| Porphyra haitanensis is economically important algae that is just one of several artificially cultivated seaweeds;however it is also endemic to China.The taxonomic classification is Porphyra and Porphyra er Bangiaceae,Bangiales,Protoflorideophyceae,Rhodophyta.To date,there have been numerous studies,both domestic and foreign,that have investigated seaweed polysaccharides.However,most of these studies focused on Porphyra yezoensis while there was little attention given to P.haitanensis polysaccharides.P.haitanesis has been largely ignored in these polysachharide studies for numerous reasons,including,time and labor intensive extraction and purification process.Additionally,there are parameters that are harder to control such as production place and harvest time may have large effect on the chemical composition of P.haitabesis.Due to these obstacles,it has become obvious to the research community what needs to be done,however investigations into production these polysaccharides in a more affective manner while maintaining the important biological properties is a big charge and it is currently not being addressed systematically.Thereby produce more or less effect on the aspects of extraction process,structure change and physiological activity,etc,which have not been systematically studied at home and abroad.This study used P.haitanensis as the subject.The research evaluates the nutrient density and flavor desirability relative to different harvest time component,and investigates the optimal preparation procedure,the separation,purification,structure identification as well as the biological activity of P.haitanensis polysaccharides.Significant findings are reported in the subsequent sections.Nutrient content of P.haitanensis was analyzed during different harvest periods.This data indicates that there is a difference in quality and taste dependent on harvest time.It was hypothesized that the concentrations of basic nutrients,minerals,fibers,the water-holding capacity and the swelling capacity were may play a role in this;therefore these parameters were compared.Additionally,for thoroughness,volatile components of P.haitanensis among different harvest periods were analyzed using an electronic nose and GC-MS.The results showed that there were differences in volatile components of P.haitanensis dependent on harvest periods.These differences were also distinguished using the electronic nose technique.Additionally,the total measurable amount of volatile components increased with the delay of harvest time.Interestingly,this data suggest that the use of the electronic nose could predict the harvest time(and quality)of P.haitanensis.(2)35,38,51 and 55 kinds of compounds were identified through GC-MS.Using normalization method of peak area,these components were identified to account for 97.01%,97.69%,95.43%and 96.72%of the effective peak area,respectively.The two large contents are aldehyde ketones and alkanes.The main volatile substances of are P.haitanensis are 8-heptadecen,nonanal and hexaldehyde.Despite different harvest periods,the stability of volatile components of P.haitanensis remained consistent,as they were detectable by both GC-MS and the electronic nose.Overall,there was little difference in the volatile components between the first-harvesting and the second-harvesting of P.haitanensis;while there was large difference in volatile components between the third-harvesting and the fourth-harvesting of P.haitanensis.Based on this data and from the perspective of product quality stability,the first-harvesting or the second-harvesting of P.haitanensis will provide the most desirable raw material.A preliminary single factor experiment identified 4 principal factors involved in a response surface experiment These factors included liquid to material ratio,pH,microwave assisted processing time and temperature.Different levels were set for response surface study.Quadratic polynomial mathematical model for the extraction of P.haitanensis polysaccharides was established by using response surface method,which can be used to optimize the extraction process of P.haitanensis polysaccharides.The results showed that the ratio of liquid to material,pH,microwave assisted processing time,extraction temperature and their quadratic term had a significant effect on polysaccharide extraction rate.Quadratic polynomial mathematical model affecting the extraction rate of P.haitanensis polysaccharides was established using response surface analysis to optimize the extraction technologies of P.haitanensis polysaccharides.This model was based on the conditions of liquid to material ratio,water extraction pH,microwave assisted processing time and extraction temperature and the specific values were 60mL/g,8.30,5.01 min and 95.91 ?,respectively.In this condition,the yield of P.haitanensis polysaccharide was 17.11%.Considering the practicality of the experiment,liquid to material ratio,pH,microwave assisted processing time and temperature were adjusted to 60mL/g,8.0,5min and extraction 90 ?,from the calculated optimal,respectively.Verification experiments were carried