Isolation, fractionation and anticoagulant activity of a sulfated galactan extracted from the green algae Penicillus capitatus / Isolamento, fracionamento e atividade anticoagulante da galactana sulfatada extraída da alga verde Penicillus capitatus

ABSTRACT: Marine algae are natural sources of macromolecules known as sulfated polysaccharides. This class of compounds has attracted the interest of Pharmaceutical Sciences due to its pharmacological anticoagulant, antiplatelet and antithrombotic properties. Therefore, this study evaluated the anticoagulant potential of sulfated polysaccharides extracted from the algae Penicillus capitatus. The extracted sulfated polysaccharides were purified, partially characterized and their anticoagulant activity was evaluated. The extraction process followed by ethanol precipitation resulted in five fractions. Among the analyzed fractions, F44 contained highest concentration of sulfated polysaccharides. After the purified fraction F23, F44 displayed in vitro anticoagulant activity in a time testing for activated partial thromboplastin time and prothrombin time. The preferential mechanism effect was based on interactions between thrombin and factor Xa. Additional studies on structure pharmacological are required to test the viability of the use of sulfated polysaccharides as therapeutic agents.

RESUMO: As algas marinhas são fontes naturais de macromoléculas conhecidas como polissacarídeos sulfatados. Esta classe de compostos atraiu o interesse das Ciências Farmacêuticas devido às suas propriedades farmacológicas como anticoagulante, antiplaquetária e antitrombótica. Portanto, este estudo tem como objetivo avaliar o potencial anticoagulante de polissacarídeos sulfatados extraídos de algas de Penicillus capitatus. Os polissacarídeos sulfatados extraídos foram purificados, parcialmente caracterizados e sua atividade anticoagulante foi avaliada. O processo de extração seguido pela precipitação com etanol resultou em cinco frações. Entre as frações analisadas, F44 foi a maior concentração de polissacarídeos sulfatados. Após a purificação, as frações F23 e F44 mostraram atividade anticoagulante in vitro em um teste de tempo de tromboplastina parcialmente ativada e tempo de protrombina. Seu mecanismo preferencial é baseado nas interações entre trombina e fator Xa. Estudos adicionais sobre a estrutura farmacológica são necessários para testar a viabilidade do uso como agente terapêutico.

Interventional effects of prunella vulgaris sulfated polysaccharide on CCl4-induced hepatic fibrosis in rats / 天津医药

Objective To investigate the intervention effects of prunella vulgaris sulfated polysaccharide (PVSP) on carbon tetrachaloride(CCl4)-induced hepatic fibrosis in rat.Methods The 40% CCl4was used to induce hepatic fibrosis in rat model, then successful model rats were randomly divided into 3 groups, with 10 rats in each group, respectively, the model group(Model),the high dose PVSP group(PVSP-H:400 mg/kg)and the low dose PVSP group(PVSP-L:100 mg/kg). The blank group and solvent group were also established.The serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were determined by automatic biochemical analyzer.HE staining and Sirius red staining were used to examine the degree of hepatic fibrosis.The expression levels of collagen typeⅠ(Col-Ⅰ)and α-smooth muscle actin(α-SMA)mRNA were detected by qRT-PCR.Col-Ⅰand α-SMA in hepatic tissues were detected by immunohistochemistry staining.Results There were no significant changes in serum expressions of ALT and AST and mRNA proteins of Col-Ⅰand α-SMA in liver tissues between the blank group and Solvent group.Compared with the model group,the serum levels of ALT and AST were significantly decreased in the PVSP-H and PVSP-L groups (P<0.05). HE staining and Sirius red staining showed that PVSP could significantly reduce the degree of hepatic fibrosis.The expression of Col-Ⅰand α-SMA mRNA were decreased in the PVSP-H and PVSP-L groups(P<0.05).Immunohistochemistry showed that the expressions of Col-Ⅰ and α-SMA in hepatic tissues were decreased by PVSP (P<0.05), and the effect was dose-dependent. Conclusion PVSP has a protective effect on CCl4-induced hepatic fibrosis in rats, which may be related with the inhibiting expressions of Col-Ⅰand α-SMA,reducing secretion of collagen and promoting extracellular matrix degradation.

