Study On The Structure-activity Relationship Of Antioxidant Tea Polysaccharides

Tea polysaccharide?TPS?is a kind of acidic glycoprotein obtained from tea,which has a variety of bioactivities including antioxidant activity.The biological activities and structure of TPS are diverse in different source materials.Two kinds of TPS which are Yingshuang?T1?with high antioxidant activity and Yunnan daye?T2?with low antioxidant activity were screen out in the early study of the laboratory.In this study,T1 and T2 were separated and purified by DEAE-52 cellulose column chromatograp Hy and Sephadex G-150 gel column chromatograp Hy,and then four homogeneous polysaccharides were obtained separately.We choose the neutral homogeneous polysaccharide?T1-1 and T2-1?and the homogeneous polysaccharides with the highest yield?T1-3 and T2-3?for the further study.It was found that T1-1,T2-1,T1-3 and T2-3 showed a single symmetric peak respectively,which indicate that they were pure polysaccharides without impurities and could be used for the further research of antioxidant activity and chemical structure.The main results are as follows: 1.The results of comparative studies on antioxidant activities of four homogeneous polysaccharidesCapacity of ·OH scavenging: In the hydroxyl radical scavenging assay,the IC50 values of T1 and T2 were 1.25 mg/m L and 2.66 mg/m L respectively,while T1-1 and T2-1 had no capacity of ·OH scavengings,the IC50 of T1-3 and T2-3 were 1.94 mg/m L and 3.09 mg/m L respectively.TPS obtained from Yingshuang has the stronger scavenging abilities of ·OH than that from Yunnan daye.Capacity of O₂^-· scavenging: In the superoxide anion free radical scavenging assay,the IC50 of T1 and T2 were 0.60 mg/m L and 1.43 mg/m L respectively,while the IC50 of T1-1 and T2-1 were 1.05 mg/m L and 1.17 mg/m L respectively.T1-3 and T2-3 do not have the ability to scavenge O₂^-·,and the effect of Yingshuang on removing O₂^-· is better than that of Yunnan daye.Capacity of DPPH scavenging: In the DPPH scavenging assay,T1 and T2 had IC50 values of 0.06 mg/m L and 0.11 mg/m L respectively,while T1-1 and T2-1 had IC50 values of 6.55 mg/m L and 8.01 mg/m L respectively.T1-3 and T2-3 had IC50 values of 3.43 mg/m L and 3.65 mg/m L respectively.The DPPH scavenging effect of Yingshuang was better than that of Yunnan daye.The results of FICP: in the chelating power of ferrous ions assay,IC50 of T1 and T2 were 0.24 mg/m L and 0.31 mg/m L respectively,while IC50 of T1-1 and T2-1 were 0.10 mg/m L and 0.15 mg/m L respectively.The chelating effect of Yingshuang on Fe²⁺ was better than that of Yunnan daye.Diversity analysis showed that,crude polysaccharides T1 and T2 existed significant difference on the DPPH scavenging?P<0.05?,whether the same grade components from the different varieties?T1 and T2,T1-1 and T2-1,T1-3 and T2-3?or the different grade components from the same variety?T1-1 and T1-3,T2-1,T2-3?had highly significant differences on the capacity of ·OH,O₂^-·,DPPH scavenging and Fe²⁺ chelating respectively?P<0.01?,The antioxidant activity of Yingshuang was stronger than that of Yunnan daye.2.Basic composition of tea polysaccharidesThere were significant differences in the contents of neutral sugars,uronic acids and proteins between the same grades of different varieties?T1 and T2,T1-1 and T2-1,T1-3 and T2-3??P<0.01?.The correlation analysis between the basic composition and antioxidant activity of tea polysaccharides showed that the semi-inhibitory concentration of hydroxyl radical?