R2 (TAC) = +1.03 + 0.3114B + 0.3152D + 0.3727AB <sup>−</sup> 0.0386AD + 0.0247A<sup>2</sup> <sup>−</sup> 0.0311B2 + 0.0148C2 <sup>−</sup> 0.1208D2 (4)

A model reduction was achieved by neglecting the insignificant model terms. Equation (4) represents the model equation showing the correlation between the TAC (R2) and the variables in coded units. The regression equation's b coefficients showed that the ethanol concentration and extraction time positively affected the anthocyanins extraction. The interaction between citric acid and ethanol concentration (AB) had an appreciably positive effect on anthocyanins extraction. In contrast, citric acid concentration (A2) and temperature (C2) had a more negligible contribution. Furthermore, moderately negative effects on the anthocyanins yield were shown by the interaction between citric acid concentration and extraction time (AD), between ethanol concentration and ethanol concentration (B2), and between time and time (D2).

The 3D surface plots of significant interaction effects display the ethanol concentration, acid, time, and temperature of extraction and the interaction among various factors influencing the conventional extraction of red onion skins anthocyanins, characterized by curved surfaces, as exhibited in Figure 1B(a–d).

In examining the effects of ethanol concentration and citric acid, it was noticed that the TAC increased as the ethanol concentration increased to 50% and the citric acid concentration was over 0.86% (Figure 1B(a)). According to the surface graphs, the concentration of anthocyanins was not influenced by temperature variation and extraction time but was influenced by ethanol concentration (Figure 1B(b,c)). The yield of anthocyanins constantly improved as the extraction time and ethanol concentration increased simultaneously, according to an analysis of the impacts of both variables (Figure 1B(d)).

The variation in the output response range concerning the reference point revealed how sensitive the response was to that variable. This plot helped to identify the factor that most influenced the TAC extraction response. The perturbations graph showing each independent variable's impact revealed that time extraction and ethanol concentration significantly affected the TAC. To a lesser extent, the temperature also influenced it (Figure 2b).

#### *3.3. Optimization and Validation of the Extraction Parameters*

The model recommended optimal factors based on maximizing the response desirability to validate the model equation. A desirability score of 1 (0.929) indicated that all selected conditions were correct (Figure 3, Table 4). The optimal conditions for generating the highest extraction of anthocyanins and the highest antioxidant activity were 0.87% citric acid, 60% ethanol, 25 ◦C, and an extraction time of 180 min.

**Figure 3.** Optimization desirability bar chart (**a**) and ramps (**b**).

**Table 4.** Validation of the mathematical model.


The model predicted the maximum concentration of antioxidant activity and anthocyanins were 35.45 mM TE/g DW and 1.43 mg C3G/g DW, respectively. At the same time, the experimental data showed immediate responses to those predicted by the model, particularly 37.20 mM TE/g DW and 1.43 mg C3G/g DW (Table 4).

#### **4. Discussion**

This study optimized the conventional extraction process parameters to extract anthocyanins from red onion skins and enhance their antioxidant activity. Four variables (ethanol concentration, citric acid, temperature, and time) were used to optimize the extraction parameters screened by CCD. Under the optimum conditions, the antioxidant activity was at the highest level at an ethanol concentration of around 60% and a low temperature (25 ◦C). However, Corrales et al. [14] reported that extracting red grape skins at 70 ◦C with 50% ethanol concentration increased the extract's antioxidant activity. These different results could potentially be due to variations in the principles or reaction times used to measure the antioxidant activity [15]. Our findings for red onion skin extracts' antioxidant activity approach those of Viera et al. [16]. The most significant DPPH radical scavenging activity, measured by the authors as 116.58 ± 4.9 mol TE/g DW, was found in red onion skin extracts obtained by conventional extraction under ideal conditions of 80% ethanol and 120 min of extraction at 25 ◦C. In a different study, Ifesan [17] asserted that the activity to scavenge DPPH radicals was highest in an onion skin extract obtained by conventional extraction (maceration for 24 h at 25 ◦C with 80% ethanol), reaching a value of 27.76 ± 0.91 μg TE/mL. Prokopov et al. [18] used 70% aqueous ethanol, 15 min as the extraction time, and 45 ◦C to find that the extract of red onion skins exposed to ultrasound had higher antioxidant activity (490.54 ± 9.43 mM TE/g DW). Additionally, to assess the optimum antioxidant activity of onion solid waste extracts, Khiari et al. [19] utilized a conventional extraction method (40 ◦C, 6 h). In comparison to our findings, the 90% ethanol acidified with 0.1% HCl produced a decreased antiradical activity (0.32 ± 0.02 mM TE/g DW).

