Antioxidative effects and mechanism study of bioactive peptides from

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Bioactive Constituents, Metabolites, and Functions

Antioxidative effects and mechanism study of bioactive peptides from defatted walnut (Juglans regia L.) meal hydrolysate Jianyong Sheng, Xiaoyu Yang, Jitang Chen, Tianhao Peng, Xiquan Yin, Wei Liu, Ming Liang, Jiangling Wan, and Xiangliang Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05722 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 1, 2019

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Journal of Agricultural and Food Chemistry

Antioxidative effects and mechanism study of bioactive peptides from defatted walnut (Juglans regia L.) meal hydrolysate Jianyong Sheng a†, Xiaoyu Yang a†, Jitang Chen a, Tianhao Peng a, Xiquan Yin b, Wei Liu a, Ming Liang b*, Jiangling Wan a,* and Xiangliang Yang a a

National Engineering Research Center for Nanomedicine, College of Life

Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, People’s Republic of China b

Joint Laboratory for The research of Modern Preparation Technology-

Huazhong University of Science and Technology and Infinitus, Guangzhou, Guangdong 510663, People’s Republic of China *Corresponding

author.

Ming

Liang,

Tel:

+86-20-80734744.

E-mail:

[email protected] * Corresponding author. Jiangling Wan, Tel: +86-27-67849580; fax: +86-2787792234. E-mail: [email protected]. ORCD ID: https://orcid.org/0000-00031414-0947 † Jianyong Sheng and Xiaoyu Yang contributed equally to this work.

1

ACS Paragon Plus Environment


1

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Abstract

2

The peptide components of defatted walnut (Juglans regia L.) meal hydrolysate

3

(DWMH) remain unclear, hindering the investigation of biological mechanisms and

4

exploitation of bioactive peptides. The present study aims to identify the peptide

5

composition of DWMH, followed by to evaluate in vitro antioxidant effects of selected

6

peptides and investigate mechanisms of antioxidative effect. Firstly, more than 1,000

7

peptides were identified by de novo sequencing in DWMH. Subsequently, a scoring

8

method was established to select promising bioactive peptides by structure based screening.

9

Eight brand new peptides were selected due to their highest scores in two different batches

10

of DWMH. All of them showed potent in vitro antioxidant effects on H2O2-injured nerve

11

cells. Four of them even possessed significantly stronger effects than DWMH, making the

12

selected bioactive peptides useful for further research as new bioactive entities. Two

13

mechanisms of hydroxyl radical scavenging and ROS reduction were involved in their

14

antioxidative effects at different degrees. The results showed peptides possessing similar

15

capacity of hydroxyl radical scavenging or ROS reduction may have significantly different

16

in vitro antioxidative effects. Therefore, comprehensive consideration of different

17

antioxidative mechanisms were suggested in selecting antioxidative peptides from DWMH.

18

Keywords

19

defatted walnut meal hydrolysate; de novo sequencing; antioxidant peptides; hydroxyl

20

radical scavenging; ROS reduction

21 2


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22

1. Introduction

23

Walnut (Juglans regia L.) has been consumed for nutritional or health purpose since

24

ancient times [1-3]. Walnut oil is one of the main products of walnut. After oil extraction,

25

a large quantity of press-cakes, mostly defatted walnut meal containing abundant amounts

26

of proteins, are produced [4]. To make the full use of walnut resources, the defatted walnut

27

meal is further hydrolyzed in the presence of digestive enzymes to produce defatted walnut

28

meal hydrolysate (DWMH), making it easier to be absorbed in the gastro-intestinal tract.

29

DWMH is a complicated mixture of which the main components are peptides. Recent

30

research of DWMH showed high antioxidant and antihypertensive effects, which are

31

beneficial to human health [5-8]. More importantly, the effect of DWMH on learning and

32

memory improvement seems promising in the aging society [9].

33

Although precise mechanisms for memory-enhancing effects remain unknown, the

34

relief of oxidative stress or free radical damage is a possible pathway. With regard to

35

DWMH, it has been reported that some antioxidative peptides of DWMH improve learning

36

and memory of mice [5, 10]. The antioxidative mechanisms of DWMH have been

37

extensively investigated, including hydroxyl radical scavenging, reactive oxygen species

38

(ROS) elimination and so on [11]. However, the material basis of the antioxidative effect

39

is far from elucidated. We hypothesize that different peptide components of DWMH

40

achieve their antioxidant effects by various mechanisms. In reality, the antioxidant effects

41

of individual peptide components of DWMH have been few explored, to say nothing of the

42

exact antioxidant mechanisms of each component. The structure-activity relationship of 3



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43

bioactive peptides from DWMH is far from clarified. Therefore, it is necessary to figure

44

out the total peptide components of DWMH and investigate the antioxidative mechanisms

45

of each component [12].

46

To our knowledge, successful identification of all the peptide composition of DWMH

47

has not been reported yet. The inaccessibility of protein database of walnut (Juglans regia

48

L.) makes it impossible to apply the commonly used database search method for identifying

49

peptides and proteins [13]. Moreover, various proteases were used in the production of

50

DWMH, making diverse cutting sites in the protein. Hence, it is inconvenient to achieve

51

the peptide sequencing in the common way. Thus, another method- de novo sequencing

52

was considered. De novo sequencing refers to sequencing a novel genome where there is

53

no reference sequence available for alignment. Sequence reads are assembled as contigs,

54

and the coverage quality of de novo sequence data depends on the size and continuity of

55

the contigs [14]. The advantages of de novo sequencing include characterization of protein

56

biomarkers or proteome of several species lacking of any gene or protein database [15, 16].