·OH?scavenged by tea polysaccharides was extremely significantly negatively correlated with the content of neutral sugar and protein?P<0.01?,and significantly negatively correlated with the content of uronic acid?P<0.05?.The semi-inhibitory concentration of superoxide anion free radical scavenging?O₂^-·?was extremely significantly negatively correlated with the content of neutral sugar and protein?P<0.01?.The semi-inhibitory concentration of DPPH radical scavenging was extremely significantly negatively correlated with the content of neutral sugars and uronic acids?P < 0.01?.The semi-inhibitory concentration of Fe²⁺ was extremely negatively correlated with the content of neutral sugars and uronic acids?P<0.01?.3.The structure of TPSInfrared spectrum?IR?analysis showed that T1-1 and T2-1 were neutral polysaccharides,while T1-3 and T2-3 were acidic polysaccharides,and all these four homogeneous polysaccharides containe ?-glycosidic bonds and dextran structure.Ultraviolet spectrum?UR?analysis showed that all these four homogeneous polysaccharides still containe proteins after separation and purification.Raman spectrum analyses showed that CC,CCC and COC are the main skeleton structures of the four homogeneous polysaccharides.High performance gel permeation chromatograp Hy?HPGPC?showed that the molecular weight of tea polysaccharides ranged from 0.5 KD to 135.8 KD.We established a precolumn derivatization HPLC method for the determination of monosaccharide composition of tea polysaccharides.We found the best condition was: chromatograp Hic column,TC-C18?4.6 mm×250 mm,5?m,Agilent Technologies Inc.USA?,the wavelength for UV detection was 250 nm.The mobile p Hase A was pure acetonitrile,and the mobile p Hase B was 10% acetonitrile which contained 0.045% KH2PO4 and 0.05% triethylamine buffer?p H 7.0?.Elution was carried out at a flow rate of 1.0 ml/min at 35? using a gradient elution of 94-94-88-88% corresponded to 0-4-5-20 minutes respectively.It showed that PMP derivatization HPLC is a more comprehensive,rapid and accurate method for the determination of monosaccharide composition compared with GC.High performance liquid chromatograp Hy?HPLC?analysis showed that ether T1-1 or T2-1 was consisted of mannose?Man?,glucose?Glc?,xylose?Xyl?,galactose?Gal?and arabinose?Ara?in a molar ratio of 0.4:15.6:0.2:5.6:0.7 and 0.7:7:0.2:5.8:2.2 respectively.While T1-3 and T2-3 was consisted of ribose?Rib?,rhamnose?Rha?,glucuronic acid?Glc A?,galacturonic acid?Gal A?,glucose?Glc?,galactose?Gal?and arabinose?Ara?in a molar ratio of 0.5:1.7:1.1:8.6:2.0:3.9:3.8 and 0.4:1:0.9:8.7:2.0:3:2.9 respectively.The results of methylation and nuclear magnetic resonance?NMR?showed that T1-1 and T2-1 had similar chemical structural units,both of them were consisted of a main chain of ?4)-Galp-?1?and ?4,6?-Glcp-?1? and a side chain of ?5?-Araf-?1 ?,?6?-Glap-?1?,?6?-Glcp-(1?,Glcp-(1? and Galp-?1? existed in nonreducing end of branched chains.T1-3 and T2-3 had similar chemical structural units,they were consisted of ?4?-Glc A-?1?,?4?-Galp-?1?,?2,4?-Rhap-?1? and?4,6?-Glcp-?1?and a side chain of ?5?-Araf-?1?,?6?-Glap-?1?,?6?-Glcp-(1?,Glcp-(1? and Galp-?1? existed in non-reducing end of branched chains.All these four tea polysaccharides are polymerized with 1,4-linked main chain and 1,6-linked residues as branched chains.Scanning electron microscopy?SEM?analysis showed that the spatial configuration of T1 and T2 were flat and lamellar structure,while T1-1 and T2-1 were clumps or lamellar aggregate,T1-3 and T2-3 were irregular granular or lamellar aggregate.The results of congo red test showed that neither crude polysaccharides nor the purified polysaccharides had triple helical,but random coil.4.The analysis of structure-activity relationshipMannose,rhamnose,galacturonic acid,glucuronic acid,galactose and arabinose are related to the antioxidant activities of tea polysaccharides closely.NMR analysis showed that all the above monosaccharides are the important components of main chain and branch chain of tea polysaccharides,and the antioxidant activities of tea polysaccharides are the synergistic effects of these monosaccharides.It was found that the antioxidant activity of tea polysaccharides which contained more branches and branches with ?6?-?-D-Glap-(1? was higher than others.