Blanching Pre-Treatment Promotes High Yields, Bioactive Compounds, Antioxidants, Enzyme Inactivation and Antibacterial Activity of ‘Wonderful’ Pomegranate Peel Extracts at Three Different Harvest Maturities
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Plant Material
2.3. Sample Preparation and Blanching Procedure
2.4. Phytochemical Extraction
2.5. Determination of Phytochemicals and Antioxidant Activity
2.5.1. Extract Yield
2.5.2. Total Phenolic Content (TPC)
2.5.3. Total Tannin Content (TTC)
2.5.4. Total Flavonoid Content (TFC)
2.5.5. Total Anthocyanin Content (TAC)
2.5.6. Ascorbic Acid Content (Vitamin C)
2.5.7. 2,2-Diphenyl-1-picryl Hydrazyl (DPPH) Antioxidant Assay
2.5.8. Ferric Ion Reducing Antioxidant Power (FRAP) Antioxidant Assay
2.5.9. 2,2-Azino-Bis (3-Ethylbenzothiazoline-6-sulphonic Acid) (ABTS) Antioxidant Assay
2.6. Enzyme Activity Evaluation
2.6.1. Sample Preparation
2.6.2. Polyphenol Oxidase (PPO) Assay
2.6.3. Peroxidase (POD) Assay
2.7. Ultra-Liquid Chromatography Mass Spectrometry
2.7.1. Determination of Individual Phenolic Acids and Flavonoid Concentration
2.7.2. Data Acquisition
2.8. Microdilution Antimicrobial Assay
2.9. Statistical Analyses
3. Results
3.1. Polyphenol Analysis (Extract Yield, TPC, TTC, TFC, TAC, and Vitamin C)
3.2. Antioxidant Activity (DPPH, FRAP, and ABTS)
3.3. Enzyme Activity (PPO and POD)
3.4. Antibacterial Activity
3.5. Correlation Matrix and Principal Component Analysis
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Harvest Maturity | Treatment | Extract Yield (%) | TPC | TTC | TFC | TAC | Vit C | DPPH | ABTS | FRAP | PPO | POD |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Unripe | Unblanched | 28.1 ± 0.6 c | 12.1 ± 0.1 b | 0.9 ± 0.1 b | 0.8 ± 0.1 d | 0.09 ± 0.00 b | 38.3 ± 0.3 e | 279.1 ± 5.6 c | 721.8 ± 1.2 c | 541.1 ± 89.2 b | 0.5 ± 0.0 b | 3.8 ± 0.0 b |
Blanched | 31.3 ± 0.6 ab | 14.0 ± 0.0 a | 1.0 ± 0.1 a | 1.1 ± 0.1 c | 0.10 ± 0.00 a | 31.0 ± 0.1 f | 359.2 ± 0.0 a | 912.2 ± 0.0 a | 802.5 ± 4.3 a | 0.2 ± 0.1 e | 1.5 ± 0.0 d | |
Ripe | Unblanched | 29.5 ± 0.8 bc | 10.6 ± 0.2 d | 0.8 ± 0.0 c | 1.2 ± 0.0 c | 0.07 ± 0.00 e | 45.2 ± 0.1 d | 243.9± 2.4 d | 718.8 ± 2.4 c | 478.0 ± 74.0 c | 0.4 ± 0.0 c | 3.0 ± 0.0 c |
Blanched | 33.8 ± 1.4 a | 12.2 ± 0.1 b | 1.1 ± 0.1 a | 1.5 ± 0.0 b | 0.08 ± 0.00 c | 53.9 ± 0.5 c | 319.2 ± 4.2 b | 778.8 ± 2.4 b | 525.3 ± 15.8 b | 0.3 ± 0.0 d | 1.5 ± 0.0 d | |
Over Ripe | Unblanched | 26.8 ± 1.0 d | 9.5 ± 0.0 e | 0.6 ± 0.0 d | 1.4± 0.1 b | 0.07 ± 0.00 e | 77.7 ± 0.2 a | 208.8 ± 2.4 e | 612.2 ± 0.0 d | 370.1 ± 3.6 d | 0.6 ± 0.0 a | 4.5 ± 0.0 a |
Blanched | 30.9 ± 0.3 b | 11.6 ± 0.0 c | 0.9 ± 0.1 b | 1.8 ± 0.0 a | 0.08 ± 0.00 d | 70.5 ± 0.1 b | 316.7 ± 2.4 b | 614.7 ± 0.6 d | 447.7 ± 98.8 c | 0.5 ± 0.0 b | 1.5 ± 0.0 d |
