dietary antioxidants, peroxidation and cardiovascular ...

The Journal of Nutrition, Health & Aging© Volume 6, Number 3, 2002

THE JOURNAL OF NUTRITION, HEALTH & AGING©

DIETARY ANTIOXIDANTS, PEROXIDATION AND CARDIOVASCULAR RISKS M. BARBASTE, B. BERKÉ, M. DUMAS, S. SOULET, J.-CL. DELAUNAY, C. CASTAGNINO, V. ARNAUDINAUD, C. CHÈZE, J. VERCAUTEREN EA 491*, Laboratoire de Pharmacognosie, Faculté de Pharmacie, Université Victor Segalen Bordeaux 2, BP 80, 146, rue Léo Saignat, 33076 Bordeaux cedex, France.Phone: 33 (5) 57 57 12 59, Fax: 33 (5) 56 96 09 75 – Email : [email protected] * With the financial support of MENRT and Conseil Régional d’Aquitaine which are warmly acknowledged.

Abstract: Most of the many epidemiological studies in the field strongly suggest that an equilibrated diet such as the so-called "mediterranean diet", is associated with protective effects against major diseases, and particularly, against cardiovascular risks. Since many reports also consider “reactive oxygen species” or “free radical oxidations” to be responsible for the accompanying disorders of most pathologies as well as for ageing, it is conceivable that natural plant metabolites such as polyphenols, are likely to play an important role in insuring this protection. Indeed, not only their presence, in particularly high amounts and varieties in foods of such a diet, but also, inter alia, their very potent antioxidant or radical scavenging properties, make polyphenols best accounting for the parodoxical part of the “french paradox”. Therefore, many efforts have been made to assess the mechanisms for such a cardiovascular disease protection. Whatever convincing were the polyphenols properties demonstrated by many in vitro experiments to support those theories, quite a great number of the results appeared somewhat contradictory when transposed to humans, in the in vivo situation. Some people totally refute this explanation, thinking that health benefits, as far as alcoholic beverages are concerned, originate from ethanol but also, with no doubt, some polyphenols even revealing to be “prooxidants”. Key words: Nutrition, elderly, reactive oxygen species, polyphenols, dietary antioxidants, radical scavenging properties, mechanisms and paradoxical issues, cardioprotective effects; french paradox, aging.

Introduction

1 - Dietary antioxidants: biochemical aspects

In the past decades, a large number of epidemiological studies came to the suggestion that a well balanced diet is associated with improved health benefits (1). People from around the Mediterranean sea are considered as having one of the most famous balanced diet that was named the "Mediterranean diet" (2). These dietary habits and the related ones (3), indeed are largely synonymous with the best prevention against cardiovascular diseases (CVD) (4, 5). It is worthy of note that the same observation is being made for other degenerative diseases such as cancer (6-8)) or Alzheimer (9-11) but, we will focus only on the cardiovascular question. This phenomenon, having been especially observed for French people, was thus named the "French paradox" (12, 13). The French situation is paradoxical because, while having a rather high intake of saturated fat (positively related to high rate CVD), and submitted to the same unfavourable stress factors as other European country people (14), they still have the lowest rate (about one third of the average) mortality from CVD. Epidemiological and clinical studies were designed to understand the mechanisms. Since these studies are so numerous and so different in their approach and in their conclusion (15-18), it could be wise to refer to some of the most recent bibliographical reviews (19-23) that help to clarify the question and to forge its own opinion.

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1.1 Obviousness of the role of dietary antioxidants Due to the huge interest for human health in understanding the impact of the diet on CVD incidence, many other studies have tried, in the meantime, to unravel the underlying pharmacological and biochemical explanations. What came out from the investigations run at a molecular level, was the constant presence of antioxidants in this type of diet (14, 2427). It became rapidly obvious that these chemicals could be the most potent support for the observed health benefits. 1.2 The biological bases of CVD Without wanting to penetrate in detail this question, it is significant to recall some of the fundamental mechanisms that are necessary to have a good understanding of the means by which the antioxidants could then interact. Physiopathology of cardiovascular diseases is not as much different as for any other “degenerative disease of aging such as cancer, immune-system decline and brain dysfunction ” (28). The more specific involved phenomena are atherosclerosis, hypertension and inflammation. Whatever the mechanism considered, it is always largely the consequence of an oxidative process resulting from some “chain reaction peroxidations”. This observation should be at the basis of the main biological and biochemical measurements used to investigate the development of the pathology in a patient.


DIETARY ANTIOXIDANTS, PEROXIDATION AND CARDIOVASCULAR RISKS 1.2.1 Cholesterol Thus, for instance, the atherosclerotic risks were thought to possibly result from molecular cholesterol deposits when present in too large excess in the plasma (29), as well as from oxidized cholesteryl derivatives. This led to the preparation of endogenous cholesterol synthesis inhibitors (fibrates and statines) as new therapeutical tools (30) to lower the plasma cholesterol. However, one can ask the questions to know if this was the best approach? Is it so much the plasma cholesterol level as the oxidized part of it the most deleterious event to atherosclerotic plaque formation? Don’t we need cholesterol molecules to be inserted in every bilayer membranes of our cells to be the most functional ones? For which reason (keeping apart the familial hypercholesterolemia disease) should we kick out of our blood stream most of these necessary molecules while our own body is programmed to produce 2 g per day in case of insufficient dietary support? For some scientists, like Steinberg, who says : "The cholesterol controversy is over. Why did it take so long?" (31), the answer is clearly yes. 1.2.2 Lipoproteins While it is generally well accepted that the atherogenicity of the low density lipoproteins (LDL) is mainly due to their oxidized forms (Ox-LDL) (32, 33) (by oxidation of either the proteic or the lipidic part of it), they all (LDL, VLDL) have been identified as the “bad cholesterol”, without making any difference with their oxidized part (Ox-LDL). This classification led to the big difference in the biological parameters, between the high density lipoproteins (HDL) considered as the “good cholesterol” and the “LDL”. Thus, it has been shown, later, that HDL was “the principal vehicle for circulating plasma lipid hydroperoxides”, suggesting that HDL lipids, more easily oxidized, in vivo, than those in LDL, but also more rapidly eliminated by the liver, could play a beneficial role by introducing a lag time in the build-up of oxidized-LDL (34). Finally, it was demonstrated that HDL was more prone than LDL to in vitro oxidation and that the modified-HDL was a “cytotoxic particle ” (35, 36). Other results demonstrated that HDL could even be protected by LDL against radical oxidation in the presence of reactive oxygen species (ROS) such as OH. or O2.- free radicals). The conclusions of the authors are very wise: “This observation addresses new questions about the interaction between HDL and LDL, especially the possibility of a reciprocal protection” (37). In the same manner as for cholesterol, is it so much the increased amount of LDL as the oxidized part of any LDL level, the main risk factor in coronary artery disease (CAD)? With no doubt, oxidized-lipoproteins are the forms that can initiate atherosclerotic processes through the cellular activation and migration leading to the tissular endothelial lesion but also, that can perpetuate the chain oxidation in the presence of oxygen. Don’t we need these native proteins to carry the fatty building blocks to the cells, to allow their division

