ANTIOXIDANTS THAT ARE NATURALLY FOUND IN FOODS

 

CAROTENOIDS

 

VITAMIN  E

 

PHENOLIC COMPOUNDS

 

ANTIOXIDANT ENZYMES

 

OTHER ANTIOXIDANTS

 

VITAMIN C

 

SELENIUM

 

 

CAROTENOIDS

 

 

            Carotenoids are among the most common natural pigments, and more than 600different compounds have been characterized until now, with β-carotene as the most prominent. Carotenoids are responsible for many of the red, orange, and yellow hues of plant leaves, fruits, and .owers, as well as the colors of some birds, insects, fish, and crustaceans. Only plants, bacteria, fungi, and algae can synthesize carotenoids, but many animals incorporate them from their diet. Carotenoids serve as antioxidants in animals, and the socalled provitamin A carotenoids are used as a source for vitamin A. Carotenoids attracted attention, because a number of epidemiological studies have revealed that an increased consumption of a diet rich in carotenoids is correlated with a diminished risk for several degenerative disorders, including various types of cancer, cardiovascular or ophthalmological diseases. The preventive effects have been associated with their antioxidant activity, protecting cells and tissues from oxidative damage. Carotenoids also influence cellular signaling and may trigger redox sensitive regulatory pathways.

 

            Structures of carotenoids

           

The unique structure of carotenoids determines their potential biological functions and actions. Most carotenoids can be derived from a 40-carbon basal structure, which includes a system of conjugated double bonds. The central chain may carry cyclic end groups which can be substituted with oxygen-containing functional groups. Based on their composition, carotenoids are divided in two classes, carotenes containing only carbon and hydrogen atoms, and oxocarotenoids (xanthophylls) which carry at least one oxygen atom.

 

            The pattern of conjugated double bonds in the polyene backbone of carotenoids determines their light absorbing properties and influences the antioxidant activity of carotenoids. According to the number of double bonds, several cis / trans (E/Z) configurations are possible for a given molecule. Carotenoids tend to isomerize and form a mixture of mono- and poly-cis-isomers in addition to the all-trans form. Generally, the all-trans form is predominant in nature.

 

            Carotenoids are lipophilic molecules which tend to accumulate in lipophilic compartments like membranes or lipoproteins. The lipophilicity of these compounds also influences their absorption, transport and excretion in the organism.

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             Antioxidant activity––singlet oxygen quenching, peroxyl radical scavenging

           

As an attribute to aerobic life the human organism is exposed to a variety of different prooxidants capable to damage biologically relevant molecules, such as DNA, proteins, carbohydrates, and lipids. Among the various defense strategies, carotenoids are most likely involved in the scavenging of two of the reactive oxygen species, singlet molecular oxygen (O2), and peroxyl radicals. Further, they are e.ective deactivators of electronically excited sensitizer molecules which are involved in the generation of radicals and singlet oxygen.

           

The interaction of carotenoids with O2 depends largely on physical quenching which involves direct energy transfer between both molecules. The energy of singlet molecular oxygen is transferred to the carotenoid molecule to yield ground state oxygen and a triplet excited carotene. Instead of further chemical reactions, the carotenoid returns to ground state dissipating its energy by interaction with the surrounding solvent. In contrast to physical quenching, chemical reactions between the excited oxygen and carotenoids is of minor importance, contributing less than 0.05% to the total quenching rate. Since the carotenoids remain intact during physical quenching of O2 or excited sensitizers, they can be reused several fold in such quenching cycles. Among the various carotenoids, xanthophylls as well ascarotenes proved to be efficient quenchers of singlet oxygen interacting with reaction rates that approach difusion control .

           

The efficacy of carotenoids for physical quenching is related to the number of conjugated double bonds present in the molecule which determines their lowest triplet energy level.β-Carotene and structurally related carotenoids have triplet energy levels close to that of O2 enabling energy transfer. In addition to β-carotene,also zeaxanthin, cryptoxanthin, and a-carotene, all of which are detected in human serum and tissues, belong to the group of highly active quenchers of O2. The most efficient carotenoid is the open ring carotenoid lycopene, which contributes up to 30% to total carotenoids in humans.

           

For clinical use, β-carotene is applied to ameliorate the secondary effects of the hereditary photosensitivity disorder erythropoietic protoporphyria. It is suggested that the carotenoid intercepts the reaction sequence that leads to the formation of singlet oxygen; the latter is thought to be the damaging agent responsible for the skin lesions observed in this disease.

           

Among the various radicals which are formed under oxidative conditions in the organism, carotenoids most eficiently react with peroxyl radicals. They are generated in the process of lipid peroxidation, and scavenging of this species interrupts the reaction sequence which .nally leads to damage in lipophilic compartments. Due to their lipophilicity and specific property to scavenge peroxyl radicals, carotenoids are thought to play an important role in the protection of cellular membranes and lipoproteins against oxidative damage. The antioxidant activity of carotenoids regarding the deactivation of peroxyl radicals likely depends on

the formation of radical adducts forming a resonance stabilized carbon-centered radical.

           

A variety of products have been detected subsequent to oxidation of carotenoids, including carotenoid epoxides and apo-carotenoids of di.erent chain length. It should be noted that these compounds might possess biological activities and interfere with signaling pathways when present in unphysiologically high amounts.

           

The antioxidant activity of carotenoids depends on the oxygen tension present in the system. At low partial pressures of oxygen such as those found in most tissues under physiological conditions, β-carotene was found to inhibit the oxidation. In contrast, the initial antioxidant activity of β-carotene is followed by a prooxidant action at high oxygen tension. It has been suggested that prooxidant e.ects of b-carotene may be related to adverse efects observed under the supplementation of high doses of β-carotene.

 

 

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     Pro-oxidant chemistry of Carotenoids

 

            The antioxidant potency of these compounds in a variety of different in vitro assays has recently been shown to correlate quantitatively with their computer calculated ionization potential, thus providing quantitative structure activity relationships.

           

However, experimental studies with an important member of this series of antioxidant molecules, i.e. β-carotene, at present provide perhaps the best example of unexpected health risks related to the use of an antioxidant as beneficial health supplement. Observational epidemiologic studies indicate that diets high in carotenoids rich fruits and vegetables as well as increased serum levels of β-carotene are associated with a decreased risk of lung cancer. Based on these observations large human intervention trials with heavy smokers receiving β-carotene supplements were undertaken. The Finnish intervention study was the first to report increased, instead of the expected reduced levels of lung cancer incidence in the population of heavy smokers receiving β-carotene supplements for several years. As a result, another large intervention trial stopped the active intervention 21 months earlier than planned. Interim efficacy results of this study showed the same tendency of increased risk of lung cancer and also increased overall mortality in the group receiving the β-carotene supplements versus placebo. Similar to the effects of β-carotene on lung cancer risk in heavy smokers, this study also reported an increased lung cancer risk due to β-carotene supplementation in asbestos-exposed workers.

           

The mechanism by which β-carotene increases lung cancer risks in both heavy smokers and asbestos workers is at present unclear, although some hypotheses and initial results have been reported. One possible explanation suggests the formation of reactive oxidative β- carotene metabolites. Especially the high oxygen pressure in the lungs may favor pro-oxidative metabolism of β-carotene and the interaction between reactive oxygen species, derived from tobacco smoke or induced in the lung upon asbestos exposure, may result in  β-carotene (auto)oxidation leading to toxic β-carotene metabolites. Also induction of cytochrome P450 activity may result in formation of increased β-carotene oxidation. A more extended hypothesis explaining how increased β-carotene oxidation may result in the increased lung tumor risks is summarized in Fig 5. The hypothesis suggests that oxidative β-carotene metabolites structurally resemble retinal and affect retinoid signalling resulting in reduced retinoid levels and suppression of RAR β gene expression, the latter representing a tumor suppress or gene. Furthermore, the whole process also induces increased expression of c-jun and c-fos genes resulting in higher levels of activator protein-1 (AP-1). Increased expression of c-Jun and c-Fos proteins has been reported for several mitogenic stimuli and tumor promoting agents, and is indeed observed in tobacco-smoke exposed ferrets supplemented with highdose β-carotene.

 

 

           

             

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Of importance to notice is that these pro-oxidative tumor promoting effects of     β-carotene are especially observed upon high dose supplementation in heavy smokers. β-carotene does not exert this tumor risk enhancing effect in former smokers. These observations support the hypothesis that a direct interaction between cigarette smoke and β- carotene is required for the tumor promoting effects of β-carotene in heavy smokers corroborating a role for oxidative β-carotene metabolites.In asbestos workers this β-carotene oxidation may be stimulated by the inflammatory process known to be induced in asbestos-exposed lungs, since inflammatory cells isolated from non-smokers with asbestosis are known to release significantly increased amounts of reactive oxygen species compared to cells recovered from control individuals.

 

            Carotenoids are efficient antioxidants protecting plants against oxidative damage. They are also part of the antioxidant defence system in animals and humans. Due to their unique structure it can be suggested that they possess specific tasks in the antioxidant network such as protecting lipophilic compartments or scavenging reactive species generated in photooxidative processes. They may further act as light filters and prevent oxidative stress by diminishing light exposure. The possible role of carotenoids as prooxidants and the implication of their prooxidant activity in adverse reactions remains to be elucidated.

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Beta Carotene

Source and Nature:

            Carotenoids are pigmented micronutrients present in fruits and vegetables.

 

            Carotenoids are precursors of vitamin A and have antioxidant effects. While over 600 carotenoids have been found in the food supply, the most common forms are alpha-carotene, beta-carotene, lycopene, crocetin, canthaxanthin, and fucoxanthin. Beta-carotene is the most widely studied. It is composed of two molecules of vitamin A (retinol) joined together. Dietary beta-carotene is converted to retinol at the level of the intestinal mucosa.

Mechanisms of Action:

            The antioxidant function of beta-carotene is due to its ability to quench singlet oxygen, scavenge free radicals and protect the cell membrane lipids from the harmful effects of oxidative degradation. The quenching involves a physical reaction in which the energy of the excited oxygen is transferred to the carotenoid, forming an excited state molecule. Quenching of singlet oxygen is the basis for beta-carotene's well known therapeutic efficacy in erythropoietic protoporphyria (a photosensitivity disorder). The ability of beta-carotene and other carotenoids to quench excited oxygen, however, is limited, because the carotenoid itself can be oxidized during the process (autoxidation). Burton and Ingold  and others have shown that beta-carotene autoxidation in vitro is dose-dependent and dependent upon oxygen concentrations. At higher concentrations, it may function as a pro-oxidant and can activate proteases.

            In addition to singlet oxygen, carotenoids are also thought to quench other oxygen free radicals. It is also suggested that beta carotene might react directly with the peroxyl radical at low oxygen tensions; this may provide some synergism to vitamin E which reacts with peroxyl radicals at higher oxygen tensions.

Figure.6.

b-carotene (CAR) + LOO˙               LOO-CAR˙  

LOO-CAR˙+ LOO˙                  LOO-CAR-OOL

           

        Carotenoids also have been reported to have a number of other biologic actions, including immuno-enhancement; inhibition of mutagenesis and transformation; and regression of premalignant lesions.

 

Chemical structure of β-carotene:

 

 

 

 Figure.7.Chemical structure of beta carotene

 

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Toxicity of the β-carotene:

 

            The Scandanavian Alpha-tocopherol, Beta-Carotene Cancer Prevention Study (ATBC) showed that a suppletion with 20 mg β-carotene resulted in an 18% increase in lung cancer incidence. In the classical toxicological approach the ‘no-observed effect level’ (NOEL) in a lab animal is divided by a safety factor of 10 to find the NOEL in human. A second factor of 10 is used to account for interindividual differences. The 20 mg β-carotene as used in the ATBC trial is apparently not a safe dose in human. If we nevertheless (viz.it is not the NOEL) divide this dose by 10 for the interindividual differences, this would result in an assumed safe dose of maximally 2 mg per day. This is below the average daily intake of 3 mg in the Netherlands. The classical toxicological approach is apparently not valid here. We have to accept that safety factors for food components might be much smaller than we approbate for drugs. Plasma/serum levels (or even better tissue levels) of the antioxidant should guide the decisions on safety of nutraceuticals. In exceeding normal plasma/serum β-carotene levels (in the ATBCstudy 17.5 times) additional research should be required.

