Research Article | Open Access
Inhibition of Neurotoxic Secretory Phospholipases A2 Enzymatic, Edematogenic, and Myotoxic Activities by Harpalycin 2, an Isoflavone Isolated from Harpalyce brasiliana Benth
Secretory phospholipases A2 (sPLA2) exert proinflammatory actions through lipid mediators. These enzymes have been found to be elevated in many inflammatory disorders such as rheumatoid arthritis, sepsis, and atherosclerosis. The aim of this study was to evaluate the effect of harpalycin 2 (Har2), an isoflavone isolated from Harpalyce brasiliana Benth., in the enzymatic, edematogenic, and myotoxic activities of sPLA2 from Bothrops pirajai, Crotalus durissus terrificus, Apis mellifera, and Naja naja venoms. Har2 inhibits all sPLA2 tested. PrTX-III (B. pirajai venom) was inhibited at about 58.7%, Cdt F15 (C. d. terrificus venom) at 78.8%, Apis (from bee venom) at 87.7%, and Naja (N. naja venom) at 88.1%. Edema induced by exogenous sPLA2 administration performed in mice paws showed significant inhibition by Har2 at the initial step. In addition, Har2 also inhibited the myotoxic activity of these sPLA2s. In order to understand how Har2 interacts with these enzymes, docking calculations were made, indicating that the residues His48 and Asp49 in the active site of these enzymes interacted powerfully with Har2 through hydrogen bonds. These data pointed to a possible anti-inflammatory activity of Har2 through sPLA2 inhibition.
The flavonoids are a group of plant secondary metabolites widely distributed in nature. They are divided in two main classes: the flavonoids and the isoflavonoids. Many pharmacological activities have been described for both classes such as anti-inflammatory and antioxidant, antiallergic, antiviral, and anticancer [1, 2]. Many works have shown the phospholipase A2 (PLA2) inhibitory effects of flavonoids such as rutin , morin , and quercetin , however, only few works have been shown the antiphospholipasic A2 activity of isoflavonoids .
The PLA2 s are a class of enzymes (EC 184.108.40.206) that catalyzes the hydrolysis of the sn-2 ester bond of phospholipids to produce lysophospholipids and free fatty acids, which are substrates for the synthesis of proinflammatory eicosanoids and platelet aggregating factor (PAF) [7, 8], in addition to reactive oxygen species produced during the synthesis of eicosanoids (which play a role as a positive feedback of the enzymatic active of the PLA2) . They are divided in two major groups: cytosolic phospholipase A2 (cPLA2) and secretory phospholipase A2 (sPLA2). The last are divided in ten groups as discussed elsewhere . In many inflammatory diseases, as in sepsis, atherosclerosis, and rheumatoid arthritis, group IIA sPLA2 are found in the inflammatory areas and play a role not fully understood up to date [11, 12].
The sPLA2 present in animal venoms share structural features with mammalian (including human) group IIA sPLA2, mainly in the active site, being for that reason useful tools for the study of sPLA2 inhibitors [7–12]. The exogenous administration of these enzymes to experimental animals provokes an inflammatory response similar to that observed with administration of endogenous sPLA2, besides more specific responses as myonecrosis [5, 13].
Current anti-inflammatory therapies include nonsteroidal anti-inflammatory drugs that inhibit either LOX or COX-1/2 enzymes and have serious side effects such as gastrointestinal ulceration, bleeding, and cardiovascular complications. In addition to these problems, COX-1/2 and LOX inhibitors cannot regulate the production of the PAF, which continues causing inflammation [7, 8]. Effective inhibitors of sPLA2 could be capable of depleting the downstream proinflammatory metabolites of arachidonic acid as well as PAF, without the adverse effects of the current corticosteroids therapy since these enzymes are secreted only in pathological conditions [7–10].
Harpalyce brasiliana Benth. (Fabaceae) is a Brazilian folk medicine, popularly known as “raiz-de-cobra” (Port. Lit. snakeroot). Its roots have been used in the Northeast of Brazil to treat snakebite , while its leaves are claimed to be anti-inflammatory (personal unpublished data). In this paper, the effect of harpalycin 2 in structure and enzymatic, edematogenic, and myotoxic activities of four sPLA2 isolated from animal venoms was evaluated. In addition, the analysis of the interaction between harpalycin 2 and the active site of the tested sPLA2 was performed by docking calculations.
