This was an experimental study conducted at the Physical Education School, Federal University of Ouro Preto in the Exercise Physiology Laboratory, approved by the Human Research Ethics Committee under protocol no. 25402813.2.1001.5150. We submitted the present study to the Brazilian Registry of Clinical Trials (ReBEC), and it was approved under number RBR-2x56pw8, registered January 15th, 2021. To participate in the study, the participants were made aware of the study objectives and the possible benefits and risks. All provided informed written consent. This study adheres to CONSORT guidelines .
The sample size was calculated based on a previous study by our research group , that used the same dosage of HPβ-CD-Ang-(1-7) in individuals submitted to eccentric exercise protocol, we used the biochemical marker creatine kinase (CK) for the sample calculation. The analysis were performed using the Bioestat software (5.3), the CK showed a variation of 197 U / L ± 159U / L in the study, the statistical power was of 0.8, and a significance level of 0.05, the sample calculation resulted in 11 volunteers per group.
The inclusion criteria were involvement in cycling training programs for at least 12 months and carrying out at least four MTB training sessions per week.
Participants were excluded if they had used supplements with a potential stimulatory effect on the cardiovascular system (such as caffeine and guarana powder). Has had current injuries or in the past six months, has not been willing to abstain from intense exercise 24 hours before the test. Participants were tested at the same time of day for each of the experimental visits.
The first author of the study performed the interventions and recruited the volunteers. Participants were recruited in local community from June to August 2016. The data assessment started in August 2016 and ended in October 2016, after reaching the estimated number of participants.
Altogether, 21 cyclists of both sex (3 female and 18 male), volunteered for this study. For the final analysis, 14 volunteers (2 female and 12 male) were considered eligible (see CONSORT flow diagram, Fig. 1). Average age was 29 ± 5 years; Average body weight was 71 ± 7 kg; Average height was 1.70 ± 0.07 m; Average body mass index was 24 ± 2 kg/m2.
The distribution of the formulation was double-blind and randomized. Participants received the formulation of HPβ-CD-Placebo or HPβ-CD-Ang-(1-7) (1.75 mg), 50% of subjects randomly used HPβ-CD-Placebo in the first session and HPβ-CD-Ang-(1-7) in the second session or vice versa. The double blind and the randomization of the experiments were performed by the corresponding researcher. The same, assigned a code for each participant. The code was revealed to other researchers only at the time of data interpretation.
A single dose of the HPβ-CD-Placebo or formulation HPβ-CD-Ang-(1-7) was given orally, both in capsule form, three hours before the beginning of each test. The capsules were identical in color, size and without flavor, ensuring the blinding of the participants.
During the intervention, we requested that physical training, food intake, and sleep hours be maintained. The volunteers were subjected to two days of tests with a seven-day interval between them, which can be considered a safe washout time. Upon return to the laboratory, we ask from the subjects to determine if there were adverse reactions and if they had maintained their diet and physical training routines.
Physical exercise protocol using the leg ergometer cycle
The test protocol was based on previous studies with MTB athletes . The tests were performed using a leg ergometer cycle (Biotec 2100, CEFISE Biotechnology), with 10 minutes of warm up and a load of 12.5 W for both genders. After warming up, a continuous progressive load test was performed with an initial load of 25W for women and 37.5W for men. The load was increased by 12.5 W every three minutes for both genders. The test finished when the individual reached voluntary exhaustion or when the rotation could not be kept at 70 rpm. At the end of each stage, heart rate (HR), ratings of perceived exertion (RPE), maximal oxygen uptake (VO2), and respiratory exchange coefficient (REC) were collected.
The formulation was developed by the Laboratory of Hypertension and Laboratory of Chemical of the University of Federal of Minas Gerais; the details were described previously . This compound (HPβCD/Ang-(1-7)) was patented (BR 10 2016 0244064/Federal University of Minas Gerais/Federal University of Ouro Preto).
Oral supplementation with HPβCD-angiotensin-(1-7) (1.75mg) or HPβCD-placebo (1.75mg) was administered 3 hours before the test protocol. The 3h time was pre-established considering the peak action of angiotensin-(1-7) which has a window of action between 2 to 6 hours .
