Microwave-Assisted Ethanolic and Aqueous Extraction of Antioxidant Compounds from Pomegranate Peel and Broccoli Leaves By-Products †

: The aim of the present work was to study the effect of solvent (50%:50% ethanol:water; 100% ethanol, and 100% water) on the extraction of total phenolic content (TPC) and total antioxidant activity (TAC) from pomegranate peel and broccoli leaves through microwave-assisted extraction (MWAE). In pomegranate peel, TPC after 100% aqueous MWAE (119.6 g GAE/kg dw) increased 1.6-fold more than 100% ethanolic extraction (74.2 g GAE/kg dw). Regarding broccoli leaves, TPC after 100% aqueous MWAE (28.4 g GAE/kg dw) increased 3.4-fold more than 100% ethanolic extraction (8.5 g GAE/kg dw). In this sense, TAC reported similar behavior in broccoli leaves’ extraction (DPPH and ABTS values ranged between 1.1–3.5 g TE/kg dw and 2.4–3.8 g TE/kg dw, respectively), while no great differences were found among solvents during the pomegranate peel extraction (DPPH and ABTS values ranged between 3.5–3.7 g TE/kg dw and 3.7–4.3 g TE/kg dw, respectively). Therefore, aqueous MWAE could be considered as a green technology to recover TPC from horticultural by-products. This could be of great interest for industry, and should contribute to the problem of food loss.


Introduction
Considering that the food supply chain can be divided into the primary production, processing, retail/distribution, and consumption stages, FAO defines 'food loss' as food discarded along the two mentioned first stages.During the pomegranate processing chain, around 43% can be catalogued as non-edible parts (peel and the carpel membranes) [1].Pomegranate by-products are very rich in bioactive compounds such as ellagitannins [2].On the other hand, harvesting broccoli generates a huge number of leaves, and stalks, which are catalogued as by-products.Furthermore, compared to broccoli florets or stalks, broccoli leaves, which could represent more than 50% of the total plant, have a higher total phenolic content (TPC), and more other bioactive compounds related to their antioxidant activity than the rest of the broccoli by-products [3].
Green extraction technologies of the target bioactive compounds have recently been developed, including ultrasound-, microwave-, and enzyme-assisted extractions, among others [1,3,4].These technologies use green solvents, minimizing environmental and health disorders.
In view of the above, the aim of the present work is to observe the effect of greensolvents (50%:50% ethanol:water; 100% ethanol, and 100% water) on the extraction of TPC and Total Antioxidant Activity (DPPH and ABTS) from pomegranate peel and broccoli leaves through microwave-assisted extraction (MWAE).

Microwave-Assisted Extraction (MWAE)
Prior to the target compounds MWAE, pre-treatments (drying and grinding steps) were carried out to obtain a non-heterogeneous material.Samples were freeze-dried using a Telstar® LyoBeta (Terrassa, Spain).The fixed variables were as follows: (i) particle size (<56 µm), (ii) solid-liquid ratio (1:10), (iii) time (13 min), and (iv) temperature (50 • C).The continuous variables were the solvent (50%-50% ethanol:water; 100% ethanol, and 100% water).Subsequently, the extraction was carried out by a microwave device (Milestone, Ethos up, Italy).Once the extraction of all the samples was completed, the samples were centrifugated and the extracts were stored (−80 • C) until further analysis.The codification and combination of the variables are detailed in Table 1.α P = pomegranate by-products; B = broccoli; FD: freeze-drying.

Spectrophotometric Analysis
The determination of TPC was carried out according to the previously described method [5], with some modifications [6].The TPC was calculated using a gallic acid standard and expressed as g of gallic acid equivalent per kg of dried weight (g GAE/kg dw).
The Total Antioxidant Capacity (TAC) was analyzed by DPPH and ABTS assay.For the DPPH assay [7], 194 µL of DPPH solution was added to 21 µL of extract in a 96-well plate (darkness incubation: 30 min at room temperature).Then, the absorbance was measured (515 nm) and data were expressed as g of Trolox Equivalents (TE)/kg dw.The ABTS assay was carried out following the previously described method [7].For that, 200 µL of the activated ABTS solution (32 µM) was added to 11 µL of extract in a 96-well plate, darkness-incubated (20 min at room temperature) and measured (414 nm).

Statistics
Statistical analyses were carried out using the statistical package.Each sample was analyzed in triplicate for each analysis.One-way ANOVA test used "solvent" for each food by-product.Tukey test was used for means comparison (95% confidence level).Pearson correlation using XLSTAT Premium 2016 (Addingsoft, Barcelona, Spain) was conducted.

