Enhanced “Greener” and Sustainable Ultrasonic Extraction of Bioactive Components from Waste Wild Apple (Malus sylvestris (L.) Mill.) Fruit Dust: The Impact of Pretreatment with Natural Deep Eutectic Solvents

Abstract

Significant depletion of natural resources, coupled with increased environmental pollution resulting from the constant evolution of global industrialization, poses a considerable problem. Therefore, it is unsurprising that sustainable “green” chemistry and technology are gathering the worldwide scientific community, whose common goal is to find applicable solutions for the abovementioned problems. This paper combined the ultrasonic extraction method (a form of “green” technology) with natural deep eutectic solvents (NADESs, a type of “green” solvent) for the production of extracts from an industrial by-product (discarded waste wild apple dust). Waste wild apple dust was pretreated with different NADESs in order to explore the pretreatment benefits regarding ultrasonic extraction of bioactive compounds. Among all solvents used, aqueous propylene glycol was chosen as the best system, which, combined with Reline NADES pretreatment, provided the highest TPC and TFC values, together with the best antioxidant activities. UHPLC-DAD-MS analyses of extracts revealed the presence of natural organic acids, quercetin and kaempferol derivatives, tannins, and flavones. Following this procedure, valorization of agro-industrial apple herbal waste resulted in obtaining extracts with high potential for utilization in different industrial branches (food and pharmaceutical industries), contributing to both cleaner production and reduced environmental impact.

1. Introduction

Agro-industrial waste comprises diverse plant materials (roots, stems, seeds, skin, peel, pomace, etc.) that are rich in valuable bioactive compounds, including carotenoids, polyphenols, dietary fibers, vitamins, enzymes, and oils [1]. Moreover, specific agricultural wastes are regarded as inexpensive, polyphenol-rich feedstock. These phytochemicals find applications across various industrial sectors, particularly in the production and development of functional foods, pharmaceuticals, nutraceuticals, cosmeceuticals, and modified textile fibers and materials [2]. Moreover, agro-industrial waste is utilized for soil fertilization and production of animal feed, bioenergy, and biofuels, thus reducing both environmental and economic impact related to waste disposal [3]. Since waste utilization is considered crucial towards achieving more sustainable development, it is necessary to gather as much information as possible in terms of the origin of specified plant wastes, their chemical compositions, and adequate extraction techniques for obtaining value-added products [2].

Wild apple (Malus sylvestris (L.) Mill.), a member of the Rosaceae family, is commonly found as a free-growing shrub in central European forests. From an ethnopharmacological perspective, it is more commonly referred to as a medicinal plant. Wild apple fruit (Mali fructus) is notably sour and generally regarded as unsuitable for raw consumption. However, owing to its high content of diverse bioactive compounds, it is valued as a raw material for producing teas, tinctures, medicinal wines, schnapps, and other beverages. In European folk medicine, it has a multitude of uses, more specifically for the alleviation of different medical conditions (pain, migraines, high blood pressure, diarrhea, vomiting, sleep deprivation, etc.).

Discarded Mali fructus dust, an agro-industrial by-product from filter tea factories, remains underutilized despite being rich in valuable functional bioactive compounds that are extensively used in nutritional, pharmaceutical, cosmetic, and chemical industries. Therefore, waste wild apple dust represents a suitable material for producing extracts enriched with a broad spectrum of hydrophilic and lipophilic antioxidants [4]. However, research focused on the composition of waste wild apple fruit dust from different regions is quite limited, despite the fact that this environmentally polluting waste may be successfully implemented in the production of animal feed, compost, and liquid and gaseous biofuels.

Conventional approaches for extracting plant-based bioactive constituents include maceration, organic solvent extraction, and steam distillation [5,6,7]. Although effective for producing extracts, solid–liquid extraction methods significantly increase production costs due to the high demand for plant materials and solvents, long extraction times, heating energy requirements, and extensive pre- and post-extraction steps, rendering these methods cost-inefficient [5,6,7].

The obstacles mentioned above can be surpassed by focusing on

(a)

Developing newer “green” extraction techniques;

(b)

Selecting the appropriate extraction agent.

Newer “green” extraction techniques must be convenient, economical, safe, and nontoxic and have a great potential to replace existing traditional extraction methods, ensuring long-term sustainability. These new techniques increase the efficiency and quality of the extract; reduce the consumption of time, solvent, and other materials; do not cause thermal damage to the compounds; and are environmentally friendly [5]. These modern, sophisticated techniques include extraction in the presence of different irradiations (microwave and ultrasound), supercritical fluids, subcritical water, enzymes, and safer “greener” solvents like water, ionic liquids, and eutectic mixtures [5,8,9,10]. Among these, ultrasound-assisted extraction (UAE) is particularly segregated as quite an efficient approach for the extraction of polyphenolics due to numerous advantages and benefits. Specifically, UAE is cheap, easily manageable and operable, fast, widely available, potent for the extraction of various natural compounds from different feedstocks, energy-saving, and cost-effective and allows for the application of different solvents or solvent systems. The produced extracts contain a higher concentration of the desired biocompound; therefore, they enable adequate scaling up for industrial purposes [11,12,13,14]. Since ultrasonic cavitation bubbles ensure enhanced penetration of solvent molecules into the selected feedstock, degrading its cellular wall and causing migration of the intracellular matter into the solvent (producing an extract), it is necessary to pay strict attention to factors that directly influence UAE’s success. These factors include the nature of the selected feedstock, ultrasonic intensity, temperature, time, and type of solvent and its properties, such as polarity, hydrophobicity, viscosity, pH, etc. [13,14,15,16]. Furthermore, the remaining post-extraction cellulosic material can be separated and exploited as a waste biomass for obtaining different products (biofuels, energy, bio-construction materials, fertilizers, and food for domesticated animals), so this double industrial-scale valorization of plant waste would increase the profit even more in the long term [17].

Another important focus is shifted towards selecting the appropriate extraction agent, crucial for maximizing the potency of the extract. Although water is regarded as both an “ideal” and “green” extraction solvent, it is adequate to successfully extract only polar and hydrophilic biocompounds. As an alternative, organic solvents demonstrate superior extraction potential towards polyphenolic compounds due to adequate solubilization power. Since industrial organic solvents are used in larger quantities (characterized by problematic properties such as flammability, toxicity, and higher polluting potential toward the environment), operational risks due to heating and hot vapors for workers require additional investment in regard to ensuring safety (special labor suits, ventilation, and protective gear such as gloves, masks, hats, and goggles), making the process even more expensive. Inadequate solvent for extraction leads to lower extraction efficiency due to restriction of plant cell walls, which makes the process less effective and poses a risk for future stability of extracted compounds in the final product [5,7]. Moreover, the market must comply with the demands of consumers who express a negative attitude toward applying bio-derived pharmaceutical or chemical products that contain additives of synthetic origin.