Considering toxicity and adverse responses in humans, the present study used a dose 16 times lower (1.75mg) than the dose used in a study conducted in cancer patients (400 μg/kg) that showed no collateral effects. In addition, a previous study in healthy younger  showed protective effects against muscle damage using the same dose without side-effects.
Blood samples were collected from the antecubital vein by a skilled phlebotomist using standard technical venipuncture. Approximately 12 ml were collected in vacutainer tubes. Immediately after collection, the blood was centrifuged at 3.000 rpm for ten minutes, and the serum transferred to Eppendorf tubes stored at –80 °C. Aliquots were used to measure non-esterified fatty acids (NEFA) and creatine kinase (CK).
Blood lactate levels
Lactate levels were measured in each participant's index finger by placing one drop (5 μl) of blood on a BM-Lactate reagent strip (Roche Diagnostics GmbH, D68298 Mannheim, Germany) and introducing it into the Accutrend® Lactate meter (Roche Diagnostics GmbH, D-68298 Mannheim, Germany). Lactate was measured immediately at the end of each test.
Non-esterified fatty acids (NEFAs)
NEFAs were analyzed according to the specific colorimetric method using the Randox® kit (Randox Laboratories, Oceanside, CA, USA).
Mechanical work and efficiency
To calculate mechanical work and efficiency, the equations above were used . Work is described as follows: w = time (min) × load (kg) × wheel circumference (m) × rotation (rpm), in kg.m., while mechanical efficiency was calculated using the equation:
where the perfect machine constant is the energy spent by a machine without loss of efficiency to perform work (1 kcal = 426.4 kg.m) in %.
Evaluation of BP and HR
Blood pressure was measured with an aneroid sphygmomanometer (Missouri®, Brazil) and stethoscope (Missouri®, Brazil) before and immediately after (20 seconds) the end of the test protocol. HR was measured with Polar RS800 (POLAR, Finland) at rest, along the steps and at the peak of maximum physical test effort.
Evaluation of subjective rating of perceived exertion (RPE)
Participants evaluated their fatigue using the subjective rating of perceived exertion with reference to the Borg Scale . RPE was determined at each stage completed by the volunteer.
Evaluation of maximum oxygen consumption
The aerobic capacity was determined in the HPβ-CD-placebo and HPβ-CD-Ang-(1-7) conditions using open-circuit spirometry on VO2000® (VO2000, MedGraphics®️, Saint Paul, Minessota-USA) equipment during the leg ergometer cycle physical test calibrated before each test. The ventilatory equivalent for oxygen (VE/VO2), ventilatory equivalent for carbon dioxide (VE/VCO2), and respiratory exchange ratio (RER) were recorded at each stage completed to determine maximal oxygen uptake and respiratory exchange quotient. The average of the final two minutes of the test was used to determine the relative VO2 .
The main objective and primary outcome of the present study was to evaluate the effect of the formulation on the physical performance of cyclists, in response to this, we evaluated the total exercise time on a leg cycle ergometer, maximum oxygen consumption, mechanical work and mechanical efficiency. The secondary outcomes were the biochemical parameters (NEFA, lactate and RQ) and the rates of perceived exertion using the Borg scale. We obtained the primary and secondary results at the beginning of the test, during and immediately after (20 seconds) of the progressive load protocol until voluntary fatigue. All tests were performed by the first author, who was unaware of the participants' allocation.
Data normality was tested using the D'Agostino & Pearson test. The data that were normally distributed were compared using the paired t-test. For non-normal data, the Wilcoxon matched pairs signed rank test was used. Information regarding data normality was added to the figures. Data were expressed as mean ± standard deviation (SD) and the significance level was p <0.05 for all tests. For the evaluation throughout the stages of the physical test, a regression using equations of straight line were also used. To run the test, we chose the fitting method of least squares regression without weighting. The null hypothesis was that one curve would fit the two conditions (HPβ-CD-Placebo and HPβ-CD-Ang-(1-7). The curve comparison method was the extra sum-of-squares F-test and the p-value was fixed at 0.05. The measurement of effect size used was Cohen’s d (Cohen's d = (M2 - M1) ⁄SDpooled). The effect size was evaluated based on Cohen’s guidelines: small (0.2), medium (0.5), and large (0.8). The lower 95% CI of the mean and the upper 95% CI of the mean for both groups were added to the figure legend.