Absorption Mechanisms Of Anthocyanins In Red Wine With Human Intestinal Caco-2 Cells

Anthocyanins are responsible for the color of red wine,also influencing the wine’s sensory quality directly or indirectly,and contribute to some biological and health-promoting features such as:antioxidant activity,cardiovascular protection,anti-inflammatory effects,cancer and antidiabetic prevention.After red wine ingestion,anthocyanins can be absorbed into the blood rapidly and distributed quickly to the human organs and tissues.However,they have been shown to present a reduced bioavailability.Consequently,it is of great importance to understand the digestion and absorption of anthocyanins in the human digestive system.The main results are as follows:（1）A fast and efficient dispersive liquid-liquid microextraction（DLLME）method was developed for extraction of phenolic compounds from wine followed by HPLC determination in this study.After different extraction solvents and conditions were evaluated,the fastest and highest efficiency extraction of 12 phenolic compounds including gallic acid,protocatechuic acid,chlorogenic acid,catechin,vanillic acid,caffeic acid,syringic acid,epicatechin,p-coumaric acid,trans-ferulic acid,quercetin,and kaempferol was achieved by using 1000μL of ethyl acetate as an extraction solvent and 500μL of acetonitrile as a dispersive solvent.The mixtures of solvents were mixed with 1000μL of wine sample for 10 s of extraction time.The extractions were carried out two times.Under the optimal condition,the extraction recoveries of 12 phenolic compounds were in a range from 76.56 to 137.74%.Meanwhile,the linearity,sensitivity,and precision of an optimized HPLC analysis method were evaluated.The results showed that each phenolic compound had a good regression coefficient.The limits of detections and quantifications for all the 12 compounds were at 0.001 to 0.133 and0.004 to 0.433 mg/L,respectively.The relative standard deviations of intra-and inter-day determinations of the 12 compounds were below 6.58 and 4.11%,respectively.（2）This part investigated the stability of wine anthocyanins under simulated gastrointestinal p H and temperature conditions.Results showed that a total of 20anthocyanins were identified in wine and most of these anthocyanins remained stable under simulated gastric digestion process.However,a dramatic concentration decrease happened to these anthocyanins during simulated intestinal digestion.The stability of anthocyanins in digestive process appeared to be related to their structure.The methoxy group in the B-ring enhanced the stability of anthocyanins,whereas hydroxyl group resulted in a reduction of their stability.Acylation decreased the stability of malvidin 3-O-glucoside.Pyruvic acid conjugation enhanced the structural stability of pyranoanthocyanins,whereas acetaldehyde attachment weakened their stability.A commercial malvidin 3-O-glucoside standard was used to investigate anthocyanin degradation products under simulated digestion process,and syringic acid,protocatechuic acid and vanillic acid were confirmed to be the degradation products via anthocyanin chalcone conversion path.Protocatechuic acid,vanillic acid,and syringic acid in wine experienced a significant concentration decrease during digestion process.However,wine model solution revealed that phenolic acids remained stable under gastrointestinal conditions.（3）This study has investigated the hunman gut microbiota metabolism of malvidin3-O-glucoside and Malavidin 3-O-（6-O-acetyl）-glucoside using a anaerobic batch-culture fermentation system.Most anthocyanins have disappeared after 4 h incubation while remained constant without gut microbiota.There are an increase of Malavidin3-O-（6-O-acetyl）-glucoside was observed.（4）The Caco-2 cell model was evaluated by light microscopy,electron microscopy,transmembrane resistance,propranolole hydrochloride and phenol red permeability.The results showed that light microscopy and TEM could visually observe the close connection between cells.The transmembrane resistance was greater than 800（?）/cm²,phenol red absorption coefficient was less than 1.0×10^-6,propranolole hydrochloride absorption coefficient was more than 2×10^-6,these results indicated that the cells have reached a close connection.In summary,Caco-2 cell model has been successfully established.（5）Caco-2 cell monolayer model was used to research the effects of time,concentration,inhibitor（phlorizin and phloretin）and the presence of other compounds in the red wine on the absorption of anthocyanins.Results showed that anthocyanin could cross the membrane of epithelial cells by passive diffusion and through transporters（SGLT1 and GLUT2）.The absorption of anthoyanins in red wine is lower than malvidin 3-O-glucoside standard.Above all,Anthocyanin crossed the membrane of epithelial cells by active transporters.The active transporters related to anthocyanin absorption include hexose transporters（SGLT1and GLUT2）.The degradation and absorption of anthocyanin is closely related to their structure,p H,gut microbiota,and wine matrix.