Optimization of the Preparation Process of Glucuronomannan Oligosaccharides and Their Effects on the Gut Microbiota in MPTP-Induced PD Model Mice.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, and accumulating evidence suggests a link between dysbiosis of the gut microbiota and the onset and progression of PD. In our previous investigations, we discovered that intraperitoneal administration of glucuronomannan oligosaccharides (GMn) derived from Saccharina japonica exhibited neuroprotective effects in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. However, the complicated preparation process, difficulties in isolation, and remarkably low yield have constrained further exploration of GMn. In this study, we optimized the degradation conditions in the preparation process of GMn through orthogonal experiments. Subsequently, an MPTP-induced PD model was established, followed by oral administration of GMn. Through a stepwise optimization, we successfully increased the yield of GMn, separated from crude fucoidan, from 1~2/10,000 to 4~8/1000 and indicated the effects on the amelioration of MPTP-induced motor deficits, preservation of dopamine neurons, and elevation in striatal neurotransmitter levels. Importantly, GMn mitigated gut microbiota dysbiosis induced by MPTP in mice. In particular, GM2 significantly reduced the levels of Akkermansia, Verrucomicrobiota, and Lactobacillus, while promoting the abundance of Roseburia and Prevotella compared to the model group. These findings suggest that GM2 can potentially suppress PD by modulating the gut microbiota, providing a foundation for the development of a novel and effective anti-PD marine drug.

Comparison of anti-tumor activities and underlying mechanisms of glucuronomannan oligosaccharides and its sulfated derivatives on the hepatocarcinoma Huh7.5 cells.

Hepatocellular carcinoma (HCC) is an aggressive tumor triggered by various factors such as virus infection and alcohol abuse. Glucuronomannan polysaccharide (Gx) is a subtype of fucoidans that possesses many bioactivities, but its anti-tumor activities in HCC have not been reported. In this paper, the anti-tumor effects of glucuronomannan oligosaccharides (Gx) and its sulfated derivatives (GxSy) on hepatocarcinoma Huh7.5 cells were investigated. The anti-proliferation, anti-metastasis activities, and underlying mechanism of Gx and GxSy on Huh7.5 cells were analyzed and compared by MTT, wound healing, transwell, and western blotting assays, respectively. Results showed that the best anti-proliferation effects were G4S1 and G4S2 among 13 drugs, which were 38.67% and 30.14%, respectively. The cell migration rates were significantly inhibited by G2S1, G4S2, G6S2, and unsulfated Gn. In addition, cell invasion effects treated with G4S1, G4S2, and G6S1 decreased to 48.62%, 36.26%, and 42.86%, respectively. Furthermore, sulfated G4 regulated the expression of (p-) FAK and MAPK pathway, and sulfated G6 down-regulated the MAPK signaling pathway while activating the PI3K/AKT pathway. On the contrary, sulfated G2 and unsulfated Gx had no inhibited effects on the FAK-mTOR pathway. These results indicated that sulfated Gx derivatives have better anti-tumor activities than unsulfated Gx in cell proliferation and metastasis process in vitro, and those properties depend on the sulfation group levels. Moreover, degrees of polymerization of Gx also played a vital role in mechanisms and bioactivities. This finding shows the structure-activity relationship for developing and applying the marine oligosaccharide candidates.

Glucuronomannan GM2 from Saccharina japonica Enhanced Mitochondrial Function and Autophagy in a Parkinson's Model.

Parkinson's disease (PD), one of the most common neurodegenerative disorders, is caused by dopamine depletion in the striatum and dopaminergic neuron degeneration in the substantia nigra. In our previous study, we hydrolyzed the fucoidan from Saccharina japonica, obtaining three glucuronomannan oligosaccharides (GMn; GM1, GM2, and GM3) and found that GMn ameliorated behavioral deficits in Parkinsonism mice and downregulated the apoptotic signaling pathway, especially with GM2 showing a more effective role in neuroprotection. However, the neuroprotective mechanism is unclear. Therefore, in this study, we aimed to assess the neuroprotective effects of GM2 in vivo and in vitro. We applied GM2 in 1-methyl-4-phenylpyridinium (MPP+)-treated PC12 cells, and the results showed that GM2 markedly improved the cell viability and mitochondrial membrane potential, inhibited MPP+-induced apoptosis, and enhanced autophagy. Furthermore, GM2 contributed to reducing the loss of dopaminergic neurons in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice through enhancing autophagy. These data indicate that a possible protection of mitochondria and upregulation of autophagy might underlie the observed neuroprotective effects, suggesting that GM2 has potential as a promising multifunctional lead disease-modifying therapy for PD. These findings might pave the way for additional treatment strategies utilizing carbohydrate drugs in PD.

