Structural characterization of new malvidin 3-glucoside-catechin aryl/alkyl-linked pigments

The condensation reaction between malvidin 3-glucoside and catechin mediated by isobutyraldehyde, benzaldehyde, and isovaleraldehyde was conducted in model solutions at two pH values (1.5 and 3.2). The formation of new alkyl/aryl-linked adducts corresponding to the structures malvidin 3-glucoside-isobutylcatechin, malvidin 3-glucoside-benzylcatechin, and malvidin 3-glucoside-3-methylbutylcatechin was respectively observed from each aldehyde. The structural characterization of these new structures has been elucidated by 1D and 2D NMR, mass spectrometry, and UV-vis techniques. These new adducts showed the same lambda(max) in the visible region at 540 nm, which is bathochromically shifted 15 nm when compared with the original anthocyanin (lambda(max) = 525 nm).     

Assessment of protein quality methodology for infant formulas

Rat bioassay was used to assess the protein quality of powdered infant formulas and to evaluate the feasibility of using modified casein diets (containing the same source and level of fat and carbohydrate contributed by the infant formulas) as reference standards. Modification of the casein diet to match the milk-based formulas caused a significant reduction in weekly protein efficiency ratios (PER) and net protein ratios (NPR) at the third and fourth weeks. Modification of the casein diet to stimulate the soy-based formulas had no significant effect on NPR values; PER values were more varied. When PER and NPR values of the powdered milk-based formulas were expressed relative to the unmodified reference standard, the relative values were lower than when each matched reference was used. With few exceptions, the relative weekly PER values of the soy-based formulas were similar regardless of the standard used. The relative NPR values of the formulas had a pattern similar to the relative PER values. The data indicate that protein quality evaluation of infant formulas using rat bioassay warrants the use of matched casein reference diets for each type of formula.     

New phenolic compounds formed by evolution of (+)-catechin and glyoxylic acid in hydroalcoholic solution and their implication in color changes of grape-derived foods

The reaction between (+)-catechin and glyoxylic acid in model solution system was investigated by LC/DAD and LC/ESI-MS analyses. The formation of phenolic compounds exhibiting absorption maxima near 300 nm and presenting a shoulder around 350 nm was observed. Their structures consisted of a (+)-catechin unit with one or two aldehyde groups linked at positions 6, 8 or 6 and 8 of the A ring. In addition, new yellow pigments exhibiting UV-visible spectra similar to those of xanthylium salts with absorption maxima at 450 and 280 nm were also detected. The major yellow compound was isolated and identified by ESI-MS and 1D and 2D NMR spectroscopy. The implication of these compounds in color change and browning observed during aging of grape-derived beverages is also discussed.     

New insights into an ancient antibrowning agent: formation of sulfophenolics in sodium hydrogen sulfite-treated potato extracts

The effect of sodium hydrogen sulfite (S), used as antibrowning agent, on the phenolic profile of potato extracts was investigated. This extract was compared to one obtained in the presence of ascorbic acid (A). In the presence of A, two major compounds were obtained, 5-O-caffeoylquinic acid (5-CQA) and 4-O-caffeoyl quinic acid. With S, their 2'-sulfo-adducts were found instead, the structures of which were confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. Also, for minor caffeoyl derivatives and quercetin glycosides, the corresponding sulfo-adducts were observed. Feruloyl and sinapoyl derivatives were not chemically affected by the presence of S. Polyphenol oxidase (PPO) was thought to be responsible for the formation of the sulfo-adducts. This was confirmed by preparing 2'-sulfo-5-O-caffeoyl quinic acid in a model system using 5-CQA, sodium hydrogen sulfite, and PPO. This sulfo-adduct exhibited a small bathochromic shift (λmax 329 nm) as compared to 5-CQA (λmax 325 nm) and a strong hypochromic shift with an extinction coefficient of 9357±395 M(-1) cm(-1) as compared to 18494±196 M(-1) cm(-1), respectively. The results suggest that whenever S is used as an antibrowning agent, the O-quinone formed with PPO reacts with S to produce sulfo-O-diphenol, which does not participate in browning reactions.     

