Cytotoxic Cyclotides from Anchietea pyrifolia, a South American Plant Species.

Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from Anchietea pyrifolia roots, a South American medicinal plant from the family Violaceae. Fourteen putative cyclotides were annotated by LC-MS. Among these, three new bracelet cyclotides, anpy A-C, and the known cycloviolacins O4 (cyO4) and O17 (cyO17) were sequenced through a combination of chemical and enzymatic reactions followed by MALDI-MS/MS analysis. Their cytotoxic activity was evaluated by a cytotoxicity assay against three human cancer cell lines (colorectal carcinoma cells: HCT 116 and HCT 116 TP53-/- and breast adenocarcinoma, MCF 7). For all assays, the IC50 values of isolated compounds ranged between 0.8 and 7.3 µM. CyO17 was the most potent cyclotide for the colorectal cancer cell lines (IC50, 0.8 and 1.2 µM). Furthermore, the hemolytic activity of anpy A and B, cyO4, and cyO17 was assessed, and the cycloviolacins were the least hemolytic (HD50 > 156 µM). This work sheds light on the cytotoxic effects of the anpy cyclotides against cancer cells. Moreover, this study expands the number of cyclotides obtained to date from Brazilian plant biodiversity and adds one more genus containing these molecules to the list of the Violaceae family.

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

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

Anchietea pyrifolia A. St.-Hil. as a Cardiovascular-Endowed Species: A Whole-Biological Investigation.

Although leaves of Anchietea salutaris are used in Brazilian traditional medicine, there is no available data in the literature proving its efficacy and safety. Thus, the aim of the study was to perform a meticulous botanical, phytochemical, toxicological, and pharmacological investigation of A. salutaris in Wistar rats. At first, a morphoanatomical characterization of Anchietea pyrifolia leaves and stems was performed. Then, a purified infusion (ethanol-soluble fraction obtained from A. pyrifolia [ESAP]) was obtained followed by its chemical profile elucidation. Furthermore, an acute toxicity test was performed, and the acute and prolonged diuretic and hypotensive effects were also evaluated in Wistar rats. Finally, the vasodilatory responses of ESAP in mesenteric vascular beds were investigated. The main secondary metabolites identified from ESAP were O-glycosylated flavonoids, chlorogenic acids, and phenylpropanoic acid derivatives. ESAP did not promote any toxic effects in female rats nor increased urinary excretion in male rats after a single exposure. However, ESAP significantly reduced renal elimination of sodium, potassium, and chloride after prolonged treatment. An ESAP highest dose promoted significant acute hypotension without affecting blood pressure levels after prolonged use. Furthermore, its cardiovascular effects seem to be related with the calcium-activated potassium channel activation in resistance vessels.

Development and characterization of eleven microsatellite loci for the tropical understory tree Paypayrola blanchetiana Tul. (Violaceae).

Microsatellites markers were developed for Paypayrola blanchetiana (Violaceae), a near-dispersing forest tree forming aggregated populations, to investigate genetic diversity and gene flow among subpopulations in a fragmented environment. Next generation sequencing (Illumina platform) was used to develop ten nuclear microsatellite loci and one plastid microsatellite locus that amplify in P. blanchetiana. Polymorphism was tested in two subpopulations separated by a distance of approximately 11 km. The identified loci contained between two and five alleles per locus. Observed heterozygosity ranged between 0.063 and 0.563 in both subpopulations, while expected heterozygosity ranged from 0.063 to 0.567 in the first, and 0.063-0.627 in the second subpopulation. The microsatellites are among the first in the family Violaceae and will be useful for population genetic studies in this species. Amplification was successful in one further Paypayrola species from Amazonia, which suggest a wider usefulness of the present markers.

Tangled banks: A landscape genomic evaluation of Wallace's Riverine barrier hypothesis for three Amazon plant species.

Wallace's Riverine Barrier hypothesis is one of the earliest biogeographic explanations for Amazon speciation, but it has rarely been tested in plants. In this study, we used three woody Amazonian plant species to evaluate Wallace's Hypothesis using tools of landscape genomics. We generated unlinked single-nucleotide polymorphism (SNP) data from the nuclear genomes of 234 individuals (78 for each plant species) across 13 sampling sites along the Rio Branco, Brazil, for Amphirrhox longifolia (8,075 SNPs), Psychotria lupulina (9,501 SNPs) and Passiflora spinosa (14,536 SNPs). Although significantly different migration rates were estimated between species, the population structure data do not support the hypothesis that the Rio Branco-an allopatric barrier for primates and birds-is a significant genetic barrier for Amphirrhox longifolia, Passiflora spinosa or Psychotria lupulina. Overall, we demonstrated that medium-sized rivers in the Amazon Basin, such as the Rio Branco, are permeable barriers to gene flow for animal-dispersed and animal-pollinated plant species.