Harvest Maturity/Treatment | ||||||
---|---|---|---|---|---|---|
Bioactive Compounds | Unripe | Ripe | Over Ripe | |||
Unblanched | Blanched | Unblanched | Blanched | Unblanched | Blanched | |
Punicalin α and β | 185 ± 3 d | 414 ± 6 a | 228 ± 5 c | 342 ± 9 b | 237 ± 4 c | 436 ± 16 a |
α-Punicalagin | 598 ± 13 b | 618 ± 12 b | 562 ± 13 c | 610 ± 11 b | 523 ± 10 d | 659 ± 19 a |
β-Punicalagin | 696 ± 18 a | 678 ± 18 b | 592 ± 33 d | 644 ± 35 c | 514 ± 19 d | 641 ± 38 b |
Ellagic acid | 308 ± 18 bc | 279 ± 16 c | 324 ± 22 b | 297 ± 34 bc | 306 ± 17 bc | 363 ± 36 a |
Catechin | 99 ± 2 b | 151 ± 6 a | 55 ± 4 d | 84 ± 4 c | 93 ± 3 c | 80 ± 3 c |
Epicatechin | 12 ± 1 c | 227 ± 1 a | 123 ± 1 c | 10 ± 1 c | 20 ± 1 a | 16 ± 2 b |
Gallic acid | 139 ± 2 c | 85 ± 1 d | 13 ± 4 c | 163 ± 4 b | 165 ± 6 b | 200 ± 9 a |
Harvest Maturity | Treatment | Gram Negative | Gram Positive | ||
---|---|---|---|---|---|
Escherichia coli | Klebsiella pneumonia | Staphylococcus aureus | Bacillus subtilis | ||
Unripe | Unblanched | 310 | 310 | 310 | 310 |
Blanched | 160 | 160 | 160 | 160 | |
Ripe | Unblanched | 310 | 310 | 310 | 310 |
Blanched | 160 | 160 | 160 | 160 | |
Over Ripe | Unblanched | 310 | 310 | 310 | 310 |
Blanched | 160 | 160 | 160 | 160 | |
Streptomycin (µg/mL) | 1.6 | 1.6 | 0.8 | 1.6 | |
Solvent Control (70% Ethanol) | - | - | - | - |
EY | TPC | TTC | TFC | TAC | Vit C | DPPH | FRAP | PPO | POD | Pα&β | αPun | βPun | EA | Cat | GA | E. c | K. p | S.a | B. s | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
EY | 1 | |||||||||||||||||||
TPC | 0.61 | 1 | ||||||||||||||||||
TTC | 0.83 | 0.88 | 1 | |||||||||||||||||
TFC | 0.29 | 0.92 | 0.70 | 1 | ||||||||||||||||
TAC | 0.39 | −0.23 | −0.08 | −0.40 | 1 | |||||||||||||||
Vit C | −0.24 | −0.74 | −0.61 | −0.70 | 0.74 | 1 | ||||||||||||||
DPPH | 0.77 | 0.93 | 0.88 | 0.80 | 0.11 | −0.50 | 1 | |||||||||||||
FRAP | 0.24 | 0.27 | −0.18 | −0.39 | 0.97 | 0.75 | 0.07 | 1 | ||||||||||||
PPO | −0.55 | 0.02 | −0.16 | 0.26 | −0.90 | −0.46 | −0.31 | −0.90 | 1 | |||||||||||
POD | −0.86 | −0.52 | −0.57 | −0.24 | −0.64 | 0.01 | −0.74 | −0.54 | 0.72 | 1 | ||||||||||
Pα&β | 0.67 | 0.54 | 0.48 | 0.39 | 0.65 | 0.07 | 0.78 | 0.62 | −0.69 | −0.93 | 1 | |||||||||
αPun | 0.65 | 0.69 | 0.71 | 0.56 | 0.34 | −0.22 | 0.85 | 0.38 | −0.61 | −0.65 | 0.74 | 1 | ||||||||
βPun | 0.48 | 0.87 | 0.84 | 0.83 | −0.33 | −0.73 | 0.80 | −0.30 | 0.01 | −0.24 | 0.29 | 0.76 | 1 | |||||||
EA | −0.09 | −0.41 | −0.32 | −0.45 | 0.59 | 0.54 | −0.17 | 0.74 | −0.72 | −0.07 | 0.17 | 0.34 | −0.13 | 1 | ||||||
Cat | 0.09 | 0.71 | 0.39 | 0.85 | −0.31 | −0.45 | 0.58 | −0.32 | 0.39 | −0.21 | 0.39 | 0.22 | 0.42 | −0.64 | 1 | |||||
