Nutritional Antioxidants: Mechanisms of Action, Analyses of Activities and Medical Applications
Jacob Vaya1
and Michael Aviram 2*
1Laboratory of Natural Medicinal Compounds, Migal - Galilee Technological Center, Kiryat Shmona 10200, Israel and 2The Lipid Research Laboratory, Rambam Medical Center, Technion Faculty of Medicine and Rappaport Institute for Research in the Medical Sciences, Haifa, 31096 Israel
*Address
correspondence to this author at the Head,
Abstract: The important role of dietary antioxidants in maintaining the integrity of living organisms is gaining ever-increasing recognition.
New data are constantly gathered to show the role of oxidative stress and the involvement of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the pathogenesis of degenerative diseases. These diseases are associated with a disturbance in the necessary balance between oxidation and reduction status in blood and tissues, leading to oxidation of lipids, proteins and nucleic acids. Such oxidative damage is accompanied by changes in macromolcules structure and function and by the manifestation of clinical disorders such as cardiovascular diseases and cancer. Hence, widespread research is being conducted aiming to investigate the possible effects and mechanisms of action of dietary antioxidants in these diseases.
The present review focuses on the types of the various natural antioxidants, their tissue distribution, bio-availability, mechanisms of actions and therapeutical applications. Antioxidants may exert their effect on biological systems by different mechanisms including electron donation (as reducing agents), metal ion chelation (thereby eliminating potential free radicals), sparing of antioxidants (co-antioxidants) or by genes expression regulation. The various methods currently in use for determination of antioxidant activities and the role of antioxidants in the management of human diseases are discussed.
1. Introduction
Antioxidants are a group of substances which, when present at low concentrations, in relation to oxidizable substrates, significantly inhibit or delay oxidative processes, while often being oxidized themselves. The applications of antioxidants are widespread in the industry and are in use in preventing polymers from oxidative degradation, rubber and plastic from losing strength, gasoline from autoxidation, synthetic and natural pigments from discoloration and as additives to cosmetics, food (especially food with high fat content), beverages and baking products.
In recent years there has been an increased interest in the application of antioxidants to medical treatment as information is constantly gathered linking the development of human diseases to oxidative stress. The generally accepted hypothesis is that in any biological system an important balance must be maintained between the formation of reactive oxygen and nitrogen species (ROS and RNS, respectively) and their removal (Fig. 1). ROS and RNS are formed regularly as a result of normal organ functions, or as a result of excess oxidative stress. The reactive species superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radical (HO•), nitrogen oxide (NO•), peroxynitrite (ONOO-) and hypochlorous acid (HOCl), are all products of normal metabolic pathways of the human organs, but under certain conditions, when in excess they can exert an harmful compounds. Superoxide, the most important source of initiating radicals in vivo, is produced in mitochondria during electron chain transfer and it regularly leaks outside of the mitochondria. To maintain an oxido/redox balance, organs protect themselves from the toxicity of excess ROS/RNS in different ways, including the use of endogenous and exogenous antioxidants (Fig. 1).
Fig. (1). Oxidation/Reduction Balance.
Naturally occurring antioxidants, of high or low molecular weight, can differ in their composition, their physical and chemical properties and in their mechanism and site of action. They can be divided into the following categories:
1. Enzymes, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase, attenuate the generation of reactive oxygen species by removing potential oxidants or by transforming ROS/RNS into relatively stable compounds. SOD which was discovered in the late 60s [1], catalyzes the transformation of the superoxide radical into hydrogen peroxide, which can then be further transformed by the enzyme catalase into water and molecular oxygen. While superoxide anion in itself is not particularly reactive, it can reduce transition metal ions, such as iron, and it is converted to one of the most reactive radicals-the hydroxyl radical. Thus, elimination of superoxide can attenuate the formation of the harmful hydroxyl radical. Glutathione peroxidase (GPx) reduces lipid peroxides (ROOH), formed by the oxidation of polyunsaturated fatty acid (PUFA), to a stable, non-toxic molecule-hydroxyl fatty acid (ROH). Together with phospholipases GPx can also convert phospholipid hydroperoxides (PL-OOH) into phospholipid hydroxide (PL-OH) [2,3].
2. High Molecular Weight Proteins, such as albumin, ceruloplasmin, transferrin and haptoglobin, which are all present in plasma, bind to redox active metals and limit the production of metal-catalyzed free radicals [4]. Albumin and ceruloplasmin can bind copper ions, and transferrin binds free iron. Haptoglobin binds heme-containing proteins and can thus clear them from the circulation. Both free and heme-associated proteins have pro-oxidant properties due to their reaction with H2O2 to form ferryl species which can easily initiate lipid peroxidation [5].
3. Low Molecular Weight Antioxidants, are subdivided into lipid-soluble antioxidants (tocopherol, carotenoids, quinones, bilirubin and some polyphenols) and water-soluble antioxidants (ascorbic acid, uric acid and some polyphenols). They delay or inhibit cellular damage mainly through their free radical scavenging property.
