Extraction, purification and properties of water-soluble polysaccharides from mushroom Lepista nuda

Accepted Manuscript Extraction, purification and properties of polysaccharides from mushroom Lepista nuda

water-soluble

Xu Shu, Yanfen Zhang, Jinxia Jia, Xiaojie Ren, Yufeng Wang PII: DOI: Reference:

S0141-8130(18)35890-2 https://doi.org/10.1016/j.ijbiomac.2019.01.214 BIOMAC 11647

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

31 October 2018 13 January 2019 31 January 2019

Please cite this article as: X. Shu, Y. Zhang, J. Jia, et al., Extraction, purification and properties of water-soluble polysaccharides from mushroom Lepista nuda, International Journal of Biological Macromolecules, https://doi.org/10.1016/j.ijbiomac.2019.01.214

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ACCEPTED MANUSCRIPT Extraction, purification and properties of water-soluble polysaccharides from muschroom Lepista nuda Xu Shu, Yanfen Zhang, Jinxia Jia, Xiaojie Ren, Yufeng Wang * College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

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ABSTRACT: Lepista nuda has high nutritional and medicinal value, especially has high antioxidant, antitumor and antiviral activities. Based on results of single factor experiment,

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response surface methodology was used to optimize the extraction conditions of polysaccharides

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(LNP). Macroporous resin D301 was used to decolorization, and its technological conditions were determined by orthogonal experiment. In addition, two novel polysaccharides (LNP-1, LNP-2)

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were purified by DEAE Cellulose-52 and Sephadex G-150 chromatography. The molecular weight of LNP-1 is 11703 Da, mainly composed of mannose, glucose, galactose, xylose, arabinose and

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fucose, while the molecular weight of LNP-2 is 13369 Da, mainly composed of mannose, glucose, galactose, arabinose and fucose. The polysaccharide contents of crude LNP, LNP-1 and LNP-2 were 70.60%, 87.71% and 81.20% respectively, and the protein contents were 1.72%, 0.97% and

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0.68% respectively. Their sulfuric contents were 3.39%, 5.02% and 8.64%, and uronic acid

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contents were 3.66%, 5.56% and 6.80%, respectively. Further studies showed that their ability to chelate iron ions, scavenge DPPH (1,1-diphenyl-2-picrylhydrazyl) free radicals and scavenge

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superoxide anion radicals were 70.09%, 55.94% and 36.64% respectively. They showed strong ability to scavenge free radicals in a concentration-dependent manner. LNP is expected to be

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developed as a new efficacy factor in the food industry. Keywords: Lepista nuda; polysaccharide; extraction; purification; antioxidant Running title: Lepista nuda Polysaccharide

* Corresponding author. E-mail address: [email protected] (Yufeng Wang);

[email protected] (Xu Shu).

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1. Introduction Lepista nuda is an edible and medicinal fungus [1]. Its fruiting body is lavender, and its round size is (6.1-8)×(4.0-5) μm [2]. This is a rare wild mushroom, which contains excellent food components [3]. Its fruiting body contains many nutrients, including 56.39% protein, 45.0% total

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sugar, 3.87% fat, and 7.92% fiber [4-7], with anti-vascular sclerosis, anti-virus, anti-gallstone, and

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anti-diabetes functions [8-11]. Its inhibits sarcoma in mice by 90% [12] and regulates glucose

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metabolism and promotes nerve conduction [13]. The current researches are mainly focused on its chemical components [14], biological characteristics [15] and cultivation methods [16].

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Polysaccharide is the basic substance for life-sustaining activities [17]. It is formed by the

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polymerization of monosaccharide through glycosidic bonds. There are many kinds of polysaccharides and their structures are complex. So far, more than 300 kinds of polysaccharides

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have been discovered as energy resources and structural substances of life [18-21].

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Polysaccharides can not only be used as nutrients to show good immunoregulatory activity, but also as drugs to show good efficacy, such as reducing blood lipids [22, 23], antioxidant [24, 25],

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antiviral and so on [26-28]. A large number of Studies have shown that polysaccharides can also

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eliminate various reactive oxygen species and inhibit lipid peroxidation [26-28]. Lepista nuda polysaccharides are abundant in content, but their research is limited. Lepista nuda polysaccharides were mainly extracted from different organic compounds. Researchers had preliminarily revealed their antibacterial and antioxidant activities [8, 10, 14]. In this study, the water extraction and alcohol precipitation method was used to extract polysaccharides from mushroom Lepista nuda. Chromatography was used to separate and purify the polysaccharides. Their molecular weight and monosaccharide composition were investigated by high performance

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ACCEPTED MANUSCRIPT gel permeation chromatography (HPGPC). In addition, their structure was characterized by ultraviolet (UV), infrared (IR), scanning electron microscope (SEM) and nuclear magnetic resonance spectroscopy (NMR). Finally, their antioxidant activities in vitro were evaluated by their scavenging experiments on different free radicals.

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2. Materials and methods

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2.1 Materials

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Fresh mushroom Lepista nuda were obtained from Yunnan Shenshan Fungus Industry Co., Ltd. (Dali, China). Unless otherwise specified, all chemicals and reagents used in the experiments

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are of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,

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China). Deionized water was used throughout the experiments and purified through a Mill-Q water purification system from Millipore (Bedford, MA, USA).

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2.2 Preparation of crude LNP

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Clean Lepista nuda thalli (100.0 g) were lyophilized by a Model 2K-XL Lyophilizer (Virtis Corporation, America), grinded with a Model ZN-100 Grinder (Zhongnan Pharmaceutical

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Machinery Factory, Shanghai, China), and filtered through 20-mesh sieve. Thalli powders (3.0 g)

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were extracted continuously with deionized water. The extract was treated as follows: extraction time, 1-4 h; extraction temperature, 75-95 °C; extraction times, 1-4 times; and solvent to raw material extraction ratio, 10.0-60.0 mL/g. Each water extract was added to excessive absolute ethyl alcohol (3:1; v/v) to form precipitation. The precipitates were separated by centrifugation (Model 5804R, Eppendorf Corporation, Germany) at 3000 ×g for 15 min at 25 °C and then lyophilized. Therefore, crude LNP was obtained and stored at 4 °C. 2.3 Response surface experimental design

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ACCEPTED MANUSCRIPT On the basis of single factor experiment, the optimum extraction conditions of crude LNP were determined by response surface test. The single factor experiment was carried out with the extraction rate of polysaccharide as the index [29]. Three main factors, extraction temperature, extraction time, and solvent to raw material extraction ratio, were selected for response surface

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methodology (RSM). A seventeen run response surface experimental design with three factors and

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three levels were implemented.