Therefore, further improvement in terms of finding more suitable and safer extracting agents is justified. Selected “green” solvents must exhibit a polyphenolic extraction capacity equivalent to or higher than that of organic solvents but must be cheap and exert minimal environmental impact. A specific class of “green” solvents is natural deep eutectic solvents (NADESs), prepared from safe, natural, and easy-to-handle compounds. In fact, the most common constituents of NADESs are primary metabolites found in plants. Adequate tailored proportions of these compounds enable intermolecular interactions (where hydrogen bonding is the most dominant among them), thus providing mixtures with desirable physicochemical properties and a strong ability to dissolve a wide range of compounds with diverse polarities [10,18,19,20,21,22,23,24,25]. Compared to conventional organic solvents, environmentally friendly NADESs offer several benefits in terms of lower cost, easy preparation, safer handling, availability of starting components, high biodegradability potential, and non-toxicity [10,18,19,20,21,22,23,24,25]. An additional beneficial NADESs feature is their potential regarding chemical transformations of cellular walls and tissues in plant-derived agricultural materials (e.g., hemicellulose hydrolysis), leaning towards an increased amount of extracted polyphenols [26,27]. This reflects a direct significant advantage regarding facilitated extraction of a wide group of plant-derived bioactive compounds and secondary metabolites, i.e., phenolic acids, flavonoids, isoflavones, catechins, polysaccharides, curcuminoids, proanthocyanidins, anthocyanins, xanthones, terpenes, tannins, lignins, pectins, alkaloids, etc. [10,18,19,20,21,22,23,24,25]. Therefore, NADESs are regarded as acceptable agents for contributing to the sustainable development of different technological areas and industries. Among common NADESs, honey is the most well-known example. A special ratio of its constituents (monosaccharides and disaccharides) enables intermolecular interactions and hydrogen bonds forming between these molecules, making it a stable mixture. Honey and honey-mimicking NADESs have shown potential to increase the bioactivity of some traditional medicines [28].

The aims and novelties of this study are

(a)

Development of novel, “greener” and environmentally friendly UAE methodologies using crude herbal powder (waste wild apple fruit dust) and four different solvent systems (distilled water, aqueous 37% (v/v) ethanol, aqueous 38% (v/v) propylene glycol, or aqueous 38% (v/v) glycerol);

(b)

Choosing the best solvent system for the UAE procedure using NADES-pretreated herbal powder in order to enhance the UAE efficiency;

(c)

UHPLC-DAD-MS/MS identification and comparison of bioactive components in produced extracts;

(d)

Comparison of their total phenolic content (TPC) and total flavonoid content (TFC) values, as well as antioxidant activity of produced extracts using DPPH and ABTS assays;

(e)

Evaluation and comparison of energy demands and environmental impacts of suggested UAE methodologies.

To the best of the authors’ knowledge, neither UAE of bioactive compounds from waste wild apple fruit dust obtained in Serbia (pretreated with several NADESs) in aqueous alcoholic solutions, nor identification and comparison of compounds in these extracts, nor a detailed comparison of their polyphenolic content, antioxidant activity, and energy consumption and the environmental impacts of these UAE methodologies have previously been reported in the literature.

2. Materials and Methods

2.1. Plant Material

Wild apple herbal powder, a waste by-product from local filter tea production, was supplied by the company “Jeligor” doo, Svrljig, Serbia (43°24′55.4″ N 22°07′12.4″ E). The waste plant material was delivered in a sealed paper package, protected from light and moisture. The by-product consisted of material picked in 2021 and discarded after the processing and unit operations used in the company (specifically cutting, grinding, sieving, and fractionation). The powder reached a moisture content of 8.9 ± 0.2% after oven-drying with hot air at 105 °C for 120 min. The mean particle size was less than 0.3 mm, unsuitable for packaging in tea bags.

2.2. Chemicals

For NADES preparation: Choline chloride (≥98%, Sigma Aldrich, St. Louis, MO, USA), urea (99.5%), distilled water (Zorka-Pharma, Šabac, Serbia), glucose, fructose, oxalic acid·2H₂O (99%, Centrohem, Stara Pazova, Serbia), and glycerol (Ph Eur-grade, MeiLab, Belgrade, Serbia). All reagents were used without further purification.

For UAE: Distilled water and absolute ethanol (Zorka-Pharma, Šabac, Serbia), propylene glycol, and glycerol (Ph Eur grade, MeiLab, Belgrade, Serbia).

For TPC determination: Folin–Ciocalteu’s reagent (Carlo Erba Reagents, Val de Reuil, France), sodium carbonate, calcium chloride dihydrate, distilled water, acetic acid (Zorka Pharma, Šabac, Serbia), and gallic acid (97%, Merck, Darmstadt, Germany).

For TPC determination: Methanol (HPLC-grade, Sigma Aldrich, Munich, Germany), AlCl₃ and CH₃COOK (p.a.-grade, Sigma Aldrich, Munich, Germany), and quercetin (p.a.-grade, Merck WGK, Darmstadt, Germany).

For UHPLC-DAD-MS/MS analysis: Purified distilled water, formic acid, and methanol (HPLC- or LC/MS-grade, Fisher Chemical, Fair Lawn, NJ, USA). Reference standards: Chlorogenic acid, citric acid, hyperoside (quercetin-3-O-galactoside), isoquercitrin (quercetin-3-O-glucoside), kaempferol, malic acid, rosmarinic acid, rutin (quercetin-3-O-rutinoside), tiliroside, and quercetin (all p.a.-grade, Sigma Aldrich, St. Louis, MO, USA).

For antioxidant activity: Methanol and ethanol (HPLC-grade), ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), potassium persulfate (K₂S₂O₈), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (all p.a.-grade, Sigma-Aldrich GmbH, Steinheim, Germany).

2.3. NADES Preparation Procedure

Honey-mimicking and ChCl-based NADESs were prepared following procedures described in the literature [28,29]. The chosen constituents in determined amounts (

Table 1. List of prepared NADESs and their constituents and properties.

NADES Name	Constituent Used for NADES Preparation				Molar Ratio	MNADES, g∙mol−1	ρ, g cm−3 (20 °C)	η, mPa∙s (20 °C)
	1	2	3	4
Honey-mimicking	Glucose	Fructose	Sucrose	Water	0.178:0.178:0.0146:1.72	99.9585	1.31	8990
Reline	Choline chloride	Urea	-	-	1:2	86.58	1.1829	3195
Oxaline	Choline chloride	Oxalic acid· 2H2O	-	-	1:1	132.845	1.120	330
Glyceline	Choline chloride	Glycerol	-	-	1:2	107.936	1.1951	490

) were mixed in a one-necked round-bottomed flask, placed on a rotary evaporator, and held at 70 °C for 60 min until a homogeneous, transparent liquid was formed. The prepared NADESs were transferred into well-closed dark glass bottles, capped, and kept in a desiccator with CaCl₂ until pretreatment experiments. All NADES remained viscous, homogeneous, and colorless liquids upon cooling, except Reline, which became a white semi-solid with time.

Density and dynamic viscosity for NADES were measured using a DMA 4500 Anton Paar densitometer and a rotational viscometer (Visco Basic Plus, ver. 0.8, Fungilab S.A., Barcelona, Spain), respectively. The molecular mass for NADESs (M_NADES) was calculated using Equation (1):

M_NADES = M_C1 × x_C1 + M_C2 × x_C2 + M_C3 × x_C3 + M_C4 × x_C4/x_C1 + x_C2 + x_C3+ x_C4

(1)

where M_NADES, M_C1, M_C2, and M_C3 denote the molecular mass of NADES and the first, second, third, and fourth compounds, respectively, while x_C1, x_C2, and x_C3 refer to the molar ratio of the compounds in order.

