Antifatigue Potential of Loquat Leaf Extract in Physical Stress in C2C12 Myotubes and In Vivo Models

Recent studies suggest that oxidative stress could be one of the mechanisms contributing to fatigue. Te purpose of the present study was to determine the antifatigue potential of loquat leaf ( Eriobotrya japonica Lindl., EJ) and its component, chlorogenic acid (CA) in C2C12 myotubes and treadmill stress test (TST)


Introduction
Fatigue is a term used to describe a decrease in physical performance and is related to an increase in the actual/ perceived difculty in performing a task or exercise.It is a common symptom of many physical, neurological, and psychiatric disorders, including depression, human immunodefciency virus infection, Parkinson's disease, cancer, multiple sclerosis, aging, and other potential physical illnesses [1].Fatigue can be categorized into three types, namely, pathological, physiological, and psychological fatigue [2].Pathological fatigue is often experienced by people with chronic illnesses such as those with liver dysfunction, diabetes, and gastrointestinal diseases and can severely impair functional activity and quality of life [3].Physiological fatigue is a state of reduced mental or physical capability which occurs due to the accumulation of metabolic products in the blood which induces fatigue [2].Psychological fatigue occurs due to emotional conficts, anxiety, or boredom [3].Currently, there are several theories regarding the mechanisms which cause fatigue.Te metabolic causes of fatigue include a decrease in phosphocreatine levels, accumulation of protons (acidemia), depletion of glycogen in the muscles and decrease in blood glucose concentrations, and increase in specifc amino acids in the plasma [4].Also, fatigue is caused by the accumulation of lactate through the activation of lactate dehydrogenase (LDH) and the production of cortisol by the adrenocorticotropic hormone in the anterior pituitary gland, whereas it is improved by energy production through glycolysis and the citrate cycle which is activated by hexokinase and citrate synthase.A rapidly emerging concept that has recently received attention is the role of the free radicals in fatigue and the benefts of antioxidants in relieving fatigue [5,6].Te increased oxidative stress during excessive exercise induces fatigue by increasing the release of free radicals and infammatory cytokines [7,8].Terefore, it can be inferred that symptoms of fatigue can improve through the consumption of antioxidants that increase superoxide dismutase (SOD) and catalase activities and regulate infammatory cytokines.
Terapeutic drugs used to relieve fatigue provide temporary benefts and are also associated with side efects.Te antifatigue efect of natural antioxidants has been reported in [9].Terefore, components of natural products are worth investigating, as they are associated with fewer side efects and could be efective in treating or preventing fatigue.
In traditional medicine, loquat leaf (Eriobotrya japonica Lindl., EJ) is used to treat infammatory diseases [10] and has neuroprotective, antiallergic, antioxidant, and antiarthritic properties [11][12][13][14].Furthermore, EJ contains many antioxidants such as chlorogenic acid (CA), quercetin-3-sambubioside, euscaphic acid, and ursolic acid [15].In a study by Jung et al. [15], EJ and CA demonstrated antioxidative and antiinfammatory properties.Terefore, in this study, we hypothesized that EJ might exhibit antifatigue potential through its antioxidative actions.We used the C2C12 myotubes, the treadmill stress test (TST), and the forced swimming test (FST) to investigate the antifatigue potential of EJ and CA and its mechanism.Tese results can be utilized as basic experimental data for the development of health nutraceuticals and herbal medicines to combat fatigue.

Preparation of EJ Extracts.
EJ extracts were provided by COSMAX NBT, INC. (Seongnam, Republic of Korea).Te EJ solution was extracted using water (EJ water extract, WEJ) or ethanol (EJ ethanol extract, EEJ).Each of the extracted solutions was fltered, concentrated, mixed with dextrin, and spray-dried.

Ultrahigh Performance Liquid Chromatography (UHPLC)
and Mass Spectrometry (MS) Analysis.Liquid chromatography/MS (LC/MS) analyses were carried out using an LTQ Orbitrap XL Fourier transform mass spectrometer (Termo Fisher Scientifc, Inc., Waltham, MA, USA) coupled to an Accelar ultrahigh pressure liquid chromatography system (Termo Fisher Scientifc, Inc.).Te chromatographic separation of metabolites was carried out using an ACQ-UITY UPLC ® BEH C 18 column and mobile phases A (water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid).Each compound was detected with a photodiode array at 200 ∼ 500 nm.Te MS analysis was performed with polarity switching with the following parameters for the MS/ MS scan: m/z range of 150−1,500.High-resolution mass spectra were acquired on the LTQ Orbitrap XL (Supplementary Figure 1).To determine the Kovats retention indices, we proceeded as follows: a sample of 2 μl of a mixture of a standard solution of linear alkanes C8-C20 in chromatograph gas was injected into the same chromatographic column with the same conditions of analysis as in the case of basil volatile oil.After confrmation (based on MS spectra) of the linear alkane structures separated by the gas chromatographic analysis, we determined the retention time for each component of the mixture (Supplementary Table 1).

