Chemico-Biological Interactions 220 (2014) 94–101

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The biflavonoid morelloflavone inhibits the enzymatic and biological activities of a snake venom phospholipase A2 Jaime Andrés Pereañez a,⇑, Arley Camilo Patiño a, Vitelbina Núñez a,b, Edison Osorio c a

Programa de Ofidismo/Escorpionismo, Facultad de Química Farmacéutica, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia Escuela de Microbiología Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia c Grupo de Investigación en Sustancias Bioactivas, Facultad de Química Farmacéutica, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia b

a r t i c l e

i n f o

Article history: Received 3 December 2013 Received in revised form 12 June 2014 Accepted 16 June 2014 Available online 1 July 2014 Keywords: Morelloflavone Snakebite Local tissue damage Docking Phospholipase A2

a b s t r a c t The biflavonoid morelloflavone has been reported as inhibitor of secretory PLA2s (phospholipases A2 from human synovial and bee venom sources); however, its capacity to interact and inhibit snake venom PLA2 activities has not been described. In this work we tested the inhibitory ability of morelloflavone on the enzymatic, anticoagulant, myotoxic and edema-inducing activities of a PLA2 isolated from Crotalus durissus cumanensis venom. The biflavonoid displayed IC50 values of 0.48 mM (95% Confidence intervals: 0.45–0.51) and 0.38 mM (95% Confidence intervals: 0.36–0.40) on the PLA2 enzymatic activity, when either aggregated or monodispersed substrates were used, respectively. In addition, morelloflavone inhibited in a time-dependent manner and irreversibly the PLA2 enzymatic activity. When mice were injected with PLA2 preincubated (preincubation assay) with 0.13, 0.63 and 1.26 mM of the biflavonoid, the myotoxic activity induced by the PLA2 was inhibited up to 63%. Nevertheless, these values decreased up to 38% when the morelloflavone was injected into muscle after PLA2. Moreover, morelloflavone inhibited, in a concentration-dependent manner, edema-forming activity of the PLA2 in the footpad. Morelloflavone also inhibited the anticoagulant activities of the PLA2 in concentration-dependent mode. In order to have insights on the mode of action of morelloflavone, intrinsic fluorescence studies were performed. Results of these assays suggest that morelloflavone interacts directly with the PLA2. These findings were supported by molecular docking results, which suggested that morelloflavone forms hydrogen bonds with residues Gly33, Asp49, Gly53 and Thr68 of the enzyme. In addition, our results suggested a p–p stacking interaction between rings A of morelloflavone with that of the residue Tyr52, and Van der Waals interactions with Gly32, His48 and Ala56. Our molecular modeling results suggest that morelloflavone may occupy part of substrate binding cleft of the PLA2. Morelloflavone is a candidate for the development of inhibitors to be used in snakebite envenomation. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Envenomation by snakebites is 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]. In the case of snakes of the family Viperidae, which inflict the vast majority of accidents in the Americas, the pathophysiology of envenomation includes both local and systemic manifestations associated with hemorrhage, necrosis, edema, hypovolemia, nephrotoxicity, coagulopathy and cardiovascular shock [2]. This complex clinical picture is the result of the action of various venom components, predominantly proteinases, both metallo- and serine proteinases,

⇑ Corresponding author. Tel.: +57 42196649. E-mail address: [email protected] (J.A. Pereañez). http://dx.doi.org/10.1016/j.cbi.2014.06.015 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

phospholipases A2, C-type lectin-like proteins, and other minor components [3]. The most important and abundant muscledamaging components in snake venoms are phospholipases A2 (PLA2; EC 3.1.1.4). These enzymes hydrolyze the sn-2 ester bond of glycerophospholipids, releasing a fatty acid and a lysophospholipid [4]. In addition, PLA2 can also induce several pharmacological effects such as edema, modulation of platelet aggregation, as well as neurotoxicity, myotoxicity and anticoagulation [4,5]. The therapy for snakebite envenomations has been based on the intravenous administration of antivenoms [6,7]. However, it has been demonstrated that antivenoms have a limited efficacy against the local tissue damaging activities of venoms [8,9]. Thus, it is important to search for alternative sources of venom inhibitors, either synthetic or natural, that would complement the action of antivenoms, particularly regarding neutralization of local tissue damage.

