RESUMO
Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally comparable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclotides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bioinsecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.
Assuntos
Ciclotídeos , Drosophila melanogaster , Inseticidas , Proteínas de Plantas , Violaceae , Animais , Humanos , Sequência de Aminoácidos , Ciclotídeos/química , Ciclotídeos/isolamento & purificação , Ciclotídeos/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Inseticidas/química , Inseticidas/isolamento & purificação , Inseticidas/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Violaceae/química , Eritrócitos/efeitos dos fármacosRESUMO
Cyclotides are a unique family of stable and cyclic mini-proteins found in plants that have nematicidal and anthelmintic activities. They are distributed across the Rubiaceae, Violaceae, Fabaceae, Cucurbitaceae, and Solanaceae plant families, where they are posited to act as protective agents against pests. In this study, we tested the nematicidal properties of extracts from four major cyclotide-producing plants, Oldenlandia affinis, Clitoria ternatea, Viola odorata, and Hybanthus enneaspermus, against the free-living model nematode Caenorhabditis elegans. We evaluated the nematicidal activity of the cyclotides kalata B1, cycloviolacin O2, and hyen D present in these extracts and found them to be active against the larvae of C. elegans. Both the plant extracts and isolated cyclotides exerted dose-dependent toxicity on the first-stage larvae of C. elegans. Isolated cyclotides caused death or damage upon interacting with the worms' mouth, pharynx, and midgut or membrane. Cycloviolacin O2 and hyen D produced bubble-like structures around the C. elegans membrane, termed blebs, implicating membrane disruption causing toxicity and death. All tested cyclotides lost their toxicity when the hydrophobic patches present on them were disrupted via a single-point mutation. The present results provide a facile assay design to measure and explore the nematicidal activities of plant extracts and purified cyclotides on C. elegans.
Assuntos
Ciclotídeos , Fabaceae , Nematoides , Violaceae , Animais , Antinematódeos/farmacologia , Caenorhabditis elegans , Ciclotídeos/farmacologia , Ciclotídeos/química , Fabaceae/química , Extratos Vegetais/química , Proteínas de Plantas/químicaRESUMO
In this study, phytoremediation potential of toxic metals like selenium (Se) and lanthanum (La) by transformed hairy roots was investigated and compared with plantlets under in-vitro conditions. Agrobacterium rhizogenes A4RS could induce hairy roots with higher biomass in 5-7 days of infection on in-vitro leaves of Hybanthus enneaspermus. The ICP-OES data indicated that the hairy roots were able to accumulate both selenium and lanthanum efficiently compared to plantlets. The hairy roots and plantlets show optimum absorption at 50 ppm under both individual and combined metal supply. The metal accumulation performances increased by 13.6% (La) and 10.9% (Se) in hairy roots with combined metal supply (La and Se) compared to individual supply (La or Se) conditions. The Se accumulated more than La, but the La accumulation percentage was found to increase substantially under combined metal supply conditions, shows the suitability and potential of hairy roots for phytoremediation of La and Se.
Assuntos
Selênio , Violaceae , Plantas Geneticamente Modificadas , Lantânio , Adsorção , Biodegradação Ambiental , Raízes de Plantas/microbiologia , Agrobacterium/genéticaRESUMO
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.
Assuntos
Produtos Biológicos , Ciclotídeos , Proteínas de Plantas , Violaceae , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Brasil , Linhagem Celular Tumoral , Ciclotídeos/química , Ciclotídeos/isolamento & purificação , Ciclotídeos/farmacologia , Humanos , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Espectrometria de Massas em Tandem , Violaceae/químicaRESUMO
Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1' sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs.
Assuntos
Cisteína Endopeptidases/metabolismo , Ligases/metabolismo , Peptídeos Cíclicos/metabolismo , Proteínas de Plantas/metabolismo , Violaceae/metabolismo , Mutagênese/fisiologiaRESUMO
Cyclotides are plant-derived peptides characterized by an â¼30-amino acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ciclotídeos/farmacologia , Descoberta de Drogas , Plantas Medicinais/química , Violaceae/química , Sequência de Aminoácidos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , Ciclotídeos/química , Ciclotídeos/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em TandemRESUMO
Cyclotides are plant-derived peptides that have attracted interest as biocides and scaffolds for the development of stable peptide therapeutics. Cyclotides are characterized by their cyclic backbone and cystine knot framework, which engenders them with remarkably high stability. This study reports the cystine knot-related peptidome of Rinorea bengalensis, a small rainforest tree in the Violaceae family that is distributed from Australia westward to India. Surprisingly, many more acyclic knotted peptides (acyclotides) were discovered than cyclic counterparts (cyclotides), with 32 acyclotides and 1 cyclotide sequenced using combined transcriptome and proteomic analyses. Nine acyclotides were isolated and screened against a panel of mammalian cell lines, showing they had the cytotoxic properties normally associated with cyclotide-like peptides. NMR analysis of the acyclotide ribes 21 and 22 and the cyclotide ribe 33 confirmed that these peptides contained the cystine knot structural motif. The bracelet-subfamily cyclotide ribe 33 was amenable to chemical synthesis in reasonable yield, an achievement that has long eluded previous attempts to synthetically produce bracelet cyclotides. Accordingly, ribe 33 represents an exciting new bracelet cyclotide scaffold that can be subject to chemical modification for future molecular engineering applications.