57

In the present work, we firstly identified all peptide components of DWMH by de

58

novo sequencing. To select several peptides that have similar biological effects with

59

DWMH, we then established a rational scoring method by taking the accuracy of

60

sequencing, detection sensitivity and structure representation into consideration.

61

Subsequently, in vitro antioxidative effects of eight selected peptides were evaluated on

62

H2O2-injured SH-SY5Y cells. The antioxidant mechanisms of each selected peptide were

63

investigated by measuring hydroxyl radical scavenging capacity and intracellular ROS of 4


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64

cells treated by peptides. All the eight brand new peptides showed quite strong antioxidant

65

capacity and improved the proliferation of oxidative damaged nerve cells by various

66

antioxidant mechanisms. The results of present study could bring new insight in screening

67

of bioactive peptides in DWMH and further investigation of structure-activity relationship.

68 69

2. Materials and methods

70

2.1 Materials

71

Defatted walnut meal hydrolysate (DWMH, batch No. 9002 & 9003, marked as

72

DWMH1 & DWMH2) was provided by Infinitus Co. Ltd. in Guangdong, China), which

73

was hydrolyzed by compound proteases and alkaline protease from defatted walnut meal.

74

Peptide components of DWMH (purity were more than 95%) were synthesized by Hefei

75

National Peptide Biotechnology Co. Ltd in Anhui, China. Fetal bovine serum (FBS) and

76

all the buffers or medium for cell culture were purchased from Life tech Inc. in Carlsbad,

77

CA, USA. Cell culture plates were bought from Corning Inc. in NY, USA. Cell Counting

78

Kit-8 (CCK-8) and other chemicals were obtained from Sigma-Aldrich.

79

2.2 HPLC-FTMS analysis

80

HPLC-FTMS analysis was performed according to a method reported previously [17],

81

and several changes were made to suit our condition. LTQ Orbitrap Elite include Ultimate

82

HPLC, DAD detector, LTQ linear ion trap MS and Orbitrap FTMS, which was controlled

83

by Xcalibur software (Version 2.0.7). The HPLC analysis was carried out using a Thermo

84

C18 column (100 × 3 mm, 1.7 μm particle size). The mobile phase comprised 0.1% (v/v) 5



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85

formic acid water (A) and acetonitrile (B) using a gradient program of 595% B from 0 to

86

50 min. DWMH or peptide components of DWMH was dissolved in deionized water at a

87

concentration of 5 mg/mL and then centrifuged at 10,000 g for 30 min. Afterwards, 10 μL

88

of supernatant was collected and injected into HPLC-FTMS system.

89

The MS experiments were performed to get an accurate MS and MS2 of the new

90

analogue. The ionization source was operated in the positive ionization mode with the flow

91

rates of the sheath gas and auxiliary gas at 40 and 10 arb. unit, respectively, capillary

92

temperature at 300℃, ion spray source capillary at 3.5 kV, source current at 100 μA. Six

93

scan events were selected in the MS experiment. Scan event 1 was used for full scan with

94

scan range from 100 to 1000 m/z and resolution 60,000. Scan event 2 to 5 were used to

95

produce MS2r through dependent scan selecting the 1th to 5th most intense ions in scan

96

event 1 and resolution 15,000. Collision energy was set at 35 V using High Energy

97

Collision Dissociation (HCD).

98

2.3 De novo sequencing

99

The method of de novo sequencing was based on previous research [18, 19]. The

100

HPLC-FTMS data of DWMH were acquired and converted by a software of pXtract 2.0

101

[20], and subsequently processed by pNovo 3 [21, 22] (Institute of Computing Technology,

102

Chinese Academy of Sciences, China) for de novo sequencing. The upper limit of missing

103

restrict enzyme cutting sites were set as 3 in pNovo 3, with the precursor mass tolerance

104

and fragment mass tolerance limited to ± 20 ppm. Moreover, carbamidomethyl (C) was set

105

as the fixed modification. Oxidation (M) and Gln→Pyro-Glu (Q) were set as the variable 6


Page 7 of 33


106

modifications. The results of pNovo 3 were transferred to pBuild 2.0 for further analysis.

107

Each peptide sequence in the results was scored by pNovo 3 according to the confidence

108

of every amino acid on this sequence. Results with a score over 45 were selected due to

109

their high reliabilities. Meanwhile, peptide sequences with an abundance in MS spectrum

110

above 1.67E+14 were chosen because they were easy to be detected.

