Subjects
Twelve male college gymnasts (aged 20.5 ± 0.3 y; height 169.4 ± 1.6 cm; weight 63.4 ± 1.8 kg) volunteered to participate in this study. Prior to the recruitment of volunteers, the study protocol was approved by Institutional Review Board of University of Taipei, Taipei, Taiwan. A detailed explanation of the study procedures, including the supplements to be received and potential risks might be involved, was informed to all participants prior to study. All subjects completed a written informed screening questionnaire after explanation. Participants had any form of health problem were precluded. All participants were asked not to change their dietary habit and not to participate in any form of training activity since a week before trial until the end of blood sample collection.
Experimental design
This study used a placebo-controlled, double-blind, crossover design with a two-week washout period. Participants were randomly assigned to one of two parallel groups, initially in 1:1 ratio, to receive either Rb1 or Placebo under supervision of a lab staff to ensure subject compliance. The Rb1 and Placebo were made by the lab manager in liquid form and were identical in appearance. They were placed in a container and consecutively numbered for each participant according to the randomization schedule. Each participant was assigned an order number and received the drink in the corresponding container. A computer-generated list of random numbers was used for allocation of the participants. A research staff assigned participants to interventions. Except for the interventions, staff was kept blind to supplement assignment of the participants. Staff that takes outcome measurements and staff that delivers the intervention was different. Staffs and participants were maintained masked to outcome measurements and trial results. Before the experiment began, all participants engaged in parallel squatting and their 85 % maximum lower-limb muscle strength (85 % of 1-RM) was assessed. Baseline assessments was conducted under overnight fasted condition. Following a 30-min warm up period consisting of a 5-min warm up, a 10-min full-body stretching, a 5-min lower body stretching, a 5-min low intensity squat practice and a 5-min rest, all participants were challenged with a single bout of high-intensity lower-limb resistance exercise. In brief, this resistance exercise consisted of 6 sets of parallel squats with a resting interval of 60 s between sets [13]. Each exercise set comprised of a 10-parallel squat repetitions at 85 % one-repetition maximal (1-RM). All participants were instructed to complete each set of squat repetitions within 60 s.
Rb1 supplementation
Following exercise challenge, each participant was instructed to consume a solution containing ginsenoside Rb1 (or a placebo solution) for 5 consecutive days. Rb1 and Placebo drinks were made available by 0900 am in the morning. Participants orally received the drink once daily in the lab, under supervision of a staff at 0900 am during the 5-day recovery period. The Rb1, provided by NuLiv Science (Walnut, CA, USA), was extracted from Asian ginseng. Rb1 content is confirmed by Prof Tsu-Chung Chang in Department of Biochemistry, National Defense Medical College, Taiwan [14]. Rb1 crystal was solubilized by a 58 % alcohol solution into a concentrate. They were then withdrawn by pipetting into 50 ml of drinking water to achieve a final dose of 1 ng/kg body weight for each participants. The placebo group consumed same volume of drinking water, which contained an identical amount of alcohol solution. Participants were unable to tell the difference between Rb1 and Placebo drinks by appearance and taste. The experimental procedure is shown in Fig. 1. The dosage used in this study is equivalent to approximately 0.1 g of ginseng (wet weight), which is considered low dose.
Samples collection
Blood samples were collected from fingertip and measured immediately for glucose concentration. Serum sample was used for insulin and cortisol measurements. A total of 200 μl of blood was collected from the fingertip for serum preparation, before and 10-min, 1 day, 3 day, and 5 days after resistance exercise challenge [13]. Each blood sample was centrifuged at 4000 rpm for 5 min. Supernatants were used as serum samples for insulin and cortisol measurements. They were frozen at −80 °C and analyzed within a week.
Biochemical analysis
Fasting blood glucose was analyzed using a glucose-oxidase method with the One-Touch glucometer (LifeScan, Milpitas, CA, USA). All hormones were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Diagnostics Systems Laboratories, Inc., Webster, TX, USA) according to the standard procedures provided by the manufacturer. In brief, serum samples (depending on the requirements for specific kits) were loaded into 96-well plates coated with specific antibodies. Once antigens were bound with the specific primary antibody, the secondary antibody was added to form the antibody-antigen-antibody complex. The enzyme was then added and conjugated to the secondary antibody, and the plates were finally developed by adding enzyme substrate to generate visual light signals. The visual light signals were detected by ELISA analyzer (Tecan Genios, Salzburg, Austria). The intra-assay coefficient of variances (CV) for insulin and cortisol were 2.03 and 5.90 %, respectively.
Heart rate variability (HRV)
HRV of all participants was assessed before (09:00), after exercise (11:50) and on the Day 1, Day 3 and Day 5 during recovery period (09:00). HRV was assessed in a quiet, dim environment at room temperature. Prior to assessment, participants were seated and instructed to relax for 5 min. An autonomic nervous system analyzer (Telemedicine Equipment Co., Ltd., Taiwan) was used to acquire a 5-min R-R interval data. HRV-HF was considered to be a measure of parasympathetic nervous system activity, whereas HRV-LF/HF was considered to be a measure of sympathetic nervous system activity [15].
Statistical analyses
Two-way ANOVA with repeated measure (Rb1 supplementation and time) was performed to assess mean differences in all variables. Fisher’s least significant difference test was performed for post hoc comparison. Paired t-test was used to compare difference in area under the curve (AUC) of HRV-LF/HF. All values were expressed as mean ± standard error (SE). The statistical significance was set at 5 % of type I error.
