Immunomodulating and antioxidant effects of polysaccharide conjugates from the fruits of Ziziphus Jujube on Chronic Fatigue Syndrome rats

Carbohydrate Polymers 122 (2015) 189–196

Contents lists available at ScienceDirect

Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Immunomodulating and antioxidant effects of polysaccharide conjugates from the fruits of Ziziphus Jujube on Chronic Fatigue Syndrome rats Aiping Chi a,∗ , Chenzhe Kang a , Yan Zhang b , Liang Tang a , Huanhuan Guo a , Hong Li a , Kunru Zhang a a b

Laboratory of Nutrition and Hygiene, Shaanxi Normal University, Xi’an 710062, China Department of Pharmacology, University of California, Irvine, CA 92697, USA

a r t i c l e

i n f o

Article history: Received 4 October 2014 Received in revised form 15 December 2014 Accepted 30 December 2014 Available online 14 January 2015 Keywords: Ziziphus Jujube Polysaccharide conjugates Immune Antioxidant

a b s t r a c t To detect the treatment effect of the fruits of Ziziphus Jujube in Chronic Fatigue Syndrome (CFS). Jujube polysaccharide conjugates (JPC) were isolated from the fruits of Z. Jujube. General physicochemical properties of JPC were analyzed. A four-week rats CFS model was established and JPC were orally administrated, the behavior experiments were conducted after CFS. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and the levels of malondialdehyde (MDA) in serum were elevated and T lymphocyte proliferation, CD4+ /CD8+ ratio and natural killer (NK) cells activity were analyzed. JPC markedly improved behaviors of CFS rats, also decreased MDA levels in serum, and elevated T lymphocyte proliferation, CD4+ /CD8+ ratio and natural killer (NK) cells activities. This suggests that JPC can improve the immune system and antioxidant activity of CFS rats and might be regarded as a biological response modifier. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Chronic fatigue syndrome (CFS) is a fatigue syndrome characterized by numerous symptoms appearing in various body systems (Meeus et al., 2011). CFS was closely related with immunosuppression resulting from physical and psychological fatigues. Many studies have demonstrated the potential involvements of the central and autonomic nervous systems, as well as a state of generalized immune activation and selective immune dysfunction in patients with CFS (Pariante, 2009; Prinsen et al., 2012; Klimas & Koneru, 2007; White, Light, Hughen, Vanhaitsma, & Light, 2012). Meanwhile, oxidative stress is associated with symptom expression in patients with CFS (Maes, Kubera, Uytterhoeven, Vrydags, & Bosmans, 2011). Free radicals may cause lipid peroxidation of cell membranes and thus alter the interactions between lipids and proteins, which deactivates enzyme activities. Increased oxidative stress and decreased antioxidant defenses are positively related to extent of syndrome in CFS (Gupta, Vij, & Chopra, 2010).

In recent years, pre-clinical studies and clinical trials on CFS have been conducted in China with a special focus on treating CFS using the traditional Chinese medicines (Adams, Wu, Yang, Tai, & Vohra, 2009; Liu, Zhang, & Li, 2011). Ziziphus Jujube mainly grows in the Yellow River basin in China, its fruits are not only a kind of popular food but also one of the typical Chinese medicines for treating CFS (Chen, Moriya, Yamakawa, Takahashi, & Kanda, 2010; Vahedi, Fathi Najafi, & Bozari, 2008; Wang, Liu, Zheng, Fan, & Cao, 2011). Recent studies have shown that polysaccharides of Jujube were able to stimulate immune function and improve antioxidant activities (Zhang et al., 2013; Bing, 2011). However, it’s not clear on the relationship between Jujube polysaccharide conjugates (JPC) and CFS, and whether it’s main active component to cure CFS. In the present study, JPC were isolated and orally administrated to CFS rats in order to reveal the functional mechanism. The results showed that JPC can ameliorate the immune function and alleviate the lipid peroxidation damage in CFS rats. 2. Materials and methods 2.1. Materials and reagents

∗ Corresponding author. Tel.: +86 29 13008408400; fax: +86 02985310156. E-mail address: [email protected] (A. Chi). http://dx.doi.org/10.1016/j.carbpol.2014.12.082 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

Jujube was obtained from the Xin-yuan Jujube Company (Shanxi province, China). Ginsenoside tablets (Production number:

190

A. Chi et al. / Carbohydrate Polymers 122 (2015) 189–196

Z20026643; Purity: 25%) were purchased from a local medicine store. DEAE-52 cellulose and Sephadex G-150 were purchased from Beijing Dingguo Reagent Co., China. Mannose (Man), ribose (Rib), rhamnose (Rha), d-glucuronic acid (GlucA) and d-galacturonic acid (GalcA), glucose (Glu), xylose (Xyl), galactose (Gal), arabinose (Ara), and Fucose (FUC) were obtained from Sigma (St. Louis, USA). Trifluoroacetic acid (TFA) and 1-Phenyl-3-methyl-5pyrazolone (PMP) were purchased from the Shanghai Ziyi Reagent Co., China. 3-[4,5-dimethyl-2-thiazole]-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), RPMI1640, phosphatebuffered saline (PBS), and fetal bovine serum (FBS) were from Gibco BRL (Gaithersburg, USA). Ficoll-Plaque Plus solution was purchased from Pharmacia Co., USA.

