Chocolate: Modern Science Investigates an Ancient Medicine Procyanidin Content and Variation in Some Commonly Consumed Foods1 John F. Hammerstone, Sheryl A. Lazarus2 and Harold H. Schmitz Analytical and Applied Sciences Group, Mars, Incorporated, Hackettstown, NJ 07840 ABSTRACT Procyanidins are a subclass of flavonoids found in commonly consumed foods that have attracted increasing attention due to their potential health benefits. However, little is known regarding their dietary intake levels because detailed quantitative information on the procyanidin profiles present in many food products is lacking. Therefore, the procyanidin content of red wine, chocolate, cranberry juice and four varieties of apples has been determined. On average, chocolate and apples contained the largest procyanidin content per serving (164.7 and 147.1 mg, respectively) compared with red wine and cranberry juice (22.0 and 31.9 mg, respectively). However, the procyanidin content varied greatly between apple samples (12.3–252.4 mg/serving) with the highest amounts on average observed for the Red Delicious (207.7 mg/serving) and Granny Smith (183.3 mg/serving) varieties and the lowest amounts in the Golden Delicious (92.5 mg/serving) and McIntosh (105.0 mg/serving) varieties. The compositional data reported herein are important for the initial understanding of which foods contribute most to the dietary intake of procyanidins and may be used to compile a database necessary to infer epidemiological relationships to health and disease. J. Nutr. 130: 2086S—2092S, 2000. KEY WORDS:
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procyanidins
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quantification
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food composition
Simple phenols consist of one aromatic ring containing at least one hydroxyl group, whereas polyphenols consist of more than one aromatic ring with each containing at least one hydroxyl group. Flavonoids are a subclass of polyphenols that have a C6-C3-C6 backbone structure (Fig. 1). One group of flavonoids, the procyanidins, are composed of flavan-3-ol monomers and their respective oligomers, commonly bonded through a 436 or 438 linkage (Fig. 2). In addition to these simple forms, they have been reported as containing gallic acid esters or as doubly linked forms as shown in Figure 3 (Haslam 1998). Although it has been known for some time that polyphenols are ubiquitous in the plant kingdom, only recently have they begun to be studied in foods. Furthermore, with some exceptions, most of the polyphenol research in food composition has been limited to simple flavonoids and phenolic acids, rather than a comprehensive investigation of all classes of polyphenols present. In addition to incomplete compositional information, commercial standards are not available for most complex polyphenols. Both of these factors contribute to poor quantitative data, and consequently, there is large variability reported for dietary intake levels, which can confound the ability to infer epidemiological relationships with health and disease.
Oligomeric procyanidins represent one class of polyphenols in which qualitative and quantitative information with regard to food composition is especially lacking. This is due in large part to analytical constraints and lack of appropriate methodology suitable for reliable characterization and quantification of these compounds. However, recent advances in analytical methodology have allowed for the separation and quantification of the procyanidin oligomers in cocoa and chocolate (Adamson et al. 1999, Hammerstone et al. 1999), and this same method has been applied to the separation of proanthocyanidin oligomers in a variety of plant foods (Lazarus et al. 1999a). This was made possible by the use of normal phase HPLC instead of the more commonly used reversed phase methodology, which is not capable of resolving the higher oligomeric forms (Hammerstone and Lazarus 2000, Wilson 1981). Oligomeric procyanidins have attracted increasing attention in the fields of nutrition and medicine due to their potential health benefits observed in vitro and in vivo. Most prominently, procyanidin oligomers have been shown to have potent antioxidant activity and the ability to scavenge reactive oxygen and nitrogen species (Ariga and Hamano 1990, Arteel and Sies 1999, Hagerman et al. 1998). In addition, recent research suggests these phytochemicals may modulate immune function and platelet activation (Mao et al. 1999, Packer et al. 1999, Rein et al. 2000, Sanbongi et al. 1997). Thus, in the present study, we extended the work of Adamson et al. (1999), in which the procyanidins were quantified in cocoa and chocolate to include other commonly consumed foods, such as apples, wine and cranberry juice, which are
1 Presented at the symposium “Chocolate: Modern Science Investigates an Ancient Medicine,” held February 19, 2000, during the 2000 Annual Meeting and Science Innovation Exposition at the American Association for the Advancement of Science in Washington, D.C. Published as a supplement to The Journal of Nutrition. Guest editors for the supplement publication were John W. Erdman, Jr., University of Illinois at Urbana-Champaign; Jo Wills, Mars, United Kingdom and D’Ann Finley, University of California, Davis. 2 To whom reprint requests should be addressed.
0022-3166/00 $3.00 © 2000 American Society for Nutritional Sciences.
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FIGURE 1
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The C15 (C6-C3-C6) basic skeleton of a flavonoid.
believed to be significant contributors to the dietary intake of procyanidin oligomers. MATERIALS AND METHODS Chemicals and reagents. HPLC-grade methylene chloride, methanol, acetone and acetic acid were obtained from VWR Scientific (Bridgeport, NJ). HPLC-grade water was obtained from a Milli-Q (Millipore, Bedford, MA) water purification system. (⫺)-Epicatechin was purchased from Sigma Chemical Co. (St. Louis, MO). Procyanidin oligomers were purified from cocoa beans, and a composite standard was prepared as described previously by Adamson et al. (1999). Sample collection. Five samples each of dark chocolate (DOVE Chocolate, M&M/Mars, Hackettstown, NJ), Cabernet Sauvignon wine (Woodbridge; Robert Mondavi, Woodbridge, CA), cranberry juice (Ocean Spray, Lakeville-Middleboro, MA) and Golden Delicious, Red Delicious, Granny Smith and McIntosh apples were purchased from five grocery and wine stores in eastern Pennsylvania and western New Jersey. Only four of the five Red Delicious apples were used for analysis because the fifth apple was found to have a rotten core and was not suitable for consumption. Sample preparation. The chocolate was prepared for analysis as described by Adamson et al. (1999). In brief, the lipid was extracted from 15 g of sample through exhaustive extraction with hexane (4 ⫻ 45 ml). One gram of the dried, lipid-free solids was extracted with 5 ml of an extraction solvent composed of acetone, water and acetic acid in a ratio of 70:29.5:0.5 (v/v/v), respectively. The resulting slurry was pelleted at 1500 ⫻ g, and then the supernatant was filtered through a 0.45-m nylon filter. For the determination of percent fat composition, AOAC Official Method 920.177 was used (AOAC International 1996). Each of the five chocolate samples were extracted and analyzed in duplicate. Wine samples were prepared using two methods. In the first, 300 ml of wine was concentrated by rotary evaporation under partial pressure at 42°C to a volume of 75 ml before HPLC analysis without further manipulation. The second method involved the use of a 20-ml solid phase extraction column (Supleco, Belefonte, PA) packed with 5 g of Sephadex LH-20 (Sigma Chemical Co., St. Louis, MO) in water. Fifty milliliters of wine was loaded onto a gravity-fed column, and then the column was rinsed with 10 ml of 20% aqueous methanol (v/v). Finally, the procyanidins were rinsed from the column with 75 ml of the extraction solvent. The resulting eluate was concentrated by rotary evaporation to a volume of ⬃2 ml, and the solution was brought to volume in a 5-ml volumetric flask with the extraction solvent and filtered through a 0.45-m nylon filter. Each of the five wine samples were prepared and analyzed in duplicate for the solid phase extraction procedure, and each was analyzed once when prepared via rotary evaporation. The five cranberry juice samples were prepared in duplicate using the solid phase extraction procedure as described above for wine. Again, an initial 50-ml sample was concentrated through the procedure to a final volume of 5 ml and filtered through a 0.45-m nylon filter. Apples were cored using a number 14 cork bore, weighed, frozen with liquid nitrogen and freeze dried. After drying, the samples were weighed again, and the percent moisture was calculated. The freeze-
FIGURE 2 Representative structures of flavan-3-ol monomers and their dimers. When R1 ⫽ OH and R2 ⫽ H, the monomer is (⫺)-epicatechin. When R1 ⫽ H and R2 ⫽ OH, then the monomer is (⫹)-catechin.
