GC-MS-based Metabolomics Unravels Metabolites across Larval Development and Diapause of a Specialist Insect.

Plant-insect interactions are a driving force into ecosystem evolution and community dynamics. Many insect herbivores enter diapause, a developmental arrest stage in anticipation of adverse conditions, to survive and thrive through seasonal changes. Herein, we investigated the roles of medium- to non-polar metabolites during larval development and diapause in a specialist insect herbivore, Chlosyne lacinia, reared on Aldama robusta leaves. Varying metabolites were determined using gas chromatography-mass spectrometry (GC-MS)-based metabolomics. Sesquiterpenes and steroids were the main metabolites putatively identified in A. robusta leaves, whereas C. lacinia caterpillars were characterized by triterpenes, steroids, fatty acids, and long-chain alkanes. We found out that C. lacinia caterpillars biosynthesized most of the identified steroids and fatty acids from plant-derived ingested metabolites, as well as all triterpenes and long-chain alkanes. Steroids, fatty acids, and long-chain alkanes were detected across all C. lacinia instars and in diapausing caterpillars. Sesquiterpenes and triterpenes were also detected across larval development, yet they were not detected in diapausing caterpillars, which suggested that these metabolites were converted to other molecules prior to the diapause stage. Our findings shed light on the chemical content variation across C. lacinia development and diapause, providing insights into the roles of metabolites in plant-insect interactions.

Dereplication of Lantana trifolia L. leaves and fruits by UFLC-DAD-(+)-ESI-MS/MS and its antifungal and cytotoxic activities.

INTRODUCTION: Lantana trifolia L. (Verbenaceae) is a shrubby plant. In folk medicine, its leaves are used in the form of infusions and syrups to treat angina, coughs, and colds; they are also applied as tranquilizer. Previous studies have reported the antimicrobial potential of the compounds present in L. trifolia leaves. OBJECTIVES: To report the anti-Candida activities of the fractions obtained from the fruits and leaves of two L. trifolia specimens. METHODS: The L. trifolia fractions were submitted to UFLC-DAD-(+)-ESI-MS/MS, and the data were analyzed by using multivariate statistical tools (PCA, PLS-DA) and spectral similarity analyses based on molecular networking, which aided dereplication of the bioactive compounds. Additionally, NMR analyses were performed to confirm the chemical structure of some of the major compounds in the fractions. RESULTS: The ethyl acetate fractions presented MIC values lower than 100 µg mL-1 against the three Candida strains evaluated herein (C. albicans, C. tropicalis, and C. glabrata). Fractions FrPo AcOEt, FrPe AcOEt, and FrPe nBut had MIC values of 1.46, 2.93, and 2.93 µg mL-1 against C. glabrata, respectively. These values resembled the MIC value of amphotericin B, the positive control (0.5-1.0 µg mL-1), against this same strain. Cytotoxicity was measured and used to calculate the selectivity index. CONCLUSION: On the basis of our data, the most active fractions in the antifungal assay were more selective against C. glabrata than against non-infected cells. The analytical approach adopted here allowed us to annotate 29 compounds, nine of which were bioactive (PLS-DA results) and belong to the class of phenolic compounds.

Neuroprotective Properties of Chlorogenic Acid and 4,5-Caffeoylquinic Acid from Brazilian arnica (Lychnophora ericoides) after Acute Retinal Ischemia.

