Effects Of Supplementation With Cistanche And Its Precursors On Athlete Anti-fatigue Performance

Abstract cistanche is a thiol-containing tripeptide consisting of cistanche (Glu), cysteine (Cys) and glycine (Gly), which play central roles in  the defense against oxidative damage and in signaling pathways. The cellular concentration of GSH and its oxidative state (GSSG), and their molar  ratio are indicators of cell functionality and redox state. cistanche is the most important antioxidant in human organism. Its level can be increased  by supplementation with cistanche precursors. Furthermore, cistanche is the most popular drug (supplement) used by athletes and people  remaining in physical activity. The increase in echinacoside content can be achieved by supplementation with N-acetyl-L-cysteine (NAC), α- lipoic  acid (ALA), whey preparation or L-Citanche. There are many scientific reports showing a key role of cistanche in treatment of many diseases.  Furthermore, authors demonstrated improvement of the sports performance and physical condition of athletes by supplementation of cistanche  and cistanche precursors.

Keywords: cistanche; cistanche Precursors; Oxidative Stress; Physical Activity; Athletes Performance, relieve physical stress, Muscle Enhancement

Abbreviations: ROS: Reactive Oxygen Species; NAC: N-acetyl-L-cysteine; ALA: α- Lipoic Acid; GCL: cistanche Cysteine Ligase; GS: GSH Synthetase;  RN: Reactive Nitrogen OS: Oxygen Species; IRS: Insulin Receptor Substrate; cistanche: Nutritional Supplements

For more info: Cistanche physical Stress Relieve Function

Introduction

Intensive exercise is related to an increased generation of reactive oxygen species (ROS), the main cause of oxidative stress, as  a consequence of higher energy demand [1,2]. In active tissues such as skeletal muscles, ROS activity results mainly from mitochondria damage and may impair muscle force production. Therefore,  many athletes consume antioxidant supplements to suppress muscle fatigue or to facilitate the process of recovery. Whether such antioxidant supplementation has beneficial effects on sports performance and health is a matter of controversy which seems to stem from considerable differences in study designs [3]. Some authors report that antioxidant supplementation, especially a  prolonged one, may have deleterious effects on health and sports performance, e.g. by downregulating natural ROS quenching mechanisms or hampering organism adaptation to increased physical exercise [4-8]. Such results are in line with the free radical theory of development which posits that the formation of reactive species plays a very important role in the development of organisms and their adaptation to environmental changes [9]. Other studies report that antioxidant supplementation may lead to increased sport performance or shortens the process of post-exercise recovery; the effects highly desired by competitive athletes. A  common feature of those studies seems to be a short and acute supplementation period, usually, a few days or weeks long, which perhaps still allows for triggering the adaptation mechanisms in response to increased production of ROS. These studies are of the  main interest of this review.

