Proanthocyanidins analysis

HPLC separation, as described above, is restricted to rather simple compounds that represent only a small proportion of flavonoids. Indeed, proanthocyanidin analysis becomes increasingly difficult as their molecular weight increases, due to the larger number of possible structures, smaller amounts of each individual compound, and poorer resolution of the chromatographic profiles. This is especially true of grape proanthocyanidins, which, unlike those of apple or cacao consisting only of epicatechin units, are based on four major... 

The various MS methods to determine the molecular composition of the constituent monomeric units in proanthocyanidins oligomers are summarized in Ref. 257. Contributions focusing on proanthocyanidin analysis via the HPLC-MS protocol included a wide range of plant-derived foods and beverages, and are summarized in Refs. 12, 258-261. In addition, references to additional significant contributions in this area are readily available via several of the excellent electronic search engines that are at our disposal. 

Figure 8.3 Sample extraction and purification steps for proanthocyanidin analysis.

KIEHNE A, LAKENBRINK c and ENGLHARDT u H (1997) Analysis of proanthocyanidins in tea samples , Z Lebensm Unters ForschA, 205, 153-7. 

Knowledge of the identity of phenolic compounds in food facilitates the analysis and discussion of potential antioxidant effects. Thus studies of phenolic compounds as antioxidants in food should usually by accompanied by the identification and quantification of the phenols. Reversed-phase HPLC combined with UV-VIS or electrochemical detection is the most common method for quantification of individual flavonoids and phenolic acids in foods (Merken and Beecher, 2000 Mattila and Kumpulainen, 2002), whereas HPLC combined with mass spectrometry has been used for identification of phenolic compounds (Justesen et al, 1998). Normal-phase HPLC combined with mass spectrometry has been used to identify monomeric and dimeric proanthocyanidins (Lazarus et al, 1999). Flavonoids are usually quantified as aglycones by HPLC, and samples containing flavonoid glycosides are therefore hydrolysed before analysis (Nuutila et al, 2002). 

LAZARUS s E, ADAMSON G E, HAMMERSTONE J F and SCHMITZ H H (1999) High-performance liquid chromatography/Mass spectrometry analysis of proanthocyanidins in foods and beverages, JAgric Food Chem, 47, 3693-701. 

Thousands of polyphenols from fruits (grapes, apples, etc.), vegetables (horse beans), and teas have been identified, many having good coloring properties, especially anthocyanins and some flavonoids. Well-documented reviews discuss the coloring capacities of some polyphenols including procyanidins. - Detailed presentations of anthocyanin and flavonoid properties and analysis are included in Sections 2.3, 4.3, and 6.3. The soluble proanthocyanidins of the colored horse bean Viciafaba L. seed coats were isolated and separated by solvent partition. 

The chemical characteristics of the proanthocyanidins were elucidated by total oxidation and partial degradation in the presence of phloroglucinol followed by HPLC analysis. The native extract of proanthocyanidins contained (+) gallocatechin, (-) epigallocatechin, (h-) catechin, and (-) epicatechin units. ... 

Nakamura, Y., Tsuji, S., and Tonogai, Y, Analysis of proanthocyanidins in grape seed extracts, health foods and grape seed oils, J. Health Set, 49, 45, 2003. 

Sun B, Spranger MI (2005) Review quantitative extraction and analysis of grape and wine proanthocyanidins and stilbenes. Ciencia Tec Vitiv 20 59-89... 

Kennedy JA, Jones GP (2001) Analysis of proanthocyanidin cleavage products following acid-catalysis in the presence of excess phloroglucinol. J Agric Food Chem 49 1740-1746... 

Lazarus, S.A. et al.. High-performance liquid chromatography/mass spectrometry analysis of proanthocyanidins in food and beverages, Meth. EnzymoL, 335, 46, 2001. 

There are many branches to the flavonoid biosynthetic pathways, with the best characterized being those leading to the colored anthocyanins and proanthocyanidins (PAs) and the generally colorless flavones, flavonols, and isoflavonoids. Genes or cDNAs have now been identified for all the core steps leading to anthocyanin, flavone, and flavonol formation, as well as many steps of the isoflavonoid branch, allowing extensive analysis of the encoded enzymes (Table 3.1). In addition, several DNA sequences are available for the modification enzymes that produce the variety of structures known within each class of compound. 

Thiolysis also proved useful for the analysis of derived tannins. Methylmethine-linked tannin-like compounds resulting from acetaldehyde-mediated condensation of flavanols (see Section 5.5.S.2) yielded several adducts when submitted to acid-catalyzed cleavage in the presence of ethanethiol, providing information on the position of linkages in the original ethyl-linked compounds. " Thiolysis of red wine extracts released benzylthioether derivatives of several anthocyanin-flavanol adducts, indicating that such structures were initially linked to proanthocyanidins. However, some of the flavonoid derivatives present in wine (e.g., flavanol-anthocyanins ) are resistant to thiolysis, while others (e.g., flavanol-ethyl anthocyanins) were only partly cleaved. Thiolysis, thus, appears as a rather simple, sensitive, and powerful tool for quantification and characterization of proanthocyanidins, but provides mostly qualitative data for their reaction products. 

ESI-MS in the positive ion mode enabled to detect [M + H] ions up to the galloylated pentamer. Analysis of wine proanthocyanidins showed the presence of additional series of species with 16 mass unit differences to signals given by grape seed procyanidins. These were attributed to the presence of (epi)gallocatechin units in mixed procyanidin prodelphinidin... 

Cheynier, V. and Fulcrand, H., Analysis of proanthocyanidins and complex polyphenols. In Methods in Polyphenol Analysis (eds C. Santos-Buelga and G. Williamson), Royal Society of Chemistry, London, 2003, p. 282. 

X-ray and CD analysis. The structure of procyanidin B-1 was unequivocally confirmed by x-ray analysis of its deca-(9-acetyl derivative by Weinges, one of the pioneers in the field of proanthocyanidin chemistry. One of the most powerful methods to establish the absolute configuration at C-4 of the T-unit in dimeric A- and B-type proanthocyanidins remains the chiroptical method via application of the aromatic quadrant rule. This has been repeatedly demonstrated by the author s own work and several other contributions listed in Refs. 7-12. 

Conformational analysis of proanthocyanidins is, in principle, concerned with the conformation of the pyran heterocycle and with the phenomenon of conformational isomerism due to... 

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. 

This unit is composed of three separate procedures describing proanthocyanidin extraction from plant tissue (see Basic Protocol 1), purification (see Basic Protocol 2), and subsequent analysis by reversed-phase HPLC (see Basic Protocol 3). These protocols have been developed and used for analysis of grape skins, grape berries, and grape seeds. Without modification, wine, apples, and pears have also been analyzed using these procedures. 

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