2.5. Single-pass intestinal perfusion studies

Sprague-Dawley rats (male, 200–250 g) were fasted overnight with free access to water. The rats were divided into different groups at random before the experiments. The surgical procedure for the single-pass intestinal perfusion experiments was performed as previously described15, 25. The process was as follows: the rats were anesthetized with an intraperitoneal injection of sodium pentobarbital. The surgery was performed under a surgical lamp to keep the body temperature at 37 °C. After the abdomen was opened by a median incision of about 3 cm, the duodenum, jejunum, ileum, and colon was exposed and cannulated with flexible tube (approximately 10 cm) and then ligated at both ends. The surgery was performed gently to minimize the damage and keep blood circulation intact. A wet gauze was placed on the exposed intestinal segment to maintain moisture.

In this study, we explored the absorption of Hup-A in four different intestinal segments. The visible Peyer׳s patches (PPs) in ileum were ligatured with silk thread before the perfusion experiment in order to study the effect of ligature of PPs on the ileal absorption of Hup-A SMEDDS. In order to investigate whether the absorption of Hup-A was dose-dependent, the Hup-A SMEDDS and Hup-A suspension were dispersed in Krebs–Ringer׳s buffer at a low, middle and high drug concentration (5, 10 and 20 μg/mL) as the perfusion solution, and the perfusion solution was placed in a 37 °C water bath to keep the temperature.

At the beginning, in order to clean out any residual debris in the intestine, the isolated intestinal segment was rinsed with normal saline solution (37 °C) at a flow rate of 0.5 mL/min. The experimental intestinal segment was perfused with the perfusion solution at a flow rate of 0.25 mL/min for 30 min in order to achieve absorption equilibrium utilizing a peristaltic pump (HL-2; Shanghai Qingpu-Huxi Instruments Factory, Shanghai, China). The intestinal perfusion samples were collected at 10-min intervals for 90 min. All samples including perfusion samples were collected from inlet and outlet drug perfusion solution at different time points. All samples were filtered and analyzed by HPLC. All glass vials were weighted, respectively, before and after the perfusion experiment. At the end of the experiment, the length and radius of the perfused intestinal segments were carefully measured.

The gravimetric method was used to calculate the correction perfusion fluid volume change caused by intestinal moisture absorption. The 0.5 mL perfusion solution was put in the tube that had been weighed and the solution weight was used to calculate the perfusion solution density (ρ_in). Similarly, the 0.5 mL solution from all intestinal perfusion samples was put into tubes that had been weighed, and the intestinal perfusion samples density (ρ_out) was calculated. The K_a and P_app of Hup-A were calculated according the following Eqs. (1)–(5)26, 27:

where m_in and m_out are the weight (g) of the inlet perfusion solution and outlet perfusion solution, respectively; ρ_in and ρ_out are the density (g/mL) of the inlet perfusion solution and outlet perfusion solution, respectively; V_in and V_out are the volume (mL) of the inlet perfusion solution and outlet perfusion solution, respectively; C_in and C_out are the concentration (μg/mL) of the drug in the inlet perfusion solution and outlet perfusion solution, respectively. Q is the perfusion flow rate (0.25 mL/min); V is the volume (mL) of the perfused intestinal segment; l is the length (cm) of the perfused intestinal segment; r is the radius (cm) of the perfused intestinal segment.