Conformational study of red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) by tryptophan fluorescence and differential scanning calorimetry

Fluorescence and differential scanning calorimetry (DSC) were used to study changes in the conformation of red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) under various environmental conditions. The possible relationship between fluorescence data and DSC characteristics was also discussed. Tryptophan fluorescence and fluorescence quenching analyses indicated that the tryptophan residues in KPI, exhibiting multiple fluorophores with different accessibilities to acrylamide, are largely buried in the hydrophobic core of the protein matrix, with positively charged side chains close to at least some of the tryptophan residues. GdnHCl was more effective than urea and SDS in denaturing KPI. SDS and urea caused variable red shifts, 2-5 nm, in the emission λ(max), suggesting the conformational compactness of KPI. The result was further supported by DSC characteristics that a discernible endothermic peak was still detected up to 8 M urea or 30 mM SDS, also evidenced by the absence of any shift in emission maximum (λ(max)) at different pH conditions. Marked decreases in T(d) and enthalpy (ΔH) were observed at extreme alkaline and/or acidic pH, whereas the presence of NaCl resulted in higher T(d) and ΔH, along with greater cooperativity of the transition. Decreases in T(d) and ΔH were observed in the presence of protein perturbants, for example, SDS and urea, indicating partial denaturation and decrease in thermal stability. Dithiothreitol and N-ethylmaleimide have a slight effect on the thermal properties of KPI. Interestingly, a close linear relationship between the T(d) (or ΔH) and the λ(max) was observed for KPI in the presence of 0-6 M urea.     

Quantification of phenolic compounds during red winemaking using FT-MIR spectroscopy and PLS-regression

We present a rapid method to quantify phenolic compounds all during the red winemaking process using Fourier transform mid-infrared (FT-MIR) spectroscopy and chemometrics. To get the reference values, we used the usual UV–vis spectroscopy methods, and the compounds studied were evaluated as total phenolic compounds (TPC), total anthocyanins (TA), and condensed tannins (CT). Sampling from five different grape varieties (Merlot, Tempranillo, Syrah, Cari?ena, and Cabernet sauvignon), harvested at different ripening states, and monitored over 10 days of vinification produced a total of 600 spectra. These were used to build and validate four different predictive models by partial least-squares (PLS) regression. The spectral regions selected for each model were between 979 and 2989 cm(–1), and when selecting the most suitable one in each case, good values of performance parameters were obtained (R2(val) > 0.95 and RPD > 4.0 for TPC; R2(val) > 0.90 and RPD > 3.0 for TA; R2(val) < 0.8 and RPD < 3.0 for CT). Furthermore, also more specific PLS regression models for each phenolic parameter and each grape variety were developed using different regions with results similar to those obtained when dealing with all of the grape varieties. It is concluded that FT-MIR spectroscopy together with multivariate calibration could be a rapid and valuable tool for wineries to carry out the monitoring of phenolic compound extraction during winemaking.     

Use of a simplified HPLC-UV analysis for soyasaponin B determination: study of saponin and isoflavone variability in soybean cultivars and soy-based health food products

Soyasaponins are phytochemicals of major interest for health. Their identification and quantification remain difficult owing to the large number of structural isomers in soybeans and the lack of stable standards. In this study, a rapid method using high performance liquid chromatography (HPLC) using a UV detector (205 nm) was developed to identify and quantify soyasaponins belonging to group B and compare them with isoflavones in different soy materials. 2,3-Dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soyasaponins were determined using external calibration or a molecular mass ratio after alkaline hydrolysis to cleave their DDMP moieties. The detection limit of soyasaponin I, used as a reference molecule to simplify the analysis, was 0.065 micromol/g. Soyasaponin contents in seven soybean varieties ranged from 13.20 to 42.40 micromol/g in the germ and from 2.76 to 6.43 micromol/g in the cotyledons. The within-day and between-days variation coefficients did not exceed 7.9 and 9.0%, respectively, for the major soyasaponins. Soyasaponin B quantification in different soy-based health supplements was reported along with measurements of their isoflavone content to provide information on the variability of these bioactive compounds among different types of soy food materials.     

