Antimicrobial activity of ethanol extract from leaves of Casearia sylvestris

Casearia sylvestris Sw., also known as 'guaatonga,' is a medicinal plant with broad use in South America. Among the popular applications attributed to this plant are anti-inflammatory, anticancer, antimicrobial, and antiulcer activities. Despite the broad popular use of this plant as a phytopharmaceutical agent, there are few studies about the antimicrobial potential of guaatonga. In this work, we have studied the antimicrobial potential of an ethanol extract obtained from C. sylvestris leaves against three yeasts, two filamentous fungus, six Gram-negative bacteria, and two Gram-positive bacteria. Through two chromatographic steps using a Sephadex LH-20 column and RP-HPLC, we isolated and characterized two derived compounds of gallic acid: isobutyl gallate-3,5-dimethyl ether (IGDE) and methyl gallate-3,5-dimethyl ether (MGDE). Both compounds showed antimicrobial activity. IGDE was much more efficient than MGDE in inhibiting yeasts (Candida albicans, Candida tropicalis, and Candida guilliermondii) and Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus). This fact is probably associated with the higher hydrophobicity degree of IDGE compared with MGDE.46534735

Purification and characterization of an N-acetylglucosamine-binding lectin from Koelreuteria paniculata seeds and its effect on the larval development of Callosobruchus maculatus (Coleoptera : Bruchidae) and Anagasta kuehniella (Lepidoptera : Pyralidae)

This study describes the purification of an N-acetylglucosamine-binding lectin from Koelreuteria paniculata seeds and its effects on the larval development of Callobruchus maculatus and Anagasta kuehniella. The lectin (KpLec) was characterized and isolated by gel filtration, affinity column, and reverse phase chromatography. SDS-PAGE indicated that this lectin is a dimer composed of subunits of 22 and 44 kDa. The N terminus exhibited 40% similarity with Urtiga dioica agglutinin. KpLec was tested for anti-insect activity against C. maculatus and A. kuehniella. With regard to C. maculatus, an artificial diet containing 0.7 and 1% KpLec produced LD50 and ED50 value, respectively. However, for A. kuenhiella, an artificial diet containing 0.65% KpLec produced an LD50, whereas 0.2% KpLec produced an ED50. The transformation of genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.51102980298

Pharmacological Study of Edema and Myonecrosis in Mice Induced by Venom of the Bushmaster Snake (Lachesis muta muta) and Its Basic Asp49 Phospholipase A(2) (LmTX-I)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Previous in vitro studies show that Lachesis muta venom and its purified Asp49 phospholipase A(2), named as LmTX-I, display potent neurotoxic and myotoxic activities. Here, an in vivo study was conducted to investigate some pharmacological effects of the venom or its LmTX-I toxin, after intra-muscular injection in tibialis anterior (TA) and following subplantar injection in hind paws of mice. Findings showed that LmTX-I increased plasma creatine kinase activity and produced strong myonecrosis and inflammatory reactions in TA muscle. In addition to these effects, the venom also induced intense local hemorrhage. Pre-treatment of the venom with EDTA (5 mM) significantly inhibited the edema and hemorrhage. Histological examination showed that L. muta venom caused inner dermal layer thickening in the pad hind paw. In addition, there was marked inflammatory cell infiltration, particularly of neutrophils, and hemorrhage. LmTX-I also demonstrated edema-forming activity, which was inhibited by pretreatment with indomethacin.276384391Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Isolation and biochemical characterization of a galactoside binding lectin from Bauhinia variegata candida (BvcL) seeds

A new lectin (BvcL) from seeds of a primitive Brazilian Caesalpinoideae, the Bauhinia variegata candida was purified and biochemical characterized. BvcL was isolated by gel filtration chromatography on Sephadex G75 and affinity chromatography on immobilized D-lactose column. SDS-PAGE showed that BvcL under non-reducing condition presents two bands of 68 and 32 kDa and a single band of 32 kDa in reducing condition. However, only one band was seen in native PAGE. The hemagglutination activity of BvcL was not specific for any human blood group trypsin-treated erythrocytes. Carbohydrate inhibition analysis indicated that BvcL is inhibited by lactose, galactose, galactosamine and other galactoside derivates. Amino acid analysis revealed a large content of Ser, Gly, Thr, Asp and Glu and low concentrations of Met, Cys and His. Intrinsic fluorescence of BvcL was not significantly affected by sugar binding galactose; and aromatic-region CD is unusually high for plant lectins. The N-terminal amino acid sequence of 17 residues showed 90% sequential homology to galactose-specific legume lectins of the subfamily Caesalpinoideae.26319320

