Optimization of Phytochemical-Rich Citrus maxima Albedo Extract Using Response Surface Methodology
Abstract
:1. Introduction
2. Results
2.1. Optimization of Extraction Conditions Using Response Surface Methodology
2.1.1. Model Fitting, Analysis of Variance, and Validation
2.1.2. The Effect of Extraction Conditions on Total Flavonoid Contents
2.2. Analysis of Phytochemical Profile
2.3. Analysis of Volatile Compounds
2.4. TPCs, TFCs, Antioxidant Activities, and Enzyme Inhibitory Activities
2.5. Genotoxicity Testing Using Bacterial Reverse Mutation Test (Ames Test)
3. Discussion
4. Materials and Methods
4.1. Chemicals and Reagents
4.2. Sample Selection, Preparation, and Extraction
4.3. Determination of Total Flavonoid Contents
4.4. Determination of Total Phenolic Contents
4.5. Determination of Antioxidant Properties
4.6. Determination of Enzyme Inhibitory Activities
4.7. Determination of Phytochemical Profiles Utilizing Liquid Chromatography–Electrospray Ionization–Tandem Mass Spectrometry (LC–ESI–MS/MS)
4.8. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis
4.9. Bacterial Reverse Mutation Test (Ames Test)
4.10. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
References
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Run | X1: Ethanol (% v/v) | X2: Temperature (°C) | X3: Time (h) | TPCs (mg GAE/g DW) | TFCs (mg QE/g DW) | |||
---|---|---|---|---|---|---|---|---|
Coded | Uncoded | Coded | Uncoded | Coded | Uncoded | Experimental | Experimental | |
1 | −1 | 50 | 1 | 70 | 0 | 4 | 13.02 ± 0.03 f | 3.26 ± 0.01 c |
2 | −1 | 50 | 0 | 50 | 1 | 6 | 1.84 ± 0.17 k | 3.07 ± 0.01 d |
3 | 0 | 70 | 1 | 70 | −1 | 2 | 20.35 ± 0.01 c | 1.76 ± 0.02 i |
4 | 0 | 70 | 1 | 70 | 1 | 6 | 20.59 ± 0.04 b | 1.51 ± 0.01 j |
5 | 0 | 70 | 0 | 50 | 0 | 4 | 11.39 ± 0.01 i | 1.43 ± 0.02 k |
6 | 0 | 70 | 0 | 50 | 0 | 4 | 11.34 ± 0.00 i | 1.40 ± 0.01 k |
7 | 1 | 90 | 1 | 70 | 0 | 4 | 12.16 ± 0.01 h | 2.87 ± 0.02 e |
8 | 0 | 70 | 0 | 50 | 0 | 4 | 11.38 ± 0.03 i | 1.46 ± 0.02 k |
9 | −1 | 50 | 0 | 50 | −1 | 2 | 4.47 ± 0.029 j | 3.71 ± 0.03 b |
10 | 1 | 90 | −1 | 30 | 0 | 4 | 15.46 ± 0.03 e | 2.08 ± 0.05 h |
11 | 0 | 70 | −1 | 30 | 1 | 6 | 11.36 ± 0.01 i | 2.34 ± 0.02 g |
12 | 1 | 90 | 0 | 50 | 1 | 6 | 11.34 ± 0.02 i | 1.45 ± 0.04 k |
13 | −1 | 50 | −1 | 30 | 0 | 4 | 15.79 ± 0.03 d | 4.50 ± 0.01 a |
14 | 1 | 90 | 0 | 50 | −1 | 2 | 21.88 ± 0.04 a | 1.48 ± 0.02 jk |
15 | 0 | 70 | −1 | 30 | −1 | 2 | 12.92 ± 0.03 g | 2.55 ± 0.05 f |
Source | TPCs | TFCs | ||||
---|---|---|---|---|---|---|
Coefficient | p-Value | Significance | Coefficient | p-Value | Significance | |
Model | 11.38 | 0.4440 | ns | 1.30 | 0.0030 | ** |
X1 | 0.7000 | 0.7033 | −0.8347 | 0.0005 | ** | |
X2 | 3.24 | 0.1085 | −0.2574 | 0.0590 | ||
X3 | −1.16 | 0.5326 | −0.1415 | 0.2386 | ||
X1X2 | −0.1325 | 0.9591 | 0.5069 | 0.0195 | * | |