dried samples were ground to a fine powder using a high-speed laboratory mill (Janke and Kunkel GmbH, Staufen, Germany). In duplicate, 2 g of the pulverized sample were extracted with 5 ml of the extraction solvent, and the resulting slurry was pelleted by centrifugation; the supernatant was filtered through a 0.45-m nylon filter. Separation and quantification of procyanidins. Sample extracts were analyzed by HPLC using the method described by Adamson et al. (1999). In brief, chromatographic analyses were performed using an HP 1100 Series HPLC (Hewlett Packard, Palo Alto, CA) equipped with an autoinjector, quaternary HPLC pump, column heater, diode array detector, fluorescence detector and HP ChemStation for data collection and manipulation. Fluorescence detection was recorded at excitation wavelength of 276 nm and emission wavelength of 316 nm and UV detection at 280 nm. Normal phase separations of the procyanidin oligomers were performed using a Phenomenex (Torrance, CA) 5-m Lichrosphere silica column (250 ⫻ 4.6 mm) at 37°C with a 5-l injection volume. The ternary mobile phase consisted of A) dichloromethane, B) methanol and C) acetic acid and water (1:1 v/v). Separations were effected by a series of linear gradients of B into A with a constant 4% C at a flow rate of 1 ml/min as follows: elution starting with 14% B in A; 14 –28.4% B in A, 0 –30 min; 28.4 –39.2% B in A, 30 – 45 min; and 39.2– 86% B in A, 45–50 min.
FIGURE 3 (A) A flavan-3-ol monomer conjugated with a gallic acid ester. (B) The structure of a doubly linked procyanidin dimer.
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TABLE 1 Average percent recovery of procyanidin oligomers from five concentrations of standards after solid phase extraction Recovery standards in 14% aq. EtOH
Oligomer (wt.% of composite standard)
Recovery of standards in 100% water %
98.7 ⫾ 2.9 106.8 ⫾ 9.0 100.9 ⫾ 10.5 96.9 ⫾ 9.1 91.3 ⫾ 7.2 85.4 ⫾ 8.6 78.9 ⫾ 11.0 75.2 ⫾ 15.2 67.2 ⫾ 16.0 81.3 ⫾ 24.6 89.4 ⫾ 5.0
Monomer (10.9) Dimer (15.4) Trimer (11.2) Tetramer (11.9) Pentamer (11.8) Hexamer (12.9) Heptamer (6.1) Octamer (6.5) Nonamer (8.9) Decamer (4.1) Total (99.7) Values are ⫾
94.5 ⫾ 3.9 95.5 ⫾ 4.8 95.5 ⫾ 7.3 93.8 ⫾ 9.2 92.9 ⫾ 13.0 92.3 ⫾ 17.7 90.2 ⫾ 21.5 94.7 ⫾ 20.9 99.3 ⫾ 36.5 122.5 ⫾ 21.6 91.0 ⫾ 6.8
SEM.
Composite standard stock solutions (0.4, 1, 2, 4 and 8 mg/ml) were made using commercial (⫺)-epicatechin and procyanidin dimers through decamers purified from cocoa beans (Adamson et al. 1999). This standard was used to prepare a calibration curve for the wine concentrated via rotary evaporation, the chocolates and the apples. For the samples prepared using solid phase extraction, the standards were prepared as described later. Calibration curves were generated using a quadratic fit for the relationship of area sum of the fluorescence signal versus concentration for the peaks corresponding to each oligomeric class. Recovery from solid phase extraction procedure. Composite standard stock solutions (2, 5, 10, 20 and 40 mg in 50 ml) were prepared in 100% water and 14% aqueous ethanol (v/v) to mimic cranberry juice and wine matrices, respectively. The stock solutions were applied to the solid phase extraction column and washed as described earlier for wine and cranberry juice. Again, the final volume was brought to 5 ml to give final concentrations of 0.4, 1, 2, 4 and 8 mg/ml and used to calculate recovery and generate calibration curves used for wine and cranberry juice.
RESULTS Variation in extraction techniques. Modifications to the extraction were required to take into account variations in sample matrices. For example, the chocolate samples required the removal of lipid components to ensure complete extraction of the procyanidins. Similarly, the apple samples required the removal of moisture to ensure proper homogenization of the samples before extraction. For each sample, the procyani-
din content was determined on either a defatted or dry weight basis (chocolate and apple, respectively); then, the concentration in the original starting material was calculated. The wine samples were prepared two ways (rotary evaporation or solid phase extraction) to compare feasibility and reliability of concentration techniques. Similarly, the cranberry juice samples were prepared using solid phase extraction for concentration of the procyanidins and to remove excessive sugar. It has been noted that the continuous loading of oligosaccharides (e.g., corn syrups) significantly reduces column longevity, thus making it desirable to remove them before analysis. Recovery studies from solid phase extraction. To determine the recovery of the procyanidins from solid phase extraction, standard solutions were prepared at known concentrations. The solutions were prepared in either 100% water or 14% aqueous ethanol to reflect the matrices for cranberry juice and wine, respectively. As can be seen in Table 1, the average percent recovery for total procyanidins was similar for the two solid phase extraction methods using standards prepared in pure water compared with aqueous alcohol. It should be noted that the recovery for the higher oligomers was more variable than that for the lower oligomers. This could in part be attributed to matrix differences; however, another explanation may be that the higher oligomers (decamer: low standard 0.008 mg/ml) are near or less than their limit of quantification (decamer 0.06 mg/ml; monomer 0.03 mg/ml) as reported by Adamson et al. (1999) compared with the lower oligomers (monomer: low standard 0.079 mg/ml). Comparison of wine sample preparations. There was little variation between wine samples and between the two methods used for concentration. Although both methods gave similar oligomeric profiles, the solid phase extraction procedure had the advantage of the greatest concentration factor. This allowed for consistent quantification of the procyanidin oligomers through octamer. In contrast, rotary evaporation allowed only consistent quantification through the hexamer. Therefore, the quantitative data reported herein refer to the results obtained using the solid phase extraction method only. Variability of analytical method and samples. To determine whether the variability seen in total procyanidin content was due to variations in the samples or the analytical method, the coefficient of variation was determined for both. As can be seen in Table 2, the coefficient of variation between duplicates was ⬍8% for all samples except the cranberry juice. The cranberry juice had a slightly higher coefficient of variation (10.77%), which is in agreement with the recovery studies for solid phase extraction previously discussed (Table 1). In contrast, the coefficient of variation between the samples in each food group was notably higher. For the wine and cranberry
TABLE 2 Average percent coefficient of variation of procyanidin content between duplicate analyses of the same sample and between different samples of each food product Apple variety Red wine
Cranberry juice
Chocolate
Granny Smith
McIntosh
Red Delicious
Golden Delicious
2.6 24.0
3.1 9.2
7.1 49.8
% CV Duplicates Samples
6.7 7.0
10.8 10.0
2.1 12.1
2.8 33.3
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TABLE 3 Comparison of total procyanidin content in foods and beverages Low
High
Average ⫾
SEM
mg/serving Chocolate Red wine Cranberry juice All apples
140.2 20.3 27.0 12.3
181.2 24.3 35.6 252.4
164.7 ⫾ 19.8 22.0 ⫾ 1.5 31.9 ⫾ 3.2 147.1 ⫾ 57.0
These data are based on five samples of chocolate, red wine and cranberry juice and samples from 19 apples, each run in duplicate.
