Structural characterization, antioxidant and hepatoprotective activities of polysaccharides from Sophorae tonkinensis Radix

Accepted Manuscript Title: Structural characterization, antioxidant and hepatoprotective activities of polysaccharides from Sophorae tonkinensis Radix Authors: Liangliang Cai, Shanshan Zou, Dengpan Liang, Libiao Luan PII: DOI: Reference:

S0144-8617(17)31500-X https://doi.org/10.1016/j.carbpol.2017.12.083 CARP 13145

To appear in: Received date: Revised date: Accepted date:

12-8-2017 1-11-2017 31-12-2017

Please cite this article as: Cai, Liangliang., Zou, Shanshan., Liang, Dengpan., & Luan, Libiao., Structural characterization, antioxidant and hepatoprotective activities of polysaccharides from Sophorae tonkinensis Radix.Carbohydrate Polymers https://doi.org/10.1016/j.carbpol.2017.12.083 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.

Structural

characterization,

antioxidant

and

hepatoprotective

activities of polysaccharides from Sophorae tonkinensis Radix Liangliang Cai, Shanshan Zou, Dengpan Liang, Libiao Luan*

Highlights Two polysaccharides had been isolated from Sophorae tonkinensis Radix.

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Preliminary characterization of the two polysaccharides was investigated.

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In vitro antioxidant activities of the two polysaccharides were studied.

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Polysaccharides reduced acetaminophen-induced liver injury in mice.

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Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009, China

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ABSTRACT

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In current study we present two polysaccharides, STRP1 and STRP2, purified from Sophorae tonkinensis Radix via column chromatography. Structural analyses indicated that STRP1 and STRP2 were consisted of mannose, rhamnose, glucuronic acid, glucose, galactose and arabinose in a similar molar ratio with main backbones of (1→3)-linked-α-D-Gal and (1→4)-linked-α-D-Glc, while average molecular weights were 1.30×104 and 1.98×105 Da, respectively. We observed a strong chelating ability on ferrous ions; substantial radical scavenging activities on DPPH, hydroxyl and superoxide anion radicals in vitro; and significant attenuation on acetaminophen-induced hepatic oxidative damage in mice for STRP1 and STRP2. The promising data on these polysaccharides showcase the need to further develop novel natural antioxidant and liver-protecting drugs.

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Keywords: Sophorae tonkinensis Radix polysaccharides; structural characterization; antioxidant activity; hepatoprotective activity.

1. Introduction

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Sophorae tonkinensis Radix (S. tonkinensis) is the processed lateral root of Sophora subprostrata (Leguminosae) which is widely distributed over the southwestern provinces of China (Cho, Chuang, & Chen, 1986). This plant is commonly used as a traditional Chinese medicine, Abbreviations: STRP, Sophorae tonkinensis Radix polysaccharides; APAP, acetaminophen; FT-IR, fourier transform infrared; HPLC, high performance liquid chromatography; HPGPC, high performance gel permeation chromatography; GC-MS, gas chromatography and mass Spectrometry; NMR, nuclear magnetic resonance spectrometer; SEM, Scanning electron microscopy; H&E, hematoxylin-eosin; AST, aspartate aminotransferases; MDA, malondialdehyde; ROS, reactive oxygen species; GSH, generation and increasing liver glutathione; GPx, glutathione peroxidase; T-SOD, total superoxide dismutase; CAT, catalase; DPPH, 1,1-Diphenyl-2-picrylhydrazyl. *Corresponding authors at: Department of Pharmaceutics, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China. E-mail address: [email protected]

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under the name Shan-dou-gen, for the treatment of acute pharyngolaryngeal infections and sore throat. Previous phytochemical studies revealed that its major constituents are quinolizidine alkaloids, flavonoids, and triterpenoids (Ding, Huang, & Chen, 2006; X. N. Li et al., 2008). Current researches have demonstrated that Sophorae tonkinensis Radix possesses multiple pharmacological activities involving anti-tumor, anti-gastrelcoma and gastric acid secretion inhibition (Ding et al., 2006). However, little attention has been paid to polysaccharides from Sophorae tonkinensis Radix and their associated bioactivities. Polysaccharides are natural polymers that exist universally in plants, animals and microorganisms. In the past decades, polysaccharides were found to play a critical role in the growth and development of living organisms (Cosgrove, 2005). In particular, plant polysaccharides have attracted increasing attentions due to diverse biological and physiological activities, such as anti-tumor effects (J. H. Xie et al., 2013; Zhu et al., 2016), antioxidant (Y. Huang, Li, Wan, Zhang, & Yan, 2015; Tseng, Yang, & Mau, 2008), anti-diabetic (Liu et al., 2013), anti-coagulation (Wijesinghe, Athukorala, & Jeon, 2011), and enhancing human immunity (Jeong, Jeong, Lee, & Kim, 2015; Yongxu & Jicheng, 2008). Most of polysaccharides have been proved to be natural and nontoxic, ideally for producing healthcare foods or medicines. The promising bioactivities of polysaccharides subsequently make it the focus of investigation as new drugs. Reactive oxygen species (ROS), including singlet oxygen (1O2), hydrogen peroxide (H2O2) as well as superoxide anion (O2•−) and hydroxyl (•OH) free radicals, can be generated during normal cellular metabolic processes such as respiration or various chemical reactions (e.g. ionizing and ultraviolet-light radiations) (Fan et al., 2014). However, excessively high levels of ROS may compromise tissues and induce oxidative injuries through biomolecule damages, causing serious age-related disorders or other severe diseases such as cognitive impairment, Parkinson’s disease, cancer and liver diseases (J. E. Li, Fan, Qiu, Li, & Nie, 2015; Zeng et al., 2016). Liver is an important organ for metabolizing endogenous and exogenous substances. Liver damage, a widespread health problem, can be induced by alcohol, chemicals, drugs, infections and other exogenous substances, resulting in impairment in these crucial metabolic functions. Acetaminophen (APAP), a commonly used over-the-counter antipyretic and analgesic agent, is associated with few adverse effects when taken under therapeutic doses while overdose can lead to severe hepatic injuries in human and animals. An APAP-induced mouse model is commonly used since it not only recapitulates similar physiological and genetic profiles to human but presents the advantage to be relatively economic in case of maintaining long-term studies (Kurahashi et al., 2016; Z et al., 2014). Recently, this model was used to evaluate the hepatoprotective activities of plant extracts or drugs. Several studies showed that oxidative stress was an important factor in the pathogenesis of APAP-induced liver injury and therefore natural products, containing antioxidants, protected the liver against liver injury by APAP (Jadeja, Urrunaga, Dash, Khurana, & Saxena, 2015). Accordingly, we hypothesized that polysaccharides, important constituents of Sophorae tonkinensis Radix, also have antioxidant and hepatoprotective effects. However, to our knowledge, there is limited literature on the purification, characterization, antioxidant and hepatoprotective activities of water-soluble polysaccharides from Sophorae tonkinensis Radix. Here, we obtained the crude Sophorae tonkinensis Radix polysaccharides (STRP) using water extraction and ethanol precipitation method, followed by column chromatography purification. As a result, two purified polysaccharides (STRP1 and STRP2) were obtained with preliminary characterizations via Fourier transform infrared (FT-IR) spectroscopy, ultraviolet (UV)