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Medicinal plants represent a vital source of novel bioactive compounds with several pharmacological activities, and constitute possible alternatives for inhibiting venom components which, eventually, might complement the therapeutic action of conventional antivenom therapy [10,11]. Morelloflavone (Fig. 1), is a compound that consists of a naringenin covalently linked to a luteolin, that is a flavanone-(C-3 ? C-800 )-flavone biflavonoid. Morelloflavone possesses a wide range of biological activities, such as antiHIV activity by inhibiting both HIV-1 reverse transcriptase (HIV-1 RT) in vitro and HIV-1 (strain LAV-1) in human lymphocytes [12]. It also inhibits 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase) [13] and melanin production [14], also having antioxidant activity against LDL-peroxidation [15], and ameliorates atherosclerosis in mice [16], among other activities. Morelloflavone has also been reported as a potent inhibitor of secretory PLA2s (from human synovial and bee venom sources); in addition, this compound exerts potent anti-inflammatory effects in mice [17]. In our ongoing efforts to search for snake venom PLA2s inhibitors, in this work morelloflavone was purified from Garcinia madruno extracts, and tested in its capacity to inhibit enzymatic and toxic activities of a PLA2 isolated from the venom of Colombian Crotalus durissus cumanensis rattlesnake. Moreover, we explored the mode of action of morelloflavone using a variety of techniques.

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2.2. Animals Swiss Webster male mice, 18–20 g body weight, were used for the in vivo assays. All experiments were conducted in accordance with guidelines of the Universidad de Antioquia Ethics Committee (Medellín, Colombia). Indicators of pain in mice were evaluated as described Morton and Griffiths [22] and Carstens and Moberg [23]. Briefly, if an animal presented weight loss 15% body weight, hunched posture, rough haircoat, and/or inability to eat or drink, they were killed prior to the planned experiment termination with an over exposition to Isoflurane vapors. Each parameter had a scale from 0 through 5. Then, total score was recorded and the severity of pain was classified as follow: 0–5: normal; 6–10: monitor carefully, consider analgesics; 11–15: suffering, provide relief, observe regularly; and 16–20 Severe pain, sacrifice animal. For all injection, measurement and blood collection procedures, the animals were anesthetized with isoflurane.

2.3. Plant Material The aerial parts of G. madruno were collected in Medellín (Colombia). This sample was identified by M.Sc. Fernando Alzate. A voucher specimen (Alz-3030) was deposited at the Herbarium of Universidad de Antioquia (HUA).

2. Materials and methods 2.4. Extraction and Isolation 2.1. Purification of the PLA2 C. durissus cumanensis venom was obtained from four specimens coming from Meta, in the south east region of Colombia, and kept in captivity at the serpentarium of the Universidad de Antioquia (Medellín, Colombia). PLA2 was purified by molecular exclusion chromatography on Sephadex G-75, followed by reverse-phase HPLC on C-18 column eluted at 1.0 mL/min with a gradient from 0% to 100% acetonitrile in 0.1% trifluoroacetic acid (v/v). Absorbance in effluent solution was recorded at 280 nm [18]. Snake venom PLA2s are enzymes that are able to resist extreme chemical conditions, such as low pH, higher ion concentrations and temperature, among others [18,19]. In addition, several studies have demonstrated that snake venom PLA2s can be purified by HPLC using acetonitrile without changes in their enzyme and biological activities [18,20,21].

The dried material of G. madruno (1.8 kg) was successively extracted with hexane (3  8 L), EtOAc (3  8 L) and MeOH (3  8 L). After evaporation, a portion of the EtOAc extract (50 g) was fractionated by Vacuum liquid chromatography (VLC) on silica gel (6–35 lm; 8  10 cm) and eluted with a hexane, hexane–EtOAc, EtOAc–MeOH gradient solvent system to give eight main fractions (A–H) on the basis of their thin layer chromatography (TLC, silica gel, hexane–EtOAc 8:2; methanolic ferric chloride as a visualization reagent) behavior. Fraction C (7.72 g) was subjected to column chromatography (CC) on silica gel (40–63 lm; 2  40 cm) using hexane–EtOAc (8:2) as the solvent to give five fractions (C 1–5). Fraction 4–5, called BF, was rechromatographed by preparative TLC, eluting with EtOAc– Ether–BuOH (6:3:1) to give several compounds between them morelloflavone (0.23 g; Rf 0.50). The 1H, 13C-NMR, COSY, HMQC, and HMBC spectra were recorded on a Mercury 400F (400 MHz/ 100 MHz) spectrometer in CD3OD or DMSO-d (Agilent Technologies, Santa Clara, CA, USA). These spectra were used to validate the identity of morelloflavone by comparing with those spectra reported in the literature [24]. The purity of morelloflavone isolated was about 98%.

2.5. Inhibition of PLA2 activity using phosphatidylcholine as aggregated substrate

Fig. 1. Structure of morelloflavone ((2R,3S)-20 -(3,4-Dihydroxyphenyl)-5,50 ,7,70 -tetrahydroxy-2-(4-hydroxyphenyl)-2,3-dihydro-4H,40 H-3,80 -bichromene-4,40 -dione). The structure was built using the program Avogadro 1.1.0 [23].

This activity was assayed according to the method reported by Dole [25], with titration of free fatty acids (FA) released from phosphatidylcholine (from dried egg yolk, Sigma) suspended in 1% Triton X-100, 0.1 M Tris–HCl, 0.01 M CaCl2, pH 8.5 buffer, using 20 lg/ 10 lL of PLA2. The time of reaction was 15 min at 37 °C. The amount of protein was selected from the linear region of activity curves. For inhibition experiments, several concentrations of morelloflavone were pre-incubated with the enzyme for 30 min at 37 °C before PLA2 activity determination. The results are indicated as inhibition percentage, where 100% activity is the activity induced by PLA2 alone.