GA | −0.01 | −0.54 | −0.28 | −0.57 | 0.73 | 0.84 | −0.27 | 0.76 | −0.64 | −0.04 | 0.09 | 0.16 | −0.31 | 0.75 | −0.60 | 1 | ||||
E. c | −0.85 | −0.67 | −0.71 | −0.47 | −0.56 | 0.06 | −0.87 | −0.47 | 0.62 | 0.93 | −0.94 | −0.79 | −0.45 | −0.01 | −0.39 | −0.08 | 1 | |||
K. p | −0.85 | −0.67 | −0.71 | −0.47 | −0.56 | 0.06 | −0.87 | −0.47 | 0.62 | 0.93 | −0.94 | −0.79 | −0.45 | −0.01 | −0.39 | −0.08 | 1.00 | 1 | ||
S. a | −0.85 | −0.67 | −0.71 | −0.47 | −0.56 | 0.06 | −0.87 | −0.47 | 0.62 | 0.93 | −0.94 | −0.79 | −0.45 | −0.01 | −0.39 | −0.08 | 1.00 | 1.00 | 1 | |
B. s | −0.85 | −0.67 | −0.71 | −0.47 | −0.56 | 0.06 | −0.87 | −0.47 | 0.62 | 0.93 | −0.94 | −0.79 | −0.45 | −0.01 | −0.39 | −0.08 | 1.00 | 1.00 | 1.00 | 1 |
No. | Tentative ID | Retention Time | M-H | MSE Fragment Ions | UV Max | Elemental Formula | References |
---|---|---|---|---|---|---|---|
Phenolic acids | |||||||
Hydroxybenzoic acids | |||||||
1 | Gallic acid * | 4.72 | 169.0146 | 169.014, 125.025, 124.017 | 270,259 | C7H5O5 | Standard |
2 | Ellagic acid * | 14.24 | 300.9969 | 213.597, 137.983, 49.474 | 254,364 | C14H5O8 | Standard |
3 | Ellagic acid hexoside | 11.06 | 463.0539 | 463.053, 300.989, 165.021,114.6995 | - | C20H15O13 | [41] |
4 | Ellagic acid pentoside | 13.59 | 433.0307 | 433.038, 303.7598, 300.995, 299.997, 201.556, 126.237 | 254,361 | C19H13O12 | [41,42] |
Organic acids | |||||||
5 | Citric acid * | 2.52 | 191.0198 | 191.0198, 173.008, 111.008,87.008, 67.017 | - | C5H7O7 | Standard |
Flavonoids | |||||||
Flavanols | |||||||
6 | (+)-Catechin * | 8.31 | 289.0733 | 289.071, 245.082, 203.072, 109.028 | C15H13O6 | Standard | |
7 | (−)-Epicatechin * | 10.19 | 289.0733 | 289.071, 245.082, 203.072, 109.028 | C15H13O6 | Standard | |
Flavones | |||||||
8 | Apigenin-7-O-glucoside Flavonols | 11.07 | 431.1906 | 161.041, 153.091 | 253, 361 | C20H20O10 | [42] |
9 | Kaempferol-3-O-rutinoside | 15.95 | 593.1494 | 593.142, 523.421, 440.063, 316.023, 300.998, 285.033, 211.911, 125.025, 101.031, 80.779 | 275,360 | C27H29O15 | [43] |
10 | Punicalin α and β * | 4.985 | 781.0506 | 781.022, 779.000,783.055, 784.065 | 270, 259 | C34H21O22 | Std |
11 | α Punicalagin * | 6.46 | 1083.0547 | 1083.060, 781.035, 600.986, 541.027, 300.997 | 258,378 | C48H27O30 | Std |
12 | β Punicalagin * | 7.69 | 1083.0558 | 1083.060, 781.035, 600.986, 541.027, 300.997 | 258,378 | C48H27O30 | Std |
13 | Punicalagin isomer | 5.305 | 1083.0547 | 1083.060, 781.035, 600.986, 541.027, 300.997 | 258,378 | C48H27O30 | [41,42] |
14 | Punicalagin derivative | 6.44 | 541.0341 | 541.027, 300.997 | 258,378 | - | [44] |
15 | Galloyl- hexahydroxydiphenol-dehydrohexahydroxydiphenol-hexoside | 6.83 | 951.0479 | 951.042, 907.087, 820.125, 783.062, 300.997, 275.021, 249.035, 102.241 | 255, 230 | C52 H23O19 | [42,44] |
