A Comparison of Methods For Quantifying Oligomeric Proanthocyanidins From Grape Seed Extracts

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1 A Comparison of Methods For Quantifying Oligomeric Proanthocyanidins From Grape Seed Extracts ANDREW L. WATERHOUSE ~*, STEVE IGNELZI 2, and JOSEPH R. SHIRLEY ~,3 Grape seed extract (GSE) has become popular in recent years as a nutritional supplement that possesses antioxidant activity. These extracts contain a heterogeneous mixture of monomers, oligomers, and polymers composed of proanthocyanidin (or flavan-3-ol) subunits. The common colorimetric methods for analyzing the procyanidin concentration and/or composition of grape seed extracts and products containing it can give only crude information on the distribution of the sizes of the components. A normal phase HPLC method has proven to be very applicable to analyzing grape seed extract material and products thereof, and monomer, oligomer (2-7 subunits) and polymeric (8-24 subunits, and ~24+ subunits) fractions can be discriminated. Values ranged from 5% to 30% for monomers, 17% to 63% oliogomers, 11% to 39% polymers and 2% to 50% for the large (24+) polymers. When specific attributes can be defined for particular size fractions, this method can provide the information needed to compare different samples for their relative potential effect. KEY WORDS: grape seed extract, oligomeric procyanidins, antioxidant, HPLC, polyphenol, tannin, flavan-3-ol Red wine consumption has been inversely correlated to coronary heart disease in many epidemiological studies, such as Renaud's French Paradox study [6]. A proposed mechanism for this correlation involves the antioxidant activity of some of the phenolic compounds found in red wine, the flavanoids [1]. However, absorption of these compounds is probably limited by molecular size, as no other large molecules are absorbed without a specific transport mechanism [9]. This appears to be the case because tannin-containing foods are not toxic, yet the polymeric tannins are known to be quite toxic when absorbed directly into the blood stream [12]. The major flavonoids found in red wine are the flavan-3-ols and their oligomers and polymers, and these arise by extraction of Vitis vinfera skins, seeds and if present, stems, during winemaking [11]. The specific phenolic compounds found in grape seeds are the catechol (dihydroxy) forms of the flavan-3-ols: the monomeric catechins, including catechin, epicatechin, and epicatechin gallate (Fig. 1) and the oligomers, procyanidins, and polymers thereof (Fig. 2). Grape seeds do not contain any of the gallocatechins (3"4"5"-trihydroxy B-ring) or the oligomers and polymers thereof, the prodelphinidins [7]. Grape seeds also do not contain any of the red flavonoid pigments known as anthocyanins, nor the flavonols. There are numerous methods that could be applied to the analysis of the phenolic material in grape seed extract (GSE). Some test only for the presence of phenolic substances, while others specifically test for the presence of flavan-3-ol functional groups, while yet oth- 1Department of Viticulture and Enology, University of California, Davis, CA 95616; 2Industrial Labs, Denver, CO; and 3Current Address: Trinchero Family Estates, St. Helena, CA. *Corresponding author [Fax: ; alwaterhouse@ ucdavis.edu]. Acknowledgements: The authors gratefully thank Second Nature Technologies and Polyphenolics LLC for their support, and we would like to remember Barry K. Saltzman for his enthusiastic interest and encouragement. Manuscript submitted for publication 11 August 1999; revised 2 August Copyright 2000 by the American Society for Enology and Viticulture. All rights reserved. 383 ers separate the mixture based on different chromatographic behaviors. The traditional industry-standard methods to analyze the content of grape seed extracts have employed colorimetric tests, which produce a single result for total procyanidin content, sometimes combined with a polymer precipitation test. While these methods can provide a reasonable estimate of the total flavanol content, they discriminate only marginally between materials with different size or degrees of polymerization distributions, and thus their analytical value for determining the size distribution of the procyanidins in a sample is extremely limited. Colorimetric methods: The Bates-Smith method is currently a common procedure for the analysis of analysis of grape seed extracts. It utilizes the unique ability of the proanthocyanidins, flavan-3-ol oligomers and polymers, to produce cyanidin and/or delphinidin under acid hydrolysis conditions and thus a unique red color. The term proanthocyanidin is derived from this anthocyanidin production. Procyanidins yield cyanidin from catechol B-rings and prodelphinidins (an insignificant from in grape seeds) yield delphinidin from gallol B-rings. The red color is quantified by absorbance and the results are expressed as 'procyanidolic value'. This number is the change in extinction coefficient at 550 nm of a one percent solution (w/v) of the product after its acid hydrolysis and conversion to cyanidin (change in absorbance X 100). For fairly pure grape seed extract, this number is often ca. 80 to 100 and is commonly misinterpreted as meaning percent procyanidin: i.e., the percent of sample that is procyanidolic material. The Porter method generates a Porter value unit or 'PVU', which is also an extinction coefficient measured as for the Bates-Smith method [4]. It is based on the Bate-Smith method with the addition of ferric ammonium sulfate as a catalyst for the acid hydrolysis. This improves the conversion of the procyanidins to cyani-

2 ~ 384- WATERHOUSE et al. HO "q ffl'~ Catechin I HOCO::,,~ o ~ O H Epicatechin Gallic Acid Epicatechin-3-O-gallate Fig. 1. The three monomeric subunits of the procyanidins and gallic acid. din and significantly reduces the variability of the assay. The Folin-Ciocalteau method uses a phosphomolybdic-phosphotungstic reagent to react with phenolic materials and produce a colorimetric response at 765- nm [13]. The response of the Folin-Cioucalteau assay is attenuated by prior oxidation of the sample. While there are potential interferences to this method, they are well described and are not found to an appreciable extent in grape seeds. They include other reducible substances such as reducing sugars, ascorbic acid, and protein (and sulfur dioxide when phenolics are present). The colorimetric response is compared to a standard curve based on gallic acid, and the final results are expressed in gallic acid equivalents (GAE). This is a general method for measuring the mass of phenolics in grape seeds; catechin gives a 'per gram' response 1.10 times that of gallic acid and epicatechin's relative response is 0.97 [10]. Thus the Folin- Ciocalteau assay also responds to the monomeric flavan-3-ols as well as the proanthocyanidins. It also responds to all other phenolic compounds depending on the number of phenoxyl groups. A method to separate condensed tannins by size has been described by Rigaud et al., and this procedure provides direct data on a sample's size composition [8]. It is a normal phase HPLC method which uses an unmodified silica column with an acidic dichloromethane mobile phase and a gradient of increasing methanol. The procyanidins are separated based on the hydrogen bonding interactions between the silica hydroxyls and the phenolic hydroxyls with the larger the molecules having more extensive interactions and thus slower elution. Molecular size estimates are generated from a chromatogram of unfermented cacao bean extract. This extract is a relatively simple mixture of epicatechin and the oligomers and polymers thereof. H Epicatechin?H HO o,,,j~ ;HI :! il O" Extension Units HO O... ~ n Epicatechin /,, I 7]'- ~ \ Gallate un ~ O \ HO ~0... Epicatechin " Catechin,,~~~' Terminal -,~ v " Unit Fig. 2. An example procyanidin, showing each possible subunit and the distinction between extension and terminal units. Only 4-,8 linkages are shown. Adequate resolution to approximately 10 subunits can be achieved. The retention time data can be used to calculate a function that reflects the relationship between the number of subunits and the retention time of the molecule. This formula can then be used describe the amounts of the different monomer, oligomer and polymer fractions of more complicated flavan-3-ol mixtures, including GSE. We will discuss this chromatographic procedure and other methods of analysis in more detail. We will also compare the results of this procedure with two standard colorimetric assays. Materials and Methods The GSEs tested were provided by various commercial and academic sources. One of the powders was extracted in 1967 from California Chenin blanc seeds. The grape variety is unknown for the other samples. Of the other nine powders, two were of Italian origin, one is actually an extract of French maritime pine bark, one is from a French grape seed extract, and the rest are from California or from anonymous suppliers. Three of the four pills tested were purchased in from retail stores in California and one was purchased at a pharmacy in France. The Folin-Ciocalteau reagent was purchased from Sigma (St. Louis, MO). HPLC grade solvents and other reagents were purchased from Fisher Scientific (Pittsburgh, PA). Porter method: Samples were dissolved at 1% w/v in 95% methanol and treated with hydrochloric acid in the presence of ferric salts as described previously [5]. Results are expressed as the change in the extinction

