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Structural elucidation and antioxidant activity of a novel polysaccharide (TAPB1) from Tremella aurantialba
Food Hydrocolloids 43 (2015) 459e464
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Structural elucidation and antioxidant activity of a novel polysaccharide (TAPB1) from Tremella aurantialba Xiuju Du a, Yang Zhang a, Hongmei Mu c, Zhiwei Lv a, Yan Yang b, Jingsong Zhang b, * a
College of Life Science, Liaocheng University, Liaocheng, Shandong 252059, PR China Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201106, PR China c College of Agricultural Science, Liaocheng University, Liaocheng, Shandong 252059, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 May 2014 Accepted 4 July 2014 Available online 30 July 2014
A novel water-soluble acidic heteropolysaccharide (designated as TAPB1), with a mean molecular weight of 7.6 105 Da, was obtained from fruiting bodies of Tremella aurantialba. The heteropolysaccharide had a carbohydrate content of ~97.6% and a negative response to the Bradford test. Monosaccharide composition analysis showed that TAPB1 comprised D-mannose, D-xylose, and D-glucuronic acid in the ratio 3.1:2.9:1.2, with trace amounts of D-galactose, D-glucose, and D-galacturonic acid. Methylation data and nuclear magnetic resonance spectral analysis indicated that TAPB1 contained an a-(1 / 3)-linked mannopyranosyl backbone, partially substituted at the O-4 positions with a side chain composed of two xyloses and at the O-2 position with a side chain comprising xylose and glucuronic acid. Antioxidant assays showed that TAPB1 exerted a significant scavenging effect on superoxide radicals and H2O2 in a dose-dependent manner. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Tremella aurantialba polysaccharide Chemical structure Methylation NMR Antioxidant activity
1. Introduction Polysaccharides, which widely exist in microorganisms, plants, and animals, possess a variety of biological activities, such as immunostimulating, antitumor, antioxidant, antihyperlipidemic, and antidiabetic activities (Schepetkin & Quinn, 2006; Zhang, Cui, Cheung, & Wang, 2007). Previously, the presence of polysaccharides in fungi has been reported, and such polysaccharides exhibit strong antioxidant effects that are related to their healthprotecting functions (Tsai, Song, Shih, & Yen, 2007; Tseng, Yang, & Mau, 2008). Moreover, most polysachcharides derived from fungi are comparatively nontoxic and do not cause significant side effects (Schepetkin & Quinn, 2006). Therefore, searching for fungal polysaccharides with antioxidant activity has become the focus of research. Tremella aurantialba, known as Jin'er in China, has been used as food and folk medicine for many years. Polysaccharides from this fungus reportedly exhibit numerous potent biological and pharmacological functions, including immunostimulating (Li, Yang, & Zhu, 2000; Du, et al., 2009; Du, Zhang, Yang, et al., 2010), antidiabetic (Qu et al., 1998; Kiho et al., 2001), antihyperlipidemic (Wang,
* Corresponding author. Tel./fax: þ86 21 62200754. E-mail address: [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.foodhyd.2014.07.004 0268-005X/© 2014 Elsevier Ltd. All rights reserved.
Qu, Chu, Li, & Tian, 2002; Zhang, Qu, & Zhang, 2004), antitumor (Yuan, Zhang, & Chen, 1996), and antioxidant (Du, Zhang, Liu, et al., 2010; Deng & Qu, 2007) functions. Polysaccharide structure should be determined to further investigate the relationship between the chemical structures and biological activities of polysaccharides in this fungus. In our previous work, the structure and immunostimulating activity of a polysaccharide fraction, termed TAPA1, has been reported (Du et al., 2009). In the present study, another polysaccharide, named TAPB1, was isolated from the fruiting bodies of T. aurantialba, and the chemical structure of TAPB1 was investigated. Moreover, the in vitro antioxidant activities of TAPB1 on superoxide anion radicals and hydrogen peroxide (H2O2) were preliminarily investigated. 2. Materials and methods 2.1. Materials and reagents The fruiting bodies of T. aurantialba Bandoni and Zang were provided by Kunming Edible Fungi Institute of General National Supply and Marketing Cooperative, China. Dextrans and monosaccharide standards, namely, D-Gal, D-Glc, D-Ara, L-Fuc, L-Rha, DFru, D-Man, D-Xyl, D-GlcA, and D-GalA, were from SigmaeAldrich (USA). DEAE-Sepharose Fast Flow and High-Resolution Sephacryl S500 were purchased from Amersham Pharmacia Company. All
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other reagents were from Chinese sources and were of analytical grade. 2.2. General methods Fourier transform infrared (FT-IR) spectroscopy of TAPB1 mixed with dry KBr was performed in the 4000 cm1 to 400 cm1 region (Nexus Euro FT-IR instrument). Total content of polysaccharides was determined by phenolesulfuric acid method (Dubois Gillis, Hamilton, Rebers, & Smith, 1956). The concentration of proteins was measured according to Bradford's method (Bradford, 1976).
adjusted to pH 4.8 with 0.04 M hydrochloric acid (HCl), following the addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (20 mg), the stirred reaction mixture was kept at pH 4.8 over 1 h. 2 mL of sodium (2 M) borodeuteride (NaBH4) was added up to the solutions, and the reaction mixture was maintained for 1.5 h at 50 C. Then the pH was adjusted to 5.0 with HCl. The solution was dialyzed for 48 h (10 kDa MWCO membrane) and freeze-dried. The reduction process of carboxyl was repeated three times to guarantee the complete reduction of all uronic acid units. Consequently, the carboxyl-reduced product TAPB1-R was obtained. 2.7. Methylation analysis
2.3. Preparation of the homogeneous polysaccharide TAPB1 T. aurantialba fruiting bodies were powdered and extracted twice with ethanol to remove the lipid material. The residue was air dried and extracted thrice with boiling water. The combined aqueous extracts were separated into four fractions based on their molecular weights by ultrafiltration using hollow-fiber membranes of different pore sizes. The >500 kDa fraction, designated as TAP50w, was fractionated over a DEAE-Sepharose Fast Flow column (XK26 100 cm) by initially eluting with water and then with a NaCl gradient (0 Me2.0 M). Five fractions (TAPA, TAPB, TAPC, TAPD, and TAPE) were monitored for carbohydrate content by phenol sulfuric acid reaction (Dubois et al., 1956). Fraction TAPB was further purified by gel-permeation chromatography on HighResolution Sephacryl S-500 column (XK16 100 cm) eluted with 0.2 M NaNO3 at a flow rate of 0.5 mL min1. Two polysaccharide peaks were detected using a refractive index detector (RID-10A, Shimadzu, Japan), and the fraction forming the symmetrical second peak was collected and designated as TAPB1. 2.4. Monosaccharide analysis Monosaccharide components and percentage composition were determined using high-performance anion-exchange pulsedamperometric detection chromatography (HPAECePAD), as reported in our previous study (Du et al., 2009). TAPB1 (2 mg) was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 110 C for 5 h. A Dionex LC30 was equipped with a CarboPac™ PA20 column (3 mm 150 mm), and the monosaccharides were monitored using a pulsed amperometric detector (Dionex). The column was eluted with 2 mM NaOH (0.45 mL min1). D-Gal, D-Glc, D-Ara, L-Fuc, L-Rha, D-Fru, D-Man, D-Xyl, D-GlcA, Rib, and D-GalA were used as standards. The absolute configurations of the monosaccharides were determined using a procedure described by Gerwig (Gerwig, Kamerling, & Vliegenthart, 1979). 2.5. Determination of homogeneity and molecular mass The homogeneity and molecular mass of TAPB1 were determined by gel-permeation chromatography (GPC) on a HighResolution Sephacryl S-500 column (XK16 100 cm), using dextran T-80, 150, 270, 410, 670 and 2000 as references. The column temperature was kept at 30 C and the signals were detected using a refractive index detector (RID). The mobile phase consisted of 0.2 M NaNO3, and the sample was prepared as 0.2% (w/v). 30 mL of sample solutions was injected into the machine and eluted at 0.5 mL min1. 2.6. Carboxyl group reduction The reduction of the carboxyl group was performed according to the method of Liu et al. (Liu et al., 2008) with minor modification. Briefly, TAPB1 (10 mg) dissolved in distilled water (2 mL) was
