3.1. Statistical Analysis and Fitting of the Model
In order to ensure that the RSM mathematical models are reliable in the prediction of MAE conditions for TPC, flavonoids, proanthocyanidins, and the antioxidant capacity from the skin of the macadamia, different statistical analyses of variation including “lack of fit”,
R squared, Predicted Residual Sum of Square (PRESS),
F ratio, and Prob > F were identified and examined and the results are shown in
Table 2. The “lack of fit” is able to calculate whether the model has the expected impact, and the R squared value is able to assess the proportion of variation that occurs in the response that is able to be accounted for by the model, rather than by random error, therefore an R squared value that nears 1 indicates that the model is a strong predictor of the response [
24]. Results (
Table 2) showed that “lack of fit” for phenolic compounds, flavonoids, proanthocyanidins, and four antioxidant assays were significantly higher than 0.05, meaning that the models for phenolic compounds and antioxidant properties were fitted and reliable for prediction of the actual values. Furthermore, R squared values for phenolic compounds and antioxidant properties were higher than 0.82, indicating that at least 82% of the predicted values could be matched with the actual values.
Table 2.
Analysis of variance for the determination of model fitting.
Table 2.
Analysis of variance for the determination of model fitting.
| TPC | Flavon-oids | Proantho-cyanidins | Antioxidant Capacity |
---|
ABTS | DPPH | CUPRAC | FRAP |
---|
Lack of fit | 0.167 | 0.892 | 0.979 | 0.136 | 0.989 | 0.525 | 0.239 |
R2 | 0.95 | 0.93 | 0.98 | 0.93 | 0.92 | 0.82 | 0.86 |
Adjusted R2 | 0.87 | 0.81 | 0.95 | 0.79 | 0.79 | 0.49 | 0.62 |
PRESS | 5129 | 439 | 57 | 234,529 | 186,966 | 800,865 | 17,548 |
F ratio of Model | 11.13 | 7.60 | 31.98 | 6.85 | 6.70 | 2.51 | 3.54 |
Prob > F | 0.01 | 0.02 | 0.001 | 0.02 | 0.02 | 0.16 | 0.09 |
The PRESS value shows how well the predictive model fits each point in the design. The
F Ratio is the test statistic for a test of whether the model differs significantly from a model where all predicted values are the response mean. Lastly, the Prob >
F is able to measure the probability of actually obtaining an F ratio that is as high as the one that is being observed, in the case where all parameters are zero, except for the intercept. Smaller Prob >
F values specify that the observed F ratio is highly unlikely [
24]. The results (
Table 2) showed that the PRESS, the
F ratio and “Prob >
F” for phenolic compounds, flavonoids, proanthocyanidins, and antioxidant properties all supported that the mathematical models for these responses are reliable for prediction of the values of these responses.
The results (
Figure 2) further showed the correlation between the predicted values and the actual values. As seen from
Figure 2, the predicted values for phenolic compounds, flavonoids and proathocyanidins were linear to their actual values, indicating a close relationship and further supporting that the mathematical models were reliable predictors for these responses.
Figure 2.
The correlation between the predicted and the actual values for TPC, flavonoids, and proanthocyanidins.
Figure 2.
The correlation between the predicted and the actual values for TPC, flavonoids, and proanthocyanidins.
The values (
Y) for phenolic compounds, flavonoids and proanthocyanidins from the macadamia skin could be fitted to the below second-order polynomial Equations (3)–(5):
Figure 3 further illustrated the correlation between the predicted values and the actual values for the four types of antioxidant assays including DPPH, ABTS, FRAP, and CUPRAC. The predicted values were found to be linear with the actual values, with the R squared value for DPPH, ABTS, FRAP, and CUPRAC, of 0.93, 0.92, 0.86, and 0.82, respectively. These results further supported that the mathematical models were also appropriate for the prediction of the antioxidant values in the current study.
Figure 3.
Correlation between the predicted and the actual values for ABTS total antioxidant capacity, DPPH free radical scavenging capacity, cupric reducing antioxidant power (CUPRAC), and ferric reducing antioxidant power (FRAP).
Figure 3.
Correlation between the predicted and the actual values for ABTS total antioxidant capacity, DPPH free radical scavenging capacity, cupric reducing antioxidant power (CUPRAC), and ferric reducing antioxidant power (FRAP).
The models could be fitted to the following second-order polynomial Equations (6)–(9):
3.2. Effect of Extraction Independent Variables on Phenolic Compounds, Flavonoids, and Proanthocyanidins
The impact of MAE radiation time, power and sample-to-solvent ratio on the extraction of phenolic compounds (TPC) is shown in
Figure 4 and
Table 3.