2.4. Pretreatment of Plant Material

Wild apple herbal powder was used in two different ways: in its original form as crude (raw) powder and as NADES-pretreated powder.

The crude powder was unmodified, stored in its original package until use. Pretreatment procedure: The powder was immersed with the selected NADES (Honey-mimicking, Reline, Oxaline, or Glyceline) in a powder-to-NADES ratio of 1:5 g/g and left at room temperature for 24 h, followed by separating the solid and liquid phases through centrifugation in a TH16B centrifuge (Hong Kong, China) at 4000 rpm for 10 min.

2.5. UAE Procedures

2.5.1. UAE Procedure Using Crude Herbal Powder

The first set of UAE experiments (

Table 2. Applied UAE procedures (identical operating conditions: 30 min; liquid-to-solid ratio: 15 mL/g; 60 °C; 150 W).

Experiment No.	Type of Herbal Powder	NADES Used for Pretreatment	Extracting Solvent
1	Crude	None	Distilled water
2	Crude	None	Aqueous 37% (v/v) ethanol
3	Crude	None	Aqueous 38% (v/v) glycerol
4	Crude	None	Aqueous 38% (v/v) propylene glycol
5	Pretreated	Honey-mimicking	Aqueous 38% (v/v) propylene glycol
6	Pretreated	Reline	Aqueous 38% (v/v) propylene glycol
7	Pretreated	Oxaline	Aqueous 38% (v/v) propylene glycol
8	Pretreated	Glyceline	Aqueous 38% (v/v) propylene glycol

, Experiments 1–4) was conducted to determine the most efficient extraction solvent. UAE was performed in a manner similar to that described in detail in Nikolić et al. [14]. Crude powder was placed in a 50 mL glass flask. After adding the selected solvent (distilled water, aqueous 37% (v/v) ethanol, aqueous 38% (v/v) propylene glycol, or aqueous 38% (v/v) glycerol) in a liquid-to-solid ratio of 15 mL/g, the flask was connected with a reflux condenser and placed in an ultrasonic bath (Sonic, Niš, Serbia) under the following initial screening conditions: constant ultrasonic power of 150 W (operating frequency of 40 kHz), temperature of 60 °C, and duration of 30 min. The obtained mixture was centrifuged in a TH16B centrifuge (Hong Kong, China) at 4000 rpm for 10 min, separating the extract from the solid material. The liquid extract (3 mL) was dried at 105 °C in a laboratory oven until a constant weight was achieved. The dry weight of the analyzed sample was determined. The extracts were refrigerated at 4 °C until further analysis.

2.5.2. UAE Procedure Using NADES-Pretreated Herbal Powder

After separation from NADES after centrifugation, NADES-pretreated herbal powders were subjected to UAE with aqueous 38% (v/v) propylene glycol as the extraction solvent (Table 2, experiment no. 5–8). In a 50 mL glass flask, the selected pretreated powder was mixed with aqueous 38% (v/v) propylene glycol in a liquid-to-solid ratio of 15 mL/g. The flask was connected with a reflux condenser and placed in an ultrasonic bath. The following steps are identical to those described in Section 2.5.1.

2.6. Qualitative and Quantitative Analyses

2.6.1. UHPLC-DAD-MS/MS Analysis

Qualitative determination of the extracts’ composition was achieved using ultra-high- performance liquid chromatography (UHPLC) on a Hypersil gold C₁₈ column (50 × 2.1 mm, 1.9 μm) at 25 °C using a Dionex Ultimate 3000 UHPLC+ system equipped with a diode array detector (DAD) and the LCQ Fleet Ion Trap Mass Spectrometer (Thermo Fisher Scientific, USA), as detailed in our previous paper [14].

The mobile phase consisted of two solvents: 0.1% aqueous formic acid solution (A) and methanol (B). At the determined flow rate (0.25 mL/min), the following gradient program was used: 0–2 min (10–30% B), 2–4 min (30–35% B), 4–5 min (35–40% B), 5–8 min (40–50% B), 8–11 min (60–90% B), followed by an isocratic segment 11–14 min (90% B), 14–14.1 min (90–10% B), finishing with the isocratic run until 20 min (10% B).

The injected volume of the analyzed extract was 4 µL. Absorption spectra were recorded on a DAD with a total spectral range between 200 and 800 nm. Mass spectrometric analysis was performed using a 3D ion trap with electrospray ionization (ESI) in both negative and positive ion mode. The ESI source parameters were source voltages of 4.5 kV and 5 kV, capillary voltages of 41 V and 49 V, and tube lens voltages of −95 V and 100 V for negative and positive polarity mode, respectively, a capillary temperature of 350 °C, and nitrogen sheath and auxiliary gas flows of 32 and 8 arbitrary units for both modes. Additional source ionization MS spectra were acquired through full-range acquisition of m/z 100–900, with a tandem mass spectrometry analysis performed using a data-dependent scan—with the collision-induced dissociation of detected molecular ion peaks ([M − H]⁻ and [M + H]⁺) tuned at 30 eV. Xcalibur software (version 2.1) was used for instrument control, data acquisition, and data analysis.

The assignation of the detected compounds was based on their retention times and UV–Vis and MS spectra from the corresponding UHPLC chromatograms. Identification of some detected compounds was also provided using reference standards: chlorogenic acid, citric acid, hyperoside (quercetin-3-O-galactoside), isoquercitrin (quercetin-3-O-glucoside), kaempferol, malic acid, rosmarinic acid, rutin (quercetin-3-O-rutinoside), tiliroside, and quercetin. The corresponding data were compared with the available literature using absorption UV–Vis and mass spectra.

2.6.2. Determination of TPC and TFC

TPC, referring to the concentration of soluble phenolic compounds in the produced extracts, was determined using the modified Folin–Ciocalteu method with gallic acid as the standard, as described in detail in Kocovic et al. [30]. First, the calibration curve was provided using a series of standard solutions of gallic acid (concentrations 25, 50, 100, 200, 400, 500, and 1000 µg/mL). In a test tube, 50 μL of liquid sample (produced extract or standard solution) was diluted with 150 μL of distilled water, followed by adding 1 mL of Folin–Ciocalteu reagent. The mixture was vortexed and kept still at room temperature for 5 min. Subsequently, 800 μL of 7.5% previously prepared aqueous sodium carbonate solution was added, followed by incubation of the test tubes in the dark for 60 min with occasional shaking. The absorbance was measured at 760 nm. TPC was quantified as mg of gallic acid equivalents per gram of dry wild apple powder (DAP) used for extract preparation (mg GAE/g DAP), determined from the calibration curve (Equation (2)):

Absorbance = 0.0028 × Total phenols + 0.0810 (R² = 0.9988)

(2)

TFC was quantified according to the modified method with quercetin as a standard [3]. First, two calibration curves were designed by employing standard solutions in different concentrations (31.25, 62.5, 125, 250, 500, and 1000 µg/mL). In a test tube, 1 mL of liquid sample (produced extract or selected standard solution) was diluted with 200 µL of 10% AlCl₃ methanolic solution, 200 μL of 1M potassium acetate aqueous solution, and 5.6 mL of distilled water. After vortexing, the test tubes were incubated at room temperature for 30 min, allowing AlCl₃ to react with flavonoids and form a yellow-colored complex. The absorbance was measured at 415 nm. TFC was quantified as mg of quercetin equivalents per g of DAP used for extract preparation (mg QE/g DAP), determined from the calibration curve (Equation (3)):

Absorbance = 0.0019 × Total flavonoid content + 0.0575 (R² = 0.9989)

(3)

2.6.3. Antioxidant Activity Assessment

The antioxidant activity assessment was performed via DPPH and ABTS assays.

