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Characterization and Measurement of Anthocyanins by UV-Visible Spectroscopy

Anthocyanin pigment content has a critical role in the color quality of many fresh and processed fruits and vegetables. Thus, accurate measurement of anthocyanins, along with their degradation indices, is very useful to food technologists and horticulturists in assessing the quality of raw and processed foods. Since many natural food colorants are anthocyanin derived (e.g., grape-skin extract, red-cabbage extract, purple-carrot extract), the same measurements can be used to assess the color quality of these food ingredients. In addition, there is intense interest in the anthocyanin content of foods and nutraceuticals because of possible health benefits such as reduction of coronary heart disease (Bridle and Timberlake, 1996), improved visual acuity (Timberlake and Henry, 1988), antioxidant activities (Takamura and Yamagami, 1994; Wang et al., 1997), and anticancer activities (Karaivanova et al., 1990; Kamei et al., 1995). Substantial quantitative and qualitative information can be obtained from the spectral characteristics of anthocyanins. The protocols described in this unit rely on the structural transformation of the anthocyanin chromophore as a function of pH, which can be measured using optical spectroscopy. The pH-differential method, a rapid and easy procedure for the quantitation of monomeric anthocyanins, is first described (see Basic Protocol 1). In addition, other auxiliary spectrophotometric techniques are used to measure the extent of anthocyanin polymerization and browning (see Basic Protocol 2). TOTAL MONOMERIC ANTHOCYANIN BY THE pH-DIFFERENTIAL METHOD Anthocyanin pigments undergo reversible structural transformations with a change in pH manifested by strikingly different absorbance spectra (Fig. F1.2.1). The colored oxonium form predominates at pH 1.0 and the colorless hemiketal form at pH 4.5 (Fig. F1.2.2). The pH-differential method is based on this reaction, and permits accurate and rapid measurement of the total anthocyanins, even in the presence of polymerized degraded pigments and other interfering compounds.

UNIT F1.2

BASIC PROTOCOL 1

2.0 1.8 1.6

Absorbance

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 260 360 pH 4.5 460 Wavelength (nm)

pH 1.0

560

660

760

Figure F1.2.1 Spectral characteristics of purified radish anthocyanins (acylated pelargonidin-3sophoroside-5-glucoside derivatives) in pH 1.0 and pH 4.5 buffers. Contributed by M. Mónica Giusti and Ronald E. Wrolstad

Current Protocols in Food Analytical Chemistry (2001) F1.2.1-F1.2.13 Copyright © 2001 by John Wiley & Sons, Inc.

Anthocyanins

F1.2.1

R1 OH HO O O-gly O-gly

quinonoidal base: blue pH = 7

R1 OH

-H+

R2

HO

O

+

R2 O-gly

O-gly

flavylium cation (oxonium form): orange to purple pH = 1 +H2O -H+

R1 R1 HO OH O-gly O-gly O OH R2 O-gly

carbinol pseudo-base (hemiketal form): colorless pH = 4.5 chalcone: colorless pH = 4.5 H O

OH R2 O-gly

HO

O

Figure F1.2.2 Predominant structural forms of anthocyanins present at different pH levels.

Materials 0.025 M potassium chloride buffer, pH 1.0 (see recipe) 0.4 M sodium acetate buffer, pH 4.5 (see recipe) 1. Turn on the spectrophotometer. Allow the instrument to warm up at least 30 min before taking measurements. 2. Determine the appropriate dilution factor for the sample by diluting with potassium chloride buffer, pH 1.0, until the absorbance of the sample at the vis-max (Table F1.2.1) is within the linear range of the spectrophotometer (i.e., for most spectrophotometers the absorbance should be less than 1.2). Divide the final volume of the sample by the initial volume to obtain the dilution factor (DF; for example see step 7).

IMPORTANT NOTE: In order to not exceed the buffer's capacity, the sample should not exceed 20% of the total volume.

3. Zero the spectrophotometer with distilled water at all wavelengths that will be used (vis-max and 700 nm).

Many spectrophotometers will allow for a rapid baseline correction to zero by using baseline adjust.

Characterization and Measurement of Anthocyanins by UV-Visible Spectroscopy

4. Prepare two dilutions of the sample, one with potassium chloride buffer, pH 1.0, and the other with sodium acetate buffer, pH 4.5, diluting each by the previously determined dilution factor (step 2). Let these dilutions equilibrate for 15 min.

F1.2.2

Current Protocols in Food Analytical Chemistry

Table F1.2.1

Reported Molar Absorptivity of Anthocyanins

Anthocyanina Cyanidin (Cyd) Cyd Cyd-3-ara

Solvent system

vis-max (nm)

Molar Reference absorptivity () 24600 34700 44400 44460 30175 35000 34300 44900 46200 46230 30200 26900 25740 34300 18800 7000 28840 3600 21200 15100 20100 19000 37150 32360 37150 38020 32360 34670 38020 32360 Schou, 1927 Ribereau-Gayon, 1959 Zapsalis and Francis, 1965 Fuleki and Francis, 1968a Niketic-Aleksic and Hrazdina, 1972 Brouillard and El Hache Chahine, 1980 Siegelman and Hendricks, 1958 Sakamura and Francis, 1961 Zapsalis and Francis, 1965 Fuleki and Francis, 1968a Swain, 1965 Jurd and Asen, 1966 McClure, 1967 Siegelman and Hendricks, 1958 Heredia et al., 1998 Figueiredo et al., 1996 Swain, 1965 Figueiredo et al., 1996 Figueiredo et al., 1996 Figueiredo et al., 1996 Figueiredo et al., 1996 Figueiredo et al., 1996 Hrazdina et al., 1977 Hrazdina et al., 1977 Hrazdina et al., 1977 Hrazdina et al., 1977 Hrazdina et al., 1977 Hrazdina et al., 1977 Hrazdina et al., 1977 Hrazdina et al., 1977

