Study On The Depolymerization And Optimization Of Grape Seed Polymeric Proanthocyanidins

Proanthocyanidins are chemically composed of monomeric flavan-3-ol units linked by interflavan bonds,with different degree of polymerization(DP).It has excellent biological activity in terms of antioxidation,scavenging free radicals,which can be used for preparation of anti-tumor,anti-inflammatory,cardiovascular protection drugs.Pine husk,cranberry,grape seed plant-derived materials have abundant PC content.Among them,grape seed extract(GSPE)has become the most important source of industrial preparation of PC.Grape seed PC consist of monomers(gallic acid,catechins,epicatechins),oligomers(OPC,DP 2-4)and polymers(PPC,DP above 5),with the DP increase the bioavailability decreased dramatically.Depolymerize PPC to B-type dimer can increase its bioavailability and enhance the economic benefits simultaneousely.In This study,we chosed commercially available GSPE as the research object,establish the hydrochloric acid-vanillin method to determinate the content and DP of PC,used UPLC method to qualitative and quantitative analysis the monomer and dimer.For the PPC obtained from ethyl acetate-water two-phase extraction,we explored the reaction mechanism of hydrochloric acid depolymerization,and the nucleophile EC/HCl(C/HCl)depolymerization.Based on the content of dimer as the response value,Central Composite Design(CCD)Response Surface Method experiments with four variables(material ratio,HCl concentration,reaction time,reaction temperature)and five levels was designed to optimize the reaction conditions.The main research conclusions are as follows:1.The PC content of GSPE was 95%(R2=0.9995),and mDP was 3.56(R2=0.9996).The UPLC method analyze the components of GSPE,including gallic acid(0.3%,M/M),catechin(1.74%),epicatechin(1.29%),B1(0.19%),B2(0.72%)B3(0.08%),B4(0.04%),ECG(0.15%),the RSD%of each standard curve was less than 0.5%,square R2 was greater than 0.999.The trimer and the higher polymer(88.7%)were difficult to separate by Reversed-phase liquid chromatography.2.The total yield of GSPE extraction was 99.2%.The extraction phase accounted for 19.3%,the mDP was 2.21;the raffinate phase accounted for 80.6%,the mDP was 5.65,and the raffinate phase contained no monomers or dimers.3.For the nucleophile EC/HCl(C/HCl)depolymerization reaction,when the reaction conditions was follow as material ratio 1,HCL 0.1 mol/L,time 20 min,temperature 40 0C,the mDP of the product was decreased to 1.74,and the contents of B2 and B4 in the product were 1.96 mg/mL and 0.243 mg/mL respectively,which ratio was approximately 8:1;For the nucleophile C depolymerization reaction,Under the same reaction conditions,mDP of the product was decreased to 1.68,the contents of B1 and B3 in the product were 2.18 mg/mL and 0.253 mg/mL respectively,which ratio was approximately 8.6:1;As a comparison,the efficiency of hydrochloric acid depolymerization was not obvious,the mDP of the product was 4.56,and the content of monomer and dimer is low.4.High yield of proanthocyanidin B2 and B1 as the ultimate aim were determined by the level of four independent variables.The optimum condition of depolymerization and theoretical maximum values of yield were predicted using polynomial models.The forecasted optimum theoretical yield of proanthocyanidin B2 was 3.35 mg/mL,when the condition as follow:mass ratio 2.8,HCl 0.06mon/L,time 16 min,temperature 36?.Under these conditions,the experimental value was 3.32 mg/mL,relative error 1.19%.And,the optimum theoretical yield of proanthocyanidin B1 was 3.64 mg/mL,when the condition as follow:mass ratio 2.8,HCl 0.07mon/L,time 17 min,temperature 34?.Under these conditions,the experimental value was 3.75 mg/mL,relative error 3.02%.The R2 of these two models were 0.981 and 0.975 respectively,and the P-values were all lower than 0.001,indicating that the model has a high degree of fitting and has a significant effect.The nucleophile EC/HCl(C/HCl)depolymerization method we developed can be capable of efficiently depolymerized PPC under mild conditions.By optimizing the reaction conditions,the content of the target dimer can be selectively increased,demonstrated that is a simple and efficient method and has great promotion potential.