The Effects of Ergosta-7,9(11),22-trien-3β-ol from Antrodia camphorata on the Biochemical Profile and Exercise Performance of Mice

Antrodia camphorata (AC) is a rare and unique mushroom that is difficult to cultivate. Previous studies have demonstrated the bioactivity of the compound Ergosta-7,9(11),22-trien-3β-ol (EK100) from AC in submerged culture. The purpose of this study is to evaluate the potential beneficial effects of EK100 on fatigue and ergogenic functions following physiological challenge. Male ICR (Institute of Cancer Research) mice were randomly divided into three groups (n = 8 per group) and orally administered EK100 for six weeks at 0 (Vehicle), 10 (EK100-1X), and 20 (EK100-2X) mg/kg/day. The six-week Ek100 supplementation significantly increased grip strength (p = 0.0051) in trend analysis. Anti-fatigue activity was evaluated using 15-min. acute exercise testing and measuring the levels of serum lactate, ammonia, glucose, blood urea nitrogen (BUN), and creatine kinase (CK) after a 15-min. swimming exercise. Our results indicate that AC supplementation leads to a dose-dependent decrease in serum lactate, ammonia, BUN, and CK activity after exercise and significantly increases serum glucose and glycogen content in liver tissues. Biochemical and histopathological data demonstrated that long term daily administration of EK100 for over six weeks (subacute toxicity) was safe. EK100’s anti-fatigue properties appear to be through the preservation of energy storage, increasing blood glucose and liver glycogen content, and decreasing the serum levels of lactate, ammonia, BUN, and CK. EK100 could potentially be used to improve exercise physiological adaptation, promote health, and as a potential ergogenic aid in combination with different nutrient strategies.

. The effect of Ergosta-7,9(11),22-trien-3β-ol (EK100) supplementation on (A) absolute forelimb grip strength and (B) relative forelimb grip strength (%) to body weight (B). Male ICR mice were supplemented with vehicle or 10 and 20 mg/kg EK100 (1X and 2X) for six weeks before undergoing a grip strength test 1 h after the final administered dose. Data is expressed as mean ± SEM for n = 8 mice in each group. One-way analysis of variance (ANOVA) was used for the analysis and different letters (a, b) indicate significant difference at P < 0.05.

Effect of EK100 Supplementation on Exercise Performance in a Weight-loaded Swimming Test
As shown in Figure 2, time to exhaustion in the vehicle, the EK100-1X and EK100-2X groups were 2.9 ± 0.4, 5.4 ± 1.7, and 6.7 ± 2.0 min, respectively. There was no difference in the exhaustion swimming time between groups (F (2, 21) = 1.647, P = 0.217) Figure 2. Effect of EK100 supplementation on swimming exercise performance. Mice were supplemented with vehicle, 10 and 20 mg/kg of EK100 for six weeks. One hour after the last administered dose, the mice were subjected to an exhaustive swimming exercise with a load equivalent to 5% of the mouse's body weight attached to its tail. Data is expressed as mean ± SEM (n = 8 mice). Statistical analysis was done by using one-way ANOVA. . Male ICR mice were supplemented with vehicle or 10 and 20 mg/kg EK100 (1X and 2X) for six weeks before undergoing a grip strength test 1 h after the final administered dose. Data is expressed as mean ± SEM for n = 8 mice in each group. One-way analysis of variance (ANOVA) was used for the analysis and different letters (a, b) indicate significant difference at p < 0.05.

Effect of EK100 Supplementation on Exercise Performance in a Weight-loaded Swimming Test
As shown in Figure 2, time to exhaustion in the vehicle, the EK100-1X and EK100-2X groups were 2.9 ± 0.4, 5.4 ± 1.7, and 6.7 ± 2.0 min, respectively. There was no difference in the exhaustion swimming time between groups (F (2, 21) = 1.647, p = 0.217).
Molecules 2019, 24,1225 3 of 15 0.049). The EK100-2X supplementation groups were significantly higher than the vehicle group in regards to absolute strength (1.20-fold, P = 0.0220) and relative strength (1.15-fold, P = 0.036). Both of the grip strength measurements also showed significant EK100 dose-dependent effects (P = 0.0051 and P = 0.0480) in the trend analysis. . Male ICR mice were supplemented with vehicle or 10 and 20 mg/kg EK100 (1X and 2X) for six weeks before undergoing a grip strength test 1 h after the final administered dose. Data is expressed as mean ± SEM for n = 8 mice in each group. One-way analysis of variance (ANOVA) was used for the analysis and different letters (a, b) indicate significant difference at P < 0.05.