Structural characteristics and anti-complement activities of polysaccharides from Sargassum hemiphyllum.

Three polysaccharides (SH-1, SH-2 and SH-3) were purified from a brown macroalgea, Sargassum hemiphyllum. The autohydrolysis products from each polysaccharide were separated to three fractions (S fractions as oligomers, L fractions as low molecular weight polysaccharides and H fractions as high molecular weight polysaccharides). Mass spectroscopy of S fractions (SH-1-S, SH-2-S and SH-3-S) showed that these three polymers all contained short stretches of sulfated fucose. The structures of L fractions (SH-1-L, SH-2-L and SH-3-L) were determined by nuclear magnetic resonance (NMR). SH-1-L was composed of two units, unit A (sulfated galactofucan) and unit B (sulfated xylo-glucuronomannan). Unit A contained a backbone of (1, 6-linked ß-D-Gal) n1, (1, 3-linked 4-sulfated α-L-Fuc) n2, (1, 3-linked 2, 4-di-sulfated α-L-Fuc) n3, (1, 4-linked α-L-Fuc) n4 and (1, 3-linked ß-D-Gal) n5, accompanied by some branches, such as sulfated fuco-oligomers, sulfated galacto-oligomers or sulfated galacto-fuco-oligomers. And unit B consisted of alternating 1, 4-linked ß-D-glucuronic acid (GlcA) and 1, 2-linked α-D-mannose (Man) with the Man residues randomly sulfated at C6 or branched with xylose (Xyl) at C3. Both SH-2-L and SH-3-L were composed of unit A and their difference was attributed to the ratio of n1: n2: n3: n4: n5. Based on monosaccharide analysis, we hypothesize that both SH-1-H and SH-2-H contained unit A and unit B while SH-3-H had a structure similar to SH-3-L. An assessment of anti-complement activities showed that the sulfated galactofucan had higher activities than sulfated galacto-fuco-xylo-glucuronomannan. These results suggest that the sulfated galactofucans might be a good candidate for anti-complement drugs.

Preparation and Neuroprotective Activity of Glucuronomannan Oligosaccharides in an MPTP-Induced Parkinson's Model.

Parkinson's disease (PD), characterized by dopaminergic neuron degeneration in the substantia nigra and dopamine depletion in the striatum, affects up to 1% of the global population over 50 years of age. Our previous study found that a heteropolysaccharide from Saccharina japonica exhibits neuroprotective effects through antioxidative stress. In view of its high molecular weight and complex structure, we degraded the polysaccharide and subsequently obtained four oligosaccharides. In this study, we aimed to further detect the neuroprotective mechanism of the oligosaccharides. We applied MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to induce PD, and glucuronomannan oligosaccharides (GMn) was subsequently administered. Results showed that GMn ameliorated behavioral deficits in Parkinsonism mice. Furthermore, we observed that glucuronomannan oligosaccharides contributed to down-regulating the apoptotic signaling pathway through enhancing the expression of tyrosine hydroxylase (TH) in dopaminergic neurons. These results suggest that glucuronomannan oligosaccharides protect dopaminergic neurons from apoptosis in PD mice.

Structural Features of Sulfated Glucuronomannan Oligosaccharides and Their Antioxidant Activity.