TAPB1 was insoluble in Me2SO, which was in agreement with the Me2SO insolubility of polysaccharide TAPA1 from T. aurantialba fruiting bodies in our previous work. Vacuum-dried TAPB1 (4 mg) was initially acetylated with a mixture of Ac2O and pyridine, and subsequently dialyzed, and lyophilized to yield TAPB1-ac. The samples (TAPB1-ac and TAPB1-R) were methylated according to the method used in our previous report (Du et al., 2009). The disappearance of the OH band (3200 cm1 to 3700 cm1) in the FT-IR spectrum was used to confirm complete methylation. The permethylated polysaccharides were hydrolyzed by treatment with HCO2H (88%), distilled water, and TFA in the ratio 3:1:2 for 5 h at 100 C. Subsequently, partially methylated sugars in the hydrolysate were reduced with NaBH4 and acetylated with Ac2O, and the partially methylated alditol acetates (PMAAs) were analyzed by gas chromatographyemass spectrometry(GCeMS) as described by Ye et al. (Ye, et al., 2008). 2.8. Nuclear magnetic resonance (NMR) spectroscopy The freeze-dried TAPB1 (30 mg) was maintained over P2O5 in a vacuum for several days and deuterium exchange was performed thrice with 0.5 mL D2O. 1H NMR, 13C NMR, correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear overhauser enhancement spectroscopy (NOESY), heteronuclear multipleequantum correlation spectroscopy (HMQC), and heteronuclear multiple bond correlation (HMBC) spectra were recorded in D2O at 500 MHz (for 1H NMR) or 125 MHz (for 13C NMR) using a Bruker Avance 500 spectrometer. 13C chemical shifts were acquired in relation to 2,2-dimethyl-2-silapentane-5-sulfonic acid (d 0.00 ppm) calibrated externally, and semi-heavy water (d 4.32 ppm) was used as the internal reference signal for 1H at 70 C. 2.9. Antioxidant activity assay 2.9.1. Preparation of tested samples TAPB1 (5 mg) was initially dissolved in 1 mL of 0.05 M phosphate-buffered saline (PBS; pH 7.8). After centrifugation at 400 g for 30 min, the suspension was further diluted with PBS buffer at concentrations of 0.1, 0.5, 1.0, 1.5, 2.0, and 2.5 mg mL1. 2.9.2. Superoxide anion-scavenging activity assay The superoxide anion radical assay of TAPB1 was performed according to the pyrogallolechemiluminescence assay as reported in our previously study (Du, Mu, Zhou, Zhang, & Zhu, 2013). Briefly, different test agents at 2 mL and pyrogallic acid (0.625 mM) at 8 mL were added to the wells of a 96-well microplate. PBS and ascorbic acid were served as the negative and positive controls, respectively. Luminolesodium carbonate buffer solution mixture at 150 mL (pH 10.2) was pumped into the wells of a 96-well microplate. The signal from each well was monitored and recorded every 0.6 s and maintained for 30 s. The scavenging effect for superoxide anion radical was calculated according to the following formula:
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Fig. 1. Spectrum of HPAEC-PAD for standard monosaccharides and TAPB1 (a) Spectrum of HPAEC-PAD for 11 standard monosaccharides (1. L-Fucose; 2. L-Rhamnose; 3. D-Arabinose; 4. D-Galactose; 5. D-Glucose; 6. D-Xylose; 7. D-Mannose; 8. D-Fructose; 9. Ribose; 10. D-GalA; 11. D-GlcA); (b) Spectrum of HPAEC for TAPB1(1. D-Xylose; 2. D-Mannose; 3. D-GlcA).
Scavenging rate (％) ¼ (A0-A1)/A0 100, in which A0 was the luminescence value of the negative control without tested sample. A1 was the luminescence values of tested sample. Each sample was repeated 3 times. 2.9.3. H2O2-scavenging activity assay The H2O2-scavenging activities were determined according to the chemiluminescence method used in our previous work (Du et al., 2013). H2O2 (1%) at 10 mL and different test agents at 40 mL were added to the wells of a 96-well microplate. Double-distilled water and ascorbic acid were used as the negative and positive controls, respectively. Each sample was repeated 3 times. The scavenging rate for H2O2 radical were performed according to the method in superoxide anion-scavenging activity assay (Section 2.9.2). 2.9.4. Statistical analysis The data obtained were expressed as mean ± SD of three determinations and were analyzed statistically by ANOVA. The significance of any differences between groups was evaluated using the Student's t-test. All computations were performed using statistical software. 3. Results and discussion 3.1. Structural analysis A novel water-soluble polysaccharide from T. aurantialba fruiting bodies, designated as TAPB1, was purified by ultrafiltration and a series of chromatographic steps. TAPB1 showed a negative response to the Bradford test (Bradford, 1976) and showed no absorption at 280 or 260 nm in the UV scanning spectrum, indicating the absence of proteins and nucleic acids. TAPB1 appeared as a single symmetrical peak on Sephacryl S-500 high-resolution chromatography, indicating that TAPB1 was a homogeneous polysaccharide. TAPB1 had an estimated molecular weight of
7.6 105 Da according to GPC results, and had a carbohydrate content of 97.6%, as measured by the phenolesulfuric acid method (Dubois et al., 1956). Monosaccharide composition analysis by HPAECePAD showed that TAPB1 comprised D-mannose, D-xylose, and D-glucuronic acid in a ratio of 3.1:2.9:1.2, along with trace amounts of D-galactose, D-glucose, and D-galacturonic acid (Fig. 1). Absolute configuration analysis showed that the mannose, xylose, and glucuronic acid residues all had the D configuration (Gerwig et al., 1979). The FT-IR spectrum of TAPB1 (Fig. 2) displayed an absorption peak at 1733.1 cm1, which is a characteristic of the C]O stretching vibration, suggesting that TAPB1 may be an acidic polysaccharide and/or may contain O-acetyl groups. The strong broad absorption peak at 3423.1 cm1 was due to the hydroxyl stretching vibration of the polysaccharide, and the peak at 2923.6 cm1 was due to the CeH stretching vibration band (Du et al., 2009). Methylation analysis of polysaccharides was performed to investigate the inter-glycosidic linkages between monosaccharide residues. The fully methylated products were hydrolyzed, converted into PMAAs, and analyzed by GCeMS. As shown in Table 1, we detected 1-substituted, 1,2-disubstituted and 1,3-disubstituted xylopyranose residues and 1,3-disubstituted, 1,2,3-trisubstituted, and 1,3,4-trisubstituted mannopyranose residues by methylation analysis of TAPB1. GCeMS analysis of reduced T. auriantalba polysaccharide (TAPB1-R) revealed one additional peak that was consistent with that of 2,3,4,6-tri-O-Me4-glucitol, indicating the presence of 1-substituted GlcA residues in TAPB1. The 1H NMR (500 MHz) spectrum of TAPB1 (Fig. 3) at 70 C mainly contained signals for five anomeric protons at d 4.45 to d 5.25. One signal at d 2.02 corresponded to the 1H signal for the CH3 moiety of an acetyl group (Serrato et al., 2008). Other sugar protons were in the region of d 4.38 to d 3.37. The results of 1H NMR (Fig. 3), 13 C NMR(125 MHz, Fig. 4), and 13Ce1H HMQC (Fig. 5) analyses indicated that the repeating unit of the polysaccharide comprised seven sugar residues, which were designated as AeG in the 1H NMR spectrum according to the decreasing chemical shifts of the anomeric protons.
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Fig. 2. The FT-IR spectroscopy of TAPB1.
