Descripción textual de los caso de uso del sistema

Extensive reviews of analytical methods for anthocyanins (Francis, 1982; Jackman et al., 1987b; Strack and Wray, 1994) and other flavonoids (Williams and Harborne, 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.

The complexity of polyphenolic composition of plant materials is such that complete identification has not been accomplished for many fruits. Often, compounds are partially characterized with respect to their compound class. Standards are often not available, but estimates of quantities can be made on the basis of reference compounds from the same class. Total polyphenolic levels can be obtained by sum-ming the individual compounds within a class and then sumsum-ming the different classes.

3.5.1 EXTRACTIONAND FRACTIONATION

Anthocyanins and other polyphenolics have their highest concentrations in the epi-dermal tissue of grapes and many berry fruits (blueberries, chokeberries, cowberries).

TX69027-ch03-Frame Page 99 Wednesday, January 16, 2002 7:10 AM

Extensive disintegration and extraction of the skin and outer layers are needed for quantitative analysis of the flavonoids. In other fruits, such as raspberries and straw-berries, the anthocyanins are more evenly distributed in the fruit tissue, and the flavonoids, to a much higher extent, dissolve in the juice obtained with homogeni-zation of the fruit tissue. When quantitative assays are to be achieved, these fruits should be extracted just prior to analysis. If the primary interest is analysis of wines, juices and certain nutraceutical preparations, analysis may be performed directly on the samples after proper dilution (Wang et al., 1996; Frankel et al., 1998; Larrauri et al., 1999; Saint-Cricq de Gaulejac et al., 1999; Burns et al., 2000; Wang and Lin, 2000). Abuja et al. (1998) analyzed spray-dried elderberry extracts after dissolving the material in salt-containing phosphate buffer.

When preparing samples for analysis, care should be taken to prevent degrada-tion of flavonoids and other phenolics prior to and during extracdegrada-tion. The use of antioxidants and reducing agents to prevent oxidation of phenolic compounds during extraction has been done in some investigations (Meyer et al., 1997). However, when antioxidant activities of berries are to be measured, any added antioxidants will interfere with the results and should be avoided. Freezing and subsequent grinding in liquid nitrogen is a method used to avoid oxidation during sample preparation (Labarbe et al., 1999; Skrede et al., 2000a), whereas extraction under anaerobic conditions to avoid oxidation has also been used (Meyer et al., 1997). When analyses are based on frozen material, care must be taken to avoid storage beyond several months. Several studies are based on freeze-dried grape and berry materials (Meyer et al., 1998; Häkkinen et al., 1999; Kähkönen et al., 1999; Lu and Foo, 1999; Saint-Cricq de Gaulejac et al., 1999), and the lyophilized samples are often finely ground prior to extraction. When possible, grinding of fresh grapes, grape skins and berries directly in the extraction medium is convenient (Strigl et al., 1995; Lapidot et al., 1998; Kalt et al., 1999a; Labarbe et al., 1999). Enzymic treatment of berry homo-genates prior to extraction has been shown to decrease extraction yields (Heinonen et al., 1998). With many commodities, special precautions need to be taken to inactivate endogenous polyphenol oxidase (Skrede et al., 2000a).

Flavonoids and phenolic acids are hydrophilic compounds with high solubility in alcohol. The most widely used extraction media are aqueous acetone (Heinonen et al., 1998; Kähkönen et al., 1999; Labarbe et al., 1999; Karadeniz et al., 2000;

Skrede et al., 2000a), aqueous methanol (Meyer et al., 1997; Heinonen et al., 1998;

Lapidot et al., 1998; Kalt et al., 1999a) and aqueous ethanol (Strigl et al., 1995; Lu and Foo, 1999). Extraction is usually repeated until the pulp is colorless. After filtration or centrifugation, the extracts are combined and evaporated. Acetone extracts may be partitioned with chloroform, which removes carotenoids and lipids along with the bulk of the acetone (Karadeniz et al., 2000; Skrede et al., 2000a).

