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The Effect of Sundarbans Honey vs Energy Drinks on Exercise, Fatigue, and Antioxidant Defense via NRF2/HO-1 Pathway – An Experiment in Mice

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15 July 2024

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16 July 2024

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Abstract
Excessive oxidative stress and exercise-induced fatigue reduce physical performance and can lead to dysfunction in antioxidant systems within muscular and hepatic tissues. Integrating natural substances with exercise may mitigate fatigue via anti-oxidant effects. This study aims to compare and evaluate the effects of exercise and supplementation with Sundarbans honey vs energy drinks on the strength, anxiety physiological profiles, and hepatic antioxidant capacity in mice undergoing extraneous exercise and fatigue. Forty-eight four-week-old Swiss albino mice were randomly assigned to one of eight groups: (1) Control, (2) Speed™ (Sp), (3) Royal Tiger™ (RT), (4) Sundarbans Honey (SH), (5) Exercise (Ex), (6) Exercise + Sundarbans Honey (Ex+SH), (7) Exercise + Speed™ (Ex+Sp), and (8) Exercise + Royal Tiger™ (Ex+RT). Supplements were administered via oral gavage: Sp (2.5 ml/kg BW), RT (2.5 ml/kg BW), and SH (1 g/kg BW), four times a week for 60 days. Mice in the Ex, Ex+SH, Ex+Sp, and Ex+RT groups underwent daily swimming and running. The strength, fatigue, anxiety levels, lipid profiles, and antioxidant biomarkers were evaluated post-treatment. The SH, Ex, and Ex+SH groups exhibited significantly enhanced physical performance (p<0.05). Plasma lipid profiles, including LDL, triglycerides, total cholesterol, and fatigue-related biomarkers (ALP, AST, ALT, LDH, CK, LA, BUN, and glucose) were markedly increased in the Sp and RT groups but decreased in the SH, Ex, and Ex+SH groups compared to control (p<0.05). Exercise-induced fatigue elevated the Nrf2/HO-1 antioxidant defense system in liver tissue. IL-6 levels were reduced in the SH, Ex, and Ex+SH groups (p<0.05). These findings indicate that regular exercise combined with SH supplementation increases strength and improves lipid profiles, reducing exercise-induced oxidative stress and fatigue via modulation of the Nrf2/HO-1 pathway in liver tissue. These results highlight SH supplementation’s potential as a complementary strategy to enhance exercise outcomes and mitigate exercise-induced stress.
Keywords: 
Subject: Medicine and Pharmacology  -   Dietetics and Nutrition

1. Introduction

Natural substances and energy drinks (EDs) are investigated for their potential to reduce fatigue and improve performance. EDs, highly caffeinated and carbonated beverages, have been widely consumed since the 1960s [1], particularly among young adults, teenagers, children, and athletes [2]. While they can have benefits, EDs also pose risks due to ingredients like caffeine, taurine, and sugars [3]. Studies have shown that EDs can cause adverse effects such as anxiety, elevated heart rate, type 2 diabetes, and sleep disturbances [4,5,6]. They may also induce oxidative stress, reducing antioxidant defenses and increasing reactive oxygen species (ROS), which damage proteins, lipids, and nucleic acids [7,8,9]. Excessive ROS triggers the Nrf2/HO-1 pathway, a key antioxidant defense mechanism [10,11,12].
Chronic fatigue can lead to serious health issues and affects many people who do not receive adequate care [13,14,15]. It is associated with aging, anxiety, infections, and diseases like multiple sclerosis and Parkinson’s [16,17]. Exercise-induced fatigue can decrease physical activity and cause cardiovascular and muscular disorders [18,19]. Exercise fatigue involves energy depletion, metabolic waste accumulation, and increased oxidative stress [20,21]. Biomarkers of this fatigue include muscular injury and ATP depletion [22]. Research continues to explore the mechanisms and management of exercise-induced fatigue. Natural honey, particularly Sundarbans honey, has antioxidant and anti-inflammatory properties. This honey, collected by Apis dorsata bees in Bangladesh’s Sundarbans mangrove forest, contains beneficial compounds like sugars, amino acids, and polyphenols [23]. Regular exercise combined with honey supplementation may enhance muscle health and mitigate fatigue.
This study aims to compare the effects of exercise with Sundarbans honey supplementation versus energy drinks on muscle strength, exercise-induced fatigue, and hepatic antioxidant capacity in Swiss albino mice.

2. Methods

2.1. Preparation or Collection of Honey Samples and Energy Drinks

Multifloral honey samples were gathered from the world’s biggest mangrove forest, Sundarban in Bangladesh’s south-west region. To prevent contamination during the experiment, 10 g of honey was dissolved in 50 mL of distilled water. The liquid was then stored in a covered container. Speed™ and Royal Tiger™ Energy Drinks were purchased from Dhaka, Bangladesh.

2.2. Animal Maintenance and Experimental Design

Forty-eight Swiss albino mice aged four weeks (body weight: 25-30 g) were randomly assigned into eight groups: 1. Control, 2. Speed™ (Sp), 3.Royal Tiger™ (RT), 4. Sundarbans honey (SH) 5.Exercise (Ex), 6.Exercise + Sundarbans honey (Ex+SH), 7.Exercise + Speed™ (Ex+Sp), 8.Exercise + Royal Tiger™ (Ex+RT). Each group consisted of six mice and was kept at room temperature (25±2ºC) with a natural light and dark cycle in the animal research Laboratory of the Department of Biochemistry and Molecular Biology, University of Rajshahi. Speed™, Royal tiger™, and Sundarbans honey were given to Sp, RT, SH, Ex+SH, Ex+Sp, and Ex+RT groups of mice at doses of 2.5 ml/kg BW, 2.5 ml/kg BW, and 1 g/kg BW, respectively, using an oral gavage tube 4 days a week for 60 days [9,24]. The control group mice received only distilled water as a vehicle. Additionally, for 60 days, we trained the Ex, Ex+SH, Ex+Sp, and Ex+RT groups of mice to swim in a water tank for 5 minutes and to run a voluntary wheel for about 10 minutes per day. The time gap between the two training sessions was three hours. Throughout the experiment, all mice were given free access to normal food and water. The Institute of Biological Sciences at Rajshahi University in Bangladesh granted ethical approval for the study protocol (No: 415(27)/320/IAMEBBC/IBSc).

