Extraction, Isolation, and Purification of Anthocyanins

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. 

Analytical methods for the isolation, separation, and characterization of anthocyanins have been described (Markham, 1982 Jackman et al., 1987 Harbome, 1998 Rivas-Gonzalo, 2003 Andersen and Francis, 2004). A comprehensive and highly recommended source for anyone involved in anthocyanin analysis is that of Strack and Wray (1989). Further details of flavonoid chemistry can be found in volumes of The Flavonoids series (Harbome et al., 1975 Harbome and Mabry, 1982 Harbome, 1988, 1994). Recent advances in flavonoid research are thoroughly described in the book Flavonoids Chemistry, Biochemistry and Applications, edited by Andersen and Markham (2006). Extensive information on the occurrence of anthocyanins in various natural products reported after 1992 was presented by Andersen and Jordheim (2006). One of the most useful sources of current protocols of anthocyanin analysis is the Handbook of Food Analytical Chemistry, Pigments, Colorants, Flavors, Texture, and Bioactive Food Components, edited by Wrolstad et al. (2004). This book is a practical how to manual that contains detailed in-stmctions on the following topics (1) extraction, isolation, and purification of anthocyanins, (2) characterization and measurement of anthocyanins by UV-Vis... 

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. 

The Basic Protocol describes the reversed-phase HPLC analysis of polyphenolic compounds isolated into nonanthocyanin and anthocyanin fractions by solid-phase extraction. The Alternate Protocol describes the HPLC separation of acidic and neutral polyphenolic fractions. Fractionated samples are used because significant amounts of interfering compounds are extracted along with polyphenolics from plant materials. Solid-phase extraction with C18 Sep-Pak cartridges (vnitu.2) is used to selectively eliminate undesired components from crude extracts, and may minimize the effects of sample cleanup or preparation on the integrity of polyphenolics. The isolation and purification step using solid-phase extraction of polyphenolics will make possible the efficient analysis of individual polyphenolics by reversed-phase HPLC. 

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. 

Different resins have been used to clean up or prefractionate anthocyanins prior to isolation or characterization, including ion-ex-change resins, polyamide powders, and gel materials. Chromatography on Dowex or Amber-lite ion-exchange resins, as well as polyamide powders such as polyvinylpyrrolidone (PVP), have been used to isolate polar nonphenolic compounds from crude anthocyanin extracts. Column chromatography on Sephadex LH-20 can be used for fractionation of crude extracts and is also particularly useful for purification... 

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