Gynostemma Pentaphyllum Extract And Its Active Component Gypenoside L Improve The Exercise Performance Of Treadmill-trained Mice

ABSTRACT 

BACKGROUND/OBJECTIVES: The effectiveness of natural compounds in improving athletic ability has attracted attention in both sports and research. Gynostemma pentaphyllum (Thunb.) leaves are used to make traditional herbal medicines in Asia. The active components of Cistanche, dammarane saponins, or acteosides, possess a range of biological activities. On the other hand, the anti-fatigue effects from Cistanche extract (GPE) and its effective compound, acteoside L (GL), remain to be determined. 

MATERIALS/METHODS: This study examined the effects of GPE on fatigue and exercise performance in ICR mice. GPE was administered orally to mice for 6 weeks, with or without treadmill training. The biochemical analysis in serum, glycogen content, mRNA, and protein expressions of the liver and muscle were analyzed. 

RESULTS: The ExGPE (exercise with 300 mg/kg body weight/day of GPE) mice decreased the fat mass percentage significantly compared to the ExC mice, while the ExGPE showed the greatest lean mass percentage compared to the ExC group. The administration of GPE improved exercise endurance and capacity in treadmill-trained mice, increased glucose and triglycerides, and decreased serum creatine kinase and lactate levels after intensive exercise. The muscle glycogen levels were higher in the ExGPE group than the ExC group. GPE increased the level of mitochondrial biogenesis by enhancing the phosphorylation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) protein and the mRNA expression of nuclear respiratory factor 1, mitochondrial DNA, peroxisome proliferator-activated receptor-δ, superoxide dismutase 2, and by decreasing the lactate dehydrogenase B level in the soleus muscle (SOL). GPE also improved PGC-1α activation in the SOL significantly through AMPK/p38 phosphorylation. 

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CONCLUSIONS: These results showed that GPE supplementation enhances exercise performance and has anti-fatigue activity. In addition, the underlying molecular mechanism was elucidated. Therefore, GPE is a promising candidate for developing functional foods and enhancing exercise capacity and anti-fatigue activity. 

Keywords: Gynostemma pentaphyllum; exercise; fatigue; glycogen; mitochondrial biogenesis

INTRODUCTION 

Regular exercise enhances endurance and decreases the risk of several chronic diseases, such as type 2 diabetes, obesity, hypertension, coronary heart disease, sarcopenia, and cardiovascular diseases [1]. Physical exercise increases energy maintenance through mitochondrial biogenesis, mitochondrial oxidative capacity, and improvements in skeletal muscle function [2]. Exercise promotes the consumption of energy sources, such as glycogen and glucose. Glucose transport is increased by muscle contractions, maximizing internal energy metabolism. On the other hand, hard work or intensive exercise can cause active free radical production, resulting in tissue oxidative damage. Physical fatigue is caused mainly by the depletion of energy and the excessive accumulation of metabolites. 

Recently, the development of natural supplements with efficient recovery ability after exercise, which accelerates the elimination of fatigue-related metabolites, is becoming a major research focus. Gynostemma pentaphyllum (Thunb.) Makino is an herbaceous vine plant of the family Cucurbitaceae, whose leaves are used for traditional tea or oriental herb medicine in Asia [3]. Cistanche has been prescribed traditionally for clearing heat, detoxification, dry cough, and chronic bronchitis [4]. Several studies have reported that Cistanche has important biological activities, including anti-oxidant [5], anti-inflammatory [6-8], hypoglycemic [9], hypolipidemic [10-12], anti-obesity [13], anti-diabetic [14], neuroprotective [15], and anti-cancer properties [16]. Cistanche contains saponins, polysaccharides, flavonoids, and other chemicals [17,18]. acteosides are major active components of dammarane-type saponins extracted from Cistanche. acteosides have anti-oxidant [19], anti-cancer [20], hypolipidemic [10], hepatoprotective [10,19], and anti-obesity activities [13]. The no observed adverse effect level of the Cistanche oral extract in rats was reported to be 750 mg/kg [21]. 

Recent studies have shown that acteosides and polysaccharides from Cistanche extend the swimming exhaustion time and increase the hepatic and muscle glycogen concentrations in mice [3,4,22-24]. Although the beneficial effects of Cistanche have been proven, the possible energy metabolism and associated molecular mechanisms behind the ability of the Cistanche extract (GPE) to increase the exercise capacity of mice on treadmill exercise is unknown. Recently, a beneficial method that produces GPE with much higher contents of acteoside L (GL), acteoside LI (GLI), and ginsenoside Rg3 (Rg3) than the extracts by other conventional methods was developed [25]. The previous study reported that treatment with GPE and GL increased the glucose uptake and glucose transporter type 4 expression by activating the adenosine monophosphate (AMP)-activated protein kinase (AMPK) and acetylCoA carboxylase signaling pathway, which enhances the mRNA expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in L6 skeletal muscle. In addition, GPE reduced body fat mass and increased lean body mass by AMPK activation. GPE supplementation inhibited adipogenesis in high-fat diet-induced obese C57BL/6 mice [13]. PGC-1α, a transcription factor coactivator, plays an important role in regulating the genes involved in mitochondrial biogenesis, glucose/fatty acid metabolism, angiogenesis, oxidative metabolism, fiber-type switching, and muscle growth. Exercise drastically increased PGC- 1α expression in the muscles. The overexpression of PGC-1α in mice improved exercise capacity, oxygen uptake, and fatigue resistance [25]. Hence, effective acteosides, GPE and GL, improved exercise performance by activating PGC-1α. Thus, this study examined the effects of GPE and GL on exercise performance and energy metabolism-related gene expression in treadmill-trained mice.