The capacity to neutralize free radicals was evaluated using DPPH radicals, following a previously established methodology [31]. Initially, a DPPH solution was prepared in methanol at a concentration of 0.05 mg/mL and stored in a dark container under refrigeration until the experiments commenced. Subsequently, a series of standard solutions of the tested extracts and reference standards was prepared in methanol at concentrations of 31.25, 62.5, 125, 250, 500, and 1000 μg/mL. In each experiment, 200 μL of the prepared extract or standard solutions was combined with 2 mL of the DPPH solution in test tubes. The mixtures were then vigorously mixed and incubated in the dark for 30 min. Following incubation, the absorbance of the solutions was measured at 517 nm relative to the control.

The ability to neutralize free radicals was also assessed using ABTS radicals, based on a previously described method [32] with modifications. During the experimental preparation, a mixture of 7 mM ABTS and 2.45 mM potassium persulfate was incubated at room temperature in the absence of light for 24 h. This solution was diluted with ethanol until an absorbance of 0.700 ± 0.02 was achieved at 734 nm. A volume of 300 μL of the extract or standard solution was mixed with 600 μL of the ABTS solution. The mixture was then incubated at room temperature for 30 min. The absorbance was measured at 734 nm. Ascorbic acid and Trolox were utilized as positive controls in both experiments.

The concentration of DPPH or ABTS radicals was calculated using Equation (4):

%Inhibition = 100 × (A_c − A_s)/A_c

(4)

where A_c represents the absorbance of the control (containing all reagents except the tested extract or standard) and A_s denotes the absorbance of the sample. The derived values were used to construct a nonlinear calibration curve, enabling the determination of the concentration of the tested sample required to inhibit 50% of the DPPH or ABTS radicals (IC₅₀).

2.6.4. Statistical Analysis

All results are reported as mean ± standard deviation from three independent measurements. Statistical comparisons of the obtained values (TPC, TFC, DPPH, and ABTS) were conducted through a one-way analysis of variance (ANOVA) combined with Tukey’s multiple comparison test at the 95% confidence level in SPSS 23.0 (IBM, New York, NY, USA) program.

3. Results and Discussion

3.1. UHPLC-DAD-MS Analyses of Extracts

The UHPLC chromatograms of the extracts obtained after UAE with different extraction solvents, i.e., distilled water, aqueous 37% (v/v) ethanol, aqueous 38% (v/v) propylene glycol or aqueous 38% (v/v) glycerol (Table 2, experiment no. 1–4), are shown in Figure 1. The list of detected compounds is shown in

Table 3. List of detected compounds in extracts. Abbreviation meanings: UAE/water—extract produced after UAE with distilled water; UAE/aEtOH—extract produced after UAE with aqueous 37% (v/v) ethanol; UAE/aGLYC—extract produced after UAE with aqueous 38% (v/v) glycerol; UAE/aPPG—extract produced after UAE with aqueous 38% (v/v) propylene glycol. MB—MassBank record, https://massbank.eu/ (accessed on 13 August 2025).

Peak No.	tR, min	λmax, nm	Molecular Ion [M − H]− m/z	MS/MS Fragment Ions	UAE/ Water	UAE/ aEtOH	UAE/ aGLYC	UAE/ aPPG	Assignment	Reference
1	0.76	-	191	173, 129, 117, 85(100%), 73, 57	+	+	+	+	Quinic acid	[33]
2	0.78	-	195	129(100%)	+	+	+	+	-	-
3	0.88	-	133	115(100%), 87	+	+	+	+	Malic acid	Standard
4	0.95	-	191	173, 111(100%), 99	+	+	+	+	Citric acid	Standard
5	1.04	-	179	161, 143, 131, 119, 113, 89(100%)	+	+	+	+	Glucose	MB:KO000804
6	2.14	220 261 297	153	109(100%)	+	+	+	+	Protocatechuic acid	[34]
7	2.30	327 290sh	353	191(100%), 179, 173	+	+	+	+	Neochlorogenic acid	[35]
8	3.60	329 290sh	515	455, 425, 353(100%), 191, 179	+	-	-	-	Dicaffeoyllquinic acid, isomer	[35]
9	3.90	305 286sh	337	267, 249(100%), 163	+	+	+	+	3-O-p-cumaroyl-quinic acid	[35]
10	4.60	-	515	455, 425, 353(100%), 191	+	+	+	+	Dicaffeoyllquinic acid, isomer	[35]
11	5.00	325 303sh	353	191(100%)	-	+	-	-	Chlorogenic acid, cis isomer	[35]
12	5.10	325 303sh	353	191(100%)	+	+	+	+	Chlorogenic acid	Standard
13	5.40	278	577	-	+	+	+	+	Procyanidin B2	[36]
14	5.60	325 303sh	353	191(100%), 179, 173, 135	+	+	+	+	Cryptochlorogenic acid, isomer	[35]
15	6.45	313 296sh	337	191, 173(100%), 163	+	+	+	+	4-O-p-coumaroyl-quinic acid	[37]
16	6.73	-	563	517(100%)	+	+	+	+	Apigenin-penthosyl-hexoside	[38]
17	8.15	-	447	285(100%)	-	+	+	+	Kaempferol glucoside/galactoside	[39]
18	8.27	-	515	469, 435, 353(100%), 273, 167	-	+	-	-	Dicaffeoyllquinic acid, isomer	[35]
19	8.57	300	515	469(100%), 353	+	+	+	+	Dicaffeoyllquinic acid, isomer	[35]
20	8.69	258 356	463	301(100%)	+	+	+	+	Hyperoside	Standard
21	8.87	258 356	463	301(100%)	+	+	+	+	Isoquercitrin	Standard
22	8.86	258 355	609	-	+	+	+	+	Rutin	Standard
23	9.10	287	567	273(100%)	+	+	+	+	Phloretin pentosyl hexoside	[39]
24	9.53	354 258	433	301(100%)	+	+	+	+	Quercetin pentoside	[39]
25	9.65	287	435	273(100%)	+	+	+	+	Phloridzin	MB:BML0059328.4.2022
26	9.69	-	359	-	+	+	+	+	Rosmarinic acid	Standard
27	9.72	287	481	435(100%)	+	+	+	+	Derivative of phloridzin, tent.
28	9.90	258 352	447	301(100%)/300, 285/284	-	+	+	+	Quercitrin	[40]
29	10.88	257 372	301	-	+	+	+	+	Quercetin	Standard
30	11.49	268 317	593	447, 307, 285(100%)	+	+	+	+	Tiliroside	Standard
31	11.63	-	273	167, 123	-	+	+	+	Phloretin	[39]
32	12.00	-	285	-	-	+	-	-	Kaempferol	Standard

.