Table 3.
Analysis of variance for the experimental results on TPC, flavonoids, and proanthocyanidins.
Table 3.
Analysis of variance for the experimental results on TPC, flavonoids, and proanthocyanidins.
Parameter | DF | TPC | Flavonoids | Proanthocyanidins |
---|
Estimate | Prob > |t| | Estimate | Prob > |t| | Estimate | Prob > |t| |
---|
β0 | 1 | 35.24 | 0.0008 * | 32.64 | <0.0001 * | 18.93 | <0.0001 * |
β1 | 1 | −1.73 | 0.5872 | 2.71 | 0.1168 | 1.57 | 0.0624 |
β2 | 1 | −5.70 | 0.1141 | −1.94 | 0.2322 | −3.34 | 0.0038 * |
β3 | 1 | −21.63 | 0.0008 * | −1.91 | 0.2383 | 2.10 | 0.0241 * |
β12 | 1 | −9.58 | 0.0724 | −8.21 | 0.0097 * | −2.15 | 0.0683 |
β13 | 1 | −0.37 | 0.9329 | −1.60 | 0.4648 | 1.34 | 0.2102 |
β23 | 1 | 12.95 | 0.0278 | −0.09 | 0.9644 | 9.98 | 0.0001 * |
β11 | 1 | −7.03 | 0.1702 | −10.54 | 0.0041 * | −7.47 | 0.0006 * |
β22 | 1 | 8.35 | 0.1153 | −8.97 | 0.008 * | 0.27 | 0.7939 |
β33 | 1 | 20.69 | 0.0053 * | −5.46 | 0.0484 * | 7.13 | 0.0007 * |
Figure 4.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on TPC (mg GAE/g).
Figure 4.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on TPC (mg GAE/g).
The results showed that microwave radiation time and power in the tested ranges did not significantly affect the extraction efficiency of TPC; however, the sample-to-solvent ratio was found to have a statistically significant impact on the extraction efficiency of TPC (
p < 0.05). The TPC extraction efficiency decreased when a higher sample-to-solvent ratio was applied. These findings were supported by the previous studies on prune [
25], apple pomace [
26],
Melissa officinali [
27], and
Eucalyptus robusta [
12]. The impact of sample-to-solvent ratio on the extraction yield of TPC can be explained by the increase in the density of the sample in the solvent, which resulted in lower extraction efficiency [
12].
The impact of MAE radiation time, power, and sample-to-solvent ratio on the extraction efficiency of flavonoids from macadamia skin is illustrated in
Table 3 and
Figure 5. The results indicated that all three extraction parameters, within the tested ranges, did not have a significant impact on the extraction efficiency of flavonoids, but the interaction between time and power significantly affected extraction efficiency of flavonoids (
p < 0.05). Previous studies also found that MAE power and sample-to-solvent ratio did not significantly affect the extraction efficiency of flavonoids, but reported that the MAE time did have a significant impact [
12]. The difference can be explained by the narrow range of the tested conditions; this narrow time range was not long enough to give a significant difference.
Figure 5.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on flavonoids (mg RE/g).
Figure 5.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on flavonoids (mg RE/g).
The impact of MAE radiation time, power and sample-to-solvent ratio on the extraction efficiency of proanthocyanidins is outlined in
Table 3 and
Figure 6. It can be seen from
Table 3 that MAE time did not have a significant impact, but the power and the sample-to-solvent ratio had a significant impact on the level of extracted proanthocyanidins (
p < 0.05). The higher the power or the sample-to-solvent ratio applied, the lower the extraction efficiency of proanthocyanidins was achieved (
Figure 6). These findings were also in agreement with the previous study on
Eucalyptus robusta [
12]. The increase of power resulted in a lowering of the extraction efficiency that can be explained by the degradation of proanthocyanidins at higher temperatures caused by the higher power.
Figure 6.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on proanthocyanidins (mg CAE/g).
Figure 6.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on proanthocyanidins (mg CAE/g).
3.3. Effect of Extraction Independent Variables on the Antioxidant Capacity of Macadamia Tetraphylla Skin
Four antioxidant assays were used in this study to determine the antioxidant capacity of the extracts prepared under a variety of extraction conditions from the skin of the macadamia. This is because each antioxidant assay has its own advantages and limitations [
28]. For example, ABTS antioxidant assay can be used over a large pH range, with a variety of solvents. Whereas, many antioxidants that typically react with peroxyl radicals may react slower, or not at all, with DPPH [
29]. In addition, FRAP antioxidant assay only measures the reducing capability of the sample, based upon the ferric ion [
29]. Therefore, more than one antioxidant assays were applied to obtain a better estimation for the antioxidant capacity.
