Unusual Sesquiterpenes from Streptomyces olindensis DAUFPE 5622.

In nature, the vast majority of sesquiterpenes are produced by type I mechanisms, and glycosylated sesquiterpenes are rare in actinobacteria. Streptomyces olindensis DAUFPE 5622 produces the sesquiterpenes olindenones A-G, a new class of rearranged drimane sesquiterpenes. Olindenones B-D are oxygenated derivatives of olindenone A, while olindenones E-G are analogs glycosylated with dideoxysugars. 13C-isotope labeling studies demonstrated olindenone A biosynthesis occurs via the methylerythritol phosphate (MEP) pathway and suggested the rearrangement is only partially concerted. Based on the structures, one potential mechanism of olindenone A formation proceeds by cyclization of the linear terpenoid precursor, likely occurring via a terpene cyclase-mediated type II mechanism whereby the terminal alkene of the precursor is protonated, triggering carbocation-driven cyclization followed by rearrangement. Diphosphate hydrolysis may occur either before or after cyclization. Although a biosynthetic route is proposed, the terpene cyclase gene responsible for producing olindenones currently remains unidentified.

Data Base similarity (DBsimilarity) of natural products to aid compound identification on MS and NMR pipelines, similarity networking, and more.

INTRODUCTION: We developed Data Base similarity (DBsimilarity), a user-friendly tool designed to organize structure databases into similarity networks, with the goal of facilitating the visualization of information primarily for natural product chemists who may not have coding experience. METHOD: DBsimilarity, written in Jupyter Notebooks, converts Structure Data File (SDF) files into Comma-Separated Values (CSV) files, adds chemoinformatics data, constructs an MZMine custom database file and an NMRfilter candidate list of compounds for rapid dereplication of MS and 2D NMR data, calculates similarities between compounds, and constructs CSV files formatted into similarity networks for Cytoscape. RESULTS: The Lotus database was used as a source for Ginkgo biloba compounds, and DBsimilarity was used to create similarity networks including NPClassifier classification to indicate biosynthesis pathways. Subsequently, a database of validated antibiotics from natural products was combined with the G. biloba compounds to identify promising compounds. The presence of 11 compounds in both datasets points to possible antibiotic properties of G. biloba, and 122 compounds similar to these known antibiotics were highlighted. Next, DBsimilarity was used to filter the NPAtlas database (selecting only those with MIBiG reference) to identify potential antibacterial compounds using the ChEMBL database as a reference. It was possible to promptly identify five compounds found in both databases and 167 others worthy of further investigation. CONCLUSION: Chemical and biological properties are determined by molecular structures. DBsimilarity enables the creation of interactive similarity networks using Cytoscape. It is also in line with a recent review that highlights poor biological plausibility and unrealistic chromatographic behaviors as significant sources of errors in compound identification.

An anchored experimental design and meta-analysis approach to address batch effects in large-scale metabolomics.

Untargeted metabolomics studies are unbiased but identifying the same feature across studies is complicated by environmental variation, batch effects, and instrument variability. Ideally, several studies that assay the same set of metabolic features would be used to select recurring features to pursue for identification. Here, we developed an anchored experimental design. This generalizable approach enabled us to integrate three genetic studies consisting of 14 test strains of Caenorhabditis elegans prior to the compound identification process. An anchor strain, PD1074, was included in every sample collection, resulting in a large set of biological replicates of a genetically identical strain that anchored each study. This enables us to estimate treatment effects within each batch and apply straightforward meta-analytic approaches to combine treatment effects across batches without the need for estimation of batch effects and complex normalization strategies. We collected 104 test samples for three genetic studies across six batches to produce five analytical datasets from two complementary technologies commonly used in untargeted metabolomics. Here, we use the model system C. elegans to demonstrate that an augmented design combined with experimental blocks and other metabolomic QC approaches can be used to anchor studies and enable comparisons of stable spectral features across time without the need for compound identification. This approach is generalizable to systems where the same genotype can be assayed in multiple environments and provides biologically relevant features for downstream compound identification efforts. All methods are included in the newest release of the publicly available SECIMTools based on the open-source Galaxy platform.

Coral microbiome manipulation elicits metabolic and genetic restructuring to mitigate heat stress and evade mortality.

Beneficial microorganisms for corals (BMCs) ameliorate environmental stress, but whether they can prevent mortality and the underlying host response mechanisms remains elusive. Here, we conducted omics analyses on the coral Mussismilia hispida exposed to bleaching conditions in a long-term mesocosm experiment and inoculated with a selected BMC consortium or a saline solution placebo. All corals were affected by heat stress, but the observed "post-heat stress disorder" was mitigated by BMCs, signified by patterns of dimethylsulfoniopropionate degradation, lipid maintenance, and coral host transcriptional reprogramming of cellular restructuration, repair, stress protection, and immune genes, concomitant with a 40% survival rate increase and stable photosynthetic performance by the endosymbiotic algae. This study provides insights into the responses that underlie probiotic host manipulation. We demonstrate that BMCs trigger a dynamic microbiome restructuring process that instigates genetic and metabolic alterations in the coral host that eventually mitigate coral bleaching and mortality.