Glucuronomannan oligosaccharides (Gs) were derived from fucoidan, which was extracted from the brown alga Sargassum thunbergii. Sulfated glucuronomannan oligosaccharides (SGs) were obtained by the sulfation of Gs. NMR techniques were used to reveal that the order of sulfation was Man-C6 > Man-C4 > Man-C1R > GlcA-C3 > Man-C3 > GlcA-C2. Finally, the antioxidant activities (hydroxyl radical scavenging activity, superoxide radical scavenging activity, reducing power and DPPH radical scavenging activity) of Gs and SGs were determined. The findings showed that the higher the degree of polymerization, the better the activity, except for the hydroxyl radical scavenging activity. In addition, the higher the sulfate content, the lower the activities for the reducing power and DPPH radical scavenging activity. Opposite results were found for the superoxide radical scavenging activity. Finally, compared with fucoidan, most Gs and SGs had higher antioxidant activity, suggesting that they might be good candidates for antioxidants.

Oligosaccharides from Sargassum thunbergii inhibit osteoclast differentiation via regulation of IRF-8 signaling.

Osteoporosis (OP) is a systemic bone degenerative disease characterized by low bone mass and deteriorated microarchitecture of bone tissue, causing high morbidity and mortality rates. Bone resorption by overactivated osteoclasts (OCs) is the main cause of osteoporosis. Glucuronomannan and its oligomers (Gs) and their sulfated derivatives (SGs) were previously prepared. The anti-osteoporosis activities of these glycans were evaluated. Firstly, we determined the viability of RAW264.7 by CCK-8 test. Nextly, we investigated the inhibitory effects of Gs and SGs on the differentiation of RAW264.7 cells into OCs using tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring staining, qualitative reverse-transcription polymerase chain reaction(qRT-PCR) and western blotting. TRAP staining revealed that Gs significantly blocked RANKL-induced OC generation while SGs did not exhibit this ability. F-actin staining assays demonstrated that Gs inhibits RANKL-induced actin ring formation. qRT-PCR analyses indicated that Gs dose-dependently inhibited the expression of OCs marker genes including Trap, NFATc1, c-Fos, DC-Stamp and ATP60 during the differentiation process, while SGs did not suppress. Regarding the mechanism of Gs, it was found that Gs suppressed osteoclastogenesis via inhibiting the degradation of IRF-8 and interfering with NF-κB pathway activation. Together, these results suggest that Gs have the ability to inhibit osteoclastogenesis by modulating IRF-8 signaling.

Sulphated glucuronomannan tetramer and hexamer from Sargassum thunbergii exhibit anti-human cytomegalovirus activity by blocking viral entry.

Human cytomegalovirus (HCMV) remains a major public health burden worldwide. The anti-HCMV activity of glucuronomannan oligosaccharides (Gs) and sulphated glucuronomannan oligosaccharides (SGs) was investigated. Among these Gs and SGs, G4S1 and G6S1 (higher sulphated glucuronomannan tetramer and hexamer) showed satisfactory anti-HCMV activity starting at 50 µg/mL and 10 µg/mL, respectively. The results of the morphology, western blotting, qPCR and TCID50 assay showed that they prevented lytic cytopathic changes, inhibited the expression of IE1/2 and UL44, and reduced the UL123 copy number and virus titre significantly. It was interesting to note that degree of sulphation and polymerization was more important for anti-HCMV activity. Moreover, the anti-HCMV activities of G4S1 and G6S1 were stable when stored at 4 °C, -20 °C, and -80 °C for at least three months and mainly occurred in the early stage of HCMV infection through the negative charge of the sulphate groups and the interaction between SGs and the host cells.

Sulfated glucuronomannan hexasaccharide G6S1 enhanced lipolysis and lipophagy via PPARα pathway.

