Suaeda maritima polysaccharides attenuate LPS-induced inflammation of RAW264.7 cells and antioxidative activity.

Our research focused on extracting polysaccharides from Suaeda maritima (SMP) to obtain crude polysaccharides (SMP-C), which were subsequently purified into SMP-F1 and SMP-F2. SMPs were evaluated for anti-inflammatory effects and SMP-F1 showed the highest inhibitory effects on nitric oxide (NO) production. The monosaccharide composition analysis of SMP-F1 (molecular weight of 112.2 × 103 g/mol) revealed predominant levels of glucose (45.4 %), arabinose (20.5 %), mannose (14.2 %), and galactose (12.7 %). The primary backbone of SMP-F1 consisted of (1 â†’ 4)-D-glucopyranoside, (1 â†’ 4,6)-D-glucopyranoside, (1 â†’ 3)-D-mannopyranoside, (1 â†’ 3,6)-D-mannopyranoside, and (1 â†’ 5)-L-arabifuranoside. In addition, we hydrolysed SMP-F1 to SMP-H1, SMP-H2, and SMP-H3 and investigated their anti-inflammatory effects on RAW264.7 macrophages. Following SMP-F1 hydrolysis, SMP-H3 (molecular weight of 25.8 × 103 g/mol) exhibited superior anti-inflammatory properties compared to SMP-H1 and SMP-H2, demonstrating a significant decrease in NO production. SMP-H3 also demonstrated a remarkable reduction in the secretion of inflammatory mediators including NO, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines including tumour necrosis factor-alpha (TNF-α), interleukin (IL-1ß and IL-6), while increasing IL-10 expression. Furthermore, SMP-H3 significantly inhibited LPS-stimulated cluster of differentiation (CD) 11b and CD40 expression. Our subsequent investigation unveiled the involvement of SMP-H3-activated macrophages in the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Additionally, SMP-H3 exhibited antioxidant activity by scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide, and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) free radicals. These findings suggest the potential of SMP-H3 as an ingredient in the development of alternative drugs or functional foods.

In Vitro Anti-Inflammatory Activity and Structural Characteristics of Polysaccharides Extracted from Lobonema smithii Jellyfish.

Crude polysaccharides were extracted from the white jellyfish (Lobonema smithii) using water extraction and fractionated using ion-exchange chromatography to obtain three different fractions (JF1, JF2, and JF3). The chemical characteristics of four polysaccharides were investigated, along with their anti-inflammatory effect in LPS-stimulated RAW264.7 cells. All samples mainly consisted of neutral sugars with minor contents of proteins and sulphates in various proportions. Glucose, galactose, and mannose were the main constituents of the monosaccharides. The molecular weights of the crude polysaccharides and the JF1, JF2, and JF3 fractions were 865.0, 477.6, 524.1, and 293.0 kDa, respectively. All polysaccharides were able to decrease NO production, especially JF3, which showed inhibitory activity. JF3 effectively suppressed iNOS, COX-2, IL-1ß, IL-6, and TNF-α expression, while IL-10 expression was induced. JF3 could inhibit phosphorylated ERK, JNK, p38, and NF-κB p65. Furthermore, flow cytometry showed the impact of JF3 on inhibiting CD11b and CD40 expression. These results suggest that JF3 could inhibit NF-κB and MAPK-related inflammatory pathways. The structural characterisation revealed that (1→3)-linked glucopyranosyl, (1→3,6)-linked galactopyranosyl, and (1→3,6)-linked glucopyranosyl residues comprised the main backbone of JF3. Therefore, L. smithii polysaccharides exhibit good anti-inflammatory activity and could thus be applied as an alternative therapeutic agent against inflammation.

Inhibitory effects of polysaccharides from Korean ginseng berries on LPS-induced RAW264.7 macrophages.

