Isolation of anthocyanins

Fortunately, the appearance in the last years of new, more accurate methods and sophisticated equipments permitted the isolation of anthocyanins on a preparative scale and allowed the identification of extremely complex and stable anthocyanins. 

Schwarz, M. et al.. Application of high-speed conntercurrent chromatography to the large-scale isolation of anthocyanins, Biochem. Eng. J., 14, 179, 2003. 

A combined technique has been developed for the preparative isolation of anthocyanins from blackcurrant (Ribes nigrum) fruits. Frozen berries (370 g) were extracted three times with 11 of methanol containing 0.1 per cent TFA at 4°C for 6 h. The combined exracts were concentrated, diluted to 250 ml with water and extracted with ethyl acetate. The aqueous phase was concentrated to 100 ml and applied in an ion-exchange column. The column... 

C. Froytog, R. Slimestad and O.M. Andersen, Combination of chromatographic techniques for the preparative isolation of anthocyanins — applied on blackcurrant (Ribes nigrum) fruits. J. Chromatogr.A 825 (1998) 89-95. 

Degenhardt, A. et al., Preparative isolation of anthocyanins by high-speed countercurrent chromatography and application of the color activity concept to red wine. J. Agric. Food Chem. 48, 5812, 2000. 

This protocol describes isolation of anthocyanins, using cherries as an example, as well as how a pure anthocyanin, cyanidin 3-(2"-glucosyl-6"-rhamnosylglucoside) (S.15 Fig. FI.4.1) is treated before NMR experiments are performed. In this protocol, 20 mg S.15 is dissolved in 0.5 ml of 95 5 (v/v) CD3OD/CF3COOD. Refer to unitfli for further details on extraction, purification, and isolation of anthocyanins. Common NMR solvents for anthocyanins are given in Table Fl.4.8. 

Paper chromatography may be used as one step for separation and/or isolation of anthocyanins from complex samples. Thin-layer chromatography, performed on cellulose or silica gel plates, was in the first period of use a convenient extension of paper chromatography. The introduction of new stationary phases and the development of instrumentation offering the possibility of quantitative analysis provided new possibilities for method developments. The advantages of TLC are the simplicity in terms of instrumentation, sample preparation and data evaluation. 

Hong, V. and Wrolstad, R.E., Use of HPLC separation/photodiode array detection for characterization of anthocyanins, J. Agric. Food Chem., 38, 708, 1990. Osmianski, J. and Lee, C.Y., Isolation and HPLC determination of phenolic compounds in red grapes. Am. J. Enol. Vitic., 41, 204, 1990. 

The total number of different anthocyanins reported to be isolated from plants was 539. However, the number of anthocyanins found in foods is much smaller. Although a large number of papers were published regarding anthocyanin composition in several foods, investigators in most studies used only chromatographic and chemical behaviors as bases for identification. In this chapter, we considered only papers in which identification was based at least on mass spectrometry (MS). In fact, the use of only MS and UV-visible information can easily lead to misidentifi-cation as the following example shows. 

Research into the copigmentation of anthocyanins started as early as 1913 when Willstatter and Everest determined the chemical structure of cyanidin 3-glucoside isolated from blue cornflowers and red roses, and attributed the color changes to different pH levels in cell saps. This theory, however, was questioned and in 1916, Willstatter and Zollinger,revising the previous work, proposed a new theory according to which the colors of the anthocyanins varied significantly by the effects... 

Choung, M.-G. et al., Isolation and determination of anthocyanins in seed coats of black soybean (Glycine max (L.) Merr.), J. Agric. Food Chem., 49, 5848, 2001. 

Salas, E. et al.. Isolation of flavanol-anthocyanin adducts by countercurrent chromatography, J. Chrom. ScL, 43, 488, 2005. 

A biphasic solvent system composed of tert-butyl methyl ether, -butanol, acetonitrile, and water (2 2 1 5) acidified with triflnoroacetic acid has been applied to fractionate anthocyanins. The npper (organic) phase acts as the stationary phase and the lower (aqneons) as the mobile phase. HSCCC has been applied to obtain several anthocyanin fractions from wine, red cabbage, black cnrrants, chokeber-ries, " bilberries (Vaccinium myrtillus) acylated anthocyanins, and also isolate individnal anthocyanins from wine. ... 

