RESUMO
The present study sought to evaluate the protective effect of Epigallocatechin-3-gallate (EGCG) and commercial green tea (GT) on eroded dentin using in vitro and in situ experimental models. For the in vitro experiment, matrix metalloproteinases (MMPs) were extracted from demineralized human coronary dentin powder (citric acid, pH 2.3) and assessed via a colorimetric assay and electrophoresis in gelatin. The gels were exposed to buffers with: control (no treatment), 0.05% sodium fluoride (NaF), 0.12% chlorhexidine digluconate (CHX), GT infusion, and 0.1% EGCG, and their respective activity was analyzed by zymography. For the in situ experiment, 20 healthy volunteers (aged 20-32 years) participated in this single-center, blind, crossover study. The subjects wore upper removable devices containing four human dentin blocks. Erosive challenge (coke-1 min) was performed four times/day/5 days. Blocks were treated for 1 min with: control (No treatment), 0.05% NaF, 0.1% EGCG, and GT. Thereafter, the specimens were subjected to stylus profilometry and SEM. ANOVA was used to evaluate dentin roughness and wear, with a significance level of 5%. In the zymography analysis, 0.12% CHX, GT, and 0.1% EGCG were found to inhibit the action of MMPs; however, in the colorimetric assay, only green tea inhibited the activity of MMPs. There were no significant differences observed in dentin roughness or wear (p > 0.05). Herein, EGCG and GT inhibited the activity of endogenous proteases, resulting in protection against erosion-induced dentin damage; however, they could not prevent tooth tissue loss in situ.
Assuntos
Catequina , Erosão Dentária , Catequina/farmacologia , Estudos Cross-Over , Dentina , Humanos , Chá , Erosão Dentária/prevenção & controleRESUMO
Abstract The present study sought to evaluate the protective effect of Epigallocatechin-3-gallate (EGCG) and commercial green tea (GT) on eroded dentin using in vitro and in situ experimental models. For the in vitro experiment, matrix metalloproteinases (MMPs) were extracted from demineralized human coronary dentin powder (citric acid, pH 2.3) and assessed via a colorimetric assay and electrophoresis in gelatin. The gels were exposed to buffers with: control (no treatment), 0.05% sodium fluoride (NaF), 0.12% chlorhexidine digluconate (CHX), GT infusion, and 0.1% EGCG, and their respective activity was analyzed by zymography. For the in situ experiment, 20 healthy volunteers (aged 20-32 years) participated in this single-center, blind, crossover study. The subjects wore upper removable devices containing four human dentin blocks. Erosive challenge (coke-1 min) was performed four times/day/5 days. Blocks were treated for 1 min with: control (No treatment), 0.05% NaF, 0.1% EGCG, and GT. Thereafter, the specimens were subjected to stylus profilometry and SEM. ANOVA was used to evaluate dentin roughness and wear, with a significance level of 5%. In the zymography analysis, 0.12% CHX, GT, and 0.1% EGCG were found to inhibit the action of MMPs; however, in the colorimetric assay, only green tea inhibited the activity of MMPs. There were no significant differences observed in dentin roughness or wear (p > 0.05). Herein, EGCG and GT inhibited the activity of endogenous proteases, resulting in protection against erosion-induced dentin damage; however, they could not prevent tooth tissue loss in situ.
