Separation, Purification, Component Identification, and Bioactivity Analysis of Polyphenols from Aronia melanocarpa

Abstract

Aronia melanocarpa (black chokeberry) is rich in bioactive compounds. In this study, polyphenols from A. melanocarpa were extracted using both ultrasound-assisted deep eutectic solvent (DES) and conventional organic solvents (80% methanol and 60% ethanol). The crude extracts were purified using AB-8 macroporous resin. The purified fractions were analyzed by ultrahigh performance liquid chromatographyquadrupole time-of-flighttandem mass spectrometry (UPLC-QTOF-MS/MS), identifying and quantifying 49 polyphenolic compounds, including 16 anthocyanins, 3 procyanidins, 20 flavonoids, and 10 phenolic acids. The DES extract exhibited the highest total polyphenol content (84.56 ± 3.85 mg/g), significantly surpassing the conventional extracts. Antioxidant activities were evaluated in vitro by assessing the scavenging capacities against hydroxyl, DPPH, and ABTS⁺ radicals. The DES extract demonstrated superior antioxidant activity in all assays. Furthermore, the DES-extracted A. melanocarpa polyphenols (AMP) and a blueberry polyphenol (BP) extract underwent in vitro simulated digestion. Postdigestion, the AMP extract showed significantly stronger inhibitory effects on α-glucosidase and pancreatic lipase activities compared to BP, indicating its greater potential for hypoglycemic and hypolipidemic applications. Results demonstrate that ultrasound-assisted DES extraction is a highly efficient and green method for obtaining polyphenol-rich extracts from A. melanocarpa with enhanced bioactivity, providing a scientific basis for its utilization in functional foods.

1. Introduction

Aronia melanocarpa (Michx. Elliott), commonly known as black chokeberry, is a perennial deciduous shrub in the Rosaceae family. It is native to eastern North America and widely cultivated in Europe and the former Soviet Union. Black chokeberry has emerged as an important economic crop worldwide. It contains abundant phytochemicals, including anthocyanins, proanthocyanidins, flavonoids, polysaccharides, and organic acids. − These bioactive compounds have multiple health benefits; they have antioxidant properties, regulate immune function, protect against cardiovascular and cerebrovascular disorders, have a hepatorenal protective effect, are anti-inflammatory, and have potential anticancer properties. , Among the phytochemicals in A. melanocarpa, polyphenols are core functional components. The synergistic relationship between polyphenol content and biological activity is a current research focus.

Isolating and purifying bioactive compounds from crude plant extracts is essential for further research and application. Conventional extraction methods for plant polyphenols, such as those organic solvents (e.g., methanol, ethanol, acetone), are well-established. However, they often face limitations including prolonged extraction times, relatively low selectivity and yield for certain polar polyphenols, high solvent consumption, and environmental and safety concerns due to solvent volatility and toxicity. , Recent advancements have therefore focused on developing greener and more efficient extraction techniques. Among these, deep eutectic solvent (DES) has emerged as promising alternatives. DES are typically formed by mixing a hydrogen bond donor and a hydrogen bond acceptor, causing mixtures with low volatility, low toxicity, high biodegradability, and tunable physicochemical properties. The tailored composition of DES allows for enhanced interactions with target polyphenols, potentially leading to higher extraction efficiencies and selectivity compared to conventional solvents. Specifically, ultrasound-assisted DES extraction synergistically combines the advantages of DES with the mechanical effects of ultrasound, which disrupts cell walls and improves mass transfer, further boosting extraction yield and reducing processing time. Given these superior attributesefficiency, environmental friendliness, and the potential to better preserve bioactive compoundswe selected ultrasound-assisted DES extraction as the primary method in this study to comprehensively evaluate its performance against traditional organic solvents for obtaining A. melanocarpa polyphenols (AMP).