0.1% HCl in ethanol 0.1% HCl in ethanol 15:85 0.1 N HCl/ethanol 15:85 0.1 N HCl/ethanol

510.5 547 538 535 520 508.5 530 535 535 535 530 510 520 530 512 510 523 522 538 528 538 536 524 528 528 530 528 530 526 528

Cyd-3,5-diglu

0.1 N HCl Methanolic HCl

Cyd-3-gal

0.1% HCl in methanol 15:85 0.1 N HCl/ethanol 15:85 0.1 N HCl/ethanol 15:85 0.1 N HCl/ethanol HCl in methanol Aqueous buffer, pH 1 0.1 N HCl 1% HCl in methanol 10% ethanol, pH 1.5 Aqueous buffer, pH 0.9 1% HCl Aqueous buffer, pH 0.9 Aqueous buffer, pH 0.9 Aqueous buffer, pH 0.9 Aqueous buffer, pH 0.9 Aqueous buffer, pH 0.9 Methanolic HCl Methanolic HCl Methanolic HCl Methanolic HCl Methanolic HCl Methanolic HCl Methanolic HCl Methanolic HCl

Cyd-3-glu

Cyd-3-rut Cyd-3-sam-5-glu Cyd-3-sam-5-glu + sinapic + caffeic + malonic Cyd-3-sam-5-glu + sinapic + ferulic Cyd-3-sam-5-glu + sinapic + ferulic + malonic Cyd-3-sam-5-glu + sinapic + p-coum + malonic Cyd-3-soph-5-glu Cyd-3-soph-5-glu + malonic Cyd-3-soph-5-glu + sinapic Cyd-3-soph-5-glu + di-sinapic Cyd-3-soph-5-glu + ferulic Cyd-3-soph-5-glu + di-ferulic Cyd-3-soph-5-glu + p-coumaric Cyd-3-soph-5-glu + di-p-coumaric Delphinidin (Dpd) Dpd

0.1% HCl in ethanol

522.5

34700

Schou, 1927

continued

F1.2.3

Current Protocols in Food Analytical Chemistry

Table F1.2.1

Reported Molar Absorptivity of Anthocyanins, continued

Anthocyanina Dpd-3-glu Malvidin (Mvd) Mvd Mvd-3,5-diglu

Solvent system 1% HCl in methanol 10% ethanol, pH 1.5 0.1% HCl in ethanol 0.1% HCl in ethanol 0.1% HCl in ethanol 0.1% HCl in ethanol 0.1 N HCl 0.1% HCl in methanol 0.1% HCl in methanol 0.1 N HCl Methanol, pH 1.0 10% ethanol, pH 1.5 0.1% HCl in methanol

vis-max (nm) 543 520 520 557 519 545 520 546 538 520 535 520 536

Molar Reference absorptivity () 29000 23700 37200 36200 10700 10300 37700 13900 29500 28000 36400 20200 30200 Asen et al., 1959 Heredia et al., 1998 Schou, 1927 Ribereau-Gayon, 1959 Schou, 1927 Ribereau-Gayon, 1959 Niketic-Aleksic and Hrazdina, 1972 Somers, 1966 Koeppen and Basson, 1966 Niketic-Aleksic and Hrazdina, 1972 Metivier et al., 1980 Heredia et al., 1998 Koeppen and Basson, 1966 Schou, 1927 Giusti et al., 1999 Giusti et al., 1999 Swain, 1965 Dangles et al., 1993 Jorgensen and Geissman, 1955 Wrolstad et al., 1970 Swain, 1965 Swain, 1965 Giusti et al., 1999 Giusti et al., 1999 Giusti et al., 1999 Giusti et al., 1999 Dangles et al., 1993 Giusti et al., 1999 Giusti et al., 1999 Giusti et al., 1999 Giusti et al., 1999 Dangles et al., 1993 Giusti et al., 1999 Giusti et al., 1999

continued

Mvd-3-glu

Mvd-3-glu + p-coum Pelargonidin (Pg) Pg

Pg-3,5-diglu Pg-3-(dicaffeoylglu)-soph-5-glu Pg-3-glu

0.1% HCl in ethanol 0.025 M potassium chloride buffer, pH 1.0 0.1% HCl in methanol HCl in methanol Aqueous buffer, pH 0.8 1% HCl in H2O

504.5 505 524 510 512 496

17800 18420 19780 32360 28000 27300 36600 22390 31620 15600 17330 32080 39591 18000­20000 25370 30690 24140 29636 18000-20000 28720 34889

Pg-3-rut-5-glu + p-coumaric

Pg-3-soph-5-glu

Pg-3-soph-5-glu + ferulic

Pg-3-soph-5-glu caffeoyl derivatives Pg-3-soph-5-glu + p-coumaric

1% HCl 1% HCl in ethanol 0.025 M potassium chloride buffer, pH 1.0 0.1% HCl in methanol 0.025 M potassium chloride buffer, pH 1.0 0.1% HCl in methanol Aqueous buffer, pH 0.8 0.025 M potassium chloride buffer, pH 1.0 0.1% HCl in methanol 0.025 M potassium chloride buffer, pH 1.0 0.1% HCl in methanol Aqueous buffer, pH 0.8 0.025 M potassium chloride buffer, pH 1.0 0.1% HCl in methanol

