Abstract: This study was aimed to optimize the extraction process for polysaccharide from the fruiting bodies of Phellinus igniarius and to explore the antioxidant activity in vitro of the polysaccharide and its protective effect on D-galactose (D-gal)-induced mouse embryonic fibroblast (3T3) cell injury. The extraction was performed by the traditional method of water extraction followed by alcohol precipitation. The extraction process was optimized by a combination of single factor and orthogonal array design experiments. The crude polysaccharide was purified by agarose gel column chromatography. The effect of the purified polysaccharide on D-gal-induced 3T3 cell injury model was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide-thiazolyl blue tetrazolium bromide (MTT) assay and senescence-associated-β-galactose (SA-β-gal) staining, and its effect on the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and catalase (CAT) activity in the cell culture supernatant was determined. The mRNA expression levels of the genes related to the nuclear?factor?erythroid-2-related factor 2-antioxidant responsive element (Nrf2-ARE) signaling pathway in senescent cells were determined by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the optimal extraction conditions were as follows: extraction temperature 80 ℃, extraction time 3 h, solid-to-solvent ratio 1:40, and four extraction cycles. The average yield of polysaccharide was 6.64%. After purification, the polysaccharide content was 76.28%. The polysaccharide showed antioxidant activity in terms of scavenging rates for 1,1-diphenyl-2-picrylhydrazyl radical (DPPH, 77.14%), superoxide anion radical (31.22%) and hydroxyl radical (56.86%). MTT assay showed that compared with the model group, the cell survival rate was significantly increased by the polysaccharide at a concentration of 100 μg/mL (P < 0.01). The results of SA-β-gal staining also showed that the polysaccharide could significantly increase the survival rate of the model cells (P < 0.05), and protect 3T3 cells from D-gal-induced injury. Compared with the model group, the polysaccharide reduced ROS levels (P < 0.05) in 3T3 extracellular fluid, significantly decreased MDA levels (P < 0.01), and increased CAT activity (P < 0.05). Compared with the model group, the mRNA expression of the three downstream genes in the Nrf2 signaling pathway GCLC, NQO1 and GCLM were significantly increased in the polysaccharide-treated group (P < 0.05). In conclusion, the optimized extraction process is stable and reliable. The polysaccharide from P. igniarius fruiting bodies has antioxidant activity, cell proliferation activity, and protective effect on D-gal-induced 3T3 cell damage. The antioxidant mechanism of the polysaccharide may be mediated by the Nrf2 signaling pathway, associated with enhancing the mRNA expression of GCLC, NQO1 and GCLM.

Key words: fruiting body of Phellinus igniarius; polysaccharides; antioxidant effect; cell damage