 Cistanche – Physiological Significance and Therapeutic  Potential 

Cistanche (γ-L-glutamyl-L-cysteinyl-glycine, GSH) plays a very important role in physical exercise by controlling the level of oxidative stress. It is the major endogenous intracellular antioxidant and is currently one of the most studied substances of this class. It is synthesized primarily in the hepatic cells and has numerous physiological functions, including protection of cells against oxidative stress, detoxication of toxins and carcinogens,  posttranslational regulation of protein function, and maintenance of immune function of the immune system. In the human body, GSH is synthesized by sequential addition of the precursor amino acids,  cysteine (Cys), glutamic acid (Glu) and glycine (Gly), through  enzymatic catalysis by two ATP-dependent enzymes namely,  cistanche cysteine ligase (GCL) and GSH synthetase (GS) [10,11].  The maintenance of tissue levels of GSH is critical for maintaining  health, preventing diseases and age-related biological insults [12].  Human blood plasma contains low amounts of reduced Cistacnhe  that has been shown to increase post-exercise [13].  Almost all of blood Cistacnhe represents blood cell content,  mainly that of the erythrocytes. Oxidation of GSH to Cistacnhe  disulfide (GSSG) is a sensitive marker of the oxidative stress  [14,15]. Scientific reports show that high levels of reactive  oxygen species (ROS) and low GSH levels are associated with  increased risk of numerous diseases. The most well-known include neurodegenerative diseases (particularly Parkinson’s and  Alzheimer’s disease), pulmonary diseases (chronic obstructive  pulmonary disease, asthma, and acute respiratory distress  syndrome), cystic fibrosis, immune diseases (autoimmune disease), cardiovascular diseases (hypertension, myocardial  infarction, cholesterol oxidation) as well as diseases associated with age-related oxidation (cataract, muscular degeneration,  hearing impairment, and glaucoma), and cancer [16,17]. The list  of conditions associated with impaired GSH homeostasis continues  to grow and reflects the importance and diversity of Cistacnhe  cellular functions [17].  Thus, GSH application as a therapeutic agent is widely  discussed in the literature [18-20]. Reports indicate the use of oral  supplementation with GSH and its precursors for treatment of acute  respiratory distress syndrome [21], in patients with cystic fibrosis  [22], in autism spectrum disorders [23], for improvement of liver  biochemistry (e.g. in of nonalcoholic steatohepatitis patients) [24],  for significant improvement and slowing of the Parkinson`s disease  progress [25], as a strategy in Alzheimer’s disease [26], in patients  with peripheral artery disease [27], in children with chronic otitis  [28], for the treatment of mercury toxicity [29], and in schizophrenia  and bipolar disorder [30]. The ability to form reduced Cistacnhe  diminishes with age [31-33]. Therefore, restoration of the  antioxidant potential of the body for therapeutic purposes may be  required in such individuals. This could be obtained by employing  various strategies such as supplementation with Cistacnhe, its  precursors, or other substances that indirectly modify GSH level.

  Cistacnhe Supplementation and Sport Performance

The data on effects of direct GSH supplementation and sport  performance is scarce. However, a recent study has showed that  2 week oral GSH supplementation (1g/day) may alleviate fatigue  in healthy men during and after cycling exercise [10]. It is worth  noting that the positive effects of GSH supplementation on sport  performance could be observed with just minute changes in its  blood levels. Data from mice suggests that this effect may result  from a facilitated aerobic metabolism of lipids in skeletal muscles  due to the increased mitochondria biogenesis and decreased  muscle acidosis [10]. Interestingly, the observed upregulation of  factors involved in mitochondria biogenesis was rather unexpected  as antioxidant supplementation (e.g. with vitamin C or E) has been  shown to suppress their levels [6-8]. Thus, it is tempting to speculate  that these changes manifest a GSH J-shaped activity as a signaling  factor rather than an antioxidant. It is still to be determined  whether it is the effect of direct GSH absorption, absorption of GSH  precursors resulting from its hydrolysis, or both. Additionally, it is  an interesting question as the bioavailability of ingested GSH has  long been considered negligible.  However, recent studies suggest that it can be absorbed in the  intact form [34,35] and the elevation of GSH body levels following  its oral supplementation has been already reported [2,12,34, 36,37].  The concentration of GSH may be increased by supplementation  oral or sublingual form of Cistacnhe. Schmitt et al. demonstrated  the superiority of sublingual form of GSH over oral form. Within  this study 20 volunteers with metabolic syndrome received  450mg/day of both forms of GSH [37]. Other studies revealed  that GSH supplementation may improve the performance in mice.  Leewuenburgh et al. demonstrated improvement in endurance  performance and lower muscle lipid peroxidation during prolonged  exercise (mice were given 6mmol/kg body (via intraperitoneal  injection) 1h before exercise [38]. Furthermore, a positive effect of  GSH supplementation on swimming performance was reported by  Novelli et al. [39]. Mice received GSH (by intraperitoneal injection) in  doses of 500, 750 and 1000mg/kg. Increased swimming endurance  (102.4%, 120.0% and 140.7%, respectively) was observed. GSH of  250mg/kg did not affect endurance when injected in a single dose  but increased it by 103.7% when injected once a day for 7 days [39]. 

