PHARMACOLOGY AND TOXICOLOGY

 

INHIBITORY EFFECTS OF PLANT PHENOLIC COMPOUNDS ON ENZYMATIC AND CYTOTOXIC ACTIVITIES INDUCED BY A SNAKE VENOM PHOSPHOLIPASE A₂

 

EFECTOS INHIBITORIOS DE COMPUESTOS FENÓLICOS DE PLANTAS SOBRE LA ACTIVIDAD ENZIMÁTICA Y CITOTOXICA INDUCIDA POR UNA FOSFOLIPASA A₂ DE VENENO DE SERPIENTE

 

 

Jaime A. PEREAÑEZ^1*; Vitelbina NÚÑEZ^1,2; Arley C. PATIÑO¹; Mónica LONDOÑO¹; Juan C. QUINTANA¹

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: andres.pereanez@siu.udea.edu.co.

 

Received: 23 February 2010
Accepted: 25 April 2011

 

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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 A₂ (PLA₂), using egg yolk as substrate. The IC50 values are between 0.38 – 3.93 mM. These polyphenolic compounds also inhibit the PLA₂ enzymatic activity when synthetic substrate is used. Furthermore, these compounds decreased the cyotoxic effect induced by a myotoxic PLA₂; 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 A₂, molecular docking.

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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 A₂ (PLA₂) 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, estos compuestos polifenólicos disminuyen el efecto citotóxico inducido por la fosfolipasa A₂ 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 PLA₂ 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 A₂, modelamiento molecular.

Key words: alpha.

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INTRODUCTION

Snakebites represent a relevant public health issue in many regions of the world, particularly in tropical and subtropical countries of Africa, Asia, Latin America and Oceania (1). The pathophysiological effects observed in ophidian bites combine the action of several enzymes, proteins and peptides, which include phospholipases A₂, hemorrhagic metalloproteases and other proteolytic enzymes, coagulant components, neurotoxins, cytotoxins and cardiotoxins, among others (2). Phospholipases A₂ (PLA₂; EC 3.1.1.4) are enzymes that abundantly occur in snake venoms with crucial action in the hydrolysis of phospholipids. PLA₂ can also induce several pharmacological effects such as edema, modulation of platelet aggregation, as well as neurotoxic, anticoagulant and myotoxic effects (3, 4). To explain the susceptibility of a tissue to a particular PLA₂ enzyme, the presence of ''target sites'' on the surface of target cells was proposed (3). These target sites are recognized by specific ''pharmacological sites'' on PLA₂ molecules. These pharmacological sites are independent of, but sometimes overlapping with, the active site of the enzyme (3).

Myotoxic PLA₂s bind to acceptors in the plasma membrane (target sites), which might be lipids or proteins, and which may differ in their affinity for the PLA₂s. Upon binding, myotoxic PLA₂s disrupt the integrity of the plasma membrane by catalytically dependent (phosphoipid hydrolysis) or independent mechanisms (interaction of pharmacological site with cell membrane). As a consequence, there is a loss in the control of permeability to ions and macromolecules. The most critical event is a prominent Ca²⁺ influx, which initiates a complex series of degenerative events associated with hypercontraction and mechanical damage of plasma membrane, activation of calpains and cytosolic Ca²⁺-dependent PLA₂s, Z line loss, and mitochondrial Ca²⁺ overload (5). These events occur rapidly, provoking necrosis in muscle cells. The role of the catalytic activity in the induction of this effect depends of a particular enzyme. Therefore, alkylation of PLA₂ by BPB, which is bound specifically in the His48 of the catalytic site, abolishes their enzymatic activity and reduces several pharmacological activities (anticoagulant, myotoxic, cytotoxic, edema-forming), suggesting their dependence on the integrity of this site. However, the effect of this modification on other pharmacological activities is less remarkable for some enzymes. These observations suggest that, despite the evidences of different sites, hydrolytic activity plays a considerable role in some biological effects (6).

The therapy for snakebites has been based on the intravenous administration of equine or ovine antivenoms (7). However, it has been demonstrated that this therapy generally has a limited efficacy against the local tissue damaging activities of venoms (8). Thus, there is a need to search for inhibitors and approaches that may be useful to complement conventional antivenom therapy.

