Keywords
Red Dragon Fruit (RDF), Strenuous exercise, MDA, Improve function cell, Fatigue
Red Dragon Fruit (RDF), Strenuous exercise, MDA, Improve function cell, Fatigue
There are several other enhancements
1. In the abstract, the conclusions are adjusted to the results of the study
2. Methods, Study Design, and Histopathology are described in detail
3. Results, explanation of the legend from figure 1 and 2
4. Conclusion explanation of the objectives and research results obtained
5. A new author was also added.
See the authors' detailed response to the review by Farzaneh Taghian
See the authors' detailed response to the review by Ermita I. Ibrahim Ilyas
Increased frequency, intensity, and duration of regular physical exercise improves performance and delays fatigue in daily work.1,2 All living things, except those that are anaerobic, require oxygen to produce energy efficiently. Oxygen is an essential component of cellular metabolism. Exercise causes an increase in oxygen consumption by 10-12 times in the body, causing oxidative damage to the lipids of various tissues.3–7 Moderate to high-intensity exercise can result in an increase in reactive oxygen species (ROS), free radicals in the body, which is characterized by an increase in malondialdehyde (MDA), and a decrease in superoxide dismutase (SOD) which is an endogenous antioxidant to suppress excess of free radicals. Several studies showed that reactive oxygen species (ROS) formed due to tissue hypoxia during muscle contraction have an adaptive physiological role during physical exercise. Oxidative stress is an imbalance between free radicals and antioxidants. Endogenous antioxidants cannot neutralize free radicals if they are formed excessively.8,9 Oxidative stress causes damage to muscle cells and lungs, known as oxidative damage. It is the breakdown of biomolecules that make up cells due to reactions with free radicals.10 Strenuous physical exercise will increase the growth of the free radicals found in muscle and liver tissue 2 to 3 times in experimental animals, which also will increase ROS. As a defense action, the body will be countered by the endogenous antioxidant system,11 which is known as oxidative stress. This can be seen based on the ability of antioxidants in the tissue to neutralize ROS,12 particularly the antioxidants produced by the body known as endogenous antioxidants that come from outside or exogenous antioxidants. These antioxidants come from food, such as fruit. Red dragon fruit (RDF) has been proven to protect the tissue from damage caused by ROS in the body.8,9,13 This study aimed to examine effect of strenuous exercise on changes malondialdehyde and superoxide dismutase and by ingestion red dragon fruit extract improves MDA, SOD, muscle and lung tissue delay fatigue in rats (Rattus norvegicus).
Animal models, particularly rodents, are widely used in biological sciences, and the findings of animal research are traditionally projected to human response similar to physiological stimuli.14 This article was reported in line with the ARRIVE guidelines. The study was a randomized post-test-only control group approved by the Animal Research Ethics Committee, Department of Biology - Faculty of Mathematics and Science, Universitas Sumatera Utara (approval number 0005/KEPH-FMIPA/2020).
In this study, we used 25 three-month-old male rats with an average weight of 200 g. The rats were obtained from the Animal House Unit of the Biology Laboratory, Universitas Sumatera Utara, Indonesia. All rats were maintained in groups in experimental animal cages in the laboratory. The cage (30 cm × 20 cm × 10 cm) was made of plastic and covered with fine wire mesh. The cage base was covered with rice husks with a thickness of 0.5–1 cm, which were replaced every day during the study. The room light was controlled to deliver a 12 h light/12 h dark cycle, the temperature was set to 25–27 °C, and the humidity of the room was adjusted to a normal range of 35–50%. The rats were fed standard rat pellets and given tap water ad libitum.
We used an in vitro experimental method with a true experimental design and a randomised post-test for the control group. Simple random sampling was used to categorise the laboratory rats into five groups as follows: group K1 with no activity and no RDF; group K2 subjected to strenuous exercise without RDF (Red Dragon Fruit); and groups P1, P2, and P3 subjected to strenuous exercise and treated with 75, 150, and 300 mg kg−1 body weight of RDF extract, respectively. In the fruit market, it is easy to find RFP fruit, acquired from farmers in Indonesia, was peeled, washed, cut into small pieces, and then dried in a drying cabinet. Next, the fruit was blended using a blender, and the extract was obtained by the maceration method with 96% ethanol, which was distilled by 10 times the weight of RDF. The RDF powder was stored in a container with 96% ethanol (ratio of 1:7, fruit powder: ethanol) and then soaked for 3 d. The RDF was macerated using a rotary evaporator at 45 °C until the extract thickened. The macerated RDF was extracted using 96% ethanol. The remaining extract was then evaporated in a water bath until a thick extract was obtained. Next, 100 mg RDF extract was weighed and crushed using a pestle and mortar. Subsequently, carboxymethylcellulose Na solution (0.5% w/v) was slowly added until a homogeneous extract was obtained, and the resulting volume was 10 mL. This final RFP extract was administered to the rats at appropriate dosages; specifically, rats weighing 200 g were fed 1.5, 3.0, or 6.0 mL of the RFP extract suspension, which corresponded to doses of 75, 150, or 300 mg kg−1 body weight, respectively.
