Optimization of the Green Chemistry-like Extraction of Phenolic Compounds from Grape (Vitis labrusca L.) and Blackberry (Rubus fruticosus L.) Seeds with Concomitant Biological and Antioxidant Activity Assessments
Abstract
:1. Introduction
2. Results and Discussion
2.1. Effect of Extraction Time and Solid-to-Solvent Ratio on the Total Phenolic Composition and Antioxidant Activity
2.2. Chemical Composition of the Optimized and Lyophilized Extracts
2.3. Phenolic Composition by LC-MS
2.4. Antihemolytic Activity
2.5. Intracellular ROS Generation and Cell Viability
3. Material and Methods
3.1. Chemical Reagents and Cells
3.2. Vegetable Material
3.3. Obtaining Hydroalcoholic Extracts and Extraction Optimization
3.4. Phenolic Composition by LC-MS/MS and UV-VIS Spectrophotometry
3.5. Antioxidant Activity
3.6. Toxicity Profiling and Cellular Antioxidant Activity
3.7. In Vitro Antihemolytic Effect
3.8. Statistical Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
Appendix A
Appendix B
Coded Values | Actual Values | ||
---|---|---|---|
Time (min) | Mass/Volume Ratio (w/v) | Time (min) | Mass/Volume Ratio (w/v) |
−1 | −1 | 40 | 1:40 |
−1 | 1 | 40 | 1:60 |
1 | −1 | 60 | 1:40 |
1 | 1 | 60 | 1:60 |
−1.41 | 0 | 35.9 | 1:50 |
1.41 | 0 | 64.1 | 1:50 |
0 | −1.41 | 50 | 1:35.9 |
0 | 1.41 | 50 | 1:64.1 |
0 | 0 | 50 | 1:50 |
0 | 0 | 50 | 1:50 |
0 | 0 | 50 | 1:50 |
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Tests | Independent Variables (Original and Coded Values) | Total Phenolic Content (mg GAE/100 g) | Flavonoids (mg QE/100 g) | Condensed Tannins (mg CTE/100 g) | Ortho-Diphenolics (mg [ACE]/100 g) | DPPH (mg AAE/100 g) | Fe2+ Chelating Capacity (mg EDTAE/100 g) | FRAP (mg AAE/100 g) | |
---|---|---|---|---|---|---|---|---|---|
Time (min) | 1:X (g/mL) | ||||||||
1 | 40 (−1) | 1:40 (−1) | 2916 ± 7 cde | 62.9 ± 1.7 f | 3242 ± 95 e | 554 ± 20 e | 5870 ± 98 def | 66.1 ± 1.8 c | 9032 ± 328 b |
2 | 40 (−1) | 1:60 (1) | 3609 ± 24 a | 78.8 ± 2.8 b | 3928 ± 315 de | 744 ± 24 bc | 7069 ± 544 a | 55.5 ± 4.9 d | 10,023 ± 98 a |
3 | 60 (1) | 1:40 (−1) | 2914 ± 61 de | 55.9 ± 2.1 g | 3885 ± 353 de | 697 ± 40 cd | 5676 ± 250 f | 45.4 ± 4.1 e | 7518 ± 241 de |
4 | 60 (1) | 1:60 (1) | 2747 ± 117 f | 74,1 ± 1.6 c | 4982 ± 438 bc | 700 ± 78 cd | 6173 ± 36 cde | 85.3 ± 5.3 a | 7294 ± 234 e |
5 | 35.9 (−1.41) | 1:50 (0) | 3623 ± 105 a | 72.7 ± 3.0 cde | 5561 ± 1092 b | 963 ± 64 a | 6650 ± 141 ab | 46.7 ± 2.6 e | 9596 ± 308 a |
6 | 64.1 (1.41) | 1:50 (0) | 3036 ± 45 bc | 69.4 ± 2.6 de | 5030 ± 538 bc | 801 ± 54 b | 5832 ± 79 def | 65.1 ± 6.5 c | 8360 ± 190 c |
7 | 50 (0) | 1:35.9 (−1.41) | 2825 ± 12 ef | 68.5 ± 3.3 e | 3993 ± 449 de | 892 ± 11 a | 5517 ± 80 f | 36.1 ± 2.4 f | 7807 ± 166 d |
8 | 50 (0) | 1:64.1 (1.41) | 3010 ± 51 bcd | 73.6 ± 3.9 cd | 5695 ± 267 ab | 753 ± 43 bc | 6679 ± 511 ab | 74.2 ± 3.7 b | 7565 ± 579 de |
