Ten healthy female subjects were recruited for this study by the sporting center of Palermo university (CUS Palermo). Informed consent was mandatory to participate to this study. The institutional Review Board of Palermo University approved the research protocol (Comitato Etico Palermo 1, N. 04, 2018) and all procedures were carried out in accordance with the Declaration of Helsinki (2000). Before data collection, the recruited participant had to fill an anamnesis questionnaire to exclude smokers, those taking medications (including contraceptive pills) or nutritional supplements, or had recent musculoskeletal injures. Moreover, the participants had to report if they were amenorrhoeic. During the first assessment procedures, the participants had to be in the luteal phase of their menstrual cycle, since this seems to correspond to the lowest physiological ROS production period in women . All participants were considered active. Each participant regularly practiced resistance training 3 to 4 times per week. Of the ten subjects who began the study, two did not complete it and were excluded from the final analysis. The characteristics of the remaining eight participants were 23.25 ± 2.95 years of age, 54.13 ± 9.05 kg, 157.75 ± 0.66 cm with a BMI of 21.69 ± 0.66 kg/m2.
This is a randomized, double-blind, placebo controlled and crossover design (Fig. 1) study. The participants were randomly divided into two groups, designated as OFI and Placebo. The OFI group received 50 ml of cold-concentrated juice from peeled fresh fruits of Sicilian Opuntia Ficus Indica (NopalRed, Nopal Company srl, Biancavilla, CT, Italy), diluted with water to 170 ml; the Placebo group received 170 ml of a beverage containing the same ingredients of the OFI juice as reported in the commercial preparation, except for the antioxidant Vitamin C and the bioactive phytochemical indicaxanthin. Each participant was instructed to consume either the OFI or Placebo juice along with their breakfast, every day for 3 days before the maximal effort test and continue to take it for 2 consecutive days after the second testing procedure. The supplementation also occurred in the testing days. One week before starting the supplementation, all participants were tested (see following sections) in order to determine a baseline measure. In this occasion, covered bottles of OFI or Placebo were distributed to all participants who were instructed about the procedure of supplement intake.
Each variable of interest was collected following specific time points: 1) baseline (BL), 2) pre-test, 3) post-test, 4) 24-h and 5) 48-h after the end of the effort test.
Following a wash out period of 15 days, the supplementation order was reversed and the same procedures as above described, were repeated (Fig. 1). Before the testing sessions, the participants completed a 7-day food diary, from which emerged that their caloric and nutrient ingestion (1881 ± 123 Kcal/die composed of 58% carbohydrate of which 10% sugars, 27% lipids and 15% proteins) was in accordance with the daily intake levels of nutrients recommended for the Italian population . Fruit intake was limited to the OFI juice during the supplementation periods. All participants were invited to follow the same dietary plan 1 week before the testing session in order to limit the influence of nutrition on the analysed parameters.
Plasma hydroperoxides concentration, antioxidant capacity, skin carotenoid score and heart rate variability were collected at baseline (BL), day 3 (pre-test and post-test), day 4 (24 h post-test) and day 5 (48 h post-test) of each period. *Indicates the assessment points.
Cardio-pulmonary maximal effort test evaluation
All tests were performed between 8 and 10 a.m., after a 12-h overnight fast. Stature and body mass were measured at baseline using a stadiometer and an electronic weighing scale, respectively (SECA, Germany). All participants performed a graded exercise test to exhaustion, on a cycle ergometer. Each participant cycled for 4 min at a constant work rate of 50 W. Thereafter, the cycle ergometer work rate increased by 25 W ∙ min-1 until the participant reached her limit of tolerance. The pedal rate was maintained at 60 rpm throughout the test. A disposable facemask with a 50–70 ml dead space (Cosmed V2, Cosmed Srl, Italy) was used to collect exhaled air throughout the test. Oxygen uptake (VO2) was recorded with a breath-by-breath measurement system (Cosmed Quark CPET, Cosmed Srl, Italy) and maximal oxygen uptake (VO2max) was defined as the highest consecutive 30 s averaged value achieved during the test. The flow meter and gas analysers were calibrated before each test, according to the manufacturer’s instructions. Heart rate was accurately recorded using a short-range radio telemetry system (Polar Electro Oy, Finland). At test cessation an active recovery period was performed at constant lode on the cycle ergometer, pedaling at 25 W at 40 rpm for 3 min, followed by a 2 min passive rest. HR and VO2 were monitored throughout exercise, recovery time and rest. The test was stopped when one or more of the following criteria were met: 1) attainment of a VO2max plateau < 2.2 ml ∙ Kg-1 ∙ min-1; 2) participants were unable to maintain the required work rate.
