The effect of Opuntia ficus‐indica juice supplementation on oxidative stress, cardiovascular parameters, and biochemical markers following yo‐yo Intermittent recovery test

Abstract The aim of the study was to investigate the effect of a flavonoid‐rich fresh fruit juice on cardiovascular, oxidative stress, and biochemical parameters during the yo‐yo intermittent recovery test (YYIRT). Twenty‐two healthy males subjects participated in this study divided into two groups: An experimental group (EG: n = 11) who consumed the antioxidant supplement and a control group (CG: n = 11). All participants performed two test sessions at 07:00 hr before and after 2 weeks of supplementation with Opuntia ficus‐indica juice. Blood samples were taken before (P1) and immediately (P2) after the YYIRT. Our results showed that following the 2,2‐diphenyl‐1‐ picrylhydrazyl (DPPH ●) test, the Opuntia ficus‐indica juice has an antioxidant capacity for capturing free radicals (p < .05) and reducing oxidative stress related to exercise. Concerning biochemical and cardiovascular parameters, our results showed a significant increase on total cholesterol (TC) (p < .01), triglycerides (TG) (p < .05), high‐density lipoprotein (HDL) (p < .01), low‐density lipoprotein (LDL) (p < .01), creatine kinase (CK) (p < .01), lactate deshydrogenase (LDH) (p < .01), glucose (GLC) (p < .01), systolic (SBP), and diastolic blood pressure (DBP) (p < .01) immediately after exercise. However, TC (p < .05), TG (p < .05) and LDL (p < .05), the maximal heart rate (HRmax), the CK (p < .05), and LDH (p < .01) as well as the malondialdehyde (MDA) (p < .01) demonstrated a significant decrease after supplementation of Opuntia ficus‐indica juice before and immediately after YYIRT. However, no significant effect on HDL (p > .05), GLC (p > .05) levels nor the SBP and DBP (p > .05) was observed after supplementation with Opuntia ficus‐indica juice. The supplementation leads to an improvement on YYIRT performance (The total distance covered during the YYIRT,VO 2max, VMA) and the rating of perceived exertion (RPE). Opuntia ficus‐indica juice has a potent antioxidant activity that reduces total and LDL‐cholesterol with only a moderate lowering of HDL‐cholesterol and oxidative stress. Moreover, supplementation decreases muscle damage caused by the endurance exercise.


investigated the antioxidant activity of
Opuntia ficus-indica juice, by the DPPH • test. The juice from purple color has higher antioxidant activity that those from fruits of yelloworange markers of stress color (Maataoui et al., 2006). Strenuous exercise was reported to increase the generation of reactive oxygen species (ROS) by cells, which may lead to cellular damage (Jówko, Długołęcka, Makaruk, & Cieśliński, 2015). These ROS are considered as a potent oxidation stress indicator in biological systems (El Abed et al., 2011;Wang, Wu, Wu, & Wei, 2013). Hammouda, Chaouachi, Ferchichi, Kallel, & Souissi (2012)) proved a significant increase in blood lactate and an increase in biochemical markers of muscle damage (i.e., creatine kinase (CK) and LDH) and lipid profile during YYIRT.
Although, research suggests that antioxidant supplementation has a potent role in postexercise recovery in athletes (Jówko et al., 2015).
Overall, the best performance is generally observed in the afternoon. Thus, studies in the domain continue to find strategies to counteract this decrease of performance in the morning.
Thus, 2-week or 4-week of antioxidant supplementation is recommended after acute exercise in order to delay muscle recovery (Jówko et al., 2015;Knab et al., 2013). However, these findings concerning the training adaptation after antioxidant supplementation was not confirmed by other studies (Higashida, Kim, Higuchi, Holloszy, & Han, 2011;Yfanti et al., 2010). In this context, the supplement of antioxidants pre-exercise has been recently a topic of current debate and maybe should be re-evaluated (Knab et al., 2013). These studies did not take into consideration the impact of natural antioxidant fruit supplementation, especially on Opuntia ficus-indica juice.
But one study mentioned in the literature in vivo. It is of Deldicque et al. (2013) who demonstrated that chronic Opuntia ficus-indica supplementation (i.e., specific extract fruit: capsule) could be an important nutritional strategy for muscle glycogen resynthesis post-exercise in healthy subjects. No study found in the literature that looks into the impact of an Opuntia ficus-indica supplementation on cardiovascular parameters and bio markers of oxidative stress after physical exercise.
It is critical therefore to investigate the outcome of 2 weeks supplementation with Opuntia ficus-indica juice on cardiovascular parameters oxidative stress, and biochemical markers of muscle damage following endurance exercise. The aim of this study was to evaluate the effect of 2-week Opuntia ficus-indica juice supplementation on biochemical parameters, oxidative stress markers, muscle damage, and glucose (GLC) in healthy subjects. We hypothesized that Opuntia ficus-indica juice has an antioxidant action and its supplementation improves performance following the YYIRT with the modification of the antioxidant profile.

