Study On The Preparation,pharmacology And Toxicology Of Spirulina Polysaccharide Extract

Spirulina polysaccharide extract was made of a natural active polysaccharide extracted from spirulina and have an immunoregulatory activities.In order to provide the oretical basis of the clinical application of Spirulina polysaccharide extract on the healthy ecological aquaculture of Penaeus vannamei,this study was carried out to investigate the preparation technology of Spirulina polysaccharide extract,acute toxicity test in mice,30-day feeding trial in rats,and immunoregulatory effects of the polysaccharide extract on Penaeus vannamei in vivo and in vitro.Methods:（1）The Spirulina polysaccharide was extracted by enzymatic assisted hot water extraction.Firstly,a certain amount of Spirulina powder was soaked in 95%ethanol for decolorization,then the sediment was added a certain amount of distilled water and papain and take a water bath after enough mixing.Secondly,the filtrate of the polysaccharide extract was concentrated after filtering,then added 5 times volume of 95%ethanol solution for alcohol precipitation.Finaly,the precipitate was added silica in the proportion of 4:1,then granulated.In order to determine the optimal extraction process,the single factor and orthogonal design test were studied,in which the content of polysaccharide of extraction was chosen as the index of evaluation and the ratio of material-liquid,enzyme content,extraction temperature and time was chosen as the influence factors.（2）In the acute toxicity test,20 mice were randomly divided into 5 groups,including 20 g/kg·BW,10 g/kg·BW,5g/kg·BW,2.50 g/kg·BW,1.25 g/kg·BW and the animals were given suspension of Spirulina polysaccharide extract by gavage in three times within24 hours after the begining of the experiment.Then mice were observed whether poisoning or death in 7 days and killed on the 8th day for observing pathological change of the internal organs.If the mice did not die,a maximum tolerance test would was performed.（3）In the 30-day feeding test of rats,80rats were randomly divided into four groups,including high dose group（20g/kg·BW）,medium dose group（10 g/kg·BW）,low dose group（5 g/kg·BW）and blank control group,and the animals were given continuously Spirulina polysaccharide extract in the way of mixed feeding for 30 days.Then the observation continued for 7 days after discontinuation.10 rats were randomly selected from each group on the 31th day and the 8th day after drug withdral to determine hematology and blood biochemical indexes.The major organs were dissected to calculate the organ index and histopathological examination.（4）The CCK8 method was used to screen the safe concentration of Spirulina polysaccharide extract on the blood lymphocytes of Penaeus vannamei,and then its effect on the immune function of the blood lymphocytes of Penaeus vannamei was observed.The test group of Spirulina polysaccharide extract,blank control group and Astragalus polysaccharide（200μg/m L）positive drug group were set up,the cells of each group were treated correspondingly and incubated to 12 h,the cells were collected,and ACP,AKP,POD activities were measured.（5）To investigate the immunomodulatory effect of Spirulina polysaccharide extract on Penaeus vannamei,Spirulina polysaccharide extract of 1.0 g/kg,2.0 g/kg,or 4.0 g/kg was added to the basic feed named low,medium and high dose drug test groups,blank control groups,and Astragalus polysaccharide（2.0 g/kg）drug controls were set up respectively,Penaeus vannamei were fed continuly for 28 days.Then the hemolymph of each group was collected on the 7th,14th,21th and 28th day respectively post treatment and the activities of SOD,ACP,AKP and PO were measured.Ammonia nitrogen stress test was conducted in shrimp after 28-day feeding of the drugs.NH₄Cl was used to adjust ammonia nitrogen concentration.After 72 hours of ammonia nitrogen stress test,the cumulative mortality of shrimp in each group was statisticed,and the activities of SOD,ACP,AKP and PO were measured.Results:（1）The Spirulina polysaccharide was extracted by enzymatic assisted hot water extraction,the content of polysaccharide was 34.38%.After orthogonal optimization,the results showed that the four factors affecting the extraction of polysaccharide from Spirulina were:extraction temperature>enzyme content>extraction time>material-liquid ratio.The optimal extraction process is A₁B₁C₁D₃,that is,the water bath temperature is 70℃,the water bath time is 2h,the material-liquid ratio is 1:10,and the enzyme content is7.50%of the weight of algal flour.（2）The results of the acute toxicity test showed that there was no death of the mice during the test,and no abnormal lesions were found in the autopsy.The results showed that Spirulina polysaccharide extract was practically non-toxic.（3）The results of 30-day feeding test of rats showed that during the period of administration and after stopping the drug,there was no poisoning or death in each experimental group,no abnormality in clinical manifestations and organ index and histopathological examination results,and no abnormal change in hematological and biochemical indexes.（4）The effect of Spirulina polysaccharide extract on blood lymphocytes of Penaeus vannamei showed that ACP,AKP,POD activities of blood lymphocytes exposed to Spirulina polysaccharide extract of 50μg/m L,100μg/m L,200μg/m L,and 400μg/m L can extremely significant（P<0.01）or significant（P<0.05）were increased when compared with the blank control group.（5）In the 28-day feeding test and ammonia nitrogen stress test,the activities of ACP,AKP,SOD,and PO in Penaeus vannamei were significantly improved（P<0.05）by administering a diet supplemented with Spirulina polysaccharide extract.And in the ammonia nitrogen stress test,the cumulative mortality of prawns fed with Spirulina polysaccharide extract was significantly reduced.Conclusion:The content of polysaccharide was increased by using enzymatic assisted hot water extraction;Spirulina polysaccharide extract has no acute toxic effect and has not caused toxic side effects after long-term continuous administration,so it is safe;The extract could improve the non-specific immunity of Penaeus vannamei by improving the activities of immune related enzymes and had significant anti ammonia nitrogen stress effect.