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Inhibitory Effects of Plant Phenolic Compounds on Enzymatic and Cytotoxic Activities Induced by a Snake Venom Phospholipase A

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Inhibitory Effects of Plant Phenolic Compounds on Enzymatic and Cytotoxic Activities Induced by a Snake Venom Phospholipase A 295 VITAE, REVISTA DE LA FACULTAD DE QUÍMICA FARMACÉUTICA ISSN 0121-4004 / ISSNe 2145-2660. Volumen 18 número 3, año 2011. Universidad de Antioquia, Medellín, Colombia. págs. 295-304 INHIBITORY EFFECTS OF PLANT PHENOLIC COMPOUNDS ON ENZYMATIC AND CYTOTOXIC ACTIVITIES INDUCED BY A SNAKE VENOM PHOSPHOLIPASE A 2 EFECTOS INHIBITORIOS DE COMPUESTOS FENÓLICOS DE PLANTAS SOBRE LA ACTIVIDAD ENZIMÁTICA Y CITOTOXICA INDUCIDA POR UNA FOSFOLIPASA A 2 DE VENENO DE SERPIENTE Jaime A. PEREAÑEZ 1*, Vitelbina NÚÑEZ 1,2 , Arley C. PATIÑO 1, Mónica LONDOÑO 1, Juan C. QUINTANA 1 Received: 23 February 2010 Accepted: 25 April 2011 ABSTRACT Polyphenolic compounds have shown to inhibit toxic effects induced by snake venom proteins. In this work, we demonstrate that gallic acid, ferulic acid, caffeic acid, propylgallate and epigallocatechingallate inhibit the enzymatic activity of a phospholipase A 2 (PLA 2), using egg yolk as substrate. The IC50 values are between 0.38 – 3.93 mM. These polyphenolic compounds also inhibit the PLA 2 enzymatic activity when synthetic substrate is used. Furthermore, these compounds decreased the cyotoxic effect induced by a myotoxic PLA 2; specifically, epigallocatechin gallate exhibited the best inhibitory capacity with 90.92 ± 0.82%, while ferulic acid showed the lowest inhibitory activity with 30.96 ± 1.42%. Molecular docking studies were performed in order to determine the possible modes of action of phenolic compounds. All polyphenols showed hydrogen bonds with an active site of enzyme; moreover, epigallocatechingallate presented the strongest binding compared with the other compounds. Additionally, a preliminary structure-activity relationship analysis showed a correlation between the IC50 and the topological polar surface area of each compound (p = 0.0491, r = -0.8079 (-0.9878 to -0.2593)), which indicates the surface area required for each molecule to bind with the majority of the enzyme. Furthermore, our results show that propylgallate and epigallocatechingallate are two novel natural products with anti-myotoxic potential. The topical application of these plant polyphenols at the bite site could lead to prevent myotoxicity; however, further in vivo studies would be necessary to confirm the in vitro results. Key words: Snake bite, phenolic compounds, local tissue damage, phospholipase A 2, molecular docking. RESUMEN Los compuestos fenólicos han mostrado inhibir los efectos tóxicos inducidos por proteínas de veneno de serpiente. En éste trabajo, nosotros demostramos que el ácido gálico, el ácido ferúlico, el ácido cafeico, el propilgalato y el epigalocatequingalato inhiben la actividad enzimática de una fosfolipasa A 2 (PLA 2) usando yema de huevo como sustrato. Los valores de IC50 están entre 0,38 – 3,93 mM. Los compuestos mencionados también inhiben la actividad enzimática cuando un sustrato sintético es usado. Además, 1 Programa de Ofidismo/Escorpionismo. Universidad de Antioquia. A.A. 1226. Medellín, Colombia. 2 Escuela de Microbiología. Universidad de Antioquia. A.A. 1226. Medellín, Colombia. * Corresponding autor: [email protected] 296 et al estos compuestos polifenólicos disminuyen el efecto citotóxico inducido por la fosfolipasa A 2 miotóxica, el epigalocatequingalato exhibe la mejor capacidad inhibitoria con 90,92 ± 0,82%, mientras que el ácido ferúlico muestra la menor actividad inhibitoria con 30,96 ± 1,42%. Con el fin de determinar los posibles mecanismos de acción de los compuestos fenólicos, realizamos estudios de modelamiento molecular. Todos los polifenoles muestran puentes de hidrogeno con el sitio activo de la enzima; además el epigalocatequingalato presenta una unión más fuerte con la PLA 2 que los otros compuestos. Adicionalmente, un análisis preliminar de relación estructura actividad muestra una correlación entre los valores de IC50 y el área superficial polar topológica (p = 0,0491, r = -0,8079 (-0,9878 a -0,2593)), la cual indica el área superficial requerida por cada molécula para unirse a la enzima. Además, nuestros resultados muestran al propilgalato y el epigalocatequingalato como dos nuevos productos naturales con potencial antimiotóxico. La aplicación tópica de estos polifenoles en el sitio de mordedura podría llevar a la prevención de la miotoxicidad; sin embargo, posteriores investigaciones in vivo serían necesarias para confirmar los resultados in vitro . Palabras clave: Accidente ofídico, compuestos fenólicos, daño tisular local, fosfolipasa A 2, modelamiento molecular. INTRODUCTION and macromolecules. The most critical event is a prominent Ca 2+ influx, which initiates a complex Snakebites represent a relevant