Effect of propolis supplementation on athletic performance, body composition, inflammation, and oxidative stress following intense exercise: A triple‐blind randomized clinical trial

Abstract Background Emerging evidence indicates that propolis as a novel potential antioxidant has unique benefits. This study aimed to evaluate the effect of propolis on oxidative stress, inflammation, body composition, and athletic performance in healthy active subjects. Methods This clinical trial was conducted on 54 male military cadets. Eligible subjects were randomly allocated to receive a single dose of 450 mg propolis twice daily for four weeks or a matching placebo containing microcrystalline cellulose. Cooper 12‐min run test and running‐based anaerobic sprint test were performed to measure aerobic and anaerobic performance. Blood samples were obtained immediately after Cooper's test to evaluate oxidative stress and inflammation status. Fat mass and fat‐free mass were analyzed using bioelectrical impedance. Results Mean changes in fat mass, fat‐free mass, anaerobic powers, fatigue index, and VO2 max did not differ significantly between the two groups after the adjustment for baseline values (P‐value>0.05). A significant change was observed in plasma levels of IL‐6 (−1.43 ± 0.11pg/mL), total oxidant status (−3.9 ± 0.2µmol/L), total antioxidant capacity (164 ± 12 µmol/L), malondialdehyde (−0.52 ± 0.03µmol/L), oxidative stress index (−0.45 ± 0.04), and glutathione (48.72±2µmol/L) in the propolis group compared with the placebo group after the adjustment for baseline values and weight changes (P‐value<0.05). Although IL‐10 concentrations had no significant changes in both groups, the ratio of IL‐6/IL‐10 significantly reduced in the propolis group compared with the placebo group (−0.174 ± 0.015 versus. 0.051 ± 0.014; P‐value: 0.041). Conclusions Our results indicated that propolis might have beneficial effects on oxidative stress and inflammation following intense activities in healthy male subjects.


| INTRODUC TI ON
Intense physical activities can increase oxygen consumption in the active muscles 10-15 times more than in the resting state (Joyner & Case y, 2015). The rise in oxygen delivery to active skeletal muscles is required to produce adenosine triphosphate (ATP) through the electron transport chain (ETC) to continue the activities (Miyazaki et al., 2001). The ETC is located in the inner mitochondrial membrane, which is not only the principal site of ATP production in skeletal muscles but also a potential source of reactive oxygen species (ROS) production (Leeuwenburgh & Heinecke, 2001). In addition to ETC, xanthine oxidase, lipoxygenase, phospholipase A 2 , myostatin, and catecholamines also contribute to ROS formation in contracting muscles (Powers & Jackson, 2008;Steinbacher & Eckl, 2015).
Some researchers have suggested that low ROS concentrations in skeletal muscles can increase the muscle's ability to produce force (Reid, 2001). However, what is certain is that ROS in high concentrations results in muscle force decline and fatigue (Powers et al., 2016).
In addition, overproduction of ROS following intense activates can cause irreversible damage to intracellular organelles, inflammation, and eventually muscle breakdown (Suzuki et al., 2020). The susceptibility of skeletal muscle to oxidative stress depends on the antioxidant defense system capability. This system comprises nonenzymatic antioxidants, such as glutathione (GSH), and enzymatic antioxidants such as superoxide dismutase (SOD) and catalase (CAT). Nutritional status influences the capacity and ability of the body's antioxidant defense to neutralize ROS (Chow, 1979). In addition, dietary antioxidants can modulate the immune response and inflammatory pathways which can help the enhancement of physical performance and recovery from exercise in athletes (Mason et al., 2020).
Propolis is a sticky substance produced by honey bees (Apis mellifera L.) from buds and various plants' exudates. Numerous polyphenol compounds have been identified in propolis from different geographical regions of the world (Huang et al., 2014). Propolis has long been used as a popular therapeutic agent to promote the body's health and treat wounds and infections (Sforcin, 2016). Recent studies have shown the antioxidant, anti-inflammatory, and immunomodulatory activities of propolis from different parts of the world (Franchin et al., 2018;Wang et al., 2018).
Animal studies support that propolis and its derivatives can ameliorate exercise-induced damage through enhancing the antioxidant defense system and suppressing nuclear factor-kappa B (NF-κB) (Kwon et al., 2014;Shen et al., 2013). Quercetin, the predominant flavonoid compound identified in propolis (Zheng et al., 2017), has been shown to promote exercise performance (Davis et al., 2010) and muscle mitochondrial biogenesis (Islam et al., 2020). This evidence indicates that propolis might help athletes protect their muscles against exercise-induced oxidative and inflammatory damage and improve exercise performance. The current trial aimed to assess the effect of propolis supplementation on pro/anti-inflammatory cytokines, antioxidant/oxidant status, and anaerobic/aerobic endurance among active subjects.

