Study The Relationship Between The Extraction Technology And The Activity On Agrocybe Cylindracea Polysaccharide

To design single-factor experiments and orthogonal test, in the purpose of choosing an optimum technology which not only can maintain the good biological activity of Agrocybe cylindracea Polysaccharide (ACP) but also can improve its extraction yield. Using different methods to remove the protein of ACP, in order to determine the best technology of removing protein. ACP separated by column chromatography and its fractions for molecular weight determination, acquair the molecular weight distribution feature of the ACP which have the better biological activity, to establish a simple product quality evaluation criteria on ACP. Provide the reference for establishing the product quality evaluation system about the other fungal polysaccharide.1 Effects of Single-factor conditions on the extraction percent and activity in Agrocybe cylindracea PolysaccharideDesign single-factor experiments, fix other factors, change the conditions of one factor, choose the extraction percent of polysaccharides and it's activity as the evaluation indexes to determine the optimum conditions for this factor, and provide the reference for the orthogonal experimental elements of choice are: extraction temperature, pH value, as well as the methods of breaking up cells .the results showed that: choose pH 7.0, pH 7.5, pH 8.0, as the three-level of orthogonal test on pH value factor. Extraction temperature 70℃, 80℃, 90℃can be used as the three-level of orthogonal test on temperature factor. Microwave 6 min, power 400 w; ultrasonic treatment 30 min; freeze-thawing three times were chosen as three-level of orthogonal test on the cell disruption methods factor.2 Effects of Different Extraction technologies on the extraction percent and activity in Agrocybe cylindracea Polysaccharidebased on single-factor pre-trial conclusion, according to L9(33) orthogonal test table to design three factors and three-level orthogonal experiments, choose the extraction percent of polysaccharides and it's activity as the evaluation indexes to determine the best extraction process. The results showed that: To improve the yield of ACP the optimum technology was: temperature 90℃, pH value was pH 7.5,the m- ethod of broking the cells was repeated Freeze-thawing three times; The best technology that can heighten the content of ACP was: temperature 80℃and pH 7.5 with 30 min of ultrasonic cell disruption; To enhance the ACP's anti-lipidperoxidation activities, the optimum extraction conditions was: temperature 80℃, pH 7.5, Freeze-thawing three times for cell disruption; To promote NO production of macrophage cells, the best extraction conditions was: temperature 80℃, pH 7.5, ultrasonic 30 min. Integrated multi-factor analysis, we can conclude that high temperature and strong alkali are disadvantage for maintaining the biological activity of polysaccharide, freeze-thawing treatment can significantly increase the polysaccharide yield, and can maintain the activity of polysaccharide in a better way, but it violate the industrial production that save energy and reduce the cost principles, consequently according to the results of orthogonal test, the extraction of polysaccharides at 80℃, pH 7.5, 30 min of ultrasonic cell disruption is considered as an optimum technology.3 Effect of the inhibiting action on lipidperoxidation of Agrocybe cylindracea polysaccharideswith different removal of protein methodTo remove protein from ACP,five schemes including Sevag, Enzyme-Sevag, Trichloroaceticacid, Trichloroaceticacid- Sevag, Isoelectric point were used in the experiment. Determined the polysaccharides conserving percentage,removed protein percentage and the activity of the polysaccharides as the evaluation indexes. In order to compare the effects of the different removal of protein methods and used the primary pure polysaccharides to do the active tests to determine the best process of removing protein. The results showed that: as far as the polysaccharides conserving percentage,removed protein percentage and the activity of the polysaccharides are concerned, the method of Sevag is better than other methods.If just from the anti-lipidperoxidation activity of polysaccharides, the unremoval protein is better than removal protein.4 The relationship between molecular weight and activity of polysaccharideACP was separated by SephadexG-100 chromatography and DEAE-cellulose chromatography, the isolated polysaccharide components were analysised by HPLC, and compared with HPLC chromatogram of dextran standards to obtained their molecular weight distribution scope; use the components to do the active tests (anti-lipid peroxidation test and induced NO production of macrophage cells by ACP test), select the components whith better activity, then determine the molecular weight distribution scope of the ACP which have the better biological activity. Analyse the relationship between the range of molecular weight distribution of polysaccharide and the biological activity of polysaccharide can find that in terms of anti-lipidperoxidation activity of ACP, ACP2-3's activity is best, its molecular weight range is 5.37×105-1.35×106, through analysis the molecular weight distribution of the six components of the polysaccharide and its activity, we can conclude that the ACP with molecular weight of 105 orders of magnitude have the better anti-lipid- peroxidation activity, molecule which is too large or too small, all can affect its activity.For the activity of stimulating NO production of macrophage cells, ACP2-1's activity is the worst, its molecular weight range is 5.50×104-3.16×105, ACP2-4's activity is the best with molecular weight range of 7.59×105-4.90×106 . ACP2-1's molecular weight is smaller than the other fractions, while the ACP2-4s'is relatively large.So we can conclude that the ACP with large molecular weight stimulate macrophages produce NO more strongly than the ACP with little molecular weight, but whether the molecular weight of polysaccharides and the activity of NO production has the positive relation need us do further study.In terms of the results of the test, we can conclude that the ACP with different molecular weight has different biological activity, so in the industrial process we must pay more attention on the process technology, according to the purpose of the polysaccharides, design the appropriate process technology to ensure that can extracte more polysaccharides with better activity.