Polyphenols in chocolate: is there a contribution to human health?

Food Research International 33 (2000) 449±459

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Polyphenols in chocolate: is there a contribution to human health? Jan Wollgast *, Elke Anklam European Commission, DG Joint Research Centre, Institute for Health and Consumer Protection, Food Products and Consumer Goods Unit, I-21020 Ispra (Va), Italy Received 3 February 2000; accepted 28 February 2000

Abstract Recently, polyphenols have gained much more attention, owing to their antioxidant capacity (free radical scavenging and metal chelating) and their possible bene®cial implications in human health, such as in the treatment and prevention of cancer, cardiovascular disease, and other pathologies. Cocoa is rich in polyphenols particularly in catechins (¯avan-3-ols) and procyanidins. Polyphenol contents of cocoa products such as dark chocolate, milk chocolate and cocoa powder have been published only recently. However, the data vary remarkably due to the quantity of cocoa liquor used in the recipe of the cocoa products but also due to the analytical procedure employed. For example, results obtained by a colourimetric method were 5±7 times higher for the same type of product than results obtained by high performance liquid chromatography (HPLC). In 1994, the per head consumption of chocolate and chocolate confectionery in the European Union ranged from 1.3 kg/year in Portugal to 8.8 kg/year in Germany. In general, consumers in the Northern countries consume on average more than people in the South. Thus, chocolate can be seen as a relevant source for phenolic antioxidants for some European population. However, this alone does not imply, that chocolate could be bene®cial to human health. Some epidemiological evidence suggests a bene®cial e€ect to human health by following a polyphenol-rich diet, namely rich in fruits and vegetables and to a less obvious extent an intake of tea and wine having a similar polyphenol composition as cocoa. In many experiments cellular targets have been identi®ed and molecular mechanisms of disease prevention proposed, in particular for the prevention of cancer and cardiovascular diseases as well as for alleviating the response to in¯ammation reactions. However, it has to be demonstrated, whether polyphenols exert these e€ects in vivo. One pre-requisite is that the polyphenols are absorbed from the diet. For monomeric ¯avonoids such as the catechins, there is increasing evidence for their absorption. For complex phenols and tannins (procyanidins) these questions have to be addressed for the future. Some indication for the absorption of procyanidins derive from studies with the human colon cancer cell line Caco-2, believed to be a valuable model for passive intestinal absorption as proposed for polyphenols. However, it has to be clari®ed which concentration is e€ective and what concentrations can be expected from food intake. Another open question is related to polyphenol metabolism. For example, much e€ort has been invested to show antioxidative e€ects of free unbound polyphenols, especially of catechins and the ¯avonol quercetin. However, only a very small part can be found in plasma in the free form but conjugated or even metabolised to several phenolic acids and other ring scission products. From the papers reviewed, it is as yet to early to give an answer to the question, whether chocolate and/or other sources rich in catechins and procyanidins are bene®cial to human health. Even though some data are promising and justify further research in the ®eld, it has to be shown in future, whether the intake of these functional compounds and/or their sources is related to measurable e€ects on human health and/or the development of diseases. # 2000 Elsevier Science Ltd. All rights reserved.

1. Introduction A primary role of the diet lies in providing sucient nutrients to meet metabolic requirements and in giving the consumer a feeling of satisfaction and well-being through hedonistic attributes. However, it can also contribute to achieve optimal health and development

* Corresponding author. Tel.: +39-0332-785995; fax: +39-0332785707. E-mail address: [email protected] (J. Wollgast).

as well as play an important role in reducing the risk or delaying the development of disease, such as cardiovascular disease (CVD), cancer and other age-related diseases. Thus, at least in the industrialised world, nutrition concepts are progressing from `adequate nutrition' to `optimal nutrition' (Bellisle, Diplock, Hornsta, Koletzko, Roberfroid, Salminen et al., 1998). For instance, there is much evidence that people with high dietary intakes of fruits and vegetables are less likely to develop cancer than people who have low dietary intakes of these foods. Block, Patterson and Subar (1992) have reviewed approximately 200 epidemiological studies that examined the

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relation between fruit and vegetable intake and several cancers, and their results are remarkably consistent. Among non-smokers, cancers of the breast and colon are the most malignancies in Western societies. The risk of colon cancer may be more responsive than that of other cancers to changes in the diet (Willet, 1989). Epidemiological studies provide some, but not conclusive evidence that animal fat or meat intake is associated with risk of colon cancer (Henderson, Ross & Pike, 1991; Willet, 1989). Inverse associations have been found repeatedly between consumption of fruit and vegetables but not of cereal products and the risk of colon cancer. This weakens the hypothesis that dietary ®bre reduces the incidence of colon cancer or at least demonstrates it to be much too simpli®ed. Speci®c fractions of ®bre may be active agents. Other candidates include antioxidants, such as vitamins C and E, or phytochemicals (Willet, 1989). In fact, the wide range of food products available to today's consumer o€ers a wide variety of complex food components, both nutritive and non-nutritive. It is the position of the American Dietetic Association (ADA) that speci®c substances in foods (e.g. phytochemicals as naturally occurring components and functional food components) may have a bene®cial role in health as part of a varied diet (Bloch & Thomson, 1995). Among phytochemicals, polyphenols constitute one of the most numerous and widely distributed groups of substances in the plant kingdom, with more than 8000 phenolic structures currently known (Bravo, 1998). Polyphenols have gained much attention, recently, owing to their antioxidant capacity (free radical scavenging and metal chelating) and their possible bene®cial implications in human health, such as in the treatment and prevention of cancer, cardiovascular disease, and other pathologies. Cocoa is rich in polyphenols particularly in catechins (¯avan-3-ols) and procyanidins (Fig. 1). The total polyphenol content of the bean is estimated to be 6±8% by weight of the dry bean (ZumbeÂ, 1998). However, the quantitative determination of polyphenols in chocolate and cocoa powder has been

