Structure and immunomodulatory activity of microparticulate

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Functional Structure/Activity Relationships

Structure and immunomodulatory activity of microparticulate mushroom sclerotial #-glucan prepared from Polyporus rhinocerus Chaoran Liu, and Peter Chi Keung Cheung J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03206 • Publication Date (Web): 25 Jul 2019 Downloaded from pubs.acs.org on July 27, 2019

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Journal of Agricultural and Food Chemistry

Structure and immunomodulatory activity of microparticulate

1

mushroom sclerotial

2

prepared from Polyporus rhinocerus

3 4

Chaoran Liu 1,2, Peter C.K. Cheung *, 2

5 6

1

Shenzhen Institute of Standards and Technology, Shenzhen, China

7

2

Food and Nutritional Sciences, School of Life Sciences, The Chinese University of

8

Hong Kong, Shatin, New Territories, Hong Kong SAR, China

9

Running title: Structure and immunomodulatory activity of microparticulate

10

mushroom )*

11 12 13 14 15 16 17 18 19 20 21 22 1

ACS Paragon Plus Environment


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ABSTRACT: In this study, an immunologically active novel microparticulate

24

mushroom )*

25

emulsification and cross-linking method. PRA-1 was a hyper-branched + /0, + /1,*

26

)*2*

27

rhinocerus. PRA-1 had a rod-like conformation, while PRA-1p exhibited monodisperse

28

and homogeneous spherical conformation with a diameter ranging from 0.3-2.0 7' in

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water. PRA-1p significantly induced NO and ROS production as well as the

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morphological changes of murine macrophages (RAW 264.7 cells) and up-regulated

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their phagocytic activity. Furthermore, PRA-1p treatment markedly enhanced the

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secretion of cytokines including CTACK, G-CSF, MCP-1, = * > MIP-2, RANTES,

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sTNFRI and TIMP-1. Activation of RAW 264.7 cells triggered by PRA-1p was

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associated with activation of iNOS,

35

the novel PRA-1p derived from the mushroom sclerotia of P. rhinocerus has potential

36

application as an immunostimulatory agent.

(PRA-1p) was prepared using an alkali-soluble glucan PRA-1 by

with a degree of branching of 0.89 isolated from the sclerotia of Polyporus

*@A ERK and AKT. This work suggests that

37 38

KEYWORDS: mushroom sclerotia, Polyporus rhinocerus microparticulate -glucan,

39

macrophage activation, polysaccharides

40 41 42 43 44 2


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INTRODUCTION

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Bioactive polysaccharides derived from edible or medicinal mushrooms have been

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demonstrated to possess a variety of therapeutic properties including antitumor,

48

antidiabetic

49

polysaccharides, )*

50

(IRMs) or immunomodulators.5, 6 As )*

51

they are classified as pathogen-associated molecules that can be recognized by pattern

52

recognition receptors (PRRs) on the surfaces of innate immune cells mainly including

53

macrophages, monocytes and dendritic cells.7-9

and

Several )*

54

effects.1-4

immunomodulating

Among

these

mushroom

are studied extensively as immune response modifiers are nondigestible in the human body,

from mushrooms have been developed into prescription drug used

55

clinically with documented evidences on inducing host-mediated antitumor immune

56

responses.10-13 These medicinal )*

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krestin from Trametes versicolor and schizophyllan from Schizophyllum commune.14

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They all shared a common structure of a (1 / 3)-linked main chain substituted at O-6

59

by a single unit of )*2*

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branching.10 It has been found that the immunomodulatory effects of

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are closely correlated to their physical state including molecular weight, water

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solubility and degree of branching.15 It has been reported that different preparations of

63

)*

64

mechanism of their immunomodulatory action. A comparative study found that

65

although both soluble and particulate )*

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the particulate )*

(

include lentinan from Lentinus edodes,

with different molecular weight and degree of 0*)*2*

s, including the particulate and the soluble ones, could also influence the

could bind to the Dectin-1 receptor, only

was able to activate it.16 In another research, it was found that 3



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0*)*2*

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derived from Candida albicans showed higher stimulating ability in a

