Hydrolysis of anthocyanins

Hydrolysis of anthocyanins to yield anthocyanidins might be another important step during analysis. Hydrolysis is either accomplished by acidic hydrolysis of crude or purified extracts or by one-step extraction/hydrolysis protocols. 

For extraction and hydrolysis of anthocyanins weigh in accurately 0.01-5 g source material (dependent on the expected content) into a 500 mL roimd neck flask and add approximately 100 mL methanol/water/HCl=93/5/2 (vol/ vol/vol) and blend carefully with an ultra-turrax. 

The main problem of submission of anthocyanins to UVA IS spectroscopy is the diversity of possible structures with different absorption properties and the lack of reliable calibration procedures. Hydrolysis of anthocyanins to anthocyanidins prior to measurement reduces possible UVA IS absorption variations in both, X max and e, to few well described structures. Yet another advantage of hydrolysis is that most anthocyanidins but only few anthocyanins are commercially available as reference standards for method calibration. The main disadvantages of acidic hydrolysis are that, oligomeric proanthocyanidins present in the sample are also hydrolyzed to anthocyanidins yielding possibly over-estimation of the original monomeric anthocyanin content. 

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. 

The hydrolysis of zeaxanthin esters by a carboxyl ester lipase indeed enhanced both the incorporation of zeaxanthin in the micellar phase and uptake of zeaxanthin by Caco-2 cells. As mentioned earher, carotenoids can also be linked to proteins by specific bindings in nature and these carotenoid-protein complexes may slow the digestion process and thus make their assimilation by the human body more difficult than the assimilation of free carotenoids. Anthocyanins are usually found in a glycosylated form that can be acetylated and the linked sugars are mostly glucose, galactose, rhamnose, and arabinose. 

Potential enzymes involved in anthocyanin metabolism — The lactase phlorizin hydrolase (LPH EC 3.2.1.108) present only in the small intestine on the outside of the brush border membrane and the cytosolic P-glucosidase (CBG EC 3.2.1.1) found in many tissues, particularly in liver, can catalyze the deglycosylation (or hydrolysis) of polyphenols. LPH may play a major role in polyphenol metabolism... 

Many methods are currently available for the qualitative analysis of anthocyanins including hydrolysis procedures," evaluation of spectral characteristics, mass spectroscopy (MS), " nuclear magnetic resonance (NMR), and Fourier transform infrared (FTIR) spectroscopy. - Frequently a multi-step procedure will be used for... 

This is the classical method of extracting anthocyanins from plant materials. This procedure involves maceration or soaking of the plant material in methanol containing a small concentration of mineral acid (e.g., HC1). Methanol extraction is a rapid, easy, and efficient method for anthocyanin extraction. However, a crude aqueous extract with several contaminants is obtained, and methanol evaporation can result in hydrolysis of labile acyl linkages, which is aggravated by the presence of HC1. 

Either Basic Protocol 1 or the Alternate Protocol should first be conducted in analyzing an unknown sample. Because of its ease and simplicity, sample preparation of anthocyanins and their HPLC separation on silica Cl8 columns (see Basic Protocol 1) is usually the preferred choice, unless the presence of acylated anthocyanins is anticipated, in which case the protocol described for acylated anthocyanins is used (see Alternate Protocol). If the anthocyanin profile is inconsistent with previously published chromatograms, or if there are extraneous unidentified peaks, then simplification is recommended (see Basic Protocol 3). Acid hydrolysis will simplify the chroma-... 

Figure FI. 3.4 shows HPLC chromatograms for anthocyanidins generated from acid hydrolysis of concord grape and strawberry juices. Extraneous peaks may be present because of incomplete hydrolysis, and degradation and polymerization of the labile aglycons even more of a problem. For acylated anthocyanins, higher yields of anthocyanidins will be achieved if the sample is first saponified (see Basic Protocol 3) and then subjected to acid hydrolysis (see Basic Protocol 2).

However, due to the artifacts resulting from oxidation, hydrolysis of esters or ethers, or isomerization of phenolics during pretreatment of wines, as well as due to the low recovery rates of some phenolics, analysis of wine phenolics via direct injection of the filtered wine into the chromatographic column is often selected (80,82-84). For the red wine and musts (80), which were injected directly into the HPLC without sample preparation, a ternary-gradient system was often employed for phenolic compounds. Twenty-two phenolic compounds, including 10 anthocyanins, were analyzed from red wine. The separation of cinnamic acid derivatives (313 nm),... 

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