2.3. Pharmacokinetic Study

Male ICR mice (7-weeks-year-old, 30–35 g) were purchased from Samtako Co. (Osan, Kyunggi-do, Korea). Animals were acclimatized for 1 week in an animal facility at Kyungpook National University. Food and water were available ad libitum. The overall experimental scheme is shown in Figure 2.

Overall experimental scheme for studies using RGE and 15 ginsenosides.

To investigate the effect of amoxicillin on the intestinal metabolism of ginsenosides, mice received amoxicillin (n = 9, 20 mg/kg/day, dissolved in water at 2 mL/kg, twice daily) for 3 days via oral gavage. Before blood sampling, mice were anesthetized using isoflurane (isoflurane vaporizer to 2% with oxygen flow at 0.8 L/min) for 5 min. Blood sampling was performed using a sparse sampling method via the right or left retro-orbital vein under isoflurane anesthesia at 24 and 48 h through the heparinized capillary tube (Heinz Herenz, Hamburg, Germany). The last blood sampling was performed via abdominal artery using a heparin-treated 1-mL syringe (Jung Lim Co. Ltd., Choong-Buk, Korea) under anesthesia with isoflurane at 72 h after the first dose of amoxicillin solution (time schedule and blood sampling volume are given in Table 3). After the centrifugation of the blood samples at 10,000× g for 1 min, 30 μL aliquots of plasma were stored at −80 °C for the analysis of plasma amoxicillin concentration. Aliquots (200 μL) of an IS (0.05 ng/mL berberine in acetonitrile) were added to 30 μL of plasma samples. After that, the mixture was vortexed for 15 min and centrifuged at 16,000× g for 5 min. After centrifugation, 200 μL of the supernatant was transferred to a clean tube and dried under a nitrogen stream at 40 °C. The residue was reconstituted using 100 μL of 50% acetonitrile supplemented with 0.1% formic acid, and a 2-μL aliquot was injected into the LC–MS/MS system.

Blood sampling method for the effect of amoxicillin on ginsenoside pharmacokinetics in mice.

RO–right: retro-orbital blood sampling—right eye under anesthesia with isoflurane. RO–left: retro-orbital blood sampling—left eye under anesthesia with isoflurane. AA: abdominal artery blood sampling under anesthesia with isoflurane.

Among the amoxicillin treated mice, six mice received RGE in a single dose (2 g/kg suspended in water at 2 mL/kg) via oral gavage 2 h after the last amoxicillin administration on 4th day and then returned to their metabolic cages with food and water ad libitum and urine and feces samples were collected for 48 h. The urine and feces samples were weighed, and 30 μL aliquots of urine and 100 μL aliquots of 10% feces homogenates were stored at −80 °C until the analysis of the ginsenosides. Blood sampling was performed using a sparse sampling method via the right or left retro-orbital vein under anesthesia with isoflurane at 0, 2, 4, and 8 h after the RGE administration through the heparinized capillary tube. The last blood sampling was performed via abdominal artery using a heparin-treated 1-mL syringe under isoflurane anesthesia at 24 and 48 h after the RGE administration (time schedule and blood sampling volume was given in Table 3). After the centrifugation of the blood samples at 10,000× g for 1 min, 30-μL aliquots of plasma were stored at −80 °C until the analysis of the ginsenosides. For the comparison, six mice received water (2 mL/kg) via oral gavage for 3 days and, on 4th day, mice received RGE in a single dose (2 g/kg suspended in water at 2 mL/kg) via oral gavage 2 h after the last water administration and then returned to their metabolic cages with food and water ad libitum and urine and feces samples were collected for 48 h.

Aliquots (200 μL) of an IS (0.05 ng/mL berberine in methanol) were added to 30 μL of plasma or urine samples. Aliquots (600 μL) of an IS methanol solution containing 0.05 ng/mL berberine were added to 100 μL of 10% feces homogenate samples. After that, the mixture was vortexed for 15 min and centrifuged at 16,000× g for 5 min. After centrifugation, 200 μL of the supernatant was transferred to a clean tube and dried under a nitrogen stream at 40 °C. The residue was reconstituted using 100 μL of 70% methanol supplemented with 0.1% formic acid, and a 10-μL aliquot was injected into the LC–MS/MS system.

Mice received amoxicillin (n = 12, 20 mg/kg, dissolved in water at 2 mL/kg, twice daily) for 3 days via oral gavage. From the 4th day, mice from the LAB + amoxicillin treatment group received LAB (2 g/kg suspended in water at 2 mL/kg, once daily) for 7 days via oral gavage. Mice from the amoxicillin treatment group received water (2 mL/kg, once daily) for 7 days via oral gavage. Subsequently, on the 10th day, mice received RGE in a single dose (2 g/kg suspended in water at 2 mL/kg) via oral gavage 2 h after the last LAB or water administration and then returned to their metabolic cages with food and water ad libitum and urine and feces samples were collected for 48 h. The urine and feces samples were weighed, and 30 μL aliquots of urine and 100 μL aliquots of 10% feces homogenates were stored at −80 °C until the analysis of the ginsenosides.

Blood sampling was performed using a sparse sampling method via the right or left retro-orbital vein under isoflurane anesthesia at 0, 2, 4, and 8 h after the RGE administration through the heparinized capillary tube. The last blood sampling was performed via abdominal artery using heparin-treated 1 mL syringe under isoflurane anesthesia at 24 and 48 h after the RGE administration (time schedule and blood sampling volume was given in Table 4). After centrifugation of the blood samples at 10,000× g for 1 min, 30 μL aliquots of plasma were stored at −80 °C until the analysis of the ginsenosides. Subsequent protocols were identical to the amoxicillin treated group.

Blood sampling method for the effect of LAB on ginsenoside pharmacokinetic study in mice.

RO–right: retro-orbital blood sampling—right eye under anesthesia with isoflurane. RO–left: retro-orbital blood sampling—left eye under anesthesia with isoflurane. AA: abdominal artery blood sampling under anesthesia with isoflurane.