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OLIGOMERIC AND POLYMERIC PROCYANIDINS FROM GRAPE SEEDS

CORINNE PRIEUR, JACQUES RIGAUD, VERONIQUE CHEYNIER and MICHEL MOUTOUNET

I.N.R.A., Institut des Produits de la Vigne, Laboratoire des PolymZres et des Techniques Physicochimiques, 2 Place Viala, 340K1 Montpellier cedex, France

(Receioed 6 Jonuory 1994)

Key Word Index- Yitis oinifera; Vitaceae; grape.; procyanidins; condensed tannins; flavan-3-01s.

Abstract-Five tannin fractions were isolated from grape (Yitis oinijizra) seeds. HPLC analysis of the degradation products released by thioacidolysis showed that all fractions consisted of catechin, epicatechin and epicatechin gallate units. Epicatechin was the major component in the extended chain. Catechin was more abundant in terminal units than in extension units. The proportion of galloyllated units varied from 13% to 29% as the M, increased. Mean degrees of polymerization ranging from 2.3 to 15.1 (by thiolysis) and from 2.4 to 16.7 (by GPC) were determined.

INTRODUCTION

Condensed tannins (proanthocyanidins) are essential grape constituents, responsible for part of the organolep- tic properties of wines and, as such, have been extensively studied. The structures of grape dimeric and trimeric proanthocyanidins have been recently elucidated [l-8]. They mostly consist of (+)-catechin and (-)epicatechin units, linked by C-4-C-8 or C-4-C-6 bonds and some- times esterified by gallic acid on the epicatechin moiety(ies), although prodelphinidins, i.e. (epi)gallo- catechin oligomers [3], and catechin-3-0-gallate derivat- ives [7J have also been reported.

However, the lower M, procyanidins are usually pre- sent in plant tissues in relatively low concentrations compared to that of larger oligomers and polymers [9]. Besides, astringency and tanning properties depend on the number of 0-dihydroxyphenolic groups on the tannin molecule [lo, 1 l] and increase both with the degree of polymerization [lo, 12-153 and with the rate of galloyl- ation [14, IS]. The purpose of the present work was to isolate and characterize the structure of oligomeric and polymeric tannins from grape seeds.

RESULTS AND DKXXJSSION

Among the numerous techniques developed to separ- ate and analyse condensed tannins, the use of nonnal- phase HPLC has recently bee.n shown to achieve elution of procyanidins according to their degree of polymeriza- tion, without derivatization [ 163. Condensed tannins isolated from grape seeds (Vitis oinifera, var. Alicante Bouchet) were analysed by using this method and eluted in increasing M, order as described by Rigaud et al. [ 163. Replacement of formic acid (2% in the original solvent system) by TFA (0.005%) was found to minimize hydroly-

tic degradation and subsequent rearrangements during concentration of the collected fractions. In addition, the elution gradient was slightly modified in order to improve resolution of the larger oligomers and polymers (5 units and above), which appeared as two broad, unresolved humps in the previous work. Application of this chro- matographic technique on the preparative scale yielded five fractions (I-V), which only slightly overlapped when injected on the analytical HPLC system after a second purification step.

The gross structure of a proanthocyanidin polymer is characterized by the nature of its constitutive extension and terminal flavan-3-01 units and its degree of poly- merization (DP), i.e. average number of units in the polymer. Among the methods available to investigate the structures of condensed tannins, acid-catalysed degrada- tion in the presence of toluene-cx-thiol [ 171 is of particular interest as it distinguishes between extension units (re- leased as their benzylthioethers) and terminal units (re- leased as flavan-3-01s). Analysis of the resulting solution by ‘H NMR [18] or HPLC [19, 203 gives access to the composition and average M, of the proanthocyanidin. Another advantage of the method is the retention of the ester moieties of 0-gallates [21, 223. Also, the HPLC method developed by Rigaud et al. [20] can be applied directly after thiolysis without sample purification and makes it possible to work with very small amounts of substrate.

The five fractions obtained by preparative HPLC were analysed using this method. Pure solutions of (+)- catechin, ( - )-epicatechin, ( - )-epicatechin-3-0-gallate and of their benzylthioethers were similarly incubated with the thiolysis reagents to evaluate the extent of epimerization, as reported by Porter [21]. Under our experimental conditions, only epicatechin and catechin were epimerized. The rate of conversion was ca 15% for

781

782 C. PRIEUR et al.

epicatechin and 4% for catechin, so that the proportion of catechin in terminal units may be slightly overestima- ted. The thiolytic products of trimers Cl and Bl-3-0- gallate were also quantitatively analysed using this pro- cedure. Thioacidolysis of Cl yielded epicatechinbenzyl- thioether and epicatechin in the ratio 2.1: 1 and that of BI-3-O-gallate released epicatechin-3-O-gallate benzyl- thioether and catechin in the ratio 1.08: I. No phloroglu- cinol was detected after thiolysis, signifying that cleavage of the C-4 to C-10 bond reported by McGraw et al. [23] did not occur under our relatively mild conditions. Thus, the applied thioacidolysis procedure was shown to be reliable.

