Studies on Antiviral and Immuno-Regulation Activity of Low Molecular Weight Fucoidan from Laminaria japonica

The antiviral activity in vitro and in vivo and the effect of the immune system of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica(LMW fucoidans) were investigated in order to examine the possible mechanism. In vitro, I-type influenza virus, adenovirus and Parainfluenza virus I were used to infect Hep-2, Hela and MDCK cells, respectively. And 50% tissue culture infective dose was calculated to detect the antiviral activity of two LMW fucoidans. The results indicated that compared with the control group, 2 kinds of LMW fucoidans had remarkable antiviral activity in vitro in middle and high doses, while at low doses, the antiviral activity of 2 kinds of LMW fucoidans was not statistically different from that in the blank control group. And there was no statistically difference between two LMW fucoidans in antiviral activity. In vivo, LMW fucoidans could prolong the survival time of virus-infected mice, and could improve the lung index of virus-infected mice significantly, which have statistical differences with the control group significantly(p 0.01). However, the survival time of the two LMW fucoidans was not statistically significant(p 0.05). In this study, it was shown that both of two LMW fucoidans(LF1, LF2) could increase the thymus index, spleen index, phagocytic index, phagocytosis coefficient and half hemolysin value in middle and high doses, which suggested that LMW fucoidans could play an antiviral role by improving the quality of immune organs, improving immune cell phagocytosis and humoral immunity.     

Mechanism Study of Endothelial Protection and Inhibits Platelet Activation of Low Molecular Weight Fucoidan from Laminaria japonica

Several studies have indicated that fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica could inhibit the activation of platelets directly by reducing the platelet aggregation. To explore the direct effect of LMW fucoidan on the platelet system furthermore and examine the possible mechanism, the endothelial protection and inhibits platelet activation effects of two LMW fucoidan were investigated. In the present study, Endothelial injury model of rats was made by injection of adrenaline(0.4 mg kg-1) and human umbilical vein endothelial cells were cultured. v WF level was be investigated in vivo and in vitro as an important index of endothelial injury. LMW fucoidan could significantly reduce v WF level in vascular endothelial injury rats and also significantly reduce v WF level in vitro. The number of EMPs was be detected as another important index of endothelial injury. The results showed that LMW fucoidan reduced EMPs stimulated by tumor necrosis factor. In this study, it was found that by inhibiting platelet adhesion, LMW fucoidan played a role in anti-thrombosis and the specific mechanism of action is to inhibit the flow of extracellular Ca2+. All in a word, LMW fucoidan could inhibit the activation of platelets indirectly by reducing the concentration of EMPs and v WF, at the same time; LMW fucoidan inhibited the activation of platelets directly by inhibiting the flow of extracellular Ca2+.     

Ultrasonic Extraction of Polysaccharides from Laminaria japonica and Their Antioxidative and Glycosidase Inhibitory Activities

In the present study, ultrasonic extraction technique(UET) is used to improve the yield of polysaccharides from Laminaria japonica(LJPs). And their antioxidative as well as glycosidase inhibitory activities are investigated. Box-Behnken design(BBD) combined with response surface methodology(RSM) is applied to optimize ultrasonic extraction for polysaccharides. The optimized conditions are obtained as extraction time at 54 min, ultrasonic power at 1050 W, extraction temperature at 80℃ and ratio of material to solvent at 1:50(g m L-1). Under these optimal ultrasonic extraction conditions, an actual experimental yield(5.75% ± 0.3%) is close to the predicted result(5.67%) with no significant difference(P 0.05). Vitro antioxidative and glycosidase inhibitory activities tests indicate that the crude polysaccharides(LJP) and two major ethanol precipitated fractions(LJP1 and LJP2) are in a concentration-dependent manner. LJP2(30%-60% ethanol precipitated polysaccharides) possesses the strongest α-glucosidase inhibitory activity and moderate scavenging activity against hydroxyl radicals(66.09% ± 2.19%, 3.0 mg m L-1). Also, the inhibitory activity against α-glucosidase(59.08% ± 3.79%, 5.0 mg m L-1) is close to that of acarbose(63.99% ± 3.27%, 5.0 mg m L-1). LJP1(30% ethanol precipitated polysaccharides) exhibits the strongest scavenging activity against hydroxyl radicals(99.80% ± 0.00%, 3.0 mgm L-1) and moderate α-glucosidase inhibitory activity(47.76% ± 1.92%, 5.0 mg m L-1). LJP shows the most remarkable DPPH scavenging activity(66.20% ± 0.11%, 5.0 mg m L-1) but weakest α-glucosidase inhibitory activity(37.77% ± 1.30%, 5.0 mg m L-1). However, all these LJPs exert weak inhibitory effects against α-amylase. These results show that UET is an effective method for extracting bioactive polysaccharides from seaweed materials. LJP1 and LJP2 can be developed as a potential ingredient in hypoglycemic agents or functional food for the management of diabetes. This study provides scientific evidence and advances in the preparation technology and a hypoglycemic activities evaluation method for seaweed polysaccharides, especially glycosidase inhibition in combination with an antioxidative activity evaluation method.     

