Isolation and characterization of ellagic acid derivatives isolated from Casearia sylvestris SW aqueous extract with anti-PLA2 activity

Toxicon 52 (2008) 655–666

Contents lists available at ScienceDirect

Toxicon journal homepage: www.elsevier.com/locate/toxicon

Isolation and characterization of ellagic acid derivatives isolated from Casearia sylvestris SW aqueous extract with anti-PLA2 activity Saulo L. Da Silva a, *, Andrana K. Calgarotto b, Jamal S. Chaar c, Se´rgio Marangoni b a

´ polis, Universidade Fed. de Sa ˜o Joa ˜o Del Rei, CEP 35501-296 Divino ´polis, MG, Brazil Campus Avançado de Divino Depto de Bioquı´mica, IB, Universidade Est. de Campinas, CEP 13569-970 Campinas, SP, Brazil c Depto de Quı´mica, ICE, Universidade Fed. do Amazonas, CEP 69077-000 Manaus, AM, Brazil b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 March 2008 Received in revised form 14 July 2008 Accepted 15 July 2008 Available online 5 August 2008

The Casearia sylvestris SW (Flacourtiaceae) is utilized in folk medicine (Brazil and all Latin American) to treat several pathologic processes as inflammation, cancer, microbial infection and snake bites. Studies showed that C. sylvestris aqueous extract can inhibit many toxic effects caused by snake venoms (or caused by phospholipase A2 isolated) from different species, mainly of Bothrops genus. Inhibition of enzymatic and myotoxic activities, decrease of edema formation and increase of the survival rate of rats injected with lethal doses of bothropic venoms are some toxic effects inhibited by C. sylvestris. In this study, four ellagic acid derivatives from aqueous extracts of C. sylvestris were isolated, characterized, and tested against effects from both total venom and PLA2 (Asp 49 BthTX-II) from the venom of Bothrops jararacussu. The isolated compounds were as follows: ellagic acid (A), 30 -O-methyl ellagic acid (B), 3,30 -di-O-methyl ellagic acid (C), 3-O-methyl-30 , 40 -methylenedioxy ellagic acid (D). The inhibition constant values (Ki) for enzymatic activity, as well the IC50 values found in the edematogenic and myotoxic activities, indicate that the ellagic acid is the best inhibitor of these activities, while compounds C and D are the substances with lowest capacity on inhibiting these same effects. Our results show that the presence of hydroxyls at position 3 or 30 (compounds A and B) increases the capacity of these derivatives on inhibiting these toxic effects. However, the presence of methoxyl groups at position 3 or 30 reduced, but did not completely inhibit the capacity of compounds C and D on inhibiting all the toxic effects studied. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Ellagic acid PLA2 Bothrops jararacussu Casearia sylvestris Enzymatic activity

1. Introduction Medicinal plants have been used in folk medicine for many pharmacological applications. Compounds with biological activities such as flavonoids, tanins, saponins and other, have been isolated from different plants and maybe used for the study and design of new pharmaceuticals. The utilization of vegetal extracts against ophidian accidents is an ancient option and this approach has been widely utilized * Corresponding author. Rua Fortunato Badan, 103 Jardim Silvaˆnia, CEP 13806-679 Moji Mirim, SP, Brazil. Tel.: +55 37 32211267; fax: +37 3221 1614. E-mail address: [email protected] (S.L. Da Silva). 0041-0101/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2008.07.011

in isolated or indigenous communities (Mors et al., 2000; Otero et al., 2000; Borges et al., 2001). During recent years, vegetal extracts have been used with the aim of identifying alternative or complementary substances for anti-serum in ophidian accidents (Pereira et al., 1992; Martz, 1992; Borges et al., 2001; Da Silva et al., 2007). Many compounds such as aristolochic acid, ellagic acid, flavonoids, retinoids and other plants from nature have been isolated, structure characterized and their capacities to inhibit PLA2 determined (Vishwanath et al., 1987; Fawzy et al., 1988; Glaser and Jacobs, 1986; Glaser et al., 1995; Rogerio et al., 2006; Lansky and Newman, 2007; Chandra et al., 2007). The Casearia sylvestris SW (Flacourtiaceae) is a plant popularly known as ‘‘guaçatonga’’ or ‘‘cha´-de-bugre’’ and is

656

S.L. Da Silva et al. / Toxicon 52 (2008) 655–666

geographically distributed in the whole Latin America and particularly along the whole extension of Brazilian territory (Lorenzi and Matos, 2002; Felfili et al., 2002; Barbosa et al., 2005; Hack et al., 2005). Many etnobotanic activities have been applied to this plant. Karaja´ Indians from Brazil prepare a maceration of this plant bark to treat diarrhea and the Shipibo-Conibo Indians of Peru use a decoction of the bark for the treatment of diarrhea, chest colds and influenza. Other Indian tribes in Brazil smash the roots or seeds of guaçatonga to treat wounds and topical leprosy (Taylor, 2002). Some studies have confirmed different popular medicinal properties of C. sylvestris as being antiinflammatory (Raslan et al., 2002), anti-ulcerous (Esteves et al., 2005), anti-cancer (Mans et al., 2000), anti-microbial (Da Silva et al., 2006, 2008a) and cytotoxic (Da Silva et al., 2008b). The indigenous populations throughout the Amazon rainforest have used guaçatonga for a long time as a snakebite medicine (Ruppelt et al., 1991; Alves, 2000; Mors et al., 2000). Borges et al. (2000) showed that the toxic effects caused by total venom of different Bothrops species and its purified PLA2 can be inhibited by the C. sylvestris aqueous extract. According to these authors, this extract is able to inhibit the enzymatic and myotoxic activities, decrease edema formation and increase the survival tax of rats injected with lethal doses of bothropic venoms. Borges et al. (2001) observed that the C. sylvestris aqueous extract also inhibits the hemorrhagic metalloproteinase activities and increases the time of plasmatic coagulation caused by the venom of various Bothrops species. In addition, Cavalcante et al. (2007) showed that the C. sylvestris aqueous extract also inhibits neuromuscular paralysis activity and neutralizes the damage to muscles in rats subdued by the action of C. durissus terrificus chrotoxine, Bothrops jararacussu bothropstoxin, B. pirajai piratoxin and B. moojeni myotoxinII. Mattos et al. (2007) showed that the C. sylvestris aqueous extract inhibits responses induced by ovalbumin or acetic acid. However, in all these studies, the authors used the brute aqueous material of C. sylvestris and did not identify the chemical substances that could be correlated to the inhibition of the toxical effects of several venoms or purified PLA2 (Borges et al., 2000, 2001; Mattos et al., 2007; Cavalcante et al., 2007). The aim of this study was to find, isolate and identify the substances that could be associated with ability of the C. sylvestris to inhibit the edema formation, enzymatic and myotoxic activities caused by B. jararacussu venom and B. jararacussu PLA2 (Asp 49 BthTX-II). The aqueous extract was submitted to chromatography and the structures of the substances capable of inhibiting the enzymatic activity were elucidated. Information about the structure of these compounds and their inhibition of toxic effects may aid in the understanding of viable mechanisms of action of the isolated substances against B. jararacussu venom and PLA2. 2. Material and methods 2.1. Chemicals The 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero3-phosphoglycerol (HPGP) substrate was provided by