513 516 496 508 504 511 498 497 506 506 507 498 506 508

F1.2.4

Current Protocols in Food Analytical Chemistry

Table F1.2.1

Reported Molar Absorptivity of Anthocyanins, continued

Anthocyanina

Solvent system

vis-max (nm) 508 508 508 508 511 532 532 532 520 532 532 531 536 512 520 535 546 520

Molar Reference absorptivity () 33010 39785 31090 39384 37200 40800 46100 46070 36654 48400 48400 48340 11300 14100 33040 23440 12900 18900 Giusti et al., 1999 Giusti et al., 1999 Giusti et al., 1999 Giusti et al., 1999 Schou, 1927 Sakamura and Francis, 1961 Zapsalis and Francis, 1965 Fuleki and Francis, 1968a Niketic-Aleksic and Hrazdina, 1972 Sakamura and Francis, 1961 Zapsalis and Francis, 1965 Fuleki and Francis, 1968a Somers, 1966 Heredia et al., 1998 Niketic-Aleksic and Hrazdina, 1972 Swain, 1965 Somers, 1966 Heredia et al., 1998

Pg-3-soph-5-glu + p-coumaric + 0.025 M potassium malonic chloride buffer, pH 1.0 0.1% HCl in methanol Pg-3-soph-5-glu + ferulic + 0.025 M potassium malonic chloride buffer, pH 1.0 0.1% HCl in methanol Peonidin (Pnd) Pnd 0.1% HCl in ethanol 15:85 0.1 N HCl/ethanol Pnd-3-ara 15:85 0.1 N HCl/ethanol 15:85 0.1 N HCl/ethanol Pnd-3,5-diglu Pnd-3-gal 0.1 N HCl 15:85 0.1 N HCl/ethanol 15:85 0.1 N HCl/ethanol 15:85 0.1 N HCl/ethanol Pnd-3-glu Petunidin (Ptd) Ptd-3,5-diglu 0.1% HCl in methanol 10% ethanol, pH 1.5 0.1 N HCl HCl in methanol 0.1% HCl in methanol 10% ethanol, pH 1.5

Ptd-3-glu

aAbbreviations: ara: arabinoside; gal: galactoside; glu: glucoside; rut: rutinoside; sam: sambubioside; soph: sophoroside.

5. Measure the absorbance of each dilution at the vis-max and at 700 nm (to correct for haze), against a blank cell filled with distilled water.

All measurements should be made between 15 min and 1 hr after sample preparation, since longer standing times tend to increase observed readings. Absorbance readings are made against water blanks, even if the samples are in buffer or bisulfite solutions, as buffer or bisulfite absorbance is nil at the measured wavelengths. The authors have compared the values obtained by using water as a blank as compared with buffer or bisulfite as blanks in different systems and have found no difference in the final values obtained for monomeric and/or polymeric anthocyanin content; on the other hand, reading the diluted samples against the corresponding buffer and/or bisulfite solution is more time-consuming and extends the procedure unnecessarily. The samples to be measured should be clear and contain no haze or sediments; however, some colloidal materials may be suspended in the sample, causing scattering of light and a cloudy appearance (haze). This scattering of light needs to be corrected for by reading at a wavelength where no absorbance of the sample occurs, i.e., 700 nm.

Anthocyanins

F1.2.5

Current Protocols in Food Analytical Chemistry

Table F1.2.2 in Naturea

Molecular Weights of Anthocyanidins, Anthocyanins, and Acylating Groups Commonly Found

Anthocyanidins Hex Hex ­H2Ob Acd + 1 hex Acd + 2 hex Acd + 3 hex Pent Pent ­H2Ob Acd + 1 pent Acd + 1 hex + 1 pent Rhamnose Rutinose Rutinose ­H2Ob Acd + rutinose Acd + rutinose + 1 hex Acd + rutinose + 1 pent Common acylating groups

Pelargonidin 271 180.2 162.2 433.2 595.4 757.6 150.0 132.0 403.0 565.2 164.2 326.2 308.2 579.2 741.4 711.2

Cyanidin Peonidin Delphinidin 287 180.2 162.2 449.2 611.4 773.6 150.0 132.0 419.0 581.2 164.2 326.2 308.2 595.2 757.4 727.2 ­H2Ob 301 180.2 162.2 463.2 625.4 787.6 150.0 132.0 433.0 595.2 164.2 326.2 308.2 609.2 771.4 741.2 303 180.2 162.2 465.2 627.4 789.6 150.0 132.0 435.0 597.2 164.2 326.2 308.2 611.2 773.4 743.2

Petunidin 317 180.2 162.2 479.2 641.4 803.6 150.0 132.0 449.0 611.2 164.2 326.2 308.2 625.2 787.4 757.2

Malvidin 331 180.2 162.2 493.2 655.4 817.6 150.0 132.0 463.0 625.2 164.2 326.2 308.2 639.2 801.4 771.2

p-Coumaric acid Caffeic acid Ferulic acid Sinapic acid Acetic acid Propionic acid Malonic acid Succinic acid

164.2 180.2 194.2 224 82 96.1 104.1 118.1

146.2 162.2 176.2 206 64 78.1 86.1 100.1

aAbbreviations: hex: hexose; pent: pentose; acd: anthocyanidin. b-H O indicates a dehydrated sugar (water is lost upon forming a glycosidic bond). 2

6. Calculate the absorbance of the diluted sample (A) as follows: A = (A vis-max ­ A700)pH 1.0 ­ (A vis-max ­ A700)pH 4.5 7. Calculate the monomeric anthocyanin pigment concentration in the original sample using the following formula: Monomeric anthocyanin pigment (mg/liter) = (A × MW × DF × 1000)/( × 1) where MW is the molecular weight (Table F1.2.2), DF is the dilution factor (for example, if a 0.2 ml sample is diluted to 3 ml, DF = 15), and is the molar absorptivity (Table F1.2.1).

