Working standards for naringenin and ranolazine were purchased from Sigma-Aldrich. HPLC-grade acetonitrile, methanol, water, and dimethyl sulfate were bought from Merck. All other materials and chemicals were of analytical reagent grade.

Animals used in the investigation

The animal ethics committee (IAEC/TRS/PT/22/26) authorized all animal research and ensured that they were carried out in accordance with the institution's policies for the care and use of laboratory animals. Six male Wistar rats (180–220 g) were confined in cages with unrestricted access to fresh water and food in typical controlled environments for a minimum of 7 days. The animals received a 12-h fast well before the program began, and they were denied access to food and drink while it was being conducted. The broiler chicks were bought from nearby farms and kept in carefully regulated circumstances.

Analysis techniques

A modified version of the approach recently reported by Nalawade et al. (2010) was used to quantify the plasma levels of ranolazine. A pump (LC-20AD), C18 column (Zorbax, Agilent) 75*4.6 mm, 5 m particle size, and a dual-wavelength ultraviolet (UV)-visible detector made up of a Shimadzu HPLC system (Shimadzu, Tokyo, Japan) (SPD-10A). Data collection and processing were carried out using LC solution software (Tokyo, Japan). The mobile phase was composed of acetonitrile and water (80:20 v/v) and 0.2 percent acetic acid.

Plasma extraction of ranolazine

The plasma was removed by centrifugation at 4000 rpm after the blood was drawn into plastic tubes containing K2 EDTA as an anticoagulant. Around 0.4 mL of plasma was combined with 2 mL of ethyl acetate for 5 min before the mixture was centrifuged at 4000 rpm for 5 min at room temperature. The organic layer was then removed, and using a nitrogen stream, it was evaporated to dryness at 50 °C. In order to perform chromatographic analysis, the residue was concentrated with 0.150 mL of the mobile phase.

Ranolazine's pharmacokinetics in SDS and the impact of naringenin

Group I received ranolazine 14 mg/kg therapy, Group II received naringenin 25 mg/kg treatment, and Group III received ranolazine 14 mg/kg treatment followed by naringenin 25 mg/kg treatment after 30 min. Oral gavages were used to provide each medication to the appropriate group. Following treatment, blood samples were taken from the retro-orbital plexus at 0.0, 0.5, 1, 2, 3, 4, 4.5, 5, 6, 8, 12, and 18 h. The concentration of ranolazine in the plasma was then calculated using the RP-HPLC technique.

Ranolazine's pharmacokinetics in MDS and the impact of naringenin

Rats with MDS were given the same medications for 7 days. On the 8th day, the blood sample was collected from the retro-orbital plexus at 0.0, 0.5, 1, 2, 3, 4, 4.5, 5, 6, 8, 12, and 18 h. The ranolazine concentration in plasma was estimated by the RP-HPLC method.

Naringenin's impact on ranolazine intestinal absorption in rat-everted sac

The gastrointestinal P-gp transit of compounds from the mucosal to the serosal surface was investigated using the everted sac technique [24]. Pentobarbital was given to male Wistar rats (weight 180–220 g) at a dose of 30 mg/kg (s.c.). An abdominal incision was made midline. The rats were killed by puncturing their hearts. A glass rod was used to quickly remove and evert the small intestine, which had a length of 10–12 cm from the Treitz ligament to the ileocaecal junction. The 2 mL of a pH 7.4 Krebs–Henseleit bicarbonate buffer solution containing 0.4 percent glucose was added to the sacs. For sample collection, one end was ligated and the other end was ligated with a needle. The sac was incubated at 37 °C in a glass container with 25 mL of the same buffer solution and was gassed at a ratio of 95/5. Ranolazine (50 µM) solution was added to the glass jar after 5 min, and the preparation was then incubated once more. At 10, 20, 30, 40, 50, and 60 min, 0.5 mL of sample was taken from the sac and 0.5 mL of buffer was added. The process was repeated with more sacs. Verapamil (10, 50, 100, and 200 µM) and Naringenin (25, 50, 100, and 200 µM) were present. The RP-HPLC technique was used to measure the ranolazine transfer from the mucosal to the serosal side at 225 nm.

Naringenin's impact on the intestinal absorption of ranolazine from the chicken everted sac

Activity evaluation was done by the new P-gp model in the intestinal sac everted by a chick. For the study, we employed this model. To prepare and mount tissue, I strictly adhered to the process outlined in Tanaka K.et al. [25]. Animals were killed, and their intestines were harvested from the duodenal loop's proximal end to its distal end immediately (2 min); the caecal attachment was withdrawn, and ice-cold ringer solution was used for flushing. The intestine was everted, the sleeves were shortened to 1.5 cm, and by securing the tissue to stainless steel rods precisely one centimeter apart over grooves.

Calibration curve standards and quality control (QC) sample preparation

Methanol was used to dissolve precisely weighted ranolazine, creating a standard primary solution with a 1 mg/mL concentration. By sequential diluting the primary solution with methanol: water (50:50), the secondary working solution with concentrations of 50, 100, 200, 1000, 5000, 10,000, 20,000, 30,000, 40,000, and 50,000 ng/mL was produced. 100 µL of blank rat plasma and 5 µL of each working solution were combined to create the calibrator standards, which had nominal values of 2.5, 5, 10, 50, 250, 500, 1000, 1500, 2000, and 2500 ng/mL. The same method was used to create the quality control (QC) samples from a separated stock solution at concentrations of 15, 80, 900, and 2100 ng/mL.

Statistic evaluation

The data were presented as mean SEM. Standard statistical procedures were used to determine the significance at p < 0.05 level using GraphPad Prism 8.0 (GraphPad Prism Software Inc., San Diego, USA).