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Volume 7 Number 1 January 2016 Pages 1–612 This is an Accepted Manuscript, which has been through the

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 Page 1 of 30 Food & Function
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DOI: 10.1039/C7FO00244K

Isolation of a novel bioactive protein from an edible mushroom

Pleurotus eryngii and its anti-inflammatory potential
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Biao Yuana,b, Liyan Zhaoa, Kanyasiri Rakariyathamb, Yanhui Hanb, Zili

Food & Function Accepted Manuscript

Gaob, Benard Muinde Kimatua, Qiuhui Hua,∗, Hang Xiaob,*

a
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu
210095, China
b
Department of Food Science, University of Massachusetts, Amherst, MA 01002, USA

*Corresponding to:

Qiuhui Hu, College of Food Science and Technology, Nanjing Agricultural University,
Nanjing, Jiangsu 210095, China: Tel +86-25-84399086; email qiuhuihu@njau.edu.cn.

Hang Xiao, Department of Food Science, University of Massachusetts, Amherst, MA

01003: Tel +1 413 545 2281; email hangxiao@foodsci.umass.edu
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Graphical Abstract:
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DOI: 10.1039/C7FO00244K
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Page 2 of 30
 Page 3 of 30 Food & Function
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Abstract
Edible mushrooms are rich sources of bioactive components. In this study, a bioactive

protein, PEP, was isolated from an edible mushroom, Pleurotus Eryngii, through
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(NH4)2SO4 precipitation and ion-exchange chromatograph. Proteomic analysis by

Food & Function Accepted Manuscript

matrix assisted laser desorption ionization-time of flight mass spectrometry showed

that PEP was a novel protein with molecular weight of 40kDa. PEP exhibited

anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophages by inhibiting the

overproduction of pro-inflammatory mediators including nitric oxide (NO), cytokine

IL-1β and IL-6. It was further demonstrated that these anti-inflammatory effects of

PEP were associated with the inhibition of inducible nitric oxide synthase (iNOS)

expression, and the deactivation of nuclear factor-kappa B (NF-κB) and

mitogen-activated protein kinase (MAPK) pathways. Our results demonstrated that

PEP might be a good candidate for anti-inflammation in the gastrointestinal tract,

especially in the colon.

Keywords: Pleurotus eryngii, anti-inflammation, protein, nitric oxide, iNOS

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INTRODUCTION
Edible mushrooms such as Pleurotus eryngii are consumed worldwide. 1-3 Apart

from their flavor and taste, the fruiting bodies of edible mushrooms are considered a
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rich source of proteins, carbohydrates, fibers, vitamins, minerals and antioxidants. 1-3

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Mushrooms are also recognized as functional foods due to their bioactive components

with potential health-promoting effects. 4 Several studies have demonstrated the

anti-inflammatory activity of extracts from Pleurotus sp. fruiting bodies. 5, 6

It is well recognized that there is a strong association between inflammation and

various types of chronic diseases such as cancer. Inflammation is considered as a major

risk factor for the pathogenesis of multiple chronic diseases. Macrophages are

important immune cells regulating inflammation and host defense by secretion of

pro-inflammatory cytokines (e.g. IL-1β and IL-6), Chemokines, NO, and reactive

oxygen species (ROS). 7 However, excessive activation of macrophages may lead to

damaging effects, such as septic shock, which can lead to multiple organ dysfunction

syndrome and even death. 8 The persistence of pro-inflammatory activity may result in

the development of chronic inflammation, such as rheumatoid arthritis, psoriasis, and

inflammatory bowel diseases. 8 Thus, appropriate modulation of macrophage

activation is a good strategy to prevent undesirable inflammatory effects. Targeting

inflammation has been considered as a promising diet-based strategy for prevention of

inflammation-driven chronic diseases such as cancer.

