Đề tài Structure elucidation and antioxidant activity of a novel polysaccharide isolated from Tricholoma matsutake

International Journal of Biological Macromolecules 47 (2010) 271–275 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journa l homepage: www.e lsev ier .com Structure elucidation and antioxidant activity of from Tr Xiang Di Zha Qun Sun a Key Laborator ge of L b Key Laborato du 61 a r t i c l Article history: Received 20 M Received in re Accepted 19 A Available onlin Keywords: Polysaccharide Antioxidant as Tricholoma ma Tricho ectra, opy. hes a galac MP-A en pe trong age s 1. Introduction Oxidation is essential to many organisms for the production of energy to fuel biological processes. However, the uncontrolled pro- duction of s of many dis processes w and natural from free ra extracted fr tsugae andC erties by th Tricholom genus Trich such as Ch in oriental used as a t ing prescri for several polysacchar towards an ∗ Correspon Environment o sity, Wangjian fax: +86 28 85 E-mail add 1 These auth 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 0141-8130/$ – doi:10.1016/j.uperoxide anion free radicals is involved in the onset eases such as cancer, atherosclerosis and degenerative ith aging [1]. Thus, it is essential to develop effective antioxidants so that they can protect the human body dicals and many chronic diseases [2]. Polysaccharides om mushrooms, such as Lentinus edodes, Ganoderma ordyceps sinensis, have also exhibited antioxidant prop- eir free radical scavenging ability [3,4]. a matsutake is a kind of fungi belonging to Sub- oloma and is widely distributed in Asian countries, ina, Japan, and Korea. As a traditional edible fungus countries, it has been consumed as a vegetable and raditional Chinese medicine in single and compound- ptions for the prevention and treatment of diseases thousand years [5]. As an extract from T. matsutake, ides (TMP) have showed strongly bioactive properties tioxidant and anti-tumor [6]. Thus the determination ding author at: Key Laboratory of Biological Resource and Ecological f the Ministry of Education, College of Life Sciences, Sichuan Univer- g Road 29#, Chengdu 610064, PR China. Tel.: +86 28 85460487; 460487. ress: biostart8083@yahoo.cn (J. Zhao). ors contributed equally to this research. 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 fromSigma–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. see front matter © 2010 Elsevier B.V. All rights reserved. ijbiomac.2010.04.010icholoma matsutake nga,1, Jie Tanga,1, Mei Caob,1, Chun-xiao Guoa, Xia a, Su Fenga, Zhi-rong Yanga, Jian Zhaoa,b,∗ y of Biological Resource and Ecological Environment of the Ministry of Education, Colle ry of Sichuan Academy of Medical Sciences, Sichuan Provincial People’s Hospital, Cheng e i n f o arch 2010 vised form 17 April 2010 pril 2010 e 27 April 2010 structure say tsutake a b s t r a c t In this study, structural features of by a combination of infrared (IR) sp magnetic resonance (NMR) spectrosc d-glucopyranose residue which branc mainly composed of an (1→3)--d- residue. The antioxidant activity of T ing DPPH− radical scavenging, hydrog results indicated that TMP-A showed s TMP-A could attenuate PC12 cell dam/ locate / i jb iomac a novel polysaccharide isolated nga, Jing Zhonga, Jie Zhanga, ife Sciences, Sichuan University, Chengdu 610064, PR China 0072, PR China loma matsutake polysaccharide (TMP-A) were investigated gas chromatography–mass spectrometry (GC–MS), nuclear The results indicated that TMP-A had a backbone of 1,4-- t O-6 based on the experimental results. The branches were topyranose residue, and terminated with -d-xylopyranose was evaluated with several biochemical methods, includ- roxide scavenging, superoxide anion radical scavenging. The antioxidant. In the in vitro antioxidant assay byMTTmethod, ignificantly caused by hydrogen peroxide. © 2010 Elsevier B.V. All rights reserved. 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 (200g) of T. matsutake were soaked with 95% E boiling wat concentrate supernatan crude polys (Staub [7])w to a DEAE-c and eluted s eluate was 0M NaCl e a Sephadex sutake poly processes a starting ma 2.3. Measur composition High pe was carried bratedwith T-10). The software). T 2M trifluor nism of aci by co-distil sis was com for thin lay ously. Deve solution (8: system (85 diphenylam dissolved in tiated by ad chloro-silic Dong [11,12 at a tempe [13]. 