In Vitro Anti-Toxoplasma Activity of Extracts Obtained from Tabebuia rosea and Tabebuia chrysantha: The Role of ß-Amyrin.

Toxoplasmosis is a parasitic disease caused by the protozoan Toxoplasma gondii that is highly prevalent worldwide. Although the infection is asymptomatic in immunocompetent individuals, it severely affects immunocompromised individuals, causing conditions such as encephalitis, myocarditis, or pneumonitis. The limited therapeutic efficacy of drugs currently used to treat toxoplasmosis has prompted the search for new therapeutic alternatives. The aim of this study was to determine the anti-Toxoplasma activity of extracts obtained from two species of the genus Tabebuia. Twenty-six extracts, 12 obtained from Tabebuia chrysantha and 14 from Tabebuia rosea, were evaluated by a colorimetric technique using the RH strain of T. gondii that expresses ß-galactosidase. Additionally, the activity of the promising extracts and their active compounds was evaluated by flow cytometry. ß-amyrin was isolated from the chloroform extract obtained from the leaves of T. rosea and displayed important anti-Toxoplasma activity. The results show that natural products are an important source of new molecules with considerable biological and/or pharmacological activity.

Around the world in 40 million years: Phylogeny and biogeography of Tecomeae (Bignoniaceae).

Intercontinental disjunct distributions can arise from vicariance, long distance dispersal, or both. Tecomeae (Bignoniaceae) are a nearly cosmopolitan clade of flowering plants providing us with an excellent opportunity to investigate global distribution patterns. While the tribe contains only about 57 species, it has achieved a distribution that is not only pantropical, but also extends into the temperate zones in both the Northern and Southern hemispheres. This distribution is similar to the distribution of its sister group, a clade of about 750 spp. that includes most remaining taxa in Bignoniaceae. To infer temporal and spatial patterns of dispersal, we generated a phylogeny of Tecomeae by gathering sequence data from chloroplast and nuclear markers for 41 taxa. Fossil calibrations were used to determine divergence times, and ancestral states were reconstructed to infer its biogeographic history. We found support for a South American origin and a crown age of the tribe estimated at ca. 40 Ma. Two dispersal events seem to have happened during the Eocene-Oligocene, one from South America to the Old World, and another from South America to North America. Furthermore, two other dispersal events seem to have taken place during the Miocene, one from North America to Asia, and another from Australia to South America. We suggest that intercontinental dispersal via land bridges and island hopping, as well as sweepstakes of long distance dispersal from the Eocene to the present explain the global distribution of Tecomeae.

Effects of heterospecific pollen on stigma behavior in Campsis radicans: Causes and consequences.

PREMISE: Pollinator sharing of co-flowering plants may result in interspecific pollen receipt with a fitness cost. However, the underlying factors that determine the effects of heterospecific pollen (HP) are not fully understood. Moreover, the cost of stigma closure induced by HP may be more severe for plants with special touch-sensitive stigmas than for plants with non-touch-sensitive stigmas. Very few studies have assessed HP effects on stigma behavior. METHODS: We conducted hand-pollination experiments with 10 HP donors to estimate HP effects on stigma behavior and stigmatic pollen germination in Campsis radicans (Bignoniaceae) at low and high pollen loads. We assessed the role of phylogenetic distance between donor and recipient, pollen size, and pollen aperture number in mediating HP effects. Additionally, we observed pollen tube growth to determine the conspecific pollen-tube-growth advantage. RESULTS: Stigma behavior differed significantly with HP of different species. Pollen load increased, while pollen size decreased, the percentage of permanent closure and stigmatic germination of HP. Stigmatic HP germination increased with increasing aperture number. However, HP effects did not depend on phylogenetic distance. In addition, conspecific pollen had a pollen-tube-growth advantage over HP. CONCLUSIONS: Our results provide a good basis for understanding the stigma-pollen recognition process of plant taxa with touch-sensitive stigmas. We concluded that certain flowering traits drive the HP effects on the post-pollination period. To better understand the impact of pollinator sharing and interspecific pollen transfer on plant evolution, we highlight the importance of evaluating more factors that determine HP effects at the community level.

