Stability to light, heat, and hydrogen peroxide at different pH values and DPPH radical scavenging activity of acylated anthocyanins from red radish extract

The stability of red radish extract to light, heat, and hydrogen peroxide at different pH values (3, 5, and 7) was examined, in which major anthocyanins were pelargonidin glycosides acylated with a combination of p-coumaric, ferulic, or caffeic acids. The light irradiation (fluorescence light, 5000 lx; at 25 degrees C) indicated that the red radish extract was more stable at lower pH than at higher pH. The HPLC analyses revealed that diacylated anthocyanins in the extract were more stable to light at pH 3 than monoacylated anthocyanins. No significant difference in degradation rates of acylated anthocyanins at pH 5 was observed, whereas anthocyanins acylated with p-coumaric or ferulic acids were more stable at pH 7 than ones with caffeic acids. The stability to heat (at 90-95 degrees C) showed a tendency similar to that for light. The number of intramolecular acyl units contributes to stability to light and heat at lower pH, whereas the characteristics of intramolecular acyl units influence the stability at higher pH. The degradation behavior of red radish extract to H2O2 were almost the same to those of light and heat, depending on the pH. However, HPLC analyses revealed that the stability of individual acylated anthocyanins were independent of the pH. These data suggest that the characteristics, the number, and the binding site of intramolecular acyl units affect the stability of anthocyanin to H2O2. DPPH radical scavenging activity of all acylated anthocyanins was higher than those of pelargonidin and perlargonidin-3-glucoside. The activity of acylated anthocyanins mostly depended on the activity of intramolecular acyl units (caffeic acid > ferulic acid > p-coumaric acid). However, the activity was highly affected by the binding site of intramolecular acyl units even if anthocyanins have common acyl units.     

Identification and characterization of anthocyanins by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry in common foods in the United States: vegetables, nuts, and grains

Anthocyanins in common foods in the United States, other than fruits and berries, were identified and characterized by high-performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry coupled with diode array detection. Of all of the 40+ vegetables, nuts, and grains screened, seven vegetables, one nut, and one grain were found to contain anthocyanins; the number of anthocyanins detected varied from two in pistachio nuts to 34 in red radishes. The individual anthocyanins were identified by comparing their mass spectrometric data and retention times with those of standards, published data, and reference food samples. In all of the samples analyzed, except for sorghum, only six common anthocyanidins (delphinidin, cyanidin, pelargonidin, petunidin, peonidin, and malvidin) were found as their glycosides. Anthocyanins in certain vegetables such as red cabbage and red radish were highly conjugated with sugars and acylated groups, and thus, their structures were very complicated. Eight different either aliphatic or aromatic acylated groups (acetoyl, coumaroyl, malonoyl, p-hydroxybenzoyl, feruoyl, caffeoyl, sinapoyl, and oxaloyl) were identified in the anthocyanins. In addition to glucose, six other sugar moieties (galactose, xylose, rhamnose, rutinose, sambubiose, and laminaribiose) were observed. Three varieties of sorghum were found to contain 3-deoxyanthocyanidins and their derivatives as major anthocyanins. A number of new anthocyanins were identified in the foods studied. This paper presents complete HPLC profiles and MS spectrometric data, obtained under the same experimental conditions, for common vegetables, pistachio nuts, and sorghum that contain anthocyanins.     

effect of dose size on bioavailability of acylated and nonacylated anthocyanins from red cabbage (Brassica oleracea L. Var. capitata)