Catechin, epicatechin and epicatechin-3-O-gallate were the only monomers released, indicating that grape seed tannins were procyanidins. Complete acid degradation of the crude procyanidin extract released cyanidin but no delphinidin.

The retention times for catechin, epicatechin and epi- catechin gallate were, respectively, 13.6, 17.4 and 22.1 min. Those of the benzylthioethers were 33.9 and 35.4 min for catechin, 37.1 min for epicatechin and 39.9 min for epicatechin gallate. Epicatechin-3-0-gallate and its benzylthioether were identified by hydrolysis with tannase followed by HPLC analysis of the released degalloylated compound, respectively, epicatechin and epicatechin benzylthioether. Identities of the four benz- ylthioethers were confirmed by reduction with Raney nickel, followed by HPLC analysis of the resulting flavan- 3-01s [S, 203. No gallic acid was released by thiolysis, confirming that the ester moieties of 0-gallates are re- tained [8,21,22].

The yield from thiolysis degradation was calculated as the ratio between the summed concentrations of the released units (flavan-3-01s and benzylthioethers) and the initial procyanidin concentration. The results obtained on the fractions purified by preparative HPLC, showed that the first eluting ones were highly contaminated by non UV-absorbing material. Therefore, further puritica- tion by chromatography on Sephadex LH20 was per- formed, yielding for each original fraction a water frac- tion and a methanol fraction. The water effluents ob- tained from fractions I-IV gave no colouration with vanillin-HCI or complete acid hydrolysis and released no flavan-3-01 derivative when submitted to thiolysis. Hence, they were shown to contain no procyanidin and dis- carded. On the contrary, the water effluent of fraction V (Vb) contained condensed tannins which were excluded from Sephadex LH20 and, thus, presumably larger than the adsorbed tannins; this was further analysed along with the five methanol effluents (la-Va). The yield from thiolysis degradation determined for each of the six purified fractions varied from 70% to 65%. It was similar to that calculated using trimer Cl (72.2%) and, thus, considered acceptable.

The compositional data obtained by thiolysis degrada- tion of each procyanidin fraction is presented in Table 1. Catechin, epicatechin and epicatechin-3-O-gallate were found in each fraction both as extension and terminal units. Epicatechin predominated in the extended chains

Table I. Composition of grape seed procyanidin

fractions, determined by HPLC following thiolysts

degradation

Terminal units Extension units

Fraction Cat EC EcG Cat EC EcG

la

IIa

tIIa

IVa

Va

Vb

15.5 17.7 11.0 17.8 35.8 2.2

IO.1 7.9 9.8 13.8 49.5 8.9

5.9 4. I 8.5 8.6 53.4 19.5

4.2 2.6 6.0 8.4 56.5 22.2

2.5 1.4 3.0 6.6 59.3 27.2

1.8 1.1 2.4 6.2 60.5 27.9

Cat, EC, EcG are the abbreviations for catechin,

epicatechin and epicatechin gallate units. Extension

units were measured as benzylthitwther derivatives.

All amounts represent relative concentrations (in

mols). For convenience of comparison, total concen-

tration in each fraction was fixed at 100.

and was the major component of all tannin fractions. Catechin was relatively more abundant in terminal units than in extension units.

Structural characteristics (i.e. cixtrans ratio, mean degree of galloylation, M, and mean DP), calculated from the results of thioacidolysis and GPC analyses, for the six procyanidin fractions are summarized in Table 2.

The extent of galloylation increased from 13.2%, in fraction I, to 18.7% in fraction II, and reached cu 30% in the last ones. The amount of gallic acid released by enzymatic degradation using tannase was systematically lower than expected from the amount ofgalloylated units detected after thiolysis. The observed discrepancy in- creased from fraction I to fraction V, suggesting that galloylated moieties become less and less accessible to enzymatic hydrolysis as the polymer size increases. This may also be due to inhibition of the enzyme by larger M,

tannins. To our knowledge, estimation of the degree of galloylation in higher M, condensed tannins is reported here for the first time.

The average degree of polymerization calculated from thioacidolysis data increased from 2.3, in fraction la, to 15.1, in fraction Va. Fraction Vb showed the largest DP of all (18.6) as expected from its exclusion from Sephadex LH20. Similar results, with average M, increasing from 1264 to 9474 were obtained by GPC of the acetylated fractions. Note that, since fraction Vb could not be redissolved after concentration, acetylation and M, deter- mination by GPC were performed directly on fraction V, without purification. Assuming for each acetylated monomeric unit a M, of 500 and for each acetylated galloyl a M, of 236, and given the proportion of galloyla- ted molecules in each fraction, this corresponds to aver- age degrees of polymerization ranging from 2.4 to 16.7. Slightly higher values determined by GPC than by HPLC have already been reported [24] and attributed to the globularity of polymers which results in larger M, by using GPC [25].

