Purification and characterization of the biological effects of phospholipase A2 from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA₂ isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the São Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA₂ proteins (named BcPLA₂1, BcPLA₂2 and BcPLA₂3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA₂1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA₂1 showed high amino acid sequence identity with PLA₂ group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA₂ and 1POC_A). In addition, BcPLA₂1 also showed significant overall homology to bee PLA₂. The enzymatic activity induced by native BcPLA₂1 (20 μg/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA₂1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA₂ from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA₂1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA₂, a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration.     

Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A2 and l-amino acid oxidase

Some proteins present in snake venom possess enzymatic activities, such as phospholipase A₂ and l-amino acid oxidase. In this study, we verify the action of the Bothrops marajoensis venom (BmarTV), PLA₂ (BmarPLA₂) and LAAO (BmarLAAO) on strains of bacteria, yeast, and Leishmania sp. The BmarTV was isolated by Protein Pack 5PW, and several fractions were obtained. Reverse phase HPLC showed that BmarPLA₂ was isolated from the venom, and N-terminal amino acid sequencing of sPLA₂ showed high amino acid identity with other lysine K49 sPLA₂s isolated from Bothrops snakes. The BmarLAAO was purified to high molecular homogeneity and its N-terminal amino acid sequence demonstrated a high degree of amino acid conservation with others LAAOs. BmarLAAO was able to inhibit the growth of P. aeruginosa, C. albicans and S. aureus in a dose-dependent manner. The inhibitory effect was more significant on S. aureus, with a MIC = 50 μg/mL and MLC = 200 μg/mL. However, the BmarTV and BmarPLA₂ did not demonstrate inhibitory capacity. BmarLAAO was able to inhibit the growth of promastigote forms of L. chagasi and L. amazonensis, with an IC₅₀ = 2.55 μg/mL and 2.86 μg/mL for L. amazonensis and L. chagasi, respectively. BmarTV also provided significant inhibition of parasitic growth, with an IC₅₀ of 86.56 μg/mL for L. amazonensis and 79.02 μg/mL for L. chagasi. BmarPLA₂ did not promote any inhibition of the growth of these parasites. The BmarLAAO and BmarTV presented low toxicity at the concentrations studied. In conclusion, whole venom as well as the l-amino acid oxidase from Bothrops marajoensis was able to inhibit the growth of several microorganisms, including S. aureus, Candida albicans, Pseudomonas aeruginosa, and Leishmania sp.     

cDNA cloning, structural, and functional analyses of venom phospholipases A2 and a Kunitz-type protease inhibitor from steppe viper Vipera ursinii renardi

Snake venom phospholipases A₂ (PLA₂s) display a wide array of biological activities and are each characteristic to the venom. Here, we report on the cDNA cloning and characterization of PLA₂s from the steppe viper Vipera ursinii renardi venom glands. Among the five distinct PLA₂ cDNAs cloned and sequenced, the most common were the clones encoding a basic Ser-49 containing PLA₂ (Vur-S49). Other clones encoded either ammodytin analogs I1, I2d and I2a (designated as Vur-PL1, Vur-PL2 and Vur-PL3, respectively) or an ammodytoxin-like PLA₂ (Vurtoxin). Additionally, a novel Kunitz-type trypsin inhibitor for this venom species was cloned and sequenced. Comparison of these PLA₂ and Kunitz inhibitor sequences with those in the sequence data banks suggests that the viper V. u. renardi is closely related to Vipera ammodytes and Vipera aspis. Separation of V. u. renardi venom components by gel-filtration and ion-exchange chromatography showed the presence of many PLA₂ isoforms. Remarkably, the most abundant PLA₂ isolated was Vur-PL2 while Vur-S49 analog was in very low yield. There are great differences between the proportion of cDNA clones and that of the proteins isolated. Two Vur-PL2 isoforms (designated as Vur-PL2A and Vur-PL2B) indistinguishable by masses, peptide mass fingerprinting, N-terminal sequences and CD spectroscopy were purified from the pooled venom. However, when rechromatographed on cation-exchanger, Vur-PL2A showed only one peak corresponding to Vur-PL2B, suggesting the existence of conformers for Vur-PL2. Vur-PL2B was weakly cytotoxic to rat pheochromocytoma PC12 cells and showed both strong anticoagulant and anti-platelet activities. This is the first case of a strong anticoagulating ammodytin I analog in Vipera venom.     