| Participants
Twenty two healthy male athletes (Table 1) volunteered for the study participated in this study. In randomized order, participants were divided into two groups; an experimental group (EG: n = 11) and control group (CG: n = 11). After receiving a detailed explanation of the protocol, they gave written consent to participate in this study. This study protocol was in agreement with the Helsinki Declaration for human experimentation and was approved by the university ethics committee. The participants were also selected based on their chronotype and on the basis of their answers to self-assessment questionnaire of Horne & Ostberg (1975). They had an intermediate chronotype (i.e., sleep duration between 23:00 ± 1:00 and 07:00 ± 1:00 hr) and kept standard times for eating prior to the beginning of the study (Abedelmalek, Chtourou, Souissi, & Tabka, 2015).
The exclusion criteria of this study were the use of tobacco products, alcohol consumption, sleep disorder, and use of any dietary supplements in 2 weeks prior to the study. Additionally, subjects suffering

| Experimental design
The experimental procedure is illustrated in Figure 1

| Dietary intake
The participants were asked to keep a similar diet for the treatment periods except refrain the use of any antioxidant products. Subjects filled out a 24-hr dietary record during this period. The total dietary intake (i.e., vitamins, macro nutrient, and energy) was estimated with a dedicated picture book and calculated, using the NUTRISOFT BILNUT (Version 2.01 Paris, France) (based on national food tables) (Kunachowicz, Nadolna, Przygoda, & Iwanow, 2005).

| Antiradical capacity by DPPH •
The tests, using 2, 2-diphenyl-1-picrylhydrazyl (DPPH • ) radical are the most of the methods applied in the determination of antiradical activity. One of the reasons is that this method is simple and sensible. DPPH • assays are based along the same rule as that described by Brand-Williams, Cuvelier, & Berset (1995), but the analytic protocols differ in more parameters (i.e., absorbance, reaction time, the reference solution). DPPH • , a purple-colored free radical, was decreased into the yellow-colored diphenylpicryl hydrazine. The method used in our study on the Opuntia ficus-indica juice is that described by Tuberoso, Kowalczyk, Sarritzu, & Cabras (2007). An ethanolic solution (2.5 mg/100 ml) of the stable DPPH • radical was prepared and applied for the assay. Opuntia ficus-indica juice is dissolved in 10 ml of ethanol and it was placed into test tubes. Then, it was added to samples at 250 μl of the ethanolic solution. The test tubes were incubated for 30 min, and the optical density was read at 520 NM against the ethanol in a UV spectrometer (Genesys-10, Madison, USA). The radical scavenging activity was estimated utilizing the following recipe:

| Sample and treatments
Opuntia ficus-indica fruit with purple color was selected from a local market in Tunisia in the springtime of 2013. Only fruit without external injuries were chosen, washed, and peeled manually. To extract the juice, the pulp was pressed using an industrial mixer (Kenwood, 700W, China) and was then drawn through a strainer to remove seed enough to get the maximum of Juice. During 2 weeks of treatment, Opuntia ficus-indica juice supplementation, subjects were asked to receive of 150 ml/daily.

| The yo-yo intermittent recovery test level-1
According to the procedures suggested by Krustrup et al. (2003), the YYIRT was performed. It consisted of 20-m shuttle runs performed at increasing velocities with 10 s of active recovery at a distance of 5-m until exhaustion. The goal of the trial was seen when the participant twice failed to hit the front line in time or he felt unable to make out another shuttle. The total distance covered during the YYIRT was considered as the test score.

| Rate of perceived exertion (RPE)
Before performing exercise testing, the RPE (Borg, 1982) scales were explained to each participant. The scale presents a 15-point scale ranging from 6 (very, very light) to 20 (very, very hard). Thereby, during exercise testing, overall RPE, and physical stress was assessed.
RPE is considered as a method of measure of training in response to different modes of exercise (Impellizzeri, Rampinini, Coutts, Sassi, & Marcora, 2004).

| Blood samples and analyses
Blood samples were taken in the antecubital vein, sitting, before (P1), and 3 min after (P2) completing the YYIRT during two experimental conditions (i.e., before supplementation and after supplementation) (Hammouda et al., 2012;Jówko et al., 2015). The test venous blood samples were drawn into heparinized and EDTA test tubes (4 ml), centrifuged (for 20 min at 3000 RPM) and frozen, and stored at -80 C until analysis of selected blood markers.  To 800 μl of 0.4% (w/v) TBA was added 200 μl of the supernatant and all mix was incubated in a shaking water bath at 90°C for 10 min and then put directly into ice water. The concentration of MDA (determined at 532 NM) was expressed in micro mole per liter of plasma.