Plant extracts constitute a rich source of pharmacologically active compounds, some of which have been reported to be an alternative to antagonizing the activity of various crude venoms and purified toxins (9-11). However, only a few of those chemical compounds have been isolated and identified as active components (12-14); from those compounds, a considerable number has been classified as polyphenols (15-17), which is a group of chemical substances found in plants and microorganisms, characterized by the presence of more than one phenol unit per molecule. Polyphenols are generally divided into hydrolyzable tannins (gallic acid esters of glucose and other sugars) and phenylpropanoids, such as lignins, flavonoids, and condensed tannins, among others. These compounds are one of the most versatile from the plant kingdom, they present effects such as the inhibition of HIV and the inhibition of human simplex virus (HSV), as well as antioxidant, bactericidal, antihelmintic, and antihepatoxic activities, among others (18).

Hence, the aim of this study was to demonstrate the inhibitory ability of the following phenolic compounds on the enzymatic and cytotoxic activities of snake venom PLA₂: gallic acid, ferulic acid, caffeic acid, propylgallate, and epigallocatechingallate (shown in figure 1). For this purpose, we tested the inhibitory capacity of these compounds on PLA₂ from the crotoxin complex (CB isolated from the Colombian Crotalus durissus cumanensis rattlesnake). This toxin is responsible for the neurotoxicity and local/systemic myotoxicity effects in the snakebite inflicted by this species. In order to determine the possible mode of action of these compounds; we have performed molecular docking studies and preliminary structure-activity relationship analysis.

 

MATERIALS AND METHODS

Chemicals and reagents

Caffeic acid, ferulic acid, propylgallate, gallic acid, tannic acid and epigallocatechingallate were purchased from Sigma and used without further purification. In all cases, compounds were diluted in 3% DMSO in PBS. The other reagents used in this work were purchased from Sigma and Merck, and their purity level was the highest available. Due to the capacity of tannic acid to precipitate proteins and its ability to inhibit snake venom proteins (19- 21), it was used as control for inhibition in all assays, and the other phenolic compounds were always compared with it.

Isolation of PLA₂

Crotalus durissus cumanensis venom was obtained from four specimens maintained in captivity at the serpentarium of the Universidad de Antioquia (Medellin, Colombia). PLA₂ was purified through molecular exclusion chromatography on Sephadex G-75 and reverse-phase HPLC on C-18 column eluted at 1.0 mL/min with a gradient from 0 to 100% of acetonitrile in 0.1% trifluoroacetic acid (v/v). The absorbance in the effluent solution was recorded at wavelength of 280 nm (21).

Inhibition of the phospholipase A₂ activity using egg yolk as substrate

PLA₂ activity was assayed according to the method established by Dole (22), with titration of free fatty acids released from egg yolk phospholipids, which were suspended in 1% Triton^® X-100, 0.1 M Tris-HCl, 0.01 M CaCl₂, pH 8.5 buffer, using 15 µg/10 µL of PLA₂. The time of reaction was 15 min at 37°C. The protein sample was selected from the linear region of activity curves. For inhibition experiments, 0.5, 1, 2 and 4 mM of each compound were pre-incubated for 30 min at 37°C before the PLA₂ activity determination. Results are indicated as inhibition percentage, where 100% is the activity induced by PLA₂ alone. Tannic acid was taken as control for inhibition. The IC50 was determined from the linear portion of the response dose curves.

Inhibition of phospholipase A₂ activity using 4-nitro-3-octanoyloxy-benzoic acid (4N3OBA) as substrate

The measurements of the enzymatic activity using the linear substrate 4N3OBA were performed according to the method described by Holzer and Mackessy (23), and adapted for a 96-well ELISA plate. The standard assay contained 200 µL of buffer (10 mM Tris-HCl, 10 mM CaCl2, 100 mM NaCl, pH 8.0), 20 µL of 10 mM of substrate (4NO3BA), 20 µL of the sample (20 µg PLA₂ or 20 µg PLA₂ + 2 mM of each compound), and 20 µL of water. The negative control consisted only of buffer. The inhibitory effect of the molecules on PLA₂ activity was determined through the co-incubation of the enzyme with each compound for 30 min at 37°C. After the incubation period, the sample was added to the assay and the reaction was monitored at 425 nm for 40 min (at 10 min intervals) at 37°C. The quantity of chromophore released (4-nitro- 3-hydroxy benzoic acid) was proportional to the enzymatic activity, and the initial velocity (Vo) was calculated considering the absorbance measured right after 20 min. Tannic acid was used as control for inhibition.

