Label-free quantitative ¹H NMR spectroscopy to study low-affinity ligand–protein interactions in solution: A contribution to the mechanism of polyphenol-mediated astringency - Fig 1

<p><b>Molecular structures of catechins and structurally related polyphenolic compounds.</b> (-)-epicatechin (a), (-)-epigallocatechin (b), (-)-epicatechin-3-gallate (c), (-)-epigallocatechin-3-gallate (d), methyl gallate (e), and quercetin-3-O-rutinoside (f).</p

Quantitation of Sweet Steviol Glycosides by Means of a HILIC-MS/MS-SIDA Approach

Meeting the rising consumer demand for natural food ingredients, steviol glycosides, the sweet principle of Stevia rebaudiana Bertoni (Bertoni), have recently been approved as food additives in the European Union. As regulatory constraints require sensitive methods to analyze the sweet-tasting steviol glycosides in foods and beverages, a HILIC-MS/MS method was developed enabling the accurate and reliable quantitation of the major steviol glycosides stevioside, rebaudiosides A–F, steviolbioside, rubusoside, and dulcoside A by using the corresponding deuterated 16,17-dihydrosteviol glycosides as suitable internal standards. This quantitation not only enables the analysis of the individual steviol glycosides in foods and beverages but also can support the optimization of breeding and postharvest downstream processing of <i>Stevia</i> plants to produce preferentially sweet and least bitter tasting <i>Stevia</i> extracts

Unbound (-)-epigallocatechin gallate (EGCG) in pure buffer (reference), 10% (v/v) human whole saliva, 1% (w/v) carboxymethyl cellulose (CMC), and a combination of both.

<p>Unbound EGCG was quantified by single qHNMR measurements and is expressed as mole fractions. Error bars denote the standard deviation as obtained from integrating all the distinct, non-overlaid proton signals of EGCG. Data points behind means are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184487#pone.0184487.s003" target="_blank">S3 Table</a>.</p

Label-free quantitative ¹H NMR spectroscopy to study low-affinity ligand–protein interactions in solution: A contribution to the mechanism of polyphenol-mediated astringency

<div><p>Nuclear magnetic resonance (NMR) spectroscopy is well-established in assessing the binding affinity between low molecular weight ligands and proteins. However, conventional NMR-based binding assays are often limited to small proteins of high purity and may require elaborate isotopic labeling of one of the potential binding partners. As protein–polyphenol complexation is assumed to be a key event in polyphenol-mediated oral astringency, here we introduce a label-free, ligand-focused ¹H NMR titration assay to estimate binding affinities and characterize soluble complex formation between proteins and low molecular weight polyphenols. The method makes use of the effects of NMR line broadening due to protein–ligand interactions and quantitation of the non-bound ligand at varying protein concentrations by quantitative ¹H NMR spectroscopy (qHNMR) using electronic reference to access in vivo concentration (ERETIC 2). This technique is applied to assess the interaction kinetics of selected astringent tasting polyphenols and purified mucin, a major lubricating glycoprotein of human saliva, as well as human whole saliva. The protein affinity values (<i>BC</i>_<i>50</i>) obtained are subsequently correlated with the intrinsic mouth-puckering, astringent oral sensation imparted by these compounds. The quantitative NMR method is further exploited to study the effect of carboxymethyl cellulose, a candidate “anti-astringent” protein binding antagonist, on the polyphenol–protein interaction. Consequently, the NMR approach presented here proves to be a versatile tool to study the interactions between proteins and low-affinity ligands in solution and may find promising applications in the discovery of bioactives.</p></div

Pairwise comparison matrix to assess the relative astringency of polyphenols.

<p>Pairwise comparison matrix to assess the relative astringency of polyphenols.</p

Decoding the Nonvolatile Sensometabolome of Orange Juice (Citrus sinensis)

Activity-guided fractionation in combination with the taste dilution analysis, followed by liquid chromatography–tandem mass spectrometry and nuclear magnetic resonance experiments, led to the identification of 10 polymethoxylated flavones (PMFs), 6 limonoid glucosides, and 2 limonoid aglycones as the key bitterns of orange juice. Quantitative studies and calculation of dose-over-threshold factors, followed by taste re-engineering, demonstrated for the first time 25 sensometabolites to be sufficient to reconstruct the typical taste profile of orange juices and indicated that not a single compound can be considered a suitable marker for juice bitterness. Intriguingly, the taste percept of orange juice seems to be created by a rather complex interplay of limonin, limonoid glucosides, PMFs, organic acids, and sugars. For the first time, sub-threshold concentrations of PMFs were shown to enhance the perceived bitterness of limonoids. Moreover, the influence of sugars on the perceived bitterness of limonoids and PMFs in orange juice relevant concentration ranges was quantitatively elucidated

Development and Application of a Stable Isotope Dilution Analysis for the Quantitation of Advanced Glycation End Products of Creatinine in Biofluids of Type 2 Diabetic Patients and Healthy Volunteers