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2.4 Decolorization of LNP 2.4.1 Adsorbents

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AB-8, D101, HPD100, D301, D941, D900 were supplied by Cangzhou Bon Adsorber

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Technology Co., Ltd. (Cangzhou, China). Table 1 showed their physical and chemical properties. During the synthesis process, some monomers and porogens are trapped in the pores of the

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resin. Therefore, MARs pretreatment is considered necessary. AB-8, D101 and HPD100 and other

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macroporous adsorbent resins were soaked in anhydrous ethanol for 3-4 hours, then washed several times with deionized water until ethanol was clean without turbidity. The macroporous ion

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exchange resins D301, D941, D900 were immersed in saturated NaCl solution of twice volume for

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24 hours, washed three times with deionized water, then immersed in NaOH, HCl, NaOH solution of 1 mol/L for 4 hours in turn, and washed to pH neutrality after each immersion. 2.4.2 Static adsorption and desorption tests Static adsorption tests were carried out as follows. In short, the pretreated resin was introduced into the 100 mL Erlenmeyer flask. Subsequently, about 20 mL of crude LNP solution (1.0 mg/mL, in deionized water as solvent) was adjusted to pH 7 and added to each flask. Shake the flask (100 rpm) in the incubator shaker (HYL-A, Taicang Experiment Equipment Co., Taicang,

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ACCEPTED MANUSCRIPT China) at 25 °C for 24 hours. The absorbance value at 300 nm was measured, and the retention rate and decolorization rate of polysaccharides were calculated. In the single factor experiment, the best decolorizing resin was selected, and the dosage of resin, shake speed, decoloring time and decoloring temperature were considered. The resin dosage

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was 1.0-3.0 g; the rotating speed of the shake was 60-180 rpm; the decolorizing time was 0.5-2.5 h;

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the decoloring temperature was 30-50°C. The absorbance value at 300 nm was determined, and

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the retention rate and decolorizing rate of polysaccharides were calculated. 2.4.3 Orthogonal experimental design

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On the basis of single factor experiment, orthogonal experiment was designed. Four factors

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and three levels of L9(34) experiments were designed to optimize decolorization (Table 2) [30]. By comparing the retention rate and decolorizing rate, the optimum conditions for the static

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decolorization of polysaccharides were determined. The retention rate and decolorization rate of

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polysaccharides were divided by the maximum value of the corresponding series and then multiplied by the item score of 100 to calculate the total score.

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2.4.4 Dynamic adsorption and desorption tests

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Based on physical performance of resins, the best resin and detergent were chosen with the dynamic adsorption experiments by static adsorption. The relationship between decolorization effect and flow rate and sample load was revealed by dynamic decolorization. Decolorization was carried out at the flow rate of 0.23 BV/h, 0.46 BV/h, 0.69 BV/h, 0.92 BV/h (1 BV = 130 mL), respectively. The sample loading was measured at 1.46 BV every 0.15 BV to study the effect of the sample loading on decolorization. 2.5 Purification of LNP

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ACCEPTED MANUSCRIPT The crude polysaccharides obtained from the optimum macroporous resin were dissolved in deionized water (15.0 mg/mL) and applied to DEAE Cellulose-52 column (φ26 × 500 mm) with 10 mL solution. Then the crude polysaccharides were gradually eluted with 0.1, 0.3, and 0.5 mol/L sodium chloride solution at a flow rate of 0.27 BV/h. The content of polysaccharide was

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determined by phenol-sulfuric acid method with glucose as the standard and eluent (5 mL/tube)

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was collected automatically. Two polysaccharides were obtained for further purification.

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The purified polysaccharides were dissolved in deionized water (10.0 mg/mL) and solution (3.0 mL) were applied to the Sephadex G-150 column at a flow rate of 0.27 BV/h. Two

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polysaccharides, LNP-1 and LNP-2, were obtained by water dialyzed and lyophilization.

2.6.1 Molecular weight determination

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2.6 Characterization of LNP

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The molecular weight of LNP was determined by high performance gel permeation

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chromatography (HPGPC) technique using HPLC (Water 600, Waters Corporation, USA), equipped with a Waters Ultrahydroge linear (φ78 mm × 300 mm) and a model 2414 refractive

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index detector (RID) [22, 31]. Then the 20 µL sample was injected into the detector and eluted

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with deionized water at a flow rate of 0.9 mL/min. The standard curves were drawn according to the elution time plotted against the logarithm of molecular weight on standard dextran T-5, T-10, T-70, T-580, T-2000. According to the retention time of LNP, the molecular weight was obtained by standard curve equation. 2.6.2 Monosaccharide composition analysis The monosaccharide composition of LNP was analyzed by high performance liquid chromatography (HPLC, Shimadzu LC-20AD, Shimadzu Corporation, Japan). The area

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ACCEPTED MANUSCRIPT normalization method was used to calculated the molar ratio of monosaccharides [32]. Trifluoroacetic acid (TFA) was used to hydrolyze 5.0 mg/mL sample for 2 h, then methanol was added to the sample and evaporated at 50 °C. The sample was dissolved in deionized water. Then, the above solution was derivatized by PMP. Methanol solution of NaOH and PMP was added in

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turn, heated in 70°C water for 100 min, then neutralized in 0.3 mol/L HCl solution, and steamed at

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50 °C. PMP was removed by adding 1 mL deionized water and chloroform, filtered and detected

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by HPLC. Finally, the curve of polysaccharide composition was obtained and the composition of polysaccharide was calculated.

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2.6.3 UV spectroscopy

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The 0.8 mg sample was dissolved in 1.0 mL deionized water and analyzed by UV spectrophotometer (EU-2600R, Shanghai Onra Instrument Co., Ltd, China) in the range of

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2.6.4 Infrared spectroscopy

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200-400 nm.