Table 4.
Analysis of variance for the experimental results on antioxidant capacity.
Table 4.
Analysis of variance for the experimental results on antioxidant capacity.
Parameter | DF | ABTS | DPPH | CUPRAC | FRAP |
---|
Estimate | Prob > |t| | Estimate | Prob > |t| | Estimate | Prob > |t| | Estimate | Prob>|t| |
---|
β0 | 1 | 323.27 | 0.0002 * | 318.49 | 0.0001 * | 592.38 | 0.0004 * | 82.93 | 0.0003* |
β1 | 1 | 20.70 | 0.3471 | 21.91 | 0.2828 | 37.22 | 0.4303 | 12.59 | 0.0765 |
β2 | 1 | −52.63 | 0.0461 * | −29.54 | 0.1656 | −32.20 | 0.4916 | −14.56 | 0.0498 * |
β3 | 1 | −70.44 | 0.0167 * | −74.33 | 0.0095 * | −72.19 | 0.1572 | −5.79 | 0.3531 |
β12 | 1 | −126.42 | 0.0065 * | −79.34 | 0.0274 * | −108.36 | 0.1378 | −16.93 | 0.088 |
β13 | 1 | −12.44 | 0.6778 | −23.26 | 0.4079 | 6.64 | 0.9181 | 6.26 | 0.4695 |
β23 | 1 | 41.21 | 0.204 | 25.51 | 0.3673 | 28.89 | 0.6578 | 15.18 | 0.1163 |
β11 | 1 | −122.97 | 0.0086 * | −134.60 | 0.004 * | −213.63 | 0.0205 * | −20.69 | 0.0555 |
β22 | 1 | 23.08 | 0.4675 | −11.09 | 0.6963 | −76.77 | 0.2834 | −12.30 | 0.1997 |
β33 | 1 | −6.30 | 0.8387 | 36.33 | 0.2333 | 91.13 | 0.2131 | 11.34 | 0.2316 |
The impact of MAE radiation time, power, and sample-to-solvent ratio on the ABTS antioxidant capacity of the macadamia skin is represented in
Table 4 and
Figure 7. The results showed that the MAE radiation time did not have a significant impact, but the MAE power and the sample-to-solvent ratio did have a significant impact on the ABTS antioxidant capacity of the macadamia skin extract (
p < 0.05). The higher the MAE power and sample-to-solvent ratio that were applied, the lower the antioxidant capacity obtained was. The results also showed that the interaction between time
x sample-to-solvent ratio, and power
x sample-to-solvent ratio did not have a significant impact, but the interaction between MAE time
x power had a significant impact on ABTS antioxidant capacity of the macadamia skin extract.
Figure 7.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on ABTS antioxidant capacity (µM TE/g).
Figure 7.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on ABTS antioxidant capacity (µM TE/g).
Figure 8.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on DPPH antioxidant capacity (µM TE/g).
Figure 8.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on DPPH antioxidant capacity (µM TE/g).
The results (
Table 4 and
Figure 8) illustrated the impact of MAE radiation time, power, and sample-to-solvent ratio on the DPPH free radical scavenging capacity of the macadamia skin extract. MAE radiation time and power were found not to significantly affect the DPPH, but the sample-to-solvent ratio did significantly affect the DPPH free radical scavenging capacity of the macadamia skin extract (
p < 0.05). As seen in the ABTS assay, the interaction between time
x sample-to-solvent ratio, and power
x sample-to-solvent ratio did not have a significant impact, but the interaction between MAE time
x power had a significant impact on ABTS antioxidant capacity of the macadamia skin extract (
p < 0.05).
Figure 9.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on CUPRAC (µM TE/g).
Figure 9.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on CUPRAC (µM TE/g).
Figure 10.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on FRAP antioxidant capacity (µM TE/g).
Figure 10.
Impact of time (2.5–5.5 min), power (30%–70%, 360–840 W) and sample-to-solvent ratio (2–8 g/100 mL) on FRAP antioxidant capacity (µM TE/g).
