Studies On The Isolation And Purification Of Polysaccharides From Three Insects And Their Immunomodulating Activities

Polysaccharide is one kind of important large molecules in organism and has manyphysiological functions, such as immunomodulating activities, anti-tumor, anti-virus,anti-aging and so on, which indicates its promising application in food and medicine fields.However, most of the researches about polysaccharide are focusing on epiphytes and plants,especially traditional herbal medicine, only a few polysaccharides from the animals werestudied and the polysaccharide of insects is rare. The insect is the biggest biocenose andunexploited resource in the world. The studies on insect polysaccharide will increase the valueof insects and deepen the comprehensive utilization of insects, then lay the foundation fordeveloping new food and medicine from the insects.The extraction and purification of polysaccharides from three insects, silkworm (Bombyxmori Linnaeus) pupa, Blaps rhynchoptera Fairmaire and cockroach (Periplaneta americanaLinnaeus) were systematically studied in this dissertation. Meanwhile, investigation was madeon the immunological regulation of three crude polysaccharides on immune system in mice andacute toxicity experiment. Main contents and results are listed as follows:(1) Extraction methodThree methods were employed in extraction process of polysaccharides from the materialsin order to select the best extraction. The optimum conditions for the extraction were obtainedby using orthogonal design L₉ (3⁴). As result, alkaline-solution is the best method forextraction of silkworm pupa polysaccharide (SPPS).The optimum conditions are: alkalinesolution concentration 0.02mol/L, temperature 80℃, time 3 hours. In case of cockroachespolysaccharide (CPS), enzyme is the best method. The optimum conditions are: papain 200μg/g, temperature 50℃, time 2 hours. For Blaps rhynchoptera Fairmaire polysaccharide(BFPS),enzyme is the best method. The optimum conditions are: papain 200μg/g, temperature 50℃,time 1 hour. Under these conditions, the average content of polysaccharide in three extracts is27.9%, 6.0%, 8.0%, respectively.(2) Removal of protein and color Sevage method, enzyme decomposition with Sevage method, trichloroacetic acid (TCA)sedimentation were used to liminate the protein from the SPPS before purification. Based onexperimental results, it was suitable to adopt TCA sedimentation to deproteination. After that,51.27% protein of SPPS could be removed, the Sevage method is 25.48% and enzymedecomposition with Sevage method 19.41%. In addition, the color of polysaccharide solutionturned from deep brown to light yellow after oxydol treatment, which suggests oxydol can bemore efficient than active carbon as decolorant. The result was the same after the other twocrude polysaccharides were treated with TCA and oxydol.(3) Isolation and purificationThe DEAE-Sepharose ion exchange chromatography and SephadexG-100 gelchromatography were applied to further purify the crude polysaccharides. Two fractions weregained from SPPS, named as PSPPS-1and PSPPS-2, one from BFPS, PBFPS-4, one from CPS,PCPS-2. The purity determination showed PCPS-2 was a pure product. It has 45.84%polysaccharide and 39.15% protein, which indicated PCPS-2 is polysaccharide-proteincomplex. The other fractions need to be determined and analysed.(4) Immunomodulating activities and acute toxicity experimentThe immunomodulating activities of three crude polysaccharides in mice indicated thatSPPS has evident nonspecific, cellular and humoral immunity, that BFPS and CPS can enhancenonspecific and humoral immunity, but have no remarkable effect on cellular immunity. Acutetoxicity test showed the maximal tolerance dose (MTD) of SPPS and CPS is the same,500mg/kg. The Median lethal dose (LD₅₀) of BFPS is 492.43 mg/kg.