Lusitropic effect of Aurantii Fructus Immaturus extract on isolated mouse ventricular myocardium

Impaired myocardial relaxation is a key feature of diastolic dysfunction-associated heart failure, including heart failure with preserved ejection fraction (HFpEF), and the development of agents with lusitropic activity is highly desired. In the present study, we investigated the direct acute effects of the methanolic extract from Aurantii Fructus Immaturus (Kijitsu) on myocardial relaxation and explored its active constituents and underlying mechanisms. Using isolated mouse right ventricular myocardial preparations, relaxation was evaluated by measuring the 90% relaxation time (RT₉₀). Kijitsu extract shortened RT₉₀ in a concentration-dependent manner, with a maximal reduction of 17.2 ± 2.6%. Among the eight major compounds identified by HPLC analysis, synephrine and nobiletin each exhibited lusitropic effects when administered alone. Pharmacological investigations demonstrated that the effects of Kijitsu extract and synephrine were abolished by the β-adrenergic receptor antagonist propranolol and the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor cyclopiazonic acid (CPA), whereas the effect of nobiletin was abolished only by CPA. Coadministration experiments resulted in enhanced lusitropic effects partially reproducing the lusitropic effect of Kijitsu extract; the lusitropic efficacy was in the order of synephrine alone < synephrine + nobiletin < synephrine + flavonoids. Furthermore, Kijitsu extract significantly improved impaired relaxation in diabetic mice exhibiting diastolic dysfunction. These findings suggest that Kijitsu extract promotes myocardial relaxation through cooperative activation of the intracellular cAMP-SERCA pathway mediated by synephrine and flavonoids, providing novel evidence that natural compounds can modulate myocardial relaxation.