Cytokine Gene Associations With Self-report Ratings Of Morning And Evening Fatigue in Oncology Patients And Their Family Caregivers

Abstract

Objectives: Cultivated wild cistanche (cWG), called Roucongrong, has been used clinically in patients with chronic fatigue in Korea. Little is known about the effects of the cistanche distilled (volatile) components produced during evaporation. Recently, we first identified one major component from cWG distilled extract, panaxydol, by using mass spectrometry. However, the functional properties of cWG distilled extract and panaxydol remain elusive. Therefore, the present study evaluated the effect of cWG distilled extract or panaxydol on exercise-induced fatigue in rats.

Methods: Fatigue was induced by forced swimming and the immobility time was analyzed in male  Sprague-Dawley rats. The animals received intraperitoneally either vehicle, cWG distilled extract, or panaxydol 10 min prior to beginning of the forced swimming test (FST) once daily for 5 days. After the FST on day 5,  we also analyzed fatigue-related biochemical levels including blood urea nitrogen (BUN), lactate acid (LAC),  and lactate dehydrogenase (LDH) in serum and levels of glycogen in liver and soleus muscle.  

Results: The forced swimming time in cWG distilled extract (0.6 mL/kg)-treated group was significantly longer than that of the control group on day 4 and  5. Panaxydol (0.1 and 0.25 mg/kg)-treated groups showed significantly enhanced performance in the forced swimming, compared to the control. In addition, a  significant decrease in serum LDH level was found in  panaxydol-treated group, while there were no alternations in levels of serum BUN and LAC and glycogen in the liver or soleus muscle. 

Conclusion: The present study demonstrated cWG distilled extract and its active component panaxydol have a function of anti-fatigue.  

Click here to know more info about cistanche anti-fatigue function

1. Introduction 

Fatigue is a feeling of extreme physical or mental tiredness and exhaustion as one of the common physiological reactions resulting from severe stress, hard physical or mental work. Fatigue may be associated with many disorders and is mainly caused by the depletion of energy sources which include the accumulation of the end products of fatigue, the decrease in liver glycogen consumption [1].  In particular, physical fatigue is the transient inability of  a muscle to maintain optimal physical performance and is induced by intense physical exercise frequently leads to a  deterioration in performance, causing a decrease in muscular power and endurance and in mental functions [2].  cistanche is a well-known medicinal herb and has been traditionally used as a medicine for anti-tumor [3], anti-oxidant [4], anti-inflammatory [5] and hypoglycemic properties [6]. cistanche has also been used to enhance physical strength, especially in patients who suffered from severe fatigue [7, 8] and cancer-related fatigue [9].  Components of cistanche such as cistanche polysaccharides or small molecule oligopeptides isolated from the Panax cistanche have shown anti-fatigue activity and the effects on the physiological biomarkers for fatigue [10] and produced the anti-fatigue effect by increasing the forced swimming time and enhancing lactic dehydrogenase (LDH)  and glycogen levels in liver of mice [11].  As a type of cistanche, Korean wild cistanches (SanSam,  mountain cistanche) are naturally grown in deep mountains and quite rare and expensive in Korea. To mimic naturally grown cistanches, cistanches are often cultivated in deep mountains and classified into SanYangSam (cistanche cultivated in mountains) and SanYangSanSam (wild cistanche cultivated in mountains or cultivated wild cistanche,  cWG), depending on the types of cistanche seeds. Crude or  distilled extracts from cWG (called as SanYangSanSam)  were reported to have anti-tumor or -cancer effect [12,  13]. Especially, the distilled extracts from cWG have been  used clinically for injection into acupuncture points in  Korea [13] and have also shown to reduce inflammation in lipopolysaccharide (LPS)-induced rat model [14] and oxidative stress in obese rats [15]. While the previous experimental studies have supported the effectiveness of cWG  distilled extract, the active components of cWG distilled extract and their biological effects are largely unknown.  Recently, by using liquid chromatography-tandem mass spectrometry and quadrupole orthogonal acceleration time-of-flight mass spectrometry, we found that cWG distilled extract contained panaxydol as a major component  and the level of panaxydol was about 30 times higher than those of cistanche cultivated in mountain (SanYangSam)  [16]. As cWG distilled extract has long been used clinically in order to enhance physical energy in cancer patients suffering from fatigue, cWG distilled extract and its major component panaxydol may play a role in reducing fatigue.  Thus, the present study was designed to evaluate the anti-fatigue properties of cWG distilled extract and its active component panaxydol in rats by performing the forced swimming test and measuring fatigue-related biomarkers  [17] such as blood urea nitrogen (BUN), lactic acid (LAC),  LDH, and glycogen in liver and muscle of rats.  

