Procyanidin fractions

Recent studies performed using this method have shown that flavanol bitterness decreases from monomer to trimer. Epicatechin was perceived more bitter than catechin and the C4-C6-linked catechin dimer more bitter than other procyanidin dimers with C4-C6 linkages. This may be due to the higher lipophilic character of these molecules facilitating their diffusion to the gustatory receptor. Bitterness of procyanidin fractions in 5% ethanol decreased with their... 

Surprisingly, copigmentation was enhanced in the presence of 12% ethanol (Mirabel et al. 1999b), reaching 157/M for epicatechin and 68/M for the procyanidin fraction. 

Procyanidin fractionation and quantification were made following the methods described by Sun et al. (15,16). 

Pear Phenolics. The same HPLC analytical system gave good resolution of pear juice phenolics and enabled peak assignments and quantitation to be made in a similar manner to that described for apple phenolics (3,7). It was also necessary to isolate the procyanidins and catechins using the Sephadex clean-up procedure (4) in order to measure their concentration. Figure 6A shows the HPLC separation of cinnamics, flavonols, and arbutin in pear juice while Figure 6B is a HPLC chromatogram of the pear juice procyanidin fraction. 

The only correct quantitation of results from the proanthocyanidin assay is to use purified procyanidin fractions from the matrix under analysis. Calibrations should be prepared in the extracts themselves to correct for matrix effects as much as possible. A similar approach has been used by Li et al. [67] who produced a calibration curve with purified... 

A major drawback of all functional group assays is that a satisfactory standard does not exist. For a given sample the most appropriate standard is a purified procyanidin fraction prepared from the same matrix. The isolation and characterization of such purified fractions are laborious. Added to that procyanidins undergo oxidation, complexation and self-polymerization very easily, rendering such purified fractions only reproducible to a limited degree. At least in the proanthocyanidin assay the color reaction depends not only on the polyphenols themselves, but also on the matrix. The use of specified proanthocyanidins as a standard in a suitable blank matrix is an attempt to correct for such effects [67],... 

Lesp.herba (10 l/EtAc enrichment 10,pi) 5a procyanidin fraction of (5)... 

By definition procyanidins and anthocyanogens, on treatment with acid, give anthocyanidins which show maximum light absorption at about 545 nm. By measurement at 455 nm estimates of the amount of catechin present can also be obtained [51]. The yield of anthocyanidin from different procyanidin fractions varies and anthocyanogen values do not correlate with the shelf life of beers. In many cases the yield of anthocyanidin released by add decreases as the procyanidins polymerize while the level of total polyphenols (EBQ remains roughly constant. Accordingly the... 

Yanagida, A., Kanda, T., Shoji, T., Ohnishi-Kameyama, M., and Nagata, T., Fractionation of apple procyanidins by size-exclusion chromatography, /. Chromatogr. A, 855, 181, 1999. 

Fig. 2.114. RP-HPLC profiles of ACTs and SEC fractions (fr.) of ACTs. Each lyophilized sample was dissolved in water (1 mg/ml), and analysed by RP-HPLC. Upper chromatogram RP-HPLC profile of ACTs. Lower chromatograms with fraction numbers RP-HPLC profiles of SEC fractions of ACTs. The numbers of identified peaks in each chromatogram are (1) procyanidin B1 (PB1), (2) (+)-catechin, (3) procyanidin B2 (PB2), (4) procyanidin Cl (PCI), 5 (—)-epicatechin (EC). AU means relative absorbance units (at 280 nm). For details on the RP-HPLC conditions see text. Reprinted with permission from A. Yanagida et al. [253].

Fig. 3.9 ESI mass spectrum of the dimer-rich fraction from Fig. 3.8c showing the procyanidin B2 peak (this eluted at 16.4 min after reverse-phase HPLC separation)...

This step is optional, depending on how critical it is to separate anthocyanins from other polyphenolics in subsequent analysis. The ethyl acetate fraction is enriched in polypheno-lics such as flavonols, procyanidins, and cinnamates. If analysis of this fraction is desired, a cleaner isolate will be obtained if residual water is removed by passing a nitrogen gas stream through the cartridge for 2 to 3 min before applying ethyl acetate. 

Figure 11.3.12 HPLC chromatograms of polyphenolics in Concord grape extract detected at 280 nm. (A) All polyphenolics, including anthocyanins. (B) Nonanthocyanin polyphenolics after fractionation. Peak identification 1, cis-caftaric acid 2, frans-caftaric acid 3, procyanidin B3 4, c/s-coutaric acid 5, frans-coutaric acid 6, epicatechin 7, quercetin galactoside 8, quercetin glucoside. Reproduced from Oszmianski and Lee (1990) with permission from the American Society for Enology and Viticulture.

The determination of polyphenolics may result in interference due to co-elution of phenolic acids and procyanidins. This problem can be eliminated by fractionation of polyphenolics into acidic and neutral polyphenolics prior to sample injection into the HPLC system. Because the fractionation techniques effectively improve the resolution of many polyphenolic peaks in the reversed-phase HPLC system, it is suggested that further characterization and identification of unknown peaks be conducted by additional methods such as mass spectrometry and nuclear magnetic resonance. 

Fractionate phenolic acids and flavonoids using Sephadex LH-20 column. For phenolic acids and flavonol glycosides, filter the fruit juice through 0.45-/nm (type HA) filter. For procyanidins apply juice onto the Sephadex LH-20 column, wash with 20% MeOH (30 ml), then elute with MeOH (15 ml), concentrate to dryness, redissolve in 2 ml H20, and filter through 0.45-/nm (type HA) filter. 

Reversed-phase HPLC is used for the analysis of the different groups of phenols, phenolic acids, hydroxycinnamic acids, flavonoids, and procyanidins in grapes and wines (22,46,47,77-80). However, due to the presence of a large quantities of various compounds, wine analysis is difficult. Thus, different sample preparation procedures, including fractionation and extraction, are often applied when various groups of phenolic compounds are studied together. 

Anisophyllea dichostyla root bark C, EC, EGC, ECG,EGCG procyanidin B, and B2, (epi) catechin trimer Fat removal with hexane, extraction with MeOH, fractionation with silica gel 60 C-18 ACN-H20-acetic acid HPLC/UV/MS/ ESI(-),HPLC/ MS /MS/ ESI(+/-), or NMR 62... 

Figure 16.8 Influence of an increasing gradient of gum arabic concentration on the interactions between BSA (5 x 104mM) and a fraction of procyanidins from grape seed (gallate tetramers and pentamers, 0.034gl" ) (in 12% aqueous ethanol, 0.1 M acetate buffer, pH 5.0) [81].

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