public health series of degenerative events associated with hy- issue in many regions of the world, particularly in percontraction and mechanical damage of plasma tropical and subtropical countries of Africa, Asia, membrane, activation of calpains and cytosolic Latin America and Oceania (1). The pathophysi- 2+ Ca -dependent PLA 2s, Z line loss, and mitochon- ological effects observed in ophidian bites combine drial Ca 2+ overload (5). These events occur rapidly, the action of several enzymes, proteins and peptides, provoking necrosis in muscle cells. The role of the which include phospholipases A 2, hemorrhagic catalytic activity in the induction of this effect de- metalloproteases and other proteolytic enzymes, pends of a particular enzyme. Therefore, alkylation coagulant components, neurotoxins, cytotoxins of PLA 2 by BPB, which is bound specifically in the and cardiotoxins, among others (2). Phospholipases His48 of the catalytic site, abolishes their enzy- A2 (PLA 2; EC 3.1.1.4) are enzymes that abundantly matic activity and reduces several pharmacologi- occur in snake venoms with crucial action in the cal activities (anticoagulant, myotoxic, cytotoxic, hydrolysis of phospholipids. PLA 2 can also induce edema-forming), suggesting their dependence on several pharmacological effects such as edema, the integrity of this site. However, the effect of this modulation of platelet aggregation, as well as neuro- modification on other pharmacological activities is toxic, anticoagulant and myotoxic effects (3, 4). To less remarkable for some enzymes. These observa- explain the susceptibility of a tissue to a particular tions suggest that, despite the evidences of different PLA2 enzyme, the presence of “target sites” on the sites, hydrolytic activity plays a considerable role in surface of target cells was proposed (3). These target some biological effects (6). sites are recognized by specific “pharmacological The therapy for snakebites has been based on the sites” on PLA molecules. These pharmacological 2 intravenous administration of equine or ovine anti- sites are independent of, but sometimes overlapping venoms (7). However, it has been demonstrated that with, the active site of the enzyme (3). this therapy generally has a limited efficacy against Myotoxic PLA 2s bind to acceptors in the plasma the local tissue damaging activities of venoms (8). membrane (target sites), which might be lipids or Thus, there is a need to search for inhibitors and proteins, and which may differ in their affinity approaches that may be useful to complement con- for the PLA 2s. Upon binding, myotoxic PLA 2s ventional antivenom therapy. disrupt the integrity of the plasma membrane by Plant extracts constitute a rich source of pharma- catalytically dependent (phosphoipid hydrolysis) or cologically active compounds, some of which have independent mechanisms (interaction of pharmaco- been reported to be an alternative to antagonizing logical site with cell membrane). As a consequence, the activity of various crude venoms and purified there is a loss in the control of permeability to ions toxins (9-11). However, only a few of those chemical 297 compounds have been isolated and identified as Hence, the aim of this study was to demonstrate active components (12-14); from those compounds, the inhibitory ability of the following phenolic a considerable number has been classified as po- compounds on the enzymatic and cytotoxic lyphenols (15-17), which is a group of chemical activities of snake venom PLA 2: gallic acid, substances found in plants and microorganisms, ferulic acid, caffeic acid, propylgallate, and characterized by the presence of more than one epigallocatechingallate (shown in figure 1). For this phenol unit per molecule. Polyphenols are generally purpose, we tested the inhibitory capacity of these divided into hydrolyzable tannins (gallic acid esters compounds on PLA 2 from the crotoxin complex of glucose and other sugars) and phenylpropanoids, (CB isolated from the Colombian Crotalus durissus such as lignins, flavonoids, and condensed tan- cumanensis rattlesnake). This toxin is responsible for nins, among others. These compounds are one of the neurotoxicity and local/systemic myotoxicity the most versatile from the plant kingdom, they effects in the snakebite inflicted by this species. In present effects such as the inhibition of HIV and order to determine the possible mode of action of the inhibition of human simplex virus (HSV), as these compounds; we have performed molecular well as antioxidant, bactericidal, antihelmintic, and docking studies and preliminary structure-activity antihepatoxic activities, among others (18). relationship analysis. Figure 1. Structures of the phenolic compounds and the enzyme used in this study. A. Gallic acid. B. Ferulic acid. C. Caffeic acid. D. Propylgallate. E. Epigallocatechingallate. Compound structures were built by means of ChemSketch
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