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spectroscopy, high performance liquid chromatography (HPLC), high performance gel permeation chromatography (HPGPC), gas chromatography and mass spectrometry (GC-MS), nuclear magnetic resonance spectrometer (NMR) and scanning electron microscopy (SEM). Additionally, the in vitro antioxidant activities (via various radical scavenging methods) and in vivo hepatoprotective activities in mice (via evaluation of the antioxidant enzymes activity) were extensively investigated for both STRP1 and STRP2. The favorable results of our investigation provide a robust basis to the development of novel polysaccharide antioxidants. 2. Materials and methods

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2.1. Materials and reagents

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Sophorae tonkinensis Radix was purchased from a local market in Guangxi, China. All the samples were identified by Doctor Hongbing Zhang in China Pharmaceutical University, China. DEAE-52 cellulose was obtained from Whatman (Balston, UK). SephacrylTM S-200 was purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). NaBH4, NaOH, KBr, concentrated sulfuric acid, trifluoroacetic acid (TFA), formaldehyde, dichloromethane (DCM), phenol and methyl iodide were purchased from Nanjing Chemical Reagent CO., Ltd (Nanjing, China). Bovine serum albumin (BSA), coomassie brilliant blue (CBB) and N-acetylcysteine were purchased from Shanghai Aladdin-Reagent Co., Ltd (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Shanghai Ling Feng Chemical Reagent Co., Ltd (Shanghai, China). 1-phenyl-3-methyl-5-pyrazolone (PMP) was from Xiya Chemical Industry Co., Ltd (Shandong, China). L-Rhamnose, D-Glucose and D-Galactose were from Beijing Zhongke Biochemical Technology Co., Ltd (Beijing, China). D-Mannose, L-Arabinose and D-Glucuronic acid were from the National Institutes for Food and Drug Control (Beijing, China). Dextran standards (4320, 12600, 60600, 110000 and 289000 Da) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd (Shanghai, China). 1,1-Diphenyl-2-picrylhydrazyl (DPPH), ferrous chloride, ferrous sulfate, pyrogallic acid, ferrozine and ascorbic acid (Vc), ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). APAP was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Aspartate aminotransferases (AST), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GPx), total superoxide dismutase (T-SOD) and catalase (CAT) assay kits were all obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Reactive oxygen species (ROS) ELISA assay Kit was purchased from Nanjing Jin Yibai Biological Science and Technology Co., Ltd (Nanjing, China). 2.2. Extraction of crude polysaccharide

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Sophorae tonkinensis Radix was washed by distilled water and dried in an oven at 70 °C. The dried Sophorae tonkinensis Radix was smashed using a universal high-speed smashing machine and sieved through a 60-mesh screen. Then the powder was extracted with 95% ethanol to remove impurities and small lipophilic molecules. Finally, the residue was separated by filtration and dried at 40 C in a hot air oven to obtain the pretreated dry sample powder, which (50.0 g) was then immersed with distilled water (1150 mL) for 3 h at 100 C. After centrifugation at 4000 rpm for 15 min, the residue was extracted repeatedly twice, and all supernatants were combined and concentrated to 100 mL volume. Subsequently, anhydrous ethanol (400 mL) was added to the concentrated supernatants with constant stirring to achieve a final concentration of 80% ethanol 3

and kept at 4 C overnight. The resulting precipitate (crude polysaccharide) was collected by centrifugation. The crude polysaccharide was transferred into bottles and treated with 1/5th the volume of the sample of the Sevage reagent (butyl alcohol/chloroform = 1/4, v/v) to remove proteins. This treatment was repeated until no remaining of white denatured proteins in the interphase. The crude Sophorae tonkinensis Radix polysaccharide was stored at -20 C after freeze drying. The extraction yield was calculated by the weight ratio of crude Sophorae tonkinensis Radix polysaccharides and dried Sophorae tonkinensis Radix powder. 2.3. Separation and purification of STRP

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Crude polysaccharide was purified based on the reported method with some modifications (S. C. Li, Yang, Ma, Yan, & Guo, 2015). Crude polysaccharide (1 g) was re-dissolved in 100 mL distilled water. After centrifugation (3500 rpm for 20 min)，the supernatants were filtered through 0.45 μm of filter. 10 mL filtrates were applied to a column (2.6 cm × 50 cm) of DEAE-52 cellulose and stepwise eluted with distilled water, 0.1, 0.3 and 0.5 M NaCl solutions at a flow rate of 1 mL/min. The fractions were collected at 5 min intervals with a fraction collector (BS-100A; Shanghai Huxi, China), and measured by the phenol-sulfuric acid method (Q. Wu et al., 2015). Two major fractions were pooled, desalted, concentrated and freeze-dried to yield the polysaccharide powder. Solutions of the polysaccharide fractions (20 mg/mL, 5 mL) were loaded onto a SephacrylTM S-200 gel column (2.6 × 50 cm). The column was eluted with distilled water at a flow rate of 0.5 mL/min. The eluent was collected and monitored under the same conditions as those used for the DEAE-52 cellulose column. The polysaccharide fractions were analyzed, concentrated, and freeze-dried as previously described. 2.4. Analysis of carbohydrate and protein

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Total carbohydrate was determined by phenol-sulfuric acid method using glucose as the standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1955). Protein was determined by Commassie Brilliant Blue G-250 and with Bovine serum albumin as the standard (Bradford, 1976). All the data were detected by a UV-2450 instrument (Shimadzu, Tokyo, Japan). The polysaccharide was dissolved to 1 mg/mL solution and scanned from 200 to 400 nm with an UV spectrophotometer.

