Proanthocyanidins standards

First, the qualitative and quantitative variability of the amount of catechins and proanthocyanidins present in plant extracts used for different studies is probably the most significant. This might be due to the use of different procedures of extraction, quantification and structural elucidation. In most cases, even the lack of rigorous phytochemical characterization and quantification of active compoimd(s) constitutes a severe limitation on the rehabihty of the results. The lack of commercially available pure standards (particularly for some proanthocyanidins) represents an additional problem that has so far hampered the execution of rigorous SAR studies. This hmitation means that although a munber of in vitro or in vivo studies have been carried out by using more or less pure standards of catechins or with plant extracts containing both catechins and proanthocyanidins, only a handful of authors have... 

Fig. 3.2 Extraction efficiencies of grape seed proanthocyanidins in different solvents. Vertical bars represent standard deviation (adapted with permission from [23])...

Standardization. Because the analysis reveals the presence of many different substances in one result (in the case of wine, this could mean thousands of substances if one takes into account the diversity of proanthocyanidins), the only practical standard is a single substance. In the cases of wine and tea, the accepted standard is gallic acid. It is a particularly good standard because it is relatively inexpensive in pure form and is stable in its dry form. Other substances have been used, and in principle any phenol could be used, but gallic acid is strongly recommended for the above reasons and the fact that the use of a single standard makes it easier to compare data. 

Identify proanthocyanidin subunits by comparing their retention factors to those shown in Table 11.4.1. After identification, quantify using a (+)-catechin standard. 

Subunits that react with phloroglucinol are derived from proanthocyanidin extension subunits. Subunits that have not reacted with phloroglucinol are derived from proanthocyanidin terminal subunits or were present as monomeric flavan-3-ols. For quantitation, the sample peak areas of the individual subunits are compared with a(+)-catechin standard, and individual quantities are determined using the relative response factors shown in Table 11.4.1. 

A colorimetric assay involves the oxidative cleavage of proanthocyanidins with ferrous sulfate. To 0.5 mL of aqueous plant extract is added a 5-mL portion of an acidic solution of ferrous sulfate (77 mg of FeS04.7H20 dissolved in 500 mL of 2 3 HCl/ -butanol). The tubes are loosely covered and placed in a water bath at 95°C for 15 min. The absorbance is read at 530 nm. The concentration of proanthocyanidins is expressed as cyanidin equivalents (used for the standard curve). The molecular extinction coefficient emoi that can be used to convert the absorbance values to a concentration is equal to 34700 L mol cm 1. 

An alternative colorimetric method relies on the reaction with vanillin under acidic conditions. A 2-mL aliquot of a freshly prepared solution of vanillin (1 g/100 mL) in 70% sulfuric acid is added to 1 mL of aqueous plant extract. The mixture is incubated in a 20°C-waterbath and after exactly 15 min. the absorbance at 500 nm read. The concentration of proanthocyanidins is expressed as (+)-catechin equivalents (used for the standard curve). This assay is specific for flavonols. As a consequence, when using this assay to determine the concentration of condensed tannins, widely distributed monomeric flavonols, such as catechin (1.39) and epicatechin (1.90), can interfere (Hagerman and Butler, 1989). 

Saskatoon berries and used as external standards (Hellstrom et al., 2006). Difficulties in obtaining standards remain a major hindrance for chromatographic analysis of proanthocyanidins. 

Catechin extension units of proanthocyanidins react with toluene-a-thiol to yield 3,4-tran -(+)-catechin benzylthioether (—)-epicatechin in the extension units of proanthocyanidins reacts with toluene-a-thiol to form 3,4-tran -(—)-epicatechin benzylthioether and 3,4-cA-(—)-epicatechin benzylthioether (Gu et al., 2003a). The response factors of catechin and catechin benzylthioether were the same using 280 nm detection, as well as for epicatechin and epicatechin benzylthioether. Thus, catechin and epicatechin can be used as standards to quantify their benzylthioethers. For procyanidins that consisted of catechin and epicatechin, the mean DP (degree of polymerization) can be calculated using the following equation. 

For convenience, (+)-catechin rather than purified procyanidins is used to standardize the assay, although this leads to a considerable overestimation. For example, the use of (+)-catechin instead of purified proanthocyanidins as a standard for the analysis of Sericea lespedeza forage crop approximately doubled proanthocyanidin estimations in the samples [52]. 

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],... 