2. Nutritional Antioxidants Bioavailability
2.1 Types and Tissue Distribution of Antioxidants
Among the lipid-soluble, low molecular weight antioxidants, the most important group is the tocopherols which are represented chiefly by -tocopherol (-TOH, vitamin E) [6]. -TOH is a highly effective antioxidant in the lipid phase of cell membranes, capable of breaking the chain reaction by scavenging a peroxyl radical [7]. The tocoperoxyl radical (TO•) generated can be stabilized, either by donating a second electron to form a quinone derivative, or by being recycled back to -TOH by vitamin C.
Low density lipoprotein (LDL), the major cholesterol carrier in plasma is consisted of an average of six molecules of -TOH per LDL particle, which together with ubiquinol, -carotene and lycopene, comprise the antioxidant pool in LDL. A second large family of ROS quenchers, the carotenoids, have a typical conjugated polyene structure, and trap singlet oxygen (1O2) most efficiently. The antioxidative function of carotenoids in mammals however is still unclear [8], with clinical trials showing conflicting results [9-14].
-Carotene or lycopene may react with peroxyl radical to form a carbon-centered radical, which can be stabilized as a result of its high delocalization possibilities. The antioxidative activity of carotenoids seems to change according to the oxygen pressure, from an efficient antioxidant at low pressure to a pro-oxidant at high pressure [15]. Ubiquinol, another effective lipid-soluble antioxidant, inhibits lipid peroxidation and, like vitamin C, can also re-generate the -TO• radical to form the active tocopherol [16].
The major water-soluble low molecular weight antioxidants in human plasma are vitamin C and uric acid. Vitamin C acts as a strong antioxidant in the plasma and presents a synergistic effect with other antioxidants (co-antioxidant). Uric acid is formed during purine metabolism, and it also presents strong antioxidant activity toward ROS in aqueous phase [17] (Fig. 2).
Fig. (2). The structure of naturally
occurring lipophilic and hydrophilic plasma antioxidants
Both tocopherols and ubiquinol are secondary metabolites of the phenol and polyphenol groups. Some of the polyphenols are lipid soluble but most of them are water soluble. Polyphenols biosynthesis in the plant kingdom is through the shikimate, mevalonate and phenylpropanoid pathways. The type and levels of phenols and polyphenols produced in plants varies greatly depending on genetic factors, environmental conditions, germination, degree of ripeness, processing and storage. Polyphenols are partly responsible for the astringency and bitterness associated with some foods and beverages. More than 8000 phenolic compounds are known [18], of which almost 2/3 belong to the flavonoid family, all sharing an aromatic ring(s) substituted with at least one free hydroxyl group (Fig. 3). Some simple phenols have a simple structure of low molecular weight, with one aromatic ring (e.g., thymol, resorcinol), whereas others have a complex structure, forming complex polymers (e.g., lignin, tannin). Phenols and polyphenols, in addition to the basic phenolic structure, are frequently conjugated to mono-, di- or more sugar residues (mostly in positions 3 or 7), forming glycosides. In some polyphenols, the basic structure is associated with other functional groups, such as lipids, amines, or carboxylic acids. The major subclasses of phenols and polyphenols are shown in (Fig. 3).
Fig. (3). Phenol and polyphenol antioxidants.
Lignin provides the main structural element of plants. It is composed of phenyl propanoid units (C6-C3), synthesized through the shikimic acid pathway. Lignin is associated with cell wall carbohydrates and confers rigidity and toughness to the cell wall. Tannins are polyphenols which occur in vascular plant tissues and they exist in two major forms: condensed and hydrolyzable. The condensed tannins exist as oligomers and polymers of anthocyanidins (Fig. 3), and the hydrolyzable tannins consist of gallic acids which is bind to carbohydrate, forming an ester. As mentioned above, the flavonoid family is the major group among the phenol compounds with more than 5000 known compounds. They are common in fruits, vegetables, seeds, cereals, nuts, wines and tea, and share the common structure of two aromatic rings (rings A and B) and an heterocyclic ring C, with at least one hydroxyl group attached to these rings (Fig. 3). Their roles in plants are thought to involve pigmentation (anthocyanines), enzyme activity regulation, protection from UV irradiation, chelation agents of transition metal ion and also reducing agents.
2.2. Bioavailability of Antioxidants
The in vivo antioxidant activity of phenols is largely dependent on their bioavailability. This in turn, depends on several factors, including their release from the food matrix in the gut, their stability in the gut flora, modifications in the intestine (glycosylation), absorption through the intestinal wall to the blood stream, stability in the liver and accessibility to the tissue in the target site. Flavonoids can be detected in the urine and plasma of humans after consumption of flavonoids- rich food [19,20].
