Accepted Manuscript

Title: Comparative study of physicochemical properties and

bioactivity of Hericium erinaceus polysaccharides at different
solvent extractions

Authors: Jing-Kun Yan, Zhi-Chao Ding, Xianli Gao, Yao-Yao

Wang, Yan Yang, Di Wu, He-Nan Zhang

PII: S0144-8617(18)30399-0
DOI: https://doi.org/10.1016/j.carbpol.2018.04.019
Reference: CARP 13473

To appear in:

Received date: 29-1-2018

Revised date: 23-3-2018
Accepted date: 3-4-2018

Please cite this article as: Yan, Jing-Kun., Ding, Zhi-Chao., Gao, Xianli., Wang, Yao-
Yao., Yang, Yan., Wu, Di., & Zhang, He-Nan., Comparative study of physicochemical
properties and bioactivity of Hericium erinaceus polysaccharides at different solvent
extractions.Carbohydrate Polymers https://doi.org/10.1016/j.carbpol.2018.04.019

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Submitted to: Carbohydrate Polymers

(Original Research MS)

Comparative study of physicochemical properties and bioactivity of Hericium erinaceus

polysaccharides at different solvent extractions

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Jing-Kun Yana, , Zhi-Chao Ding a, Xianli Gao a, Yao-Yao Wang a, Yan Yang b,c
, Di Wu b,c
,

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He-Nan Zhangb,c,*

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a
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School of Food & Biological Engineering, Jiangsu University, Zhenjiang, 212013, China
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b
National Engineering Research Center of Edible Fungi, Key Laboratory of Edible Fungi
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Resources and Utilization (South), Ministry of Agriculture, China

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c
Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, 201403,
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China
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 Corresponded authors: JK Yan, Tel: +08615952819661, E-mail: jkyan_27@163.com,

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jkyan27@ujs.edu.cn; HN Zhang, Tel: +08618516187330, E-mal: henanhaoyun@126.com

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Highlights

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  Four different solvents were used to extract Hericium erinaceus polysaccharides

(HEPs).

 Extraction solvents had significant effects on physicochemical properties of HEPs.

 HEP-C extracted with citric acid showed stronger bioactivities than HEP-W and

HEP-S.

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 Citric acid can be used in food processing and for the extraction of bioactive PSs.

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Abstract

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In this study, hot water, 0.9% NaCl, citric acid, and 1.25 M NaOH/0.05% NaBH4 were

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separately used for the extraction of water-soluble H. erinaceus polysaccharides (HEPs;
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HEP-W, HEP-S, HEP-C, and HEP-A) from the fruit body of Hericium erinaceus. The
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physicochemical properties and biological activities were then investigated and compared.
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Results showed that the extraction solvents exhibited significant effects on the extraction
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yields, molecular weights, monosaccharide compositions, preliminary structural

characteristics, microstructures of HEPs and on their contents, such as neutral sugar, uronic
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acid, protein, and β-(1→3)-glucan. In vitro antioxidant activity assays indicated that HEP-C
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extracted with citric acid solution showed stronger scavenging abilities on hydroxyl and
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DPPH radicals and antioxidant capacities than HEP-W and HEP-S. Moreover, HEP-C

exhibited the strongest inhibitory effects on α-glycosidase and α-amylase activities. Therefore,
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HEP-C extracted with citric acid can be developed as a potential bioactive ingredient for

applications in food, medicine, and cosmetics industries.

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Keywords: Hericium erinaceus fruit body; Water-soluble polysaccharides; Solvent

extractions; Physicochemical properties; In vitro antioxidant activity

Chemical compounds studied in this article:

Sodium chloride (PubChem CID: 5234)

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Sodium hydroxide (PubChem CID: 14798)

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Sodium borohydride (PubChem CID: 22959485)

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N-butyl alcohol (PubChem CID: 263)

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Chloroform (PubChem CID: 6212)

Trifluoroacetic acid (PubChem CID: 6422)

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Pyridine (PubChem CID: 1049)
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Acetic acid (PubChem CID: 176)

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Ethanol (PubChem CID: 702)

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Acetic anhydride (PubChem CID: 7918)

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1. Introduction
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Hericium erinaceus (Bull.) Pers., which is generally called Houtou or Shishigashira in

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China and Yamabushitake in Japan, is an edible mushroom that can also be used in medicinal

practices belonging to the Aphyllophorales, Hydnaecase, and Hericium families (Friedman,

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2015; Thongbai et al., 2015). H. erinaceus has been commonly prescribed in traditional

Chinese medicine of its benefits to human health. It has been used in traditional folk medicine

and consumed as a tonic food in China, Korea, and Japan (Wang et al., 2014). Over the past

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decade, H. erinaceus has attracted increasing attention in the fields of functional foods and

biomedicine. H. erinaceus exhibits a broad spectrum of physiological and health-promoting

functions, such as anti-aging and anticancer functions and functions against gastritis and

metabolic diseases (Khan, Tania, Liu, Rahman, 2013; Wang et al., 2014). Modern

pharmacological studies have demonstrated that H. erinaceus polysaccharides (HEPs)

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represent a major class of bioactive ingredients of H. erinaceus and have multiple health

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benefits, including immunomodulatory (Shen et al., 2017), antitumor (Kim et al., 2011),

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antigastritic (Wang, Gao, Xu, Gao, 2015), antioxidative and hepatoprotective (Han, Ye, Wang,