The HPLC conditions were as following: the column temperature was 25 ◦ C, the UV absorbance was measured at 250 nm, and the flow phase was 0.02 M sodium acetate, the flow rate was 1 mL/min. 2.5. Amino acid analysis The sample (20 mg) was hydrolyzed under vacuum at 110 ◦ C in 6 M hydrochloric acid for 24 h. The hydrolysis products were detected with an amino acid analysis analyzer (Hitachi L-8900, Japan) with a group of standard amino acids as the markers. The content of each kind of amino acids was calculated using the standard curves. 2.6. Experimental animals and establishment of CFS model

2.2. Isolation and purification of polysaccharide Jujube fruits were dried and mashed into powder, and soaked in 95% alcohol (1: 5, w/v) at room temperature for 2 h. After the mixture was filtered, the residues were dried by airing and then extracted in hot water (1:10, w/v) at 80 ◦ C three times, 1 h each time. The extracted solution was concentrated to 40% of the original volume in a rotary evaporator under reduced pressure, and the free proteins were deproteinized seven times using the Savage method (Staub, 1965), and then polysaccharides were precipitated with four-fold volume of ethanol at 4 ◦ C for 24 h. Subsequent dialysis against distilled water for 24 h gave rise to the retentate by the membranes with molecular weight cut-off of 10 kDa. The retentate was collected and lyophilized to afford crude polysaccharide conjugates. Crude polysaccharide conjugates were dissolved in distilled water (1:10, w/v), and then injected into a column (2.0 × 80 cm) of DEAE-52 cellulose and eluted with distilled water at rate of 0.8 mL/min and eluate was collected with automatic fraction collector (5.0 mL per tube), and polysaccharides were detected by the phenol-sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), and protein eluted was determined automatically by monitoring absorbance at 280 nm (Chen, Zhang, & Xie, 2005). The main elution peaks were collected and precipitated with ethanol and dried. Then the fractions were dissolved in the lowest possible volume of 0.1 mol/L NaCl solution, added into a column (2.0 cm × 40 cm) of Sephadex G-150 and eluted with 0.1 mol/L NaCl solution. The purified fractions were collected as described above. 2.3. General analysis Total carbohydrate content was determined according to the phenol-sulfuric acid method using glucose as a standard (Dubois et al., 1956). Moisture was determined by drying sample at 110 ◦ C for 2 h and calculated the amount of evaporated water, and the ash content was measured by incinerating JPC overnight in a muffle furnace at 550 ◦ C and weighing the residue (Fernández-Torres et al., 2005). Infrared (IR) spectroscopy was analyzed using a Tensor 27 Bruker instrument. JPC was ground with KBr powder and then pressed into pellets for IR measurement in the frequency range of 500–4000 cm−1 (Jing et al., 2014). 2.4. Monosaccharide compositions analysis The monosaccharide compositions were analyzed according to the previous method (Yang, Lv, Tian, & Zhao, 2010). Briefly, JPC was hydrolyzed into 2 mL of 3 M TFA at 100 ◦ C for 6 h to release monosaccharides, which were derivatized with 40 ␮L of 0.5 M PMP and 40 ␮L of 0.3 M NaOH at 70 ◦ C for 2 h. The analysis of the PMP derivatives was performed by an HPLC (LC-2010A, Shimadzu, Japan) system and a RP-C18 column (4.6 mm × 250 mm, 5 ␮m, Venusil, USA) was used for the separation of monosaccharide.

Total sixty Sprague-Dawley rats (180–220 g), 6–8 week old, were purchased from Experimental Animal Centre of Chinese Traditional Medicine Institute (Xi’an, China). All animals were acclimatized for at least one week prior to use and maintained in a temperature-controlled environment (23 ± 2 ◦ C) with humidity 55% ± 15% and 12 h light-dark cycle with free access to water and standard rodent chow. All animals were treated in accordance with the Guidelines of the Principle of Laboratory Animal Care (NIH Publication, revised 1985). Animals were randomly divided into six groups with ten rats each, i.e., normal control group, CFS control group, positive group (400 mg/kg BW ginsenoside) and JPC treatment groups (100, 200 and 400 mg/kg BW JPC, named I, II and III, respectively). Ginsenoside and JPC were dissolved in water and given to rats in the respective groups by oral gavage for 30 days, and those in normal control and CFS control groups were given the same volume of vehicle (normal saline) alone. Ginsenoside tablets were chosen as positive control medicine in present study because it was a kind of recognized medicine to cure CFS patients in Chinese medicine market and known for neuroprotection (Cao et al., 2012). Based on some previous reports (Lalremruta, & Prasanna, 2012; Sachdeva, Kuhad, & Chopra, 2011; Takashi et al., 2006), electricshock, restraint-stress and cold-water-swim were used to mimic the multiple-factor nosogenesis of CFS. Electric-shock: The circuit board (20 × 30 cm2 ) was made with the corrosion-resistant copper material, and loaded on the bottoms of the cages of CFS control group, positive control group, and JPC treatment groups (I, II, III). Connected to the circuit board and transformer to adjust voltage meters, these rats were fed two times every day and circuit boards were energized four times (3 min each time) at indefinite time during rat’s diet. Restraint-stress: The rats in CFS control group, positive control group and JPC treatment groups (I, II, III) were placed individually in PVC tubes (20.0 cm in length, 5.0 cm in diameter) for 2 h every other day. The front wall of each tube was perforated, so that rat could breathe. The rat’s tail extended through the rear door of tube and was fixed to the tube by adhesive tape. Cold-WaterSwim: After the restraint-stress, rats were placed in 21 ◦ C water for 30 min every day. 2.7. Assays of rats behaviors Morris-water-maze test, Open-field test and Tail-suspension test were carried out to evaluate the behaviors of rats. Those tests were widely used to screen anti-fatigue and anti-depressant medicines and have a high reliability. Morris-water-maze was performed in a cylindrical pool (15 cm in diameter, 60 cm in height) filled with 24 ◦ C water as Morris’s report (1984). The pool was divided into four quadrants and a circular escape platform (12 cm in diameter) submerged 2 cm underneath the surface of water in 2nd quadrant. From the 24th day, all rats were trained for four consecutive days and were randomly placed into one of other three quadrants for each training trial in order to learn to find the escape