Apoptotic Mechanisms of Human Liver Cancer HepG-2 Cells Induced by Undaria pinnatifida Sulfated Polysaccharides / 中国药学杂志

OBJECTIVE: To investigate the mechanism of human liver cancer HepG-2 cells apoptosis induced by Undaria pinnatifida sulfated polysaccharides S-UPPSIB. METHODS: The anti-proliferation effects of S-UPPSIB on HepG-2 cells was by MTT assay. [Ca2+]i in HepG-2 cells was detected by laser cofocal scaning microscopy (LCSM). The apoptosis rate and protein expression level of Bcl-2, Bax, Cyt-C and p53 were detected by flow cytometry (FCM). Caspase Assay Kit was used to detected the activities of Caspase-3 and -9. RESULTS: S-UPPSIB could inhibit the proliferation of HepG-2 cells and the IC50 was 50.09 μgmL-1. With the increase of drug delivery dosage, apoptosis rate also increased, and the significant regulating effects of S-UPPSIB on Ca2+ and its associated channel proteins were observed. The [Ca2+]i level, the protein expression level of Cyt-c, p53 and the activities of Caspase-3 and -9 were all increased remarkably (P<0.05). The ratio of Bcl-2 to Bax was reduced. CONCLUSION: Undaria pinnatifida sulfated polysaccharides S-UPPSIB can effectively inhibit the proliferation of human liver cancer HepG-2 cells by inducing apoptosis of HepG-2 cells through mitochondrial pathway.

Anti-inflammatory activity of a sulfated polysaccharide isolated from an enzymatic digest of brown seaweed Sargassum horneri in RAW 264.7 cells

BACKGROUND/OBJECTIVES: Sargassum horneri is an edible brown alga that grows in the subtidal zone as an annual species along the coasts of South Korea, China, and Japan. Recently, an extreme amount of S. horneri moved into the coasts of Jeju Island from the east coast of China, which made huge economic and environmental loss to the Jeju Island. Thus, utilization of this biomass becomes a big issue with the local authorities. Therefore, the present study was performed to evaluate the anti-inflammatory potential of crude polysaccharides (CPs) extracted from S. horneri China strain in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. MATERIALS/METHODS: CPs were precipitated from S. horneri digests prepared by enzyme assistant extraction using four food-grade enzymes (AMG, Celluclast, Viscozyme, and Alcalase). The production levels of nitric oxide (NO) and pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-1β were measured by Griess assay and enzyme-linked immunosorbent assay, respectively. The levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPKs) were measured by using western blot. The IR spectrums of the CPs were recorded using a fourier transform infrared spectroscopy (FT-IR) spectrometer. RESULTS: The polysaccharides from the Celluclast enzyme digest (CCP) showed the highest inhibition of NO production in LPS-stimulated RAW 264.7 cells (IC₅₀ value: 95.7 µg/mL). Also, CCP dose-dependently down-regulated the protein expression levels of iNOS and COX-2 as well as the production of inflammatory cytokines, including TNF-α and IL-1β, compared to the only LPS-treated cells. In addition, CCP inhibited the activation of NF-κB p50 and p65 and the phosphorylation of MAPKs, including p38 and extracellular signal-regulated kinase, in LPS-stimulated RAW 264.7 cells. Furthermore, FT-IR analysis showed that the FT-IR spectrum of CCP is similar to that of commercial fucoidan. CONCLUSIONS: Our results suggest that CCP has anti-inflammatory activities and is a potential candidate for the formulation of a functional food ingredient or/and drug to treat inflammatory diseases.

Sperm and Egg Jelly Coat from Sea Urchin Lytechinus variegatus Collected in Rio de Janeiro Contain Distinct Sialic Acid-Rich Polysaccharides

This work found the occurrence of a distinct sialic acid-rich polysaccharide in the sperm surface of the sea urchin Lytechinus variegatus, which differed significantly from a similar molecule found in the egg jelly. The sperm polysaccharide extracted by protease digestion was purified using anion exchange chromatography and characterized using agarose gel electrophoresis, gas chromatography/mass spectrometry and NMR spectroscopy. This polysaccharide was highly sulfated and composed almost exclusively of N-acetylneuraminic acid. In contrast, the sialic acid-rich polysaccharide from the egg jelly was composed of N-glycolylneuraminic acid and contains several other hexoses in its structure. This new molecule could help to characterize in further detail the mechanism of fertilization in the sea urchin model system. Sulfated polysaccharides from the jelly coat of sea urchins showed species-specificity in inducing the sperm acrosome reaction, providing an example of a signal transduction event regulated by the sulfated polysaccharide. The new sialic acid-rich polysaccharide found in the sperm head could represent a new molecule involved in the biology of the sea urchin fertilization.