Extraction of initial extracts with hexane may also be done to remove fat and fat-soluble compounds (Lu and Foo, 1999). The organic component of the extraction solvent is removed by evaporation under vacuum, taking care to keep the temperature low to prevent oxidation of the polyphenols. Samples are finally dissolved and diluted with acidified (1% v/v HCl) water or other solvents, depending upon the require-ments of subsequent analysis. It is often convenient to directly extract with the

Flavonoids from Berries and Grapes 101

solvent to be used in the analytical protocol (Prior et al., 1998; Iversen, 1999; Kalt et al., 1999a).

By using sequential extraction with different solvents, various groups of polyphenols may be separated directly during the extraction procedure (Saint-Cricq de Gaulejac et al., 1999). Bomser et al. (1996) reported an extraction scheme in which crude bilberry extracts were fractionated into purified total anthocyanins, procyanidins and various fractions of other phenolic compounds using a sequence of organic solvents (1% HCl acidified methanol, ethyl acetate, hexane and chloro-form). By extracting wine with ethyl acetate, flavonols, flavanols and phenolic acids (organic phase) were separated from the anthocyanin-containing aqueous phase (Ghiselli et al., 1998). Further extraction of the organic phase with ethyl acetate at various pH values facilitated fractionation of the extract into subclasses of flavanols, flavonols and phenolic acids.

Solid-phase extraction (SPE) has been used for purifying extracts and samples prior to analysis. By using C-18 cartridges, sugar and acids are removed from crude extracts (Kähkönen et al., 1999; Skrede et al., 2000a). Furthermore, anthocyanins can be separated from other flavonoids with ethyl acetate elution (Kondo et al., 1999;

Skrede et al., 2000a). This procedure is convenient if anthocyanins are interfering with the analysis of other polyphenolics. Lapidot et al. (1998) used SPE for purifying pigments from the mobile phase after chromatography.

3.5.2 HYDROLYSIS OF GLYCOSIDES AND ESTERS

Acid hydrolysis of anthocyanins (1–2 N HCl at 100°C for 30 min) will cleave the glycosidic substituents (Francis, 1982; Skrede et al., 2000a), permitting identification of anthocyanidins and individual sugars. The anthocyanidins generated are unstable, and samples should be flushed with nitrogen, stored on ice and analyzed without delay (Hong and Wrolstad, 1990). Similar conditions have been used to hydrolyze other phenolic glycosides from berry extracts using HCl and varying temperatures and times (Hertog et al., 1992; McDonald et al., 1998; Häkkinen et al., 1999). For the quantification and characterization of individual units of proanthocyanidins, acidic hydrolysis with phenyl-methanethiol (thiolysis) has been used (Labarbe et al., 1999).

With this method, proanthocyanidin units are released as flavan-3-ols, and degree of polymerization and molecular weight of the proanthocyanidins can be determined.

For those anthocyanins that contain acyl substituents, the aliphatic and aromatic acids are readily released by alkaline saponification, 10% KOH for 15 min at room temperature (Rodriguez-Saona et al., 1998). The individual organic acids and the anthocyanin glycoside produced can be subsequently identified by various chromato-graphic procedures. Alkali has also been used to cleave conjugated caffeic acid and p-coumaric acid (Burns et al., 2000).

3.5.3 TOTAL PHENOLICS

The phenolics of grapes and berry fruits constitute a large group of compounds with various structural complexities. Thus, total phenolics encompass the more simple phenolic acids along with the various classes of flavonoids, some occurring

TX69027-ch03-Frame Page 101 Wednesday, January 16, 2002 7:10 AM

as glycosides (anthocyanins, flavonols), some as esters (cinnamates, some antho-cyanins) and some in various polymeric forms (flavanols, procyanidins). For many applications, a simple measure of the total amounts of phenolics is often desired.

The most common method available for this purpose is a spectroscopic method based on the Folin-Ciocalteau reagent (Slinkard and Singleton, 1977). The results are usually expressed as GAEs, however, this choice is somewhat arbitrary, and other reference standards have been used depending upon the composition of the sample and the purpose of the investigation (Ghiselli et al., 1998; Velioglu et al., 1998). It should be remembered that the method measures the number of potentially oxidizable phenolic groups. The number of phenolic groups per molecule will vary greatly both within and among different phenolic compound classes. This procedure still provides a very useful index for phenolic content, but it would not be expected to correlate with the actual weight of phenolics present. A number of investigations, however, have shown high correlation between total phenolics measured by this method and antioxidant activity as measured by various procedures.