Complete chloroplast genome of Campsis grandiflora (Thunb.) schum and systematic and comparative analysis within the family Bignoniaceae.

BACKGROUND: Plants belonging to the Bignoniaceae family have a wide distribution in the tropics and large populations around the world. However, limited information is available about Bignoniaceae. This study aimed to obtain more research information about Bignoniaceae plants and provide data support for the study of plant plastid genomes. METHODS AND RESULTS: In the present study, we focused on the chloroplast genome bio-information of Campsis grandiflora. The chloroplast DNA of C. grandiflora was extracted, sequenced, assembled, and annotated with corresponding software. Results show that the complete chloroplast genome of C. grandiflora is 154,303 bp in length and has a quadripartite structure with large single copy of 85,064 bp and a small single copy of 18,009 bp separated by inverted repeats of 25,615 bp. A total of 110 genes in C. grandiflora comprised 79 protein-coding genes, 27 transfer RNA genes, and 4 ribosomal RNA genes. The distribution of simple sequence repeats and long repeat sequences was determined. We carried out phylogenetic analysis based on homologous amino acid sequence among 45 species derived from Bignoniaceae. Compared with the chloroplast genome of A. thaliana, an inversion was identified in that of C. grandiflora, which result in the incomplete clpP gene. CONCLUSIONS: The chloroplast genomes were used for molecular marker, species identification, and phylogenetic studies. The outcome strongly supported that C. grandiflora and genus Incarvillea formed a cluster within Bignoniaceae. This study identified the unique characteristics of the C. grandiflora cp. genome, thus providing theoretical basis for species identification and biological research.

Genetic diversity and verbascoside content in natural populations of Pyrostegia venusta (Ker Gawl.) Miers.

BACKGROUND: Pyrostegia venusta (Ker Gawl.) Miers occurs in threatened biodiversity hotspots of Cerrado and Atlantic forest biomes in Brazil and is used in traditional medicine to treat various respiratory and skin diseases. METHODS AND RESULTS: This study (i) examined the genetic diversity and structure of six natural populations of P. venusta from different Brazilian regions using sequence-related amplified polymorphism (SRAP) markers; and (ii) compared the intra- and inter-populational levels of the bioactive component verbascoside using high-performance liquid chromatography. The population from Nova Mutum, Mato Grosso, presented the highest genetic variability (Nei index H = 0.2759; Shannon index I = 0.4170; 85.14% polymorphic loci), whereas the population from Araxá, Minas Gerais, presented the lowest genetic variability (H = 0.1811; I = 0.2820; 70.27% polymorphic loci). The intra-populational variability (79%) was significantly higher (p = 0.001) than the inter-populational variability (21%). The populations were clustered into two groups but their genetic differentiation was not associated with geographical origin (Mantel test, r = 0.328; p > 0.05). The verbascoside content significantly differed (p > 0.05) among the six populations and between the individuals from each population. The highest verbascoside levels (> 200 µg/mg extract) were detected in populations from Araxá and Serrana, while the lowest verbacoside levels were detected in populations from Paranaíta and Sinop. CONCLUSIONS: This is the first report on the use of SRAP markers to analyze genetic variability in the family Bignoniaceae. Our findings shall help to better understand the genetic and chemical diversity of P. venusta populations, as well as provide useful information to select the most appropriate individuals to prepare phytomedicines.

High-Resolution Mass Spectrometry Identification and Characterization of Flavonoids from Fridericia chica Leaves Extract with Anti-Arbovirus Activity.

