Journal Pre-proofs

A novel Hericium erinaceus polysaccharide: structural characterization and

prevention of H2O2-induced oxidative damage in GES-1 cells

Bingwu Liao, Chunhui Zhou, Tingting Liu, Yangyan Dai, Huihua Huang

PII: S0141-8130(19)35132-3
DOI: https://doi.org/10.1016/j.ijbiomac.2019.11.027
Reference: BIOMAC 13808

To appear in: International Journal of Biological Macromole-

cules

Received Date: 5 July 2019

Revised Date: 21 October 2019
Accepted Date: 5 November 2019

Please cite this article as: B. Liao, C. Zhou, T. Liu, Y. Dai, H. Huang, A novel Hericium erinaceus
polysaccharide: structural characterization and prevention of H2O2-induced oxidative damage in GES-1 cells,
International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.11.027

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 A novel Hericium erinaceus polysaccharide: structural characterization and

prevention of H2O2-induced oxidative damage in GES-1 cells

Bingwu Liao a, Chunhui Zhou b, Tingting Liu c, Yangyan Dai a, Huihua Huang a,†

a
School of Food Science and Engineering, South China University of Technology,

Guangzhou 510641, China

b
School of Food and Biotechnology, Guangdong Industry Polytechnic College,

Guangzhou 510300, China

c
Guangdong Apollo Group Co., Ltd, Guangzhou 510665, China

†
Corresponding author. Tel./fax: +86-20-87112851. E-mail address: [email protected]
Abstract

In this paper, the structural characteristics and bioactivity of a novel polysaccharide

from H. erinaceus (HEPN) were investigated, wherein, physico-chemical

characterization demonstrated that HEPN with an average molecular weightof 12.713

kDa was composed of mannose (5.13%), glucose (43.02%), and galactose (51.85%).

The models in vitro for preventing oxidative damage of human gastric epithelium

(GES-1) cells were established and used to investigate the preventive effects of HEPN

from oxidative damage. It was found that HEPN could significantly prevent GES-1 cells

against H2O2-induced oxidative damage by promoting cell proliferation, inhibiting cell

necrosis, reducing ROS levels, regulating mitochondrial membrane potential and

maintaining mitochondrial membrane permeability. These results indicated HEPN can

effectively prevent gastric cell damage in vitro and suggested the potential application

of HEPN as bioactive ingredient for healthy foods.

Keywords: Hericium erinaceus polysaccharide; structural characterization; GES-1 cells;

oxidative damage; mitochondrial dysfunction.

1. Introduction

Since the active polysaccharides were developed, researches on their biological

activities have been numerous in the scientific community. Typically, olive leaf

polysaccharides (OLP) displayed strong DPPH-radical scavenging activity (IC50 =

34.80 μg/mL) and have attractive antibacterial activity against S.enterica and M.luteus

with inhibition zones of 23.5 and 21.5 mm, respectively [1]; a novel polysaccharide

(FCPW80-2) with a molecular weight of 1.21 × 105 Da was first isolated from Ficus

carica could not only up regulate the expression of p-p65 and p-IκB-α, but also cause

the translocation of nuclear factor-kappa B (NF-κB) p65 from cytosol to nuclei in

RAW264.7 macrophages [2].

As a kind of polysaccharide with good efficacies, Hericium erinaceus polysaccharide

(HEP) is also a hotspot of nutritional sciences in the present. The application of modern

food processing technology in polysaccharides provides many new ways for the deep

processing of HEP, among them, the industrial application of microwave, ultrasonic

wave and membrane separation are becoming more and more widespread [3-8].

Moreover, modern pharmacological studies have shown that HEP represents a large

class of bioactive substances of H. erinaceus with a variety of health benefits, including

immunomodulatory, antioxidative, antitumor, antigastritic and antihyperlipidemic

activities [9-13]. Therefore, in terms of H. erinaceus, the innovation of its processing

technologies and the development of its functional activities are potentially useful for

the food-borne pharmaceutical industry.

 In recent years, many researches have focused more on the resistance of H. erinaceus

or HEP to various gastric ulcers or gastric cancers. Gastric ulcer (GU) is a complex

multifactorial disease that develops from a variety of aggressive factors, including

Helicobacter pylori infection, non-steroidal anti-inflammatory drugs, steroids, alcohol,

and stress. The pathophysiology of GU mainly involves by cellular damage, mucosal

damage, inducing inflammatory signals and triggering oxidative stress [14-17].

Currently, GU is alleviated mostly by inhibiting gastric acid secretion through chemical

drugs, neutralizing the acid through antacid drugs, or impeding cellular apoptosis

through cytoprotective drugs [18, 19]. Previous studies have confirmed that, a novel

polysaccharide (EP-1) derived from H. erinaceus mycelium culture resists chronic

atrophic gastritis by inducing cell cycle arrest [20]; Hericium erinaceus polysaccharide

has gastroprotective effect on ethanol-induced gastric mucosal injury in rats, and also

has potential to prevent duodenal ulcer and promote productions of short-chain fatty

acids (SCFAs) [21]; a novel polysaccharide-protein (HEG-5) purified from fermented

mycelium of H. erinaceus inhibits the growth of human gastric cancer cells (SGC-7901)

via cell cycle arrest and apoptosis induction [22].

Although researches on active polysaccharides are getting better and better, there are

still research directions are worth exploring, such as the field of preventive pharmacy.

The focal point of preventive pharmacy mainly focuses on the response of cells and

organisms to chemical substances (such as drugs, carcinogens, reactive oxygen species,

etc.), using various chemical and biological methods, combining with clinical samples

and data to study pathogeneses of major chronic non-communicable diseases [23-25].

However, the preventive effects of natural products on chronic diseases were only

reported in a few literatures. Typically, an active sapogenin cycloastragenol (CAG)

from astragaloside IV (AST) could effectively prevent isoproterenol-induced cardiac

fibrosis by suppressing the NLRP3 inflammasome pathway [26]; animal experiments

have demonstrated long-term treatment with lotus seedpod procyanidins (LSPCs) could

prevent cognitive deficits and oxidative damage were caused by extremely low

frequency electromagnetic field (ELF-EMF), and predict that LSPCs may have

potential in treatment of neurodegenerative diseases [27].

The present study aims to explore the initial application and possible biological

mechanisms of HEP in the field of preventive pharmacy. In this study, a novel

polysaccharide (HEPN) was purified from H. erinaceus and its structural characteristics

were investigated by physical and chemical methods. Then, the cytotoxicity of HEPN

and its preventive effects on H2O2-induced oxidative damage in human gastric

epithelium (GES-1) cells were studied. Specifically, the study also evaluated the cell

proliferation, cell death, intracellular reactive oxygen species and mitochondrial

function of the research objects. In summary, the valuable findings provided evidences

for understanding structure and biological activities of Hericium erinaceus

polysaccharides, and suggested that HEPN can be applied as potential feedstock for

preventive drugs.

