| The winemaking process generates large amounts of solid organic waste and by-products,including grape pomace(74%),wine lees(14%),and dewatered sludge(12%).Grape polyphenols are mainly found in grape pomace,including stems,skins and seeds.Grape pomace from white winemaking are not subject to maceration and thus keep nearly all the polyphenols of grapes,so they represent important sources of bioactive compounds.Oligomeric polyphenols,as the most powerful natural antioxidants,have been highly concerned by the scientific community during the last three decades.Grape polyphenols are presented essentially in the form of polymers with very low bioavailability,while bioactive oligomers are presented in a very small proportion.This fact,together with the limitations of separation techniques,makes it very difficult for large preparation of oligomers and their individual compounds.In this project,the Vitis vinifera grape was chosen to conduct the experiments.First of all,detailed phenolic composition of white grape pomace was measured by ultrahigh-performance liquid chromatography-tandem mass spectrometry.Then,the acid-catalyzed degradation of polymers into oligomers was studied and the innovative methods for large preparation of oligomers based on the optimized reaction conditions were established.High-speed counter-current chromatography was used for further fractionation and the separation of the degradation oligomers into various individual compounds with different polymerization degrees and different structural features was also preformed by HPLC.On the basis of these results,the effect of different individual compounds on in vitro antioxidant activity and proliferative activity towards malignant glioma U87 cells were investigated.The execution of this project will fill the gaps of largely obtaining bioactive individual compounds in the scientific community.The development of the method by ultrahigh-performance liquid chromatography-fourier transform ion cyclotron resonance mass spectrometry and ultrahigh-performance liquid chromatography-triple quadruple mass spectrometry has been performed for determination of the detailed phenolic composition in the skin,seed and stem of white grapes.Results confirmed that the developed method was more effective and sensitive for simultaneous determination of the major phenolic compounds in white grape pomace.The optimized method of ultrahigh-performance liquid chromatography-fourier transform ion cyclotron resonance mass spectrometry allowed to identify up to 35 phenolic compounds in grape pomace,which has,as far as we know,been reported this grapevine variety for the first time.Seeds,skins and stems exhibited different qualitative phenolic profiles.These results provided useful information for recovery of phenolic antioxidants from different parts of white grape pomace.To develop an efficient method for large preparation of various individual polyphenols from grape skins and seeds,an optimized preparative high-speed counter-current chromatography(HSCCC)condition with two-phase solvent system composed of Hex-EtOAc-H2O(1:50:50,v/v)was used to separate grape skin and stems polyphenols into various fractions.Both the tail-head and head-tail elution modes were used with a flow rate of 3.0 mL/min and a rotary speed of 950 rpm.Phloroglucinolysis-UPLC was preformed to analyze the structural characterization of procyanidins in each polyphenol fraction from HSCCC.Afterwards,a preparative-HPLC separation was applied to isolate individual polyphenols in each fraction from HSCCC.Total of 8 fractions(Fraction A to H)were obtained from grape skin extract by HSCCC.After preparative-HPLC isolation,15 individual compounds from skins were obtained,most of which presented high yields and purity(all over 90%).The HSCCC method followed with preparative-HPLC appeared to be convenient and economical,constituting an efficient strategy for the isolation of grape skin and stem procyanidins.Compared to grade seed procyanidins fractionated by HSCCC,oligomers in grape skin and stem have a lower proportion in oligomeric procyanidins(OPCs)but higher proportion in polymeric procyanidins(PPCs).Furthermore,the content of each individual procyanidins isolated from HPLC in grape skin is less than in grade seedPolymeric polyphenols were the major constituents of polyphenols,while they have poor bioactivity.To develop an efficient method for degradation of grape seed and skin polyphenol polymers into oligomers,an optimized sulphurous acid degradation conditions combining single-factor design,orthogonal test and nonlinear multiple regression analysis for grape seed with the temperature of 60?,reaction time of 60 min and sample-sulphurous acid ratio of 1:0.2,and for grape skin with the temperature of 40?,reaction time of 60 min and sample-sulphurous acid ratio of 1:0.2,were established.Afterwards,HSCCC and prep-HPLC were used to fractionate and isolate individual oligomers from the degradation products.Total of 13 dimeric or trimeric procyanidins were obtained,and most of them presented high yield.The proposed method provides a new way for large preparation of oligomeric procyanidins from naturally abundant and wasted polymeric ones.To develop a semisynthetic approach for converting polymeric polyphenols to oligomers,grape seed and skin PPCs were simultaneously reacted with catechin(C)and epicatechin(EC)under acid condition.Combining response surface methodology(RSM)and single-factor design,an optimized semisynthetic condition was confirmed with the ratio of PPCs with C and EC of 1:1:1,temperature of 40?,reaction time of 20 min and 0.1M methanolic HCl.Then High-speed counter-current chromatography(HSCCC)was applied to obtain three fractions from semisynthetic products and preparative-HPLC was used to isolate individual procyanidins.10 B-type procyanidins including dimers and trimers were got with high yield and high purity.The developed semisynthesis combined with separation method was efficient to obtain individual OPCs in preparative scale.To determine the antioxidant activities and their structure-relationship of 13 individual procyanidins,DPPH,ABTS and FRAP assays were investigated.The antioxidant activity of the individual procyanidins increased gradually as their DP increased.And galloylated procyanidins exhibited higher activities than nongalloylated ones.To further establish a structure-activity relationship for procyanidins,we verified the cyto-protective effect of 13 grape procyanidins,ranging from monomers to trimers against H2O2-induced oxidative stress in PC-12 neuroblastoma cells.Our study demonstrated some procyanidins were able to significantly protect PC-12 cells from the H2O2-induced cytotoxicity suggesting they possess neuroprotective effects against oxidative stress.Procyanidins' protective effects against oxidative stress mainly depended on their polymerization degree in addition to their structural features.A positive correlation was found between procyanidins' polymerization degree and the protective effect against oxidative stress in PC-12 cells.The presence of 3-or 3'-galloylated groups in the C-ring of procyanidin molecules significantly increased their protective activity as well.These results demonstrated that galloylated high-molecular-mass procyanidins would be of more interesting as promising antioxidant natural compounds.This work for the first time demonstrated the structure-activity relationships of 13 procyanidins' antioxidative stress activity,which could have a significant impact on future development of procyanidins for healthy food products or drugs to treat disease such as neurodegenerative disorders.The effect of oligomer procyanidins(F2)(F2,degree of polymerization 2-15),a natural fraction isolated from grape seeds on the biological behavior of glioblastoma cells was investigated before.The results suggest that F2 may be a promising candidate for the development of novel anti-tumor therapeutics.Based this previous work,the anti-glioma activity and their structure-activity relationship of 13 individual procyanidins was evaluated using proliferation assay.As the DPs increased,the anticancer activity of procyanidins increased.The PPCs,trimer C1,T2 and galloylated procyanidins could effectively inhibit the growth of U-87 cells. |
PDF Full Text Request