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2.6. Inhibition of PLA2 activity using 4-nitro-3-octanoyloxybenzoic acid (4N3OBA) as monodispersed substrate

above. Results are shown in percentage of inhibition, taking toxin and PBS injections as 100% and 0% of activity, respectively.

The measurements of enzymatic activity using the monodispersed substrate 4N3OBA were performed according to the method described by [26] and adapted for a 96-well ELISA plate. The standard assay contained 200 lL of buffer (10 mM Tris–HCl, 10 mM CaCl2, 100 mM NaCl, pH 8.0), 20 lL of 10 mM of substrate (4NO3BA), 20 lL of sample (20 lg PLA2 or 20 lg PLA2 + several concentrations of morelloflavone) and 20 lL of water. The negative control was only buffer. The inhibitory effect of morelloflavone on PLA2 activity was determined through co-incubation of the enzyme with each concentration of the 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 at 20 min.

2.10. Inhibition of edema-inducing activity

2.7. Dialysis study to test the reversibility of the inhibition Two ml of the reaction mixtures containing 20 lg of PLA2 and 0.35 or 7.0 mM of morelloflavone were pre-incubated along with 10 mM Tris–HCl, 10 mM CaCl2, 100 mM NaCl, pH 8.0 for 30 min. An aliquot from these mixtures were assayed for PLA2 activity before dialysis. The remaining mixture was dialyzed against 20 mL 10 mM Tris–HCl, 10 mM CaCl2, 100 mM NaCl, pH 8.0 at 4 °C in dialysis tubing (Molecular weight cutoff 3500) for 24 h with two buffer changes. Then, dialyzed solution was frozen at 20 °C and lyophilized. Finally, the PLA2 activity was calculated to 20 lg of lyophilized powder. 2.8. Effect of time on the inhibition of the PLA2 activity Mixtures of 20 lg of PLA2 and 3.5 or 7 mM morelloflavone in a total volume of 100 lL were pre-incubated for 0, 15 and 30 min at 37 °C. Then the inhibition of the PLA2 activity was measured as described above. 2.9. Inhibition of myotoxic activity 2.9.1. Inhibition of myotoxic activity in preincubation assays Myotoxic activity of the PLA2s was estimated by determining the plasma activity of creatine kinase (CK; EC2.7.3.2) in groups of three mice (18–20 g body weight), after an intramuscular injection (in the gastrocnemius) of 10 lg of PLA2, either alone, or preincubated with 0.126, 0.63 and 1.26 mM morelloflavone for 30 min at 37 °C. Control groups received an identical injection (100 lL) of either PLA2 alone or 3% DMSO in PBS, pH 7.2, alone. After 3 h, blood samples were collected from the tail into heparinized capillary tubes, and the plasma CK activity of plasma was determined by a kinetic assay (Weiner Lab, CK-NAC (Creatine kinase activated by N-acetyl cysteine) UV-AA. This kit use creatine phosphate a substrate for CK). Controls of 0–100% toxicity consisted of PBS and toxin, respectively. Experiments were carried out in duplicate. 2.9.2. Inhibition of myotoxic activity by independent administration of morelloflavone The ability of morelloflavone to inhibit myotoxicity by independent in situ administration was evaluated. Mice received an i.m. injection 3 lg of PLA2. Thirty seconds later 50 lL of 0.126, 0.63 or 1.26 mM morelloflavone were injected at the same site. Control animals received either PLA2 alone or 3% DMSO in PBS. After 3 h, plasma creatine kinase activity was determined as described

