Optimization of Flavonoids Extraction Process in Panax notoginseng Stem Leaf and a Study of Antioxidant Activity and Its Effects on Mouse Melanoma B16 Cells
Abstract
:1. Introduction
2. Results and Discussion
2.1. Effect of Surfactant Types on Extraction Efficiency of SLPF
2.2. Single Factor Experiments
2.3. Model Fitting and Optimization of SLPF
2.3.1. Model Fitting
0.010BC − 0.001BD − 0.076CD − 0.003A2 − 0.002B2 − 0.683C2 − 0.010D2
2.3.2. Analysis of Response Surface
2.3.3. Validation of the Model
2.4. Antioxidant Activities of the P. notoginseng Stem Leaf Extracts
2.5. Effect of Extracts on B16 Cell Activity
3. Materials and Methods
3.1. Plant Materials
3.2. Chemicals and Reagents
3.3. Optimization of Extraction Condition Selection
3.4. Experimental Design of RSM
3.5. Total Flavonoid Assay
3.6. In Vitro Antioxidant Activity Assays
3.6.1. Reducing Power Assay
3.6.2. DPPH Radical Scavenging Capacity
3.6.3. Detection of ABTS+ Scavenging Assay
3.6.4. Assay of Hydroxyl Radical Scavenging Activity
3.6.5. Detection of Superoxide Anion Scavenging Activity
3.7. Cell Experiment
3.7.1. Cell Culture
3.7.2. α-MSH Treatment
3.7.3. Intracellular Tyrosinase Activity
3.7.4. Melanin Content Assay
3.8. Statistical Analysis
4. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
ABTS | 2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid |
BBD | Box-Behnken design |
DPPH | 2,2-diphenyl-1-picrylhydrazyl |
RSM | Response surface methodology |
SLPF | stem and leaf of P. notoginseng flavonoid |
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Sample Availability: Samples of the extracts are available from the authors. |
Std. Order | Run Order | Variable | Total Flavonoids Y (%) | ||||
---|---|---|---|---|---|---|---|
A | B | C | D | Observed | Predicted | ||
25 | 1 | 50 (0) | 50 (0) | 2 (0) | 20:1 (0) | 2.127 | 2.117 |
8 | 2 | 50 (0) | 50 (0) | 2.5 (1) | 25:1 (1) | 1.384 | 1.422 |
11 | 3 | 40 (−1) | 50 (0) | 2 (0) | 25:1 (1) | 1.781 | 1.776 |
22 | 4 | 50 (0) | 60 (1) | 2 (0) | 15:1 (−1) | 1.615 | 1.662 |
12 | 5 | 60 (1) | 50 (0) | 2 (0) | 25:1 (1) | 1.369 | 1.364 |
20 | 6 | 60 (1) | 50 (0) | 2.5 (1) | 20:1 (0) | 1.579 | 1.562 |
1 | 7 | 40 (−1) | 40 (−1) | 2 (0) | 20:1 (0) | 1.441 | 1.448 |
6 | 8 | 50 (0) | 50 (0) | 2.5 (1) | 15:1 (−1) | 1.794 | 1.844 |
10 | 9 | 60 (1) | 50 (0) | 2 (0) | 15:1 (−1) | 1.949 | 1.922 |
17 | 10 | 40 (−1) | 50 (0) | 1.5 (−1) | 20:1 (0) | 1.535 | 1.584 |
9 | 11 | 40 (−1) | 50 (0) | 2 (0) | 15:1 (−1) | 1.335 | 1.306 |
21 | 12 | 50 (0) | 40 (−1) | 2 (0) | 15:1 (−1) | 1.630 | 1.626 |
27 | 13 | 50 (0) | 50 (0) | 2 (0) | 20:1 (0) | 2.127 | 2.117 |
2 | 14 | 60 (1) | 40 (−1) | 2 (0) | 20:1 (0) | 1.762 | 1.811 |
16 | 15 | 50 (0) | 60 (1) | 2.5 (1) | 20:1 (0) | 1.711 | 1.663 |
7 | 16 | 50 (0) | 50 (0) | 1.5 (−1) | 25:1 (1) | 1.973 | 1.924 |
23 | 17 | 50 (0) | 40 (−1) | 2 (0) | 25:1 (1) | 1.694 | 1.678 |
28 | 18 | 50 (0) | 50 (0) | 2 (0) | 20:1 (0) | 2.145 | 2.117 |
14 | 19 | 50 (0) | 60 (1) | 1.5 (−1) | 20:1 (0) | 1.666 | 1.683 |
29 | 20 | 50 (0) | 50 (0) | 2 (0) | 20:1 (0) | 2.074 | 2.117 |
15 | 21 | 50 (0) | 40 (−1) | 2.5 (1) | 20:1 (0) | 1.669 | 1.618 |
4 | 22 | 60 (1) | 60 (1) | 2 (0) | 20:1 (0) | 1.496 | 1.490 |
13 | 23 | 50 (0) | 40 (−1) | 1.5 (−1) | 20:1 (0) | 1.832 | 1.846 |
18 | 24 | 60 (1) | 50 (0) | 1.5 (−1) | 20:1 (0) | 1.879 | 1.885 |
5 | 25 | 50 (0) | 50 (0) | 1.5 (−1) | 15:1 (−1) | 1.627 | 1.590 |
19 | 26 | 40 (−1) | 50 (0) | 2.5 (1) | 20:1 (0) | 1.634 | 1.659 |