2. Material and Methods
Secretory PLA2 from Bothrops pirajai (PrTx-III) and Crotalus durissus terrificus (Cdt F15) were purified as described by Toyama et al. [15, 16], respectively. sPLA2 from Apis mellifera venom was purchased from BIOMOL International. Bovine pancreas and Naja naja venom sPLA2 were purchased from Sigma-Aldrich. The COX-1, COX-2, LOX 15hrc, and LOX 15syP1 came from Cayman Chemical. Other salts, reagents, solvents were ultrapure grade, HPLC grade, or sequencing grade purchase from the BIORAD, Sigma-Aldrich (Supelco) and Pharmacia.
2.2. Plant Material
Leaves of Harpalyce brasiliana Benth. were collected at the Chapada do Araripe, Barbalha (Ceará, Brazil) by Prof. E. R. Silveira. Botanical authentication was made by Prof. E. P. Nunes of the Department of Biology, Federal University of Ceará. Voucher specimen (number: 32525) has been deposited at the Prisco Bezerra Herbarium (EAC), Department de Biology, Federal University of Ceará, Fortaleza (Ceará, Brazil).
2.3. General Procedures
The mass spectra were obtained on a Hewlett-Packard 5971 mass spectrometer by electron impact ionization (70 eV). 1H and 13C NMR spectra were recorded on a Bruker Avance DRX-500 (500 MHz for 1H and 125 MHz for 13C); chemical shifts were expressedin scale and were referenced to residual DMSO (2.5 and 39.5 ppm). Silica Gel 60 (Merck, 70–230 mesh) was used for analytical TLC. Column chromatographies were performed over silica gel (Merck, 60 F254 230–400 mesh).
2.4. Extraction and Isolation of Harpalycin 2
Leaves of Harpalyce brasiliana were pulverized and extracted with EtOH at room temperature. The solvent was removed under reduced pressure which produced a dark viscous extract (HBFE). Liquid-liquid partition of a water suspension of HBFE (110 g) using petrol ether, CHCl3, EtOAc, and n-BuOH yielded five fractions after solvent evaporation: HBFEEp (24.5 g), HBFEC (22.4 g), HBFEA (6.8 g), HBFEB (30.4 g), and HBFEAq (21.2 g).
Flash chromatography of HBFEC (12.0 g) using n-hexane and EtOAc as binary mixtures of increasing polarity afforded 30 fractions, which were pooled in 9 fractions after thin layer chromatography (TLC) analysis. HBFEC (10–12) presented a yellow precipitate, yielding 120.0 mg after recrystallization. NMR and Mass-spectrometric analysis showed the structure of the isoflavone harpalycin 2 (Har2). The fractions HBFEC (8-9) and HBFEC (13–17) were purified, using the same method, yielding more 200.0 mg of Har2.
2.5. Inhibition of sPLA2 Activity
sPLA2 activity was measured following the protocols described by Hernandez-Oliveira et al.  and modified by Toyama et al.  for 96-well plate. The standard assay mixture contained 200 mL of buffer (10 mM Tris-HCl, 10 mM CaCl2, 100 mM, and NaCl, pH 7.8), 20 μL of substrate (4-nitro-3-octanoyloxy-benzoic acid (4N3OBA) 1 mg/mL, manufactured by BIOMOL, USA), 20 μL of water, and 20 μL of sPLA2 solution (1 mg/mL). Enzymatic activity was calculated based on the increase in absorbance at 425 nm after 20 min, at 37°C, as a direct result of the cleavage of the synthetic substrate. All assays were done using and absorbance was measured using a SpectraMax 340 multiwell plate reader (Molecular Devices, Sunnyvale, CA). Evaluation of Har2 effect on sPLA2 enzymatic activity was performed after incubation of Bothrops pirajai (PrTx-III), Crotalus durissis terrificus (Cdt F15), Apis mellifera (purified, without the mellitin component), and Naja naja sPLA2 with Har2 at equal mass (1 : 1; w : w) for a period of 30 minutes. The final concentration of the inhibitor in the reaction mixture was the same of the substrate due to the kinetic behavior of the sPLA2.
