Roger Remy Dresch

ACL-I, a lectin from the marine sponge Axinella corrugata: isolation, characterization and chemotactic activity.

The lectin from the marine sponge Axinella corrugata (ACL-I) was purified by affinity chromatography on rabbit erythrocytic stroma incorporated into a polyacrylamide gel followed by gel filtration on Ultrogel AcA 44 column. Purified ACL-I is a hexameric glycoprotein with a Mr of 82.3 kDa estimated by SDS-PAGE and 78.5 kDa by FPLC on Superose 12 HR column. The pI of lectin is 6.3 and ACL-I is constituted of 13.9 kDa similar subunits some of them linked by disulphide bridges. This lectin agglutinates native rabbit, goat and dog erythrocytes and in less extent human erythrocytes. The hemagglutinating activity is independent of Ca(2+), Mg(2+) and Mn(2+), but it is strongly inhibited by carbohydrates containing N-acetyl groups. ACL-I is stable up to 70 degrees C for 30 min, with optimum pH between 7 and 8, and it is also resistant to enzymatic proteolysis in vitro. In the presence of reducing or denaturant agents, the lectin activity decreases. ACL-I displays chemotactic effect on rat neutrophil in vitro which is inhibited by N-acetyl-d-glucosamine.

Detecção de atividade lectínica e atividade hemolítica em extratos de esponjas (Porifera) nativas da costa atlântica do Brasil

Aqueous extracts of twenty species of sea sponges of the Brazilian Atlantic coast were tested with the aim of searching the presence of lectinic and hemolytic activity. Hemagglutinating activity for human erythrocytes and for distinct animals were found in 12 of the 20 tested extracts. The extracts of Axinella corrugata, Chondrilla nucula, Chondrosia collectrix, Cinachyrella alloclada and Guitarra sp1. were the ones that presented highest hemagglutinating activity. Ten of the 12 hemagglutinating extracts had the activity inhibited by one or more sugars or glycoproteins. The lectin from Chondrilla nucula was resistant to thermal denaturation when heated up to 100 oC for 60 minutes. Hemolytic activity was only found in the extracts from Petromica citrina and Acervochalina sp. The species of sea sponges that showed major potential for futures studies of their lectins were Axinella corrugata, Chondrilla nucula and Chondrosia collectrix, due to the highest hemagglutinating activity presented by their extracts, allied to the highest specific activity.

Biological activities of ACL-I and physicochemical properties of ACL-II, lectins isolated from the marine sponge Axinella corrugata

Lectin II from the marine sponge Axinella corrugata (ACL-II) was purified by affinity chromatography on rabbit erythrocytic stroma incorporated into a polyacrylamide gel, followed by gel filtration on Ultrogel AcA 44 column. Purified ACL-II is a lectin with an Mr of 80 kDa and 78 kDa, estimated by SDS-PAGE and by FPLC on Superose 12 HR column, respectively. ACL-II mainly agglutinates native rabbit erythrocytes and this hemagglutinating activity is independent of Ca(2+), Mg(2+) and Mn(2+), but is inhibited by d-galactose, chitin and N-acetyl derivatives, with the exception of GalNAc. ACL-II is stable for up to 65 °C for 30 min, with a better stability at a pH range of 2 to 6. In contrast, ACL-I displays a strong mitogenic and cytotoxic effect.

Appraisal of the Antichemotactic Activity of Flavonoids on Polymorphonuclear Neutrophils

Flavonoids are polyphenols that are ubiquitous in plants and frequently consumed in the diet. They are suggested to have many beneficial actions on human health, including anti-inflammatory activity. Their properties have been studied in a number of cell types, but little is known about their effects on neutrophil biology. Consequently, we selected 25 flavonoids with different structural features to evaluate their in vitro inhibition of rat polymorphonuclear neutrophil (PMN) chemotaxis, employing a modified Boyden chamber. Migratory activity was measured towards a chemotactic stimulant, formyl-Met-Leu-Phe or lipopolysaccharide. Furthermore, the cytotoxic effect of flavonoids on PMNs was determined by the release of cytosolic lactate dehydrogenase (LDH). Ten flavonoids significantly retarded the migration of PMNs with at least one of the concentrations tested in a range between 0.625 and 100 µM; the best antichemotactic agents were flavone, flavonol, quercetin and rutin. None of the flavanones evaluated presented any significant inhibition of migration in this assay. Our findings indicated that non-hydroxylated flavones possess a better antichemotactic activity when compared to flavones with hydroxy groups. The presence of a sugar moiety in rutin did not produce any increase in this effect, when compared to the respective aglycone analogue. Finally, none of the flavonoids exhibited cell toxicity and for many of these flavonoids this is the first report of the inhibition of PMN chemotaxis.