111

2.4 Scoring method for de novo results

112

Four sub-scores were given for each selected peptide sequence. The first one, score A

113

was based on the abundance in MS spectrum. The logarithm of a value of abundance was

114

first calculated. Using the min-max scaling normalization method, we then took the

115

normalization processing measures to the logarithm of abundance and got score A which

116

are mapped to [60,100]. The second score, score B, was based on the confidence of de novo

117

sequencing. Similarly, a linear normalization method was used to convert the value of

118

pNove confidence score into the interval of [60,100]. The third sub-score, score C, was

119

related to the ion match quality of peptide-spectrum match (PSM), which was manually

120

checked. All the selected peptides were categorized as perfect match, good match or poor

121

match, and given the score C of 100, 95 or 60, respectively. The fourth and last sub-score,

122

score D, was associated with the amino acid composition. The content of specific amino

123

acids (Glu, Arg, Asp, Gly) in a peptide was calculated by dividing the number of specific

124

amino acids by the total number of amino acids. Using the min-max scaling normalization

125

method, score D mapped to [80,100] was obtained from the content of specific amino acids.

126

Subsequently, different weights were given to the above four sub-scores, with 40%, 20%, 7



Page 8 of 33

127

20% and 20% to score A, score B, score C and score D, respectively. Finally, the total score

128

of each selected peptide was calculated by adding up the four weighted sub-scores.

129

2.5 Hydroxyl radical scavenging capacity

130

The hydroxyl radical scavenging capacity of DWMH, peptide components of DWMH

131

or the reductive glutathione (GSH) was determined in a similar method to the previous

132

report [24, 25]. Briefly, 2 mL of 1.8 mM FeSO4 solution and 1.5 mL of 1.8 mM salicylic

133

acid-ethanol solution were added to 1 mL of 1 mg/mL sample solution in deionized water.

134

Then the mixture was incubated at 37℃ for 30 min. Subsequently, 200 μL of H2O2 solution

135

was added. After incubation for 10 min, the absorbance of samples at the wavelength of

136

510 nm were detected by a UV-VIS spectrophotometer (UV-1750, SHIMADZU, Japan).

137

In the control group, pure deionized water was used instead of DWMH or DWMH derived

138

peptide solution. All tests were performed in triplicate. The hydroxyl radical scavenging

139

capacity of each samples was calculated using the following equation:

140

Percentage of hydroxyl radical scavenged (%) = [(𝐴𝑐 ― 𝐴𝑠)/ 𝐴𝑐)] × 100

141

where As is the absorbance of sample of DWMH or DWMH derived peptides; Ac is the

142

absorbance of the control group.

143

2.6 Protective effect on H2O2-injured SH-SY5Y cells

144

SH-SY5Y cells were obtained from Xiamen University in Fujian, China. The cell

145

culture medium was Dulbecco's Modified Medium containing 1% of nonessential amino

146

acid, 1% of L -glutamine, 1% of penicillin (100 IU/mL) and streptomycin (100 mg/mL)

147

and 10% of fetal bovine serum. Cells were grown at 37 °C in an atmosphere of 95% relative 8


Page 9 of 33


148

humidity and 5% CO2. Prior to the experiment, SH-SY5Y cells were seeded onto a 96-well

149

plate at a density of 6,000 cells/cm2 and incubated for 24 h. Then, 600 μM H2O2 solution

150

was added to the cells. After incubation for another 24 h, cells were treated with the sterile

151

solution of GSH, DWMH or DWMH-derived peptides at the concentration of 300 μg/mL

152

for 24 h at 37℃. Cell viability was determined using CCK-8 method as previously

153

described [23].

154

2.7 Measurement of intracellular amount of ROS

155

We used 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA), a fluorescent probe

156

for reactive oxygen species (ROS), to quantify intracellular ROS. SH-SY5Y cells were

157

seeded onto a 24-well plate at a density of 1×105 cells/cm2, and then cultured and treated

158

with solutions of peptides or DWMH in the same way as described in section 2.6.

159

Subsequently, solutions of peptides or DWMH were discarded and cells were washed with

160

PBS. Then 1 mL of 10 μM DCFH-DA solution was added to each well of cell culture plate

161

and incubated for 20 min. After treated with pancreatic enzyme, cells were detached from

162

cell culture plate and washed twice using PBS. The suspensions of cells in PBS were

163

analyzed using a flow cytometer (CytoFLEX S, Beckman Coulter, USA). The mean

164

fluorescent intensity was recorded to represent the concentration of intracellular ROS. The

165

intracellular ROS amount of each group was compared.

166

2.8 Statistical analysis.

167

Statistical analysis of the data was performed using one-way analysis of variance

168

(ANOVA), followed by the least significance difference (LSD) multiple comparison test, 9



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169

with SPSS 21.0 statistical software (SPSS, Inc., Chicago, IL). The results were presented

170

as the mean ± standard error of the mean (SEM). Values with p < 0.05 were considered

171

significant.

172 173

3. Results

174

3.1 HPLC-FTMS detection of DWMH

175

Two batches of DWMH products (DWMH1 & DWMH2) were characterized by

176

MALDI-TOF mass spectrometry MS. Complicated compositions of DWMH were

177

illustrated by the dense peaks in spectrums (Fig. S1). Then the DWMH products were

178

determined by HPLC-MS-FTMS of high specificity and resolution. The chromatogram of

179

DWMH1 and DWMH2 were shown in Fig. 1. Up to 7322 and 7250 tandem mass spectrums

180

were collected for DWMH1 and DWMH2, respectively.

181 182

Figure 1. HPLC-FTMS chromatograms of (A) DWMH1 and (B) DWMH2.