Groups of three mice (18–20 g) received a subcutaneous injection of 50 lL of PLA2 (containing 10 lg, corresponding to two minimum edematogenic doses) on the right footpad. The left footpad received 50 lL of 3% DMSO in PBS as control. Inhibition studies were performed by pre-incubating 0.25, 0.5 or 1.0 mM morelloflavone with PLA2 for 30 min at 37 °C. After injection, the progression of edema was evaluated with a caliper at intervals of 1, 2, 3, 6, 24 h and expressed in millimeters. Control animals received 1.00 mM of morelloflavone alone. Pain severity was evaluated was tested 15 min before each measure of the progression of edema, as described in Section 2.2. 2.11. Inhibition of anticoagulant activity Twenty-five micrograms of PLA2 in 25 lL PBS were mixed with 75 lL of different concentrations of morelloflavone (0.1, 0.5 or 1.0 mM), and pre-incubated for 30 min at 37 °C. After, 100 lL of mixtures were added to 0.5 mL of plasma, and incubated for 10 min at 37 °C. Plasma aliquots incubated with PBS or 1.0 mM morelloflavone were used as controls. Then, coagulation times were recorded after addition of 0.1 mL of 0.25 M CaCl2 [27]. 2.12. Intrinsic fluorescence The relative intrinsic fluorescence intensity of PLA2 with and without morelloflavone was monitored with Perkin-Elmer spectrofluorometer. A reaction mixture of 3.0 mL in 1 cm path length quartz cuvette contained 100 mM Tris HCl buffer (pH 7.4), 5 mM calcium, PLA2 (30 lg/ml) and 0.5 and 1.0 mM morelloflavone. Fluorescence spectra were measured between 300 and 500 nm after excitation at 280 nm. Three spectra were taken for each sample and all spectra were corrected by subtraction of buffer blanks. 2.13. Molecular docking studies The program Avogadro 1.1.0 [28] was used to build the morelloflavone molecule and to improve its overall structure by an energy minimization process based on the MMF94 force field by means of a steepest-descent algorithm in 500 steps. Molecular docking was carried out on a personal computer using Autodock Vina [29]. The PLA2 (PDB code 2QOG) from Crotalus durissus terrificus showed 57% homology in the N-terminal with the PLA2 used in these studies [18]. Protein was used without water molecules. The structure of the protein was prepared using the Protein Preparation module implemented in the Maestro program. First, hydrogen atoms were automatically added to each protein according to the chemical nature of each amino acid, on the basis of the ionized form, expected in physiological condition. This module also controls the atomic charges assignment. Second, each 3D structure of the protein was relaxed through constrained local minimization, using the OPLS (Optimized Potentials for Liquid Simulations) force fields in order to remove possible structural mismatches due to the automatic procedure employed to add the hydrogen atoms. 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. The a-carbon of His48 was used as center of the grid (X = 44.981, Y = 27.889 and Z = 46.392), whose size was 24 Å3. Exhaustiveness = 20. Then, the ligand poses with best affinity were chosen, and a visual inspection of the interactions at the active site was performed and recorded. Molegro Molecular Viewer (MMV 2.5.0, http://

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www.clcbio.com/products/molegro/#molecular-viewer) and UCSF Chimera (www.cgl.ucsf.edu/chimera/) were used to generate docking images. 2.14. Statistical analysis In order to determine significant differences among the concentrations of morelloflavone used in the inhibition of PLA2, myotoxic and edema-forming activity, two-way ANOVA followed by Bonferroni’s test was applied. To determine significant differences between control and compounds in the inhibition of anticoagulant activity, an ANOVA followed by Dunnett’s test was applied. In all cases, a difference with a p < 0.05 was considered significant. Results are shown as mean ± SEM of n indicated in each case. 3. Results 3.1. Inhibition of PLA2 activity Morelloflavone inhibited dose-dependently the PLA2 activity when either aggregated or monodispersed substrates were used (Fig. 2). When compared both substrates, at all concentrations of inhibitor used statistical differences were not detected (p > 0.05). The biflavonoid displayed median inhibitory concentrations (IC50) of 0.48 mM (95% Confidence Intervals: 0.45–0.51) when aggregated was used. In contrast, the IC50 was 0.38 mM (95% Confidence intervals: 0.36–0.40) when monodispersed substrate was used.

Fig. 3. Effect of time on the inhibition of the PLA2 activity. Three point five and 7.0 mM of morelloflavone were pre-incubated for 0, 15 and 30 min with 20 lg of PLA2. n = 5. ⁄Represent statistical differences as compared with the inhibition percentages of 7.0 mM values. Results are shown as mean ± SEM.

Whereas, these concentrations displayed 46.5 ± 2.4 and 60.3 ± 2.8 inhibition percentages, after dialysis process. Statistical differences were not revealed before and after dialysis study (p > 0.05).

3.2. Dialysis study and effect of time on the inhibition of the PLA2 activity

3.3. Inhibition of myotoxic activity

When 3.5 and 7.0 mM of morelloflavone were pre-incubated for 0, 15 and 30 min with the PLA2, an inhibition of the enzyme activity were detected in a time-dependent way. At 15 and 30 min statistical differences were observed between the concentrations used (p < 0.01) (Fig. 3). On the other hand, before dialysis 3.5 and 7.0 mM showed 49.4 ± 7.1 and 61.1 ± 2.0 inhibition percentages.

In both assays (preincubation and independent administration), the inhibition of myotoxic activity induced by the PLA2 was dose dependent (Fig. 4). Inhibition was more effective when enzyme and morelloflavone were incubated prior to the test than when they were administered independently (p < 0.01 at all doses tested).

Fig. 2. Inhibition of enzymatic activity of the venom PLA2. A constant amount of PLA2 (20 lg) was incubated, for 30 min at 37 °C, with either PBS or various concentrations of morelloflavone, and the enzymatic activity of the mixtures was assessed on 4NO3BA or phosphatidylcholine, as described in Section 2. Results are expressed in% inhibition, n = 6. Results are shown as mean ± SEM.