           

Health effects of the β-carotene:

 

            Surveys suggest an association between diets rich in beta-carotene and vitamin A and a lower risk of some types of cancer

 

             There is evidence that a higher intake of green and yellow vegetables or other food sources of beta-carotene and/or vitamin A may decrease the risk of lung cancer. However, a number of studies that tested the role of beta-carotene supplements in cancer prevention did not find it to be protective. In a study of 29,000 men, incidence of lung cancer was greater in the group of smokers who took a daily supplement of beta-carotene.

            The Carotene and Retinol Efficacy Trial, a lung cancer chemoprevention trial that provided randomized subjects with supplements of beta-carotene and vitamin A, was stopped after researchers discovered that subjects receiving beta-carotene had a 46% higher risk of dying from lung cancer than those who did not receive beta-carotene . The Institute of Medicine (IOM) states that “beta-carotene supplements are not advisable for the general population,” although they also state that this advice “does not pertain to the possible use of supplemental beta-carotene as a provitamin A source for the prevention of vitamin A deficiency in populations with inadequate vitamin A nutriture”.

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Lycopene:

 

 

Chemical structure:

 

           Figure.8. Chemical structure of lycopene

 

     Lycopene is the pigment principally responsible for the characteristic deep-red color of ripe tomato fruits and tomato products. It has attracted attention due to its biological and physicochemical properties, especially related to its effects as a natural antioxidant.            Although it has no provitamin A activity, lycopene does exhibit a physical quenching rate constant with singlet oxygen almost twice as high as that of -carotene. This makes its presence in the diet of considerable interest. Increasing clinical evidence supports the role of lycopene as a micronutrient with important health benefits, because it appears to provide protection against a broad range of epithelial cancers. Tomatoes and related tomato products are the major source of lycopene compounds, and are also considered an important source of carotenoids in the human diet.

             Undesirable degradation of lycopene not only affects the sensory quality of the final products, but also the health benefit of tomato-based foods for the human body. Lycopene in fresh tomato fruits occurs essentially in the all-trans configuration. The main causes of tomato lycopene degradation during processing are isomerization and oxidation. Isomerization converts all-trans isomers to cis-isomers due to additional energy input and results in an unstable, energy-rich station. Determination of the degree of lycopene isomerization during processing would provide a measure of the potential health benefits of tomato-based  foods. Thermal processing (bleaching, retorting, and freezing processes) generally cause some loss of lycopene in tomato-based  foods. Heat induces isomerization of the all-trans to cis forms. The cis-isomers increase with temperature and processing time. In general, dehydrated and powdered tomatoes have poor lycopene stability unless carefully processed and promptly placed in a hermetically sealed and inert atmosphere for storage. A significant increase in the cis-isomers with a simultaneous decrease in the all-trans isomers can be observed in the dehydrated tomato samples using the different dehydration methods. Frozen foods and heat-sterilized foods exhibit excellent lycopene stability throughout their normal temperature storage shelf life. Lycopene bioavailability (absorption) can be influenced by many factors. The bioavailability of cis-isomers in food is higher than that of all trans isomers. Lycopene bioavailability in processed tomato products is higher than in unprocessed fresh tomatoes. The composition and structure of the food also have an impact on the bioavailability of lycopene and may affect the release of lycopene from the tomato tissue matrix. Food processing may improve lycopene bioavailability by breaking down cell walls, which weakens the bonding forces between lycopene and tissue matrix, thus making lycopene more accessible and enhancing the cis-isomerization.

            More information on lycopene bioavailability, however, is needed. The pharmacokinetic properties of lycopene remain particularly poorly understood. Further research on the bioavalability, pharmacology, biochemistry, and physiology must be done to reveal the mechanism of lycopene in human diet, and the in vivo metabolism of lycopene. Consumer demand for healthy food products provides an opportunity to develop lycopene rich food as new functional foods, as well as food-grade and pharmaceutical-grade lycopene as new nutraceutical products. An industrial scale, environmentally friendly lycopene extraction and purification procedure with minimal loss of bioactivities is highly desirable for the foods, feed, cosmetic, and pharmaceutical industries. High-quality lycopene products that meet food safety regulations will offer potential benefits to the food industry.

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Lutein:

 Figure.9. Chemical structure of lutein

            Lutein is a complex compound which belongs to the carotenoid family that plants are able to synthesize, but the human body can’t.

            Lutein has many uses in the body. For some time researchers have been looking at the role lutein and other antioxidants play in protecting the skin against the effects of the sun. The sun´s rays contain UVA radiation. This radiation can cause the formation of free radicals in cells that have been shown to be a first step in the chain leading to cancer; they may also be responsible for the aging process that affects skin. It has been estimated that sun exposure is the largest factor contributing to skin aging caused by external factors.

            Lutein is found in the skin and perhaps, not by chance, in highest concentrations in nasal skin - the part of the body that is often the most exposed to sunlight. The chemical structure of lutein gives it its antioxidant power and therefore it can protect against free radical damage caused by UVA radiation.

Several leafy vegetables contain large quantities of lutein including spinach, swiss chard and kale.

 

Other carotenoids chemical structures are shown in the figure;

 Figure.10. Chemical structure of other carotenoids

 

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VITAMIN E (alpha tocopherol)

 

 

vitamin E

          

 Figure.11. Chemical structure of Vitamin E

 

            Vitamin E is a generic term that includes all entities that exhibit the biological activity of natural vitamin E, d-alpha-tocopherol. In nature, eight substances have been found to have vitamin E activity: d-alpha-, d-beta-, d-gamma- and d-delta-tocopherol (which differ in methylation site and side-chain saturation; and d-alpha-, d-beta-, d-gamma- and d-delta-tocotrienol. Also, the acetate and succinate derivatives of the natural tocopherols have vitamin E activity, as do synthetic tocopherols and their acetate and succinate derivatives.

 

Of all these, d-alpha-tocopherol has the highest biopotency, and its activity is the standard against which all the others must be compared. It is the predominant isomer in plasma.

 

             Vitamin E is an essential nutrient that functions as an antioxidant in the human body. It is essential, by definition, because the body cannot manufacture its own vitamin E and thus it must be provided by foods and supplements.

 

            Tocopherols are present in oils, nuts, seeds, wheat germ and grains. Absorption is believed to be associated with intestinal fat absorption. Approximately 40% of the ingested tocopherol is absorbed. Most tocopherols enter the blood via lymph where they are associated with chylomicrons. Vitamin E was shown to be stored in adipose tissue. Phospholipids of the mitochondria & endoplasmic reticulum & plasma membranes possess affinities for alpha tocopherol & the vitamin tends to concentrate in these sites.

 

Mechanisms of Action:

 

            Vitamin E is more appropriately described as an antioxidant than a vitamin. This is because, unlike most vitamins, it does not act as a co-factor for enzymatic reactions.

 

            Also, deficiency of vitamin E does not produce a disease with rapidly developing symptoms such as scurvy or beriberi. Overt symptoms due to vitamin E deficiency occur only in cases involving fat malabsorption syndromes, premature infants and patients on total parenteral nutrition. The effects of inadequate vitamin E intake usually develop over a long time, typically decades, and have been linked to chronic diseases such as cancer and atherosclerosis.

 

            Hence, its main function is to prevent the peroxidation of membrane phospholipids, and avoids cell membrane damage through its antioxidant action. The lipophilic character of tocopherol enables it to locate in the interior of the cell membrane bilayers. Tocopherol-OH can transfer a hydrogen atom with a single electron to a free radical, thus removing the radical before it can interact with cell membrane proteins or generate lipid peroxidation. When tocopherol-OH combines with the free radical, it becomes tocopherol-O·, itself a radical. When ascorbic acid is available, tocopherol-O· plus ascorbate (with its available hydrogen) yields semidehydroascorbate (a weak radical) plus tocopherol-OH. By this process, an aggressive ROI is eliminated and a weak ROI (dehydroascorbate) is formed, and tocopherol-OH is regenerated. Despite this complex defence system, there are no known endogenous enzymatic antioxidant systems for the hydroxyl radical.

 

Alpha tocopherol + LOO˙         Alpha tocopherol˙+ LOOH

Alpha tocopherol˙+ LOO˙          LOO-alpha tocopherol

 

            Vitamin E also stimulates the immune response. Some studies have shown lower incidence of infections when vitamin E levels are high, and vitamin E may inhibit cancer initiation through enhanced immunocompetence.

 

            Vitamin E also has a direct chemical function. It inhibits the conversion of nitrites in smoked, pickled and cured foods to nitrosamines in the stomach. Nitrosamines are strong tumour promoters.

 

            Alpha-tocopherol has been shown to be capable of reducing ferric iron to ferrous iron (i.e. to act as a pro-oxidant). Moreover, the ability of alpha-tocopherol to act as a pro-oxidant (reducing agent) or antioxidant depends on whether all of the alpha-tocopherol becomes consumed in the conversion from ferric to ferrous iron or whether, following this interaction, residual alpha-tocopherol is available to scavenge the resultant ROI .

 

 

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Chemical structure                    

 

                                                           

            Vitamin E is the overall collective term for all biological active tocopherols and tocotrienols and their derivatives which exhibit the biological activity of R,R,R-α-tocopherol, the natural occurring vitamer with the highest activity. The chemical structure of the tocopherols is shown in Fig. 12.

           

-tocopherol.

 

 

 


           

           

   

    The molecule can be divided into two parts, a chroman head and a phytyl chain. The phytyl chain intercalates with fatty acid residues of phospholipids, while the chroman head—responsible for the actual antioxidant effect— faces the cytosol, although the chroman ring is stil located in the hydrophobic zone of the lipid bilayer. An important characteristic of vitamin E is the presence of three asymmetrical carbon atoms in the phytyl tail. The biological half-life of the eight stereoisomers of a vitamer greatly differs. The half-life also affects the number of methyl groups on the chroman head, since the half-lifeof the different vitamers also greatly differs. The superior biological activity of R,R,R-α-tocopherol resides in its long retention in the body compared with the other vitamers such as R,R,R--tocopherol or to the other stereoisomers such as S,S,S-α-tocopherol. This is due to the high affinity of R,R,R-α-tocopherol for transfer and binding proteins .

           

            After ingestion of vitamin E, it has to be incorporated in micelles for efficient absorption. In the gastrointestinal tract vitamin E esters are hydrolysed to free vitamin E and this is transported via the lymph to blood. The absorption greatly depends on the composition of diet, i.e. the amount of fat consumed. All body tissues, in particular all cell membranes and subcellular organelles (mitochondria, cell nuclei, endoplasmatic reticulum) are supplied with vitamin E.

 

            Recently, another metabolite of a vitamin E, has gained much attention. 2,7,8-trimethyl-2-(β-carboxyethyl)- 6-hydroxychroman, a metabolite from -tocopherol that is formed by β-oxidation of the phytyl chain (Fig. 13), was reported to possess a strong natriuretic effect.

            -tocopherol has a natriuretic effect.

 

 

 

 

 

                       

 

 

It was claimed that this metabolite is identical to a putative hormone proposed four decades ago, that controls the body’s pool of extracellular fluid. This is an important determinant in hypertension, congestive heart failure and cirrhosis.Surprisingly, the comparable metabolite formed from α-tocopherol does not display a natriuretic effect, suggesting a great specificity of the metabolite of -tocopherol.          

