Esterification Of Flavan-3-Ols And Characteristics Of Oil-Soluble Grape Seed Proanthocyanidin

Grape seed proanthocyanidins,composed of flavan-3-ols of different degrees of polymerization,are natural food antioxidants.Natural proanthocyanidins?PC?in grape seed are usually polyhydroxy derivatives which exhibit hydrophilic character,but they are weakly lipophilic and difficult to apply in lipid and oil food systems.In order to improve the lipophilicity of GSP,flavan-3-ols were modified structurally through chemical and enzymatic esterification,and obtained lipophilic flavan-3-ol esterification product.Results indicated that the lipophilicity of flavan-3-ol enhanced.The structure of flavan-3-ol enzymatic acylation products was also investgated.Base on the chemical and enzymatical esterification of flavan-3-ols,GSP was acylated by enzymatic esterification to obtain oil-soluble GSP?GSP-o?.The main results are as follows:1.Chemical esterification of flavonoids with Flavan-3-ols?catechin,epicatechin,epigallocatechin and epicatechin gallate?were acylated by chemical methods,and the effects of fatty acid chloride carbon chain length,reaction solvent,reaction temperature and reaction time on the chemical esterification of flavan-3-ol were researched.The results showed that the optimal esterification conditions were 5 mg of flavan-3-ol and 5 mg of palmitoyl chloride mixed in ethyl acetate,and sodium hydrogencarbonate was added,the mixture was incubated at 40°C with magnetic stirring for 10 h.The flavan-3-ol chemical esterification product analyzed by HPLC-MS had multiple esterification peaks and the composition was complicated.The lipid solubility of flavan-3-ol esters by the chemical esterification is remarkably improved,but the oxidation activity is decreased.2.The esterification of monomeric and oligomeric flavan-3-ols with long-chain fatty acids?lauric acid?yielded mainly mono-,di-,and trilauroylated derivatives.The specific structures of the synthesized flavan-3-ol derivatives were confirmed by ¹H and ¹³C NMR as 3?-O-lauroyl catechin,3?-O-lauroyl epicatechin,3?,5?-2-O-lauroyl epigallocatechin,3?,3?,5?-3-O-lauroyl epicatechin gallate,3?,3?-2-O-lauroyl procyanidin A2,3?,3?-2-O-lauroyl procyanidin B1,and3?,3?,3?-3-O-lauroyl procyanidin C1.These derivatives had higher lipophilicity than parent flavan-3-ols.The antioxidant activity of all flavan-3-ol derivatives was superior to that of parent flavan-3-ols in terms of DPPH and ABTS radical scavenging capacities.3.The lipase-catalyzed synthesis of GSP-o by means of direct esterification of GSP and lauric acid in an organic solvent was investigated in the present work.We found that Lipozyme TLIM was the most efficient lipase in ethanol as a solvent.An optimum conversion rate of87.86%was achieved at 46.1°C,after 13.67 h when lauric acid/GSP at a mass ratio of 1.23 was used.The changes of FT-IR spectra confirmed that esterification of GSP occurred.The presence of mono-,di-,and trilauroylated derivatives in GSP-o was confirmed by HPLC-MSMS,and four lauroylated derivatives were identified as 3?,5?-2-O-lauroyl epigallocatechin,3?-O-lauroyl catechin,3?-O-lauroyl epicatechin,and 3?,3?,5?-3-O-lauroyl epicatechin gallate.GSP-o had higher lipophilicity than GSP.4.An acute oral toxicity test proved GSP-o to be actually nontoxic.The antioxidant activity of GSP-o was superior to that of parent flavan-3-ols in terms of DPPH and ABTS radical scavenging capacities.0.04%GSP-o could significantly increase the frying stability of palm oil and the effect was higher than BHT and TBHQ.GSP-o can significantly reduce the lipid oxidation,and its performance is equivalent to TBHQ.And GSP-o is particularly prominent for inhibiting the secondary oxidation and unsaturated fatty acids.5.The addition of GSP-o in oil before frying produced a higher inhibition effect on the acrylamide formation than BHA,BHT and TBHQ in potato crisps and French fries.The 0.1%and 0.05%GSP-o treatments in potato crisps and French fries,respectively,were regarded as the optimal method,achieving 81.5%and 84.7%reduction of the acrylamide content.The inhibitory mechanism of GSP-o against acrylamide formation may be by both reducing lipid oxidation in the acrolein pathway and decreasing reducing sugar levels in the asparagine pathway.This method could not only effectively reduce the acrylamide level but also maintain reasonable sensory attributes.6.The in vitro anti-cancer activity of lipophilic grape seed proanthocyanidin?GSP-o?against PC3 human prostate cancer cells was evaluated by MTT assay,flow cytometry and immunoblot analysis.And in vivo anti-cancer efficacy was assessed by PC3 xenograft growth in nude mice via oral gavage coin oil or one supplemented with 0.3%or 1%GSP-o?w/v?.Ki67and cleaved caspase 3 immunostaining were performed in tumor tissues.The results showed that GSP-o effectively inhibited the proliferation of PC3 cells by inducing apoptosis.Treatment with GSP-o resulted in an increase in the percentage of PC3 cells in sub G1 phase and a decrease in the percentage of cells in the S-phase.GSP-o induced cell cycle arrest by down-regulating the expression of cyclin D1 and CDK4,and significantly up-regulated the tumor suppressor p21 and p27 in PC3 cells.Furthermore,activation of cleaved fragments of caspases 3 and 9 as well as PARP indicated that GSP-o-induced apoptosis is caspase-dependent.Upstream of caspase cascade,GSP-o increased the cytochrome c release in cytoplasm.And after treatment with GSP-o,the Bcl-2/Bax ratio also decreased in PC3 cells.In tumor studies,GSP-o inhibits the growth of PC3 prostate cancer xenografts in nude mice by inhibiting tumor cell proliferation and inducing apoptosis.Our findings suggest that GSP-o is an effective anti-prostate cancer component and deserves further study.