Acylated Anthocyanins from Red Cabbage and Purple Sweet Potato Can Bind Metal Ions and Produce Stable Blue Colors.

Red cabbage (RC) and purple sweet potato (PSP) are naturally rich in acylated cyanidin glycosides that can bind metal ions and develop intramolecular π-stacking interactions between the cyanidin chromophore and the phenolic acyl residues. In this work, a large set of RC and PSP anthocyanins was investigated for its coloring properties in the presence of iron and aluminum ions. Although relatively modest, the structural differences between RC and PSP anthocyanins, i.e., the acylation site at the external glucose of the sophorosyl moiety (C2-OH for RC vs. C6-OH for PSP) and the presence of coordinating acyl groups (caffeoyl) in PSP anthocyanins only, made a large difference in the color expressed by their metal complexes. For instance, the Al3+-induced bathochromic shifts for RC anthocyanins reached ca. 50 nm at pH 6 and pH 7, vs. at best ca. 20 nm for PSP anthocyanins. With Fe2+ (quickly oxidized to Fe3+ in the complexes), the bathochromic shifts for RC anthocyanins were higher, i.e., up to ca. 90 nm at pH 7 and 110 nm at pH 5.7. A kinetic analysis at different metal/ligand molar ratios combined with an investigation by high-resolution mass spectrometry suggested the formation of metal-anthocyanin complexes of 1:1, 1:2, and 1:3 stoichiometries. Contrary to predictions based on steric hindrance, acylation by noncoordinating acyl residues favored metal binding and resulted in complexes having much higher molar absorption coefficients. Moreover, the competition between metal binding and water addition to the free ligands (leading to colorless forms) was less severe, although very dependent on the acylation site(s). Overall, anthocyanins from purple sweet potato, and even more from red cabbage, have a strong potential for development as food colorants expressing red to blue hues depending on pH and metal ion.

Molar absorptivities (ε) and spectral and colorimetric characteristics of purple sweet potato anthocyanins.

Purple sweet potato, a source of acylated cyanidin and peonidin derivatives, is commercially available as a food colorant. Our objectives were to determine molar absorptivities (ε), spectral and colorimetric properties of purple sweet potato anthocyanins. Anthocyanins were isolated by semi-preparative HPLC, weighed, dried, and redissolved in acidic methanol or water. Anthocyanins were diluted in pH 1-9; ε, spectra, and color were measured on the methanolic and aqueous solutions. Higher ε were obtained in 0.1% HCl methanol (10,797-31,257 L/(mol × cm)) than in aqueous solution pH 1 (8861-24,303 L/(mol × cm)). Peonidin-3-sophoroside-5-glucoside had greatest ε in pH 1, but in alkaline pH, ε of acylated Peonidin-3-sophoroside-5-glucoside derivatives were greatest. Generally monoacylation decreased ε while diacylation increased ε. Location of acylation also affected ε of two Peonidin isomers (pH 1: 15,999 and 21,011 L/(mol × cm)). All anthocyanins expressed red-pink hues (330°-13.2°) in acidic pH and blues (230°-262°) in alkaline pH.

Black goji as a potential source of natural color in a wide pH range.

Lycium ruthenicum Murr. is a traditional Chinese herb widely distributed in Tibet. The fruit, known as black goji, is popular in traditional Chinese medicine. The objective of this study was to investigate its anthocyanin profile (by HPLC coupled to PDA and MS detectors) and the colorimetric and spectrophotometric properties. Black goji extracts contained abundant petunidin derivatives, with cis and trans isomers of petunidin-3-p-coumaroyl-rutinoside-5-glucoside. The colorimetric and spectrophotometric traits of black goji anthocyanins were significantly impacted by solid-phase-extraction, pH, and acylation. MCX cartridge removed considerable polyphenolics from fruit extracts, but attenuated the saturation of color expression. Petunidin-3-trans-p-coumaroyl-rutinoside-5-glucoside contributed most of the color expression of the black goji extract, and showed superior stability compared to other extracts over time. Acylation strengthened the petunidin derivatives color retention, and enhanced the color intensity and stability. Black goji anthocyanins produced various vivid hues over wide ranges of pH, making them promising candidates for natural colorants.

Encapsulation of purple corn and blueberry extracts in alginate-pectin hydrogel particles: Impact of processing and storage parameters on encapsulation efficiency.