Polysaccharides isolated from Korean ginseng berries (GBPs) have shown beneficial effects such as immunomodulatory, anti-inflammatory, anti-cancer, and anti-diabetic properties. However, little is known about anti-inflammatory effects of GBPs. Thus, the purpose of this study was to investigate anti-inflammatory properties of four fractions of GBPs, namely GBP-C, GBP-F1, GBP-F2, and GBP-F3, in macrophages. Their toxicities and effects on NO production in RAW264.7 cells were assessed by culturing cells with various concentrations of GBPs and stimulating cells with LPS. Furthermore, expression levels of inflammatory mediators, cytokines, cell surface molecules, and immune signaling pathways were evaluated in LPS-stimulated macrophages using different fractions of GBPs at 450 µg/mL. These GBPs activated LPS-stimulated RAW264.7 cells to significantly reduce NO production. They suppressed the expression of mRNA and cell surface molecules via MAPK and NF-κB pathways. Collectively, results revealed that all four GBP fractions showed anti-inflammatory effects, with GBP-F1 having a more efficient anti-inflammatory effect than GBP-C, GBP-F2, and GBP-F3. The structure of GBP-F1 mainly consists of 1 → 3)- Araf, 1 → 4)- Glcp, and 1 → 6)-Galp glycosidic linkages. These results demonstrate that GBPs can be employed as alternative natural sources of anti-inflammatory agents.

Preparation, Characterization, and Biological Properties of Hydroxyapatite from Bigeye Snapper (Priancanthus tayenus) Bone.

The optimum condition of acid hydrolysis for hydroxyapatite extraction from bigeye snapper (Priancanthus tayenus) bone and the effects of extraction time (10-60 min) and HCl concentration (2.0-5.0% w/v) on yield and hydroxyapatite properties were determined. The optimum extracted condition was found using 5% HCl for 60 min, which was 13.4% yield; 19.8 g/100 g Ca content; 9.6 g/100 g P content; 2.1 Ca/P ratio; L*, a*, b*; and ΔE as 84.5, 2.8, 16.5, and 15.6, respectively. The using of 5% NaOH solution was optimum for hydroxyapatite precipitation from the extracted solution. The characteristic and biological properties of the obtained hydroxyapatite were studied. Fourier transform infrared spectroscopy and X-ray diffraction results showed a good comparison between the extracted and commercial hydroxyapatite. The microstructure of the extracted hydroxyapatite from a scanning electron microscope showed an irregular and flat-plate shape, large surface area, and roughness. The extracted hydroxyapatite was non- and low-cytotoxicity at a concentration of 50 and 100-400 µg/mL, respectively. Bovine serum albumin (BSA) adsorption and desorption of hydroxyapatite was studied. An increasing BSA concentration, hydroxyapatite amount, and adsorption time significantly increased protein adsorption on hydroxyapatite. Protein desorption from BSA-loaded hydroxyapatite showed an increase of release initially in the first 4 days and became a steady release rate until 14 days.

Sulphation and Hydrolysis Improvements of Bioactivities, and Immuno-Modulatory Properties of Edible Amanita hemibapha Subspecies javanica (Corner and Bas) Mucilage Polysaccharide as a Potential in Personalized Functional Foods.

In this study, the mucilage polysaccharide (MP) from Amanita hemibapha subspecies javanica was prepared by hot water extraction and ethanol precipitation and then fractionated using anion-exchange chromatography equipped with a DEAE Sepharose fast flow column. The most immune-enhancing polysaccharide fraction 2 (MPF2) was subjected to a structural modification such as hydrolysis or over-sulphation. The sulphate and molecular weight (Mw) of over-sulphated (OS1-3) and hydrolysed (HS1-3) derivatives of MPF2 differed between 9.85% and 14.2% and 32.8 and 88.1 × 103 g/mol, respectively. Further, the immune-enhancing properties of MPF2 and its derivatives were tested on RAW264.7 and NK cells through various in vitro assays. Interestingly, a low molecular weight of HS1-3 significantly increased the nitric oxide (NO) production (p < 0.05) more than MPF2, indicating that Mw is a major factor in RAW264.7 cell stimulation. In addition, RAW264.7 cells produced various cytokines by up-regulating mRNA expression levels and the activation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. On the other hand, OS1-3-treated natural killer (NK) cells induced cytotoxicity in HepG2 cells through the expression of IFN-γ, Grandzyme-B, perforin, NKp30, and FasL. These results demonstrated that sulphate derivatives play an important role in NK cell activation. Further, this study also explores how polysaccharide binds to RAW264.7 and NK cells. MPF2 and HS3 may activate RAW264.7 cells via binding to TLR4 receptors, and OS2 could be activated through the CR3 signalling pathways.