Molecular Probes (USA). B. jararacussu venoms (vBj) were purchased from a private serpentarium (Batatais, SP, Brazil). The Asp 49 BthTX-II (bjPLA2) isoform was isolated from B. jararacussu venoms. All other chemicals used herein were purchased from Sigma Co. (USA), Aldrich Co. (USA) and Merck Co. (Germany). 2.2. Preparation of the leaf powder and extraction The aqueous extract from leaves of C. sylvestris was obtained from fresh leaves collected in the city of Campinas, SP, Brazil, in March 2005. This plant was identified by the botanists from the herbarium of the Biological Institute of UNICAMP, where a voucher was deposited under the number UEC 118743. Leaves of C. sylvestris were washed with water and the fleshy portion was collected, dried at 40  C in a hot air oven and finely powdered. The powder was used for extraction. The aqueous extract was prepared by homogenizing the leaves powder (400 g) in 2 L of warm water (50  C) and allowing to stand for 24 h. After filtration through Whatman No. 1 paper, the filtrate was lyophilized. The lyophilized aqueous extract (34.75 g) was re-extracted with 150 mL of methanol and centrifuged at 30,000g for 20 min. The pellet was discarded and the supernatant was concentrated under reduced pressure and the residue was stored at 20  C until use. 2.3. Column chromatography on silicic acid The total extract (TE) was obtained using 8.3 g of the residue obtained from the aqueous extract of the C. sylvestris dissolved in methanol (50 mL). This solution was immobilized on silicic acid (20 g) by lyophilization, and subjected to column chromatography (CC), using a 38  4.5 cm glass column filled with silica gel 60 (mesh size: 70–230) in DCM (dichloromethane) to a level 5 cm from the top. The immobilized extract was added to the free volume at the head of the column. After bedding down of the gel material, fractionation was conducted by successive applications of 250 mL of each system: hexane (F1), DCM (F2), and methanol (1, 2, 5, 10, 20, 30, 50 and 100%) in DCM (F3–F10). Ten fractions (F1–F10) with 250 mL each were collected, and the solvent was removed by rotary evaporation in vacuo at 35  C. The fractions were dried and stored at 20  C. All fractions were individually analyzed and we selected only those with the capacity to inhibit PLA2 activity. These selected fractions were submitted to analytical and semi-preparative HPLC studies. 2.4. Analytical high performance liquid chromatography Analytical HPLC was conducted on a Shimadzu HPLC system (Kyoto, Japan) liquid chromatograph fitted with a Waters mBondapak C18 reversed-phase column (250  3.9 mm, 5 mm). Selected fractions of C. sylvestris (and TE) were dissolved in methanol (5.0 mL) and, when necessary, were further diluted prior to injection (20 mL) into the HPLC. The mobile phase consisted of 2% acetic acid in water (solvent A) and methanol (solvent B) with the following gradient: 95% A for 2 min, to 75% A in 8 min, to 60% A in 10 min, to 50% A in10 min and 0% A until

S.L. Da Silva et al. / Toxicon 52 (2008) 655–666

657

The selected fractions from analytical HPLC were submitted to semi-preparative HPLC. Semi-preparative HPLC was conducted on Shimadzu HPLC system liquid chromatograph fitted with a Waters mBondapak C18 reversed-phase column (10 mm i.d.) similar to that used for analytical HPLC. Acetonitrile was used instead of methanol as a mobile phase with a flow rate of 3 mL/min. Peaks eluting from the column were collected on an Agilent HP 220 Microplate Sampler. Each major peak was isolated and solvent was removed by lyophilization. The peaks were tested for their capacity to inhibit PLA2 and those that were selected had their molecular structure elucidated by RMN.

injections, the mixtures containing bjPLA2 (or vBj) and the inhibitors were pre-incubated for 10 min at 37  C. Negative control groups were injected with 50 mL of 3% DMSO in PBS (pH 7.2). Control groups for all compounds were performed through the i.d. injection of 50 mL of a solution containing 80 mM of ellagic acid derivative compounds dissolved in 3% DMSO in PBS (pH 7.2). The edema progression was evaluated with a low pressure pachymeter (Mitutoyo, Japan) at various time intervals after injection (0.5, 1, 2, 4, 6, 24 h) (Souza et al., 2008; Calgarotto et al., 2008) The IC50 values (concentration that inhibits 50% of edema-inducing activity caused by bjPLA2) were calculated from dose–response curves produced by the percentage values of the edema volume reduction caused by bjPLA2, in regard to the positive control, when utilizing increasing concentrations (0.1–1000 mM) of each ellagic acid derivative (compounds A–D). The curves shown are those demonstrating the compounds with highest and lowest IC50 values.

2.6. NMR spectroscopy

2.9. Myotoxic activity

IR spectra were recorded on a Bomem MB100 spectrometer, KBr, nmax, cm1. 1H and 13C NMR spectra were obtained on a Bruker AVANCE 400 spectrometer, operating at 9.4 T, observing 1H at 400.13 MHz and 13C at 100.61 MHz. 1 H and 13C chemical shifts d are reported in ppm relative to TMS with an internal reference in mixed solvents or indirectly via pure solvent signals: CHD2OD ¼ 3.28 ppm for 1H; CD3OD ¼ 49.08 ppm for 13C; DMSO-d5 ¼ 2.52 ppm for 1H; and DMSO-d6 ¼ 39.56 for 13C. All reagents and solvents used were previously purified and dried.

Swiss male mice (18–22 g) were intramuscularly injected in the right gastrocnemius muscle with 50 mL of a solution containing 25 mg of PLA2 purified from B. jararacussu dissolved in 3% DMSO (Dimethyl Sulfoxide) in PBS (phosphate-buffered saline – pH 7.2) (positive control). The whole venom solution (vBj) contained 50 mg dissolved in 3% DMSO, in PBS (phosphate-buffered saline – pH 7.2). Inhibition studies were performed by injecting 50 mL of a mixed solution composed of PLA2 (or vBj) and each ellagic acid derivative compound (80 mM) dissolved in 3% DMSO in PBS (pH 7.2). Prior to the injections, the mixtures containing PLA2 (or vBj) and the inhibitors were pre-incubated for 10 min at 37  C. Negative controls received 50 mL of 3% DMSO–PBS alone. Control groups for all compounds were performed by the i.d. injection of 50 mL of a solution containing only the ellagic acid derivative compound (80 mM) dissolved in 3% DMSO in PBS (pH 7.2). Mice were bled from the tail at 3 h after injections and blood was collected into heparinized capillary tubes. Plasma creatine kinase activity was determined using the 47-UV Kit (Sigma Chemical Co.). Activity was expressed in units/L, with 1 unit corresponding to the production of 1 mM of NADH/min at 30  C (Da Silva et al., 2008c; Villar et al., 2008). The IC50 values (concentration that inhibits 50% of myotoxic activity caused by bjPLA2) were calculated from dose–response curves produced by the values of percentage reduction in the myotoxic activity of bjPLA2, compared to the positive control, when utilizing increasing concentrations (0.1 mM and 1000 mM) of each ellagic acid derivative (compounds A–D). The curves shown are those for the highest and lowest IC50 values of the compounds.

