Structure, physical property and antioxidant activity of catechin grafted Tremella fuciformis polysaccharide

Structure, physical property and antioxidant activity of catechin grafted Tremella fuciformis polysaccharide

Accepted Manuscript Title: Structure, physical property and antioxidant activity of catechin grafted Tremella fuciformis polysaccharide Author: Jun Li...

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Accepted Manuscript Title: Structure, physical property and antioxidant activity of catechin grafted Tremella fuciformis polysaccharide Author: Jun Liu Chen-guang Meng Ye-hua Yan Ya-na Shan Juan Kan Chang-hai Jin PII: DOI: Reference:

S0141-8130(15)30120-3 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.11.027 BIOMAC 5532

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

6-8-2015 8-11-2015 11-11-2015

Please cite this article as: J. Liu, C.-g. Meng, Y.-h. Yan, Y.-n. Shan, J. Kan, C.-h. Jin, Structure, physical property and antioxidant activity of catechin grafted Tremella fuciformis polysaccharide, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.11.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A purified TPS fraction with the molecular weight of 5.82 × 105 Da was obtained. Catechin-g-TPS was prepared with the grafting ratio of 265 mg CAE/g. Catechin-g-TPS was characterized by FT-IR, 1H NMR, TGA, XRD and SEM. Catechin-g-TPS exhibited higher antioxidant activity in vitro than TPS.

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Structure, physical property and antioxidant activity of catechin grafted Tremella fuciformis polysaccharide

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Jun Liu*[email protected], Chen-guang Meng, Ye-hua Yan, Ya-na Shan, Juan Kan, Chang-hai Jin College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, Jiangsu, China Tel: +86-514-87978158, Fax: +86-514-87313372.

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Abstract In this study, structural characterization, physical property and antioxidant activity of catechin grafted Tremella fuciformis polysaccharide (catechin-g-TPS) were investigated. Crude polysaccharides were isolated from the fruit bodies of T. fuciformis and further purified on DEAE-52 and Sepharose CL-4B chromatography to afford a main purified fraction (named TPS). The molecular weight of TPS was determined as 5.82 × 105 Da by HPLC. Then, the free radical mediated grafting of catechin onto TPS was achieved by using a redox system. As compared with the unmodified TPS, catechin-g-TPS showed new bands within the range of 1300–1600 cm−1 in FT-IR spectrum, and exhibited new signals at around δ6.00 and 6.80ppm in 1H NMR spectrum. Thermogravimetric analysis indicated the thermal stability of catechin-g-TPS was higher than TPS. X-ray diffraction spectrum of catechin-g-TPS exhibited two sharp narrow diffraction peaks at 14.2 and 32.1°, corresponding to the crystalline peaks of catechin. Scanning electron microscopy observation revealed the surface of TPS was smooth, whereas the surface of catechin-g-TPS was much rough. These results all confirmed the successful grafting of catechin onto TPS. Moreover, catechin-g-TPS had higher 2,2-diphenyl-1- picrylhydrazyl radical scavenging activity and reducing power as compared to TPS.

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Keywords Antioxidant; Catechin; Grafted; Tremella fuciformis polysaccharide

1. Introduction Tremella fuciformis, also known as silver ear or white jelly fungus, is a traditional Chinese edible and medicinal mushroom. In the past decades, polysaccharides from T. fuciformis has received increasing attention due to its diverse pharmacological activities, such as cytokine-stimulating [1], antioxidant [2], anti-tumor [3], anti-diabetic [4], anti-radiation [5] and anti-obesity effects [6]. The chemical structures of T. fuciformis polysaccharides (TPS) are consisted of a linear (1→3)-linked α-D-mannose backbone with mainly β-D-xylose and β-D-glucuronic acid in the side chains [7, 8]. In recent years, the chemical modification of polysaccharides has provided an opportunity to develop new agents with potential therapeutic functions [9–11]. As reported, TPS can be modified