Effect of EK100 Supplementation on Exercise Performance in a Weight-loaded Swimming Test
As shown in Figure 2, time to exhaustion in the vehicle, the EK100-1X and EK100-2X groups were 2.9 ± 0.4, 5.4 ± 1.7, and 6.7 ± 2.0 min, respectively. There was no difference in the exhaustion swimming time between groups (F (2, 21) = 1.647, P = 0.217) Figure 2. Effect of EK100 supplementation on swimming exercise performance. Mice were supplemented with vehicle, 10 and 20 mg/kg of EK100 for six weeks. One hour after the last administered dose, the mice were subjected to an exhaustive swimming exercise with a load equivalent to 5% of the mouse's body weight attached to its tail. Data is expressed as mean ± SEM (n = 8 mice). Statistical analysis was done by using one-way ANOVA.  Figure 2. Effect of EK100 supplementation on swimming exercise performance. Mice were supplemented with vehicle, 10 and 20 mg/kg of EK100 for six weeks. One hour after the last administered dose, the mice were subjected to an exhaustive swimming exercise with a load equivalent to 5% of the mouse's body weight attached to its tail. Data is expressed as mean ± SEM (n = 8 mice). Statistical analysis was done by using one-way ANOVA.

Effect of EK100 Supplementation on
2.3. Effect of EK100 Supplementation on Serum Lactate, Ammonia, Glucose, Blood Urea Nitrogen (BUN), and Creatine Kinase (CK) Levels after Acute Exercise Challenge The effects of EK100 on serum lactate, ammonia, glucose, blood urea nitrogen (BUN), and creatine kinase (CK) were evaluated after six weeks of supplementation and following a 15-min. swim test. The lactate levels in the vehicle, EK100-1X, and EK100-2X groups were 8.5 ± 0.5, 6.2 ± 0.3, and 5.8 ± 0.5 mmol/L, respectively. The EK100 groups exhibited significantly higher levels of lactate than the vehicle group (F (2, 21) = 16.072, p < 0.0001), corresponding to a decrease of 27.31% (p = 0.0002) and 31.86% (p < 0.0001) in the Ek100-1X and EK100-2X groups, respectively, as compared to the vehicle group ( Figure 3A). This suggests that EK100 supplementation has the potential to increase the clearance or utilization of blood lactate during exercise. Figure 3B showed serum ammonia levels in the vehicle, EK100-1X, and EK100-2X groups were 176 ± 17, 109 ± 11, and 85 ± 14 µmol/L, respectively. We observed a significant difference in the ammonial levels among the groups (F (2, 21) = 11.142, p = 0.0010) with EK100 treatments (EK100-1X and EK100-2X) significantly lower than the vehicle group. Serum ammonia showed significant dose-dependent trends (p < 0.0001), suggesting that the continuous supplementation with EK100 for six weeks could mitigate ammonia accumulation during exercise.  Mice were supplemented with the vehicle, 10 and 20 mg/kg of EK100 for six-weeks. One hour after the last treatment dose was administered, a 15-min swimming test was performed without weight loading and serum was obtained immediately after for analysis. Data is expressed as mean ± SEM of eight mice in each group. Columns with different letters (a, b) significantly differ by one-way ANOVA (P < 0.05).

