Optimization of Solvent-Free Microwave-Assisted Hydrodiffusion and Gravity Extraction of Morus nigra L. Fruits Maximizing Polyphenols, Sugar Content, and Biological Activities Using Central Composite Design
Abstract
:1. Introduction
2. Results and Discussion
2.1. Design of the Experiments (DoE) Analysis
2.2. MHG Optimization and Model Validation
2.3. Application of the HPLC-MS/MS Method to the Validated MHG Run
2.4. Comparison between MHG Validation Run Extract and Conventional Solvent Extracts
3. Materials and Methods
3.1. Plant Material
3.2. Chemicals
3.2.1. HPLC Analysis
3.2.2. Biological Studies
3.3. Extraction Procedures
3.3.1. Microwave Hydrodiffusion and Gravity (MHG) Extraction
3.3.2. Conventional Solvent Extraction
3.4. Design of the Experiments (DoE)
3.4.1. Central Composite Design
- (1)
- Extraction yield %, calculated as the weight of lyophilized extract per dry weight of fruit.
- (2)
- Total phenolic content (TPC), determined as reported in Section 3.6.
- (3)
- Total flavonoid content (TFC), determined as reported in Section 3.7.
- (4)
- Total anthocyanin content (TAC), determined as reported in Section 3.8.
- (5)
- Total sugar content (TSC), determined as reported in Section 3.9.
- (6)
- α-Glucosidase inhibition, determined as reported in Section 3.10.
- (7)
- Lipase inhibition, determined as reported in Section 3.11.
- (8)
- Xanthine oxidase inhibition, determined as reported in Section 3.12.1.
- (9)
- DPPH radical scavenging, determined as reported in Section 3.12.2.
3.4.2. Optimization and Validation
3.5. HPLC-ESI-MS/MS Analysis of the Validation MHG Run
3.6. Total Phenolic Content (TPC)
3.7. Total Flavonoid Content (TFC)
3.8. Total Anthocyanin Content (TAC)
3.9. Total Sugar Content (TSC)
3.10. α-Glucosidase Inhibition
3.11. Lipase Inhibition
3.12. Free Radical Scavenging Activity
3.12.1. Xanthine/Xanthine Oxidase Inhibition
3.12.2. DPPH Radical Scavenging
3.13. Moisture Content
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Response | Best Model a | R2 | R2adj | R2pred | Mallow’s Cp | p-Value regr b | p-Value LOF b |
---|---|---|---|---|---|---|---|
EY% | Y = −2.83 + 0.36P + 0.519T − 1.487P2 − 0.006T2 | 0.972 | 0.953 | 0.872 | 4.66 | *** | * |
TPC (mg GAE/100 g) | Y = −3393 + 4135P +127.9T − 1343 P2 − 2.61 T2 + 39.4 P*T | 0.936 | 0.873 | 0.623 | 6 | ** | ns |
TFC (mg RE/100 g) | Y = 1352 + 99P − 16.4T + 10.76 P*T | 0.654 | 0.506 | 0.00 | 2.08 | * | ns |
TAC (mg CGE/100 g) | Y = 361 + 1008P − 13.01T − 327.6 P2 | 0.934 | 0.902 | 0.814 | 5.01 | *** | * |
TSC (mg GE/100 g) | Y = 33,210 − 4034P − 495T + 239 P*T | 0.652 | 0.477 | 0.00 | 2.27 | ns | ** |
AGHi IC50 (µg/mL) | Y = 442 − 578P + 15.1T +230P2 − 8.9P*T | 0.726 | 0.360 | 0.00 | 6.52 | ns | ** |
Li IC50 (µg/mL) | Y = 10,447 − 8735P + 2260P2 | 0.823 | 0.779 | 0.625 | 4.11 | ** | ns |
XOi IC50 (µg/mL) | Y = 2045 − 1348P − 33.8T + 304.7P2 + 0.455T2 | 0.913 | 0.855 | 0.58 | 4.02 | ** | ** |
DPPH IC50 (µg/mL) | Y = 481.6 − 63.6P − 1.79T | 0.598 | 0.498 | 0.273 | 1.37 | * | ns |
Run. | MAE Conditions | Composite Desirability | Responses Optimized with Desirability | Desirability Function | 95% Interval of Confidence | 95% Interval of Prediction | |
---|---|---|---|---|---|---|---|
Power (W/g) | Time (min) | ||||||
12 | 1.86 | 31.07 | 0.83 | EY% | Maximize | 12.4–14.9 | 11.1–16.2 |
TPC | Maximize | 2906–3901 | 2379–4428 | ||||
TAC | Maximize | 622–806 | 525–903 | ||||