Quantum Chemistry Calculations for Metabolomics.

A primary goal of metabolomics studies is to fully characterize the small-molecule composition of complex biological and environmental samples. However, despite advances in analytical technologies over the past two decades, the majority of small molecules in complex samples are not readily identifiable due to the immense structural and chemical diversity present within the metabolome. Current gold-standard identification methods rely on reference libraries built using authentic chemical materials ("standards"), which are not available for most molecules. Computational quantum chemistry methods, which can be used to calculate chemical properties that are then measured by analytical platforms, offer an alternative route for building reference libraries, i.e., in silico libraries for "standards-free" identification. In this review, we cover the major roadblocks currently facing metabolomics and discuss applications where quantum chemistry calculations offer a solution. Several successful examples for nuclear magnetic resonance spectroscopy, ion mobility spectrometry, infrared spectroscopy, and mass spectrometry methods are reviewed. Finally, we consider current best practices, sources of error, and provide an outlook for quantum chemistry calculations in metabolomics studies. We expect this review will inspire researchers in the field of small-molecule identification to accelerate adoption of in silico methods for generation of reference libraries and to add quantum chemistry calculations as another tool at their disposal to characterize complex samples.

Comprehensive Metabolome Analysis of Fermented Aqueous Extracts of Viscum album L. by Liquid Chromatography-High Resolution Tandem Mass Spectrometry.

Fermented aqueous extracts of Viscum album L. are widely used for cancer treatment in complementary medicine. The high molecular weight compounds viscotoxins and lectins are considered to be the main active substances in the extracts. However, a vast number of small molecules (≤1500 Da) is also expected to be present, and few studies have investigated their identities. In this study, a comprehensive metabolome analysis of samples of fermented aqueous extracts of V. album from two host tree species (Malus domestica and Pinus sylvestris), both prepared by two pharmaceutical manufacturing processes, was performed by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). A total of 212 metabolites were putatively annotated, including primary metabolites (e.g., amino acids, organic acids, etc.) and secondary metabolites (mostly phenolic compounds). A clear separation between V. album samples according to the host tree species, but not due to manufacturing processes, was observed by principal component analysis. The biomarkers responsible for this discrimination were assessed by partial least squares-discriminant analysis. Because V. album extracts from different host trees have different clinical applications, the present work highlights the possibility of characterizing the metabolome for identification and traceability of V. album fermented aqueous extracts.

Extending compound identification for molecular network using the LipidXplorer database independent method: A proof of concept using glycoalkaloids from Solanum pseudoquina A. St.-Hil.

INTRODUCTION: Molecular networks are now established as the method of choice for tandem mass spectrometry dereplication and similarity-based structure elucidation. Node identification can be used to start the propagation of the structure elucidation of unknown compounds progressively. OBJECTIVE: To demonstrate the capabilities of using the LipidXplorer data results along with molecular networking to identify nodes and aid sequential structure elucidation of unknown compounds. MATERIAL AND METHODS: Molecular fragmentation query language (MFQL) files were written to identify glycoalkaloids based on known structures described for Solanum species. A dataset generated from liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis of Solanum pseudoquina sample were submitted to dereplication on both LipidXplorer software and Global Natural Products Social Molecular Network (GNPS) online system. The resulting attribute table from GNPS calculations was merged with the LipidXplorer results and this merged file was used for network visualisation in Cytoscape. Nodes in the molecular network were labelled using the LipidXplorer identifiers, thus assisting the structure elucidation of unidentified compounds. RESULTS: The combination of the LipidXplorer glycoalkaloids list and GNPS analysis was used in Cytoscape to label nodes in the molecular network. The analysis of the network using these labelled starting points triggered the structure elucidation of closely related nodes leading to the identification of 30 compounds using the LipidXplorer output and four purified and structure elucidated compounds, including a new glycoalkaloids identified as 3-O-(ß-d-xylopyranosyl)-(20R,25S)-22,26-epimino-16-acetyl-cholesta-5,22(N)-diene. CONCLUSION: A significant compound identification completely based on molecular formula and fragmentation queries was achieved. This new and effective approach could help researches to expand the identification rate of compounds in dereplication studies using molecular networks.

Dereplication of plant phenolics using a mass-spectrometry database independent method.

