Ultrasonic disruption extraction, characterization and bioactivities of polysaccharides from wild Armillaria mellea

Journal Pre-proof Ultrasonic disruption extraction, characterization and bioactivities of polysaccharides from wild Armillaria mellea

Ruizhan Chen, Xing Ren, Wei Yin, Juan Lu, Li Tian, Lun Zhao, Ruping Yang, Shujun Luo PII:

S0141-8130(19)38360-6

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.196

Reference:

BIOMAC 13977

To appear in:

International Journal of Biological Macromolecules

Received date:

15 October 2019

Revised date:

13 November 2019

Accepted date:

24 November 2019

Please cite this article as: R. Chen, X. Ren, W. Yin, et al., Ultrasonic disruption extraction, characterization and bioactivities of polysaccharides from wild Armillaria mellea, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/ j.ijbiomac.2019.11.196

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© 2019 Published by Elsevier.

Journal Pre-proof

Ultrasonic disruption extraction, characterization and bioactivities of polysaccharides from wild Arimillaria mellea Ruizhan Chen*, Xing Ren , Wei Yin, Juan Lu, Li Tian, Lun Zhao, Ruping Yang, Shujun Luo College of Chemistry, Changchun Normal University, Changchun 130032, PR China ABSTRACT

An efficient ultrasonic disruption extraction (UDE) of polysaccharides from Arimillaria mellea (AM) were optimized by response surface methodology (RSM). Under optimum conditions: ultrasonic power 915.00 W, temperature 69.27℃ and time 39.13 min, the crude polysaccharides (AMPs) yield was

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19.5%. Two purified fractions AMPs-1-1 and AMPs-2-1 were obtained through anion-exchange and gel chromatography. AMPs-1-1 was a heteropolysaccharide with average molecular weights of 1.23 ×

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105 Da, and composed of Glc, Gal and GlcA with mole percentages of 89.06%, 9.59% and 1.34%, respectively, owning a backbone structure of (1)-β-D-Glcp, (13,6)--D-Glcp and (13)-β-D-Glcp

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residues. AMPs-2-1 was a heteropolysaccharide with average molecular weights of 6.76 × 10 4 Da, and composed of Glc, Gal, GlcA and Man with mole percentages of 65.28%, 22.87%, 2.87% and 8.98%,

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containing a main backbone chain of (13,6)--D-Glcp and (16)-β-D-Glcp residues. AMPs-2-1 possessed obviously antioxidant activities in terms of stronger scavenging activity against DPPH and

lymphocytes and RAW264.7

lP

ABTS+, higher FRAP and ORAC value than AMPs-1-1. AMPs-2-1 could promote splenocyte macrophages proliferation and enhanced the phagocytosis of

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macrophages, exhibited significant immunomodularory activities. These results suggested that UAE is an effective extract technology, and AMPs-2-1 could be explored as potential natural antioxidants and

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immunomodulator agents.

Keywords: Physicochemical properties; Arimillaria mellea polysaccharides; Biological activities, Carbohydrate chemistry

Nonstandard abbreviations: UDE, ultrasonic disruption extraction; HRE, hot reflux extraction; AM,

Arimillaria mellea; RSM, response surface methodology; AMPs, Arimillaria mellea polysaccharides extracted by UDE method; AMPs-1-1 and AMPs-2-1, purified polysaccharides from AMPs; Glc, glucose; Gal, galactose; GlcA, glucuronic acid; Man, mannose; DPPH, 1, 1-diphenyl-2-picryl-hydrazyl; ABTS+, 2,2-azinobis 3-ethylbenzothiazoline-6-sulfonate radical; FRAP, Ferric reducing antioxidant power; ORAC, oxygen radical absorbance capacity; HPGPC, high performance gel permeation chromatography; HPLC, high performance liquid chromatography; GC-MS, gas chromatograph-mass spectrometer;

NMR,

nuclear

magnetic

resonance;

Trolox,6-hydroxy-2,5,7,8-

tetramethychroman-2-carboxylic acid. 

Corresponding author at: R Chen, College of Chemistry, Changchun Normal University, Changchun, Jilin, PR

China. E-mail addresses: [email protected] 1

Journal Pre-proof 1. Introduction A Arimillaria mellea (AM), one of the most popular and wild edible mushroom, have become attractive as a source of bioactive compounds including polysaccharides, proteins, amino acid, fatty acid, sterol, minerals and vitamins [1]. Polysaccharides extracted from AM, possess various pharmacological activities, including immunomodulatory, antitumor and antioxidant activities [2]. Polysaccharides, as a kind of natural biological macromolecules, existed in almost all organisms have attracted broad attention and have been widely used in

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food, feed, medicine and pharmaceutics, due to their non-cytotoxic, biodegradable properties,

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a wide range of biological functions and pharmacology activities [3], such as energy storage, structure support, cell regulation, immunomodulatory, anti-tumor, antioxidant, anti-

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inflammatory, anti-viral and so on [4-5]. Extraction techniques are an important step for

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preparation and utilization of polysaccharides as it could have a great influence on the

lP

extraction yields, physicochemical properties, and bioactivities of polysaccharides [6]. The conventional extraction method of polysaccharide is hot reflux extraction (HRE) [7].

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However, HRE requires high extraction temperature and long extraction time, which may cause the degradation of polysaccharide and decrease of pharmacological activities [8].

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Recently, some novel techniques have been developed to extract polysaccharide, such as ultrasonic-assisted

extraction

[9],

high-pressure

ultrasonic-assisted

extraction

[10],

microwave-assisted extraction [11], enzyme-assisted extraction [12], ultrahigh pressure extraction [13]. Among different innovative extraction methods, UDE has been recognized as a most promising extraction techniques and environment-friendly extraction technique. Ultrasonic can effectively disrupt cell walls, reduce particle-size, enhance mass transfer of the cell contents and improve the extraction yield, reduce the energy and extraction time along with obtaining a higher bioactivity [14]. The extraction method can improve the extraction efficiency of polysaccharide, which may improve the bioactivity of polysaccharide [15]. Thus, developing an optimized novel extraction technology plays a key role in the industrialize production and application of polysaccharide. 2

Journal Pre-proof The

biological

activities

of

polysaccharides

have

been

correlated

with the

physicochemical characterization [16]. However, little work has been reported on the isolation, purification, characterization, antioxidant and immunostimulatory activitiy and structure-activity relationship of polysaccharide from AM. In this study, the extraction conditions of UDE of AMPs was optimized by using RSM. Two polysaccharide fractions were isolated and purified by DEAE-Sepharose Fast Flow and Sephadex G-100 chromatography. Furthermore, the physicochemical properties, antioxidant

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and immunomodulatory activities of two polysaccharides were investigated. The results can

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promote the application of AMPs to pharmaceutical industries.