Male Swiss mice (20–25 g) obtained from the Animal Facilities of Federal University of Ceará were used in this study. The animals were maintained under standard conditions (°C; 12 h light/dark cycle) with food and water ad libitum. All experiments with animals were guided in accordance with Brazilian laws for Care and Use of Laboratory Animals and all the study protocols were approved by Committee of Ethics from Federal University of Ceará (Fortaleza, Brazil) protocol number 68/08.
2.7. Neutralization of the Edema Inducing Activity
Neutralization of sPLA2-induced paw edema by Har2 was performed according to Iglesias et al. , using male Swiss mice (20–25 g, ). The edema was induced by a single subplantar injection of 25 μL of sPLA2 (25 μg/paw). Paw volume was measured immediately before the injection of the samples and at selected time intervals thereafter (30, 60, 120, 240, and 480 minutes) using a plethysmometer (Ugo Basile, Italy). All samples were dissolved in sterile PBS. Results were expressed as the increase in paw volume (μL) and calculated by subtracting the basal volume. Evaluation of Har2 effect on sPLA2 edema-inducing activity was carried out after incubation of Bothrops pirajai (PrTx-III), Crotalus durissis terrificus (Cdt F15), Apis mellifera (purified, without the mellitin component), and Naja naja sPLA2 with Har2 at equal mass (1 : 1; w : w) for 30 minutes at 37°C. The negative controls were performed by administration of Har2 (25 μg/paw). These values were subtracted from the volume of the paws treated with sPLA2 incubated with Har2 for clarity reasons.
2.8. Neutralization of Myotoxic Activity
Plasma creatine kinase (CK) activity was measured using a CK-UV kinetic kit (Sigma Chemical Co.). Native sPLA2 (1 mg/mL) or those previously incubated with Har2, as described above, were injected intramuscularly (25 μL) in the gastrocnemius of male Swiss mice (20–25 g, ). The control group was injected with sterile PBS, and the negative control with Har2. After 3 hours, a blood sample was collected from the tails using heparinized capillary tubes and centrifuged for plasma separation. CK activity was determined in triplicate using 4 μL of plasma according to the manufacturer’s instructions, and its activity was expressed in U/L.
2.9. Characterization of IC50 of Har2 against Several sPLA2 and Inflammatory Enzymes
The inhibitory capability of Har2 against COX-1/2, LOX 15hrc, and 15syP1, bovine pancreas, and human group V sPLA2 was investigated according to the manufacturer’s instructions (Cayman Chemical). All assays were carried out using and the data measured using a SpectraMax 340 multiwell plate reader (Molecular Devices, Sunnyvale, CA). Har2 was added in different concentrations and IC50 values were calculated using GraphPad Prism 5.0.
2.10. Circular Dichroism Spectroscopy
Native sPLA2, and Har2-treated sPLA2 were dissolved in 10 mM sodium phosphate buffer (pH 7.4) and final protein concentrations were adjusted to 8.7 mM. After centrifugation at 4000 g for 5 min, samples of 20 μL were injected into molecular exclusion column TSK G4000SWXL (0.7 × 300 mm) coupled in the LC-2000Plus Series HPLC Systems (Jasco, USA), which have been previously equilibrated with same buffer used for the preparation of the samples sPLA2, Har2-treated sPLA2. In this case, the chromatographic run of each samples was simultaneously monitored using a CD-2095 Circular Dichroism HPLC detector (Jasco, USA), FP-2020 Fluorescence detector, and UV-2075 190 to 600 nm detector. Circular dichroism spectra were obtained by adjust the wavelength range 220–260 nm and 260–320 nm to measure the presence of random coil and tertiary protein folding, respectively. Data collection was performed with a bandwidth of 1 nm, response time of 1 s at room temperature with 100 nm/min scanning speed. The fluorescence detection was adjusted specifically for monitoring the fluorescence emission of tryptophan, which was measured between 300 and 450 nm after excitation at 280 nm.