The strenuous exercise given to all rats involved a morning swim between 08 – 09 AM for 20 minutes a day three times a week for four weeks.15 The rats were treated with RDF extract every day for four weeks respectively at half an hour before the strenuous exercise. All rats completed the strenuous exercise test. At the end of the study, the results were obtained in the fourth week of exercise testing until the maximum exercise was swimming until almost drowning.
One of the biomarkers of oxidative stress is a high level of malondialdehyde (MDA) and decreased SOD activity due to excessive lipid peroxidation processes in cells. One way to control excessive oxidative stress is by consuming antioxidants from food (exogenous antioxidants); one source of exogenous antioxidants is RDF, which consists of Group P1 treated with 75 mg kg−1.bw; Group P2 with 150 mg kg−1.bw; and Group P3 with 300 mg kg−1.bw of RDF extract.
The consumption of RDF extract suppresses the increase in free radicals due to strenuous exercise. It increases SOD, an endogenous antioxidant, so oxidative stress does not occur, and repair mitochondrial cell function has fatigue delaying effect.
All of the rats completed the strenuous exercise course. They experienced maximal physical activity, i.e., swimming, until they almost drowned. Blood for MDA and SOD was taken consecutively was assessed with enzyme-linked immune sorbent assay (ELISA) method and spectrophotometry with a wavelength of 450 nm. The assessment was done by using mouse malondialdehyde ELISA kit (Brand Bioassay TL, catalogue: EO625Mo). The SOD kit (Brand Bioassay TL, catalogue: EO168Ra) rat super oxidase dismutase ELISA kit was determined using the equation obtained from the standard curve.16–18
Muscle soleus and lung tissue samples were collected by performing a biopsy to determine the degree of muscle damage based on haematoxylin and eosin (H&E) staining. The soleus muscle tissues of the rats were collected and fixed with 10% formalin for 24 h. The muscle and lung tissues were embedded in paraffin, sectioned to a 4 μm thickness, and stained via H&E staining. The stained sections were then examined under a light microscope (400× magnification) with 10 fields of view to determine the degree of damage concerning inflammatory cells and necrosis. The examination was conducted by a pathologist who was blinded to the applied treatment.
Normality was assessed with Shapiro-Wilk test (p>0.05). Data Analysis was done by one-way analysis of variance (ANOVA) to indicate the effect of treatments for each group. The data were analysed with SPSS version 25 software and presented in tabulated and graphical forms as means and standard deviation. Significant differences were determined at p<0.05. The Post Hoc Bonferroni test was conducted after the significant results were obtained.
During consumption of the antioxidant RDF extract, all rats were accustomed to reducing stress-related disorders and seemed to be in good condition. No rats were poisoned, and there were no deaths in the experiment period.
A normality test indicated that the data are normally distributed (Table 1).
Strenuous exercise followed by RDF extract ingestion was compared for fatigue in terms of duration and time; before (24.55±1.38 minute) and after (95.31±7.82 minute) and led to a significant difference of 39% (p<0.01).
The results of One-Way ANOVA test for groups K2, P1, P2, and P3 showed significant differences (Table 2). It is known that the measurement of MDA levels is a marker for assessing the increase in free radical production in rats treated with physical activity.
Groups | MDA level (μg/dL) | p-value |
---|---|---|
K1 | 0.4191±0.2080bc | p<0.05 |
K2 | 0.5471±0.0399c | |
P1 | 0.3120±0.1357ab | |
P2 | 0.3159±0.0377ab | |
P3 | 0.2531±0.0284a |
MDA expression (Table 2 and Figure 1) was decreased after treatment with RDF extract (0.4191 vs 0.5471 vs 0.3120 vs 0.3159 vs 0.2531 μg/dL). The P3 group had the lowest score compared to the other groups. This study showed a significant reduction between groups.