X1X3 | −0.7980 | 0.7584 | 0.1534 | 0.3520 | ||
X2X3 | −1.92 | 0.4669 | −0.0102 | 0.9482 | ||
5.17 | 0.0711 | 1.13 | 0.0008 | *** | ||
−2.44 | 0.3494 | 0.7478 | 0.0049 | ** | ||
1.00 | 0.6919 | −0.0038 | 0.9817 | |||
R2 | 0.5923 | 0.9686 | ||||
R2 adjusted | 0.0703 | 0.9122 | ||||
Lack of fit | 1.59 | <0.0001 | **** | 0.4086 | ns |
Phenolics | Amount (µg/g DW) |
---|---|
Non-acid hydrolysis | |
Hesperidin | 161.03 ± 5.03 |
Acid hydrolysis | |
Apigenin | 41.96 ± 1.29 |
Naringenin | 3430.41 ± 135.02 |
Sinapic acid | 142.32 ± 11.92 |
Peak Number with Possibility Score of More Than 50% | Retention Time (min) | Name | Formula | Area (%) |
---|---|---|---|---|
1 | 8.683 | Limonene | C10H16 | 4.05 |
2 | 30.060 | Phosphonic acid, (phydroxyphenyl)- | C6H7O4P | 4.28 |
3 | 37.934 | Catecholborane | C6H5BO2 | 9.26 |
Types | Analyses | Values |
---|---|---|
Antioxidant activities | DPPH (µmol TE/g DW) | 15.35 ± 0.47 |
FRAP (µmol TE/g DW) | 9.36 ± 0.06 | |
ORAC (µmol TE/g DW) | 675.34 ± 22.16 | |
Enzyme inhibitions | AChE (% inhibition) # | 14.57 ± 1.17 |
BChE (% inhibition) # | 26.76 ± 1.17 | |
BACE-1 (% inhibition) $ | 54.61 ± 5.08 | |
Lipase (% inhibition) # | 20.89 ± 0.35 | |
α-Glucosidase (% inhibition) # | 45.40 ± 1.73 | |
α-Amylase (% inhibition) # | 35.38 ± 2.24 |
Doses (µg/Plate) | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
---|---|---|---|---|---|---|---|---|---|---|
Revertant Colonies | MR | Revertant Colonies | MR | Revertant Colonies | MR | Revertant Colonies | MR | Revertant Colonies | MR | |
Neg | 87.67 ± 4.61 | 1.00 (−) | 71.17 ± 2.73 | 1.00 (−) | 373.33 ± 4.46 | 1.00 (−) | 9.67 ± 1.11 | 1.00 (−) | 8.67 ± 0.94 | 1.00 (−) |
10 | 85.00 ± 3.00 | 0.97 (−) | 69.50 ± 3.15 | 0.98 (−) | 374.50 ± 6.80 | 1.00 (−) | 11.00 ± 1.15 | 1.14 (−) | 9.17 ± 0.37 | 1.06 (−) |
100 | 88.33 ± 4.85 | 1.01 (−) | 71.50 ± 2.99 | 1.00 (−) | 376.50 ± 4.79 | 1.01 (−) | 10.33 ± 0.94 | 1.07 (−) | 9.33 ± 0.75 | 1.08 (−) |
500 | 83.67 ± 3.14 | 0.95 (−) | 73.17 ± 5.34 | 1.03 (−) | 374.67 ± 4.50 | 1.00 (−) | 10.00 ± 1.00 | 1.03 (−) | 8.67 ± 1.37 | 1.00 (−) |
1000 | 86.17 ± 2.41 | 0.98 (−) | 71.33 ± 2.36 | 1.00 (−) | 373.67 ± 4.03 | 1.00 (−) | 9.83 ± 1.07 | 1.02 (−) | 9.17 ± 1.34 | 1.06 (−) |
2000 | 85.50 ± 4.19 | 0.98 (−) | 69.00 ± 3.27 | 0.97 (−) | 374.00 ± 7.85 | 1.00 (−) | 10.67 ± 1.11 | 1.10 (−) | 10.00 ± 0.82 | 1.15 (−) |
4-NQO | 1047.33 ± 32.20 | 11.95 (+) | ||||||||
NaN3 | 1146.67 ± 22.82 | 16.11 (+) | 271.17 ± 5.81 | 28.05 (+) | ||||||
MMC | 1108.67 ± 19.92 | 2.97 (+) | ||||||||
9-AA | 776.67 ± 33.02 | 89.62 (+) |
Doses (µg/plate) | TA98 | TA100 | TA102 | TA1535 | TA1537 | |||||
---|---|---|---|---|---|---|---|---|---|---|
Revertant Colonies | MR | Revertant Colonies | MR | Revertant Colonies | MR | Revertant Colonies | MR | Revertant Colonies | MR | |