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by methods of acetylation and sulfation with enhanced immunostimulating, antiviral and antioxidant activities [12–15]. However, little information on the chemical modification of TPS by other methods is available. Recently, the application of graft copolymerization for the modification of polysaccharides has received a particular focus [16]. Graft copolymerization of functional groups, especially phenolic compounds onto polysaccharides can improve the desired biological activities of polysaccharides as well as widen the field of their potential applications [17–19]. Catechin is one of the main phenolic compounds in green tea with various pharmacological effects including antioxidant, anti-diabetic, anti-inflammatory, anti-mutagenic, anti-carcinogenic and antimicrobial activities [20]. Increasing evidence has highlighted that catechin can be grafted onto polysaccharides such as chitosan, dextran and inulin to enhance the antioxidant, anti-diabetic and antitumor activities of original polysaccharides [21–25]. The aim of this study is to graft catechin onto TPS in order to improve the antioxidant activity of TPS. Firstly, the crude TPS was extracted from the fruit bodies of T. fuciformis by hot water and further purified on anion-exchange and gel filtration chromatography. Then, the purified TPS fraction was grafted with catechin by a free radical mediated method. The structures and physical properties of TPS before and after graft copolymerization were characterized by Fourier-transform infrared (FT-IR), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), X-ray diffraction (XRD) and scanning electron microscopy (SEM). Finally, antioxidant activity in vitro of TPS and its grafted copolymer was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and reducing power assays. Our results provide a novel approach to enhance the antioxidant activity of TPS. 2. Materials and methods 2.1. Materials and reagents The dried fruit bodies of T. fuciformis were purchased from local market (Jiangsu, China). Samples were ground into powder using a milling machine, passed through a 40-mesh sieve and stored at −20 °C prior to extraction. Ascorbic acid (Vc), (+)-catechin hydrate, Folin–Ciocalteu reagent and DPPH were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were of analytical grade. 2.2. Extraction of crude TPS The extraction of TPS was carried out according to the method of Chen with some modifications [2]. Briefly, 200 g of T. fuciformis powder was extracted with 4 L of distilled water at 80 °C for 8 h. After filtration to remove debris fragments, the filtrate was centrifuged at 10, 000 rpm for 30 min. The resultant supernatant was collected, concentrated and mixed with three volumes of dehydrated ethanol at 4 °C overnight. The precipitate of obtained dispersion was collected by centrifugation at 10, 000 rpm for 15 min and followed by deproteinization with 1/5 volume of Sevag reagent (CHCl3-BuOH = 5/1, v/v) for 5 times. The deproteinized solution was then dialyzed against distilled water, concentrated and lyophilized to afford crude TPS. 2.3. Purification of TPS The crude TPS was purified sequentially by chromatography of DEAE-52 and Sepharose CL-4B. Crude TPS solution (10 mg/ml, 10 ml) was applied to a DEAE-52 column (2.6 × 30 cm), and stepwise eluted with distilled water, 0.1, 0.3 and 0.5 M NaCl solutions at a flow rate of 1 ml/min. Eluate was collected automatically (10 ml/tube) and the carbohydrate content was determined by the phenol–sulfuric acid method. As a result, two fractions (F-1 and F-2) were obtained.

