Ultrasound extraction optimization, structural features, and antioxidant activity of polysaccharides from Tricholoma matsutake

Ultrasound extraction optimization, structural features, and antioxidant activity of polysaccharides from Tricholoma matsutake 松茸多糖的超声波提取优化、结构特性和抗氧化活性研究 1 first-author 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:duxiuju@lcu.edu.cn †E-mail:duxiuju@lcu.edu.cn duxiuju@lcu.edu.cn 1 2 1 College of Life Science, Liaocheng University, Liaocheng 252059, China College of Life Science, Liaocheng University, Liaocheng 252059, China Agricultural Science Institute of Liaocheng, Liaocheng 252000, China Agricultural Science Institute of Liaocheng, Liaocheng 252000, China An ultrasonic-assisted technique was employed to extract crude polysaccharide from Tricholoma matsutake fruiting bodies. Single-factor tests and orthogonal experimental design ( L 9 (3 3 )) were used to obtain the optimal extraction conditions. Results showed that the optimal parameters were as follows: ultrasonic temperature, 40 °C; ultrasonic time, 50 min; water to raw material ratio, 25 ml/g; ultrasonic frequency, 45 kHz; and ultrasonic power, 100 W. Three novel T. matsutake polysaccharide (TMP) fractions (TMP30, TMP60, and TMP80) were isolated and purified from TMP by stepwise alcohol precipitation. Their preliminary structural features were determined by high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD) and Fourier transform infrared spectrophotometer (FT-IR) analyses. Furthermore, their in vitro antioxidant activity was investigated in terms of a reducing power assay and the scavenging rates of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals. The order of the various fractions based on their antioxidant activity was TMP80 > TMP > TMP60 > TMP30. These findings suggested that novel polysaccharide fractions from T. matsutake , especially TMP80, could be promising active macromolecules for biomedical use. 目的 2 对超声提取松茸多糖（TMP）的工艺条件进行优化，对TMP进一步分离纯化，并对纯化多糖（TMP30、TMP60和TMP80）的结构和抗氧化活性进行评估。 创新点 2 首次对超声辅助制备的TMP进行了乙醇分级，对其进行了结构和抗氧化活性评估，并筛选出了抗氧化活性最强的部位（TMP80）。 方法 2 采用单因素试验和正交试验（ L 9(3 3 )）对超声提取TMP的工艺条件进行了优化；采用不同的乙醇终浓度对TMP分离纯化为3种多糖TMP30、TMP60和TMP80；采用高效阴离子色谱（HPAEC-PAD）和傅里叶红外（FT-IR）等技术对3种多糖的理化性质进行了结构表征；利用还原力测定、二苯三硝基苯肼（DPPH）和羟自由基的清除作用对它们的抗氧化活性进行了评估。 结论 2 正交试验结果表明，超声提取TMP的最优工艺条件为：超声温度40 °C，超声时间50 min，水原料比例25 ml/g，超声波频率45 kHz和超声波功率100 W（表1和表2）。在此条件下的TMP得率为8.06%。采用乙醇分级法对TMP进一步分离纯化，制备得到3种多糖TMP30、TMP60和TMP80。HPAEC-PAD和FT-IR的结果表明，TMP30、TMP60和TMP80均主要由岩藻糖（L-Fuc）、半乳糖（D-Gal）、葡萄糖（D-Glc）、木糖（D-Xyl）和甘露糖（D-Man）组成，它们具有相同的单糖组成，但含有不同的摩尔比，且均有β-构型（图2、图3和表3）。抗氧化活性结果表明，它们的能力高低顺序为：TMP80>TMP>TMP60>TMP30（图4和表4），TMP80抗氧化活性最高，为松茸多糖的进一步开发利用提供重要的科学依据。 Tricholoma matsutake polysaccharide Orthogonal test design Ultrasound-assisted extraction Monosaccharide composition Antioxidant activity 松茸多糖 正交试验设计 超声辅助提取 单糖组成 抗氧化活性 Introduction 1 Tricholoma matsutake , known in China as Songrong, is a kind of ectomycorrhizal symbiotic fungus belonging to the subgenus Tricholoma . It contains a variety of nutrients, such as polysaccharides, proteins, volatile flavoring substances, and minerals. It is one of the most precious mushrooms in the world due to its unique taste and flavor, and its pharmacological properties (Kim et al., 2010a 2010a ). The fruiting bodies of T. matsutake have been used for the prevention and treatment of diseases for 1000 years in Asian countries, including Korea, Japan, and China (Yin et al., 2011 2011 ; You et al., 2013 2013 ). Recent studies have shown that water-soluble T. matsutake polysaccharides (TMPs) from its fruiting bodies are one of its main active components, and have a range of pharmacological effects such as antitumour (Yang et al., 2010 2010 ; You et al., 2013 2013 ), immunobiological (Hoshi et al., 2008 2008 ), antioxidative (Kim et al., 2010b 2010b ; You et al., 2013 2013 ), hematopoietic, and anti-mutagenic activity (Bohn and BeMiller, 1995 1995 ). However, until now, no detailed studies have been carried out on the physicochemical characteristics and antioxidative capacities of the three polysaccharide fractions fractionated by stepwise alcohol precipitation from T. matsutake fruiting bodies. Compared with conventional extraction methods, the ultrasound-assisted extraction (UAE) technique has many benefits, such as increased extraction yield, accelerated speed of extraction, shortened extraction duration, economical power consumption, and reduced use of energy and solvents (You et al., 2014 2014 ; Chemat et al., 2017 2017 ). UAE has proved recently to be a green and economically viable alternative to conventional extraction techniques for natural products and foods (You et al., 2014 2014 ; Chemat et al., 2017 2017 ). Also, compared with other test-design methods, orthogonal designs not only need fewer experiments, but also provide more information. Thus, an orthogonal design has been widely adopted in the optimization of experimental factors involved in extraction (Xu et al., 2005 2005 ; Ye et al., 2013 2013 ). In the present study, UAE was employed to extract the crude TMP from