Inhibition of cytotoxic activity

Cytotoxic activity of the purified PLA₂ and its inhibition was assayed on murine myotubes obtained from C2C12 s keletal muscle myoblast (ATCC CRL-1772) grown in 96-well plates, as previously described (24). The toxin alone, or mixed with compounds at concentrations equi valent to IC50, obtained from PLA₂ activity inhibition assays, was incubated for 30 min at 37°C. Then, aliquots of 150 µL (containing 20 µg of toxin + compounds diluted in Dubelcco's Modified Eagle's Medium) were applied to the cultures. After 3h at 37°C, a supernatant aliquot was collected for determination of lactic dehydrogenase activity (LDH; EC 1.1.1.27) released from damaged cells using a kinetic assay (Wiener LDH-P UV). Tannic acid was used as control for inhibition. Additional controls consisted of cells incubated with compounds in the absence of toxins. Results are shown as the percentage of inhibition, considering toxin and culture medium to be 100 and 0% of activity, respectively.

Molecular docking and physicochemical properties

Molecular docking was carried out using a Molegro Virtual Docker (MVD) (25). MVD is based on a differential evolution algorithm; docking scoring function, E_score; and the solution of the algorithm takes into account the sum of the intermolecular interaction energy between the ligand and the protein (E_inter), and the intramolecular interaction energy of the ligand (E_intra). Compound structures were built and minimized by means of ChemSketch 12.0, a software from ACD/Labs and available at http://www.acdlabs.com/download/chemsketch/download.html. The structure of PLA₂ (PDB code 2QOG) from Crotalus durissus terrificus that showed 57% of homology with the PLA₂ from C. d. cumanensis (21), which was used in this study, was uploaded without water molecules. When necessary, bonds, bond orders, hybridizations, and hydrogen atoms were added, charges were assigned (a formal charge of +2 for Ca ion) and flexible torsions of ligands were detected. Then, an automatic procedure was used to detect possible binding cavities. During this process, the maximum number of cavities was fixed to 5, the grid resolution was 0.80 Å, and the probe size was 1.2 Å; while the other parameters were set to default. Two cavities were detected, and the cavity around the catalytic site (with a volume of approximately 80.38 ų) was used for docking calculations using the MolDock- Optimizer as the search algorithm. During docking, the grid resolution was set to 0.3 Å, while the binding site radius was set to 14 Å. RMSD thresholds for multiple cluster poses was set at < 1.00 Å. The docking algorithm was set at a maximum of 1,500 iterations with a simplex evolution population size of 50 and a minimum of 10 runs. The ligand configurations with minor E_score were chosen, and a visual inspection of the interactions at the active site was performed and recorded. In order to perform a preliminary structure-activity relationship study, several physicochemical properties of each compound were obtained from Molinspiration by means of the ''Calculation of Molecular Properties and Drug-likeness'' tool, available at http://www.molinspiration.com/cgi-bin/properties.

Statistical analysis

In order to determine the IC50 of each compound in the inhibition of PLA₂ activity assay, the lineal portion of dose-response curve was used, and a simple lineal regression analysis was performed. To determine the significant differences between compounds and tannic acid in the same assay, a two-way ANOVA was performed, followed by a Bonferron's test. To determine the significant differences between compounds and tannic acid in the cytotoxicity inhibition assay, an ANOVA was performed, followed by a Dunnett's test, and a difference was considered significant when p < 0.05. A non-parametric correlation was carried out using the Spearman method between the TPSA (Topological Polar Surface Area) and the PLA₂ inhibition of each compound. In all cases, results are shown as the mean ± SEM of n indicated in each case.

 

RESULTS AND DISCUSSION

Myonecrosis is a commonly found in snakebites, and it is caused by PLA₂, one of the most important and abundant muscle damaging components present in snake venoms. The action of these enzymes over membrane phospholipids includes the release of fatty acids such as the arachidonic acid, which is a precursor of pro-inflammatory eicosanoids (26); moreover, such degradation can lead to destabilization of the phospholipids bilayer (4).

