Tài liệu Comparison of the structural characterization and biological

World J Microbiol Biotechnol (2012) 28:2029–2038 DOI 10.1007/s11274-012-1005-6 ORIGINAL PAPER Comparison of the structural characterization and biological activity of acidic polysaccharides from Cordyceps militaris cultured with different media Fengyao Wu • Hui Yan • Xiaoning Ma • Junqiang Jia • Guozheng Zhang • Xijie Guo Zhongzheng Gui • Received: 14 October 2011 / Accepted: 18 January 2012 / Published online: 18 March 2012 Ó Springer Science+Business Media B.V. 2012 Abstract Two acidic polysaccharide fractions, CM-jdCPS2 and CM-jd(Y)-CPS2, were isolated from the fruiting bodies of cultured Cordyceps militaris grown on solid rice medium and silkworm pupa, respectively, by hot-water extraction, ethanol precipitation and fractionation using ion-exchange column (DEAE-cellulose-52) and gel-filtration column (Sephadex G-100) chromatography. Their structural characterizations were performed by gas chromatography and fourier-transform infrared spectroscopy. Some differences existed between their structures, which indicated that culture media could influence the structure of polysaccharides of C. militaris. The antioxidant activities of CM-jd-CPS2 and CM-jd(Y)-CPS2 were evaluated by various methods in vitro. They had strong 2,2-diphenyl-1picrylhydrazyl radical-scavenging activity and ferrous ionchelating capacity, but moderate reducing power. The antioxidant activities of CM-jd(Y)-CPS2 were slightly higher than those of CM-jd-CPS2. These two acidic fractions were evaluated for proliferation of mouse splenocyte activity in vitro. They both possessed does-dependent mitogenic effects on mouse splenocytes, and could synergistically promote murine T- and B-lymphocytes induced by Con A and LPS. CM-jd(Y)-CPS2 exhibited stronger stimulatory activities upon immunomodulation than CM-jd-CPS2. These results are beneficial for the F. Wu Á H. Yan Á X. Ma Á J. Jia Á G. Zhang Á X. Guo Á Z. Gui (&) Jiangsu University of Science and Technology, Zhenjiang 212018, China e-mail: [email protected] J. Jia Á G. Zhang Á X. Guo Á Z. Gui Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China interpretation of the connection between polysaccharide structures and their biological activities. Keywords Cordyceps militaris Á Media Á Polysaccharide Á Structure Á Antioxidant Á Splenocyte proliferation Á In vitro Introduction Cordyceps militaris is an entomogenous fungus that has been used as a traditional Chinese medicine for centuries. Studies have shown that it possesses similar constituents and pharmacological efficacy to those of Cordyceps sinensis (Wei et al. 2004). These include immunomodulation (Kuo et al. 2001; Kim et al. 2008), anti-tumor (Bok et al. 1999; Lin and Chiang 2008), and antioxidant effects (Liu et al. 1997). In recent years, C. militaris has been considered to be a suitable alternative to C. sinensis, which is scarce due to problems with its artificial culture and over-exploitation (Gui and Zhu 2008). Polysaccharides from fungi used for medicinal purposes have aroused wide interest because of their health benefits, such as antioxidant capacity (Liu et al. 1997; Leung et al. 2009), and immunomodulatory activity (Wasser 2002). Some mushroom polysaccharides such as lentinan and schizophyllan have been applied or are in clinical trials as immunomodifiers and adjuvant drugs for cancer therapies (Zhang et al. 2007; Yang and Zhang 2009). It has been reported that the bioactivities of polysaccharides are related to their chemical composition, glycosidic linkages, conformation, molecular weight, and degree of branching (Methacanon et al. 2005). As one of the major bioactive constituents of C. militaris, several studies have been 123 2030 conducted to ascertain the structures and bioactivities of different polysaccharides isolated from it. In recent few years, investigators have explored different types of media to artificially culture C. militaris. These include silkworm pupa, solid rice medium, germinated soybean medium, and soybean broth. Studies have shed light on the structural characterizations, antioxidant activity and immunomodulation of polysaccharides obtained from fruiting bodies of