Infrared spectrum (IR) was carried out in 4000-400 cm-1 band by using infrared

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spectrophotometer (Nicolet IR200, Nicolet Co., USA). The 1.0 mg sample was milled together

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with 100.0 mg spectral grade KBr powder and pressed into 1 mm particles [33]. 2.6.5 Scanning electron microscopy The morphology of polysaccharides was observed by the scanning electron microscope (SU8010, Hitachi Co., Japan) at 10kV acceleration voltage. The magnification was 100 to 10000 times. Freeze-dried polysaccharide samples were coated with thin layer of gold and then examined under high vacuum conditions [34]. 2.6.6 Nuclear magnetic resonance spectroscopy

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ACCEPTED MANUSCRIPT Nuclear magnetic resonance (NMR) was conducted by NMR spectrometer (INOVA 500, Varian Co., USA). The 20 mg samples were dissolved in 0.5 mL of heavy water at 25 °C, and the frequency was 500 MHz. 2.7 Physicochemical properties of LNP

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2.7.1 Total polysaccharide content

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The content of polysaccharides was determined by phenol sulfuric acid method. The

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absorbance of 0.1 mg/mL solution mixed with 6% phenol and concentrated sulfuric acid was measured at 490 nm. The total polysaccharide content was calculated by the standard curve of

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glucose [35].

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2.7.2 Protein content

The content of protein was determined by bicinchoninic acid method (BCA). Take bovine

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serum albumin (BSA) as the standard (Bradford, 1976). The 0.005 g sample was dissolved with 1

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mol/L NaOH and mixed with BCA working reagent. The absorbance at 562 nm was measured by the Model 752 UV spectrophotometer, The protein content was calculated by BSA standard curve

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[36].

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2.7.3 Sulfate group content

The content of sulfate group was determined by cesium chloride gelatin colorimetry method. The standard curve was drawn with potassium sulphate as standard material. 1.0 mg sample was dissolved in HCl, TCA and barium chloride gelatin solution. The absorbance at 360 nm was obtained and the content of sulfate group was calculated [37]. 2.7.4 Glucuronic acid content The content of glucuronic acid was determined by sulfuric acid-carbazole method. The

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ACCEPTED MANUSCRIPT standard curve was drawn on the basis of glucuronic acid. The polysaccharide sample (1.0 mg/mL) was mixed with carbazole ethanol to obtain its absorbance at 530 nm. The glucuronic acid content was then calculated according to the standard curve [38]. 2.8 In vitro antioxidant activity of LNP

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2.8.1 Scavenging ability on superoxide anion radical

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The scavenging ability on superoxide anion radical was determined by pyrogallol oxidation

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method [39]. We added 4.5 mL Tris-HCl buffer (50 mmol/L, pH=8.2) and 0.1 mL pyrogallol-hydrochloric acid solution to the samples at different concentrations. The absorbance

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was obtained at 325 nm and marked as A1. The absorbance value A2 was measured again under the

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same condition after 4 minutes. The autooxidation rate S1 was calculated with A1 and A2. The equivalent distilled water instead of the polysaccharide solution was used as the control group. Do

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the same operations to measure A1, A2, and S2. The scavenging activity of superoxide anion radical

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was calculated:

S  ( A2  A1 ) / 4

(1)

P%  (S2  S1 ) / S2 100

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(2)

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where S is the auto-oxidation rate; P is superoxide anion radical scavenging rate. 2.8.2 Scavenging ability on DPPH radical The scavenging capacity of DPPH radical was identified by DPPH method [40]. An ethanol DPPH solution (0.4 mmol/L) was prepared. The initial absorbance of DPPH in ethanol was determined at 517 nm. The aliquot (0.2 mL) of each sample was added to 2.0 mL ethanol DPPH solution. After 30 min incubation in the dark for 30 °C, the absorbance value was measured at 517 nm. The scavenging capacity of DPPH radical was calculated:

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ACCEPTED MANUSCRIPT DPPH radical scavenging activity%  [ A0  ( A1  A2 )] / A0 100

(3)

where A0 is the absorbance of the control (deinoized water instead of sample); A1 is the absorbance of the sample; A2 is the absorbance of the sample only (ethanol instead of reaction solution). 2.8.3 Chelating ability on ferrous ion

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The chelating activity of ferrous ion was determined by ferrous chelating method [41]. In

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short, mix 2.0 mL sample with 100 mL FeCl2 (ferrous chloride, 2 mmol/L), 2.75 mL deinoized

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water and 100 mL ferrozine (5 mmol/L). The solution was incubated at 25 °C for 10 min., and the absorbance was measured at 562 nm, and EDTA-2Na (ethylenediamine tetracetic acid disodium

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salt) was used as the positive control. The chelating activity of ferrous ions was calculated: (4)

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Ferrous ion chelating activity%  A0  ( A1  A2 )/ A0 100

where A0 is the absorbance of the control (deinoized water instead of sample); A1 is the absorbance

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of the sample; A2 is the absorbance of the sample only (deinoized water instead of ferrous

2.9 Statistical analysis

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chloride).

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All experiments were repeated three times per replicate (three fingers per replicate). The data

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were expressed as the means ± standard deviations of triplicate determinations (n = 3 × 3). One-way ANOVA and Duncan’s multiple range test were performed using Statistical Analysis System software to assess the significance of the differences between the mean values. The significance of the level was set at p < 0.05. 3 Results and discussion 3.1 Single-factor experiments for extracting crude LNP 3.1.1 Effect of extraction time on the polysaccharide extraction rate

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ACCEPTED MANUSCRIPT In order to study the effect of extraction time on the extraction rate of LNP, different extraction time (1, 2, 3, and 4 h) was selected. The extraction temperature, extraction times and solvent-material ratio were fixed at 90 °C, 3 times, and 40.0 mL/g, respectively. As shown in Fig.1a, with the extension of extraction time, the extraction of LNP increased first and then

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decreased. When the extraction time was less than 3 hours, the extraction rate of polysaccharides

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increased significantly with the extension of extraction time (p<0.05). When the extraction time

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exceeded 3 h, the extraction rate of polysaccharides decreased slightly. When the extraction time was less than 3 h, the crude polysaccharide could not be completely dissolved and the extraction

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rate was low. With the extension of extraction time, the extraction rate also increased. However, if

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the extraction time was longer than 3 hours, the glycoside bonds of polysaccharides would be unstable and ease to break, and the decomposition of polysaccharide would lead to the decrease of

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extraction rate.

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In terms of extraction time, the extraction rate of polysaccharides in 1 h, 2 h and 3 hours was significantly different (p<0.05), but there was no significantly difference in the extraction rate of

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polysaccharides in 3 and 4 hours (p>0.05). Therefore, we chose 3 hours as the center point of the

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response surface experiment.