Recently, we demonstrated that the PLA₂ used in this study exhibited the above mentioned effects, among others (21). This enzyme is a component of the crotoxin complex of the Colombian Crotalus durissus cumanensis rattlesnake venom, which is a heterodimeric complex that is formed by a basic PLA₂ known as CB, and an acidic nonenzymatic component known as crotapotin, which increases the pharmacological activity of PLA₂ (acting as chaperone protein for the enzyme, preventing the binding of PLA₂ to non-specific sites) (27, 28). Crotoxin is responsible for neurotoxicity, renal failure, edema, and local and systemic myotoxicity in cases of snakebites inflicted by the South American Crotalus durissus rattlesnake (29). However, CB alone also induces these effects, and it belongs to the group IIA PLA₂s, which shares the general characteristics shown in figure 1F: the structure is formed by three long α helixes (two of which are antiparallel), two β wings and a calcium-binding loop (figure 1F). These proteins have a variable length ranging from 119 to 134 amino acids. Their antiparallel α helixes (residues 37-57 and 90-109, respectively) define the hydrophobic channel, with the assistance of the N-terminal helix (residues 1-12). This region leads the substrate to the active site, which is formed by four residues: His48, Asp49, Tyr52 and Asp99; from which, the combination of Asp49 with Tyr28, Gly30 and Gly32 forms the calcium-binding loop, which is responsible of coordinating the Ca²⁺ required during catalysis. In addition, there is an interfacial binding surface, which mediates the adsorption of the enzyme onto the lipid-water interface of the phospholipids membrane bilayer (3, 30).

Tannins, the secondary metabolites of plants, are mostly water-soluble phenolic compounds that can produce the common phenolic reactions and can precipitate alkaloids, gelatin, and other proteins (18). According to their structures, tannins are categorized as hydrolysable tannins, condensed tannins, or complex tannins (18). Hydrolysable tannins are the esters of the 3, 4, 5-trihydroxyl benzoic acid (gallic acid), which are esterified to a core polyol, and the galloyl groups may be further esterified or oxidatively cross-linked to form more complex structures. The implication of the enzymatic activity is a key step in the induction of myonecrosis, inflammation and neurotoxicity induced by PLA₂ (5, 31, 32). As it is shown in figure 2, tannic acid (control for inhibition) showed an excellent inhibitory capacity of PLA₂ activity (IC50 = 0.59 mM). Likewise, epigallotechingallate exhibited similar inhibitory activity (IC50 = 0.38 mM). In addition, this compound did not reveal significant differences with respect to tannic acid (control for inhibition) at the concentrations used. At the highest concentration used, gallic acid and its derivative, propylgallate, did not exhibit significant differences with respect to control for inhibition. They presented the following IC50 values: 1.84 mM and 1.84 mM, respectively.

 

Contrastively, the cinnamic acid derivatives, ferulic acid and caffeic acid showed the lowest inhibitory ability. They exhibited significant differences with respect to tannic acid (control for inhibition) at all the concentrations used (p > 0.05). Additionally, these compounds presented the following IC50 values: 3.93 mM and 1.40 mM, respectively.

Similarly, all compounds inhibited PLA₂ activity when the synthetic substrate (4N3OBA) was used. However, as it is shown in table 1, epigallocatechingallate presented the best inhibitory ability, while ferulic acid exhibited the lowest inhibitory capacity.

Due to the abundance of the PLA₂ in the venom of viperids/crotalids, and to the large amount of venom injected during snakebite accidents, these myotoxins are undoubtedly central to the development of myotoxicity, which occurs in two clinical patterns: local and systemic myotoxicity (33). The action of PLA₂ may result in irreversible lesions and even amputation of the affected limb (29). Additionally, it has been demonstrated that antivenoms generally have a limited efficacy against the local tissue damaging activities of venoms (7). Thus, there is a need to search for inhibitors and approaches that may be useful to complement conventional antivenom therapy. The use of cell cultures, such as rodent lines of skeletal muscle myoblasts/myotubes to evaluate miotoxicity of these PLA₂ enzymes, appears to correlate well with their in vivo myotoxic activity (24). This correlation was used to demonstrate the inhibitory capacity of phenolic compounds on the cytotoxic activity induced by PLA₂. As it is shown in figure 3, all compounds showed significant differences with respect to Tannic acid (control for inhibition).

 

However, the best inhibitory capacity was shown by epigallocatechingallate, with an inhibition of 90.92 ± 0.82 %. The lowest inhibitory activity was presented by ferulic acid with 30.96 ± 1.42 %; whereas, gallic acid, propylgallate and caffeic acid exhibited inhibition levels of 75.20 ± 0.75%, 41.34 ± 1.45%, and 56.50 ± 0.57%, respectively. At the concentration used, all compounds did not show cytotoxic effect on myotubes (data not show).