| Statistical analysis
Statistical tests were processed using STATISTICA Software (StatSoft, France). Data were reported as mean ± ES. Once the normality was confirmed, using the Shapiro-Wilk-W-test parametric tests were performed. YYIRT parameter (i.e., VO 2max , distance, HR and the RPE scores) were analyzed using a two-way ANOVA with repeated measures (2 (group) × 2 (conditions)). Biochemical parameters (i.e., total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, LDH, CK, GLC), blood pressure and MDA data were studied utilizing a threeway ANOVA with repeated measures (2 (group) × 2 (conditions) × 2 (points of measurement)). The Bonferroni post hoc test was performed whenever significant effects or a significant interaction was found using ANOVA. Anthropometric parameters were examined by a paired Student's t-test. To assess the data practical significance, effect Size was calculated as partial eta-squared, (η p 2 ). The level of statistical significance was set at p < .05.

| The anti-radical activity of Opuntia ficus-indica juice
The t student test for independent samples showed a significant dose- Thus, the Opuntia ficus-indica introduced an anti-radical activity significantly higher compared to the ethanol solution ( Figure 2).

| The YYIRT performances
The analysis of variance ANOVA revealed a significant group effect (F = 6.36; η 2 p = 0.54; p < .05) and a significant condition effect (F = 6.59; η 2 p = 0.59; p < .05) for the total distance traversed during the YYIRT and, VO 2max and VMA estimated. However, no significant interaction (F = 2.83; η 2 p = 0.04; p > .05) was observed between these two factors (group × conditions). The post hoc analysis demonstrated that the Opuntia ficus-indica juice supplementation leads to an improvement for the VO 2max , VMA and the total distance covered during the YYIRT for EG (p < .05) ( Table 2).

| The RPE scores
Statistical analysis showed a significant group effect (F = 8.391, p < .05) and a condition effect (F = 15.92; p < .01) for the RPE scales.
However, the interaction between these two factors (group × conditions) was not significant (p > .05).
The post hoc analysis revealed that the RPE scales registered following the YYIRT present a significant decrease after Opuntia ficusindica juice supplementation for EG.

| The cardiovascular parameters
The cardiovascular parameters are presented in Table 3. The ANOVA revealed that the supplementation was associated with an increase in the HRmax (F = 9.92; η 2 p = 0.65; p < .05) with no significant group effect (F = 3.36; η 2 p = 0.04; p > .05). Likewise, there was a significant interaction (group × conditions) (F = 6.14; η 2 p = 0.49; p < .05). Concerning point of measurement (before and after YYIRT), the analysis revealed a significant effect for DBP (F = 45.14; η 2 p = 0.78; p < .01) and SBP (F = 114.31; η 2 p = 0.52; p < .01). However, no significant effect neither group, nor supplementation and no significant interaction (group × conditions × points of measurement) was observed for SBP or DBP (p > .05). The post hoc analysis showed that DBP and SBP values registered present a significant increase after YYIRT compared with baseline for both EG and CG.

| The biochemical markers
The biochemical markers are presented in the Table 4

| DISCUSSION
Our results indicated that following the DPPH • test the Opuntia ficus-indica juice has an antioxidant capacity for catching free radicals. Several studies showed that Opuntia ficus-indica juice has a more important antioxidant capacity than other fruits (Seeram et al., 2008;Yahia & Mondragon-Jacobo, 2011). These findings support our pick of the variety studied. Cactus pear contains compounds with antiradical action (i.e., phenolics, flavonoids and pigment compounds) (Maataoui et al., 2006;Tesoriere et al., 2008). These compounds were found to possess a high potential for capturing free radicals. Consistent with previous studies, the evaluation of the antioxidant activity indicate T A B L E 3 Cardiovascular parameters (mean ± SE) measured Before (P1) and After (P2) the YYIRT, before and after supplementation EG CG Before supplementation that the Opuntia ficus-indica juice studied contain substances which are able to inhibit the action of free radicals as DPPH • (Maataoui et al., 2006;Tesoriere et al., 2008). The choice of the DPPH • method is in accordance with that of Fernandez-Lopez et al. (Bloomer & Cole, 2009).