Effect of kilning on the antioxidant and pro-oxidant activities of pale malts

Pale malts were prepared using standard and rapid kilning regimes that differed in the temperature and moisture profiles in the kiln. Samples were taken over the last 9 h of kilning, that is, at 18, 20, 22, 25, and 27 h. Antioxidant activity, assessed by redox potential, scavenging of the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS*+), and ferric reducing/antioxidant power (FRAP), increased at moisture levels below 6.7% for both regimes. The 27 h malt exposed to the rapid regime (moisture content of 4.8%) had a higher activity than the 27 h standard regime sample (moisture content of 4.8%). None of the malts scavenged oxygen. Pro-oxidant activity profiles were different for the malts obtained using each regime and, at 27 h, the rapid procedure gave malt with higher activity. Levels of (+)-catechin and ferulic acid (the most abundant phenolic compounds identified) generally increased as the moisture content of malt fell below 6.7%. Differences in antioxidant and pro-oxidant activities of the 27 h malts are partly attributed to the Maillard reaction, as evidenced by lower L* and higher b* values and higher levels of Maillard-derived flavor compounds, in the sample obtained by the rapid procedure. Levels of lipid-derived flavor compounds were significantly higher after 27 h of kilning using the rapid procedure.     

Pharmacokinetics of quercetin absorption from apples and onions in healthy humans

A high-throughput method for the extraction and analysis of quercetin in human plasma using 96-well SPE and LC-(ESI)MS/MS (7 min/run) is described. Quercetin exists as a range of glycosides in foods. The dominant types of quercetin glycosides vary depending on genetics (i.e., species and cultivar). Dietary sources include onions and apples (i.e., the peel). Herein the quercetin glycoside composition was determined in a composite standard of dried apple peel and in onion powder. The predominant forms of quercetin in apple peel include quercetin O-arabinoside, 3-O-galactoside, 3-O-glucoside, and 3-O-rhamnoside. In the onion powder, quercetin occurred as the quercetin 3,4'-O-glucoside and 4'-O-glucoside. Pharmacokinetics relating to absorption (C(max), t(max), and AUC(0-24?h)) and elimination (k(el) and t(1/2)) were compared after the consumption of apple peel powder (AP), onion powder (OP), or a mixture of the apple peel and onion powder enriched applesauce (MP) by healthy volunteers (eight females and eight males). The enriched applesauce delivered ～100 mg of quercetin aglycone equivalents. Consumption of the OP resulted in C(max) = 273.2 ± 93.7 ng/mL, t(max) = 2.0 ± 1.7 h, and t(1/2) = 14.8 ± 4.8 h, whereas the AP resulted in C(max) = 63.8 ± 22.4 ng/mL, t(max) = 2.9 ± 2.0 h, and t(1/2) = 65.4 ± 80.0 h. The MP resulted in an intermediate response with C(max) = 136.5 ± 45.8 ng/mL, t(max) = 2.4 ± 1.5 h, and t(1/2) = 18.7 ± 6.8 h. Consumption of the OP led to faster absorption, higher concentration, and greater bioavailability as compared to the AP. No significant gender-related differences were observed in the absorption of quercetin, whereas significant gender-related differences in the elimination half-time (t(1/2)) were observed.     

北京市菜地土壤重金属现状分析与评价

近年来北京蔬菜种植业得到迅速发展,已成为农业经济的支柱产业之一。为了解北京地区菜地以重金属为主要指标的土壤环境质量现状,采集北京市主要蔬菜生产区表层土壤(0~20cm),测定镉(Cd)、铅(Pb)、铜(Cu)、铬(Cr)、锌(Zn)、镍(Ni)、汞(Hg)含量和基础理化性质。依照《土壤环境质量标准》和《土壤环境监测技术规范》采用单项和综合污染指数法进行评价,结果表明,与土壤背景值相比,大兴、昌平、密云、延庆、房山、顺义、通州区内Cd、Cr明显富集,顺义区、房山区Cd质量分数平均值接近于《食用农产品产地环境质量评价标准》限量值(0.40 mg/kg),但未超过《土壤环境质量标准》中二级限量值(0.60 mg/kg)。Cd、Cu、Zn、Cr单项污染指数平均值处于0.7~1.0,属于尚清洁(警戒限)水平,且土壤Cd质量分数变异系数极高,40%以上样点存在"轻度"和"中度"污染,设施菜地较裸露菜地相比存在较高Cd污染风险。综上,北京市菜地土壤重金属环境质量指标总体安全,处于非污染状态,但存在一定程度的Cd、Cr、Cu、Zn累积污染风险。     