Chemical analysis, antioxidant, antichemotactic and monoamine oxidase inhibition effects of some pteridophytes from Brazil

Background: Ferns are a group of plants that have been little explored from a chemical and biological perspective but that have interesting potential, occurring in various parts of the world. Objective: This work investigates the chemical profile and the biological effects of ferns from Brazil. Materials and Methods: Analyses were performed using rapid performance liquid chromatography (RP-LC) with a diode array detector (DAD). Extracts were tested for their in vitro antioxidant activity, by the total reactive antioxidant potential method and for their antichemotactic potential, by the Boyden chamber method. Cytotoxic effects were assessed by lactate dehydrogenase levels, while the monoamine oxidase (MAO) assay was carried out using a fluorescence-based method. Results: Different chemical compositions were found for the studied ferns, such as Asplenium gastonis, in which hesperidin was identified in its extract, while A. serra showed the presence of xanthone mangiferin. The most samples with highest antioxidant activity were the Asplenium serra, Lastreopsis amplissima and Cyathea dichromatolepis extracts, at 10 μg/mL. High antichemotactic activity was found for A. serra (94.06%) and Didymochlaena truncatula (93.41%), at 10 μg/mL. The extracts showed no cytotoxicity at the highest concentration. Against MAO-A, D. truncatula (82.61%), Alsophila setosa (82.21%), Cyathea phalerata (74.07%) and C. delgadii (70.32%) were the most active extracts (100 μg/mL). Conclusion: The hypothesis was considered that phenolics and triterpenes are responsible for these pronounced activities.

In vitro Antioxidant and Enzymatic Approaches to Evaluate Neuroprotector Potential of Blechnum Extracts without Cytotoxicity to Human Stem Cells.

Background: Investigation of selected plant extracts on multi-targets related to neurodegeneration, such as monoamine oxidases (MAO), cholinesterase enzymes, and antioxidant activities (AOA) is a useful tool for identification of new scaffolds. Objective: This work investigated biological effects of three Blechnum methanol extracts from Brazil and chemical profile of the most active sample. Materials and Methods: AOA included scavenging of hydroxyl and nitric oxide radicals, also lipid peroxidation inhibition. Enzymatic modulation of Blechnum binervatum, Blechnum brasiliense, and Blechnum occidentale extracts on MAO and cholinesterases was conducted. Moreover, total phenol content was performed with all samples, and high-performance liquid chromatography-diode array detection mass spectrometry HPLC-DAD-MS analysis was carried out with B. brasiliense. Possible toxic effects were evaluated on Wistar rats polymorphonuclear cells (PMN) and human stem cells. Results: B. brasiliense extract presented the highest phenolic amount (9.25 g gallic acid equivalent/100 g extract) and lowest IC50values (112.3 ± 2.61 and 176.1 ± 1.19 μg/mL) against hydroxyl radicals and on lipid peroxidation, respectively, showing strong AO effects. On nitric oxide assay and cholinesterase inhibition, all extracts were considered inactive. MAO-A selective action was evidenced, being B. brasiliense powerful against this enzyme (IC50: 72.7 μg/mL), followed by B. occidentale and B. binervatum (IC50: 130.85 and 165.2 μg/mL). No cytotoxic effects were observed on PMN and human stem cells treated with Blechnum extracts. HPLC-DAD-MS analysis of B. brasiliense allowed the identification of chlorogenic and rosmarinic acids. Conclusion: Our results especially highlight B. brasiliense, with pronounced phenols content and strong effects on selected targets related to neurodegeneration, being characterized as a natural safe source of bioactive hydroxycinnamic acids. SUMMARY Blechnum crude extracts showed high phenolic amounts and valuable IC50 values on targets related with neurodegenerative disorders Blechnum brasiliense was the most active sample, with strong radical scavenging and lipid peroxidation inhibition, also with monoamine oxidases: A selective modulation No cytotoxic effects were observed on polymorphonuclear cells rat cells and human stem cells treated with Blechnum extracts High-performance liquid chromatography-diode array detection-mass spectrometry analysis of Blechnum brasiliense allowed the identification of hydroxycinnamic derivatives: Chlorogenic and rosmarinic acids. Abbreviations used: IC50: half maximal inhibitory concentration; MAO: monoamine oxidase; MAO-A: monoamine oxidase isoform A; MAO-B: monoamine oxidase isoform B; HO•: hydroxyl radical.

