Changbai Mountain ginseng (Panax ginseng
C.A. Mey, CMG) is a wild ginseng that grows in the Changbai Mountain Range in Jilin Province, the highest mountains (2750 m) in Northeastern China. CMG is harvested from the local forests and has been used as a traditional Chinese medicine (TCM) for more than a century. Recently, the Chinese government has developed large wild ginseng growing bases, where traditional Chinese herbs are produced, to meet the Good Agricultural Practices (GAP) standards and encourage the development of the pharmaceutical industry in Northern China [1
]. In general, the two major species of ginseng are Asian (Panax ginseng
) and American (Panax quinquefolius
]. In previous reports, a common mixture of active ingredients was demonstrated to be ginsensosides [3
]. The benefits of ginseng are attributed to bioactive compounds, such as ginsenosides, volatile oils, polyphenols, flavonoids, polysaccharides, and vitamins [6
]. In previous reports on physiological functions, ginseng exhibited anti-cancer [11
], immunomodulatory [12
], and antidiabetic [13
] effects, and cardiovascular improvement [14
] in delivering ginseng ginsenosides. Ginseng roots vary from different regions, and differences in environmental factors could influence the genotypes [15
]. Thus, CMG could be more bioactive than ginseng grown in other locations. The aim of the current study was to evaluate the anti-fatigue activities of ginseng polysaccharides, as well as the neutral, acidic portions and ginseng polysaccharides of CMG [16
]. However, there is a lack of research to support the anti-fatigue effects of the major compounds of CMG, which could improve exercise performance. In this study, we identified the major compound of CMG extract and evaluated the ergogenic, anti-fatigue, and health promotion effects of CMG supplementation using our previously established in vivo platform [17
The beneficial effects of ginsenoside species have been well demonstrated in many studies. However, the function of ginsenoside Ro, an oleanane-type saponin, has not been sufficiently investigated. The place of origin and weather could influence ginseng trace elements and secondary metabolites, which could explain why CMG differs from other types of ginseng and has a high content of ginsenoside Ro. In general, programmed exercise training is required to increase grip strength [20
]; however, we found that ginsenoside Ro supplementation benefited grip strength even though the test animals underwent no training intervention. Thus, long-term ginsenoside Ro supplementation could benefit the muscle explosive force under non-training conditions. However, few studies have investigated the use of ginseng supplementation to improve muscle strength. According to a previous study, dammarane steroid, a ginseng steroid that is present in many ginseng species, has anti-inflammatory effects on skeletal muscle following a bout of muscle-damaging exercise [21
]. Thus, ginsenoside Ro may have an anti-inflammation effect and enhance skeletal muscle performance. Ginseng has a wide range of benefits that could promote physical performance and recovery capacity from interval exercise [22
]. We suggest that ginsenoside Ro may improve endurance performance in the absence of training. Further investigation is required to clarify which types of saponins could benefit exercise training for endurance and explosive force performance. In a previous report, 20(R)-ginsenoside Rg3 was demonstrated to increase weight-loaded swimming time [23
]. Moreover, ginsenoside Ro may act as a potential ergogenic aid for exercise supplementation.
Exercise-induced muscle fatigue can be evaluated with biochemical indicators, including lactate, ammonia, glucose, CK, and BUN levels [24
]. The clearance of lactate is thought to reduce peripheral neuromuscular fatigue and have positive effects on muscle function [26
]. After acute exercise, relaxation is significantly affected by the blood lactate clearance rate. Approximately 75% of the total amount of lactate produced is used for oxidative production of energy in the exercising body, and lactate could be utilized for the de novo synthesis of glucose in the liver [27
]. During exercise, muscle utilizes glucose from glycogen by intramuscular glycogenolysis and by increased glucose uptake. Regardless, aerobic and resistance exercises increase glucose transporter type 4 (GLUT4) abundance and translocation, thereby increasing serum glucose uptake by a pathway that is not dependent on insulin [28
]. Ammonia, an important metabolite during energy metabolism for exercise, is generated by different sources. Accumulation of ammonia in the blood and brain during exercise can negatively affect the central nervous system and cause fatigue. The present data suggest that continuous supplementation with CMG extract for four weeks could decrease lactate, ammonia, and BUN accumulation, economize serum glucose levels and, thus, CMG extract could have anti-fatigue activity. The effect on CK serum response appears to be related to the magnitude of eccentric contractions involved in the activity and the subsequent extent of muscle disruption [30
]. High-intensity exercise challenge can physically or chemically cause tissue damage and muscular cell necrosis [31
]. Thus, ginsenoside Ro could reduce muscle damage after exercise.