juice samples, the differences between samples could be attributed to the analytical method rather than to bottle-to-bottle variations. One would expect variations between juice and wine samples to occur; however, it is likely that the samples purchased for this study were from the same production season and underwent similar processing, thus minimizing betweensample variations. Conversely, the variation in procyanidin levels in chocolate and apples must have been a result of sampling as exemplified by the range of procyanidin levels determined in the four foods (Table 3). Total procyanidin content in foods and beverages. Figure 4 illustrates the total procyanidin content determined for the
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individual food and beverage samples expressed as mg/g for chocolate and apples and g/L for wine and cranberry juice. The total content was derived by summing the concentrations for each oligomeric class, monomer through decamer. As can be seen, chocolate contains a significantly larger amount of procyanidins by weight (3.79 – 4.9 mg/g) compared with the other foods and beverages. By contrast, the cranberry juice had the lowest concentration, ranging from 0.13 to 0.15 g/L. Additionally, it is interesting to note the contribution of the monomers versus oligomers to the total procyanidin content. On average, monomers contributed the most to the total procyanidin content in chocolate (1.08 mg/g monomers in 4.45 mg/g total procyanidins) and red wine (0.05 g/L monomers in 0.21 g/L total procyanidins). In contrast, monomers contributed the least to the total procyanidin content in cranberry juice (0.01 g/L monomers in 0.14 g/L total procyanidins) and in apples (Granny Smith, 0.14 mg/g monomers in 1.01 mg/g total procyanidins; Red Delicious, 0.15 mg/g monomers in 1.04 mg/g total procyanidins; McIntosh, 0.06 mg/g monomers in 0.66 mg/g total procyanidins; and Golden Delicious, 0.05 mg/g monomers in 0.49 mg/g total procyanidins). Figure 5 shows the total procyanidin content calculated for an individual serving size. This was calculated using the actual (label) serving size of the chocolate bar (36.9 g) and standard serving sizes for the wine (3.5 oz) and cranberry juice (8 oz). For the apple samples, a serving size was considered to be a whole apple and calculated using the wet weight of each cored apple with skin. As a result, the serving size varied for each
FIGURE 4 Total procyanidin content of each food and beverage sample expressed as mg/g or g/L. Each column represents the different samples, and the error bars indicate the standard deviation between duplicate analyses.
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FIGURE 5 Total procyanidin content of each food and beverage sample as expressed in mg per serving. Each column represents the different samples, and the error bars indicate the standard deviation between duplicate analyses.
apple sample (Granny Smith 153.74 –237.30 g, McIntosh 140.27–180.69 g, Red Delicious 173.46 –256.40 g and Golden Delicious 156.49 –244.44 g). When accounting for serving size, the differences between the procyanidin content in chocolate and apples are equivocal. In addition, although cranberry juice had the lowest concentration by weight, the wine offers the least amount of procyanidins per serving. Finally, the procyanidin content was also expressed on an energy basis through calculations using the caloric density for each food promulgated in the United States Department of Agriculture Nutrient Database for Standard Reference, Release 12 (March 1998). Although chocolate has a significantly higher concentration of procyanidins by weight than the other foods and beverages, apples tend to offer more procyanidins per kilocalorie (1.3 mg/kcal) on average than chocolate (0.9 mg/kcal), red wine (0.3 mg/kcal) and cranberry juice (0.2 mg/kcal). DISCUSSION In the present study, procyanidin oligomers were isolated and purified from the seeds of Theobroma cacao and used as standards for quantifying the procyanidin content in chocolate, wine, apples and cranberry juice. It is well known that the primary flavan-3-ols in cocoa are (⫺)-epicatechin and (⫹)catechin (Clapperton et al. 1992, Osakabe et al. 1998, Porter et al. 1991). In addition, the procyanidin oligomers in cocoa mainly consist of (⫺)-epicatechin as their monomeric unit, as
demonstrated by the fact that procyanidins B2 and B5 are the major dimers present (Porter et al. 1991). Furthermore, there are eight possible dimeric stereoisomers of (⫹)-catechin and (⫺)-epicatechin with either the 436 or 438 linkage, in addition to the doubly linked conformations. It is not clear whether these stereoisomers have similar fluorescent yields for a given concentration. Recently, Carando et al. (1999) reported slight differences for the responses of dimers B1 through B4. Therefore, the use of oligomers from cocoa may introduce a slight bias in the quantification of non– cocoa-based procyanidins. Furthermore, it has been shown that the fluorescence detector is insensitive to procyanidins containing a gallic acid ester and/or the gallocatechins as a monomeric unit, and hence, neither fluorescence nor the cocoa-based standard would be applicable to these classes (Hammerstone and Lazarus, 2000). However, this is not an issue in the present study because all of the foods and beverages analyzed contain (⫺)epicatechin and (⫹)-catechin as the monomeric units. Because there are many stereoisomers possible for a given oligomeric class, it is necessary to identify which peaks in the HPLC chromatogram belong to each class. Hence, the analytical method used in the present study allowed for the elution of the procyanidin oligomers in discrete groupings according to degree of polymerization. For each food and beverage, these groupings were confirmed with the use of HPLC coupled with mass spectrometry as recently described by Hammerstone et al. (1999) for chocolate, Lazarus et al.