Bioactive compounds isolated from various mushrooms species showed

promising inhibition activities against colon, breast, lung and liver cancers. 9, 10 Recent
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studies have indicated that polysaccharides, peptides and proteins from mushroom had

potentials in anti-inflammation, immunomodulation, and cancer inhibition. 11-13 Our

previous studies have identified polysaccharides and proteins from Pleurotus eryngii
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with immune-modulatory and anti-cancer effects. 14, 15 In the present study, a novel 40

Food & Function Accepted Manuscript

kDa protein, designated as PEP, was isolated from the aqueous extract of P. eryngii for

the first time. We further characterized the anti-inflammatory potential of this novel

protein in cell culture models.

METHODS

Reagents and antibodies

RPMI-1640 medium and fetal bovine serums (FBS) were purchased from GIBCO

(Grand Island, NY, USA). DEAE Sepherose fast flow ion exchange resin was

purchased from GE healthcare (Uppsala, Sweden). Dimethyl Sulphoxide (DMSO),

3-(4,5-dimethyl2-thizolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained

from Sigma-Aldrich (St. Louis, MO, USA). The antibodies used in this study for

inducible nitric oxide synthase (iNOS) (#2982), cyclooxygenase-2 (COX-2) (#4842),

phospho-IκB alpha (p-IκBα) (#2859), phospho-Jun N terminal kinase (p-JNK) (#9255),

JNK (#9252), phospho-p38 MAPK (p-p38) (#4511), p38 (#8690), phospho-p44/42

MAPK (p-ERK1/2) (#4370), ERK1/2 (#9194) were obtained from Cell Signaling

Technology (Beverly, MA, USA). Antibodies for phospho-NF-kappaB p65 (p-NF-κB)

(sc-136548), p65 (sc-56735) were obtained from Santa Cruz (Santa Cruz, CA, USA).

Anti β-actin antibodies (A5441) were obtained from Sigma-Aldrich. Purification of

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mushroom protein PEP

The protein from Pleurotus eryngii (PEP) was purified according to the

previously reported method with modifications. 16 One hundred gram of freeze-dried

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powdered fruiting bodies of P. eryngii was incubated with 2,000 mL of ice-cold 5%

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acetic acid (including 50 mM β-mercaptoethanol) overnight. The soluble proteins in the

supernatant were precipitated by the addition of ammonium sulfate to 60-80%

saturation, and collected by centrifugation (14,000 g, 20 minutes, 4 oC: Thermo

Scientific, Waltham, USA). The precipitate was dialyzed against 10 mM Tris/HCl (pH

8.0) solutions at 4 oC for 48 hours during which dialysis solution was changed every 12

hours. The dialysate was applied to a DEAE-Sepharose fast flow column that was

previously equilibrated with 10 mM Tris/HCl (pH 8.0). The column was washed with

200 mL equilibration buffer and then eluted with increasing concentrations of NaCl

solution. The protein fractions collected were subjected to anti-inflammation assay in

lipopolysaccharides (LPS)-treated RAW264.7 macrophages as described below. The

fraction with the highest bioactivity as a single peak at 280 nm absorbance was pooled.

The purity of the protein was analyzed by sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE) and visualized by Coomassie blue staining. The purified

protein was freeze dried and stored at -20 oC for further analysis.

Determination of amino acid sequence

The protein band was excised from the polyacrylamide gel and the gel slice was

cut into pieces. After de-staining, the gel pieces were dried under vacuum and digested

with sequencing grade trypsin overnight. The proteolytic sample was subject to
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matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass

spectrometry (MS). Peptide mass fingerprints were sequenced using the Joint Genome

Institution (JPI) MycoCosm database (The fungal genomics resource)

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(http://genome.jgi.doe.gov/).

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Cell culture

Macrophage RAW 264.7 was obtained from the American Type Culture

Collection (ATCC, Rockville, MD, USA). RAW 264.7 macrophage cells were

maintained in RMPI-1640 media supplemented with 10% heat-inactivated fetal bovine

serum (FBS). All media were supplemented with penicillin (100 units mL-1),

streptomycin (100 mg/mL), L-glutamine (4.5 mg/mL) and glucose (4.5 mg/mL),

followed by incubation at 37 oC in a humidified atmosphere containing 5% CO2 and 95%

air.