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2.5. Monosaccharide composition analysis The HPLC analysis of PMP-derived monosaccharides was carried out on a Shimadzu LC-2010 HPLC system equipped with a quaternary gradient pump unit, an UV detector, an auto-sampler and a column oven. The analytical column was a BDS Hypersil C18 column (250

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mm  4.6 mm, 5 μm, Thermo Scientific, Sunnyvale, Calif. United States of America). The PMP derivatives elution was performed with a mixture of 0.05 M phosphate buffer (pH 6.8) and acetonitrile in a ratio of 84:16 (v/v) with a flow rate of 1 mL/min at 40 C. The UV absorbance of the effluent was set at 250 nm. The polysaccharide sample (5.0 mg) was hydrolyzed with 5 mL trifluoroacetic acid (TFA, 4M) at 110 C in an oven for 8 h. Then the PMP derivatization of monosaccharides was carried out following the reported method (Yang, Wang, Wang, Mei, & Zhao, 2005). Firstly, 200 μL of hydrolyzed polysaccharide sample or monosaccharide standard solution was mixed with 240 μL of

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0.3 M sodium hydroxide. Then, the mixture was added with 240 μL of 0.5 M methanolic solution of PMP and mixed thoroughly by a vortex mixer. After the solution was shaken for 10 s, the mixture was kept at 70 C for 2 h in an oven. The mixture was neutralized with 240 μL of 0.3 M hydrochloric acid solution after being cooled to 25 C. Then, added 1 mL of chloroform to the mixture and shook it vigorously. The chloroform layer was discarded and the extraction process was repeated three times. Finally, the aqueous layer was filtered through a 0.45 μm membrane for HPLC analysis. The derivatization procedures of both hydrolysate and monosaccharide standard samples should be carried out under the same condition.

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2.6. Molecular weight determination The molecular weight (Mw) of polysaccharides was determined by HPGPC according to the reported method with slight modifications (Jian Hua Xie et al., 2016). Briefly, polysaccharide samples (25.2 mg) were dissolved in distilled water (5 mL), passed through a 0.45 μm filter, and

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applied to a Ultrahydrogel TM Linear gel-filtration chromatography column (7.8  300 mm). The column was maintained at the temperature of 40 C and eluted with deionized water at a flow rate of 0.8 mL/min with the injection volume of 20 μL. The column was calibrated with the Dextran standards of known molecular weight (4320, 12600, 60600, 110000, 289000 Da). The Mw of polysaccharides was estimated by reference to the calibration curve made above.

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2.7. Scanning electron microscopy (SEM) analysis

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2.8. Methylation analysis

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The molecular morphologies of polysaccharides were observed using a scanning electronic microscope (SEM) (SU8010, Hitachi High-Technologies Co., Tokyo, Japan). The samples were coated with a thin gold layer and placed on the substrate, and the images were observed at a voltage of 5.0 kV with 10000-fold magnification under high vacuum.

Methylation analysis was conducted using the method of Ciucanu and Kerek with some modifications (Ciucanu & Kerek, 1984). Polysaccharide samples (5.0 mg) were dried overnight at

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40 C in a vacuum oven and dissolved in DMSO (0.5 mL) by sonication at room temperature. Methyl iodide (0.3 mL) and finely powdered dry sodium hydroxide (20 mg) were added to a stirred solution of the polysaccharide dissolved in DMSO under nitrogen at room temperature. The mixture was stirred for two hours. The methylated polysaccharides were extracted with DCM. The DCM phase was washed three times with 1 mL of water and dried with a stream of nitrogen. The methylated polysaccharide was converted into partially methylated alditol acetates (PMAA) by hydrolysis and acetylation. The PMAA was analyzed by GC–MS (Agilent 7890/5975C, Agilent Technologies, Palo Alto, Calif, United States of America) attached to a HP-5 capillary column (30

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m × 0.25 mm i.d., 0.25 μm film thickness). The injector temperature was 260 C, and the ion source temperature was 200 C. The column temperature program was 80 C for 1 min, followed by a 8 C/min ramp up to 250 C followed by held for 5min. Helium was the carrier gas with a flow rate of 0.9 mL/min. PMAA was assigned by their fragment ions in GC-MS and molar ratios were evaluated with the peak areas and the response factors. 2.9. FT-IR and NMR analysis Two milligrams of polysaccharide fractions were mixed with 400 mg of dried KBr to make a

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tablet. The absorption spectra of the tablet was measured with a FT-IR Spectrometer (Tensor 27, Bruker, Karlsruhe, Germany) from 4000 to 400 cm-1 at 0.2 cm/s according to the reported method (Jouraiphy et al., 2008). For NMR measurements polysaccharide fractions were dried in a vacuum over P2O5, and then exchanged with deuterium by lyophilizing with D2O for three times. The deuterium-exchanged polysaccharide (20 mg) was dissolved in D2O (1mL, 99.8%). Spectrum was recorded with a Bruker AV-300 spectrometer (Bruker Instrumental Inc, Billerica, Massachusetts, United States of America). The 1H NMR spectrum was recorded fixing the HOD signal at d 4.70

2.10. Analysis of in vitro antioxidant activity of polysaccharide fractions 2.10.1. DPPH radical scavenging activity assay

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ppm at 30 C.

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The activity of the polysaccharide fractions on scavenging DPPH radical was analyzed according to the reported method (Jian Hua Xie et al., 2015). Briefly, polysaccharide powder was dissolved in distilled water (0.05~1.0 mg/mL). 2 mL of a freshly prepared DPPH ethanol solution

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(810-5 g/mL) was added to 2 mL of the sample. The mixture was incubated at room temperature for 30 min in the dark, and the absorbance was measured at 517 nm. In this study, ascorbic acid (Vc) served as positive control. The scavenging activity was calculated with the following equation: Scavenging activity (%) = [A0 - (A1 - A2)]/A0 × 100% Where A0 was the absorbance of DPPH solution without any sample, A1 was the absorbance of a mixture solution of the sample and DPPH, and A2 was the absorbance of the absorbance of the sample solution without DPPH. The experiment was repeated three times, each with duplicate samples.

