| This work was supported by Marine Industry Research Special Funds for Public Welfare Projects—Part experiment content of "Exploration,Preservation and utilization of germ plasm resources on several important officinal marine organisms".This dissertation is divided into five major parts.In the first chapter,this chapter reviewed the system in scattered research progress of four genera of officinal marine organisms.From the second chapter to the sixth chapter is specific research content and correlated conclusion as follows:There are four segments in the second chapter about antipyretic,anti-inflammatory and anti-oxidative activity of Thais luteostoma extract.First segment,T.luteostoma was divided into shell and soft tissue as the samples.The soft tissue had been homogenized 3 times and It was centrifuged at 4? and 8000 r/min for 10 min,after which,supernatant was frozen drying 12 h to obtain freeze-dried powder.After the soft tissue was removed,shells were dried to remove moisture under 60 ? and crushed into powder.The soft tissue was divided into visceral mass and uropod.Partial visceral mass was in boiling water bath for 30 minutes,after which,supernatant was frozen drying 12 h to obtain freeze-dried powder;the extraction method of uropod is the same as that of visceral mass.Seven samples were prepared.In the second segment,the rat model of yeast-induced fever was used to investigate the antipyretic effect of T.luteostoma,and in the same time the concentration variation of PGE2,cAMP,Na+/Ca2+,TNF?,IL-1?,IL-2,IL-6 was also observed in the study.The results showed that TLSH-1,TLST-1,TLST-2,TLST-3 and TLST-5 significantly inhibited yeast induced pyrexia in rats at post-administration,and exhibited more lasting effects as compared to aspirin,and TLSH-1 had the better antipyretic activity than TLST-1,and part of its antipyretic effect may be mediated through the suppression of production of PGE2,cAMP,Na+/Ca2+,TN?,IL-1?,IL-2 and IL-6.In the third segment,the mouse model of auricle swell induced by dimethylbenzene,the mouse model of hind paw edema induced by carrageenan and the rat model of hind paw edema induced by carrageenan were used to investigate the anti-inflammatory effect of TLSH-1 and TLST-1,and in the same time the concentration variation of PGE2,TNF?,IL-1?,IL-2,IL-6,NO,MDA and SOD was also tested.The results showed that TLSH-1 and TLST-1 could significantly reduce against dimethylbenzene induced auricle swell and carrageenan induced paw edema in mice or rats(P<0.05);and they markedly reduced levels of PGE2,TNFoc,IL-1?,IL-2,IL-6,NO,MDA and increased the vitality of SOD.In the fourth segment,the radical-scavenging activity in our study was evaluated by a standard spectrophotometric assay using the DPPH radical in a 96-well microplate.The result showed that TLST-1,TLSH-2 and TLSH-3 had anti-oxidative activity.There are two segments in the third chapter about bio-activity evaluation on Sipunculus nudus.First segment,S.nudus was dissect and divided into somatopleura-viscera and coelomic fluid.Somatopleura-viscera was cut into pieces,and homogenized after two times freeze-thaw cycles.Somatopleura-viscera and coelomic fluid were centrifuged to obtain supernatant and precipitate.The supernatant of somatopleura-viscera was frozen drying 12 h to obtain freeze-dried powder,and the precipitate of somatopleura-viscera was extracted in water;both supernatant and precipitate of coelomic fluid was frozen drying 12 h to obtain freeze-dried powder.Four samples were prepared.In the second segment,inhibition rates of platelet aggregation in vitro were determined in the model of platelet aggregation induced by adenosine diphosphate.Their anticoagulative effects were also evaluated on prothrombin time(PT),activated partial thromboplastin time(APTT)and thrombin time(TT).The model of artery-vein bypass thrombosis was established to observe their antithrombotic effect.The antioxidant effects were investigated by DPPH free radical scavenging capability test.The fibrinolytic activity was determined by fibrin plate.The result showed that the extracts had significant inhibition effect on platelet aggregation,and could obviously prolong PT,APTT and TT,inhibited the artery-vein bypass,reduced the thrombus weight significantly(wet or dry).And the extracts of S.nudus also could eliminate DPPH free radicals and dissolve fibrin in different degree.There are two segments in the fourth chapter about bio-activity evaluation on Inimicus japonicas livers.First segment,fresh and healthy I.japonicas livers were first washed in distilled