Sulfated Undaria pinnatifida polysaccharide inhibits the formation of kidney stones by inhibiting HK-2 cell damage and reducing the adhesion of nano­calcium oxalate crystals.

OBJECTIVE: The formation of kidney stone is closely related to cell injury and crystal adhesion. METHOD: The sulfur trioxide-pyridine method was used to sulfate raw Undaria pinnatifida polysaccharide (UPP) with a molecular weight (Mw) of 8.33 kDa. Four polysaccharides with the sulfate group (-OSO3-) contents of 1.59% (UPP0), 6.03% (UPP1), 20.83% (UPP2), and 36.39% (UPP3) were obtained. The antioxidant activity of the four UPPs, the difference in oxidative damage inflicted by nano-CaOx monohydrate (nano-COM) on human proximal tubular epithelial (HK-2) cells before and after protection by UPPs, and the inhibitory effect on nano-COM adhesion were explored. RESULTS: Structural characterization showed that sulfation was successful. As the -OSO3- content in the UPPs was increased, the antioxidant activity and capability of the UPPs to regulate the growth of calcium oxalate (CaOx) crystals gradually increased. The damage caused by nano-COM crystals to HK-2 cells under protection by UPPs was weakened. This effect enhanced cell viability, enabled the maintenance of good cell morphology, reduced reactive oxygen species (ROS) levels, and inhibited the decrease in mitochondrial membrane potential, as well as decreased the eversion of phosphatidylserine (PS) and the expression of the adhesion proteins osteopontin (OPN), heat shock protein (HSP 90), and Annexin A1 (ANXA1). The adhesion of nano-COM to HK-2 cells was inhibited under the protection by UPPs. CONCLUSION: UPP3 with the highest content of -OSO3- presented the best antioxidant activity and crystal regulation ability, while UPP2 with the second highest -OSO3- content showed optimal cell protection ability and crystal adhesion inhibition ability. The biological activity of UPPs was regulated by Mw and -OSO3- content. UPP2 with moderate -OSO3- content may become a potential drug for preventing CaOx stones.

Exploration of interaction between angiotensin I-converting enzyme (ACE) and the inhibitory peptide from Wakame (Undaria pinnatifida).

The interaction between angiotensin I-converting enzyme (ACE) and the inhibitory peptide KNFL from Wakame was explored using isothermal titration calorimetry, multiple spectroscopic techniques and molecular dynamics simulations, and an inhibition model was established based on free energy binding theory. The experiments revealed that the binding of KNFL to ACE was a spontaneous exothermic process driven by enthalpy and entropy and occurred via multiple binding sites to form stable complexes. The complexes may be formed through multiple steps of inducing fit and conformational selection. The peptide KNFL had a fluorescence quenching effect on ACE and its addition not only affected the microenvironment around the ACE Trp and Tyr residues, but also increased the diameter and altered the conformation of ACE. This study should prove useful for improving our understanding of the mechanism of ACE inhibitory peptides.

Affinity Purification of Angiotensin Converting Enzyme Inhibitory Peptides from Wakame (Undaria Pinnatifida) Using Immobilized ACE on Magnetic Metal Organic Frameworks.

Angiotensin-I-converting enzyme (ACE) inhibitory peptides derived from marine organism have shown a blood pressure lowering effect with no side effects. A new affinity medium of Fe3O4@ZIF-90 immobilized ACE (Fe3O4@ZIF-90-ACE) was prepared and used in the purification of ACE inhibitory peptides from Wakame (Undaria pinnatifida) protein hydrolysate (<5 kDa). The Fe3O4@ZIF-90 nanoparticles were prepared by a one-pot synthesis and crude ACE extract from pig lung was immobilized onto it, which exhibited excellent stability and reusability. A novel ACE inhibitory peptide, KNFL (inhibitory concentration 50, IC50 = 225.87 µM) was identified by affinity purification using Fe3O4@ZIF-90-ACE combined with reverse phase-high performance liquid chromatography (RP-HPLC) and MALDI-TOF mass spectrometry. Lineweaver-Burk analysis confirmed the non-competitive inhibition pattern of KNFL, and molecular docking showed that it bound at a non-active site of ACE via hydrogen bonds. This demonstrates that affinity purification using Fe3O4@ZIF-90-ACE is a highly efficient method for separating ACE inhibitory peptides from complex protein mixtures and the purified peptide KNFL could be developed as a functional food ingredients against hypertension.

Rapid analytical approach for bioprofiling compounds with radical scavenging and antimicrobial activities from seaweeds.

