Anti-platelet activity of infusion of tithonia diversifolia's leaves

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORTithonia diversifolia, also known as "Mexican arnica", has been used in traditional medicine to treat inflammatory refractory with absence of citotoxicity. The possible health risks associated with the consumption of ingestion of the infusion (tea) plant makes it is necessary to identify the potential pharmacological activity or toxicity to prove certain plants that are acclimated in Brazil. Considering the limited number of pharmacological studies regarding the Tithonia diversifolia, the aim of this study was evaluate the effects of this infusion in platelet aggregation. Venous blood was collected with informed consent from healthy volunteers who denied taking any medication in the previous 14 days. Whole blood was transferred into polypropylene tubes containing one-tenth of final volume of acid citrate dextrose (ACD-C; citric acid 3%, trisodium citrate 4%, glucose 2%; 1:9 v/v) and centrifuged at 200g for 15 min. Platelet rich plasma was added of wash buffer solution (NaCl 140mM, KCl 5mM, sodium citrate 12mM, glucose 10mM and saccharose 12mM; pH 6; 5:7 v/v) and centrifuged at 800g for 12 min at 20°C. Platelet pellet was gently resuspended in Krebs-Ringer solution and counts were performed on a Neubauer chamber. Aggregation assay was carried out with 400 μL of platelet suspension (1.2x10 8 platelets/mL) in a cuvette at 37°C with constant stirring. Platelet suspension was incubated for 3 min with aqueous extract infusion (0.6-20μg/mL) prior to addition of thrombin (100 mU/mL). Percentage of platelet aggregation was recorded with an aggregometer (Chrono-log Lumi-Aggregometer model 560-Ca, USA). Our results show an inhibition of thrombin induced platelet aggregation in the presence of 0.6-20 ug/mL Tithonia diversifolia infusion leaves. The Tithonia diversifolia infusion leaves inhibits thrombin induced washed platelet aggregation.Tithonia diversifolia, also known as "Mexican arnica", has been used in traditional medicine to treat inflammatory refractory with absence of citotoxicity. The possible health risks associated with the consumption of ingestion of the infusion (tea) plant makes it is necessary to identify the potential pharmacological activity or toxicity to prove certain plants that are acclimated in Brazil. Considering the limited number of pharmacological studies regarding the Tithonia diversifolia, the aim of this study was evaluate the effects of this infusion in platelet aggregation. Venous blood was collected with informed consent from healthy volunteers who denied taking any medication in the previous 14 days. Whole blood was transferred into polypropylene tubes containing one-tenth of final volume of acid citrate dextrose (ACD-C; citric acid 3%, trisodium citrate 4%, glucose 2%; 1:9 v/v) and centrifuged at 200g for 15 min. Platelet rich plasma was added of wash buffer solution (NaCl 140mM, KCl 5mM, sodium citrate 12mM, glucose 10mM and saccharose 12mM; pH 6; 5:7 v/v) and centrifuged at 800g for 12 min at 20°C. Platelet pellet was gently resuspended in Krebs-Ringer solution and counts were performed on a Neubauer chamber. Aggregation assay was carried out with 400 μL of platelet suspension (1.2x10 8 platelets/mL) in a cuvette at 37°C with constant stirring. Platelet suspension was incubated for 3 min with aqueous extract infusion (0.6-20μg/mL) prior to addition of thrombin (100 mU/mL). Percentage of platelet aggregation was recorded with an aggregometer (Chrono-log Lumi-Aggregometer model 560-Ca, USA). Our results show an inhibition of thrombin induced platelet aggregation in the presence of 0.6-20 ug/mL Tithonia diversifolia infusion leaves. The Tithonia diversifolia infusion leaves inhibits thrombin induced washed platelet aggregation1124127FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORsem informaçãosem informaçãosem informaçã