cultured C. militaris grown on a particular medium (Ohta et al. 2007; Leung et al. 2009). However, an investigation comparing these properties between polysaccharides isolated from one strain of C. militaris grown on different media has not been completed. We investigated if there are differences with respect to structural characterization, antioxidant activity and splenocyte proliferation between acidic polysaccharides obtained from the fruiting bodies of cultured C. militaris grown on two media. The results will be helpful for revealing connections between the structure and function of polysaccharides. World J Microbiol Biotechnol (2012) 28:2029–2038 collected as crude CM-jd-CPS and CM-jd(Y)-CPS (cultured on solid rice medium and silkworm pupa, respectively) and then lyophilized. Isolation and purification of polysaccharides Materials and methods Each crude precipitate (100 mg), CM-jd-CPS and CMjd(Y)-CPS was dissolved in 10 mL distilled water, centrifuged at 8,000 rpm for 10 min at room temperature, and loaded onto a DEAE-cellulose-52 column (2.6 9 30 cm). After loading with sample, the column was eluted with distilled water, and 0.1, 0.2 and 0.3 mol/L NaCl (each at 80 mL) at 1.0 mL/min. The eluate was collected at 4 min/ tube. This process was monitored by the phenol–sulfuric acid method (Xu et al. 2005). The resulting fraction (5 mg) was loaded onto a Sephadex G-100 column (2.6 9 30 cm) for further purification, then eluted with distilled water. The flow rate was 0.5 mL/min. Consequently, the homogeneous fractions, CM-jd-CPS2 and CM-jd(Y)-CPS2, were obtained and lyophilized. They were used in the subsequent structural and bioactive studies. Fungal strains and materials Ultraviolet spectroscopy of polysaccharides The C. militaris strain used was CM-jd, which was conserved by our research team. Fresh fruiting bodies grown on a solid rice medium and silkworm pupa were obtained. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid (vitamin C), ferrozine, potassium ferricyanide, ferrous chloride and ferric chloride were purchased from Bio Basic Inc (Toronto, Canada). RPMI-1640 medium, fetal calf serum (FCS) and dimethylsulfoxide (DMSO) were from Gibco Laboratories (Gaithersburg, MD, USA). Concanavalin A (Con A), lipopolysaccharide (LPS) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St Louis, MO, USA). All other reagents were obtained from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). All reagents were of analytical grade. CM-jd-CPS2 and CM-jd(Y)-CPS2 were dissolved in distilled water to 0.1 mg/mL. They were scanned with a UV spectrophotometer (UV-2450, Shimadzu, Beijing, China) at wavelengths from 800 to 200 nm. Extraction of polysaccharides The dried powder of C. militaris fruiting bodies (100 g) cultured on solid rice medium and silkworm pupa were defatted with ethanol for 10 h twice at 70°C, and exhaustively extracted twice with 20 volumes of water at 80°C, each time for 10 h. Extracts were concentrated under reduced pressure to 100 mL, deproteinated by the Sevag method (Wu et al. 2011) and dialyzed against distilled water for 4 days to remove low-molecular-weight compounds. Crude polysaccharides were obtained through precipitation with ethanol to a final concentration of ethanol of 90%, and left overnight at 4°C. Precipitates were 123 Gas chromatography (GC) analyses Derivatives of acid hydrolytic products from CM-jd-CPS2 and CM-jd(Y)-CPS2 were analyzed by GC to identify their monosaccharide components. Each fraction (10 mg) was placed in an ampoule. It was hydrolyzed with 3 mL of 4 mol/L trifluoroacetic acid (TFA) at 115°C for 12 h. The ampoule was sealed under a nitrogen atmosphere. The acidolysis solution was dried with a stream of N2 at 65°C in a water bath. The solid residual was re-dissolved in methanol (1 mL) and then distilled at 65°C with a stream of N2. This process was repeated thrice to remove the acid. The solid hydrolysate was mixed with hydroxylamine hydrochloride (8 mg) and inositol (10 mg) as internal standard. They were then re-dissolved in pyridine (0.5 mL) and allowed to react with shaking at 95°C for 30 min. The sample was then cooled to room temperature. Acetic anhydride (0.5 mL) was added to