Identification of New Anti-inflammatory Peptides from Zein Hydrolysate

Subscriber access provided by RMIT University Library

Article

Identification of New Anti-inflammatory Peptides from Zein Hydrolysate after Simulated Gastrointestinal Digestion and Transport in Caco-2 Cells Qiufang Liang, Meram Chalamaiah, Xiaofeng Ren, Haile Ma, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04562 • Publication Date (Web): 01 Dec 2017 Downloaded from http://pubs.acs.org on December 1, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

Identification of New Anti-inflammatory Peptides from Zein Hydrolysate after Simulated Gastrointestinal Digestion and Transport in Caco-2 Cells Qiufang Lianga,b, Meram Chalamaiahb, Xiaofeng Rena,b, Haile Maa, Jianping Wub

a

School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road,

Zhenjiang, Jiangsu 212013, China b

Department of Agricultural, Food and Nutritional Science (AFNS), 4-10 Ag/For

Centre, University of Alberta, Edmonton, Alberta, Canada

Corresponding Author Dr. Jianping Wu E-mail: [email protected]; Fax: +1 780 492 4265; Tel: +1 780 492 6885

1

ACS Paragon Plus Environment


1

Page 2 of 32

ABSTRACT

2

Chronic inflammation is an underlying contributor to various chronic diseases.

3

The objectives of this study were to investigate the anti-inflammatory activity of zein

4

hydrolysate after simulated gastrointestinal digestion and Caco-2 cell absorption, and

5

to identify novel anti-inflammatory peptides after transport across Caco-2 cells. Three

6

zein hydrolysates were prepared and further digested using gastrointestinal proteases;

7

their transports were studied in Caco-2 cells. Anti-inflammatory activity was studied

8

in endothelial EA.hy926 cells. Three zein hydrolysates and their digests significantly

9

decreased

the

expression

of

tumor

necrosis

factor-α

(TNF-α)

induced

10

pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) by 37.3-66.0%.

11

Eleven novel peptides with 5-9 amino acid residues were sequenced; three peptides

12

showed strong anti-inflammatory activity by inhibiting the VCAM-1 by 54-38.9% and

13

intercellular cell adhesion molecule-1 (ICAM-1) by 36.5-28.6% at 0.2 mM. A new

14

approach to identify novel anti-inflammatory peptides that could survive

15

gastrointestinal digestion and absorption is developed.

16

KEYWORDS: zein hydrolysate, bioactivity, simulated gastrointestinal digestion,

17

peptide bioavailability

18 19 20 21 22 2


Page 3 of 32


23

INTRODUCTION

24

Maize is the third most widely cultivated cereal in the world. Zein, a byproduct

25

of corn starch processing, is the main storage protein accounting for ~ 50% of total

26

endosperm protein.1 Zein is often used as a coating material, a drug carrier, or in

27

tissue engineering;2 zein is also claimed as a good source of bioactive peptides. Zein

28

derived bioactive peptides were shown to possess antioxidant,3 angiotensin converting

29

enzyme (ACE) inhibitory,4 antihepatotoxic,5 anti-cancer, immunomodulatory6

30

activities, and facilitating alcohol metabolism.7 The anti-inflammatory activity of a

31

corn gluten hydrolysate was suggested in a rat model of experimental colitis,8

32

although characterization of the responsible peptides has not been reported. Bioactive

33

peptides released from food proteins have great potential as functional

34

food/nutraceutical ingredients for improving human health.

35

Endothelial cells, lining the inner layer of blood vessels, play a vital role in

36

vascular biology, such as regulation of blood vessel tone, hemostasis, neutrophil

37

recruitment, hormone trafficking, and fluid filtration.9 Vascular inflammation is a key

38

factor that contributes to endothelial dysfunction and has been associated with a

39

variety of disease states including atherosclerosis, diabetes, coronary artery disease,

40

hypertension

41

pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α) phosphorylates nuclear

42

factor κappa B (NF-kB), which leads to increased expression of intercellular adhesion

43

molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the

44

release of monocyte chemoattractant protein-1 (MCP-1) in endothelial cells.11 These

and

hypercholesterolemia.10

Under

3


inflammatory

conditions,


45

adhesion molecules then recruit leukocytes (monocytes and macrophages) to the site