Fig. 4. Inhibition of PLA2-induced myotoxicity by morelloflavone in either preincubation or independent administration experiments. Three lg of PLA2 and different concentrations of morelloflavone were used, n = 6. ⁄Represent statistical differences as compared with the inhibition percentages of the preincubation assay. Results are shown as mean ± SEM.

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Fig. 5. Inhibition of edema-forming activity of the PLA2 by morelloflavone. A constant amount of the PLA2 was incubated for 30 min at 37° with either of 3% DMSO in PBS or various concentrations of morelloflavone. Then aliquots of 50 lL of the mixtures, containing 10 lg of the PLA2, were injected s.c. into the right footpads of mice. Controls included mice (Negative control) injected with 1.0 mM morelloflavone. Edema was assessed at various time intervals by measuring the increase in footpad thickness (n = 6). ⁄Represent statistical differences as compared with the PLA2. Results are shown as mean ± SEM.

3.4. Inhibition of edema-inducing activity The highest edema-inducing activity of the toxin was observed two hours after injection (Fig. 5). At this time, at 0.25, 0.5 and 1.0 mM morelloflavone, edema caused by PLA2 was reduced about 18%, 32% and 40%, respectively (p < 0.001). Significant edemainducing activity was not shown by 1.0 mM morelloflavone (the highest concentration of morelloflavone used in this assay). At 6 h and 24 h, statistical differences between all groups were not detected (p > 0.05). At all times, the pain severity in animals never was above 5 (normal), thus they never received analgesics. 3.5. Inhibition of anticoagulant activity Plasma incubated with PBS followed by CaCl2 addition showed clotting times of 6.23 ± 0.05 min. When plasma was incubated with the PLA2, and then CaCl2 was added, clotting time was 13.64 ± 0.36 min. At all concentrations of morelloflavone used in this assay, statistical differences were observed regarding the anticoagulant effect induced by PLA2 (p > 0.01) (Fig. 6). At 1.0 mM, morelloflavone did not display coagulant effect, showing comparable coagulation times with negative control in which plasma was incubated with PBS. 3.6. Intrinsic fluorescence To determine structural changes induced by morelloflavone on the PLA2, the intrinsic fluorescence of the enzyme with and without morelloflavone (0.75 or 1.5 mM) was recorded. Morelloflavone caused a moderate decrease in fluorescence intensity in a concentration-dependent manner (Fig. 7). These results suggest a slight structural modification in the protein and a direct interaction of morelloflavone with the PLA2. 3.7. Molecular docking studies Docked solution with the lowest binding energy was selected and described. The observed binding free energy of morelloflavone

Fig. 6. Inhibition of anticoagulant activity of the PLA2 by morelloflavone. Twentyfive micrograms of PLA2 were mixed with different concentrations of morelloflavone, and pre-incubated for 30 min at 37 °C. Afterward, 100 lL of the mixtures were added to 0.5 mL of plasma, and incubated for 10 min at 37 °C. The coagulation was induced by the addition of 0.1 mL of 0.25 M CaCl2. Finally coagulation times were recorded (n = 6). ⁄Represents statistical significant difference respect to PLA2 alone. Results are shown as mean ± SEM.

was 7.6 kcal/mol. Docking results suggested that OH moieties from 30 00 and 4000 of morelloflavone could form hydrogen bonds with residues Gly33 and Asp49, respectively. In the same way, hydroxyls from 500 and 700 of morelloflavone could form hydrogen bonds with residues Gly53 and Thr68, respectively (Fig. 8A). In addition, our results suggested a p–p stacking interaction between rings A of morelloflavone with that of the residue Tyr52. Additional Van der Waals interactions with Gly32, His48 and Ala56 were also detected.

4. Discussion Phospholipase A2-induced myotoxicity occurs in two clinical patterns: local and systemic myotoxicity [30]. The action of these enzymes may result in irreversible lesions, which in addition to edema, hemorrhage and blistering may lead to permanent tissue loss, disability or amputation of the affected limb [1,2]. Neutralization of these activities by antibodies present in antivenoms is a difficult task because some of these effects appear within few minutes after envenomation [8]. Consequently, the use of synthetic/natural PLA2 inhibitors that could be administered in the field directly at the same site of venom injection may represent a valid alternative to confront this difficult problem. In this direction, we assayed the ability of the morelloflavone to inhibit enzymatic and toxic activities of a snake venom PLA2. Morelloflavone inhibited the enzymatic activity of the snake venom PLA2 with IC50 values of 0.48 mM and 0.38 mM, when either aggregated or monodispersed substrates were used. Nevertheless, these values are higher than that reported for Gil et al. [17] for the inhibition of human recombinant synovial and bee venom enzymes (IC50 = 0.9 and 0.6 lM, respectively). This is likely due to the use of different models to measure the PLA2 enzymatic activity or, alternatively, to differences in the PLA2s tested. It is known that human synovial, snake and bee venom enzymes have different catalytic behavior [31]. In addition, the inhibitory effect of morelloflavone on the PLA2 enzyme activity was timedependent and irreversible, as evident by dialysis experiment. On the other hand, we also tested the inhibitory capacity of morelloflavone on the myotoxic effect induced by the PLA2. In both (preincubation and independent administration) assays morelloflavone moderately inhibited the myotoxic activity of the enzyme.

