Extraction, characterization and immunomodulatory property of pectic polysaccharide from pomegranate peels: Enzymatic vs conventional approach

Molecular characteristics, structural properties and immunostimulatory activities of polysaccharides from pomegranate peel were evaluated after 0.1 M HCl, Cellic CTec2 and buffer extractions. The isolated polysaccharides were mainly formed of neutral sugars (32.1%-51.1%) and uronic acids (19.9%-30.8%) as well as varying levels of proteins (15.0%-39.5%). Different levels of sugars including glucose (44.9%-68.1%), galactose (14.6%-19.4%), mannose (3.4%-18.1%), arabinose (3.1%-18.1%) and rhamnose (3.5-6.0%) constructed the structure of isolated polysaccharides. The average molecular weight (Mw) of polysaccharides differed notably and ranged from 422.5 × 103 to 18,631.8 × 103 g/mol. Polysaccharide molecules obtained using Cellic CTec2 enzyme induced RAW264.7 murine macrophage cells to release considerable amount of inflammatory mediators including nitric oxide, IL-1β, TNF-α, IL-6 and IL-10. The polysaccharide stimulation of macrophage cells initiated through NF-κB and MAPKs signaling pathways and activation of p-NF-κB, p-JNK, p-ERK and p-p38 proteins. The most potent immunostimulatory polysaccharides were consisted of (1 → 3)-linked glucose, (1 → 6)-linked galactose, (1 → 4)-linked mannose and (1 → 4)-linked arabinose residues with branching points at C6 and C3. These results indicated that the lower molecular weight of polysaccharides isolated with Cellic CTec2 enzyme could be one of the major determinant structural characteristics in stimulating macrophage cells.