The Antioxidant and Anti-Inflammatory Properties of Merremia umbellata Extract
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemical Reagents
2.2. Plant Collection, Voucher Specimen Information, and Extract Preparation Method
2.3. Determination of the Total Phenolic and Total Flavonoid Contents
2.4. Determination of the Antioxidant Activity
2.5. Cell Culture and Treatment
2.6. MTT Assay
2.7. Nitric Oxide Assay
2.8. RNA Isolation, cDNA Synthesis, and Quantitative RT-PCR Analysis
2.9. Protein Extraction and Western Blot Analysis
2.10. UHPLC-PDA-QTOF/MS Analysis
2.11. Quantitative Analysis of Major Components
2.12. Molecular Docking Analysis
2.13. Statistical Analysis
3. Results
3.1. Antioxidant Properties of the MU Extract
3.2. Effect of the MU Extract on Cytotoxicity in LPS-Stimulated RAW264.7 Macrophages
3.3. Effect of MU Extract on LPS-Induced NO Production and Pro-Inflammatory Enzyme Expression in RAW264.7 Macrophages
3.4. Effect of MU Extract on LPS-Induced TLR4/NF-κB Signaling Activation in RAW264.7 Macrophages
3.5. Constituents of MU Extract
3.6. Identification of TLR4 and Compounds from MU Extract
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene | Sequence (5′→3′) |
---|---|
β-actin | F 1: AGA TGA CCC AGA TCA TGT TTG AGA |
R 2: CAC AGC CTG GAT GGC TAC GT | |
COX-2 | F: CAT ACT CAA GCA GGA GCA TCC |
R: ACC GCT CAG GTG TTG CAC GTA GTC | |
IL-1β | F: GCA ACT GTT CCT GAA CTC AAC T |
R: ATC TTT TGG GGT CCG TCA ACT | |
IL-6 | F: TCG GAG GCT TAA TTA CAC ATG TTC |
R: TGC CAT TGC ACA ACT CTT TTC T | |
NOS2 | F: CAG CTG GGC TGT ACA AAC CTT |
R: CAT TGG AAG TGA AGC GTT TCG | |
TNF-α | F: TGG CCT CCC TCT CAT CAG TT |
R: CAG GCT TGT CAC TCG AAT TTT G | |
TLR4 | F: GCA GAA AAT GCC AGG ATG ATG |
R: AAC TAC CTC TAT GCA GGG ATT CAA G |
DPPH (% Inhibition) | ABTS (% Inhibition) | TPC (mg GAE c) | TFC (μg CE d) | |
---|---|---|---|---|
DPPH a | 1 | 0.982 ** | 0.979 ** | 0.953 ** |
ABTS b | 1 | 0.969 ** | 0.931 ** | |
TPC | 1 | 0.979 ** | ||
TFC | 1 |
No. | tR, min | Molecular Formula | UV | Tentative Identification | Class | Negative Mode | Positive Mode | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Detected Ion [M–H]− | Calculated Ion [M–H]− | Error (ppm) | Major Fragment Ions | Detected Ion [M+H]+ | Calculated Ion [M+H]+ | Error (ppm) | Major Fragment Ions | ||||||
1 | 2.98 | C18H24O12 | 266, 271, 277 | Unknown | Unknown | 431.1189 | 431.1190 | −0.7 | 327, 165 | 455.1133 | – | – | _ |
2 | 3.12 | C16H18O9 | 326 | Chlorogenic acid a | Phenolic acid | 353.0877 | 353.0873 | 1.1 | 191 | 355.1019 | 355.1029 | −2.8 | 163 |
3 | 3.43 | C9H8O4 | 323 | Caffeic acid a | Phenolic acid | 179.0344 | 179.0344 | 0.0 | 135 | – | – | – | – |
4 | 3.79 | C18H28O9 | – | Unknown | Miscellaneous | 387.1664 | 387.1655 | 2.3 | – | – | – | – | – |
