Study On Extraction,Structure Characterization And Anti-tumor Activity Of Polysaccharide From Perilla Frutescens Var.frutescens

Perilla frutescens var.frutescens is a kind of medicinal and food homologous plant with rich nutrition,unique taste,extensive cultivation resources,widely used in food,medicine and other fields.Studies have found that P.frutescens var.frutescens have anti-oxidant,anti-bacterial,anti-allergy,anti-tumor,reducing blood glucose and lipid effects.In this study,using P.frutescens var.frutescens as raw materials,a new water-soluble polysaccharide was extracted from P.frutescens var.frutescens by water extraction and alcohol precipitation method.After that,the structure of the polysaccharide was characterized and the anti-tumor activity in vivo was investigated.In this study,the optimal extraction conditions of P.frutescens var.frutescens polysaccharide were obtained by single factor experiment and response surface experiment:solid-liquid ratio of 1:30,extraction time of 4 h,extraction for 3 times,alcohol concentration of 60%,the yield of polysaccharides was the highest,reaching 5.22±0.17%.After deproteinization by Sevag method,the small molecules were removed by using a 100k Da dialysis bag,and then purified by Sephadex G-200 gel column.The homogeneous component of P.frutescens var.frutescens polysaccharide(PFP)was obtained after the eluting peak was collected and lyophilized.In the structural characterization part,the basic components of PFP were detected firstly,and the results showed that the total sugar content of PFP was 89.73±0.23%,the protein content was 1.56±0.31%,the uronic acid content was 8.93±0.37%,without reducing sugar.The molecular weight by high performance liquid chromatography for testing,the results for the PFP molecular weight of 1.18×10~6Da,Fourier infrared spectroscopy,ion chromatography and nuclear magnetic resonance analysis results show that PFP is a pyranose,containingα-andβ-glycosidic bond,mainly consists of rhamnose,arabinose,galactose,glucose,xylose,and galacturonic acid composition,mole ratio was 0.13:0.55:1.40:1.00:0.13:0.22.The results of circular dichroism and Congo red showed that there was no triple helix structure in PFP.After that,the physical and chemical characteristics of PFP were analyzed by scanning electron microscopy,thermogravimetric analysis,differential thermal scanning and X-ray diffraction.The results showed that PFP had lamellar structure,smooth surface but with fragments,good thermal stability,without crystallization phenomenon,is a kind of amorphous structure.The anti-tumor activity of P.frutescens var.frutescens polysaccharide was investigated by establishing H22 tumor-bearing mouse model.Annexin V-FITC/PI double staining assay showed that P.frutescens var.frutescens polysaccharide could induce apoptosis of H22 tumor cells.PI single staining assay showed that it inhibited the proliferation of H22 tumor cells by arresting in the S phase.JC-1 assay showed that it could decrease mitochondrial membrane potential of tumor cells.HE staining showed that it could destroy tumor cells and protect liver cells.These results will provide a theoretical basis for the development of safe and effective natural anti-liver cancer drugs using P.frutescens var.frutescens polysaccharides as raw materials.