important cellular antioxidant.For every mole of oxidized glutathione (GSSG), one mole of NADPH is required to reduce GSSG to GSH. The enzyme forms a FAD bound homodimer The homodimer. glutathione reductase is conserved between all kingdoms. In bacteria, yeasts, and animals, one glutathione reductase gene is found; however, in plant genomes, two GR genes are encoded. Drosophila and Trypanosomes do not have any GR at all. In these organisms, glutathione reduction is performed by either the thioredoxin or the trypanothione system, respectively.

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Biological antioxidants Non-Enzymes Non Lipid-soluble antioxidants Tocopherols Carotenoids Quinones Bilirubin Polyphenols

Water-soluble antioxidants Ascorbic acid Uric acid Glutathione Cysteine Creatinine

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TocopherolsVitamin E exists in eight different forms, four tocopherols and four tocotrienols. All feature a chromanol ring, with a hydroxyl group that can donate a hydrogen atom to reduce free radicals and a hydrophobic side chain which allows for penetration into biological membranes. Both the tocopherols and tocotrienols occur in , and forms, determined , by the number and position of methyl groups on the chromanol ring.

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Vitamin E (Tocopherol) Tocopherol or Vitamin E is a fat-soluble vitamin in eight forms that is an important antioxidant. Vitamin E is often used in skin creams and lotions because it is believed to play a role in encouraging skin healing and reducing scarring after injuries such as burns. Natural vitamin E exists in 8 different forms, 4 tocopherols and 4 tocotrienols. All isomers have a chromanol ring, with a OH group, which can donate a H atom to reduce free radicals and a hydrophobic side chain, which allows for penetration into biological membrane. Sources: Vegetable oils Margarine and salad dressing Seeds and nuts23

TocopherolsTocopherols (or TCP) are a class of chemical compounds of which manyhave vitamin E activity. It is a series of organic compounds consisting of various methylated phenols. Because the vitamin activity was first identified in 1936 from a dietary fertility factor in rats, it was given the name "tocopherol" from the Greek words [birth], and , [to bear or carry] meaning in sum "to carry a pregnancy," with the ending "-ol" signifying its status as a chemical alcohol. alphaalpha-Tocopherol is the main source found in supplements and in the European diet, while gamma-tocopherol is the most common form in the American diet. The compound -tocopherol, a common form of tocopherol added to food products, is denoted by the E number E307. Tocotrienols, which are related compounds, also have vitamin E activity. All of these various derivatives with vitamin activity may correctly be referred to as "vitamin E." Tocopherols and tocotrienols are fat-soluble antioxidants but also seem to have many other functions in the body.

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Tocopherols (or TCP) have proven to be effective inhibitors of thepropagation step of lipid peroxidation. Each tocopherol molecule can react with 2 peroxyl radicals, as shown below: TCP + LOO TCP + LOOH TCP + LOO LOO-TCP The first product is the -tocopheroxyl radical, which is a resonance-stabilized, oxygen-centered radical. The TCP radical can react with another peroxyl radical to form a stable adduct, which has been isolated. TCP can scavenge hydroxyl radicals and quench singlet oxygen. It inhibits the cyclooxygenase pathway, reducing the formation of prostaglandins It also prevents formation of nitrosamines from nitrites. Transport and storage of TCP depend on the presence of Se, these 2 antioxidants are synergistic in their radical scavenging action and inhibition of neoplastic events.25

Vitamin E (Tocopherol) Vitamin E is a fat-soluble antioxidant. Its main action is to stop the chain reaction of free radicals producing more free radicals. It protects the vulnerable components of the cells and their membranes from destruction. It prevents the oxidation of the PUFAs. Vitamin E may also reduce the risk of heart disease by protecting LDL against oxidation. Vitamin E may also block the formation of nitrosamines, which are carcinogens formed in the stomach from nitrites consumed in the diet. It also may protect against the development of cancers by enhancing immune function.

