*2.8. Oral Pharmacokinetic Study in Rats*

To determine whether quercetin affects the intestinal efflux mediated by BCRP, we divided the male rats into two groups: A sulfasalazine (a substrate of BCRP) control group and a quercetin pretreatment plus sulfasalazine group (*n* = 4, each). Considering the similar expression levels of intestinal BCRP between male and female rats, male rats were used in this study [30,31]. Briefly, overnight fasted male SD rats were anesthetized by intramuscular administration of 50 mg/kg tiletamine HCl/zolazepam HCl (Zoletil®) (Vibrac, TX, USA) and 10 mg/kg xylazine HCl (Rompun®, Bayer, Puteaux, France). While the rats were anesthetized, the femoral artery (for blood sampling) and vein (for supplementing body fluids) were catheterized using polyethylene tubing (PE 50; Clay Adams, Parsippany, NJ, USA). Upon recovery from anesthesia (i.e., after 4 h), quercetin was administered by oral gavage at 10 mg/kg (or 0 mg/kg in the case of the sulfasalazine control group; DMSO/polyethylene glycol 400/saline [1:4:5 (v/v/v)]). The pretreatment dose of quercetin was determined based on the compound solubility in the dosing vehicle and the likely daily dose of human dietary supplement. Fifteen minutes after the pretreatment, a dosing solution containing sulfasalazine at 2 mg/kg was administered by oral gavage. Blood samples (150 µL) were collected at 5, 15, 30, 60, 120, 240, 360, and 480 min after the sulfasalazine administration. Immediately after each blood collection, an identical volume of saline was intravenously provided to the animal to compensate for fluid loss. To prevent blood clotting during blood collection, the cannula was filled with 25 IU/mL heparinized saline. The plasma fraction was separated from the blood samples by centrifugation (16,100 × g for 5 min at 4 ◦C) and stored at −80 ◦C until the LC-MS/MS assay.