Determination of nitric oxide

RAW 264.7 cells were seeded in 96-well plate (at the density of 50 × 104 cells

mL-1 in 200 µL medium in each well). After 24 hours of incubation, these cells were

treated with lipopolysaccharide (LPS) (1 µg/mL) together with different concentrations

of the P. eryngii protein PEP for 24 hours. Aliquot of cell culture medium (150 µL) was

mixed with 100 µL of cold Griess reagent (2% sulfanilamide and 0.2%

naphthylethylenediamine dihydrochloride in 3% (v/v) phosphoric acid) and incubated

at room temperature for 30 minutes. The absorbance at 540 nm was read in a microplate

reader (Elx800TM absorbance microplate reader, BioTek Instrument, Winooski, VT,

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USA). A cell-free solution was used as the blank. Sodium nitrite concentration ranging

from 0 to 100 µM was used as the standard as we previously reported. 17

Quantification of cytokines
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RAW 264.7 cells (5 × 106 cells well-1) were seeded in 6-well plates. After 24 hours,

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cells were treated with 1 µg/mL LPS alone or with serial concentration of PEP in 2.5

mL of complete culture medium. After another 24 hours of incubation, the culture

medium was collected for the determination of the levels of IL-1β and IL-6 by

commercial ELISA kits as we previously described, 18, 19 according to the

manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Quantification of cellular ROS

RAW 264.7 cells were seeded in fluorescence micro titer 96-well black plates (50

× 104 cells mL-1 in 200 µL of complete medium) and incubated in a humidified

incubator containing 5% CO2 and 95% air at 37 oC for 24 hours. Culture medium was

then replaced with medium containing PEP and incubated for 24 hours. After the

removal of the culture medium, the cells were washed with phosphate-buffered saline

(PBS) (pH 7.4). Subsequently, 100 µL of 10 µM 2ʹ,7ʹ-dichlorofluorescin-diacetate

(DCFH-DA) in PBS (pH 7.4) was added and the cells were incubated in the dark for 30

minutes, and then 100 µL of 0.3mM t-BHP in PBS (pH 7.4) was added to the cells.

During the incubation of 1 hour, DCFH was oxidized by intracellular reactive oxygen

species (ROS) to form 2’,7’-dichlorofluorescin (DCF), a fluorescent product, which

was monitored at the excitation wavelength (Ex) of 485 nm and the emission
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wavelength (Em) of 528 nm using a fluorescence microplate reader (Elx800TM

absorbance microplate reader, BioTek Instrument, Winooski, VT, USA).

Western blot analysis

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Whole cell lysates were prepared as previously described. 20-22 RAW264.7

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macrophages were treated with the P. eryngii protein PEP for specific time periods. For

whole-cell lysate preparation, the cells were washed with cold PBS and lysed in RIPA

buffer (Tris-HCl pH 7.2, 25 mM; SDS 0.1%; Triton X-100 1%, sodium deoxycholate

1%; NaCl 0.15M; ethylenediaminetetraacetic acid (EDTA) 1 mM) (Boston

Bioproducts, Ashland, MA, USA) containing protease inhibitor (inhibitor of proteinase,

phosphatase inhibitor I and phosphatase inhibitor II). For western blot analysis, an

equal amount of cellular protein was separated on 8% sodium dodecyl

sulfate-polyacrylamide gels (SDS-PAGE) by electrophoresis. Proteins in the gels were

transferred onto a nitrocellulose membrane. The membrane was then blocked with the

blocking buffer from Odyssey and sequentially incubated with primary antibodies and

fluorescent dye-labeled secondary antibodies before imaging with the Odyssey CLx

infrared imaging system (Li-Cor Bioscence, Nebraska, USA).

Statistical Analysis

Data were presented as Means ± Standard Deviation (SD) for the indicated

number of independently performed experiments. One-way factorial analysis of

variance (ANOVA) (more than two groups) or Student's t-test (two groups) was used to

assess the mean difference between treatment groups and the control group. A p-value
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of < 0.05 was considered statistically significant.

