Enhanced Preparative-Scale Extraction from Graševina Grape Pomace Using Ultrasound-Assisted Extraction and Natural Deep Eutectic Solvents

Abstract

This research paper presents an advanced exploration into the utilization of ultrasound-assisted extraction (UAE) combined with natural deep eutectic solvents (NADESs) to achieve higher concentrations of polyphenols from Graševina grape pomace. Focusing on optimizing extraction parameters to maximize the yield of polyphenols, this study evaluates their potential industrial applications, particularly within the food and cosmetics sectors. The effectiveness of betaine/glucose (BGlc) NADESs in producing stable, ready-to-use extracts with enhanced bioavailability and eco-friendly attributes is underscored. The integration of UAE with BGlc NADESs has shown significant scalability and applicability for industrial use, as evidenced by the extracts’ collagenase-inhibitory effects, determined using a ninhydrin-based colorimetric assay showing the significant inhibition of gelatine degradation and scratch tests on cultured skin cells, demonstrating enhanced cell migration and wound healing, indicating their potential in anti-aging cosmetic products. Additionally, the results from PAMPA tests demonstrated that NADES extraction significantly enhances the intestinal absorption of polyphenols from grape pomace extracts compared to conventional solvents, highlighting the potential of NADESs to improve the bioavailability of these compounds and offering promising implications for their application in the food industry. Furthermore, the research highlights the practicality of directly incorporating these extracts into products, such as anti-aging creams and functional foods, supporting sustainability initiatives within the cosmetic and food industries. This work aims to provide a comprehensive guide to green extraction techniques on a preparative scale, showcasing the versatility and innovative applications of NADES-extracted compounds across various industries, thereby paving the way for the development of eco-conscious and effective products.

1. Introduction

In the last few decades, the world has been witnessing a growing interest in environmental, safety, and economic issues. This evolution in thinking and approaches within the industry and science has led to the application of innovative techniques that emphasize sustainability and ecological responsibility in sectors such as the food, pharmaceutical, and cosmetic industries. To achieve this transformation, numerous principles and guidelines have been developed, such as the 12 principles of green chemistry and the 12 principles of green engineering, which serve as guidelines for achieving sustainability in these sectors [1,2].

One of the key aspects of this sustainable revolution is the concept of green extraction. Green extraction represents a new approach to the design of extraction processes aimed at reducing energy consumption, ensuring high-quality products, and promoting the use of alternative solvents and renewable energy sources. Conventional extraction methods, often employing organic solvents, are notorious for their high energy consumption and long extraction times, posing risks to the environment. The central aim of green extraction is not only to obtain the target product but also to create by-products instead of waste. This strategy underlines the essence of the circular economy, where the valorization of by-products promotes resource efficiency and sustainability, transforming what was once considered waste into valuable materials for various industrial applications. This is particularly important in the context of the wine industry, where grape pomace often represents a significant form of waste [3,4,5]. Grape pomace is a mixture of grape seeds, skins, and stems rich in biologically active phenolic compounds. These compounds have proven beneficial effects on human health, including their role in preventing chronic diseases and promoting gastrointestinal health [6,7]. Graševina, extensively cultivated in Croatia’s Slavonija and Podunavlje regions, plays a key role in national wine production. Winemaking generates pomace, about 20–30% of the grapes’ weight. Globally, wine production waste amounts to 5–10 million tons annually, as per FAO and WHO. One of the innovative approaches that stands out in the field of green extraction is the application of eutectic solvents, also known as deep eutectic solvents (DESs). A DES is a mixture of hydrogen bond acceptor(s) and donor(s), characterized by properties such as non-flammability, non-volatility, and high viscosity. When natural compounds are used to create a DES, such a solvent is called a natural deep eutectic solvent (NADES). What sets NADES apart from other solvents is their biodegradability and safety for use in various industries, including cosmetics, pharmaceuticals, and the food industry [8,9,10].