C2C12 Myoblasts Cell
Culture.Murine myoblast C2C12 cells were grown in Dulbecco's modifed eagle medium (DMEM; Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum and antibiotics at 37 °C in a 5% CO 2 atmosphere.To diferentiate the C2C12 cells, 80% of the confuent culture was replaced with DMEM containing 2% horse serum, and the medium was replaced with fresh medium every other day for 4 days.

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide (MTT) Assay.Te MTT tetrazolium assay was used to check cytotoxicity by analyzing the intracellular metabolic activity in which MTT is converted into a purple formazan product.Te diferentiated C2C12 cells (3 × 10 5 /well) were plated on a 24-well plate, stabilized for 30 min, and treated with WEJ, EEJ, and CA for 24 h at 37 °C.After changing to a fresh medium, the plates were treated with MTT (0.5 mg/ ml) for 4 h.Insoluble formazan crystals were solubilized in dimethyl sulfoxide.Te amount of formazan was quantitated by absorbance at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

ELISA.
Te produced contents of cytokines were quantitated by the ELISA method [16].
2.6.Animals.Te ICR mice (male, 4 weeks old), purchased from the Dae-Han Experimental Animal Center (Chungbuk, Republic of Korea), were stabilized in individual cages for one week at room temperature (20-23 °C), with 50-60% humidity and a 12 h light and 12 h dark cycle.Te mice were treated in accordance with the current law and animal care standards provided in the Guide for the care and use of laboratory animals approved by the Animal Ethics Committee of Kyung Hee University (KHUASP (SE)-20-263).

FST.
In the FST, time spent by the mice swimming or being immobile is measured and increased immobility time indicates fatigue.Mice were subjected to an FST initially, to determine the diferences between the individual mice.Te mice were divided into 7 groups based on the immobility time: normal, control (distilled water, D.W.), WEJ (25, 50, and 100 mg/kg), EEJ (50 mg/kg), and CA (1 mg/kg).According to the previous study [11], the concentrations of WEJ were determined to be 25, 50, and 100 mg/kg.Te CA (1 mg/kg) concentration was also determined according to an earlier study [17].WEJ, EEJ, and CA were orally administered to the mice daily for 28 days.Te FST was then carried out on the last day as previously reported [18].

2
Journal of Food Biochemistry 2.8.TST.Te TST was conducted as previously reported [19].Briefy, the treadmill speed was started at 0 m/min and slowly increased (10 m/min at 10 min, 16 m/min at 10 min, and 21 m/min at 10 min).On the 28th day, the mice were made to exercise until exhaustion (10 m/min at 5 min and 40 m/min at 30 min).Te oral administration of D.W., WEJ, EEJ, and CA was carried out daily for 28 days.
2.9.Analysis of Fatigue-Related Biochemical Indicators in Serum, Muscle, or Liver.Blood, muscle (extensor digitorum longus (EDL), tibialis anterior (TA), soleus, and gastrocnemius muscles), or liver were collected immediately after performing FST and TST.Te serum was obtained by centrifugation of blood of the mice, and muscle and liver samples were quickly collected.A glycogen colorimetric assay kit (Biovision Inc., Milpitas, California, USA) was used to measure the levels of glycogen.Te levels of citrate synthase were analyzed using a commercially available kit (MyBiosource Inc., San Diego, California, USA).Te levels of lactate, SOD, malondialdehyde (MDA), and catalase were quantitated using assay kits (DoGenBio, Seoul, South Korea).Te levels of free fatty acids and cortisol were also quantitated using assay kits (Abcam, Cambridge, UK and Sigma-Aldrich Co., St. Louis, MO, USA), respectively.A DRI CHEM NX500 analyzer (Fujiflm, Tokyo, Japan) was used to quantify the levels of creatine kinase (CK), alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), glucose, and LDH.