reported only very recently: using the colourimetric Folin±Ciocalteau method, Waterhouse, Sirley and Donovan (1996) have found 8.4 mg polyphenols/g dark chocolate, 5.0 mg polyphenols/g milk chocolate and 20 mg polyphenols/g cocoa powder given as gallic acid equivalents whereas Vinson, Proch and Zubik (1999) found higher values (36.5, 15 and 65 mg/g, respectively) using the same method but catechin as a standard and the results are referred to defatted dry weight of the samples. These values are high compared to the results obtained by Adamson, Lazarus, Mitchell, Prior, Cao, Jacobs et al. (1999) using a modi®ed normal phase high performance liquid chromatography (NP-HPLC) method for the quanti®cation of catechins and procyanidins, milk chocolate having 0.7 mg polyphenols/g and dark chocolate having 1.7 mg polyphenols/g. Arts, Hollman and Kromhout (1999) measured the content of catechins only by reversed phase HPLC (RP-HPLC) and found dark chocolate having 0.5 mg/g and milk chocolate having about 0.2 mg/g, respectively. Richelle, Tavazzi, Enslem and O€ord (1999) reported higher levels of only epicatechin in dark chocolate (2 mg/g) but the employed method had not been mentioned. Results from quantitative analysis of chocolates and cocoa powders are summarised in Table 1. Chocolate is not a standardized product, however, considering the great discrepancy, it is obvious, that the reported results depend very much on the employed method for quanti®cation and that a standardized procedure would be desirable. It is striking, that results obtained by the colourimetric method are generally much higher than results obtained by HPLC methods. Catechins from chocolate contributed 20% of the total catechin intake in a representative sample of the Dutch population (Arts et al., 1999). In 1994, the per head consumption of chocolate and chocolate confectionery in the European Union ranged from 1.3 kg/year in Portugal to 8.8 kg/year in Germany with people on the Northern countries in average consuming more than people in the South (Stutzer, 1998). Thus, chocolate can

Fig. 1. Structure of common polyphenols found in chocolate and other cocoa products.

Results are referred to defatted dry samples.

95% Aqueous methanol Methanolic HCl 70% Aqueous methanol 70% Aqueous methanol Methanolic HCl 75% Aqueous acetone 75% Aqueous acetone 95% Aqueous methanol Methanolic HCl 70% Aqueous acetone 70% Aqueous methanol Methanolic HCl 70% Aqueous acetone n.a. 95% Aqueous methanol Methanolic HCl 70% Aqueous acetone 70% Aqueous methanol Methanolic HCl 70% Aqueous acetone

Cocoa powder Cocoa powders Cocoa powder (non-alkalinised) Cocoa powders (instant) Cocoa powders Cocoa powder (non-alkalinised) Cocoa powder (instant) Dark chocolate Dark chocolates Dark chocolate Dark chocolate Dark chocolate Dark chocolate Dark chocolate Milk chocolate Milk chocolate Milk chocolate Milk chocolate Milk chocolate Milk chocolate

a

Extraction solvent

Source Folin±Ciocalteau (gallic acid standard) Folin±Ciocalteau (catechin standard) Folin±Ciocalteau (gallic acid standard) Folin±Ciocalteau (gallic acid standard) RP±HPLC (catechin standards) RP±HPLC (epicatechin standard) RP±HPLC (epicatechin standard) Folin±Ciocalteau (gallic acid standard) Folin±Ciocalteau (catechin standard) Modi®ed NP±HPLC (procyanidin standards) RP±HPLC (catechin standards) RP±HPLC (catechin standards) Modi®ed NP±HPLC (procyanidin standards) n.a. Folin±Ciocalteau (gallic acid standard) Folin±Ciocalteau (catechin standard) Modi®ed NP±HPLC (procyanidin standards) RP±HPLC (catechin standards) RP±HPLC (catechin standards) Modi®ed NP±HPLC (procyanidin standards)

Analysis method 20 mg/g Total polyphenols 6519 mg/g Total polyphenolsa Ca 58 mg/g Total polyphenols 6.462.44 mg/g Total polyphenols 2.96±3.27 mg/g Catechin and epicatechin Ca 3 mg/g Epicatechin 0.260.05 mg/g Epicatechin 8.4 mg/g Total polyphenols 36.55 mg/g Total polyphenolsa 1.70.08 mg/g total procyanidins 0.5 mg/g Catechin and epicatechin 0.48±1.37 mg/g Catechin and epicatechin 0.80.08 mg/g Catechin and epicatechin 2 mg/g Epicatechin 5 mg/g Total polyphenols 155.8 mg/g Total polyphenolsa 0.70.17 mg/g Total procyanidins 0.16 mg/g Catechin and epicatechin 0.15±0.16 mg/g Catechin and epicatechin 0.20.05 mg/g Catechin and epicatechin

