G. pentaphyllum was purchased from Hunan Tea Group Co., Ltd. (Changsha, China). Professor Zhou Ribao was responsible for identifying the plant species. A voucher specimen was deposited at the College of Medicine, Hunan University of Traditional Chinese Medicine (HUCM) Herbarium under number 20210301-1. GPE and GL were obtained from BTC Corporation (Ansan, Korea) [2627]. Briefly, the dried leaves of G. pentaphyllum were extracted with hot water and a 50% aqueous ethanol solution. The resulting extracts were filtered and evaporated to dryness. The GL content in GPE was analyzed by high-performance liquid chromatography [2627]. The developed GPE contained much higher GL, GLI, and Rg3 contents than the products from conventional extraction methods (18 mg, 14 mg, and 1.5 mg, respectively, per g of G. pentaphyllum leaf extract). Creatine monohydrate (CrM) (Sigma-Aldrich Inc., St. Louis, MO, USA) was used as the positive control.

The Animal Care and Use Committee of Hallym University approved this study (Hallym 2019-28). Male ICR mice (5 weeks) were supplied by Doo Yeol Biotech Co., Ltd. (Seoul, Korea) and fed a standard laboratory diet (Cargill Agri Purina, Inc., Seongnam, Korea) and with access to tap water ad libitum. The mice were acclimatized for 1 week before the experiments and housed under a constant room temperature (23 ± 3°C) and humidity (50 ± 10%) controlled room with a 12 h light/dark cycle (lights on from 8:00 AM to 8:00 PM). A 2-way experiment (Exercise X Treatment) was designed for the 6 groups (n = 10 per group in each test): sedentary + vehicle (SC), sedentary + 300 mg/kg body weight (BW)/day GPE (SGPE), exercise + vehicle (ExC), exercise + 300 mg/kg BW/day GPE (ExGPE), exercise + 7 mg/kg BW/day GL (ExGL), and exercise + 75 mg/kg BW/day creatine monohydrate (ExCrM). GPE, GL, and CrM were dissolved in a saline solution and administered daily by oral gavage for 6 weeks. Treadmill training was conducted using a single-lane rodent treadmill (Exer-3R Treadmill; Columbus Instruments, Columbus, OH, USA) supplied with a shocking grid at the rear. The mice were selected based on their exercise compliance and subjected to weekly training sessions involving running speeds of 10 m/min over a 15-min period with a 10° inclination to the treadmill environment acclimatization. The exercise duration was increased gradually from 15 to 40 min per week (Fig. 1). The bodyweight, dietary, and social behaviors were monitored during the administration period and exercise.

An exercise endurance test was conducted after 6 weeks of GPE, GL, and CrM administration and exercise. The exercise capacity was determined by increasing the treadmill speed from a 10 m/min period with 10° inclination in 1 m increments every 1 min until exhaustion (> 25 m/min maximum speed). The point of exhaustion was defined as when mice remained in continuous contact with the shocking grid for 10 s. The exercise capacity was expressed as the work done in Joules (kg m²s⁻²).

The whole-body composition (fat mass and lean body mass percentage) was measured using dual-energy X-ray absorptiometry (DEXA, PIXlmus^TM; GE Lunar, Madison, WI, USA) before one day the end of the experiment. After 16 h fasting, the mice were then anesthetized using tribromoethanol diluted with tertiary amyl alcohol. Blood samples were taken from the orbital venous plexus of the mice using capillary tubes. Blood was then centrifuged at 5,000 rpm for 10 min to separate the serum, which was stored at −70°C until further analysis. The visceral organs, including the quadriceps femoris muscle (QF), gastrocnemius muscle (GA), SOL, extensor digitorum longus muscle (EDL), and liver were excised and weighed accurately after sacrifice via a cervical dislocation. The organs were kept at −70°C until further analysis.

Serum levels of glucose, triglyceride (TG), total cholesterol (CHOL), low-density lipoprotein-cholesterol (LDL-CHOL), high-density lipoprotein-cholesterol (HDL-CHOL), blood urea nitrogen (BUN), and creatinine (CREA), as well as the serum activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured using a blood chemistry autoanalyzer (KoneLab 20XT, Thermo Fisher Scientific, Vantaa, Finland). The serum lactate levels were analyzed using commercial enzyme-linked immunosorbent assay kits (Abcam, Cambridge, MA, USA) according to the manufacturer's instructions.

For each mouse, 100 mg of liver and muscle (GA) was weighed, transferred to a tube, and homogenized in 1 mL of ice-cold phosphate-buffered saline. After centrifugation at 12,000 rpm for 10 min at 4°C, the supernatant was decanted and kept on ice. The glycogen content in the liver and muscle was analyzed using a commercial kit (Abcam), according to the manufacturer's instructions.

