| Objective: The method of Enteromorpha polysaccharides sulfate modified and its effect and mechanism on hypoglycemic activity.Methods:1. The EP was got by cellulase-catalyzed extraction, and dimethyl formamide method and chlorosulfonic acid-pyridine method were applied to desulfurization acidification modification and sulphation modification to gain TEP and SEP respectively.2. The Phenol-sulfuric acid method, barium sulphate turbidimetry, liquid chromatography, gas chromatography and infrared spectrum were used to analyse the amount of sulfate group, the molecular weight of polysaccharides, the composition of monosaccharide and the function of the polysaccharide respectively.3. In order to investigate the ability of glucose uptake caused by the amount of sulfate group, we designed four EP groups, including 1×10-3 g/m L(HEP)?1×10-4g/m L(MEP) ? 1×10-5 g/m L(LEP) ? 1×10-6 g/m L(SEP), and four TEP groups,including 1×10-3 g/m L(HTEP)?1×10-4 g/m L(MTEP)?1×10-5 g/m L(LTEP)?1×10-6g/m L(STEP), and four SEP groups, including 1×10-3 g/m L(HSEP)?1×10-4 g/m L(MSEP)?1×10-5 g/m L(LSEP)?1×10-6 g/m L(SSEP),the group with inhibitor of HY294002(Blank+?Model+?Metformin+?HTEP+?MTEP+?LTEP+?STEP+?HEP+?MEP+?LEP+?SEP+?HSEP+?MSEP+?LSEP+?SSEP+),and two positive control groups, such as metformin and insulin, and one as normal control.4. The model of insulin resistance in Hep G2 was built to analyze the effects of decreased blood glucose. Groups were designed as four EP groups, including1×10-3 g/m L(HEP)?1×10-4 g/m L(MEP)?1×10-5 g/m L(LEP)?1×10-6 g/m L(SEP),and four TEP groups, including 1×10-3 g/m L(HTEP)?1×10-4 g/m L(MTEP)?1×10-5g/m L(LTEP)?1×10-6 g/m L(STEP), and four SEP groups, including 1×10-3 g/m L(HSEP)?1×10-4 g/m L(MSEP)?1×10-5 g/m L(LSEP)?1×10-6 g/m L(SSEP), and two control groups, such as normal control and model group and one as positive control group of metformin. The m RNA expression levels of Akt and GSK-3? by RT-PCR and protein expression levels were calculated by Western Blotting in Hep G2, then discussed probable mechanism of changing blood glucose with inhibitor treatment.Result:1. The method of sulfuric acid modification on EP The different reaction time in acidification of the desulfurization experiments had an effect on polysaccharide and 3h is the best reaction time. The different reaction quantity of chlorosulfonic acid of polysaccharide sulfate content had an effect on polysaccharide in Sulfating experiment, which suggested 3ml as the best addition amount of chlorosulfonic acid.2. Analysis the composition and structure of EP With gelatin-barium sulfate turbidimetry, the polysaccharide content of EP is84.3%, TEP is 82.2%, and SEP is 83.4%;The results of gas phase analysis showed that all polysaccharide contain rhamnose(Rha), xylose(Xyl), mannose(Man), glucose(Glu), galactose(Gal), the highest monosaccharide is Rha. The mole ratio of Rha, Xyl, Man, Glu, Gal is13.5:4:0.9:2.9:1 in EP, it contains a small amount of Ara; The mole ratio of Rha, Xyl,Man, Glu, Gal is 21.2:5.5:0.9:1.4:1 in TEP; and The mole ratio of Rha, Xyl, Man,Glu, Gal is 14:4.2:1:1.2:1 in SEP, it contains a small amount of Ara. And FTIR results showed that TEP?EP?SEP are alpha- pyran polysaccharide with sulfate.3. Research the effects of EP Through the glucose consumption test of Hep G2 cells, we found that the GC of Insulin, Metformin, HEP, MEP, LEP, HTEP, HSEP, MSEP, LSEP, SSEP groups were higher than blank group(P<0.05). Compared with Metformin group, the blank, SEP, MTEP, LTEP, STEP were significant lower(P<0.05), while insulin group was higher(P< 0.05); Compared with the Insulin group, the blank,Metformin, HEP, MEP, LEP, SEP, STEP, MTEP, LTEP, STEP, LSEP, SSEP were lower(P<0.05).The experience of setting up an insulin resistance model, 1×10-3 mmol/L was succeed in insulin resistance in Hep G2 cells after 36 h and it was stable within 60 h.The bright red lipid drops significantly increased in the insulin resistance model with Oil staining. The RT-PCR showed that the expression of IR in insulin resistance was lower than the Blank(P<0.05); While the expression of Glu T2 was higher than the blank(P<0.05).Through the glucose consumption test of Hep G2 cells after setting up Insulin resistance Model, compared with Blank, the GC of Model, EP, TEP, SEP were lower(P<0.05); and compared with the Model group, the Metformin, HTEP, the four groups of EP, and the four groups of SEP were higher(P<0.05); Compared with Metformin group, the GC of Model, the four groups of EP, the four groups of TEP, MSEP, LSEP, SSEP were significant lower(P<0.05).4. Research the antidiabetic mechanism of EP Observe the expression of Akt, we found that compared with blank, all of the other groups were lower(P<0.05); Compared with the Model, all of the other groups were higher(P< 0.05); Compared with Metformin, the expression of Akt in HEP, HTEP, HSEP were lower(P< 0.05), the expression of Akt in the experiment without HY294002 were higher than the experiment with HY294002(P<0.05).Compared with blank group, the expression of GSK-3? in other groups were lower(P<0.05); Compared with the model group, the expression of GSK-3? of Metformin, HEP, HTEP, HSEP were higher(P<0.05); Compared with Metformin,the expression of GSK-3? of HEP, HTEP, HSEP were lower(P<0.05). The expression of GSK-3? in the experiment without HY294002 were higher than the experiment with HY294002(P<0.05).Conclusion:The effect of Enteromorpha polysaccharides is not only associated with the main structure of polysaccharide, but also related to its sulfate group, and the strength is positively related to the content of sulfate groups.1. Two different methods applied to Enteromorpha polysaccharides sulfate group affected the amount of sulfate group but the main structure.2. The enhanced ability of up taking glucose showed in EP, TEP and SEP, and the trend is increased with the content of polysaccharide sulfate.3. All of EP, TEP, and SEP could improve insulin resistance and protect, to some extent, regular function of glycometabolism, and the strength was positively related to the content of sulfate groups.4. The expression of Akt, GSK-3? in PI3K/Akt pathway with inhibitor of HY294002 that had increased significantly than groups without inhibitor. It suggests that the pathway is inhibited when insulin resistance happened, which could be improved by increasing the expression of Akt and GSK-3?, and the strength was positively related to the content of sulfate groups. |
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