16 | Galloyl- hexahydroxydiphenol -dehydrohexahydroxydiphenol-hexoside (Granatin B) | 12.43 | 951.071 | 951.044, 933.079, 915.9995, 763.081, 614.803, 464.044, 341.011, 302.006, 300.999, 273.002, 169.013, 1233.011 | 273 | C52H23O19 | [42,44] |
17 | Granatin A | 8.34 | 799.0454 | 799.0454, 800.0641, 802.0781 | C34H23O23 | [45,46] | |
18 | Digalloyl- hexa hydroxydiphenol-gluconic acid (punigluconin) | 8.60 | 801.0732 | 801.073, 781.3245 | - | C41H21O18 | [46] |
19 | Digalloyl- hexahydroxydiphenol-hexoside (Pedunculagin II)) | 9.37 | 785.0775 | 785.021, 781.795, 635.099, 483.122, 419.050, 345.086, 301.995, 300.9982, 275.032, 165.021, 125.132 | 264,370 | C34H25O22 | [41,42,45] |
20 | Galloyl-hexahydroxydiphenol-hexoside | 9.76 | 633.0686 | 633.075, 597.082, 464.051, 301.9998, 275.0219, 125.024 | 260,360 | C27H22O18 | [44,47] |
21 | Digalloyl-gallagyl-hexoside | 9.99 | 1085.0889 | 933.081, 783.088, 633.0696, 540.536, 450.989, 301.996, 300.997, 275.014, | 257,370 | C48H29O30 | [41,47] |
22 | Bis-hexahydroxydiphenol-hexoside (pedunculagin I) | 8.83 | 783.0746 | 783.567, 635.087, 541.022, 453.103, 392.036, 291.016, 203.0396 | 260,370 | C34H23O22 | [41,44] |
Unknown | |||||||
23 | Unknown a | 1.58 | 217.0461 | 217.042, 191.0211, 173.006 | - | C12H9O4 | - |
24 | Unknown b | 2.37 | 353.0731 | 353.0742, 191.0211, 173.0062 | - | C12H17O12 | - |
25 | Unknown c | 12.81 | 539.2146 | 541.038, 492.1699, 405.014, 328.021, 302.001, 300.998, 222.005, 169.011, 52.256 | 254,230 | C26H35O12 | - |
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Magangana, T.P.; Makunga, N.P.; la Grange, C.; Stander, M.A.; Fawole, O.A.; Opara, U.L. Blanching Pre-Treatment Promotes High Yields, Bioactive Compounds, Antioxidants, Enzyme Inactivation and Antibacterial Activity of ‘Wonderful’ Pomegranate Peel Extracts at Three Different Harvest Maturities. Antioxidants 2021, 10, 1119. https://doi.org/10.3390/antiox10071119
Magangana TP, Makunga NP, la Grange C, Stander MA, Fawole OA, Opara UL. Blanching Pre-Treatment Promotes High Yields, Bioactive Compounds, Antioxidants, Enzyme Inactivation and Antibacterial Activity of ‘Wonderful’ Pomegranate Peel Extracts at Three Different Harvest Maturities. Antioxidants. 2021; 10(7):1119. https://doi.org/10.3390/antiox10071119
Chicago/Turabian StyleMagangana, Tandokazi Pamela, Nokwanda P. Makunga, Chris la Grange, Maria A. Stander, Olaniyi Amos Fawole, and Umezuruike Linus Opara. 2021. "Blanching Pre-Treatment Promotes High Yields, Bioactive Compounds, Antioxidants, Enzyme Inactivation and Antibacterial Activity of ‘Wonderful’ Pomegranate Peel Extracts at Three Different Harvest Maturities" Antioxidants 10, no. 7: 1119. https://doi.org/10.3390/antiox10071119