183

3.2 De novo sequencing of DWMH

184

After the data of HPLC-MS/MS was analyzed by the de novo sequencing software 10


Page 11 of 33


185

pNovo 3, a total of 2,212 and 1,536 peptides were acquired for DWMH1 and DWMH2,

186

respectively. Peptides ranking top 15 in abundance, which is related to the limit of detection,

187

were sorted in a descending order (Table S1 and Table S3). A confidence score of each

188

peptide sequence was calculated by the software, indicating the reliability of the result.

189

Peptides ranking top 15 in confidence from DWMH1 and DWMH2 were listed in Table

190

S2 and Table S4, respectively.

191

3.3 Selection of peptide components

192

Peptides ranking top 15 in either credibility or relative abundance from DWMH1 or

193

DWMH2 are first selected (Table 1 & Table 2). Four sub-scores of each peptide, including

194

score A, score B, score C and score D, were based on relative abundance in MS spectrum,

195

credibility of de novo sequencing, ion match quality of the result and content of specific

196

amino acids, respectively. Total score of each peptide was the sum of its score A, score B,

197

score C and score D, with the weights of 40%, 20%, 20% and 20% respectively. Peptides

198

ranking top 12 in total score were chosen from DWMH1 and DWMH2, respectively. Due

199

to the variation of manufacturing process, the top12 results of DWMH1 were not strictly

200

content with those of DWMH2. After all, the top12 results of DWMH1 and DWMH2 had

201

eight peptides (P1, P16, P18, P21, P22, P24, P26 & P27) in common, which were shown

202

in Tables S1-S4.

203

The eight selected peptides with high representativeness among different batches of

204

DWMH are synthesized and characterized by HPLC-FTMS (Fig. S2 & Fig. S3).

205

According to the isoelectric point (pI) (Tables S1-S4), P1 and P24 are positively charged 11



206

while the other six peptides are negatively charged in neutral pH biological fluids.

207

Moreover, after search the sequences of eight selected peptides in RCSB database, no

208

match was found.

209

Table 1. Scoring of selected de novo sequencing results of DWMH1 Abundance

No.

Page 12 of 33

Sequence (N –C)

