Microbiomes and metabolomes of dominant coral reef primary producers illustrate a potential role for immunolipids in marine symbioses.

The dominant benthic primary producers in coral reef ecosystems are complex holobionts with diverse microbiomes and metabolomes. In this study, we characterize the tissue metabolomes and microbiomes of corals, macroalgae, and crustose coralline algae via an intensive, replicated synoptic survey of a single coral reef system (Waimea Bay, O'ahu, Hawaii) and use these results to define associations between microbial taxa and metabolites specific to different hosts. Our results quantify and constrain the degree of host specificity of tissue metabolomes and microbiomes at both phylum and genus level. Both microbiome and metabolomes were distinct between calcifiers (corals and CCA) and erect macroalgae. Moreover, our multi-omics investigations highlight common lipid-based immune response pathways across host organisms. In addition, we observed strong covariation among several specific microbial taxa and metabolite classes, suggesting new metabolic roles of symbiosis to further explore.

Old Meets New: Mass Spectrometry-Based Untargeted Metabolomics Reveals Unusual Larvicidal Nitropropanoyl Glycosides from the Leaves of Heteropterys umbellata.

The Aedes aegypti (Diptera: Culicidae) mosquito is the vector of several arboviruses in tropical and subtropical areas of the globe, and synthetic pesticides remain the most widely used combat strategy. This study describes the investigation of secondary metabolites with larvicidal activity from the Malpighiaceae taxon using a metabolomic and bioactivity-based approach. The workflow initially consisted of a larvicidal screening of 394 extracts from the leaves of 197 Malpighiaceae samples, which were extracted using solvents of different polarity, leading to the selection of Heteropterys umbellata for the identification of active compounds. By employing untargeted mass spectrometry-based metabolomics and multivariate analyses (PCA and PLS-DA), it was possible to determine that the metabolic profiles of different plant organs and collection sites differed significantly. A bioguided approach led to the isolation of isochlorogenic acid A (1) and the nitropropanoyl glucosides karakin (2) and 1,2,3,6-tetrakis-O-[3-nitropropanoyl]-beta-glucopyranose (3). These nitro compounds exhibited larvicidal activity, possibly potentialized by synergistic effects of their isomers in chromatographic fractions. Additionally, targeted quantification of the isolated compounds in different extracts corroborated the untargeted results from the statistical analyses. These results support a metabolomic-guided approach in combination with classical phytochemical techniques to search for natural larvicidal compounds for arboviral vector control.

Reaction of the Phytochemistry Community to Green Chemistry: Insights Obtained Since 1990.

This review article aims to study how phytochemists have reacted to green chemistry insights since 1990, the year when the U.S. Environmental Protection Agency launched the "Pollution Prevention Act". For each year in the period 1990 to 2019, three highly cited phytochemistry papers that provided enough information about the experimental procedures utilized were sampled. The "greenness" of these procedures was assessed, particularly for the use of solvents. The highly hazardous diethyl ether, benzene, and carbon tetrachloride did not appear in the papers sampled after 2010. Advances in terms of sustainability were observed mainly in the extraction stage. Similar progress was not observed in purification procedures, where chloroform, dichloromethane, and hexane regularly have been employed. Since replacing such solvents in purification procedures should be a major goal, potential alternative approaches are discussed. Moreover, some current initiatives toward a more sustainable phytochemical research considering aspects other than only solvents are highlighted. Although some advances have been achieved, it is believed that natural products chemists can play a major role in developing a novel ecological paradigm in chemistry. To contribute to this objective, six principles for performing natural products chemistry consistent with the guidelines of green chemistry are proposed.

Untargeted Metabolomics Sheds Light on the Diversity of Major Classes of Secondary Metabolites in the Malpighiaceae Botanical Family.