By replacing inefficient and hazardous solvents, NADESs represent a key strategy for achieving environmentally friendly industrial processes. NADESs can readily dissolve phenolic compounds, enhancing their accessibility, whereby the choice of NADES components, the molar ratios of the latter, and water content allow for the customization of solvent properties. Most NADES-related patents come from academia, with industrial viability yet to be proven [11]. The cosmetics sector, spearheaded by firms such as Naturex and Gattefosse, adopts NADESs as substitutes for conventional solvents like water, which enables the elimination of often-needed hazardous stabilizers. Building on the discussion of commercial applications by companies like Naturex and Gattefosse in utilizing NADES extracts for cosmetic products, it is pertinent to highlight that a core tenet of green processing across food, agriculture, and extraction methodologies centers on minimizing energy use through cutting-edge techniques.

Ultrasound is widely used in industry to enhance various processes, such as drying, pasteurization, microbial inactivation, emulsification, homogenization, and sieving [12,13,14], including biomass pretreatment, cellulose conversion, and sugar production [15]. It boosts these processes by increasing forces, inducing cavitation, and generating reactive radicals [16,17]. The main principle of ultrasonic work is based on the creation of cavitation bubbles and the implosion of gas bubbles within the treated liquid medium, causing chemical and physical changes in the cell walls [18]. Parameters such as frequency, applied amplitude, ultrasonic power, and cycles from the physical point of view coupled with the geometry of the device (the diameter of the probe, the length of the probe, and immersion depth) are main drivers that determine ultrasonic work.

Ultrasound-assisted extraction (UAE) is one of the most commonly used applications of this environmentally friendly and non-thermal technology. UAE significantly reduces the extraction time to just a few minutes while ensuring reliability and reducing both solvent and energy consumption [12,19,20]. The type of solvent, extraction time, solvent-to-solid ratio, pH, and temperature have influence on the applied ultrasound within the extraction process [21,22]. At the industrial scale, the number of companies utilizing ultrasound as an extraction technique has significantly increased. One of them is Arkopharma, a French company specializing in dietary supplements based on plant extracts [23]. At an industrial scale, the combination of NADESs and UAE has not yet been realized, highlighting the need for further research, particularly in the context of scaling up.

This innovative approach aims to apply green extraction principles to extract polyphenols from grape pomace, optimizing the process using UAE and natural deep eutectic solvents (NADESs), focusing on their integration for safe application in the cosmetic and food industry. The additional goal is to scale up the extraction process to a pilot level. First, the impacts of UAE in continuous and batch extraction processes at a 5 L scale were investigated. After selecting the optimal extraction method, parameters affecting the efficiency of the process, such as time, type of solvent, and the power of alternative energy sources, were further optimized using response surface methodology. Finally, the optimized extract was produced and validated for application in the cosmetic and food industry through various tests, including skin healing assessments (scratch tests), anti-aging activity tests (the inhibition of collagenase activity), and permeability testing through an artificial small intestine membrane (PAMPA).

2. Materials and Methods

2.1. Chemicals and Materials

All chemicals and standards were purchased from Sigma (St. Louis, MO, USA) and Fisher Bioreagents (Fair Lawn, NJ, USA). The normal human keratinocyte cell line, HaCaT, was purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany), fetal bovine serum (FBS) was purchased from GIBCO Life Technologies (Paisley, UK), and trypsin-EDTA was purchased from Sigma-Aldrich (St. Louis, MI, USA). Cells were cultured in BioLite Petri dishes (Thermo Fisher Scientific, Drive Rochester, NY, USA) in a humidified atmosphere with 5% CO₂ at 37 °C in an incubator. Individual experiments were performed in 96-well plates (Thermo Fisher Scientific, USA). A CellTiter 96^®® AQueous One Solution Cell Proliferation assay was purchased from Promega (Madison, WI, USA), while measurement was performed on the microplate reader (Tecan, Switzerland).

Vitis vinifera cv. Graševina grape pomace was obtained from the Croatian native grape cultivar Kutjevo d.d. in September 2022. Fresh grape pomace was vacuum dried at 70 °C until constant mass was achieved, milled using an IKA MultiDrive control (Staufen, Germany), and stored at 25 °C in a desiccator until further use.

2.2. Determination of TP Content

TP content was determined by the Folin–Ciocalteu method, as briefly described by Singleton et al. [24]. The absorbance was measured at 760 nm, and results were expressed as mg of gallic acid equivalent per g of grape pomace (mg_GAE g⁻¹). Spectrophotometric analyses were conducted in triplicate.