Plant extracts are complex mixtures that are difficult to characterize, and mass spectrometry is one of the main techniques currently used in dereplication processes. Fridericia chica is a species with medicinal uses in Latin American countries, used in the treatment of inflammatory and infectious diseases. Extracts of this plant species are characterized by the presence of anthocyanidins. In this study, using high-resolution mass spectrometry coupled with liquid chromatography, it was possible to determine the molecular formula of thirty-nine flavonoids. Fragmentation analysis, ultraviolet spectrum and nuclear magnetic resonance data allowed the partial characterization of the structures of these compounds. The spectral dataset allowed the identification of a series of flavones in addition to the desoxyanthocyanidins common in extracts of the species. The occurrence of some of the proposed structures is uncommon in extracts of species of the Bignoniaceae family, and they are reported for the first time in the extract of this species. Quantitative analyses of total flavonoids confirmed the high content of these constituents in the species, with 4.09 ± 0.34 mg/g of dry plant material. The extract under study showed low in vitro cytotoxicity with CC50 ≥ 296.7 ± 1.4 µg/mL for Vero, LLC-MK2 and MRC-5 cell lines. In antiviral activity assays, inhibition of the cytopathic effects of Dengue, Zika and Mayaro viruses was observed, with EC50 values ranging between 30.1 and 40.9 µg/mL. The best result was observed against the Mayaro virus, with an EC50 of 30.1 µg/mL.

RNA-Seq reveals different responses to drought in Neotropical trees from savannas and seasonally dry forests.

BACKGROUND: Water is one of the main limiting factors for plant growth and crop productivity. Plants constantly monitor water availability and can rapidly adjust their metabolism by altering gene expression. This leads to phenotypic plasticity, which aids rapid adaptation to climate changes. Here, we address phenotypic plasticity under drought stress by analyzing differentially expressed genes (DEG) in four phylogenetically related neotropical Bignoniaceae tree species: two from savanna, Handroanthus ochraceus and Tabebuia aurea, and two from seasonally dry tropical forests (SDTF), Handroanthus impetiginosus and Handroanthus serratifolius. To the best of our knowledge, this is the first report of an RNA-Seq study comparing tree species from seasonally dry tropical forest and savanna ecosystems. RESULTS: Using a completely randomized block design with 4 species × 2 treatments (drought and wet) × 3 blocks (24 plants) and an RNA-seq approach, we detected a higher number of DEGs between treatments for the SDTF species H. serratifolius (3153 up-regulated and 2821 down-regulated under drought) and H. impetiginosus (332 and 207), than for the savanna species. H. ochraceus showed the lowest number of DEGs, with only five up and nine down-regulated genes, while T. aurea exhibited 242 up- and 96 down-regulated genes. The number of shared DEGs among species was not related to habitat of origin or phylogenetic relationship, since both T. aurea and H impetiginosus shared a similar number of DEGs with H. serratifolius. All four species shared a low number of enriched gene ontology (GO) terms and, in general, exhibited different mechanisms of response to water deficit. We also found 175 down-regulated and 255 up-regulated transcription factors from several families, indicating the importance of these master regulators in drought response. CONCLUSION: Our findings show that phylogenetically related species may respond differently at gene expression level to drought stress. Savanna species seem to be less responsive to drought at the transcriptional level, likely due to morphological and anatomical adaptations to seasonal drought. The species with the largest geographic range and widest edaphic-climatic niche, H. serratifolius, was the most responsive, exhibiting the highest number of DEG and up- and down-regulated transcription factors (TF).

Do s genes or deleterious recessives control late-acting self-incompatibility in Handroanthus heptaphyllus (Bignoniaceae)? A diallel study with four full-sib progeny arrays.