2.3. Measurement of Muscle Strength Using Inverted Screen Test

In the inverted screen test or grip test, mice were placed in the center of a 43 cm-wide inverted wire mesh made of 12 mm squares and 1 mm diameter wire and surrounded by a 4 cm-deep wooden beading. The screen rotated 180 degrees in two seconds and the timer started recording the mice’s falling times. The screen was set up 40-50 cm over a cushioned surface. The scoring system was implemented by the previous method [25].

2.4. Measurement of Muscle Strength Using Weights Test

The weights test apparatus included seven weights, each of which was a ball of coiled fine-gauge stainless steel wire (about 7g) and steel chain links weighing around 13g. Possible weight masses included 20, 33, 46, 59, 72, 85, and 98g. Mice were grasped by the middle of their tails to support the first weight (20g) placed on the laboratory table. Once the mice had clutched the wire scale collector with their forepaws, a timer was set and the mouse was elevated until the link was free of the bench. The final score was determined by multiplying the number of links in the heaviest chain sustained for three seconds [25,26].

2.5. Forced Swim Test

The endurance efficacy (until exhaustion) was assessed using a forced swim test by previous protocol [27,28]. Each mouse was individually placed in a cylindrical swimming pool 65 cm long and 20 cm in radius, with a weight equal to 5% of BW attached to the root of its tail. The water was leveled at 40 cm and the temperature of 25±2ºC. The mice’s stamina was determined by their swimming times from the start until exhaustion.

2.6. Measuring Anxiety-Like Behavior with the EPM

In general, the elevated plus maze (EPM) is utilized to evaluate anxiety-like behavior in mice. The EPM is a laboratory apparatus in the shape of a plus, featuring two open arms measuring 50 × 10 cm each, two closed arms measuring 50 × 10 × 40 cm, and a vertical dimension of 50 cm [29]. Five minutes after being placed in the center of the maze with one of the closed arms facing them, the mice were permitted to investigate. The total amount of time each mouse spent in both arms throughout the test session was recorded. After each test, the maze was cleaned with 70% ethanol to prevent smell cues from impacting the results.

2.7. Biochemical Assays

2.7.1. Chemicals and Kits

Commercial kits from Human Diagnostic, Germany, were used to test the enzymatic activity of alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatine kinase (CK), and serum biochemical parameters such as lactic acid (LA), blood urea nitrogen (BUN), glucose, total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). The activities of catalase (CAT), glutathione reductase (rGR), and superoxide dismutase (SOD) were determined in liver tissue homogenates using an ELISA kit (Cayman Chemical, USA). The liver tissue homogenate was also used to measure the protein concentrations of Nuclear factor erythroid 2-related factor 2 (Nrf2), Heme oxygenase-1 (HO-1), and Interleukin 6 (IL-6) using ELISA (Elabscience, USA) by manufacturer guidelines.

2.7.2. Collection of Blood Serum

Day after the exhaustion, strength, and behavioral tests, blood samples were collected from the thoracic arteries of mice immediately following the 15-minute exhaustive swimming exercise, after anesthetization with diethyl ether [28]. For coagulation, the blood was kept at room temperature for 30 minutes and, after centrifugation at 4000 rpm for 15 minutes at 4 °C, the serum was extracted and stored at −80 °C until the analyses were performed

2.7.3. Liver Tissue Homogenate Preparation

All experimental animals were sacrificed by cervical dislocation under moderate Diethyl ether anesthesia. Mice’s liver tissue was removed, washed with ice-cold physiological saline, and stored at -80°C until analysis. To prepare the liver tissue homogenate, 200 mg of the liver was combined with 9 times of cold physiological saline and centrifuged at 2500×g for 10 minutes using a homogenizer. The supernatant (top, roughly clear layer) was carefully collected in a separate microtube and frozen for further analysis.

2.7.4. Biochemical Analysis of Serum and Liver Tissue

An analyzer (Humalyzer 3000, Germany) was used to assess serum indices using commercially available kits following the manufacturer’s procedure. The activity of CAT, rGR, SOD, Nrf2, HO-1, and IL-6 was determined using a microplate reader (Mikura Ltd., UK). All serum samples were examined in duplicate, and mean values were obtained.

2.8. Statistical Analysis

Data were presented as Mean ± SEM (standard error of the mean). Using the Shapiro-Wilk and Kolmogorov-Smirnov tests, we found that all of the data passed the normality test, indicating that they are normally distributed. The statistical significance of the parameters of eight groups was determined using Welch’s t-test (when comparing two groups) and ordinary one-way ANOVA (when comparing all experimental groups with the control group), as specified. p<0.05 were considered statistically significant. Data were processed and graphs were created using GraphPad Prism 8.01 (GraphPad Software, La Jolla, CA).

3. Results

3.1. Effects of Exercise, SH, and Energy Drinks (EDs) Supplementation on Mice’s Physical Performances

Mice in the Ex and SH groups exhibited significantly greater muscle strength (Figure 1A and 1B) and endurance capability (Figure 1C) compared to sedentary controls [F (7, 40) = 12.09, p <0.0001, F (7, 40) = 32.22, p <0.0001, F (7, 40) = 525.8, p <0.0001)]. Further comparisons revealed that the mice exposed to Ex+SH had a significant increase in scores of physical performance (p < 0.01), especially in endurance capability (p < 0.0001), about those exposed to Ex only. Conversely, mice exposed to Ex+Sp and Ex+RT showed a significant decrease in physical performance (p < 0.01) that was also more prominent for the endurance capability (p < 0.0001), compared to those exposed to Ex.

3.2. Effects of Exercise and Supplementation of SH and EDs on Anxiety-Like Behavior in Mice

The effect of exercise and supplementation with SH and EDs on anxiety-like behavior is illustrated in Figure 2. Mice supplemented with Sp and RT exhibited anxiety-like symptoms and spent more time in the closed arms than open arms of the EPM compared to control mice [F(7, 40) = 50.65, p <0.0001). Inversely, Mice treated with SH and Ex spent more time in the open arms than closed arms compared to control mice [F (7, 40) = 44.57, p<0.0001)].

3.3. Effects of Exercise and Supplementation with SH and EDs on Lipid Profile in Mice

The influence of exercise and its combined ingestion of SH and EDs on the plasma lipid profile is presented in Table 1. The analyses showed that LDL, TG, and TC levels were considerably higher in the SH and RT groups compared to the control group, but significantly decreased in the SH and Ex groups [F (7, 40) = 74.27, p<0.0001, F (7, 40) = 284.0, p<0.0001 and F (7, 40) = 136.8, p<0.0001 respectively]. Additionally, mice in the EX+SH group had significantly lower LDL, TG, and TC lipid profiles compared to the Exgroup (p<0.05). It was found that HDL levels were significantly lower in Sp and RT group mice than in the control group [F (7, 40) = 220.5, p<0.0001).