2. Materials and methods

2.1. Materials and reagents

 Hericium erinaceus was purchased from theplanting base of H. erinaceus (Gutian

County, Fujian Province, China). Diethylaminoethyl (DEAE)-52 was acquired from

Whatman International Ltd. (Maidstone, Kent, UK) and standard monosaccharides

(including glucuronic acid, rhamnose, arabinose, fucose, xylose, mannose, glucose,

galactose and inositol) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

(Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum

(FBS), phosphate-buffered saline (PBS, pH 7.2±0.2), and streptomycin/penicillin

double-resistance solutions were acquired from Gibco brand of Thermo Fisher

Scientific Inc. (Waltham, MA, USA). Hydrogen peroxide (H2O2) was obtained from

Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Cell counting kit-8 (CCK-8)

assay was purchased from MedChemExpress LLC (Monmouth Junction, NJ, USA).

Hoechst 33342/PI Double Stain Kit, Reactive Oxygen Species (ROS) Assay Kit and

Mitochondrial Membrane Potential Kit (JC-10 Assay) were obtained from Beijing

Solarbio Science & Technology Co., Ltd. (Beijing, China). Mitochondrial PT Pore

Assay Kit was purchased from Cayman Chemical Company (Ann Arbor, MI, USA).

All other reagents used in this study were of analytical grade.

2.2. Extraction and purification of polysaccharides from H. erinaceus

Methods for extracting and purifying polysaccharides from H. erinaceus were

referred to the previous study in our laboratory with minor modifications [28].

Therefore, the similarities (i.e., methods for preparing crude polysaccharides) were not

repeated here. And the differences were as follows, briefly, the crude polysaccharides
from H. erinaceus were dissolved in distilled water and separated by an ultrafiltration

membrane with a molecular weight cut-off of 10 kDa, then the separated components

were separately freeze-dried to obtain two components including HEP-A (<10 kDa, its

yield was 1.98 ± 0.17%) and HEP-B (ı10 kDa, its yield was 6.73 ± 0.19%). About

250 mg of HEP-B was dissolved in 10 mL of ultrapure water and fractionated by

DEAE-52-cellulose column (2.6 cm × 30 cm). The column was eluted with distilled

water and NaCl-gradient solution (0.10, 0.30 and 0.50 mol·L-1, respectively) at a flow

rate of 1.0 mL/min. Then, the total sugar contents of all fractions were measured by the

phenol-sulfuric acid method. Finally, the HEPN eluting with distilled water was

collected, dialyzed, freeze-dried and available for the subsequent experiments.

2.3. Molecular weight determination

The average molecular weight of HEPN was measured by high performance gel

permeation chromatography (HPGPC, Waters, USA). Briefly, 5 mg of the sample was

dissolved in 1 mL of 0.02 M phosphate solution. After passing through microporous

filter (0.45 μm), 20 μL of the prepared sample solution was injected into the

Chromatography system. The HPGPC system was eluted by KH2PO4 (0.02 mol·L-1, 0.6

mL/min) and the column temperature was set at 35 ć. The various standard dextrans

(from 5.0 kDa to 670 kDa) were used for the calibration curve and the molecular weight

was evaluated on the basis of the standard dextran.

2.4. Morphological observation

 Usually, the morphology of polysaccharides was analyzed by scanning electron

microscope (SEM) [29]. The freeze-dried HEPN powders coating with gold layer were

spread on the specimen holder by double-sided adhesive tape and observed by SEM

(EM-30 Plus, COXEM, Korea) at an accelerating voltage of 20.0 kV. In addition, the

magnification of SEM image was set as ×500 and ×1000.

2.5. Monosaccharide composition analysis

The monosaccharide composition of polysaccharides was determined by gas

chromatography (GC) as described previously, with slight revisions [30, 31]. Briefly,

10 mg of HEPN was hydrolyzed with 4.0 mL of 2 mol·L-1 trifluoroacetic acid (TFA) at

110 ć for 6 h, and then the solution was dried totally. The aforementioned

hydrolysates were mixed with hydroxylamine hydrochloride (10 mg), inositol (1 mg)

and pyridine (1.0 mL) at 90 ć for 30 minutes. After which, the above resulting

solution was mixed with 0.5 mL of acetic anhydride and incubated at 90 ć for 30

minutes.

Next, the solution was filtered through 0.22 μm of microporous membrane (organic

phase) and analyzed by Agilent 6890N gas chromatograph system (Agilent

Technologies, USA). The initial temperature was set to 120 ć, linearly increased to

160 ć at 2 ć/min, and linearly increased to 250 ć at 3 ć/min and then kept for

5 minutes at 250 ć. Moreover, the temperature of injector and detector were set at

250 ć. A set of monosaccharides (glucuronic acid, rhamnose, arabinose, fucose,

xylose, mannose, glucose and galactose) were used as standards and inositol (with

concentration of 1.0 mg/mL) was used as the internal standard.

2.6. Methylation analysis

The methylation of HEPN was carried out according to previous reports [32-34] with

minor modifications. The HEPN powders were vacuum dried with phosphorus

pentoxide (P2O5) for two days, and after that twenty milligrams of dried HEPN was

dissolved in DMSO (6 mL), mixed with NaOH (100 mg), and kept at room temperature

for 30 minutes. 1 mL of CH3I was added to the above mixture in an ice bath, and then

stirred for 30 minutes at room temperature. The methylation was terminated by addition

of 0.5 mL of distilled water, and excess methyl iodide were distilled off under reduced

pressure at room temperature, after which, the products were dialyzed for 24 h and

concentrated to dryness under reduced pressure. The above processes were repeated

five times until the methylation was exhaustive confirmed by infrared spectrums.

After the permethylated HEPN was hydrolyzed with 2 mL of 2 mol·L-1 TFA at 110 ć

for 2 h, the hydrolysates were reduced with 30 mg of NaBH4 at room temperature and

further acetylated with 2 mL of acetic anhydride at 100 ć for 2 h to generate the

partially methylated alditol acetates. The products were dissolved in chloroform,

filtered through 0.45 μm of organic filter and detected by GC-MS (7890, Agilent, USA)

equipped with HP-5 capillary column.

2.7. Ultraviolet spectrum scan and infrared spectra analysis

 The HEPN samples were subjected to spectral scanning by a UV-Vis

spectrophotometer (UV-180, Shimadzu, Japan) with a wavelength range of 200-400

nm, and resulting in 200 spectral points. The freeze-dried HEPN samples were mixed

with spectroscopic grade KBr powder and pressed into pellets, and the transmittance of

mixtures in the infrared region of 4000-400 cm-1 was analyzed by Fourier transform

infrared (FT-IR) spectrometer (VERTEX-33, Bruker, Germany).

2.8. Nuclear magnetic resonance (NMR) spectroscopy

Approximately 50 mg of HEPN powder was dissolved in 1.0 mL of heavy water (D2O)

in an NMR tube, after which, the 1H spectrum and 13C spectrum of HEPN were recorded

by a NMR spectrometer (AVANCE III HD 600, Bruker, Germany) at 25 ć, and data

analysis was executed by MestreNova 5.3.0 software (Mestrelab Research S.L.).