Recent studies have concentrated on optimizing extraction and purification methods to enhance polyphenol yield and purity. , Ultrasound-assisted DES extraction has been successfully used to extract polyphenols from a variety of materials, including pomegranate seeds, bayberry, apple pomace, and grapes. Adsorption techniques using a range of materials are effective for enriching polyphenols. Among such materials, macroporous resins have been widely adopted in natural product purification because of their adsorption and separation properties. These resins offer notable advantages, including high enrichment efficiency, mild desorption conditions, strong resistance to interference from inorganic compounds, and reusability. Therefore, they are commonly used for the purification of functional components and represent the most frequently used approach for isolating and purifying AMP. For example, Lin et al. used ultrasound-microwave-assisted neutral aqueous DES extraction to obtain anthocyanins from A. melanocarpa, achieving an extraction efficiency of 97.05 ± 0.85% after purification with AB-8 macroporous resin. Similarly, Gao et al. demonstrated that the use of XAD-7 macroporous resin significantly enhanced the purity of AMP from 11.62 to 64.37%. Santos-Martín et al. purified A. melanocarpa pomace extract using an acrylic resin adsorption–desorption method, achieving a 96% anthocyanin recovery rate in the final extract.

Polyphenols are often complexed with other components in plant cells. Therefore, it is important to elucidate the polyphenol components bound to dietary fiber. This study focused on the separation and purification of AMP obtained through microwave-assisted DES extraction. The purified products were identified and quantified using ultrahigh performance liquid chromatography quadrupole time-of-flight – tandem mass spectrometry (UPLC–QTOF–MS/MS). The ability of the AMP extract and a similar extract from blueberry to inhibit α-glucosidase and pancreatic lipase was evaluated, and their in vitro antioxidant activity was assessed through chemical assays. The results of these analyses provided experimental evidence for the bioactive properties of AMP.

2. Materials and Methods

2.1. Plant Material

Fresh Aronia melanocarpa (black chokeberry) fruits were collected in October 2024 from Jiangsu Traditional Chinese Medicine Science and Technology Park (on campus training base), Taizhou, Jiangsu Province, China. The plant material was identified by the Prof. Chengzhong Li at the School of Modern Agriculture, Jiangsu Agri-animal Husbandry Vocational College. A voucher specimen (AM2024063) has been deposited at the campus herbarium. The samples were cleaned, freeze-dried prior to extraction. Fresh blueberries were purchased from local supermarket in Taizhou City for comparative analysis.

2.2. Reagents and Instruments

α-Amylase (porcine pancreas) and α-glucosidase were purchased from the Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Pepsin, trypsin, pancreatic lipase, bile salt, folinic acid, p-nitrophenyl-α-d-glucopyranoside (PNPG), the hydroxyl radical assay kit, the DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS⁺ (2,2′-azino-bis­(3-ethylbenzothiazoline-6-sulfonic acid)) radical assay kits were obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). The macroporous resins (AB-8, D101, DM-130, and NKA-9) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Acetonitrile, formic acid, and salicylic acid were supplied by Sigma-Aldrich (St Louis, MO).

2.3. Extraction of AMP

Portions of A. melanocarpa powder (10 g) were mixed with DES, 80% methanol, or 60% ethanol at a solid-to-liquid ratio of 1:40 (w/v). The DES consisted of choline chloride and lactic acid at a molar ratio of 1:3, containing 80% (v/v) water. This specific DES formulation was selected based on its demonstrated high efficiency in extracting polyphenolic compounds, attributed to its strong hydrogen-bonding capacity and suitable polarity, which promote effective solvation of target polyphenols. Furthermore, the addition of 80% water was intended to reduce viscosity, enhance mass transfer, and improve the contact between the solvent and plant matrix. ,

Extraction was performed under ultrasound-assisted conditions at a controlled temperature of 40 °C for 40 min, with an ultrasonic power of 240 W. The temperature was maintained to prevent thermal degradation of thermolabile polyphenols while promoting solubility and diffusion. The ultrasound power and duration were optimized to ensure effective cell wall disruption and sufficient mass transfer without causing excessive degradation of bioactive compounds. The solid-to-liquid ratio of 1:40 was chosen to ensure adequate solvent volume for complete solute transfer while maintaining extraction efficiency. The extract was then centrifuged at 5000g and 4 °C for 10 min, and the supernatant was filtered through a 0.45 μm organic membrane filter before further use.

