The Structural Difference of Isobaric N-Glycans of Two Microalgae Samples Reveals Taxonomic Distance.

Microalgae of the Chlorella clade are extensively investigated as an environmentally friendly source of renewable biofuels and high-value nutrients. In addition, essentially unprocessed Chlorella serves as wholesome food additive. A recent study on 80 commercial Chlorella preparations revealed an unexpected variety of protein-linked N-glycan patterns with unprecedented structural features, such as the occurrence of arabinose. Two groups of products exhibited a characteristic major N-glycan isobaric to the Man2GlcNAc2XylFuc N-glycan known from pineapple stem bromelain, but tandem mass spectrometry (MS/MS) analysis pointed at two types of N-glycan different from the bromelain structure, as well as from each other. Here we report the exact structures of these two novel N-glycan structures, elucidated by nuclear magnetic resonance spectroscopy and MS/MS, as well as on their phylogenetic context. Despite their humble size, these two N-glycans exhibited a very different design with structural features unrelated to those recently described for other Chlorella-clade strains. The major glycans of this study presented several novel structural features such as substitution by arabinose or xylose of the internal N-acetylglucosamine, as well as methylated sugars. ITS1-5.8S-ITS2 rDNA barcode analyses revealed that the xylose-containing structure derived from a product primarily comprising Scenedesmus species, and the arabinose-containing glycan type related to Chlorella species (SAG211-34 and FACHB-31) and to Auxenochlorella. This is another example where characteristic N-glycan structures distinguish phylogenetically different groups of microalgae.

Isolation and Characterization of Acetylcholinesterase Inhibitors from Piper longum and Binding Mode Predictions.

Restoration of cholinergic function is considered a rational approach to enhance cognitive performance. Acetylcholinesterase inhibitors are still the best therapeutic option for Alzheimer's disease. The fruits of Piper longum have been used in traditional medicines for the treatment of memory loss. It was demonstrated that the dichloromethane extract of these fruits is able to inhibit acetylcholinesterase. Thus, the aim of this study was to identify the contained acetylcholinesterase inhibitors. The active zones were presented via TLC-bioautography, and five compounds were isolated in the process of a bioassay-guided phytochemical investigation. Their structures were characterized as piperine, methyl piperate, guineenisine, pipercide, and pellitorine using spectroscopy and spectrometry methods (UV, IR, MS, 1H-, and 13C-NMR). In vitro acetylcholinesterase inhibitory activities of the isolates and their IC50 values were determined via a colorimetric assay. Three of them exhibited enzyme inhibitory activities, with piperine being the most potent compound (IC50 of 0.3 mM). In order to investigate the binding mode of the tested compounds, docking studies were performed using the X-ray crystal structure of acetylcholinesterase from Tetronarce californica with the Protein Data Bank code 1EVE. The content of the active compounds in the extract was determined by a developed HPLC method. Piperine was present in the maximum quantity in the fruits (0.57%), whereas methyl piperate contained the minimum content (0.10%).

Antiplasmodial activity of triterpenes isolated from the methanolic leaf extract of Combretum racemosum P. Beauv.