2.3. Development of an Environmentally Friendly Method for Extraction of Polyphenols from Graševina Grape Pomace Using NADESs

2.3.1. Selection of Optimal Extraction Method and Preparation of Extracts

To determine the most effective method for extracting polyphenolic compounds from grape pomace, water was used as a solvent, and various extraction conditions were tested. The comparison involved three methods: (i) solid-to-liquid extraction with stirring at room temperature (conventional extraction); (ii) UAE using an ultrasonic probe system for batches (bUAE); and (iii) a continuous (cUAE) process with a flow cell (Hielscher FC2T600K; cUAE), an ultrasonic processor (UIP1000hdT) operating at a frequency of 20 kHz, and various probes connected to a B4-1.4 booster with orientation as an amplifier and an applied amplitude cycle of 100% and 90%, without cooling.

Parameters that were set to determine the optimal extraction method are presented in Table S2. Further, batch extraction was conducted using a batch process with different ultrasonic probe diameters (22 mm, 34 mm, and 50 mm). The solid–liquid ratio used for extractions was constant, but a comparison was made for different scales. For optimization purposes, water was used as a solvent. After each method, the extracts were centrifuged, the supernatant was separated, and it was stored at +4 °C until further analysis.

2.3.2. Preparation of NADESs

The NADESs were synthesized at certain molar ratios of betaine (B) as a hydrogen bond acceptor (HBA) to glucose (Glc) as a hydrogen bond donor (HBD). These two components were placed in a 1:1 molar ratio, with 30 and 65% (v/v) of water, in a flat-bottomed glass bottle, and then placed in a Shaker–Incubator ES-20/60 (Biosan, Riga, Latvia), stirred, and heated to 50 °C for 2 h until a homogeneous, transparent, colorless liquid was formed.

2.3.3. Extraction Method Optimization Using NADES

In 300 mL of B:Glc (1:1) NADES or water, a specific amount of dry grape pomace was added. A Hielscher FC2T600K flow cell, an ultrasonic processor UIP1000hdT operating at a frequency of 20 kHz, and a probe connected to a B4-1.4 booster with amplification orientation were employed, with various amplitudes, without cooling. The optimization of the UAE of polyphenols from grape pomace was performed via Box–Behnken design. The influence of the independent variables such as the percentage of applied amplitude (X1, 20–100%), water content in the NADES (X2, 30–100%, v/v), extraction time (X3, 2–15 min), and solid-to-liquid content (X4, 0.02–0.1 g/mL) on the dependent variable, extracted polyphenolic content (Y), was investigated (Table S3). Twenty-seven experiments were performed with 3 center points per block to optimize the extraction method. Design-Expert (Version 7.0.0., Suite 480, Minneapolis, MN, USA) software was used for the analysis of variance (ANOVA) to obtain the quadratic polynomial mathematical model, which was established to describe the interaction of process parameters on the extraction of polyphenolic compounds (Table S4). After the treatment, the sample was filtered using vacuum filtration. The obtained extract’s volume was measured and stored in a refrigerator for further use. The variable process parameters included the NADES ratio, amplitude strength, time, and dry grape pomace mass.

2.4. Evaluation of Biological Activities of Prepared Extracts

2.4.1. HPLC Analyses

Polyphenols were identified and quantified using an HPLC system (1260 Infinity II, Agilent, Santa Clara, CA, USA) coupled with a diode array detector (UV/DAD, 1260 Infinity II, Agilent, USA) and an automatic sampler (1260 Infinity II, Agilent, USA). Separation was achieved on a Poroshell 120 SB C18 column (150 mm, 4.6 mm, 5 µm; Agilent, USA) using H₂O with 0.25% AcOH (A) and ACN (B) as the mobile phases, as previously described by Panić et al., 2021 [25].