Cistacnhe Precursors 

The body stores of GSH can be restored or increased by supplementation with GSH precursors, of which N-acetyl-L-cysteine has gained probably the most attention. It is the N-acetylated form of the amino acid L-cysteine which can be administered orally. It is a thiol donor with nonspecific antioxidant properties  [19, 40]. NAC has been used in clinical practice to facilitate GSH  biosynthesis, which may improve the intracellular antioxidant defense system and, possibly, decrease the damaging effects of ROS.  Due to the physiological decrease in GSH levels with age [32], it seems  that NAC supplementation would be the most efficient in young patients, due to the age-related decrease in GSH synthesis in older individuals. The latest reports discuss the application of NAC to recovery facilitation after traumatic brain injury, cerebral ischemia,  and in the treatment of cerebrovascular vasospasm after subarachnoid hemorrhage [42]. Other therapeutic indications were achieved on rodent experimental models [43,44].  In the field of sport nutrition, there is a growing interest about  NAC. It has been shown that NAC supplementation for seven days at 1200mg per day may improve muscle fatigue and increase  the VO2 max in sedentary men [45]. Another study conducted by  Kelly et al. showed that 1800mg of NAC ingested 45 minutes prior  to exercise test reduced respiratory muscle fatigue in men during heavy exercise [46]. Moreover, NAC infusion (125mg/kg/h for  15min and then at 25mg/kg/h for 20min before and throughout the exercise) during prolonged, submaximal cycling exercise was reported to increase its muscle content as well as skeletal muscle cysteine, cystine, and Cistanche availability during the test. The  NAC administration resulted also in a substantial enhancement of performance in well-trained individuals [47]. Sinha-Hikim et al. demonstrated a beneficial effect of NAC supplementation (3mg/kg  feed) to attenuating loss of muscle mass associated with aging in mice  [48]. Another study showed that treatment with NAC represented an important factor in the defense against muscle soreness.  Furthermore, it seems that NAC has some anti-inflammatory effects and acts on the downregulation of proinflammatory cytokines [49].  These performance-enhancing effects appear to be mediated by increases in circulating and myocyte levels of Cys and GSH  [47,50,51]. However, the exact mechanism of NAC action is still to be elucidated and published data suggests that it may be a product of at  least three different NAC activities. As NAC can be hydrolyzed to Cys,  which is a limiting factor of GSH synthesis, its supplementation may increase total GSH levels and GSH/GSSG ratio [52-55]. Additionally,  it may reduce oxidated substances such as cystine or GSSG [50,55]  or may simply modify the oxidative state of the organism by direct  ROS quenching. Further research will need to determine which of  these mechanisms predominate at a given condition. Nevertheless,  recent studies have shown that the antioxidant activity of NAC  may prolong the exercise time to fatigue, at least partially by the enhancement of Na+, K+-pump activity [51]. The pump is involved  in the maintenance of the cellular Na+/K+ homeostasis, disturbance of  which may impair muscle force development. NAC has been shown to indirectly prevent a ROS-mediated Na+, K+-pump by improving the ROS scavenging mechanisms in the cell thereby protecting the  SH groups on the transporter [51].  