Anti-Thrombin, Anti-Adhesive, Anti-Migratory, and Anti-Proliferative Activities of Sulfated Galactans from the Tropical Green Seaweed, Udotea flabellum.

In this study, sulfated polysaccharide-rich extracts were isolated from 22 tropical seaweeds (4 red, 11 brown, and 7 green) found in northeastern Brazil, and evaluated for the role of anticoagulant agents. Fifteen of the extracts showed anticoagulant activity, including all the extracts from green seaweeds. Udotea flabellum (a green seaweed) extract was the most potent, requiring an amount of only 3 µg to double the plasma coagulation time in the activated partial thromboplastin time test. A similar result was obtained with 1 µg of heparin. Two sulfated homogalactans with anticoagulant activity, F-I (130 kDa) and F-II (75 kDa), were isolated from this extract using several bio-guided purification steps. Their anticoagulant activity, as well as properties related to antitumor activity (anti-proliferative, anti-adhesive, and anti-migratory), were accessed. Their anticoagulant activities were close to that of heparin. We found that F-I and F-II (0.5⁻10 µg/mL) were not able to directly inhibit thrombin. In the presence of anti-thrombin, F-I (0.5 µg/mL) was more effective than heparin (0.5 µg/mL) in inhibiting thrombin, while F-II showed similar effects as heparin. F-I and F-II also inhibited B16-F10 (murine melanoma cells) adhesion, migration, and proliferation on a fibronectin-coated surface, but not on laminin- or collagen I-coated surfaces. Except for the antiproliferative activity, the other effects of F-I and F-II were eliminated upon their desulfation (~50%), indicating that the degree of sulfation is not as important for F-I and F-II anti-proliferative activity as the sulfation position. Taken together, the results provide strong evidence for the potential utility of sulfated galactans from U. flabellum, making these compounds an interesting option for future investigations that aim to design new anticoagulant/antitumor agents.

A low-molecular-weight galactofucan from the seaweed, Spatoglossum schröederi, binds fibronectin and inhibits capillary-like tube formation in vitro.

A low-molecular-weight (LMW) heterofucan (designated fucan B) was obtained from the brown seaweed, Spatoglossum schröederi, and its activity as an inhibitor of capillary-like tube formation by endothelial cells (ECs) was analyzed. Chemical, infrared and electrophoretic analyses confirmed the identity of fucan B. In contrast to other LMW fucans, fucan B (0.012-0.1 mg/mL) inhibited ECs capillary-like tube formation in a concentration-dependent manner. In addition, fucan B (0.01-0.05 mg/mL) did not affect ECs proliferation. Fucan B also inhibited ECs migration on a fibronectin-coated surface, but not on laminin- or collagen-coated surfaces. Biotinylated fucan B was used as a probe to identify its localization. Confocal microscopy experiments revealed that biotinylated fucan did not bind to the cell surface, but rather only to fibronectin. Our findings suggest that fucan B inhibits ECs capillary-like tube formation and migration by binding directly to fibronectin and blocking fibronectin sites recognized by cell surface ligands. However, further studies are needed to evaluate the in vivo effects of fucan B.

Antibacterial, Antiproliferative, and Immunomodulatory Activity of Silver Nanoparticles Synthesized with Fucans from the Alga Dictyota mertensii.

In this study, we aimed to synthesize silver nanoparticles containing fucans from Dictyota mertensii (Martius) Kützing using an environmentally friendly method and to characterize their structure as well as antiproliferative, immunomodulatory, and antibacterial effects. Fucan-coated silver nanoparticles (FN) were characterized by Fourier-transform infrared analysis, dynamic light scattering, zeta potential, atomic force microscopy, energy dispersive X-ray spectroscopy, and inductively coupled plasma emission spectrometry. They were evaluated for their effect on cell viability, minimum inhibitory bactericidal concentration, and release of nitric oxide and cytokines. The FN were successfully synthesized using an environmentally friendly method. They were size-stable for 16 months, of a spherical shape, negative charge (-19.1 mV), and an average size of 103.3 ± 43 nm. They were able to inhibit the proliferation of the melanoma tumor cell line B16F10 (60%). In addition, they had immunomodulatory properties: they caused an up to 7000-fold increase in the release of nitric oxide and cytokines (IL-10; IL-6 and TNF-α) up to 7000 times. In addition, the FN showed inhibitory effect on Gram-positive and -negative bacteria, with MIC values of 50 µg/mL. Overall, the data showed that FN are nanoparticles with the potential to be used as antitumor, immunomodulatory, and antibacterial agents.