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2. Materials and methods 2.1. Materials and reagents

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Samples of wild AM were collected in Fusong County, Jilin Province, China. Fresh AM

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was sun-dried, ground into fine powder, and then pretreated with petroleum ether and 95% ethanol to remove some colored materials and small molecule materials. Male Kunming mice

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(8-12 weeks old) were purchased from Pharmacology Experimental Center of Jilin University.

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Fluorescein sodium (FL), 2,2’-azobis (2-methylpropionamidine) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic

acid

(Trolox),

2,2’-azino-bis

(3-

ethylbenzthiazoline-6-sulfonic) acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Concanavalin A (ConA), lipopolysaccharide

(LPS),

D-mannose

(Man), D-ribose (Rib), L-rhamnose (Rha),

D-glucuronic acid (GlcA), D-galacturonic acid (GalA), D-glucose (Glc), D-xylose (Xyl), D-galactose (Gal), L-arabinose (Ara), D-fucose (Fuc), T-series Dextran (T-10, T-50, T-100, T-200 and T-500) were purchased from Sigma-Aldrich (Germany). DEAE-Sepharose Fast Flow and Sephadex G-100 were obtained from Amersham Biosciences Co (USA). RPMI-1640 medium was provided from Gibco Invitrogen Co (USA). Bovine serum albumin (BSA) was purchased from Kaiyang Biochemistry Co (China). RAW264.7 murine 3

Journal Pre-proof macrophage cells obtained from Keygen Co (China). All other reagents were of grade AR. 2.2. Extraction of AMPs The UDE was performed with a SL-2010N multi-frequency ultrasonic cell crushing apparatus intelligent temperature control, multi-frequency ultrasonic processor, selected power, time and temperature (Shun Liu Apparatus Co. Ltd). The extracts were filtered, concentrated, precipitated by addition of dehydrated ethanol to a final concentration of 75% (v/v) and kept at 4 ℃ overnight. The precipitates were collected by centrifugation,

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deproteinated by the Sevag method [17], dialyzed and lyophilized to get the crude

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polysaccharides (AMPs). The polysaccharide content of AMPs was measured by the phenol-sulfuric method using Glc as a standard [18]. The AMPs yield (Y, %) was calculated

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as: Y (%) = (WP/WA)  100, where WP is the weight of polysaccharides, and WA is the weight

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of AM.

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2.3. Experimental design

Based on the single-factor experiments, a three-factor three-level BBD was chosen to

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evaluate the combined effect of three independent variables: ultrasonic power (X1), extraction temperature (X2) and extraction time (X3). The ranges of independent variables, levels, and

second-order model: 𝑌

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responses are presented in Table 1S. The experimental data was fitted to the following polynomial

= 𝛽0 + ∑3i=1 𝛽𝑖 𝑋i + ∑3i=1 𝛽ii 𝑋i2 + ∑3i=1 ∑3j=1 𝛽ij 𝑋i 𝑋j ， where Y is the

response variables (yields of AMPs); 0, i, ii and ij are the regression coefficients for the intercept, linearity, square and interaction terms; Xi and Xj are the independent coded variables, respectively. All experiments were performed in triplicates, and the experimental results were presented as mean ± standard deviation (SD). The experimental data were analyzed using Design Expert software (version 7.0, USA). The values of R2, adjusted-R2 of models were evaluated to check the model adequacies. The P < 0.05 was considered to be statistically significant.

4

Journal Pre-proof 2.4. Scanning electron microscope (SEM) analysis In order to investigate the influence of UDE on the microstructure of samples and to understand the extraction mechanisms, the dried powders of the untreated sample and the extracted residues after UDE (or HRE) were observed by SU8000TMP SEM (Hitachi, Japan). The dried powder samples were sputtered with gold under reduced pressure and determined by SEM at a 10 kV of acceleration voltage. 2.5. Isolation and purification of AMPs

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AMPs were re-dissolved in distilled water, filtrated, the supernatants were applied on a DEAE-Sepharose Fast Flow column (20 × 250 mm), eluted with a linear gradient from 0‒0.8

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M NH4HCO3 solution at a flow rate of 0.8 mL/min. The obtained fractions were combined

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according to the total carbohydrate content quantified by the phenol-sulfuric acid method, two

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main fractions (named AMPs-1 and AMPs-2) were collected, dialyzed against distilled water for 48 h and concentrated, then further purified by a Sephadex G-100 (20 × 500 mm)

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size-exclusion chromatography. The relevant fractions were collected, dialyzed against

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distilled water, concentrated and finally lyophilized to obtain two purified polysaccharides, namely AMPs-1-1 and AMPs-2-1.

