High-Resolution Liquid Chromatography-Mass Spectrometry-Based Metabolomics for the Classification of Chuquiraga (Barnadesioideae, Asteraceae): New Phenylpropanoid Derivatives as Chemical Markers for Chuquiraga spinosa.

Despite their relatively poorly investigated phytochemistry, species of the genus Chuquiraga are widely commercialized. The present study reports the use of a high-resolution liquid chromatography-mass spectrometry-based metabolomics approach coupled with exploratory and supervised multivariate statistical analyses for species classification and chemical marker identification of four species of Chuquiraga (C. jussieui, C. weberbaueri, C. spinosa, and Chuquiraga sp.) from Ecuador and Peru. Based on these analyses, a high percentage of correct classifications (87% to 100%) allowed the prediction of the taxonomic identity of Chuquiraga species. Through the metabolite selection process, several key constituents with the potential to be chemical markers were identified. Samples of C. jussieui displayed alkyl glycosides and triterpenoid glycosides as discriminating metabolites, while Chuquiraga sp. displayed high concentrations of p-hydroxyacetophenone, p-hydroxyacetophenone 4-O-glucoside, p-hydroxyacetophenone 4-O-(6-O-apiosyl)-glucoside, and quinic acid ester derivatives as the main metabolites. Caffeic acid was characteristic for C. weberbaueri samples, whereas C. spinosa displayed higher concentrations of the following new phenylpropanoid ester derivatives: 2-O-caffeoyl-4-hydroxypentanedioic acid (24), 2-O-p-coumaroyl-4-hydroxypentanedioic acid (34), 2-O-feruloyl-4-hydroxypentanedioic acid (46), 2,4-O-dicaffeoylpentanedioic acid (71), and 2-O-caffeoyl-4-O-feruloylpentanedioic acid (77).

Metabolomic and Gene Expression Studies Reveal the Diversity, Distribution and Spatial Regulation of the Specialized Metabolism of Yacón (Smallanthus sonchifolius, Asteraceae).

Smallanthus sonchifolius, also known as yacón, is an Andean crop species commercialized for its nutraceutical and medicinal properties. The tuberous roots of yacón accumulate a diverse array of probiotic and bioactive metabolites including fructooligosaccharides and caffeic acid esters. However, the metabolic diversity of yacón remains unexplored, including the site of biosynthesis and accumulation of key metabolite classes. We report herein a multidisciplinary approach involving metabolomics, gene expression and scanning electron microscopy, to provide a comprehensive analysis of the diversity, distribution and spatial regulation of the specialized metabolism in yacón. Our results demonstrate that different metabolic fingerprints and gene expression patterns characterize specific tissues, organs and cultivars of yacón. Manual inspection of mass spectrometry data and molecular networking allowed the tentative identification of 71 metabolites, including undescribed structural analogues of known bioactive compounds. Imaging by scanning electron microscopy revealed the presence of a new type of glandular trichome in yacón bracts, with a distinctive metabolite profile. Furthermore, the high concentration of sesquiterpene lactones in capitate glandular trichomes and the restricted presence of certain flavonoids and caffeic acid esters in underground organs and internal tissues suggests that these metabolites could be involved in protective and ecological functions. This study demonstrates that individual organs and tissues make specific contributions to the highly diverse and specialized metabolome of yacón, which is proving to be a reservoir of previously undescribed molecules of potential significance in human health.

Untargeted LC-MS metabolomic studies of Asteraceae species to discover inhibitors of Leishmania major dihydroorotate dehydrogenase.