Several attempts have been made to obtain a more complete picture of the phenolic composition of a sample. For this purpose some researchers propose the conductance of several complementary assays [106]. More popular is the formation of ratios which are claimed to correlate with the relative degree of polymerization. Considering the complex reaction scheme of these assays, interpretations of such ratios must be performed very carefully. In our opinion, some of them more likely reflect the specific proanthocyanidin structures than actual relative degrees of polymerization. In the last few years the following ratios have been described in the literature dimethylaminocinnamaldehyde / proanthocyanidin ratio for wine and grape tissue [149], vanillin / dimethylaminocinnamaldehyde ratio for wine and purified standards [96] and proanthocyanidin / vanillin ratio for plums [95] as well as purified proanthocyanidins from various plant sources [155]. 

Sephadex LH-20 (Pharmacia) is a support for gel chromatography and is widely used in isolation protocols for proanthocyanidins. It is composed of dextran molecules which are three-dimensionally cross-linked with hydroxypropyl groups [205]. Proanthocyanidins are separated by filtration (molecule sieve effect of the gel) and adsorption (hydrogen bonding). Retention increases primarely with increasing ( ) molecular weight and C4—>C6 linked procyanidins are more retarded than their C4—>C8 linked counterparts [206-207]. Sephadex LH-20 has first been used for sample clean up in the analysis of apple ciders by Lea and Timberlake [208]. So far, no pre-packed columns exist on the market. All methods described in the literature work with different column dimensions (see Tab. (6)). Standardization would help to make results more comparable. Some validation procedures have been performed for two protocols [185,209],... 

The main problem of submission of anthocyanins to UVA IS spectroscopy is the diversity of possible structures with different absorption properties and the lack of reliable calibration procedures. Hydrolysis of anthocyanins to anthocyanidins prior to measurement reduces possible UVA IS absorption variations in both, X max and e, to few well described structures. Yet another advantage of hydrolysis is that most anthocyanidins but only few anthocyanins are commercially available as reference standards for method calibration. The main disadvantages of acidic hydrolysis are that, oligomeric proanthocyanidins present in the sample are also hydrolyzed to anthocyanidins yielding possibly over-estimation of the original monomeric anthocyanin content. 

After appropriate extraction/hydrolysis as detailed earlier we determine in the first instance the content of anthocyanidins by UVAHS spectroscopy in accordance with most pharmaeopoeial and indnstry standards. To oin-understanding overestimation due to hydrolysis of proanthocyanidins is not eonsidered a major issue because of low natural contents in berries. Even if in rare eases proanthocyanidins are measnred as anthocyanidins, the health... 

Pressurized liquid extraction (PLE), also known as accelerated solvent extraction (ASE) or subcritical solvent extraction, uses solvents utilized in standard hquid extraction at high pressures and temperatures above their boiling points to shorten the extraction time, reduce solvent usage, and increase extraction efficiency. At elevated temperatures (50-200°C), diffusivity increases, mass transfer improves, solubility increases, and the interactions between solute and matrices is minimized. At high pressures (500-3,000 psi), the penetration of the solvent is also improved (Richter et al. 1996). Subcritical water extraction (SWE), used to recover catechins and proanthocyanidins from wine pomace and sequential extraction at different temperatures (50 C, 100 C, and 150°C) was reported to have the highest recovery, but different flava-... 

The vanillin procedure involves reaction of an aromatic aldehyde, vanillin, with the meta-substituted ring of flavanols to yield a red adduct. Although the vanillin assay is an easy and cheap assay to measure flavanols and proanthocyanidins, several aspects of the procedure need to be carefully considered to obtain reliable results. Catechin is commonly used to standardize the vanillin reaction, although both proanthocyanidins and catechin react with vanillin at different rates. Therefore,... 

Proanthocyanidins from ten cultivars grown in a replicated (Randomized Complete Block Design-RCBD) field trial were quantified from fruit harvested over three dates. The proanthocyanidins were quantified using die standard vanillin/sulphuric acid analysis (24,25). Analysis of variance indicated both... 

Data expressed as mg/kg fresh weight standard deviation. PA - proanthocyanidins PC - procyanidins PD -prodelphidins PP - propelargonidins A - indicates existence of A-type proanthocyanidins. 

Mammalian metabolites are usually assumed to have the same stereochemistry as the substance fed, but this can only be determined following isolation of the metabolite. Even when a synthetic and stereochemically-defined standard is available it does not define the stereochemistry of a substance in plasma or urine. Because of this uncertainty, in this chapter we define a large portion of flavan-3-ols are present in foods in the form of oligomers and polymers (proanthocyanidins). Galloylation and polymerization appear to have dramatic effects on the extent of intestinal absorption. 

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