RESUMO
Soybean toxin (SBTX) is a protein isolated from soybean seeds and composed of two polypeptide subunits (17 and 27 kDa). SBTX has in vitro activity against phytopathogenic fungi such as Cercospora sojina, Aspergillus niger, and Penicillium herguei, and yeasts like Candida albicans, C. parapsilosis, Kluyveromyces marxiannus, and Pichia membranifaciens. The present study aimed to analyze in vitro whether SBTX causes any side effects on non-target bacterial and mammalian cells that could impede its potential use as a novel antifungal agent. SBTX at 100 µg/mL and 200 µg/mL did not hinder the growth of the bacteria Salmonella enterica (subspecies enterica serovar choleraesuis), Bacillus subtilis (subspecies spizizenii) and Staphylococcus aureus. Moreover, SBTX at concentrations up to 500 µg/mL did not significantly affect the viability of erythrocytes, neutrophils, and human intestinal Caco-2 cells. To study whether SBTX could induce relevant alterations in gene expression, in vitro DNA microarray experiments were conducted in which differentiated Caco-2 cells were exposed for 24 h to 100 µg/mL or 200 µg/mL SBTX. SBTX up-regulated genes involved in cell cycle and immune response pathways, but down-regulated genes that play a role in cholesterol biosynthesis and platelet degranulation pathways. Thus, although SBTX did not affect bacteria, nor induced cytotoxity in mammalian cells, it affected some biological pathways in the human Caco-2 cell line that warrants further investigation.
Assuntos
Antifúngicos/farmacologia , Glicoproteínas/farmacologia , Proteínas de Soja/farmacologia , Animais , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Eritrócitos/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Neutrófilos/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Transcriptoma/efeitos dos fármacosRESUMO
Jaburetox (JBTX) is an insecticidal and antifungal peptide derived from jack bean (Canavalia ensiformis) urease that has been considered a candidate for developing genetically modified crops. This study aimed to perform the risk assessment of the peptide JBTX following the general recommendations of the two-tiered, weight-of-evidence approach proposed by International Life Sciences Institute. The urease of C. ensiformis (JBU) and its isoform JBURE IIb (the JBTX parental protein) were assessed. The history of safe use revealed no hazard reports for the studied proteins. The available information shows that JBTX possesses selective activity against insects and fungi. JBTX and JBU primary amino acids sequences showed no relevant similarity to toxic, antinutritional or allergenic proteins. Additionally, JBTX and JBU were susceptible to in vitro digestibility, and JBU was also susceptible to heat treatment. The results did not identify potential risks of adverse effects and reactions associated to JBTX. However, further allergen (e.g. serum IgE binding test) and toxicity (e.g. rodent toxicity tests) experimentation can be done to gather additional safety information on JBTX, and to meet regulatory inquiries for commercial approval of transgenic cultivars expressing this peptide.
Assuntos
Antifúngicos/toxicidade , Inseticidas/toxicidade , Proteínas de Plantas/toxicidade , Medição de Risco , Urease/toxicidade , Animais , Antifúngicos/química , Canavalia/enzimologia , Biologia Computacional , Fungos/efeitos dos fármacos , Insetos/efeitos dos fármacos , Inseticidas/química , Proteínas de Plantas/química , Isoformas de Proteínas/química , Isoformas de Proteínas/toxicidade , Proteólise , Urease/químicaRESUMO
Plant proteins are emerging as an alternative to conventional treatments against candidiasis. The aim of this study was to better understand the mechanism of action of Mo-CBP2 against Candida spp, evaluating redox system activity, lipid peroxidation, DNA degradation, cytochrome c release, medium acidification, and membrane interaction. Anti-candida activity of Mo-CBP2 decreased in the presence of ergosterol, which was not observed with antioxidant agents. C. albicans treated with Mo-CBP2 also had catalase and peroxidase activities inhibited, while superoxide dismutase was increased. Mo-CBP2 increased the lipid peroxidation, but it did not alter the ergosterol profile in live cells. External medium acidification was strongly inhibited, and cytochrome c release and DNA degradation were detected. Mo-CBP2 interacts with cell membrane constituents, changes redox system enzymes in C. albicans and causes lipid peroxidation by ROS overproduction. DNA degradation and cytochrome c release suggest apoptotic or DNAse activity. Lipid peroxidation and H+-ATPases inhibition may induce the process of apoptosis. Finally, Mo-CBP2 did not have a cytotoxic effect in mammalian Vero cells. This study highlights the biotechnological potential of Mo-CBP2 as a promising molecule with low toxicity and potent activity. Further studies should be performed to better understand its mode of action and toxicity.
