Extraction, Radical Scavenging Activities, and Chemical Composition Identification of Flavonoids from Sunflower (Helianthus annuus L.) Receptacles
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials and Chemicals
2.2. Content Determination of TFSR
2.3. Response Surface Optimization of Extraction Conditions
2.4. Preparation of the Crude Extract and Fractions
2.5. Radical Scavenging/Reducing Assays
2.5.1. DPPH Radical Scavenging Capacity Assays
2.5.2. ABTS Radical Scavenging Capacity Assay
2.5.3. Iron Ion Reducing Ability Assay
2.6. Preliminary Purification of EAF by Polyamide Resin
2.7. Qualitative Analysis
2.7.1. Chromatographic Conditions
2.7.2. Mass Spectrometry Conditions
2.8. Statistical Analysis
3. Results and Discussion
3.1. Response Surface Optimization
3.1.1. Statistical Analysis and Model Fitting
3.1.2. Interaction Analysis
3.1.3. Verification of Predictive Model
3.2. The Flavonoids Contents of the Extracts
3.3. Assay of Radical Quenching
3.3.1. DPPH Radical Scavenging Assay
3.3.2. ABTS Free Radical Scavenging Assay
3.3.3. Iron Ion Reducing Assay
3.4. Polyamide Resin Preliminary Purification Results
3.5. UHPLC-HRMS/MS Analysis Results
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
Abbreviations
TFSR | Total flavonoids in sunflower receptacles |
UHPLC-HRMS/MS | Ultimate 3000 Nano LC System coupled to a Q Exactive HF benchtop Orbitrap mass spectrometer |
DPPH | 2,2-Diphenyl-1-picrylhydrazyl |
ABTS | 2,2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) |
BHT | Butylated hydroxytoluene |
PEF | Petroleum ether fraction |
EAF | Ethyl acetate fraction |
nBUF | n-Butanol fraction |
WAF | Water fraction |
Df | Degree of freedom |
RT | Retention time |
Rt | Retention time |
Fr | Fragment |
CUPRAC | Cupric ion reducing antioxidant capacity |
ANOVA | One-way analysis of variance |
Appendix A. Details of Comparison with Standards
Appendix A.1. Chromatographic Conditions
Appendix A.2. Mass Spectrometry Conditions
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NO. | A Extraction Time (h) | B Ethanol Concentration (%) | C Material to Liquid Ratio (v/w) | D Extraction Temperature (°C) | Y Extraction Yield (%) |
---|---|---|---|---|---|
1 | 2 | 90 | 20 | 85 | 0.21 |
2 | 3 | 60 | 20 | 85 | 0.90 |
3 | 2 | 60 | 20 | 65 | 1.08 |
4 | 2 | 90 | 25 | 65 | 0.35 |
5 | 3 | 60 | 15 | 65 | 0.93 |
6 | 2 | 60 | 20 | 65 | 1.06 |
7 | 2 | 30 | 20 | 45 | 0.41 |
8 | 1 | 60 | 25 | 65 | 0.71 |
9 | 3 | 30 | 20 | 65 | 0.60 |
10 | 2 | 30 | 15 | 65 | 0.51 |
11 | 2 | 30 | 25 | 65 | 0.59 |
12 | 1 | 60 | 20 | 85 | 0.71 |
13 | 2 | 60 | 20 | 65 | 1.00 |
14 | 2 | 60 | 20 | 65 | 1.00 |
15 | 2 | 60 | 25 | 45 | 0.72 |
16 | 2 | 90 | 15 | 65 | 0.35 |
17 | 2 | 90 | 20 | 45 | 0.34 |
18 | 3 | 60 | 20 | 45 | 0.84 |
19 | 2 | 60 | 15 | 85 | 0.76 |
20 | 2 | 30 | 20 | 85 | 0.62 |
21 | 2 | 60 | 20 | 65 | 1.07 |
22 | 3 | 60 | 25 | 65 | 0.94 |
23 | 1 | 30 | 20 | 65 | 0.55 |
24 | 1 | 60 | 15 | 65 | 0.70 |
25 | 2 | 60 | 25 | 85 | 0.77 |
26 | 2 | 60 | 15 | 45 | 0.51 |
27 | 3 | 90 | 20 | 65 | 0.56 |
28 | 1 | 60 | 20 | 45 | 0.64 |
29 | 1 | 90 | 20 | 65 | 0.22 |
Source | Sum of Squares | Df | Mean-Square | F-Value | p-Value | Significance |
---|---|---|---|---|---|---|
Model | 1.800 | 14 | 12.86 | 79.78 | <0.0001 | * * * |
A | 0.127 | 1 | 12.65 | 78.47 | <0.0001 | * * * |
B | 0.130 | 1 | 13.01 | 80.67 | <0.0001 | * * * |
C | 0.008 | 1 | 0.79 | 4.93 | 0.0434 | * |
D | 0.022 | 1 | 2.20 | 13.67 | 0.0024 | * * |
AB | 0.022 | 1 | 2.20 | 13.62 | 0.0024 | * * |
AC | 0.022 | 1 | 0.00 | 0.01 | 0.9172 | |
AD | 0.000 | 1 | 0.00 | 0.02 | 0.8785 | |
BC | 0.000 | 1 | 0.14 | 0.85 | 0.3712 | |
BD | 0.001 | 1 | 2.85 | 17.70 | 0.0009 | * * * |