BACKGROUND AND AIMS: Genetically controlled self-incompatibility (SI) mechanisms constrain selfing and thus have contributed to the evolutionary diversity of flowering plants. In homomorphic gametophytic SI (GSI) and homomorphic sporophytic SI (SSI), genetic control is usually by the single multi-allelic locus S. Both GSI and SSI prevent self pollen tubes reaching the ovary and so are pre-zygotic in action. In contrast, in taxa with late-acting self-incompatibility (LSI), rejection is often post-zygotic, since self pollen tubes grow to the ovary, where fertilization may occur prior to floral abscission. Alternatively, lack of self fruit set could be due to early-acting inbreeding depression (EID). The aim of our study was to investigate mechanisms underlying the lack of selfed fruit set in Handroanthus heptaphyllus in order to assess the likelihood of LSI versus EID. METHODS: We employed four full-sib diallels to study the genetic control of LSI in H. heptaphyllus using a precociously flowering variant. We also used fluorescence microscopy to study the incidence of ovule penetration by pollen tubes in pistils that abscised following pollination or initiated fruits. KEY RESULTS: All diallels showed reciprocally cross-incompatible full sibs (RCIs), reciprocally cross-compatible full sibs (RCCs) and non-reciprocally compatible full sibs (NRCs) in almost equal proportions. There was no significant difference between the incidences of ovule penetrations in abscised pistils following self- and cross-incompatible pollinations, but those in successful cross-pollinations were around 2-fold greater. CONCLUSIONS: A genetic model postulating a single S locus with four S alleles, one of which, in the maternal parent, is dominant to the other three, will produce RCI, RCC and NRC full sib situations each at 33 %, consistent with our diallel results. We favour this simple genetic control over an EID explanation since none of our pollinations, successful or unsuccessful, resulted in partial embryo development, as would be expected under a whole-genome EID effect.

The Influence of Anthocyanidin Profile on Antileishmanial Activity of Arrabidaea chica Morphotypes.

Arrabidaea chica Verlot (crajiru) is a plant used in folk medicine as an astringent, anti-inflammatory, wound healing and to treat fungal and viral diseases such as measles chickenpox and herpes. Arrabidaea chica has several morphotypes recognized but little is known about its chemical variability. In the present study the anthocyanidin profile of A. chica morphotypes collected in two seasons (summer and winter) have been examined and their activity against Leishmania infection compared. High-performance liquid chromatography coupled to a diode-array detector (HPLC-DAD-UV) and by tandem mass spectrometry with electrospray ionization (ESI-MS/MS) were used for anthocyanidin separation and identification. Antileishmanial activity was measured against promastigote forms of Leishmania amazonensis. Multivariate analysis, principal component analysis (PCA) and Pearson's correlation were performed to classify morphotypes accordingly to their anthocyanidin profile. The presence of 6,7,3',4'-tetrahydroxy-5-methoxyflavylium (3'-hydroxy-carajurone) (1), carajurone (2), 6,7,3'-trihydroxy-5,4'-dimethoxy-flavylium (3'-hydroxy-carajurin) (3) and carajurin (4), and three unidentified anthocyanidins were detected. Two different groups were recognized: group I containing 3'-hydroxy-carajurone; and group II with high content of carajurin. Among anthocyanidins identified in the extracts, only carajurin showed significant statistical correlation (p = 0.030) with activity against L. amazonensis. Carajurin could thus be considered as a pharmacological marker for the antileishmanial potential of the species.

Iridoids and Amino Acid Derivatives from the Paraguayan Crude Drug Adenocalymma marginatum (ysypó hû).

The crude drug ysypó hû (Adenocalymma marginatum DC., Bignoniaceae) is used traditionally by the Guarani of Eastern Paraguayan as a male sexual enhancer. The aim of the present study was to identify the main constituents of the crude drug and to evaluate the in vitro inhibitory activity towards the enzyme phosphodiesterase-5 (PDE-5). The main compounds were isolated by counter-current chromatography (CCC). The metabolites were identified by spectroscopic and spectrometric means. The chemical profiling of the extracts was assessed by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS). The crude extract and main isolated compounds were tested for their PDE-5 inhibitory activity using commercial kits. The iridoid theviridoside and 4-hydroxy-1-methylproline were isolated as the main constituent of the crude drug. Four chlortheviridoside hexoside derivatives were detected for the first time as natural products. Chemical profiling by HPLC-MS/MS led to the tentative identification of nine iridoids, six phenolics, and five amino acids. The crude extracts and main compounds were inactive towards PDE-5 at concentrations up to 500 µg/mL. Iridoids and amino acid derivatives were the main compounds occurring in the Paraguayan crude drug. The potential of ysypó hû as a male sexual enhancer cannot be discarded, since other mechanisms may be involved.