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Free Radicals Cause Many Diseases, Antioxidants Quench Free Radicals

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The Actions of Free Radicals and Antioxidants (contd)

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CarotenoidsCarotenoidsare tetraterpenoid organic pigments that are naturally occurring in the chloroplasts and chromoplasts of plants and some other photosynthetic organisms like algae, some types of fungus some bacteria and at least one species of aphid. Carotenoids are generally not manufactured by species in the animal kingdom, although one species of aphid is known to have acquired the genes for synthesis of the carotenoid torulene from fungi, by the known phenomenon of horizontal gene transfer.

There are over 600 known carotenoids; they are split into two classes, xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons, and contain no oxygen). Carotenoids in general absorb blue light. They serve two key roles in plants and algae: they absorb light energy for use in photosynthesis, and they protect chlorophyll from photodamage. In humans, four carotenoids (beta-carotene, alpha-carotene, gamma-carotene, and betacryptoxanthin) have vitamin A activity (meaning they can be converted to retinal), and these and other carotenoids can also act as antioxidants. In the eye, certain other carotenoids (lutein and zeaxanthin) apparently act directly to absorb damaging blue and near-ultraviolet light, in order to protect the macula lutea.

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Biosynthetic pathways for the conversion of -carotene in different organisms. K, -carotene ketolase. H, -carotene hydroxylase.33

CarotenoidsCarotenoids are bleached when exposed to radicals such as those that arise during lipid peroxidation, which indicates that these pigments must also intercept active oxygen species. Their long, conjugated, double-bond systems make them excellent substrates for radical attack. Carotenoids are very rapidly bleached when exposed to the trichloromethylperoxyl radical (CCl3OO), which is generated during the pulse radiolysis of chloroform in the presence of oxygen. -Carotene is an effective antioxidant at various oxygen tensions. -Carotene is a better antioxidant at 15 torr (2% oxygen) than at 150 torr (20% oxygen), and that it acts as a prooxidant at 760 torr. -Carotene may react directly with the peroxyl radical (LOO) to form a resonance-stabilized, carbon-centered radical. CAR + LOO LOO-CAR Carotenoids should be able to quench at least 2 peroxyl radicals as does tocopherol LOO-CAR + LOO LOO-CAR-OOL 34

CarotenoidsHowever, the quenching ability need not stop here, but could continue with the formation of multiple resonance-stabilized, carbon-centered radicals on a single carotene molecule, followed by radical-radical quenching with the addition of another peroxyl radical. LOO-CAR-OOL + LOO (LOO)2-CAR-OOL + LOO (LOO)2-CAR-(OOL)2 Although, the interaction of -carotene and radicals is still unclear, some of the products formed from the reaction of peroxyl radicals with -carotene have recently been described. The products are primarily carbonyl derivatives of carotene, along with some epoxides.

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QuinonesA quinone is a class of organic compounds that are formally "derived from aromatic compounds [such as benzene or naphthalene] by conversion of an even number of CH= groups into C(=O) groups with any necessary rearrangement of double bonds," resulting in "a fully conjugated cyclic dione structure." The class includes derivatives of heterocyclic aromatic compounds. The prototypical member of the class are 1,4-benzoquinone or cyclohexadienedione, often called simply quinone (whence the name of the class). Other important examples are 1,2-benzoquinone (ortho-quinone), 1,4naphthoquinone and 9,10-anthraquinone. Quinones are often oxidized derivatives of aromatic compounds and are often readily made from reactive aromatic compounds with electron-donating substituents such as phenols and catechols which increase the nucleophilicity of the ring and contributes to the large redox potential needed to break aromaticity. (Quinones are conjugated but not aromatic).