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2013; Zhang et al., 2012), antihyperlipidemic (Shang et al., 2015), and antihyperglycemic

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activities (He et al., 2017). Therefore, the specific structures and sugar compositions of HEPs
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are potentially useful for the dietary application of H. erinaceus as functional foods.
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In the recent years, a series of bioactive HEPs with different molecular weights and
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chemical structures have been extracted and isolated from the mushroom fruit bodies,
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mycelia, and fermentation broths of natural and cultured H. erinaceus (Friedman, 2015; He et

al., 2017). As the first and most important step, extraction plays an important role in the
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characterization and utilization of bioactive HEPs from H. erinaceus. HEPs existed as

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structural components of the fungal cell wall, and the selection of suitable extraction methods
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depends on the cell wall structure (Zhang et al., 2011). In general, water extraction has been

widely used as a classic method for the extraction and preparation of HEPs and other
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polysaccharides (PS) in the laboratory and industry application because of its dominant

advantages, such as simplicity in application, low cost, safety, and environment-friendly

properties (Nie & Xie, 2011; Yang, Zhao, Lv, 2008). High temperature can accelerate the

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dissolution of PS from the cell walls. Meanwhile, saline, acidic, and diluted alkali solutions

are often used for PS extraction. These solutions break the cell walls from the outer layer to

the inner layer under mild and strong conditions (Nie, Zhang, Li, Xie, 2013). For example,

Gottschalk et al. (1994) prepared a capsular PS and long-chain lipopolysaccharides of

Actinobacillus pleuropneumoniae serotype 1 as antigens for the serodiagnosis of swine

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pleuropneumonia by using a saline boiled extract. Zhang’s group established a new and

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environment-friendly isolation method to obtain lentinan from the fruiting bodies of Lentinus

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edodes through alkali extraction with 1.25 M NaOH/0.05% NaBH4 solution. Their results

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showed that the isolated lentinan had a yield of up to 5% and had a triple-helix conformation

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(Wang, Xu, Zhang, 2008; Zhang, Zhang, Cheng, 1999). Imbs et al. (2015) obtained the
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fucose-containing sulfated PS with excellent antioxidant activity from Fucus evanescens
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through hot acidic extraction and extraction with aqueous calcium chloride solution. Peasura
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et al. (2015) extracted sulfated PS from Ulva intestinalis by using distilled water, 0.1 M HCl,
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and 0.1 M NaCl at 80 °C and different extraction times. They found that solvent extraction

effectively enhances antioxidant activity by a distinct molecular weight and chemical

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characteristic of the sulfated PS. Yao et al. (2017) compared hypolipidemic and antioxidant
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capacities of PS obtained from Laminaria japonica by using different extraction media (water,
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citric acid, sulfuric acid, hydrochloric acid, and phosphoric acid), and their results showed

that extraction with water acidified with citric acid increases PS yield and bile salt adsorption
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rate and enhances free radical scavenging ability and antioxidant capacity. Thus, solvent

extraction has a significant effect on PS yield and on the physicochemical properties, and

structural characteristics, biological activities, and functionalities of PSs. However, to the best

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of our knowledge, no study or only a few study has reported the comprehensive evaluation of

different solvent extractions on the physicochemical properties and bioactivities of HEPs

from H. erinaceus.

Therefore, in the present study, we aimed to extract water-soluble HEPs from the

fruiting body of H. erinaceus by using different extraction solvents (water, saline, acid, and

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alkaline solvents). The effects of extraction solvents on the physicochemical properties of

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HEPs were characterized using the total carbohydrate, protein, uronic acid, and β-1,3-glucans

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contents, as well as the monosaccharide composition, molecular weight, and preliminary

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structural features. The antioxidant and hyperglycemic activities of these HEPs in vitro were

also evaluated and compared.

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2. Materials and methods

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2.1. Materials and Chemicals

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The fruiting body of H. erinaceus was obtained from Shanghai Guosen Biotechnology

Co., Ltd. (Shanghai, China). The strain used was H0605, which was obtained from the
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Herbarium of Edible Fungi Culture Collection Center Branch of the China Culture Collection
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of Agricultural Microorganisms. The fruit body (500 g) was ground into powder, passed
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through a 60 mesh sieve, and then treated with refluxing petroleum ether twice for 6 h for the

removal of lipids and pigments. The residue was air dried and sealed in airtight plastic bags
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before use. Monosaccharide standards [D-Arabinose (D-Ara), D-glucose (D-Glu), D-galactose

(D-Gal), D-mannose (D-Man), L-rhamnose (L-Rha), and D-xylose (D-Xyl)], glucuronic acid,

gallic acid, trifluoroacetic acid (TFA), curdlan, acarbose, porcine pancreatic α-amylase,

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α-glucosidase, p-nitrophenyl-α-D-glucopyranoside (pNPG), hydrogen peroxide (H2O2),

1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2΄-azinobis (3-ethylbenzothiazoline-6-sulphonic

acid) (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 2, 4,

6-tris(2-pyridyl)-s-triazine were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO,

USA). Sodium chloride, sodium hydroxide, sodium borohydride, potassium persulphate,

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ferrous sulfate, aniline blue, citric acid, methanol, ethanol, chloroform, n-butyl alcohol, acetic

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acid, petroleum ether, acetic anhydride, pyridine, sulfuric acid, carbazole,

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hydroxylammonium chloride and phenol were of analytical grade (AR) purchased from local

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suppliers.

