Study On Physicochemical Properties,Activity Screening And Quality Evaluation Of Polysaccharides From Polygonatum Kingianum

Objective:Polygonatum kingianum is a perennial herb of the family Liliaceae.It is a large-scale medicinal material produced by the genus Lily.The previous research of the research group showed that the polysaccharide of scutellariae can significantly reduce the fasting blood glucose level of diabetic mice and significantly improve the glucose tolerance of diabetic mice.Reduce postprandial blood glucose,lower serum total cholesterol and triglycerides,increase the relative content of high-density lipoprotein and lower the level of low-density lipoprotein,significantly increase the content of liver and muscle glycogen,and alleviate the pathological changes of kidney in diabetic mice.Repairing islet damage caused by diabetes,these results indicate that Polygonatum kingianum polysaccharide has a certain therapeutic effect on diabetes.In this study,the quality control,separation and purification,chemical composition,structural characterization,in vitro antioxidant and hypoglycemic activities of polysaccharides from Polygonatum kingianum were studied,and the quality equivalence of polysaccharides from Polysaccharides and the traditional“Nine Steamed Nine”system were studied.The transformation of polysaccharides was studied in order to lay the foundation for the development and utilization of polysaccharides.Method:（1）Collecting 12 batches of Polygonatum kingianum from different producing areas,using infrared Fourier transform infrared spectrometer to establish infrared fingerprints to study the overall quality uniformity;comparing the difference and quality of polysaccharides in three kinds of legal bases by using infrared spectroscopy and PMP-HPLC Uniformity;ultraviolet determination of total polysaccharide content;（2）Using water extraction and alcohol precipitation to obtain total polysaccharide（PKS）of P.chinensis,using infrared,ultraviolet light,HPLC,reagent method to analyze the physicochemical properties of polysaccharides and establish quality control methods;PKS purified by fractional alcohol precipitation to obtain 50 sites%,70%,90%and 95%precipitates were deproteinized,dialyzed to remove small molecules,and purified by DEAE-Sepharose FF ion exchange column and Sephadex G-100 column chromatography to obtain a homogeneous polysaccharide named PKS-1.PKS-2,PKS-4,PKS-5.The monosaccharide composition,relative molecular mass and structure were studied by PMP-HPLC,high performance gel permeation chromatography and infrared spectroscopy respectively.（3）The in vitro hypoglycemic activity of PKS,PKS-1,PKS-2,PKS-4 and PKS-5 was screened by the adipocyte glucose uptake model and the Hep G2 cell glucose consumption model;superoxide anion radicals and hydroxyl groups were used.Antioxidant activity of PKS,PKS-1,PKS-2,PKS-4 and PKS-5 was evaluated by free radical and DPPH free radical scavenging experiments;（4）Infrared spectroscopy combined with PCA analysis was used to identify the chemical constituents in the processing of“Nine Steamed Nine”system.The changes of polysaccharide composition and content in the processing were further determined by high performance gel chromatography and ultraviolet spectroscopy.Result:（1）Infrared fingerprints of 12 batches of Polygonati rhizoma were established,and there were 7 infrared fingerprint peaks,which were 3353.6 cm^-1,2933.2 cm^-1,1623.8 cm^-1,1419.4cm^-1,1128.2 cm^-1,respectively.1051.0 cm^-1,825.4 cm^-1,the similarity coefficient of 12batches of medicinal materials were all greater than 0.9,indicating that the quality of the raw materials of scutellariae was uniform.The IR average spectrum and the second derivative map of the three legal bases of Polygonatum kingianum polysaccharides were significantly different.PCA,PLS-DA and HCA analysis could distinguish the three primary sources of the Polygonatum,and the Polygonatum fine HCA was grouped into one type,PCA,PLS-The DA scatter is more concentrated,indicating that the quality of the polysaccharide is higher.Pre-column derivatization-HPLC analysis showed that there were differences in the monosaccharide composition of the polysaccharides in the three legal bases,and the common components were D-mannose,D-ribose,L-rhamnose,D-galacturonic acid,D-Glucuronic acid,D-galactose,D-glucose,D-xylose,D-arabinose and L-fucose.The results of the content determination showed that the polysaccharide content of the tested samples met the requirements of the 2015 edition of the Chinese Pharmacopoeia.