Studies On Hypolipidemic Effect Of Phascolosoma Esculenta And Its Polysaccharides

Phascolosoma esculenta,a marine invertebrate animal,is classified to the Sipuncula.It is a special species in China.It is recognized as a kind of delicious and traditional seafood and has been used as a special dish with pharmacological benefits for a variety of physiological functions for 300years.In order to develop its potential value as a health food and enlarge its application,this thesis focused on the exploration of hypolipidemic effect for Phascolosoma esculenta.The results of animal experiments indicated that Phascolosoma esculenta had significantly hypolipidemic effect on high-fat diet-fed mice.It had been reported by literature that a variety of polysaccharides had hypolipidemic effect.Therefore,this thesis investigated the hypolipidemic effect of Phascolosoma esculenta polysaccharides for further.First of all,Phascolosoma esculenta polysaccharides were extracted,followed by,in vitro antioxidant experiment and cells experiment were carried out.Finally,animal experiments were carried out and all mice were sacrificed for analysis.It was expected to provide data support on hypolipidemic application of Phascolosoma esculenta,and provide preliminary basis for its development and expand the application scope.The main work of this thesis can be summarized as follows:?1?Preliminary studies on hypolipidemic effect of Phascolosoma esculenta:To investigate the hypolipidemic effects of Phascolosoma esculenta on high-fat diet-fed mice,Kunming mice were randomly divided into seven groups namely the normal group?N?,the negative control group?NC,hyperlipemia model?,the positive control group 1?PC1,Soybean lecithin soft capsule?,the positive control group 2?PC2,Lipid-Lowering tablets?,the low-dose Phascolosoma esculenta powder group?PE-L,0.0675g/kg?,the middle-dose Phascolosoma esculenta powder group?PE-M,1.35g/kg?,the high-dose Phascolosoma esculenta powder group?PE-H,2.7 g/kg?.The body weight and liver weight of mice were weighed respectively.The concentration of serum triglyceride?TG?,cholesterol?TC?,high-density lipoprotein cholesterol?HDL-C?,low-density lipoprotein cholesterol?LDL-C?and liver TG,TC,and the activities of liver Aspartate aminotransferase?AST?,Alanine aminotransferase?ALT?,Superoxide dismutase?SOD?were analyzed.Liver coefficient and Arteriosclerosis Index?AI?were calculated.The experimental data indicated that liver coefficient of PE groups was significantly lower than that of the NC group?p<0.01?,the levels of serum TG,TC,LDL-C and AI were significantly lower than those of the NC group?p<0.01?,and the level of HDL-C was significantly higher than that of the NC group?p<0.01 or p<0.05?,and the levels of liver TG,TC were significantly lower than those of the NC group?p<0.01?.It can be concluded that Phascolosoma esculenta has a significantly hypolipidemic effect.Phascolosoma esculenta also can regulate the lipids metabolism disorder,reduce the accumulation of lipids on mice effectively,thus prevent the form and development of atherosclerosis.Phascolosoma esculenta significantly reduced the activities of liver AST and ALT?p<0.01 or p<0.05?.It showed that Phascolosoma esculenta has a liver protective effect,which was attributed to improve liver injury caused by hyperlipemia.Phascolosoma esculenta significantly increased the activity of liver SOD?p<0.01?.It showed that Phascolosoma esculenta can improve the anioxidant capacity,which was attributed to promote the activiy of anti-oxidation enzyme.The results suggested that Phascolosoma esculenta played a role of hypolipidemic effect may be related to its antioxidant.?2?Extraction of Phascolosoma esculenta polysaccharides:Polysaccharides were isolated from Phascolosoma esculenta by hot water extration and enzyme digestion,extraction yield was 63.09%.Polysaccharides content was31.08%measured by phenol-sulfuric acid method.?3?In vitro study on the antioxidant activities of Phascolosoma esculenta polysaccharides:The in vitro antioxidant activities were evaluated with the DPPH free radical scavenging assay,superoxide anion radical scavenging assay,hydroxyl free radical scavenging assay,ferrous ion cheating assay and measure of total phenol.The results of study showed that the DPPH free radical scavenging rate of 1.0 mg/mL Phascolosoma esculenta polysaccharides was 56.51%and its 50%effective concentration(EC₅₀)was1.16 mg/mL.The superoxide anion free radical scavenging rate was 43.30%.The hydroxyl free radical scavenging rate was 23.75%and its EC₅₀0 was 2.30mg/mL.The ferrous ion chelating rate was 18.59%and its EC₅₀0 was 6.41mg/mL.The concentration of total polyphenol using gallic acid equivalent was 36.63±5.08 mg/g.All data together would suggest that Phascolosoma esculenta polysaccharides possessed