2.4.2. Inhibition of Collagenase Activity

To determine the inhibitory effect of grape pomace extracts on collagenase activity, experiments were performed according to the method described by Zhang et al. [26]. Five different concentrations of polyphenols in NADES as well as in conventional solvent ethanol were tested to the investigate possible inhibitory effect on collagenase activity of gelatine as a substrate. Briefly, 1.9 mg mL⁻¹ of gelatine was dissolved in reaction buffer, and polyphenol-enriched extract was added to the mixture. To initiate hydrolysis, collagenase (1 mg mL⁻¹) was added to the gelatine solution and the digestion reaction was conducted at 37 °C for 10 min. Then, the reaction mixture was mixed with an equal volume of quenching buffer to stop the reaction. For color development and measurement, ninhydrin reagent was added to the quenched sample and heated at 80 °C for 10 min in a water bath. The inhibition of gelatine hydrolyzation was detected on a spectrophotometer at 545 nm in triplicate. A sample containing only ninhydrin but without gelatine and collagenase was used as blank. As positive control solution of gallic acid was used and distilled water was used as a negative control. Amino acids and small peptides obtained with gelatine hydrolyzation created a complex with ninhydrin, resulting in purple colorization, and could be detected at 545 nm wavelength. The inhibition of collagenase activity should have prevented the degradation of gelatine so that ninhydrin would not have had a substrate to bind on and colorization would not have appeared. The lesser intensity of purple coloring meant the higher inhibition of collagenase activity. Results were expressed as mg per mL of leucine which was used as a standard for the measurement of amino acid degradation.

2.4.3. Scratch Test

The scratch test is used to investigate the impact of various factors, such as chemicals, on cell migration, i.e., wound healing [27]. The experiment began by culturing HaCaT cells in a monolayer. The monolayer was then scratched with a straight line in the center of the plate using a pipette tip, mimicking a wound. The incision was marked with a marker on the external side of the plate. Images of the wound formation were immediately captured using a microscope camera. The plates were then exposed to extracts in NADES and only NADES (BGlc). Untreated cells were used as a control. The plates were incubated for 48 h. A microscope was used to monitor the cell growth at the incision site, i.e., wound healing under the influence of the extract in NADES.

2.4.4. Permeability Simulation by the PAMPA Method

The PAMPA (Parallel Artificial Membrane Permeability Assay) test was used to predict the intestinal absorption of polyphenols from grape pomace extracts into the intestinal membrane [28]. The PAMPA test consists of donor and acceptor wells filled with a buffer and separated by an artificial membrane with a filter through which components are transported by passive diffusion. Artificial membranes serve as an ideal system for mimicking gastrointestinal or other types of biological membranes in assessing the absorption potential of individual compounds [29]. A Multi-Screen Permeability Plate Assembly with a 96-well filtration plate (Millipore, MA, USA) was used as the carrier of the artificial membrane and as the acceptor plate. The filter in each well of the filtration (donor) plate was coated with 5 μL of a solution of L-α-phosphatidylcholine in 1% (w/v) dodecane. The donor plate was then placed onto the acceptor plate, which was pre-filled with 300 μL of a phosphate-buffer solution at pH 7.4. Next, 125 µL of extract was added to each well of the donor plate. The plate system was sealed with a plastic cover to prevent evaporation and incubated for 24 h at room temperature. After incubation, the volumes in the donor and acceptor wells were measured, and the concentrations of TPs on the acceptor plate were determined as previously described.

The apparent permeability coefficients (P_app) were calculated using the following equations:

$$\%T=100\times \frac{C_R\times V_R}{C_D\times V_R}$$

(1)

$$P_{app}=\frac{V_D\times V_R}{(V_D+V_R)\times S\times t}\ln \left[\frac{100\times V_D}{100\times V_D-\%T\times \left(V_D+V_R\right)}\right]$$

(2)

V_D and V_R are the volumes of the donor and receiving solutions, respectively (mL); C_D and C_R are the concentrations of TP in the donor and receiving, respectively; S is the surface area of the artificial membrane (0.28 cm², according to the manufacturer); t is the incubation time (s); and %T is the extent of transport across the membrane.

2.5. Statistical Analysis

The estimation of model coefficients using nonlinear regression analysis, statistical significance analysis (ANOVA) of the examined parameters on the observed processes, and numerical optimization of the investigated process parameters were conducted using the Design Expert 10.0.3 software package. A visual, three-dimensional representation of the parameters affecting the examined process was created using the STATISTICA 8.0 software package.

3. Results and Discussion

3.1. The Selection of Extraction Method

The research goal was to optimize the green extraction of polyphenols from Graševina grape pomace. Ultrasound was chosen as the extraction method and we initially sought to determine the most effective ultrasound extraction method, considering its potential for industrial application to obtain ready-to-use extracts that could be easily transferred to the industry. UAE was chosen as the technique considering its both non-thermal and eco-friendly characteristics coupled with benchmarked applicative characteristics in industry. The effects associated with ultrasound energy include increased convective forces, acoustic cavitation, and the generation of highly reactive radicals, enhancing solubility and accelerating the dissolution of target molecules from biomass.

