International Journal of Biological Macromolecules 47 (2010) 271–275 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Structure elucidation and antioxidant activity of a novel polysaccharide isolated from Tricholoma matsutake Xiang Ding a,1 , Jie Tang a,1 , Mei Cao b,1 , Chun-xiao Guo a , Xia Zhang a , Jing Zhong a , Jie Zhang a , Qun Sun a , Su Feng a , Zhi-rong Yang a , Jian Zhao a,b,∗ a Key Laboratory of Biological Resource and Ecological Environment of the Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, PR China b Key Laboratory of Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, Chengdu 610072, PR China article info Article history: Received 20 March 2010 Received in revised form 17 April 2010 Accepted 19 April 2010 Available online 27 April 2010 Keywords: Polysaccharide structure Antioxidant assay Tricholoma matsutake abstract In this study, structural features of Tricholoma matsutake polysaccharide (TMP-A) were investigated by a combination of infrared (IR) spectra, gas chromatography–mass spectrometry (GC–MS), nuclear magnetic resonance (NMR) spectroscopy. The results indicated that TMP-A had a backbone of 1,4-␤- d-glucopyranose residue which branches at O-6 based on the experimental results. The branches were mainly composed of an (1→ 3)-␣-d-galactopyranose residue, and terminated with ␣-d-xylopyranose residue. The antioxidant activity of TMP-A was evaluated with several biochemical methods, includ- ing DPPH − radical scavenging, hydrogen peroxide scavenging, superoxide anion radical scavenging. The results indicated that TMP-A showedstrong antioxidant. In the in vitro antioxidant assay by MTT method, TMP-A could attenuate PC12 cell damage significantly caused by hydrogen peroxide. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Oxidation is essential to many organisms for the production of energy to fuel biological processes. However, the uncontrolled pro- duction of superoxide anion free radicals is involved in the onset of many diseases such as cancer, atherosclerosis and degenerative processes with aging [1]. Thus, it is essential to develop effective and natural antioxidants so that they can protect the human body from free radicals and many chronic diseases [2]. Polysaccharides extracted from mushrooms, such as Lentinus edodes, Ganoderma tsugae and Cordyceps sinensis, have also exhibited antioxidant prop- erties by their free radical scavenging ability [3,4]. Tricholoma matsutake is a kind of fungi belonging to Sub- genus Tricholoma and is widely distributed in Asian countries, such as China, Japan, and Korea. As a traditional edible fungus in oriental countries, it has been consumed as a vegetable and used as a traditional Chinese medicine in single and compound- ing prescriptions for the prevention and treatment of diseases for several thousand years [5]. As an extract from T. matsutake, polysaccharides (TMP) have showed strongly bioactive properties towards antioxidant and anti-tumor [6]. Thus the determination ∗ Corresponding author at: Key Laboratory of Biological Resource and Ecological Environment of the Ministry of Education, College of Life Sciences, Sichuan Univer- sity, Wangjiang Road 29#, Chengdu 610064, PR China. Tel.: +86 28 85460487; fax: +86 28 85460487. E-mail address: biostart8083@yahoo.cn (J. Zhao). 1 These authors contributed equally to this research. of the structure of polysaccharides was necessary to establish the relationship between the biological activities and the struc- ture. In this work, one novel water-soluble polysaccharide was extracted and purified from the fruiting bodies of T. matsutake using a DEAE-cellulose column chromatography and a Sephadex G-100 column chromatography. Its chemical structures were char- acterized for the first time. The antioxidant activity of TMP-A was evaluated by various antioxidant assay and MTT method. The result of this study introduced T. matsutake as a possible valuable source which helped to exhibit unique antioxidant prop- erties. 