Anthocyanins other solvents

This mechanism can be extrapolated to other enolizable precursors potentially present in wine, including yeast metabolites such as a-ketoglutatic acid and 2-hydroxybutan-2-one, but also to acetone, which can react with anthocyanins during solvent extraction proced-ures. The resulting products are pyranoanthocyanins as presented in Figure 5.11, with Rs = CH2-COOH, R4 = COOH R3 = H, R4 = CHOH CH3 R3 = H, R4 = CH3. 

Supercritical fluid extraction (SEE) is another modern separation technology usually employed to extract lipophilic compounds such as cranberry seed oil, lycopene, coumarins, and other seed oils. Anthocyanins generally and glycosylated anthocyanins in particular were considered unsuitable for SEE due to their hydrophilic properties, since SEE is applicable for non-polar analytes. However, a small amount of methanol was applied as co-solvent to increase CO2 polarity in anthocyanin extraction from grape pomace. New applications of SEE for anthocyanin purification have been reported for cosmetic applications from red fruits. ... 

The wavelength of maximum absorption and the molar absorptivity are very dependent on pH, buffer, temperature, solvent, and the presence of other materials that may interact with anthocyanins. In addition, anthocyanin absorption follows a linear relationship with concentration only when present at low levels therefore considerable dilution is usually necessary. Absorbance normally should vary from 0.2 to 1.0 unit in order to obey Lambert-Beer s law. However, absorbance values as high as 1.5 to 2.0 absorbance units may be valid for sophisticated new instruments. 

Purification of anthocyanin-containing extracts is often necessary for further structural identification. Since none of the solvents used for extraction is specific for anthocyanins, considerable amounts of other compounds may be also extracted and concentrated. The variety and concentration of other compounds will depend on the solvent and methodologies used. The presence of extraneous materials could affect the stability and/or analysis of anthocyanins. Therefore, the next step toward anthocyanin characterization is the purification of those extracts. 

When appreciable amounts of pectin, proteins, lipids, unwanted polyphenols, or other compounds are suspected to be present in anthocyanin-containing extracts, some of them can be precipitated or the anthocyanins may be crystalhzed and separated from the others. Pectin and proteins can be removed by organic solvents such as methanol and acetone in order to reduce their solubility, then precipitated and separated by centrifugation. Gelatin was used to remove proanthocyanidin due to its high molecular weight. Anthocyanins were reported to be precipitated early by lead acetate to achieve isolation from other materials. ... 

To obtain anthocyanins closer to their natural state, a number of researchers have performed the initial extraction using neutral solvents such as 60% methanol, n-butanol, cold acetone, acetone/methanol/water mixtures, or simply water (Jackman et al., 1987). Others have isolated anthocyanin pigments with mix-... 

C18 solid-phase extraction is used to fractionate polyphenolics for their identification and characterization. This technique can eliminate interfering chemicals from crude extracts and produce desirable results for HPLC or other analytical procedures. To obtain a sufficient volume for all analyses, several separations by solid-phase extraction may be performed. The individual fractions need to be combined and dissolved in solvents appropriate for HPLC analysis. In Basic Protocol 2, the application of a current of nitrogen gas for the removal of water from the C18 cartridge is an important step in the selective fractionation of polyphenolics into non-anthocy-anin and anthocyanin fractions. After the collection of non-anthocyanin polyphenolics, no additional work is necessary to elute anthocyanins bound to the C18 solid phase if anthocyanins are not to be determined. 

It is important to remember the few restrictions imposed by electrospray when considering an LC-MS analysis. Common solvents like methanol, water, acetonitrile and volatile salts (below 25 mM) like ammonium acetate and ammonium bicarbonate are acceptable in the mobile phase, whereas phosphate salts/buffers, mineral acids or other nonvolatile components cannot be used. Unfortunately, this conflicts with many of the routine mobile phases used for the analysis of phenolic compounds and anthocyanins, necessitating changes in methods when going from LC to LC-MS analyses. 

Fig. 4 Thin-layer chromatograms and visible absorption spectra of the hydrolyzed gardenia yellow extracted from commercial foods under TLC/scanning densitometry. (A) Hydrolyized gardenia yellow standard. (B) Candy containing gardenia yellow and anthocyanin. (C) Crocetin. Plate RP-18 (E. Merck). Solvent system acetonitrile-tetrahydrofuran-0.1 mol/L oxalic acid (7 8 7). Other conditions see Fig. 1.

Since water is perhaps the most environmentally friendly solvent available at high purity and low cost, it has been exploited for the extraction of avoparcin in animal tissue (53), fungicides in agricultural commodities (54), fragrances from clove (55), antioxidative compounds from sage (56), anthocyanins and total phenolics from dried red grape skin (48), and other bioactive compounds from plants (57). See also chapters 5 and 6 in this book uid a few review articles (51,58). 

Alternatively berries can be freeze dried prior to extraction. Dried, berry anthocyanin extracts are dissolved in methanol or ethanol containing 0.1-2.0 % cone. HCl, kept for 5 minutes in an ultrasonic bath and finally cleared by filtration. Deny juice is best acidified with HCl (0.1-2.0 %) for stabilization and diluted with an appropriate solvent. If anthocyanins are extracted from dietary supplements or from processed food products (i.e., beny jam or ice cream), repeated extraction with mixtures of polar organic solvents and aqueous acids is usually appropriate. The aqueous part is most often necessary for dissolution of the other ingredients (e.g., sugars, proteins). 

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