Antiprotozoal Activity of Plants Used in the Management of Sleeping Sickness in Angola and Bioactivity-Guided Fractionation of Brasenia schreberi J.F.Gmel and Nymphaea lotus L. Active against T. b. rhodesiense.

Folk medicine is widely used in Angola, even for human African trypanosomiasis (sleeping sickness) in spite of the fact that the reference treatment is available for free. Aiming to validate herbal remedies in use, we selected nine medicinal plants and assessed their antitrypanosomal activity. A total of 122 extracts were prepared using different plant parts and solvents. A total of 15 extracts from seven different plants exhibited in vitro activity (>70% at 20 µg/mL) against Trypanosoma brucei rhodesiense bloodstream forms. The dichloromethane extract of Nymphaea lotus (leaves and leaflets) and the ethanolic extract of Brasenia schreberi (leaves) had IC50 values ≤ 10 µg/mL. These two aquatic plants are of particular interest. They are being co-applied in the form of a decoction of leaves because they are considered by local healers as male and female of the same species, the ethnotaxon "longa dia simbi". Bioassay-guided fractionation led to the identification of eight active molecules: gallic acid (IC50 0.5 µg/mL), methyl gallate (IC50 1.1 µg/mL), 2,3,4,6-tetragalloyl-glucopyranoside, ethyl gallate (IC50 0.5 µg/mL), 1,2,3,4,6-pentagalloyl-ß-glucopyranoside (IC50 20 µg/mL), gossypetin-7-O-ß-glucopyranoside (IC50 5.5 µg/mL), and hypolaetin-7-O-glucoside (IC50 5.7 µg/mL) in B. schreberi, and 5-[(8Z,11Z,14Z)-heptadeca-8,11,14-trienyl] resorcinol (IC50 5.3 µg/mL) not described to date in N. lotus. Five of these active constituents were detected in the traditional preparation. This work provides the first evidence for the ethnomedicinal use of these plants in the management of sleeping sickness in Angola.

Surface sensing triggers a broad-spectrum antimicrobial response in Pseudomonas aeruginosa.

Interspecies bacterial competition may occur via cell-associated or secreted determinants and is key to successful niche colonization. We previously evolved Pseudomonas aeruginosa in the presence of Staphylococcus aureus and identified mutations in the Wsp surface-sensing signalling system. Surprisingly, a ΔwspF mutant, characterized by increased c-di-GMP levels and biofilm formation capacity, showed potent killing activity towards S. aureus in its culture supernatant. Here, we used an unbiased metabolomic analysis of culture supernatants to identify rhamnolipids, alkyl quinoline N-oxides and two siderophores as members of four chemical clusters, which were more abundant in the ΔwspF mutant supernatants. Killing activities were quorum-sensing controlled but independent of c-di-GMP levels. Based on the metabolomic analysis, we formulated a synthetic cocktail of four compounds, showing broad-spectrum anti-bacterial killing, including both Gram-positive and Gram-negative bacteria. The combination of quorum-sensing-controlled killing and Wsp-system mediated biofilm formation endows P. aeruginosa with capacities essential for niche establishment and host colonization.

Metabolomics of Myrcia bella Populations in Brazilian Savanna Reveals Strong Influence of Environmental Factors on Its Specialized Metabolism.

Environmental conditions influence specialized plant metabolism. However, many studies aiming to understand these modulations have been conducted with model plants and/or under controlled conditions, thus not reflecting the complex interaction between plants and environment. To fully grasp these interactions, we investigated the specialized metabolism and genetic diversity of a native plant in its natural environment. We chose Myrcia bella due to its medicinal interest and occurrence in Brazilian savanna regions with diverse climate and soil conditions. An LC-HRMS-based metabolomics approach was applied to analyze 271 samples harvested across seven regions during the dry and rainy season. Genetic diversity was assessed in a subset of 40 samples using amplified fragment length polymorphism. Meteorological factors including rainfall, temperature, radiation, humidity, and soil nutrient and mineral composition were recorded in each region and correlated with chemical variation through multivariate analysis (MVDA). Marker compounds were selected using a statistically informed molecular network and annotated by dereplication against an in silico database of natural products. The integrated results evidenced different chemotypes, with variation in flavonoid and tannin content mainly linked to soil conditions. Different levels of genetic diversity and distance of populations were found to be correlated with the identified chemotypes. These observations and the proposed analytical workflow contribute to the global understanding of the impact of abiotic factors and genotype on the accumulation of given metabolites and, therefore, could be valuable to guide further medicinal exploration of native species.