Recent studies indicate that anthocyanin intake conveys a variety of health benefits, which depend on absorption and metabolic mechanisms that deliver anthocyanins and their bioactive metabolites to responsive tissues. The anthocyanin bioavailability of red cabbage (Brassica oleracea L. var. capitata) was evaluated as reflected by urinary excretion of anthocyanins and anthocyanin metabolites. Twelve volunteers consumed 100, 200, and 300 g of steamed red cabbage (containing 1.38 micromol of anthocyanins/g of cabbage) in a crossover design. Anthocyanin concentration in cabbage extract and urine was measured by HPLC-MS/MS. Six nonacylated and 30 acylated anthocyanins were detected in red cabbage, and 3 nonacylated anthocyanins, 8 acylated anthocyanins, and 4 metabolites were present in urine. Mean 24 h excretion of intact anthocyanins increased linearly from 45 (100 g dose) to 65 nmol (300 g dose) for acylated anthocyanins and from 52 (100 g dose) to 79 nmol (300 g dose) for nonacylated anthocyanins. Urinary recovery of intact anthocyanins (percent of anthocyanin intake) decreased linearly from 0.041% (100 g dose) to 0.020% (300 g dose) for acylated anthocyanins and from 0.18% (100 g dose) to 0.09% (300 g dose) for nonacylated anthocyanins. Anthocyanin metabolites consisted of glucuronidated and methylated anthocyanins. The results show that red cabbage anthocyanins were excreted in both intact and metabolized forms and that recovery of nonacylated anthocyanins in urine was >4-fold that of acylated anthocyanins.     

Properties of 3-deoxyanthocyanins from sorghum

There is increasing interest in natural food colorants with functional properties. Anthocyanins from black, brown (containing tannins), and red sorghums were characterized by spectrophotometric and HPLC techniques. The antioxidant activity and pH stability of the anthocyanins were also determined. Sorghum brans had 3-4 times higher anthocyanin contents than the whole grains. Black sorghum had the highest anthocyanin content (average = 10.1 mg/g in bran). The brown and red sorghum brans had anthocyanin contents of 2.8-4.3 mg/g. Only 3-deoxyanthocyanidins were detected in sorghum. These compounds are more stable to pH-induced color change than the common anthocyanidins and their glycosides. Additionally, crude sorghum anthocyanin extracts were more stable than the pure 3-deoxyanthocyanidins. The antioxidant properties of the 3-deoxyanthocyanidins were similar to those of the anthocyanins. Pigmented sorghum bran has high levels of unique 3-deoxyanthocyanidins, which are stable to change in pH and have a good potential as natural food pigments.     

Interactions between grape anthocyanins and pyruvic acid, with effect of pH and acid concentration on anthocyanin composition and color in model solutions.

The formation of vitisin A, an anthocyanin formed naturally in small quantities in maturing port wines, was studied in model wine solutions at a range of pH values (2.0-4.5) and pyruvate concentrations [molar ratios of pyruvic acid to total anthocyanins (PA/TA) ranging from 12.20 to 172.40]. Additionally, the effect of vitisin A formation on the color changes of these model wines was evaluated. Vitisin A was formed through the interaction between malvidin 3-glucoside and pyruvic acid, and vitisin A in acylated forms, having the 6-position of the sugar acylated with acetic acid (3-acetylvitisin A) and p-coumaric acid (3-p-coumarylvitisin A), formed through the interaction between pyruvic acid and malvidin 3-acetylglucoside and malvidin 3-p-coumarylglucoside, respectively; their identities were confirmed by spectral analysis and FABMS. The maximum formation of these new anthocyanin derivatives was at pH 2. 7-3.0, at the higher pyruvic acid concentration (PA/TA of 172.40 units). The vitisins A caused changes in the color of the solution and expressed about 11 times (pH 3) to 14 times (pH 2) more color than the normal anthocyanins. On aging, the model solutions changed from a bluish red, attributable to the main anthocyanins present, to a slightly more orange red, attributable to the vitisin compounds. The aged models containing vitisins A were all much redder than the more red-brown color of the models aged without pyruvic acid.     

Phytochemical composition and pigment stability of Açai (Euterpe oleracea Mart.)