Lychnophora is a genus of South American flowering plants in the daisy family, popularly known as "Brazilian arnica". It is used in traditional medicine as an anti-inflammatory and analgesic agent, whose active components are derived from chlorogenic acid (CGA) and C-flavonoids. Since the drugs currently used are ineffective to treat glaucoma, agents with antioxidant and anti-inflammatory properties may represent new alternatives in preventing cellular lesions in retinal ischemia. In this study, we report the neuroprotective effects of CGA and 4,5-di-O-[E]-caffeoylquinic (CQA) acid, isolated from Lychnophora plants, in a rodent glaucoma model. Wistar rats were administered intravitreally with 10 µg CGA or CGA, and then subjected to acute retinal ischemia (ISC) by increasing intraocular pressure (IPO) for 45 minutes followed (or not) by 15 minutes of reperfusion (I/R). Qualitative and quantitative analyses of neurodegeneration were performed using hematoxylin-eosin or Fluoro-Jade C staining protocols. All retinas submitted to ISC or I/R exhibited matrix disorganization, pyknotic nuclei, and pronounced vacuolization of the cytoplasm in the ganglion cell layer (GCL) and inner nuclear layer (INL). Pretreatment with CGA or CQA resulted in the protection of the retinal layers against matrix disorganization and a reduction in the number of vacuolized cells and pyknotic nuclei. Also, pretreatment with CGA or CQA resulted in a significant reduction in neuronal death in the GCL, the INL, and the outer nuclear layer (ONL) after ischemic insult. Our study demonstrated that CGA and CQA exhibit neuroprotective activities in retinas subjected to ISC and I/R induced by IPO in Wistar rats.

Phytochemical Composition and Protective Effect of Vernonanthura polyanthes Leaf against In Vivo Doxorubicin-Mediated Toxicity.

Vernonanthura polyanthes (Spreng.) A.J. Vega & Dematt. (syn.: Vernonia polyanthes Less) is popularly known as "assa-peixe" and its leaves are used in folk medicine mainly to treat respiratory diseases. In this study, we evaluated the cytogenotoxic and anticytogenotoxic potential of the V. polyanthes leaf aqueous extract (VpLAE) and its n-butanol fraction (n-BF) in the presence or absence of doxorubicin (DXR) (pre-, co-, and post-treatments) on a murine model for 24 h or 120 h. The micronucleus test (MN) and the comet assay were used to assess the cytogenotoxic and anticytogenotoxic potential of VpLAE and n-BF (250, 500, and 1000 mg/kg) administered via gavage to Swiss Webster mice. The chemical profiles of VpLAE and n-BF were assessed by liquid chromatography coupled to mass spectrometry, and their metabolites were putatively identified. Lastly, the possible biological activities related to the (anti) cytogenotoxicity of the compounds were predicted using the PASS online webserver. The in vivo results showed that different doses of VpLAE and n-BF did not present cytotoxic activity; however, the MN test revealed a slight mutagenic activity for the 24 h treatments. Moderate genotoxic effects were demonstrated for all treatments in the comet assay. Regarding anticytotoxicity and antimutagenicity, VpLAE and n-BF presented a high cytoprotective potential against DXR toxic effects. In the co-treatment, VpLAE reduced the DXR genotoxicity by ~27%, and n-BF did not demonstrate antigenotoxic potential. In contrast, an antigenotoxic effect was observed for both VpLAE and n-BF in the pre- and post-treatments, reducing DXR genotoxicity by ~41% and ~47%, respectively. Chemical analysis of VpLAE and n-BF showed the presence of eight phenolic compounds, including seven chlorogenic acids and a flavonoid. The PASS online tool predicted antimutagenic, anticancer, antineoplastic, chemoprotective, antioxidant, and radical scavenging activities for all constituents identified in VpLAE and n-BF. V. polyanthes leaves presented a protective effect against DXR cytogenotoxicity. In general, VpLAE and n-BF showed a greater antigenotoxic potential in the pre- and post-treatments. The metabolites putatively identified in VpLAE and n-BF exhibited antioxidant and chemoprotective potential according to computational prediction analysis. Altogether, our results highlight the potential application of V. polyanthes to protect against toxic manifestations induced by DXR.

Intra-clade metabolomic profiling of MAR4 Streptomyces from the Macaronesia Atlantic region reveals a source of anti-biofilm metabolites.