Concerning glycogen, in our present study, ginsenoside Ro may have influenced hepatic and muscle glycogen metabolism. Previously, it has been reported that ginsenoside suppresses hepatic glucose production through inhibiting the small heterodimer partner (SHP) gene expression [32
] or increasing hepatic glycogen storage [33
]. Ginseng, or ginsenosides associated with acetyl CoA carboxylase kinase (AMPK) activation, switches off anabolic pathways, including glycolysis, lipolysis, glycogen synthesis, and protein synthesis in the liver [34
]. There are at least three mechanisms to regulate the glucose uptake of skeletal muscle. To begin with, glucose delivery to the skeletal muscle cells; then, glucose transport through the cell surface membrane; and, finally, flux through intracellular metabolism [35
]. During short-term exercise, the muscle glycogen is the preferred carbohydrate fuel for both aerobic and anaerobic metabolism. When exercise is prolonged, glycogen stores in muscle and liver are depleted during exercise, and blood glucose utilization becomes the main carbohydrate fuel during exercise [36
]. Therefore, we suggested that CMG-1X supplementation was the proper dose for recommendation as an ergogenic aid. Our present data also showed that CMG-5X could increase the serum glucose levels by decreasing glycogen storage. We suggested that the ginsenosides may increase glycolysis [37
]. During exercise, both serum glucose and muscle glycogen are important fuels. The increase of muscle glucose uptake during exercise depends upon the delivery of glucose (capillary perfusion and plasma glucose concentration) and the permeability of the muscle membrane to glucose [35
]. In addition to muscle glycogen content, serum glucose level is the other energy utilization index. Therefore, CMG could modulate the muscle glycogen storage and ginsenoside Ro could be as an ergogenic supplement. It had been reported that ginseng saponin decreases plasma triglyceride levels and inhibits atheroma formation in animals with hypercholesterolemia [38
]. The reason why enhanced nutrition absorption with TP was increased by CMG extract supplementation could be that the ginsenosides were biotransformed by intestinal bacteria, which further improved intestinal absorption and bioactivity and diminished the toxicity of the metabolite [39
However, there were still some imitations of this study. The major limitation of the study is that we cannot extrapolate our results to the human because only animal blood of tissue samples were included in this study. Another limitation of the study is the lack of information on the major compound of ginsenoside profiling data from fresh or dried material or during growth of Changbai Mountain Ginseng in different seasons.
4. Materials and Methods
4.1. Preparation of Changbai Mountain Ginseng (CMG) Extract
CMG specimens were cut into small pieces and soaked in ethanol at ambient temperature for seven days. The extracts were decanted and filtered through Whatman No. 2 filter paper (Sigma, St. Louis, MO, USA), and the filtrates were concentrated in a rotary evaporator before being lyophilized. The yield of CMG was 0.4% (1.96 g/470.5 g × 100).
4.2. Liquid Chromatographic-Mass Spectrometry (LC-MS) Analysis of CMG Extract
One hundred microliters of CMG extract powder (670 mg/mL) were dissolved in milli-Q water. Before being loaded into SPE (polymeric reversed-phase solid-phase-extraction cartridges; 200 mg/3 mL; StrataX, Phenomenex, Denver, CO, USA), the CMG extract liquid was added into 600 μL methanol and then eluted out with 10%–100% of acetonitrile. The system included an HPLC equipped with a Thermo Finnigan model LXQ linear ion trap mass spectrometer (San Jose, CA, USA) operated in negative ion electrospray mode. A YMC Hydrosphere C18 analytical column (2.0 × 150 mm, 5 μm, YMC, Kyoto, Japan) was maintained at room temperature, and a flow-rate of 0.2 mL/min was used. The mobile phase consisted of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B). Gradients were programmed as follows: 21% B for 0–5 min, 21%–34% B for 5–40 min, holding at 34% B for 40–60 min, 34%–38% B for 60–70 min, 38%–100% B for 70–80 min, and holding at 100% B for 80–85 min. A ginseng ginsenosides standard kit (Sigma, G-015, St. Louis, MO, USA) was prepared for injection volumes of 10 µL.