The study results compared the MDA of rats after ingestion of RDF extract, which was tested with the Post Hoc test - Bonferroni. In the K2 vs. P1 group, there was a significant difference of p<0.05, the K2 vs. P2 group had a significant difference of p<0.05, and the K2 vs. P3 group had an increased significant difference of p<0.01.
The free radicals in the body are balanced with endogenous defense mechanisms, and the body will produce antioxidants with an anti-free radical effect. In this study, the K2 group performed physical activity and SOD levels were 0.4632±0.2449 ng/mL. There was an increase in SOD levels in the K1 group (0.8647±0.1744 ng/mL) that did not perform physical activity. The increase in SOD continued with RDF extract treatment in groups K1 (1.3499±0.1359 ng/mL), P2 (1.9370±0.0236 ng/mL) and P3 (1.9521±0.0239 ng/mL). The three groups were given RDF treatment and showed significant differences (p<0.05), analysed with the One-Way ANOVA test (Table 3 and Figure 2).
Groups | SOD level (ng/mL) | p-value |
---|---|---|
K1 | 0.8647±0.1744b | p<0.05 |
K2 | 0.4632±0.2449a | |
P1 | 1.3499±0.1359c | |
P2 | 1.9370±0.0236d | |
P3 | 1.9521±0.0239d |
The histopathological examination were observed under a microscope. It was seen that in group K1 changes in muscle and lung tissue did not occur. Ingroup K2 the changes were very significant, and many inflammatory cells and necrosis were observed in both the muscles and lungs. In contrast to P1, the P2 and P3 groups showed a decrease in inflammatory cells. In addition, in these two groups compared to P1, the lungs in the intra-alveolar and the alveolar sacs were dilated, and tissue repair was shown by the hyalinization process. Results showed changes in free radicals that could damage tissue in the positive control group K2. In contrast, the histopathological features of the P1, P2, P3 groups showed lung tissue and muscle cell repair, after being given RDF (Figures 3 and 4).
Free radicals in the skeletal muscles cause muscle fatigue. The free radicals significantly reduce muscle strength, contributing to muscle fatigue during prolonged training.7,19,20 The role of oxidants in muscle fatigue has been investigated in various animal models in vitro and situ during exercise. Oxidants are detectable in muscle at low levels during rest and at higher levels during contractions. RNS depress force production but do not appear to cause fatigue of healthy muscle. In contrast, muscle-derived ROS contribute to fatigue because loss of function can be delayed by ROS- specific antioxidants.21–23 A study showed that exogenous antioxidants derived from food to capture ROS slowed down muscle fatigue, and enzymatic and nonenzymatic antioxidants delayed muscle fatigue during contraction. In the study, the subject characteristics have been standardized in accordance with WHO, adjusted to the provisions of the criteria24–26 in the Research Guideline for Evaluating the Safety and Efficacy of Herbal Medicines.
In skeletal muscles, antioxidants are enzymatic (e.g., Glutathione peroxidase (GPx) and catalase) and nonenzymatic (for example, GSH, uric acid, bilirubin, vitamin E, vitamin C, etc.) function as an integrated antioxidant complex that acts to capture ROS.27,28 These intracellular antioxidants are usually present in cells, cytoplasm, and organelles (for example, mitochondria) whose role is to protect muscle fibres from damage caused by ROS.27,29–31 Endogenous free radicals are formed as a normal response to the chain reaction of respiration in the body. The free radicals in the body are balanced by an endogenous defense system mechanism,32 in which the body produces antioxidants that have an anti-free radical effect. One of the endogenous antioxidants is SOD, which is the body's first line of defense against ROS activation.8 When the level of ROS rises beyond the endogenous defense capacity, oxidative instability, known as oxidative stress, occurs.9,29 Oxidative stress conditions due to free radicals will cause lipid peroxidation of cell membranes and damage cell membrane organization. One of the biomarkers of oxidative stress is a high level of MDA33 and decreased SOD activity due to excessive lipid peroxidation processes in cells.9 One way to control excessive oxidative stress is by consuming antioxidants from food (exogenous antioxidants).34 One source of exogenous antioxidants is RDF that can be found in Indonesia.