Natural products produced by plants are one of the most investigated natural sources, which substantially contributed to the development of the natural products field. Even though these compounds are widely explored, the literature still lacks comprehensive investigations aiming to explore the evolution of secondary metabolites produced by plants, especially if classical methodologies are employed. The development of sensitive hyphenated techniques and computational tools for data processing has enabled the study of large datasets, being valuable assets for chemosystematic studies. Here, we describe a strategy for chemotaxonomic investigations using the Malpighiaceae botanical family as a model. Our workflow was based on MS/MS untargeted metabolomics, spectral searches, and recently described in silico classification tools, which were mapped into the latest molecular phylogeny accepted for this family. The metabolomic analysis revealed that different ionization modes and extraction protocols significantly impacted the chemical profiles, influencing the chemotaxonomic results. Spectral searches within public databases revealed several clades or genera-specific molecular families, being potential chemical markers for these taxa, while the in silico classification tools were able to expand the Malpighiaceae chemical space. The classes putatively annotated were used for ancestral character reconstructions, which recovered several classes of metabolites as homoplasies (i.e., non-exclusive) or synapomorphies (i.e., exclusive) for all sampled clades and genera. Our workflow combines several approaches to perform a comprehensive evolutionary chemical study. We expect it to be used on further chemotaxonomic investigations to expand chemical knowledge and reveal biological insights for compounds classes in different biological groups.

Identification of a Prenyl Chalcone as a Competitive Lipoxygenase Inhibitor: Screening, Biochemical Evaluation and Molecular Modeling Studies.

Cyclooxygenase (COX) and lipoxygenase (LOX) are key targets for the development of new anti-inflammatory agents. LOX, which is involved in the biosynthesis of mediators in inflammation and allergic reactions, was selected for a biochemical screening campaign to identify LOX inhibitors by employing the main natural product library of Brazilian biodiversity. Two prenyl chalcones were identified as potent inhibitors of LOX-1 in the screening. The most active compound, (E)-2-O-farnesyl chalcone, decreased the rate of oxygen consumption to an extent similar to that of the positive control, nordihydroguaiaretic acid. Additionally, studies on the mechanism of the action indicated that (E)-2-O-farnesyl chalcone is a competitive LOX-1 inhibitor. Molecular modeling studies indicated the importance of the prenyl moieties for the binding of the inhibitors to the LOX binding site, which is related to their pharmacological properties.

Cyclotides from Brazilian Palicourea sessilis and Their Effects on Human Lymphocytes.

Cyclotides are plant-derived peptides found within five families of flowering plants (Violaceae, Rubiaceae, Fabaceae, Solanaceae, and Poaceae) that have a cyclic backbone and six conserved cysteine residues linked by disulfide bonds. Their presence within the Violaceae species seems ubiquitous, yet not all members of other families produce these macrocyclic peptides. The genus Palicourea Aubl. (Rubiaceae) contains hundreds of neotropical species of shrubs and small trees; however, only a few cyclotides have been discovered hitherto. Herein, five previously uncharacterized Möbius cyclotides within Palicourea sessilis and their pharmacological activities are described. Cyclotides were isolated from leaves and stems of this plant and identified as pase A-E, as well as the known peptide kalata S. Cyclotides were de novo sequenced by MALDI-TOF/TOF mass spectrometry, and their structures were solved by NMR spectroscopy. Because some cyclotides have been reported to modulate immune cells, pase A-D were assayed for cell proliferation of human primary activated T lymphocytes, and the results showed a dose-dependent antiproliferative function. The toxicity on other nonimmune cells was also assessed. This study reveals that pase cyclotides have potential for applications as immunosuppressants and in immune-related disorders.

A green and sustainable method for monitoring the chemical composition of soybean: an alternative for quality control.

INTRODUCTION: Soybean is one of the most important crops in the world, an important source of isoflavones, and used to treat various chronic diseases. High-performance liquid chromatography (HPLC), associated with multivariate experiments and green solvents, is increasingly used to develop comprehensive elution methods for quality control of plants and derivatives. OBJECTIVE: The work aims to establish a HPLC fingerprinting method for soybean seeds employing Green Chemistry Principles, a sustainable solvent with low toxicity, and a comprehensive experimental design that reduces the number of experiments. MATERIALS AND METHODS: The fingerprinting method was optimised through Design of Experiments by evaluating seven chromatographic variables: initial percentage of ethanol (X1), final percentage of ethanol (X2), temperature (X3), percentage of acetic acid in water (X4), flow rate (X5), run time (X6), and stationary phase (X7). The dependent variable was the number of peaks (n). RESULTS: An initial factorial design for screening purposes indicated that the most significant quantitative parameters to separate soybean metabolites were X1 and X3. The conditions were optimised by a Doehlert design, to obtain a HPLC-PAD (photodiode array detector) fingerprinting of the polar extract of soybean seeds with the markers identified by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). The optimum fingerprinting method was determined as 5-55% of ethanol in 30 min, at 35°C, and flow rate of 1 mL/min, by employing a phenyl-hexyl column (150 mm × 4.6 mm). CONCLUSION: The developed green method enabled markers of soybean to be separated and identified and could be an eco-friendlier alternative for soybean quality control that covered seven Green Analytical Chemistry Principles.