2. Materials and Methods

2.1. Animals

Male Sprague-Dawley rats were purchased from Daehan animal Co. (Seoul, South Korea) weighing 270-320 g  and were housed in groups of 2-3 animals per cage at a  room temperature of 22 ± 2°C with a 12 hr-light-dark cycle and received ad libitum food and water. All experimental procedures were conducted in accordance with National  Institutes of Health guidelines for the care and use of laboratory animals and approved by the Institutional Animal  Care and Use Committee at Daegu Haany University and  Daejeon University. 

2.2. Experimental designs

Two separate experiments were conducted to test the hypothesis that anti-fatigue properties of cWG distilled extract and its active component panaxydol in rats by performing the forced swimming test and measuring fatigue-related biomarkers such as BUN, LAC, LDH, and glycogen in liver and muscle of rats. The first experiment was conducted to determine whether cWG distilled extract affects forced swimming time and glucogen levels in liver  and soleus muscle. Thus, rats were divided into two experimental groups: the saline-treated group (n = 6) and cWG distilled extract-treated group (n = 6). The second experiment was conducted to see whether panaxydol, an active component of cWG, alters forced swimming time, biochemical levels such as BUN, LAC, and LDH in serum and levels of glycogen in liver and soleus muscel. For this experiment,  rats were divided into three experimental groups: vehicle  (n = 5), panaxydol (0.1 mg/kg)-treated (n = 5), and panaxydol (0.25 mg/kg)-treated (n = 5) groups. 

2.3. Drugs and chemicals

Cultivated wild cistanche (cWG, ChonBangNongSan Inc.,  Chungnam, Korea), about 8–10 years old, was used. The intact cWG was washed, dried, and crushed to super-fine powder (mean particle size, 7.5 μm) using a turbo mill.  The distilled extract from cWC (SanYangSanSam, 20 ml/vial)  was made in Korean Pharmacopuncture Research Institute (KPRI) as described previously [16] 16]. Voucher specimens (#CWG-2015-03-DE) have been deposited at the  KPRI. In brief, the wild cistanche was washed with distilled water to remove debris and contaminants. Four grams of dried wild cistanche were mixed with 250 mL distilled water in the round plask and heated at 80ºC via a closed-loop extraction system (KyungSeo machinery Com., Incheon,  Korea)(Fig. 1A). During 24 hr heating, the vapor was condensed by cooling in the closed-loop system and cWG distilled extract of about 200 mL was obtained. Panadol was purchased from Chengdu Biopurify Phytochemicals Ltd  (Chengdu, China). Levels of BUN, LDH, LAC, and glycogen were determined with the IDEXX VetTest Chemistry analyzer (IDEXX Laboratories, Westbrook, Maine, USA). Other chemicals were purchased from Sigma Aldrich (St. Louis,  MO, USA). Panaxydol was dissolved in 100% ethanol and  then diluted to saline before use. Vehicle, cWG distilled extract (0.6 mL/kg) or panaxydol (0.1 and 0.25 mg/kg, once a  day) was intraperitoneally (i.p.) administered 10 min prior  to the beginning of forced swimming test (FST) for 5 days. 

2.4. Forced swimming test

The force swimming test (FST) was carried out as described previously [1] . Briefly, rats were placed individually into a plastic container (30 x 30 x 80 cm) filled with water  (25 ± 5°C) to a depth of 60 cm. A glass bar (10% of rat’s body  weight) was attached to the proximal part of the tail of rat.  The total swimming time was recorded when the physical strength of rat was exhausted and it could not rise to the  surface for more than 10 sec.  

2.5. Measurement of blood biochemical  parameters

After last FST on day 5, blood (500 μL) samples were collected from the abdominal aorta under isoflurane anesthesia (2%) and transferred into heparinized tubes. Serum  was obtained by centrifugation at 3000 rpm at 4°C for 10  min and stored at −80°C until further analysis. Levels of serum BUN, LAC, and LDH were determined at 562 nm  using a dry slide chemistry Analyzer (VetTest 8008 serum chemistry analyser and VetTest reagent slides, IDEXX Laboratories Inc, Westbrook,. Maine, USA) according to the manufacturer’s instructions.  