3.4. Effect of Exercise and Supplementation with SH and EDs on Fatigue-Related Biomarkers In Mice

Results of serum analyses of ALP, AST, ALT, LDH, CK, LA, BUN, and glucose after exhaustive swimming are provided in Table 2. In comparison to the control group, the activities of ALP, AST, and ALT were significantly decreased in the SH and Ex groups and significantly increased in the Sp and RT groups, after swimming-induced exhaustion [F (7, 40) = 24.55, p<0.0001, F (7, 40) = 50.59, p<0.0001 and F (7, 40) = 23.82, p<0.0001 respectively]. The supplementation with SH in the Ex group resulted in a significant decrease in serum LDH and CK activity compared to the control group (p<0.05). In addition, our data reports that LA, BUN, and glucose were substantially higher in Sp and RT-treated mice and significantly lower in SH and EX-treated animals compared to control mice [F (7, 40) = 264.8, p<0.0001, F (7, 40) = 18.64, p<0.0001 and F (7, 40) = 26.93, p<0.0001 respectively]. Overall, all of the fatigue-related biomarkers were significantly lower in the Ex+SH group mice but significantly higher in the Ex+Sp and Ex+RT groups, when compared to the Ex group (p<0.05).

3.5. Exercise, SH, and EDs Influence Antioxidant Enzymes in Mice’s Liver after Exhaustion

The levels of hepatic antioxidant enzymes CAT, rGR, and SOD were significantly elevated in the liver of mice treated with SH and Ex and decreased in mice treated with Sp and RT, compared to control mice (p<0.05). This difference was also statistically significant, as indicated by the high F-values [F (7, 40) = 94.74, p<0.0001 for CAT, F (7, 40) = 590.5, p<0.0001 for rGR, and F (7, 40) = 164.7, p<0.0001 for SOD). The Ex+SH group of mice exhibited remarkably elevated levels of antioxidant enzyme activity in their liver tissue, while the Ex+Sp and Ex+RT groups of mice showed significantly reduced enzymatic activity, relative to the Ex group (p<0.001). Table 3

3.6. Exercise, SH, and EDs Affect on NRF2 and HO-1 Protein Levels in Mice Liver Tissue after Exercise-Induced Fatigue

Prolonged exercise and fatigue dramatically increase the levels of NRF2 and HO-1 in the liver of mice in both SH-treated and Ex groups while significantly reducing these protein levels in mice treated with Sp and RT, compared to the control group [F (7, 40) = 86.11, p<0.0001, F (7, 40) = 279.3, p<0.0001] (Figure 3). Additionally, these protein levels in liver tissue were considerably higher in Ex+SH and significantly lower in Ex+SP and Ex+RT groups compared to Ex group mice (p<0.05).

3.7. Exercise, SH, and EDs Influence on IL-6 Levels in Mice Liver Tissue after Exhaustion

Interleukin-6 is a key pro-inflammatory cytokine whose pleiotropic function includes acute phase protein synthesis in the liver and lymphocyte differentiation. The hepatic levels of IL-6 were significantly higher in Sp and RT groups and significantly lower in SH and EX groups, compared to control mice [F (7, 40) = 83.46, p<0.0001] (Figure 4). Interestingly, we found that IL-6 levels were significantly lower in the Ex+SH group mice than in the Ex group (p<0.01).

4. Discussion

Our results show that both regular exercise and SH supplementation improve muscle strength, endurance against fatigue, and antioxidant defense in mice. However, mice supplemented with SP and RT energy drinks showed no changes in physical parameters. Given the adverse effects of prescription drugs and uncontrolled ED consumption by athletes, young adults, and children, testing natural substance supplementation is relevant for sports and health sciences. Excessive exercise and fatigue reduce physical performance and may lead to various diseases.
Physiologically, fatigue results from energy depletion and increased intracellular ROS, impairing muscle contraction and neuromotor control [30,31]. Strength and swimming tests were used to assess anti-fatigue effects and musculoskeletal conditions in our study [32]. Exercise combined with SH supplementation significantly improved physical performance compared to exercise alone. Conversely, performance scores dropped significantly in groups supplemented with EDs (Ex+Sp and Ex+RT). SH supplementation with exercise improved fatigue biomarkers (ALP, AST, ALT, LDH, CK, LA, BUN) in mice blood post-exercise, reflecting enhanced physiological parameters and musculoskeletal adaptation.
Endurance performance relies on cardiovascular function, associated with plasma lipid levels (LDL, HDL, TG, cholesterol) [33,34]. Chronic ED consumption increased LDL, TG, and total cholesterol while decreasing HDL levels, indicative of poor cardiovascular health [35]. EDs also induced anxiety-like behaviors in mice treated with Sp and RT, as shown by decreased time in the open arm of the EPM test [36]. Conversely, exercise and SH supplementation reduced anxiety, increasing time in the open arm.
From another perspective, exercise-induced excessive ROS generation can oxidize proteins, lipids, or nucleic acids, while it decreases the cell’s defense against free radicals played by antioxidant enzymes such as CAT, GPX, GST, GR, and SOD [37,38,39], being considered a main cause of liver damage and inadequate recovery [40,41]. Remarkably, our findings revealed that the mice consuming EDs had reduced activity of CAT, SOD, and rGR in liver tissue, after exercise-induced exhaustion; whereas, mice in exercise and SH supplementation regimes showed an increased antioxidant enzyme activity in the liver tissue after exhaustion. Moreover, when compared to mice only exercising, those on exercise plus RT supplementation regime exhibited a significantly higher antioxidant enzyme activity in liver tissue. Conversely, the mice who exercised under supplementation with EDs exhibited a significantly lower antioxidant hepatic enzyme activity than mice exercising without supplementation, after the exercise-induced exhaustion test.
Further, our results show regular physical activity with supplementation SH reduces exercise-induced oxidative stress while significantly elevating the hepatic levels of Nrf2 and HO-1 – which compose an essential defense system against oxidative stress in cells [42]. Under physiological conditions, the primary transcriptional factor in the antioxidant system tissue-wide Nrf2 is bound to Kelch-like ECH-associated protein 1 (KEAP1) [43,44]. During high-intensity exercise and oxidative stress states, NRF2 separates from KEAP1 and enters the nucleus to interact with antioxidant response element (ARE) sequences present in genes that encode for detoxification enzymes such as SOD, CAT, GPx, GST, NADH, and HO-1 [45,46,47]. Upregulation of HO-1 expression downstream of NRF2 activation increases its activity of removing toxic heme, producing iron ions and carbon monoxide-carbon monoxide, conferring to the NRF2/HO-2 a potent antioxidant system [48,49]. Together, our results indicate that supplementation with SH had an additional effect on the improvements in antioxidant capability acquired with regular exercise. However, when mice consumed EDs - Sp and RT - on a chronic basis, either alone or combined with exercise, we observed a downregulation in hepatic the NRF2/HO-1 antioxidant system. These results corroborate the effects found for the supplementation with SH versus EDs reported in the serum analyses of fatigue-associated biomarkers, where animals treated with ED exhibited a higher oxidative stress profile.
Furthermore, oxidative stress can damage cellular membranes and lead to the expression of pro-inflammatory cytokines like interleukin (IL)-6 [50]. Whereas upregulation in the NRF2/HO-1 system contributes to protection against free radicals, suppression of Nrf2 expression increases IL-6 expression [51,52]. Interestingly, IL-6 expression was significantly higher in the mice undergoing ED supplementation with or without exercise. Meanwhile, mice who exercised and/or consumed SH had a substantial reduction in the levels of IL-6, indicating an improvement in the inflammatory response to exercise-induced oxidative stress.