T. matsutake fruiting bodies. Based on single-factor tests, an orthogonal experiment ( L 9 (3 3 )) was used to optimize extraction conditions for UAE. We also focused on the preliminary determination of the structural features of three novel polysaccharides TMP30, TMP60, and TMP80, isolated and fractionated from TMP using different final concentrations of alcohol precipitation. In addition, the in vitro antioxidant capabilities of the fractions were estimated so as to seek new natural functional ingredients for use in the food and pharmaceutical industries. Materials and methods 1 Materials and reagent equipment 2 T . matsutake fruiting bodies were purchased from the Kunming Edible Fungi Institute of General National Supply and Marketing Cooperative of the People’s Republic of China Company (Kunming, China). Monosaccharide standards D -glucose ( D -Glc), D -galactose ( D -Gal), D -arabinose ( D -Ara), L -fucose ( L -Fuc), D -mannose ( D -Man), L -rhamnose ( L -Rha), D -fructose ( D -Fru), D -xylose ( D -Xyl), D -GlcA, and D -GalA were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,2-Diphenyl-1-picrylhydrazyl free radical (DPPH•) was purchased from the Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were bought from Sinopharm Chemical Reagent Co., Ltd., (Shanghai, China) and were of analytical reagent (AR) grade. Extraction optimization and fractionation of polysaccharide TMP 2 2.2.1 Extraction of TMP and yield determination Dried raw material (460 g) was ground into a powder and then pretreated with 85% ethanol at room temperature for 24 h to degrease and remove some small molecular materials, oligosaccharides, and some colored materials. The solid residue was separated from the solvent by centrifugation (1776 g , 10 min) and dried in air. Each pretreated sample (1.0 g) was extracted in an ultrasonic cleaner (KQ-100VDB double frequency numerical control ultrasonic cleaner, Kunshan Ultrasound Instrument Co., China) with distilled water. The extraction procedure was repeated once. The combined supernatant was concentrated to 1/5 of the original volume with a rotary evaporator. Dehydrated ethanol was slowly added to the concentrated supernatant to 80% (v/v) of the final alcohol content, then kept at 4 °C overnight. After centrifugation, dialysis, and lyophilization, the crude polysaccharide was obtained and named TMP. Single-factor experiments were performed under the following conditions: ultrasonic frequency (45 and 80 kHz), ultrasonic power (from 60 to 100 W), extraction temperature (from 40 to 80 °C), ultrasonic time (from 20 to 60 min), and ratio of water to raw material (from 15 to 35 ml/g). The yield (%) of TMP was calculated using the following formula: yield= M 1 / M 0 ×100%, where M 1 was the weight (g) of crude TMP, and M 0 was the weight (g) of pretreated material. 2.2.2 Optimization for UAE parameters of the polysaccharide TMP Based on the results of the single-factor experiments, two parameters (ultrasonic power and ultrasonic frequency) were selected at 100 W and 45 kHz, respectively. An orthogonal L 9 (3 3 ) test design in the extraction mode was applied to optimize the other extraction parameters. The independent variables, including the water to raw material ratio ( A ), ultrasonic time ( B ), and extraction temperature ( C ) at three levels in the extraction process, are shown in Table 1 1 . Nine-extraction tests (Table 2 2 ) were carried out and the crude TMPs were obtained according to the method mentioned above in Section 2.2.1, weighed, and then their extraction yields were calculated. Level Water to raw material ratio ( A , ml/g) Ultrasonic temperature ( B , °C) Ultrasonic time ( C , min) 1 20 50 30 2 25 60 40 3 30 70 50 k 1 , k 2 , and k 3 refer to the sums of the test results (extraction yield) corresponding to the numbers in the 1st, 2nd, and 3rd columns, respectively. R refers to the result of extreme difference analysis. TMP: T. matsutake polysaccharide 2 No. 3 Level 2 Yield of TMP (%) Water to raw material ratio ( A ) Ultrasonic temperature ( B ) Ultrasonic time ( C ) 1 1 1 1 6.13 2 1 2 2 6.42 3 1 3 3 7.87 4 2 1 2 6.51 5 2 2 3 7.53 6 2 3 1 7.60 7 3 1 3 6.33 8 3 2 1 6.73 9 3 3 2 7.22 5 k 1 6.81 6.32 6.82 k 2 7.21 6.89 6.72 k 3 6.76 7.56 7.24 R 0.45 1.24 0.52 2.2.3 Preparation and fractionation of three novel polysaccharides TMP30, TMP60, and TMP80 Under the optimal conditions, crude TMP was obtained from the pretreated dry powder of T. matsutake . A stepwise ethanol precipitation assay was used to fractionate the TMP (Du et al., 2013 2013 ; Zhao et al., 2013 2013 ). Briefly, dehydrated ethanol was slowly added to the concentrated supernatant to final alcohol concentrations of 30%, 60%, and 80%. Accordingly, three purified fractions termed TMP30, TMP60, and TMP80 were obtained. Monosaccharide compositions of three polysaccharide fractions 2 Each sample of polysaccharide (2 mg) was hydrolyzed with 2 mol/L trifluoroacetic acid (TFA) at 110 °C for 5 h. Ten kinds of monosaccharides, D -Glc, D -Gal, D -Ara, L -Fuc, L -Rha, D -Fru, D -Xyl, D -Man, D -GlcA, and D -GalA were used as standards. The column was eluted with 2 mol/L NaOH at a flow rate of 0.45 ml/min. The monosaccharide composition and the percentages of various polysaccharides were determined by high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD), using a CarboPac™ PA20 column (3 mm×150 mm) eluted with 2 mmol/L NaOH at a flow rate of 0.45 ml/min (Du et al., 2009 2009 ; 2013 2013 ). Fourier transform infrared analysis 2 Infrared (IR) analysis was performed using a Fourier transform infrared spectrophotometer (FT-IR, Scimitor 800, Varian, USA). Two milligrams of each polysaccharide sample and 100 mg of KBr were mixed, ground, and pressed into a tablet in an agate mortar. IR spectra were recorded in the frequency range of 4000–400 cm −1 (Ye et al., 2009 2009 ). Antioxidant activity assays 2 The reducing power and DPPH and hydroxyl radical-scavenging activity of the crude TMP and its fractions (TMP30, TMP60, and TMP80) were determined according to our previous report (Du et al., 2013 2013 ). Briefly, the reducing power assay was performed using the K 3 Fe(CN) 6 reduction method (Wang et al., 2012 2012 ), DPPH radical-scavenging activity was determined following Ardestani and Yazdanparast ( 2007 2007 ) with minor modifications, and the hydroxyl radical-scavenging activity was measured by the salicylic acid method (Smironff and Cumbes, 1989 1989 ). General methods 2 The total sugar content was measured using anthrone-sulfonic acid with glucose as a standard (Zhang, 1999 1999 ). The protein content was determined by Coomassie brilliant blue method Bradford using bovine serum albumin as the standard (Murphey et al., 1989 1989 ). The reducing sugar content was estimated by the 3,5-dinitrosalicylic acid (DNS) colorimetry method (Zhang, 1999 1999 ). Statistical analysis 2 All data are presented as mean±standard deviation (SD) and were analyzed by the analysis of variance (ANOVA) method. The significance of any differences between groups was evaluated using Student’s t -test. All computations were performed using STST (Nanjing Agricultural University, Nanjing, China). All experiments were carried out three times. Results and discussion 1 Effect of ultrasonic-assisted extraction conditions on the yield of TMP 2 3.1.1 Ultrasonic frequency Firstly, to assess the influence of ultrasonic frequency on the yield of TMP, different ultrasonic frequencies (45 and 80 kHz) were set. Other parameters were set as follows: ultrasonic power, 80 W; ultrasonic time, 30 min; ultrasonic temperature, 70 °C; water to raw material ratio, 25 ml/g. The results are shown in Fig. 1a 1a . When the ultrasonic frequency was set at 45 or 80 kHz, the yields of TMP were 5.55% and 5.38%, respectively, indicating that different ultrasonic frequencies affected the yield of TMP. So the optimal ultrasonic frequency was selected at 45 kHz for the orthogonal test. 3.1.2 Ultrasonic power Different ultrasonic powers have been shown to affect the yield of polysaccharides (Hu et al., 2016 2016 ). To investigate the influence of ultrasonic power on the extraction yield of TMP, in this study, the ultrasonic power was set at 60, 70, 80, 90, or 100 W while other parameters were set as follows: ultrasonic frequency, 45 kHz; ultrasonic time, 30 min; ultrasonic temperature, 70 °C; water to raw material ratio, 25 ml/g. The yield of TMP increased with increasing ultrasonic power from 60 to 100 W, increased only slowly from 80 to 90 W, and then reached a peak at 100 W (Fig. 1b 1b ). Thus, the optimal ultrasonic power was chosen as 100 W in orthogonal tests. 3.1.3 Water to raw material ratio It is reported that the extraction yield of polysaccharides is influenced by the ratio of water to raw material (Huang and Ning, 2010 2010 ). In this study, different ratios of water to raw material (15, 20, 25, 30, and 35 ml/g) were used to investigate their effects on the extraction yield of TMP. The yield of TMP rose clearly with an increasing ratio of water to material from 15 to 25 ml/g and reached a maximum at a ratio of 25 ml/g (Fig. 1c 1c ). However, when the ratio rose above 25 ml/g, the yield started to decline slowly. Thus, the optimal ratio of water to raw material was chosen as 25 ml/g. 3.1.4 Ultrasonic time The ultrasonic time usually significantly affects the extraction yield of polysaccharide (Zhao et al., 2013 2013 ). To assess its effect on the extraction yield of TMP, the ultrasonic time was set at 20, 30, 40, 50, or 60 min, while other extraction conditions were set as follows: ultrasonic frequency, 45 kHz; ultrasonic temperature, 70 °C; ultrasonic power, 80 W; water to raw material ratio, 25 ml/g. The extraction yield of TMP obviously increased within the initial 40 min, reached the maximum, and then decreased slightly from 40 to 60 min (Fig. 1d 1d ). Thus, the optimal ultrasonic time was chosen as 40 min. 3.1.5 Ultrasonic temperature Increasingly, studies have reported that ultrasonic temperature plays an important role in the extraction yield of polysaccharides (Hu et al., 2016 2016 ). In the present study, the