RESUMEN
Callingcard Vine (Entada polystachya (L.) DC. var. polystachya - Fabaceae) is a common plant in coastal thickets from western Mexico through Central America to Colombia and Brazil, especially in Amazon biome. It has been popularly used as a urinary burning reliever and diuretic. However, the plant chemical constituents are poorly understood and Entada spp. genotoxic potential have not been previously investigated. In the present study we determined the chemical composition of the aqueous E. polystachya crude seed extract (EPCSE) and evaluated the cytotoxic, genotoxic and mutagenic properties of EPCSE in Salmonella typhimurium and Chinese hamster ï¬broblast (V79) cells. Cytotoxic activity was also evaluated in tumor cell lines (HT29, MCF7 and U87) and non-malignant cells (MRC5). The chemical analysis by High Resolution Mass Spectrometry (HRMS) of EPCSE indicated the presence of saponin and chalcone. The results of the MTT and clonal survival assays suggest that EPCSE is cytotoxic to V79 cells. Survival analysis showed higher IC50 in non-tumor compared with tumor cell lines. EPCSE showed induction of DNA strand breaks as revealed by the alkaline comet assay and micronucleus test. Using the modified comet assay, it was possible to detect the induction of oxidative DNA base damage by EPCSE in V79 cells. Consistently, the extract induced increase lipid peroxidation (TBARS), superoxide dismutase (SOD) and catalase (CAT) activities in V79 cells. In addition, EPCSE induced mutations in S. typhimurium TA98 and TA100 strains, confirming a mutagenic potential. Taken together, our results suggest that EPCSE is cytotoxic and genotoxic to V79 cells and mutagenic to S. typhimurium. These properties can be related to the pro-oxidant ability of the extract and induction of DNA lesions. Additionally, EPCSE could inhibit the growth of tumor cells, especially human colorectal adenocarcinoma (HT29) cell line, and can constitute a possible source of antitumor natural agents.
Asunto(s)
Antineoplásicos , Fabaceae , Cricetinae , Animales , Humanos , Mutágenos/toxicidad , Daño del ADN , Cricetulus , Ensayo Cometa , Línea Celular Tumoral , Extractos Vegetales/toxicidad , ADNRESUMEN
Using a rat model of peritonitis, we herein report the inflammatory effect induced by the lectin isolated from Vatairea guianensis (VGL) seeds in the context of interactions between VGL and both toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1). Peritoneal macrophages were stimulated with VGL for dose-dependent gene expression and release of TNF-α. In vivo results showed that VGL (1 mg/kg; intraperitoneal) induced peritonitis in female Wistar rats. Leukocyte migration, macrophage activation, and protein leakage were measured 3 and 6 hours after induction. In vitro, peritoneal macrophages were stimulated with VGL for gene expression and TNF-α dosage (mean ± SEM (n = 6), analysis of variance, and Bonferroni's test (P < .05)). In silico, VGL structure was applied in molecular docking with representative glycans. It was found that (a) VGL increases vascular permeability and stimulates leukocyte migration, both rolling and adhesion; (b) lectin-induced neutrophil migration occurs via macrophage stimulation, both in vitro and in vivo; (c) lectin interacts with TLR4 and TNFR1; and (d) stimulates TNF-α gene expression (RT-PCR) and release from peritoneal macrophages. Thus, upon lectin-glycan binding on the cell surface, our results suggest that VGL induces an acute inflammatory response, in turn activating the release of peritoneal macrophages via TNF-α and TLR and/or TNFR receptor pathways.
Asunto(s)
Fabaceae/química , Glicoconjugados/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Lectinas de Plantas/farmacología , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glicoconjugados/química , Leucocitos/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Peritonitis/inducido químicamente , Peritonitis/metabolismo , Peritonitis/patología , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Ratas Wistar , Receptores Tipo I de Factores de Necrosis Tumoral/química , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A novel lectin from seeds of Clathrotropis nitida (CNA) was purified and characterized. CNA is a glycoprotein containing approximately 3.3% carbohydrates in its structure. CNA promoted intense agglutination of rabbit erythrocytes, which was inhibited by galactosides and porcine stomach mucin (PSM). The lectin maintained its hemagglutinating activity after incubation in a wide range of temperatures (30-60 °C) and pH (6.0-7.0), and its binding activity was dependent on divalent cations (Ca(+2) and Mg(+2)). SDS-PAGE showed an electrophoretic profile consisting of a single band of 28 kDa, as confirmed by electrospray ionization mass spectrometry, which indicated an average molecular mass of 27,406 ± 2 Da and the possible presence of isoforms and glycoforms. In addition, CNA exhibited no toxicity to Artemia sp. nauplii and elicited reversible and dose-dependent vasorelaxation in precontracted aortic rings. CNA was successfully immobilized on chitosan beads and was able to capture PSM in solution. This study demonstrated that CNA is a lectin that has potential as a biotechnological tool in glycomics and glycoproteomics applications.
