Pre-exercise supplementation with curcuma xanthorrhiza roxb has minimal impact on red blood cell parameters but reduces oxidative stress: a preliminary study in rats
Article information
Abstract
[Purpose]
This study examined the effects of longterm pre-exercise Curcuma xanthorriza Roxb supplementation on red blood cell indices along with circulating malondialdehyde (MDA) and superoxide dismutase (SOD) levels in response to endurance exercise to address previously inconsistent findings.
[Methods]
Male Wistar rats (Rattus norvegicus; n = 20, aged 12–16 weeks) were divided equally into an exercise-only group (C) and three groups supplemented with Curcuma extract at dosages of 6.75 (T1), 13.50 (T2), and 20.25 mg (T3). Curcuma extract supplementation was administered for 28 d immediately prior to exercise.
[Results]
Following 28 d of exhaustive swimming, the hematocrit and erythrocyte count increased by 15% (p = 0.06). Pre-exercise Curcuma supplementation did not significantly affect mean corpuscular volume or mean corpuscular hemoglobin concentration. Longterm exercise intervention resulted in elevated MDA levels by 41% (p <0.001), while Curcuma supplementation (13.50 mg) attenuated this increase by 16.6% (p = 0.09). Additionally, Curcuma supplementation resulted in a dose-dependent increase in SOD levels, with an 82.6% increase observed at 20.25 mg (p = 0.028).
[Conclusion]
Our preliminary findings indicated that pre-exercise supplementation with Curcuma extract had a negligible effect on changes in red blood cell markers, but it mitigated the increase in oxidative stress induced by exercise training. Our future research direction will involve applying the findings to humans.
INTRODUCTION
Exercise until exhaustion leads to a reduction in hematocrit mass due to intravascular hemolysis [1], resulting from mechanical rupture when erythrocytes pass through capillaries in contracting muscles [2]. This phenomenon is known as sports anemia, a non-clinical form of anemia considered part of the adaptation process to endurance exercise [2]. The increase in hematocrit mass due to endurance exercise causes elevated blood viscosity in mice [3], compromising exercise capacity [4]. This effect may be mitigated by bioactive compounds from natural sources. However, the effect of high-content bioactive supplements on hematocrit levels in relation to endurance exercise is inconsistent. For instance, studies have reported the use of ubiquinol supplementation to prevent exercise-induced decreases in hematocrit levels [5]. However, another study demonstrated that hematocrit levels following 3 months of training remained unchanged in the group supplemented with antioxidants [6].
Contracting skeletal muscles during exercise is one of the stimuli resulting in reactive oxidative species production [7]. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels play crucial roles in maintaining the homeostasis of the redox system. MDA levels in the circulation serve as a prevalent marker of oxidative stress, with studies indicating that a prolonged exercise duration correlates with elevated MDA levels, reflecting increased lipid peroxidation [8]. Moreover, an increase in SOD levels, functioning as a metalloenzyme to neutralize excessive free radicals, has been observed after prolonged endurance training [9]. The oxidative state in body may also be influenced by red blood cell levels, which are the primary oxygen carriers in the body [10].
Curcuma xanthorriza Roxb, commonly referred to Javanese turmeric, is recognized for its abundance of phenolic groups, as highlighted in a previous study [11]. Recent research has demonstrated that long-term supplementation with curcumin, the main compound found in turmeric, significantly reduces creatine kinase levels in the circulation, indicating its potential to mitigate muscle damage [12]. A recent meta-analysis also showed that the use of curcumin alleviates the increase in inflammatory markers caused by exercise [13] by modulating inflammatory pathway signals and suppressing the production of inflammatory mediators [14]. However, despite these findings, there remains a gap in understanding the impact of pre-exercise Curcuma extract supplementation on red blood cell indices and oxidative status following exercise. Therefore, the primary objective of our study was to elucidate the effects of pre-exercise Curcuma extract supplementation on red blood cell profiles, as a part of resolving the inconsistent outcomes of previous studies [5,6]. Additionally, we aimed to evaluate its potential antioxidant properties by assessing MDA and SOD levels post-exercise. The findings of the current study serve as a preliminary basis for developing interventions involving Curcuma supplements to regulate hematological profiles and the redox state following a period of exercise training in human trials. Such interventions hold promise to address anemia-related issues in endurance athletes.