effect of different temperatures (40, 50, 60, 70, and 80 °C) on the extraction yield of TMP was tested. The results indicated that the extraction yield significantly increased when the temperature increased from 40 to 60 °C, suddenly decreased at 70 °C, then increased at 80 °C (Fig. 1e 1e ). Therefore, the preferred ultrasonic temperature was selected as 60 °C. Optimization for extraction of TMP 2 The results of orthogonal tests and extreme difference analysis are presented in Table 2 2 . Here, the k and R values were calculated according to nine-extraction yields of TMP. The R values of the three factors were 0.45 ( A ), 1.24 ( B ), and 0.52 ( C ). Accordingly, the influence of extraction factors on yield decreased in the order B > C > A (ultrasonic temperature > ultrasonic time > ratio of water to raw material). That is to say, the ultrasonic temperature was found to be the most important determinant of the extraction yield. According to the k values of the three extraction factors, the optimal parameters would be the combination A 2 B 3 C 3 , where 1‒3 are levels. Integrating the results of the single-factor experiments with those of orthogonal tests, the optimal UAE conditions for TMP were obtained as follows: ratio of water to raw material, 25 ml/g; ultrasonic temperature, 40 °C; ultrasonic time, 50 min; ultrasonic frequency, 45 kHz; and ultrasonic power, 100 W. Moreover, through verified tests, the highest extraction yield of polysaccharide TMP was 8.06%. The UAE technique in recent years has been widely applied due to its high efficiency and extraction rate (Li et al., 2013 2013 ). Compared with the traditional hot-water extraction (HWE) method, the optimization results in this paper showed that UAE had increased TMP yield by 164%, and reduced the operation time and temperature by 320% and 137%, respectively (Yin et al., 2009 2009 ). Furthermore, determination of the optimal conditions for UAE may help the industrialization or up-scaling of the UAE process. So pilot-plant testing of extraction of TMP would be our next step (Achat et al., 2012 2012 ). However, it has been reported that ultrasound might lead to degradation of natural products (Pingret et al., 2012 2012 ). The main focus of this paper was screening for more bioactive TMPs. Moreover, our unpublished results showed that the TMPs extracted by the UAE technique had stronger bioactivity than those obtained by the HWE method. Whether TMPs extracted by UAE are degraded will be the subject of a future study. Characterization of the three polysaccharide fractions 2 3.3.1 Composition of the three polysaccharides The crude TMP extracted from T . matsutake fruiting bodies was obtained by a series of purification procedures such as ethanol infusion, ultrasonic extraction, and ethanol sedimentation. The total sugar, reducing sugar, polysaccharide, and protein content of the TMP were 35.81%, 4.54%, 31.27%, and 7.48%, respectively. After fractionation from the crude TMP using a sequence of different final concentrations of alcohol precipitation, three different polysaccharides (TMP30, TMP60, and TMP80) were successfully obtained. They accounted for 45.38%, 30.51%, and 24.11% of the TMP, respectively (Table 3 3 ). Their physicochemical properties, including their total sugar and protein content, and monosaccharide component, are summarized in Table 3 3 . The properties of polysaccharides, such as their composition and molar ratio, make an important contribution to their bioactivity (Lv et al., 2009 2009 ). In the present study, HPAEC-PAD was applied to identify and quantify the monosaccharide composition of various polysaccharides under the same analytical conditions. 2 Component 4 Content (%) a TMP30 TMP60 TMP80 TMP  Yield 45.38 30.51 24.11 8.06  Total sugar 41.71 57.49 20.55 35.81  Reducing sugar 4.12 4.41 4.58 4.54  Polysaccharide b 37.59 53.08 15.97 31.27  Protein content 5.73 3.52 11.63 7.48  Monosaccharide    L -Fucose 9.3 6.6 8.1    D -Galactose 26.8 17.6 21.2    D -Glucose 40.1 42.3 43.0    D -Xylose 2.6 12.1 4.2    D -Mannose 16.4 21.1 23.6 The HPAEC-PAD results of 10 monosaccharide standards are shown in Fig. 2a 2a , and the monosaccharide compositions of TMP30, TMP60, and TMP80 in Figs. 2b–2d 2b–2d , respectively. By matching the retention time with those of monosaccharide standards, five peaks were identified in the three fractions in the order L -Fuc, D -Gal, D -Glc, D -Xyl, and D -Man. That is to say, TMP30, TMP60, and TMP80 had the same monosaccharide composition, but different molar ratios. Comparing Fig. 2b 2b with Fig. 2a 2a , TMP30 was found to consist of L -Fuc, D -Gal, D -Glc, D -Xyl, and D -Man in a molar ratio of 9.3:26.8:40.1:2.6:16.4 (Table 3 3 and Fig. 2 2 ). Similarly, TMP60 and TMP80 contained the same monosaccharide components in a molar ratio of 6.6:17.6:42.3:12.1:21.1 and 8.1:21.2:43.0: 4.2:23.6, respectively (Figs. 2c and 2d 2c and 2d , and Table 3 3 ). Obviously, glucose was the primary monosaccharide in the three polysaccharide fractions, with molar ratios of 40.1%, 42.3%, and 43.0%, respectively (Table 3 3 and Fig. 2 2 ). However, one unknown peak was detected (Fig. 2d 2d ), associated with a retention time at 9.51 min, which might imply the presence of a new compound in TMP80. Peaks: 1, L -fucose; 2, L -rhamnose; 3, L -arabinose; 4, D -galactose; 5, D -glucose; 6, D -xylose; 7, D -mannose; 8, D -fructose; 9, D -GalA; 10, D -GluA. HPAEC-PAD: high-performance anion-exchange chromatography with pulsed-amperometric detection. nC refers to the unit “nano-Coulomb” that represents electrical signal value 3.3.2 FT-IR analysis The structural features of the four polysaccharide fractions (TMP30, TMP60, TMP80, and TMP) were further analyzed by FT-IR spectra (Fig. 3 3 ). The FT-IR spectra of the four fractions were similar (Fig. 3 3 ), indicating that they had a similar structure, which was in accordance with the monosaccharide composition analysis by HPAEC-PAD. The broad and strong bands at 3395‒3386 cm −1 were assigned to the O–H stretching vibration. The small peaks at 2931‒ 2922 cm −1 were due to the stretching and bending vibrations of C–H (Hu et al., 2016 2016 ). Bands at both 1650‒1635 cm −1 and 1417‒1406 cm −1 were observed, corresponding to symmetric and asymmetric stretching, respectively, of C=O (Afshari et al., 2015 2015 ). In addition, the absorptions at 1073‒1071 cm −1 (Fig. 3 3 ) indicated a pyranose form of sugar (Wang et al., 2016 2016 ). A weak absorption at 897 cm −1 was observed in spectra of the four polysaccharide fractions, suggesting that pyranoses existed in the β-configuration in TMP30, TMP60, TMP80, and TMP (Luo et al., 2011 2011 ). Antioxidant activity 2 3.4.1 Reducing power The reducing power is generally used to evaluate the potential antioxidant activity of polysaccharides (Devasagayam et al., 2004 2004 ). Reducing power was determined by measuring the formation of Prussian blue at 700 nm. A higher absorbance value at 700 nm indicated a relative stronger reduction force. It was clear that the reducing power of the four polysaccharide fractions increased with increasing concentration in the range of 62.5‒1200 µg/ml (Fig. 4a 4a ). The RP 0.5AU values (defined as the effective concentration at which the absorbance was 0.5 for reducing power) of TMP30, TMP60, TMP80, and TMP were 4.33, 4.30, 1.39, and 2.15 mg/ml, respectively (Table 4 4 ). Therefore, the reducing power of the four fractions could be listed in the following order: TMP80 > TMP > TMP60 > TMP30. To some extent, this represents the order of potential antioxidant activity of the four fractions. EC 50 value: the effective concentration at which the antioxidant activity was 50%. RP 0.5AU value: the effective concentration at which the absorbance was 0.5 for reducing power 2 Fraction 2 EC 50 value (mg/ml) 2 RP 0.5AU value (mg/ml) DPPH-scavenging activity assay Hydroxyl-scavenging activity assay TMP30 0.86 3.73 4.33 TMP60 0.83 3.58 4.30 TMP80 0.26 2.53 1.39 TMP 0.72 3.06 2.15 (a) Reducing power; (b) Scavenging activity to DPPH radical; (c) Scavenging activity to hydroxyl radical. Data are presented as the mean values ( n =3) 3.4.2 DPPH radical-scavenging activity DPPH radical-scavenging activity is also commonly used to represent a compound’s antioxidant activity. A higher scavenging rate of DPPH radicals indicates that the antioxidant ability of a material is stronger (Brand-Williams et al., 1995 1995 ). Various fractions showed a dose-dependent scavenging effect at the tested dosage (62.5‒1500 µg/ml) (Fig. 4b 4b ). EC 50 values, defined as the effective concentration at which the antioxidant activity is 50%, generally serve as important indicators of radical-scavenging activity. Lower EC 50 values indicate stronger radical-scavenging activity. The EC 50 values of TMP30, TMP60, TMP80, and TMP were 0.86, 0.83, 0.26, and 0.72 mg/ml, respectively (Table 4 4 ). Thus, the order of the four fractions in terms of their DPPH-scavenging activity was TMP80 > TMP > TMP60 > TMP30, which was the same as that of the reducing power. 3.4.3 Hydroxyl radical-scavenging activity A hydroxyl radical-scavenging assay is also generally applied to assess the antioxidant effect of polysaccharides because hydroxyl free radicals may directly or indirectly cause tissue damage and induce many human diseases (Tursun et al., 2010 2010 ). The results for TMP30, TMP60, TMP80, and TMP are shown in Fig. 4c 4c . TMP80 exhibited the strongest hydroxyl radical-scavenging ability. The EC 50 of TMP80 was 2.53 mg/ml, which was significantly lower than that of TMP30 (3.73 mg/ml), TMP60 (3.58 mg/ml), and TMP (3.06 mg/ml). That is to say, the order of the four fractions based on their hydroxyl radical-scavenging activity was TMP80 > TMP > TMP60 > TMP30 (Fig. 4c 4c and Table 4 4 ), which was similar to that of reducing power and DPPH radical-scavenging effects. Conclusions 1 In this study, UAE technology was employed to prepare crude TMP from T. matsutake fruiting bodies. Single-factor tests and orthogonal experimental design ( L 9 (3 3 )) were used to obtain the optimal extraction conditions. Results showed that the extraction