INTRODUCTION: Dereplication, an approach to sidestep the efforts involved in the isolation of known compounds, is generally accepted as being the first stage of novel discoveries in natural product research. It is based on metabolite profiling analysis of complex natural extracts. OBJECTIVE: To present the application of LipidXplorer for automatic targeted dereplication of phenolics in plant crude extracts based on direct infusion high-resolution tandem mass spectrometry data. MATERIAL AND METHODS: LipidXplorer uses a user-defined molecular fragmentation query language (MFQL) to search for specific characteristic fragmentation patterns in large data sets and highlight the corresponding metabolites. To this end, MFQL files were written to dereplicate common phenolics occurring in plant extracts. Complementary MFQL files were used for validation purposes. RESULTS: New MFQL files with molecular formula restrictions for common classes of phenolic natural products were generated for the metabolite profiling of different representative crude plant extracts. This method was evaluated against an open-source software for mass-spectrometry data processing (MZMine®) and against manual annotation based on published data. CONCLUSION: The targeted LipidXplorer method implemented using common phenolic fragmentation patterns, was found to be able to annotate more phenolics than MZMine® that is based on automated queries on the available databases. Additionally, screening for ascarosides, natural products with unrelated structures to plant phenolics collected from the nematode Caenorhabditis elegans, demonstrated the specificity of this method by cross-testing both groups of chemicals in both plants and nematodes.

Athenolide A, a New Steroidal Lactone from the Leaves of Athenaea martiana (Solanaceae) Determined by Means of HPLC-HR-MS-SPE-NMR Analysis.

Athenaea (Solanaceae) is an endemic genus belonging to the Brazilian Atlantic Rainforest. Recently, botanical investigations suggested the re-evaluation of the generic status of the genus Athenaea as a synonym of Aureliana. In this study, the first investigation of the Athenaea genus performed on Athenaea martiana by means of HPLC-HR-MS-SPE-NMR combined with high-resolution radical scavenging profile led to identification of several phenolic acids as radical scavengers: protocatechuic acid (1), 4-hydroxybenzoic acid (2), caffeic acid (3), vanillic acid (4), and ferulic acid (6). Additional analysis revealed a new steroidal lactone, named athenolide A (9). Their structures were elucidated by extensive use of NMR spectroscopy as well as HR-MS. Chemotaxonomic considerations based on these results supported the chemical relationships between the Athenaea and Aureliana genera, in agreement with the recent botanical findings.

Dual high-resolution inhibition profiling and HPLC-HRMS-SPE-NMR analysis for identification of α-glucosidase and radical scavenging inhibitors in Solanum americanum Mill.

Solanum americanum is one of the most prominent species used to treat type 2 diabetes in Guatemala. In our ongoing efforts to find antidiabetic and antioxidative compounds from natural sources, an ethyl acetate extract of this medicinal herb was investigated using dual high-resolution α-glucosidase/radical scavenging inhibition profiling. The high-resolution biochromatograms obtained by this technique were used to target subsequent structural elucidation by HPLC-HRMS-SPE-NMR analysis towards the bioactive constituents. This led to identification of 4-hydroxybenzoic acid (1) and 3-indolecarboxylic acid (6) associated with radical scavenging activity, and the amide alkaloids N-trans-p-coumaroyloctopamine (3), N-trans-p-feruloyloctopamine (4), N-trans-p-coumaroyltyramine (8) and N-trans-p-feruloyltyramine (9) correlated with α-glucosidase inhibitory activity as well as radical scavenging activity. Further analysis revealed a new lactone, methyl 5-ethyl-4-hydroxy-5-methyl-2-oxotetrahydro-2H-pyran-4-carboxylate (7) and a new steroid with a rare F ring (11). Corchorifatty acid B (12) was reported for the first time in the Solanaceae family. Their structures were elucidated by extensive use of 1D and 2D NMR spectroscopy as well as HRMS analysis.

Absolute Configuration of (-)-Centratherin, a Sesquiterpenoid Lactone, Defined by Means of Chiroptical Spectroscopy.

(-)-Centratherin is a bioactive sesquiterpenoid lactone, whose absolute configuration (AC) was not established, but has been proposed based on those of germacrane precursors. To verify this proposal, the experimental electronic circular dichroism (ECD), electronic dissymmetry factor (EDF), optical rotatory dispersion (ORD), vibrational circular dichroism (VCD), and vibrational dissymmetry factor (VDF) spectra of (-)-centratherin have been analyzed with the corresponding density functional theoretical predictions. These analyses suggest the AC of naturally occurring (-)-centratherin to be (6R,7R,8S,10R,2'Z).