Phylogeny of the Neotropical tribe Jacarandeae (Bignoniaceae).

PREMISE: The tribe Jacarandeae includes Jacaranda (49 species) and Digomphia (3 species), two genera of trees and woody shrubs with Neotropical distribution. Jacarandeae is sister to the rest of the Bignoniaceae, but not much is known about interspecific and intergeneric relationships within this group. METHODS: We reconstructed the phylogeny of Jacarandeae using chloroplast (ndhF, rpl32-trnL, trnL-F) and nuclear (ETS, PPR62) markers. Evolutionary relationships within Jacarandeae were inferred using Bayesian, Maximum Likelihood, and species tree approaches. The resulting phylogenetic framework was used as the basis to interpret the evolution of key morphological character states (i.e., stamen and calyx traits) and revise the infra-generic classification of the group. RESULTS: Jacaranda and Digomphia belong to a well-supported clade, with Digomphia nested within Jacaranda. We propose the necessary taxonomic changes to recognize monophyletic taxa, including a broadly circumscribed Jacaranda divided into four sections: (1) Jacaranda sect. Nematopogon, species previously included in Digomphia and united by divided staminode apices and spathaceous calyces; (2) Jacaranda sect. Copaia, species with monothecal anthers and cupular calyces; (3) Jacaranda sect. Jacaranda, species with monothecal anthers and campanulate calyces; and (4) Jacaranda sect. Dilobos, species with dithecal anthers and cupular calyces, and including more than half of the species of the genus, all restricted to Brazil. CONCLUSIONS: As circumscribed here, Jacarandeae includes only a broadly defined Jacaranda divided into four sections. Each section is defined by a unique combination of anther and calyx morphologies.

Characterization of inhibitory constituents of NO production from Catalpa ovata using LC-MS coupled with a cell-based assay.

An effective screening method for inhibitors of NO production in natural products using LC-QTOF MS/MS coupled with a cell-based assay was proposed. The ethyl acetate fraction of Catalpa ovata exhibited a strong inhibitory effect on NO production in lipopolysaccharide-induced BV2 microglia cells. We attempted to identify the active constituents of C. ovata by using LC-QTOF MS/MS coupled with a cell-based assay. Peaks at approximately 14-15 min on the MS chromatogram were estimated to be the bioactive constituents. A new iridoid compound, 6-O-trans-feruloyl-3ß-hydroxy-7-deoxyrehamaglutin A (4), and nine known compounds (1-3, 5-10) were isolated from the ethyl acetate fraction of C. ovata by repeated column chromatography. Compounds 3, 4, 5, 7, and 8 significantly attenuated lipopolysaccharide-stimulated NO production in BV2 cells. Our results indicate that LC-QTOF MS/MS coupled with a cell-based NO production inhibitory assay successfully predicted active compounds without a time-consuming isolation process.

New cycloartanes and new iridoids from Dolichandrone spathacea collected in the mangrove forest of Soc Trang province, Vietnam.

