Fig 1.
MALDI-TOF spectrum of polysaccharide.
MALDI-TOF spectrum shows Gaussian ion distribution from m/z: 200 to 2200. The hexose species are detected in this sample until m/z 2149 (n = 13). The matrix used for the polysaccharide is 2,5-dihydroxybenzoic acid (DHB).
Fig 2.
1H-NMR spectra of the polysaccharide recorded in D2O.
Proton NMR of the polysaccharide displays characteristic signals between δ3.3 and δ5.6, typical of saccharides. The highly shielded signal at δ 5.5 is assignable to α-anomeric protons of glucose.
Fig 3.
1H-NMR spectra of the polysaccharide recorded after acid hydrolysis in D2O.
The NMR spectrum recorded after acid hydrolysis using 2% DCl for 1 hour at 100°C, shows characteristic signals of hexose carbohydrates. The spectrum clearly shows the nature of monomers and that the polysaccharide is composed of glucose.
Fig 4.
2D DOSY MAP of the polysaccharide recorded in D2O.
The NMR DOSY experiment displays a low molecular weight oligosaccharide around 1000 g/mol. The x-axis corresponds to the chemical shifts (classical proton NMR) and the y-axis corresponds to the diffusion coefficients.
Fig 5.
Possible repeating units of C. borivilanum glucans showing pre-dominantly Glu1→6Glu Linkage.
Using mass spectroscopy technique coupled with a CID unit, the single peaks from the MALDI-TOF MS spectrum were fragmented. The sugar linkage was identified predominantly to be Glu1→6Glu, composed of glycans.
Fig 6.
A. Effect of polysaccharide on yeast chronological lifespan. Histogram showing area under the survival curve at different doses of polysaccharide extract (ng/ml), compared to the vehicle control (-ve) and a positive control 10 μg/ml Resveratrol (+ve) for S. cerevisiae. Error bars are standard deviation from the mean AUC of eight experiments. For statistical significance see Table 1. B: The effect of polysaccharide on yeast Chronological Lifespan requires a functional TOR pathway. Histograms showing area under the survival curve at 10ng/ml of polysaccharide extract in BY4741 and BY4741 containing deletions of TOR1, SNF1, GCN5 and SIR2. Deletions represent replacement of the coding region with the KanMX selection casette. Error bars are standard deviations from the mean AUC of three measurements.
Fig 7.
Effect of polysaccharide on C.elegans lifespan.
Survival curves for C.elegans treated with 10μg/ml polysaccharide extract (blue line), 10μg/ml Resveratrol (yellow line) or vehicle control (green line).
Table 1.
Effect of polysaccharide fraction treatment on median chronological lifespan and AUC in yeast.
Fig 8.
Effect of the polysaccharide treatment on keratinocyte proliferation.
C. borivilanium polysaccharide displays 47% proliferation at 25μg/mL. HB-EGF, used as a positive control, displays 60% proliferation at 0.001μg/mL. *p < 0.05.
Fig 9.
Effect of the polysaccharide treatment on hyaluronic acid synthesis in HaCaT keratinocytes.
The induction of hyaluronic acid in HaCaT keratinocytes when C. borivilanium polysaccharide is added to culture medium. A two-fold induction of hyaluronic acid is observed with 16 μg/mL of C. borivilanium polysaccharide.
Fig 10.
Histological analysis for effect of polysaccharide on tissue morphology.
Microscopic examination of histological paraffin sections (H & E staining) showed standard and consistent morphology of reconstructed human epidermis (RHE). Thin and broad arrows indicate intact stratum corneum and Epidermis respectively.
Fig 11.
Expression of CD44 and HA in human reconstructed epidermis after treatment with polysaccharide.
Proteins were quantified using ELISA in two separate EpiSkinTM D6 tissue samples and at three different concentrations of polysaccharide (1, 10 and 100 μg/mL). Control was the tissue sample treated with solvent DMSO.
Fig 12.
Western blot analysis of pAKT and GAB1 in human reconstructed epidermis after treatment with polysaccharide at a concentration of 100μg/ml.