A screening method for the identification of glycosylated flavonoids and other phenolic compounds using a standard analytical approach for all plant materials

A screening method was developed for the systematic identification of glycosylated flavonoids and other phenolic compounds in plant food materials based on an initial, standard analytical method. This approach applies the same analytical scheme (aqueous methanol extraction, reverse phase liquid chromatographic separation, and diode array and mass spectrometric detection) to every sample and standard. This standard approach allows the cross-comparison of compounds in samples, standards, and plant materials previously identified in the published literature. Thus, every analysis contributes to a growing library of data for retention times and UV/vis and mass spectra. Without authentic standards, this method provides provisional identification of the phenolic compounds: identification of flavonoid backbones, phenolic acids, saccharides, and acyls but not the positions of the linkages between these subclasses. With standards, this method provides positive identification of the full compound: identification of subclasses and linkages. The utility of the screening method is demonstrated in this study by the identification of 78 phenolic compounds in cranberry, elder flower, Fuji apple peel, navel orange peel, and soybean seed.     

Arabica and robusta coffees: identification of major polar compounds and quantification of blends by direct-infusion electrospray ionization-mass spectrometry

Considering that illegal admixture of robusta coffee into high-quality arabica coffee is an important task in coffee analysis, we evaluated the use of direct-infusion electrospray ionization-mass spectrometry (ESI-MS) data combined with the partial least-squares (PLS) multivariate calibration technique as a fast way to detect and quantify arabica coffee adulterations by robusta coffee. A total of 16 PLS models were built using ESI± quadrupole time-of-flight (QTOF) and ESI± Fourier transform ion cyclotron resonance (FT-ICR) MS data from hot aqueous extracts of certified coffee samples. The model using the 30 more abundant ions detected by ES+ FT-ICR MS produced the most accurate coffee blend percentage prediction, and thus, it was later successfully employed to predict the blend composition of commercial robusta and arabica coffee. In addition, ESI± FT-ICR MS analysis allowed for the identification of 22 compounds in the arabica coffee and 20 compounds in the robusta coffee, mostly phenolics.     

Evaluation of some carotenoids in grapes by reversed- and normal-phase liquid chromatography: a qualitative analysis

Carotenoids in grapes of three Port winemaking cultivars were investigated. Extracts were obtained with n-hexane/diethyl ether mixtures (0/100; 20/80; 50/50; 100/0) and analyzed by normal and reversed phase HPLC-DAD. Selection and identification of peaks were based on spectroscopic characteristics - lambda(max), (%III/II) and k' values, leading to 28 probable carotenoids. Using pure standards, it was possible to identify seven compounds previously described (neochrome, neoxanthin, violaxanthin, flavoxanthin, zeaxanthin, lutein, and beta-carotene), one more type of neochrome reported here, for the first time, and in addition, two geometrical isomers of lutein and beta-carotene were tentatively described. The remaining 17 need to be further identified. High polarity solvent mixtures lead to qualitatively richer chromatograms. Reversed-phase separations allowed the detection of flavoxanthin and the possible geometrical isomer(s) of beta-carotene. Under normal phase, zeaxanthin was detected, and neochromes were better separated from neoxanthin. Extraction with 50/50 n-hexane/diethyl ether mixtures and reversed-phase conditions was the best combination for analysis of the carotenoids, known as precursors of compounds with high aroma impact in wines.     

