Jailma Almeida-Lima

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.

BC nanofibres: In vitro study of genotoxicity and cell proliferation

Nanomaterials have unusual properties not found in the bulk materials, which can be exploited in numerous applications such as biosensing, electronics, scaffolds for tissue engineering, diagnostics and drug delivery. However, research in the past few years has turned up a range of potential health hazards, which has given birth to the new discipline of nanotoxicology. Bacterial cellulose (BC) is a promising material for biomedical applications, namely due its biocompatibility. Although BC has been shown not to be cytotoxic or genotoxic, the properties of isolated BC nanofibres (NFs) on cells and tissues has never been analysed. Considering the toxicity associated to other fibre-shaped nanoparticles, it seems crucial to evaluate the toxicity associated to the BC-NFs. In this work, nanofibres were produced from bacterial cellulose by a combination of acid and ultrasonic treatment. The genotoxicity of nanofibres from bacterial cellulose was analysed in vitro, using techniques previously demonstrated to detect the genotoxicity of fibrous nanoparticles. The results from single cell gel electrophoresis (also known as comet assay) and the Salmonella reversion assays showed that NFs are not genotoxicity under the conditions tested. A proliferation assay using fibroblasts and CHO cells reveals a slight reduction in the proliferation rate, although no modification in the cell morphology is observed.

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.

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.

Evaluating the possible genotoxic, mutagenic and tumor cell proliferation-inhibition effects of a non-anticoagulant, but antithrombotic algal heterofucan

Fucan is a term used to denominate a family of sulfated polysaccharides rich in L‐fucose. They are extracted mainly from brown seaweeds and echinoderms. The brown seaweed Spatoglossum schröederi (Dictyotaceae) synthesizes three heterofucans named A, B and C. Our research group purified a non‐anticoagulant heterofucan (fucan A) which displays antithrombotic activity in vivo. However, its in vitro toxicity has yet to be determined. This work presents the evaluation of the potential cytotoxicity, mutagenicity and genotoxicity of this fucan. After 48 h incubation fucan A cytotoxicity was determinate using MTT assay. Tumor‐cell (HeLa, PC3, PANC, HL60) proliferation was inhibited 2.0–43.7%; at 0.05–1 mg ml−1 of the heterofucan, the 3T3 non‐tumor cell line proliferation was also inhibited (3.3–22.0%). On the other hand, the CHO tumorigenic and RAW non‐tumor cell lines proliferation were not affected by this molecule (0.05–1 mg ml−1). We observed no mutagenic activity in Salmonella reversion assay when bacterial strains TA97a, TA98, TA100 and TA102 (with and without S9) were used. Comet assay showed that fucan A had no genotoxic effect (from 20 to 1000 µg ml−1) on CHO cells. In conclusion, this study indicates that the S. schröederi fucan A was not found to be genotoxic or mutagenic compound; thus it could be used in new antithrombotic drug development. Copyright

Evaluation of sulfated polysaccharides from the brown seaweed Dictyopteris justii as antioxidant agents and as inhibitors of the formation of calcium oxalate crystals.

Oxalate crystals and other types of crystals are the cause of urolithiasis, and these are related to oxidative stress. The search for new compounds with antioxidant qualities and inhibitors of these crystal formations is therefore necessary. In this study, we extracted four sulfated polysaccharides, a fucoglucoxyloglucuronan (DJ-0.3v), a heterofucan (DJ-0.4v), and two glucans (DJ-0.5v and DJ-1.2v), from the marine alga Dictyopteris justii. The presence of sulfated polysaccharides was confirmed by chemical analysis and FT-IR. All the sulfated polysaccharides presented antioxidant activity under different conditions in some of the in vitro tests and inhibited the formation of calcium oxalate crystals. Fucan DJ-0.4v was the polysaccharide that showed the best antioxidant activity and was one of the best inhibitors of the crystallization of calcium oxalate. Glucan DJ-0.5v was the second most potent inhibitor of the formation of oxalate crystals, as it stabilized dehydrated oxalate crystals (less aggressive form), preventing them from transforming into monohydrate crystals (more aggressive form). The obtained data lead us to propose that these sulfated polysaccharides are promising agents for use in the treatment of urolithiasis.