3-Ishwarone, a rare ishwarane sesquiterpene from Peperomia scandens Ruiz & Pavon: structural elucidation through a joint experimental and theoretical study.

3-Ishwarone, (1), a sesquiterpene with a rare ishwarane skeleton, was isolated from Peperomia scandens Ruiz & Pavon (Piperaceae). Its structure was unambiguously determined by 1D- and 2D-NMR and infrared analyses, as well as by comparative theoretical studies which involved calculations of 13C-NMR chemical shifts, using the Density Functional Theory (DFT) with the mPW1PW91 hybrid functional and Pople's 6-31G(d) basis set, and of vibrational frequencies, using the B3LYP hybrid functional and triple ζ Dunning's correlation consistent basis set (cc-pVTZ), of (1) and three of its possible diastereomers, compounds 2-4.

Development of a new sodium diclofenac certified reference material using the mass balance approach and ¹H qNMR to determine the certified property value.

Certified reference materials (CRMs) are essential tools to guarantee the metrological traceability of measurement results to the International System of Units (SI), which means the accuracy and comparability of results over time and space. In the pharmaceutical area, only a few CRMs are available and the use of (non-certified) reference materials is a much more common practice. In this paper, the studies on a new candidate CRM of sodium diclofenac based on the ISO Guides 34:2009 and 35:2005 are described. The project steps included characterization, homogeneity test, stability studies, and uncertainties estimation. In the characterization, the mass fractions of organic, inorganic, and volatile impurities were determined, and the results were cross-checked by independent reference methods or interlaboratorial study. The API mass fraction was calculated by mass balance and cross-checked by quantitative proton nuclear magnetic resonance (¹H qNMR). The paper also presents a Monte Carlo simulation to estimate the measurement uncertainty as an approach to validate the GUM results in ¹H qNMR. The homogeneity between batch units was verified, and the candidate CRM stability under transport and storage conditions was evaluated in short- and long-term stability studies. The CRM certified property value and corresponding expanded uncertainty, obtained from the combined standard uncertainty multiplied by the coverage factor (k=2), for a confidence level of 95%, was (999.76+0.10) mg g⁻¹.

Antiherpetic activity of a flavonoid fraction from Ocotea notata leaves

This study describes the isolation of a flavonoid fraction from leaves of Ocotea notata (Nees & Mart.) Mez, Lauraceae, the identification of six major compounds (an A-type proanthocyanidin trimer [3], isoquercitrin [4], reynoutrin [5], miquelianin [6], quercitrin [7], afzelin [8]) and four minor compounds (catechin [1], epicatechin [2], quercetin [9], kaempferol [10]) present in the fraction and its activity against the Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The 50% effective concentrations values (EC50) calculated from the dose-response curve and the selectivity indices (SI) against the virus were: EC50 35.8 µg/mL and SI 5.5 to HSV-1 and EC50 23.5 µg/mL and SI 8.5 to HSV-2. The flavonoid fraction was more active against HSV-2 than HSV-1. The mechanisms of antiviral action of the flavonoid fraction against the virus were also evaluated. The percentage inhibition (PI) obtained for HSV-2 was higher than 90% in the following assays: virucidal, pre-treatment of cells, treatment of cells after viral adsorption and treatment of cells after viral penetration. For HSV-1, the flavonoid fraction had no effect in pre-treatment of cells and showed 60% of inhibition in virucidal assay.

Tratamento clínico da obesidade / Clinical treatment of obesity

RESUMO: A prevalência da obesidade tem aumentado progressivamente e seu tratamento é imprescindível, pois ela está associada ao aparecimento de diversas doenças crônicas. Dentre as opções terapêuticas para a obesidade, o tratamento clínico é fundamental, sendo parte integrante inclusive do tratamento cirúrgico e tem como objetivo mudar o estilo de vida do indivíduo obeso, melhorando seu padrão alimentar e também estimulando a prática de atividade física. Para isso é preciso identificar as falhas tanto no comportamento alimentar do paciente, quanto outros erros nos seus hábitos de vida. Não existem evidências que indique qual a composição mais adequada da dieta para se promover uma maior perda de peso, porém uma alimentação equilibrada parece promover mais saúde e bem estar do que dietas altamente restritivas em algum tipo de nutriente e ser igualmente eficaz em promover perda de peso. Apesar de ser uma das enfermidades metabólicas mais antigas da humanidade, as opções farmacológicas para tratamento da obesidade são limitadas e apresentam diversos efeitos colaterais. Além disso, as mudanças de estilo de vida e do hábito alimentar promovem resultado mais duradouro quando comparados ao tratamento farmacológico isolado. O tratamento farmacológico deve auxiliar o paciente a mudar seu hábito alimentar e não simplesmente reduzir a sensação de fome e a ingestão alimentar...