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Afterwards, F-1 fraction was concentrated, dialyzed and further purified on a Sepharose CL-4B column (1.6 × 60 cm) to afford purified TPS. 2.4. Determination the molecular weight of TPS A Shimadzu LC-20A HPLC system (Shimadzu, Kyoto, Japan) equipped with a refractive index detector (RID) was used to determine the molecular weight of purified TPS. The tested sample was eluted with 0.1 M Na2SO4 solution on a TSK-GEL G3000SWxl column (7.5 × 300 mm, Tosoh Corp., Tokyo, Japan) at the flow rate of 0.8 ml/min. The column was calibrated with pullulan standards of Shodex standard P-82 (Showa denko Co. Ltd., Japan). 2.5. Synthesis of catechin grafted TPS (catechin-g-TPS) The synthesis of catechin-g-TPS was carried out by a redox system [19]. Briefly, 0.5 g of purified TPS, 0.1 g of Vc and 0.8 g of catechin were dissolved in 50 ml distilled water in a 0.5 L three-necked round bottom flask. A slow stream of oxygen free nitrogen gas was passed through the flask for 30 min. Then, 2 ml of 5 M H2O2 solution was added into the mixture to initiate the reaction. The reaction was conducted under a continuous flow of nitrogen gas for 12 h. Finally, the reaction mixture was dialyzed and lyophilized to afford catechin-g-TPS. The grafting ratio of catechin-g-TPS was measured by Folin–Ciocalteu method and expressed as mg of catechin equivalents per g (mg CAE/g) of grafted copolymer. 2.6. Characterization of catechin-g-TPS FT-IR spectra were recorded in the frequency range of 4000–400 cm–1 on a Varian 670 IR equipped with a Varian 610-IR microscope (Varian Inc., USA) in KBr pellet. 1H NMR spectra were measured at 25 °C using an AVANCE-600 spectrometer (Bruker Inc., Germany) operating at 600 MHz. Thermogravimetric analysis was conducted with a Pyris 1 TGA instrument (PerkinElmer Ltd., USA). Experiment was carried out by heating 3–5 mg of sample from 30 to 800 °C in nitrogen flow at a heating rate of 10 °C/min. XRD measurements were performed on an D8 Advance powder X-ray diffractometer (Bruker Inc., Germany) with Ni-filtered Cu Kα radiation (λ = 1.5406 Å) with scattering angles (2θ) of 5–80°. The voltage and current were maintained at 40 kV and 40 mA, respectively. SEM images were taken on an S-4800 microscope (Hitachi Ltd., Japan) at the accelerating voltage of 15 kV. Samples used for SEM observation were dried under vacuum, mounted on metal stubs and sputter-coated with gold. 2.7. Determination the antioxidant activity in vitro of catechin-g-TPS 2.7.1. Determination of DPPH radical scavenging activity The DPPH free radical scavenging activity was measured by the method of Shimada et al. [26]. Briefly, 0.2 ml of 0.4 mM DPPH in methanol was mixed with 1.8 ml of water and 1.0 ml of sample solution at different concentrations (0.05–1 mg/ml). The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. Then, the absorbance of the mixture was measured at 517 nm by using a Lambda 35 spectrophotometer (PerkinElmer Ltd., USA). Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The DPPH radical scavenging activity was calculated using the following equation: Scavenging activity (%) = [1 – (A1 – A2)/A0] × 100 (4) where A0 is the absorbance of the control (water instead of sample), A1 is the absorbance of the sample, and A2 is the absorbance of the sample only (water instead of DPPH). 2.7.2. Determination of reducing power The reducing power was determined according to the method of Oyaizu [27]. Reaction was carried out in a mixture containing 2.5 ml of sample solution at different concentrations (0.05–1 mg/ml),

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2.5 ml of 0.1 M sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide (w/v). The mixture was incubated at 50 °C for 20 min. After 2.5 ml of 10% trichloroacetic acid (w/v) was added, the mixture was centrifuged at 5000 rpm for 10 min. The upper layer (5 ml) was mixed with 0.5 ml of 0.1% ferric chloride (w/v), and the absorbance was measured at 700 nm. Higher absorbance indicates higher reducing power. 2.8. Statistical analysis. Data were expressed as mean ± standard deviation (SD). The Duncan test and one-way analysis of variance (ANOVA) were used for multiple comparisons by the SPSS 13.0 software package. Difference was considered to be statistically significant if p < 0.05. 3. Results and discussion 3.1. Purification and molecular weight of TPS The traditional water extraction method has been demonstrated as an effective way to extract polysaccharides from the fruit bodies of T. fuciformis [2]. Thus, TPS was extracted with hot water in this study. The extraction yield of crude TPS was 2.96%. The obtained crude TPS was then fractionated on a DEAE-52 chromatography to afford two polysaccharide fractions (F-1 and F-2) as shown in Fig. 1a. Further purification of F-1 on another Sepharose CL-4B chromatography yielded only one carbohydrate peak (Fig. 1b). The recovery rate of purified TPS fraction based on the amount of crude TPS were 65.2%. The molecular weight of purified TPS was determined by HPLC. As shown in Fig. 2, the purified TPS fraction showed only one symmetrical peak, indicating that no other polysaccharide was present in the sample. The molecular weight of TPS fraction was estimated as 5.82 × 105 Da.