2.4. Methyl The pol methyl iod After comp was depoly at 100 ◦C in using the m 2.5. UV and TMP-A w analysis of polysacchar were run in 2.6. Nuclea 1H NMR ian Unity IN 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 ing t aven ging he a is th ontro were aven abil ined rcen s fol /A co l gro e abs ance study eterm erox the eroxi effe ance and use ell li 2 ce ined eat-i tics midi ntio his s trati m. Af H2O awn ther oluti 0.5m ed to de (D ount reme AD) e inh bsor ance s the tatis data eplictOH, the residue was dried and then extracted with er for three times (6h for each). After the filtrate was d, dialyzed (MWCO 5000, Sigma), and centrifuged, the t was added with 3 volumes of 95% EtOH to precipitate accharides (32.8 g, recovery 16.4%). After Sevag method asused for thedeproteination, TMP(8g)was subjected ellulose column (Tris–HCl, pH 7.0, 4.5 cm×50 cm, Cl−) tepwise with 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 1.0M NaCl. The monitored by the phenol-sulfuric acid method [8]. The luation was concentrated, lyophilized and purified on G-100 column (2.6 cm×60 cm). The resulting T. mat- saccharide, named TMP-A, was obtained by the above nd the yield rate of TMP-A was 0.22% (0.432g) for the terial. ement of molecular weight and monosaccharide analysis of TMP-A rformance gel permeation chromatography (HPGPC) out tomeasuremolecularweight. The columnwas cali- standardT-seriesDextran (T-500, T-110, T-70, T-40and data were processed with Waters GPC (Millennium32 hepolysaccharideTMP-A (5.0mg)washydrolyzedwith oacetic acid (TFA) at 110 ◦C for 6h on the mecha- d-catalyzed hydrolysis [9]. Excess acid was removed lation with methyl alcohol (MeOH) after the hydroly- pleted. One part of the hydrolysate (1.0mg) was used er chromatography (TLC) analysis as described previ- loping solvent: acetoacetate–pyridine–ethanol–water 5:1.5:1); the developer system: diphenylamine–aniline % phosphoric acid solution 140mL containing 8mL ine, 8 g aniline) [10], and the other (1.0mg) was pyridine (0.2mL). The derivatization reaction was ini- dition of hexamethyl-disilazane (0.2mL) and trimethyl ane (0.2mL) according to the method described by ]. The resulting supernatant was examined by GC–MS rature program of 50–230 ◦C with a rate of 2 ◦C/min ation analysis ysaccharide, TMP-A (10mg), was methylated using ide (MeI) according to the Hakomori method [14]. lete methylation, the permethylated polysaccharide merized with 90% aqueous formic acid (3mL) for 10h a sealed tube. The methylated sugars were derivatized ethod described and analyzed by GC–MS. infrared (IR) spectra analysis as tested in UV from 200 to 600nm and infrared the samples was obtained by grinding a mixture of ide with dry KBr and then pressing in a mold. Spectra the 4000–400 cm−1 region. r magnetic resonance (NMR) experiment spectra and 13C NMR spectra were recorded on a Var- OVA 400/45 in D2O with tetramethylsilane as internal accord age sc scaven trol is t sample itive c (BHT) 2.8. Sc The determ The pe lated a blank) contro was th absorb in the 2.9. D Sup ing to of sup enging absorb system Vc was 2.10. C PC1 mainta 10% h antibio in a hu 2.11. A In t concen Mediu before withdr for ano stock s tion of aspirat sulfoxi the am measu (Bio-R damag is the a absorb and A i 2.12. S All three ro themethod described by Braca et al. [15]. The percent- ging activity was calculated by the following formula: effect (%) = (1−A sample/A control)×100,whereA con- bsorbance of control (DPPH solution without sample), A e test sample (DPPH solution plus test sample or pos- l) [16]. Vitamin C (Vc) and butylated hydroxytoluene used as a positive control in the study. ging effect on hydroxyl radicals ity of the TMP-A to scavenge hydrogen peroxide was according to the method of Smirnoff and Cumbes [17]. tage of scavenging of hydrogen radicals was calcu- lows: scavenging effect (%) = [1− (A sample−A sample ntrol]×100, where A control was the absorbance of the up in the hydroxyl radicals generation system, A sample orbance of the test group and A sample blank was the of the samples only. Vc was used as a positive control . ination of superoxide anion scavenging activity ide anion scavenging activity was measured accord- pyrogallol’s autoxidation method [18]. The inhibition de anion production was calculated as follows: scav- ct (%) = (A−B)/A×100, where A is the change speed of of the control group in the superoxide anion generation B is the change speed of absorbance of the test sample. d as a positive control in the study. nes and culture lls (ATCC, American