           

A similar difference in efficacy between vitamins and their metabolites is also found in the inhibition of cyclooxygenase. -Tocopherol and one of its major metabolites -CEHC were effective inhibitors of this enzyme, while α-tocopherol only had a minor effect. It was hypothesised that this anti-inflammatory effect may contribute to disease prevention by -tocopherol.

 

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            Antioxidant activity

 

            The best-studied activity of vitamin E is the protection of membranes against free radical damage. In the antioxidant activity of tocopherol, a radical abstracts a hydrogen atom from the aromatic hydroxyl group anda chromanoxyl radical is formed (Fig. 14).

 

           

           

           

 

 

 

           

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

 

           

 

    This chromanoxyl radical is fairly stable due to delocalization of the unpaired electron. The oxygen in the heterocyclic ring of the chroman ring is fixed in such a position that there is a considerable overlap between the 2p-type orbital of a lone pair of the oxygen and the aromatic system. This permits stabilisation of the chromanoxyl radical by interaction of the unpaired electron with a lone pair of that oxygen . In this way the degree of delocalization is enhanced.

           

The chromanoxyl radical can be converted back into tocopherol in several ways, e.g. by the interaction with vitamin C  or reduced glutathione (GSH). The reaction of GSH and the chromanoxyl radical is catalysed by a free radical reductase. Despite this interplay with other antioxidants, tocopherol can be converted into a quinone. By this oxidation, one of the rings of the chroman headis opened (Fig. 14). This quinone can be reduced into a hydroquinone  It has been reported that the antioxidant activity of this hydroquinone is superior to that of tocopherol. Provocatively, it has been stated that α-tocopherol may serve as a reservoir of α-tocopheryl hydroquinone. Experiments using deuterated _-tocopheryl quinonedemonstrate that α-tocopheryl quinone can be converted into α-tocopherol in man. During oxidation, numerous other products, including dimers, are formed.

 

            Beside oxidation of the chroman head, a conversion directly linked to the antioxidant function of tocopherol, tocopherols undergo ω-oxidation and subsequent β-oxidation of the phytyl chain . Several metabolites generated via the latter route have been described.

           

 Insertion of vitamin E into lipid bilayers decreases membrane fluidity. This decrease is similar to that observed after cholesterol enrichment. More recently, other non-antioxidant effects of tocopherol have been reported. R,R,R-_α-tocopherol stimulates protein phosphatase 2A in a concentration-dependent way. This activation may lead to dephosphorylation and inactivation of protein kinase C, a key enzyme in the proliferation of a number of cells. This pathway is suggested to be involved in the inhibition of the proliferation of vascular smooth muscle cells by R,R,R-α-tocopherol. Interestingly, R,R,R-β-tocopherol has no effect on proliferation of these cells. This difference in effect of both vitamers cannot be explained by a difference in uptake in the cells nor by a difference in free radical scavenging activity since these properties are very similar for both compounds.

 

            The work of Azzi et al. suggests that the antioxidant effect of vitamin E is not the primary action of vitamin E. It is a challenging thought that vitamin E is in the first place a hormone-like compound that controls several cellular functions. Due to its oxidisability, it would act as a sensor of oxidative stress rather than a direct antioxidant. So the primary effect of scavenging of free radicals would not be to detoxify these radicals but rather to trigger the cellular response toward oxidative stress.

           

The new and diverse biological effects of vitamin E,advocate a re-evaluation of the effect of vitamin E administration. In this re-evaluation the metabolism and the biological effects of the metabolites should be included. Also the difference between the vitamers and the stereoisomers of the vitamers have to be considered. As discussed above the kinetics, efficacy, metabolism and toxicity of these vitamers may greatly differ.

 

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Toxicity

           

The recommended daily allowance of vitamin E for an adult is 10 mg R,R,R-α-tocopherol equivalents per day. It is generally believed that α-tocopherol is relatively safe in a ‘therapeutic’ daily dose up to 300 mg. The major adverse effect reported was that oral intake of high levels of vitamin E can exacerbate an impaired blood coagulation due to vitamin K deficiency caused by malabsorption or anticoagulant therapy. Recently, we have found that tocopherols and several tocopherol esters inhibit glutathione S-transferase P 1-1 (GST P 1-1). It is known that GST P1-1 is present in the skin. Mice lacking GST P1-1 have an increased risk for skin tumorgenesis. Interestingly, vitamin E has been reported to be a complete tumour promoter in mouse skin. Combining these data, it is tempting to suggest that the promoter effect of vitamin E might be caused by GSTP1-1 inhibition. It can be calculated that the concentration of tocopherol esters in numerous cosmetic products is sufficient for blocking GST P1-1. This urges to evaluate the potential risk of the application of these products.

 

            As mentioned above, quinones are metabolites from vitamin E generated when it exerts its antioxidant activity. In general, quinones have to be regarded as cytotoxic due to their ability to generate oxygen radicals, their ability to oxidise cellular components, and their ability to form Michael adducts with cellular thiols. The Michael addition of thiols to quinines depends on the nature, the number and the position of substituents on the quinone. α-Tocopheryl quinone, the fully substituted tocopherol quinone, is incapable of forming these adducts with thiols, while α-tocopheryl and α-tocopheryl quinone, that are only partially substituted, do. It has been found that ααα-tocopherol quinine and α-tocopheryl quinone are far more cytotoxic in cell culture than α-tocopheryl quinone. This difference in toxicity can be explained by the difference in ability of these different quinines to form Michael adducts. This effect of substituents on the toxicity of α-tocopheryl quinone parallels that of the effect of subtituents on the toxicity of N acetaminophen quinonimide. The toxicity of the fully substituted quinonimide is far less compared with the partially or non-substituted derivatives. Additionally, it has been shown that α-tocopheryl quinone can antagonise the reduction of the chromanoxyl radical of tocopherol by the GSH-dependent free radical reductase.

 

The difference in toxicity between α-tocopheryl quinoneand the other tocopheryl quinones is in line withthe results of a study performed in 1930 with natural oils that were treated with FeCl3 to destroy vitamin E. Although at that time it was not realised that tocopheryl quinones were formed, they undoubtedly were. Processed cod liver oil supported growth in experimental animals, whereas processed germ oil was highly toxic. Cod liver oil mainly contains α-tocopherol, whereas germ oil also contains α and ß-tocopherol. The contrasting biological effect between both processed oils may now be explained by the difference in toxicity of the different quinones formed.

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    Pro-oxidant chemistry:

 

 

 

 

 

 

Vitamin E is a term used to describe a family of tocopherols of which α-tocopherol (Fig. 15) is the most abundant and important member. Observational epidemiologic studies provide the basis for relating intake of vitamin E rich food to decreased incidence of risk of mortality due to cardiovascular diseases. However, generally results from large-scale intervention studies are inconclusive reporting adverse as well as beneficial effects or no effects at all of daily supplementation with α-tocopherol. Vitamin E intakes provided protection against LDL cholesterol oxidation and reduced risk of heart disease. Human intervention studies in which smoking male volunteers were exposed during 5–8 years to daily supplementation with vitamin E did not reveal any effect on the overall mortality of male smokers, but did show increased mortality resulting from hemorrhagic stroke.

           

The inconsistency in the effects of vitamin E may be related to the complex function and chemical behavior of vitamin E, being able to have an anti-oxidant, neutral or prooxidant effect. This dualistic behavior is best described by the mechanism depicted in Fig. 15. The pathway depicted implies that increased levels of α-tocopherol result, upon subsequent oxidative stress, in increased levels of α-tocopherol radicals. These α-tocopherol radicals can initiate processes of for example lipid peroxidation by themselves. When antioxidant networks are balanced, this pro-oxidant action of vitamin E radicals is inhibited by co-antioxidants which can reduce the radical back to vitamin E. Increasing only the levels of α-tocopherol, may, especially under conditions of increased oxidative stress, result in increased levels of α-tocopherol radicals which can no longer be efficiently detoxified by the co-antioxidants. This provides the possibility for the pro-oxidant toxicity of the α-tocopherol radical. This biochemical rationale explains why foods containing comparably small levels of vitamin E but also co-antioxidants provide greater health benefits than vitamin E supplements. The observed improvement of α-tocopherol levels in red blood cell membranes in vivo upon prolonged green tea catechin consumption also supports this idea of antioxidant networks and the hypothesis that the levels of co-antioxidants are important for maintaining high levels of α-tocopherol. These results clearly point at the importance of balanced antioxidant networks and the risks related to unbalanced networks when only one member of such an antioxidant network is increased.

           

In addition to its use as a functional food ingredient or supplement, vitamin E is often used in anti-sun products and other skin cosmetics. In view of this it is of importance that vitamin E has been shown to be a complete tumor promotor in a mouse skin model. Upon treatment of the skin with the tumor initiator dimethylbenz[a]anthracene (DMBA), subsequent promotion by topical application of vitamin E showed that vitamin E can act as a complete tumor promotor in DMBA treated mouse skin with an efficiency approaching that of the standard tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA). The authors state that it may be prudent to avoid repetitive or prolonged topical exposure of human skin to antioxidants like vitamin E, especially in the case of co-exposure to chemical carcinogens or tumor initiators. In addition, several other studies have reported vitamin E to be a tumor initiating and tumor promoting carcinogen. For example, vitamin E has been reported to enhance the induction of intestinal tumorigenesis by 1,2-dimethylhydrazine. The chronic subcutaneous administration of vitamin E to mice and rats was reported to result in tumor induction and continuous oral administration of vitamin E to mice was reported to significantly increase liver cancer incidence .In spite of this, some large intervention studies report the complete absence of any significant effect of vitamin E supplementation on both lung tumor incidence but also on overall mortality, and even revealed increased mortality resulting from hemorrhagic stroke again illustrating the controversial results observed upon vitamin E supplementation. The mechanism of tumor promotion by vitamin E is at present unknown, but may also be related to increased formation of α-tocopherol radicals which, when not efficiently scavenged by other antioxidants, may act as reactive radical species themselves. Alternatively, it may be related to the Cu2+-dependent pro-oxidant action of vitamin E. In in vitro systems alfa-tocopherol induced oxidative DNA damage in the presence of Cu2+, caused by vitamin E mediated copper-dependent reactive oxygen species formation. Furthermore, Van Haaften et al. (2001) showed that vitamin E inhibits GST P1-1 activity with an IC50 of less than 1 _M. This inhibitory potency might result in an increased risk for skin tumorigenesis because it has been shown that mice lacking GST P1-1, which is normally abundant in the skin, have an increased risk for skin tumorgenesis.

           

Finally, similar controversial results have been reported for the effects of vitamin E (and other antioxidants) on the cardiotoxicity of doxorubicin, or the lung toxicity of bleomycin. Both these anticancer drugs are believed to generate their dose-limiting toxic effects by means of generation of reactive oxygen species. Co-administration of vitamin E has been investigated as a means to overcome the dose-limiting toxic side effects.

 

        In addition to studies reporting protective effects and/or the absence of effects  also potentiation of oxidative doxorubicin and bleomycin toxicity by vitamin E has been reported: Shinozawa reported that treatment of ICR mice with vitamin E and doxorubicin resulted in significant reduction in the survival time as compared to treatment with doxorubicin alone. The mechanism behind the potentiating pro-oxidant effect of antioxidants like vitamin E in the case of these tumor drugs may be related to the fact that generation of reactive oxygen species by the drugs may be in part mediated by drug-Fe2+ complexes reducing O2, resulting in superoxide anion radical formation and drug- Fe3+ complexes. Subsequent reduction of the drug-Fe3+ complex by antioxidants may support thecatalytic redoxcycle of the drug-Fe complex.