Immunosuppressive Activities of Fucoidan from Laminaria japonica

Effects of fucoidan from Laminariajaponica on 2,4-dinitrochlorobenzene induced de-layed-type hypersensitivity (DTH) reaction and the serum levels of IgG, IgM, complement C3 and C4 were investigated in the present study. Results showed that oral administration of fucoidan atdose of 1.50 and 300 mg/(kg*d) for 9 days before the hapten challenge significantly inhibited 2,4-dinitrochlorobenzene induced delayed-type hypersensitivity reaction; and also inhibited the humoral immunity. Serum C3 and C4 levels were markedly reduced by fucoidan at dose of 1.50 and 300 mg/kg; and serum IgG and IgM levels were reduced by fucoidan at dose of 300 mg/kg. The inhibitory effects of fucoidan on delayed-type hypersensitivity suggested that it may be potential medication for chronic inflammatory diseases in the future.     

Isolation and characterization of a fucoidan-degrading bacterium from Laminaria japonica

Fucoidan, a polysaccharide containing abundant fucose and sulfate ester group, was prepared from Laminaria japonica. In order to obtain fucoidan-degrading enzyme, bacteria capable of degrading fucoidan were screened from kelp. A bacterial strain named RC2-3 was obtained, which degraded fucoidan by the maximum extent of 54% ± 1.3%, the highest among all bacterial isolates. High-performance size exclusion chromatography(HPSEC) showed that the molecular weight of fucoidan was gradually reduced by RC2-3 with culturing time, suggesting the production of fucoidan-degrading enzyme by RC2-3. Phylogenetic analysis of partial 16S ribosomal RNA gene(16S rDNA) sequence showed that RC2-3 belonged to the family Flavobacteriaceae. However, it showed different physiological and biochemical characteristics from the known Flavobacteriaceae members producing fucoidan-degrading enzyme, thus RC2-3 was proposed to be a new member of this family.     

The Effect of Hydrolysis with Neutrase on Molecular Weight,Functional Properties,and Antioxidant Activities of Alaska Pollock Protein Isolate

In this study, the Alaska pollock protein isolate(APPI) was hydrolyzed by Neutrase for 20, 40, 80, 120, 160, 200, and 240 min. Hydrolysates with different molecular weights were produced and they were named as H1–H7. Furthermore, the effects of hydrolysis on the average molecular weights, functional properties(solubility, oil-holding capacities, foaming activities, and emulsifying properties), and antioxidant activities(1, 1-diphenyl-2-picrylhydrazyl, superoxide, and hydroxyl free radical-scavenging activities) were determined. It was found that when the degree of hydrolysis(DH) increased, the average molecular weights of the hydrolysates decreased significantly. The functional properties of APPI were also significantly improved. The hydrolysates of APPI exhibited better solubility, emulsifying activities, and foaming activities. Hydrolysates with low molecular weights(1 kDa) had better solubility, oil-holding capacities, and emulsifying activities, while hydrolysates with higher molecular weights(1 kDa) had better foaming activities. In addition, the hydrolysates exhibited excellent antioxidant properties, while the inhibition values of 1, 1-diphenyl-2-picryl hydroxyl(DPPH), superoxide, and hydroxyl free radical-scavenging activities, were 85.22%, 53.56%, and 75.00% respectively, when the concentration of the hydrolysates was 5.0 mg mL~(-1). The lower the average molecular weight was, the higher was the antioxidant activity. These results indicated that hydrolysis with Neutrase is an effective method for improving the functional and antioxidant properties of APPI. The hydrolysates of APPI displayed great potentials to be used as natural antioxidants in protein-rich aqueous foods such as nutrient supplements and sports beverages.     