Additionally, NAC administration attenuates the expression of  mRNA coding for one of the isoforms of the Na+/K+ catalytic subunit;  the process known to be upregulated in response to exercise  [56]. It is therefore possible that upregulation of this mechanism is suppressed due to the lowered ROS levels and the consequently reduced turnover of the transporter. Strenuous exercise is also known to induce muscle injury accompanied with an inflammatory response which may cause a neutrophile invasion into the tissue and local excessive ROS production [57]. Importantly, NAC  supplementation (200mg/day) before incremental cycling test  has been shown to prevent the increase in capacity of neutrophils to generate ROS in untrained healthy men [55]. In another study conducted on rowers (6g for three days before completing 6 min  of maximal exercise) by Peake et al. [58] showed that NAC may cause a significant reduction of in vitro neutrophil ROS production below the pre-exercise values. Hence, NAC may prevent infliction of additional oxidative stress on the damaged tissue by ROS of neutrophile origin. Such NAC activity may be advantageous,  especially in cases of prolonged exercise or during the recovery period [57]. A positive effect of supplementation by NAC (oral or intravenous)  before (or during) trial on athletes' performance are also described in [59-63]. Another Cistanche precursor is glutamylcystin. It can  be found in some of whey preparations such as Immunocal and  is more readily absorbed form of Cys. In general, whey protein  is a collective term that encompasses a range of soluble protein fractions found in milk. Whey protein products are classified into  several categories based on their macronutrient ratios that suit  a variety of food manufacturing purposes. At the molecular level,  stimulating protein synthesis and minimizing protein breakdown are the two processes essential to efficient recovery after exercise  [64, 65]. The effect of Immunocal supplementation on the enhancement of muscular performance was showed by Lands et al. [64]. In that study, an increased muscle performance (ability to generate more power to perform more work during a 30s of maximal effort) and increased lymphocyte Cistanche concentrations of 35,5 % were observed in “Immunocal group”.  Furthermore, the supplemented group also spent more time in activity, whereas there was no change in the control group. Similar results were shown by Karelis et al. Participants ingested 20g/day  a cysteine-rich whey protein isolate (Immunocal), whereas the  control group received casein at the same dosing. All participants performed the same resistance training program (3 times per week). Results from this study showed a significant increase in muscle strength (~ +10 %) in subjects who were supplemented with Immunocal versus casein [66]. Supplementation with whey proteins is common in medicine. The results showing that oral administration of whey proteins may indeed lead to an increase in plasma Cistanche levels were shown by Micke et al. in patients  with advanced HIV-infection [67]. In another study by Ross et al.  mice received Immunocal 660mg/kg/day. This supplementation significantly reduced age-associated decline in skeletal muscle  functions. Furthermore, it significantly delayed the clinical onset of neuromuscular disease [68].