           

Altogether, supplementation with an antioxidant like vitamin E may not always exert the protective effect aimed for. The ultimate balance between potentiating and protective effects may depend on the subtle redox equilibrium within the cells and the balance within the complete cellular antioxidant network.

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PHENOLIC COMPOUNDS

 

            Much work has been carried out in recent years on the beneficial effect of phenolic compounds as natural antioxidants which help to neutralize free radicals. In fact, researchers have focused their attention on the pathological role of free radicals in a variety of diseases, among which the most important are atherosclerosis and cancer. Thus, among the components of the so-called ‘Mediterranean Diet’, phenolic compounds have received increased attention as epidemiological studies have shown that consumption of foods and beverages rich in phenolics is correlated with reduced incidence of heart disease.

           

PHENOLIC ACIDS

 

Also they are known as phenolcarbon acids. Compounds which are in synamic acids group have structure between C3 –C6 . Synamic acids that especially found in fruits are cafeic, cumaric and ferrulic acid. Phenolic acids are not found at their free forms in living plant tissue, but they occur by thr hydrolysis during the processing.

 

Carboxil groups of phenolic acids are generally found as alcohol and phenol esters and sometimes with other compounds.

 

For example; synamic acids in fruits are found as D-cuinic acid and glucose esters. The most popular derivative of the synamic acid is clorojenic acid which is occured by combining of caffeic acid and cuinic acid. In addition; synamic acids should be found as malic and tartaric acid esters (Figure 16 )

 

The other group of the phenolic acids are benzoic acids which have structure between C6-C1.

 

The derivatives of benzoic acids are found less than synamic acid derivatives or never found. The most important benzoic acid derivative structure is shown at Fig. 16

 

 

        

 

 Fig.16.Benzoik and cinamik acids' chemical structure

 

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POLYPHENOLS(FLAVONOIDS):

 

            Fruit and vegetable intake is associated with a reduced risk of cancer and cardiovascular disease. While these protective effects have been primarily attributed to beta-carotene and ascorbate, phenolic constituents may also play a role. Flavonoids are a broad class of low molecular weight, secondary plant phenolics characterized by the flavan nucleus. Widely distributed in the leaves, seeds, bark and flowers of plants, over 4,000 flavonoids have been identified to date. In plants, these compounds afford protection against ultraviolet radiation, pathogens, and herbivores. The anthocyanin copigments in flowers attract pollinating insects and are responsible for the characteristic red and blue colors of berries, wines, and certain vegetables—major sources of flavonoids in the human diet. Although dietary intake varies considerably among geographic regions and cultures, it is estimated to be 23 mg daily in the Netherlands.

           

The protective effects of flavonoids in biological systems are ascribed to their capacity to transfer electrons free radicals, chelate metal catalysts activate antioxidant enzymes reduce alpha-tocopherol radicals and inhibit oxidases. Although this multi dimensional effect is likely responsible for the consistent overall effectiveness of these compounds in diverse experimental systems, it poses difficulties in delineating structure-activity relationships (SAR). A number of structure-activity studies employ oxidases and/or transition metals as ROS generators, both of which confound the identification of relationships between chemical structure and free radical scavenging. Over the past fifteen years, a considerable number of in vitro studies have sought to arrive at a common heirarchy of flavonoids in terms of substitutions and antioxidant activity. These data enable a better understanding of the antioxidant and prooxidant effects of flavonoids, and offer reasonable predictions of the influence of structural modifications that ensue during metabolism..

 

 

Classification and chemical structure

 

            Flavonoids are benzo-gama-pyrone derivatives consisting of phenolic and pyrane rings (Fig. 17) and are classified according to substitutions (Fig. 18). Dietary flavonoids differ in the arrangements of hydroxyl, methoxy, and glycosidic side groups, and in the conjugation between the A- and B- rings.

 

 

 

 


    

    During metabolism, hydroxyl groups are added, methylated,sulfated or glucuronidated. In food, flavonoids exist primarilyas 3-O-glycosides and polymers. Several types of higher structure exist, and polymers comprise a substantial fraction of dietary flavonoid intake. Enzymatic oxidation of green tea leaves (Camellia sinensis) during fermentation to black tea results in polymerization of flavanols to tannins and other complex compounds. Condensed tannins, or proanthocyanidins, consist of flavanol units. Of these compounds, the procyanidins are most relevant to the human diet; these compounds consist of (+)-catechin and (-)- epicatechin  monomers. The ß4 → 6 and ß4→8 linked procyanidin dimers, trimers, and oligomers occur in red wines ,grape seeds, apples and cocoa. Proanthocyanidins may reach high molecular weights, consisting of up to 17 flavanol units. Esters of gallic acid are known as hydrolyzable tannins or gallotannins. The galloyl moieties of these tannins and of the monomeric catechins in green tea are partly responsible for the chelating  and radical scavenging properties of these compounds.

 

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Metin Kutusu: Fig. 18. Classification, structure, food sources, and Trolox equivalent antioxidant activities (TEAC) of dietary flavonoids. Higher TEAC values reflect greaterantioxidant capability. A free 3-hydroxyl group and 3_,4_-catechol (dihydroxy) structure, a 2–3 double bond, and a 4-oxo group endow the flavonoid withactivity superior to isoforms lacking these features. Glycosidic substitution decreases TEAC.
 

 

 


 

 

 

 Absorption and metabolism

 

            Understanding the biodynamics of flavonoids after oral administration is fundamental to appropriate extrapolation of existing SAR information to preventive nutrition. In addition to structural and physico-chemical attributes of the nascent compound, the absorption, pharmacokinetics, biotransformation, and the relative activities of metabolites are critical determinants of biological effects in organisms. In vitro data effectively and consistently demonstrate the antioxidant efficacy of structurally diverse flavonoids under many circumstances of oxidative stress. However, the current understanding of absorption and metabolism in humans is limited to a small number of dietary flavonoids. To integrate new knowledge of SAR into the areas of human nutrition and medicine, future research must elucidate

 

  1. the extent of absorption in relation to structure,

ii.      pharmacokinetics in humans,

iii.    characterization of flavonoid metabolites,

iv.    SAR and health effects of these metabolites.

 

Most dietary flavonoids occur in food as O-glycosides. The most common glycosidic unit is glucose, but other examples include glucorhamnose, galactose, arabinose, and rhamnose. Not surprisingly, the β-linkage of these sugars resists hydrolysis by pancreatic enzymes, so it had long been assumed that intestinal microbiota were responsible for beta-hydrolysis of sugar moieties. However, two β-endoglucosidases capable of flavonoid glycoside hydrolysis have since been characterized in the human small intestine, including lactase phlorizin hydrolase and a nonspecific cytosolic enzyme believed to deglycosylate flavonoids to allow a site for conjugation. Spencer et al reported that luteolin-7-glucoside, kaempferol-3- glucoside and quercetin-3-glucoside are hydrolyzed and absorbed by the small intestine, supporting β-glucosidase activity. During passage across everted intestine, luteolin- 3-glucoside also undergoes complete hydrolysis to luteolin aglycone, in addition to its methyl- and sulfate conjugates. In another study, cell-free extracts from human small intestine cleaved flavonoid 4’- and 7-monoglucosides, but did not modify a series of rhamnoglucosides and diglucosides, suggesting differences in flavonoid bioavailability according to the location and structure of the sugar moiety. Although some evidence suggests that anthocyanin glycosides are absorbed intact, the hydrolytic removal of glucose and rutinose from quercetin is well documented. Though quercetin represents but one of hundreds of dietary flavonoids, it is among the most abundant, potent, and widely studied and provides insight into the absorption and metabolism of these polyphenols. It is not surprising that absorption kinetics vary considerably among foods, owing to the heterogeneity of sugars and other functional groups about the flavan nucleus. Absorption may also depend on dosage, vehicle of administration, antecedent diet, sex differences, and microbial population of the colon. Separate locations of uptake also suggestdifferent metabolic fates of rutinosides versus glucosides, as the liver may play a larger role in the metabolism of flavonoids absorbed in the small intestine compared to metabolism of compounds taken up by the colon.

 

            For hydrolysis and absorption of some flavonoid glycosides,enteric bacteria are indispensable. The requirement of colonic microflora for hydrolysis of rutinosides may explain the low bioavailability of rutin (quercetin-3-rutinoside) compared to quercetin-3-glucoside in human studies. Following oral administration of rutin, quercetin is gradually recovered in plasma of subjects with an intact colon ,but is undetectable in plasma of ileostomy patients. In the latter investigation, a glucose moiety increased absorption of quercetin in the small intestine to 52%, compared to 24% for the aglycone and 17% for rutin. Quercetin glucoside, but not rutin, has been reported to interact with epithelial glucose transporters, offering a possible explanation for the rapid uptake and bioavailability of glucosides.

           

Due to molecular size, absorption of polymeric flavonoids across the intestinal epithelium requires preliminary degradation to smaller, low molecular weight compounds. Procyanidin dimers and trimers, but not oligomers averaging 7 units, are capable of translocating across the small intestinal epithelium. Thus, it is possible that degree of polymerization is less predictive of antioxidant activity in vivo compared to in vitro, and the value of the latter research is limited in describing the role of proanthocyanidins in human nutrition. Since these molecules generally consist of (+)-catechin and (-)-epicatechin subunits, it is conceivable that catechins are predominant degradation products. Caecal bacteria  and the low gastric pH contribute to this process. The latter investigation demonstrated the hydrolysis of proanthocyanidin oligomers of 3 to 6 units into catechin dimers and free catechins after 3.5 hr in the gastric environment. Beyond 3 catechin units, susceptibility to degradation increased proportionally to the degree of polymerization. Although these observations suggest that catechins are responsible for the health effects of high-molecular weight proanthocyanidins, 3.5 hr exceeds the normal human gastric emptying rate of 30 to 90 min, and the contribution of acid hydrolysis is probably less signify cant than subsequent metabolic events.

           

 Aside from hydrolysis of flavonoid glycosides, cecal microflora participate in degradation of polymers and scission of monomeric flavonoids to monophenolic acids. Metabolism of quercetin by intestinal bacteria produces 3,4-dihydroxyphenylacetic acid and phloroglucinol via cleavage of the C3-C4 bond of the heterocycle. Following oral administration of rutin to rats, phenylacetic acids,   3,4-dihydroxytoluene, and 3-(m-hydroxyphenyl)propionic acid are recovered in urine. Tannins are degraded by cultured colon flora to similar aromatic compounds. A study incubated cultures of human colonic bacteria with 14C-labeled proanthocyanidins, averaging 7ß-linked flavanol units, under anaerobic conditions resemblingthe enteric environment. After 48 hr of incubation, nearly all of the substrate was degraded into monohydroxylated phenylacetic, phenylpropionic and phenylvaleric acids as determined by gas chromatography coupled to mass spectrometry. These metabolites are similar to the phenolic acid degradation products of catechin and procyanidin dimers. Rice-Evans and coworkers proposed a metabolic scheme for quercetin in which the flavone heterocycle is cleaved to phenolic acids subject to subsequent dehydroxylation, O-methylation, or β-oxidation to benzoic acid derivatives. Miyake and colleagues promulgated a metabolic scheme for orally administered eriocitrin, a flavone diglycoside from lemons, after oral administration to rats. The native eriocitrin was undetectable in plasma, but metabolites were conjugated to glucuronic acid or sulfate and were characterized as eriocitrin aglycone, 4’-methoxy eriodictyol (hesperetin), 3’-methoxy eriodictyol (homoeriodictyol), and 3,4 dihydroxyhydrocinnamic acid. Although the relative antioxidative activities of these metabolites were not directly evaluated, the susceptibility of plasma to oxidative damage decreased significantly in their presence.