in

MS

spectrum

Score A

Confidence of de novo sequencing

Score

Ion match

Score

B

quality

C

Content of specific amino acid

Score

Total

D

score

P1

VEGNLQVLRPR

6.13E+15

100.00

22.9

72.23

good

100.00

0.37

92.54

92.95

P2

QLQVLRPR

3.05E+15

99.67

11

63.87

good

100.00

0.25

88.47

90.34

P3

QLPR

1.93E+15

98.40

5.5

60.00

too short

60.00

0.25

88.47

81.06

P4

VNLVNQHKLPL

6.76E+14

95.66

27.1

75.18

good

100.00

0

80.00

89.30

P5

LGLLPSFSNAPR

4.74E+14

95.01

16.9

68.01

not good

60.00

0.17

85.76

80.76

P6

LLANEPR

3.83E+14

94.62

13.6

65.69

not good

60.00

0.29

89.83

80.95

P7

NLPLL

3.70E+14

94.56

11.4

64.15

too short

60.00

0

80.00

78.65

P8

VVDSEGKWEL

3.63E+14

94.53

22.3

71.81

60.00

0.4

93.56

82.88

P9

VLRGDA

3.17E+14

94.28

10.2

63.30

not good

60.00

0.5

96.95

81.76

AVLDLSNHANQLD

2.71E+14

93.99

42

85.66

good

100.00

0.16

85.42

91.81

60.00

0.3

90.17

81.04

60.00

0

80.00

80.16

60.00

0.34

91.53

85.71

P10

lack information

lack

P11

LVAVTVEDEL

2.60E+14

93.92

15.7

67.17

P12

LSLLPSYQPTSP

2.38E+14

93.76

24.4

73.29

P13

QNGAGFEWV

2.08E+14

93.51

48.2

90.02

P14

LSTVNSQNLPLL

1.52E+14

92.80

36.3

81.65

good

100.00

0

80.00

89.45

P15

TLMMSEEL

1.39E+14

92.78

11.6

64.29

bad

60.00

0

80.00

77.97

P16

LAGNPHQQQQN

3.09E+13

90.04

62.4

100.00

complete

100.00

0.09

83.05

92.63

EAQFGQQHVSGAGQ

6.24E+12

87.13

60.3

98.52

complete

100.00

0.29

89.83

92.52

95.00

0.28

89.49

91.75

P17

information lack information bad continuity

almost

P18

HNLDTQTESDV

1.05E+13

88.08

59.7

98.10

P19

SLLDTNNNANQLDQNPR

4.93E+6

61.55

56.9

96.13

complete

100.00

0.18

86.10

81.07

P20

NTNNNANQLDQNPR

2.11E+6

60.00

55.2

94.94

complete

100.00

0.15

85.08

80.00

P21

AGNDGFEYVTLK

9.27E+13

92.04

60.4

98.59

95.00

0.34

91.53

93.84

P22

WSVWEQELEDR

1.24E+13

88.38

47.4

89.46

complete

100.00

0.46

95.59

92.36

P23

NSALYVPHWNLNAH

8.52E+11

83.50

50.5

91.63

complete

100.00

0

80.00

87.73

P24

QQRQQQGL

1.37E+13

88.56

49.9

91.21

complete

100.00

0.25

88.47

91.36

QNPDDEFRPQGQ

5.16E+12

86.78

46.9

89.10

complete

100.00

0.42

94.24

91.38

P25

complete

almost complete

12


Page 13 of 33


P26

AELQVVDHLGQTV

1.13E+13

88.21

50.9

91.92

P27

EQEEEESTGRMK

1.40E+12

84.41

47.2

89.31

P28

QQERHHGQQQ

4.49E+13

90.72

46.5

88.82

P29

TMFQQQSQHPAQPPR

1.60E+12

84.66

46.5

P30

WSVWEQEL

5.10E+11

82.57

46.1

complete

100.00

0.23

87.80

91.23

95.00

0.59

100.00

90.63

complete

100.00

0.3

90.17

92.09

88.82

complete

100.00

0.07

82.37

88.10

88.54

complete

100.00

0.25

88.47

88.43

almost complete

210

13



211 No.

Table 2. Scoring of selected de novo sequencing results of DWMH2

Sequence (N –C)

Abundan

Scores

Confidence

Scores

Ion

ce in MS

A

of de novo

B

quality

spectrum P1

VEGNLQVLRPR

P31

DVARVALAP

P32

EPDVLALLQG

P4

VNLVNQHKLPL

P5

LGLLPSFSNAPR

P6

LLANEPR

P7

NLPLL

P8

VVDSEGKWEL

P9

VLRGDA

P11

LVAVTVEDEL

P33

RLDWVEA

P34

PEEVLALA

P14 P35

Page 14 of 33

LSTVNSQNLPLL QPDVTQVFEFQ

P36

VQLVDDNGDNVFDWV

P16

LAGNPHQQQQN

P18

HNLDTQTESDV

9.59E+15 2.78E+15 7.09E+14 6.86E+14 6.12E+14 5.05E+14 4.44E+14 4.36E+14 4.21E+14 2.98E+14 2.34E+14 1.95E+14 1.83E+14 1.70E+14 1.67E+14 3.45E+13 1.19E+13

match

Scores

Content

C

specific

sequencing

of

Scores

Total

D

scores

amino acid

100.00

21.7

70.30

good

100.00

0.37

91.04

92.27

97.77

10.2

61.59

bad

60.00

0.23

86.87

80.80

60.00

0.30

88.96

80.99

60.00

0

80.00

80.92

60.00

0.17

85.07

80.32

60.00

0.29

88.66

80.35

continuity 95.30

15.2

65.38

bad continuity

95.24

26.7

74.09

95.04

16.6

66.44

low

peak

intensity lack information 94.69

13

63.71

lack information

94.46

11

62.20

too short

60.00

0

80.00

78.22

94.43

21.7

70.30

lack

60.00

0.4

91.94

82.22

information 94.36

10

61.44

good

100.00

0.5

94.93

89.02

93.74

33.5

79.24

lack

60.00

0.3

88.96

83.14

93.31

8.1

60.00

good

100.00

0.43

92.84

87.89

92.98

14

64.47

lack

60.00

0.25

87.46

79.58

60.00

0

80.00

81.82

60.00

0.19

85.67

78.30

information

information 92.86

36.3

81.36

lack information

92.73

8.6

60.38

bad continuity

92.70

32.8

78.71

good

100.00

0.34

90.15

90.85

89.86

60.9

100.0

complete

100.00

0.09

82.69

92.48

87.94

55.4

almost

95.00

0.28

88.36

91.01

0 95.83

complete

P21

AGNDGFEYVTLK

1.11E+14

91.96

54.4

95.08

complete

100.00

0.34

90.15

93.83

P26

AELQVVDHLGQTV

1.34E+13

88.15

45.3

88.18

complete

100.00

0.23

86.87

90.27

P37

MMRPDEDEQEGAGRQ

4.91E+11

82.19

51.8

93.11

complete

100.00

0.60

97.91

91.08

P38

DTNNNANQLDQNPR

2.22E+6

60.00

51.5

92.88

complete

100.00

0.22

86.57

79.89

P39

LAGNPDDEFTKQGPSQEYEQ

88.49

50.5

92.12

almost

95.00

0.34

90.15

90.85

100.00

0.67

100.0

89.87

H P40

DDDLREGQL

1.62E+13 3.36E+12

complete 85.66

31.9

78.03

complete

0 14


Page 15 of 33

P41


LRHRADTQTESDV

3.59E+13

89.93

47.1

89.55

almost

95.00

0.39

91.64

91.21

complete

P27

EQEEEESTGRMK

1.73E+12

84.46

48.5

90.61

complete

100.00

0.59

97.61

91.43

P24

QQRQQQGL

1.94E+13

88.82

46.3

88.94

complete

100.00

0.25

87.46

90.81

P42

SDALYVPHWARNAH

1.07E+12

83.59

46

88.71

complete

95.00

0.15

84.48

88.07

P22

WSVWEQELEDR

1.33E+13

88.14

43.6

86.89

complete

100.00

0.46

93.73

91.38

P43

LSTVNSQNLPLL

2.51E+12

85.13

45.7

88.48

complete

100.00

0.08

82.39

88.23

P44

LCVQQSGSNLFSGFDVCFL

79.85

45.6

88.41

almost

100.00

0.16

84.78

85.58

1.34E+11

complete

212 213 214

3.4 Hydroxyl radical scavenging capacity Hydroxyl radicals scavenging is one of antioxidant mechanisms. Two batches of

215

DWMH exhibited similar capacities of hydroxyl radical scavenging with approximately 44%

216

of hydroxyl radical scavenged at the concentration of DWMH at 1.0 mg/mL (Fig. 2). The

217

hydroxyl radical scavenging capacities of P1, P18, P21, P22 and P27 were similar to that

218

of DWMH. Although P16, P24 and P26 possessed significantly lower hydroxyl radical

219

scavenging capacities compared to DWMH, they eliminated similar amount of the

220

hydroxyl radical as GSH.