Antiophidic Solanidane Steroidal Alkaloids From Solanum Campaniforme

Three new solanidane alkaloids bearing a 22,23-epoxy ring (1-3) and four known compounds were isolated from leaves of Solanum campaniforme. The structures were determined using spectroscopic techniques, including 1Dand 2D NMR, and HRESIMS experiments. The antiophidic activity of the alkaloids was tested against Bothrops pauloensis venom. Compounds 1-3 completely inhibited myotoxicity without inhibiting phospholipase A 2 activity of the venom, while hemorrhage and skin necrosis were significantly reduced in the presence of alkaloids 1 and 2. © 2011 The American Chemical Society and American Society of Pharmacognosy.741021682173Wink, M., (2003) Phytochemistry, 64, pp. 3-19Nino, J., Correa, Y.M., Mosquera, O.M., (2009) Pharm. Biol., 47, pp. 255-259Ikeda, T., Tsumagari, H., Honbu, T., Nohara, T., (2003) Biol. Pharm. Bull., 26, pp. 1198-1201Kodoru, S., Grierson, D.S., Van De Venter, M., Afolayan, A.J., (2007) Pharm. Biol., 45, pp. 613-618Sun, L., Zhao, Y., Li, X., Yuan, H., Cheng, A., Lou, H., (2010) Toxicol. in Vitro, 24, pp. 1504-151Emmanuel, S., Ignacimuthu, S., Perumalsamy, R., Amalraj, T., (2006) Fitoterapia, 77, pp. 611-612Pandurangan, A., Khosa, R.L., Hemalatha, S., (2010) J. Asian Nat. Prod. Res., 12, pp. 691-695Fenner, R., Betti, A.H., Mentz, L.A., Rates, S.M.K., (2006) Rev. Bras. Cienc. Farm., 42, pp. 369-394Harrison, R.A., Hargreaves, A., Wagstaff, S.C., Faragher, B., Lalloo, D.G., (2009) PLoS Negl. Trop. Dis., 3, p. 569Mors, W.B., Nascimento, M.C., Pereira, B.M.R., Pereira, N.A., (2000) Phytochemistry, 55, pp. 627-642Correa-Netto, C., Teixeira-Araujo, R., Aguiar, A.S., Melgarejo, A.R., De-Simone, S.G., Soares, M.R., Foguel, D., Zingali, R.B., (2010) Toxicon, 55, pp. 1222-1235Gutiérrez, J.M., Rucavado, A., Chaves, F., Díaz, C., Escalante, T., (2009) Toxicon, 54, pp. 958-975Caron, E.J., Manock, S.R., Maudlin, J., Koleski, J., Theakston, R.D., Warrell, D.A., Smalligan, R.D., (2009) Toxicon, 54, pp. 779-83Cotrim, C.A., De Oliveira, S.C., Diz Filho, E.B., Fonseca, F.V., Baldissera, Jr.L., Antunes, E., Ximenes, R.M., Toyama, M.H., (2011) Chem. Biol. Interact., 15, pp. 9-16Lomonte, B., León, G., Angulo, Y., Rucavado, A., Núñez, V., (2009) Toxicon, 54, pp. 1012-1028Coelho, R.M., Souza, M.C., Giotto, M.H.S., (1998) Phytochemistry, 49, pp. 893-897Lawson, D.R., Green, T.P., Haynes, L.W., Miller, A.R., (1997) J. Agric. Food Chem., 45, pp. 4122-4126Chiesa, F.A.F., Moyna, P., Alcaloides Esteroidales (2004) Farmacognosia: Da Planta Ao Medicamento, pp. 869-883. , In, 5 th ed. Simões, C. M. O. Schenkel, E. P. Gosmann, G. Mello, J. C. P. Mentz, L. A. Petrovick, P. R. Ed.a da UFRGS: Porto AlegreDraper, R.W., Puar, M.S., Vater, E.J., McPhail, A.T., (1998) Steroids, 63, pp. 135-140Jiang, Z.H., Wang, J.R., Li, M., Liu, Z.Q., Chau, K.Y., Zhao, C., Liu, L., (2005) J. Nat. Prod., 68, pp. 397-399Daneluttea, A.P., Costantina, M.B., Delgadob, G.E., Braz-Filho, R., Kato, M.J., (2005) J. Braz. Chem. Soc., 16, pp. 1425-1430Slimestad, R., Fossen, T., Verheul, M.J., (2008) J. Agric. Food Chem., 56, pp. 2436-2441Friedman, M., Lee, K.-R., Kim, H.-J., Lee, I.-S., Kozukue, N., (2005) J. Agric. Food Chem., 53, pp. 6162-6169Milner, S.E., Brunton, N.P., Jones, P.W., Brien, N.M.O., Collins, S.G., Maguire, A.R., (2011) J. Agric. Food Chem., 59, pp. 3454-3484Corrêa, L.C., Marchi-Salvador, D.P., Cintra, A.C.O., Sampaio, S.V., Soares, A.M., Fontes, M.R.M., (2008) Biochim. Biophys. Acta, 1784, pp. 591-599Cintra-Francischinelli, M., Pizzo, P., Angulo, Y., Gutiérrez, J.M., Montecucco, C., Lomonte, B., (2010) Toxicon, 55, pp. 590-596Montecucco, C., Ornella, R., Caccin, P., Rigoni, M., Carli, L., Morbiato, L., Muraro, L., Paoli, M., (2009) Toxicon, 54, pp. 561-564Fernandes, C.A., Marchi-Salvador, D.P., Salvador, G.M., Silva, M.C., Costa, T.R., Soares, A.M., Fontes, M.R., (2010) J. Struct. Biol., 171, pp. 31-43Silva, J.O., Fernandes, R.S., Ticli, F.K., Oliveira, C.Z., Mazzi, M.V., Franco, J.J., Giuliatti, S., Sampaio, S.V., (2007) Toxicon, 50, pp. 283-291Esmeraldino, L.E., Souza, A.M., Sampaio, S.V., (2005) Phytomedicine, 12, pp. 570-57

