Optimization of Ultrasound-Assisted Extraction of Phloretin and Other Phenolic Compounds from Apple Tree Leaves (Malus domestica Borkh.) and Comparison of Different Cultivars from Estonia
Abstract
:1. Introduction
2. Materials and Methods
2.1. Selection of Apple Tree Cultivars
2.2. Collection of Apple Tree Leaves and Preparation of Samples
2.3. Extraction Procedure
2.4. RSM Design for the Optimization of Extraction
2.5. Determination of Total Phenolic Content
2.6. DPPH Free Radical Scavenging Activity
2.7. Identification and Quantification of Polyphenols by LC-MS Method
2.8. Statistical Analysis
3. Results and Discussion
3.1. Optimization of Ultrasound-Assisted Extraction of Phloretin
3.2. Polyphenols Content and Radical Scavenging Activity of Apple Leaves Extracts from Different Cultivars
3.3. Identification and Quantification of Individual Phenolic Compounds in Apple Leaves Extracts
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Material | Apple Cultivar | Solvent | Sample to Solvent Ratio (mg/mL) | Extraction Procedure | Total Phenolic Content * | Phloridzin Content * | Phloretin Content * | Reference |
---|---|---|---|---|---|---|---|---|
Lyophilized leaf powder | Aldas | 70% Ethanol | 25 | Extraction in US bath at 60 °C for 40 min. | 163.35 ± 4.36 | 106.01 ± 4.23 | 1.81 ± 0.07 | [8] |
Auksis | 98.81 ± 1.51 | 108.90 ± 4.32 | 1.52 ± 0.06 | |||||
Ligol | 107.93 ± 2.94 | 114.43 ± 4.72 | 2.40 ± 0.09 | |||||
Lodel | 159.86 ± 4.02 | 109.51 ± 4.62 | 1.40 ± 0.06 | |||||
Lyophilized leaf powder | Szampion | Acidified 30% Methanol (1% HCl) | 20 | 15 min in US bath, 24 h at 4 °C, and 15 min in sonication water bath. | 73.33 | Dihydrochalcones 36.76 | [22] | |
Ozarak Gold | 115.82 | 89.85 | ||||||
Lyophilized leaf powder | Red Fuji | Petroleum Ether | 67 | 3 successive extractions with the different solvents by boiling for 2 h each time. | 7.23 ± 0.78 | n.a. ** | n.a. | [23] |
Ethyl Acetate | 25.17 ± 1.52 | n.a. | n.a. | |||||
75% Ethanol | 56.74 ± 2.80 | 66.1 | n.a. | |||||
Lyophilized leaf powder | Ozarak Gold | Acidified 80% Methanol (1% acetic acid) | 50 | 15 min in US bath, 24 h at 4 °C, and 15 min in US bath. | 160.93 | 110.15 ± 2.43 | n.a. | [12] |
Dried leaves at 55 °C | Red Chief | 50% Methanol | 50 | Vortexing for 2 min repeated 2 times after renewing the solvent. | 24.10 ± 0.11 | 21.90 ± 0.02 | 0.17 ± 1.13 | [24] |
70% Methanol | 24.48 ± 0.29 | 18.79 ± 0.08 | 0.12 ± 0.29 | |||||
50% Ethanol | 25.38 ± 0.38 | 21.07 ± 0.06 | 0.13 ± 0.07 | |||||
70% Ethanol | 30.38 ± 0.50 | 24.43 ± 0.05 | 0.15 ± 0.05 | |||||
Air-dried leaves powder | Golden | Ethanol | 500 | Extraction 5 times with ethanol (1 L × 1, 500 mL × 4) at room temperature for 24 h each time. | 37.1 | 52.0 | n.a. | [25] |