The use of mass spectroscopic analyses for characterization of anthocyanins has increased dramatically over the past decade. Most reports cite the use of HPLC coupled to MS detectors or isolating individual pigments prior to the mass spectroscopic analysis. - - " ... 

Gasiorowski, K. et al., Antimutagenic activity of anthocyanins isolated from Aronia melanocarpa fruits, Cancer Lett., 119, 37, 1997. 

Rodriguez-Saona, L.E. and Wrolstad, R.E., Extraction, isolation, and purification of anthocyanins, in Current Protocols in Food Analytical Chemistry, Wrolstad, R.E., et al., Eds., John Wiley Sons, New York, 2001, 1. 

Du, Q., Jerz, G., and Winterhalter, P., Isolation of two anthocyanin sambubiosides from bilberry (Vaccinium myrtillus) by high-speed counter-current chromatography, J. Chromatogr A, 1045, 59, 2004. 

Strack, D. et al., Cyanidin 3-oxalylglncoside in orchids, J. BioscL, 41, 707, 1986. Choung, M.-G. et al.. Isolation and determination of anthocyanins in seed coats of black soybean (Glycine max (L.) Merr.), J. Agric. Food Chem., 49, 5848, 2001. Covey, T., Analytical characteristics of the electrospray ionization process, in Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry, ACS Symposium Series, Snyder, A.P. and Anaheim, C. A., Eds., Washington, D.C., 1995, chap. 2. 

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. 

Novel pyranoanthocyanins have also been isolated and identified in blackcurrant (Ribes nigrum) seed using HPLC, 2D NMR and ES-MS. Blackcurrant seeds were extracted with acetone-water (70 30, v/v) and the components of the extract were separated in a polyamide column followed by HPLC-DAD. The new pigments were finally separated in an MCI-HP20 column. The chemical structures of anthocyanins 1-2 and the novel pyranoanthocyanins 3-6 with the pyrano[4,3,2-de]-l-bcn/opyrylium core structure are shown in Fig. 2.110. It was stated that the analytical method developed separated well the novel pyranoanthocyanins [245],... 

Choung M, Baek 1-Y, Kang S-T et al (2001) Isolation and determination of anthocyanins in seed coats of black soybean (Glycine max (L.) Merr.). J Agric Food Chem 49 5848-5851 Lee JH, Kang NS, Shin S-O et al (2009) Characterization of anthocyanins in the black soybean (Glycine max L.) by HPLC-DAD-ESI/MS analysis. Food Chem 112 226-231 Springob K, Nakajima 1, Yamazaki M et al (2003) Recent advances in the biosynthesis and accumulation of anthocyanins. Nat Prod Rep 20 288-303... 

Absorption spectra have also been used in the reexamination of pH-dependent color and structural transformations in aqueous solutions of some nonacylated anthocyanins and synthetic flavylium salts." ° In a recent study, the UV-Vis spectra of flower extracts of Hibiscus rosasinensis have been measured between 240 and 748 nm at pH values ranging from 1.1 to 13.0." Deconvolution of these spectra using the parallel factor analysis (PARAFAC) model permitted the study of anthocyanin systems without isolation and purification of the individual species (Figure 2.21). The model allowed identification of seven anthocyanin equilibrium forms, namely the flavylium cation, carbinol, quinoidal base, and E- and Z-chalcone and their ionized forms, as well as their relative concentrations as a function of pH. The spectral profiles recovered were in agreement with previous models of equilibrium forms reported in literature, based on studies of pure pigments. 

Chandler, V.R. et al.. Two regulatory genes of the maize anthocyanin pathway are homologous isolation of B using R genomic sequences. Plant Cell, 1, 1175, 1989. 

The total number of different anthocyanins reported to be isolated from plants in this review is 539 (Appendix A). This number includes 277 anthocyanins that have been identified later than 1992. Several previously reported anthocyanins have for the first time received complete structural elucidation, and some structures have been revised. The majority of anthocyanins with the most complex structures and highest molecular masses have been reported in the period of this review. 

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