221 222

Figure 2. Hydroxyl radical scavenging capacities of DWMH and peptides derived from

223

DWMH. Values marked with same letter were not significantly different (p > 0.05). 15



Page 16 of 33

224 225

3.5 Protective effect on H2O2-injured SH-SY5Y cells

226

H2O2 was used to induce oxidative damage of SH-SY5Y cells [26]. H2O2 solution can

227

effectively injured cells in a dose-dependent manner within the range of 2001,000 μM

228

(Fig. S4). 600 μM was chosen as the concentration of H2O2 solution because approximately

229

65% of cells survived after the treatment which was appropriate for the proliferation test.

230

Then the effect of concentration of tested peptide solutions on cell viability was

231

investigated. All tested peptide solute ons induced proliferation of cells in a dose-dependent

232

manner in the range of 150300 μg/mL (Fig. S5). Hence, the concentration of peptide

233

solutions were set as 300 μg/mL in this study. After treatment of DWMH-derived peptides

234

or DWMH, the viability of SH-SY5Y cells was significantly higher than control group (p

235

＜ 0.05), indicating the protection effect of DWMH or selected peptides in oxidative

236

damaged SH-SY5Y cells (Fig. 3). More importantly, the protective effect of P1, P18, P24

237

or P26 was similar to that of GSH and significantly stronger than that of DWMH (p＜0.05).

238

However, the other four peptides possessed similar effect with DWMH and significantly

239

weaker than GSH.

240

16


Page 17 of 33


241 242

Figure 3. Protective effects of DWMH and peptides derived from DWMH on H2O2

243

induced oxidative damage in SH-SY5Y cells. Values marked with same letter were not

244

significantly different (p > 0.05).

245

3.6 ROS elimination in H2O2-injured SH-SY5Y cells

246

Reduction of intracellular ROS is another antioxidant mechanism. A nearly 10-fold

247

increase of intracellular ROS amount was seen for SH-SY5Y cells treated by H2O2 solution,

248

indicating the raise of ROS level is a manifestation of oxidative damage. After treatment

249

of DWMH or DWMH-derived peptides except for P22, the intracellular ROS amount

250

significantly decreased (Fig. 4), indicating their abilities to reduce intracellular ROS. More

251

importantly, P1, P16, P18, P21, P26 or P27 showed significantly stronger ability of

252

intracellular ROS elimination compared with DWMH or GSH (p＜0.05).

17



Page 18 of 33

253 254

Figure 4. Amount of reactive oxygen species (ROS) in H2O2-injured SH-SY5Y cells after

255

treatment of DWMH or peptides derived from DWMH. The intracellular amount of ROS

256

was represented by fluorescent intensity of DCFH, a fluorescent probe for intracellular

257

ROS. Values marked with same letter were not significantly different (p > 0.05).

258 259

3.7 Summary of antioxidant mechanisms

260

We investigated two antioxidant mechanisms, hydroxyl radical scavenging and ROS

261

reduction of all the selected peptides and DWMH. Different mechanisms were involved in

262

antioxidative effects of different peptides. As shown in the Table 3, both above mentioned

263

antioxidant mechanisms were involved in antioxidant effects of P1, P16, P18, P21 and

264

DWMH. On the contrary, only one of the above mentioned mechanism accounted for the

265

antioxidant effect of P22, P24, P26 and P27.

266

For P24 and P26 which possessed significantly stronger in vitro antioxidative effects

267

than DWMH, only one of the above mentioned antioxidative mechanism was involved. For

268

P16, P22 and DWMH which had similar in vitro antioxidative effects, the above mentioned 18


Page 19 of 33

269


two mechanisms accounted for the effects in different proportions.

270 271

Table 3. Antioxidant effects and mechanisms of eight selected peptides from DWMH. No. of peptide

Content of specific amino acid (%)

In vitro antioxidant effect

Capacity of hydroxyl radical scavenging

Capacity of ROS reduction

P1

37

++

+

++

P16

9

+

-

++

P18

28

++

+

++

P21

34

+

+

++

P22

46

+

+

-

P24

25

++

-

+

P26

23

++

-

++

P27

59

+

+

++

DWMH1

——

+

+

+

DWMH2

——

+

+

+

272

+ means the antioxidative effect via certain mechanism is significantly different from the

273

control or similar to DWMH, ++ means the antioxidative effect via certain mechanism is

274

significant stronger than that of DWMH, - means the antioxidative effect via certain

275

mechanism is significant weaker than that of DWMH or not significantly different from

276

the control.