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2.2. Polysaccharide extraction and isolation
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We separately used hot water, 0.9% NaCl, citric acid (pH 3.0), and 1.25 M
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NaOH/0.05% NaBH4 solutions to extract HEPs from the fruiting body of H. erinaceus. 50.0
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g of pretreated powder was extracted twice with 500 mL of distilled water at 95 °C for 8 h,

500 mL of 0.9% NaCl aqueous solution at 95 °C for 8 h, 500 mL of citric acid aqueous
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solution (pH 3.0) at 95 °C for 8 h, or 500 mL of 1.25 M NaOH/0.05% NaBH4 aqueous

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solution at 25 °C for 3 h. After centrifugation (DL-6C, Shanghai Anting Scientific

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Instrument Factory, China), the resulting supernatants (1000 mL) of the citric acid and 1.25

M NaOH/0.05% NaBH4 solutions were neutralized by adding 0.5 M NaOH and 36% acetic
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acid, respectively. Afterward, the four aqueous extracts were concentrated to 300 mL using a

rotary evaporator (RE-2000, Shanghai Yarong Biochemical Instrument Factory, China),

respectively, and precipitated with 4 volumes of 95% ethanol at 4 °C for 12 h, followed by

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centrifugation at 5000 rpm for 20 min. Four precipitates were washed three times with

absolute ethanol. Then, the washed precipitates were dissolved in distilled water,

deproteinized with a Sevag reagent (Staub, 1956), dialyzed with 3500 Da molecular weight

cutoff membrane against distilled water for 72 h, and freeze-dried (Alpha 2-4, Martin Christ

Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) for the acquisition of

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partially purified PSs, namely, HEP-W (4.05 g), HEP-S (4.83 g), HEP-C (4.20 g), and

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HEP-A (5.88 g). The detailed extraction and isolation procedures of HEPs are summarized

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in Fig. 1.

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A
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Fig. 1. Schematic routes for PS extraction and isolation of from the fruiting body of H.

erinaceus by using different solvent extractions.

2.3. General physicochemical properties of HEPs

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 The total carbohydrate, uronic acid, and protein contents of HEPs were determined by

the phenol–sulfuric acid method using glucose as a standard (Dubois, Gilles, Hamilton,

Rebers, & Smith, 1956), via sulfuric acid–carbazole method using glucuronic acid as a

standard (Bitter, & Muir, 1962), and via the Bradford method using bovine serum albumin

(BSA) as a standard (Bradford, 1976), respectively. Total phenolic content was determined by

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Folin-Ciocalteu assay reagent using gallic acid as a reference (Siu, Chen, Wu, 2014). The

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extraction yield (%) of HEPs was calculated by the following equation: extraction yield (%,

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w/w) = [weight of dried HEPs (g)/weight of raw materials (g)] × 100%. The intrinsic

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viscosity [η] of HEPs in water was determined at 25±0.1 °C with an Ubbelohde capillary

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viscometer (type ϕ 0.4-0.5 mm, Shanghai Shenyi Glass Instrument Factory, China). The
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kinetic energy correlation was kept negligible.
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2.4. Determination of molecular weight and molecular weight distribution (MWD)

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The weight–average molecular weight (Mw), number–average molecular weight (Mn),

and MWD (Mw/Mn) of the HEPs were determined using high-performance size exclusion
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chromatography coupled to a multiangle laser light scattering (HPSEC–MALLS, DAWN

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HELLOS II λ=658 nm; Wyatt Technologies Corporation, USA). HPSEC–MALLS was

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performed on an Agilent 1260 HPLC system (Agilent, USA) equipped with an Agilent

G1362A refractive index detector (RID, Agilent) and two SEC columns (OHpak SB-806 M
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HQ and SB-805 HQ, 8 mm Ф×30 cm, Shodex, Japan) in a series at 25 °C. A total of 0.1 M

NaCl was used as the elution solvent at a flow rate of 0.5 mL/min. Moreover, HEPs were

dissolved in this solvent at 1.0 mg/mL and filtered through 0.45 μm membranes (Millipore,

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USA) prior to injection. The dn/dc value used was 0.125. The on-line ASTRA 6.1 software

package (Wyatt Technologies, USA) was used for data collection and analysis.

2.5. Monosaccharide composition analysis

A total of 10 mg freeze-dried HEP samples were hydrolyzed with 5.0 mL of 2 M TFA at

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100 °C for 8 h in a sealed tested tube. After the samples were evaporated dry under reduced

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pressure, the resulting hydrolysate was treated with 10 mg HONH2·HCl and 1.0 mL of

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pyridine at 90 °C for 30 min and then treated with 1.0 mL of acetic anhydride at 90 °C for 30

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min. The acetylated derivatives were filtered through 0.45 μm membranes (Millipore, USA)

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and analyzed by gas chromatography (GC) on an Agilent 7890A instrument (HP-5
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fused-silica capillary column, 30 m×0.25 mm×1 μm) and a flame ionization detector (FID).
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The column temperature was held at 180 °C for 3 min, then increased to 280 °C at 10 °C/min,
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and held for 2 min. The monosaccharide standards were also derived and measured according
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to the same procedure as above.

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2.6. UV–Vis and FT–IR spectroscopic analyses

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UV–Vis spectra of the HEPs (1.0 mg/mL) were recorded in a spectrophotometer

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(Carry-100, Varian, USA) in the wavelength range of 190–400 nm at room temperature.