Secretory phospholipase A2 enzymes as pharmacological targets for treatment of disease

Phospholipase A₂ (PLA₂) cleave phospholipids preferentially at the sn-2 position, liberating free fatty acids and lysophospholipids. They are classified into six main groups based on size, location, function, substrate specificity and calcium requirement. These classes include secretory PLA₂ (sPLA₂), cytosolic (cPLA₂), Ca²⁺-independent (iPLA₂), platelet activating factor acetylhydrolases (PAF-AH), lysosomal PLA₂ (LyPLA₂) and adipose specific PLA₂ (AdPLA₂). It is hypothesized that PLA₂ can serve as pharmacological targets for the therapeutic treatment of several diseases, including cardiovascular diseases, atherosclerosis, immune disorders and cancer. Special emphasis has been placed on inhibitors of sPLA₂ isoforms as pharmacological moieties, mostly due to the fact that these enzymes are activated during inflammatory events and because their expression is increased in several diseases. This review focuses on understanding how sPLA₂ isoform expression is altered during disease progression and the possible therapeutic interventions to specifically target sPLA₂ isoforms, including new approaches using nano-particulate-based strategies.     

In Vitro Antiplasmodial Activity of Phospholipases A2 and a Phospholipase Homologue Isolated from the Venom of the Snake Bothrops asper

The antimicrobial and antiparasite activity of phospholipase A₂ (PLA₂) from snakes and bees has been extensively explored. We studied the antiplasmodial effect of the whole venom of the snake Bothrops asper and of two fractions purified by ion-exchange chromatography: one containing catalytically-active phospholipases A₂ (PLA₂) (fraction V) and another containing a PLA₂ homologue devoid of enzymatic activity (fraction VI). The antiplasmodial effect was assessed on in vitro cultures of Plasmodium falciparum. The whole venom of B. asper, as well as its fractions V and VI, were active against the parasite at 0.13 ± 0.01 µg/mL, 1.42 ± 0.56 µg/mL and 22.89 ± 1.22 µg/mL, respectively. Differences in the cytotoxic activity on peripheral blood mononuclear cells between the whole venom and fractions V and VI were observed, fraction V showing higher toxicity than total venom and fraction VI. Regarding toxicity in mice, the whole venom showed the highest lethal effect in comparison to fractions V and VI. These results suggest that B. asper PLA₂ and its homologue have antiplasmodial potential.     

Morinda citrifolia Inhibits Both Cytosolic Ca2+-dependent Phospholipase A2 and Secretory Ca2+-dependent Phospholipase A2

This study investigated the effects of the methanol extracts of Morinda citrifolia containing numerous anthraquinone and iridoid on phospholipase A₂ (PLA₂) isozyme. PLA₂ activity was measured using various PLA₂ substrates, including 10-pyrene phosphatidylcholine, 1-palmitoyl-2-[¹⁴C]arachidonyl phosphatidylcholine ([¹⁴C]AA-PC), and [³H]arachidonic acid (AA). The methanol extracts suppressed melittin-induced [₃H]AA release in a concentration-dependent manner in RAW 264.7 cells, and inhibited cPLA₂/sPLA₂-induced hydrolysis of [¹⁴C]AA-PC in a concentration- and time-dependent manner. A Dixon plot showed that the inhibition by methanol extracts on cPLA₂ and sPLA₂ appeared to be competitive with inhibition constants (K_i) of 3.7µg/ml and 12.6µg/ml, respectively. These data suggest that methanol extracts of Morinda citrifolia inhibits both Ca²⁺-dependent PLA₂ such as, cPLA₂ and sPLA₂. Therefore, Morinda citrifolia may possess anti-inflammatory activity secondary to Ca²⁺-dependent PLA₂ inhibition.     

Synthesis and evaluation of sesquiterpene lactone inhibitors of phospholipase A2 from Bothrops jararacussu

Several sesquiterpene lactone were synthesized and their inhibitive activities on phospholipase A₂ (PLA₂) from Bothrops jararacussu venom were evaluated. Compounds Lac01 and Lac02 were efficient against PLA₂ edema-inducing, enzymatic and myotoxic activities and it reduces around 85% of myotoxicity and around 70% of edema-inducing activity. Lac05-Lac08 presented lower efficiency in inhibiting the biological activities studied and reduce the myotoxic and edema-inducing activities around only 15%. The enzymatic activity was significantly reduced. The values of inhibition constants (K_I) for Lac01 and Lac02 were approximately 740 μM, and for compounds Lac05-Lac08 the inhibition constants were approximately 7.622-9.240 μM. The enzymatic kinetic studies show that the sesquiterpene lactones inhibit PLA₂ in a non-competitive manner. Some aspects of the structure-activity relationships (topologic, molecular and electronic parameters) were obtained using ab initio quantum calculations and analyzed by chemometric methods (HCA and PCA). The quantum chemistry calculations show that compounds with a higher capacity of inhibiting PLA₂ (Lac01-Lac04) present lower values of highest occupied molecular orbital (HOMO) energy and molecular volume (VOL) and bigger values of hydrophobicity (LogP). These results indicate some topologic aspects of the binding site of sesquiterpene lactone derivatives and PLA₂.     