2. Materials and methods 2.1. Chemicals The fruiting bodies of T. matsutake were collected in Xiaojing country of Sichuan Province, China, and were authenticated by Prof. Sao-rong Ge (College of Life Sciences, Sichuan University, Chengdu, China). At the same time, a voucher specimen had been preserved in Key Laboratory for Biological Resource and Ecolog- ical Environment of Education Ministry, College of Life Sciences, Sichuan University. DEAE-cellulose 52 and Sephadex G-100 were purchased from Sigma–Aldrich (mainland, China). Monosaccharide standards, Dextran T-500, T-110, T-70, T-40, and T-10, were pur- chased from Beijing Biodee Biotechnology Co., Ltd. (Beijing, China). All other reagents used were of analytical grade. 0141-8130/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2010.04.010 272 X. Ding et al. / International Journal of Biological Macromolecules 47 (2010) 271–275 2.2. Extraction, purity and fractionation of polysaccharides from T. matsutake After the fruiting bodies (200 g) of T. matsutake were soaked with 95% EtOH, the residue was dried and then extracted with boiling water for three times (6 h for each). After the filtrate was concentrated, dialyzed (MWCO 5000, Sigma), and centrifuged, the supernatant was added with 3 volumes of 95% EtOH to precipitate crude polysaccharides (32.8 g, recovery 16.4%). After Sevag method (Staub [7]) was used for thedeproteination, TMP(8 g) was subjected to a DEAE-cellulose column (Tris–HCl, pH 7.0, 4.5 cm× 50 cm, Cl − ) and eluted stepwise with 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 1.0 M NaCl. The eluate was monitored by the phenol-sulfuric acid method [8]. The 0 M NaCl eluation was concentrated, lyophilized and purified on a Sephadex G-100 column (2.6 cm × 60cm). The resulting T. mat- sutake polysaccharide, named TMP-A, was obtained by the above processes and the yield rate of TMP-A was 0.22% (0.432 g) for the starting material. 2.3. Measurement of molecular weight and monosaccharide composition analysis of TMP-A High performance gel permeation chromatography (HPGPC) was carried out to measure molecular weight. Thecolumn was cali- brated withstandard T-seriesDextran (T-500,T-110, T-70,T-40 and T-10). The data were processed with Waters GPC (Millennium32 software). Thepolysaccharide TMP-A(5.0 mg) was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 110 ◦ C for 6 h on the mecha- nism of acid-catalyzed hydrolysis [9]. Excess acid was removed by co-distillation with methyl alcohol (MeOH) after the hydroly- sis was completed. One part of the hydrolysate (1.0 mg) was used for thin layer chromatography (TLC) analysis as described previ- ously. Developing solvent: acetoacetate–pyridine–ethanol–water solution (8:5:1.5:1); the developer system: diphenylamine–aniline system (85% phosphoric acid solution 140 mL containing 8 mL diphenylamine, 8 g aniline) [10], and the other (1.0 mg) was dissolved in pyridine (0.2 mL). The derivatization reaction was ini- tiated by addition of hexamethyl-disilazane (0.2 mL) and trimethyl chloro-silicane (0.2 mL) according to the method described by Dong [11,12]. The resulting supernatant was examined by GC–MS at a temperature program of 50–230 ◦ C with a rate of 2 ◦ C/min [13]. 2.4. Methylation analysis The polysaccharide, TMP-A (10 mg), was methylated using methyl iodide (MeI) according to the Hakomori method [14]. After complete methylation, the permethylated polysaccharide was depolymerized with 90% aqueous formic acid (3 mL) for 10 h at 100 ◦ C in a sealed tube. The methylated sugars were derivatized using the method described and analyzed by GC–MS. 2.5. UV and infrared (IR) spectra analysis TMP-A was tested in UV from 200 to 600 nm and infrared analysis of the samples was obtained by grinding a mixture of polysaccharide with dry KBr and then pressing in a mold. Spectra were run in the 4000–400 cm −1 region. 2.6. Nuclear magnetic resonance (NMR) experiment 1 H NMR spectra and 13 C NMR spectra were recorded on a Var- ian Unity INOVA 400/45 in D 2 O with tetramethylsilane as internal standard. 2.7. Determination of 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH − ) scavenging activity of TMP-A The DPPH − radical scavenging activity of TMP-A was measured according to the method described by Braca et al. [15]. The percent- age scavenging activity was calculated by the following formula: scavenging effect (%)= (1 − A sample/A control) × 100, where A con- trol is the absorbance of control (DPPH solution without sample), A sample is the test sample (DPPH solution plus test sample or pos- itive control) [16]. Vitamin C (Vc) and butylated hydroxytoluene (BHT) were used as a positive control in the study. 