Innovative omics-based approaches for prioritisation and targeted isolation of natural products - new strategies for drug discovery.

Covering: 2013 to 2019 The exploration of the chemical diversity of extracts from various biological sources has led to major drug discoveries. Over the past two decades, despite the introduction of advanced methodologies for natural product (NP) research (e.g., dereplication and high content screening), successful accounts of the validation of NPs as lead therapeutic candidates have been limited. In this context, one of the main challenges faced is related to working with crude natural extracts because of their complex composition and the inadequacies of classical bioguided isolation studies given the pace of high-throughput screening campaigns. In line with the development of metabolomics, genomics and chemometrics, significant advances in metabolite profiling have been achieved and have generated high-quality massive genome and metabolome data on natural extracts. The unambiguous identification of each individual NP in an extract using generic methods remains challenging. However, the establishment of structural links among NPs via molecular network analysis and the determination of common features of extract composition have provided invaluable information to the scientific community. In this context, new multi-informational-based profiling approaches integrating taxonomic and/or bioactivity data can hold promise for the discovery and development of new bioactive compounds and return NPs back to an exciting era of development. In this article, we examine recent studies that have the potential to improve the efficiency of NP prioritisation and to accelerate the targeted isolation of key NPs. Perspectives on the field's evolution are discussed.

Quantitative CE analysis of punicalagin in Combretum aculeatum extracts traditionally used in Senegal for the treatment of tuberculosis.

Mycobacterium tuberculosis is the causative agent of tuberculosis, an infectious bacterial disease, which most commonly affects the lungs. In the search for novel active compounds or medicines against tuberculosis, an ethnopharmacological survey combined with a host-pathogen assay has recently highlighted the potency of an aqueous extract of Combretum aculeatum. C. aculeatum is used in traditional medicine and has demonstrated a significant in vitro antimycobacterial activity. Punicalagin, an ellagitannin, was isolated and found to be related to the biological activity of the extract. An analytical method for the evaluation of punicalagin in C. aculeatum was developed by capillary electrophoresis. After method optimization, the quantification of punicalagin was achieved for the evaluation of various plant extracts to determine the content of punicalagin related to the extraction modes and conditions, origin of the plant material, and harvesting period. The developed method demonstrated that the leaves presented the highest punicalagin content compared to the seeds and stems. A decoction of 30 min in boiling water was found to be the best extraction mode of C. aculeatum.

Discovery of Lipid Peroxidation Inhibitors from Bacopa Species Prioritized through Multivariate Data Analysis and Multi-Informative Molecular Networking.

A major goal in the discovery of bioactive natural products is to rapidly identify active compound(s) and dereplicate known molecules from complex biological extracts. The conventional bioassay-guided fractionation process can be time consuming and often requires multi-step procedures. Herein, we apply a metabolomic strategy merging multivariate data analysis and multi-informative molecular maps to rapidly prioritize bioactive molecules directly from crude plant extracts. The strategy was applied to 59 extracts of three Bacopa species (B. monnieri, B. caroliniana and B. floribunda), which were profiled by UHPLC-HRMS2 and screened for anti-lipid peroxidation activity. Using this approach, six lipid peroxidation inhibitors 1‒6 of three Bacopa spp. were discovered, three of them being new compounds: monnieraside IV (4), monnieraside V (5) and monnieraside VI (6). The results demonstrate that this combined approach could efficiently guide the discovery of new bioactive natural products. Furthermore, the approach allowed to evidence that main semi-quantitative changes in composition linked to the anti-lipid peroxidation activity were also correlated to seasonal effects notably for B. monnieri.

