Antimicrobial and antibiofilm activities of Inga cylindrica trypsin inhibitor.

Inga cylindrica, a tropical fruit tree of the Fabaceae family (subfamily Mimosoideae), is native to South America. The seeds from this family are an essential source of trypsin inhibitors, which display promising bioactivity for increasing host defense against pathogens. The aim of the present study was to characterize the antimicrobial and antibiofilm activities of the trypsin inhibitor extracted from I. cylindrica seeds, ICTI. ICTI demonstrated antifungal activity with a minimum inhibitory concentration (MIC) of 32.11 µmol.L-1, and a minimum fungicidal concentration (MFC) of 32.1 µmol.L-1, against Cryptococcus gattii, Candida albicans, Candida glabrata and Candida guilliermondii. Combining ICTI with Amphotericin B had a significant synergistic effect, reducing the concentration of the antibiotic by 75% for C. albicans and 94% for C. gatti. The significant increase (16 x) in activity observed with ergosterol (1.01 mol.L-1) for C. albicans and C. gatti, and the lack of activity against bacterial strains, suggests that ICTI interferes with the integrity of the fungal cell membrane. Treatment with ICTI at 10 x MIC resulted in a 51% reduction in biofilm formation and a 56% decrease in mature biofilm colonies for C. albicans. Finally, ICTI displayed no toxicity in the in vivo Galleria mellonella model. Given its antifungal and antibiofilm properties, ICTI could be a promising candidate for the development of new antimicrobial drug prototypes.

Inflammatory responses induced in mice by lectin from Talisia esculenta seeds.

A novel lectin from Talisia esculenta seeds (TEL) has recently been purified and characterized. In this study we investigated the proinflammatory activity of TEL in mice using both the air-pouch and peritoneal cavity as well as paw oedema models. TEL (10-40 microg) induced significant neutrophil and mononuclear cell recruitment when injected into either mouse air-pouch or peritoneal cavity. The neutrophil accumulation into the air-pouch was dose- and time-dependent with a maximal response at 16 h, returning to control levels at 72 h whereas maximal mononuclear cell accumulation was observed at 24 h after TEL injection. The same profile of neutrophil accumulation was observed when this lectin was injected into mouse peritoneal cavity, although the maximal mononuclear cell recruitment was observed 48 h after TEL injection. Additionally, TEL (12.5-200 microg/paw) caused a dose-dependent mice paw, as evaluated at 4 h after the lectin injection. D-mannose, better than D-glucose, significantly inhibited TEL-induced neutrophil migration into the peritoneal cavity or air-pouch. D-galactose had no effect on TEL-induced neutrophil migration in either cavity studied. On the other hand, D-mannose slightly inhibited the TEL-induced paw oedema, whereas neither D-glucose nor D-galactose affected this phenomenon. In conclusion, our data show that TEL induces neutrophil and mononuclear cell accumulation by a mechanism related to their specific sugar-binding properties.

Properties of a Kunitz-type trypsin inhibitor from Delonix regia seeds against digestive proteinases of Anagasta kuehniella (Z.) and Corcyra cephalonica (S.) (Lepidoptera: Pyralidae).

DrTI was effective against trypsin-like enzymes from A. kuehniella and C. cephalonica, however an artificial diet was insufficient to affect the survival and body weight of either insect. The inhibitor stimulated chymotrypsin-like enzymes and probably induced the synthesis of enzymes insensitive to TLCK in neonate larvae.

Estudo in vitro da atividade do extrato etanólico de sementes de bacupari (Rheedia gardneriana Planch. & Triana) e das frações no crescimento de Streptococcus mutans / In vitro study of the activity of ethanol extract from bacupari (Rheedia gardneriana Planch. & Triana) seeds and its fractions on Streptococcus mutans growth

Streptococcus mutans, principal microrganismo da cavidade oral, desempenha papel preponderante na formação de placas dentárias, sendo considerado o agente etiológico primário da cárie. Rheedia gardneriana, conhecida popularmente como bacupari, é uma planta utilizada com fins medicinais para o tratamento de diversas patologias, e por apresentar atividade antimicrobiana de compostos das folhas contra bactérias Gram-positivas e Gram-negativas. O objetivo do presente trabalho foi avaliar o efeito de extrato de semente de R. gardneriana sobre a cepa S. mutans UA159. Os testes foram conduzidos com o extrato etanólico bruto e as frações obtidas com os solventes diclorometano, etanol-água, metanol e hexano, em ensaios de inibição in vitro. O extrato bruto (100 por cento) apresentou halos de inibição com diâmetro similar ao obtido com solução de digluconato de clorexidina 0,12 por cento, usada como controle. Os ensaios com a fração diclorometano exibiram atividade inibitória 35 por cento menor comparado com o controle, enquanto nenhum efeito antimicrobiano foi observado com a fração etanol-água. Contrariamente, os resultados obtidos com as frações hexânica e metanólica demonstraram claramente a atividade antimicrobiana por inibição do crescimento bacteriano. Na fração metanólica a formação de halos de inibição foi similar ao do controle. Estes dados apresentam atividade antimicrobiana de R. gardneriana contra S. mutans.