The absorption of polyphenol depends primarily on factors related to their basic structure, including the degree of glycosylation, their molecular size and their level of conjugation with other polyphenols. Flavonoids (glycosides and free aglycones) are prone to degradation by gut microorganisms [21,22]. Hence, only a small proportion of the digested flavonoids reach the blood-stream [23]. Absorption of glycosides through the intestinal tract in mammals requires first the hydrolysis of the sugar residue [24]. As mammals lack the appropriate -glycosidases, hydrolysis to release free aglycones occurs mostly, in the large intestine, by cecal microflora which, at the same time, also degrade dietary phenols. A major site for polyphenol metabolism is the liver [24], and some flavonoid metabolism occurs also in the intestinal walls and kidneys.
Different flavonoids access their target sites in different ways. For example, while only less than 1% of catechin and quercetin exposed to LDL binds to the lipoproteins, under the same experimental conditions, more than 80% of the lipophilic isoflavan glabridin binds to LDL [25,26].
3. Mechanism of Action of Antioxidants
Two principle mechanisms of action have been proposed for antioxidants [27]. The first is a chain-breaking mechanism, by which the primary antioxidant donates an electron to the free radical present in the system (e.g., lipid radical).
The second mechanism involves removal of ROS/RNS initiators (secondary antioxidants) by quenching chain-initiating catalysts. The present review section will discuss the mechanism of action of primary and secondary antioxidants, and of those compounds which by themselves can act as antioxidants, but under certain conditions they can also prevent other pro-oxidants from initiating free radical reaction acting as co-antioxidants. The effect of some antioxidants on gene expression will be also discussed.
3.1. Electron Donation
Primary antioxidants are compounds which are able to donate hydrogen atom rapidly to a lipid radical, forming a new radical, more stable than the initial one [28-30]. Biological organs contain many polyunsaturated fatty acids (PUFA), such as linoleic, linolenic and arachidonic acids, mainly in the form of esters with phospholipids, triglycerides, or with cholesterol. These PUFA can undergo lipid peroxidation which can be interrupted by antioxidants by the donation of electrons. The mechanism of lipid peroxidation is shown in (Fig. 4). In the first step, hydrogen atom is abstracted from the PUFA (represented in Fig. 4 as linoleic acid) [30,31], by initiators (such as enzymes) or by ROS, such as a hydroxyl radical (HO), peroxyl radical (ROO), alkoxyl radical (RO) or alkyl radical (R) generated in the biological system [32]. The initiator abstracts hydrogen atom from the allylic position of the fatty acid (the most labile position), and this is followed by a rapid isomarization of one of the double bonds to yield a trans configuration, thereby forming two conjugated double bonds and a new radical in either position 13 or 9 (in Fig. 4, position 13 is shown). In the presence of molecular oxygen (O2), a rapid reaction can take place between the fatty acid alkyl radical and the molecular oxygen, forming a peroxyl radical which can then further abstract hydrogen atom from a new linoleic acid, to form linoleic hydroperoxide and a new linoleyl radical. The last two reactions are known as the propagation steps in lipid peroxidation and cause chain reaction.
Fig. (4). Mechanism of linoleic acid
peroxidation and reactive oxygen species (ROS) formation.
Primary antioxidants, such as flavonoids, tocopherol and ascorbic acid, can stop chain reaction by donating an electron to the peroxyl radical of the fatty acid, and thus stops the propagation steps. Enzymes such as glutathione peroxidase (GPx) can also act as antioxidants, by reducing oxidized lipids and phospholipid hydroperoxide (ROOH, and PL-OOH) to their corresponding alcohols (ROH, PL-OH) [33].
Any compound which can react with the initiating radical (or inhibit the initiating enzyme), or reduce the oxygen level (without generating reactive radical species), can be considered as a secondary antioxidants.
Inhibition or progression of lipid peroxidation can also be catalyzed by other mechanisms, e.g., reaction of PUFA with singlet oxygen (1O2), an energetically exited molecular oxygen, or with metal ions. Thus, PUFA (RH) can react directly with singlet oxygen to form lipid hydroperoxide (Fig. 4, Reaction II).
In addition, the presence of transition metal ions, such as copper or iron, in either oxidative state (Cu+1, Cu+2, Fe+2, Fe+3), can re-initiate lipid peroxidation and form species with much higher activity than the starting materials (Fig. 4, Reactions III and IV). Thus, in a reaction catalyzed by metal ion fatty acid hydroperoxide can break down to form alkoxyl radical (RO•), which is much more reactive than the initial hydroperoxide (Fig. 4, Reaction III). This alkoxyl radical can then re-initiate lipid peroxidation, or it can be hydrolyzed to from aldehyde molecules. Some of the most common methods for the detection of oxidative stress are based on measuring the amount of intermediates or of fragmentation products of such reactions and involve the spectrophotometric measurement of conjugated dienes (CD) at 234 nm, the measurement of lipid peroxides (PD) generated, or the measurement of malondialdehyde equivalents formed by reaction with thiobarbituric acid (TBA).