continue the acetylation reaction at 95°C for 30 min. Upon reaction completion, the solution was mixed with methanol (2 mL), and dried with a stream of N2. The derivatives were dissolved in chloroform (1 mL) and analyzed by GC. The derivation of mixed standard World J Microbiol Biotechnol (2012) 28:2029–2038 monosaccharides (rhamnose, arabinose, xylose, mannose, glucose, galactose) was operated using the same method as described above. GC was undertaken on an Agilent 6820 Gas Chromatography system (Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector (FID), through a fused-silica capillary column (0.23 mm 9 30 m). N2 was used as the carrier gas, and H2 as the burning gas. The sample injection volume was 1 lL at a N2 flow of 50 mL/min at a split ratio of 50:1. The injection temperature and FID detector were controlled at 230°C. The column temperature was first fixed at 130°C for 20 min, increased to 190°C at 5°C/min, and maintained for 20 min, then increased to 230°C at 5°C/min, and fixed for 10 min. Infrared (IR) analyses IR spectrometry of CM-jd-CPS2 and CM-jd(Y)-CPS2 was done in the 4,000–400 cm-1 wavenumber range. The dried sample (1–2 mg) was pressed into KBr (100–200 mg) disks, and then scanned with a fourier-transform infrared (FTIR) spectrometer (Tensor 27; Bruker, Billerica, MA, USA). 2031 incubated at 50°C for 20 min. Then, 2 mL of trichloroacetic acid (10%, w/v) was added to the mixture to terminate the reaction, and centrifuged at 5,000 rpm for 10 min at room temperature. The supernatant (2.5 mL) was mixed with distilled water (2.5 mL) and 0.5 mL ferric chloride (0.1%, w/v) and allowed to stand for 10 min. The absorbance was measured at 700 nm. Vc was used as the positive control. Increased absorbance of the reaction mixture indicated increased reducing power. Ferrous ion-chelating capacity assay Ferrous ion-chelating capacity was determined according to the method of Decker and Welch (1990) with some modification. The reaction mixture, containing 3 mL of each sample with different concentrations (2, 4, 6 and 8 mg/mL), 0.05 mL ferrous chloride solution (2 mmol/L), and 0.2 mL of ferrozine solution (5 mmol/L), was shaken vigorously and incubated at room temperature for 10 min. The absorbance of the mixture was measured at 562 nm. Ethylenediamine tetraacetic acid disodium salt (EDTA-2Na) was used as the positive control. The ferrous ion-chelating capacity of the sample was calculated using the following formula: In vitro antioxidant assay Chelating capacity ð%Þ ¼ 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay DPPH radical-scavenging capacity was assayed according to the method of Luo et al. (2009). with some modifications. Each sample (2 mL) at different concentrations (2, 4, 6 and 8 mg/mL) was mixed with a solution of 0.04 mg/mL DPPHÁ (2 mL) in ethanol. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then, it was centrifuged at 5,000 rpm for 10 min at room temperature. The absorbance of the supernatant was measured at 517 nm. Vitamin C (Vc) was used as the positive control. DPPH radical-scavenging capacity was calculated using the following formula: Scavenging capacity ð%Þ ¼ 1 À ðA1 À A2 Þ Â 100 A0 where A0 is the absorbance of the control (ethanol instead of sample), A1 is the absorbance of the sample, and A2 is the absorbance of the blank (which was obtained by replacing the DPPHÁ ethanol solution with ethanol). A0 À ðA1 À A2 Þ Â 100 A0 where A0 is the absorbance of the control (water instead of sample), A1 is the absorbance of the sample, and A2 is the absorbance of the blank (which was obtained by replacing the FeCl2 solution with distilled water). Measurement of immunomodulatory activity Preparation of mouse spleen cells Male Kunming mice were killed by cervical dislocation. The spleens were removed, minced and washed through a sterilized copper mesh (200 mesh) by RPMI-1640 (5 mL) to obtain a suspension of single spleen cells. The suspension was centrifuged at 1,500 rpm for 5 min at 4°C to obtain precipitated cells. Erythrocytes in precipitated cells were lysed with TrisNH4Cl solution (0.14 mol/L NH4Cl and 20 mmol/L Tris) for 2–3 min. The lysed solution was centrifuged at above condition, washed twice with