9C | 50 (0) | 1:50 (0) | 3087 ± 72 b | 70.1 ± 2.7 cde | 6384 ± 368 a | 787 ± 80 b | 6388 ± 91 bc | 61.4 ± 1.3 cd | 8661 ± 342 bc |
10C | 50 (0) | 1:50 (0) | 3063 ± 100 b | 69.2 ± 2.3 de | 4547 ± 411 cd | 636 ± 16 d | 5786 ± 293 ef | 64.2 ± 7.2 c | 8305 ± 202 c |
11C | 50 (0) | 1:50 (0) | 2884 ± 89 e | 84.9 ± 3.0 a | 4022 ± 128 de | 648 ± 12 d | 6237 ± 153 bcd | 61.7 ± 2.9 cd | 8660 ± 249 bc |
p-Value (homocedasticity) | 0.503 | 0.999 | 0.942 | 0.914 | 0.709 | 0.954 | 0.97 | ||
p-Value (one-way ANOVA) | <0.001 | <0.001 | <0.001 | <0.001 | <0.001 | <0.001 | <0.001 |
Tests | Independent Variables (Original and Coded Values) | Total Phenolic Content (mg GAE/100 g) | Flavonoids (mg QE/100 g) | Condensed Tannins (mg CTE/100 g) | Ortho-Diphenolics (mg ACE/100 g) | DPPH (mg AAE/100 g) | Fe2+ Chelating Capacity (mg EDTAE/100 g) | FRAP (mg AAE/100 g) | |
---|---|---|---|---|---|---|---|---|---|
Time (min) | 1:X (g/mL) | ||||||||
1 | 40 (−1) | 1:40 (−1) | 923 ± 11 h | 46.2 ± 1.6 e | 579 ± 44 f | 253 ± 32 e | 1769 ± 87 f | 144 ± 8 f | 3060 ± 53 f |
2 | 40 (−1) | 1:60 (1) | 1167 ± 6 g | 58.1 ± 1.7 cd | 637 ± 50 ef | 258 ± 3 e | 2246 ± 197 cde | 227 ± 1 a | 3823 ± 143 d |
3 | 60 (1) | 1:40 (−1) | 1067 ± 25 cd | 55.9 ± 1.8 d | 594 ± 42 f | 350 ± 35 cd | 2152 ± 147 de | 143 ± 4 f | 3437 ± 92 e |
4 | 60 (1) | 1:60 (1) | 1243 ± 13 a | 65.7 ± 1.0 a | 1001 ± 112 ab | 367 ± 13 bcd | 1229 ± 190 g | 215 ± 9 b | 4258 ± 148 a |
5 | 35.9 (−1.41) | 1:50 (0) | 1143 ± 9 de | 60.9 ± 0.6 b | 856 ± 19 cd | 426 ± 66 a | 2470 ± 173 bc | 186 ± 7 c | 3893 ± 78 cd |
6 | 64.1 (1.41) | 1:50 (0) | 1202 ± 8 b | 63.8 ± 1.6 a | 840 ± 40 cd | 409 ± 31 ab | 2366 ± 194 cd | 185 ± 5 c | 3958 ± 93 bcd |
7 | 50 (0) | 1:35.9 (−1.41) | 1073 ± 26 g | 59.4 ± 1.6 bc | 750 ± 45 de | 420 ± 42 ab | 2098 ± 93 e | 120 ± 4 g | 3551 ± 92 e |
8 | 50 (0) | 1:64.1 (1.41) | 1170 ± 21 cd | 60.3 ± 0.4 bc | 1114 ± 141 a | 326 ± 18 d | 2760 ± 127 a | 235 ± 5 a | 4295 ± 317 a |
9C | 50 (0) | 1:50 (0) | 1105 ± 20 f | 58.4 ± 1.4 c | 872 ± 112 bcd | 382 ± 49 abcd | 2264 ± 78 cde | 175 ± 2 d | 3951 ± 80 bcd |
10C | 50 (0) | 1:50 (0) | 1187 ± 30 bc | 59.7 ± 1.1 bc | 872 ± 20 bcd | 372 ± 12 abcd | 2706 ± 94 ab | 166 ± 5 e | 4153 ± 166 ab |
11C | 50 (0) | 1:50 (0) | 1129 ± 10 ef | 61.1 ± 0.9 b | 926 ± 96 bc | 391 ± 15 abc | 2634 ± 79 ab | 183 ± 3 cd | 4088 ± 209 abc |
p-Value (homocedasticity) | 0.949 | 0.976 | 0.889 | 0.699 | 0.948 | 0.79 | 0.879 | ||
p-Value (one-way ANOVA) | <0.001 | <0.001 | <0.001 | <0.001 | <0.001 | <0.001 | <0.001 |
Model Components | Regression Coefficients | Standard Error | t-Value | p-Value | −95% Confidence | +95% Confidence |
---|---|---|---|---|---|---|
Total phenolic content | ||||||
Mean | 3012 | 29.6 | 102 | <0.001 | 2951 | 3073 |
(1) Time (min)(L) | −212 | 18.1 | −11.7 | <0.001 | −249 | −175 |
Time (min)(Q) | 140 | 21.6 | 6.5 | <0.001 | 95.7 | 185 |
(2) Ratio (x/s)(L) | 99 | 18.1 | 5.4 | <0.001 | 61.3 | 136 |