A. Chi et al. / Carbohydrate Polymers 122 (2015) 189–196

191

platform in the 2nd quadrant, and animals were allowed a maximum of 60 s to locate and mount the platform. On the 28th day, time of finding platform of each rat was calculated from the starting at three different quadrants. Number of crossing over the area where platform was previously hidden was recorded within 1 min. Openfield test was carried out on the 29th day as previous report (Zou, Yuan, Lv, & Tu, 2010). Briefly, the field in test box (length 60 cm, width 60 cm, depth 65 cm) was divided into nine grids, and the locomotion of rats was recorded with an animal Behavior Videotracking System. Number of crossing through the adjacent grids and number of standing with hind legs were recorded within 3 min. The crossing criteria were that more than three claws step into next grid. Tail-suspension test was carried out on the 30th days. The tails of rats were fixed on a horizontal board (1 m in height from ground) and the motionless time of rats was recorded within 6 min.

culture for 8 min and 30 ␮L of citric acid (1 mol/L) was added to stop the reaction. The absorbance values (A) at 570 nm were measured using the above-mentioned microplate reader. NK cell activities were determined by the following equation: NK cell activities (%) = (Atest − Ablank control )/(Amaximum control − Ablank control )×100%.

2.8. Serum analysis and preparations of peripheral blood mononuclear cells (PBMC)

Crude polysaccharides were extracted from dried Jujube fruits (1000 g) and fractionated on DEAE-52 cellulose column. One main eluted peak was obtained (Fig. 1A). It was collected and further purified using Sephadex G-150 column. One main fraction was collected and named JPC with a yield of 3.16% (w/w). As shown in Fig. 1B, the polysaccharide detected curve of JPC at 490 nm by the phenol-sulfuric acid method was symmetrical with the curve at 280 nm. The result showed that JPC are probably the protein-bound polysaccharide. The present results suggested that contents of total carbohydrate, ash and moisture in JPC were 63.29%, 4.68% and 9.75%, respectively. JPC was not soluble in organic solvents such as ethanol, ether, acetone, and chloroform but easily soluble in water. Monosaccharide composition of JPC was determined by HPLC. The result from Fig. 2B suggests that JPC was composed of Man, Rib, GlucA, GalcA, Glu, Xyl, Gal and Ara in the molar percentages of 5.3%, 3.1%, 3.6%, 11.4%, 13.4%, 14.5%, 23.4%, and 25.1% (mol %), respectively. Total ratio of uronic acid was 15% from the result of monosaccharide composition of JPC. As shown in Fig. 3, amino acid analysis indicated that the protein of JPC consisted of eight amino acids (␮g/mg): Glu 4.64, Cys 16.52, Ile 15.28, Leu 14.37, Tyr 18.26, Phe 16.64, Lys 17.19, His14.03, respectively. Total protein content of JPC was determined as 11.69% by above analysis of the amino acids. IR spectrum of JPC exhibited some different absorption peaks (Fig. 4). The strong band at 3419.81 cm−1 region was attributed to the stretching vibration of O H and N H of JPC, and that at 2917.35 cm−1 was due to the C H stretching vibration absorption. The absorption bands at 1703.16 cm−1 were attributed to the stretching vibration of uronic acids, which were in line with the result of monosaccharide composition assay. The band at 1645.92 cm−1 would be due to the stretching vibration of C O and –CHO, and the absorptions around 1386.24 cm−1 represented internal C H deformation. The absorption peak at 907.62 cm−1 was characteristic of ␤ anomeric configuration while the absorption at 1016.75 cm−1 was typical for the pyranose form (He et al., 2013; Wang et al., 2013).

Rats were anesthetized with 25% urethane (i.p. 0.5 mL/100 g) and blood was drawn to prepare serum by centrifugation at 3000 rpm at 4 ◦ C for 10 min. Activities of SOD, GSH-Px and levels of MDA were determined using commercially available kits from the Nanjing Jiancheng Biological Company (Nanjing, China), and the levels of interleukin 2 (IL-2), interleukin 4 (IL-4) and interleukin 10 (IL-10) by enzyme-linked immunosorbent assay (ELISA) kit (Bio-tek ELX808, USA). Meanwhile 10 mL of the anticoagulated blood was diluted with PBS at a ratio of 1:1. The diluted sample (20 mL) was put in a 50 mL centrifuge tube together with an equal volume of Ficoll-Plaque Plus solution. The tube was then centrifuged at 2000 rpm for 15 min at 18 ◦ C. The layer of Mononuclear Cells was sucked with pipette and put into another centrifuge tube with five times volume of RPMI-1640 medium plus 10% FBS, then centrifuged at 1500 rpm for 10 min at 18 ◦ C. The supernatant was discarded, and PBMCs were resuspended in the same volume of RPMI-1640 medium plus FBS. The cell number was counted, and the viability of cells was checked by trypan blue exclusion assay. 2.9. Assay of T lymphocyte subgroup and proliferation As previously described (Diaz-Romero, Vogt, & Weckbecker, 2002), the isolated PBMCs were incubated for 30 min at 4 ◦ C with 10 ␮L of anti-CD3 and anti-CD4, or anti-CD3 and anti-CD8 antibodies (Caltag Co., USA). Then cells were washed twice with PBS and resuspended in 1% paraformaldehyde. CD4+ and CD8+ T cell counts were determined by flow cytometry (Millipore Corporation, USA). PBMCs were suspended at a final density of 2 × 106 cells/mL in RPMI-1640 medium supplemented with 10% FBS, and then were seeded into a 96-well plate (100 ␮L/well) in the presence of Con A (5 ␮g/mL) and cultured at 37 ◦ C in 5% CO2 incubator. After 72 h of incubation, 10 ␮L of MTT was added to each well and the plate was incubated for an additional 4 h, and then 100 ␮L of DMSO was added to each well. Finally, the absorbance values (A) at 570 nm were measured using a Zenyth 3100 microplate reader (Anthos, Austria). 2.10. Assay of NK cell activities Based on a previous report (Lin-Na, Zhi-Wei, Yan, Hua, & Yan, 2012), PBMCs (2 × 106 cells/mL) and K562 cells (2 × 105 cells/mL) were seeded into 96-well plates with a ratio of 50:1. In addition, K562 cells were added with 100 ␮L of RPMI-1640 medium as blank control and 100 ␮L of 1% NP-40 as maximum control, respectively. After that, the plates were incubated at 37 ◦ C in 5% CO2 incubator for 4 h. Next, 100 ␮L of lactate dehydrogenase (LDH) was added to