RPMI-1640 medium, and adjusted to 5 9 106 cells/mL in the RPMI-1640 medium supplemented with 10% of FBS (Fetal bovine serum), penicillin (100 U/mL) and streptomycin (100 lg/mL). Reducing power assay Assay of splenocyte proliferation Reducing power was determined by the method of Tsais et al. (2006). Briefly, 1 mL of different concentrations of samples (2, 4, 6 and 8 mg/mL) in phosphate buffer (0.2 mol/L, pH 6.6) was mixed with 2 mL potassium ferricyanide (1%, w/v), and Isolated splenocytes (100 lL/well) were seeded onto a 96-well plate in the presence or absence of three concentrations (50, 100 and 200 lg/mL) of each sample (100 lL). 123 2032 World J Microbiol Biotechnol (2012) 28:2029–2038 Con A (5 lg/mL) was used as the positive control. After incubation for 48 h at 37°C in a humidified incubator containing 5% CO2, MTT solution (5 mg/mL; 20 lL/well) was added and the plate incubated for a further 4 h. After removing MTT by centrifugation at 1,000 rpm for 5 min at 4°C, the formazan precipitate was solubilized in DMSO (100 lL/well). The absorption of each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader (EL310, Bio-TKE Instruments, Winooski, VT, USA) at 570 nm. CPS2. These acidic fractions were collected and applied to a Sephadex G-100 gel filtration column, each giving a single elution peak in Fig. 1c, d, which elucidated the homogeneity of the fraction. The UV absorption spectra (Fig. 2) of CM-jd-CPS2 and CM-jd(Y)-CPS2 showed no absorption at 260 and 280 nm, indicating that nucleic acids and proteins were absent in these polysaccharides. Assay of Con A- or LPS-induced splenocyte proliferation GC analyses of CM-jd-CPS2 and CM-jd(Y)-CPS2 Splenocytes (prepared by following the procedure described above) were seeded onto a 96-well plate (100 lL/ well), and mixed with serial dilutions of each sample (50, 100 and 200 lg/mL). Cells were incubated in the presence of Con A (5 lg/mL) or LPS (10 lg/mL) for 48 h in a humidified incubator containing 5% CO2 at 37°C. MTT solution (5 mg/mL) was added to each well (20 lL/well), and the plate incubated at 37°C for a further 4 h. The medium was removed and the formazan crystals formed dissolved by adding DMSO (100 lL/well). The absorption of each well was measured using an ELISA reader at 570 nm. Control experiments were undertaken without polysaccharide samples. The alditol acetates derived from the acetylation of CM-jdCPS2, CM-jd(Y)-CPS2 hydrolysate and standard monosaccharide were measured by GC (Fig. 3). Mixtures of monosaccharide and inositol were completely separated (Fig. 3a). The peaks emerged in the order: rhamnose, arabinose, xylose, mannose, glucose, galactose and inositol. Figure 3b, c show that three types of monosaccharide (mannose, glucose and galactose) were identified in the hydrolysate of CM-jd-CPS2 and CM-jd(Y)-CPS2 on the basis of the retention time and correction factor of standard monosaccharides. According to the peak area, the mole ratio of mannose:glucose:galactose in CM-jd-CPS2 was 1.52:8.53:1.00, and in CM-jd(Y)-CPS was 3.11:1.00:2.12. These two fractions comprised the same kinds of monosaccharide, but there was a large difference in the mole ratio of each type of monosaccharide in their polysaccharides. The content of glucose was highest in CM-jd-CPS, but that of mannose and galactose much lower. Conversely, mannose comprised the highest content in CM-jd(Y)-CPS, but glucose content was the lowest. Statistical analyses All treatments and assays were carried out in triplicate for three separate experiments. Values are mean ± SD. The statistical significance was analyzed by Student’s t test and regression analysis and the data were fitted by using the Expert Design 7.1.3 for Windows software (SPSS Inc., USA). Comparison of the structural characterizations of CM-jd-CPS2 and CM-jd(Y)-CPS2 IR analyses of CM-jd-CPS2 and CM-jd(Y)-CPS2 Results Isolation and purification of CM-jd-CPS and CM-jd(Y)CPS Through hot-water extraction and ethanol precipitation, the crude polysaccharides CM-jd-CPS and CM-jd(Y)-CPS were isolated from the fruiting bodies of C. militaris cultured with solid rice medium and silkworm pupa, respectively. Figure 1a, b show the elution profiles of deproteinized