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particulate form than in a solubilized form.15 However, researches that focus on the

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underlying mechanism on how water-insoluble )*

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immune cells and changes their morphological properties are rare.

mediates the

activation of

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Polyporus rhinocerus is a well-known medicinal mushroom belonging to the

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Polyporacea family in China and Southeast Asia.17 Sclerotium is a compact aggregate

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of mycelia and is one of the developmental stages in the mushroom life cycle. The

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sclerotia of P. rhinocerus, which are rich in )*

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and prevention of various human diseases including gastric ulcer, chronic hepatitis, and

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cancer in Asia.17 Previous study found that )*

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weight of the sclerotia of P. rhinocerus.7 The potent bioactivity of alkali-soluble

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polysaccharide and water-soluble polysaccharide-protein complex (PPC) isolated from

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the sclerotia of P. rhinocerus have been compared in our previous studies.8 It was found

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that the water-soluble PPC which has a structure of a heteroglycan and protein was

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more immunopotent than the alkali-soluble homoglycan +)*

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vitro, which might be explained by their differences on water solubility and molecular

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conformation in an aqueous medium.8, 17-19 Alkali-soluble polysaccharides are usually

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aggregated in an aqueous medium and thus have fewer chances to contact with the

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immune cell surface, not to mention stimulating the cell responses.

have a long history in treatment

constituted for more than 70% dry

, both in vivo and in

In the present study, we aim at preparing an immunologically active microparticulte

86 87

)*

using an alkali-soluble glucan PRA-1 obtained from the sclerotia of P.

88

rhinocerus by employing the emulsification and cross-linking method. Accordingly, 4


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the primary structure of PRA-1 was elucidated by methylation analysis. The

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morphological properties of PRA-1 and PRA-1p were examined by Fourier transform

91

infrared spectroscopy (FTIR), transmission electron microscopy (TEM), scanning

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electron microscopy (SEM) and laser light scattering (LLS) spectroscopy. The

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immunostimulating action such as nitric oxide (NO) production, reactive oxygen

94

species (ROS) generation, phagocytic activity, cytokine profile and the related

95

molecular mechanisms were further studied. This work provides fundamental

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information of a novel microparticulate mushroom sclerotial )*

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rhinocerus which has potential for further biomedical applications.

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MATERIALS AND METHODS

from P.

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Chemicals and Antibodies. Analytical grade reagents including ethyl acetate and

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acetone were purchased from Duksan Pure Chemicals Co. (South Korea). Ethanol was

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purchased from Merck Millipore (USA). Lipopolysaccharides (LPS), Thiazolyl blue

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tetrazolium bromide (MTT) and FITC-labeled dextran were purchased from Sigma-

103

Aldrich (USA). The primary anti-mouse monoclonal antibodies and horseradish

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peroxidase (HRP) conjugated secondary antibody used in Western blot analysis was

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purchase from Cell Signaling Technology (USA).

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Preparation of PRA-1. The dried Mushroom sclerotia of P. rhinocerus, a

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commercial product originated from mainland China, were purchased from Chinese

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herbal store in Hong Kong. After the removal of the peel, the sclerotia were pulverized

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into powders to pass through a screen with an aperture of 0.5 mm by using a cyclotech

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mill (Tecator, $G

H

Sweden). Powder of sclerotia of P. rhinocerus were firstly 5



111

defatted in ethyl acetate (1 h, 3 times) and then acetone (1 h, 3 times) at 60 oC before

112

being extracted with boiling water. After removing the hot water-soluble extract by

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centrifugation (4000 g for 10 min), the water-insoluble residue was extracted with 1M

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NaOH at room temperature, the supernatant was neutralized to pH 7.0 with 0.5 M acetic

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acid and the precipitate (alkali-soluble polysaccharide fraction PRA-1) was obtained by

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centrifugation (4000 g for 10 min) and washed with distilled water to remove salt.