Extraction, Structural Characterisation, and Immunomodulatory Properties of Edible Amanitahemibapha subspecies javanica (Corner and Bas) Mucilage Polysaccharide as a Potential of Functional Food.

This research aimed to extract mucilage polysaccharides (MP) from Amanita hemibapha subspecies javanica (Corner and Bas), and further fractionate them using anion-exchange chromatography, yielding two fractions (MPF1 and MPF2). The crude extract, and fractions mainly consisted of carbohydrates (83.5-93.2%) with minor amounts of proteins (5.40-7.20%), and sulphates (1.40-9.30%). Determination of the monosaccharide composition revealed that glucose was the major unit, followed by galactose, mannose, rhamnose, and arabinose. The average molecular weight (MW) of the crude extract and fractions was in the range 104.0-479.4 × 103 g/mol. Interestingly, the crude extract, and fractions did not cause any toxic effect in RAW264.7 cells. However, they stimulated the RAW264.7 cells to release nitric oxide and cytokines through the activation of nuclear factor-kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) pathways via cell surface TLR4. Structural analysis of the most immunestimulating extract fraction, MPF2, revealed that the main backbone consisted of α-D-(1→6)-glucopyranoside. These results suggest that the MPs derived from A. hemibapha subspecies javanica (Corner and Bas) are potent in enhancing immunity; hence, they can be used as a functional ingredient in food products.

Studies on structural properties and immune-enhancing activities of glycomannans from Schizophyllum commune.

Polysaccharides were extracted from Schizophyllum commune (a common mushroom) and their structural and immune-enhancing properties were investigated. Crude and fractions (F1 and F2) were composed of sugars (50.3-82.8%), proteins (1.46-20.1%), and sulfates (1.33-7.01%). Monosaccharide compositions of Cr and F1 were mainly composed of glucose (75.5% and 88.2%) with small amounts of mannose, galactose and xylose whereas the F2 was mainly composed of manose (55.2%) with minor amounts of galactose, glucose, and xylose. Their immune-enhancing activities were tested using RAW264.7 cells. Proliferation activity of RAW264.7 cells was over 100% after treatment with these polysaccharides. In addition, RAW264.7 cells produced large amounts of nitric oxide and various cytokines by up-regulating mRNA expression levels and the activation of nuclear factor-kappa (NF-κB) and mitogen-activated protein kinase (MAPK) after treatment with these polysaccharides. In addition, RAW264.7 cells were activated mainly through CR-3 and TLR-4 receptors. The backbone of F2 with excellent immune-enhancing activity was mainly linked by (1→3)-linked-mannopyranosyl and (1→2,3)-linked-mannopyranosyl residues.

Effect of sulfation and partial hydrolysis of polysaccharides from Polygonatum sibiricum on immune-enhancement.

The aqueous polysaccharide from Polygonatum sibiricum was extracted and fractionated using anion-exchange chromatography to obtain F1 fraction. The F1 was chemically sulfated and partially acid-hydrolyzed for the production of its over-sulfated (OS1,2,3) and hydrolyzed (HP1,2,3) derivatives, in which the sulfate content of OS1,2,3 was 7.5-17.1%, and the Mw of HP1,2,3 ranged from 18.2 × 103 to 57.3 × 103 g/mol. Considerable RAW264.7 cell activation was observed by HP1,2,3 with NO production of 34.9, 44.3 and 42.7 µM, respectively, as well as the mRNA expression of cytokines (IL-1ß, IL-6, IL-10 and IL-12). NK cell cytotoxicity against HT-29 cell was facilitated by OS1,2,3 treatment with the increased gene expressions of INF-γ, Granzyme-B, perforin, NKG2D, and FasL. RAW264.7 cells appeared to be activated via MR and TLR4 mediated signaling pathway, but CR3 and TRL2 might play a main role in stimulating NK cells. Overall, the present study suggests the potential application of polysaccharides from P. sibiricum in functional foods and pharmacological industries.

Molecular structures, chemical properties and biological activities of polysaccharide from Smilax glabra rhizome.