46

of inflammation and enhance the plaque formation between endothelial cells and

47

vascular smooth muscle cells that eventually contributes to increased blood pressure

48

and initiation of atherosclerosis.10, 12 Therefore, targeting inflammation of endothelial

49

cells is a strategy to reduce endothelial cells’ inflammation thus improve endothelial

50

function. A number of food bioactive components were reported to exert

51

anti-inflammatory activity. Recently, milk-derived hydrolysates have been reported to

52

exhibit anti-inflammatory activity in endothelial cells by down regulation of

53

expression of VCAM-1, ICAM-1 and E-selectin.13 Furthermore, IRW, IQW, GWNI

54

and GW peptides isolated from egg ovotransferrin, and VPP peptide derived from

55

casein showed anti-inflammatory effects in endothelial cells.14-16

56

Bioactive peptides composed of amino acids are susceptible to proteases that

57

present in the gastrointestinal tract; some peptides render inactive while more potent

58

peptides may also be released after the proteolytic digestion.17 Therefore it is vital to

59

study the stability of bioactive peptides in the gastrointestinal tract. As the ethical

60

regulation on animal studies have become more stringent, simulated gastrointestinal

61

digestion, which is considered to be simple, rapid, and inexpensive, has been widely

62

used for assessment of the stability of a wide range of food derived bioactive peptides

63

such as ham peptides,18 loach protein hydrolysates,19 etc. As the absorption of

64

peptides mainly occurs through the epithelium of the small intestine after

65

gastrointestinal digestion, another key factor affecting the bioactivity of bioactive

66

peptides is their transport through the gut epithelium.20 Caco-2 cell line, a human 4


Page 4 of 32

Page 5 of 32


67

intestinal epithelial cell model derived from a colon carcinoma, is a well-established

68

model of intestinal absorption in vitro.21 Recently, Caco-2 monolayers have been

69

successfully used to study the transport of some purified peptides and protein

70

hydrolysates.22-23 Since studies related to anti-inflammatory effect of zein derived

71

peptides are scanty, the objectives of this study were to investigate the

72

anti-inflammatory activity of zein hydrolysate after simulated gastrointestinal

73

digestion and Caco-2 cell absorption, and to identify novel anti-inflammatory peptides

74

after transport across Caco-2 cells.

75

MATERIALS AND METHODS

76

Chemicals

77

Neutral protease (from bacteria Bacillus subtilis) was obtained from BIA-CAT

78

(Troy, NY, USA). Zein, Alcalase® 2.4L (from Bacillus licheniformis, Subtilisin A),

79

thermolysin (from Geobacillus stearothermophilus) were bought from Sigma

80

Chemical Co. (St.Louis, MO, USA). Recombinant human TNF-α was obtained from

81

R&D System (Minneapolis, MN, USA). Fetal bovine serum (FBS) and Dulbecco’s

82

modified eagle medium (DMEM) were bought from Gibco/Invitrogen (Carlsbad, CA,

83

U.S.A.). The antibodies of VCAM-1 and ICAM-1 were purchased from Santa Cruz

84

Biotechnologies (Santa Cruz, CA, USA). α-Tubulin antibody (rabbit polyclonal

85

antibody) was purchased from Abcam (Cambridge, MA, USA). Goat anti-rabbit and

86

donkey anti-mouse fluorochrome-conjugated secondary antibodies were obtained

87

from Licor (Licor Biosciences, Lincoln, NE, USA). All other chemicals and reagents

88

used were analytical grade. 5



89

Enzymatic hydrolysis of zein

90

Zein powder was dissolved in distilled water to obtain 50 mg/mL protein

91

concentration. The pH and the temperature of enzymes (Alcalase, neutral protease,

92

and thermolysin) were adjusted according to the manufacturer’s recommendations

93

(Table 1). The enzymes were added at a ratio of 50:1 (w/w, zein to protease) to start

94

the hydrolysis. The hydrolysis was performed in a jacketed beaker connected to a

95

circulating water bath for maintaining constant temperature and Titrando (Metrohm,

96

Herisan, Switzerland) for maintaining constant pH with 0.5 M NaOH. After 3 h of

97

hydrolysis, the mixture was kept in boiling water for 10 min and centrifuged at

98

10,000g for 15 min. The supernatant was concentrated and freeze-dried for further

99

use.