Enzymatic And Structural Characterization Of New Pla2 Isoform Isolated From White Venom Of Crotalus Durissus Ruruima

This work reports the structural and enzymatic characterization of a new sPLA2 from the white venom of Crotalus durissus ruruima, nominated PLA2A. The homogeneity of the PLA2A fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14,299.34 Da. Structural investigation, through circular dichroism spectroscopy, revealed that PLA2A has a high content of alpha helix and beta-turn structures, 45.7% and 35.6% respectively. Its amino acid sequence, determined by Edman degradation and "de novo amino acid sequencing", exhibited high identity to PLA2 Cdt F15 from Crotalus durissus terrificus. The enzymatic investigation, conducted using the synthetic substrate 4-nitro-3-(octanoyloxy)-benzoic acid, determined its Vmax (7.56 nmoles/min) and KM (2.76 mM). Moreover, PLA2A showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Intrinsic fluorescence measurements suggested that Ca2+ induced a significant increase of PLA2A fluorescence, whereas its replacement for Mg2+, Mn2+, Sn2+ and Cd2+ apparently induced no structural modifications. The optimal pH and temperature for the enzymatic activity of PLA2A were 8.4 and 40 °C, respectively, and the minimal concentration of p-BPB and crotapotin that significantly inhibited such activity was 0.75 mM and 0.4 μM, respectively. In addition, PLA2A showed a significant antibacterial effect that was not strictly dependent on the enzymatic activity of such sPLA2. © 2008 Elsevier Ltd. All rights reserved.531104114Bercovici, D., Chudzinski, A.M., Dias, W.O., Esteves, M.I., Hiraichi, E., Oishi, N.Y., Picarelli, Z.P., Raw, I., A systematic fractionation of Crotalus durissus terrificus venom (1987) Mem. Inst. Butantan (São Paulo), 49, pp. 69-78Barrio, A., Estudio electroforético del veneno de serpiente de cascabel (1954) Cienc. Investig., 10, pp. 368-370Bon, C., Bouchier, C., Choumet, V., Faure, G., Jiang, M.S., Lambezat, M.P., Radvanyi, F., Saliou, B., Crotoxin, half-century of investigations on a phospholipase A2 neurotoxin (1989) Acta Physiol. Pharmacol. Latinoam., 39, pp. 439-448Chen, Y.H., Wang, Y.M., Hseu, M.J., Tsai, I.H., Molecular evolution and structure-function relationships of crotoxin-like and asparagine-6-containing phospholipases A2 in pit viper venoms (2004) Biochem. J., 381 (PART 1), pp. 25-34Chang, L.S., Lin, S.R., Chang, C.C., Probing calcium ion-induced conformational changes of Taiwan cobra phospholipase A2 by trinitrophenylation of lysine residues (1994) J. Protein Chem., 16, pp. 51-57Chang, L.S., Wu, P.F., Liou, J.C., Chiang-Lin, W.H., Yang, C.C., Chemical modification of arginine residues of Notechis scutatus scutatus notexin (2004) Toxicon, 44, pp. 491-497Chu, Y.P., Cheng, Y.C., Yang, C.C., Chang, L.S., The structural events