Assuntos
Ciclotídeos/síntese química , Cistina/química , Violaceae/química , Linhagem Celular Tumoral , Ciclotídeos/química , Eritrócitos/efeitos dos fármacos , Humanos , Extratos Vegetais/química , Proteínas de Plantas/química , Proteômica , Queensland , TranscriptomaRESUMO
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.
Assuntos
Ciclotídeos/efeitos dos fármacos , Ciclotídeos/metabolismo , Fabaceae/química , Linfócitos/metabolismo , Solanaceae/química , Violaceae/química , Brasil , Ciclotídeos/química , Humanos , Linfócitos/química , Linfócitos/efeitos dos fármacos , Magnoliopsida , Espectrometria de Massas , Folhas de Planta/química , Folhas de Planta/metabolismoRESUMO
To elucidate the origin and migration history of the "Mansen elements," a group of temperate grassland plants mainly distributed in northeastern Asia, phylogeographic analyses based on chloroplast DNA markers and double-digest restriction site-associated DNA sequencing (ddRAD-seq) data were performed on Viola orientalis, one of the representative species of the group. Phylogenetic analyses using ddRAD-seq data revealed that the populations of V. orientalis were clustered into five clades, among which the continental clades made of populations from Russia and Korea diverged more than 100,000 years earlier than the Japanese clades. The Japanese clade likely diverged during the last glacial period, followed by a further post-glacial divergence into the Kyushu and the Honshu subclades. Our study demonstrated that V. orientalis originated in the continental area of northeastern Asia and, during the last glacial period, has spread southward through the Korean Peninsula across the Japanese Islands. This finding supports the previously proposed evolutionary hypothesis regarding the origin and migration routes of the Mansen elements.
Assuntos
Viola , Violaceae , Variação Genética , Pradaria , Filogenia , Filogeografia , Plastídeos , Análise de Sequência de DNARESUMO
BACKGROUND AND AIMS: Hybanthus austrocaledonicus (Violaceae) is a nickel (Ni) hyperaccumulator endemic to New Caledonia. One of the specimens stored at the local herbarium had a strip of bark with a remarkably green phloem tissue attached to the sheet containing over 4 wt% Ni. This study aimed to collect field samples from the original H. austrocaledonicus locality to confirm the nature of the green 'nickel-rich phloem' in this taxon and to systematically assess the occurrence of Ni hyperaccumulation in H. austrocaledonicus and Hybanthus caledonicus populations. METHODS: X-ray fluorescence spectroscopy scanning of all collections of the genus Hybanthus (236 specimens) was undertaken at the Herbarium of New Caledonia to reveal incidences of Ni accumulation in populations of H. austrocaledonicus and H. caledonicus. In parallel, micro-analytical investigations were performed via synchrotron X-ray fluorescence microscopy (XFM) and scanning electron microscopy with X-ray microanalysis (SEM-EDS). KEY RESULTS: The extensive scanning demonstrated that Ni hyperaccumulation is not a characteristic common to all populations in the endemic Hybanthus species. Synchrotron XFM revealed that Ni was exclusively concentrated in the epidermal cells of the leaf blade and petiole, conforming with the majority of (tropical) Ni hyperaccumulator plants studied to date. SEM-EDS of freeze-dried and frozen-hydrated samples revealed the presence of dense solid deposits in the phloem bundles that contained >8 wt% nickel. CONCLUSIONS: The occurrence of extremely Ni-rich green phloem tissues appears to be a characteristic feature of tropical Ni hyperaccumulator plants.
Assuntos
Níquel , Violaceae , Nova Caledônia , Níquel/análise , Floema , Folhas de Planta/químicaRESUMO
Hybanthus enneaspermus is an Indian folk medicinal herb that has been widely used as a libido enhancer. This plant belongs to the Violaceae plant family, which ubiquitously contains disulfide-rich cyclic peptides named cyclotides. Cyclotides are an expanding plant-derived peptide family with numerous interesting bioactivities, and their unusual stability against proteolysis has attracted much attention in drug design applications. Recently, H. enneaspermus has been reported to be a rich source of cyclotides, and hence, it was of interest to investigate whether cyclotides contribute to its aphrodisiac activity. In this study, we evaluated the in vivo aphrodisiac activity of the herbal powder, extract, and the most abundant cyclotide, hyen D, extracted from H. enneaspermus on rats in a single dose regimen. After dosing, the sexual behaviors of male rats were observed, recorded, analyzed, and compared with those of the vehicle group. The results show that the extract and hyen D significantly decreased the intromission latency of sexually naïve male rats and the extract improved a range of other measured sexual parameters. The results suggest that the extract could enhance libido as well as facilitate erectile function in male rats and that the cyclotide hyen D could contribute to the libido-enhancing activity of this ethnomedicinal herb.