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(regeneration) to occur? 1.2.3 Fatty acids Very early in the last century, the hypothesis was put that atherosclerosis and resultant vascular diseases might be due to a deficiency of essential fatty acids in the diet, rather than to an excess of dietary saturated fats (38). This proposition was anticipating for many years the idea, slowly becoming currently accepted, that polyunsaturated fatty acids (linolenic ω-3 series, namely) are not only conferring many health advantages over saturated ones but also, that their higher oxidizability risks “may be minimized by the concomitant intake of an antioxidant, like vitamin E” (39). We will see below, how other dietary antioxidants could be play this role. 1.3 The relevant biological parameters The situation, from the biochemical point of view, is unclear. It is most probable that the biological parameters considered today as relevant to the CVD (LDL, VLDL/ HDL ratios? Blood cholesterol? Fibrinogen levels? Factor VII? Platelets response to aggregation induced by collagen or ADP? Effect on isolated LDL oxidizability? Prolonged lag time to copper-catalyzed oxidation of LDL (40)? Phenolic compounds in LDL by the Folin-Denis method? Phenolic acids in urine? Arterial lipid infiltration, remodelling, and aging, diffusion imaging by nuclear magnetic resonance (41)?, …), will have to be adjusted in the coming years according to the increasing of our knowledge of the mechanisms. As we will see below, the pending questions are not trivial at all: it is just a matter of taking into account, in the most integrated fashion, what makes one of the most exciting and difficult thing to understand: the human beings fighting against peroxidation, against aging 2 - Dietary antioxidants: chemical aspects 2.1 Dietary antioxidants 2.1.1 Wine antioxidants Studies, at the end of past century, have provided much evidence linking "French paradox" to the wine drinking (42). Indeed, when Creasy et al. (from the Cornell Institute, New York) published in the “New York Times” at mid-august 1990, that resveratrol, a phytoalexin produced by grape was in the largest amounts in Bordeaux red wines and that it was very potent at lowering plasma cholesterol, the news was made available all around the world within a day. This was the starting point for hundreds of works on isolation, structure elucidation, synthesis and biological properties assessment of wine polyphenolics (43-53). There is a sound rationale for considering these polyphenols as important contributors to the dietary anti-oxidant intake for moderate wine dinkers: they are many in quantity (more than 3g/l, for red wines) as well as in various categories (54, 55) and they possess very important in vitro biological properties, including very interesting NOmediated vasorelaxation (56). In most of the epidemiological


THE JOURNAL OF NUTRITION, HEALTH & AGING© studies, red wine has been observed to be more effective in lowering the risk of cardiovascular disease than any other vegetable foods (57-59). Even, more recently, strong correlations have been established between wine drinking and lower all-cause mortality (60, 61). “Wine drinkers seemed to be at lower risk than beer drinkers in both sexes … results tend to confirm that intermediate alcohol consumption is a component and contributor to a low coronary risk Lifestyle” (62). 2.1.2 Other dietary antioxidants Besides of the special case made to wine, antioxidants are widely distributed in plant kingdom from which they are specific secondary metabolites. Thus, they are found in many other foodstuffs (red fruits, vegetables, olive oil, cocoa and tea drinks) of the “Mediterranean diet”. It is finally, the total antioxidants from the whole plant sources from our diet together that make this class of compounds standing apart among others. They all display radical more or less scavenging and anti-oxidant properties (43, 63, 64) and interesting biological activities, regarding the CVD, which have been demonstrated many times in vitro: anti-atherosclerotic (43, 65), anti-thrombotic (66, 67), anti-inflammatory (68, 69), supporting the beneficial health effects. 2.2 Chemical features of antioxidants The biological properties, indeed, are strongly related to their physico-chemical properties such as reducing potential, lipo- or hydrosolubility, etc., which also deeply depend on their chemical structure. The peculiar points addressed in the following part are dealing with their oxidation state: reduced form (phenolic structure) and oxidised form (quinone structure), since their chemical properties change thoroughly between these two forms. Clearly, polyphenols are important metabolites but to better understand what is hidden behind this question, it is necessary to have information on the relevant key features of their structures. 2.2.1 Structure of antioxidants, a huge diversity: We will not display thoroughly the structures of the antioxidants but we want to stress the point that apart from thiols (amino-acids and Allium cepa metabolites: alliine, etc) and polyenes (carotenoids) that will not be discussed here, they all are polyphenols. A large number of reviews (70-77) exist, to which interested readers can refer to, the most recent one being on polyphenols as health sources (78). They all feature at least an hydroxylated aromatic ring ("benzenic") and have a mixed biogenetic origin: the shikimate (mevalonate) and the acetate (pyruvate) pathways (figure 1). Further polyphenols such as stilbenoids are forming a class among which the famous resveratrol and its oligomers, tannins also designated as " procyanidins " or " proanthocyanidins " (see dimer B3), because they form flavylium (anthocyanidins) upon acidic cleavage (hydrolysis) and oxidation, but also, " hydrolysable tannins" such as gallo- or ellagitannins, abundant

