Euscaphic acid relieves fatigue by enhancing anti-oxidative and anti-inflammatory effects

Abstract Background Oxidative stress and inflammation are involved in chronic fatigue. Euscaphic acid (EA) is an active compound of Eriobotrya japonica (Loquat) and has anti-oxidative effect. Methods The goal of present study is to prove whether EA could relieve fatigue through enhancing anti-oxidant and anti-inflammatory effects in in vitro/in vivo models. Results EA notably improved activity of superoxide dismutase (SOD) and catalase (CAT), while EA reduced levels of malondiadehyde (MDA) and inflammatory cytokines without cytotoxicity in H2O2-stimulated in myoblast cell line, C2C12 cells. EA significantly reduced levels of fatigue-causing factors such as lactate dehydrogenase (LDH) and creatin kinase (CK), while EA significantly incresed levels of anti-fatigue-related factor, glycogen compared to the H2O2-stimulated C2C12 cells. In treadmill stress test (TST), EA significantly enhanced activities of SOD and CAT as well as exhaustive time and decreased levels of MDA and inflammatory cytokines. After TST, levels of free fatty acid, citrate synthase, and muscle glycogen were notably enhanced by oral administration of EA, but EA decreased levels of lactate, LDH, cortisol, aspartate aminotransferase, alanine transaminase, CK, glucose, and blood urea nitrogen compared to the control group. Furthermore, in forced swimming test, EA significantly increased levels of anti-fatigue-related factors and decreased excessive accumulations of fatigue-causing factors. Conclusions Therefore, the results indicate that potent anti-fatigue effect of EA can be achieved via the improvement of anti-oxidative and anti-inflammatory properties, and this study will provide scientific data for EA to be developed as a novel and efficient component in anti-fatigue health functional food.


Introduction
Fatigue is a complex pathological and physiological phenomenon with many etiologies, which can be divided into mental and physical fatigue [1,2]. Fatigue is a frequent condition experienced by healthy individuals as well as patients. Coronavirus Disease 2019 (COVID- 19), which is currently prevalent worldwide, is in an international public health emergency [3]. The most common symptoms of patients with COVID-19 are shortness of breath, cough, fever, and fatigue [4].
Reactive oxygen species (ROS) are formed because of the electron receptivity of O 2 containing many free radicals and highly reactive molecules and are scavenged by activation of anti-oxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) [5]. ROS have been known as a defense response against microbial and virus invasion, which acts as a weapon of immune cells during inflammatory reactions [6].
The appropriate amount of ROS is beneficial for muscle repair, but ROS of relative high levels increased by repetitive muscle contraction due to exhaustive exercise causes oxidative stress [7]. Oxidative stress is caused by an imbalance between production and elimination of ROS, increased ROS leads to potentially damaged DNA, protein, and lipids and causes mitochondrial dysfunction and cell damage that play a key role in various inflammatory diseases [8,9]. Furthermore, oxidative stress is associated with the infections caused by COVID-19 and other respiratory viruses [10]. An excessive oxidative stress increases production and expression of inflammatory cytokines including tumor necrosis factor (TNF)-a, interleukin (IL)-8, IL-6, IL-4, and IL-1b [11]. It has been reported that the inflammatory cytokine level of fatigue patients is always higher than that of normal people [12]. Oxidative stress-induced inflammatory cytokine production leads to muscle fatigue [13][14][15]. Anti-oxidants and antiinflammatory agents such as Korean traditional medicines, natural compounds, and nutrients have been reported for improving endurance capacity, and such materials are being applied in delaying and relieving the degree of fatigue [16].
Euscaphic acid (EA) is an active compound of Eriobotrya japonica (Loquat, EJ) and has anti-oxidative and anti-inflammatory effects in in vitro model [17,18]. Because oxidative stress and inflammatory reactions are closely related to fatigue, we speculated that EA may has an anti-fatigue effect. However, the anti-fatigue effects of EA have not been reported yet. The goal of this study was to prove whether EA could relieve fatigue through enhancing anti-oxidant and anti-inflammatory effects in myoblast C2C12 cells in vitro model and treadmill stress test (TST) and forced swimming test (FST) in vivo models.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay MTT was used to assess C2C12 cell cytotoxicity by EA. The differentiated C2C12 cell was seeded into 24-well plates and then were treated with EA for 24 h. MTT was then added to each well as previously performed [19]. Living cells convert the water-soluble MTT to an insoluble purple formazan. The formazan is then solubilized, and its concentration was determined by optical densitometer (at 540 nm).