completion of the run. The flow rate was 1 mL/min. The elutions were detected at 280 nm with a diode array UV detector (HP 1040M). 2.5. Semi-preparative HPLC and fraction collection

2.7. PLA2 purification from B. jararacussu venom B. jararacussu venoms were purchased from a private serpentarium (Batatais, SP, Brazil). B. jararacussu PLA2 was isolated on Sephadex G-75, followed by cation-exchange chromatography. The column was previously equilibrated with 0.05 M ammonium bicarbonate buffer, pH 8.0. Elution was carried out with a continuous gradient up to a concentration of 0.5 M ammonium bicarbonate. Absorbance of the effluent solution was recorded at a wavelength of 280 nm (Souza et al., 2008; Da Silva et al., 2008c). PLA2 homogeneity was assessed by native and SDS-PAGE and reverse-phase HPLC. In this study, was utilized the faction II known as Asp 49 BthTX-II was utilized. This phospholipase will be denominated in this paper as just PLA2. All reagents were purchased from or Sigma Aldrich Co. (USA). 2.8. Edema-inducing activity Edema was induced in the right foot pad of male Swiss mice (18–22 g) by i.d. injection of 50 mL of a solution containing 50 mg of bjPLA2 purified from B. jararacussu venom dissolved in 3% DMSO (Dimethyl Sulfoxide) in PBS (phosphate-buffered saline – pH 7.2) (positive control). A solution with 100 mg of the whole venom (vBj) was dissolved in 3% DMSO. Inhibition studies were performed by i.d. injection of 50 mL of a solution containing a mixture of bjPLA2 (or vBj) and 80 mM of each ellagic acid derivative compound dissolved in 3% DMSO in PBS (pH 7.2). Prior to the

2.10. Enzymatic activities The measurement of the enzymatic activity using the micellar substrate 1-hexadecanoyl-2-(1-pyrenedecanoyl)sn-glycero-3-phosphoglycerol (HPGP) was carried out through the microtiter plate assay (Souza et al., 2008; Da Silva et al., 2008d). Each ellagic acid derivative compound was tested in final concentrations of 0.0, 0.1, 0.2, 0.3 and

658

S.L. Da Silva et al. / Toxicon 52 (2008) 655–666

0.4 mM. Seven wells of a 96-well microtiter plate were used for each assay, resulting in six measurement repetitions of the enzymatic activity for each final concentration of the inhibitor. Thus, for each assay with different concentrations of the inhibitor, 100 mL of Solution A in assay buffer (27 mM bovine serum albumin, 50 mM KCl, 1 mM CaCl2, 50 mM Tris–HCl, pH 8.0) were added to seven wells, followed by the addition of a volume of 26.5 mM ellagic acid derivative compounds (1–4 mL, according to the assay concentration) dissolved in DMSO. For control reactions1–4 mL of DMSO were used alone. Solution B had the same composition as Solution A, however, with the presence of PLA2 (0.5 mg/mL) or vBj (1.0 mg/mL) and was delivered in 100 mL samples to seven wells except for the first one. Instead of Solution B, an additional 100 mL-samples of Solution A was added to the first of the seven wells in the assay. Solution B was prepared immediately prior to each set of assays to avoid loss of enzymatic activity. The assay was rapidly initiated after the addition of Solution B by the addition of 0.5– 50 mL-volumes of Solution C (53 mM HPGP vesicles in assay buffer) with a repeating pipettor to the seven wells. The final concentration of HPGP varied from 0.125 to 10 mM. The final volume of the assay was 265 mL and, when necessary, the wells received an extra portion of Solution A in order to complete this volume. The fluorescence (excitation ¼ 342 nm, emission ¼ 395 nm) was read with a microtiter plate spectrophotometer (Fluorocount, Packard Instruments). Control reactions without enzyme or inhibitor were run for all assays and the initial velocity was calculated from the initial slope of fluorescence versus time, for each concentration of the substrate used. The significance of differences between groups was evaluated using the Student’s t-test. P-value < 0.05 was considered to be significant. 2.11. Statistical analysis Results are presented as the mean values  S.D. obtained with the indicated number of tested animals. The statistical significance of differences between groups was evaluated using Student’s unpaired t-test. A P-value < 0.05 was considered to indicate significance. 3. Results and discussion 3.1. Isolation and characterization of ellagic acid derivatives Ten fractions were obtained from a column chromatography on silicic acid (Fig. 1). All the fractions, after solvent evaporation, were submitted to enzymatic PLA2 inhibition testing utilizing HPGP vesicles (1-hexadecanoyl-2-(1pyrenedecanoyl)-sn-glycero-3-phosphoglycerol). Only the fractions F5, F6 and F9 (eluted with 5, 10 and 50% of methanol on DCM, respectively) inhibited the enzymatic activity. The F5 and F6 fractions showed very similar chromatographic profiles and were grouped. Fig. 1 shows the HPLC analytic chromatographic profile obtained from C. sylvestris total extract (TE), with the pool of fractions, F5 and F6, and the fraction F9. The isolated fractions F5–F6 and F9 were submitted to chromatography again utilizing semi-preparative HPLC. All

Fig. 1. Analytical HPLC chromatograms as monitored by UV absorption at 280 nm for the C. sylvestris total extract (TE) of the leaves and for the fractions F5–F6 and F9, as obtained by column chromatography of the extract on silicic acid. The peaks A–D correspond to the compounds shown in Fig. 2.

peaks isolated from F5–F6 or F9, were tested according to their capacity to inhibit bjPLA2. Only the substances that corresponded to the four peaks detached in Fig. 1 (compounds A–D) were able to inhibit the enzymatic activity. The substances were isolated and these structures were elucidated through NMR (Nuclear Magnetic Resonance – 13C and 1H). The following compounds were characterized (Fig. 2): ellagic acid (A) (M.M. 302.19): IR (KBr, nmax, cm1): 3762, 3426, 1700; NMR 1H (400 MHz – CHD2OD/DMSO-d5, ppm) d: 7.60 (2H: H-5, H-50 ); 8.80 (2H: OH-4a, OH-8a); 12.10 (2H: OH-3a, OH-7a). NMR 13C (100 MHz – CD3OD/DMSO-d6, ppm) d: 113.60 (C1, C10 ); 136.80 (C2, C20 ); 140.38 (C3, C30 ); 148,74 (C4, C40 ); 111.54 (C5, C50 ); 108.80 (C6, C60 ); 161.28 (C100, C200 ). 30 -O-Methyl ellagic acid (B) (M.M. 316.02): IR (KBr, nmax, cm1): 3752, 3430, 1704; NMR 1H (400 MHz – CHD2OD/DMSO-d5, ppm) d: 4.09 (3H: OCH3-3) 7.54 (1H: H-5); 7.48 (1H: H-50 ); 8.72 (1H: OH-8a) 8.82 (1H: OH-4a); 12.15 (1H: OH-3a). NMR 13C (100 MHz – CD3OD/DMSO-d6, ppm) d: 112.50 (C1, C10 ); 141.90 (C2, C20 ); 140.68 (C3, C30 ); 152.47 (C4, C40 ); 111.84 (C5, C50 ); 113.08 (C6, C60 ); 159.40 (C100, C200 ); 61.24 (3-OCH3). 3,30 -Di-O-methyl ellagic acid (C) (M.M. 330.25): IR (KBr, nmax, cm1): 3732, 3432, 1700; NMR 1H (400 MHz – CHD2OD/DMSO-d5, ppm) d: 4.06 (6H: OCH3-3, OCH3-30 ) 7.52 (2H: H-5, H-50 ); 8.68 (1H: OH-8a) 8.78 (1H: OH-4a). NMR 13C (100 MHz – CD3OD/DMSO-d6, ppm) d: 111.70 (C1, C10 ); 141.30 (C2, C20 ); 140.18 (C3, C30 ); 152.17 (C4, C40 ); 111.49 (C5, C50 ); 112.08 (C6, C60 ); 158.50 (C100, C200 ); 60.89 (3-OCH3, 30 -OCH3). 3-O-Methyl-30 ,40 -methylenedioxy ellagic acid (D) (M.M. 328.23): IR (KBr, nmax, cm1): 3762, 3426, 1700, 1070, 950. NMR 1H (400 MHz – CHD2OD/DMSO-d5, ppm) d: 4.04 (3H: OCH3-3), 6.36 (2H: CH2-3a), 7.52