The water-soluble crude polysaccharides, extracted from the rhizome of Smilax glabra, were fractionated using an anion exchange chromatography, yielding two fractions, F1 and F2. The crude and fractions (F1 and F2) mainly consisted of carbohydrates (66.7%-91.1%), proteins (7.30%-23.9%) and minor amount of sulfates (1.60%-9.40%). Glucose was the major monosaccharide unit of the polysaccharides with different levels of sugar constituents including galactose, arabinose, rhamnose and mannose. The molecular weight (Mw) of crude and fractions ranged from 32,102-6.3 × 103 g/mol. The crude and fractions could stimulate RAW264.7 cells to release nitric oxide and cytokines via up-regulation of their mRNA expression by the activation of NF-κB and MAPKs pathways. The related pattern recognition receptors (PRRs) on the surface of the cells appeared to be TLR2 and CR3. The GC-MS analysis revealed that the main backbone of the most immune-enhancing F2 was (1 → 4)-linked glucose and galactose chain with minor linkages of (1 → 6)-galactose, (1 → 3)-mannose, (1 → 2)-rhamnose and (1 → 5)-arabinose with some branches at C-3 and C-4 rhamnose, or C-6 galactose.

Extraction, characterization and immunomodulatory property of pectic polysaccharide from pomegranate peels: Enzymatic vs conventional approach.

Molecular characteristics, structural properties and immunostimulatory activities of polysaccharides from pomegranate peel were evaluated after 0.1 M HCl, Cellic CTec2 and buffer extractions. The isolated polysaccharides were mainly formed of neutral sugars (32.1%-51.1%) and uronic acids (19.9%-30.8%) as well as varying levels of proteins (15.0%-39.5%). Different levels of sugars including glucose (44.9%-68.1%), galactose (14.6%-19.4%), mannose (3.4%-18.1%), arabinose (3.1%-18.1%) and rhamnose (3.5-6.0%) constructed the structure of isolated polysaccharides. The average molecular weight (Mw) of polysaccharides differed notably and ranged from 422.5 × 103 to 18,631.8 × 103 g/mol. Polysaccharide molecules obtained using Cellic CTec2 enzyme induced RAW264.7 murine macrophage cells to release considerable amount of inflammatory mediators including nitric oxide, IL-1ß, TNF-α, IL-6 and IL-10. The polysaccharide stimulation of macrophage cells initiated through NF-κB and MAPKs signaling pathways and activation of p-NF-κB, p-JNK, p-ERK and p-p38 proteins. The most potent immunostimulatory polysaccharides were consisted of (1 → 3)-linked glucose, (1 → 6)-linked galactose, (1 → 4)-linked mannose and (1 → 4)-linked arabinose residues with branching points at C6 and C3. These results indicated that the lower molecular weight of polysaccharides isolated with Cellic CTec2 enzyme could be one of the major determinant structural characteristics in stimulating macrophage cells.

Water-soluble polysaccharides from Ulva intestinalis: Molecular properties, structural elucidation and immunomodulatory activities.

Water-soluble sulfated polysaccharides extracted from Ulva intestinalis and fractionated using DEAE Sepharose fast flow column to identify their molecular properties and macrophage cells stimulating activities. Crude and fractions (F1 and F2) were formed of neutral sugars (58.7-74.7%), sulfates (6.2-24.5%), uronic acids (4.9-5.9%) and proteins (3.2-10.4%). Different levels of sugar constituents including rhamnose (30.1-39.1%), glucose (39.0-48.4%), galactose (0.0-15.8%), xylose (8.5-11.3) and arabinose (0.0-5.1%). The molecular weight (Mw) of crude and fractionated polysaccharides ranged from 87.1 × 103 to 194.1 × 103 (g/mol). Crude polysaccharides were not toxic to RAW264.7 cells and fractions induced cell proliferation. Fraction F1 stimulated RAW264.7 cells to release considerable amounts of nitric oxide, IL-1ß, TNF-α, IL-6, IL-10 and IL-12 cytokines. The main backbone of the most immunostimulating polysaccharide (F1) was consisted of mixed linkages of (1 â†’ 2)-linked rhamnose and (1 â†’ 2)-linked glucose residues.