Enzyme-linked immunosorbent assay (ELISA)
The amount of cytokine in cell supernatant and serum was estimated by ELISA according to previous study [19].

Animals
Male, 4-week-old ICR mice weighing approximately 20 $ 23 g were purchased from the Dae-Han Experimental Animal Center (Eumsung, Chungbuk, Republic of Korea). Mice were kept in individual cages (n ¼ 5/cage) for 1 week at 22 ± 2 C, a 12:12 h light: dark cycle (lights on at 08:00 AM), and 50 ± 5% humidity. All experimental procedures were conducted in accordance with current laws and guidelines for the care and use of laboratory animals approved by the Animal Ethics Committee of Kyung Hee University [KHUASP (SE)-20-263].

TST
The mice in each group were forced to run on the treadmill for 30 min once a week for 4 weeks (Supplementary Figure 1). Each mouse begins running at 10 m/min for 10 min, which serves as warm-up and familiarization. Subsequently, the treadmill speed is increased to 16 m/min for 10 min and next to 21 m/min for 10 min. Each mouse begins running at 10 m/ min for 5 min and next the treadmill speed is increased by 5-10 m/min each minute until exhaustion [20]. The dosage of EA was determined to be 1 mg/kg by reference a previous report [21]. EA (1 mg/kg) was orally administered daily. Distilled water (D.W.) was administered orally for 28 days as a control group. The exhaustive time of TST was evaluated on the last day of this experiment.

FST
FST was performed once a week for 4 weeks according to a previous report [22]. On the 28th day, FST was performed for 6 min. The immobility time was measured for 4 min after delay of 2 min. EA (1 mg/kg) was orally administered daily. D.W. was administered orally for 28 days as a control group.

Statistical analysis
The statistical analysis of data was done using SPSS 12.0 software. In vitro experimental results were expressed as mean-± standard error mean (SEM) from the results of at least three independent experiments in duplicate. In vivo experimental results were presented as the mean ± SEM (n ¼ 5/ group). To analysis the anti-fatigue effects of EA, one-way analysis of variance (ANOVA), followed by Turkey multiple comparison test and the independent t-test were used. P-value less than 0.05 was considered statistically significant.

