| OBJECT:To research the purification conditions of EP; To compare the different of monosaccharide composition, molecular weight, functional group and active ingredients from different EP, which extracted by different preparation methods; To research antidiabetics and its related gene of EP, which extracted by different preparation methods; To explore the relationship bewteen the EP’s primary structure and antidiabetics.METHOD: 1. Take the enteromorpha which is produced by fujian province as the research object. The EP is extracted by ultrasonic or cellulose enzymatic. Then remove protein by neutral protease, remove salts by materialto dialysis bag, remove pigment by DA201 resin. Combinated with water extract-alcohol precipitation to extract the UEP and EEP.2. DEAE-52 cellulose and Sephadex-100 will be used to purify and separate the EP, then the principal components of UEP and EEP will be getted. Analysis the principal components of UEP and EEP about molecular size by HPLC, monosaccharide composition by gas chromatographic, functional group by infrared spectrum,polysaccharide content by phenol sulfuric acid method, sulfate radical content by barium sulfate turbidimetry, uronic acid content by m-hydroxydiphenyl.3. Using Hep G2 cells, cultured with high sugar medium. Taking insulin and metformin as positive control group, observe whether EP can reduce glucose levels in the culture medium.4. set up an insulin resistance model, Taking metformin as the positive control group, observe whether the EP can reduce glucose levels in the culture medium. Then detect the expression level of HNFs, IR, GLUTs through RT-PCR.RESULT:1. The extraction, separation and purification of EP With purification by DAEA-52, both UEP and EEP can be divided into five components. And their most component are the first one. The mole ratio of UEP’s five components is 146:27:40:3:1, and the principal component called UEP1 accounted for 67.28 percent of UEP. The mole ratio of EEP’s five components is 89:18.5:27:8.5:1, and the principal component called EEP1 accounted for 61.80 percent of EEP. After purification by Sephadex G-100, the and recovery rate of UEP1 and EEP1 are respectively 98.85%, 99.12%.EP is a white powder solid. It is insoluble in methanol, ethanol, acetone and other organic solvents. It is difficute to dissolve. The maximum solubility of UEP and EEP in water is 40 mg/m L. After purification and separation, the maximum solubility of UEP1 and EEP1 in water is 20 mg/m L.The aqueous solution of EP can turn into form gel, and the solution-gel transition phenomenon is thermal reversible.2. Analysis the structure and composition of EPFTIR analysis showed that both UEP1 and EEP1 were free serum alpha-pyran EP which are rich in sulfate groups. Both of them contain uronic acid, carboxylic acid, ether linkage. The average molecular weight of UEP1 is 79547, when the average molecular weight of EEP1 is 520160. Whether UEP1 or EEP1, the highest monosaccharide is Rha. The mole ratio of Rha, Xyl, mans, Glu, Gal is 3.96:1.09:0.24:0.34:0.16 in UEP1, while the mole ratio of Rha, Xyl, mans, Glu, Gal is 3.08:1:0.17:0.30:0.11 in EEP1.With phenol-sulfuric acid method, the polysaccharide content of UEP1 is 98.59%, while EEP1 is 97.0%. With gelatin-barium sulfate turbidimetry, the sulfate radical content of UEP1 is 17.74%, while EEP1 is 20.71%. With m-hydroxyldiphenyl, the uronic acid content of UEP1 is 22.20%, while EEP1 is 21.76%.3. Research the antidiabetics and its mechanism of EP extracted by different preparation methodThrough the glucose consumption test of Hep G2 cells, deducting the effect of cell apoptosis. We found that compared with Blank, the GC of Insulin, Metformin, HUEP1, MUEP1, LUEP1, SUEP1, EHEH1, MEEP1, LEEP1, SUEP1 were higher(P<0.05). Compared with Metformin, the GC of Blank, LUEP1, SUEP1, SEEP1 were lower(P<0.05), the GC of Insulin, HEEP1, MEEP1 were higher(P<0.05). Compared with Insulin, the GC of Blank, Metformin, MUEP1, LUEP1, SUEP1, SEEP1 were lower(P<0.05), the GC of MEEP1 were higher(P<0.05).Setting up an insulin resistance model, we found that Hep G2 cells start to produce insulin resistance when using insulin with 1×10-3mmol/L. The model is stable within 60 