Microanalysis and preliminary pharmacokinetic studies of a sulfated polysaccharide from Laminaria japonica

A rapid,sensitive and reproducible high performance liquid chromatography(HPLC) method with post-column fluorescence derivatization has been developed to determine the amount of low-molecularweight sulfated polysaccharide(GFS) in vivo. The metabolism of GFS has been shown to fit a two component model following its administration by intravenous injection,and its pharmacokinetic parameters were determined to be as follows: half-time of distribution phase(t_(1/2α))=11.24±2.93 min,half-time of elimination phase(t_(1/2β))=98.20±25.78 min,maximum concentration(C_(max))=110.53 μg/mL and peak time(T_(max))=5 min. The pharmacokinetic behavior of GFS was also investigated following intragastric administration. However,the concentration of GFS found in serum was too low for detection,and GFS could only be detected for up to 2 h after intragastric administration(200 mg/kg body weight). Thus,the bioavailability of GFS was low following intragastric administration because of the metabolism of GFS. In conclusion,HPLC with postcolumn derivatization could be used for quantitative microanalysis and pharmacokinetic studies to determine the presence of polysaccharides in the serum following intravenous injection.     

In-vitro anticoagulant activity of fucoidan derivatives from brown seaweed Laminaria japonica

Fucoidan, a group of sulfated heteropolysaccharides, was extracted from Laminaria japonica, an important economic alga species in China. The anticoagulant activity of fucoidan and its derivatives (including sulfated, phosphorylated, and aminated fucoidan) was examined using in-vitro anticoagulant systems. The correlation between chemical variations within the fucoidan group and anticoagulant activity was determined. The in-vitro anticoagulant properties of fucoidan and its derivatives were determined by measuring activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). The results indicate anticoagulant activity in all samples using APTT and TT assays; however, only the fucoidan derivatives affected the PT assay. Thus, the fucoidan derivatives were able to inhibit both intrinsic and extrinsic blood coagulants. Fucoidan (FPS) and its derivatives presented better anticoagulant activity than low molecular weight fucoidan (DFPS) and its derivatives, suggesting that molecular weight and proper conformation are contributing factors for anticoagulant activity of polysaccharides. Amino groups have a positive charge and can thus change the charge density of fucoidan. Accordingly, among the tested samples, aminated fucoidan (NF) was the most active reflecting the importance of charge density for anticoagulant activity. Available data obtained using in-vitro models suggest that the sulfate content, sulfate/total-sugar ratio, molecular weight, and the substituted group of fucoidan are important factors for anticoagulant activity but that the influence of sulfate, phosphate and amino groups on anticoagulant activity was different.     

Anti-proliferative activity of phlorotannin extracts from brown algae Laminaria japonica Aresch

In this study,we evaluated the anti-proliferative activity of phlorotannins derived from brown algae Laminaria japonica Aresch extracts on the human hepatocellular carcinoma cell (BEL-7402) and on murine leukemic cells (P388) by MTT assay.Cells were incubated with 100 μg/mL of the phlorotannin extract (PE) for 48 h.The inhibitory rate of PE on BEL-7402 and P388 cells was 30.20±1.16% and 43.44±1.86%,respectively,and the half-inhibitory concentration of PE (IC50) on P388 and BEL-7402 cells was 120 μg/mL and >...     

Anti-proliferative activity of phlorotannin extracts from brown algae Laminaria japonica Aresch

In this study, we evaluated the anti-proliferative activity of phlorotannins derived from brown algae Laminaria japonica Aresch extracts on the human hepatocellular carcinoma cell (BEL-7402) and on murine leukemic cells (P388) by MTT assay. Cells were incubated with 100 μg/mL of the phlorotannin extract (PE) for 48 h. The inhibitory rate of PE on BEL-7402 and P388 cells was 30.20±1.16% and 43.44±1.86%, respectively, and the half-inhibitory concentration of PE (IC₅₀) on P388 and BEL-7402 cells was 120 μg/mL and >200 μg/mL, respectively. Microscopic observation shows that the morphologic features of tumor cells treated with PE and 5-fluorouracil are markedly different from the normal control group. The inhibitory rate of fraction A₂ isolated from PE by sephadex LH-20 for BEL-7402 and P388 cells at the sample concentration of 70.42 μg/mL was 61.96±7.02% and 40.47±8.70%, respectively. The apoptosis peak for fraction A₂ was the most profound of all fractions used in the flow cytometry assay. The results indicate that the anti-proliferative of this algal extract is associated with the total phlorotannin content.     