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3.2. Synthesis of catechin-g-TPS The synthesis of catechin-g-TPS was performed by a free radical mediated reaction. The reaction mechanism involves a complex radical reaction between catechin and TPS. In particular, the interaction between Vc and H2O2 redox pair induces the formation of hydroxyl radicals, which attack the hydrogen atoms on TPS to form many reactive sites. The subsequent linkage of catechin onto TPS involves reaction with these reactive sites [19]. The grafting ratio of catechin-g-TPS was determined as 265 mg CAE/g by Folin-Ciocalteu method. 3.3. Characterization of catechin-g-TPS 3.3.1 FT-IR spectrum of catechin-g-TPS The FT-IR spectra of TPS and catechin-g-TPS are presented in Fig. 3. The strong broad absorption peak at 3407 cm−1 was due to the hydroxyl stretching vibration of the polysaccharide, and the peak at 2933 cm−1 was due to C–H stretching vibration band. Characteristic absorption peak at 1723 cm−1 was corresponded to C=O stretching vibration, suggesting that TPS may be an acidic polysaccharide and/or may contain O-acetyl groups. The couple of bands at 1611 and 1423 cm−1 were attributed to asymmetric and symmetric stretching modes of COO− group [28]. Other bands at 1252 and 1073 cm−1 were assigned to C–H variable angle vibration and C–O–C stretching vibration in saccharide ring. These typical absorption peaks were also observed in catechin-g-TPS, indicating that no major functional group transformations happened during the grafting process. Notably, some new bands appeared in the range of 1300–1600 cm−1, attributing to C=C stretching vibrations of aromatic ring [29]. This suggested that catechin was successfully grafted onto TPS.

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3.3.2. 1H NMR spectrum of catechin-g-TPS

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The 1H NMR spectra of TPS and catechin-g-TPS are shown in Fig. 4. For TPS, two anomeric protons at δ5.18 and 5.10 ppm were assigned to α-D-annopyranose. Signal for anomeric protons at δ4.35ppm was contributed to β-D-xylopyranose. However, the signal for β-D-glucopyranuronic acid was not detected due to overlap with HOD peak. Moreover, signals at δ2.02 and 1.16 ppm were corresponded to the CH3 moiety of acetyl group and rhamnan residues, respectively. Catechin-g-TPS preserved all the proton signals of TPS. In addition, new proton signals were also observed in the 1H NMR spectrum of catechin-g-TPS. The signals at around δ6.80ppm were attributed to H–2’, H–5’ and H–6’ of catechin B ring [20]. The small signal at δ6.00ppm was assigned to H–6 of catechin A ring. These results further confirmed the successful grafting of catechin onto TPS.

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3.3.3. TGA of catechin-g-TPS Thermal stability of TPS and catechin-g-TPS was studied by TGA, as it can provide a quantitative measurement of mass change in materials along with temperature. Thermal gravimetric curves of TPS and catechin-g-TPS are shown in Fig. 5a. TPS and catechin-g-TPS both showed two stages of weight loss. The first stage of weight loss was corresponded to the loss of absorbed and structural water of polymers. The second stage of weight loss was corresponded to the depolymerization, decomposition and combustion of the polymers. DTG curves (Fig. 5b) also showed the temperatures for the rapidest weight loss of TPS and catechin-g-TPS appeared at 318 and 326 °C, respectively. Notably, TPS degraded more rapidly than catechin-g-TPS, suggesting that the thermal stability of TPS was lower than catechin-g-TPS.

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3.3.4. XRD analysis of catechin-g-TPS XRD is a rapid analytical technique widely used for phase identification of a crystalline material. In general, crystalline materials show sharp narrow diffraction peaks while amorphous components exhibit broad peaks. As shown in Fig. 6, The XRD profile of TPS exhibited two broad diffraction peaks at 9.7 and 24.1°, indicating that TPS was amorphous in nature. By contrast, catechin-g-TPS showed two additional sharp narrow diffraction peaks at 14.2 and 32.1°, which was corresponded to the crystalline peaks of catechin [24]. This result suggested that catechin-g-TPS had higher crystallinity than TPS.

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3.3.5. SEM of catechin-g-TPS SEM is a qualitative tool to analysis the surface morphology of polysaccharides. The SEM images of TPS and catechin-g-TPS are shown in Fig. 7. Both TPS and catechin-g-TPS exhibited the piece-shaped morphology. However, the surfaces of TPS and catechin-g-TPS were quite different. The surface of TPS was smooth, whereas the surface of catechin-g-TPS was much rough. The rough surface of catechin-g-TPS may be closely related to the grafted catechin residues.

