Untargeted Metabolomics Toward Systematic Characterization of Antioxidant Compounds in Betulaceae Family Plant Extracts
Abstract
:1. Introduction
2. Results
2.1. Metabolite Profiling and Antioxidant Activity Assay-Guided Screening of Plant Extracts
2.2. Effect of the Alnus firma Ethanol Extracts (AFEE) on H2O2-Induced HDF Cell Damage
2.3. Bioactivity Assay for A. Firma SPE Fraction Eluates
2.4. Metabolite Profiling of SPE MeOH Eluates of A. firma Extracts
2.5. Preparative HPLC Sub-Fractionation (15 and 31 Min) of 40% MeOH SPE Eluates and Antioxidant Assays
2.6. Bioactivity Validation of Proposed Metabolites
3. Discussion
4. Materials and Methods
4.1. Chemicals and Reagents
4.2. Sample Information and Preparation
4.3. Metabolite Profiling of Plants from Betulaceae
4.4. Solid-Phase Extraction (SPE) for A. Firma Extracts
4.5. Metabolite Profiling and Multivariate Analysis of A. Firma SPE Samples
4.6. Bioactivity Assays
4.6.1. ABTS Radical Scavenging Assay of Plant Extracts Obtained from the Betulaceae Family
4.6.2. ABTS Radical Scavenging Assay for SPE Eluates of Extracts from A. firma
4.7. Cell Culture and Maintenance
XTT Cell Viability Assay using Extracts of A. firma Plants
4.8. Combined Preparative HPLC Analysis and ABTS Assay of 40% SPE Eluates of A. Firma Extracts
4.9. Analysis of Bioactive Prep-HPLC Sub-Fractions Using UHPLC-LTQ-IT-MS/MS and UHPLC-Q-Orbitrap-MS
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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No. a | Putative Identification b | UHPLC-LTQ-IT-MS/MS | |||||
---|---|---|---|---|---|---|---|
RT c (min) | m/z Nega d | m/z Posi e | M.W. f | MSn Fragment Pattern | UV(nm) | ||
1 | Ethyl gallate | 6.72 | 197 | 199 | 198 | 159 [posi] g | 217 |
2 | Myricetin-3-O-galactoside | 7.04 | 479 | 481 | 480 | 319 [posi] | 215,357 |
3 | N.I. 1 | 7.24 | 343 | 345 | 344 | 269 | 247 |
5 | Myricetin-3-O-pentoside | 7.44 | 449 | 451 | 450 | 319 [posi] | 218,355 |
6 | Quercetin-3-O-glucoside | 7.57 | 463 | 465 | 464 | 301 > 179, 151 | 207,255 |
8 | N.I. 2 | 7.72 | 413 | 415 | 414 | 311 | - |
9 | Quercetin-3-O-glucuronide | 7.73 | 477 | 479 | 478 | 301 | - |
11 | Hirsutoside | 8.02 | 491 | 493 | 492 | 311 | - |
13 | N.I. 3 | 8.03 | 537 | - | - | - | - |
14 | Luteolin-7-O-glucuronide | 8.22 | 461 | 463 | 462 | 285, 267 | - |
16 | Pinosylvin diglucoside | 8.27 | 581[M+FA]− | 537 | 536 | 375 > 213 [posi] | 214,274 |
19 | N.I. 6 | 8.47 | 325[M+FA]− | 303[M + Na]+ | 280 | 113 | - |
21 | N.I. 7 | 8.74 | 327 | 329 | 328 | - | - |
22 | Platyphyllonol | 8.79 | 313 | 315 | 314 | 297 [posi] | 205,366 |
Fractions | No a | Putative Identification | UHPLC-LTQ-IT-MS/MS | UHPLC-Q-Orbitrap-MS | ID h | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
RT b (min) | m/z Nega c | m/z Posi d | M.W. e | MSn Fragment Pattern | UV (nm) | RT (min) | m/z Nega | M.F.b g | Δppm | ||||
15 min | 1 | Ethyl gallate | 6.59 | 197 | 199 | 198 | 159 [posi] f | 217 | 5.10 | 197.0449 | C9H10O5 | 2.2 | STD |
2 | Myricetin-3-O-galactoside | 6.95 | 479 | 481 | 480 | 319 [posi] | 215,357 | 5.34 | 479.0837 | C21H20O13 | –0.3 | STD | |
3 | N.I. 1 | 7.17 | 343 | 345 | 344 | 269 | 247 | - | - | - | - | - | |
8 | N.I. 2 | 7.60 | 413 | 415 | 414 | 311 | - | - | - | - | - | - | |
9 | Quercetin-3-O-glucuronide | 7.60 | 477 | 479 | 478 | 301 | - | 5.87 | 477.0685 | C21H18O13 | –0.2 | STD | |
