Anti-Inflammatory, Cytotoxic, and Genotoxic Effects of Soybean Oligopeptides Conjugated with Mannose
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Preparation and Characterization of Soybean Oligopeptide (OT), Allulose-Conjugated OT (OT-AL), and D-Mannose-Conjugated OT (OT-Man)
2.3. Cell Culture
2.4. Cytotoxicity Assay
2.5. Hemolysis Assay
2.6. Measurement of Nitric Oxide (NO)/Nitrite
2.7. Pro-Inflammatory Cytokine Determination
2.8. Immunoblotting
2.9. Mutagenicity Testing of the Oligopeptide and Its Mannose-Conjugated Form Using the Salmonella Mutation Assay
2.10. In Vivo Genotoxicity Evaluation of the Oligopeptide and Its Mannose-Conjugated Form Using the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster
2.11. Statistical Analysis
3. Results
3.1. Cytotoxicity of the Oligopeptide and Its Conjugated Forms
3.2. The Effect of the Oligopeptide and Its Conjugated Forms on Nitric Oxide (NO) Production in LPS-Treated Macrophages
3.3. Effect of the Oligopeptide and Its Conjugated Forms on iNOS and COX-2 Levels in LPS-Treated RAW 264.7 Cells
3.4. Effect of OT-AL and OT-Man on the Secretion of IL-6 and TNF-α in LPS-Treated RAW 264.7 Cells
3.5. Mutagenicity of Oligopeptide (OT), D-Mannose (Man), and Mannose Conjugated Oligopeptide (OT-Man)
3.6. In Vivo Genotoxicity Analysis in Drosophila Using SMART
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Cell Lines | Oligopeptide (OT) (µg/mL) | Allulose (AL) (µg/mL) | D-mannose (Man) (µg/mL) | |||
---|---|---|---|---|---|---|
IC20 | IC50 | IC20 | IC50 | IC20 | IC50 | |
HepG2 | >1000 | >1000 | >1000 | >1000 | >1000 | >1000 |
HEK293 | >1000 | >1000 | >1000 | >1000 | >1000 | >1000 |
LX-2 | >1000 | >1000 | >1000 | >1000 | >1000 | >1000 |
RAW 264.7 | 60 | 510 | >1000 | >1000 | >1000 | >1000 |
3T3-L1 Pre-adipocyte | >1000 | >1000 | >1000 | >1000 | >1000 | >1000 |
3T3-L1 Mature-adipocytes | >1000 | >1000 | >1000 | >1000 | >1000 | >1000 |
Cell Lines | Oligopeptide–Allulose (OT-AL) (µg/mL) | Oligopeptide–D-mannose (OT-Man) (µg/mL) | ||
---|---|---|---|---|
IC20 | IC50 | IC20 | IC50 | |
HepG2 | >1000 | >1000 | >1000 | >1000 |
HEK293 | >1000 | >1000 | >1000 | >1000 |
LX-2 | >1000 | >1000 | >1000 | >1000 |
RAW 264.7 | 270 | >1000 | 450 | >1000 |
3T3-L1 Pre-adipocyte | >1000 | >1000 | >1000 | >1000 |
3T3-L1 Mature-adipocytes | >1000 | >1000 | >1000 | >1000 |
Extracts (µg/mL) | % Cell Viability (n = 5) |
---|---|
0 | 100 |
Oligopeptide (OT) | |
100 | 114 ± 20.76 |
200 | 110 ± 23.32 |
400 | 117 ± 20.97 |
800 | 120 ± 24.60 |