in oak tree (figure 2). One can even include other metabolites as “essential” as vitamins: tocopherols (Vit. E) and Vitamin K, or coenzymes: quinones (ubiquinones) are present in plants. Vitamin C is very much related to polyphenols, by its enol function of ascorbyl residue that gives it all the same features. By far not all polyphenolics are known yet even though new ones are discovered every year: castavinols (79), vitisins (8082), astringin (51, 83), stilbenoid oligomers (84). The crude polyphenolic content from one plant is so much specific that it was shown to be a "biological tracer" of it. This method allows us to discriminate between plant species and, as the best example with no precedent, clones and cultivars of grapevines (85-87). 2.2.2 The physico-chemical properties of polyphenols Besides their ability to complex metal ions, that all are implicated in biological systems as cofactors of enzymes with “red-ox” functions (iron, copper, …) that could partly explain their properties (88), they share two other important characteristics (89) which both are due to the phenolic nucleus itself, i.e.: - The ability to complex macromolecules such as proteins (tanning) or polysaccharides, and - The antioxidant and radical scavenging properties. 2.2.2.1 Tanning properties Polyphenols interacting strongly with proteins is known for a very long time. It is because they transform living skins into indestructible leathers, by making strong complexes with the proteins (collagens) and glycoproteins that they are named "tannins". Investigation in the field is really difficult. Butler and Hagerman have done the most determinant approach (90, 91) at the turn of the eighties but researchers have designed new experiments to better understand which mechanisms are concerned by the establishment of such strong bindings (9296). Preliminary conclusions, implicating the hydrophobic interactions mainly with proline (97), however, need to be reconsidered on the basis of recent results obtained by modern NMR techniques analysis (NOEsy) of tetrapeptide models (GGPG) that did not confirm any interaction of catechin or dimer B3 (catechin-4α→8-catechin) with the proline residue (98, 99). However, interesting observation was made when e p i g a l l o c a t e c h i n - 3 - -gallate was added to a solution of bradykynin (100) whose conformation was differently structured. This question is undoubtedly a very important one and well deserves all the efforts done by many teams to solve it. Indeed, fundamental but not fully understood parameters that regulate the action of polyphenols are hidden behind this property: • First of all, it is necessary to unravel this question if one expects to prove that polyphenols are available in vivo (actually absorbed from the gut) even in the presence of the proteins of the bolus, to reach the biological site where they should exert their properties, then to improve this "bioavailability" (101).

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DIETARY ANTIOXIDANTS, PEROXIDATION AND CARDIOVASCULAR RISKS Figure 1 Biogenetic pathway to flavonoids and acid-phenols OH

COOH COUMARINES

OH

OH

HO

HO

O 2

OH

HO OH

COOH

COOH

shikimic ac.

R

OH

OH + 3 x C2 (acetates)

O

HO

OH

O

O OH O benzo-γ-pyrones "CHROMONES" FLAVONOIDS

O

OH

OH O

HO

p-hydroxy cinnamic ac.

O

OH O

FLAVONOIDS

ACID-PHENOLS

OH

benzo-γ-pyrones "2-phenylchromones"

O CHALCONES

OH

+ O

HO

OH

OH OH flavanols CONDENSED TANNINS or "procyanidins"

OH benzo-pyrylium ANTHOCYANINS

Figure 2 Further categories of polyphenols as antioxidants

HO HO

O

O

OH OH

O HO

HO

ELLAGITANNINS

OH

HO HO

OH

O CO

HO OH resveratrols STILBENOIDS

OH

OH

CO

C O

O

HO

O

O

C

HO HO

OH

OH

OH

OH

HO

HO

OH

O

OH OH

Dimer B3

Figure 3 Phenolics as “essential” metabolites CH3

CH3 H3 C

OH

OH

CH3

O

H

CH3

3

CH3

HO CH3

Vit. E ( -tocophérol)

OH

Vit. K 1(20)

Partial proof of this importance can come from the experience done with the wine phenolics in the absence or in the presence of alcohol. It is claimed that this later solvent would give them a higher rate of resorption by diminishing the interaction they have with bolus proteins (102). • The binding to proteins may explain why polyphenols inhibit almost every enzyme that they are exposed to in vitro (103): for some authors, for example, it is part of the mechanism that makes polyphenols so efficient to prevent LDL-oxidation (104), inhibitors of human DNA ligase i (105) or topoisomerases (106), glucosyltransferases implicated in the cariogenicity which is considered to be strongly associated with the synthesis of extracellular water-insoluble glucans (107, 108), collagenase (109), etc. • Astringency is a direct observable effect of the tanninproteins interaction: the feeling of loss of lubrication (real dryness) developing in the mouth after absorption of any astringent molecule is the result of a binding reaction of this molecule with salivary proteins. It was proposed by Bate-Smith (110) that this astringency develops via precipitation of proteins

212

H3 CO

H

O

O

10

H3 CO OH

Ubiquinol-10

HO

CH2OH H OH OH

L-ascorbic acid

and mucopolysaccharides in the mucous secretions, which are surrounded by a mantel of water molecules. Upon precipitation, the lubrication disappears because of the disappearance of the “cortège” of these water molecules. The mechanisms of these interactions are not clear but it is perhaps worth noting that the binding of polyphenols to simple peptides or to Proline-rich proteins (PRPs), such as salivary proteins, are strongly under control of hydrophobic effects. The most important parameters that seem to be important to favour the binding are: molecular size of the polyphenols, their conformational flexibility, the number and position of phenolic nuclei and water solubility (111). A good water solubility disfavours the polyphenol-protein complexation; an increase of the molecular size and of the conformational flexibility favour it (97, 112, 113). This peculiar property is probably responsible for part of the special antioxidant properties, unexplained till now.