           

Using the Trolox (a water-soluble α-tocopherol analog)- equivalent antioxidant capacity (TEAC) assay, Rice-Evans and coworkers compared radical scavenging activitiesof phenolic acids occurring in higher plants. This method assesses the hydrogen donating ability of flavonoids in the aqueous phase by evaluating 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) cation (ABTS.+) scavenging, and activity is expressed as the millimolar concentration of Trolox (TEAC = 1) equivalent to the activity of a 1 mM solution of the experimental compound. All phenolic acids demonstrated lower TEAC values than flavonoids, and hydroxycinnamates were generally more active than most hydroxyphenylacetates and hydroxybenzoates. The quercetin metabolite 3,4-dihydroxyphenylacetic acid (TEAC = 2.19) was substantially weaker than quercetin (TEAC = 4.7) but remained over twice as effective as vitamin E. The putative 4-dehydroxy metabolite of quercetin (TEAC = 0.90) was much less effective than both quercetin and 3,4-hydroxyphenylacetic acid. While this evidence supports that scission of the flavonoid heterocycle produces compounds with lower activity, these metabolites retain a radical scavenging capacity comparable to vitamin E. Structure-activity comparisons suggest that antioxidant activity of phenolic acids depends on the number and orientation of hydroxyl groups relative to the electron-withdrawing CO2H, CH2CO2H, or (CH)2CO2CH functional group .

           

Interactions between flavonoids and cytochromes P450 are complex and are reviewed elsewhere. Hydroxylation of flavonoids by CYP1A isozymes yields 3’4’-dihydroxylated derivatives that retain the flavan nuclear structure .However, flavonoids are known to inhibit various P450 isozymes, including CYP1A. Selectivity for P450 isoforms is governed by hydroxyl and methoxy substitutionon the 3’ and 4’ position of the B-ring .Since a requisite for microsomal hydroxylation is a maximum of one B-ring hydroxyl group, it is conceivable that the increase in OH groups after phase I metabolism of flavonoids with relatively low activity may give rise to transient compounds with greater antioxidant capability, as the aromatic OH is a critical determinant of hydrogen donation and free radical scavenging by phenolic compounds. Methylation by catechol-O methyltransferase requires a free B-ring OH and mitigates both the antioxidant and prooxidant activity of the flavonoid .

           

 Despite a paucity of metabolic and pharmacokinetic data, existing evidence supports that flavonoids are structurally altered in vivo. Whether phenolic acids or flavonoid isoforms predominate is unclear. Quercetin is recovered in plasma following oral adminstration to humans, but is minimally detected in urine .Walle and coworkers demonstrated quercetin absorption after an oral dose of 100 mg quercetin aglycone ranges from 36 to 53%. Warden and colleagues reported only 1.68% of 400 mg orally administered tea catechins was recovered in plasma, urine, and feces. Collectively these observations suggest extensive biotransformation by tissues and/or intestinal microbiota. Phase II conjugation in the liver and enterocytes gives rise to glucuronides, sulfates ,methyl conjugates and small quantities of free aglycones. Considering that the reduction potential of the B-ring is lower than that of the A-ring, conjugation at the 3’- or 4’-position is likely to increase the reduction potential such that hydrogen donation is less thermodynamically favorable as opposed to A-ring conjugation. A report by Sanz and coworkers that a 7-glucuronide does not interfere with antioxidant effects in rat liver microsomes is consistent with this premise.

 

Although peak concentrations of flavonoids typically occur approximately 2 hr after ingestion of a test food, one study reported peak levels at 24 hr following an oral dose of epicatechin. In healthy volunteers, the half-life of quercetin ranges from 20 to 72 hr. Provided that- sufficient dietary intake is sustained, this long half-life is conducive to accumulation in plasma and a concomitant decline in oxidant status. However, chronic intake of high levels may result in a compensatory decrease in absorption, suggesting a steady state mechanism at the gut level. For example, Manach and colleagues reported that in rats fed a high flavonol diet prior to treatment of pharmacological doses of quercetin, absorption was significantly reduced compared to animals previously maintained on low-quercetin diets. Other dietary components, namely proteins and iron, may theoretically impair absorption by forming complexes with polyphenols. Due to the affinity of flavonoid hydroxyl groups for proline residues, the antioxidant capacity of catechin gallates in vitro is attenuated by the presence of proteins such as β-casein. This suggests proteins in the food itself, the digestive milieu, and the bloodstream may potentially mask the biological activity of polyhydroxylated flavonoids. However, addition of milk to black tea had no effect on the absorption of quercetin or kaempferol in a group of healthy individuals. The exact mechanisms of flavonoid absorption and metabolism remain uncertain, and appear to depend on the type of flavonoid and other variables. Nonetheless, consumption of these compounds decreases plasma oxidant status in a dose-dependent manner. Thiobarbituric acid reactive substances (TBARS) and other plasma indices of oxidative stress used in such studies do not always rule out the possibility that the flavonoid is working indirectly (enzyme interactions, metal chelation) versus directly (radical scavenging), but these lines of evidence demonstrate the bioavailability and antioxidant efficacy of flavonoids in humans.

 

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 Structural features and antioxidant activity

 

The antioxidant activity of flavonoids and their metabolites in vitro depends upon the arrangement of functional groups about the nuclear structure. The past 15 years of SAR research has generated several consistent lines of evidence supporting the role of specific structural components as requisites for radical scavenging, chelation and oxidant activity. The nutritional application of this information requires extensive investigation of flavonoid metabolism, systematic analysis of foods for flavonoid content and composition, and controlled comparison of antioxidant activity of structural isoforms in vivo. Ultimately, SAR may refine current dietary recommendations of fruits, vegetables and other plant foods.

 

Hydroxyl groups

 

The spatial arrangement of substituents is perhaps a greater determinant of antioxidant activity than the flavan backbone alone. Consistent with most polyphenolic antioxidants, both the configuration and total number of hydroxyl groups substantially influence several mechanisms of antioxidant activity . Free radical scavenging capacity is primarily attributed to the high reactivities of hydroxyl substituents that participate in the following reaction:

 

 F-OH +R·                 F-O· + RH 

 

             The B-ring hydroxyl configuration is the most significant determinant of scavenging of ROS and RNS. Hydroxyl groups on the B-ring donate hydrogen and an electron to hydroxyl, peroxyl, and peroxynitrite radicals, stabilizing them and giving rise to a relatively stable flavonoid radical. Among structurally homologous flavones and flavanones, peroxyl and hydroxyl scavenging increases linearly and curvilinearly, respectively, according to the total number of OH groups.

 

A 3’4’-catechol structure in the B-ring strongly enhances lipid peroxidation inhibition. This arrangement is a salient feature of the most potent scavengers of peroxyl, superoxide, and peroxynitrite radicals. For example, the peroxyl radical scavenging ability of luteolin substantially exceeds kaempferol; both have identical hydroxyl configurations, but kaempferol lacks the B-ring catechol. Peroxynitrite scavenging by catechin is mainly ascribed to its B-ring catechol. Oxidation of a flavonoid occurs on the B-ring when the catechol is present, yielding a fairly stable ortho-semiquinone radical through facilitating electron delocalization. Flavones lacking catechol or o-trihydroxyl (pyrogallol) systems form relatively unstable radicals and are weak scavengers.

           

The significance of other hydroxyl configurations is less clear, but beyond increasing total number of hydroxyl groups, A-ring substitution correlates little with antioxidant activity. A 5-OH may contribute to antioxidant effects, and may explain why genestein exhibits a higher TEAC (Fig. 18) and greater peroxynitrite scavenging ability. A 5,7-m-dihydroxy arrangement increases TEAC, but loss of a free 6-OH group by methylation does not modify inhibition of Fe(II)/ascorbate- and CCl4-induced lipid peroxidation by flavonoids. Compared to the B-ring hydroxylation pattern, the impact of the A-ring arrangement on antioxidant activity is of questionable significance.

 

            The flavonoid heterocycle contributes to antioxidant activity by

 

1.       the presence of a free 3-OH, and

2.       permitting conjugation between the aromatic rings.

 

 The closed C-ring itself may not be critical to the activity of flavonoids, given that chalcones are active antioxidants. Free radical scavenging by flavonoids is highly dependent on the presence of a free 3-OH. Flavonoids with a 3-OH and 3’,4’ catechol are reported to be 10-fold more potent than ebselen, a known RNS scavenger, against peroxynitrite. The superiority of quercetin in inhibiting both metaland nonmetal-induced oxidative damage is partially ascribed to its free 3-OH substituent, which is thought to increase the stability of the flavonoid radical. The torsion angle of the B-ring with respect to the rest of the molecule strongly influences free radical scavenging ability. Flavonols and flavanols with a 3-OH are planar, while the flavones and flavanones, lacking this feature, are slightly twisted. Planarity permits conjugation, electron dislocation, and a corresponding increase in flavonoid phenoxyl radical stability. Removal of a 3-OH abrogates coplanarity and conjugation, thereby compromising scavenging ability.

 

 Quercetin exhibits a TEAC of approximately 4.7, whereas luteolin has a value of 2.1, supporting the role of the 3-OH group in free radical scavenging. Compared to the flavonols quercetin, myricetin, and kaempferol, the flavone luteolin is a very weak scavenger of DPPH (2,2- diphenyl-1-picrylhydrazyl radical). Substitution of 3-OH by a methyl or glycosyl group completely abolishes the activity of quercetin and kaempferol against ß-carotene oxidation in linoleic acid. It is postulated that B-ring hydroxyl groups form hydrogen bonds with the 3-OH, aligning the B-ring with the heterocycle and A-ring. Eliminating this hydrogen bond effects a minor twist of the B-ring, compromising electron delocalization capacity. Due to this intramolecular hydrogen bonding, the influence of a 3-OH is potentiated by the presence of a 3’,4’-catechol, explaining the potent antioxidant activity of flavan- 3-ols and flavon-3-ols that possess the latter feature.

           

Compared to the aqueous TEAC assay, experiments of lipid peroxidation provide limited information concerning relationships between structure and antioxidant mechanism. Protection of lipids against oxidative damage can be ascribed to

 

 

1.     scavenging of hydroxyl, peroxyl, or synthetic radicals,

2.    termination of chain reactions in the lipid phase, involving peroxyl radicals and hydroperoxide,

3.     chelation of divalent cations used to initiate oxidative events in vitro,

4.     interactions with other initiators, such as ascorbate, which may reduce and recycle the flavonoid radical or vice-versa.

 

Despite the disparity among methods of assessing activity, there is broad agreement that hydroxyl groups endow flavonoids with substantial radical scavenging ability.

 

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 O-methylation

           

The differences in antioxidant activity between polyhydroxylated and polymethoxylated flavonoids are most likely due to differences in both hydrophobicity and molecular planarity. Quercetin is a potent peroxyl radical scavenger, followed by O-methylated and O-glycosylated derivatives. Suppression of antioxidant activity by O-methylation may reflect steric effects that perturb planarity. Although the ratio of methoxy to hydroxyl substituents does not necessarily predict the scavenging ability of a flavonoid, the B-ring is particularly sensitive to the position of the methoxy group. Alternating a 6’-OH/4’-OMe configuration to 6’-OMe/4’-OH completely abolishes the scavenging of DPPH by inducing coplanarity .

           

DPPH is not a naturally occurring radical, and is relatively stable compared to the highly reactive superoxide and hydroxyl species primarily responsible for oxidative damage in biological systems. Because the multidimensional effects of flavonoids confound the correlation of chemical structure with a particular mechanism, it is not unexpected that some in vitro experiments generate data that are inconsistent with outcomes from simpler assays of aqueous radicals. For example, the half-maximal inhibitory concentration (IC50) of gardenin D (5,3’-OH/6,7,8,4’-OMe-flavone) against CCl4-induced microsomal lipid peroxidation is considerably lower than (+)catechin, but the latter exhibits greater TEAC values than O-methylated flavonoids. The results of the former study may reflect other physiologically relevant parameters of antioxidant activity, such as the lipophilicity and membrane partitioning ability afforded by methoxy groups .