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2.6. Analysis for physicochemical characteristics of the polysaccharides The content of total carbohydrate was determined by phenol-sulphuric acid method using glucose as a standard [18]. The uronic acid content was measured by a modified hydroxydiphenyl assay with GlcA as a standard [19]. Protein content was determined by the Bradford method using BSA as standard [20]. The molecular weight and homogeneity of the sample was measured by HPGPC. 10 L of the sample solution (2 mg/mL) was applied to Waters 2695 HPLC system equipped with a ultrahydrogel 500 column (10 m, 7.8  300 mm, Waters, USA), eluted with 0.05 M Na2SO4 solution at a flow rate of 0.6 mL/min, and detected by a refractive index detector (Waters 2414, USA), the column temperature was 35℃. The average molecular weight was calculated according to the standard curve established by standard dextran, and the equation of the

5

Journal Pre-proof standard curve was: logMw = -0.2019t + 9.9649, R2 = 0.9978 (where Mw represented the molecular weight, while t represented retention time). Sample (2 mg) was hydrolyzed with 2.5 M TFA (1 mL) at 100℃ for 8 h. The mixture was vacuum evaporated with methanol to dryness, and the procedure was repeated till TFA to be removed. The released monosaccharides from sample or standard monosaccharides mixture (0.2 mL) were dissolved in 0.5 mL NaOH (0.3 M), and 0.5 mL methanol solution of PMP (0.5 M) was added. The mixture was stirred for 1 h at 75℃. Subsequently neutralized

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with 0.5 M HCl, the resulting solution was extracted three times with 1 mL chloroform each

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time, and the aqueous layer was syringe filtered using a 0.45 m nylon filter for HPLC analysis. The monosaccharide contents in the samples were determined using HPLC system

-p

(Waters 2695, USA), with a UV detector (Waters 2487, USA) and a high performance

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carbohydrate analysis column (4 μm, 250 × 4.6 mm, Waters, USA). The HPLC condition was

lP

as follows: column temperature, 30°C; detection wavelength, 248 nm; mobile phase, acetonitrile (A), KH2PO4 (3.8 mM) containing 3.5 mM triethylamine (pH 7.5) (B), elution

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gradient, 94%–94%–91%–91% B from 0–5–8–30 min; flow rate, 0.8 mL/min; injection volume, 20 L. Identification of sugar configuration was done by comparison with retention

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time of the derivatives of reference standard monosaccharide. The molar ratios of monosaccharides were quantified by comparing their integration values of peak area to a calibrated standard curve.

The methylation analysis procedure was carried out according to the method described by Luo et al. [21]. The methylated products were hydrolyzed, reduced and converted into partially methylated alditol acetate, which were then analyzed by GC-MS on an Agilent 7890/5975C instrument (Agilent Technologies Co. Ltd., USA) with a HP-5 column (0.25 m, 30 m  0.25 mm). The temperature program was set at 160℃ (held for 2 min) to 180℃ at the rate of 5℃/min, and 180–250℃ (held for 5 min) at a rate of 3℃/min. N2 was used as the carrier gas and the flow rate was kept at 0.8 mL/min. The injection volume was 1 L. Infrared (IR) spectra were obtained using a fourier transform infrared (FT-IR) 6

Journal Pre-proof spectrometer (Nicolet Nexus 470, Thermo Fisher Scientific, USA). The sample was mixed with KBr powder and pressed into pellets for IR measurement at a wave number range of 4000–400 cm-1. AMPs-1-1 or AMPs-2-1 sample (30 mg) was dissolved in 2 mL D2O at 25°C, and filtered through 0.45 μm membrane. The 1H and 13C nuclear magnetic resonance (NMR) of spectra were recorded by Bruker Avance AV600 NMR spectrometer (Bruker, Germany). 2.7. Antioxidant activity assay

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The total antioxidant activity of sample was determined by using a FRAP assay according to the reported method [22]. the antioxidant power of sample was expressed as the equivalent

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to that of 1 μM FeSO4·7H2O. The DPPH· and ABTS+ scavenging activities were determined

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by the method of Chen et al. [23]. The ORAC of sample was estimated according to the

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reported method [24], the ORAC value was calculated and expressed as M of Trolox equivalent (TE)/g of sample using the calibration curve of Trolox at different concentrations

lP

(20–200 μM).

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2.8. Immunomodulatory activity assay

ConA-induced splenic lymphocyte proliferation was used to evaluate T lymphocyte

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proliferative activities and B lymphocyte proliferative activities were performed by using the LPS-induced proliferation as described by Liu et al. [25]. The effect of different polysaccharide on the RAW264.7 macrophages proliferation was carried out using MTT assay as the description of Li et al. [26]. The phagocytic activities of RAW264.7 macrophages were determined using neutral red assay as described by Di et al. [4]. 3. Results and discussion 3.1. Response surface optimization of extraction conditions The BBD matrix and the variation of extraction yield of AMPs with X1 , X2 and X3 are presented in Table 1S. A second-order polynomial model for establishing relationship between independent and the yield (Y) of AMPs was as following: Y (%) = 19.00 + 1.89X1 + 1.34X2 - 0.33X3 - 4.40X12 - 1.05X22 - 2.69X32 + 1.19X1X2 - 0.09X1X3 7

Journal Pre-proof - 1.17X2X3. The analysis of variance (ANOVA) for the response surface quadratic model is shown in Table 2S. The quadratic regression model had a high F-value (F = 56.8) and a very low P-value (P < 0.0001), indicating that the model was highly statistically significant. The coefficient of determination (R2) and the adjusted determination coefficient (Adj R2) are 0.986 and 0.969, which implied that 98.6% of the variations could be explained by the fitted model, and only 3.1% of the total variation could not be explained by the model. The lack of fit

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F-value of 2.68 and the associated P-value of 0.18 indicated that the lack of fit statistic was not significant (P > 0.05), which implied that it was not significant and a 18.00% chance

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could occur due to noise. A low value of coefficient of the variation (C.V.% = 3.94) clearly

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indicated that the experimental values of regression model was precise and reliable. These

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results suggested that the developed model could adequately represent the real relationship among the parameters chosen. Table 2S shows that the linear coefficients (X1 and X2),

lP

quadratic term coefficients (X12 and X32) and cross product coefficients (X1X2 and X2X3)

significant (P > 0.05).