INTRODUCTION: Interesting data about the family Asteraceae as a new source of Leishmania major dihydroorotate dehydrogenase (LmDHODH) inhibitors are presented. This key macromolecular target for parasites causing neglected diseases catalyzes the fourth reaction of the de novo pyrimidine biosynthetic pathway, which takes part in major cell functions, including DNA and RNA biosynthesis. OBJECTIVES: We aimed to (1) determine LmDHODH inhibitor candidates, revealing the type of chemistry underlying such bioactivity, and (2) predict the inhibitory potential of extracts from new untested plant species, classifying them as active or inactive based on their LC-MS based metabolic fingerprints. METHODS: Extracts from 150 species were screened for the inhibition of LmDHODH, and untargeted UHPLC-(ESI)-HRMS metabolomic studies were carried out in combination with in silico approaches. RESULTS: The IC50 values determined for a subset of 59 species ranged from 148 µg mL-1 to 9.4 mg mL-1. Dereplication of the metabolic fingerprints allowed the identification of 48 metabolites. A reliable OPLS-DA model (R2 > 0.9, Q2 > 0.7, RMSECV < 0.3) indicated the inhibitor candidates; nine of these metabolites were identified using data from isolated chemical standards, one of which-4,5-di-O-E-caffeoylquinic acid (IC50 73 µM)-was capable of inhibiting LmDHODH. The predictive OPLS model was also effective, with 60% correct predictions for the test set. CONCLUSION: Our approach was validated for (1) the discovery of LmDHODH inhibitors or interesting starting points for the optimization of new leishmanicides from Asteraceae species and (2) the prediction of extracts from untested species, classifying them as active or inactive.

Investigation of the Anti-Leishmania (Leishmania) infantum Activity of Some Natural Sesquiterpene Lactones.

Leishmaniases are neglected infectious diseases caused by parasites of the 'protozoan' genus Leishmania. Depending on the parasite species, different clinical forms are known as cutaneous, muco-cutaneous, and the visceral leishmaniasis (VL). VL is particularly fatal and the therapy presents limitations. In the search for new anti-leishmanial hit compounds, seven natural sesquiterpene lactones were evaluated against promastigotes and intracellular amastigotes of Leishmania (Leishmania) infantum, a pathogen causing VL. The pseudoguaianolides mexicanin I and helenalin acetate demonstrated the highest selectivity and potency against intracellular amastigotes. In addition, promastigotes treated with helenalin acetate were subject to an ultrastructural and biochemical investigation. The lethal action of the compound was investigated by fluorescence-activated cell sorting and related techniques to detect alterations in reactive oxygen species (ROS) content, plasma membrane permeability, and mitochondrial membrane potential. Helenalin acetate significantly reduced the mitochondrial membrane potential and the mitochondrial structural damage was also confirmed by transmission electron microscopy, displaying an intense organelle swelling. No alteration of plasma membrane permeability or ROS content could be detected. Additionally, helenalin acetate significantly increased the production of nitric oxide in peritoneal macrophages, probably potentiating the activity against the intracellular amastigotes. Helenalin acetate could hence be a useful anti-leishmanial scaffold for further optimization studies.

ent-Pimarane and ent-Kaurane Diterpenes from Aldama discolor (Asteraceae) and Their Antiprotozoal Activity.

Aldama discolor (syn.Viguiera discolor) is an endemic Asteraceae from the Brazilian "Cerrado", which has not previously been investigated for its chemical constituents and biological activity. Diterpenes are common secondary metabolites found in Aldama species, some of which have been reported to present potential antiprotozoal and antimicrobial activities. In this study, the known ent-3-α-hydroxy-kaur-16-en-18-ol (1), as well as three new diterpenes, namely, ent-7-oxo-pimara-8,15-diene-18-ol (2), ent-2S,4S-2-19-epoxy-pimara-8(3),15-diene-7ß-ol (3) and ent-7-oxo-pimara-8,15-diene-3ß-ol (4), were isolated from the dichloromethane extract of A. discolor leaves and identified by means of MS and NMR. The compounds were assayed in vitro against Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani, Plasmodium falciparum and also tested for cytotoxicity against mammalian cells (L6 cell line). The ent-kaurane 1 showed significant in vitro activity against both P. falciparum (IC 50 = 3.5 µ M) and L. donovani (IC 50 = 2.5 µ M) and ent-pimarane 2 against P. falciparum (IC 50 = 3.8 µ M). Both compounds returned high selectivity indices (SI >10) in comparison with L6 cells, which makes them interesting candidates for in vivo tests. In addition to the diterpenes, the sesquiterpene lactone budlein A (5), which has been reported to possess a strong anti-T. b. rhodesiense activity, was identified as major compound in the A. discolor extract and explains its high activity against this parasite (100% growth inhibition at 2 µ g/mL).