2.11. Statistical analysis Data are presented as the mean ± standard deviation (SD). Statistical differences were evaluated by one-way ANOVA using a t-test (between groups). P values <0.05 was considered significant. 3. Results 3.1. Components of JPC

3.2. Effects of JPC on behaviors of CFS rats As shown in Table 1, Platform-time and Motionless-time were significantly higher (p < 0.05 or p < 0.01) but Platform-Times, Crossing-times and Stand-times were markedly lower (p < 0.05) in CFS control group than those in normal group. According to Table 1, Platform-time and Motionless-time in JPC treatment groups were found to be significantly reduced in a dose-dependent manner compared with those in CFS control group. However, Platform-times, Crossing-times and Stand-times in JPC treatment groups were significantly increased in a dose-dependent manner as compared with that. In addition, it was found that ginsenoside treatment also exhibited effects on all of the compared items. The results showed

192

A. Chi et al. / Carbohydrate Polymers 122 (2015) 189–196

Fig. 1. Profile for fractionation and purification of JPC on DEAE-52 cellulose column (A) and Sephadex G-150 column (B).

Fig. 2. The HPLC chromatograms of PMP-labeled standard monosaccharides (A) and component monosaccharides released from JPC (B). JPC was hydrolyzed into component monosaccharides with TFA at 100 ◦ C for 6 h and then labeled with PMP. The PMP-labeled monosaccharides were separated and identified by HPLC-UV at 250 nm. 1 Man; 2 Rib; 3 Rha; 4 GlucA; 5 GalcA; 6 Glu; 7 Xyl; 8 Gal; 9 Ara; 10 FUC (internal standard).

Fig. 3. Amino acid composition of JPC. JPC was hydrolyzed in hydrochloric acid and then detected with an amino acid analysis analyzer as described in the Materials and methods. The content of each kind of amino acids was calculated using the standard curves.

A. Chi et al. / Carbohydrate Polymers 122 (2015) 189–196

193

Fig. 4. IR spectrum of JPC. IR spectroscopy (KBr pellets) was analyzed using a Tensor 27 Bruker instrument in a range of 500–4000 cm−1 .

that no significant difference between JPC treatment groups and positive control group. 3.3. Effects of JPC on serum antioxidant status in CFS rats As shown in Table 2, the SOD and GSH-Px activities were markedly decreased (p < 0.05) while MDA level was significantly increased (p < 0.01) in CFS control group rats when compared with those in normal control group. However, SOD activity level of rats in group II and group III and GSH-Px activities in all of JPC treatment groups were markedly increased (p < 0.05) in comparison with those of CFS control group. In contrast, MDA levels of rats in all of JPC treatment groups only in group III was significantly decreased (p < 0.05). In addition, SOD and GSH-Px activities of rats in positive control group were significantly increased (p < 0.05) in comparison with those in CFS control group. Moreover, the results showed that both JPC and Ginsenoside had obvious antioxidant activities.

levels of group III rats were significantly decreased. The counts of CD4+ T cells and the ratio of CD4+ /CD8+ in CFS control group were significantly lower than those in normal control group as well as those of group II and group III. In addition, no significant difference was found regarding counts of CD4+ T cells and the ratio of CD4+ /CD8+ between positive control group and CFS control group. Moreover, those results showed that JPC treatment had better effect on increasing the counts of CD4+ T cell and the ratio of CD4+ /CD8+ than Ginsenoside treatment. T cell proliferation and NK cell activity were significantly decreased in CFS control group when compared with those in normal control group. The results also showed that T cells proliferation was significantly increased in group III rats as well as NK cell activity was markedly elevated in group II and group III. In addition, NK cells activity was significantly increased in positive control group rats as compared with those in CFS control group, no significant difference regarding T cell proliferation between both groups.

