Đề tài Khoa học cordyceps militaris polysaccharides can enhance the immunity and antioxidation activity in immunosuppressed mice

Carbohydrate Polymers 89 (2012) 461– 466 Contents lists available at SciVerse ScienceDirect Carbohydrate Polymers jo u rn al hom epa ge: www.elsev ier .com Cordyc ce t activity Mi Wang ang Ying Lib, a Institute of Tr tural U b Department o cience a r t i c l Article history: Received 18 Ja Received in re Accepted 8 Ma Available onlin Keywords: Cordyceps mili Cyclophospha immunosuppr Immunomodulation Anti-oxidation activity in vivo nd re 0 ma s we dy w ters w duced leen imm perox the malondialdehyde levels in vivo. © 2012 Elsevier Ltd. All rights reserved. 1. Introdu In rece polysacchar and used a 2008). Amo fungus belo used as a cr is known a pharmacolo medicine Co Cai, 2004). T functions a are most pr Various been report and antitum agent, and the major a rides from C antioxidant ulatory (Ch ∗ Correspon ∗∗ Correspon E-mail add 0144-8617/$ – doi:10.1016/j.ction nt years, many natural polysaccharides and ide-protein complexes were isolated from fungi s a source of therapeutic agents (Novak & Vetvicka, ng them, Cordyceps militaris, an entomopathogenic nging to the class Ascomycetes, has been extensively ude drug and a folk tonic food in East Asia. C. militaris s the Chinese rare caterpillar fungus and has similar gical activities to the well-known Chinese traditional rdyceps sinensis (Gai, Jin, Wang, Li, & Li, 2004; Zheng & he beneficial effects of Cordyceps on renal and hepatic nd immunomodulation-related antitumour activities omising and deserve further attention (Paterson, 2008). bioactive constituents from the Cordyceps species have ed, such as cordycepin, polysaccharides, antibacterial our adenosine derivatives, ophicordin, an antifungal L-tryptophan. Polysaccharides are considered one of ctive components of Cordyceps. Purified polysaccha- . militaris have numerous biological activities, such as (Li, Li, Dong, & Tsim, 2001; Li et al., 2003), immunomod- eung et al., 2009; Kim et al., 2008), antitumour (Park, ding author. Tel.: +86 25 84395203; fax: +86 25 84398669. ding author. Tel.: +86 21 34293460; fax: +86 21 34293396. resses: ylhu@njau.edu.cn (Y.l. Hu), feiqun@gmail.com (F.Q. Xue). Kim, Lee, Yoo, & Cho, 2009; Rao, Fang, Wu, & Tzeng, 2010), and anti-inflammatory (Rao et al., 2010). Previous studies on the immunomodulatory and antioxidant effects of C. militaris polysaccharides (CMPs) in in vitro systems have been conducted. CMPs can induce the functional activation of macrophages through the upregulation of cytokine expression and nitric oxide (NO) release (Lee et al., 2010), induce T-lymphocyte proliferation and secretion of interleukin (IL)-2, IL-6, and IL-8 (Chen, Zhang, Shen, & Wang, 2010), and stimulate the phagocytosis of macrophages in vitro. These results confirm the important role of CMPs in triggering immune responses. The CMPs fractions P70- 1 and CBP-1 were found to exhibit hydroxyl radical-scavenging activity in vitro (Yu et al., 2007, 2009). In the present study, the fruiting body of C. militaris came from Shanghai, which has been scarcely investigated. Successive tests were conducted to evaluate the immune activation and reac- tive oxygen species (ROS)-scavenging activity of CMP in vivo. The details are reported in the current study. 