Purple corn (PC) and blueberry (BB) extracts were encapsulated in alginate-pectin hydrogel particles to protect anthocyanins (ACNs) from degradation. Combinations of alginate to pectin ratios at 82 to 18% and 43 to 57% and total gum concentrations (TGC) at 2.2% and 2.8% TGC were prepared to encapsulate both PC and BB ACN. The alginate-pectin hydrogel particles containing PC or BB extracts were produced by dripping solution into pH 1.2 buffer. Blueberry extract encapsulation efficiency was significantly higher than that of purple corn extract due to ACN chemical structure differences and the compatibility between the ACN structures and alginate-pectin hydrogel structure at the low pH environment. Effect of initial ACN concentration in droplets, particle shape, alginate to pectin ratio, TGC, ACN source, and curing bath conditions on encapsulation efficiency after curing (EEm) was investigated. The initial ACN concentration and particle shape didn't influence the EEm, while the alginate to pectin ratio, TGC, ACN source and the pH of the curing bath showed significant effect on the EEm. The EEm was improved from 26% to 65% for PC ACN and from 48% to 116% for BB ACN by augmenting curing bath with ACN at various concentrations. The ACN retention during storage (ARs) in hydrogel particles stored in pH 3.0 buffer was improved at low temperature and high particle weight to solution volume ratio. Higher amount of ACN was retained in the hydrogel particles when spherical particles were used. Encapsulation in hydrogel particles significantly reduced the anthocyanin photodegradation upon exposure to fluorescence light. The degradation of ACN was described with a first-order kinetics with half-life values of 630 h for encapsulated PC ACN and 58 h for PC ACN aqueous solution. Hydrogel production and subsequent storage conditions can be optimized to increase the anthocyanin delivered to human body using the low pH beverages such as fruit juices as a delivery vehicle.

Assessment of the color modulation and stability of naturally copigmented anthocyanin-grape colorants with different levels of purification.

Grape skins or their by-products from wine production are rich sources of anthocyanins and various colorless phenolics, depending on the grape variety. Phenolics have strong antioxidant and anthocyanin stabilizing properties and help to produce functional anthocyanin colorants with improved stability. This study aimed to assess differences in color expression and stability of anthocyanin colorants from red grape varieties naturally copigmented and with different levels of purity and to compare them to synthetic FD&C Red No. 3. Model juice systems were prepared at pH 3.5 with anthocyanins and phenolic copigments extracted from four Vitis vinifera grape varieties ('Tempranillo', 'Syrah', 'C. Sauvignon', and 'Graciano') both crude and purified by C18 solid phase extraction. Attention was focused on differential colorimetry and phenolic composition related to the color. Degradation kinetics of total color were also studied during storage of 17 days in darkness at 25 °C. Grape variety significantly influenced pigment yield, proportion of acylation, and proportion of copigments:pigments ratios in crude extracts; purification modulated the copigment:pigment ratios. This proportion was related to perceptible color variability among colorants and to different stabilities. With the same pigment content, grape varieties richer in skin copigments and higher copigment/pigment ratios ('Syrah' and 'Tempranillo') produced more intensely colored crude extracts whose tonalities ranged from reddish ('Graciano') to red-bluish ('Syrah'), depending on the proportion of acylation. Increasing the purity of the pigments diminished the color variability due to variety, making them less vivid and visually more similar to one another and also to the synthetic colorant. Degradation kinetic studies showed that unpurified grape colorants had higher color stability over time, with the greatest stabilizing effects achieved with varieties richer in skin flavonols ('Tempranillo' and 'Syrah').

The effect of pigment matrix, temperature and amount of carrier on the yield and final color properties of spray dried purple corn (Zea mays L.) cob anthocyanin powders.

Spray drying is an economic technique to produce anthocyanin-based colorants. High pigments yields with minimum color degradation are desirable to maximize quality and profits. This study evaluated the impacts of purple corncob (PCC) anthocyanin extraction matrices (hot water, 40% ethanol, C18 purified), drying inlet temperature (130, 150, 170°C) and amount of carrier (2%, 5%, 10% maltodextrin) on the yields and quality of PCC anthocyanin powders. Monomeric and polymeric anthocyanins, color properties (CIELch, haze), and pigments composition before and after spray drying were determined. The yield and final color quality of spray dried PCC anthocyanins were affected (p<0.05) by all parameters evaluated. The pigment matrix, inlet temperature, and carrier amount had biggest impacts on product water solubility, pigments degradation and yield, respectively. The optimal combination of hot water extracts spray dried with 5% maltodextrin at 150°C gave the highest pigment yield (∼90%) with good solubility with the least color loss.