CM-jd-CPS and CM-jd(Y)-CPS on a DEAEcellulose-52 ion-exchange column. In each profile, the first peak (which was eluted with distilled water) was ascribed to a neutral polysaccharide fraction. The main peak subsequently eluted with NaCl solution was an acidic polysaccharide fraction termed CM-jd-CPS2 and CM-jd(Y)- 123 In the IR spectrum of CM-jd-CPS2 (Fig. 4a), the largest absorption band (at 3,396 cm-1), was ascribed to the stretching of the hydroxyl group and the hydrogen bond within or between the molecules. The peak at 2,927 cm-1 was attributed to the C–H stretching band of the saccharide, and the weak peaks between 1,400 and 1,200 cm-1 ascribed to the C–H bending vibration. The bands at 1,651 and 1,541 cm-1 were assigned to the stretching vibration of the C=O bond and the bending vibration of the N–H bond, which suggested the presence of an acetamido group. The two absorption bands near 1,240 and 850 cm-1 resembled the stretching band of S=O and C–O–S, respectively, which indicated the existence of –O–SO3. There was a group of strong absorption peaks from 1,200 to 950 cm-1 which could be attributed to the ether linkage (C–O–C) and the hydroxyl present in the pyranose ring. World J Microbiol Biotechnol (2012) 28:2029–2038 2033 300 ntration of polysaccharide (m mg/ml) Concen Conce entration of polysaccharide ( (mg/ml) 350 A 300 250 200 150 100 50 B 250 200 150 100 50 0 0 0 10 20 30 40 50 60 70 80 0 90 10 C Concentration of polysaccharide (mg/ml) f Concentration of polysaccharide (mg/ml) 30 40 50 60 70 80 90 18 40 35 30 25 20 15 10 5 0 20 No. tube No. tube 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 No. tube 16 D 14 12 10 8 6 4 2 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 No. tube Fig. 1 Elution profiles of polysaccharides extracted from cultured C. militaris by column chromatography. a and b Ion exchange chromatogram of crude polysaccharides, CM-jd-CPS and CM-jd(Y)- CPS, on a DEAE-cellulose-52 column. c and d Gel filtration chromatogram of acidic polysaccharide fractions, CM-jd-CPS2 and CM-jd(Y)-CPS2, on a Sephadex G-100 column Fig. 2 Ultraviolet absorption curve of CM-jd-CPS2 and CM-jd(Y)CPS2 The peak at 761 cm-1 was the symmetric ring stretching of pyranose, which also implied that the monosaccharide in CM-jd-CPS2 was a pyranose. The presence of the a-glycosidic linkage was proven by the C–H bending vibration at 850 cm-1. The absorption peaks at 931 and 761 cm-1 suggested the existence of a-D-glucopyranose (a-D-Glcp). CM-jd(Y)-CPS2 also possessed the characteristic absorption peaks of a saccharide at 3,600–3,200, 3,000–2,800 and 1,400–1,200 cm-1 (Fig. 4b). The bands at 1,652 and 1,541 cm-1 indicated an acetamido group. The absorption band at 1,220 cm-1 was ascribed to the bending vibration of O–H in a carboxy group (–COOH). The bands of a C–O stretching vibration in the carboxy group at 1,440–1,395 cm-1 further proved the presence of a carboxy group in CM-d(Y)-CPS2. The group of strong absorption peaks from 1,200 to 950 cm-1 suggested that the monosaccharide in CM-jd(Y)-CPS2 was a pyranose. 123 2034 Fig. 3 GC profiles of standard monosaccharide (a), CM-jd-CPS2 (b) and CM-jd(Y)-CPS2 (c). a Peak identity: 1 Rhamnose (rt: 30.897); 2 Arabinose (rt: 31.336); 3 Xylose (rt: 31.803); 4 Mannose (rt: 40.963); 5 Glucose (rt: 41.530); 6 Galactose (rt: 42.978); 7 Inositol as an internal World J Microbiol Biotechnol (2012) 28:2029–2038 standard. b Peak identity: 1 Mannose (rt: 41.195); 2 Glucose (rt: 41.816); 3 Galactose (rt: 43.213); 4 Inositol as an internal standard. c Peak identity: 1 Mannose (rt: 40.866); 2 Glucose (rt: 41.376); 3 Galactose (rt: 42.830); 4 Inositol as an internal standard Fig. 4 IR spectrum of CM-jd-CPS2 (a) and CM-jd(Y)-CPS2 (b) Unlike CM-jd-CPS2, there was b-glycosidic linkage in CM-jd(Y)-CPS2 which was ensured by a C–H bending vibration at 893 cm-1(Wang et al. 2011). 