| The Angelica sinensis polysaccharides (ASP) have immunoregulatory effectson mammals, but these effects have not been reported on aquatic animals to date. Inthis dissertation, the ASP was extracted from Chinese herb Angelica sinensis (Oliv.)Diels and purified, then the structure of purified ASP are analysed. The effects ofASP on non-specific immunity of Epinephelus malabaricus are studied via oraladministration, intraperitoneal injection and in vitro test. This dissertation providestheoretical basis for the application of ASP on aquaculture.The main results and conclusion were presented as follows: The crude ASP wasextracted from A. sinensis (Oliv.) Diels via water boil and ethanol sedimentation.The coloring matters in crude ASP were removed by ethanol and acetone. Protein incrude ASP was removed using Sevag method. Then the ASP0was obtained afterdialysis and lyophilization. The ASP0was further purified using DE-52cellulosecolumn chromatography. The ASP0was eluted by distilled water,0.1M NaCl,0.2MNaCl,0.4M NaCl successively. The subfraction, ASP1, ASP2, ASP3and ASP4,were obtained. The molecular weight and structure of ASP1, ASP2and ASP3wereanalysied by high performance liquid chromatography, infrared spectrum andmethylation method. Results showed that protein content in ASP1, ASP2, ASP3andASP4were2.21%,1.38%,2.53%and2.34%respectively. Total sugar in ASP1,ASP2, ASP3and ASP4were95.10%,94.73%,81.68%and79.51%. Reducing sugarin ASP1, ASP2, ASP3and ASP4were5.75%,4.85%,4.86%and4.77%. Themolecular weight of ASP1, ASP2, ASP3were4.12×105Da,2.34×105Da,1.94×105Da. The monosaccharide in ASP1, ASP2, ASP3were Glc:Xyl:Gal=18:3:1,GlcA:Glc:Xyl:Gal=7:8:3:2and Glc:Xyl=28:1, respectively. The monosaccharideresidue of ASP1, ASP2, ASP3was β-pyranoid ring. In ASP1, Glcp is1'3,6、1'3and1'linked, Xylp and Galp is1'3linked. In ASP2, GlcAp is1'3linked, Glcpis1'3,6,1'3and1'linked. Xylp and Galp is1'3linked. In ASP3, Glcp is1'6,1'4,1'3,6,1'3and1'linked. Xylp is1'3linked.In order to test the effects of ASP on non-specific immunity and diseaseresistance of E. malabaricus, ASP0was supplemented in diet at0,500,3000mg/kg. Fish were sampled after4,8,12weeks of feeding with test diets. Results showedthat ASP0enhanced blood leucocytes phagocytosis, serum lysozyme (LZM) activity,head kidney leucocytes respiratory burst, proliferation, and phagocytosissignificantly. The ASP0reduced cumulative mortality of test fish challenged byEdwardsiella tarda. Fish fed with ASP0at3000mg/kg for12weeks achievedhigherst non-specific immunity and disease resistance.In order to test the effects of ASP on growth, non-specific immunity of E.malabaricus, ASP0was supplemented in diet at0,500,3000mg/kg and fed to testfish for101days. Results showed that ASP0exerted no significant influence onfeeding, growth, anti-bacterial activity of skin mucus, serum nitric oxide (NO), totalprotein, albumin or albumin/globulin ratio. Low dosage of ASP0enhanced skinmucus LZM activity significantly. High dosage of ASP0boosted blood leucocytes,NBT-positive cells significantly, and heightened blood leucocytes phagocytic ratioand serum LZM activity. The ASP0reduced cumulative mortality of test fishchallenged by E. tarda significantly. The protective rate of500,3000mg ASP0/kgdiet were45%and69%, respectively.In order to test the subfractions of ASP0on non-specific immunity of E.malabaricus, fish were intraperitoneal injected with ASP1, ASP2, ASP3at20mg/kgbody weight. The ASP0served as control and sterile PBS served as blank. Fish weresampled befor injection and on3,7,14,21,28d post injection. Results showed thatthe ASP0and its subfractions enhanced skin mucus LZM activity, blood leucocytesand NBT-positive cells, serum LZM activity, NO content and albumin/globulin ratiosignificantly. The skin mucus anti-bacterial activity was boosted significantly byASP0while not influenced significantly by ASP1, ASP2or ASP3. The non-specificimmunity in fish showed time-effect relationship after injected with ASP0and itssubfractions: the immune parameters elevated after ASP0and its subfractionsinjection, then decreased to blank level at28-day post injection. Fish mortality resultfrom E. tarda challenge decreased after injection with ASP0and its subfractions.The mortaliy in fish injected with ASP0was the lowest. Compared with ASP0,single injection of ASP0subfractions was not effective on non-specific immunity ofE. malabaricus.In order to test the effects of ASP0subfractions on head kidney leucocytesimmunocompetence, head kidney leucocytes were cultured in vitro with ASP1,ASP2and ASP3(0,1,10,100,1000μg/mL). The ASP0served as control. Leucocytes proliferation, respiratory burst and phagocytosis were assayed afterincubation. Results showed that the proliferation, phagocytosis and respiratory burstactivity of leucocytes incubated with ASP0and its subfraction was boosted.Leucocytes proliferation and respiratory burst activity were enhanced with theincrease of ASP0and its subfractions. Leucocytes proliferation was enhancedsignificantly by ASP0at1000μg/mL. Leucocytes respiratory burst activity wasboosted significantly by ASP2at1-1000μg/mL. Leucocytes incubated with ASP0and its subfraction showed a tendency of augment first and then decline inphagocytosis. Leucocytes phagocytosis peaked when incubated with ASP0andASP1at100μg/mL, and when incubated with ASP2and ASP3at10μg/mL.In conclusion, ASP0and its subfractions augment non-specific immunity anddesease resistance of E. malabaricus by means of augment of humoral and cellularimmunocompetence. Compared with ASP0, single use of its subfractions was noteffective on the augment of non-specific immunity. The diversities on structure andmolecular weight of ASP0subfractions may be the reason for their differentimmunocompetences. The monosaccharide residue with β pyran ring is the basis ofASP bioactivities. |
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