Preparation and potential applications of alginate oligosaccharides.

Alginate, a linear polymer consisting of ß-D-mannuronic acid (M) and α-L-guluronic acid (G) with 1,4-glycosidic linkages and comprising 40% of the dry weight of algae, possesses various applications in the food and nutraceutical industries. However, the potential applications of alginate are restricted in some fields because of its low water solubility and high solution viscosity. Alginate oligosaccharides (AOS) on the other hand, have low molecular weight which result in better water solubility. Hence, it becomes a more popular target to be researched in recent years for its use in foods and nutraceuticals. AOS can be obtained by multiple degradation methods, including enzymatic degradation, from alginate or alginate-derived poly G and poly M. AOS have unique bioactivity and can bring human health benefits, which render them potentials to be developed/incorporated into functional food. This review comprehensively covers methods of the preparation and analysis of AOS, and discussed the potential applications of AOS in foods and nutraceuticals.

Potential Food and Nutraceutical Applications of Alginate: A Review.

Alginate is an acidic polysaccharide mainly extracted from kelp or sargassum, which comprises 40% of the dry weight of algae. It is a linear polymer consisting of ß-D-mannuronic acid (M) and α-L-guluronic acid (G) with 1,4-glycosidic linkages, possessing various applications in the food and nutraceutical industries due to its unique physicochemical properties and health benefits. Additionally, alginate is able to form a gel matrix in the presence of Ca2+ ions. Alginate properties also affect its gelation, including its structure and experimental conditions such as pH, temperature, crosslinker concentration, residence time and ionic strength. These features of this polysaccharide have been widely used in the food industry, including in food gels, controlled-release systems and film packaging. This review comprehensively covers the analysis of alginate and discussed the potential applications of alginate in the food industry and nutraceuticals.

Immune activation of murine RAW264.7 macrophages by sonicated and alkalized paramylon from Euglena gracilis.

BACKGROUND: Euglena is a new super health food resource that is rich in the natural polysaccharide paramylon, a linear ß-1,3-glucan with various biological activities including activity on the immune system in different cell lines and animals. Despite these reports, the immune regulation mechanism of paramylon is still unclear. RESULTS: We investigate the signaling pathways paramylon impacts in immune macrophages. In RAW264.7 macrophages, sonicated and alkalized paramylon oligomers up-regulated inducible nitric oxide synthase (iNOS) and increased secretion of nitric oxide (NO), interleukin (IL)-6 and tumor necrosis factor (TNF)-α, in a concentration-dependent manner. In addition, paramylon activated the nuclear factor-κB(NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and inhibiting these pathways attenuated the paramylon-induced secretion of the above immune-mediators. CONCLUSIONS: These results demonstrate that Euglena gracilis paramylon modulates the immune system via activation of the NF-κB and MAPK signaling pathways and thus has potential therapeutic benefits.

Unsaturated guluronate oligosaccharide enhances the antibacterial activities of macrophages.

Alginate from marine seaweeds is receiving continuous attention owing to its wide physiological activities. Herein, we sought to elucidate possible effects of alginate-derived polyguluronate (PG) and unsaturated guluronate oligosaccharide (GOS) on antibacterial activities of macrophages. Our results showed that, in contrast to PG, GOS markedly increased the phagocytosis of IgG-opsonized Escherichia coli and Staphylococcus aureus and further inhibited the survival of intracellular bacteria in macrophages. In line with this, GOS treatment resulted in the enhanced expression of Fcγ receptors on macrophages. In addition, GOS activated NF-κB pathway, induced TNF-α secretion, and elevated the expression of inducible nitric oxide synthase and the production of nitric oxide. Meanwhile, GOS stimulated the production of reactive oxygen species in macrophages. Moreover, guluronate trimer to hexamer (G3-G6) in GOS exhibited significant activity that increased the bacterial phagocytosis of macrophages, with the pentamer (G5), displaying the highest activity. Finally, our in vivo results further confirmed that GOS but not PG significantly improved bacterial clearance in murine acute peritonitis. In conclusion, GOS enhances antibacterial activities of macrophages via modulating signaling pathways related to innate immunity, suggesting that GOS might be a promising therapeutic candidate to improve the host defense against bacterial infection.

Alginate-Derived Oligosaccharide Inhibits Neuroinflammation and Promotes Microglial Phagocytosis of ß-Amyloid.