Asunto(s)
Fabaceae/química , Lectinas de Plantas/aislamiento & purificación , Lectinas de Plantas/farmacología , Vasodilatadores/aislamiento & purificación , Vasodilatadores/farmacología , Secuencia de Aminoácidos , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Artemia/efectos de los fármacos , Quitosano , Fabaceae/genética , Hemaglutinación/efectos de los fármacos , Humanos , Proteínas Inmovilizadas/química , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Peso Molecular , Lectinas de Plantas/genética , Plantas Medicinales/química , Plantas Medicinales/genética , Conejos , Ratas , Ratas Wistar , Semillas/química , Homología de Secuencia de Aminoácido , Vasodilatadores/químicaRESUMEN
Os inibidores de proteinases são proteínas extensivamente investigadas nos tecidos de estocagem, mas pouco prospectadas em outros tecidos vegetais. O objetivo deste estudo foi detectar a presença de inibidores de serinoproteinases em extratos foliares de quinze espécies de leguminosas arbóreas da Amazônia. As espécies estudadas foram: Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata e S. polyphylla. Folhas foram coletadas, secas a 30ºC durante 48 h, trituradas e submetidas à extração com NaCl (0,15 M, 10 por cento p/v) resultando no extrato total. Ensaios foram executados para determinar a concentração de proteínas e detectar a atividade inibitória contra a tripsina e quimotripsina bovina. Os teores de proteínas bruta e solúvel nos extratos foliares variaram de 7,9 a 31,2 por cento e 1,3 a 14,8 por cento, respectivamente. A atividade inibitória sobre a tripsina e quimotripsina foi observada em todos os extratos foliares. Contudo, nos extratos de E. maximum, L. leucocephala, P. pendula, S. corrugata e S. polyphylla a inibição foi maior sobre a tripsina, enquanto o extrato de P. multijuga foi mais efetivo contra a quimotripsina. Nós concluímos que nos extratos foliares de leguminosas arbóreas têm inibidores de serinoproteinases e exibem potencial aplicações biotecnológicas.
The proteinase inhibitors are proteins extensively investigated in tissue storage, but few prospected in other plant tissues. The aim of this study was to detect the presence of serine proteinase inhibitors in leaf extracts from fifteen species of leguminous trees of the Amazon forest. The species studied were Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata and S. polyphylla. Leaves were collected, dried at 30ºC for 48 h, crushed and subjected to extraction with NaCl (0.15 M, 10 percent w/v), resulting in the total extract. Tests were performed to determine the concentration of proteins and to detect of inhibitory activity against bovine trypsin and chymotrypsin. The content of crude and soluble protein in leaf extracts varied from 7.9 to 31.2 percent and 1.3 to 14.8 percent, respectively. The inhibitory activity on trypsin and chymotrypsin was observed in all leaf extracts. However, in extracts of E. maximum, L. leucocephala, P. pendula, S. corrugata and S. polyphylla, the inhibition was greater on trypsin, while extract of P. multijuga was more effective against chymotrypsin. We conclude that leaf extracts of leguminous trees have serine proteinase inhibitors and show potential biotecnological applications.
Asunto(s)
Quimotripsina , Tripsina , ProteínasRESUMEN
Los estudios de requerimientos nutricionales son de gran importancia para identificar aquellos más importantes en el desarrollo fisiológico y crecimiento de plántulas. Con el objetivo de evaluar las exigencias nutricionales y los efectos de la omisión de macronutrientes en el crecimiento de plántulas de Aniba rosaeodora; se realizó un experimento en el vivero del INPA-Amazonas-Brasil teniendo como substrato un suelo Podozolico Rojo de baja disponibilidad de nutrientes. Se utilizaron 8 tratamientos bajo la técnica del nutriente faltante: Control (Suelo con macronutrientes), Suelo natural, y la omisión de un macronutriente por vez (-N, -P, -K, -Ca, -Mg, -S). Se evaluaron las siguientes características: tasa de crecimiento relativo (TCR), Tasa de asimilación neta (TAN), peso de la materia seca de la parte aérea (MSPA) y de las raíces (MSR), contenido de nutrientes en las hojas, concluyendo que el N, Mg y Ca, demostraron ser limitantes al crecimiento en suelo con pequeña disponibilidad; Las plántulas de A. rosaeodora presentaron un bajo requerimiento nutricional para el P, K y S. La omisión de Ca y N perjudica TCR de la especie. Los elementos más importantes para la MSPA fueron el Ca y el Mg; actuando el Mg más en el área foliar; por otro lado la omisión de azufre favorece la absorción de macronutrientes (N, P, K, Ca, Mg).
Studies of nutritional requirements are of great importance for identifying the most important nutrients in physiologic development and seedling growth. A greenhouse pot experiment was conducted at INPA to evaluate the mineral nutritional demands and the effects of macronutrient omission in the plant growth of Aniba rosaeodora Ducke. The following treatments were used: Complete (fertilization with N, P, K, Ca, Mg, S, B and Zn), Standard (nature soil), Complete without N, Complete without P, Complete without K, Complete without Ca, Complete without Mg, and Complete without S. An Ultisoil with low nutrient availability was used as a substratum. The following characteristics were evaluated, relative growth rate (RGR), net assimilation rate (NAR), plant height, diameter, dry matter production of the aerial part (DMPAP) and amount of nutrient in the dry matter of leaves. We concluded from the results that: low availability of N, Ca and Mg constraints the growth of the Aniba rosaeodora plants. Seedlings of A. rosaeodora required little P, K and S. The omission of Ca and N harmed the RGR of the species. The most important elements for DMPAP were Ca and Mg; the Mg acting more in the leaf area; on the other hand, the omission of sulfur favored the macronutrient absorption (N, P, K, Ca, Mg).
Asunto(s)
Nutrientes/análisis , Lauraceae , Bosque LluviosoRESUMEN
Plant lectins are the most studied group of carbohydrate-binding proteins. Despite the high similarity between the members of the Diocleinae subtribe (Leguminosae) group, they present differing biological activities. Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293 K. After optimization, crystals suitable for diffraction were obtained under the condition 0.1 M HEPES pH 7.5 and 3.0 M sodium formate. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 126.70, b = 66.64, c = 64.99 A, alpha = 90.0, beta = 120.8, gamma = 90.0 degrees . Assuming the presence of a dimer in the asymmetric unit, the solvent content was estimated to be about 46%. A complete data set was collected at 1.5 A resolution.