277

4. Discussion

278

DWMH were produced from degreased walnut dregs by enzymatic hydrolysis. In

279

detail, degreased walnut dregs were incubated with alcalase and protamex at 5055℃ with 19



Page 20 of 33

280

agitation for 1824 hours. After heat-inactivation of enzymes, the hydrolysates were

281

centrifuged at the rate of 8,000 rpm for 20 min. The supernatant were collected for

282

ultracentrifugation. Products of which the molecular weight were less than 10,000 Da were

283

collected and dried as DWMH. A large proportion of walnut in China was planted in Shanxi

284

or Yunnan Province. Hence, in this study, two batches of DWMH used were prepared from

285

walnut sourced from these two areas respectively. Moreover, the parameters of production

286

process including temperature and duration of hydrolysis were also slightly different. As

287

illustrated by chromatogram of HPLC-FTMS (Fig.1), the composition of DWMH is rather

288

complicated. It was reported that DWMH mainly consisted of peptides and polyphenols of

289

similar molecular weight [27], making the identification of DWMH components a difficult

290

task. Herein, we focused on figuring out the dominant ingredients, peptides of DWMH.

291

First of all, peptides contained in DWMH were isolated by HPLC-FTMS, a separation

292

and detection method with high sensitivity and resolution. Due to complexity of the

293

DWMH components, it is no surprising that more than 7,000 different mass spectrums

294

were collected by the HPLC-FTMS. Although total ion chromatogram of DWMH1 was

295

highly similar to that of DWMH2 (Fig.1), difference in collected mass spectrums revealed

296

different compositions between two batches. Then, based on the mass spectrums, de novo

297

sequencing was used to identify peptide components of DWMH, which does not need any

298

protein database. The results of de novo sequencing revealed more than 1,000 peptide

299

compositions in DWMH, which is firstly reported in total peptide sequencing of DWMH.

300

In addition, de novo sequencing results showed their were differences in peptide 20


Page 21 of 33

301


compositions between DWMH1 and DWMH2.

302

The large number of peptides acquired by de novo sequencing makes it rather difficult

303

to investigate the biological effects of all the components. Herein, a rational selection

304

criteria was created by us to help focus further research on most promising peptides in

305

mechanism study. First of all, promising peptides should be predominant in composition

306

and easily detected. In other words, the fragment ions have high abundance in the MS

307

spectrum of HPLC-MS/MS detection. Furthermore, confidence of sequencing, scored by

308

pNovo, is also important. Based on this, the peptides ranking top 15 in either relative

309

abundance or credibility score were chosen for further analysis (Table S1 Table S2).

310

Subsequently, a scoring method was proposed. Four sub-scores were given to a total

311

of 60 selected peptides (Table 1 & Table 2). Score A and score B were used to judge the

312

relative abundance and confidence of sequencing, respectively. Furthermore, score C

313

derived from manual analysis of ion match quality of HPLC-MS/MS data, supplementing

314

the judgement of reliability. Last but not least, score D was designed to reflect the content

315

of specific amino acids. The content of amino acid of protein was related to biological

316

functions. It was hypothesized that peptides resembled DWMH in structure may possess similar

317

function with DWMH. Because we knew almost nothing about the structure-activity

318

relationship of antioxidative peptides in DWMH, we used a basic structure parameter,

319

content of amino acids of high abundance. We chose Glu, Arg, Asp, Gly, which were

320

reported to be rich in amino acid composition of walnut protein[4], as parameters. We

321

regarded the relative abundance as the most important factor while the other three factors 21



Page 22 of 33

322

equally meaningful, so the weight of score A was set as 40% and weights of other scores

323

as 20% in the total score. Hence, peptides with higher total scores are easier to be detected,

324

more reliable and similar to DWMH in structure and function.

325

Eight peptides with highest total scores and were in results of DWMH1 and DWMH2

326

were selected. High purity products of eight selected peptides were obtained by solid phase

327

synthesis, and then their in vitro antioxidant effects were evaluated in H2O2-injuered SH-

328

SY5Y cells. SH-SY5Y cell model was widely used to evaluate or predict biological effects

329

of nutrients on nerve system [28, 29]. Because H2O2 can freely penetrate the cell membrane

330

and generate the highly reactive hydroxyl radical (•OH) within the cell, it has been used to

331

induce cell damage in different types of cells [30]. The results revealed that all selected

332

peptides and DWMH had protective effect on the proliferation of H2O2-injured SH-SY5Y

333

cells, some peptides had even better effect than DWMH (Fig.2). The good results also

334

indicated the success of our scoring method in selecting bioactive peptides.

335

Various mechanisms are involved in antioxidative effects, among which hydroxyl

336

radical scavenging and ROS reduction are commonly accepted. Oxidative damage induces

337

increase of intracellular ROS, which leads to cell death [31]. Therefore, we investigated

338

the antioxidative effects of all the selected peptides and DWMH via the above two

339

mechanisms (Fig.3 & Fig.4). The two mechanisms were involved in the antioxidant

340

capacities of selected peptides in various proportions (Table 3). Both of the above

341

mentioned mechanisms accounted for the antioxidative effect of DWMH. Therefore, the

342

results are biased when using the parameter of either mechanism as a single indicator to 22


Page 23 of 33


343

select bioactive peptides. It could be confirmed by P16, P22 and DWMH which had similar

344

in vitro antioxidative effects but were different in capacity of hydroxyl radical scavenging

345

or ROS reduction. Furthermore, peptides possessed similar hydroxyl radical scavenging

346

capacity, such as P16 and P18, had significantly different in vitro antioxidative effects.

