Ethyl caffeate from Verdicchio wine: Chromatographic purification and in vivo evaluation of its antifibrotic activity

Ethyl caffeate (CfE, caffeic acid ethyl ester) was extracted from dealcoholized Verdicchio, a white wine from Marche (Italy) with ethyl acetate and then purified with semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC) using an ODS2 column (25 cm x 20 mm id) at an isocratic flow of 5 mL/min (the mobile phase A was formic acid 4.5% in water and the mobile phase B was acetonitrile). The CfE extract administered intraperitoneally at 1 mumol/L in rats previously treated with 10 mg/kg dimethylnitrosamine was able to prevent the dimethylnitrosamine-induced loss in body and liver weight, as well as to reduce the degree of liver injury, as determined by alanine aminotransferase values and necroinflammatory score, after a 1-week treatment. This was associated with a reduced hepatic stellate cells activation (from 16.8 to 8.3% of smooth muscle actin positive parenchyma) and proliferation (from 11.3 to 5.5 cells/mm(2)). The collagen synthesis was also reduced: the percentage of Sirius Red positive parenchyma decreased from 21.7 to 7.2%. The CfE levels of Verdicchio wine determined with RP-HPLC-DAD were about 14 times the active levels tested in the in vivo test. CfE can be considered as a promising natural compound for future application in chronic liver disease.