pH-dependent cytotoxic effects of extracts of the marine sponge Polymastia janeirensis on cancer cell lines

Abstract The purpose of this work was to study the cytotoxic effects of marine sponge Polymastia janeirensis, which has been observed in the field to release an orange substance that is toxic to fish. The result showed that aqueous extract (pH 7.0) was highly cytotoxic to glioma (U87) and neuroblastoma (SHSY5Y) cancer cell lines (IC50 < 1.0 μg/mL). In addition, this extract showed potent antioxidant and procoagulant (decreased the clotting time by 1.7-fold) activities. Interestingly, the cytotoxic effects were pH-dependent since the viability of the cancer cells was not affected with the extract (pH 5.5). The close similarity between the aqueous extract (pH 7.0) and the orange liquid that is released by the sponge indicates that this potential chemical defence of P. janeirensis deserves further investigation.

Immunohistochemical localization of an N-acetyl amino-carbohydrate specific lectin (ACL-I) of the marine sponge Axinella corrugata.

The N-acetyl amino-carbohydrate specific lectin (ACL-I) was previously identified and purified by us from the marine sponge Axinella corrugata (phylum Porifera, class Demospongiae). The distribution of the specific lectin within the tissue of the sponge was studied by bright-field optical microscopy immunohistochemistry in order to better understand its physiological role in the sponge. Polyclonal antibodies were raised against purified ACL-I in mice and tested by Western blot technique. The immunohistochemical analysis of ACL-I in cross sections of A. corrugata showed that this lectin is found inside the denominated spherulous cells, which contain vesicles that store the lectin. Some evidence is shown that ACL-I might also be present in the extracellular matrix. It was not possible to demonstrate by the immunohistochemical technique if ACL-I is colocalized in both the plasma membrane and in the cytoplasm of the spherulous cells.

Sphingosines Derived from Marine Sponge as Potential Multi-Target Drug Related to Disorders in Cancer Development.

Haliclona tubifera, marine sponge species abundant in Brazilian coastline, presents only a few papers published in the literature. Recently, we have reported the isolation of two modified C18 sphingoid bases: (2R,3R,6R,7Z)-2-aminooctadec-7-ene-1,3,6-triol and and (2R,3R,6R)-2-aminooctadec-1,3,6-triol. In order to continue our research, in this work aimed at the biological investigation of fractions that led to the isolation of these compounds. We evaluated the cytotoxic effect of marine sponge H. tubifera fractions in glioma (U87) and neuroblastoma (SH-SY5Y) human cell lines. In addition, considering the link between cancer, imbalance of reactive oxygen species and coagulation disorders, we also investigated the in vitro effects on blood coagulation and their redox properties. We showed that the ethyl acetate (EtOAc) fraction, rich in sphingoid bases, had important cytotoxic effects in both cancer cell lines with an IC50 < 15 μg/mL and also can inhibit the production of peroxyl radicals. Interestingly, this fraction increased the recalcification time of human blood, showing anticoagulant properties. The present study indicates the sphingosines fraction as a promising source of chemical prototypes, especially multifunctional drugs in cancer therapy.

Potential use of secondary products of the agri-food industry for topical formulations and comparative analysis of antioxidant activity of grape leaf polyphenols

Abstract The aim of the present study was to develop a phytocosmetic using Vitis waste by-products, for use as a topical formulation for skin protection against ultraviolet radiation damage. The study also evaluates the free radical scavenger activity of the crude extracts of dried leaves of Vitis vinifera and Vitis labrusca, as well as the anthocyanins, flavonoid fraction and isolated compounds. Next, release and permeation studies of hydrogels were performed using Franz-type diffusion cells. Flavonoid acted more intensively in TRAP and conjugated dienes antioxidant assays, whereas anthocyanins had higher antioxidant activity in hydroxyl and nitric oxide assay. Only quercetin-3-O-glucuronide (5) was released from hydrogels, and the flavonoid retention in porcine ear skin after eight hours of permeation was below of limit of quantification for this compound. The polyphenols present in Vitis are capable of absorbing UV and visible light, justifying their potential as sunscreens for the development of a phytocosmetic.