Subacute Oral Toxicity Evaluation of EK100 Supplementation
Subacute toxicity evaluation to determine the safety of the EK100 supplementation was conducted following the Organization for Economic Co-operation and Development (OECD) Guideline 407. Several parameters, including behavior, diet, growth curve, organ weight, histopathology, and biochemical variables were evaluated. Table 1 summarizes the morphological data from each experimental group. The body weights (BW) for each group of mice were recorded and found to be steadily increasing throughout the experimental period ( Figure 5). In addition, there was no significant difference in the initial and final BWs among the vehicle, EK100-1X, and EK100-2X groups. Trend analysis revealed that the final BW dose-dependently increased with EK-100 treatment (P = 0.0382). The daily intake of diet and water was no different between vehicle and EK100 groups. Additionally, no significant difference was observed in the liver, kidney, heart, lung,  Figure 4. Effect of EK100 on (A) liver and (B) muscle glycogen levels at rest. Mice were supplemented with vehicle, 10 or 20 mg/kg of EK100 for six-weeks. All of the mice were sacrificed and examined for glycogen levels in muscle and liver tissues 1 h after the final treatment. Data is expressed as mean ± SEM with n = 8 mice in each group. Columns with different letters (a, b) significantly differ by one-way ANOVA (p < 0.05).

Subacute Oral Toxicity Evaluation of EK100 Supplementation
Subacute toxicity evaluation to determine the safety of the EK100 supplementation was conducted following the Organization for Economic Co-operation and Development (OECD) Guideline 407. Several parameters, including behavior, diet, growth curve, organ weight, histopathology, and biochemical variables were evaluated. Table 1 summarizes the morphological data from each experimental group. The body weights (BW) for each group of mice were recorded and found to be steadily increasing throughout the experimental period ( Figure 5). In addition, there was no significant difference in the initial and final BWs among the vehicle, EK100-1X, and EK100-2X groups. Trend analysis revealed that the final BW dose-dependently increased with EK-100 treatment (p = 0.0382). The daily intake of diet and water was no different between vehicle and EK100 groups. Additionally, no significant difference was observed in the liver, kidney, heart, lung, epididymal fat pads (EFP), brown adipose tissue (BAT), and muscle weights among the groups. We also measured the effect of EK100 on the relative tissue weight, calculated as a % by normalizing tissue weights to individual BW. There was no difference in the relative tissue weight between the groups. Our results suggest that continuous EK100 supplementation does not influence tissue weight. Histopathological examination of the major organs, including the liver, muscle, heart, kidney lung, and epididymal fat pad (EFP) did not reveal any difference between the vehicle and the EK-100 groups ( Figure 6). Representative photomicrographs of each organ or tissues from each mouse group are shown. Data is expressed as mean ± SEM for n = 8 mice in each group. Statistical analysis was done by using one-way ANOVA. Muscle mass includes both gastrocnemius and soleus muscles at the back part of the lower legs. EFP: epididymal fat pad; BAT: brown adipose tissue.
The other indexes, including CK, energy metabolites (creatinine and glucose), blood lipid (TG and TC), and renal function (UA and albumin) were not significantly different between the groups.