Assuntos
Candida/efeitos dos fármacos , Membrana Celular/metabolismo , Moringa oleifera/química , Proteínas de Plantas/farmacologia , Sementes/química , Esteróis/metabolismo , Animais , Antifúngicos/farmacologia , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Ergosterol/metabolismo , Glucose/farmacologia , Itraconazol/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Nistatina/farmacologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Células VeroRESUMO
In nature, plants are simultaneously challenged by biotic and abiotic stresses. However, little is known about the effects of these combined stresses for most crops. This work aimed to evaluate the responsed of the virus-resistant cowpea genotype BRS-Marataoã to the exposure of salt stress combined with CPSMV infection. Cowpea plants were exposed to 200â¯mM NaCl either simultaneously (SV plant group) or 24â¯h prior to the CPSMV infection [S(24â¯h)V plant group]. Physiological, biochemical, and proteomic analyses at 2 and 6â¯days post salt stress (DPS) revealed that cowpea significantly reprogrammed its cellular metabolism. Indeed, plant size, photosynthetic parameters (net photosynthesis, transpiration rate, stomatal conductance, and internal CO2 partial pressure) and chlorophyll and carotenoid contents were reduced in S(24â¯h)V compared to SV. Moreover, accumulation of viral particles at 6 DPS in S(24â¯h)V was observed indicating that the salt stress imposed prior to virus infection favors viral particle proliferation. Proteomic analysis showed differential contents of 403 and 330 proteins at 2 DPS and 6 DPS, respectively, out of 733 differentially abundant proteins between the two plant groups. The altered leaf proteins are involved in energy and metabolism, photosynthesis, stress response, and oxidative burst. BIOLOGICAL SIGNIFICANCE: This is an original study in which a virus-resistant cowpea genotype (BRS-Marataoã) was (i) exposed simultaneously to 200â¯mM NaCl and inoculation with CPSMV (SV plant group) or (ii) exposed to 200â¯mM NaCl stress 24â¯h prior to inoculation with CPSMV [S(24â¯h)V plant group]. The purpose was to shed light on how this CPSMV resistant cowpea responded to the combined stresses. Numerous key proteins and associated pathways were altered in the cowpea plants challenged with both stresses, but unexpectedly, the salt stress imposed 24â¯h prior to CPSMV inoculation allowed viral proliferation, turning the cowpea genotype from resistant to susceptible.
Assuntos
Comovirus/metabolismo , Genótipo , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Estresse Salino , Vigna , Proteômica , Vigna/genética , Vigna/metabolismo , Vigna/virologiaRESUMO
BACKGROUND: During the last few years, a growing number of antimicrobial peptides have been isolated from plants and particularly from seeds. Recent results from our laboratory have shown the purification of a new trypsin inhibitor, named CaTI, from chilli pepper (Capsicum annuum L.) seeds. This study aims to evaluate the antifungal activity and mechanism of action of CaTI on phytopathogenic fungi and detect the presence of protease inhibitors in other species of this genus. RESULTS: Our results show that CaTI can inhibit the growth of the phytopathogenic fungi Colletotrichum gloeosporioides and C. lindemuthianum. CaTI can also permeabilize the membrane of all tested fungi. When testing the inhibitor on its ability to induce reactive oxygen species, an induction of reactive oxygen species (ROS) and nitric oxide (NO) particularly in Fusarium species was observed. Using CaTI coupled to fluorescein isothiocyanate (FITC), it was possible to determine the presence of the inhibitor inside the hyphae of the Fusarium oxysporum fungus. The search for protease inhibitors in other Capsicum species revealed their presence in all tested species. CONCLUSION: This paper shows the antifungal activity of protease inhibitors such as CaTI against phytopathogenic fungi. Antimicrobial peptides, among which the trypsin protease inhibitor family stands out, are present in different species of the genus Capsicum and are part of the chemical arsenal that plants use to defend themselves against pathogens. © 2017 Society of Chemical Industry.
