Structural Characterization of a Novel Glucan from <i>Achatina fulica</i> and Its Antioxidant Activity

A novel glucan designated AFPS-IB was purified from <i>Achatina fulica</i> (China white jade snail) by anion-exchange and gel-permeation chromatography. Chemical composition analysis indicated AFPS-IB was composed of glucose, fucose, rhamnose, mannose, and galactose in a molar ratio of 189:2:1:1:2 and with an average molecular weight of 128 kDa. Its structural characteristics were investigated by Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC–MS), methylation analysis, nuclear magnetic resonance (NMR) spectroscopy (¹H,¹³C, H–H COSY, HSQC, TOCSY, and NOESY), and atomic force microscopy (AFM). The glucan mainly consisted of a backbone of repeating (1→4)-α-d-glucose residues with (1→6)-β-d glucosyl branches at random points on the backbone glucose. Antioxidant studies revealed AFPS-IB showed significant DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, superoxide anion (O₂^–) scavenging activities and high reduction potential. This study suggested that AFPS-IB could be a new source of dietary antioxidants

Protein-Bound Polysaccharide from <i>Corbicula fluminea</i> Inhibits Cell Growth in MCF-7 and MDA-MB-231 Human Breast Cancer Cells

<div><p>A novel protein-bound polysaccharide, CFPS-1, isolated from <i>Corbicula fluminea</i>, is composed predominantly of mannose (Man) and glucose (Glc) in a molar ratio of 3.1:12.7. The polysaccharide, with an average molecular weight of about 283 kDa, also contains 10.8% protein. Atomic force microscopy, high-performance liquid chromatography, Fourier transform infrared spectroscopy, gas chromatography/mass spectrometry, and nuclear magnetic resonance spectroscopy analyses revealed that CFPS-1 has a backbone of 1,6-linked and 1,4,6-linked-α-D-Glc, which is terminated with a 1-linked-α-D-Man residue at the O-4 position of 1,4,6-linked-α-D-Glc, in a molar ratio of 3:1:1. Preliminary <i>in vitro</i> bioactivity tests revealed that CFPS-1 effectively and dose-dependently inhibits human breast cancer MCF-7 and MDA-MB-231 cell growth, with an IC₅₀ of 243 ± 6.79 and 1142 ± 14.84 μg/mL, respectively. In MCF-7, CFPS-1 produced a significant up-regulation of p53, p21, Bax and cleaved caspase-7 and down-regulation of Cdk4, cyclin D1, Bcl-2 and caspase-7. These effects resulted in cell cycle blockade at the S-phase and apoptosis induction. In contrast, in MDA-MB-231, with limited degree of change in cell cycle distribution, CFPS-1 increases the proportion of cells in apoptotic sub-G1 phase executed by down-regulation of Bcl-2 and caspase-7 and up-regulation of Bax and cleaved caspase-7. This study extends our understanding of the anticancer mechanism of <i>C</i>. <i>fluminea</i> protein-bound polysaccharide.</p></div

Yield, chemical compositions and contents of carbohydrate, protein, <i>M</i>_W and sulfonic acid in the <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1.

<p>Yield, chemical compositions and contents of carbohydrate, protein, <i>M</i>_W and sulfonic acid in the <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1.</p

Effect of the <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1 on MCF-7 and MDA-MB-231 cell cycle progression.

<p>Cells were cultured for 24 h with CFPS-1 (0, 50, 150 and 250 μg/mL, respectively). Untreated cells (0 μg/mL) were used as control. Cell cycle distribution (%) of MCF-7 (A) and MDA-MB-231 (C) cells were determined by flow cytometry after CFPS-1 treatment; Apoptotic rate (%) of MCF-7 (B) and MDA-MB-231 (D) cells treated with CFPS-1. Apoptotic cells were identified with a TUNEL technique and counted with a light microscope (magnification, ×40). All data were expressed as mean ± SD of three experiments and each experiment included triplicate repeats. Values marked with * are significantly different from the control (<i>p</i> < 0.05).</p

The amino acid composition of the protein-bound polysaccharide CFPS-1 from <i>C</i>. <i>fluminea</i>.

<p>The amino acid composition of the protein-bound polysaccharide CFPS-1 from <i>C</i>. <i>fluminea</i>.</p

¹H and ¹³C NMR chemical shifts for the protein-bound polysaccharide CFPS-1 isolated from <i>C</i>. <i>fluminea</i> in D₂O ^a.

<p>¹H and ¹³C NMR chemical shifts for the protein-bound polysaccharide CFPS-1 isolated from <i>C</i>. <i>fluminea</i> in D₂O <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167889#t004fn001" target="_blank">^a</a>.</p

GC-MS of alditol acetate derivatives from the methylated product of the <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1.

<p>GC-MS of alditol acetate derivatives from the methylated product of the <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1.</p

Structural elucidation of the <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1.

<p><b>(A)</b>^<b>1</b><b>H NMR and (B)</b>^<b>13</b><b>C NMR spectra of CFPS-1 in D</b>_<b>2</b><b>O.</b> Chemical shifts were reported relative to internal acetone at δH 2.23 ppm for ¹H spectrum and δC 32.45 ppm for ¹³C spectrum; (C) A diagram showing the partial structures of polysaccharide portions of the protein-bound polysaccharide CFPS-1 from C. fluminea. (a): 1,6-linked -α-D-Glc; (b): 1,4,6-linked-α-D-Glc; (c): 1-linked-α-D-Man.</p

Effects of the <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1 on the expression of p21, Cyclin D1, Cdk4, p53, Bcl-2, Bax and caspase-7 proteins associated with cell cycle and apoptosis in MCF-7(A, B, C and D) and MDA-MB-231 (E, F, G and H) cells.

<p>The relative expression of protein was quantified densitometrically (%). Western blot analysis was performed in triplicate per experimental point; β-actin was used as reference control. Values marked with * are significantly different from the control (<i>p</i> < 0.05).</p

Molecular and morphological characterization of <i>C</i>. <i>fluminea</i> protein-bound polysaccharide CFPS-1.

<p>(A and B) The atomic force microscopy (AFM) images of CFPS-1. The CFPS-1 concentration was 1 μg/mL (A; B1), 10 μg/mL (B2) and 100 μg/mL (B3), respectively. Arrow: (a) single linear chain; (b) branched chain; (C) FTIR spectra of CFPS-1 (4000 cm⁻¹ to 400 cm⁻¹) were obtained from solid samples by KBr disc method using a Nicolet 5700 FT-IR spectrophotometer. FTIR spectra showed that CFPS-1 mainly contained three types of groups, namely hydroxyl group, amino group and sulfate group.</p