2.8. Scavenging effect on hydroxyl radicals The ability of the TMP-A to scavenge hydrogen peroxide was determined according to the method of Smirnoff and Cumbes [17]. The percentage of scavenging of hydrogen radicals was calcu- lated as follows: scavenging effect (%) =[1 − (A sample − A sample blank)/A control] × 100, where A control was the absorbance of the control group in the hydroxyl radicals generation system, A sample was the absorbance of the test group and A sample blank was the absorbance of the samples only. Vc was used as a positive control in the study. 2.9. Determination of superoxide anion scavenging activity Superoxide anion scavenging activity was measured accord- ing to the pyrogallol’s autoxidation method [18]. The inhibition of superoxide anion production was calculated as follows: scav- enging effect (%) =(A − B)/A × 100, where A is the change speed of absorbance of the control group in the superoxide anion generation system and B is the change speed of absorbance of the test sample. Vc was used as a positive control in the study. 2.10. Cell lines and culture PC12 cells (ATCC, American Type Culture Collection, USA) were maintained in Dulbcco’s Modified Eagle Medium, which contained 10% heat-inactivated horse serum, 5% fetal bovine serum and antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin) at 37 ◦ C in a humidified atmosphere containing 5% CO 2 . 2.11. Antioxidant activity assay In this study, PC12 cells were seeded into 96-well plates at the concentration of 5 × 10 4 cells/mL using Dulbcco’s Modified Eagle Medium. After 24 h, PC12 cells were pretreated with TMP-A for 2 h before H 2 O 2 (300 mMsolution) exposurefor 1h. Afterthe H 2 O 2 was withdrawn, cells were then further incubated in the fresh medium for another 6 h at 37 ◦ C. Then methyl thiazolyl tetrazolium (MTT) stock solution was added to each well reaching a final concentra- tion of 0.5 mg/mL. After incubating for 4 h, the supernatants were aspirated to remove untransformed MTT. Finally, 150 ␮L dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals and the amount of purple formazan was determined by the absorbance measurement at 570 nm using the Universal Microplate Reader (Bio-RAD) [19]. The damage inhibitory effect was expressed as: damage inhibitory effect (%) =[(A s − A)]/[(A 0 − A)]× 100% where A s is the absorbance in the presence of the sample and H 2 O 2 , A 0 is the absorbance of the control in the absence of the sample and H 2 O 2 , and A is the absorbance only in the presence of the H 2 O 2 . 2.12. Statistical analysis All data were presented as means ± standard deviation (SD) of three replications. Statistical analyses were performed using Stu- X. Ding et al. / International Journal of Biological Macromolecules 47 (2010) 271–275 273 Fig. 1. FTIR spectra of polysaccharide TMP-A. dent’s t-test and one-way analysis of variance. Values of P < 0.05 were considered to be a statistically significant finding. 3. Results and discussion 3.1. Extraction, purity and composition of polysaccharides The crude polysaccharide, named TMP, was obtained from the fruiting bodies of T. matsutake with a yield of 16.4%. After fractionation on DEAE-cellulose 52 and Sephadex G-100 column chromatography, 324 mg of TMP-A was obtained from the 0 M NaCl eluate and detected by the phenol-sulfuric acid assay. The homo- geneity of the polysaccharide was elucidated by the following tests. TMP-A waseluted fromgel-filtration chromatography on Sephadex G-100 column and was detected by the phenol-sulfuric acid assay as a single peak. No absorption at 280 and 260 nm in UV absorp- tion spectra of TMP-A demonstrated the absence of protein and nucleic acid in this polysaccharide and it had the same optical rotation: [␣] 20 D −1.648 ◦ (c0.5, water) in different low concentra- tion of ethanol using HK7-SGW-1 automatic optical polarimeter at room temperature. Weight-average molecular weight was around 8.89 × 10 4 Da. The three monosaccharides, d-glucose, d-galactose, d-xylose (d-Glc, d-Gal, and d-Xyl) were also identified using the hydrolysate of TMP-A by GC–MS which was in good agreement with the TLC with the ratios of 79.37:9.81:10.82.TMP-A was sup- posed to contain the d-configuration monosaccharide according to GC–MS analysis. 