Type Culture Collection, USA) were in Dulbcco’s Modified Eagle Medium, which contained nactivated horse serum, 5% fetal bovine serum and (100U/mL penicillin, 100mg/mL streptomycin) at 37 ◦C fied atmosphere containing 5% CO2. xidant activity assay tudy, PC12 cells were seeded into 96-well plates at the on of 5×104 cells/mL using Dulbcco’s Modified Eagle ter 24h, PC12 cells were pretreated with TMP-A for 2h 2 (300mMsolution) exposure for1h.After theH2O2 was , cells were then further incubated in the fresh medium 6h at 37 ◦C. Then methyl thiazolyl tetrazolium (MTT) on was added to each well reaching a final concentra- g/mL. After incubating for 4h, the supernatants were remove untransformed MTT. Finally, 150L dimethyl MSO) was added to dissolve the formazan crystals and of purple formazan was determined by the absorbance nt at 570nm using the Universal Microplate Reader [19]. The damage inhibitory effect was expressed as: ibitory effect (%) = [(As −A)]/[(A0 −A)]×100% where As bance in the presence of the sample and H2O2, A0 is the of the control in the absence of the sample and H2O2, absorbance only in the presence of the H2O2. tical analysis were presented as means± standard deviation (SD) of ations. Statistical analyses were performed using Stu- X. Ding et al. / International Journal of Biological Macromolecules 47 (2010) 271–275 273 dent’s t-tes were consid 3. Results 3.1. Extract The cru the fruiting fractionatio chromatogr eluate and geneity of th TMP-Awas G-100 colum as a single tion spectra nucleic acid rotation: [ tion of etha room temp 8.89×104 D d-xylose (d hydrolysate with the TL posed to co GC–MS ana 3.2. Structu The inte (Fig. 1) was charide and 2923.62 cm in the regio Two strong range of 12 monosacch tion at 875 linkages, w ı4.570 in th tic absorpti in the polys anomeric p (400MHz) nances in t spectrum o of -d-gluc and -d-xy signals at ı1 chem residu -d-Gl --d-G -d-Ga ylp-(1 results of methylation analysis of TMP-A. lated sugar Linkage m/z e3-Glc 1,4- 45,59,73,88,101, 133,146,159,232 e2-Glc 1,4,6- 45,59,73,88,101,116,133,146,174,232 e3-Gal 1,3- 45,59,73,89,116,146,159,191,204,233 e3-Xyl T- 45,59,73,88,101,116,133,146,174 to C-1 of →4,6)--d-Glcp-(1→; ı100.7 to C-1 of →3)--d- →; ı104.1 to C-1 of .-d-Xylp-(1→, respectively (Fig. 2). All ignment of the carbon atoms signals was shown in Table 1. r methylation according to the Hakomori method for four themethylated polysaccharidewas depolymerized and con- into partially methylated ramifications. The analysis of thylated monosaccharide was conducted by GC–MS. The ation in MS showed that fragment ion peaks were consis- ith the data of d-configuration monosaccharide fragment eaks which can be concluded that the glucose, galactose lose residues were d configuration. Methylation analysis P-A proved that the -d-glucopyranose residues were 2,3- stituted and 2,3,6-trisubsituted, the -d-galactopyranose es were 2,4,6-trisubsituted, and the -d-xylopyranose e was 2,3,4-trisubsituted (Table 2). Results methylatedFig. 1. FTIR spectra of polysaccharide TMP-A. t and one-way analysis of variance. Values of P<0.05 ered to be a statistically significant finding. and discussion ion, purity and composition of polysaccharides de polysaccharide, named TMP, was obtained from bodies of T. matsutake with a yield of 16.4%. After n on DEAE-cellulose 52 and Sephadex G-100 column aphy, 324mgof TMP-Awas obtained from the 0MNaCl detected by the phenol-sulfuric acid assay. The homo- e polysaccharidewas elucidated by the following tests. eluted fromgel-filtration chromatographyonSephadex n and was detected by the phenol-sulfuric acid assay peak. No absorption at 280 and 260nm in UV absorp- of TMP-A demonstrated the absence of protein and in this polysaccharide and it had the same optical ]20D −1.648◦ (c0.5, water) in different low concentra- nol using HK7-SGW-1 automatic optical polarimeter at erature. Weight-average molecular weight was around a. The three monosaccharides, d-glucose, d-galactose, -Glc, d-Gal, and d-Xyl) were also identified using the of TMP-A by GC–MS which was in good agreement C with the ratios of 79.37:9.81:10.82.TMP-A was sup- ntain the d-configuration monosaccharide according to lysis. re elucidation of TMP-A nsity of bands around 3408.22 cm−1 in the IR spectrum due to the hydroxyl stretching vibration of the polysac- as expected theywere broad. The bands in the region of −1 were due to C–H stretching vibration, and the bands n of 1643.29 cm−1 were due to associated