The research began by exploring the possibilities of UAE and its potential for continuous industrial-scale application. Water was selected as a solvent in the optimization of ultrasound reactors due to its versatility as a universal solvent, making it capable of dissolving a wide range of compounds, polyphenols too. Additionally, its non-toxic nature, environmental friendliness, and cost-effectiveness align with the practical considerations for the intended ultrasound-assisted process. These experiments took several different forms: (i) mixing (conventional extraction), (ii) batch extractions (bUAE), and (iii) continuous extraction using an ultrasonic probe (cUAE) (Figure 1). The choice of extraction conditions was selected by balancing between achieving optimal extraction efficiency, preserving sensitive compounds, and ensuring cost-effective and energy-efficient processes.

The results presented in Figure 1 represent the concentrations of total polyphenols (TPs) obtained at the end of the process, at 15 min for extractions without heating to compare with UAE conditions, in the ultrasonic bath, and batch extraction using a 34 mm diameter probe, and at 15 min for continuous extraction using a 34 mm diameter probe. The polyphenol contents were extracted in the following order, from highest to lowest: 34 mm probe, batch > 34 mm probe, continuous > extractions without heating (Figure 1). Furthermore, the measured final energy input for the continuous process was 191 180 Ws, while for a batch process, it was 91 915 Ws. Therefore, an ultrasonic batch reactor was selected as the extraction method.

In the industry, the shift towards unconventional methods, notably ultrasound technology, is increasingly observed due to their efficiency and potential for energy savings, primarily attributed to the cavitation phenomenon. Continuous systems are preferred due to their ability to streamline processes; nonetheless, a critical evaluation of their merits and demerits is crucial. Following the selection of the method, the choice of probe diameter becomes imperative, as it significantly affects the distribution and intensity of ultrasonic waves, thereby influencing the efficiency and effectiveness of the extraction process [18,23]. A larger probe diameter leads to a broader cavitation zone, promoting more extensive and uniform extraction throughout the sample. It enhances penetration depth, ensuring interaction with a larger volume of the extraction matrix, and influences power distribution, impacting the breakdown of cell walls and release of target compounds. Optimizing the probe diameter is crucial for achieving efficient and scalable ultrasonic-assisted extraction processes, and results are presented in Figure 2. A 50 mm probe was selected as optimal, and extraction conditions were optimized for it.

3.2. Optimization of Preparative-Scale Parameters for Ultrasound-Assisted Extraction Process

Following the identification of the optimal UAE method, the response surface methodology to further enhance the extraction process was employed. Notably, the chosen extraction solvent here was a natural deep eutectic solvent (NADES), betaine/glucose (BGlc), previously selected by Panić et al. [30], aligning with the principles of green extraction. This selection aimed to investigate potential improvements in extraction efficiency associated with the use of NADESs in order to achieve ready-to-use stable extracts [31].

The efficiency of extraction depends on various factors, including the solvents used, extraction time, the power of ultrasound, and the mass-to-solvent ratio [32]. These four independent variables were encoded at one level, resulting in the experimental design shown in

Table 1. Mean values of mass fractions of TP in extracts obtained according to the Box–Behnken experimental design.

Experiment Number	Factor 1 % NADES [%, w/w]	Factor 2 A [%]	Factor 3 t [min]	Factor 4 Mass/Solvent [g mL−1]	Response TP [mgGAE g−1]
1	35.00	100.00	8.50	0.02	12.47
2	35.00	100.00	8.50	0.10	8.88
3	0.00	60.00	15.00	0.06	13.87
4	35.00	60.00	15.00	0.10	8.95
5	70.00	60.00	2.00	0.06	19.14
6	35.00	20.00	8.50	0.02	11.40
7	35.00	60.00	8.50	0.06	13.52
8	35.00	20.00	2.00	0.06	16.28
9	0.00	60.00	2.00	0.06	11.00
10	35.00	100.00	2.00	0.06	8.84
11	35.00	100.00	15.00	0.06	12.19
12	70.00	20.00	8.50	0.06	20.94
13	35.00	20.00	15.00	0.06	17.89
14	35.00	60.00	2.00	0.02	11.78
15	35.00	60.00	8.50	0.06	14.34
16	35.00	60.00	15.00	0.02	16.54
17	0.00	100.00	8.50	0.06	12.22
18	35.00	60.00	8.50	0.06	15.73
19	0.00	20.00	8.50	0.06	7.61
20	35.00	20.00	8.50	0.10	10.78
21	35.00	60.00	2.00	0.10	12.55
22	0.00	60.00	8.50	0.02	14.11
23	70.00	60.00	15.00	0.06	20.18
24	70.00	60.00	8.50	0.10	12.59
25	0.00	60.00	8.50	0.10	7.86
26	70.00	60.00	8.50	0.02	14.42
27	70.00	100.00	8.50	0.06	6.64

