# Optimisation of Ultrasonic Conditions as an Advanced Extraction Technique for Recovery of Phenolic Compounds and Antioxidant Activity from Macadamia (Macadamia tetraphylla) Skin Waste

^{*}

## Abstract

**:**

## 1. Introduction

## 2. Experimental Section

#### 2.1. Materials

#### 2.2. Chemicals

#### 2.3. Ultrasonic-Assisted Extraction (UAE)

#### 2.4. Methods for the Determination of Chemical Properties

#### 2.4.1. Total Phenolic Content (TPC)

_{2}CO

_{3}, then mixed well on a vortex agitator and incubated in the dark at room temperature for one hour before the absorbance was measured at 760 nm using a UV spectrophotometer (Varian Australia Pty. Ltd., Victoria, Australia). A standard curve was formed through the use of gallic acid and the results were then specified in milligrams of gallic acid equivalents per gram of sample (mg GAE/g).

#### 2.4.2. Total Flavonoids

_{2}O and 0.15 mL of 5% (w/v) NaNO

_{2}were added together and left at room temperature for 6 min. Following the 6 min, 0.15 mL of 10% (w/v) AlCl

_{3}was added and left at room temperature for a further 6 min. Finally 2 mL 4% (w/v) NaOH and 0.7 mL of H

_{2}O were added, with the final solution being mixed well and left at room temperature for a further 15 min before the absorbance was measured at 510 nm using a UV spectrophotometer. A standard curve was designed through the use of rutin and the results were then specified in milligrams of rutin equivalents per gram of sample (mg RUE/g).

#### 2.4.3. Proanthocyanidins

#### 2.5. Methods for the Determination of Antioxidant Properties

#### 2.5.1. ABTS Radical Scavenging Capacity

_{2}S

_{2}O

_{8}and left at room temperature in the dark for 15 h, and then stored at −20 °C until required. The working solution was freshly prepared by mixing 1 mL of stock solution in 60 mL of methanol to obtain an absorbance value of 1.1 ± 0.02 at 734 nm. 0.15 mL of sample was added to 2.85 mL of the working solution and mixed, then left in the dark at room temperature for 2 h before its absorbance was measured at 734 nm using a UV-VIS spectrophotometer. A standard curve was designed through the application of trolox and the results were expressed as micromoles of trolox equivalents per gram of dried sample (μM TE/g).

#### 2.5.2. DPPH Radical Scavenging Activity

#### 2.5.3. Cupric Reducing Antioxidant Capacity (CUPRAC)

_{2}, 1 mL of neocuproine and 1 mL of NH

_{4}Ac were added and then 1.1 mL of diluted sample was added. After mixing well, the mixture was incubated at room temperature for 1.5 h before measuring the absorbance at 450 nm using the UV spectrophotometer. A standard curve was designed through the use of trolox and the results were expressed as micromoles of trolox equivalents per gram of sample (μM TE/g).

#### 2.5.4. Ferric Reducing Antioxidant Power (FRAP)

#### 2.6. Response Surface Methodology

_{i}values are independent variables affecting the responses Y; β

_{0}, β

_{i}, β

_{ii}and β

_{ij}are the regression coefficients for intercept, linear, quadratic, and interaction terms, respectively; and k is the number of variables.

Run | Experimental Conditions | Experimental Results | ||||||||
---|---|---|---|---|---|---|---|---|---|---|

X_{1} | X_{2} | X_{3} | TPC | Flavon-Oids | Proantho-Cyanidins | ABTS | DPPH | CUPRAC | FRAP | |

1 | 30 | 30 | 60 | 144.28 | 120.80 | 155.58 | 123.46 | 912.40 | 2678.23 | 1099.37 |

2 | 30 | 10 | 80 | 138.28 | 109.81 | 143.48 | 123.63 | 890.36 | 2524.60 | 1062.25 |

3 | 30 | 50 | 80 | 155.13 | 123.31 | 163.20 | 123.32 | 969.60 | 2848.05 | 1278.35 |

4 | 30 | 30 | 100 | 161.46 | 127.70 | 172.13 | 123.47 | 995.60 | 2857.49 | 1306.06 |

5 | 40 | 10 | 60 | 145.82 | 108.83 | 153.42 | 123.60 | 938.73 | 2398.47 | 1076.15 |

