Nowadays, athletes use supplements to enhance muscle strength and endurance. A group of sport supplements that are good candidates for antioxidant therapy are flavonoids. Flavonoids are a class of polyphenolic compounds available in the fruits and vegetables. Epidemiological studies showed that the risk of cardiovascular disease in subjects, who had a high intake of flavonoids, has been reduced. ¹ The most prominent flavonoids in fruits and vegetables are flavonols, and, of these, quercetin is the most commonly consumed in the human diet. ² Quercetin is claimed to exert many positive effects on health, including protection against various diseases such as osteoporosis, lung cancer, and cardiovascular disease. Especially the ability of quercetin, to reduce reactive species such as peroxynitrite and hydroxyl radical, is proposed to be involved in these sorts of positive effects on health. Therefore, numerous studies have been conducted on the possible pharmacological aspects of this flavonoid. ² Several in vitro researches confirmed the antioxidant effects of quercetin. ²,³,⁴ Contradictory, the studies with human subjects showed little or no effect on inflammatory and oxidative stress biomarkers.- ⁵,⁶,⁷ Particularly, in many human studies on athletes, a significant effect of quercetin supplementation was not observed in the measurement parameters such as F2-isoprostanes, antioxidant capacity, and CRP.- ³,⁸ But there are some other evidences that quercetin effects may be potentiated in the presence of certain pro-oxidants through mediating electron transfer from flavonoid quercetin and by preventing its oxidative degradation. ² For example in animal studies quercetin co-ingestion with omega-3 long-chain fatty acids, vitamin C, or vitamin E resulted in anti-inflammatory and antioxidative effects. ⁹,¹⁰ Therefore, due to the failure of quercetin alone to reduce inflammation, and reactive oxygen species in many human reports, especially those on athletes with aim of increasing their physical performance, ¹¹,¹² and confirming data on co-operative activities of quercetin with ascorbate in enhancing quercetin biological activities such as its antiviral effects or reducing the increased cutaneous vascular permeability occurring in conditions of experimentally induced inflammation, ¹³,¹⁴ this research was conducted to explore whether the efficacy of the co-ingestion of quercetin with vitamin C, could improves its anti-inflammatory and anti-oxidative stress activity in athletes with regular exercise training.

Subjects

This randomized, double-blind placebo-controlled clinical trial was conducted among 60 men physical education students volunteered for this investigation. The study was approved by the Ethics Committee of Isfahan University of Medical Sciences. This study was registered in the Iranian Registry of Clinical Trials ( www.irct.ir ) on December 23, 2011 with IRCT registration number: 01112055062N4. Healthy subjects who were fit and physically active, performing a continuous graded exercise test (GXT) on a HP cosmos treadmill (Mercury, Germany) prior to and following the intervention, with no disease and medication use were included in the study. After laboratory tests, subjects with primary abnormal tests (CRP> 3) and any drugs reactions during the study were excluded from the study. Participants were supplemented orally for 8 weeks with quercetin (Solaray, USA), vitamin C (Razi, Iran), or placebo (Pharmacy Faculty, Isfahan University of Medical Sciences, Iran). Sixty subjects were randomized using permuted block randomization to one of four groups, each containing 15 individuals: 500 mg quercetin + 250 mg vitamin C as pro-oxidant (Q+C), 500 mg of quercetin alone (Q), 250 mg of vitamin C alone (C), or placebo (Control). Written informed consent was obtained from each subject. During the study, subjects agreed to avoid the use of vitamin/mineral supplements, nutritional supplements, herbs, and drugs known to affect immune function for 3 weeks before and during the 8 weeks of study. Before and after the supplementation period, participants performed their normal exercise. The following information was also reported by each subject: diet, physical activity, power status, and gastrointestinal side effects, skin or other.

Collection and handling of blood samples

A total of 15 mL of blood was drawn fasting period before and after supplementation, and serum samples were stored at -70° C until analysis. C-reactive protein was measured by enzyme immunoturbidometric assay (Randox laboratory Ltd, Belfast, United Kingdom) and IL-6, E-selectin F2-isoprostane was determined with the solid-phase enzyme-linked immunosorbent assay kits (Bender MedSystems GmbH, Vienna, Austria).

Statistical analysis

The results are presented as mean±standard error. One-way multivariate analysis of covariance (MANCOVA) controlling for the pretest differences, followed by Dunnett′s posthoc comparison was used for multiple between group comparisons. Within group comparisons were done using paired samples t-test. Due to non-normality of the studied variables (positive skewed distribution) logarithmic transformation was done and homogeneity of covariance matrix has been tested via Box′M statistics. Analyses were performed with the SPSS version 16 (SPSS Inc, Chicago, IL) statistical package.

General mean ± SD for all study sample for age (years), weight (kg), and body mass index (BMI, kg/m²) was (21.0 ± 1.6), (67.5 ± 10.8) and (22.3 ± 3.3), respectively. Table 1 presents these variables for each studied groups. There are no statistically significant differences between groups in terms of basic characteristics. {Table 1}

Pre- and postchanges in CRP for group 1 and control was statistically significant at P<0.01 while for group 3 at P<0.1. Within group differences in interleukin 6 and F2-isoprostane only for group 1 was marginally significant (P<0.1). Also, pre- and postchanges in E-selectin was marginally significant for all study groups (P<0.1).

MANCOVA result showed that there was marginally statistical between group differences (Wilk′s λ= 0.261, F = 2.22; P<0.1). Group 1 had marginally smaller F2-isoprostane (P<0.1) and interleukin 6, than control group (P<0.05), and there were marginally differences in CRP between respondents in group 1, and 2 with the control group (P<0.1). There were no differences in E-selectin between active treatment groups and control group Table 2.{Table 2}

In this research in the supplemented athletes, the CRP and IL-6 levels in Q+C group was lower than placebo, quercetin alone and vitamin C Figure 1a and 1b. In a similar study in a two-week supplementation with quercetin + omega-3 fatty acids on trained cyclists after three day heavy exercise, quercetin-omega-3 supplement reduced inflammatory biomarkers (in particular CRP and IL-6) versus placebo, ¹⁵ while quercetin alone compared with placebo did not alter exercise-induced inflammatory markers in other studies.- ¹¹,¹² Figure 1

Estimated marginal means of concentrations of inflammatory and oxidative stress markers before and after ingestion of quercetin + vitamin C (Q+C), quercetin (Q), vitamin C (C), and control group