| Study design and participants
This randomized, triple-blind, placebo-controlled clinical trial was designed to evaluate the effect of propolis supplementation on pro/ anti-inflammatory cytokines, antioxidant/oxidant status, and exercise performance. Participants were recruited from new male cadets entering AJA University of Medical Sciences in Tehran, Iran. A total of 107 young males were enrolled in the trial, of whom 54 eligible participants underwent randomization to assign either the propolis group or the placebo group ( Figure 1). The protocol, available at the Iranian Registry of Clinical Trials (IRCT20180824040857N2), was reviewed and approved by the ethics committee at the AJA University of Medical Sciences (IR.AJAUMS.REC.1399.107). Each participant was provided verbally with information on the objectives of the trial and its benefits and possible health risks at the time of enrollment. Each participant provided written informed consent. This trial was conducted in accordance with the principles of the Helsinki Declaration.

| Eligibility criteria
Cadets, who agreed to participate in this trial, were screened based on eligibility criteria. Inclusion criteria were male gender, age of 20-30 years, body mass index (BMI) of 18.5-25 kg/m 2 , a minimum of 6 hr/wk of sports activities in the last year, and written informed consent. Those who had a history of adverse reaction to bee products, musculoskeletal dysfunction, previous musculoskeletal injuries during training or exercise, heart diseases, diabetes mellitus, metabolic diseases, regular use of antioxidant supplements (e.g., vitamin E, vitamin C, β-carotene, selenium, and propolis) or antiinflammatory drugs in the last three months, or specific dietary regimen (e.g., vegetarian diets) and energy-restricted diets, in the last six months were not eligible to participate in the trial.

| Trial randomization and blinding
Cadets who met the eligibility criteria underwent randomization after the screening visit. Randomization sequences were generated using a random-number table and opaque, sealed, numbered envelopes. Randomization was stratified according to age (20-25 years versus. 26-30 years). Eligible participants were randomly assigned K E Y W O R D S athletic performance, body composition, inflammation, oxidative stress, propolis, VO 2 max in a 1:1 ratio with a blinded manner to the intervention or control group. Participants and investigators, except the trial pharmacist, were concealed from the study-group assignment until the end of the trial and data analyses.

| Intervention
Participants in the intervention group were assigned to take ethanolic-extracted poplar propolis at a single dose of 450 mg twice daily, before lunch and dinner, for 4 weeks. In contrast, those in the control group received a matching placebo containing microcrystalline cellulose. The dose of propolis was selected according to the previous phase ΙΙ trial (Zhao et al., 2016). Propolis extract was standardized based on total polyphenols and flavonoids content, according to Bankova's recommendation (Bankova, 2005). Each propolis tablet contained 180 mg polyphenols and 134 mg flavonoids. The Reyhan Naghsh Jahan Pharmaceutical Company manufactured both the propolis and matching placebo tablets under good manufacturing practice (GMP) conditions. Adherence was estimated by counting unused tablets. During the trial, all cadets were in the same place, had the same physical activity, ate the same meals, and slept the same number of hours. Participants were also followed by face-to-face visits every week. During the intervention period, participants who were unwilling to continue the trial were sensitive or unable to take the assigned intervention (<80%), or used other dietary supplements and drugs were excluded from the trial. Among 54 eligible participants, 49 completed the 4-week intervention period.
Two subjects in the propolis group and two subjects in the placebo group were withdrawn from the trial because of poor adherence to the trial-group assignment. Also, one in the propolis group discontinued the trial for a reason unrelated to the study ( Figure 1).