Brown seaweeds are traditionally used as food in Asian countries, and they are a valuable source of bioactive compounds. Herein, a novel high-throughput methodological approach was developed for the tracing of compounds with radical scavenging and antimicrobial activities in Saccharina japonica and Undaria pinnatifida methanol extracts. The seaweed metabolites were separated by a novel high-performance thin-layer chromatography method, the bioactive bands were identified by bioautography assays. The bioactive compounds were characterized with ultra-high-performance liquid chromatography coupled with linear trap quadrupole tandem mass spectrometry. Stearidonic, eicosapentaenoic, and arachidonic acids were identified as major components having radical scavenging and antimicrobial activities. The suggested method provides a fast identification and quantification of bioactive compounds in multicomponent biological samples.

Undaria pinnatifida fucoidan nanoparticles loaded with quinacrine attenuate growth and metastasis of pancreatic cancer.

Pancreatic cancer is a devastating gastrointestinal tumor with limited Chemotherapeutic options. Treatment is restricted by its poor vascularity and dense surrounding stroma. Quinacrine is a repositioned drug with an anticancer activity but suffers a limited ability to reach tumor cells. This could be enhanced using nanotechnology by the preparation of quinacrine-loaded Undaria pinnatifida fucoidan nanoparticles. The system exploited fucoidan as both a delivery system of natural origin and active targeting ligand. Lactoferrin was added as a second active targeting ligand. Single and dual-targeted particles prepared through nanoprecipitation and ionic interaction respectively were appraised. Both particles showed a size lower than 200 nm, entrapment efficiency of 80% and a pH-dependent release of the drug in the acidic environment of the tumor. The anticancer activity of quinacrine was enhanced by 5.7 folds in dual targeted particles compared to drug solution with a higher ability to inhibit migration and invasion of cancer. In vivo, these particles showed a 68% reduction in tumor volume compared to only 20% for drug solution. In addition, they showed a higher animals' survival rate with no hepatotoxicity. Hence, these particles could be an effective option for the eradication of pancreatic cancer cells.

Screening of Human CYP1A2 and CYP3A4 Inhibitors from Seaweed In Silico and In Vitro.

Phenolic compounds and carotenoids are potential inhibitors of cytochrome P450s. Sixteen known compounds, phenolic compounds and carotenoids from seaweed were examined for potential inhibitory capacity against CYP1A2 and CYP3A4 in silico and in vitro. Morin, quercetin, and fucoxanthin inhibited the enzyme activity of CYP1A2 and CYP3A4 in a dose-dependent manner. The IC50 values of morin, quercetin, and fucoxanthin were 41.8, 22.5, and 30.3 µM for CYP1A2 and 86.6, 16.1, and 24.4 µM for CYP3A4, respectively. Siphonaxanthin and hesperidin did not show any significant effect on CYP1A2, but they slightly inhibited CYP3A4 activity at high concentrations. In silico modeling of CYP's binding site revealed that the potential inhibitors bound in the cavity located above the distal surface of the heme prosthetic group through the 2a or 2f channel of CYPs. This study presents an approach for quickly predicting CYP inhibitory activity and shows the potential interactions of compounds and CYPs through in silico modeling.

Sulfated polysaccharide from Undaria pinnatifida stabilizes the atherosclerotic plaque via enhancing the dominance of the stabilizing components.

The purpose of this study was to investigate the stable effect and mechanism of sulfated polysaccharide from Undaria pinnatifida (SPUP) on atherosclerotic plaque. The results showed that atherosclerotic plaques in the ApoE-/- mice of high-fat diet model group increased significantly without drug intervention. The content of vulnerable components (lipid, inflammatory macrophage) increased significantly, and the content of stability components (smooth muscle cell, collagen) reduced significantly. However, it could find that atherosclerotic plaque areas were decreased in a dose-dependent manner after SPUP intervention. SPUP could enhance the dominance of the stability components in plaque, and reduce the content of vulnerable component. Furthermore, SPUP could significantly reduce the matrix metalloprotein-9 content in atherosclerotic plaque. These results suggested that SPUP could stabilize atherosclerotic plaque by enhancing the dominance of the stability components content, reducing the vulnerability components content, and lowering the vulnerability index value.

Antimicrobial biopolymer formation from sodium alginate and algae extract using aminoglycosides.