NF-κB and Angiogenesis Inhibitors from the Aerial Parts of Chresta martii.

The ethyl acetate extract of the aerial parts of Chresta martii showed significant in vitro NF-κB inhibition. Bioactivity-guided isolation was undertaken using HPLC microfractionation to localize the active compounds. Different zones of the HPLC chromatogram were linked to NF-κB inhibition. In parallel to this HPLC-based activity profiling, HPLC-PDA-ESI-MS and UHPLC-TOF-HRMS were used for the early identification of some of the compounds present in the extract and to get a complete phytochemical overview. The isolation of the compounds was performed by high-speed counter-current chromatography and further semipreparative HPLC. Using this approach, 14 compounds were isolated, two of them being new sesquiterpene lactones. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, ECD, NMR, and HRMS. All isolated compounds were evaluated for their inhibitory activity of NF-κB and angiogenesis, and compound 2 showed promising NF-κB inhibition activity with an IC50 of 0.7 µM. The isolated compounds 1, 2, 5, 7, and 8 caused a significant reduction in angiogenesis when evaluated by an original 3D in vitro angiogenesis assay.

Identification and Mode of Action of a Plant Natural Product Targeting Human Fungal Pathogens.

Candida albicans is a major cause of fungal diseases in humans, and its resistance to available drugs is of concern. In an attempt to identify novel antifungal agents, we initiated a small-scale screening of a library of 199 natural plant compounds (i.e., natural products [NPs]). In vitro susceptibility profiling experiments identified 33 NPs with activity against C. albicans (MIC50s ≤ 32 µg/ml). Among the selected NPs, the sterol alkaloid tomatidine was further investigated. Tomatidine originates from the tomato (Solanum lycopersicum) and exhibited high levels of fungistatic activity against Candida species (MIC50s ≤ 1 µg/ml) but no cytotoxicity against mammalian cells. Genome-wide transcriptional analysis of tomatidine-treated C. albicans cells revealed a major alteration (upregulation) in the expression of ergosterol genes, suggesting that the ergosterol pathway is targeted by this NP. Consistent with this transcriptional response, analysis of the sterol content of tomatidine-treated cells showed not only inhibition of Erg6 (C-24 sterol methyltransferase) activity but also of Erg4 (C-24 sterol reductase) activity. A forward genetic approach in Saccharomyces cerevisiae coupled with whole-genome sequencing identified 2 nonsynonymous mutations in ERG6 (amino acids D249G and G132D) responsible for tomatidine resistance. Our results therefore unambiguously identified Erg6, a C-24 sterol methyltransferase absent in mammals, to be the main direct target of tomatidine. We tested the in vivo efficacy of tomatidine in a mouse model of C. albicans systemic infection. Treatment with a nanocrystal pharmacological formulation successfully decreased the fungal burden in infected kidneys compared to the fungal burden achieved by the use of placebo and thus confirmed the potential of tomatidine as a therapeutic agent.

Dibenzofurans and Pseudodepsidones from the Lichen Stereocaulon paschale Collected in Northern Quebec.

Chemical investigation of the methanol extract of the lichen Stereocaulon paschale collected in Nunavik, Canada, led to the isolation and identification of two new dibenzofurans (1 and 3) and 11 known lichen metabolites. The structures of the new compounds were established by analysis of 1D and 2D NMR spectroscopic and high-resolution mass spectrometric data. Herein, the first isolation of ascomatic acid dibenzofuran derivatives (1-3) from a whole lichen organism is reported. In addition, some of the isolated metabolites showed antibacterial activity against the oral pathogens Porphyromonas gingivalis and Streptococcus mutans.

Generation of Antifungal Stilbenes Using the Enzymatic Secretome of Botrytis cinerea.

The protein secretome of Botrytis cinerea was used to perform the biotransformation of resveratrol, pterostilbene, and a mixture of both. Metabolite profiling by UHPLC-HRMS revealed the presence of compounds with unusual molecular formula, suggesting the existence of new products. To isolate these products, the reactions were scaled-up, and 21 analogues were isolated and fully characterized by NMR and HRESIMS analyses. The reaction with pterostilbene afforded five new compounds, while the reaction with a mixture of pterostilbene and resveratrol afforded seven unusual stilbene dimers. The antifungal properties of these compounds were evaluated using in vitro bioassays against Plasmopara viticola. The cytological effects of the isolated antifungal compounds on the ultrastructure of P. viticola were also evaluated.