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Fig. 7. Intrinsic fluorescence spectra of the PLA2 in presence or absence of 0.7 and 1.75 mM morelloflavone. Results are shown as a mean of three independent experiments.

Fig. 8. Molecular docking results. (A) Interaction of morelloflavone with active-site residues of the PLA2. Hydrogen bonds (blue dotted lines). Ca2+ ion is shown as a blue sphere. Hydroxyl moieties involved in H-bond interactions are indicated. Ring A is also marked to indicate a p–p stacking interaction. (B) Binding of morelloflavone to substrate binding cleft. Morelloflavone occupies part of substrate binding site (hydrophobic channel) of the PLA2. Blue sphere represents Ca2+ ion. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Nevertheless, in experiments that resemble the actual situation of snakebite (independent administration) the neutralization values were lower than those obtained in the preincubation assay. However, the reduction in muscle damage is still of considerable benefit since the major limitation for the development of any clinically effective inhibitor of myotoxins is the dramatic speed at which PLA2s affects skeletal muscle [30]. Local edema is an effect frequently induced by snake venom PLA2s, which may be due to their combined effect to hydrolyze/ destabilize phospholipid membrane – resulting in the loss of membrane integrity–, as well as their metabolic activity generating proinflammatory products such as eicosanoids, which amplify the inflammatory event [32]. Gil et al. [17] demonstrated the antiinflammatory activity of morelloflavone. Nevertheless, this biflavonoid failed to modify arachidonic acid-induced ear inflammation or eicosanoid levels in ear homogenates [17]. These results proposed that anti-inflammatory mechanism is not related to its capacity to inhibit the PLA2 activity. In contrast, in our study, morelloflavone inhibited paw-edema provoked by the venom PLA2 in preincubation assay. We suggest that anti-inflammatory activity of morelloflavone, in the range of mM, is also associated with its capacity to inhibit PLA2 enzyme and maybe other mechanisms, such as COX inhibition, as proposed for other biflavonoids [33]. Our results indicate that morelloflavone may be used as a lead for developing new anti-inflammatory agents. In vitro anticoagulation is another effect induced by some snake venom PLA2s. Morelloflavone inhibited the anticoagulant activity of the PLA2 in a dose-dependent manner. The enzyme used in this study was classified as a ‘‘strong anticoagulant PLA2’’ [18]. The mechanism of this type of enzymes is related to their capacity to interact by means of their ‘‘anticoagulation site’’ with factor Xa during the coagulation cascade [34]. The human coagulation factor Xa-binding site on the surface of snake venom PLA2 was reported by Faure et al. [35], and it is composed by residues: 2, 3, 7 (helix A); 16; 18, 19 (helix B); 23, 24; 31–34 (Ca2+ loop); 53, 59, 60, 69, 70 (helix C-b-wing loop); and 118, 119, 121–124, 129–131, 133 (C-terminal segment). The change in the intrinsic fluorescence reflects conformational changes in a protein due to the interaction with a ligand [36,37]. As observed in the fluorescence spectra, morelloflavone reduced the intensity of the fluorescence spectra of the enzyme, which suggests a direct interaction between the biflavonoid and PLA2. Similar results were obtained with cholic and ursodeoxycholic acids, which also inhibited the enzyme used in this study [36]. In an effort to describe the mode of action of morelloflavone on the PLA2, a docking study was performed. Results of molecular docking suggest that morelloflavone could interact with the active site (His48, Asp49 and Tyr52) and other regions of the enzyme. Our results also propose that biflavonoid could form hydrogen bonds with residues Gly33 and Asp49. These specific interactions between morelloflavone and the enzyme could also displace the Ca2+-coordinated ion, which is required for the catalysis. In addition, the inhibitor could block the binding of the substrate to the active site of the PLA2, because as depicted in Fig. 8B, rings A and C of morelloflavone could occupy part of substrate binding site (hydrophobic channel). All of the interactions mentioned above could explain the inhibition of PLA2 enzymatic activity, and further decreasing of myotoxic and edema-forming activities. It has been reported that enzymatic activity of the PLA2s is a key step on induction of myonecrosis and inflammation [30,32]. On the other hand, molecular modeling results also suggested that hydroxyl moieties of 30 00 and 500 of morelloflavone could form hydrogen bonds with Gly33 and Gly53, respectively. These interactions could cause steric clashes to the binding to human coagulation factor Xa, and they could also explain the inhibitory capacity of the biflavonoid on the anticoagulant activity of the snake venom PLA2. For