Nonalcoholic fatty liver disease (NAFLD) is considered as the hepatic manifestation of metabolic syndrome, ranging from benign steatosis to severe non-alcoholic steatohepatitis. Recently, it has been found that lipophagy plays a pivotal role in lipid turnover, which can alleviate NAFLD in hepatocytes. In this study, we found that a highly sulfated glucuronomannan hexamer G6S1 has the ability to enhance lipophagy. When treated with G6S1, the number and the size of lipid droplet (LD) decreased significantly on hepatocytes AML12 cells. Western blot results showed that the expressions of the lipolysis-related proteins increased, while the expressions of proteins that is responsible for lipid transportation and synthesis exhibited no significant change. Immunofluorescence assay and electron microscopy results showed an increase of autophagy related protein expression level and lysosome number in hepatocytes treated with G6S1, suggesting that G6S1 could also promote lipophagy. A significant increase of peroxisome proliferator-activated receptor alpha (PPARα) expression level was detected in G6S1 treated cells, suggesting that G6S1 may promote autophagy via enhancing the expression of PPARα. In addition, these effects could be inhibited after treatment with autophagy inhibitor 3-methyladenine (3-MA) and PPARα inhibitor MK-886. These findings indicate that G6S1 can promote lipophagy via enhanced PPARα expression and can result in a slowdown of lipids accumulation.

The structure-activity relationship of the interactions of SARS-CoV-2 spike glycoproteins with glucuronomannan and sulfated galactofucan from Saccharina japonica.

The SARS-CoV-2 spike glycoproteins (SGPs) and human angiotensin converting enzyme 2 (ACE2) are the two key targets for the prevention and treatment of COVID-19. Host cell surface heparan sulfate (HS) is believed to interact with SARS-CoV-2 SGPs to facilitate host cell entry. In the current study, a series of polysaccharides from Saccharina japonica were prepared to investigate the structure-activity relationship on the binding abilities of polysaccharides (oligosaccharides) to pseudotype particles, including SARS-CoV-2 SGPs, and ACE2 using surface plasmon resonance. Sulfated galactofucan (SJ-D-S-H) and glucuronomannan (Gn) displayed strongly inhibited interaction between SARS-CoV-2 SGPs and heparin while showing negligible inhibition of the interaction between SARS-CoV-2 SGPs and ACE2. The IC50 values of SJ-D-S-H and Gn in blocking heparin SGP binding were 27 and 231 nM, respectively. NMR analysis showed that the structure of SJ-D-S-H featured with a backbone of 1, 3-linked α-L-Fucp residues sulfated at C4 and C2/C4 and 1, 3-linked α-L-Fucp residues sulfated at C4 and branched with 1, 6-linked ß-D-galacto-biose; Gn had a backbone of alternating 1, 4-linked ß-D-GlcAp residues and 1, 2-linked α-D-Manp residues. The sulfated galactofucan and glucuronomannan showed strong binding ability to SARS-CoV-2 SGPs, suggesting that these polysaccharides might be good candidates for preventing and/or treating SARS-CoV-2.

Inhibition of glucuronomannan hexamer on the proliferation of lung cancer through binding with immunoglobulin G.

The anti-lung cancer activity of oligosaccharides derived from glucuronomannan was investigated. The inhibition of A549 cell proliferation by glucuronomannan (Gn) and its oligomers (dimer (G2), tetramer (G4) and hexamer (G6)) were concentration dependent. In vivo activities on the A549-derived tumor xenografts showed the tumor inhibition of G2, G4 and G6 were 17 %, 40 % and 46 %, respectively. Organ coefficients in nude mice showed an increase in the kidney with G4, the brain with G6, and the spleen with G6. An advanced tandem mass tag labeled proteomics approach was performed. A significant differential expression was found in 59 out of the 4371 proteins, which involved the immune system. Surface plasmon resonance (SPR) studies revealed G6 was strongly bound to immunoglobulin G. This suggests that glucuronomannan hexamer inhibits the proliferation of lung cancer through its binding to immunoglobulin.

Structural analysis of a novel sulfated galacto-fuco-xylo-glucurono-mannan from Sargassum fusiforme and its anti-lung cancer activity.

