HPLC-PDA-MS and NMR characterization of C-glycosyl flavones in a hydroalcoholic extract of Citrus aurantifolia leaves with antiplatelet activity

A hydroalcholic extract of lime (Citrus aurantifolia) leaves has been developed in Cuba to be used as a nutritional supplement and phytomedicine in the form of tincture (TLL). A HPLC-PDA-ESI/MS/MS method has been used for the comprehensive analysis of C-glycosyl flavones in TLL. Six C-glycosyl flavones were characterized and, to confirm the proposed structures and to elucidate the nature of the sugar units, a preparative procedure was applied, and isolated compounds were characterized by NMR. Apigenin-6,8-di-C-beta-D-glucopyranoside (vicenin II) (1), diosmetin-6,8-di-C-beta-D-glucopyranoside (2), apigenin-8-C-beta-D-glucopyranoside (vitexin) (3), apigenin-8-C-[alpha-L-arabinopyranosyl-(1-->6)]-O-beta-D-glucopyranoside (4), apigenin-6-C-[alpha-L-arabinopyranosyl-(1-->6)]-O-beta-D-glucopyranoside (5). and apigenin-6-C- -D-glucopyranoside (isovitexin) (6) were identified in TLL and quantified by HPLCPDA. Compounds 4 and 5 were two new arabinosyl derivatives of vitexin and isovitexin. Inhibitor effect of TLL on platelet aggregation induced by physiological agonists of platelets was evaluated in human plasma. TLL inhibited significantly ADP and epinephrine-induced platelet aggregation in a concentration-dependent manner (IC(50) = 0.40 and 0.32 mg/mL, respectively).