Preparation And Determination Of Polysaccharide In Polygonatum Kingianum

Polygonati Rhizome is a traditional Chinese medicine,which has the function of invigorating kidney and nourishing dry,and is mainly used to treat the deficiency of body,heart palpitation,shortness of breath,dry cough,diabetes and so on.Polysaccharide was identified as one of the main active substance in Polygonati Rhizome.In this thesis,polysaccharide of Polygonatum kingianum planted in Puer city of Yunnan province was prepared and determined,which helped for the quality analysis and in-depth development of this herb.The main research results were obtained as follows.Three polysaccharide fractions,PK-F1?PK-F3 were extracted from olygonat kingianum by hot water and followed by alcohol precipitation in graduation.PK-F1 was then separated by Toyopearl DEAE-650M anion exchange chromatography,eluted with 0.5 mol/1 NaCl to give purified PK-P1 fraction.These agents(PKs)were supplied as the following analysis samples.The relative weight-average molecular weight(Mw)of PKs was then assayed by high performance size exclusion chromatography-refractive index detector(HPSEC-RI)assay,using glucans as reference substances.The chromatography was performed with LC-20A system and Shodex RI-101 detector,using TSK-gel G3000 PWXL column(10 ?m,7.8 mm×300 mm)with water as mobile phase,the flow rate was 0.5 ml/min;the column temperature was 30 ? and the injection volume was 20 ?l.The regression equation of glucan was calculated to be lgMw =-0.3409tR + 8.7708(R2 = 0.9974)within the linear range of 0.18?200 kD,and the accuracy,precision,repeatability and stability data fitted to the methodology requirement.The main polysaccharide fraction in the PK-F1?PK-F3 samples was 149.4,40.3 and 1.47-3.76 kD,respectively.Among them,one of the fractions(Mw 149.4 kD)was observed for the first time.The uronic acid of PKs was also determined by hydroxy diphenyl chromogenic method assay,using galacturonic acid as reference substance.The regression equation of galacturonic acid was calculated to be y = 0.0072 x-0.0674(R2 = 0.9982)within the linear range of 10.02?60.12 ?g/ml,and the accuracy,precision,repeatability,stability and recovery data fitted to the methodology requirement.The content of uronic acid in the PK-F1?PK-F3 samples was 28.3%,16.2%and 4.93%,respectively.This is the first report of the uronic acid data in the PKs.The neutral sugar content of PKs was finally determined by anthrone-sulfuric acid chromogenic method assay,according to Chinese Pharmacopoeia(2015 edition).The regression equation of glucose was calculated to be y = 0.0096 x-0.1021(R2 = 0.9984)within the linear range of 10.03?80.24 ?g/ml,and the accuracy,precision,repeatability,stability and recovery data fitted to the methodology requirement.The content of neutral total sugar in the PK-F1?PK-F3 samples was 78.0%,66.9%and 64.5%,respectively.