100

Degree of hydrolysis (DH) and yield

101

DH of zein was measured by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)

102

method.24-25 Zein was completely hydrolyzed using 6 M HCl at 115 °C for 24 h to

103

determine the total amino group. The nitrogen content was determined according to

104

the modified Kjeldahl method using LECO Truspec total CN analyser (LECO, USA).

105

The yield was calculated by the equation: % =

106

ℎ ℎ × 100

ℎ

Simulated gastrointestinal digestion

107

Zein and zein derived hydrolysates were subjected to simulated gastric and

108

intestinal digestion based on the previous method.26 Simulated gastric and intestinal

109

fluids were prepared according to the U.S. Pharmacopeia.27 Briefly, zein and three 6


Page 6 of 32

Page 7 of 32


110

zein hydrolysates were digested with gastric fluid at 1:20 (w/v) for 4 h in a shaking

111

incubator (at 150 rpm) at 37 °C. Then pH was adjusted to 6.8 and pancreatin was

112

added to form the intestinal fluid. The mixture was incubated for a further 6 h to

113

mimic the intestinal digestion. The digestion was terminated by keeping the digests in

114

boiling water for 10 min. The digests were allowed to cool down and centrifuged at

115

10,000 g for 10 min to collect the supernatant. The control (using water instead of

116

samples) was performed under the same conditions and used to correct interference

117

from the simulated digestive juices.

118

Cell culture

119

The endothelial cell line, EA.hy926 (CRL-2922TM), and human colon

120

adenocarcinoma cell line, Caco-2 (HTB-37TM), were purchased from American-type

121

culture collection (ATCC, Manassas, VA, USA). DMEM supplemented with 10%

122

FBS, 2.5% HEPES, 1% antibiotics and 1% non-essential amino acids was used as cell

123

growth medium. The medium was replaced each other day, and the cells were

124

subcultured using 0.25% trypsin-EDTA treatment. The cells were incubated in a

125

humidified atmosphere with 5% CO2 at 37 °C.

126

Measurement of cytotoxicity

127

The cell cytotoxic properties were monitored using an Alamar Blue assay

128

described by Huang et al.28 Caco-2 cells were seeded in 96-well plates at a density of

129

1×104 cell/well for 24 h. Then the cells were treated with various concentrations

130

(10-50 mg/mL) of zein hydrolysate for another 24 h in a fresh medium. After 24 h

131

treatment, the media was discarded, and the fresh medium with 10% Alamar Blue 7



132

reagent was added and incubated for 4 h at 37 °C. The fluorescence intensity of the

133

wells was measured at an emission wavelength of 590 nm and an excitation

134

wavelength of 560 nm. The viability of the treated cell was expressed as the

135

percentage as compared to untreated cells.

136

Transport studies

137

The transport experiments were performed according to the modified method

138

of Bejjani et al.29 Caco-2 cells were grown in 12-well Transwell® plates (0.4 µm pore

139

size, 12 mm diameter, 1.12 cm2 grown surface, Corning Costar Corporation,

140

MA, USA) at a concentration of 1×105 cells/cm2. The Caco-2 monolayers that showed

141

transepithelial electrical resistance values > 300 Ω/cm2 could be used for the

142

experiment. Before initiation of transport experiments, the culture medium of the

143

Caco-2 cells was replaced by HBSS buffer and the Caco-2 cells were pre-incubated

144

for 0.5 h at 37 °C. Transport of zein hydrolysate digests was performed by adding the

145

digests at 20 mg/mL (dissolved in HBSS buffer) to the apical (AP) surface. AP surface

146

samples at 0 h and basal surface (BL) samples at 0.5, 1, 2, 4 h were collected for

147

further analysis.