IMPORTANT NOTE: The MW and used in this formula correspond to the predominant anthocyanin in the sample. Use the reported in the literature for the anthocyanin pigment in acidic aqueous solvent. If the of the major pigment is not available, or if the sample composition is unknown, calculate pigment content as cyanidin-3-glucoside, where MW = 449.2 and = 26,900 (see Background Information, discussion of Molar Absorptivity). The equation presented above assumes a pathlength of 1 cm.

Characterization and Measurement of Anthocyanins by UV-Visible Spectroscopy

F1.2.6

Current Protocols in Food Analytical Chemistry

INDICES FOR PIGMENT DEGRADATION, POLYMERIC COLOR, AND BROWNING Indices for anthocyanin degradation of an aqueous extract, juice, or wine can be derived from a few absorbance readings of a sample that has been treated with sodium bisulfite. Anthocyanin pigments will combine with bisulfite to form a colorless sulfonic acid adduct (Figure F1.2.3). Polymerized colored anthocyanin-tannin complexes are resistant to bleaching by bisulfite, whereas the bleaching reaction of monomeric anthocyanins will rapidly go to completion. The absorbance at 420 nm of the bisulfite-treated sample serves as an index for browning. Color density is defined as the sum of absorbances at the vis-max and at 420 nm. The ratio between polymerized color and color density is used to determine the percentage of the color that is contributed by polymerized material. The ratio between monomeric and total anthocyanin can be used to determine a degradation index. Materials Bisulfite solution (see recipe) 0.025 M potassium chloride buffer, pH 1.0 (see recipe) 1. Turn on the spectrophotometer and allow the instrument to warm up at least 30 min before taking measurements. 2. Determine the appropriate dilution factor for the sample by diluting with 0.025 M potassium chloride buffer, pH 1.0 until the absorbance of the sample at the vis-max is within the linear range of the spectrophotometer (i.e., for most spectrophotometers the absorbance should be less than 1.2). Divide the final volume of the sample by the initial volume to obtain the dilution factor (DF; for example see step 6). 3. Zero the spectrophotometer with distilled water at all wavelengths that will be used (420 nm, vis-max, 700 nm).

Many spectrophotometers will allow for a rapid baseline correction to zero by using baseline adjust.

BASIC PROTOCOL 2

4. Dilute the sample with distilled water using the dilution factor already determined (step 2). Transfer 2.8 ml of the diluted sample to each of two cuvettes. Add 0.2 ml of bisulfite solution to one and 0.2 ml distilled water to the other. Equilibrate for 15 min.

It is critical that the pH not be adjusted to highly acidic conditions (e.g., pH 1) but rather be in the typical pH range of fruit juices and wines, or higher (e.g., pH 3). Highly acidic conditions will reverse the bisulfite addition reaction and render the measurement invalid.

5. Measure the absorbance of both samples at 420 nm, vis-max, and 700 nm (to correct for haze), against a blank cell filled with distilled water.

All measurements should be made between 15 min (see step 4) and 1 hr after sample preparation and bisulfite treatment. Longer standing times tend to increase observed readings. Absorbance readings are made against water blanks, even if the samples are in buffer or bisulfite solutions, as buffer or bisulfite absorbance is nil at the measured wavelengths. The authors have compared the values obtained by using water as a blank as compared with the use buffer or bisulfite as a blank in different systems and have found no difference in the final values obtained for monomeric and/or polymeric anthocyanin content; on the other hand, reading the samples against the corresponding buffer and/or bisulfite solution is more time-consuming and extends the procedure unnecessarily. The samples to be measured should be clear and contain no haze or sediments; however, some colloidal materials may be suspended in the sample, causing scattering of light and a cloudy appearance (haze). This scattering of light needs to be accounted for by reading at a wavelength where no absorbance of the sample occurs (i.e., 700 nm).

Anthocyanins

F1.2.7

Current Protocols in Food Analytical Chemistry

6. Calculate the color density of the control sample (treated with water) as follows: Color density = [(A420 nm ­ A700nm) + (A vis-max ­ A700 nm)] × DF where DF is the dilution factor (for example, if 0.2 ml sample diluted to 3 ml, DF = 15) 7. Calculate the polymeric color of the bisulfite bleached sample as follows: Polymeric color = [(A420 nm ­ A700 nm) + (A vis-max ­ A700 nm)] × DF 8. Calculate the percent polymeric color using the formula: Percent polymeric color = (polymeric color/color density) × 100 REAGENTS AND SOLUTIONS

Use deionized or distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Bisulfite solution Dissolve 1 g of potassium metabisulfite (K2S2O5) in 5 ml of distilled water.

This reagent must be prepared the same day as the readings; otherwise, it develops a yellow color that will contribute to the absorbance readings and interfere with the quantitation.

Potassium chloride buffer, 0.025 M, pH 1.0 Mix 1.86 g KCl and 980 ml of distilled water in a beaker. Measure the pH and adjust to 1.0 with concentrated HCl. Transfer to a 1 liter volumetric flask and fill to 1 liter with distilled water.

The solution should be stable at room temperature for a few months, but the pH should be checked and adjusted prior to use (see Critical Parameters).

Sodium acetate buffer, 0.4 M, pH 4.5 Mix 54.43 g CH3CO2Na3 H2O and 960 ml distilled water in a beaker. Measure the pH and adjust to 4.5 with concentrated HCl. Transfer to a 1 liter volumetric flask and fill to 1 liter with distilled water.

The solution should be stable at room temperature for a few months, but the pH should be checked and adjusted prior to use (see Critical Parameters).