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2.10.2. Hydroxyl radical scavenging ability

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Hydroxyl radical (•OH) scavenging activity was measured according to the reported method (S. Wu et al., 2013). In general, the reaction mixture contained 1 mL of ferrous sulfate (9 mM), 1mL of salicylic acid-ethanol (9 mM), and 7 mL of various concentrations of polysaccharide (0.05~1.0 mg/mL). The reaction was started by 1 mL of hydrogen peroxide (9 mM). After

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incubating at 37 C for 30 minutes, the absorbance of samples was measured at 510 nm, using Vc as a positive control. The hydroxyl radical scavenging activity was calculated as the following formula: Inhibition rate (%) = [A0 - (A1 - A2)]/A0 × 100% Where A0 was the absorbance of the blank (distilled water instead of the sample), A1 was the absorbance of the sample, and A2 was the absorbance of the control (distilled water instead of the H2O2). The experiment was repeated three times, each with duplicate samples. 2.10.3. Superoxide anion radical scavenging activity Superoxide anion radical scavenging activity was detected using the method described by Marklund and Marklund (Marklund, Marklund, Marklamd, & Marklamd, 2005), with a minor modification. The sample was firstly dissolved in distilled water, of which 0.3 mL of sample solution was mixed with 3.9 mL distilled water and 4.5 mL 50 mM Tris-HCl buffer (pH 8.2). The reaction mixture was incubated in a water bath at 25 C, followed by the addition of 0.3 mL 0.045 6

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2.10.4. Ferrous ion chelating ability

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The ferrous ion chelating ability was measured according to the reported method with some modifications (Fan et al., 2014). 0.8 mL sample was mixed with 50 μL ferrous chloride solution (FeCl2, 2 mmol/L) and 2.75 mL distilled water. The reaction was initiated by adding 200 μL 5 mmol/L ferrozine and the mixture was shaken vigorously and incubated at room temperature for 10 min. The absorbance at 562 nm was detected. EDTA-2Na was used as the positive control. The ferrous ion chelating activity was estimated by the following equation: Ferrous ion chelating activity (%) = [A0 - (A1 - A2)]/A0 × 100% Where A0 is the absorbance of the control (distilled water instead of sample solution), A1 is the absorbance of the sample in the reaction mixture and A2 is the absorbance of the sample only (distilled water added instead of ferrous chloride solution). The experiment was repeated three times, each with duplicate samples.

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2.11. Evaluation of in vivo hepatoprotective effect of polysaccharide fractions 2.11.1. Experimental animals and treatment

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Male ICR mice (25-30g) were purchased from the Qinglongshan Experimental Animal Breeding Farm (Nanjing, China). The animals were maintained under controlled conditions (22 ±

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2 C, 55 ± 5% humidity and 12 h light-dark cycle) with free access to standard rodent chow and water. All animals were received humane care in compliance with the institutional animal care guidelines approved by the Experimental Animal Ethical Committee, China Pharmaceutical University. After acclimatization to the laboratory conditions, the mice were randomized into following nine groups: six active testing groups (10 animals each group) including STRP1 (200, 100, 50 mg/kg) and STRP2 (200, 100, 50 mg/kg) groups as well as three control groups including normal control (NC), model control (MC), and positive control (PC) groups. Mice were weighed before the first treatment every day to calculate dosage. For the first treatment, mice in NC group were treated with physiological saline by intraperitoneal injection while mice in all other groups were treated with APAP at a dose of 400 mg/kg. After 6h, the intraperitoneal injection of polysaccharides in the six active groups was performed three times daily at an interval of 6 h, whereas NC and MC groups were treated with physiological saline and PC group with N-acetylcysteine (120 mg/kg) as controls. The entire treatment period lasted for two days. At the end of the experiment, all mice were sacrificed, and blood samples were collected by excising eyeballs. Liver tissues were promptly collected, placed in ice-cold 0.9% NaCl solution, perfused with the physiological saline solution to remove blood cells. And a portion of the liver fixed in 10% buffered formalin was used for histological assessment. The remaining tissue was 7

stored at -80 C for further use. 2.11.2. Histological evaluations The fresh liver tissue was fixed in 4% formaldehyde overnight, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Each section was photo-graphed under microscope showing the histopathological changes ( 200 magnifications). 2.11.3. Serum enzyme assay

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The blood sample was kept at room temperature for 2h. Serum was collected after centrifugation at 3500 rpm for 15min. The AST in serum was measured with a commercial reagent kit according to the manufacturer’s protocol (Xiang, Wang, Zhang, Ling, & Xu, 2012).

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2.11.4. MDA, ROS, GSH, GPx, T-SOD, and CAT content assay The liver tissues were weighed and homogenized (1:9, g/mL) in physiological saline solution.

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The homogenates were centrifuged (3000 rpm, 4 C) for 20 min, and the supernatants were collected for further analysis. MDA, GSH, GPx, T-SOD and CAT activities in liver tissues were determined using relevant test kits according to the manufacturer’s instructions. Liver ROS content was measured with the ELISA assay kit according to the manufacturer’s instruction.

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2.12. Statistic analysis

3. Results and discussion

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All statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, Calif, United States of America). Statistical comparisons were performed using one-way analysis of variance (ANOVA), followed by Tukey’s test. The level of significance was set at p < 0.05 or p < 0.01.