associated with the binding of divalent cations to beta-bungarotoxin (2005) Toxicon, 45, pp. 139-145Dal Belo, C.A., Toyama, M.H., Toyama Dde, O., Marangoni, S., Moreno, F.B., Cavada, B.S., Fontana, M.D., Boschero, A.C., Determination of the amino acid sequence of a new phospholipase A(2) (MIDCA1) isolated from Micrurus dumerilii carinicauda venom (2005) Protein J., 24 (3), pp. 147-153Dos Santos, M.C., Ferreira, L.C., Da Silva, W.D., Furtado, M.F., Characterization of the biological activities of the 'yellow' and 'white' venoms from Crotalus durissus ruruima compared with the Crotalus durissus terrificus venom. Neutralizing activity of Crotalus durissus ruruima antivenins (1993) Toxicon, 31, pp. 1459-1469Dos Santos, M.C., Assis, E.B., Moreira, T.D., Pinheiro, J., Fortes-Dias, C.L., Individual venom variability in Crotalus durissus ruruima snakes, a subspecies of Crotalus durissus from the Amazonian region (2005) Toxicon, 46, pp. 958-961Faure, G., Choumet, V., Bouchier, C., Camoin, L., Guillaume, J.L., Monegier, B., Vuilhorgne, M., Bon, C., The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus (1994) Eur. J. 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Ciênc., 22, pp. 141-150Garcia, F., Toyama, M.H., Castro, F.R., Proença, P.L., Marangoni, S., Santos, L.M., Crotapotin induced modification of T lymphocyte proliferative response through interference with PGE2 synthesis (2003) Toxicon, 42, pp. 433-437Georgieva, D.N., Betzel, C., Aleksiev, B., Genov, N., Spectroscopic investigation of calcium binding sites in the neurotoxin vipoxin and its components-relation with the X-ray structure (2000) Spectrochim. Acta A Mol. Biomol. Spectrosc., 56, pp. 2811-2816Gibbs, H.L., Prior, K.A., Weatherhead, P.J., Johnson, G., Genetic structure of populations of the threatened eastern massasauga rattlesnake, Sistrurus c. catenatus: evidence from microsatellite DNA markers (1997) Mol. 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Corrigendum To "effect Of The Synthetic Coumarin, Ethyl 2-oxo-2h-chromene-3-carboxylate, On Activity Of Crotalus Durissus Ruruima Spla2 As Well As On Edema And Platelet Aggregation Induced By This Factor" [toxicon 55 (8) (2010) 1527-1530]

[No abstract available]5806/07/1561

Quercetin As An Inhibitor Of Snake Venom Secretory Phospholipase A2

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors. © 2010 Elsevier Ireland Ltd. All rights reserved.18901/02/15916Kini, R., Excitement ahead: Structure, function and mechanism of snake venom phospholipase A2 enzymes (2003) Toxicon, 42 (8), pp. 827-840Nevalainen, T., Graham, G., Scott, K., Antibacterial actions of secreted phospholipases A2. Review (2008) Biochim. Biophys. Acta, 1781 (12), pp. 1-9Burke, J., Dennis, E., Phospholipase A2 biochemistry (2009) Cardiovasc. 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