ETHNOPHARMACOLOGICAL RELEVANCE: Combretum racemosum showed activity in previous ethnopharmacological investigations of some Combretum species used in malaria treatment in parts of West Africa. AIM OF THE STUDY: This study aimed at confirming the antimalarial potential of this plant by an activity-guided isolation of its active principles. MATERIALS AND METHODS: A crude methanolic leaf extract of Combretum racemosum and fractions thereof obtained by partition with chloroform and n-butanol were investigated for antiplasmodial activity against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of Plasmodium falciparum. Repeated chromatographic separations were conducted on the chloroform fraction to isolate bioactive compounds for further tests on antiplasmodial activity. The characterization of the isolated substances was performed by applying NMR- and MS-techniques (ESI-MS, HR-ESIMS, 1D and 2D NMR). RESULTS: The chloroform fraction (D10: IC50 = 33.8 ±â€¯1.5 µg/mL and W2: IC50 = 27.8 ±â€¯2.9 µg/mL) exhibited better antiplasmodial activity than the n-butanol fraction (D10: IC50 = 78.1 ±â€¯7.3 µg/mL and W2: IC50 = 78 ±â€¯15 µg/mL) as well as the methanolic raw extract (D10: IC50 = 64.2 ±â€¯2.7 µg/mL and W2: IC50 = 65.8 ±â€¯14.9 µg/mL). Thus, the focus of the phytochemical investigation was laid on the chloroform fraction, which led to the identification of four ursane-type (19α-hydroxyasiatic acid (1), 6ß,23-dihydroxytormentic acid (4), madecassic acid (8), nigaichigoside F1 (10)) and four oleanane-type (arjungenin (2), combregenin (5), terminolic acid (7), arjunglucoside I (11)) triterpenes, as well as abscisic acid (9). Compounds 1 and 2, 4 and 5, 7 and 8 as well as 10 and 11 were isolated as isomeric mixtures in fractions CR-A, CR-C, CR-E and CR-H, respectively. All isolated compounds and mixtures exhibited moderate to low activity, with madecassic acid being most active (D10: IC50 = 28 ±â€¯12 µg/mL and W2: IC50 = 17.2 ±â€¯4.3 µg/mL). CONCLUSION: This paper reports for the first time antiplasmodial principles from C. racemosum and thereby gives reason to the traditional use of the plant.

Random coil shifts of posttranslationally modified amino acids.

Most eukaryotic proteins are modified during and/or after translation, regulating their structure, function and localisation. The role of posttranslational modifications (PTMs) in both normal cellular processes and in diseases is already well recognised and methods for detection of PTMs and generation of specifically modified proteins have developed rapidly over the last decade. However, structural consequences of PTMs and their specific effects on protein dynamics and function are not well understood. Furthermore, while random coil NMR chemical shifts of the 20 standard amino acids are available and widely used for residue assignment, dihedral angle predictions and identification of structural elements or propensity, they are not available for most posttranslationally modified amino acids. Here, we synthesised a set of random coil peptides containing common naturally occurring PTMs and determined their random coil NMR chemical shifts under standardised conditions. We highlight unique NMR signatures of posttranslationally modified residues and their effects on neighbouring residues. This comprehensive dataset complements established random coil shift datasets of the 20 standard amino acids and will facilitate identification and assignment of posttranslationally modified residues. The random coil shifts will also aid in determination of secondary structure elements and prediction of structural parameters of proteins and peptides containing PTMs.

Correction: 19F multiple-quantum coherence NMR spectroscopy for probing protein-ligand interactions.

[This corrects the article DOI: 10.1039/C8RA09296F.].

19F multiple-quantum coherence NMR spectroscopy for probing protein-ligand interactions.

A new 19F NMR method is presented which can be used to detect weak protein binding of small molecules with up to mM affinity. The method capitalizes on the synthetic availability of unique SF5 containing compounds and the generation of five-quantum coherences (5QC). Given the high sensitivity of 5QC relaxation to exchange events (i.e. reversible protein binding) fragments which bind to the target with weak affinity can be identified. The utility of the method in early stage drug discovery programs is demonstrated with applications to two model proteins, the neurotoxic NGAL and the prominent tumor target ß-catenin.

Lupinalbin A as the most potent estrogen receptor α- and aryl hydrocarbon receptor agonist in Eriosema laurentii de Wild. (Leguminosae).