The muscle (GA) was homogenized in ice-cold lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 20 μg/mL aprotinin, 10 μg/mL anti-pain, 10 μg/mL leupeptin, 80 μg/mL benzamidine HCl, and 0.2 mM phenylethylsulfonyl fluoride). The insoluble material was removed by centrifugation at 12,000 rpm for 10 min, and the supernatant was collected for western blot analysis. The protein content of the lysates was measured using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). The proteins (50 μg) were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked for 1 h in 5% skim milk-TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) at room temperature and then incubated overnight at 4°C with anti-phospho-AMPKα (Thr172), anti-AMPKα, anti-phospho-p38 (Thr180/Tyr182), anti-p38, anti-PGC-1α, anti-β-actin (Cell Signal Technology, Beverly, MA, USA), anti-phospho-PGC-1α (R&D system Inc., Minneapolis, MN, USA), anti-NRF2, and anti-phospho-NRF2 (Ser40) (Abcam) primary antibodies. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG. The immunoreactive bands were detected with Luminata ^TM Forte Western HRP Substrate (Millipore) and quantified using an ImageQuant^TM LAS 500 imaging systems (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). β-actin was used as the internal control.

The total RNA from the skeletal muscle (SOL) was extracted using TRIzol reagent (Roche, Indianapolis, IN, USA). cDNA was synthesized from 2 μg of the total RNA using a HyperScript^TM RT master mix kit (GeneAll Biotechnology, Seoul, Korea). Real-time PCR for various genes was performed using Rotor-gene 300 PCR (Corbett Research, Mortlake, NSW, Australia) and Rotor-Gene^TM SYBR Green Kit (QIAGEN, Hilden, Germany). The data were then analyzed using the Rotor-gene 6000 series System Software program (Corbett Research), and the values were normalized to those of Gapdh. Table 1 lists the various primers used for real-time PCR analysis.

All data are reported as the mean ± SEM. One-way analysis of variance followed by Duncan's multiple comparisons test or Student's t-test were performed using SAS statistical software 9.4 (SAS Institute Inc., Cary, NC, USA). P-values < 0.05 were considered significant.

The effects of GPE supplementation on exercise performance were evaluated by administering GPE, GL, and CrM, or saline solution (SC and ExC) orally to the mice. Table 2 lists the bodyweight, fat mass, lean mass, and muscle weight. The weights of the experimental groups were monitored continuously during the 6 weeks of exercise, supplementation, or both. At the beginning of the experiment, there was no significant difference in the initial BWs among the experimental groups. The supplementation of GPE without exercise for 6 weeks induced a significant decrease in BW and fat mass, but the lean mass percentage was significantly higher in the SGPE group than the SC group. In the ExC group, significant changes were observed in the final BW gain, fat mass, and lean mass percentage, compared to the SC group. In addition, the ExGL group showed a significant decrease in BW gain, while the ExGPE group showed no significant differences in BW gain. Significant differences in the fat mass percentage among the groups were observed, which was significantly decreased compared to the ExC group, while the lean mass percentage of ExGPE showed the greatest relative increase, 84.6 ± 0.8%, which was higher than that in the ExC group. There were no significant differences in the BW, fat mass, and lean mass in the ExCrM group. In terms of muscle mass, SGPE increased the SOL and EDL muscle weight significantly compared to the SC group but did not change the QF and GA muscle weight. Moreover, the muscle masses of GA, SOL, and EDL in the ExC group were increased by 0.104, 0.013, and 0.016 g/100 g BW, respectively, compared to the SC group. No significant differences between the ExGPE and ExCrM groups were observed relative to the ExC group. On the other hand, the GA muscle weights were significantly higher in the ExGL group than the ExC group, but there were no changes in the QF, SOL, and EDL groups.

The anti-fatigue activity of GPE was determined as the endurance exercise capacity of the mice using a treadmill until exhaustion. As shown in Fig. 2A, the SGPE and ExC groups took a significantly longer time to reach exhaustion (1.31-fold and 1.74-fold, respectively) than the SC group. In addition, the ExGPE, ExGL, and ExCrM mice exercised for a longer period than those in the ExC group, demonstrating significant differences (1.46-fold with ExGPE, 1.22-fold with ExGL, and 1.37-fold with ExCrM) compared to the ExC group. GPE, GL, and CrM supplementation were associated with significantly improved exercise capacity in the ExGPE, ExGL, and ExCrM mice, respectively, compared to the vehicle treatment (Fig. 2B). These results suggest that GPE and GL supplementation enhances endurance exercise capacity.