6 | 40 | 50 | 60 | 164.29 | 124.61 | 179.62 | 123.26 | 1007.87 | 2752.79 | 1338.07 |

7 | 40 | 30 | 80 | 220.17 | 127.58 | 189.71 | 123.49 | 1082.87 | 2705.35 | 1746.36 |

8 | 40 | 30 | 80 | 173.08 | 131.57 | 191.43 | 123.42 | 1047.40 | 2903.49 | 1545.67 |

9 | 40 | 30 | 80 | 174.10 | 132.36 | 251.16 | 123.39 | 1055.47 | 2909.49 | 1795.67 |

10 | 40 | 10 | 100 | 167.55 | 128.66 | 190.67 | 123.59 | 1061.96 | 2925.49 | 1343.73 |

11 | 40 | 50 | 100 | 177.55 | 128.97 | 200.86 | 123.17 | 1056.20 | 2885.16 | 1304.95 |

12 | 50 | 30 | 60 | 170.09 | 132.89 | 196.08 | 123.27 | 1006.20 | 2754.54 | 1229.62 |

13 | 50 | 10 | 80 | 159.00 | 129.15 | 173.11 | 123.57 | 1005.13 | 2613.07 | 1164.46 |

14 | 50 | 50 | 80 | 180.45 | 151.20 | 210.82 | 123.26 | 1021.42 | 3037.35 | 1313.26 |

15 | 50 | 30 | 100 | 180.17 | 146.43 | 211.62 | 123.33 | 1035.93 | 3011.91 | 1305.24 |

_{1}(temperature, °C), X

_{2}(time, min.) and X

_{3}(power, %, W); TPC (mg GAE/g of dried weight), Flavonoids (mg RUE/g of dried weight), Proanthocyanidins (mg CE/g of dried weight), ABTS (μM TE/g of dried weight), DPPH (µM TE/g of dried weight), CUPRAC (μM TE/g of dried weight) and FRAP (μM TE/g of dried weight).

_{1}(temperature, °C), X

_{2}(time, min.) and X

_{3}(power, %, W). Thus, the function containing these three independent variables is expressed as follows:

#### 2.7. Statistical Analyses

## 3. Results and Discussion

#### 3.1. Statistical Analysis and Fitting of the Model

^{2}, Predicted Residual Sum of Square (PRESS) for the models, F ratio and Prob > F were analyzed to identify the fitting of the RSM mathematical models. “Lack of fit” shows a test assessing if the model has the appropriate effects, when that test can be conducted; whereas, R

^{2}estimates the proportion of variation in the response that can be attributed to the model rather than to random error, with an R

^{2}value near 1 indicating that the model is a good predictor of the response. PRESS 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. Finally, the Prob > F value measures the probability of obtaining an F Ratio as large as what is observed, given that all parameters except the intercept are zero. Small values of Prob > F indicate that the observed F Ratio is unlikely. Such values are considered evidence that there is at least one significant effect in the model [23].

^{2}) of the models for all three responses was close to 1 (ranging from 0.73 to 0.94), indicating that these mathematical models are reliable predictors for TPC, flavonoids and proanthocyanidins. These R

^{2}values also reveal that at least 73% of the actual values were matched with the predicted values proposed by the mathematical models. The results also outlined that “lack of fit” of the models for TPC, flavonoids and proanthocyanidins were all significantly higher than 0.05. These indicated that these models had the appropriate effects when the experiments were conducted. In addition, the F ratio of the models were found to be low, and the values of “Prob > F” ranged from 0.01 to 0.34, with these results further confirming that the RSM models were reliable in the prediction of the optimal ultrasonic extraction conditions for TPC, flavonoids and proanthocyanidins in the current study.