COMMENTARY Background Information

Anthocyanin pigments are responsible for the attractive red to purple to blue colors of many fruits and vegetables. Anthocyanins are relatively unstable and often undergo degradative reactions during processing and storage. Measurement of total anthocyanin pigment content along with indices for the degradation of these pigments are very useful in assessing the color quality of these foods. Interest in the anthocyanin content of foods and nutraceutical preparations has intensified because of their possible health benefits. They may play a role in reduction of coronary heart disease (Bridle and Timberlake, 1996) and increased visual acuity (Timberlake and Henry, 1988), and also have antioxidant (Takamura and Yamagami, 1994; Wang et al., 1997) and anticancer properties (Karaivanova et al., 1990; Kamei et al., 1995). Anthocyanins have also found considerable potential in the food industry as safe and effective food colorants (Strack and Wray, 1994); interest in this application has increased in recent years. In 1980, the annual world production had been estimated as reaching 10,000 tons from grapes alone (Timberlake, 1980). Quantitative and qualitative anthocyanin composition are important factors in determining the feasibility of the use of new plant materials as anthocyanin-based colorant sources. Frequently, it is desirable to express anthocyanin determinations in terms that can be compared with the results from different workers. The best way to express these results is in terms

Characterization and Measurement of Anthocyanins by UV-Visible Spectroscopy

F1.2.8

Current Protocols in Food Analytical Chemistry

R1 OH HO O

+

R1 OH HO R2 O O-gly OH SO3H bisulfite addition compound: colorless R2

O-gly OH flavylium cation: red

Strong acid

Figure F1.2.3 Formation of colorless anthocyanin-sulfonic acid adducts.

of absolute quantities of anthocyanins present (Fuleki and Francis, 1968a). The total anthocyanin content in crude extracts containing other phenolic materials has been determined by measuring absorptivity of the solution at a single wavelength. This is possible because anthocyanins have a typical absorption band in the 490 to 550 nm region of the visible spectra (Figure F1.2.1). This band is far from the absorption bands of other phenolics, which have spectral maxima in the UV range (Fuleki and Francis, 1968a). In many instances, however, this simple method is inappropriate because of interference from anthocyanin degradation products or melanoidins from browning reactions (Fuleki and Francis, 1968b). In those cases, the approach has been to use differential and/or subtractive methods to quantify anthocyanins and their degradation products (Jackman and Smith, 1996). The differential method (see Basic Protocol 1) measures the absorbance at two different pH values, and relies on the structural transformations of the anthocyanin chromophore as a function of pH (Fig. F1.2.1 and Fig. F1.2.2). This concept was first introduced by Sondheimer and Kertesz in 1948, who used pH values of 2.0 and 3.4 for analyses of strawberry jams (Francis, 1989). Since then, the use of other pH values has been proposed. Fuleki and Francis (1968b) used pH 1.0 and 4.5 buffers to measure anthocyanin content in cranberries, and modifications of this technique have been applied to a wide range of commodities (Wrolstad et al., 1982, 1995). The pH differential method has been described as fast and easy for the quantitation of monomeric anthocyanins (Wrolstad et al., 1995). Subtractive methods (see Basic Protocol 2) are based on the use of bleaching agents that

will decolor anthocyanins but not affect interfering materials. A measurement of the absorbance at the visible maximum is obtained, followed by bleaching and remeasuring to give a blank reading (Jackman et al., 1987). The two most used bleaching agents are sodium sulfite (Somers and Evans, 1974; Wrolstad et al., 1982) and hydrogen peroxide (Swain and Hillis, 1959). By using both of these spectral procedures, accurate measurement of the total monomeric anthocyanin pigment content can be obtained, along with indices for polymeric color, color density, browning, and degradation. To determine total anthocyanin content, the absorbance at pH 1.0 and 4.5 is measured at the vis-max (Table F1.2.1) and at 700 nm, which allows for haze correction. The bisulfite bleaching reaction is utilized to generate the various degradation indices. While monomeric anthocyanins are readily bleached by bisulfite at product pH (Fig. F1.2.3), the polymeric anthocyanin-tannin and melanoidin pigments are resistant and will remain colored. Somers and Evans (1974) used this reaction in developing spectral methods for assessing the color quality of wines. The author's laboratory has found them useful for tracking color quality in a wide range of anthocyanin-containing foods (Wrolstad et al., 1982, 1995). Absorbance measurements are taken at the vis-max and at 420 nm on the bisulfite bleached and control samples. Color density is the sum of the absorbances at the vis-max and at 420 nm of the control sample, while polymeric color is the same measurement for the bisulfite treated sample. A measure of percent polymeric color is obtained as the ratio between these two indexes. The absorbance at 420 nm of the bisulfite-treated sample is an index for browning, as the accumulation of brownish

