Study On Quality Control Of Yupingfeng Polysaccharides And Mono Polysaccharides Of Yupingfeng Prescription

YuPingFeng polysaccharides(YuPingFeng polysaccharides,YPF-P)is an effective part of the total polysaccharides of Yupingfeng powder,which has the effective of enhancing immunity,antiviral,preventing and treating liver damage and so on.However,the Quality Control standards of polysaccharides is incomplete until now(Only the total sugar content was detected),causing some fake and inferior polysaccharide products to appear repeatedly.In this test,YPF-P,Astragalus polysaccharides,Atractylodes polysaccharides and Sposknikovan were extracted and purified,and the total sugar content was determined.The weight of molecular was determined by high performance gel chromatography(HPLC-GPC),the varity of monosaccharides and the material ratio were determined by thin layer chromatography(TLC)and pre-column derivatization HPLC.Dimethylthiazole(MTT)method was used to determine the proliferation ability of chicken embryo fibroblasts(CEF)after polysaccharide treatment in vitro.And a systematic,fast and effective method of quality control of YPF-P was established.The contents of the study were as follows:(1)Polysaccharides were extracted by water extraction and alcohol precipitation,and purified by AB-8 macroporous resin.The total sugar content of purified polysaccharides was determined by phenol sulfuric acid method.(2)Using the HPLC-GPC and RID method to measure the molecular weight of these four polysaccharides.Mobile phase: ultrapure water;Flow velocity: 0.7 mL/min;Column temperature: 35 ?.(3)TLC was used to determine the monosaccharide composition of polysaccharides.Using two kinds of expansion agent to expand two times on the Silica gel G plate,heating and developing the sample(105 ?)for45 min.(4)Using pre-column derivatization HPLC to measure the monosaccharide composition of these four polysaccharides.The polysaccharide samples were hydrolyzed in trifluoroacetic acid solution,then precolumn derivatizated with1-phenyl-3-methyl-5-pyrazolone(PMP),The chromatographic separation was carried out on Wondasil C18(250mm×4.6mm,5?m)using phosphoric acid aqueous solution-acetonitrile(pH=6.9)and acetonitrile(85 : 15)as mobile phases,at a flow rate of1 mL/min,40 ?,with 254 nm detected wavelength.(5)MTT assay was used to test CEF proliferation in vitro.The best concentration was selected from the dilute YPF-P solutions,and then 4 kinds of polysaccharides were compared with the best concentration.The results showed:(1)The content of these 4 polysaccharides samples were all more than 90%.(2)Through HPLC-GPC,YPF-P,Astragalus polysaccharides,Atractylodes polysaccharides contained more than 75% of micromolecule polysaccharides(1300<Mw<5000),and Sposknikovan contained 60% of macromolecule polysaccharides(Mw?20000000).(3)Through TLC,fructose,D-glucose,D-mannose,arabinose and D-galactose could be found in four kinds of polysaccharides.(4)By pre-column derivatization HPLC,YPF-P and sposknikovan were composed of mannose,ribose,rhamnose,galactose uronic acid,glucose,galactose and Arabia sugar,whose mole ratio was4.64 : 1 : 5.88 : 2.40 : 120 : 42.9 : 69.5;Sposknikovan was composed of mannose,ribose,rhamnose,galactose uronic acid,glucose,galactose and Arabia sugar,and their mole ratio was 6.17 : 1 : 4.73 : 4.12 : 202 : 80.2 :68.9;Astragalus polysaccharides was composed of mannose,glucose,galactose and Arabia sugar,the mole ratio was 1: 388 : 17.5 : 14.7;Atractylodes polysaccharides was composed of mannose,glucose,galactose and Arabia sugar,the mole ratio was 1 : 239 : 18.6 : 19.3.(5)By MTT,The tested group of 15.63-1000?g/mL of YPF-P could significantly promote the proliferation of CEF.The best concentration was 1000 ?g/mL.The effect size order of 4 polysaccharides on CEF proliferation was YPF-P > Astragalus polysaccharides > Atractylodes polysaccharides >Sposknikovan.The test results showed that the polysaccharide extraction and purification method was stable,and the polysaccharide content and biological activity were high.The established methods for molecular weight detection,monosaccharide composition and biological activity analysis of polysaccharides were fast,efficient,stable and reliable.The experiment provided important basic data for formulating the quality control standards of YPF-P and its constituent polysaccharides,and also provided reference for the study of polysaccharide quality control methods.