Can Statistical Evaluation Tools for Chromatographic Method Development Assist in the Natural Products Workflow? A Case Study on Selected Species of the Plant Family Malpighiaceae.

Proper chromatographic methods may reduce the challenges inherent in analyzing natural product extracts, especially when utilizing hyphenated detection techniques involving mass spectrometry. As there are many variations one can introduce during chromatographic method development, this can become a daunting and time-consuming task. To reduce the number of runs and time needed, the use of instrumental automatization and commercial software to apply Quality by Design and statistical analysis automatically can be a valuable approach to investigate complex matrices. To evaluate this strategy in the natural products workflow, a mixture of nine species from the family Malpighiaceae was investigated. By this approach, the entire data collection and method development procedure (comprising screening, optimization, and robustness simulation) was accomplished in only 4 days, resulting in very low limits of detection and quantification. The analysis of the individual extracts also proved the efficiency of the use of a mixture of extracts for this workflow. Molecular networking and library searches were used to annotate a total of 61 compounds, including O-glycosylated flavonoids, C-glycosylated flavonoids, quinic/shikimic acid derivatives, sterols, and other phenols, which were efficiently separated by the method developed. These results support the potential of statistical tools for chromatographic method optimization as an efficient approach to reduce time and maximize resources, such as solvents, to get proper chromatographic conditions.

Discovery of a Beetroot Protease Inhibitor to Identify and Classify Plant-Derived Cystine Knot Peptides.

Plant peptide protease inhibitors are important molecules in seed storage metabolism and to fight insect pests. Commonly they contain multiple disulfide bonds and are exceptionally stable molecules. In this study, a novel peptide protease inhibitor from beetroot (Beta vulgaris) termed bevuTI-I was isolated, and its primary structure was determined via mass spectrometry-based amino acid sequencing. By sequence homology analysis a few peptides with high similarity to bevuTI-I, also known as the Mirabilis jalapa trypsin inhibitor subfamily of knottin-type protease inhibitors, were discovered. Hence, we assessed bevuTI-I for inhibitory activity toward trypsin (IC50 = 471 nM) and human prolyl oligopeptidase (IC50 = 11 µM), which is an emerging drug target for neurodegenerative and inflammatory disorders. Interestingly, using a customized bioinformatics approach, bevuTI-I was found to be the missing link to annotate 243 novel sequences of M. jalapa trypsin inhibitor-like peptides. According to their phylogenetic distribution they appear to be common in several plant families. Therefore, the presented approach and our results may help to discover and classify other plant-derived cystine knot peptides, a class of plant molecules that play important functions in plant physiology and are currently being explored as lead molecules and scaffolds in drug development.

Advanced Glycation End Product Inhibition by Alkaloids from Ocotea paranapiacabensis for the Prevention of Skin Aging.

A bioassay-guided study aiming at identifying inhibitors of the glycation process on the leaves of Ocotea paranapiacabensis afforded four benzylisoquinoline alkaloids (1-4), with 1 and 2 identified as new naturals products, while 3 and 4 were previously described in the literature, with 3 being identified as magnocurarine. Purification was performed by column chromatography and high-performance liquid chromatography. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, NMR, and HRMS. The process of skin aging has been recently associated with advanced glycation end products (AGEs), and strategies inhibiting their formation have been addressed by pharmaceutical companies for the development of novel antiaging compounds. Alkaloids 1-4 were evaluated for their potential to inhibit AGE formation and showed inhibition of 62.9%, 83.3%, 26.1%, and 98.2% (150 µM), respectively. The antiaging potential of compounds 1 and 4 were evaluated with a reconstructed human skin model in vitro, and results showed a decrease in dermis contraction (8.7% and 4.2% respectively for 1 and 4) when compared to the glycated control (57.4%). Additionally, absorption, distribution, metabolism, and excretion (ADME) and toxicity properties were predicted using in silico methods, and the results were considered significantly promising for alkaloids 1 and 4 to continue the development of these alkaloids with skincare properties.