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3.1. Separation and purification of polysaccharide

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The crude STRP was obtained by hot water extraction, centrifugation, 95% ethanol precipitation, and deproteinization. The yield of STRP was 7.1%. Then the crude STRP was loaded onto a DEAE-52 cellulose column, and sequentially eluted with distilled water, 0.1, 0.3 and 0.5 mol/L NaCl solutions. Two polysaccharides were separated and designated as STRP1 and STRP2 (Fig. 1a). STRP1 and STRP2 accounted for 57.2% and 35.7% of the total STRP content, respectively. The overall recovery was 92.9%, indicating that the polysaccharides had been effectively eluted from the column. STRP1 and STRP2 were further purified through SephacrylTM S-200 column chromatography and eluted with deionized water. Each polysaccharide appears to be a single, symmetrical sharp peak (Fig. 1b and c), suggesting that they were relatively homogeneous samples. Following concentration and freeze-drying, STRP1 and STRP2 powders were obtained. 3.2. Preliminary characterization of polysaccharide fractions 3.2.1. Analysis of polysaccharide and protein contents The carbohydrate contents of STRP1 and STRP2 were 97.4% and 93.7% by the phenol-sulfuric acid method. STRP1 and STRP2 had not response to the Commassie Brilliant Blue G-250 method. Moreover, both polysaccharides had no absorption at 280 and 260 nm in UV 8

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3.2.3. Molecular weight analysis by gel permeation chromatography

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scanning spectrum within 200-400 nm, indicating the absence of proteins. 3.2.2. Monosaccharide composition analysis The analysis of monosaccharide compositions commonly involves the cleavage of glycosidic linkages by complete acid hydrolysis, derivatization, and detection and quantification with GC and HPLC. In this study, the HPLC chromatograms of PMP-derived component monosaccharides released from STRP and six standard monosaccharides are shown in Fig. 2a. The monosaccharide composition of polysaccharide was identified by comparing the retention time with standards. STRP1 was composed of mannose, rhamnose, glucuronic acid, glucose, galactose, arabinose, with relative molar percentages of 5.1%, 2.5%, 1.8%, 25.1%, 42.3% and 23.2%, respectively. In comparison, STRP2 had similar components but in different relative molar percentages (mannose, rhamnose, glucuronic acid, glucose, galactose, arabinose: 1.1%, 2.2%, 5.1%, 34.1%, 23.5% and 34.0%, respectively).

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In this study, HPGPC analysis was applied to evaluate and determine the homogeneity and average molecular weights of polysaccharide fractions. As shown in Fig. 2b, the profiles of STRP1 and STRP2 all appeared as comparatively single and symmetrical peaks, which demonstrate that two polysaccharides were homogeneous fractions. The standard regression equation is Y = -0.05584 X3 + 1.855 X2 − 21.064 X + 85.648, where Y represents molecular weight (Da), and X represents retention time (min). According to the standard regression equation,

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the molecular weights of STRP1 and STRP2 were calculated to be 1.30  104 and 1.98  105 Da, respectively.

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3.2.4. SEM analysis

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The stereo-shapes of polysaccharides are typically more complex than those of nucleotides and proteins. To provide direct evidence of the chain conformations of STRP1 and STRP2, SEM was used to determine the morphologies of the polysaccharides. As shown in Fig. 2c, STRP1 has an irregular, large lamellar shape and a smooth surface, which illustrates STRP1 has an amorphous structure. A possible explanation is that a polysaccharide associated with different macromolecules and the water removal process upon lyophilization impacted morphology. In contrast, the SEM image of STRP2 revealed a rough and uneven surface consisting of large sponge-like particles. 3.2.5. Methylation and GC-MS analysis

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To acquire further structural information, methylated productions of STRP1 and STRP2 were analyzed by GC-MS. The total ion chromatograms (TICs) of STRP1 and STRP2 are shown in Fig. 3a. The signals and mass fragments were analyzed through a comparison with the database. The identification and proportions of the methylated alditol acetates of STRP1 and STRP2 are presented in Table 1. STRP1 results showed that molar ratios of 2,3,5-Me3-Ara, 3,4-Me2-Rha, 2,3,4,6-Me4-Glc, 2,3-Me2-Ara, 3,4,6-Me3-Man, 2,4,6-Me3-Gal, 2,3,6-Me3-Glc, 3,6-Me2-Glc, 2,4-Me2-Glc, and 3,4-Me2-Gal were 5.6, 1.0, 5.8, 2.3, 1.4, 8.3, 1.1, 0.8, 0.8, and 3.0 respectively according to the peak areas. Galactose based sugar residues (including 1,3-linked Gal, 1,2,6-linked Gal) were highly enriched in STRP1 in consistent with monosaccharide composition analysis. Results on STRP2 showed molar ratios of 3.9, 0.4, 1.3, 2.8, 0.4, 2.5, 3.4, 0.7, 1.0, and 1.3 for

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2,3,5-Me3-Ara, 3,4-Me2-Rha, 2,3,4,6-Me4-Glc, 2,3-Me2-Ara, 3,4,6-Me3-Man, 2,4,6-Me3-Gal, 2,3,6-Me3-Glc, 3,6-Me2-Glc, 2,4-Me2-Glc, and 3,4-Me2-Gal respectively, while glucose based sugar residues (including 1-linked Glc, 1,4-linked Glc, 1,2,4-linked Glc, 1,3,6-linked Glc) were highly enriched, in agreement with monosaccharide composition analysis. 3.2.6. FT- IR and 1H NMR analysis

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FT-IR spectra were performed to analyze the molecular properties of the purified STRP1 and STRP2 fractions. The FT-IR spectra of the purified STRP1 and STRP2 fractions were found to be similar (Fig. 3b). Each of the two polysaccharide fractions had characteristic carbohydrate absorption peaks at 4000-400 cm-1. The broad bands at 3200-3600 cm-1 were the characteristic peaks of intra- or intermolecular hydrogen bonds with O-H stretching vibration. The signal at 2930-2941 cm-1 indicated the presence of C-H stretching vibration in a methylene group (-CH2-). The strong peaks at approximately 1600 cm-1 were caused by the stretching vibration of a free carboxylic carbonyl group. The peaks at approximately 1400 cm-1 were caused by the C-H bending vibration. The peaks in the range of 1300-1000 cm-1 were characteristic of carbohydrates. Moreover, the peaks of 839.8/852.2 cm-1 in STRP1 and STRP2 show that the sugar linkage types were α-type glycosidic linkages (S. Li & Shah, 2014). These results indicated that STRP1 and STRP2 possessed typical absorption peaks of polysaccharides. The structural characteristics of STRP1 and STRP2 were further determined with NMR spectral analysis. The 1H NMR spectra show that chemical shifts of polysaccharide fractions are mainly in the region from 3.3 to 5.4 ppm. As shown in Fig. 3c, the signals in the range of δ 5.0-5.4 ppm in the 1H NMR spectrum indicated that STRP1 and STRP2 contained α-glycosidic configuration (S. Q. Huang, Li, Li, & Wang, 2011; F. Wang, Wang, Huang, Liu, & Zhang, 2015). The results were in agreement with the glycosidic linkage types by IR. The signals at δ 5.36 and 5.21 ppm were assigned to anomeric signals of arabinose residues, and the signals between δ 5.16 and δ 5.00 were assigned to anomeric signals of galactose (Pastell, Virkki, Harju, Tuomainen, & Tenkanen, 2009; Shakhmatov, Toukach, Kuznetsov, & Makarova, 2014). The strong signal at δ 4.70 ppm presented in the spectrum was attributed to HDO. In addition, the signals between δ 3.30 and 4.20 ppm represented ring protons highlighting the presence of pyranose (Poli et al., 2010). The signals at δ 1.93 and 1.21 ppm from methyl protons were also found.

