Anthocyanins common, 24

This protocol describes isolation of anthocyanins, using cherries as an example, as well as how a pure anthocyanin, cyanidin 3-(2"-glucosyl-6"-rhamnosylglucoside) (S.15 Fig. FI.4.1) is treated before NMR experiments are performed. In this protocol, 20 mg S.15 is dissolved in 0.5 ml of 95 5 (v/v) CD3OD/CF3COOD. Refer to unitfli for further details on extraction, purification, and isolation of anthocyanins. Common NMR solvents for anthocyanins are given in Table Fl.4.8. 

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. 

Wu, X. and Prior, R.L., Identification and characterization of anthocyanins hy high performance liquid chromatography-electrospray ionization-tandem mass spectrometry in common foods in the United States vegetables, nuts, and grains, J. Agric. Food Chem., 53, 3101, 2005. 

The purpose of this chapter is to provide an overview of our present knowledge about the health benehts of pigments, particularly their effects on chronic diseases. We examine the effects of lipophilic (carotenoids, chlorophylls) and hydrophilic pigments (anthocyanins and flavones-flavonols), and curcumin. Descriptive and mechanistic studies are reviewed in regard to common chronic diseases. 

Cyanidin is the most common anthocyanin in foods. In addition, anthocyanins are stabilized by the formation of complexes with other flavonoids (co-pigmentation). In the United States, the daily anthocyanin consumption is estimated at about 200 mg. Several promising studies have reported that consumption of anthocyanin-rich foods is associated with reductions of the risks of cancers - and atherosclerosis and with preventive effects against age-related neuronal and behavioral declines. These beneficial effects of anthocyanins might be related to their reported biological actions such as modulators of immune response and as antioxidants. Knowledge of anthocyanin bioavailability and metabolism is thus essential to better understand their positive health effects. 

As for anthocyanins, betalains are found in vacuoles and cytosols of plant cells. From the various natural sources of betalains, beetroot (Beta vulgaris) and prickly pear cactus (Opuntia ficus indica) are the only edible sources of these compounds. In the food industry, betalains are less commonly used as natural colorants from plant sources than anthocyanins and carotenoids, probably related to their more restricted distribution in nature. To date, red beetroot is the only betalain source exploited for use as a natural food coloring agent. The major betalain in red beetroot is betanin (or betanidin 5-0-P-glucoside). Prickly pear fruits contain mainly (purple-red) betanin and (yellow-orange) indicaxanthin and the color of these fruits is directly related to the betanin-to-indicaxanthin ratio (99 to 1, 1 to 8, and 2 to 1, respectively in white, yellow, and red fruits)." ... 

In the past decade, general interest and research activities focusing on anthocyanins have considerably increased. This increased interest is not only based on the common knowledge that these water-soluble pigments can be used as possible alternatives to artificial food colorants, but also it relates to their bioactive properties. When search-... 

FIGURE 4.3.2 Structures of common mono- and disaccharides occurring in anthocyanins from foods. 

To obtain anthocyanins closer to their natural state, a number of researchers have used neutral solvents for initial extraction such as 60% methanol, n-butanol, cold acetone, mixtures of acetone, methanol, and water, or simply water. Methanol is the most common solvent used for anthocyanin extraction. Metivier et al. (1980) compared the efficiency of extraction with three different solvents (methanol, ethanol, and water) and different acids, and found that methanol extraction was 20% more effective than ethanol and 73% more effective than water when used for anthocyanin recovery from grape pomace. 

Maceration of crnshed or gronnd material in methanol containing small amounts of HCl (<1%) is commonly used at refrigerated temperatures for times ranging from a few hours to overnight. The extracted material is usually too dilute for further analyses and the extraction procednre is usually followed by evaporation of the methanol using vacnnm and mild temperatures (30 to 40°C). Alternatively, the plant materials and solvents can be mixed well with a laboratory blender for a few minutes or a chemical-resistant stir bar for a longer time. Concentration of anthocyanin extracts can be done by rotary evaporation under vacuum conditions for volatile solvents or lyophilization for water. 