S.L. Da Silva et al. / Toxicon 52 (2008) 655–666

659

Fig. 2. Structures of the ellagic acid derivative compounds identified from C. sylvestris aqueous extract, with inhibition capacity either for B. jararacussu (vBj) total venom enzymatic activity or for its isoform, Asp 49 BthTX-II (bjPLA2).

(1H: H-5), 7.54 (1H: H-50 ), 8.70 (1H: OH-8a). NMR 13C (100 MHz – CD3OD/DMSO-d6, ppm) d: 116.08 (C1, C10 ); 131.10 (C2, C20 ); 138.25 (C3, C30 ); 149.96 (C4, C40 ); 103.79 (C5, C50 ); 110.81 (C6, C60 ); 158.02 (C100, C200 ); 60.84 (3-OCH3); 104.16 (C300 ). The 1H and 13C chemical shifts were comparable to the values described in the literature (Sato, 1987; Nawwar et al., 1994; Li et al., 1999; Atta-Ur-Rahman et al., 2001). 3.2. Edema-inducing activity The results of the edema-inducing activity assay are shown in Fig. 3. As expected, the total extract (100 mg) was capable of decreasing the percentage of induced edema, both by vBj and bjPLA2 (Borges et al., 2000). However, the results obtained with a fixed concentration of isolated compounds A–D (80 mM) demonstrated that the ellagic acid (compound A) was the most efficient compound in inhibiting the edema levels caused both by vBj and bjPLA2. After 6 h of induction, 80 mM ellagic acid decreased the edema levels to around 60% (P < 0.01) and 25% (P < 0.001) when vBj and bjPLA2, respectively, are utilized. The compounds B–D (80 mM) reduced the edema levels induced by vBj were only to around 80% (P < 0.01), when each one of the substances was present. However, when the edema was induced utilizing bjPLA2, the presence of 80 mM compound

B reduced the edema to around 40% (P < 0.05). The same concentration (80 mM) of compounds C and D only reduced the edemato around 60% (P < 0.05) after 6 h of assay. The IC50 values were 23.8  0.3; 34.0  1.0; 170.1  0.9; and 172.3  1.2 mM for the compounds A–D, respectively. Fig. 4 shows the dose–response curves obtained from the variation in the percentage reduction of edema-inducing activity for increasing concentrations of compounds A and D (higher and lower actives, respectively). 3.3. Myotoxic activity Eighty micromole of ellagic acid was able of, after 3 h of induction, decreasing the levels of myotoxic activity to 64 (P < 0.05) and 83% (P < 0.001) when we have utilized vBj and bjPLA2, respectively. Conversely, 80 mM of other compounds (B, C, and C) reduced between 8 and 20% the myotoxicity levels induced by vBj. However, the presence of 80 mM of compound B reduced the myotoxicity induced by bjPLA2, in about 78% (P < 0.001). The same concentration (80 mM) of compounds C and D only reduced the myotoxic activity induced by bjPLA2 in about 30% (P < 0.05) after 3 h after injection of these compounds (Fig. 5). The IC50 values of the myotoxic activity were 23.8  0.3; 34.0  1.0; 170.1  0.9; and 172.3  1.2 mM for the

660

S.L. Da Silva et al. / Toxicon 52 (2008) 655–666

Fig. 3. Effect of ellagic acid derivatives (80 mM) and total extract (TE) on edema-inducing activity by: (A) B. jararacussu total venom (vBj) and (B) Asp 49 BthTX-II from B. jararacussu venom (bjPLA2). The PBS control solution contains 3% of DMSO. Results are expressed as means  S.D. (n ¼ 6). The statistical significance of differences between groups was evaluated. A P-value < 0.05 was considered to indicate significance.

compounds A–D, respectively. Fig. 6 shows the dose– response curves obtained from the variation in the percentage reduction of edema-inducing activity for increasing concentrations of compounds A and D (higher and lower activities, respectively). 3.4. Enzymatic activity Borges et al. (2000) showed that aqueous extract of was capable of decreasing the percentage of enzymatic activity of both vBj and bjPLA2. In this study, the kinetic behavior study of bjPLA2 from B. jararacussu, in the presence and absence of ellagic acid derivative compounds (A–D), is shown in Fig. 7. All the curves indicate that, in the substrate concentration range used in this study (0 mM–0 mM),

the enzyme action on the micellar substrate, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol (HPGP), reflects the classical behavior of a Michaelian enzyme, not only in the presence of small concentrations of the compounds A and D (0.1–0.4 mM), but also in their absence. Fig. 7 also shows that all tested compounds are able to inhibit PLA2 enzymatic activity. However, compounds A and B (ellagic acid and 30 -O-methyl ellagic acid, respectively) were more efficient in inhibiting bjPLA2 than compounds C and D (3,30 -di-O-methyl ellagic acid and 3-Omethyl-30 ,40 -methylenedioxy ellagic acid, respectively). These observations were confirmed by kinetic parameters, as shown in Table 1. Table 1 demonstrates that in all the tests, the maximum velocity of the enzyme (Vmax) did not vary in function of the presence of compounds A–D and presented a value of around 18 nmol/min. Conversely, the presence of inhibitors induced an increase in KM values. In the presence of increasing concentrations of compounds A and B, the KM values of bjPLA2 increased from around 0.59 to around 86.68 mM (see details in Table 1). For compounds C and D, the KM values increased from 0.60 to approximately 5.81 mM (see details in Table 1). In addition, the inhibition constant values (KI) for compounds A and B are approximately 3 and 7 nM, respectively, and for compounds C and D, the inhibition constant values (KI) are approximately 45 and 48 nM, respectively (see details in Table 1). These results show that the enzymatic inhibition is competitive and that compounds A and B present a similar capacity for inhibiting PLA2, and are superior to compounds C and D. 4. Ellagic acid derivative action models: proposal

Fig. 4. The dose–response curve of percentage variation of edema-inducing activity caused by Asp 49 BthTX-II from B. jararacussu venom (bjPLA2) in regard to the positive control due to ellagic acid derivates with increasing concentration of the compound A (IC50 ¼ 23.8  0.3 mM) and compound D (IC50 ¼ 172.3  1.2 mM). Results are expressed as the means  S.D. (n ¼ 6). The statistical significance of differences between groups was evaluated. A P-value < 0.05 was considered to indicate significance.