3.4. Effects of JPC treatment on immune function in CFS rats 4. Discussion As shown in Table 3, IL-2 level was markedly decreased while IL-10 levels were significantly increased in CFS control group rats when compared with those in normal control group. When compared with CFS control group rats, no significant difference were found abut IL-4 levels of rats in JPC treatment groups, but IL-2 level of rats in groups II and III were markedly increased whereas IL-10

CFS is a fatigue-related disorder with unknown etiology and uncertain therapeutic options. Western medicine believes that stress is a major factor that contributes to chronic fatigue, as long-term chronic stress is commonly seen in CFS patients. Basic and clinical studies have shown that chronic stress may lead to

Table 1 Results of behavior test on rats CFS model. Groups (n = 10)

Normal control CFS control JPC(100 mg/kg) JPC(200 mg/kg) JPC(400 mg/kg) Ginsenoside control * ** # ## 

Morris-water-maze test

Open-field test

Time of finding platform (s)

Number of crossing

Number of crossing

Number of standing

53.48 ± 9.49 64.62 ± 11.13* 58.81 ± 10.38 56.64 ± 13.72# 55.77 ± 12.10# 54.35 ± 11.84#

4.8 ± 1.9 3.2 ± 1.6* 3.7 ± 1.1 4.6 ± 1.2 5.1 ± 1.6# 4.9 ± 1.7#

41.4 ± 7.8 33.6 ± 6.6* 36.6 ± 10.5 38.0 ± 9.2# 38.3 ± 10.3# 40.2 ± 8.8##

13.3 ± 5.4 8.7 ± 3.9* 9.5 ± 2.6  10.1 ± 3.2 12.9 ± 5.8# 13.2 ± 4.8#

p < 0.05. p < 0.01, vs the normal control. p < 0.05. p < 0.01 vs the CFS control. p < 0.05 vs the positive control.

Tail-suspension testMotionless Time (s) 105.24 ± 16.99 137.91 ± 18.21** 103.51 ± 17.44# 98.73 ± 15.68## 97.53 ± 13.72## 105.21 ± 11.69#

194

A. Chi et al. / Carbohydrate Polymers 122 (2015) 189–196

Table 2 Effects of JPC on the antioxidant status of CFS rats. Groups (n = 10) Normal control CFS control JPC JPC JPC Ginsenoside control * ** # ##

Dose (mg/kg)

SOD(U/mL)

GSH-Px(U/mL)

MDA(mmol/mL)

100 200 400 400

69.53 ± 8.36 62.72 ± 5.87* 64.48 ± 9.19 72.15 ± 6.54# 68.62 ± 7.42# 71.59 ± 5.91#

35.87 ± 4.35 28.29 ± 3.81* 34.71 ± 4.56# 35.43 ± 3.64# 36.47 ± 4.86# 38.67 ± 4.98#

5.17 ± 1.62 8.52 ± 1.39** 6.94 ± 2.05# 6.67 ± 1.31# 6.04 ± 1.26# 5.87 ± 0.95##

p < 0.05 vs Normal control group. p < 0.01 vs Normal control group. p < 0.05 vs CFS control group. p < 0.01 vs CFS control group.

behavioral changes, a variety of functional disorders, and even structural changes in tissues (Aragona, & Aragona, 1994). Many CFS animal models were used to study the mechanism of CFS, such as mental stimulation, forced exercise, herbal drugs and restraint stress protocol (Lyle et al., 2009; Shevchuk, 2007; Zou et al., 2010). The required time for CFS model construction is always more than two weeks (Ottenweller et al., 1998; Singh, Naidu, Gupta, & Kulkarni, 2002). In this study, the time of CFS model construction was more than 4 weeks and three types of stimulations (Electric-shock, Restraint-stress and Cold-water-swim) were adopted for emotional stimulation and energy consumption. CFS patients always present behavioral symptoms, such as memory deficit, inattention and disinterest in new things, disappointment or depression in depressive environment (Cook, Lange, & DeLuca, 2001; Heins, Knoop, & Bleijenberg, 2013; Heins, Knoop, Lobbestael, & Bleijenberg, 2011). Morris water maze test is often used to test the learning and memory capability of rats (Gandhi, Kelly, Wiley, & Walsh, 2000). Rodents are irrigative of water environment, and will strive to escape from water, and make the best use of any spatial memory in order to leave the water as quickly as possible. The preservation of this memory involves such brain areas as hippocampus and cerebral cortex (Fischer, Bjorklund, & Chen, 1991). Open field test reflects rat’s exploration activities and emotion in a new environment, and can be used to examine the status of animal’s anxiety and excitability. Tail suspension test reflects the animal’s emotion of disappointment and depression, and can be used to observe the physical force of rats in an uncommon posture and their psychological depression as well. As shown in Table 1, the results of behavioral measures were significant different between CFS control group and normal group, indicating that chronic stress caused physical and mental fatigue of rats in CFS group. In order to evaluate the efficacy of JPC in the present study, we chose Ginsenoside tablets as positive control which is a recognized quite expensive mono-medicine to cure CFS patients in Chinese medicine market. The results of behavioral measures revealed that JPC is comparable to Ginsenoside.