Produced extracts contained a variety of flavonoids, phenols, phenolic acids, natural organic acids, and their derivatives. Regarding UHPLC chromatograms of the extracts obtained under the identical conditions with different solvents (distilled water, 37% (v/v) ethanol, 38% (v/v) glycerol or 38% (v/v) propylene glycol), the most intense peaks correspond to natural organic acids (protocatechuic, chlorogenic, and 4-O-p-coumaroyl-quinic), quercetin derivatives (isoquercitrin and rutin), as well as phloridzin and its derivative. In fact, all of these extracts contained glucose, a variety of natural organic acids (quinic, malic, citric, protocatechuic, neochlorogenic, 3-O-p-cumaroyl-quinic, 4-O-p-coumaroyl-quinic, chlorogenic, and rosmarinic acids, two isomers of dicaffeoyllquinic acid and one cryptochlorogenic acid isomer), quercetin and its derivatives (isoquercitrin, rutin, quercetin pentoside, hyperoside), tannin procyanidin B2, flavone apigenin-penthosyl-hexoside, dihydrochalcone derivatives (phloretin pentosyl hexoside and phloridzin) and tiliroside, a kaempferol derivative and glycosidic flavonoid. Interestingly, the extract produced after UAE with distilled water contained one additional dicaffeoylquinic acid isomer but lacked another isomer, as well as the cis isomer of chlorogenic acid, quercitrin, phloretin, kaempferol, and kaempferol glucoside/galactoside. These compounds were found in the aqueous alcoholic extracts. It is known that under prolonged exposure to aqueous solutions phlorizin subjects to hydrolysis and provides phloretin and glucose [41,42].

Due to more compounds extracted after UAE with aqueous 38% (v/v) propylene glycol, plus the non-volatility and general safety of propylene glycol, together with its lower viscosity compared to glycerol [14,29], this system was chosen for further extraction experiments, using NADES-pretreated plant waste.

Figure 2 represents UHPLC-DAD and UHPLC-MS chromatograms for extracts produced after UAE with aqueous 38% (v/v) propylene glycol from waste apple dust pretreated with different NADESs (Table 2, experiment no. 5–8). The list of detected compounds in all extracts is shown in

Table 4. List of detected compounds in extracts produced after UAE with aqueous 38% (v/v) propylene glycol from waste apple dust pretreated with different NADESs: Honey-mimicking, Reline, Oxaline and Glyceline. MB—Mass bank record, https://massbank.eu/.

Peak No.	tR, min	λmax, nm	Molecular Ion [M − H]− m/z	MS/MS Fragment Ions	NADES Used for Pretreatment				Assignment	Reference
					Honey	Reline	Oxaline	Glyceline
1	0.76	-	191	173, 129, 117, 85(100%), 73, 57	+	-	-	-	Quinic acid	[33]
2	0.88	-	133	115(100%), 87	+	+	+	+	Malic acid	Standard
3	0.95	-	191	173, 111(100%), 99	+	+	+	+	Citric acid	Standard
4	1.04	-	179	161, 143, 131, 119, 113, 89(100%)	+	-	-	-	Hexose (glucose or galactose)	MB:KO000804
5	1.09	-	147	129(100%), 115, 87, 75	-	+	+	+	Citramalic acid	PubChem:1081
6	1.25	-	191	173, 115(100%), 99, 71	-	+	+	+	n.i.
7	1.55	-	179	161, 143(100%), 131, 119, 113, 89	+	+	-	-	Hexose (glucose or galactose	MB:KO000804
8	1.60	285	249	173, 111(100%)	+	-	+	+	n.i. citric or quinic acid derivative
9	1.71	-	345	309, 266, 234, 192(100%)	-	+	-	-	n.i.
10	2.20	220 261 297	153	109(100%)	+	+	+	+	Protocatechuic acid	[34]
11	2.46	327 290sh	353	191(100%), 179, 173	+	+	+	+	Neochlorogenic acid	[35]
12	3.14	270	219	111(100%)	-	-	+	-	Ethyl citrate	[41]
13	3.90	329 290sh	515	455, 425, 353(100%), 191, 179	+	+	-	+	Dicaffeoyllquinic acid, isomer	[35]
14	4.10	305 286sh	337	267, 249(100%), 163	+	+	+	+	3-O-p-cumaroyl-quinic acid	[35]
15	4.90	-	515	455, 425, 353(100%), 191	+	+	+	+	Dicaffeoyllquinic acid, isomer	[35]
16	5.00	325 303sh	353	191(100%)	+	+	+	+	Chlorogenic acid, cis isomer	[35]
17	5.27	325 303sh	353	191(100%)	+	+	+	+	Chlorogenic acid	Standard
18	5.51	278	577	-	+	+	-	+	Procyanidin B2	[36]
19	5.70	325 303sh	353	191, 179, 173(100%), 135	+	+	+	+	Cryptochlorogenic acid	[35]
20	6.55	313 296sh	337	191, 173(100%), 163	+	+	+	+	4-O-p-coumaroyl-quinic acid	[37]
21	7.33	328 297	411	353, 191(100%), 179, 173, 161, 135	-	+	+	-	Chlorogenic acid derivative	[35]
22	7.40	-	483	437(100%), 305	-	+	-	+	n.i.
23	8.13	-	619	583(100%), 289	-	+	+	+	n.i.
24	8.37	-	447	285(100%)	+	+	+	+	Kaempferol glucoside/galactoside	[39]
25	8.67	-	515	469, 435, 353(100%), 273, 167	+	+	+	+	Dicaffeoyllquinic acid, isomer	[35]
26	8.74	327 302	381	191, 179(100%), 173, 161, 135	-	-	+	-	Quinic acid derivative	[35]
27	8.86	258 356	463	301(100%)	+	+	+	+	Hyperoside	Standard
28	8.90	258 356	463	301(100%)	+	+	+	+	Isoquercitrin	Standard
29	9.09	258 355	609	-	+	+	+	+	Rutin	Standard
30	9.21	287	567	273(100%)	+	+	+	+	Phloretin pentosylhexoside	[39]
31	9.23	354 258	433	301(100%)	+	+	+	+	Quercetin pentoside	[39]
32	9.68	354 258	433	301(100%)	+	+	-	+	Quercetin pentoside	[39]
33	9.81	287	435	273(100%)	+	+	+	+	Phloridzin	MB:BML0059328.4.2022
34	9.82	287	481	435(100%)	+	+	+	+	Derivative of phloridzin, tent.
35	9.89	-	359	-	+	+	+	+	Rosmarinic acid	Standard
36	9.90	258 352	447	301(100%)/300, 285/284	+	+	+	+	Quercitrin	[40]
37	11.08	257 372	301	179(100%), 175, 165, 151	+	+	+	+	Quercetin	Standard
38	11.58	268 317	593	447, 307, 285(100%)	+	+	+	+	Tiliroside	Standard
39	11.72	-	273	167, 123	+	+	+	+	Phloretin	[39]
40	12.00	-	285	-	-	-	+	-	Kaempferol	Standard

.