The oxidation of polyunsaturated lipids results first in the formation of lipid hydroperoxide (ROOH), lipid hydroxide (ROH) and oxysterols , which further degrade to small fragments, such as aldehydes (Fig. 5). Aldehydes formed are predominately a group of 4-hydroxy-2-alkenal structure, such as 4-hydroxy-2-nonenal [35,36]. Exposure of cells to such aldehydes can result in growth inhibition, alteration in enzymatic activities and inhibition of protein synthesis [35]. These effects are the result of the high nucleophilic properties of aldehydes, which enable them to react with electrophilic sites, such as amino and thiol groups. Such reactions of aldehydes can forms Schiff bases with the -amino groups of lysine residues, and can then further generate cross-linking between lipid and protein molecules [37,38]. For example, during LDL oxidation, about half of its reactive -amino groups become masked [39], with the formation of apolipoprotein B-100-bound aldehydes (such as 4-hydroxy-2-nonenal), to the amino acids histidine and lysine residues of the LDL apo B-100 protein [38]. These LDL-protein modifications alter its charges and configuration, leading to a more negatively charged LDL with atherogenic properties.
Major aldehydes
formed during lipid peroxidation (Fig. 5)
are the 4-hydroxy-2-alkenals (I) and 4-hydroxy-2-nonenal (II). Malondial-dehyde
(III) is another aldehyde most frequently detected during lipid peroxidation.
Other compounds (Fig. 5, IV-VI) are examples of adducts formed in vivo as a result of reactions between
malondialdehyde and lysine residues (IV and V) or between 4-hydroxy-2-nonenal
and other amino acids (Cys,
3.2. Metal Chelation
Secondary antioxidants can retard the rate of radical initiation reaction by means of initiators elimination. This can be accomplished by deactivation of high energy species (e.g., singlet oxygen), absorption of UV light, scavenging of oxygen and thus reducing its concentration, chelations of metal catalyzing free radical reaction, or by inhibition of peroxidases, such as NADPH oxidase, xanthine oxidase, dopamine--hydroxy-lase or lipoxygenases.
The ability of antioxidants to chelate transition metal ions can be followed spectroscopically [40]. High molecular weight proteins bind directly or indirectly to redox active metals and thus inhibit the production of metal-catalyzed free radicals. Some low molecular weight compounds, such as polyphenols, in addition to their ability to donate hydrogen atom and thus to act as chain-breaking antioxidants, can also chelate transition metal ions and hence inhibit free radical formation [40-42].
Thus, the relative contribution of free radical scavenging or of metal ion chelation to the antioxidative effect is not clear [43,44]. Methods have been developed to distinguish between the two types of activities. Using electrochemical oxidation as a model for the scavenging reaction [42], the half peak oxidation potential (Ep1/2) can be measured [45]. Quantum chemical calculation of the differences in heat formation (Hf) between the radical and its parent antioxidant molecule (Hf) is also in use for measurement of the ability to donate an electron [46,47]. Hf value can give information about the ease with which the radical is formed, and thus distinguish between the contribution resulted from hydrogen atom donation and the effect of ion metal chelation.
Another simple, but very informative technique to differentiate between the above two potential contributors to antioxidant activity is the reaction of an antioxidant with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) [48] or with galvinoxyl [49] (Fig. 6). The principle of this method is that, in the presence of a molecule consisting of a stable free radical (DPPH), an antioxidant with the ability to donate a hydrogen atom will quench the stable free radical, a process which is associated with a changes in the absorption which can be followed spectroscopically. This simple method can be applied either when the antioxidant is in its pure form, or in a mixture (e.g., a natural extract). Using this method, it is possible to follow the kinetics of the reaction, the number of electrons an antioxidant molecule can donate, and also to estimate the structure of the oxidized antioxidant after it has donated hydrogen atom(s).
Fig. (6). Structure of
galvinoxyl, DPPH
and of quercetin reduced and oxidized forms.
Studying the interaction of the flavonol quercetin with copper ion, Brown et al [50] showed that during interaction of quercetin with transition metal, oxidation of the flavonoid occurs at the free 3-OH group, with an additional oxidation of the 4'-OH group. During the formation of a copper-flavonoid chelate, an oxo/redox reaction takes place (Fig. 6). It seems that the most important moiety of polyphenol for chelation of transition metal ions is the catechol structure at ring B (two adjacent hydroxyl groups at 3’, 4’ positions).
3.3 Co-antioxidants
Sixty years ago, Golumbic and Mattill [51] observed that while ascorbic acid (vitamin C) alone has little effect in preventing lard oil from oxidation, the combination of ascorbic acid with tocopherol (-TOH) gave rise to a strong synergistic antioxidative effect. These authors concluded that the role of ascorbic acid was to preserve -TOH from consumption. This behavior of ascorbic acid is termed a co-antioxidant effect. Since then, many other compounds have been found to produce a similar co-antioxidant effect with -TOH (Fig. 7) [52].
Fig. (7). Structure of some
co-antioxidants.
