Establishment of a Sonotrode Extraction Method and Evaluation of the Antioxidant, Antimicrobial and Anticancer Potential of an Optimized Vaccinium myrtillus L. Leaves Extract as Functional Ingredient
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents and Plant Material
2.2. Extraction of Phenolic Compounds from Bilberry Leaves by Sonotrode
2.3. Experimental Design
2.4. TPC and Antioxidant Capacity Assays (FRAP and DPPH)
2.5. HPLC-ESI-TOF-MS Analysis
2.6. Antimicrobial Analysis
2.7. Cell Cultures and In Vitro Studies
2.7.1. Cell Lines and Culture
2.7.2. In Vitro Antiproliferative Assay
2.8. Statistical Analysis
3. Results and Discussion
3.1. Fitting the Model
3.2. Analysis of Response Surfaces
3.3. Optimization of Sonotrode Parameters
3.4. Determination of Phenolic Compounds by HPLC-ESI-TOF-MS
3.5. Antimicrobial Activity of V. myrtillus L. Leaves Extract
3.6. Antitumor Activity of V. myrtillus L. Leaves Extract
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Independent Factors | Dependent Factors | |||||
---|---|---|---|---|---|---|
No | X1 | X2 | X3 | TPC (mg GAE/g d.w.) | DPPH (mg TE/g d.w.) | FRAP (mg TE/g d.w.) |
1 | 10 (−1) | 5 (−1) | 60 (0) | 171.82 | 253.76 | 267.28 |
2 | 100 (1) | 5 (−1) | 60 (0) | 130.69 | 126.96 | 148.01 |
3 | 10 (−1) | 45 (1) | 60 (0) | 164.93 | 218.73 | 243.48 |
4 | 100 (1) | 45 (1) | 60 (0) | 173.61 | 151.99 | 184.86 |
5 | 10 (−1) | 25 (0) | 20 (−1) | 170.77 | 190.50 | 272.97 |
6 | 100 (1) | 25 (0) | 20 (−1) | 135.15 | 116.10 | 164.67 |
7 | 10 (−1) | 25 (0) | 100 (1) | 165.60 | 200.43 | 257.46 |
8 | 100 (1) | 25 (0) | 100 (1) | 145.44 | 142.78 | 195.94 |
9 | 55 (0) | 5 (−1) | 20 (−1) | 179.51 | 253.43 | 271.62 |
10 | 55 (0) | 45 (1) | 20 (−1) | 175.30 | 220.19 | 291.60 |
11 | 55 (0) | 5 (−1) | 100 (1) | 174.69 | 234.33 | 289.50 |
12 | 55 (0) | 45 (1) | 100 (1) | 119.82 | 73.73 | 114.33 |
13 | 55 (0) | 25 (0) | 60 (0) | 181.86 | 220.20 | 295.80 |
14 | 55 (0) | 25 (0) | 60 (0) | 179.02 | 224.76 | 291.70 |
15 | 55 (0) | 25 (0) | 60 (0) | 180.80 | 222.77 | 288.05 |
Regression Coefficients | TPC (mg GAE/g d.w.) | DPPH (mg TE/g d.w.) | FRAP (mg TE/g d.w.) | |||
---|---|---|---|---|---|---|
Effect | p-Value | Effect | p-Value | Effect | p-Value | |
β0 | 158.9447 | 0.0000 * | 181.9114 | 0.0000 * | 225.1434 | 0.0000 * |
Linear | ||||||
β1 | −20.1117 | 0.0028 * | −86.5233 | 0.0004 * | −87.6007 | 0.0011 * |
β2 | 2.1615 | 0.1814 | −35.6410 | 0.0023 * | −21.5165 | 0.0176 * |
β3 | −8.3473 | 0.0161 * | −15.3924 | 0.0120 * | −21.3091 | 0.0179 * |
Crossed | ||||||
β12 | 24.9041 | 0.0033 * | 30.0333 | 0.0057 * | 30.3200 | 0.0160 * |
β13 | 7.7287 | 0.0329 * | 8.3744 | 0.0669 | 23.3861 | 0.0264 * |
β23 | −25.3309 | 0.0032 * | −63.6782 | 0.0013 * | −97.5754 | 0.0016 * |
Quadratic | ||||||
β11 | 14.1936 | 0.0028 * | 33.8420 | 0.0012 * | 49.9700 | 0.0016 * |
β22 | 6.1028 | 0.0147 * | 0.8747 | 0.5381 | 30.9723 | 0.0042 * |