Efeito dos ácidos hialurônicos como condroprotetores em modelo experimental de osteoartrose / Effect of hyaluronic acids as chondroprotective in experimental model of osteoarthrosis

Objetivo: analisar os efeitos do ácido hialurônico de diferentes pesos moleculares em modelo experimental de osteoartrose em coelhos. Métodos: foram alojados de modo aleatório 44 coelhos da raça California, machos, em três grupos (PR, S e P) e submetidos a ressecção do ligamento cruzado anterior do joelho direito. Decorridas três semanas do procedimento cirúrgico iniciaram-se as três injeções intra-articulares semanais de ácido hialurônico nativo (Polireumin®)-PR, ácido hialurônico de cadeia ramificada (Synvisc®)-S e soro fisiológico 0,9%-P. Todos os animais foram sacrificados após 12 semanas do ato cirúrgico e os platôs tibiais dos joelhos infiltrados foram dissecados. Cortes histológicos da cartilagem das áreas de apoio com maior espessura dos platôs tibiais foram corados com Alcian Blue pH = 1,0, Alcian Blue pH = 2,5 e Azul de Toluidina para pesquisa da quantidade de proteoglicanos. A intensidade de coloração foi quantificada em um aparelho de microscopia ZeissImager Z2 Metasystems e analisada pelo software MetaferMsearch. A análise estatística consistiu no uso dos testes Kolmorov-Smirnov, análise de variância (Anova), t de Student e qui-quadrado. O nível de significância usado foi de 5%. Resultado: o efeito condroprotetor dos ácidos hialurônicos usados no estudo foi demonstrado quando comparado ao do grupo controle, porém, feita a comparação entre si, não houve diferença estatística quanto à condroproteção. Conclusão: os ácidos hialurônicos testados obtiveram efeito condroprotetor, sem diferença estatística com relação aos diferentes pesos moleculares. .

Objective: to analyze the effects of hyaluronic acid of different molecular weights in an experimental model of osteoarthritis in rabbits. Methods: forty-four male California rabbits were divided randomly into three groups and underwent resection of the anterior cruciate ligament in his right knee. After three weeks of the surgical procedure began three weekly intra-articular injections of hyaluronic acid native (Polireumin®)-PR, hyaluronic acid branched chain (Synvisc®)-S and 0.9% saline-P. All animals were sacrificed after twelve weeks of surgery and tibial plateau infiltrated the knees were dissected. Histological cartilage of the support areas of the tibial plateaus were stained with Alcian Blue pH 1.0, Alcian Blue pH = 2.5 and toluidine blue for research on the amount of proteoglycans. The intensity of staining was quantified on a Zeiss microscope apparatus Imager Z2 MetaSystems and analyzed by software MetaferMsearch. Results: the effect of chondroprotetor hyaluronic acids used in the study was confirmed when compared to the control group, but the comparison made between them, there was no statistically significant difference regarding chondroprotetion. Conclusion: the hyaluronic acids tested had chondroprotective effect, with no statistical difference with regard to the different molecular weights. .

Effect of hyaluronic acids as chondroprotective in experimental model of osteoarthrosis.

