Cold Alkali Extractable Antioxidative Polysaccharide from Russula pseudocyanoxantha, a Novel Mushroom, Stimulates Immune Responses in RAW264.7 Cells by Regulating TLR/NF-κB Pathway

In our previous study, we have established Russula pseudocyanoxantha as a unique species, playing a crucial role in indigenous diets through ages. The research also brought attention to bioactive potential of polysaccharide fraction extracted from the unexplored food using hot water. However, residue of the conventional process still contains therapeutic bio-polymers that could further be utilized for pharmacological purposes instead of being discarded. Therefore, the current study aims to valorize the solid remnants, contributing to a deeper understanding of the novel taxon. Subsequently, the leftover was treated with cold alkali, leading to the preparation of a high-yield fraction (RP-CAP). Chemical characterization through FT-IR, GC-MS, HPTLC, and spectroscopy demonstrated presence of several monomers in the carbohydrate backbone, predominantly composed of β-glucan. Furthermore, GPC chromatogram indicated presence of a homogeneous polymer with Mw of ~129.28 kDa. Subsequently, potent antioxidant activity was noted in terms of radical scavenging (O2•−, OH•, DPPH• and ABTS•+), chelating ability, reducing power and total antioxidant activity where EC50 values ranged from 472–3600 µg/mL. Strong immune-boosting effect was also evident, as the bio-polymers stimulated murine macrophage cell proliferation, phagocytic activity, pseudopod formation, and NO as well as ROS synthesis. In-depth analysis revealed that the fraction stimulated synthesis of several inflammatory mediators, elucidating the mode of action through TLR/NF-κB pathway. Therefore, the findings collectively suggest that RP-CAP possesses great potential to serve as a health-improving component.