Exploring antioxidant potential and phenolic compound extraction from Vitis vinifera L. using ultrasound-assisted extraction

2.1 Reagents

All utilized reagents and solvents were of analytical caliber. Folin–Ciocalteu’s phenol reagent and gallic acid were procured from Merck (Darmstadt, Germany). 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin trap were sourced from Sigma Aldrich (St. Louis, MO).

2.2 Sample collection and preparation

In this study, grape pomace (Vitis vinifera L.) of the Syrah variety was used, which was collected from the L.A. CETTO Plant in Valle de Guadalupe, Ensenada, Baja California Grape pomace is obtained as a byproduct of the wine industry. Initially, the grape pomace was transported in plastic bags to the Universidad Estatal de Sonora (UES) in Hermosillo, Sonora. Subsequently, it was placed in a freezer at a temperature of −18°C until further use. A manual cleaning was performed to separate the existing stems and leaves from the raw grape pomace, leaving only the seeds and skin. The sample was then lyophilized (Labconco FreeZone 12, Kansas City, MO, USA) at −61°C, 2.4 × 10⁻⁴ Pa, for 72 h. The lyophilized grape pomace (skin and seeds) was ground using a 600 W processor and sieved through a T20 sieve (0.085 mm, Marca FICCS S.A de CV México), the entire process is depicted in Figure 1. Finally, the sample was stored in darkness at room temperature until it was subjected to the extraction process.

Figure 1

Preparation and processing of grape pomace (Vitis vinifera L.).

2.3 Statistical analysis

UAE of grape pomace was conducted using a 2^K factorial design, where k = 3 (solute:solvent ratio, 1:18 and 1:42 g:mL; power, 250 and 400 W; time, 15 and 20 min). Statistical evaluations of overall phenolic content, flavonoids, and antioxidant activity were executed through a two-way analysis of variance using NSCC v.8 for Windows. In all instances, the disparities between mean values were scrutinized via Tukey’s test, with a significance threshold of 5%.

2.4 Ultrasound-assisted extraction (UAE)

The extraction of antioxidant compounds from grape pomace was performed by ultrasonic irradiation using a high-intensity ultrasound processor, the Ultrasonic Processor Q500. The ultrasound was equipped with a sonotrode (head area of 3/4″). Sonication was performed at 20 kHz with an operation temperature of 30 ± 5°C monitored by a thermometer. The sonotrode was immersed for 2 cm in the extraction solution. Extractions were performed in duplicate in a 300-mL beaker, using 96% ethanol and ultrapure water as solvents in a 50% ratio (water 100 mL–ethanol 100 mL). Eight treatments with different extraction conditions were used (solute ratio, ultrasound power, and extraction time): treatment 1 (1:18, 250 W, and 15 min), treatment 2 (1:18, 250 W, and 20 min), treatment 3 (1:18, 400 W, and 15 min), treatment 4 (1:18, 400 W, and 20 min), treatment 5 (1:42, 250 W, and 15 min), treatment 6 (1:42, 250 W, and 20 min), treatment 7 (1:42, 400 W, and 15 min), and treatment 8 (1:42, 400 W, and 20 min). Once this process was carried out, the extracts were centrifuged (Heraeus Multifuge X3R) at 4°C at 9,000 rpm for 30 min. The ethanol contained in the supernatant was evaporated in a convection oven (Linderberg/Blue M, Asheville, NC, USA) at 40°C 72 h and subsequently lyophilized (Labconco FreeZone 12, Kansas City, MO, USA) at -61°C and 72 h to obtain the powdered extract. The entire process is depicted in Figure 2.

Figure 2

Ultrasound-Assisted Extraction (UAE) of antioxidant compounds from grape pomace (Vitis vinifera L.).

2.5 Quantification of total phenol content

Incubation procedures were carried out in test tubes by combining 50 µL of each sample with 3.0 mL of distilled water, 0.25 mL of Folin-Ciocalteau reagent, 0.75 mL of 20% Na₂CO₃, and 0.950 mL of distilled water. After a 3-minute interval, 1.0 mL of sodium carbonate solution was introduced, initiating a 60-minute reaction period conducted in darkness [28]. Subsequent spectrophotometric readings were taken at 765 nm. A calibration curve using gallic acid was established, and the results were expressed in micromoles of gallic acid equivalents per gram of dry weight (μmol GAE/g d.w). All analytical determinations were conducted in triplicate.