THE JOURNAL OF NUTRITION, HEALTH & AGING© 2.2.2.2 The antioxidant and radical scavenging properties 2.2.2.2.1 Oxygen: the vital molecule In biological systems, this property has something to see with the different forms of the oxygen molecule, the one that composes 20% of air that we breath (O2) whose ground state is a diradical with two unpaired electrons (both having the same spin) in two π* antibonding orbitals (figure 4). This molecule is used in all mammalian cells as the only one to oxidize glucose to CO 2 and H2O and to produce ATP-energy in the mitochondria. This stable state can be altered by different means, namely by photoexcitation (by UV-A or -B) to form a very reactive “singlet oxygen” or by the successive uncontrolled reductions to superoxides (superoxide anion radical O 2.— , hydroperoxyl radical HOO . ), to peroxides (hydrogen peroxide O 22— = H 2O 2 and to the 3–electrons reduced oxygen HO. = hydroxyl radical. 2.2.2.2.2 Oxygen: the mortal problem Superoxide (O 2.—) is formed in cells from oxygen by a number of reactions (during the oxidation of hydroquinones (i.e. ubiquinones), of catecholamines or thiols) and normally, it is readily converted to hydrogen peroxide (H 2O 2) by the superoxide dismutase (SOD) enzymes. However, in acidic medium, it forms the hydroperoxyl radical (HOO .) that spontaneously disproportionates (Figure 5) into hydrogen peroxide (H2O2). This latter is not a radical by itself but is very dangerous if not readily destroyed (by catalases, for instance). It penetrates any cell compartment, like simple water (H 2O) would, including the nucleus, and takes part in the formation of the most deleterious “reactive oxygen species” (ROS), the hydroxyl radical (HO.), via two major reactions known as the Haber-Weiss and the Fenton reactions that both require a transition metal such as Fe++ or Cu + (Figure 5). Other ROS are possibly derived in living systems from these primary radicals when reacting with nitrogen- and sulfurderivatives: the nitric oxide NO. (a neurotransmitter formed by NO-synthases from L-arginine), than can form peroxynitrites NOx— by reacting with superoxide anion, for instance.

2.2.2.2.3 Polyphenols as powerful reducers 2.2.2.2.3.1 The acidity of polyphenols: A phenolic function is more acidic (pKa= 8-10) than a simple aliphatic alcohol (pKa= 16-18), because of the conjugation of the resulting phenate anion with the aromatic nucleus (figure 6). 2.2.2.2.3.2 The stability of the radicals formed from polyphenols: The reduction (the transfer of a single electron) by this phenate anion to the oxygen free radical is thus an easy mechanism. The result is the formation of an aryloxy radical that is also stabilised by many resonance forms. Hence, the energy difference between an alkoxide (R-OH) and a phenoxide (Ar-OH) to form the corresponding “oxy-radicals”, the Bond Dissociation Enthalpy (BDE) of O-H bond, is largely in favour of the aryloxide, by almost 20 Kcal/mol (figure 7). 2.2.2.2.3.3 The redox potential of reduced polyphenols: These acidity and stability lead to the very specific property shared by all antioxidants, namely the polyphenols: the reducing power. One important feature to always keep in mind with this kind of very reactive entities (ROS), is that their ability to reduce or to oxidize another molecule depend on their “relative oxidizing/reducing power” (their “redox potential” couple, formed by the pair of the oxidized and the reduced forms of every entity). These values (see Table) are expressed under the E0 value (Volts) if at pH= 0 (1 M) but under the E’0 value if at physiological pH= 7 (10-7 M). They are depending on some physical parameters (pH, concentration, temperature, pressure, …). In any case, a redox potential couple will be able to reduce (deliver its electron) another couple, only if it has a lower redox potential (more negative or less positive) than it. In the reverse situation, this compound will be the reduced one. From the reported data in the table, it is clear that Vitamin C is able to reduce quercetin but not the reverse, meaning that, at least in these conditions, quercetin should not be able to “spare Vit. C” as it is sometimes purported.

Figure 4 Electronic configurations of various oxygen species

hν singlet O2, ∆g

ground state O2

1

superoxide O2-.

peroxide O22-

radical anion

hydroxyle OH.

radical

Figure 5 Formation of hydrogen peroxide and two important ways to form hydroxyl radical 2 HO2.

O2

O2-.

H2O 2

O2

OH.

OH -

(Haber-Weiss reaction)

Fe++

H2O 2

Fe+++

OH.

OH -

(Fenton reaction)

H2O 2 hydrogen peroxide

hydroxyl radical

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DIETARY ANTIOXIDANTS, PEROXIDATION AND CARDIOVASCULAR RISKS Figure 6 Polyphenols possess a noticeable acidity. Ascorbic acid is even more acidic (pKa= 4.2) pKa = 8-10

O

OH

CH2OH

O

H

CH2OH

O

O

O

H OH

OH pK = 4.2

OH

CH2OH

O

O

H

OH HO

O

+

H

O

O

O-

HO

O

L-ascorbate

L-ascorbic acid

O

L-dehydroascorbate

Figure 7 Aryloxy radicals and resonance forms OH + RO

O

+ ROH

BDE (ArO. /H.) = 85 Kcal/mol

(reduction of a radical)

BDE (RO ./H.) = 104.5 Kcal/mol O.

OH

O

O

O

+ H.

