Plant Polyphenol Antioxidants and Oxidative Stress

INES URQUIAGA and FEDERICO LEIGHTON

Laboratorio de Citología Bioquímica y Lípidos, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile

ABSTRACT

In recent years there has been a remarkable increment in scientific articles dealing with oxidative stress. Several reasons justify this trend: knowledge about reactive oxygen and nitrogen species metabolism; definition of markers for oxidative damage; evidence linking chronic diseases and oxidative stress; identification of flavonoids and other dietary polyphenol antioxidants present in plant foods as bioactive molecules; and data supporting the idea that health benefits associated with fruits, vegetables and red wine in the diet are probably linked to the polyphenol antioxidants they contain.In this review we examine some of the evidence linking chronic diseases and oxidative stress, the distribution and basic structure of plant polyphenol antioxidants, some biological effects of polyphenols, and data related to their bioavailability and the metabolic changes they undergo in the intestinal lumen and after absorption into the organism.Finally, we consider some of the challenges that research in this area currently faces, with particular emphasis on the contributions made at the International Symposium "Biology and Pathology of Free Radicals: Plant and Wine Polyphenol Antioxidants" held July 29-30, 1999, at the Catholic University, Santiago, Chile and collected in this special issue of Biological Research.

Key words: Oxidative stress; antioxidant; plant polyphenol; flavonoid; chronic diseases; diet

This review and the accompanying articles in this special issue of Biological Research reflect the content of the presentations and discussions held on the occasion of the International Symposium "Biology and Pathology of Free Radicals: Plant and Wine Polyphenol Antioxidants," July 29-30, 1999, at the Catholic University in Santiago, Chile.

Chronic diseases and oxidative stressChronic diseases constitute a major challenge for medicine and basic biology and will certainly remain so for the next decades. We have seen the emergence, in epidemic proportions, of modern chronic diseases in the latter part of the 20th century, a process that is still in progress (Wilks et al., 1998). In developing countries, this process is part of what is known as an epidemiological transition (Vio & Albala, 2000), and it is particularly striking in the Americas (Castillo-Salgado et al., 1999). Characteristically, infectious diseases are replaced by chronic or non-communicable diseases as the primary cause of morbidity and mortality. This situation is associated with changes in diet and lifestyle that contribute to the development of chronic diseases. Among the risk behaviors characteristic of the transition are excessive dietary fat intake, low intake of fruits and vegetables, sedentary life style, smoking, and environmental contamination.

A primary focus of preventive medicine is the detection and treatment of individuals at risk, and molecular tools are increasingly used to recognize risk. Today, chronic diseases are at the interface of molecular genetics and preventive medicine. For chronic diseases such as coronary heart disease, context-dependent effects are determinant; they include interactions among genes (genetic epistasis) and between genes and environmental factors (gene-environment interactions) (Ellsworth et al., 1999). Strikingly, there are some common risk factors and pathophysiological conditions that affect most diseases grouped into the category of modern chronic diseases: cardiovascular disease, hypertension, diabetes mellitus, and some forms of cancer. Oxidative stress is a central risk factor for chronic diseases.

Oxidative stress, the consequence of an imbalance of prooxidants and antioxidants in the organism, is rapidly gaining recognition as a key phenomenon in chronic diseases. It is directly involved in the pathogenic mechanism of risk factors and in the protection exerted by various environmental factors. And the quantification of oxidative stress in populations appears to be a possible indicator for the magnitude of environmental risk factors. For example, it has been proposed that the relatively high cardiovascular mortality rate in post-communist countries is the consequence of environmental conditions resulting in higher levels of oxidative stress (Ginter, 1996). Diet plays a major role in the environmental control of oxidative stress: fruits, vegetables and red wine decrease oxidative stress, whereas the occidental diet, characteristically rich in fats, induces oxidative stress (Leighton et al., 1999).

Compelling evidence has led to the conclusion that diet is a key environmental factor and a potential tool for the control of chronic diseases. After tobacco, inadequate diet and activity patterns are the most prominent contributors to mortality in USA (McGinnis and Foege, 1993). Dietary recommendations for the prevention of cancer, atherosclerosis and other chronic diseases have been established by various health agencies (Bronner, 1996; Munoz de Chavez and Chavez, 1998). More specifically, fruits and vegetables have been shown to exert a protective effect (Gillman et al., 1995; Joshipura et al., 1999; Cox et al., 2000; Strandhagen et al., 2000). The high content of polyphenol antioxidants in fruits and vegetables is probably the main factor responsible for these effects.