The 13C NMR (125 MHz, Fig. 4) results, which was supported by the both spectra 1H NMR and 13Ce1H HMQC (Fig. 5), showed seven anomeric signals at d 105.94, 105.57, 104.95, 104.70, 104.29, 103.72, and 102.65, which were designated as D, G, B, F, C, E, and A, respectively. Furthermore, downfield signals at d 175.19 and 179.25 in the 13C NMR spectrum revealed two carboxyl groups, and a signal at d 23.79 upfield revealed a CH3 moiety of an acetyl group (Serrato et al., 2008). The identities of the monosaccharide residues AeG were established based on 2D-NMR analysis involving 1He1HeCOSY, TOCSY, NOESY, 1He13C heteronuclear singleequantum correlation (HSQC), and HMBC experiments, which were used to assign the chemical shifts and anomeric configurations of the seven sugar residues present in the repeating unit.
Table 1 Results of the methylation analysis of TAPB1 and TAPB1-R by GCeMS. Glycosyl residue
Methylated sugar (as alditol acetate)
Mode of linkage
Molar ratiosa TAPB1
TAPB1-R
Man
4,6-Me2 2,6-Me2 2,4,6-Me3 2,3,4-Me3 3,4-Me2 2,4-Me2 2,3,4,6-Me4
/2,3[Manp]1/ /3,4[Manp]1/ /3 [Manp]1/ [Xylp]1/ /2[Xylp]1/ /3[Xylp]1/ [Glcp]1/
1.01 0.92 0.89 0.96 0.93 0.96 trace
0.93 0.98 0.97 0.83 1.02 1.04 0.82
Xyl
Glc a
The molar ratios of samples were calculated on basis of peak area of PMAAs.
Fig. 3. 1H NMR (500 MHz) spectrum determined in D2O at 70 C of T. aurantialba polysaccharide TAPB1: the anomeric protons are labeled A-G.
Residues A, B, and C had anomeric chemical shifts at d 5.25, 5.25, and 5.10, respectively. The 1H resonances for H1eH6 in Residue A were assigned from the cross peaks in the COSY, TOCSY, and NOESY spectra (Table 2). The carbon signals from C-1 to C-6 of residue A were identified from the HSQC spectrum (Table 2). The relatively small coupling constant values of JH-1,H-2 and JH-2,H-3 (<2 Hz) and the large coupling constant values of JH-3,H-4 (>8 Hz), JH-4,H-5 (>8 Hz), and JC-1,H-1 (~166 Hz) showed that residue A was a-Dmannose (Mondal, Chakraborty, Rout, & Islam, 2006; Rout, Mondal, Chakraborty, & Islam, 2006; Yang et al., 2007). The linkage positions were determined from the high carbon chemical shift values (Yu & Yang, 2002, pp. 901e906; Zhang, 1999, pp. 245e252). Therefore, the downfield chemical shifts of the C-1 (d 102.65), C-2 (d 75.81), and C-3 (d 75.58) carbon signals indicated that residue A was a (1 / 2,3)-a-D-mannopyranose. In addition, residues B and C were identified as (1 / 3,4)-a-D-mannopyranose and (1 / 3)-a-Dmannopyranose, respectively. Residues D, E, and F had anomeric chemical shifts at d 4.69, 4.69, and 4.54, respectively. Protons chemical shifts from H-2 to H-4 of residues D were assigned from COSY, TOCSY, and NOESY spectra. The large coupling constant values JH-1,H-2 (>7.8 Hz) and JH-1,C-1 (~160 Hz) indicated that these were all b-linked residues (Du et al., 2009; Ghosh et al., 2008), and this finding was confirmed by the intra-residue NOESY signals between H-1 and H-3, and H-1 and H5. H-5a and H-5b were assigned from the TOCSY and NOESY spectra, and their carbon signals from C-1 to C-5 were assigned from the HMBC and HSQC spectra (Table 2). The downfield chemical shifts of each residue indicated substituted positions (Yu & Yang, 2002, pp. 901e906; Zhang, 1999, pp. 245e252). Therefore, the residues D, E, and F were designated as follows: D, (1 / 3)-b-D-
Fig. 4. 13C NMR (125 MHz) spectra determined in D2O at 70 C of T. aurantialba polysaccharide TAPB1: the anomeric protons are labeled A-G.
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463
(I)
Confirmation of Sequence I was obtained by the inter-residue proton cross peaks observed in the NOESY spectrum (data not shown). The following inter-residual NOE correlations were observed between H-1 of residue A and H-3 of residue C, between H-1 of residue C and H-3 of residue B, and between H-1 of residue B and H-3 of residue A. HMBC spectral analysis revealed clear correlations between H-1 of residue G and C-3 of residue D, between C-1 of residue G and H-3 of residue D, between H-1 of residue D and C-2 of residue A, and between C-1 of residue D and H-2 of residue A, thereby establishing the following sequence (Sequence II): Fig. 5. 1He13C HSQC spectrum of TAPB1 showing anomeric atom cross-peaks. Determined in D2O at 70 C.
xylopyranose; E, 1/b-D-xylopyranose; and F, (1 / 2)-b-Dxylopyranose. Residue G had the anomeric signal at d 4.45 and a large JH-1, H-2 coupling constant value (~8.6 Hz), indicating that G was a b-linked residue (Du et al., 2009). The H-1 track (d 4.45) of residue G in the TOCSY spectrum showed the complete spin system H-1,2,3,4,5, which is a characteristic of a b-GlcA residue (Dobruchowska, Gerwig, Babuchowski, & Kamerling, 2008; Vinogradov, Petersen, Duus, & Wasser, 2004). Cross peaks between H-1 and H-2 (d 4.45/ 3.55) and between H-2 and H-3 (d 3.55/3.67) were observed in the COSY spectrum. The H-4 (d 3.78) and H-5 (d 3.92) resonances were assigned from the TOCSY and NOESY spectra. 13C resonances were assigned from the HMQC spectrum (Table 2). The downfield shifts of the C-1 (d 105.57) carbon signals indicated that residue G was a 1-linked b-D-glucuronic acid pyranose. The sequence of glycosyl residues in TAPB1 was determined from results of HMBC studies followed by confirmation from NOESY results. The HMBC spectral analysis (data not shown) revealed inter-residue connectivities between H-1 of residue A and C-3 of residue C, between C-1 of residue A and H-3 of residue C, between H-1 of residue C and C-3 of residue B, between C-1 of residue C and H-3 of residue B, between H-1 of residue B and C-3 of residue A, and between C-1 of residue B and H-3 of residue A. Such connectivities indicated that the sequence of A, B, and C residues (Sequence I) was as follows: Table 2 1 H and 13C NMR chemical shift data (d, ppm) for T. aurantialba polysaccharide TAPB1 determined in D2O at 70 C. Residue
A /2,3)-a-D-Manp-(1/ B /3,4)-a-D-Manp-(1/ C /3)-a-D-Manp-(1/ D /3)b-D-Xylp-(1/ E b-D-Xylp-(1/ F /2)-b-D-Xylp-(1/ G b-D-GlcA p-(1/ a
1
H/13C
1
2
3
4
5/5a
5.25a 102.65 5.25 104.95 5.10 104.29 4.69 105.94 4.69 103.72 4.54 104.70 4.45 105.57
4.18 75.81 3.87 72.54 3.87 72.54 4.38 76.05 3.37 76.05 3.49 78.10 3.55 75.23
3.67 76.58 3.99 75.23 3.87 75.81 3.67 86.26 3.41 76.05 3.67 76.20 3.67 76.43
3.61 69.26 3.63 76.20 3.63 69.26 3.76 70.85 3.74 70.25 3.37 67.57 3.78 70.25
3.67 71.67 3.86 73.08 3.67 71.67 3.76 63.04 3.77 63.04 3.82 63.04 3.92 76.10
The figure in bold font indicated the substituted position.
