HSCCC Separation,Phloroglucinolysis And Structure-activity Relationship Of Grape Seed Procyanidins

Isolation and separation of grape seed procyanidins according to degree of polymerization(DP)and their structure features are very difficult to achieve by the traditional separation methods,due to their structure complexity.In the product development of oligomeric procyanidins(OPCs),polymeric procyanidins(PPCs)are often discarded,being the waste of procyanidin resources.The critical problems involved the selection of suitable and effective methods for separation of grape seed proanthocyanidins according to their DP and structure features,and the utilization and development of the resources.In this paper,a new and effective method by preparative high-speed counter-current chromatography(HSCCC)coupled with or without semi-preparative HPLC was developed for large separation of various grape seed procyanidins into various fractions based on their DP and a series of individual procyanidins compounds commercially not available,with high yields and high purity.PPCs were depolymerized into highly active small molecules by phloroglucinolysis method,and the effects of antioxidant and anticancer activity based on their DP and structures were also investigated.Under the optimized HSCCC conditions with solvent system of n-hexane-ethyl acetate-water(1:50:50,v/v/v)and in the tail-head elution modes,only one-step semi-preparative HSCCC can separate grape seed proanthocyanidins into 7 fractions during 380 min.Each run of semi-preparative HSCCC separation on 400 mg of crude grape seed extract could produce high yields from 11.22 mg to 169.8 mg,accounting for 89.4%in the crude grape seed exracts.Phloroglucinolysis followed by UPLC analysis showed that the mean polymerization degrees(mDP)of these proanthocyanidins fractions were in increasing order,from 1.44 for fraction 1(F1)up to 6.95 for fraction 7(F7).The monomeric procyanidins(catechin,epicatechin,epicatechin 3-O-gallate)could be largely isolated by re-chromatography on semi-preparative HSCCC of F1 under the following conditions:solvent system of n-hexane-ethyl acetate-water(1:10:10,v/v/v)in both head-tail and tail-head elution modes.Each of the isolated compounds presented high yields(7.13-78.90 mg)and high purity(71.04-95.7%)by HSCCC separation of 200 mg F1.The established HSCCC method is also useful for one-step separation of catechins in the crude grape seed extracts.HSCCC separation on 300 mg of crude grape seed extract could produce the yields of 1.82-16.58 mg and high purity of 71.29-98.2%.Large separation of individual proanthocyanidins dimers(B1,B2,B3,B4,B8,B2-3-O-G,B1-3-O-gallate,B2-3'-O-gallate,EC-C,EC-EC(1-2))and trimers(C1 and T2)was also achieved from F2,F3 and F4 and their structures were confirmed by MS and 1H-NMR.An ultra-high performance liquid chromatographic mass spectrometric(UPLC-QqQ/MS)method has been developed and validated for the determination of thirteen procyanidins in grape seed.The composition and distribution characteristics of procyanidins in grape seed extract or fractions with distinct DP were elucidated.PPCs isolated from the crude grape seed extract were depolymerized in the presence of phloroglucinol(0.1 mol/L HCl)into highly active catechins and their nucleophile derivatives.Each of the phloroglucinolysis products was successfully separated and isolated in large amount by semi-preparative HSCCC technique under the optimized conditions based on a selection of suitable solvent system.The optimized solvent system consisted of n-hexane-ethyl acetate-water(1:80:80,v/v/v)with a combination of head-tail and tail-head elution modes.High yields(5.79-27.02 mg)and high purity(76.42-94.6%)were achieved by only one-step HSCCC of 400 mg of grape seed extracts.The structures of these products were identified by MS and 1H-NMR analysis,including epicatechin-phloroglucinol derivative,catechin-phloroglucinol derivative,epicatechin,catechin,epicatechin-3-O-gallate and epicatechin-3-O-galloyl-phloroglucinol derivative(ECGP),respectively.The antioxidant activities of procyanidins and phloroglucinolysis products were investigated by the DPPH,ABTS and FRAP assays.The correlations of antioxidant activities with the DPs and structures of procyanidins were estimated based on ABTS,DPPH and FRAP assays.The antioxidant activity of the individual procyanidins increased gradually as their DP increased.Among the fractions with different DP,the antioxidant activity of each molecule increased as the DP increased,but the antioxidant activity of the constitutive unit decreased.From the point of view of molecular structure,galloylated procyanidins exhibited higher acitivities than nongalloylated ones.The antioxidant activities of phloroglucinolysis products were firstly evaluated by DPPH,ABTS and FRAP assays.The results of the three tests demonstrated that all these phloroglucinolysis products possessed strong antioxidant activities,especially ECGP possessed the highest antioxidant properties.The anticancer activity of the grape seed procyanidins was evaluated using proliferation,invasion and migration assays and the protein expression of MMP-2 and MMP-9.The individual procyanidins and fractions with distinct DPs exhibited the obvious inhibitory effect of gastric cancer cell growth(MGC-803 and SGC-7901).As the DPs increased,the anti cancer activity of grape seed procyanidins increased.The PPCs,trimer C1 and galloylated procyanidins(F2 and B2-3'-O-gallate)could effectively inhibit the growth of MGC-803 and SGC-7901 cells.Compared the activities of procyanidin B2-3-O-gallate and B2-3'-O-gallate,it was deduced that the anticancer activity was affected by galloylated position.The grape seed procyanidins,particularly procyanidin Cl and B2-3'-O-gallate,could remarkably inhibited the invasion and migration of gastric cancer cells by reducing the protein expression of MMP-2 and MMP-9.The cytotoxicity of phloroglucinolysis products were firstly evaluated by MTT assay.Phloroglucinolysis products appeared safe and no significant cytotoxic effects in concentration range from 25 to 150 ?mol/L.