Alginate from marine brown algae has been widely applied in biotechnology. In this work, the effects of alginate-derived oligosaccharide (AdO) on lipopolysaccharide (LPS)/ß-amyloid (Aß)-induced neuroinflammation and microglial phagocytosis of Aß were studied. We found that pretreatment of BV2 microglia with AdO prior to LPS/Aß stimulation led to a significant inhibition of production of nitric oxide (NO) and prostaglandin E2 (PGE2), expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and secretion of proinflammatory cytokines. We further demonstrated that AdO remarkably attenuated the LPS-activated overexpression of toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB in BV2 cells. In addition to the impressive inhibitory effect on neuroinflammation, we also found that AdO promoted the phagocytosis of Aß through its interaction with TLR4 in microglia. Our results suggested that AdO exerted the inhibitory effect on neuroinflammation and the promotion effect on microglial phagocytosis, indicating its potential as a nutraceutical or therapeutic agent for neurodegenerative diseases, particularly Alzheimer's disease (AD).

Morphological and proteomic analyses reveal that unsaturated guluronate oligosaccharide modulates multiple functional pathways in murine macrophage RAW264.7 cells.

Alginate is a natural polysaccharide extracted from various species of marine brown algae. Alginate-derived guluronate oligosaccharide (GOS) obtained by enzymatic depolymerization has various pharmacological functions. Previous studies have demonstrated that GOS can trigger the production of inducible nitric oxide synthase (iNOS)/nitric oxide (NO), reactive oxygen species (ROS) and tumor necrosis factor (TNF)-α by macrophages and that it is involved in the nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase signaling pathways. To expand upon the current knowledge regarding the molecular mechanisms associated with the GOS-induced immune response in macrophages, comparative proteomic analysis was employed together with two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and Western blot verification. Proteins showing significant differences in expression in GOS-treated cells were categorized into multiple functional pathways, including the NF-κB signaling pathway and pathways involved in inflammation, antioxidant activity, glycolysis, cytoskeletal processes and translational elongation. Moreover, GOS-stimulated changes in the morphologies and actin cytoskeleton organization of RAW264.7 cells were also investigated as possible adaptations to GOS. This study is the first to reveal GOS as a promising agent that can modulate the proper balance between the pro- and anti-inflammatory immune responses, and it provides new insights into pharmaceutical applications of polysaccharides.

Soy protein isolate-polyguluronate nanoparticles loaded with resveratrol for effective treatment of colitis.

Polyguluronate (PG) is an acidic homopolymer of α-(1,4)-l-guluronate separated from alginate. In this study, PG was first grafted with soy protein isolate (SPI) through the Maillard reaction to obtain a new glycoprotein (SPI-PG). Then, this novel glycoprotein was used to prepare nanoparticles to encapsulate the resveratrol (RES). Our results showed that SPI-PG-RES had better pH stability, storage stability and ionic stability than SPI-RES. In vitro digestion experiments showed that the RES bioavailability of SPI-PG-RES was much higher than that of free RES and SPI-RES. Furthermore, the in vitro antioxidant capacity of SPI-PG-RES was much stronger than that of free RES and SPI-RES. In addition, SPI-PG-RES was more effective in preventing the symptoms of DSS-induced colitis than RES and SPI-RES. These results suggested that the protein nanoparticles prepared using SPI-PG were a stable and effective hydrophobic polyphenol carrier and could be applied to food-grade components in functional foods and nutritional supplements.

Enhancement of the chemical stability of nanoemulsions loaded with curcumin by unsaturated mannuronate oligosaccharide.

Unsaturated mannuronate oligosaccharide (MOS) is an acidic oligosaccharide prepared from alginate-derived polymannuronate by enzymatic depolymerization, followed by double bond formation between C-4 and C-5 at the nonreducing end. In this study, MOS was used as a stabilizer to fabricate O/W nanoemulsions loaded with curcumin (MOS-CUR) for the first time. The results revealed that the MOS-CUR showed small droplet sizes and narrow size distributions and was slightly more stable than normal oil-in-water (O/W) curcumin nanoemulsions (water-CUR). Additionally, MOS can improve the superoxide anion scavenging ability and iron ion reducing ability of the curcumin nanoemulsion system. Although the digestion behaviour of MOS-CUR and water-CUR was similar, the bioavailability of curcumin in MOS-CUR was significantly higher than that in water-CUR. All these results indicated that MOS could be used as a stabilizer for preparing nanoemulsions to easily encapsulate labile nutrients and to enhance the bioavailability and antioxidant capacity of these nutrients.

Preparation, characterization and immunomodulatory effects of unsaturated sulfated oligoguluronic acid.