6a/5b 3.78 63.47 3.75 63.47 3.66 63.47 3.72 3.81 3.78
175.19
6b 3.75 3.71 3.62
(II) Sequence II was confirmed by the following inter-residual NOE correlations. The NOESY spectrum indicated the cross peaks between H-1 of residue G and H-3 of residue D and between H-1 of residue D and H-2 of residue A. Cross peaks were observed in the HMBC spectrum between H-1 of residue E and C-2 of residue F, between C-1 of residue E and H-2 of residue F, between H-1 of residue F and C-4 of residue B, and between C-1 of residue F and H-4 of residue B, thereby establishing the following sequence (Sequence III):
(III) Sequence III was also confirmed by the following inter-residual NOE correlations. NOESY spectrum analysis revealed the cross peaks between H-1 of residue E and H-2 of residue F and between H-1 of residue F and H-4 of residue B. 3.2. Antioxidant activity Reactive oxygen species (ROS), such as superoxide anion radicals and hydrogen peroxide (H2O2), are among the main factors associated with the initiation of numerous diseases and degenerative processes during aging (Soares et al., 2009). Ascorbic acid was used as positive control in O 2 -scavenging and H2O2-scavenging assays, and its EC50 values were calculated. The values obtained for ascorbic acid showed the effective concentration at which the antioxidant activity was 50%. As shown in Fig. 6, TAPB1 can scavenge superoxide radicals and H2O2 at the concentration range of 0.1 mg mL1 to 2.5 mg mL1. Scavenging effects of TAPB1 on both ROS increased with increasing concentration. The EC50 values of TAPB1 for superoxide radicals and H2O2 were 1.04 and 1.77 mg mL1, respectively. These values showed the lower antioxidant capacity of TAPB1 compared with ascorbic acid, which had EC50 values of 23.6 and 79.6 mg mL1, respectively. 4. Conclusions TAPB1, isolated and purified from the fruiting bodies of T. aurantialba, was a hepta-polysaccharide with a molecular weight of 7.6 105 Da. Monosaccharide composition analysis showed that TAPB1 comprised D-mannose, D-xylose, and D-glucuronic acid in the ratio 3.1:2.9:1.2, along with trace amounts of D-galactose, DGlucose, and D-galacturonic acid. According to methylation data and NMR analysis, the repeating unit of TAPB1 comprised an a(1 / 3)-linked mannopyranosyl backbone. The backbone was
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Fig. 6. Scavenging effect of the polysaccharide TAPB1 on superoxide radicals and H2O2: (a) scavenging activities to superoxide radicals; (b) scavenging activities to H2O2; data are presented as the mean values (n ¼ 3).
partially substituted at the O-4 positions with a side chain composed of two xyloses and at the O-2 position with a side chain comprising xylose and glucuronic acid. Based on the results of superoxide anion radical scavenging assay and H2O2 test, TAPB1 showed significant and dosedependent scavenging effects on these ROS, suggesting its potential as an effective natural antioxidant. However, further studies are required to establish the mode and mechanisms underlying the antioxidant action of TAPB1 and to investigate the relationships between the chemical structure and the above mentioned antioxidant activities. Acknowledgments This work was supported by Natural Science Foundation of Shandong Province of China (ZR2010CL008) and Doctoral Research Start-up Fundation of Liaocheng University (31805). The authors are deeply grateful to Mrs. Liping Shi for recording the NMR spectra. References Bradford, M. M. (1976). Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248e254. Deng, Y. X., & Qu, W. J. (2007). In vitro antioxidant function of extracellular polysaccharides from Tremella aurantialba. Acta Edulis Fungi, 14(3), 50e52. Dobruchowska, J. M., Gerwig, G. J., Babuchowski, A., & Kamerling, J. P. (2008). Structural studies on exopolysaccharides produced by three different propionibacteria strains. Carbohydrate Research, 343, 726e745. Dubois, M., Gillis, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350e356. Du, X. J., Mu, H. M., Zhou, S., Zhang, Y., & Zhu, X. L. (2013). Chemical analysis and antioxidant activity of polysaccharides extracted from Inonotus obliquus sclerotia. International Journal of Biological Macromolecules, 62, 691e696. Du, X. J., Zhang, J. S., Liu, Y. F., Tang, Q. J., Jia, W., Yang, Y., et al. (2010). The antioxidant activity of various extracts from Tremella aurantialba fruiting bodies and their protective effects on PC12 cells injured by oxidation. Acta Agriculturae Shanghai, 26(2), 49e52. Du, X. J., Zhang, J. S., Yang, Y., Tang, Q. J., Jia, W., & Pan, Y. J. (2010). Purification, chemical modification and immunostimulating activity of polysaccharide from Tremella aurantialba fruiting bodies. Journal of Zhejiang University, 11(16), 437e442. Du, X. J., Zhang, J. S., Yang, Y., Ye, L. B., Tang, Q. J., Jia, W., et al. (2009). Structural elucidation and immuno-stimulating property of an acidic heteropolysaccharide (TAPA1) from Tremella aurantialba. Carbohydrate Research, 344(5), 672e678. Gerwig, G. J., Kamerling, J. P., & Vliegenthart, J. F. G. (1979). Determination of the absolute configuration of monosaccharides in complex carbohydrates by capillary g.l.c. Carbohydrate Research, 77, 1e7. Ghosh, K., Chandra, K., Roy, S. K., Mondal, S., Maiti, D., Das, D., et al. (2008). Structural studies of a methyl galacturonosyl-methoxyxylan isolated from the stem of Lagenaria siceraria (Lau). Carbohydrate Research, 343, 341e349.
Kiho, T., Kochi, M., Usui, S., Hirano, K., Aizawa, K., & Inakuma, T. (2001). Anti-diabetic effect of an acidic polysaccharide (TPA) from Tremella aurantia and its degradation product (TAP-H). Biological & Pharmaceutical Bulletin, 24(12), 1400e1403. Li, X. H., Yang, L. L., & Zhu, S. F. (2000). Effect of Tremella aurantialba on immunnocompetence in mice. Journal of Shanxi Medical University, 31(3), 206e207. Liu, C. H., Li, X. D., Li, Y. H., Feng, Y., Zhou, S., & Wang, F. S. (2008). Structural characterisation and antimutagenic activity of a novel polysaccharide isolated from Sepiella maindroni ink. Food Chemistry, 110, 807e813. Mondal, S., Chakraborty, I., Rout, D., & Islam, S. S. (2006). Isolation and structural elucidation of a water-soluble polysaccharide (PS-I) of a wild edible mushroom, Termitomyces Striatus. Carbohydrate Research, 341, 878e886. Qu, W. J., Zhang, X. F., Tang, S. X., He, X. Y., Zhuang, C. H., & Wu, Z. S. (1998). Pharmacological experiments of efficacies of Tremella aurantialba in relieving cough, reducing phlegm and decreasing blood sugar. Acta Agriculturae Shanghai, 14(1), 58e62. Rout, D., Mondal, S., Chakraborty, I., & Islam, S. S. (2006). The structure of a polysaccharide from Fraction-II of an edible mushroom, Pleurotus florida. Carbohydrate Research, 341, 995e1002. Schepetkin, I. A., & Quinn, M. T. (2006). Botanical polysaccharides: macrophage immunomodulation and therapeutic potential. International Immunopharmacology, 6, 317e333. Serrato, R. V., Sassaki, G. L., Gorin, P.,A. J., Cruz, L. M., Pedrosa, F. O., Choudhury, B., et al. (2008). Structural characterization of an acidic exoheteropolysaccharide produced by the nitrogen-fixing bacterium Burkholderia tropica. Carbohydrate Polymers, 73, 564e572. Soares, A. A., de Souza, C. G. M., Daniel, F. M., Ferrari, G. P., da Costa, S. M. G., & Peralta, R. M. (2009). Antioxidant activity and total phenolic content of Agaricus brasiliensis (Agaricus blazei Murril) in two stages of maturity. Food Chemistry, 112(4), 775e781. Tsai, M. C., Song, T. Y., Shih, P. H., & Yen, G. C. (2007). Antioxidant properties of water-soluble polysaccharides from Antrodia cinnamomea in submerged culture. Food Chemistry, 104(3), 1115e1122. Tseng, Y. H., Yang, J. H., & Mau, J. L. (2008). Antioxidant properties of polysaccharides from Ganoderma tsugae. Food Chemistry, 107(2), 732e738. Vinogradov, E., Petersen, B. O., Duus, J.