Assuntos
Afrodisíacos/farmacologia , Extratos Vegetais/farmacologia , Violaceae/química , Animais , Feminino , Masculino , Ratos , Comportamento Sexual AnimalRESUMO
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.
Assuntos
Especiação Genética , Genômica , Violaceae/genética , Brasil , Fluxo Gênico , Genoma de Planta/genética , Polimorfismo de Nucleotídeo Único/genética , RiosRESUMO
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.
Assuntos
Repetições de Microssatélites/genética , Violaceae/genética , Alelos , Brasil , Mapeamento Cromossômico/métodos , Primers do DNA , Loci Gênicos/genética , Genética Populacional/métodos , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Polimorfismo Genético/genética , Especificidade da Espécie , Árvores/genéticaRESUMO
Cyclotides are macrocyclic cystine-knotted peptides most commonly found in the Violaceae plant family. Although Rinorea is the second-largest genera within the Violaceae family, few studies have examined whether or not they contain cyclotides. To further our understanding of cyclotide diversity and evolution, we examined the cyclotide content of two Rinorea species found in Southeast Asia: R. virgata and R. bengalensis. Seven cyclotides were isolated from R. virgata (named Rivi1-7), and a known cyclotide (cT10) was found in R. bengalensis. Loops 2, 5, and 6 of Rivi1-4 contained sequences not previously seen in corresponding loops of known cyclotides, thereby expanding our understanding of the diversity of cyclotides. In addition, the sequence of loop 2 of Rivi3 and Rivi4 were identical to some related noncyclic "acyclotides" from the Poaceae plant family. As only acyclotides, but not cyclotides, have been reported in monocotyledons thus far, our findings support an evolutionary link between monocotyledon-derived ancestral cyclotide precursors and dicotyledon-derived cyclotides. Furthermore, Rivi2 and Rivi3 had comparable cytotoxic activities to the most cytotoxic cyclotide known to date: cycloviolacin O2 from Viola odorata; yet, unlike cycloviolacin O2, they did not show hemolytic activity. Therefore, these cyclotides represent novel scaffolds for use in future anticancer drug design.
Assuntos
Ciclotídeos/química , Violaceae/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Extratos Vegetais/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Ciclotídeos/isolamento & purificação , Violaceae/química , Sequência de Aminoácidos , Cromatografia Líquida , Ciclotídeos/química , Folhas de Planta/química , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Caules de Planta/química , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Fluxo Gênico , Dispersão Vegetal , Rios , Violaceae/genética , Brasil , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
This study evaluated the effects of Hybanthus enneaspermus aqueous extract (HEE) as a feed supplement on growth, immune responses, haematological, and biochemical parameters of Labeo rohita and its susceptibility to Aeromonas hydrophila infection. Diets containing five variable concentrations of HEE (g kg-1) (0 [basal diet], 1.0 [H1], 2.0 [H2], 3.0 [H3], and 4.0 [H4]) were fed to fish (average weight: 17.2 ± 0.21 g) for 6 weeks. Growth parameters, immune parameters, and haemato-biochemical parameters were examined 6 weeks post-feeding. The results showed that growth parameters, such as final weight (39.47 ± 3.1 g) and specific growth rate (1.92 ± 0.2), were significantly higher in the H3 group than in the control. Immunological parameters such as serum lysozyme (24.73 ± 2.16 U mg-1), phagocytic activity (19.14± 0.26%), and respiratory burst activity (0.307 ± 0.005) were significantly higher in the H3 group, compared with the control. Among the haematological parameters examined, red blood cells, white blood cells, neutrophils, total serum protein, and albumin levels were significantly higher in the H3 group. However, HEE administration had no significant effect on haematocrit, haemoglobin, monocytes, or eosinophils levels. Blood biochemical parameters, including superoxide dismutase and alkaline phosphatase activities, were higher (P < 0.05) in the H3 group than in the control; however, the opposite result was found for aspartate transaminase, alanine transaminase, glucose, and cholesterol. Supplementation with 3 g kg-1 HEE increased the activities of intestinal digestive enzyme (amylase, protease, and lipase) in fish. Moreover, fish fed diets supplemented with 3 g kg-1 HEE exhibited the highest disease resistance against A. hydrophila infection. These results suggest that dietary administration of HEE (at 3 g kg-1) has several positive effects on growth, immune and haematological responses in L. rohita.
