Purification and characterization of antioxidant peptides from Alcalase

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Bioactive Constituents, Metabolites, and Functions

Purification and characterization of antioxidant peptides from Alcalase-hydrolyzed soybean (Glycine max L.) hydrolysate and their cytoprotective effects in human intestinal Caco-2 cells Qiaozhi Zhang, Xiaohong Tong, Yang Li, Huan Wang, Zhongjiang Wang, Baokun Qi, Xiaonan Sui, and Lianzhou Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01235 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 3, 2019

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Journal of Agricultural and Food Chemistry

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Purification and characterization of antioxidant peptides from

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Alcalase-hydrolyzed soybean (Glycine max L.) hydrolysate and their

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cytoprotective effects in human intestinal Caco-2 cells

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Qiaozhi Zhang1, Xiaohong Tong1, Yang Li1*, Huan Wang1, Zhongjiang Wang1, Baokun Qi1, Xiaonan Sui1*,

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Lianzhou Jiang1,2*

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1 College 2 National

of Food Science, Northeast Agricultural University, Harbin, 150030, China

Research Center of Soybean Engineering and Technology, Harbin, 150030, China

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*To whom correspondence should be addressed.

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Tel.: +86 451 55190716

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Fax: +86 451 55190716

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Email: [email protected] (Yang Li)

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[email protected] (Xiaonan Sui)

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[email protected] (Lianzhou Jiang)

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Running title: Purification and characterization of antioxidant and cytoprotective soybean peptides

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Abstract

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This study aimed to purify and identify antioxidant peptides from the low-molecular-weight

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fraction (SPH-I, MW 0.05).

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Thereafter, cytoprotective effect of synthesized peptides was assayed by determining their

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ROS-quenching abilities in H2O2-stressed Caco-2 cells. DCFH-DA is a non-polar dye that can be

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taken up by Caco-2 cells and deacetylated to more polar DCFH by cellular esterases. Upon

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generation of ROS, intracellular DCFH was oxidized to form the highly fluorescent DCF 37. ROS-

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mediated oxidative damage is considered as a culprit in the development of various intestinal

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mucosal disorders, like inflammatory bowel diseases and associated cancers 14. As shown in Fig.

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5B, exposure to H2O2 alone dramatically exacerbated the intracellular ROS production (1.8-fold,

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compared with NC), while all synthesized peptides dose-dependently decreased the induced ROS

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level, implying their ability to protect oxidant-stressed Caco-2 cells. Interestingly, unlike the

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results of chemical antioxidant assays, considerable variations were observed among peptides.

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SPH-IC and SPH-ID exhibited statistically higher ROS-quenching abilities than SPH-IA and SPH-

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IB (p < 0.05). At 100 μM, SPH-IC and SPH-ID repressed 85.9% and 96.2% of the H2O2-induced

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ROS generation, respectively. Fluorescence imaging presented similar results, where cells

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pretreated with 100 μM of SPH-IC and SPH-ID showed more faint signals in comparison to those

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treated with SPH-IA and SPH-IB (Fig. 5C). The higher contents of hydrophobic and antioxidant

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AA residues in SPH-IC and SPH-ID constitute an underlying explanation. As previously indicated,

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hydrophobic AA residues, like Phe (F), Ile (I) and Val (V), could facilitate entry of peptides into

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cells by interaction with membrane lipid bilayers and terminate radical chain reactions through

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proton donation. Moreover, Asp (D) and aromatic AAs, namely, Trp (W), Tyr (Y) and Pro (P),

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were credited as antioxidant AAs and could enhance antioxidant efficacy of peptides via directly

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transferring proton to electron deficient radicals 27,38. Additionally, peptides with positive charges

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(SPH-ID, net charge: +1) were preferentially membrane permeable and might be taken up by

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mitochondria to regulate associated oxidative processes 39. Therefore, it was speculated that those

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aforementioned AA residues and associated structures contributed to the better performance of

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SPH-IC and SPH-ID in modulating ROS-related oxidative damage.

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Subsequently, protective effect of synthesized peptides against H2O2-induced decrease of CV

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was investigated. As shown in Fig. 6A, after exposure to H2O2, Caco-2 cells exhibited significantly

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diminished CV, when compared to the NC (-57.1%, p < 0.05). Pretreatment with peptides at 50

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μM (except for SPH-IB) statistically preserved this induced loss of CV (p < 0.05), with SPH-IC

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showing the best protective potency. Next, the ability of synthesized peptides to inhibit lipid

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peroxidation was evaluated, via determining the level of MDA, a degradation product of lipid

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peroxides (Fig. 6B). As seen, with respect to the NC, H2O2-stressed Caco-2 cells showed 3.4-fold

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higher content of MDA. Howbeit, all pure peptides statistically down-regulated this induced MDA

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production (p < 0.05), indicating their potential to attenuate ROS-mediated membrane damage.