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UbiquinoneCoenzyme Q10, also known as ubiquinone, ubidecarenone, coenzyme Q, and abbreviated at times to CoQ10, CoQ, Q10, or Q, is a 1,4-benzoquinone, where Q refers to the quinone chemical group, and 10 refers to the number of isoprenyl chemical subunits in its tail. This oil-soluble, vitamin-like substance is present in most eukaryotic cells, primarily in the mitochondria. It is a component of the electron transport chain and participates in aerobic cellular respiration, generating energy in the form of ATP. 95% of the human bodys energy is generated this way. Therefore, those organs with the highest energy requirements such as the heart, liver and kidney have the highest CoQ10 concentrations. There are three redox states of Coenzyme Q10: fully oxidized (ubiquinone), semiquinone (ubisemiquinone), and fully reduced (ubiquinol).

Coenzyme Q10 was first discovered by Prof. Fredrick L. Crane and colleagues at the University of WisconsinMadison Enzyme Institute in 1957. In 1958, its chemical structure was reported by Dr. Karl Folkers and coworkers at Merck; in 1968, Folkers became a Professor in the Chemistry Department at the University of Texas at Austin.

Prof. Fredrick L. Crane

38 Dr. Karl Folkers

UbiquinoneAbsorption CoQ10 is a crystalline powder that is insoluble in water. Absorption follows the same process as that of lipids and the uptake mechanism appears to be similar to that of vitamin E, another lipid-soluble nutrients. This process in the human body involves the secretion of pancreatic enzymes and bile into the small intestines that facilitate emulsification and micelle formation that is required for the absorption of lipophilic substances. Food intake (and the presence of lipids) stimulates bodily biliary excretion of bile acids and greatly enhances the absorption of CoQ10. Exogenous CoQ10 is absorbed from the small intestinal tract and is best absorbed if it is taken with a meal. Serum concentration of CoQ10 in fed condition is clearly higher than in fasting conditions. Metabolism Data on the metabolism of CoQ10 in animals and humans are limited. A study with 14C-labeled CoQ10 in rats showed most of the radioactivity in the liver 2 hours after oral administration, but it should be noted that CoQ9 is the predominant form of coenzyme Q in rats. It appears that CoQ10 is metabolized in all tissues, while a major route for its elimination is biliary and fecal excretion. After the withdrawal of CoQ10 supplementation, the levels return to normal within a few days, irrespective of the type of formulation used. Factors affecting CoQ10 levels Various factors reduce the concentration of CoQ10 in different organs; the following are known: Use of statins (HMG CoA reductase inhibitor) reduce CoQ10 levels. Aging, in individuals older than 20 years, reduces CoQ10 levels in internal organs. UV exposure reduces CoQ10 levels in the skin. UQ in its reduced form (UQH2) has long been known to inhibit lipid peroxidation. Although UQH2 might function by reducing the -tocopheroxy radical back to -tocopherol, UQH2 can also function in the absence of -tocopherol, presumably by acting directly on either peroxyl or alkoxyl radicals. 39

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The mode of action of ubiquinol as an inhibitor of lipid peroxidation and its relationship of that of vitamin E. Theavailable information is consistent with the conclusion that ubiquinol acts primarily by preventing the formation of lipid peroxyl radicals (LOO), whereas vitamin E is generally believed to exert its effect mainly by quenching these radicals. Ubiquinol may exhibit its preventive effect by reducing the initiating perferryl radical, with the formation of ubisemiquinone and H202. The possibility that ubiquinol may act by quenching L, yielding LH, has also been considered but seems unlikely in view of the very high rate at which L is known to react with 02, giving rise to LOO. In addition, ubiquinol may act by eliminating LOO, either directly or through the regeneration of vitamin E from the tocopheroxyl radical, a process that otherwise must rely on access to water-soluble antioxidants such as ascorbate.