123 From the results described above, it could be concluded that CM-jd-CPS2 was a type of sulfated polysaccharide containing an acetamido group. The monosaccharide in CM-jd-CPS2 was World J Microbiol Biotechnol (2012) 28:2029–2038 2035 a pyranose, which was connected by an a-glycosidic linkage. CM-jd(Y)-CPS2 was a type of carboxylated polysaccharide containing an acetamido group. The monosaccharide was a pyranose, which was linked by a b-glycosidic linkage. for both of them was much lower than that of Vc. This finding suggested that CM-jd-CPS2 and CM-jd(Y)-CPS2 had moderate reducing power. Ferrous ion-chelating activity of CM-jd-CPS2 and CM-jd(Y)-CPS In vitro antioxidant activities of CM-jd-CPS2 and CM-jd(Y)-CPS2 In the present study, the ferrous ion-chelating capacity of antioxidants was detected by inhibiting the formation of red-colored ferrozine–Fe2? complexes. At 8 mg/mL, the ferrous ion-chelating activities of CM-jd-CPS2 and CMjd(Y)-CPS2 were 72 and 89%, respectively, and both activities were concentration-related (Fig. 5c). However, overall, CM-jd(Y)-CPS2 possessed higher ferrous ion-chelating activity than CM-jd-CPS2 with, on average, &20% higher activity at each concentration. The positive control EDTA-2Na had excellent ferrous ion-chelating activities (100% at each concentration tested). CM-jd(Y)-CPS2 exhibited strong Fe2?-chelating activities at 4, 6 and 8 mg/ mL, but that of CM-jd-CPS2 was comparatively weak. DPPH radical-scavenging activity of CM-jd-CPS2 and CM-jd(Y)-CPS2 CM-jd-CPS2 and CM-jd(Y)-CPS2 exerted concentrationdependent DPPHÁ-scavenging activity (Fig. 5a). At 8 mg/ mL, DPPHÁ-scavenging activity for CM-jd-CPS2 and CMjd(Y)-CPS2 reached &94%. However, at a low concentration (2 mg/mL), the scavenging activity of CM-jd-CPS2 was higher than that of CM-jd(Y)-CPS2. Compared with the positive control (Vc), the scavenging activity of these two fractions were slightly lower than Vc at each concentration. These results indicated that CM-jd-CPS2 and CM-jd(Y)-CPS2 had strong DPPHÁ-scavenging activities. In vitro immunomodulatory activity of CM-jd-CPS2 and CM-jd(Y)-CPS2 Reducing power of CM-jd-CPS2 and CM-jd(Y)-CPS2 Effect of CM-jd-CPS2 and CM-jd(Y)-CPS2 on splenocyte proliferation Samples showed a dose-dependent reducing capacity (Fig. 5b). At 8 mg/mL, the reducing power of CM-jd-CPS2 was 0.985, for CM-jd(Y)-CPS it was 1.214, and for Vc it was 2.108. The reducing power of CM-jd(Y)-CPS2 was higher than that for CM-jd-CPS2, but the reducing power B 100 95 Absorbency Scavenging capacity (%) A 90 85 CM-jd-CPS2 CM-jd(Y)-CPS2 j ( ) Vc 80 75 70 2 4 6 8 Concentration (mg/ml) 2.2 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 CM-jd-CPS2 CM jd CPS2 CM-jd(Y)-CPS2 Vc 2 4 6 8 Concentration (mg/ml) C 120 Chelating capacity (%) Fig. 5 The antioxidant activities of CM-jd-CPS2 and CM-jd(Y)-CPS2. a DPPH radical scavenging activities with Vc as the positive control. b The reducing power assay with Vc as the positive control. c The ferrous ion chelating capacity with EDTA-2Na as the positive control. Values are represented as mean ± SD (n = 3) A colorimetric assay using MTT for cell proliferation was carried out to evaluate the effect of CM-jd-CPS2 and 100 80 60 CM-jd-CPS2 CM-jd(Y)-CPS2 40 EDTA 20 0 2 4 6 8 Concentration (mg/ml) 123 2036 World J Microbiol Biotechnol (2012) 28:2029–2038 Table 1 Effect of CM-jd-CPS2 and CM-jd(Y)-CPS2 on mouse splenocyte proliferation Preparation Dose (lg/mL) Control A570 – 5 Positive control (Con A) CM-jd-CPS 0.256 ± 0.001 0.376 ± 0.007 50 0.265 ± 0.003 100 0.279 ± 0.006a 200 0.293 ± 0.006a 50 0.283 ± 0.008a 100 200 0.306 ± 0.003b 0.362 ± 0.002b CM-jd(Y)-CPS a P \ 0.01 when compared with the control group Discussion P \ 0.05 when compared with the control group b compared with the Con A control group (P \ 0.01). The stimulatory effects of CM-jd-CPS2 and CM-jd(Y)-CPS2 upon lymphocyte proliferation induced by LPS were nearly identical, and were both significantly increased (P \ 0.01) at 200 lg/mL. These results suggested that CM-jd-CPS2 and CM-jd(Y)-CPS2 could dose-dependently promote murine T- and B-cell proliferation induced by specific mitogens, whereas CM-jd(Y)-CPS2 possessed a stronger stimulatory activity to T cells than CM-jd-CPS2. Table 2 Effects of CM-jd-CPS2 and CM-jd(Y)-CPS2 on Con A or LPS induced mouse splenocyte proliferation Preparation Control (Con A) Dose (lg/mL) 5 Control (LPS) 10 CM-jd-CPS 50 Con A (A570) LPS(A570) 0.376 ± 0.007 – – 0.390 ± 0.007 0.380 ± 0.005 0.398 ± 0.009c a 0.415 ± 0.004c 200 a 0.407 ± 0.004 0.425 ± 