OBJECTIVE: to analyze the effects of hyaluronic acid of different molecular weights in an experimental model of osteoarthritis in rabbits. METHODS: forty-four male California rabbits were divided randomly into three groups and underwent resection of the anterior cruciate ligament in his right knee. After three weeks of the surgical procedure began three weekly intra-articular injections of hyaluronic acid native (Polireumin(®))-PR, hyaluronic acid branched chain (Synvisc(®))-S and 0.9% saline-P. All animals were sacrificed after twelve weeks of surgery and tibial plateau infiltrated the knees were dissected. Histological cartilage of the support areas of the tibial plateaus were stained with Alcian Blue pH 1.0, Alcian Blue pH = 2.5 and toluidine blue for research on the amount of proteoglycans. The intensity of staining was quantified on a Zeiss microscope apparatus Imager Z2 MetaSystems and analyzed by software MetaferMsearch. RESULTS: the effect of chondroprotetor hyaluronic acids used in the study was confirmed when compared to the control group, but the comparison made between them, there was no statistically significant difference regarding chondroprotetion. CONCLUSION: the hyaluronic acids tested had chondroprotective effect, with no statistical difference with regard to the different molecular weights.

OBJETIVO: analisar os efeitos do ácido hialurônico de diferentes pesos moleculares em modelo experimental de osteoartrose em coelhos. MÉTODOS: foram alojados de modo aleatório 44 coelhos da raça California, machos, em três grupos (PR, S e P) e submetidos a ressecção do ligamento cruzado anterior do joelho direito. Decorridas três semanas do procedimento cirúrgico iniciaram­se as três injeções intra­articulares semanais de ácido hialurônico nativo (Polireumin®)­PR, ácido hialurônico de cadeia ramificada (Synvisc®)­S e soro fisiológico 0,9%­P. Todos os animais foram sacrificados após 12 semanas do ato cirúrgico e os platôs tibiais dos joelhos infiltrados foram dissecados. Cortes histológicos da cartilagem das áreas de apoio com maior espessura dos platôs tibiais foram corados com Alcian Blue pH = 1,0, Alcian Blue Ph = 2,5 e Azul de Toluidina para pesquisa da quantidade de proteoglicanos. A intensidade de coloração foi quantificada em um aparelho de microscopia ZeissImager Z2 Metasystems e analisada pelo software MetaferMsearch. A análise estatística consistiu no uso dos testes Kolmorogov­Smirnov, análise de variância (Anova), t de Student e qui­quadrado. O nível de significância usado foi de 5%. RESULTADO: o efeito condroprotetor dos ácidos hialurônicos usados no estudo foi demonstrado quando comparado ao do grupo controle, porém, feita a comparação entre si, não houve diferença estatística quanto à condroproteção. CONCLUSÃO: os ácidos hialurônicos testados obtiveram efeito condroprotetor, sem diferença estatística com relação aos diferentes pesos moleculares.

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 α5ß1, 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.

Freshwater plants synthesize sulfated polysaccharides: heterogalactans from Water Hyacinth (Eicchornia crassipes).

Sulfated polysaccharides (SP) are found mainly in seaweeds and animals. To date, they have only been found in six plants and all inhabit saline environments. Furthermore, there are no reports of SP in freshwater or terrestrial plants. As such, this study investigated the presence of SP in freshwaters Eichhornia crassipes, Egeria densa, Egeria naja, Cabomba caroliniana, Hydrocotyle bonariensis and Nymphaea ampla. Chemical analysis identified sulfate in N. ampla, H. bonariensis and, more specifically, E. crassipes. In addition, chemical analysis, FT-IR spectroscopy, histological analysis, scanning electron microscopy (SEM) and energy-dispersive X-ray analysis (EDXA), as well as agarose gel electrophoresis detected SP in all parts of E. crassipes, primarily in the root (epidermis and vascular bundle). Galactose, glucose and arabinose are the main monosaccharides found in the sulfated polysaccharides from E. crassipes. In activated partial thromboplastin time (APTT) test, to evaluate the intrinsic coagulation pathway, SP from the root and rhizome prolonged the coagulation time to double the baseline value, with 0.1 mg/mL and 0.15 mg/mL, respectively. However, SP from the leaf and petiole showed no anticoagulant activity. Eichornia SP demonstrated promising anticoagulant potential and have been selected for further studies on bioguided fractionation; isolation and characterization of pure polysaccharides from this species. Additionally in vivo experiments are needed and are already underway.