Two new cycloartanes, named dolichandrone A (1) and dolichandrone B (2), as well as two new iridoids, named [6-O-[(E)-4-methoxycinnamoyl]-1ß-hydroxy-dihydrocatalpolgenin (3) and 6-O-[(E)-4-methoxycinnamoyl]-1α-hydroxy-dihydrocatalpolgenin (4), together with four known iridoids (5-8), were isolated from the leaves and barks of Dolichandrone spathacea. Their structures were elucidated by means of extensive analysis of their HRESIMS, 1D and 2D NMR spectroscopic data. All of these compounds have been isolated for the first time from this plant. Compounds 1, 2, 5, and 7 were evaluated for their cytotoxic activity in vitro against four human cancer cell lines KB, Lu, HepG2, and MCF7. The results showed that only compound 2 exhibited a good cytotoxicity against KB cell line with IC50 of 18.77 µM.

Involvement of Opioid System, TRPM8, and ASIC Receptors in Antinociceptive Effect of Arrabidaea brachypoda (DC) Bureau.

Arrabidaea brachypoda (DC) Bureau is a medicinal plant found in Brazil. Known as "cipó-una", it is popularly used as a natural therapeutic agent against pain and inflammation. This study evaluated the chemical composition and antinociceptive activity of the dichloromethane fraction from the roots of A. brachypoda (DEAB) and its mechanism of action. The chemical composition was characterized by high-performance liquid chromatography, and this fraction is composed only of dimeric flavonoids. The antinociceptive effect was evaluated in formalin and hot plate tests after oral administration (10-100 mg/kg) in male Swiss mice. We also investigated the involvement of TRPV1 (transient receptor potential vanilloid 1), TRPA1 (transient receptor potential ankyrin 1), TRPM8 (transient receptor potential melastatin 8), and ASIC (acid-sensing ion channel), as well as the opioidergic, glutamatergic, and supraspinal pathways. Moreover, the nociceptive response was reduced (30 mg/kg) in the early and late phase of the formalin test. DEAB activity appears to involve the opioid system, TRPM8, and ASIC receptors, clearly showing that the DEAB alleviates acute pain in mice and suggesting the involvement of the TRPM8 and ASIC receptors and the opioid system in acute pain relief.

Demographical history and palaeodistribution modelling show range shift towards Amazon Basin for a Neotropical tree species in the LGM.

BACKGROUND: We studied the phylogeography and demographical history of Tabebuia serratifolia (Bignoniaceae) to understand the disjunct geographical distribution of South American seasonally dry tropical forests (SDTFs). We specifically tested if the multiple and isolated patches of SDTFs are current climatic relicts of a widespread and continuously distributed dry forest during the last glacial maximum (LGM), the so called South American dry forest refugia hypothesis, using ecological niche modelling (ENM) and statistical phylogeography. We sampled 235 individuals of T. serratifolia in 17 populations in Brazil and analysed the polymorphisms at three intergenic chloroplast regions and ITS nuclear ribosomal DNA. RESULTS: Coalescent analyses showed a demographical expansion at the last c. 130 ka (thousand years before present). Simulations and ENM also showed that the current spatial pattern of genetic diversity is most likely due to a scenario of range expansion and range shift towards the Amazon Basin during the colder and arid climatic conditions associated with the LGM, matching the expected for the South American dry forest refugia hypothesis, although contrasting to the Pleistocene Arc hypothesis. Populations in more stable areas or with higher suitability through time showed higher genetic diversity. Postglacial range shift towards the Southeast and Atlantic coast may have led to spatial genome assortment due to leading edge colonization as the species tracks suitable environments, leading to lower genetic diversity in populations at higher distance from the distribution centroid at 21 ka. CONCLUSION: Haplotype sharing or common ancestry among populations from Caatinga in Northeast Brazil, Atlantic Forest in Southeast and Cerrado biome and ENM evince the past connection among these biomes.

Generic delimitations, biogeography and evolution in the tribe Coleeae (Bignoniaceae), endemic to Madagascar and the smaller islands of the western Indian Ocean.