Streptococcus mutans, which is the main microorganism of the oral cavity, plays a preponderant role in dental plaque formation and is considered the primary etiologic agent regarding caries. Commonly known as "bacupari", Rheedia gardneriana is a plant used for medicinal purposes in the treatment of several pathologies; besides, its leaves have compounds that present antimicrobial activity against Gram-positive and Gram-negative bacteria. The aim of this work was to evaluate the effect of R. gardneriana seed extract on S. mutans strain UA159. The tests were carried out with crude ethanol extract and the fractions obtained with the solvents dichloromethane, ethanol-water, methanol, and hexane in in vitro inhibition assays. The crude extract (100 percent) presented inhibition halos with diameter similar to that obtained by using 0.12 percent chlorhexidine digluconate solution as control. Assays with the fraction dichloromethane showed an inhibitory activity 35 percent lower than that of the control, whereas no antimicrobial effect was observed with the ethanol-water fraction. Conversely, the results obtained with the fractions hexane and methanol clearly demonstrated antimicrobial activity by inhibiting the bacterial growth. In the methanol fraction, the formation of inhibition halos was similar to that in the control. These data present antimicrobial activity of R. gardneriana against S. mutans.

Isolation and characterization of a lectin from Annona muricata seeds.

A lectin with a high affinity for glucose/mannose was isolated from Annona muricata seeds (Annonaceae) by gel filtration chromatography on Sephacryl S-200, ion exchange chromatography on a DEAE SP-5 PW column, and molecular exclusion on a Protein Pak Glass 300 SW column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) yielded two protein bands of approximately 14 kDa and 22 kDa. However, only one band was seen in native PAGE. The Mr of the lectin estimated by fast-performance liquid chromatography-gel filtration on Superdex 75 was 22 kDa. The lectin was a glycoprotein with 8% carbohydrate (neutral sugar) and required divalent metal cations (Ca2+, Mg2+, and Mn2+) for full activity. Amino acid analysis revealed a large content of Glx, Gly, Phe, and Lys. The lectin agglutinated dog, chicken, horse, goose, and human erythrocytes and inhibited the growth of the fungi Fusarium oxysporum, Fusarium solani, and Colletotrichum musae.

Biochemical characterization of a lectin from Delonix regia seeds.

A lectin from Delonix regia (DRL) seeds was purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on diethylaminoethyl-Sepharose and reverse-phase high-performance liquid chromatography on a C18 column. Hemagglutinating activity was monitored using rat erythrocytes. DRL showed no specificity for human erythrocytes of ABO blood groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein in the presence of 0.1 M of dithiothreitol (DTT) and in nonreducing conditions. Native-PAGE showed that DRL is a monomer with a molecular mass of about 12 kDa, as determined by denaturing gel electrophoresis and gel filtration chromatography. An amino acid composition revealed the absence of cysteine residues, the presence of 1 mol methionine/mol protein and a high proportion of acidic amino acids and glycine. The N-terminal sequence of DRL was determined by Edman degradation, and up to 16 amino acid residues showed more than 90% homology with other lectins from the Leguminosae family. The optimal pH range for lectin activity was between pH 8.0 and 9.0, and the lectin was active up to 60 degrees C. The lectin required Mn2+ for hemagglutinating activity and remained active after reduction with 0.1 M of DTT, but lost activity in the presence of 8 M of urea. Sodium metaperiodate had no effect on the activity of DRL.

Purification and properties of storage proteins (vicilins) from cowpea (Vigna unguiculata) seeds which are susceptible or resistant to the bruchid beethe Callosobruchus maculatus

Vicilins (7S storage proteins) from seeds of cowpea (Vigna unguiculata) cultivars which are susceptible or resistant to the bruchil veetle C. maculatus were purified by size-exclusion and ion-exchange chromatography. The vicilins were partially characterized by polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions, by Western blotting and by amino acid analysis. The variant vicilins from C. maculatus-resistant seeds do not differ appreciably from vicilins from susceptible seeds by these criteria except that they are more strongly bound to DEAE-Sepharose, suggesting differences in charge between the various molecules