Quantity

Table 1 Polyphenol content (mg/g) in coca powder, dark chocolate, and milk chocolate according to the applied analytical procedure

Waterhouse et al. (1996) Vinson et al. (1999) Serra Bonvehi and Ventura Coll (1997) Serra Bonvehi and Ventura Coll (1997) Vinson et al. (1999) Serra Bonvehi and Ventra Coll (1997) Serra Bonvehi and Ventura Coll (1997) Waterhouse et al. (1996) Vinson et al. (1999) Adamson et al. (1999) Arts et al. (1999) Vinson et al. (1999) Adamson et al. (1999) Richelle et al. (1999) Waterhouse et al. (1996) Vinson et al. (1999) Adamson et al. (1999) Arts et al. (1999) Vinson et al. (1999) Adamson et al. (1999)

Reference

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be seen as a relevant source for phenolic antioxidants for the European population. If a food or dietary component can be proven to confer bene®cial health e€ects to the consumer it becomes functional in its nature (ZumbeÂ, 1998). However, no universally accepted de®nition for functional foods exists. The ADA de®ned functional foods ``as any modi®ed food or food ingredient that may provide health bene®t beyond the traditional nutrients it contains'' (Bloch & Thomson, 1995). In a European consensus document, Bellisle et al. (1998) used the following working de®nition for functional foods: ``A food can be regarded as `functional' if it is satisfactorily demonstrated to a€ect bene®cially one or more target functions in the body, beyond adequate nutritional e€ects in a way that is relevant to either an improved state of health and well-being and/or reduction of risk of disease. Functional foods must remain foods and they must demonstrate their e€ects in amounts that can normally be expected to be consumed in the diet: they are not pills or capsules, but part of a normal food pattern''. The term ``satisfactorily demonstrated'' leaves open space for discussion. In any case, to evaluate the bene®cial health e€ects of polyphenols from cocoa or from chocolate and other cocoa products an integrated approach is required. The tasks considered in this paper are (i) the epidemiological evidence for health bene®ts of polyphenols and polyphenol-rich food, (ii) the data on polyphenol bioavailability, and (iii) proposed e€ects of polyphenols in disease prevention. Due to the abundant scienti®c literature on polyphenolic compounds, as far as distinguishable the focus in this paper is on chocolate and cocoa as well as catechins and procyanidins as the predominant cocoa polyphenols. Since chocolate and cocoa has not been objects much of research in this context until few years ago, data on other sources rich in catechins and procyanidins, such as green and black tea, red wine, and a procyanidin-rich extract from the bark of Pinus maritima, known as the nutritional supplement Pycnogenol1, are included as well. 2. Epidemiological evidence for health bene®cial e€ects of polyphenols Results from epidemiological studies on ¯avonoids and the risk of the development of cancer remain inconclusive. Knekt, Jaervinen, Seppaenen, Helioevaara, Teppo, Pukkala et al. (1997) observed an inverse association among approximately 10,000 Finish men and women between the intake of ¯avonoids and incidence of all sites of cancers combined. The sex- and ageadjusted relative risk of all sites of cancers combined

between the highest and lowest quartiles of ¯avonoid intake was 0.80, mainly due to the low relative risk for lung cancer (0.54). However, Hertog (1996) pointed out in a recent review of epidemiology of ¯avonoids that ¯avonoid intake was not related to the incidence of cancer in the two prospective studies conducted by them. It has to be mentioned that in all studies ¯avonoid intake was calculated from the determination of the content of only ®ve ¯avonols and ¯avones in foods. The average intake in the Netherlands was calculated to be approximately 23 mg/day with quercetin being the most predominant at 16 mg/day. The estimated ¯avonoid intake was highest in Japan at 64 mg/day and lowest in Finland at 6 mg/day. However, tea and wine (as well as cocoa) are rich sources of ¯avanols, proanthocyanidins and anthocyanins, all being strong antioxidants in vitro, rather than of ¯avonols and ¯avones. Results of epidemiological studies for single ¯avonoidrich beverages such as green and black tea, and wine could not con®rm consistent cancer-protective e€ects. Hertog (1996) concluded that the foods often associated with low cancer rates in epidemiological studies, such as green-yellow vegetables and cruciferous vegetables, are not important sources of ¯avonols and ¯avones and that those ¯avonoids only play a minor role in the explanation of the cancer protective e€ect of vegetables and fruits. Moreover, the more consistently reported inverse relationship between consumption of onions as particular rich in quercetin and the risk of cancerdevelopment could also be explained by other potential anti-carcinogens present in onions, for example diallysulphides (Hertog, 1996). As with cancers, an increasing body of epidemiological evidence links high intakes of antioxidants with reduced risk of cardiovascular disease (CVD). The evidence is strongest for vitamin E, limited but promising for bcarotene, and inconsistent for vitamin C. However, protective e€ects of vitamin E may be evident only at high doses±much more than can be obtained from a normal diet (Langseth, 1995). So far, the association between the intake of ¯avonoids and the risk of CVD, namely coronary heart disease (CHD) and stroke, has been investigated only in one prospective cohort study, the Zupthen Elderly Study (Hertog, Feskens, Hollman, Katan & Kromhout, 1993; Keli, Hertog, Feskens & Kromhout, 1996). The CHD mortality was approximately 65% lower in the highest tertile of ¯avonoid intake compared with the lowest tertile of ¯avonoid intake. The inverse relationship between ¯avonoid intake and ®rst myocardial infarction was less pronounced as well as the intakes of tea, onions, and apples in relation to CHD mortality (Hertog et al., 1993). Dietary ¯avonoids were inversely associated with stroke incidence after adjustment for potential confounders, including antioxidant vitamins (Keli et al., 1996). In other studies, tea consumption