Fourier transform–infrared (FT-IR) spectra of HEPs were determined using a Nexus 670
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FT–IR spectrometer (Thermo Nicolet Co., USA) in the wavenumber range of 500–4000 cm−1

with KBr pellets referenced against air.

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2.7. β-1,3-glucan content determination

The total β-1,3-glucans contents of HEPs were determined using the reported methods

(Ko & Lin, 2004; Nitschke et al., 2011). The working solution was previously prepared by

mixing 127 mL of distilled water, 37 mL of glycine/NaOH buffer (1.0 M glycine, 1.25 M

NaOH), 13.1 mL of 2 M HCl, and 25 mL of dye solution (5.0 g/L aniline blue). Afterward,

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we stirred the solution evenly and then stored it in the dark for 12 h to decolorize it from blue

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to yellow. Meanwhile, the HEP samples were predissolved in distilled water and kept at 4 °C

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for 12 h for complete hydration. A total of 0.4 mL of the sample and 3.6 mL of the working

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solution were mixed into a 5 mL centrifuged tube and incubated at 50 °C for 30 min. After

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the tubes were cooled to 25 °C, the fluorescence intensity was measured using a Cary Eclipse
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fluorescence spectrophotometer (Agilent, USA). The fluorescence was measured at an
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emission wavelength of 502 nm, excitation wavelength of 398 nm, and spectral bandwidth of
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20 nm. The total β-1,3-glucans contents were derived from the calibration curve obtained by
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curdlan in the 0–15 μg/mL concentration range (R2=0.9989).

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2.8. Scanning electronic microscope (SEM) observation

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The freeze-dried HEPs were fixed onto a copper stub. After sputtering with a layer of
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gold, the SEM images were observed and recorded using a S4800 SEM (JEOL Ltd., Japan)

under a high vacuum condition at an accelerating voltage of 15 kV and image magnification

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of 500 ×.

2.9. Antioxidant activity assays in vitro

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 The antioxidant activities in vitro of HEP-W, HEP-S, HEP-C, and HEP-A were

evaluated by calculating the hydroxyl radical scavenging activity, DPPH radical scavenging

activity, Trolox equivalent antioxidant capacity (TEAC) assay, and ferric reducing ability of

plasma (FRAP) assay. For all assays, the HEP samples were predissolved in distilled water

and measured at different concentrations (0-3.0 mg/mL) against distilled water as the blank

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control and the vitamin C (Vc) as a positive antioxidant reference.

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For the adapted hydroxyl radical method, a volume of 0.2 mL of the tested solution

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(sample or control) was added into an equal volume of an aqueous solution of 5 mM FeSO 4.

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Then, a volume of 0.2 mL of 1% (v/v) H2O2 was added into the mixture under continuous

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stirring. After stirring and incubation at room temperature for 60 min, the absorbance of the
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resulting solution was measured at 510 nm by a UV-1600 Spectrophotometer (Beijing Ruili
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Analytical Instrument Co., Ltd., China). The hydroxyl radical scavenging activity (%) was
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estimated by the following formula: scavenging activity (%) = (1-A/A0) × 100%, where A,
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and A0 are the absorbance values of the sample and blank control, respectively.

Concerning the DPPH scavenging assay, a volume of 2 mL of the solution was added
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into 2 mL 0.1 mM DPPH solution in methanol. The reaction mixture was stirred and
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incubated at 25 °C for 30 min in the dark. The absorbance of the resulting solution was
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measured at 517 nm using a UV-1600 Spectrophotometer. The DPPH radical scavenging

activity was calculated based on the following equation: scavenging activity (%) = [1− (A1 −
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A2)/A0]×100%, where A0, A1, and A2 are the respective absorbance values of the blank

control, the sample with DPPH solution, and the sample with distilled water.

As for TEAC assay, the TEAC of HEPs was tested against ABTS·+ radical, which was

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generated from the oxidation of ABTS by potassium persulphate. The TEAC values were

derived from the calibration curve generated with Trolox (0-30 µM). In the FRAP assay,

ferric to ferrous ion reduction at low pH led to the formation of a colored

ferrous-tripyridyltriazine complex, and the FRAP values of the samples were obtained by

calibration with ferrous sulphate (0-30 µM). Details of the operating conditions and methods

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were described as previously reported (Leung, Zhao, Ho, & Wu, 2009).

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2.10. In vitro antihyperglycemic activity assays

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The anti-hyperglycemic activity in vitro of HEPs was evaluated with α-glucosidase and

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α-amylase inhibitory activity assays. For the α-glucosidase inhibition test, pNPG, which can
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be decomposed by α-glucosidase for the release of p-nitrophenol and can be detected by a
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color reagent at 405 nm, was used as substrate. For the two assays, the HEP samples were
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previously dissolved in distilled water at various concentrations against distilled water as the
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blank and acarbose as positive control. The details of the operating conditions and methods

were described in details before (Yan et al., 2017).

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2.11. Statistical analysis

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All experiments are conducted in three replicates and the mean ±standard deviation (SD)

is used in the analysis. The statistical analysis was performed by Student’s t-test and analysis
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of variance (ANOVA) using OriginPro Software Version 8.0 (OriginLab Corp., MA, USA).

P<0.05 indicated statistically significant differences.