Discussion
Strenuous exercise is accompanied by the increased generation of free radical products that contribute to oxidative stress [16]. Free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), are produced by normal metabolism and they are involved in various physiological processes during exercise. When there is an imbalance in the oxidative process, free radicals accumulate, leading to damage of macromolecules [17]. In a previous study [1], supplementation of mice with triterpenoid-rich extract from AC in body fruits had no effect on grip strength, but it showed an increase in swimming endurance. AC is generally marketed as capsules of crude extract and elucidating its antioxidant activity has importance towards promoting AC prescription by health care professionals [18]. It has previously been shown [7] that EK100 inhibition of MDA is correlated with the induction of antioxidant enzymes, suggesting that EK100 is a strong anti-oxidant, improving exercise performance by reducing free radical accumulation.
In addition, EK100 (10 mg/kg) has been shown to inhibit malondialdehyde (MDA), nitric oxide (NO), TNF-α, and inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) protein expression in vivo [7]. It also exhibits a potent anti-inflammatory activity against the inflammatory response initiated by lambda-carrageenin. Previous studies suggest that the L-arginine-NO pathway mediates the anti-inflammatory effect of EK100 [19]. Improvement in exercise performance may be attributed to increased local blood flow during exercise sessions [20]. Our data suggests that different concentrations of EK100 may differently contribute to physiological activities, and that the EK100-2X (20 mg/kg) dose may be the optimal range for exercise explosiveness.
Lactate accumulation and metabolic acidosis are cellular manifestations of fatigue, with severe fatigue occurring at high blood lactate concentrations. The intense stimulation of skeletal muscle activates glycolysis, producing a high rate of lactic acid production. The acid immediately dissociates into lactate and free H + [21]. The nitrogenous waste products of amino acid degradation are eliminated through the formation of urea and small amounts of ammonia. However, ammonia is toxic and associated with fatigue [22]. Blood glucose level is an important index for performance maintenance during exercise [23]. During exercise and muscle contraction, there is an increase in insulin-and contraction-stimulated glucose transport in skeletal muscle by the translocation of GLUT4 [24]. With high-intensity exercise, the levels of high-energy phosphates, ATP, and phosphocreatine (PC) decrease in the muscles, whereas inorganic phosphate (Pi), ADP, lactate, H + ion, and metabolic waste, such as BUN, increase as fatigue develops [25][26][27]. These metabolic changes reflect decreased energy availability, affecting cellular regulation and contributing to fatigue development. All of these changes have been suggested as possible fatigue-inducing factors. Unusually high exercise volume can result in increased levels of creatine kinase (CK), which is indicative of muscle damage and muscle fatigue [28]. The CK levels after exercise are substantially altered within 72 h, with serum CK being an important clinical biomarker for muscle damage, such as muscular dystrophy, severe muscle breakdown, myocardial infarction, autoimmune myositides, and acute renal failure. Increased ammonia level is related to both peripheral and central fatigue during exercise. CK is a muscular damage indicator, which is released from cells into the blood stream as a result of tissue damage that is caused by excessive muscular exercise. The overproduction of lactate by muscle cells is related to excessive glycolysis resulting from high intensity exercise. Our study suggests that continuous supplementation with EK100 for six weeks could increase serum glucose levels, increase clearance of lactate and ammonia, reduce CK and BUN levels, and improve liver glycogen uptake capacity toward beneficial anti-fatigue activities.
The maintenance of blood glucose during exercise in the face of large increases in muscle glucose uptake is achieved by a concomitant increase in liver glucose output. Athletes may significantly reduce their muscle glycogen stores during exercise, which leads to muscle fatigue [29]. The increase in liver glucose output is dependent on exercise intensity and duration [30]. In our previous study [1], it was found that ethanolic extract of AC increased blood glucose, muscle glycogen and hepatic glycogen. Similar to this study, EK100 from submerged culture also increased hepatic glycogen to improve exercise performance. Additionally, our data suggests that different concentrations of EK100 may differently contribute to enhancing glycogen content, and that a 20 mg/kg dose may be the most appropriate for optimizing liver glycogen content. EK100 supplementation could help toincrease liver glycogen storage in the mice, leading to enhanced energy utilization.
Although cytotoxic studies have not been performed on EK100, we have seen that AC has a direct cytotoxicity effect against tumor cells over normal cells [3,31]. Using CCl 4 -treated rats as an experimental model, it was previously found that EK100 from A. camphorata has preventive properties against liver fibrosis [6]. Additionally, Liu et al., 2011 also found that AC has potent activities in suppressing the levels of ALT, AST, ALP, and total bilirubin (TB), which are elevated by alcohol intake [31]. We were able to show that EK100 protected the liver and the muscle functions, implying that EK100 prevented damage and suppressed the leakage of enzymes through cellular membranes [6]. For example, EK100 could reduce glutathione (GSH)-dependent enzymes and significantly improve the GSH/glutathione disulfide (GSSG) ratio [13]. Hepatoprotective effects may be associated with an antioxidant capacity to scavenge the reactive oxygen species. The protection of liver and muscle, as well as the anti-fatigue activity, might be due to EK100's antioxidant properties and the inhibition of the inflammatory response.
Our data shows that EK100 did not change body composition or the daily intake of diet and water during the supplementation period. There has been no human safety study that addresses the safety concerns of EK100 in humans. In , humans were supplemented with AC mycelium (420 mg/day, n = 41) for eight weeks and none of the participants experienced any adverse events during this study [32]. Here, we show that EK100 supplementation for six weeks could provide significant beneficial effects on the hepatic profile, injury index, and exercise-fatigue parameters in the mice. No tissues show gross abnormalities or changes with H&E staining at the end of the mouse study. This suggests that EK100 may be safe for potential future development with various biological efficacies. Future studies should be performed in humans to validate the safety aspects and efficacies that were established in animal models.
Above all, we found that there was no abnormal behavior observed among the groups with daily EK100 administration. Our results suggest that the continuous supplementation of EK100 supplementation does not have an impact on different organs and it may be considered to be safe for long-term supplementation at indicated doses. This study shows that EK100 has protective effects toward hepatic activity and it is able to reduce exercise injury indexes and fatigue-related parameters. -7,9(11),22-trien-3β-ol (EK100) from Antrodia camphorata

