Isolation,Purification And Biological Evaluation Of Enzymatic Hydrolysate Of Polysaccharide From Porphyra Haitanensis

Porphyra haitanensis is a species of red algae,commonly known as zicai and wucai.It has been widely cultured in the southern coastal areas such as Zhejiang and Fujian provinces.This seaweed plant has high edible and medicinal value,due to its high contents of protein,polysaccharide and vitamin.Thus,it is very popular,and has become a research hotspot.Recent literature reports showed that the molecular weight of Porphyra haitanensis polysaccharides was higher,and the biological activity was lower.In order to improve the application value of polysaccharide from Porphyra haitanensis,in this thesis,the crude polysaccharide from Porphyra haitanensis was enzymatically hydrolyzed by pectinase.Then,isolated and purified with DEAE-52 and Sephadex G-100.In addition,their chemical components,structural characterization,bioactivities were also investigated.The main results that we have obtained are as follows:1.Crude polysaccharide(CPH)was obtained from Porphyra haitanensis by water decoction and alcohol precipitation.Five enzymes,including glycosylase,pectinase,cellulase,a-amylase and lipase were screened for the hydrolysis of crude polysaccharides employing DPPH scavenging activity as the indicator.The results showed that pectinase-hydrolyzed polysaccharides possess the highest DPPH scavenging ability.The degradation conditions were optimized using a Box-Behnken response surface design,The optimum enzymolysis conditions were obtained as:pectinase concentration was 50 U/mL,pH is 5.0,and temperature is 47.5 ?,under these conditions,hydrolysis was carried out for 2 h and hydrolysate was named as DCPH.2.The binding capacities against cholic acid and antioxidant activity of DCPH were superior to those of CPH.DCPH has stronger binding ability to sodium taurocholate and chenodeoxycholic acids,suggesting its possible cholesterol-lowering activity.The IC50 on DPPH scavenging ability by CPH,DCPH,was 17.55 mg/mL,7.26 mg/mL,respectively.The IC50 of CPH,DCPH on hydroxyl radical scavenging ability was 12.32 mg/mL,7.83 mg/mL,respectively.3.Initially,DCPH was separated into three fractions by DEAE Cellulose-52 column under the concentrations of 0.3 mol/L,0.5 mol/L,0.7 mol/L NaCl.Then,the Sephadex G-100 column was applied for further purification.Finally,three fractions were obtained,and named as P1,P2 and P3,respectively.4.The chemical composition and structure characterization of P1,P2 and P3 were performed.Compared to CPH and DCPH,total sugar and sulfate content of P1,P2 and P3 were both increased,whereas protein content decreased.The UV-visible spectrum showed that those samples do not contain nucleic acid and protein.The FT-IR spectra showed similar frequency bands,indicating that functional groups were less affected during the degradation and purification.Analysis of IR,as well as 1H NMR and 13C NMR spectra indicated the presence of a and P-glycosidic bonds in those three polysaccharide samples.The monosaccharide compositions of CPH,DCPH,P1,P2 and P3 were analyzed by gas chromatography-mass spectrometry(GC-MS).The results showed that:galactose is the major constituent in CPH,DCPH,P1,P2 and P3,along with small amounts of glucose,mannose,arabinose,fucose,xylose,Rhamnose.xylose.The molecular weight of P1,P2 and P3 were determined to be 300.3 kDa,130.4 kDa,115.1 kDa by HPGPC,compared to CPH(523 kDa),there was a significant decrease in molecular weight.5.The IC50 on DPPH scavenging ability by P1,P2,P3 was found to be 6.28 mg/mL,5.82 mg/mL,and 5.17 mg/mL,respectively.The IC50 of P1,P2,P3 on hydroxyl radical scavenging ability was determined as 7.05 mg/mL,5.87 mg/mL,and 6.53 mg/mL,respectively.Ferric iron reducing power is P3>P2>P1.6.We selected the macrophage cell line RAW264.7 as cellular model.(1)MTT assay for cell proliferation:the proliferation capacity of cells increased when the concentration was within the range of 12.5-100 ?g/mL.The effect of 24 h stimulation was better than 48 h;P2 and P3 have a stronger promoting effect,when incubated with 100 ?g/mL for 24 hours;the cell proliferation capacity of P2 and P3 was 5.6 and 4.8 times of the blank group.(2)Neutral red method:compared with the blank control group,P1,P2 and P3 can significantly enhance the phagocytosis in dose-dependent manner.(3)Griess reagent:the concentration of NO released were 98.46,127.06,110.28,140.78,146.28 ?mol/L when incubated with CPH,DCPH,P1,P2 and P3(400?g/mL)for 48 hours,while the concentration of NO in the control group was 14.6 ?mol/L,showing that it remarkably increased NO release compared to the control group.