5. Limitations

We understand that the utilization of SH, Sp, and RT as investigative tools presents limitations for the determination of the specific chemical components responsible for modulating fatigue. Additionally, while our assessment focused on evaluating the levels of antioxidant enzymes Nrf2 and HO-1 within liver tissue, the absence of examination of these biochemical parameters in muscle tissue represents another limitation, as it precludes a comprehensive understanding of their systemic effects.

6. Practical Applications

The study underscores practical implications for athletes, fitness enthusiasts, and those interested in natural approaches to enhance performance and combat fatigue. Sundarbans honey supplementation, when paired with regular exercise, offers a comprehensive strategy to optimize athletic capabilities. Athletes and fitness enthusiasts can integrate Sundarbans honey into their routines to capitalize on its natural carbohydrates, antioxidants, and bioactive compounds. Unlike energy drinks, Sundarbans honey sustains energy levels without the potential drawbacks of caffeine and excessive sugar, thereby supporting prolonged physical endurance and faster recovery while fostering overall health benefits such as reduced inflammation and strengthened immune function.
Furthermore, Sundarbans honey activates the Nrf2/HO-1 pathway, which helps mitigate oxidative stress, potentially lowering the risk of various diseases. Tailoring supplementation and exercise protocols to individual needs is crucial for achieving optimal results. Continued research into optimal dosages and underlying mechanisms could further broaden the therapeutic applications of Sundarbans honey, extending beyond fatigue management to personalized strategies aimed at enhancing health and performance.

7. Conclusions

Our study demonstrated that combining SH supplementation and regular EX enhances physiological markers and physical performance in mice. That includes improvements in muscle strength, endurance, and antioxidant capability, and reductions in anxiety-like behavior and lipid profiles. Our results also elucidated that exercise and sH supplementation can mitigate fatigue and reduce pro-inflammatory responses through the activation of the NRF2/HO-1 pathway, safely promoting health. Conversely, the study revealed that consumption of Sp and RT energy drinks, widely available in Bangladesh, may adversely affect biochemical markers of oxidative stress, posing potential risks to public health.
Based on the results gleaned from this study, further investigations should explore the precise mechanisms underlying the interaction between exercise and SH supplementation in modulating the NRF2/HO-1 pathway within different tissues, including the brain, to alleviate fatigue and oxidative stress. Additionally, clinical studies are warranted to validate the translatability of these findings into practical interventions for enhancing physical performance and managing fatigue in athletes, individuals engaged in regular exercise, or at risk of disease. Moreover, future research endeavors should delve into optimizing the dosage and duration of SH supplementation, as well as the intensity and frequency of exercise, to delineate the most efficacious regimen for achieving optimal outcomes.

Author Contributions

Conceptualization, K.K.B., J.H.; methodology, A.A., K.H.H., M.K.; formal analysis, A.A., K.H.H.; investigation A.A., K.H.H., J.M., M.K., M.H.M.; validation, K.H.H.; data curation, A.A., K.H.H., J.M., M.K., M.H.M., G.G.d.A.; writing—original draft preparation, G.G.d.A., P.F.d.A-N, A.A., K.K.B., J.H., H.İ.C., N.L.B.; writing—review and editing, K.K.B., H.İ.C., N.L.B. P.F.d.A-N, G.G.d.A.; supervision, J.H., H.İ.C., N.L.B.; project administration, J.H. H.İ.C., funding acquisition, N.L.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was approved by the Ethical Committee of the Institute of Biological Sciences at Rajshahi University in Bangladesh (No. 415(27)/320/IAMEBBC/IBSc).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available for research purposes upon reasonable request to the corresponding author.