31 min | 14 | Luteolin-7-O-glucuronide | 8.08 | 461 | 463 | 462 | 285, 267 | - | 6.32 | 461.0734 | C21H18O12 | 0.2 | STD |
16 | Pinosylvin diglucoside | 8.13 | 581 [M+COOH]− | 537 | 536 | 375 > 213 [posi] | 214,274 | 6.44 | 581.1887 | C26H32O12 | –0.8 | CCD | |
21 | N.I. 7 | 8.61 | 327 | 329 | 328 | - | - | - | - | - | - | - | |
22 | Platyphyllonol | 8.64 | 313 | 315 | 314 | 297 [posi] | 205,366 | 6.53 | 313.1449 | C19H22O4 | 0.5 | CCD | |
49 | N.I. 17 | 8.36 | 447 | 449 | 448 | 317 | - | - | - | - | - | - | |
50 | N.I. 18 | 8.53 | 405 | 407 | 406 | 303 | 295 | - | - | - | - | - | |
51 | Myricetin | 8.97 | 317 | 319 | 318 | 289 | - | 6.38 | 317.0669 | C15H10O8 | –0.2 | STD | |
52 | N.I. 19 | 8.97 | 287 | 289 | 288 | 153 [posi] | - | - | - | - | - | - | |
53 | N.I. 20 | 7.94 | 542 | - | - | 466 | 215,247,362 | - | - | - | - | - |
Compounds | EC50 (µg/mL) | Regression Curve | R2 |
---|---|---|---|
Ethyl gallate (1) | 61.6 | y = –0.0048x + 0.6745 | 0.9872 |
Quercetin-3-O-glucuronoide (9) | 64.6 | y = −0.0048x + 0.6745 | 0.9872 |
Myricein-3-O-galactoside (2) | 144.9 | y = −0.0024x + 0.7120 | 0.9933 |
Quercetin-3-O-glucoside (6) | 176.1 | y = −0.0019x + 0.6958 | 0.9953 |
Luteolin-7-O-glucuronide (14) | 215.3 | y = −0.0016x + 0.6913 | 0.9959 |
Myricetin (51) | 221.3 | y = −0.0016x + 0.7154 | 0.9997 |
No. | Family | Genus | Species | Collection Area | Collection Date |
---|---|---|---|---|---|
1 | Betulaceae | Alnus | firma | Sin-ri, Goryeong-eup, Goryeong-gun, Gyeongsangbuk-do | 2014-07-23 |
2 | Betulaceae | Alnus | hirsuta | Sangjung-ri, Geumgwang-myeon, Anseong-si, Gyeonggi-do | 2014-07-25 |
3 | Betulaceae | Alnus | japonica | Yonggi-ri, Gibuk-myeon, Buk-gu, Pohang-si, Gyeongsangbuk-do | 2014-07-30 |
4 | Betulaceae | Betula | schmidtii | Icheon-ri, Sangbuk-myeon, Ulju-gun, Ulsan | 2014-08-01 |
5 | Betulaceae | Betula | dahurica | Ungyo-ri, Bangnim-myeon, Pyeongchang-gun, Gangwon-do | 2014-08-08 |
6 | Betulaceae | Betula | pendula | Sogye-ri, Hwanggan-myeon, Yeongdong-gun, Chungcheongbuk-do | 2014-08-14 |
7 | Betulaceae | Carpinus | cordata | Apgok-ri, Bongsan-myeon, Hapcheon-gun, Gyeongsangnam-do | 2014-07-24 |
8 | Betulaceae | Carpinus | turczaninowii | Jiro-ri, Byeongyeong-myeon, Gangjin-gun, Jeollanam-do | 2014-08-12 |
9 | Betulaceae | Carpinus | laxiflora | Seonheul-ri, Jocheon-eup, Jeju-si, Jeju special self-governing province | 2014-08-24 |
10 | Betulaceae | Corylus | heterophylla | Apgok-ri, Bongsan-myeon, Hapcheon-gun, Gyeongsangnam-do | 2014-07-24 |
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Lee, S.; Oh, D.-G.; Singh, D.; Lee, H.J.; Kim, G.R.; Lee, S.; Lee, J.S.; Lee, C.H. Untargeted Metabolomics Toward Systematic Characterization of Antioxidant Compounds in Betulaceae Family Plant Extracts. Metabolites 2019, 9, 186. https://doi.org/10.3390/metabo9090186
Lee S, Oh D-G, Singh D, Lee HJ, Kim GR, Lee S, Lee JS, Lee CH. Untargeted Metabolomics Toward Systematic Characterization of Antioxidant Compounds in Betulaceae Family Plant Extracts. Metabolites. 2019; 9(9):186. https://doi.org/10.3390/metabo9090186
Chicago/Turabian StyleLee, Sunmin, Dong-Gu Oh, Digar Singh, Hye Jin Lee, Ga Ryun Kim, Sarah Lee, Jong Seok Lee, and Choong Hwan Lee. 2019. "Untargeted Metabolomics Toward Systematic Characterization of Antioxidant Compounds in Betulaceae Family Plant Extracts" Metabolites 9, no. 9: 186. https://doi.org/10.3390/metabo9090186