Study of chromatographic retention of natural terpenoids by chemoinformatic tools.

The study of chromatographic retention of natural products can be used to increase their identification speed in complex biological matrices. In this work, six variables were used to study the retention behavior in reversed phase liquid chromatography of 39 sesquiterpene lactones (SL) from an in-house database using chemoinformatics tools. To evaluate the retention of the SL, retention parameters on an ODS C-18 column in two different solvent systems were experimentally obtained, namely, MeOH-H2O 55:45 and MeCN-H2O 35:75. The chemoinformatics approach involved three descriptor type sets (one 2D and two 3D) comprising three groups of each (four, five, and six descriptors), two different training and test sets, four algorithms for variable selection (best first, linear forward, greedy stepwise, and genetic algorithm), and two modeling methods (partial least-squares regression and back-propagation artificial neural network). The influence of the six variables used in this study was assessed in a holistic context, and influences on the best model for each solvent system were analyzed. The best set for MeOH-H2O showed acceptable correlation statistics with training R(2) = 0.91, cross-validation Q(2) = 0.88, and external validation P(2) = 0.80, and the best MeCN-H2O model showed much higher correlation statistics with training R(2) = 0.96, cross-validation Q(2) = 0.92, and external validation P(2) = 0.91. Consensus models were built for each chromatographic system, and although all of them showed an improved statistical performance, only one for the MeCN-H2O system was able to separate isomers as well as to improve the performance. The approach described herein can therefore be used to generate reproducible and robust models for QSRR studies of natural products as well as an aid for dereplication of complex biological matrices using plant metabolomics-based techniques.

In Silico prediction and experimental evaluation of furanoheliangolide sesquiterpene lactones as potent agents against Trypanosoma brucei rhodesiense.

As a continuation of our earlier study on the in vitro antiprotozoal activity of 40 natural sesquiterpene lactones (STLs), we extended the set of tested compounds from our laboratories to 59. On the basis of this extended data set, further enriched by literature data for 10 compounds tested under the same conditions, our quantitative structure-activity relationship (QSAR) analyses for activity against T. brucei rhodesiense (etiologic agent of human African trypanosomiasis, or sleeping sickness) were continued, and the QSAR model thus obtained with 69 structures was used to predict the activity of a virtual library of 1,750 STL structures. As a major result from these calculations, furanoheliangolide-type compounds, a subclass of STLs hitherto untested against T. brucei rhodesiense, were predicted to have an exceptionally high level of in vitro activity. Four representative compounds of this type, goyazensolide, 4,5-dihydro-2',3'-epoxy-15-deoxygoyazensolide, budlein A, and 4,15-isoatriplicolide tiglate, were therefore tested. They displayed 50% inhibitory concentrations (IC50s) of 0.07, 0.20, 0.07, and 0.015 µM, respectively, so that the in silico prediction was experimentally confirmed. 4,15-Isoatriplicolide tiglate is the most potent STL against T. b. rhodesiense found. Furanoheliangolide STLs were thus identified as interesting leads against this parasite which deserve more detailed investigations.

In vitro leishmanicidal activities of sesquiterpene lactones from Tithonia diversifolia against Leishmania braziliensis promastigotes and amastigotes.

Natural compounds represent a rich and promising source of novel, biologically active chemical entities for treating leishmaniasis. Sesquiterpene lactones are a recognized class of terpenoids with a wide spectrum of biological activities, including activity against Leishmania spp. In this work, a sesquiterpene lactone-rich preparation-a leaf rinse extract (LRE) from Tithonia diversifolia-was tested against promastigote forms of L. braziliensis. The results revealed that the LRE is a rich source of potent leishmanicidal compounds, with an LD50 value 1.5 ± 0.50 µg·mL-1. Therefore, eight sesquiterpene lactones from the LRE were initially investigated against promastigote forms of L. braziliensis. One of them did not present any significant leishmanicidal effect (LD50 > 50 µg·mL-1). Another had a cytotoxic effect against macrophages (4.5 µg·mL-1). The five leishmanicidal compounds with the highest level of selectivity were further evaluated against intracellular parasites (amastigotes) using peritoneal macrophages. Tirotundin 3-O-methyl ether, tagitinin F, and a guaianolide reduced the internalization of parasites after 48 h, in comparison with the negative control. This is the first report on sesquiterpene lactones that have potent leishmanicidal effects on both developmental stages of L. braziliensis.