Structural studies have shown that the phenolic hydroxyl of compounds such as vitamin E, aristolochic acid, rosmarinic acid and quercetin forms a hydrogen bond with His 48 and Asp 49 amino acids of the PLA2 extracted from various sources and inhibit these enzymes (Chandra et al., 2002a,b; Ticli et al., 2005; La¨ttig et al., 2007). His 48 and Asp 49 amino acids are essential for enzymatic activity in

S.L. Da Silva et al. / Toxicon 52 (2008) 655–666

661

Fig. 5. Effect of ellagic acid derivatives (80 mM) and total extract (TE) on myotoxic activity induced by: (A) B. jararacussu total venom (vBj) and (B) Asp 49 BthTX-II from B. jararacussu venom (bjPLA2). The PBS control solution contains 3% of DMSO. Results are expressed as means  S.D. (n ¼ 6). The statistical significance of differences between groups was evaluated. A P-value < 0.05 was considered to indicate significance.

the calcium-dependent phospholipases A2; if the inhibitors are bonded to these amino acids the PLA2 catalytic efficiency can be changed (Arni and Ward, 1996; Berg et al., 2001). Moreover, structural studies show that the benzopyrone ring of the flavonoids (such quercetin) and the phenantrenic ring of aristolochic acid also bind to PLA2 through hydrophobic interactions with Phe 5, Trp 31 and Phe 98 residues next to the enzyme active site (Chandra et al., 2002a,b; La¨ttig et al., 2007). Studies have shown that pomegranate seeds are enriched with the ellagic acid, 3,30 -di-O-methyl ellagic acid and other polyphenolics and are capable of inhibiting inflammatory processes, as well as other physiopathologic states (Lansky and Newman, 2007). Rogerio et al. (2006) showed that ellagic acid inhibits synovial human PLA2

Fig. 6. The dose–response curve of percentage variation in myotoxic activity caused by Asp 49 BthTX-II from B. jararacussu venom (bjPLA2) in regard to the positive control due to ellagic acid derivates with increasing concentrations of the compound A (IC50 ¼ 15.9  07 mM) and compound C (IC50 ¼ 231.1  2.1 mM). Results are expressed as means  S.D. (n ¼ 6). The statistical significance of differences between groups was evaluated. A P-value < 0.05 was considered to indicate significance.

activity (IC50 ¼ 20 nM), but does not inhibit the platelet aggregation factor. However, Glaser et al. (1995) also show the ellagic acid inhibits synovial human PLA2 (IC50 ¼ 33 nM), but is inactive for inflammatory cellular processes using human neutrophils and murine macrophages. Taking this information together with our results, ellagic acids appear to have a great capacity to inhibit PLA2 of groups IIA and IIB. The ellagic acid (compound A) is formed by the fusion of two hydroxy-benzopyrone structures, creating a system of four conjugated rings with a high degree of electronic resonance (Fig. 8). Apparently, these hydroxy-benzopyrone structures help the flavonoids to strongly inhibit the PLA2 (Gil et al., 1994; Lindahl and Tagesson, 1997; Kim et al., 2001; La¨ttig et al., 2007). Moreover, La¨ttig et al. (2007) showed that the presence of a phenolic hydroxyl in the C7 position of the benzopyrone ring of quercetin is responsible for the binding of this flavonoid to the His 47 amino acid in human secretory PLA2. Based on the structural information, described in the literature (Chandra et al., 2002a,b; Ticli et al., 2005; La¨ttig et al., 2007), and on our experimental results, compounds A–D appear to compete with the substrate (HPGP) for the active site (competitive inhibition); as such we propose a model for the mechanism of inhibition of B. jararacussu bjPLA2 ellagic acid derivatives isolated from C. sylvestris aqueous extract as shown in Fig. 9. The experimental results show that the ellagic acid (compound A) and the 30 -O-methyl ellagic acid (compound B) isolated from C. sylvestris aqueous extract, are the most efficient for inhibiting PLA2. The compounds 3,30 -di-Omethyl ellagic acid and 3-O-methyl-30 ,40 -methylenedioxy ellagic acid (compounds C and D, respectively), whilst to inhibiting the PLA2, have a lower efficiency when compared to the compounds A and B. Fig. 2 shows that the compounds A and B present hydroxyl groups at positions C3 and C30 , however, the compounds C and D have methoxyl groups at these positions. Apparently, the polarity of groups binding to the positions C3 and C30 of the ellagic acid derivatives is an important factor in their PLA2 inhibitory capacities. The

662

S.L. Da Silva et al. / Toxicon 52 (2008) 655–666

Fig. 7. Effect of substrate concentration on the kinetics of PLA2 activity of the Asp 49 BthTX-II from B. jararacussu venom (bjPLA2). The enzyme kinetics were studied under the influence of different concentrations (0, 0.1, 0.2, 0.3, 0.4 mM) of the compounds A–D. Ellagic acid (A), 30 -O-methyl ellagic acid (B), 3,30 -di-Omethyl ellagic acid (C), 3-O-methyl-30 ,40 -methylenedioxy ellagic acid (D). Results are expressed by the mean  S.D of three independent experiments in six replicates (n ¼ 6). The statistical significance of differences between groups was evaluated. A P-value < 0.05 was considered to indicate significance.

polar groups might favor the reduction of enzymatic activity; probably, the bond group in C3 (or C30 ) has a position in a polar region of the active site of the PLA2. As such, the hydroxyls at C3 or C30 of the ellagic acid derivative should favor the hydrogen bond formation between hydroxyls at C4 and C40 with the amino acids His 48 and Asp 49 and can also facilitate the interaction between benzopyrone rings and the hydrophobic amino acids at the active site (Phe 5, Trp 31 and Phe 98 – Fig. 8a and b). On the other hand, when the C3 (or C30 ) methoxyls of compounds C and D are positioned at this polar region of the active site a repulsive force is created that destabilize the complex formed with the PLA2 and reduces the inhibitory capacity of their enzymatic activity. This repulsive interaction should have a low intensity because the both compounds (C and D) can cause the inhibition of the enzyme (Fig. 5). This repulsion probably causes a change leading to better positioning of the compounds C and D inside the active site, but does not cause the complete inhibition of PLA2 Thus, it is possible that the compounds C and D, in contrast to, A and B do not form hydrogen bond between hydroxyls in C4 or C40 with the amino acids His 48 and Asp 49, and can establish only hydrophobic interactions of

benzopyrone rings conjugated with the amino acids, Phe 5, Trp 31 and Phe 98 of PLA2 (Fig. 8c and d). The activity of compound B (30 -O-methyl ellagic acid) was lower than that of compound A, but significantly higher than those of compounds C and D. Possible due to the manner in which compound B enters the PLA2 active site. In contrast to compound A, compound B has a methoxylated position C30 and the possibility for only one orientation (where C3 is positioned in polar region, Fig. 8) that favors the hydrogen bonding with the active site. When compound B directs its hydroxyl to C40 and the methoxyl to C30 is directed to the active site, these appear to provide the same repulsive strength present in compounds C and D (Fig. 8c and d) and decrease the PLA2 inhibition efficiency of compound B. More structural studies are needed to confirm these ideas and there are some attempts to cocrystallize the B. jararacussu PLA2 with ellagic acid to verify how the interactions are established between these two substances. 5. Conclusions According to previously demonstrated data, the C. sylvestris aqueous extract is capable of inhibiting various toxic effects provoked by venom from different snakes