3.1.2 Effect of extraction temperature on the polysaccharide extraction rate To estimate the effect of extraction temperature on the extraction rate of LNP, we chose different extraction temperature (75, 80, 85, 90, and 95 °C) and fixed the extraction time, extraction times, and the ratio of solvent to material ratio at 3 h, 3 times, and 40.0 mL/g, respectively. As shown in Fig. 1b, the extraction rate of polysaccharides increased significantly when the temperatures rose from 75 to 90 °C, and reached its maximum at 90 °C. With the

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ACCEPTED MANUSCRIPT increase of temperature, the thermal diffusion of molecules increased and the dissolution of polysaccharides was promoted. However, when the extraction temperature continued to rise, the polysaccharides were degraded and lost after ethanol treatment, and the extraction rate decreased. Statistical results showed that there were significant differences in extraction rates under 75,

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80, 85 and 90 °C conditions (p<0.05). We determined 90 °C as the center point of the response

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surface experiment.

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3.1.3 Effect of extraction times on the polysaccharide extraction rate

Different extraction times (1, 2, 3, and 4 times) were selected to investigate the effect of

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extraction times on the extraction rate of LNP. The extraction time, extraction temperature, and the

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solvent to material ratio were fixed at 3 h, 90 °C, and 40.0 mL/g, respectively. As shown in Fig.1c, with the increase of extraction times, the solubility and the extraction rate of polysaccharides

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increased gradually. When the polysaccharide extraction times reached three times, the

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polysaccharides were almost completely extracted, and the extraction rate did not change. There were significant differences in the extraction rates of two, three and four times

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(p<0.05), but there was no significant difference in the extraction rates of three and four times

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(p>0.05) , so three times was the best extraction times. 3.1.4 Effect of solvent to material ratio on the polysaccharide extraction rate The effect of solvent/material ratio on the extraction rate of LNP were investigated by selecting 10:1, 20:1, 30:1, 40:1, 50:1 and 60:1 mL/g as different solvent/material rate. The extraction time, extraction temperature, and extraction times were fixed at 3 h, 90 °C, and 3 times, respectively. As shown in Fig. 1d, with the increase of solvent volume, polysaccharides are dissolved faster, resulting in higher extraction rate. When the solvent exceeded a certain volume,

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ACCEPTED MANUSCRIPT the polysaccharide solution is saturated, and the effect of the solvent was no longer obvious. Statistical results showed that the extraction rates of 30:1, 40:1, 50:1, 60:1 mL/g were significantly different (p<0.05), but the extraction rates of 10:1, 20:1 and 30:1 mL/g were not significantly different (p>0.05). Therefore, we chose the ratio of solvent to material equal 40:1

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mL/g as the center point of response surface experiment.

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3.2 Optimization of LNP extracting conditions

took the extraction rate of LNP as the response value.

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According to the single factor test results, a response surface method was designed, which

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The extraction temperature, extraction time and the ratio of solvent to material were

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optimized, and the optimal extraction conditions were obtained. The experimental design and results were given in Table 3a.

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The multivariate quadratic regression model equation is obtained through regression analysis

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(Table 3b):

Y=5.36+0.30A+0.055B-0.0075C+0.21AB-0.075AC-0.25BC-0.32A2-0.26B2-0.22C2

(5)

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When the response surface value (Y) reached the maximum, the corresponding extraction

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temperature, extraction time and the ratio of solvent to material were 93.67 °C, 3.63 h and 35.17 mL/g, respectively. At the same time, the extraction rate of polysaccharides is 5.49%, which is the best condition for extracting polysaccharides. The experiment proved that the extraction rate of polysaccharides was 5.76%, which was close to the theoretical prediction value. The results showed that the model had high accuracy and strong predictive ability for the extraction of polysaccharides from the Lepista nuda. 3.3 Static decolorization of crude LNP

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ACCEPTED MANUSCRIPT 3.3.1 Single-factor experiments for decolorizing crude LNP The effect of resin dosage (1.0, 1.5, 2.0, 2.5, and 3.0 g) on the decolorization of polysaccharides is shown in Fig. 2a. When the dosage of resin was 2.0 g, the pigment molecules were completely adsorbed and removed, and the rising trend of decoloring rate gradually flattened.

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However, the retention rate of polysaccharides was opposite to that of decolorization. According

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to the compound score calculated by polysaccharide retention rate and decoloring rate, 2.5 g resin

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was selected as the best experimental factor.

Fig. 2b showed the effect of shaking speed (60, 90, 120, and 180 rpm) on the decolorization

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of polysaccharides. The decoloring rate increased with the increase of shaking speed. When the

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shaking speed was too slow, the contact between polysaccharide and resin was not enough, resulting in a worse decolorization efficiency. When the rotational speed is higher than 120 rpm,

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the decoloring rate would no longer change. On the contrary, with the increase of shaking speed,

the best factor level.

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the retention rate of polysaccharide decreased. Considering the total score, we chose 120 rpm as

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The effects of different decoloring time (0.5, 1.0, 1.5, 2.0, and 2.5 h) on the polysaccharide

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decolorization of LNP were studied. As shown in Fig. 2c, with the prolongation of decoloring time, the decoloring rate increased significantly and the retention rate of polysaccharides decreased sharply. When the decoloring time exceeded 1.5 h, the pigment molecule was completely adsorbed, the polysaccharide molecule is further adsorbed, the decoloring rate changed little, but the retention rate of polysaccharides changed significantly. Therefore, we chose 1.5 hours as the best decoloring time in order to ensure the best decolorization effect and less loss of polysaccharides. The effect of decoloring temperature (30, 35, 40, 45, and 50 °C) on the decolorization of

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ACCEPTED MANUSCRIPT polysaccharides was shown in Fig. 2d. With the increase of temperature, the decoloring rate gradually increased, and the retention rate of polysaccharides fluctuated at about 60%, indicating that temperature had no effect on the retention rate of polysaccharides. Compared with the comprehensive evaluation score, 45 °C was determined as the best factor level.

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3.3.2 Optimization of the LNP decolorization conditions

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On the basis of the single factor experiment, orthogonal test was designed. Four independent

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variables in this experiment included resin dosage (A), shaking speed (B), decoloring time (C), and decoloring temperature (D). The orthogonal test results are shown in Table 2. Orthogonal

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range analysis showed that decoloring time had the greatest influence on the decolorization

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process, followed by resin dosage, shaking speed and decoloring temperature. The best combination of process parameters was A2B2C2D3. Under the optimum conditions, the verification

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experiments were carried out. The actual retention rate was 79.02%, and the decoloring rate was

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86.29%.

