Structural characterization and biological activity of an α-glucan from the mollusk Marcia hiantina (Lamarck, 1818).

Marcia hiantina (Mollusca, Bivalvia) (Lamarck, 1818), is an edible clam mainly distributed along the tropical coastal regions. Recent researches have demonstrated that clams can possess compounds, including polysaccharides, with a wide range of biological actions including antioxidant, immunomodulatory and antitumor activities. Here an α-glucan was isolated from M. hiantina by hot water, purified by anion exchange chromatography, and its structure was characterized by a combination of multiple nuclear magnetic resonance (NMR) methods (1D 1H, 1H-1H COSY, 1H-1H TOCSY, 1H-1H NOESY, 1H-13C HSQC and 1H-13C HSQC-NOESY spectra), gas chromatography-mass spectrometry, and high performance size exclusion chromatography (HPSEC). The analysis from NMR, monosaccharide composition, methylation analyses and HPSEC combined with multi-angle light scattering (MALS) of M. hiantina-derived α-glycan confirmed a branched polysaccharide exclusively composed of glucose (Glc), mostly 4-linked in its backbone, branched occasionally at 6-positions, and having a molecular weight of ~ 570 kDa. The mollusk α-glucan was subjected to four cell-based assays: (i) viability of three cell lines (RAW264.7, HaCaT, and HT-29), (ii) activity on lipopolysaccharide (LPS)-induced prostaglandin production in RAW264.7 cells, (iii) inhibitory activities of in H2O2- and LPS-induced reactive oxygen species (ROS) production in HMC3 cells, and (iv) HaCaT cell proliferation. Results have indicated no cytotoxicity, potent inhibition of both H2O2- and LPS-induced ROS, and potent cell proliferative activity.

Biocompatibility and structural characterization of glycosaminoglycans isolated from heads of silver-banded whiting (Sillago argentifasciata Martin & Montalban 1935).

Glycosaminoglycans (GAGs) were extracted from heads of silver-banded whiting (SBW) fish and subjected to preliminary biocompatibility testing per ISO 10993: intracutaneous irritation, maximization sensitization, systemic toxicity, and cytotoxicity. When the GAG solution was injected intradermally, the observed irritation was within ISO limits and comparable to a marketed control. There was no evidence of sensitization, systemic toxicity, or cellular toxicity on the test organisms treated with the GAG mixture from SBW fish heads. Fractionation by size-exclusion chromatography has shown three distinct fractions: F1 as low molecular weight hyaluronic acid (190 kDa), F2 (82 kDa) and F3 (64 kDa), both as chondroitin sulfates. Structural characterization by 1D and 2D nuclear magnetic resonance spectroscopy and disaccharide analysis have shown sulfation ratios at positions C4:C6 of the F2 and F3 fractions respectively as 70:20% and 50:30%, and the balance of non-sulfated and 4,6-di-sulfated units. The preliminary results here suggest that GAG-based extracts from SBW fish heads are suitable alternative products to be used in soft tissue augmentation, although further long-term biocompatibility studies are still required.

Skin regeneration effect of the Glycosaminoglycans from Liparis tessellatus eggs.

A purified Glycosaminoglycans (GAGs) extract from L. tessellatus eggs was enzyme-hydrolyzed and then fractionated with DEAE-Sepharose column chromatography. The fractions were subsequently subjected to skin regeneration effects analysis against skin fibroblast (CCD-986sk) cell lines. Fraction 3 is evidently to have tyrosinase inhibition activity by 20.1% at concentration of 50mg/mL. DOPA oxidation assay, collagenase inhibition activity assay, fibroblast proliferation assay, and production of type I C-peptide assays were done to further proof the skin regeneration effect of GAGs fractions. Results revealed that fraction 3 has effective skin regeneration activities at a concentration of 200mg/mL.

Hyaluronidase-inhibitory activities of glycosaminoglycans from Liparis tessellatus eggs.

Polysaccharide fractions isolated from L. tessellatus eggs were purified and eluted using the DEAE-sepharose fast flow column. These were collected, tested and pooled based on their sugars content: F1, F2, and F3 which contain 26.8, 23.3, and 20.2% sulfated glycans; 34.5, 38.2, and 45.0% uronic acids; and 23.5, 19.0, and 7.5% acetylhexosamines and hexosamines, respectively. Hyaluronidase inhibitory effects of the fractions are in the order F3>F2>F1>Ascorbic acid, with F3 having the highest inhibition among the fractions and that of the standard, ascorbic acid. The electrospray ionization tandem mass spectrometry (ESI-MS/MS) confirmed the presence of uronic acids on F3, which could be a 0,2A2 fragment plus loss of methyl group which is very common among non-methylated, sulfated disaccharides.

Enzyme-assisted extraction of anticoagulant polysaccharide from Liparis tessellatus eggs.

This study aimed to recover a heparin-like anticoagulant polysaccharide from Liparis tessellatus eggs (PLE) by using enzyme-assisted extraction technique. Extraction experiments were carried out using three different enzymes (Alcalase®2.4 L, Flavourzyme®500 MG, and Protamex®) under different conditions of temperature (45, 50, and 55°C), pH (6.5, 7.0, and 7.5), incubation time (24, 36, and 48 h), and enzyme to substrate ratio (E/S=0.5, 1.0, and 1.5%, w/w), which were combined according to a D-optimal design. Statistical analysis of extraction results allowed identifying the variables with greater influence on the extraction yield, and selecting the conditions that maximize the PLE extraction. The best extraction results were achieved when using the Protamex® enzyme in an E/S ratio of 1.34% (w/w), pH 6.60, 47.40°C, during 26.50 h. Under these conditions, a polysaccharide yield of 2.10% (w/w) was obtained. Clotting time measurements, activated partial thromboplastin time, and prothrombin time for evaluation of the anticoagulant properties of PLE were determined and showed increasing activities in correlation with the concentrations used. In the final step, the heparin-like nature of PLE was confirmed by digestion with heparinases I, II, and III, which showed ΔDiHS-0S, ΔDiHS-6S, ΔDiHS-diS1, and ΔDiHS-diS2 at compositions of 0.04, 0.03, 0.35, and 0.24 mol/g, respectively.