3 GRAPE SEED EXTRACTS m 385 k-- i i2ol !_ ' ' ]10, il J ' ' 4o;i' r 40 : 70, Retention Fig. 3. Normal-phase HPLC chromatogram of fresh cacao bean extract with peaks labelled with degree of polymerization. coefficient of the solution at 550 nm X 100. Folin-Ciocalteau (FC) method: The solid was dissolved into methanol and analyzed as described [ 13]. Standards were prepared from gallic acid, mixed with Folin-Ciocalteau reagent; later sodium carbonate was added, and the absorbance was measured at 765 nm. The final results are expressed as percent GAE calculated by measuring the GAE response from a sample, dividing by the weight used in the analysis, and multiplying by 100. Thus, a sample which gives the same response as gallic acid on a mass basis would have a result of 100% GAE. Normal phase HPLC method: Samples were dissolved in methanol at 1000 mg/l and filtered with through a PTFE 0.45-gm membrane. Samples (25 gl) of these solutions were injected for analysis by HPLC using a method previously described [8]. Briefly, the stationary phase was silica gel and the mobile phase dicholormethane containing small amounts of water and formic acid with a methanol gradient. Peaks were detected by UV absorbance at 280 nm and spectra were obtained on peaks to confirm their flavan-3-ol identity. This analysis was carried out on a Hewlett Packard 1090 system (Palo Alto, CA) with a diode array detector using a LiChrosphere Si-60 (5 gm, 250 X 4 mm) column (EM Science, Gibbstown, NJ). Catechin was chosen as the reference standard. The average response to 100 ppm catechin was 1327 milli-absorbance units X seconds area. Ranges of procyanidin DP sizes were selected for peak area summation based on the retention times of a fresh cacao sample with easily identifiable DP sizes (Fig. 3). Time Results and Discussion Colorimetric methods: The Bate-Smith method of analysis for proanthocyanidins can result in variable conversion efficiencies. This has been attributed to variations in the concentrations of metals, which are known to have catalytic effects on the acid-hydrolysis reaction. As mentioned before, the Porter method improves on the Bate-Smith assay by providing an ample amount of ferric ammonium sulfate to maximize the conversion of the proanthocyanidins. Surprisingly the Bate-Smith method is still widely used in the analysis of commercial grape seed extract. In many cases, the Bates-Smith procedure is combined with a salt precipitation test to detect the amount of. highly polymerized material. While this combination of procedures does... I ' " provide some information about the av- erage molecular size of the analyte, the ability of the method to define the molecular size of the constituents in the sample is quite limited. Neither the Bate-Smith nor the Porter methods respond to the catechins, the monomeric flavan-3-ols, nor do they respond to the terminal units of the oligomeric or polymeric flavan-3-ols. Neither substrate is able to generate the C-4 carbo-cation necessary for the conversion to an anthocyanidin. Thus the monomeric catechins give no response, dimeric procyanidins give a 50% response, trimeric procyanidins give a 66.6% response, and very large polymers give close to a 100% response. This results in a relatively low response from grape seed extracts with low average DP, while samples with very high average DP values give a high response. Thus, GSE which has a high concentration of the oligomeric substances, thought by some to have more physiological activity, actually has a weaker response than material which is largely polymer. A major advantage of the Porter method (and of the Bate-Smith method) is its relative lack of interference from other common plant components, since only flavan-3-ol containing samples can produce anthocyanidins under the defined conditions. Delphinidin gives a different proportional response than cyanidin at 550 nm, but as there are no prodelphinidins in grape seed extracts, this is not a concern here. Our analyses of commercial and experimental grape seed extracts had a range from 202 to 320 PVU, with an outlier at 15. Analyses of prepared supplement doses have responses from 117 to 324 PVU (Table 1). These latter values are dependent on the assumption that the labeled concentration was accurate: i.e., a "dose" of 100 mg/pill was the actual amount present. In addition, replicate analysis of one sample (A1) over 10 different days indicated a coefficient of variation of 10%. For the Folin-Ciocalteau method, we compared the response of the powders to pure gallic acid and express the results as a percentage of the response of gallic