(stabilisation by conjugation)

aryloxy radicals

Table of E’O Standard redox potentials (calculated at “physiological” pH 7.0, [H+]= 10-7 M) oxidative part of the couple

reductive part of the couple

2e-

CH3 CHO

E'0 = -0.58 V

2 H + + 2e -

H2

E'0 = -0.42 V (pH= 7.0)

NAD+ + 2 H+ + 2e -

NADH + H+

CH3 COOH + 2

NADP O

+

+

+ 2H

+

-

+ 2e

H

NADPH + H OH H3CO H

10

+ 2 H+ + 2eO Ubiquinone10 (Q10 )

H3CO

E'0 = -0.32 V +

H3CO

10

OH

Ubiquinol-10 (Q10 H2 )

sense of reduction

H3CO

H+

E'0 = -0.324 V

E'0 = +0.06 V

ascorbyl radical + H+ + e-

ascorbate (Vit. C)

quercetin radical + H+ + e-

quercetin

E'0 = +0.33 V

tocopheryl radical + H+ + e-

tocopherol (Vit. E)

E'0 = +0.48 V

1/

2O2

+ 2 H + + 2e -

O 2 + H+ + e

The acid-base and redox properties of the aryloxy radicals derived from selected flavonoids could be studied (by pulse radiolysis) of aqueous solutions (114). They were shown to be pH-dependent, as expected, but also to vary with the overall structure of the flavonoids. The German team who did make a very nice work in measuring these couples has made very interesting observations: “Two structural criteria became apparent: all substances containing the B-ring catechol group and the 2,3-double bond have a higher redox potential than

214

E'0 = +0.14 V

H2O

E'0 = +0.82 V

HO2

E'0 = +1.06 V

ascorbate and are consequently able to oxidize it to th e ascorbyl radical” (see figure 8). “With fisetin and kaempferol having values very similar to ascorbate, only the flavanone dihydro-quercetin was capable of reducing the ascorbyl radical, thus fulfilling the so-called ''ascorbate-protective'' function, originally proposed by Szent-Gyorgyi. While flavonoids are effective radical scavengers, these rather high redox potentials for most flavonols may explain their occasional prooxidant behaviour” (115).


THE JOURNAL OF NUTRITION, HEALTH & AGING© Figure 8 Various flavonoids with discrepancies in their redox potentials OH HO

O

2

3

OH

B

HO

OH

O

OH

2

3

O

OH

fisetin

OH

B

HO

O

OH

2

3

O

OH

kaempferol

B OH

OH HO

OH

2

3

O

OH

quercetin

It is quite surprising, however, to have these values working opposite to what can be drawn from the following results, making quercetin one of the fastest reducing agent of superoxide while B-ring monosubstituted derivatives were measured “with substantially higher redox potentials”: The rates of the superoxide radical ( O 2 .— ) reactions with flavonoids depend on the redox potential and on the charge of the flavonoids (pH). The highest rates are measured for the reduction by quercetin and rutin, whereas the lowest are those for the B-ring monosubstituted derivatives, with substantially higher redox potentials. Uncharged catechin at pH 7 reacts at k= 6.6x104.M-1.s-1, whereas the rate at pH 10, where catechin is doubly negatively charged, is approximately 4 times lower, k= 1.8 x104.M–1.s–1 (116). The singlet oxygen quenching rate constants for a range of hydroxycinnamic acids in acetonitrile and D 2O solutions were measured using time resolved near infrared phosphorescence in order to establish their antioxidant activity (117). The relative antioxidant activities, against radicals generated in the aqueous phase, were evaluated in vitro for many other polyphenols: gallocatechins, epigallocatechin gallate, purpurogallin, theaflavins (118, 119) from green and black teas, from fruits, vegetables and wine. The results show the importance of the positions and extents of hydroxylation of the A and B rings to the total antioxidant activity: compounds with 3',4' dihydroxy substituents in the B ring, such as quercetin and cyanidin (figure 9), and conjugation between the A and B rings, have antioxidant potentials four times that of the water soluble vitamin E analogue (Trolox). Removing the ortho-dihydroxy substitution, as in kaempferol, or the potential for electron delocalisation by reducing the 2,3 double bond in the C ring, as in catechin and epicatechin, decreases the antioxidant activity by more than 50%, but these structures are still more effective than α-tocopherol or Vitamin C (120). A special structural feature was evidenced recently: it concerns the presence of methoxyle(s) ortho-positioned to the

O

B OH OH

O

dihydroquercetin

phenolic group. In polymethoxy phenols, as in ubiquinols, the hydrogen atom abstraction revealed to be surprisingly easy. It was found that 2-methoxyphenol was almost entirely intramolecularly hydrogen bonded even in strong hydrogen accepting (HBA) solvents (121). H

O

CH3 H

H3CO intramolecular hydrogen bonds

10

H3CO

CH3 H

O

Ubiquinol-10 (Q10H 2)

This intramolecularly hydrogen bonding prevents the formation of strong hydrogen bonds with solvent molecules or polar solutes. Thus, phenolic hydrogen atoms remain available for hydrogen atom abstraction even in strong HBA environments (122). This is important in the LDL particles where they have to perform their antioxidant function and may explain why a simple modification of the chemical structure (methylation of catechol ring in flavanols, that often occurs) can deeply modify the polyphenol’s properties. 3 - Dietary antioxidants: biological implications 3.1 Peroxidation: at the membrane and cell structure levels Most of these ROS will rapidly react (oxidise/reduce) with organic molecules in the surroundings (lipids, for instance), thus creating an “oxidized” state in the cell. It is worthy of note that the most reactive of all ROS is HO. whose half-life is in the order of 10–9 s Other ones have a mean half-life of about the s e c o n d. They initiate the process of lipid peroxidation by abstraction of a hydrogen atom from an unsaturated aliphatic lipid side-chain (arachidonic acid, here) to yield first, alkyl radicals that add rapidly the diradical O2 (if present) to form lipid peroxyl radicals eventually giving rise by an autocatalytic chain reaction to a lipid hydroperoxide (figure 10).