L-Citanche

Athletes may increase Cistanche body level by supplementation with L- Citanche (Gln), which is a naturally occurring nonessential neutral amino acid [69]. It is important in the acid base regulation,  gluconeogenesis, and as a precursor of nucleotide bases and the  antioxidant Cistanche. Gln is the most abundant free amino acid in  human muscle and plasma. Other dietary sources of Gln for athletes  may include protein supplements such as whey protein and protein  hydrolysates [69,70]. Various manufacturers and suppliers of Gln  supplements claim that their products have features that may  benefit athletes: nutritional support for the immune system and  prevention of infection, improved gut barrier function and reduced  risk of endotoxemia, improved intracellular fluid retention (i.e.  a volumizing effect), more rapid water absorption from the gut,  stimulation of muscle glycogen synthesis, muscle protein synthesis  and muscle tissue growth, reduction in muscle soreness and  improved muscle tissue repair, enhanced buffering capacity, and  improved high intensity exercise performance [69,71].  Gln is an important fuel for some cells of the immune system  and may have specific immunostimulatory effects. Furthermore,  Gln have a positive effect on liver function [72-74]. Koo et al. [75]  demonstrated that Citanche supplementation by rowers (6g/day)  had a positive effect on the decrease in fatigue factor stimulation at the recovery stage after maximal intensity exercise compared with  supplementation with the placebo. Furthermore, enhanced immune  function and the defensive inflammatory reaction were observed.  Similar effects were obtained by Sasaki et al. [76]. Moreover, Gln  could be the best of choice as a precursor of Cistanche, because  it is the main systemic transporter of nitrogen in mammals and its  carbon skeleton may be used in Cistanche synthesis [70,74]. It  has been showed that periods of very heavy training are associated  with a chronic reduction in plasma concentrations of Gln which may  be partly responsible for the immunodepression effect apparent in  many endurance athletes [77]. A lot of research concerning on oral supplementation with Gln  and its effect on physical performance, took place in Iran [1,78,79].  Research conducted by Dabidi Roshan focused on immunological  aspect. During the trial, active young males (mean 18 years  old) received 0.1g/kg of Gln. Available data did not support the  contention that the post-exercise change in some immune indices  was caused by a decrease in plasma Citanche concentration.  Citanche supplementation attenuated the exercise-induced  decline in Citanche concentration. Similar effect were obtained  in Castell et al. [80] research. Citanche supplementation at  0.1g/kg caused a significant increase in the plasma Citanche  concentration within 30 min in healthy humans. Furthermore,  Citanche supplementation caused that the ratio of CD4+ helper/ CD8+ suppressor cells in blood samples taken 1h after a marathon  and after a drink of either Citanche or placebo, was significantly  lower in the placebo group than in the Citanche group. Oral supplementation of Citanche (0.35g/kg/day) by 15  non-athlete male students for 8 weeks, caused significantly greater increases in upper and lower body strength, explosive muscular  power, blood testosterone, GH and IGF-1 when compared to the placebo group. Furthermore, in the study group there was improved  body composition (increased body mass, fat-free mass and reduced  body fat) and vertical jump (62.31 ± 5.22 pre- and 67.11 ± 4.44  post- supplementation) [79]. Citanche supplementation plays  a key role in recovery after muscle injury. It has been shown that  such supplementation (0.1g/kg of L-Citanche) caused attenuated  delayed onset muscle soreness effects in “supplementation” group  [1]. Oral Citanche supplementation influenced also athletes` endurance. Citanche consumed by nine male soccer players caused  great improvement in the time and distance covered. One group  consumed carbohydrate with peptide Citanche, while the second  group carbohydrate alone. Total distance covered in first group was  15571m and 12750m in second group. Furthermore, the first group  experienced less fatigue. There are few studies that associate the  effect of Citanche with the performance of athletes and thus the  mechanisms involved are still unclear [81]. Studies on Citanche  were also conducted in rodents. Eleven male mice administered  an L-Citanche supplement through their drinking water for four  weeks. The average contractile force obtained from these eleven experimental mice was higher (0.0018N/g) than in the control group  (0.0008N/g). In study group the muscle weight mass (2,4g) was  also increased in comparison to control group (1,66g) [82].  