hours. The bright red lipid drops significantly increased in the insulin resistance model with Oil red O staining.Through improving the insulin resistance model, deducting the effect of cell apoptosis. We found that compared with Blank, the GC of Model, all doses of UEP1 and EEH1 were lower(P<0.05). Compared with Model, the GC of other groups were higher(P<0.05). Compared with Metformin, the GC of Model, HUEP1, MUEP1, LUEP1, SUEP1, LEEP1, SEEP1 were lower(P<0.05).Observe the expression of hepatic cells factor HNFs. The first one is HNF4α. Compared with Blank, the GC of other groups were lower(P<0.05). Compared with Model, the GC of other groups were higher(P<0.05). Compared with Metformin, the GC of MUEP1, LUEP1, SUEP1, MEEP1, LEEP1, SEEP1 were lower(P<0.05). The second one is HNF1α. Compared with Blank, the GC of other groups were lower(P<0.05). Compared with Model, the GC of Metformin, HUEP1, MUEP1, HEEP1, MEEP1, LEEP1 were higher(P<0.05). Compared with Metformin, the GC of MUEP1, LUEP1, SUEP1, LEEP1, SEEP1 were lower(P<0.05). The third one is HNF1β. Compared with Blank, the GC of other groups were lower(P<0.05). Compared with Model, the GC of Metformin, HUEP1, MUEP1, LUEP1, HEEP1, MEEP1, LEEP1, SEEP1 were higher(P<0.05). Compared with Metformin, the GC of MUEP1, LUEP1, SUEP1, MEEP1, LEEP1, SEEP1 were higher(P < 0.05). The last one is HNF6. Compared with Blank, the GC of other groups were lower(P<0.05). Compared with Model, the GC of other groups were higher(P<0.05). Compared with Metformin, the GC of LUEP1, SUEP1, MEEP1, LEEP1, SEEP1 were lower(P<0.05).Observe the expression of IR. Compared with Blank, the GC of other groups were lower(P<0.05). Compared with Model, the GC of other groups were higher(P<0.05). Compared with Metformin, the GC of LUEP1 was higher(P<0.05), the GC of SUEP1, MEEP1, LEEP1, SEEP1 were lower(P<0.05).Observe the expression of GLUT4. Compared with Blank, the GC of other groups were lower(P<0.05). Compared with Model, the GC of other groups were higher(P<0.05). Compared with Metformin, the GC of HUEP1, MUEP1, HEEP1 were higher(P<0.05).Observe the expression of GLUT2. Compared with Blank, the GC of other groups were higher(P<0.05). Compared with Model, the GC of other groups were higher(P<0.05). Compared with Metformin, the GC of EP’s groups were lower(P<0.05).Conclusion:1. With purification by DAEA-52, both UEP and EEP can be divided into five components, and their most component are the first one. The first component is about 60%. It shows single peak when purified with Sephadex-100. The polysaccharide content of component which elutioned by 0.4mol/L Na Cl is the highest. The polysaccharide content decreases with Na Cl concentration.2. FTIR analysis showed that both UEP1 and EEP1 were free serum alpha-pyran EP which are rich in sulfate groups. Both of them are mixture with different molecular weight of EP. The average molecular weight of UEP1 is lower than EEP1. The rhamnose content was the highest. Both UEP1 and EEP1 was composed of rhamnose, xylose, mannose, glucose, galactose and a small quantity of arabinose. But UEP1 also contain ribose and fucose. The monosaccharide composition is different bewteen UEP1 and EEP1.3. Setting up an insulin resistance model by insulin with 1×10-3mmol/L. The bright red lipid drops significantly increased in the insulin resistance model with Oil red O staining, while the normal cells only have little lipid drop. RT-PCR showed that the expression of HNF 4α, HNF 1α, HNF 1β, HNF 6, IR and GLUT4 reduce, and the expression of GLUT2 is up regulation. The model is stable within 60 hours. It can be used to research drugs about its effect of T2 DM.4. EP can lower blood glucose. The effect of antidiabetic is better than insulin and metformin. The antidiabetic effect of EEP1 is excellent than UEP1. Maybe it is beacuse the molecular weight of EEP1 is larger than UEP1. Raising the number of the insulin receptor might have something to do with it.5. The mechanism of EP improving insulin resistance contain three parts. The first one, raising the expression quantity of HNFs. The second one, raising the expression quantity of GLUT4. The third one, reducing the expression quantity of GLUT2. |
PDF Full Text Request