Abstract: 【Aim】To promote the development and utilization of the traditional Chinese medicine Aspongopus chinensis, and to explore the effects of A. chinensis polysaccharides on human breast cancer HCC1937 cells.【Methods】Single factor experiment and orthogonal test were used to explore the optimal conditions for the extraction of A. chinensis polysaccharides by water extraction and alcohol precipitation. The optimal liquid to material ratio, extraction temperature, extraction time and extraction times were determined based on the extraction rates of A. chinensis polysaccharides. The effects of A. chinensis polysaccharides (0, 2, 4, 6, 8 and 10 mg/mL) on the cell proliferation of human breast cancer HCC1937 cells at 24 and 48 h after treatment were detected by CCK-8 method. At 0, 24 and 48 h after treatment with 0, 4 and 8 mg/mL of A. chinensis polysaccharides, the HCC1937 cell morphology was observed under inverted microscope and at 24 and 48 h the HCC1937 cell apoptosis was detected by Hoechst 33258 staining. The cell cycle and the expression of apoptosis-related proteins of HCC1937 cells at 24 h after treatment with 0, 4 and 8 mg/mL of A. chinensis polysaccharides were detected by flow cytometry and Western blotting, respectively. 【Results】The results of orthogonal experiments indicated that the optimum conditions for the extraction of A. chinensis polysaccharides were 40 mL/g of liquid to material ratio, 90 ℃ of extraction temperature, 3 h of extraction time, and three times of extraction. The extraction rate of A. chinensis polysaccharides was the highest (5.067%±0.071%) under the above conditions. A. chinensis polysaccharides at 4, 6, 8 and 10 mg/mL significantly inhibited the proliferation of HCC1937 cells. A. chinensis polysaccharides at 8 mg/mL blocked the cell cycle of HCC1937 cells at the S phase. A. chinensis polysaccharides at 4 and 8 mg/mL promoted the apoptosis of HCC1937 cells, upregulated the expression of pro-apoptotic proteins CASPASE-3, CASPASE-8 and BAX, and downregulated the expression of anti-apoptotic protein BCL-2. 【Conclusion】The highest extraction rate of A. chinensis polysaccharides was 5.067%±0.071% by water extraction and alcohol precipitation method under the optimum conditions of 40 mL/g of liquid to material ratio, 90 ℃ of extraction temperature, 3 h of extraction time, 3 times of extraction. A. chinensis polysaccharides have the activities of inhibiting human breast cancer HCC1937 cells in vitro and may trigger apoptosis through the death receptor pathway and the mitochondrial pathway.

Key words: Aspongopus chinensis, polysaccharide, water extraction and alcohol precipitation method, HCC1937, apoptosis