The extraction process is determined by the values of the extraction parameters employed while extracting bioactive compounds from plant matrices. Likewise, the different polarities of the compounds extracted using an experimental model may have an unpredictable effect on the extraction conditions. As a result, the extractions were conducted using solvents with varying polarities and varying the water and ethanol proportions. Adding water to ethanol may enhance the yield of anthocyanins extraction [20], and the resulting extracts are simple to introduce into biological systems. Highly glycosylated phenolics found in red onion skins cannot be extracted entirely only with pure organic solvents and require the application of mixtures containing water and acids. Water plays an important role in the swelling of plant material. In contrast, ethanol is responsible for disrupting the bonding between the solutes and the plant matrix, thus enabling a better mass transfer of the compounds. Therefore, a mixture of water and ethanol as solvent agents displays a synergistic effect that facilitates phenolics extraction. In addition, the citric acid used in a solvent mixture ruptures the cell membranes and releases phenol compounds [21]. The flavylium cation is the dominant species at pH = 3 or lower and contributes to the purple, orange, and red colors. A high solubility of anthocyanins in water is obtained by lowering the pH, increasing the structure transfer to the flavylium cation, and enhancing the stability. Hence, to optimize the extraction, citric acid is added to the extraction blend to acidify the medium [22]. When protic polar solvents such as ethanol are utilized, the acidification of the solvent improves the capacity to extract phenolics. The phenol–phenolate equilibrium moves toward the less polar phenyl form when the medium is acidified, making organic solvent extraction easier [23]. For anthocyanins, acidified ethanol is frequently used, which denatures the cell membranes while dissolving and stabilizing them [24]. Even anthocyanins that are structurally dependent on the medium's pH could be extracted through acidification, which changes their solubility characteristics and affects their stability [23]. Therefore, the use of weak organic acids, such as citric acid, is recommended besides the addition of water to maximize the effectiveness of solvent extraction. As previously noted, acids are typically used for effective anthocyanin extraction. To avoid the degradation or change of the native forms of the phenolic compounds, weak organic acid citric acid at the concentration of 0.05–2.64% was chosen in the experiments. It is also important to mention that the ethanol and citric acid combination was preferred as a food-grade solvent component for phenolics extraction. For example, the extraction of anthocyanins in conditions such as 30% ethanol with 3% of citric acid and 24 h at room temperature has been reported to give good results for blueberry leaves extraction [25]. In the past, mixtures of ethanol and citric acid were employed to extract phenolic compounds, particularly anthocyanins [26–28], and ethanol and its combination with citric acid have been reported to contribute to a successful extraction.

The CCD findings revealed that ethanol concentration and extraction time positively affected anthocyanins extraction. The findings support the results of Khazaei et al. [29], who found that by increasing the ethanol percentage and time, the TAC increased, which indicated a positive effect on anthocyanins extraction. In a Box–Behnken optimization study [30], 90% aqueous glycerol extracts of red onions under optimum sonication conditions with a 90/1 solvent/solid ratio at 45 ◦C for 60 min generated a high concentration of total pigments (1.87 ± 0.39 mg C3G/g DW). The TAC of the red onion skin extract reported by Bordin Viera et al. [31] was greater than that obtained in the present study, ranging from 0.82 to 4.31 mg C3G/g DW. Using ultrasound extraction, the anthocyanin yield was greater as the ethanol concentration increased (60–80%).