Diterpenoids with inhibitory activity of nitrite production from Croton floribundus.

ETHNOPHARMACOLOGICAL RELEVANCE: Croton floribundus Spreng. (Euphorbiaceae), popularly known as Capixinguí, stands out due to its widespread use in traditional medicine to treat wounds, syphilis, hemorrhoids, eye diseases and as a purgative. AIM OF THE STUDY: To characterize clerodanes diterpenes from C. floribundus and to evaluate the effects of the fraction and diterpenes (1-5) on inhibition of nitrite production. MATERIALS AND METHODS: The hydroethanolic root extract of C. floribundus was fractionated on a solid phase extraction column to obtain the fraction named Fr80%. From this, five compounds were obtained and characterized. The absolute configuration of compound 1 was determined by a combination of electronic and vibrational circular dichroism spectroscopies. Additionally, compounds 1-5 were evaluated for their inhibitory effects on nitrite production induced by lipopolysaccharide (LPS) in RAW 264 macrophage cell. RESULTS: Five clerodane diterpenoids were characterized, and the absolute stereochemistry of 1 was established as 3R,4R,5R,8R,9R,10S,12S. The IC50 values obtained through inhibition of nitrite production were 28.52 ±â€¯2.21 µM (1), 40.26 ±â€¯2.79 µM (2), 25.47 ±â€¯2.16 µM (3), 35.78 ±â€¯2.93 µM (4) and 40.58 ±â€¯4.78 µM (5). In the tested concentrations, the samples presented low toxicity in macrophages. CONCLUSIONS: Four new diterpenes were characterized from C. floribundus, these being croflorins A-D (1-4) and a known halimane (5). These compounds exhibited inhibitory effect on nitrite production.

Natural Products: Perspectives and Challenges for use of Brazilian Plant Species in the Bioeconomy.

The development of our society has been based on the use of biodiversity, especially for nutrition, medicines and beauty. Brazil is the nation with the largest biodiversity in the world, with a rich chemical diversity, which is a potential source for bioeconomy. Considering the chemical and biological diversity of the Brazilian territory, we would like to highlight the value of secondary metabolites from Brazilian biodiversity with potential application for new products and technologies and the importance of scientific programs to support the sustainable use of biodiversity.

Synthesis, Racemic X-ray Crystallographic, and Permeability Studies of Bioactive Orbitides from Jatropha Species.

Orbitides are small cyclic peptides with a diverse range of therapeutic bioactivities. They are produced by many plant species, including those of the Jatropha genus. Here, the objective was to provide new structural information on orbitides to complement the growing knowledge base on orbitide sequences and activities by focusing on three Jatropha orbitides: ribifolin (1), pohlianin C (7), and jatrophidin (12). To determine three-dimensional structures, racemic crystallography, an emerging structural technique that enables rapid crystallization of biomolecules by combining equal amounts of the two enantiomers, was used. The high-resolution structure of ribifolin (0.99 Å) was elucidated from its racemate and showed it was identical to the structure crystallized from its l-enantiomer only (1.35 Å). Racemic crystallography was also used to elucidate high-resolution structures of pohlianin C (1.20 Å) and jatrophidin (1.03 Å), for which there was difficulty forming crystals without using racemic mixtures. The structures were used to interpret membrane permeability data in PAMPA and a Caco-2 cell assay, showing they had poor permeability. Overall, the results show racemic crystallography can be used to obtain high-resolution structures of orbitides and is useful when enantiopure samples are difficult to crystallize or solution structures from NMR are of low resolution.

Inhibition of Breast Cancer Cell Migration by Cyclotides Isolated from Pombalia calceolaria.