| Anthropometric assessment
Before and after the intervention period, body weight, fat mass (FM), and fat-free mass (FFM) were measured using the bioelectrical F I G U R E 1 Screening, Randomization, Treatment, and Follow-up impedance analysis (BIA) technique (Tania BC-418 machine, Tania Corp., Tokyo, Japan). All participants were asked to be well hydrated and to abstain from exercising and caffeine-containing products in the 6 hr and 24 hr before the BIA measure, respectively (Kyle et al., 2004). Height was measured using a portable stadiometer (Seca 213, Hamburg, Germany) without shoes in a standing position to the nearest 1 cm. Then, BMI was computed by dividing weight in kilograms by height in meters squared.

| Exercise performance assessment
Aerobic endurance was measured using the Cooper 12-min run test.
This test was designed by Dr. Ken Cooper to evaluate the aerobic capacity of the US military (Cooper, 1968). Cooper's test is a valid and reliable method of estimating the maximum oxygen uptake (VO 2 max) (Bandyopadhyay, 2015). Before and after the intervention period, participants conducted the 12 min of continuous running in the 400-m running track, and then, covered distance was used to calculate VO 2 max according to Cooper's equation as follows: We also used the running-based anaerobic sprint test (RAST) to measure anaerobic endurance before and after the intervention period. This test provides a valid and reliable method for estimating anaerobic power and fatigue index (Zagatto et al., 2009). The RAST test comprises six consecutive 35-m sprints with a rest time of 10 s between sprints. Participants conducted the RAST test, and time spent in each attempt was automatically recorded using an elec- In the familiarization session, all participants were informed about doing sports tests and were motivated to do them with maximal effort. They also were abstained from intense physical activities in the 24 hr before sports tests. All participants had a five-minute warm-up period of light aerobic activities prior to performing exercise tests and were verbally encouraged throughout performing exercise tests.

| Biochemical assessment
Heparinized blood samples were taken from the cubital vein immediately after Cooper's test. Then, samples were centrifuged at 3,000 rpm for 10 min at 4°C, and supernatants were used to assess inflammation, antioxidants, and oxidative stress. Plasma levels of the interleukin (IL) 6 and IL-10 were measured based on the biotin double antibody sandwich method using commercial enzyme-linked immunosorbent assay (ELISA) kits (Diaclone, Besancon, France).
Then, the ratio of the IL-6 and IL-10 was used to indicate the degree of inflammation status. Plasma levels of total antioxidants capacity (TAC), GSH, and total oxidant status (TOS) were measured based on the enzymatic colorimetric method using commercial kits (ZellBio GmbH, Ulm, Germany). The ratio of the TAC to TOS was used to compute the oxidative stress index (OSI) as follows (Buico et al., 2009): OSI =100 × (TOS/TAC). In addition, malondialdehyde (MDA) concentrations as an indicator of the degree of oxidative damage were assessed by the colorimetric reagent kit (ZellBio GmbH, Ulm, Germany). All tests were read using an ELISA reader (Stat Fax 2,100, Awareness Technology, Inc., USA).