All extracts produced after UAE with aqueous 38% (v/v) propylene glycol from plant material pretreated with different NADESs contained a variety of natural organic acids (malic, citric, protocatechuic, neochlorogenic, 3-O-p-cumaroyl-quinic, 4-O-p-coumaroyl-quinic, chlorogenic, cryptochlorogenic, and rosmarinic acids: two isomers of dicaffeoyllquinic acid and one chlorogenic acid cis isomer), quercetin and its derivatives (isoquercitrin, rutin, quercetin pentoside, quercitrin, hyperoside), dihydrochalcone derivatives (phloretin pentosyl hexoside, phloretin, and phloridzin), kaempferol glucoside/galactoside and its glycosidic flavonoid tiliroside. Only extracts produced after UAE with aqueous 38% (v/v) propylene glycol from plant material pretreated with Honey-mimicking and Reline NADESs contained hexoses (glucose or galactose). Only extract produced after UAE with aqueous 38% (v/v) propylene glycol from plant material pretreated with Honey-mimicking NADES contained quinic acid yet lacked citramalic acid found in other extracts. After Reline and Oxaline NADESs pretreatment of plant waste, the obtained UAE extracts contained more chlorogenic, dicaffeoyllquinic, and quinic acid derivatives. Only extract produced after UAE with aqueous 38% (v/v) propylene glycol from plant material pretreated with Oxaline NADES contained kaempferol and ethyl citrate, yet it lacked procyanidin B2 and quercetin pentoside, found in other extracts produced from NADES-pretreated plant material.

Protocatechuic acid and its derivatives exhibited potent biological activities (antibacterial, antiviral, anti-inflammatory, anticancer, antiulcer, antiaging, antifibrotic, anti-sclerotic, and hepatoprotective) [43]. Chlorogenic acid, a water-soluble compound derived from caffeic and quinic acids, and its isomers expressed numerous properties. According to several animal and cell-based studies, these compounds have demonstrated beneficial impacts due to exhibited potent antioxidant, antibacterial, antitumor, anti-inflammatory, antidyslipidemic, antihypertensive, antidiabetic, and neuroprotective effects [44,45]. Quinic acid expressed antiviral, anti-neuroinflammatory, antioxidative, and radioprotective activities [46,47]. 4-O-p-coumaroyl-quinic acid, a quinic acid derivative, exhibited potent antifungal effects, suggesting potential antifungal drug development [48]. Isoquercitrin exhibits anti-inflammatory, antidiabetic, neuroprotective, atheroprotective, anticancer, and antioxidant effects [49,50]. Rutin (sophorin or rutoside) exhibits anti-inflammatory, antiapoptotic, antidepressant, antioxidant, anticancer, antidiabetic, cognition-enhancing, cardiovascular, and neuroprotective properties [51,52]. Phloretin and its derivatives (phlorizin and phloretin pentosylhexoside) exhibit antioxidant, anti-inflammatory, antidiabetic, antiproliferative, antimetastatic, and antiangiogenic activities [53,54].

Citramalic acid, a promising wrinkle-reducing agent, is rarely found in plants [55], but was identified in UAE extracts prepared from waste material pretreated with Reline, Oxaline, and Glyceline NADESs. Ethyl citrate, a flavoring agent, surface-active agent in foods, and material for the production of pharmaceutical coatings and plastics, was only detected in UAE extract from waste material pretreated with Oxaline. Cryptochlorogenic (4-caffeoylquinic) acid was only detected in UAE extracts from NADES-pretreated material. This natural phenolic acid demonstrated anti-inflammatory, antioxidant, and cardioprotective effects [56].

3.2. TPC, TFC, and Antioxidant Activity in Waste Wild Apple UAE

One-way ANOVA revealed statistically significant differences in both TPC (F(7) = 441230, p < 0.001) and TFC (F(7) = 113208.493, p < 0.001) values according to the type of extract. Pairwise comparisons of the means using Tukey HSD revealed significant differences between TPC and TFC values of the produced extracts (

Table 5. TPC, TFC and antioxidant activity of different UAE/aqueous extracts.

Extract from Experiment No.	NADES Used for Pretreatment	Extracting Solvent	TPC (mg GAE/g DAP)	TFC (mg QE/g DAP)	TFC/TPC Ratio (%)	DPPH (IC50 µg/g DAP)	ABTS (IC50 µg/g DAP)
1	None	Distilled water	161.615 ± 0.385 a	35.528 ± 0.735 a	0.22 ± 0.004 c	291.067 ± 9.025 g	596.251 ± 24.372 f
2	None	Aqueous 37% (v/v) ethanol	292.641 ± 0.588 c	37.009 ± 0.321 a	0.1265 ± 0.00084 a	142.301 ± 3.564 d,e	258.491 ± 8.392 e
3	None	Aqueous 38% (v/v) glycerol	290.974 ± 0.588 c	54.324 ± 0.893 b	0.1867 ± 0.0027 b	152.716 ± 4.07 e,f	216.734 ± 8.024 d
4	None	Aqueous 38% (v/v) propylene glycol	252.513 ± 0.222 b	62.657 ± 0.160 c	0.2481 ± 0.00042 d	154.682 ± 4.006 e,f	188.134 ± 5.859 d
5	Honey	Aqueous 38% (v/v) propylene glycol	802.922 ± 1.444 d	243.285 ± 1.352 d	0.303 ± 0.00114 e	171.420 ± 14.915 f	276.536 ± 21.912 e
6	Reline	Aqueous 38% (v/v) propylene glycol	975.655 ± 1.426 g	590.271 ± 1.864 g	0.605 ± 0.001 h	27.915 ± 1.429 b	33.824 ± 3.362 a,b
7	Oxaline	Aqueous 38% (v/v) propylene glycol	894.421 ± 0.792 f	451.682 ± 0.723 f	0.505 ± 0.00036 g	49.861 ± 3.742 c	53.834 ± 4.213 b
8	Glyceline	Aqueous 38% (v/v) propylene glycol	824.463 ± 0.815 e	324.838 ± 1.567 e	0.394 ± 0.0015 f	131.412 ± 9.845 d	137.627 ± 11.963 c
Ascorbic acid						5.409 ± 0.576 a	5.502 ± 0.626 a
Trolox						6.702 ± 0.689 a	8.310 ± 0.876 a

TPC—Total phenolic content; TFC—Total flavonoid content; GAE—gallic acid equivalent; QE—quercetin equivalent; DAP—dry apple powder. Row values (different small letters) are significantly different at the 95% confidence level by Tukey’s multiple range test.

).

It is well known that herbal drugs contain a complex mixture of different primary and secondary metabolites. Additionally, the importance of ecology and environmental protection is growing. In this study, we sought to integrate the rational use of all parts of herbal materials with environmentally friendly solvents, extraction methods, and pretreatment strategies. The results clearly demonstrate that wild apple fruit dust represents a promising source of phenolic and flavonoid compounds when subjected to appropriate pretreatment and UAE.

The absence of the NADES-assisted pretreatment step resulted in a lower degree of polyphenolic extraction. Both aqueous ethanol and glycerol were demonstrated to be the most adequate extracting media with similar extraction capacity (extracting 1.8 times more polyphenolics than pure water), followed by aqueous propylene glycol (1.56 times more extracted polyphenolics than water). However, aqueous propylene glycol enabled extracting the highest amount of flavonoids, as seen by significant differences in TFC. These findings are expected, as polyphenolic flavonoids exhibit greater solubility in polar solvents compared to water. Since aqueous propylene glycol provided roughly 1.7 times higher flavonoid content than pure water, this system was chosen as the extraction medium for further UAE experiments.

