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.

Discovery of a natural cyan blue: A unique food-sourced anthocyanin could replace synthetic brilliant blue.

The color of food is critical to the food and beverage industries, as it influences many properties beyond eye-pleasing visuals including flavor, safety, and nutritional value. Blue is one of the rarest colors in nature's food palette-especially a cyan blue-giving scientists few sources for natural blue food colorants. Finding a natural cyan blue dye equivalent to FD&C Blue No. 1 remains an industry-wide challenge and the subject of several research programs worldwide. Computational simulations and large-array spectroscopic techniques were used to determine the 3D chemical structure, color expression, and stability of this previously uncharacterized cyan blue anthocyanin-based colorant. Synthetic biology and computational protein design tools were leveraged to develop an enzymatic transformation of red cabbage anthocyanins into the desired anthocyanin. More broadly, this research demonstrates the power of a multidisciplinary strategy to solve a long-standing challenge in the food industry.

The influence of acylation, metal binding and natural antioxidants on the thermal stability of red cabbage anthocyanins in neutral solution.

The main red cabbage anthocyanins (pigments) are cyanidin glycosides bearing one or two acyl groups derived from hydroxycinnamic acids (HCAs). Through π-stacking interactions with the cyanidin chromophore, the HCA residues have a deep influence on the color expressed and its stability. In this work, a series of non-, mono- and diacylated anthocyanins were investigated in neutral solution (pH 7 and 8), where the pigments exhibit purple to blue colors. Under such conditions, the gradual color loss observed is a combination of two distinct processes involving the cyanidin nucleus: reversible water addition and irreversible autoxidation. By acidification to pH < 2, the colorless forms stemming from water addition (hemiketal and chalcones) are converted to the red flavylium ion, thereby permitting the selective monitoring of the irreversible contribution. The kinetics of color loss and of true pigment degradation could thus be recorded for each pigment. The influence of iron - cyanidin binding and of antioxidants (caffeic acid, N-acetylcysteine) was also investigated. A complete kinetic analysis combining the anthocyanin colored and colorless forms and the degradation products is provided. Overall, it appears that acylation is critical to color stability. For instance, the nonacylated pigment is rapidly bleached as a result of fast water addition and its iron complex is too unstable to provide protection. By contrast, the diacylated pigments are efficiently protected against hydration but much more moderately against autoxidation, which on the other hand is inhibited by efficient iron binding and addition of N-acetylcysteine. Finally, the diacylated pigments are much more resistant to bleaching by hydrogen peroxide (possibly produced by cyanidin autoxidation) and bisulfite (a common food preservative).

Solid phase fractionation techniques for segregation of red cabbage anthocyanins with different colorimetric and stability properties.

Red cabbage anthocyanin extract contains cyanidin derivatives as various non-, mono-, and di-acylated forms capable of expressing various colors from red to blue. The objective of this study was to develop a simple solid phase extraction (SPE) method to fractionate and segregate di-acylated anthocyanins which have greater stability and more desirable blue colorimetric properties. Two different sorbents with different separation chemistries: SCX (Strong Cation Exchange) and C18 sorbents were evaluated. Red cabbage anthocyanins were fractionated and eluted from the SCX cartridge using combinations of different buffers (pH 6-8) and MeOH (20-70%) or eluted from the C18 cartridge using different percentages of acidified (0.01% HCl) MeOH (30-100%) or EtOH (10-100%). With SCX SPE, washing the loaded pigments with a mixture of buffer pH 6 + 20% MeOH followed by elution with buffer pH 8 + 70% MeOH proved most effective in segregating the di-acylated pigments; however, some anthocyanins were permanently retained by the sorbent. When using the C18 cartridge, washing bound pigments with 32% MeOH or 18.5% EtOH and eluting with 100% alcohol respectively resulted in high (> 93%) recovery of di-acylated pigments. The C18 cartridge with MeOH eluent was the most effective for isolating the target di-acylated red cabbage anthocyanins that produced desirable blue colors with increased stability.

Unraveling the molecular mechanisms of color expression in anthocyanins.

Anthocyanins are a broad family of natural dyes, increasingly finding application as substitutes for artificial colorants in the food industry. In spite of their importance and ubiquity, the molecular principles responsible for their extreme color variability are poorly known. We address these mechanisms by computer simulations and photoabsorption experiments of cyanidin-3-O-glucoside in water solution, as a proxy for more complex members of the family. Experimental results are presented in the range of pH 1-9, accompanied by a comprehensive systematic computational study across relevant charge states and tautomers. The computed spectra are in excellent agreement with the experiments, providing unprecedented insight into the complex behavior underlying color expression in these molecules. Besides confirming the importance of the molecule's charge state, we also unveil the hitherto unrecognized role of internal distortions in the chromophore, which affect its degree of conjugation, modulating the optical gap and in turn the color. This entanglement of structural and electronic traits is also shared by other members of the anthocyanin family (e.g. pelargonidin and delphinidin) highlighting a common mechanism for color expression across this important family of natural dyes.

