Novel unimolecular bivalent glycoconjugates were assembled combining several functionalized capsular polysaccharides of Streptococcus pneumoniae and Neisseria meningitidis to a carrier protein by using an effective strategy based on the Ugi 4-component reaction. The development of multivalent glycoconjugates opens new opportunities in the field of vaccine design, but their high structural complexity involves new analytical challenges. Nuclear Magnetic Resonance has found wide applications in the characterization and impurity profiling of carbohydrate-based vaccines. Eight bivalent conjugates were studied by quantitative NMR analyzing the structural identity, the content of each capsular polysaccharide, the ratios between polysaccharides, the polysaccharide to protein ratios and undesirable contaminants. The qNMR technique involves experiments with several modified parameters for obtaining spectra with quantifiable signals. In addition, the achieved NMR results were combined with the results of colorimetric assay and Size Exclusion HPLC for assessing the protein content and free protein percentage, respectively. The application of quantitative NMR showed to be efficient to clear up the new structural complexities while allowing the quantitative assessment of the components.

Salvadora persica L. (toothbrush tree, Miswak) is well recognized in most Middle Eastern and African countries for its potential role in dental care, albeit the underlying mechanism for its effectiveness is still not fully understood. A comparative MS and NMR metabolomics approach was employed to investigate the major primary and secondary metabolites composition of S. persica in context of its organ type viz., root or stem to rationalize for its use as a tooth brush. NMR metabolomics revealed its enrichment in nitrogenous compounds including proline-betaines i.e., 4-hydroxy-stachydrine and stachydrine reported for the first time in S. persica. LC/MS metabolomics identified flavonoids (8), benzylurea derivatives (5), butanediamides (3), phenolic acids (8) and 5 sulfur compounds, with 21 constituents reported for the first time in S. persica. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) of either NMR or LC/MS dataset clearly separated stem from root specimens based on nitrogenous compounds abundance in roots and is justifying for its preference as toothbrush versus stems. The presence of betaines at high levels in S. persica (9−12 μg/mg dry weight) offers novel insights into its functioning as an osmoprotectant that maintains the hydration of oral mucosa. Additionally, the previously described anti-inflammatory activity of stachydrine along with the antimicrobial effects of sulfonated flavonoids, benzylisothiocynate and ellagic acid derivatives are likely contributors to S. persica oral hygiene health benefits. Among root samples, variation in sugars and organic acids levels were the main discriminatory criterion. This study provides the first standardization of S. persica extract using qNMR for further inclusion in nutraceuticals.

Ceramides (CERs) play a major role in skin barrier function and direct replacement of depleted skin CERs,due to skin disorder or aging, has beneficial effects in improving skin barrier function and skin hydration.Though, plants are reliable source of CERs, absence of economical and effective method of hydrolysis toconvert the dominant plant sphingolipid, glucosylceramides (GlcCERs), into CERs remains a challenge.This study aims at exploring alternative GlcCERs sources and chemical method of hydrolysis into CERsfor dermal application. GlcCERs isolated from lupin bean (Lupinus albus), mung bean (Vigna radiate) andnaked barley (Hordium vulgare) were identified using ultra high performance liquid chromatographyhyphenated with atmospheric pressure chemical ionization - high resolution tandem mass spectrometer(UHPLC/APCI-HRMS/MS) and quantified with validated automated multiple development-high perfor-mance thin layer chromatography (AMD-HPTLC) method. Plant GlcCERs were hydrolyzed into CERs withmild acid hydrolysis (0.1 N HCl) after treating them with oxidizing agent, NaIO4,and reducing agent,NaBH4. GlcCERs with 4,8-sphingadienine, 8-sphingenine and 4-hydroxy-8-sphingenine sphingoid baseslinked with C14 to C26 -hydroxylated fatty acids (FAs) were identified. Single GlcCER (m/z 714.5520)was dominant in lupin and mung beans while five major GlcCERs species (m/z 714.5520, m/z 742.5829,m/z 770.6144, m/z 842.6719 and m/z 844.56875) were obtained from naked barley. The GlcCERs con-tents of the three plants were comparable. However, lupin bean contains predominantly (> 98 %) a singleGlcCER (m/z 714.5520). Considering the affordability, GlcCER content and yield, lupin bean would bethe preferred alternative commercial source of GlcCERs. CER species bearing 4,8-sphingadienine and 8-sphingenine sphingoid bases attached to C14 to 24 FAs were found after mild acid hydrolysis. CER specieswith m/z 552.4992 was the main component in the beans while CER with m/z 608.5613 was dominantin the naked barley. However, CERs with 4-hydroxy-8-sphingenine sphingoid base were not detected inUHPLC-HRMS/MS study suggesting that the method works for mainly GlcCERs carrying dihydroxy sph-ingoid bases. The method is economical and effective which potentiates the commercialization of plantCERs for dermal application.