Two new bracelet cyclotides from roots of Pombalia calceolaria with potential anticancer activity have been characterized in this work. The cyclotides Poca A and B (1 and 2) and the previously known CyO4 (3) were de novo sequenced by MALDI-TOF/TOF mass spectrometry (MS). The MS2 spectra were examined and the amino acid sequences were determined. The purified peptides were tested for their cytotoxicity and effects on cell migration of MDA-MB-231, a triple-negative breast cancer cell line. The isolated cyclotides reduced the number of cancer cells by more than 80% at 20 µM, and the concentration-related cytotoxic responses were observed with IC50 values of 1.8, 2.7, and 9.8 µM for Poca A (1), Poca B (2), and CyO4 (3), respectively. Additionally, the inhibition of cell migration (wound-healing assay) exhibited that CyO4 (3) presents an interesting activity profile, in being able to inhibit cell migration (50%) at a subtoxic concentration (2 µM). The distribution of these cyclotides in the roots was analyzed by MALDI imaging, demonstrating that all three compounds are present in the phloem and cortical parenchyma regions.

Mass spectrometry for characterization of homologous piperidine alkaloids and their activity as acetylcholinesterase inhibitors.

RATIONALE: Piperidine alkaloids from Senna spectabilis constitute a rare class of natural products with several biological activities. However, the absence of chromophores makes their structural elucidation by conventional methods a great challenge. In this context, mass spectrometry emerges as a powerful tool for metabolomics studies. METHODS: The piperidine alkaloids (-)-cassine and (-)-spectaline and the semisynthetic derivatives (-)-3-O-acetylcassine and (-)-3-O-acetylspectaline were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the positive mode and electron ionization mass spectrometry (EI-MS). ESI fragmentation studies were performed with a quadrupole time-of-flight instrument; N2 was used as collision gas. The acetylcholinesterase inhibitory activity of the investigated compounds was evaluated by bioautography and microplate screening assays. RESULTS: ESI-MS/MS and EI-MS provided valuable and complementary information about the structure of the piperidine compounds. Collision-induced dissociation experiments (MS/MS) revealed that neutral elimination of water or acetic acid is the major fragmentation pathway, which agrees with the stereochemistry proposed for (-)-cassine and (-)-spectaline and the semisynthetic derivatives (-)-3-O-acetylcassine and (-)-3-O-acetylspectaline. CONCLUSIONS: The ESI-MS/MS and EI-MS studies allowed us to propose fragmentation mechanisms for piperidine alkaloids and derivatives. Therefore, mass spectrometry is an important tool for characterizing the structure of these compounds and for supporting further metabolomics studies.

The potential contribution of the natural products from Brazilian biodiversity to bioeconomy.

The development of our society has been based on the use of biodiversity, especially for medicines and nutrition. Brazil is the nation with the largest biodiversity in the world accounting for more than 15% of all living species. The devastation of biodiversity in Brazil is critical and may not only cause the loss of species and genes that encode enzymes involved in the complex metabolism of organisms, but also the loss of a rich chemical diversity, which is a potential source for bioeconomy based on natural products and new synthetic derivatives. Bioeconomy focus on the use of bio-based products, instead of fossil-based ones and could address some of the important challenges faced by society. Considering the chemical and biological diversity of Brazil, this review highlights the Brazilian natural products that were successfully used to develop new products and the value of secondary metabolites from Brazilian biodiversity with potential application for new products and technologies. Additionally, we would like to address the importance of new technologies and scientific programs to support preservation policies, bioeconomy and strategies for the sustainable use of biodiversity.

Biologically Active Orbitides from the Euphorbiaceae Family.

A comprehensive overview of natural orbitides isolated from Euphorbiaceae species and their most relevant biological activities are presented. Euphorbiaceae is a large and diverse family, which comprises about 300 genera, and is known as an important source of medicines and toxins. Several classes of secondary metabolites have been described for this taxon, however, orbitides have been broadly reported in Jatropha and Croton genera. Additionally, the latex is documented as the main source of orbitides in this family. Based on their structural and functional diversity, orbitides present a large variety of biological activities described as cytotoxicity, antimalarial, antibacterial, antifungal, enzymatic inhibition, and immunosuppressive, although the mechanism of action still needs to be further investigated. In recent years, the discovery of bioactive cyclic peptides from different sources has grown exponentially, making them promising molecules in the search for new drug leads. This review also highlights the attempts made by many researchers to organize the orbitides nomenclature and amino acid numbering, as well the important progress recently achieved in the biosynthetic study area.