β33 | 12.1282 | 0.0038 * | 26.2820 | 0.0020 * | 19.1176 | 0.0110 * |
R2 | 0.9978 | 0.9980 | 0.9992 | |||
p Model | 0.0018 * | 0.0065 * | 0.0024 * | |||
p Lack of fit | 0.1814 | 0.1249 | 0.5379 |
Parameter | Optimal Conditions | ||
---|---|---|---|
Ethanol (%) | 30 | ||
Time (min) | 5 | ||
Amplitude (%) | 55 | ||
TPC | DPPH | FRAP | |
Predicted value (mg/g d.w.) | 195.59 ± 6.76 | 276.85 ± 10.98 | 301.55 ± 18.96 |
Empirical value (mg/g d.w.) | 217.03 ± 4.92 | 271.13 ± 5.84 | 312.21 ± 9.30 |
Coefficient of variation (%) | 7.35 | 1.48 | 2.46 |
Peak No. | Retention Time (min) | m/z Exp. | m/z Calc. | Molecular Formula | Error (ppm) | Score | Proposed Compound | Quantification (mg/g d.w.) |
---|---|---|---|---|---|---|---|---|
Phenolic acids and derivatives | ||||||||
2 | 3.61 | 285.0608 | 285.0610 | C12H14O8 | −0.7 | 99.26 | Dihydroxybenzoic acid pentose | 0.30 ± 0.03 |
3 | 4.42 | 343.1034 | 343.1029 | C15H20O9 | 1.5 | 99.98 | Dihydro-caffeoyl-O-hexoside | 1.01 ± 0.07 |
4 | 4.61 | 341.0869 | 341.0873 | C15H18O9 | −1.2 | 93.96 | Caffeoyl-O-hexoside | <LOQ |
5 | 4.68 | 515.1406 | 515.1401 | C22H28O14 | 1 | 96.08 | Chlorogenoyl hexose | 0.51 ± 0.04 |
6 | 4.96 | 433.0981 | 433.0982 | C17H22O13 | −0.2 | 99.21 | Gallic acid di-pentoside I | <LOQ |
8 | 5.02 | 417.1037 | 417.1033 | C17H22O12 | 1 | 99.99 | Dihydroxybenzoic acid di-pentoside isomer a | <LOQ |
9 | 5.05 | 417.1034 | 417.1033 | C17H22O12 | −0.2 | 99.95 | Dihydroxybenzoic acid di-pentoside isomer b | <LOQ |
10 | 5.12 | 707.1827 | 707.1823 | C32H36O18 | 0.6 | 99.20 | Chlorogenic acid dimer isomer a | 2.54 ± 0.07 |
11 | 5.25 | 707.1826 | 707.1823 | C32H36O19 | 0.4 | 93.54 | Chlorogenic acid dimer isomer b | 7.49 ± 0.14 |
12 | 5.68 | 353.0862 | 353.0873 | C16H18O9 | −3.1 | 100 | Chlorogenic acid * | 90.66 ± 0.40 |
13 | 5.95 | 707.1813 | 707.1823 | C32H36O18 | −1.4 | 95.65 | Chlorogenic acid dimer isomer c | 14.20 ± 0.24 |
14 | 6.26 | 707.1829 | 707.1823 | C32H36O18 | 0.9 | 99.90 | Chlorogenic acid dimer isomer d | 2.26 ± 0.06 |
15 | 6.38 | 691.1888 | 691.1874 | C32H36O17 | 2 | 94.75 | Methyl 5-(6-caffeoyl-glucopyranosyl)-caffeoylquinic acid | 1.67 ± 0.09 |
18 | 7.05 | 337.0909 | 337.0923 | C16H18O8 | −4.2 | 100 | Coumaroylquinic acid isomer a | 1.85 ± 0.10 |
19 | 7.28 | 337.0908 | 337.0923 | C16H18O8 | −4.4 | 99.80 | Coumaroylquinic acid isomer b | 2.75 ± 0.13 |
21 | 7.70 | 367.1023 | 367.1029 | C17H20O9 | −1.6 | 99.70 | Feruloylquinic acid | <LOQ |
24 | 8.33 | 551.1400 | 551.1401 | C25H28O14 | −0.2 | 99.86 | Caffeoyl hexosyl trihydroxymethoxyphenyl propanoic acid | 1.70 ± 0.10 |
27 | 9.00 | 705.1643 | 705.1651 | C32H34O18 | −2.3 | 98.26 | Subulatin | <LOQ |
43 | 11.81 | 411.1650 | 411.1655 | C20H28O9 | −1.2 | 99.94 | Coumaric acid-malonyl-hexoside | 7.35 ± 0.21 |
Flavonoids and derivatives | ||||||||
7 | 5.00 | 305.0660 | 305.0661 | C15H14O7 | −0.3 | 99.70 | Epigallocatechin | 0.87 ± 0.08 |