Screening of danofloxacin residue in bovine tissue by terbium-sensitized luminescence on c18 sorbent strips

Danofloxacin (DANO) residue in bovine muscle was screened at 200 ng/g by terbium-sensitized luminescence (TSL) directly measured on 10 × 6 mm C18 sorbent strips. The analyte was first adsorbed on sorbent surface by immersion in defatted homogenates. After reagent application and desiccation, TSL was directly measured on sorbent surfaces at λ(ex) = 273 nm and λ(em) = 546 nm. The luminescence intensity was linearly dependent on DANO concentration in the 0-1000 ng/g range (R(2) = 0.9967). A threshold was established at x(200) - 3σ(200), where x(200) and σ(200) are the mean and standard deviation, respectively, of the DANO signals at 200 ng/g. Among 48 blind samples randomly fortified at 0-1000 ng/g, 45 were screened correctly and 3 negative samples were presumed positive. This simple screening protocol has the potential to significantly reduce sample numbers and hence improve sample throughput and save assay costs.     

Tetrahydrofolic Acid is a potent suicide substrate of mushroom tyrosinase

The coenzyme tetrahydrofolic acid is the most rapid suicide substrate of tyrosinase that has been characterized to date. A kinetic study of the suicide inactivation process provides the kinetic constants that characterize it: λ(max), the maximum apparent inactivation constant; r, the partition ratio or the number of turnovers made by one enzyme molecule before inactivation; and k(cat) and K(m), the catalytic and Michaelis constants, respectively. From these values, it is possible to establish the ratio λ(max)/K(m), which represents the potency of the inactivation process. Besides acting as a suicide substrate of tyrosinase, tetrahydrofolic acid reduces o-quinones generated by the enzyme in its action on substrates, such as l-tyrosine and l-DOPA (o-dopaquinone), thus inhibiting enzymatic browning.     

Orally administered delphinidin 3-rutinoside and cyanidin 3-rutinoside are directly absorbed in rats and humans and appear in the blood as the intact forms

Four components of black currant anthocyanins (BCA), delphinidin 3-O-beta-rutinoside (D3R), cyanidin 3-O-beta-rutinoside (C3R), delphinidin 3-O-beta-glucoside (D3G), and cyanidin 3-O-beta-glucoside (C3G), were found to be directly absorbed and distributed to the blood and excreted into urine as the glycosylated forms. In a rat study, following oral administration of purified D3R, C3R, and C3G (800 micromol/kg of body weight), the anthocyanins were detected in the plasma and the C(max) values were 580 +/- 410, 850 +/- 120, and 840 +/- 190 nmol/L, respectively, 0.5-2.0 h after administration. In a human study, when a mixture of BCA [6.24 micromol (3.58 mg) consisting of 2.75 micromol (1.68 mg) of D3R, 2.08 micromol (1.24 mg) of C3R, 1.04 micromol (0.488 mg) of D3G, and 0.37 micromol (0.165 mg) of C3G/kg of body weight)] was orally ingested by eight volunteers, D3R, C3R, D3G, and C3G were detected in the plasma and urine. The plasma C(max) values were 73.4 +/- 35.0, 46.3 +/- 22.5, 22.7 +/- 12.4, and 5.0 +/- 3.7 nmol/L, respectively, 1.25-1.75 h after intake, and the cumulative excretion of the four compounds in urine in the period 0-8 h after intake was 0.11 +/- 0.05% of the dose ingested. These results indicate that 3-O-beta-rutinosyl anthocyanins were directly absorbed and distributed to the blood.     

Recommendations for establishment of reference standards for recombinant-DNA-derived proteins and polypeptides

An integrated approach is presented for the establishment of purified protein and peptide reference standard materials suitable for both biological and chemical assays. Preliminary considerations, handling and storage conditions, and a variety of chemical methods for defining the reference standards are examined in detail. Of the chemical methods, liquid chromatography (LC), amino acid analysis, Kjeldahl protein assay, electrophoresis, and ion chromatography are key assays in determining the potency, purity, and identity of the reference standard preparation. Finally, the role of liquid chromatography in assessing reference standards for identity and chemical purity is examined and correlated with other methods.     