Royal | 34.5 | 20.3 | n.a. | |||||
Apple tree wood dried 22 h at 50 °C, then 4 h at 103 °C | King Jonagold | 60% Ethanol | 5 | Microwave-assisted extraction, 100 °C for 20 min. | Core: 23.8 ± 0.8 Bark: 38.7 ± 1.4 Root: 44.4 ± 2.2 | 6.89 ± 0.55 20.92 ± 1.09 25.21 ± 1.26 | 0.195 ± 0.006 0.275 ± 0.006 0.399 ± 0.020 | [26] |
Run | Extraction Time (A, min) | Sonicator Amplitude (B, %) | Sample Weight per 100 mL Solvent (C, g) | |||
---|---|---|---|---|---|---|
Real Value | Coded Level | Real Value | Coded Level | Real Value | Coded Level | |
1 | 30 | 1 | 55 | 0 | 10 | 1 |
2 | 17.5 | 0 | 55 | 0 | 5.5 | 0 |
3 | 30 | 1 | 55 | 0 | 1 | −1 |
4 | 5 | −1 | 100 | 1 | 5.5 | 0 |
5 | 17.5 | 0 | 55 | 0 | 5.5 | 0 |
6 | 17.5 | 0 | 55 | 0 | 5.5 | 0 |
7 | 17.5 | 0 | 100 | 1 | 10 | 1 |
8 | 30 | 1 | 10 | −1 | 5.5 | 0 |
9 | 17.5 | 0 | 10 | −1 | 10 | 1 |
10 | 5 | −1 | 55 | 0 | 10 | 1 |
11 | 17.5 | 0 | 10 | −1 | 1 | −1 |
12 | 17.5 | 0 | 55 | 0 | 5.5 | 0 |
13 | 5 | −1 | 10 | −1 | 5.5 | 0 |
14 | 17.5 | 0 | 100 | 1 | 1 | −1 |
15 | 30 | 1 | 100 | 1 | 5.5 | 0 |
16 | 17.5 | 0 | 55 | 0 | 5.5 | 0 |
17 | 5 | −1 | 55 | 0 | 1 | −1 |
Quantifier m/z | Qualifier | ||||||
---|---|---|---|---|---|---|---|
Compound | Retention Time (min) | Parent Ion [M+H]+ | Parent Ion [M−H]− | CE * V | Production m/z | CE V | Production m/z |
Chlorogenic acid | 6.00 | 355 | −13 | 166 | −41 | 117 | |
p-Coumaric acid | 9.00 | 165 | −12 | 147 | −20 | 119 | |
Caffeic acid | 6.65 | 179 | 17 | 135 | 32 | 88 | |
Epicatechin | 7.77 | 291 | −14 | 139 | −15 | 123 | |
Phloridzin | 15.14 | 437 | −39 | 107 | −12 | 275 | |
Phloretin | 19.65 | 275 | −15 | 107 | −11 | 169 | |
Rutin | 13.65 | 611 | −20 | 303 | −12 | 465 | |
Quercetin-3-glucoside | 13.75 | 465 | −13 | 303 | −55 | 153 | |
Quercetin-3-galactoside | 13.45 | 465 | −13 | 303 | −55 | 153 | |
Kaempferol-3-glucoside | 12.26 | 449 | −53 | 153 | −13 | 287 | |
Quercetin-3-rhamnoside (quercitrin) | 15.34 | 447 | 25 | 301 | 45 | 271 |
Cultivar | Total Polyphenols | Flavonols | DPPH Radical Scavenging Activity | ||
---|---|---|---|---|---|
mg GA eq./g 1 | mg ChlA eq./g 2 | mg Q eq./g 3 | % 4 | mg GA eq./g 1 | |
Cortland | 37.92 c | 43.46 d | 8.90 c | 30.4 c | 6.54 c |
±0.38 | ±0.22 | ±0.08 | ±0.5 | ±0.12 | |
Karksi renett | 43.90 b | 50.76 b | 10.10 b | 40.1 b | 9.19 b |
±0.83 | ±0.53 | ±0.08 | ±1.1 | ±0.26 | |
Liivi kuldrenett | 37.49 c | 39.60 e | 9.37 d | 30.2 c | 6.46 c |
±1.29 | ±1.26 | ±0.48 | ±0.5 | ±0.18 | |
Paide taliõun | 42.04 b | 46.20 c | 7.47 d | 37.9 b | 8.35 b |
±0.33 | ±0.12 | ±0.09 | ±0.1 | ±0.08 | |
Talvenauding | 37.00 c | 39.60 e | 9.37 c | 28.9 c | 6.06 c |
±1.65 | ±1.26 | ±0.48 | ±1.2 | ±0.32 | |
Tellissaare | 57.74 a | 71.06 a | 12.23 a | 48.4 a | 11.42 a |
±0.99 | ±1.25 | ±0.05 | ±0.8 | ±0.23 | |