METHODS
Curcuma extract
The raw materials of C. xanthorriza Roxb were sourced from the local market in Purworejo, Semarang Province, Indonesia, which is renowned for its high-quality Curcuma. The procedure of Curcuma extraction in the current study followed our previously described method, which yielded curcumin levels of approximately 27% [15]. A total 2.5 kg of Curcuma powder was macerated using a mixture of 70% ethanol and water in a ratio of 1:10 for 2 h at a temperature of approximately 60°C. Subsequently, the mixture was filtered, and the residue was re-extracted using the same extraction process, followed by ethanol extraction with a liquid-liquid hexane solvent. The extract was then subjected to evaporation at approximately 60°C, followed by sterilization at 140°C for 2 s, and drying using a vacuum drying system (Memmert, model VO 400, Schwabach, Germany).
Study design
The current study employed a pre-test–post-test control group design conducted at the Laboratory for Research and Animal Testing Universitas Muhammadiyah Semarang, Indonesia. The animal housing was standardized and controlled to ensure a stress-free environment. This was achieved by maintaining an ambient temperature of 22°C, a 12-h light/dark cycle, and periodically changing the bedding. The minimum sample size was calculated using G*Power software version 3.1, with an estimated effect size of 0.9, considering the bioactive properties of Curcuma extract. A minimum of five animals for each group were required. The administration of supplements, outcome assessments, and data analysis were conducted in a blinded manner to minimize potential deviations in the intervention. Twenty male Wistar rats (Rattus norvegicus) were randomly assigned to one of four groups: a control group (C, n = 5) receiving only exercise, and three treatment groups (T1, T2, and T3) receiving pre-exercise Curcuma extract doses of 6.75 mg (n = 5), 13.5 mg (n = 5), and 20.25 mg (n = 5), respectively. The dosages were determined based on our previous findings, which demonstrated that 750 mg/day of Curcuma extract (equivalent to 13.5 mg in rats) had minimal effects on hematocrit levels in soccer athletes [16]. Consequently, we increased the dosage in the current study. The lowest dose was included to assess the dose-response trend across three dosage levels. A randomization method was conducted using an online random number generator (GraphPad, San Diego, CA, USA). The Wistar rats, bred by the Laboratory for Research and Animal Testing at Universitas Muhammadiyah Semarang, were aged 12–16 weeks and weighed an average of 160–200 g, representing young adulthood. Rats exhibiting signs of poor physical health, such as physical inactivity, food intake deprivation, and extreme weight loss, were excluded. Individual cages were utilized to monitor food consumption, with each rat receiving 40 g of standard laboratory food daily and ad libitum access to drinking water. Food leftovers were measured to assess consumption.
Prior to the study, the rats underwent 10 d of acclimatization to the swimming training regimen until exhaustion, with swimming sessions conducted daily in the morning for 28 d thereafter. Swimming sessions were terminated when signs of physical exhaustion, such as weakening movements and partial submersion of the head, were observed. Curcuma extract supplementation (~2 mL) was administered orally immediately prior to each exercise session. Ethics approval was obtained from the Institutional Review Board of the Faculty of Health and Nursing Science, Universitas Muhammadiyah Semarang (approval number 001/KE/04/2023).
Hematological analysis
Before blood was drawn, the rats were anesthetized using ether (Indochemical Citra Kimia, Semarang, Indonesia). Blood samples were collected from the orbital plexus on day 1 before exercise and day 28 after exercise. The samples were promptly transferred into 0.5 mL ethylenediaminetetraacetic acid tubes and then homogenized. Red blood cell indices were the primary outcomes. The erythrocyte count (number of cells/μm), hematocrit (%), mean corpuscular volume (MCV; fL), mean corpuscular hemoglobin (MCH; pg), and MCH concentration (MCHC; %) were automatically counted using a Sysmex XN-1000™ Hematology Analyzer (Sysmex Corporation, Kobe, Japan).
For MDA assessments, serum samples were assayed using enzyme-linked immunosorbent assays (ELISAs) based on the double-antibody sandwich method (MyBioSource, Vancouver, Canada). Samples and standards were incubated at 37°C for 90 min. Subsequently, the samples and biotinylated polyclonal detection antibody were added to the ELISA plate wells and incubated at 37°C for 60 min. Following this, the wells were washed with phosphate-buffered saline (PBS) and avidin-peroxidase conjugates were added, followed by further incubation at 37°C for 30 min. A tetramethylbenzidine substrate solution was then added to the wells for color development after thorough washing with PBS to remove the enzyme conjugate. The MDA concentration was calculated and is expressed as mmol/mL.