4 ~ WATERHOUSE et al. Table 1. Porter and Folin responses for the powdered procyanidin grape seed extracts as well as the grape seed extract supplement pills. Not all samples replicated. See text for description of the variability of the analysis. Porter Folin (Porter value units) (% Gallic acid equivalents) Powders A A B C D E F Pills G H X M N P Q acid. In our samples, the same powders gave responses from 46% to 111% GAE (Table 1). Replicate analysis of one sample indicated a 6.4% coefficient of variation on this assay. Surprisingly, the responses by the Folin and Porter methods do not have a high correlation, it being less than 0.4. It appears that some samples with high Folin and low Porter values have phenolic material that is not Porter-responsive, such as low molecular weight proanthocyanidins and monomers or non-flavan-3-ol phenolic material. The samples with high Porter values and low Folin values are likely to have high average molecular weight and be partially oxidized. This is very pronounced in a 30-year-old sample (F) that has a very low Folin value (46.0% GAE), but a relatively high Porter value (264). HPLC methods: Chromatographic methods by definition separate the components of a mixture. Thus, instead of having one result from an analysis, ideally there would be one result for each chemically distinct component. However, GSE is such a complex mixture that this cannot be achieved today. The term 'oligomer' is defined functionally by the largest molecule in a mixture with increasing degrees of polymerization (DP) that can be separately identified. Because this definition is functional, it is variable, and in the case of procyanidins varies between 7 for GSE and 11 for cocoa tannins using the normal phase HPLC method described herein. Larger molecules that have higher DPs and thus elude resolution or separation and are defined as polymers. Polymeric flavan-3- ols are referred to as condensed tannins. The term tannin refers to a material's ability to 'tan' animal hide, which is done by reacting with the proteins of the animal's skin. The polymers are known to be effective in this regard, but monomeric, and presumably small oligomers, are not useful. Grape seed extract, (GSE) usually contains all sizes of the flavan-3-ols, from monomers to oligomers and polymers. The oligomers and polymers are generally formed by linkages between the 4 and 8 (4-~8) (Fig. 2) and/or 4 and 6 (4-~6) position between adjacent subunits, however, other linkage types are known [2]. Reverse phase high performance liquid chromatography (HPLC) procedures can provide excellent baseline resolution for flavan-3-ol monomers, dimers, and some trimers, but it is not capable of separating the larger molecules [14]. These methods separate according to polarity differences rather than molecular size so further information about the identity of the components requires additional testing, such as comparison with a standard. This is particularly difficult with flavan-3-ol oligomers and polymers, since only the monomers and a very few dimers are available. The small number of standards is a particularly vexing problem because there are an immense number of possibilities of procyanidin polymers even at moderate DP due to the variety of subunits and linkages possible. In GSEs there are three possible subunits for the polymers and two possible linkages. The number of possible combinations quickly becomes astronomic as DP increases to moderate lengths. Since the polarity of the procyanidins all falls into a relatively small range, it is only reasonable to expect that a few dozen could be separated by RP-HPLC, since all have similar polarity. Because there are three possible subunits in GSE -- catechin, epicatechin, and epicatechin gallate -- a polymer of DP = n will have 3 n possible subunit combinations. This is multiplied times the number of different linkages, two due to 4-~8 and 4-+6 bonds, so a polymer with a DP = n will have 2 (n-l) different linkage combinations. Thus, the total number of possible combinations assuming the standard subunits and linkages for a polymer of DP = n is 3 n 2 (nz), SO a simple dimer has 18 possible forms, while a longer decamer has roughly 30 million possibilities. GSE appears to have a distribution of DP averages between 1 and 30 units, so the number of individual compounds is virtually impossible to measure. Consequently, attempts to separate these mixtures into their individual components will not be successful. In practice, by RP-HPLC it is usually possible to distinguish peaks of the monomers and a few of the smaller oligomers, but the larger material appears to elute as an undifferentiated broad peak [14]. Gel permeation chromatography (GPC) is a chromatographic method that separates based on molecular size and has been used to separate procyanidin mixtures from plant extracts. Unfortunately, for the procyanidin mixtures, GPC has some significant drawbacks. First, the resolution is fairly low; i.e., it doesn't discriminate to the extent of individual DP sizes. Second, it has been necessary to derivatize samples prior to analysis, so this method is quite tedious and has