2.4. Determination of Polyphenol Content

The polyphenol content was measured using the modified Folin–Ciocalteu method. A calibration curve was prepared using gallic acid (0.002–0.01 mg/mL). The polyphenol content, as determined from the regression equation of the calibration curve (y = 14.355x + 0.0561, R ² = 0.9993), is expressed as mg gallic acid. In this method, 2 mL AMP was mixed with 0.5 mL Folin – Ciocalteu phenol reagent. Then, 1.5 mL 10% sodium carbonate solution was added, and the mixture was shaken thoroughly before completing the volume to 10 mL with water. After incubation for 2 h, the absorbance of the mixture was measured at 765 nm using a UV–visible spectrophotometer (BIOMATE3S, Thermo Scientific, Madison, WI).

2.5. Screening of Macroporous Resins

Four types of macroporous resins (AB-8, D101, DM-130, and NKA-9) were pretreated as follows: First, each resin was soaked in absolute ethanol at room temperature for 24 h, then 5% v/v HCl solution was added and the mixture was kept at room temperature for a further 12 h. Each resin was then washed with distilled water until the pH returned to neutral, and then soaked in 5% w/v NaOH solution for 12 h before washing again with distilled water. Finally, 2 g of each pretreated macroporous resin was weighed and combined with 40 mL AMP solution. The adsorption conditions were 20 °C with shaking at 160 r/min in the dark for 24 h. The adsorption capacity and AMP adsorption rate were calculated using formulas () and (). After adsorption, the AMP solution was removed, and each adsorption-saturated macroporous resin was washed thoroughly with 70% v/v ethanol until no AMP solution remained on the surface. The desorption conditions were 30 °C with shaking at 120 r/min in the dark for 24 h. The desorption capacity and AMP desorption rate were calculated using formulas () and ().

$$\mathrm{a}\mathrm{dsorption}\mathrm{capacity}(\mathrm{mg}/\mathrm{g})=\frac{(C_0-C_1)\times V_0}{W}$$ 1

$$\mathrm{a}\mathrm{dsorption}\mathrm{rate}(\%)=\frac{C_0-C_1}{C_0}\times 100$$ 2

$$\mathrm{d}\mathrm{esorption}\mathrm{capacity}(\mathrm{mg}/\mathrm{g})=\frac{C_2\times V_1}{W}$$ 3

$$\mathrm{d}\mathrm{esorption}\mathrm{rate}(\%)=\frac{C_2}{C_0-C_1}\times 100$$ 4

Where C ₀, C ₁, and C ₂ represent the initial concentration of AMP, the AMP concentration after adsorption, and the AMP concentration after desorption (mg/mL), respectively; V ₀ and V ₁ represent the volume before and after adsorption of AMP (mL), respectively; and W represents the dry weight of the resin (g).

2.6. Separation and Purification of AMP Using Macroporous Resin

The crude AMP extracts obtained from DES extraction and conventional organic solvent extraction (80% methanol and 60% ethanol) were purified using AB-8 macroporous resin by column chromatography. ,− Briefly, the activated macroporous resin was mixed with the crude AMP extract and incubated at 25 °C with constant shaking (160 r/min) for 12 h to achieve complete adsorption. The resin was then packed into the column at half of the bed volume (1/2 BV). Next, three bed volumes (BV) of ultrapure water were added to remove choline chloride, lactic acid, proteins, sugars, and other polar compounds. Polyphenols were eluted with two BV of 70% v/v methanol acidified with 0.1% v/v formic acid to obtain the crude polyphenol solution. The methanol was removed by low-pressure rotary evaporation at 45 °C. The resulting concentrate was freeze-dried to obtain purified polyphenol powder, which was stored at −20 °C in the dark until further analysis. The DES-extracted blueberry polyphenols (BP) were purified following the same protocol.