A Cyclotide Isolated from Noisettia orchidiflora (Violaceae).

Biologically active cyclotides have been found on some flowering plants species and are involved in the role of the plant protection. As part of studies focusing on peptides from Brazilian plant species, we are reporting the detection by LC-MS of several cyclotides from leaves and stems of Noisettia orchidiflora (Violaceae). From stems it was possible to isolate and characterize a cyclotide named Nor A. Its primary structure (amino acid sequence) was established by MALDI-TOF-MS, based on the y- and b-type ion series, after reduction and alkylation reactions, as well as enzymatic digestion using the enzymes endoproteinase glutamic acid (endoGlu-C), trypsin, and chymotrypsin. Furthermore, the amino acid analysis was also described.

Pinpointing disulfide connectivities in cysteine-rich proteins.

A simple MD-based protocol is presented to accurately predict both the sequence and order of disulfide bond formation in proteins containing multiple cysteine residues. It provides a detailed description of their dynamical and structural features, which can be used to perform ensemble-averaged ECD calculations. Plant cyclotides are used as model compounds.

Wide but not impermeable: Testing the riverine barrier hypothesis for an Amazonian plant species.

Wallace's riverine barrier hypothesis postulates that large rivers, such as the Amazon and its tributaries, reduce or prevent gene flow between populations on opposite banks, leading to allopatry and areas of species endemism occupying interfluvial regions. Several studies have shown that two major tributaries, Rio Branco and Rio Negro, are important barriers to gene flow for birds, amphibians and primates. No botanical studies have considered the potential role of the Rio Branco as a barrier, while a single botanical study has evaluated the Rio Negro as a barrier. We studied an Amazon shrub, Amphirrhox longifolia (A. St.-Hil.) Spreng (Violaceae), as a model to test the riverine barrier hypothesis. Twenty-six populations of A. longifolia were sampled on both banks of the Rio Branco and Rio Negro in the core Amazon Basin. Double-digest RADseq was used to identify 8,010 unlinked SNP markers from the nuclear genome of 156 individuals. Data relating to population structure support the hypothesis that the Rio Negro acted as a significant genetic barrier for A. longifolia. On the other hand, no genetic differentiation was detected among populations spanning the narrower Rio Branco, which is a tributary of the Rio Negro. This study shows that the strength of riverine barriers for Amazon plants is dependent on the width of the river separating populations and species-specific dispersal traits. Future studies of plants with contrasting life history traits will further improve our understanding of the landscape genetics and allopatric speciation history of Amazon plant diversity.

Minimum sample sizes for population genomics: an empirical study from an Amazonian plant species.

High-throughput DNA sequencing facilitates the analysis of large portions of the genome in nonmodel organisms, ensuring high accuracy of population genetic parameters. However, empirical studies evaluating the appropriate sample size for these kinds of studies are still scarce. In this study, we use double-digest restriction-associated DNA sequencing (ddRADseq) to recover thousands of single nucleotide polymorphisms (SNPs) for two physically isolated populations of Amphirrhox longifolia (Violaceae), a nonmodel plant species for which no reference genome is available. We used resampling techniques to construct simulated populations with a random subset of individuals and SNPs to determine how many individuals and biallelic markers should be sampled for accurate estimates of intra- and interpopulation genetic diversity. We identified 3646 and 4900 polymorphic SNPs for the two populations of A. longifolia, respectively. Our simulations show that, overall, a sample size greater than eight individuals has little impact on estimates of genetic diversity within A. longifolia populations, when 1000 SNPs or higher are used. Our results also show that even at a very small sample size (i.e. two individuals), accurate estimates of FST can be obtained with a large number of SNPs (≥1500). These results highlight the potential of high-throughput genomic sequencing approaches to address questions related to evolutionary biology in nonmodel organisms. Furthermore, our findings also provide insights into the optimization of sampling strategies in the era of population genomics.

Identification and determination of the inulin content in the roots of the Northeast Brazilian species Pombalia calceolaria L.