2.6 Quantification of total flavonoids

In this procedure, the AlCl₃ test was conducted [29]. A 500 μL aliquot was withdrawn from each respective sample and mixed with 2 mL of water and 150 µL of a 10% (w/v) AlCl₃ solution. Following a 10-minute incubation in the absence of light at room temperature, 1 mL of 1 M sodium hydroxide (NaOH) and 1.2 mL of distilled water were added to the mixture. After another 15-minute incubation in the dark at room temperature, the sample was analyzed using a spectrophotometer, with detection performed at a wavelength of 430 nm. Quercetin served as the reference standard in this procedure. Consequently, results are expressed in terms of micromoles of quercetin equivalent per gram of dry weight (μmol QE/g d.w) [30].

2.7 Antioxidant capacity ABTS assay

The antioxidant capacity of a sample can be measured by the ABTS method, which consists of the color change from green to blue produced by the radical cation. To do this, 19.2 mg of ABTS was dissolved in 5 mL of water and then propagated by oxidation with potassium persulfate (K₂S₂O₈, 4.95 mM), for 12 h at 25°C, protecting from light. Then, to reach an absorbance of 0.7 at 734 nm, the ABTS was prepared at a concentration of 0.2 M, adjusted to pH 7.4. A reaction of 200 mL of the ABTS prepared with 20 mL of sample was carried out, and after 30 min, the absorbance was read at 734 nm. The antioxidant activity was reported as a percentage inhibition of ABTS radicals. Eq. 1 was obtained to determine the sweeping capacity.

where Abs₀ is the absorbance of water (control); Abs₁ is the absorbance of the sample with ABTS; Abs₂ is the absorbance of water with ABTS.

The Trolox Equivalent Antioxidant Capacity (TEAC) assay is performed by comparing a reference antioxidant standard (Trolox^®), which is based on the ability of a sample to reduce the ABTS radical. A curve was made in the range of 100 to 800 μmol·mL⁻¹ of Trolox^®, and the absorbance was measured in a microplate reader at 734 nm. Values were expressed as micromoles of Trolox^® equivalent per g of sample (μmol TE/g d.p.).

2.8 Antioxidant capacity FRAP assay

The assessment of ferric reduction capacity was conducted utilizing the Ferric Reducing Antioxidant Power (FRAP) assay. The FRAP reagent was meticulously prepared using an acetate buffer with a pH of 3.6, comprising a 10 mM solution of 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) in 40 mM hydrochloric acid (HCl) and a 20 mM solution of iron (III) chloride, with these components being combined in volumetric ratios of 10:1:1 (v/v/v) [31]. The experimental treatments underwent diverse dilution protocols. After aliquoting 20 µL of the test sample, 280 µL of the FRAP reagent was introduced. In tandem, a standard Trolox solution was meticulously prepared and subsequently subjected to analogous conditions. The ensuing chemical reaction was sustained for a duration of 30 min, subsequent to which the absorbance was quantified at a wavelength of 638 nm. The final quantification was expressed in terms of micromoles of Trolox equivalent per gram of dry weight (µmol TE/g d.w).

2.9 DPPH radical scavenging assay

The assessment of DPPH radical scavenging efficacy was conducted following the methodology delineated by Hu et al. [32]. Initial preparations involved the dilution of experimental treatments at varying concentrations. Subsequently, each concentration (50 µL) was meticulously combined with a methanolic DPPH solution (0.2 mL) set at a concentration of 0.4 mM. This amalgam was subsequently introduced into dedicated wells within a 96-well microplate. The ensuing reaction was orchestrated under light-free conditions, maintaining a controlled temperature of 25°C for a duration of 30 minutes. Upon completion of the reaction, quantification of absorbance transpired at a specific wavelength of 515 nm. To provide a contextual reference, a standard Trolox solution underwent meticulous preparation and concomitant exposure to identical reaction conditions [31]. Moreover, the antioxidant concentration corresponding to a 50% inhibition of the DPPH radical (IC₅₀ DPPH) was ascertained. This determination was achieved through the construction of a graphical representation depicting the scavenging percentage vis-à-vis the concentration of the antioxidant (mg·mL⁻¹) under scrutiny, facilitated by the implementation of linear regression analysis [28].