3.4. Antioxidant activity of catechin-g-TPS The model of scavenging the stable DPPH radical is a widely used method for evaluating the free radical scavenging ability of natural compounds. Fig. 8a shows the DPPH radical scavenging activity of TPS and catechin-g-TPS at different concentrations. The DPPH radical scavenging activity of TPS and catechin-g-TPS increased dependently with concentrations, and was 10.60% and 52.27% at 1 mg/ml, respectively. Obviously, the scavenging activity of TPS was significantly

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lower than that of catechin-g-TPS. However, the scavenging activity of these polymers against DPPH radical was less than that of catechin and Vc at the same concentrations. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. As shown in Fig. 8b, the reducing power of TPS and catechin-g-TPS increased with increasing concentrations. Moreover, catechin-g-TPS exhibited higher reducing power than TPS. At the concentration of 1 mg/ml, the reducing power of catechin was 1.53. It has been reported that the antioxidant activity is generally associated with the presence of electron-donating groups or hydrogen atoms [30]. The potent antioxidant activity of catechin is connected to the ability to trap free radicals by donation of the phenolic hydrogen atom in the A and B rings [31]. Thus, when catechin was grafted onto TPS, the antioxidant activity of TPS was greatly enhanced.

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4. Conclusion In this work, the modification of TPS with catechin was achieved by a free radical mediated grafting method. The spectroscopic results of FT-IR and NMR both confirmed the successful grafting. The thermal stability and crystallinity of catechin-g-TPS were enhanced as compared to the unmodified TPS. Moreover, catechin-g-TPS showed higher antioxidant activity than TPS, suggesting the antioxidant ability of TPS could be enhanced by grafting with antioxidant molecules. Our results indicated that catechin-g-TPS could be used in food and pharmaceutical industries as a novel antioxidant. Acknowledgements This work was supported by Grants-in-Aid for scientific research from the National Natural Science Foundation of China (No. 31101216 and 31571788), Natural Science Foundation of Jiangsu Province (No. BK20151310), Postgraduate Innovation Project of Jiangsu Province (No. SJLX15_0671), Innovation and Entrepreneurship Training Program for College Students in Jiangsu Province (No. 201511117094X), Jiangsu Provincial Government Scholarship for Overseas Studies, and High Level Talent Support Program of Yangzhou University. References [1] Q.P. Gao, R. Seljelid, H.Q. Chen, R. Jiang, Characterization of acidic heteroglycans from Tremella fuciformis Berk. with cytokine stimulating activity, Carbohydr. Res. 228 (1996) 135–142. [2] B. Chen, Optimization of extraction of Tremella fuciformis polysaccharides and its antioxidant and antitumour activities in vitro, Carbohydr. Polym. 81 (2010) 420–424. [3] S. Ukai, K. Hirose, T. Kiho, C. Hara, T. Irikura, Antitumor activity on sarcoma 180 of the polysaccharides from Tremella fuciformis Berk, Chem. Pharm. Bull. 20 (1972) 2293–2294. [4] E.J. Cho, H.J. Hwang, S.W. Kim, J.Y. Oh, Y.M. Baek, J.W. Choi, S.H. Bae, J.W. Yun, Hypoglycemic effects of exopolysaccharides produced by mycelial cultures of two different mushrooms Tremella fuciformis and Phellinus baumii in ob/ob mice, Appl. Microbiol. Biotechnol. 75 (2007) 1257–1265. [5] W. Xu, X. Shen, F. Yang, Y. Han, R. Li, D. Xue, C. Jiang, Protective effect of polysaccharides isolated from Tremella fuciformis against radiation-induced damage in mice, J. Radiat. Res. 53 (2012) 353–60. [6] H.J. Jeong, S.J. Yoon, Y.R. Pyun, Polysaccharides from edible mushroom hinmogi (Tremella fuciformis) inhibit differentiation of 3T3-L1 adipocytes by reducing mRNA expression of PPARγ, C/EBPα, and leptin, Food Sci. Biotechnol. 17 (2008) 267-273.

