Optimization of Astilbin Extraction from the Rhizome of Smilax glabra, and Evaluation of Its Anti-Inflammatory Effect and Probable Underlying Mechanism in Lipopolysaccharide-Induced RAW264.7 Macrophages
Abstract
:1. Introduction
2. Results and Discussion
2.1. Box-Behnken Design Analysis
Run | Extract Time (A, min) | Ethanol Concentration (B, %) | Temperature (C, °C) | Liquid-Solid Ratio (D, mL/g) | EYA (mg/g) | |
---|---|---|---|---|---|---|
Observed | Predicted | |||||
1 | 40(1) | 55(0) | 65(0) | 10(−1) | 12.25 | 12.13 |
2 | 40(1) | 55(0) | 65(0) | 30(1) | 14.35 | 14.26 |
3 | 40(1) | 60(1) | 65(0) | 20(0) | 13.91 | 14.00 |
4 | 40(1) | 55(0) | 75(1) | 20(0) | 14.40 | 14.22 |
5 | 10(−1) | 55(0) | 55(−1) | 20(0) | 13.60 | 13.75 |
6 | 25(0) | 55(0) | 55(−1) | 10(−1) | 12.04 | 12.06 |
7 | 10(−1) | 55(0) | 65(0) | 30(1) | 14.06 | 13.99 |
8 | 25(0) | 55(0) | 65(0) | 20(0) | 14.17 | 14.39 |
9 | 25(0) | 55(0) | 65(0) | 20(0) | 14.50 | 14.39 |
10 | 25(0) | 55(0) | 75(1) | 10(−1) | 12.80 | 12.81 |
11 | 25(0) | 50(−1) | 65(0) | 10(−1) | 12.61 | 12.73 |
12 | 25(0) | 55(0) | 75(1) | 30(1) | 14.20 | 14.40 |
13 | 25(0) | 55(0) | 55(−1) | 30(1) | 14.05 | 14.27 |
14 | 40(1) | 50(−1) | 65(0) | 20(0) | 12.52 | 12.66 |
15 | 10(−1) | 50(−1) | 65(0) | 20(0) | 14.04 | 14.17 |
16 | 40(1) | 55(0) | 55(−1) | 20(0) | 12.87 | 13.03 |
17 | 25(0) | 50(−1) | 55(−1) | 20(0) | 13.78 | 13.46 |
18 | 25(0) | 60(1) | 65(0) | 10(−1) | 11.47 | 11.54 |
19 | 25(0) | 50(−1) | 65(0) | 30(1) | 13.76 | 13.66 |
20 | 10(−1) | 55(1) | 65(0) | 20(0) | 12.32 | 12.41 |
21 | 25(0) | 50(−1) | 75(1) | 20(0) | 13.76 | 13.79 |
22 | 25(0) | 60(1) | 55(−1) | 20(0) | 13.37 | 13.14 |
23 | 25(0) | 55(0) | 65(0) | 20(0) | 14.64 | 14.39 |
24 | 25(0) | 60(1) | 65(0) | 30(1) | 14.57 | 14.42 |
25 | 25(0) | 55(0) | 65(0) | 20(0) | 14.31 | 14.39 |
26 | 25(0) | 55(0) | 65(0) | 20(0) | 14.33 | 14.39 |
27 | 10(−1) | 55(0) | 75(1) | 20(0) | 13.62 | 13.43 |
28 | 25(0) | 60(1) | 75(1) | 20(0) | 13.55 | 13.68 |
29 | 10(−1) | 55(0) | 65(0) | 10(−1) | 12.43 | 12.32 |
Source | Sum of Squares | df | Mean Square | F-Value | p-Value (prob>) | Remarks |
---|---|---|---|---|---|---|
Sequntial model sum of squares | ||||||
Mean | 5306.33 | 1 | 5306.33 | |||
Linear | 11.52 | 4 | 2.88 | 6.89 | 0.0008 | |
2FI | 4.10 | 6 | 0.68 | 2.07 | 0.1086 | |
Quadratic | 5.29 | 4 | 1.32 | 28.19 | <0.0001 | Suggested |
Cubic | 0.35 | 8 | 0.043 | 0.84 | 0.6032 | Aliased |
Residual | 0.31 | 6 | 0.052 | |||
Total | 5327.90 | 29 | 183.72 | |||
Lack of fit tests | ||||||
Linear | 9.91 | 20 | 0.50 | 14.90 | 0.0089 | |
2FI | 5.81 | 14 | 0.42 | 12.48 | 0.0128 | |
Quadratic | 0.52 | 10 | 0.052 | 1.57 | 0.3510 | Suggested |
Cubic | 0.18 | 2 | 0.089 | 2.66 | 0.1839 | Aliased |
Pure error | 0.13 | 4 | 0.033 | |||
Model summary statistics | ||||||
Source | Std. Dev. | R2 | Adjusted R2 | Predicted R2 | Press | Remarks |
Linear | 0.65 | 0.5344 | 0.4568 | 0.3588 | 13.83 | |