Note: amplitude—A. time—t. TP—total polyphenol content.

.

According to the obtained results, as shown in Figure 3, the highest concentration of TP was achieved in experiment 12. A BGlc with 30% of water content (v/v, %) was applied. The extraction was conducted at an amplitude of 20%, for 8.5 min, and with a mass-to-volume ratio of 0.06. The lowest polyphenol concentration was obtained in experiment 27, where a NADES with 30% water, 100% amplitude, 8.5 min of extraction, and a mass-to-volume ratio of 0.06 was used.

The TP content ranged from 6.64 to 20.94 mg g⁻¹ dry weight. Additionally, the RSM model was evaluated, and ANOVA was used to determine the statistical significance of the quadratic model and the regression coefficients for each response. The results are summarized in Table S1.

The determined optimal conditions for extracting polyphenols from grape pomace using NADES BGlc (1:1) are as follows: a NADES ratio of 70%, an amplitude strength of 28.13%, an extraction time of 13.21 min, and a mass-to-solvent ratio of 0.04. The obtained concentration of TP after extraction under these optimal conditions was 21.6 mg_GAE g⁻¹ experimentally, while when it was theoretically predicted, the value was. 21.1 mg_GAE g⁻¹. The collected experimental data were used to create three-dimensional response surface plots for the TP yield as a function of the NADES content, time, amplitude, and mass-to-solvent ratio. According to Figure 3, it is evident that the concentration of TP increased with the higher content of NADES in the solvent. The highest concentrations were achieved at a NADES content of 70%, while the lowest concentrations were obtained when extraction was performed with water, as has been previously noted [25]. The TP yield was also influenced by amplitude and the mass-to-volume ratio. The yield of TPs increased with decreasing amplitude and decreasing mass-to-volume ratio. Extraction time had a positive influence but did not have a significant impact on polyphenol extraction yield. Similarly, the response-surface plots showed that total anthocyanin content increased with increasing water content, from 10 to 30%; it seems that mass transfer between the solid and liquid phases is hampered by high solvent viscosity in NADESs with 10% water, while anthocyanins are much less stable in NADESs with higher water contents. Panic et al. have also reported on the relationship between water content in NADESs and anthocyanin stability [25], while Cvjetko Bubalo et al. noticed that solvent viscosity is influenced by water content, and thus impacts upon mass transfer [33]. MW and US irradiation also decrease the viscosity of NADESs. Extraction time had a significant effect on total anthocyanin content. Increasing the extraction time led to decreased anthocyanin content, indicating that long extraction times and prolonged heating increase degradation [34].

Furthermore, Dai et al. reported polyphenol yields using conventional solvents like ethanol and water mixtures, with yields ranging significantly based on the material and extraction conditions [35]. The comparison highlights NADESs as a promising alternative that can achieve competitive or superior yields, especially considering their environmental and operational benefits. While much of the existing research on NADES and UAE has been conducted at laboratory scales, studies focused on scaling up (e.g., Ruesgas-Ramón et al.) provide valuable insights into the challenges and opportunities of larger-scale operations [36]. Our findings indicate that with careful optimization, NADES-based UAE processes can be effectively scaled, aligning with the industry’s move towards more sustainable extraction methods.

Comparing the outcomes from laboratory-scale experiments, where 26.07 mg_GAE g⁻¹ of polyphenols was extracted [30], to the results obtained in the preparative scale in this study, which recorded a similar yield of 21.6 mg_GAE g⁻¹ of polyphenols, suggests that the NADES process holds promise for effective extraction on larger scales.