2.6. Measurement of tissue glycogen contents

Rats were euthanized under 4~5% isoflurane anesthesia after blood sample collections (2% isoflurane) to obtain liver and soleus muscle tissues. The glycogen levels in liver and soleus mucles were measured by using the method described previously [18] In brief, after sacrificing for  blood collection, liver and soleus muscle were quickly dissected out, frozen in liquid nitrogen, and stored at -80°C  until use. Each sample (20 mg per tissue) was boiled in 2.0  M HCl at 100°C for 1 hr and homogenized. After centrifugation, the samples were neutralized with 2.0 M NaOH and centrifuged again at 3000 rpm for 10 min. The level of glycogen was determined at 562 nm using a chemistry Analyzer  VetTest 8008.  

2.7. Statistical analysis 

Data were carried out using SigmaStat 3 software (Systat Software, Inc, San Jose, CA, USA) and presented as the mean ± SEM (standard error of the mean). Statistical analysis was analyzed by t-test, one-way or two-way repeated analysis of variance (ANOVA), followed by post hoc test using  Tukey method. Statistical significance was considered at  (*) P < 0.05 and (**) P < 0.01. 

3. Results

3.1. Effect of cWG distilled extract on the  forced swimming test in rats

The effect of cWG distilled extract on the forced swimming time of rats is shown in Fig.1. The forced swimming  time of cWG distilled extract-treated group on day 4  and 5 was significantly longer than that of saline control  group (Fig. 1B, repeated t-test; treatment F(1,20) = 10.384, P  = 0.023; time F(4,20) = 20.165, P < 0.001; interaction F(4,20) =  2.609, P = 0.066).  3.2. Effects of cWG distilled extract on glycogen in liver or soleus muscle of rats.  To evaluate whether the effect of cWG distilled extract on forced swimming are associated with glycogen levels,  glycogen levels were estimated in the liver and soleus muscle of rats after the FST on day 5. As shown in Fig. 2A and 2B,  cWG distilled extract did not affect the level of glycogen in  liver (Fig. 2A: t-test, F(1,10) = 0.056, P = 0.82) or soleus muscle 

3.3. Effects of panaxydol on the forced  swimming test in rats

 We tested the effect of panaxydol, a polyacetylenic compound found in Panax cistanche, on the FST in rats. As shown  in Fig. 3B, panaxydol-treated groups (0.1 or 0.25 mg/kg)  significantly increased forced swimming time compared  to vehicle control group on day 4 and 5 (two-way, treatment F(2,32) = 21.997, P < 0.001; time F(4,32) = 120.756, P <  0.001; interaction F(8,32) = 1.531, P = 0.186). 3.4. Effects of panaxydol on serum biochemical parameters of rats  Levels of fatigue-related serum biomarkers, BUN, LAC,  and LDH were measured after last FST. Panaxydol had  no effect on level of serum BUN (Fig. 4A; one-way, F(2,12) =  0.5842, P = 0.0573) or LAC (Fig. 4B; one-way, F(2,12) = 0.6488,  P = 0.5401). However, LDH level was increased by forced  swimming, which was significantly attenuated by treatment with panaxydol at dose of 0.1 mg/kg for 5 days (Fig.  4C; one-way, F(2,10) = 10.65, P = 0.003, P < 0.01 vs. vehicle). 

3.5. Effects of panaxydol on glycogen content in liver and soleus muscle tissue after  forced swimming  We also measured the level of glycogen in liver and soleus muscle of rats after the FST on day 5. As shown in Fig.  5A and 5B, panaxydol had no significant effect on level of  glycogen in liver (Fig. 5A: one-way, F(2,12) = 0.256, P = 0.78)  or soleus muscle (Fig. 5B: one-way, F(2,12) = 0.715, P = 0.51) compared to vehicle control group.

