Experimental animals

Healthy male Sprague–Dawley rats weighing about 200 g-250 g were used for the study. The animals were housed in large spacious cages. Food and water were given ad libitum. The animal house was well ventilated and under a 12 h light/dark cycle at the ambient temperature of 25-30 °C, throughout the experimental period. Rats were allowed to adapt to their environmental conditions for at least 10 days before the initiation of experiment. All experiments and protocols described in the study were approved by the Animal Ethics Committee (Project approval number: UPM/FPSK/PADS/BR-UUH/00484) of the Faculty of Medicine and Health Science, Universiti Putra Malaysia, Malaysia.

The HFHC diet was formulated according to Imam et al. [17], with minor modifications. Every kg of the HFHC formulation contained 500 g ground standard rat chow, 25 g of cholesterol, 200 ml palm oil, 60 g fine sugar, 200 g Nespray® full cream milk and 50 g of starch (See Additional file 1 for diet composition). This HFHC pellet was dried in an incubator at 60 °C for 24 h, cut into small equal sized pieces and fed to the rats.

The rats were randomly divided into nine groups of seven rats each; the normal control (NC) received normal pellet, while the control group received HFHC and the STATIN groups received HFHC + oral gavage of 10 mg/kg/day simvastatin. The aqueous leaf extract (AL) and aqueous methanolic leaf extract (AML) groups were given HFHC + oral gavage of 500, 250 or 125 mg/kg/day/rat of the respective extracts.

During the experiment, weekly body weights of the rats were recorded, while at the end of the experimental period (7 weeks), the animals were fasted overnight and sacrificed by dissection method. Blood (10 ml) was collected by venous puncture after an overnight fast, and centrifuged at 3000 rpm for 10 min at 4 °C to separate the serum. The serum was transferred into 1.5 ml tubes (eppendoff) and stored at −20 °C until analysis. The liver, kidney, heart, brain, spleen and lungs were excised immediately, washed with ice-cold saline, dried with filter paper, and then weighted prior to storage in formalin-free tissue fixation, RCL2 at −80 °C.