An economic analysis from our prior work highlights that while the initial setup costs for using NADES may be higher due to the need for specialized equipment, the long-term operational costs are significantly lower. This is attributed to reduced energy consumption and minimal waste treatment requirements, making NADESs a viable option for industrial applications [10].

3.3. Biological Activity Evaluation of Grape Pomace Extracts Prepared in Optimal Conditions

In the pursuit of understanding and harnessing the full potential of grape pomace extract in NADESs, our study delved into the preliminary biological activity evaluation of extracts prepared under optimal conditions. The biological evaluation of optimal extracts holds paramount significance in both the food and cosmetic industries, as it provides a comprehensive understanding of the potential benefits and safety considerations associated with these extracts. In this context, examining the biological properties of optimal extracts emerges as a pivotal step in the development and formulation of products for the food and cosmetic sectors.

To date, the Graševina grape pomace extract in BGlc has been assessed on the HaCaT cell line, and cytotoxicity was determined through an in vitro assay specific to the HaCaT cell line (keratinocytes). The extract prepared with BGlc exhibited a stimulatory effect on HaCaT cell proliferation, showing a potential increase of up to 40% depending on the extract volume ratio. This suggests a proportional relationship between polyphenol concentration and the stimulatory effect, making them promising for application in the cosmetic industry [30]. In this study, we further explored additional methods of biological activity and compared them with the previously used ethanol extract in the research by Panić et al. [30].

In this research, various biological evaluations were undertaken to assess the potential applications of extracts obtained under optimal conditions at the preparative scale in the cosmetic and food industry. The study involved the examination of collagenase activity inhibition, in vitro wound healing through a scratch test, and the simulation of polyphenol permeability from grape pomace extracts across an artificial membrane using the PAMPA (Parallel Artificial Membrane Permeability Assay) method simulating the gastrointestinal tract barrier.

First, HPLC analysis of the NADEES extract prepared under optimal conditions and the ethanol extract used as a control was conducted, and the results are presented in

Table 2. Polyphenolic content (mg g _dw⁻¹ of pomace) in prepared extract. Specific polyphenol contents and total polyphenol content were expressed as the means (n = 3) ± S.D.

Extracts
Compound	GPBGlc	GPEtOH
Epigallocatechin	0.235 ± 0.010	0.279 ± 0.001
Catechin	0.739 ± 0.006	0.820 ± 0.002
Epicatechin	0.750 ± 0.005	0.471 ± 0.009
Gallic acid	0.549 ± 0.008	-
Procyanidin B1	1.017 ± 0.005	0.505 ± 0.002
Procyanidin B2	0.265 ± 0.022	-
Procyanidin B3	0.966 ± 0.003	1.197 ± 0.030
Procyanidin C1	0.258 ± 0.053	-

.

Considering compatibility and in comparison with previous work [29], no significant differences were observed in biologically active compounds. Different solvents dissolve various types of compounds, and a synergistic effect is possible, making it worthwhile to further investigate the biological activity.

The potential inhibition of collagenase was examined for the fixed concentration of polyphenols in the grape extract. The results indicate that the negative control had the lowest collagenase inhibition, followed by the positive control. Among the extracts, the BGlc extract demonstrated values of statistically higher (p < 0.05) inhibition compared to the EtOH extract (Figure 4).

The findings presented in our study align with prior research emphasizing the significance of natural extracts in addressing skin aging processes, particularly collagenase activity [37,38].

So far, several research articles have compared the impact of NADES on collagenase activity, with findings indicating notable results. Specifically, NADES formulations based on ChCl/Urea and ChCl/glycerol exhibited significant inhibition values for collagenase (91.1% and 92.7%, respectively) [38]. However, it is important to mention that choline chloride is restricted for use in cosmetics, which means these NADES formulations might not be the best choice for the final cosmetic product [10].

Moreover, we want to investigate the impact of extracts on in vitro wound healing. Cell migration is an important step in wound healing. Tests that can assess cell migration are very useful for evaluating in vitro wound healing. A gap in the keratinocyte monolayer, mimicking wound formation, was created using the scratch test [39]. This experiment examined the impact of the extract obtained in the NADES and the solvent itself on cell migration, compared to the control (cells only in a monolayer). Figure 5 shows the results of the scratch test.

