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

Abstract

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 (t_R) 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.