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Again, SPH-IC and SPH-ID displayed higher inhibitory effects when compared to SPH-IA and

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SPH-IB. In addition to eliminating primary/secondary oxidation products, enhancement of the

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intrinsic antioxidant defense system is another key action of cytoprotective candidates 40. GSH is

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a key antioxidant that plays a pivotal role in the regulation of detoxification and antioxidation in

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all eukaryotic cells 14. As seen in Fig. 6C, incubation with H2O2 markedly reduced the GSH content

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by 52.17%, while only SPH-IC and SPH-ID significantly improved the cellular GSH level (p
1.0 mg/mL.

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Values were expressed as mean ± SD from triplicate experiments.

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Figure 5. Impact of synthesized antioxidant peptides on (A) Cell viability, (B) and (C)

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Intracellular ROS generation in Caco-2 cells. For assaying cell viability, Caco-2 cells were

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treated with each synthesized peptide at various concentrations from 25-100 μM for 24 h; For

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ROS determination, Caco-2 cells were incubated with each synthesized peptide at indicated

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concentrations for 24 h, followed by stimulation with 1 mM H2O2 for 3 h. NC, negative control;

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PC, positive control. Values were expressed as the mean ± SD from quintuplicate experiments.

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Bars with different letters indicated statistical differences (p < 0.05, Duncan’s test).

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Figure 6. Cytoprotective effects of synthesized antioxidant peptides against H2O2-induced

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oxidative stress in Caco-2 cells. (A) Cell viability (B) Lipid peroxidation; (C) Intracellular GSH

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content, (C) CAT activity, (D) GR activity, and (F) Secretion level of IL-8. Caco-2 cells were

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pretreated with each synthesized peptide at 50 μM for 24 h, followed by incubation with 1 mM

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H2O2 for an extra 3 h. NC, negative control; PC, positive control; CAT, catalase, GR, glutathione

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reductase. Values were expressed as the mean ± SD from quintuplicate experiments. Bars with

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different letters indicated statistical differences (p < 0.05, Duncan’s test). *p < 0.05, **p < 0.01,

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***p< 0.001, Student’s t-test.

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Table 1 Characteristics of peptides purified from SPH-I and identified by nano-LC-ESI-MS/MS

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Peptide

MW

Net Intensity (% of area) Theoretical pI Hydrophobicity

sequence

(Da)

VVFVDRL

847

Accessions Charge Uncharacterized protein OS=Glycine max

8.75×108 (21.4%)

6.74

9.01

0 GN=GLYMA_01G047700 PE=4 SV=1 Uncharacterized protein OS=Glycine max

VIYVVDLR

976

8.41×108 (20.5%)

6.67

8.89

0 PE=4 SV=1 Uncharacterized protein OS=Glycine max

IYVVDLR

877

5.93×108

(14.5%)

6.67

9.35

0 PE=4 SV=1 Uncharacterized protein OS=Glycine max

IYVFVR

795

5.73×108

(14.0%)

9.92

5.25

+1 GN=GLYMA_05G127700 PE=4 SV=1

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Peptides were identified by searching against the UniProt soybean (Glycine max) database (http://www.uniprot.org).

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Theoretical pI, hydrophobicity, and net charge of each peptide was calculated by using PepDraw (http://pepdraw.com).

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Table 2 Chemical antioxidant activity of synthesized peptides

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Peptide sequence

DPPH radical scavenging activity

ABTS•+ radical scavenging activity ORAC

FRAP

VVFVDRL

14.9 ± 0.7b (17.6 ± 0.8AB)

3.14 ± 0.1b (3.71 ± 0.1BC)

136 ± 1.9a (161 ± 4.1A)

63.1 ± 1.2b (74.5 ± 1.4A)

VIYVVDLR

16.1 ± 0.5b (16.5 ± 0.5B)

3.34 ± 0.2ab (3.42 ± 0.2C)

140 ± 2.0a (143 ± 2.1C)

53.4 ± 1.2c (54.7 ± 1.2B)

IYVVDLR

17.8 ± 0.9a (20.3 ± 1.0A)

3.72 ± 0.3a (4.24 ± 0.4A)

139 ± 1.0a (158 ± 1.1B)

68.9 ± 1.4a (78.6 ± 1.6A)

IYVFVR

14.4 ± 0.6b (18.1 ± 0.7AB)

3.22 ± 0.1b (4.05 ± 0.1AB)

136 ± 3.8a (171 ± 4.8A)

62.9 ± 0.5b (79.0 ± 0.6A)

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ABTS•+ radical scavenging activity was expressed as both mM TE/mg peptide (outside brackets) and mM TE/μM peptide (inside brackets).

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DPPH radical scavenging activity and ORAC were expressed as both μM TE/mg peptide (outside brackets) and μM TE/μM peptide (inside brackets).

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FRAP was expressed as both mM Fe2+/mg peptide (outside brackets) and mM Fe2+/μM peptide (inside brackets).

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Values represent as the mean ± SD from triplicate experiments. Different superscript letters in the same group indicate significant difference (p < 0.05,

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Duncan’s test).

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

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Purification and characterization of antioxidant peptides from Alcalase

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