2.11. Molecular Modeling (Docking)
The structural optimization of the harpalycin 2 ligand was initially achieved using the AM1 method  implemented in the BioMedCache program (BioMedCache, 1989) with default values for the convergence criteria. Docking calculations were performed with the GOLD 4.0 program  in order to obtain the relative in silico affinities of the Har2 ligand with respect to the sPLA2 targets. The sPLA2 structures were taken from the RCSB Protein Data Bank (http://www.pdb.org/), under the PDB ID: 1GMZ, 2QOG, 1PSH, and 1POC, respectively, for PrTX-III, Cdt F15, Naja, and Apis.
The docking calculations were performed taking advantage of the flexibility of the Har2 ligand, by activating its rotational degrees of freedom. The active site was defined as all atoms within a radius of 10.0 from the residue 48 (His or Asp), which is an important residue according to the literature [21, 22].
2.12. Statistical Analysis
Results were expressed as the mean ± SEM of replicated experiments. The significance of differences between means was assessed by an analysis of variance, followed by a Dunnett’s test where several experimental groups were compared with the control group. The confidence limit for significance was set at .
3. Results and Discussion
Natural products from plants are of potential interest for the treatment of a number of inflammatory diseases. They serve as template molecules for the development of new drugs and prototypes . Flavonoids are polyphenolic compounds widely distributed in plants. Due to their various effects on immune and inflammatory systems, these compounds are currently of great pharmacological interest . There are several reports demonstrating that flavonoids are able to inhibit PLA2 activity, arachidonic acid release, and the formation of arachidonic acid metabolites [3, 4, 13]. In particular, isoflavones have also been reported to show anti-inflammatory activities, including inhibition of phospholipases A2 and COX-1/2 [17, 18]. Harpalycin 2 (Har2) was isolated as a white amorphous solid with m.p. 232.6–234.4°C. Its molecular formula of C21H18O7 was established by the molecular ion at m/z 382 Daltons in the MS spectrum. Structure elucidation was performed by spectroscopic means, including 1D and 2D NMR, and comparison with the data from literature . The structure of Har2 is shown in Figure 1.
Group IIA secretory phospholipases A2 may be catalytically active or inactive depending on the amino acid residue 49. In catalytically active isoforms, this residue is occupied by an aspartic acid . Cotrim et al.  showed through docking calculations that the chemical treatment of crotoxin B (Cdt F15) with quercetin lead to an inhibition of enzymatic activity due to the fact that quercetin binds in the vicinity of His48 and Asp49 residues. Herein, Har2 inhibits all sPLA2 tested when the treatment was made before the substrate addiction, with percentages of inhibition at about 58.7% for PrTX-III, at 78.8% for Cdt F15, at 87.7% for Apis, and at 88.1% for Naja secretory phospholipase A2 (Figure 2). These percentages of inhibition were greater than p-bromophenacyl bromide (~40%), a well-known sPLA2 inhibitor .
Neutralization of edema induced by exogenous sPLA2 administration performed in mice paws by harpalycin 2 showed significant inhibition of the edema initial step induced by PrTX-III, Cdt F15, Apis, and Naja (Figure 3). This first step is correlated with histamine/serotonin involvement . The edema-inducing effect of sPLA2 s could be attributed to their ability to hydrolyse phospholipids; however Lys49 phospholipase A2-homologues can also induce edema in the absence of PLA2 activity, which implies a different mechanism of action for this pharmacological effect . In this case, all sPLA2 tested were catalytic active and the partial inhibition of edema formation may be correlated to the inhibition of phospholipids catalysis by the isoflavone.