2. Materials and methods 2.1. Material Dry cultured C. militaris was obtained from Shanghai Dianzhi Bioengineering Corp. (Shanghai, China). The material (No. 06- 01-0727) was identified by Associate Researcher X.H. Gao of the Research Group of Dong Chong Xia Cao, Shanghai see front matter © 2012 Elsevier Ltd. All rights reserved. carbpol.2012.03.029eps militaris polysaccharides can enhan in immunosuppressed mice a,b, Xin Yu Mengb, Rui Le Yangb, Tao Qina, Xiao Y Yuan liang Hua,∗, Fei Qun Xueb,∗∗ aditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricul f Pharmacy, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural S e i n f o nuary 2012 vised form 28 February 2012 rch 2012 e 19 March 2012 taris polysaccharides mide-induced ession a b s t r a c t To evaluate the immune activation a taris polysaccharides (CMP) in vivo, 9 mice in the three experimental group injection and 17.5, 35, or 70 mg/kg bo cytic index, and biochemical parame CMP was able to overcome the CY-in thymus indices, and enhanced the sp improve the antioxidation activity in dismutase, catalase, and glutathione / locate /carbpol he immunity and antioxidation Wangb, Ke Yu Zhangb, Chen Zhong Feib, niversity, Nanjing 210095, PR China s, Shanghai 200241, PR China active oxygen species scavenging activity of Cordyceps mili- le BALB/c mice were randomly divided into six groups. The re given cyclophosphamide at 80 mg/kg/d via intraperitoneal eight CMP via gavage. The lymphocyte proliferation, phago- ere measured. The results show that the administration of immunosuppression, significantly increased the spleen and lymphocyte activity and macrophage function. CMP can also unosuppressed mice, significantly increase the superoxidase idase levels and the total antioxidant capacity, and decrease 462 M. Wang et al. / Carbohydrate Polymers 89 (2012) 461– 466 Academy of Agricultural Sciences, Shanghai, China. RPMI 1640 was purchased from Gibco. The T-cell mitogen concanavalin A (ConA) (DMSO) w Engineering diphenyltet Co. Assay ondialdehy (SOD), and the Nanjing Cyclophosp Medicine C albumin, Co purchased f membrane All chemica 2.2. Polysac Polysacc described ( der of cultu subsequent 2 h. The resu and filtered aqueous so reduced pr centrifugati then suspen (w/w) poly anthrone w 2.3. Animal Male BA from Shan Academy o vided with in a rodent acclimatiza were appro of Agricultu groups cons used for ph test, 3 were the other ex to their env mice were groups and 70 mg/kg bo to 3, the ot intraperiton istered as fo CMP groups administere istered via g last drug ad ficed via de were excise The thymu formula, ind 2.4. Lymph The mou ficed mice homogenised, and passed through a 40 m nylon cell strainer to obtain single-cell suspensions in accordance with the method used n, So e we esusp m su rp., C lin. S onta or 3 ted f ntrif . DM unt red o agoc func ce te proc ly in speci inal .1% N ible nd th ted a OD1 t2 OD1 gocy , B is oche orga repa salin , and , T-A e MD thod chiy idati as m Benz omol atisti data ndow ana ’s te nsid ults fect o sple ogno dice ose was purchased from Sigma. Dimethyl sulfoxide as acquired from the Yixin Institute of Chemical (Jiangsu, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5- razolium bromide (MTT) was purchased from Amresco kits for the total antioxidant capacity (TAOC), mal- de (MDA), catalase (CAT), superoxidase dismutase glutathione peroxidase (GSH-Px) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). hamide (CY) was purchased from Jiangsu Hengrui o., Ltd. (Lianyungang, Jiangsu, China). Bovine serum omassie Brilliant Blue G-250, and cellulose sacks were rom Sigma Chemical Co. (St. Louis, MO, USA). The filter was purchased from Millipore Corp. (Billerica, MA, USA). ls used in the experiments were of analytical grade. charide extraction harides from C. militaris were prepared as previously Li, Yang, & Tsim, 2006; Yu et al., 2007). The dried pow- red C. militaris was defatted with ethanol for 10 h and ly extracted three times with hot water (100 ◦C) for lting suspension was centrifuged (8000 × g for 20 min) through a 0.45 m membrane (Millipore). The filtered lution was concentrated to a specific volume under essure. The dark brown precipitate was collected via on and washed twice with ethanol. The precipitate was ded in water and lyophilized to yield CMP with 41.2% saccharide content, which was