Anthocyanin and polyphenolic compounds present in a?ai (Euterpe oleracea Mart.) were determined and their respective contribution to the overall antioxidant capacity established. Color stability of a?ai anthocyanins against hydrogen peroxide (0 and 30 mmol/L) over a range of temperatures (10-30 degrees C) was also determined and compared to common anthocyanin sources. Additionally, stability in a model beverage system was evaluated in the presence of ascorbic acid and naturally occurring polyphenolic cofactors. Cyanidin 3-glucoside (1040 mg/L) was the predominant anthocyanin in a?ai and correlated to antioxidant content, while 16 other polyphenolics were detected from 4 to 212 mg/L. Red grape anthocyanins were most stable in the presence of hydrogen peroxide, while a?ai and pigments rich in acylated anthocyanins displayed lower color stability in a temperature-dependent manner. In the presence of ascorbic acid, acylated anthocyanin sources generally had increased color stability. A?ai was recognized for its functional properties for use in food and nutraceutical products.     

Stability and enhancement of berry juice color

Attractive color is one of the main sensory characteristics of fruit and berry products. Unfortunately, the color of red juices is unstable and easily susceptible to degradation, leading to a dull and weak juice color. This study was designed to investigate the color stability and copigmentation of four different berry juices enhanced by phenolic acids and commercial color enhancers. Phenolic acid enrichment improved and stabilized the color of the berry juices during storage. The commercial color enhancers immediately produced an intensive color to the juices, which, however, was not very stable. The color enhancement was intensive in strawberry and raspberry juices and effective in lingonberry and cranberry juices. Sinapic acid induced the strongest color in strawberry juice. Ferulic and sinapic acids improved raspberry juice color equally. Rosmarinic acid enhanced the color of lingonberry and cranberry juices the most. The addition of the simple cinnamic acids produced novel peaks to the end of the high-performance liquid chromatography chromatogram, indicating a formation of new compounds. It can be assumed that sinapic and ferulic acids formed new intramolecular copigmentation compounds with berry anthocyanins whereas rosmarinic acid stabilized anthocyanins intermolecularly.     

Effect of red grapes co-winemaking in polyphenols and color of wines

The red grapes co-winemaking effect on phenolic fraction and wine color has been studied for the first time, where Monastrell was comacerated and cofermentated with Cabernet Sauvignon and Merlot. Changes in the relative abundance of anthocyanins were observed as well as hyperchromic shifts at 530 and 620 nm; these effects remain constant after aging. Co-winemaking also favored copigmentation, giving way to more stable anthocyanins and facilitating their polymerization. With regard to color evolution, the mixture of Monastrell with Merlot grapes was more appropriate than with Cabernet Sauvignon for aging wines in oak barrels. The extent of copigmentation was more important in young wines than in aged wines. This is mainly due to the self-anthocyanin monomer reactions in the case of young wines, whereas in aged wines copigmentation is mainly due to the reaction between the anthocyanins and other polyphenolic cofactors. Discriminant analysis showed the possibility of differentiating wines according to the aging time and the type of wine, with color parameters (color intensity, OD 620 nm, and OD 520 nm) being the most important discrimination variables in the first case and petunidin-3-glucoside and peonidin-3-glucoside contents in the second case.     

Identification of reaction products of acylated anthocyanins from red radish with peroxyl radicals

Red radish anthocyanin extract, which consists of 12 known acylated anthocyanins, was reacted with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) to generate peroxyl radicals under acidic pH conditions at 37 degrees C. The reaction products were isolated using preparative HPLC, and their chemical structures were determined to be p-hydroxybenzoic acid (1), 6-O-(E)-p-coumaroyl-2-O-beta-d- glucopyranosyl-alpha-d-glucopyranoside (3), p-coumaric acid (4), 6-O-(E)-feruloyl-2-O-beta-d-glucopyranosyl-alpha-d-glucopyranoside (5), and ferulic acid (6). Some products were not identified. HPLC analyses of the mixture of acylated pelargonidin isolated from red radish and AAPH revealed that the acylated pelargonidins possess the radical scavenging ability on some common sites even if the characteristics of the intramolecular acyl units are different. Degradation rates of acylated pelargonidins and the formation rates of the resulting reaction products were found to be quite different.     