After the application of different pretreatments, the total amount of phenolic and flavonoid compounds increases 3–4 times, with the Reline pretreatment proving to be the best. Quantitatively, TPC ranged from 161.615 ± 0.385 mg GAE/g DAP (control, water extraction without pretreatment) to 975.655 ± 1.426 mg GAE/g DAP following Reline pretreatment, representing an approximately six-fold enhancement. Similarly, TFC demonstrated a marked increase, from 35.528 ± 0.735 mg QE/g DAP in the control to a maximum of 590.271 ± 1.864 mg QE/g DAP with Reline, highlighting the exceptional efficiency of this pretreatment. Oxaline and Glyceline pretreatments also yielded substantial increases in both TPC and TFC, achieving 894.421 ± 0.792 and 824.463 ± 0.815 mg GAE/g DAP for TPC, and 451.682 ± 0.723 and 324.838 ± 1.567 mg QE/g DAP for TFC, respectively. These findings position Reline as the most potent enhancer of polyphenol and flavonoid extraction, followed by Oxaline and Glyceline. Notably, the TFC/TPC ratio was highest in the Reline-treated sample (0.605%), suggesting a more favorable extraction of flavonoids relative to total phenolics.

According to DPPH and ABTS assays, the antioxidant activity was determined, with the IC₅₀ value being monitored. As the IC₅₀ value increases, the antioxidant activity decreases. NADES-pretreated samples demonstrated overall better antioxidant activity (Table 5). Antioxidant capacity showed a pronounced correlation with phenolic content. The IC₅₀ values for DPPH dropped significantly from 291.067 ± 9.025 µg/g DAP (control, water) to just 27.915 ± 1.429 µg/g DAP with Reline pretreatment, reflecting a more than tenfold increase in activity. ABTS assay results paralleled this trend, with IC₅₀ values improving from 596.251 ± 24.372 to 33.824 ± 3.362 µg/g DAP. While Oxaline and Glyceline also significantly enhanced antioxidant activity (DPPH IC₅₀: 49.861 ± 3.742 and 131.412 ± 9.845 µg/g DAP; ABTS IC₅₀: 53.834 ± 4.213 and 137.627 ± 11.963 µg/g DAP, respectively), Reline consistently outperformed all other treatments. The use of Honey-mimicking NADES for the pretreatment of the waste material also showed beneficial effects, increasing TPC and TFC to 802.922 ± 1.444 and 243.285 ± 1.352 mg/g DAP, respectively, although the antioxidant activity (DPPH IC₅₀: 171.420 ± 14.915 µg/g) was moderate compared to NADES-based pretreatments. Control samples extracted with conventional solvents such as ethanol (292.641 ± 0.588 mg GAE/g DAP, 37.009 ± 0.321 mg QE/g DAP), glycerol (290.974 ± 0.588 and 54.324 ± 0.893), or propylene glycol (252.513 ± 0.222 and 62.657 ± 0.160), without pretreatment, resulted in significantly lower yields of bioactive compounds and weaker antioxidant capacities (e.g., DPPH IC₅₀ values between 142.301 and 154.682 µg/g DAP). Overall, these results underscore the pivotal role of appropriate pretreatments—particularly those involving NADESs—in maximizing the recovery of phenolic and flavonoid constituents from wild apple fruit dust and enhancing their antioxidant potential. Reline emerges as the most effective strategy, followed by Oxaline, Glyceline, and Honey-mimicking NADES, thereby providing a clear ranking of pretreatment efficacy in this context.

3.3. Differences in Extraction Efficiencies of Applied UAE/Aqueous Alcoholic Systems

In general, extraction of bioactive components from plant-derived feedstocks is considered a solid–liquid process. Two main factors that dictate the extraction efficiency are the nature (composition) of both the extracting compounds and the used solvent system [14]. Following Fick’s law, the diffusion process is driven by the concentration gradient. According to the Stokes–Einstein equation (Equation (5)),

D = k_B × T/6π × η × r_s

(5)

where D is diffusion, k_B is the Boltzmann’s constant, T is temperature, η is viscosity and r_s is the effective radius of the diffusing molecule; it is obvious that higher viscosity of the extracting solvent inhibits the diffusion rate, while heating improves the overall diffusion process of the solvent into the plant matrix. Moreover, the higher number of H-bonding interactions established between molecules of solvents and components targeted for extraction directly enhances the extraction [57]. The properties of the pure solvents are listed in

Table 6. The properties of the pure solvents.

Property/Solvent		Glycerol	Ethanol	Propylene Glycol	Water	Reference
Molar Mass, g·mol−1		92.09	46.07	76.09	18
ρ, g·cm−3	20 °C	1.2611	0.7893	1.0361	0.99821	[58]
	25 °C	1.257	0.787	1.033		[59]
η, mPa·s	20 °C		1.203		1.002	[58]
	25 °C	934	1.074	40.4	0.89	[58]
	50 °C	152	0.694	11.3	0.547	[58]
Partition coefficient (log P) a		–1.76	–0.31	-0.92		[59]
			–0.30			[58]
Dipole moment		2.56	1.69	2.25	1.8546	[58]
		4.21	1.69	3.63		[59]
Dielectric constant b		46.53	25.3	27.5	80.2	[58]
pKa a		14.15	15.5	14.8	14	[58]

^a At 25 °C; ^b At 20 °C.

.

At 25 °C, pure ethanol and water have similar dynamic viscosity values of 1.074 mPa⋅s and 0.89 mPa⋅s [58]. However, pure propylene glycol and glycerol are more viscous than water, with dynamic viscosity values of 40.4 mPa⋅s and 934 mPa⋅s, respectively [58]. As a result, when these alcohols are mixed with water, the overall mixture’s viscosity is expected to rise, thus making the diffusion process more difficult. Dynamic viscosity values for the aqueous alcoholic solutions are calculated using (Equation (6)):

μ_MIX = (x_A × μ_A + x_B × μ_B)⁻¹

(6)

where μ_MIX, μ_A, and μ_B are the viscosities of the aqueous alcoholic solutions and pure components A and B, while x_A and x_B are the molar fractions of the components. At 25 °C, calculated dynamic viscosity values for the aqueous 37 vol.% ethanol, 38 vol.% propylene glycol and 38 vol.% glycerol solutions are 0.9141 mPa⋅s, 1.0207 mPa⋅s and 1.0247mPa⋅s, which are 2.7%, 14.68% and 15.13% higher than those of pure water.

However, dielectric constant values of alcohols (25.3 at 20 °C for ethanol, 27.5 at 30 °C for propylene glycol, and 46.53 at 20 °C for glycerol) are lower than that of pure water (80.1 at 20 °C) [58,59]. Therefore, all alcohols will reduce the dielectric constant of pure water. As a result, aqueous alcoholic solutions will be less polar and more efficient for the extraction of polyphenolics from the plant matrices [60].

The observed hierarchy of efficacy among the tested NADES-based pretreatments (Reline > Oxaline > Glyceline > Honey-mimicking) can be attributed to differences in solvent composition, hydrogen-bonding dynamics, and physicochemical interactions with target phytochemicals.