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Bilirubin(formerly referred to as hematoidin is the yellow breakdown product of normal heme hematoidin) catabolism. Heme is found in hemoglobin, a principal component of red blood cells. Bilirubin is excreted in bile and urine, and elevated levels may indicate certain diseases. It is responsible for the yellow color of bruises, urine, and the yellow discoloration in jaundice. Bilirubin consists of an open chain of four pyrrole-like rings (tetrapyrrole). In heme, by contrast, these four rings are connected into a larger ring, called a porphyrin ring. Bilirubin is very similar to the pigment phycobilin used by certain algae to capture light energy, and to the pigment phytochrome used by plants to sense light. All of these contain an open chain of four pyrrolic rings. Like these other pigments, some of the double-bonds in bilirubin isomerize when exposed to light. This is used in the phototherapy of jaundiced newborns: the E,E-isomer of bilirubin formed upon light exposure is more soluble than the unilluminated Z,Z-isomer. The naturally occurring isomer is the Z,Z-isomer. This compound can effectively inhibit lipid peroxidation in both homogeneous solution and liposomes. Bilirubin is almost as effective an antioxidant as -tocopherol in the latter system.

Bilirubin

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Polyphenols

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BioavailabilityGallic acid and isoflavones are the most well-absorbed polyphenols, followed by catechins, flavanones, and quercetin glucosides, but with different kinetics. The least well-absorbed polyphenols are the proanthocyanidins, galloylated tea catechins, and anthocyanins. Compared with the effects of polyphenols in vitro, the effects in vivo, although the subject of ongoing research, are limited and vague. The reasons for this are 1) the absence of validated in vivo biomarkers, especially for inflammation or carcinogenesis; 2) long-term studies failing to demonstrate effects with a mechanism of action, specificity or efficacy; and 3) invalid applications of high, unphysiological test concentrations in the in vitro studies, which are subsequently irrelevant for the design of in vivo experiments. To overcome this problem of bioavailability, an alternative could be liposome encapsulation. For instance, liposomes have been prepared encapsulating baicalin and tested in mice. In rats, polyphenols absorbed in the small intestine may be bound in protein-polyphenol complexes modified by intestinal microflora enzymes, allowing derivative compounds formed by ring-fission to be better absorbed.

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Flavonoids and gallocatechins, which are plant polyhydroxybenzenes, are fair to excellent electron donors. One-electron reduction potentials of avonoid radicals at pH 7 (TABLE 1), which are equal to the oxidation potentials of parent avonoids, range from 0.33 V for quercetin to 0.72 V for hesperidin. On the basis of lower reduction potentials, all avonoids may fully restitute any DNA base (E7 = 1.29 V for the most oxidizable base, guanosine) or protein amino acid radical (E7 = 0.9 V for the most vulnerable, tyrosine). Alkyl peroxyl radicals with high E7 = 1.06 V are inactivated to hydroperoxides by electron transfer from lower oxidation potential avonoids. Some avonoids, epigallocatechin (E7 = 0.42 V), epigallocatechin gallate (E7 = 0.43 V) and quercetin (E7 = 0.33 V), may repair vitamin E radical (E7 = 0.48 V), thus maintaining desirable concentration of this important physiological antioxidant. 47

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Flavonoids and gallocatechins are very efficient scavengers of the superoxide radical (TABLE 2). The reactivities of avonoids and gallocatechins with the superoxide radical are high, as expected from their excellent electron donating abilities. The highest rate constants were measured for theaavins, k theaavins, 7 1 1, ~10 M s very probably because of favorable charge separation within the benzocycloheptenone moiety in these black tea derivatives. However, it should be emphasized that even the poorest electron donor, galangin, with the inactivation rate constant of k = 8.8 102 M1 s1, still efficiently scavenges superoxide radical. 49

. Functional groups important to the antioxidant activity of catechin monomers, dimers (theaflavins) and polymers (thearubigins): example, epicatechin gallate (ECG).

