Cnidium officinale polysaccharide enhanced RAW 264.7 cells activation and NK-92 cells cytotoxicity against colon cancer via NF-κB and MAPKs signaling pathways.

In this study, Cnidium officinale-derived polysaccharides were isolated and investigated for their immune enhancing and anticancer activities. The isolated crude and its fractions, such as F1 and F2, contain carbohydrates (51.3-63.1%), sulfates (5.4-5.8%), proteins (1.5-7.1%), and uronic acids (2.1-26.9%). The molecular weight (Mw) of the polysaccharides ranged from 59.9 to 429.0 × 103 g/mol. The immunostimulatory activity of the polysaccharides was tested on RAW 264.7 cells, and the results showed that the F2 treatment notably enhanced pro-inflammatory activity in RAW 264.7 cells by increasing NO production and the expression of various cytokines. Furthermore, the influence of polysaccharide treatment on natural killer cells (NK-92) anticancer activities was investigated using a colon cancer cell line (HCT-116). Crude polysaccharide and its fractions showed no direct cytotoxicity to NK-92 and HCT-116 cells. However, the treatment of F2 showed an enhancement of NK-92 cells cytotoxicity against HCT-116 cells by upregulating the mRNA expression of IFN-γ, TNF-α, NKGp44, and granzyme-B. The western blot results showed that the induced RAW 264.7 cells activation and NK-92 cells cytotoxicity occur via NF-κB and MAPK signaling pathways. Overall, C. officinale-derived polysaccharides show potential as immunotherapeutic agents capable of enhancing pro-inflammatory macrophage signaling and activating NK-92 cells; thus, they could be useful for biomedical applications.

Structural properties and immune-enhancing activities of galactan isolated from red seaweed Grateloupia filicina.

A water-soluble polysaccharide (GFP) was isolated from Grateloupia filicina and fractionated using a DEAE Sepharose Fast Flow column to evaluate immunostimulatory activity. Carbohydrates (62.0%-68.4%) and sulfates (29.3%-34.3%) were the major components of GFP and its fractions (GFP-1 and GFP-2), with relatively lower levels of proteins (4.5%-15.4%) and uronic acid (1.4%-3.9%). The average molecular weight (Mw ) for GFP and its fractions was calculated between 98.2%-243.7 kDa. The polysaccharides were composed of galactose (62.1%-87.2%), glucose (4.5%-33.2%), xylose (3.1%-5.3%), mannose (1.4%-2.2%), rhamnose (1.2%-2.0%), and arabinose (0.9%-1.7%) units connected through →3)-Galp-(1→, →4)-Galp-(1→, →2)-Galp-(1→, →6)-Galp-(1→, →3,4)-Galp -(1→, →3,6)-Galp-(1→, →4,6)-Galp-(1→, →3,4,6)-Galp-(1→, →2,3)-Galp-(1→, →2,4)-Galp-(1→, →4)-Glcp-(1→, →6)-Glcp-(1→ and →4,6)-Glcp-(1→residues. The isolated polysaccharides effectively induced RAW264.7 murine macrophages by releasing nitric oxide (NO) and various cytokines via nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Further, the expression of toll-like receptor-2 (TLR-2) and TLR-4 in RAW264.7 cells indicated their activation through TLR-2 and TLR-4 binding receptors. Among the polysaccharides, GFP-1 highly stimulated the activation of RAW264.7 cells, which was mainly constituted of (→1) terminal-D-galactopyranosyl, (1→3)-linked-ᴅ-galactopyranosyl, (1→4)-linked-ᴅ-galactopyranosyl and (1→3,4) -linked-ᴅ-galactopyranosyl residues. These findings demonstrate that GFP-1 from G. filicina are effective at stimulating the immune system and this warrants further investigation to determine potential biomedical applications.

Extraction, structural elucidation and immunostimulating properties of water-soluble polysaccharides from wheat bran.

A water-soluble polysaccharide was extracted from wheat bran (WBP) and investigate their structural characteristics and immunostimulatory activities. The chemical composition of WBP and purified fraction (WBP-F) mainly consists of neutral sugars (91.2 ± 1.2 and 98.7 ± 1.2%), proteins (8.6 ± 0.3 and 0.2 ± 0.1%) and uronic acids (0.7 ± 0.1 and 0.6 ± 0.1%). The molecular weight (Mw ) of WBP and WBP-F was calculated as 911.7 and 510.2 × 103  g/mol, respectively. The WBP-F stimulates the RAW264.7 cells through the production of nitric oxide and various cytokines. The treatment of WBP-F facilitated the phosphorylation of P38, JNK, ERK, and NF-ƘB in RAW264.7 cells suggesting that they might stimulate RAW264.7 cells through the activation of NF-ƙB and MAPKs pathways. Furthermore, the structural details of WBP-F were studied by GC-MS and NMR spectrum, which confirms that the main backbone consists of 4-α-D-linked glucopyranosyl residues with branching points at C-6. PRACTICAL APPLICATIONS: Wheat bran is a potential source of health-promoting compounds. It has been reported that polysaccharides of wheat bran containing numerous beneficial activities. In this study, the wheat bran polysaccharide was extracted, fractionated and investigated their immunostimulatory activities. The results found in this study revealed that the purified polysaccharide from wheat bran potentially enhanced the RAW264.7 cells activation. Hence, these polysaccharides could be utilized as a potent immunity-enhancing agent in food and pharmaceutical industries.