Some researcher reported that NK cell activities and CD4+ /CD8+ ratio of CFS patients were decreased (Bansal, Bradley, Bishop, KianiAlikhan, & Ford, 2012;Chen et al., 2009; Robertson et al., 2005). In the present study, the results showed that JPC treatment at the medium and high doses dramatically improved NK cell activities, T cell proliferation, CD4+ /CD8+ ratio and CD4+ counts in CFS rats. In addition, we found JPC treatment dose-dependently enhanced IL-2 levels and reduced IL-10 levels as compared with those in CFS control group, though the changes were only significant at the medium or high dose levels. CD4+ T cells contain T helper 1 (Th1) and T helper 2 (Th2) cells and Th1/Th2 balance is involved in immunoregulation (Nijs, & Frémont, 2008). Th1 cells secrete IL-2 which activates T cell proliferation and NK cell activities, and IL-4 and IL-10 are from Th2 cells and activate B cells which support humoral immune response. Our results showed that JPC administration may enhance cellular immune function of CFS rats, but not humoral immune response. SOD and GSH-Px are two major components of antioxidative system in organisms and function to detoxify O2 − or H2 O2 . Increased oxidative stress and decreased antioxidant defenses are related to the extent of symptomatology in CFS patients (Maes et al., 2011; Gupta et al., 2010; Kennedy et al., 2005). Maes et al. (2011) found that H2 O2 and MDA levels in plasma were significantly higher in patients with CFS than in normal controls. Similarly, in the current study, SOD and GSH-Px activities in the CFS control group were markedly decreased and MDA levels were significantly increased in comparison with rats in normal control group. The results also showed that both JPC and Ginsenoside administrations significantly increased SOD and GSH-Px activities and decreased MDA levels. Many studies have reported that plant polysaccharides could improve reproduction of beneficial microorganisms in animal intestines, which could improve immune function by synthesizing vitamins and stimulating immunoglobulin activities (Chengfu et al., 2010; Chi, Chen, Wang, Xiong, & Li, 2008; Han et al., 2012). Other studies suggested that polysaccharides, which have complex molecular structures, could activate immune cells through some

Table 3 Effects of JPC treatment on immune function in CFS rats. Groups (n = 10) Normal Control CFS Control JPC JPC JPC Ginsenoside control * ** # ##

Dose (mg/kg)

100 200 400 400

p < 0.05 vs Normal control group. p < 0.01 vs Normal control group. p < 0.05 vs CFS control group. p < 0.01 vs CFS control group.

IL-2(pg/mL)

IL-4(pg/mL)

IL-10(pg/mL)

CD4+ (109 /L)

CD8+ (109 /L)

CD4+ /CD8+

T cells proliferation(A570 )

NK cells activity(%)

3.53 ± 1.01 2.98 ± 0.56* 3.21 ± 0.79 3.95 ± 0.86# 4.51 ± 1.03# 3.07 ± 1.01

6.87 ± 2.45 7.29 ± 1.92 6.57 ± 2.11 7.21 ± 3.02 6.95 ± 1.59 8.03 ± 1.76

35.17 ± 6.41 38.52 ± 8.26* 36.72 ± 7.72 36.45 ± 8.18 35.81 ± 5.04# 37.65 ± 6.73

4.05 ± 0.77 2.84 ± 0.82** 3.02 ± 0.94 3.65 ± 1.04# 3.82 ± 0.93# 2.96 ± 0.81

2.39 ± 0.68 2.76 ± 1.03 2.42 ± 0.84 2.15 ± 0.73 1.97 ± 0.85 2.56 ± 0.79

1.59 ± 0.82 1.13 ± 0.65* 1.29 ± 0.74 1.64 ± 0.92# 1.82 ± 0.98## 1.26 ± 0.85

0.213 ± 0.071 0.148 ± 0.053* 0.162 ± 0.037 0.169 ± 0.086 0.185 ± 0.049# 0.157 ± 0.046

34.82 ± 3.77 26.96 ± 4.71* 28.27 ± 3.88 31.15 ± 5.21# 32.46 ± 6.07# 31.95 ± 6.73#