J. Wollgast, E. Anklam / Food Research International 33 (2000) 449±459

showed no relation to the prevalence of CHD. However, the mean tea consumption was very low in those studies. In contrast, wine consumption has been found more consistently to be related to a lower risk of CHD (Hertog, 1996). In another study (Lee & Pa€enberger Jr., 1998), consumption of sugar candy and chocolate was associated with greater longevity of approximately 1 year. It could not be di€erentiated between consumption of sugar candy and chocolate, but the authors suggested the presence of phenolic antioxidants in chocolate as a plausible explanation for the observed e€ect. However, the correlation is not very strong and greater consumption of candy and chocolate was not associated with progressively lower mortality. It is evident that the science of epidemiology has inherent limits. Although epidemiology is very e€ective in identifying strong links between an environmental factor and disease (e.g. the link between smoking and lung cancer), it is less e€ective in discerning weaker associations (Taubes, 1995; Langseth, 1996). The link between fruit and vegetable intake and several cancers can be considered to be a strong one in magnitudes like smoking and lung cancer or alcohol and oral/oesophageal cancers (Block et al., 1992). However, many associations between diet and disease are relatively subtle. It may be impossible to determine from epidemiology alone, whether such relatively weak associations are real or whether they re¯ect some type of subtle bias or measurement error (Langseth, 1996). This might be the case for associations between intake of a single nutrient or nonnutrient and disease as well as consumption of a single food commodity and disease. Bellisle et al. (1998) suggest that human intervention studies in particular bioavailability studies and dose-response studies, in combination with the development and application of biomarkers, might be a more successful research strategy. 3. Bioavailability of polyphenols Reliable data on the contents of polyphenols in food is still scarce. In the 1970s the average daily intake in the United States has been estimated to be between 1 and 1.1 g/day (expressed as glycosides), depending on the season (KuÈhnau, 1976). However, these data are believed to be too high as this estimate su€ers from unreliable analytical data and a poor determination of food intake (Hollman, 1997). Recently, Hertog et al. (1993) calculated the ¯avonoid intake in the Dutch diet, based on only ®ve ¯avonols and ¯avonons, and found it to be 23 mg/day. In Denmark, ¯avonoid intake has been estimated to be 28 mg/day, in this case based on ¯avonols, ¯avonons, and ¯avanons (Bravo, 1998). It is striking that these estimations do not include catechins (¯avanols) and/or

453

proanthocyanidins. Intake of these compounds may be of great quantitative relevance, since their presence is predominant in tea, wine and cocoa products and these items are frequently consumed in Western countries. It is important to know not only a person's daily intake or intake with a particular food item of dietary polyphenols but also the bioavailability of those ingested polyphenols, since their nutritional signi®cance and potential systemic e€ects will greatly depend on their behaviour in the digestive tract (Bravo, 1998). However, only very little information is available on the absorption and subsequent distribution, metabolism and excretion of polyphenols in humans. The absorption and metabolism of food phenolics are determined primarily by their chemical structure, which depends on factors such as the degree of glycosylation/acylation, their basic structure (i.e., benzene or ¯avone derivatives), conjugation with other phenolics, molecular size, degree of polymerisation, and solubility (Bravo, 1998). Catechins and procyanidins can only be found as aglykones in plants and plant-derived food products and thus, molecular size and solubility might be the determining properties for absorption. After absorption of ¯avonoids, the subsequent metabolism and excretion of ¯avonoids is rather well known from animal studies, but only few data in humans are available. Catechin, however, has been the object of many studies in di€erent mammalian species (Heilmam & Merfort, 1998). Hydroxyl groups of the intact molecule are conjugated with glucuronic acid or sulphate in the liver. In addition, methylation may occur. The conjugates have been found in urine but excretion in bile of glucuronides and sulphates seems to be important as well. Micro-organisms in the colon hydrolyse conjugates which is supposed to enable absorption of the liberated aglykones. Thus, conjugates can be reabsorbed and enter an enterohepatic cycle. However, these microorganisms also substantially degrade the ¯avonoid moiety by cleavage of the heterocyclic ring, leading to di€erent phenolic acids. In the case of catechin, di€erent d-phenylvalerolactones have been found, a class of intermediate metabolites that could not have been detected as metabolites of other ¯avonoids (Heilmam & Merfort, 1998). The phenolic acids are absorbed and excreted with urine. A wide range of mammalian species has shown considerable species variation in this secondary metabolism (Griths, 1982; Heilmam & Merfort, 1998; Hollman, 1997). Although evidence of absorption and metabolism of polyphenols in the gut exists, less is known about the eciency of such uptake and the permanence of phenolic compounds or their conjugates and derivatives in the body. Blood concentrations of total catechins of 0.17 mmol/l after ingestion of black tea and up to 0.55 mmol/l after green tea were reported recently (Van het Hof, Kivitis, Weststrate & Tijburg, 1998). These data agree with another estimation that the absorption of tea catechins correspond to about 0.2±0.9% of the ingested