2.10 Antioxidant capacity expressed in Trolox equivalent

The Trolox equivalent antioxidant capacity (TEAC) assay is predicated upon the intrinsic ability of a given specimen to effectuate the reduction of the ABTS radical in relation to a well-established reference (Trolox^®). The construction of a calibration curve spanned the analytical range of 100–800 μmol·mL⁻¹ Trolox^® concentrations. The monitoring of absorbance occurred at a specific wavelength of 734 nm, with the resulting measurements translated into quantifications denoted as μmol of Trolox^® antioxidant equivalence per gram of the assessed sample [33].

3.1 Quantification of phenolic compounds

The quantification of phenolic compounds extracted from grape pomace in the UAE was carried out using the Folin–Ciocalteu method. This methodology has been reported for determining total phenolic compounds. Quantitative determination was executed via spectrophotometric analysis, a technique founded upon a redox reaction involving the phenolic constituents inherent to the specimen and the Folin–Ciocalteu reagent. This reagent contains a mixture of sodium tungstate and sodium molybdate in phosphoric acid, resulting in yellow phosphomolybdotungstic acid. This acid can be reduced by the phenolic groups in the sample, leading to an intense blue color. The colored product exhibits a maximum absorption at 760 nm [34]. Figure 3 illustrates the outcomes derived from the quantitative assessment of total phenolic content, thereby substantiating the presence of these compounds within the extracts acquired through distinct treatment modalities. It is noteworthy that Treatment 6 evinced a diminished concentration of total phenolic content, ranging between 50 and 60 μmol GAE/g d.w. Statistical scrutiny revealed that for the majority of treatment conditions, no statistically significant disparities (p > 0.05) were discerned as a consequence of the interplay among the three assessed variables, namely solute:solvent ratio, power, and time. The obtained results delineated a concentration spectrum spanning from 50 to 80 μmol GAE/g d.w., surpassing the value documented by Drevelegka and Goula [21], who performed UAE of grape pomace and obtained a maximum yield of 33.88 μmol GAE/g d.w. with a solute:solvent ratio of 8 g:mL, 40% amplitude, and a 10 min extraction time using 50% v/v ethanol. Their investigation encompassed the analysis of yield, activities, and the extraction process of antioxidants from grape pomace, culminating in the deduction that grape pomace indeed constitutes a viable and sustainable reservoir of inherent natural antioxidants and phenolic compounds. The outstanding performance achieved in this study can be primarily attributed to exposure to UAE, but above all, to the particle size of the sample prior to extraction. The use of a sieve size of 0.085 mm, as opposed to the 0.3 mm used in the mentioned experiment, may have led to a greater surface area of contact. The above achieved an increase in the efficiency of UAE.

Figure 3

The quantification of total phenols within grape pomace (Vitis vinifera L.) extracts acquired through distinct UAE treatments is presented. Each data point is denoted as the mean ± standard deviation (n = 4). The utilization of varying letters (^{a, b, c}) indicates noteworthy differences in statistical significance (p < 0.05) among the depicted means.

In another study conducted by González-Centeno et al. [35], the effect of different variables on grape pomace extraction was evaluated, including ultrasound frequency (40, 80, and 120 kHz), ultrasound power (50, 100, and 150 W), and extraction time (5, 15, and 25 min) on total phenols. They found that a frequency of 40 kHz, a power of 150 W, and a 25-minute extraction time allowed for a maximum of 32.31 mmol GAE/g d.w. for total phenols. However, they observed a decrease in total phenolic compounds when increasing the ultrasound frequency to moderate values (70–80 kHz), and the highest quantity of phenols was obtained at low frequencies as ultrasound power increased. The observed disparities in the results can be attributed, primarily, to the particle size employed in the UAE technique. In the mentioned study, particles with an average size of 7 mm were utilized. Second, the differences may be linked to the power used in the process; whereas the mentioned study employed a maximum power of 150 W, in the current work, levels of 250 and 400 W were utilized. It is speculated that higher power in the UAE could have intensified the cavitation effect, resulting in increased efficiency in the extraction of bioactive compounds. On the other hand, Shirsath et al. [36] highlighted that applying frequencies in the range of 18–40 kHz can achieve sufficient cell wall disruption to enhance mass transfer, thereby aiding in compound extraction.