3.2. Structure elucidation of TMP-A The intensity of bands around 3408.22 cm −1 in the IR spectrum (Fig. 1) was due to the hydroxyl stretching vibration of the polysac- charide and asexpected they were broad. The bands inthe region of 2923.62 cm −1 were due to C–H stretching vibration, and the bands in the region of 1643.29 cm −1 were due to associated water [20]. Two strong absorption bands at 1075.03cm −1 , 1041.64 cm −1 in the range of 1200–1000 cm −1 in the IR spectrum suggested that the monosaccharide in TMP-A had a pyranose-ring [21]. The absorp- tion at 875.43 cm −1 indicated that TMP-A had ␤-glucopyranose linkages, which was indicated by the anomeric proton signals at ı4.570 in the 1 H NMR (400 MHz) [22]. Moreover, the characteris- tic absorptions at 799.73 cm −1 indicated ␣-configurations existing in the polysaccharide [23], which was in good agreement with the anomeric proton signals at ı5.182, ı5.107, ı5.060 in the 1 HNMR (400 MHz) spectrum. According to the literature [24], the reso- nances in the region of 98–106 ppm in the 13 C NMR (200 MHz) spectrum of TMP-A were attributed to the anomeric carbon atoms of ␤-d-glucopyranose (␤-d-Glcp), ␣-d-galactopyranose (␣-d-Galp) and ␣-d-xylopyranose (␣-d-Xylp). In the anomeric carbon region, signals at ı105.2 could be attributed to C-1 of →4)-␤-d-Glcp-(1→; Fig. 2. The 13 C NMR spectra of polysaccharide TMP-A. Table 1 13 C NMR chemical shift data (ı, ppm) for polysaccharide TMP-A. Sugar residues Chemical shifts, ı (ppm) C 1 C 2 C 3 C 4 C 5 C 6 →4)-␤-d-Glcp-(1→ 105.239 69.370 78.160 72.941 78.750 63.636 →4,6)-␤-d-Glcp-(1→ 105.423 69.370 78.426 72.121 79.598 63.636 →3)-␣-d-Galp-(1→ 100.762 70.820 77.388 75.439 80.717 63.258 ␣-d-Xylp-(1→ 104.194 71.047 75.638 74.305 72.121 Table 2 GC–MS results of methylation analysis of TMP-A. Methylated sugar Linkage m/z 2,3,6-Me 3 -Glc 1,4- 45,59,73,88,101, 133,146,159,232 2,3-Me 2 -Glc 1,4,6- 45,59,73,88,101,116,133,146,174,232 2,4,6-Me 3 -Gal 1,3- 45,59,73,89,116,146,159,191,204,233 2,3,4-Me 3 -Xyl T- 45,59,73,88,101,116,133,146,174 ı105.4 to C-1 of →4,6)-␤-d-Glcp-(1→; ı100.7 to C-1 of →3)-␣-d- Galp-(1→; ı104.1 to C-1 of ␣ d-Xylp-(1→, respectively (Fig. 2). All the assignment of the carbon atoms signals was shown in Table 1. After methylation according to the Hakomori method for four times, the methylated polysaccharide was depolymerized and con- verted into partially methylated ramifications. The analysis of the methylated monosaccharide was conducted by GC–MS. The information in MS showed that fragment ion peaks were consis- tent with the data of d-configuration monosaccharide fragment ions peaks which can be concluded that the glucose, galactose and xylose residues were d configuration. Methylation analysis for TMP-A proved that the ␤-d-glucopyranose residues were 2,3- bis-substituted and 2,3,6-trisubsituted, the ␣-d-galactopyranose residues were 2,4,6-trisubsituted, and the ␣-d-xylopyranose residue was 2,3,4-trisubsituted (Table 2). Results methylated linkage analysis of TMP-A indicated that (1 → 4)-linked-␤-d- glucopyranose was one of the largest amounts residue of the polysaccharide structure, the branched residue was (1 → 4,6)- linked -␤-d-glucopyranose revealing that (1 → 4)-linked-␤-d- glucopyranose should be possible to form the backbone structure. The relative amounts of (1 → 4,6)-linked-␤-d-glucopyranose indi- cating that approximate branch ratios could theoretically be 12.5%, namely on average one branching point for each eight residues of backbone. Residues of branch structure were (1 → 3)-linked-␣-d- galactopyranose and terminated with ␣-d-xylopyranose residue. It is concluded that a repeating unit of TMP-A has a backbone of (1 → 4)-␤-d-glucopyranose residues which branches at O-6 based 274 X. Ding et al. / International Journal of Biological Macromolecules 47 (2010) 271–275 Fig. 3. Predicted chemical structure of polysaccharide TMP-A. Fig. 4. DPPH − radical scavenging effect of TMP-A from Tricholoma matsutake. on the experimental results. The branch was supposed to be the composition of an (1 → 3)-␣-d-galactopyranose residue and one terminated with ␣-d-xylopyranose residue. The predicted struc- ture of the novel polysaccharide TMP-A was shown in Fig. 3. 