Effect of copigments and grape cultivar on the color of red wines fermented after the addition of copigments

The prefermentation addition of copigments led to significantly different red wines according to the copigment structure (flavonol or hydroxycinnamic acid) and the grape cultivar [Tempranillo (= Cencibel) or Cabernet Sauvignon]. The flavonol rutin enhanced copigmentation and anthocyanin extraction, improving the red color, but the hydroxycinnamic acids (especially caffeic acid) had converse results. The above effects were higher in Cabernet Sauvignon wines, particularly if rutin or p-coumaric acid was used. These wines showed the highest copigmentation as they contained more anthocyanins and flavonols, whereas the coumaroylated anthocyanins of Tempranillo wines could have prevented the action of the added copigments. After 21 months, the main pyranoanthocyanins found were the malvidin-3-glucoside 4-vinylphenol and the malvidin-3-glucoside 4-vinylcatechol (pinotin A) adducts. The results suggested that the former adduct was primarily generated following enzymatic decarboxylation of p-coumaric acid during fermentation, whereas pinotin A was formed through a pure chemical reaction, which depended on the concentration of free caffeic acid during aging.     

LC-PDA-ESI/MS(n) identification of new anthocyanins in purple Bordeaux radish ( Raphanus sativus L. variety)

An LC-PDA-ESI/MS(n) profiling method was used to identify the anthocyanins of purple Bordeaux radish and led to the assignment of 60 anthocyanins: 14 acylated cyanidin 3-(glucosylacyl)acylsophoroside-5-diglucosides, 24 acylated cyanidin 3-sophoroside-5-diglucosides, and 22 acylated cyanidin 3-sophoroside-5-glucosides. The identifications were supported by the presence of 3-sophoroside-5-diglucoside and 3-sophroside-5-glucoside of cyanidin in the alkaline-hydrolyzed extract. A reliable method to identify the anthocyanins containing 3-(glucosylacyl)acylsophorosyl functions is described. The tentative identifications were obtained from tandem mass data analysis and confirmed by high-resolution mass measurements. Further assignments were made for some anthocyanins from a comparison of the mass and UV-vis data and elution order with those of the anthocyanins in the authors' polyphenol database and from consideration of the structural characteristics of the anthocyanins from similar plants and similar anthocyanins in the literature. The presence of 38 acylated cyanidin 3-sophoroside-5-diglucosides and around 10 acylated cyanidin 3-sophoroside-5-malonylglucosides in plants is reported here for the first time.     

Phytochemical stability and color retention of copigmented and processed muscadine grape juice

Muscadine (Vitis rotundifolia) grape juice was assessed for color and phytochemical stability as influenced by anthocyanin copigmentation with a water-soluble rosemary extract, fortification with ascorbic acid, and processing by heat or high hydrostatic pressure (HHP). The roles of polyphenolic cofactors in the presence and in the absence of ascorbic acid were assessed as a means to improve the overall processing stability of the juice. Addition of rosemary extract from 0 to 0.4% (v/v) readily formed copigment complexes with anthocyanins and resulted in concentration-dependent hyperchromic shifts from 10 to 27% that corresponded to increased antioxidant activity. The presence of ascorbic acid was generally detrimental to juice quality, especially in the presence of rosemary extract, and resulted in overall anthocyanin, ascorbic acid, and antioxidant activity losses. Although thermal and high-pressure processing methods were detrimental to juice quality, HHP resulted in greater losses after processing, likely due to action from residual oxidase enzymes. Although physicochemical attributes were enhanced by copigmentation with rosemary extract, methods to inactivate residual enzymes should be addressed prior to copigmentation to prevent degradation of anthocyanins in the presence of ascorbic acid.     