S.L. Da Silva et al. / Toxicon 52 (2008) 655–666

663

Table 1 Kinetic parametersa obtained for the reaction of PLA2 (Asp 49 BthTX-II) from B. jararacussu in the presence and absence of ellagic acid derivative compounds using the substrate HPGP Inhibitor concentrations (mM) 0.0

0.1

0.2

0.3

0.4

Compound A Vmax KM KI

18,29  0.56 0.59  0.02

18.20  0.45 21.65  0.25 2.82  0.56

18.28  0.98 41.56  0.31 2.90  0.65

18.30  0.21 65.63  0.18 2.74  0.22

18.17  0.11 86.68  0.25 2.76  0.32

Compound B Vmax KM KI

18.27  0.43 0.61  0.05

18.28  0.22 9.55  0.28 6.82  0.81

18.31  0.87 18.08  0.11 6.98  1.03

18.27  1.01 27.36  0.21 6.84  1.10

18.19  0.78 35,66  0.28 6.96  0.93

Compound C Vmax KM KI

18.36  0.65 0.63  0.10

18.25  1.12 1.91  0.31 44.61  1.25

18.27  0.86 3.20  0.35 45.20  0.99

18.31  0.71 4.65  0.32 43.52  1.31

18.25  0.10 5.81  0.18 45.18  1.15

Compound D Vmax KM KI

18.27  0.89 0.60  0.08

18.18  0.67 1.85  0.23 47.82  0.97

18.19  0.57 3.09  0.18 48.21  1.31

18.19  0.90 4.28  0.31 49.01  1.29

18.29  0.18 5.54  0.11 48.64  0.81

The Vmax values are given in nmol/min. KM values are given in mM. KI values are given in nM. The statistical significance of differences between groups was evaluated. A P-value < 0.05 was considered to indicate significance. a Average of three independent determinations; six replicates; values are mean  S.D.

(Borges et al., 2000, 2001; Mattos et al., 2007; Cavalcante et al., 2007). To aid in elucidating the mode of action of C. sylvestris on venom effects, we sought to isolate, purify and characterize chemical compounds that are linked to the mode of action of the aqueous extract of this plant on ophidian toxins, principally on snake venoms of the Bothrops genus. Thus, we have isolated four compounds of C. sylvestris that are associated with the inhibition of B. jararacussu venom toxic effects and an isoform of its PLA2s (Asp 49 BthTX-II). In this study, some compounds were isolated that are capable of inhibiting some effects caused by whole venom and by one of the PLA2 isoforms (Asp 49 BthTX-II). These isolated compounds were characterized as ellagic acid and three of its derivatives (30 -O-methyl ellagic acid, 3,30 -di-Omethyl ellagic acid and 3-O-methyl-30 ,40 -methylenedioxy ellagic acid). The kinetic parameters, obtained from the study of enzymatic kinetics, showed that all compounds

compete for the bjPLA2 active site and inhibit the enzyme in a competitive manner. Moreover, all the substances were capable of inhibiting the myotoxic activities and formation of induced edema, both by PLA2 and total venom, but at different levels (Figs. 3–5). It is important observe that other studies have already demonstrated that ellagic acid is a potent group II PLA2 inhibitor (Glaser et al., 1995; Chandra et al., 2007), but does not efficiently inhibit other important points in inflammatory processes such as synthesis of platelet aggregation factor (PAF) or neutrophil and macrophage activation (Marshall et al., 1990). Our results show that ellagic acid is also no efficient inhibitor group II PLA2 and some effects caused by it (edema-inducing formation and myotoxic activity). The information available in the literature together with our results suggests a theoretical model in which the ellagic acid hydroxyl at C4 interacts directly (or through a water

Fig. 8. Ellagic acid and quercetin structures. The structures of ellagic acid are similar to the quercetin and demonstrate similarities between the C7 and C4 hydroxyls. Both compounds present a hydroxy-benzopyrone.

664

S.L. Da Silva et al. / Toxicon 52 (2008) 655–666

Fig. 9. Models of the proposed mechanisms for B. jararacussu PLA2 inhibition by ellagic acid and its derivatives. These models, the Phe 98 and Trp 31 amino acids, associate with each other through hydrophobic interactions with the p electronic cloud from the upper part of the conjugated benzopyrone ring system of the ellagic acid, while the Phe 5 amino acid is associated with the lower part of the benzopyrone ring by hydrophobic interactions (Fig. 8). The hydroxyl in C4 (similar to C7 of the quercetin) can interact directly with His 48 and Asp 49 through hydrogen bonds (A) or through a water molecule (B).

molecule) with the His 48 and Asp 49 amino acids of PLA2, establishing hydrogen bonds similar to those made between the quercetin hydroxyl at C7 and the human synovial PLA2 (La¨ttig et al., 2007). Furthermore, the ellagic acid polycyclic aromatic rings bind to Phe 5, Trp 31 and Phe 98 amino acids through hydrophobic interactions (Fig. 9). Apparently, the efficiency of compounds A–D in inhibiting the PLA2 activity is related to the groups bonding of the groups at positions C3 and C30 (Fig. 2). The compounds with hydroxyls in this position (compounds A and B) were capable of inhibiting the toxic effects of isolated PLA2 and

also the total venom of B. jararacussu with more efficiency than the compounds with methoxyls (compounds C and D). It is possible that the polar groups at C3 and C300 help the formation of stronger complexes and with more intense interactions (hydrogen bonds with the amino acids His 48 and Asp 49 and hydrophobic interactions with Phe 5, Trp 31 and Phe 98 amino acids) between the ellagic acid derivates and the enzyme (Fig. 10c and b). However, the apolar groups in C3 and C300 (compounds C and D) establish a repulsive force (Fig. 10c and d) that cause a small change in the position of C and D in regard to

Fig. 10. The derivatives of ellagic acid may present polar groups (Fig. 8a and b) or non-polar groups (Fig. 8c and 8d) in C3 and/or C30 . Apparently, the polar groups (hydroxyls in C3 or C30 ) favor either the formation of hydrogen bindings to His 48 and Asp 49, or the hydrophobic interactions with Phe 5, Trp 31 and Phe 98 (derivatives A and B). When non-polar groups (methoxyl) are in C3 and/or C30 , a repulsive strength occurs in the polar region that moves the ellagic acid derivative and inhibits the formation of a hydrogen bond. The hydrophobic interactions are maintained; however, this occurrence decreases the inhibition potential of the derivatives, C and D.