Tian, Q. et al., Screening for anthocyanins using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry with precursor-ion analysis, product-ion analysis, common-neutral-loss analysis, and selected reaction monitoring, J. Chromatogr. A, 1091, 72, 2005. 

Anthocyanins are the deeply colored glycosides of the aglycone anthocyanidins. Only the six aglycones shown in Figure 13.6 are common, although others occur sporadically [11,12]. 

Anthocyanins are Che most easily assayed and commonly studied derivatives of aromatic amino acids (Figure 1). Glyphosate drastically reduces accumulation of anthocyanin flavonoids in treated tissues (6, 19) (Figure 3). Levels of rutin and procyanidin, both flavonoids, are reduced in glyphosate-treated buckwheat hypocotyls (6). Glyphosate would presumably similarly affect levels of flavonoids and flavonoid derivatives that are known to be allelochemicals. 

Food and plant phenolics are commonly detected using DAD detectors (Tan and others 2008). Photodiode array detection allows collection of the entire UV spectrum during the elution of a chromatographic peak, which makes it possible to identify a phenolic compound by its spectra. Simple phenols, phenolic acids, flavanones, benzophenones, isoflavones, and flavan-3-ols have maximum absorbance at 280 nm, hydroxycinnamic acids at 320 nm, flavonols, flavones, and dihydroflavonols at 365 nm, and anthocyanins at 520 nm (Ibern-G6mez and others 2002 Merken Hand Beecher 2000). Hydrolyzable tannins show a characteristic shoulder at 300 nm, suitable for identifying them (Arapitsas and others 2007). For stilbenes, maximum absorbance of trans-forms are at 306 nm and at 285 nm for cA-forms (Lamuela-Raventos and others 1995). 

Flavanones In some cases, flavanones produced by CHI will accumulate to sizeable amounts instead of being diverted away to form flavonols, anthocyanins, and flavanols (see Fig. 5.4). These flavanone products, hesperetin and naringenin being the most common, are frequently encountered in citrus fruits and juices (USDA Flavonoids Database Release 2.1,2007). In most of these cases, essentially no flavonols or anthocyanins are encountered the flavonoid pathway is essentially blocked at the F3H step. 

Anthocyanins are extracted commercially using either acidified water or alcohol. The extract is then vacuum evaporated to produce a commercial colour concentrate. The raw materials can be blackcurrants, hibiscus, elderberry, red cabbage or black grape skins. The most commonly used commercially are black grape skins, which can be obtained as a by-product. 

Numerous CE separations have been published for synthetic colours, sweeteners and preservatives (Frazier et al., 2000a Sadecka and Polonsky, 2000 Frazier et al., 2000b). A rapid CZE separation with diode array detection for six common synthetic food dyes in beverages, jellies and symps was described by Perez-Urquiza and Beltran (2000). Kuo et al. (1998) separated eight colours within 10 minutes using a pH 9.5 borax-NaOH buffer containing 5 mM /3-cyclodextrin. This latter method was suitable for separation of synthetic food colours in ice-cream bars and fmit soda drinks with very limited sample preparation. However the procedure was not validated for quantitative analysis. A review of natural colours and pigments analysis was made by Watanabe and Terabe (2000). Da Costa et al. (2000) reviewed the analysis of anthocyanin colours by CE and HPLC but concluded that the latter technique is more robust and applicable to complex sample types. Caramel type IV in soft drinks was identified and quantified by CE (Royle et al., 1998). 

Wu X, Beecher GR, Holden JM et al (2006) Concentrations of anthocyanins in common foods in the United States and estimation of normal consumption. J Agric Pood Chem 54 4069 075... 

The most frequently used detection method for HPLC is UV spectrophotometry. Routine detection in HPLC is typically based on measurement of UV absorption, or visible absorption in the case of anthocyanins. No single wavelength is ideal for all classes of flavonoids since they display absorbance maxima at distinctly different wavelengths. The most commonly used wavelength for routine detection has been 280 nm, which represents a suitable compromise. 

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