Evaluation of Rhamnetin as an Inhibitor of the Pharmacological Effect of Secretory Phospholipase A2.

Rhamnetin (Rhm), 3-O-methylquercetin (3MQ), and Rhamnazin (Rhz) are methylated derivatives of quercetin commonly found in fruits and vegetables that possess antioxidant and anti-inflammatory properties. Phospholipase A2 (PLA2) displays several important roles during acute inflammation; therefore, this study aimed at investigating new compounds able to inhibit this enzyme, besides evaluating creatine kinase (CK) levels and citotoxicity. Methylated quercetins were compared with quercetin (Q) and were incubated with secretory PLA2 (sPLA2) from Bothrops jararacussu to determine their inhibitory activity. Cytotoxic studies were performed by using the J774 cell lineage incubated with quercertins. In vivo tests were performed with Swiss female mice to evaluate decreasing paw edema potential and compounds' CK levels. Structural modifications on sPLA2 were made with circular dichroism (CD). Despite Q and Rhz showing greater enzymatic inhibitory potential, high CK was observed. Rhm exhibited sPLA2 inhibitory potential, no toxicity and, remarkably, it decreased CK levels. The presence of 3OH on the C-ring of Rhm may contribute to both its anti-inflammatory and enzymatic inhibition of sPLA2, and the methylation of ring A may provide the increase in cell viability and low CK level induced by sPLA2. These results showed that Rhm can be a candidate as a natural compound for the development of new anti-inflammatory drugs.

Evaluation of macroalgae sulfated polysaccharides on the Leishmania (L.) amazonensis promastigote.

The sulfated polysaccharides from Solieria filiformis (Sf), Botryocladia occidentalis (Bo), Caulerpa racemosa (Cr) and Gracilaria caudata (Gc) were extracted and extensively purified. These compounds were then subjected to in vitro assays to evaluate the inhibition of these polysaccharides on the growth of Leishmania (L.) amazonensis promastigotes. Under the same assay conditions, only three of the four sulfated polysaccharides were active against L. amazonensis, and the polysaccharide purified from Cr was the most potent (EC50 value: 34.5 µg/mL). The polysaccharides derived from Bo and Sf demonstrated moderate anti-leishmanial activity (EC50 values of 63.7 µg/mL and 137.4 µg/mL). In addition, we also performed in vitro cytotoxic assays toward peritoneal macrophages and J774 macrophages. For the in vitro cytotoxicity assay employing J774 cells, all of the sulfated polysaccharides decreased cell survival, with CC50 values of 27.3 µg/mL, 49.3 µg/mL, 73.2 µg/mL, and 99.8 µg/mL for Bo, Cr, Gc, and Sf, respectively. However, none of the sulfated polysaccharides reduced the cell growth rate of the peritoneal macrophages. These results suggest that macroalgae contain compounds with various chemical properties that can control specific pathogens. According to our results, the assayed sulfated polysaccharides were able to modulate the growth rate and cell survival of Leishmania (L.) amazonensis promastigotes in in vitro assays, and these effects involved the interaction of the sulfated polysaccharides on the cell membrane of the parasites.

The extract of the jellyfish Phyllorhiza punctata promotes neurotoxic effects.

Phyllorhiza punctata (P. punctata) is a jellyfish native to the southwestern Pacific. Herewith we present the biochemical and pharmacological characterization of an extract of the tentacles of P. punctata. The tentacles were subjected to three freeze-thaw cycles, homogenized, ultrafiltered, precipitated, centrifuged and lyophilized to obtain a crude extract (PHY-N). Paralytic shellfish poisoning compounds such as saxitoxin, gonyautoxin-4, tetrodotoxin and brevetoxin-2, as well as several secretory phospholipase A(2) were identified. PHY-N was tested on autonomic and somatic neuromuscular preparations. In mouse vas deferens, PHY-N induced phasic contractions that reached a peak of 234 ± 34.7% of control twitch height, which were blocked with either 100 µ m of phentolamine or 1 m m of lidocaine. In mouse corpora cavernosa, PHY-N evoked a relaxation response, which was blocked with either L-N(G) -Nitroarginine methyl ester (0.5 m m) or 1 m m of lidocaine. PHY-N (1, 3 and 10 µg ml(-1) ) induced an increase in tonus of the biventer-cervicis neuromuscular preparation that was blocked with pre-treatment of galamine (10 µ m). Administration of 6 mg kg(-1) PHY-N intramuscularly produced death in broilers by spastic paralysis. In conclusion, PHY-N induces nerve depolarization and nonspecifically increases neurotransmitter release.

Quercetin as an inhibitor of snake venom secretory phospholipase A2.

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors.

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 A(2) and l-amino acid oxidase. In this study, we verify the action of the Bothrops marajoensis venom (BmarTV), PLA(2) (BmarPLA(2)) 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 BmarPLA(2) was isolated from the venom, and N-terminal amino acid sequencing of sPLA(2) showed high amino acid identity with other lysine K49 sPLA(2)s 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 microg/mL and MLC=200 microg/mL. However, the BmarTV and BmarPLA(2) did not demonstrate inhibitory capacity. BmarLAAO was able to inhibit the growth of promastigote forms of L. chagasi and L. amazonensis, with an IC(50)=2.55 microg/mL and 2.86 microg/mL for L. amazonensis and L. chagasi, respectively. BmarTV also provided significant inhibition of parasitic growth, with an IC(50) of 86.56 microg/mL for L. amazonensis and 79.02 microg/mL for L. chagasi. BmarPLA(2) 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.

Purification and biological effects of Araucaria angustifolia (Araucariaceae) seed lectin.

This paper describes the purification and characterization of a new N-acetyl-d-glucosamine-specific lectin from Araucaria angustifolia (AaL) seeds (Araucariaceae) and its anti-inflammatory and antibacterial activities. AaL was purified using a combination of affinity chromatography on a chitin column and ion exchange chromatography on Sephacel-DEAE. The pure protein has 8.0kDa (SDS-PAGE) and specifically agglutinates rabbit erythrocytes, effect that was independent of the presence of divalent cations and was inhibited after incubation with glucose and N-acetyl-d-glucosamine. AaL showed antibacterial activity against Gram-negative and Gram-positive strains, shown by scanning electron microscopy. AaL, intravenously injected into rats, showed anti-inflammatory effect, via carbohydrate site interaction, in the models of paw edema and peritonitis. This lectin can be used as a tool for studying bacterial infections and inflammatory processes.

Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity.

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.

Effects of morin on snake venom phospholipase A2 (PLA2).

Flavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA2 isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to study the effect of flavonoids on PLA2. We observed that a treatment of PLA2 with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA2 possess a second pharmacological site which does not affect or depend on the enzymatic activity.