Polysaccharide (HFSGF) was purified from Sargassum fusiforme. Autohydrolysis and gel column chromatography were performed to fractionate HFSGF into three components (HFSGF-S, HFSGF-L and HFSGF-H). Compositional analysis, mass spectrometry and nuclear magnetic resonance spectroscopy were used to elucidate the structural features of HFSGF. HFSGF-S was a mixture of sulfated galacto-fuco-oligomers, from the branches terminal ends; in HFSGF-L, the branches of HFSGF, was a sulfated galactofucan, containing a backbone of 1,3-linked α-L-fucan sulfated at C2/4 and/or C4 and interspersed with galactose (Gal); and in HFSGF-H, the backbone of HFSGF, was composed of alternating 1,2-linked α-D-mannose (Man) and 1,4-linked ß-D-glucuronic acid (GlcA), branched with sulfated galactofucan or sulfated fucan, 1,3-linked α-L-fucan sulfated at C2/4 and/or C4 and partly interspersed with Gal. Some fucose (Fuc) residues were also partially branched with xylose (Xyl). The anti-lung cancer activities of HFSGF-L and HFSGF-H against human lung cancer A549 cells in vitro and A549 xenograft tumor growth in vivo were determined. HFSGF-H had higher activity in vitro (IC50 ~12 mg/mL for 24 h) and in vivo (tumor inhibition ~51%.) than HFSGF-L, indicating that HFSGF-H might be a leading compound for a potential new therapeutics for the treatment of lung cancer.

Sulfated polysaccharides of the Vietnamese brown alga Sargassum aquifolium (Fucales, Sargassaceae).

A fucoidan preparation named FSA was isolated from the brown alga Sargassum aquifolium collected from the coastal waters of Vietnam. l-Fucose, d-galactose, d-mannose, d-glucuronic acid, d-xylose, and sulfate were found to be the main constituents of FSA. The preparation was fractionated by anion-exchange chromatography on DEAE-Sephacel eluted stepwise with 0.5, 1.0, 1.5, and 2.0 M NaCl to give four fractions differing in monosaccharide composition and degree of sulfation. Their NMR spectra were too complex to be completely interpreted. Fractions 1.0 M and 1.5 M were analyzed by methylation before and after desulfation. In addition, desulfated 1.0 M was fractionated by anion-exchange chromatography into six fractions according to the uronic acid content. They were characterized by methylation and NMR spectral data, and three structurally different polysaccharides were identified. One of them has a core of alternating 2-linked α-d-Manp and 4-linked ß-d-GlcpA residues, about a half of the former bearing single α-l-Fucp or ß-d-Xylp at position 3. The second polymer is a (1 â†’ 3)-ß-d-glucopyranuronan partially substituted with single ß-d-Xylp or single α-l-Fucp at position 4. The third polysaccharide is a xylo(fuco)galactan having a linear core of alternating 4-linked α-d-Gal and 3-linked ß-d-Gal residues. The latter bear single ß-d-Xylp or a short chain of 4-linked ß-d-Xyl, 6-linked ß-d-Gal, and variously linked α-l-Fuc. In FSA, these polysaccharides are sulfated at different positions and devoid of regularity. Fractions of FSA possess anticoagulant, cytotoxic, and antitumor activities, which increase with the degree of sulfation. The most sulfated fraction 2.0 M that contains mainly a sulfated fucogalactan, is about half as active as anticoagulant as the standard low-molecular mass heparin (enoxaparin).

Structure of a shear-thickening polysaccharide extracted from the New Zealand black tree fern, Cyathea medullaris.

A shear-thickening water-soluble polysaccharide was purified from mucilage extracted from the fronds of the New Zealand black tree fern (Cyathea medullaris or 'mamaku' in Maori) and its structure characterised. Constituent sugar analysis by three complementary methods, combined with linkage analysis (of carboxyl reduced samples) and 1H and 13C nuclear magnetic resonance spectroscopy (NMR) revealed a glucuronomannan comprising a backbone of 4-linked methylesterified glucopyranosyl uronic acid and 2-linked mannopyranosyl residues, branched at O-3 of 45% and at both O-3 and O-4 of 53% of the mannopyranosyl residues with side chains likely comprising terminal xylopyranosyl, terminal galactopyranosyl, non-methylesterified terminal glucopyranosyl uronic acid and 3-linked glucopyranosyl uronic acid residues. The weight-average molecular weight of the purified polysaccharide was ∼1.9×10(6) Da as determined by size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS). The distinctive rheological properties of this polysaccharide are discussed in relation to its structure.