Fractionation of High-Amylose Maize Starches by Differential Alcohol Precipitation and Chromatography of the Fractions

Three high-amylose maize starches (HAS) and a common corn starch (CCS) were subjected to differential alcohol precipitation using isoamyl alcohol and 1-butanol to obtain fractions designated as amylose (AM), amylopectin (AP), and intermediate material (IM). For each starch, IM had a blue value and an iodine binding wavelength maximum (λ_max) between the λ_max of the respective AM and AP. Size-exclusion chromatography (SEC) showed similarities in the AM from CCS and HAS. HAS AP had higher blue values and iodine binding λ_max values than CCS AP. SEC of the intact HAS AP and IM both showed large proportions of material eluting after the void volume (45–85%) when compared to CCS AP and IM. Chain length (CL) distributions of debranched AP and IM indicated that these fractions from each starch were highly branched, and that AP had a shorter average chain length than IM. Consequently, the differential precipitation behavior of the HAS AP and IM appears dependent on general branching structure rather than size. We conclude that in both CCS and HAS, AP and IM are subsets of the branched molecules with AP as the predominant fraction. For HAS, AP and IM include molecules of a size typical for AM and contain a higher proportion of chains that are longer than those of CCS AP. Differential alcohol precipitation is a useful method of separating amylose, amylopectin, and intermediate material from HAS.     

A mass spectrometric validated high-performance liquid chromatography procedure for the determination of folates in foods

A series of five food reference materials (RM) that had certified values of folate concentrations and five frozen food samples were analyzed for 5-methyltetrahydrofolic acid (5-MTHFA) and folic acid (FA) using a high-performance liquid chromatography (HPLC) method with fluorescence detection that was validated using an HPLC mass spectrometry (MS) method with electrospray ionization. Identical sample specimens were extracted and analyzed in triplicate using both instrumental methods, and a comparison was made of the mean values of 5-MTHFA and FA resulting from these determinations. The analytes were isolated on either a high capacity strong anion exchange solid phase extraction column (HPLC method) or a phenyl Bond Elut column (MS method) prior to analyses. For quantification of the analytes by MS, (13)C-labeled 5-MTHFA and FA were added to samples as internal standards prior to enzymatic digestion and conversion of the polyglutamate forms of 5-MTHFA to the monoglutamic acid. Quantification of FA and 5-MTHFA using the HPLC analysis was carried out using external standards. With the exception of one RM (pig liver), the values established for 5-MTHFA using these methods were highly comparable. In determining the variance associated with these two procedures, it was observed that the mean relative standard error for 5-MTHFA was 12 (range, 2-27%) and 11% (range, 5-25%) for the HPLC and MS methods, respectively. FA was detected in only three of the samples, and the values obtained for it by either method were similar. This is the first paper that describes a mass spectrometric method used in the validation of an HPLC determination of food folates across a wide range of sample matrixes. The comparable values for 5-MTHFA and FA suggest that HPLC analysis with fluorescent detection may be used to accurately quantify folates present in a variety of food matrixes.     

Antioxidant Activity and Characterization of Phenolic Compounds from Bacaba ( Oenocarpus bacaba Mart.) Fruit by HPLC-DAD-MS(n)

The phytochemicals in fruits have been shown to be major bioactive compounds with regard to health benefits. Bacaba ( Oenocarpus bacaba Mart.) is a native palm fruit from the Brazilian savannah and Amazon rainforest that plays an important role in the diet of rural communities and is also a source of income for poor people. This paper reports the characterization and analyses of phenolics from bacaba fruit extract. The total phenolic content of bacaba fruit amounted to 1759.27 ± 1.01 mg GAE/100 g, the flavonoid content was 1134.32 ± 0.03 mg CTE/100 g, and the anthocyanin content was 34.69 ± 0.00 mg cyn-3-glc/100 g. The antioxidant activity was evaluated through different assays [ORAC, FRAP, DPPH, TEAC, and cellular antioxidant assay (CAA) assays] and revealed a significant antioxidant capacity for bacaba in comparison to the data available in the literature. The assignment of the phenolic compounds using HPLC-DAD-MS(n) was based on the evaluation of their UV-vis absorption maxima (λ(max)) and mass spectral analyses, and 14 compounds were tentatively identified. The results suggest that bacaba fruits are a promising source of phenolics.