Allulose | |
800 | 108 ± 17.96 |
Oligopeptide–Allulose (OT-AL) | |
100 | 104 ± 19.21 |
200 | 110 ± 22.12 |
400 | 111 ± 25.05 |
800 | 113 ± 24.14 |
D-mannose | |
800 | 106 ± 13.26 |
Oligopeptide–D-mannose (OT-Man) | |
100 | 99 ± 12.60 |
200 | 111 ± 20.32 |
400 | 114 ± 17.88 |
800 | 120 ± 16.60 |
Extracts (µg/mL) | % Hemolysis (n = 5) |
---|---|
0.05% triton x-100 | 100 ± 0.00 |
Oligopeptide (OT) | |
100 | 1.98 ± 0.11 |
200 | 2.01 ± 0.16 |
400 | 1.76 ± 0.13 |
800 | 1.65 ± 0.27 |
Allulose | |
800 | 1.76 ± 0.26 |
Oligopeptide–Allulose (OT-AL) | |
100 | 2.19 ± 0.23 |
200 | 2.26 ± 0.16 |
400 | 2.56 ± 0.26 |
800 | 2.75 ± 0.37 |
D-mannose | |
800 | 1.84 ± 0.42 |
Oligopeptide–D-mannose (OT-Man) | |
100 | 2.14 ± 0.09 |
200 | 2.38 ± 0.12 |
400 | 2.54 ± 0.23 |
800 | 2.98 ± 0.32 |
Treatment | Dose (per Plate) | His+ Revertant Colonies per Plate | |||
---|---|---|---|---|---|
TA100 | TA98 | ||||
+S9 | −S9 | +S9 | −S9 | ||
DMSO | - | 131.3 ± 11.0 | 122.8 ± 6.2 | 38.5 ± 3.8 | 30.7 ± 5.0 |
2-AA | 0.5 µg | 599.7 ± 8.7 | - | 325.8 ± 16.2 | - |
AF-2 | 0.01 µg | - | 527.3 ± 15.0 | - | - |
AF-2 | 0.1 µg | - | - | - | 293.0 ± 10.3 |
Oligopeptide (OT) | 0.04 mg | 133.3 ± 6.7 | 128.2 ± 4.5 | 34.8 ± 6.5 | 30.2 ± 1.8 |
0.2 mg | 144.0 ± 10.0 | 131.2 ± 3.8 | 38.2 ± 5.8 | 31.3 ± 7.0 | |
1 mg | 161.5 ± 6.5 | 147.5 ± 7.8 | 44.3 ± 6.3 | 34.5 ± 4.8 | |
5 mg | 228.2 ± 8.8 (k) | 191.8 ± 0.2 (k) | 62.8 ± 9.8 (k) | 43.5 ± 1.8 (k) | |
D-Mannose (Man) | 0.04 mg | 127.5 ± 2.5 | 135.2 ± 5.2 | 37.7 ± 5.7 | 30.5 ± 2.5 |
0.2 mg | 140.3 ± 2.3 | 137.5 ± 4.5 | 37.8 ± 3.8 | 30.8 ± 4.8 | |
1 mg | 139.0 ± 0.7 | 135.2 ± 4.5 | 36.5 ± 7.5 | 27.8 ± 6.5 | |
5 mg | 138.5 ± 4.2 | 129.7 ± 4.7 | 38.5 ± 7.8 | 28.7 ± 6.0 | |
Mannose-conjugated oligopeptide (OT-Man) | 0.04 mg | 139.5 ± 7.2 | 136.0 ± 5.7 | 37.5 ± 4.8 | 28.8 ± 6.2 |
0.2 mg | 137.3 ± 4.0 | 128.2 ± 5.2 | 39.3 ± 9.0 | 29.0 ± 4.7 | |
1 mg | 145.2 ± 4.8 | 127.2 ± 1.8 | 41.2 ± 8.2 | 32.5 ± 4.5 | |
5 mg | 146.3 ± 5.0 (k) | 140.8 ± 9.2 (k) | 45.0 ± 8.7 | 31.3 ± 4.7 |
Samples | Number of Wings | Frequency of Mutant Spots per Individual (Number of Spots) # | ||||
---|---|---|---|---|---|---|
Small Single (1–2 Cells) | Large Single (>2 Cells) | Twin | Total Spots | |||
DI (negative control) | 40 | 0.63 (25) | 0.05 (2) | 0.00 (0) | 0.68 (27) | |
20 mM urethane | 40 | 6.10 (244) + | 3.40 (136) + | 0.68 (27) + | 10.18 (407) + | |
OT | 62.5 µg/mL | 40 | 0.50 (20) − | 0.00 (0) − | 0.00 (0) − | 0.50 (20)− |
125 µg/mL | 40 | 0.50 (20) − | 0.00 (0) − | 0.00 (0) − | 0.50 (20) − | |