Upon comparing the images on both sides of the display, with the left side representing a mimicked wound and the right side showcasing the healing process after 48 h, it is evident that cells treated with extract in NADES effectively bridged the gap. Notably, there were more cells in the treated group compared to the untreated cells (control). Even cells treated with NADES alone exhibited growth, contributing to wound healing. These observations strongly suggest that the extract in NADES (BGlc) and NADES itself play a role in promoting cell migration and wound healing, highlighting their potential application in the cosmetics industry.

Similarly, Silva et al. [40] tested the influence of hydrophobic DES based on menthol and saturated fatty acids. This formulation also enhanced wound healing. However, it is crucial to acknowledge a limitation in this experiment. The in vitro cell culture environment did not fully replicate an in vivo surgical wound. While in vitro results are promising, the transition to in vivo studies is crucial for a comprehensive understanding of their effectiveness and safety in wound healing of real scars [41].

Furthermore, simulations to assess the permeability of polyphenols from grape pomace extracts through an artificial membrane that mimics the gastrointestinal tract were conducted. The PAMPA method was employed to investigate the passive transport of polyphenols, particularly in the natural deep eutectic solvent (NADES) BGlc and ethanol, across the small intestine epithelium and into the bloodstream. The apparent permeability coefficients (Papp) were determined before and after in vitro small intestinal simulation, revealing that the permeability of polyphenols in BGlc was 2.5 times higher than that in ethanol (

Table 3. Permeability coefficient (cm s⁻¹) calculated for the different systems. Presented values followed by different lower-case letters (a,b) are significantly different (p < 0.05) as measured by Tukey’s HSD test. GPBGlc—grape pomace extract in NADES BGlc obtained under optimal conditions. GPEtOH—grape pomace extract in aqueous ethanol (70%. v/v).

	Permeability (10−11 cm s−1)
GPBGlc	5.15 ± 0.47 a
GPEtOH	1.99 ± 0.11 b

).

This enhanced permeability is attributed to potential interactions between the NADES and the phospholipid bilayer of the artificial membrane. Although the observed permeability falls into the category of low absorption, with values less than 1 × 10⁻⁶ cm s⁻¹ [42], the results obtained by PAMPA still go in favor of the extraction in the NADES compared to a conventional solvent.

Similarly, in a previous study published by [43], the focus was on optimizing and validating sustainable routes for the extraction and valorization of polyphenols from red grape pomace. The investigation included formulations using NADESs, particularly betaine combined with citric acid (BCa). The permeation performance of BCa was found to be superior in Franz cells. Passive diffusion through the membrane is influenced by various factors, including lipophilicity, pH value, hydrogen bonding, and molecular weight. Our results on polyphenol permeability are in accordance with the literature [44]. Improved permeability, referring to the enhanced ability of polyphenols to penetrate through the gastrointestinal tract, results in the greater efficacy and utilization of active compounds in the extract, particularly in applications such as food.

Our previous research has also explored potential applications of NADES-extracted compounds beyond the food and cosmetic industries. This includes their use in pharmaceuticals, nutraceuticals, and agricultural products, highlighting the versatility and broad market opportunities for these extracts [10].

4. Conclusions

In conclusion, the successful integration of ultrasonic assisted extraction (UAE) with betain/glucose (BGlc) natural deep eutectic solvents (NADESs) has established its applicability for industrial use, demonstrating consistent scalability. The observed collagenase-inhibitory effects in these extracts further indicate their promising potential in the cosmetic industry. With grape pomace extract showcasing stability in NADESs and favorable attributes such as enhanced bioavailability and eco-friendly characteristics, these extracts present themselves as convenient, ready-to-use solutions suitable for cosmetics, food supplements, and diverse food applications. This underscores NADESs’ effectiveness as solvent systems for extracting bioactive compounds, creating possibilities for innovative applications across industries.

The practical feasibility of directly incorporating our extracts into cosmetic or food products, eliminating the need for traditional solvent evaporation, aligns seamlessly with sustainability initiatives prevalent in the cosmetic and food industry. This not only underscores the environmental friendliness of our approach but also highlights the potential seamless integration of these extracts into cosmetic and food formulations, paving the way for the development of effective products.