Prediction of wine color attributes from the phenolic profiles of red grapes (Vitis vinifera)

Knowledge about the relation between grape and wine phenolics is of key interest for the wine industry with respect to being able to predict wine quality from analyses of grapes. Prediction of the phenolic composition and color of experimentally produced red wines from the detailed phenolic composition of the corresponding grapes was investigated using a multivariate approach. Grape extracts and wines were produced from 55 different grape samples, covering 8 different Vitis vinifera cultivars: Alicante, Merlot, Syrah, Cinsault, Grenache, Carignan, Cabernet Sauvignon, and Mourvedre. The phenolic composition of the grapes and wines showed that the average ratios between wine and grape phenolics ranged from 0.25 to 7.9 for the different phenolic compounds. Most interestingly, the average ratios were low for anthocyanins (0.31) and tannins (0.32), intermediate for (+)-catechin (0.75) and polymeric pigments (0.98), and high for gallic acid (7.9). Individual wine phenolics in general correlated well with several grape phenolics, indicating that a multivariate approach might be advantageous for prediction of wine phenolics from grape phenolics analysis. However the use of multivariate prediction of individual wine phenolics from the complete grape phenolic composition only improved the prediction of wine polymeric pigments, whereas wine anthocyanins were predicted with the same precision as from the direct relation with grape anthocyanins. Prediction of color attributes of pH normalized experimental wines from the phenolic profiles of grapes was accomplished using a multivariate approach. The correlation between predicted and measured total wine color was high ( r = 0.958) but was very similar to the correlation coefficient obtained for the direct relation between grape anthocyanins and total wine color ( r = 0.961). Color due to copigmentation, color due to anthocyanins, and color intensity were also predicted well.     

Preparative isolation of anthocyanins by high-speed countercurrent chromatography and application of the color activity concept to red wine

Red pigments were isolated from wine and grape-skin extracts using preparative high-speed countercurrent chromatography (HSCCC) and identified by NMR and MS techniques. Four solvent systems were developed in order to separate anthocyanins with different polarities. Malvidin-3-glucoside was the major component present in young red wines, and up to 500 mg of pure malvidin-3-glucoside could be obtained from a single bottle of a red wine. Other isolated pigments were the malvidin- and peonidin-3,5-diglucosides, as well as acetyl-, coumaroyl-, and caffeoyl-derivatives of anthocyanins. Furthermore, condensed red wine pigments formed from malvidin-3-glucoside (vitisin A and acetylvitisin A) were isolated on a preparative scale. Isolated compounds were used as standards for quantification of anthocyanins in a range of red wines. The "color activity concept" was applied to red wine, and visual detection thresholds were determined for some of the isolated anthocyanins. Mono-glucosides were found to exhibit lower visual detection thresholds than di-glucosides and acylated anthocyanins.     

Anthocyanin color behavior and stability during storage: effect of intermolecular copigmentation

Intermolecular copigmentation reactions are significantly responsible for the manifold color expression of fruits, berries, and their products. These reactions were investigated with five anthocyanins and five phenolic acids acting as copigments. The stability of the pigment-copigment complexes formed was studied during a storage period of 6 months. The study was conducted using a UV-visible spectrophotometer to monitor the hyperchromic effect and the bathochromic shift of the complexes. The greatest copigmentation reactions took place in malvidin 3-glucoside solutions. The strongest copigments for all anthocyanins were ferulic and rosmarinic acids. The immediate reaction of rosmarinic acid with malvidin 3-glucoside resulted in the biggest bathochromic shift (19 nm) and the strongest hyperchromic effect, increasing the color intensity by 260%. The color induced by rosmarinic acid was not very stable. The color intensity of pelargonidin 3-glucoside increased greatly throughout the storage period with the addition of ferulic and caffeic acids.     

Influence of procyanidins on the color stability of oenin solutions

The aim of the present work was to specify the influence of the polymerization degree on the color stability of anthocyanins using model solutions under higher thermal conditions simulating rapid food aging. Results showed that an increase in polymeric degree improves the color stability of oenin. Solutions containing a catechin tetramer, purified from brown rice, displayed a remarkable stability. Flavanols as monomers, (+)-catechin and (-)-epicatechin, appeared to decrease stability with the formation of a xanthylium salt leading to yellowish solutions. For the dimers, procyanidin B2 and B3, different behaviors on red color stability have been observed corresponding to their different susceptibility to cleavage upon heating. In the presence of the trimeric procyanidin C2, the red color appeared more stable. However, the HPLC chromatograms showed a decrease in the amplitude of the peaks of oenin and procyanidin C2. Concomitantly, a new peak appeared with a maximal absorption in the red region. This newly formed pigment probably came from the condensation of oenin and procyanidin C2.     