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dose (Lee, Wang, Li, Chen, Sun, Gobbo et al., 1995). Mainly conjugates of the administered catechins were present in plasma. After a single oral application of 0.5, 1 and 2 g catechin in human volunteers, the peak plasma concentration has been reached after 1±2 h (Balant, Burki, Wermeille & Golden, 1979). Independently of the doses, 0.5% of the catechin has been found in the free unbound form in plasma and the same amount of free catechin was excreted in the urine. However, 25% of the administered doses have been found as di€erent metabolites, indicated by an intact A ring. The elimination half-life was estimated to be 1±1.5 h (Balant et al., 1979). This is in agreement with the observation in rats after being fed 10 mg of 14C labelled catechin of which 34% have been excreted with bile and 20% with urine (Shaw & Griths, 1980). Very recently, Richelle et al. (1999) studied plasma kinetics of epicatechin in man after consumption of 40 and 80 g of black chocolate. Epicatechin increased markedly after chocolate consumption, reaching a maximum between 2 and 3 h. The maximal concentration and area under the curve of plasma kinetics correlate very well with the dose of chocolate. It has been concluded that epicatechin is absorbed from chocolate and is rapidly eliminated from plasma. Attainable plasma levels found were 0.7 mmol/l (free epicatechin and epicatechin conjugates) from 80 g of black chocolate containing 164 mg of epicatechin. Data on the permanence of polyphenols in the body are of great importance, because some of the physiologic e€ects of food polyphenols depend on their circulating levels (e.g. their antioxidant capacity). Van het Hof et al. (1998) reported that maximum blood levels of tea catechins occurred 2 h after tea ingestion and elimination half-life varied between 4.8 and 6.9 h for green and black tea catechins. In contrast, quercetin concentrations after ingestion of onions reached maximum plasma levels after 3.3 hours and the elimination half-life was 16.8 h (Hollman, 1997). Attention should be given to the fact that in most cases, and mainly because of the diculty in their analysis and characterisation, the study of digestive fate and physiologic e€ects of insoluble polyphenols Ð highly polymerised or bound tannins Ð is usually neglected (Bravo, 1998; Heilmam & Merfort, 1998). Relaying on the few data mostly obtained from in vitro studies, Heilmann and Merfort (1998) suggest the microbial depolymerisation of procyanidins to give the catechin subunits as the ®rst step in the colon. Subsequently, catechins are either absorbed or further metabolised by the colonic micro¯ora as desribed above. However, the metabolic fate in vivo of procyanidins and whether procyanidins can be absorbed as intact molecules has yet to be determined (Heilmam & Merfort, 1998). Facino, Carini, Aldini, Berti, Rossoni, Bombardelli et al. (1998) demonstrated an increased antioxidant activity

of plasma and decreased ischemia/reperfusion damage in rats after being fed a procyanidin-enriched diet for 3 weeks. This indicates that procyanidins are absorbed from the diet, at least in experimental rats. In a relatively new approach, studies using 14C-labelled oligomers and cultured human colon cells (Caco-2) indicate that the monomer catechin is taken up passively through tight junctions. There is more limited uptake of dimers and trimers by the same route, but the higher oligomers and polymers appear to enter by transcytosis as it is known to happen for carageens, b-lactoglobulin and -lactalbumin (Cli€ord, 1999). Some authors (Bravo, 1998; Bravo, Abia, Eastwood & Saura-Calixto, 1994) have suggested a classi®cation of polyphenols for nutritional purposes in `extractable' polyphenols (EPP) and `non-extractable' polyphenols (NEPP). EPP are low- and intermediate-molecular-mass phenolics including some hydrolysable tannins and proanthocyanidins that can be extracted using di€erent solvents, such as water, ethanol, methanol and in some cases aqueous acetone. NEPP are high-molecular-weight compounds or phenols bound to dietary ®bre or protein that remain insoluble in the usual solvents. Results from in vitro tests with digestive enzymes and from animal studies suggest the non-availability of some polyphenolic compounds, mainly NEPP. NEPP from carob pod concentrate, which is rich in highly polymerised condensed tannins, have been extensively (98%) recovered in the faeces of rats, con®rming the resistance of these compounds to intestinal digestion and/or absorption as well as degradation by colonic micro¯ora. Conversely, EPP were excreted only in minor amounts, suggesting that extensive digestion and/or absorption as well as microbial degradation of these phenolic compounds occur (Bravo, 1998). A similar observation has also been reported by Cli€ord (1999), namely that anaerobic gut ¯ora micro-organisms extensively degraded monomer, dimer and some higher oligomers. Due to the fact, that some polyphenols form complexes with proteins it has been suggested that addition of milk to black tea reduces the bioavailability of tea polyphenols. This was corroborated by the observation that ingestion of green or black tea signi®cantly increased the total plasma antioxidant capacity, but addition of milk to tea abolished the e€ect (Sera®ni, Ghiselli & Ferro Luzzi, 1996). However, in this study the concentration of polyphenols in plasma was not determined. More recently, it was found that addition of milk to green or black tea had no e€ect on the concentrations of catechins or quercetin in blood when compared with plain tea (Hollman, Tijburg & Yang, 1997; Van het Hof et al., 1998). This di€erence in the observations has been attributed to the fact, that in the study of Sera®ni et al. (1996) much higher amounts of added milk had been used (Van het Hof et al., 1998). However, the inhibitory e€ect of milk on plasma anti-