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(1999a) for wine and apple and Prior et al. (unpublished observations) for cranberry juice. The applicability of the analytical method to each food sample has been confirmed, so the total procyanidin content was determined taking into account the contributions from each oligomeric class (monomer through decamer, where applicable). Not surprisingly, the procyanidin content was different for each of the types of apples, with the Red Delicious (207.7 mg/serving) and Granny Smith (183.3 mg/serving) apples containing the most procyanidins on average and the McIntosh (105.0 mg/serving) and Golden Delicious (92.5 mg/serving) apples containing the least. Variations between apple cultivars have been noted previously by Sanoner et al. (1999). In addition to large variations between cultivars, there also were large differences in the procyanidin concentration of apples within each cultivar. One possible explanation for these variations could be the maturity of the apples at harvest time, because it is known that procyanidin content decreases on ripening (Macheix et al. 1990). Another possible explanation could be related to the ratio between pulp and skin, because procyanidins are more concentrated in the skin than the pulp (Guyot et al. 1998). Although more modest than the variation in apples, there also were small differences noted among the chocolate samples even though they are from the same manufacturer. Because the cocoa liquor component of chocolate is commonly formulated and sourced from the same geographical regions, little or no variation in the procyanidin content would be expected. However, genetic variations within a geographical region and differences in postharvest practices between farmers result in differences in procyanidin content of the cocoa raw material (Clapperton et al. 1992, Kim and Keeney 1984). In addition, because chocolate can contain varying amounts of cocoa ingredient that has been formulated from a unique blend of different cocoa sources, it would be expected that additional variations would be observed among commonly consumed chocolate products. In contrast to apple and chocolate, little variation was noted among the wine samples. Previously, large variations in red wine procyanidin concentrations have been reported and have been attributed to aging (Carando et al. 1999, Waterhouse and Walzem 1998), geographical location (Carando et al. 1999, Frankel et al. 1995), enological practices (Kovac et al. 1992, Waterhouse and Walzem 1998), types of red wine (Frankel et al. 1995, Salagoı¨ty-Auguste and Bertrand 1984) and fining (Waterhouse and Walzem 1998). However, it is important to note that the five samples analyzed in the current study were from the same manufacturer and vintage year, thus minimizing variability. Similar to wine, little variation was observed among the five cranberry juice samples. Although the procyanidin content in cranberries has not been extensively studied, one would expect that varietal selection, juice clarification, maturity and geographical location may cause considerable variation as observed for other foods and beverages. It is apparent that the foods analyzed in the current study, in addition to other commonly consumed foods such as tea (Bronner and Beecher 1998), can contain significant levels of procyanidins. As such, procyanidins may contribute significantly to the total dietary intake of flavonoids; however, most intake studies to date have focused on a limited number of simple flavonoids. For example, Hertog et al. (1993), Rimm et al. (1996) and Yochum et al. (1999) applied compositional data for three flavonols and two flavones in fruits, vegetables and beverages to Dutch and U.S. food consumption surveys to estimate dietary flavonoid intake. The compositional data used
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for these studies reported the flavonoid content of Granny Smith and Golden Delicious apples as 0.024 and 0.025 mg/g, respectively (Hertog et al. 1992). However, the procyanidin content for these apples were determined in this study to be 1.01 and 0.491 mg/g, respectively. Thus, the total flavonoid content for apple was significantly underestimated and possibly could have been for other foods in this study as well. Similarly, Arts et al. (1999) examined the dietary flavan-3-ol intake in a Dutch population and found that chocolate and tea contributed 20 and 55%, respectively. However, their methodology only accounted for the monomeric flavan-3-ol derivatives and neglected the oligomers that are abundant in chocolate and only minor in tea (Lazarus et al. 1999b). Importantly, the use of select flavonoids to infer epidemiological relationships to health and disease could be confounded by the fact that different flavonoids may exhibit varying physiological effects. Extensive research has been conducted investigating the relationship between flavonoid structure and biological activity, especially as it relates to antioxidant properties, and found that the hydroxylation pattern is a key factor (Rice-Evans et al. 1996). In contrast, little is known regarding the impact of the degree of polymerization of the procyanidins and their biological properties. Recently, Arteel and Sies (1999) and Bearden et al. (2000) investigated the effectiveness of cocoa procyanidins in vitro to scavenge peroxynitrite and inhibit LDL oxidation, respectively, and both found that antioxidant activity was influenced by oligomeric size. Also, Mao et al. (1999) studied the ability of the procyanidins to modulate interleukin-2 in vitro and found the higher oligomers inhibited interleukin-2 expression in stimulated cells, whereas the monomer had no effect. These preliminary studies suggest that the physiological effects of the procyanidins may be affected by the degree of polymerization, and therefore, the oligomeric composition should be included when determining the flavonoid content of foods. In conclusion, the current study demonstrated that commonly consumed foods and beverages, including chocolate, apples, cranberry juice and wine, contain substantial amounts of procyanidins. In addition, these results suggest that significant variation in procyanidin content can exist within these foods. This research represents an initial investigation to understand which foods may contribute most to the dietary intake of procyanidins. However, a more extensive sampling plan would have to be implemented to compile a comprehensive database of foods and beverages that contain procyanidins. Given that these compounds have exhibited the ability to influence health in in vitro and in vivo studies, it would be important to use this compositional data in food consumption surveys to define intake levels in populations and to determine whether epidemiological associations with health and disease end points may be present. ACKNOWLEDGMENT The authors gratefully acknowledge F. Plog for assisting in sample preparation.
LITERATURE CITED Adamson, G. E., Lazarus, S. A., Mitchell, A. E., Prior, R. L., Cao, G., Jacobs, P. H., Kremers, B. G., Hammerstone, J. F., Rucker, R. B., Ritter, K. A. & Schmitz, H. H. (1999) HPLC method for the quantification of procyanidins in cocoa and chocolate samples and correlation to total antioxidant capacity. J. Agric. Food Chem. 47: 4184 – 4188. AOAC International (1996) Official Methods of Analysis Ether Extraction of Confectionery: 920.177, 16th ed., AOAC International, Gaithersburg, MD. Ariga, T. & Hamano, M. (1990) Radical scavenging action and its mode in procyanidins B-1 and B-3 from azuki beans to peroxyl radicals. Agric. Biol. Chem. 54: 2499 –2504.
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Arteel, G. E. & Sies, H. (1999) Protection against peroxynitrite by cocoa polyphenol oligomers. FEBS Lett. 462: 167–170. Arts, I.C.W., Hollman, P.C.H. & Kromhout, D. (1999) Chocolate as a source of tea flavonoids. Lancet 354: 488. Bearden, M. M., Pearson, D. A., Rein, D., Chevaux, K. A., Carpenter, D. R., Keen, C. L. & Schmitz, H. (2000) Potential cardiovascular health benefits of procyanidins present in chocolate and cocoa. In: Caffeinated Beverages: Health Benefits, Physiological Effects and Chemistry (Parliament, T. H., Ho, C. T. & Schieberle, P., eds.), pp. 177–186. American Chemical Society, Washington, D.C. Bronner, W. E. & Beecher, G. R. (1998) Method for determining the content of catechins in tea infusions by high-performance liquid chromatography. J. Chromatogr. A 805: 137–142. Carando, S., Teissedre, P.-L., Pascual-Martinez, L. & Cabanis, J.-C. (1999) Levels of flavan-3-ols in French wines. J. Agric. Food Chem. 47: 4161– 4166. Clapperton, J., Hammerstone, J.F., Romanczyk, L. J., Yow, S., Lim, D. & Lockwood, R. (1992) Polyphenols and Cocoa Flavour. Proceedings of the 16th International Conference of Groupe Polyphenols: Lisbon, Portugal, Groupe Polyphenols, Norbonne, France, Tome II: 112–115. Frankel, E. N., Waterhouse, A. L. & Teissedre, P. L. (1995) Principal phenolic phytochemicals in selected California wines and their antioxidant activity in inhibiting oxidation of human low-density lipoproteins. J. Agric. Food Chem. 43: 890 – 894. Guyot, S., Marnet, N., Laraba, D., Sanoner, P. & Drilleau, J.-F. (1998) Reversed-phase HPLC following thiolysis for quantitative estimation and characterization of the four main classes of phenolic compounds in different tissue zones of a French cider apple variety (Malus domestica var. Kermerrien). J. Agric. Food Chem. 46: 1698 –1705. Hagerman, A. E., Riedl, K. M., Jones, G. A., Sovik, K. N., Ritchard, N. T., Hartzfeld, P. W. & Riechel, T. L. (1998) High molecular weight plant polyphenolics (tannins) as biological antioxidants. J. Agric. Food Chem. 46: 1887–1892. Hammerstone, J. F. & Lazarus, S. A. (2000) HPLC/MS analysis of flavonoids in foods and beverages. In: Caffeinated Beverages: Health Benefits, Physiological Effects and Chemistry (Parliament, T. H., Ho, C. T. & Schieberle, P., eds.). pp. 374 –384. American Chemical Society, Washington, D.C. Hammerstone, J. F., Lazarus, S. A., Mitchell, A. E., Rucker, R. & Schmitz, H. H. (1999) Identification of procyanidins in cocoa and chocolate using high performance liquid chromatography/mass spectrometry. J. Agric. Food Chem. 47: 490 – 496. Haslam, E. (1998) Polyphenols: structure and biosynthesis. In: Practical Polyphenolics From Structure to Molecular Recognition and Physiological Action (Haslam, E., ed.), pp. 10 – 83, Cambridge University Press, Cambridge, U.K. Hertog, M. G., Feskens, E. J., Hollman, P. C., Katan, M. B. & Kromhout. D. (1993) Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet 342: 1007–1011. Hertog, M. G., Hollman, P. C. & Katan, M. B. (1992) Content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in the Netherlands. J. Agric. Food Chem. 40: 2379 –2383. Kim, H. & Keeney, P. G. (1984) (⫺)-Epicatechin content in fermented and unfermented cocoa beans. J. Food Sci. 49: 1090 –1092.