This study presents the most complete generic phylogenetic framework to date for the tribe Coleeae (Bignoniaceae), which is endemic to Madagascar and the other smaller islands in the western part of the Indian Ocean. The study is based on plastid and nuclear DNA regions and includes 47 species representing the five currently recognized genera (including all the species occurring in the western Indian Ocean region). Bayesian and maximum likelihood analyses supported (i) the monophyly of the tribe, (ii) the monophyly of Phylloctenium, Phyllarthron and Rhodocolea and (iii) the paraphyly of Colea due to the inclusion of species of Ophiocolea. The latter genus was also recovered paraphyletic due to the inclusion of two species of Colea (C. decora and C. labatii). The taxonomic implications of the mutual paraphyly of these two genera are discussed in light of morphological evidence, and it is concluded that the two genera should be merged, and the necessary new nomenclatural combinations are provided. The phylogenetic framework shows Phylloctenium, which is endemic to Madagascar and restricted to dry ecosystems, as basal and sister to the rest of the tribe, suggesting Madagascar to be the centre of origin of this clade. The remaining genera are diversified mostly in humid ecosystems, with evidence of multiple dispersals to the neighboring islands, including at least two to the Comoros, one to Mauritius and one to the Seychelles. Finally, we hypothesize that the ecological success of this tribe might have been triggered by a shift of fruit-dispersal mode from wind to lemur.

A new phenylethanoid glycoside from Incarvillea compacta.

A new phenylethanoid glycoside, 3'''-O-methylcampneoside I (1), was isolated from the 90% ethanolic extract of the roots of Incarvillea compacta, together with three known compounds, campneoside I (2), ilicifolioside A (3), and campneoside II (4). Their structures were determined spectroscopically and compared with previously reported spectral data. Compound 1 existed as epimers and displayed better 1,1-diphenyl-2-picrylhydrazyl (DPPH)-free radical scavenging activity using di-tert-butyl-4-methylphenol (BHT) as the positive control. In addition, pretreatment of human HepG2 cells with compound 1 significantly increased the viability on CCl4-induced cell death.

Optimization of Betulinic Acid Extraction from Tecomella undulata Bark Using a Box-Behnken Design and Its Densitometric Validation.

Betulinic acid (BA) is a pentacyclic triterpenoid acid obtained from the stem bark of Tecomella undulata Seem. (Bignoniaceae). Development of an efficient extraction method for the isolation of BA is important as it has a wide range of pharmacological activity. A Box-Behnken design (BBD) was used to investigate the effect of extraction variables such as temperature (30-60 °C), time (4-8 h) and solvent to drug ratio (300-500 mL/100 g) on the maximization of BA yield and its quantification using validated densitometric high performance thin layer chromatography coupled with ultraviolet detection (HPTLC-VIS). A quadratic polynomial model was found to best fit the model with R² = 0.99. The optimized Soxhlet extraction yielded 2.449% w/w of BA at a temperature 53.86 °C, time 6.38 h and solvent to drug ratio 371 mL/100 g. BA in Tecomella undulata bark was detected at Rf value of 0.65 at 510 nm using the solvent system toluene-ethyl acetate-glacial acetic acid (8.5:1.5:0.02 v/v/v). The analytical method was validated and the linear regression analysis reflects good linear relationship (R² = 0.9902). Lower %RSD and SEM suggested that the developed HPTLC-VIS method was precise, accurate and robust. Therefore, these economical techniques are very efficient and promising for the extraction and quantification of pharmaceutically important BA.

Phylogeny and biogeography of Tynanthus Miers (Bignonieae, Bignoniaceae).