Therapeutic potential of biodegradable microparticles containing Punica granatum L. (pomegranate) in murine model of asthma.

OBJECTIVE AND DESIGN: Among the options for treatment of diseases affecting the respiratory system, especially asthma, drug delivering systems for intranasal application represent an important therapeutic approach at the site of inflammation. The present study aimed to evaluate the therapeutic effect of biodegradable microparticles formed by poly lactic-co-glycolic acid (PLGA) containing encapsulated pomegranate extract on a murine model of asthma. MATERIAL: The extract was acquired from the leaves of P. granatum and characterized qualitatively by HPLC. A w/o/w emulsion solvent extraction-evaporation method was chosen to prepare the microparticles containing pomegranate encapsulated extract (MP). TREATMENT: OVA-sensitized BALB/c mice were used as asthma model and treated with dexamethasone and P. granatum extract in solution form or encapsulated into microparticles. RESULTS: MP were able to inhibit leukocytes' recruitment to bronchoalveolar fluid, especially, eosinophils, decreasing cytokines (IL-1ß and IL-5) and protein levels in the lungs. CONCLUSIONS: This approach can be used as an alternative/supplementary therapy based on the biological effects of P. granatum for managing inflammatory processes, especially those with pulmonary complications.

Ethnobotany, chemistry, and biological activities of the genus Tithonia (Asteraceae).

The genus Tithonia is an important source of diverse natural products, particularly sesquiterpene lactones, diterpenes, and flavonoids. The collected information in this review attempts to summarize the recent developments in the ethnobotany, biological activities, and secondary metabolite chemistry of this genus. More than 100 structures of natural products from Tithonia are reported in this review. The species that has been most investigated in this genus is T. diversifolia, from which ca. 150 compounds were isolated. Biological studies are described to evaluate the anti-inflammatory, analgesic, antimalarial, antiviral, antidiabetic, antidiarrhoeal, antimicrobial, antispasmodic, vasorelaxant, cancer-chemopreventive, cytotoxic, toxicological, bioinsecticide, and repellent activities. A few of these studies have been carried out with isolated compounds from Tithonia species, but the majority has been conducted with different extracts. The relationship between the biological activity and the toxicity of compounds isolated from the plants of this genus as well as T. diversifolia extracts still remains unclear, and mechanisms of action remain to be determined.

Antimicrobial activity of diterpenes from Viguiera arenaria against endodontic bacteria.

Six pimarane-type diterpenes isolated from Viguiera arenaria Baker and two semi-synthetic derivatives were evaluated in vitro against a panel of representative microorganisms responsible for dental root canal infections. The microdilution method was used for the determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia, Prevotella buccae, Fusobacterium nucleatum, Bacteroides fragilis, Actinomyces naeslundii, Actinomyces viscosus, Peptostreptococcus micros, Enterococcus faecalis and Aggregatibacter actinomycetemcomitans. The compounds ent-pimara-8(14),15-dien-19-oic acid, its sodium salt and ent-8(14),15-pimaradien-3ß-ol were the most active, displaying MIC values ranging from 1 to 10 µg mL-1. The results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new chemicals for use as a complement to instrumental endodontic procedures.

Effect of the Andean Geography and Climate on the Specialized Metabolism of Its Vegetation: The Subtribe Espeletiinae (Asteraceae) as a Case Example.