The search for new and effective strategies to reduce bacterial biofilm formation is of utmost importance as bacterial resistance to antibiotics continues to emerge. The use of anti-biofilm agents that can disrupt recalcitrant bacterial communities can be an advantageous alternative to antimicrobials, as their use does not lead to the development of resistance mechanisms. Six MAR4 Streptomyces strains isolated from the Madeira Archipelago, at the unexplored Macaronesia Atlantic ecoregion, were used to study the chemical diversity of produced hybrid isoprenoids. These marine actinomycetes were investigated by analysing their crude extracts using LC-MS/MS and their metabolomic profiles were compared using multivariate statistical analysis (principal component analysis), showing a separation trend closely related to their phylogeny. Molecular networking unveiled the presence of a class of metabolites not previously described from MAR4 strains and new chemical derivatives belonging to the napyradiomycin and marinone classes. Furthermore, these MAR4 strains produce metabolites that inhibit biofilm formation of Staphylococcus aureus and Marinobacter hydrocarbonoclasticus. The anti-biofilm activity of napyradiomycin SF2415B3 (1) against S. aureus was confirmed.

Natural Products Diversity in Plant-Insect Interaction between Tithonia diversifolia (Asteraceae) and Chlosyne lacinia (Nymphalidae).

The chemical ecology of plant-insect interactions has been driving our understanding of ecosystem evolution into a more comprehensive context. Chlosyne lacinia (Lepidoptera: Nymphalidae) is an olygophagous insect herbivore, which mainly uses host plants of Heliantheae tribe (Asteraceae). Herein, plant-insect interaction between Tithonia diversifolia (Heliantheae) and Chlosyne lacinia was investigated by means of untargeted LC-MS/MS based metabolomics and molecular networking, which aims to explore its inherent chemical diversity. C. lacinia larvae that were fed with T. diversifolia leaves developed until fifth instar and completed metamorphosis to the adult phase. Sesquiterpene lactones (STL), flavonoids, and lipid derivatives were putatively annotated in T. diversifolia (leaves and non-consumed abaxial surface) and C. lacinia (feces, larvae, pupae, butterflies, and eggs) samples. We found that several furanoheliangolide-type STL that were detected in T. diversifolia were ingested and excreted in their intact form by C. lacinia larvae. Hence, C. lacinia caterpillars may have, over the years, developed tolerance mechanisms for STL throughout effective barriers in their digestive canal. Flavonoid aglycones were mainly found in T. diversifolia samples, while their glycosides were mostly detected in C. lacinia feces, which indicated that the main mechanism for excreting the consumed flavonoids was through their glycosylation. Moreover, lysophospholipids were predominately found in C. lacinia samples, which suggested that they were essential metabolites during pupal and adult stages. These findings provide insights into the natural products diversity of this plant-insect interaction and contribute to uncovering its ecological roles.

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.

Neuroprotective effects of chlorogenic acid against oxidative stress in rats subjected to lithium-pilocarpine-induced status epilepticus.

Epilepsy is a condition marked by sudden, self-sustained, and recurring brain events, showcasing unique electro-clinical and neuropathological phenomena that can alter the structure and functioning of the brain, resulting in diverse manifestations. Antiepileptic drugs (AEDs) can be very effective in 30% of patients in controlling seizures. Several factors contribute to this: drug resistance, individual variability, side effects, complexity of epilepsy, incomplete understanding, comorbidities, drug interactions, and no adherence to treatment. Therefore, research into new AEDs is important for several reasons such as improved efficacy, reduced side effects, expanded treatment options, treatment for drug-resistant epilepsy, improved safety profiles, targeted therapies, and innovation and progress. Animal models serve as crucial biological tools for comprehending neuronal damage and aiding in the discovery of more effective new AEDs. The utilization of antioxidant agents that act on the central nervous system may serve as a supplementary approach in the secondary prevention of epilepsy, both in laboratory animals and potentially in humans. Chlorogenic acid (CGA) is a significant compound, widely prevalent in numerous medicinal and food plants, exhibiting an extensive spectrum of biological activities such as neuroprotection, antioxidant, anti-inflammatory, and analgesic effects, among others. In this research, we assessed the neuroprotective effects of commercially available CGA in Wistar rats submitted to lithium-pilocarpine-induced status epilepticus (SE) model. After 72-h induction of SE, rats received thiopental and were treated for three consecutive days (1st, 2nd, and 3rd doses). Next, brains were collected and studied histologically for viable cells in the hippocampus with staining for cresyl-violet (Nissl staining) and for degenerating cells with Fluoro-Jade C (FJC) staining. Moreover, to evaluate oxidative stress, the presence of malondialdehyde (MDA) and superoxide dismutase (SOD) was quantified. Rats administered with CGA (30 mg/kg) demonstrated a significant decrease of 59% in the number of hippocampal cell loss in the CA3, and of 48% in the hilus layers after SE. A significant reduction of 75% in the cell loss in the CA3, shown by FJC+ staining, was also observed with the administration of CGA (30 mg/kg). Furthermore, significant decreases of 49% in MDA production and 72% in the activity of SOD were seen, when compared to animals subjected to SE that received vehicle. This study introduces a novel finding: the administration of CGA at a dosage of 30 mg/kg effectively reduced oxidative stress induced by lithium-pilocarpine, with its effects lasting until the peak of neural damage 72 h following the onset of SE. Overall, the research and development of new AEDs are essential for advancing epilepsy treatment, improving patient outcomes, and ultimately enhancing the quality of life for individuals living with epilepsy.