The use of UAE for obtaining bioactive compounds from grape pomace has proven to be useful, particularly in the extraction of phenolic compounds. A study published by Luo et al. [27] presented the UAE of phenols from grape pomace. The results showed that ultrasound extraction significantly increased the phenolic content compared to other conventional methods. Additionally, higher antioxidant activity was observed in the extracts obtained through the UAE. Another study conducted by Medina-Torres et al. [26] evaluated the influence of ultrasound extraction parameters, such as temperature and extraction time, on the phenolic content of grape pomace. They found that increasing the temperature and extraction time led to a higher phenolic content in the extracts. Moreover, the use of suitable solvents and optimization of ultrasound extraction parameters can further improve the yield of extracted phenols. Regarding the antioxidant capacity of the extracts, Turker and Isleroglu [25] conducted a study to investigate the antioxidant capacity of the UAE phenolic compounds (13.99 ± 1.8 μg GAE/g d.w) from grape pomace using 80% amplitude and 20 min. The results demonstrated that ultrasound extraction enhanced the antioxidant activity of the extracted phenols, indicating their potential as protective agents against oxidative damage.

The manifestation of antioxidant activity has been documented in bioactive compounds derived from diverse botanical origins, as evidenced by findings from a study conducted by Beres et al. [37]. In this study, the antioxidant activity of Pinot Noir grape pomace flour from Brazil was evaluated. The grape pomace flours were sieved and classified as coarse (G; particle diameter, >355 μm), which exhibited 0.2163 ± 0.0014 mg GAE/g d.w, medium (M; particle diameter, 354–250 μm) with 0.2671 ± 0.0028 mg GAE/g d.w., and fine (F; particle diameter, <249 μm) with 0.43 ± 0.049 GAE/g d.w. The assessment of ABTS scavenging capacity revealed a discernible augmentation in antioxidant activity concurrent with a reduction in particle size. This observation culminates in the deduction that there exists a positive correlation between the diminution of particle size and the concurrent enhancement of antioxidant activity. Therefore, it is important to consider particle size in the UAE process.

3.2 Quantification of total flavonoids

The determination of total flavonoid content within the grape pomace extracts was undertaken in accordance with the established procedure outlined by Domínguez-Perles et al. [38]. This is a spectrophotometric assay based on the formation of complexes with aluminum and relies on the formation of an Al–Flavonoid complex. First, the flavonoid is nitrated (with NaNO₂) at C-3 or C-4 of an unsubstituted or sterically unhindered aromatic ring. Then, AlCl₃ is added, where the hydroxyl groups at C-3 and C-5 of the B ring coordinate with an Al atom, forming a yellow-colored complex. Subsequently, an alkaline compound (NaOH) is added, and the reaction is stopped, resulting in an intense orange color that is measured at 510 nm. The results are reported as micromoles equivalents of quercetin per gram of dry sample (μmol QE/g d.w), which are calculated from a quercetin standard curve.

Quantification of total flavonoids confirmed the presence of these bioactive compounds in grape pomace extracts. The results are shown in Figure 4, indicating that the different treatments exhibited a concentration of total flavonoids within the range of 2.5–4 μmol QE/g d.w. It is noteworthy that the application of treatment 1 yielded the highest amount of flavonoids (p < 0.05), ranging from 3.7 to 4 μmol QE/g d.w. These findings align with a study conducted by Drosou et al. [39], where a recovery of grape pulp extract resulted in a flavonoid content of 4.36 ± 1.2 μmol QE/g d.w. The authors reported that the sample underwent a process of grinding and subsequent air drying, leading to the inference that the drying procedure and the concomitant augmentation in surface area due to the grinding process collectively contributed to the enhanced retrieval of these flavonoid compounds. Ozkan et al. [40] reported the total flavonoid content in fresh grapes (hybrid of V. labrusca-V. vinifera L.) to be 4.6 ± 0.2 μmol QE/g d.w. This content was influenced by the applied drying process (p > 0.05), as samples subjected to lyophilization showed a higher flavonoid content compared to those subjected to hot air drying at 60°C with an air velocity of 2 m·s⁻¹, demonstrating the beneficial effect of lyophilization on extraction. Additionally, Gulati and Datta [41] report that the sonication process enhances the rate of water removal within the material’s surface by creating microscopic channels through compression and expansion. Hence, in the present study, UAE was employed for improved extraction of bioactive compounds. They also mention that the outer surface can act as an impermeable barrier, restricting water vapor transfer to the surrounding environment and potentially causing the waxy outer surface to become vitreous, thereby limiting water mobility and prolonging drying time when ultrasound is used.