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3. Results and discussion

3.1. Extraction yields and physicochemical properties of HEPs

The extraction yields and physicochemical properties of the four water-soluble HEPs

from the fruit body of H. erinaceus obtained through different extraction methods are

summarized in Table 1. The extraction methods had significant influences on the extraction

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yields and physicochemical properties. Depending on the extraction solvent, the yield of

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HEPs varied (approximately 8.10%–11.76%). Among these HEPs, HEP-A had the highest

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yield (11.76%), followed by HEP-S (9.66%), HEP-C (8.84%), and HEP-W (8.10%). In

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general, insoluble PSs can be effectively released from the cell wall and converted into

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soluble PSs through the disruption of the hydrogen bonds between the cellulose and
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hemicellulose during the alkali extraction of the cell wall materials, which would help
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increase the final recovery (Lahaye & Robic, 2007). Moreover, the HEP yield was lower with
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citric acid than with saline and alkaline at the same extraction time and temperature because
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of partial acid hydrolysis. In the process of extraction, the citric acid solution, especially at

95 °C, can degrade the long-chain PSs into short-chain PSs to some extent; even free sugars
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may be not precipitated by ethanol, leading to the lower HEP yields obtained (Yuliarti et al.,
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2008). Moreover, the lowest extraction yield of HEPs was obtained during water extraction
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because water only dissolves long-chain soluble PS. This result was in agreement with those

of Peasura et al. (2015), who reported lower yield with acid extraction than with alkali
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extraction.

The highest total sugar content was obtained from HEP-C (81.51%) via citric acid

extraction, and the lowest was obtained from HEP-W (72.30%) via hot water extraction.

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These results may be due to the fact that the glycosidic bonds in the PS chain are easily

destroyed by citric acid (Yuliarti et al., 2015). The total protein content in the four HEPs

except for HEP-A was low (1.90%–2.81%) because of the reduction of the isolated proteins

in these HEPs via solvent extraction. Specifically, HEP-A extracted with alkali solution had

the highest protein content (8.31%), corresponding to the release of proteins by the breakage

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of hydrogen bridges in the alkaline solution. This result also revealed that HEP-A may be a

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polysaccharide-protein complex (Yuliarti et al., 2015), which was verified by the UV–Vis

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spectrum analysis (data not shown). Uronic acid content (glucoronic acid equivalent) was

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highest in HEP-S (16.96%), followed by HEP-C (14.99%), HEP-W (13.98%), and HEP-A

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(12.14%), which indicated that the four HEPs were all acidic. Additionally, the four HEPs
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had small quantities of polyphenol (0.54%–0.77%). On the basis of the findings above, we
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can conclude that these differences may be attributed to the selective type of extraction
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method and the use of extraction solvent. The results were in agreement with those of the
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previous studies (Peasura et al., 2015; Yuliarti et al., 2015).

Table 1. Extraction yields, chemical properties, and compositions of PSs from H. erinaceus
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by using different solvent extractions.

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Sample HEP-W HEP-S HEP-C HEP-A

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Yield (%) 8.10±0.23 9.66±0.81 8.84±0.14 11.76±0.31

Carbohydrate (wt%) 72.30±1.70 73.97±2.30 81.51±2.56 79.17±2.76
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Protein (wt%) 2.81±0.18 1.90±0.04 1.97±0.32 8.31±0.13

Uronic acid (wt%) 13.98±1.51 16.96±2.83 14.99±1.04 12.14±1.47
Polyphenols (wt%) 0.77±0.02 0.62±0.02 0.64±0.03 0.54±0.02
β-1,3-glucans (mg/g fruit body) a 0.23±0.05 0.38±0.05 1.26±0.08 23.21±0.75
Sugar composition (molar ratios) (mol%)

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L-Rhamnose 9.9 1.7 9.0 3.3
D-Arabinose 1.6 1.0 2.0 2.4
D-Mannose 1.0 1.0 1.0 1.0
D-Glucose 35.5 30.7 40.7 66.5
D-Galactose 6.1 4.6 7.5 9.5
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The total β-1,3-glucans content was determined by an aniline blue fluorescence assay.

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In the current work, a fluorescence assay based on the aniline blue dye, which binds

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specifically to β-(1→3)-glucans, was used for the determination of the total β-(1→3)-glucan

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contents of the four HEPs at different solvent extractions. As shown in Table 1, the extraction

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medium had significant influence on the total β-(1→3)-glucan contents. HEP-A extracted
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with alkali solution showed the highest content (23.21 mg/g fruit body), followed by HEP-C
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(1.26 mg/g fruit body), HEP-S (0.38 mg/g fruit body), and HEP-W (0.23 mg/g fruit body).
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The β-(1→3)-glucan constitutes one of the structural macromolecules in the cell wall of
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mushrooms or fungus; thus, the alkali solution can readily cause the cell wall to swell, which

can destroy the hydrogen bonds in the cell walls, leading to the increased release of
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β-(1→3)-glucans. Consequently, the alkali solution resulted in the highest content of

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β-(1→3)-glucan; similar results were also observed in the previous studies (Ko & Lin, 2004;
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Zhang, Xu & Zhang, 2005).

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Fig. 2. GC profiles of nitrile acetates of (a) monosaccharide standards, (b) HEP-W, (c) HEP-S,
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(d) HEP-C, and (e) HEP-A. Peaks: 1, L-Rha (Rt: min); 2, D-Ara (Rt: min); 3, D-Xyl (Rt:

min); 4, D-Man (Rt: min); 5, D-Glc (Rt: min); 6, D-Gal (Rt: min).