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significantly affected the yield of AMPs (P < 0.01). The other terms (X3, X1X3) were not

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The three-dimensional (3D) response surface and two dimensional (2D) contour plots were obtained using Design Expert. The effects of power and temperature on the yield of AMPs are shown in Fig. 1a (3D) and b (2D), respectively. It can be seen that the yield enhanced rapidly with the increasing of power and reached to the peak value at 916 W. This occurrence might be the reason that higher power facilitates the disruption of fungi tissues and cell walls, which suggested that the increasing power could enhance yield. However, the yield decreased rapidly with the increase of the power beyond 916 W. Excessive high power would cause an increase in the bubble numbers in the solvent during cavitation, which may reduce the efficiency of the ultrasonic energy transmitted into the medium[27]. At the same time, excessive high power also caused polysaccharide depolymerization, which would result in a decrease of the extraction yield [8]. With the increase of temperature from 60 to 71℃, the

8

Journal Pre-proof yield was gradually increased from 12.7% to 18.7%, and then decreased gradually from 71 to 80℃. This is probably because that higher temperature could cause opening cell matrix, increase the diffusivity of the solvent, decrease the solvent viscosity, and improve the solubility of polysaccharides. However, a lower yield was found at 80℃ than that at 71℃. It has to be assumed that during the extraction process of polysaccharides degradation occurs more than the production increase from 71 to 80℃. High temperature led to the decrease of surface tension and the increase of vapour pressure within micro bubbles, causing the

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damping of the ultrasound wave [28]. It indicated that higher yield could be obtained when

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the moderate power and temperature were selected.

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Fig. 1c and d shows the combined effect of power and time on the extraction yield when temperature was kept at 0 levels. In the present study, both the power and time have a positive

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impact on the yield, in the power range of 750 to 937.5 W and in the time range of 30 to 39.5

lP

min. The maximum AMPs yield 19.0% was achieved at 937.5 W, 39.5 min, respectively. The increase of AMPs yield is due to the phenomenon of acoustic cavitation that can be

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intensified with the increase of power and time. Therefore, higher power and longer time are beneficial to the extraction of AMPs. However, higher power, longer ultrasound time might

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induce the degradation of polysaccharides and diminish the extraction yield, because of frequent asymmetric collapse of microbubbles. Similar correlation between yield, ultrasound time and temperature was established by Chen et al. [29]. Fig. 1e and f shows the combined effect of extraction temperature and time. It can be seen that the yield of AMPs increased rapidly with the increase of time, and reached the peak value rapidly at 41.5 min, but beyond 41.5 min, the yield decreased with the increasing of time. This result indicated that the time had a different extent of influence on yield at different temperature. The yield of AMPs showed an escalating trend with the increase of temperature. Higher mass transfer rate and solubility might be the primary factors responsible for these changes. Therefore, higher temperature and moderately long time are advantageous to the extraction of AMPs. 9

Journal Pre-proof According to response surface plots, contour plots and variance analysis, it can be concluded that optimal extraction conditions of AMPs were power 915 W, temperature 69.27℃ and time 39.13 min. Under these optimal conditions, the model predicted a maximum response of 19.5%. In ensure the predicted result was not biased toward the practical value, three verification experiments were performed by using following modified optimal conditions: power 915 W, temperature 69℃, time 39 min, and water to raw material ratio 40 mL/g. A mean value of 18.89 ± 0.43% (n = 3) was gained, which was very close to the

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predicted value of 19.5%, indicating that the model is valid for the extraction process.

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3.2. Scanning electron microscopy (SEM) analysis

The extraction yield was related to the physical changes of the plant tissues. In order to

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understand UDE mechanism, the microstructures of AM fruiting bodies tissue before and

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after UDE (or HRE) were observed by SEM, and the results are showed in Fig. 2. Compared

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with untreated raw material, different extraction methods produced remarkably different physical change of the plant tissues. The untreated raw material kept almost all cell walls of

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tissues intact (Fig. 2a and b), and microstructure of tissues had little damage after extraction by HRE (Fig. 2c and d), which may be the results of long heating time. As compared to HRE,

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the AM tissue after extracted by UDE was partially split into thin and fragmented branches formed of few chain strands or loose structures (Fig. 2e and f). The reason for this impact may be that ultrasonic cavitation and and its several physical effects (shear forces, shock waves, turbulence, micro-jets and increasing of pressure) led to change of plant tissue, break down cell walls, reducing particle size, which resulted in the effective and easy extraction of polysaccharide with the aid of UDE. The SEM analysis provided strong evidence of the high AMPs yield when the UDE method was used. 3.3. Isolation and purification of AMPs The polysaccharide extracts were obtained from AM under the optimal UDE condition. After ethanol precipitation, deproteinization, concentrate, dialysis and lyophilization, AMPs were obtained. The yield of AMPs was 18.89% based on the dried AM. AMPs were first

10

Journal Pre-proof separated using a DEAE-Sepharose Fast Flow column chromatography, two fractions corresponding to AMPs, AMPs-1 and AMPs-2 were clearly separated in the elution profile of the anion-exchange chromatogram (Fig. 3a). Then each fraction was further purified by Sephadex G-100 size-exclusion chromatography, and two main fractions, named AMPs-1-1 and AMPs-2-1 obtained at yields (based on the amount of AMPs) of 31.5% and 19.2%, respectively. 3.4. Physicochemical characteristics of AMPs-1-1 and AMPs-2-1

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The physicochemical properties of AMPs-1-1 and AMPs-2-1 are summarized in Table 1. The results showed that AMPs-1-1 contained 91.2% of total carbohydrate, 2.17% of protein,

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and 1.28% of uronic acid, while AMPs-2-1 contained 88.5% of total carbohydrate, 4.02% of

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protein and 3.36% of uronic acid. The homogeneity and average molecular weight of

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AMPs-1-1 and AMPs-2-1 determined by HPGPC are shown in Fig. 3b and c, the HPGPC profiles of AMPs-1-1 and AMPs-2-1 exhibited a single and symmetrically peak, respectively,

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which indicated that they were heteropolysaccharides with high purity. The average molecular

respectively.