Ø., & Wasser, S. (2004). The structure of the glucuronoxylomannan produced by culinary-medicinal yellow brain mushroom (Tremella mesenterica Ritz.:Fr., Heterobasidiomycetes) grown as one cell biomass in submerged culture. Carbohydrate Research, 339, 1483e1489. Wang, H., Qu, W., Chu, S., Li, M., & Tian, C. (2002). Studies on the preventive and therapeutic effects of the polysaccharide of Tremella aurantialba mycelia on diet-induced hyperlipidemia in mice. Acta Nutrimenta Sinica, 24, 431e432. Yang, Y., Zhang, J., Liu, Y., Tang, Q., Zhao, Z., & Xia, W. (2007). Structural elucidation of a 3-O-methyl-d-galactose-containing neutral polysaccharide from the fruiting bodies of Phellinus igniarius. Carbohydrate Research, 342, 1063e1070. Ye, L., Zhang, J., Ye, X., Tang, Q., Liu, Y., Gong, C., et al. (2008). Structural elucidation of the polysaccharide moiety of a glycopeptide (GLPCW-II) from Ganoderma lucidum fruiting bodies. Carbohydrate Research, 343, 746e752. Yu, D. Q., & Yang, J. S. (2002). A manual of analytical chemistry VII. Beijing: Chemical Industry Press. Yuan, X., Zhang, P., & Chen, S. (1996). Preliminary study on chemical properties of a soluble polysaccharide from the mushroom Tremella aurantia, and its oncosuppressive activity. Journal of Fudan University (Science Technology), 35, 703e706. Zhang, W. J. (1999). Biochemical research technology on glycoconjugates (2nd ed.). Hangzhou: Zhejiang University Press. Zhang, M., Cui, S., Cheung, P., & Wang, Q. (2007). Antitumor polysaccharides from mushrooms: a review on their isolation process, structural characteristics and antitumor activity. Trends in Food Science & Technology, 18, 4e19. Zhang, W., Qu, W., & Zhang, X. (2004). The anti-hyperglycemic activity of polysaccharides from Tremella aurantialba mycelium. Acta Nutrimenta Sinica, 26(4), 300e303.
Contents lists available at ScienceDirect
Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Structural elucidation and antioxidant activity of a novel polysaccharide (TAPB1) from Tremella aurantialba Xiuju Du a, Yang Zhang a, Hongmei Mu c, Zhiwei Lv a, Yan Yang b, Jingsong Zhang b, * a
College of Life Science, Liaocheng University, Liaocheng, Shandong 252059, PR China Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201106, PR China c College of Agricultural Science, Liaocheng University, Liaocheng, Shandong 252059, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 May 2014 Accepted 4 July 2014 Available online 30 July 2014
A novel water-soluble acidic heteropolysaccharide (designated as TAPB1), with a mean molecular weight of 7.6 105 Da, was obtained from fruiting bodies of Tremella aurantialba. The heteropolysaccharide had a carbohydrate content of ~97.6% and a negative response to the Bradford test. Monosaccharide composition analysis showed that TAPB1 comprised D-mannose, D-xylose, and D-glucuronic acid in the ratio 3.1:2.9:1.2, with trace amounts of D-galactose, D-glucose, and D-galacturonic acid. Methylation data and nuclear magnetic resonance spectral analysis indicated that TAPB1 contained an a-(1 / 3)-linked mannopyranosyl backbone, partially substituted at the O-4 positions with a side chain composed of two xyloses and at the O-2 position with a side chain comprising xylose and glucuronic acid. Antioxidant assays showed that TAPB1 exerted a significant scavenging effect on superoxide radicals and H2O2 in a dose-dependent manner. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Tremella aurantialba polysaccharide Chemical structure Methylation NMR Antioxidant activity
1. Introduction Polysaccharides, which widely exist in microorganisms, plants, and animals, possess a variety of biological activities, such as immunostimulating, antitumor, antioxidant, antihyperlipidemic, and antidiabetic activities (Schepetkin & Quinn, 2006; Zhang, Cui, Cheung, & Wang, 2007). Previously, the presence of polysaccharides in fungi has been reported, and such polysaccharides exhibit strong antioxidant effects that are related to their healthprotecting functions (Tsai, Song, Shih, & Yen, 2007; Tseng, Yang, & Mau, 2008). Moreover, most polysachcharides derived from fungi are comparatively nontoxic and do not cause significant side effects (Schepetkin & Quinn, 2006). Therefore, searching for fungal polysaccharides with antioxidant activity has become the focus of research. Tremella aurantialba, known as Jin'er in China, has been used as food and folk medicine for many years. Polysaccharides from this fungus reportedly exhibit numerous potent biological and pharmacological functions, including immunostimulating (Li, Yang, & Zhu, 2000; Du, et al., 2009; Du, Zhang, Yang, et al., 2010), antidiabetic (Qu et al., 1998; Kiho et al., 2001), antihyperlipidemic (Wang,
* Corresponding author. Tel./fax: þ86 21 62200754. E-mail address: [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.foodhyd.2014.07.004 0268-005X/© 2014 Elsevier Ltd. All rights reserved.
Qu, Chu, Li, & Tian, 2002; Zhang, Qu, & Zhang, 2004), antitumor (Yuan, Zhang, & Chen, 1996), and antioxidant (Du, Zhang, Liu, et al., 2010; Deng & Qu, 2007) functions. Polysaccharide structure should be determined to further investigate the relationship between the chemical structures and biological activities of polysaccharides in this fungus. In our previous work, the structure and immunostimulating activity of a polysaccharide fraction, termed TAPA1, has been reported (Du et al., 2009). In the present study, another polysaccharide, named TAPB1, was isolated from the fruiting bodies of T. aurantialba, and the chemical structure of TAPB1 was investigated. Moreover, the in vitro antioxidant activities of TAPB1 on superoxide anion radicals and hydrogen peroxide (H2O2) were preliminarily investigated. 2. Materials and methods 2.1. Materials and reagents The fruiting bodies of T. aurantialba Bandoni and Zang were provided by Kunming Edible Fungi Institute of General National Supply and Marketing Cooperative, China. Dextrans and monosaccharide standards, namely, D-Gal, D-Glc, D-Ara, L-Fuc, L-Rha, DFru, D-Man, D-Xyl, D-GlcA, and D-GalA, were from SigmaeAldrich (USA). DEAE-Sepharose Fast Flow and High-Resolution Sephacryl S500 were purchased from Amersham Pharmacia Company. All
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other reagents were from Chinese sources and were of analytical grade. 2.2. General methods Fourier transform infrared (FT-IR) spectroscopy of TAPB1 mixed with dry KBr was performed in the 4000 cm1 to 400 cm1 region (Nexus Euro FT-IR instrument). Total content of polysaccharides was determined by phenolesulfuric acid method (Dubois Gillis, Hamilton, Rebers, & Smith, 1956). The concentration of proteins was measured according to Bradford's method (Bradford, 1976).