Figure 9 Various flavonoids with discrepancies in their redox potentials catechol ring OH O

HO

OH

kaempferol

3' OH

O 2

HO OH

O

OH

4' OH

3 OH

O

B

O

HO

A

OH

OH O 2

HO

C

3 OH

OH

OH OH

quercetin

215

OH

C

cyanidin

OH

catechin


DIETARY ANTIOXIDANTS, PEROXIDATION AND CARDIOVASCULAR RISKS Figure 10 Lipid peroxidation and " physiological " responses cellular messengers R'

R'

R' OO

R'

O

R'

R

H

H

O O H R

H R

R

O arachidonic acid

H 2O

R'

R'

H

H

chain reaction oxidation R

PGH 2

OH

HOOC

Prostacyclin PGI2

O H

H

AGGREGATION --VASODILATATION

OH

O

INFLAMMATION

COOH

VASOCONSTRICTION

R'

O OH

Thromboxane A 2

..... ! !

H

O2

O O

R R radicals dienes

COOH

O

OH

hydroperoxide

OH

HO

R

R alkylperoxy radical

leucotrienes

H H

oxidation sites

H

cascade of reactions

H

prostaglandines

H

physiological actions

OH

COOH

LTB 4

neuroglandines

R' H H

(in the brains)

When produced in an integer cell from eicosanoids (arachidonic acid), under the control of enzymes (cyclo, lipooxigenases) they give rise to chemicals (prostaglandins, thromboxanes, leucotrienes) that are very potent cellular messengers (cytokins) (123, 124). Even if corresponding to a dysfunction state, these reactions (inflammation) insure the defence of the cell. 3.1.1 The advantage of dietary polyunsaturated fatty acids In pathological situations, due to the action of ROS that are overproduced (HO., etc), oxidation runs quickly (less than µs timescale) at any carbon site of cell structures (bilayer membrane). If oxidation occurs on saturated fatty acids of the membrane (thin “broken” arrows, figure 10), the m a n y peroxidized molecules that can be formed would have no “biological meaning” to any receptor in the cell that would thus be definitely damaged and most of all, in which peroxidationchain would continue without any chance to deliver any signal. If, by a well balanced diet, our membranes have incorporated some unsaturated fatty acids, uncontrolled oxidations by ROS, would still occur in a manner “guided” towards the specific carbon sites situated between two double bonds (thick “plain” arrows, figure 10). These special features (twice in allylic position) in eicosanoid structures make them the most oxidizable sites and, in the absence of any antioxidant, would lead mainly to the same oxidized metabolites as those formed in the case of an enzymatic control (cytokins). Thus, chemical oxidation would still be meaningful for biological systems, insuring the defence of the organism. 3.2 Dietary antioxidants in biological systems 3.2.1 Polyphenols as antioxidants If a molecule is to avoid the formation of such radicals with efficacy, it has to compete with the molecules of the cell structure and to react more rapidly than any of them. That is to say, it must reduce (transfer of a single electron) the ROS (here,

216

OH

anti-THROMBOTIC CHIMIOTACTISM +++ (poly neutro + eosino)

??

HO.) before it reacts with the lipid: in such a case, it can be seen as an antioxidant (figure 11). All the various properties (hydrosolubility, acidity, redox potential(s)), defined above apply. 3.2.2 Polyphenols as radical scavengers When a radical is already formed on the lipid, it immediately reacts with oxygen (O2) to form a peroxyl radical (ROO.). This later is able to oxidise any fatty acid in the membrane to yield a new radical that reacts also with O 2 and so on: it is an autooxidation chain reaction. Only a “radical scavenger” can reduce it and, doing so, can stop the damaging effects (figure 12). 3.2.3 The advantage of dietary antioxidants These reactivities are “THE” main chemical properties of phenolic compounds under their “reduced form”. They are, by far (10 5 to 10 6 times), more efficient to develop these properties, than the saturated fatty acids and even than unsaturated ones (10 2 to 10 4 times). Because of the much higher stability (thus, the less reactivity) of the resulting aryloxy radicals, a n t i o x i d a n t s would avoid completely peroxidations to propagate. The unique question is the necessary presence of these antioxidants on the site of production of the ROS (which is deeply related to food intake on the long term scale). The evolution would then correspond to a transfer of ROS entities to oxidized but stabilized polyphenolics that would migrate then to the water phase (see figure 14), in which Vitamin C is able to totally detoxify these radicals. The neat result of such properties, exerted in biological systems, would be to avoid oxidation of any structure cells, keeping it in the “normal” state, whatever the production of ROS.


THE JOURNAL OF NUTRITION, HEALTH & AGING© Figure 11 Polyphenols as antioxidants are more readily oxidized than arachidonic acid H2O OH

O

O H

+

OH

OH

OH

aryloxy radicals

OH R H

H

R

R'

H2O HO

H

R'

H

R R' dienyl radicals

R

H H

R

R'

H

R'

H

O arachidonic acid

oxidation sites

Figure 12 Polyphenols as radical scavengers by reduction of a preformed radical O OH

O

OH

OH

+H

R'

R'

R'

RO

O2

ROH

H

H

H H R

OH aryloxy radicals

O peroxy radicals

oxidation chain reaction

R

R alkyl radical

H

R

OH O

O

O H OH

R

OH O

R

R H

H

R'

R'

H hydroperoxides

R'

R'

3.3 The biological problems 3.3.1 Oxidative stress and ageing or pathology: There is now a strong body of evidence showing the relationship between oxidative damages in the tissues (due mainly to oxidants such as oxygen-centred free radicals) and ageing (125, 126) or most of the chronic pathologies (28, 127). It is generally agreed that the “visible part” of pathologies such as neurodegenerative ones (Parkinson (128), Alzheimer (129)) are mainly resulting from repeated oxidative stresses, but also, that perturbations in lipid metabolism, namely, excess of fatty acids in the diet or their peroxidation, are cause of atherosclerosis leading to coronary arterial disease or associated with arthritis, atherogenesis and cancer. Such perturbations or disorders happen when cellular mechanisms (enzymatic: peroxidases, catalases, superoxide dismutases, or non enzymatic ones: Vitamins E and C, etc.), dedicated to fight against the pro-oxidant states of the tissues are overflowed, either because of the too high level of ROS production or by the loss of their efficacy. In such a context, it becomes more and more understandable to consider that antioxidants or radical scavengers may play a general and powerful protective role against them.