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oxidant activity could be also due to a reduction of antioxidants other than catechins or quercetin, such as the condensed tannins (thea¯avins and thearubigens) (Hollman et al., 1997). Probably even more important to the bioavailability of polyphenols, are some protein fractions in the saliva, in particular to complex phenolics and tannins (CPT). These include the so-called salivary proline-rich proteins (RPRs) and salivary histatins. Both fractions have a much higher anity to CPT than for example enzymes or bovine serum albumin and gelatine (Bacon & Rhodes, 1998). In model solutions, it has been shown that complexes of CPT and salivary RPRs and histatins, respectively, remain stabile in conditions similar to those in the stomach. The complexes of CPT and salivary histatins remained stable also under model conditions similar to that in the small intestine (Naurato, Wong, Lu, Wroblewski & Bennick, 1999). In contrast, the complex of CPT and salivary RPRs showed decreased stability under the same conditions, in particular in the presence of bile salts (Lu & Bennick, 1998). Interestingly, in some mammals (e.g. in rats) it has been demonstrated that a diet rich in CPT caused an increased production of salivary RPRs by the parotid glands, whereas the production of histatins seem to be independent of CPT consumption (Jansman, Frohlich, & Marquardt, 1994; Lu & Bennick, 1998). Whether this is valid also in humans has to be established. In conclusion, there are still very few data of the fate of ¯avonoids in humans available. The signi®cance of results obtained in laboratory animals for humans is mostly unclear, because animal studies have shown considerable species variation in metabolism of ¯avonoids. High plasma levels of ¯avonoids are not found. The data available do not indicate whether this is attributable to ecient metabolism or uptake by other tissues. Only the liver has been investigated as a metabolic organ. Other tissues such as the intestine wall and kidneys may play a role. In any case, ¯avonoids are found in plasma. According to Hollman (1997) the limited data available indicate that concentrations are potentially high enough to give biological e€ects, at least for monomeric ¯avonoids. The question of absorption of proanthocyanidins and the e€ects on bioavailability that could have proteins in foods (e.g. milk-proteins in milk chocolate) and of the saliva, respectively, must be addressed in the future. 4. Putative health e€ects of polyphenols from cocoa and sources with similar polyphenol composition Studies on health e€ects of polyphenols from cocoa or of chocolate and/or other cocoa products are still scarce. Kondo, Hirano, Matsumoto, Igarashi and Itakura (1996) have reported an increased antioxidant

455

capacity of blood plasma after consumption of black chocolate. Since reactive oxygen species (ROS) are known to be involved in immune responses, e.g. by activation of transcription factors NF-kB and AP-1, Sanbongi, Suzuki and Sakane (1997) studied the e€ects of antioxidants from chocolate, cacao liquor polyphenol (CLP), on human immune functions in vivo. CLP inhibited both hydrogen peroxide and superoxide anion production in activated granulocytes as well as in normal human peripheral blood lymphocytes and macrophages. In addition, CLP inhibited mitogen-induced lymphocyte proliferation and polyclonal immunoglobuline (Ig) production by B cells in a dose-dependent manner. These e€ects are believed to be at least partly due to reduced production of interleukin-2 (IL-2) by T lymphocytes and Sanbongi et al. (1997) could show that CLP inhibited both IL-2 mRNA expression of and IL-2 secretion by T cells. In addition to these two publications, extensive studies on cocoa extracts, consisting of monomeric catechins and oligomeric procyanidins with 2±18 monomeric units have been conducted. However, so far their results have been only published in a US patent application (Romanczyk, Hammerstone, Buck, Post, Cipolla, Micceland et al. (1997). Therein, they suggest anti-atherogenic, anti-carcinogenic, anti-in¯ammatory, immunemodulating, and anti-microbial activity properties of cocoa extracts. In vitro cocoa procyanidins have been shown to be antioxidative as well as chelators of copper and iron and thereby preventing LDL from oxidation. In addition procyanidins inhibited cyclo-oxygenase 1 and 2 (COX-1 and COX-2), and lipoxygenase. By enhancing levels of nitric oxide (NO), having been identi®ed as the endothelial-derived relaxing factor (EDRF), derived from constitutive endothelial nitric oxide synthase (eNOS), procyanidins could cause vasodilatation. Indeed, Romanczyk et al. (1997) suggest, that although the polyphenolic compounds inhibit the oxidation of LDL, the more comprehensive e€ect is their multidimensional e€ects on atherosclerosis via NO. Bene®cial e€ects of NO modulation include regulation of blood pressure, lowering NO a€ected hypercholesterolemia, inhibition of platelet aggregation and monocyte adhesion, all of which are involved in the progression of atherosclerosis. Interestingly, the e€ects were seen only with oligomeric procyanidins and of these in particular oligomers of 2±10 sub-units with the most e€ective being the dimer, trimer, tetramer, and pentamer (Romanczyk et al., 1997). With respect to anti-in¯ammatory and immune-modulating activity the discussed mechanisms are inhibition of phospholipase A2, cyclo-oxygenase and lipoxygenase thereby decreasing levels of the in¯ammatory prostaglandin PGE2 (Romanczyk et al., 1997). Romanczyk et al. (1997) examined anti-carcinogenic properties of cocoa extracts, using several human cancer