Effects of sulfated fucan from the sea cucumber Stichopus japonicus on natural killer cell activation and cytotoxicity.

The aqueous crude sulfated fucan (SF) from Stichopus japonicus was extracted and fractionated using anion-exchange chromatography to obtain four fractions (F1, F2, F3 and F4) and to investigate their NK cell activation and cytotoxicity. The most potent NK cell cytotoxicity (45% at 250µg/mL) against HeLa cells was observed by F1 treatment, on the other hand, F3 and F4 treatment exhibited strong NK cell cytotoxicity (31-34% at 250µg/mL) against HepG2 and HT-29 cells. The SF treatment enhanced the activation of NK cells through the mRNA expression of IFN-γ, an activating receptor (NKp30), lysing proteins (perforin and granzyme-B) as well as a death ligand (FasL). However, the treatment of the SF derivatives, deproteinated-F1 and desulfated-F3 (DP-F1 and DS-F3), markedly lowered the levels of NK cell cytotoxicity and mRNA expression of the activating factors, suggesting that the protein and sulfate were pivotal for the interaction between the SF and NK cells. The antibody neutralization test revealed that complement receptor-3 (CR3) may be a critical receptor involved in NK cell activation by the SF.

Structural characterization of immunostimulating protein-sulfated fucan complex extracted from the body wall of a sea cucumber, Stichopus japonicus.

The water soluble protein-sulfated fucan (PSF) complex isolated from the body wall of Stichopus japonicus was fractionated using an anion-exchange chromatography to obtain four purified fractions (F1, F2, F3, and F4) and to investigate their structural characteristics and immuno-enhancing activities. The crude PSF and fractions mostly consisted of neutral sugars, proteins and sulfates in various proportions. Fucose was their major monosaccharide unit with small portion of mannose, glucose and galactose. The molecular weight (Mw) of the crude PSF and fractions ranged from 323.6×103 to 3630.0×103g/mol. The crude PSF, F2, and F3 were potent stimulator of RAW264.7 cells inducing marked nitric oxide (NO) and cytokines production. The glycosidic linkage of polysaccharides was evaluated using GC-MS and confirmed by 2D-NMR. The main backbone of highly immunostimulating F3 fraction was (1→3)-α-l-linked fucosyl residue with sulfation at C-2 and/or C-4.

Structural effects of sulfated polysaccharides from Codium fragile on NK cell activation and cytotoxicity.

The effects of sulfates and proteins of the sulfated polysaccharide-F2 (SP-F2) from Codium fragile on the NK cell activation and cytotoxicity were systematically investigated. The SP-F2 treatment significantly increased both NK cell proliferation (129%/100µg/mL) and their potent cytotoxic effects against HeLa cells (46%). The SP-F2 treatment appeared to enhance NK cell activation through the expression of the activating receptor, NKp30; the secretion of the cytokine, IFN-γ and the release of the lysing proteins, perforin and granzyme-B. However, the treatment of the SP-F2 derivatives, deproteinated and desulfated-F2 (DP-F2 and DS-F2), markedly lowered the mRNA expression levels of IFN-γ, granzyme-B, NKp30 and FasL, suggesting that the proteins and sulfates were essential for the interaction between the SP-F2 and NK cells. The antibody neutralization test revealed that CR3 might be a critical receptor involved in SP-F2 NK cell activation.

RAW264.7 Cell Activating Glucomannans Extracted from Rhizome of Polygonatum sibiricum.

Water-soluble polysaccharides isolated from the rhizome of Polygonatum sibiricum and fractionated using ion-exchange chromatography were investigated to determine their structure and immunostimulating activity. Crude and fractions (F1 and F2) consisted of carbohydrates (85.1~88.3%) with proteins (4.51~11.9%) and uronic acid (1.79~7.47%), and included different levels of mannose (62.3~76.3%), glucose (15.2~20.3%), galactose (4.35~15.3%), and arabinose (4.00~7.65%). The crude contained two peaks with molecular weights (Mw) of 151×103 and 31.8×103, but F1 and F2 exhibited one major peak with Mw of 103×103 and 628×103, respectively. Little immunostimulatory activity was observed by the crude; however, F1 and F2 significantly activated RAW264.7 cells to release nitric oxide and various cytokines, suggesting they were potent immunostimulators. The backbone of the most immunostimulating fraction (F1) was (1→4)-manno- and (1→4)-gluco-pyranosyl residues with galactose and glucose attached to O-6 of manno-pyranoside.