26 | 8.64 | 531.1337 | 531.1339 | C25H24O13 | −0.4 | 99.98 | 6′′-O-Malonylglycitin | <LOQ |
32 | 9.77 | 595.1298 | 595.1299 | C26H28O16 | −0.2 | 94.74 | Quercetin3-O-arabinosylgalactoside | <LOQ |
34 | 10.09 | 447.0934 | 447.0927 | C21H20O11 | 1.6 | 96.17 | Kaempferol 3-O-glucoside | <LOQ |
35 | 10.13 | 609.1462 | 609.1456 | C27H30O16 | 1 | 100 | Quercetin-rutinoside isomer a * | <LOQ |
36 | 10.29 | 609.1461 | 609.1456 | C27H30O16 | 0.8 | 99.73 | Quercetin-rutinoside isomer b * | 3.59 ± 0.27 |
37 | 10.39 | 463.0885 | 463.0877 | C21H20O12 | 1.7 | 99.84 | Quercetin 3-O-galactoside isomer a | 3.53 ± 0.36 |
38 | 10.53 | 463.0885 | 463.0877 | C21H20O12 | 0.9 | 99.55 | Quercetin 3-O-galactoside isomer b | 3.73 ± 0.40 |
39 | 10.83 | 477.0645 | 477.0669 | C21H18O13 | 5 | 99.99 | Quercetin-3-glucuronide | 7.27 ± 0.50 |
40 | 11.24 | 433.0751 | 433.0771 | C20H18O11 | −4.6 | 99.46 | Quercetin-3-arabinoside | 1.86 ± 0.13 |
41 | 11.41 | 505.0970 | 505.0982 | C23H22O13 | −2.4 | 99.95 | Quercetin 3-(2″-acetylgalactoside) isomer a | 4.37 ± 0.22 |
42 | 11.66 | 447.0921 | 447.0927 | C21H20O11 | −1.3 | 99.72 | Quercetin-3-O-rhamnoside | <LOQ |
44 | 11.90 | 505.0966 | 505.0982 | C23H22O13 | −3.2 | 80.48 | Quercetin 3-(2″-acetylgalactoside) isomer b | 0.28± 0.01 |
45 | 11.95 | 491.0803 | 491.0826 | C22H20O13 | −4.7 | 90.78 | Isorhamnetin-glucuronide | 0.21± 0.05 |
46 | 11.99 | 579.1350 | 579.1350 | C26H28O15 | 0 | 95.15 | Quercetin-3-O-R-arabinofuranoside | 0.17 ± 0.01 |
47 | 12.15 | 505.0988 | 505.0982 | C23H22O13 | 1.2 | 99.99 | Quercetin 3-(2″-acetylgalactoside) isomer c | 0.12 ± 0.01 |
48 | 12.49 | 489.1040 | 489.1033 | C23H22O12 | 1.4 | 97.29 | Kaempferol 3-O-acetyl-glucoside | 0.89 ± 0.03 |
49 | 12.71 | 519.1134 | 519.1139 | C24H24O13 | −1 | 99.66 | Isorhamnetin-acylated-hexoside | <LOQ |
50 | 13.35 | 591.1363 | 591.1350 | C27H28O15 | 2.2 | 99.98 | Quercetin-HMG-rhamnoside | 2.94 ± 0.04 |
51 | 15.09 | 329.0653 | 329.0661 | C17H14O7 | −2.4 | 99.78 | 3′.7-Dimethylquercetin | <LOQ |
Condensed tannins | ||||||||
16 | 6.72 | 577.1337 | 577.1346 | C30H26O12 | −1.6 | 99.98 | Procyanidin dimer | 2.51 ± 0.18 |
22 | 7.75 | 879.1778 | 879.1773 | C45H36O19 | 0.6 | 89.69 | Procyanidin-prodelphinidin trimer(1 A-type bond) | <LOQ |
23 | 8.29 | 865.1976 | 865.1980 | C45H38O18 | −0.5 | 91.43 | Procyanidin trimer | 1.15 ± 0.09 |
25 | 8.57 | 863.1823 | 863.1825 | C45H36O18 | 0.2 | 90.32 | Procyanidin trimer (1A-type bond) | 1.33 ± 0.10 |
28 | 9.11 | 739.1647 | 739.1663 | C39H32O15 | −2.2 | 93.04 | Cinchonain II isomer a | <LOQ |
29 | 9.35 | 739.1680 | 739.1663 | C39H32O15 | 2.3 | 99.20 | Cinchonain II isomer b | 0.05 ± 0.06 |
30 | 9.43 | 451.1025 | 451.1029 | C24H20O9 | −0.9 | 99.97 | Cinchonain I a | 0.31 ± 0.12 |
33 | 9.86 | 577.1337 | 577.1346 | C30H26O12 | 0.9 | 88.65 | Procyanidin dimer | 0.60 ± 0.06 |
Lignans | ||||||||