Improving the screening of potato breeding lines for specific nutritional traits using portable mid-infrared spectroscopy and multivariate analysis.

Efficient selection of potato varieties with enhanced nutritional quality requires simple, accurate and cost effective assays to obtain tuber chemical composition information. In this study, 75 Andean native potato samples from 7 Solanum species with different colors were characterized and quantified for their anthocyanin, phenolics and sugar content using traditional reference methods. IR (infrared) spectra of potato extracts were collected using a portable infrared system and partial least squares regression (PLSR) calibration models were developed. These models were validated using both full cross-validation and an independent sample set giving strong linear correlation coefficients of prediction (rPred)>0.91 and standard error of prediction (SEP) of 24mg/100g phenolics, 7mg/100g monomeric anthocyanins, 0.1g/100g reducing sugars and 0.12g/100g sucrose. Overall, portable infrared system with PLSR showed great potential to facilitate potato breeding and certain aspects of crop management, material selection for potato processing and related research by providing alternative prediction models.

Molar absorptivity (ε) and spectral characteristics of cyanidin-based anthocyanins from red cabbage.

Red cabbage extract contains mono and di-acylated cyanidin (Cy) anthocyanins and is often used as food colorants. Our objectives were to determine the molar absorptivity (ε) of different red cabbage Cy-derivatives and to evaluate their spectral behaviors in acidified methanol (MeOH) and buffers pH 1-9. Major red cabbage anthocyanins were isolated using a semi-preparatory HPLC, dried and weighed. Pigments were dissolved in MeOH and diluted with either MeOH (0.1% HCl) or buffers to obtain final concentrations between 5×10(-5) and 1×10(-3) mol/L. Spectra were recorded and ε calculated using Lambert-Beer's law. The ε in acidified MeOH and buffer pH 1 ranged between ~16,000-30,000 and ~13,000-26,000 L/mol cm, respectively. Most pigments showed higher ε in pH 8 than pH 2, and lowest ε between pH 4 and 6. There were bathochromic shifts (81-105 nm) from pH 1 to 8 and hypsochromic shifts from pH 8 to 9 (2-19 nm). Anthocyanins molecular structures and the media were important variables which greatly influenced their ε and spectral behaviors.

Polyphenol extraction optimisation from Ceylon gooseberry (Dovyalis hebecarpa) pulp.

Originally from Asia, Dovyalis hebecarpa is a dark purple/red exotic berry now also produced in Brazil. However, no reports were found in the literature about phenolic extraction or characterisation of this berry. In this study we evaluate the extraction optimisation of anthocyanins and total phenolics in D. hebecarpa berries aiming at the development of a simple and mild analytical technique. Multivariate analysis was used to optimise the extraction variables (ethanol:water:acetone solvent proportions, times, and acid concentrations) at different levels. Acetone/water (20/80 v/v) gave the highest anthocyanin extraction yield, but pure water and different proportions of acetone/water or acetone/ethanol/water (with >50% of water) were also effective. Neither acid concentration nor time had a significant effect on extraction efficiency allowing to fix the recommended parameters at the lowest values tested (0.35% formic acid v/v, and 17.6 min). Under optimised conditions, extraction efficiencies were increased by 31.5% and 11% for anthocyanin and total phenolics, respectively as compared to traditional methods that use more solvent and time. Thus, the optimised methodology increased yields being less hazardous and time consuming than traditional methods. Finally, freeze-dried D. hebecarpa showed high content of target phytochemicals (319 mg/100g and 1,421 mg/100g of total anthocyanin and total phenolic content, respectively).

Anthocyanins contents, profiles, and color characteristics of red cabbage extracts from different cultivars and maturity stages.

