Ruth Medeiros Oliveira

Biological activities of sulfated polysaccharides from tropical seaweeds

Sulfated polysaccharides from 11 species of tropical marine algae (one edible specie of Rhodophyta, six species of Phaeophyta and four species of Chlorophyta) collected from Natal city coast (Northeast of Brazil) were evaluated for their anticoagulant, antioxidant and antiproliverative in vitro activities. In the activated partial thromboplastin time (APTT) test, which evaluates the intrinsic coagulation pathway, seven seaweeds presented anticoagulant activity. Dictyota cervicornis showed the highest activity, prolonging the coagulation time to double the baseline value in the APTT with only 0.01 mg/100 microl of plasma, 1.4-fold lesser than Clexane, a low molecular weight heparin. In the protrombin time (PT) test, which evaluates the extrinsic coagulation pathway, only Caulerpa cupresoides showed anticoagulant activity. All species collected showed antioxidant activities. This screening emphasized the great antioxidant potential (total capacity antioxidant, power reducing and ferrous chelating) of four species: C. sertularioide; Dictyota cervicornis; Sargassum filipendula and Dictyopteris delicatula. After 72 h incubation, HeLa cell proliferation was inhibited (p<0.05) between 33.0 and 67.5% by S. filipendula; 31.4 and 65.7% by D. delicatula; 36.3 and 58.4% by Caulerpa prolifera and 40.2 and 61.0% by Dictyota menstrualis at 0.01-2mg/mL algal polysaccharides. The antiproliferative efficacy of these algal polysaccharides were positively correlated with the sulfate content (r=0.934). Several polysaccharides demonstrated promising antioxidant, antiproliferative an/or anticoagulant potential and have been selected for further studies on bioguided fractionation, isolation and characterization of pure polysaccharides from these species as well as in vivo experiments are needed and are already in progress.

Antioxidant and Antiproliferative Activities of Heterofucans from the Seaweed Sargassum filipendula

Fucan is a term used to denominate a type of polysaccharide which contains substantial percentages of l-fucose and sulfate ester groups. We obtained five heterofucans from Sargassum filipendula by proteolytic digestion followed by sequential acetone precipitation. These heterofucans are composed mainly of fucose, glucose, glucuronic acid, galactose and sulfate. These fucans did not show anticoagulant activity in PT and aPTT tests. Their antioxidant activity was evaluated using the follow tests; total antioxidant capacity, scavenging hydroxyl and superoxide radicals, reducing power and ferrous ion [Fe(II)] chelating. All heterofucans displayed considerable activity, especially SF-1.0v which showed the most significant antioxidant potential with 90.7 ascorbic acid equivalents in a total antioxidant capacity test and similar activity when compared with vitamin C in a reducing power assay. The fucan antiproliferative activity was performed with HeLa, PC3 and HepG2 cells using MTT test. In all tested conditions the heterofucans exhibited a dose-dependent effect. The strongest inhibition was observed in HeLa cells, where SF-1.0 and SF-1.5 exhibited considerable activity with an IC50 value of 15.69 and 13.83 μM, respectively. These results clearly indicate the beneficial effect of S. filipendula polysaccharides as antiproliferative and antioxidant. Further purification steps and additional studies on structural features as well as in vivo experiments are needed to test the viability of their use as therapeutic agents.

Anticoagulant, Antioxidant and Antitumor Activities of Heterofucans from the Seaweed Dictyopteris delicatula

In the present study, six families of sulfated polysaccharides were obtained from seaweed Dictyopteris delicatula by proteolytic digestion, followed by acetone fractionation and molecular sieving on Sephadex G-100. Chemical analyses demonstrated that all polysaccharides contain heterofucans composed mainly of fucose, xylose, glucose, galactose, uronic acid, and sulfate. The fucans F0.5v and F0.7v at 1.0 mg/mL showed high ferric chelating activity (∼45%), whereas fucans F1.3v (0.5 mg/mL) showed considerable reducing power, about 53.2% of the activity of vitamin C. The fucan F1.5v presented the most prominent anticoagulant activity. The best antiproliferative activity was found with fucans F1.3v and F0.7v. However, F1.3v activity was much higher than F0.7v inhibiting almost 100% of HeLa cell proliferation. These fucans have been selected for further studies on structural characterization as well as in vivo experiments, which are already in progress.