Study on antithrombotic and antiplatelet activities of low molecular weight fucoidan from Laminaria japonica

The antithrombotic and antiplatelet effects of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica were compared in order to examine the influence of chemical character on their antithrombotic activity and the possible mechanism. Both LMW fucoidan fractions exhibited favorable antithrombotic activity in an Fecl3-induced arterial thrombosis. The antithrombotic activity of LMW fucoidan was related with decrease of TXB2 and whole blood viscosity and hematocrit. LMW fucoidan showed a correlation between anticoagulant, antiaggregant and antithrombotic effects in vivo. For LMW fucoidan, antithrombotic activity required high dose of 5-10 nmol kg-1, concomitantly with increase in anticoagulant activity and inhibition of platelet aggregation. Administration of LMW fucoidan significantly promoted the 6-keto-PGF1α content and decreased the TXB2 content, indicating its inhibition of tissue factor pathway and regulation of metabolism of arachidonic acid. By comparison, highly sulfated fucoidan LF2 with Mw 3900 seemed to be a more suitable choice for antithrombotic drug for its antithrombotic activity accompanied with specific inhibitory activity on platelet aggregation, low anticoagulant activity and low hemorrhagic risk in vivo.     

Preliminary Analysis of Population Genetic Diversity of Cultivated Laminaria japonica Sporophyte via AFLP Technique

The amplified fragment length polymorphic DNA (AFLP) technique was adopted to estimate the population genetic polymorphism among 30 sporophytes of Laminaria japonica collected from a cultivating farm in Rongcheng,China.Three methods were used for genomic DNA extraction from Laminaria japonica sporophyte and only the products obtained using the improved genomic DNA extraction kit method proved qualified for AFLP analysis.The parameters of the method were optimized.Samples of forty milligrams and the cell lysis time of 120 min were suggested to replace the parameters recommended by the manufacturer.Thirty individuals of Laminaria japonica from the same cultivating site were investigated using one pair of selective primers.A total of 21 loci were obtained and 17 of them were polymorphic.The mean percent age of polymorphic loci of this population was 80.95%.The Nei's gene diversity (H) within this population was 0.3028 and the average Shannon's Information index (I) was 0.4498.A genetic distance matrix among different individuals was constructed as well.Through this study,an applicable AFLP genetic analysis working system for Laminaria japonica sporophyte was established.The results of this research also revealed a high level of genetic diversity within the studied population.     

Cryopreservation of Gametophytes of Laminaria japonica （Phaeophyta） Using Encapsulation-Dehydration with Two-Step Cooling Method

Gametophytes of Laminaria japonica were cryopreserved in liquid nitrogen using encapsulation-dehydration with two-step cooling method. Gametophytes cultured at 10℃ and under continuous irradiance of 30 μmol m-2 s-1 for 3 weeks were encapsulated in calcium alginate beads. The beads were dehydrated in 0.4 molL-1 sucrose prepared with seawater for 6 h, desiccated in an incubator set at 10℃ and 70% relative humidity for 4 h, pre-frozen at either -40℃ or -60℃ for 30 min, and stored in liquid nitrogen for ＞24 h. As high as 43% of survival rate was observed when gametophytes were thawed by placing the beads in 40℃seawater and re-hydrated in 0.05 molL-1 citrate sodium prepared using 30‰ NaC1 7 d later. More cells of male gametophytes survived the whole procedure in comparison with female gametophytes. The cells of gametophytes surviving the preservation were able to grow asexually and produce morphologically normal sporophytes.     

Effects of blue light on gametophyte development of Laminaria japonica （Laminariales, Phaeophyta）

1 INTRODUCTION Light acts as an important ambient factor to regulate plant growth. Several critical points in life history for many marine algae are controlled by light. Photobiology of algae has been a discipline of particular interest for a long time (Dring, 1988). Many light effects on algae are photoperi- odic-regulated, such as sporangia formation in red algae Porphyra tenera (Rentschler, 1967) and As- paragopsis armata (Oza, 1976), and erect thalli de- velopment in brown algae Scy…     

Effects of Suspended Culture of the Seaweed Laminaria japonica Aresch on the Flow Structure and Sedimentation Processes

The culture of suspended kelp, such as Laminaria japonica Aresch, has arisen in nearshore areas for approximately 30 years since the 1980 s. This long-term activity has significant impact on the regional hydrodynamic and sedimentary environments. In this study the impact was investigated, based on synchronized multi-station data from continuous observations made within and around the culture area. In total, three current velocity profiles were identified inside and on the landward side of the culture area. Based on the current velocity profiles we calculated the boundary layer parameters, the fluxes of erosion/deposition, and the rate of sediment transport in different times at each observation site. Comparison between culture and non-culture periods showed that the presence of suspended kelp caused the reduction in the average flow velocity by approximately 49.5%, the bottom friction velocity by 24.8%, the seabed roughness length by 62.7%, and the shear stress and the flux of resuspended sediment by approximately 50%. From analyses in combination with the corresponding vertical variation of the suspended sediment distribution, it is revealed that the lifted sediments by resuspension is mixed with the upper suspended material, which will modify the regional distribution of suspended sediment. These changes in flow structure and sediment movement will accelerate seabed siltation, which corresponds to the changes in seabed erosion/deposition. However, under the influences of the seasonal changes in kelp growth the magnitude of change with the seabed siltation was not obvious inside the culture area, but a fundamental change was apparent around the culture area.     