148

UPLC analysis

149

The samples collected from the AP and BL surfaces were analyzed using an

150

Acquity Ultra-Performance Liquid Chromatograph (UPLC) system. 15 µL sample

151

was loaded on an Acquity UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm,

152

Waters, Milford, MA, USA). Mobile phases were solvent A (0.1% trifluoroacetic acid

153

in Milli-Q water) and solvent B (0.1% trifluoroacetic acid in acetonitrile). The 8


Page 8 of 32

Page 9 of 32


154

peptides were eluted with a gradient of solvent A (100-75% in 25 min, 75-50% in

155

25-35 min) at 0.3 mL/min. The detection wavelength was 220 nm. Transport percent

156

was expressed as the percentage of total peak area calculated at different time points

157

in the BL surface as compared to 0 h in the AP surface.

158

EA. hy926 cells experiment

159

The EA. hy926 cells with passage number < 12 were grown in 48-well plates.

160

The cells reaching 80−90% confluence were treated with various concentrations (2.5

161

mg/mL of hydrolysates and digests, 0.2 mM and 3.0 mM of synthetic peptides) of the

162

samples for 18 h. Then the cells were stimulated with TNF-α at 10 ng/mL and

163

incubated for an additional 6 h in order to induce inflammation.

164

Measurement of ICAM-1 and VCAM-1

165

The level of ICAM-1 and VCAM-1 was determined by Western blot analysis

166

according to our previous study.14 After the treatment period, the culture medium of

167

the EA. hy926 cells was discarded and the boiling Laemmle buffer containing 0.2%

168

TritonX-100 and 50 µM dithiothreitol was added to lysate the cells. The cell lysates

169

were then run on 9% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The

170

gels were transferred onto nitrocellulose membranes, and immunoblotted with

171

anti-ICAM-1/anti-VCAM-1 antibodies. The concentration of antibody to α-tubulin

172

used was 0.4 µg/mL, while all other antibodies were 0.1 µg/mL. The protein bands

173

were scanned using Licor Odyssey BioImager (Licor Biosciences, Lincoln, NB, USA)

174

and quantified by densitometry using Image Studio Lite 5.2. The expression of

175

ICAM-1 and VCAM-1 were normalized using the control α-tubulin. All the data were 9



176

expressed as the percentage change of the corresponding positive control (cells treated

177

with TNF-α alone).

178

Identification of anti-inflammatory peptides

179

The peptides were subjected to liquid chromatography−electrospray ionization

180

tandem mass spectrometry (LC−ESI-MS/MS) analysis using Waters ACQUITY

181

UPLC coupled to Waters Micromass Q-TOF MS Premier Instrument. The sample (5

182

µL) was desalted by loaded onto a peptides trap column (180 µm×20 mm, Symmetry

183

C18 nanoAcquity column, Waters) and desalted at 10 µL/min for 5 min using 1%

184

acetonitrile in water (containing 0.1% formic acid). Then the desalted sample was

185

loaded onto a nano analytical column (150 mm×75 µm, Atlantis d C18 nanoAcquity

186

column, Waters) and separated by a gradient of acetonitrile with 0.1% formic acid

187

(1-6% in 0-2 min, 6-25% in 2-25 min, 25-45% in 25-40 min, 45-75% in 40-45 min,

188

75-95% in 45-50 min, keeping at 95% in 50-55 min) at 350 µL/min. The mass

189

spectrometer was operated in a positive mode with capillary voltage 3.6 kV, source

190

temperature 100 °C, scanning m/z range 200-1000 in MS mode and 50-1990 in

191

MS/MS mode. The MS/MS data were processed by Peaks Viewer 4.5 (Bioinformatics

192

Solutions Inc., Waterloo, ON, Canada) in combination with manual de novo

193

sequencing. Identified peptide sequences were synthesized, validated using

194

LC-MS/MS and its purity (>98%) was further validated using HPLC by Genscript

195

Corp (Piscataway, NJ, USA). All synthetic peptides were further used for

196

anti-inflammatory assays.

197

Statistical analysis 10


Page 10 of 32

Page 11 of 32


198

The data were presented as the mean ± standard deviation of 4-6 determinations.

199

Statistical analyses were performed using one-way analysis of variance and

200

differences with P values

Identification of New Anti-inflammatory Peptides from Zein Hydrolysate

Recommend Documents