Rhodioloside

Rhodioloside, whose molecular structure is shown in Fig. 1, was provided in lyophilized powder form by the Traditional Chinese and Tibetan Medicine Lab of the Medical College of Qinghai University. Analytically pure thionine was purchased from Sigma, US. SABC-AP immunohistochemical reagent kit, Bcl-2 antibody, Bax antibody, p53 antibody, and total protein quantitative assay kit were all procured from Nanjing Jiancheng Bioengineering Institute. FACS420 FCM was a product of Becton Dickinson Corporation, US.

This study was a randomized, double-blind, crossover clinical trial conducted at the Beijing Sport University and Peking University. Four groups of resistance exercise-untrained volunteers and two groups of resistance exercise-trained volunteers of similar age and BMI were recruited as participants from these two universities, and their somatotype profile is shown in Table 1.
The recruitment and screening process for the young male resistance exercise-untrained volunteers began 3 months prior to the formal human study with an initial measurement screen conducted to select suitable subjects from a large number of potential subjects. The volunteers (including placebo, CAF, RHO, RHO+CAF groups) who were eventually recruited for resistance exercise-training initially bench pressed approximately 25.21 ± 3.10, 24.79 ± 3.45, 24.58 ± 3.34, 25.00 ± 3.02 kg at one-repetition maximum, deep squatted approximately 36.25 ± 4.20, 36.04 ± 3.76, 35.83 ± 4.17, 35.63 ± 4.28 kg at one-repetition maximum, with MVICs of 179.17 ± 14.50, 180.25 ± 13.43, 177.42 ± 14.29, 178.67 ± 13.13 N·m and maximal repetitions of 60% 1RM bench press of 10.42 ± 1.08, 10.25 ± 0.97, 10.33 ± 1.07, 10.17 ± 1.11 times. These young male volunteers were primarily general college students who did not yet have experience with resistance exercise training. These forty-eight healthy, resistance exercise-untrained volunteers who willingly participated in the study were randomly divided into four groups (n = 12) to take the corresponding treatment while training, numbered 1–4: (1) placebo, (2) CAF, (3) RHO and (4) RHO+CAF.
Similarly, the recruitment process of the resistance exercise-trained volunteers started three months before the official human experiment. These resistance exercise-trained volunteers were mainly from Beijing Sport University, and they all had more than 5 years of resistance training experience; although they had more than three different sports-specific skills, they all had fitness and bodybuilding-specific skills, so their daily training process included not only aerobic training but also anaerobic training. These resistance exercise-trained volunteers had an initial state bench press 1RM of approximately 139.58 ± 3.17 kg (placebo) and 139.79 ± 3.61 kg (CAF+RHO), while the initial deep squat 1RM values were 192.08 ± 3.82 kg (placebo) and 191.67 ± 3.59 kg (CAF+RHO). Meanwhile, the initial MVIC values were 684.08 ± 10.85 kg (placebo) and 682.58 ± 11.33 kg (CAF+RHO), while the maximal repetitions of 60% 1RM bench press values were 17.92 ± 1.16 kg (placebo) and 18.17 ± 0.83 kg (CAF+RHO), all of which indicated a suitable strength training base and athletic performance. Due to the low number of resistance exercise-trained volunteers, we were only able to recruit 24 of them. These healthy volunteers with more than 5 years of resistance training experience were divided into 2 groups (n = 12) while training, and the corresponding treatments were numbered 5–6: (5) placebo and (6) RHO + CAF.
In order to maintain the scientific integrity of our study, and in accordance with the relevant experimental ethical requirements of the Beijing Sport University, we reached agreements and signed commitments with the young male resistance exercise-untrained volunteers and resistance exercise-trained volunteers three months prior to the formal conduct of human experiments that they would not be self-administered CAF and RHO until all the experiments were completed. In addition, subjects were repeatedly and periodically reminded throughout the study not to use CAF and RHO autonomously to ensure that the experimental process was free of unnecessary interference.
The specific experimental procedure is shown in Figure 1. All the participants had stated that they had not consumed any caffeine, tea, additional nutritional supplements, ergogenic aids or alcohol-containing products that could significantly affect their muscular performance spanning 3 months before this study, and they were also non-smokers. Additionally, all the participants had no neuromuscular or musculoskeletal disorders, and they were able to perform the bench press and the deep squat exercises. The group of participants with zero resistance training experience had not participated in any upper body exercises, including the bench presses, or lower body exercises, including the deep squats, before the study [41 (link)]. CAF capsules were purchased from Nutricost, UT, USA, containing 200 mg caffeine anhydrous for each capsule, and the pharmaceutical excipients were rice flour and gelatin (capsule). The CAF drug powder from the capsules was accurately weighed to the desired amount and then taken by the participants at a dose of 3 mg/kg. RHO was purchased from Beijing Tong Ren Tang Pharmaceuticals, Beijing, China, and was administrated as the recommended dose (2.4 g daily, containing 12 mg rhodioloside in total, and the pharmaceutical excipients are starch and gelatin (capsule)) for 30 days. RHO should be taken early in the day because it can disrupt sleep or produce vivid dreams (not nightmares), and it is contraindicative in heated conditions. RHO should not be used in patients with bipolar disorder who are prone to becoming manic when taking antidepressants or stimulants because of its activating antidepressant effect [42 (link)]. Therefore, when we recruited the participants, we avoided recruiting those who were taking antidepressants or stimulants, and we asked all the participants to take RHO on an empty stomach 30 min before breakfast and lunch. For the control group, we removed the drug powder from the CAF or RHO capsules. The participants were to intake the empty capsules (without RHO or CAF inside while indistinguishable from the other supplements given) without being informed [43 (link)].
It is important to note that in order to avoid the impacts of the placebo effect, the placebo group also drank the same amount of water when taking empty CAF and RHO capsules. Similarly, for the CAF single dosing group, they also needed to take empty RHO capsules, and for the RHO single dosing group, they were also required to take empty CAF capsules to avoid the placebo effect.
Participants did not attend any other strength training, use any other nutritional supplements, participate in any other relevant scientific research projects or have any health problems during the study period. Moreover, participants were fully informed of the study procedures, including any risks of participation in the study, before providing the written informed agreement. The experimental methods were conducted in accordance with the Declaration of Helsinki and were approved by the Human Research Ethics Committee of the Beijing Sport University (Ethics Committee No. 2022053H).

Echinacoside (standard), rhodioloside (standard), poliumoside (standard), verbascoside (standard), isoacteoside (standard), geniposidic acid (standard), leonurusoside (standard), catalpol (standard), phenol, 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), catechin, ascorbic acid, forinol and acetonitrile (HPLC grade) were used. The rest of the reagents, such as methanol, were analytically pure. All reagents were purchased from Chengdu Pfeiffer Biotechnology Co., Ltd., Chengdu, China.