Several studies have been published suggesting a mechanism by which ascorbic acid acts as co-antioxidant and enhances -TOH activity [53-55]. Tappel [53] suggested that, in a mixture of ascorbic acid and -TOH, ascorbic acid could reduce oxidized -TOH in vivo. Using pulse radiolysis, it was shown indeed that the ascorbate anion formed could reduce different types of phenoxyl radicals [56], including the -tocopheroxyl radical (-TO•) [57]. This latter observation was confirmed by ESR spectroscopy [58,59]. Studying the effects of the medium by which peroxidation was initiated, Doba T et al [54] showed that ascorbic acid was a satisfactory co-antioxidant in the presence of -TOH when lipid peroxidation was initiated in the aqueous phase. When the peroxidation was initiated in the lipid phase, ascorbic acid acted as an excellent coantioxidant and demonstrated synergism with -TOH.
Bowry et al [52] examined the co-antioxidative activity of various radical scavenger compounds, including bilirubin, aminophenol derivatives, catechol derivatives, various quinols, and 6-palmityl ascorbate (Fig. 7). Like ascorbic acid, all these compounds were also very effective. Interestingly, while the female hormone estradiol was not active as a co-antioxidant, 2-hydroxy estradiol, the catabolic product of estradiol which contains an additional hydroxyl group at the aromatic ring to form a catechol structure, was very active, almost as much as ascorbic acid (Fig. 7). Another very active cellular co-antioxidant is the tryptophan metabolite, 3-hydroxyanthranilic acid, whose molecule contains a primary amino group, [60]. It seems that the high co-antioxidant activity of 3-hydroxyanthranilic acid is due to the two adjacent amino and phenolic hydroxyl groups, since these authors also found that 2-aminophenol showed the same co-antioxidant activity as did 3-hydroxyanthranilic acid.
Fig. (7). Structure of some
co-antioxidants.
The number of known natural amino antioxidants (aniline derivatives) is very limited, compared with the group of phenols and polyphenols. Comparison of the co-antioxidant activity of phenols with molecules of similar structure, in which part or all the hydroxyl groups are replaced by amine groups (e.g., catechol with 2-aminophenol or 2-aminoaniline) could be of interest, since indications in the literature showed the superiority of aminophenols over phenols as antioxidants [61,62].
In a series of publications [55,63] it was demonstrated that -TOH can act as pro- or as anti-oxidant in the LDL particles, and the type of activity (anti- vs. pro-oxidants) depends on the rate by which -TOH is consumed. Under oxidative stress, the initially formed peroxyl radical ROO• reacts with -TOH (Fig. 8, Reaction I) to generate a single -TO•, which is a lipophilic species and as such, it is incapable of escaping from the hydrophobic core of LDL. In the presence of an endogenous co-antioxidant, such as ubiquinol, the co-antioxidant within the LDL core donates an electron to -TO•, and thus regenerates -TOH (Fig. 8, Reaction II), while the ubiquinol radical is converted to a stable molecule-ubiquinone. In the absence of co-antioxidants, or after their consumption, and also under conditions of a high flux of initiating peroxyl radicals generation (high copper concentration ions or free radical generating compounds such as AAPH), a second ROO• react with -TO• to form a stable compound (Fig. 8, Reaction III).
Fig. (8). The mechanism of vitamin E (a-TOH)-mediated LDL lipid peroxidation.
In contrast, under conditions which generate a mild flux of initiating peroxyl radicals, or in the presence of low concentrations of copper or iron ions, the lipophilic -TO• trapped in the LDL particle is unlikely to react with a second ROO•, since it is generated at a too low rate. Thus, a reaction between -TO• and PUFA molcule in the LDL core (linoleic, arachidonic) is most likely to occur, resulting in the abstraction of an hydrogen atom from the allylic methylene group in the PUFA molecule (Fig. 8, Reaction IV), and thus initiates a new chain reaction, while -TO• is converted into -TOH [55,63-65].
The mechanism of pro-oxidation in LDL was termed tocopherol-mediated peroxidation (TMP). The effect of a co-antioxidant, such as ascorbic acid, on the above mechanism is to eliminate -TO• from the LDL core at an early stage of LDL oxidation, before it reacts with PUFA (RH). An efficient co-antioxidant (such as ascorbic acid) reacts strongly with the peroxyl radical and allows the co-antioxidant radical formed to escape rapidly from the core of the LDL particle into the aqueous layer.
3.4. Gene Expression
Antioxidants possess the ability to donate electrons and thereby act as reducing agents, to chelate metal ions and thereby remove potential radical initiators and to facilitate antioxidant activity by other compounds (co-antioxidants). Antioxidants can also affect directly or indirectly the expression of genes in tissues. A number of genes are regulated by changes in the cellular redox status [66]. Cellular redox status is determined, among other things, by the type and concentration of ROS/RNS present in the tissue as well as by the type and levels of antioxidants. ROS such as H2O2 [67,68], UV radiation [69], HOCl [70], singlet oxygen [71] and also oxidized LDL [72] were all studied for their ability to stimulate the expression of genes and protein activity, in order to identify redox-sensitive genes. Among the genes affected are amphiregulin (AP-1), c-fos, c-jun:, c-myc, and MAP kinase. Oxidative stress was shown to increase Type III and Type IV collagen mRNA, the trascription of collagenase and the expression of ferritin. Antioxidants thus, may modulate signal transduction pathways and gene expression through their reducing properties, rather than through their ability (under certain conditions) to generate ROS, or through their metabolic products. It was shown that only polyphenols with relatively high antioxidant potential were able to induce c-foc mRNA, while those lacking potent antioxidant properties were inactive [73]. Induction of c-fos and c-jun mRNAs by phenolic antioxidants is mediated by an antioxidant response element (ARE), in a specific, and dose-dependent manner.