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cell lines. Interestingly, the e€ects were seen only with oligomeric procyanidins and of these in particular oligomers of 5±12 sub-units with the most e€ective being the pentamer. It is suggested that the mechanisms by which procyanidins exert anti-carcinogenic activity include inhibition of DNA strand breaks, DNA-protein crosslinks and free radical oxidation of nucleotides due to their antioxidative activity as well as inhibition of enzyme activities of cyclo-oxygenase 2 (COX-2) and DNAtopoisomerase II. Moreover, procyanidins modulate nitric oxide production by macrophages, possessing an inducible nitric oxide synthase (iNOS), and thereby a€ecting ribonuclease reductase, the enzyme that converts ribonucleotides to deoxyribonucleotides necessary for DNA synthesis. Inhibition of DNA synthesis may be an important way in which macrophages and other tissues possessing iNOS can inhibit the growth of rapidly dividing tumour cells or infectious bacteria. Tea catechins have been demonstrated to inhibit nitrosation, modulate carcinogen-metabolising enzymes, contribute to trapping of ultimate carcinogens, and inhibit cell proliferation-related activities (Yang, Laihshun, Lee & Landau, 1996). In addition, they inhibit the formation of ROS and the promotion-related enzymes, such as epidermal ornithin decarboxylase, protein kinase C, lipoxygenase and cyclo-oxygenase (Huang & Ferraro, 1992; Yang et al., 1996). Moreover, Naasani, Seimiya and Tsuruo (1998) demonstrated that epigallocatechin gallate (EGCG), a major tea catechin, strongly and directly inhibits telomerase, both in a cell free system and in living cells. In addition, the continued growth of two representative human cancer cell lines showed life span limitations accompanied with telomere shortening, chromosomal abnormalities and expression of the senescence-associated b-galactosidase in the presence of non-toxic concentrations of EGCG (Naasani et al., 1998). As for chocolate (Kondo et al., 1996), an increase of antioxidant capacity of plasma has been reported as well after consumption of wine (Leake, 1998) and tea (Miura, Watanabe, Tomita, Sano & Tomita, 1994). In addition, it has been reported that polyphenols from wine, liquorice and other plant sources are incorporated into LDL particles and thereby decreasing their ability for being oxidised when isolated from plasma (Aviram & Fuhrman, 1998). In one study (Fuhrman, Lavy & Aviram, 1995), isolated LDL from human plasma showed a decrease in oxidisability after 2 weeks of consumption of daily 400 ml red wine. However, in contrast to these studies, De Rijke, Demacker, Assen, Sloot, Katan and Stalenhoef (1996) and Sharpe, McGrath, McClean, Young and Archbold (1995) observed no e€ect on oxidisability of LDL ex vivo of consumption of 550 ml daily for 4 weeks and 200 ml for 10 days, respectively. In addition, ¯avonoids have been shown to restore a-tocopherol in LDL particles, possibly by

scavenging free radicals and thereby protecting a-tocopherol from being consumed by these free radicals, or they may convert a-tocopheroxyl radicals back into atocopherol, like it is suggested for ascorbic acid (Leake, 1998). Fitzpatrick, Bing and Rohdewald (1998) showed that procyanidins form the bark of Pinus maritima (Pycnogenol1) with a procyanidin composition similar to that of chocolate and wine, increased NO levels in vascular cell walls. From the observation that superoxide dismutase (SOD) did not signi®cantly alter Pycnogenol-induced relaxations, they concluded that endothelium derived relaxation is not a result of superperoxide scavenging but of increased synthesis of NO. In addition to its endothelium-dependent vascular e€ects, Pycnogenol1 also showed a weak inhibitory e€ect on the angiotensinconverting enzyme (ACE) resulting in a mild hypotensive e€ect (Fitzpatrick et al., 1998). In contrast to the observation of iNOS inducing activities of cocoa polyphenols, Pycnogenol1 showed a signi®cantly decreased NO generation on activated macrophages (Virgili, Kobuchi & Packer, 1998a). It was concluded that this e€ect was due to the combination of several di€erent biological activities of Pycnogenol1, i.e. its ROS and NO scavenging activity, inhibition of iNOS activity, and inhibition of iNOS-mRNA expression. However, the same researchers reported elsewhere (Virgili, Kobuchi & Packer, 1998b) a biphasic e€ect of Pycnogenol1 on NO production by macrophages. 10 mg/ml slightly but signi®cantly stimulated the formation of oxides of nitrogen, whereas higher concentrations up to 100 mg/ml signi®cantly inhibit the generation of NO. This result was con®rmed by the observation on the enzymatic activity of iNOS, being slightly potentiated by the low level and inhibited by higher levels. They hypothesised that in vivo this could result in a long-term generation of a ¯ow of NO from macrophages at levels not compromising the viability of the cell itself (Virgili et al., 1998b). Hoer, Rimpler and Heinrich (1994) demonstrated an anti-secretory activity of procyanidins, in particular with a degree of polymerisation higher than 8, from Guazuma ulmifolia on cholera toxin-induced chloride secretion in rabbit distal colon mounted in an Ussing chamber. Since the extract did not inhibit PGE2induced chloride secretion, an indirect anti-secretory mechanism has been proposed, probably due to a protective layer of procyanidins along the intestinal lumen. 5. Conclusion Can the conclusion be drawn from the above summarized data that chocolate and/or other sources rich in catechins and procyanidins are bene®cial to human health, e.g. preventing cardiovascular disease and cer-