Antimicrobial biopolymers provide a biodegradable, sustainable, safe, and cheap approach to drug delivery and wound dressing to control bacterial infection and improve wound healing respectively. Here, we report a one-step method of making antimicrobial alginate polymer from sodium alginate and aqueous extract of Wakame using antibiotic aminoglycosides. Thin layer chromatography of commercially available sodium alginate and Wakame extract showed similar oligosaccharide profiles. Screening of six aminoglycosides showed that kanamycin disulfate and neomycin sulfate produces the highest amount of biopolymer; however, kanamycin disulfate produces the most malleable and form fitting biopolymer. Image texture analysis of biopolymers showed similar quantification parameters for all the six aminoglycosides. Weight of alginate polymer as a function of aminoglycoside concentration follows a growth model of prion protein, consistent with the aggregating nature of both processes. Slow release of antibiotics and the resulting zone of inhibition against E. coli DH5α were observed by agar well diffusion assay. Inexpensive method of production and slow release of antibiotics will enable diverse applications of antimicrobial alginate biopolymer reported in this paper.

Arsenic speciation and biotransformation by the marine macroalga Undaria pinnatifida in seawater: A culture medium study.

Freshwater and marine organisms are capable of metabolizing arsenic (As) efficiently and regulating the As biogeochemical cycles. In this study, Undaria pinnatifida was exposed to As(V) (0, 0.1, and 1 µM) and phosphate (P; 1 and 10 µM) in seawater under laboratory-controlled conditions for up to seven days to analyze As biotransformation. The growth rates and chlorophyll fluorescence of the alga were unaffected by As stress, and statistically insignificant differences were observed among the cultures (p > 0.05). As(V) was readily accumulated by this macroalga through phosphate transporters, transformed intracellularly, and excreted into the medium, depending on the As(V) to P molar ratios. The concentration of As(V) and biotransformed species As(III) and DMAA(V) varied significantly in the algal cultures on the basis of the exposure period (p < 0.05). The concentration of As(III) was initially higher but decreased with the incubation period, whereas the concentration of DMAA(V) increased gradually. At the end of the incubation, 0.04 and 0.32 µM DMAA(V) were recorded in the media containing 0.1 and 1 µM As(V) with a constant 1.0 µM P, respectively. The results also indicated that the cellular uptake of As(V) and subsequent release of DMAA(V) were inhibited by P in the medium. The biotransformation was consistent with the As(V) detoxification mechanism based on reduction and methylation, which was enhanced by the lower As(V) to P molar ratios. These findings can be helpful in understanding the contribution of macroalgae to As biogeochemistry in marine environments and the potential risks of As dietary intake.

Degradation of Fucoxanthin to Elucidate the Relationship between the Fucoxanthin Molecular Structure and Its Antiproliferative Effect on Caco-2 Cells.

Fucoxanthin has an antiproliferative effect on cancer cells, but its detailed structure⁻activity correlation has not yet been elucidated. To elucidate this correlation, fucoxanthin was degraded by ozonolysis. The degraded compounds of fucoxanthin obtained by ozonolysis were purified by HPLC and analyzed by NMR. The polyene chain of fucoxanthin was cleaved by ozonolysis, and the fucoxanthin was divided into two types of cyclohexyl derivatives, one with a ß,γ-epoxy ketone group and the other with an allenic bond. In order to elucidate the structure⁻activity correlation, Caco-2 cells (human colorectal carcinoma) were treated with fucoxanthin degradation compounds. It was found that the entire structure of fucoxanthin is not essential for its antiproliferative effect and that even a partial structure exerts this effect.

Production of biodiesel from carbon sources of macroalgae, Laminaria japonica.

As aquatic biomass which is called "the third generation biomass", Laminaria japonica (also known as Saccharina japonica) consists of mannitol and alginate which are the main polysaccharides of algal carbohydrates. In this study, oleaginous yeast (Cryptococcus curvatus) was used to produce lipid from carbon sources derived from Laminaria japonica. Volatile fatty acids (VFAs) were produced by fermentation of alginate extracted from L. japonica. Thereafter, mannitol was mixed with VFAs to culture the oleaginous yeast. The highest lipid content was 48.30%. The composition of the fatty acids was similar to vegetable oils. This is the first confirmation of the feasibility of using macroalgae as a carbon source for biodiesel production.

Zinc and cadmium removal by biosorption on Undaria pinnatifida in batch and continuous processes.