Frei B , Higdon J V J. Nutr. 2003;133:3275S-3284S

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Structures of catechins, theaflavins, and caffeine

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The mechanism of the inactivation of the superoxide radical by avonoids results in the formation of avonoid phenoxyl radical and hydrogen peroxide. The rate constants of the inactivation of the superoxide radical by avonoids are low, k ~102-107 M1 s1, in comparison with typical radical reactions (k ~109 M1 s1). However, this is enough for very efficient in vivo scavenging of the superoxide radical. Given that the reactivities of the superoxide with the biological molecules are of the order of ~1 s1 or less, 26 even at ~106 M concentrations avonoids would favorably compete for the superoxide (k = 106 M107 M1 s1 = 10 s1).

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Singlet oxygen, 1O2, is a byproduct of biological oxidation reactions and a component of photochemical smog. It may oxidize various biomolecules causing cell death and mutations. Flavonoids are efficient scavengers of singlet oxygen (TABLE 3), very probably by a chemical reaction. The rate constants of the reactions of 1O2 with avonoids, k = 104 108 M1 s1 are comparable to that of vitamin E, k = 5108 M1s1. Higher rate constants were measured for avonoids with lower oxidation potentials, e.g. k (1O2 + epigallocatechin gallate) = 2.2108 M1s1, indicating that a polar transition state is involved in the quenching.54

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Ascorbic acidAscorbic acidis a sugar acid with antioxidant properties. Its appearance is white to light-yellow crystals or powder, and it is water-soluble. One form of ascorbic acid is commonly known as vitamin C. The name is derived from a- (meaning "no") and scorbutus (scurvy), the disease caused by a deficiency of vitamin C. In 1937, the Nobel Prize for chemistry was awarded to Walter Haworth for his work in determining the structure of ascorbic acid (shared with Paul Karrer, who received his award for work on vitamins), and the prize for Physiology or Medicine that year went to Albert Szent-Gyrgyi for his studies of the biological functions of L-ascorbic acid. At the time of its discovery in the 1920s, it was called hexuronic acid by some researchers. It is an enzyme cofactor in tyrosine oxidation. It creates volatile compounds when mixed with glucose and amino acids.

Albert Szent-Gyrgyi 56

Ascorbate

usually acts as an antioxidant by being available for energetically favorable oxidation. Many oxidants (typically, reactive oxygen species) such as the hydroxyl radical (formed from hydrogen peroxide), contain an unpaired electron, and thus are highly reactive. This can be highly damaging to humans and plants at the molecular level due to their possible interaction with nucleic acids, proteins, and lipids. These free radical interactions are damaging since they result in a whole chain of free radical reactions. More specifically, the interaction of an initial free radical (often reactive oxygen species) with another molecule changes that molecule itself into a free radical, which then reacts with other molecules, also turning them into free radicals. Ascorbate can terminate these chained radical reactions by serving as a stable (electron + proton) donor in interactions with free radicals, being converted into the radical ion called "semidehydroascorbate" and then dehydroascorbate. The net reaction is RO + C6H7O6- ROH + C6H6O6-. The oxidized forms of ascorbate are relatively unreactive, and do not cause cellular damage. They can be converted back to ascorbate by cellular enzymes. However, being a good electron donor, excess ascorbate in the presence of free metal ions can not only promote, but also initiate free radical reactions, thus making it a potentially dangerous prooxidative compound in certain metabolic contexts.

Reduced form

Oxidized form

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Uric acidUric acid (or urate)is a heterocyclic compound of carbon, nitrogen, oxygen, and hydrogen with the formula C5H4N4O3. Uric acid is created when the body breaks down purine nucleotides. Uric acid may be a marker of oxidative stress, and may have a potential therapeutic role as an antioxidant. On the other hand, like other strong reducing substances such as ascorbate, uric acid can also act as a prooxidant, particularly at elevated levels. Thus, it is unclear whether elevated levels of uric acid in diseases associated with oxidative stress such as stroke and atherosclerosis are a protective response or a primary cause. For example, some researchers propose hyperuricemia-induced oxidative stress is a cause of metabolic syndrome. On the other hand, plasma uric acid levels correlate with longevity in primates and other mammals. This is presumably a function of urate's antioxidant properties.