Kovac, V., Alonso, E., Bourzeix, M. & Revilla, E. (1992) Effect of several enological practices on the content of catechins and proanthocyanidins of red wines. J. Agric. Food Chem. 40: 1953–1957. Lazarus, S. A., Adamson, G. E., Hammerstone, J. F. & Schmitz, H. H. (1999a) High performance liquid chromatography/mass spectrometry analysis of proanthocyanidins in food stuffs. J. Agric. Food Chem. 47: 3693–3701. Lazarus, S. A., Hammerstone, J. & Schmitz, H. H. (1999b) Chocolate contains additional flavonoids not found in tea. Lancet. 354: 1825. Macheix, J.-J., Fleuriet, A. & Billot, J. (1990) Changes and metabolism of phenolic compounds in fruits. In: Fruit Phenolics (Macheix, J.-J., Fleuriet, A. & Billot, J., eds.), pp. 149 –237, CRC Press, Inc., Boca Raton, FL. Mao, T. K., Powell, J. J., van de Water, J., Keen, C. L., Schmitz, H. H. & Gershwin, M. E. (1999) The influence of cocoa procyanidins on the transcription of interleukin-2 in peripheral blood mononuclear cells. Int. J. Immunother. XV: 23–29. Osakabe, N., Yamagishi, M., Sanbongi, C., Natsume, M., Takizawa, T. & Osawa, T. (1998) The antioxidative substances in cacao liquor. J. Nutr. Sci. Vitaminol. 44: 313–321. Packer, L., Rimbach, G. & Virgili, F. (1999) Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (Pinus maritima) bark, pycnogenol. Free Radic. Biol. Med. 27: 704 –724. Porter, L. J., Ma, Z. & Chan, B. G. (1991) Cacao procyanidins: major flavonoids and identification of some minor metabolites. Phytochemistry. 30: 1657– 1663. Rein, D., Paglieroni, T. G., Wun, T., Pearson, D. A., Schmitz, H. H., Gosselin, R. & Keen, C. L. (2000) Cocoa inhibits platelet activation and function. Am. J. Clin. Nutr. (in press). Rice-Evans, C. A., Miller, N. J. & Paganga, G. (1996) Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20: 933–956. Rimm, E. B., Katan, M. B., Ascherio, M. D., Stampfer, M. J. & Willett, W. C. (1996) Relation between intake of flavonoids and risk of coronary heart disease in male health professionals. Ann. Intern. Med. 125: 384 –389. Salagoı¨ty-Auguste, M.-H. & Bertrand, A. (1984) Wine phenolics: analysis of low molecular weight components by high performance liquid chromatography. J. Sci. Food. Ag. 35: 1241–1247. Sanbongi, C., Suzuki, N. & Sakane, T. (1997) Polyphenols in chocolate, which have antioxidant activity, modulate immune functions in humans in vitro. Cell Immunol. 177: 129 –136. Sanoner, P., Guyot, S., Marnet, N., Molle, D. & Drilleau, J.-F. (1999) Polyphenol profiles of French cider apple varieties (Malus domestica sp.). J. Agric. Food Chem. 47: 4847– 4853. Waterhouse, A. L. & Walzem, R. L. (1998) Nutrition of grape phenolics. In: Flavonoids in Health and Disease (Rice-Evans, C. A. & Packer, L., eds.), pp. 359 –385, Marcel Dekker, New York, NY. Wilson, E. L. (1981) High-pressure liquid chromatography of apple juice phenolic compounds. J Sci Food Agric. 32: 257–264. Yochum, L., Kushi, L. H., Meyer, K. & Folsom, A. R. (1999) Dietary flavonoid intake and risk of cardiovascular disease in postmenopausal women. Am. J. Epidemiol. 149: 943–949.
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procyanidins
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quantification
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food composition
Simple phenols consist of one aromatic ring containing at least one hydroxyl group, whereas polyphenols consist of more than one aromatic ring with each containing at least one hydroxyl group. Flavonoids are a subclass of polyphenols that have a C6-C3-C6 backbone structure (Fig. 1). One group of flavonoids, the procyanidins, are composed of flavan-3-ol monomers and their respective oligomers, commonly bonded through a 436 or 438 linkage (Fig. 2). In addition to these simple forms, they have been reported as containing gallic acid esters or as doubly linked forms as shown in Figure 3 (Haslam 1998). Although it has been known for some time that polyphenols are ubiquitous in the plant kingdom, only recently have they begun to be studied in foods. Furthermore, with some exceptions, most of the polyphenol research in food composition has been limited to simple flavonoids and phenolic acids, rather than a comprehensive investigation of all classes of polyphenols present. In addition to incomplete compositional information, commercial standards are not available for most complex polyphenols. Both of these factors contribute to poor quantitative data, and consequently, there is large variability reported for dietary intake levels, which can confound the ability to infer epidemiological relationships with health and disease.
Oligomeric procyanidins represent one class of polyphenols in which qualitative and quantitative information with regard to food composition is especially lacking. This is due in large part to analytical constraints and lack of appropriate methodology suitable for reliable characterization and quantification of these compounds. However, recent advances in analytical methodology have allowed for the separation and quantification of the procyanidin oligomers in cocoa and chocolate (Adamson et al. 1999, Hammerstone et al. 1999), and this same method has been applied to the separation of proanthocyanidin oligomers in a variety of plant foods (Lazarus et al. 1999a). This was made possible by the use of normal phase HPLC instead of the more commonly used reversed phase methodology, which is not capable of resolving the higher oligomeric forms (Hammerstone and Lazarus 2000, Wilson 1981). Oligomeric procyanidins have attracted increasing attention in the fields of nutrition and medicine due to their potential health benefits observed in vitro and in vivo. Most prominently, procyanidin oligomers have been shown to have potent antioxidant activity and the ability to scavenge reactive oxygen and nitrogen species (Ariga and Hamano 1990, Arteel and Sies 1999, Hagerman et al. 1998). In addition, recent research suggests these phytochemicals may modulate immune function and platelet activation (Mao et al. 1999, Packer et al. 1999, Rein et al. 2000, Sanbongi et al. 1997). Thus, in the present study, we extended the work of Adamson et al. (1999), in which the procyanidins were quantified in cocoa and chocolate to include other commonly consumed foods, such as apples, wine and cranberry juice, which are
1 Presented at the symposium “Chocolate: Modern Science Investigates an Ancient Medicine,” held February 19, 2000, during the 2000 Annual Meeting and Science Innovation Exposition at the American Association for the Advancement of Science in Washington, D.C. Published as a supplement to The Journal of Nutrition. Guest editors for the supplement publication were John W. Erdman, Jr., University of Illinois at Urbana-Champaign; Jo Wills, Mars, United Kingdom and D’Ann Finley, University of California, Davis. 2 To whom reprint requests should be addressed.