Anthocyanins

F1.2.9

Current Protocols in Food Analytical Chemistry

degradation products increases the absorption in the 400 to 440 nm range. The absorption of these compounds are in general not affected by the addition of a bisulfite solution. Molar absorptivity Regardless of the method used for anthocyanin quantitation, the determination of the amount present requires an absorptivity coefficient. Absorptivity coefficients have been reported as the absorption of a 1% solution measured through a 1-cm path at the vis-max, or as a molar absorption coefficient. Absorptivity coefficients of some known anthocyanins have been reported by different researchers (Table F1.2.1). Through the years, there has been a lack of uniformity on the values of absorptivity reported, mainly due to the difficulties of preparing crystalline anthocyanin, free from impurities, in sufficient quantities to allow reliable weighing under optimal conditions (Fuleki and Francis, 1968a; Francis, 1982; Giusti et al., 1999). Other problems are that the anthocyanin mixtures may be very complicated, and not all absorptivity coefficients may be known. Even when they are known, it is necessary to first evaluate if the objective is the estimation of total anthocyanin content or the determination of individual pigments, and then to decide which absorption coefficient(s) to use. The absorptivity is dependent not only on the chemical structure of the pigment but also on the solvent used; preferably, the coefficient used should be one obtained in the same solvent system as the one used in the experiment. If the identity of the pigments is unknown, it has been suggested that it can be expressed as cyanidin-3-glucoside, since that is the most abundant anthocyanin in nature (Francis, 1989). Spectral characteristics Substantial information can be obtained from the spectral characteristics of anthocyanins (Fig. F1.2.1). Two distinctive bands of absorption, one in the UV-region (260 to 280 nm) and another in the visible region (490 to 550 nm) are shown by all anthocyanins. The different aglycons have different vis-max, ranging from 520 nm for pelargonidin to 546 nm for delphinidin, and their monoglucosides exhibit their vis-max at about 10 to 15 nm lower (Strack and Wray, 1989). The shape of the spectrum may give information regarding the number and position of glycosidic substitutions and number of cinnamic acid acylations. The ratio between the absorbance at 440 nm and the absorbance at the vis-max is almost twice as

much for anthocyanins with glycosidic substitutions in position 3 as compared to those with substitutions in positions 3 and 5 or position 5 only. The presence of glycosidic substitutions at other positions (e.g., 3,7-diglycosides) can be recognized because they exhibit a different spectral curve from those of anthocyanins with common substitution patterns. The presence of cinnamic acid acylation is revealed by the presence of a third absorption band in the 310 to 360 nm range (Figure F1.2.1), and the ratio of absorbance at 310 to 360 nm to the absorbance at the visible vis-max will give an estimation of the number of acylating groups (Harborne, 1967; Hong and Wrolstad, 1990). The solvent used for spectral determination will affect the position of the absorption bands, and therefore must be taken into consideration when comparing available data.

Critical Parameters and Troubleshooting

The pH of buffers should always be checked and adjusted prior to use. The use of buffers with lower or higher pH levels will result in under- or overestimations of the pigment content. The accuracy of the results will be greatly affected by the accuracy of the volumetric measurements. Make sure that any volumetric flasks or pipets used for obtaining the appropriate dilutions are calibrated correctly. For the methodologies described in this unit, all spectral measurements should be made between 15 min and 1 hr after the dilutions have been prepared. The observed readings tend to increase with time. When working with several different samples, it may be acceptable to use one common approximate vis-max that is typical of all samples (i.e., 520 nm). The visible absorbance peak is broad, and measuring a few nanometers off vis-max will not significantly alter the estimated final values. Serial dilutions are recommended to ensure accurate measurements of highly concentrated, high density, or dried samples. Perform a weight-by-volume dilution with distilled water to obtain a single-strength solution (e.g., usually around 10° Brix for fruit juices; UNIT H1.4), followed by a second dilution using 0.025 M potassium chloride buffer, pH 1.0. Both dilution factors must be considered when calculating monomeric anthocyanin content. For example, 1 g of a 75° Brix juice concentrate was diluted to a final volume of 10 ml with distilled water (dilution factor = 10; assuming

Characterization and Measurement of Anthocyanins by UV-Visible Spectroscopy

F1.2.10

Current Protocols in Food Analytical Chemistry

Table F1.2.3

Anthocyanin Content of Some Common Fruits and Vegetables

Source Apples (Scugog) Bilberries Blackberries Black currants Blueberries Red cabbage Black chokeberries Cherries Cranberries Elderberry Grapes Kiwi Red onions Plum Red radishes Black raspberries Red Raspberries Strawberries Tradescantia pallida (leaves)

Pigment content Reference (mg/100 g fresh weight) 10 300­320 83­326 130­400 25­495 25 560 4­450 60­200 450 6­600 100 7­21 2­25 11­60 300­400 20­60 15­35 120 Mazza and Miniati, 1993 Mazza and Miniati, 1993 Mazza and Miniati, 1993 Timberlake, 1988 Mazza and Miniati, 1993 Timberlake, 1988 Kraemer-Schafhalter et al., 1996 Kraemer-Schafhalter et al., 1996 Timberlake, 1988 Kraemer-Schafhalter et al., 1996 Mazza and Miniati, 1993 Kraemer-Schafhalter et al., 1996 Mazza and Miniati, 1993 Timberlake, 1988 Giusti et al., 1988 Timberlake, 1988 Mazza and Miniati, 1993 Timberlake, 1988 Shi et al., 1992

a density of 1 g/ml for juice). Then, the appropriate dilution factor for the sample was determined by diluting 0.2 ml of the solution with 2.8 ml of 0.025 M potassium chloride buffer, pH 1.0 (dilution factor = 15). To calculate monomeric anthocyanin content, color density, or polymeric color, the dilution factor to use would be: DF = (10 × 15) = 150. The methodologies used to measure color density and polymeric color were developed for fruit juices, which naturally have an acidic pH. If the material to be measured has a pH in the neutral or alkaline range, the pH of the solution should be lowered with a weak acid. In these cases, the authors recommend the use of a 0.1 M citric acid buffer, pH 3.5, instead of distilled water to prepare the different dilutions. Some potential interfering materials are other red pigments: FD&C Red No. 40, FD&C Red No. 3, cochineal, and beet powder (betalain pigments). The presence of alternative colorants may be suspected if the vis-max at pH 1.0 is high (550 nm, more typical of betalain pigments), or if a bright red coloration is found at pH 4.5 (potential presence of artificial dyes). The presence of ethanol does not interfere with the assay at the levels typically encountered in wines (10% to 14%).

