| Diabetes Mellitus is a metabolic disease caused by insufficient insulin secretion and insulin resistance.In recent years,diabetes has become another disease that seriously threatens human health after cancer and cardiovascular diseases.Although there are many drugs clinically that can effectively slow down the development of diabetes,long-term medication will inevitably cause certain toxic and side effects to the human body.Therefore,it is very important to find a safe,efficient and non-toxic natural hypoglycemic drug.Fructus arctii is the dried and mature fruit of Arctium lappa L of the Compositae family.It is a natural plant resource with the same food and medicine.fructus arctii has many functions such as anti-diabetes,anti-inflammatory,anti-obesity,anti-tumor,anti-bacterial,immune regulation and nourishing stomach.fructus arctii polysaccharide is its extremely important biologically active substance.However,there are few reports on the activity of fructus arctii polysaccharides,so the research on the structure and pharmacological activity of fructus arctii polysaccharides is very important.In this project,fructus arctii was used as raw material to obtain fructus arctii polysaccharides by ultrasonic-assisted water extraction and alcohol precipitation method,and based on single-factor experimental results,a response surface optimization experiment was designed to optimize the extraction process.The total sugar content,protein content and uronic acid content of FAP were determined,and FAP was purified by DEAE-cellulose column to obtain four fructus arctii polysaccharides.The four fructus arctii polysaccharides were subjected to in vitro anti-oxidation and hypoglycemic experiments,and the more active component FAP-W(water eluting component)was screened,the structure of FAP-W was characterized,and the insulin resistance HepG2 cell model was established.Using fructus arctii polysaccharide FAP-W for protection,study the protective effect of fructus arctii polysaccharide on diabetes,in order to provide new ideas and theoretical basis for the use of fructus arctii polysaccharide in the prevention and treatment of diabetes.The specific research results are as follows1.Extraction,separation,process optimization and content determination of FAP crude polysaccharide from Fructus arctiiUltrasound-assisted water extraction and alcohol precipitation method was used to extract fructus arctii polysaccharides.The optimal extraction parameters of fructus arctii polysaccharides were obtained through single factor experiment and response surface optimization method:ultrasonic time 63min,extraction temperature 84℃,material-to-liquid ratio 1:25 g/mL Under these conditions,the yield of fructus arctii polysaccharide was 14.02%.In addition,the primary and secondary order affecting the yield of fructus arctii polysaccharides is:ultrasonic time>extraction temperature>liquid-to-material ratio.The total sugar content of fructus arctii polysaccharide is 64.27%,the protein content is 19.38%,and the uronic acid content is 5.32%.Then,four components were purified by DEAE cellulose column to obtain the purified fructus arctii polysaccharide:FAP-W,FAP-S1,FAP-S2,FAP-S3.2.Research on hypoglycemic and antioxidant activity of FAP-W,FAP-S1,FAP-S2,FAP-S3FAP-W,FAP-S1,FAP-S2,FAP-S3 all have certain antioxidant capacity and hypoglycemic activity.Among them,FAP-W has higher antioxidant activity.At 3.2 mg/mL,DPPH free radicals,the scavenging rates of hydroxyl radical and superoxide anion were 58.89%,63.61%,82.53%,and the total reducing power was 0.1975.At the same time,FAP-W also has high hypoglycemic activity.At 20mg/mL,the inhibition rates of α-glucosidase and α-amylase were 55.25%and 54.12%,respectively.3.FAP-W structure characterizationFAP-W has no obvious absorption peaks at 260 nm and 280 nm,indicating that FAP-W does not contain nucleic acids and proteins;the results of infrared spectroscopy and nuclear magnetic resonance spectroscopy indicate that FAP-W is a β-pyranose.The results of dextran gel penneation chromatography showed that the molecular weight of FAP-W was 1.99×104 Da.The monosaccharide composition results show that FAP-W is mainly composed of mannose,glucose,galactose and arabinose,and its mole percentage is 3.4:23.59:21.27:47.72.The atomic force and Congo red test results show that FAP-W does not contain a triple helix structure.Scanning electron microscopy results show that FAP-W has a uniform texture,and its freeze-dried form is in the form of square regular crystals.4.FAP-W’s cell hypoglycemic test resultsFAP-W can up-regulate the glucose consumption of IR-HepG2 cells.The glucose consumption of the FAP-W high-dose group increased by 83.17%compared with the IR group(p<0.001);FAP-W can promote the synthesis of glycogen in cells,The glycogen content of the FAP-W high-dose group was increased by 9.46%compared with the IR group(p<0.001);at the same time,FAP-W reduced the triglyceride level in the cells,and the FAP-W high-dose group had a higher triglyceride content than the IR group reduced by 20.96%(p<0.001);FAP-W can enhance the activity of hexokinase and pyruvate kinase in IR-HepG2 cells,and the level of hexokinase in the FAP-W high-dose group was increased by 32.53%compared with the IR group(p<0.001);FAP-W can enhance the activity of superoxide dismutase in IR-HepG2 cells.The level of superoxide dismutase in the FAP-W high-dose group was 76.49%higher than that in the IR group(p<0.001),FAP-W can reduce the content of malondialdehyde and lower the level of reactive oxygen species.The content of malondialdehyde in the FAP-W high-dose group was 34.06%lower than that in the IR group(p<0.001).FAP-W,a polysaccharide component extracted and purified from fructus arctii,has excellent performance in antioxidant and hypoglycemic activity.This indicates that the fructus arctii polysaccharide is a potential active substance of traditional Chinese medicine.This study provides a new idea and theoretical basis for the research and development of the pharmacological activity of the fructus arctii polysaccharide. |
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