Elucidating the neurotoxicity of the star fruit.

Caramboxin: Patients suffering from chronic kidney disease are frequently intoxicated after ingesting star fruit. The main symptoms of this intoxication are named in the picture. Bioguided chemical procedures resulted in the discovery of caramboxin, which is a phenylalanine-like molecule that is responsible for intoxication. Functional experiments in vivo and in vitro point towards the glutamatergic ionotropic molecular actions of caramboxin, which explains its convulsant and neurodegenerative properties.

Microbial transformation of the sesquiterpene lactone tagitinin C by the fungus Aspergillus terreus.

The biotransformation of the sesquiterpene lactone tagitinin C by the fungus Aspergillus terreus MT 5.3 yielded a rare derivative that was elucidated by spectrometric methods. The fungus led to the formation of a different product through an unusual epoxidation reaction between C4 and C5, formation of a C3,C10 ether bridge, and a methoxylation of the C1 of tagitinin C. The chemical structure of the product, namely 1ß-methoxy-3α-hydroxy-3,10ß-4,5α-diepoxy-8ß-isobutyroyloxygermacr-11(13)-en-6α,12-olide, is the same as that of a derivative that was recently isolated from the flowers of a Brazilian population of Mexican sunflower (Tithonia diversifolia), which is the source of the substrate tagitinin C. The in vitro cytotoxic activity of the substrate and the biotransformed product were evaluated in HL-60 cells using an MTT assay, and both compounds were found to be cytotoxic. We show that soil fungi may be useful in the biotransformation of sesquiterpene lactones, thereby leading to unusual changes in their chemical structures that may preserve or alter their biological activities, and may also mimic plant biosynthetic pathways for production of secondary metabolites.

The protective role of Lychnophora ericoides Mart. (Brazilian arnica) in 1,2-dimethylhydrazine-induced experimental colon carcinogenesis.

Aberrant crypt foci (ACF) and colon rectal mucosal epithelial cell proliferation have been shown to be increased in patients with colon cancer and have been largely used for early detection of factors that influence colorectal carcinogenesis in rats. Fifty male Wistar rats were randomly divided into 5 groups. The groups G1 to G4 were given 4 injections of the carcinogen 1,2-dimethylhydrazine (DMH). The G2 group received Lychnophora ericoides (LE) extracts for 6 wk. The groups G3 and G4 received LE for 4 wk and 2 wk, respectively, at the postinitiation and initiation phases of colonic carcinogenesis. The group G5 was the control. Forty-two days after the first injections of DMH for the neoplasic induction, we observed a statistically significant decrease in the number of aberrant crypt foci (ACF) and an attenuation of the increase in cell proliferation induced by DMH in all the LE-treated groups. Thus, we concluded that Lychnophora ericoides extracts were effective against the development of cancer. These data suggest that LE has a protective influence on the process of colon carcinogenesis, suppressing both the initiation and the promotion of colonic carcinogenesis.