RESULTS

Isolation of Mushroom Protein PEP

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Mushrooms have been reported to contain many bioactive components including

Food & Function Accepted Manuscript

proteins.9 In this study, we isolated PEP from an edible mushroom Pleurotus eryngii

as depicted in Figure 1a. The water extractable proteins were precipitated by

(NH4)2SO4 (60-80%), and then subjected to the DEAE Sepherose Fast Flow

chromatography. Four peaks (DE1, DE2, DE3, and DE4) were eluted from the

chromatography (Fig. 1c). Based on the anti-inflammation assay in LPS-stimulated

macrophages, protein from peak DE3 showed the strongest anti-inflammatory potential

in comparison with proteins from other peaks (data not shown). The homogeneity of

the purified protein from peak DE3 was confirmed by SDS-PAGE, which showed that

the protein appeared as a single band with a molecular weight about 40 kDa (Fig. 1b).

The purified protein from peak DE3 was named PEP. The purity of PEP was greater

than 95% based on SDS-PAGE (as estimated by Coomassie blue staining). The

abundance of PEP in the mushroom was about 35.0 ± 0.7 mg PEP per 10 g mushroom

powder determined by the bicinchoninic acid assay.

To identify the protein, PEP band from SDS-PAGE gel was excised, digested by

trypsin, and characterized by peptide mass fingerprinting based on the matrix-assisted

laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS).

From the Joint Genome Institution MycoCosm database (The fungal genomics

resource) (http://genome.jgi.doe.gov/), no homologous proteins of PEP could be

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identified. However, the peptide fingerprinting of PEP showed considerable similarity

with that of protein Pleery1 (Protein ID: 1310667,

http://genome.jgi.doe.gov/cgi-bin/dispGeneModel?db=Pleery1&id=1310667). The
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score of mascot search matching was above 97 and the sequence coverage was 58%

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(Fig. 2). The highlighted amino acids represented the amino acids sequences shared

between PEP and Pleery1. Given the fact that Pleery1 (48 kDa) and PEP (40 kDa)

have different molecular weight, we concluded that PEP could not be identified as

Pleery1, and therefore PEP was considered as a novel protein purified from Pleurotus

eryngii.

PEP inhibited LPS-stimulated inflammatory response in macrophages.

To establish the anti-inflammatory potential of PEP, PEP was subjected to

LPS-treated macrophages to determine its effect on LPS-stimulated inflammatory

response. The results showed that PEP at the concentrations up to 200 µg/mL did not

produce significant inhibitory effects (<10%) on the growth of the macrophages (p <

0.05). Using the concentration range established, the effects of PEP on LPS-induced

production of inflammatory mediators including NO, IL-1β and IL-6 were examined.

As expected, treatment with 1 µg/mL LPS caused a significant increase in NO and

pro-inflammatory cytokine production of the macrophages (p < 0.05). The production

of NO was increased by LPS treatment by 6-fold compared to the negative control

cells. The treatment with PEP decreased the levels of NO production in LPS-stimulated

macrophages in a dose-independent manner (Fig. 3b). For example, PEP at 50, 100, and

200 µg/mL led to 16.1, 58.1, and 83.2% inhibition on NO production. LPS
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significantly up-regulated the protein expression of iNOS, which was responsible for

the increased NO production in LPS-treated macrophages (Fig. 3c) (p < 0.05). PEP

caused a dose-dependent decrease on the protein expression level of iNOS in

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LPS-treated macrophages, i.e. PEP at 50 µg/mL led to 52.6 % decrease on iNOS

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expression while PEP at 100 and 200 µg/mL abolished the iNOS expression. Similarly,

LPS stimulated the production of pro-inflammatory cytokines such as IL-1β and IL-6,

and PEP treatment caused dose-dependent inhibition on these two cytokines.

Specifically, PEP at 50, 100, and 200 µg/mL caused 17.8, 31.5, and 65.8% inhibition

on IL-1β, and 41.2, 55.1, and 76.3% inhibition on IL-6, respectively (Fig 4a, 4b).

PEP inhibited t-BHP-induced ROS production in macrophages.

