Screening for pesticide residues in cocoa (Theobroma cacao L.) by portable infrared spectroscopy.

Rapid assessment of pesticide residues ensures cocoa bean quality and marketability. In this study, a portable FTIR instrument equipped with a triple reflection attenuated total reflectance (ATR) accessory was used to screen cocoa beans for pesticide residues. Cocoa beans (n = 75) were obtained from major cocoa growing regions of Peru and were quantified for pesticides by gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS). The FTIR spectra were used to detect the presence of pesticides in cocoa beans or lipid fraction (butter) by using a pattern recognition (Soft Independent Modeling by Class Analogy, SIMCA) algorithm, which produced a significant discrimination for cocoa nibs (free or with pesticides). The variables related to the class grouping were assigned to the aliphatic (3200-2800 cm-1) region with an interclass distance (ICD) of 3.3. Subsequently, the concentration of pesticides in cocoa beans was predicted using a partial least squares regression analysis (PLSR), using an internal validation of the PLRS model, the cross-validation correlation coefficient (Rval = 0.954) and the cross-validation standard error (SECV = 14.9 mg/kg) were obtained. Additionally, an external validation was performed, obtaining the prediction correlation coefficient (Rpre = 0.940) and the standard error of prediction (SEP = 16.0 µg/kg) with high statistical performances, which demonstrates the excellent predictability of the PLSR model in a similar real application. The developed FTIR method presented limits of detection and quantification (LOD = 9.8 µg/kg; LOQ = 23.1 µg/kg) with four optimum factors (PC). Mid-infrared spectroscopy (MIR) offered a viable alternative for field screening of cocoa.

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.

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.

Ex Vivo and In Vivo Assessment of the Penetration of Topically Applied Anthocyanins Utilizing ATR-FTIR/PLS Regression Models and HPLC-PDA-MS.

Anthocyanins are natural colorants with antioxidant properties, shown to inhibit photoaging reactions and reduce symptoms of some skin diseases. However, little is known about their penetration through the stratum corneum, a prerequisite for bioactivity. The aim was to investigate anthocyanin penetration from lipophilic cosmetic formulations through the skin using a porcine ear model and human volunteers. ATR-FTIR/PLS regression and HPLC-PDA-MS were used to analyze anthocyanin permeation through the stratum corneum. Penetration of all anthocyanins was evident and correlated with molecular weight and hydrophilicity. Lower-molecular-weight (MW) anthocyanins from elderberry (449-581 Da) were more permeable within the skin in both ex vivo and in vivo models (Kp = 2.3-2.4 × 10-4 cm h-1) than the larger anthocyanins (933-1019 Da) from red radish (Kp = 2.0-2.1 × 10-4 cm h-1). Elderberry and red radish anthocyanins were found at all levels of the stratum corneum and at depths for activity as bioactive ingredients for skin health.

The NCI-N87 Cell Line as a Gastric Epithelial Model to Study Cellular Uptake, Trans-Epithelial Transport, and Gastric Anti-Inflammatory Properties of Anthocyanins.

Anthocyanins are ubiquitous plant pigments with reported antioxidant, anti-inflammatory, and anti-cancer activities. To better understand these benefits, metabolism of anthocyanins requires further evaluation, especially in the stomach. Mammalian cell cultures provide useful models for investigating compound metabolism and absorption, but they are generally maintained at physiological pH. The NCI-N87 cell line is an acid-stable model of the gastric epithelium used to study gastric drug metabolism. The objective of this work was to investigate the uptake, trans-epithelial transport, and anti-inflammatory activity of anthocyanins by the NCI-N87 cell line. The cells formed a coherent monolayer, stable ≤32 days post confluency. Minimal effects on monolayer integrity were observed when the pH of the apical chamber was adjusted to pH 3.0, 5.0, or 7.4. Anthocyanins were transported across the NCI-N87 cell monolayer at 37 °C, but not at 0 °C, suggesting a facilitated process. Chokeberry anthocyanins (0-1500 µM) were not cytotoxic. At apical pH 3.0, they had anti-inflammatory properties by significantly attenuating IL-8 secretion when added to medium before, during, and after incubation with IL-1ß. These results suggest that the NCI-N87 cell line is a physiologically relevant model for in vitro studies of the transport, anti-inflammatory and potential anti-carcinogenic activities of anthocyanins in gastric tissue.

