Extraction and Determination of Polar Bioactive Compounds from Alfalfa (Medicago sativa L.) Using Supercritical Techniques
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Plant Material
2.3. Extractions Plan According to Box-Behnken Design (BBD)
2.4. Supercritical Fluid Extraction of M. sativa at Quarter-Technical Plant
2.5. Maceration
2.6. Total Phenolics Content (TPC)
2.7. Total Flavonoids Content (TFC)
2.8. Supercritical Fluid Chromatography
2.9. Preparation of Standards and Samples Solutions for SFC Determination
3. Results and Discussion
3.1. SFE-scCO2–Result of the Optimization
- for yield–temperature and solvent flow rate,
- for TPC–temperature, pressure, solvent flow rate, linear correlation between pressure and flow rate,
- for TFC–pressure, solvent flow rate, linear correlation between temperature and pressure and squared pressure. It can be noticed that pressure has a crucial impact on the TFC content.
3.2. Result of SFC Analysis
4. Conclusions
Author Contributions
Acknowledgments
Conflicts of Interest
Abbreviations
ANOVA | analysis of variance |
ASE | accelerated solvent extraction |
BBD | Box-Behnken design |
DM | dry mass/dry matter |
F (kg/h−1) | solvent flow rate |
GAE | gallic acid equivalent |
LOF | lack of fit |
m (g) | mass of the extract |
M (kg) | mass of the feedstock |
MAE | microwave solvent extraction |
MSPD | matrix solid phase dispersion |
P (MPa) | pressure |
R2 | coefficient of determination |
RU | rutin equivalent |
RSM | response surface methodology |
scCO2 | supercritical carbon dioxide |
SE | Soxhlet extraction |
SFC | supercritical fluid chromatography |
SFE | supercritical fluid extraction |
SF | supercritical fluid |
SFE-scCO2 | supercritical fluid extraction - supercritical carbon dioxide |
SPE | solid phase extraction |
T (K) | temperature |
TPC | total phenolics content |
USAE | ultrasound assisted extraction |
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Box-Behnken Design | Results | |||||
---|---|---|---|---|---|---|
T, K | P, MPa | F, kg h−1 | Y, % DM | TPC, mg GAE/g DM | TFC, mg RU/g DM | |
E1 | 333.15 (0) | 80.00 (1) | 7.00 (1) | 4.53 | 6.32 | 13.16 |
E2 | 353.15 (1) | 80.00 (1) | 5.00 (0) | 5.76 | 11.86 | 11.42 |
E3 | 353.15 (1) | 20.00 (−1) | 5.00 (0) | 3.47 | 5.38 | 0.00 |
E4 | 313.15 (−1) | 50.00 (0) | 3.00 (−1) | 1.27 | 26.19 | 2.11 |
E5 | 333.15 (0) | 50.00 (0) | 5.00 (0) | 4.19 | 9.65 | 8.96 |
E6 | 353.15 (1) | 50.00 (0) | 3.00 (−1) | 4.58 | 17.00 | 7.13 |
E7 | 333.15 (0) | 50.00 (0) | 5.00 (0) | 3.50 | 11.58 | 8.37 |
E8 | 333.15 (0) | 50.00 (0) | 5.00 (0) | 3.67 | 14.42 | 11.19 |
E9 | 313.15 (−1) | 80.00 (1) | 5.00 (0) | 2.03 | 18.97 | 0.32 |
E10 | 333.15 (0) | 20.00 (−1) | 3.00 (−1) | 3.39 | 7.83 | 0.36 |
E11 | 313.15 (−1) | 20.00 (−1) | 5.00 (0) | 1.47 | 19.01 | 8.96 |
E12 | 313.15 (−1) | 50.00 (0) | 7.00 (1) | 2.72 | 17.59 | 12.15 |
E13 | 333.15 (0) | 80.00 (1) | 3.00 (−1) | 1.89 | 28.08 | 3.41 |
E14 | 333.15 (0) | 20.00 (−1) | 7.00 (1) | 3.31 | 9.01 | 0.36 |
E15 | 353.15 (1) | 50.00 (0) | 7.00 (1) | 6.72 | 7.87 | 10.86 |
T, K | TPC, mg GAE/mL | TFC, mg RU/mL | ΣTPC + TFC, mg/mL |
---|---|---|---|
313.15 | 8.51 | 8.49 | 17.00 |
333.15 | 10.98 | 10.34 | 21.32 |
353.15 | 28.16 | 3.26 | 31.42 |
Output Variables | Regression Equations (Significant Model Terms Are Colored) | Coefficient of Determination, R2 | Model F-Value | Lack of Fit F-Value, LOF | p Value of the Model | |