2.9. Cell culture and CCK-8 assay

The human gastric epithelium (GES-1) cells were built by American Type Culture

Collection (ATCC, Rockville, MD, USA) and purchased from Fenghui Biotechnology

Co., Ltd (Changsha, China).

GES-1 cells were cultured in DMEM containing 10% (v/v) fetal bovine serum,

penicillin-streptomycin (100 U/mL-100 μg/mL) double-resistance solutions at 37 ć

in a 5% CO2 humidified atmosphere [35, 36]. In this experiment, the CCK-8 assay kit

was used to detect cell viability and cytotoxicity. Briefly, GES-1 cells were seeded at a

density of 3×104 cells/mL into 96-well microplates (Corning Incorporated, Costar®,

USA) until the cells were adherent and then treated with different concentrations of

treatments (HEPN or H2O2). After the aforementioned steps, 10 μL of CCK-8 was

pipetted into each well (note that this step does not create bubbles) and the microplates

were incubated for 2 h [37].

Finally, the absorbance was recorded on Synergy™ Neo2 Multi-Mode Microplate

Reader (BioTek Instruments, Inc., Winooski, VT, USA) at 450 nm. The cell viability

was calculated by the absorbances of the treated cells (A) and control cells (B), as the

following formula:

Cell viability (%) = A/B ×100%

2.10. Cell modeling

The GES-1 cells at logarithmic growth phase were plated (at a density of 3×104

cells/mL) and cultured for 6 h until the cells were adherent. Then, the medium was

removed and the cells were replaced with new medium containing HEPN (0-1000

µg/mL) for different treatment times (0, 3, 6, 12 and 24 h). After which, the previously

pretreated cells were damaged with 600 μmol·L-1 H2O2 for 12 h, among them, the

concentration and treat-time of H2O2 were based on the previous research results of our

laboratory [28].

The aim of cell modeling was to screen out the optimal treat-time of HEPN used to

construct in vitro prevention models for GES-1 cells. Furthermore, the cells were

grouped as follows: (A) control group, in which, no HEPN, no H2O2, only cells; (B)

blank group, in which, containing 600 μmol·L-1 H2O2 and cells; (C) prevention groups,

in which, containing different concentrations of HEPN, 600 μmol·L-1 H2O2 and cells.
2.11. Cell proliferation and death assessment

In this experiment, cell proliferation and death were assessed primarily by measuring

cell viability, observing cell morphology, and determining the ratio of dead cells. The

cell viability was measured by CCK-8 assay kit according to the manufacturer’s

instructions, and the specific steps were same as Section 2.9. The cell morphology was

observed by an inverted fluorescence microscope (Olympus IX73, Japan) with bright

field. The ratio of dead cells was determined by Hoechst 33342/PI Double Stain Kit (in

this step, the cells were stained only with propidium iodide to study necrotic cells)

according to the manufacturer’s instructions, and the necrotic cells were viewed with

an inverted fluorescence microscope (Olympus IX73, Japan) (488 nm excitation, 650

nm emission).

2.12. Determination of ROS generation

The level of intracellular ROS was assessed by measuring the degree of oxidation of

2, 7-dichlorofluorescein diacetate (DCFH-DA) according to the past reports [38].

Moreover, DCFH-DA is by far the most commonly used and sensitive probe for

detecting intracellular ROS. Briefly, cell grouping and cell modeling were identical to

Section 2.10, after which, all of the differently treated cells were washed three times

with PBS and incubated with serum-free culture medium containing 10 μmol·L-1

DCFH-DA in a humidified atmosphere (5% CO2, 37 ć) for 45 minutes. Subsequently,

the cells were digested with 0.25% trypsin-EDTA and formulated into cell suspensions
which were centrifuged at 1000 r/min for 5 minutes and the supernatants were removed.

Then, the cells were resuspended with PBS and divided into two parts, the fluorescence

images of one part were shot by an inverted fluorescence microscope (Olympus IX73,

Japan) (485 nm excitation, 525 nm emission), and the fluorescent intensities of another

part were measured by Synergy™ Neo2 Multi-Mode Microplate Reader (485 nm

excitation, 525 nm emission).

2.13. Measurement of mitochondrial membrane potential (ƸΨm )

JC-10 is an ideal fluorescent probe widely used to detect mitochondrial membrane

potential (ƸΨm) of cells or animal tissues. The JC-10 aggregates in mitochondrial

matrix produce red fluorescence under high-ƸΨm conditions; correspondingly, the JC-

10 monomers in mitochondrial matrix produce green fluorescence under low-ƸΨm

conditions [39].

Briefly, the different groups of treated cells were incubated with 0.5 mL of JC-10

working solution for 20 minutes at 37 ćin dark, detached with 0.25% trypsin-EDTA

and centrifuged at 600 g for 5 minutes. After which, the collected cells were washed

twice times and resuspended with JC-10 buffer solution and then divided into two parts,

the cell counting of one part were performed immediately by flow cytometry (Beckman

Coulter CytoFLEX, USA) (490 nm excitation, 530 nm emission), and the fluorescent

intensities of another part were measured by Synergy™ Neo2 Multi-Mode Microplate

Reader (490 nm excitation, 530 nm emission).

2.14. Detection of mitochondrial permeability transition pore (mPTP) opening

The opening of mPTP in GES-1 cells was evaluated by calcein-AM/cobalt assay

method. The basic theory of this method is as follows, the measurement of mPTP

opening was adapted for whole cells using a calcein-AM/cobalt quenching technique,

and this technique uses calcein-AM to stain entire cell, followed by a treatment with

cobalt chloride (CoCl2) to quench the calcein fluorescence outside of mitochondrial

matrix. If the inner mitochondrial membrane (IMM) is intact, cobalt is unable to

penetrate the IMM and the mitochondrial matrix exhibits green fluorescence. If the

IMM is compromised, the calcein fluorescence is quenched and no fluorescence is

observed [40, 41].

Briefly, the different groups of treated cells were incubated with Hank’s Balanced

Salt Solution (HBSS) containing 10 μmol·L-1 calcein-AM and 100 μmol·L-1 CoCl2 for

15 minutes at 37 ć in dark, detached with 0.25% trypsin-EDTA and centrifuged at

600 g for 5 minutes. After which, the collected cells were washed twice times and

resuspended with HBSS and then divided into two parts, the detection methods of each

part were identical to Section 2.13.

2.15. Statistical analysis

All the assays were carried out in triplicates and all data were expressed as mean ±

standard deviation (S.D.). The statistical significance was determined using one-way

ANOVA followed by Tukey's post-hoc test. A value of p < 0.05 was considered as the

level of significant, and p < 0.01 was accepted as the level of highly significant.
3. Results and discussion

3.1. Purification of polysaccharides from H. erinaceus

The crude polysaccharides were extracted from the dried fruiting bodies of H.

erinaceus by “hot water extraction and ethanol precipitation” method [42, 43],

separated by centrifugal ultrafiltration and further purified by DEAE-52-cellulose

column chromatography using distilled water and NaCl-gradient solution as eluents (as

shown in Fig. 1A). Then, the major fraction (HEPN) eluting with distilled water was

obtained, moreover, HEPN may be a kind of neutral polysaccharide carrying little

negative charge according to the elution characteristics of polysaccharides.