Polyguluronic acid (PG) is a type of polysaccharide found in edible brown seaweeds. This study synthesized a new sulfated saccharide (SOGA) by sulfating PG-derived unsaturated oligoguluronic acid (OGA). The molecular weight and degree of sulfate-group substitution of SOGA were 1.6 kDa and 1.03, respectively. The structures of PG, OGA, and SOGA were elucidated using FT-IR and NMR spectroscopy. Furthermore, the immunomodulatory effects of PG, OGA, and SOGA on LPS-triggered RAW264.7 and BV2 cells were evaluated. SOGA, but not PG or OGA, significantly reduced the LPS-stimulated overproduction of proinflammatory mediators and suppressed the activation of corresponding signalling pathways. Also, SOGA could actively regulate immune balance by inhibiting apoptosis and pyroapoptosis. These results suggested that SOGA is a potential therapeutic agent for the prevention of diseases associated with immune disorders owing to its remarkable immunomodulatory effects, and that sulfate groups in the carbohydrate chain play a crucial role for its bioactivities.

Preparation, characterization and macrophage-stimulating activity of polyguluronate nanoliposomes.

Polyguluronate (PG) consists entirely of α-L-guluronic acid derived from alginate, which is an acidic polysaccharide extracted from brown algae. PG has a short half-life and is easily degraded by microorganisms, resulting in decreased activity and thus its application in the medical field. In this study, polyguluronate liposomes (PGLs) were prepared to improve the macrophage-stimulating activity of PG. The morphology, encapsulation efficiency, particle size distribution, zeta potential and stability of the PGLs were characterized. Results showed that PGLs were uniformly round with an encapsulation efficiency of 77.76 ± 0.89%, a particle size of 63.96 ± 3.98 nm and a zeta potential of -53.4 ± 1.75 mV. The stability studies showed that PGLs should be stored in a neutral environment at 4 °C. The macrophage-stimulating activity of PGLs was better than that of PG. This study provides a promising carrier for the further application of PG in food or medicine.

Unsaturated guluronate oligosaccharide used as a stabilizer of oil-in-water nanoemulsions loaded with bioactive nutrients.

Unsaturated guluronate oligosaccharide (GOS) is generated via alginate-derived polyguluronate (PG) degradation by alginate lyase, followed by formation of a double bond between C-4 and C-5 at the nonreducing end. In this study, GOS was first used as a stabilizer to fabricate O/W nanoemulsions loaded with resveratrol (GOS-RES). Our results revealed that both the GOS-RES and normal O/W resveratrol nanoemulsions (water-RES) showed small droplet sizes and narrow size distributions under certain experimental conditions. However, the particle size and stability of the GOS-RES were slightly greater than those of the water-RES in acidic and neutral environments and at high temperatures. Furthermore, the GOS-RES exhibited a better sustained release effect for resveratrol than the water-RES. Moreover, the GOS-RES showed a significant superoxide radical scavenging effect. All these results demonstrated that GOS has good prospects for preparing nanoemulsions to encapsulate hydrophobic nutrients, which could be applied as food-grade components in beverages and other foods.

Inhibitory Effects of Macelignan on Tau Phosphorylation and Aß Aggregation in the Cell Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder mainly affecting old population. In this study, two Tau overexpressing cell lines (SH-SY5Y/Tau and HEK293/Tau), N2a/SweAPP cell line, and 3× Transgene (APPswe/PS1M146V/TauP301L) mouse primary nerve cell lines were used as AD models to study the activity and molecular mechanism of macelignan, a natural compound extracted from Myristica fragrans, against AD. Our study showed that macelignan could reduce the phosphorylation of Tau at Thr 231 site, Ser 396 site, and Ser 404 site in two overexpressing Tau cell lines. It also could decrease the phosphorylation of Tau at Ser 404 site in mouse primary neural cells. Further investigation of its mechanism found that macelignan could reduce the phosphorylation of Tau by increasing the level of autophagy and enhancing PP2A activity in Tau overexpressing cells. Additionally, macelignan could activate the PERK/eIF2α signaling pathway to reduce BACE1 translation, which further inhibits the cleavage of APP and ultimately suppresses Aß deposition in N2a/SweAPP cells. Taken together, our results indicate that macelignan has the potential to be developed as a treatment for AD.

Alginate-Derived Mannuronate Oligosaccharide Attenuates Tauopathy through Enhancing Autophagy.