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activity of catechin grafted inulin, Int. J. Biol. Macromol. 64 (2014) 76–83. [25] W. Zhu, Z. Zhang, Preparation and characterization of catechin-grafted chitosan with antioxidant and antidiabetic potential, Int. J. Biol. Macromol. 70 (2014) 150–155. [26] K. Shimada, K. Fujikawa, K. Yahara, T. Nakamura, Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion, J. Agric. Food Chem. 40 (1992) 945–948. [27] M. Oyaizu, Studies on products of browning reactions: Antioxidative activities of products of browning reaction prepared from glucosamine, Jpn. J. Nutr. 44 (1986) 307–315. [28] Z. Košťálová, Z. Hromádková, A. Ebringerová, Structural diversity of pectins isolated from the Styrian oil-pumpkin (Cucurbita pepo var. styriaca) fruit, Carbohydr. Polym. 93 (2013) 163–171. [29] Y.M. Chen, T.M. Tsao, C.C. Liu, P.M. Huang, M.K. Wang, Polymerization of catechin catalyzed by Mn-, Fe- and Al-oxides, Colloids Surf. B Biointerfaces 81 (2010) 217–223. [30] M. Leopoldini, T. Marino, N. Russo, M. Toscano, Antioxidant properties of phenolic compounds: H-atom versus electron transfer mechanism, J. Phys. Chem. A 108 (2004) 4916–4922. [31] K. Kondo, M. Kurihara, N. Miyata, T. Suzuki, M. Toyoda, Mechanistic studies of catechins as antioxidants against radical oxidation, Free Radic. Biol. Med. 27 (1999) 855–863.

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Fig. 1 Stepwise elution curve of crude TPS on DEAE-52 column (a) and elution curve of main polysaccharide fraction (F-1) on Sepharose CL-4B column. Fig. 2 HPLC chromatogram of purified TPS on TSK-GEL G3000SWxl column. Fig. 3 FT-IR spectra of TPS and catechin-g-TPS. Fig. 4 1H NMR spectra (600 Hz, D2O) of TPS and catechin-g-TPS. Fig. 5 TGA (a) and DTG (b) curves of TPS and catechin-g-TPS. Fig. 6 XRD spectra of TPS and catechin-g-TPS. Fig. 7 SEM micrographs of TPS (a) and catechin-g-TPS (b) at magnification of 400×. Fig. 8 The DPPH scavenging activity (a) and reducing power (b) of TPS (-△-), catechin-g-TPS

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(-●-), catechin (-◆-) and Vc (-□-). Data are presented as means ± SD of triplicates.

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te

0.6 0.4 0.2

Ac ce p

Absorbance at 490nm

0.6

ip t

1.6

Concentration of NaCl (M)

(a)

0

0

342 343

10

20

30

40

50

Number of tube

Fig. 1

9

Page 9 of 16

3000

ip t

2000

1000

cr

Response (nRIU)

4000

0 5

10 Time (min)

344

te

d

M

an

Fig. 2

20

Ac ce p

345

15

us

0

10

Page 10 of 16

TPS 917 800

2933

1252

3407

1073

us

Catechin-g-TPS

1519

an

2934

1616

3000

2500

2000

610

1449 1240 1370

1500

800

10711036

1000

500

-1

Ac ce p

te

Fig. 3

d

Wavenumber (cm )

346 347

3500

M

3390

4000

611

cr

Transmittance (%)

1611 1423

ip t

1723

11

Page 11 of 16

us

cr

ip t

TPS

10.0

9.0

8.0

7.0

an

Catechin-g-TPS

6.0

5.0

4.0

3.0

2.0

1.0

0

M

ppm

348

te

d

Fig. 4

Ac ce p

349

12

Page 12 of 16

(a)

100

Catechin-g-TPS

cr

40 TPS

20 0 200

400

600

an

0

ip t

60

us

Weight (%)

80

800

Temperature (°C)

M

350

Catechin-g-TPS

Ac ce p

te

DTG (%/min)

d

(b)

0

200

TPS

400

600

800

Temperature (°C)

351 352

Fig. 5

13

Page 13 of 16

1200

TPS Catechin-g-TPS

cr

600

us

Intensity

800

an

400

M

200 0 20

40 2θ (deg)

60

80

353

Ac ce p

Fig. 6

te

d

0

354

ip t

1000

14

Page 14 of 16

b

ip t

a

te

d

M

an

us

Fig. 7

Ac ce p

356

cr

355

15

Page 15 of 16

120 100

ip t

80 60 40

cr

Scavenging activity (%)

(a)

20

0

0.2

us

0 0.4

0.6

0.8

1

0.8

1

an

Concentration (mg/ml) 357

M

1.6

d

1.2

te

0.8 0.4

Ac ce p

Absorbance at 700 nm

(b)

0

0

358 359

0.2

0.4

0.6

Concentration (mg/ml)

Fig. 8

360

16

Page 16 of 16