           

Steric obstruction of the 3’4’-catechol structure by 4’-Omethylation significantly compromises antioxidant capability. For example, 4’-O-methylation of quercetin to tamarixetin decreases percentage inhibition of ferrous sulfateinduced lipid peroxidation from 98.0% to _2.6%. Kaempferol-3’,4’-dimethylether exhibits approximately half the peroxyl scavenging activity of kaempferol.Multiple A-ring methoxy groups also reverse the positive effect of a B-ring catechol, as inhibition of formation of the oxidation product malondialdehyde (MDA) by flavones with A-ring ortho-dimethoxy or trimethoxy structures is not enhanced by this element. This information suggests that isorhamnetin, the 3’-methoxy metabolite of quercetin detected in humans, is a less effective antioxidant than the parent compound. O-methylation enhances antioxidant activity in some microsomal systems, but microsomal peroxidation assays permit multiple mechanisms of antioxidant activity, such as

 

1.   recycling endogenous microsomalα-tocopherol,

2.  undergoing biotransformation by NADPH-activated cytochrome P450 to a more or less effective derivative,

3.   chelating iron,

4.   phenoxyl radical reduction and recycling by ascorbate.

 

 These cooperative actions may explain greater activity of flavonoids in these systems.

Moreover, given that each mechanism may be influenced by structure, it is not unexpected that some of these investigations report outcomes that do not paralel data from simpler, sensitive assays such as the TEAC method and the ORAC (total oxyradical absorbance capacity) assay developed by Cao et al.. The latter employs hydroxyl and peroxyl radicals representative of ROS in the cellular environment. Synthetic radicals used in some methods do not always partition sufficiently into a membrane to which a relatively lipophilic polymethoxylated flavonoid has localized. Based on the foregoing, it is rational to conclude that the influence of O-methylation depends on the method of evaluation, the type of radical used, and whether the oxidizable substrate is a lipid structure in which lipophilicity may contribute to total antioxidant activity.

 

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The 2–3 double bond and 4-oxo function

           

            A distinguishing feature among the general flavonoid structural classes in Fig. 15 is the presence or absence of an unsaturated 2–3 bond in conjugation with a 4-oxo function.

 

Aside from the 3’,4’- catechol, 3-OH and overall hydroxylation pattern of quercetin, several studies have sought to determine the significance of 2–3 unsaturation and a 4-carbonyl group. Experiments of catechins and anthocyanidins suggest that these may be dispensable provided that other structural criteria are fulfilled. For example, the TEAC of quercetin (4.7) and cyanidin (4.44) differ by a narrow magrin of 0.36 Trolox equivalents. In a systematic study of 33 flavonoids, Burda and Oleszek  identified no consistent correlation between 2–3 unsaturation and antioxidant activity in a methanol solution of DPPH. However, comparison of quercetin with taxifolin suggests that in flavonoids fulfilling other structural criteria, the 4-oxo and double bond distinguishes the better antioxidant. Quercetin is a more potent inhibitor of ferrous sulfate-induced MDA formation than taxifolin; both structures have a 4-oxo group, but taxifolin is saturated between carbons 2 and 3. Flavonoids with a 2–3 double bond in conjugation with a 4-carbonyl group exhibit lower IC50 values (indicative of stronger antioxidant activity) in a microsomal system compared to those with saturated heterocycles .

           

The majority of research supports that flavonoids lacking one or both features are less potent antioxidants than those with both elements. Conjugation between the A- and Brings permits a resonance effect of the aromatic nucleus that lends stability to the flavonoid radical and is therefore critical in optimizing the phenoxyl radical-stabilizing effect of a 3’,4’-catechol. The premise that flavanols are more effective free radical scavengers than flavones may be ascribed to the greater number of hydroxyl groups and 3-OH in the former. Hydroxyl and peroxyl scavenging capacities of genestein and 6-OH-flavone, respectively, are lower than the activity of vitamin E as determined by ORAC ,but both structures lack a 3’4’-catechol, 3-OH, and multiple B-ring OH groups. The TEAC of quercetin (4.7) is almost twice that of (+)-catechin (2.4), illustrating the significance of both 2–3 unsaturation and a carbonyl at position 4. Apigenin (TEAC = 1.45) and naringenin (TEAC =1.53) exhibit a much smaller difference in ABTS.+ scavenging, so 2,3-unsaturation may be less important than the 4-oxo itself. Although other structural elements must essentially be considered, free radical scavenging by flavonoids is variably enhanced by the presence of both elements.

 

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 Carbohydrate moieties

 

            Aglycones are more potent antioxidants than their corresponding glycosides. Daidzein and genestein aglycones exhibit greater TEAC values (1.25 and 2.9) than their 7-glycosides (1.15 and 1.24, respectively). Genistin is also inferior to its aglycone, genestein, in attenuating peroxynitrite-induced oxidation of LDL. Plumb and coworkers reported that the antioxidant properties of flavonol glycosides from tea decreased as the number of glycosidic moieties increased. Aside from mere presence and total number, the position and structure of the sugar play an important role. Ioku and colleagues evaluated the effect of quercetin and several of its glycosides in a liposomal phospholipid suspension exposed to aqueous ROS, where inhibition of hydroperoxidation of methyl linoleate was measured to determine peroxyl radical scavenging activity. Luteolin and quercetin aglycones significantly exceeded their 3-,4’- and 7-O-glucosides in retarding the accumulation of hydroperoxides in membrane bilayers, but a 4’-sugar was more suppressive than 3- or 7 substitution. Since C-glycosylation in the A-ring also decreases activity, this negative effect may stem from the properties of the sugar itself, rather than displacement of a free OH. As in O-methylation, steric effects imparted by 4’-glycosylation exact a particularly suppressive influence through blocking the B-ring catechol.

           

In the diet, flavonoid glycosidic moieties occur most frequently at the 3- or 7- position, but an A-ring sugar results in a greater diminution of activity than 3-glycosylation in the heterocycle. O-glycosylation at carbon 7, but not carbon 3, weakens the antioxidant effect of flavonoids in rat mitochondria, but no difference between 3- and 7- glucosides of quercetin was detected against hydroperoxide in phospholipid bilayers. It is plausible that the relative influence of 3- and 7-glycosylation is ruled by other structural considerations. It is also important to acknowledge that a glycosyl substituent, regardless of position and structure, seldom confers an antioxidant advantage over the aglycone. Like methylation, O-glycosylation interferes with the coplanarity of the B-ring with the rest of the flavonoid and the ability to delocalize electrons. Though glycosides are usually weaker antioxidants than aglycones, bioavailability is sometimes enhanced by a glucose moiety .

           

Rutinose is a unique case in that addition of this disaccharide to quercetin to form rutin does not consistently decrease antioxidant ability. Rutin is only marginally weaker than quercetin in attenuating Fe(II)-induced MDA formation in liposomes and ascorbic acid-induced lipid peroxidation in rat mitochondria. In addition, Mora and colleagues reported only a minor difference in IC50 of ferrous sulfate-induced oxidative damage by rutin (19.5 uM) compared to quercetin (17.6 uM). However, the TEAC of quercetin is nearly twice that of rutin. The reason why the suppressive effect of rutinose is less pronounced in the aforementioned experiments is unclear and may involve differences in methodology.

             Whether the sugar moiety is glucose, rhamnose, or rutinose is also relevant. For example, compared to rutinose, arhamnose moiety on quercetin significantly reduces scavenging of radicals generated by stimulated human neutrophils. Aside from occupying free OH groups necessary for hydrogen abstraction and radical scavenging, any sugar substituent is capable of

 

1.  diminishing coplanarity of the B-ring relative to the rest of the flavonoid

2.  lending hydrophilicity and altering access to lipid peroxyl and alkoxyl radicals during propagation of LPO in membranes.

 

In light of the aforementioned evidence that glycosidic bonds are often cleaved at the gut level, the influence of sugar moieties on antioxidant properties is of questionable significance in humans. Based on cumulative evidence, removal of the glycosidic substituent by enteric enzymes or bacteria is likely to increase the activity of dietary flavonoids in vivo.

 

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 Degree of polymerization

 

            Polymeric flavonoids present a complex extension of SAR that is poorly understood. Tannins comprise a substantial fraction of daily flavonoid intake among Western cultures in the form of black tea, red wine and cocoa. Grape seed extract, currently marketed as a dietary antioxidant supplement, consists primarily of oligomeric and dimeric catechins. Due to the relative complexity and diversity of tannins, less is known regarding structureactivity relationships. Procyanidin dimers and trimers are more effective than monomeric flavonoids against superoxide anion, but the activities of dimers and trimers differ little. Tetramers exhibit greater activity against peroxynitrite- and superoxide- mediated oxidation than trimers, while heptamers and hexamers demonstrate significantly greater superoxide scavenging properties than trimers and tetramers. It appears that to a point, increasing degree of polymerization enhances the effectiveness of procyanidins against a variety of radical species. Extensive conjugation between 3-OH and B-ring catechol groups, together with abundant ß 4   8 linkages, endow a polymer with significant radical scavenging properties by increasing the stability of its radical.

           

The clinical efficacy of dimeric, trimeric and oligomeric procyanidins is supported by controlled human trials. In a randomized, placebo-controlled crossover study, 20 human volunteers receiving a standardized diet were supplemented with 300 mg grape procyanidins daily over 4 days. By day 5, total serum antioxidant activity had increased by 13%. Red wine, containing predominantly procyanidin dimers, has been reported to increase serum antioxidant activity and may contribute to the French Paradox by reducing LDL oxidation and atheroma formation. From this evidence, it is conceivable that flavonoid dimers play a unique, protective role in the human diet. Unlike larger procyanidins, dimers and trimers are more resistant to acid hydrolysis in the stomach and may be absorbed intact without scission of the ß-linkage. Though it is rational to attribute the health effects of red wine to procyanidins, further research is necessary to elucidate SAR of other polyphenolic constituents of wine. A reproducible heirarchy of structure-activity relationships of procyanidins and other tannins has yet to materialize.

 

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Structural features of metal-chelating flavonoids

           

            The chelating properties of flavonoids and tannins contribute to their antioxidant activity. By removing and neutralizing iron ions from iron-loaded hepatocytes, flavonoids inhibit oxidative damage. Chelation of a divalent cation does not necessarily render the flavonoid inactive, as the complex retains ROS scavenging activity. The clinical utility of this knowledge is promising in cases of oxidative stress associated with iron overload, which has been demonstrated in iron-overloaded rats. In these animals, supplemental rutin resulted in significant reductions in peroxidation of liver microsomes and oxygen radical production by phagocytes, while minimal effects were found among animals with normal iron status.

           

Hydroxyl radicals are the most reactive and detrimental ROS in biological systems. Free ferrous iron is quite sensitive to oxygen and gives rise to ferric iron and superoxide, thereby generating hydrogen peroxide. Reaction of ferrous iron with hydrogen peroxide generates the hydroxyl radical, which may subsequently oxidize surrounding biomolecules. In this process, known as the Fenton reaction,hydroxyl radical production is directly related to the concentration of copper or iron. In pathological states involving iron overload or impaired sequestering of iron by transport or storage proteins, Fenton chemistry is an important generator of ROS in vivo. Both quercetin and rutin are highly effective chelators of transition metals, suggesting little difference between aglycones and glycosides in the ability to complex metals. Fenton-induced oxidation is strongly inhibited by flavonoids with 3’,4’- catechol, 4-oxo, and 5-OH arrangements. Chelating complexes with divalent cations may form between the 5-OH and 4-oxo group, or between the 3’- and 4’-OH. By virtue of both metal-chelating properties and radical scavenging ability, polyhydroxylated flavonoids may offer considerable benefit as inhibitors of the Fenton reaction in vivo. That these polyphenols are often more effective inhibitors of metal-induced oxidation compared to non-metalinduced oxidation lends support to the role of metal chelation in flavonoid inhibition of free radical damage, which may be more significant than previously thought.