Comprehensive colorimetric study of anthocyanic copigmentation in model solutions. Effects of pH and molar ratio

New colorimetric variables have been defined in the uniform CIELAB color space to assess the quantitative and qualitative color changes induced by copigmentation and their incidence on visual perception. The copigmentation process was assayed in model solutions between malvidin 3-glucoside and three phenolic compounds (catechin, epicatechin, and caffeic acid) as a function of the pH and the pigment/copigment molar ratio. Along the pH variation, the greatest magnitude of copigmentation was obtained at pH 3.0, being significantly higher with epicatechin and caffeic acid. At high acidic pH, the main contribution of copigmentation to the total color was qualitative, whereas between pH 2.0 and 4.0, the main colorimetric contribution was quantitative. The contribution of epicatechin and caffeic acid to the color changes was more marked for the quantitative characteristics. On contrast, particularly at higher pH values, the qualitative contribution was more important in catechin copigmented solutions. Increasing copigment concentration induced perceptible color changes at molar ratios higher than 1:2, consisting in a bluish and darkening effect of the anthocyanin solutions. Among the different CIELAB attributes, hue difference was the best correlated parameter with the increase of copigment concentration, proving the relevance of this physicochemical phenomenon on the qualitative changes of anthocyanin color.     

Anthocyanin composition of black carrot (Daucus carota ssp. sativus var. atrorubens Alef.) cultivars Antonina, Beta Sweet, Deep Purple, and Purple Haze

This study aimed to identify the pigment composition of black carrot (Daucus carota ssp. sativus var. atrorubens Alef.) cultivars Antonina, Beta Sweet, Deep Purple, and Purple Haze. Cyanidin 3-xylosyl(glucosyl)galactosides acylated with sinapic acid, ferulic acid, and coumaric acid were detected as major anthocyanins by high-performance liquid chromatography with diode array detection (HPLC-DAD) and with electrospray ionization multiple mass spectrometry (HPLC-ESI-MS(n)) analyses. The preparative isolation of these pigments was carried out by means of high-speed countercurrent chromatography (HSCCC). The color activity concept was applied to the isolated anthocyanins at three pH values. Cyanidin 3-xylosyl(sinapoylglucosyl)galactoside was found to exhibit a lower visual detection threshold and a higher pH stability than cyanidin 3-xylosyl(feruloylglucosyl)galactoside and cyanidin 3-xylosyl(coumaroylglucosyl)galactoside. The color parameters of the fresh roots of the four cultivars were described by the CIELab coordinates L* (lightness), C* (chroma), and h(ab) (hue angles). Total phenolics varied among the cultivars and ranged from 17.9 to 97.9 mg gallic acid equivalents (GAE)/100 g fresh weight (fw). For the content of monomeric anthocyanins, values between 1.5 and 17.7 mg/100 g fw were determined.     

Effect of storage temperature on the stability of anthocyanins of a fermented black carrot (Daucus carota var. L.) beverage: shalgam

The effect of temperature on the stability of shalgam anthocyanins stored at 4, 25, and 40 degrees C for 90 days was investigated. The effect of pasteurization and sorbate addition on the anthocyanin stability as compared to control was also studied. The monomeric anthocyanin content and color density decreased with increasing time as a function of storage temperature whereas the percent polymeric color and browning increased. The same trends were observed in control, pasteurized, and sorbate-added shalgam samples. Shalgam anthocyanins consisted of two nonacylated and three acylated cyanidin derivatives. Acylated anthocyanins were more stable when compared to nonacylated ones at all storage temperatures. The activation energies, 11.11-11.64 kcal/mol, were calculated from the reaction rate constants evaluated taking first-order reaction kinetics. The highest anthocyanin retention was observed at 4 degrees C storage temperature with a half-life between 231 and 239 days.