Antioxidant, UV Protection, and Antiphotoaging Properties of Anthocyanin-Pigmented Lipstick Formulations.

Consumer demand for foods and cosmetics containing naturally derived ingredients has been increasing. Naturally derived anthocyanins (ACNs), from fruits and vegetables, were previously demonstrated to provide a wide range of hues as lipstick colorants with high stability. Therefore, the objective of this study was to evaluate the use of ACNs as bioactive pigments in lipstick formulations. Commercially available sources of nonacylated and acylated derivatives of the six major ACN aglycones were incorporated into a commercial lipstick base. The ACN-containing lipsticks were evaluated for their ability to act as ultraviolet (UV) absorbers [a source of sun protection factor (SPF)], free radical scavengers against 2,2-diphenyl-1-picrylhydrazyl (DPPH), and preventers of melanin formation through tyrosinase inhibition. All formulas showed increased UV absorption over the lipstick base, and acylated ACNs contributed to the highest in vitro SPF (UVB) values (≥15.8) in formulations. All formulas exhibited high inhibition of DPPH free radicals and inhibition of melanin production by tyrosinase at microgram per milligram concentrations similar to or less than kojic acid (2.41 ± 0.06 µg/mg). This is physiologically relevant because lipstick use is on average 24 mg/day. This study suggests the potential for ACNs to be used as biologically active ingredients in lipstick formulations by acting as antioxidants and UV-protection and antiaging compounds.

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.

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.

Metabolic fingerprinting for diagnosis of fibromyalgia and other rheumatologic disorders.

Diagnosis and treatment of fibromyalgia (FM) remains a challenge owing to the lack of reliable biomarkers. Our objective was to develop a rapid biomarker-based method for diagnosing FM by using vibrational spectroscopy to differentiate patients with FM from those with rheumatoid arthritis (RA), osteoarthritis (OA), or systemic lupus erythematosus (SLE) and to identify metabolites associated with these differences. Blood samples were collected from patients with a diagnosis of FM (n = 50), RA (n = 29), OA (n = 19), or SLE (n = 23). Bloodspot samples were prepared, and spectra collected with portable FT-IR and FT-Raman microspectroscopy and subjected to metabolomics analysis by ultra-HPLC (uHPLC), coupled to a photodiode array (PDA) and tandem MS/MS. Unique IR and Raman spectral signatures were identified by pattern recognition analysis and clustered all study participants into classes (FM, RA, and SLE) with no misclassifications (p < 0.05, and interclass distances > 2.5). Furthermore, the spectra correlated (r = 0.95 and 0.83 for IR and Raman, respectively) with FM pain severity measured with fibromyalgia impact questionnaire revised version (FIQR) assessments. Protein backbones and pyridine-carboxylic acids dominated this discrimination and might serve as biomarkers for syndromes such as FM. uHPLC-PDA-MS/MS provided insights into metabolites significantly differing among the disease groups, not only in molecular m/z+ and m/z- values but also in UV-visible chromatograms. We conclude that vibrational spectroscopy may provide a reliable diagnostic test for differentiating FM from other disorders and for establishing serologic biomarkers of FM-associated pain.

Stereochemistry and glycosidic linkages of C3-glycosylations affected the reactivity of cyanidin derivatives.

The impact of glycosylation on anthocyanin stability has largely been associated with sugar type, site, and size, with glycosyl stereochemistry being under-explored. Seven cyanidin-3-glycosides were isolated by HPLC, diluted in pH 1-9, mixed with bisulfite or ascorbic acid at pH 3, and stored for 8 weeks (25 °C, dark). Spectral changes, half-lives, and bleaching rates were determined. Cyanidin-3-galactoside was more reactive (susceptible to hydration and bleaching) than cyanidin-3-glucoside. The 1 → 2 disaccharides exhibited greater λvis-max (≤16 nm), resistance to hydration, and bleaching compared to 1 → 6 disaccharides.The 1 → 6 disaccharides had similar λvis-max (∼2 nm) to the monosaccharides but slightly improved resistance to hydration and bleaching. The tri-glycosylated anthocyanin had the greatest stability and its spectral and bleaching characteristics was intermediate to 1 → 2 and 1 → 6 disaccharides. The 1 → 2 disaccharides generally exhibited lower half-lives compared to monosaccharides; whereas, 1 → 6 disaccharides exhibited higher stability. These findings highlight the role of glycosyl assembly on anthocyanin reactivity and stability.