The four HEPs were completely hydrolyzed by TFA, converted into nitrile acetates, and
17
then subjected to GC analysis; the obtained GC results are shown in Table 1 and Fig. 2.

According to the monosaccharide standards, the four HEPs were all composed of L-Rha,

D-Ara, D-Man, D-Glc, and D-Gal in different molar percentages. This finding suggested that

the four HEPs extracted from H. erinaceus were heteropolysaccharides with different

chemical compositions. Particularly, D-Glc was a predominantly monosaccharide in these

T
HEPs. Zhang et al. (2012) reported that three fractions of the endopolysaccharides (HEP40,

IP
HEP60, and HEP80) from the mycelium of H. erinaceus all comprised glucose, galactose,

R
mannose, arabinose, xylose, rhamnose, and fucose with varying molar ratios. Zhu et al. (2014)

SC
also found that the HEP monosaccharide compositions of HEPs extracted from the fruits of H.

U
erinaceus by hot water are not considerably different from those extracted by
N
enzyme-assisted methods, although they differ significantly with respect to molar ratio.
A

Consequently, the variation in the monosaccharide composition of HEPs may be attributed to

M

the differences in the source of raw materials and extraction methods and isolation procedures
ED

used.

In the current work, we performed HPSEC–MALLS–RID analysis to determine Mw, Mn,

PT

and MWD of the four HEPs, and the results are illustrated in Table 2 and Fig. 3. As shown in
E

Fig. 3, two peaks were observed for each HEP in the HPSEC–MALLS curves regardless of
CC

the extraction solvent used. Differences in the elution profiles reflect differences in the

molecular size. The results suggested that these HEPs possessed two MWDs that mainly
A

consisted of high- and low-Mw fractions. HEP-W extracted with hot water presented two

main peaks with the Mws of 597.6 and 314.7 kDa, and the high fraction of ~72% was the

dominant based on the peak area. Similarly, HEP-S obtained by saline extraction also showed

18
two peaks with Mws of 449.4 and 442.7 kDa, and the high Mw fraction of ~73% was the main

composition. Alternatively, the Mws of HEPs extracted with acid and alkali were significantly

lower than those of HEPs obtained by hot water and saline. This result suggests that the PS

chains may have been destroyed and degraded to some extent during the acid and alkaline

extractions, then converted a portion of the high-Mw fractions into the low-Mw fractions,

T
thereby increasing the amounts of low-Mw fractions in the HEP molecules. For example, the

IP
Mws of HEP-C extracted with citric acid were 263.6 and 229.8 kDa with peak areas of 28%

R
and 72%, respectively. The same results were also obtained in our previous studies (Wang,

SC
Pei, Ma, Cai, Yan, 2014; Yan et al., 2009).

chromatograms
U
N
HWP-W HEP-S HEP-C HEP-A

LS
A
1.0
M
Relative Scale

0.5
ED
PT

0.0
25.0 30.0 35.0 40.0
time (min)
E

Fig. 3. HPSEC–MALLS curves of HEP-W, HEP-S, HEP-C, and HEP-A in 0.1 M NaCl
CC

aqueous solution at 25 °C.

A

As shown in Table 2, different extraction solvents had significant effects on the intrinsic

viscosity ([η]) of the four HEPs at the same conditions. Specifically, HEP-C extracted with

citric acid solution had the lowest [η] value (135.60 mL/g) than others possibly because of

19
the acidic degradation of HEPs during the extraction. Thus, acidic conditions have significant

effect on the distribution of molecular weights of polysaccharide (Shu & Lung, 2004), which

is in agreement with the result of HPSEC–MALLS–RID analysis.

Table 2. Molecular characteristics of PSs from H. erinaceus by using different solvent

extractions.

T
Sample [η] (mL/g) Mw (×103 Da) a Mn (×103 Da) b Mw/Mn c %/Area

IP
HEP-W 658.98 597.6 582.3 1.03 72.2

R
314.7 204.4 1.54 27.8

SC
HEP-S 620.24 449.4 413.2 1.09 73.6
442.7 295.2 1.50 26.4
HEP-C 135.60 229.8
263.6 U
218.4
162.1
1.05
1.63
72.0
28.0
N
HEP-A 242.27 256.8 249.7 1.03 67.9
A
320.8 263.0 1.22 32.1
M

a
Weight average molecular weight.
ED

b
Number average molecular weight.

c
Molecular weight distribution.
E PT

3.2. Preliminary structural features and microstructures of HEPs

CC

FT–IR spectroscopy is often used for the identification of characteristic organic groups

in PSs. Fig. 4 displays the FT–IR spectra of HEP-W, HEP-S, HEP-C, and HEP-A. No
A

obvious difference was observed among the characteristic organic groups of HEPs obtained

from different extraction solvents. The result indicated that the different solvent extraction

has no effect on sugar structure. The broad and strong absorption peak at approximately 3330

20
cm−1 was assigned to the O–H stretching vibration, while the weak band at approximately

2921 cm−1 was attributed to the C–H stretching vibration. The two bands at approximately

1645 and 1417 cm−1 corresponded to the asymmetric and symmetric stretching of the

carboxylate anions groups, further indicating that these HEPs are acidic (Manrique, & Lajolo,

2002). The strong extensive absorption peaks located at 1150, 1077, and 1030 cm−1 were

T
ascribed to the stretching vibrations of the C–O–H side groups and the C–O–C glycosidic

IP
band vibration of a pyranose ring. These results further confirm that the four extracted HEPs

R
contained pyranose sugar (Kacurakova et al., 2000). In addition, the characteristic absorption

SC
bands at approximately 890 and 908 cm−1 indicate that β-glycosidic bonds are the main bond

type in these HEPs.