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 M-r 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.224185865566

Neurotoxic and myotoxic actions from Lachesis muta muta (surucucu) whole venom on the mouse and chick nerve-muscle preparations

Lachesis genus is one of the less studied among others from Viperidae's genera, mainly due to difficulties in obtaining the venom. Accidents by Lachesis snakes cause severe envenoming syndrome, eventually leading victims to shock. This work is part of a comprehensive study aimed at studying the venom and its effects. Herein the neurotoxicity and myotoxicity of L. muta muta venom were investigated on mouse phrenic nerve-diaphragm (PNDp) and chick biventer cervicis (BCp) preparations. For both preparations the time required to venom produces 50% neuromuscular blockade was indirectly concentration-dependent, being for PNDp: 117.6 +/- 6.5 min (20 mu g/ml), 70.1 +/- 8.6 min (50 mu g/ml) and 43.6 +/- 3.8 min (100 mu g/ml), and for BCp: 28 +/- 1.8 min (50 mu g/ml), 30.4 +/- 2.3 min (10 mu g/ml), 50.4 +/- 4.3 min (5 mu g/ml) and 75.2 +/- 0.7 min (2 mu g/ml), (n = 5/dose). In BCp, a venom dose of 50 mu g/ml significantly reduced contractures elicited by exogenous acetylcholine (55 mu M) and KCl (20 mM), as well as increased the release of creatine kinase (442.7 +/- 39.8 IU/l in controls vs 4322.6 +/- 395.2 IU/l, after 120 min of venom incubation (P < 0.05). Quantification of myonecrosis in BCp indicated the doses 50 and 10 mu g/ml as significantly myotoxic affecting 59.7 +/- 6.2%, and 20.8 +/- 1.2% of fibers, respectively, whereas 5 and 2 mu g/ml that affected 13.5 +/- 0.8% and 5.4 +/- 0.6% of fibers, were considered weakly- and non-myotoxic, respectively. We concluded that there are neurotoxins present in the venom, the concentration of which governs its pre- (if low) or postsynaptic (if high) activity. Since myotoxicity in the avian preparation is negligible at lower venom doses, but not neurotoxicity, we suggest that this effect may contribute minimum to the venom neurotoxic effect. The BCp is more sensible than PNDp to Lachesis in. muta venom. (c) 2005 Elsevier Ltd. All rights reserved.46222222

Functional characterization of a basic D49 phospholipase A(2) (LmTX-I) from the venom of the snake Lachesis muta muta (bushmaster)

The whole venom of Lachesis muta muta is preponderantly neurotoxic but moderately myotoxic on the chick biventer cervicis preparation (BCp). We have now examined these toxic activities of a basic phospholipase A(2), LmTX-I, isolated from the whole venom. LmTX-I caused a significant concentration-dependent neuromuscular blockade in the BCp. The time to produce 50% neuromuscular blockade was 14.7 +/- 0.75 min (30 mu g/ml). 23.6 +/- 0.9 min (10 mu g/ml). 34 +/- 1.7 min (2.5 mu g/ml) and 39.2 +/- 3.6 min (1 mu g/ml), (n = 5/concentration; p < 0.05). Complete blockade with all tested concentrations was not accompanied by inhibition of the response to ACh. At the highest concentration. LmTX-I (30 mu g/ml) significantly reduced contractures elicited by exogenous KCl (20 mM), increased the release of creatine kinase (1542.5 +/- 183.9 IU[L vs 442.7 +/- 39.8 IU/L for controls after 120 min, p < 0.05), and induced the appearance of degenerating muscle fibers (similar to 15%). Quantification of myonecrosis indicated 14.8 +/- 0.8 and 2.0 +/- 0.4%, with 30 and 10 mu g/ml venom concentration, respectively, against 1.07 +/- 0.4% for control preparations. The findings indicate that the basic PLA(2) present on venom from L. m. muta (LmTX-1) possesses a dominant neurotoxic action on isolated chick nerve-muscle preparations, whereas myotoxicity was mainly observed at the highest Concentration used (30 mu g/ml). These effects of LmTX-I closely reproduce the effects of the whole venom of L. m. muta in chick neuromuscular preparations. (c) 2006 Elsevier Ltd. All rights reserved.47775976

Ability of rabbit antiserum against crotapotin to neutralize the neurotoxic, myotoxic and phospholipase A(2) activities of crotoxin from Crotalus durissus cascavella snake venom