The Andean mountains are 'center stage' to some of the most spectacular examples of plant diversifications, where geographic isolation and past climatic fluctuations have played a major role. However, the influence of Andean geography and climate as drivers of metabolic variation in Andean plants is poorly elucidated. Here, we studied the influence of those factors on the metabolome of the subtribe Espeletiinae (Asteraceae) using liquid chromatography coupled to high-resolution mass spectrometry data of over two hundred samples from multiple locations. Our results demonstrate that metabolic profiles can discriminate Espeletiinae taxa at different geographic scales, revealing inter- and intraspecific metabolic variations: at the country level, segregation between Colombian and Venezuelan taxa was observed; regionally, between páramo massifs; and locally, between páramo complexes. Metabolic differences in Espeletiinae were mainly explained by geographic isolation, although differences in taxonomic genera, temperature, and elevation, were also important. Furthermore, we found that different species inhabiting the same páramo complex showed stronger chemical similarities than the same species at different locations, corroborating that geographic isolation represents the main driver of metabolic change in Espeletiinae. The current study serves as a starting point to fill in the gaps in how Andean geography and climate have shaped the metabolism of its vegetation and reveal the potential of untargeted metabolomics to study the environmental physiology of plants.

Pimarane-type diterpenes: antimicrobial activity against oral pathogens.

Seven pimarane type-diterpenes re-isolated from Viguiera arenaria Baker and two semi-synthetic pimarane derivatives were evaluated in vitro against the following main microorganisms responsible for dental caries: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis and Lactobacillus casei. The compounds ent-pimara-8(14),15-dien-19-oic acid (PA); ent-8(14),15-pimaradien-3beta-ol; ent-15-pimarene-8beta,19-diol; ent-8(14),15-pimaradien-3beta-acetoxy and the sodium salt derivative of PA were the most active compounds, displaying MIC values ranging from 2 to 8 microg mL(-1). Thus, this class of compounds seems promising as a class of new effective anticariogenic agents. Furthermore, our results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new natural compounds that could be employed in the development of oral care products.

Metabolomic and gene expression approaches reveal the developmental and environmental regulation of the secondary metabolism of yacón (Smallanthus sonchifolius, Asteraceae).

Acting as chemical defense or signaling compounds, secondary metabolites (SMs) play an essential role in the evolutionary success of many angiosperm plant families. However, the adaptive advantages that SMs confer, and the influence of environmental and developmental factors on SMs expression, remains poorly understood. A study of taxa endemic to the variable Andean climate, using a metabolomics approach, may provide further insight. By analyzing gene expression patterns and metabolic fingerprints, we report herein the developmental and environmental regulation of the secondary metabolism of Smallanthus sonchifolius (yacón), a medicinal Andean plant. Our results demonstrate a clear developmental stage dependent regulation of the secondary metabolism of yacón leaves wherein the metabolic diversity increases with plant age. However, environmental factors seem to regulate biosynthetic pathways, creating differences in the expression of chemical classes, pointing to an association between transcription levels of relevant genes and the relative amounts of more than 40 different metabolites. This study suggests that the secondary metabolism of yacón is regulated by a complex interplay between environmental factors and developmental stage and provides insight into the regulatory factors and adaptive roles of SMs in Andean taxa.

Pimaradienoic acid inhibits vascular contraction and induces hypotension in normotensive rats.

The present investigation was designed to investigate the effect of the diterpene ent-pimara-8(14),15-dien-19-oic acid (pimaradienoic acid, PA) on smooth muscle extracellular Ca(2+) influx. To this end, the effect of PA on phenylephrine- and KCl-induced increases in cytosolic calcium concentration ([Ca(2+)](c)), measured by the variation in the ratio of fluorescence intensities (R340/380 nm) of Fura-2, was analysed. Whether bolus injection of PA could induce hypotensive responses in conscious normotensive rats was also evaluated. PA inhibited the contraction induced by phenylephrine (0.03 or 10 micromol L(-1)) and KCl (30 or 90 mmol L(-1)) in endothelium-denuded rat aortic rings in a concentration dependent manner. Pre-treatment with PA (10, 100, 200 micromol L(-1)) attenuated the contraction induced by CaCl(2) (0.5 nmol L(-1) or 2.5 mmol L(-1)) in denuded rat aorta exposed to Ca(2+)-free medium containing phenylephrine (0.1 micro mol L(-1)) or KCl (30 mmol L(-1)). Interestingly, the inhibitory effect displayed by PA on CaCl(2)-induced contraction was more pronounced when KCl was used as the stimulant. Phenylephrine- and KCl-induced increases in [Ca(2+)](c) were inhibited by PA. Similarly, verapamil, a Ca(2+)-channel blocker, also inhibited the increase in [Ca(2+)](c) induced by either phenylephrine or KCl. Finally, bolus injection of PA (1-15 mg kg(-1)) produced a dose-dependent decrease in mean arterial pressure in conscious normotensive rats. The results provide the first direct evidence that PA reduces vascular contractility by reducing extracellular Ca(2+) influx through smooth muscle cellular membrane, a mechanism that could mediate the hypotensive response induced by this diterpene in normotensive rats.

Trypanocidal activity of pimarane diterpenes from Viguiera arenaria (Asteraceae).

Five structurally related pimarane diterpenes isolated from the roots of Viguiera arenaria and a further compound obtained by chemical derivatization were evaluated in vitro against the trypomastigote forms of Trypanosoma cruzi. The natural compound ent-15-pimarene-8 beta,19-diol and the derivative ent-8(14),15-pimaradiene-3beta-acetoxy showed the highest trypanocidal activity, displaying IC(50) values of 116.5 +/- 1.21 and 149.3 +/- 1.07 microM, respectively, while the positive control, violet gentian, showed an IC(50) of 76 microM. Based on the results, it can be concluded that minor structural differences among the tested diterpenes influence significantly the trypanocidal activity, thus bringing new perspectives to the establishment of structure-activity relationships among this type of metabolites to the treatment of Chagas' disease.

Further sesquiterpene lactones from Viguiera robusta and the potential anti-inflammatory activity of a heliangolide: inhibition of human neutrophil elastase release.

In addition to known heliangolides, a new eudesmanolide was isolated from the leaf rinse extract of Viguiera robusta (Asteraceae). Structural elucidation was based on spectral analysis. It is the first report on eudesmanolides in members of the subgenus Calanticaria of Viguiera. In this work, the main isolated compound, the furanoheliangolide budlein A, besides its previously reported in vitro and in vivo anti-inflammatory activities, inhibited human neutrophil elastase release. The inhibition was at the concentration of (16.83 +/- 1.96) microM for formylated bacterial tripeptide (fMLP) stimulation and (11.84 +/- 1.62) microM for platelet aggregation factor (PAF) stimulation, being slightly less active than the reference drug parthenolide. The results are important to demonstrate the potential anti-inflammatory activities of sesquiterpene lactones and corroborate the previous studies using other targets.

Antimicrobial activity of kaurane diterpenes against oral pathogens.

Two kaurane diterpenes, ent-kaur-16(17)-en-19-oic acid (KA) and 15-beta-isovaleryloxy-ent-kaur-16(17)-en-19-oic acid (KA-Ival), isolated from Aspilia foliacea, and the methyl ester derivative of KA (KA-Me) were evaluated against oral pathogens. KA was the most active compound, with MIC values of 10 microg mL(-1) against the following microorganisms: Streptococcus sobrinus, Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, and Lactobacillus casei. However, KA did not show significant activity against Streptococcus salivarius and Enterococcus faecalis, with MIC values equal to 100 and 200 microg mL(-1), respectively. Our results show that KA has potential to be used as a prototype for the discovery of new effective anti-infection agents against microorganisms responsible for caries and periodontal diseases. Moreover, these results allow to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to studies on the structure-activity relationship of this type of metabolites with respect to caries and periodontal diseases.

Natural products as inhibitors of Leishmania major dihydroorotate dehydrogenase.