A polysaccharide was extracted from the roots of Pombalia calceolaria, a plant used in folk medicine in Northeastern Brazil, by decoction followed by precipitation with methanol, yielding a concentration of 13.0% w/w, and purification with acetone. The molar mass peak was estimated to be 4.0×10(3)Da using gel permeation chromatography (GPC). Polarized light photomicrography of histological sections revealed the presence of inulin in the cortical parenchyma. The chemical composition of inulin was identified by 1D and 2D NMR and FT-IR spectroscopy and the findings were compared with the literature. This is the first time inulin has been identified on FT-IR and NMR for the species Pombalia calceolaria.

Hybanthus calceolaria poisoning in cattle.

Hybanthus calceolaria, also known as "papaconha" or "ipepacuanha," is a herbaceous plant found in northeastern Brazil, which is often implicated by farmers as the cause of neurological signs in livestock grazing. Several poisoning outbreaks associated with the ingestion of this plant were observed in cattle in the municipalities of Colônia de Gurguéia in the state of Piauí and Sirinhaém in the state of Pernambuco, Brazil. The main clinical signs were ataxia, recumbency, and myokymia. No significant lesions were observed during necropsy or on histological examination. The disease was experimentally reproduced by the administration of 2 daily doses of 40 g/kg/body weight of the fresh green plant containing fruits. The plants without fruits were nontoxic, which is in accordance with the farmers' information, as it was stated that the poisoning only occurs when the plant is fruiting.

Neutral dynamics and cluster statistics in a tropical forest.

The neutral theory of biodiversity attributes community structure to the effects of chance alone, assuming that all species and individuals are demographically equivalent. Here we present a spatially explicit version of the neutral theory and test it against the Barro Colorado Island (BCI) data. Monitoring the dynamics of clusters, we show that the effect of local heterogeneities (e.g., microtopography) is weak, making a spatially homogenous model plausible. We then compare the cluster statistics of the three most frequent species with the patterns obtained from neutral dynamics, examining two families of recruitment kernels: one that interpolates between a limited distance and panmictic dispersal (local-global) and one that assumes a scale-free Cauchy kernel. The results rule out the local-global dispersal model and show that the spatial patterns fit very nicely those obtained from the fat-tailed kernel. Our work emphasizes the importance of spatiotemporal cluster dynamics as an instrument for detecting the factors that govern community assembly.

Viola arguta (Violaceae), una especie de la medicina tradicional ecuatoriana. Elementos de diagnóstico, ensayos fitoquímicos y actividades biológicas / Viola arguta (Violaceae), a species of traditional Ecuadorian medicine. Diagnostic elements, phytochemical assays and biological activities

Ecuador se reconoce desde la Constitución del año 1998 como un país pluricultural y multiétnico y que además está ubicado en uno de los sitios de mayor megabiodiversidad del planeta. Dentro de esa biodiversidad, Viola arguta es una planta endémica del país, que crece en la zona cordillerana, a altitudes de 1500 a 3500 msm, en las provincias de Zamora-Chinchipe, Azuay, Bolívar, Cañar, Carchi, Chimborazo, Cotopaxi, Imbabura, Loja, Morona-Santiago, Pichincha. Conocida como violeta del campo o violeta roja, es utilizada tradicionalmente para el tratamiento de diversas afecciones, entre ellas algunas relacionadas con la capacidad antiinflamatoria que se le atribuye. Hasta donde sabemos, no existen hasta el momento datos botánicos que permitan su reconocimiento, ni información sobre su composición química y actividades biológicas que justifiquen su empleo en terapéutica. A fin de proveer elementos de diagnóstico que contribuyan a su identificación, y eventual control de calidad, así como contribuir a su estudio fitoquímico se encara el análisis micrográfico de esta especie, así como se efectúan los ensayos fitoquímicos preliminares, detección de metabolitos secundarios característicos del género Viola y la determinación de la acción antioxidante y actividad antiinflamatoria.