Statistical Analysis.
For all statistical analyses, the SPSS 12.0 (IBM Corp., Armonk, NY, USA) software was utilized.
To verify the normality of the data, the Shapiro-Wilk test was used.An independent t-test was performed for a comparison between the normal (or blank) and control groups, and a oneway ANOVA test was performed for a comparison of three or more groups such as the control and drug groups.In vivo (n � 5/group) and in vitro data were expressed as mean-± standard error mean (SEM).In vitro data were indicated as the results of three independent experiments.A P value less than 0.05 meant that an efect was observed.Te infammatory responses induced by the infammatory cytokines are closely related to fatigue, and their high levels are indicative of chronic fatigue.In this study, H 2 O 2 stimulation remarkably augmented the secretion of infammatory cytokines, interleukin (IL)-6, and tumor necrosis factor (TNF)-α compared with the unstimulated cells.Treatment with WEJ, EEJ, or CA signifcantly decreased the levels of IL-6, and TNF-α increased by H 2 O 2 stimulation (Figures 1(d) and 1(e), P < 0.05).WEJ dose-dependently regulated the fatigue-related biochemical indicators.Furthermore, WEJ, EEJ, or CA showed no cytotoxicity to C2C12 myotubes (Figure 1(f )).

Efect of EJ on Oxidation
Oxidative stress is known to be one of the factors triggering fatigue.To investigate the antioxidative efect of EJ, the activities of SOD and catalase were analyzed using the respective assay kits.Te stimulation with H 2 O 2 signifcantly decreased the activities of SOD and catalase compared with unstimulated cells (Figures 2(a) and 2(b), P < 0.05).However, treatment with WEJ, EEJ, or CA showed a signifcant increase in the activities of SOD and catalase which were decreased by H 2 O 2 (Figures 2(a) and 2(b), P < 0.05).In addition, WEJ remarkably increased the activities of SOD and catalase (Figures 2(a) and 2(b), P < 0.05).MDA is a direct indicator of oxidative stress and a biomarker of fatigue.In H 2 O 2 -stimulated C2C12 myotubes, WEJ, EEJ, or CA treatment decreased the levels of MDA signifcantly (Figure 2(c), P < 0.05).

Efect of EJ on Immobility and Exhaustion Time in Animal
Models.Next, to investigate the antifatigue efect of EJ, we conducted experiments in vivo in mice [18].In the FST, the immobility time of the mice administered WEJ, EEJ, or CA was signifcantly reduced compared with the control group at 28 days (Figure 3(a), P < 0.05).In addition, WEJ remarkably decreased the immobility time (Figure 3(a), P < 0.05).Furthermore, the antifatigue efect of EJ was also investigated by performing the TST.Te time to exhaustion of the mice administered with WEJ, EEJ, or CA was signifcantly prolonged compared with the control group (Figure 3(b), P < 0.05).Tere was no signifcant change in the body weight of the WEJ, EEJ, or CA groups compared to the control group (Supplementary Figure 2).

Efect of EJ on Metabolic Factors Associated with Fatigue
after FSTand TST.We investigated the antifatigue efects of EJ by analyzing metabolic factors associated with fatigue.Following exercise, the levels of lactate, LDH, MDA, or cortisol increased in the serum.Te levels of lactate, LDH, MDA, and cortisol were quantitated in the serum of the mice administered with WEJ, EEJ, or CA after performing the FST and TST.As seen in Figure 4, the serum levels of lactate, LDH, MDA, and cortisol in the control group were remarkably augmented compared with the normal group (P < 0.05).Te lactate, LDH, MDA, and cortisol levels in the serum of the mice administered WEJ or CA remarkably decreased signifcantly compared to the control group (Figure 4, P < 0.05).EEJ remarkably decreased the serum levels of LDH, MDA, and cortisol (Figure 4).Te lactate levels were decreased by EEJ, but not signifcantly (Figure 4).
Te levels of fatigue-related blood biochemical indicators were measured after the FST and TST.Te serum levels of BUN, ALT, AST, and CK in the control mice were remarkably augmented when compared with the normal group, but WEJ, EEJ, or CA-administration resulted in a signifcant decrease in these levels compared to the control group (Table 1, P < 0.05).

