MALDI-TOF MS Combined With NMR And HPLC Analysis Of Vegetable Tannins With Their Antioxidant Activities

In order to screen and exploit vegetable tannins which have importantproperties in practical application,convenient and reliable methods arerequired to determine vegetable tannins structure.In the present study,wehave studied the following several aspects on the chemical structure andantioxidant activity of vegetable tannins from several plants in south China:1.The effect of different reaction conditions on cyanidin from degradation ofproanthocyanidin extracted from Bruguiera gymnorrhiza calyces and sideproducts were studied by high performance liquid chromatography-diode arraydetector (HPLC-DAD) for the first time.The cyanidin and corresponding sideproduct formed during butanol/HCI hydrolysis were separated and quantifiedby HPLC-DAD.The degradation products of proanthocyanidin were effectgreatly by water content of reaction solvents.The yield of cyanidin increasedwith the increasing the water content (from 5% to 15%) of reaction solvents,but the yield of side products declined.The yieldof cyanidin and side productsdecreased with the increasing the water content when the water content wasabove 15%.However,there is no great effect of proanthocyanidinconcentration and reaction time on the yield of cyanidin and side products.HPLC-DAD is a powerful method for the analysis of the content and structureunit of proanthocyanidin.2.Types of structural units,degree of polymerization and the meanmolecular weight of proanthocyanidin extracted from B.gymnorrhiza calyxwere characterized by matrix-assisted laser desorption/ionization time-of-flightmass spectrometry (MALDI-TOF MS).Crude cyanidin products were preparedfrom proanthocyanidin of B.gymnorrhiza by means of butanol/HCI reaction forthe first time.In addition,the effects of crude cyanidin products on freeradical-scavenging and antioxidant activity were determined by using1,1-diphenyl-2-picryhydrazyl radical (DPPH·) scavenging activity and ferric reducing/antioxidant power (FRAP) model systems.Catechin/epicatechin(procyanidin) was the basic unit occurring in procyanidin of B.gymnorrhiza.The average degree of polymerization was 7.5,and mean molecular mass was2081.60.Crude cyanidin products showed a very good DPPH radicalscavenging activity (IC₅₀,the half-inhibition concentration was 43.89μg/L) andferric reducing/antioxidant power (7.72 mmol AAE/g).A new method forpreparing cyanidin and anthocyanidin from easilyavailable proanthocyanidinby means of butanol/HCI reaction was established.3.Tannins from Prunus salicina,Syzygium cuminic and Canarium albumwere charactered using MALDI-TOF MS combined with NMR and HPLCanalysis for the first time.(1) The content of total phenolics and extractablecondensed tannins in P.salicina fruit were 82.89±13.12 mg/g and 14.31±9.27 mg/g respectively.Epicatechin was the basic units occurring in P.salicinafruit condensed tannins,A-type and B-type linkage were most commonlybetween the structural units of polymers.The average degree ofpolymerization (DP) of condensed tannins was 5.3,and the mean molecularweight was 1583.7.The effects of tannins from P salicina fruit on freeradical-scavenging were determined by DPPH radical scavenging activity andthe IC₅₀ value was 57.98μg/mL.(2) Hydrolysable tannins in S.cuminic fruitstone were indentified as ellagitannins that were composed of gallic acid andellagic acid,linked to a sugar moiety.Condensed tannins in S.cuminic fruitskin were identified as B-type oligomers of epiafzelechin (propelargonidin) witha degree of polymerization up to eleven.The antioxidant activity,measured bytwo vitro models:DPPH radical scavenging activity and ferricreducing/antioxidant power.Tannins extracted from S.cumini fruit showed avery good DPPH radical scavenging activity and ferric reducing/antioxidantpower.The results indicate promising the fruit of S.cumini for the utilization assignificant source of natural antioxidant.(3) The predominance of signalsrepresentative of procyanidins and prodelphinidins with 2,3-cisstereochemistry of condensed tannins was determined in the stem bark of C. album.In addition,epicatechin and epigallocateclin polymers with galloylatedprocyanidin or prodelphinidin were also observed.The average DP and theaverage molecular weight were 5.3 and 1578.25,respectively.TheMALDI-TOF MS,NMR and HPLC provide the rapid and ideal method forcharacterization of polydispersed vegetable tannins.Tannins extracted fromleaves,twigs and stem bark all showed very good DPPH radical scavengingactivity (IC₅₀ of 56.86,62.31 and 54.80μg/mL) and ferric reducing power (4.28,3.74 and 4.49 mmol AAE/g dried tannins).