*3.2. Effect of the Extraction Parameters on TPC*

This study aimed to find the ideal parameters for extracting phenolic compounds from red grape skins. The TPC ranged from 24.67 to 43.97 mg/g DW based on the experimental design depicted in Table 2. Data presented in Table 3 revealed that for TPC, a Model F-value of 2495.65 implies that the model is significant, and *p*-values less than 0.05 imply that the model terms were significant. Model terms are considered significant if their *p*-values are less than 0.0500. In this case, A, B, C, D, AC, AD, BC, BD, CD, A2, C2, and D2 are significant model terms.

$$\text{R2 (TPC)} = +37.41 + 3.19\text{A} + 2.88\text{B} + 3.10\text{C} - 1.10\text{D} - 0.1614\text{AB} - 0.9895\text{AC} + 5.39\text{AD} - 0.9180\text{BC} + 5.21\text{BD} - \tag{3}$$

$$\text{2.07}\text{CD} - 2.42\text{A}^2 - 0.6054\text{C}^2 - 2.89\text{D}^2$$

The model equation presenting the correlation between the R2 (TPC) and the variables in coded units is revealed in Equation 3. The regression equation's b coefficients showed that the ethanol concentration and extraction time positively affected the phenolic compounds extraction.

The interactions between citric acid concentration and time of extraction (AD) and between ethanol concentration and extraction time (BD) had an appreciably positive effect on TPC extraction. On the contrary, citric acid concentration (A2) and extraction time (D2) had an appreciably negative contribution. Furthermore, interactions between citric acid concentration and ethanol concentration (AB) and acid citric concentration and temperature (AC) of extraction, as well as ethanol concentration and temperature (BC), had a moderately negative effect on the TPC yield.

Figure 1(Ba–Bf) shows the tridimensional surface plots and 3D surfaces of the interactions between the concentrations of citric acid and ethanol, temperature, and extraction time that have the greatest influence on the extraction of TPC. The TPC increased when the ethanol concentration approached 85% and the citric acid concentration reached over 2%, according to an analysis of the impacts of ethanol concentration and citric acid concentration (Figure 1(Ba)). According to the surface graphs, citric acid concentration and ethanol concentration had a greater impact on polyphenol concentration than did extraction duration. A decrease in phenolic compounds content is observed at an increased concentration of citric acid and extraction time (Figure 1(Bb)). Additionally, the increase in TPC with an increasing temperature was observed at low (<25 min) and moderate (35 min) extraction time concentrations. However, the effect of the temperature almost disappeared at a higher extraction time (>65 min) (Figure 1(Bc)). Figure 1(Bd) demonstrates a considerable increase in TPC with a decreasing temperature and increasing ethanol concentration in the solvent composition.

Moreover, the perturbations graph (Figure 2B) exhibiting the effects of each independent variable on the TPC revealed that both citric acid and ethanol concentration had a significant influence on increasing the TPC.

Following the conventional extraction, the highest content of total phenolic compounds, 43.97 mg EAG/g DW, was obtained for the extraction with 50% ethanol and 0.1% citric acid after 25 min of extraction at 60 ◦C. Katalini´c et al. [38] reported content of 45.0 ± 26.3 mg EAG/g grape skin following a conventional extraction with ethanol (ethanol/water 80/20, at 60 ◦C, for 60 min). On the other hand, Negro et al. [39] obtained a content of 33.3 ± 0.3 mg EAG /100 g DW in the skin of red grapes by extraction with 80% ethanol with acetic acid. Poudel et al. [40] determined a content of 8.47 ± 0.20 (mg/g EAG) in the skin of the *Ebizuru* grape variety by 80% methanol extraction with 1N HCl. Tournour et al. [41] reported a content ranging from 69.30 to 131.70 mg/g EAG for different grape cultivars following a conventional magnetic stirring extraction for 48 h using an 80% ethanolic solution.

### *3.3. Effect of Extraction Parameters on AOA*

The recorded values of antioxidant activity ranged from 15.95 to 20.98 mM TE/g DW according to the influence of various variables (Table 2). For the AA parameter, the model is suggested to be significant by the model's Model F-value of 1241.30, and *p*-values less than 0.0500 indicate that model terms are significant. In this case, A, B, C, D, AB, AC, AD, BC, BD, CD, A2, B2, C2, and D2 are significant model terms.

Equation (4) reveals the model equation for the relationship between the antioxidant activity (R3) and variables expressed in coded units.

$$\begin{array}{l} \text{R3 (AOA)} = +17.21 - 0.8438\text{A} - 0.9931\text{B} - 0.6835\text{C} - 0.0497\text{D} - 11.84\text{AB} - 0.4843\text{AD} - 0.4184\text{BC} - \text{(4)}\\ \qquad 0.8576\text{BD} + 0.5237\text{CD} + 1.66\text{A}^2 - 0.1089\text{B}^2 - 0.0656\text{C}^2 + 0.5114\text{D}^2 \end{array} \tag{4}$$

The regression equation's b coefficients showed that among all the variables, the time of extraction had a minor negative effect on antioxidant activity. The interaction between citric acid concentration and ethanol concentration (AB) significantly negatively impacted the AOA of the red grape skin extract. The interactions between citric acid concentration and time of extraction (AD), ethanol concentration and temperature (BC), ethanol concentration and time of extraction (BD), quadratic ethanol concentration (B2), and quadratic temperature (BD) were all found to have a small negative impact (C2). Additionally, the interaction between temperature and time (CD) and quadratic extraction duration had a moderate impact on the extract's antioxidant activity (D2). Additionally, the antioxidant activity of the red grape skins extract is significantly positively influenced by quadratic citric acid content (A2).