Antioxidants, like other signaling molecules, induce specific activity, which depends not only on the structure of the stimulus, but also on the target place; the specific tissue or the cell type. When tissue interacts with an antioxidant, the signal formed must then translate into a biological response, and as tissues differ in their composition, the biological effect or response also vary. Antioxidants can thus mediate activities of biological systems, directly or indirectly, through involvement in one of the multiple stages associated with signal transduction pathways.
4. Measurement of Antioxidant Activity
The interpretation of results obtained from in vitro measurements of antioxidant activity of a compound or of a crude plant extract, must be dealt with caution as the antioxidative effect of a tested compound may vary considerably with the method and conditions used. Thus, selection of the appropriate assay to be used should be based on the intended application of the antioxidant.
Factors which influence the efficiency of an antioxidant in vivo are complex and require consideration of its bioavailability, the site of action, the type of ROS that the antioxidant must react with, its pharmacokinetic character, its stability, its toxicity and its possible synergistic behavior with other compounds. One must take all these limitations into account when extrapolating the inhibitory effect of an antioxidant in vitro to an in vivo application.
Methods to examine antioxidant activity of a sample can be divided in principle into two major categories: 1) Measuring its ability to donate an electron (or hydrogen atom) to a specific ROS or to any electron acceptor. 2) Testing its ability to remove any source of oxidative initiation, e.g., inhibition of enzymes, chelation of transition metal ions and absorption of UV radiation.
Antioxidant function in biological systems is much more complicated than a simple free radical scavenging process. An antioxidant may affect biological system by suppressing the formation of ROS and RNS, by affecting enzyme activities [74], by inducing biosynthesis of other defense enzymes, and thereby affecting other endogenous antioxidants [75], by preserving NO activity [76], or by sequestering transition metal ions. It is apparent that some antioxidants have more than one mechanism for their effect on biological systems.
In many methods of detecting antioxidant activity, the ability of an antioxidant to retard the oxidation of PUFA such as linoleic acid (C-18:2), exposed to oxidative stress, is determined by exposing the linoleic acid to heat, oxygen, light, or to free radical generators.
The following assays are commonly used for measurements of lipid peroxidation:
4.1. Conjugated Diene assay [77]
This method allows dynamic quantification of conjugated dienes (CD) formed as a result of initial PUFA oxidation by measuring UV absorbance at = 234 nm. The principle of this assay is that, during linoleic acid oxidation, the double bonds are converted into conjugated double bonds, which are characterized by a strong UV absorption at 234 nm.
4.2. Lipid Peroxide (PD) Assay
When linoleic acid is oxidized, oxidation starts at its allylic position in a non-specific reaction, to form an unstable mixture of lipid peroxides. The total amount of lipid peroxides can be detected iodometrically by using the method originally developed by El-Saadani et al [78]. The amount of lipid peroxides accumulated first reaches a maximum, thereafter declining (decomposition phase) while forming aldehydes.
4.3. Thiobarbituric Acid Reactive Substances (TBARS) Assay [79]
During lipid peroxidation, lipid peroxides are formed, with a subsequent formation of peroxyl radicals, followed by a decomposition phase to yield the aldehydes such as hexanal, malondialdehyde (MDA) and 4-hydroxynonenal. This assay is based on the detection of a stable product which is formed between aldehydes and thiobarbituric acid (TBA) in the aqueous phase.
4.4. Linoleyl Hydroperoxide
(L-OOH) and Linoleyl Hydroxide (L-OH) Assay [80]
During the initial stage of lipid peroxidation, hydroperoxides and hydroxides of esterified fatty acids are mainly formed, with minor amounts of free oxidized fatty acids. The position of the hydroperoxide and hydroxide along the fatty acid chain depends on the type of fatty acid and on the nature of the oxidative stress. Various approaches have been suggested for the quantitative determination of L-OOH and of L-OH but the most direct one is by HPLC. This method is specific and does not require any derivatization, as does the more sensitive GC-MS methods which involves esterification, silylation and, in some instances, also a reduction of the double bonds.
4.5. The -Carotene-Linoleic Acid System [81]
A modification of the above assays is based on the determination of the coupled oxidation of -carotene and linoleic acid. This assay is simple, reproducible and time-efficient for a rapid evaluation of antioxidant properties.