0022-3166/00 $3.00 © 2000 American Society for Nutritional Sciences.
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FIGURE 1
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The C15 (C6-C3-C6) basic skeleton of a flavonoid.
believed to be significant contributors to the dietary intake of procyanidin oligomers. MATERIALS AND METHODS Chemicals and reagents. HPLC-grade methylene chloride, methanol, acetone and acetic acid were obtained from VWR Scientific (Bridgeport, NJ). HPLC-grade water was obtained from a Milli-Q (Millipore, Bedford, MA) water purification system. (⫺)-Epicatechin was purchased from Sigma Chemical Co. (St. Louis, MO). Procyanidin oligomers were purified from cocoa beans, and a composite standard was prepared as described previously by Adamson et al. (1999). Sample collection. Five samples each of dark chocolate (DOVE Chocolate, M&M/Mars, Hackettstown, NJ), Cabernet Sauvignon wine (Woodbridge; Robert Mondavi, Woodbridge, CA), cranberry juice (Ocean Spray, Lakeville-Middleboro, MA) and Golden Delicious, Red Delicious, Granny Smith and McIntosh apples were purchased from five grocery and wine stores in eastern Pennsylvania and western New Jersey. Only four of the five Red Delicious apples were used for analysis because the fifth apple was found to have a rotten core and was not suitable for consumption. Sample preparation. The chocolate was prepared for analysis as described by Adamson et al. (1999). In brief, the lipid was extracted from 15 g of sample through exhaustive extraction with hexane (4 ⫻ 45 ml). One gram of the dried, lipid-free solids was extracted with 5 ml of an extraction solvent composed of acetone, water and acetic acid in a ratio of 70:29.5:0.5 (v/v/v), respectively. The resulting slurry was pelleted at 1500 ⫻ g, and then the supernatant was filtered through a 0.45-m nylon filter. For the determination of percent fat composition, AOAC Official Method 920.177 was used (AOAC International 1996). Each of the five chocolate samples were extracted and analyzed in duplicate. Wine samples were prepared using two methods. In the first, 300 ml of wine was concentrated by rotary evaporation under partial pressure at 42°C to a volume of 75 ml before HPLC analysis without further manipulation. The second method involved the use of a 20-ml solid phase extraction column (Supleco, Belefonte, PA) packed with 5 g of Sephadex LH-20 (Sigma Chemical Co., St. Louis, MO) in water. Fifty milliliters of wine was loaded onto a gravity-fed column, and then the column was rinsed with 10 ml of 20% aqueous methanol (v/v). Finally, the procyanidins were rinsed from the column with 75 ml of the extraction solvent. The resulting eluate was concentrated by rotary evaporation to a volume of ⬃2 ml, and the solution was brought to volume in a 5-ml volumetric flask with the extraction solvent and filtered through a 0.45-m nylon filter. Each of the five wine samples were prepared and analyzed in duplicate for the solid phase extraction procedure, and each was analyzed once when prepared via rotary evaporation. The five cranberry juice samples were prepared in duplicate using the solid phase extraction procedure as described above for wine. Again, an initial 50-ml sample was concentrated through the procedure to a final volume of 5 ml and filtered through a 0.45-m nylon filter. Apples were cored using a number 14 cork bore, weighed, frozen with liquid nitrogen and freeze dried. After drying, the samples were weighed again, and the percent moisture was calculated. The freeze-
FIGURE 2 Representative structures of flavan-3-ol monomers and their dimers. When R1 ⫽ OH and R2 ⫽ H, the monomer is (⫺)-epicatechin. When R1 ⫽ H and R2 ⫽ OH, then the monomer is (⫹)-catechin.
dried samples were ground to a fine powder using a high-speed laboratory mill (Janke and Kunkel GmbH, Staufen, Germany). In duplicate, 2 g of the pulverized sample were extracted with 5 ml of the extraction solvent, and the resulting slurry was pelleted by centrifugation; the supernatant was filtered through a 0.45-m nylon filter. Separation and quantification of procyanidins. Sample extracts were analyzed by HPLC using the method described by Adamson et al. (1999). In brief, chromatographic analyses were performed using an HP 1100 Series HPLC (Hewlett Packard, Palo Alto, CA) equipped with an autoinjector, quaternary HPLC pump, column heater, diode array detector, fluorescence detector and HP ChemStation for data collection and manipulation. Fluorescence detection was recorded at excitation wavelength of 276 nm and emission wavelength of 316 nm and UV detection at 280 nm. Normal phase separations of the procyanidin oligomers were performed using a Phenomenex (Torrance, CA) 5-m Lichrosphere silica column (250 ⫻ 4.6 mm) at 37°C with a 5-l injection volume. The ternary mobile phase consisted of A) dichloromethane, B) methanol and C) acetic acid and water (1:1 v/v). Separations were effected by a series of linear gradients of B into A with a constant 4% C at a flow rate of 1 ml/min as follows: elution starting with 14% B in A; 14 –28.4% B in A, 0 –30 min; 28.4 –39.2% B in A, 30 – 45 min; and 39.2– 86% B in A, 45–50 min.
FIGURE 3 (A) A flavan-3-ol monomer conjugated with a gallic acid ester. (B) The structure of a doubly linked procyanidin dimer.
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TABLE 1 Average percent recovery of procyanidin oligomers from five concentrations of standards after solid phase extraction Recovery standards in 14% aq. EtOH
Oligomer (wt.% of composite standard)
Recovery of standards in 100% water %
98.7 ⫾ 2.9 106.8 ⫾ 9.0 100.9 ⫾ 10.5 96.9 ⫾ 9.1 91.3 ⫾ 7.2 85.4 ⫾ 8.6 78.9 ⫾ 11.0 75.2 ⫾ 15.2 67.2 ⫾ 16.0 81.3 ⫾ 24.6 89.4 ⫾ 5.0
Monomer (10.9) Dimer (15.4) Trimer (11.2) Tetramer (11.9) Pentamer (11.8) Hexamer (12.9) Heptamer (6.1) Octamer (6.5) Nonamer (8.9) Decamer (4.1) Total (99.7) Values are ⫾
94.5 ⫾ 3.9 95.5 ⫾ 4.8 95.5 ⫾ 7.3 93.8 ⫾ 9.2 92.9 ⫾ 13.0 92.3 ⫾ 17.7 90.2 ⫾ 21.5 94.7 ⫾ 20.9 99.3 ⫾ 36.5 122.5 ⫾ 21.6 91.0 ⫾ 6.8
SEM.