Polyphenols, Natural Antioxidants in Food and Beverages

Polyphenols are present in a variety of plants utilized as important components of both human and animal diets (Bravo, 1998; Chung et al., 1998; Crozier et al., 2000). These include food grains such as sorghum, millet, barley, dry beans, peas, pigeon peas, winged beans, and other legumes; fruits such as apples, blackberries, cranberries, grapes, peaches, pears, plums, raspberries, and strawberries; and vegetables such as cabbage, celery, onion and parsley also contain a large quantity of polyphenols. Phenolic compounds are also present in tea and wine. Forages such as crownvetch, lespedeza, lotus, sainfoin, and trefoil are also reported to contain polyphenolic compounds.

Diets containing an abundance of fruit and vegetables are protective against a variety of diseases, particularly cardiovascular disease and cancer. The primary nutrients thought to provide the protection afforded by fruit and vegetables are the antioxidants (Eastwood, 1999). Potter (1997) reviewed 200 epidemiological studies, the majority of which showed a protective effect of increased fruit and vegetable intake. When the role of individual antioxidants, vitamins C and E, and carotenoids, is examined by epidemiological studies or supplementation trials, the results are not as clear-cut as those obtained for fruit and vegetables and are often disappointing. Potter’s conclusion was that fruit and vegetables provide the best polypharmacy against the development of a chronic disease, considering that they contain a vast array of antioxidant components such as polyphenols.

Diets rich in fruits and vegetables, such as vegetarian and Mediterranean diets, contain a large quantity of polyphenols. Dietary habits consistent with protection from coronary heart disease have been considered too restrictive (high in polyunsaturated fats and/or vegetarian); however, the diet in some Mediterranean countries, such as France, Spain and Italy, is varied and characterized by a low consumption of butter and high consumption of bread, vegetables, fruit, cheese, vegetable fat, and wine: the so called Mediterranean type diet. In addition, other foods high in saturated fat are eaten; 14-15 % of energy intake corresponds to saturated fat (Renaud and de Lorgeril, 1992; Segasothy and Phillips, 1999). Certainly, a high consumption of vegetables constitutes a healthy habit observed in Mediterranean countries in conjunction with moderate wine consumption. The univariate correlation coefficients between coronary heart disease mortality and the intake of various foodstuffs in a study based on statistics from the 21 most industrialized wine-drinking countries were as follows: vegetables, -0.48 (P<.05); vegetable fats, -0.44 (P<.05); fruit, -0.28 (NS); dairy products, 0.66 (P<.001); and wine, -0.87 (P<.001) (Renaud and Ruf, 1994). In this study, the protective effect of wine appears to be much more convincing than that of other vegetable foods. Indeed, wines are rich in polyphenols, especially red wine.There is no accurate information available on the dietary intake of polyphenols because their content in plant foods varies greatly, even among cultivars of the same species. The presence of polyphenols in plant foods is largely influenced by genetic factors and environmental conditions. Other factors, such as germination, degree of ripening, variety, processing, and storage, also influence the content of plant phenolics (Bravo, 1998).

Hertog et al. (1993) measured the flavonoid content of fruits, vegetables, and beverages important in the Dutch diet and reported a dietary flavonoid intake of approximately 26 mg per day in this population. The flavonoid content of the diet was defined as the sum of quercetin, kaempferol, myricetin, apigenin, and luteolin. The investigators found that 95% of total dietary flavonoids were from two compounds, 63% from quercetin and 32% from kaempferol, and that the seasonal variation in the flavonoid content of food was low. The major dietary contributor to flavonoid intake was black tea (61%) followed by onions (13%) and apples (10%). Knekt et al. (1996) also reported that flavonoid intake ranged from 0 to 41.4 mg per day in the Finnish population, and the main sources of flavonoids were apples and onions. Both groups found an inverse association between intake of dietary flavonoids and cardiovascular disease. These studies, however, consider only the intake of some specific flavonoids and do not look at other phenolic compounds. Thus, an accurate estimation of total polyphenolic intake is not available.

Structure of Plant Polyphenols

Phenolic compounds, or polyphenols, constitute one of the most numerous and widely-distributed groups of substances in the plant kingdom, with more than 8,000 phenolic structures currently known. Polyphenols are products of the secondary metabolism of plants. The expression "phenolic compounds" embraces a considerable range of substances that possess an aromatic ring bearing one or more hydroxyl substituents. Most of the major classes of plant polyphenol are listed in Table I, according to the number of carbon atoms of the basic skeleton. The structure of natural polyphenols varies from simple molecules, such as phenolic acids, to highly polymerized compounds, such as condensed tannins (Harborne, 1980).

