Modulatory role of rooibos on exercise-induced oxidative stress and performance while exploring influences on the human metabolome and effect of genetic variations

Abstract

Phase I - Establishment of an exercise-induced oxidative stress model for testing herbal interventions, such as Rooibos Background: Despite general clarity on the importance of oxidative stress to human health, it is widely acknowledged that this phenomenon is not yet well enough understood to allow for effective clinical manipulation. Oxidative stress was conceptualised several decades ago and was first defined by Sies and co-workers, as the imbalance of pro-oxidative molecules and antioxidants within a system. In recent years, it has been redefined as a cellular condition caused by an imbalance between the production and accumulation of reactive species in the cells/tissues and the ability of a biological system to detoxify these reactive species/products. Many in vitro and animal models have been developed over the years to conduct oxidative stress-based research, but to date, there is to our knowledge, no standardised human intervention models. This may in part be because, beyond biological variation, it is much more difficult to control lifestyle factors in a human population than in an experimental animal population. Furthermore, it remains challenging to generate oxidative stress in a predictable and controllable fashion in a human subject without undue risk of further harm. It is, however, extremely important that this phenomenon be further studied to understand the role of free radicals and antioxidants in physiology, but also for possible development of preventative and/or therapeutic interventions. Especially, after many years of research studies, only a few therapeutic interventions could be directly attributed to the research done on oxidative stress, with one of the major factors frustrating these efforts being the lack of standardised human models. Even though the in vitro and animal models are helpful, they do not generally translate well to humans in the field of oxidative stress. Methods: This study, therefore, aimed to standardise an exercise model to induce oxidative stress which may serve as a repeatable and controllable non-pathology-related method of inducing oxidative stress. In order to use this as a research model, it would, also require that other factors impacting oxidative stress be controlled. This was achieved by recruiting thirty young apparently healthy adult male participants and assessing their physical activity, dietary intake, medicinal or recreational drug use and screening for general pathological conditions which may contribute to oxidative stress. To induce oxidative stress in a controlled fashion, the exercise intervention was designed to include sprints to exhaustion. In addition to this, the trial was designed as a crossover trial, meaning that participants would take part in both the placebo and Rooibos intervention sessions at different times with a washout period in between. These dietary interventions (Rooibos and placebo) were taken before commencement of the exercise regimes, while markers of oxidative stress were measured after consumption with a standardised snack, during exercise and after exercise. Exercise performance parameters (time, distance, power, work, heart rate and oxygen uptake) were captured and measured by the Wattbike monitor, while exercise-induced oxidative stress damage blood biomarkers for lipid peroxidation (TBARS and CDs), protein oxidation (protein carbonyl), DNA damage, fatigue and muscle damage (lactate, creatine kinase and lactate dehydrogenase), blood redox status (GSH, GSSG, GSH/GSSG ratio) and biochemical analytes were analysed using respective assays and analyser. Results and Conclusion: In our proposed model, a controlled Wattbike exercise regime reliably and repeatably induced oxidative stress in the study population in a manner that has a low likelihood of causing an adverse event. The exercise regime caused an increase in oxidised glutathione, an important endogenous antioxidant and induced lipid peroxidation in study participants irrespective of the dietary oral intervention. It was also established that herbal interventions do not complicate the model and thus can be successfully introduced along with the model. Rooibos modulated the oxidative stress response, by allowing relevant oxidative stress markers to return to baseline levels more rapidly than placebo. As such we suggest that our proposed model demonstrates that exercise can be used to induce oxidative stress reliably and controllably in a selected human population. In addition, our results demonstrate that oral interventions do not complicate the onset of oxidative stress. This, therefore, opens the way for further studies to refine the model and standardise a human oxidative stress model for clinical testing within the context of sport and exercise. Phase II – The modulatory role of a standardised fermented Rooibos beverage to serve as an ergogenic aid using a sub-maximal exercise regime in adult males Background: Excessive production and/or accumulation of reactive species, particularly reactive oxygen and nitrogen species during exercise may result in or cause oxidative stress, exercise-induced ailments and/or overall poor exercise performances and recovery. Many athletes and sport/fitness professionals have been and still use non-specific treatments and preventative approaches to mitigate and alleviate exercise-induced ailments. These approaches may include synthetic drugs, which can be very costly and have various side effects. However, recent studies have shown that the use of natural products such as plant-derived polyphenolic constituents could be the game changer in an attempt to minimise the effects of oxidative stress and exercise-induced ailments experienced during certain physical activities. As a result, many athletes and sport professionals are increasingly ingesting these naturally derived phytochemicals and including them in their daily diet as a means to modulate exercise-induced ailments and also as health boosting and/or disease prevention strategies. Rooibos is a well-known indigenous herbal tea in South Africa with a growing demand worldwide because of its reported health promoting bioactive properties. Aim: The current study aimed to assess if a standardised fermented Rooibos beverage could modulate exercise-induced oxidative stress and improve exercise performance outcomes. Methods: Using a blinded randomised, cross-over placebo-controlled design, thirty healthy adult males consumed 375 mL of the standardised Rooibos or placebo beverages before completing an exercise regime. The regime included a modified sub-maximal exercise test followed by repeated sprints (up to 10 sets) on a Wattbike. Blood samples were collected at various