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3.3. In vitro antioxidant activities of polysaccharide fractions 3.3.1. DPPH scavenging effect

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The antioxidant activities of the extracted polysaccharide fractions were investigated by measuring their scavenging effects as hydrogen donors on the DPPH radical. This radical has a characteristic absorbance at 517 nm which exhibits a purple color that tends to fade when the radical is scavenged by the antioxidant (Gülçin, 2006). As shown in Fig. 4a, all the samples had a noticeable effect on scavenging DPPH free radicals, especially at high concentrations. However, the scavenging activities of STRP1 and STRP2 were lower than those of Vc at all concentrations tested in the current study. Between the polysaccharide samples, the scavenging activity of STRP1 was significantly higher than that of STRP2. At the concentration of 1.0 mg/mL, the scavenging effects of STRP1 and STRP2 on DPPH radical were 77.30 ± 1.42% and 59.62 ± 1.22%, respectively. For most employed concentrations, the antioxidant activity of STRP1 is greater than that of STRP2 fractions. This could ascribe to the shorter chain length of low molecular weight 10

STRP1. 3.3.2. Hydroxyl radical scavenging ability

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Hydroxyl radicals have free access to cell membranes and can cause tissue damage. Thus, the scavenging of these specific radicals may prevent tissue injury (Chen, Liu, Xiao, Huang, & Liu, 2016). The scavenging activities of STRP1 and STRP2 on hydroxyl radicals are shown in Fig. 4b. The scavenging effects of STRP1 and STRP2 varied significantly in different concentrations of polysaccharides. Higher scavenging activities were found when the content of polysaccharide increased, presenting dosage dependent relations. At the concentration of 1.0 mg/mL, the scavenging effects of STRP1 reached 74.02 ± 1.48%, only 15% lower than that of Vc, and both more effective than STRP2 (61.85 ± 1.86%). It is reported that the mechanism underlying hydroxyl radical scavenging by polysaccharides involves the interaction of hydrogen with radicals followed by a termination of the radical chain reaction. Unfortunetely, the finer details of this mechanism have not been elucidated (Shi, Zhang, & Yang, 2013). 3.3.3. Superoxide anion radical scavenging activity

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Superoxide anion radicals are weak oxidants and thus harmless to the body. However, these radicals in combination with hydroxyl molecules may damage DNA and other biomolecules. As an initial free radical, superoxide radical is formed from mitochondrial electron transport systems. In addition, it can create other strong free radicals that may cause various types of diseases (Vaz et al., 2011; Zhang & Liang, 2015). Hence, the in vitro superoxide anion free radical scavenging activities of the STRP1 and STRP2 were also investigated. As illustrated in Fig. 4c, all concentrations of the STRP1 and STRP2 solutions showed obvious scavenging activity towards superoxide anion radicals in a concentration-dependent manner, especially at higher concentrations (0.4~1.0 mg/mL). The scavenging superoxide anion of STRP2 was lower than that of STRP1 and Vc at the concentrations ranging from 0.05 to 1.0 mg/mL. At the concentration of 1.0 mg/mL, the scavenging effects of STRP1 and STRP2 on superoxide anion radicals were 77.93 ± 1.87% and 58.73 ± 1.96%, respectively. In contrast, Vc achieved 90.09% activity at the same concentration of 1.0 mg/mL. Similar to the explanation of the scavenging activities on DPPH radicals, the result could be mainly attributed to the shorter chain length of the low molecular weight STRP1.

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3.3.4. Ferrous ion chelating ability

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Chelation is an important biological process, as iron is an essential metallic element for respiration, oxygen transport, and the activity of many enzymes for metabolism. However, excessive iron catalyses the oxidation of protein, lipids and other components (Y. Wang, Mao, & Wei, 2012). Ferrous ions (Fe2+) chelation may render important antioxidative effects by retarding metal-catalyzed oxidation and has been applied to measure a compound potential antioxidant activity (Gülçin, Huyut, Elmastaş, & Aboul-Enein, 2010). The chelating activities at various concentrations of STRP1 and STRP2 were examined and compared with a chelating standard (EDTA-2Na), as shown in Fig. 4d. The Fe2+ chelating activities of the polysaccharides were concentration-dependent. At 2 mg/mL, the chelating ratio of STRP1 and STRP2 were 82.31 ± 2.45% and 55.03 ± 2.14%, respectively. At concentrations of 0.05~2 mg/mL, the EDTA-2Na chelating ratio increased from

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76.19 to 98.02%, while that of STRP1 increased from 13.98 to 82.31%. The data indicates that STRP1 has an effective capacity for chelating the ferrous ion while the acidic polysaccharide STRP2 has low chelating capacities. Altogether, whether the structures can be related to antioxidant activities remain to be determined in future studies. 3.3.5. Correlation of structure and antioxidant activity