Procyanidins from grape seeds 783

Table 2. Characteristics of grape seed procyanidin fractions

Fraction Ia IIa IIIa IVa Va Vb

15.5: trans ratio 2.O:l 3.2:1 5.9:1 6.9:l 10.1:111.5:1 % of galloylation 13.2 18.7 28.0 28.2 30.2 30.3 DP (by thiolysis) 2.3 3.6 5.4 7.8 14.4 18.6 M, (by GPC)* 1264 2312 3622 6458 9474 DP (by GPC) 2.4 4.2 604 11.4 16.7

*Of the acetylated fractions.

The dominant stereochemistry in grape seed conden- sed tannins was cis. The cis: truns ratio increased with DP, ranging from 2: 1 to 11.5: 1, because catechin was rela- tively more abundant in terminal units.

Characterization of the crude procyanidin extract by thiolytic degradation indicated an average DP of 3.2, with catechin, epicatechin and epicatechin-gallate re- leased in the ratio of 1: 3 : 1. The proportions of mono- mers, oligomers and polymers in the crude grape seed extract were evaluated from their peak areas by normal- phase HPLC on the analytical scale. The monomers were eluted first as three individual peaks followed by procyan- idin groups, starting from lower M, procyanidins, eluted as successive peaks. The latter were collected from the analytical HPLC column, concentrated to dryness and characterized by thiolysis degradation. Given the larger extinction coefficient of galloyl esters at 280 nm (2.11 times higher than that of non-esterified flavan-3-ols, in mol), the proportion of galloylated units in each fraction was taken into account in the calculation of concentra- tions from peak areas at 280 nm. Monomers together represented 1 l%, oligomers (from dimers to pentamers) ca 34% and polymeric compounds (DP> 16) 55% of the eluted flavan-3-01s (in mol of monomer units).

EXPERIMENTAL

Material. (+ )-Catechin and ( - )-epicatechin were pur- chased from Fluka. Epicatechin-3-O-gallate, and pro- cyanidin oligomer standards were isolated from grape seeds and identified as described earlier [8, 203. The benzylthioethers of catechin, epicatechin, and epi- catechin-3-0-gallate were obtained by thiolysis of pro- cyanidin oligomers and purified by semi-prep. reverse- phase HPLC. Cyanidin and delphinidin chlorides were from Sarsyntex. All solvents were HPLC grade.

Extraction and isolation of procyanidin fractions. Grape seeds were obtained from berries of Y. oinifera, var. Alicante Bouchet, harvested at commercial maturity. They were ground under liq. N,, extracted with 60% Me,CO and centrifuged to remove plant debris. The Me&O supematant was then taken to dryness under vacuum, dissolved in MeGH and filtered to get a crude extract of procyanidins. Fractionation of the MeOH soln was achieved by normal-phase HPLC using a Lichros- pher Silo0 column from Merck (5 pM particle size, 250 x 4 mm) protected with a guard column of the same

material. The elution conditions were as follows:

flow-rate, 1 mlmin- t; temp. 26”; mobile phase A, CH,CI,-MeOH-H,O-TFA (10:86: 2:0.005), mobile phase B, CH&I,-MeGH-H,O-TFA (82: 18:2:0.005); linear gradients from 0 to 40% A in 50 mitt, from 40 to 55% A in 5 min, and from 55 to 100% A in 5 min, followed by washing for 5 min and reconditioning of the column. The chromatograms were monitored at 280 nm using a UV detector. Similar sepns were achieved at the prep. scale using Spherisorb Si-60 Merck (25~40pm particle size) packed in a 4 x 24 cm column and eluted with the same gradient as above at 24 ml min- ‘. The five frs collected were rechromatographed on the prep. HPLC system to reduce overlapping, taken to dryness, dissolved in MeOH and finally purified by chromatography on Sephadex LH20 using H,O and 60% Me&O as eluents, prior to lyophilization.

