Anthocyanins hydrolysis

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. 

Just as with anthocyanin analysis, the advent of HPLC/mass spectrometer coupling made it possible to avoid acid hydrolysis for flavonoid identification. Maata... 

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... 

Saponification or hydrolysis with a base (10% KOH for 8 min) will not affect the glycosidic bond, but it will break down the ester linkages of acylating groups. This procedure can be used to determine with certainty which anthocyanins are... 

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. 

Suggested changes would be to increase the percentage of solution A at the beginning if retention times are excessively long or decrease it at 20 min if there is incomplete resolution. It is not unusual to have minor amounts of unhydrolyzed anthocyanins glycosides in the sample. If amounts are excessive, increase the hydrolysis time or decrease the amount of sample subjected to hydrolysis. 

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-... 

---