Figure 4

Quantification of flavonoids in grape pomace (Vitis vinifera L.) extracts obtained by the different UAE treatments. Each data point is denoted as the mean ± standard deviation (n = 4). The utilization of varying letters (^{a, b, c}) indicates noteworthy differences in statistical significance (p < 0.05) among the depicted means.

Peixoto et al. [42] assessed the phenolic profile inherent to grape pomace derived from Vitis vinifera L., encompassing the constituents present in the skins and seeds as well as their amalgamation. The results indicated the presence of flavonoids and flavan-3-ols (catechin, epicatechin, and pro anthocyanidins). Furthermore, it has been reported that grape skins exhibit high concentrations of anthocyanins (7.9 ± 0.1 μg·g⁻¹), highlighting these by-products as an important source of bioactive molecules. Numerous studies have reported that flavonoids have received attention due to their diverse properties with medical relevance, such as their use as antiallergic, anti-inflammatory, antioxidant, antitumor, antimicrobial, estrogenic, and enzyme-inhibiting agents [43].

3.3 Antioxidant capacity-ABTS assay

Grape pomace stands out as a notable biodegradable residue originating from the viticultural sector, encompassing grape seeds, skins, and stalks. It inherently harbors substantial quantities of phenolic compounds, flavonoids, and anthocyanins, which collectively exhibit robust antioxidant attributes [44]. Within the scope of the ongoing investigation, the evaluation of the antioxidant efficacy inherent to grape pomace extracts was undertaken employing the ABTS assay. This spectrophotometric assay is based on electron transfer and measures the ability of an antioxidant sample to reduce a color-changing oxidant. Precisely, this assay entails the quantification of color diminution that ensues upon the addition of an antioxidant to the blue-green chromophore, ABTS. The role of the antioxidant is to effectuate the reduction of ABTS^•+ to ABTS, thereby resulting in the discoloration of the solution. This alteration in color intensity was subsequently measured at a wavelength of 734 nm [45]. Figure 5 displays the ABTS results, confirming the presence of antioxidant activity in the ethanolic extracts of grape pomace obtained through the UAE. The extracts derived from the array of eight treatment modalities did not manifest statistically significant variances (p > 0.05) when subjected to evaluation at a concentration of 781.25 μg·mL⁻¹. However, when the concentration was increased to 1,562.5 and 3,125 μg·mL⁻¹, significant differences (p < 0.05) were observed between the treatments. A clear effect of the solute:solvent ratio (g:mL) factor was observed when applying a concentration of 6,250 μg·mL⁻¹, treatments 1, 2, 3, and 4 did not exhibit significant differences (p > 0.05), exhibiting 25–35% ABTS radical scavenging when using a solute:solvent ratio of 1:18 g:mL. This same behavior was observed when evaluating the antioxidant capacity of treatments 5,6,7 and 8 (1:42 g:mL solute:solvent ratio), showing results within a range of 30–45% ABTS radical scavenging. These results indicate that the highest solute:solvent ratio results in a higher antioxidant capacity against the ABTS radical.

Figure 5

Antioxidant capacity of grape pomace extract by ABTS using Trolox as a reference antioxidant. The utilization of varying letters (^{a, b, c}) indicates noteworthy differences in statistical significance (p < 0.05) among the depicted means.

The antioxidant capacity of dried Isabel black grapes (a hybrid of V. labrusca L.V. vinifera L.) using different drying methods was also investigated, with the ABTS value of Isabel black grapes reported as 70.01 μmol TE/g d.w. The most effective drying method was found to be lyophilization [40]. Following a study by Breksa et al. [46], which reported ABTS values of 16 commercial raisins (Vitis vinifera L.) ranging from 7.7 to 60.9 μmol TE/g d.w., it can be concluded that the lyophilization drying method effectively retains compounds and enhances antioxidant capacity.

The results of the ABTS assay were consistent with findings reported by Zhang and Tsao [47], who explained that phenolic compounds and flavonoids possess effective radical scavenging activity. The authors also postulated that the observed antioxidant activity could potentially be attributed to the quantity and specific arrangement of hydroxyl groups within the molecular structure. Quercetin, which contains a 3-hydroxy group, exhibits relatively higher activity in neutralizing free radicals compared to those without this structure. In the present study, a quercetin standard curve was constructed, and the extracts were compared to this standard. The presence of antioxidant activity in a compound or sample inhibits the oxidation process and may also exhibit antiradical activity by inhibiting the activity of free radicals [45]. Based on the above, it can be inferred that the analyzed extracts in the present study have the potential to be used as antioxidants.