Assuntos
Capsicum/química , Fungicidas Industriais/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Doenças das Plantas/microbiologia , Extratos Vegetais/farmacologia , Sementes/química , Inibidores da Tripsina/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colletotrichum/efeitos dos fármacos , Colletotrichum/crescimento & desenvolvimento , Colletotrichum/metabolismo , Fungicidas Industriais/química , Fungicidas Industriais/isolamento & purificação , Fungicidas Industriais/metabolismo , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Fusarium/metabolismo , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificação , Inibidores da Tripsina/metabolismoRESUMO
Proteins extracted from Capsicum annuum L. fruits were initially subjected to reversed-phase chromatography on HPLC, resulting in eight peptide-rich fractions. All the fractions obtained were tested for their ability to inhibit porcine trypsin and amylase from both human saliva and from larval insect in vitro. All fractions were also tested for their ability to inhibit growth of the phytopathogenic fungi. Several fractions inhibited the activity of human salivary amylase and larval insect amylase, especially fraction Fa5. No fraction tested was found to inhibit trypsin activity, being Fa2 fraction an exception. Interestingly fraction Fa5 also displayed high antimicrobial activity against the species of the Fusarium genus. Fraction Fa5 was found to have two major protein bands of 17 and 6.5 kDa, and these were sequenced by mass spectrometry. Two peptides were obtained from the 6.5-kDa band, which showed similarity to antimicrobial peptides. Fraction Fa5 was also tested for its ability to permeabilize membranes and induce ROS. Fraction Fa5 was able to permeabilize the membranes of all the fungi tested. Fungi belonging to the genus Fusarium also showed an increase in the endogenous production of ROS when treated with this fraction. Antimicrobial peptides were also identified in the fruits from other Capsicum species.
Assuntos
Anti-Infecciosos , Capsicum/química , Inibidores Enzimáticos , Frutas/química , Fusarium/crescimento & desenvolvimento , Peptídeos , Proteínas de Plantas , alfa-Amilases/antagonistas & inibidores , Animais , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Humanos , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , SuínosRESUMO
Lectins are proteins that bind to specific mono- or oligosaccharides. This study aimed to evaluate the antinociceptive and anti-inflammatory effects of the lectin from the red marine alga Solieria filiformis. The animals (n = 6) were pretreated with S. filiformis lectin 30 min before they were given the nociceptive or inflammatory stimulus. The antinociceptive activity was evaluated in Swiss mice using the abdominal writhing, formalin, and hot plate tests. The anti-inflammatory properties were evaluated in Wistar rats using carrageenan-induced peritonitis and paw edema induced by different phlogistic agents. The S. filiformis lectin toxicity was assayed through its application in mice (7 days). S. filiformis lectin significantly reduced the number of abdominal writhings and reduced the paw licking time in the second phase of the formalin test (p < 0.05), but it did not prolong the reaction time in the hot plate test (p > 0.05). Furthermore, S. filiformis lectin reduced neutrophil migration in a peritonitis model and reduced paw edema induced by carrageenan, dextran, and serotonin (p < 0.05). Additionally, the administration of S. filiformis lectin resulted in no signs of systemic damage. Thus, S. filiformis lectin appears to have important antinociceptive and anti-inflammatory activities and could represent a potential therapeutic agent for future studies.
Assuntos
Analgésicos/isolamento & purificação , Anti-Inflamatórios não Esteroides/isolamento & purificação , Lectinas/isolamento & purificação , Rodófitas/química , Analgésicos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Feminino , Lectinas/farmacologia , Masculino , Camundongos , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Ratos , Ratos WistarRESUMO
Herein described is the biochemical characterisation, including in vitro and in vivo assays, for a proteinase inhibitor purified from Clitoria fairchildiana seeds (CFPI). Purification was performed by hydrophobic interaction and gel filtration chromatography. Kinetic studies of the purified inhibitor showed a competitive-type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3 × 10(-10) and 1.5 × 10(-10)M, respectively, displaying a tight binding property. SDS-PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15 kDa under non-reducing conditions. However, MALDI-TOF analysis demonstrated a molecular mass of 7.973 kDa, suggesting that CFPI is dimeric in solution. The N-terminal sequence of CFPI showed homology with members of the Bowman-Birk inhibitor family. CFPI remained stable to progressive heating for 30 min to each temperature range of 37 up to 100 °C and CD analysis exhibited no changes in spectra at 207 nm after heating at 90 °C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2-10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited significant inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on A. kuehniella development.