Tiina | 35.67 c | 37.28 e | 8.70 c | 31.0 c | 6.65 c |
±0.52 | ±0.08 | ±0.08 | ±1.1 | ±0.25 |
Phenolic Compounds | Cortland | Karksi Renett | Liivi Kuldrenett | Paide Taliõun | Talvenauding | Tellissaare | Tiina |
---|---|---|---|---|---|---|---|
Chlorogenic acid | 227 ± 7 cd | 296 ± 14 b | 178 ± 2 d | 208 ± 3 d | 246 ± 7 c | 204 ± 2 de | 338 ± 11 a |
p-Coumaric acid | 18 ± 1 d | 25 ± 1 b | 25 ± 1 b | 30 ± 1 a | 18 ± 1 d | 23 ± 1 c | 24 ± 1 bc |
Caffeicacid | 15 ± 1 d | 23 ± 1 b | 16 ± 1 d | 27 ± 1a | 20 ± 1c | 17 ± 1 cd | 20 ± 1 bc |
Phloridzin | 17,696 ± 211 c | 20,059 ± 173 b | 15,491 ± 39 d | 18,476 ± 64 c | 11,511 ± 406 f | 31,654 ± 148 a | 12,351 ± 373 e |
Phloretin | 292 ± 7 e | 492 ± 3 b | 466 ± 6 c | 726 ± 7 a | 298 ± 5 e | 505 ± 4 b | 360 ± 2 d |
Quercetin- 3-glucoside | 940 ± 11 c | 1554 ± 8 b | 637 ± 16 e | 447 ± 20 g | 515 ± 19 f | 1915 ± 15 a | 849 ± 12 d |
Quercetin- 3-galactoside | 220 ± 11 d | 896 ± 30 a | 553 ± 9 b | 360 ± 8 c | 409 ± 13 c | 859 ± 16 a | 540 ± 14 b |
Quercetin-3-rhamnoside | 2248 ± 32 cd | 1994 ± 23 e | 3530 ± 44 b | 1988 ± 1 e | 2133 ± 70 d | 2289 ± 31 c | 4290 ± 11 a |
Rutin | 198 ± 3 c | 426 ± 4 a | 86 ± 1 e | 48 ± 1 f | 143 ± 4 d | 313 ± 2 b | 21 ± 1 g |
Kaempferol- 3-glucoside | 979 ± 10 d | 943 ± 15 e | 1377 ± 6 b | 809 ± 8 f | 866 ± 38 f | 1162 ± 21 c | 1680 ± 34 a |
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Ben-Othman, S.; Kaldmäe, H.; Rätsep, R.; Bleive, U.; Aluvee, A.; Rinken, T. Optimization of Ultrasound-Assisted Extraction of Phloretin and Other Phenolic Compounds from Apple Tree Leaves (Malus domestica Borkh.) and Comparison of Different Cultivars from Estonia. Antioxidants 2021, 10, 189. https://doi.org/10.3390/antiox10020189
Ben-Othman S, Kaldmäe H, Rätsep R, Bleive U, Aluvee A, Rinken T. Optimization of Ultrasound-Assisted Extraction of Phloretin and Other Phenolic Compounds from Apple Tree Leaves (Malus domestica Borkh.) and Comparison of Different Cultivars from Estonia. Antioxidants. 2021; 10(2):189. https://doi.org/10.3390/antiox10020189
Chicago/Turabian StyleBen-Othman, Sana, Hedi Kaldmäe, Reelika Rätsep, Uko Bleive, Alar Aluvee, and Toonika Rinken. 2021. "Optimization of Ultrasound-Assisted Extraction of Phloretin and Other Phenolic Compounds from Apple Tree Leaves (Malus domestica Borkh.) and Comparison of Different Cultivars from Estonia" Antioxidants 10, no. 2: 189. https://doi.org/10.3390/antiox10020189
APA StyleBen-Othman, S., Kaldmäe, H., Rätsep, R., Bleive, U., Aluvee, A., & Rinken, T. (2021). Optimization of Ultrasound-Assisted Extraction of Phloretin and Other Phenolic Compounds from Apple Tree Leaves (Malus domestica Borkh.) and Comparison of Different Cultivars from Estonia. Antioxidants, 10(2), 189. https://doi.org/10.3390/antiox10020189