Excessive exposure to reactive oxygen species (ROS) causes oxidative stress,

which may lead to damage to essential cellular macromolecules such as proteins,

lipids and DNA, increasing the risk of several human diseases such as cancer,

inflammatory and neurodegenerative diseases25. As shown in Figure 4, t-BHP

increased the accumulation of intra-cellular ROS in macrophages by 5-fold in

comparison with the negative control cells, whereas pre-treatment with PEP resulted in

a dose-dependent reduction of ROS accumulation in macrophages. PEP at 50, 100 and

200 µg/mL decreased the intracellular ROS accumulation in macrophages by 28.4,

38.4 and 70.1%, respectively (Fig. 4c).

κB and MAPK pathways in LPS-treated

PEP inhibited activation of NF-κ

macrophages
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NF-κB is an essential transcription factor involved in the production of

pro-inflammatory cellular mediators. The effect of PEP on NF-κB activation was

examined by western blotting analysis. As shown in the Figure 5, an increase in the

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levels of p-IκB and p-NF-κB p65 was observed after stimulation with LPS, suggesting

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that NF-κB signaling was activated by LPS treatment in macrophages. PEP treatment

caused a dramatic inhibition on the activation of NF-κB signaling, i.e., PEP at 50

µg/mL decreased the levels of p-IκB and p-NF-κB p65 by 75.4 and 64.6% in

LPS-stimulated macrophages, respectively. Moreover, PEP at 100 and 200 µg/mL

abolished LPS-induced increase in the levels of p-IκB and p-NF-κB p65.

The MAPK, including ERK, JNK, and p38, signaling pathways play an important

role in regulating the synthesis and secretion of inflammatory mediators in

LPS-stimulated macrophages.26 The activated MAPKs by LPS were found to be

involved in the over production of NO and pro-inflammatory cytokines.27 Since

phosphorylation is essential in the activation of MAPKs, the effect of PEP on the

phosphorylation of ERK, JNK and p38 MAPK was examined. As displayed in Figure 6,

the expression levels of phosphorylated ERK, JNK and p38 MAPK were significantly

increased by LPS stimulation, indicating the activation of these MAPKs by LPS (p <

0.05). PEP treatments inhibited the LPS-induced phosphorylation of ERK, JNK and

p38 in macrophages. However, the inhibitory effects of PEP on activation of MAPKs

were not as strong as that observed on the activation of NF-κB pathway.

Discussion

Edible mushrooms have been considered as a rich resource of bioactive

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components.10, 29 While polysaccharides from mushrooms have been frequently studied,

only a few studies have been focused on bioactive proteins from mushroom. 30 Several

bioactive proteins from edible mushroom have recently been purified. A protein
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(LEP91-3A2) was isolated from Lentinula edodes liquid mycelia culture supernatant,

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and showed antitumor effect against several kinds of cancerous cells. 29 A family of

fungal proteins (FIP-vvo, FIP-gts, FIP-fve et.al) have been identified from Volvariella

volvacea, Ganoderma tsugae, Flammulina velutipes, and they have been

demonstrated to exert anti-tumor and immune-modulatory properties. 11, 16, 31 In the

present study, using step precipitation by (NH4)2SO4 and ion-exchange chromatography,

for the first time, we successfully purified a novel protein, PEP, from an edible

mushroom Pleurotus eryngii. To establish the potential bioactivity of this novel protein,

we conducted a series of experiments to determine its anti-inflammatory potential.

Macrophages are extraordinarily versatile cells and play a critical role in the

host’s defense against bacterial infection because of their phagocytic, cytotoxic and

intracellular killing ability. 32, 33 However, the overproduction of inflammatory

mediators by macrophage can cause severe damage to the host, such as septic shock

and autoimmune diseases. Macrophages can be activated by various signals to

stimulate many intracellular cascades involving the cytokines and chemokines.