Inducing inflammatory response in RAW264.7 and NK-92 cells by an arabinogalactan isolated from Ferula gummosa via NF-κB and MAPK signaling pathways.

The polysaccharide isolated from F. gummosa (FGP) was found homogenous with a weight average molecular weight (Mw) of 50.0 × 103 g/mol and radius of gyration (Rg) of 105.3 nm. The FGP was an arabinogalactan with a backbone formed of →6)-ß-Galp-1→ residues having random branching points at C-3 extended with either ß-Galp-(1→3)-ß-Galp-(1→ or α-Araf-(1→ side chain residues. FGP exhibited proliferative effect on RAW264.7 cells and induced macrophages to exert proinflammatory response releasing NO and up-regulating the transcription of cytokines including TNF-α, IL-1ß, IL-6 and IL-12. The FGP induced NK-92 cells to up-regulate the expressions of TNF-α, IFN-γ, granzyme-B, perforin, NKG2D and FasL. The presence of p-NF- κB, p-ERK, p-JNK and p-p38 in RAW264.7 and NK-92 cells indicated their activation through NF-κB and MAPKs signaling pathways. These findings suggested that polysaccharides from F. gummosa are potent in boosting immune system and thus may be considered for further studies of biomedical applications.

Sulfated galactan from Halymenia dilatata enhance the antioxidant properties and prevents Aeromonas hydrophila infection in tilapia fish: In vitro and in vivo study.

The structural characterization and pharmaceutical perspective of sulfated galactan from Halymenia dilatata (Hd-SG) were reported in this study. The Hd-SG consists of carbohydrate (58.5 ± 0.9%), sulfate (28.7 ± 0.9%) and protein (2.7 ± 0.2%). The existence of carbon (28.14%), hydrogen (5.50%), nitrogen (0.51%) and sulfur (8.26%) was confirmed in CHNS analysis. The Hd-SG was mainly comprising of galactose and mannose connected by (1 â†’ 4)-glycosidic linkages, and it shows the molecular weight of 900.9 × 103 g/mol in high-performance size exclusion chromatography (HPSEC). The Hd-SG exhibited the dose depended on antioxidant activities. The in vitro and in vivo studies proved the antibacterial efficacy of Hd-SG against Aeromonas hydrophila. The pre-treated Oreochromis fish with Hd-SG (2.0 g/0.1 kg of feed) showed the highest survival, antioxidant, and improved histological changes than the fish infected with A. hydrophila alone. These results concluded that the isolated Hd-SG has extensive therapeutic properties, and it can be used as preventive medicine.

The activation of NF-κB and MAPKs signaling pathways of RAW264.7 murine macrophages and natural killer cells by fucoidan from Nizamuddinia zanardinii.

Polysaccharides from Nizamuddinia zanardinii were extracted using water at elevated temperature and fractionated by a DEAE Sepharose FF column yielding four fractions (F1-F4). Crude and fractions were composed of neutral sugars (50.8-57.4%), proteins (10.8-18.1%), sulfates (7.5-17.3%) and uronic acids (3.5-7.7%). Various levels of galactose (13.4-44.4%), fucose (34.1-40.1%), mannose (14.1-33.2%) and xylose (7.4-15.2%) formed the building blocks of the polysaccharide structures. The weight average molecular weights (Mw) of polysaccharides varied between 40.3 and 1254.4 × 103 g/mol. F3 polysaccharide was the most active fraction stimulating RAW264.7 murine macrophage cells to secrete NO, TNF-α, IL-1ß and IL-6, and activating NK cells to release TNF-α, INF-γ, granzyme-B, perforin, NKG2D and FasL through NF-κB and MAPKs signaling pathways. Highly-branched F3 polysaccharide mainly consisted of (1 â†’ 2)-Fucp, (1 â†’ 2,3)-Manp, (1 â†’ 3)-Galp, (1 â†’ 2)-Manp, (1 â†’ 3)-Manp, (1 â†’ 2,3,4)-Manp and (1 â†’ 2,3,6)-Manp residues with great amount of (→1)-Fucp and (→1)-Xylp. Sulfates substituted at C-2 of fucose and galactose residues. Overall, fucoidan from N. zanardinii showed immense potency in boosting immune system through macrophages and NK cells activations and therefore suitable for further exploration in immune-mediated biomedical applications.

Isolation, structural elucidation and immuno-stimulatory properties of polysaccharides from Cuminum cyminum.

The aim of the present study was to evaluate the effect of water-soluble polysaccharides from Cuminum cyminum to induce inflammatory response in immune cells and understand their underlying mechanisms. Weight average molecular weight (Mw) of polysaccharides varied between 191.4-512.2 × 103 g/mol. Polysaccharides induced RAW264.7 cells to release nitric oxide and express TNF-α, IL-1ß, IL-6 and IL-12 inflammatory cytokines. Polysaccharides activated NK-92 cells to produce TNF-α, IFN-γ, perforin, granzyme B, NKG2D and FasL. Activations of RAW264.7 and NK-92 cells were through NF-κB and MAPKs signal pathways indicated by the presence of phosphorylated NF-κB, ERK, JNK and p38 proteins. The polysaccharide structure was mainly constituted of →4)-Galp-(1→, →3)-Galp-(1→, →2)-Arap-(1→ and →2)-Arap-(1→ glycosidic linkages. Overall results suggested that polysaccharides from C. cyminum possessing lower MW and greater expanded conformation more effectively stimulate RAW264.7 and NK-92 cells and thus could be considered for further studies on their biomedical applications.

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.

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.