time, extraction temperature, and water to raw material ratio were the main factors influencing the yield of TMP. The optimal extraction conditions consisted of an ultrasonic temperature of 40 °C, an ultrasonic time of 50 min, and a water to raw material ratio of 25 ml/g. Under the optimal conditions, the yield of TMP was 8.06%. Three novel polysaccharide fractions, TMP30, TMP60, and TMP80, were isolated and purified from the crude TMP fraction using the final concentration of alcohol precipitation at 30%, 60%, and 80%, respectively. Their physicochemical properties, including total sugar and protein content, monosaccharide composition, and molar ratio, were studied because of their possible association with antioxidant capacities. Their chemical constituent and structural characteristics were determined using HPAEC-PAD and FT-IR analyses. The results showed that TMP30, TMP60, and TMP80 had almost the same monosaccharide composition (they all consisted mainly of L -Fuc, D -Gal, D -Glc, D -Xyl, and D -Man) but in different ratios. FT-IR spectra showed that pyranoses might exist in the β-configuration in TMP30, TMP60, TMP80, and TMP. Furthermore, compared with other polysaccharides, one unknown peak in the HPAEC-PAD spectrum of TMP80 was detected, which might imply the presence of a new compound in this fraction, worthy of further investigation. The in vitro antioxidant activity of the polysaccharides was investigated based on a reducing power assay and scavenging of DPPH and hydroxyl radicals. The results of antioxidant activity assays showed that the various polysaccharide fractions exhibited increasing antioxidant activity with increasing dosage within the range tested. The order of reducing power was TMP80>TMP>TMP60>TMP30, which was the same as that of the DPPH and hydroxyl radical-scavenging capacities. In other words, TMP80 exhibited the strongest antioxidant activity among the fractions tested. These results indicated that fractionation by stepwise ethanol precipitation is an effective way to select polysaccharides with enhanced antioxidant activity. The differences in antioxidant activity might have been due to the physicochemical properties of the fractions, such as their total sugar and protein content, monosaccharide composition, and molar ratios. Antioxidants can protect against excessive free radicals that have been implicated in the etiology of many diseases (Devasagayam et al., 2004 2004 ). The findings reported here suggested that three polysaccharide fractions extracted from T. matsutake , especially TMP80, could be developed as ingredients in healthy and functional food to prevent or alleviate oxidative stress. The structural features of TMP80, especially their structure-function relationships, will be the focus of our continuing work. References S Achat , V Tomao , K Madani , 等 . Direct enrichment of olive oil in oleuropein by ultrasound-assisted maceration at laboratory and pilot plant scale . Ultrason Sonochem , 2012 . 19 ( 4 ): 777 - 786 . 22281378 DOI: 10.1016/j.ultsonch.2011.12.006 http://doi.org/10.1016/j.ultsonch.2011.12.006 . Direct enrichment of olive oil in oleuropein by ultrasound-assisted maceration at laboratory and pilot plant scale K Afshari , V Samavati , SA Shahidi . Ultrasonic-assisted extraction and in-vitro antioxidant activity of polysaccharide from Hibiscus leaf . Int J Biol Macromol , 2015 . 74 558 - 567 . 25150112 DOI: 10.1016/j.ijbiomac.2014.07.023 http://doi.org/10.1016/j.ijbiomac.2014.07.023 . Ultrasonic-assisted extraction and in-vitro antioxidant activity of polysaccharide from Hibiscus leaf A Ardestani , R Yazdanparast . Antioxidant and free radical scavenging potential of Achillea santolina extracts . Food Chem , 2007 . 104 ( 1 ): 21 - 29 . DOI: 10.1016/j.foodchem.2006.10.066 http://doi.org/10.1016/j.foodchem.2006.10.066 . Antioxidant and free radical scavenging potential of Achillea santolina extracts JA Bohn , JN BeMiller . (1→3)-β- D -Glucans as biological response modifiers: a review of structure-functional activity relationships . Carbohydr Polym , 1995 . 28 ( 1 ): 3 - 14 . DOI: 10.1016/0144-8617(95)00076-3 http://doi.org/10.1016/0144-8617(95)00076-3 . (1→3)-β-D-Glucans as biological response modifiers: a review of structure-functional activity relationships W Brand-Williams , ME Cuvelier , C Berset . Use of a free radical method to evaluate antioxidant activity . LWT-Food Sci Technol , 1995 . 28 ( 1 ): 25 - 30 . DOI: 10.1016/S0023-6438(95)80008-5 http://doi.org/10.1016/S0023-6438(95)80008-5 . Use of a free radical method to evaluate antioxidant activity F Chemat , N Rombaut , A Sicaire , 等 . Ultrasound assisted extraction of food and natural products. Mechanisms, techniques, combinations, protocols and applications. A review . Ultrason Sonochem , 2017 . 34 540 - 560 . 27773280 DOI: 10.1016/j.ultsonch.2016.06.035 http://doi.org/10.1016/j.ultsonch.2016.06.035 . Ultrasound assisted extraction of food and natural products. Mechanisms, techniques, combinations, protocols and applications. A review TPA Devasagayam , JC Tilak , KK Boloor , 等 . Free radicals and antioxidants in human health: current status and future prospects . J Assoc Phys India , 2004 . 