5 GRAPE SEED EXTRACTS m 387 been used very infrequently with procyanidin mixtures [15]. Recently, a preliminary report indicated better performance with multiple columns and strong polar aprotic solvents, which eliminate the need for derivatization [3]. Nonetheless, the normal phase method has several advantages over gel permeation chromatography. The unmodified silica column has a higher apparent efficiency, and it is relatively inexpensive [8]. Simple procyanidin mixtures of varying DP are well separated with this method. For instance, cacao procyanidins are composed exclusively of epicatechin and one linkage type; thus at each DP there is only one component, i.e., one peak for the monomers, one peak for the dimers, one peak for the trimers, etc. So, the chromatogram is a series of peaks of components of increasing size, as in Figure 2. The peaks are well resolved up to about DP = 10 with the system that was employed. The retention times of these peaks can be used to calibrate retention time vs. molecular size. Thus, the sizes of the components of complex mixtures such as GSE can be ascertained from this "size" calibration. As epicatechin comes out before catechin and epicatechin gallate, the range of monomers can be described as the beginning of epicatechin elution to the beginning of the epicatechin dimer (E-E) elution. Similarly the range of dimers can be described as the beginning of E-E elution to the beginning of E-E-E elution, and so on. Grape seed extracts are significantly more complex as they contain so many different subunits as shown in Figure 4. Very approximate molecular size estimates of the larger fractions can be accomplished by developing a function based on the retention time of the first 10 polymers in the cacao chromatogram. The retention time can be extrapolated for a given number of subunits. Using this extrapolation technique, it is possible to estimate the absorbance due to monomer, dimer, trimer, tetramer, etc., up to 24 subunits based on the function y = 0.38 X e -s 4x where x is the retention time observed and y is the approximate size in number of subunits. It should be stressed that this template refers to non-galloylated subunits, i.e., polymers composed of only catechin and epicatechin subunits. Each gallate group appears to increase retention by about one half mau! ~ 10 2 ~0 30 -z~() ~0... i~0 ~ min - 7 -"... " ~- Fig. 4. Normal-phase HPLC chromatogram of one GSE sample showing the estimated degree of polymerization cut-off points for the integration of the different-sized fractions listed in Table 2. as much as an equivalent flavonoid unit would. This is expected because gallates have three additional phenolic hydroxyl groups. Further analysis of the chromatogram can take the form of dividing the chromatogram along arbitrary size (time) points for quantification of the amount of material in a certain size range. Here we have arbitrarily divided the chromatograms into the sizes of monomers, dimers to heptamers (oligomers), octamers to 24-mers (polymers) and material >24 units. It is clear that the dividing line for 24 units is not very precise in terms of molecular size, but such a dividing line is quite useful for comparisons of samples in one set. The concentration of different sized fractions is measured by summing the absorbance X time area of a particular time (size) range. This can be converted to catechin equivalents by calibration with catechin. The data is then expressed in milligrams per gram (or liter depending on the physical state of the sample) catechin equivalents (CE) for each of the polymeric groups/classes chosen. It should be noted that gallic acid will co-elute with epicatechin, probably because they have similar size/ hydrogen bonding ratios. Gallic acid has three phenolic hydroxyls and a carboxylic acid group (188 Daltons) and epicatechin has four phenolic hydroxyls and one alkoxy group (290 Daltons). If gallic acid is present, it will add to the procyanidin monomer count, and gallic acid is not a procyanidin. If necessary, this interference can be accounted for by determining the gallic acid concentration using reverse phase HPLC, as well as the flavan-3-ol monomer (catechin, epicatechin and epicatechin gallate) levels themselves. As noted above, the presence of the gallated units causes an aberration in the elution time of molecules with a specific degree of polymerization. The molecular weight of catechin and epicatechin is 290 Daltons. The gallation of epicatechin adds 170 Daltons to the molecule along with three more phenolic hydroxyls to interact with the silica