| Statistical analysis
Data were analyzed using SPSS software, version 16 (SPSS Inc., Chicago, IL, USA). We estimated that a sample of 27 subjects in each study arm would provide a power of 80% to detect an effect size of 1.4 gr/L in GSH concentrations response to propolis intake (Zhao et al., 2016) and a withdrawal rate of 10% at a significance level of 5% (two-tailed). Kolmogorov-Smirnov test was used to examine the normal distribution of quantitative data. Within-group comparisons were done using paired Student's t test for normally distributed data and Wilcoxon rank-sum test for ordinal or nonnormally distributed data. Independent Student's t test for normally distributed data, Mann-Whitney U test for ordinal or non-normally distributed data, and the chi-square test or Fisher's exact test for nominal data were also used to ascertain between-group comparisons. We estimated the adjusted effects of propolis using analysis of Covariance (ANCOVA) test with baseline values as covariates.
P-values of less than 0.05 were considered to indicate statistically significant differences.

| RE SULTS
The participants had a mean age of 24.21 ± 2.09 years and a BMI of 23.52 ± 1.31 kg/m 2 . The mean age and BMI of participants were 24.21 ± 1.98 years and 23.82 ± 1.06 kg/m 2 in the propolis group and 24.20 ± 2.24 years and 23.22 ± 1.47 kg/m 2 in the placebo group.
There were no significant differences in age and BMI between the two groups (P-value >0.05). The mean compliance rate was 96.9% for propolis and 97.1% for placebo. Participants throughout the trial reported no adverse effects.
The adjusted mean changes in the anthropometric characteristics are shown in Table 1 The adjusted mean changes in the biochemical parameters measured immediately after Cooper's test are shown in Table 3.
The baseline values did not differ significantly between the two groups (P-value>0.05). The mean IL-6, IL-6/IL-10, MDA, and TOS significantly reduced, while GSH and TAC significantly increased in the propolis group at the end of the trial. But, these variables did not have significant changes in the placebo group.   waist circumference among patients with cardiometabolic risk factors (Mujica et al., 2017). Also, Zakerkish et al. showed that the daily intake of 1,000 mg/day of propolis for three months had no effects on weight in diabetic subjects (Zakerkish et al., 2019). Conversely, Samadi et al. reported that the administration of 900 mg/day of propolis in diabetic subjects over a period of 3 months reduced weight and BMI while did not affect waist circumference (Samadi et al., 2017). This controversy can be related to confounders' effect, such as changes in energy intake and physical activity throughout the study of Samadi et al.