3.2. Molecular weight and morphology of HEPN

In the present research, HEPN was further purified from HEP-B (ı10 kDa), and the

average molecular mass of HEPN was estimated to be 12.713 kDa with a single

absorption peak based on the HPGPC result (as shown in Fig. 1B), additionally, the

total carbohydrate of HEPN was determined to be 87.63 ± 2.05% (w/w) by the

corresponding chemical analysis. The possible causes for this result was different from

other researchers' previous reports [44-45] are the different separation methods,

varieties and growing environments of the studied H. erinaceus. As shown in Fig. 1C,

the SEM images showed that the morphology of HEPN-aggregates at different

magnifying power (500× and 1000×), and indicated that HEPN-aggregates with bright

lusters and exhibited irregular, unevenly distributed granules. At high magnification

(1000×), partial details of HEPN-aggregates could be observed, the partial detailed

information showed that granular HEPN-aggregates were concave, wrinkled and

relatively fluffy. The above results indicate that the interaction force between HEPN-

aggregates was relatively weak, which may be closely related to the molecular

arrangements and the glycosidic bonds of HEPN.

3.3. Monosaccharide composition and methylation analysis of HEPN

The monosaccharide composition of HEPN was evaluated by GC system based on

the differences in retention time of different monosaccharide standards. As shown in

Fig. 2A (monosaccharide standards) and Fig. 2B (HEPN), HEPN was constituted of

mannose (5.13%), glucose (43.02%), and galactose (51.85%). Among them, glucose

and galactose account for the main proportion of HEPN, thereby predicting that HEPN

was a kind of hetero-polysaccharide containing a large amount of aldehyde groups [46].

Methylation analysis is one of the most important research methods for knowing the

type of glycosidic bond in polysaccharides, which complements and perfects the

structural information were not explicitly stated in monosaccharide composition

analysis. Generally, this method mainly involves methylation reaction and GC-MS

analysis of partially methylated alditol acetates (PMAAs). In this research, the peaks

were classified according to total ion chromatograph as well as the standard data in the

CCRC Spectral Database for PMAAs, and the results of methylation analysis for HEPN

were presented in Fig. 2C and Table 1. It can be concluded that the peaks were identified

as 2, 3, 4, 6-Me4-Glcp, 2, 3, 6-Me3-Glcp, 2, 3, 4-Me3-Glcp, 2, 3, 4-Me3-Manp, 2, 4-

Me2-Manp, 2, 3, 4-Me3-Galp with different molar ratios of 24.67: 5.98: 10.35: 0.79:

3.43: 54.78, correspondingly, their retention time at 6.780, 7.653, 3.350, 5.594, 8.518

and 7.802 minutes. The results suggested that HEPN contained six linkage forms: (1

ė)-linked Glc, (1ė4)-linked Glc, (1ė6)-linked Glc, (1ė6)-linked Man, (1ė3,6)-

linked Man and (1ė6)-linked Gal. Besides, the degree of branching (DB) of HEPN was

calculated as 28.10% judging by the following formula:

DB = (NT+NB) / (NT+NB+NL)

where NT, NB and NL represented the number of the terminal residues (T-Glcp), branch

residues (1,3,6-Manp) and linear residues (1,4-Glcp, 1,6-Glcp, 1,6-Manp and 1,6-Galp),

respectively [47].

3.4. Spectroscopic characteristics and NMR analysis of HEPN

In general, the spectroscopic data of polysaccharides were expressed in the form of

an ultraviolet spectrum and an infrared spectrum. The UV spectrum of HEPN showed

no absorption peak in the wavelength of 260 nm and 280 nm (Fig. 3A), which indicated

that there were no impurity of nucleotides or proteins. As shown in Fig. 3B, the FT-IR

spectrum of HEPN can reveal its major functional groups and chemical boundaries. The

characteristic peaks at 3407.1, 2923.0 and 1647.1 cm-1 may be due to the stretching

vibration of the hydroxyl stretching vibration, C-H stretching vibration and bound water,

respectively [48]. The peak around 1422.5 cm-1 was attributed to the presence of

carbonyl groups, and, in-plane bending vibration of C-H caused the characteristic peak

at the wavenumber of 1368.4 cm-1. Moreover, the peaks at 1047.3 cm-1 and 574.7 cm-1
revealed the characteristics of glycosidic bonds and α-configurations, respectively [47].

NMR spectroscopy is one of the most commonly used characterization methods for

analyzing detailed structural information of unknown polysaccharides. And the

characteristic signals of carbohydrates in such spectra are attributed to monosaccharide

composition, glycosidic bonds and chemical shifts [49].

The structural characteristics of HEPN were further confirmed by NMR spectral

analysis and the signals of HEPN in 1H and 13C NMR spectra were presented in Fig. 3C

(1H NMR spectrum) and Fig. 3D (13C NMR spectrum). Simultaneously, the anomeric

hydrogen proton signals and the anomeric carbon signals in the NMR spectra were

further analyzed in combination with the results of monosaccharide composition

analysis, methylation analysis and previous reports. As shown in Fig. 3C, the proton

signals of HEPN in the hydrogen spectrum were focused on the range of 3.10 to 4.94

ppm, besides, the proton signals also appeared sporadically at 1.10, 1.11, and 1.77 ppm.

Among them, the chemical shift of most protons were in the range of 3~4 ppm due to

the shielding effect of hydroxyl groups, and serious overlap between signals led to

difficulty in analysis; additionally, the chemical shift of anomeric hydrogen were in the

range of 4.5~5.5 ppm, the two anomeric hydrogen signals (δ 4.63 and 4.94) in this range

corresponded with β-D-Glc and α-D-Gal, respectively. As shown in Fig. 3D, the

anomeric carbon signals of HEPN in the carbon spectrum were focused on the range of

60.77 to 75.94 ppm, besides, the carbon signals also appeared sporadically at 15.74,

103.01 and 167.18 ppm. Generally, the anomeric carbon signals were mostly in the

range of 95~110 ppm, where the signal at 103.01 ppm suggested the presence of β-D-
Glc. In summary, the NMR results were basically consistent with the results of

methylation analysis.

3.5. Construction of models in vitro for preventing oxidative damage of GES-1 cells

In the field of traditional Chinese medicine (TCM), researches on the prevention and

treatment of diseases through active ingredients have been in existence for a long time,

and most of the preventive treatments on diseases involve cell models or animal models

[50, 51]. Additionally, the active ingredients need to be tested for their toxicities before

being used in cell biology researches. Hence, based on the above theory, the

cytotoxicity assay and cell modeling were completed in this study.

As described in Fig. 4A, the cell viabilities of GES-1 cells were inhibited at the

concentration of HEPN above 1000 μg/mL, but at the concentration of HEPN less than

1000 μg/mL, the HEPN had no toxicity to the GES-1 cells, which possibly due to the

increase of viscosity of polysaccharides hindered cells from absorbing nutrients in the

extracellular matrixc (ECM). Furthermore, when the concentration of HEPN was nearly

500 μg/mL, the proliferation rate of GES-1 cells was relatively highest.