Acknowledgments

The Department of Biochemistry and Molecular Biology at Rajshahi University provided laboratory equipment, chemicals, and kits to help our research. We appreciate their support. We express our gratitude to those who took part in this research.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Nowak, D.; Jasionowski, A. Analysis of the consumption of caffeinated energy drinks among Polish adolescents. Int. J. Environ. Res. Public Health. 2015, 12, 7910–7921. [Google Scholar] [CrossRef]
  2. Trapp, G.S.; Allen, K.L.; O’Sullivan, T.; Robinson, M.; Jacoby, P.; Oddy, W.H. Energy drink consumption among young Australian adults: associations with alcohol and illicit drug use. Drug Alcohol Depend. 2014, 134, 30–37. [Google Scholar] [CrossRef] [PubMed]
  3. Heckman, M.A.; Sherry, K.; De Mejia, E.G. Energy drinks: an assessment of their market size, consumer demographics, ingredient profile, functionality, and regulations in the United States. Compr. Rev. Food Sci. Food Saf. 2010, 9, 303–317. [Google Scholar] [CrossRef]
  4. Richards, G.; Smith, A.P. A review of energy drinks and mental health, with a focus on stress, anxiety, and depression. J. Caffeine Res. 2016, 6, 49–63. [Google Scholar] [CrossRef] [PubMed]
  5. Holubcikova, J.; Kolarcik, P.; Madarasova Geckova, A.; Reijneveld, S.A.; van Dijk, J.P. Regular energy drink consumption is associated with the risk of health and behavioural problems in adolescents. Eur. J. Pediatr. 2017, 176, 599–605. [Google Scholar] [CrossRef] [PubMed]
  6. Grasser, E.K.; Yepuri, G.; Dulloo, A.G.; Montani, J.P. Cardio-and cerebrovascular responses to the energy drink Red Bull in young adults: a randomized cross-over study. Eur. J. Nutr. 2014, 53, 1561–1571. [Google Scholar] [CrossRef] [PubMed]
  7. Reis, R.; Charehsaz, M.; Sipahi, H.; Ekici, A.I.D.; Macit, C.; Akkaya, H.; Aydın, A. Energy drink induced lipid peroxidation and oxidative damage in rat liver and brain when used alone or combined with alcohol. J. Food Sci. 2017, 82, 1037–1043. [Google Scholar] [CrossRef]
  8. Díaz, A.; Treviño, S.; Guevara, J.; Muñoz-Arenas, G.; Brambila, E.; Espinosa, B. . Aguilar-Alonso, P. Energy drink administration in combination with alcohol causes an inflammatory response and oxidative stress in the hippocampus and temporal cortex of rats. Oxid Med Cell Longev. 2016. [CrossRef]
  9. Al-Basher, G.I.; Aljabal, H.; Almeer, R.S.; Allam, A.A.; Mahmoud, A.M. Perinatal exposure to energy drink induces oxidative damage in the liver, kidney and brain, and behavioral alterations in mice offspring. Biomed Pharmacother. 2018, 102, 798–811. [Google Scholar] [CrossRef]
  10. de Assis, G.G.; Cięszczyk, P. Exercise − A Unique Endogenous Regulator of Irisin, BDNF, Leptin and Cortisol against Depression. Balt. J. Heal. Phys. Act. 2020, 12, 1–8. [Google Scholar] [CrossRef]
  11. Murawska-Ciałowicz, E.; Wiatr, M.; Ciałowicz, M.; de Assis, G.G.; Borowicz, W.; Rocha-Rodrigues, S.; Paprocka-Borowicz, M.; Marques, A. Bdnf Impact on Biological Markers of Depression—Role of Physical Exercise and Training. Int. J. Environ. Res. Public Health 2021, 18, 7553. [Google Scholar] [CrossRef] [PubMed]
  12. de Assis, G.G.; Hoffman, J.R.; Bojakowski, J.; Murawska-Ciałowicz, E.; Cięszczyk, P.; Gasanov, E.V. The Val66 and Met66 Alleles-Specific Expression of BDNF in Human Muscle and Their Metabolic Responsivity. Front. Mol. Neurosci. 2021, 14, 1–12. [Google Scholar] [CrossRef] [PubMed]
  13. Chu, L.; Valencia, I.J.; Garvert, D.W.; Montoya, J.G. Onset patterns and course of myalgic encephalomyelitis/chronic fatigue syndrome. Front. pediatr. 2019, 7, 12. [Google Scholar] [CrossRef] [PubMed]
  14. Papanicolaou, D.A.; Amsterdam, J.D.; Levine, S.; McCann, S.M.; Moore, R.C.; Newbrand, C.H. ; .. Kales, A. Neuroendocrine aspects of chronic fatigue syndrome. Neuroimmunomodulation. 2004, 11, 65–74. [Google Scholar] [CrossRef] [PubMed]
  15. Campbell, David. “The myth of compassion fatigue.” The violence of the image. Routledge. 2020, 97-124.
  16. Hsiao, C.Y.; Hsu, Y.J.; Tung, Y.T.; Lee, M.C.; Huang, C.C.; Hsieh, C.C. Effects of Antrodia camphorata and Panax ginseng supplementation on anti-fatigue properties in mice. J Vet Med Sc. 2018, 80, 284–291. [Google Scholar] [CrossRef] [PubMed]
  17. Tharakan, B.; Dhanasekaran, M.; Brown-Borg, H.M.; Manyam, B.V. Trichopus zeylanicus combats fatigue without amphetamine-mimetic activity. Phytother. Res. 2006, 20, 165–168. [Google Scholar] [CrossRef] [PubMed]
  18. Ament, W.; Verkerke, G.J. Exercise and fatigue. Sports Med. 2009, 39, 389–422. [Google Scholar] [CrossRef] [PubMed]
  19. Wang, F.; Wang, X.; Liu, Y.; Zhang, Z. Effects of exercise-induced ROS on the pathophysiological functions of skeletal muscle. Oxid Med Cell Longev. 2021, 1–5. [Google Scholar] [CrossRef] [PubMed]
  20. Hu, M.; Han, M.; Zhang, H.; Li, Z.; Xu, K.; Kang, H. . Liu, W. Curcumin (CUMINUP60®) mitigates exercise fatigue through regulating PI3K/Akt/AMPK/mTOR pathway in mice. Aging (Albany NY). 2023, 15, 2308. [Google Scholar] [CrossRef]
  21. Allen, D.G.; Lamb, G.D.; Westerblad, H. Skeletal muscle fatigue: cellular mechanisms. Physiol. Rev. 2008, 88, 287–332. [Google Scholar] [CrossRef]
  22. Finsterer, J.; Drory, V.E. Wet, volatile, and dry biomarkers of exercise-induced muscle fatigue. BMC Musculoskelet. Disord. 2016, 17, 1–13. [Google Scholar] [CrossRef] [PubMed]
  23. Islam, M.R.; Pervin, T.; Hossain, H.; Saha, B.; Hossain, S.J. Physicochemical and antioxidant properties of honeys from the Sundarbans mangrove forest of Bangladesh. Prev Nutr Food Sci. 2017, 22, 335. [Google Scholar] [CrossRef] [PubMed]
  24. Eraslan, G.; Kanbur, M.; Silici, S.; Karabacak, M. . Beneficial effect of pine honey on trichlorfon induced some biochemical alterations in mice. Ecotoxicol. Environ. Saf. 2010, 73, 1084–1091. [Google Scholar] [CrossRef] [PubMed]