Neg | 89.83 ± 3.72 | 1.00 (−) | 85.83 ± 3.29 | 1.00 (−) | 375.00 ± 4.00 | 1.00 (−) | 10.00 ± 1.15 | 1.00 (−) | 9.50 ± 0.96 | 1.00 (−) |
10 | 92.00 ± 3.27 | 1.02 (−) | 84.50 ± 2.57 | 0.98 (−) | 380.33 ± 5.65 | 1.01 (−) | 11.33 ± 1.11 | 1.13 (−) | 9.67 ± 0.94 | 1.02 (−) |
100 | 94.17 ± 3.58 | 1.05 (−) | 82.00 ± 2.38 | 0.96 (−) | 374.33 ± 4.35 | 1.00 (−) | 10.33 ± 1.11 | 1.03 (−) | 9.33 ± 0.94 | 0.98 (−) |
500 | 95.17 ± 2.54 | 1.06 (−) | 81.50 ± 3.64 | 0.95 (−) | 383.50 ± 5.62 | 1.02 (−) | 10.67 ± 1.37 | 1.07 (−) | 10.67 ± 1.25 | 1.12 (−) |
1000 | 90.17 ± 1.46 | 1.00 (−) | 81.83 ± 4.41 | 0.95 (−) | 374.00 ± 3.70 | 1.00 (−) | 10.83 ± 1.57 | 1.08 (−) | 11.17 ± 0.90 | 1.18 (−) |
2000 | 90.33 ± 3.54 | 1.01 (−) | 82.83 ± 6.79 | 0.97 (−) | 374.83 ± 5.01 | 1.00 (−) | 11.17 ± 0.69 | 1.12 (−) | 10.83 ± 1.07 | 1.14 (−) |
2-AA | 1134.67 ± 53.80 | 12.63 (+) | 1006.67 ± 40.24 | 11.73 (+) | 1118.00 ± 23.20 | 2.98 (+) | 369.83 ± 8.39 | 36.98 (+) | 200.83 ± 6.44 | 21.14 (+) |
Assay | Assay Components | ||||
---|---|---|---|---|---|
Enzyme | Substrate | Indicator | Extract | Detection Wavelength | |
AChE | 100 μL of 0.25 µg/mL AChE 1 | 50 μL of 0.32 mM ACh | 10 µL of 16 mM DTNB | 40 µL | 412 nm |
BChE | 100 μL of 1.5 µg/mL BChE 2 | 50 μL of 0.4 mM BCh | |||
Lipase | 100 µL of 5 µg/mL lipase 3 | 50 μL of 0.2 mM DMPTB | |||
BACE-1 | BACE-1 FRET assay kit (Sigma-Aldrich, St. Louis, MO, USA) following manufacturer’s recommendations | λex = 320 nm λem = 405 nm | |||
α-Amylase | 100 µL of 50 mg/mL α-amylase 4 | 50 µL of 30 mM pNPM | 50 µL | 405 nm | |
α-Glucosidase | 100 µL of 0.1 U/mL α-glucosidase 5 | 50 µL of 2 mM pNPG + 160 µL KPB (pH 7) | 50 µL |
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Inthachat, W.; Temviriyanukul, P.; On-Nom, N.; Kanoongon, P.; Thangsiri, S.; Chupeerach, C.; Suttisansanee, U. Optimization of Phytochemical-Rich Citrus maxima Albedo Extract Using Response Surface Methodology. Molecules 2023, 28, 4121. https://doi.org/10.3390/molecules28104121
Inthachat W, Temviriyanukul P, On-Nom N, Kanoongon P, Thangsiri S, Chupeerach C, Suttisansanee U. Optimization of Phytochemical-Rich Citrus maxima Albedo Extract Using Response Surface Methodology. Molecules. 2023; 28(10):4121. https://doi.org/10.3390/molecules28104121
Chicago/Turabian StyleInthachat, Woorawee, Piya Temviriyanukul, Nattira On-Nom, Panyaporn Kanoongon, Sirinapa Thangsiri, Chaowanee Chupeerach, and Uthaiwan Suttisansanee. 2023. "Optimization of Phytochemical-Rich Citrus maxima Albedo Extract Using Response Surface Methodology" Molecules 28, no. 10: 4121. https://doi.org/10.3390/molecules28104121
APA StyleInthachat, W., Temviriyanukul, P., On-Nom, N., Kanoongon, P., Thangsiri, S., Chupeerach, C., & Suttisansanee, U. (2023). Optimization of Phytochemical-Rich Citrus maxima Albedo Extract Using Response Surface Methodology. Molecules, 28(10), 4121. https://doi.org/10.3390/molecules28104121