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.

Impact of location, type, and number of glycosidic substitutions on the color expression of o-dihydroxylated anthocyanidins.

Anthocyanins express many hues depending on environmental factors and structural aspects, of which aglycone structure and acylation have been considered most impactful. Effects of different glycosylations on anthocyanins' colors are less known. Twelve cyanidin and 3-deoxy-cyanidin (luteolinidin) derivatives were isolated from red cabbage and black carrot hydrolysates and from black sorghum, diluted in pH 1-9, and analyzed by spectrophotometry and colorimetry. Location, number, and structure of glycosylations affected λmax and spectral shape of o-dihydroxylated anthocyanins, playing important roles on color expression. Generally, glycosylation of cyanidin decreased its λmax (≤27 nm), greatest decreases by 3-monoglycosylation. All cyanidin-glycosides appeared red in pH 1-3 and paled in pH 4-6. However, cyanidin-3-glycosides did not decolor completely like 3,5-glycosides. In alkaline pH, glycosylation patterns affected color more greatly: Cy-3-glycosides expressed maroon-purple hues (300-20°), 5-glycosides were green (100-115°), and only 3,5-glycosides expressed blue (240-250°). Luteolinidin derivatives shifted from yellow to red-purple hues with increasing pH.

Effects of Growth Temperature and Postharvest Cooling on Anthocyanin Profiles in Juvenile and Mature Brassica oleracea.

The effects of growth temperatures on anthocyanin content and profile were tested on juvenile cabbage and kale plants. The effects of cold storage time were evaluated on both juvenile and mature plants. The anthocyanin content in juvenile plants ranged from 3.82 mg of cyanidin-3,5-diglucoside equivalent (Cy equiv)/g of dry matter (dm) at 25 °C to 10.00 mg of Cy equiv/g of dm at 16 °C, with up to 76% diacylated anthocyanins. Cold storage of juvenile plants decreased the total amount of anthocyanins but increased the diacylated anthocyanin content by 3-5%. In mature plants, cold storage reduced the total anthocyanin content from 22 to 12.23 mg/g after 5 weeks of storage in red cabbage, while the total anthocyanin content increased after 2 weeks of storage from 2.34 to 3.66 mg of Cy equiv/g of dm in kale without having any effect on acylation in either morphotype. The results obtained in this study will be useful for optimizing anthocyanin production.

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.

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.

Flavanol and procyanidin content (by degree of polymerization 1-10) of chocolate, cocoa liquors, cocoa powders, and cocoa extracts: first action 2012.24.

An international collaborative study was conducted on an HPLC method with fluorescent detection for the determination of flavanols and procyanidins in chocolate and cocoa-containing materials. The sum of the oligomeric fractions with degree of polymerization 1-10 was the determined content value. Sample materials included dark and milk chocolates, cocoa powder, cocoa liquors, and cocoa extracts. The content ranged from approximately 2 to 500 mg/g (defatted basis). Thirteen laboratories--representing commercial, industrial, and academic institutions in six countries--participated in this interlaboratory study. Fourteen samples were sent as blind duplicates to the collaborators. Results for 12 laboratories yielded repeatability RSD (RSDr) values below 10% for all materials analyzed, ranging from 4.17 to 9.61%. Reproducibility RSD (RSDR) values ranged from 5.03 to 12.9% for samples containing 8.07 to 484.7 mg/g material analyzed. In one sample containing a low content of flavanols and procyanidins (approximately 2 mg/g), the RSDR was 17.68%.

Determination of flavanol and procyanidin (by degree of polymerization 1-10) content of chocolate, cocoa liquors, powder(s), and cocoa flavanol extracts by normal phase high-performance liquid chromatography: collaborative study.

An international collaborative study was conducted on an HPLC method with fluorescent detection (FLD) for the determination of flavanols and procyanidins in materials containing chocolate and cocoa. The sum of the oligomeric fractions with degree of polymerization 1-10 was the determined content value. Sample materials included dark and milk chocolates, cocoa powder, cocoa liquors, and cocoa extracts. The content ranged from approximately 2 to 500 mg/g (defatted basis). Thirteen laboratories representing commercial, industrial, and academic institutions in six countries participated in the study. Fourteen samples were sent as blind duplicates to the collaborators. Results from 12 laboratories yielded repeatability relative standard deviation (RSDr) values that were below 10% for all materials analyzed, ranging from 4.17 to 9.61%. The reproducibility relative standard deviation (RSD(R)) values ranged from 5.03 to 12.9% for samples containing 8.07 to 484.7 mg/g. In one sample containing a low content of flavanols and procyanidins (approximately 2 mg/g), the RSD(R) was 17.68%. Based on these results, the method is recommended for Official First Action for the determination of flavanols and procyanidins in chocolate, cocoa liquors, powder(s), and cocoa extracts.