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Preparation of particulate PRA-1. The particulate PRA-1 (PRA-1p) was prepared

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using the native PRA-1 by emulsification and cross-linking method firstly described to

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form chitosan nanoparticles with some modifications.20 In brief, PRA-1 was firstly

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dissolved in DMSO at a concentration of 30 mg/mL. Then, 500 7! of the PRA-1

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solution was slowly added into 25 mL liquid paraffin containing 3% span 80 followed

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by 30 min sonication (Model VC 600 processor, Sonics & Materials Inc., Newton, USA)

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under a fixed frequency of 20 kHz and a power of 600W to obtain a stable dispersion.

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Afterwards, 0.5 mL of glutaraldehyde was added as a cross-linking agent to the

125

dispersion to harden the formed droplets, followed by 30 min sonication under a fixed

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frequency of 20 kHz and a power of 600W. The dispersion was stirred using a magnetic

127

stirrer for 3 h and then it was subjected to centrifugation at 1000 g for 15 min. The

128

precipitate was washed with petroleum ether followed by methanol and finally with

129

acetone for five times. PRA-1p obtained was then lyophilized and stored in the

130

refrigerator at 4 °C.

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Chemical Composition Analysis. The amount of the total carbohydrates in PRA-1

132

were determined by the phenol-sulfuric acid assay and the amount of the total proteins 6


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133

were determined by the BCA protein assay as described previously.21, 22 Content of total

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uronic acids in PRA-1 was analyzed colorimetrically using meta-hydroxydiphenyl-

135

sulfuric acid.23

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Monosaccharide composition analysis. PRA-1 sample was firstly acid hydrolyzed

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into monosaccharides followed by reduction and acetylation to yield the sugar

138

derivatives of alditol acetates according to the method described previously.24 The

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derivatives were separated and analyzed by gas chromatography according to the

140

method described previously.25 The column, the gas chromatograph (GC), the mass

141

spectrometry (MS) and the conditions used were the same as previously reported. 7

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Linkage Analysis by Methylation. The glycosidic linkages in PRA-1 was analyzed

143

by methylation as previously described with some modifications26 The column, oven

144

temperature, MS detector condition used were the same as previously reported.7 Each

145

partially methylated alditol acetates (PMAA) was identified according to the literature

146

database.27

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Transmission electron microscopy. PRA1 and PRA-1p were dissolved in distilled

148

water at a concentration of 5 mg/mL and were heated to 80 °C in water bath for 2 h

149

with constant stirring. The samples were prepared using holey carbon film (200 mesh,

150

Beijing Zhongjingkeyi Technology, China), which was supported by a copper grid, for

151

the analysis by transmission electron microscopy (TEM) (H-7650, Hitachi, Japan).

152

After filtration through a 0.22 7' nylon syringe filter, a droplet of the sample was

153

deposited on the specimen, which was finally dried in air at ambient temperature and

154

humidity. Molecular morphology of the prepared samples was performed on TEM at 7



155

an accelerating voltage of 80 kV.

156

Scanning electron microscopy. Droplets of PRA1 and PRA-1p samples (5 mg/mL)

157

were deposited on the silicon wafer and allowed to dry in air. The test samples were

158

sputtered with a gold layer and analyzed by SEM (Hitachi S-3400N) which was

159

performed at an accelerating voltage of 25 kV.

160

Size distribution. The size distribution of PRA-1p in aqueous solution (1 mg/mL)

161

was measured using laser light scattering spectroscopy with a Mastersizer equipment

162

(Malvern Panalytical, Malvern, UK).

163

Infrared spectrophotometric analysis. The infrared spectra of PRA-1 and PRA-1p

164

were recorded using a Fourier Transform Infrared Spectrometer (FTIR, Nicolet 670) in

165

the range of 4000-400 cm-1 using the KBr-disk method. Briefly, the PRA-1 and PRA-

166

1p samples were blended with KBr powder and pressed into transparent pellet for the

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FTIR measurement.