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.

Influence of cyanidin glycosylation patterns on carboxypyranoanthocyanin formation.

Anthocyanins can condense with compounds having enolizable groups to form pyranoanthocyanins. These pigments are less susceptible to degradation and color changes associated with nucleophilic addition common to anthocyanins. This study aimed to evaluate the impact of glycosylation patterns of anthocyanins on carboxypyranoanthocyanin formation. Nine cyanidin derivatives were isolated by semi-preparative HPLC. Pyruvic acid was added to induce pyranoanthocyanin formation. Composition (HPLC-MS/MS), spectra (absorbance 380-700 nm), and color (CIEL*c*h*) of solutions were monitored during 31 days storage at 25 °C. Cyanidin-3-glycosides with 1 → 6 disaccharides produced the highest pyranoanthocyanin yield (∼31%), followed by Cyanidin-3-monoglycosides (∼20%); 1 → 2 disaccharides produced the least proportions of pyranoanthocyanins (5-7%). Cyanidin-3-arabinoside converted to pyranoanthocyanins but degraded quickly (3% yield) under these conditions. No pyranoanthocyanins were formed from Cyanidin-3-sophoroside-5-glucoside. Glycosyl bonds were more critical than the size of the substitution alone, further supported by Cyanidin-3-(glucosyl)-(1 → 6)-(xylosyl-(1 → 2)-galactoside) yield (11%). Pyranoanthocyanins were hypsochromically shifted and had higher hue angles than their respective anthocyanins.

Cis-Trans Configuration of Coumaric Acid Acylation Affects the Spectral and Colorimetric Properties of Anthocyanins.

The color expression of anthocyanins can be affected by a variety of environmental factors and structural characteristics. Anthocyanin acylation (type and number of acids) is known to be key, but the influence of acyl isomers (with unique stereochemistries) remains to be explored. The objective of this study was to investigate the effects of cis-trans configuration of the acylating group on the spectral and colorimetric properties of anthocyanins. Petunidin-3-rutinoside-5-glucoside (Pt-3-rut-5-glu) and Delphinidin-3-rutinoside-5-glucoside (Dp-3-rut-5-glu) and their cis and trans coumaroylated derivatives were isolated from black goji and eggplant, diluted in pH 1-9 buffers, and analyzed spectrophotometrically (380-700 nm) and colorimetrically (CIELAB) during 72 h of storage (25 °C, dark). The stereochemistry of the acylating group strongly impacted the spectra, color, and stability of the Dp and Pt anthocyanins. Cis acylated pigments exhibited the greatest λmax in all pH, as much as 66 nm greater than their trans counterparts, showing bluer hues. Cis acylation seemed to reduce hydration across pH, increasing color intensity, while trans acylation generally improved color retention over time. Dp-3-cis-p-cou-rut-5-glu exhibited blue hues even in pH 5 (C*ab = 10, hab = 256°) where anthocyanins are typically colorless. Cis or trans double bond configurations of the acylating group affected anthocyanin spectral and stability properties.

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').

Time, Concentration, and pH-Dependent Transport and Uptake of Anthocyanins in a Human Gastric Epithelial (NCI-N87) Cell Line.