250 µg/mL | 40 | 0.28 (11) − | 0.00 (0) − | 0.00 (0) − | 0.28 (11) − | |
500 µg/mL | 40 | 0.28 (11) − | 0.03 (1) i | 0.00 (0) − | 0.30 (12) − | |
1000 µg/mL | 40 | 0.43 (17) − | 0.00 (0) − | 0.00 (0) − | 0.43 (17) − | |
2000 µg/mL | 40 | 0.45 (18) − | 0.03 (1) i | 0.00 (0) − | 0.48 (19) − | |
5000 µg/mL | 40 | 0.35 (14) − | 0.03 (1) i | 0.00 (0) − | 0.38 (15) − | |
Man | 62.5 µg/mL | 40 | 0.40 (16) − | 0.03 (1) i | 0.00 (0) − | 0.43 (17)- |
125 µg/mL | 40 | 0.50 (20) − | 0.03 (1) i | 0.00 (0) − | 0.53 (21) − | |
250 µg/mL | 40 | 0.43 (17) − | 0.03 (1) i | 0.00 (0) − | 0.45 (18) − | |
500 µg/mL | 40 | 0.25 (10) − | 0.03 (1) i | 0.00 (0) − | 0.28 (11) − | |
1000 µg/mL | 40 | 0.25 (10) − | 0.03 (1) i | 0.00 (0) − | 0.28 (11) − | |
2000 µg/mL | 40 | 0.23 (9) − | 0.00 (0) − | 0.00 (0) − | 0.23 (9) − | |
5000 µg/mL | 40 | 0.18 (7) − | 0.00 (0) − | 0.00 (0) − | 0.18 (7) − | |
OT-Man | 62.5 µg/mL | 40 | 0.25 (10) − | 0.00 (0) − | 0.00 (0) − | 0.25 (10) − |
125 µg/mL | 40 | 0.25 (10) − | 0.00 (0) − | 0.00 (0) − | 0.25 (10) − | |
250 µg/mL | 40 | 0.30 (12) − | 0.00 (0) − | 0.00 (0) − | 0.30 (12) − | |
500 µg/mL | 40 | 0.25 (10) − | 0.00 (0) − | 0.00 (0) − | 0.25 (10) − | |
1000 µg/mL | 40 | 0.43 (17) − | 0.00 (0) − | 0.00 (0) − | 0.43 (17) − | |
2000 µg/mL | 40 | 0.13 (5) − | 0.00 (0) − | 0.00 (0) − | 0.13 (5) − | |
5000 µg/mL | 40 | 0.18 (7)− | 0.00 (0)− | 0.00 (0)− | 0.18 (7)− |
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Pitchakarn, P.; Buacheen, P.; Taya, S.; Karinchai, J.; Temviriyanukul, P.; Inthachat, W.; Chaipoot, S.; Wiriyacharee, P.; Phongphisutthinant, R.; Ounjaijean, S.; et al. Anti-Inflammatory, Cytotoxic, and Genotoxic Effects of Soybean Oligopeptides Conjugated with Mannose. Foods 2024, 13, 2558. https://doi.org/10.3390/foods13162558
Pitchakarn P, Buacheen P, Taya S, Karinchai J, Temviriyanukul P, Inthachat W, Chaipoot S, Wiriyacharee P, Phongphisutthinant R, Ounjaijean S, et al. Anti-Inflammatory, Cytotoxic, and Genotoxic Effects of Soybean Oligopeptides Conjugated with Mannose. Foods. 2024; 13(16):2558. https://doi.org/10.3390/foods13162558
Chicago/Turabian StylePitchakarn, Pornsiri, Pensiri Buacheen, Sirinya Taya, Jirarat Karinchai, Piya Temviriyanukul, Woorawee Inthachat, Supakit Chaipoot, Pairote Wiriyacharee, Rewat Phongphisutthinant, Sakaewan Ounjaijean, and et al. 2024. "Anti-Inflammatory, Cytotoxic, and Genotoxic Effects of Soybean Oligopeptides Conjugated with Mannose" Foods 13, no. 16: 2558. https://doi.org/10.3390/foods13162558