Zn(II) and Cd(II) removal by biosorption using Undaria pinnatifida was studied in batch and dynamic systems. The kinetic uptake follows a pseudo second order rate equation indicating that the rate limiting step is a chemical reaction. The equilibrium data are described by the Langmuir isotherm in mono-component solutions. In binary solutions, the Jain and Snowyink model shows that most of the active sites are exclusively accessible to cadmium ions without competition with the zinc ions. The dynamic studies show that the biosorbent has higher retention and affinity for Cd(II) than for Zn(II) in both mono- and bi-component systems. SEM-EDX analysis indicates that the active sites are heterogeneously distributed on the cell wall surface. FT-IR spectrometry characterization shows that carboxylic groups and chemical groups containing N and S contribute to Zn(II) and Cd(II) uptake by U. pinnatifida. According to these results calcium-treated U. pinnatifida is a suitable adsorbent for Zn(II) and Cd(II) pollutants.

Dynamic Cr(III) uptake by Macrocystis pyrifera and Undaria pinnatifida biomasses

Background: The increased industrial activity has resulted in the discharge of large amount of pollutants including non-degradable metals into the environment. Chromium is produced in several industrial processes and it can be found in the environment in two stable oxidation states, Cr(VI) and Cr(III). Cr(VI) is more hazardous due to its carcinogenic and mutagenic effects on living organisms. Although much less toxic, Cr(III) can also exert genotoxic effects under prolonged or severe exposure. It can be separated from the solution by precipitation but biosorption using brown algae seems to be an effective and sustainable treatment technique owing to its cost-effectiveness and environmental friendly characteristics. Macrocystis pyrifera and Undaria pinnatifida are two marine brown macroalgae with high capability of removing heavy metals including Cr(III) in batch mode of operation. In this work packed bed biosorption of Cr(III) by M. pyrifera and U. pinnatifida biomasses was evaluated. Results: The shapes of the breakthrough curves were rather different for each biomaterial. Parameters like the breakthrough time (t b) andzone mass transfer (MTZ) showed that U. pinnatifida has greater affinity for Cr(III). The maximum adsorption capacity at the exhaustion operating time (t e) demonstrated that M. pyrifera has higher retention capacity of Cr(III). The experimental data were fitted to Thomas, Yoon-Nelson and Dose-Response models. The best correlation coefficient (0.94 or 0.96) was obtained with Dose-Response that accurately describes the uptake behaviour of Cr(III) on the seaweed biomasses under different experimental conditions. The FT-IR spectra evidenced that Cr(III) adsorption occurred mainly by interaction between metal and carboxylate groups present on both the seaweed surfaces. Conclusions: M. pyrifera and U. pinnatifida biomasses are efficient biosorbents for Cr(III) adsorption under a continuous mode of operation although differences between uptake capacities suggest different mechanisms involved in the biosorption.

Isolation and characterization of Microbulbifer species 6532A degrading seaweed thalli to single cell detritus particles.

To reduce the volume of seaweed wastes and extract polysaccharides, seaweed-degrading bacteria were isolated from drifting macroalgae harvested along the coast of Toyama Bay, Japan. Sixty-four bacterial isolates were capable of degrading "Wakame" (Undaria pinnatifida) thallus fragments into single cell detritus (SCD) particles. Amongst these, strain 6532A was the most active degrader of thallus fragments, and was capable of degrading thallus fragments to SCD particles within a day. Although the sequence similarity of the 16S rRNA gene of strain 6532A was 100% similar to that of Microbulbifer elongatus JAMB-A7, several distinct differences were observed between strains, including motility, morphology, and utilization of D: -arabinose and gelatin. Consequently, strain 6532A was classified as a new Microbulbifer strain, and was designated Microbulbifer sp. 6532A. Strain 6532A was capable of degrading both alginate and cellulose in the culture medium, zymogram analysis of which revealed the presence of multiple alginate lyases and cellulases. To the best of our knowledge, this is the first study to directly demonstrate the existence of these enzymes in Microbulbifer species. Shotgun cloning and sequencing of the alginate lyase gene in 6532A revealed a 1,074-bp open reading frame, which was designated algMsp. The reading frame encoded a PL family seven enzyme composed of 358 amino acids (38,181 Da). With a similarity of 74.2%, the deduced amino acid sequence was most similar to a Saccharophagus enzyme (alg 7C). These findings suggest that algMsp in strain 6532A is a novel alginate lyase gene.

In situ ATR-IR spectroscopic and electron microscopic analyses of settlement secretions of Undaria pinnatifida kelp spores.