Nauclea latifolia: biological activity and alkaloid phytochemistry of a West African tree.

Covering up to 2016Nauclea latifolia (syn. Sarcocephalus latifolius, Rubiaceae), commonly called the African pincushion tree, is a plant widely used in folk medicine in different regions of Africa for treating a variety of illnesses, including malaria, epilepsy and pain. N. latifolia has not only drawn the interest of traditional healers but also of phytochemists, who have identified a range of bioactive indole alkaloids in its tissue. More recently, following up on the traditional use of extracts in pain management, a bio-guided purification from the roots of the tree led to the identification of the active ingredient as tramadol, available as a synthetic analgesic since the 1970s. The discovery of this compound as a natural phytochemical was highlighted worldwide. This review focuses on the correlation between extracted compounds and pharmacological activities, paying special attention to infectious diseases and neurologically-related disorders. A critical analysis of the data reported so far on the natural origin of tramadol and its proposed biosynthesis is also presented.

Conjugation of N-acylhydrazone and 1,2,4-oxadiazole leads to the identification of active antimalarial agents.

Malaria, caused by several Plasmodium species, is the major life-threatening parasitic infection worldwide. Due to the parasite resistance to quinoline based drugs, the search for antimalarial agents is necessary. Here, we report the structural design, synthesis and antiparasitic evaluation of two novel series of 1,2,4-oxadiazoles in conjugation to N-acylhydrazones, both groups recognized as privileged structures, as well as the studies on the antimalarial activity of 16 previous described analogues. By varying substituents attached to the phenyl ring, it was possible to retain, enhance or increase the antiparasitic activity in comparison to the nonsubstituted derivatives. Replacement of substituted aryl rings by ferrocenyl and cinnamyl moieties attached in the N-acylhydrazone ablated the antiparasitic response, evidencing the structural features associated with the activity. Active compounds exhibited in vitro potency similar to mefloquine, but not all inhibited ß-hematin formation. Additionally, the active compounds displayed low cytotoxicity in HepG2 cells and did not cause hemolysis in uninfected erythrocytes. In Plasmodium berghei-infected mice, the compounds reduced parasitemia but exhibited limited efficacy in increasing mice survival when compared to chloroquine, suggesting that pharmacological improvement is still necessary.

Targeted Isolation of Indolopyridoquinazoline Alkaloids from Conchocarpus fontanesianus Based on Molecular Networks.

A dichloromethane-soluble fraction of the stem bark of Conchocarpus fontanesianus showed antifungal activity against Candida albicans in a bioautography assay. Off-line high-pressure liquid chromatography activity-based profiling of this extract enabled a precise localization of the compounds responsible for the antifungal activity that were isolated and identified as the known compounds flindersine (17) and 8-methoxyflindersine (18). As well as the identification of the bioactive principles, the ultra-high-pressure liquid chromatography-high-resolution mass spectrometry metabolite profiling of the dichloromethane stem bark fraction allowed the detection of more than 1000 components. Some of these could be assigned putatively to secondary metabolites previously isolated from the family Rutaceae. Generation of a molecular network based on MS(2) spectra indicated the presence of indolopyridoquinazoline alkaloids and related scaffolds. Efficient targeted isolation of these compounds was performed by geometric transfer of the analytical high-pressure liquid chromatography profiling conditions to preparative medium-pressure liquid chromatography. This yielded six new indolopyridoquinazoline alkaloids (5, 16, 19-22) that were assigned structurally. The medium-pressure liquid chromatography separations afforded additionally 16 other compounds. This work has demonstrated the usefulness of molecular networks to target the isolation of new natural products and the value of this approach for dereplication. A detailed analysis of the constituents of the stem bark of C. fontanesianus was conducted.

