2.10. Animals and In Vivo Measurement of Blood Pressure

All animal experiments were executed in accord with the guidelines for the Care and Use of Laboratory Animals under a protocol approved by the Institutional Animal Care and Use Committee of National Taiwan Ocean University, Keelung, Taiwan. The approval number for the ethical clearance was 96,023. Eighteen male SHRs aged 7 weeks were purchased from the National Laboratory Animal Center, Taipei, Taiwan. The SHRs were housed in cages with a maintained light–dark cycle of 12 h. The constant temperature and humidity in the animal room were controlled at 23 ± 1 °C and 55% ± 5%, respectively. The SHRs were fed a standard laboratory diet (Rodent Laboratory Chow Diet 5001, PMI Nutrition International, Brentwood, MO, USA). Tap water was freely available to the rats for eight weeks before the beginning of the experimental period. At the age of 15 weeks, the SHRs (body weight = 350 ± 5 g, SBP = 173.0 ± 4.2 mm Hg, diastolic blood pressure (DBP) = 150.0 ± 3.7 mm Hg) were divided into two experimental groups (both n = 6) that were respectively administered PN-1 dissolved in 2 mL of saline by gastric intubation at doses of 30 and 60 mg of powder/rat BW (350 g, equivalent to 85.7 and 171.4 mg of powder/kg BW or 10 and 20 mg peptide/rat BW). Equal volumes of saline were given to the control group (n = 6) during the trial. The SBP and DBP of each SHR were measured at 2, 4, 6, 8, and 24 h after oral administration. Each rat was placed in a thermostatic box at 45 °C for 5 min to determine the SBP, DBP, and heart rate by using the tail-cuff method (BP-98, Softron, Tokyo, Japan). The results are shown as means and SDs.