Polymannuronate (PM) is an acidic polysaccharide prepared from alginate, contained in edible brown seaweeds. An unsaturated mannuronate oligosaccharide (MOS) is an enzymatically depolymerized oligosaccharide prepared from PM. The effects of MOS on attenuating tauopathy were studied in HEK293/Tau cells and primary triple transgenic (3×Tg) neurons. MOS inhibited heparin-induced aggregation of the Tau-K18 oligomer and suppressed the levels of phosphorylated Tau protein. MOS treatment reduced the activity of glycogen synthase kinase-3ß (GSK-3ß) by decreasing its phosphorylation levels on the sites of Y216 and increasing phosphorylation levels on the sites of S9. MOS treatment increased the ratio of LC3-II/LC3-I levels and reduced the expression of p62, indicating an increase in autophagy. Finally, MOS-induced decrease in Tau protein expression was attenuated by the addition of an autophagy inhibitor, confirming the involvement of autophagy. These data support MOS as a promising functional food or potential pharmaceutics for attenuating Tau protein-related disease.

Optimization of preparation conditions and in vitro sustained-release evaluation of a novel nanoemulsion encapsulating unsaturated guluronate oligosaccharide.

Unsaturated guluronate oligosaccharide (GOS) was prepared from alginate-derived homopolymeric blocks of guluronic acid by alginate lyase-mediated depolymerization. In this study, a GOS-based water-in-oil-in-water (W1/O/W2) nanoemulsion was prepared, and different influencing factors were investigated. First, linseed oil was selected as the optimal carrier oil. Then, other optimal conditions of the GOS nanoemulsion were determined based on response surface methodology (RSM). Under the optimal conditions, the obtained GOS nanoemulsion showed a spherical structure with an average particle size of 273.93 ±â€¯8.91 nm, and its centrifugal stability was 91.37 ±â€¯0.45 %. Moreover, the GOS nanoemulsion could achieve the aim of sustained release in vitro and be stably stored at 4°C for at least 5 days. This work prepared a novel GOS-based W1/O/W2 nanoemulsion that may effectively address the storage difficulties of unsaturated GOS and provides a valuable contribution to the application of GOS in the food and medicine fields.

Unsaturated mannuronate oligosaccharide ameliorates ß-amyloid pathology through autophagy in Alzheimer's disease cell models.

Unsaturated mannuronate oligosaccharide (MOS) is an enzymatic depolymerization product from alginate-derived polymannuronate (PM). In this study, we investigated for the first time the potential therapeutic effect of MOS on Alzheimer's disease (AD) and its molecular mechanism in N2a-sw cells and 3×Tg-AD primary cortex neurons. Our results showed that MOS ranges from mannuronate dimer to mannuronate undecamer (M2-M11) with an unsaturated nonreducing terminal structure and with a double bond and 1,4-glycosidic linkages. It significantly inhibited the aggregation of amyloid-ß (Aß)1-42 oligomer, decreased expression of Aß1-42 and reduced levels of amyloid precursor protein (APP) and BACE1. It promoted the autophagy, which involves the inactivation of mTOR signaling pathway and the facilitation of the fusion of autophagosomes and lysosomes. Finally, autophagy inhibitors blocked MOS' anti-AD actions, confirming the involvement of autophagy. In conclusion, MOS from seaweed alginate might be a promising nutraceutical or natural medicine for AD therapy.

Genipin Attenuates Tau Phosphorylation and Aß Levels in Cellular Models of Alzheimer's Disease.

Alzheimer's disease (AD) is a devastating brain disorder characterized by neurofibrillary tangles and amyloid plaques. Inhibiting Tau protein and amyloid-beta (Aß) production or removing these molecules is considered potential therapeutic strategies for AD. Genipin is an aglycone and is isolated from the extract of Gardenia jasminoides Ellis fruit. In this study, the effect and molecular mechanisms of genipin on the inhibition of Tau aggregation and Aß generation were investigated. The results showed that genipin bound to Tau and protected against heparin-induced Tau fibril formation. Moreover, genipin suppressed Tau phosphorylation probably by downregulating the expression of CDK5 and GSK-3ß, and activated mTOR-dependent autophagy via the SIRT1/LKB1/AMPK signaling pathway in Tau-overexpressing cells. In addition, genipin decreased Aß production by inhibiting BACE1 expression through the PERK/eIF2α signaling pathway in N2a/SweAPP cells. These data indicated that genipin could effectively lead to a significant reduction of phosphorylated Tau level and Aß generation in vitro, suggesting that genipin might be developed into an effective therapeutic complement or a potential nutraceutical for preventing AD.

Macrophage-stimulating activity of European eel (Anguilla anguilla) peptides in RAW264.7 cells mediated via NF-κB and MAPK signaling pathways.