Structural characterization of sulfated arabinans extracted from Cladophora glomerata Kützing and their macrophage activation.

Water-soluble sulfated heteropolysaccharides were extracted from Cladophora glomerata Kützing and fractionated by ion-exchange chromatography, which yielded two subfractions, F1 and F2. The crude and fractionated polysaccharides (F1 and F2) mostly consisted of carbohydrates (62.8-74.5%) with various amounts of proteins (9.00-17.3%) and sulfates (16.5-23.5%), including different levels of arabinose (41.7-54.4%), galactose (13.5-39.0%), glucose (0.80-10.6%), xylose (6.84-13.4%), and rhamnose (0.20-2.83%). Based on the size exclusion chromatography (SEC) profiles, the crude and fractions mainly contained one peak with shoulders having molecular weight (Mw) ranges of 358-1,501 × 10(3). The F1 fraction stimulated RAW264.7 cells to produce considerable amounts of nitric oxide and cytokines compared to the crude and F2 fraction. The backbone of the most potent immunostimulating fraction (F1) was α-(1→4)-L-arabinopyranoside with galactose and xylose residues as branches at O-2 position, and sulfates mainly at O-2 position as well.

Characterization and immunomodulatory activities of polysaccharides from Spirogyra neglecta (Hassall) Kützing.

Sulfated polysaccharides (SP) isolated from freshwater green algae, Spirogyra neglecta (Hassall) Kützing, and fractionated SPs were examined to investigate their molecular characteristics and immunomodulatory activity. The crude and fractionated SPs (F1, F2, and F3) consisted mostly of carbohydrates (68.5-85.3%), uronic acids (3.2-4.9%), and sulfates (2.2-12.2%) with various amounts of proteins (2.6-17.1%). D-galactose (23.5-27.3%), D-glucose (11.5-24.8%), L-fucose (19.0-26.7%), and L-rhamnose (16.4-18.3%) were the major monosaccharide units of these SPs with different levels of L-arabinose (3.0-9.4%), D-xylose (4.6-9.8%), and D-mannose (0.4-2.3%). The SPs contained two sub-fractions with molecular weights (Mw) ranging from 164 × 10(3) to 1460 × 10(3) g/mol. The crude and fractionated SPs strongly stimulated murine macrophages, producing considerable amounts of nitric oxide and various cytokines via up-regulation of their mRNA expression by activation of nuclear factor-kappa B and mitogen-activated protein kinases pathways. The main backbone of the most immunoenhancing SP was (1→3)-L-Fucopyranoside, (1→4,6)-D-Glucopyranoside, and (1→4)-D-Galactopyranoside.

Exopolysaccharides from lactic acid bacteria: structural analysis, molecular weight effect on immunomodulation.

Exopolysaccharides (EPS) obtained from the culture medium of Lactobacillus confusus TISTR 1498 were investigated to determine their molecular characteristics and the effect of molecular weight (Mw) on immunomodulatory activity. The EPS mainly consisted of carbohydrates (81.9±2.4%) with only one type of monosaccharide, D-glucose, which was mostly connected by α-(1→6) glycosidic linkages. The EPS itself was unable to stimulate RAW264.7 cells to produce pro-inflammatory mediator nitric oxide (NO) and cytokines. However, considerable stimulation of RAW264.7 cells was observed by the low Mw of EPSs having Mw values≤70×10(3)g/mol. The partially hydrolyzed EPS stimulated RAW264.7 cells to induce considerable NO and various cytokine production such as TNF-α, IL-1ß, IL-6 and IL-10 via up-regulation of their mRNA expression. In addition, the degradation Iκ-B and the phosphorylation of c-Jun NH2-terminal kinase (JNK) were facilitated by BW-30 and MW-40, suggesting that the partially hydrolyzed EPS stimulated RAW264.7 cells through the activation of NF-κB and JNK pathways.