Anti-Inflammatory and Antioxidant Effects of Anthocyanins of Trifolium pratense (Red Clover) in Lipopolysaccharide-Stimulated RAW-267.4 Macrophages
Abstract
1. Introduction
2. Materials and Methods
2.1. Preparation of Anthocyanin Fractions from Red Clover
2.2. Cell Culture
2.3. Measurement of Intracellular ROS
2.4. Quantitative Polymerase Chain Reaction (qPCR) Analysis
2.5. Enzyme-Linked Immunosorbent Assay (ELISA) for TNFα
2.6. Western Blot Analysis
2.7. Identification and Quantification of Red Clover Polyphenols Using HPLC/ESI-MS Analysis
2.8. Statistical Analysis
3. Results
3.1. RC and RCA Decreased the Expression of Genes Related to Pro-Inflammatory Markers
3.2. RC and RCA Inhibited LPS-Induced ROS Production
3.3. RCA Inhibited the Activation of NF-kB in LPS-Induced RAW 264.7 Cells
3.4. Identification and Quantification of Red Clover Polyphenols
4. Discussion
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Gene | Forward | Reverse |
---|---|---|
COX2 | GCCTACTACAAGTGTTTCTTTTTGCA | CATTTTGTTTGATTGTTCACACCAT |
GAPDH | GGTGGTCTCCTCTGACTTCAACA | GTTGCTGTAGCCAAATTCGTTGT |
IL1β | GTCACAAGAAACCATGGCACAT | GCCCATCAGAGGCAAGGA |
iNOS | AATCTTGGAGCGAGTTGTGG | CAGGAAGTAGGTGAGGGCTTG |
MCP1 | CTTCTGGGCCTGCTGTTCA | CCAGCCTACTCATTGGGATCA |
NOX1 | TTCACAGTTATTCATATCATTGC | AGAGAACAGAAGCGAGAG |
NRF2 | CTCGCTGGAAAAAGAAGTG | CCGTCCAGGAGTTCAGAGG |
TNFα | GGCTGCCCCGACTACGT | ACTTTCTCCTGGTATGAGATAGCAAAT |
Number | RT (min) | Isoflavone Derivatives | Amount (μg/g dw) | [M-H]-m/z |
---|---|---|---|---|
1 | 27.1 | Luteolin 7-O-β-D-glucoside | 368.5 ± 14.7 | 449, 287 |
2 | 28.7 | unknown tetrahydroxyflavone glucoside | 2394.3 ± 28.0 | 449, 287 |
3 | 29.2 | unknown tetrahydroxyflavone glucoside | 1356.2 ± 18.0 | 449, 287 |
4 | 31.5 | Isoquercitrin-6″-O-malonate | 1677.9 ± 20.9 | 551, 303 |
5 | 32.3 | Pratensein-7-O-β-D-glucoside | 682.1 ± 6.3 | 463, 301 |
6 | 33.4 | Genistin | 12892.3 ± 35.6 | 433, 271, 153 |
7 | 33.9 | Hyperoside | 3103.0 ± 20.9 | 465, 303 |
8 | 34.3 | Isoquercetriin | 8940.1 ± 16.6 | 465, 303 |
9 | 35.0 | Apigenin-7-O-β-D-glucoside | 512.1 ± 1.7 | 433, 271 |
10 | 36.1 | Pseudobaptigenin | 5809.6 ± 14.0 | 283 |
11 | 37.0 | unknown tetrahydroxyflavone glucoside | 24560.7 ± 60.7 | 449, 287 |
12 | 38.4 | Kaempferol or Luteolin | 5022.7 ± 37.8 | 287 |
13 | 38.9 | Gemosteom-7-O-β-D-glucoside-6″-O-malonate | 17933.8 ± 123.6 | 519, 271 |
14 | 39.3 | unknown tetrahydroxyflavone glucoside | 2691.2 ± 13.6 | 449, 287 |
15 | 40.2 | 3-methylquercetin-7-O-β-D-glucoside | 7838.7 ± 7.6 | 479, 317 |
16 | 42.7 | Kaempferol or Luteolin | 12876.9 ± 16.1 | 287 |