S.L. Da Silva et al. / Toxicon 52 (2008) 655–666

A and B, blocking the formation of hydrogen between the hydroxyls in C4 (or C40 ) with the His 48 and Asp 49 amino acids, but does not blocking the establishment of hydrophobic interactions with Phe 5, Trp 31 and Phe 98 amino acids. Thus, the PLA2 – compound C (or D) is weaker than the PLA2 – compound A (or B). However, it must be highlighted that more structural studies must be carried out to further the knowledge about how the interactions between ellagic acid (and its derivatives) and the PLA2s are established. Acknowledgements We would like to thank CAPES and CNPq (Brazilian agencies) for their financial support. Conflict of interest The authors declare that this manuscript do not have any conflict of interest. References Alves, T.M.A., 2000. Biological screening of Brazilian medicinal plants. Memo´rias do Instituto Oswaldo Cruz 95, 367–373. Arni, R.K., Ward, R.J., 1996. Phospholipase A2 – a structural review. Toxicon 34, 827–841. Atta-Ur-Rahman, Ngounou, F.N., Choudhary, M.I., Malik, S., Makhmoor, T., Nur-E-Alam, M., Zareen, S., Lontsi, D., Ayafor, J.F., Sondengam, B.L., 2001. New antioxidant and antimicrobial ellagic acid derivatives from Pteleopsis hylodendron. Planta Medica 67, 335–339. Barbosa, R.I., Nascimento, S.P., Amorin, P.A.F., Silva, R.F., 2005. Notas sobre a composiça˜o arbo´reo-arbustiva de uma fisionomia das savanas de Roraima, Amazoˆnia Brasileira. Acta Botanica Brasilica 19, 323–329. Berg, O.G., Gelb, M.H., Tsaı´, M.D., Jain, M.K., 2001. Interfacial enzymology: the secreted phospholipase A2-paradigm. Chemical Reviews 101, 2613–2653. Borges, M.H., Soares, A.M., Rodrigues, V.M., Oliveira, F., Fransheschi, A.M., Rucavado, A., Giglio, J.R., Homsi-Brandeburgo, M.I., 2001. Neutralization of proteases from Bothrops snake venoms by the aqueous extract from Casearia sylvestris (Flacourtiaceae). Toxicon 39, 1863–1869. Borges, M.H., Soares, A.M., Rodrigues, V.M., Andriao-Escarso, S.H., Diniz, H., Hamaguchi, A., Quintero, A., Lizano, S., Gutierrez, J.M., Giglio, J.R., Homsi-Brandeburgo, M.I., 2000. Effects of aqueous extract of Casearia sylvestris (Flacourtiaceae) on actions of snake and bee venoms and on activity of phospholipases A2. Comparative Biochemistry and Physiology 127, 21–30. Calgarotto, A.K., Damico, D.C.S., Ponce-Soto, L.A., Baldasso, P.A., Da Silva, S. L., Souza, G.H.M.F., Eberlin, M.N., Marangoni, S., 2008. Biological and biochemical characterization of new basic phospholipase A2 BmTX-I isolated from Bothrops moojeni snake venom. Toxicon 51, 1509–1519. Cavalcante, W.L.G., Campos, T.O., Pai-Silva, M.D., Pereira, P.S., Oliveira, C.Z., Soares, A.M., Gallacci, M., 2007. Neutralization of snake venom phospholipase A2 toxins by aqueous extract of Casearia sylvestris (Flacourtiaceae) in mouse neuromuscular preparation. Journal of Ethnopharmacology 112, 490–497. Chandra, V., Kaur, P., Jasti, J., Betzel, C., Srinivasan, A., Singh, T.P., 2002a. First structural evidence of a specific inhibition of phospholipase A2 by a-tocopherol (vitamin E) and its implications in inflammation: crystal structure of the complex formed between phospholipase A2 and a-tocopherol at 1.8 A resolution. Journal of Molecular Biology 320, 215–222. Chandra, V., Jasti, J., Kaur, P., Srinivasan, A., Betzel, C., Singh, T.P., 2002b. Structural basis of phospholipase A2 inhibition for the synthesis of prostaglandins by the plant alkaloid aristolochic acid from a 1.7 A crystal structure. Biochemistry 41, 10914–10919. Chandra, J.N.N.S., Ponnappa, K.C., Sadashiva, C.T., Priya, B.S., Nanda, B.L., Gowda, T.V., Vishwanath, B.S., Rangappa, K.S., 2007. Chemistry and structural evaluation of different phospholipase A2 inhibitors in arachidonic acid pathway mediated inflammation and snake venom toxicity. Current Topics in Medicinal Chemistry 7, 787–800.