Red cabbage (Brassica oleracea L.) is an excellent source of food colorant. This study aimed to evaluate the anthocyanin pigment contents and profiles from seven red cabbage cultivars at two maturity stages (8 weeks apart) and evaluate their color characteristics and behavior under acidic and neutral pH. Anthocyanin concentrations ranged from 1111 to 1780 mg Cy3G/100 g DM and did not increase with time. Cultivar and maturation affected pigment profile. Some varieties accumulated ≥30% of diacylated pigments, and proportions of monoacylated pigments decreased with time. Extracts from selected varieties at first harvesting time produced colors similar (λmax = 520 nm and ΔE = 6.1-8.8) to FD&C Red No. 3 at pH 3.5. At pH 7, extracts from the second harvest with s higher proportion of diacylation produced λmax ≃ 610 nm, similar to FD&C Blue No. 2. Cultivar selection and maturation affected color and stability of red cabbage extracts at different pH values.

Characterization and quantitation of anthocyanins and other phenolics in native Andean potatoes.

Andean potatoes are gaining popularity not only for their appealing colors and culinary uses but also for their potential higher content of polyphenolic compounds. The objective of this study was to identify potato varieties with increased phenolic content. This was achieved through characterization and quantitation of the phenolic composition in 20 varieties of native Andean potatoes from 4 different Solanum species with different colors. Major quantitative and qualitative differences among evaluated samples were more dependent on the coloration of the extracted sample rather than on the species. The most predominant anthocyanidins were petunidin-3-coumaroylrutinoside-5-glucoside and pelargonidin-3-coumaroylrutinoside-5-glucoside in purple and red potato extracts, respectively, while chlorogenic acid and its isomers were the main phenolic compund (43% of the total phenolic content). Our study suggested that the appropriate selection of native potatoes could provide new sources of polyphenolics with health promoting properties and natural pigments with increased stability for food applications.

NMR-based metabolomic investigation of bioactivity of chemical constituents in black raspberry (Rubus occidentalis L.) fruit extracts.

Black raspberry (Rubus occidentalis L.) (BR) fruit extracts with differing compound profiles have shown variable antiproliferative activities against HT-29 colon cancer cell lines. This study used partial least-squares (PLS) regression analysis to develop a high-resolution (1)H NMR-based multivariate statistical model for discerning the biological activity of BR constituents. This model identified specific bioactive compounds and ascertained their relative contribution against cancer cell proliferation. Cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside were the predominant contributors to the extract bioactivity, but salicylic acid derivatives (e.g., salicylic acid glucosyl ester), quercetin 3-glucoside, quercetin 3-rutinoside, p-coumaric acid, epicatechin, methyl ellagic acid derivatives (e.g., methyl ellagic acetyl pentose), and citric acid derivatives also contributed significantly to the antiproliferative activity of the berry extracts. This approach enabled the identification of new bioactive components in BR fruits and demonstrates the utility of the method for assessing chemopreventive compounds in foods and food products.

Susceptibility of anthocyanins to ex vivo degradation in human saliva.

Some fruits and their anthocyanin-rich extracts have been reported to exhibit chemopreventive activity in the oral cavity. Insights regarding oral metabolism of anthocyanins remain limited. Anthocyanin-rich extracts from blueberry, chokeberry, black raspberry, red grape, and strawberry were incubated ex vivo with human saliva from 14 healthy subjects. All anthocyanins were partially degraded in saliva. Degradation of chokeberry anthocyanins in saliva was temperature dependent and decreased by heating saliva to 80 °C and after removal of cells. Glycosides of delphinidin and petunidin were more susceptible to degradation than those of cyanidin, pelargonidin, peonidin and malvidin in both intact and artificial saliva. Stability of di- and tri-saccharide conjugates of anthocyanidins slightly, but significantly, exceeded that of monosaccharide compounds. Ex vivo degradation of anthocyanins in saliva was significantly decreased after oral rinsing with antibacterial chlorhexidine. These results suggest that anthocyanin degradation in the mouth is structure-dependent and largely mediated by oral microbiota.

Effect of black raspberry ( Rubus occidentalis L.) extract variation conditioned by cultivar, production site, and fruit maturity stage on colon cancer cell proliferation.