Measurement of hydroperoxides concentration and plasma antioxidant capacity
Oxidative stress evaluation was performed with a portable integrated analytical system composed of a photometer and a mini-centrifuge (FRAS 4 Evolvo; H&D Srl, Parma, Italy) as indicated in the study design section. Samples of whole capillary blood were centrifuged for 1:30 min immediately after being collected with a finger puncture, and 10 μL of plasma was used for measuring the concentration of hydroperoxides (using d-ROMs testing) and antioxidant capacity (using PAT testing). The test of active oxygen metabolites (d-ROMs; Diacron International Srl, Grosseto, Italy) measures increases in red color intensity after the addition of a small quantity of plasma to a solution of N,N-diethylparaphenylendiamine (chromogen), buffered to pH 4.8. Such coloring is attributed to the cation radical formation of the amines via oxidation, which is due to alkoxyl and peroxyl radicals. The latter derive from the reaction of the Fe2+ and Fe3+ ions released by proteins in acidic conditions, as created in vitro. The results are expressed as Units Carratelli (U. CARR), and it has been experimentally established that 1 U. CARR corresponds to 0.08 mg/dL H2O2. The normal values of a d-ROMs test range from 250 U to 300 U. CARR (i.e., between 20 and 24 mg/dL H2O2). The plasma antioxidant test (PAT; Diacron International Srl) measures the capacity of a plasma sample to reduce the iron of a colored complex containing ferric ions to its colorless ferrous derivative. The chromatic change of this reaction was photometrically measured at 505 nm, and the results were expressed in U. Cor, in which 1 U. Cor corresponds to 1.4 μmol/L of ascorbic acid used as a standard for reducing the iron. The normal value of a PAT test is > 2200 U. Cor. To maintain consistency, the same set of kits was used for all assessments, and the same operator using the same calibrated machine carried out the evaluations.
Measurement of skin carotenoid score
Skin carotenoid score (SCS) was measured using a portable device (Pharmanex BioPhotonic Scanner S3, NuSkin, Provo, Utah, USA) based on the Resonance Raman Spectroscopy (RRS). All participants placed the palm of their hand against a light window of the device, twice and held it there for 90 s. During the warm-up process, a black calibration cap was placed over the light window and the scanners self-calibrated using a patented process. When a low intensity laser monochromatic light interacts with some molecules, these diffuse the light emitting a new higher wavelength, monochromatic light that can be revealed by a scanner converting Raman intensity in counts. Because of their conjugated carbon backbone molecular structure, carotenoids possess characteristic vibrational/rotational energy levels that make them particularly well suited for RRS, strongly absorbing in the blue wavelength region and emitting in the green region. The green light emitted from the skin is captured by a highly sensitive light detector. The quantity of dermal carotenoids revealed by RRS can be considered as a marker of the individual antioxidative network and its measurement was applied to assess the body redox state of each participant . Scanner signals are visualized as a coloured scale going red (poor carotenoid score, < 19,000) to dark blue (high carotenoid score, > 50,000). When Raman intensity count difference was 2000 score between scanner scores, participants were scanned a third time. This was done to minimize the individual variation in the scanner score.
Analysis of heart rate variability and recovery
HRV parameters were collected at baseline, immediately after the maximal graded test, 24 h and 48 h after the end of maximal effort test. Heart rate recovery was measured at 2 min (HRR2) after the end of the test. These measurements were performed with the subjects in a supine position on an examination table for 10 min, during which RR interval recordings were acquired using a portable heart rate monitor (Polar V800, Polar, Finland). The last 5 min of the RR recording were analysed by means of Kubios HRV 2.2 software . HRV time and frequency domains were retrieved . The mean squared differences of successive RR intervals (rMSSD) were retained for the time domain, while the very low frequency (VLF < 0.04 Hz), low frequency (LF from 0.04 to 0.15 Hz) and high frequency (HF from 0.15 to 0.40 Hz) components, in absolute (ms2) and in normalized units (nu) [LFnu: 100 x LF / (total power – VLF), and HFnu: 100 x HF / (total power – VLF)] were retained for the frequency domain. From the values of LF and HF, the LF/HF ratio was determined . The validity of the HRV procedure derived from V800 Polar heart rate monitor is reported elsewhere .
Data are reported as means and standard deviations. Normal distribution of the data was verified through the Shapiro-Wilk test. A power analysis was calculated through G*Power v18.104.22.168 (power 0.80; α 0.05; effect size 0.5) estimating a sample of 8 participants. A two-way ANOVA for repeated measures (2 treatments × 4 time-measurements) was used to compare all parameters and identify the source of variation and time-treatment interactions. Sidak’s correction was conducted to compare the OFI and Placebo groups. Furthermore, a one-way within participant’s analysis of variance (ANOVA) was conducted to check for differences in-between times (Pre-test, Post-test, 24 h-post-test, 48 h-post-test). Post-hoc analysis with the Tukey’s test was also perform to detect differences between groups. Effect size using Cohen’s d, for time, treatment and time x treatment interactions have been also reported. Level of statistical significance was set at a p-value < 0.05. All analyses were performed with Jamovi (The jamovi project (2021). jamovi (Version 1.6) [Computer Software]. Retrieved from https://www.jamovi.org).