17 | 6.95 | 553.1544 | 553.1557 | C25H30O14 | −2.4 | 98.82 | Ligustrosidic acid | |
20 | 7.56 | 553.1555 | 553.1557 | C25H30O14 | −0.4 | 99.80 | Ligustrosidic acid | |
Other compounds | ||||||||
1 | 0.45–0.60 | 191.0545 | 191.0556 | C7H12O6 | −4.8 | 99.00 | Quinic acid | |
31 | 9.57 | 535.1448 | 535.1452 | C25H28O13 | −0.7 | 98.95 | Coumaroyl iridoid (I) | |
Sum of phenolic acids | 134.28 ± 2.73 | |||||||
Sum of flavonoids | 35.79 ± 2.11 | |||||||
Sum of phenolic compounds | 170.07 ± 4.84 |
Strain Type | Bilberry Leaves Extract | Sorbic Acid (E-200) | |||
---|---|---|---|---|---|
MIC * (mg/mL) | MBC/MFC * (mg/mL) | MIC * (mg/mL) | MBC/MFC * (mg/mL) | ||
Gram-positive | L. monocytogenes | 3.12 | 6.25 | 3.12 | 6.25 |
L. innocua | 3.12 | 6.25 | 3.12 | 6.25 | |
S. aureus | 0.4 | 0.8 | 0.8 | 1.56 | |
E. faecalis | 3.12 | 6.25 | 3.12 | 6.25 | |
B. cereus | 0.4 | 0.8 | 1.56 | 3.12 | |
Gram-negative | S. enterica | 25 | 50 | 3.12 | 6.25 |
E. coli | 25 | 50 | 3.12 | 6.25 | |
S. sonnei | 25 | 50 | 6.25 | 12.5 | |
P. aeruginosa | 25 | 50 | 1.56 | 3.12 | |
Fungi | C. sake | 25 | 50 | 12.5 | 25 |
Z. bailii | 50 | >50 | 6.25 | 12.5 | |
P. expansum | 50 | >50 | 6.25 | 12.5 | |
A. niger | 50 | >50 | 6.25 | 12.5 |
Cellular Line | IC50 (μg/mL) | |
---|---|---|
Bilberry Leaves Extract | 5-Fluorouracil | |
HT-29 (Human grade II colorectal adenocarcinoma) | 213.2 ± 2.5 | 10.3 ± 0.2 |
T-84 (Human colorectal carcinoma) | 1140.3 ± 5.2 | 27.0 ± 1.6 |
SW-837 (Human grade IV rectum adenocarcinoma) | 936.5 ± 4.6 | 19.4 ± 0.7 |
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Gil-Martínez, L.; Aznar-Ramos, M.J.; del Carmen Razola-Diaz, M.; Mut-Salud, N.; Falcón-Piñeiro, A.; Baños, A.; Guillamón, E.; Gómez-Caravaca, A.M.; Verardo, V. Establishment of a Sonotrode Extraction Method and Evaluation of the Antioxidant, Antimicrobial and Anticancer Potential of an Optimized Vaccinium myrtillus L. Leaves Extract as Functional Ingredient. Foods 2023, 12, 1688. https://doi.org/10.3390/foods12081688
Gil-Martínez L, Aznar-Ramos MJ, del Carmen Razola-Diaz M, Mut-Salud N, Falcón-Piñeiro A, Baños A, Guillamón E, Gómez-Caravaca AM, Verardo V. Establishment of a Sonotrode Extraction Method and Evaluation of the Antioxidant, Antimicrobial and Anticancer Potential of an Optimized Vaccinium myrtillus L. Leaves Extract as Functional Ingredient. Foods. 2023; 12(8):1688. https://doi.org/10.3390/foods12081688
Chicago/Turabian StyleGil-Martínez, Lidia, María José Aznar-Ramos, Maria del Carmen Razola-Diaz, Nuria Mut-Salud, Ana Falcón-Piñeiro, Alberto Baños, Enrique Guillamón, Ana María Gómez-Caravaca, and Vito Verardo. 2023. "Establishment of a Sonotrode Extraction Method and Evaluation of the Antioxidant, Antimicrobial and Anticancer Potential of an Optimized Vaccinium myrtillus L. Leaves Extract as Functional Ingredient" Foods 12, no. 8: 1688. https://doi.org/10.3390/foods12081688