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Molecules 2021, 26, 1516 11 of 13 FRAP Assay The FRAP assay, consists of measuring the reduction of ferric-tripyridyltriazine (FeIII– TPTZ) to a ferrous complex (ferrous-tripyridyltriazine, FeII–TPTZ) by the presence of an antioxidant compound with an acetate buffer at pH 3.6. In the process a colour varia- tion (blue-colored product) occurs which is measured with a UV/Vis spectrophotometer at 593 nm (Jasco V-630). From each sample, 300 μL of the supernatant (prepared as de- scribed before) were used and mixed with 3 mL of the FRAP reagent solution. This last solution was prepared by using 2.5 mL of 2,4,6-tripyridyl-s-triazine (10 mM), 25 mL of acetate buffer (300 mM) at pH 3.6, in HCl (37% v/v) and 2.5 mL of FeCl3-6H2O (20 mM) in distilled water [36]. Trolox was applied as reference and the results were expressed in mg Trolox equivalent/g of Curcuma longa L. oil extracted (mg TE/g). Each sample was tested in triplicate. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) Assay The ABTS assay is based on the reaction of a solution of ABTS (7 mM) with potassium persulphate (2.45 mM) in the dark to generate the ABTS+ radical. This solution was diluted by a phosphate buffer salt (PBS) solution at pH 7.4 until an absorbance of 0.7 at 734 nm. Then, 30 μL of supernatant of each sample were added to a 3 mL of the last solution. After 6 min, the absorbance was measured by a UV/Vis spectrophotometer (Jasco V-630) [36]. Trolox was the standard reference and results were presented in mg Trolox equivalent/g Curcuma longa L. oil extracted (mg TE/g). All samples were measured in triplicate. 3.4. Statistical Analysis The statistical analysis was performed using IBM SPSS software (Version 24, Inc. Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to validate the experimental design and to study the differences between the MAE and Soxhlet extraction for each analysed parameter. The values of the significant differences were identified by Duncan’s multiple range test and significance was accepted at α = 0.05. The results were shown as mean ± SD (standard deviation) by performing 3 measurements for each analysis. 4. Conclusions The optimization of the extraction yield of Curcuma longa L. oil was carried out by microwave-assisted extraction using the Box-Behnken experimental design with response surface methodology. The optimum conditions for the extraction were 29.99 min, 160 W and a Curcuma longa L./EtOH ratio of 1:20 g/mL, and the yield of extracted oil was 10.32 ± 0.69%. This yield value was higher than that obtained by the conventional Soxhlet method (8.44 ± 0.17%). It was also concluded that the treatments applied for the extraction of the Curcuma longa L. oil influenced the TPC and antioxidant activity. The data from the three applied assays (DPPH, ABTS and FRAP) revealed higher phenolic content and higher antioxidant activity (in Curcuma longa L. oil when it was extracted using the MAE method. Therefore, these results demonstrated that MAE is a viable and more environmentally friendly method for the extraction of Curcuma longa L. oil. Author Contributions: Conceptualization, R.F.-M. and S.C.M.F.; methodology, R.F.-M.; writing— original draft preparation, R.F.-M., S.C.M.F. and J.L.; supervision, J.L. and M.A.A.; funding acquisition, S.C.M.F. and J.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Basque Country Government (IT 1008-16). Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. Acknowledgments: R.F.-M. would like to acknowledge the financial support of the Basque Country Government (scholarship of young researchers training and IT 1008-16). S.C.M.F. is the recipient of anPDF Image | Microwave-Assisted Extraction of Curcuma
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