Harpalycin 2 was able to inhibit the myotoxic activity of the venom secretory phospholipases A2 tested in this study (Figure 4), which is a pharmacological activity shared by several types of snake venom sPLA2 . This model, when studied using Asp49 sPLA2, is interesting for evaluation of the interfacial activation of the sPLA2s, since myotoxicity is directed linked with enzymatic activity in the catalytic sPLA2 isoforms . Recently, involvement of potassium, ATP, calcium, and purinergic receptors in the myotoxic activity of snake venom sPLA2 was described by Cintra-Francischinelli et al. . The authors emphasized the role of the purinergic receptor P2X and its inhibitors in the extent of muscle tissue damage, giving an additional explanation to the finding that the antitrypanosomal drug suramin provides protection from the toxic effect of the Lys49 myotoxins of the Bothrops jararacussu and Bothrops asper venoms. Suramin also binds to P2X channels, and this property could, at least in part, account for its myotoxic inhibitory activity. Several flavonoids and isoflavones antagonize purinergic receptors  which could explain in part the inhibitory effect of these compounds in the myotoxic activity of snake venom sPLA2.
In order to elucidate the anti-inflammatory activity of harpalycin 2, we further examined the ability of Har2 to inhibit the enzymatic activity of COX-1/2 and LOX 15hrc and 15syP1 as well as bovine pancreas PLA2 and human group V PLA2. Har2 showed lower IC50 values for both bovine and human sPLA2 than for COX-1/2 and LOX enzymes, as shown in Table 1. These results showed that harpalycin 2 has probably more affinity for sPLA2 than for other proinflammatory enzymes.
|Note: PrTX-III (piratoxin-III); BPPLA2 (bovine pancreas PLA2); HGVPLA2 (human group V PLA2); COX-1 (cyclooxygenase-1); COX-2 (cyclooxygenase-2); LOX 15HRC (Lipoxygenase 15hrc); LOX 15SY (Lipoxygenase 15sy). N/D (Not determined).|
Aiming to understand how harpalycin 2 interacts with these sPLA2s, CD spectroscopy, fluorescence analysis, and docking calculations were performed. Fluorescence profile analysis of Har2, sPLA2s, and Har2:sPLA2s showed significant changes in the spectral fluorescence profile among Har2 and the sPLA2 treated with Har2. CD analysis of native and Har2-treated sPLA2 revealed that the treatment induced discrete unfolding of sPLA2, which did not modify the tridimensional structure of the proteins (See supplementary material, available at doi: 10.1155/2012/987517).
The sPLA2 structures were represented by the PDB IDs 1GMZ, 2QOG, 1PSH, and 1POC, respectively, for PrTX-III, Cdt F15, Naja, and Apis. The calculated docking score values (GOLD scores) for these targets were 33.93, 44.12, 55.26, and 51.23, respectively, for PrTX-III, Cdt F15, Naja, and Apis. One can see in Figure 5 that an interesting trend between the in silico (docking scores) and in vitro (percentages of inhibition) results was observed. This means that greater stability (the most positive docking score values) of the complex between the harpalycin 2 and the four sPLA2 enzymes is related to greater inhibition percentage of the enzymatic activities. Thus, harpalycin 2 has inhibitory capacity against the enzymatic, edematogenic, and myotoxic activities from neurotoxic venom secretory phospholipases A2, probably due to the interaction with residues 48 and 49 in the active site of these toxins. Comparing the amino acid sequences alignment performed by Clustal X  among the four sPLA2, we found that the Apis has a more specific sequence (with an identity of 24% as compared with PrTX-III) while the other three sequences are similar to each other (identity values of 44% for Naja and 57% for Cdt F15, when compared to PrTX-III), Figure 6(a). Considering the secondary and tertiary structures, the difference between the Apis and the others is more relevant. Therefore, we performed a structural alignment only for PrTX-III, Cdt F15, and Naja, using PyMOL , as shown in Figure 6(b). The great similarity among these three PLA2 is probably related to their evolutionary origin from snake venoms, where PrTX-III and Cdt F15 are classified as GIIA, while Naja is classified as GIA). Using the 1PSH (Naja) target as example, because it represents the best result, a detailed inspection for the molecular reasons of the good inhibition behavior of the harpalycin 2 in this target can be found in Figure 7. The most important residues in the active site, including HIS48 and ASP49, are labeled and are involved in polar interactions with harpalycin 2.