a considerably good antioxidant capacity in vitro.?4?Study on the effect of Phascolosoma esculenta polysaccharides on3T3-L1 preadipocyte:To investigate the effect of Phascolosoma esculenta polysaccharides on 3T3-L1 preadipocyte,MTT method was carried out,and the suitable concentration of Phascolosoma esculenta polysaccharides were estimated.After that,Dexamethasone,3-isobutyl-a-methylxanthine and insulin were used to induce 3T3-L1 preadipocyte differentiation into adipocyte.3T3-L1 preadipocyte was randomly divided into two groups namely the control group and the treated group?PEP?.Cells using oil red O staining were subjected to observe the effects Phascolosoma esculenta polysaccharides on 3T3-L1 preadipocyte after cells differentiation for 8 days.Subsequently,isopropyl alcohol solution was measured for its differentiation rate.Then the concentration of triglyceride was measured with the assay kit,the protein expression of SirT1 and PPAR?were detected by Western blot.The results showed that the optimum concentration of Phascolosoma esculenta polysaccharides was 0.1 mg/mL,which had no obvious effect on cell proliferation.When compared with the control group,0.1 mg/mL Phascolosoma esculenta polysaccharides could inhibit the formation of adipocyte,reduce differentiation rate of 3T3-L1 preadipocyte and the concentration of triglyceride on adipocyte,upregulate SirT1 protein expression,downregulate PPAR?protein expression.Evidences collected from experiments indicated that Phascolosoma esculenta polysaccharides could inhibit the differentiation of 3T3-L1 preadipocyte into adipocyte.?5?Study on hypolipidemic effect of Phascolosoma esculenta polysaccharides:To explore the hypolipidemic effects of Phascolosoma esculenta polysaccharides,Kunming mice were randomly divided into seven groups namely the normal group?N?,the negative control group?NC,hyperlipemia model?,the positive control group?PC,Soybean lecithin soft capsule?,the Phascolosoma esculenta powder group?PE,1.35 g/kg?,the low-dose Phascolosoma esculenta polysaccharides group?PEP-L,0.2 g/kg?,the middle-dose Phascolosoma esculenta polysaccharides group?PEP-M,0.4g/kg?,the high-dose Phascolosoma esculenta polysaccharides group?PEP-H,0.8 g/kg?.The body weight and liver weight of mice were weighed respectively.The concentration of serum TG,TC,HDL-C,LDL-C and liver TG,TC,and the activities of AST,ALT,SOD,and the concentration of serum Malondialdehyde?MDA?was analyzed.The data indicated that liver coefficient of PE and PEP groups was significantly lower than that of the NC group?p<0.01?,the levels of serum TG,TC,LDL-C and AI were significantly lower than those of the NC group?p<0.01?,and the level of HDL-C was significantly higher than that of the NC group?p<0.01 or p<0.05?,and the levels of liver TG,TC were significantly lower than those of the NC group?p<0.01 or p<0.05?.It can be concluded that Phascolosoma esculenta and its polysaccharides possess a significantly hypolipidemic effect,can regulate the lipids metabolism disorder,reduce the accumulation of lipids on mice effectively,and then prevent atherosclerosis.Phascolosoma esculenta and its polysaccharides significantly reduced the activities of AST and ALT on serum and liver?p<0.01 or p<0.05?,suggesting that Phascolosoma esculenta and its polysaccharides have the protective effects on liver injury by hyperlipemia.Phascolosoma esculenta and its polysaccharides significantly reduced the serum level of MDA?p<0.01?,and increase the activity of serum SOD?p<0.05?significantly.It can be concluded that Phascolosoma esculenta and its polysaccharides could promote the activity of anti-oxidation enzyme and reduce the level of peroxidation in body.Therefore,Phascolosoma esculenta and its polysaccharides maybe play a role of hypolipidemic effect by the way of antioxidant.This thesis demonstrated that oral administration of Phascolosoma esculenta and its polysaccharides had outstanding hypolipidemic effect,could improve and adjust the lipids metabolism disorder,reduce the risk of atherosclerosis,improve the body's antioxidant capacity and protect liver.This is agreement with the activity of the known clinical used health products Soybean lecithin soft capsule.The results of this thesis suggested that Phascolosoma esculenta had a potential value to be a source of hypolipidemic functional health foods,and it could be developed into a kind of a potential anti-hyperlipidemic drug for hyperlipidemic patients,using for prevention and treatment of atherosclerosis and arterial thrombosis as well as other associated cardiovascular diseases.In summary,the present investigation for the first time confirmed the hypolipidemic effects of Phascolosoma esculenta and its polysaccharides,yet the mechanism and the bioactive ingredient remained to be further confirmed.