Isolation and characterization of two new Lys49 PLA2s with heparin neutralizing properties from Bothrops moojeni snake venom

Among the proteins and peptides already characterized in Bothrops moojeni venom, two novel phospholipases A₂ (PLA₂) have been purified and fully sequenced by ESI-MS/MS techniques. Both of them belong to the enzymatically non-active Lys49 variants of PLA₂. They consist of 122 amino acids and share a characteristic sequence in their C-terminal region composed of clusters of basic amino acids known to interact with heparin. Thus, as already established, heparin can be used as an antidote to antagonize some myotoxic PLA₂s from venoms of Bothrops genus. The two PLA₂ variants were shown to interact in vitro with unfractionated heparin (UFH) and low molecular weight heparin (LMWH), neutralizing their anticoagulant properties. Although the influences of PLA₂s from snake venoms on the blood coagulation system are known, their use to antagonize the anticoagulant effect of heparin in vitro or in vivo has never been proposed. These finding recommend diagnostic and therapeutic applications, which are currently investigated.     

Immunome and venome of Bothrops jararacussu: A proteomic approach to study the molecular immunology of snake toxins

A combination of anti-bothropic and anti-crotalic sera has been reported to be more effective in neutralizing the effects of Bothrops jararacussu venom than anti-bothropic serum alone. The role of proteins from B. jararacussu venom in the horse immune response was evaluated via the analysis of cross-reactivity with homologous and heterologous sera. Many of the proteins in B. jararacussu venom were identified via 2D gel electrophoresis. Western blots revealed that anti-jararacussu showed higher reactivity to l-aminoxidase (LAOs) and snake venom metalloproteinase, (SVMPs) and weaker reactivity towards Snake venom serine proteases (SVSPs), PLA₂, C-type lectin and cysteine-rich proteins. Anti-jararaca preferentially recognized LAOs, SVMPs and SVSPs. Both of these sera failed to recognize low-molecular weight proteins. Anti-crotalic serum clearly recognized LAOs, C-type lectin, SVSP, cysteine-rich proteins, SVMP and Asp49-PLA₂. The cross-reactivity with anti-PLA₂ revealed the immunoreactivity of these antibodies to proteins with molecular masses in a range that is poorly recognized by other studied anti-sera. Our results suggest that the contribution of anti-crotalic serum to the neutralization of B. jararacussu by may be due to its cross-reactivity with proteins such as C-type lectins, SVSPs, Asp49-PLA₂. These results also reinforce the importance of neutralizing the highly toxic proteins inclusive those with low immunogenicity in commercial antivenom production to obtain a highly protective serum against snake venoms.     

Purification, characterization, and cDNA cloning of acidic platelet aggregation inhibiting phospholipases A2 from the snake venom of Vipera lebetina (Levantine viper)

Two novel acidic phospholipase A₂s (PLA₂) were isolated by size exclusion chromatography and reversed-phase chromatography from the crude Vipera lebetina venom. The molecular masses of VLPLA₂-1 (13,704 Da) and VLPLA₂-2 (13,683 Da) and their internal tryptic peptides were determined by MALDI-TOF mass-spectrometry. When tested in human platelet-rich plasma, both enzymes showed a potent inhibitory effect on aggregation induced by ADP and collagen. Chemical modification with p-bromophenacylbromide abolished the enzymatic activity of PLA₂; its anti-platelet activity was fully inhibited in case of collagen as inducer and partially inhibited in case of ADP as inducer. The complete cDNAs encoding PLA₂ were cloned from a single venom gland cDNA library. Complete amino acid sequences of the VLPLA₂ were deduced from the cDNA sequences. The full-length cDNA sequences of the VLPLA₂ possess 615 bp and encode an open reading frame of 138 amino acids that include signal peptide (16 amino acids) and mature enzyme (122 amino acids). The VLPLA₂s have significant sequence similarity to many other phospholipase A₂s from snake venoms. The phylogenetic analysis on the basis of the amino acid sequence homology demonstrates that VLPLA₂s grouped with other Asp49 PLA₂s and they appear to share a close evolutionary relationship with the European vipers.     