A Lactose-Binding Lectin from the Marine Sponge Cinachyrella Apion (Cal) Induces Cell Death in Human Cervical Adenocarcinoma Cells

Cancer represents a set of more than 100 diseases, including malignant tumors from different locations. Strategies inducing differentiation have had limited success in the treatment of established cancers. Marine sponges are a biological reservoir of bioactive molecules, especially lectins. Several animal and plant lectins were purified with antitumor activity, mitogenic, anti-inflammatory and antiviral, but there are few reports in the literature describing the mechanism of action of lectins purified from marine sponges to induce apoptosis in human tumor cells. In this work, a lectin purified from the marine sponge Cinachyrella apion (CaL) was evaluated with respect to its hemolytic, cytotoxic and antiproliferative properties, besides the ability to induce cell death in tumor cells. The antiproliferative activity of CaL was tested against HeLa, PC3 and 3T3 cell lines, with highest growth inhibition for HeLa, reducing cell growth at a dose dependent manner (0.5–10 µg/mL). Hemolytic activity and toxicity against peripheral blood cells were tested using the concentration of IC50 (10 µg/mL) for both trials and twice the IC50 for analysis in flow cytometry, indicating that CaL is not toxic to these cells. To assess the mechanism of cell death caused by CaL in HeLa cells, we performed flow cytometry and western blotting. Results showed that lectin probably induces cell death by apoptosis activation by pro-apoptotic protein Bax, promoting mitochondrial membrane permeabilization, cell cycle arrest in S phase and acting as both dependent and/or independent of caspases pathway. These results indicate the potential of CaL in studies of medicine for treating cancer.

Heterofucan from Sargassum filipendula Induces Apoptosis in HeLa Cells

Fucan is a term used to denominate a family of sulfated polysaccharides rich in sulfated l-fucose. Heterofucan SF-1.5v was extracted from the brown seaweed Sargassum filipendula by proteolytic digestion followed by sequential acetone precipitation. This fucan showed antiproliferative activity on Hela cells and induced apoptosis. However, SF-1.5v was not able to activate caspases. Moreover, SF-1.5v induced glycogen synthase kinase (GSK) activation, but this protein is not involved in the heterofucan SF-1.5v induced apoptosis mechanism. In addition, ERK, p38, p53, pAKT and NFκB were not affected by the presence of SF-1.5v. We determined that SF-1.5v induces apoptosis in HeLa mainly by mitochondrial release of apoptosis-inducing factor (AIF) into cytosol. In addition, SF-1.5v decreases the expression of anti-apoptotic protein Bcl-2 and increased expression of apoptogenic protein Bax. These results are significant in that they provide a mechanistic framework for further exploring the use of SF-1.5v as a novel chemotherapeutics against human cervical cancer.

Chitooligosaccharides antagonize the cytotoxic effect of glucosamine

Chitooligosaccharides (COS) are partially hydrolyzed compounds derived from chitosan that exhibit a number of biological activities, including antitumor, antibacterial and antifungal properties. In this work, we examined the cytotoxicity of pure COS and oligomers A, B and C (solutions composed of different amounts of COS) produced by enzymatic hydrolysis using a crude enzyme extract produced by the fungus Metarhrizium anisopliae. The antiproliferative effect of these molecules was analyzed using tumor cell lines (HepG2 and HeLa cells) and in a normal cell line (3T3). The antioxidant activity was analyzed in several in vitro experiments. Glucosamine showed higher toxicity (approximately 92%) to all cell lines studied. However, the oligomers obtained after hydrolysis demonstrated no toxic effects on the normal cells (3T3). Furthermore, we showed that a small amount of other COS can decrease the cytotoxic effect of glucosamine against 3T3 cells, indicating that glucosamine could be used as an antitumor drug in the presence of other COS. In addition, different effects were found in antiproliferative assays, which depended on the COS composition in the oligomers (A, B and C), showing that a combination of them may be essential for developing antineoplastic drugs. Superoxide anion scavenging was the main antioxidant activity demonstrated by the COS and oligomers. This activity was also dependent on the oligomer composition of the chitosan hydrolysates. Further work will identify the ideal proportions of COS and glucosamine for maximizing the effects of these biological activities.

Ipomoea asarifolia neutralizes inflammation induced by Tityus serrulatus scorpion venom