Method performance and multi-laboratory assessment of a normal phase high pressure liquid chromatography-fluorescence detection method for the quantitation of flavanols and procyanidins in cocoa and chocolate containing samples.

The quantitative parameters and method performance for a normal-phase HPLC separation of flavanols and procyanidins in chocolate and cocoa-containing food products were optimized and assessed. Single laboratory method performance was examined over three months using three separate secondary standards. RSD(r) ranged from 1.9%, 4.5% to 9.0% for cocoa powder, liquor and chocolate samples containing 74.39, 15.47 and 1.87 mg/g flavanols and procyanidins, respectively. Accuracy was determined by comparison to the NIST Standard Reference Material 2384. Inter-lab assessment indicated that variability was quite low for seven different cocoa-containing samples, with a RSD(R) of less than 10% for the range of samples analyzed.

Discriminating between cultivars and treatments of broccoli using mass spectral fingerprinting and analysis of variance-principal component analysis.

Metabolite fingerprints, obtained with direct injection mass spectrometry (MS) with both positive and negative ionization, were used with analysis of variance-principal components analysis (ANOVA-PCA) to discriminate between cultivars and growing treatments of broccoli. The sample set consisted of two cultivars of broccoli, Majestic and Legacy, the first grown with four different levels of Se and the second grown organically and conventionally with two rates of irrigation. Chemical composition differences in the two cultivars and seven treatments produced patterns that were visually and statistically distinguishable using ANOVA-PCA. PCA loadings allowed identification of the molecular and fragment ions that provided the most significant chemical differences. A standardized profiling method for phenolic compounds showed that important discriminating ions were not phenolic compounds. The elution times of the discriminating ions and previous results suggest that they were common sugars and organic acids. ANOVA calculations of the positive and negative ionization MS fingerprints showed that 33% of the variance came from the cultivar, 59% from the growing treatment, and 8% from analytical uncertainty. Although the positive and negative ionization fingerprints differed significantly, there was no difference in the distribution of variance. High variance of individual masses with cultivars or growing treatment was correlated with high PCA loadings. The ANOVA data suggest that only variables with high variance for analytical uncertainty should be deleted. All other variables represent discriminating masses that allow separation of the samples with respect to cultivar and treatment.

UV spectral fingerprinting and analysis of variance-principal component analysis: a useful tool for characterizing sources of variance in plant materials.

UV spectral fingerprints, in combination with analysis of variance-principal components analysis (ANOVA-PCA), can differentiate between cultivars and growing conditions (or treatments) and can be used to identify sources of variance. Broccoli samples, composed of two cultivars, were grown under seven different conditions or treatments (four levels of Se-enriched irrigation waters, organic farming, and conventional farming with 100 and 80% irrigation based on crop evaporation and transpiration rate). Freeze-dried powdered samples were extracted with methanol-water (60:40, v/v) and analyzed with no prior separation. Spectral fingerprints were acquired for the UV region (220-380 nm) using a 50-fold dilution of the extract. ANOVA-PCA was used to construct subset matrices that permitted easy verification of the hypothesis that cultivar and treatment contributed to a difference in the chemical expression of the broccoli. The sums of the squares of the same matrices were used to show that cultivar, treatment, and analytical repeatability contributed 30.5, 68.3, and 1.2% of the variance, respectively.

Identification and quantification of flavonoids of Mexican oregano (Lippia graveolens) by LC-DAD-ESI/MS analysis.

LC-DAD-ESI/MS was used to identify 23 flavonoids in the extract of Mexican oregano (Lippia graveolens H.B.K.), a spice and herb, used in the USA and Mexico. The identification of luteolin-7-O-glucoside, apigenin 7-O-glucoside, phloridzin, taxifolin, eriodictyol, scutellarein, luteolin, quercetin, naringenin, pinocembrin and galangin was confirmed by direct comparison with standards. Identification of 6-hydroxyluteolin, two 6-hydroxyluteolin 7-O-glycosides, three pentahydroxyflavanone hexosides, scutellarein 7-O-hexoside, 3-hydroxyphloretin hexoside, and three other flavones, was made by detailed analysis of their UV and mass spectral data. The identification of the flavonoid glycosides was further confirmed through detection of their aglycones following hydrolysis of the samples. The concentration of the identified flavonoids in three samples was also estimated. This is the first report of detection of over 20 flavonoids, including chalcones, in this plant material.