Efect of EJ on Factors Related to Improvement in Fatigue
after the FSTand TST.Te glucose, free fatty acids, glycogen, and citrate synthase contents were measured after the FST and TST to investigate whether EJ increased the levels of the factors associated with improvement in fatigue.As shown in Figure 5(a), the glucose levels of the control group were remarkably augmented compared with the normal group.Journal of Food Biochemistry citrate synthase contents compared to the control group after the FST or TST (Figures 5(b) and 5(d), P < 0.05).Te glycogen content after the FST and TST was increased by EEJ and CA, but not signifcantly (Figure 5(c)).Te glycogen contents in the liver were remarkably augmented by the administration of WEJ, EEJ, or CA compared to the control group (Figure 5(e), P < 0.05).

Efect of EJ on Fatigue-Related Infammatory Cytokines in
Serum after the FST and TST.To investigate whether EJ played a role in the down regulation of the infammatory cytokines, the levels of IL-1β, IL-4, IL-6, and TNF-α in serum were analyzed using the ELISA method after the FST and TST.As seen in Figure 6, the serum levels of infammatory cytokines in the control group were remarkably augmented compared with the normal group (P < 0.05).Te serum levels of infammatory cytokines in the mice administered WEJ, EEJ, or CA showed a signifcant decrease compared with the control group (Figure 6, P < 0.05).

Efect of EJ on SOD and Catalase Activities after the FST and TST.
In the in vitro test, EJ augmented the activities of SOD and catalase (Figure 2).To confrm the antioxidative efect of EJ in the in vivo test, the SOD and catalase activities were analyzed after the FST and TST.As shown in Figures 7(a)-7(c), the SOD activities in the serum, liver, and muscle of the control mice were remarkably lower compared with the normal group (P < 0.05).However, the SOD activities in the serum, liver, and muscle of the WEJ or CA group were remarkably augmented compared with the control group after the FST and TST (Figures 7(a)-7(c), P < 0.05).Te EEJ only increased the SOD activity after TST compared with the control group (Figures 7(a)-7(c)).Furthermore, the administration of WEJ, EEJ, or CA signifcantly upregulated the levels of catalase in the liver compared with the control group after the FST (Figure 7(d), P < 0.05).WEJ (100 mg/kg) signifcantly increased the catalase activity compared with the control group after the TST (Figure 7(d)).