The second-order contour plots were designed to predict the correlation between the independent and dependent variables, as seen in Figure 1C. The same correlation is used to highlight the synergistic effects of the studied independent variables on the values of antioxidant activity for the red grape skin extract. The correlative effect of the selected independent three-dimensional response area can describe the extract's antioxidant activity. Figure 1(Ca–Cd) shows the extraction parameters that affect antioxidant activity. The maximum antioxidant activity was obtained after 25 min of extraction at a concentration of ethanol of about 85%. Antioxidant activity increases as citric acid concentrations increase (Figure 1(Ca,Cb)). Shorter extraction times and higher citric acid concentrations also positively impacted the DPPH free radical-scavenging capacity, as evidenced by the plots. AOA decrease was observed at an increased ethanol concentration (Figure 1(Cc)). However, at higher extraction times (≥45 min) and at a moderate ethanol concentration (64%) an increase in AOA was observed (Figure S1(Cc)). Higher temperatures increase the solubility of phenolic compounds, leading to an increase in AOA. Nonetheless, using higher extraction temperatures and a longer extraction time, the extracted phenolic compounds began to degrade and, after reaching equilibrium, reduced AOA concentrations (Figure 1(Cd)). Similar results were obtained by Li et al. [9] who found a negative correlation between increasing ethanol concentration and increasing temperature, indicating that the antioxidant activity of AOA decreased as the ethanol concentration and temperature increased. Moreover, curve D from the perturbations graph has played a significant role in the determination of AOA in the perturbation graph, demonstrating the sizeable influence of the time of extraction value. Additionally, curves B and C will have less impact on extraction than curve A (Figure 2C).

After 25 min of extraction at 25 ◦C, the extraction with 85% ethanol and 2% citric acid produced the highest AOA of 24.67 mM TE/g DW. For the same grape variety but harvested in 2012, Constantin et al. [42] reported 4.89 ± 0.02 μM TE/g DW by the ABTS assay. Rockenbach et al. [12] obtained an average of 2.076 mM TE/100 g DW for different grape varieties from Brazil. The pinot noir and Isabel varieties had the highest levels of antioxidant activity. In a study conducted by Kupe et al. [43], nine '*Karaerik*' grape clones' peel samples showed DPPH radical scavenging between 1.08 and 1.34 mM TE /100 g FW. After model validation, for extraction, Li et al. [9] used the following parameters: 49% ethanol/51 C/15 min and obtained an AOA of 41.78 ± 1.13 mg TE/g.

#### *3.4. Extraction Parameter Optimization and Validation*

To verify the model equation, the model suggested the best factors based on maximizing response desirability (Figure 3, Table 4). The ramp graphs are labeled with a specific point that represents the optimal level for the variable under study. The value of the desirability function varies from the value zero, outside the imposed limits, to the value 1 or a value close to 1. The program's objective is to maximize the function, starting at a random point and aiming for the steepest slope possible [44]. A score of 1 (0.926) meant that all chosen conditions were true. The ideal conditions for maximizing phenolic compounds extraction and antioxidant activity were 0.85% citric acid, 85% ethanol, a temperature of 57.39 ◦C, and an extraction time of 52.14 min.

**Figure 3.** The optimization desirability bar chart (**A**) and ramps (**B**).

**Table 4.** The mathematical model's validation.


According to the model, the maximal levels of anthocyanins, total phenolic compounds, and antioxidant activity were 2.25 mg C3G/g DW, 37.41 mg GAE/g DW, and 17.2 mM TE/g DW, respectively. The experimental findings (Table 4) demonstrated fast responses to the model's predictions. Three extractions were carried out under those predicted variables to verify the model. In a study conducted by Li et al. [9], using a Box– Behnken design by RSM optimized the extraction parameters (48.80% ethanol at 50.79 ◦C for 14.82 min) of grape skin for obtaining the highest yields of the TPC (15.24 mg GAE/g) and TAC (3.46 mg CGE/100 g).

#### **4. Conclusions**

To obtain peel extracts from *Băbească neagră* grapes with a high yield of phenolic compounds and high levels of antioxidant activity, the conventional solvent extraction process variables (citric acid concentration—0.85%, ethanol concentration—85%, temperature—57.39 ◦C, and extraction time—52.14 min) were optimized using a CCD and response surface methodology. The maximum concentrations of anthocyanins (2.25 mg C3G/g DW), total phenolic compounds (37.41 mg GAE/g DW), and DPPH radical scavenging activity (17.2 mM TE/g DW) in the experiment were obtained from the interaction of time, temperature, acid, and solvent concentrations. The optimized extraction process may be a potentially efficient way to obtain valuable extracts from affordable, natural sources like grape skins with potential antioxidants and a high yield of anthocyanins and other phenolic compounds. These results demonstrate proper factor combinations and a unique solvent mixture for obtaining a high yield of phenolic compounds and, particularly, anthocyanins.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/inventions8020059/s1.

**Author Contributions:** Conceptualization, D.S., O.E.C. and G.R.; methodology, D.S., G.H. and O.E.C.; software, O.E.C. and I.A.; validation, O.E.C. and I.A.; formal analysis, G.H. and D.S.; investigation, D.S.; resources, G.E.B.; data curation G.R. and N.S.; writing—original draft preparation D.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 "Dunărea de Jos" University of Galati (SCDS-UDJG) http://www.cssd-udjg.ugal.ro/ (accessed on 9 April 2022) that will be held on the 9–10 June 2022, in Galati, Romania.

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

#### **References**


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