BackgroundSkin ageing is associated with structure-functional changes in the extracellular matrix, which is in part caused by proteolytic degradation. Since cysteine cathepsins are major matrix protein-degrading proteases, we investigated the age-dependent expression of elastolytic cathepsins K, S, and V in human skin, their in vitro impact on the integrity of the elastic fibre network, their cleavage specificities, and the release of bioactive peptides.MethodsCathepsin-mediated degradation of human skin elastin samples was assessed from young to very old human donors using immunohistochemical and biochemical assays, scanning electron microscopy, and mass spectrometry.ResultsElastin samples derived from patients between 10 and 86 years of age were analysed and showed an age-dependent deterioration of the fibre structure from a dense network of thinner fibrils into a beaded and porous mesh. Reduced levels of cathepsins K, S, and V were observed in aged skin with a predominant epidermal expression. Cathepsin V was the most potent elastase followed by cathepsin K and S. Biomechanical analysis of degraded elastin fibres corroborated the destructive activity of cathepsins. Mass spectrometric determination of the cleavage sites in elastin revealed that all three cathepsins predominantly cleaved in hydrophobic domains. The degradation of elastin was efficiently inhibited by an ectosteric inhibitor. Furthermore, the degradation of elastin fibres resulted in the release of bioactive peptides, which have previously been associated with various pathologies.ConclusionCathepsins are powerful elastin-degrading enzymes and capable of generating a multitude of elastokines. They may represent a viable target for intervention strategies to reduce skin ageing.

Knipholone (1) and knipholone anthrone (2), isolated from the Ethiopian medicinal plant Kniphofia foliosa Hochst. are two phenyl anthraquinone derivatives, a compound class known for biological activity. In the present study, we describe the activity of both 1 and 2 in several biological assays including cytotoxicity against four human cell lines (Jurkat, HEK293, SH-SY5Y and HT-29), antiplasmodial activity against Plasmodium falciparum 3D7 strain, anthelmintic activity against the model organism Caenorhabditis elegans, antibacterial activity against Aliivibrio fischeri and Mycobacterium tuberculosis and anti-HIV-1 activity in peripheral blood mononuclear cells (PBMCs) infected with HIV-1c. In parallel, we investigated the stability of knipholone (2) in solution and in culture media. Compound 1 displays strong cytotoxicity against Jurkat, HEK293 and SH-SY5Y cells with growth inhibition ranging from approximately 62–95% when added to cells at 50 μM, whereas KA (2) exhibits weak to strong activity with 26, 48 and 70% inhibition of cell growth, respectively. Both 1 and 2 possess significant antiplasmodial activity against Plasmodium falciparum 3D7 strain with IC50 values of 1.9 and 0.7 μM, respectively. These results complement previously reported data on the cytotoxicity and antiplasmodial activity of 1 and 2. Furthermore, compound 2 showed HIV-1c replication inhibition (growth inhibition higher than 60% at tested concentrations 0.5, 5, 15 and 50 μg/ml and an EC50 value of 4.3 μM) associated with cytotoxicity against uninfected PBMCs. The stability study based on preincubation, HPLC and APCI-MS (atmospheric-pressure chemical ionization mass spectrometry) analysis indicates that compound 2 is unstable in culture media and readily oxidizes to form compound 1. Therefore, the biological activity attributed to 2 might be influenced by its degradation products in media including 1 and other possible dimers. Hence, bioactivity results previously reported from this compound should be taken with caution and checked if they differ from those of its degradation products. To the best of our knowledge, this is the first report on the anti-HIV activity and stability analysis of compound 2.