Neurobiological activity of Parawixin 10, a novel anticonvulsant compound isolated from Parawixia bistriata spider venom (Araneidae: Araneae).

The neurobiological activity of Parawixin 10, isolated from Parawixia bistriata spider venom, was investigated. Cannulas were implanted in the lateral ventricles of Wistar rats (200-250 g, n=6-8 per group) to perform anticonvulsant and behavioral assays, and synaptosomes from cerebral cortices of male Wistar rats were used for neurochemical studies. The results indicate that pretreatment with Parawixin 10 prevents the onset of seizures induced with kainic acid, N-methyl-D-aspartate, and pentylenetetrazole in a dose-response manner. Lower doses of Parawixin 10 significantly increased the latency to onset of kainic acid-, pentylenetetrazole-, and N-methyl-D-aspartate-induced seizures. There were maximum increases of 79% in L-[(3)H]glutamine uptake and 40% in [(3)H]glycine uptake; [(3)H]GABA uptake did not change. The findings demonstrate that this novel compound from P. bistriata venom exerts a pharmacological effect on the glutamatergic and glycinergic systems.

Plant Metabolites Involved in the Differential Development of a Heliantheae-Specialist Insect.

Balanced nutritional intake is essential to ensure that insects undergo adequate larval development and metamorphosis. Integrative multidisciplinary approaches have contributed valuable insights regarding the ecological and evolutionary outcomes of plant-insect interactions. To address the plant metabolites involved in the larval development of a specialist insect, we investigated the development of Chlosyne lacinia caterpillars fed on Heliantheae species (Tithonia diversifolia, Tridax procumbens and Aldama robusta) leaves and determined the chemical profile of plants and insects using a metabolomic approach. By means of LC-MS and GC-MS combined analyses, 51 metabolites were putatively identified in Heliantheae species and C. lacinia caterpillars and frass; these metabolites included flavonoids, sesquiterpene lactones, monoterpenoids, sesquiterpenoids, diterpenes, triterpenes, oxygenated terpene derivatives, steroids and lipid derivatives. The leading discriminant metabolites were diterpenes, which were detected only in A. robusta leaves and insects that were fed on this plant-based diet. Additionally, caterpillars fed on A. robusta leaves took longer to complete their development to the adult phase and exhibited a greater diapause rate. Hence, we hypothesized that diterpenes may be involved in the differential larval development. Our findings shed light on the plant metabolites that play roles in insect development and metabolism, opening new research avenues for integrative studies of insect nutritional ecology.

Acaricidal Activity of Annonaceae Plants for Dermanyssus gallinae (Acari: Dermanyssidae) and Metabolomic Profile by HPLC-MS/MS.

The poultry red mite Dermanyssus gallinae (De Geer) is the most important haematophagous ectoparasite in the poultry industry. The use of synthetic acaricides for this control is presenting risks related to human food. In this sense, plant secondary metabolites are promising for controlling this pest. Thus, this study aimed to evaluate the acaricidal activity of Duguetia lanceolata A.St.-Hil. (stem bark), Xylopia emarginata Mart. (stem bark), and Xylopia sericea A.St.-Hil. (stem bark and fruits) against D. gallinae. Additionally, the secondary metabolite profile of the X. emarginata was analysed by UFLC-DAD-ESI(+)-MS/MS (micrOTOF-QII) and data analysis was performed using the Molecular Networking. In a topical application test, all plant species tested showed bioactivity, in that order of toxicity with the respective probability survival: X. emarginata (stem bark) (0.28) > X. sericea (stem barks) (0.35) > X. sericea (fruits) and D. lanceolata (stem bark) (0.47). The most promising results were found for X. emarginata (LC50 = 331.769 µg/cm2). It is noteworthy that the LC50 of the insecticide cypermethrin was 1234.4 µg/cm2, which was 73% higher than that of X. emarginata. The metabolomic profile of X. emarginata revealed the presence of alkaloids, amides, terpenoids, and phenolic compounds. This is the first report of X. emarginata acaricidal activity against D. gallinae and exploratory chemical analysis by untargeted metabolomics and the molecular network of this plant.