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weights of AMPs-1-1 and AMPs-2-1 were estimated to be 1.23 × 105, 6.76 × 104 Da,

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The monosaccharide compositions of AMPs-1-1 and AMPs-2-1 were measured by HPLC after hydrolyzed and further derived with PMP. As shown in Fig. 3d, 10 PMP-labeled standard monosaccharides were rapidly separated within 30 min by the improved HPLC condition. The chromatogram profiles of two purified polysaccharides are shown in Fig. 3e and f, by comparing the retention time with those of standard monosaccharide, the monosaccharide compositions in AMPs-1-1 and AMPs-2-1 were identified and the molar ratios were calculated with reference to a standard curve. AMPs-1-1 was composed of Glc, Gal and GlcA with mole percentages of 89.06%, 9.59% and 1.34%, respectively. The AMPs-2-1 was mainly composed of Glc, Gal, Man and GlcA in molar percentages of 65.28%, 22.87%, 2.87% and 8.98%. However, Man existed in AMPs-2-1 while not in AMPs-1-1. These results indicated that Glc was the major monosaccharide component of AMPs-1-1 and AMPs-2-1. Many researches have indicated that polysaccharides obtained by different 11

Journal Pre-proof methods have a lot of differences, for example, polysaccharide content, monosaccharide compositions, molecular weight distribution, protein and uronic acid content [30]. The investigation suggests that the ultrasonic wave might degrade the high-molecular-weight polysaccharides into the low-molecular polysaccharides, which might lead to the differences in monosaccharide compositions and biological activities. Types and ratios of glycosidic linkages of the fraction AMPs-1-1 and AMPs-2-1 are shown in Table 2. The results indicated that two polysaccharide fractions had different

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glycosidic linkages. AMPs-1-1 possessed seven kinds of linkages, such as 1-linked-Glcp, 1,3-linked-Glcp, 1,3,6-linked-Glcp, 1,6-linked-Glcp, 1,6-linked-Galp, 1,3,6-linked-Galp, molar percentages of 17.47%, 34.96%, 26.64%, 9.98%, 6.47%, 3.12%

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1,3-linked-GlcAp with

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and 1.35%, respectively. The results also showed that 1,3-linked-glucosyl was the largest

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amount residue in AMPs-1-1, revealing that 1,3,6-linked-Glcp, 1-linked-Glcp and 1,3-linked-glucosyl should be possible to form the backbone structure. AMPs-2-1 was mainly of

1,4-linked-Manp,

1-linked-Glcp,

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composed

1,3-linked-Glcp,

1,3,6-linked-Glcp,

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1,6-linked-Glcp, 1,3-linked-Galp, 1,6-linked-Galp, 1,3,6-linked-Galp and 1,3-linked-GlcAp in ratio percentages of 10.16%, 7.42%, 8.50%, 31.34%, 13.27%, 3.53%, 12.22%, 10.29% and

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3.27%, respectively. According to the linkage analysis, Glc was the main component in AMPs-2-1 which revealed that 1,3,6-linked-Glcp and 1,6-linked-Glcp residues existed in the backbone structure of AMPs-2-1.

FT-IR spectra of AMPs-1-1 and AMPs-2-1 were recorded at the range of 400–4000 cm-1 (Fig. 3g and h). The strong and broad absorption peaks at 3416 cm-1 (AMPs-1-1) and 3398 cm-1 (AMPs-2-1), and weak absorption bands at 2941 cm−1 (AMPs-1-1) and 2926 cm-1 (AMPs-2-1) were assigned to the stretching vibration of O-H and C-H in the molecules of polysaccharides, respectively [31]. The absorption peaks at 1750 cm-1 (AMPs-1-1) and 1739 cm-1 (AMPs-2-1) were attributed to C=O stretching vibration, which indicates the existence of uronic acids [32]. The relatively strong band at around 1643 cm-1 (AMPs-1-1) and 1632 cm-1 (AMPs-2-1) was caused by C=O stretching vibration of the carbonyl group. The bands at 1446 and 1421 cm-1 in AMPs-1-1 and AMPs-2-1 were assigned to C–H variable angle 12

Journal Pre-proof vibration and C–O–C stretching vibration in saccharide ring, and the absorption peak at around 1417 cm-1 was the characteristic IR absorptions of protein in AMPs-2-1. The weak absorption peak at 1258 cm-1 in AMPs-2-1 was attributed to the asymmetric C-O-C stretching vibration, suggesting the presence of -OCH3 [33]. The relatively strong absorption peaks at 1043, 1022 cm-1 were caused by a pyranose form of sugar in AMPs-1-1 and AMPs-2-1 [34]. The absorption bands at 892 and 836 cm-1 demonstrated the presence of β-glycoside and α-glycoside linkages in AMPs-1-1 and AMPs-2-1 [35]. The absorption peak at 873 cm-1

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suggested the presence of mannose residues in AMPs-2-1 [36]. These results confirm that AMPs-1-1 and AMPs-2-1 are the highly purified polysaccharides containing uronic acid and

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proteins.

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The 1H NMR and 13C NMR spectra of AMPs-1-1 and AMPs-2-1 are presented in Fig. 4a

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to 4d. The chemical shifts (δ) of H and C assignment of anomeric proton and anomeric carbon of AMPs-1-1 and AMPs-2-1 are summarized in Table 2, which are assigned on the glycosidic

lP

linkages. The signals of AMPs-1-1 and AMPs-2-1 at δ 3.0–5.5 ppm (Fig. 4a and b) and δ 60– 110 ppm (Fig. 4c and d) were assigned the typical distribution of NMR signals of the

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polysaccharides [37]. The signals at δ 5.38 (AMPs-1-1) and 5.48 ppm (AMPs-2-1), and δ

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177.72-179.46 (AMPs-1-1) and 179.02-181.55 ppm (AMPs-2-1) showed that uronic acid were present in two polysaccharides, similar to polysaccharide extracted from other plants [38]. The signals at δ 4.97, 4.43, 5.01, 5.04 ppm, and δ 99.04, 104.35, 99.24, 97.46 ppm in AMPs-1-1 were attributed to the H1 and C1 of (1)- -D-Glcp, (13)- -D-Glcp, (16)-α-D-Glcp, and (13,6)--D-Glcp, respectively. The signals at δ 5.04, 103.28 ppm (in AMPs-1-1) were tentatively deduced to be anomeric protons and anomeric proton of (13)- -D-GlcAp. The chemical shifts of anomeric protons at δ 4.51, 4.54 ppm, and those of anomeric carbons at δ 104.97, 104.53 ppm in AMPs-1-1, were identified as (16)- -D-Galp, (13,6)- -D-Galp, respectively. The strong signals at δ 4.70 and 4.71 ppm were assigned to the solvent proton peak of AMPs-1-1 and AMPs-2-1. The anomeric proton and carbon signals at δ 4.62 (102.14), 4.37 (104.37), 5.05 (98.42) and 5.00 (99.52) ppm in AMPs-2-1 were

