Study On The Antioxidant Activity Of Porphyra Haitanensis Polysaccharide And The Preparation And Properties Of Its Entrapped Resveratrol Microcapsule

Porphyra haitanensis is a kind of economic algae,which is widely cultured in the coastal areas of China.It has a delicious taste and is rich in nutrients,thus is deeply favored by consumers.As an important nutritional component of Porphyra haitanensis,Porphyra haitanensis polysaccharide has a variety of biological activities.In order to further study the biological activity of Porphyra haitanensis polysaccharide,and extend the its application,in this thesis,the components of the crude Porphyra haitanensis polysaccharide?CPHP?were separated and purified.The chemical composition and structure of the purified fractions?PHP1,PHP2 and PHP3?were characterized.The in vitro antioxidant activity of these purified components of Porphyra haitanensis polysaccharide was also investigated.A combination of PHP2 with Ovalbumin was employed as the material for the entrapment of resveratrol to prepare microcapsules?OPR?.The stability and activity of OPR microcapsules were studied.The main results that we have obtained are follows:1.The crude polysaccharide from Porphyra haitanensis?CPHP?of was prepared by water extraction and alcohol precipitation method.The CPHP were separated by cellulose DE-52and purified by Sephadex G-100.Finally,3 polysaccharide components?PHP1,PHP2 and PHP3?were collected.The extraction rate of CPHP was 10.53%.Yield of PHP1,PHP2 and PHP3 was 11.44%,16.39%and 8.97%,respectively.2.The total sugar contents of CPHP,PHP1,PHP2 and PHP3 are 53.11%,64.53%,66.89%and 65.12%respectively.The four polysaccharides contain a certain amount of sulfate and a very small amount of protein.The results of monosaccharide composition assay by GC-MS and the determination of 3,6-anhydro-galactose content indicated,that the four polysaccharide components are mainly composed of galactose and 3,6-anhydro-galactose,almost no other monosaccharides are present.The molecular weights of the four polysaccharides were determined to be 538,499,523 and 797 kDa by HPGPC.The infrared spectra of the four polysaccharides were similar,which indicated that the configuration of the polysaccharides had not changed during the separation and purification.¹H NMR spectra assays revealed that the fracations PHP1,PHP2 and PHP3 all contained 6-sulfated?-L-galactose units,3,6-anhydro-?-L-galactose units and?-D-galactose units.3.The results of in vitro free radical scavenging experiments showed that IC₅₀ values of CPHP,PHP1,PHP2 and PHP3 against DPPH free radical were 5.68,5.99,5.34 and 4.49mg/mL;their IC₅₀ values againist superoxide anion free radical were 5.22,5.48,4.82 and 4.24mg/mL and those data for hydroxyl free radical scavenging were 5.44,6.15,5.22 and 4.84mg/mL;the order of ferric reducing power was the same as that for the three kind of free radicals:PHP3>PHP2>CPHP>PHP1.4.The results of antioxidant stress assays in a RAW 264.7 macrophage cell model showed that the polysaccharide samples had marked protective activity against oxidative stress.Under the intervene of CPHP,PHP1,PHP2 and PHP3?400?g/mL?,the MDA content in RAW 264.7macrophages stimulated by H₂O₂ decreased by 0.72,0.63,0.96 and 1.07 nmol/mg prot,respectively;the SOD activity increased by 3.97,3.13,6.55 and 6.7 U/mg prot;the CAT activity increased by 1.32,0.95,1.41 and 1.51 U/mg prot;the GSH-Px activity increased by8.12,6.18,8.71 and 9.71 U/mg prot;The ROS level decreased by 124.05%,112.94%,123.94%and 142.02%,respectively.5.Ovalbumin?OVA?-PHP2 composite particles were prepared by a complex coacervation method.The OVA-PHP2 composite particle dispersion possesses a large absolute value of?potential,a small and uniform particle size,suggesting a good stability and a potential to become the microencapsulation shell material.A microcapsule entrapping resveratrol in OVA-PHP2 composite particles?OPR?was prepared,and the conditions for the preparation of OPR microcapsules were optimized by response surface methodology.The entrapment rate of resveratrol reached 95.38%,with a drug loading rate of 2.89%.The average particle size of OPR was 256.83 nm?pH=7?,with a PDI value of 0.4123.The analysis of pH stability,UV irradiation stability and storage stability of the microcapsules embedding resveratrol indicated that using protein-polysaccharide system?OVA-PHP2?is superior to using OVA alone as embedding system in both dispersion stability and the protection on resveratrol.The results of in vitro digestion simulation experiment showed that OPR released resveratrol slower in stomach and more rapidly in intestine when compared OVA-resveratrol microcapsules?OR?.Resveratrol in OPR released in intestine was 20.07%higher than that released in stomach.6.Both OPR and OR possess superior DPPH radical scavenging ability to that of free resveratrol.The IC₅₀ values of unentrapped resveratrol,OR and OPR against DPPH radical were 35.77,29.88 and 24.99?g/ml,respectively.Antitumor activity assays indicated that the inhibition rates of OPR?692.04?g/mL,containing resveratrol 20?g/mL?on Hela cells were30.59%and 36.69%after 24 h and 48 h incubation,respectively,and the IC₅₀ values were1114.87?g/ml and 927.49?g/ml respectively;at the same concentration,the inhibition rates of OPR on HepG2 cells were 29.76%and 39.63%after 24 h and 48 h incubation respectively,and the IC₅₀ values were 1145.59 and 842.06?g/mL respectively.