Composite standard stock solutions (0.4, 1, 2, 4 and 8 mg/ml) were made using commercial (⫺)-epicatechin and procyanidin dimers through decamers purified from cocoa beans (Adamson et al. 1999). This standard was used to prepare a calibration curve for the wine concentrated via rotary evaporation, the chocolates and the apples. For the samples prepared using solid phase extraction, the standards were prepared as described later. Calibration curves were generated using a quadratic fit for the relationship of area sum of the fluorescence signal versus concentration for the peaks corresponding to each oligomeric class. Recovery from solid phase extraction procedure. Composite standard stock solutions (2, 5, 10, 20 and 40 mg in 50 ml) were prepared in 100% water and 14% aqueous ethanol (v/v) to mimic cranberry juice and wine matrices, respectively. The stock solutions were applied to the solid phase extraction column and washed as described earlier for wine and cranberry juice. Again, the final volume was brought to 5 ml to give final concentrations of 0.4, 1, 2, 4 and 8 mg/ml and used to calculate recovery and generate calibration curves used for wine and cranberry juice.
RESULTS Variation in extraction techniques. Modifications to the extraction were required to take into account variations in sample matrices. For example, the chocolate samples required the removal of lipid components to ensure complete extraction of the procyanidins. Similarly, the apple samples required the removal of moisture to ensure proper homogenization of the samples before extraction. For each sample, the procyani-
din content was determined on either a defatted or dry weight basis (chocolate and apple, respectively); then, the concentration in the original starting material was calculated. The wine samples were prepared two ways (rotary evaporation or solid phase extraction) to compare feasibility and reliability of concentration techniques. Similarly, the cranberry juice samples were prepared using solid phase extraction for concentration of the procyanidins and to remove excessive sugar. It has been noted that the continuous loading of oligosaccharides (e.g., corn syrups) significantly reduces column longevity, thus making it desirable to remove them before analysis. Recovery studies from solid phase extraction. To determine the recovery of the procyanidins from solid phase extraction, standard solutions were prepared at known concentrations. The solutions were prepared in either 100% water or 14% aqueous ethanol to reflect the matrices for cranberry juice and wine, respectively. As can be seen in Table 1, the average percent recovery for total procyanidins was similar for the two solid phase extraction methods using standards prepared in pure water compared with aqueous alcohol. It should be noted that the recovery for the higher oligomers was more variable than that for the lower oligomers. This could in part be attributed to matrix differences; however, another explanation may be that the higher oligomers (decamer: low standard 0.008 mg/ml) are near or less than their limit of quantification (decamer 0.06 mg/ml; monomer 0.03 mg/ml) as reported by Adamson et al. (1999) compared with the lower oligomers (monomer: low standard 0.079 mg/ml). Comparison of wine sample preparations. There was little variation between wine samples and between the two methods used for concentration. Although both methods gave similar oligomeric profiles, the solid phase extraction procedure had the advantage of the greatest concentration factor. This allowed for consistent quantification of the procyanidin oligomers through octamer. In contrast, rotary evaporation allowed only consistent quantification through the hexamer. Therefore, the quantitative data reported herein refer to the results obtained using the solid phase extraction method only. Variability of analytical method and samples. To determine whether the variability seen in total procyanidin content was due to variations in the samples or the analytical method, the coefficient of variation was determined for both. As can be seen in Table 2, the coefficient of variation between duplicates was ⬍8% for all samples except the cranberry juice. The cranberry juice had a slightly higher coefficient of variation (10.77%), which is in agreement with the recovery studies for solid phase extraction previously discussed (Table 1). In contrast, the coefficient of variation between the samples in each food group was notably higher. For the wine and cranberry
TABLE 2 Average percent coefficient of variation of procyanidin content between duplicate analyses of the same sample and between different samples of each food product Apple variety Red wine
Cranberry juice
Chocolate
Granny Smith
McIntosh
Red Delicious
Golden Delicious
2.6 24.0
3.1 9.2
7.1 49.8
% CV Duplicates Samples
6.7 7.0
10.8 10.0
2.1 12.1
2.8 33.3
PROCYANIDIN CONTENT AND VARIATION
TABLE 3 Comparison of total procyanidin content in foods and beverages Low
High
Average ⫾
SEM
mg/serving Chocolate Red wine Cranberry juice All apples
140.2 20.3 27.0 12.3
181.2 24.3 35.6 252.4
164.7 ⫾ 19.8 22.0 ⫾ 1.5 31.9 ⫾ 3.2 147.1 ⫾ 57.0
These data are based on five samples of chocolate, red wine and cranberry juice and samples from 19 apples, each run in duplicate.
juice samples, the differences between samples could be attributed to the analytical method rather than to bottle-to-bottle variations. One would expect variations between juice and wine samples to occur; however, it is likely that the samples purchased for this study were from the same production season and underwent similar processing, thus minimizing betweensample variations. Conversely, the variation in procyanidin levels in chocolate and apples must have been a result of sampling as exemplified by the range of procyanidin levels determined in the four foods (Table 3). Total procyanidin content in foods and beverages. Figure 4 illustrates the total procyanidin content determined for the
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individual food and beverage samples expressed as mg/g for chocolate and apples and g/L for wine and cranberry juice. The total content was derived by summing the concentrations for each oligomeric class, monomer through decamer. As can be seen, chocolate contains a significantly larger amount of procyanidins by weight (3.79 – 4.9 mg/g) compared with the other foods and beverages. By contrast, the cranberry juice had the lowest concentration, ranging from 0.13 to 0.15 g/L. Additionally, it is interesting to note the contribution of the monomers versus oligomers to the total procyanidin content. On average, monomers contributed the most to the total procyanidin content in chocolate (1.08 mg/g monomers in 4.45 mg/g total procyanidins) and red wine (0.05 g/L monomers in 0.21 g/L total procyanidins). In contrast, monomers contributed the least to the total procyanidin content in cranberry juice (0.01 g/L monomers in 0.14 g/L total procyanidins) and in apples (Granny Smith, 0.14 mg/g monomers in 1.01 mg/g total procyanidins; Red Delicious, 0.15 mg/g monomers in 1.04 mg/g total procyanidins; McIntosh, 0.06 mg/g monomers in 0.66 mg/g total procyanidins; and Golden Delicious, 0.05 mg/g monomers in 0.49 mg/g total procyanidins). Figure 5 shows the total procyanidin content calculated for an individual serving size. This was calculated using the actual (label) serving size of the chocolate bar (36.9 g) and standard serving sizes for the wine (3.5 oz) and cranberry juice (8 oz). For the apple samples, a serving size was considered to be a whole apple and calculated using the wet weight of each cored apple with skin. As a result, the serving size varied for each
FIGURE 4 Total procyanidin content of each food and beverage sample expressed as mg/g or g/L. Each column represents the different samples, and the error bars indicate the standard deviation between duplicate analyses.