3.3.2 The “time-scale” These two complementary antioxidant and radica l scavenging properties are the ones that make polyphenols the most fascinating molecules but also, with the most difficult reactivity to understand. The peculiar question we are facing is due to the possible interferences between “living cell metabolism”, reactive oxygen species (ROS) and dietary antioxidants. Most of the inconsistencies between studies aiming at assessing the action of dietary antioxidants on CVD result from a kinetic problem: the “ t i m e s c a l e” of each independent mechanism implied in the settlement of CV pathology (very low pace phenomena) and the radical oxidations (ultra fast) and that are very much different from each other, for instance: a) The one for the reactive oxygen species to react further (less than microseconds) b) The one for inflammatory response to appear at a cellular level (seconds) c) The one for inflammatory response to appear at a tissue level (minutes) d) The one for the peroxides to noticeably decrease the total “antioxidant” level of plasma (days) e) The one for the “plasma total antioxidant capacity” depletion to produce visible disorders (weeks)

217


DIETARY ANTIOXIDANTS, PEROXIDATION AND CARDIOVASCULAR RISKS f) The one for atheromatous plaque to form and to injure the wall vessel (years) g) The one for myocardial infarction to occur (decades)

However, in the corresponding corn oil-in-water emulsions, all tea catechins, GA, and PG were prooxidants at 5 and 20 mu M by accelerating hydroperoxide and hexanal formation. In contrast, in soy lecithin Liposomes oxidized at 50 degrees C, EGCG and PG were the best antioxidants, followed by EC, EGC, ECG, catechin, and GA at 20 mu M. In liposomes oxidized at 37 degrees C with 10 mu M cupric acetate, catechin and EC were better antioxidants than ECG, but EGCG, EGC, PG, and GA promoted lipid oxidation. The improved antioxidant activity observed for tea catechins in liposomes compared to emulsions can be explained by the greater affinity of the polar catechins toward the polar surface of the lecithin bilayers, thus affording better protection. The marked variation in activity among tea catechins may be partly explained by their different reducing potentials, stabilities, and relative partitions between phases in different lipid systems” (134, 135).

In other words, how is it possible to measure/evaluate accurately the impact of an antioxidant (µs) on a physiological system in which the oxidation process is not even likely to occur? In the meantime, from the very first event to the fatal one, so many other important mechanisms could play a role and could be more relevant to understand the total phenomenon. Till now, in the in vivo situation, we only are able to investigate events that are producing the medium timescale parameters: from d to e, while all are important to consider. 3.3.3 The “pro-oxidant” activity of dietary polyphenols All these reducing and antioxidant properties reveal in vitro to be very much dependent: • on the nature of the oxidisable matter (lipids, proteins, etc.), • on the medium in which it stands (water (pH) or lipid bilayer membranes, or in any phase in between), • on the nature of the oxidant (radical, anion-radical, singlet oxygen, nitric oxide (130)), etc. (131-133). It seems that results become as contradictory as for the human epidemiological data (see above). For example, one can read from literature relating works to investigate the biological phenomena by simulation in vitro:

3.3.4 Vitamin E as a “pro-oxidant” Haslam recently said: “ Vitamins , minerals and other nutritional supplements do not work in isolation ; they are not drugs . There is a delicate system of checks and balances with other nutrients” (111). This assertion is very wise but, most of all, it reminds us that we cannot play as a Quack (charlatan) with such reactive molecules before knowing all the involved mechanisms. For example, Vitamin E itself, the most potent physiological radical scavenger in the LDL-cholesterol emulsions, was demonstrated to be pro-oxidant: there is a process by which the tocopheryl radical, issued from the reduction of a lipid peroxy radical, rather than to reduce a second one and to form non radicalar species, can abstract an hydrogen atom, oxidize a lipid molecule (figure 13). Vitamin E is thus initiating a new peroxidation chain reaction named “TMP” (tocopherol mediated peroxidation process) (136, 137). This process, hopfully is inhibited by Vit. C, in the aqueous phase and by ubiquinol-10 in the lipidic phase of LDL particles because they have lowest reduction potentials:

“In corn oil triglycerides oxidised at 50° C, epigallocatechin (EGC), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG) were better antioxidants than epicatechin (EC) and catechin at 140 M. Used as reference compounds, gallic acid (GA) was more active than propyl gallate (PG), and both were more effective than EC and catechin. OH

OH OH

O

HO

OH

OH O

HO

OH OH

OH O

HO

OH O

OH

OH 3 O

OH

O

OH

OH

O

OH

OH

OH

epigallocatechin= EGC

OH

epigallocatechin-3-O-gallate= EGCG

epicatechin-3-O-gallate= ECG

Figure 13 The “tocopherol-mediated peroxidation” (TMP) process first step of protection R' alkylperoxy radical H

Tocopherol Mediated Peroxidation process CH3 Phyt H 3C O

O O

R

O

HO

OH

Vit. C R' OH

CH3 H 3C

C O

R

R1 tocopherols

CH2 OH

HO

H

CH3

O

O

O H R hydroperoxy FA

diene radical

HO

R' H

R'

H3CO

Phyt CH3

O

H H

R1

R

218

CH3

H3CO

Phyt OH

Q 10-H2


THE JOURNAL OF NUTRITION, HEALTH & AGING© ArO. + Q-10-H2 ---> ArOH + Q-10-H. = semiquinone radical (1) Q-10-H. + O2 ---> Q-10 (quinone form) + O2.- (+H+) (2) Superoxide anion O 2 .- that is formed in step (2) simultaneously to the quinone (Q-10), is a water-soluble species that escapes LDL particle and thus stops the TMP process. 3.3.5 Supplementation in Vitamin E: contradictory results Most experiences with Vitamin E have used a l p h a t o c o p h e r o l, because it is the most abundant and the most available one out of the eight possible tocopherols or tocotrienols. CH3