BackgroundSelenium (Se) exerts its biological activity largely via selenoproteins, which are key enzymes for maintaining the cellular redox homeostasis. However, besides these beneficial effects there is also evidence that an oversupply of Se might increase the risk towards developing metabolic disorders. To address this in more detail, we directly compared effects of feeding distinct Se compounds and concentrations on hepatic metabolism and expression profiles of mice.MethodsMale C57BL6/J mice received either a selenium-deficient diet or diets enriched with adequate or high doses of selenite, selenate or selenomethionine for 20 weeks. Subsequently, metabolic parameters, enzymatic activities and expression levels of hepatic selenoproteins, Nrf2 targets, and additional redox-sensitive proteins were analyzed. Furthermore, 2D-DIGE-based proteomic profiling revealed Se compound-specific differentially expressed proteins.ResultsWhereas heterogeneous effects between high concentrations of the Se compounds were observed with regard to body weight and metabolic activities, selenoproteins were only marginally increased by high Se concentrations in comparison to the respective adequate feeding. In particular the high-SeMet group showed a unique response compromising higher hepatic Se levels in comparison to all other groups. Accordingly, hepatic glutathione (GSH) levels, glutathione S-transferase (GST) activity, and GSTpi1 expression were comparably high in the high-SeMet and Se-deficient group, indicating that compound-specific effects of high doses appear to be independent of selenoproteins.ConclusionsNot only the nature, but also the concentration of Se compounds differentially affect biological processes.General significanceThus, it is important to consider Se compound-specific effects when supplementing with selenium.

Trigonelline (3-carboxy-1-methyl pyridinium) was identified as a relevant bioactivity and taste imparting component in Balanites aegyptiaca fruit, using 1H NMR of crude extracts without any fractionation or isolation step. The structural integrity of trigonelline was established within the extract matrix via1H NMR, 1H–1H COSY, HMQC and HMBC and by comparison with authentic standard. A quantitative 1H NMR method (qHNMR) was used to determine trigonelline concentrations in the peel and pulp of B. aegyptiaca fruit of 8 and 13 mg g−1, respectively. Trigonelline so far has not been reported from B. aegyptiaca or its genus as it easily escapes LC–MS based detection. Its discovery provides novel insight into the balanite fruits antidiabetic properties as the compound is known for a pronounced hypoglycemic effect. In addition, it is likely to impart the perceptible bitter taste portion to balanites sweet bitter taste. UPLC–MS of the crude extract additionally revealed the fruit flavonoid pattern showing quercetin/isorhamnetin flavonol conjugates in addition to epicatechin, the latter being present at much lower levels.

HPLC-UV and MS/MS studies of impurity profiles of original (Xenical®, F. Hoffmann-La Roche Ltd., Switzerland) and generic (Cobese™, Ranbaxy Laboratories Limited, India, and Orsoten, KRKA, Russia) products were carried out. The drug and related impurities were extracted by dissolving commercial samples in ethanol. The generic formulations contained higher levels of impurities than the original product. Impurity profiles (HPLC-MS/MS) of the generic samples are similar among themselves, whilst different in comparison to the impurity profile of the original product. The number of detected impurities for generics (14 impurities in Cobese™ and 13 impurities in Orsoten) is higher than for the original product (3 impurities in Xenical®). Based on these analyses the overall analytical quality follows the order Xenical® (best) > Orsoten > Cobese™.