2.7. In Vitro Antioxidant Activity

2.7.1. ABTS⁺ Radical Scavenging Activity

The ABTS⁺ radical scavenging activity was evaluated using a kit. In brief, 7 μL AMP/BP solution and 280 μL ABTS working solution were mixed and then allowed to react at room temperature for 6 min. The absorbance of the mixture was determined at 734 nm. The ABTS⁺ radical scavenging rate was estimated using formula , as follows:

$$\mathrm{r}\mathrm{adical}\mathrm{scavenging}\mathrm{rate}(\%)=\frac{1-(A_x-A_y)}{A_0}\times 100\%$$ 5

Where A _x is the absorbance of the reaction mixture, A _y is the absorbance of the control (without working solution), and A ₀ is the absorbance of the blank (with anhydrous ethanol instead of sample).

2.7.2. Hydroxyl Radical Scavenging Rate

The hydroxyl radical scavenging activity of the extracts was determined using a kit, which employed a colorimetric method. The 0.03% v/v H₂O₂ standard solution, Fe²⁺ substrate solution, salicylic acid substrate solution, and Griess chromogenic agent were prepared according to the kit’s instructions. All these reagents were prewarmed at 37 °C for 3 min. Then, a reaction mixture was prepared by mixing 0.2 mL AMP solution, 0.2 mL Fe²⁺ substrate solution, and 0.4 mL salicylic acid substrate solution. The mixture was incubated at 37 °C for 1 min, and then the chromogen was added to terminate the reaction. The absorbance of the mixture was measured at 517 nm. The hydroxyl radical scavenging rate was calculated using formula , as follows:

$$\mathrm{h}\mathrm{ydroxyl}\mathrm{radical}\mathrm{scavenging}(\%)=(1-\frac{A_y-A_x}{A_c{A}_0})\times 100\%$$ 6

Where A _y is the absorbance of the control group (without Fe²⁺ substrate solution), and A _x is the absorbance of the test group (with AMP extract but no H₂O₂ solution), A_c is the absorbance of the test group (AMP with H₂O₂ solution), and A ₀ is the absorbance of the blank (with ethanol instead of the AMP extract).

2.7.3. DPPH Radical Scavenging Rate

The DPPH radical scavenging rate was evaluated using a commercial kit. In brief, 400 μL AMP solution and 600 μL DPPH working solution were mixed, the mixture was incubated at 25 °C for 30 min in the dark, centrifuged for 5 min at 4000 r/min, and then the absorbance at 517 nm was determined. The DPPH radical scavenging rate was estimated using formula .

2.8. UPLC–QTOF–MS/MS Analysis of AMP

2.8.1. Qualitative Analysis of AMP by UPLC–QTOF–MS/MS

Chromatographic separation was performed on a Waters UPLC system equipped with an ACQUITY BEH C18 column (2.1 mm × 100 mm × 1.7 μm) maintained at 30 °C. The mobile phase consisted of (A) 0.1% formic acid in water and (B) acetonitrile, with a flow rate of 0.4 mL/min. The gradient program was: 5% B (0–0.01 min), 5–50% B (0.01–6.01 min), 50–95% B (6.01–18.01 min), 95% B (18.01–21.01 min), 95–5% B (21.01–35.01 min), and 5% B (35.01–38.01 min). The injection volume was 1 μL.

Mass spectrometric detection was carried out on a XEVO G2-XS QTOF mass spectrometer (Waters) equipped with an electrospray ionization source. Data were acquired in both positive and negative ion modes to comprehensively capture different polyphenol classes. The optimal operating parameters were as follows: Capillary voltage of 2.5 kV (positive)/2.2 kV (negative); cone voltage of 40 V; ion source temperature of 120 °C; desolvation gas (N₂) temperature of 450 °C at a flow rate of 800 L/h; cone gas flow of 50 L/h. Full-scan data were collected over the m/z range of 50–1200 with a scan time of 0.2 s. For MS/MS analysis, data-dependent acquisition mode was used with a collision energy ramp of 10–40 eV to fragment the top 3 most intense ions in each scan.