3.5.4 ANTHOCYANINS

The determination of monomeric anthocyanins is based on the ability of anthocya-nins to shift in color from bright red/bluish at pH 1 to nearly colorless at pH 4.5 (Wrolstad, 1976; Wrolstad et al., 1982). In contrast, the polymeric anthocyanin forms retain considerable color at pH 4.5. The absorbency at the absorbance maximum of the sample at pH 4.5 is subtracted from that at pH 1, and the total monomeric anthocyanins are calculated based on the molecular weight and extinction coefficient of the most prevalent anthocyanin. Total anthocyanin indices that also include the polymeric pigments are determined from the absorbency at pH 1, omitting measure-ment at pH 4.5 (Burns et al., 2000).

3.5.5 TOTAL FLAVANOLS

Total flavanols may be determined by the spectrophotometric vanillin method using (+)-catechin as the reference (Saito et al., 1998). Corrections must be made for any anthocyanins present (Broadhurst and Jones, 1978; McMurrough and Baert, 1994).

Furthermore, procyanidins are transformed into anthocyanidins when treated at high temperature in acidic water-free conditions (Porter et al., 1986; Simonetti et al., 1997; Saint-Cricq de Gaulejac et al., 1999). For anthocyanin-rich samples, complete separations may be difficult to achieve, and interference between the anthocyanidins formed during the reaction and those naturally occurring in the samples provides confounding factors and limitations for this method (Skrede, 2000b).

3.5.6 HPLC ANALYSIS

High-performance liquid chromatography (HPLC) is the method for detection, iden-tification and also quaniden-tification of flavonoids, phenolic acids and their derivatives.

With this method, the sample is applied and eluted through a chromatographic column under specific conditions designed for optimum separation and resolution so that each compound or group of compound passes through the column with a

Flavonoids from Berries and Grapes 103

different speed. With a detector at the column outlet, each compound generates a characteristic signal proportional to the amount present. By comparison with known substances either mixed with the sample (internal standard) or analyzed in a separate run (external standard), the analysis can be made quantitatively. When analyzing plant material, however, the detailed identity of each compound is seldom known, and compounds are often grouped by their typical absorbance maxima: phenolic acids and flavanols (280 nm), flavonols (260 or 365 nm) and anthocyanins (520 nm) (Meyer et al., 1997; Häkkinen et al., 1999; Skrede et al., 2000a). With a diode-array detector available, the entire UV visible spectrum of the compound may be matched with spectra of standard compounds (Larrauri et al., 1999), so that peak identification becomes more reliable.

There is a large number of chromatographic systems published for the separation of flavonoids and phenolic acids. Most methods use reversed-phase chromatography on a C-18 column equipped with a corresponding pre-column. The mobile phases are aqueous organic solvents applied most often as linear gradients with increasing proportions of the organic fractions. Elution systems are based on aqueous phos-phoric acid mixed with ethanol (Frankel et al., 1998; Lapidot et al., 1998), acetonitrile (Häkkinen et al., 1999) or acetonitrile and methanol (Karadeniz et al., 2000; Skrede et al., 2000a); aqueous formic acid and methanol (Strigl et al., 1995; Cabrita and Andersen, 1999; Kalt et al., 1999b); acetonitrile (Strigl et al., 1995; Iversen, 1999);

or both methanol and acetonitrile (Ghiselli et al., 1998). Also, aqueous acetic acid and acetonitrile (Larrauri et al., 1999; Lu and Foo, 1999) as well as aqueous trifluor-acetic acid (TFA) and acetonitrile (Burns et al., 2000) have been used for the separation of the various groups of flavonoids and phenolic acids by HPLC.

Electrospray mass spectroscopy (ESMS) is a recent auxiliary technique shown to be very effective for more positive anthocyanin identification. The ESMS unit can be coupled directly to the HPLC outlet (Ghiselli et al., 1998) or, alternatively, used as an off-line method (Skrede et al., 2000a). A typical HPLC chromatogram and the corre-sponding ESMS spectra of highbush blueberries (V. corymbosum) anthocyanins are shown in Figure 3.11, with the explanatory peak assignments given in Table 3.5.

3.6 ANTIOXIDANT ACTIVITY OF FLAVONOIDS AND