column. In every instance the proportion of gallated compounds of the individual polymers increase as the DP increases. Thus the apparent amount with a particular DP value can increase or decrease, with the notable exception of the dimers. This is due to the added molecular weight of the gallated procyanidins in the dimer of epicatechin gallate (EG-EG). In this case the two gallic acid moieties together add up to the molecular weight of 340 Daltons, slightly more than one epicatechin or catechin subunit. Thus the EG-EG dimer actually elutes in the trimer region, thus lowering the count of dimers and increasing the count of trimers. The dimers (most if not all) with only one gallate would elute in the dimer region or in-between-note the complex peak pattern of GSE compared to cacao extract. For instance, there will be trimers having two or

6 ~ 388- WATERHOUSE et al three gallate moieties causing them to elute in the tetramer region, thereby decreasing the trimer count and increasing the tetramer count. There will be tetramers with two, three or four gallate moieties that would cause them to fall into the pentamer and possibly even hexamer region, and so on. However in the case of the dimers, there is not a gallated moiety that will cause an increase of dimer count. The only gallated monomer (epicatechin gallate) elutes in the monomer region and not the dimer region. These ambiguities in ascertaining degree of polymerization from retention time would be just as problematic with any separation based on size, such as gel permeation chromatography. While catechin has the same extinction coefficient at 280 nm as epicatechin, epicatechin gallate is significantly different, with a response about 2.11 times as high as that of the non-galloylated species [8]. This can lead to higher overall responses from GSE's with higher degrees of galloylation. The difference in overall concentration can appear excessively high when compared to procyanidolic values, which do not respond to the gallate moieties at all. Thus, the total amount of catechin equivalents is typically higher than 1 g/g because the gallated components have a higher response than the non-gallated catechin standard. The procedure is reproducible with the variability being similar to chromatographic procedures. When an epicatechin standard was analyzed repeatedly (9 Table 2. Integrated peak areas (280 nm) of material eluted during specific DP ranges based on elution times of cacao standard. Replication using different solvent for solution gave similar results (+ 10%); replication with identical solvent gave nearly identical results (+ 3%). Normal phase HPLC results Approximate DP ranges Sum Powders percentage (%) mg/g A A B C D E F G H X* Pills M N P Q Ave Max Min * Sample X not included in the statistical analysis. times), the coefficient of variation was less than 4%. The quantification of the oligomer and polymer fraction is more variable, largely due to the variation in the manual integration process. Based on 12 replicates the average variation in the oligomer and polymer peaks was 8%. The samples A1 and A2, from the same supplier, had a predominant oligomer (DP = 2-7) fraction with decreasing amounts of polymer and high polymer (Table 2). From the complexity of the chromatogram, i.e., many peaks in each DP range, it appeared that gallate esters were present in nearly all samples. Sample B had a very high oligomer fraction, 57% of the total, and the largest proportion amongst all the powders. The Pill sample M, which was apparently produced using material from the same manufacturer, had a similar high proportion of the oligomer fraction, although the other fractions were not as similar. Sample B also had the highest total response. Sample C had only 32% of the total in the oligomer fraction and the largest amount (39%) in the polymer fraction (DP 8-24). Sample D was not grape seed extract, but pine bark extract. Its chromatogram was distinctly different from the others, being much simpler, since pine bark lacks the gallate esters. Otherwise, it was not remarkable except for the low total amount. However, the lack of the highly light-absorbing gallate esters would result in the smaller peak areas seen. Sample E was of median character, having proportions and a total close to the average. Sample F was quite notable for the very low amount of monomer and oligomer, and the high proportion of very large polymer. This sample also had a very low Folin response and high Porter response, suggesting that it was highly oxidized. This is not surprising since the sample was over 30 years old and had been stored at room temperature over that time. Sample G had a high proportion of monomers and oligomers, while sample H had higher proportions of the polymer and large polymer. Sample