time points and analysed for serum total polyphenolic content and antioxidant capacity, oxidative lipid and -protein damage markers, muscle damage markers, while various exercise performance outputs were recorded. Results: Study results indicated an increasing trend in plasma total polyphenol content and a reduction trend in oxidative lipid and -protein damage in participants who consumed the Rooibos. Study results also showed an improved total and reduced glutathione level (p<0.05) and decreased (p<0.05) levels of creatine kinase, aspartate aminotransferase and alanine aminotransferase when Rooibos was consumed before embarking on the exercise regime. Exercise sub maximal (75 – 80%) test outcomes indicated participants exercised for longer (14.05% increase) enabling them to cover a greater distance (13.60% increase) when they consumed Rooibos compared to placebo. Those participants also achieved a higher work rate and cycled with more (3.65% increase) power (W) and greater (2.35%) work (Kj) during the Rooibos intervention trial, while their heart rate and VO2 relative oxygen consumption differences between the Rooibos and placebo trial was higher by 2.40% and 5.53%, respectively. this was less than overall improvement in endurance performance measures in terms of mean gain of 14.05% in time to withdrawal and 13.60% increased distance completed. After a 24 h recovery, serum lactate levels showed a decreasing trend, although not significant, when Rooibos was consumed. Conclusion: These study findings not only support Rooibos’ known bioactivities but also make novel contributions to the field of sport and exercise and Rooibos as potential ergogenic aid to the relevant communities. Phase III – The influence of exercise-induced oxidative stress and Rooibos consumption on the human serum metabolome Background: Changes in the human metabolome because of different stimuli have been observed for many decades. Exercise is an external stimulus which causes disturbance to the metabolome and the whole body’s homeostasis which often requires an immediate response that subsequently alters the metabolomic profile. Application of metabolomics to investigate the altered metabolome offers novel opportunities for a better understanding and evaluating an organisms’ response to several challenges such as drugs/therapeutics, intense stress or exercise, food nutrients or any other stimulus to the metabolome. However, research on the metabolite shift and metabolic processes that occur and possibly underscore positive effects/outcomes after consumption of phytochemical polyphenolic-rich compounds such as Rooibos, remains unknown. Aims: To evaluate the influence of a standardised fermented Rooibos and placebo beverages on plasma metabolites or metabolic pathways in thirty healthy adult males following a submaximal exercise regime. Methods: Collected blood samples at the study time points, 0 h (baseline), immediately after exercise (IAE) and 24 h post-exercise, were analysed and quantified using nuclear magnetic resonance (NMR) and liquid chromatography mass spectrometry (LC-MS) platforms. The study data were analysed as placebo vs Rooibos at the different study time points (0 h, IAE and 24 h post-exercise). The study participant group was also further separated into two subgroups, i.e. average sprint performers (ASP) and high sprint performers (HSP). Results: Metabolites did not differ significantly at the time points (0 h, IAE and 24 h post-exercise) when comparing the placebo vs Rooibos interventions for the participant group. However, when comparing the data from the subgroups, specifically the ASP, consumption of fermented Rooibos significantly increased plasma tetradecanoylcarnitine and acetylcarnitine at time points IAE and 24 h post-exercise, respectively. In the HSP group, aspartic acid was significantly reduced at time point IAE, while pipecolic acid was significantly increased 24 h post-exercise when Rooibos was consumed. Conclusion: In this study, fermented Rooibos consumption positively influenced the study subgroups' metabolic process, particularly the energy metabolic pathways as indicated by a significant increase in the mentioned plasma metabolites which all play key roles in the energy production process and the recovery time of exercised muscles. Phase IV – The influence of genetic variations in genes related to oxidative stress and injury on exercise outputs following the consumption of an acute dose of a standardised fermented Rooibos beverage Introduction: Antioxidant enzymes help to mitigate exercise-induced damage, however, genetic variations in genes encoding these antioxidant enzymes, do affect the effectiveness of these enzymes. During exercise or physical activity, this may subsequently influence the extent of the induced oxidative stress damage and/or other exercise-induced ailments in individuals. Aim: To determine a possible link between selected genetic variants in genes involved in oxidative stress, inflammation and exercise injury risk with oxidative stress status and muscle damage markers in a healthy, active male population. Methods: Blood samples were collected directly after completion of the exercise test regime for the application of pathology-supported genetic testing (PSGT). The real-time polymerase chain reaction (PCR) using TaqMan endpoint genotyping assays was used to detect genetic variants associated with inflammation (TNF-α -308 G>A), oxidative stress (MnSOD 47 T > C, V16Al), and exercise injury risks [COL1A1(G > T), GDF5 (T > C), CASP8 (CTTACT > del), MIR608 (C>G) and COL5A1 genes genetic variants (i) C > T, (ii) del > AGGG, (iii) ATCT > del, and (iv) A > T)] in thirty physically active healthy males. Results: When compared the baseline level and 24 h post-exercise biochemical biomarker results, participants with the mutant homozygote genotype of most analysed genes’ genetic variants had increased plasma oxidative lipid (TBARS, CDs) and -protein (PC) damage, and increased serum muscle damage (CK, LDH and lactate) levels. Those with the wild-type genotype and to an extent, the heterozygote genotype, had decreased levels of the analysed damage biomarkers. Conclusion: Study results suggest wild-type genotypes may have positive influences and/or protect against exercise-induced muscle and oxidative stress damage, while the mutant homozygous may negatively influence the extent of muscle and oxidative damage. Hence, it is reasonable to hypothesise that individuals with the mutated allele (mutant homozygote genotypes) may be more susceptible to muscle and oxidative damage, than those with wild-type and to an extent, heterozygote genotypes. The use of PSGT in combination with blood biochemistry markers has the potential to improve the quality of suggested dietary interventions through a comprehensive approach that involves genetic and biochemical analyses.