ETHNOPHARMACOLOGICAL RELEVANCE Envenoming caused by scorpion sting is a serious public health problem. In Brazil, 13,038 accidents caused by venomous animals have been reported. Of this total, 53% of the cases and 14 deaths were caused by scorpions. Furthermore, Tityus serrulatus (Buthidae) is the most dangerous scorpion due to the high toxicity of its venom. The treatment is the common supportive therapy and the serum therapy, but some people do not have access to both therapies and seek healing through the use of medical plants. AIM OF THE STUDY This study evaluated the ability of the crude extract and fractions from the leaves of Ipomoea asarifolia in neutralizing the main biological effects caused by Tityus serrulatus envenoming in mice. MATERIALS AND METHODS BALB/c mice were pretreated (i.v.) with 100 μλ of aqueous extracts and fractions dichloromethane, ethyl acetate, and n-butanol (CH₂Cl₂, EtOAc, and n-BuOH, respectively) of Ipomoea asarifolia, rutin or saline. Then, the animals received 100 μλ (i.p.) of venom of Tityus serrulatus (0.8 mg/kg). After six hours, the peritoneal lavage was performed with PBS and the number cells were determined using a Neubauer chamber. The supernatants were collected for determination of cytokines, such as IL-6, IL-12, and IL-1β. RESULTS The aqueous extract, fractions and rutin, at all doses, significantly reduced cell migration, which was endorsed by the reduction of the levels of certain cytokines. CONCLUSION This is the first study that demonstrated the potential effect of Ipomoea asarifolia against inflammation caused by Tityus serrulatus venom, suggesting that these extracts and/or their bioactive molecules, especially the flavonoid rutin, have potential use in the therapy of this envenomation.

Fucan effect on CHO cell proliferation and migration

Fucan is a term used to denominate sulfated L-fucose rich polysaccharides. Here, a heterofucan, named fucan B, was extracted from the Spatoglossum schröederi seaweed. This 21.5 kDa galactofucan inhibited CHO-K1 proliferation and migration when fibronectin was the substrate. Fucan B derivatives revealed that such effects depend on their degree of sulfation. Fucan B did not induce cell death, but promoted G1 cell cycle arrest. Western blotting and flow cytometry analysis suggest that fucan B binds to fibronectin and activates integrin, mainly integrin α5β1, which induces FAK/RAS/MEK/ERK activation. FAK activation inhibits CHO-K1 migration on fibronectin and ERK blocks cell cycle progression. This study indicates that fucan B could be applied in developing new antitumor drugs.

Baccharis trimera (Less.) DC Exhibits an Anti-Adipogenic Effect by Inhibiting the Expression of Proteins Involved in Adipocyte Differentiation

Baccharis trimera (Less.) DC (gorse) is a plant popularly used for the treatment of obesity. In this study, we prepared three B. trimera extracts aqueous extract (AE), decoction (AE-D), and methanol extract (ME) and investigated their antioxidant effects in six different tests and their anti-adipogenic effect in 3T3-L1 cells. The extracts showed a dose-dependent antioxidant activity in all tests. AE was the most potent antioxidant in copper and ferric ion chelation assays, whereas AE-D was the most potent in superoxide and hydroxyl radical scavenging assays, reducing power assay, and total antioxidant capacity analysis. Only ME showed a cytotoxic effect against 3T3-L1 cells. Lipid accumulation decreased in 3T3-L1 adipocytes in the presence of AE and AE-D extracts (0.5 to 1.0 mg/mL). In addition, the extracts dramatically attenuated the levels of adipogenic transcriptional factors, including CCAAT enhancer-binding protein α (C/EBPα), CCAAT enhancer-binding protein β (C/EBPβ), and gamma receptors by peroxisome proliferators (PPARγ), during adipogenesis. AE-D (1.0 mg/mL) caused an approximately 90% reduction in the levels of these molecules. We propose that B. trimera has an anti-adipogenic effect and could be used in the development of functional foods.

Antiproliferative Activity of Fucan Nanogel

Sulfated fucans comprise families of polydisperse natural polysaccharides based on sulfated L-fucose. Our aim was to investigate whether fucan nanogel induces cell-specific responses. To that end, a non toxic fucan extracted from Spatoglossum schröederi was chemically modified by grafting hexadecylamine to the polymer hydrophilic backbone. The resulting modified material (SNFuc) formed nanosized particles. The degree of substitution with hydrophobic chains was close to 100%, as estimated by elemental analysis. SNFfuc in aqueous media had a mean diameter of 123 nm and zeta potential of −38.3 ± 0.74 mV, as measured by dynamic light scattering. Nanoparticles conserved their size for up to 70 days. SNFuc cytotoxicity was determined using the MTT assay after culturing different cell lines for 24 h. Tumor-cell (HepG2, 786, H-S5) proliferation was inhibited by 2.0%–43.7% at nanogel concentrations of 0.05–0.5 mg/mL and rabbit aorta endothelial cells (RAEC) non-tumor cell line proliferation displayed inhibition of 8.0%–22.0%. On the other hand, nanogel improved Chinese hamster ovary (CHO) and monocyte macrophage cell (RAW) non-tumor cell line proliferation in the same concentration range. The antiproliferative effect against tumor cells was also confirmed using the BrdU test. Flow cytometric analysis revealed that the fucan nanogel inhibited 786 cell proliferation through caspase and caspase-independent mechanisms. In addition, SNFuc blocks 786 cell passages in the S and G2-M phases of the cell cycle.