<i>N</i>-(1-Methyl-4-oxoimidazolidin-2-ylidene) α-amino acids were recently identified in roasted meat as so far unknown advanced glycation end products (AGEs) of creatinine. For the first time, this paper reports on the preparation of ¹³C-labeled twin molecules of six <i>N</i>-(1-methyl-4-oxoimidazolidin-2-ylidene) α-amino acids and the development of a stable isotope dilution analysis (SIDA) for their simultaneous quantitation in meat, plasma, and urine samples by means of HPLC-MS/MS. Method validation demonstrated good precision (<14% RSD) and accuracy (97–118%) for all analytes and a lower limit of quantitation of 1 pg injected onto the column. The SIDA was applied to monitor plasma appearance and urinary excretion of these AGEs in type 2 diabetes mellitus patients (DM, <i>n</i> = 7) and healthy controls (<i>n</i> = 10) prior to and after ingestion of a bolus of processed beef meat. Interestingly, the basal concentration of <i>N</i>-(1-methyl-4-oxoimidazolidin-2-ylidene) aminopropionic acid was elevated in plasma and urine of DM patients compared to healthy individuals. Further, ingestion of processed meat led to a significantly higher concentration of this AGE in biofluids from DM patients when compared to healthy controls. These findings suggest a favored in vivo formation, as demonstrated by physiological model incubations of creatinine and carbohydrates (37 °C, pH 7.4), or a more efficient dietary up-take of <i>N</i>-(1-methyl-4-oxoimidazolidin-2-ylidene) α-amino acids in hyperglycemic diabetes patients

Antioxidative Compounds from <i>Garcinia buchananii</i> Stem Bark

An aqueous ethanolic extract of the stem bark of <i>Garcinia buchananii</i> showed strong antioxidative activity using H₂O₂ scavenging, oxygen radical absorbance capacity (ORAC), and Trolox equivalent antioxidant capacity (TEAC) assays. Activity-guided fractionation afforded three new compounds, isomanniflavanone (<b>1</b>), an ent-eriodictyol-(3α→6)-dihydroquercetin-linked biflavanone, 1,5-dimethoxyajacareubin (<b>2</b>), and the depsidone garcinisidone-G (<b>3</b>), and six known compounds, (2″<i>R</i>,3″<i>R</i>)-preussianon, euxanthone, 2-isoprenyl-1,3,5,6-tetrahydroxyxanthone, jacareubin, isogarcinol, and garcinol. All compounds were described for the first time in <i>Garcinia buchananii.</i> The absolute configurations were determined by a combination of NMR, ECD spectroscopy, and polarimetry. These natural products showed high in vitro antioxidative power, especially isomanniflavanone, with an EC₅₀ value of 8.5 μM (H₂O₂ scavenging), 3.50/4.95 mmol TE/mmol (H/L-TEAC), and 7.54/14.56 mmol TE/mmol (H/L-ORAC)

UPLC-ESI-TOF MS-Based Metabolite Profiling of the Antioxidative Food Supplement Garcinia buchananii

Comparative antioxidative analyses of aqueous ethanolic extracts from leaf, root, and stem of Garcinia buchananii revealed high activity of all three organs. To investigate the metabolite composition of the different parts of <i>G. buchananii</i>, an untargeted metabolomics approach using UPLC-ESI-TOF MS with simultaneous acquisition of low- and high-collision energy mass spectra (MS^e) was performed. Unsupervised statistics (PCA) highlighted clear differences in the metabolomes of the three organs. OPLS-DA revealed (2<i>R</i>,3<i>S</i>,2″<i>R</i>,3″<i>R</i>)-GB-1, (2<i>R</i>,3<i>S</i>)-morelloflavone, and (2<i>R</i>,3<i>S</i>)-volkensiflavone as the most decisive marker compounds discriminating leaf from root and stem extract. Leaves represent the best source to isolate GB-1, morelloflavone, and volkensiflavone. Root extract is the best organ to isolate xanthones and stem bark extract the best source to isolate (2<i>R</i>,3<i>S</i>,2″<i>R</i>,3″<i>R</i>)-manniflavanone; the identified polyisoprenylated benzophenones are characteristic compounds for the leaf organ. Morelloflavone, volkensiflavone, and garcicowin C were isolated for the first time from <i>G. buchananii</i>, identified via MS, NMR, and CD spectroscopy, and showed in H₂O₂ scavenging, H/L-TEAC, and H/L-ORAC assays moderate to strong in vitro antioxidative activities

Retroarray analysis of HERV transcriptional activity in MF.

<p>HERV activity profiles representing pairs of lesion (A) and non-malignant (B) skin tissue specimens (digitally aligned). Each sample pair (n=17) was derived from an individual patient (MF 1-12, psoriasis 13-17). HERV subgroups representing a skin-specific core transcription profile (HERV-E, HERV-F, HERV-W, ERV-9, HERV-K(HML-4)) are emphasized with red letters. A panel of housekeeping genes (Ubiquitin, GAPDH, RPL19, β-Actin, HPRT) served as internal controls (for detailed information on methodology, see [77,79]). Each positive spot on the microarray may represent multiple HERV loci of one subgroup of multicopy HERV elements with sufficient sequence similarity that individual elements cannot be distinguished. Weak signals may be unrecognizable in the printed figure.</p