out under the adjusted conditions.Five parallels were set,the average yield of P.haitanensis polysaccharides was 16.38%±0.12%and lower the predicted value was only 0.73%.The 2 yield values were very close indicating that the process is stable,feasible,and suitable for extraction of P.haitanensis polysaccharides.Polysaccharides PP? and PP? with higher antioxidant activity isolated from P.haitanensis polysaccharide were separated and purified by DEAE-52 cellulose anion-exchange chromatography and Sephadex G-100 gel column chromatography.The basic structures of the polysaccharides were identified using IR,GC-MS and NMR.Primarily,specific chemical groups,monosaccaride composition within polysaccharides,configuration and the connection position of sugar residues,etc.were located.The results showed that,the IR spectra show 3 pure polysaccharides with homogeneous charges,characteristics and molecular weight distributions.These compounds are named PP ?-G,PP? G-1 and PP ? G-2 and were further eluted through G-100 chromatography.The 3 polysaccharides all had an absorption peak with characteristics of polysaccharides;PP? and PP? contain a sulfate radical.PP?-G,PP?G-1 and PP?G-2 have pyranose,which might contain 3,6-inner ether bridge.GC analysis showed that PP?-G,PP?G-1 and PP?G-2 were all mainly composed of galactose,and the galactose content of the three pure polysaccharides were 88.57%,96.67%and 97.64%,respectively.Additionally,the 3 polysaccharides also contained xylose and glucose.Analysis of NMR spectroscopy showed that P.haitanensis polysaccharide PPIVG-2 was composed of 5 monosaccharides with the signal intensity ratio of 8.3:3.1:1.3:2.7:1 and contain little uronic acid.Also,PP? glycoside residue contained both?-type and ?-type pyranose.The clearance affect of P.haitanensis polysaccharides with different molecular weights on active oxygen free radical(·OH)clearance rate,superoxide anion(·O2-)and DPPH were studied.The activities of glutathione peroxidase(GSH-Px),catalase(CAT),superoxide dismutase(SOD)activity as well as malondialdehyde(MDA)concentration in the liver,brain tissue and blood of the fed mice were measured.The effects of P.haitanensis polysaccharides on axons,axon secondary bifurcation number,total axial length,maximum axial length and the expression of nerve cell proteins was studies.Specific proteins that were studied include,BDNF,ptrkb,pAkt and pGsk3?in mice with cerebral cortex nerve cells damage which were induced by phencyclidine(PCP),are analyzed to further study the physiological activity of P.haitanensis polysaccharides.The results showed that:(1)The experiment testing antioxidant activities in vitro show polysaccharides with different molecular weights all had antioxidant abilities and had a clearance affect on hydroxyl free radical(·OH),superoxide anion(·O2-)and DPPH radical.The clearance abilities had a dose-dependent relationship with polysaccharides concentration and all the clearance abilities were improved with the increase of polysaccharides concentration.There were significant differences in the antioxidant activities of polysaccharides with different relative molecular weights among different antioxidant systems.When the concentration was 10mg/mL,PPIV had a clearance rate of 60.56%,60.25%and 39,72%on hydroxyl free radical(·OH),superoxide anion(·O2-)and DPPH radical,respectively.PP?(MW:50,000-100,000)and PP?(MW:50,000-100,000)with relatively smaller molecular weights had better clearance affect on hydroxyl free radical(·OH),superoxide anion(·O2-)and DPPH radical than the polysaccharides with the other three molecular weights.(2)P.haitanensis polysaccharides PP? and PP? had strong clearance ability on radicals,as the result the antioxidant activities of these two polysaccharides were used in subsequent in vivo studies.After 60 days when the mice were injected with D-galactose continuously,the activities of SOD,GSH-Px and CAT in the liver tissue,brain tissue and blood of the mice in the aging model group all decreased significantly.MDA content,on the other had,significantly increased.Moderate and high dose groups of PP? and PP? could significantly improve the SOD,GSH-Px and CAT activities in the liver tissue,brain tissue and blood to varying degrees.Similar doses also reduced the MDA content.Taken together these data suggest a preferable antioxidant effect.However,most of the antioxidant indexes of PPIV group were obviously better than those of PP? group.(3)P.haitanensis polysaccharide group had an improved morphology compared to the PCP injured group.Specifically there was a more uniform morphology and compact intranuclear cytoplasm.This could provide certain protective effect to the nerve cell somata and promote the growth of somata neuronal axon.The mechanism of the protective effect of P.haitanensis polysaccharide on the nerve cells might promote the secretion of BDNF and then act through the pAkt,Gsk3 pathways,which result in promoting the growth of primary neuronal axons as well as suppressing or reducing the growth of nerve cells caused by PCP injury. |
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