| Objective:Through the extraction,purification,fingerprinting and anti-tumor activity of the Anemone raddeana Regel polysaccharides,the total mass analysis of the Anemone raddeana Regel polysaccharides was carried out,which provided a scientific reference for the material research,development and research of the Anemone raddeana Regel polysaccharides to inhibit intestinal cancer.Methods:The extraction of the polysaccharides from the Anemone raddeana Regel was carried out by water extraction and alcohol precipitation.The extraction process of the polysaccharides from the Anemone raddeana Regel was optimized by Box-Behnken response surface method.The decolorization of the Anemone raddeana Regel polysaccharides was carried out by activated carbon decolorization method.The decolorization process of the Anemone raddeana Regel polysaccharides was optimized by Box-Behnken response surface methodology.Deproteinization of Anemone raddeana Regel polysaccharides by TCA method,and optimization of deproteinization process of Anemone raddeana Regel polysaccharides by Box-Behnken response surface methodology.The monosaccharide composition of the Anemone raddeana Regel polysaccharides from 12 producing areas was studied and the pre-column derivatization fingerprints of the Anemone raddeana Regel were established.The data were simplified and optimized by principal component analysis,cluster analysis and canonical correlation analysis.Treatment,selection of representative indicators to evaluate the quality difference of Anemone raddeana Regel polysaccharides from different regions.The effect of Anemone raddeana Regel polysaccharide on the proliferation of intestinal cancer cell line DLD-1was determined by CCK8 method.The effect of Anemone raddeana Regel polysaccharide on the apoptosis of intestinal cancer DLD-1 cells was detected by flow cytometry,and the AO-EB double staining fluorescent colorimetry was used to observe the difference.The effects of Anemone raddeana Regel polysaccharides on the apoptosis of intestinal cancer DLD-1 cells were detected by RT-PCR.The mRNA levels of C-myc,Cyclin D1,MMP2 and MMP9 related genes were detected by RT-PCR.Results : The optimal extraction process of the Anemone raddeana Regel polysaccharides was: liquid-to-liquid ratio 10:1,extraction time 120 min,extraction times2 times,under which the extraction rate of the Anemone raddeana Regel polysaccharideswas 2.04±0.03%;the best decolorization process of the Anemone raddeana Regel polysaccharides was: activated carbon addition The amount is 7.3%,the number of decolorization is 3 times,the decolorization temperature is 90?.Under this condition,the decolorization rate of the Anemone raddeana Regel polysaccharide is 97.26±0.91%,and the two-headed polysaccharide solution can be completely decolorized.The optimal deproteinization process of the Anemone raddeana Regel polysaccharide is TCA concentration of 20%.The reaction time was 19 min,and the deproteinization temperature was 52?.Under this condition,the deproteinization rate of the Anemone raddeana Regel polysaccharide was 94.92±1.64%.The Anemone raddeana Regel polysaccharide was purified by the above method,and the purity was analyzed after purification,and the purity was over 90%.A common pattern of pre-column derivatization fingerprints of Anemone raddeana Regel polysaccharides was established,and 10 common peaks were identified,and 7common peaks were identified,namely D-mannose,D-ribose,L-rhamnose,D-The similarity of glucose,D-galactose,L-arabinose,L-fucose,12 batches of Anemone raddeana Regel polysaccharides was 0.890-0.978,indicating that the monosaccharide composition of the Anemone raddeana Regel polysaccharides in 12 producing areas was consistent.11 monosaccharides,but the content of each component is different.Cluster analysis results When the Euclidean distance is 20,the Anemone raddeana Regel polysaccharides in 12 producing areas can be clustered into two types,S1 and S4 are one type,and the rest are one type;when the Euclidean distance is 5,the latter can be aggregated into Class 2,S5,S6,S11,and S12 are grouped into one class,and S2,S8,S3,S7,S9,and S10 are grouped into one class.It is indicated that the PCA results show that the cumulative contribution rate of the first three components reaches 87.973%,and the three factors can be selected to comprehensively evaluate the Anemone raddeana Regel polysaccharides.In the study of the effect of different drug concentrations and different time on the proliferation of colon cancer DLD-1 cells by CCK-8 method,it was found that the inhibition rate of colon cancer DLD-1 cells increased with the increase of drug concentration.The cell inhibition rate reached the maximum after 24 hours of action.Flow cytometry was used to determine the effect of Anemone raddeana Regel polysaccharideson the apoptosis rate of intestinal cancer DLD-1 cells.It was found that the apoptosis rate increased with the increase of drug concentration(P<0.01).AO-EB double-stained fluorescent colorimetry showed that the effect of different concentrations of the Anemone raddeana Regel polysaccharide on the apoptosis of intestinal cancer DLD-1 cells was that the orange condensed cells increased significantly with the increase of drug concentration,and the number of apoptosis was also increased(P<0.01).The mRNA levels of C-myc,Cyclin D1,MMP-2 and ?-Catenin related genes were detected by RT-PCR.Compared with the control group,the cells were supplemented with pentafluorouracil and different concentrations of the Anemone raddeana Regel polysaccharides,and the gene MMP-2.The relative expression levels of ?-catenin,Cyclin D1 and C-MYC were all decreased(P<0.01),and the relative expression of gene MMP-2 was significantly decreased(P<0.01).Conclusion : The Box-Behnken response surface method was used to extract,decolorize and deproteinize the Anemone raddeana Regel polysaccharides,and the regression model has good fitting degree.It can be used for the extraction,separation and purification of the polysaccharides from the Anemone raddeana Regel.Combining HPLC fingerprints with cluster analysis,Principal component analysis for pattern recognition is an effective evaluation method for the quality control of Anemone raddeana Regel polysaccharides.CCK-8 method,AO-EB double staining fluorescent colorimetry,flow cytometry and RT-PCR showed that the Anemone raddeana Regel polysaccharides had a certain inhibitory effect on colon cancer DLD-1 cells.The above research results can provide a reliable scientific reference for the study of anti-tumor activity of Anemone raddeana Regelpolysaccharides,and lay a foundation for the development and application of polysaccharides. |
PDF Full Text Request