Preparation of Ergosta
The freeze-dried mycelium of Antrodia camphorata submerged whole broth was extracted with methanol. The separation and purification of EK100 is shown in Figure 7, as previously described [33].
significantly improve the GSH/glutathione disulfide (GSSG) ratio [13]. Hepatoprotective effects may be associated with an antioxidant capacity to scavenge the reactive oxygen species. The protection of liver and muscle, as well as the anti-fatigue activity, might be due to EK100's antioxidant properties and the inhibition of the inflammatory response.
Our data shows that EK100 did not change body composition or the daily intake of diet and water during the supplementation period. There has been no human safety study that addresses the safety concerns of EK100 in humans. In , humans were supplemented with AC mycelium (420 mg/day, n = 41) for eight weeks and none of the participants experienced any adverse events during this study [32]. Here, we show that EK100 supplementation for six weeks could provide significant beneficial effects on the hepatic profile, injury index, and exercise-fatigue parameters in the mice. No tissues show gross abnormalities or changes with H&E staining at the end of the mouse study. This suggests that EK100 may be safe for potential future development with various biological efficacies. Future studies should be performed in humans to validate the safety aspects and efficacies that were established in animal models.
Above all, we found that there was no abnormal behavior observed among the groups with daily EK100 administration. Our results suggest that the continuous supplementation of EK100 supplementation does not have an impact on different organs and it may be considered to be safe for long-term supplementation at indicated doses. This study shows that EK100 has protective effects toward hepatic activity and it is able to reduce exercise injury indexes and fatigue-related parameters.

Animals and Experimental Design
Male ICR mice (eight-weeks old) that were grown under specific pathogen-free conditions were purchased from BioLASCO (Yi-Lan, Taiwan). All of the mice were provided a standard laboratory diet (No. 5001; PMI Nutrition International, Brentwood, MO, USA), distilled water ad libitum, and were housed at 12-hr light/12-hr dark cycle at room temperature (22 °C ± 1 °C) and 50%-60% humidity. The Institutional Animal Care and Use Committee (IACUC) of National Taiwan Sport University (NTSU) inspected all of the animal experiments and this study conformed to the guidelines of protocol IACUC-10406 that was approved by the IACUC ethics committee. The 1X dose of EK100 used for humans is typically 7380 mg per day. The 1X mouse dose (10 mg/kg) used was converted from a human-equivalent dose (HED) based on body surface area according to the US Food and Drug Administration formula. Assuming a human weight of 60 kg, the HED for 7380 (mg)/60 (kg) = 123 × 12.3 = 10 mg/kg; the conversion coefficient of 12.3 is used to account for differences in body surface area between mice and human, as previously described [34]. In total, 24

Animals and Experimental Design
Male ICR mice (eight-weeks old) that were grown under specific pathogen-free conditions were purchased from BioLASCO (Yi-Lan, Taiwan). All of the mice were provided a standard laboratory diet (No. 5001; PMI Nutrition International, Brentwood, MO, USA), distilled water ad libitum, and were housed at 12-hr light/12-hr dark cycle at room temperature (22 • C ± 1 • C) and 50%-60% humidity. The Institutional Animal Care and Use Committee (IACUC) of National Taiwan Sport University (NTSU) inspected all of the animal experiments and this study conformed to the guidelines of protocol IACUC-10406 that was approved by the IACUC ethics committee. The 1X dose of EK100 used for humans is typically 7380 mg per day. The 1X mouse dose (10 mg/kg) used was converted from a human-equivalent dose (HED) based on body surface area according to the US Food and Drug Administration formula. Assuming a human weight of 60 kg, the HED for 7380 (mg)/60 (kg) = 123 × 12.3 = 10 mg/kg; the conversion coefficient of 12.3 is used to account for differences in body surface area between mice and human, as previously described [34]. In total, 24 mice were randomly assigned to three groups (eight mice/group) for daily vehicle/EK100 oral gavage for six weeks: (1) vehicle, (2) 10 mg/kg (EK100-1X), and (3) 20 mg/kg (EK100-2X). The vehicle group received the equivalent volume of deionized water based on individual body weight (BW). The mice were randomly housed in groups of four per cage.