Anti-oxidative and anti-inflammatory effects of EA in H 2 O 2 -stimulated C2C12 cells
EA was isolated from EJ extract. The amount of EA in EJ was about 0.1-1 mg/g (Supplementary Figure 2). The purity of EA was over 95%. EA isolated from EJ was used in the present study.
First, we performed the MTT assay to determine the concentration of EA without cytotoxicity in C2C12 cells. As shown in Fig. 1A, EA (0.01, 0.1, and 1 lg/ml) did not show the cytotoxic effect to C2C12 cells. Thus, the doses of 0.01, 0.1, and 1 lg/ml were used in the next experiment to observe the dose-dependent anti-fatigue effect of EA.
To observe whether EA could regulate oxidative stress in C2C12 cells, effects of EA on activities of SOD and CAT and levels of MDA were investigated in H 2 O 2 -stimulated C2C12 cells. Compared to the H 2 O 2 -stimulated C2C12 cells, treatment with EA significantly increased the activities of SOD and CAT, which were also dose-dependent (Figure 1(B,C), p < 0.05). Increased MDA levels refer to the oxidative stress in a cell or organism [23]. The results indicated that EA notably reduced the amount of MDA in the H 2 O 2 -stimulated C2C12 cells in a dose-dependent manner (Figure 1(D), p < 0.05). In addition, EA significantly reduced production of TNF-a and IL-6 compared to the H 2 O 2 -stimulated C2C12 cells (Figure 1(E,F), p < 0.05).
To observe whether EA would exert anti-fatigue effects through its anti-oxidant and ant-inflammatory effects, we analyzed the levels of fatigue-related factors in the H 2 O 2stimulated C2C12 cells. As shown in Table 1, EA significantly reduced the levels of fatigue-causing factors such as LDH and CK compared to the H 2 O 2 -stimulated C2C12 cells, while EA significantly incresed the levels of anti-fatigue-related factor, glycogen compared to the H 2 O 2 -stimulated C2C12 cells (p < 0.05). These effects were also concentration dependent.
Anti-oxidative effect of EA after TST Oxidative stress could significantly affect the etiology of chronic fatigue syndrome and that anti-oxidants might be a possible treatment [24]. An anti-fatigue effect of EA was observed by performing TST. In TST fatigue animal model, the effects of EA on the anti-fatigue and enzymatic anti-oxidant activities were shown in Figure 2. The exhaustive time of EA-administered mice was notably longer than that of the  control mice (Figure 2(A), p < 0.05). Next, our results showed that the activity of SOD and CAT was notably enhanced by the oral administration of EA compared to the control mice ( Figure 2(B,C), p < 0.05). Furthermore, activity of SOD was notably higher in the serum, muscle, and liver of EA-administered mice than that of control group. MDA is a product of oxidation of polyunsaturated fatty acids. MDA has frequently been used as an indicator of fatigue in response to exercise [25]. In this study, the MDA levels were significantly decreased in the EA-administrated mice compared to the control mice (Figure 2(D), p < 0.05). The body weights had no significant changes among the groups until the end of the experiment. (Supplementary Figure 3(A)).

Anti-inflammatory effect of EA in serum after TST
To observe whether EA would regulate the amount of inflammatory cytokine through its anti-oxidant effect, ELISA was performed for serum inflammatory cytokine after TST. In the control mice, the amount of TNF-a, IL-6, IL-4, and IL-1b was notably increased compared to the normal mice, while the EA-administered mice exserted a significant decreased in the amount of these cytokines compared to the control mice ( Figure 3, p < 0.05).

Regulatory effect of EA on fatigue-related factors after TST
To observe whether EA could increase the amount of factors related to fatigue improvement, levels of muscle glycogen, free fatty acid, citrate synthase, and glucose were measured after TST by each assay kit and a DRI CHEM NX500 analyzer. The amount of muscle glycogen, free fatty acid, and citrate synthase in serum and muscle was notably enhanced by oral administration of EA compared to the control mice ( Table 2, p < 0.05). The amount of serum glucose was higher in the control mice than normal mice, while the amount of serum glucose in EA-administered mice was notably lower than control mice ( Table 2, p < 0.05).
To observe whether EA could decrease the amount of fatigue-causing factors, lactate, levels of LDH, cortisol, ALT, AST, CK, and BUN in serum was measured after TST. As a result, the amount of serum lactate, LDH, cortisol, ALT, AST, CK, and BUN in the control mice was notably enhanced compared to the normal mice ( Table 2, p < 0.05). However, the amount of serum lactate, LDH, cortisol, ALT, AST, CK, and BUN in the EA-administered mice was lower than that in the control mice (Table 2, p < 0.05).

Anti-fatigue effect of EA after FST
Finally, FST was performed to confirm the anti-fatigue effect of EA. In the FST, EA notably decreased the immobility time compared to the control mice (Table 3, p < 0.05). The body weights had no significant changes among the groups until the end of the experiment. (Supplementary Figure 3B). The amount of SOD, CAT, free fatty acid, glycogen, citrate synthase, MDA, glucose, lactate, LDH, cortisol, ALT, AST, CK, BUN, and inflammatory cytokines in serum, muscle, and liver was analyzed after the last FST. As shown in Table 3, EA notably increased the amount of SOD, CAT, muscle glycogen, citrate synthase, and free fatty acid, while the excessive accumulations of MDA, glucose, lactate, LDH, cortisol, ALT, AST, CK, BUN, and inflammatory cytokines were notably reduced by oral adiminstration of EA (p < 0.05).