measured using vitriol- ith anhydrous glucose as the standard control. and experimental design LB/c mice (8 weeks old, 18 h to 20 g) were purchased ghai Slac Laboratory Animal Center of the Chinese f Sciences (Shanghai, China). The animals were pro- water and mouse chow ad libitum and were housed facility at (22 ± 1) ◦C with a 12 h light-dark cycle for tion. All procedures involving animals and their care ved by the Ethics Committee of the Chinese Academy ral Sciences. The mice were randomly divided into 6 isting of 15 mice each. Three mice from each group were agocytic index determination in the carbon clearance used for lymphocyte proliferation, and 9 were used for periments. All animals were allowed one week to adapt ironment before the treatment. Two groups of healthy used as normal control (NS group) and positive control treated once daily with physiological saline solution and dy weight CMP, respectively, for 18 days. From days 1 her four groups of mice were given 80 mg/kg/d CY via eal injection. From days 4 to 18, the mice were admin- llows: model group, physiological saline solution; three , 17.5, 35, or 70 mg/kg body weight CMP. CY (0.2 ml) was d via intraperitoneal injection. The others were admin- avage in 0.2 ml solutions. Twenty-four hours after the ministration, the animals were weighed and then sacri- capitation. The heart, liver, kidney, spleen, and thymus d; the spleen and thymus were immediately weighed. s and spleen indices were calculated according to the ex (mg/g) = (weight of thymus or spleen)/body weight. ocyte proliferation assay se spleens were aseptically removed from the sacri- using scissors and forceps in 0.1 M cold PBS, gently by Yua mixtur were r mediu gen Co penicil plate c tured f incuba was ce carded shaken measu 2.5. Ph The clearan to the venous Blood the ret 2 ml 0 UV-vis liver a calcula K = lg where Pha weight 2.6. Bi The were p logical 10 min protein and th ric me acid (U autoox level w assay ( in nan 2.7. St All for Wi tistical Scheffe was co 3. Res 3.1. Ef The and pr mus in with thng, Li, Li, and Dai (2006). The trythrocytes in the cell re washed via hypo-osmostic haemolysis, and the cells ended to a final density of 5 × 106 cells/ml in RPMI 1640 pplemented with 10% newborn bovine serum (Invitro- arlsbad, CA, USA), 100 U/ml streptomycin, and 100 U/ml pleen cells (100 l/well) were seeded into a 96-well ining ConA (8 g/ml). The spleen cells were then cul- days in 5% CO2 atmosphere at 37 ◦C, and then further or 4.5 h with 10 l MTT (5 mg/ml) per well. The plate uged at 200 × g for 15 min, and the supernate was dis- SO (100 l) was added to each well, which was then il all crystals dissolved. The absorbance at 570 nm was n a microplate reader (Thermo Multiskan MK3, USA). ytic index tion of the macrophage cells was assessed via a carbon st performed on three mice from each group according edure of Wang et al. (2011). Each mouse was intra- jected with diluted India ink at 100 l/10 g body weight. mens were collected at 2 min (t1) and 10 min (t2) from venous plexuses, and 20 l blood was then mixed with a2CO3. The absorbance at 600 nm was measured on a spectrophotometer with 0.1% Na2CO3 as the blank. The e spleen were weighed, and the phagocytic index was s follows: − lg OD2 − t1 was for t1 and OD2 was for t2. tic index ˛ = 3√K × A/(B + C), where A is the body the liver weight, and C is the spleen weight. mical assay n homogenates (including the liver, heart, and kidney) red in a 0.1 g/ml wet weight of ice-cold isotonic physio- e. The samples were centrifuged at 2000 × g at 4 ◦C for the supernates were used for the measurement of the OC, MAD, CAT, SOD, and GSH-Px levels. The SOD activity A and TAOC levels were measured via spectrophotomet- s. The MDA level was detected using 2-thiobarbituric ama & Mihara, 1978). The SOD activity was analysed via on of