665

Da Silva, S.L., Figueredo, P.M.S., Yano, T., 2006. Antibacterial and antifungal activities of volatile oils from Zanthoxylum rhoifolium leaves. Pharmaceutical Biology 44, 657–659. Da Silva, S.L., Figueiredo, P.M.S., Yano, T., 2007. Chemotherapeutic potential of the volatile oils from Zanthoxylum rhoifolium Lam leaves. European Journal of Pharmacology 576, 180–188. Da Silva, S.L., Chaar, J.D.S., Damico, D.C.S., Figueiredo, P.D.M.S., Yano, T., 2008a. Antimicrobial activity of ethanol extract from leaves of Casearia sylvestris. Pharmaceutical Biology 46, 347–351. Da Silva, S.L., Chaar, J.S., Figueiredo, P.M.S., Yano, T., 2008b. Cytotoxic evaluation of essential oil from Casearia sylvestris Sw on human cancer cells and erythrocytes. Acta Amazonica 38, 107–112. Da Silva, S.L., Calgarotto, A.K., Maso, V., Damico, D.C.S., Baldasso, P.A., Veber, C.L., Villar, J.A.F.P., Oliveira, A.R.M., Comar Jr., M., Oliveira, K.M.T., Marangoni, S., 2008c. Molecular modeling and inhibition of phospholipase A2 by polyhydroxy phenolic compounds. European Journal of Medicinal Chemistry, doi:10.1016/j.ejmech.2008.02.043. Da Silva, S.L., Comar Jr., M., Oliveira, K.M.t, Chaar, J.S., Bezerra, E.R.M., Calgarotto, A.K., Baldasso, P.A., Veber, C.L., Villar, J.A.F.P., Oliveira, A.R. M., Marangon, S., 2008d. Molecular modeling of the inhibition of enzyme PLA2 from snake venom by dipyrone and 1-phenyl-3-methyl5-pyrazolone. International Journal of Quantum Chemistry 108, 2576–2585. Esteves, I., Souza, I.R., Rodrigues, M., Cardoso, L.G.V., Santos, L.S., Sertie, J. A.A., Perazzo, F.F., Lima, L.M., Schneedorf, J.M., Bastos, J.K., Carvalho, J. C.T., 2005. Gastric antiulcer and anti-inflammatory activities of the essential oil from Casearia sylvestris Sw. Journal of Ethnopharmacology 101, 191–196. Fawzy, A.A., Vishwanath, B.S., Franson, R.C., 1988. Inhibition of human non-pancreatic phospholipases A2 by retinoids and flavonoids. Mechanism of action. Agents & Actions 25, 394–400. Felfili, J.M., Nogueira, P.E., Silva Jr., M.C., Morimon, B.S., Delitti, W.B.C., 2002. Composiça˜o florı´stica e fitossociolo´gica do cerrado sentido restrito no municı´pio de A´gua Boa – MT. Acta Botanica Brasilica 16, 103–112. Gil, B., Sanz, M.J., Terencio, M.C., Gunasegaran, R., Paya, M., Alcaraz, M.J., 1994. Morelloflavone, a novel biflavonoid inhibitor of human secretory phospholipase A2 with anti-inflammatory activity. Biochemical Pharmacology 53, 733–740. Glaser, K.B., Jacobs, R.S., 1986. Molecular pharmacology of manoalide. Inactivation of bee venom phospholipase A2. Biochemical Pharmacology 35, 449–453. Glaser, K.B., Sung, M.-L.A., Hartman, D.A., Lock, Y.W., Bauer, J., Walter, T., Carlson, R.P., 1995. Cellular and topical in vivo inflammatory murine models in the evaluation of inhibitors of phospholipase A2. Skin Pharmacology 8, 300–308. Hack, C., Longhi, S.J., Boligon, A.A., Murari, A.B., Pauleski, D.T., 2005. Ana´lise fitossociolo´gica de um fragmento de floresta estacional decidual no municı´pio de Jaguari, RS. Cieˆncia Rural 35, 1083–1091. Kim, H.P., Pham, H.T., Ziboh, V.A., 2001. Flavonoids differentially inhibit guinea pig epidermal cytosolic phospholipase A2. Prostaglandins, Leukotrienes, and Essential Fatty Acids 65, 281–286. Lansky, E.P., Newman, R.A., 2007. Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. Journal of Ethnopharmacology 109, 177–206. La¨ttig, J., Bo¨hl, M., Fischer, P., Tischer, S., Tietbo¨hl, C., Menschikowski, M., Gutzeit, H.O., Metz, P., Pisabarro, M.T., 2007. Mechanism of inhibition of human secretory phospholipase A2 by flavonoids: rationale for lead design. Journal of Computer-Aided Molecular Design 21, 473– 483. Li, X.-C., Elsohly, H.N., Hufford, C.D., Clark, A.M., 1999. NMR assignments of ellagic acid derivatives. Magnetic Resonance in Chemistry 37, 856–859. Lindahl, M., Tagesson, C., 1997. Flavonoids as phospholipase A2 inhibitors: importance of their structure for selective inhibition of group II phospholipase A2. Inflammation 21, 347–356. Lorenzi, H., Matos, F.J.A., 2002. Plantas Medicinais no Brasil: Nativas e exo´ticas. Instituto Plantarum de Estudos da Flora LTDA, Nova Odessa, SP, pp. 23–24. Mans, D.R.A., Rocha, A.B., Schwartsmann, G., 2000. Anti-cancer drug discovery and development in Brazil: targeted plant collection as a rational strategy to acquire candidate anticancer compounds. Oncologist 18, 185–198. Martz, W., 1992. Plants with a reputation against snakebite. Toxicon 30, 1131–1142. Mattos, E.S., Frederico, M.J.S., Colle, T.D., Pieri, D.V., Peters, R.R., Piovezan, A.P., 2007. Evaluation of antinociceptive activity of Casearia sylvestris and possible mechanism of action. Journal of Ethnopharmacology 112, 1–6.

666

S.L. Da Silva et al. / Toxicon 52 (2008) 655–666

Marshall, L.A., Sung, A., Franson, R., Chang, J.Y., 1990. Pharmacological differences of phospholipases A2 purified from human synovial fluid of patients with rheumatoid arthritis and human platelets. The FASEB Journal A1146. Mors, W.B., Nascimento, M.C., Pereira, B.M.R., Pereira, N.A., 2000. Plant natural products active against snake bite – the molecular approach. Phytochemistry 55, 627–642. Nawwar, M.A.M., Hussein, S.A.M., Merfort, I., 1994. NMR spectral analysis of polyphenols from Punica granatum. Phytochemistry 36, 793–798. ˜ ez, V., Barona, J., Fonnegra, R., Jime´nez, S.L., Osorio, R.G., Otero, R., Nu´n Saldarriaga, M., Dı´az, A., 2000. Snakebites and ethnobotany in the northwest region of Colombia. Part III: neutralization of the haemorrhagic effect of Bothrops atrox venom. Journal of Ethnopharmacology 73, 233–241. Pereira, B.M.R., Gonçalves, L.C., Pereira, N.A., 1992. Abordagem farmacolo´gica de plantas recomendadas pela medicina folclo´rica como antiofı´dicas. III – atividade antiedematogeˆnica. Revista Brasileira de Farma´cia 73, 85–86. Raslan, D.S., Jamal, C.M., Duarte, D.S., Borges, M.H., De Lima, M.E., 2002. Anti-PLA2 action test of Casearia sylvestris Sw. Bollettino Chimico Farmaceutico 141, 457–460. Rogerio, A.P., Fontanari, C., Melo, M.C.C., Ambrosio, S.R., De Souza, G.E.P., Pereira, P.S., Franca, S.C., Costa, F.B., Albuquerque, D.A., Faccioli, L.H., 2006. Anti-inflammatory, analgesic and anti-oedematous effects of Lafoensia pacari extract and ellagic acid. Journal of Pharmacy and Pharmacology 58, 1265–1273.

Ruppelt, B.M., Pereira, B.E., Gonçalves, L.C., Pereira, N.A., 1991. Pharmacological screening of plants recommended by folks medicine as antisnake venom-I. Analgesic and anti-inflammtory activities. Memo´rias do Instituto Oswaldo Cruz 86, 203–205. Sato, T., 1987. Spectral differentiation of 3,3-di-O-methylellagic acid from 4,4-di-O-methylellagic acid. Phytochemistry 26, 2124–2125. Souza, A.D.L., Rodrigues-Filho, E., Souza, A.Q.L., Pereira, J.O., Calgarotto, A. K., Maso, V., Marangoni, S., Da Silva, S.L., 2008. Koningins, phospholipase A2 inhibitors from endophytic fungus Trichoderma koningii. Toxicon 51, 240–250. Taylor, L., 2002. Herbal Secrets of the Rainforest: Technical Data Report for Guaçatonga. Sage Press, Austin, TX, pp. 1–4. Ticli, F.K., Hage, L.I.S., Cambraia, R.S., Pereira, P.S., Magro, A.J., Fontes, M.R. M., Sta´beli, R.G., Giglio, J.R., França, S.C., Soares, A.M., Sampaio, S.V., 2005. Rosmarinic acid, a new snake venom phospholipases A2 inhibitor from Cordia verbenacea (Boraginceae): antiserum action potentiation and molecular interaction. Toxicon 46, 318–327. Villar, J.A.F.P., Lima, F.T.D., Veber, C.L., Oliveira, A.R.M., Calgarotto, A.K., Marangoni, S., da Silva, S.L., 2008. Synthesis and evaluation of nitrostyrene derivative compounds, new snake venom phospholipase A2 inhibitors. Toxicon 51, 1467–1478. Vishwanath, B.S., Appu Rao, A.G., Gowda, T.V., 1987. Interaction of phospholipase A2 from Vipera russelli venom with aristolochic acid: a circular dichroism study. Toxicon 25, 939–946.