Characterization of polymeric procyanidins. The pro- cyanidin frs dissolved in MeOH (1 g I- ‘) were introduced together with an equal vol. (20 ~1) of a 5% soln of toluene- z-thiol in MeOH containing HCI (0.2 M) into glass ampoules. After sealing the mixt. was shaken and heated at 90” for 2 min. The hydrolysed soln was used directly for HPLC analysis. The elution conditions were as follows: flow-rate 1 ml min - ‘: temp. 30”; column, Spherisorb ODS-2 (250 x 4 mm); solvent A, 2.5% HOAc in H,O; solvent B, MeCN-solvent A (4: 1); linear gradients from 5 to 50% A in 35 min and from 50 to 40% A in 5 min, followed by washing and re-equilibrating the column; detection, UV 280 nm. All analyses were performed in triplicate. Calibration curves (based on peak area) were established using flavan-3-01 and benzylthioether stand- ards. When not commercially available, the latter were prepd by semi-prep. HPLC (250 x 8 mm column). The elution conditions were the same as used for analyt. HPLC, but the flow rate was 2 mlmin- ‘. Enzymatic hydrolysis with tannase and complete acid hydrolysis were carried out as described in ref. [S]. The anthocyan- ins formed were chromatographed by reverse-phase HPLC with diode array detection and identified by co- injn with cyanidin and delphinidin standards.

GPC analysis. This was performed on peracetate deriv- atives 1261 using TSK G 2500 Hxl and TSk G 3000 Hxl columns (Tosohaas) connected in series. The elution was isocratic with THF at 1 mlmin-I. Detection was at 254 nm and 30”. The GPC system was calibrated with 11 polystyrene standards (M, 162-50 000).

REFEREKCES

1. Weinges, K. and Piretti, M. V. (1971) Liebigs Ann. Chem. Dtsch. 748, 218.

2. Lea, A. G. H., Bridle, P., Timberlake, C. F. and Singleton, V. L. (1979) Am. J. Enol. Vitic. 30, 289. Czochanska, Z., Foo, L. Y. and Porter, L. J. (1979) Phytochemistry 18, 18 19. Haslam, E. (1980) Phytochemistry 19, 1577. Boukharta, M. (1988) Ph.D. Thesis. I.N.P.L. (Nancy, France). Ozmianski, J. and Sapis, J. C. (1989) J. Agric. Food Chem. 37, 1293.

784 C. PRIEUR et al.

7. Lee, C. Y. and Jaworski, A. W. (1990) Am. J. Enol. Vitic. 38, 277.

8. Ricardo da Silva, J. M., Rigaud, J.. Cheynier, V., Cheminat, A. and Moutounet, M. (1991) Phyto- chemistry 30, 1259.

9. Czochanska, Z., Foo, L. Y., Newman, R. H. and

Porter, L. J. (1980) J. Chem. Sot. Perkin Truns ! 2278. 10. Haslam, E. (1974) Biochem. J. 139, 285. 11. Haslam, E. and Lilley, T. H. (1988) Crit. Rec. Food

Sci. Nutr. 27. 1. 12. Lea, A. G. H. and Arnold, G. M. (1978) .I. Sci. Food

Agric. 29. 478. 13. Porter, L. J. and Woodruffe, J. (1984) Phytochemistry

23, 1255. 14. Gkuda, T., Mori, K. and Hatano, T. (1985) Chem.

Pharm. Bull. 33, 1424. 15. Cheynier, V., Rigaud, J. and Ricardo da Silva, J. M.

(1992) in Plant Polyphenols: Synthesis, Properties, Significance (Hemingway, R. W. and Laks, P. E., eds), p. 281. Plenum Press, New York.

16. Rigaud, J., Escribano-Bailon, M. T., Prieur, C., Sou-

quet, J.-M. and Cheynier, V. (1993) J. Chromatogr. 654, 255.

17. Thompson, J., Tanner, D., Haslam, E. and Tanner, R. J. N. (1972) J. Chem. Sot. Perkin Trans I 1387.

18. Cai, Y., Evans, F. J., Roberts, M. F., Phillipson, J. D., Zenk, M. H. and Glebas, Y. Y. (1991) Phyfochemistry 30, 2033.

19. Shen, Z., Haslam, E., Falsham, C. P. and Begley, M. J. (1986) Phytochemistry 25, 2629.

20. Rigaud, J., Perez-llzarbe, J., Ricardo da Silva, J. M. and Cheynier, V. (1991) J. Chromutogr. 540, 401.

21. Porter, L. J. (1988) in The Flaoonoids (Harborne, J. B., ed.), p. 21. Chapman and Hall, London.

22: Nonaka, G., Sakai, R. and Nishioka, I. (1984) Phyto- chemistry 23, 1753.

23. McGraw, G. W., Steynberg, J. P. and Hemingway, R. W. (1993) Tetrahedron Letters 34, 987.

24. Koupai-Abyazani, M. R., Muir, A. D., Bohm, B. A., Towers, G. H. N. and Gruber, M. Y. (1993) Phyto- chemistry 34, 113.

25. Porter, L. J. (1984) Rec. Latinam. Quim. 15, 43. 26. Williams, V. M., Porter, L. J. and Hemingway, R. W.

(1983) Phytochemistry 22, 569.