Extraction Methods of Anthocyanin Pigments

Method Characteristics

0.001% HCl in methanol

0.001% HCl in ethanol 0.001% HCl in water Methanol acidified with citric acid Water acidified with acetic acid Water with 1000 ppm of SO2

It is the most effective method but HCl is corrosive and methanol has toxic effects in human health 80% as effective as methanol 27% as effective as methanol It is the most effective of the organic acids In efficiency is followed by citric, tartaric and hydrochloric acid Extraction is better than that obtained by the use of the traditional extraction which involves ethanol: acetic acid: water system

Source: Adapted from Delgado-Vargas et al. (2000).17

may adulterate their products to achieve a better visual appearance, introducing other sources of colors and consequently a commercial advantage; analysis of anthocya-nins has been undertaken to detect adulteration in these products. The methodology is based on the fact that each fruit has a characteristic anthocyanin pattern;35 this approach has been used in the quality control of prune juice, cherry jams (which frequently are prepared with red cherries, i.e., less expensive fruits), blackberry jams, and wines.3637 In this respect, the HPLC C18 methodology combined with multivari-ate statistical methods is suggested as suitable for routine analysis of red wines.38

6. Methodological Aspects a. Extraction

Anthocyanins are polar molecules and consequently are more soluble in polar than in nonpolar solvents. However, it is clear that solubility depends on several factors, including certain media conditions. At a pH value where the anthocyanin molecule is nonionized, anthocyanins could be soluble in ether39 and are not stable in neutral or alkaline solutions; thus, the conventional methods of anthocyanin extraction involve the use of acidic solvents (Table 8.7).17 As can be noticed, the extraction systems have been modified to produce better yields but have been compromised by safety concerns. Hydrochloric acid serves to maintain a low pH that simultaneously favors the formation of the anthocyanin-colored forms; however, this is a strong acid, which can alter the native form of anthocyanins by breaking some weak associations with metals and copigments, for example. Some anthocyanins contain aliphatic dicarboxyl acyl groups such as malonic, malic, and oxalic. These acids are susceptible to diluted solutions of hydrochloric acid; thus, weaker acids (e.g., citric acid or acetic acid) are used in the extraction procedure (Table 8.7).3,40 Consequently, the use of a weak organic acid is recommended for the extraction of complex anthocyanins. In addition, the use of ethanol or aqueous SO2 or bisulfite solutions has been introduced to obtain concentrates with good quality characteristics; in fact, this system is used to extract the anthocyanins from sunflower hulls using sulfurous water (1000 ppm SO2). Complete extraction is reached in 1 h, suggesting that the interaction of anthocyanins with HSO- ions is responsible for improved anthocyanin solubility easing their diffusion through cell walls.41

b. Separation

In crude extracts, anthocyanins are commonly mixed with considerable quantities of extraneous material such as other polyphenols, pectins, and sugars. As can be expected, the study, characterization, and use of these anthocyanins imply the use of chromatographic techniques to purify them. In anthocyanin purification it is common to employ insoluble polyvinylpyrrolidone (PVP), polyamide-PVP, Sepha-dex G-25 or LH-20, reverse-phase C18, weak anion exchange (e.g., Amberlite CG-50), polyethylene glycol dimethacrylate, and cellulose-type resins. The gel Toyopearl HW-40F has been used to separate anthocyanins, and resolution is better than with Sephadex LH-20.42

Methodologies of separation involve adsorption in paper chromatography, thin-layer chromatography (TLC), droplet countercurrent chromatography, and, today, HPLC and high-performance capillary chromatography to solve complex mixtures, which permit their separation and quantitation. As can be imagined, solvent systems vary and must be adapted to the particular extract being analyzed.1,4

c. Characterization

Spectroscopy. Anthocyanins are colored pigments and they are commonly studied by UV-visible spectrophotometry; this methodology gives very valuable information about the structure of anthocyanins (acylation, glycosylation, presence of methoxyl groups, and copigments) (Table 8.8).1317 On the other hand, different methodologies have been employed to elucidate the anthocyanin structure (Table 8.9).17 Particularly important is the introduction of the diode array detector (DAD) coupled with the HPLC, which is commonly used in most laboratories and industries. In general, the use of nuclear magnetic resonance (NMR) in one or two dimensions permits complete structural characterization of anthocyanins. In particular, proton NMR has been used to study the copigmentation phenomena; the main drawback of NMR is that large quantities of purified material are required. When only small amounts of material are available, it is common to use MS. This methodology was generalized after the introduction of the fast atom bombardment (FAB), which permits the ionization of polar and unstable molecules; this ionization chamber has been followed by improvements such as electrospray and thermospray. In addition, HPLC coupled with MS was recently introduced; this is the methodology of choice in the study of natural products including anthocyanins. HPLC/MS using atmospheric pressure ionization (API), electrospray ionization (ESI), and MS, and ion trap multiple mass spectrometry (MS/MS) has been successfully applied to identify antho-cyanins. In addition, matrix-assisted laser desorption/ionization (MALDI) MS (MALDI-MS) has been recently evaluated for anthocyanin determination in red wine and fruit juices resulting in highly qualitative and quantitative reproducible results; this was the first study of applications in food.43 In any case, the classical hydrolysis

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