Development and validation of multiplex PCR assay for differentiating tunas and billfishes.

Commercially available tunas and billfishes are generally processed as steaks, making it difficult to visually distinguish between the two. We developed and validated species-specific primers to prevent the adulteration of tunas by billfishes. Tunas and billfishes primers were designed on the cytochrome oxidase subunit I. Multiplex PCR bands obtained were 579 bp, 291 bp and 114 bp for tunas, billfishes and internal control. Sensitivity was determined to be 5 ng for tunas and billfishes. A total of 50 samples were monitored: 49 for tunas and 1 for billfish. As a result of the monitoring, the fake tunas did not show due to the agreement between product name and the raw material of the wrapping paper. Our results indicate that the species-specific primers developed in this study are suitable for differentiating tunas and billfishes. The newly developed multiplex PCR assay is a time and cost effective technique for determining the authenticity of tunas and billfishes.

Structural Features and Anti-coagulant Activity of the Sulphated Polysaccharide SPS-CF from a Green Alga Capsosiphon fulvescens.

Previously, we reported that the sulphated polysaccharides (SPS)-CF, a water-soluble polysaccharide isolated and purified from Korean green alga Maesaengi (Capsosiphon fulvescens, Chlorophyta), is a glucuronogalactomannan based mainly on the monosaccharide composition determined by high-performance liquid chromatography (HPLC) analysis after 1-phenyl-3-methyl-5-pyrazolone (PMP) labelling of sugars in the acid (trifluoroacetic acid (TFA)) hydrolyzates of SPS-CF, which showed mannose (55.4 mol %), galactose (25.3 mol %) and glucuronic acid (16.3 mol %) as major sugars (Na et al., Int Immunopharmacol 10:364-370, 2010). However, the results of the present study re-performed for monosaccharide composition of this polysaccharide using, in addition to HPLC of PMP-labelled sugars, other separation methods, i.e. high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), gas chromatography with flame ionising detection (GC-FID) and thin-layer chromatography (TLC), clearly demonstrated that the most prominent neutral monosaccharides of SPS-CF are xylose (38.6-49.4 mol %) and rhamnose (39.6-45 mol %), while mannose and galactose are present at a much lesser extent or in negligible amount. These extensive monosaccharide analyses, correlation nuclear magnetic resonance (NMR), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) measurements confirmed the sulphated glucuronorhamnoxylan (ulvan) type of SPS-CF polysaccharide, whose backbone is composed of alternating sequence of 4-linked L-rhamnose-3-sulphate and D-xylose residues (ulvobiose U3s) carrying monomeric D-glucuronic acid or D-glucuronic acid-3-sulphate on O-2 of some L-rhamnose-3-sulphate units as the side chains. The SPS-CF exhibited significant in vitro anti-coagulant activity by which the activated partial thromboplastin time (aPTT) and thrombin time (TT) were significantly prolonged. The results of this study demonstrated that the ulvan SPS-CF isolated from Korean Maesaengi C. fulvescens can be considered a potential anti-coagulant agent.

Purification, characterization and immunostimulating activity of water-soluble polysaccharide isolated from Capsosiphon fulvescens.

A water-soluble polysaccharide (SPS-CF) was isolated and purified from Korean Capsosiphon fulvescens by dilute acid extraction, ethanol precipitation, and DEAE-cellulose ion exchange chromatography. The purified SPS-CF was shown to be a glucuronogalactomannan with a molecular mass of 385 kDa and the monosaccharide composition of the SPS-CF was determined to be mannose (55.4% in mole percentage), galactose (25.3%), glucuronic acid (16.3%), and arabinose (0.8%). Fourier-transform infrared and elemental analysis indicated that the purified SPS-CF is a sulfated polysaccharide containing significant amount of sulfate esters (5.7% in mass). Enzyme Linked Immunosorbent Assay showed that the SPS-CF significantly stimulates the release of the pro-inflammatory cytokines, TNF-alpha and IL-6, in a dose-dependent manner. RT-PCR analysis demonstrated that the SPS-CF also induced a more than two-fold increase in the expression of iNOS and COX-2, responsible for the induction of NO and PGE2, respectively, at 5 microg/ml in RAW264.7 murine macrophages. These results suggest that the sulfated SPS-CF isolated from C. fulvescens has potent immunostimulating activity.