（2）The physicochemical properties and content of total polysaccharide PKS of P.chinensis showed that PKS was an acidic total polysaccharide containing various monosaccharide compositions,which contained impurities such as protein and pigment.The purity of polysaccharide in PKS was calculated by establishing a standard curve to be 67.2.%,the protein content was 17.1%,and the furfural content was 21.4%.Six homogeneous polysaccharides PKS-1,PKS-2,PKS-3,PKS-4,PKS-5 and PKS-6 were isolated and purified from the total polysaccharide PKS of Lycium chinense Maxim.The molecular weights were determined by high performance gel chromatography.-1 85017.83Da,PKS-2 266084.76 Da,PKS-3 474799.22 Da,PKS-4 14049.15Da,PKS-5 7694.85Da,PKS-6 3237.50Da.None of the impurities such as protein and nucleic acid were detected by UV detection;PMP-HPLC derivatization results indicated that PKS-1～PKS-5 mainly consisted of D-mannose,D-galacturonic acid,D-glucuronic acid and D-galactose.,D-arabinose,D-fucose and other monosaccharides,and the composition and content of monosaccharides vary greatly.IR showed that PKS-1～3 had absorption at 874 cm-1,890 cm-1 and 890 cm-1,indicating that all three polysaccharides haveβ-glycosidic bonds（C1-H bending vibration）,PKS-4～5Absorption peaks at 820 cm-1 and 831 cm-1 indicate that both polysaccharides have alpha glycosidic bonds.The results of the Congo red experiment indicated that PKS-1,PKS-2,PKS-3,PKS-4,and PKS-5 did not have a triple helix configuration.The PKS-4 nuclear magnetic analytic structure is composed of five different types of fructose,which areβ-（2→1,6）-D-Fruf,β-（2→6）-D-Fruf,β-（2→1）-D-Fruf andβ-（2→）-D-Fruf,and glucose is linked to the ketose chain in the form ofα-D-（1→）Glcp.（3）In vitro hypoglycemic screening results showed that total polysaccharide PKS and homogenous polysaccharides PKS-1,PKS-2,PKS-4,and PKS-5 could significantly promote glucose consumption in Hep G2 cells,and did not promote glucose uptake by adipocytes..The in vitro antioxidant results indicated that PKS-1,PKS-2,PKS-4,PKS-5 and PKS all had certain total reducing power,DPPH free radical and-OH scavenging ability.（4）The IR average spectrum and the second derivative map of different processed products are obviously different.PCA pattern recognition can distinguish different kinds of processed phlegm and yellow essence.The results of HPGPC show that the polysaccharides in the scutellariae are mainly composed of uniform polysaccharides PKS-4 and PKS-5.After the preparation of the nine steamed nine crops,the polysaccharide composition was mainly composed of small molecule homopolysaccharide PKS-6.The reason was that the uniform polysaccharides PKS-4 and PKS-5 were gradually hydrolyzed into PKS-6 with the increase of the number of processing.The results of the content determination showed that the content of polysaccharide in the direct dried product of the scutellaria baicalensis was the highest.With the increase of the number of processing,the content of polysaccharide decreased first and then stabilized.Conclusion:This study systematically isolated,purified and physicochemical properties of polysaccharides from Polygonatum kingianum,and further screened its hypoglycemic and antioxidant activities.In addition,the quality equivalence of the polysaccharides of the legal base species and the changes of polysaccharides in the processing of the“Nine Steamed Nine”system were compared,which provided a basis for the quality evaluation of the subsequent polysaccharides and the development of functional foods.