26 | 27 | 50 (0) | 50 (0) | 2 (0) | 20:1 (0) | 2.114 | 2.117 |
24 | 28 | 50 (0) | 60 (1) | 2 (0) | 25:1 (1) | 1.486 | 1.522 |
3 | 29 | 40 (−1) | 60 (1) | 2 (0) | 20:1 (0) | 1.697 | 1.649 |
Source | Sum of Squares | DF | Mean Square | F Value | p Value Prob > F | Significance |
---|---|---|---|---|---|---|
Model | 1.589 | 14 | 0.114 | 53.850 | <0.0001 | *** |
A | 0.031 | 1 | 0.031 | 14.757 | 0.0018 | ** |
B | 0.011 | 1 | 0.011 | 5.026 | 0.0417 | * |
C | 0.046 | 1 | 0.046 | 21.826 | 0.0004 | *** |
D | 0.006 | 1 | 0.006 | 2.750 | 0.1195 | |
AB | 0.068 | 1 | 0.068 | 32.313 | <0.0001 | *** |
AC | 0.040 | 1 | 0.040 | 18.891 | 0.0007 | *** |
AD | 0.263 | 1 | 0.263 | 124.984 | <0.0001 | *** |
BC | 0.011 | 1 | 0.011 | 5.165 | 0.0393 | * |
BD | 0.009 | 1 | 0.009 | 4.360 | 0.0555 | |
CD | 0.143 | 1 | 0.143 | 67.750 | <0.0001 | *** |
A2 | 0.487 | 1 | 0.487 | 230.873 | <0.0001 | *** |
B2 | 0.386 | 1 | 0.386 | 182.935 | <0.0001 | *** |
C2 | 0.189 | 1 | 0.189 | 89.704 | <0.0001 | *** |
D2 | 0.409 | 1 | 0.409 | 194.231 | <0.0001 | *** |
Residual | 0.030 | 14 | 0.002 | |||
Lack of Fit | 0.027 | 10 | 0.003 | 3.858 | 0.1025 | |
Pure Error | 0.003 | 4 | 0.001 | |||
Cor Total | 1.619 | 28 | ||||
Std. Dev. | 0.046 | R2 | 0.982 | |||
Mean | 1.728 | R2Adj | 0.964 | |||
C.V. (%) | 2.657 | R2Pred | 0.902 | |||
PRESS | 0.158 | RSN | 24.553 |
Name | Goal | Lower Limit | Upper Limit | Lower Weight | Upper Weight |
---|---|---|---|---|---|
A: Ultrasound time (min) | is in range | 40 | 60 | 1 | 1 |
B: Ethanol concentration (%) | is in range | 40 | 60 | 1 | 1 |
C: SDS content (%) | is in range | 1.5 | 2.5 | 1 | 1 |
D: Liquid material ratio (v/w) | is in range | 15 | 25 | 1 | 1 |
Total flavonoids content (%) | maximize | 1.33502 | 100 | 1 | 1 |
Category | Ultrasound time (min) | Ethanol concentration (%) | SDS content (%) | Liquid material ratio (v/w) | Total flavonoids content (%) |
Predicted value | 51.84 | 48.68 | 1.88 | 19.81:1 | 2.13 ± 0.008a |
Experimental value | 52 | 48.7 | 1.9 | 20:1 | 2.10 ± 0.017a |
Surfactant | SDS | Triton X-100 | Tween-20 | Tween-80 | Span-20 |
---|---|---|---|---|---|
HLB | 40 | 14.6 | 16.7 | 15 | 8.6 |
Types | Anionic | Nonionic | Nonionic | Nonionic | Nonionic |
Factors | Codes | Levels | ||
---|---|---|---|---|
−1 | 0 | 1 | ||
Ultrasound time (min) | A | 40 | 50 | 60 |
Ethanol concentration (%) | B | 40 | 50 | 60 |
SDS content (%) | C | 1.5 | 2 | 2.5 |
Liquid material ratio (v/w) | D | 15:1 | 20:1 | 25:1 |
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Dai, C.-Y.; Liu, P.-F.; Liao, P.-R.; Qu, Y.; Wang, C.-X.; Yang, Y.; Cui, X.-M. Optimization of Flavonoids Extraction Process in Panax notoginseng Stem Leaf and a Study of Antioxidant Activity and Its Effects on Mouse Melanoma B16 Cells. Molecules 2018, 23, 2219. https://doi.org/10.3390/molecules23092219
Dai C-Y, Liu P-F, Liao P-R, Qu Y, Wang C-X, Yang Y, Cui X-M. Optimization of Flavonoids Extraction Process in Panax notoginseng Stem Leaf and a Study of Antioxidant Activity and Its Effects on Mouse Melanoma B16 Cells. Molecules. 2018; 23(9):2219. https://doi.org/10.3390/molecules23092219
Chicago/Turabian StyleDai, Chun-Yan, Peng-Fei Liu, Pei-Ran Liao, Yuan Qu, Cheng-Xiao Wang, Ye Yang, and Xiu-Ming Cui. 2018. "Optimization of Flavonoids Extraction Process in Panax notoginseng Stem Leaf and a Study of Antioxidant Activity and Its Effects on Mouse Melanoma B16 Cells" Molecules 23, no. 9: 2219. https://doi.org/10.3390/molecules23092219