Highly acylated anthocyanins may not respond to pH changes the same way as anthocyanins with no or few acylating groups, and may not decolor as much as nonacylated or monoor diacylated anthocyanins do at pH 4.5.

Anticipated Results

The anthocyanin content of different common fruits and vegetables is presented in Table F1.2.3. Anthocyanin-containing fruit or vegetable juices typically have pigment content ranging from 50 to 500 mg/liter. Anthocyaninbased natural colorants and nutraceuticals may have a much higher pigment concentration, on the order of a few grams/liter. Fresh fruit or vegetable juices should have a low percentage of polymeric color (usually less than 10%), while processed samples and materials subjected to storage abuse will be much higher (30% or more). This is highly variable, dependent on the commodity, processing conditions, and storage history. Always express anthocyanin pigment content in terms of the specific anthocyanin used for calculation, and specify molecular weight and utilized.

Anthocyanins

F1.2.11

Current Protocols in Food Analytical Chemistry

Time Considerations

Quantitation of anthocyanins can be achieved in <1 hr. It is necessary to wait for the spectrophotometer to warm up, and for the diluted samples to equilibrate at least 15 min. The absorbance readings take a few minutes.

Hong, V. and Wrolstad, R.E. 1990. Use of HPLC separation/photodiode array detection for characterization of anthocyanins. J. Agric. Food Chem. 38:708-715. Hrazdina, G., Iredale, H., and Mattick, L.R. 1977. Anthocyanin composition of Brassica oleracea cv. Red Danish. Phytochemistry 16:297-301. Jackman, R.L. and Smith, J.L. 1996. Anthocyanins and betalains. In Natural Food Colorants, 2nd ed. (G.A.F.Hendry and J.D. Houghton, eds.) Chpt. 8. Blackie & Son, Glasgow, Scotland. Jackman, R.L., Yada, R.Y., and Tung, M.A. 1987. A review: Separation and chemical properties of anthocyanins used for their qualitative and quantitative analysis. J. Food Biochem. 11:279-308. Jorgensen, E.C. and Geissman, T.A. 1955. The chemistry of flower pigmentation in Antirrhinum majus color genotypes. III. Relative anthocyanin and aurone concentrations. Biochem. Biophys. 55:389-402. Jurd, L. and Asen, S. 1966. The formation of metal and "co-pigment" complexes of cyanidin 3-glucoside. Phytochemistry 5:1263-1271. Kamei, H., Kojima, T., Hasegawa, M., Koide, T., Umeda, T., Yukawa, T., and Terabe, K. 1995. Suppression of tumor cell growth by anthocyanins in vitro. Cancer Invest. 13:590-594 Karaivanova, M., Drenska, D., and Ovcharov, R. 1990. A modification of the toxic effects of platinum complexes with anthocyans. Eksp. Med. Morfol. 29:19-24. Koeppen, B.H. and Basson, D.S. 1966. The anthocyanin pigments of Barlinka grapes. Phytochemistry 5:183-187. Kraemer-Schafhalter, A., Fuchs, H., Strigl, A., Silhar, S., Kovac, M., and Pfannhauser, W. 1996. Process consideration for anthocyanin extraction from Black Chokeberry (Aronia meloncarpa ELL). In Proceedings of the Second International Symposium on Natural Colorants, INF/COL II (P.C. Hereld, ed.), pp. 153-160. S.I.C. Publishing Col., Hamden, Ct. Mazza, G. and Miniati, E. 1993. Introduction. In Anthocyanins in fruits, vegetables, and grains. (G. Mazza and E. Miniati, eds). CRC Press, Boca Raton, Fla. McClure, J.W. 1967. Photocontrol of Spirodela intermedia flavonoids. Plant Phys. 43:193-200. Metivier, R.P., Francis, F.J., and Clydesdale, F.M. 1980. Solvent extraction of anthocyanins from wine pomace. J. Food Sci. 45:1099-1100. Niketic-Aleksic, G. and Hrazdina, G. 1972. Quantitative analysis of the anthocyanin content in grape juices and wines. Lebensm. Wiss. U. Technol. 5:163-165. Ribereau-Gayon, P. 1959. Recherches sur les anthocyannes des vegetaux. Application au genre Vitis. Doctoral dissertation, p.118. University of Bordeaux. Libraire Generale de l'Ensignement, Paris.

