Fractionation anthocyanin extraction

Different resins have been used to clean up or prefractionate anthocyanins prior to isolation or characterization, including ion-ex-change resins, polyamide powders, and gel materials. Chromatography on Dowex or Amber-lite ion-exchange resins, as well as polyamide powders such as polyvinylpyrrolidone (PVP), have been used to isolate polar nonphenolic compounds from crude anthocyanin extracts. Column chromatography on Sephadex LH-20 can be used for fractionation of crude extracts and is also particularly useful for purification... 

Purification of anthocyanins extracted fi om berries is not deemed necessary if measurement of UV/VlS-absorption (>500 nm) for quantification is used. Except in cases of adulteration with red or blue food colorants the measurement wave lengths are selective enough to quantify anthocyanins in crude extracts. If berry anthocyanins are extracted from complex matrices (dietary supplements or food products) removal of other ingredients extracted is advantageous [125, 126]. For structural elucidation, for the analysis of the specific glycosidation/acylation pattern or the requirement to enrich anthocyanins from low content sources, crude extracts need to be purified and eventually fractionated [63, 67, 114, 118, 127-131]. 

ESE has several additional advantages over conventional solvent extraction, such as the possibility of extract fractionation and a higher extraction flexibility, which is offered by the possibility of modifying solvent dissolution capability just by changing operational conditions as dissolved CO2, and temperature and pressure, which can also be explored. The presented work protocols for anthocyanins extraction use solvents and techniques considered as acceptable and generally regarded as safe in the food and pharmaceutical industries [17]. 

A novel pigment has been isolated from the petals of Rosa hybrida with complex chromatographic techniques and the structure was elucidated with spectroscopic methods and high resolution fast-atom bombardement mass spectrometry, lH NMR, and FTIR. Anthocyanins were extracted from 7.9 kg of petals of Rosa hybrida cv. M me Violet with 80 per cent aqueous ACN containing 0.1 per cent TFA. The extract was purified in a Sephadex LH-20 column, and the fraction eluted with 80 per cent ACN was further fractionated in a HP-20 column using water, 15, 20 and 30 per cent ACN as mobile phases. The last fraction was lyophilized and separated by preparative RP-HPLC using an ODS column (50 X 5 cm i.d.). Solvents were 0.5 per cent aqueous TFA (A) and water-methanol... 

TOTAL MONOMER (MAF). RED (RPAF) AND YELLOW-BROWN POLYMER (YBPAF) ANTHOCYANIN FRACTIONS OF COMMERCIAL ANTHO-CYANIN-RICH EXTRACTS... 

Fig. 4.2 HPLC-DAD chromatograms of anthocyanins from soluble and insoluble extracts of black, red-brown, and brown soybean seed coats at 520 nm. (a, d) Black (Clark), (b, e) red-brown (Mil), and (c, f) brown (MlOO) seed coats, (a and b) Pulverized fresh seed coats extracted with 80% methanol in water, (d-f) Insoluble pulverized seed coat fraction extracted with 1-butanol/HCl (19 1) 1% SDS. Compound identifications were based on comparison of retention times and absorption spectra to authentic standards. Peak 1, unknown peak 2, delphinidin-3-O-galactoside peak 3, delphinidin-3-O-glucoside peak 4, cyanidin-3-O-galactoside peak 5, cyanidin-3-O-glucoside peak 6, petunidin-3-O-glucoside peak 7, pelargonidin-3-O-glucoside peak 8, peonidin-3-O-glucoside and peak 9, malvidin-3-O-glucoside...

Reddivari, L., Vanamala, J., Chintharlapalli, S., Safe, S. H., Miller, J. C. Jr. (2007b). Anthocyanin fraction from potato extracts is cytotoxic to prostate cancer cells through activation of caspase-dependent and caspase-independent pathways. Carcinogenesis, 28, 2227-2235. 

In a polyphenolic extract, anthocyanins can interfere with other polyphenolics such as pro-cyanidins during HPLC analysis and hence should be removed prior to analysis. Anthocyanins from crude polyphenolic extracts can be removed as described in Basic Protocol 2. The ethyl acetate used for elution of phenolic compounds other than anthocyanins is removed using a rotary evaporator at 20°C. The non-anthocyanin polyphenolics are dissolved in deionized distilled water and the pH is adjusted to 7.0 with NaOH as described in Alternate Protocol 2 or the method developed by Oszmianski and Lee (1990a). In the latter method, polyphenolics were fractionated into three groups neutral fraction A (flavanols and other polar phenolics), neutral fraction B (flavonols), and acidic phenolics. Polyphenolic extracts were adjusted to pH 7.0 with NaOH... 