A. Chi et al. / Carbohydrate Polymers 122 (2015) 189–196

signaling pathways (Leung, Liu, Koon, & Fung, 2006; Peng, Zhang, Zeng, & Kennedy, 2005; Roy et al., 2009). In summary, JPC treatments showed similar efficacy to ginsenoside administration in treating CFS rats. However, the mechanism of them was not same pathway: JPC might perfectly improve the immune function and antioxidant activities whereas ginsenoside likely enhance antioxidant activities and NK cell activity in CFS patients. Moreover, JPC has better advantage to develop a medicine of treating CFS because jujube fruits have very lower price and much more source than ginseng in China. Further studies of JPC are under investigation. Acknowledgments We are grateful to Dr. Xing-bin Yang (College of Food Engineering and Nutritional Science, Shaanxi Normal University) for his skillful technical assistance in conducting the Monosaccharide compositions analysis. This work was supported by the Fundamental Research Funds for the Central Universities (GK201402046) and the National Science Foundation of China (no. 30900710 and no. 31071035). References Adams, D., Wu, T., Yang, X., Tai, S., & Vohra, S. (2009). Traditional Chinese medicinal herbs for the treatment of idiopathic chronic fatigue and chronic fatigue syndrome. Cochrane Database of Systematic Reviews, 4, CD006348. http://dx.doi.org/ 10.1002/14651858.CD006348. pub2 Aragona, M., & Aragona, F. (1994). Chronic stress and histological effects on the human central nervous system and other organs. Functional Neurology, 9, 121–131. Bansal, A. S., Bradley, A. S., Bishop, K. N., Kiani-Alikhan, S., & Ford, B. (2012). Chronic fatigue syndrome, the immune system and viral infection. Brain, Behavior, and Immunity, 26, 24–31. Bing, W. (2011). Chemical characterization and Ameliorating effect of polysaccharide from Chinese jujube on intestine oxidative injury by ischemia and reperfusion. International Journal of Biological Macromolecules, 48, 386–391. Cao, Y., Hu, Y., Liu, P., Zhao, H. X., Zhao, X. J., & Wei, Y. M. (2012). Effects of a Chinese traditional formula Kai Xin San (KXS) on chronic fatigue syndrome mice induced by forced wheel running. Journal of Ethnopharmacology, 139, 19–25. Diaz-Romero, J., Vogt, G., & Weckbecker, G. (2002). A small-volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four-color flow cytometry. Cytometry, 47, 265–275. Chen, H., Zhang, M., & Xie, B. (2005). Components and antioxidant activity of polysaccharide conjugate from green tea. Food Chemistry, 90, 17–21. Chen, R., Moriya, J., Luo, X., Yamakawa, J., Takahashi, T., Sasaki, K., et al. (2009). Hochu-ekki-to combined with interferon-gamma moderately enhances daily activity of chronic fatigue syndrome mice by increasing NK cell activity, but not neuroprotection. Immunopharmacology and Immunotoxicology, 31, 238–245. Chen, R., Moriya, J., Yamakawa, J., Takahashi, T., & Kanda, T. (2010). Traditional Chinese medicine for chronic fatigue syndrome. Evidence-based Complementary and Alternative Medicine, 7, 3–10. Chengfu, Y., Changdong, W., Youquan, B., Li, C., Xiuning, H., Zhiwei, W., et al. (2010). Antioxidative and immunoprotective effects of Pyracantha fortuneana (Maxim.) Li polysaccharides in mice. Immunology Letters, 133, 14–18. Chi, A. P., Chen, J. P., Wang, Z. Z., Xiong, Z. Y., & Li, Q. X. (2008). Morphological and structural characterization of a polysaccharide from Gynostemma pentaphyllum Makino and its anti-exercise fatigue activity. Carbohydrate Polymers, 74, 868–874. Cook, D. B., Lange, G., & DeLuca, J. (2001). Relationship of brain MRI abnormalities and physical functional status in chronic fatigue syndrome. International Journal of Neuroscience, 107, 1–6. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–356. Fernández-Torres, R., Pérez-Bernal, J. L., Bello-López, M. A., Callejón-Mochón, M., Jiménez-Sánchez, J. C., & Guiraúm-Pérez, A. (2005). Mineral content and botanical origin of Spanish honeys. Talanta, 65, 686–691. Fischer, W., Bjorklund, A., & Chen, K. (1991). NGF improves spatial memory in aged rodents as a function of age. Journal of Neuroscience, 11, 889–906. Gandhi, C. C., Kelly, R. M., Wiley, R. G., & Walsh, T. J. (2000). Impaired acquisition of a Morris water maze task following selective destruction of cerebellar purkinje cells with OX7-saporin. Behavioural Brain Research, 109, 37–47. Gupta, A., Vij, G., & Chopra, K. (2010). Possible role of oxidative stress and immunological activation in mouse model of chronical fatigue syndrome and its attenuation by olive extract. Journal of Neuroimmunology, 226, 1–7. Han, X. Q., Chen, B. C. L., Dong, C. X., Yang, Y. H., Ko, C. H., Yue, G. G. L., et al. (2012). Isolation, structure characterization, and immunomodulating activity of