0.003d 50 0.403 ± 0.006a 0.403 ± 0.006c 100 200 b 0.415 ± 0.007c 0.430 ± 0.001d 100 CM-jd(Y)-CPS a 0.396 ± 0.002 0.430 ± 0.005 0.440 ± 0.003b P \ 0.05 when compared with the Con A control group b P \ 0.01 when compared with the Con A control group c P \ 0.05 when compared with the LPS control group d P \ 0.01 when compared with the LPS control group CM-jd(Y)-CPS2 upon splenocyte proliferation (Table 1). In comparison with the control group (without sample treatment), promoting effects increased with increasing sample concentration (50–200 lg/mL). CM-jd(Y)-CPS2 produced statistically significant promotion (P \ 0.01) of proliferation of mouse splenocytes at 200 lg/mL, which was close to that of the positive control group (Con A). CM-jd-CPS2 showed weaker stimulating activity than that of CM-jd(Y)-CPS2. Effect of CM-jd-CPS2 and CM-jd(Y)-CPS2 on Con A- or LPS-induced splenocyte proliferation The effects of CM-jd-CPS2 and CM-jd(Y)-CPS2 on lymphocyte proliferation induced by Con A or LPS were investigated. The stimulatory effects of the two samples upon proliferation were higher with increasing doses (Table 2). The proliferation of lymphocytes induced by Con A was promoted by &17% by CM-jd(Y)-CPS2 at 200 lg/mL, which was a statistically significant promotion 123 Cultivated fruiting bodies of C. militaris have been sold as drug materials and healthfood products in China and South East Asia in recent years. With the growing demand of its fruiting bodies, culturing it with silkworm pupa was not sufficient to match market requirements. Hence, solid rice medium (which is relatively plentiful and not affected by seasonal changes) was used to cultivate the fruiting bodies of C. militaris on a large scale. However, there is no report comparing the bioactive constituents contained in fruiting bodies cultured by these two media. As one of its main components, polysaccharides have attracted wide attention. Polysaccharides have the greatest potential for structural variability to carry biological information, so it is important to determine their structure and bioactivity. In the last few years, structural characterizations of several polysaccarides obtained from cultured C. militaris have been reported. An acidic polysaccharide isolated from C. militaris grown on germinated soybeans was found to be composed of galactose, arabinose, xylose, rhamnose and galacturonic acid (Ohta et al. 2007). CPS-2 was isolated from cultured C. militaris, and primarily comprised rhamnose, glucose and galactose in a molar ratio of 1:4.46:2.43 (Yu et al. 2004). Lee et al. (2010) reported the structural properties of CPSN Fr II, which was obtained from the cultured mycelia of C. militaris. CPSN Fr II was a 1,6-branched-glucogalactomannan with a b-linkage and random coil conformation. The interpretation of structural differences between the results described above may be because they were different strains of C. militaris. Otherwise, different culture media (silkworm pupa, solid rice medium, and broth) may contribute to the differences between the structures of the polysaccharide. In the present study, two acidic polysaccharides, CM-jd-CPS2 and CMjd(Y)-CPS2, were extracted from the fruiting bodies of the same strain of C. militaris cultivated on solid rice medium and silkworm pupa, respectively. Structure elucidation showed that CM-jd-CPS2 and CM-jd(Y)-CPS2 comprised mannose, glucose and galactose, but with different proportions. CM-jd-CPS2 had a large proportion of glucose, whereas CM-jd(Y)-CPS2 had a large proportion of World J Microbiol Biotechnol (2012) 28:2029–2038 mannose. The IR spectrum revealed that the monosaccharide within them was a pyranose, that CM-jd-CPS2 was connected by an a-glycosidic linkage, and that CM-jd(Y)CPS2 was connected by a b-glycosidic linkage. In addition, CM-jd-CPS2 was a type of sulfated acidic polysaccharide containing an acetamido group, whereas CM-jd(Y)-CPS2 was a kind of carboxylated polysaccharide. These results indicated that the differences between their structures were due to different culture media. Hence, we showed that culture media could influence the structure of polysaccharides of C. militaris. Polysaccharides and their derivatives