2FI | 0.57 | 0.7244 | 0.5712 | 0.4870 | 11.06 | |
Quadratic | 0.22 | 0.9696 | 0.9391 | 0.8505 | 3.22 | Suggested |
Cubic | 0.23 | 0.9856 | 0.9329 | -0.1926 | 25.72 | Aliased |
Source | Sum of Squares | df | Mean Square | F-Value | p-Value (prob>) |
---|---|---|---|---|---|
Model | 20.91 | 14 | 1.49 | 31.85 | <0.0001 |
A | 4.408 × 10−3 | 1 | 4.408 × 10−3 | 0.094 | 0.7637 |
B | 0.14 | 1 | 0.14 | 2.91 | 0.1100 |
C | 0.57 | 1 | 0.57 | 12.20 | 0.0036 |
D | 10.81 | 1 | 10.81 | 230.54 | <0.0001 |
AB | 2.42 | 1 | 2.42 | 51.56 | <0.0001 |
AC | 0.57 | 1 | 0.57 | 12.16 | 0.0036 |
AD | 0.055 | 1 | 0.055 | 1.18 | 0.2962 |
BC | 0.010 | 1 | 0.010 | 0.21 | 0.6513 |
BD | 0.95 | 1 | 0.95 | 20.27 | 0.0005 |
CD | 0.093 | 1 | 0.093 | 1.98 | 0.1808 |
A2 | 1.59 | 1 | 1.59 | 34.01 | <0.0001 |
B2 | 2.22 | 1 | 2.22 | 47.27 | <0.0001 |
C2 | 0.53 | 1 | 0.53 | 11.40 | 0.0045 |
D2 | 3.35 | 1 | 3.35 | 71.37 | <0.0001 |
Residual | 0.66 | 14 | 0.047 | ||
Lack of fit | 0.52 | 10 | 0.052 | 1.57 | 0.3510 |
Pure error | 0.13 | 4 | 0.033 | ||
Cor toal | 21.56 | 28 |
2.2. Optimization of Extraction Conditions of Astilbin
2.3. Verification of Predictive Model
2.4. Effects of Astilbin on RAW264.7 Cell Viability and Production of Pro-Inflammatory Mediators
2.5. Effects of Astilbin on NF-κB and MAPKs Pathways
3. Materials and Methods
3.1. Plant & Materials
3.2. Extraction Procedure
3.3. Determination of Astilbin
3.4. Experimental Design
3.5. Determination of the Anti-Inflammatory Efficiency of Astilbin
3.5.1. Cell Culture and Treatment
3.5.2. Cell Viability Assay
3.5.3. Measurement of Nitric Oxide Production
3.5.4. Determination of Production of Pro-Inflammation Cytokines
3.5.5. RNA Extractions and Quantitative Real-Time PCR
3.5.6. Western Blot Analysis
3.6. Statistical Analysis
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Lu, C.-L.; Zhu, Y.-F.; Hu, M.-M.; Wang, D.-M.; Xu, X.-J.; Lu, C.-J.; Zhu, W. Optimization of Astilbin Extraction from the Rhizome of Smilax glabra, and Evaluation of Its Anti-Inflammatory Effect and Probable Underlying Mechanism in Lipopolysaccharide-Induced RAW264.7 Macrophages. Molecules 2015, 20, 625-644. https://doi.org/10.3390/molecules20010625
Lu C-L, Zhu Y-F, Hu M-M, Wang D-M, Xu X-J, Lu C-J, Zhu W. Optimization of Astilbin Extraction from the Rhizome of Smilax glabra, and Evaluation of Its Anti-Inflammatory Effect and Probable Underlying Mechanism in Lipopolysaccharide-Induced RAW264.7 Macrophages. Molecules. 2015; 20(1):625-644. https://doi.org/10.3390/molecules20010625
Chicago/Turabian StyleLu, Chuan-Li, Yan-Fang Zhu, Meng-Mei Hu, Dong-Mei Wang, Xiao-Jie Xu, Chuan-Jian Lu, and Wei Zhu. 2015. "Optimization of Astilbin Extraction from the Rhizome of Smilax glabra, and Evaluation of Its Anti-Inflammatory Effect and Probable Underlying Mechanism in Lipopolysaccharide-Induced RAW264.7 Macrophages" Molecules 20, no. 1: 625-644. https://doi.org/10.3390/molecules20010625