195

a hyperbranched polysaccharide from the fruiting bodies of ganoderma sinense. Journal of Agricultural and Food Chemistry, 60, 4276–4281. Heins, M. J., Knoop, H., & Bleijenberg, G. (2013). The role of the therapeutic relationship in cognitive behaviour therapy for chronic fatigue syndrome. Behaviour Research and Therapy, 51, 368–376. Heins, M. J., Knoop, H., Lobbestael, J., & Bleijenberg, G. (2011). Cildhood maltreatment and the response to cognitive behavior therapy for chronic fatigue syndrome. Journal of Psychosomatic Research, 71, 404–410. He, L., Ji, P., Cheng, J., Wang, Y., Qian, H., Li, W., et al. (2013). Structural characterization and immunostimulatory activity of a novel protein-bound polysaccharide produced by Hirsutella sinensis. Food Chemistry, 141, 946–953. Jing, Y., Huang, L., Lv, W., Tong, H., Song, L., Hu, X., et al. (2014). Structural characterization of a novel polysaccharide from pulp tissues of Litchi chinensis and its immunomodulatory activity. Journal of Agricultural and Food Chemistry, 62, 902–911. Kennedy, G., Spence, V. A., McLaren, M., Hill, A., Underwood, C., & Belch, J. J. (2005). Oxidative stress levels are raised in chronic fatigue syndrome and are associated with clinical symptoms. Free Radical Biology and Medicine, 39, 584–589. Klimas, N. G., & Koneru, A. O. (2007). Chronic fatigue syndrome: inflammation, immune function, and neuroendocrine interactions. Current Rheumatology Reports, 9, 482–487. Lalremruta, V., & Prasanna, G. S. (2012). Evaluation of protective effect of Aegle marmelos Corr. in an animal model of chronic fatigue syndrome. Indian Journal of Physiology and Pharmacology, 44, 351–356. Leung, M. Y., Liu, C., Koon, J. C., & Fung, K. P. (2006). Polysaccharide biological response modifiers. Immunology Letters, 105, 101–114. Lin-Na, L., Zhi-Wei, G., Yan, Z., Hua, Q., & Yan, H. (2012). Polysaccharide extracted from Rheum tanguticum prevents irradiation-induced immune damage in mice. Asian Pacific Journal of Cancer Prevention, 13, 1401–1405. Liu, Y., Zhang, H. G., & Li, X. H. (2011). A Chinese herbal decoction, Danggui Buxue Tang, improves chronic fatigue syndrome induced by food restriction and forced swimming in rats. Phytotherapy Research, 25, 1825–1832. Lyle, N., Gomes, A., Sur, T., Munshi, S., Paul, S., Chatterjee, S., et al. (2009). The role of antioxidant properties of Nardostachys jatamansi in alleviation of the symptoms of the chronic fatigue syndrome. Behavioural Brain Research, 202, 285–290. Maes, M., Kubera, M., Uytterhoeven, M., Vrydags, N., & Bosmans, E. (2011). Increased plasma peroxides as a marker of oxidative stress in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Medical Science Monitor, 17, 11–15. Meeus, M., Eupen, I. V., Baarle, E. V., Boeck, V. D., Luyckx, A., Kos, D., et al. (2011). Symptom fluctuations and daily physical activity in patients with chronic fatigue syndrome: A case-control study. Archives of Physical Medicine and Rehabilitation, 92, 1820–1826. Morris, R. (1984). Developments of a water-maze procedure for studying spatial learning in the rat. Journal of Neuroscience Methods, 11, 47–60. Nijs, J., & Frémont, M. (2008). Intracellular immune dysfunction in myalgic encephalomyelitis/chronic fatigue syndrome: state of the art and therapeutic implications. Expert Opinion on Therapeutic Targets, 12, 281–289. Ottenweller, J. E., Natelson, B. H., Gause, W. C., Carroll, K. K., Beldowicz, D., Zhou, X. D., et al. (1998). Mouse running activity is lowered by Brucella abortus treatment: A potential model to study chronic fatigue. Physiology & Behavior, 63, 795–801. Pariante, C. M. (2009). Chronic fatigue syndrome and the immune system: Findings in search of meanings. Brain, Behavior and Immunity, 23, 325–326. Peng, Y. F., Zhang, L. N., Zeng, F. B., & Kennedy, J. F. (2005). Structure and antitumor activities of the water-soluble polysaccharides from Ganoderma tsugae mycelium. Carbohydrate Polymers, 59, 385–392. Prinsen, H., de Vries, I. J., Torensma, R., Pots, J. M., Mulder, S. F., van Herpen, C. M., et al. (2012). Humoral and cellular immune responses after influenza vaccination in patients with chronic fatigue syndrome. BMC Immunology, 13, 71. Robertson, M. J., Schacterle, R. S., Mackin, G. A., Wilson, S. N., Bloomingdale, K. L., Ritz, J., et al. (2005). Lymphocyte subset differences in patients with chronic fatigue syndrome, multiple sclerosis and major depression. Clinical and Experimental Immunology, 141, 326–332. Roy, S. K., Das, D., Mondal, S., Maiti, D., Bhunia, B., Maiti, T., et al. (2009). Structural studies of an immunoenhancing water-soluble glucan isolated from hot water extract of an edible mushroom, Pleurotus florida, cultivar Assam Florida. Carbohydrate Research, 18, 2596–2601. Sachdeva, A. K., Kuhad, A., & Chopra, K. (2011). Epigallocatechin gallate ameliorates behavioral and biochemical deficits in rat model of load-induced chronic fatigue syndrome. Brain Research Bulletin, 86, 165–172. Shevchuk, N. A. (2007). Possible use of repeated cold stress for reducing fatigue in chronic fatigue syndrome: A hypothesis. Behavioral and Brain Functions, 3, 55. Singh, A., Naidu, P. S., Gupta, S., & Kulkarni, S. K. (2002). Effect of natural and synthetic antioxidants in a mouse model of chronic fatigue syndrome. Journal of Medicinal Food, 5, 211–220. Staub, A. M. (1965). Removal of protein-Sevag method. Methods in Carbohydrate Chemistry, 5, 5–6. Takashi, T., Fei, Y., Shi-jie, Z., Junji, M., Hiroyuki, S., Shigeto, M., et al. (2006). Beneficial effect of brewers’ yeast extract on daily activity in a murine model of chronic fatigue syndrome. Evidence-based Complementary and Alternative Medicine, 3, 109–115. Vahedi, F., Fathi Najafi, M., & Bozari, K. (2008). Evaluation of inhibitory effect and apoptosis induction of Zyzyphus Jujube on tumor cell lines, an in vitro preliminary study. Cytotechnology, 56, 105–111. Wang, B. N., Liu, H. F., Zheng, J. B., Fan, M. T., & Cao, W. (2011). Distribution of phenolic acids in different tissues of jujube and their antioxidant activity. Journal of Agricultural and Food Chemistry, 59, 1288–1292.

196

A. Chi et al. / Carbohydrate Polymers 122 (2015) 189–196

Wang, Y., Fang, J., Ni, X., Li, J., Liu, Q., Dong, Q., et al. (2013). Inducement of cytokine release by GFPBW2, a novel polysaccharide from fruit bodies of Grifola frondosa, through dectin-1 in macrophages. Journal of Agricultural and Food Chemistry, 61, 11400–11409. White, A. T., Light, A. R., Hughen, R. W., Vanhaitsma, T. A., & Light, K. C. (2012). Differences in metabolite-detecting, adrenergic, and immune gene expression after moderate exercise in patients with chronic fatigue syndrome, patients with multiple sclerosis, and healthy controls. Psychosomatic Medicine, 74, 46–54.

Yang, X. B., Lv, Y., Tian, L. M., & Zhao, Y. (2010). Composition and systemic immune activity of the polysaccharides from an herbal tea (Lycopus Iucidus Turcz). Journal of Agricultural and Food Chemistry, 58, 6075–6080. Zhang, J., Chen, J., Wang, D., Hu, Y., Zhang, C., Qin, T., et al. (2013). Immune-enhancing activity comparison of sulfated ophiopogonpolysaccharide and sulfated jujube polysaccharide. International Journal of Biological Macromolecules, 52, 212–217. Zou, J., Yuan, J., Lv, S., & Tu, J. (2010). Effects of exercise on behavior and peripheral blood lymphocyte apoptosis in a rat model of chronic fatigue syndromn. Journal of Huazhong University of Science and Technology. Medical sciences, 30, 258–264.