Insights in the knowledge of two Argentine medicinal species of the genus Hybanthus (Violaceae) / Contribución al estudio de dos especies medicinales argentinas del género Hybanthus (Violaceae)

A differential morphoanatomical study was performed in two Argentine species: Hybanthus parviflorus and Hybanthus bigibbosus. Fresh material fixed in acetoalcoholic formalin and material from herbarium were employed. Paradermal and longitudinal sections of stems and transversal sections of leaves were prepared and stained with aqueous alcoholic safranin. The hydrochloric fluoroglucyn test was performed. Known and novel characters (distribution of vascular bundles of the primary stem and type and relative length of cover hairs and cuticular ornamentation of the adaxial epidermis, respectively) prove to be of value for the recognition of these species.Furthermore, the intestinal propulsor activity, related to the popular uses of these species was also assessed. Infusions of the whole plant, their roots and the 50 percent EtOH extracts of the aerial parts of H. parviflorus and H. bigibbosus were tested by the activated charcoal method. The extracts of H. parviflorus proved to have a significant activity which would allow validating its popular use.

Se encaró el estudio morfoanatómico diferencial de dos especies argentinas: Hybanthus parviflorus e Hybanthus bigibbosus. Se trabajó con material fresco fijado en formalina aceto-alcohólica y de herbario. Se realizaron preparados paradermales y cortes longitudinales y transversales de tallos y transversales de hoja empleando safranina alcohólico-acuosa y el test de floroglucina clorhídrica. Caracteres conocidos (distribución de haces vasculares en tallo primario) y novedosos (tipología y longitud relativa de tricomas tectores; ornamentación cuticular de la epidermis adaxial) poseen valor diferencial para el reconocimiento de estas especies.Asimismo se determinó la actividad de propulsión intestinal de ambas especies, relacionada con su uso popular. Se ensayaron las infusiones de la raíz y de las partes aéreas y el extracto EtOH 50 por ciento de las partes aéreas de H. parviflorus e H. bigibbosus, empleando el método del carbón activado. Los extractos de H. parviflorus presentaron una actividad significativa que permitiría validar su uso popular.

Does human-induced habitat transformation modify pollinator-mediated selection? A case study in Viola portalesia (Violaceae).

Pollinator-mediated selection is one of the most important factors driving adaptation in flowering plants. However, as ecological conditions change through habitat loss and fragmentation, the interactions among species may evolve in new and unexpected directions. Human-induced environmental variation is likely to affect selection regimes, but as yet no empirical examples have been reported. In the study reported here, we examined the influence of human-induced habitat transformation on the composition of pollinator assemblages and, hence, pollinator-mediated selection on the flower phenotype of Viola portalesia (Violaceae). Our results indicate that pollinator assemblages differed substantially in terms of species composition and visitation rate between nearby native and transformed habitats. Similarly, the insect species that contributed most to visitation rates differed between plant populations. While the magnitude and sign of pollinator-mediated selection on flower length and width did not differ between sites, selection for flower number lost significance in the transformed habitat, and a significant pattern of disruptive selection for flower shape, undetected in the native habitat, was present in the transformed one. Overall, the results of this study suggest that human-induced habitat change may not only modify the species composition of pollinator assemblages, relaxing the selection process on some flower characters, but they may also create new opportunities for fitness-trait covariation not present in pristine conditions.

Cyclotide proteins and precursors from the genus Gloeospermum: filling a blank spot in the cyclotide map of Violaceae.

Cyclotides are disulfide-rich plant proteins that are exceptional in their cyclic structure; their N and C termini are joined by a peptide bond, forming a continuous circular backbone, which is reinforced by three interlocked disulfide bonds. Cyclotides have been found mainly in the coffee (Rubiaceae) and violet (Violaceae) plant families. Within the Violaceae, cyclotides seem to be widely distributed, but the cyclotide complements of the vast majority of Violaceae species have not yet been explored. This study provides insight into cyclotide occurrence, diversity and biosynthesis in the Violaceae, by identifying mature cyclotide proteins, their precursors and enzymes putatively involved in their biosynthesis in the tribe Rinoreeae and the genus Gloeospermum. Twelve cyclotides from two Panamanian species, Gloeospermum pauciflorum Hekking and Gloeospermum blakeanum (Standl.) Hekking (designated Glopa A-E and Globa A-G, respectively) were characterised through cDNA screening and protein isolation. Screening of cDNA for the oxidative folding enzymes protein-disulfide isomerase (PDI) and thioredoxin (TRX) resulted in positive hits in both species. These enzymes have demonstrated roles in oxidative folding of cyclotides in Rubiaceae, and results presented here indicate that Violaceae plants have evolved similar mechanisms of cyclotide biosynthesis. We also describe PDI and TRX sequences from a third cyclotide-expressing Violaceae species, Viola biflora L., which further support this hypothesis.