Analysis Of Polysaccharides In Qingzhuan Dark Tea And Study On Antioxidation Of Iron Complex

Objectives:1.An LC-MS/MS method was established to analyze and detect the monosaccharide types and contents of polysaccharides from Qingzhuan Dark Tea in different years.2.Study on preparation and antioxidant effect of Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex.Methods:1.The polysaccharide is extracted by water extraction and alcohol precipitation method,the protein is removed by Sevage method to obtain the polysaccharide of Qingzhuan Dark Tea.2.LC-MS/MS method was used to analyze the components and contents of monosaccharides in polysaccharides of Qingzhuan Dark Tea in different years（2years,5 years and 11 years）.（1）Based on LC-MS/MS,the method of detecting polysaccharide monosaccharides in Qingzhuan Dark Tea was established:The optimized chromatographic condition was achieved on a Shimadzu C18 column（5.0μm,250 mm×4.6 mm i.d.）.The aqueous phase A（5 m M aqueous ammonium acetate）and organic phase B（acetonitrile）were used to elute the targets with the isocratic elution（0-60 min:18%B）.The flow rate of the chromatographic system was 0.9m L/min and the column temperature was set to 27℃.ESI ion source（positive ion mode）was equipped to mass spectrometer detector and multiple reaction monitoring（MRM）mode was used for the determination of 1-phenyl-3-methyl-5-pyrazolone（PMP）derived monosaccharides.Carry out methodological investigation and verification of specificity,linearity and minimum limit of quantification,precision and accuracy,stability and repeatability.（2）Sample preparation of monosaccharide in Qingzhuan Dark Tea polysaccharide:Qingzhuan Dark Tea crude polysaccharide is hydrolyzed by hydrochloric acid to obtain Qingzhuan Dark Tea monosaccharide hydrolysate.The hydrolysate is subjected to PMP derivatization,the acid hydrolysis conditions and derivatization conditions are optimized through a single factor test.（3）Determination of sample content in polysaccharides of Qingzhuan Dark Tea in different years:The volume of the sample solution after derivatization of the monosaccharide of Qingzhuan Dark Tea polysaccharide in different years and the volume of internal standard solution after derivatization were mixed thoroughly at 5:1,passed through a 0.22μm filter membrane,then subjected to liquid quality analysis.Samples from different years were injected 6 times,the peak area was recorded and the content of each monosaccharide was calculated.3.Preparation of Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex and analysis of physical and chemical properties:（1）Preparation of Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex:Qingzhuan Dark Tea polysaccharide is prepared in an alkaline environment under the condition of magnetic stirrer at 60℃with ferric chloride to prepare Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex;Through a single factor test,investigate the effect of p H,temperature,time and the mass ratio of Qingzhuan Dark Tea polysaccharide and sodium citrate on the iron content of the complex;Through ultraviolet-visible spectroscopy,fourier infrared spectroscopy on Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex for structural identification;（2）Analysis of physicochemical properties of Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex:Observe the color,state and solubility of the complex of Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）in different solvents;By adding potassium thiocyanate solution or sodium hydroxide solution dropwise to the Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex,the stability of Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex is analyzed.4.Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex antioxidant experiment:By observing Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex to DPPH free radical scavenging ability,superoxide anion free radical scavenging ability,hydroxyl radical scavenging ability and inhibitory ability of lipid peroxidation evaluated the antioxidant effect of Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex.Results:1.The results of LC-MS/MS analysis of monosaccharide components and contents in polysaccharides of Qingzhuan Dark Tea in different years（2 years,5 years and 11 years）:（1）PMP-labeled monosaccharide derivatives,in the positive ion mode,show a strong molecular ion peak[M+2PMP-H₂O+H]⁺m/z 175.20[PMP+H]⁺secondary fragment,identified 9 kinds of monosaccharides（Man,Rib,Rha,Gal UA,Glu,Gal,Xyl,Ara,Fuc）in different years（2 years,5 years and 11 years）of Qingzhuan Dark Tea.D-Man,D-Glu,D-Gal,MS transition is m/z 511.40→175.20,D-Rib,D-Xyl,L-Ara,MS transition is m/z 481.36→175.20,L-Rha,L-Fuc,MS transition is m/z 495.20→175.20 and D-Gla UA,D-Glu A,MS transition is m/z525.40→175.20.Methodological verification results show that the optimized LC-MS/MS conditions in this research have good specificity;All 9 analytes have a good（r>0.9900）linear relationship in the range of 20.0～1000.0μg/m Land LLOQ 20μg/m L;The RSD value of the analytes intraday precision is 0.47%-3.55%,the recovery rate is 85.3%-113.6%;The RSD value of the precision for three consecutive days is 2.47%-14.79%and the recovery rate is 85.6%-114.6%;Stability results show that after storage at 4℃for 24 h and 48 h,the RSD of the analytes are 0.28%-9.71%and 1.10%-13.66%,respectively;The repeatability results show that the RSD values of Qingzhuan Dark Tea polysaccharides in different years are less than 7.27%.