Presynaptic action of Bothriopsis bilineata smargadina (forest viper) venom in vitro

In this work, we examined the neuromuscular activity of Bothriopsis bilineata smargadina (forest viper) venom in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations the venom caused concentration-dependent (0.1-30 μg/ml) neuromuscular blockade that was not reversed by washing, with 50% blockade occurring in 15-90 min. Muscle contractures to exogenous acetylcholine and KCl were unaffected by venom, but there was a slight increase in creatine kinase release after 120 min (from 80 ± 15 to 206 ± 25 U/ml, n = 6, p < 0.05). In mouse phrenic nerve-diaphragm preparations, the venom (1, 10 and 30 μg/ml) produced marked facilitation (∼120% increase above basal) at the highest concentration followed by neuromuscular blockade; the effects at lower concentrations were considerably less marked. Venom increased the quantal content values after 15 and 30 min followed by significant inhibition at ≥90 min. However, venom did not alter the muscle membrane resting potential or the response to exogenous carbachol. In both preparations, incubation at 22 °C instead of 37 °C delayed the onset of blockade, as did inhibition of venom PLA₂ activity. In curarized mouse preparations, the venom produced only muscle facilitation. These results indicate that B. b. smargadina venom causes neuromuscular blockade in vitro by a presynaptic mechanism involving PLA₂.     

Absence of phospholipase A2 in most Crotalus horridus venom due to translation blockage: comparison with Crotalus horridus atricaudatus venom

To investigate the peculiar absence of phospholipases A₂ (PLA₂s) in most Crotalus horridus (CH) venom, we cloned and sequenced the venom PLA₂s of three CH specimens from different regions. The results revealed that all the venom glands contained mRNAs that encoded an acidic PLA₂ (designated as either CH-E6 or CH-E6’). The predicted CH-E6 from the Iowan CH and CH-E6’ from the South Carolinian CH differed by only one amino acid residue, while the PLA₂ cDNA cloned from the Kentuckian CH contained an early stop codon instead of a Tyr²² codon. Only the individual South Carolinian CH venom was found to contain the CH-E6’ protein whose mass was confirmed by MALDI-TOF analysis. Our results suggest that low PLA₂ expression levels in most CH venom can be attributed to translation blockage. We also purified two acidic PLA₂s and canebrake toxin from the pooled venom of Crotalus horridus atricaudatus (neurotoxic CH subspecies). One of the acidic PLA₂s was identical to CH-E6 and showed high lipolytic activity and weak anti-platelet activities. The possibility that C. h. atricaudatus could be a hybrid between CH and Crotalus scutulatus is discussed.     

Identification and characterization of phospholipase A2 inhibitors from the serum of the Japanese rat snake, Elaphe climacophora

Two distinct phospholipase A₂ (PLA₂) inhibitory proteins (PLIs) were purified from the serum of the Japanese rat snake, Elaphe climacophora. The 150-kDa inhibitor, a trimer of a 50-kDa subunit, specifically inhibited the basic PLA₂ purified from the venom of Gloydius brevicaudus, whereas the 120-kDa one composed of two distinct 25-kDa subunits, A and B, inhibited both the acidic and basic PLA₂s of G. brevicaudus. On the basis of their amino acid sequences, these inhibitors were assigned as PLIβ and PLIγ, respectively. A PLIα homolog (PLIα-like protein; PLIα-LP) having an apparent molecular weight of 50-kDa and composed of 15-kDa subunits was also purified from the E. climacophora serum. This homolog was immunoreactive with antibody raised against the G. brevicaudus PLIα, but lacked in the inhibitory activity toward the acidic and basic PLA₂s. The cDNAs encoding PLIα-LP, PLIβ, PLIγ-A, and PLIγ-B were cloned from liver RNA, and their nucleotide sequences were compared with those of other venomous and non-venomous snakes.     

Effect of a recombinant Lys49PLA2 myotoxin and Lys49PLA2-derived synthetic peptides from Agkistrodon species on membrane permeability to water

The aim of this work was to study the effect of recombinant ACL myotoxin, a Lys49PLA₂ from Agkistrodon contortrix laticinctus snake venom and Lys49PLA₂-derived synthetic peptides corresponding to the region 115-129 of venom of the two different Agkistrodon species on water permeability in the toad urinary bladder. The water flow through the membrane was measured gravimetrically in bag preparations of the bladder. The addition of recombinant ACL myotoxin-MBP (maltose binding protein) fusion protein (10 nM) to the bathing solution significantly increased (above 60%) the water transport compared with the control hemibladders. The addition of the Lys49PLA₂-derived synthetic peptides in several concentrations to the bathing solution did not affect the water transport across membrane. These results suggest that the ACL myotoxin effect on water transport is not related to the cytotoxic C-terminal region.     