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tain cancers as leading causes of death in the industrialized world? Some epidemiological evidence suggests a bene®cial e€ect to human health by following a polyphenol-rich diet, namely rich in fruits and vegetables and to a less obvious extent tea and wine. The antioxidative and radical scavenging activities as well as metal-chelating properties and the interaction with proteins of polyphenols have been suggested to be the driving criteria to explain the putative disease preventive character of polyphenols (Haslam, 1996). In many experiments, mostly in vitro models, cellular targets have been identi®ed and molecular mechanisms of disease prevention proposed, in particular for the prevention of cancer and cardiovascular diseases as well as for alleviating the response to in¯ammation reactions. However, it has to be shown, whether polyphenols exert these e€ects in vivo. One prerequisite is that the polyphenols are absorbed from the diet. For monomeric ¯avonoids such as the catechins, there is increasing evidence for their absorption and milk proteins do not seem to impair absorption (Hollman et al., 1997; Van het Hof et al., 1998). For complex phenols and tannins (procyanidins), present in relatively signi®cant amounts in red wine, black tea and cocoa (chocolate), these questions have to be addressed to the future. Some indication that absorption of procyanidins takes place derive from studies with the human colon cancer cell line Caco-2, believed to be a valuable model for passive intestinal absorption as proposed for polyphenols (Cli€ord, 1999). Presumed that polyphenols reach their target destination in the body to exert their bene®cial health e€ects described from in vitro experiments on human cells and isolated enzymes, it has to be clari®ed which concentration is e€ective and what concentrations can be expected from food intake. For example, Romanczyk et al. (1997) have shown that the e€ective dose of oligomeric cocoa procyanidins would be above 10 mM sometimes up to 100 mM in the case of human cancer cell proliferation. Monomeric catechins have reached plasma concentrations of 1 mM or less after intake of remarkable amounts (80 g) of dark chocolate (Richelle et al., 1999) or tea, respectively. For the oligomeric procyanidins absorption has not been investigated to date. However, the above mentioned e€ective doses could very well be reached in the gastro-intestinal tract and thus, protect against ulcers and gastric or colon carcinomas. Another open question is related to polyphenol metabolism. For example, much e€ort has been invested to show antioxidative e€ects of free unbound polyphenols, especially of catechins and the ¯avonol quercetin. However, only a very small part can be found in plasma in the free form. Mostly, glucuronidation and sulphatisation and to a lesser extent O-methylation occur in the liver and possibly in the gut and kidneys. Colonic microorganisms degrade ¯avonoids to yield phenolic acids

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and other ring scission products. Thus, for example, it could be that the conjugates have a lower antioxidative activity than the free ¯avonoids as it has been reported for glycosides and methoxylates (Cook & Samman, 1996). However, studies on the bioactivity of conjugates and metabolites have not been published to date. Considering the research on cocoa and chocolate, respectively, milk chocolate compared to dark (black) chocolate has been less of an object of investigation. Amounts of polyphenols in milk chocolate are smaller than in dark chocolate due to the lower amount of cocoa liquor used in milk chocolate (ca. 10±15%) compared to black chocolate (ca. 30±50%). In addition, milk proteins, especially caseins being relatively proline-rich, may impair absorption of procyanidins due to complexation. Thus, dark chocolate seems a priori to have a higher potential in being bene®cial to human health. However, the role of salivary proline-rich proteins being even stronger complexing agents to procyanidins (Haslam, 1996) has to be taken into consideration as well. It is yet to early to give an answer to the question, whether chocolate and/or other sources rich in catechins and procyanidins are bene®cial to human health and thereby becoming functional in their nature. Even though some data are promising and justify further research in the ®eld, it has to be shown in future, whether the intake of these functional compounds and/or their sources is related to measurable e€ects on human health and/or the development of diseases. In any case, the relationship between nutrition and health gains public acceptance and thus, the market for functional foods grows (Bellisle et al., 1998). However, there is a risk of premature or even aggressively drawn conclusions regarding phytochemicals and functional foods and their relation to human health due to commercial interest. Obviously, the ®rst priority must be to conduct research to develop mechanisms of action that directly relate to healthy physiologic processes, disease development, or both (Waterhouse, German, Walzem, Hansen & Kasim-Karakas, 1999). A very promising research strategy to ®nally evaluate the health bene®t of a food item or an isolated compound lies in the development and application of validated and accepted biomarkers, which are clearly related to disease (Bellisle et al., 1998; Halliwell, 1999). Biomarkers of oxidative DNA damage and lipid peroxidation can be used to establish the role of antioxidants in this protection and the optimal intake of these antioxidants. This concept is based on the well accepted presumptions that oxidative DNA damage is a signi®cant contributor to age-related development of some cancers and that lipid peroxidation plays a key role in the development of cardiovascular disease (Halliwell, 1999). Probably the most promising biomarkers for oxidative damage are various families of isoprostanes and of multiple DNA base oxidation products, in

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particular of 8-hydroxydeoxyguanosine (8-OHdG) in human ¯uids, such as urine and plasma (Diplock, Charleux, Crozier-Willi, Kok, Rice-Evans, Roberfroid et al., 1998; Halliwell, 1999). The biomarker approach could make the identi®cation of the diets/foods that decrease DNA and lipid damage in humans, and the subsequent isolation of the protective factors from them possible (Halliwell, 1999). Taking the summarised data in this paper into account, chocolate being a rich source of polyphenolic antioxidants, seems to be a worthy candidate for such biomarker studies.

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