    TABLE I The major classes of phenolic compounds in plants

Number of carbon atoms	Basic skeleton	Class	Examples
6	C6	Simple phenols Benzoquinones	Catechol, hydroquinone 2,6-Dimethoxybenzoquinone
7	C6-C1	Phenolic acids	Gallic, salicylic
8	C6-C2	Acetophenones  Tyrosine derivatives  Phenylacetic acids	3-Acetyl-6-methoxybenzaldehyde  Tyrosol  p-Hydroxyphenylacetic
9	C6-C3	Hydroxycinnamic acids Phenylpropenes Coumarins Isocoumarins Chromones	Caffeic, ferulic  Myristicin, eugenol Umbelliferone, aesculetin Bergenon Eugenin
10	C6-C4	Naphthoquinones	Juglone, plumbagin
13	C6-C1-C6	Xanthones	Mangiferin
14	C6-C2-C6	Stilbenes  Anthraquinones	Resveratrol  Emodin
15	C6-C3-C6	Flavonoids  Isoflavonoids	Quercetin, cyanidin  Genistein
18	(C6-C3)2	Lignans  Neolignans	Pinoresinol  Eusiderin
30	(C6-C3-C6)2	Biflavonoids	Amentoflavone
n	(C6-C3)n  (C6)n  (C6-C3-C6)n	Lignins  Catechol melanins  Flavolans (Condensed Tannins)

Flavonoids represent the most common and widely distributed group of plant phenolics. Their common structure is that of diphenylpropanes (C6-C3-C6) and consists of two aromatic rings linked through three carbons that usually form an oxygenated heterocycle (Harborne, 1980). Figure 1 shows the basic structure and the system used for the carbon numbering of the flavonoid nucleus. Structural variations within the rings subdivide the flavonoids into several families: flavonols, flavones, flavanols, isoflavones, antocyanidins and others. These flavonoids often occur as glycosides, glycosylation rendering the molecule more water-soluble and less reactive toward free radicals. The sugar most commonly involved in glycoside formation is glucose, although galactose, rhamnose, xylose and arabinose also occur, as well as disaccharides such as rutinose. The flavonoid variants are all related by a common biosynthetic pathway, incorporating precursors from both the shikimate and the acetate-malonate pathways (Crozier et al., 2000). Further modification occurs at various stages, resulting in an alteration in the extent of hydroxylation, methylation, isoprenylation, dimerization and glycosylation (producing O- or C-glycosides).Phenolic compounds act as antioxidants with mechanisms involving both free radical scavenging and metal chelation. They have ideal structural chemistry for free radical-scavenging activities, and have been shown to be more effective antioxidants in vitro than vitamins E and C on a molar basis (Rice- Evans et al., 1997).

Figure 1: Flavonoids (C6-C3-C6) Basic structure and system used for carbon numbering of the flavonoid nucleus. Structural variations within the rings subdivide the flavonoids into several families.

 

Biological Effects of Polyphenols

Polyphenols exhibit a wide range of biological effects as a consequence of their antioxidant properties. They inhibit LDL oxidation in vitro (Frankel et al., 1993). Moreover, LDL isolated from volunteers supplemented with red wine or red wine polyphenols show reduced susceptibility to oxidation (Fuhrman et al., 1995; Nigdikar et al., 1998). Thus, polyphenols probably protect LDL oxidation in vivo with significant consequences in atherosclerosis. and also protect DNA from oxidative damage with important consequences in the age-related development of some cancers (Halliwell, 1999). In addition, flavonoids have antithrombotic and anti-inflammatory effects (Gerritsen et al., 1995; Muldoon and Kritchevsky, 1996). The antimicrobial property of polyphenolic compounds has been well documented (Chung et al., 1998).

Several types of polyphenols (phenolic acids, hydrolysable tannins, and flavonoids) show anticarcinogenic and antimutagenic effects. Polyphenols might interfere in several of the steps that lead to the development of malignant tumors, inactivating carcinogens, inhibiting the expression of mutant genes and the activity of enzymes involved in the activation of procarcinogens and activating enzymatic systems involved in the detoxification of xenobiotics (Bravo, 1998). However, some polyphenols have been reported to be mutagenic in microbial assays and co-carcinogens or promoters in inducing skin carcinogenesis in the presence of other carcinogens (Chung et al., 1998). This latter possibility warrants further research.

Several studies have shown that in addition to their antioxidant protective effect on DNA and gene expression, polyphenols, particularly flavonoids, inhibit the initiation, promotion and progression of tumors, possibly by a different mechanism.