347

The commonly used method to identify bioactive peptides was bioassay-guided

348

purification coupled with de novo sequencing. In this way, active peptides are identified

349

during the purification process [32]. Briefly, peptide components of different molecular

350

weight are initially isolated by size-exclusion chromatography and collected. Then the

351

activities of different fractions are evaluated. Several fractions of strong biological effects

352

are selected to be further separated and identified by LC-MS/MS or de novo sequencing

353

[33]. The results obtained by this kind of method could be biased, which was proved above.

354

It may due to the fact that a component of strong bioactivity surrounded with components

355

of extremely low biological effects in the same fraction could be buried. More importantly,

356

it is far from rigorous to use single parameter of bioactive effects as an indicator to select

357

bioactive peptides. Comprehensive consideration of different antioxidative mechanisms

358

were recommended in selection antioxidative peptides from DWMH.

359

As we know, cysteine, tyrosine, tryptophan and methionine are common antioxidative

360

amino acids. However, it is inappropriate to choose amino acids of high antioxidative effect

361

as parameters in our selection criteria. In the eight selected peptides, only P27 was

362

composed of methionine and none of them consisted of cysteine. In addition, only P21 and

363

P22 were composed of tyrosine and tryptophan, respectively. However, P21, P22 or P27 23



Page 24 of 33

364

did not show greater antioxidative capacity than others (Fig. 3). Moreover, P1, P18, P24

365

or P26 without any common antioxidative amino acids possessed similar in vitro

366

antioxidative capacity compared to GSH. In this study, we chose content of amino acids of

367

high abundance as a basic structure parameter. Though it was a parameter had nothing to

368

do with the antioxidative effect at the first beginning, the results showed that it worked in

369

selecting peptides of strong hydroxyl radical scavenging capacity (Table 3). Furthermore,

370

the results of antioxidative effects suggested that it was hard to attribute the antioxidative

371

effect of a peptide to a single antioxidative amino acid residue. Thus, choosing amino acids

372

of high antioxidative effect as parameters in the peptide selection of DWMH or other

373

products may be misleading.

374

Furthermore, parameters based on structure-activity relationship could be a useful

375

supplement to current method of bioactive compounds selection. The results of our first

376

attempt to select bioactive peptide from DWMH using structure parameters is not that

377

satisfying but meaningful. No direct relationship was found between the content of specific

378

amino acids (Glu, Arg, Asp, Gly) and in vitro antioxidative effect. However, the content

379

of specific amino acids correlated well with hydroxyl radical scavenging capacity (Table

380

3). Of course, more complex and complicated structure parameters including specific

381

amino acid consequences or conformation of peptides should be taken into consideration

382

and tested in a larger sample. We believed that the final success of structure based screening

383

would be realized with continuous optimization based on feedback.

384

In the present study, all peptide components of DWMH were identified by de novo 24


Page 25 of 33


385

sequencing. Eight brand new peptides were selected by a rational selection criteria and

386

proved to have strong protective effect on H2O2-injured SH-SY5Y cells. Two different

387

mechanisms, hydroxyl radical scavenging and ROS reduction were involved in their

388

antioxidant effects at various degrees. Peptides possessing similar capacity of hydroxyl

389

radical scavenging or ROS reduction may have significantly different in vitro antioxidative

390

effects. Therefore, comprehensive consideration of different antioxidative mechanisms

391

were suggested in selecting antioxidative peptides from DWMH. Moreover, parameters

392

based on structure-activity relationship could be a useful supplement to current method of

393

bioactive compounds selection.

394 395

Acknowledgments

396

We are thankful for financial support from the National Natural Science Foundation

397

of China (No. 81703443) and China Postdoctoral Science Foundation (No. 2017M622461).

398

Conflict of interest statement

399 400

The authors declare no competing financial interest. Table of contents

25



Page 26 of 33

401 402

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Figure 1. HPLC-FTMS chromatograms of (A) DWMH1 and (B) DWMH2. 140x65mm (300 x 300 DPI)



Figure 2. Hydroxyl radical scavenging capacities of DWMH and peptides derived from DWMH. Values marked with same letter were not significantly different (p > 0.05). 74x65mm (300 x 300 DPI)


Page 30 of 33

Page 31 of 33


Figure 3. Protective effects of DWMH and peptides derived from DWMH on H2O2 induced oxidative damage in SH-SY5Y cells. Values marked with same letter were not significantly different (p > 0.05). 74x64mm (300 x 300 DPI)



Figure 4. Amount of reactive oxygen species (ROS) in H2O2-injured SH-SY5Y cells after treatment of DWMH or peptides derived from DWMH. The intracellular amount of ROS was represented by fluorescent intensity of DCFH, a fluorescent probe for intracellular ROS. Values marked with same letter were not significantly different (p > 0.05). 74x65mm (300 x 300 DPI)


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Table of contents


Antioxidative effects and mechanism study of bioactive peptides from

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