九香虫多糖提取条件筛选及其对人乳腺癌HCC1937细胞的抑制作用

【目的】推进中药九香虫Aspongopus chinensis开发与利用，探究九香虫多糖对人乳腺癌HCC1937细胞的作用效果。【方法】采用单因素实验和正交试验探究水提醇沉法提取九香虫多糖的最优条件，基于九香虫多糖提取率来确定最佳液料比、提取温度、提取时间和提取次数；CCK-8法检测0, 2, 4, 6, 8和10 mg/mL九香虫多糖作用后24和48 h时对人乳腺癌HCC1937细胞增殖的影响；0, 4和8 mg/mL九香虫多糖处理HCC1937细胞，0, 24和48 h时倒置显微镜下观察HCC1937细胞形态并于24和48 h时利用Hoechst 33258染色检测HCC1937细胞凋亡，24 h时利用流式细胞术检测HCC1937细胞的细胞周期并利用Western blot检测凋亡相关蛋白的表达。【结果】正交试验结果表明，九香虫多糖的最佳提取条件为：液料比40 mL/g、提取温度90℃、提取时间3 h和提取次数3次，在上述条件下九香虫多糖提取率最高，为5.067%±0.071%。4, 6, 8和10 mg/mL九香虫多糖能够显著抑制HCC1937细胞增殖；8 mg/mL九香虫多糖阻...

Extraction conditions of polysaccharides fromAspongopus chinensis(Hemiptera: Dinidoridae) and their inhibitory effects on human breast cancer HCC1937 cells

【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.