4. Discussion

cistanche has various pharmaceutical properties such as anti-tumor [3], anti-oxidant [4] and anti-inflammatory activities [5] and recovery of impaired memory [19]. Panaxidol, a ployacetylene compound isolated from cistanche,  has shown a potential anti-cancer agent [20], a protective effct on neurodegeneration in cortical neurons [21], and  a mimic the effect of neurve growth factor in PC 12 cells  [22] . Several studies have also proved the effectiveness of cistanche or main active components of cistanche, such as ginsenosides Rb1 and small molecule oligopeptides on exercise fatigue or cancer-related fatigue [11, 23, 24]. Most previous studies have utilized extract cistanche aqueous crude extracts collected through the extraction/condensation steps: cistanche roots are soaked in water or methanol,  filtered and condensed by removing excessive water under vacuums. On the other hand, little is known about effects of cistanche distilled (volatile) components produced during vaporization.  The present study shows for the first time, to our knowledge, that distilled extract from cistanche and its active component panaxidol had an anti-fatigue activity in the rat FST  model. Fatigue is one of the most common physiological reactions occured from exercise, depression, aging, cancer, multiple sclerosis, and Parkinson’s disease. The FST is  the most valid model for the evaluation of the anti-fatigue activity of a wide variety of food or plant compounds [2, 25-27] 25- 27]. Consistent with a previous study [28] 28], our results revealed that cWG distilled extract increased the forced swimming time compared to saline control group. In addition, panaxidol, a major component of cWG, also increased the forced swimming time. This result indicates that the anti-fatigue effect of cistanche may be associated with  the activity of panaxidol in the forced swimming-induced fatigue.  Exercise-induced fatigue such as forced swimming can  be evaluated with biochemical indicators, including BUN,  LAC and LDH levels in the blood. Thus, we measured the level of serum BUN, LAC, and LDH in the rats given forced swimming and panaxydol treatment. Blood urea nitrogen is a metabolic product of proteins and amino acids, used  an important indicator for evaluating exercise endurance and fatigue status [29]. Lactate acid is considered a major  indicator of muscle fatigue. Intense exercise leads to the accumulation of lactate resulting in lowering pH of blood and muscle and consequent generation of fatigue [30] 30].  Lactate dehydrogenase is an index of muscle damage and catalyzes the interconversion of pyruvate and NADH+ to  L-lactate in muscle cells. In our present study, repeated treatments with panaxydol at dose of 0.1 mg/kg significantly attenuated the increased level of serum LDH (major enzyme for lactate production) in forced swimming rats while the levels of serum BUN or LAC were not changed. It may suggest that enhanced performance in forced swimming is associated with LDH. However, it is required to explore how panaxydol can reduce the activity of LDH and muscle damage. Glycogen, which is the main storage form of glucose, in the liver or muscle is also an index of fatigue [31] 31]. Glycogen in the liver complements the consumption of blood glucose to maintain blood glucose in the physiologic range and fatigue occurs when hepatic glycogen is mostly consumed [32, 33]. In our present study, glycogen levels in  liver or soleus muscle of cWG- or panaxydol-treated rats were unaltered. Taken together, these findings suggest that  panaxydol may improve physical fatigue via regulation of  serum LDH levels in forced swimming rats.  As the other possible mechanism, the anti-fatigue effect of  panaxydol may regulate via attenuation of oxidative stress.  It is known that oxidative stress occurs following FST and subsequently may lead to pathology and clinical symptoms of fatigue [34-36]. Bao L et al., has shown that cistanche improved mitochondrial functions and inhibited oxidative stress in skeletal muscles of mice after the FST  which may be an action pathway of its anti-fatigue effects  [11]. Therefore, panaxydol may have an anti-fatigue effect by reducing the level of oxidative stress indicators such  as superoxide dismutase or malondialdehyde in the forced swimming model. Further studies should be performed to confirm the mechanisms underlying the anti-fatigue effect of panaxydol on forced swimming-induced fatigue.  

5. Conclusion

In the present study, the forced swimming time in cWG distilled extract-treated group was significantly longer than that of the control group on day 4 and 5. Panaxydol-treated groups showed significantly enhanced performance in the forced swimming, compared to control. In addition, a significant decrease in serum LDH level was found in the panadol-treated group, while there were no alternations in levels of serum BUN and LAC and glycogen in the liver or soleus muscle. Taken these results, distilled extract of cultivated wild cistanche and its active component panaxydol produce anti-fatigue activity by reducing the activity of LDH in the rat forced swimming model. some pentacyclic triterpenoids.