Knowledge about the settlement of marine organisms on substrates is important for the development of environmentally benign new methods for control of marine biofouling. The adhesion to substrates by spores of Undaria pinnatifida, a kelp species that is invasive to several countries, was studied by scanning electron and transmission electron microscopies (SEM/TEM) as well as by in situ attenuated total reflection infrared (ATR-IR) spectroscopy. The IR spectra showed that adhesive secretion began approximately 15 min after initial settlement and that the adhesive bulk material contained protein and anionic polysaccharides. Energy dispersive X-ray microanalysis of the adhesive identified sulphur and phosphorus as well as calcium and magnesium ions, which facilitate the gelation of the anionic polysaccharides in the sea water. The adhesive may be secreted from Golgi bodies in the spore, which were imaged by TEM of spore thin sections. Additionally, an in situ settlement study on TiO(2) particle film by ATR-IR spectroscopy revealed the presence of phosphorylated moieties directly binding the substrate. The presence of anionic groups dominating the adhesive suggests that inhibition of spore adhesion will be favoured by negatively charged surfaces.

Isolation and characterization of marine bacterial strain degrading fucoidan from Korean Undaria pinnatifida Sporophylls.

In spite of an increasing interest in fucoidans as biologically active compounds, no convenient commercial sources with fucoidanase activity are yet available. A marine bacterial strain that showed confluent growth on a minimal medium containing fucoidan, prepared from Korean Undaria pinnatifida sporophylls, as the sole carbon source was isolated and identified based on a 16S rDNA sequence analysis as a strain of Sphingomonas paucimobilis, and named Sphingomonas paucimobilis PF-1. The strain depolymerized fucoidan into more than 7 distinct lowmolecular- mass fucose-containing oligosaccharides, ranging from 305 to 3,749 Da. The enzyme activity was shown to be associated with the whole cell, suggesting the possibility of a surface display of the enzyme. However, a whole-cell enzyme preparation neither released the monomer Lfucose from the fucoidan nor hydrolyzed the chromogenic substrate p-nitrophenyl-alpha-L-fucoside, indicating that the enzyme may be an endo-acting fucoidanase rather than an alpha-L-fucosidase. Therefore, this would appear to be the first report on fucoidanolytic activity by a Sphingomonas species and also the first report on the enzymatic degradation of the Korean Undaria pinnatifida sporophyll fucoidan. Moreover, this enzyme activity may be very useful for structural analyses of fucose-containing polysaccharides and the production of bioactive fucooligosaccharides.

Degradation of the seaweed wakame (Undaria pinnatifida) by a composting process with the inoculation of Bacillus sp. HR6.

Disposal of the seaweed wakame (Undaria pinnatifida) by inoculating the halotolerant bacterium Bacillus sp. HR6 was examined in an experimental scale composting system. Strain HR6 was effective in initiating the composting process of wakame, and there was a rapid increase in temperature to over 54.9-55.7 degrees C after 18-20 h. The composting process of wakame could be carried out despite a high NaCl content, 28.2 mg/g, although lower salinity resulted in a shorter lag time and higher weight reduction. In a larger scale composting process with aeration, two peaks of temperature change were found which corresponded well to oxygen consumption and CO2 emission during the process. The pH increased to 8.83 and organic materials were reduced to 93.4% after 72 h. The initial N and C contents were 3.9 and 34.0%, respectively, both of which decreased during the composting process. The changes in the viable cell numbers suggested that strain HR6 predominated before 24 h and other microorganisms including HR6 were present in a mixed state during the later period of composting. The total content of alginate (TA), 32.2% in the initial stage, decreased to 29.2% after 72 h, while water soluble alginate (WSA) increased, indicating that the solubilization and decomposition of alginate had occurred during the composting process.

Algae -- a poor man's HAART?

Drawing inferences from epidemiologic studies of HIV/AIDS and in vivo and in vitro HIV inhibition by algae, we propose algal consumption as one unifying characteristic of countries with anomalously low rates. HIV/AIDS incidence and prevalence in Eastern Asia ( approximately 1/10000 adults in Japan and Korea), compared to Africa ( approximately 1/10 adults), strongly suggest that differences in IV drug use and sexual behavior are insufficient to explain the 1000-fold variation. Even in Africa, AIDS/HIV rates vary. Chad has consistently reported low rates of HIV/AIDS (2-4/100). Possibly not coincidentally, most people in Japan and Korea eat seaweed daily and the Kanemba, one of the major tribal groups in Chad, eat a blue green alga (Spirulina) daily. Average daily algae consumption in Asia and Africa ranges between 1 and 2 tablespoons (3-13 g). Regular consumption of dietary algae might help prevent HIV infection and suppress viral load among those infected.