Date of Award

5-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Food, Nutrition and Packaging Sciences

Advisor

Haley-Zitlin, Vivian

Committee Member

Jiang , Xiuping

Committee Member

Brooks , James

Abstract

The purpose of this study was to investigate if the molecular weight and degree of sulfation of chondroitin sulfate (CS) has an impact on its in vitro absorption and anti-inflammatory activity.
For absorption studies, Caco-2 cells were incubated with eight CS samples of differing molecular weights (7 kDa - 35 kDa). The amount of CS transported into the basolateral side of the Caco-2 monolayer was quantitatively determined to calculate the permeability coefficients (Peff). The permeability coefficients of the eight different CS samples across Caco-2 cell monolayers were assessed. For anti-inflammatory studies, RAW 264.7 murine macrophage cells were pre incubated with the CS samples for an hour followed by addition of bacterial lipopolysaccharide (LPS). The anti-inflammatory activity of CS samples was measured by the ability of CS samples to inhibit expression of a panel of inflammatory cytokines- tumor necrosis factor-alpha (TNF-á), Interleukin-1 beta (IL-1â) and Interleukin-6 (IL-6). These inflammatory markers were quantitatively measured using ELISA and inhibition of nitric oxide (NO) production was measured using Griess reagent assay.
Of the eight samples evaluated, four had a Peff value of 15 x 10-6 cm sec-1 or higher indicating moderate to high absorption. Two of the four samples with higher Peff values were high molecular weight compounds (~ 35 kDa). At concentrations of 5 µg/ml as well as 15 µg/ml, CS samples significantly inhibited expression of LPS induced TNF-á. Expression of IL-6 was inhibited by some of the CS samples at 15 µg/ml concentration but not at 5µg/ml. Under the experimental conditions, IL-1â and NO were not useful in estimating the anti-inflammatory activity of the CS samples. Statistical analysis which examined the relation between molecular weight and each of these inflammatory markers revealed no correlation (p<0.05).
Within the CS molecular weight range used in this experiment, the absorption of CS samples did not have a correlation with their molecular weights but, interestingly, correlation was observed between the absorption and percentage of 6-sulfated disaccharide in the CS samples. CS samples used in this study appeared to inhibit some of the inflammatory cytokines but no correlation seemed to exist between the molecular weights and anti-inflammatory activity of these samples.

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