4.6 Reaction with 1,1-Diphenyl-2-Picryl-Hydrazyl (DPPH) [48,82]
This assay measures the free radical scavenging
capacity of a compound. DPPH is a molecule containing a stable free radical
(Fig. 6). In the presence of an
antioxidant which can donate an electron to DPPH, the purple color which is
typical to free DPPH radical decays, and the change in absorbancy at 517nm is
followed either spectrophotometrically or by detecting changes in the
concentration of the starting materials, using HPLC analysis [25]. This simple test can provide
information on the ability of a compound to donate a hydrogen atom, on the
number of electrons a given molecule can donate, and on the mechanism of
antioxidant action. In cases where the structure of the electron donor is not
known (e.g., a plant extract), this method can afford data on the reduction
potential of the sample, and hence can be helpful in comparing the reduction
potential of unknown materials. A similar method, using a different stable
radical (galvinoxyl) was introduced by Tsuchiya et al [82].
4.7 Cyclic Voltametry (CV)
This technique, developed by Kohen et al.[45], measure the antioxidant activity of tissue or plasma [83] on the bases of the reducing properties of the tested sample, by measuring their oxidation potential (E1/2, between 0-1 V).
4.8. Radical Stability Calculation by Quantum Mechanical Parameters
The DPPH (or to galvinoxyl) method and the CV method analyze the net ability of an antioxidant to donate an electron without interfering with other possible properties of the antioxidant (which might contribute to the total antioxidant activity), such as its ability to chelate metal ions. Calculation of heat formation differences (Hf) between radicals and their parent compound is performed, using the PM3 semi-empirical Hamiltonian for energy optimized species [84]. The Hf value represents the relative stability of a radical with respect to its parent compound and it enables a comparison between the stability of radical achieved after hydrogen abstraction from parent molecule (toward radical formation) from alternative positions within an individual molecule, as well as between molecules [42,47].
5. Role of Antioxidants in Oxidative Stress in Human Diseases
Reactive oxygen and nitrogen species (ROS and RNS, respectively) are endogenous intermidiates constantly produced in the human body and essential parts of its functions. They are components in signaling cascade involved in cellular functions such as proliferation, inflammation and adhesion. Violation of the necessary balance of cellular oxido/redox status toward a more oxidative stress, results in pathological manifestations.
The extensive list of disorders and pathogenesis in which radicals and oxidants have been implicated is still growing. As research on the role and involvement of ROS and RNS advances, more and more biological functions are being found to be associated with these species. An excess of oxidative stress can lead to the oxidation of lipids and proteins, which is associated with changes in their structure and functions. ROS/RNS also cause DNA damage which is associated with the development of cancer, cardiovascular diseases, cataract, neurological disorders and lung disease. Aging is also thought to occur as a result of a constant exposure of the organs to ROS/RNS, with a cumulative damage, through the entire life, along with a gradually decreasing repair capacity and increasing degenerative changes in the organs [85]. Nutritional antioxidants could maintain the necessary oxido/redox balance within the organ.
5.1 Atherosclerosis
Atherosclerosis is the main cause of mortality and morbidity in the western world. It is characterized by the accumulation of cholesterol, lipid peroxides and oxysterols in the arterial wall, and it is the main cause of heart atack and stroke.
Atherosclerosis is a multifactorial disease, and aside from genetic susceptibility, various risk factors are involved. Major theories for the initiation of atherosclerosis include endothelial injury and lipid infiltration [86,87]. Lipoprotein retention in the arterial wall (binding to extracellular matrix proteoglycans), as well as lipoprotein oxidation are two major hypothesis attributed to the pathogenesis of atherosclerosis.
Incubation of low density lipoprotein (LDL) with endothelial cells, which resulted in the formation of oxidized LDL that was taken up at an enhanced rate by macrophages [88,89]. It was also shown that oxidized LDL promotes toxic injury to arterial cells in culture [90]. Despite many publications on oxidized LDL and atherosclerosis, the central causative role of Ox-LDL in atherogenesis are still controversial [91].
However, the atherogenicity of Ox-LDL is supported by in vitro and in vivo studies. Oxidized LDL is taken up by macrophages at an enhanced rate via the macrophage scavenger receptors, and unlike the uptake of native LDL, the uptake of Ox-LDL is not down-regulated by cellular cholesterol accumulation [92-95].
Cholesterol-laden macrophages contain unesterified cholesterol, cholesteryl esters and oxysterols and it is characteristic of the early atherosclerotic lesions [96].
A large body of
evidence suggests that Ox-LDL is more atherogenic than native LDL not only because of its contribution to
foam cell formation, but also as a result of its effects on the secretion of
potent substances from arterial wall cells, and its cytotoxicity towards
endothelial cells and smooth muscle cells [94,97]. Ox-LDL can cause the release
of macrophage interleukins, apolipoprotein
E and proteases [98] and of smooth muscle cell
growth factors and cytokines. It can induce the release of endothelial cell-derived
monocyte chemoattractant protein 1 (MCP-1) [99], colony-stimulating factors
(CSF), endothelium-derived relaxation factors [100], and other tissue factors.