Discussion
In the present study, we used C2C12 cells, TST, and FST to evaluate the anti-fatigue effect of EA and typical fatigue-related biochemical factors were determined. The data indicated that EA improved anti-fatigue effect through enhancing anti-oxidative and anti-inflammatory activities.
Fatigue is a phenomenon caused by oxidative stress and inflammatory reactions. Oxidative stress is caused by free radicals and ROS, which increases the inflammatory reaction and worsens fatigue. SOD catalyzes the conversion of superoxide radicals to H 2 O 2 which acts a key role in defense against superoxide radicals. CAT catalyzes the H 2 O 2 to form H 2 O and O 2 [26]. Yu et al. [27] found that ingestion of exogenous anti-oxidants can improve overall physiological conditions while reducing oxidative stress caused by exercise. You et al. [27] reported that oxidative stress can have a major impact on the causes of chronic fatigue syndrome, and that antioxidants may be possible treatments. Many antioxidants such as anwulignan, phenolic compounds, and Moringa oleifera are used to recover from fatigue, and most of them have been reported to have anti-fatigue effects by increasing the activity of SOD and CAT [28][29][30]. MDA is an important lipid peroxidation production, a sensitive indicator that accesses oxidative stress and causes fatigue [31]. Tan et al. [32] reported that ginsenoside Rb1 showed the effects of reversion that it decreased the amount of MDA and enhanced the activity of SOD in the fatigue animal model. Excessive oxidative stress increases the production of inflammatory cytokines [11]. It has been reported that  inflammatory cytokines are increased in fatigue patients compared to normal subjects [12]. Anti-TNF has been reported as an effective treatment for fatigue in inflammatory bowel disease, rheumatoid arthritis, and sarcoidosis, and TNF regulators reduce the inflammatory response by blocking TNF-a [33]. Although these drugs are aimed at inflammatory disease activity as opposed to symptoms of fatigue, the benefits of a crossover condition indicate that the mechanism of fatigue may be similar or may be related to the inflammatory response in the body [33]. In this study, the results indicated that EA enhanced the activities of SOD and catalase, while reduced the amount of MDA and inflammatory cytokines (TNF-a, IL-6, IL-4, and IL-1b) in in vitro and in vivo models. Therefore, we suggest that EA has anti-oxidative and anti-inflammatory effects in in vivo. Fatigue is accompanied by prolonged endurance exercises and causes the tissue and organ damages, such as hepatic dysfunction, acute kidney damage, and muscle damage [34,35]. Several markers are used for tissue and organ damages, such as AST and ALT for hepatic damage, CK and LDH for muscle damage, and BUN and creatinine for renal damage [24]. During oxidative stress and long-term exercise, cortisol is secreted by adrenocorticotropic hormone, causing fatigue [36]. Accumulation of various metabolites, such as lactate, calcium, or ammonia, the depletion of glycogen, free fatty acid, and citrate synthase, which are the main factors of energy production system, and induction of oxidative stress during endurance exercise cause fatigue [24,37,38]. The amount of serum glucose was higher in the control mice than that of normal mice in TST and FST [20]. In the present study, EA increased the amount of muscle glycogen, citrate synthase, and free fatty acid, while reduced the amount of lactate, LDH, cortisol, glucose, ALT, AST, CK, and BUN in the TST and FST models. Therefore, these results suggest that EA has an anti-fatigue effect by enhancing anti-oxidative and anti-inflammatory activities.
In conclusion, these data indicate that potent anti-fatigue effect of EA might be achieved via improvement of anti-oxidative and anti-inflammatory properties. Furthermore, the anti-fatigue effect of EA presented here can be used as basic data for the development of pharmaceutical compounds and health function food.

Author contributions
HJ Jeong and HM Kim contributed to the study conception and design. Material preparation, data collection, and analysis were performed by HY Kim, H Jung, M Kweon, J Kim, SY Choi, HJ Ahn, and CS Park. The first draft of the manuscript was written by HY Kim, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.

Data availability statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.