As described in Fig. 4B, the cells in the logarithmic growth phase were selected as

research objects and the cell viability was measured by CCK-8 assay. It was found that

the cell viability of blank group (i.e., untreated group containing cells, 0 μg/mL HEPN

and 600 μmol·L-1 H2O2) was non-fluctuating significantly with different culture-time,

compared with the control group. In terms of the prevention groups, the changes of cell

viability as described below: the cell viability was increased gradually with prolonged
treatment time at the same concentration of HEPN, meanwhile, when the treatment time

reached or above 12 h, the cell viability was almost no longer increased; besides, there

are no uniform regularity in the changes of cell viability under the same treatment time,

among them, when the treatment time reached 12 h, the cell viabilities at 500, 750 and

1000 μg/mL HEPN were 67.04 ± 2.03%, 66.82 ± 3.21% and 64.53 ± 2.02%, respectively.

Therefore, it can be indicated that the effect of high concentration of HEPN on cell

viability appeared negative growth rather than continuous improvement based on the

foregoing data. In summary, the HEPN (its treatment time reached 12 h, and its

concentration is less than or equal to 500 μg/mL) can be adopted as the suitable

conditions to construct prevention models for GES-1 cells in vitro.

3.6. Effects of HEPN on cell proliferation, cell death and ROS generation of GES-1 cells

in prevention models in vitro

In our current research, the preventive effects of HEPN on H2O2-induced oxidative

damage in GES-1 cells were evaluated by cell proliferation, cell death and ROS

generation. Generally, the cell proliferation of different groups can be explained by cell

viability and cell morphology (as shown in Fig. 4B and Fig. 4C). The cell viabilities of

different groups (control group, blank group, 125 μg/mL HEPN, 250 μg/mL HEPN and

500 μg/mL HEPN) were 100 ± 2.98%, 47.89 ± 3.25%, 48.55 ± 2.34%, 56.09 ± 3.44%,

67.04 ± 2.03%, respectively (Fig. 4B). Moreover, the cell morphology of different

groups were also different, the GES-1 cells of blank group were stimulated by H2O2,

and their morphology were changed distinctly, for instance, nuclear contraction and cell
shedding. However, compared with the control group, the cells of prevention groups

proliferated, stretched and regained normal morphology as increasing concentration of

HEPN (Fig. 4C).

As shown in Fig. 4D, the fluorescence distribution and fluorescence intensity of the

stained cells (staining with PI) were observed by an inverted fluorescence microscope.

It was found that the necrotic cells of each group were successfully stained and showed

red fluorescence, wherein, high concentration (500 μg/mL) of HEPN can effectively

inhibit cell necrosis, and correspondingly, this phenomenon appeared in the

microscopic field of view was the decrease of fluorescent particles.

As described in Fig. 5, the GES-1 cells of each group were successfully loaded with

DCFH-DA probes and showed green fluorescence of different densities and intensities.

Wherein, the comparison-results between the blank group and the prevention groups

showed that the pro-oxidant (H2O2) induced a large amount of ROS in the cells, while,

the HEPN inhibited or blocked the ROS generation to some extent. And the similar

results have been confirmed in the cells’ fluorescence intensity, as shown in the

histogram in Fig. 5, the H2O2 caused a significant elevation of intracellular ROS levels

significantly in comparison to the control group (##p < 0.01) and the HEPN (250 μg/mL,

500 μg/mL) pretreatment restrained the intracellular ROS elevation significantly

compared with the blank group (*p < 0.05, **p < 0.01).

These results indicate that HEPN can scavenge H2 O2-induced ROS, additionally, it is

worth noting that ROS is an important regulator of cell function, high levels of ROS

can damage cell lipids, DNA and proteins, while low levels of ROS have proliferative
effects on cells [52-54].

3.7. HEPN alleviates H2O2-induced mitochondrial dysfunction of GES-1 cells in

prevention models in vitro

Many diseases are related to the ROS-caused oxidative stress, such as various

gastrointestinal diseases, furthermore, the mitochondria are the main site of ROS

generation in vivo, wherefore it is particularly important to study the mitochondrial

function of research objects [55-57]. In our study, the mitochondrial function was

reflected by mitochondrial membrane potential ( Ƹ Ψm) and mitochondrial

permeability transition pore (mPTP) opening.

In terms of ƸΨm, its basic theory is as follows, the JC-10 monomers showed green

fluorescence and high-intensity of green fluorescence indicated the low-ƸΨm as well

as early apoptosis. And the cytometric analysis showed that the percentage of cells

loaded with JC-10 monomers in different groups (control group, blank group, 125

μg/mL HEPN, 250 μg/mL HEPN and 500 μg/mL HEPN) were 16.12%, 75.67%, 71.00%,

42.42% and 39.00%, respectively (Fig. 6). Furthermore, the similar results have been

confirmed in the cells’ fluorescence intensity, as shown in the histogram in Fig. 6, the

HEPN (250 μg/mL, 500 μg/mL) pretreatment significantly improved ƸΨm, compared

with the blank group (**p < 0.01, **p < 0.01); suggesting HEPN can prevent GES-1

cells from H2O2-induced mitotoxicity. In terms of (mPTP) opening, its basic theory is

as follows, the calcein retained in the cells, causing the mitochondria to emit green

fluorescence and low-intensity of green fluorescence indicated active-mPTP as well as

mitochondrial dysfunction. And the cytometric analysis showed that the percentage of

cells stained with calcein in different groups (control group, blank group, 125 μg/mL

HEPN, 250 μg/mL HEPN and 500 μg/mL HEPN) were 33.27%, 10.80%, 18.00%, 28.39%

and 32.15%, respectively (Fig. 7). Moreover, the similar results have been confirmed

in the cells’ fluorescence intensity, as shown in the histogram in Fig. 7, the HEPN (250

μg/mL, 500 μg/mL) pretreatment significantly enhanced mean fluorescent intensity of

calcein, compared with the blank group (*p < 0.05, **p < 0.01); indicating that HEPN

prevented H2O2-induced mPTP opening.

The results of this section suggested that HEPN may be involved in maintaining the

integrity and functionality of mitochondria in GES-1 cells. It is well known that the

dissipation of mitochondrial membrane potential (MMP) is thought to be an early event

in mitochondrial damage and apoptosis cascades [58]. This theory was confirmed in the

present study, meanwhile, HEPN showed significant resistance to H2O2-induced

mitochondrial damage of GES-1 cells, which provides a theoretical basis for revealing

and discovering the protective effect of natural polysaccharides on GU.