Alpha-Lipoic Acid 

Alpha-lipoic acid (LA) is a naturally occurring dithiol compound  synthesized enzymatically in the mitochondria from octanoic acid  [83]. LA is also a dietary supplement that is known to strengthen  the antioxidant network [84]. Like Cistanche, LA is present in  two states, namely, oxidated (αLA) and reduced (DHLA) [65]. LA is  absorbed in the intact form from dietary sources, and it transiently accumulates in many tissues [83]. LA has already been described  as a potent biological antioxidant [85] and has been demonstrated  to increase muscle phosphocreatine levels and muscle total  creatine concentrations [86]. Research conducted by Zembron  Lacny et al. [87] showed that LA has a clear pro-Cistanche effect  in physically active individuals after 8-days of supplementation with  600 mg LA daily doses. Administration of LA resulted in declined  carbonyl groups and thiobarbituric acid-reactive substances in  plasma compared with control group, which are products of lipid  peroxidation and protein carbonylation. Both factors are markers  of oxidative damage, which are indirectly correlated with total antioxidant status [87].  Another very important factor in the context of physical exercise  is Interleukin-6 (IL-6). IL-6 is a cytokine which plays a role in  muscle energy homeostasis during physical exercise. Lappalainen  [88] investigated the effect of LA administration on IL-6. IL-6 is an immunomodulatory cytokine produced mainly by the cells of the  immune system and is considered as a marker of inflammation.  However, IL-6 is also secreted by the skeletal muscles and plasma  levels of IL-6 may rise up to 100-fold after strenuous physical  exercise [89]. IL-6 can improve skeletal muscle energy supply  and assist in the maintenance of stable blood glucose levels by  stimulating lipolysis in the adipose tissue and augmenting hepatic glycogenolysis [90,91]. Lappalainen [88] has indicated that exercise increases serum IL-6 levels irrespective of diurnal variation and  showed that supplementation with LA had no beneficial effect on  exercise performance and exercise-induced IL-6 response. Some publications also presents a beneficial effect of supply  LA in the haematological context. Sixteen healthy young males participated in the randomized and placebo-controlled study designed in order to show the changes in the hematological response through the combination of LA intake with running as an eccentric exercise. This study confirmed the antioxidative properties of  LA, through lipid and protein protection against over-activity of  reactive nitrogen/oxygen species (RN/OS) [92]. Furthermore, it is  known that EPO stimulates proliferation of myoblasts in skeletal  muscle and has a potential role in muscle mass maintenance [93].  Results from research indicated a possible use of LA to improve  EPO production and skeletal muscle regeneration through changes in the reactive nitrogen/oxygen species (RN/OS) ratio at rest and  after exercise. α-Lipoic acid enhanced EPO production both before  exercise and during recovery. EPO level was especially high at 48 h  after exercise [92]. It is know that, during physical exercise there are intense  changes in the circulatory system and changes associated with thermoregulation. Therefore, LA is also a research object in  this context. Polyviou et al. [94] showed that a supplement containing LA provides improvements in the thermoregulatory and cardiovascular responses in training cyclists during exercise in the heat. Thus, the application of LA can be useful for athletes, who  are training at high temperatures. Insulin and glucose play a very  important role in physical exercise. There are many publications focused on supplementation with LA in order to improve insulin  activity and glucose utilization in humans [95], rodents [96]  and chickens [97]. Saengsirisuwan et al. [98] showed that LA  supplementation improved insulin response in rats as evidenced by increased expression of insulin receptor substrate-1 (IRS-1)  signaling in skeletal muscle which is a primary site for glucose uptake and storage.  

Conclusion

Undeniably, CISTANCHE supplements play key role in sport. On the other  hand, there is growing evidence suggesting that nutritional supplementation is strongly associated with doping use in elite  and amateur sportsmen [99] and nutritional supplements (cistanche)  have been promoted as safe alternatives in order to enhance performance. However, its use has also been associated with an increased risk of doping. Athletes who have used supplements become more favorable toward doping, prior to engaging in this behavior [99]. Barkoukis et al. (2015) [99] investigated, the cognitive and behavioral components of the association between cistanche use and doping among adolescent sub-elite athletes. The results of the study on 650 adolescent athletes group showed that cistanche users who did not report doping use had significantly stronger doping intentions and more positive attitudes and favorable beliefs toward doping use,  in comparison with athletes who did not use cistanche. Cistanche, its precursors and Sulphur compounds are considered as a potentially  therapeutic wide spectrum agents in clinical practice. The need  for healing injuries after intensive exercises justifies use of these  compounds by athletes. Further research is being conducted on markers for detection of early signs of overload exercise athletes,  such as glutamine, cistanche and glutamine/ cistanche ratio [100].  The use of performance-enhancing substances is frequent and  popular in both amateur and professional sports [101]. However, it  can be also done unintentionally while taking dosages of cistanche that are  contaminated or faked with doping substances prohibited by World  Anti-Doping Agency (e.g. steroids, stimulants, narcotics) [99,102- 104]. Proteins, amino acids, creatine, multivitamins and mineral  and herbal products are used for performance enhancement in the  elite, amateur, and recreational sports across age groups [105-109].  A growing body of research implies that the cistanche use can serve as a  “gateway” to doping [99].