13

Journal Pre-proof attributed to the H1 and C1 of (1)- -D-Glcp, (13)- -D-Glcp, (13,6)--D-Glcp and (16)- -D-Glcp, respectively. The signals in AMPs-2-1 at 5.09 (101.58), 4.47 (104.44) and 4.54 (104.62) ppm were assigned to the H1 (C1) of (13)-α-D-Galp, (16)- -D-Galp and (13,6)- -D-Galp, respectively. Moreover, in AMPs-2-1 spectra H1/C1 peaks characteristic for 1,3-linked- -GlcAp and CH3O- -1,3-GlcAp were observed, at δ 5.05/102.60 and 5.12/102.14 ppm, respectively. The chemical shift of δ 4.94 and 103.70 ppm of AMPs-2-1 was identified as (14)- -D-Manp. According to relevant literature report [39], the signals of

of

AMPs-1-1 and AMPs-2-1 between δ 3.6 and 4.1 ppm may be attributed to ring protons the

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presence of pyranose (H2–H5), the signals appearing at δ 55–86 ppm can be assigned to

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sugars C2-C6. The signals at δ 2.00 (in AMPs-2-1) and 1.48 ppm (in AMPs-1-1) were corresponded to the COO-CH3 group of an uronic acid with methyl-esterified carboxyl group

re

[40]. Based on the NMR and methylated analysis results, it can be concluded that both of

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AMPs-1-1 and AMPs-2-1 contained pyran-type glucose with α and  configurations, the major backbone of AMPs-1-1 was composed of (1)- -D-Glcp, (13,6)--D-Glcp and

-D-Glcp.

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3.5. Antioxidant activities

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(13)- -D-Glcp, while AMPs-1-1 was mainly composed of (13,6)--D-Glcp and (16)-

The antioxidant capacities of two polysaccharides are shown in Fig. 5a. It can be clearly seen that the polysaccharides extracted from wild AM by UDE had remarkable antioxidant activities and antioxidant capacities depend largely on the concentration of two polysaccharides. At the concentration of 1.00 mg/mL, the FRAP values of AMPs-1-1 and AMPs-2-1 were 1147.98  54.92 M and 1832.13  68.47 M, respectively. The capability of antioxidants of AMPs-2-1 was significantly higher than that of AMPs-1-1. By comparing the physicochemical characterizations of AMPs-2-1 and AMPs-1-1, we found that Man was observed in AMPs-2-1, but not in AMPs-1-1; furthermore AMPs-2-1 with lower molecular weight had relative higher contents of Gal, GlcA, and protein than that of AMPs-1-1, resulting in stronger antioxidant activity. It has been reported that ultrasonic wave might 14

Journal Pre-proof appropriately degrade the high-molecular-weight polysaccharides and enhance their antioxidant activities [41]. Likewise, some other reports have demonstrated that polysaccharides with higher contents of uronic acid and Gal, lower molecular weight were found to have stronger antioxidant capacity [42]. Thus, it could be concluded that AMPs-2-1 had stronger antioxidant activity than AMPs-1-1, which might be due to its higher contents of Gal, GlcA, Man, and protein. As shown in Fig. 5b, both AMPs-1-1 and AMPs-2-1 exhibited remarkable scavenging

of

activities on DPPH. In a relatively low concentration range of 0.1 to 1.00 mg/mL, the

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scavenging activities of AMPs-1-1 and AMPs-2-1 increased significantly with increasing concentrations. At the concentration of 1.00 mg/mL, scavenging activities of AMPs-1-1,

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AMPs-2-1 and Vc were 56.24  1.15%, 77.01  0.87% and 88.15  1.98%, respectively. The

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scavenging activity of AMPs-2-1 was higher than AMPs-1-1 at the same conditions, but was

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lower than that of Vc. The stronger DPPH scavenging activity of AMPs-2-1 might be partially due to the lower molecular weight and higher content of uronic acid and protein.

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These results were similar to the previous literatures [43]. The ABTS+ scavenging activities of the two polysaccharide fractions are shown in Fig. 5c.

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It was observed that the scavenging activities on ABTS+ were increased significantly with the increased concentrations of the polysaccharides in the order of AMPs-2-1 and AMPs-1-1. At 2.0 mg/mL, the scavenging activities were 49.72  1.88%, 68.91  1.75% and 99.02  2.79% for AMPs-1-1, AMPs-2-1 and Trolox, respectively. The present study showed that AMPs-2-1 displayed higher ABTS+ scavenging activity than AMPs-1-1, indicating that AMPs-2-1 could serve as an electron donor and react with free radicals to terminate radical chain reactions. Previous research has demonstrated that the presence of large amounts of uronic acid in polysaccharides could result in higher levels of ABTS+ scavenging [44]. These results also demonstrated that the structure of polysaccharide might be change during UDE process, which can improve biological activity. However, the UDE treatment could alter the structure of polysaccharides, which depends of the process conditions and of the polysaccharide nature 15

Journal Pre-proof [45]. The total antioxidant activities of AMPs-1-1 and AMPs-2-1 were examined by ORAC assay and the results are shown in Fig. 5d. Two polysaccharide fractions showed a dose-dependent effect at the concentrations tested. Moreover, AMPs-2-1 exhibited higher ORAC value (2126.53  44.66 M TE/g than that of AMPs-1-1 (1791.05  35.46 M TE/g), at the concentration of 0.05 mg/mL, and the similar results were observed in DPPH, ABTS+ scavenging activities. These results indicated that the polysaccharides from wild AM contain

of

a diversity of antioxidant active groups that have ability to scavenge a higher peroxyl radicals

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as well as ROS. In this study, the molecular weight, chemical composition and structure of AMPs-2-1 were different from that of AMPs-1-1, which might explain their different

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antioxidant activity. These might due to the differences in polysaccharide species and change

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in chemical structure induced by ultrasonic treatments [46]. Therefore, UDE was proven a more effective technique with shorter extraction time, higher extraction yield and greater

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antioxidant activity compared to conventional extraction method. The methodologies

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developed in this study will provide useful information for the producing and using of AMPs as a potential natural antioxidant used in pharmaceutical industry.