Compound identification was based on the accurate mass measurement with an error of less than 5 ppm; interpretation of key fragment ions in the MS/MS spectra; the comparison of retention times and mass spectra with those of authentic standards when available (e.g., cyanidin-3-O-galactoside, procyanidin B1, chlorogenic acid, quercetin-3-O-rutinoside); and the matching against online databases (MassBank, PubChem) and published literature.

2.8.2. Quantitative Analysis of AMP by UPLC–QTOF–MS/MS

The instrumental, chromatographic, and mass spectrometric conditions were the same as those described in Section . Targeted quantification was performed using the ACQUITY UPLC H-Class System coupled with a tunable UV detector (Waters) with the external standard method, based on a database of 56 reference substances and polyphenol metabolites from MassLynx application software. Standard curves were established to quantify each component. The content of polyphenolic compounds was calculated using formula .

$$\mathrm{p}\mathrm{olyphenolic}\mathrm{content}(\mu \mathrm{g}/\mathrm{mL})=\frac{C\times V}{m\times {10}^3}$$ 7

where C represents the concentration of solution, V represents volume of methanol, and m is the weight of sample.

The method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy. The LOD and LOQ, defined as signal-to-noise ratios of 3 and 10, respectively, were in the ranges of 0.01–0.05 μg/mL and 0.03–0.15 μg/mL for the quantified compounds. Intraday and interday precision (expressed as relative standard deviation, RSD) were less than 5 and 8%, respectively. The average recovery rates for spiked samples ranged from 92 to 105%, confirming the accuracy of the method.

The content of each identified polyphenol in the AMP extracts was calculated based on its corresponding calibration curve. For compounds without a commercial standard, quantification was performed using the calibration curve of a structurally similar standard.

2.9. In Vitro Digestion Treatments

The in vitro digestion of AMP and BP extracts was performed following a well-established protocol with modifications. Briefly, the digestion process sequentially simulated oral, gastric, and intestinal phases using three distinct simulated fluids: (1) Simulated salivary fluid (SSF), pH 6.7: Composed of α-amylase (150 U/mL) in a KCl-based electrolyte solution to initiate starch breakdown. (2) Simulated gastric fluid (SGF), pH 2.0: Comprised of pepsin (2000 U/mL) in a NaCl-based electrolyte solution to simulate protein digestion under acidic conditions. (3) Simulated intestinal fluid (SIF), pH 7.6: Contained pancreatin (trypsin activity, 100 U/mL) and bile salts (10 mM) in a KH₂PO₄-based electrolyte solution to mimic the final digestion and micelle formation stage. , For the in vitro digestion experiments, the polyphenol extracts obtained via the ultrasound-assisted DES method were selected for both Aronia melanocarpa (AMP) and blueberry (BP). This selection was based on two primary considerations: (1) The DES extraction method yielded high polyphenol content and antioxidant activity (as demonstrated in Sections and 3.2), representing the most bioactive form of the extracts for subsequent bioactivity evaluation. (2) Using the extracts from the same (and superior) extraction method for both species ensures a fair and comparable assessment of their intrinsic hypoglycemic and hypolipidemic potentials after digestion, independent of variations introduced by different extraction efficiencies.

Specifically, a portion (0.1 g) of DES-extracted AMP or BP was mixed with 5 mL water, then 1 mL SSF was added. The mixture was oscillated at 36 °C and 120 r/min for 10 min, then immediately inactivated by boiling in a water bath for 3 min. After cooling to room temperature, the pH was adjusted to 2.0, and 7.5 mL SGF was added. The solution was oscillated at 36 °C and 120 r/min for 100 min, followed by enzyme inactivation in a boiling water bath for 3 min. The mixture was then centrifuged at 4 °C and 6500 r/min for 15 min, and the supernatant was collected to obtain the gastric digests of AMP and BP. After cooling to room temperature, the pH was adjusted to 7.60 and 7.5 mL SIF was added. The mixture was shaken again under the same conditions as the gastric digestion. The final supernatant collected after centrifugation was designated as the intestinal digest (ID) for subsequent analyses.

