Structure Characterization And Antioxidant Activity Of Polysaccharides From Blackened Jujube(Ziziphus Jujuba Mill.)

Blackened jujube is a product made from red jujube fermented under certain temperature and humidity conditions.It is black-brown in color,sweet in taste,soft,and rich in aroma.Polysaccharides are natural polymers consisting of aldoses or ketoses linked together by glycosidic bonds.Jujube polysaccharide is an important component of jujube nutrients.Fermentation of jujube results in changes in its taste and active ingredients elevated polysaccharide content,and enhanced antioxidant activity.There have been many studies on jujube polysaccharides,but the structure and antioxidant activity of blackened jujube polysaccharides have not been reported.Clarifying the structure of blackened jujube polysaccharides is of great significance to study their conformational relationships and to reveal the mechanism of blackening of red jujube and its quality regulation.In this project,we extracted and purified blackened jujube polysaccharides from blackened jujube and characterized the structure of the purified polysaccharides.Also,the antioxidant activity of different polysaccharides was analyzed by chemical level antioxidant assay and cellular level antioxidant assay.The main study contents and results are as follows:(1)Extraction,isolation,and purification of blackened jujube polysaccharideThe crude polysaccharide(BJP)of blackened jujube was obtained by water bath alcohol precipitation method,ethyl acetate removal of fat-soluble substances,and Sevage method of protein removal.The separation of BJP by chromatography on a DEAE-52 cellulose column and a Sephadex G-100 column yielded one neutral polysaccharide(BJP-0)and four acidic polysaccharides(BJP-1,BJP-2,BJP-3,BJP-4),which were recovered in the following order:2.45%,1.13%,5.56%,13.35%,and 4.41%.(2)Structural identification of blackened jujube polysaccharideAs detected by UV spectroscopy,there were no obvious absorption peaks at 260 and 280nm for the purified five fractions,indicating that the five polysaccharides contained less nucleic acids and proteins.The results of infrared spectroscopy showed that the functional groups of the five components were similar,the characteristic absorption peaks of polysaccharides were found at 3600-3200 cm^-1,2970-2840 cm^-1,1640-1540 cm^-1,1430-1290cm^-1,1200-1000 cm^-1.It is speculated that all five polysaccharides are pyranose,and BJP-0may have ?-type glycosidic bonds,and BJP-2,BJP-3,and BJP-4 may have ?-type glycosidic bonds.The average molecular weight of blackened jujube polysaccharide was determined by HPGPC method,and the results were as follows:BJP-0(1.50×10⁴Da),BJP-1(1.36×10⁴Da),BJP-2(1.33×10⁵Da),BJP-3(1.19×10⁵Da),and BJP-4(1.23×10⁵Da).The monosaccharide composition of blackened jujube polysaccharides was determined by the HPAEC method with the following results:BJP-0(Ara:Gal:Glc:Man:Xyl:Fru=76.71:11.29:5.35:0.78:4.56:1.31),BJP-1(Ara:Gal:Glc:Man:Xyl:Fru:Gal A=44.46:39.27:7.33:1.92:1.38:2.04:3.59),BJP-2(Rha:Ara:Gal:Glc:Xyl:Fru:Gal A=0.43:13.37:61.57:0.71:0.54:0.58:22.80),BJP-3(Rha:Ara:Gal:Glc:Man:Xyl:Gal A=10.38:18.88:9.93:1.68:0.08:1.57:57.48),BJP-4(Ara:Gal:Glc:Man:Fru:Gal A=34.74:15.38:3.02:3.85:9.58:33.42).Since BJP-3 was the most abundant in the five purified fractions,it was analyzed by methylation and NMR spectroscopy.In the methylation analysis,ten derivatives were identified and characterized,including Araf(1?,?5)Araf(1?,?2)Rhap(1?,?2,4)Rhap(1?,Galp(1?,?3,4)Galp(1?,?4,6)Galp(1?,?4)Galp A(1?,Xylp(1?and?4)Manp(1?in molar ratios of 10.45:6.52:4.85:3.46:6.16:1.29:1.82:57.70:1.44:3.06.Nuclear magnetic resonance spectroscopy analysis combined with methylation analysis results presume that BJP-3 contains two units:T-?-D-Galp(1?4)-?-D-Gal Ap(1?OMe)and T-?-L-Araf(1?5)-?-L-Araf(1?.(3)Antioxidant activity of blackened jujube polysaccharideAntioxidant activity at the chemical level:The antioxidant activity of crude polysaccharide of blackened jujube was compared with the five fractions after purification using Vc as control.In the DPPH radical scavenging assay,the IC₅₀values of BJP,BJP-0,BJP-1,BJP-2,BJP-3 and BJP-4 were 0.24,0.79,0.38,0.68,1.19 and 0.54 mg/m L,respectively.In the ABTS⁺radical scavenging assay,the IC₅₀values of BJP,BJP-0,BJP-1,BJP-2,BJP-3 and BJP-4 were 0.16,0.91,0.31,0.44,0.99 and 0.58 mg/m L,respectively.The results of the total reducing power assay showed that the absorbance of BJP,BJP-0,BJP-1,BJP-2,BJP-3,and BJP-4 at a concentration of 1 mg/m L were 0.448,0.112,0.263,0.194,0.094,and 0.127,respectively.The antioxidant activity of BJP was the best in all three experiments.Antioxidant activity at the cellular level:The protective effect of BJP against H₂O₂-induced oxidative damage in HUVEC cells was mainly investigated.The evaluation was performed by MTT assay,cell morphology observation,MMP assay,Hoechst 33258 cell apoptosis assay,and ROS level assay.The results showed that BJP was able to increase cell survival,protect cell morphology,inhibit mitochondrial dysfunction,reduce apoptosis and decrease ROS levels,indicating that BJP has a good protective effect on HUVEC cells.(4)Product standard of blackened jujube polysaccharideThe product standard of blackened jujube polysaccharide was drafted.Requirements were made in terms of sensory indexes and physicochemical indexes,which provided a basis for the product to go to market.