Consistent with the literatures, our results demonstrated that LPS induced macrophage

activation and increased the production of inflammatory mediators and cytokines,

including NO, IL-6 and IL-1β. The pro-inflammatory cytokines IL-1β and IL-6 are

largely produced under inflammatory conditions and function as messengers that

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stimulate the inflammatory process. Nitric oxide (NO) has been considered as a

pro-inflammatory mediator that induces the inflammation due to its over-production

in abnormal situations. 34 IL-1β is a pro-inflammatory cytokine that has also been

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implicated in inflammation conditions. 35 IL-6 is strongly involved in experimentally

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induced autoimmune diseases, and increased level of IL-6 was observed in

rheumatoid arthritis and other chronic inflammation disease. 36 Thus, the

identification of anti-inflammatory substances that can modulate the production of

aforementioned pro-inflammatory mediators could be an efficient way to manage

inflammatory conditions. Previous study showed that extracts from several kinds of

edible mushroom had potential anti-inflammatory activities through the inhibition of

NO production and mRNA expression of IL-1β and IL-6. 37 Our results demonstrated

that PEP from edible mushroom effectively inhibited LPS-induced overproduction of

NO, IL-1β and IL-6 in macrophages, which suggesting the potential of PEP as an

anti-inflammatory agent.

LPS is well known to activate two downstream pathways: the MyD88 and

TRIF-dependent pathways. LPS activates NF-κB and MAPK signaling through a

MyD88-dependent pathway, which leads to increased production of inflammatory

cytokines. 38 In the MyD88 pathway, the phosphorylated MAPKs act as transcriptional

activators of AP-1 expression. Numerous studies have demonstrated that natural

compounds suppress the inflammatory response by inhibiting the production of

pro-inflammatory mediators.19, 31 Our results demonstrated that PEP has an inhibitory

effect on the protein expression of iNOS. Furthermore, PEP showed a strong inhibition
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on the activation of NF-κB by decreasing the levels of phosphorylation of IκB-α and

p65. PEP showed moderate inhibition on the activation of three major MAPKs (EKR,

JNK, and p38). This suggested that inhibition of LPS-induced inflammation by PEP
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was mainly through the inhibition of iNOS and activation of NF-κB pathway, and the

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inhibition of MAPK pathway might play a less important role. LPS induced

inflammation in macrophages through its interaction with toll-like receptors (TLR).

Several studies have demonstrated that the involvement of TLR in recognizing

bioactive proteins that displayed various biological activities via TLR-mediated signal

pathway. 39, 40 For example, an immune-modulatory protein, PCP from Poria Cocos

was found to function as an immune stimulator through TLR4 in peritoneal

macrophages. 40 Future investigation is warranted to determine the potential role of

PEP in interacting with TLR in terms of inhibition of TLR-mediated inflammation

response.

As macromolecules, most orally ingested protein and peptide-based bioactive

food components are not able to enter systemic circulation intact. Therefore, the

gastrointestinal tract might be their suitable target tissues for biological functions, e.g.

by direct interaction with intestinal epithelial cells. A challenge for protein and

peptide-based bioactive food components is to maintain their bioactivities in the

gastrointestinal tract where they might be altered and degraded by digestive enzymes.

45
The colon has attracted attention as a potential target site for protein or

peptide-based bioactive agents because of its lower proteolytic activity compared to

the upper gastrointestinal tract. 46, 47 Recently, several colon specific delivery systems
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have been developed to transport bioactive proteins or peptides to the colon, such as

coating with pH-dependent polymers, design of timed-release dosage forms, and the

utilization of carriers degrades exclusively by colonic bacteria. 45, 47, 48 Based on our
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results, PEP might be a good candidate for delivery to the colon for its

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anti-inflammatory effects.

Conclusion

In this study, a 40 kDa protein, PEP was purified from Pleurotus eryngii

mushroom, and its anti-inflammatory effects on LPS-stimulated macrophages were

determined. Our results showed that PEP inhibited LPS-induced overproduction of

pro-inflammatory mediators mainly through inhibition of iNOS expression and NF-κB

activation. Our results suggested that PEP might be a good candidate for colon-specific

delivery to maximize its beneficial effects.

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Conflict of interest.

The authors declare that there are no conflicts of interest.