Assuntos
Clitoria/química , Inseticidas , Inibidores de Proteases , Sementes/química , Animais , Bovinos , Quimotripsina/análise , Inseticidas/química , Inseticidas/isolamento & purificação , Inseticidas/farmacologia , Cinética , Larva/efeitos dos fármacos , Larva/metabolismo , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Tripsina/análiseRESUMO
Antimicrobial peptides (AMPs) are produced by a range of organisms as a first line of defense against invaders or competitors. Owing to their broad antimicrobial activity, AMPs have attracted attention as a potential source of chemotherapeutic drugs. The increasing prevalence of infections caused by Candida species as opportunistic pathogens in immunocompromised patients requires new drugs. Lecythis pisonis is a Lecythydaceae tree that grows in Brazil. The AMPs produced by this tree have not been described previously. We describe the isolation of 12 fractions enriched in peptides from L. pisonis seeds. Of the 12 fractions, at 10 µg/ml, the F4 fraction had the strongest growth inhibitory effect (53.7%) in Candida albicans, in addition to a loss of viability of 94.9%. The F4 fraction was separated into seven sub-fractions by reversed-phase chromatography. The F4.7' fraction had the strongest activity at 10 µg/ml, inhibiting C. albicans growth by 38.5% and a 69.3% loss of viability. The peptide in F4.7' was sequenced and was found to be similar to plant defensins. For this reason, the peptide was named L. pisonis defensin 1 (Lp-Def1). The mechanism of action that is responsible for C. albicans inhibition by Lp-Def1 includes a slight increase of reactive oxygen species induction and a significant loss of mitochondrial function. The results described here support the future development of plant defensins, specifically Lp-Def1, as new therapeutic substances against fungi, especially C. albicans.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Magnoliopsida/embriologia , Peptídeos/farmacologia , Sementes/química , Antifúngicos/química , Antifúngicos/isolamento & purificação , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Cromatografia de Fase Reversa , Eletroforese em Gel de Poliacrilamida , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos/isolamento & purificação , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cry8Ka5 is a mutant protein from Bacillus thuringiensis (Bt) that has been proposed for developing transgenic plants due to promising activity against coleopterans, like Anthonomus grandis (the major pest of Brazilian cotton culture). Thus, an early food safety assessment of Cry8Ka5 protein could provide valuable information to support its use as a harmless biotechnological tool. This study aimed to evaluate the food safety of Cry8Ka5 protein following the two-tiered approach, based on weights of evidence, proposed by ILSI. Cry1Ac protein was used as a control Bt protein. The history of safe use revealed no convincing hazard reports for Bt pesticides and three-domain Cry proteins. The bioinformatics analysis with the primary amino acids sequence of Cry8Ka5 showed no similarity to any known toxic, antinutritional or allergenic proteins. The mode of action of Cry proteins is well understood and their fine specificity is restricted to insects. Cry8Ka5 and Cry1Ac proteins were rapidly degraded in simulated gastric fluid, but were resistant to simulated intestinal fluid and heat treatment. The LD50 for Cry8Ka5 and Cry1Ac was >5000 mg/kg body weight when administered by gavage in mice. Thus, no expected relevant risks are associated with the consumption of Cry8Ka5 protein.
