Animals and surgery
Male ICR mice (4 weeks old, n = 40) and Sprague–Dawley rats (6 weeks old, n = 20) were obtained from Central Lab Animal Inc. (Seoul, Korea), kept in air-conditioned room, and acclimated for at least 7 days. The mice were divided into four groups; one group received distilled water (DW) (Control group, n = 10), and the other groups received three different dosages of HX108-CS (10, 50 and 100 mg/kg, n = 10 per group) solution in water orally. Then, the rats were divided into two groups; one received DW (Control group, n = 10), and the other group received HX108-CS (100 mg/kg, n = 10) solution in the same way as mice. Before the exercise test, the animals were given either DW or HX108-CS for 2 weeks. In the rats, they were anesthetized with zoletil 50 (10 mg/kg, i.p.; Vibac Laboratories, Carros, France) for surgery. For the blood analysis, a silicone catheter was inserted into the jugular vein and fixed by a 35 mm silk thread. The exteriorized distal end of the catheter was fixed at the animal’s nape as described in a previous study . Three days after surgery, the rats were kept individually in their cages until the start of the running tests for full recovery. All experimental procedures were approved in accordance with the institutional animal care and use guidelines of the Animal Experimental Center at Seoul National University.
The procedures for handling and caring for the animals were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Laboratory Animal Resources at Seoul National University (SNU-100705-6).
Preparation of HX108-CS
Total water-soluble extract of the dried fruits, HX108-CS was prepared from these hardy kiwifruits. Briefly, the dried Schisandra chinensis and Chaenomeles sinensis fruits were mixed at a 2:1 ratio by weight and extracted by boiling in DW for 3 hours. The extract was filtered with Whatman filter paper (No. 2, 110 nm), and concentrated using a rotary evaporator, followed by a freeze-drying process. Powdered HX108-CS was dissolved in DW at a concentration of 200 mg/ml and stored at −80°C until use.
Cell differentiation and sample treatment (in vitro)
C2C12 cell line (kindly donated by Prof. Kang, Seoul, Korea) was routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% heat inactivated fetal bovine serum (FBS), 100 IU/ml penicillin G and 100 μg/ml streptomycin in a humidified 5% CO2 atmosphere at 37°C. The medium was changed three times a week. The cells were harvested and reseeded when reached by 50%.
For lactate measurement, C2C12 cells were seeded on 24-well plates (2.5 x 104 cells/500 μl) in DMEM medium. C2C12 cells were treated with HX108-CS (250, 500 and 1000 μg/ml) for 30 min before NaN3 (sodium azide) treatment. After 30 min, NaN3 was treated to cells and incubated for 6.5 hrs. After 7 hrs, supernatant of cells was harvested and used for lactate measurement. For LDH and CK activities measurement, C2C12 cells were differentiated for 5 days. Briefly, C2C12 cells were seeded on 6-well plates (2 x 105 cells/2 ml) in DMEM medium. To induce differentiation, the medium was replaced with DMEM containing 2% horse serum prior to the cells reaching confluence. The medium was changed every day. Differentiated C2C12 cells were treated with HX108-CS (250, 500, and 1,000 μg/ml) and 2, 4-dinitrophenyl (DNP) for 1 hr. After 1 hr, supernatant of cells was harvested and used for LDH and CK measurement.
Swim-to-exhaustion exercise test
Each of the mice had a weight attached (10% body weight) to the tail for the duration of the swim-to-exhaustion exercise. The mice were assessed to be fatigued when they failed to rise to the surface of the water to breathe within 5 sec. Swimming time was recorded as minute for each mice. Blood samples were taken by capillary glass tubes from eye venous pool of mice when they were no longer able to continue to swim. Throughout the course of this experiment, one researcher who did not know the grouping of the mice determined the duration of swimming time to exhaustion. The swimming exercise was carried out in a tank (32 × 50 × 35 cm), filled with water to 25 cm depth and maintained at a temperature of 33 ± 1°C.
Progressive treadmill exercise protocol
Two hours before the experiment, the rats were fasted by removing chow to minimize the glucose effect from consumption just prior to the measurement. A week before the day of experiment, all animals were familiarized to the treadmill, starting with walking on the treadmill (5 m/min) and then running at 15 m/min, 0% grade for 20 min/day, 5 days/week. The day of the experiment, the rats were placed on the treadmill at 0% slope and the speed was gradually increased up to 25 m/min for 30 min.
Measurements of lactate, LDH and CK activity
In vitro study, lactate production, LDH and CK activity were measured using commercial kit (Lactate assay, Bioassay systems, USA; LDH assay, Takara, Japan; CK assay, Bioassay systems, USA). For lactate measurement, the working reagent (assay buffer, enzyme, NAD, PMS, MTT) was added into plate and tapped the plate to mix. After 0 min and 20 min, the absorbance at 565 nm was read using a 96-well plate reader. For LDH assay, the reaction mixture was added into plate and incubated for 30 min. After incubation, the absorbance at 490 nm was read using a 96-well plate reader. For CK assay, the plate was incubated at 37°C for 10 min. After 10 min and 40 min, the absorbance at 340 nm was read using a 96-well plate reader.
In vivo study, fatigue related parameters were evaluated as described previously  with some modifications. Lactate concentration (Lactate pro LT-1710, ARKRAY Inc., JAPAN) was determined from whole blood samples before, during (20 and 25 min) and immediately after exercise and recovery phases (3, 6, 9, 15 and 30 min after exercise) on the treadmill. On the other hand, LDH and CK levels were measured from whole blood samples before, immediately after exercise and recovery phases (15 and 30 min after exercise). At least 30 μl of blood sample was taken from the jugular vein catheter to measure blood lactate, LDH and CK activities. Blood CK and LDH activities were assayed using commercially available kits (LDH assay, Takara, Japan; CK assay, Bioassay systems, USA).
Data were presented as mean ± S.E.M, and differences between vales were compared with two-way repeated measure analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons with the pre-exercise value as a control. Differences between groups were tested with unpaired Student’s t-test by using Origin 8.0. P values less than 0.05, which were calculated as one-tailed P values, were considered to be statistically significant.