In the dynamic decolorization experiment, the retention rate of polysaccharides increased

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sharply with the increase of loading volume, while the decoloring rate decreased sharply. With the

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increase of flow rate, the retention rate of polysaccharides increased and the decoloring rate decreased. The optimum flow rate was 0.46 BV/h, and the retention and decoloring rates of polysaccharides were 71.21% and 81.07%, respectively. The retention rate and decoloring rates of polysaccharides were 72.33% and 80.81%, respectively when the loading amount was 0.92 BV. 3.4 Purification of LNP According to the elution curves of DEAE-52 column, three peaks (LNP-1, LNP-2, and LNP-3) were obtained (Fig. 3a). The extraction rates of LNP-1 and LNP-2 were significantly

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ACCEPTED MANUSCRIPT higher than those of LNP-3. Because of their similar properties, we chose LNP-1 and LNP-2 with high content and purity as the further purification by SephadexG-150 column. As shown in Fig. 3b and Fig. 3c, LNP-1 and LNP-2 were all single components after purification. The extraction rates were 47.54% and 57.38% respectively.

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3.5 Structure characterization

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3.5.1 Molecular weight

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The molecular weight was determined by high performance gel permeation chromatography. As shown in Fig. 4a and Fig. 4b, the number average molecular weights of LNP-1 and LNP-2

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were 3011 Da and 3297 Da respectively. Their weight average molecular weights were 11703 Da

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and 13369 Da respectively. The absorption peak percentages of the two components were close to 100%, indicating that the purity of LNP-1 and LNP-2 was high.

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Compared with hericium erinaceus polysaccharide with an average molecular weight of

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17000 Da, the molecular weight of Lepista nuda polysaccharide was larger, while that of lentinus polysaccharide was the largest, reaching 800000 Da [42, 43].

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3.5.2 Monosaccharide composition

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The monosaccharide composition of polysaccharides was determined by high performance liquid chromatography (HPLC) after acid hydrolysis and pre-column derivatization. Fig. 4c was a HPLC chromatogram of various standard monosaccharide samples used as a reference. Different monosaccharide standards had corresponding retention time, which were mannose (tR=15.734), ribose (tR=20.141), rhamnose (tR=21.314), glucuronic acid (tR=23.601), galacturonic acid (tR=27.534), glucose (tR=33.621), galactose (tR=38.744), xylose (tR=40.231), arabinose (tR=42.341) and fucose (tR=48.321).

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ACCEPTED MANUSCRIPT The monosaccharide compositions of LNP-1 and LNP-2 were shown in Fig. 4d and Fig. 4e, respectively. LNP-1 was mainly composed of mannose, glucose, galactose, xylose, arabinose and fucose with molar ratios of 19.0, 33.5, 18.0, 4.6, 21.0 and 3.9, while LNP-2 was mainly composed of mannose, glucose, galactose, arabinose and fucose with molar ratios of 23.5, 11.4, 34.2, 21.4

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and 9.5.

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3.5.3 UV spectrum

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The crude LNP, LNP-1 and LNP-2 were scanned by UV spectrometer at 200 to 400 nm band. As shown in Fig. 4f, Fig. 4g and Fig. 4h, a weak absorption peak of crude LNP at 260 nm

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indicated that crude polysaccharides from Lepista nuda contained very few proteins. However,

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LNP-1 and LNP-2 had no absorption peak at 260 nm, which meant that all proteins were removed during the purification process. In addition, no absorption peak were found in crude LNP, LNP-1

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and LNP-2 at 280 nm, indicating that they contained no nucleic acid.

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3.5.4 FT-IR spectrum

Their infrared spectra were shown in Fig. 4i and Fig. 4j. For LNP-1, the strong peak at 3392

CE

cm-1 is attributed to the tensile vibration of hydroxyl groups. The peak value at 2933 cm-1 belongs

AC

to C-H tensile vibration of sugar alkyl. The absorption peak of 1647 cm-1 is caused by the tensile vibration of C=O. Tensile vibration peak of C-O appears at 1417 cm-1, indicating that LNP-1 may contain carboxyl group. Because of the change of C-H angular vibration, the peak value is 1354 cm-1. The absorption band at 1245 cm-1 belongs to the tensile vibration of C-N and N-H, and the symmetrical tensile vibration of S=O reaches its peak at 1149 cm-1. The absorption peak at 1078 cm-1 represents C-O-C tensile vibration or C-O-H deformation vibration. The absorption peak at 1026 cm-1 belongs to the pyranose ring, while the C-H bond on the deformation vibration of α-

17

ACCEPTED MANUSCRIPT pyran ring produces an absorption peak at 800 cm-1 [44]. For LNP-2, the strong and broad peaks at 3371 cm-1 belong to the tensile vibration of hydroxyl groups. The C-H tensile vibration of glycosyl alkyl peaked at 2933 cm-1. The tensile vibration of C=O produces an absorption peak at 1649 cm-1. The absorption peak at 1419 cm-1

PT

belongs to C-O tensile vibration of carboxyl group. The vibration peak of C-H is at 1371 cm-1. The

RI

absorption peak at 1246 cm-1 belongs to C-N and N-H tensile vibration. The tensile vibration of

SC

S=O bond and sulfate symmetric group is 1149 cm-1. The absorption peak at 1078 cm-1 is caused by C-O-C tensile vibration and C-O-H deformation vibration. The absorption peak at 1041 cm-1 is

NU

the vibration absorption peak of the pyranose ring, and the absorption peak at 800 cm-1 is the C-H

MA

of the α-pyran ring [45].

From the above analysis, we can see that the peaks and structures of LNP-1 and LNP-2 are

D

basically similar. They are carboxyl and sulfate-containing polysaccharides. In addition, they are

PT E

acid polysaccharides of furanose type and pyranose type. Compared with the infrared spectra of lentinan, Lepista nuda polysaccharides has similar

CE

vibration peaks of hydrogen bond, acetamide bond, amino bond, and pyran glycoside bond [46].