The origin of Neotropical biodiversity represents a key question in evolutionary biology. Despite the attempts to decipher the role of ecological and historical factors to present-day distribution patterns, robust phylogenetic studies of Neotropical clades are still needed before a comprehensive picture of the origin of Neotropical biodiversity can be achieved. Tynanthus Miers (Bignonieae, Bignoniaceae) is a well-circumscribed genus of Neotropical lianas with species that are narrowly distributed, except from a few taxa. The genus is characterized by a clove odor, small bilabiate flowers with the two upper lobes almost fused, and fruits with raised margins, all of which represent morphological synapomorphies for this clade. Other distinctive characters are the thecae reflexed forward, the densely pubescent ovaries and the poorly-developed nectariferous disk. The circumscription of the genus has remained constant over the years, despite the problematic limits of most genera of tribe Bignonieae. In this study, we reconstruct the phylogeny of Tynanthus based on two plastid (ndhF and rpl32-trnL) and one nuclear (pepC) markers and use this phylogenetic framework to investigate the biogeographical history of the genus. Our phylogenetic hypothesis provides further support for the monophyly of Tynanthus, and strongly supports a series of infra-generic clades. Most species are reconstructed as monophyletic, while T. cognatus and T. polyanthus are paraphyletic. Biogeographic reconstructions suggest that Tynanthus originated between 9.4 and 21.5Mya, most likely at approximately 15.3Mya. The MRCA of the genus was likely broadly distributed through lowland Amazonia, Western South America and Central America and diversified in the Neotropics during the Miocene. Closely related species are generally distributed within the same biogeographic area, suggesting that niche conservatism has played an important role in the diversification history of the group.

Secondary phloem diversity and evolution in Bignonieae (Bignoniaceae).

BACKGROUND AND AIMS: Phloem evolution has been explored in the literature across very broad scales, either for vascular plants as a whole or for major plant groups, such as the monocotyledons or the former dicotyledons. However, it has never been examined in a way that would elucidate evolutionary shifts leading to the diversification of phloem in single lineages. Therefore, the present study explores in detail the patterns of phloem evolution in the tribe Bignonieae (Bignoniaceae). This group represents a particularly good model for phloem studies since it is known to have a very conspicuous and diverse phloem. METHODS: A total of 19 phloem characters were coded in 56 species from all 21 genera currently recognized in the tribe Bignonieae, accounting for phloem wedge growth and for all the anatomical cell diversity encountered in the phloem. Phloem evolution was explored by reconstructing ancestral character states using maximum-likelihood assumptions with a time-calibrated molecular phylogeny for the group. Directionality and the effect of phylogenetic transformations in the current variation of quantitative traits and evolutionary correlations of selected discrete phloem traits were also tested under a maximum-likelihood approach. KEY RESULTS: Individual phloem features are quite diverse in the tribe, but generally conserved within smaller clades. Contrasting phloem patterns were found when comparing major groups, with certain lineages having the phloem marked by a background of phloem fibres where all other cells are embedded, tangentially arranged sieve tubes and sieve-tubecentric parenchyma. In contrast, other lineages exhibited a scarcely fibrous phloem, regularly stratified phloem, sieve tube elements in radial or diffuse arrangement, and diffuse parenchyma. We found signals of directional evolution in fibre abundance and number of sieve areas, which increased in the 'Fridericia and allies extended clade' and decreased in the 'Multiples of four extended clade', resulting in no signal of directionality when the whole Bignonieae was considered. In contrast, no indication of directional evolution was found for the axial parenchyma, either in single clades within Bignonieae or in the entire tribe. Positive correlation was found between sieve element length and both sieve plate type and the presence of a storied structure. Correlated evolution was also found between fibre abundance and several traits, such as sieve tube arrangement, sieve plate type, parenchyma arrangement, ray lignification and number of companion cells. CONCLUSIONS: The secondary phloem of Bignonieae is extremely diverse, with sister lineages exhibiting distinct phloem anatomies derived from contrasting patterns of evolution in fibre abundance. Fibre abundance in the tribe has diversified in correlation with sieve tube arrangement, sieve tube morphology, number of companion cells and parenchyma type. The results challenge long-standing hypotheses regarding general trends in cell abundance and morphological cell evolution within the phloem, and demonstrate the need to expand studies in phloem anatomy both at a narrow taxonomic scale and at a broad one, such as to families and orders.