C18 solid-phase extraction is used to fractionate polyphenolics for their identification and characterization. This technique can eliminate interfering chemicals from crude extracts and produce desirable results for HPLC or other analytical procedures. To obtain a sufficient volume for all analyses, several separations by solid-phase extraction may be performed. The individual fractions need to be combined and dissolved in solvents appropriate for HPLC analysis. In Basic Protocol 2, the application of a current of nitrogen gas for the removal of water from the C18 cartridge is an important step in the selective fractionation of polyphenolics into non-anthocy-anin and anthocyanin fractions. After the collection of non-anthocyanin polyphenolics, no additional work is necessary to elute anthocyanins bound to the C18 solid phase if anthocyanins are not to be determined. 

A method for fractionation of polyphenoiics into non-anthocyanin and anthocyanin fractions from red grapes by solid-phase extraction using disposable C18 cartridges. 

The Basic Protocol describes the reversed-phase HPLC analysis of polyphenolic compounds isolated into nonanthocyanin and anthocyanin fractions by solid-phase extraction. The Alternate Protocol describes the HPLC separation of acidic and neutral polyphenolic fractions. Fractionated samples are used because significant amounts of interfering compounds are extracted along with polyphenolics from plant materials. Solid-phase extraction with C18 Sep-Pak cartridges (vnitu.2) is used to selectively eliminate undesired components from crude extracts, and may minimize the effects of sample cleanup or preparation on the integrity of polyphenolics. The isolation and purification step using solid-phase extraction of polyphenolics will make possible the efficient analysis of individual polyphenolics by reversed-phase HPLC. 

Additional reagents and equipment for fractionating crude polyphenolics by solid-phase extraction into anthocyanin and nonanthocyanin fractions unit 11.2)... 

This fractionation step may be optional. Some samples can be directly analyzed by HPLC after filtration (step 2) without solid-phase extraction. Anthocyanins that can be detected at 280 nm can interfere with the separation of some polyphenolics. If the analyst is interested in nonanthocyanin polyphenolics, and especially if plant materials containing high levels of anthocyanins are being analyzed, this fractionation technique should be utilized. 

Figure 11.3.12 HPLC chromatograms of polyphenolics in Concord grape extract detected at 280 nm. (A) All polyphenolics, including anthocyanins. (B) Nonanthocyanin polyphenolics after fractionation. Peak identification 1, cis-caftaric acid 2, frans-caftaric acid 3, procyanidin B3 4, c/s-coutaric acid 5, frans-coutaric acid 6, epicatechin 7, quercetin galactoside 8, quercetin glucoside. Reproduced from Oszmianski and Lee (1990) with permission from the American Society for Enology and Viticulture.

Seabra, I.J. Braga, M.E.M. Batista, M.T. de Sousa, H.C. 2010. Effect of solvent (CO2/ ethanol/Fl20) on the fractionated enhanced solvent extraction of anthocyanins from elderberry pomace. J. Supercrit. Fluids 54 145-152. 

Evidence of such adducts in wine fractions has been provided, as detailed in Chapter 9A. These include F-A+ (Alcalde-Eon et al. 2006 Boido et al. 2006) and F-A-A+ (Alcalde-Eon et al. 2006) adducts based on different flavanol and anthocyanin units and (F) -A+ adducts deriving from different flavanols monomers and oligomers (Hayasaka and Kennedy 2003). Proanthocyanidins arising from these reactions cannot be distinguished from those extracted from grapes. However, detection of F-A+ adducts without prior fractionation (Morel-Salmi et al. 2006) confirmed the occurrence of the acid-catalyzed interflavanic bond breaking process in wines. 

Alterations in the capillary filtration of macromolecules are well documented in diabetic patients and experimental diabetes. Various flavonoids, including anthocyanins and ginkgo biloba extracts, have been shown to be effective against experimentally induced capillary hyperfiltration. Cohen-Boulakia et al." demonstrated that anthocyanins were effective in preventing the increase in capillary filtration of albumin and the failure of lymphatic uptake of interstitial albumin in male rats with streptozotocin-induced diabetes. In an earlier study, Valensi and coworkers demonstrated in a placebo-controlled trial that a purified micronized flavonoid fraction (Daflon 500 mg) can improve and even normalize capillary filtration of albumin in diabetic patients. 

Koide, T., Hashimoto, Y., Kamei, H., Kojima, T., Hasegawa, M., and Terabe, K., Antitumor effect of anthocyanin fractions extracted from red soybeans and red beans in vitro and in vivo, Cancer Biother. Radiopharmac., 12, 227-280, 1997. 

Extensive reviews of analytical methods for anthocyanins (Francis, 1982 Jackman et al., 1987b Strack and Wray, 1994) and other flavonoids (Williams and Harbome, 1994) as well as phenolic acids (Herrmann, 1989) have been published. In these reviews, extraction procedures, methods for fractionation of groups of polyphenols and the identification and quantification of individual components are presented. Here, a brief presentation of more recently published methods for grape and berry polyphenolic analyses is given with respect to their relationship to antioxidant activity and health benefits. 

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