168

Nitrite determination. RAW 264.7 cells in 6-well plates (5×104 cell/mL) were

169

preincubated for 24 h. After 72-h stimulation with PRA1 (100 7 I'!, or PRA-1p (100

170

7 I'!, or LPS (100 ng/mL), the cell culture medium were collected. Nitrite oxide

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production in the culture medium was determined by the Greiss reagent system

172

(Promega) according to the manufacturer’s instructions.

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Phagocytosis of FITC-labeled dextrans. RAW 264.7 cells (5×104 cells/mL) were

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incubated with PRA1 (100 7 I'!, PRA-1p (100 7 I'!, and LPS (100 ng/mL)

175

individually. After 24 h, cells were collected and the live cell count was performed by

176

trypan blue dye exclusion assay. About 1x105 live cells were incubated in 1 mL medium 8


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containing FITC-labeled dextrans (1mg/mL) at 37 °C for 1 h. After washing with PBS

178

(3 times) to remove the extra FITC-labeled dextrans, cells were analyzed by a CXP 500

179

flow cytometry (Beckman Coulter, Miami, FL).

180

Reactive Oxygen Species (ROS) generation. The intercellular ROS generation in

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RAW 264.7 cells treated by PRA1 (100 7 I'!, PRA-1p (100 7 I'!, and LPS (100

182

ng/mL) individually was determined by 2’, 7’-Dichlorodihydrofluorescein diacetate

183

(DCFH-DA) assay. DCFH-DA that measures hydrogen peroxide is able to penetrate

184

through the cell membrane and is rapidly deacetylated and finally oxidized in the

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presence of the intracellular hydrogen peroxide to a highly fluorescent 2’, 7’-

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Dichlorodihydrofluorescein. Briefly, the treated RAW 264.7 cells were harvested using

187

trypsin and subjected to centrifugation (800 g for 5 min) to form a pellet. The cells

188

(1x105) then were incubated in DMEM medium containing DCFH-DA (10 7=, at

189

37 °C for 30 min. Then the cells were washed for three times using PBS and the

190

fluorescent signal was immediately measured by a fluorescence microscopy with the

191

excitation and emission wavelength set at 488 and 525 nm, respectively (Carl Zeiss

192

PALM inverted microscope).

193

Determination of Cytokine Profile. RAW 264.7 cells (5×104 cells/mL) were

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incubated with 100 7 I'! PRA-1p for 24h and then the cell culture medium were

195

collected. The RayBio® Mouse Cytokine Antibody Array II (RayBiotech) was used to

196

detect the relative levels of cytokines secreted by RAW 264.7 cells according to the

197

manufacturer’s instructions.

198

Western Blot Analysis. RAW 264.7 cells (105 cells/well) were plated in a 6-well 9



199

plate and incubated with PRA-1 (100 7 I'!, or PRA-1p (100 7 I'!, or LPS (100

200

ng/mL) for 24h. The protein in RAW 264.7 cells were extracted following the method

201

described previously with some modifications.7 The protein concentration was

202

determined using BCA reagent (Pierce). An equal amount of denatured proteins were

203

subjected to western blot analysis for iNOS, (*

204

AKT and -actin as described previously.7

*@A

*@A p-ERK, ERK, p-AKT,

205

Statistical Analysis. Results were represented as mean ± standard deviation (SD).

206

Experiments were performed in triplicate unless specified otherwise. Difference

207

between two groups was analyzed by two-tailed Student’s t test and P < 0.05 (*) or P

macrophage inflammatory protein-1>

32.39 ± 0.37

MIP-2

macrophage inflammatory protein-2

30.48 ± 0.08

MCP-1

monocyte chemoattractant protein-1

13.84 ± 0.20

641 642

#

The fold changes of the cytokines/chemokines are relative to the control.

643 644 645 646 647 648 649 650 35




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Structure and immunomodulatory activity of microparticulate

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