3.4 Antioxidant capacity – DPPH assay

The assessment of the antioxidant potential inherent in grape pomace extracts was additionally conducted via the DPPH assay. This technique employs spectrophotometric analysis to ascertain the capacity of compounds within the sample to effectuate the reduction of DPPH, a radical that is typically absent in biological contexts. This system utilizes a stable free radical, meaning that it provides information about its radical scavenging or antioxidant capacity. In this case, the color intensity decrease is related to the concentration of antioxidants in the sample and is determined at 515 nm [45].

The antioxidant activity can be considered one of the most notable bioactivities of phenolic compounds derived from grape pomace. It depends on the number of hydroxyl groups present in the specific molecule, and its activity can be enhanced by steric interference or hindrance [48]. The results of the assay (Figure 6) indicated low DPPH radical scavenging (%) when tested at 781.25 μg·mL⁻¹. However, as the concentration increased to 1,562.5 μg·mL⁻¹, the antioxidant capacity of the extracts from all treatments increased, reaching approximately 40–50%. Further increasing the concentration to 3,125 μg·mL⁻¹ resulted in an even higher DPPH radical scavenging of 65–80%. The extracts obtained from treatment 1 exhibited the highest antioxidant capacity at concentrations of 1,562.5 and 3,125 μg·mL⁻¹, and they also had the highest flavonoid content. Finally, when analyzing the effect of a concentration of 6,250 μg·mL⁻¹, a substantial increase in the antioxidant effect of all treatments on DPPH radical scavenging (%) was observed.

Figure 6

The antioxidant capacity of grape pomace extract was evaluated through the DPPH assay, with Trolox employed as a reference antioxidant. The differentiation of letters (^{a, b, c}) conveys the presence of statistically significant disparities (p < 0.05) among the represented values.

It is noteworthy to mention that the antioxidant properties of grape pomace extracts have been supported by similar outcomes derived from the pomace of red wine grape cultivars (Cabernet Franc) and white wine grape cultivars (Chardonnay). These extracts, formulated utilizing an ethanol concentration of 80% as the extraction solvent, exhibited significant DPPH radical scavenging capacities. Specifically, the assessment revealed that one milligram of red wine grape pomace and white wine grape pomace exhibited the capacity to quench 66.1 ± 0.6 and 67.4 ± 4.1% of DPPH radicals within the reaction medium, respectively. Notably, the comparison of these pomace extracts did not yield statistically significant distinctions (p > 0.05) [49]. Thus, it is discerned that the incorporation of grape pomace into the dietary landscape contributes substantively to the augmentation of cardiovascular well-being within the intestinal milieu. This beneficial impact can be attributed to the orchestration of factors such as blood lipid and glucose regulation, facilitation of appetite control, amelioration of inflammatory and oxidative stress responses, and the cultivation of a favorable composition of gut microbiota [11].

3.5 IC₅₀ concentration

The IC₅₀ value holds pervasive utility as a metric for the quantification of the antioxidant potential inherent in natural extracts. Grape pomace, a consequential by-product of the winemaking sector, has garnered noteworthy consideration owing to its discernible health-promoting attributes stemming from the abundance of phenolic compounds endowed with antioxidant attributes. The IC₅₀ outcomes are detailed in Table 1. The DPPH and ABTS assays are commonly employed to assess the radical scavenging and reducing abilities of extracts. This research aimed to determine the IC₅₀ values of grape pomace extracts using these assays, providing valuable insights into their antioxidant potential. In this investigation, different concentrations of the grape pomace extracts were mixed with DPPH and ABTS solutions and incubated for a specified time.

The assessment involved measuring absorbance at a specific wavelength, followed by calculating the percentage inhibition of DPPH and ABTS. Subsequently, a dose–response curve was generated, allowing for the determination of the IC₅₀ value. It is pertinent to note that multiple studies have indicated that the bioactive compound content and antioxidant activity of grape pomace extracts, obtained through UAE, can be influenced by various factors, including power, time, and their interactive effects [1]. The IC₅₀ results for ABTS are shown in Table 1. It can be observed that in a solute:solvent ratio of 1:18, lower IC₅₀ values were obtained in the extracts of treatments where ultrasound was applied for 15 min, while for a time of 20 min, the IC₅₀ values were higher. On the other hand, when using a solute:solvent ratio of 1:42, similar IC₅₀ values were obtained for treatments 7 and 8, indicating no effect of ultrasound time.

