| Diabetes is a chronic metabolic disease with elevated blood glucose level as a clinical diagnostic indicator.Long-term metabolic disorders can cause multiple system damage and oxidative damage.Studies have found that Urtica plants have a wide range of pharmacological activities such as lowering blood glucose,anti-oxidation,regulating blood lipids,anti-inflammatory,antibacterial,analgesic,and anti-prostatic hyperplasia.Polysaccharide is one of the main active components of urtica in anti-diabetic and antioxidant effects.Therefore,it is of great significance for the study of Urtica polysaccharide and its hypoglycemic activity.In this paper,Urtica fissa E.pritz.is used as the raw material,the semi-biomimetic extraction method is used to extract PUF,and the total glucose,uronic acid and protein content of the crude polysaccharide are determined;Four polysaccharides(PUF1,PUF2,PUF3,PUF4)were separated from PUF by DEAE cellulose column chromatography and the antioxidant and hypoglycemic activities of the four polysaccharides were evaluated and the purity,molecular weight,monosaccharide composition and structure of PUF3 were analyzed;the effect of PUF3 on glucose metabolism and antioxidant mechanism of IR-HepG2 cells was studied.The specific research content is as follows:1.Extraction and purification of Urtica polysaccharideThe pH1,pH2,material-to-liquid ratio,extraction temperature,and extraction time were optimized by response surface analysis,and the best process parameters were obtained:pH1 was 3,pH2 was 8,the material-liquid ratio was 1:16,and the extraction temperature at 65℃,extraction time is 77min,PUF yield is 7.04%,total glucose,uronic acid,and protein content are 78.48%,16.12%,5.34%,respectively.Using DEAE cellulose column chromatograpHy,PUF was eluted with various concentrations of NaCl solution(0,0.1,0.3,0.5 mol/L)to obtain PUF1,PUF2,PUF3,PUF4,respectively,and the yield of each component polysaccharide was 32.3%,21.8%,29.6%,14.3%.2.Antioxidant and hypoglycemic activity of Urtica polysaccharides in vitroPUF1,PUF2,PUF3,PUF4 in vitro antioxidant experiments show that nettle polysaccharides have a certain scavenging effect on DPPH free radicals,hydroxyl free radicals,and superoxide anions.The scavenging rate of DPPH free radicals is up to 69.59%,the scavenging rate of hydroxyl radicals is up to 65.41%,and the scavenging rate of superoxide anions is up to 52.46%,which has relatively little effect on the total reducing power.PUF1,PUF2,PUF3,PUF4 in vitro hypoglycemic experiments showed that 4 kinds of nettle polysaccharides have a certain inhibitory effect on α-glucosidase and α-amylase.The highestα-glucosidase inhibitory activity reached 69.48%,and the order of strength was:PUF3>PUF1>PUF4>PUF2.The highest α-amylase inhibitory activity reached 89.45%,and the order of strength was:PUF3>PUF2>PUF1>PUF4.The results show that PUF3 has the strongest hypoglycemic activity.3.Structural identification of the polysaccharide PUF3 of UrticaUltraviolet full-wavelength scanning shows that PUF3 does not contain nucleic acid and protein;combined with high-performance gel permeation chromatography(HPGPC)analysis,the molecular weight of PUF3 is 3.08×105Da;the monosaccharide composition analysis of PUF3 shows that PUF3 is composed of mannose,ribose,It is composed of rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,arabinose,and fucose,and the molar ratio is 1.32:0.65:14.76:2.49:5.20:3.13:27.13:20.90:1.16.The Congo Red test showed that PUF3 does not have a three-strand helical conformation;scanning electron microscopy results show that PUF3 has an irregular sheet structure with dense small holes on the surface;atomic force microscopy results show that multiple polysaccharide molecular chains are entangled in irregular sheets;IR Spectroscopy and nuclear magnetic resonance analysis showed that PUF3 is a pyran-type polysaccharide,with glycosidic bonds of α and βconfigurations,and characteristic peaks of polysaccharides.4.Study on the hypoglycemic activity of PUF3 on IR-HepG2 cellsThe optimal insulin concentration for constructing IR-HepG2 cell model is 1×10-7mol/L,and the action time is 48h.Compared with the Control group,the IR group had a significant reduction in glucose consumption and no cytotoxicity,indicating that the IR-HepG2 cell model was successfully constructed.Set up blank group(Control),model group(IR),metformin group(Met),PUF3 high-dose group(PUF3-H),PUF3 medium-dose group(PUF3-M),PUF3 low-dose group(PUF3-L).Compared with the IR group,the PUF3-H,PUF3-M,and PUF3-L groups can all increase the glucose consumption of cells to varying degrees(p<0.05,p<0.01).In addition,after the intervention of PUF3,in the PUF3-H group,the levels of malondialdehyde(MDA)and triglyceride(TG)were down-regulated compared with the IR group(p<0.01)(p<0.05),hexokinase(HK),The content of pyruvate kinase(PK)and glycogen were up-regulated(p<0.05)(p<0.01)(p<0.05)compared with IR group,and SOD activity was significantly improved.The effects of PUF3 on ROS were observed using the fluorescent probe DCFH-DA combined with a fluorescent inverted microscope.The results showed that the fluorescence intensity of PUF3 high,medium,and low dose groups all decreased to different degrees.In summary,this article has carried out separation,purification and activity study on Urtica polysaccharides,and the results show that PUF3 has strong antioxidant activity and in vitro hypoglycemic activity.The results show that Urtica polysaccharide can reduce insulin resistance by affecting the glucose consumption of IR-HepG2 cells;by increasing HK and PK content,promoting glycogen synthesis,and reducing TG content to regulate glucose and lipid metabolism;by reducing MDA content,enhancing SOD activity,Reduce ROS levels to improve oxidative stress. |
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