  25. Deacon, R.M. Measuring the strength of mice. J Vis Exp. 2013, 76, e2610. [Google Scholar] [CrossRef]
  26. Pavithra, V.; Janhavi, P.; Natasha, J.; Neelam, R.; Mrityunjaya, M.; Selvi, M.K.; Ravindra, P.V. A blend of cod liver oil and virgin coconut oil improves the endurance performance in mice. Sport Sci. Health. 2022, 18, 1229–1241. [Google Scholar] [CrossRef]
  27. Huang, C.C.; Hsu, M.C.; Huang, W.C.; Yang, H.R.; Hou, C.C. Triterpenoid-rich extract from Antrodia camphorata improves physical fatigue and exercise performance in mice. J Evid Based Complementary Altern Med. 2012. [CrossRef]
  28. Hsiao, C.Y.; Hsu, Y.J.; Tung, Y.T.; Lee, M.C.; Huang, C.C.; Hsieh, C.C. Effects of Antrodia camphorata and Panax ginseng supplementation on anti-fatigue properties in mice. J Vet Med Sc. 2018, 80, 284–291. [Google Scholar] [CrossRef] [PubMed]
  29. Aktar, S.; Jahan, M.; Alam, S.; Mohanto, N.C.; Arefin, A.; Rahman, A. . Saud, Z.A. Individual and combined effects of arsenic and lead on behavioral and biochemical changes in mice. Biol. Trace Elem. Res. 2017, 177, 288–296. [Google Scholar] [CrossRef]
  30. do Nascimento Salvador, P.C.; Nascimento, E.M.F.; Antunes, D.; Guglielmo, L.G.A.; Denadai, B.S. Energy metabolism and muscle activation heterogeneity explain V ̇ O 2 ̇V_O_2 slow component and muscle fatigue of cycling at different intensities. Exp Physiol. 2023, 108, 503–517. [Google Scholar] [CrossRef]
  31. Belluardo, N.; Westerblad, H.; Mudó, G.; Casabona, A.; Bruton, J.; Caniglia, G.; Ibáñez, C.F. Neuromuscular junction disassembly and muscle fatigue in mice lacking neurotrophin-4. Mol. Cell. Neurosci. 2001, 18, 56–67. [Google Scholar] [CrossRef]
  32. Al-Sayegh, N.; Al-Obaidi, S.; Nadar, M. Smoking impact on grip strength and fatigue resistance: implications for exercise and hand therapy practice. J Phys Act Health. 2014, 11, 1025–1031. [Google Scholar] [CrossRef] [PubMed]
  33. Georgoulis, M.; Chrysohoou, C.; Georgousopoulou, E.; Damigou, E.; Skoumas, I.; Pitsavos, C.; Panagiotakos, D. Long-term prognostic value of LDL-C, HDL-C, lp (a) and TG levels on cardiovascular disease incidence, by body weight status, dietary habits and lipid-lowering treatment: the ATTICA epidemiological cohort study (2002–2012). Lipids Health Dis. 2022, 21, 141. [Google Scholar] [CrossRef] [PubMed]
  34. Van Horn, L.; McCoin, M.; Kris-Etherton, P.M.; Burke, F.; Carson, J.A.S.; Champagne, C.M. . Sikand, G. The evidence for dietary prevention and treatment of cardiovascular disease. J Am Diet Assoc. 2008. 108, 287–331. [CrossRef]
  35. Famurewa, A.; Folawiyo, A.M.; Epete, M.A.; Onuoha, M.; Igwe, E. Consumption of caffeinated energy drink induces alterations in lipid profile and hepatic aminotransferases in experimental rats. J Chem Pharm Res. 2015, 7, 363–369. [Google Scholar]
  36. Belo-Silva, A.E.; Silva, N.K.D.G.T.; Marianno, P.; de Araújo Costa, G.; da Rovare, V.P.; Bailey, A. . Camarini, R. Effects of the combination of chronic unpredictable stress and environmental enrichment on anxiety-like behavior assessed using the elevated plus maze in Swiss male mice: Hypothalamus-Pituitary-Adrenal Axis-mediated mechanisms. Horm. Behav. 2024, 162, 105538. [Google Scholar] [CrossRef] [PubMed]
  37. Tripathi, B.N.; Mehta, S.K.; Amar, A.; Gaur, J.P. Oxidative stress in Scenedesmus sp. during short-and long-term exposure to Cu2+ and Zn2+. Chemosphere. 2006, 62, 538–544. [Google Scholar] [CrossRef] [PubMed]
  38. Margonis, K.; Fatouros, I.G.; Jamurtas, A.Z.; Nikolaidis, M.G.; Douroudos, I.; Chatzinikolaou, A. . Kouretas, D. Oxidative stress biomarkers responses to physical overtraining: implications for diagnosis. Free Radic Biol Med. 2007, 43, 901–910. [Google Scholar] [CrossRef] [PubMed]
  39. Bloomer, R.J.; Goldfarb, A.H.; Wideman, L.; McKenzie, M.J.; Consitt, L.A. Effects of acute aerobic and anaerobic exercise on blood markers of oxidative stress. J Strength Cond Res. 2005, 19(2), 276–285. [Google Scholar] [CrossRef] [PubMed]
  40. Peake, J.M.; Suzuki, K.; Coombes, J.S. The influence of antioxidant supplementation on markers of inflammation and the relationship to oxidative stress after exercise. J Nutr Biochem. 2007, 18, 357–371. [Google Scholar] [CrossRef]
  41. Rezzani, R.; Franco, C. Liver, Oxidative Stress and Metabolic Syndromes. Nutrients 2021, 13, 1–4. [Google Scholar] [CrossRef]
  42. Loboda, A.; Damulewicz, M.; Pyza, E.; Jozkowicz, A.; Dulak, J. Role of Nrf2/HO-1 System in Development, Oxidative Stress Response and Diseases: An Evolutionarily Conserved Mechanism. Cell. Mol. Life Sci. 2016, 73, 3221–3247. [Google Scholar] [CrossRef] [PubMed]
  43. Bao, J.; Ding, R. , Zou, L.; Zhang, C.; Wang, K.; Liu, F.;... He, C. Forsythiae fructus inhibits B16 melanoma growth involving MAPKs/Nrf2/HO-1 mediated anti-oxidation and anti-inflammation. Am. J. Chin. Med. 2016, 44, 1043–1061. [Google Scholar] [CrossRef] [PubMed]
  44. Liu, C.; Xu, H.; Fu, S.; Chen, Y.; Chen, Q.; Cai, Z. ;... Wang, Z. Sulforaphane ameliorates bladder dysfunction through activation of the Nrf2-ARE pathway in a rat model of partial bladder outlet obstruction. Oxid Med Cell Longev. 2016. [Google Scholar] [CrossRef] [PubMed]
  45. Prabu, S.M.; Muthumani, M. RETRACTED ARTICLE: Silibinin ameliorates arsenic induced nephrotoxicity by abrogation of oxidative stress, inflammation and apoptosis in rats. Mol. Biol. Rep. 2012, 39, 11201–11216. [Google Scholar] [CrossRef]
  46. Sarkar, S.; Mukherjee, S.; Chattopadhyay, A.; Bhattacharya, S. Low dose of arsenic trioxide triggers oxidative stress in zebrafish brain: expression of antioxidant genes. Ecotoxicol Environ Saf. 2014, 107, 1–8. [Google Scholar] [CrossRef] [PubMed]