（2）When the concentration of PMP methanol solution is 0.5 mol/L,it shows a peak symmetry,no tailing phenomenon and the response value of each monosaccharide is large,indicating that the monosaccharide derivation is more at this concentration complete;When the HCl concentration is 2 mol/L,the reaction time is 2 h and the reaction temperature is 90℃,the peak area of each monosaccharide is large and the polysaccharide hydrolysis is relatively complete.（3）The ratio of the monosaccharides in 2 years,5 years and 11 years,Man:Rib:Rha:Gal UA:Glu:Gal:Xyl:Ara:Fuc is0.20:0.15:0.13:0.35:1.02:0.50:0.12:0.43:0.11;0.28:0.25:0.19:0.71:1.58:1.30:0.24:0.94:0.23;0.87:0.32:0.52:2.70:4.60:4.10:0.33:2.84:0.27.Among them,Glu,Gal,Ara,Gal UA four kinds of monosaccharides are the main components of Qingzhuan Dark Tea polysaccharides,Rib and Fuc are two kinds of monosaccharides not reported in Qingzhuan Dark Tea polysaccharides.In addition,the content of 9monosaccharides in polysaccharide samples of Qingzhuan Dark Tea in different years was 11 years>5 years>2 years.2.Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex preparation physical and chemical properties analysis results:（1）The iron content of Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex was determined by o-phenanthroline ultraviolet spectrophotometry and Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex was obtained mass fraction of iron in the compound is 32.20%;According to single factor test,the optimal complexing conditions of Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）were determined:time 1 h,temperature 60℃,p H 8 and Qingzhuan Dark Tea polysaccharide mass ratio of sodium is 2:1;UV-visible spectroscopy results show that TPS and Fe（Ⅲ）have reacted to form a complex;Fourier infrared spectroscopy results show that at 861 cm^-1,Qingzhuan Dark Tea polysaccharide iron（Ⅲ）has the characteristic absorption peak ofβ-Fe OOH,which confirms the successful complexation of Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex,the basic skeleton of the polysaccharide structure after complexation has not changed;（2）Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex is red-brown powder,odorless and tasteless,easily soluble in water（the dissolved aqueous solution is neutral）,difficult to dissolve in organic solvents such as absolute ethanol,anhydrous methanol,ether,etc.;In the aqueous solution of Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex,potassium thiocyanate solution and sodium hydroxide solution are added dropwise no change occurred.3.Antioxidant research results show that:The scavenging activity of DPPH free radicals,hydroxyl free radicals,superoxide anion free radicals and the inhibition rate of lipid peroxidation are better than Qingzhuan Dark Tea polysaccharides.The maximum scavenging rate of DPPH free radicals by Qingzhuan Dark Tea polysaccharide and Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）complex are21.56%and 82.23%,respectively.The maximum scavenging rates for hydroxyl radicals are 36.76%and 60.62%,respectively.The maximum scavenging rates for superoxide anion radicals were 52.75%and 84.21%,respectively.The inhibition rates of lipid peroxidation were 32.01%and 81.9%,respectively.Conclusions:1.LC-MS/MS method for determining the composition and content of monosaccharides in Qingzhuan Dark Tea polysaccharides is accurate,fast,and efficient;2.Qingzhuan Dark Tea polysaccharide sample contains 9 monosaccharides of mannose,ribose,rhamnose,galacturonic acid,glucose,galactose,xylose,arabinose and fucose.The main components are glucose,galactose,arabinose and 4 kinds of monosaccharides of galacturonic acid;The longer storage time of Qingzhuan Dark Tea,the higher content of 9 kinds of monosaccharides in tea polysaccharides;3.Qingzhuan Dark Tea polysaccharides are modified by Fe³⁺,which increases the structural stability and can significantly increase the antioxidant activity in vitro;Qingzhuan Dark Tea polysaccharide-iron（Ⅲ）derivative have a scavenging effect on DPPH radicals,hydroxyl radicals,superoxide anion radicals and lipids the inhibitory effect of peroxidation was better than that of Qingzhuan Dark Tea polysaccharide.