| D ISCUSS I ON
Our trial shows that propolis supplementation improves TAC and GSH and reduces TOS, MDA, and OSI following intense exercise.
These concentrations in diabetic subjects (Hesami et al., 2019). Propolis contains high amounts of phenolic compounds which directly react with and quench free radicals. In addition, propolis and its derivatives have been shown to activate the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway, which mainly regulates the expression of a large battery of genes related to endogenous antioxidants (Jin et al., 2015). In this line, propolis has been shown in vivo to increase the levels of heme oxygenase-1 (HO-1), glutamate-cysteine ligase (GCL: the rate-limiting enzyme for the biosynthesis of GSH), and thioredoxin reductase 1 (TrxR1) (Zhang et al., 2015). The activation of the Nrf2 pathway not only up-regulates the GCL mRNA expression but also facilitates the cellular uptake of cystine as the rate-limiting precursor for the synthesis of GSH via enhancing the cystine/glutamate amino acid antiporter (Xc-system) (Correa et al., 2011).
Our trial shows that propolis supplementation reduces the IL-6 concentrations and IL-6/IL-10 ratio in plasma following intense exercise. Intense physical activities stimulate inflammatory cytokines, which attenuate muscle force production, immune response, and recovery times (Nemet et al., 2002). There are some inconsistencies regarding the effect of propolis on inflammatory cytokines in previous clinical trials. Zakerkish et al.'s study showed that propolis supplementation reduced TNFα while had no effect on IL-6 and IL-1β among diabetic subjects (Zakerkish et al., 2019). The study of Zhao et al. also reported that propolis reduced TNFα concentrations, whereas increased IL-1β and IL-6 levels among diabetic subjects (Zhao et al., 2016). Fukuda et al. found that propolis supplementation (226.8 mg/day for 8 weeks) had no effect on TNFα and IL-6 concentrations among diabetic subjects (Fukuda et al., 2015). Khayyal et al. found that propolis supplementation (260 mg/day for 8 weeks) reduced TNFα, intercellular adhesion molecule-1 (ICAM-1), IL-6, and IL-8 and increased IL-10 in asthmatic patients (Khayyal et al., 2003). Afsharpour et al. reported that propolis supplementation (1,500 mg/day for 8 weeks) reduced TNFα among diabetic subjects (Afsharpour et al., 2017). Taken together, a recent metaanalysis revealed a significant reduction in IL-6, TNFα, and highsensitivity C-reactive protein (hs-CRP) concentrations by 17.96 pg/ ml, 34.08 pg/ml, and 1.16 pg/ml, respectively, following propolis supplementation. Nonetheless, no significant reduction was reported in IL-1β concentrations with propolis consumption (Shang et al., 2020). Collectively, propolis may be a promising candidate to treat acute and chronic inflammatory diseases (Franchin et al., 2018;. Our results failed to show a significant improvement in aerobic and anaerobic endurance. Scientific evidence on the effect of antioxidants on exercise performance is limited and conflicting. In a 1-week randomized trial, Davis et al. found that the daily administration of 1,000 mg of quercetin, which is identified in propolis, increased VO 2 max (3.9%) and bike-ride times to fatigue (13.2%) among untrained subjects (Davis et al., 2010). In a 3-week randomized trial, Jourkesh et al. reported that a daily intake of 400 mg of vitamin E along with 1,000 mg of vitamin C improved VO 2 max. However, it had no significant effect on anaerobic power among male students (Jourkesh et al., 2007). Conversely, the administration of 1,000 mg/ day of vitamin C for 4 weeks had no significant effect on VO 2 max among recreationally active men (Roberts et al., 2011). Likewise, in another clinical trial, Paulsen et al. reported that the daily intake of 1,000 mg of vitamin C and 235 mg of vitamin E for 11 weeks did not affect VO 2 max on healthy subjects (Paulsen et al., 2014). Taken together, it seems that long-term antioxidant supplementation may attenuate its favorable effects on exercise performance. In this line, a body of evidence suggests that high levels of antioxidants in skeletal muscles can attenuate exercise adaptations and mitochondrial biogenesis by suppressing redox signaling pathways related to exercise-induced ROS production (Cobley et al., 2015;Strobel et al., 2011). Consistent with this hypothesis, it has been shown that ROS can modulate the force production in unfatigued skeletal muscles (Powers & Jackson, 2008).
The lack of access to an ergo-spirometer for direct evaluation of VO 2 max via a breath-by-breath gas analyzing system was a limitation of this study. However, we used the Cooper test to evaluate the aerobic capacity of the military (Cooper, 1968). This test is a valid and reliable method for the estimation of VO 2 max in the field (Bandyopadhyay, 2015). Another limitation of this study is that it was conducted only among men and the results may not be generalizable to women. This trial's strengths were the triple-blind design, high compliance rates, matched control, the similarity in terms of living place, physical activities, meals, and sleep duration among all participants, minimal drop-out rates, and adjustment for potential confounding factors.
In conclusion, our findings demonstrate that supplementation with propolis might have beneficial effects on oxidative stress and inflammation status following intense physical activities while not affecting athletic performance in healthy active subjects. Further studies are needed to ascertain the effect of propolis on exercise performance in trained and nontrained subjects.

ACK N OWLED G M ENTS
The authors would like to thank the AJA University of Medical Sciences for the financial support of this research. We also sincerely appreciate all those who participated in this study.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflict of interest.

E TH I C S A PPROVA L
The trial protocol was approved by the research ethics committee by the ethics committee at the AJA University of Medical Sciences (IR. AJAUMS.REC.1399.107). All patients provided written consent for participation in this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.