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3.6. Immunomodulatory activities

T lymphocyte activity can be evaluated by treating the splenocyte with ConA, whereas B lymphocyte activity can be assessed by LPS-induced B lymphocyte proliferation [47]. The proliferation of T and B lymphocyte is a crucial indicator in immune response system. The effects of AMPs-1-1 and AMPs-2-1 on splenic lymphocyte proliferation through mitogen (Con A or LPS) stimulation were investigated. As shown in Fig. 6a, when the effects on T lymphocyte proliferation were assessed for the AMPs-1-1 and AMPs-2-1 in the presence of ConA, a significant concentration-dependent increase in T lymphocyte proliferation were all observed which were all higher than that treated with ConA alone, indicating a promotion on ConA-induced lymphocyte proliferation by all the two fractions. Fig. 6b showed that two fractions stimulated LPS-induced B lymphocyte proliferation in the dose range of 50-250

16

Journal Pre-proof g/mL, it was obvious that AMPs-1-1 and AMPs-2-1 all significantly increased the proliferation of B-lymphocytes (P < 0.01, or P < 0.001) compared with the LPS control. These results suggested that two fractions strongly promoted ConA-induced T lymphocyte and LPS-induced B cell proliferation at 50–250 g/mL (P < 0.05, P < 0.01, or P < 0.001); The higher proliferation rat on splenocytes of AMPs-1-1 and AMPs-2-1 might be attributed to their high content of Glu and Gal. While AMPs-2-1 displayed stronger effects, which implied that AMPs-2-1 might be beneficial for existing (1→3,6)--linked Glc residues in the

of

backbone structure and lower molecular weight. These findings are consistent with the

ro

previous observations [48].

The effects of AMPs-1-1 and AMPs-2-1 on RAW264.7 macrophages proliferation were

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investigated, and the results are shown in Fig. 6c. In the concentration range of 50–250

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g/mL, both two fractions could promote the proliferation of RAW264.7 macrophages

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compared with the control group (P < 0.05 or P < 0.01). The proliferation effect for AMPs-1-1 occurred in a concentration dependent manner, while AMPs-2-1 had the highest

na

proliferation ratio at concentration 150 g/mL, but the effect decreased when the concentration increased. Although the effect of AMPs-2-1 at 150 μg/mL was higher than at

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250 μg/mL, but there was no significant difference between two groups. These results suggested that AMPs-1-1 and AMPs-2-1 could promote the proliferation of RAW264.7 macrophages. Especially, AMPs-2-1 exhibited significantly (P < 0.05 or P < 0.01) higher proliferation activity of RAW264.7 macrophages than AMPs-1-1, which mainly might due to the lower molecular weight and higher molar proportion of Gal and protein when compared with AMPs-1-1. However, some contrary findings showed that higher molecular weight of polysaccharides, led to higher immunomodulatory activity [26]. Macrophages play an essential role in the host defense, including phagocytosis of pathogens and apoptotic cell, and production of cytokines. Stimulating macrophage proliferation is one way to activate macrophages [49]. The above results indicated that the polysaccharide from AM, could activate peritoneal macrophages and enhance proliferation activity.

17

Journal Pre-proof The effects of AMPs-1-1 and AMPs-2-1 on RAW264.7 murine macrophages were carried out by the neutral red phagocytosis assay and the results are presented in Fig.6d. AMPs-1-1 and AMPs-2-1 could significantly enhance the RAW264.7 macrophages in the concentration range of 50–250 g/mL, compared with the control group. In the concentration range of 50– 250 g/mL, AMPs-2-1 significantly stimulated the phagocytic activity of RAW264.7 macrophage in a dose-dependent manner compared with the control group (P < 0.01, or P < 0.001). However, the maximum value of AMPs-1-1 promoted phagocytic activity of

of

RAW264.7 macrophages was observed at 150 g/mL (P < 0.01), but the effect decreased

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when the polysaccharide concentration increased. At each determination concentration, AMPs-2-1 showed higher phagocytic activities than the AMPs-1, which might be related to

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the higher contents of Man, such result was relatively agreed to Li et al. [50]. All these results

composition,

molecular

weight,

glycosidic-linkage

composition,

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monosaccharide

re

indicated that the immunomodulatory activity of polysaccharide could be influenced by the

4. Conclusion

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conformation. These finding are in accord with those of previous reports [51].

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A maximum extraction yield (19.5%) of AMPs was obtained using the RSM with optimized UDE conditions as follows: 915 W, 69.27°C, and 39.13 min. Two homogeneous polysaccharides, namely AMPs-1-1 and AMPs-2-1 with average molecular weight of 1.23 × 105 and 6.76 × 104 Da, were isolated and purified from AMPs, which they contain α- and β-type glycosidic linkages with pyranose ring. AMPs-1-1 consisted of Glc (89.06%), Gal (9.59%) and GlcA (1.34%), and possessed a majorly backbone of (1)-β-D-Glcp, (13,6)--D-Glcp and (13)-β-D-Glcp residues, while AMPs-2-1 consisted of Glc (65.28%), Gal (22.87%), GlcA (2.87%) and Man (8.98%), contained a main backbone of (13,6)--D-Glcp and (16)-β-D-Glcp residues. Bioactivity tests showed that AMPs-1-1 and AMPs-2-1 exerted significant antioxidant activities and immunostimulatory effects, especially AMPs-2-1 showed stronger scavenging activities for DPPH, ABTS+, higher

18

Journal Pre-proof FRAP and ORAC value, while AMPs-2-1 exhibited better promoting lymphocyte and macrophage proliferation and increasing macrophage phagocytosis than AMPs-1-1. These results suggested that UAE is an effective polysaccharide extract technology, and AMPs-2-1 could be explored as a novel potential antioxidants and immunomodulator agents used in the functional food and pharmaceutical industries. Acknowledgements

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This study was supported by Program of the 13th Five Science and Technology Research

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349–357.