Reline demonstrated superior extraction efficiency for phenolic and flavonoid compounds, likely due to its strong hydrogen-bond acceptor (HBA) capacity and the ability to disrupt plant cell wall matrices through urea’s nucleophilic properties [61]. Urea’s small molecular size and high polarity facilitate deeper penetration into biomass, enhancing solubilization of hydroxyl-rich phenolic compounds via synergistic interactions between the HBA (choline chloride) and hydrogen-bond donor (HBD; urea) [62,63]. This aligns with studies showing urea-containing DESs exhibit exceptional solvation capabilities for aromatic and polar metabolites due to their ability to form extensive hydrogen-bond networks with phenolic hydroxyl groups [64]. In contrast, Oxaline (composed of choline chloride and oxalic acid), while effective due to its acidic pH (favorable for protonating phenolic hydroxyl groups and improving solubility), may partially degrade labile antioxidants during extraction, thereby reducing its net efficacy compared to Reline [65]. Despite its biocompatibility, Glyceline suffers from higher viscosity, which limits mass transfer rates and access to intracellular metabolites [66]. Honey and its biomimetic deep eutectic solvent, though rich in natural sugars and weak organic acids, lack the tunable solvation power of DESs, as its heterogeneous composition results in non-specific interactions and lower extraction selectivity for target antioxidants [67,68].

The enhanced antioxidant activity observed with Reline-pretreated extracts correlates with its ability to preserve redox-active phenolic structures during extraction. Urea’s stabilizing effect prevents oxidative degradation of flavonoids, while choline chloride’s quaternary ammonium group facilitates π-π interactions with aromatic rings, optimizing electron transfer capacity in radical scavenging assays [61,64]. This is consistent with findings that Reline extracts exhibit higher tyrosinase inhibition and acetylcholinesterase modulation compared to traditional solvents, underscoring its role in maintaining bioactive conformations [61]. Furthermore, Reline’s weak basic (almost neutral) pH minimizes acid hydrolysis of glycosidic bonds in flavonoid conjugates, preserving aglycone structures critical for antioxidant potency [69]. While Oxaline’s acidity enhances initial phenolic liberation, it may also hydrolyze esterified antioxidants (e.g., chlorogenic acid derivatives), reducing measurable activity—a trade-off noted in DESs with extreme pH profiles [65]. These mechanistic insights align with prior reports that DESs with balanced HBA/HBD ratios and moderate polarity maximize both extraction yield and bioactivity retention, positioning Reline as an optimal solvent for pretreatments targeting redox-active phytochemicals [62,70].

3.4. Evaluation and Comparison of the Proposed UAE Methodologies

In order to classify a newly designed methodology suitable to be proposed for scaling up on an industrial level, it is necessary to establish and strictly evaluate both economic and environmental specific factors [14]. The parameters regarding the energy demands and environmental impact of the designed UAE methodologies are listed in

Table 7. Comparison of the proposed UAE methodologies.

Experiment No.	1	2	3	4	5	6	7	8
Extracting solvent	Distilled water	Aqueous 37% (v/v) ethanol	Aqueous 38% (v/v) glycerol	Aqueous 38% (v/v) propylene glycol	Aqueous 38% (v/v) propylene glycol	Aqueous 38% (v/v) propylene glycol	Aqueous 38% (v/v) propylene glycol	Aqueous 38% (v/v) propylene glycol
NADES used for pretreatment	None	None	None	None	Honey	Reline	Oxaline	Glyceline
Mass of water (g) per g p.m.	15	9.43	9.28	9.28	9.28	9.28	9.28	9.28
Mass of organic solvent (g) per g p.m.	0	4.38	7.19	5.91	5.91	5.91	5.91	5.91
Mass of solvent system (g) per g p.m.	15	13.81	16.47	15.19	15.19	15.19	15.19	15.19
Specific heat capacity (kW·h/kg·K)	0.0150	0.0163	0.0137	0.0148	0.0148	0.0148	0.0148	0.0148
TPC (mg GAE/g DE)	161.615	292.641	290.974	252.513	802.922	975.655	894.421	824.463
TPC per kW·h (mg GAE/g DE/kW·h)	2154.87	3901.88	3879.65	3366.84	10705.63	13008.73	11925.61	10992.84
TPC per kW·h/K (mg GAE/g DE/kW·h/K)	0.485	0.878	0.873	0.758	2.41	2.93	2.685	2.475
TPC per kW·h·K (mg GAE/g DE/kW·h·K)	53,842.04	97,493.35	96,937.99	84,124.71	267,493.46	325,039.46	297,976.36	274,669.85
TFC (mg QE/g DE)	35.528	37.009	54.324	62.657	243.285	590.271	451.682	324.838
TFC per kW·h (mg QE/g DE/kW·h)	473.71	493.45	724.32	835.43	3243.8	7870.28	6022.43	4331.17
TFC per kW·h/K (mg QE/g DE/kW·h/K)	0.107	0.111	0.163	0.188	0.73	1.77	1.36	0.98
TFC per kW·h·K (mg QE/g DE/kW·h·K)	11,836.15	12,329.5	18,098.04	20,874.18	81,050.4	196,648.8	150,477.9	108,219.78

.

Due to unchanged operating conditions (effective electric power 150 W, extraction temperature 60 °C, time consumption 30 min), thermal conductance (0.000450248 kW/K), electricity consumption (0.075 kW·h), heat capacity (0.000225124 kW·h/K) and mass of emitted CO₂ (60 g) were identical for all UAE methodologies.

Choosing distilled instead of deionized water contributes to the reduced cost.

All chosen alcohols (ethanol, propylene glycol, and glycerol) are known as available solvents, applied in different products for human consumption [14,29]. Moreover, these solvents do not require being separated from the solid material, further providing energetic savings after the scaling-up of the process.

The choice of the most appropriate alcohol must be related to the extraction efficiency. From Table 7, it is clear that the NADES-associated pretreatment enhanced the extraction, thus providing higher TPC and TFC values.

Although the ethanolic solution was the most efficient, its use for the production of pharmaceuticals, cosmetics, and food products can be problematic due to several reasons (evaporation and flammability, contact irritation, and forbidden consumption due to medical conditions or religious beliefs).

Since the UAE system with aqueous 38% (v/v) propylene glycol is more recommended for future use, the choice of the most appropriate NADES is the next step. Based on the provided TPC and TFC values, as well as demonstrated antioxidative activities of the extracts, Reline is the most effective NADES-based choice in this regard.

4. Conclusions

NADESs were tested as pretreatment media for waste wild apple fruit dust (filter tea factory by-product). Pretreatments significantly enhanced the UAE of flavonoids and phenols, as well as the antioxidant activity of extracts obtained from wild apple waste, with ChCl-based NADESs being more efficient pretreatment media than honey-mimicking NADES. Quantitatively, significant increases in both TPC and TFC values were demonstrated; from 161.6 mg GAE/g DAP and 35.53 mg QE/g DAP (control, UAE using water as solvent and crude plant waste dust) to 975.6 mg GAE/g DAP and 590.271 ± 1.864 mg QE/g DAP (UAE using aqueous 38% (v/v) propylene glycol as solvent and Reline-pretreated plant waste dust). Therefore, a UAE system combining Reline-pretreatment of plant waste with aqueous propylene glycol as the extraction solvent is recommended for optimization and further studies. By utilizing wild apple waste as a source of highly valuable bioactive compounds, this study also points to opportunities for innovation in the production of food, pharmaceutical, and cosmetic products and promoting circular economy practices within the tea processing sector.