52 794 - 804 . . Free radicals and antioxidants in human health: current status and future prospects XJ Du , Y Yang , LB Ye , 等 . Structural elucidation and immuno-stimulating property of an acidic heteropolysaccharide (TAPA1) from Tremella aurantialba . Carbohydr Res , 2009 . 344 ( 5 ): 672 - 678 . 19223030 DOI: 10.1016/j.carres.2009.01.021 http://doi.org/10.1016/j.carres.2009.01.021 . Structural elucidation and immuno-stimulating property of an acidic heteropolysaccharide (TAPA1) from Tremella aurantialba XJ Du , HM Mu , S Zhou , 等 . Chemical analysis and antioxidant activity of polysaccharides extracted from Inonotus obliquus sclerotia . Int J Biol Macromol , 2013 . 62 ( 11 ): 691 - 696 . 24145301 DOI: 10.1016/j.ijbiomac.2013.10.016 http://doi.org/10.1016/j.ijbiomac.2013.10.016 . Chemical analysis and antioxidant activity of polysaccharides extracted from Inonotus obliquus sclerotia H Hoshi , B Iijima , Y Ishihara , 等 . Absorption and tissue distribution of an immunomodulatory α- D -glucan after oral administration of Tricholoma matsutake . J Agric Food Chem , 2008 . 56 ( 17 ): 7715 - 7720 . 18680305 DOI: 10.1021/jf801123k http://doi.org/10.1021/jf801123k . Absorption and tissue distribution of an immunomodulatory α-D-glucan after oral administration of Tricholoma matsutake J Hu , XJ Jia , XB Fang , 等 . Ultrasonic extraction, antioxidant and anticancer activities of nove lpolysaccharides from Chuanxiong rhizome . Int J Biol Macromol , 2016 . 85 277 - 284 . 26712703 DOI: 10.1016/j.ijbiomac.2015.12.046 http://doi.org/10.1016/j.ijbiomac.2015.12.046 . Ultrasonic extraction, antioxidant and anticancer activities of nove lpolysaccharides from Chuanxiong rhizome SQ Huang , ZX Ning . Extraction of polysaccharide from Ganoderma lucidum and its immune enhancement activity . Int J Biol Macromol , 2010 . 47 ( 3 ): 336 - 341 . 20371373 DOI: 10.1016/j.ijbiomac.2010.03.019 http://doi.org/10.1016/j.ijbiomac.2010.03.019 . Extraction of polysaccharide from Ganoderma lucidum and its immune enhancement activity SS Kim , JS Lee , JY Cho , 等 . Effects of C/N ratio and trace elements on mycelial growth and exo-polysaccharide production of Tricholoma matsutake . Biotechnol Bioproc E , 2010 . 15 ( 2 ): 293 - 298 . DOI: 10.1007/s12257-008-0226-x http://doi.org/10.1007/s12257-008-0226-x . Effects of C/N ratio and trace elements on mycelial growth and exo-polysaccharide production of Tricholoma matsutake SS Kim , JS Lee , JY Cho , 等 . Process development for mycelial growth and polysaccharide production in Tricholoma matsutake liquid culture . J Biosci Bioeng , 2010 . 109 ( 4 ): 351 - 355 . 20226376 DOI: 10.1016/j.jbiosc.2009.10.010 http://doi.org/10.1016/j.jbiosc.2009.10.010 . Process development for mycelial growth and polysaccharide production in Tricholoma matsutake liquid culture Y Li , AS Fabiano-Tixier , V Tomao , 等 . Green ultrasound-assisted extraction of carotenoids based on the bio-refinery concept using sunflower oil as an alternative solvent . Ultrason Sonochem , 2013 . 20 ( 1 ): 12 - 18 . 22884112 DOI: 10.1016/j.ultsonch.2012.07.005 http://doi.org/10.1016/j.ultsonch.2012.07.005 . Green ultrasound-assisted extraction of carotenoids based on the bio-refinery concept using sunflower oil as an alternative solvent Q Luo , J Zhang , L Yan , 等 . Composition and antioxidant activity of water-soluble polysaccharides from Tuber indicum . J Med Food , 2011 . 14 ( 12 ): 1609 - 1616 . 21877953 DOI: 10.1089/jmf.2011.1659 http://doi.org/10.1089/jmf.2011.1659 . Composition and antioxidant activity of water-soluble polysaccharides from Tuber indicum Y Lv , XB Yang , Y Zhao , 等 . Separation and quantification of component monosaccharides of the tea polysaccharides from Gynostemma pentaphyllum by HPLC with indirect UV detection . Food Chem , 2009 . 112 ( 3 ): 742 - 746 . DOI: 10.1016/j.foodchem.2008.06.042 http://doi.org/10.1016/j.foodchem.2008.06.042 . Separation and quantification of component monosaccharides of the tea polysaccharides from Gynostemma pentaphyllum by HPLC with indirect UV detection JM Murphey , SE Spayd , JR Powers . Effect of grape maturation on soluble protein characteristics of Gewurztraminer and White Riesling juice and wine . Am J Enol Viticult , 1989 . 40 ( 3 ): 199 - 207 . . Effect of grape maturation on soluble protein characteristics of Gewurztraminer and White Riesling juice and wine D Pingret , G Durand , A Fabiano-Tixier , 等 . Degradation of edible oil during food processing by ultrasound: electron paramagnetic resonance, physicochemical, and sensory appreciation . J Agric Food Chem , 2012 . 60 ( 31 ): 7761 - 7768 . 22804736 DOI: 10.1021/jf301286f http://doi.org/10.1021/jf301286f . Degradation of edible oil during food processing by ultrasound: electron paramagnetic resonance, physicochemical, and sensory appreciation N Smironff , QJ Cumbes . Hyroxyl radical scavenging activity of compatible solutes . Phytochemistry , 1989 . 