Salicylic acid and heat acclimation pretreatment protects Laminaria japonica sporophyte (Phaeophyceae) from heat stress

Possible mediatory roles of heat acclimation and salicylic acid in protecting the sporophyte of marine macroalga Laminaria japonica (Phaeophyceae) from heat stress were studied. Heat stress resulted in oxidative injury in the kelp blades. Under heat stress significant accumulation of hydrogen peroxide (H₂O₂) and malonaldehyde (MDA), a membrane lipid peroxidation product, and a drastic decrease in chlorophyll a content were recorded. Activity of the enzymatic antioxidant system was drastically affected by heat stress. The activity of superoxide dismutase (SOD) was significantly increased while peroxidase (POD), catalase (CAT) and glutathione peroxidase (GPX) were greatly inhibited and, simultaneously, phenylalanine ammonia-lyase was activated while polyphenol oxidase (PPO) was inhibited. Both heat acclimation pretreatment and exogenous application of salicylic acid alleviated oxidative damage in kelp blades. Blades receiving heat acclimation pretreatment and exogenous salicylic acid prior to heat stress exhibited a reduced increase in H₂O₂ and MDA content, and a lower reduction in chlorophyll a content. Pretreatment with heat acclimation and salicylic acid elevated activities of SOD, POD, CAT, GPX and PPO. Considering these results collectively, we speculate that the inhibition of antioxidant enzymes is a possible cause of the heat-stress-induced oxidative stress in L. japonica, and enhanced thermotolerance may be associated, at least in part, with the elevated activity of the enzymatic antioxidant system.     

Polysaccharides in Sipunculus nudus: Extraction Condition Optimization and Antioxidant Activities

Marine organisms constitute unlimited resource of bioactive substances due to their high biodiversity and represent a valuable source of new compounds. This study optimized the alkali-extraction conditions and antioxidant activities of soluble polysaccharides from the body wall of Sipunculus nudus. The effects of solid-liquid ratio, extraction duration, extraction temperature, and alkali concentration on the yield of S. nudus polysaccharides(SNP) were examined, according to which the optimal combination of extraction parameters was obtained by an orthogonal test. The relative influencing importance of different extraction parameters on the yield of SNP followed the order as solid-liquid ratio extraction temperature alkali concentration extraction duration. The highest extraction yield, 1.98%, was achieved under an optimal extraction condition: temperature 60℃, solid-liquid ratio 1:6 g mL~(-1), duration 5 h, and alkali(Na OH) mass fraction 6%. The in vitro antioxidant activities examination showed that extracted SNP under this optimized condition had strong power in reducing certain hydroxyl and superoxide radical scavenging abilities. The promising results showed that extracted SNP could be a potent natural antioxidant.     

Researches on the Internal Molecular Weight Uniformity of Chitosan Biomaterials

Uniform molecular weight(Mw)chitosan(CS)is highly demanded in medical biomaterial industry.This present article described heterogeneous degradation of CS in aqueous HCl/ethanol solution,in which progress uniform Mw CS was successfully prepared.The Mw distribution of CS was measured by gel permeation chromatography(GPC)analysis.Moreover,the structure and properties of degraded CS were characterized by Fourier transform infrared spectroscopy(FT-IR),nuclear magnetic resonance spectroscopy(^1H NMR),X-ray diffraction(XRD)and thermogravimetric(TG)analysis.In addition,the biocompatibility of degraded CS was also assessed by hemolysis rate(HR)measurement.The Mw of CS dramatically decreased from 246 KDa to 76 k Da at the initial 30 min,and stabilized at 18 kDa after 24 h.GPC analysis results showed that the degraded CS molecular become homogenization.FT-IR and 1 H NMR analysis confirmed the basic structure of CS molecular backbone was not destroyed during this progress.Besides,the water solubility of CS was not significantly influenced by this reaction.Moreover,the XRD analysis revealed that crystallinity of degraded CS increased from 70.32% to 99.25%with time.The TG analysis showed improved thermal stability of degraded CS.HR measurement demonstrated the degraded CS possessed excellent biocompatibility.This simple and efficient heterogeneous degradation would open up a new route to produce uniform Mw CS.