Figure 1
Single-factor experimental result for TPC and TFC. DW, dry weight; TFC, total flavonoid content; TPC, total phenolic content.
Table 1
BBD and experimental values of response factors obtained in the analysis.
| Standard order | Run order | Input factors | Responses | |||||
|---|---|---|---|---|---|---|---|---|
| A (%) | B (min) | C (°C) | TPC | TFC | MCA (%) | ARA (%) | ||
| 4 | 1 | 70 | 60 | 40 | 4.92 ± 0.32 | 10.05 ± 0.55 | 58.21 ± 5.00 | 67.14 ± 0.51 |
| 3 | 2 | 30 | 60 | 40 | 3.72 ± 0.15 | 3.67 ± 0.04 | 91.38 ± 0.27 | 23.16 ± 0.51 |
| 5 | 3 | 30 | 40 | 30 | 4.13 ± 0.07 | 5.45 ± 0.30 | 89.94 ± 1.30 | 33.94 ± 0.40 |
| 1 | 4 | 30 | 20 | 40 | 4.11 ± 0.08 | 5.67 ± 0.36 | 84.63 ± 1.32 | 41.97 ± 0.20 |
| 11 | 5 | 50 | 20 | 50 | 4.95 ± 0.32 | 8.07 ± 0.47 | 77.19 ± 4.66 | 57.85 ± 0.22 |
| 7 | 6 | 30 | 40 | 50 | 3.80 ± 0.25 | 4.55 ± 0.04 | 78.19 ± 3.37 | 26.64 ± 0.71 |
| 10 | 7 | 50 | 60 | 30 | 5.10 ± 0.63 | 7.33 ± 0.18 | 78.39 ± 1.48 | 51.25 ± 5.41 |
| 8 | 8 | 70 | 40 | 50 | 4.84 ± 0.91 | 8.88 ± 0.15 | 60.31 ± 0.90 | 74.85 ± 0.40 |
| 14 | 9 | 50 | 40 | 40 | 4.83 ± 0.24 | 7.52 ± 0.36 | 68.13 ± 7.14 | 50.97 ± 1.41 |
| 9 | 10 | 50 | 20 | 30 | 5.11 ± 0.29 | 6.81 ± 0.36 | 73.73 ± 8.50 | 58.78 ± 0.54 |
| 2 | 11 | 70 | 20 | 40 | 5.20 ± 0.41 | 10.10 ± 0.76 | 59.02 ± 4.24 | 73.91 ± 0.51 |
| 15 | 12 | 50 | 40 | 40 | 4.60 ± 0.72 | 7.07 ± 0.43 | 65.50 ± 10.57 | 48.17 ± 1.55 |
| 13 | 13 | 50 | 40 | 40 | 5.01 ± 0.30 | 6.98 ± 0.55 | 68.04 ± 6.61 | 45.30 ± 1.51 |
| 6 | 14 | 70 | 40 | 30 | 5.39 ± 0.29 | 10.14 ± 0.31 | 55.80 ± 5.34 | 74.51 ± 0.51 |
| 12 | 15 | 50 | 60 | 50 | 4.67 ± 0.34 | 7.21 ± 0.07 | 69.98 ± 10.83 | 43.23 ± 0.22 |
[i] A, B and C are solvent concentration, time and temperature, respectively; units of TPC and TFC are mg GAE · g−1 DW and mg RE · g−1 DW, respectively.
[ii] ARA, anti-radical activity; BBD, Box–Behnken design; DW, dry weight; GAE, gallic acid equivalents; MCA, metal chelating activity; RE, rutin equivalents; TFC, total flavonoid content; TPC, total phenolic content.
Table 2
Results of the ANOVA for model fitting and optimisation of extraction as per the three-factor BBD of RSM.
| Source | p values | |||
|---|---|---|---|---|
| TPC (quadratic) | TFC (linear) | MCA (quadratic) | DPPH (linear) | |
| Model | 0.0027* | <0.0001* | 0.0002* | <0.0001* |
| A-concentration | 0.0001* | <0.0001* | <0.0001* | <0.0001* |
| B-Time | 0.0736 | 0.18 | 0.5736 | 0.0007* |
| C-Temperature | 0.0181* | 0.5536 | 0.0828 | 0.1511 |
| AB | 0.7295 | 0.1162 | ||
| AC | 0.4973 | 0.0095* | ||
| BC | 0.4104 | 0.0308* | ||
| A2 | 0.0051* | 0.3132 | ||
| B2 | 0.5835 | 0.0051* | ||
| C2 | 0.2644 | 0.0494* | ||
| Residual | ||||
| Lack of fit | 0.8729 | 0.1821 | 0.3174 | 0.4079 |
| R2 values | ||||
| R2 | 0.9699 | 0.9288 | 0.989 | 0.9621 |
| Predicted R2 | 0.9156 | 0.9094 | 0.9692 | 0.9517 |
| Adjusted R2 | 0.8274 | 0.8513 | 0.8578 | 0.9313 |
ANOVA, analysis of variance; BBD, Box–Behnken design; DPPH, 2,2-diphenyl-1-picrylhydrazyl; MCA, metal chelating activity; RSM, response surface methodology; TFC, total flavonoid content; TPC, total phenolic content.
For each response, the optimisation analysis is discussed here.
Figure 2
TPC 3D surface plots for interactive effect of (A) solvent concentration and extraction time, (B) solvent concentration and extraction temperature and (C) extraction temperature and extraction time. TPC, total phenolic content.
Figure 3
TFC3D surface plot for the interactive effect of solvent concentration and extraction time. DW, dry weight; TFC, total flavonoid content.
Figure 4
ARA 3D surface plot for interactive effect of solvent concentration and extraction.
Figure 5
MCA 3D surface plots for interactive effect of (A) solvent concentration and extraction time, (B) solvent concentration and extraction temperature, and (C) extraction temperature and extraction time. MCA, metal chelating activity.
Table 3
Predicted and observed response values and their corresponding percent errors.
| Responses | Predicted value | Observed value | Error (%) |
|---|---|---|---|
| TPC (mg GAE · g−1 DW) | 5.27 | 4.937 | 6.318 |
| TFC (mg RE · g−1 DW) | 9.869 | 9.333 | 5.431 |
| MCA (%) | 54.366 | 50.378 | 7.335 |
| DPPH (%) | 73.804 | 79.458 | −7.661 |
[i] DPPH, 2,2-diphenyl-1-picrylhydrazyl; DW, dry weight; GAE, gallic acid equivalents; MCA, metal chelating activity; RE rutin equivalents; TFC, total flavonoid content; TPC, total phenolic content.