Literature Cited

Asen, S., Stuart, N.W., and Siegelman, H.W. 1959. Effect of various concentrations of nitrogen, phosphorus and potassium on sepal color of Hydrangea macrophylla. Am. Soc. Hort. Sci. 73:495-502. Bridle, P. and Timberlake, C.F. 1996. Anthocyanins as natural food colors-selected aspects. Food Chem. 58:103-109. Brouillard, R. and El Hache Chahine, J.M. 1980. Chemistry of anthocyanin pigments. 6. Kinetic and thermodynamic study of hydrogen sulfite addition to cyanin. Formation of a highly stable Meisenheimer-type adduct derived from a 2phenylbenzopyrylium salt. J. Am. Chem. Soc. 102:5375-5378. Dangles, O., Saito, N., and Brouillard, R. 1993. Anthocyanin intramolecular copigment effect. Phytochemistry 34:119-124. Figueiredo, P., Elhabiri, M., Saito, N., and Brouillard, R. 1996. Anthocyanin intramolecular interactions. A new mathematical approach to account for the remarkable colorant properties of the pigments extracted from Matthiola incana. J. Am. Chem. Soc. 118:4788-4793. Francis, F.J. 1982. Analysis of anthocyanins. In Anthocyanins as Food Colors (P. Markakis, ed.) pp. 182-205. Academic Press, New York. Francis, F.J. 1989. Food colorants: Anthocyanins. Crit. Rev. Food Sci. Nutr. 28:273-314. Fuleki, T. and Francis, F.J. 1968a. Quantitative methods for anthocyanins. 1. Extraction and determination of total anthocyanin in cranberries. J. Food Sci. 33:72-78. Fuleki, T. and Francis, F.J. 1968b. Quantitative methods for anthocyanins. 2. Determination of total anthocyanin and degradation index for cranberry juice. J. Food Sci. 33:78-82. Giusti, M.M., Rodriguez-Saona, L.E., Baggett, J.R., Reed, G.L., Durst, R.W., and Wrolstad, R.E. 1998. Anthocyanin pigment composition of red radish cultivars as potential food colorants. J. Food Sci. 63:219-224. Giusti, M.M., Rodriguez-Saona, L.E., and Wrolstad, R.E. 1999. Spectral characteristics, molar absorptivity and color of pelargonidin derivatives. J. Agric. Food Chem. 47:4631-4637. Harborne, J.B. 1967. Comparative Biochemistry of the Flavonoids. Academic Press, London. Characterization and Measurement of Anthocyanins by UV-Visible Spectroscopy Heredia, F.J., Francia-Aricha, E.M., Rivas-Gonzalo, J. C., Vicario, I.M., and Santos-Buelga, C. 1998. Chromatic characterization of anthocyanins from red grapes. I. pH effect. Food Chem. 63:491-498.

F1.2.12

Current Protocols in Food Analytical Chemistry

Sakamura, S. and Francis, F.J. 1961. The anthocyanins of the American cranberry. J. Food Sci. 26:318-321. Schou, S.A. 1927. Light absorption of several anthocyanins. Helv. Chim. Acta. 10:907-915. Siegelman, H.W. and Hendricks, S.B., 1958. Photocontrol of alcohol, aldehyde and anthocyanin production in apple skin. Plant Phys. 33:409413. Somers, T.C. 1966. Grape phenolics: the anthocyanins of Vitis vinifera, var. Shiraz. J. Sci. Food Agric. 17:215-219. Somers, T.C. and Evans, M.E. 1974. Wine quality: Correlations with colour density and anthocyanin equilibria in a group of young red wines. J. Sci. Food Agric. 25:1369-1379. Strack, D. and Wray, V. 1989. Anthocyanins. In Methods in Plant Biochemistry, Vol. I, Plant Phenolics (P.M. Dey and J.B. Harborne. eds.). Academic Press, San Diego. Strack, D. and Wray, V. 1994. The anthocyanins. In The Flavonoids: Advances in Research Since 1986. (J.B. Harborne, ed.). Chapman and Hall. Swain, T. 1965. Analytical methods for flavonoids. In Chemistry and Biochemistry of Plant Pigments (T.W. Goodwin, ed.). Academic Press, London. Swain, T. and Hillis, W.E. 1959. The phenolic constituents of Prunus domestica. I. The quantitative analysis of phenolic constituents. J. Sci. Food Agric. 10:63-68. Takamura, H. and Yamagami, A. 1994. Antioxidative activity of mono-acylated anthocyanins isolated from Muscat Bailey A grape. J. Agric. Food Chem. 42:1612-1615. Timberlake, C.F 1980. Anthocyanins [related to beverages]--occurrence, extraction and chemistry [coloring material]. Food Chem. 5:69-80. Timberlake, C.F. 1988. The biological properties of anthocyanin compounds. NATCOL Quarterly Bulletin 1:4-15. Timberlake, C.F. and Henry, B.S., 1988. Anthocyanins as natural food colorants. Prog. Clin. Biol. Res. 280:107-121.

Wang, H., Cao, G., and Prior, R.L., 1997. Oxygen radical absorbing capacity of anthocyanins. J. Agric. Food Chem. 45:304-309 Wrolstad R.E. 1976. Color and pigment analyses in fruit products. Oregon St. Univ. Agric. Exp. Stn., Bulletin 624:1-17. Wrolstad, R.E., Putnam, T.P., and Varseveld, G.W. 1970. Color quality of frozen strawberries: Effect of anthocyanin, pH, total acidity and ascorbic acid variability. J. Food Sci. 35:448-452. Wrolstad, R.E., Culbertson, J.D., Cornwell, C.J., and Mattick, L.R. 1982. Detection of adulteration in blackberry juice concentrates and wines. J. Assoc. Off. Anal. Chem. 65:1417-1423. Wrolstad, R.E., Hong, V., Boyles, M.J., and Durst, R.W. 1995. Use of anthocyanin pigment analysis for detecting adulteration in fruit juices. In Methods to Detect Adulteration in Fruit Juice and Beverages, Vol. I (S. Nagy and R.L. Wade, ed.). AgScience Inc., Auburndale, Fla. Zapsalis, C. and Francis, F.J. 1965. Cranberry anthocyanins. J. Food Sci. 30:396-399.

Key References

Giusti et al.,1999. See above. Compares the molar absorptivity of many anthocyanins in different solvent systems. Somers and Evans, 1974. See above. Spectral methods are described for generating several color quality indices for wines. Wrolstad et al., 1982. See above. Description of the pH differential method for determination of total anthocyanins and indices for anthocyanin degradation as applied to fruit juices and wines.

Contributed by M. Mónica Giusti, University of Maryland College Park, Maryland Ronald E. Wrolstad, Oregon State University Corvallis, Oregon

Anthocyanins

F1.2.13

Current Protocols in Food Analytical Chemistry

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