Wine contains many compounds that apparently exhibit anti-cancer properties, including gallic acid, caffeic acid, ferulic acid, catechin, quercetin and resveratrol, among others. Gallic acid is antimutagenic with the Ames’ test (Hour et al., 1999) and hepato protective for carbon tetrachloride toxicity (Kanai and Okano,1998). In an experiment with transgenic mice that spontaneously develop skin tumors, the addition of red wine solid extract to their diet led to a marked delay in tumor development (Clifford et al., 1996).

Caffeic and ferulic acids react with nitrite in vitro and inhibit nitrosamine formation in vivo. They inhibit the formation of skin tumors induced by 7,12-dimethyl-benz(a) anthracene in mice (Kaul and Khanduja, 1998). They also inhibit tyrosine nitration mediated by peroxynitrite (Pannala et al., 1998).

Resveratrol has been extensively studied. It has been isolated from several sources and shown to inhibit the development of preneoplastic lesions in rat mammary gland tissue in cultures in the presence of carcinogens; it also inhibits skin tumors in mice (Clifford et al., 1996; Jang et al., 1997). Other researchers have shown that the combination of resveratrol and quercetine exerts a synergic effect in the inhibition of growth and proliferation of human oral squamous carcinoma cells (ElAttar and Virji, 1999). In this study, however, the best result was observed with diluted red wine. Since resveratrol and quercetin are present in low concentrations, other polyphenols could also be responsible for this effect and for the potentiation of cell growth inhibition.

Polyphenol Bioavailability and Metabolism

The knowledge of absorption, biodistribution and metabolism of polyphenols is partial and incomplete, yet it is sufficient to state that in general, some polyphenols are bioactive compounds that are absorbed from the gut in their native or modified form. They are subsequently metabolized with products detected in plasma that retain at least part of the antioxidant capacity and then excreted. Experimental studies in animals support the previous general statement (Das and Griffiths, 1969; Das and Sothy, 1971; Griffiths and Smith, 1972; Manach et al., 1995; Manach et al., 1997; Piskula and Terao, 1998; Morand et al., 1998; Okushio et al., 1999a; 1999b). In humans, studies aim at identifying native compounds and their metabolites in plasma and urine after the administration of test meals or drinks. These studies also support the initial general statement. Many of the studies performed with humans are centered on the detection of quercetin after the consumption of onions, tea, and apple juice (Hollman et al., 1996,1997; Aziz et al., 1998; Manach et al., 1998; Lean et al., 1999; McAnlis et al., 1999).

Some of these studies have addressed the question of the biological activity of rutin and quercetin metabolites, such as the ability of quercetin and isorhamnetin to inhibit copper induced LDL oxidation (Manach et al., 1998; Morand et al., 1998). These authors state that the plasma metabolites retain antioxidant activity.

After green tea consumption, epigallocatechin gallate and epicatechin gallate are detected in plasma and urine (Yang et al., 1998). Red wine consumption leads to the accumulation of o-methylcatechin, a catechin metabolic product, in plasma (Donovan et al. 1999). These findings should be considered important initial contributions to the identification of the various bioavailable polyphenols present in tea and wine, as well as the identification of their metabolites. Pietta et al. (1998) employed green tea to attempt an overall evaluation of absorption and metabolism. They detected green tea flavanols in plasma and some monohydroxy and dihydroxybenzoic acids in urine, accounting for approximately 15% of the polyphenols administered. These phenolic acids would result from bacterial metabolization of catechin and quercetin in the gut. The intestinal flora has enzymes that cleave the benzopyranosic ring (Das and Griffiths, 1969; Winter et al., 1989).Methylation in one or more phenolic hydroxyls is another possibility in polyphenol metabolism, having been observed for catechin, epicatechin and green tea flavonoids (Piskula and Terao, 1998; Okushio et al., 1999a, 1999b). This reaction is apparently mediated by catechol-O-methyl transferase, an enzyme present in liver and kidney. Epicatechin, methylated and conjugated with glucuronic acid and sulfate, appears as the plasma metabolite with the longer half life, after a single dose of epicatechin to rats (Piskula and Terao, 1998). In rats receiving 0.2% quercetin in their diet for three weeks, the most abundant metabolite was the glucuronic acid and sulfate conjugate of isorhamnetin, the 3' methylation product of quercetin (Morand et al., 1998).

Sulfate and glucuronic acid conjugation, which leads to increased water solubility, is a common strategy for drug metabolism, and in general for xenobiotic metabolism, the products can be more easily eliminated into the urine. Polyphenol glucuronidation occurs in the intestine and in the liver (Sfakianos et al., 1997; Piskula and Terao, 1998; Morand et al., 1998), whereas sulfation apparently occurs only in the liver (Shali et al., 1991; Piskula and Terao, 1998).