TPC | Flavon-Oids | Proantho-Cyanidins | Antioxidant Capacity | ||||
---|---|---|---|---|---|---|---|

ABTS | DPPH | CUPRAC | FRAP | ||||

Lack of fit | 0.998 | 0.198 | 0.987 | 0.380 | 0.647 | 0.618 | 0.980 |

R^{2} | 0.73 | 0.94 | 0.76 | 0.94 | 0.97 | 0.87 | 0.94 |

Adjusted R^{2} | 0.24 | 0.84 | 0.32 | 0.84 | 0.91 | 0.65 | 0.84 |

PRESS | 3573 | 1353 | 7804 | 0.21 | 12,605 | 540,637 | 123,793 |

F ratio of Model | 1.50 | 9.41 | 1.72 | 8.92 | 17.52 | 3.83 | 9.25 |

Prob > F | 0.34 | 0.01 | 0.29 | 0.01 | 0.00 | 0.08 | 0.01 |

**Figure 2.**Correlation between the predicted and the experimental values for TPC, flavonoids, and proanthocyanidins.

^{2}values of the models for DPPH, ABTS, FRAP and CUPRAC were 0.94, 0.97, 0.87 and 0.94, respectively. These results revealed a close correlation between the predicted values and experimental values with at least 87% of experimental values for antioxidant capacity matching with their responding predicted values. The values for “lack of fit” of the models for all four antioxidant assays were all significantly higher than 0.05, meaning that these models had the appropriate effect when conducting the experiments. In addition, the values of “Prob > F” were significantly lower than 0.05, and these further confirmed that the RSM models were adequate in the prediction of optimal ultrasonic extraction conditions for all four antioxidant properties in the current study. The models could be fitted to the following second-order polynomial formulas Equations (6)–(9):

**Figure 3.**Correlation between the predicted and the experimental values for ABTS total antioxidant capacity, DPPH free radical scavenging capacity, cupric reducing antioxidant power (CUPRAC) and ferric reducing antioxidant power (FRAP).

#### 3.2. Effect of Extraction Independent Variables on TPC, Flavonoids and Proanthocyanidins

**Figure 4.**Impact of temperature (60–90 °C), time (10–30 min) and power (60%–100%) on TPC (mg GAE/g).

**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| | ||

β_{0} | 1 | 189.115 | <0.0001 * | 130.503 | <0.0001 * | 210.765 | <0.0001 * |

β_{1} | 1 | 11.320 | 0.121 | 9.756 | 0.002 * | 19.656 | 0.059 |

β_{2} | 1 | 8.347 | 0.227 | 6.455 | 0.009 * | 11.728 | 0.205 |

β_{3} | 1 | 7.781 | 0.255 | 5.579 | 0.016 * | 11.321 | 0.219 |

β_{12} | 1 | 1.151 | 0.898 | 2.138 | 0.374 | 4.498 | 0.709 |

β_{13} | 1 | −1.776 | 0.844 | 1.661 | 0.482 | −0.253 | 0.983 |

β_{23} | 1 | −2.115 | 0.815 | −3.868 | 0.138 | −4.004 | 0.739 |

β_{11} | 1 | −15.350 | 0.146 | 3.525 | 0.183 | −17.700 | 0.196 |

β_{22} | 1 | −15.552 | 0.142 | −5.661 | 0.056 | −20.412 | 0.146 |

β_{33} | 1 | −9.761 | 0.323 | −2.074 | 0.405 | −9.212 | 0.472 |

_{0}: Intercept; β

_{1}, β

_{2}and β

_{3}: Linear regression coefficients for temperature, time and power; β

_{12}, β

_{13}, and β

_{23}: Regression coefficients for interaction between temperature x time, temperature x power and time x power; β

_{11}, β

_{22}, and β

_{33}: Quadratic regression coefficients for temperature x temperature, time x time and power x power.

**Figure 5.**Impact of temperature (60–90 °C), time (10–30 min) and power (60%–100%) on flavonoids (mg RUE/g).

**Figure 6.**Impact of temperature (60–90 °C), time (10–30 min) and power (60%–100%) on proanthocyanidins (mg CAE/g).

#### 3.3. Effect of Extraction Independent Variables on the Antioxidant Capacity of Macadamia Tetraphylla Skin

Parameter | DF | ABTS | DPPH | CUPRAC | FRAP | ||||
---|---|---|---|---|---|---|---|---|---|

Estimate | Prob>|t| | Estimate | Prob>|t| | Estimate | Prob>|t| | Estimate | Prob>|t| | ||

β_{0} | 1 | 123.433 | <.0001 * | 1061.911 | <.0001 * | 2839.442 | <.0001 * | 1695.903 | <.0001 * |

β_{1} | 1 | −0.057 | 0.039 * | 37.592 | 0.001 * | 63.562 | 0.153 | 33.318 | 0.328 |

