Flavan-3-ol monomers

For phloroglucinolysis, a solution of 0.1 N HCl in MeOH, containing 50 g/L phloroglucinol and 10 g/L ascorbic acid, is prepared. The PA of interest is reacted in this solution at 50°C for 20 min and then combined with 5 volumes of 40 mM aqueous sodium acetate to stop the reaction. After acid-catalyzed cleavage in the presence of phloroglucinol, the fraction is depolymerized and the terminal subunits released as flavan-3-ol monomers and the extension subunits released as phloroglucinol adducts of flavan-3-ol intermediates. These products are then separated and quantified by HPLC [25]. 

The PAs, or condensed tannins, are polymers synthesized from flavan-3-ol monomer units. The phlobaphenes are 3-deoxy-PAs formed from flavan-4-ol monomers. The biosynthesis of both types of PAs follows the biosynthetic route of anthocyanins from chalcones through to the branch points to flavan-3-ol and flavan-4-ol formation. In this section, the specific enzymes forming the monomers are discussed, along with a discussion on the polymerization process. Although the chemistry of tannins is described in detail elsewhere in this book, it is useful to briefly mention the nature of the monomer subunit types and the polymer forms. 

Gacon, K., Peleg, H., and Noble, A.C., Bitterness and astringency of flavan-3-ol monomers, dimers and trimers. Food Qual. Prefer. 7, 343, 1996. 

This purification step is designed to remove impurities from the proanthocyanidin extract. It utilizes liquid-liquid extraction to remove lipophilic material and monomeric flavan-3-ols, and also adsorption chromatography to remove more hydrophilic material such as organic acids, sugars, and residual flavan-3-ol monomers. Following the steps in this protocol, purified and powdered proanthocyanidins are obtained. 

For example, extract the proanthocyanidin mixture with chloroform to remove chlorophyll, carotenoids, and waxy material. Use ethyl acetate if substantial amounts of flavan-3-ol monomers are present. Tissues that would benefit from a chloroform extraction include leafy tissues that contain chlorophyll (i.e., tea leaves) and seeds that contain oils (i.e., grape seeds). Ethyl acetate would be useful in plants such as apples, berries, grapes, and teas (i.e., tissues known to contain significant amounts offlavan-3-ol monomers). 

The potential impurities will vary according to the plant tissue extracted, and therefore the exact washing volume will vary. It is important to determine the impurities present and their retention properties on the column to minimize impurities in the final proanthocyanidin and maximize proanthocyanidin recovery. For this step, the use of a spectrophotometer is helpful in monitoring the eluate. Some typical impurities and monitoring wavelengths include organic acids (215 nm), flavan-3-ol monomers (280 nm), hydroxycinnamic acids (320 nm), andflavonols (365 nm). Anthocyanins are observable in the visible spectrum. 

It is important to purify proanthocyanidins, particularly for determining their conversion yield. It is also advantageous to do so to eliminate extraneous material that might otherwise react with the proanthocyanidins. A combination of liquid-liquid extraction and adsorption chromatography is effective in removing impurities. The use of chloroform in liquid-liquid extraction is very effective in removing fat-soluble compounds such as carotenoids, chlorophyll, oils, and waxes. These compounds would be expected in leafy plant tissues (carotenoids and chlorophyll) as well as seeds and fruits (oils and waxes). Ethyl acetate is effective in the selective removal of flavan-3-ol monomers, which are also typically present with proanthocyanidins. 

It is important to monitor the time that the proanthocyanidin is allowed to react with the phloroglucinol solution. The products formed are not stable under acidic conditions, and it is therefore critically important that the reaction not exceed 20 min. Of particular concern are the flavan-3-ol monomers, which degrade more rapidly than the phloroglucinol adducts (Kennedy and Jones, 2001). Excessive degradation of the flavan-3-ol monomers will result in reduced amount of terminal subunits. This in turn will reduce the conversion yield and increase the average degree of polymerization calculated. 

Because of the varied nature of the plant tissues from which the proanthocyanidin extracts are derived, it is difficult to anticipate the expected outcome. As an example of how these procedures can be adapted to specific tissues and analyses, using grape tissues, fruit is harvested and the tissues of interest (e.g., skins and seeds) are removed from the remainder of the berry. They are rinsed well and then extracted as whole tissues using the conditions described in these protocols. For grape skins, aliquid-liq-uid extraction with chloroform has been successful in the removal of chlorophyll and waxes, yet no extraction with ethyl acetate has been performed because of the small proportion of flavan-3-ol monomers (Kennedy et al., 2001). For grape seeds, these protocols have... 