4. Conclusion

In conclusion, a novel polysaccharide HEPN with an average molecular weight of

12.713 kDa was purified from H. erinaceus, and HEPN was constituted of mannose

(5.13%), glucose (43.02%), and galactose (51.85%). The main linkage types of HEPN

were 2, 3, 4, 6-Me4-Glcp, 2, 3, 6-Me3-Glcp, 2, 3, 4-Me3-Glcp, 2, 3, 4-Me3-Manp, 2, 4-

Me2-Manp, 2, 3, 4-Me3-Galp with different molar ratios of 24.67: 5.98: 10.35: 0.79:
3.43: 54.78, which based on the combined results of monosaccharide composition,

methylation and NMR analysis. Furthermore, the suitable concentration (500 μg/mL)

of HEPN had no apparent cytotoxic effect on human gastric epithelium (GES-1) cells,

and could also prevent gastric cell damage caused by peroxidation via inhibiting ROS

generation and alleviating mitochondrial dysfunction. These findings indicated that

HEPN might be used as potential feedstock for healthy foods or preventive drugs.

Conflicts of interest

None.

Acknowledgments

The authors gratefully acknowledge Guangdong Science and Technology Planning

Project (Grant No. 2013B090600008) as well as Guangdong Apollo Group Co., Ltd.

(Guangzhou, China).

References

[1] Khemakhem, I. , Abdelhedi, O. , Trigui, I. , Ayadi, M. A. , & Bouaziz, M. . (2018). Structural,

antioxidant and antibacterial activities of polysaccharides extracted from olive leaves. International

Journal of Biological Macromolecules, 106, 425-432.

[2] Du, J. , Li, J. , Zhu, J. , Huang, C. , Bi, S. , & Song, L. , et al. (2018). Structural characterization

and immunomodulatory activity of a novel polysaccharide from ficus carica. Food & Function, 9,

3930-3943.

[3] Remón, J. , Matharu, A. S. , & Clark, J. H. . (2018). Simultaneous production of lignin and

polysaccharide rich aqueous solutions by microwave-assisted hydrothermal treatment of rapeseed

meal. Energy Conversion and Management, 165, 634-648.

[4] Wang, H. , Li, Y. , Ren, Z. , Cong, Z. , Chen, M. , & Shi, L. , et al. (2018). Optimization of the

microwave-assisted enzymatic extraction of Rosa roxburghii Tratt. polysaccharides using response

surface methodology and its antioxidant and α- d -glucosidase inhibitory activity. International

Journal of Biological Macromolecules, 112, 473-482.

[5] Guo, H. , Kong, F. , & Yan, C. . (2017). Optimization of polysaccharide ultrasonic extraction

conditions using purple sweet potato tubers based on free radical scavenging and glycosylation

inhibitory bioactivities. Pharmacognosy Magazine, 13(51), 504-511.

[6] Ghavi, P. P. . (2015). Modeling and optimization of ultrasound-assisted extraction of

polysaccharide from the roots of althaea officinalis. Journal of Food Processing and Preservation,

6 (39), 2107-2118.

[7] Wang, Y. , Hu, X. , Han, J. , Ni, L. , Tang, X. , & Hu, Y. , et al. (2016). Integrated method of

thermosensitive triblock copolymer–salt aqueous two phase extraction and dialysis membrane

separation for purification of lycium barbarum polysaccharide. Food Chemistry, 194, 257-264.

[8] Ma, F. W. , Kong, S. Y. , Tan, H. S. , Wu, R. , Xia, B. , & Zhou, Y. , et al. (2016). Structural

characterization and antiviral effect of a novel polysaccharide psp-2b from prunellae spica.

Carbohydrate Polymers, 152, 699-709.

[9] Sheng, X. , Yan, J. , Meng, Y. , Kang, Y. , Han, Z. , & Tai, G. , et al. (2017). Immunomodulatory

effects of hericium erinaceus derived polysaccharides are mediated by intestinal immunology. Food

& Function, 8(3), 1020-1027.

[10] Han, Z. H. , Ye, J. M. , & Wang, G. F. . (2013). Evaluation of in vivo antioxidant activity of

hericium erinaceus polysaccharides. International Journal of Biological Macromolecules, 52, 66-

71.

[11] Kim, S. P. , Kang, M. Y. , Kim, J. H. , Nam, S. H. , & Friedman, M. . (2011). Composition and

mechanism of antitumor effects of hericium erinaceus mushroom extracts in tumor-bearing mice.

Journal of Agricultural and Food Chemistry, 59(18), 9861-9869.

[12] Choi, W. S. , Jang, D. Y. , Nam, S. W. , Park, B. S. , Lee, H. S. , & Lee, S. E. . (2012).

Antiulcerogenic activity of scoparone on HCl/ethanol-induced gastritis in rats. Journal of the Korean

Society for Applied Biological Chemistry, 55(2), 159-163.

[13] Shang, H. M. , Song, H. , Shen, S. J. , Yao, X. , & Ding, G. D. . (2015). Effects of dietary

polysaccharides from the submerged fermentation concentrate of hericium caput-medusae (bull.:fr.)

pers. on fat deposition in broilers. Journal of the Science of Food and Agriculture, 95(2), 267-274.

[14] Calam, J. , & Baron, J. H. . (2001). Pathophysiology of duodenal and gastric ulcer and gastric

cancer. British Medical Journal, 323(7319), 980-982.

[15] Malfertheiner, P. , Chan, F. K. L. , & Mccoll, K. E. L. . (2009). Peptic ulcer disease. The Lancet,

374(9699), 1449-1461.

[16] Ben Barka, Z. , Tlili, M. , Alimi, H. , Ben Miled, H. , Ben Rhouma, Khémais, & Sakly, M. , et

al. (2017). Protective effects of edible rhus tripartita (ucria) stem extract against ethanol-induced

gastric ulcer in rats. Journal of Functional Foods, 30, 260-269.

[17] Hasebe, T. , Harasawa, S. , & Miwa, T. . (2010). Changes in gastric function during healing of

gastric ulcers. Digestive Endoscopy, 6(4), 342-348.

[18] Handa, O. , Naito, Y. , Fukui, A. , Omatsu, T. , & Yoshikawa, T. . (2014). The impact of non-

steroidal anti-inflammatory drugs on the small intestinal epithelium. Journal of Clinical

Biochemistry & Nutrition, 54(1), 2-6.

[19] Garro, M. F. , Salinas Ibáñez, A. G. , Vega, A. E. , Arismendi Sosa, A. C. , Pelzer, L. , & Saad,

J. R. , et al. (2015). Gastroprotective effects and antimicrobial activity of lithraea molleoides and

isolated compounds against helicobacter pylori. Journal of Ethnopharmacology, 176, 469-474.

[20] Wang, M. , Gao, Y. , Xu, D. , & Gao, Q. . (2015). A polysaccharide from cultured mycelium

of hericium erinaceus and its anti-chronic atrophic gastritis activity. International Journal of

Biological Macromolecules, 81, 656-661.

[21] Wang, X. Y. , Yin, J. Y. , Zhao, M. M. , Liu, S. Y. , Nie, S. P. , & Xie, M. Y. . (2018).

Gastroprotective activity of polysaccharide from hericium erinaceus against ethanol-induced gastric

mucosal lesion and pylorus ligation-induced gastric ulcer, and its antioxidant activities.

Carbohydrate Polymers, 186, 100-109.