The influence of MAE radiation time, power and sample-to-solvent ratio on the cupric ion reducing antioxidant capacity (CUPRAC) of the macadamia skin is represented in
Table 4 and
Figure 9. The results (
Table 4) revealed that MAE radiation time, power, and sample-to-solvent ratio did not have a significant impact on the CUPRAC of the macadamia skin (
p > 0.05). Similarly, there was no significant impact between the interaction of MAE time
x power, time
x sample-to-solvent ratio, and power
x sample-to-solvent ratio of the macadamia skin extract (
p > 0.05).
Finally, the results (
Table 4 and
Figure 10) indicated the impact of MAE radiation time, power, and sample-to-solvent ratio on the FRAP of the macadamia skin extract. MAE power was found to have a significant impact on the FRAP of the macadamia skin extract; whereas, MAE radiation time and sample-to-solvent ratio did not have a significant effect. The results (
Table 4) also showed that there was no significant impact between the interaction of MAE time
x power, time
x sample-to-solvent ratio, and power
x sample-to-solvent ratio on FRAP of the macadamia skin extract (
p > 0.05).
3.4. Optimisation and Validation of Microwave Extraction Conditions
The predicted mathematical models of this study indicated that the optimal extraction conditions for the highest level of TPC, flavonoids, proanthocyanidins, and antioxidant properties were MAE time of 4.5 min, power of 30% (360 W), and a sample-to-solvent ratio of 5 g/100 mL. To ensure that the results of the predicted conditions were matched with the results when these conditions were applied in reality, the sample of macadamia skin was extracted under the recommended conditions in triplicates. The actual results and the predicted results are shown in
Table 5. As can be seen from the
Table 5, all the experimental values for TPC, flavonoid, proanthocyanidin, and antioxidant properties were not significantly different to their predicted values (
p > 0.05), indicating that these predicted conditions were valid and could be applied for maximum recovery of phenolic compounds and antioxidant properties from the macadamia skin.
Table 5.
Validation of the predicted values for TPC, flavonoids, proanthocyanidins, and antioxidant potential.
Table 5.
Validation of the predicted values for TPC, flavonoids, proanthocyanidins, and antioxidant potential.
| Values |
---|
Predicted | Experimental (n = 3) |
---|
TPC (mg GAE/g) | 51.13 ± 13.86 a | 44.75 ± 2.34 a |
Flavonoids (mg RE/g) | 28.08 ± 6.64 a | 29.10 ± 1.04 a |
Proanthocyanidins (mg GAE/g) | 22.95 ± 3.05 a | 33.60 ± 0.48 b |
ABTS (µM TE/g) | 434.36 ± 92.71 a | 361.60 ± 14.22 a |
DPPH (µM TE/g) | 355.74 ± 84.61 a | 292.78 ± 17.63 a |
CUPRAC (µM TE/g) | 572.60 ± 201.70 a | 459.80 ± 51.75 a |
FRAP (µM TE/g) | 92.73 ± 26.29 b | 297.03 ± 24.74 b |
Under these extraction conditions, approximately 45 mg of TPC, 29 mg of flavonoids and 33 mg of proanthocyanidins could be extracted from one gram of dried macadamia skin. Alasalvar and Shahidi [
30] reported that one gram of macadamia kernel contained 1.56 mg of the phenolic compounds, meaning that the level of phenolic compounds in the macadamia skin is significantly higher than that in the kernel. In addition, Yang [
31] reported the flavonoid content of the macadamia kernel was 1.379 mg/g, which was less than 5% of the flavonoids available in the skin of the macadamia, revealing that macadamia skin is the waste, but it is a rich source of phenolic compounds. Furthermore, Alasalvar and Shahidi [
30] also reported a FRAP antioxidant value for macadamia kernel was 0.42 mM/100 g or 4.2 µM TE/g, which is also significantly lower than the FRAP values found in macadamia skin in the current study. Therefore, these findings further confirmed that macadamia skin is a rich source of phenolic content and is also a potent source of antioxidants in comparison with its kernel.
In comparison with the conventional extraction method at optimal conditions of 90 °C, 20 min, and sample-to-solvent ratio of 5 g/100 mL, which could extract 96 mg of TPC, 24 mg of flavonoids, and 97 mg of proanthocyanidins from a dried gram of macadamia skin [
32], MAE method under these optimal conditions gave significant lower levels of TPC, flavonoids and proanthocyanidins. The reason for the low recovery yields of bioactive compounds when using MAE method can be explained by the heat, which was generated during extraction process. As the bioactive compounds from the macadamia skin were sensitive to the high temperature, thus they were partially degraded during the extraction process.