Assuntos
Proteínas de Bactérias/efeitos adversos , Endotoxinas/efeitos adversos , Inocuidade dos Alimentos , Proteínas Hemolisinas/efeitos adversos , Proteínas Mutantes/efeitos adversos , Testes de Toxicidade Aguda/métodos , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Sequência de Aminoácidos , Animais , Aspartato Aminotransferases/sangue , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Contagem de Células Sanguíneas , Colesterol/sangue , Biologia Computacional , Creatinina/sangue , Endotoxinas/genética , Feminino , Proteínas Hemolisinas/genética , Insetos , Inseticidas , Dose Letal Mediana , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/genética , Tamanho do Órgão/efeitos dos fármacos , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Triglicerídeos/sangue , Ureia/sangueRESUMO
Studies have contested the innocuousness of Bacillus thuringiensis (Bt) Cry proteins to mammalian cells as well as to mammals microbiota. Thus, this study aimed to evaluate the cytotoxic and antimicrobial effects of two Cry proteins, Cry8Ka5 (a novel mutant protein) and Cry1Ac (a widely distributed protein in GM crops). Evaluation of cyto- and genotoxicity in human lymphocytes was performed as well as hemolytic activity coupled with cellular membrane topography analysis in mammal erythrocytes. Effects of Cry8Ka5 and Cry1Ac upon Artemia sp. nauplii and upon bacteria and yeast growth were assessed. The toxins caused no significant effects on the viability (IC50 > 1,000 µg/mL) or to the cellular DNA integrity of lymphocytes (no effects at 1,000 µg/mL). The Cry8Ka5 and Cry1Ac proteins did not cause severe damage to erythrocytes, neither with hemolysis (IC50 > 1,000 µg/mL) nor with alterations in the membrane. Likewise, the Cry8Ka5 and Cry1Ac proteins presented high LC50 (755.11 and >1,000 µg/mL, resp.) on the brine shrimp lethality assay and showed no growth inhibition of the microorganisms tested (MIC > 1,000 µg/mL). This study contributed with valuable information on the effects of Cry8Ka5 and Cry1Ac proteins on nontarget organisms, which reinforce their potential for safe biotechnological applications.
Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas Mutantes/genética , Plantas Geneticamente Modificadas/genética , Animais , Artemia/efeitos dos fármacos , Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Endotoxinas/administração & dosagem , Eritrócitos/efeitos dos fármacos , Proteínas Hemolisinas/administração & dosagem , Humanos , Linfócitos/efeitos dos fármacos , Proteínas Mutantes/administração & dosagem , Controle Biológico de VetoresRESUMO
Among the Bauhinia species, B. cheilantha stands out for its seed protein content. However, there is no record of its nutritional value, being used in a nonsustainable way in the folk medicine and for large-scale extraction of timber. The aim of this study was to investigate the food potential of B. cheilantha seeds with emphasis on its protein quality to provide support for flora conservation and use as raw material or as prototype for the development of bioproducts with high added socioeconomic value. B. cheilantha seeds show high protein content (35.9%), reasonable essential amino acids profile, low levels of antinutritional compounds, and nutritional parameters comparable to those of legumes widely used such as soybean and cowpea. The heat treatment of the seeds as well as the protein extraction process (to obtain the protein concentrate) increased the acceptance of diets by about 100% when compared to that of raw Bc diet. These wild legume seeds can be promising alternative source of food to overcome the malnutrition problem faced by low income people adding socioeconomic value to the species.
Assuntos
Bauhinia/química , Clima Desértico , Medicina Tradicional , Valor Nutritivo , Sementes/química , Testes de Toxicidade , Aminoácidos/análise , Animais , Lectinas/metabolismo , Masculino , Camundongos , Ratos , Ratos Wistar , Sementes/enzimologia , Tripsina/metabolismo , Urease/metabolismoRESUMO
The marine ecosystem is able to provide enormous biomolecule diversity that could be used for treatment of various diseases. In this highly competitive environment, organisms need chemical barriers to reduce or avoid microorganism contamination. Among the molecules that protect these animals the antimicrobial peptides (AMPs) are included. In the present study, crude extracts of coral coral specimens Carijoa riisei, Muriceopsis sulphurea, Neospongodes atlantica, Palythoa caribeorum, Phyllogorgia dilatata and Plexaurella grandiflora were challenged against multiple Grampositive and -negative bacteria showing different activities. P. dilatata crude extract showed the antibacterial activity, and was ammonium-sulfate (0-40%) fractionated, being able to control the growth of K. pneumoniae, S. flexineri and S. aureus. Rich-fraction was further purified by using Amicon® Ultra Centrifugal 10 kDa associated with reversed-phase HPLC chromatography (C18), producing the peptide named Pd-AMP1. Pd-AMP1 was able to inhibit S. aureus development. Mass spectrometry analyses showed a monoisotopic mass of 5372.66 Da and N-terminal sequence showed no significant match with databank. In this view, the prospecting of protein biomolecules and biotechnological potential from marine animals is still little explored and may serve as an alternative to common antibiotics.