The IC₅₀ values obtained from the DPPH assay demonstrated a consistent pattern among the various treatments, except for treatment 1, where a distinct departure was evident. This discrepancy emphasizes the notable influence of the increased flavonoid content facilitated by treatment 1 in the extraction of bioactive compounds from grape pomace. These findings align cohesively with the established correlation illustrating the cumulative content of total phenolic and flavonoid compounds, coupled with the resultant antioxidant capacity displayed by the obtained extracts [50]. It is important to emphasize that there is evidence from research supporting the applications of grape pomace extracts in the treatment of various conditions. Among these, the antitumor activity of Bulgarian Mavrud grape pomace extracts and their antioxidant and antitumor effects against breast cancer have been highlighted. Dose–response curves were systematically interrogated within the cellular milieu of mammary gland type A adenocarcinoma (MCF-7) and the triple-negative breast cancer cell line (MDA MB-231) [51]. Concurrently, recent inquiries have elucidated the transient ramifications inherent to grape pomace extracts on mitochondrial functionality while concurrently distinguishing their impact from that exerted on the viability of HepG2 cells. Notably, the observed effects encompassed the amplification of mitochondrial respiration, with particular emphasis on the augmentation of the fraction of oxygen consumption dedicated to fueling adenosine triphosphate (ATP) synthesis. These discernible physiological alterations were accompanied by a conspicuous augmentation in the cellular antioxidative capacity [33].

3.6 Antioxidant capacity expressed in Trolox equivalent

The assessment of the antioxidant potential intrinsic to grape pomace extracts was further conducted through the utilization of the Ferric Reducing Antioxidant Power (FRAP) assay. This methodology entails a spectrophotometric technique designed to quantify the capability of a given sample to effectuate the reduction of ferric iron. The mechanism underlying this assay is predicated upon the reduction of the ferric iron and the ensuing transformation of the TPTZ complex into its ferrous form, a process executed under low pH conditions. The degree of reduction is quantified by monitoring the alteration in absorbance exhibited at a wavelength of 638 nm [45].

The observed reduction of ferric iron within grape pomace extracts serves as a corroborative indicator of the presence of constituents endowed with antioxidant activity. Table 2 shows the results, indicating values ranging from 26 to 45 µM TE/g d.w. It can be observed that treatments 1 and 7 were statistically equal in terms of Trolox equivalent (µmol TE/g d.w.) concerning FRAP, reflecting the effect of time (15 min). In the case of treatments 5, 6, and 8, they were statistically equal, demonstrating the effect of the solute:solvent ratio (1:42). In a study conducted by Zhu et al. [52], the antioxidant properties of grape pomace extracts were evaluated, and it was indicated that the reducing power of the compounds is closely related to their electron transfer capacity. Therefore, we can infer that the compounds present in the sample are antioxidants, as according to the values reported in this study, they can reduce Fe + by donating electrons, thus demonstrating a positive correlation between the content of bioactive compounds in the sample and its Fe + reduction capacity. The reduction of the ferric complex is increased by the phenolic content present in the grape pomace extract [53].

The ABTS results reveal a statistically significant difference among treatments, primarily attributed to the effect of the solute:solvent ratio. Lower Trolox equivalent values (µmol TE/g d.w.) are predominantly observed in treatments employing a 1:18 solute:solvent ratio. Conversely, in the case of DPPH, no significant differences (p > 0.05) were discerned among the results of the applied treatments, with values hovering around 225 µmol TE/g d.w. These outcomes align with those documented for grape pomace flour from Niagara Rosada (255 μg·mL⁻¹) and Niagara Rosada 50% Bordo 50% (292.9 μg·mL⁻¹) [6]. On the contrary, these values are surpassed by those reported for Syrah (360.41 µmol TE/g d.w) and Chardonnay (453.02 µmol TE/g d.w) extracts obtained using a hydroalcoholic solvent (ethanol/water 1:1 v/v) [4]. Notably, in grape pomace seeds of the Mavrud variety, the highest antioxidant activity was observed (653.77 μmol TE/g), indicating that grape seeds are inherently richer in antioxidant compounds [51].