Li | Minimize | 1476–2536 | 597–3415 | ||||
XOi | Minimize | 0–98.4 | 0–240 |
Run. | Point Type a | Coded Variables b | Uncoded Variables | Absolute Values | |||
---|---|---|---|---|---|---|---|
ET (min) | MP (W/g) | ET (min) | MP (W/g) | ET (min) | MP (W) | ||
1 | F | −1 | −1 | 15 | 1 | 15 | 500 |
2 | F | −1 | 1 | 15 | 2.4 | 15 | 1200 |
3 | F | 1 | −1 | 45 | 1 | 45 | 500 |
4 | F | 1 | 1 | 45 | 2.4 | 45 | 1200 |
5 | A | 0 | −1.41 | 30 | 0.7 | 30 | 355 |
6 | A | 0 | 1.41 | 30 | 2.7 | 30 | 1345 |
7 | A | −1.41 | 0 | 8.8 | 1.7 | 8.8 | 850 |
8 | A | 1.41 | 0 | 51.2 | 1.7 | 51.2 | 850 |
9 | C | 0 | 0 | 30 | 1.7 | 30 | 850 |
10 | C | 0 | 0 | 30 | 1.7 | 30 | 850 |
11 | C | 0 | 0 | 30 | 1.7 | 30 | 850 |
No. | Compound | Concentration (mg/kg ± RSD%) |
---|---|---|
Anthocyanins | ||
1 | Delphindin-3,5-diglucoside | nd |
2 | Delphindin-3-galactoside | nd |
3 | Cyanidin-3-glucoside | 6016.72 ± 3.1 |
4 | Petunidin-3-glucoside | nd |
5 | Pelargonidin-3-rutinoside | 68.52 ± 3.2 |
6 | Pelargonidin-3-glucoside | 108.81 ± 4.5 |
7 | Malvidin-3-galactoside | nd |
Flavonols | ||
8 | Quercetin | 46.49 ± 1.7 |
9 | Rutin | 222.9 ± 1.2 |
10 | Isoquercitrin | 94.4 ± 2.1 |
11 | Quercitrin | nd |
12 | Hyperoside | 207.67 ± 0.6 |
13 | Isorhamnetin | nd |
14 | Myricetin | nd |
15 | Kaempferol | 1.35 ± 4.3 |
16 | Kaempferol-3-glucoside | 7.85 ± 3.0 |
Flavan-3-ols | ||
17 | (+)-Catechin | 1.92 ± 0.9 |
18 | (-)- Epicatechin | nd |
19 | Procyanidin A2 | nd |
20 | Procyanidin B2 | nd |
Dihydrochalcones | ||
21 | Phloridzin | 1.41 ± 2.0 |
22 | Phloretin | 1.12 ± 1.2 |
Flavanones | ||
23 | Hesperidin | nd |
24 | Naringin | nd |
Phenolic acids | ||
25 | Neochlorogenic acid | 57.5 ± 1.2 |
26 | Chlorogenic acid | 565.6 ± 1.7 |
27 | Gallic acid | 4.0 ± 0.8 |
28 | p-Hydroxybenzoic acid | 0.4 ± 1.1 |
29 | 3-Hydroxybenzoic acid | nd |
30 | Caffeic acid | 3.9 ± 2.7 |
31 | Vanillic acid | 8.0 ± 1.3 |
32 | Syringic acid | 2.2 ± 1.12 |
33 | p-Coumaric acid | 8.8 ± 2.6 |
34 | Ferulic acid | 1.6 ± 3.3 |
35 | 3,5-Dicaffeoylquinic acid | 5.0 ± 2.6 |
36 | Ellagic acid | 51.66 ± 1.04 |
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Mustafa, A.M.; Mazzara, E.; Abouelenein, D.; Angeloni, S.; Nunez, S.; Sagratini, G.; López, V.; Cespi, M.; Vittori, S.; Caprioli, G.; et al. Optimization of Solvent-Free Microwave-Assisted Hydrodiffusion and Gravity Extraction of Morus nigra L. Fruits Maximizing Polyphenols, Sugar Content, and Biological Activities Using Central Composite Design. Pharmaceuticals 2022, 15, 99. https://doi.org/10.3390/ph15010099
Mustafa AM, Mazzara E, Abouelenein D, Angeloni S, Nunez S, Sagratini G, López V, Cespi M, Vittori S, Caprioli G, et al. Optimization of Solvent-Free Microwave-Assisted Hydrodiffusion and Gravity Extraction of Morus nigra L. Fruits Maximizing Polyphenols, Sugar Content, and Biological Activities Using Central Composite Design. Pharmaceuticals. 2022; 15(1):99. https://doi.org/10.3390/ph15010099
Chicago/Turabian StyleMustafa, Ahmed M., Eugenia Mazzara, Doaa Abouelenein, Simone Angeloni, Sonia Nunez, Gianni Sagratini, Víctor López, Marco Cespi, Sauro Vittori, Giovanni Caprioli, and et al. 2022. "Optimization of Solvent-Free Microwave-Assisted Hydrodiffusion and Gravity Extraction of Morus nigra L. Fruits Maximizing Polyphenols, Sugar Content, and Biological Activities Using Central Composite Design" Pharmaceuticals 15, no. 1: 99. https://doi.org/10.3390/ph15010099