Statistical analysis
Statistical analysis was performed using SPSS version 27.0 (IBM, Armonk, NY, USA). Differences among groups were evaluated using one-way analysis of variance followed by a least significant difference post-hoc test. Data are presented as the mean ± standard deviation. A type 1 error threshold of p ≤0.05 was considered significant, indicating a 5% probability that the result supporting the hypothesis would be untrue, while p <0.1 was considered as moderately significant.
RESULTS
Food consumption and body weight
No animals died during the current study, and all presented data were obtained from a total of animals (n = 20). Food intake and body weight gain over the 5-week experimental period are depicted in Figure 1A–B. The food intake and body weight remained consistent during the study; thus, they were not confounding variables.
Food consumption and body weight of the animals during the study.
Weekly food intake (A) and body weight (B) of the animals. C: exercise-only intervention group (n = 5), T1: Curcuma extract supplementation group with a dosage of 6.75 mg (n = 5), T2: Curcuma extract supplementation group with a dosage 13.50 mg (n = 5), and T3: Curcuma extract supplementation group with a dosage 20.25 mg (n = 5).
Red blood cell indices
Table 1 illustrates the impact of pre-exercise Curcuma extract treatment on red blood cell indices. Following a 28-d swimming intervention, the hematocrit level increased by approximately 15% (p = 0.056). Of note, the administration of Curcuma extract normalized the hematocrit levels. The MCV remained unchanged in both the control and pre-exercise Curcuma-supplemented groups (p >0.05). The hemoglobin content, as indicated by the MCH and MCHC values, exhibited a similar pattern of response between the pre-exercise Curcuma extract intervention and swimming-only intervention. The maximal exercise intervention increased the erythrocyte count by 15% (p = 0.059), and this erythrocyte count remained unchanged in the pre-exercise Curcuma extract group.
Effect of pre-exercise Curcuma extract supplementation on the red blood cell indices.
MDA levels
Table 2 illustrates the influence of pre-exercise Curcuma extract on MDA levels pre- and post-intervention. Maximal-intensity swimming induced a significant increase in MDA levels by approximately 41% (p <0.001). However, this elevation was attenuated with the administration of a low dose of Curcuma extract (p = 0.19), moderately decreasing by approximately 17% (p = 0.09) with a dosage of 13.5 mg. Notably, the highest dosage did not exhibit any additional effect (p = 0.77).
Effect of pre-exercise Curcuma extract supplementation on the circulating MDA levels.
SOD levels
The impact of pre-exercise Curcuma extract on SOD levels pre- and post-intervention is presented in Table 2. Following long-term maximal-intensity swimming, SOD levels showed no significant change (p = 0.15). However, supplementation with pre-exercise Curcuma extract notably increased by approximately 63% at dosage of 6.75 mg (p = 0.001). Furthermore, this increase continued with higher dosages, with SOD levels increasing by 71 (p = 0.028) and 82.6% (p = 0.028) at 13.5 and 20.25 mg, respectively.
DISCUSSION
Approximately 5.7% of athletes experience anemia [17], with a higher prevalence observed among athletes undergoing aerobic activities [18,19]. To contribute to this field, we investigated the effects of pre-exercise Curcuma extract supplementation on red blood cell profiles. Red blood cells, which serve as carriers of oxygen, undergo changes during exercise because of the increased oxygen demand in active muscles [2]. Our findings can be summarized as follows: (1) endurance exercise training increased the number of red blood cells by 15%; (2) pre-exercise Curcuma extract supplementation had minimal effect on the changes in the number of red blood cells induced by exercise; (3) Curcuma supplementation prior to exercise substantially reduced oxidative stress levels.