U
N
A
M
ED
E PT

Fig. 4. FT–IR spectra of (a) HEP-W, (b) HEP-S, (c) HEP-C, and (d) HEP-A.
CC

Fig. 5 displays the SEM images of the four HEPs (HEP-W, HEP-S, HEP-C, and HEP-A)
A

at magnifications of 500×. As shown in Fig. 5, the surface morphologies of the four HEPs

extracted using different solvents presented significant differences in shape and size. HEP-W

extracted with hot water showed a massive and rough surface with many aggregates, forming

21
large tracts of PS (Fig. 5a). Similarly, HEP-S extracted by saline solution presented clumpy

surfaces with a small part of lumpish particles (Fig. 5b). However, HEP-C possessed

relatively loose and flaky surfaces (Fig. 5c), which indicated that citric acid may have

partially disrupted PS and decreased its size. This result was in line with those of

HPSEC–MALLS–RID analysis. Compared with citric acid, the alkali solution had a more

T
drastic effect on the cell walls of the fruiting bodies, and the disruption of the cells increased

IP
the contact area between the solid and liquid phases. This phenomenon leads to the relatively

R
rough surface of HEP-A, with on a reticular layer and a nonuniform size (Fig. 5d). Thus, the

SC
SEM analysis verified that the alkali extraction produced the highest yield (Table 1).

(a) (b)
U
N
A
M
ED
PT

(c) (d)
E
CC
A

Fig. 5. Scanning electron micrographs (500 ×) of (a) HEP-W, (b) HEP-S, (c) HEP-C, and (d)

22
HEP-A.

3.3. In vitro antioxidant activity of HEPs

The DPPH radical is a stable free radical that has been widely used for the estimation of

the free-radical scavenging capacities of antioxidants (Matthaus, 2002). Fig. 6a shows the

T
DPPH radical scavenging ability of the four HEPs (HEP-W, HEP-S, HEP-C, and HEP-A)

IP
extracted from H. erinaceus by using different extraction solvents. The four HEPs showed

R
clear scavenging ability on the DPPH radical at all tested concentrations (0–3.0 mg/mL). At

SC
3.0 mg/mL, the scavenging abilities of HEP-W, HEP-S, HEP-C, HEP-A, and Vc on DPPH

U
radical were 62.31%, 62.42%, 67.97%, 68.26%, and 95.24%, respectively. Moreover, the IC50
N
values of HEP-W, HEP-S, HEP-C, HEP-A and Vc were calculated to be about 1.62, 1.55,
A

1.07, 1.29, 0.26 mg/mL, respectively. HEP-C and HEP-A showed the high DPPH scavenging
M

but much weaker than that of Vc. The possible reason for this result may be the PS
ED

degradation by acidic and alkali treatments, which resulted in PS with low molecular weight

(Table 2) and with enhanced hydrogen-donating ability. The DPPH radical scavenging ability
PT

of antioxidants may be related to the hydrogen-donating capacity. Notably, HEP-C with the
E

lower molecular weight showed stronger a DPPH radical scavenging ability than HEP-A.
CC

Similarly, Zhang et al. (2013) reported that two PSs, that is, UFP and MFP, with decreased

molecular weights extracted from Flammulina velutipes by ultrasonic and microwave

A

treatments, respectively, showed higher DPPH radical scavenging abilities possibly because

of the strong hydrogen-donating ability. Zhang et al. (2012) also prepared three fractions of

the endopolysaccharides (HEP40, HEP60, and HEP80) from the mycelium of H. erinaceus

23
grown on tofu whey by gradient concentrations of ethanol. The scavenging effects of HEP40,

HEP60, and HEP80 (1.25 mg/mL) on DPPH radical were approximately 43%, 31%, and 60%,

respectively, which was weaker than those of HEPs reported in this work. These results

revealed that more bioactive PSs may be extracted using an acid or alkali solution than hot

water, which was further verified by Wang et al. (2014).

T
R IP
SC
U
N
A
M
ED
E PT
CC

Fig. 6. Antioxidant activities in vitro of HPE-W, HEP-S, HPE-C, and HPE-A via the (a)

scavenging ability on DPPH, (b) ·OH, (c) TEAC assay, and (d) FRAP assay. Each value is
A

expressed as mean ± SD (n =3).

Hydroxyl radicals are typical reactive oxygen species generated in the body and can

24
easily damage cells (Yuan, Zhang, Fan, Yang, 2008). Thus, the removal of hydroxyl radical is

crucial to antioxidant defense in cells or food systems, and hydroxyl radical scavenging is

extremely important in the evaluation of antioxidant activity in vitro. Fig. 6b presents the

hydroxyl radical scavenging ability of the four HEPs using Vc as the positive control. The

scavenging ability of these HEPs on the hydroxyl radical dose dependently increased with the

T
increase in HEP concentration from 0 mg/mL to 3.0 mg/mL. At 3.0 mg/mL, the scavenging

IP
abilities of HEP-W, HEP-S, HEP-C, and HEP-A on the hydroxyl radical were 69.05%,

R
69.91%, 79.37%, and 79.11%, respectively, which were stronger than that of the result

SC
reported by Zhang et al. (2012), but less than that of Vc (90.43%). The results suggested that