pyrogallol (Marland & Marklund, 1974). The TAOC easured using the ferric reducing/antioxidant power ie & Strain, 1996). The enzyme activity was expressed es per milligram of protein. cal analysis are presented as the mean ± SD, analysed using SPSS s version 15.0 (SPSS Inc., Chicago, IL, USA). The sta- lysis was evaluated via one-way ANOVA followed by st to detect the intergroup differences. A P < 0.05 values ered statistically significant. f CMP on mouse spleen and thymus indices en and thymus indices can reflect the immune function sis of an organism. As shown in Fig. 1, the spleen and thy- s of the model group remarkably decreased compared of the normal group (P < 0.05). CMP increased the spleen M. Wang et al. / Carbohydrate Polymers 89 (2012) 461– 466 463 Fig. 1. Effects mpar group. Values and thymu manner at 1 the CY-indu 3.2. Effect o Spleen l the mechan in Fig. 2, th remarkably (P < 0.05). C liferation in 35, and 70 m CMP is dire 3.3. Effect o system Carbon c of CMP on model grou CMP effecti dose-depen phagocytic 4.51 to 4.74 function in 3.4. Antioxi 3.4.1. Effect of the immu Fig. 4 sh (P < 0.01) in trol group. relative to t 3.4.2. Effect of the immu Fig. 5 sho hearts, liver groups. CM activity com 3.4.3. Effect organs of th Fig. 6 s (P < 0.01) in control gro activity com Effect mmu 7 s 1) in l grou OC a Effect mmu 8 sh hear oups red t cussi is a c ent i uno n, 2 ssion tive by C n re ine w ongly mmu ation e th er p ts th e sy acrop ld, 20 iffere romo ing v In prof CMP on the internal organ indices of the CY-induced mice. *P < 0.05, **P < 0.01 co are means ± SD. s indices in the CY-treated mice in a dose-dependent 7.5, 35, and 70 mg/kg, indicating that CMP can reverse ced atrophy of immune organs. f CMP on cellular immunity in mice ymphocyte proliferation was examined to understand ism of the immunoregulatory activity of CMP. As shown e spleen lymphocyte proliferation of the model group decreased compared with that of the normal group MP significantly increased the spleen lymphocyte pro- CY-treated mice in a dose-dependent manner at 17.5, g/kg compared with the model group, suggesting that ctly mitogenic for mouse splenocytes. f CMP on the phagocytic activity of the macrophage learance tests were performed to determine the effect macrophage activation. The phagocytic index ˛ of the p was lower compared with that of the NS group (Fig. 3). vely increased the ˛ value of the CY-treated mice in a dent manner. At the high CMP dose (70 mg/kg/d), the activity was restored to above the normal level (from ), demonstrating that CMP can enhance the macrophage CY-treated mice. dant activity of CMP in vivo of CMP on the activity of SOD in the different organs nosuppressed mice ows that CY significantly reduced the SOD activity the hearts, livers and kidneys compared to the NS con- All CMP doses significantly increased the SOD activity he model group (P < 0.01). of CMP on the activity of CAT in the different organs 3.4.4. of the i Fig. (P < 0.0 contro the TA 3.4.5. of the i Fig. in the trol gr compa 4. Dis CY tant ag to imm & Bara suppre protec caused CMP ca In l are str their i stimul improv in canc sugges immun and m Boqwa from d of mac activat 2006).nosuppressed mice ws the marked reductions CAT activity (P < 0.01) in the s and kidneys of mice in the CY-treated and NS control P (17.5, 35, and 70 mg/kg) significantly increased CAT pared to the model group (P < 0.01). of CMP on the activity of GSH-Px in the different e immunosuppressed mice hows the significant reductions in GSH-Px activity the hearts, livers and kidneys of the CY-treated and NS ups. All CMP doses significantly increased the GSH-Px pared to the model group (P < 0.01). to induce t upregulatio and IL-1) as the prod ner. They p nitric oxide secretion o enzymatic the current the spleen index ˛ in can also en function in ed