Evaluating the possible anticoagulant and antioxidant effects of sulfated polysaccharides from the tropical green alga Caulerpa cupressoides var. flabellata

Seaweeds are a source of several biopolymers widely used in cosmetics, food, and pharmaceuticals. Among them are sulfated polysaccharides, which have several biological/pharmacological activities, such as antioxidant and anticoagulant activities. In the present study, four sulfated polysaccharides, denominated CCB-F0.3, CCB-F0.5, CCB-F1.0, and CCB-F2.0, were obtained from the chlorophyte Caulerpa cupressoides var. flabellata through proteolytic digestion, followed by acetone fractionation and molecular sieving in Sephadex G-100. Chemical analyses showed that CCB-F0.5 had the highest sulfate/sugar ratio (0.73), whereas CCB-F1.0 exhibited the lowest ratio (0.23). Polysaccharides from C. cupressoides displayed a heterogeneous constitution of monosaccharides, with galactose as the main sugar unit (except for CCB-F2.0). The presence of sulfated polysaccharides was confirmed by electrophoretic and infrared analyses. Sulfated polysaccharides showed no activity in superoxide and hydroxyl radical scavenging; however, they did demonstrate total antioxidant capacity and ferrous chelating activity. Caulerpa polysaccharides also exhibited anticoagulant activity in the intrinsic (activated partial thromboplastin time (aPTT) test) and extrinsic pathway (prothrombin time (PT) test). In the aPTT test, all polysaccharides displayed considerable dose-dependent activity. A significant result was the aPTT activity of the polysaccharides CCB-F0.3 and CCB-F0.5, which was similar to that of Clexane®, a commercial low molecular weight heparin. In addition, CCB-F0.3 and CCB-F0.5 showed PT activity. Sulfated polysaccharides from C. cupressoides are therefore promising antioxidant agents in preventing the formation of reactive oxygen species and for their possible use in anticoagulant therapy.

Evaluation of acute and subchronic toxicity of a non-anticoagulant, but antithrombotic algal heterofucan from the Spatoglossum schröederi in Wistar rats

Fucan is a term used to denominate a family of sulfated polysaccharides rich in L-fucose. The brown alga Spatoglossum schroederi, Dictyotaceae, synthesizes three heterofucans named A, B, and C. Fucan A is a non-anticoagulant heterofucan which possesses potent antithrombotic (in vivo) and antiproliferative (in vitro) activities. However, its toxicity in vivo has not been determined. The present study examined the acute and subchronic toxicity of the fucan A in Wistar rats after subcutaneous administration. After that, the animals were killed and examined. The results showed in the acute study that fucan A did not cause general adverse effects and mortality in the concentrations 0, 20, 100, 1000, and 2000 µg/g body weight per rat for seven days. Regarding the subchronic study, the data showed that the fucan A did not cause any change in hematological and biochemistry parameters, as well as in the morphology, and in the size of the rats organs analyzed at a concentration of 20 µg/g body weight per rat during a 62-day period. In conclusion, this study indicates this heterofucan is a compound with potential pharmacological value that has no toxicity in vivo.

Methanolic Extracts from Brown Seaweeds Dictyota cilliolata and Dictyota menstrualis Induce Apoptosis in Human Cervical Adenocarcinoma HeLa Cells

Carcinoma of the uterine cervix is the second most common female tumor worldwide, surpassed only by breast cancer. Natural products from seaweeds evidencing apoptotic activity have attracted a great deal of attention as new leads for alternative and complementary preventive or therapeutic anticancer agents. Here, methanol extracts from 13 species of tropical seaweeds (Rhodophytas, Phaeophyta and Chlorophyta) collected from the Northeast of Brazil were assessed as apoptosis-inducing agents on human cervical adenocarcinoma (HeLa). All extracts showed different levels of cytotoxicity against HeLa cells; the most potent were obtained from the brown alga Dictyota cilliolata (MEDC) and Dictyota menstrualis (MEDM). In addition, MEDC and MEDM also inhibits SiHa (cervix carcinoma) cell proliferation. Studies with these two extracts using flow cytometry and fluorescence microscopy showed that HeLa cells exposed to MEDM and MEDC exhibit morphological and biochemical changes that characterize apoptosis as shown by loss of cell viability, chromatin condensation, phosphatidylserine externalization, and sub-G1 cell cycle phase accumulation, also MEDC induces cell cycle arrest in cell cycle phase S. Moreover, the activation of caspases 3 and 9 by these extracts suggests a mitochondria-dependent apoptosis route. However, other routes cannot be ruled out. Together, these results point out the methanol extracts of the brown algae D. mentrualis and D. cilliolata as potential sources of molecules with antitumor activity.

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.