  47. Lu, T.H.; Tseng, T.J.; Su, C.C.; Tang, F.C.; Yen, C.C.; Liu, Y.Y. . Chen, Y.W. Arsenic induces reactive oxygen species-caused neuronal cell apoptosis through JNK/ERK-mediated mitochondria-dependent and GRP 78/CHOP-regulated pathways. Toxicol. Lett. 2014, 224, 130–140. [Google Scholar] [CrossRef]
  48. Agarwal, A.; Bolisetty, S. Adaptive responses to tissue injury: role of heme oxygenase-1. Trans. Am. Clin. Climatol. Assoc. 2013, 124, 111. [Google Scholar] [PubMed]
  49. Weiss-Sadan, T.; Ge, M.; Hayashi, M.; Gohar, M.; Yao, C.H.; de Groot, A.; Harry, S.; Carlin, A.; Fischer, H.; Shi, L.; Wei, T.Y.; Adelmann, C.H.; Wolf, K.; Vornbäumen, T.; Dürr, B.R.; Takahashi, M.; Richter, M.; Zhang, J.; Yang, T.Y.; Vijay, V.; Fisher, D.E.; Hata, A.N.; Haigis, M.C.; Mostoslavsky, R.; Bardeesy, N.; Papagiannakopoulos, T.; Bar-Peled, L. NRF2 Activation Induces NADH-Reductive Stress, Providing a Metabolic Vulnerability in Lung Cancer. Cell Metab. 2023, 35, 487–503. [Google Scholar] [CrossRef]
  50. Singh, M.K.; Yadav, S.S.; Yadav, R.S.; Chauhan, A.; Katiyar, D.; Khattri, S. Protective effect of Emblica-officinalis in arsenic induced biochemical alteration and inflammation in mice. SpringerPlus. 2015, 4, 1–8. [Google Scholar] [CrossRef]
  51. Wruck, C.J.; Streetz, K.; Pavic, G.; Götz, M.E.; Tohidnezhad, M.; Brandenburg, L.O. . Pufe, T. Nrf2 induces interleukin-6 (IL-6) expression via an antioxidant response element within the IL-6 promoter. J. Biol. Chem. 2011, 286, 4493–4499. [Google Scholar] [CrossRef]
  52. Chen, D.; Li, Z.; Bao, P.; Chen, M.; Zhang, M.; Yan, F. . Ye, J. Nrf2 deficiency aggravates Angiotensin II-induced cardiac injury by increasing hypertrophy and enhancing IL-6/STAT3-dependent inflammation. Biochim Biophys Acta. 2019, 1865, 1253–1264. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Effect of exercise with the supplementation of Sundarbans Honey and the Eds Speed™ and Royal Tiger™ on mice’s strength (A) Inverted screen test or grip test and (B) Weights test, and endurance capability (C) Forced swim test. Control, Sp, RT, SH, Ex, Ex+SH, Ex+Sp, and Ex+RT groups of mice were expressed as mean with SEM values, where n = 6 for each group of mice. Significantly different means were performed by Welch’s t-test (****p < 0.0001 ***p < 0.001, **p < 0.01, *p < 0.05) and ordinary one-way ANOVA (***p <0.0001).
Figure 1. Effect of exercise with the supplementation of Sundarbans Honey and the Eds Speed™ and Royal Tiger™ on mice’s strength (A) Inverted screen test or grip test and (B) Weights test, and endurance capability (C) Forced swim test. Control, Sp, RT, SH, Ex, Ex+SH, Ex+Sp, and Ex+RT groups of mice were expressed as mean with SEM values, where n = 6 for each group of mice. Significantly different means were performed by Welch’s t-test (****p < 0.0001 ***p < 0.001, **p < 0.01, *p < 0.05) and ordinary one-way ANOVA (***p <0.0001).
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Figure 2. . Percentage of time spent by mice in the open arm and closed arm of the EPM. The time spent in open arms and closed arms of control, Sp, RT, SH, Ex, Ex+SH, Ex+Sp, and Ex+RT groups of mice is expressed in box plots. The data were expressed as Mean ± SEM (where n = 6). The central line that crosses the boxes and the upper and lower bounds of the boxes represent the maximum, minimum, and median values. Using Welch’s t-test, groups were compared (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05) and ordinary one-way ANOVA (p<0.0001).
Figure 2. . Percentage of time spent by mice in the open arm and closed arm of the EPM. The time spent in open arms and closed arms of control, Sp, RT, SH, Ex, Ex+SH, Ex+Sp, and Ex+RT groups of mice is expressed in box plots. The data were expressed as Mean ± SEM (where n = 6). The central line that crosses the boxes and the upper and lower bounds of the boxes represent the maximum, minimum, and median values. Using Welch’s t-test, groups were compared (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05) and ordinary one-way ANOVA (p<0.0001).
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Figure 3. Hepatic NRF2 and HO-1 concentrations in mice after exhaustive swimming. (A) NRF2 ng/mg (B) HO-1 ng/mg. Control, Sp, RT, SH, Ex, Ex+SH, Ex+Sp, and Ex+RT groups of mice were presented as mean with SEM values, where n = 6 for each group of mice. Significantly different means were performed by Welch’s t-test (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05) and ordinary one-way ANOVA (***p <0.0001).
Figure 3. Hepatic NRF2 and HO-1 concentrations in mice after exhaustive swimming. (A) NRF2 ng/mg (B) HO-1 ng/mg. Control, Sp, RT, SH, Ex, Ex+SH, Ex+Sp, and Ex+RT groups of mice were presented as mean with SEM values, where n = 6 for each group of mice. Significantly different means were performed by Welch’s t-test (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05) and ordinary one-way ANOVA (***p <0.0001).
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Figure 4. Liver tissue IL-6 levels in experimental mice after exhaustive swimming. Control, Sp, RT, SH, Ex, Ex+SH, Ex+Sp, and Ex+RT groups of mice were presented as mean with SEM values, where n = 6 for each group of mice. Significantly different means were performed by Welch’s t-test (****p < 0.0001, ***p < 0.001, **p < 0.01,) and ordinary one-way ANOVA (***p <0.0001).
Figure 4. Liver tissue IL-6 levels in experimental mice after exhaustive swimming. Control, Sp, RT, SH, Ex, Ex+SH, Ex+Sp, and Ex+RT groups of mice were presented as mean with SEM values, where n = 6 for each group of mice. Significantly different means were performed by Welch’s t-test (****p < 0.0001, ***p < 0.001, **p < 0.01,) and ordinary one-way ANOVA (***p <0.0001).
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Table 1. Effect of exercise with treatment of SH and EDs on plasma lipid profile in mice.
Table 1. Effect of exercise with treatment of SH and EDs on plasma lipid profile in mice.