[22] Zan, X. , Cui, F. , Li, Y. , Yang, Y. , Wu, D. , & Sun, W. , et al. (2015). Hericium erinaceus

polysaccharide-protein HEG-5 inhibits SGC-7901 cell growth via cell cycle arrest and apoptosis.

International Journal of Biological Macromolecules, 76, 242-253.

[23] Zellmer, W. A. . (1975). Preventive health care and pharmacy. American Journal of Hospital

Pharmacy, 32(8), 791.

[24] Willcox, J. K. , Ash, S. L. , & Catignani, G. L. . (2004). Antioxidants and prevention of chronic

disease. Critical Reviews in Food Science and Nutrition, 44(4), 275-295.

[25] Selkoe, D. J. . (2007). Developing preventive therapies for chronic diseases: lessons learned

from alzheimer's disease. Nutrition Reviews, 65(s3), S239-S243.

[26] Wan, Y. , Xu, L. , Wang. Y., X. , Tuerdi, N. , Ye, M. , & Qi, R. . (2018). Preventive effects of

astragaloside iv and its active sapogenin cycloastragenol on cardiac fibrosis of mice by inhibiting

the nlrp3 inflammasome. European Journal of Pharmacology, 833, 545-554.

[27] Duan, Y. , Wang, Z. , Zhang, H. , He, Y. , Lu, R. , & Zhang, R. , et al. (2013). The preventive

effect of lotus seedpod procyanidins on cognitive impairment and oxidative damage induced by

extremely low frequency electromagnetic field exposure. Food & Function, 4(8), 1252-1262.

[28] Liao B.W., & Huang, H. H. . (2019). Structural characterization of a novel polysaccharide from

hericium erinaceus and its protective effects against H2O2-induced injury in human gastric

epithelium cells. Journal of Functional Foods, 56, 265-275.

[29] Jia, S. , Yu, H. , Lin, Y. , & Dai, Y. . (2007). Characterization of extracellular polysaccharides

from nostoc flagelliforme cells in liquid suspension culture. Biotechnology and Bioprocess

Engineering, 12(3), 271-275.

[30] Yang, S. , Li, Y. , Jia, D. , Yao, K. , & Liu, W. . (2017). The synergy of box-behnken designs

on the optimization of polysaccharide extraction from mulberry leaves. Industrial Crops and

Products, 99, 70-78.

[31] Zhang, J. Q. , Li, C. , Huang, Q. , You, L. J. , Chen, C. , & Fu, X. , et al. (2019). Comparative

study on the physicochemical properties and bioactivities of polysaccharide fractions extracted from

fructus mori at different temperatures. Food & Function, 10, 410-421.

[32] Nie, S. P. , Cui, S. W. , Phillips, A. O. , Xie, M. Y. , Phillips, G. O. , & Al-Assaf, S. , et al.

(2011). Elucidation of the structure of a bioactive hydrophilic polysaccharide from cordyceps

sinensis by methylation analysis and nmr spectroscopy. Carbohydrate Polymers, 84(3), 894-899.

[33] Panda, B. C. , Maity, P. , Nandi, A. K. , Pattanayak, M. , Manna, D. K. , & Mondal, S. , et al.

(2017). Heteroglycan of an edible mushroom pleurotus cystidiosus: structural characterization and

study of biological activities. International Journal of Biological Macromolecules, 95, 833-842.

[34] Li, Q. M. , Wang, J. F. , Zha, X. Q. , Pan, L. H. , Zhang, H. L. , & Luo, J. P. . (2017). Structural

characterization and immunomodulatory activity of a new polysaccharide from jellyfish.

Carbohydrate Polymers, 159, 188-194.

[35] Yang, R. , Lin, Q. , Gao, H. B. , & Zhang, P. . (2014). Stress-related hormone norepinephrine

induces interleukin-6 expression in ges-1 cells. Brazilian Journal of Medical and Biological

Research, 47(2), 101-109.

[36] Lian, H. G. , Cui, J. F. , Wang, Y. , Liu, J. , Wang, J. , & Shen, H. T. , et al. (2014).

Downregulation of rad51 participates in ota-induced dna double-strand breaks in ges-1 cells in vitro.

Toxicology Letters, 226(2), 214-221.

[37] Liu, Y. S. , Zhang, Y. , Jia, K. , Dong, Y. , & Ma, W. Y. . (2015). Metformin inhibits the

proliferation of a431 cells by modulating the pi3k/akt signaling pathway. Experimental &

Therapeutic Medicine, 9(4), 1401-1406.

[38] Galant, L. S. , Braga, M. M. , De Souza, D. , De Bem, A. F. , Sancineto, L. , & Santi, C. , et al.

(2017). Induction of reactive oxygen species by diphenyl diselenide is preceded by changes in cell

morphology and permeability in saccharomyces cerevisiae. Free Radical Research, 51(7-8): 657-

668.
[39] Yokokura, S. , Yurimoto, S. , Matsuoka, A. , Imataki, O. , Dobashi, H. , & Bandoh, S. , et al.

(2014). Calmodulin antagonists induce cell cycle arrest and apoptosis in vitro and inhibit tumor

growth in vivo in human multiple myeloma. BMC Cancer, 14, 882.

[40] Onishi, A. , Miyamae, M. , Kaneda, K. , Kotani, J. , & Figueredo, V. M. . (2012). Direct

evidence for inhibition of mitochondrial permeability transition pore opening by sevoflurane

preconditioning in cardiomyocytes: comparison with cyclosporine a. European Journal of

Pharmacology, 675(1-3), 40-46.

[41] He, F. , Wu, Q. , Xu, B. , Wang, X. , Wu, J. , & Huang, L. , et al. (2017). Suppression of stim1

reduced intracellular calcium concentration and attenuated hypoxia-reoxygenation induced

apoptosis in h9c2 cells. Bioscience Reports, 37, BSR20171249.

[42] Qing, G. E. , Zhang, A. Q. , & Sun, P. L. . (2010). Isolation, purification and structural

characterization of a novel water-soluble glucan from the fruiting bodies of phellinus baumii pilát.

Journal of Food Biochemistry, 34(6), 1205-1215.

[43] Wang, L. , Liu, F. , Wang, A. , Yu, Z. , Xu, Y. , & Yang, Y. . (2017). Purification,

characterization and bioactivity determination of a novel polysaccharide from pumpkin (cucurbita

moschata) seeds. Food Hydrocolloids, 66, 357-364.

[44] Zhang, A. Q. , Sun, P. L. , Zhang, J. S. , Tang, C. H. , Fan, J. M. , & Shi, X. M. , et al. (2007).

Structural investigation of a novel fucoglucogalactan isolated from the fruiting bodies of the fungus

hericium erinaceus. Food Chemistry, 104(2), 451-456.

[45] Li, Q. Z. , Wu, D. , Zhou, S. , Liu, Y. F. , Li, Z. P. , & Feng, J. , et al. (2016). Structure

elucidation of a bioactive polysaccharide from fruiting bodies of hericium erinaceus in different

maturation stages. Carbohydrate Polymers, 144, 196-204.