The flavoenzyme dihydroorotate dehydrogenase (DHODH) catalyzes the fourth reaction of the de novo pyrimidine biosynthetic pathway, which exerts vital functions in the cells, especially within DNA and RNA biosynthesis. Thus, this enzyme stands out as a new key molecular target for parasites causing Neglected Diseases (NDs). Focused on contributing to the development of new therapeutic alternatives for NDs, in this study, for the first time, a screening of 57 natural products for in vitro inhibition of Leishmania major DHODH (LmDHODH) was carried out, including cross validation against the human DHODH (HsDHODH). A subset of natural products consisting of 21 sesquiterpene lactones (STLs) was submitted to QSAR studies. Additionally, thermostability studies by differential scanning fluorimetry (DSF) were performed to determine whether the STLs are effectively or not binding to the enzyme. The IC50 values against LmDHODH varied from 27 to 1200 µM; only irrelevant inhibition was obtained on HsDHODH. DSF assays confirmed binding of STLs to LmDHODH; moreover, it is suggested that such inhibitors might act in a different site other than the active site. A reliable QSAR model based on molecular descriptors was obtained (R2: 0.83; Q2CV: 0.69 and Q2EXT/F2: 0.66) indicating that stronger inhibition requires a balanced distribution of the hydrophobic regions across the molecular surface, as well as higher width and lower hydrophobicity of the molecules. A pharmacophore-based 3D-QSAR approach also afforded a useful model (R2: 0.72; Q2CV: 0.50 and Q2EXT/F2: 0.62), which confirmed the importance of proper orientation of the ligands, molecular surface features and shape for stronger inhibition, reflecting properties of a putative common binding site. These data indicated for the first time that natural products can actually inhibit LmDHODH and highlighted some metabolites as potentially interesting starting points for the discovery of more potent LmDHODH inhibitors, ultimately aiming at new effective therapeutic alternatives for leishmaniasis and, possibly, other NDs caused by trypanosomatids.

Budlein A, a Sesquiterpene Lactone From Viguiera robusta, Alleviates Pain and Inflammation in a Model of Acute Gout Arthritis in Mice.

Background: Gout is the most common inflammatory arthritis worldwide. It is a painful inflammatory disease induced by the deposition of monosodium urate (MSU) crystals in the joints and peri-articular tissues. Sesquiterpene lactones (SLs) are secondary metabolite biosynthesized mainly by species from the family Asteraceae. It has been demonstrated that SLs present anti-inflammatory, analgesic, antitumoral, antiparasitic, and antimicrobial activities. In this study, we aimed at evaluating the efficacy of the SL budlein A in a model of acute gout arthritis in mice. Methods: Experiments were conducted in male Swiss or male LysM-eGFP mice. Animals were treated with budlein A (1 or 10 mg/kg) or vehicle 30 min before stimulus with MSU (100 µg/10 µL, intra-articular). Knee joint withdrawal threshold and edema were evaluated using electronic von Frey and caliper, respectively, 1-15 h after MSU injection. Leukocyte recruitment was determined by counting cells (Neubauer chamber), H&E staining, and using LysM-eGFP mice by confocal microscopy. Inflammasome components, Il-1ß, and Tnf-α mRNA expression were determined by RT-qPCR. IL-1ß and TNF-α production (in vitro) and NF-κB activation (in vitro and in vivo) were evaluated by ELISA. In vitro analysis using LPS-primed bone marrow-derived macrophages (BMDMs) was performed 5 h after stimulation with MSU crystals. For these experiments, BMDMs were either treated or pre-treated with budlein A at concentrations of 1, 3, or 10 µg/mL. Results: We demonstrated that budlein A reduced mechanical hypersensitivity and knee joint edema. Moreover, it reduced neutrophil recruitment, phagocytosis of MSU crystals by neutrophils, and Il-1ß and Tnf-α mRNA expression in the knee joint. In vitro, budlein A decreased TNF-α production, which might be related to the inhibition of NF-κB activation. Furthermore, budlein A also reduced the IL-1ß maturation, possibly by targeting inflammasome assembly in macrophages. Conclusion: Budlein A reduced pain and inflammation in a model of acute gout arthritis in mice. Therefore, it is likely that molecules with the ability of targeting NF-κB activation and inflammasome assembly, such as budlein A, are interesting approaches to treat gout flares.