This data corroborates both ethnopharmacological uses of this plant by Brazilian northeast population: the treatment of snake bites, as sPLA2 s are the main toxins in these venoms and, and as anti-inflammatory, due to the main role of sPLA2 in the inflammatory cascade of events. The data also pointed to a possible anti-inflammatory activity of this isoflavone mainly in disorders which involve sPLA2, such as asthma and rheumatoid arthritis.
|Cdt F15:||Crotoxin B from Crotalus durissus terrificus|
|gVPL:||Group V phospholipase|
|PAF:||Platelet activation factor|
|sPL:||Secretory phospholipase .|
R. M. Ximenes and M. M. Rabello have contributed equally to this work.
This work was financially supported by CAPES (Conselho de Aperfeiçoamento de Pessoal de Ensino Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo), and FUNCAP (Fundação Cearense de Apoio a Pesquisa). The authors would like to thank Sidney Ann Pratt for language revision.
The results of CD spectra and chromatographic analysis between harpalycin 2 and the four venom phospholipases A2 tested here are described in Supplementary material.
- A. García-Lafuente, E. Guillamón, A. Villares, M. A. Rostagno, and J. A. Martínez, “Flavonoids as anti-inflammatory agents: implications in cancer and cardiovascular disease,” Inflammation Research, vol. 58, no. 9, pp. 537–552, 2009.
- B. H. Havsteen, “The biochemistry and medical significance of the flavonoids,” Pharmacology and Therapeutics, vol. 96, no. 2-3, pp. 67–202, 2002.
- M. Lindahl and C. Tagesson, “Flavonoids as phospholipase A2 inhibitors: importance of their structure for selective inhibition of group II phospholipase A2,” Inflammation, vol. 21, no. 3, pp. 347–356, 1997.
- C. V. Iglesias, R. Aparicio, L. Rodrigues-Simioni et al., “Effects of morin on snake venom phospholipase A2 (PLA2),” Toxicon, vol. 46, no. 7, pp. 751–758, 2005.
- C. A. Cotrim, S. C. B. De Oliveira, E. B. S. Diz-Filho et al., “Quercetin as an inhibitor of snake venom secretory phospholipase A2,” Chemico-Biological Interactions, vol. 189, no. 1-2, pp. 9–16, 2011.
- K. K. Dharmappa, R. Mohamed, H. V. Shivaprasad, and B. S. Vishwanath, “Genistein, a potent inhibitor of secretory phospholipase A2: a new insight in down regulation of inflammation,” Inflammopharmacology, vol. 18, no. 1, pp. 25–31, 2010.
- G. Lambeau and M. H. Gelb, “Biochemistry and physiology of mammalian secreted phospholipases A2,” Annual Review of Biochemistry, vol. 77, pp. 495–520, 2008.
- B. B. Boyanovsky and N. R. Webb, “Biology of secretory phospholipase A2,” Cardiovascular Drugs and Therapy, vol. 23, no. 1, pp. 61–72, 2009.
- B. L. Nanda, A. Nataraju, R. Rajesh, K. S. Rangappa, M. A. Shekar, and B. S. Vishwanath, “PLA2 mediated arachidonate free radicals: PLA2 inhibition and neutralization of free radicals by anti-oxidants—a new role as anti-inflammatory molecule,” Current Topics in Medicinal Chemistry, vol. 7, no. 8, pp. 765–777, 2007.
- J. E. Burke and E. A. Dennis, “Phospholipase A2 biochemistry,” Cardiovascular Drugs and Therapy, vol. 23, no. 1, pp. 49–59, 2009.
- K. Hamaguchi, H. Kuwata, K. Yoshihara et al., “Induction of distinct sets of secretory phospholipase A2 in rodents during inflammation,” Biochimica et Biophysica Acta, vol. 1635, no. 1, pp. 37–47, 2003.
- K. K. Dharmappa, R. V. Kumar, A. Nataraju, R. Mohamed, H. V. Shivaprasad, and B. S. Vishwanath, “Anti-inflammatory activity of oleanolic acid by inhibition of secretory phospholipase A2,” Planta Medica, vol. 75, no. 3, pp. 211–215, 2009.
- B. Gil, M. J. Sanz, M. C. Terencio, R. Gunasegaran, M. Payá, and M. J. Alcaraz, “Morelloflavone, a novel biflavonoid inhibitor of human secretory phospholipase A2 with anti-inflammatory activity,” Biochemical Pharmacology, vol. 53, no. 5, pp. 733–740, 1997.