Discussion
In this study, we demonstrated that EJ and CA have an antifatigue efect through their ability to decrease the levels of metabolic factors associated with fatigue and increase the levels of substances which play a role in improving fatigue in the C2C12 myotubes in vitro model and the FST and TST in the in vivo models.
Te metabolic factors involved in the pathophysiology of fatigue include lactate, LDH, MDA, BUN, ALT, AST, CK, and cortisol, whereas recovery from fatigue is induced by glucose, free fatty acids, glycogen, and citrate synthase [4].Accumulation of lactate by LDH reduces the pH of the blood and muscle tissue, damages various organs, and is one of the causes of fatigue [20].During exercise, MDA increased by oxidative stress causes muscle damage and fatigue [21][22][23].BUN is another sensitive index of fatigue status, and BUN levels were found to increase signifcantly after excessive exercise [24].Physiological changes which occur during fatigue are closely associated with increases in AST and ALT levels suggestive of liver failure [25].Te serum levels of CK are increased by exercise-induced muscle damage [26].An increase in cortisol is associated with stress.During longterm exercise, the release of cortisol in the adrenal cortex is induced by an increase in the secretion of the adrenocorticotropic hormone in the anterior pituitary resulting in fatigue [27].Fatigue is induced by a lack of energy due to mitochondrial dysfunction [28].Terefore, energy storage and supply are important factors in exercise endurance.When exercising without an energy source (glucose, free fatty acids, and glycogen), physical fatigue increases, resulting in a signifcant reduction in endurance [4,29].A defciency of citrate synthase could cause fatigue due to lactate accumulation in the blood, and a signifcant decrease in citrate synthase was shown in chronic fatigue syndrome (CFS) patients compared to healthy controls [30].Many substances with antifatigue efects have been shown to decrease the levels of lactate, LDH, MDA, BUN, ALT, AST, CK, and cortisol and increase the levels of glycogen, free fatty acids, and citrate synthase [1-6, 18, 25].Previous studies have shown that EJ improves muscle function and reduces muscle loss [31,32].In the present study, EJ and CA remarkably decreased the levels of lactate, LDH, MDA, BUN, ALT, AST, CK, and cortisol, whereas they augmented the glycogen, free fatty acids, and citrate synthase levels.Terefore, these results indicate that EJ and CA could have contributed to exercise performance by ameliorating fatigue.
Glucose is the predominant energy source during exercise.Decreases in blood glucose levels lead to fatigue and impair endurance exercise [33].Gibb et al. [34] reported that exercise-induced fatigue results in a decrease of glucose levels, while triglycerides are degraded and stored in the form of free fatty acids in the blood.In contrast, other researchers reported that the FST and TST increased the serum glucose levels and decreased serum free fatty acids, and that many substances with antifatigue efects decreased the glucose levels and increased the free fatty acids levels [4,19,35].Also, the previous result indicated that blood glucose levels continued to rise steadily during exercise and gradually decreased to levels similar to the baseline during the recovery period (1-2 h after exercise cessation) [36].In this study, blood was collected immediately after the cessation of FST and TST, and glucose levels increased in the control group, while EJ reduced glucose levels.Terefore, these results indicate that EJ can regulate the increase in glucose levels induced by exercise.Moreover, when exerciseinduced stress increases, cortisol can supply the body with glucose, increasing blood glucose levels [37].Exerciseinduced exhaustion has been reported to decrease the availability of fatty acids and improve insulin sensitivity in glucose metabolism [38].Rau et al. [39] reported that stress induces hyperglycemia and stress-induced hyperglycemia releases stress hormones and cytokines.Terefore, we speculate that EJ might alleviate stress-induced hyperglycemia.However, further investigation is necessary to determine whether EJ has an antihyperglycemic property in hyperglycemia animal models such as hyperglycemic mice or obese diabetic mice.
Oxidative stress causes mitochondrial dysfunction and cell damage, leading to infammatory disorders, and is a major cause of fatigue [40].In the CFS patients, the levels of antioxidants (SOD and catalase) were remarkably decreased, while there was an increase in the generation of reactive oxygen species (ROS) [41].Excessive oxidative stress induces a rise in infammatory markers, such as IL-1β, IL-4, IL-6, and TNF-α [42].Blundell et al. [43] reported that the levels of infammatory cytokines in CFS patients were augmented compared to the normal control.Oxidative stress after strenuous exercise resulted in increased TNF-α levels, resulting in muscle fatigue [29].Terefore, substances that decrease the levels of infammatory cytokines by increasing SOD and catalase activities could play a role in reducing fatigue [44].In vitro and in vivo data indicate that CA has antioxidant activity and reduces oxidative stress in a variety of diseases [45].According to Jiang et al. [46], CA reduces infammation and oxidative stress while also preventing cell damage.Moreover, CA reduced expression of IL-1β, TNF-α, and IL-6 via the inhibition of nuclear factor (NF)-κB activation, which in turn reduced rat liver fbrosis and infammation [45].
Te muscles of a mouse include the EDL, TA, soleus, and gastrocnemius muscles [47,48].Among them, both EDL and soleus muscles are representative examples of fast-twitch and slow-twitch muscles [48].Fast-twitch muscles are primarily utilized during short bursts of intense exercise, while slowtwitch muscles are predominantly used during prolonged or endurance activities [49].Te rate of glycogen accumulation varies depending on the muscle type such as fast-twitch and slow-twitch muscles [50].Following exercise, the levels of citrate synthase decreased in the EDL muscles; on the contrary, the levels of citrate synthase increased in the soleus muscles [51].During exercise, stanozolol administration induced a signifcant increase of SOD activity in both EDL and soleus muscles [52].In the current study, we demonstrated that citrate synthase content and SOD activity decreased in the muscle tissues (EDL, TA, soleus, and gastrocnemius muscles) of the control group, with no change in glycogen content.In contrast, EJ increased glycogen and citrate synthase contents and SOD activity within the muscle tissues (EDL, TA, soleus, and gastrocnemius muscles).Terefore, further research based on muscle tissue types should be conducted to elucidate the detailed efects of EJ on muscle biomarkers.In the CFS mice group, compared to the acute-exercise-treated mice group, hepatic glycogen levels signifcantly decreased, but muscle glycogen levels remained unchanged [53].An antifatigue material, Sarcodon imbricatus signifcantly enhanced glycogen levels in the liver of acute-exercise-treated mice [53].Tis response was similar to our research fndings.Published literature indicates that fatigue was one of the most common symptoms of COVID-19, and in many patients even after recovery.Fatigue caused by COVID-19 is pathological fatigue, and as mentioned above, the biomarkers for the pathophysiology of fatigue are similar.Terefore, we assume that EJ might have the ability to alleviate pathological fatigue caused by COVID-19.However, because exercise-induced physiological fatigue can be distinguished from pathological and psychological fatigue, further research on pathological and psychological fatigue models should be conducted to confrm the improvement efect of EJ on COVID-19-induced pathological fatigue.
In the present study, administration of EJ and CA decreased the levels of IL-1β, IL-4, IL-6, and TNF-α and increased the activities of SOD and catalase in C2C12 myotubes and after FST and TST in animal models.Terefore, these data suggest that EJ and CA display antifatigue potential through their ability to increase antioxidative activity.Additionally, we confrm that CA plays an important role in fatigue by being an active component of EJ.In conclusion, this study showed that EJ and CA can signifcantly relieve excessive fatigue.Terefore, we suggest that EJ may have a potential therapeutic role in relieving fatigue in people sufering from the fatigue symptom.Values are the mean ± SEM. n � 5 per group.WEJ, EJ water extract; EEJ, EJ ethanol extract; and CA, chlorogenic acid.# P < 0.05, signifcantly diferent from the normal mice.* P < 0.05, signifcantly diferent from the control mice.
10 Journal of Food Biochemistry