adjusted to pH 4.8 with 0.04 M hydrochloric acid (HCl), following the addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (20 mg), the stirred reaction mixture was kept at pH 4.8 over 1 h. 2 mL of sodium (2 M) borodeuteride (NaBH4) was added up to the solutions, and the reaction mixture was maintained for 1.5 h at 50 C. Then the pH was adjusted to 5.0 with HCl. The solution was dialyzed for 48 h (10 kDa MWCO membrane) and freeze-dried. The reduction process of carboxyl was repeated three times to guarantee the complete reduction of all uronic acid units. Consequently, the carboxyl-reduced product TAPB1-R was obtained. 2.7. Methylation analysis
2.3. Preparation of the homogeneous polysaccharide TAPB1 T. aurantialba fruiting bodies were powdered and extracted twice with ethanol to remove the lipid material. The residue was air dried and extracted thrice with boiling water. The combined aqueous extracts were separated into four fractions based on their molecular weights by ultrafiltration using hollow-fiber membranes of different pore sizes. The >500 kDa fraction, designated as TAP50w, was fractionated over a DEAE-Sepharose Fast Flow column (XK26 100 cm) by initially eluting with water and then with a NaCl gradient (0 Me2.0 M). Five fractions (TAPA, TAPB, TAPC, TAPD, and TAPE) were monitored for carbohydrate content by phenol sulfuric acid reaction (Dubois et al., 1956). Fraction TAPB was further purified by gel-permeation chromatography on HighResolution Sephacryl S-500 column (XK16 100 cm) eluted with 0.2 M NaNO3 at a flow rate of 0.5 mL min1. Two polysaccharide peaks were detected using a refractive index detector (RID-10A, Shimadzu, Japan), and the fraction forming the symmetrical second peak was collected and designated as TAPB1. 2.4. Monosaccharide analysis Monosaccharide components and percentage composition were determined using high-performance anion-exchange pulsedamperometric detection chromatography (HPAECePAD), as reported in our previous study (Du et al., 2009). TAPB1 (2 mg) was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 110 C for 5 h. A Dionex LC30 was equipped with a CarboPac™ PA20 column (3 mm 150 mm), and the monosaccharides were monitored using a pulsed amperometric detector (Dionex). The column was eluted with 2 mM NaOH (0.45 mL min1). D-Gal, D-Glc, D-Ara, L-Fuc, L-Rha, D-Fru, D-Man, D-Xyl, D-GlcA, Rib, and D-GalA were used as standards. The absolute configurations of the monosaccharides were determined using a procedure described by Gerwig (Gerwig, Kamerling, & Vliegenthart, 1979). 2.5. Determination of homogeneity and molecular mass The homogeneity and molecular mass of TAPB1 were determined by gel-permeation chromatography (GPC) on a HighResolution Sephacryl S-500 column (XK16 100 cm), using dextran T-80, 150, 270, 410, 670 and 2000 as references. The column temperature was kept at 30 C and the signals were detected using a refractive index detector (RID). The mobile phase consisted of 0.2 M NaNO3, and the sample was prepared as 0.2% (w/v). 30 mL of sample solutions was injected into the machine and eluted at 0.5 mL min1. 2.6. Carboxyl group reduction The reduction of the carboxyl group was performed according to the method of Liu et al. (Liu et al., 2008) with minor modification. Briefly, TAPB1 (10 mg) dissolved in distilled water (2 mL) was
TAPB1 was insoluble in Me2SO, which was in agreement with the Me2SO insolubility of polysaccharide TAPA1 from T. aurantialba fruiting bodies in our previous work. Vacuum-dried TAPB1 (4 mg) was initially acetylated with a mixture of Ac2O and pyridine, and subsequently dialyzed, and lyophilized to yield TAPB1-ac. The samples (TAPB1-ac and TAPB1-R) were methylated according to the method used in our previous report (Du et al., 2009). The disappearance of the OH band (3200 cm1 to 3700 cm1) in the FT-IR spectrum was used to confirm complete methylation. The permethylated polysaccharides were hydrolyzed by treatment with HCO2H (88%), distilled water, and TFA in the ratio 3:1:2 for 5 h at 100 C. Subsequently, partially methylated sugars in the hydrolysate were reduced with NaBH4 and acetylated with Ac2O, and the partially methylated alditol acetates (PMAAs) were analyzed by gas chromatographyemass spectrometry(GCeMS) as described by Ye et al. (Ye, et al., 2008). 2.8. Nuclear magnetic resonance (NMR) spectroscopy The freeze-dried TAPB1 (30 mg) was maintained over P2O5 in a vacuum for several days and deuterium exchange was performed thrice with 0.5 mL D2O. 1H NMR, 13C NMR, correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear overhauser enhancement spectroscopy (NOESY), heteronuclear multipleequantum correlation spectroscopy (HMQC), and heteronuclear multiple bond correlation (HMBC) spectra were recorded in D2O at 500 MHz (for 1H NMR) or 125 MHz (for 13C NMR) using a Bruker Avance 500 spectrometer. 13C chemical shifts were acquired in relation to 2,2-dimethyl-2-silapentane-5-sulfonic acid (d 0.00 ppm) calibrated externally, and semi-heavy water (d 4.32 ppm) was used as the internal reference signal for 1H at 70 C. 2.9. Antioxidant activity assay 2.9.1. Preparation of tested samples TAPB1 (5 mg) was initially dissolved in 1 mL of 0.05 M phosphate-buffered saline (PBS; pH 7.8). After centrifugation at 400 g for 30 min, the suspension was further diluted with PBS buffer at concentrations of 0.1, 0.5, 1.0, 1.5, 2.0, and 2.5 mg mL1. 2.9.2. Superoxide anion-scavenging activity assay The superoxide anion radical assay of TAPB1 was performed according to the pyrogallolechemiluminescence assay as reported in our previously study (Du, Mu, Zhou, Zhang, & Zhu, 2013). Briefly, different test agents at 2 mL and pyrogallic acid (0.625 mM) at 8 mL were added to the wells of a 96-well microplate. PBS and ascorbic acid were served as the negative and positive controls, respectively. Luminolesodium carbonate buffer solution mixture at 150 mL (pH 10.2) was pumped into the wells of a 96-well microplate. The signal from each well was monitored and recorded every 0.6 s and maintained for 30 s. The scavenging effect for superoxide anion radical was calculated according to the following formula:
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Fig. 1. Spectrum of HPAEC-PAD for standard monosaccharides and TAPB1 (a) Spectrum of HPAEC-PAD for 11 standard monosaccharides (1. L-Fucose; 2. L-Rhamnose; 3. D-Arabinose; 4. D-Galactose; 5. D-Glucose; 6. D-Xylose; 7. D-Mannose; 8. D-Fructose; 9. Ribose; 10. D-GalA; 11. D-GlcA); (b) Spectrum of HPAEC for TAPB1(1. D-Xylose; 2. D-Mannose; 3. D-GlcA).