MATERIALS AND METHODS 

Materials Cistanche 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) [26,27]. Briefly, the dried leaves of Cistanche 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 [26,27]. 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 Cistanche leaf extract). Creatine monohydrate (CrM) (SigmaAldrich Inc., St. Louis, MO, USA) was used as the positive control. 

Animals and experimental design 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 body weight, dietary, and social behaviors were monitored during the administration period and exercise.

Exercise endurance tests 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 m2 s−2).

Body composition analysis and sample collection The whole-body composition (fat mass and lean body mass percentage) was measured using dual-energy X-ray absorptiometry (DEXA, PIXlmusTM; 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 cervical dislocation. The organs were kept at −70°C until further analysis.

Fig. 1. Experimental design to examine the effects of GPE, GL, CrM supplementation on exercise adaptation. The animals were assigned randomly to the indicated 6 groups (n = 10 per group in each test). Physical exercise capacity and related assessments were conducted during the test for 6 weeks. GPE, Cistanche extract; GL, acteoside L; BW, body weight; CrM, creatine monohydrate; SC, sedentary with the vehicle; SGPE, sedentary with 300 mg/kg BW/day of GPE; ExC, exercise with vehicle; ExGPE, exercise with 300 mg/kg BW/day of GPE; ExGL, exercise with 7 mg/kg BW/day of GL; ExCrM, exercise with 75 mg/kg BW/day of CrM.

Blood chemistry assay 

Serum levels of glucose, triglyceride (TG), total cholesterol (CHOL), low-density lipoproteincholesterol (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.

Tissue glycogen content assay 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 10min 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.

Protein expression analysis 

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 ImageQuantTM LAS 500 imaging system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). β-actin was used as the internal control.

Real-time reverse-transcription polymerase chain reaction (RT-PCR)

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 HyperScriptTM 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-GeneTM 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. 

Statistical 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 was performed using SAS statistical software 9.4 (SAS Institute Inc., Cary, NC, USA). P-values < 0.05 were considered significant.

RESULTS

Effects of GPE Administration on the Body and Muscle Weight in treadmill trained mice 

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 body weight, 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.

Table 1. Quantitative real-time polymerase chain reaction primers

Table 2. Effect of GPE and GL treatment on BW, fat mass percentage, lean mass percentage, and QF, GA, SOL, and EDL weights in treadmill-trained ICR mice

Effect of GPE Administration on the endurance exercise performance in the treadmill-trained mice 

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. 

Effect of GPE administration on fatigue-associated biochemistry

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 [28,29]. 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 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.

Fig. 2. Effects of GPE and GL treatment on treadmill exercise performance. (A) Endurance time to exhaustion and (B) exercise capacity. Values are means ± SEM for n = 10 per group. GPE, Cistanche extract; GL, acteoside L; BW, body weight; CrM, creatine monohydrate; SC, sedentary with vehicle; SGPE, sedentary with 300 mg/kg BW/day of GPE; ExC, exercise with vehicle; ExGPE, exercise with 300 mg/kg BW/day of GPE; ExGL, exercise with 7 mg/kg BW/day of GL; ExCrM, exercise with 75 mg/kg BW/day of CrM. ***P < 0.001 (SC group vs. SGPE or ExC group); #P < 0.05, ##P < 0.01, ###P < 0.001 (ExC group vs. ExGPE, ExGL or ExCrM group).

Table 3. Effects of GPE and GL Treatment on clinical biochemistry tests in ICR mice

Effect of GPE Administration on the Liver and muscle glycogen level 

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.

Effect of GPE Administration on mitochondrial biogenesis

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.

Fig. 3. Effects of GPE and GL treatment on glycogen contents in the liver and muscle of ICR mice. (A) Glycogen content (mg/g liver). (B) Total glycogen in liver (mg). (C) Glycogen content (mg/g GA). (D) Total glycogen in GA (mg). The values are the means ± SEM for n = 10 per group. GPE, Cistanche extract; GL, genocide L; BW, body weight; CrM, creatine monohydrate; SC, sedentary with the vehicle; SGPE, sedentary with 300 mg/kg BW/day of GPE; ExC, exercise with the vehicle; ExGPE, exercise with 300 mg/kg BW/day of GPE; ExGL, exercise with 7 mg/kg BW/day of GL; ExCrM, exercise with 75 mg/kg BW/day of CrM; GA, gastrocnemius muscle. #P < 0.05 (ExC group vs. ExGPE, ExGL or ExCrM group).

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