U
the four HEPs exhibited strong scavenging ability on the hydroxyl radical, and HEP-C
N
extracted with citric acid showed the highest hydroxyl radical scavenging ability among these
A

HEPs, which was consistent with the result of DPPH radical scavenging assay. Similar results
M

were also found in the previous studies (Lu et al., 2013; Yao et al., 2017).
ED

Figs. 6c and d show the in vitro antioxidant capacities of the four HEPs. The capacities

were evaluated via the TEAC (on ABTS·+ radical scavenging activity) and FRAP assays (on
PT

ferric reducing activity). In the TEAC assay, HEP-A with a TEAC value of 32.2 μmol
E

Trolox/g sample was obtained by alkali extraction. It had the highest antioxidant capacity
CC

among the tested HEP samples possibly because it had the highest protein content (8.31% vs.

1.90%–2.81%; Table 1). In the FRAP assay, HEP-C extracted with citric acid solution
A

significantly showed the highest antioxidant capacity with a FRAP value of 44.82 μmol

Fe2+/g sample. These results suggested that extraction medium affected the antioxidant

activities of HEPs. HEPs extracted from H. erinaceus through acid or alkali extraction

25
possessed higher sugar and protein contents (Table 1) and lower intrinsic viscosities and

molecular weights (Table 2) than those extracted through other methods, thereby exhibiting

enhanced antioxidant capacities. This result was in line with those of DPPH and hydroxyl

radical scavenging activities. Additionally, the total β-(1→3)-glucan contents had certain

effects on the antioxidant activities of the HEPs.

T
3.4. Inhibitory effects of HEPs on α-glycosidase and α-amylase

IP
In individuals with type 2 diabetes, α-glycosidase and α-amylase can hydrolyze starch to

R
glucose during the PS catabolism, leading to a significant increase in the postprandial blood

SC
glucose (Omojokun, 2015). Thus, α-glycosidase and α-amylase inhibition assays can be

U
regarded as simple and effective strategies for the evaluation of hypoglycemic activities of
N
bioactive compounds in vitro (Lordan et al., 2013). Fig. 7 shows the inhibitory effects of the
A

four HEPs on the α-glycosidase and α-amylase activities by using acarbose as the positive
M

reference. The four HEPs had similar inhibitory effects on α-glycosidase and α-amylase
ED

activities, and they were dose-dependently increased as the HEP concentration was increased

from 0 mg/mL to 5.0 mg/mL. HEP-C extracted with citric acid solution exhibited the
PT

strongest inhibitory effects on the α-glycosidase and α-amylase activities at all tested
E

concentrations, whereas were weaker than that of acarbose. At 5.0 mg/mL, the inhibitory
CC

abilities of HEP-C on α-glycosidase and α-amylase were 73.66% and 65.92%, respectively,

which suggested that HEP-C possessed stronger inhibitory ability against α-glycosidase than
A

that of α-amylase under the same concentrations. This variation may be attributed to the

different mechanisms of action between α-glycosidase and α-amylase, and a moderated

inhibition of α-amylase and a stronger α-glycosidase inhibition should be preferred (Cho,

26
Han, & You, 2011). Similarly, Zhao et al. (2017) reported that the PS from Dioscorea

hemsleyi obtained using different extraction techniques inhibited both α-glycosidase (IC50

27.41–274.36 μg/mL) and α-amylase (IC50 3.66–47.57 μg/mL); however, the quantities

required for α-amylase inhibition were much higher than those required for α-glycosidase. In

contrast, Li et al. (2017) found that the Sargassum pallidum PSs showed weaker inhibitory

T
effects on α-glycosidase than α-amylase. The α-glycosidase and α-amylase inhibitory

IP
activities may be related to the sugar and protein contents, monosaccharide composition,

R
molecular weight, and chemical structures of HEPs. Therefore, on the basis of the above

SC
results, we conclude that the citric acid extraction is more useful in extracting HEPs from H.

U
erinaceus with prominent antioxidant and hypoglycemic activities than the other extraction
N
solvents.
A
M
ED
E PT
CC

Fig. 7. Inhibitory effects of HPE-W, HPE-S, HPE-C, and HPE-A on the (a) α-glycosidase and

(b) α-amylase activity. Each value is expressed as mean ± SD (n =3).

A

4. Conclusions

In summary, hot water, 0.9% NaCl, citric acid, and 1.25 M NaOH/0.05% NaBH4 were

27
used for the extraction of water-soluble PSs (HEP-W, HEP-S, HEP-C, and HEP-A) from the

fruit body of H. erinaceus. The results showed that the extraction solvents had significant

effects on the extraction yields, physicochemical properties, preliminary structural

characteristics, and microstructures of the HEPs. HEP-C extracted with citric acid showed

stronger antioxidant and α-glycosidase and α-amylase inhibitory activities in vitro than

T
HEP-W and HEP-S and had a lower molecular weight. Therefore, citric acid as a safe food

IP
additive that can be widely used in food processing and for the extraction of bioactive PS.

R
Further investigations on the isolation, purification, and structural characterization of HEP-C

SC
are ongoing.

U
N
Acknowledgements
A
This work was supported financially by the National Natural Science Foundation of
M

China (31671812), the Key Research & Development Program (Modern Agriculture) of

Jiangsu Province (BE2017362), Jiangsu Overseas Research & Training Program for
ED

University Prominent Young & Middle-aged Teachers and Presidents.

PT

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