Acknowledgements
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This work was partially supported by the National Natural Science Foundation

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of China under Grant No. 31471927, and China Agriculture Research System

(CARS-24), and the Priority Academic Program Development of Jiangsu Higher

Education Institutions (PAPD).

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FIGURE LEGENDS

Figure 1. (a) The purification procedure of Pleurotus eryngii protein PEP (b) SDS-PAGE analysis

of purified target protein, left lane is the standard marker, right lane is the protein PEP (c) Elution
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profile of P. eryngii protein PEP on DEAE-Sepherose Fast Flow cellulose chromatograph. The

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slanting dotted line across the chromatograph indicated linear 0-0.5 M NaCl concentration

gradient in Tris/HCl buffer (pH 8.0) used to elute fractions DE1, DE2, DE3 and DE4 at a Flow

rate, 1.0 mL min-1 and fraction size is 10 mL.

Figure 2. The amino acid sequence of the potential PEP. The highlighted amino acids represent

the shared amino acids between PEP and the protein Pleery1.

Figure 3. (a) Cytotoxic effect of PEP on RAW 264.7 cells. The viability of control cells was set as

the reference with a value of 100%. Results were presented as mean ± standard deviation (SD)

from six replicates. No significant different was found between any treatment and the control

group. (b) Effect of PEP on LPS-stimulated NO production in RAW 264.7 macrophages. Results

were presented as mean ± SD from six replicates. (c) Inhibitory effects of PEP on LPS-induced

protein of iNOS in RAW264.7 macrophages.

Figure 4. (a) Effect of PEP on LPS-induced production of IL-1β. (b) Effect of PEP on

LPS-induced production of IL-6. (c) Effect of PEP on t-BHP induced production of ROS in RAW

264.7 macrophages. * p < 0.05, ** p < 0.01 indicated statistically significantly differences from

LPS-treated positive control group.

Figure 5. Inhibitory effects of PEP on LPS-induced NF-κB activation in RAW 264.7 macrophages.

The numbers underneath the blots represent band intensity that was normalized to β-actin and

measured by Image J software (means of three independent replicates). The standard deviations
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DOI: 10.1039/C7FO00244K

were all within 15% of the means.

Figure 6. Inhibitory effect of PEP on the phosphorylation of p38, JNK, ERK in RAW 264.7

macrophages. The numbers underneath the blots represent band intensity that was normalized to
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β-actin and measured by Image J software (means of three independent replicates). The standard

Food & Function Accepted Manuscript

deviations were all within 15% of the means.
 Page 21 of 30 Food & Function
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DOI: 10.1039/C7FO00244K

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Food & Function Accepted Manuscript

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Page 25 of 30

a)

b)

25kDa
30kDa
46kDa
58kDa
80kDa
175kDa
Figure 1.

Marker PEP
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c)
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Figure 2.
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Page 26 of 30
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Figure 3.
a) b)
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Food & Function Accepted Manuscript

c)
1.0 393.6 186.4* 34.9* 0.5*

iNOS 130 kDa

β-actin 45 kDa

PEP (µg/mL) - - 50 100 200

LPS - + + + +
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c）
a)
Figure 4.
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b)
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Page 28 of 30
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Figure 5.

1.0 75.6 18.6 * 5.0* 1.1*

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p-IκB 40 kDa

Food & Function Accepted Manuscript

1.0 14.7 5.2 * 1.7* 0.1*

p-NF-κB p65 65 kDa

β-actin 45 kDa

PEP (µg/mL) - - 50 100 200

LPS - + + + +
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Figure 6.

1.0 4.0 3.8 2.0* 2.7*

43 kDa
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p-p38

Food & Function Accepted Manuscript

p38 43 kDa

1.0 4.9 3.6 3.0* 3.3*

p-ERK 42/44 kDa

ERK 42/44 kDa

1.0 2.6 2.0 2.3 1.7*

p-JNK 46/54 kDa

JNK 46/54 kDa

β-actin 45 kDa

PEP (µg/mL) - - 50 100 200

LPS - + + + +