Black raspberries have been shown to inhibit multiple stages of oral, esophageal, and colon cancer. The objective of this study was to evaluate how black raspberry extract variability conditioned by horticultural factors affected the antiproliferative activity of 75 black raspberry extracts using an in vitro colon cancer cell model. HT-29 cells grown in 96-well plates were treated with freeze-dried extracts at 0.6 and 1.2 mg of extract/mL of medium. Percent cell growth inhibition for each concentration of the extracts was determined using the sulforhodamine B assay. All extracts significantly inhibited the growth of HT-29 colon cancer cells in a dose-dependent manner. Cell proliferation was significantly influenced by cultivar, production site, and stage of maturity. The lack of correlation between growth inhibition and extract total phenolic and total monomeric anthocyanin assays suggested horticultural parameters influence bioactivity in a complex manner.

Selective removal of the violet color produced by anthocyanins in procyanidin-rich unfermented cocoa extracts.

Cacao (Theobroma cacao L.) is rich in procyanidins, a large portion of which degrades during the natural fermentation process of producing cocoa powder. Recent advances in technology have enabled scientists to produce unfermented cocoa powder, preserving the original profile of procyanidins present in cocoa and allowing for the development of highly concentrated procyanidin-rich extracts. During this process, the anthocyanins naturally present in unfermented cocoa remain intact, producing a violet color in the final extract. The objective of this study was to selectively remove the violet color in procyanidin-rich extracts produced from unfermented cocoa powder, while maintaining the stability and composition of procyanidins present in the matrix. Several processing parameters, including pH fluctuations, enzymatic treatments, and the addition of potassium meta-bisulfite, were explored to influence the color of procyanidin-rich extracts throughout a 60-d shelf life study. The addition of potassium meta-bisulfite (500 ppm) was found to be the most effective means of removing the violet color present in the treated extracts (L*= 71.39, a*= 8.44, b*= 9.61, chroma = 12.79, and hue = 48.8˚) as compared to the control (L*= 52.84, a*= 11.08, b*= 2.24, chroma = 11.28, and hue = 11.4˚). The use of potassium meta-bisulfite at all treatment levels (200, 500, and 1000 ppm) did not show any significant detrimental effects on the stability, composition, or amount of procyanidins present in the extracts over the shelf life period as monitored by UV-Vis spectrophotometry and HPLC-MS. This research will enable the food industry to incorporate highly concentrated procyanidin-rich extracts in food products without influencing the color of the final product.

Extraction and normal-phase HPLC-fluorescence-electrospray MS characterization and quantification of procyanidins in cranberry extracts.

Cranberries are a major source of procyanidins, an abundant class of bioactive polyphenols found in nature. The objective of this study was to apply a protocol for extraction and normal-phase high-performance liquid chromatograph-fluorescence-mass spectroscopy separation and quantification of procyanidins in cranberry extracts containing varying processing aids and/or methods of production. Cranberry extracts were best extracted using an acetone/water technique versus an acid/alkaline extraction. Procyanidins were semi-purified with C-18 and Sephadex LH-20 SPE cartridges (GE Healthcare Life Sciences, Piscataway, N.J., U.S.A.). Characterization and quantification of procyanidins up to octamers and higher molecular weight compounds, including separation of the A- and B-type dimers to tetramers was achieved.

Improving the screening process for the selection of potato breeding lines with enhanced polyphenolics content.

Efficient selection of potato varieties with enhanced nutritional quality requires simple, rapid, accurate, and cost-effective assays to obtain tuber chemical composition information. Our objective was to develop simple protocols to determine phenolics, anthocyanins, and antioxidant capacity in polyphenolic extracts of potatoes using Fourier transform infrared spectroscopy combined with multivariate techniques. Lyophilized potato samples (23) were analyzed. Polyphenolic compounds were extracted from potatoes and applied directly applied onto a three-bounce ZnSe crystal for attenuated total reflectance measurements in the infrared region of 4000 to 700 cm (-1). Robust models were generated (r > or = 0.99) with standard error of cross-validation values of 4.17 mg gallic acid equivalent/100 g (total phenolics), 0.87 mg pelargonidin-3-glucoside/100 g (monomeric anthocyanins), and 130.8 mumol Trolox equivalent/100 g (antioxidant capacity) potato powder. In addition, classification models discriminated potato samples at the species and variety level. Application of a simple infrared spectroscopic protocol allowed simultaneous rapid quantification of specific nutritional components in potatoes and efficient selection of value-added potato varieties.