Before supplementation
It is well accepted that the physical exercise is characterized by an increment in the volume of oxygen consumed (Powers & Jackson, 2008) which leads to an increase in ROS production and causes oxidative stress and muscle damage (Hammouda et al., 2012). As a result, an elevation of LDH and CK levels will be observed after prolonged exercise. Likewise, Baird, Graham, Baker, &Bickerstaff (2012) andCeci et al. (2014) showed that acute exercise provokes micro-lesions in the active muscle. This damage causes an increase in plasma concentrations of CK and LDH which leads to the decrease of the performance (Brancaccio, Lippi, & Maffulli, 2010). Indeed, the increase of CK rate indicates that the athlete is more vulnerable to injury and muscle damage caused by an intense physical activity. Our results illustrate that CK levels are significantly higher P2 above P1, before supplementation for EG as well as CG. Similarly, our findings showed that plasma concentrations of LDH recorded on P2 are significantly higher than those at P1 in EG and CG. Indeed, the study of Bloomer & Cole (2009)  Regarding blood pressure, our results indicate that the values of DBP and SBP recorded following the YYIRT are significantly higher than those at rest for the EG as well as CG.
Concerning supplementation, Opuntia ficus-indica juice leads to an improvement on YYIRT performance (i.e., VO 2max , VMA). In concordance with previous research (Hammouda et al., 2013), it seems that the enhancement of the YYIRT performance presented in this study is essentially due to the decrease of muscle damage and the rating of perceived exertion (RPE Nevertheless, no significant difference was observed in CG. These data coincide with those found by Schmitt, Fouillot, Nicolet, & Midol (2008) who recorded a decrease in HR (−9 beats/min) for elite athletes after taking an extract of Opuntia ficus-indica (5 g/kg body weight). Likewise, the supplementation of Opuntia ficus-indica juice has no effect on the DBP or in the SBP. In this context, few studies in the field have focused on the measurement of blood pressure after antioxidant supplementation (Champagne, 2006;Saneei, Salehi-Abargouei, Esmaillzadeh, & Azadbakht, 2014 This supplement decreases the oxidative damage and improves the antioxidant status. In this context, the study of Jówko et al. (2011) found that dietary with antioxidant supplementation (green tea extract) could enhance antioxidant status and reduce tissue damage from oxidative stress occurred during the exercise. The production of free radicals in response to physical exercise is an important source of muscle damage. Thus, to protect themselves from oxidative stress and maintaining the redox balance in the most difficult periods of a sports season in mixed disciplines (aerobic, anaerobic), the athlete must increase the nutritional content of antioxidants to neutralize free radicals (Jówko et al., 2011;Teixeira, Valente, Casal, Marques, & Moreira, 2009).
Concerning HDL-cholesterol concentrations, our results revealed a significant increase after YYIRT in both EG and CG. Similarly, the TC and TG levels after supplementation is significantly lower than before supplementation in the EG. However, no significant difference was observed in CG. According to Wolfram, Kritz, Efthimiou, Stomatopoulos, & Sinzinger (2002), consumption of a variety of the Opuntia ficus-indica for 8 weeks causes a decrease in TC (12%), LDL cholesterol (15%), the apolipoprotein B (9%), TG (12%), GLC (11%), and insulin (11%).
Another study showed that Opuntia ficus-indica reduced cholesterol levels in human blood and altered the composition of LDL (Stintzing & Carle, 2005).
For GLC values, our data indicated that the two groups behaved in the same manner. Indeed, these concentrations increased significantly with YYIRT for the EG and even for CG before as well as after supplementation. Cicero, Derosa, & Gaddi (2004) showed that the Opuntia ficus-indica supplements can also be effective in reducing GLC levels. In the same way, the work of Feugang et al. (2006), showed that Opuntia ficus-indica contain fiber and pectin, which can reduce the level of GLC. Likewise, Trejo-González et al. (1996) demonstrated the hypoglycemic activity of the extracts of Opuntia ficus-indica on diabetic individuals.

| Limits of study
This study has some obvious limitations. The first concerns samples taken. It will be better that we make a belated sample to know the impact of Opuntia ficus-indica juice supplementation on recovery. In addition, the ideal would be the addition of another group; a placebo group or group who receives another antioxidant supplementation.
One more limitation is that, in the present work, we did not carry away the effect of Opuntia ficus-indica supplementation in the recovery period. No women were available for the study. Another limitation about methods to access antioxidant activity (FRAP, ABTS). Future studies should highlight other markers of oxidative stress (i.e., carbonated protein, glutathione, total antioxidant status etc.).

| CONCLUSION
In conclusion, we conclude that 14 days of supplementation with Opuntia ficus-indica juice has a potent antioxidant activity makes it possible to reduce total and LDL-cholesterol with only a moderate lowering of HDL-cholesterol and oxidative stress. Moreover, supplementation decreases muscle damage caused by the endurance exercise.