Figure 1.11 Less common flavan-3-ol monomers ( -)-epiafzelechin and ( + )-afzelechin.

Figure 2.1 Structure of flavan-3-ol monomers and dimers. (A)(—)-Epicatechin with = OH and R2 = H or ( + )-catechin with Ri = H and R2 = OH , (B) procyanidin (4p - 8)-dimer (C) procyanidin (4p -> 6)-dimer.

Table I. Maximum Intensity (MAX) andTotal Duration (TOT) of Bitterness and Astringency of Flavan-3-ol monomers, dimers and trimers (n= 18 judges x 2 reps) (19)...

TABLE 6.6. Positive-Ion ESI Tandem Mass Product Ions of Flavan-3-ol monomers and PAs Dimers and Oligomers"... 

In grapes, flavan-3-ol monomers and proanthocyanidins, which are flavan-... 

Procyanidin derivatives (i.e. galloylated procyanidins) and flavan-3-ol monomers are not mentioned. 

There is no final consensus on whether procyanidin biosynthesis is controlled thermodynamically or enzymatically. In either case proanthocyanidins are synthesized through sequential addition of flavan-3,4-diol units (in their reactive forms as carbocations or quinone methides) to a flavan-3-ol monomer [218]. Based on the latest findings there is some evidence that different condensation enzymes might exist which are specific for each type of flavan-3,4-diol [64] and that polymer synthesis would be subject to a very complex regulatory mechanism [63]. But so far, no enzyme synthetase systems have been isolated and enzymatic conversion of flavanols to proanthocyanidins could not be demonstrated in vitro [219]. If biosynthesis was thermodynamically controlled, the variation in proanthocyanidin composition could be explained by synthesis at different times or in different compartments [64], The hypothesis of a thermodynamically controlled biosynthesis is based on the fact that naturally and chemically synthesized procyanidin dimers occur as a mixture of 4—>8 and 4—>6 linked isomers in approximate ratios of 3-4 1 [220]. Porter [164] found analogous ratios of 4—>8 and 4—>6 linkages in proanthocyanidin polymers. 

On principle, pre- and post-column derivatization can be performed. Derivatization of complex samples prior to chromatographic separation is more problematic because matrix effects may alter reactions. Therefore, pre-column derivatization procedures are less suitable for quantitative analysis. Nevertheless quantitative procedures are described in the literature. Piretti et al. [71-72,247] analyzed peracetylated flavan-3-ol monomers and procyanidin dimers among other compounds in apple tissue after acetylation on a nitrile stationary phase under normal-phase conditions. Tarnai et al. [13] used the same approach in the analysis of procyanidins from cherry tissue. Incomplete acetylation was never observed [71], but so far validation data are not available. 

Some derivatization reactions which are frequently used for the structural elucidation of procyanidins have been adopted for HPLC analysis. Koupai-Abyazani et al. [231] developed a qualitative HPLC procedure to separate flavan-3-ol monomers and phloroglucinol adducts. The reaction is based on the acid degradation of procyanidins in the presence of phloroglucinol. The procedure has been used for quantitation of polymeric proanthocyanidins from sainfoin leaves [63]. HPLC analysis of benzylthioethers after acid degradation of procyanidins in the presence of toluene-a-thiol has so far only been used for qualitative analysis [250-251],... 

Micellar electrokinetic capillary chromatography (MECC) has recently been evaluated for the analysis of procyanidins from hawthorn [257] and flavan-3-ol monomers from green tea [258]. In view of the relatively insensitive detection methods (see section detection) it is very doubtful that this technology will replace HPLC in the near future, because only very small sample amounts can be analyzed. 

Fig. (7). UV spectra and absorbance features of flavan-3-ol monomer, phenolic acids and tlavonoids.

Incorporation of labeled phenylalanine and cinnamic acid into dimeric proanthocyanidins occurs asymmetrically, with differences in the amount of precursors incorporated into the two portions of the molecule (Stafford, 1989). Further, the pool of free flavan-3-ol monomers including (+ )-catechin (3) and (— )-epicatechin (30) contained much higher levels of radioactive label than the comparable initiating (or terminating) unit. Tritium label at the C-2 position is retained, whereas that at the C-3 position is lost in the dimers (Stafford, 1989). 

PAs are complex polyphenolic polymers of plant origin encompassing a large span of polymerization degrees (DP). They are described by the presence of three-ring flavan-3-ol monomer units linked by C—C and in some... 

Table 57.2 Distribution of flavan-3-ol monomers in food products... 

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