Figure 5 :
Figure 5: Efect of EJ on glucose, free fatty acid, glycogen, and citrate synthase in the serum and muscle after the FSTand TST.After the last FST and TST, serum and muscle samples were obtained from each mouse.Levels of (a) glucose and (b) free fatty acid in serum were analyzed by a DRI CHEM NX500 analyzer.Levels of (c) glycogen and (d) citrate synthase in muscle were measured with each kit.(e) Levels of glycogen in liver were measured with glycogen kit.Values are the mean ± SEM. n � 5 per group.WEJ, EJ water extract; EEJ, EJ ethanol extract; and CA, chlorogenic acid.# P < 0.05, signifcantly diferent from the normal mice.* P < 0.05, signifcantly diferent from the control mice.

Figure 6 :
Figure6: Efect of EJ on fatigue-related infammatory cytokines in the serum after the FST and TST.After the last FST and TST, serum samples were obtained from heart.Levels of (a) IL-1β, (b) IL-4, (c) IL-6, and (d) TNF-α were analyzed by the ELISA method.Values are the mean ± SEM. n � 5 per group.WEJ, EJ water extract; EEJ, EJ ethanol extract; and CA, chlorogenic acid.# P < 0.05, signifcantly diferent from the normal mice.* P < 0.05, signifcantly diferent from the control mice.

Figure 7 :
Figure 7: Efect of EJ on SOD activities in the serum, liver, and muscle after the FST and TST.After the last FST and TST, samples were analyzed for SOD and catalase activities.Te activities of SOD in (a) serum, (b) liver, and (c) muscle.(d) Te activity of catalase in liver.Values are the mean ± SEM. n � 5 per group.WEJ, EJ water extract; EEJ, EJ ethanol extract; and CA, chlorogenic acid.# P < 0.05, signifcantly diferent from the normal mice.* P < 0.05, signifcantly diferent from the control mice.
Fatigue-Related Biochemical Indicators in H 2 O 2 -Stimulated C2C12 Myotubes.Initially, to investigate the antifatigue efects of EJ, the levels of fatigue-related biomarkers in H 2 O 2 -stimulated C2C12 myotubes were determined.Te levels of LDH and CK were signifcantly augmented by H 2 O 2 stimulation.However, the levels that were increased by H 2 O 2 stimulation were remarkably decreased by treatment with WEJ, EEJ, or CA (Figures1(a) and 1(b), P < 0.05).Glycogen levels were signifcantly augmented by treatment with WEJ, EEJ, or CA, compared with the levels seen with H 2 O 2 stimulation (Figure1(c), P < 0.05).

Table 1 :
Efect of EJ on fatigue-causing factors in serum after FST and TST.After the last FST and TST, serum samples were obtained from the heart.Levels of BUN, ALT, AST, and CK were analyzed with each kit.Values are the mean ± SEM. n � 5 per group.WEJ, EJ water extract; EEJ, EJ ethanol extract; CA, chlorogenic acid.# P < 0.05, signifcantly diferent from normal mice.