Study On Isolation,Structure Characterization And Hypoglycemic Activity Of Pueraria Lobata Polysaccharides

Pueraria lobata is a medicine and food homologous vine approved by the Ministry of Health of China,which not only has medicinal value,but also has the effect of nutrition and health care.Pueraria lobata is a potential candidate for hypoglycemia and edible Chinese herbal medicine in ancient Chinese medicine prescriptions and proprietary Chinese medicines.At present,the compound preparations and health products listed with Pueraria lobata as king medicine are Pueraria balsam pear chromium capsule,propolis Pueraria soft capsule,Pueraria chewable tablet and Pueraria powder and so on.As a potential effective constituent or active ingredient,Pueraria lobata has attracted much attention from scholars and has been widely used in health care and green food.Based on the research status of Pueraria lobata polysaccharides,this paper will extract and purify the active homogeneous polysaccharides from Pueraria lobata,analyze the structure by a variety of physical and chemical methods,and study its hypoglycemic activity in vitro and its molecular mechanism based on HepG2 cells.Pueraria lobata crude polysaccharides were obtained by water extraction and alcohol precipitation,and five homogeneous polysaccharides were isolated and purified by macroporous resin HP-20,DEAE cellulose and Sephadex G-200 gel chromatography column,and the overall yields were PLP-1(0.13%),PLP-2(0.18%),PLP-3(0.03%),PLP-4(0.01%)and PLP-5(0.08%),respectively.The relative average molecular weight was determined as PLP-1(16.2 kDa),PLP-2(11.0 kDa),PLP-3(6.2 kDa),PLP-4(3.8 kDa)and PLP-5(1.6 kDa)by high performance gel permeation chromatography.The total sugar contents of homogeneous polysaccharides determined by phenol-sulfuric acid method were PLP-1(96.6%),PLP-2(98.4%),PLP-3(94.3%),PLP-4(94.4%)and PLP-5(96.8%).The protein contents of homogeneous polysaccharides determined by Bradford method were PLP-1(2.4%),PLP-2(1.8%),PLP-3(2.4%),PLP-4(4.7%)and PLP-5(4.0%).Homogeneous polysaccharides were hydrolyzed,reduced and acetylated to form glycol acetate derivatives.GC-MS analysis showed that five homogeneous polysaccharides(PLP-1,PLP-2,PLP-3,PLP-4,PLP-5)were all composed of glucose,but the connection mode was different.Therefore,in order to clarify the structure of homogeneous polysaccharides in more detail,PLP-1 with the highest molecular weight was selected for in-depth study.Methylation analysis and nuclear magnetic resonance spectra(1H-NMR,13C-NMR,DEPT-135°,HSQC and HMBC)showed that the main chain of PLP-1 was mainly composed of →3)-α-D-Glcp(1→ and →4)-β-D-Glcp(1→ with a relative molar ratio of 7:1.Atomic force microscope observation showed that PLP-1 is spirally coiled in spatial configuration.In addition,the in vitro insulin resistance model of HepG2 cells was successfully established by 0.5 mmol/L palmitic acid combined with 30 mmol/L glucose for 8 hours.The stability of the model was investigated at 8 h,16 h,24 h and 32 h,respectively.The results showed that the insulin resistance HepG2 cell model lasted for 24 h.Metformin hydrochloride(2 mM)was selected as the positive control.Homogeneous polysaccharides were incubated with insulin resistant HepG2 cells at high(500 μg/mL),medium(250 μg/mL)and low(125 μg/mL)concentrations for 10 hours,respectively.The high and medium concentrations of homogenized polysaccharides(PLP-1,PLP-2,PLP-3)were screened to have hypoglycemic activity in vitro by glucose assay kit.In order to further verify the hypoglycemic activity of homogenized polysaccharides,PLP-1(500 μg/mL)was selected to study its mechanism via real-time fluorescence quantitative PCR,Western blot and immunofluorescence.The results showed that PLP-1 upregulated the mRNA and protein expression levels of PI3 K,AKT and GSK-3β,and down-regulated the mRNA and protein expression levels of FoxO1,PCK2 and G6 Pase in insulin resistant HepG2 cells.In FoxO1 immunofluorescence staining,PLP-1 can reduce the expression of FoxO1 and inhibit nuclear translocation.These studies show that PLP-1 may be involved in the regulation of PI3K/AKT/GSK-3β and PI3K/AKT/FoxO1 signaling pathways to promote glycogen synthesis and improve glucose metabolism disorders,and provide scientific basis for the development and use of Pueraria lobata polysaccharide as an alternative functional food or clinical adjuvant therapy to improve metabolic syndrome in diabetic people.