European eel (Anguilla anguilla) is considered to be a vital commercial fish species. In this study, the effect and molecular mechanism of bioactive peptides from European eel on macrophage-stimulating activity in RAW264.7 cells were investigated. Eel peptide (EP) markedly induced NO and iNOS production and promoted TNF-α and IL-6 secretion in a concentration-dependent manner. Moreover, EP dose-dependently activated NF-κB and MAPK signaling pathways in RAW264.7 cells. In addition, EP was purified using a Sephadex A-25 column and a Bio-Gel P-6 column, and the fraction (Fr-1-1) showing the strongest NO-inducing activity was obtained. Then, the molecular weights of the components in Fr-1-1 were analyzed by LC-MS/MS and found to range from 700 to 1900 Da for the majority of components, which suggested that Fr-1-1 mainly consisted of peptides containing 8-20 amino acid residues. Overall, our results indicated that EP from Anguilla anguilla activated macrophages and could be used as a potential nutraceutical or pharmaceutical.

The inhibitory activity of alginate against allergic reactions in an ovalbumin-induced mouse model.

Marine seaweed polysaccharides have been considered as a potential resource for antiallergic therapy. Alginate is an acidic linear polysaccharide and soluble dietary fiber that was extracted from brown algae, Laminaria japonica. The molecular weight of alginate was 108 kDa, and its water solution exhibited non-Newtonian characteristics, including viscoelasticity and shear-thinning behavior. The ability of alginate to inhibit allergic reactions was investigated in ovalbumin (OVA)-induced BALB/c mice, which have been widely used as a mouse model of egg allergy. The results showed that alginate could effectively attenuate the occurrence of allergic reactions, including improving the integrity of the intestinal epithelial villi and inhibition of mast cell degranulation in the jejunum, in OVA-induced mice. Moreover, after treatment with alginate, the levels of IgE, histamine and IL-4 in OVA-induced mice were remarkably decreased, and the levels of IFN-γ were markedly increased. In addition, the number of Treg cells in spleen tissues in OVA-induced mice was increased by alginate, and the OVA-induced differentiation of Th0 cells into Th2 cells was significantly inhibited. These results demonstrate that alginate possesses potential antiallergic activities in a mouse model of egg allergy, which might provide important evidence that alginate, extracted from Laminaria japonica, can be developed into a novel functional food for inhibiting egg allergy.

Specific Degradation of Endogenous Tau Protein and Inhibition of Tau Fibrillation by Tanshinone IIA through the Ubiquitin-Proteasome Pathway.

Alzheimer's disease (AD) is a common neurodegenerative disease which is partly characterized by the aggregation of hyperphosphorylated Tau proteins forming neurofibrillary tangles that promote AD pathogenesis. In this study, we investigated the effects of tanshinone IIA (Tan IIA) isolated from Salvia miltiorrhiza on Tau degradation in the treatment of AD. The results showed that Tan IIA reduced the Tau expression and attenuated Tau phosphorylation in N2a cells, Tau-overexpressing cells, and 3×Tg-AD mouse primary neuron cells. Moreover, Tan IIA increased polyubiquitinated Tau accumulation and induced proteasomal degradation of the Tau protein. Additionally, Tan IIA became bound to the Tau protein and inhibited the formation of heparin-induced Tau fibrils. In summary, Tan IIA can increase polyubiquitinated Tau accumulation and induce the proteasomal degradation of the Tau protein and the binding of Tan IIA to the Tau protein, inhibiting the formation of Tau fibrils. Tan IIA may be further explored as a potential candidate for AD treatment.

Characterization and Neuroprotection Potential of Seleno-Polymannuronate.

Seleno-polymannuronate (Se-PM) was prepared from alginate-derived polymannuronate (PM) through a sulfation followed by a selenylation replacement reaction. The organic selenium content of Se-PM was 437.25 µg/g and its average molecular weight was 2.36 kDa. The neuroprotection effect of Se-PM and corresponding molecular mechanisms were investigated. Our results showed that, comparing to both sulfated PM (S-PM) and PM, Se-PM remarkably inhibited the aggregation of Aß1-42 oligomer in vitro and significantly reduced the APP and BACE1 protein expression in N2a-sw cells, highlighting the critical function of the selenium presented in Se-PM. Moreover, Se-PM decreased the expression of cytochrome c and the ratio of Bax to Bcl-2, and enhanced the mitochondrial membrane potential in N2a-sw cells. These results suggested that Se-PM treatment can markedly inhibit N2a-sw cell apoptosis and promote N2a-sw cell survival and that Se-PM might be a potential therapeutic agent for the prevention of neurodegeneration owing to its remarkable neuroprotection effect.