| As the third major bioactive substances,polysaccharides play many biological activities,such as: antioxidant、anti-cancer、promotion of immunity、regulation of intestinal flora,etc.However,the biological activities is closely related to its structure which could be directly affected by the molecular weights(MWs)of polysaccharides,and further affected the absorption and distribution in vivo.In this study,we selected three kinds of mannosan with different molecular weights(3MWs-M),(MWs of YM-50,YM-70,YM-90 are 172.9 k Da 、87.09 k Da、54.05 k Da,separately),to study their biological characteristics and cell absorption mechanism in vitro,and preliminarily explore their distribution characteristics in vivo.Our results would provide basic data for the structure-activity relationship between polysaccharide and molecular weights for their application.Firstly,the biological characteristics of 3MWs-M were studied by SEM 、acidification of/with artificial gastric juice、hemolysis、cytotoxicity and antioxidant tests.The changes of structure and molecular weights of three kinds of mannan were detected after 4 h acidification in artificially prepared gastric juice;One percent erythrocyte suspension was prepared for detection the hemolysis of three kinds of mannan;The cytotoxicity of three kinds of mannan was detected by MTT assay on Caco-2 cell,and the antioxidant capacities were analyzed by the examinations of total reducing ability、hydroxyl radical scavenging ability and DPPH radical scavenging ability.The results showed that the microscopic morphology of three kinds of mannanans were different,which the surface of YM-50 was flat and smooth without cracks and pores;the surface of YM-70 was relatively smooth but with crisscrosses of cracks and particles attached;the surface of YM-90 was honeycomb-like and porous.Acidification with artificial gastric juice did not affect the basic structural of sugar groups in/of the three kinds of mannan,but could degrade them into smaller molecular polysaccharides,even monosaccharides.The smaller the molecular weights of mannan were,the easier the molectular weights were degraded.The hemolysis rates of the three kinds of mannan with different molecular weights met the requirements of Chinese pharmacopoeia with slightly fluctuated,which all of them were less than 5%.The results of cytotoxicity showed that the inhibition ratio of YM-50 on Caco-2 cell increased with the concentration increasing,while the cytotoxicities of YM-70 and YM-90 decreased which had certain proliferation effects on Caco-2 cell;The antioxidant capacities of the three kinds of mannan were all positively,correlated with sugar concentration.The lower the molecular weights of mannan are,the stronger the antioxidant activity are.Secondly,fluorescence staining mannan was used to directly observe the absorption process of polysaccharides into Caco-2 cell.In vitro,the absorptions,the expressions of proteins and RNAs of transporters of the three kinds of mannan were studied by established/developed the Caco-2 cell model containing M cells,and the absorption tested with the everted gut nacs to analyze the absorption mechanism.Results show that YM-50 labeled FITC didn’t present within 90 minutes,while the fluorscents of YM-70 and YM-90 labeled FITC gradually appeared with the testing time,and closely transferred into the cytoplasm and nuclear of Caco-2 cell during30～60 min,the bright spots overlaped the fluorscents with nuclei labeled DAPI and mannosan labeled FITC could be found at 90 min,these results indicated that the mannan with larger molecular weights hardly move into the cell,meanwhile the mannosan with smaller molecular weights might enter the cytoplasm and nucleus to exert biological activity.The Caco-2 cell model containing M cells was established successfully when the resistance of layer of cells stabilized at about 700 Ω,the permeability of fluorescein sodium didn’t penetrate through the cells layer,the M typical microvilli,a tight junction between cells and the secretion follicles were appeared,and the polarity ratio of alkaline phosphatase was above 2.The apparent permeability coefficients(Papps)of the three kinds of mannan intestinal cell models detected by the Caco-2 cell model showed that two absorption peaks of YM-90 and YM-50 were found separately,and one low peak of YM-70 absorption was found,and proved that the bioavailabilities of all three kinds of mannan could reach 100%;The results of mannan absorption tested with the everted gut nacs showed that YM-50didn’t be absorbed through guts,then caused the sugar effloxation from the jejunum,while,YM-70 and YM-90 could be absorbed,and the smaller the molecular weights were,the easier them wer absorbed from the jejunum and ileum.RT-PCR results showed that the three kinds of mannan could cause the changes of expression amounts of the transporters,i.e.,SGLT1,GLUT2 and Clathrin.The fluctuations of m RNA amount of three kinds of absorption transfer factor in YM-50 were slower,while,the changes of YM-70,YM-90 reached the peak in 60 min,30 min separately,it confirmed that the m RNA amount of three kinds of absorption transfer factor increased with the molecular weights decreasing,of which the expression amount of GLUT2 was the highest,then was Clathrin,the SGLT1 was the lowest.Western Blot results showed that the absorption of YM-50 was mainly mediated by the transporters of GLUT2 and Clathrin,while the absorptions of YM-70 and YM-90 mainly depended on GLUT2,which indicated that sugar transporters involved in mannanans of different molecular weights were different.The results of three kinds of sugar absorption tests in this chapter also indicate that different methods have different effects on detecting sugar absorption.Finally,KM mice were administrated three kinds of mannan solutions to study their absorption and distribution in vivo.The results showed that YM-50 with high molecular weights couldn’t be absorbed into the blood,and had obvious hypoglycemic effect.YM-50 may be distributed in the spleen through endocytosis,and could flow in the intestinal lumen/cavity.However,YM-70 and YM-90 with low molecular weights could be absorbed into the blood and increase blood glucose,which are mainly distributed in the liver,lung and kidney with rich blood in vivo,then,unabsorbed polysaccharides flowed to the intestinal lumen of the jejunum and ileum.In summary,this study demonstrated that polysaccharides with different molecular weights have direct effects on the absorptions and biological activities via the biological characteristics,absorptions and distribution of mannan with different molecular weights in vivo.These data provide basic data for the development and application of mannan. |