the reason that, these residues are involved in the binding of that coagulation factor [35]. In our screening assay (in vitro), we tested several concentrations of morelloflavone (lM through mM), nevertheless those of the range of lM did not inhibit the venom PLA2. However, we decided to test some concentrations of morelloflavone in the range of mM. The results are promising; however, we are looking for a strategy to improve the inhibitory concentrations of morelloflavone (or its derivative) on in vitro and subsequently in vivo effects of our target. 5. Conclusion In conclusion, we report the inhibitory activity of morelloflavone on the enzymatic, myotoxic, edema-forming and anticoagulant activities induced by a snake venom PLA2. Our results suggest that this compound could interact with the active site and other regions of the enzyme, blocking its catalytic cycle and the binding of substrate. In addition, our results propose that morelloflavone is a candidate for the development of inhibitors to be used in snakebite envenomation and inflammatory pathologies. Conflict of Interest The authors declare that there are no conflicts of interest associated with this work. Transparency Document The Transparency document associated with this article can be found in the online version.

Acknowledgements The authors acknowledge the Comité para el Desarrollo de la Investigación (CODI) of the Universidad de Antioquia for financial support from project CIQF-177 and the Estrategia de Sostenibilidad 2013-2014 of the both Investigation Groups. The authors thank Raquel Agudelo for her collaboration in some experiments. References [1] J.M. Gutiérrez, D. Williams, H.W. Fan, D.A. Warrell, Snakebite envenoming from a global perspective: towards an integrated approach, Toxicon 51 (2010) 80–92. [2] D.A. Warrel, Epidemiology, clinical features and management of snake bites in Central and South America, in: J. Campbell, W.W. Lamar (Eds.), Venomous Reptiles of the Western Hemisphere, Vol. 2, Cornell University Press, Ithaca, New York, 2004, pp. 706–761. [3] J.J. Calvete, Proteomic tools against the neglected pathology of snake bite envenoming, Expert. Rev. Proteomics 8 (2011) 739–758. [4] R.M. Kini, Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes, Toxicon 42 (2003) 827–840. [5] D.A. Six, E.A. Dennis, The expanding superfamily of phospholipase A(2) enzymes: classification and characterization, Biochim. Biophys. Acta 1488 (2000) 1–19. [6] C. Bon, The serum – therapie was discovered 100 years ago, Toxicon 34 (1996) 142–143. [7] J.M. Gutiérrez, G. León, B. Lomonte, Y. Angulo, Antivenoms for snakebite envenoming, Inflamm. Allergy – Drug Targets 10 (2011) 369–380. [8] J.M. Gutiérrez, G. León, G. Rojas, B. Lomonte, A. Rucavado, F. Chaves, Neutralization of local tissue damage induced by Bothrops asper (terciopelo) snake venom, Toxicon 36 (1998) 1529–1538. [9] J.M. Gutiérrez, B. Lomonte, G. León, A. Rucavado, F. Chaves, Y. Angulo, Trends in snakebite envenomation therapy: scientific, technological and public health considerations, Curr. Pharm. Des. 13 (2007) 2935–2950. [10] A.M. Soares, F.K. Ticli, S. Marcussi, M.V. Lourenço, A.H. Januário, S.V. Sampaio, J.R. Giglio, B. Lomonte, P.S. Pereira, Medicinal plants with inhibitory properties against snake venoms, Curr. Med. Chem. 12 (2005) 2625–2641.