5 | 3.89 | C19H30O8 | 240 | Roseoside a | Miscellaneous | 431.1925 [M+HCOO]− | 431.1917 [M+HCOO]− | 1.9 | 281, 153 | 387.2002 | 387.2019 | −4.4 | 207 |
6 | 4.04 | C15H20O7 | – | Unknown | Unknown | 311.1136 | 311.1131 | 1.6 | – | – | – | – | – |
7 | 4.23 | C12H18O4 | 219, 310 | Unknown | Unknown | 225.1131 | 225.1127 | 1.8 | – | 227.1279 | 227.1283 | −1.8 | 209 |
8 | 4.31 | C14H24O17 | 280, 284 | Unknown | Unknown | 463.0916 | 463.0935 | −4.1 | – | – | – | – | – |
9 | 4.49 | C17H24O10 | 219 | Unknown | Unknown | 387.1296 | 387.1291 | 1.3 | 361, 300 | – | – | – | – |
10 | 4.54 | C27H30O16 | 255, 353 | Quercetin–O–dihexoside | Flavonoid glycoside | 609.1464 | 609.1456 | 1.3 | 300, 271 | 611.1594 | 611.1612 | −2.9 | 303 |
11 | 4.60 | C27H30O16 | 255, 352 | Rutin a | Flavonoid glycoside | 609.1458 | 609.1456 | 0.3 | 300, 271 | 611.1612 | 611.1612 | 0.0 | 465, 303 |
12 | 4.70 | C21H20O12 | 260, 349 | Hyperoside a | Flavonoid glycoside | 463.0873 | 463.0877 | −0.9 | 300, 271, 255 | 465.1029 | 465.1033 | −0.9 | 303 |
13 | 4.77 | C21H20O12 | 254, 350 | Isoquercitrin a | Flavonoid glycoside | 463.0872 | 463.0877 | −1.1 | 300, 271, 255 | 465.103 | 465.1033 | −0.6 | 303 |
14 | 4.87 | C21H20O11 | 268, 347 | Luteolin–O–hexoside | Flavonoid glycoside | 447.0929 | 447.0927 | 0.4 | 285 | 449.107 | 449.1084 | −3.1 | 287 |
15 | 5.05 | C20H18O11 | 270, 342 | Quercetin–O–pentoside | Flavonoid glycoside | 433.0772 | 433.0771 | 0.2 | 300, 271, 255 | 435.0919 | 435.0927 | −1.8 | 303 |
16 | 5.12 | C25H24O12 | 325 | 3,4–Dicaffeoylquinic acid a | Phenolic acid | 515.1191 | 515.1190 | 0.2 | 353, 191 | 517.1343 | 517.1346 | −0.6 | 197, 163 |
17 | 5.22 | C25H24O12 | 327 | 3,5–Dicaffeoylquinic acid a | Phenolic acid | 515.1185 | 515.1165 | 3.9 | 353, 191 | 517.1335 | 517.1346 | −2.1 | 499, 163 |
18 | 5.28 | C21H20O11 | 255, 345 | Quercitrina | Flavonoid glycoside | 447.093 | 447.0927 | 0.7 | 300, 271, 255 | 449.1086 | 449.1084 | 0.4 | 303 |
19 | 5.34 | C22H22O12 | 267, 336 | Isorhamnetin–O–hexoside | Flavonoid glycoside | 477.1033 | 477.1033 | 0.0 | 314, 299, 271 | 479.117 | 497.119 | −4.2 | 317, 303 |
20 | 5.41 | C20H18O10 | 265, 346 | Kaempferol–O–pentoside | Flavonoid glycoside | 417.0823 | 417.0822 | 0.2 | 284, 255, 227 | 419.0963 | 419.0978 | −3.6 | 287 |
21 | 5.51 | C25H24O12 | 327 | 4,5–Dicaffeoylquinic acid a | Phenolic acid | 515.119 | 515.1190 | 0.0 | 353, 191 | 517.1345 | 517.1346 | −0.2 | 327 |
22 | 5.80 | C21H20O10 | 263, 344 | Afzelin a | Flavonoid glycoside | 431.0973 | 431.0978 | −1.2 | 285, 255, 227 | 433.1144 | 433.1135 | 2.1 | 287 |
23 | 6.07 | C17H17NO3 | 287, 312 | Paprazine [20] | Tyramine (Alkaloid) | 282.1133 | 282.1130 | 1.1 | 161, 119 | 284.1289 | 284.1287 | 0.7 | 147 |
24 | 6.28 | C18H19NO4 | 285, 318 | Feruloyltyramine | Tyramine (Alkaloid) | 312.1244 | 312.1236 | 2.6 | 178 | 314.1392 | 314.1392 | 0.0 | 177 |