2.10. Evaluation of Hypoglycemic and Hypolipidemic Activity In Vitro

2.10.1. α-Glucosidase Inhibition Rate

The inhibition of α-glucosidase activity determined as described by Sun et al. with minor changes. Twenty μL of sample solution, 100 μL of phosphate-buffered saline (PBS; 0.1M, pH = 7.2), and 20 μL of α-glucosidase (0.25 U/mL) were mixed in the wells of a 96-well plate. The reaction mixture was incubated at 37 °C for 10 min and then p-nitrophenyl-β-d-glucopyranoside (pNPG) solution (2.5 mM, 20 μL, pH = 6.8) was added. The mixture was incubated at 37 °C for 10 min, and then Na₂CO₃ solution (0.2 M, 40 μL) was added to terminate the reaction. The absorbance at 405 nm was measured. The inhibition rate of α-glucosidase was calculated according to formula .

$$\mathrm{i}\mathrm{nhibition}\mathrm{rate}(\%)=\frac{A_m-(A_b-A_c)}{A_m}\times 100$$ 8

Where A _m, A _b, A _c represent the absorbance of the control group, the sample group, and the blank group, respectively.

2.10.2. Inhibition of Pancreatic Lipase Activity

The pancreatic lipase activity inhibition assay was conducted following an established protocol with slight modifications. First, pancreatic lipase was dissolved in Tris-HCl buffer (pH = 8.0) to prepare a 1000 U/mL enzyme solution. Subsequently, 50 μL of 1000 U/mL and 50 μL of gastrointestinal digestive solution preparation in Section were mixed. The mixture was incubated at 37 °C for 10 min. Subsequently, p-Nitrophenyl Phosphate (pNPP) substrate (2 mM) was added. The reaction mixture was incubated at 37 °C for 10 min, then the reaction was terminated by boiling for 5 min. After centrifugation, the supernatant was transferred to a 96-well plate and the absorbance was measured at 405 nm. The inhibition rate (%) was calculated using formula .

$$\mathrm{i}\mathrm{nhibition}\mathrm{rate}(\%)=\frac{1-(A_b-A_c)}{A_m}\times 100$$ 9

Where A _b, A _c, and A _m represent the absorbance of the control group (sample but no substrate), the sample group (all reagents), and the blank group (all reagents except the tested sample), respectively.

2.11. Statistical Analysis

Multiple comparisons were assessed via one-way or two-way ANOVA, followed by Tukey’s post hoc test. All data are means ± standard deviation obtained from three parallel experiments. Origin 2021 (OriginLab Corporation, Northampton, MA) and IBM SPSS 22.0 (SPSS Inc., Chicago, IL) were used to draw figures and conduct statistical analyses to detect significant differences. For comparisons among groups, the significance level was α = 0.05 (*P < 0.05 indicates statistically significant difference).

3. Results and Discussion

3.1. Screening of Macroporous Adsorption Resins

Different macroporous resins have different adsorption and desorption properties for natural products because of their specific characteristics, i.e., their polarity, specific pore size, and surface area. In this study, to select the most appropriate resin for enrichment of AMP, the adsorption and desorption performances of four resins were compared. The adsorption rate and adsorption capacity of these resins for AMP are shown in Figure . The results show that AB-8 resin demonstrated the highest adsorption rate (91.2%) and the highest desorption rate (82.32%). Among the four resins, AB-8 also showed the highest adsorption mass (0.67 g) and had the highest desorption capacity for AMP (0.61 g). The adsorption mass of AB-8 was significantly higher than that of the other resins (P < 0.05). Polyphenolic compounds are characterized by multiple hydroxyl groups and glycosidic bond structures and relatively low molecular polarity. Thus, they adsorb best onto weakly polar or nonpolar resins. Through a comparison of static adsorption and desorption properties, AB-8 was identified as the most suitable resin, and so it was selected for the purification experiment.

1.