water [20]. absorption bands at 1075.03 cm−1, 1041.64 cm−1 in the 00–1000 cm−1 in the IR spectrum suggested that the aride in TMP-A had a pyranose-ring [21]. The absorp- Table 1 13C NMR Sugar →4)- →4,6) →3)- -d-X Table 2 GC–MS Methy 2,3,6-M 2,3-M 2,4,6-M 2,3,4-M ı105.4 Galp-(1 the ass Afte times, verted the me inform tent w ions p and xy for TM bis-sub residu residu.43 cm−1 indicated that TMP-A had -glucopyranose hich was indicated by the anomeric proton signals at e 1H NMR (400MHz) [22]. Moreover, the characteris- ons at 799.73 cm−1 indicated -configurations existing accharide [23], which was in good agreement with the roton signals at ı5.182, ı5.107, ı5.060 in the 1H NMR spectrum. According to the literature [24], the reso- he region of 98–106ppm in the 13C NMR (200MHz) f TMP-A were attributed to the anomeric carbon atoms opyranose (-d-Glcp), -d-galactopyranose (-d-Galp) lopyranose (-d-Xylp). In the anomeric carbon region, 05.2 could be attributed to C-1 of →4)--d-Glcp-(1→; linkage an glucopyran polysaccha linked -- glucopyran The relative cating that namely on backbone. R galactopyra It is conclu (1→4)--dFig. 2. The 13C NMR spectra of polysaccharide TMP-A. ical shift data (ı, ppm) for polysaccharide TMP-A. es Chemical shifts, ı (ppm) C1 C2 C3 C4 C5 C6 cp-(1→ 105.239 69.370 78.160 72.941 78.750 63.636 lcp-(1→ 105.423 69.370 78.426 72.121 79.598 63.636 lp-(1→ 100.762 70.820 77.388 75.439 80.717 63.258 → 104.194 71.047 75.638 74.305 72.121alysis of TMP-A indicated that (1→4)-linked--d- ose was one of the largest amounts residue of the ride structure, the branched residue was (1→4,6)- d-glucopyranose revealing that (1→4)-linked--d- ose should be possible to form the backbone structure. amounts of (1→4,6)-linked--d-glucopyranose indi- approximate branch ratios could theoretically be 12.5%, average one branching point for each eight residues of esidues of branch structure were (1→3)-linked--d- nose and terminated with -d-xylopyranose residue. ded that a repeating unit of TMP-A has a backbone of -glucopyranose residues which branches at O-6 based 274 X. Ding et al. / International Journal of Biological Macromolecules 47 (2010) 271–275 Fig. Fig. 4. DPP on the expe compositio terminated ture of the n 3.3. Determ DPPH is enging acti there is a di idants. Fig. polysacchar that the IC about 3.0m effect on sca tity. Howev Vc. 3.4. Scaven Hydroxy Thus, hydro living syste enging effec and IC50 va trations, TM a concentra cation poly effects than 3.5. Determ Fig. 6 illu 1, 2, 3, 4, an Vc. At all th varying deg ydroxyl radical scavenging effect of TMP-A from Tricholoma matsutake. uperoxide radical scavenging effect of TMP-A from Tricholoma matsutake.3. Predicted chemical structure of polysaccharide TMP-A. H− radical scavenging effect of TMP-A from Tricholoma matsutake. rimental results. The branch was supposed to be the n of an (1→3)--d-galactopyranose residue and one with -d-xylopyranose residue. The predicted struc- ovel polysaccharide TMP-A was shown in Fig. 3. ination of DPPH radical scavenging activity of TMP-A a useful reagent for investigating the free radical scav- vities of various samples. It is noticeable by eye that scolouration from purple to yellow induced by antiox- Fig. 5. H Fig. 6. S4 illustrates the scavenging activity of the purified ide samples on the DPPH radical. These results showed 50 value of TMP-A for eliminating DPPH radicals was g/mL, which indicated that TMP-A have a noticeable vengingDPPH radical, especially at high additionquan- er, the inhibiting ability was lower than that of BHT and ging effect of hydroxyl radical by TMP-A l radical can easily cause tissue damage or cell death. xyl radical removing is important for the protection of ms. Fig. 5 shows the percentage hydroxyl radical scav- ts of TMP-A at the dose of 0.5, 1, 2, 3, 4, 5 and 10mg/mL lue of TMP-A was about 7.1mg/mL. At the test concen- P-A exhibited scavenging effect on hydroxyl radicals in tion-dependent manner which showed that the purifi- saccharides had weaker hydroxyl radical scavenging Vc of same dose. ination of superoxide anion scavenging effect strates the superoxide radical scavenging effect of 0.5, d 5mg/mL of TMP-A in comparison to the same doses of e concentrations, the polysaccharide samples exhibited rees of antioxidant effect and IC50 value of TMP-A was Fig. 7. TM about 3.6m rides had w same dose. 3.6. Antioxi In MTT A on PC12 After pretr PC12 cell c a dose dep 35.4%, 76.1P-A attenuated PC12 cell damage induced by h