No 5001

Thirty-two 8-week-old female ICR mice in a specific pathogen-free condition were obtained from the BioLASCO (Yi-Lan, Taiwan). The mice were housed in an animal room at a constant temperature (22 ± 1 °C) and humidity (50%–60%) under a 12:12 h light-dark cycle with standard laboratory diet (No. 5001; PMI Nutrition International, Brentwood, MO, USA). The distilled water was provided ad libitum. A total of eight cages housing ICR female mice (n = 4 animals per cage) were administrated in the current study. All animal experiments were reviewed and approved by the Animal Care and Use Committee (IACUC) on the ethics of animal experiments at the University of National Taiwan Sport, and this study conformed to guidelines of protocol IACUC-10508 for animal welfare.
After one week of acclimation, the animals were randomly divided into the four groups (n = 8 per group in each test) for oral gavage treatment with CAP once a day for 28 consecutive days: (1) vehicle control; (2) 205 mg/kg CAP (CAP-1X); (3) 410 mg/kg CAP (CAP-2X); and (4) 1025 mg/kg CAP (CAP-5X). Vehicle or CAP was administrated by oral gavage. The control group received the vehicle at the same dosage volume of solution equivalent to body weight (BW). The food intake and water consumption were monitored daily, and BW was recorded weekly.

The RC extract (T. pratense) was purchased from NOW FOOD (IL, USA). Female ICR mice (6 weeks old) grown under specific pathogen-free conditions were purchased from Liaoning Changsheng Biotechnology (Shenyang City, China). The animal protocol (IACUC-19003) was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin Sport University, Changchun City, China. All mice were provided a standard laboratory diet (No. 5001; PMI Nutrition International, Brentwood, MO, USA) and distilled water ad libitum. Housing conditions included a 12-h light-dark cycle and air maintained at room temperature (22°C ± 1°C) with 30–40% humidity. The 1X dose of RC used for humans is typically 1,500 mg per day. The 1X mouse dose (308 mg/kg) we used was converted from a human-equivalent dose (HED) based on body surface area according to the US Food and Drug Administration formula: Assuming a human weight of 60 kg, the HED is 1,500 (mg)/60 (kg) = 25 × 12.3 = 308 mg/kg; the conversion coefficient 12.3 is used to account for differences in body surface area between mice and humans, as previously described. In total, 32 mice were randomly assigned to four groups (eight mice/group) for daily vehicle/RC oral gavage for 6 weeks. The four groups were vehicle, 308 (RC-1X), 615 (RC-2X), and 1,538 (RC-5X) mg/kg. The vehicle group received an equivalent volume of solution base.

Female ICR mice (8 weeks old) grown under specific pathogen-free conditions were purchased from BioLASCO (Yi-Lan, Taiwan). All mice were provided a standard laboratory diet (No. 5001; PMI Nutrition International, Brentwood, MO, USA) and distilled water ad libitum and were housed under a 12-hr light/dark cycle at room temperature (22 °C ± 1 °C) and 50%-60% humidity. The Institutional Animal Care and Use Committee (IACUC) of National Taiwan Sport University (NTSU) inspected all animal experiments, and this study conformed to the guidelines of protocol IACUC-10527 approved by the IACUC ethics committee.
The experimental design is depicted in Fig. 1. After 1 week of acclimatization, 32 mice were divided randomly into two groups. The normal group (n=8) was fed a standard chow diet (control), and the experimental group (n=24) received a high-fructose diet (HFD). The 24 experimental mice were divided into three groups (n=8/each group): (1) HFD with sedentary control (HFD), (2) HFD with aquatic strength exercise training (HFD+SE) and (3) HFD with aquatic aerobic exercise training (HFD+AE). Food intake and water consumption were recorded daily, and all animals were weighed weekly.

Six-week old male institute of cancer research (ICR) mice were obtained from BioLASCO Taiwan (Yi-Lan Breeding Center, Yi-Lan County, Taiwan). All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan Sport University (IACUC-11019). The mice were adapted to the environment before the experiment and provided food ad libitum for 2 weeks. All animals were provided a standard laboratory diet (No. 5001; PMI Nutrition International, Brentwood, MO, USA) and distilled water ad libitum, and maintained in a 12-h light/12-h dark cycle at room temperature (22 ± 2 °C) and 60–70% humidity. In total, 50 mice were randomly assigned to 5 groups (10 mice/group): (1) vehicle (vehicle control or water only), (2) isocaloric (0.93 g casein/kg/mice/day), (3) SPSPE-1X (0.99 g/kg/mice/day), (4) SPSPE-2X (1.98 g/kg/mice/day), and (5) SPSPE-5X (4.95 g/kg/mice/day). All mice received samples with SPSPE by oral gavage for four consecutive weeks, and body weight, water consumption, and food intake were recorded each week.