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GlutathioneGlutathione (GSH)is a tripeptide that contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side-chain. It is an antioxidant, preventing damage to important cellular components caused by reactive oxygen species such as free radicals and peroxides. Thiol groups are reducing agents, existing at a concentration of approximately 5 mM in animal cells. Glutathione reduces disulfide bonds formed within cytoplasmic proteins to cysteines by serving as an electron donor In the process, glutathione is converted to its oxidized form glutathione disulfide (GSSG). donor. Glutathione is found almost exclusively in its reduced form, since the enzyme that reverts it from its oxidized form, glutathione reductase, is constitutively active and inducible upon oxidative stress. In fact, the ratio of reduced glutathione to oxidized glutathione within cells is often used as a measure of cellular toxicity.

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Glutathione

exists in reduced (GSH) and oxidized (GSSG) states. In the reduced state, the thiol group of cysteine is able to donate a reducing equivalent (H++ e-) to other unstable molecules, such as reactive oxygen species. In donating an electron, glutathione itself becomes reactive, but readily reacts with another reactive glutathione to form glutathione disulfide (GSSG). Such a reaction is possible due to the relatively high concentration of glutathione in cells (up to 5 mM in the liver). GSH can be regenerated from GSSG by the enzyme glutathione reductase. In healthy cells and tissue, more than 90% of the total glutathione pool is in the reduced form (GSH) and less than 10% exists in the disulfide form (GSSG). An increased GSSG-to-GSH ratio is considered indicative of oxidative stress.

Glutathione has multiple functions: It is the major endogenous antioxidant produced by the cells, participating directly in the neutralization of free radicals and reactive oxygen compounds, as well as maintaining exogenous antioxidants such as vitamins C and E in their reduced (active) forms. Regulation of the nitric oxide cycle, which is critical for life but can be problematic if unregulated Through direct conjugation, it detoxifies many xenobiotics (foreign compounds) and carcinogens, both organic and inorganic. This includes heavy metals such as mercury, lead, and arsenic. It is essential for the immune system to exert its full potential, e.g., (1) modulating antigen presentation to lymphocytes, thereby influencing cytokine production and type of response (cellular or humoral) that develops, (2) enhancing proliferation of lymphocytes, thereby increasing magnitude of response, (3) enhancing killing activity of cytotoxic T cells and NK cells, and (4) regulating apoptosis, thereby maintaining control of the immune response. It plays a fundamental role in numerous metabolic and biochemical reactions such as DNA synthesis and repair, protein synthesis, prostaglandin synthesis, amino acid transport, and enzyme activation. Thus, every system in the body can be affected by the state of the glutathione system, especially the immune system, the nervous system, the gastrointestinal system and the lungs.

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A lower E indicates that less energy is required for hydrogen or electron donation and is one factor in determining antioxidant activity. Tea catechins and theaavins have E values comparable to that of tocopherol (vitamin E), but higher than ascorbate (vitamin C) (Table 1), which is a superior hydrogen donor (antioxidant) to tea polyphenols. Even with very high intakes of tea or tea extracts, plasma and intracellular concentrations of tea catechins and polyphenols in humans are likely to be 100 to 1000 times lower than those of other physiological antioxidants, such as ascorbate, urate and glutathione (Table 2). Thus, the relative importance of tea catechins and polyphenols as radical and oxidant scavengers in vivo may be minor, based on their reduction potentials and concentrations achieved in plasma and tissues. 61