are emerging as new options for combating oxidative stress-mediated disorders (Huang et al. 2009). The antioxidant properties of polysaccharides are very relevant to their health-protecting and anti-cancer functions. These two acidic fractions could efficiently scavenge the stable free radical DPPHÁ. This was attributed to their electron-transfer or hydrogen-donating ability. It has been suggested that the hydroxyl (–OH) group in polysaccharides can donate electrons to reduce the radicals to a more stable form or react with the free radicals to terminate the radical chain reaction (Leung et al. 2009). CM-jd-CPS2 and CM-jd(Y)-CPS2 showed moderate reducing power. This may have been due to the –OH group and certain reducing groups in their structures. The presence of reductants is associated with reducing power. Reductants have been shown to exert antioxidant actions by breaking the free-radical chain by donating a hydrogen atom (Zhang et al. 2010a, b). The results of the ferrous ionchelating capacity assay showed that CM-jd-CPS2 and CM-jd(Y)-CPS2 exhibited strong Fe2?-chelating activities at high concentrations. It has been demonstrated that compounds with metal-chelating activities usually contain two or more of the following functional groups: –OH, –SH, –COOH, –PO3H2, –C=O, –NR2, –S– and –O– (Yuan et al. 2005). Accordingly, the ferrous ion-chelating capacities of the two acidic polysaccharides were partially accounted for by the presence of –OH, C=O, –S– and –O– groups in their structure. Moreover, it has been reported that the antioxidant capacity of polysaccharides is also strongly dependent upon the type and organization of the monosaccharide, the linkage pattern of the main chain (a or b) and the branching configuration (Liu et al. 2007). This could explain the differences of reducing power and ferrous ion-chelating capacity between CM-jd-CPS2 and CM-jd(Y)-CPS2. The therapeutic effects of C. militaris have been shown to be mediated through reinforcement of the immune system (Won and Park 2005). The spleen is one of the major immune organs, and contains T-and B-lymphocytes. Splenocyte proliferation is related to the improvement of immunity of T- and B-lymphocytes (Qiao et al. 2010). In the present study, CM-jd-CPS2 and CM-jd(Y)-CPS2 had direct mitogenic effects on mouse splenocytes, which could 2037 strengthen the immunological response. CM-jd(Y)-CPS2 showed mitogenic effect, but it was not comparable than CM-jd-CPS2. These acidic polysaccharides affected the Con A- and LPS-induced splenocyte proliferation. Administration of CM-jd(Y)-CPS2 at high concentrations was found to significantly increase the proliferation of splenocytes. CM-jd-CPS2 was the one to have mitogenic effect but this effect was not comparable with CM-jd(Y)CPS2. The two acidic fractions strongly increased proliferation of splenocytes with LPS at 200 lg/mL. It is known that the combination of polysaccharides and Con A can significantly promote the proliferation of T-lymphocytes (Yuan et al. 2005; Zhao et al. 2006; Zhang et al. 2010a, b). These results demonstrated that CM-jd-CPS2 and CM-jd(Y)-CPS2 could synergistically promote the induction of murine T- and B-lymphocytes by specific mitogens. CM-jd(Y)-CPS2, which was obtained from silkworm pupacultivated C. militaris, possessed stronger stimulatory activity on immunomodulation than CM-jd-CPS2. It has been reported that many factors influence the activities of polysaccharides, including monosaccharide composition, glycosyl residues, chain conformation and molecular mass (Bao et al. 2001; Huang et al. 2007). Consequently, the different immunomodulatory activities of the two acidic polysaccharides may have been due to differences in their structures. Also, some researches suggested that acidic polysaccharides are likely to be more bioactive than neutral polysaccharides because the acidic groups form associations with target biomolecules through electronic interactions (Tadera et al. 2003). It was found that the acidic polysaccharide from Tanacetum vulgare L. showed higher immuno-activities than the neutral polysaccharide fraction (Won and Park 2005; Xie et al. 2007). 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