In this study, the endogenous antioxidants in the body were superoxide dismutase (SOD), and they are unable to neutralize free radicals. This condition results in an imbalance of free radicals and antioxidants, leading to oxidative damage, as reported in previous studies.35 Unstabilized oxidative stress produces free radicals, which can damage muscle tissue and lungs and cause impaired cell function, which is involved in muscle fatigue. RDF treatment can increase SOD significantly (p<0.05) and function as a good source of several natural antioxidants, such as betalain, polyphenols, and ascorbic acid, as evidenced in previous studies.36,37 During strenuous exercise, the increase in ROS formation during contractile activity is directly related to increased oxygen consumption. This condition results in a 50 or 100 fold increase in mitochondrial activity in the formation of superoxide in skeletal muscle during aerobic contraction.38,39 An increase in oxidative stress, as observed, leads to an increase in lipid peroxidation accompanied by a decline in SOD level activity, as the antioxidants are given depending on the dose affect the increase in SOD levels.28 This improvement in oxidative status suggests that the natural antioxidants in the extract with high doses were responsible for delaying fatigue in this study, as reported in previous studies.40,41 In this study, it was found that the higher the dose given, the greater the SOD, as shown in group P3 that was on treatment so that this SOD level could neutralize free radicals. The SOD enzyme is the first defense system against free radicals. Thus, moderate-intensity regular exercise has been shown to increase antioxidant defenses by increasing the activity of endogenous antioxidant enzymes, such as SOD, glutathione peroxidase, and catalase.42,43 These enzymes can suppress or inhibit the formation of free radicals by breaking the chain reaction so that the product is more stable. This process is known as the antioxidant chain-breaking reaction.
RDF is rich in antioxidants, such as phenol and flavonoid compounds. Phenolic compounds that function as antioxidants neutralize free radicals and peroxide radicals to inhibit lipid oxidation effectively. Flavonoids are exogenous antioxidants that are beneficial in preventing cell damage due to oxidative stress. Its role is to donate hydrogen ions to neutralize the toxic effects from free radicals due to exercise. RDF consumption can also increase the VO2max value.44
The relationship between the provision of antioxidants after treatment with RDF extract is that the administration of exogenous antioxidants helps suppress the spread of free radicals in the body because antioxidants can come from within the body (endogenous) or come from outside the body (exogenous), simultaneously suppressing free radicals due to exercise.
Anthocyanin is one type of flavonoid widely found in dragon fruit,45 which is able to improve mitochondrial function by influencing free radicals. Anthocyanins can suppress the occurrence of lipid peroxidation as an inflammatory response due to free radicals, thereby suppressing the production of MDA.46
An increase in the free radicals in the body causes an imbalance between oxidants and antioxidants. This condition leads to oxidative stress. The earliest known and widely studied cell or tissue mechanism is lipid peroxidation. RDF extract contains anthocyanin pigments which function as antioxidants.18,47,48 Anthocyanins can play a role in inhibiting free radicals that occur due to strenuous exercise. This study examined the provision of RDF extract comprising anthocyanins, one of the types contained in flavonoids, which provides a response to inflammation in the muscles and lung tissue. The presence of anthocyanins repairs damaged tissue so that physiological mitochondrial function returns, as anthocyanins can suppress the occurrence of lipid peroxidation and suppress MDA production so that MDA levels decrease.49,50 Anthocyanins can quickly bind metal ions to form a stable anthocyanin-metal complex. This means that anthocyanins bind to the transitioned ion metal to prevent highly toxic and reactive hydroxyl reactions. In the end, anthocyanins can suppress lipid peroxidation and suppress MDA production to reduce MDA levels.
Strenuous exercise causes an increase in ROS, resulting in increased free radical levels, leading to oxidative stress to occur. Ingesting RDF extracts suppresses the increase. The group that was given RDF doses of 150 mg, and 300 mg performed better than the group with a dose of 75 mg in responding to oxidative stress with strenuous exercise. RDF extract dose resulted in decreased oxidative stress, repaired muscle and lung tissue.
Figshare: Datasets, https://doi.org/10.6084/m9.figshare.15074544.v5.51 This project contains the following underlying data:
Figshare: ARRIVE checklist for ‘The effect of ingestion of Red dragon fruit extract on levels of malondialdehyde and superoxide dismutase after strenuous exercise in rats (Rattus norvegicus)’, https://doi.org/10.6084/m9.figshare. 15074544.v5.51
Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).
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Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Sport and nutrition
Is the work clearly and accurately presented and does it cite the current literature?
Partly
Is the study design appropriate and is the work technically sound?
Partly
Are sufficient details of methods and analysis provided to allow replication by others?
Partly
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Partly
Are the conclusions drawn adequately supported by the results?
No
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Exercise Physiology
Is the work clearly and accurately presented and does it cite the current literature?
No
Is the study design appropriate and is the work technically sound?
No
Are sufficient details of methods and analysis provided to allow replication by others?
No
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
No
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Sport and nutrition
Alongside their report, reviewers assign a status to the article:
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Version 1 18 Oct 21 |
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