25 | 6.43 | C23H24O12 | 271, 330 | Unknown | Flavonoid glycoside | 491.1188 | 491.1190 | −0.4 | 329, 271 | 493.1334 | 493.1346 | −2.4 | 270, 242 |
26 | 6.53 | C30H26O13 | 266, 314 | Tiliroside | Flavonoid glycoside | 593.1301 | 593.1295 | 1.0 | 447, 284 | 595.1445 | 595.1452 | −1.2 | 287 |
27 | 6.69 | C34H30O15 | 330 | 3,4,5–tricaffeoylquinic acid a | Phenolic acid | 677.1519 | 677.1506 | 1.9 | 515, 439, 315 | 679.1646 | 679.1663 | −2.5 | 317 |
28 | 7.28 | C16H17NO2 | 275 | Unknown | Alkaloid | 254.1185 | 254.1181 | 1.6 | 161 | 256.1338 | 256.1338 | 0.0 | – |
29 | 7.84 | C18H16O10S | 272, 333 | Unknown | Flavonoid sulfate | 423.0385 | 423.0386 | −0.2 | 343, 328, 313, 298, 285, 270 | 425.054 | 425.0542 | −0.5 | 345, 331 |
30 | 8.92 | C42H66O14 | – | Unknown | Triterpenoid saponin | 793.4399 | 793.4374 | 3.2 | 631 | – | – | – | – |
31 | 9.55 | C19H18O6 | – | Unknown | Unknown | – | – | – | – | 343.1189 | 343.1182 | 2.0 | 313, 287 |
32 | 9.58 | C16H14O5 | 288, 326 | Unknown | Flavonoid phytoalexin | 285.0769 | 285.0763 | 2.1 | 164 | 287.092 | 287.0919 | 0.3 | 153 |
No. | Compound Name | Contents in MU Extract a (mg g−1) | Linear Equations b | Correlation Coefficient (r2) | LOD c (ng mL−1) | LOQ c (ng mL−1) |
---|---|---|---|---|---|---|
16 | 3,4-Dicaffeoylquinic acid | 4.91 | y = 6100.3x − 1185.4 | 0.9999 | 134 | 406 |
17 | 3,5-Dicaffeoylquinic acid | 4.29 | y = 7585.3x + 87.11 | 0.9999 | 164 | 498 |
18 | Quercitrin | 7.89 | y = 7297.1x − 1050.2 | 0.9999 | 87 | 262 |
21 | 4,5-Dicaffeoylquinic acid | 2.66 | y = 15177x − 2183.9 | 0.9999 | 119 | 360 |
Binding Ligand | Amino Acid Involved Interaction | Binding Score (kcal/mol) |
---|---|---|
3,4-Dicaffeoylquinic acid | ASN44, TYR46, PHE45, LYS47, PRO28, PRO49, ASN51, ASP50 | −3.65 |
3,5-Dicaffeoylquinic acid | LYS47, HIS68, GLU94, GLY70, SER71, SER73, TYR72 | −3.78 |
Quercitrin | TYR46, LEU43, PHE45, ASP50, ASN51, PRO28 | −5.19 |
4,5-Dicaffeoylquinic acid | PRO28, CYS29, ASN51, ASP50, LEU52, PHE54, PRO53, NAG1, FUL805 | −4.68 |
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Lee, S.C.; Ahn, J.; Kim, J.; Lee, J.-Y.; Kim, J.; Uddin, M.S.; Lee, S.W.; Kim, C.Y. The Antioxidant and Anti-Inflammatory Properties of Merremia umbellata Extract. Antioxidants 2023, 12, 2037. https://0-doi-org.brum.beds.ac.uk/10.3390/antiox12122037
Lee SC, Ahn J, Kim J, Lee J-Y, Kim J, Uddin MS, Lee SW, Kim CY. The Antioxidant and Anti-Inflammatory Properties of Merremia umbellata Extract. Antioxidants. 2023; 12(12):2037. https://0-doi-org.brum.beds.ac.uk/10.3390/antiox12122037
Chicago/Turabian StyleLee, Sunny Chung, Jongmin Ahn, Jina Kim, Joo-Yeon Lee, Juhae Kim, Md. Salah Uddin, Sang Woo Lee, and Choon Young Kim. 2023. "The Antioxidant and Anti-Inflammatory Properties of Merremia umbellata Extract" Antioxidants 12, no. 12: 2037. https://0-doi-org.brum.beds.ac.uk/10.3390/antiox12122037