The non-selective precipitation on snake venom proteins and the chelating property of cofactors required by these enzymes are two possible modes of action attributed to polyphenols, especially those with complex structures (15, 16, 34, 35). However, by means of molecular docking studies it has been demonstrated that polyphenols (such as chlorogenic acid, curcumin, 1,3,5-trihydroxy benzene, 1,3-dihydroxy benzene and 2,4,6-trihydroxy acetophenone) inhibit PLA₂ by interacting with the enzyme active site (36, 37). In order to explain the differences among the inhibitory effects induced by the polyphenols used in this study, a molecular docking analysis was performed. As it is shown in figure 4, all compounds could be perfectly adjusted in the active site of PLA₂. In addition, all polyphenols showed a H-bonding interaction with Asp49. This bond could promote the destabilization of the calcium coordination, and it could cause a displacement of this cation from the calcium binding loop, which is essential for the enzymatic activity since it helps to polarize the sn2 ligation of the glycerophospholipids that will be hydrolyzed (27, 37). With the exception of propylgallate, all inhibitors presented a hydrogen bond with His48 that blocks water activation, which is important for a further basic general catalysis mechanism involved in hydrolysis of glicerophospholipids (30, 38). Additional H-bonding interactions were shown between caffeic acid and Cys29, and among epigallocatechingallate, Ala23 and Ala102. Moreover, the propylgallate carbonated side chain showed hydrophobic interactions with Leu6 and Phe5. Finally, as it is exposed in table 2, MolDock score values showed that epigallocatechingallate presented the strongest interaction energy, whereas gallic acid presented the least E_score (table 2). Nevertheless, this is a contradictory result, because gallic acid was the second more potent compound in the inhibition assays.

The best inhibitory ability of epigallocatechingallate on enzymatic and cytotoxic effects of PLA₂ could be explained by its physicochemical properties, which are presented in table 1. TPSA is the sum of the contributions to the molecular (usually van der Waals) surface area of polar atoms, such as oxygen, nitrogen and their attached hydrogens (39). TPSA indicates the surface area required to bind with the majority of the target receptor (PLA₂ in this case). As it is shown in figure 5, when a non-parametric correlation analysis between TPSA and IC50 values was performed, a significant correlation was observed (p = 0.0491, r = -0.8079 (-0.9878 to -0.2593)). Similar results were obtained with other polyphenolic compounds (flavonoids and isoflavones) for inhibiting telomerase and aromatase (40). The H bond donors and acceptors pattern of epigallocatechingallate is undoubtedly another property that should be considered. These donors and acceptors are known to play an important role in the contribution of water solubility by donning/accepting hydrogen bonds from water molecules and adding polarity to the structures. They also play an important role in drug-receptor interactions. Epigallocatechingallate has a great capacity of forming hydrogen bond interactions, providing a high affinity for PLA₂ (as it is shown in table 2, E_inter value). Therefore, this compound showed the lowest interaction energy (affinity) for the enzyme. And, this molecule also has four rotatable bonds, which may give more number of degrees of freedom for interacting with the PLA₂, therefore, it has more possibilities to form a stable complex. In fact, this is supported by the highest value of internal energy shown by epigallocatechingallate (table 2).

 

Polyphenolic compounds, such as f lavonoids, have already been reported to be PLA₂ inhibitors (41). Polyphenols (such as rosmarinic acid, aristolochic acid and α-tocoferol (vitamin E)) have also inhibited PLA₂s from snake venoms (17, 42, 43). Moreover, ferulic acid, caffeic acid and gallic acid have shown an inhibitory ability against the activities induced by whole snake venoms (15, 44). However, these compounds had not been evaluated on purified PLA₂s. Furthermore, from the results of this study, it can be concluded that propylgallate and epigallocatechingallate are two novel natural products with anti-myotoxic potential.

 

CONCLUSIONS

The use of plant extracts and other substances in different forms (poultices, steams, baths, among others) at the bite site is a common strategy used in the traditional medicine of several countries (10, 11). However, the efficacy of some of these practices have not been evaluated in controlled assays. In this study, some polyphenols that are present in different plants demonstrated to inhibit various activities induced by snake venom PLA₂. The topical application of these plant polyphenols directly at the bite site should produce, to some extent, the desired anti-venom effects, particularly the prevention of myotoxicity, which generally cannot be cured through the administration of antivenom. However, further in vivo investigation is be necessary to confirm the in vitro results.

 

ACKNOWLEDGEMENTS

The authors would like to thank Paola Rey Suarez for her technical help in general. We are also very grateful to Dr. Rene Thomsen for granting us a trail license for the Molegro Virtual Docker, University of Aarhus, Denmark. This project was partly supported by Universidad de Antioquia and COLCIENCIAS (project 393-2006).

 

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