AC

3.5.5 SEM analysis

Fig. 4k and Fig. 4l portrayed their surface morphology in SEM images. LNP-1 had a smooth surface and a small number of cracks. The surface of the sample was uneven and there were gaps between the crystals. LNP-2 was fibrous and had a rough surface, which was due to the irregular shape of the molecules, resulting in different molecular aggregation. Generally speaking, the interactions between LNP-1 and LNP-2 polysaccharides were mainly repulsive, but less attractive. 3.5.6 NMR spectrum

18

ACCEPTED MANUSCRIPT Fig. 4m and Fig. 4n showed the 1H NMR spectra of LNP-1 and LNP-2. In the 1H NMR spectrum, the signals of polysaccharide were concentrated between δ 3.4 and 5.4 ppm. The NMR signals of hydrogen protons on α configuration glycoside anomeric carbon generally occurred between δ 5.0 and 5.5 ppm, while the isomers of β configuration glycosides occurred between δ

PT

4.5 and 5.0 ppm. For LNP-1 and LNP-2, there were two types of glycoside bonds (α- and

RI

β-configurations), in which β-constitution was dominant. LNP-1 contained mannose (δ 3.72, δ

SC

3.90 ppm), glucose (δ 5.01, δ 5.08), galactose (δ 3.87 ppm), xylose (δ 1.92, δ 4.27 ppm), arabinose (δ 3.33, δ 3.42, δ 3.51ppm), and fucose (δ 1.25, δ 5.16 ppm). LNP-2 contained mannose (δ 3.71, δ

NU

3.90 ppm), glucose (δ 4.54, δ 5.01, δ 5.08 ppm), galactose (δ 3.86 ppm), arabinose (δ 3.33, δ 3.41,

MA

δ 3.52 ppm), and fucose (δ 1.24, δ 5.15 ppm), which were consistent with the monosaccharide composition analysis. In addition, there was no hydrogen proton signal at δ 5.40 ppm, indicating

D

that LNP-1 and LNP-2 were pyranose, which was consistent with infrared spectroscopy analysis

PT E

[47, 48]. Fig. 4o and Fig. 4p showed

13

C NMR spectrua of LNP-1 and LNP-2. Six signal peaks (δ

CE

105.02, δ 102.45, δ 101.49, δ 99.25, δ 98.27 and δ 97.67 ppm) appeared in the isomeric carbon 13

C NMR spectra, indicating that there were six

AC

region (δ 95-110 ppm) of LNP-1 in the

monosaccharides in LNP-1. LNP-2 had five signals (δ 105.02, δ 102.79, δ 101.47, δ 100.90, δ 98.29 ppm) in the allogenic carbon region. Among them, the α-type was in the range of δ 97-101 ppm and the β-type was in the range of δ 103-106 ppm. Therefore, LNP-1 and LNP-2 had both α-configuration and β-configuration, and β-conformation was the main part. C6 substitution appeared in LNP-1 and LNP-2, indicating that they had a 1→6 glycosidic bond. Therefore, LNP-1 and LNP-2 were mainly β-1,6-pyranose [49].

19

ACCEPTED MANUSCRIPT 3.6 Physicochemical properties The physicochemical properties of crude LNP, LNP-1 and LNP-2 were determined by physical and chemical methods. The contents of polysaccharides in crude LNP, LNP-1 and LNP-2 were 70.60%, 87.71% and 81.20% respectively. The protein content was 1.72%, 0.97% and 0.68%

PT

respectively. The contents of sulfuric acid were 3.39%, 5.02% and 8.64%. The contents of uronic

RI

acid were 63.66%, 5.56% and 6.80% respectively.

3.7.1 Superoxide anion radical scavenging activity

SC

3.7 LNP in vitro antioxidant activities

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In order to analyze the antioxidant activity of LNP in vitro, three parameters were evaluated.

MA

As shown in Fig. 5a, the scavenging activity of LNP on superoxide anion radicals increased from 0 to 4.0 mg/mL in a concentration dependent manner. The scavenging activities of Vc, crude LNP,

D

LNP-1, and LNP-2 on superoxide anion radicals were significantly different at the experimental

PT E

concentration. The scavenging activity of ·O2- increased with increasing concentration. When the concentration was 4.0 mg/mL, the scavenging activity of crude LNP on superoxide anion radicals

CE

was 36.64 ± 1.18% higher than that of LNP-1 (24.67 ± 0.55%) and LNP-2 (23.42 ± 1.20%), but

AC

much lower than that of Vc (62.91 ± 1.00%). Compared to polysaccharides from Mushroom, Pleurotus ostreatus and Chicken leg mushroom, their scavenging superoxide anion free radicals showed similar trend and increased with concentration. When the concentration was 4.0 mg/mL, the superoxide anion free radicals scavenging activities of polysaccharides from Mushroom, Pleurotus ostreatus and Chicken leg mushroom were 21.5%, 30.04% and 6.9%, respectively. Therefore, the in vitro antioxidant activity of LNP was stronger than that of polysaccharides from Mushroom and Chicken leg mushroom

20

ACCEPTED MANUSCRIPT [50]. 3.7.2 DPPH radical scavenging activity Fig. 5b depicted the scavenging activity of LNP on DPPH radicals. The scavenging activity of Vc was significantly higher than that of LNP. Among the polysaccharides, the activity of

PT

scavenging DPPH radicals by crude LNP was the strongest, followed by LNP-1 and LNP-2. As

RI

the concentration increased from 0 to 4.0 mg/mL, the scavenging activities of samples on DPPH

SC

radicals increased gradually. When the concentration of polysaccharide reached 4.0 mg/mL, the DPPH radical scavenging rates of crude LNP, LNP-1, and LNP-2 were 55.94 ± 0.78%, 42.65 ±

NU

4.05%, and 43.06 ± 3.86% respectively.

MA

The scavenging activity of LNP on DPPH radicals was similar to that of polysaccharides from Agrocybe cylindracea, and was much higher than that of polysaccharides from Pleurotus

D

ostreatus. When the concentration of polysaccharides was 4.0 mg/mL, the scavenging rate of

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DPPH radicals by polysaccharides from Pleurotus ostreatus, Flammulina velutipe, and Agrocybe cylindracea was 41.82%, 53,21% and 56.76%, respectively [51].

CE

3.7.3 Ferrous ion chelating activity

AC

As shown in Fig. 5c, the chelating activity of LNP on ferrous ions was also in concentration dependent manner. The chelating activity of LNP was significantly lower than that of Vc. The Fe2+ chelating ability of the four samples was Vc, crude LNP, LNP-1, and LNP-2 in descending order. With the increase of concentration, the chelating activity of Vc on Fe2+ increased, and tended to be gentle at 1.0 mg/mL, and the chelating activity of LNP increased. When the solution concentration reached 4.0 mg/mL, the iron chelating rate of crude LNP was 70.09 ± 2.39%, higher than that of LNP-1 (50.62 ± 1.01%) and LNP-2 (46.78 ± 3.08%).

21

ACCEPTED MANUSCRIPT Compared to polysaccharides from Agaricus bisporus, the chelating activity was stable with the increase of concentration, and the chelating rate of polysaccharides from Agaricus bisporus was close to 80% [52]. When the concentration of the solution was 4.0 mg/mL, the chelating rate of polysaccharides from Black Ganoderma was only 20%, lower than that of LNP [53]. Therefore,

PT

there are differences in the chelating activity of ferrous ions among different edible fungus. In

RI

general, the ferrous ion chelating activity of LNP was stronger than most of the others.