Influences of sulfated polysaccharide from pine (Pinus massoniana) pollen on the regulation of [ Ca2+] i in T lymphocytes / 中国药理学通报

Aim Tostudytheinfluencesofsulfated polysaccharides ( SPPM60-D) on the regulation of free calcium concentration ( [ Ca2+] i ) of T lymphocytes of mice in vitro and explore the mechanisms involved. Methods Polysaccharides(PPM60)wereextracted from masson pine pollen with hot water and 60% etha-nol. PPM60-D was separated and purified from PPM60 with Sephacryl S-400HR. Sulfated polysaccharides ( SPPM60-D ) were derivated by chlorosulfonic acid-pyridine method and the [ Ca2+] i of T lymphocytes were measured by fluorescence spectrophotometer. IL-2 and IL-4 were measured by ELISA kits. Results ConAandSPPM60-Dcouldincrease[Ca2+]iinT lymphocytes by 211. 5% and 201. 8% respectively ( P<0. 01). 2-APB, LY294002, U73122 and verapamil rather than TAK-242 could significantly inhibit the in-crease of [ Ca2+] i induced by SPPM60-D. SPPM60-D could significantly increase the level of IL-2 and IL-4 in supernatant ( P <0. 01 ) . 2-APB rather than TAK-242 could significantly inhibit the increase of cyto-kines.Conclusion ItisspeculatedthatSPPM60-D could increase [ Ca2+ ] i via TCR/CD3-PI3K-PLC-IP3 R-Ca2+ signal pathway through TCD/CD3 receptor in T lymphocytes so that it could improve the level of IL-2 and IL-4 in supernatant in T lymphocytes.

Effect and mechanism of Prunella vulgaris sulfated polysaccharide on angiogenesis in hepatocellular carcinoma / 中国肿瘤临床

Objective:A study was conducted to investigate the effect of Prunella vulgaris sulfated polysaccharide (PVSP) on the expression of angiogenic growth factors (bFGF, VEGF, and IL-8) and angiogenesis in hepatocellular carcinoma. Methods:ELISA as-say was used to observe the effects of PVSP and the negative control drug Lentinan (LNT, non-sulfate radical drug) on secretions of the angiogenic growth factors, namely, bFGF, VEGF, and IL-8, in HepG2 cells in vitro. In an in vivo experiment, the microvessel density in hepatocellular carcinoma tissue sections treated with PVSP and LNT was calculated, analyzed, and compared with the microvessel den-sity in the phosphate-buffered saline (PBS) control. Results:Compared with the PBS control group, PVSP at 200μg/mL inhibited bF-GF secretion (P<0.01), whereas LNT failed to affect bFGF secretion. Neither PVSP nor LNT affected the secretions of VEGF and IL-8. In vivo results showed that PVSP at 200 mg/kg reduced the microvessel density in tumor tissue sections (P=0.03), whereas LNT at 10 mg/kg failed to affect microvessel density. Conclusion:Inhibition of bFGF secretion is a probable mechanism underlying the preven-tive effect of PVSP on hepatocellular carcinoma angiogenesis.

Role of the NO/K ATP pathway in the protective effect of a sulfated-polysaccharide fraction from the algae Hypnea musciformis against ethanol-induced gastric damage in mice

Seaweeds are the most abundant source of polysaccharides such as alginates and agar, as well as carrageenans. This study aimed to investigate the gastroprotective activity and the mechanism underlying this activity of a sulfated-polysaccharide fraction extracted from the algae Hypnea musciformis (Wulfen) J.V. Lamour. (Gigartinales-Rhodophyta). Mice were treated with sulfated-polysaccharide fraction (3, 10, 30, and 90 mg/kg, p.o.) and, after 30 min, they were administered 50% ethanol (0.5 mL/25 g, p.o.). After 1 h, gastric damage was measured using a planimeter. In addition, samples of the stomach tissue were obtained for histopathological examination and for assays to determine the glutathione and malondialdehyde levels. Other groups of mice were pretreated with N G-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.p.), aminoguanidine (100 mg/kg, i.p.), or glibenclamide (10 mg/kg, i.p.). After 30 min to the aminoguanidine group and 1 h to the other groups, sulfated-polysaccharide fraction (30 mg/kg, p.o.) was administered and gastric damage was induced as described above. Sulfated-polysaccharide fraction prevented ethanol-induced gastric injury in a dose-dependent manner. However, treatment with L-NAME or glibenclamide reversed this gastroprotective effect. Administration of aminoguanidine did not influence the effect of sulfated-polysaccharide fraction. Our results suggest that sulfated-polysaccharide fraction exerts a protective effect against ethanol-induced gastric damage via activation of the NO/K ATP pathway.