Chemical Diversity in a Stingless Bee-Plant Symbiosis.

Bees are essential pollinators on earth, supporting forest equilibrium and human agriculture. The chemistry of the stingless bee-plant symbiosis is a complex and not completely understood phenomenon. Here, we combined untargeted tandem mass spectrometry, molecular networking, and multivariate statistical analysis to investigate the chemical diversity in colonies of the stingless bee Scaptotrigona depilis. Flavonoids were the most representative and diverse group of plant metabolites detected, indicating the importance of these biologically active natural products to the bees. We unveiled the metabolome, mapped the distribution of plant metabolites in stingless bee colonies, and digitized the chemical data into a public database.

Online identification of chlorogenic acids, sesquiterpene lactones, and flavonoids in the Brazilian arnica Lychnophora ericoides Mart. (Asteraceae) leaves by HPLC-DAD-MS and HPLC-DAD-MS/MS and a validated HPLC-DAD method for their simultaneous analysis.

Lychnophora ericoides Mart. (Asteraceae, Vernonieae) is a plant, endemic to Brazil, with occurrence restricted to the "cerrado" biome. Traditional medicine employs alcoholic and aqueous-alcoholic preparations of leaves from this species for the treatment of wounds, inflammation, and pain. Furthermore, leaves of L. ericoides are also widely used as flavorings for the Brazilian traditional spirit "cachaça". A method has been developed for the extraction and HPLC-DAD analysis of the secondary metabolites of L. ericoides leaves. This analytical method was validated with 11 secondary metabolites chosen to represent the different classes and polarities of secondary metabolites occurring in L. ericoides leaves, and good responses were obtained for each validation parameter analyzed. The same HPLC analytical method was also employed for online secondary metabolite identification by HPLC-DAD-MS and HPLC-DAD-MS/MS, leading to the identification of di- C-glucosylflavones, coumaroylglucosylflavonols, flavone, flavanones, flavonols, chalcones, goyazensolide, and eremantholide-type sesquiterpene lactones and positional isomeric series of chlorogenic acids possessing caffeic and/or ferulic moieties. Among the 52 chromatographic peaks observed, 36 were fully identified and 8 were attributed to compounds belonging to series of caffeoylferuloylquinic and diferuloylquinic acids that could not be individualized from each other.

Comprehensive untargeted metabolomics of Lychnnophorinae subtribe (Asteraceae: Vernonieae) in a phylogenetic context.

Members of the subtribe Lychnophorinae occur mostly within the Cerrado domain of the Brazilian Central Plateau. The relationships between its 11 genera, as well as between Lychnophorinae and other subtribes belonging to the tribe Vernonieae, have recently been investigated upon a phylogeny based on molecular and morphological data. We report the use of a comprehensive untargeted metabolomics approach, combining HPLC-MS and GC-MS data, followed by multivariate analyses aiming to assess the congruence between metabolomics data and the phylogenetic hypothesis, as well as its potential as a chemotaxonomic tool. We analyzed 78 species by UHPLC-MS and GC-MS in both positive and negative ionization modes. The metabolic profiles obtained for these species were treated in MetAlign and in MSClust and the matrices generated were used in SIMCA for hierarchical cluster analyses, principal component analyses and orthogonal partial least square discriminant analysis. The results showed that metabolomic analyses are mostly congruent with the phylogenetic hypothesis especially at lower taxonomic levels (Lychnophora or Eremanthus). Our results confirm that data generated using metabolomics provide evidence for chemotaxonomical studies, especially for phylogenetic inference of the Lychnophorinae subtribe and insight into the evolution of the secondary metabolites of this group.

Metabolomic analysis applied to chemosystematics and evolution of megadiverse Brazilian Vernonieae (Asteraceae).