3.3. Determination of DPPH radical scavenging activity of TMP-A DPPH is a useful reagent for investigating the free radical scav- enging activities of various samples. It is noticeable by eye that there is a discolouration from purple to yellow induced by antiox- idants. Fig. 4 illustrates the scavenging activity of the purified polysaccharide samples on the DPPH radical. These results showed that the IC 50 value of TMP-A for eliminating DPPH radicals was about 3.0 mg/mL, which indicated that TMP-A have a noticeable effect on scavenging DPPH radical,especially at highaddition quan- tity. However, the inhibiting ability was lower than that of BHT and Vc. 3.4. Scavenging effect of hydroxyl radical by TMP-A Hydroxyl radical can easily cause tissue damage or cell death. Thus, hydroxyl radical removing is important for the protection of living systems. Fig. 5 shows the percentage hydroxyl radical scav- enging effects of TMP-A at the dose of 0.5, 1, 2, 3, 4, 5 and 10 mg/mL and IC 50 value of TMP-A was about 7.1 mg/mL. At the test concen- trations, TMP-A exhibited scavenging effect on hydroxyl radicals in a concentration-dependent manner which showed that the purifi- cation polysaccharides had weaker hydroxyl radical scavenging effects than Vc of same dose. 3.5. Determination of superoxide anion scavenging effect Fig. 6 illustrates the superoxide radical scavenging effect of 0.5, 1, 2, 3, 4, and 5 mg/mL of TMP-A in comparison to the same doses of Vc. At all the concentrations, the polysaccharide samples exhibited varying degrees of antioxidant effect and IC 50 value of TMP-A was Fig. 5. Hydroxyl radical scavenging effect of TMP-A from Tricholoma matsutake. Fig. 6. Superoxide radical scavenging effect of TMP-A from Tricholoma matsutake. Fig. 7. TMP-A attenuated PC12 cell damage induced by hydrogen peroxide. about 3.6 mg/mL. Results showed that the purification polysaccha- rides had weaker hydroxyl radical scavenging effects than Vc of same dose. 3.6. Antioxidant activity analysis of TMP-A by MTT In MTT experiments we determined protection effect of TMP- A on PC12 cell from hydrogen peroxide (H 2 O 2 ) induced injury. After pretreated with 1.25, 2.5, 5, 10 mg/mL of TMP-A, the PC12 cell could been protected from H 2 O 2 (300 mM) injury in a dose dependent way, with the cell viability rate of 12.1%, 35.4%, 76.1%, 85.2%, respectively (Fig. 7). Thus, we confirmed X. Ding et al. / International Journal of Biological Macromolecules 47 (2010) 271–275 275 that TMP-A may attenuate the injury on PC12 cells induced by H 2 O 2 . 4. Conclusions According to the results above, it was concluded that the novel polysaccharide obtained from T. matsutake is a heteropolysac- charide, namely TMP-A and the purified polysaccharide prepared (TMP-A) was confirmed of high purity. The present study also showed that TMP-A consisted of three monosaccharides, namely d-Glc, d-Xyl and d-Gal and their ratios were 8:1:1 by GC–MS. Structure study demonstrated that TMP-A has a backbone of (1 → 4)-␤-d-glucopyranose residues which branches at O-6 based on the experimental results. The branches were mainly composed of an (1 → 3)-␣-d-galactopyranose residue, and terminated with ␣-d-xylopyranose residue. Purification polysaccharides prepared in our work are confirmed of high purity. Anti-oxidation test in vitro shows that it possesses strong free radical scavenging activity, which may be comparable to Vc and BHT. In PC12 cell antioxidant effect assay, we found that TMP-A could significantly attenuate PC12 cell damage caused by hydrogen peroxide. Overall, T. matsutake may be one ideal sources of antioxidants develop- ment. Acknowledgements The authors thank Dr. PuSu (Department ofChemistry of Medic- inal Natural Products and Key Laboratory of Drug Targeting of Education Ministry PRC, West China College of Pharmacy, Sichuan University) for helpful assistance in NMR experiments. 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