Scavenging rate (％) ¼ (A0-A1)/A0 100, in which A0 was the luminescence value of the negative control without tested sample. A1 was the luminescence values of tested sample. Each sample was repeated 3 times. 2.9.3. H2O2-scavenging activity assay The H2O2-scavenging activities were determined according to the chemiluminescence method used in our previous work (Du et al., 2013). H2O2 (1%) at 10 mL and different test agents at 40 mL were added to the wells of a 96-well microplate. Double-distilled water and ascorbic acid were used as the negative and positive controls, respectively. Each sample was repeated 3 times. The scavenging rate for H2O2 radical were performed according to the method in superoxide anion-scavenging activity assay (Section 2.9.2). 2.9.4. Statistical analysis The data obtained were expressed as mean ± SD of three determinations and were analyzed statistically by ANOVA. The significance of any differences between groups was evaluated using the Student's t-test. All computations were performed using statistical software. 3. Results and discussion 3.1. Structural analysis A novel water-soluble polysaccharide from T. aurantialba fruiting bodies, designated as TAPB1, was purified by ultrafiltration and a series of chromatographic steps. TAPB1 showed a negative response to the Bradford test (Bradford, 1976) and showed no absorption at 280 or 260 nm in the UV scanning spectrum, indicating the absence of proteins and nucleic acids. TAPB1 appeared as a single symmetrical peak on Sephacryl S-500 high-resolution chromatography, indicating that TAPB1 was a homogeneous polysaccharide. TAPB1 had an estimated molecular weight of
7.6 105 Da according to GPC results, and had a carbohydrate content of 97.6%, as measured by the phenolesulfuric acid method (Dubois et al., 1956). Monosaccharide composition analysis by HPAECePAD showed that TAPB1 comprised D-mannose, D-xylose, and D-glucuronic acid in a ratio of 3.1:2.9:1.2, along with trace amounts of D-galactose, D-glucose, and D-galacturonic acid (Fig. 1). Absolute configuration analysis showed that the mannose, xylose, and glucuronic acid residues all had the D configuration (Gerwig et al., 1979). The FT-IR spectrum of TAPB1 (Fig. 2) displayed an absorption peak at 1733.1 cm1, which is a characteristic of the C]O stretching vibration, suggesting that TAPB1 may be an acidic polysaccharide and/or may contain O-acetyl groups. The strong broad absorption peak at 3423.1 cm1 was due to the hydroxyl stretching vibration of the polysaccharide, and the peak at 2923.6 cm1 was due to the CeH stretching vibration band (Du et al., 2009). Methylation analysis of polysaccharides was performed to investigate the inter-glycosidic linkages between monosaccharide residues. The fully methylated products were hydrolyzed, converted into PMAAs, and analyzed by GCeMS. As shown in Table 1, we detected 1-substituted, 1,2-disubstituted and 1,3-disubstituted xylopyranose residues and 1,3-disubstituted, 1,2,3-trisubstituted, and 1,3,4-trisubstituted mannopyranose residues by methylation analysis of TAPB1. GCeMS analysis of reduced T. auriantalba polysaccharide (TAPB1-R) revealed one additional peak that was consistent with that of 2,3,4,6-tri-O-Me4-glucitol, indicating the presence of 1-substituted GlcA residues in TAPB1. The 1H NMR (500 MHz) spectrum of TAPB1 (Fig. 3) at 70 C mainly contained signals for five anomeric protons at d 4.45 to d 5.25. One signal at d 2.02 corresponded to the 1H signal for the CH3 moiety of an acetyl group (Serrato et al., 2008). Other sugar protons were in the region of d 4.38 to d 3.37. The results of 1H NMR (Fig. 3), 13 C NMR(125 MHz, Fig. 4), and 13Ce1H HMQC (Fig. 5) analyses indicated that the repeating unit of the polysaccharide comprised seven sugar residues, which were designated as AeG in the 1H NMR spectrum according to the decreasing chemical shifts of the anomeric protons.
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Fig. 2. The FT-IR spectroscopy of TAPB1.
The 13C NMR (125 MHz, Fig. 4) results, which was supported by the both spectra 1H NMR and 13Ce1H HMQC (Fig. 5), showed seven anomeric signals at d 105.94, 105.57, 104.95, 104.70, 104.29, 103.72, and 102.65, which were designated as D, G, B, F, C, E, and A, respectively. Furthermore, downfield signals at d 175.19 and 179.25 in the 13C NMR spectrum revealed two carboxyl groups, and a signal at d 23.79 upfield revealed a CH3 moiety of an acetyl group (Serrato et al., 2008). The identities of the monosaccharide residues AeG were established based on 2D-NMR analysis involving 1He1HeCOSY, TOCSY, NOESY, 1He13C heteronuclear singleequantum correlation (HSQC), and HMBC experiments, which were used to assign the chemical shifts and anomeric configurations of the seven sugar residues present in the repeating unit.
Table 1 Results of the methylation analysis of TAPB1 and TAPB1-R by GCeMS. Glycosyl residue
Methylated sugar (as alditol acetate)
Mode of linkage
Molar ratiosa TAPB1
TAPB1-R
Man
4,6-Me2 2,6-Me2 2,4,6-Me3 2,3,4-Me3 3,4-Me2 2,4-Me2 2,3,4,6-Me4
/2,3[Manp]1/ /3,4[Manp]1/ /3 [Manp]1/ [Xylp]1/ /2[Xylp]1/ /3[Xylp]1/ [Glcp]1/
1.01 0.92 0.89 0.96 0.93 0.96 trace
0.93 0.98 0.97 0.83 1.02 1.04 0.82
Xyl
Glc a
The molar ratios of samples were calculated on basis of peak area of PMAAs.
Fig. 3. 1H NMR (500 MHz) spectrum determined in D2O at 70 C of T. aurantialba polysaccharide TAPB1: the anomeric protons are labeled A-G.
Residues A, B, and C had anomeric chemical shifts at d 5.25, 5.25, and 5.10, respectively. The 1H resonances for H1eH6 in Residue A were assigned from the cross peaks in the COSY, TOCSY, and NOESY spectra (Table 2). The carbon signals from C-1 to C-6 of residue A were identified from the HSQC spectrum (Table 2). The relatively small coupling constant values of JH-1,H-2 and JH-2,H-3 (<2 Hz) and the large coupling constant values of JH-3,H-4 (>8 Hz), JH-4,H-5 (>8 Hz), and JC-1,H-1 (~166 Hz) showed that residue A was a-Dmannose (Mondal, Chakraborty, Rout, & Islam, 2006; Rout, Mondal, Chakraborty, & Islam, 2006; Yang et al., 2007). The linkage positions were determined from the high carbon chemical shift values (Yu & Yang, 2002, pp. 901e906; Zhang, 1999, pp. 245e252). Therefore, the downfield chemical shifts of the C-1 (d 102.65), C-2 (d 75.81), and C-3 (d 75.58) carbon signals indicated that residue A was a (1 / 2,3)-a-D-mannopyranose. In addition, residues B and C were identified as (1 / 3,4)-a-D-mannopyranose and (1 / 3)-a-Dmannopyranose, respectively. Residues D, E, and F had anomeric chemical shifts at d 4.69, 4.69, and 4.54, respectively. Protons chemical shifts from H-2 to H-4 of residues D were assigned from COSY, TOCSY, and NOESY spectra. The large coupling constant values JH-1,H-2 (>7.8 Hz) and JH-1,C-1 (~160 Hz) indicated that these were all b-linked residues (Du et al., 2009; Ghosh et al., 2008), and this finding was confirmed by the intra-residue NOESY signals between H-1 and H-3, and H-1 and H5. H-5a and H-5b were assigned from the TOCSY and NOESY spectra, and their carbon signals from C-1 to C-5 were assigned from the HMBC and HSQC spectra (Table 2). The downfield chemical shifts of each residue indicated substituted positions (Yu & Yang, 2002, pp. 901e906; Zhang, 1999, pp. 245e252). Therefore, the residues D, E, and F were designated as follows: D, (1 / 3)-b-D-
Fig. 4. 13C NMR (125 MHz) spectra determined in D2O at 70 C of T. aurantialba polysaccharide TAPB1: the anomeric protons are labeled A-G.
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463
(I)
Confirmation of Sequence I was obtained by the inter-residue proton cross peaks observed in the NOESY spectrum (data not shown). The following inter-residual NOE correlations were observed between H-1 of residue A and H-3 of residue C, between H-1 of residue C and H-3 of residue B, and between H-1 of residue B and H-3 of residue A. HMBC spectral analysis revealed clear correlations between H-1 of residue G and C-3 of residue D, between C-1 of residue G and H-3 of residue D, between H-1 of residue D and C-2 of residue A, and between C-1 of residue D and H-2 of residue A, thereby establishing the following sequence (Sequence II): Fig. 5. 1He13C HSQC spectrum of TAPB1 showing anomeric atom cross-peaks. Determined in D2O at 70 C.