28 ( 4 ): 1051 - 1060 . DOI: 10.1016/0031-9422(89)80182-7 http://doi.org/10.1016/0031-9422(89)80182-7 . Hyroxyl radical scavenging activity of compatible solutes K Tursun , R Zhan , H Zhang , 等 . Study on antioxidant activity of Acroptilon repens . Lett Biol , 2010 . 21 ( 3 ): 406 - 411 . (in Chinese) DOI: 10.3969/j.issn.1009-0002.2010.03.025 http://doi.org/10.3969/j.issn.1009-0002.2010.03.025 . Study on antioxidant activity of Acroptilon repens NN Wang , Y Zhang , XP Wang , 等 . Antioxidant property of water-soluble polysaccharides from Poria cocos Wolf using different extraction methods . Int J Biol Macromol , 2016 . 83 103 - 110 . 26601761 DOI: 10.1016/j.ijbiomac.2015.11.032 http://doi.org/10.1016/j.ijbiomac.2015.11.032 . Antioxidant property of water-soluble polysaccharides from Poria cocos Wolf using different extraction methods YF Wang , ZW Yang , XL Wei . Antioxidant activities potential of tea polysaccharide fractions obtained by ultrafiltration . Int J Biol Macromol , 2012 . 50 ( 3 ): 558 - 564 . 22230610 DOI: 10.1016/j.ijbiomac.2011.12.028 http://doi.org/10.1016/j.ijbiomac.2011.12.028 . Antioxidant activities potential of tea polysaccharide fractions obtained by ultrafiltration F Xu , M Jabasini , Y Baba . Screening of mixed poly (ethylene oxide) solutions for microchip separation of double-stranded DNA using an orthogonal design approach . Electrophoresis , 2005 . 26 ( 15 ): 3013 - 3020 . 16007697 DOI: 10.1002/elps.200410434 http://doi.org/10.1002/elps.200410434 . Screening of mixed poly (ethylene oxide) solutions for microchip separation of double-stranded DNA using an orthogonal design approach S Yang , XD Ren , JX Sheng , 等 . Preparation and the antitumor activity in vitro of polysaccharides from Tricholoma matsutake . W J Microbiol Biotechnol , 2010 . 26 ( 3 ): 497 - 503 . DOI: 10.1007/s11274-009-0196-y http://doi.org/10.1007/s11274-009-0196-y . Preparation and the antitumor activity in vitro of polysaccharides from Tricholoma matsutake C Ye , N Han , FK Teng , 等 . Extraction optimization of polysaccharides of Schisandrae Fructus and evaluation of their analgesic activity . Int J Biol Macromol , 2013 . 57 ( 6 ): 291 - 296 . 23511049 DOI: 10.1016/j.ijbiomac.2013.03.025 http://doi.org/10.1016/j.ijbiomac.2013.03.025 . Extraction optimization of polysaccharides of Schisandrae Fructus and evaluation of their analgesic activity LB Ye , JS Zhang , Y Yang , 等 . Structural characterisation of a heteropolysaccharide by NMR spectra . Food Chem , 2009 . 112 ( 4 ): 962 - 966 . DOI: 10.1016/j.foodchem.2008.07.017 http://doi.org/10.1016/j.foodchem.2008.07.017 . Structural characterisation of a heteropolysaccharide by NMR spectra XL Yin , QH You , ZH Jiang . Extraction and purification of Tricholoma matsutake polysaccharides . China Brewing , 2009 . 211 ( 10 ): 171 - 173 . . Extraction and purification of Tricholoma matsutake polysaccharides XL Yin , QH You , ZH Jiang . Optimization of enzyme assisted extraction of polysaccharides from Tricholoma matsutake by response surface methodology . Carbohydr Polym , 2011 . 86 ( 3 ): 1358 - 1364 . DOI: 10.1016/j.carbpol.2011.06.053 http://doi.org/10.1016/j.carbpol.2011.06.053 . Optimization of enzyme assisted extraction of polysaccharides from Tricholoma matsutake by response surface methodology LJ You , Q Gao , MY Feng , 等 . Structural characterisation of polysaccharides from Tricholoma matsutake and their antioxidant and antitumour activities . Food Chem , 2013 . 138 ( 4 ): 2242 - 2249 . 23497882 DOI: 10.1016/j.foodchem.2012.11.140 http://doi.org/10.1016/j.foodchem.2012.11.140 . Structural characterisation of polysaccharides from Tricholoma matsutake and their antioxidant and antitumour activities QH You , XL Yin , CW Ji . Pulsed counter-current ultrasound-assisted extraction and characterization of polysaccharides from Boletus edulis . Carbohydr Polym , 2014 . 101 ( 1 ): 379 - 385 . 24299786 DOI: 10.1016/j.carbpol.2013.09.031 http://doi.org/10.1016/j.carbpol.2013.09.031 . Pulsed counter-current ultrasound-assisted extraction and characterization of polysaccharides from Boletus edulis WJ Zhang . Biochemical Research Technology on Glycoconjugates, 2nd Ed , 1999 . Zhejiang University Press, Hangzhou , (in Chinese) . Biochemical Research Technology on Glycoconjugates, 2nd Ed Z Zhao , X Xu , Q Ye , 等 . Ultrasound extraction optimization of Acanthopanax senticosus polysaccharides and its antioxidant activity . Int J Biol Macromol , 2013 . 59 ( 4 ): 290 - 294 . 23628583 DOI: 10.1016/j.ijbiomac.2013.04.067 http://doi.org/10.1016/j.ijbiomac.2013.04.067 . Ultrasound extraction optimization of Acanthopanax senticosus polysaccharides and its antioxidant activity Compliance with ethics guidelines Yun CHEN, Xiu-ju DU, Yang ZHANG, Xin-hua LIU, and Xuan-dong WANG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1600239 Project supported by the Natural Science Foundation of Shandong Province of China (No. ZR2015CL002) and the Doctoral Research Startup Foundation of Liaocheng University (No. 31805), China 2016-05-24 2016-09-12 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 18