Exhaustive endurance training has been shown to increase the absolute blood volume because it stimulates erythropoiesis, resulting in red blood cell volume expansion [20]. The increase in the number of erythrocytes at the end of intervention showed a linear trend with the increasing hematocrit level, suggesting that the hematocrit increase was partly driven by elevated red blood cell production. Regular endurance training of consistent intensity induces hematological adaptations, including enhanced red blood cell production, as part of the physiological response of body to sustained exercise [21]. Pre-exercise Curcuma supplementation showed a neglected alteration on hematocrit and erythrocyte levels. This phenomenon may be associated with the potential of Curcuma to improve vascular endothelial function, thereby promoting endothelial vasodilation [22]. The mechanism may involve attenuation of the redox-signaling pathway, limiting the bioavailability of nitric oxide, which is critical for endothelial function [22,23] and resulting in plasma volume expansion. In this study, we anticipated a significant reduction in hematocrit levels because of the expected increase in plasma volume following Curcuma extract supplementation. However, the lack of a pronounced effect may be attributed to insufficient dosage, the timing of supplementation, or intervention duration, which should be addressed in future research. Curcuma extract was administered immediately prior to exercise training, potentially influencing its bioavailability in plasma. Considering that curcumin concentration peaks 1–2 h following oral ingestion, this timing may have impacted its effectiveness [24].
A transient increase in MDA levels induced by exhaustive training has been reported to stimulate antioxidant enzyme activity mediated by the activation of redox-sensitive signaling pathways [25]. This regular exercise intervention potentially upregulates the antioxidant defense capacity to produce endogenous antioxidant enzymes [9]. Our current findings revealed that Curcuma supplementation potentially induced cellular antioxidant upregulation, resulting in increasing the circulating levels of SOD, an antioxidant enzyme. This increase in endogenous antioxidant levels was proportional to the supplementation dosage. To the best of our knowledge, this is the first report that Curcuma extract decreases MDA levels in response to exhaustive exercise, and these findings support the use of Curcuma extract as a source of antioxidants. In our previous study, the curcumin content, comprising approximately 27% of Curcuma extract, was hypothesized to play a pivotal role in modulating the redox balance [15]. This result provides a scientific basis for the use of Curcuma supplementation to alleviate the delayed-onset muscle soreness, which is mostly caused by increasing oxidative stress levels [26]. Our findings indicated that higher doses of Curcuma extract supplementation did not result in further reductions in MDA levels, suggesting a threshold effect in its ability to neutralize excessive free radical production. The current study identified 13.50 mg as the optimal Curcuma supplementation dose for mitigating exercise-induced MDA levels in rats.
The relationship between food intake and growth is typically linear. However, we observed an anomaly in the group receiving the highest dosage of Curcuma extract supplementation, which exhibited a slightly contrasting trend with body weight. We hypothesized that Curcuma extract supplementation may control body weight increases through various mechanism, as discussed in a previous report [27]. Further research involving human subjects is needed to validate our hypothesis.
Herein, we present findings regarding the impact of pre-exercise Curcuma extract supplementation on rats. The current study has several limitations. Using rats is the first limitation. Future research should explore the application of this supplementation in untrained individuals undergoing an exercise training program. Second, the absence of a statistically significant difference in hematocrit levels in the Curcuma-extract-treated groups limited the strength of the study’s conclusions. However, the near-significant increase in hematocrit levels observed in the control group suggests that Curcuma extract may have mitigated the exercise-induced increase in hematocrit levels, indicating a potential normalizing effect. Third, further investigation is required to evaluate the effect of Curcuma supplementation on additional oxidative stress markers to generalize its free radical neutralizing properties. Fourth, higher doses of Curcuma extract and the timing of supplementation prior to exercise training should be explored to confirm the optimal regimen for mitigating exercise-induced free radical production. Fifth, the type and intensity of exercise training should also be considered in future research. Comparing different exercise intensities may provide a more comprehensive understanding of the effects of pre-exercise Curcuma extract supplementation on hematological indices and oxidative stress markers, thereby offering clearer insights into its efficacy as a supplement.
In conclusion, we found that Curcuma extract supplementation reduced several hematological indices and oxidative parameters. Curcuma supplementation potentially accelerates recovery after exercise training by mitigating excessive free radical production induced by such training. However, the effect of pre-exercise training supplementation with Curcuma on red blood cell indices requires further exploration. The limited sample size of the current study may have influenced the statistical significance of several outcomes.
Acknowledgements
We would like to express our gratitude to the authorities for granting permission to conduct this study at the Laboratory for Research and Animal Testing, Universitas Muhammadiyah Semarang. The author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The manuscript version submitted has been approved by all authors. The datasets used and/or analyzed will be available on written request.