- G. L. Da Silva, M. I. L. Machado, F. J. D. A. Matos, and R. Braz-Filho, “A new isoflavone isolated from Harpalyce brasiliana,” Journal of the Brazilian Chemical Society, vol. 10, no. 6, pp. 438–442, 1999.
- M. H. Toyama, D. G. De Oliveira, L. O. S. Beriam, J. C. Novello, L. Rodrigues-Simioni, and S. Marangoni, “Structural, enzymatic and biological properties of new PLA2 isoform from Crotalus durissus terrificus venom,” Toxicon, vol. 41, no. 8, pp. 1033–1038, 2003.
- M. H. Toyama, P. D. Costa, J. C. Novello et al., “Purification and amino acid sequence of MP-III 4R D49 phospholipase A2 from Bothrops pirajai snake venom, a toxin with moderate PLA2 and anticoagulant activities and high myotoxic activity,” Protein Journal, vol. 18, no. 3, pp. 371–378, 1999.
- S. Hernandez-Oliveira, M. H. Toyama, D. O. Toyama, S. Marangoni, S. Hyslop, and L. Rodrigues-Simioni, “Biochemical, pharmacological and structural characterization of a new PLA2 from Crotalus durissus terrificus (South American Rattlesnake) venom,” Protein Journal, vol. 24, no. 4, pp. 233–242, 2005.
- D. O. Toyama, S. Marangoni, E. B. S. Diz-Filho, S. C. B. Oliveira, and M. H. Toyama, “Effect of umbelliferone (7-hydroxycoumarin, 7-HOC) on the enzymatic, edematogenic and necrotic activities of secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus collilineatus venom,” Toxicon, vol. 53, no. 4, pp. 417–426, 2009.
- M. J. S. Dewar, E. G. Zoebisch, E. F. Healy, and J. J. P. Stewart, “AM1: a new general purpose quantum mechanical molecular model,” Journal of the American Chemical Society, vol. 107, no. 13, pp. 3902–3909, 1985.
- G. Jones, P. Willett, R. C. Glen, A. R. Leach, and R. Taylor, “Development and validation of a genetic algorithm for flexible docking,” Journal of Molecular Biology, vol. 267, no. 3, pp. 727–748, 1997.
- H. M. Verheij, J. J. Volwerk, E. H. Janse et al., “Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism,” Biochemistry, vol. 19, no. 4, pp. 743–750, 1980.
- D. L. Scott and P. B. Sigler, “Structure and catalytic mechanism of secretory phospholipases A2,” Advances in Protein Chemistry, vol. 45, pp. 53–88, 1994.
- M. Cintra-Francischinelli, P. Caccin, A. Chiavegato et al., “Bothrops snake myotoxins induce a large efflux of ATP and potassium with spreading of cell damage and pain,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 32, pp. 14140–14145, 2010.
- B. C. Zychar, C. S. Dale, D. S. Demarchi, and L. R. C. Gonçalves, “Contribution of metalloproteases, serine proteases and phospholipases A2 to the inflammatory reaction induced by Bothrops jararaca crude venom in mice,” Toxicon, vol. 55, no. 2-3, pp. 227–234, 2010.
- L. C. Corrêa, D. P. Marchi-Salvador, A. C. O. Cintra, S. V. Sampaio, A. M. Soares, and M. R. M. Fontes, “Crystal structure of a myotoxic Asp49-phospholipase A2 with low catalytic activity: insights into Ca2+-independent catalytic mechanism,” Biochimica et Biophysica Acta, vol. 1784, no. 4, pp. 591–599, 2008.
- S. P. H. Alexander, “Flavonoids as antagonists at A1 adenosine receptors,” Phytotherapy Research, vol. 20, no. 11, pp. 1009–1012, 2006.
- J. D. Thompson, T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins, “The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools,” Nucleic Acids Research, vol. 25, no. 24, pp. 4876–4882, 1997.
- W. L. DeLano, The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, Calif, USA, 2002, http://pymol.sourceforge.net.
Copyright © 2012 Rafael M. Ximenes et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.