Radio-protective effect of sulfated polysaccharide purified from Ecklonia cava against small intestinal stem cells of gamma-ray irradiated mice / 동물의과학연구지

Our previous research on sulfated polysaccharide purified from Ecklonia cava, a brown alga found in Jeju island, Korea, showed that sulfated polysaccharides modulate the apoptotic threshold of intestinal cells, thereby preventing intestinal damage caused by ionizing radiation. In this study, we investigated the ability of sulfated polysaccharide to augment restoration of small intestinal stem cells from gamma-ray-induced damage. In our results, sulfated polysaccharide treatment increased the numbers of Ki-67-positive cells as well as inducible nitric oxide synthase (iNOS)-expressing cells in the small intestine compared with those of irradiated only mice. Meanwhile, exposure to irradiation increased the number of paneth cells, which are frequently associated with intestinal inflammation, whereas sulfated polysaccharide treatment reduced the number of paneth cells in the small intestinal crypt. Conclusively, our data suggest that reduction of iNOS-expressing cells and paneth cells in sulfated polysaccharide-treated mice contributes to the inhibition of radiation-induced intestinal inflammation.

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 schröederi, 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 rat's 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.

In Vitro Anti-influenza Virus Activities of Sulfated Polysaccharide Fractions from Gracilaria lemaneiformis / 中国病毒学·英文版

In this paper, in vitro anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were investigated. Cytotoxicities and antiviral activities of Gracilaria lemaneiformis polysaccharides (PGL), Gracilaria lemaneiformis polysaccharide fraction-1 (GL-1), Gracilaria lemaneiformis polysaccharide fraction-2 (GL-2) and Gracilaria lemaneiformis polysaccharide fraction-3 (GL-3) were studied by the Methyl thiazolyl tetrazolium (MTT) method, and the inhibitory effect against Human influenza virus H1-364 induced cytopathic effect (CPE) on MDCK cells were observed by the CPE method. In addition, the antiviral mechanism of PGL was explored by Plaque forming unit (PFU), MTT and CPE methods. The results showed: i) Cytotoxicities were not significantly revealed, and H1-364 induced CPE was also reduced treated with sulfated polysaccharide fractions from Gracilaria lemaneiformis; ii) Antiviral activities were associated with the mass percentage content of sulfate groups in polysaccharide fractions, which was about 13%, in polysaccharides (PGL and GL-2) both of which exhibited higher antiviral activity; iii) A potential antiviral mechanism to explain these observations is that viral adsorption and replication on host cells were inhibited by sulfated polysaccharides from Gracilaria lemaneiformis. In conclusion, Anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were revealed, and the antiviral activities were associated with content of sulfate groups in polysaccharide fractions.

Antiviral activity and its antiviral mechanism of a sulfated polysaccharide from Sargassum patens against respiratory syncytial virus (RSV) / 中国海洋药物

Objective To detect the antiviral effect and its antiviral mechanism of the sulfated polysaccharide SP2 isolated from the brown alga Sargassum patens C. Ag. against RSV.Methods The antiviral activity,cytotoxicity and its possible antiviral mechanism were assayed by cytopathogenic effect (CPE) inhibition assay,MTT assay and plaque reduction assay. Reduction assay when it was added during virus adsorption. It did not show any antiviral activity against influenza A and B viruses and parainfluenza type 3 virus up to the concentration of treatment of cells with SP2 did not protect the cells from RSV infection. This polysaccharide addition assay showed that when SP2 was present only during virus adsorption phase at 37℃, it could significantly inhibit RSV-induced plaque formation. Conclusions SP2 might be a potent substance for treating RSV infection. The mode of action of SP2 might depend on inhibiting RSV attachment to host cells.

Antioxidative activity of polysaccharide fraction from Por-phyra haitanensis on oxidative stress in mice irradiated by 60Co / 中国海洋药物

Sulfated polysaccharide from Porphyra haitanensis showed inhibitory effect on the lipid peroxidation in vitro. In the present study, the changes in the antioxidative enzyme activity, lipid peroxidation, and total antioxidative capacity (T-AOC) in different organs after 60 Co irradiation were detected in mice by using biochemical methods. Increased endogenous lipid peroxidation and decreased contents of T-AOC as well as decreased activities of super-oxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were observed in mice after 60Co irradiation. Intraperitoneal administration of polysaccharide fraction F2 significantly decreased the lipid peroxidation. F2 treatment increased T-AOC and the activities of SOD and GSH-Px in all organs tested in 60Co irradiated mice. It is concluded that the sulfated polysaccharide fraction F2 from Porphyra haitanensis can be used in compensating the decline in antioxidative capacity arising from radiotherapy and thereby reduced the risks of lipid peroxidation.

