*4.4. Preparation of RA-3 Solution*

Fresh working solutions of RA-3 (50 and 100 mg/kg body weight) were prepared in 2% Tween 20. These concentrations were based on our previous studies on the compound [14–16].

#### *4.5. Investigation of the Antihyperuricemic and Nephroprotective Potential of RA-3*

The adenine and gentamicin induced hyperuricemia and nephrotoxicity rat model, slightly modified from that described by Meng et al. [34] was used to evaluate the antihyperuricemic and nephroprotective effect of the triterpene (RA-3). Sprague Dawley rats of either sex (150–200 g) were randomly divided into two major groups; normal and model groups. The rats in the model group were orally administered with adenine (150 mg/kg body weight) and intraperitoneally injected with gentamicin (40 mg/kg body weight) daily for fourteen days to induce hyperuricemia and nephrotoxicity.

Following induction of nephrotoxicity, the animals in the model group were randomly divided into four groups (II-V) of at least five rats per group (*n* = 5). The normal control (I) and model control (II) groups received drinking water and 2% Tween 20 (vehicle, p.o), respectively, throughout the experimental period. While the positive control group (III) received allopurinol (10 mg/kg, body weight, p.o), the experimental groups IV and V were orally administered with RA-3 at 50 and 100 mg/kg, respectively. The respective drugs were administered daily for a further fourteen days period. All drug administrations were performed between 8:00 and 9:00 AM and the animals were treated in the same order throughout the experimental period. At the end of the experimental period, the rats were fasted for eight hours and euthanized under anesthesia (Pentobarbital at 30 mg/kg body weight, ip). A complete loss of sensation was confirmed by pedal withdrawal reflex before any procedure could be conducted on the rats. The rats were quickly dissected, blood and kidney samples were immediately collected. The collected tissues samples were used for estimation of some biochemical parameters and histopathological indications, respectively.