Assuntos
Antozoários/química , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/isolamento & purificação , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Brasil , Humanos , Dados de Sequência Molecular , Peptídeos/isolamento & purificaçãoRESUMO
The antimicrobial, antioxidant, and anticholinesterase activities of ethanolic seed extracts of twenty-one plant species from Brazilian semiarid region were investigated. The extracts were tested for antimicrobial activity against six bacteria strains and three yeasts. Six extracts presented activity against the Gram (-) organism Salmonella choleraesuis and the Gram (+) organisms Staphylococcus aureus and Bacillus subtilis. The MIC values ranged from 4.96 to 37.32 mg/mL. The Triplaris gardneriana extract presented activity against the three species, with MIC values 18.8, 13.76, and 11.15 mg/mL, respectively. Five extracts presented antioxidant activity, with EC50 values ranging from 69.73 µ g/mL (T. gardneriana) to 487.51 µ g/mL (Licania rigida). For the anticholinesterase activity, eleven extracts were capable of inhibiting the enzyme activity. From those, T. gardneriana, Parkia platycephala and Connarus detersus presented the best activities, with inhibition values of 76.7, 71.5, and 91.9%, respectively. The extracts that presented antimicrobial activity were tested for hemolytic assay against human A, B, and O blood types and rabbit blood. From those, only the Myracrodruon urundeuva extract presented activity (about 20% of hemolysis at the lowest tested concentration, 1.9 µg/mL). Infrared spectroscopy of six representative extracts attested the presence of tannins, polyphenols, and flavonoids, which was confirmed by a qualitative phytochemical assay.
Assuntos
Anacardiaceae/química , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Inibidores da Colinesterase/farmacologia , Extratos Vegetais/farmacologia , Animais , Bacillus subtilis/efeitos dos fármacos , Brasil , Colinesterases/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Extratos Vegetais/química , Extratos Vegetais/classificação , Coelhos , Salmonella/efeitos dos fármacos , Sementes/químicaRESUMO
The objective of this study was to evaluate the reactivity of an in-house antigen, extracted from a strain of C. posadasii isolated in northeastern Brazil, by radial immunodiffusion and Western blotting, as well as to establish its biochemical characterization. The protein antigen was initially extracted with the use of solid ammonium sulfate and characterized by 1-D electrophoresis. Subsequently, it was tested by means of double radial immunodiffusion and Western blotting. A positive reaction was observed against the antigen by both immunodiagnostic techniques tested on sera from patients suffering from coccidioidomycosis. Besides this, two immunoreactive protein bands were observed and were revealed to be a ß-glucosidase and a glutamine synthetase after sequencing of the respective N-terminal regions. Our in-house Coccidioides antigen can be promising as a quick and low-cost diagnostic tool without the risk of direct manipulation of the microorganism.
Assuntos
Antígenos de Fungos/imunologia , Coccidioides/imunologia , Coccidioidomicose/diagnóstico , Coccidioidomicose/imunologia , Testes Imunológicos/métodos , Sequência de Aminoácidos , Antígenos de Fungos/química , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Peso MolecularRESUMO
In this study, the antifungal activity of peptides extracted from Adenanthera pavonina seeds was assessed. Peptides were extracted and fractionated by DEAE-Sepharose chromatography. The non-retained D1 fraction efficiently inhibited the growth of the pathogenic fungi. This fraction was later further fractionated by reversed-phase chromatography, resulting in 23 sub-fractions. All separation processes were monitored by tricine-SDS-PAGE. Fractions H11 and H22 strongly inhibited the growth of Saccharomyces cerevisiae and Candida albicans. Fraction H11 caused 100% death in S. cerevisiae in an antimicrobial assay. The complete amino acid sequence of the peptide in fraction P2 was determined, revealing homology to plant defensins, which was named ApDef1. Peptides from fraction H22 were also sequenced.
