Research On The Hypo-cholesterolemic Activity And The Extraction Purification, Primary Structure Of Polysacchairdes From Equisetum Hiemale L

This research was sponsored by the Key Agriculture Project entitled “Demonstrationof Functional Egg Industrialization (NO.20100243)” sponsored by Jilin ProvincialScience and Technology Department. In this research, the preventive effect againsthypercholesterolemic and the extraction, purification, primary structure and in vitrocardiovascular protective effect of polysaccharides from E. hiemale L. were studied.Firstly, response surface methodology (RSM) based on a Box-Behnken design (BBD)was employed to optimize the preparation parameters of polysaccharides with high DPPHscavenging activity from E. hiemale L. stems. Three independent variables such as finalconcentration of ethanol (%), ratio of water to raw material, extraction temperature (°C)were investigated. Analysis of variance (ANOVA) showed that the contribution ofmathematical models obtained from multiple regression analysis was significant for theresponses. A relatively high yield of polysaccharides (3.01±0.04%) with high DPPHscavenging activity (40.18±2.20%, at the concentration of0.5mg/mL of reactionsolution) was obtained under these conditions: final concentration of ethanol90%, ratioof water to raw material24.4:1, extraction temperature86.9°C, which in turn proved thevalidity of the prediction models.The preventive activity against hypercholesterolemic of the obtained crude E. hiemaleL. polysaccharides (CEPS) was evaluated compared to the aqueous extract of E. hiemaleL.(AE) in high fat diet induced hyperlipidemic rats. The oral administration of CEPS andAE at the dose of10,20,40gbw/day were found to ameliorate the hyperlipidemiccondition by dose-dependently reducing serum concentrations of triglycerides (TG), totalcholesterol (TC), low-density lipoprotein cholesterol (LDL-C), coronary heart diseases(CHD) risk ratio and by dose-dependently increasing serum high-density lipoproteincholesterol (HDL-C) content. Further, the oral administration of CEPS and AEdose-dependently reduced liver triglyceride and cholesterol content. Additionally the CEPS and AE treatment dose-dependently increased fecal bile acids, triglyceride andcholesterol excretion. All above activities reached significant levels when high dose ofCEPS and AE (40mg/100gbw/day) were administered to the rats fed a high fat diet(P<0.05). The results suggest that both CEPS and AE inhibited the increase oftriglyceride and cholesterol in rats fed high fat diet. And one of the mechanisms may bedue to the promotion of fecal cholesterol and bile acids excretion.Polyamide adsorption resin chromatography was employed to remove protein andpigments existing in the crude polysaccharides, which resulted in a decrease of proteincontent by95.08%and a decrease of pigments content by66.10%. After this primarypurification procedure, the polysaccharides retention rate was as high as90.37while theDPPH scavenging activity of the polysaccharides was reduced by29.07%. Fouriertransform-infrared (FT-IR) spectroscopy showed that both of the original polysaccharidesand the polysaccharides underwent primary purification procedure (DEPS) had distinctiveabsorptions of polysaccharides and the original polysaccharides contained more silicon.Then the polysaccharides obtained from primary purification procedure (DEPS) wasfurther purified by fractional precipitation and DEAE-52anion exchange chromatography.After the fractional precipitation by ethanol of different final concentrations (30,50,70,90%), one polysaccharides fraction with high yield and DPPH scavenging activity wasobtained and named as DEPS-1, which underwent further purification by DEAE-52fastflow anion exchange chromatography and one neutral fraction (DEPS-α) was obtained.The HPGPC profile of showed a single and symmetrical sharp peak, indicating thatDEPS-α was a homogeneous polysaccharide. The purity of DEPS-α was determined to be71.12%.The primary structure of DEPS-α was studied. The ultraviolet-visible absorptionspectrum of DEPS-α indicated DEPS-α had few protein, nucleic acid and pigments. Andthe FT-IR spectroscopy of DEPS-α showed that DEPS-α had distinctive absorptions ofα-polysaccharides. The number-average molecular weight, weight-average molecularweight, Z-average molecular weight,(Z+1)-average molecular weight of DEPS-α weredetermined and calculated to be707384Da,850566Da,1217579Da,2215167Da,respectively using HPLC and GPC software. The monosaccharide composition of DEPS-αwas determined by GC-MS after it was hydrolyzed by trifluoroacetic acid and derivatized to acetylate aldononitriles. The results showed that DEPS-α was made up of Arabinose,Xylose, Glucose and Galactose with the molecular ratio of3.8∶2.0∶18.1∶1.8.In vitro activity assays including in vitro bile acid-binding assay, in vitro anti-serumlipoprotein oxidation assay and in vitro anticoagulate assay showed DEPS-αdose-dependently inhibited Cu2+-induced serum lipoprotein oxidation and prolongedactivated partial thromboplastin time (APTT), indicating that the polysaccharide havepotential cardiovascular protective activities, though it didn’t exhibit in vitro bile acidbinding activity.