Studies On Isolation, Purification, Structure And Function Of Polysaccharides From Rape Pollen

In recent years,pollen has been used as a form of full nutrition food all over the world.In China,the fundamental research and applicative research on pollen have been deeply developed for many years,and many pleased results were acquired,even some aspects have entered leading level in the international research.Rape pollen,which has the biggest output in China,is well-known for its bioactive functions such as anticancer,enhancing immunity,et al.Making full use of rape pollen resource and doing systematic research on Rape Pollen make it possible that a new level in development and utilization,and will bring a great of economic and social benefit.However,the maximal extraction percent of rape pollen polysaccharides currently reported was only 1.450%.In order to better improve the extraction percent of rape pollen polysaccharides and find the best method of breaking wall,three method that were temperature difference-machine,temperature difference-ultrasonic and Superfine comminution were used.On the basis of systematic research of extraction,isolation,purification,physiochemical properties and primary structure of Rape Pollen polysaccharides,antioxidation research of Rape Pollen polysaccharides was estimated, which gave the foundation for the further research and development.The main research and results were as follows;1.The contents of proteins,fats,flavonoids,several metal elements such as calcium,magnesium, zinc,copper,iron in rape pollen were determined.The contents of proteins and fats were found higher in rape pollen.Besides,the contents of metal elements which were measured were rich. This provides scientific reference for the further study and development of rape pollen resources.2.Temperature difference-machine,temperature difference-ultrasonic and Superfine comminution were used to break wall of rape pollen.Wall-breaking effects of the fomer two method were far inferior to Superfine comminution.Wall-breaking rate of Superfine comminution could reach over 99% comminution3.Using only glucose as the standard,the optimum way of coloration in the process of determining rape pollen polysaccharides content by phenol-sulfuric acid method was obtained as follows;1.0 mL 5% phenol solution was added to 2.0 mL rape pollen polysaccharides solution and mixed.Then 5 mL concentrated sulfuric acid was added quickly drop by drop.After set at ambience for 20 minute,the solution was detected at wavelength of 482 nm.4.The extraction of polysaccharides by water from rape pollen was studied.The effects of four factors such as temperature,time,ratio of solid-liquid and times of extraction were investigated.The results showed that the effect of temperature was the most important,time was the second,times was the third,and the ratio of solid-liquid was the least.Through orthogonal test,the optimum conditions of extraction were obtained on the basis of single factor test.The results were as follows;90℃for 4h and 3 times extraction,ratio of solid-liquid 1;8.The extraction percent of polysaccharides was 4.48% on the optimum conditions.5.Protein in the crude rape pollen polysaccharides was removed by Sevag method and trichloroacetic acid sedimentation respectively.Removal effect of the latter was higher than that of the former,and meanwhile the loss rate of the latter was lower than that of the former.6.The primary purification of the refining rape pollen polysaccharides were carried out by DEAE-cellulose chromatography.Neutral polysaccharides D₁ and acidic polysaccharides D₃ were mainly obtained in this process.Then Neutral polysaccharides D₁₁and acidic polysaccharides D₃₁were achieved by further purifing D₁ and D₃ respectively with Sephacryl S-200 chromatography.The identification of purification with Sephacryl S-300 chromatography and HPLC implied that only D₁₁was homogeneous polysaccharides.Its molecular weight was 80271 dalton,estimating by gel filtration.The monosaccharide compositions of polysaccharides D₁₁and D₃₁were analyzed by gas chromatography.They were composed of rhamnose, arabinose,xylose,mannose,glucose,galactose and fucose at least.No typical absorption of nucleic acid or protein in polysaccharides D₁₁was observed in UV spectrum.Infrared spectrum of polysaccharides D₁₁and D₃₁indicates that they had typical characteristic absorption peaks of polysaccharides.7.Antioxidant abilities of crude and refining rape pollen polysaccharides were measured as DPPH scavenging activity,activity against hydroxyl radical and superoxide anion-scavenging activity.The investigation demonstrated that they possessed free radical scavenging activity. With increasing of polysaccharide concentration,action effects were increased.Action effects of refining rape pollen polysaccharides were better than crude rape pollen polysaccharides.