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The antioxidant activities of polysaccharides are generally considered to be strongly correlated with their structural characteristics, including monosaccharide composition, glycosidic linkage, molecular weight and chain conformation (Jahanbin, Gohari, Moini, Emamdjomeh, & Masi, 2011). Few studies of regarding the structure and antioxidant activity relationships of these polysaccharides have been reported with difficulties encountered to extrapolate the structures of polysaccharides to their antioxidant activities. Nevertheless, some relationships can be inferred from current study as follows. STRP1 was composed of galactose, while STRP2 was mainly composed of glucose. STRP1 displayed higher antioxidant activities than STRP2. Some studies have demonstrated that the potential antioxidant activities of polysaccharides were attributed to the rich in galactose, which were consistent with our findings (Fan et al., 2014). It is well established that the molecular weights of polysaccharides are closely associated with their biological activities (Soltani, Kamyab, & Enshasy, 2013).The molecular weight of STRP1 (1.30×104 Da) was lower than that of STRP2 (1.98×105). The antioxidant activities of the two polysaccharide fractions were affected by their molecular weight, which was in agreement with the findings of xing et al, who reported that the antioxidant activity for low Mw polysaccharides were more pronounced than that of high Mw polysaccharides (Xing et al., 2005). According to the FT-IR spectra, STRP1 and STRP2 mainly contained a-glycosidic linkages. The antioxidant activities of STRP1 and STRP2 were attributed to their a-glycosidic linkages, which was different from the results obtained by Mateosaparicio et al. Their studies found that the β-glycosidic linkages had the stonger antioxidant activity compared with α-glycosidic linkages (Mateosaparicio, Mateospeinado, Jiménezescrig, & Rupérez, 2010). Therefore, the antioxidant activities of STRP1 might be related to relatively higher galactose content, smaller molecular weight, and a-glycosidic linkages content. The underlying relevance between structure and function of polysaccharides can be very complicated. The antioxidant activity of polysaccharides was mainly affected by their monosaccharide composition, molecular weight, glycosidic linkage, chain conformation and advanced structure (Cheng et al., 2013). Which factor played a leading role in the antioxidant activity remained to be further investigated. 3.4. Polysaccharide fractions protect against APAP-induced acute liver injury in mice

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3.4.1. Effects of polysaccharide fractions on liver histopathology In the current study, liver histological study was used to determine the protective effect of polysaccharide fractions on APAP-induced liver injury. As shown in Fig. 5a, liver tissues of the NC group represented the normal liver structure. Liver tissues of MC group exhibited obvious pathological changes, including extensive hemorrhage, necrosis and neutrophil infiltration (Fig. 5b). In contrast, treatments of STRP1 and STRP2 significantly reduced APAP-induced pathological changes (Fig. 5d-i), especially in the high-dose group of STRP1, where the hepatic histology was comparable to the normal control group (Fig. 5f). Importantly, the N-acetylcysteine 12

treatment had similar restoring effects on the APAP-induced pathological changes (Fig. 5c). These results suggested that STRP1 and STRP2 had obviously reduction in APAP-induced morphological changes. 3.4.2. Serum enzyme assay

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Enzyme activity in serum was commonly used for the investigation of early liver damage (Nyblom, Berggren, Balldin, & Olsson, 2004). As shown in Fig. 6, significant increase of AST activities were observed in mice after they were treated with APAP (MC groups), indicating that liver damage had occurred. The AST activity reached 190.23 ± 27.15 U/L in STRP1 groups at the dosage of 200 mg/kg, with 55.13% lower than that in MC groups (p < 0.01), and 29.82% lower than that in STR2 groups (200 mg/kg, p < 0.01). These results testified that the alleviated effects

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in serum enzyme of STRP1 surpassed that of STRP2. Our results demonstrated that APAP-induced liver injury, as reflected by increased levels of AST, was significantly blocked by STRP1 and STRP2 treatments. In conjunction with previous histopathology analysis (STRP1 and STRP2 attenuated APAP-induced liver injury), we observed the significant protective effects of STRP1 and STRP2 against APAP-induced liver injury.

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3.4.3. Effects of polysaccharide fractions on ROS, MDA, and GSH content

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Studies showed that oxidative stress as a result of ROS is associated with the pathogenesis of liver injury (Copple, Jaeschke, & Klaassen, 2010); MDA, one of the most frequently used indicators of lipid peroxidation, was also used to assess the oxidative stress (Yoshida, Umeno, & Shichiri, 2013); in addition, it is reported that the intracellular levels of some nonenzymatic antioxidants, such as GSH, influence the activities of the enzymatic antioxidants (Elango, Samuel, & Chinnakkannu, 2006). Accordingly, we studied the effects of STRP1 and STRP2 on APAP-induced liver ROS, MDA and GSH contents in this study. As shown in Fig. 7a and c, APAP treatment markedly increased hepatic ROS and MDA levels by 88.76% and 229.24% as compared with those of the NC group, respectively (p < 0.01). Interestingly, treatment with STRP1 and STRP2, on the other hand, showed dose-dependent inhibition in this elevation (p < 0.01). As shown in Fig. 7b, the GSH level in APAP-treated mice was markedly decreased by 63.98% as compared with that of NC group (p < 0.01). However, treatments with STRP1 and STRP2 in APAP-treated mice significantly increased the hepatic GSH level in a dose-dependent manner (p < 0.01). 3.4.4. Effects of polysaccharide fractions on activities of antioxidant enzymes

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The antioxidant enzymes play an important role in maintaining the intracellular redox balance. The decline of several enzyme activities (SOD, GPx, and CAT) was often used as biochemical marker for monitoring early oxidative stress in vivo (X. M. Li, Ma, & Liu, 2007). As shown in Fig. 7d-f, significant decreases in the hepatic activities of T-SOD, GPx, CAT were observed in APAP-induced liver injury mice as compared to those of the NC group (p< 0.01), respectively, indicating that early damage was occurred in liver. As shown in Fig. 7d, in 200 mg/kg STRP1 treated dosage group, the T-SOD activities reached 248.87 ± 32.08 U/mg prot, with 23.22% higher than that of STRP2 treated equivalent

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dose group (p < 0.01); and 94.01% higher than that of the MC group (128.22 ± 18.52 U/mg prot, p < 0.01). Furthermore, the hepatic T-SOD activity in the mice treated with STRP1 at the dosage of 200 mg/kg was more approximate to that of the NC groups (299.54 ± 27.07 U/mg prot), even better than the PC group (211.39 ± 30.17 mg prot). Fig. 7e showed that the GPx activities in the mice treated with STRP1 and STRP2 at the dosage of 200 mg/kg reached 1001.79 ± 82.88 and 898.91 ± 96.51 U/mg prot, higher than that of the MC groups (747.02 ± 91.889 U/mg prot, p < 0.01), respectively. Additionally, the hepatic CAT activities of dosage groups treated with STRP1 and STRP2 at dosage of 200 mg/kg reached 52.75 ± 6.46 and 44.31 ± 4.97 U/mg prot, respectively, which was 69.78% and 42.61% higher than that of the MC group (31.07 ± 5.45 U/mg prot, p < 0.01) (Fig. 7f). As mentioned above, all the results suggested that STRP1 and STRP2 might have protective effects against APAP - induced liver injury in mice through their antioxidant activity to protect biological systems against the oxidative stress in multiple mechanisms, including free radicals scavenging activities, increasing the activities of a number of antioxidant enzymes and decreasing the lipid peroxidation. Although it is well accepted that the hepatoprotective effect of polysaccharides is associated with their antioxidant activity, the exact molecular mechanisms and signaling pathways involved are not clear. Thus, the detailed mechanisms need to be further researched. 4. Conclusions