Standardized LC×LC-ELSD Fractionation Procedure for the Identification of Minor Bioactives via the Enzymatic Screening of Natural Extracts.

To identify natural bioactive compounds from complex mixtures such as plant extracts, efficient fractionation for biological screening is mandatory. In this context, a fully automated workflow based on two-dimensional liquid chromatography (2D-LC × LC) was developed, allowing for the production of hundreds of semipure fractions per extract. Moreover, the ELSD response was used for online sample weight estimation and automated concentration normalization for subsequent bioassays. To evaluate the efficiency of this protocol, an enzymatic assay was developed using AMP-activated protein kinase (AMPK). The activation of AMPK by nonactive extracts spiked with biochanin A, a known AMPK activator, was enhanced greatly when the fractionation workflow was applied compared to screening crude spiked extracts. The performance of the workflow was further evaluated on a red clover (Trifolium pratense) extract, which is a natural source of biochanin A. In this case, while the crude extract or 1D chromatography fractions failed to activate AMPK, semipure fractions containing biochanin A were readily localized when produced by the 2D-LC×LC-ELSD workflow. The automated fractionation methodology presented demonstrated high efficiency for the detection of bioactive compounds at low abundance in plant extracts for high-throughput screening. This procedure can be used routinely to populate natural product libraries for biological screening.

Preparative Scale MS-Guided Isolation of Bioactive Compounds Using High-Resolution Flash Chromatography: Antifungals from Chiloscyphus polyanthos as a Case Study.

In natural product research, the efficient purification of molecules from large amounts of complex extracts is a key element. In this regard, an integrative strategy for efficient MS-guided isolation of antifungal compounds has been developed. First, off-line HPLC antifungal activity-based profiling and HPLC-PDA-MS profiling were used to localize the compounds of interest on the analytical scale. Then, the analytical gradient was geometrically transferred to the flash chromatographic level. Finally, an MS-triggered isolation of the localized bioactive molecules was realized using high-resolution flash chromatographic columns (15 µm spherical particles) coupled to a single quadrupole mass spectrometer via a splitter system. This isolation strategy was applied for the MS-targeted purification of antifungal principles from the liverwort Chiloscyphus polyanthos. This rational methodology has high potential for the targeted large-scale purification of bioactive compounds, avoiding the need to repeat a given bioassay at each isolation step. Seven sesquiterpene lactones were isolated, of which five were found to be bioactive and one was reported as a new compound. The absolute configuration of some compounds was established for the first time by electronic circular dichroism spectroscopy.

Rational and Efficient Preparative Isolation of Natural Products by MPLC-UV-ELSD based on HPLC to MPLC Gradient Transfer.

In natural product research, the isolation of biomarkers or bioactive compounds from complex natural extracts represents an essential step for de novo identification and bioactivity assessment. When pure natural products have to be obtained in milligram quantities, the chromatographic steps are generally labourious and time-consuming. In this respect, an efficient method has been developed for the reversed-phase gradient transfer from high-performance liquid chromatography to medium-performance liquid chromatography for the isolation of pure natural products at the level of tens of milligrams from complex crude natural extracts. The proposed method provides a rational way to predict retention behaviour and resolution at the analytical scale prior to medium-performance liquid chromatography, and guarantees similar performances at both analytical and preparative scales. The optimisation of the high-performance liquid chromatography separation and system characterisation allows for the prediction of the gradient at the medium-performance liquid chromatography scale by using identical stationary phase chemistries. The samples were introduced in medium-performance liquid chromatography using a pressure-resistant aluminium dry load cell especially designed for this study to allow high sample loading while maintaining a maximum achievable flow rate for the separation. The method has been validated with a mixture of eight natural product standards. Ultraviolet and evaporative light scattering detections were used in parallel for a comprehensive monitoring. In addition, post-chromatographic mass spectrometry detection was provided by high-throughput ultrahigh-performance liquid chromatography time-of-flight mass spectrometry analyses of all fractions. The processing of all liquid chromatography-mass spectrometry data in the form of an medium-performance liquid chromatography x ultra high-performance liquid chromatography time-of-flight mass spectrometry matrix enabled an efficient localisation of the compounds of interest in the generated fractions. The methodology was successfully applied for the separation of three different plant extracts that contain many diverse secondary metabolites. The advantages and limitations of this approach and the theoretical chromatographic background that rules such as liquid chromatography gradient transfer are presented from a practical viewpoint.