X did not have measurable amounts of any identifiable components, so it was not considered in the comparisons or the determination of averages, etc. Among the pill samples, M had a high proportion of monomers at 26%, while the others had lower amounts of monomers ranging from 13-16%. Sample M also had a very large fraction in the oligomer form, over 60% compared to the others, all in the 40% range. Conversely, M had less than 13% in the two polymer fractions, while the others all had about 40% in these forms. In terms of overall quantity, the pills were similar except for Q, which had half as much material absorbing light at 280 nm. With some notable exceptions there appeared to be some trends in the relationships between the different analyses of the various samples. The samples that have relatively low DP profiles by HPLC tend to respond more strongly to the Folin-Ciocalteau assay and relatively more weakly on the Porter assay. Lower DP flavan-3-ols react well with the Folin-Ciocalteau re-

7 GRAPE SEED EXTRACTS m 389 agent, giving a response that is very similar to gallic acid on a mass basis. The samples that have relatively higher DP profiles tend to respond less to the Folin- Ciocalteau assay, possibly as a result of oxidation. Conclusions Due to complexity of the composition of grape seed extracts and related proanthocyanidin extracts, existing colorimetric methods cannot provide adequate information on their composition if there is a need to know the true oligomer content of the samples. The acid-based anthocyanidin-producing assays respond indiscriminately to proanthocyanidin content regardless of molecular size, and if there is any discrimination it is to provide a larger response to the high molecular weight polymers. The normal phase HPLC method provides an accurate and reproducible means to assess the true oligomer content of a grape seed proanthocyanidin sample. Such a method will provide the industry and researchers with a workable tool to analyze the chemical composition of these preparations. Literature Cited 1. Frankel, E. N., J. Kanner, et al. Inhibition of oxidation of human lowdensity lipoprotein by phenolic substances in red wine. Lancet 341: (1993). 2. Guyot, S., J. Vercauteren, and V. Cheynier. Structural determination of colourless and yellow dimers resulting from (+)-catechin coupling catalysed by grape polyphenoloxidase. Phytochemistry 42:1279 (1996). 3. Kernan, M. R., and D. Karr. Size exclusion chrmoatography/mass spectrometry and MS/MS fragmentation of proanthocyanidin polymers using electrospray ionization. In: Third Tannin Conference. G. G. Gross, R. W. Hemingway, and T. Toshida (Eds.). Phytochemical Connections, Alexandria, Louisiana (1998). 4. Porter, L. J. Flavans and proanthocyanidins. In: The flavonoids. Advances in research since J. B. Harborne (Ed.). pp Chapman and Hall, London (1988). 5. Porter, L. J., R. Y. Wong, et al Conformational analysis of flavans: proton NMR and molecular mechanical (MM2) studies of the benzpyran ring of 3',4',5,7-tetrahydroxyflavan-3-ols: the crystal and molecular structure of the procyanidin (2R,3S,4R)-3',4',5,7-tetramethoxy-4-(2,4,6- trimethoxyphenyl)flavan-3-ol. J. Chem. Res. Synop. (3):86-87 (1986). 6. Renaud, S., and M. de Lorgeril. Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet 339: (1992). 7. Ricardo da Silva, J. M., J. Rigaud, et al Procyanidin dimers and trimers from grape seeds. Phytochemistry 4: (1991). 8. Rigaud, J., M. T. Escribano-Bailon, et al Normal-phase high-performance liquid chromatographic separation of procyanidins from cacao beans and grape seeds. J. Chromatogr. A 654: (1993). 9. Sherwood, L. Human Physiology. West Publishing, New York (1993). 10. Singleton, V. L. Analytical fractionation of the phenolic substances of grapes and wine ans some practical uses of such analyses. In: Chemistry of Winemaking. A. D. Webb (Ed.). pp American Chemical Society, Washington, DC (1974). 11. Singleton, V. L. Grape and Wine Phenolics; Background and Prospects. In: Proc. Symposium Univ. Calif. Davis, Grape & Wine Centennial, pp Department of Viticulture and Enology, University of California, Davis, CA (1982). 12. Singleton, V. L.,. and F. H. Kratzer. Toxicity and related physiological activity of phenolic substances of plant origin. J. Agric. Food Chem. 17: (1969). 13. Singleton, V. L., and J. A. Rossi. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16: (1965). 14. Waterhouse, A. L., S. F. Price, and J. D. McCord. Reversed-phase high-performance liquid chromatography methods for analysis of wine polyphenols. Metho. Enzymol. 299: (1998). 15. Williams, V. M., L. J. Porter, and R. W. Hemmingway. Molecular weight profiles of proanthocyanidin polymers. Phytochemistry 22: (1983).

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