The cytotoxicity of Ox-LDL toward endothelial cells can cause functional
changes in the cells that allow the penetration of monocytes and LDL into the
sub-endothelial space, which can then accelerate the formation of the
atherosclerotic lesions [101]. Ox-LDL, unlike native LDL,
is chemoattractive to monocytes, causing their movement into the intima and
preventing their exit back to the circulation [102].
Since the effect of dietary antioxidants on cardiovascular diseases have been the subject of many recent reviews [103-108], such a discussion is beyond the scope of the present review. The effect of dietary antioxidants on the development of human atherosclerosis is also controversial, and a number of contradictory examples have been published. Supplementation of -tocopherol to humans has been shown to reduce the risk of ischemic heart disease [109], and vitamin E levels in plasma has been shown to be negatively correlated with that risk [110,111]. On the other hand, the GISSI-Prevenzione trial [112] showed that -tocopherol has no protective effect against cardiovascular disease.
Although oxidative stress accelerates atherosclerosis under certain conditions ROS can play important beneficial physiological functions. Biological systems maintain reduced state (high GSH/GSSG ratio), but simultaneously require oxidants in localized sites (e.g., during apoptosis). ROS can damage cellular tissues, but at the same time the presence of certain ROS in specific sites is essential for the proper function of some system (e.g., gene expression). Most of the research on the role of antioxidants in cardiovascular diseases has focused on testing a single pure compound, usually to examine its ability to scavenge free radicals and thereby to prevent lipid peroxidation. However, antioxidant contribution in vivo goes far beyond scavenging free radical. Moreover, a single antioxidant is usually not present alone in biological systems but acts in combination with other antioxidants and hence the “protective effect of a diet is not equivalent to a protective effect of antioxidants in diet” [91].
5.2. Cancer
Reactive oxygen and nitrogen species, such as superoxide anion, hydrogen peroxide, hydroxyl radical and nitrogen oxide and their biological metabolites also play an important role in carcinogenesis. ROS induce DNA damage, as the reaction of free radicals with DNA includes strand breaks, base modification and DNA-protein cross-links. An example of base modification is the formation of 8-oxoguanine [113] which induces Guanine:Cytidine to Thymine:Adenine transver-sions at the DNA replication stage [114], an important process in carcinogenesis and tumor development.
Phenolic antioxidants exert anticarcinogenic activity, presumably through the induction of phase II detoxifying enzymes (such as glutathione S-transferases and quinone reductase), which provides some explanation for phenolic antioxidants prevention of tumor initiation. At the same time, under certain conditions they can promote tumor initiation [115,116]. Antitumor activity by phenolic antioxidants may be explained by the inhibition of AP-1 activity through induction of Fra expression [115,116]. Phenols, such as butylated hydroxy anisol (BHA), are capable of activating mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase 2 (ERK2), and c-jun N-terminal kinase 1 (JNK1).
Antioxidants can decrease oxidative stress-induced carcinogenesis by a direct scavenging of ROS [117], and/or by inhibiting cell proliferation secondary to the inhibition of protein phosphory-lation [115,118]. Ascorbic acid (vitamin C) consumption has been inversely associated with all subtypes of gastric cancer in a significant dose-response manner, with a risk reductions between 40-60% [119].
-Carotene has been found to be also very active in reducing cancer risk, particularly in the intestinal type of cancers, while tocopherol shows less definite effects . The administration of a mixture of the above three antioxidants revealed the highest reduction in risk of developing noncardia cancer. In another large trial of the -TOH, -Carotene Cancer Prevention Study Group [11], the effect of supplementing -TOH and -carotene, alone or in combination, on the incidence of lung cancer and an other cancers was examined. The results indicate that -TOH does not reduce cancer incidence; while, unexpectedly, among smokers receiving -carotene a higher incidence of lung cancer was observed, in comparison to a placebo receiving subjects.
No evidence of an interaction between -TOH and -carotene with respect to the incidence of lung cancer was observed. In respect to other types of cancer, fewer cases of prostate cancer were diagnosed among those who received -TOH, while -carotene had no effect. Tomato’s lycopene was shown to reduce prostate and lung cancer.
While individuals with high intakes of fruits and vegetables experience a lower risk of developing cancer, results from the administration of pure compounds are less definite. In a trial performed with about 40,000 women aged 45 years or older, supplementation with -carotene for 2 years shows no statistical significant differences in incidence of cancer [120].
A study was conducted in Japan to examine geographic associations between plasma antioxidant levels and gastric cancer risk [121]. Plasma concentrations of the major carotenoids, -TOH, and ascorbic acid among 634 men aged 40-49 years were measured and correlated with mortality rate from gastric cancer. It was found that -carotene and -TOH were inversely related to gastric cancer rates and this correlation was even stronger for -carotene and lycopene.
Tocopherol and epigallocatechin gallate were found to delay the onset of UV-induced skin cancer in mice when applied topically to the skin [122]. Daily topical use of super virgin olive oil after sun-bathing could also delay and reduce UV-induced skin cancer development in human skin.
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