---|---|---|---|---|---|---|
Y, % DM | coded | Y = 3.7744 + 1.6305A + 0.3180 B + 0.7674 C + 0.4325 AB + 0.1725 AC + 0.68 BC − 0.0308 A2 − 0.5733 B2 + 0.0647 C2 | 0.94 | 8.87 | 4.31 | 0.0135 |
uncoded | Y = −13.5295 + 0.0753 T − 0.2224 P − 1.7857 F + 0.00072 TP + 0.0043 TS + 0.0113 PF–0.000077 T2 − 0.0006 P2 + 0.017 F2 | |||||
TPC, mg GAE/g DM | coded | TPC = 11.8833 − 4.9563 A + 3 B − 4.7887 C + 1.63 AB − 0.1325 AC − 5.735 BC + 3.1371 A2 − 1.2154 B2 + 2.1421 C2 | 0.96 | 12.78 | 0.98 | 0.0059 |
uncoded | TPC = 997.7188 − 5.5927 T − 0.1921 P − 1.8669 F + 0.0027 TP − 0.0033 TS − 0.09558 PF + 0.0078 T2 − 0.00135 P2 + 0.5355 F2 | |||||
TFC, mg RU/g DM | coded | TFC = 9.51 + 0.7337 A + 2.33 B + 2.94 C + 5.02 AB–1.58 AC + 2.44 BC–0.2958 A2–4.04 B2–1.15 C2 | 0.92 | 6.20 | 3.57 | 0.0293 |
uncoded | TFC = −30.68185+0.308521 T–2.46040P + 15.44277F + 0.008358 TP − 0.039437 TF + 0.040625 PF–0.0000740T2 − 0.004484 P2 − 0.287083F2 |
Response Surface Methodology–Graphical Results | |||
---|---|---|---|
Response Surface Plot as a Function of: Pressure (P) and Temperature (T) | Response Surface Plot as a Function of: Solvent Flow Rate (F) and Temperature (T) | Response Surface Plot as a Function of: Pressure (P) and Solvent Flow Rate (F) | |
Y, % DM | |||
TPC, mg GAE/g DM | |||
TFC, mg RU/g DM |
Output Variables | Optimal Conditions | Predicted Value | Actual Value | Confidence Interval | |||
---|---|---|---|---|---|---|---|
T, K (°C) | P, MPa | F, kg h−1 | |||||
Desirability-1.0 | −95% | 95% | |||||
Yield, % DM | 352.99 (79.80) | 78.55 | 6.83 | 7.08 | 7.39 | 6.72 | 7.42 |
TPC, mg GAE/g DM | 353.15 (80) | 71.74 | 3.32 | 21.92 | 20.20 | 20.82 | 23.02 |
TFC, mg RU/g DM | 351.51 (78.40) | 78.29 | 6.72 | 15.32 | 14.89 | 14.55 | 16.09 |
Analytes | Range [mg/mL] | Regression Curve | r2 | LOD [µg/g] | LOQ [µg/g] |
---|---|---|---|---|---|
Flavon | 0.001–1 | y = 1334023x − 3680 | 0.9998 | 1 | 3.3 |
Salicylic acid | 0.05–1 | y = 32304x − 1279 | 0.9992 | 20 | 66 |
Ferulic acid | 0.02–1 | y = 308257x + 1274 | 0.9990 | 10 | 33 |
Naringenin | 0.02–1 | y = 181025x − 4073 | 0.9990 | 10 | 33 |
Apigenin | 0.02–1 | y = 505689x − 5334 | 0.9985 | 10 | 33 |
Samples | Flavonoids Content μg/g DM (n = 3) | Phenolic Acids Content μg/g DM (n = 3) | |||
---|---|---|---|---|---|
Flavon | Naringenin | Apigenin | Salicylic Acid | Ferulic Acid | |
Yield | 1.64 | 35.9 | 12.3 | 49.5 | 1.2 |
TPC | 16.9 | 37.8 | 15.0 | 53.4 | 4.9 |
TFC | 22.0 | 41.4 | 17.9 | 49.9 | 4.2 |
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Wrona, O.; Rafińska, K.; Walczak-Skierska, J.; Możeński, C.; Buszewski, B. Extraction and Determination of Polar Bioactive Compounds from Alfalfa (Medicago sativa L.) Using Supercritical Techniques. Molecules 2019, 24, 4608. https://doi.org/10.3390/molecules24244608
Wrona O, Rafińska K, Walczak-Skierska J, Możeński C, Buszewski B. Extraction and Determination of Polar Bioactive Compounds from Alfalfa (Medicago sativa L.) Using Supercritical Techniques. Molecules. 2019; 24(24):4608. https://doi.org/10.3390/molecules24244608
Chicago/Turabian StyleWrona, Olga, Katarzyna Rafińska, Justyna Walczak-Skierska, Cezary Możeński, and Bogusław Buszewski. 2019. "Extraction and Determination of Polar Bioactive Compounds from Alfalfa (Medicago sativa L.) Using Supercritical Techniques" Molecules 24, no. 24: 4608. https://doi.org/10.3390/molecules24244608