with the NS group; #P < 0.05, ##P < 0.01 compared with the model of CMP on the activity of TAOC in the different organs nosuppressed mice hows the remarkable reductions in TAOC activity the hearts, livers and kidneys of the CY-treated and NS ps. CMP (17.5, 35, and 70 mg/kg) significantly increased ctivity compared to the model group (P < 0.01). of CMP on the activity of MDA in the different organs nosuppressed mice ows the significant increases in MDA levels (P < 0.01) ts, livers and kidneys of the CY-treated and NS con- . All CMP doses significantly decreased the MDA levels o the model group (P < 0.01). ons ytotoxic chemotherapeutic drug that acts as an impor- n tumour treatment. However, its administration leads suppression, which may be life-threatening (Hong, Yan, 004). Traditional Chinese medications for immuno- treatment are available. In the present study, the effects of CMP in reversing the immunosuppression Y treatment were investigated. The results indicate that verse the CY-induced atrophy of immune organs. ith the usage of Cordyceps in China, Chinese medicines recommended for the ageing population to enhance ne system and prevent possible infection. Immuno- itself is regarded as one of the important strategies to e body ′ s defense mechanism in elderly people as well as atients. A significant amount of experimental evidence at polysaccharides from mushrooms enhance the host stem by stimulating natural killer cells, T-cells, B-cells, hage-dependent immune system responses (Dalmo & 08; Dennert & Tucker, 1973). Polysaccharides obtained nt natural sources represent a structurally diverse class lecules, which exert their antitumour action mostly by arious immune system responses (Schepetkin & Quinn, evious studies, Cordyceps polysaccharides were found he functional activation of macrophages through the n of cytokine expression (tumour necrosis factor alpha and nitric oxide (NO) release (Lee et al., 2010), as well uction of IL-6 and IL-10 in a dose-dependent man- romote the mRNA and protein expressions of inducible synthase, induce T-lymphocyte proliferation and the f IL-2, IL-6, and IL-8, and increase the phagocytic and activities of the acid phosphatase of macrophages. In study, the administration of CMP significantly enhanced lymphocyte proliferation and increased the phagocytic a dose-dependent manner, thereby implying that CMP hance the spleen lymphocyte activity and macrophage CY-treated mice. 464 M. Wang et al. / Carbohydrate Polymers 89 (2012) 461– 466 Fig. 2. Effect of CMP on the spleen lymphocyte proliferation in CY-treated mice. *P < 0.05, **P < 0.01 compared with the NS group; #P < 0.05, ##P < 0.01 compared with the model group. Values are means ± SD. Fig. 3. Effect of CMP on the phagocytic index in the CY-treated mice. *P < 0.05,**P < 0.01 compared with the NS group; #P < 0.05, ##P < 0.01 compared with the model group. Values are means ± SD. Fig. 4. Effect of CMP on the SOD activity in the hearts, livers and kidneys of the immunosuppressed mice. *P < 0.05, **P < 0.01 compared with the NS group; #P < 0.05, ##P < 0.01 compared with the model group. Values are means ± SD. Fig. 5. Effect of CMP on the CAT activity in the hearts, livers and kidneys of the immunosuppressed mice. *P < 0.05, **P < 0.01 compared with the NS group; #P < 0.05, ##P < 0.01 compared with the model group. Values are means ± SD. M. Wang et al. / Carbohydrate Polymers 89 (2012) 461– 466 465 Fig. 6. Effect of CMP on the GSH-Px activity in the hearts, livers and kidneys of the immunosuppressed mice. *P < 0.05, **P < 0.01 compared with the NS group; #P < 0.05, ##P < 0.01 compared with the model group. Values are means ± SD. Fig. 7. Effect o presse ##P < 0.01 com Free-rad with a num such as sup (collectively tissue dama (Simic, Berg ing amount rides have p damage in l & Chang, 1 2003). Cord the anti