The antihypertensive effect of marine sulfated polysaccharide AHD and its related mechanisms of action / 中国海洋药物

The antihypertensive effect of marine sulfated polysaccharide AHD and its underlying mechanisms were studied in renovascular hypertensive rats (RHR) [two-kidney one clip,Goldblatt(2-K 1C)J. AHD were given orally at doses of 12.5, 25,50mg?kg～-1 once a day for five weeks,simultaneously wity the initiation of the establishment of renovascular hypertensive model. Serum nitric oxide(NO) was determined with NO kit,and plasma Angiotensin E (Ang Ⅱ ) and Eneothelin (ET) Were measured by radioimmumoassays. The results indicated that AHD decreased arteial blood pressure in a dose-dependent manner. Moreover, nitric oxide (NO) contents in serum were dose-dependently elevated,accompanied by dramatic reduction in Ang Ⅱ and ET contents. These findings suggested that AHD exerted its hypotensive activity by enhancing tye release of NO,followed by reduction in production of boty ANG Ⅱ and ET in vivo.

Bioactive substances of Porphyridium and their applications / 中国海洋药物

The unicellular red alga, Porphyridium , can be artificially cultivated. Some of very useful bioactive substances such as phycobiliproteins? polyunsaturated fatty acids (PUFA) and sulfated polysaccharides, which are synthesize by Porphyridium , have promising applications. This paper deals with the study of the character, production and application of Porphyridium.

The effect of sulfated polysaccharides from brown seaweeds GS201 on neuronal survival in embryonic wistar rats / 中国海洋药物

The effects of sulfated polysaccharides from brown seaweeds GS201 on neuronal survival of cultured brain neurons were investigated in this paper.Results indicated that GS201 at concentrations of 0.01 0.1 1 10 mg?L -1 significantly enhanced the neuronal survival of both hippocampus and neurocortex.The mechanisms underlying the neurotrophic effect exerted by GS201 need to be further elucidated.

The relationships between senile plaques, neurofibrillary tangles and sulfated polysaccharides / 中国药理学通报

Two aberrant structures, extracellular senile plaques (SP) and intracellular neurofibrillary tangles (NFTs) are the characteristic neuropathological hallmarks of Alzheimers disease (AD). Amyloid ? protein (A?) and hyperphosphorylated tau protein are the major components of SP and NFTs respectively. A large body of evidence has highlighted the pivotal role of sulfated polysaccharides in the amyloidogenesis and formation of NFTs. The underlying mechanisms of the involvement of sulfated polysaccharides in the development of AD were reported to contribute to their high affinity for both A? and tau protein. Sulfated polysaccharides not only promoted the ? secretase cleavage of APP and the increased production of A? and induced the aggregation and deposition of A?, but also facilitated the phosphylation of tau and promoted tau polymerization into fibrils and tangle formation. On the other hand, the neurotrophic effects exerted by sulfated polysaccharides were also demonstrated. These notions were probably due to the inhibition of the formation of A? fibrils or to the counteraction of the abnormal phosphorylation of tau by promoting the protein phosphatase2B activity, which has been speculated to be attributed to the variation in either structural backbone or degree of sulfation or position of sulfation. Putting together, the appropriate structural modification of sulfated polysaccharides may be effective as therapeutic agents for AD.

The relationship between sulfated polysaccharides and atherosclerosis / 中国药理学通报

The atherogensis was involved in a complex pathological process. Injury to endothelial cells of blood vessels was confirmed to be the initial stage of this process. Migration to subendothelial layer and accumulation and proliferation of smooth muscle cells were attributed to various cytokines and adhesive molecules secreted by activated endothelial cells, subsequently resulting in aggregation of lymphocytes, platelets, monocytes and macrophages in the intima of artery. These cellular components ultimatedly led to the formation of a mature atherosclerotic plaque. It's quite acknowledged that a better understanding of the atherogenic events might promise us the development of new chemical entities of anti-atherosclerotic therapies. A large body of evidence has demonstrated that sulfated polysaccharides played a critical role in the development of atheroscle- rosis. The underlying mechanisms of the antiatherosclerotic activity of sulfated polysaccharides were reported to contribute to protecting against endothelial cells injury, inhibiting migration and proliferation of vascular smooth muscle cells, and reducing the adhesion of inflammatory Cells, platelets and lymphocytes. And also, the prevention of complement activation by sulfated polysaccharides could not be excluded. On the other hand, the promoting effects of sulfated polysaccharides atherosclerosis was also reported. It's therefore concluded that the relationships between atherioscierosis and sulfated polysaccharides remained to be further elucidated.