Challenges for Research on Polyphenols and their Relationship with Chronic Diseases

There are hundreds of polyphenols with antioxidant activity that are potential contributors to the antioxidant mechanisms in humans and animals in general. These compounds are excellent candidates to explain the health benefits of diets rich in fruits and vegetables, although there is still not enough information on food composition data, bioavailability, interaction with other food components and biological effects (Institute of Medicine, 1998).

Through the number of indexed scientific publications and their distribution over time, it is possible to evaluate the quantity and relative importance of scientific efforts on specific subjects. Thus, we can see in Figure 2 that great emphasis has been placed on the subject of chronic disease in recent decades. Vitamins E and C have received sustained attention in the last few decades, perhaps with a particular increment in the last five years. In contrast, the subject of oxidative stress has seen an explosive growth in recent years; 80% of the articles published on the subject have appeared in the last five years.

Figure 2: Evolution of the scientific interest in antioxidants, oxidative stress and chronic diseases. The curves correspond to the relative distribution of Medline indexed publications for the period 1965-2000, expressed in five year periods. The total number of indexed publications in the period for oxidative stress, natural antioxidants, flavonoids, chronic diseases, and vitamins E and C, was 12,083; 1,230; 2,159; 125,042 and 21,128, respectively.

There is evidence that polyphenols are metabolized by intestinal flora and that they and their metabolites are absorbed. This information is, for the moment, restricted to a few compounds. Similarly, we know that some species are metabolized after absorption. The extent, specificity and localization of polyphenol metabolism in the organism have not been established systematically. In this respect, the known chelating capacity of polyphenols raises the question of their participation in aspects related to metal metabolism and pathology (Morel et al., 1998; Núñez et al., 2000; Opazo et al., 2000; Zago et al., 2000). Another aspect of polyphenol metabolism not yet characterized systematically corresponds to its reaction with other biological antioxidants. Interactions between ascorbate and catechin have been shown (Lotito & Fraga, 2000), leading to the hypothesis that polyphenol antioxidants are part of the antioxidant network of the organism. Indeed, their ability to interact with other antioxidant radicals and peroxyl radicals can be predicted from their reduction potentials (Jovanovic et al., 1998). Attempts have been made to estimate the relative contribution of polyphenols to the total antioxidant capacity in plasma (Perez et al., 2000) but insufficient knowledge on the nature and concentration of circulating polyphenol species renders these results very uncertain. Polyphenol-SH interactions is another subject that remains to be explored systematically. In this respect, Hidalgo et al. (2000) describe the effect of redox reagents on the activity of intracellular calcium release channels in muscle and nerve cells, which raises the possibility of another target to explain the biological effects of polyphenol antioxidants.

The interaction of nitric oxide with polyphenol antioxidants is highly relevant in physiological and pathological cellular mechanisms. Atherogenesis is a process markedly dependent of lipid oxidation products that are recognized by specific receptors (Moriel et al., 2000; Rigotti, 2000). Nitric oxide, a free radical itself, participates in the atherogenic process (Rubbo et al., 2000) through membrane lipid and lipoprotein oxidation events (Boveris et al., 2000). Nitric oxide apparently regulates mitochondrial respiration and polyphenol antioxidants are also active at this level (Hodnick & Pardini, 1998; Carreras et al., 2000)

Another rapidly developing aspect of free radical metabolism is its participation in the process of mediating and regulating cellular function. Nitric oxide and superoxide anion are continuously produced in aerobic cells and regulate the mitochondrial function (Valdez et al., 2000) and these and other free radicals can modulate signal transduction pathways and gene expression (Foncea et al., 2000). Thus it seems very likely that dietary polyphenol antioxidants continuously participate in the regulation of cellular function.

ACKNOWLEDGEMENTS This work and the International Symposium "Biology and Pathology of Free Radicals: Plant and Wine Polyphenol Antioxidants" held July 29-30, 1999, at the Catholic University, Santiago, Chile, were partially supported by the Molecular Basis of Chronic Diseases Program of the Catholic University (PUC-PBMEC99).

Received: August 10, 2000. Accepted: August 30, 2000

Correponding Author: Federico Leighton. Dep. Biología Celulay y Molecular, facultad de Ciencias Biológicas, P. Universidad Católica de Chile. Casilla 114-D, Santiago, Chile. Phone/fax: (56-2) 222-2577. E-mail: fleighto@genes.bio.puc.cl

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