Synthetic antioxidantsTrolox BHT BHA TBHQ Propyl gallate

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TroloxTrolox is Hoffman-LaRoche's trade name for 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid, a water-soluble derivative of vitamin E. It is an antioxidant, like vitamin E, and is used in biological or biochemical applications to reduce oxidative stress or damage. Trolox equivalent antioxidant capacity (TEAC) is a measurement of antioxidant strength based on Trolox, measured in units called Trolox Equivalents (TE), e.g. micromolTE/100 g. Due to the difficulties in measuring individual antioxidant components of a complex mixture (such as blueberries or tomatoes), Trolox equivalency is used as a benchmark for the antioxidant capacity of such mixture. Trolox equivalency is most often measured using the ABTS decolorization assay. Other measures include oxygen radical absorbance capacity (ORAC) and ferric reducing ability of plasma (FRAP).

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Butylated hydroxytoluene; BHTButylated hydroxytoluene (BHT), also known as butylhydroxytoluene, is a lipophilic (fat-soluble) organic compound that is primarily used as an antioxidant food additive (E number E321) as well as an antioxidant additive in cosmetics, pharmaceuticals, jet fuels, rubber, petroleum products, electrical transformer oil, and embalming fluid.The species behaves as a synthetic analogue of vitamin E, primarily acting as a terminating agent that suppresses autoxidation, a process whereby unsaturated (usually) organic compounds are attacked by atmospheric oxygen. BHT stops this autocatalytic reaction by converting peroxy radicals to hydroperoxides. It effects this function by donating a hydrogen atom:

RO2. + ArOH ROOH + ArO. RO2. + ArO. nonradical productswhere R is alkyl or aryl, and where ArOH is BHT or related phenolic antioxidants. One can see that each BHT consumes two peroxy radicals.

IUPAC name 2,6-bis(1,1-dimethylethyl)-4-methylphenol Other names 2,6-di-tert-butyl-4-methylphenol 2,6-di-tert-butyl-p-cresol (DBPC) 3,5-di-tert-butyl-4-hydroxytoluene BHT

Microscopic Image of BHT (White Crystalline Powder) Showing Individual Crystals (Courtesy: Jack Reed, Department of Entomology, Mississippi State University)

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Butylated hydroxytoluene (BHT) is a phenolic antioxidant. It has been shown to inhibit lipid peroxidation. It causes lung injury and promotes tumors in mice, but this may be due to a metabolite of BHT, 6- tert-butyl-2-[2-(2-hydroxymethyl)-propyl]-4-methylphenol. Metabolites of BHT have also been reported to induce DNA strand breaks and internucleosomal DNA fragmentation (a characteristic of apoptosis) in cultured cells. In rats, a single intraperitoneal injection of BHT (60 mg/kg body mass) results in a significant increase in nuclear DNA methyl transferase activity in the liver, kidneys, heart, spleen, brain and lungs. Incubation of alveolar macrophages with BHT significantly reduced the level of TNF- which may explain the mechanism by which this antioxidant reduces inflammation. Preincubation of aspirin-treated platelets with BHT inhibits the secretion, aggregation, and protein phosphorylation induced by protein kinase C activators. BHT was also found to inhibit the initiation of hepatocarcinogenesis by aflatoxin B1.

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Butylated hydroxyanisole; BHAButylated hydroxyanisole (BHA) is an antioxidant consisting of a mixture of two isomeric organic compounds, 2-tert-butyl-4hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. It is prepared from 4-methoxyphenol and isobutylene. It is a waxy solid used as a food additive with the E number E320. The primary use for BHA is as an antioxidant and preservative in food, food packaging, animal feed, cosmetics, rubber, and petroleum products. BHA also is commonly used in medicines, such as isotretinoin, lovastatin, and simvastatin, among others. Since 1947, BHA has been added to edible fats and fat-containing foods for its antioxidant properties as it prevents food from becoming rancid and developing off-flavors. Like butylated hydroxytoluene (BHT), the conjugated aromatic ring of BHA is able to stabilize free radicals, sequestering them. By acting as free radical scavengers, further free radical reactions are prevented.