Ratio (x/s)(Q) | −67 | 21.6 | −3.1 | 0.005 | −112 | −22.5 |
1 L to 2 L | −215 | 25.6 | −8.4 | <0.001 | −268 | −162 |
R2 | 0.905 | |||||
Adjusted R2 | 0.888 | |||||
p-value (normality of residues) | 0.763 | |||||
Free radical scavenging activity (DPPH) | ||||||
Mean. | 6171 | 52.1 | 118 | <0.001 | 6064 | 6279 |
(1) Time (min)(L) | −281 | 61.2 | −4.6 | <0.001 | −408 | −155 |
(2) Ratio (x/s)(L) | 418 | 61.2 | 6.8 | <0.001 | 292 | 544 |
R2 | 0.706 | |||||
Adjusted R2 | 0.680 | |||||
p-value (normality of residues) | 0.945 | |||||
Ferric-reducing antioxidant power (FRAP) | ||||||
Mean | 8542 | 99.6 | 85.8 | <0.001 | 8336 | 8747 |
(1) Time (min)(L) | −751 | 61.1 | −12.3 | <0.001 | −877 | −624 |
Time (min)(Q) | 254 | 72.9 | 3.5 | 0.002 | 103 | 404 |
Ra (io/s)(Q) | −396 | 72.9 | −5.4 | <0.001 | −546 | −246 |
1 L to 2 L | −304 | 86.2 | −3.5 | 0.002 | −482 | −126 |
R2 | 0.795 | |||||
Adjusted R2 | 0.765 | |||||
p-value (normality of residues) | 0.401 |
Model Components | Regression Coefficient | Standard Error | t-Value | p-Value | −95% Confidence | +95% Confidence |
---|---|---|---|---|---|---|
Total phenolic content | ||||||
Mean | 1145 | 6.6 | 173 | <0.001 | 1131 | 1159 |
(1) Time (min)(L) | 38.0 | 5.6 | 6.8 | <0.001 | 26.5 | 49.6 |
(2) Ratio (x/s)(L) | 69.7 | 5.6 | 12.5 | <0.001 | 58.2 | 81.2 |
Ratio (x/s)(Q) | −22.6 | 6.4 | −3.5 | 0.002 | −35.8 | −9.5 |
1 L to 2 L | −17.3 | 7.9 | −2.2 | 0.038 | −33.6 | −1.0 |
R2 | 0.706 | |||||
Adjusted R2 | 0.664 | |||||
p-value (normality of residues) | 0.648 | |||||
Fe2+ chelating capacity | ||||||
Mean | 177 | 1.6 | 110 | <0.001 | 173 | 180 |
Time (min)(Q) | 5.4 | 1.5 | 3.5 | 0.002 | 2.2 | 8.6 |
(2) Ratio (x/s)(L) | 39.7 | 1.4 | 29.3 | <0.001 | 36.9 | 42.5 |
R2 | 0.965 | |||||
Adjusted R2 | 0.963 | |||||
p-value (normality of residues) | 0.077 | |||||
Ferric-reducing antioxidant power (FRAP) | ||||||
Mean | 4065 | 51.6 | 78.8 | <0.001 | 3959 | 4171 |
(1) Time (min)(L) | 113 | 31.6 | 3.6 | 0.002 | 47.9 | 178 |
Time (min)(Q) | −140 | 37.7 | −3.7 | 0.001 | −218 | −62.4 |
(2) Ratio (x/s)(L) | 330 | 31.6 | 10.4 | <0.001 | 265 | 395 |
Ratio (x/s)(Q) | −142 | 37.7 | −3.8 | <0.001 | −220 | −63.8 |
R2 | 0.719 | |||||
Adjusted R2 | 0.679 | |||||
p-value (normality of residues) | 0.232 |
Parameters Evaluated | Predicted Mean Values | −95% Prediction | +95% Prediction | Experimental Mean Values | Relative Error (%) | |
---|---|---|---|---|---|---|
Grape | TPC (mg GAE/100 g) | 3847 | 3625 | 4068 | 3623 ± 40 | 5.8 |
FRAP (mg AAE/100 g) | 10,084 | 9339 | 10,829 | 9395 ± 14 | 6.8 | |
DPPH (mg AAE/100 g) | 6914 | 6167 | 7661 | 7102 ± 164 | 2.7 | |
Blackberry | TPC (mg GAE/100 g) | 1249 | 1184 | 1314 | 1295 ± 12 | 3.7 |
FRAP (mg AAE/100 g) | 4180 | 3811 | 4549 | 3804 ± 45 | 9.0 | |
Fe2+ (mg EDTA/100 g) | 241 | 225 | 256 | 231 ± 6 | 4.1 |