           

The well-established propensity of polyhydroxyflavonoids to complex redox-active metals poses a confounding factor in discerning relationships between structure and scavenging activity in lipid peroxidation assays. To rule out chelation, a nonmetal initiator such as CCl4 may be used, or the iron contamination of flavonoids may be assessed.

 

Azo-radical-induced peroxidation restricts antioxidant behavior to peroxyl scavenging, and therebyeliminates chelation as a contributing mechanism. From a physiological perspective, both chelation and free radical scavenging decrease oxidant status. However, given the high reducing power of tannins and the affinity of polyhydroxyflavonoids for iron, chronic pharmacological doses might exacerbate iron deficiency. Additional investigation is required to delineate SAR of chelating flavonoids and to evaluate adverse effects of flavonoids in human subjects with clinical iron deficiency.

 

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 Mechanisms of pro-oxidant action of flavonoids

 

            Flavonoids have been reported to be able to exert pro-oxidant chemistry including the formation instead of scavenging of radicals. Flavonoids with a phenol-type substituent pattern in their B-ring, like apigenin and naringenin (Fig.18) have been reported to result in a 30–50 times increase in the formation of reactive oxidant species when incubated in the presence of GSH and enzymes from thymus and bone marrow like peroxidases. Especially these flavonoids were found to generate increased lipid peroxidation and to act as a pro-oxidant at concentrations where other flavonoids were still active as antioxidants preventing this lipid peroxidation.

 

    The redoxcycling GSH-oxidizing pro-oxidant activity of this type of flavonoids seemed to partly correlate with the high one-electron oxidation potential of their corresponding phenoxyl radicals .

 

    The mechanism may proceed as depicted in fig.19. Enzymatic and/or chemical (auto)oxidation of the flavonoid generates the flavonoid semiquinone radical,which may be scavenged by GSH, thereby regenerating the flavonoid and generating the thiyl radical of glutathione. This thiyl radical may react with GSH to generate a disulfide radical anion which rapidly reduces molecular oxygen to superoxide anion radicals(fig.19)

 

 

 

 


 

 

            Another mechanism of flavonoid pro-oxidant activity is related to the formation of quinone type oxidation products and occurs especially when a 3’,4’-catechol type moiety is present in the flavonoid molecule. Flavonoids containing a catechol-type substituent pattern in their B-ring did not co-oxidize GSH when oxidized to their semiquinone. This may be due to their lower one-electron redox potentials .

 

Subsequent studies revealed that B-ring catechol-type flavonoids showed swift formation of their two electron oxidized quinone type metabolites, even upon their one

electron oxidation by peroxidases (fig.20)

 

           

Metin Kutusu: Figure 20. Pro-oxidant chemistry of catechole type flavonoids

 

 


 

             This implies the formation of electrophilic toxic quinone type metabolites which may be scavenged by GSH not by means of chemical reduction but rather by conjugate formation. The formation of these GSH flavonoid adducts was recently demonstrated providing evidence for the actual pro-oxidative formation of reactive quinone type metabolites from B-ring catechol flavonoids. The possible pro-oxidant toxicity of these catechol-containing compounds has recently been underlined by studies on the mutagenicity of estrogens. Metabolic activation of estrogens to redox active and/or electrophilic metabolites has been proposed as one of the mechanisms responsible for the link between estrogen exposure and the risk of developing cancer. Especially catechol (ortho-diol)-type of metabolites resulting from cytochrome P450 catalyzed hydroxylation of estrogens may be involved. The involvement of catechol-type metabolites has also been outlined to play a role in the metabolic activation of polycyclic aromatic hydrocarbons. Clearly flavonoids like quercetin, luteolin, fisetin and many others already contain the pro-oxidative catechol structural element, without the requirement for an initial bioconversion step.

 

            Oxidation of the catechols to quinones and their isomeric quinone methides (fig.20) generates potent electrophiles that could alkylate DNA. With respect to this possible pro oxidant toxicity it is of interest to notice that the mutagenic properties of the flavonoid quercetin have been demonstrated in a variety of bacterial and mammalian mutagenicity tests, and have been related to its quinone/quinone methide chemistry.

 

Interestingly, the structural requirements for good antioxidant activity match the requirements essential for pro-oxidant action and quinone methide formation.

 

            Based on these positive mutagenicity results in a variety of bacterial as well as mammalian test systems, several studies have investigated the possible carcinogenicity of especially quercetin. Several animal studies reported no tumor initiating activity. In contrast, Pamukcu et al reported induction of intestinal and bladder tumors by quercetin in male and female rats. A study from the National Toxicology Program reported some evidence of carcinogenic activity of quercetin in male F344/N rats, based on an increased incidence of renal tubular cell carcinomas. Ertu¨rk et al. reported bladder tumors in rats exposed to quercetin. Dunnick and Haily reported quercetin to show carcinogenic activity in the kidney of male F344/N rats.

 

            The relevance for the human in vivo situation as well as the mechanism behind this quercetin-mediated toxic effect remains a matter of debate. Ito suggested a possible factor of special interest to be the role of α2u globulin nephropathy in chemically induced renal carcinogenicity, a nephropathy which is observed selectively in male rats only. Such a hypothesis would be in line with the observations that increased numbers of benign tumors are often observed in male but not female rats.

 

 Another mechanism which may be of importance for carcinogenicity upon exposure to quercetin is the hypothesis that overloading the organism with quercetin may deplete the cofactor for catechol O-methyltransferases,S-adenosyl-L-methionine (SAM), because catechol

 O-methyltransferase metabolism represents an important metabolic pathway for catechol type flavonoids. Cofactor depletion may affect the methylation of catechol estrogens, thereby providing increased possibilities for estrogen mediated carcinogenesis, because accumulation of catechol-type estrogens in the kidney may stimulate their oxidation to DNA alkylating electrophilic quinones.

 

Altogether, the pro-oxidative carcinogenicity of quercetin and possible interference of hormonal factors in this carcinogenicity needs to be re-evaluated.

 

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ANTIOXIDANT ENZYMS

Superoxide dismutase (SOD)

Source and Nature:

SOD is an endogenously produced intracellular enzyme present in essentially every cell in the body.

 

Cellular SOD is actually represented by a group of metalloenzymes with various prosthetic groups.  The prevalent enzyme is cupro-zinc (CuZn) SOD, which is a stable dimeric protein (32,000 D).

 

SOD appears in three forms: (1) Cu-Zn SOD in the cytoplasm with two subunits, and (2) Mn-SOD in the mitochondrion. A third extracellular SOD recently has been described contains Copper (CuSOD).

                         2O2·+  2H +  SOD         H2O2 + O2

 

Mechanism of action:

 

SOD is considered fundamental in the process of eliminating ROI by reducing (adding an electron to) superoxide to form H2O2. Catalase and the selenium-dependent glutathione peroxidase are responsible for reducing H2O2 to H2O.

 

The respective enzymes that interact with superoxide and H2O2 are tightly regulated through a feedback system. Excessive superoxide inhibits glutathione peroxidase and catalase to modulate the equation from H2O2 to H2O .Likewise, increased H2O2 slowly inactivates CuZn-SOD. Meanwhile, catalases and glutathione peroxidase, by reducing H2O2, conserve SOD; and SOD, by reducing superoxide, conserves catalases and glutathione peroxidase. Through this feedback system, steady low levels of SOD, glutathione peroxidase, and catalase, as well as superoxide and H2O2 are maintained, which keeps the entire system in a fully functioning state.

 

SOD also exhibits antioxidant activity by reducing O2·- that would otherwise lead to the reduction of Fe3+ to Fe2+ and thereby promote ·OH formation. When the catalase activity is insufficient to metabolize the H2O2 produced SOD will increase the tissue oxidant activity. Hence, it was found that the antioxidant enzymes function as a tightly balanced system, any disruption of this system would lead to promotion of oxidation.

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Glutathione peroxidase enzyme

 

The glutathione redox cycle is a central mechanism for reduction of intracellular hydroperoxides.

Source and Nature:

 

It is a tetrameric protein 85,000-D. it has 4 atoms of selenium (Se) bound as seleno-cysteine moieties that confers the catalytic activity. One of the essential requirements is glutathione as a cosubstrate.

 

Glutathione peroxidase reduces H2O2 to H2O by oxidizing glutathione (GSH) (Equation A). Rereduction of the oxidized form of glutathione (GSSG) is then catalysed by glutathione reductase (Equation B). These enzymes also require trace metal cofactors for maximal efficiency, including selenium for glutathione peroxidase; copper, zinc, or manganese for SOD; and iron for catalase.

 

H2O2 + 2 GSH         GSSG + 2 H2O (equation A)

 

GSSG + NADPH + H+          2 GSH + NADP+ (equation B)

 

The catalase enzyme:

 

This enzyme is a protein enzyme present in most aerobic cells in animal tissues. Catalase is present in all body organs being especially concentrated in the liver & erythrocytes.  The brain, heart, skeletal muscle contains only low amounts.

 

Catalase and glutathione peroxidase seek out hydrogen peroxide and convert it to water and diatomic oxygen. An increase in the production of SOD without a subsequent elevation of catalase or glutathione peroxidase leads to the accumulation of hydrogen peroxide, which gets converted into the hydroxyl radical. Indeed research in the pathogenesis of Down’s syndrome has revealed that the existence of trisomy 21 leads to the overproduction of SOD, the gene for which is located also on chromosome 21. This finding is intriguing in that it reveals the possibility of a genetic link to the increased activity of free radicals.

                          

2 H2O2           2 H2O + O                      

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Other antioxidants

Retinoids:

Retinol, retinoic acid but not retinyl palmitate or retinyl acetate all have antioxidant properties. However, retinoids in general are not classified as antioxidants as they mainly function as antiproliferatives.

Glutathione (GSH):

GSH is synthesized intracellularly from cysteine, glycine, and glutamate.

 

In addition to its role as a substrate in GSH redox cycle, GSH is also a scavenger of hydroxyl radicals and singlet oxygen. It is capable of either directly scavenging ROI or enzymatically via glutathione peroxidase, as described previously. In addition, GSH is crucial to the maintenance of enzymes and other cellular components in a reduced state. GSH also has an important role in xenobiotic metabolism and leukotriene synthesis. It is found in millimolar concentration in all human cells.

 

The majority of GSH is synthesized in the liver, and approximately 40% is secreted in the bile. The biologic role of GSH in bile is believed to be defence against dietary xenobiotics and lipid peroxidation in the lumen of the gut and protection of the intestinal epithelium from oxygen radical attack .

CoQ10:

CoQ10 (Coenzyme Q10) is also known as ubiquinone. It is found in almost every living cell (hence the name "ubiquitous") and is essential to energy production by the mitochondria. Far beyond producing energy, CoQ10 can protect the body from destructive free radicals and enhance immune defences.

Uric acid:

Acts as an endogenous radical scavenger and antioxidant. It is present in about 0.5 mmol/L in body's fluids and is the end product of purine metabolism. Uric acid is a powerful scavenger of singlet oxygen, peroxyl radical (ROO·) and ·OH radical.

Albumin:

Depending on the fact that albumin has one sulfhydryl group per molecule, it itself scavenges several free radicals  and thus can be considered as one of the primary extracellular defense systems.

 

Albumin is an additional sacrificial antioxidant that can bind copper tightly and iron weakly to its surface. The bound metals would still be on its surface. The bound metals would still be available for participation in Haber-Weiss reaction, but any generated ·OH would immediately react with and be scavenged by albumin. The resultant protein damage is biologically insignificant because of the large amount of available albumin and free radicals would be inactivated before reacting with other more vital protein structures.