Dimeric flavonoids from Arrabidaea brachypoda and assessment of their anti-Trypanosoma cruzi activity.

The nonpolar fraction of an aqueous ethanol extract of the roots of Arrabidaea brachypoda, a Brazilian medicinal plant, demonstrated significant in vitro activity against Trypanosoma cruzi, the parasite responsible for Chagas disease. Targeted isolation of the active constituents led to the isolation of three new dimeric flavonoids (1-3), and their structures were elucidated using UV, NMR, and HRMS analysis, as well as by chemical derivatization. The anti-T. cruzi activity and cytotoxicity toward mammalian cells were determined for these substances. Compound 1 exhibited no activity toward T. cruzi, while flavonoids 2 and 3 exhibited selective activity against these trypomastigotes. Compounds 2 and 3 inhibited the parasite invasion process and its intracellular development in host cells with similar potencies to benznidazole. In addition, compound 2 reduced the blood parasitemia of T. cruzi-infected mice. This study has revealed that these two dimeric flavonoids represent potential anti-T. cruzi lead compounds for further drug development.

Chemical composition of the bark of Tetrapterys mucronata and identification of acetylcholinesterase inhibitory constituents.

The secondary metabolite content of Tetrapterys mucronata, a poorly studied plant that is used occasionally in Brazil for the preparation of a psychotropic plant decoction called "Ayahuasca", was determined to establish its chemical composition and to search for acetylcholinesterase (AChE) inhibitors. The ethanolic extract of the bark of T. mucronata exhibited in vitro AChE inhibition in a TLC bioautography assay. To localize the active compounds, biological profiling for AChE inhibition was performed using at-line HPLC-microfractionation in 96-well plates and subsequent AChE inhibition bioautography. The analytical HPLC-PDA conditions were transferred geometrically to a preparative medium-pressure liquid chromatography column using chromatographic calculations for the efficient isolation of the active compounds at the milligram scale. Twenty-two compounds were isolated, of which six are new natural products. The structures of the new compounds (9, 10, 16-18, and 20) were elucidated by spectroscopic data interpretation. Compounds 1, 5, 6, 9, and 10 inhibited AChE with IC50 values below 15 µM.

Study of phenoxy radical couplings using the enzymatic secretome of Botrytis cinerea.

Phenoxy radical coupling reactions are widely used in nature for the synthesis of complex molecules such as lignin. Their use in the laboratory has great potential for the production of high value compounds from the polyphenol family. While the enzymes responsible for the generation of the radicals are well known, the behavior of the latter is still enigmatic and difficult to control in a reaction flask. Previous work in our laboratory using the enzymatic secretome of B. cinerea containing laccases has shown that incubation of stilbenes leads to dimers, while incubation of phenylpropanoids leads to dimers as well as larger coupling products. Building on these previous studies, this paper investigates the role of different structural features in phenoxy radical couplings. We first demonstrate that the presence of an exocyclic conjugated double bond plays a role in the generation of efficient reactions. In addition, we show that the formation of phenylpropanoid trimers and tetramers can proceed via a decarboxylation reaction that regenerates this reactive moiety. Lastly, this study investigates the reactivity of other phenolic compounds: stilbene dimers, a dihydro-stilbene, a 4-O-methyl-stilbene and a simple phenol with the enzymatic secretome of B. cinerea. The observed efficient dimerization reactions consistently correlate with the presence of a para-phenol conjugated to an exocyclic double bond. The absence of this structural feature leads to variable results, with some compounds showing low conversion or no reaction at all. This research has allowed the development of a controlled method for the synthesis of specific dimers and tetramers of phenylpropanoid derivatives and novel stilbene derivatives, as well as an understanding of features that can promote efficient radical coupling reactions.