SC

4. Conclusion

In this study, the extraction and decolorization processes of the polysaccharide is effective.

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Two novel polysaccharides were isolated and purified from mushroom Lepista nuda, and

MA

characterized. LNP mainly contained two components, LNP-1 and LNP-2, which are 11703 Da and 13369 Da respectively. The monosaccharides of LNP were mainly mannose, glucose,

D

galactose, xylose, arabinose, and fucose, mainly β-1,6-pyran acid polysaccharide. According to the

PT E

free radical scavenging experiment, the superoxide anion scavenging activities of crude LNP, LNP-1 and LNP-2 were 36.64 ± 1.18%, 24.67 ± 0.55% and 23.42 ± 1.20%, respectively when the

CE

concentration of the solution was 4.0 mg/mL. The scavenging rates of crude LNP, LNP-1, and

AC

LNP-2 on DPPH free radicals were 55.94 ± 0.78%, 42.65 ± 4.05%, and 43.06 ± 3.86% respectively. The ferrous ion chelating rates of crude LNP, LNP-1, and LNP-2 were 70.09 ± 2.39%, 50.62 ± 1.01% and 46.78 ± 3.08% respectively. LNP exhibited potent antioxidant activity and could be developed as a health care product or drug. Future research will focus on its in vivo experiments, more functions, and structure-activity relationship. Acknowledgments Authors gratefully acknowledge the Natural Science Foundation of Jiangsu Province

22

ACCEPTED MANUSCRIPT (BK20171379); Jiangsu Science and Technology Supporting Special Item for Northern Jiangsu (BN2015071); the Enterprise Graduate Workstation of Nanjing Agricultural University and the Enterprise Graduate Workstation of Jiangsu Province (Nanjing Agricultural University & Jiangsu Lianyi Biological Technology Co., Ltd.); the Enterprise Graduate Workstation of Jiangsu Province

PT

(Nanjing Agriculture University & Nantong Chitsuru Foods Co., Ltd.).

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Chem. 107 (2008) 231-241.

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ACCEPTED MANUSCRIPT Table 1 The physical and chemical property of different resins Specific surface area Resin name

property

Particle size (mm)

Aperture(Å) (m2/g)

Weak-polar

0.3~1.25>90%

130~140

480~520

D101

Non-polar

0.3~1.25>90%

90~100

500~550

HPD100

Non-polar

0.3~1.25>90%

85~90

D301

Weak -alkaline

0.315~1.25≥95%

40~70

D941

Weak -alkaline

0.3~1.2>90%

D900

Weak -alkaline

0.3~1.25>90%

RI

SC

≥650 ≥550

40~60

-

-

-

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MA D PT E CE AC

30

PT

AB-8

ACCEPTED MANUSCRIPT Table 2 Design and results of orthogonal test polysaccharide Run

A

B

C

D

Overall Decoloring rate /%

1

1

1

85.95

53.39

80.98

2

1

2

2

2

84.42

78.07

94.41

3

1

3

3

3

66.39

81.20

85.75

4

2

1

2

3

81.67

86.16

97.51

5

2

2

3

1

77.70

78.59

90.81

6

2

3

1

2

73.72

74.54

86.15

7

3

1

3

2

75.25

70.23

84.54

8

3

2

1

3

71.28

78.98

87.30

9

3

3

2

1

65.78

82.11

85.92

K1

87.05

87.68

84.81

85.90

K2

91.49

90.84

92.62

88.37

K3

85.92

85.94

87.03

90.19

R

5.57

4.90

7.80

4.28

D

PT E

CE

AC

31

SC

PT

1

MA

1

RI

rating

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retention rate /%

ACCEPTED MANUSCRIPT Table 3 Response surface optimization result analysis a. Design and results of response surface experiments Extraction

solvent to Extraction

temperature °

Predictive

value%(Y)

value%(Y)

material ratio time h(B) ml/g(C)

-1

-1

0

2

1

0

1

3

1

1

0

4

0

1

1

5

-1

0

6

0

-1

7

0

0

8

0

9

-1

10

0

4.68

4.63 5.04

5.30

5.35

4.68

4.68

4.56

4.58

1

5.03

5.06

0

5.33

5.36

1

-1

5.21

5.18

1

0

4.33

4.31

0

0

5.36

5.36

0

0

5.35

5.36

-1

0

-1

4.40

4.45

0

0

0

5.36

5.36

14

1

0

-1

5.22

5.20

15

1

-1

0

4.79

4.81

16

0

-1

-1

4.58

4.58

17

0

0

0

5.41

5.36

12 13

NU MA

1

PT E

CE 0

AC

11

SC

5.08

D

1

RI

C(A)

PT

Number

Actual

32

ACCEPTED MANUSCRIPT b. Analysis of variance of regression equation Source of

sum of

Degree of

Mean F value

P value

Significant

significant

square

freedom

square

Model

2.25

9

0.25

117.08

<0.0001

A

0.73

1

0.73

342.08

<0.0001

B

0.024

1

0.024

11.31

C

0.0004500

1

0.0004500

0.21

AB

0.18

1

0.18

AC

0.022

1

0.022

BC

0.24

1

A2

0.44

1

B2

0.29

1

C2

0.21

Residual Missing items

Total deviation

0.5824

0.0101

112.20

<0.0001

0.44

205.91

<0.0001

0.29

136.61

<0.0001

1

0.21

98.28

<0.0001

0.015

7

0.002140

0.011

3

0.003833

4.41

0.0930

0.003480

4

0.0008700

CE 2.27

NU

10.51

PT E

<0.0001

D

SC

RI

0.0068

86.40

AC

Error

PT

variance

MA

0.24

16

33

not significant

ACCEPTED MANUSCRIPT a

5.5

c

5.0

c

Extraction rate/%

b 4.5

4.0

a 3.5

3.0 1.0

1.5

2.0

2.5

3.0

3.5

4.0

b

PT

Extraction time/h

d

d

c

4.5

SC

Extraction rate/%

5.0

b 4.0

a

75

NU

3.5

3.0

RI

5.5

80

85

90

95

Extraction temperature/℃

MA

c 5.5

4.0

D

4.5

PT E

Extraction rate/%

5.0

c

c

3

4

b

a

3.5

3.0

CE

1

2

Extraction times

Extraction rate/%

AC

d

cd cd

5.5

c

d

5.0

b 4.5

4.0

3.5

a 3.0 10

20

30

40

50

60

Extract solvent to material ratio(mL/g)

Fig. 1. Effects of different factors on the polysaccharide extraction rate. (a) Extraction time; (b) Extraction temperature; (c) Extraction times;(d) Extraction solvent to material ratio. Data are means ± SD (n = 3). The error bars represent the standard deviation. Values marked by the same letter are not significantly different (p < 0.05).

