Studies On Polysaccharide Containing Selenium Of Cardamine With Rich Selenium From Enshi

Cardamine is a kind of plant enriching selenium, which grows in mountainous area of Enshi, Hubei province . It not only contains rich selenium but also is a sort of plant which is edible and has medical functions. In order to develop the Cardamine resource, the dried Cardamine powder was used as material for experiment and the polysaccharide containing selenium was selected as cut-over point for study, and the isolation and purification characters for polysaccharide containing selenium from Cardamine were discussed systematically to provide theory and practice basis for developing Cardamine. Main study results are as follows. 1Evaluating determination method for polysaccharide from CardamineThe colorimetric method by phenol and sulphuric acid can be used to determine quantitatively polysaccharide from Cardamine according to accuracy, reappearance, recycle ratio and stability for determination method. At the same time, the monosaccharides forming polysaccharide from Cardamine are mainly pyranose.2 Characters about abstracting crude polysaccharide from CardamineIn order to discuss the suitable abstracting conditions for polysaccharide from Cardamine, the dried Cardamine powder 1 g used as material for experiment and the distilled water selected as abstracting reagent, influence of the single factor including temperature, time, distilled water volume, abstraction times and pH on abstraction quantity of polysaccharide was studied, then the whirly design test was carried out on the aforementioned basis to discuss comprehensive influence of factors on abstraction quantity of polysaccharide. The results showed that temperature, time, distilled water volume, abstraction times and pH all had significant influence on abstraction quantity of polysaccharide, and the optimum conditions for abstracting polysaccharide from Cardamine were temperature 90℃, time 3 h, distilled water volume 30 ml, abstraction times 5 and pH 9, so abstraction quantity of polysaccharide was up to 4.76%.3 The law for alcohol precipitating polysaccharide from CardamineIn order to discuss the characters about alcohol precipitating polysaccharide from Cardamine, first of all, the influence of alcohol concentration on the amount of polysaccharide precipitated was studied, then the influence of precipitation times on the amount of polysaccharide precipitated was studied. The results showed that using 90% alcohol to precipitate polysaccharide from Cardamine two times can precipitate polysaccharide at most, which not only purifys polysaccharide and was beneficial to the follow-up process of isolation and purification for polysaccharide.4 The isolation and purification method for accompanying precipitant from CardamineOne unknown compound is precipitated accompanying polysaccharide precipitating when alcohol precipitating crude polysaccharide solution from Cardamine enshiensis, in order to isolate the accompanying precipitate from polysaccharide, first of all, accompanying precipitate was flocculated by Na₂HPO₄ from crude polysaccharide solution, precipitate was accumulated after centrifugation, Na₂HPO₄ was added to flocculating accompanying precipitate from clear solution, the aforementioned operations were carried out again and again until no flocculation appeared in clear solution. The accompanying precipitate was soluble with distilled water, then adding acetone to aforementioned solution, the precipitate was soluble with distilled water, adding Na₂HPO₄ to the solution to flocculate accompanying precipitate, the precipitate was soluble with distilled water after centrifugation, the aforementioned operations were carried out again and again until the accompanying precipitate was determinated to be negative by phenol and sulphuric acid colorimetric method. Then the accompanying precipitate was further purified with macroporous resin (35×120mm) adsorption chromatography, its chromatography conditions were the volume of apply sample 40 ml with certain concentration, distilled water elution, and the elution flowing speed 1.2 ml/min, 30 %—80 % carbinol gradient elution. finally, the accompanying precipitate is further purified by using gel filtration chromatography Sepharose 6B (20×400mm) , its chromatography conditions were the volume of apply sample 3 ml with certain concentration, distilled water elution, and the flowing speed of elution 1.5 ml/min.5 Purity identification for accompanying precipitate and its structure describingPurity of accompanying precipitate was testified by HPLC and the structure of accompanying precipitate was determinated by mass spectrum. The results showed that the purity of accompanying precipitate was chromatograph grade, and the accompanying precipitate is a kind of macromolecular compound which has five-membered ring and six-membered ring cycloalkane parent nucleuses linking with-OH groups, and -C^=O—O- groups existing between parent nucleuses.6 The flocculation law for accompanying precipitate from CardamineIn order to discuss the flocculation characters for accompanying precipitate from Cardamine, The Na₂HPO₄ and Ca(OH)₂ selected as flocculating reagents, influence of pH value and the amount of Na₂HPO₄ and Ca(OH)₂ on flocculating accompanying precipitate and comprehensive influence of two factors aforementioned on flocculating accompanying precipitate were studied. The results showed that alkaline solution and metal ion existing in solution were two factors impacting flocculationamount while accompanying precipitate flocculating.7 Characters about pigment existing in polysaccharide solution from CardamineWhen the pigment exists in crude polysaccharide from Cardamine, in order to study the fundamental characters about the pigment removed from polysaccharide solution, the active carbon used as adsorpting reagent and the temperature, time as well as the amount of adsorpting reagent for decolorization selected as experiment factors, the single factor and orthogonal test were carried out. The results showed that the optimum conditions for decolorization were temperature 70℃, time 4 h and the amount of adsorpting reagent 0.8%. In order to reduce the loss of polysaccharide due to it adsorpted by active carbon while the pigment adsorpted by active carbon, distilled water used as desorption reagent and the temperature, time as well as the amount of desorpting reagent selected as experiment factors, the uniform test with nine levels was carried out. The results showed that the optimum conditions desorpting polysaccharide from active carbon were temperature 78℃, time 100 minand the amount of distilled water 10 times by active carbon.8 Characters about protein existing in polysaccharide solution from CardamineWhen the protein exists in crude polysaccharide from Cardamine, in order to study the fundamental characters about the protein removed from polysaccharide solution, the trichloroacetic acid used as reagent removing protein from polysaccharide solution, the single factor tests such as the concentration of trichloroacetic acid, temperature and time were carried out, then the uniform test with nine levels and two factors including trichloroacetic acid concentration and time was carried out. the results through analysis of variance and regression showed no influence of temperature on trichloroacetic acid removing protein and the influence of trichloroacetic acid concentration and treating time on removing protein was significant, at the same time, the suitable conditions for removing protein from polysaccharide were trichloroacetic acid concentration 4 % and treating time 12 h-16 h under room temperature. 9 The methods for purifying polysaccharide from CardamineIn order to discuss the methods for purifying polysaccharide from Cardamine, first of all, using cellulose DEAE-celloluse 52 chromatography isolated the neutral polysaccharide containing selenium and acid polysaccharide containing selenium, then using Sephadex G-100 chromatography purified the neutral polysaccharide containing selenium and acid polysaccharide containing selenium respectively. The neutral polysaccharide containing selenium was testified to be purified compound by HPLC, and the acid polysaccharide containing selenium was testified to be two components, one component is polysaccharide containing selenium, the other is a sort of unknown component.10 Describing the structure of polysaccharide containing selenium Se-P IIn order to describing the structure of polysaccharide containing selenium Se-P I, the structure of polysaccharide containing selenium Se-P I purified was studied initially by using chemical method and modern analysis technology such as Gas chromatography, Gas chromatography- mass spectrum, infrared spectrum and nuclear magnetic resonance spectrum. The results showed that polysaccharide containing selenium Se-P I is a kind of complicated polysaccharide which consists of D-arabinose, D-Mannose, D-galactose and D-glucose, and the ring style of these monosaccharides is pyrane which configurations are beta and alpha, and the glucose and galactose form glucoside with other monosaccharides between C -1 and C-5.