The toxicity of crotoxin, the major toxin of Crotalus durissus terrificus (South American rattlesnake) venom, is mediated by its basic phospholipase A(2) (PLA(2)) subunit. This PLA(2) is non-covalently associated with crotapotin, an acidic, enzymatically inactive subunit of the crotoxin complex. In this work, rabbit antiserum raised against crotapotin purified from Crotalus durissus eascavella venom was tested for its ability to neutralize the neurotoxicity of this venom and its crotoxin in vitro. The ability of this antiserum to inhibit the enzymatic activity of the crotoxin complex and PLA(2) alone was also assessed, and its potency in preventing myotoxicity was compared with that of antisera raised against crotoxin and PLA(2). Antiserum to crotapotin partially neutralized the neuromuscular blockade caused by venom and crotoxin in electrically stimulated mouse phrenic nerve-hemidiaphragm preparations and prevented the venom-induced myotoxicity, but did not inhibit the enzymatic activity of crotoxin and purified PLA(2). In contrast, previous findings showed that antisera against crotoxin and PLA(2) from C. d. cascavella effectively neutralized the neuromuscular blockade and PLA(2) activity of this venom and its crotoxin. The partial neutralization of crotoxin-mediated neurotoxicity by antiserum to crotapotin probably reduced the binding of crotoxin to its receptor following interaction of the antiserum with the crotapotin moiety of the complex. (c) 2007 Elsevier Ltd. All rights reserved.22124024

Biological and biochemical characterization of new basic phospholipase A(2) BmTX-I isolated from Bothrops moojeni snake venom

BmTX-I, an Asp49 phospholipase A(2), was purified from Bothrops moojeni venom after only one chromatographic step using reverse-phase HPLC on mu-Bondapak C-18 column. A molecular mass of 14238.71 Da was determined by MALDI-TCF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BmTX-I PLA(2) had a sequence of 121 residues of amino acids: DLWQFNKMIK KEVGKLPFPF YGAYGCYCGW GGRGEKPKDG TDRCCFVHDC CYKKLTGCPK WDDRYSYSWK DITIVCGEDL PCEEICECDR AAAVCFYENL GTYNKKYMKH LKPCKKADYP C and pI value 7.84, and showed a high degree of homology with basic Asp49 PLA(2) myotoxins from other Bothrops venoms. BmTX-I presented PLA2 activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca2+ and in the presence of Mg2+, Cd2+ and Mn2+ it was reduced in presence or absence of Ca2+. Crotapotin from Crotalus durissus colillineatus rattlesnake venom has significantly inhibited (P < 0.05) the enzymatic activity of BmTX-I. In vitro, the whole venom and BmTX-I caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other bothrops species. In mice, BmTX-I and the whole venom-induced myonecrosis and a systemic interieukin-6 response upon intramuscular injection. Edema-forming activity was also analyzed through injection of the venom and the purified BmTX-I into the subplantar region of the right footpad. Since BmTX-I exert a strong proinflammatory effect; the enzymatic phospholipids hydrolysis might be relevant for these phenomena. (c) 2008 Elsevier Ltd. All rights reserved.5181509151

Molecular modeling and inhibition of phospholipase A(2) by polyhydroxy phenolic compounds

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Phospholipases A(2) are enzymes responsible for the hydrolysis of membrane phospholipids that release arachidonic acid, which serves as substrate for pro-inflammatory mediators, such as prostaglandins and leucotriens. The design of specific inhibitors for PLA(2) might help in the development of new anti-inflammatory drugs. Polyhydroxy phenolic compounds, such as flavonoids, vitamin E, rosmarinic acid and aristolochic acid, are able to inhibit PLA(2) from different sources. Herein, we have studied the kinetic behavior and the capacity of inhibiting edema formation induced by PLA(2) of five different polyhydroxy phenolic compounds (two phenolic derivatives and three acetophenone hydroxylated derivatives) extracted from the venom of Crotalus adamanteus. The results showed that compounds 1,3-dihydroxy benzene, 1,3,5-trihydroxy benzene and 2,4,6-trihydroxy acetophenone were the most efficient in the inhibition of the enzymatic activity and edema induction by PLA2. It was also verified that the number of hydroxyls in each molecule is not a limiting factor for the inhibition capacity of these compounds. Molecular modeling studies indicated that the most active compounds are linked to the amino acid Asp 49 and that they destabilize the coordination of the calcium atom, which is essential to the catalytic activity. The study of potential surfaces showed that there are conditions in which the potential values must be adequate for enzyme complex fort-nation with polyhydroxy phenolic compounds. When the potential over the hydroxyl surfaces is very high, formation of stable complexes does not occur and the enzyme does not act intensely. These results might be helpful in the design of a drug that specifically inhibits PLA(2). (C) 2008 Elsevier Masson SAS. All rights reserved.441312321Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq