In Vitro and In Silico Antioxidant Activity of Novel Peptides Prepared from Paeonia Ostii ‘Feng Dan’ Hydrolysate
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents
2.2. Extraction of Bioactive Peptides from P. Ostii Seed Meal Protein (PoSMP)
2.2.1. Preparation of PoSMP
2.2.2. Preparation of Protein Hydrolysates from PoSMP
2.3. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.4. Purification and Identification of the Bioactive Peptides Extracted from PoSMP
2.4.1. Ultrafiltration
2.4.2. Ion-Exchange Chromatography
2.4.3. Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)
2.4.4. Nano Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) Analysis
2.4.5. Identification of Peptides
2.5. Synthesis of Antioxidant Peptides
2.6. Antioxidant Capacity Assays
2.6.1. DPPH (1, 1-Diphenyl-2-Picrylhydrazyl) Radical-Scavenging Assay
2.6.2. Hydroxyl Radical-Scavenging Activity Assay
2.6.3. ABTS (2, 2’-Azino-bis) Radical-Scavenging Assay
2.6.4. Superoxide Anion Radical-Scavenging Activity
2.7. Quantum Chemical Calculations
2.8. In Vitro Cytotoxicity Assay on L929 Fibroblast Cells
2.9. Statistical Analysis
3. Results and Discussion
3.1. Hydrolysis of PoSMP with Peptidases and Antioxidant Activity
3.2. SDS-PAGE of the P. ostii Seed Meal Protein Hydrolysates (PoSMPHs)
3.3. Purification and Identification of the Antioxidant Peptides
3.3.1. Purification of Alcalase Hydrolysates (AHs) using Ultrafiltration
3.3.2. Purification of F4 Using Anion Exchange Chromatography
3.3.3. RP-HPLC of Fraction P3
3.4. Identification and Characterization of Antioxidant Peptides
3.5. Computational Methodology
3.6. In Vitro Cytotoxicity Assay on L929 Fibroblast Cells
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ABTS | 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) |
AHs | Alcalase hydrolysates |
BDE | Bond Dissociation Energy |
DFT | Density Functional Theory |
DPPH | 1, 1-diphenyl-2-picrylhydrazyl |
FA | Formic Acid |
FBS | Fetal Bovine Serum |
HOMO | Highest Occupied Molecular Orbital |
IP | Ionization Potential |
LUMO | Lowest Unoccupied Molecular Orbital |
NBO | Natural Bond Orbital |
NHs | NeutraseHydrolysates |
O2- | Superoxide Anion |
OH· | Hydroxyl Radical |
PA | Proton Affinity |
PaHs | Papin Hydrolysates |
PeHs | Pepsin hydrolysates |
P. ostii | Paeonia ostii |
PoSMP | Paeonia ostii seed meal protein |
PoSMPHs | Paeonia ostii seed meal protein hydrolysates |
ROS | Reactive oxygen species |
RP-HPLC | Reversed phase high performance liquid chromatography |
TFA | Trifluoroacetic Acid |
THs | Trypsin Hydrolysates |
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Enzyme (activity) | Proteolytic activity of enzyme (U/g) | Optimal pH and temperature | Antioxidant activity | |||
---|---|---|---|---|---|---|
DPPH (%) | ABTS (%) | Hydroxyl (%) | O2−● (%) | |||
Alcalase | 20,000 | pH 9.0; 55 °C | 74.9 ± 1.6 b | 45.5 ± 1.5 a | 56.1 ± 1.8 a | 37.6 ± 1.2 a |
Trypsin | 250,000 | pH 7.5; 45 °C | 66.4 ± 2.0 c | 35.0 ± 1.8 b | 46.0 ± 1.6 c | 32.7 ± 1.6 b |
Papain | 10,000 | pH 7.5; 50 °C | 78.4 ± 0.9 a | 14.2 ± 1.1 d | 39.4 ± 1.9 d | 23.5 ± 1.1 c |
Neutrase | 50,000 | pH 7.5; 45 °C | 57.9 ± 1.7 e | 30.0 ± 2.1 c | 52.1 ± 1.2 b | 21.1 ± 1.7 c |
Pepsin | 10,000 | pH 3.0; 37 °C | 80.2 ± 1.8 a | 10.6 ± 1.3 e | 48.8 ± 1.4 c | 31.3 ± 1.4 b |
Ultrafiltration | F1 | F2 | F3 | F4 | Positive |
---|---|---|---|---|---|
DPPH (%) | 75.4 ± 2.1 a | 61.0 ± 1.9 b | 62.8 ± 2.5 b | 38.4 ± 1.0 c | 74.4 ± 2.1 a (GSH) |
ABTS (%) | 47.4 ± 1.0 c | 36.8 ± 1.2 e | 41.3 ± 1.7 d | 69.7 ± 1.0 a | 58.2 ± 1.8 b (VC) 2 |
Hydroxyl (%) | 50.2 ± 1.7 c | 49.6 ± 1.5 c | 52.4 ± 1.8 b | 68.8 ± 1.2 a | 69.2 ± 1.6 a(GSH) |
O2−● (%) 1 | 32.2 ± 1.1 c | 37.8 ± 1.3 b | 37.3 ± 1.0 b | 30.8 ± 1.4 c | 48.3 ± 1.3 a(GSH) |
FSAP | PVETVR | QEPLLR | EAAY | VLRPPLS | |||||
---|---|---|---|---|---|---|---|---|---|
Atom | Charge | Atom | Charge | Atom | Charge | Atom | Charge | Atom | Charge |
N1 | −0.88759 | N1 | −0.69881 | N1 | −0.88921 | N1 | −0.91883 | N1 | −0.88832 |
H2 | 0.38462 | H2 | 0.40074 | H2 | 0.37610 | C2 | −0.14216 | H2 | 0.37434 |
C16 | 0.69191 | C7 | 0.71086 | O8 | −0.69341 | C5 | 0.83494 | N24 | −0.68781 |
O17 | −0.66103 | O8 | −0.67434 | N9 | −0.83729 | O6 | −0.59491 | H25 | 0.42348 |
N18 | −0.65131 | N9 | -0.6504 | H10 | 0.44054 | O7 | −0.71113 | N27 | −0.80799 |
H19 | 0.41982 | H10 | 0.42203 | C19 | 0.83827 | N10 | −0.66883 | H28 | 0.34732 |
C20 | −0.1693 | O31 | −0.75823 | O20 | −0.62187 | H11 | 0.44739 | N29 | −0.88529 |
O22 | −0.75366 | H32 | 0.47055 | C22 | 0.71839 | C14 | 0.69475 | H30 | 0.39771 |
H23 | 0.48868 | C33 | −0.70866 | O23 | −0.68693 | O15 | −0.65216 | C32 | 0.72417 |
C24 | 0.68167 | C34 | 0.69293 | C38 | 0.69139 | C35 | 0.32133 | O33 | −0.67817 |
O25 | −0.65711 | O35 | −0.66451 | O39 | −0.63917 | O36 | −0.68939 | C39 | 0.72043 |
N26 | −0.63958 | N50 | −0.66717 | N55 | −0.68442 | H37 | 0.48477 | O40 | −0.67631 |
H27 | 0.44129 | H51 | 0.43862 | H56 | 0.41222 | C38 | 0.82460 | N41 | −0.48518 |
C30 | 0.72173 | N53 | −0.8806 | N58 | −0.89905 | O39 | −0.60017 | C46 | 0.70801 |
O31 | −0.69570 | H54 | 0.40155 | H59 | 0.40002 | H46 | 0.39801 | O47 | −0.67163 |
C37 | 0.81978 | N56 | −0.85604 | N61 | −0.79998 | H47 | 0.38678 | O61 | −0.76549 |
O38 | −0.58935 | H57 | 0.34416 | H62 | 0.35620 | O59 | −0.70009 | H62 | 0.48539 |
H42 | 0.38653 | C58 | 0.83508 | O109 | −0.70815 | H60 | 0.51537 | C63 | 0.84110 |
O57 | −0.70917 | O59 | −0.59617 | H110 | 0.49871 | - | - | O64 | −0.62418 |
H58 | 0.51168 | O100 | −0.71190 | H111 | 0.49870 | - | - | O118 | −0.71865 |
- | - | H101 | 0.49801 | - | - | - | - | H119 | 0.53081 |
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Wang, M.; Li, C.; Li, H.; Wu, Z.; Chen, B.; Lei, Y.; Shen, Y. In Vitro and In Silico Antioxidant Activity of Novel Peptides Prepared from Paeonia Ostii ‘Feng Dan’ Hydrolysate. Antioxidants 2019, 8, 433. https://doi.org/10.3390/antiox8100433
Wang M, Li C, Li H, Wu Z, Chen B, Lei Y, Shen Y. In Vitro and In Silico Antioxidant Activity of Novel Peptides Prepared from Paeonia Ostii ‘Feng Dan’ Hydrolysate. Antioxidants. 2019; 8(10):433. https://doi.org/10.3390/antiox8100433
Chicago/Turabian StyleWang, Min, Cong Li, Haoyu Li, Zibo Wu, Bang Chen, Yibo Lei, and Yehua Shen. 2019. "In Vitro and In Silico Antioxidant Activity of Novel Peptides Prepared from Paeonia Ostii ‘Feng Dan’ Hydrolysate" Antioxidants 8, no. 10: 433. https://doi.org/10.3390/antiox8100433
APA StyleWang, M., Li, C., Li, H., Wu, Z., Chen, B., Lei, Y., & Shen, Y. (2019). In Vitro and In Silico Antioxidant Activity of Novel Peptides Prepared from Paeonia Ostii ‘Feng Dan’ Hydrolysate. Antioxidants, 8(10), 433. https://doi.org/10.3390/antiox8100433