CH3 H 3C

CH3 HO CH3

H 3C

CH3

-tocopherol

nucleophilic occupied site

CH3

CH3

CH3

O

CH3

O

CH3 CH3

HO

be definite proofs) but also because many of the biological properties of diet flavonoids remain unknown. Most importantly, the proof of their ability to exert in vivo all the properties they show in vitro (their bioavailability) is still a matter of concern (58) and is not easy to prove (146-148). Further studies are required to determine if red wine phenolics are actually absorbed from the gut and under which form they are, if any. It has been demonstrated recently that these molecules (even the oligomeric ones) were mostly transformed by human gut flora (52, 149) in much smaller molecules (150) closely related to aspirin, which is … an “acid phenol”, commonly used as a remedy for the lifetime, in case of major circulatory dysfunctions, … So, which are the active molecules? Conclusion

-tocopherol

nucleophilic free carbon atom

Another publication, maybe, brings about the answer: “These experiences have failed to demonstrate a protective effect in heart disease. Interestingly, Vit. E appeared to offer protection only when taken up from the diet and not when taken in supplements which consist primarily of alpha-tocopherol” (138), particularly if we consider the fundamental observation made by Christen et al., that γ-tocopherol traps efficiently mutagenic electrophiles such as peroxinitrites (NOx) but not at all α-tocopherol and, that supplements in this latter were inducing a strong decrease to a 5% of the normal levels of the former (139). The mechanism to scavenge this kind of ROS is totally different from redox ones: it is a nucleophilic addition of a carbon atom of the aromatic ring onto the electrophilic peroxynitrite that detoxifies them. Such an aromatic ring with a nucleophilic carbon atom that is not substituted, is only present on the γ-tocopherol molecule. 3.3.6 Antioxidants against “pro-oxidants”: a question of perfect matching Physiological implications are evident: one needs as many as possible radical scavengers but in “equilibrated” amounts of each … Maybe, those provided by the “balanced” Mediterranean diet are the best ones ever obtained today. In such a figure, all plant polyphenols would be potentially good for health and able to confer their own part of the antioxidant protection (none of these molecules is known as toxic in acute dose. At present, it is still difficult to fully understand how is regulated such an equilibrium in the human body, but such a phenomenon could explain why results, in v i v o, are sometimes so contradictory and also, why the association of a “pool” of polyphenolic substances at the same time, results always in more potent antioxidant effects (140). This kind of synergistic protection was observed in many experiments run with polyphenols (141-145). This contradictory situation could come about because of the kind of data taken into account (epidemiological studies lead to “correlations” that, even if very strong indications, will never

We postulate that positive health benefits of d i e t a r y antioxidants could possibly best develop when each individual polyphenol in the mixture, according to its structure, to its reduction potential, to the intra vs the intermolecular bondings with the surroundings, to its solubility, etc…, could stand and detoxify ROS, at the right place in the right compartments of the cell in which they appear. If this presumed behaviour is accepted, then the largest amounts of the highest varieties of such molecules could best insure the best protection against the damages related to the perturbations of the oxygen metabolism. These molecules would thus have typically the same function as Vitamin E or C, or as ubiquinones and maybe, should they be named as such. However, this would not change anything to the reality, it just would change our mind on the mode of action of these vital molecules and maybe, would help to a better comprehension of the underlying complex phenomena. Figure 14, even though only partly, is proposed as a scheme that takes into account all these facts. The few percentages (5%) of leaks of ROS that appear in the cell (around the mitochondria), would distribute in all the cell compartments, according to their polarity (neutral, as radical hydroxyle; polar, as superoxide anion; etc.). Thus, to scavenge all of them, we need the whole series of reducing substances (maybe, thousands), from the most polar ones in the aqueous phase like Vit. C to the most lipophilic ones like Vit. E (all tocopherols, ubiquinols, ... ). In between these two extremes, all the dietary antioxidants, reputed as radical scavengers in vitro (stilbenoids, flavonoids, chalcones, anthocyanins, ... ), would assure a complementary detoxifying role in any cell compartments of intermediate polarity, either directly or eventually as a “relay” in the electron transfer chain from Vit. C to Vit. E, to regenerate them. In summary, dietary flavonoids would considerably help to drain all the deleterious species towards the aqueous phase, thus, turning down all the uncontrolled oxidative processes, keeping the cells working smoothly as the best protective effect against CVD. This remains to be proven.

219


DIETARY ANTIOXIDANTS, PEROXIDATION AND CARDIOVASCULAR RISKS Figure 14 Mode of action of polyphenolic "vitamins" (putative) OH CH3 O CH3 CH3

CH3 O

HO H

HO CH3

CH3 CH3

O

Rha-Glc-O

CH3

Vitamin E ( -tocopherol)

HO

H 3C

OCH3

CH3

HOH 2C

OH

flavanones O OH Rha-Glc-O

CH3

OCH3

O

HO OH OCH3 OH

OCH3 O

HO

HO

CH3O O chalcones

stilbenoïds OH

OGlc H anthocyanins

HO

gradient concentration of antioxidants from water to lipid compartments membranes, lipoproteins

ONO2-

ROO

O

vacuoles, cytoplasme

reticulum, mitochondria

NO

HO

O

Vitamin C

OH

Vitamin E ( -tocopherol)

HC

AQUEOUS Phase

ORGANIC Phase

H 3C

CH3

CH3

O2.-

excretion

detoxified and water-soluble forms of ROS and of antioxidants

O2

overproduction of ROS (leaks from oxygen metabolism; 5% max)

ATP ATP ATP ATP ATP

H

HO O

H H

O H OH OH

H

HO O

H H

O

H

HO O

H OH OH

H H

glucose

H OH

H OH

H OH

H OH

O H OH OH

H

HO O

H H

O H OH OH

mitochondria (energy cell production)

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