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In conclusion, polysaccharide was extracted from Sophorae tonkinensis Radix, and two purified fractions (STRP1 and STRP2) were obtained from the crude STRP through sequential purification by DEAE-52 and SephacrylTM S-200 chromatography. Then, the STRP1 and STRP2 were characterized by FT-IR, HPLC, HPGPC, GC-MS, NMR and SEM. Experimental results showed that STRP1 was mainly composed of mannose, rhamnose, glucuronic acid, glucose, galactose and arabinose, with relative molar percentage of 5.1%, 2.5%, 1.8%, 25.1%, 42.3% and

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23.2%, respectively. The average molecular weight of STRP1 was determined to be 1.30104 Da. STRP2 was mainly composed of mannose, rhamnose, glucuronic acid, glucose, galactose and arabinose, with relative molar percentage of 1.1%, 2.2%, 5.1%, 34.1%, 23.5% and 34.0%

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respectively. The average molecular weight of STRP2 was determined to be 1.98105 Da. There were characteristic infrared absorption peaks of polysaccharides in the FT-IR spectra of STRP1 and STPP2. And the backbones of STRP1 and STRP2 were mainly composed of (1→3)-linked-α-D-Gal and (1→4)-linked-α-D-Glc. Besides, the antioxidant activities and the potent protective effects against APAP-induced liver injury in mice liver were evaluated. We observed strong chelating ability on ferrous ions and radical scavenging activities on DPPH, hydroxyl and superoxide anion radicals in vitro as well as attenuation in APAP-induced hepatic oxidative damage by inhibiting MDA, ROS generation and increasing liver GSH, GPx, T-SOD, CAT levels in vivo. This study indicates that the polysaccharides from Sophorae tonkinensis Radix can be further explored as novel natural antioxidant and liver-protecting drugs.

Conflicts of interest The authors have no conflicts of interest to declare. 14

Acknowledgments

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The authors are indebted to State Key Laboratory of Natural Medicines, China Pharmaceutical University, for running the FT-IR experiments for the determination of glycosidic bond types.

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Fig.1. Anion-exchange chromatogram of the crude STRP on DEAE-52 cellulose column (a); gel filtration

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chromatograms of STRP1 (b) and STRP2 (c) on SephacrylTM S-200 column.

Fig.2. (a) Chromatograms of monosaccharide compositions; (b) The HPGPC chromatograms of STRP1 and

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STRP2; (c) Scanning electron micrographs of SRTP1 and STRP2 (10000 × ).

20

IP T SC R U N

A

Fig.3. (a) Total ion chromatograms (TICs) of STRP1 and STRP2; (b) Fourier transform infrared spectrum of

A

CC E

PT

ED

M

STRP1 and STRP2; (c) 1H NMR spectrum of STRP1 and STRP2.

Fig.4. Antioxidant activity assay of STRP1 and STRP2. (a) DPPH radical scavenging assay; (b) Hydroxyl radical scavenging ability assay; (c) Superoxide anion-scavenging activity assay; (d) Ferrous ion chelating ability assay. Values are means ± SD, n=3.

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Fig.5. Effects of STRP1 and STRP2 on histopathological changes in liver tissues (magnification 200×). (a) liver of mice in the NC group; (b) liver of mice in the MC group; (c) liver of mice in the PC group; (d-f) liver of mice

ED

injected with STRP1 at dosage of 50, 100 and 200 mg/kg, and (g-i) liver of mice injected with STRP2 at dosage of

A

CC E

PT

50, 100 and 200 mg/kg, respectively.

Fig.6. Effects of STRP1 and STRP2 on serum AST levels. The values presented are the mean ± S.E.M. (n=10 in each group). ** p< 0.01 compared with NC groups; ## p< 0.01 compared with MC groups.

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Fig.7. Effects of STRP1 and STRP2 on liver ROS, GSH, MDA, T-SOD, GPx and CAT levels. (a) ROS; (b) GSH; (c) MDA; (d) T-SOD; (e) GPx; (f) CAT. The values presented are the mean ± S.E.M. (n=10 in each group).

A

CC E

PT

** p < 0.01 compared with NC groups; # p < 0.05, ## p< 0.01 compared with MC groups.

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Table1 Methylation results of STRP1 and STRP2 Peak

Methylated sugars

Linkages

Maior mass fragments (m/z)

Molar ratios STRP1

STRP2

2,3,5-Me3-Ara

1-linked Ara

87, 101, 117, 129, 161, 233

5.6

3.9

2

3,4-Me2-Rha

1,2-linked-Rha

87, 89, 99, 115, 129,131, 189

1.0

0.4

3

2,3,4,6- Me4-Glc

1-linked Glc

87, 101, 117, 129, 145, 161, 205

5.8

1.3

4

2,3-Me2-Ara

1,5-linked Ara

87, 101, 117, 129, 189, 233

2.3

2.8

5

3,4,6-Me3-Man

1,2-linked-Man

87, 101, 117, 129, 161, 189, 233

1.4

0.4

6

2,4,6-Me3-Gal

1,3-linked Gal

87, 101, 117, 129, 161, 181, 233

8.3

2.5

7

2,3,6-Me3-Glc

1,4-linked Glc

87, 99, 117, 129, 161, 173, 233

1.1

8

3,6-Me2-Glc

1,2,4-linked Glc

87, 99, 113, 129 ,189, 233

0.8

9

2,4-Me2-Glc

1,3,6-linked Glc

87, 101, 117, 129 139, 189, 233

0.8

10

3,4-Me2-Gal

1,2,6-linked Gal

87, 99, 117, 129, 189, 233

SC R

U N A M ED PT CC E A

24

3.0

IP T

1

3.4 0.7 1.0

1.3