 

Other plasma proteins namely ceruloplasmin and transferrin have also shown antioxidant activity.

Drugs:

Several pharmaceutical agents have been found to exert an antioxidant effect :

 

·        Xanthine oxidase inhibitors: e.g. allopurinol, folic acid.

·        NADPH inhibitors: e.g. adenosine, calcium channel blockers.   

·   Inhibitors of iron redox cycling: deferoxamine, apotransferin and ceruloplasmin

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 Ascorbic acid (vitamin C)

Source and Nature:

    Ascorbic acid (vitamin C) is a water-soluble, antioxidant present in citrus fruits, potatoes, tomatoes and green leafy vegetables.

 

    Vitamin C, also known as ascorbic acid, L-ascorbic acid, dehydroascorbic acid, the antiscorbutic vitamin, L-xyloascorbic acid and L-threo-hex-2-uronic acidy-lactone, is a much talked about vitamin, with people claiming it as a cure-all for may diseases and problems - from cancer to the common cold.

 

    Humans are unable to synthesize l-ascorbic acid from d-glucose due to absence of the enzyme L-gulacolactone oxidase . Hence, humans must therefore obtain ascorbic acid from dietary sources.

Mechanism of Action:

    The chemopreventive action of vitamin C is attributed to two of its functions. It is a water-soluble chain breaking antioxidant . As an antioxidant, it scavenges free radicals and reactive oxygen molecules, which are produced during metabolic pathways of detoxification. It also prevents formation of carcinogens from precursor compounds . The structure of ascorbic acid is reminiscent of glucose, from which it is derived in the majority of mammals.

One important property is its ability to act as a reducing agent (electron donor). Ascorbic acid is a reducing agent with a hydrogen potential of +O.08V, making it capable of reducing such compounds as a molecular oxygen, nitrate and cytochromes a and c. Donation of one electron by ascorbate gives the semi-dehydroascorbate radical (DHA). Ascorbate reacts rapidly with O2·- and even more rapidly with ·OH to give DHA. DHA, itself can act as a source of vitamin C.

 

Ascorbic acid + 2O2·- + 2H+         H2O2 + DHA

 

    It has also been shown that ascorbate is more potent than a-tocopherol in inhibiting the oxidation of LDL in a cell free system . Co-incubation of LDL with ascorbate during similar oxidative condition inhibited LDL oxidation and resulted in preservation of the endogenous antioxidant in the LDL particle . The concentration of ascorbate used to inhibit LDL oxidation (40-60 mm) is well within the normal plasma range (23-85 pm).

 

    Vitamin C also contributes to the regeneration of membrane bound oxidized vitamin E. It will react with the a -tocopheroxyl radical, resulting in the generation of tocopherol in this process itself being oxidized to dehydroascorbic acid . Vitamin C supplementation in animals leads to increased plasma and tissue levels of vitamin E.

I

    In vitro studies suggest that the antioxidant properties of ascorbic acid may not increase linearly as ascorbic acid concentrations rise . Moreover, ascorbic acid alone can act as a "pro-oxidant" or reducing agent to react with copper or iron salts. Ferric iron (Fe3+) formed by the reaction,  Fe2+ + H2O2          HO + ·OH + Fe3+, is converted by ascorbic acid to ferrous (Fe2+) ion. Ferrous iron is therefore recycled to promote the conversion of more H2O2 to ·OH .

 

Vitamin C is required for

  

     Vitamin C is required in the synthesis of collagen in connective tissue, neurotransmitters, steroid hormones, carnitine, conversion of cholesterol to bile acids and enhances iron bioavailability. Ascorbic acid is a great antioxidant and helps protect the body against pollutants.

   

    Because vitamin C is a biological reducing agent, it is also linked to prevention of degenerative diseases - such as cataracts, certain cancers and cardiovascular diseases.

Ascorbic acid also promotes healthy cell development, proper calcium absorption, normal tissue growth and repair - such as healing of wounds and burns. It assists in the prevention of blood clotting and bruising, and strengthening of the walls of the capillaries.

 

    Vitamin C is needed for healthy gums, to help protect against infection, and assisting with clearing up infections, and is thought to enhance the immune system and help reduce cholesterol levels, high blood pressure and preventing arteriosclerosis.

 

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The Pro-oxidant chemistry of the natural antioxidant Vitamin C

 

    It is commonly believed that the beneficial effects of vitamin C (ascorbate)  increase with the amount of vitamin C consumed. Vitamin C is presently marketed as an antioxidant supplement, and claimed to increase resistance to diseases and oxidative stress. Several prospective studies have investigated the effect of vitamin C intake from fruits and vegetables and cancer development. Outcomes obtained have varied from an inverse association to no effect . Observational epidemiological studies suggest that antioxidant vitamins, including vitamin C, at sufficient concentration inhibit heart disease and cancer . Some recent studies have shown a significant reduction in the risk of lung cancer by increased dietary vitamin C uptake . An association between plasma vitamin C levels and lung cancer risk has been less firmly established. Vitamin C plasma levels have been reported to be not predictive of subsequent lung cancer or overall cancer mortality , or to show an only modest or non-significant beneficial effect, which may be most relevant for cancer of the digestive tract, particularly of the esophagus and the stomach . Another study has concluded that vitamin C has not demonstrated substantial efficacy in cancer chemoprevention . As a result, the in vivo beneficial effects of vitamin C supplementation can be questioned.

    In addition to its well-known antioxidant properties, ascorbate, depending on the environment and conditions in which the molecule is active, can also act as a pro-oxidant . In vitro induction of lipid peroxidation by ascorbate-iron systems is a standard test for inducing oxidative stress and testing antioxidant activity of other antioxidants. In this model system, chelation of Fe2+ by ascorbate creates an active catalyst for the production of reactive oxygen species. When Fe3+ is present, vitamin C can convert Fe3+ into Fe2+, which subsequently reacts with oxygen or hydrogen peroxide resulting in formation of superoxide anions and hydroxyl radicals (Fig. 21) .

    Also of importance in relation to the claimed anticarcinogenic and pro-oxidative action of vitamin C is that vitamin C has been reported to induce cell death, nuclear fragmentation and internucleosomal DNA cleavage in human myelogenous leukemia cell lines, all in line with the ability of high concentrations of vitamin C to induce apoptosis in various tumor cell lines. The apoptosis-inducing activity of vitamin C has been ascribed to its pro-oxidant action and is inhibited by catalase, antioxidants like N-acetylcysteine and GSH, Ca2+ depletion and Fe3+, but stimulated by H2O2, Cu2+ and iron chelators .

Fig.21. Pro-oxidant chemistry of vitamin C

 

    Especially the observations that Fe3+ inhibits ascorbate mediated apoptosis, and that iron chelators stimulate ascorbate mediated apoptosis, point at a pro-oxidant mechanism different from the generally accepted mechanism for ascorbate-Fe mediated induction of lipid peroxidation and oxidative stress depicted in Fig. 21. In agreement with this observation is that reactive oxygen species are found to inhibit apoptosis by the induction of NFkB . This inhibiting effect of oxidative stress on apoptosis may provide a mechanistic model for the vitamin C induced stimulation of apoptosis. An alternative mechanism suggested for the ascorbate-induced apoptosis is that induction of hypoxia-inducible factor 1- stabilizes p53 resulting in growth arrest or apoptosis . Because the activity of p53 is generally related to tumor suppression, this would imply that the vitamin C induced apoptosis might be a beneficial rather than toxic effect.

    Also of interest with respect to the balance between beneficial and toxic effects of vitamin C supplementation are the results from an in vivo study in which 30 healthy volunteers received dietary supplements of 500 mg of vitamin C per day for 6 weeks . The parameter used to assess the anti- and pro-oxidant effects of vitamin C was the level of modified DNA bases detected in peripheral blood lymphocytes. Whereas the level of 8-oxoguanine was found to decrease upon supplementation relative to placebo, the level of 8-oxoadenine increased. Since both 8-oxoguanine and 8-oxoadenine may represent mutagenic lesions this observation, although mechanistically unexplained, reflects that even for vitamin C supplementation the ultimate balance between anti- and prooxidative effects is more complex than commonly believed.

    Recently, the fact that vitamin C has proven to be ineffective in cancer chemoprevention has been related to vitamin C mediated formation of genotoxins from lipidhydroperoxides, even in the absence of transition metal ions . Vitamin C was reported to be even more efficient than transition metal ions at initiating the decomposition of for example 13(S)-hydroperoxy-( Z,E)-9,11-octadecadienoic acid to ,α ß-unsaturated aldehydic bifunctional electrophiles. The mechanism suggested for this vitamin C mediated prooxidant action is similar to the formation of ,α ß-unsaturated aldehyde genotoxins observed with transition metals, since vitamin C is suggested to result in peroxide decomposition by means of one electron donation . Lee et al. also indicate that their finding that vitamin C generates bifunctional electrophiles explains why hydroperoxide dependent lipid peroxidation in vitro is enhanced by vitamin C, and could help to explain why vitamin C has not demonstrated substantial efficacy in cancer chemoprevention trials.

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 Selenium

 

        A couple of years ago, selenium used to be treated as a very toxic substance, but modern science now regards it as essential - but in small quantities. An overdose or deficiency of selenium is equally bad, and good steady amounts should be available - but in small quantities.

 

Selenium is required for

 

        One of the main activities of this mineral is its anti-aging properties and its ability to help rid the body of free radicals, as well as toxic minerals such as mercury, lead and cadmium. It is helpful in fighting infections since it stimulates increased antibody response to infections, promotes more energy in the body, and while it helps with alleviating menopausal symptoms in women, it assists the male in producing healthy sperm.

 

        In certain cases selenium has also proven effective in helping to fight cold sores and shingles, which are both caused by the herpes virus. Some researchers have shown that in selenium-deficient animals a harmless virus can mutate into a virulent form capable of causing damage and death - this has also been followed up with other studies, which seem to indicate that selenium helps to keep the spread and multiplying of viruses in check.

 

        Selenium is also used against arthritis and multiple sclerosis and if provided in adequate amounts it is thought to help prevent cancer as well. Tissue elasticity and pancreatic function is also dependant on this mineral.

 

    In a study it was shown that selenium could be useful in treating certain cancers, and is also helpful in making the blood less "sticky", which is helpful in preventing heart attacks and strokes.

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Dosage

 

        The dosage is the Recommended Daily Allowance (RDA), but be aware that this dosage is the minimum that you require per day, to ward off serious deficiency of this particular nutrient. In the therapeutic use of this nutrient, the dosage is usually increased considerably, but the toxicity level must be kept in mind.

 

        In the case of microelements, such as trace elements, the amounts are very small, yet they are still important and 70 micrograms per day is taken as the required dosage.

 

Toxicity and symptoms of high Selenium intake

 

        As mentioned earlier - selenium is toxic and too large quantities may result in hair loss, tooth decay, brittle nails, white spots, poor appetite, sour taste in the mouth, loss of feeling in the hands and feet, change in skin pigmentation and the breath may have a garlic smell.

 

Best used with

 

        Selenium should always be taken with vitamins E, A and beta-carotene, and it is preferable when taking a supplement to take selenium in the form of selenocysteine or selenomethionine, which are both organic.

 

People with yeast intolerance should check the source of the selenium used in the supplement, as certain manufacturers obtain selenium from yeast.

 

When more Selenium may be required

 

    Men need more selenium than women as it is lost in the seminal fluid, and people staying in areas where the soil is poor in selenium, should also pay attention to their selenium intake.

 

Food sources of Selenium

 

Brazil nuts are excellent sources of selenium, but are also found in whole grains and shellfish.

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