Vernonia sensu lato is the largest and most complex genus of the tribe Vernonieae (Asteraceae). The tribe is chemically characterized by the presence of sesquiterpene lactones and flavonoids. Over the years, several taxonomic classifications have been proposed for Vernonia s.l. and for the tribe; however, there has been no consensus among the researches. According to traditional classification, Vernonia s.l. comprises more than 1000 species divided into sections, subsections and series (sensu Bentham). In a more recent classification, these species have been segregated into other genera and some subtribes were proposed, while the genus Vernonia sensu stricto was restricted to 22 species distributed mainly in North America (sensu Robinson). In this study, species from the subtribes Vernoniinae, Lepidaploinae and Rolandrinae were analyzed by UHPLC-UV-HRMS followed by multivariate statistical analysis. Data mining was performed using unsupervised (HCA and PCA) and supervised methods (OPLS-DA). The HCA showed the segregation of the species into four main groups. Comparing the HCA with taxonomical classifications of Vernonieae, we observed that the groups of the dendogram, based on metabolic profiling, were in accordance with the generic classification proposed by Robinson and with previous phylogenetic studies. The species of the genera Stenocephalum, Stilpnopappus, Strophopappus and Rolandra (Group 1) were revealed to be more related to the species of the genus Vernonanthura (Group 2), while the genera Cyrtocymura, Chrysolaena and Echinocoryne (Group 3) were chemically more similar to the genera Lessingianthus and Lepidaploa (Group 4). These findings indicated that the subtribes Vernoniinae and Lepidaploinae are non-chemically homogeneous groups and highlighted the application of untargeted metabolomic tools for taxonomy and as indicators of species evolution. Discriminant compounds for the groups obtained by OPLS-DA were determined. Groups 1 and 2 were characterized by the presence of 3',4'-dimethoxyluteolin, glaucolide A and 8-tigloyloxyglaucolide A. The species of Groups 3 and 4 were characterized by the presence of putative acacetin 7-O-rutinoside and glaucolide B. Therefore, untargeted metabolomic approach combined with multivariate statistical analysis, as proposed herein, allowed the identification of potential chemotaxonomic markers, helping in the taxonomic classifications.

Preservation of phenolic compounds on dried leaf infusion of Bauhinia forficata Link

Abstract Bauhinia forficata Link aqueous extract is usually recommended as a phytomedicine to reduce blood glucose levels and its biological activity has been linked to the presence of phenolic compounds from B. forficata preparations. Several drying processes are used in the production of dry herbal extracts, which may influence the chemical composition and efficacy of final herbal medicines. Due to significant chemical changes, defining appropriate drying processes is essential for phytopharmaceutical drug development. In view of this, we analyzed dried B. forficata leaf infusion (BFLI) extracts by HPLC-UV-MSn, followed by molecular networking analysis to evaluate the chemical profiles from dried extracts yielded by freeze-and spray-drying processes. The main metabolites detected included 11 ferulic/isoferulic acid derivatives and 13 glycosylated flavonoids. The qualitative chemical profiles were alike for both drying processes, whereas the relative abundance of some flavonoids was higher using spray-drying. Taken together, our results showed that freeze-and spray-drying preserved the phenolic profile of BFLI and suggested that spray-drying may be the most suitable to obtain its dried products. Along with studying the chemical profiles of dried herbal extracts, evaluating the influence of drying processes on the quality and chemical profiles of final products is pivotal and may benefit future research.

Permeability profile estimation of flavonoids and other phenolic compounds by biopartitioning micellar capillary chromatography.

This paper points out the usefulness of biopartitioning micellar chromatography (BMC) using capillary columns as a high-throughput primary screening tool providing key information about the oral absorption, skin permeability, and brain-blood distribution coefficients of 15 polyphenols (6 flavones, 2 flavonols, a flavanone, 2 flavan-3-ols, 3 phenolic acids, and a phloroglucinol) in a simple and economical way. For the compounds studied, except vicenin-2, rutin, chlorogenic acid, p-hydroxycinnamic acid, and 4-hydroxybenzoic acid, maximal oral absorption (>90%) can be expected, if there are not solubility problems or metabolic processes. On the other hand, the most retained compounds in BMC, that is, 5-hydroxyflavone, flavone, and flavanone, show the highest brain-blood distribution coefficients and skin permeability coefficients.