Structure-function relationships of anthocyanins from various anthocyanin-rich extracts on the inhibition of colon cancer cell growth.

Anthocyanins are potent antioxidants and may be chemoprotective. However, the structure-function relationships are not well understood. The objectives of this study were to compare the chemoprotective properties of anthocyanin-rich extracts (AREs) with variable anthocyanin profiles to understand the relationship between anthocyanin chemical structure and chemoprotective activity, measured as inhibition of colon cancer cell proliferation. Additionally, the chemoprotective interaction of anthocyanins and other phenolics was investigated. AREs with different anthocyanin profiles from purple corn, chokeberry, bilberry, purple carrot, grape, radish, and elderberry were tested for growth inhibition (GI 50) using a human colorectal adenocarcinoma (HT29) cell line. All AREs suppressed HT29 cell growth to various degrees as follows: purple corn (GI 50 approximately 14 microg of cy-3-glu equiv/mL) > chokeberry and bilberry > purple carrot and grape > radish and elderberry (GI 50 > 100 microg of cy-3-glu equiv/mL). Anthocyanins played a major role in AREs' chemoprotection and exerted an additive interaction with the other phenolics present. Statistical analyses suggested that anthocyanin chemical structure affected chemoprotection, with nonacylated monoglycosylated anthocyanins having greater inhibitory effect on HT-29 cell proliferation, whereas anthocyanins with pelargonidin, triglycoside, and/or acylation with cinnamic acid exerted the least effect. These findings should be considered for crop selection and the development of anthocyanin-rich functional foods.

Intact anthocyanins and metabolites in rat urine and plasma after 3 months of anthocyanin supplementation.

Anthocyanins are polyphenols responsible for most red to purple colors in plants. Human consumption of these pigments is increasing because of their potential health benefits and use as natural colorants. With more than 600 different anthocyanins found in nature, the impact of chemical structure on their absorption and metabolism needs to be investigated. Urine and plasma samples were collected from 32 rats receiving control diet or chokeberry-, bilberry-, and grape-enriched (3.85 g cyanidin 3-galatoside equivalent/kg) diet for 14 wk. Below 2 micromol/l of anthocyanins and relatively higher levels of presumable metabolites were detected by high-performance liquid chromatography-photodiode array in the plasma. In the urine the total concentration of intact anthocyanins and methylated derivatives ranged from 17.4 (bilberry) to 52.6 (chokeberry) nmol/l. The type and number of anthocyanin glycosylations affected the absorption remarkably. Detection of an acylated anthocyanin in plasma and urine suggests bioavailability of these anthocyanin derivatives that are commonly found in commercially available colorants.

Anthocyanin-rich extracts inhibit multiple biomarkers of colon cancer in rats.

The aim of the present study was to investigate the chemoprotective activity of anthocyanin-rich extracts (AREs) from bilberry (Vaccinium myrtillus L.), chokeberry (Aronia meloncarpa E.), and grape (Vitis vinifera) by assessing multiple biomarkers of colon cancer in male rats treated with a colon carcinogen, azoxymethane. Fischer 344 male rats were fed the AIN-93 diet (control) or AIN-93 diet supplemented with AREs for 14 wk. Biomarkers that were evaluated included the number and multiplicity of colonic aberrant crypt foci (ACF), colonic cell proliferation, urinary levels of oxidative DNA damage, and expression of cyclooxygenase (COX) genes. To assess the bioavailability, levels of anthocyanins in serum, urine, and feces were evaluated. Total ACF were reduced (P<0.05) in bilberry, chokeberry, and grape diet groups compared with the control group. The number of large ACF was also reduced (P<0.05) in bilberry and chokeberry ARE-fed rats. Colonic cellular proliferation was decreased in rats fed bilberry ARE and chokeberry ARE diets. Rats fed bilberry and grape ARE diets had lower COX-2 mRNA expression of gene. High levels of fecal anthocyanins and increased fecal mass and fecal moisture occurred in ARE-fed rats. There was also a significant reduction (P<0.05) in fecal bile acids in ARE-fed rats. The levels of urinary 8-hydroxyguanosine were similar among rats fed different diets. These results support our previous in vitro studies suggesting a protective role of AREs in colon carcinogenesis and indicate multiple mechanisms of action.