β_{2} | 1 | −0.171 | 0.000 * | 19.864 | 0.020 * | 132.715 | 0.017 * | 73.505 | 0.063 |

β_{3} | 1 | −0.003 | 0.878 | 35.561 | 0.002 * | 137.002 | 0.015 * | 64.595 | 0.090 |

β_{12} | 1 | −0.001 | 0.986 | −15.739 | 0.117 | 25.209 | 0.657 | −16.824 | 0.715 |

β_{13} | 1 | 0.012 | 0.705 | −13.367 | 0.169 | 19.527 | 0.730 | −32.770 | 0.485 |

β_{23} | 1 | −0.018 | 0.556 | −18.722 | 0.074 | −98.663 | 0.124 | −75.179 | 0.145 |

β_{11} | 1 | −0.006 | 0.841 | −59.469 | 0.001 * | 0.696 | 0.991 | −260.986 | 0.002 * |

β_{22} | 1 | 0.017 | 0.600 | −30.814 | 0.016 * | −84.370 | 0.189 | −230.335 | 0.004 * |

β_{33} | 1 | −0.045 | 0.195 | −14.908 | 0.146 | −14.595 | 0.803 | −199.844 | 0.007 * |

_{0}: Intercept; β

_{1}, β

_{2}, and β

_{3}: Linear regression coefficients for temperature, time and power; β

_{12}, β

_{13}, and β

_{23}: Regression coefficients for interaction between temperature x time, temperature x power and time x power; β

_{11}, β

_{22}, and β

_{33}: Quadratic regression coefficients for temperature x temperature, time x time and power x power.

**Figure 8.**Impact of temperature (60–90 °C), time (10–30 min) and power (60%–100%) on DPPH free radical scavenging capacity.

#### 3.4. Optimisation and Validation of Ultrasonic Extraction Conditions

**Table 5.**Validation of the predicted values for TPC, flavonoids, proanthocyanidins and antioxidant potential.

Values | ||
---|---|---|

Predicted | Experimental (n = 3) | |

TPC (mg GAE/g) | 190.23 ± 24.96 ^{a} | 168.22 ± 0.77 ^{a} |

Flavonoids (mg RE/g) | 131.76 ± 6.38 ^{a} | 135.01 ± 3.18 ^{a} |

Proanthocyanidins (mg CE/g) | 212.42 ± 33.18 ^{a} | 187.71 ± 14.04 ^{a} |

ABTS (µM TE/g) | 123.39 ± 0.09 ^{a} | 102.36 ± 0.16 ^{a} |

DPPH (µM TE/g) | 1064.95 ± 24.23 ^{a} | 1128.76 ± 12.33 ^{a} |

CUPRAC (µM TE/g) | 2867.35 ± 155.57 ^{a} | 2736.31 ± 22.28 ^{a} |

FRAP (µM TE/g) | 1699.88 ± 126.83 ^{a} | 1607.82 ± 7.89 ^{a} |

^{a}) are significantly different from each other (p < 0.05).

## 4. Conclusions

## Acknowledgments

## Author Contributions

## Conflicts of Interest

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## Share and Cite

**MDPI and ACS Style**

Dailey, A.; Vuong, Q.V.
Optimisation of Ultrasonic Conditions as an Advanced Extraction Technique for Recovery of Phenolic Compounds and Antioxidant Activity from Macadamia (*Macadamia tetraphylla*) Skin Waste. *Technologies* **2015**, *3*, 302-320.
https://doi.org/10.3390/technologies3040302

**AMA Style**

Dailey A, Vuong QV.
Optimisation of Ultrasonic Conditions as an Advanced Extraction Technique for Recovery of Phenolic Compounds and Antioxidant Activity from Macadamia (*Macadamia tetraphylla*) Skin Waste. *Technologies*. 2015; 3(4):302-320.
https://doi.org/10.3390/technologies3040302

**Chicago/Turabian Style**

Dailey, Adriana, and Quan V. Vuong.
2015. "Optimisation of Ultrasonic Conditions as an Advanced Extraction Technique for Recovery of Phenolic Compounds and Antioxidant Activity from Macadamia (*Macadamia tetraphylla*) Skin Waste" *Technologies* 3, no. 4: 302-320.
https://doi.org/10.3390/technologies3040302