ABSTRACT
Background: As known, inhibition of phosphodiesterase 5 (PDE5) has the therapeutic effect on male erectile dysfunction (ED), and the processed folium of Epimedium sagittatum Maxim. (PFES) characterized by 8-isopentenyl flavonoids is a famous herb for treating ED. However, the main flavonoids inhibitory activities, structure-activity relationship (SAR) and signaling pathway have been not systematically studied so that its pharmacodynamic mechanism is unclear. Methods: We aimed to initially reveal the PFES efficacy mechanism for treating ED. For the first time, 6 main 8-isopentenyl flavonoids (1-6) from PFES were isolated and identified. Then based on HPLC detection, we proposed a novel method with superior applicability compared to traditional radioisotope assay to screen inhibitors among them. We further established three-dimensional quantitative structure-activity relationship (3D-QSAR) models through CoMFA and CoMSIA to analyze the SAR for those inhibitors. Results: The results were verified by cellular effects of the screened flavonoids. Among 6 compounds, Icariin (1), 2-O"-rhamnosylicaridide II (2) and Baohuoside I (3) were identified with significant activities (IC50 = 8.275, 3.233, 5.473 mM). Then 3D-QSAR studies showed that the replacement of C8 with bulky steric groups as isopentenyl, C3 with positive charge groups and C4' with a hydrogen bond acceptor substituent could increase inhibitory effects. In contrast, the substitution of C7 with bulky steric groups or hydrophilic groups tended to decrease the efficacies. And compounds1, 2, 3 could increase cGMP level and decrease cytoplasmic Ca2+ of rat corpus cavernosum smooth muscle cells (CCSMCs)by activating PKG. Conclusion: 8-isopentenyl flavonoids could be the main pharmacodynamic substances of PFES in the treatment for ED, and some had significant PDE5A1 inhibitory activities so as to activate cGMP/PKG/Ca2+ signaling pathway in CCSMCs, that was related to the substituents at the key sites such as C8, C3, C4' and C7 in the characteristic compounds.