34

ACCEPTED MANUSCRIPT

a 90

90

C

C

Decolorizing rate/%

85

Polysaccharide retention rate/%

C

85

B

80

80

a 75

75

b b

A

70

b

70

c

65

65 1.5

2.0

2.5

3.0

PT

1.0

Resin dosage/g

RI

b

90

85

85

B

80

SC

a 75 70

C

C

C

b

75 70

c

65

A 60 55 60

80

100

d

NU

Decolorizing rate/%

a 80

120

140

65 60

Polysaccharide retention rate/%

90

55

160

180

MA

Shake speed/rpm

90 85

a

85

C

C

C

80

80

D

b

75

B

70

c

70

d

65

65

A

60

60

e

55

CE

50

1.0

1.5

2.0

50

2.5

Decolorizing time/h

90

90

85

C 80

Decolorizing rate/%

AC

d

0.5

55

B

C

85

80

B

75

75

A b

b

70

a

a

70

a

65

60

65

Polysaccharide retention rate/%

75

PT E

Decolorizing rate/%

90

Polysaccharide retention rate/%

c

60 30

35

40

45

50

Decolorizing temperature/℃

Fig. 2. Effects of different factors on the polysaccharide decolorization. (a) Resin dosage; (b) Shake speed; (c) Decolorizing time; (d) Decolorizing temperature. ■: polysaccharide retention rate/%, ●: decolorizing rate/%. Data are means ± SD (n = 3). Values marked by the same letter are not significantly different (p <0.05).

35

ACCEPTED MANUSCRIPT

2.0

0.6

Absorbance values（A490）

LNP-1

LNP-2

0.5

1.5 0.4

1.0

0.3

0.2 0.5 0.1

0.0

0

20

40

60

80

100

120

140

160

b

RI

Number of tubes-1

2.0

LNP-1

NU

1.5

SC

2.5

1.0

0.5

0.0 0

5

MA

Absorbance values（ A490）

PT

0.0

NaCl concentration（mol/L）

a

10

15

20

25

30

Number of tubes-1

c

1.5

LNP-2

1.0

AC

CE

Absorbance values（ A490）

PT E

D

2.0

0.5

0.0 0

5

10

15

Number of tubes

20

25

30

-1

Fig. 3. Purification of LNP. (a) Stepwise elution curve of crude LNP by DEAE-52 column; (b) Elution curve of LNP-1 by SephadexG-150; (c) Elution curve of LNP-2 by SephadexG-150. ▲: Absorbance values (A490), ■: NaCl concentration (mol/L).

36

ACCEPTED MANUSCRIPT

a 12

12505

LNP-1

10 8

MV

6 4 2 0

△

△

-2

0

5

10

15

20

25

30

PT

Rt/min

8

b

11791

RI

LNP-2 6

SC

1670598

MV

4

2

0

△

NU

△ △

-2

0

5

10

15

20

25

30

MA

Rt/min

c 35

D

standard monosaccharide 30

1

mAU

PT E

25

2

20

3

15

4

6

5

7

8

9

10

10

d

0 0

10

20

30

40

50

time/min

50

LNP-1 40

30

mAU

AC

CE

5

2

1 20

3

10

4

5 6

0 0

10

20

30

time/min

37

40

50

ACCEPTED MANUSCRIPT

50

e

LNP-2 40

mAU

30

3

1

20

2

10

4

5

0 0

10

20

30

40

50

f

PT

time/min

0.8

crude LNP

Abs

SC

0.6

RI

1.0

0.4

0.0 200

250

NU

0.2

300

350

400

g

MA

wavelength（nm）

PT E

Abs

0.4

LNP-1

D

0.6

0.2

250

300

350

400

wavelength（nm）

h 1.0

LNP-2 0.8

Abs

AC

CE

0.0 200

0.6

0.4

0.2

0.0 200

250

300

wavelength（nm）

38

350

400

ACCEPTED MANUSCRIPT

i

100

LNP-1

800 565

1647

2933

40

1417 1354 1245

60

3392

Transmission rate/%

80

0 4000

3000

2000

PT

1149 1078

20

1000

100

SC

j

RI

Wave number（cm-1）

LNP-2

0 4000

3000

2000

800 567

1149

MA

20

1078

1649

40

1419 1371 1246

2933

NU

60

3371

Transmission rate/%

80

1000

D

Wave number（cm-1）

k

l LNP-2

m

AC

CE

PT E

LNP-1

n LNP-2 1H NMR

1

LNP-1 H NMR

39

ACCEPTED MANUSCRIPT o

p LNP-2 13C NMR

LNP-1 13C NMR

AC

CE

PT E

D

MA

NU

SC

RI

PT

Fig. 4. Structure characterization of LNP. (a,b) Molecular weight of LNP-1 and LNP-2; (c,d,e) Monosaccharide composition of standard monosaccharide samples, LNP-1 and LNP-2; (f,g,h) UV spectrum of crude LNP, LNP-1 and LNP-2; (i,j) FT-IR spectrum of LNP-1 and LNP-2; (k,l) SEM image of LNP-1 and LNP-2; (m,n) 1H NMR spectrum of LNP-1 and LNP-2; (o,p) 13C NMR spectrum of LNP-1 and LNP-2.

40

ACCEPTED MANUSCRIPT

a

Vc crude LNP LNP-1 LNP-2

60

Clearance rate/%

50

40

30

20

0

0

1

2

PT

10

3

4

100

SC

b

RI

Polysaccharide concentration（mg/mL）

Vc crude LNP LNP-1 LNP-2

60

NU

Clearance rate/%

80

40

0

MA

20

0

1

2

3

4

100

PT E

c

D

Polysaccharide concentration（mg/mL）

Vc crude LNP LNP-1 LNP-2

60

CE

Clearance rate/%

80

40

AC

20

0

0

1

2

3

4

Polysaccharide concentration（mg/mL）

Fig. 5. In vitro antioxidant activities of LNP. (a) Superoxide anion radicals; (b) DPPH radicals; (c) Ferrous ion chelating activity.

41