water.After the large blood vessels and ligaments were removed,the livers were cut into pieces and homogenized.After freezing and melting for several times,a double-volume distilled water was added to the product which was then heated to 95 ? for 5 min to obtain the heat-stable peptides.Cooled to room temperature,it was centrifuged at 4?,after which,supernatant was frozen drying 12 h to obtain freeze-dried powder.One sample was prepared.In the second segment,immunosuppression animal model was established by cyclophosphamide induction.The immunity organs were weighed to measure the thymus index and spleen index;the clearance of charcoal particles method was used to measure mice's mononuclear macrophages functions,and sheep red blood cells immunizations was used to measure mice's humoral immune functions;mice's blood counts was measured by blood-counter system,and Enzyme-linked Immuno Sorbent Assay(ELISA)was used to measured the TNF-a,IL-1?,IL-2 and IL-6 level in blood serum;the spleen cell cycle was measured by flow cytometry,spleen and thymic tissue morphology was observed and graded with HE.The results showed that Inimicus japonicas liver extract significantly improved the thymus index and spleen index,promoted the division and proliferation of spleen cells,improved the mononuclear macrophages functions,increased the hemolysin level of blood serum and the levels of leukocytes,neutrophils,lymphocytes and monocytes,and also increased the levels of TNF-?,IL-1?,IL-2 and IL-6 in blood serum.There are two segments in the fifth chapter about bio-activity evaluation on Grateloupia asiatica.First segment,G.asiatica was extracted in water,depositing in alcohol after concentration.Supernatant and precipitate were obtained after suction filtration in 24 hours.The supernatant was concentrated without alcohol;the precipitate was washed with alcohol,and then it was frozen drying 12 h to obtain freeze-dried powder after alcohol was gotten rid of.Two samples were prepared.In the second segment,the mouse model of alloxan-induced diabetes was used to investigate the hpyerglycemic effect of G.asiatica.The results showed that G.asiatica significantly reduced blood-sugar content of alloxan-induced diabetic mice,significantly improved the alloxan-induced diabetic mice's weight,organ coefficient and lightened inflammation of pancreas,and the solution after depositing in alcohol also inhibited?-glucosidase's activity.Conclusion:This paper,by establishing the effect of antipyretic and anti-inflammatory evaluation system,is to investigate the antipyretic and anti-inflammatory activity of T.luteostoma,and the radical-scavenging activity was evaluated by a standard spectrophotometric assay using the DPPH radical in a 96-well microplate;the thrombotic activity of S.nudus extracts is evaluated by investigation of the effects on platelet aggregation and coagulation function rabbit plasma in vitro,and the effect on the model of artery-vein bypass thrombosis in rats,as well as capacity of eliminating DPPH free radicals and dissolving fibrin;influence of I.japonicas liver extract on the immune function was investigated on the basis of immunosuppressed mice;the mouse model of alloxan-induced diabetes was used to investigate the hpyerglycemic effect of G.asiatica.These studies showed that T.luteostoma extract has antipyretic and anti-inflammatory activity,and S.nudus extract had strong antithrombotic effect,and I.japonicas liver extract enhanced immune-suppressed mice's immune function,and G.asiatica could regulate blood-sugar content of alloxan-induced diabetic mice.This paper characteristics and innovations:1.Animal model was used to evaluate the antipyretic and anti-inflammatory activity of T.luteostoma extract for the first time,according to our research results,not only edible part(soft tissue)but also discarded part(shell)exhibited obvious antipyretic and anti-inflammatory activities,which provided scientific data for further exploring and developing original drug or functional product.2.Influence of Inimicus japonicas liver extract on the immune function was investigated on the basis of immunosuppressed mice for the first time;the results showed that Inimicus japonicas liver extract enhanced immunosuppressed mice's immune function.3.The mouse model of alloxan-induced diabetes was used to investigate the hpyerglycemic effect of G.asiatica;the results showed that the solution significantly reduced blood-sugar content of alloxan-induced diabetic mice after depositing in alcohol. |
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