Antioxidant Capacity and Epicatechin Bioavailability of Polyphenolic

Chapter 12

Antioxidant Capacity and Epicatechin Bioavailability of Polyphenolic-Rich Beverages (Cocoa and Teas)

Downloaded by UNIV OF LIVERPOOL on November 24, 2016 | http://pubs.acs.org Publication Date: January 15, 2000 | doi: 10.1021/bk-2000-0754.ch012

M . Richelle, T . Huynh-Ba, I. Tavazzi, V. Mooser, M. Enslen, and E . A . Offord Nestlé Research Center, P.O. Box 44,1000 Lausanne 26, Switzerland

The antioxidant capacity of cocoa and tea was investigated by measuring the inhibition of copper- or AAPH-mediated human low-density lipoprotein ( L D L ) oxidation in vitro. The extent of oxidation was determined spectrophotometrically by measuring absorbance (at 234 nm) of conjugated dienes. Both cocoa and teas produced dose-dependent inhibition of LDL oxidation which was similar with both pro-oxidant agents. Cocoa polyphenols extracted with 20% aqueous ethanol showed a stronger antioxidant capacity than those extracted with water. When prepared as ready-to-drink, the polyphenol-rich beverages such as cocoa, green and black teas exhibited a similar antioxidant capacity. In a second stage, the plasma bioavailabilty o f epicatechin from cocoa extract and chocolate was evaluated in man after a single dose administration. Plasma concentration of epicatechin increased markedly after cocoa consumption and the area under the curve of plasma kinetics correlated very well with the dose of epicatechin present in the cocoa product. The epicatechin kinetics were similar with either 20% aqueous ethanol cocoa extract or plain chocolate indicating that the food matrix did not affect the epicatechin bioavailability. Attainable epicatechin plasma values were 0.7 μΜ from 80 g of plain chocolate representing about 2% of the intake. Similar results have been described for tea. These results suggest that beverages of tea and cocoa make a significant contribution to the daily intake of polyphenolic antioxidants.

The oxidative modification of low density lipoprotein ( L D L ) is currently viewed as a pivotal step in the pathogenesis of atherosclerosis (1 - 3). Indeed, the uptake of intact L D L by the B , E receptors induces a control of intracellular content of 102

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Parliment et al.; Caffeinated Beverages ACS Symposium Series; American Chemical Society: Washington, DC, 2000.


103 cholesterol while the uptake o f oxidized L D L by scavenger receptor (4) is uncontrolled. Therefore, cholesterol accumulates into macrophages which are progressively transformed into foam cells (5). The detection of products of L D L oxidation in atherosclerotic lesions of rabbit and humans (6) also implicates oxidized L D L in the atherosclerotic process. It is important to find dietary compounds which can inhibit L D L oxidation. The method assessing the susceptibility of L D L towards in vitro oxidation is appropriate for determining the influence of dietary treatment with different fatty acid patterns or antioxidants but also for evaluating the antioxidant potential of dietary antioxidant matrices. Polyphenols constitute an important source of dietary antioxidants, being found widely in fruits, vegetables, cereals and beverages such as red wine, tea, coffee and cocoa. The antioxidant capacity of this diverse group of compounds depends on the individual structure and number of hydroxyl groups. Polyphenolic derivatives of catechins are found in significant amount in tea, from which they are readily extracted in hot water infusions. Six catechins occur in green tea leaves : catechin, gallocatechin, epicatechin, epigallocatechin, epicatechingallate and epigallocatechingallate. Overall, levels of catechins vary with leaf age, tending to be higher in young leaves. During the manufacture of black tea, enzyme-catalazed oxidation of the catechins leads to the formation of catechin quinones, which subsequently react to form the more complexly structured pigmented teaflavins and tearubinins. The approximate catechin amounts in fresh leaf, green tea and black tea are in the range of 30-35%, 10-25% and 1-9% of dry matter, respectively (7). Unfermented cocoa beans are rich in polyphenols, which comprise 12-18% of the whole bean's dry weight. Two major classes of polyphenols are present : catechins (with approximately 35% polyphenols as epicatechin) and anthocyanins, which are responsible for the characteristic purple colour of unfermented cocoa beans (8,9). During fermentation, the colour of the bean changes from purple to brown. The polyphenols themselves undergo a variety of reactions : epicatechin diffuses from its storage cells and undergoes oxidation and polymerisation to form complex tannins (10,11) leading to a reduction of epicatechin concentration to approximately 2-3 mg/g fermented cocoa beans (12). Previously, polyphenols were considered only for their colour and aroma but more recently they have been investigated for their biological activities relating to health. Cocoa polyphenols have been shown to exhibit a strong protective effect against L D L oxidation (13, 14), antimutagenic effects have been shown in the Ames test system; and studies in isolated human Τ and Β lymphocytes and granulocytes indicate that cocoa polyphenols have immunoregulatory properties (15). The objective of this study was to efficiently extract polyphenols from cocoa powder in order to evaluate their antioxidant capacity in vitro but also to compare them with food matrix such as cocoa drink, plain chocolate and tea. To produce a biological effect in vivo, it is essential that biologically relevant quantities of polyphenols are absorbed. Therefore, the plasma kinetics of epicatechin from cocoa products (20% aqueous ethanol extract and plain chocolate) were evaluated in healthy man and compared with published data for tea.