[46] Patra, S., Maity, K. K., Bhunia, S. K., Dey, B., Das, D., & Mondal, S., et al. (2010). Structural

characterization of an immunoenhancing heteropolysaccharide isolated from hot water extract of

the fresh leaves of catharanthus rosea. Carbohydrate Polymers, 81(3), 584-591.

[47] Zhang, M., Wang, G., Lai, F., & Hui, W. (2016). Structural characterization and

immunomodulatory activity of a novel polysaccharide from Lepidium meyenii. Journal of

Agricultural and Food Chemistry, 64, 1921-1931.

[48] Gao, J., Lin, L., Sun, B., & Zhao, M. (2017). A comparison study on polysaccharides extracted

from Laminaria japonica using different methods: Structural characterization and bile acid-binding

capacity. Food & Function, 8, 3043-3052.

[49] Bubb, W. A. . (2004). Nmr spectroscopy in the study of carbohydrates: characterizing the

structural complexity. Concepts in Magnetic Resonance Part A, 19A(1), 1-19.

[50] Liu, L. F. , Song, J. X. , Lu, J. H. , Huang, Y. Y. , Zeng, Y. , & Chen, L. L. , et al. (2015).

Tianma gouteng yin, a traditional chinese medicine decoction, exerts neuroprotective effects in

animal and cellular models of parkinson’s disease. Scientific Reports, 5, 16862.

[51] Zhang, L. , Wu, C. , Zhang, Y. , Liu, F. , Zhao, M. , & Bouvet, M. , et al. (2013). Efficacy

comparison of traditional chinese medicine lq versus gemcitabine in a mouse model of pancreatic

cancer. Journal of Cellular Biochemistry, 114(9), 2131-2137.

[52] Luan, S. , Yun, X. , Rao, W. , Xiao, C. , Xu, Z. , & Lang, J. , et al. (2017). Emamectin benzoate

induces ros-mediated dna damage and apoptosis in trichoplusia tn5b1-4 cells. Chemico-Biological

Interactions, 273, 90-98.

[53] Salehi, F. , Behboudi, H. , Kavoosi, G. , & Ardestani, S. K. . (2017). Chitosan promotes ros-

mediated apoptosis and s phase cell cycle arrest in triple-negative breast cancer cells: evidence for

intercalative interaction with genomic dna. RSC Advances, 7(68), 43141-43150.

[54] Aredia, F. , Czaplinski, S. , Fulda, S. , & Scovassi, A. I. . (2016). Molecular features of the

cytotoxicity of an nhe inhibitor: evidence of mitochondrial alterations, ros overproduction and dna

damage. Bmc Cancer, 16(1), 851.

[55] Indo, H. P. , Davidson, M. , Yen, H. C. , Suenaga, S. , & Majima, H. J. . (2007). Evidence of

ros generation by mitochondria in cells with impaired electron transport chain and mitochondrial

dna damage. Mitochondrion, 7(1-2), 106-118.

[56] Angélica Ruiz-Ramírez, Monserrath Chávez-Salgado, José Antonio Peñeda-Flores, Zapata, E. ,

& El-Hafidi, M. . (2011). High-sucrose diet increases ros generation, ffa accumulation, ucp2 level,

and proton leak in liver mitochondria. AJP Endocrinology and Metabolism, 301(6), E1198-207.

[57] Ross, T. , Szczepanek, K. , Bowler, E. , Hu, Y. , Larner, A. , & Lesnefsky, E. J. , et al. (2013).

Reverse electron flow-mediated ros generation in ischemia-damaged mitochondria: role of complex

i inhibition vs. depolarization of inner mitochondrial membrane. Biochimica et Biophysica Acta

(BBA)-General Subjects, 1830(10), 4537-4542.

[58] Satpute, R. M., Hariharakrishnan, J., & Bhattacharya, R. (2008). Alpha-ketoglutarate and N-

acetyl cysteine protect PC12 cells from cyanide-induced cytotoxicity and altered energy metabolism.

Neurotoxicology, 29(1), 170-178.

Figure Caption
Fig. 1. Chromatograms of HEPN from H. erinaceus by DEAE-52-cellulose (A); HPGPC (B) and

SEM images of HEPN (C).

Fig. 2. Gas chromatograms of standard monosaccharides (A), HEPN sample (B) and total ion

chromatograph of methylated HEPN (C).

Fig. 3. UV (A), FT-IR (B), 1H NMR (C) and 13C NMR (D) spectra of HEPN.

Fig. 4. Cell viabilities of basal conditions (only HEPN treatment) (A), construction of models in

vitro for preventing oxidative damage of GES-1 cells (B), effects of HEPN treatment on H2O2-

induced cell morphology of GES-1 cells in prevention models in vitro (C), effects of HEPN treatment

on H2O2-induced cell death of GES-1 cells in prevention models in vitro were assessed by PI

staining (D). Cell viabilities were determined by CCK-8 assay. Data were expressed as mean ± S.D.

(n=3).

Fig. 5. Effects of HEPN treatment on H2O2-induced ROS generation of GES-1 cells in prevention

models in vitro. Data were expressed as mean ± S.D. (n=3), the significant differences were

designed as #p < 0.05, ##p < 0.01 compared to control group; *p < 0.05, **p < 0.01 compared to

blank group.

Fig. 6. Effects of HEPN treatment on H2O2-induced mitochondrial membrane potential (ƸΨm) of

GES-1 cells in prevention models in vitro. Data were expressed as mean ± S.D. (n=3), the significant

differences were designed as #p < 0.05, ##p < 0.01 compared to control group; *p < 0.05, **p <

0.01 compared to blank group.

Fig. 7. Effects of HEPN treatment on H2O2-induced mitochondrial permeability transition pore

(mPTP) opening of GES-1 cells in prevention models in vitro. Data were expressed as mean ± S.D.

(n=3), the significant differences were designed as #p < 0.05, ##p < 0.01 compared to control group;
*p < 0.05, **p < 0.01 compared to blank group.

Table 1. Glycosidic Linkage Composition of Methylated HEPN

Residues PMAA Linkage Molar ratio (%) T/min
D-Glcp 2, 3, 4, 6-Me4-Glcp T- 24.67 6.780
2, 3, 6-Me3-Glcp 1, 4- 5.98 7.653
2, 3, 4-Me3-Glcp 1, 6- 10.35 3.350
D-Manp 2, 3, 4-Me3-Manp 1, 6- 0.79 5.594
2, 4-Me2-Manp 1, 3, 6- 3.43 8.518
D-Galp 2, 3, 4-Me3-Galp 1, 6- 54.78 7.802

Fig. 1.
Fig. 2.
 A B

Fig. 3.
Fig. 4.

Fig. 5.
Fig. 6.

Fig. 7.
Highlights

l HEPN with antioxidant activity was obtained from H. erinaceus.

l Structure of HEPN is characterized by methylation, FT-IR and NMR.

l HEPN can effectively prevent gastric cell damage in vitro.

l HEPN can alleviate H2O2-induced mitochondrial dysfunction.