CD | 0.010 | 1 | 1.01 | 6.27 | 0.0253 | * |
A2 | 0.046 | 1 | 4.61 | 28.61 | 0.0001 | * * * |
B2 | 1.360 | 1 | 136.26 | 845.13 | <0.0001 | * * * |
C2 | 0.131 | 1 | 13.15 | 81.55 | <0.0001 | * * * |
D2 | 0.239 | 1 | 23.97 | 148.63 | <0.0001 | * * * |
Residual | 0.022 | 14 | 0.1612 | |||
Lack of Fit | 0.172 | 10 | 0.1718 | 1.28 | 0.4390 | |
Pure Error | 0.005 | 4 | 0.1347 | |||
Cor Total | 1.820 | 28 | ||||
R2 = 0.9876 | ||||||
R2Adj = 0.9752 | ||||||
CV = 5.93 |
Fraction | Contents of Flavonoids (mg RTE/g DW) |
---|---|
PEF | 15. 14 ± 0.29 d |
EAF | 191.95 ± 2.94 a |
nBUF | 73.41 ± 2.04 b |
WAF | 46.46 ± 0.56 c |
Fraction | Ethanol Elution Concentration | Contents of Flavonoids (mg RTE/g DW) |
---|---|---|
A | 0% | 210.53 ± 3.01 a |
B | 30% | 363.30 ± 2.71 b |
C | 70% | 174.70 ± 2.52 c |
D | 95% | 46.96 ± 1.19 d |
No | Full Name | Ion Mode | Predicted Formula | Rt (min) | Measured (m/z) | Theoretical (m/z) | Error (ppm) | Fr (m/z) |
---|---|---|---|---|---|---|---|---|
1 | Isoquercetin | + | C21H20O12 | 8.36 | 464.09580 | 464.09548 | −0.634 | 465.10309 303.04990 137.02316 97.02859 |
2 | Hispidulin | + | C16H12O6 | 11.87 | 300.06390 | 300.06339 | −1.572 | 301.07114 147.04395 286.04630 121.02824 119.04924 |
3 | (2S,3S)-3,5,7-Trihydroxy-2-(4-hydroxyphenyl)-8-(3-methyl-2-buten-1-yl)-2,3-dihydro-4H-chromen-4-one | + | C20H20O6 | 10.02 | 356.12560 | 356.12599 | 1.099 | 357.13287 175.0750 151.03867 122.03613 108.02065 |
4 | Daidzein | + | C15H10O4 | 10.36 | 254.05780 | 254.05791 | 0.300 | 255.06511 137.02350 119.04904 121.02834 |
5 | Tricin 5-O-β-D-glucoside | + | C23H24O12 | 9.69 | 492.12620 | 492.12678 | 1.156 | 493.13382 175.03880 151.03864 79.05429 |
6 | 5-O-Methylgenistein | + | C16H12O5 | 10.54 | 284.06870 | 284.06847 | −0.948 | 285.07602 197.05949 121.02834 107.04912 |
7 | Cirsiliol | + | C17H14O7 | 11.81 | 330.07370 | 330.07395 | 0.788 | 331.08093 316.05637 121.02825 119.04902 |
8 | Scrophulein | + | C17H14O6 | 10.53 | 314.07940 | 314.07904 | −1.131 | 315.08667 297.08643 |
9 | Silibinin | + | C25H22O10 | 10.06 | 482.12210 | 482.12130 | −1.766 | 483.12943 163.03848 147.04361 |
10 | 1,5-Anhydro-6-deoxy-2-O-(6-deoxy-α-L-mannopyranosyl)-1-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-6-yl] hexitol | - | C27H30O13 | 10.4 | 562.16870 | 562.16864 | −0.131 | 561.16162 339.08701 |
11 | Pectolinarigenin | + | C17H14O6 | 12.29 | 314.07940 | 314.07904 | −1.131 | 315.08682 161.09552 |
12 | Jaceosidin | + | C17H14O7 | 27.98 | 330.07460 | 330.07395 | −1.875 | 331.19049 316.05704 |
13 | 5,2’-Dihydroxy-6,7,8,6’-tetramethoxyflave | + | C19H18O8 | 13.75 | 374.11050 | 374.10017 | −0.825 | 375.10776 345.05911 169.01268 |
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Qiao, Z.; Han, L.; Liu, X.; Dai, H.; Liu, C.; Yan, M.; Li, W.; Han, W.; Li, X.; Huang, S.; et al. Extraction, Radical Scavenging Activities, and Chemical Composition Identification of Flavonoids from Sunflower (Helianthus annuus L.) Receptacles. Molecules 2021, 26, 403. https://doi.org/10.3390/molecules26020403
Qiao Z, Han L, Liu X, Dai H, Liu C, Yan M, Li W, Han W, Li X, Huang S, et al. Extraction, Radical Scavenging Activities, and Chemical Composition Identification of Flavonoids from Sunflower (Helianthus annuus L.) Receptacles. Molecules. 2021; 26(2):403. https://doi.org/10.3390/molecules26020403
Chicago/Turabian StyleQiao, Zian, Lu Han, Xinsheng Liu, Huining Dai, Changmin Liu, Min Yan, Wannan Li, Weiwei Han, Xinlu Li, Silu Huang, and et al. 2021. "Extraction, Radical Scavenging Activities, and Chemical Composition Identification of Flavonoids from Sunflower (Helianthus annuus L.) Receptacles" Molecules 26, no. 2: 403. https://doi.org/10.3390/molecules26020403