Dereplication by HPLC-DAD-ESI-MS/MS and Screening for Biological Activities of Byrsonima Species (Malpighiaceae).

INTRODUCTION: Byrsonima species have been used in the treatment of gastrointestinal and gynecological inflammations, skin infections and snakebites. Based on their biological activities, it is important to study other organisms from this genus and to identify their metabolites. OBJECTIVES: To determine the metabolic fingerprinting of methanol and ethyl acetate extracts of four Byrsonima species (B. intermedia, B. coccolobifolia, B. verbascifolia and B. sericea) by HPLC-DAD-ESI-MS/MS and evaluate their in vitro antioxidant, anti-glycation, anti-inflammatory and cytotoxic activities. MATERIALS AND METHODS: Antioxidant activity was determined by DPPH˙, ABTS˙+ and ROO˙ scavenging assays. Anti-glycation activity was evaluated by the ability to inhibit the formation of advanced glycation endproducts (AGEs). Anti-inflammatory activity was evaluated using a murine macrophage cell line (RAW 264-7) in the presence of lipopolysaccharide (LPS). Tumour necrosis factor alpha (TNF-α) and nitrite (NO2- ) production were measured by ELISA and the Griess reaction, respectively. The compounds present in the extracts were tentatively identified by HPLC-DAD-ESI-MS/MS. RESULTS: The evaluation of the biological activities showed the potential of the extracts. The activities were assigned to the presence of glycoside flavonoids mainly derived from quercetin, quinic acid derivatives, gallic acid derivatives, galloylquinic acids and proanthocyanidins. Two isomers of sinapic acid-O-hexoside were described for the first time in a Byrsonima species. CONCLUSION: This research contributes to the study of the genus, it is the first report of the chemical composition of B. sericea and demonstrates the importance of the dereplication process, allowing the identification of known compounds without time-consuming procedures. Copyright © 2017 John Wiley & Sons, Ltd.

A trypsin inhibitor from Moringa oleifera flower extract is cytotoxic to Trypanosoma cruzi with high selectivity over mammalian cells.

In this study, Moringa oleifera flower extract and a trypsin inhibitor (MoFTI) isolated from it were evaluated for anti-protozoal activity against Trypanosoma cruzi and cytotoxicity to mammalian cells. The presence of flavonoids was remarkable in the HPLC fingerprints of the extract at 254 and 360 nm. Amino acid sequences of peptides derived from in-gel digestion of MoFTI were determined. Both the extract and MoFTI caused lysis of T. cruzi trypomastigotes with LC50/24 h of 54.18 ± 6.62 and 41.20 ± 4.28 µg/mL, respectively. High selectivity indices (7.9 to >12) for T. cruzi cells over murine peritoneal macrophages and Vero cells were found for the extract and MoFTI. The results show that MoFTI is a trypanocidal principle of the flower extract.

The potential contribution of the natural products from Brazilian biodiversity to bioeconomy

ABSTRACT The development of our society has been based on the use of biodiversity, especially for medicines and nutrition. Brazil is the nation with the largest biodiversity in the world accounting for more than 15% of all living species. The devastation of biodiversity in Brazil is critical and may not only cause the loss of species and genes that encode enzymes involved in the complex metabolism of organisms, but also the loss of a rich chemical diversity, which is a potential source for bioeconomy based on natural products and new synthetic derivatives. Bioeconomy focus on the use of bio-based products, instead of fossil-based ones and could address some of the important challenges faced by society. Considering the chemical and biological diversity of Brazil, this review highlights the Brazilian natural products that were successfully used to develop new products and the value of secondary metabolites from Brazilian biodiversity with potential application for new products and technologies. Additionally, we would like to address the importance of new technologies and scientific programs to support preservation policies, bioeconomy and strategies for the sustainable use of biodiversity.