Study On Hypolipidemic Activity And Nano-vesicles Development Of Corn Silk Polysaccharides

The corn silk polysaccharide（CSP）was seperated from the corn silk which was considered as corn by-products as well as functional food.CSP can be produced with a wide source and a high economic value.Study on the corn silk showed there was no chronic toxic effects on rats.As one of the main components of corn silk,CSP has been proved to have significant hypoglycemic,anti-fatigue,anti-oxidation,anti-tumor,hepatoprotective activity and hypolipidemic activity.The development and application of CSP are promising.However,CSP is a kind of heteroglycan with complex structure,which is difficult to carry on quality control.There was few research about the structure characterization,hypolipidemic activity and related preparations of CSP,which limited its practical application.In order to enrich the structure and bioactivity theory of natural CSP and explore its practical application,two kinds of CSP fraction were separated and purified in this study.In addition,the similarities and differences between their primary and advanced structure were analyzed,respectively,and the hypolipidemic activities of two CSP components were studied in vitro and in vivo.Furthermore,the key structural characteristics of CSP which may affect its hypolipidemic activity were discussed.Finally,the CSP noionic surfactant nano-vesicles was prepared and studied with a series of evaluation in vitro.The full study includes the following five chapters as follows:Chapter one:ReviewsThe first chapter summarized the extraction,separation and purification methods of CSP as well as its structure and bioactivities.Additionally,the current studies about the structure-bioactivities relationship of polysaccharides were analyzed and compared.In order to study the critical structure characters affecting the hypolipidemic activity of CSP,the structure and hypolipidemic activity between two fractions of corn polysaccharides were compared.Besides,a novel nonionic surfactant nano-vesicles was prepared to entrap water-soluble polysaccharides,which provides reference for the practical application of corn polysaccharides.The above contents provided a basic theoretical foundation for this task.Chaper two:Isolation and purification of CSPIn this chapter,CSP were extracted,separated and purified.The phenol sulfuric acid method was used for the determination of neutral sugar content,the Coomassie Blue method was used for determination of protein content,sulfuric acid carbazole method was applied to measure the acid sugar content and the molecular weight was determined by high performance gel chromatography.The above data were collectively evaluated the purity of the polysaccharide.CSP was separated and purified to obtain two main components including CSP-1 and CSP-2,the chemical composition analysis showed that the neutral sugar contents of CSP-1 and CSP-2 were（79.71±2.07）%and（56.49±3.06）%,respectively,the protein contents were（0.92±0.09）%（CSP-1）and（0.87±0.12）%（CSP-2）,the contents of uronic acid were（14.56±0.67）%and（37.00±1.03）%,respectively.In addition,the molecular weight of CSP-1 and CSP-2 were12.93 kDa and 19.32 kDa,respectively.These results showed that CSP-1 and CSP-2were similar in molecular weight,which belong to the same order of magnitude,and the significant difference between CSP-1 and CSP-2 was uronic acid content.CSP-1was neutral polysaccharide but CSP-2 was acid polysaccharide.Chapter three:Structure characterization of CSPIn this chapter,the primary structure of CSP-1 and CSP-2 were determined and compared,including monosaccharide composition,glycoside bond type and the kinds of glycoside bond.Secondly,the advanced structure of two components were studied including the branched chain composition and the chain conformation of polysaccharide.The monosaccharide composition of CSP-1 and CSP-2 were determined by the pre-column derivatization HPLC method.Then,the possible glycoside bond type and the structure difference between them were compared using IR spectra.The periodate oxidation was used for studying the kinds of glycoside bond existing in the CSP-1 and CSP-2.Finally,the branched chains of CSP-1 and CSP-2were determined by partial acid hydrolysis,and the chain conformation of polysaccharide was determined by the Congo red reaction which further confirmed by atomic force microscope.The results showed that the CSP-1 and CSP-2 were both contained seven kinds of monosaccharides,the hydrolysate of CSP-1 included D-mannose,L-rhamnose,D-glucose,D-galactose,L-arabinose and D-xylose,as well as D-galacturonic acid with respective molar ratios of 1.00:0.21:1.41:1.44:0.70:0.44:0.56,whereas the molar ratio of CSP-2 was 1.00:0.20:0.91:1.85:0.60:0.24:2.00,their uronic acid content was significantly different.Besides,the IR spectra showed that the glycoside bond of two natural polysaccharides wereα-configuration.The periodate oxidation results showed that the glycoside bond types in CSP-1 were 1→6 and 1→3bond,however,1→6,1→3 and 1→2 or 1→4 bond were found in the CSP-2.Additionally,the Congo red reaction and atomic force microscope showed that CSP-1and CSP-2 were non triple helix structure,but the polysaccharide chain aggregation could be observed of CSP-2.Chapter four:Study on the hypolipidemic activity of CSPIn this chapter,the hypolipidemic activities of CSP-1 and CSP-2 were evaluated by the in vitro cholate conjunction test and two kinds of hyperlipidemia mice model.During the in vitro test,sodium taurocholate and sodium glycocholate were chosen as binding reagents since they were both the main existing types of bile acid in vivo,moreover,cholestyramine was chosen as the positive drug to evaluate the conjunction rates of polysaccharide at different concentrations and different reaction times.Besides,the in vivo hypolipidemic activities of CSP were studid and compared by Poloxamer407 induced acute hyperlipidemia mice and high-fat diet induced chronic hyperlipidemia mice.The results showed that the in vitro binding rates of CSP-1 and CSP-2 in different polysaccharide concentrations and different reaction times were（47.96%⁹8.00%,54.46%¹03.50%）and（38.70%⁹0.32%,49.82%⁸7.10%）,respectively,which indicating their significant hypolipidemic activities in vitro.In addition,CSP-1 and CSP-2 showed no plasma lipid lowering activities at low doses(100 mg·kg^-1),but good lipid lowering activities in medial-dose group(300 mg·kg^-1)and high dose group(500 mg·kg^-1)in P407 induced acute hypeipilidemia mice.In the high-fat diet induced hyperlipidemia mice,CSP-1 and CSP-2 lowered plasma cholesterol（TC）and triglyceride（TG）,but did not improved fat accumulation of liver cells at low dose.While both CSP showed the same plasma-lowering activities as positive drug and improved fatty degeneration of liver tissues significantly at high dose.In summary,the hypolipidemic activities of CSP-1 and CSP-2 were not affected by the significant difference between their structures.Chapter five:Study on the CSP nano-vesiclesIn this chapter,the nonionic surfactant nano-vesicles were developed by ethanol injection method which was convenient and economical,and the prepared vesicles were of high encapsulation efficiency and good stability.The effects of cholesterol and surfactant composition on the formability of vesicles were investigated.The most suitable compositions of vhicle were confirmed span80:tween80=6:5（g/g）by examining the appearance,particle size and Zeta potential.Afterwards,the ratio of the drug-vehicle ratio,the proportion of the cholesterol and the preparation temperature were optimized by using the orthogonal design for encapsulation efficiency as the index.The best prescriptions were the temperature at 65℃,the drug vehicle ratio was 1:2and the ratio of surfactant to cholesterol was 7:1.In addition,a series of in vitro evaluations of CSP loaded nano-vesicles including particle size and Zeta potential,transmission electron microscopy（TEM）and storage stability were performed.The results showed that those nano-vesicles were hollow vesicles,the particle size was（208.55±0.26）nm with evenly distributed（PDI<0.08）,Zeta potential value was（-31.46±0.97）mV and the encapsulation efficiency was 74.60%without big change within 60 days at 4℃.