The anti-inflammatory activity of specific-sized hyaluronic acid oligosaccharides.

Severe acute inflammatory conditions may cause tissue damage, sepsis, and death. As a critical component of the extracellular matrix, hyaluronic acid (HA) has been reported to possess pro- and anti-inflammatory properties via Toll-like receptors (TLRs). In this study, we prepared different sizes and structures of HA oligosaccharides and derivatives and investigated the effects on inflammation in vitro and in vivo. Our results showed that HA tetra-saccharide was the minimum fragment to enhance inflammation, whereas HA disaccharide competitively blocked TLR4-dependent inflammation. The enzymatic HA disaccharide (ΔHA2) inhibited lipopolysaccharide (LPS)-induced inflammation. Based on structure-activity relationship analysis, we observed that anti-inflammatory activity depended on HAs polymerization degree, acetyl group, and configuration. In addition, we demonstrated that ΔHA2 reduced LPS-induced pro-inflammatory cytokines production in vivo. ΔHA2, a native metabolite of HA polysaccharides, may have a potential role against LPS-mediated inflammatory diseases.

Preparation, Characterization, and Inhibition of Hyaluronic Acid Oligosaccharides in Triple-Negative Breast Cancer.

Hyaluronic acid (hyaluronan, HA) is a critical component of the extracellular matrix and plays an important biological function of interacting with different molecules and receptors. In this study, both odd- and even-numbered HA oligosaccharides (HAOs) with specific degrees of polymerization (DP) were prepared by different hydrochloric acid hydrolyses, and their structures were characterized by means of HPLC, ESI-MS, and NMR. The data show that the odd-numbered HAOs (DP3-11) have a glucuronic acid reducing end, while the even-numbered HAOs (DP2-10) have an N-acetylglucosamine reducing end. Biological evaluations indicated that all HAOs significantly inhibited the growth and migration of triple-negative breast cancer (TNBC) MDA-MB-231 cells. Among these oligosaccharides, the HA tetrasaccharide (DP4) was confirmed to be the minimum fragment necessary to inhibit MDA-MB-231 cells. Our data suggest that HAOs have potential value in the treatment of TNBC.

Extraction, isolation and structural characterization of a novel polysaccharide from Cyclocarya paliurus.

A water soluble polysaccharide CP-III was extracted and purified from Cyclocarya paliurus. CP-III is identified as a novel pectin-like polysaccharide with molecular weight (Mw) of 72.7 kDa. The structural features of CP-III were characterized by methylation and nuclear magnetic resonance (NMR) spectroscopy. Its depolymerized fragments were analyzed by hydrophilic interaction chromatography-Fourier transform mass spectrometry (HILIC-FTMS). The main chain of CP-III is composed of →4)GalAp(α1 → and →2)Rhap(α1 → 4)GalAp(α1→, repeatedly. The residue of →4)Galp(ß1 → and →5)Araf(α1 → alternately exists on the O-4 of partial →2)Rhap(α1 → residues as side chains. On the O-3 of sectional →5)Araf(α1 → residues is a secondary branch assembled by →3)Araf(α1→. Moreover, on the non-reducing terminus of →4)Galp(ß1 → occasionally have an →5)Araf(α1 → chain. Surprisingly, a sub-branch constructed by →6)Hexp(ß1 → with a galacturonate or methyl galacturonate exists on the O-3 of certain →4)Galp(ß1 → residues in the non-reducing terminus. In addition, a terminal Xyl is located on the O-3 of fractional →4)GalAp(ß1 → residue. The highly branched polysaccharide CP-III with high water solubility can be used as food supplement and medicinal carrier in the future.

Preparation and structural characterization of regioselective 4-O/6-O-desulfated chondroitin sulfate.

The sulfation pattern plays a crucial role in chondroitin sulfate (CS) biological activity, and preparation of CS with defined structure is essential for accurate pharmacological study. In this study, we focused on the preparation of regioselective 4-O/6-O-desulfated CS derived from porcine, employing a dimethyl sulfoxide-methanol (DMSO-MeOH) method and an N-methyl-N-(trimethylsilyl) -trifluoroacetamide (MTSTFA) method CS, respectively. Results showed that the sulfate at C4 position (4-O-S) of N-acetylgalactosamine (GalNAc) was selectively removed by the DMSO-MeOH method, and the sulfate at C6 position (6-O-S) of GalNAc was selectively removed by the MTSTFA method. Structures of desulfated CS were characterized by means of FT-IR, NMR and disaccharide composition analysis. The preparations of regioselective 4-O/6-O-desulfated CS are powerful for the study of structure-activity relationship of CS.

Extraction, Isolation, Structural Characterization and Anti-Tumor Properties of an Apigalacturonan-Rich Polysaccharide from the Sea Grass Zostera caespitosa Miki.