Total Phenolic Content (mg GAE/100 g) | Flavonoids (mg QE/100 g) | Condensed Tannins (mg CTE/100 g) | Ortho-Diphenols (mg ACE/100 g) | DPPH (mg AAE/100 g) | Fe2+ Chelating Capacity (mg EDTAE/100 g) | FRAP (mg AAE/100 g) | OH Radical Capture (mg AAE/100 g) | Lipidic Peroxidation Inhibition—IC50 (mg/L) | |
---|---|---|---|---|---|---|---|---|---|
Grape | 2778 ± 218 | 61 ± 0.05 | 2609 ± 257 | 729 ± 81 | 4838 ± 654 | 59 ± 2 | 4972 ± 164 | 258,165 ± 17,568 | 124.1 |
Blackberry | 981 ± 185 | 38.1 ± 0.06 | 409 ± 154 | 233 ± 37 | 1377 ± 117 | 210 ± 8 | 1856 ± 164 | 164,279 ± 5856 | 30.6 |
Grape (mg/100 g) | Blackberry (mg/100 g) | |
---|---|---|
Phenolic acids | ||
Ferulic acid | 1.3 ± 0.2 | 0.8 ± 0.1 |
2,5- Dihydroxybenzoic acid | ND | 33.5 ± 1.6 |
3,4-Dihydroxybenzoic acid | 5.7 ± 0.1 | 34.5 ± 0.9 |
Salicylic acid | ND | 0.2 ± 0.0 |
p-Coumaric acid | 1.2 ± 0.0 | ND |
Caffeic acid | ND | 0.1 ± 0.0 |
Synaptic acid | 0.6 ± 0.0 | 0.9 ± 0.1 |
Sinapaldehyde | ND | 1.0 ± 0.0 |
Flavonoids | ||
(−)-Epicatechin | 833.1 ± 2.7 | 81.5 ± 3.4 |
(+)-Catechin | 573.8 ± 11.2 | 88.8 ± 3.4 |
Epigallocatechin-3-gallate | 785.0 ± 1.6 | 8.4 ± 0.1 |
Quercetin | 231.7 ± 1.2 | 15.2 ± 0.2 |
Quercetin-3-rutinoside | 72.6 ± 1.0 | 13.1 ± 0.6 |
Quercetin-3-glucoside | 2.5 ± 0.1 | 35.3 ± 1.6 |
Hesperidin | 9.1 ± 0.7 | 12.7 ± 0.3 |
Kaempferol | ND | 1.8 ± 0.1 |
Kaempferol-3-rutinoside | 1.5 ± 0.1 | ND |
Taxifolin | 0.4 ± 0.1 | ND |
Pinocembrine | ND | 1.0 ± 0.1 |
Galangine | ND | 0.1 ± 0.0 |
Apigenin | ND | 0.2 ± 0.0 |
Others | ||
Coniferaldehyde | ND | 0.5 ± 0.1 |
Total identified (mg/100 g) | 2519 | 329 |
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© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Junior, T.K.; de Moura, C.; Cruz, T.M.; Marques, M.B.; Carmo, M.A.V.d.; Deolindo, C.T.P.; Daguer, H.; Azevedo, L.; Granato, D. Optimization of the Green Chemistry-like Extraction of Phenolic Compounds from Grape (Vitis labrusca L.) and Blackberry (Rubus fruticosus L.) Seeds with Concomitant Biological and Antioxidant Activity Assessments. Plants 2023, 12, 2618. https://doi.org/10.3390/plants12142618
Junior TK, de Moura C, Cruz TM, Marques MB, Carmo MAVd, Deolindo CTP, Daguer H, Azevedo L, Granato D. Optimization of the Green Chemistry-like Extraction of Phenolic Compounds from Grape (Vitis labrusca L.) and Blackberry (Rubus fruticosus L.) Seeds with Concomitant Biological and Antioxidant Activity Assessments. Plants. 2023; 12(14):2618. https://doi.org/10.3390/plants12142618
Chicago/Turabian StyleJunior, Tufy Kabbas, Cristiane de Moura, Thiago Mendanha Cruz, Mariza Boscacci Marques, Mariana Araújo Vieira do Carmo, Carolina Turnes Pasini Deolindo, Heitor Daguer, Luciana Azevedo, and Daniel Granato. 2023. "Optimization of the Green Chemistry-like Extraction of Phenolic Compounds from Grape (Vitis labrusca L.) and Blackberry (Rubus fruticosus L.) Seeds with Concomitant Biological and Antioxidant Activity Assessments" Plants 12, no. 14: 2618. https://doi.org/10.3390/plants12142618