Proanthocyanidins acetone extraction

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. ... 

Flavan-3-ols (catechins, proanthocyanidins, and condensed tannins) can often be extracted directly with water. However, the composition of the extract does vary with the solvent — whether water, methanol, ethanol, acetone, or ethyl acetate. For example, it is claimed that methanol is the best solvent for catechins and 70% acetone for procyanidins. ... 

This extraction method utilizes an aqueous acetone system to extract proanthocyanidins from whole plant tissue. Acetone is preferred over other extraction solvent systems (most notably methanol) because of its ability to solubilize proanthocyanidin-containing material that is insoluble in methanol. 

Place the filtered proanthocyanidin extract into an appropriately sized round-bottom flask and remove acetone on a rotary evaporator equipped with a water aspirator or vacuum pump, at 40°C. 

The extraction of proanthocyanidins is the first step in determining their subunit composition. A number of extraction systems have been investigated in different plant tissues. The most common solvent systems are acetone and methanol with various amounts of water and with or without acid. In general, it has been found that an aqueous acetone system gives the best results in terms of total amount extracted. 

Solvents used for elution in various cases are different, and the order of use also makes differences. For fractionation of polymeric proanthocyanidins, the solvent combination used is acetone with water which is applied for both Sephadex G-50 and LH-20 [22, 154]. The elution of solvents depends on the solubility of specific phenolic components in specific solvents. Anthocyanins are not soluble in ethyl acetate however, many other polyphenolic compounds are soluble in ethyl acetate. Hence, in the protocol mentioned in the book Handbook of Food Analytical Chemistry-Pigments, Colorants, Flavors, Texture, and Bioactive Food Components for the separation of anthocyanins from the rest of the components in a polyphenolic extract, ethyl acetate is used for elution of the polyphenolic components other than anthocyanins, while the anthocyanins are eluted with acidic methanol [12]. pH is another important property affecting the separation of polyphenols through SPE. Phenolic acids have been reported to be completely ionized at pH 7.0 and un-ionized at pH 2.0 hence, this property is used as the basis for fractionation of neutral polyphenohcs at pH 7.0 and acidic polyphenohcs at pH 2.0, which helps in prevention of interference in-between polyphenohc compounds during HPLC [12]. In certain cases, combination of various SPE methods is used for the fractionation of the extracts, and then the purified extracts are further processed to be analyzed [140, 141, 150]. 

The approach we have found to be most useful for the isolation and purification of Type 1 or 2 proanthocyanidin polymers is extraction from plant tissue with acetone-water mixtures (25, 37), separation of the monomers and lower oligomers from the resulting aqueous solution with ethyl acetate, adsorption on Sephadex LH-20 of the aqueous solution diluted with an equal volume of methanol and washing with the same solvent to eliminate impurities. The polymer is then displaced with acetone-water to yield freeze-dried analytically pure material (25). In the case of procyanidins, the ethyl acetate fraction contains monomers (catechin or epicatechin), dimers, trimers, and some tetramers, whereas the LH-20 fractions contain tetramers, on up to genuinely polymeric species. 

Acetone and acetone-water mixtures have also been used for the extraction of anthocyanins,flavonols, and xanthones, or ilavanols and proanthocyanidins, 57,84,85... 

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