Study On Optimal Extraction And Antitumor Effect And Mechanism Of Rose Roxburghii Tratt Fruits Polysaccharide From Yunnan

Objective:To study the optimal extraction and antioxidant activity of Rose roxburghii Tratt fruits polysaccharide（RTFP）from Yunnan province,and explore its anti-tumor effect and mechanism in vitro.Methods:1 Optimization extraction of RTFP:With the yield of RTFP as the index,extraction time,extraction temperature,solid-liquid ratio and extraction times as the influencing factors,the optimal extraction parameters of RTFP were determined on the basis of single factor experiment combined with orthogonal experiment.2 Antioxidant activity of RTFP:With DPPH free radical and ABTS free radical scavenging rate as the index,the antioxidant capacity of RTFP was determined.3 Screening of RTFP anti-tumor activity:The inhibitory effect of RTFP on the proliferation of different malignant tumor cells（human cervical cancer Hela cells,human malignant melanoma A375 cells,human non-small cell lung adenocarcinoma A549 cells）was detected by cell proliferation assay,then,sensitive cancer cell lines were screened.4 Study on the anti-tumor mechanism of RTFP:The effects of RTFP on proliferation,migration and invasion of sensitive cell lines were detected by cell proliferation assay,scratch assay and Transwell assay.The effect of propidium iodide single dye process（PI）on the cycle of sensitive cancer cell lines was determined by flow cytometry.The effect of polysaccharide on apoptosis of sensitive cancer cell lines was detected by flow cytometry phosphoacylerine fluorescein/propyl iodide double dyeing（Annexin V-FITC/PI）.The m RNA expression of cell cycle and apoptosis related genes was detected by real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）.Protein immunoblotting（Western Blot）was used to detect the expression of gene proteins related to cell cycle and apoptosis.Results:1 Optimal extraction of RTFP:The optimal extraction parameters of RTFP were extraction time of 4 h,extraction temperature of 70℃,ratio of solid to liquid of 1:30（g/m L）,extraction for 2 times.Under these conditions,the yield of RTFP was（25.15±0.78）%.2 Antioxidant activity of RTFP:The IC₅₀of RTFP to DPPH radical and ABTS radical were 0.124 mg/m L and 0.113 mg/m L,indicating that RTFP had certain antioxidant activity.3 Screening of RTFP anti-tumor activity:The sensitivity of RTFP to Hela cells was better than that of A375 cells and A549 cells,and human cervical cancer Hela cells were selected as the sensitive cells,to carry out the effect and mechanism of RTFP against Hela cells of human cervical cancer.4 Mechanism of RTFP against Hela cells of human cervical cancer:4.1 RTFP significantly inhibited the proliferation,migration and invasion of Hela cells in a time-dependent and dose-dependent manner.4.2 RTFP may inhibit the expression of CDK-1 and Cyclin B1 m RNA and protein to arrest Hela cell cycle at G₂/M phase,in a dose-dependent manner.4.3 RTFP promoted the m RNA expression levels of apoptosis-related genes Caspase-3,Caspase-8 and Caspase-9.RTFP significantly up-regulated the expression levels of apoptosis-related genes Bax,Caspase-3,Caspase-8 and Caspase-9 protein,and down-regulated the expression of anti-apoptotic Bcl-2 protein.RTFP may induce the apoptosis of Hela cells.Conclusion:Water extraction and alcohol precipitation method were used to optimize the extraction of RTFP.In addition,RTFP inhibited the proliferation,migration and invasion of Hela cells,blocked the cells in G₂/M phase,and induced Hela cell apoptosis.These results suggest that the RTFP from Yunnan province has anti-cervical cancer activity,while has medical development value.