4.3. Animals and Experiment Design
Male ICR mice (nine weeks old) grown under specific pathogen-free conditions were purchased from BioLASCO (Yi-Lan, Taiwan). All mice were provided a standard laboratory diet (No. 5001; PMI Nutrition International, Brentwood, MO, USA) and distilled water ad libitum, and they were housed with a 12-h light/12-h dark cycle at room temperature (22 ± 1 °C) and 50%–60% humidity. The Institutional Animal Care and Use Committee (IACUC) of National Taiwan Sport University (NTSU) inspected all animal experiments, and this study conformed to the guidelines of protocol IACUC-105,020 approved by the IACUC ethics committee. In this study, the dose of CMG for humans was 24.4 mg per day (CMG extract). The mice dose (5 mg/kg) we used was converted from a human-equivalent dose (HED) based on body surface area by the following formula from the US Food and Drug Administration: assuming a human weight of 60 kg, the HED for 24.4 (mg)/60 (kg) = 0.406 × 12.3 = 5 mg/kg; the conversion coefficient 12.3 was used to account for differences in body surface area between mice and humans as recently described [41
]. In total, 24 mice were randomly assigned to three groups (eight mice/group) for daily oral CMG treatment for 4 weeks. The groups and treatment courses were as follows: vehicle; 5 mg/kg (CMG-1X); and 25 mg/kg (CMG-5X). The vehicle group received the same volume of solution equivalent to individual body weight (BW). Mice were randomly housed in groups of four per cage.
4.4. Forelimb Grip Strength Test
A low-force testing system (Model-RX-5, Aikoh Engineering, Nagoya, Japan) was used to measure the forelimb grip strength of treated mice as previously described [42
]. Forelimb grip strength was tested one hour after administration of the indicated CMG supplementation for four weeks. The forelimb grip strengths of all mice were measured on the same day.
4.5. Swimming Exercise Performance Test
The swimming endurance test was conducted after the forearm grip strength test for all mice. For the swim-to-exhaustion test, loads corresponding to 5% of the mouse BW were attached to the tail to evaluate endurance time [43
]. The swimming endurance time of each mouse was recorded from the beginning to exhaustion, determined by observing loss of coordinated movements and failure to return to the surface within 7 s. The test of swimming endurance time was performed one hour after administration of the indicated CMG supplementation for 28 days. The swimming exercise performance tests of all mice were performed on the same day as the forelimb grip strength measurement.
4.6. Determination of Fatigue-Associated Biochemical Variables
The effect of CMG supplementation on fatigue-associated biochemical indices was evaluated after exercise as previously described [43
]. The 15-min swimming test was performed one day after the forelimb grip strength and the swimming exercise performance test. After CMG supplementation for one hour, all mice underwent a 15-min swimming test without weight loading. Immediately after the 15-min test, 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. Serum lactate, ammonia, glucose, creatine kinase (CK), and blood urea nitrogen (BUN) levels were determined with an autoanalyzer (Hitachi 7060, Hitachi, Tokyo, Japan).
4.7. Clinical Biochemical Profiles
Two days after the 15-min swimming test, all mice were sacrificed with 95% CO2 asphyxiation, and their blood was immediately collected. Blood collected by cardiac puncture was centrifuged at 1500× g for 10 min at 4 °C. Serum was separated by centrifugation and the levels of clinical biochemical variables (CK, albumin, total protein (TP), BUN, creatinine, uric acid (UA), total cholesterol (TC), triacylglycerols (TG), and glucose) were measured with an autoanalyzer (Hitachi 7060, Hitachi, Tokyo, Japan).
4.8. Histology of Tissues
Liver, skeletal muscle, heart, lung, kidney, and epididymal fat pad (EFP) tissues were carefully removed, minced, and fixed in 10% formalin. Samples were embedded in paraffin and cut into 4-μm thick slices for morphological and pathological evaluations. Tissues were stained with hematoxylin and eosin (H and E) and examined under a light microscope equipped with a CCD camera (BX-51, Olympus, Tokyo) by a veterinary pathologist.
4.9. Tissue Glycogen Determination and Visceral Organ Weight
The stored form of glucose is glycogen, which mostly exists in liver and muscle tissue. Liver and muscle tissues were excised after the mice were sacrificed and weighed for glycogen content analysis as described previously [44
]. The weights of the liver, skeletal muscle, heart, lung, kidney, epididymal fat pad (EFP), and brown adipose tissue (BAT) related to visceral organs were recorded.
4.10. Statistical Analysis
All data are expressed as mean ± SD, n = 8 mice per group. Statistical differences among groups were analyzed with one-way analysis of variance (ANOVA and the Cochran-Armitage test for dose-effect trend analysis with SAS 9.0 (SAS Inst., Cary, NC, USA). p < 0.05 was considered statistically significant. Differences between groups were analyzed by one-way analysis of variance (ANOVA) using Duncan’s post-hoc test, and p values < 0.05 were considered significant.