Assuntos
Cyprinidae/crescimento & desenvolvimento , Cyprinidae/imunologia , Suplementos Nutricionais , Doenças dos Peixes/imunologia , Imunidade Inata , Extratos Vegetais/metabolismo , Violaceae/química , Aeromonas hydrophila/imunologia , Ração Animal/análise , Animais , Cyprinidae/sangue , Dieta/veterinária , Resistência à Doença , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Distribuição AleatóriaRESUMO
Cyclotides are a large family of naturally occurring plant-derived macrocyclic cystine-knot peptides, with more than 400 having been identified in species from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families. Nevertheless, their specialized distribution within the plant kingdom remains poorly understood. In this study, the diversity of cyclotides was explored through the screening of 197 plants belonging to 43 different families. In total, 28 cyclotides were sequenced from 15 plant species, one of which belonged to the Rubiaceae and 14 to the Violaceae. Every Violaceae species screened contained cyclotides, but they were only sparsely represented in Rubiaceae and nonexistent in other families. The study thus supports the hypothesis that cyclotides are ubiquitous in the Violaceae, and it adds to the list of plants found to express kalata S and cycloviolacin O12. Finally, previous studies suggested the existence of cyclotide isoforms with either an Asn or an Asp at the C-terminal processing site of the cyclotide domain within the precursor proteins. Here we found that despite the discovery of a few cyclotides genuinely containing an Asp in loop 6 as evidenced by gene sequencing, deamidation of Asn during enzymatic digestion resulted in the artifactual presence of Asp isoforms. This result is consistent with studies suggesting that peptides can undergo deamidation after being subjected to external factors, including pH, temperature, and enzymatic digestion.
Assuntos
Ciclotídeos/isolamento & purificação , Cistina/isolamento & purificação , Fabaceae/química , Proteínas de Plantas/isolamento & purificação , Rubiaceae/química , Solanaceae/química , Violaceae/química , Sequência de Aminoácidos , Ciclotídeos/química , Cistina/química , Estrutura Molecular , Proteínas de Plantas/químicaRESUMO
CONTEXT: Ionidium suffruticosum (L.) Ging (Violaceae) is an important medicinal plant widely used as a herbal traditional medicine in Ayurveda for the treatment of infertility. Currently, little pharmacological information is available on its male fertility properties following prolonged use. OBJECTIVE: To investigate I. suffruticosum leaf extracts for male fertility parameters. MATERIALS AND METHODS: The ethanol lyophilized fraction was administered orally on carbendazim-induced sub-fertility rats (250 mg/kg body weight for 28 days). The effects of fractions on rat's fertility parameters i.e., body and testes weight, sperm motility, sperm vitality, epididymal sperm counts, its morphology, enzyme and antioxidant stress and histopathology were studied and compared with clomiphene citrate. RESULTS: The sub-fertile male rats treated with I. suffruticosum leaf extract increased the body weight of 7 g, testis weight of 97 mg, increased cauda epididymal sperm counts of 34.2 × 106 sperm/mL, motility of sperm 46% and vitality 28% also increased and normal sperm morphology also improved up to 32%. The carbendazim-treated group showed loss in body weight of 33 g, testis weight of 851 mg, decreased epididymal sperm counts of 15 × 106 sperm/mL, with sluggish motility and a highly significant fall in the live sperms of about 57%. DISCUSSION AND CONCLUSION: The leaf fraction of I. suffructicosum increased the testicular weight, spermatogenesis, sperm counts, lessened sperm agglutination, and increased testicular oxidative biomarkers, SOD, and CAT. This study therefore supports the usage of I. suffructicosum in traditional medicine for infertility.
Assuntos
Epididimo/efeitos dos fármacos , Fertilidade/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/farmacologia , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos , Violaceae , Animais , Benzimidazóis/toxicidade , Peso Corporal/efeitos dos fármacos , Carbamatos/toxicidade , Masculino , Folhas de Planta , Ratos , Ratos Wistar , Motilidade dos Espermatozoides/efeitos dos fármacos , Testículo/metabolismo , Testículo/patologiaRESUMO
Cyclotides are plant-derived cyclic peptides that have a head-to-tail cyclic backbone and three conserved disulphide bonds that form a cyclic cystine knot motif. They occur in plants from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families, typically with 10-100 cyclotides in a given plant species, in a wide range of tissues, including flowers, leaves, stems, and roots. Some cyclotides are expressed in large amounts (up to 1g kg(-1) wet plant weight) and their natural function appears to be to protect plants from pests or pathogens. This article provides a brief overview of their discovery, distribution in plants, and applications. In particular, their exceptional stability has led to their use as peptide-based scaffolds in drug design applications. They also have potential as natural 'ecofriendly' insecticides, and as protein engineering frameworks.