An apigalacturonan (AGA)-rich polysaccharide, ZCMP, was isolated from the sea grass Zostera caespitosa Miki. The depolymerized fragments derived from ZCMP were obtained by either acidic degradation or pectinase degradation, and their structures were characterized by electrospray ionization collision-induced-dissociation mass spectrometry (ESI-CID-MS2) and nuclear magnetic resonance (NMR) spectroscopy. The average molecular weight of ZCMP was 77.2 kD and it consisted of galacturonic acid (GalA), apiosefuranose (Api), galactose (Gal), rhamnose (Rha), arabinose (Ara), xylose (Xyl), and mannose (Man), at a molar ratio of 51.4꞉15.5꞉6.0꞉11.8꞉4.2꞉4.4꞉4.2. There were two regions of AGA (70%) and rhamnogalacturonan-I (RG-Ι, 30%) in ZCMP. AGA was composed of an α-1,4-D-galactopyranosyluronan backbone mainly substituted at the O-3 position by single Api residues. RG-Ι possessed a backbone of repeating disaccharide units of →4GalAα1,2Rhaα1→, with a few α-L-arabinose and ß-D-galactose residues as side chains. The anti-angiogenesis assay showed that ZCMP inhibited the migratory activity of human umbilical vein endothelial cell (HUVECs), with no influence on endothelial cells growth. ZCMP also promoted macrophage phagocytosis. These findings of the present study demonstrated the potential anti-tumor activity of ZCMP through anti-angiogenic and immunoregulatory pathways.

Structural Characterization of a Hybrid Carrageenan-Like Sulfated Galactan from a Marine Red Alga Furcellaria lumbricalis.

Carrageenans are sulfated galactan isolated from marine red algae with different disaccharide forms. There are also some hybrid carrageenan-like oligomers, which are reported to possess a number of bioactivities. Here, we describe a method to study the structural characterization of a carrageenan-like sulfated galactan FB1 extracted from the red seaweed Furcellaria lumbricalis. We show the process of the general analysis of FB1, including the molecular weight, sulfate content, total sugar content, protein content, and 3,6-anhydrogalactose (3,6-AnG) content analyses. The fine structure identification methods, including desulfation and methylation, nuclear magnetic resonance (NMR), and electrospray ionization collision induced dissociation tandem mass spectrometry (ES-CID-MS/MS), are also described in detail.

Structural and compositional characteristics of hybrid carrageenans from red algae Chondracanthus chamissoi.

Two polysaccharides CCC and CCH were extracted from red algae Chondracanthus chamissoi by cold water and hot water, respectively. Characterization of the structure by chemical and spectroscopic methods showed that CCC was a hybrid carrageenan composed of κ-carrageenan (35%) and ι-carrageenan (43%) along with its precursor µ-carrageenan (22%) and CCH was an ideal κ-carrageenan. Oligosaccharides from CCH and CCC were prepared and their structural sequences determined by ES-CID-MS/MS gave further insight into the structural characteristics of hybrid carrageenans from C. chamissoi. Some hybrid oligosaccharides, e.g., κ-κ, κ-µ and κ-ι, were obtained with mild acid hydrolysis of CCC. Then, a regular even-numbered oligosaccharides generated with reductive acid hydrolysis of CCH confirmed it an ideal κ-carrageenan.

Analysis of structural heterogeneity of fucoidan from Hizikia fusiforme by ES-CID-MS/MS.

A fucoidan from the brown alga Hizikia fusiforme was extracted with hot water and fractionated by anion-exchange chromatography. The main fraction YF5 was depolymerized by partial acid hydrolysis and further purified by gel filtration chromatograph. The sequences of oligosaccharides generated were then determined by ES-CID-MS/MS, which gave directly information on the structural heterogeneity of fucoidan from H. fusiforme. It was shown that YF5 had a backbone of a repeating disaccharide unit of [→4GlcAß1,2Manα1→], in good agreement with previous report by NMR and methylation analysis. In the product-ion spectra, the unique (0.2)A type fragmentation was important to establish the presence of a 2-linked Man in the backbone structure. In addition, abundant novel heterogeneous branched structural fragments were also detected and characterized: Fuc1,3Fuc, Fuc1,3Gal, Fuc1,4GlcA, Gal1,4Gal, Gal1,4GlcA and GlcA1,4GlcA. The sulfation mainly occurred at C2 or C4 of the fucose residue and C2, C4 or C6 of the galactose residue.

Specificities of Ricinus communis agglutinin 120 interaction with sulfated galactose.

Lectins are used extensively as research tools to detect and target specific oligosaccharide sequences. Ricinus communis agglutinin I (RCA(120)) recognizes non-reducing terminal ß-D-galactose (Galß) and its specificities of interactions with neutral and sialylated oligosaccharides have been well documented. Here we use carbohydrate arrays of sulfated Galß-containing oligosaccharide probes, prepared from marine-derived galactans, to investigate their interactions with RCA(120). Our results showed that RCA(120) binding to Galß1-4 was enhanced by 2-O- or 6-O-sulfation but abolished by 4-O-sulfation. The results were corroborated with competition experiments. Erythrina cristagalli lectin is also a Galß-binding protein but it cannot accommodate any sulfation on Galß.