Total Polyphenolic Content
The effect of the solvent on the TPC extraction by MWAE in pomegranate by-products and broccoli by-products is shown in Figure 1.It should be noted that because of the polarity differences between solvents, the solubility of the key bioactive compounds into the solvent is expected to be different.Significant differences were observed among the studied solvents in both pomegranate and broccoli by-products.Also, it is important to highlight that the use of water as a solvent was the best option for TPC extraction in both food by-products under study.In pomegranate peel, TPC after 100% aqueous extraction increased 1.6-fold more than 100% ethanolic extraction.In broccoli leaf by-products, TPC after 100% aqueous extraction increased 3.4-fold more than 100% ethanolic extraction.In general, previous studies indicated that the effect of the dielectric constants of the solvents impacts the extraction of antioxidant compounds.Methanol and water present higher constants than other solvents, such as ethanol and acetone.Therefore, the extraction yield depends on the type of antioxidant compound and must be optimized each fruit and vegetable commodity and their derivatives.It is worth noting that there are other solvents with a higher capacity to extract key bioactive compounds.but these are not catalogued as green solvents [8,9].

Statistics
Statistical analyses were carried out using the statistical package.Each sample was analyzed in triplicate for each analysis.One-way ANOVA test used "solvent" for each food by-product.Tukey test was used for means comparison (95% confidence level).Pearson correlation using XLSTAT Premium 2016 (Addingsoft, Barcelona, Spain) was conducted.

Total Polyphenolic Content
The effect of the solvent on the TPC extraction by MWAE in pomegranate by-products and broccoli by-products is shown in Figure 1.It should be noted that because of the polarity differences between solvents, the solubility of the key bioactive compounds into the solvent is expected to be different.Significant differences were observed among the studied solvents in both pomegranate and broccoli by-products.Also, it is important to highlight that the use of water as a solvent was the best option for TPC extraction in both food by-products under study.In pomegranate peel, TPC after 100% aqueous extraction increased 1.6-fold more than 100% ethanolic extraction.In broccoli leaf by-products, TPC after 100% aqueous extraction increased 3.4-fold more than 100% ethanolic extraction.In general, previous studies indicated that the effect of the dielectric constants of the solvents impacts the extraction of antioxidant compounds.Methanol and water present higher constants than other solvents, such as ethanol and acetone.Therefore, the extraction yield depends on the type of antioxidant compound and must be optimized each fruit and vegetable commodity and their derivatives.It is worth noting that there are other solvents with a higher capacity to extract key bioactive compounds.but these are not catalogued as green solvents [8,9].

Total Antioxidant Capacity
The effect of the solvent on the TAC via DPPH and ABTS assay was different in pomegranate and broccoli by-product extracts obtained using MWAE.In pomegranate extracts, ethanolic and aquose extracts presented the highest DPPH assay values, followed by 50:50 ethanol:water solvent.No significant differences were observed among solvents in ABTS data in pomegranate peel.On the other hand, the highest DPPH values were observed in the extracts obtained via 50:50 ethanol:water solvent, followed by aquose

Total Antioxidant Capacity
The effect of the solvent on the TAC via DPPH and ABTS assay was different in pomegranate and broccoli by-product extracts obtained using MWAE.In pomegranate extracts, ethanolic and aquose extracts presented the highest DPPH assay values, followed by 50:50 ethanol:water solvent.No significant differences were observed among solvents in ABTS data in pomegranate peel.On the other hand, the highest DPPH values were observed in the extracts obtained via 50:50 ethanol:water solvent, followed by aquose solvent.Brocoli by-product extracts using 50:50 ethanol:water and 100% water solvent presented the highest ABTS values (Figure 2).solvent.Brocoli by-product extracts using 50:50 ethanol:water and 100% water so presented the highest ABTS values (Figure 2).Table 2 shows the correlation among phytochemical parameters (TPC and TA DPPH • and ABTS + assays).A positive and significant correlation was observed betw TPC and DPPH • , while no significant correlation was observed between TPC and AB

Conclusion
In general, it can be concluded that the type of solvent and food by-product (p granate and broccoli material) influenced the extraction of TPC, which was higher in p egranate by-products than in broccoli by-products.MWAE was demonstrated to clean, efficient, and a green alternative for the extraction of TPC and bioactive compo with TAC from pomegranate peels and broccoli leaves.Aqueous MWAE is a green a native to recover TPC and is of great interest for manufacturers, which should addres problem of food loss, as specified in the goals of global, European and national strat (2030 Agenda, European Green Deal, and the FAO Strategic Framework 2022-31).

Figure 1 .
Figure 1.Total phenolic compounds (g gallic acid equivalents/kg dw) derived through Folin assay (garnet-coloured colums: pomegranate samples; greenish columns: broccoli samples).‡ Columns with a different letter and the same color were significantly different (p < 0.05).

Figure 1 .
Figure 1.Total phenolic compounds (g gallic acid equivalents/kg dw) derived through Folin assay (garnet-coloured colums: pomegranate samples; greenish columns: broccoli samples).‡ Columns with a different letter and the same color were significantly different (p < 0.05).

Figure 2 .
Figure 2. Antioxidant activity: DPPH (filled columns) and ABTS assay (unfilled columns) ‡ Col with the different letter and same color were significantly different (p < 0.05).

Table 1 .
Codification and combination of the variables.