Anthocyanidins analysis

As we have seen above, anthocyanins comprise an aglycone fraction commonly known as anthocyanidin and a frequently acylated osidic substituent. This characteristic leads to two different approaches for the analysis of these pigments (1) a direct anthocyanin analysis without a hydrolysis stage requiring identification of a number of molecules (several hundreds in the plant kingdom) or (2) an analysis of the anthocyanidin fraction only after hydrolysis of the anthocyanins present in the medium. 

HPLC has proved to be fast and sensitive for the analyses of phenolic plant constit-nents, and is especially useful for the analysis of anthocyanins. The first application of HPLC to anthocyanin analyses was in 1975 by Manley and Shubiak and it has now become the method of choice for the separation of mixtures of anthocyanins and anthocyanidins. HPLC is now used for anthocyanin qualitative, quantitative, and preparative work, offering improved resolution compared to chromatographic procedures previously employed. It also allows for simultaneous rapid monitoring of the eluting anthocyanins. ... 

From extensive analysis of recombinant proteins, and the crystal structure of A. thaliana protein, detailed reaction mechanisms have been proposed. The ANS reaction likely proceeds via stereospecific hydroxylation of the leucoanthocyanidin (flavan-3,4-cA-diol) at the C-3 to give a flavan-3,3,4-triol, which spontaneously 2,3-dehydrates and isomerizes to 2-flaven-3,4-diol, which then spontaneously isomerizes to a thermodynamically more stable anthocyanidin pseudobase, 3-flaven-2,3-diol (Figure 3.2). The formation of 3-flaven-2,3-diol via the 2-flaven-3,4-diol was previously hypothesized by Heller and Forkmann. The reaction sequence, and the subsequent formation of the anthocyanidin 3-D-glycoside, does not require activity of a separate dehydratase, which was once postulated. Recombinant ANS and uridine diphosphate (UDP)-glucose flavonoid 3-D-glucosyltransferase (F3GT, sometimes... 

Table F1.3.4 Troubleshooting Guide for HPLC Analysis of Anthocyanins and Anthocyanidins...

Since the work of Manley and Shubiak (182), who were the first to apply HPLC to anthocyanin analysis, numerous HPLC techniques have been developed for the separation and quantification of anthocyanins and anthocyanidins. Nowadays HPLC has become the method of choice, because it offers the advantage that it is a rapid, sensitive, and quantitative method. For the peak identification and quantitative evaluation of chromatograms, the use of pure anthocyanin standards is recommended however, only a limited, but constantly increasing, number of substances is avail-... 

Paolocci F, Robbins MP, Madeo L, Arcioni S, Martens S, Damiani F. 2007. Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. Structure, expression, analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus. Plant Physiol 143 504-516. 

Anthocyanins are generally more stable at an acidic pH. Therefore, anthocyanins are commonly extracted under cold conditions using either acidic methanol or ethanol to avoid degradation1 5169 (Table 3.4). In comparison, acetone allows more reproducible extraction and avoids problems with pectins. However, it is limited by the coextraction of proanthocyanins.39 In general, ethanol is preferable as an extraction solvent, although it can require an additional step for the removal of lipid-soluble substances. SPE using Ci8, polyamide, HLB (hydrophilic lipophilic balanced stationary phases), or Amberlite has been employed for the purification of anthocyanidins prior to HPLC analysis.39-51 66 69... 

The polyphenols such as anthocyanidins might have a protective effect on atherosclerosis. This concept suggests that the Recommended Optimal Intake (ROI) is a more important factor for the antioxidation than the Recommended Dietary Allowance (RDA). Now, considering the absence of the appropriate methodology to identify anthocyanidins in the plasma, the analysis of the plasma antioxidant capacity might be a suitable index to define the optimal nutritional intake [95]. 

With HPLC methods where individual anthocyanins are separated and the method of detection is DAD, individual anthocyanin standards are preferred for quantitation, but not always available. A standard mix of the glucoside form of the six aglycones of the anthocyanidins has been commercially available. However, obtaining a consistent purity has been a challenge at times. Because of the large numbers of anthocyanins present, it is not reasonable at this point in time to have a standard for every individual anthocyanin. Thus, some assumptions and compromises must be made in any quantitative analysis of anthocyanins. Calculation of the anthocyanins containing the same anthocyanidin based upon the glucoside equivalent has been a workable compromise. 

Anthocyanins are intensely coloured plant pigments, consisting of glycosylated anthocyanidins. MS analysis of phenols, procyanidins, and anthocyanins in grapes and wines was reviewed by Flamini [79]. Some studies on the LC-MS analysis of anthocyanidins are briefly reviewed here. 

The MS analysis can be performed either by ESI-direct injection of the sample or LC separation. Table 6.18 reports the anthocyanidin C4 derivatives identified by ESI/MS of fraction 4 (point m in Fig. 6.25) of a red wine and a grape marc extract. 

Datzberger, K., Steiner, I. Washuttl, J., and Kroyer, G. 1991. Methods for the fast analysis of anthocyanins and anthocyanidins in red wine. Z. Lebensm. Unters. Forsch. 193, 462-464. 

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