xylopyranose; E, 1/b-D-xylopyranose; and F, (1 / 2)-b-Dxylopyranose. Residue G had the anomeric signal at d 4.45 and a large JH-1, H-2 coupling constant value (~8.6 Hz), indicating that G was a b-linked residue (Du et al., 2009). The H-1 track (d 4.45) of residue G in the TOCSY spectrum showed the complete spin system H-1,2,3,4,5, which is a characteristic of a b-GlcA residue (Dobruchowska, Gerwig, Babuchowski, & Kamerling, 2008; Vinogradov, Petersen, Duus, & Wasser, 2004). Cross peaks between H-1 and H-2 (d 4.45/ 3.55) and between H-2 and H-3 (d 3.55/3.67) were observed in the COSY spectrum. The H-4 (d 3.78) and H-5 (d 3.92) resonances were assigned from the TOCSY and NOESY spectra. 13C resonances were assigned from the HMQC spectrum (Table 2). The downfield shifts of the C-1 (d 105.57) carbon signals indicated that residue G was a 1-linked b-D-glucuronic acid pyranose. The sequence of glycosyl residues in TAPB1 was determined from results of HMBC studies followed by confirmation from NOESY results. The HMBC spectral analysis (data not shown) revealed inter-residue connectivities between H-1 of residue A and C-3 of residue C, between C-1 of residue A and H-3 of residue C, between H-1 of residue C and C-3 of residue B, between C-1 of residue C and H-3 of residue B, between H-1 of residue B and C-3 of residue A, and between C-1 of residue B and H-3 of residue A. Such connectivities indicated that the sequence of A, B, and C residues (Sequence I) was as follows: Table 2 1 H and 13C NMR chemical shift data (d, ppm) for T. aurantialba polysaccharide TAPB1 determined in D2O at 70 C. Residue
A /2,3)-a-D-Manp-(1/ B /3,4)-a-D-Manp-(1/ C /3)-a-D-Manp-(1/ D /3)b-D-Xylp-(1/ E b-D-Xylp-(1/ F /2)-b-D-Xylp-(1/ G b-D-GlcA p-(1/ a
1
H/13C
1
2
3
4
5/5a
5.25a 102.65 5.25 104.95 5.10 104.29 4.69 105.94 4.69 103.72 4.54 104.70 4.45 105.57
4.18 75.81 3.87 72.54 3.87 72.54 4.38 76.05 3.37 76.05 3.49 78.10 3.55 75.23
3.67 76.58 3.99 75.23 3.87 75.81 3.67 86.26 3.41 76.05 3.67 76.20 3.67 76.43
3.61 69.26 3.63 76.20 3.63 69.26 3.76 70.85 3.74 70.25 3.37 67.57 3.78 70.25
3.67 71.67 3.86 73.08 3.67 71.67 3.76 63.04 3.77 63.04 3.82 63.04 3.92 76.10
The figure in bold font indicated the substituted position.
6a/5b 3.78 63.47 3.75 63.47 3.66 63.47 3.72 3.81 3.78
175.19
6b 3.75 3.71 3.62
(II) Sequence II was confirmed by the following inter-residual NOE correlations. The NOESY spectrum indicated the cross peaks between H-1 of residue G and H-3 of residue D and between H-1 of residue D and H-2 of residue A. Cross peaks were observed in the HMBC spectrum between H-1 of residue E and C-2 of residue F, between C-1 of residue E and H-2 of residue F, between H-1 of residue F and C-4 of residue B, and between C-1 of residue F and H-4 of residue B, thereby establishing the following sequence (Sequence III):
(III) Sequence III was also confirmed by the following inter-residual NOE correlations. NOESY spectrum analysis revealed the cross peaks between H-1 of residue E and H-2 of residue F and between H-1 of residue F and H-4 of residue B. 3.2. Antioxidant activity Reactive oxygen species (ROS), such as superoxide anion radicals and hydrogen peroxide (H2O2), are among the main factors associated with the initiation of numerous diseases and degenerative processes during aging (Soares et al., 2009). Ascorbic acid was used as positive control in O 2 -scavenging and H2O2-scavenging assays, and its EC50 values were calculated. The values obtained for ascorbic acid showed the effective concentration at which the antioxidant activity was 50%. As shown in Fig. 6, TAPB1 can scavenge superoxide radicals and H2O2 at the concentration range of 0.1 mg mL1 to 2.5 mg mL1. Scavenging effects of TAPB1 on both ROS increased with increasing concentration. The EC50 values of TAPB1 for superoxide radicals and H2O2 were 1.04 and 1.77 mg mL1, respectively. These values showed the lower antioxidant capacity of TAPB1 compared with ascorbic acid, which had EC50 values of 23.6 and 79.6 mg mL1, respectively. 4. Conclusions TAPB1, isolated and purified from the fruiting bodies of T. aurantialba, was a hepta-polysaccharide with a molecular weight of 7.6 105 Da. Monosaccharide composition analysis showed that TAPB1 comprised D-mannose, D-xylose, and D-glucuronic acid in the ratio 3.1:2.9:1.2, along with trace amounts of D-galactose, DGlucose, and D-galacturonic acid. According to methylation data and NMR analysis, the repeating unit of TAPB1 comprised an a(1 / 3)-linked mannopyranosyl backbone. The backbone was
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Fig. 6. Scavenging effect of the polysaccharide TAPB1 on superoxide radicals and H2O2: (a) scavenging activities to superoxide radicals; (b) scavenging activities to H2O2; data are presented as the mean values (n ¼ 3).
partially substituted at the O-4 positions with a side chain composed of two xyloses and at the O-2 position with a side chain comprising xylose and glucuronic acid. Based on the results of superoxide anion radical scavenging assay and H2O2 test, TAPB1 showed significant and dosedependent scavenging effects on these ROS, suggesting its potential as an effective natural antioxidant. However, further studies are required to establish the mode and mechanisms underlying the antioxidant action of TAPB1 and to investigate the relationships between the chemical structure and the above mentioned antioxidant activities. Acknowledgments This work was supported by Natural Science Foundation of Shandong Province of China (ZR2010CL008) and Doctoral Research Start-up Fundation of Liaocheng University (31805). The authors are deeply grateful to Mrs. Liping Shi for recording the NMR spectra. References Bradford, M. M. (1976). Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248e254. Deng, Y. X., & Qu, W. J. (2007). In vitro antioxidant function of extracellular polysaccharides from Tremella aurantialba. Acta Edulis Fungi, 14(3), 50e52. Dobruchowska, J. M., Gerwig, G. J., Babuchowski, A., & Kamerling, J. P. (2008). Structural studies on exopolysaccharides produced by three different propionibacteria strains. Carbohydrate Research, 343, 726e745. Dubois, M., Gillis, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350e356. Du, X. J., Mu, H. M., Zhou, S., Zhang, Y., & Zhu, X. L. (2013). Chemical analysis and antioxidant activity of polysaccharides extracted from Inonotus obliquus sclerotia. International Journal of Biological Macromolecules, 62, 691e696. Du, X. J., Zhang, J. S., Liu, Y. F., Tang, Q. J., Jia, W., Yang, Y., et al. (2010). The antioxidant activity of various extracts from Tremella aurantialba fruiting bodies and their protective effects on PC12 cells injured by oxidation. Acta Agriculturae Shanghai, 26(2), 49e52. Du, X. J., Zhang, J. S., Yang, Y., Tang, Q. J., Jia, W., & Pan, Y. J. (2010). Purification, chemical modification and immunostimulating activity of polysaccharide from Tremella aurantialba fruiting bodies. Journal of Zhejiang University, 11(16), 437e442. Du, X. J., Zhang, J. S., Yang, Y., Ye, L. B., Tang, Q. J., Jia, W., et al. (2009). Structural elucidation and immuno-stimulating property of an acidic heteropolysaccharide (TAPA1) from Tremella aurantialba. Carbohydrate Research, 344(5), 672e678. Gerwig, G. J., Kamerling, J. P., & Vliegenthart, J. F. G. (1979). Determination of the absolute configuration of monosaccharides in complex carbohydrates by capillary g.l.c. Carbohydrate Research, 77, 1e7. Ghosh, K., Chandra, K., Roy, S. K., Mondal, S., Maiti, D., Das, D., et al. (2008). Structural studies of a methyl galacturonosyl-methoxyxylan isolated from the stem of Lagenaria siceraria (Lau). Carbohydrate Research, 343, 341e349.
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