Figure captions

Figures 1. Response surface plots (a, c, and e) and contour plots (b, d and f) showing the effect of ultrasonic power 23

Journal Pre-proof (X1), extraction temperature (X2) and extracting time (X3) on the yield of AMPs. Figures 2. Scanning electron micrographs of AM fruiting bodies. (a) and (b) untreated raw material (Scale bar 50 m and 10 m); (c) and (d) sample obtained after HRE (Scale bar 50 m and 10 m); (e) and (f) sample obtained UDE (Scale bar 50 m and 10 m). Magnification: 2000-fold.

Figures 3. Elution profiles of AMPs by ion exchange chromatography on a column of DEAE-Sepharose Fast Flow (a). HPGPC chromatogram of AMPs-1-1 (b) and AMPs-2-1 (c). HPLC chromatograms of PMP derivatives of 10 standard monosaccharides (d) and component monosaccharides released from AMPs-1-1 (e) and AMPs-2-1 (f) (Peaks: 1. Man; 2. Rib; 3. Rha; 4. GlcA; 5. GalA; 6. Glc; 7. Gal; 8. Xyl; 9. Ara; 10. Fuc). IR spectra of AMPs-1-1 (g) and AMPs-2-1 (h).

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Figures 4. NMR spectra of AMPs-1-1 and AMPs-2-1. (a) 1H-NMR spectrum of AMPs-1-1; (b) 1H-NMR spectrum

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of AMPs-2-1; (c) 13C-NMR spectrum of AMPs-1-1; (d) 13C-NMR spectrum of AMPs-2-1.

Figures 5. Antioxidant activity of the AMPs-1-1 and AMPs-2-1 with various methods: (a) FRAP activity, (b)

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Scavenging activity on DPPH, (c) Scavenging activity on ABTS+, (d) ORAC activity. Each value is presented as

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mean ± SD (n = 3).

Figures 6. Effect of AMPs-1-1 and AMPs-2-1 on ConA- (a) or LPS- (b) induced lymphocyte proliferation; RAW

SD (n = 3).

Statistically significant difference at the 0.05 level;  Statistically significant difference at the 0.01 level; Statistically significant difference at the 0.001 level.

na



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

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264.7 macrophages proliferation (c) and phagocytosis (d) activities of mice in vivo. Data were expressed as mean ±

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Journal Pre-proof Conflict of interest

Jo ur

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Authors declare no conflict of interest

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Journal Pre-proof Table 1 Comparison of the properties of AMPs-1-1 and AMPs-2-1 Samples Physicochemical properties AMPs-1-1

AMPs-2-1

Total sugar (%)

91.2

88.5

Protein (%)

2.17

4.02

Uronic acid content (%)

1.28

Molecular weight (Da)

3.36

1.23 × 10

5

6.76 × 104

Monosaccharide molar percentages (%) 89.06

65.28

Gal

9.59

22.87

GlcA

1.34

2.87

Man

nda

8.98

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Glc

a

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nd: not detected

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Journal Pre-proof

Table 2 Methylation analyses data and 1H and 13C NMR chemical shifts (δ) assignment of anomeric proton and anomeric carbon of AMPs-1-1 and AMPs-2-1 AMPs-1-1 Methylation product

Linkage types

AMPs-2-1

Main fragment ions (m/z) H1/C1

Molar (%)

H1/C1

Molar (%)

4)-β-D-Manp-(1

43, 71, 87, 99, 102, 113, 118, 129, 131, 161, 173, 233

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nd

4.94/103.70

10.16

2,3,4,6-Me4-Glcp

-D-Glcp-(1

43, 59, 71, 87, 101, 117, 129, 145, 161, 205

4.97/99.04

17.47

4.56/102.14

7.42

2,4,6-Me3-Glcp

3)--D-Glcp-(1

59, 71, 87, 101, 118, 129, 161, 174, 202, 234

4.43/104.35

34.96

4.37/104.37

8.50

2,4-Me2-Glcp

3,6)--D-Glcp-(1

59, 87, 101, 118, 129, 160, 189, 234, 305

5.04/97.46

26.64

5.05/98.42

31.34

2,3,4-Me3-Glcp

6)-α-D-Glcp-(1

71, 87, 99, 101, 117, 129, 161, 173, 189, 233

5.01/99.24

9.98

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nd

2,3,4-Me3-Glcp

6)-β-D-Glcp-(1

71, 87, 99, 101, 117, 129, 161, 173, 189, 233

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nd

5.00/99.52

13.27

2,4,6-Me3-Galp

3)-α-D-Galp-(1

87, 101, 118, 129, 161, 234

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nd

5.09/101.58

3.53

2,3,4-Me3-Galp

6)--D-Galp-(1

43, 87, 99, 101, 117, 129, 161, 173, 189, 233

4.51/104.97

6.47

4.47/104.44

12.22

2,4-Me2-Galp

3,6)--D-Galp-(1

59, 87, 101, 117, 129, 159, 161, 189, 234

4.54/104.53

3.12

4.54/104.62

10.29

2,4,6-Me3-Glcp

3)--D-GlcAp-(1

59, 71, 87, 101, 118, 129, 161, 174, 202, 234

5.04/103.28

1.35

5.05/102.60

3.27

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2,3,6-Me3-Manp

a

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-p

nd: not detected

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Journal Pre-proof Highlights ► An effective ultrasonic disruption extraction process was optimized by response surface method

► Two homogeneous polysaccharide components had been isolated from wild Arimillaria mellea

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► Characteristic of polysaccharide was characterized by HPGPC, HPLC, GC-MS, IR,

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NMR

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► AMPs-2-1 showed notable antioxidant and immunomodulatory activities

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5