In addition, it was observed that the extraction temperature displayed a minor effect on the extraction yield of anthocyanins. Backes et al. [32] revealed that mild temperatures and a high content of ethanol increased the yield of anthocyanins extraction. Therefore, the authors confirmed that a solvent consisting of ethanol 100% acidified with citric acid, mixed and centrifuged with a powdered sample in a solid/liquid ratio of 50 g/L for 13.74 min at 35.64 ◦C was the optimal analytical factor to increase the TAC in extracts from fig skin (a byproduct of fruit). Higher temperatures damage anthocyanins (may cause their degradation) and result in a loss in yield, according to several earlier studies [33,34].

In our study, the TAC range was close to the one obtained by Oancea and Drághici [20], that reported 0.99 mg C3G/g fresh matter for the outer skins of the Sibiu red onion (*Allium cepa* L.) cultivar after extraction at 4 ◦C for 2 h with a mixture of ethanol/acetic acid/water (50:8:42, *v*/*v*/*v*), but lower than that reported by Samir et al. [35] (20 mg C3G/100 g), who used acidified ethanol with 1.5 N HCl (85:15, *v*/*v*) by maceration at 4 ◦C for 24 h.

The concentration of anthocyanins in the red onion skin extracts obtained through conventional extraction was examined by Viera et al. [16]. The extract obtained using 60% ethanol with an extraction time of 60 min at 25 ◦C yielded a concentration of anthocyanins of 470.2 ± 16.2 mg C3G/100 g DW. Additionally, with 90 min of extraction using 70% ethanol at 40 ◦C provided a more significant amount of anthocyanins extracted from red onion skins (847.47 ± 34.23 mg C3G/100 g DW) [36]. Makris [37] obtained a higher TAC of 183.85 mg C3G/100g DW by conventional extraction under the optimal conditions of 25 ◦C and 3.7 h extraction time from onion skins extract using 60% ethanol. Furthermore, the cultivar/origin of the plant material and its extraction conditions impact the amount of the extracted bioactive compounds.

According to the results of the optimization experiment, increasing ethanol concentration and time (up to 2 h) can be favorable to achieve a higher antioxidant activity. These findings are in agreement with the study conducted by Bordin Viera et al. [31], who suggested that ethanol concentration has a significant influence on the DPPH scavenging activity. Therefore, the red onion skin extracts obtained with ethanol at 20%, 40%, and 60% presented antioxidant activities of 26.12, 44.47, and 83.27 μmol TE/g DW, respectively.

#### **5. Conclusions**

A CCD and response surface methodology was used to optimize the variables of the conventional solvent extraction process (citric acid concentration—0.87%, ethanol concentration—60%, temperature—25 ◦C, and extraction time—179.99 min) to obtain red onion skin extracts with a high content of anthocyanins and high levels of antioxidant activity. The interaction of optimal time, temperature and acid and solvent concentrations improved the extraction of the antioxidant compounds yielding higher concentrations of anthocyanins (1.43 mg C3G/g DW) and DPPH radical scavenging activity levels (37.20 mM TE/g DW).

The optimization of the extraction process proved that the conventional solvent extraction could be an effective method for obtaining valuable extracts from natural and inexpensive sources such as food by-products with potential antioxidant and free radical scavenging activities.

These findings display an economically efficient extraction considering the low cost of by-product materials. Due to the high concentration of functional bioactive components in red onion skins, these compounds have a variety of uses in the food, pharmaceutical, and nutraceutical industries.

**Author Contributions:** Conceptualization, F.S., O.E.C. and G.R.; methodology, F.S. and O.E.C.; software, O.E.C. and I.A.; validation, O.E.C. and I.A.; formal analysis, F.S.; investigation, F.S.; resources, G.E.B.; data curation G.R. and N.S.; writing—original draft preparation F.S. and I.A.; writing—review and editing, G.R. and N.S.; visualization, N.S.; supervision, G.E.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data supporting this study's findings are available from the corresponding author (G.R.) upon reasonable request.

**Acknowledgments:** The results of this work have been presented to the 10th edition of the Scientific Conference organized by the Doctoral Schools of "Dunarea de Jos" University of Galati (SCDS-UDJG) http://www.cssd-udjg.ugal.ro/ (accessed on 9 April 2022) that will be held on 9th and 10th of June 2022, in Galati, Romania.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