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FIGURE 5 Total procyanidin content of each food and beverage sample as expressed in mg per serving. Each column represents the different samples, and the error bars indicate the standard deviation between duplicate analyses.
apple sample (Granny Smith 153.74 –237.30 g, McIntosh 140.27–180.69 g, Red Delicious 173.46 –256.40 g and Golden Delicious 156.49 –244.44 g). When accounting for serving size, the differences between the procyanidin content in chocolate and apples are equivocal. In addition, although cranberry juice had the lowest concentration by weight, the wine offers the least amount of procyanidins per serving. Finally, the procyanidin content was also expressed on an energy basis through calculations using the caloric density for each food promulgated in the United States Department of Agriculture Nutrient Database for Standard Reference, Release 12 (March 1998). Although chocolate has a significantly higher concentration of procyanidins by weight than the other foods and beverages, apples tend to offer more procyanidins per kilocalorie (1.3 mg/kcal) on average than chocolate (0.9 mg/kcal), red wine (0.3 mg/kcal) and cranberry juice (0.2 mg/kcal). DISCUSSION In the present study, procyanidin oligomers were isolated and purified from the seeds of Theobroma cacao and used as standards for quantifying the procyanidin content in chocolate, wine, apples and cranberry juice. It is well known that the primary flavan-3-ols in cocoa are (⫺)-epicatechin and (⫹)catechin (Clapperton et al. 1992, Osakabe et al. 1998, Porter et al. 1991). In addition, the procyanidin oligomers in cocoa mainly consist of (⫺)-epicatechin as their monomeric unit, as
demonstrated by the fact that procyanidins B2 and B5 are the major dimers present (Porter et al. 1991). Furthermore, there are eight possible dimeric stereoisomers of (⫹)-catechin and (⫺)-epicatechin with either the 436 or 438 linkage, in addition to the doubly linked conformations. It is not clear whether these stereoisomers have similar fluorescent yields for a given concentration. Recently, Carando et al. (1999) reported slight differences for the responses of dimers B1 through B4. Therefore, the use of oligomers from cocoa may introduce a slight bias in the quantification of non– cocoa-based procyanidins. Furthermore, it has been shown that the fluorescence detector is insensitive to procyanidins containing a gallic acid ester and/or the gallocatechins as a monomeric unit, and hence, neither fluorescence nor the cocoa-based standard would be applicable to these classes (Hammerstone and Lazarus, 2000). However, this is not an issue in the present study because all of the foods and beverages analyzed contain (⫺)epicatechin and (⫹)-catechin as the monomeric units. Because there are many stereoisomers possible for a given oligomeric class, it is necessary to identify which peaks in the HPLC chromatogram belong to each class. Hence, the analytical method used in the present study allowed for the elution of the procyanidin oligomers in discrete groupings according to degree of polymerization. For each food and beverage, these groupings were confirmed with the use of HPLC coupled with mass spectrometry as recently described by Hammerstone et al. (1999) for chocolate, Lazarus et al.
PROCYANIDIN CONTENT AND VARIATION
(1999a) for wine and apple and Prior et al. (unpublished observations) for cranberry juice. The applicability of the analytical method to each food sample has been confirmed, so the total procyanidin content was determined taking into account the contributions from each oligomeric class (monomer through decamer, where applicable). Not surprisingly, the procyanidin content was different for each of the types of apples, with the Red Delicious (207.7 mg/serving) and Granny Smith (183.3 mg/serving) apples containing the most procyanidins on average and the McIntosh (105.0 mg/serving) and Golden Delicious (92.5 mg/serving) apples containing the least. Variations between apple cultivars have been noted previously by Sanoner et al. (1999). In addition to large variations between cultivars, there also were large differences in the procyanidin concentration of apples within each cultivar. One possible explanation for these variations could be the maturity of the apples at harvest time, because it is known that procyanidin content decreases on ripening (Macheix et al. 1990). Another possible explanation could be related to the ratio between pulp and skin, because procyanidins are more concentrated in the skin than the pulp (Guyot et al. 1998). Although more modest than the variation in apples, there also were small differences noted among the chocolate samples even though they are from the same manufacturer. Because the cocoa liquor component of chocolate is commonly formulated and sourced from the same geographical regions, little or no variation in the procyanidin content would be expected. However, genetic variations within a geographical region and differences in postharvest practices between farmers result in differences in procyanidin content of the cocoa raw material (Clapperton et al. 1992, Kim and Keeney 1984). In addition, because chocolate can contain varying amounts of cocoa ingredient that has been formulated from a unique blend of different cocoa sources, it would be expected that additional variations would be observed among commonly consumed chocolate products. In contrast to apple and chocolate, little variation was noted among the wine samples. Previously, large variations in red wine procyanidin concentrations have been reported and have been attributed to aging (Carando et al. 1999, Waterhouse and Walzem 1998), geographical location (Carando et al. 1999, Frankel et al. 1995), enological practices (Kovac et al. 1992, Waterhouse and Walzem 1998), types of red wine (Frankel et al. 1995, Salagoı¨ty-Auguste and Bertrand 1984) and fining (Waterhouse and Walzem 1998). However, it is important to note that the five samples analyzed in the current study were from the same manufacturer and vintage year, thus minimizing variability. Similar to wine, little variation was observed among the five cranberry juice samples. Although the procyanidin content in cranberries has not been extensively studied, one would expect that varietal selection, juice clarification, maturity and geographical location may cause considerable variation as observed for other foods and beverages. It is apparent that the foods analyzed in the current study, in addition to other commonly consumed foods such as tea (Bronner and Beecher 1998), can contain significant levels of procyanidins. As such, procyanidins may contribute significantly to the total dietary intake of flavonoids; however, most intake studies to date have focused on a limited number of simple flavonoids. For example, Hertog et al. (1993), Rimm et al. (1996) and Yochum et al. (1999) applied compositional data for three flavonols and two flavones in fruits, vegetables and beverages to Dutch and U.S. food consumption surveys to estimate dietary flavonoid intake. The compositional data used
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for these studies reported the flavonoid content of Granny Smith and Golden Delicious apples as 0.024 and 0.025 mg/g, respectively (Hertog et al. 1992). However, the procyanidin content for these apples were determined in this study to be 1.01 and 0.491 mg/g, respectively. Thus, the total flavonoid content for apple was significantly underestimated and possibly could have been for other foods in this study as well. Similarly, Arts et al. (1999) examined the dietary flavan-3-ol intake in a Dutch population and found that chocolate and tea contributed 20 and 55%, respectively. However, their methodology only accounted for the monomeric flavan-3-ol derivatives and neglected the oligomers that are abundant in chocolate and only minor in tea (Lazarus et al. 1999b). Importantly, the use of select flavonoids to infer epidemiological relationships to health and disease could be confounded by the fact that different flavonoids may exhibit varying physiological effects. Extensive research has been conducted investigating the relationship between flavonoid structure and biological activity, especially as it relates to antioxidant properties, and found that the hydroxylation pattern is a key factor (Rice-Evans et al. 1996). In contrast, little is known regarding the impact of the degree of polymerization of the procyanidins and their biological properties. Recently, Arteel and Sies (1999) and Bearden et al. (2000) investigated the effectiveness of cocoa procyanidins in vitro to scavenge peroxynitrite and inhibit LDL oxidation, respectively, and both found that antioxidant activity was influenced by oligomeric size. Also, Mao et al. (1999) studied the ability of the procyanidins to modulate interleukin-2 in vitro and found the higher oligomers inhibited interleukin-2 expression in stimulated cells, whereas the monomer had no effect. These preliminary studies suggest that the physiological effects of the procyanidins may be affected by the degree of polymerization, and therefore, the oligomeric composition should be included when determining the flavonoid content of foods. In conclusion, the current study demonstrated that commonly consumed foods and beverages, including chocolate, apples, cranberry juice and wine, contain substantial amounts of procyanidins. In addition, these results suggest that significant variation in procyanidin content can exist within these foods. This research represents an initial investigation to understand which foods may contribute most to the dietary intake of procyanidins. However, a more extensive sampling plan would have to be implemented to compile a comprehensive database of foods and beverages that contain procyanidins. Given that these compounds have exhibited the ability to influence health in in vitro and in vivo studies, it would be important to use this compositional data in food consumption surveys to define intake levels in populations and to determine whether epidemiological associations with health and disease end points may be present. ACKNOWLEDGMENT The authors gratefully acknowledge F. Plog for assisting in sample preparation.
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