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EXPERIMENTAL

PROCEDURES

Antioxidant Capacity, in vitro LDL were isolated from blood of healthy volunteers and oxidized with either 1.7 mmol copper (II) sulfate or 1.25 mmol AAPH in the presence or absence of polyphenolic-rich beverages. After incubation, the extent of LDL oxidation was determined by recording spectrophotometrically the formation of conjugated dienes (16). Extraction of cocoa polyphenols was performed by refluxing during lh defatted cocoa powder containing 10 - 12% fat in different solvents i.e. 5% aqueous methanol; 20% aqueous acetone; 20% aqueous ethanol and water. The crude extracts were concentrated and thereafter lyophilized. Their polyphenol content was determined using Folin Ciocalteu method according to Anonymous (17). The results are expressed as gallic acid equivalents (GAE). Beverages were prepared at different concentrations in order to cover the different habits of consumers from various countries i.e. green and black teas were prepared as 1.5 g, 2 g or 3 g per 220 ml of hot water and infused for 5 min. Cocoa beverages were prepared by diluting cocoa powder (97%) in 220ml hot water at 1.5% up to 3.5%, in order to cover the habits of consumers from different countries. Bioavailability of Epicatechin in Plasma Eight normolipidemic healthy male volunteers were studied on three occasions with one-week interval. Volunteers were asked to refrain from foods rich in polyphenols from the day before the test until its completion (restriction concerned the intake of tea, coffee, wine,fruitjuice, cocoa products). They consumed chocolate with bread and water, blood samples were drawn every hour over 8h. Epicatechin glucuronides and sulphates were simultaneously hydrolyzed to aglycones and determined by HPLC (18).

RESULTS Antioxidant Capacity, in vitro The antioxidant capacity was evaluated by the resistance of LDL towards oxidation. The oxidation of LDL was initiated by copper or AAPH. Conjugated diene lipid hydroperoxides were formed from the oxidation of polyunsaturated fatty acids (PUFAs) present in the LDL particles. The kinetics of oxidation were characterized by three parameters : a) the lag time or the time during which antioxidants were consumed; b) the rate of oxidation (R max) and c) the maximum production of conjugated dienes. The higher the antioxidant potential, the longer the lag time. 2

In the presence of cocoa extracts, L D L oxidation was delayed as characterized by the increase of lag time whereas the rate of oxidation as well as the maximum production of conjugated dienes remained constant (Fig. 1). This antioxidant potential was dose dependent.



105 Polyphenols were previously described to have the capacity of chelating transition metal ions. Therefore, in this in vitro L D L oxidation model where cupric ions were used as pro-oxidant agent, the protection of L D L against oxidation by cocoa polyphenols may be an artefact due to the chelation of cupric ions rather than a scavenging of the radicals produced. To exclude this possibility, A A P H which generated free radicals at a constant rate over a few hours, was used as an alternative pro-oxidant initiator. The dose dependent ability of cocoa polyphenols to protect L D L against oxidation was similar with both pro-oxidant agents confirming the antioxidant capacity of cocoa polyphenols (Data not shown). In order to study the effect of the food matrix on the antioxidant capacity of cocoa polyphenols, they were extracted from defatted cocoa powder with various solvents. The extraction yield of cocoa polyphenols increased in the following order : 5% aqueous methanol; 20% aqueous acetone, 20% aqueous ethanol and water with 10, 13, 19 and 32 mg G A E / g cocoa, respectively. The antioxidant capacity of polyphenols present in the 20% aqueous ethanolic and water extracts were evaluated (Fig. 2) when similar amounts of polyphenols were incorporated in the L D L system. At any polyphenol concentration, the ethanolic cocoa extract exhibited stronger antioxidant potential than the water extract, indicating that the solvent used for the polyphenol extraction of cocoa affected not only the polyphenol content of the extract but also the composition of polyphenols, the ones present in the ethanolic extract being more active. Finally, defatted cocoa powder and plain chocolate exhibited similar antioxidant potential indicating that at this stage, the food matrix had no effect (Data not shown). The antioxidant capacity of polyphenolic-rich beverages such as cocoa and teas were also compared. For this purpose, teas (green and black) and cocoa were prepared at different concentrations in order to cover habits of consumers of different countries. Both beverages protected L D L against in vitro oxidation in a dosedependent manner. The antioxidant potential of cocoa was similar to that of green and black teas (Fig. 3). Epicatechin Bioavailability The bioavailability of cocoa polyphenols from plain chocolate (18) was compared to a lyophilized powder of 20% aqueous ethanolic extract of defatted cocoa powder. The aim of using the extract from cocoa powder was to have a simple matrix of polyphenols which would be compared to a more complex food matrix (chocolate). Precise analysis of epicatechin showed 82 mg, 164 mg and 33 mg in 40g, 80 g plain chocolate and 11.45 g cocoa extract. Cocoa contains a wide variety of polyphenolic compounds. We determined the appearance of epicatechin in plasma as a marker of the cocoa polyphenol bioavailability. Before starting cocoa administration, plasma epicatechin at Oh (Fig. 4) was very low or most of the time undetectable indicating that volunteers did effectively refrain from a polyphenol rich diet. After cocoa intake, plasma epicatechin rose to 111 ng/ml (0.383 μΜ) with 40 g chocolate, to 203 ng (0.7 μΜ) with 80 g chocolate and to 41 ng/ml (0.141 μΜ) with cocoa extract. The clearance of epicatechin from plasma compartment was very fast (half time of 2h, 2.8h and 3h,