Analysis of flavanols in foods: what methods are required to enable meaningful health recommendations?

Flavanols and their related oligomeric compounds, the procyanidins, have received increased attention during the past decade due to their reported health benefits. On the basis of compelling data published during the past decade demonstrating that the consumption of certain flavanol-rich foods can improve markers of cardiovascular health, additional clinical, and epidemiological research is clearly warranted to establish appropriate public health recommendations. However, recommendations on the consumption of these foods appropriate for use by health professionals can only be made on the basis of clinical investigations that accurately identify and quantify--through proper analytical measurement systems--the flavanols in the foods used in these investigations. This manuscript provides an overview of the strengths, weaknesses, and limitations of commonly used analytical methods to characterize the content of flavanols in foods. Two nonspecific measurements widely used by investigators, the Folin-Ciocalteu assay and the Oxygen Radical Absorbance Capacity (ORAC) measurement, are discussed in this context, as is the use of various high-performance liquid chromatography methods that provide more specific data related to the content of flavanols in foods. A comparison of the data obtained from these analytical methods to those of the more rigorous high-performance liquid chromatography analyses demonstrates that these nonspecific methods are ill-suited for providing unequivocal data necessary to evaluate the importance of dietary flavanols in the context of improving cardiovascular health. Meaningful dietary recommendations for the consumption of flavanol-rich foods will only be made possible by additional well-designed clinical and epidemiological studies enabled by detailed compositional data obtained through use of appropriate analytical methods.

High-performance liquid chromatography separation and purification of cacao (Theobroma cacao L.) procyanidins according to degree of polymerization using a diol stationary phase.

A new chromatographic approach for separating cacao procyanidins according to their degree of polymerization has been developed. It utilizes diol stationary phase columns operating in normal phase mode with a binary gradient of acidified acetonitrile and methanol-water. Performance of the diol stationary phase was evaluated on an analytical scale utilizing classical chromatographic conditions for the normal phase separation of procyanidins according to their degree of polymerization. The new separation approach was developed on an analytical scale but further extended to the preparative scale. These newly developed analytical and preparative high-performance liquid chromatography procedures were successfully applied to the separation, as well as isolation, of cacao procyanidins from unfermented cacao seeds. The degree of polymerization associated with each molecular weight fraction was determined by mass spectrometry.

Cultivation conditions and selenium fertilization alter the phenolic profile, glucosinolate, and sulforaphane content of broccoli.

Broccoli is a food often consumed for its potential health-promoting properties. The health benefits of broccoli are partly associated with secondary plant compounds that have bioactivity; glucosinolates and phenolic acids are two of the most abundant and important in broccoli. In an effort to determine how variety, stress, and production conditions affect the production of these bioactive components broccoli was grown in the greenhouse with and without selenium (Se) fertilization, and in the field under conventional or organic farming procedures and with or without water stress. High-performance liquid chromatography/mass spectrometry was used to separate and identify 12 primary phenolic compounds. Variety had a major effect: There was a preponderance of flavonoids in the Majestic variety, but hydroxycinnamic esters were relatively more abundant in the Legacy variety. Organic farming and water stress decreased the overall production of phenolics. Se fertilization increased glucosinolates in general, and sulforaphane in particular, up to a point; above that Se fertilization decreased glucosinolate production. Organic farming and water stress also decreased glucosinolate production. These data show environmental and genetic variation in phenolics and glucosinolates in broccoli, and warn that not all broccoli may contain all health-promoting bioactive components. They further show that selection for one bioactive component (Se) may decrease the content of other bioactive components such as phenolics and glucosinolates.

Selenium enrichment of broccoli: interactions between selenium and secondary plant compounds.

Multiple components of broccoli, such as sulforaphane (Sf) and phenolic acids, may inhibit cancer. Additionally, broccoli can accumulate selenium (Se), and Se has been demonstrated to reduce the risk of cancer. Studies were conducted to determine whether enhancement of broccoli with Se would produce a plant with superior health benefits. Although increasing the concentration of Se in broccoli from <1.0 to >800 microg/g resulted in inhibition of colon cancer in rats, it also decreased the Sf content by >80% and inhibited production of most phenolic acids. The inclusion of Se-enriched broccoli in the diet of rats induced the activity of the selenoprotein thioredoxin reductase beyond the maximum activity induced by Se alone. These results emphasize the complex interactions of bioactive chemicals in a food; attempts to maximize one component may affect accumulation of another, and consumption of high amounts of multiple bioactive compounds may result in unexpected metabolic interactions within the body.