Antiplatelet aggregation and antithrombotic benefits of terpenes and flavones from hawthorn leaf extract isolated using the activity-guided method

Hawthorn is a well-known functional food; at present, increasing attention has been given to hawthorn leaf due to its numerous functional and nutritional properties. In this study, the antithrombotic properties of hawthorn leaves were evaluated using the activity-guided isolation process and high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS). A crude extract prepared in 75% ethanol was fractionated using macroporous resin D101 and polyamide chromatography to obtain three active fractions (Fr.C, Fr.C-1 and Fr.C-2). Thereafter, the inhibitory activities of these fractions were examined by platelet aggregation and antithrombus assays using a zebrafish model. Using the HPLC-QTOF-MS technique, we identified 25 compounds in the active fraction (Fr.C). The structures of these compounds were identified by comparing the retention time (tR) and mass spectral data from the previous reports and 19 reference compounds. Based on the analysis, 21 peaks were detected in the mass spectrum of Fr.C-1 and 8 peaks were detected in Fr.C-2, we found that 11 compounds in Fr.C-1 exhibited potent inhibitory effects on platelet aggregation, including nine monoterpenoids, one diterpenoid and one flavanone. Accordingly, monoterpenoids are suggested as the main anti-platelet aggregation constituents from hawthorn leaves. Particularly, compounds 10 and 24 inhibited ADP-induced platelet aggregation and delayed FeCl3-induced thrombus in zebrafish. Furthermore, interactions between compounds 10 and 24 with two ADP receptors P2Y1 and P2Y12, serving as the target for key regulators of antiplatelet aggregative activity, were investigated via molecular modeling. In addition, five flavones were obtained from the active fraction (Fr.C-2). These results indicated that monoterpenoid glycosides and some flavones were responsible for the antithrombotic activity of hawthorn leaves. Moreover, this study shows that the activity-guided isolation is a fast, efficient and systematic separation method for the identification of active compounds in natural products.