17 | 44.3 | Pratensein-7-O-β-D-glucoside-6″-malonate | 2437.5 ± 8.8 | 549, 317 |
18 | 44.9 | Pseudobaptigenin-7-O-β-D-glucoside | 2280.1 ± 8.4 | 445, 283 |
19 | 45.5 | Kaempferol or Luteolin | 2213.7 ± 9.1 | 287 |
20 | 46.7 | Glycitein | 312.1 ± 15.1 | 285, 167 |
21 | 47.5 | Pseudobaptigenin-7-O-β-D-glucoside-6″-O-malonate | 2361.1 ± 47.3 | 445, 283, 137 |
22 | 52.2 | Formononetin-7-O-β-D-glucoside-6″-O-malonate | 2231.1 ± 19.0 | 517, 269 |
23 | 53.7 | Calysosin-Glucoside-Malonate | 213.7 ± 15.9 | 533, 285, 137 |
24 | 57.0 | Prunetin-4′-O-β-D-glucoside-6″-O-malonate | 2089.3 ± 38.1 | 533, 285 |
25 | 58.7 | Formononetin | 497.3 ± 13.6 | 269, 137 |
26 | 60.2 | Biochanin A-7-O-β-D-glucoside-6″-O-malonate | 3713.8 ± 3.1 | 533, 285 |
27 | 66.8 | Biochanin A | 1059.8 ± 14.8 | 285 |
Total | 128069.5 ± 628.9 |
Number | RT (min) | Anthocyanins | Amount (μg/g dw) | [M-H]-m/z |
---|---|---|---|---|
1 | 27.5 | Delphinidin-3, 5-O-diglucoside | 139.0 ± 1.1 | 627, 465, 303 |
2 | 29.9 | Cyanidin-3-O-galactoside | 380.3 ± 2.2 | 449,287 |
3 | 31.5 | Cyanidin-3-O-glucoside | 47.7 ± 3.3 | 449,287 |
4 | 33.5 | Petunidin-3-O-galactoside | 145.5 ± 4.4 | 479,317 |
5 | 34.4 | Peonidin-3-O-galactoside | 633.6 ± 5.5 | 463,301 |
6 | 36.3 | Malvidin-3-O-galactoside | 2129.4 ± 6.6 | 493,331 |
7 | 37.3 | Petunidin-3-O-rutinoside | 123.9 ± 7.7 | 625,479,317 |
Total | 3599.5 ± 10.4 |
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Lee, S.G.; Brownmiller, C.R.; Lee, S.-O.; Kang, H.W. Anti-Inflammatory and Antioxidant Effects of Anthocyanins of Trifolium pratense (Red Clover) in Lipopolysaccharide-Stimulated RAW-267.4 Macrophages. Nutrients 2020, 12, 1089. https://doi.org/10.3390/nu12041089
Lee SG, Brownmiller CR, Lee S-O, Kang HW. Anti-Inflammatory and Antioxidant Effects of Anthocyanins of Trifolium pratense (Red Clover) in Lipopolysaccharide-Stimulated RAW-267.4 Macrophages. Nutrients. 2020; 12(4):1089. https://doi.org/10.3390/nu12041089
Chicago/Turabian StyleLee, Sang Gil, Cindi R. Brownmiller, Sun-Ok Lee, and Hye Won Kang. 2020. "Anti-Inflammatory and Antioxidant Effects of Anthocyanins of Trifolium pratense (Red Clover) in Lipopolysaccharide-Stimulated RAW-267.4 Macrophages" Nutrients 12, no. 4: 1089. https://doi.org/10.3390/nu12041089
APA StyleLee, S. G., Brownmiller, C. R., Lee, S.-O., & Kang, H. W. (2020). Anti-Inflammatory and Antioxidant Effects of Anthocyanins of Trifolium pratense (Red Clover) in Lipopolysaccharide-Stimulated RAW-267.4 Macrophages. Nutrients, 12(4), 1089. https://doi.org/10.3390/nu12041089