The serum collected 30 min after the exercise test at 6 weeks was analyzed to investigate the effects of GPE and GL on the anti-fatigue-associated indices. During intense exercise, the energy supply comes from glycogen degradation by phosphorylase and circulating glucose released by the liver. The glucose levels in serum are an important marker for exercise performance maintenance [2829]. As shown in Table 3, the serum glucose levels in the ExGPE, ExGL, and ExCrM groups were 25.1%, 38.2%, and 57.9%, respectively, compared to the ExC group. The serum TG levels were also increased significantly in the ExGPE group compared to that in the ExC group. No significant differences were observed between the groups with respect to the other indices, such as CHOL, LDL-CHOL, or HDL-CHOL levels, compared to those in the ExC groups. Intensive exercise increased the creatine kinase (CK) and LDH markers, indicating muscle damage. As shown in Table 3, the CK levels in the ExGPE group were significantly lower than those in the ExC group. The serum LDH levels were significantly higher in the ExC group. Although there was no significant difference in the LDH levels between the ExGPE group and the ExC group, the LDH levels of the ExGL and ExCrM groups were significantly lower. Compared to the ExC group, the serum lactate levels were 22.2%, 18.3%, and 30.98% lower in the ExGPE, ExGL, and ExCrM groups, respectively. No significant changes in the liver (ALT, AST, and ALP) and renal (BUN and CREA) profiles were observed in any group except in the ExGL group for BUN. Trend analysis showed that GPE and GL treatment had a remarkable effect on the blood glucose and TG levels and decreased the serum CK, LDH, and lactate levels.

The glycogen level of liver and muscle (GA) was monitored to determine the effects of GPE supplementation on the glycogen status. As shown in Fig. 3, the liver glycogen content did not differ significantly among the groups (Fig. 3A and B). On the other hand, muscle glycogen levels exhibited a significant difference among the groups. The muscle glycogen of the ExGPE, ExGL, and ExCrM groups was significantly higher than that in the ExC group (Fig. 3C and D). These results suggest that GPE and GL supplementation during the exercise increased the muscle glycogen storage in mice and maintained a hepatic glycogen content in the mice, leading to enhanced energy reserves and blood glucose maintenance. This may be one of the mechanisms of anti-fatigue effects on GPE and GL supplementation.

The muscles (GA and SOL) of the mice, which are used extensively during treadmill training, were collected to verify the effects of GPE supplementation on mitochondrial biogenesis in mice. The gene expression of the mitochondrial biogenesis transcription factors and mitochondrial DNA (mtDNA) content in the muscle tissue of administrated mice was evaluated. GPE, GL, and CrM administration greatly increased the protein expression of phosphorylated PGC-1α by 1.84-fold, 1.42-fold, and 1.65-fold, respectively, compared to the ExC group (Fig. 4A). The nuclear respiratory factor 1 (Nrf1) gene for the expression of the key mitochondrial genes was also upregulated remarkably in the SGPE and ExC groups by 15.52-fold and 4.92-fold, respectively, compared to that of the SC group. By contrast, the levels of the Nrf1 gene in the ExGPE and ExGL groups were increased 2.54-fold and 5.93-fold compared to the ExC group (Fig. 4B). On the other hand, ExCrM mice did not show any detectable changes in Nrf1 gene expression compared to the ExC group. The mtDNA content also increased markedly in the SGPE group and ExC group by 1.29-fold and 1.52-fold, respectively, compared to that of the SC group. In particular, both the ExGPE group and ExGL group were 3.06-fold higher than that in the ExC group (Fig. 4C). The ExCrM group did not affect the mtDNA content. These results suggest that the GPE and GL treatments elevated mitochondrial biogenesis by promoting PGC-1α expression.

In skeletal muscles, PGC-1α acts as a transcriptional coactivator of several exercise-related genes responsible for processes, such as lactate metabolism, fatty acid metabolism, and antioxidant response [3031]. The mRNA expression of PGC-1α-related genes, including LDH B, peroxisome proliferator-activated receptor-δ (PPARδ), and superoxide dismutase 2 (SOD2) in the muscle (SOL) were measured to investigate the molecular basis of GPE for the exercise performance effect. As shown in Fig. 5A, the SGPE and ExC group increased the mRNA expression level of Ldhb, even though there was no significant difference in Ldhb mRNA expression between the SC groups. In contrast, the Ldhb mRNA level showed a trend toward a significant decrease in the ExGPE and ExCrM groups compared to that in the ExC group. The mRNA expression of Ppard was upregulated in the muscles of the ExGPE and ExGL group compared to that in the ExC group (Fig. 5B). On the other hand, the ExCrM group showed no significant activity. The ExGPE, ExGL, and ExCrM groups also showed significantly upregulated mRNA expression of Sod2 in the muscle compared to the ExC group (Fig. 5C).

PGC-1α is induced by multiple signaling pathways typically activated by mitochondrial biogenesis during endurance exercise. These include the activation of AMPK, p38 MAP kinase (p38), Ca²⁺ mediated pathways, and reactive oxygen species (ROS)-induced signaling [323334]. Therefore, this study examined whether AMPK and p38 mediated the effects of GPE on PGC-1α activation. As shown in Fig. 6A, the ExGPE group showed 1.33 times higher AMPK phosphorylation than the ExC group. The phosphorylated p38 level was 1.81 times higher in the SGPE group than the SC group. In addition, the ExGPE, ExGL, and ExCrM groups also showed 1.8, 2.21, and 2.29 times higher levels of phosphorylated p38, respectively, than the ExC group (Fig. 6B). These results suggest that GPE and GL supplementation increased the expression of proteins involved in activating PGC-1α through AMPK and p38 phosphorylation.