Blood parameters(mg/dL)
Control
Sp
RT
SH
Ex
Ex+SH
Ex+Sp
Ex+RT
P value
LDL 60.19±1.76
 72.87±2.78a 72.3±1.63a 46.23±1.84a 46.65±1.78a 35.27±1.00b 75.53±2.08c 75.6±1.36d ap<0.01
b,c,dp<0.001
HDL 30.2±0.59 22.05±0.68a 25.21±0.80a 47.26±1.06a 49.23±0.73a 57.08±1.22b 25.01±1.00c
 27.96±0.96d ap<0.001
b,c,dp<0.001
TG 52.13±1.52 64.67±1.55a 65.1±1.16a 36.15±0.73a 38.68±0.97a 34.54±0.88b 80.91±1.02c 81.5±1.01d ap<0.001
b,c,dp<0.05
TC 160.7±2.99 229.9±6.07a 192.3±2.62a 130±2.33a 137.4±2.01a 128.7±1.30b 236.5±2.24c 216.7±7.21d ap<0.001
b,c,dp<0.01
Notes: LDL=Low-density lipoproteins, HDL= High-density lipoproteins, TG=Triglyceride, TC=Total Cholesterol. Data are expressed as Mean ± SEM, n = 6 for each group of mice. significantly different from the control mice group and b,c, and dSignificantly different among Ex vs Ex+SH, Ex vs Ex+Sp, and Ex vs Ex+RT mice group. Group comparisons were performed by Welch’s t-test and ordinary one-way ANOVA (p < 0.0001).
Table 2. Effect of exercise with the addition of SH and EDs on fatigue-related biomarkers in mice after exhaustive swimming.
Table 2. Effect of exercise with the addition of SH and EDs on fatigue-related biomarkers in mice after exhaustive swimming.
Blood parameter Control Sp RT SH Ex Ex+SH Ex+Sp Ex+RT p-value
ALP(U/L) 124.5±3.26
 142.2±4.97a 140.4±4.26a
 90.62±5.32a 99.5±4.5a 79.12±3.63b
 143.1±8.71c
 139.5±5.59d ap<0.05
b,c,dp<0.01
AST(U/L) 147.7±4.51 176.7±3.55a 162.3±3.34a
 127.8±2.38a 130.7±1.99a 122.5±2.21b
 178.5±2.81c
 176.5±4.72d ap<0.05
b,c,dp<0.05
ALT(U/L) 77.17±4.6 93.5±1.97a 92.74±4.67a 61.67±3.92a 64.5±2.80a 45.5±2.07b
 94.33±4.55c
 91.27±4.49d ap<0.05
b,c,dp<0.001
LDH
(U/L)		 2659±22.26 3107±26.1a 3079±18.36a 2135±44.02a 1715±46.56a 1112±48.27b 3183±41.54c 3103±30.03d ap<0.0001
b,c,dp<0.001
CK(U/L) 226.6±5.18 248±2.8a 247±2.77a 192.4±9.21a 160.5±5.79a 129.2±4b 243.3±12.59c 248.8±11.79d ap<0.05
b,c,dp<0.01
LA (mg/dL) 162.5±2.17 182.8±2.48a 178.7±2.18a 123.3±2.17a 107.3±2.62a 98.5±2.32b 189.3±2.52c 187.8±2.13d ap<0.001
b,c,dp<0.05
BUN mg/dL 79.3±3.74 96.6±3.18a 98.6±4.50a 67.39±2.34a 61.34±4.64a 47.69±3.05b 94.51±5.15b 95.28±7.28d ap<0.05
b,c,dp<0.01
Glucose (mg/dL) 16.33±0.47 18.41±0.51a 19.52±0.66a
 14.13±0.36a 13.22±0.33a 11.75±0.46b
 18.7±0.75c
 18.29±0.74d ap<0.05
b,c,dp<0.001
Notes: ALP=Alkaline Phosphatase, AST=Aspartate Transaminase, ALT=Alanine Transaminase, LDH=Lactate Dehydrogenase, CK=Creatine Kinase, LA=Lactic acid, BUN=Blood Urea Nitrogen, Data are expressed as Mean ± SEM, n = 6 for each group of mice. significantly different from the control mice group and b,c, and dSignificantly different among Ex vs Ex+SH, Ex vs Ex+Sp, and Ex vs Ex+RT mice groups. Group comparisons were performed by Welch’s t-test and ordinary one-way ANOVA (p< 0.0001).
Table 3. Exercise, SH, and EDs influence antioxidant enzymes in mice liver tissue after exhaustion.
Table 3. Exercise, SH, and EDs influence antioxidant enzymes in mice liver tissue after exhaustion.
Enzymes activity Control Sp RT SH Ex Ex+SH Ex+Sp Ex+RT p value
CAT (umol/mg) 31.18±0.77 25±1.30a 23.99±1.33a 47.38±1.20a 43.85±1.26a 52.96±1.34b 28.59±0.83c 29.9±0.82d ap<0.01
b,c,dp<0.001
rGR (umol/mg) 3.275±0.01 3.13±0.03a 3.11±0.03a 4.38±0.02a 4.32±0.01a 4.71±0.02b 3.33±0.04c 3.31±0.01d ap<0.01
b,c,dp<0.0001
SOD (U/mg) 3.81±0.03 3.45±0.06a 3.37±0.05a 4.63±0.06a 4.48±0.04a 4.98±0.01b 3.55±0.06c 3.51±0.02d ap<0.001
b,c,dp<0.0001
Notes: CAT=Catalase , rGR=Glutathione Reductase, SOD= Superoxide Dismutase. Data are expressed as Mean ± SEM, n = 6 for each group of mice. significantly different from the control mice group and b,c, significantly different among Ex vs Ex+SH, Ex vs Ex+Sp, and Ex vs Ex+RT mice groups. Group comparisons were performed by Welch’s t-test and ordinary one-way ANOVA (p< 0.0001).
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