Isolation and functional characterization of a new acidic PLA2 Ba SpII RP4 of the Bothrops alternatus snake venom from Argentina

An acidic protein with phospholipase A₂ activity was purified to homogeneity from the venom of the Northeast Argentinian viperid Bothrops alternatus by two chromatographic steps: a conventional gel filtration on Sephadex G-75 and reversed phase on C18 HPLC column.A molecular mass of 14185.48 Da was determined by mass spectrometry, displaying a homodimer conformation. The kinetic assay demonstrated a catalytically active phospholipase A₂ in correspondence with Asp49 PLA₂ group. The enzyme designated Ba SpII RP4 contains an amino acid composition of 121 residues and a calculated theoretical pI value of 4.88. Amino acid sequence alignments with other Bothrops PLA₂ revealed a high degree of homology sequence (90-56%). Ba SpII RP4 did not show myotoxic activity upon muscular fibers at doses up to 100 μg i.m. route injection or lethal response when it was i.p. injected at the hightest dose of 200 μg. This toxin generates slight biological activities like paw edema inflammation and a delay in the clotting time, although Ba SpII RP4 exhibited catalytic activity. The primary amino acid sequence, determined a quadruple-time of flight (Q-TOF) hybrid mass spectrometer Q-TOF Ultima from Micromass (Manchester, UK) equipped with a nano Zspray source operating in a positive ion mode and tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) “de novo amino acid sequencing”, also provides more database about the small group of the non-myotoxic PLA₂s isolated up to the present.     

Synergism between baltergin metalloproteinase and Ba SPII RP4 PLA2 from Bothrops alternatus venom on skeletal muscle (C2C12) cells

Acute muscle damage, myonecrosis, is one of the main characteristics of envenoming by Bothrops genus. In this in vitro study we investigated the role of a metalloproteinase (baltergin) and an acidic phospholipase A₂ (Ba SPII RP4) in the cytotoxicity exhibited by Bothrops alternatus venom. Baltergin metalloproteinase purified from the venom exerted a toxic effect on C2C12 myoblast cells (CC₅₀: 583.34 μg/mL) which involved morphological alterations compatible with apoptosis/anoikis. On the contrary, the most abundant PLA₂ isolated from this venom did not exhibit cytotoxicity at times and doses tested. However, when myoblasts were treated with both enzymes together, synergic activity was demonstrated. Neutralization of the venom with specific antibodies (IgG anti-baltergin and IgG anti-PLA₂) confirmed this synergism.     

Purification and renal effects of phospholipase A(2) isolated from Bothrops insularis venom.

Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A(2) protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A(2) (PLA(2)) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as IV-1 to IV-5, from which IV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA(2) of Bothrops jararaca and Bothrops asper. Phospholipase A(2) isolated from B. insularis (Bi PLA(2) ) venom (10 microg/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n=6). Bi PLA(2) increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa(+)) and chloride tubular reabsorption (%TCl(-)) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA(2) isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney.     

LmrTX, a basic PLA2 (D49) purified from Lachesis muta rhombeata snake venom with enzymatic-related antithrombotic and anticoagulant activity

A basic phospholipase A₂ (LmrTX) isoform was isolated from Lachesis muta rhombeata snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-5 Discovery^® Bio Wide column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of LmrTX was measured as 14.277.50 Da. The amino acid sequence showed a high degree of homology between PLA₂ LmrTX from L. muta rhombeata and other PLA₂ from snake venoms, like CB1 and CB2 from Crotalus durissus terrificus; LmTX-I and LmTX-II from Lachesis muta muta. LmrTX had PLA₂ activity in the presence of a synthetic substrate and alkylation of histidine residues significantly inhibited (P < 0.05) the enzymatic activity of LmrTX and its anticoagulant and antithrombotic activity. In this study, we examined the ability of the LmrTX in altering thrombus formation in living mouse, using a photochemically induced arterial thrombosis model. The control animals that did not receive protein injection showed a normal occlusion time, which was around 57 ± 7.8 min. LmrTX, the PLA₂ from L. muta rhombeata venom, caused a change in the occlusion time to 99 ± 10 min with doses of 7.5 μg/mice. Additionally, LmrTX showed the anticoagulant activity in vitro and ex vivo and prolonging the time aggregation in wash platelet induced by ADP and Thrombin.     

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

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), 3′-O-methyl ellagic acid (B), 3,3′-di-O-methyl ellagic acid (C), 3-O-methyl-3′,4′-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 3′ (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 3′ reduced, but did not completely inhibit the capacity of compounds C and D on inhibiting all the toxic effects studied.