BACKGROUND: Eriosema laurentii De Wild. (Leguminosae) is a plant used in Cameroon against infertility and gynecological or menopausal complaints. In our previous report, a methanol extract of its aerial parts was shown to exhibit estrogenic and aryl hydrocarbon receptor agonistic activities in vitro and to prevent menopausal symptoms in ovariectomized Wistar rats. METHODS: In order to determine the major estrogen receptor α (ERα) agonists in the extract, an activity-guided fractionation was performed using the ERα yeast screen. To check whether the ERα active fractions/compounds also accounted for the aryl hydrocarbon receptor (AhR) agonistic activity of the crude methanol extract, they were further tested on the AhR yeast screen. RESULTS: This study led to the identification of 2'-hydroxygenistein, lupinalbin A and genistein as major estrogenic principles of the extract. 2'-hydroxygenistein and lupinalbin A were, for the first time, also shown to possess an AhR agonistic activity, whereas genistein was not active in this assay. In addition, it was possible to deduce structure-activity relationships. CONCLUSIONS: These results suggest that the identified compounds are the major active principles responsible for the estrogenic and AhR agonistic activities of the crude methanol extract of the aerial parts of Eriosema laurentii.

2-deprenyl-rheediaxanthone B isolated from Metaxya rostrata induces active cell death in colorectal tumor cells.

Metaxya rostrata C. Presl (Metaxyaceae) is a common tree fern in Central and South America that is used for the treatment of intestinal ulcers and tumours in ethnic medicine. Using a bioactivity-guided strategy 2-deprenyl-rheediaxanthone B (XB) has been isolated as one of the active principles in this plant. XB induced loss of cell viability in colorectal cancer cell lines at IC50 concentrations of 11-23 µM. This was caused by both accumulation of cells in the G2- and S-phase as well as by induction of active cell death in a time and concentration-dependent manner. Cells exposed to XB were incapable of undergoing regular mitosis due to down-regulation of FoxM1 and absence of chromosome condensation. The apoptosis-related proteins Bcl2 and Bclxl were up-regulated so that Caspase 3 was not activated and classical apoptosis was not observed. However, XB triggered damage pathways down-stream of ATR and activated Caspase 2 causing cell death by a mechanism similar to mitotic catastrophe. Our observations are the first to show the cytotoxic activity of 2-deprenyl-rheediaxanthone B and indicate that XB is an interesting new lead compound for cancer therapy that merits further development.

Combination of bioautography with HPTLC-MS/NMR: a fast identification of acetylcholinesterase inhibitors from galbanum(†).

INTRODUCTION: In the search for new natural compounds with acetylcholinesterase (AChE) inhibitory activity this study focused on galbanum, the oleo gum-resin from Ferula gummosa Boiss., which had shown AChE inhibitory activity in a screening. OBJECTIVE: The isolation of bioactive compounds from plant extracts usually is laborious and time consuming. In an approach to accelerate the characterisation of compounds with AChE inhibitory activity, the potential of a combination of HPTLC bioautography with HPTLC-MS/NMR for the fast identification of active compounds in galbanum was studied. METHOD: Pre-fractionation of the dichloromethane extract was performed by vacuum liquid chromatography. The resulting fractions were separated by HPTLC and active zones determined by bioautography. A TLC-MS interface was used to elute the single zones from the plates directly into a mass spectrometer. The interface was also used to extract the two major active zones from HPTLC plates for off-line one- and two-dimensional NMR and quadrupole time of flight (QTOF) MS. RESULTS: The isolated compounds were identified as 7-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}-2H-chromen-2-one (auraptene) and 7-{[(1R,4aR,6S,8aS)-6-hydroxy-5,5,8a-trimethyl-2-methylenedecahydronaphthalen-1-yl]methoxy}-2H-chromen-2-one (farnesiferol A). This is the first report of these substances in F. gummosa. Their median inhibitory concentration (IC50 ) values for AChE inhibition were determined as 47 and 17 µg/mL in comparison with physostigmine as a positive control (IC50 : 0.8 µg/mL) and their concentrations in galbanum were quantified by HPLC as 3.5% and 7.9%, respectively. CONCLUSION: The study showed that HPTLC-MS/NMR can be considered as a fast and high-confidence method for dereplication of natural compounds. From the correlation of the concentration of the elucidated compounds and their IC50 values for AChE inhibition it can be concluded that auraptene and farnesiferol A are contributing to this activity of galbanum.

Secondary metabolites of Centaurea calolepis and evaluation of cnicin for anti-inflammatory, antioxidant, and cytotoxic activities.

CONTEXT: Centaurea L. (Astreaceae) species are used as herbal remedies in Turkey. Centaurea calolepis Boiss. is an endemic species of Anatolia that has not been subjected to phytochemical studies except essential oil analysis. OBJECTIVE: Secondary metabolite determination, isolation and structure elucidation of pure compounds were performed on C. calolepis. Cnicin, which is the main component of several Centaurea species, was tested for its in vitro anti-inflammatory, antioxidant and cytotoxic activities. MATERIALS AND METHODS: Chloroform and methanol extracts of the aerial parts of C. calolepis were subjected to isolation process using column chromatography. The structures of the compounds were characterized by 1D- and 2D-NMR experiments. Thin-layer chromatography and high performance liquid chromatography were used in determination of phenolics. Cnicin was subjected to a panel of cellular assays to test for inhibition of nuclear factor κB (NF-κB), inducible nitric oxide synthase (iNOS), reactive oxygen species and cytotoxicity. RESULTS: Cnicin, lucenin-2, schaftoside and 3-O-feruloylquinic acid were isolated from C. calolepis extracts. Vicenin-2, vitexin, isovitexin, homoorientin, rutin, orientin, luteolin-7-O-glycoside and chlorogenic acid were determined in fractions. Cnicin showed inhibition of NF-κB and inhibition of iNOS activity with IC50 Values of 1.8 and 6.5 µM, respectively. Cytotoxic activity of cnicin was observed toward pig kidney epithelial (LLC-PK11), human malignant melanoma (SK-MEL) and human ductal carcinoma (BT-549) cells with IC50 values of 23.3, 14.0 and 18.3 µM, respectively. DISCUSSION AND CONCLUSION: This is the first detailed report of secondary metabolites of C. calolepis. Evaluation of biological activity of cnicin establishes the potential of this compound as an anti-inflammatory and cytotoxic agent.

Rapid structural identification of cytotoxic bufadienolide sulfates in toad venom from Bufo melanosticus by LC-DAD-MS(n) and LC-SPE-NMR.

Toad venom, namely, "Chansu" in China, has been widely used for the treatment of heart failure, sores, pains, and various cancers. Upon LC-MS analysis of the venom from Bufo melanosticus collected in Indonesia, new bufadienolide sulfates were identified. For a complete characterization, the MeOH extract of the toad venom from B. melanosticus was fractionated by preparative HPLC, and the structures of five new buadienolide sulfates (1-5) along with one new bufogenin (6) were rapidly elucidated on the basis of LC-DAD-MS(n) and LC-SPE-NMR data. The in vitro growth inhibitory activity of these six compounds along with hellebrin (positive control) has been assayed by means of the MTT colorimetric assay in four human and two mouse cancer cell lines. Compound 3 and hellebrin displayed similar and marked in vitro cytotoxicity.

Total synthesis of the microtubule stabilizing antitumor agent laulimalide and some nonnatural analogues: the power of Sharpless' asymmetric epoxidation.

Three different routes are described for the synthesis of deoxylaulimalide (3), which is the immediate precursor of the marine sponge metabolite laulimalide (1). These routes mainly differ with respect to their ring closing step. Thus, route 1 uses a Still-Gennari olefination, route 2 a Yamaguchi lactonization, and route 3 an intramolecular allylsilane-aldehyde addition for establishing the macrocyclic structure. The unprotected deoxy derivative 3 was subjected to Sharpless' asymmetric epoxidation (SAE). With (R,R)-tartrate the 16,17-epoxide laulimalide (1) is formed selectively, whereas (S,S)-tartrate generates the 21,22-epoxide 142. This demonstrates the high reagent control involved in the SAE process, which in this case is used to achieve high stereo- and regioselectivity. Laulimalide and some derivatives thereof have been tested with respect to antitumor activity and compared to standard compounds paclitaxel and epothilone B.