Anthocyanins are the largest class of water soluble plant pigments and a common part of the human diet. They may have many potential health benefits, including antioxidant, anti-inflammatory, anti-cancer, and cardioprotective activities. However, anthocyanin metabolism is not well understood. Studies suggest that anthocyanins absorption may occur in the stomach, in which the acidic pH favors anthocyanin stability. A gastric epithelial cell line (NCI-N87) has been used to study the behavior of anthocyanins at a pH range of 3.0-7.4. This work examines the effects of time (0-3 h), concentration (50-1500 µM), and pH (3.0, 5.0, 7.4) on the transport and uptake of anthocyanins using NCI-N87 cells. Anthocyanins were transported from the apical to basolateral side of NCI-N87 cells in time and dose dependent manners. Over the treatment time of 3 h the rate of transport increased, especially with higher anthocyanin concentrations. The non-linear rate of transport may suggest an active mechanism for the transport of anthocyanins across the NCI-N87 monolayer. At apical pH 3.0, higher anthocyanin transport was observed compared to pH 5.0 and 7.4. Reduced transport of anthocyanins was found to occur at apical pH 5.0.

Natural Colorants: Food Colorants from Natural Sources.

The color of food is often associated with the flavor, safety, and nutritional value of the product. Synthetic food colorants have been used because of their high stability and low cost. However, consumer perception and demand have driven the replacement of synthetic colorants with naturally derived alternatives. Natural pigment applications can be limited by lower stability, weaker tinctorial strength, interactions with food ingredients, and inability to match desired hues. Therefore, no single naturally derived colorant can serve as a universal alternative for a specified synthetic colorant in all applications. This review summarizes major environmental and biological sources for natural colorants as well as nature-identical counterparts. Chemical characteristics of prevalent pigments, including anthocyanins, carotenoids, betalains, and chlorophylls, are described. The possible applications and hues (warm, cool, and achromatic) of currently used natural pigments, such as anthocyanins as red and blue colorants, and possible future alternatives, such as purple violacein and red pyranoanthocyanins, are also discussed.

Health Benefits of Purple Corn (Zea mays L.) Phenolic Compounds.

Purple corn (Zea mays L.), a grain with one of the deepest shades in the plant kingdom, has caught the attention of the food industry as it could serve as a source for alternatives to synthetic colorants. Also being rich in phenolic compounds with potential health-promoting properties, purple corn is becoming a rising star in the novel ingredients market. Although having been widely advertised as a "healthy" food, the available information on purple corn health benefits has not yet been well reviewed and summarized. In this review, we present compositional information focused on the potential functional phenolic compounds correlated to health-promoting effects. Studies evaluating potential health-benefitting properties, including in vitro tests, cell models, animal and human trials, are also discussed. This paper emphasizes research using purple corn, or its extracts, but some other plant sources with similar phenolic composition to purple corn are also mentioned. Dosage and toxicity of purple corn studies are also reviewed. Purple corn phenolic compounds have been shown in numerous studies to have potent antioxidant, anti-inflammatory, antimutagenic, anticarcinogenic, and anti-angiogenesis properties. They were also found to ameliorate lifestyle diseases, such as obesity, diabetes, hyperglycemia, hypertension, and cardiovascular diseases, based on their strong antioxidant power involving biochemical regulation amelioration. With promising evidence from cell and animal studies, this rich source of health-promoting compounds warrants additional attention to better understand its potential contributions to human health.

Bathochromic and hyperchromic effects of aluminum salt complexation by anthocyanins from edible sources for blue color development.

Use of artificial food colorants has declined due to health concerns and consumer demand, making natural alternatives a high demand. The effects of Al(3+) salt on food source anthocyanins were evaluated with the objective to better understand blue color development of metalloanthocyanins. This is one of the first known studies to evaluate the effects of food source anthocyanin structures, including acylation, with chelation of aluminum. Cyanidin and delphinidin derivatives from different plants were treated with factorial excess of Al(3+) in pH 3-6 and evaluated by spectrophotometry and colorimetry over 28 days. Anthocyanin concentration, salt ratio, and pH determined final color and intensity. Pyrogallol moieties on delphinidin showed furthest bathochromic shifts, whereas acylation promoted higher chroma. Blue color developed at lower pH when acylated anthocyanins reacted with Al(3+); hue ∼270 occurred with acylated delphinidin at pH ≥ 2.5. Highest chelate stability was found with AlCl3100-500× anthocyanin concentration. This investigation showed anthocyanin-metal chelation can produce a variety of intense violet to blue colors under acidic pH with potential for food use.