J.A. Pereañez et al. / Chemico-Biological Interactions 220 (2014) 94–101 [11] J.A. Pereañez, V. Núñez, A.C. Patiño, M. Londoño, J.C. Quintana, Inhibitory effects of plant compounds on enzymatic and activities induced by a snake phospholipase A2, Vitae 18 (2011) 295–304. [12] Y.M. Lin, H. Anderson, M.T. Flavin, Y.H. Pai, E. Mata-Greenwood, T. Pengsuparp, J.M. Pezzuto, R.F. Schinazi, S.H. Hughes, F.C. Chen, In vitro anti-HIV activity of bioflavonoids isolated from Rhus succedanea and Garcinia multiflora, J. Nat. Prod. 60 (1997) 884–888. [13] K.A. Tuansulong, N. Hutadilok-Towatana, W. Mahabusarakam, D. Pinkaew, K. Fujise, Morelloflavone from Garcinia dulcis as a novel biflavonoid inhibitor of HMG-CoA reductase, Phytother. Res. 25 (2011) 424–428. [14] D.A. Mulholland, E.M. Mwangi, N.C. Dlova, N. Plant, N.R. Crouch, P.H. Coombes, Non-toxic melanin production inhibitors from Garcinia livingstonei (Clusiaceae), J. Ethnopharmacol. 149 (2013) 570–575. [15] E. Osorio, J. Londoño, J. Bastida, Low-density lipoprotein (LDL)-antioxidant bioflavonoids from Garcinia madruno, Molecules 18 (2013) 6092–6100. [16] D. Pinkaew, N. Hutadilok-Towatana, B.B. Teng, W. Mahabusarakam, K. Fujise, Morelloflavone, a biflavonoid inhibitor of migration-related kinases, ameliorates atherosclerosis in mice, Am. J. Physiol. Heart Circ. Physiol. 302 (2012) H451–458. [17] B. Gil, M.J. Sanz, M.C. Terencio, R. Gunasegaran, M. Payá, M.J. Alcaraz, Morelloflavone, a novel biflavonoid inhibitor of human secretory phospholipase A2 with anti-inflammatory activity, Biochem. Pharmacol. 53 (1997) 733–740. [18] J.A. Pereañez, V. Núñez, S. Huancahuire-Vega, S. Marangoni, L.A. Ponce-Soto, Biochemical and biological characterization of a PLA2 from crotoxin complex of Crotalus durissus cumanensis, Toxicon 53 (2009) 534–542. [19] S.M. Munekiyo, S.P. Mackessy, Effects of temperature and storage conditions on the electrophoretic, toxic and enzymatic stability of venom components, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 119 (1998) 119–127. [20] D.C. Damico, T. Vassequi-Silva, F.D. Torres-Huaco, A.C. Nery-Diez, R.C. de Souza, S.L. Da Silva, C.P. Vicente, C.B. Mendes, E. Antunes, C.C. Werneck, S. Marangoni, LmrTX, a basic PLA2 (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity, Toxicon 60 (2012) 773–781. [21] L.J. Vargas, M. Londoño, J.C. Quintana, C. Rua, C. Segura, B. Lomonte, V. Núñez, An acidic phospholipase A2 with antibacterial activity from Porthidium nasutum snake venom, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 161 (2012) 341–347. [22] D.B. Morton, P.H. Griffiths, Guidelines on the recognition of pain, distress and discomfort in experimental animals and an hypothesis for assessment, Vet. Rec. 16 (1985) 431–436.

101

[23] E. Carstens, G.P. Moberg, Recognizing pain and distress in laboratory animals, ILAR J. 41 (2000) 62–71. [24] X.C. Li, A.S. Joshi, B. Tan, H.N. ElSohly, L.A. Walker, J.K. Zjawiony, D. Ferreira, Absolute configuration, conformation, and chiral properties of flavanone(3 ? 80 0 )-flavone bioflavonoids from Rheedia acuminata, Tetrahedron 58 (2002) 8709–8717. [25] V.P. Dole, A relation between non esterified-fatty acids in plasma and the metabolisms of glucose, J. Clin. Invest. 35 (1956) 150–154. [26] M. Holzer, S.P. Mackessy, An aqueous endpoint assay of snake venom phospholipase A2, Toxicon 34 (1996) 1149–1155. [27] J.M. Gutiérrez, B. Lomonte, F. Chaves, E. Moreno, L. Cerdas, Pharmacological activities of a toxic phospholipase A isolated from the venom of the snake Bothrops asper, Comp. Biochem. Physiol. 84C (1986) 159–164. [28] M.D. Hanwell, D.E. Curtis, D.C. Lonie, T. Vandermeersch, E. Zurek, G.R. Hutchison, Avogadro: an advanced semantic chemical editor, visualization, and analysis platform, J. Cheminform. 4 (2012) 1–17. [29] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading, J. Comput. Chem. 31 (2010) 455–461. [30] J.M. Gutiérrez, C.L. Ownby, Skeletal muscle degeneration induced by venom phospholipase A2: insights into the mechanisms of local and systemic myotoxicity, Toxicon 42 (2003) 915–931. [31] O.G. Berg, M.H. Gelb, M.D. Tsai, M.K. Jain, Interfacial enzymology: the secreted phospholipase A(2)-paradigm, Chem. Rev. 101 (2001) 2613–2654. [32] C.F. Teixeira, E.C. Landucci, E. Antunes, M. Chacur, Y. Cury, Inflammatory effects of snake venom myotoxic phospholipase A2, Toxicon 42 (2003) 947– 962. [33] H. Park, Y.H. Kim, H.W. Chang, H.P. Kim, Anti-inflammatory activity of the synthetic C–C bioflavonoids, J. Pharm. Pharmacol. 58 (2006) 1661–1667. [34] R.M. Kini, Structure–function relationships and mechanism of anticoagulant phospholipase A2 enzymes from snake venoms, Toxicon 45 (2005) 1147–1161. [35] G. Faure, V.T. Gowda, R.C. Maroun, Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics, BMC Struct. Biol. 7 (2007) 82. [36] J.A. Pereañez, V. Núñez, A.C. Patiño, Inhibitory effects of bile acids on enzymatic and pharmacological activities of a snake venom phospholipase A2 from group IIA, Protein J. 30 (2011) 253–261. [37] J.A. Pereañez, A.C. Patiño, P. Rey-Suarez, V. Núñez, I.C. Henao, C.A. Rucavado, Glycolic acid inhibits enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper snake venom: insights from docking and molecular modeling, Toxicon 71 (2013) 41–48.