IUPAC name 2-tert-Butyl-4-hydroxyanisole and 3-tert-butyl-4hydroxyanisole (mixture) Other names BOA tert-Butyl-4-hydroxyanisole (1,1-Dimethylethyl)-4-methoxyphenol tert-Butyl-4-methoxyphenol Antioxyne B

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Tertiary-butylhydroquinone; Tertiary-butylhydroquinone; TBHQtert-Butylhydroquinone (TBHQ, tertiary butylhydroquinone) is an aromatic organic compound which is a type of phenol. It is a derivative of hydroquinone, substituted with tert-butyl group. TBHQ is a highly effective antioxidant. In foods, it is used as a preservative for unsaturated vegetable oils and many edible animal fats. It does not cause discoloration even in the presence of iron, and does not change flavor or odor of the material to which it is added. It can be combined with other preservatives such as butylated hydroxyanisole (BHA). As a food additive, its E number is E319. It is added to a wide range of foods, with the highest limit (1000 mg/kg) permitted for frozen fish and fish products. Its primary advantage is enhancing storage life. It is used industrially as a stabilizer to inhibit autopolymerization of organic peroxides. It is also used as a corrosion inhibitor in biodiesel. In perfumery, it is used as a fixative to lower the evaporation rate and improve stability. It is also added to varnishes, lacquers, resins, and oil field additives.

IUPAC name 2-(1,1-Dimethylethyl)-1,4benzenediol Other name TBHQ(i)

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Both the European Food Safety Authority (EFSA) and the United States Food and Drug Administration (FDA) have evaluated TBHQ and determined that it is safe to consume at the concentration allowed in foods. The FDA sets an upper limit of 0.02% of the oil or fat content in foods. At higher doses, it has some negative health effects on lab animals, such as producing precursors to stomach tumors and damage to DNA. A number of studies have shown that prolonged exposure to high doses of TBHQ may be carcinogenic, especially for stomach tumors. Other studies, however, have shown opposite effects including inhibition against HCAinduced carcinogenesis (by depression of metabolic activation) for TBHQ and other phenolic antioxidants (TBHQ was one of several, and not the most potent). The EFSA considers TBHQ to be non-carcinogenic. A review of scientific literature concerning the toxicity of TBHQ determined that there is a wide margin of safety between the levels of intake by humans and the doses that produce adverse effects in animal studies. However, it should also be noted that the review cited in the last sentence took place some time ago (1986).

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Propyl gallatePropyl gallate, or propyl 3,4,5-trihydroxybenzoate is an ester formed by the condensation of gallic acid and propanol. Since 1948, this antioxidant has been added to foods containing oils and fats to prevent oxidation. As a food additive, it is used under the E number E310. Manufacturers add propyl gallate to food products, including vegetable oil, mayonnaise, meat, soup, dried milk, spices, candy, snack foods, vitamins and chewing gum. Propyl gallate also is a common additive to pet food. The personal care industry adds propyl gallate to perfume, soaps, lotions and moisturizers, lipstick and other make-up, hair care products, bath products, sunscreen, and toothpaste. In addition, the substance is added to adhesives and lubricants. An antioxidant that exhibits antimicrobial activity. Propyl gallate has been reported to be an effective antioxidant-based hepatoprotector, both in vitro and in vivo. It has also been shown to prevent neuronal apoptosis and block the death of neurons exposed to FeSO4/GA as well as partially protect endothelial cells against TNF-induced apoptosis. However, propyl gallate induced single strand breaks in DNA at concentrations higher than 0.25 M when it was combined with copper concentrations at 5 M and above. Another report, however, found that incubation of isolated rat hepatocytes with propyl gallate at concentrations of >1 mM induced cell killing, whereas, a lower concentration of propyl gallate > 0.5 mM resulted in a ladder formation of soluble low-molecular weight DNA fragments, characteristic of apoptosis. 70

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