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Figure 1: L D L were oxidized with cupric ions (1.7 mM) at 37°C in the absence or the presence of increasing amount of defatted cocoa powder.

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Polyphenol (ng) •Water #20% aqueous EtOH Figure 2: The antioxidant capacity of wa{er and 20% ethanolic cocoa extract was compared at similar polyphenol content. The protection of L D L against oxidation is expressed by the increase of lag time (lag time of L D L with cocoa extract minus lag time of L D L control).

1

Figure 3: Cocoa and teas bevereages were prepared at different concentrations. The protection o f L D L against oxidation by cupric ions in the presence o f 1 μΐ of each beverage was evaluated. The results mean ± SD, n= 3.



- β - 4 0 g plain chocolate - & - 8 0 g plain chocolate - A — Cocoa extract

Time (h)

Figure 4: Plasma epicatechin concentration after ingestion of 40g, 80g plain chocolate or 11.45g 20% aqueous ethanolic cocoa extract. Results are expressed as mean ± S D .

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109 respectively). Interestingly, the Cmax and area under the curve of the plasma epicatechin kinetics were proportional to the dose of chocolate ingested and was not affected by the food matrix. The relative bioavailability of epicatechin in plasma was 1.45%, 2.12% and 2.05% for 40g, 80g chocolate and cocoa extract, respectively. Interestingly, the bioavailability of epicatechin from cocoa is quite similar to that of tea epicatechins which is 2% (19).


DISCUSSION This study demonstrates an antioxidant capacity of cocoa polyphenols towards L D L oxidation. In order to study the effect of the food matrix, cocoa polyphenols were extracted from defatted cocoa powder using different solvents. Water and 20% aqueous ethanol results in a good yield of polyphenols. Although water resulted in more abundant polyphenol content in the extract compared to 20% aqueous ethanol extract, the biological activity of polyphenols present in the water extract is weaker, indicating that the solvent used for the extraction affects not only the total concentration of polyphenols but also the type of polyphenols. The physical and chemical properties of individual phenolic molecules strongly affect their antioxidant potential (20-22). In addition, these molecules could have a synergistic or an antagonistic effect when present in complex mixtures. On the other hand, the antioxidant potential of plain chocolate or cocoa powder is similar indicating that the food matrix has no effect. Therefore, it is clear from the present data that polyphenols of cocoa and teas (green and black) have an in vitro antioxidative capacity. A better understanding of the protective role of dietary antioxidants in vivo requires quantitative data on their absorption (23). Indeed after consumption, polyphenols have to cross the intestinal wall but must also resist further catabolism. The metabolism of epicatechin involves two important organs : the liver, where biotransformation enzymes convert epicatechin or their metabolites into conjugated form such as glucuronides or sulphates (24), and, the colon, where microorganisms degrade unabsorbed epicatechins (25). Plasma kinetics of epicatechin from plain chocolate (18) and 20% aqueous ethanol extract were evaluated in man after a single oral administration. These plasma concentration curves represent the net result of two opposite processes ; absorption vs elimination i.e. involving catabolism, storage in tissues and excretion in urine). Epicatechin was rapidly absorbed leading to appreciable amount in plasma (up to 0.7 μΜ). The maximal plasma concentration as well as the area under the curve were strongly related to the amount of epicatechin present in plain chocolate or extract, indicating that no saturation of the absorption is reached and that the food matrix does not affect the epicatechin absorption. In addition, similar absorption of epicatechin from tea has been previously described by Lee et al (19). Interestingly, plasma concentration of 1 μΜ which is in the biologically accepted range to achieve a biological effect (26). However, further studies are necessary to elucidate whether polyphenol antioxidants from cocoa show antioxidant in vivo.


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LITERATURE 1. 2. 3. 4. 5. 6.


USA. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

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Antioxidant Capacity and Epicatechin Bioavailability of Polyphenolic

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