Assuntos
Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fabaceae/química , Fungos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Sementes/química , Sequência de Aminoácidos , Antifúngicos/química , Peptídeos Catiônicos Antimicrobianos/química , Cromatografia Líquida , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Dados de Sequência Molecular , Extratos Vegetais/química , Alinhamento de SequênciaRESUMO
This work aimed at describing the first biochemical and structural data of a lectin belonging to Swartzieae, a primitive Legume Taxa. A lactose-binding seed lectin (SLL) was purified by affinity chromatography of crude saline extracts of Swartzia laevicarpa on immobilized lactose. The SLL agglutinated rabbit erythrocytes but not rat or human (A, B, O) erythrocytes. Lectin activity was retained after heating at 100 �C for 15 min and was best inhibited by Nacetylgalactosamine, lactose and galactose. The lectin exhibited a single electrophoretic pattern that corresponded to a molecular mass of 29,000 Da, which was confirmed by MS analysis. In addition, the lectin reacted positively with Schiff's reagent. The unique N-terminal amino acid sequence (39 residues) and the internal peptide sequence were determined by Edman degradation and MS/MS, respectively. The sequencing revealed complete homology of the SLL with legume lectins belonging to primitive groups (Dalbergieae and Sophoreae). The SLL (at 1 mg/ml) did not exhibit antifungal activity against various phytopathogens or cytotoxicity (at 100 µg/ml) towards different cancer cell lines.
Assuntos
Fabaceae/química , Fungos/efeitos dos fármacos , Hemaglutinação/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Lectinas de Plantas/farmacologia , Acetilgalactosamina/farmacologia , Sequência de Aminoácidos , Animais , Morte Celular , Cromatografia de Afinidade , Eletroforese , Galactose/farmacologia , Testes de Hemaglutinação , Humanos , Lactose/metabolismo , Dados de Sequência Molecular , Peso Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Lectinas de Plantas/isolamento & purificação , Coelhos , Ratos , Sementes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais CultivadasRESUMO
Moringa oleifera Lam. is a perennial multipurpose tree that has been successfully used in folk medicine to cure several inflammatory processes. The aim of this study was to purify and characterize a chitin-binding protein from Moringa oleifera seeds, named Mo-CBP4, and evaluate its antinociceptive and anti-inflammatory effects in vivo. The protein was purified by affinity chromatography on chitin followed by ion exchange chromatography. Acetic acid-induced abdominal constrictions assay was used for the antinociceptive and anti-inflammatory activity assessments. Mo-CBP4 is a glycoprotein (2.9% neutral carbohydrate) composed of two protein subunits with apparent molecular masses of 28 and 18 kDa (9 kDa in the presence of reducing agent). The intraperitoneal injection of Mo-CBP4 (3.5 and 10 mg/kg) into mice 30 min before acetic acid administration potently and significantly reduced the occurrence of abdominal writhing in a dose dependent manner by 44.7% and 100%, respectively. In addition, the oral administration of the protein (10 mg/kg) resulted in 18% and 52.8% reductions in abdominal writhing when given 30 and 60 min prior to acetic acid administration, respectively. Mo-CBP4, when administered by intraperitoneal route, also caused a significant and dose-dependent inhibition of peritoneal capillary permeability induced by acid acetic and significantly inhibited leukocyte accumulation in the peritoneal cavity. In conclusion, this pioneering study describes that the chitin-binding protein Mo-CBP4, from M. oleifera seeds, exhibits anti-inflammatory and antinociceptive properties and scientifically supports the use of this multipurpose tree in folk medicine.