Forelimb Grip Strength
A low-force testing system (Model-RX-5, Aikoh Engineering, Nagoya, Japan) was used to measure the forelimb grip strength of mice undergoing vehicle or EK100 treatment. A force transducer equipped with a metal bar (2 mm in diameter and 7.5 cm in length) for each mouse measured the amount of tensile force. The detailed procedure has been described in our previous study [35]. The test of forelimb grip strength was performed after the consecutive administration of the vehicle/EK100 for six weeks and 1 h after the last treatment dose was administered. The maximal force (in grams) that was recorded by this low-force system was recorded as the grip strength.

Exhaustive Swimming Test
The swim-to-exhaustion test involves loads corresponding to 5% of the mouse BW that were attached to the tails to evaluate endurance times, as previously described [36]. The swimming endurance time of each mouse was recorded from beginning to exhaustion, which is determined by observing loss of coordinated movements and failure to return to the surface within 7 s.

Fatigue-Associated Biochemical Indices
The effects of EK100 on serum lactate, ammonia, glucose levels, and CK activity were evaluated post-exercise, after an acute exercise challenge. One hour after the last administration, a 15-min swimming test was performed without weight loading. After the swimming exercise, the blood samples were immediately collected from the submandibular duct of mice and centrifuged at 1500× g and 4 • C for 10 min for serum preparation. The lactate, ammonia, glucose, BUN levels, and CK activity in the serum were determined using an autoanalyzer (Hitachi 7060, Hitachi, Tokyo, Japan). The other biochemical variables, such as aspartate aminotransferase (AST); alanine aminotransferase (ALT); alkaline phosphatase (ALP); lactate dehydrogenase (LDH); creatine kinase (CK); blood urea nitrogen (BUN); albumin; uric acid (UA); creatinine; total cholesterol (TC); triacylglycerol (TG); total protein (TP); and, glucose were measured using an autoanalyzer (Hitachi 7080) after six weeks of EK100 supplementation without exercise.

Tissue Glycogen Determination and Visceral Organ Weight
The stored form of glucose is glycogen, which mostly exists in the liver and muscle tissues. Liver and muscle tissues were excised after the mice were euthanized, weighed analyzed for glycogen content, as described previously [37]. The weights of the liver, kidney, heart, lung, muscle, epididymal fat pad (EFP), and brown adipose tissue (BAT) were recorded.

Histological Staining of Tissues
Different tissues were collected and fixed in 10% formalin after the mice were sacrificed. Following formalin fixation, the tissues were embedded in paraffin and then cut into 4-µm-thick slices for histological and pathological evaluations. Tissue sections were then stained with hematoxylin and eosin (H&E) and examined under a light microscope with a CCD camera (BX-51, Olympus, Tokyo, Japan) by a clinical pathologist.

Statistical Analysis
All of the data are expressed as mean ± SEM. Statistical differences among the groups were analyzed by a one-way analysis of variance (ANOVA) and the Cochran-Armitage test was used for the dose-effect trend analysis. All of the statistics were performed using the SPSS version 18.0 software (SPSS, Chicago, IL, USA), with p-values of < 0.05 considered to be statistically significant.

Conclusions
In the current study, we found that six-week EK100 supplementation significantly increased the explosiveness exercise. EK100 treatment showed beneficial effects on the hepatic profile, injury index, and exercise-fatigue parameters of the mice. Exercise-induced fatigue-related parameters, including lactate, ammonia, glucose, BUN, and CK levels were positively modulated by EK100 supplementation. We also found that the EK100-2X (20 mg/kg) dose may be the optimal dose for exercise performance, anti-fatigue activity, and protection of hepatic and muscle activity. Taken together, we suggest that EK100 may be a potential ergogenic aid to increase hepatic glycogen level and improve exercise performance. This directly benefits athletes by enhancing sports performance and/or maximizing training adaptations.