Adsorption and analytical experiments of selected macroporous resin. (A) Adsorption and desorption capacity; (B) Adsorption and desorption rate. Different letters indicated significant difference (P < 0.05).

3.2. Determination of Antioxidant Properties of AMP In Vitro

3.2.1. ABTS⁺ Radical Scavenging Rate

The ABTS⁺ assay has the advantage of measuring both water-soluble and fat-soluble antioxidant substances and is frequently used for evaluating antioxidant activity. As shown in Figure , the ABTS⁺ radical scavenging rate of the DES-extracted AMP (100 ± 4.04%) was significantly higher than those of the 80% methanol extract (72.8 ± 2.16%) and the 60% ethanol extract (62.3 ± 1.09%) (P < 0.05). The radical inhibition capacity of the DES-extracted AMP was 1.3–1.6 fold higher than that of AMP extracted with conventional solvents.

2.

ABTS⁺, hydroxyl radical, DPPH free radical scavenging rate of AMP among three extraction methods. Different letters indicate significant difference (P < 0.05).

3.2.2. Hydroxyl Radical Scavenging Rate

In the hydroxyl radical inhibition assay, the DES-extracted AMP demonstrated significantly higher radical scavenging capacity (96.77 ± 3.56%) than those of the 80% methanol extract (86.53 ± 2.98%) and the 60% ethanol extract (64.67 ± 2.12%) (P < 0.05). The radical inhibition capacity of the DES-extracted AMP was 1.1–1.5 fold higher than that of AMP extracted using conventional solvents.

3.2.3. DPPH Radical Scavenging Rate

The DPPH method is widely used for evaluating the antioxidant activity of plant extracts. The method is based on the reduction of alcoholic DPPH solutions at 517 nm in the presence of a hydrogen-donating antioxidant. Polyphenols are potent hydrogen donators to the DPPH radical because of their ideal structural chemistry. As shown in Figure , the DPPH radical scavenging rate of DES-extracted AMP (100 ± 2.99%) was significantly higher than those of the 80% methanol extract (94.34 ± 1.56%) and the 60% ethanol extract (87.35 ± 2.34%) (P < 0.05). The radical inhibition capacity of the DES-extracted AMP was 1.06–1.15 fold higher than that of AMP extracted using conventional solvents.

3.3. Analysis of AMP Components

The AMP extracts were purified by column chromatography under the optimized conditions described in Section . Then, UPLC–QTOF–MS/MS was carried out to identify and analyze AMP’s composition. These analyses were conducted for AMP extracted with DES under optimal conditions and for AMP extracted using conventional organic solvents. These analyses successfully identified the types and contents of constituents in AMP, including anthocyanins, proanthocyanidins, flavonoids, and glycosylated/methylated derivatives of phenolic acids (comprising dimers and polymers) (Figure A,B). Qualitative and quantitative analyses were performed by matching the data against those in the in-house database of 800 polyphenolic metabolites, on the basis of characteristic mass spectral fragmentation patterns of reference standards.

3.

(A) The total ion flow pattern of AMP extracted by DES. (B) Secondary mass spectrum of DES components in negative ion mode. (C) Heatmaps for component identification among the three extraction methods of AMP. (D) AMP component classification ring diagram.

As shown in Figure C and Table , 49 major compounds were identified, comprising 16 anthocyanins, three procyanidins, 20 flavonoids and 10 phenolic acids. Among them, 46 compounds were detected in the DES-extracted AMP, 36 in the 80% methanol-extracted AMP, and 12 in the 60% ethanol-extracted AMP. The major bioactive components of AMP were procyanidin B1, epicatechin, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, chlorogenic acid, neochlorogenic acid, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, and kaempferol-3-O-galactopyranoside. Notably, two compounds previously unreported in AMP were identified (isorhamnetin-3-O-β-d-glucoside-7-O-β-d-gentiobioside and kaempferol-3-O-gentiobioside). These two newly identified compounds were potentially associated with gentisic acid degradation and were isorhamnetin and kaempferol derivatives joined via a glycosidic bond. Quantitative analyses revealed that the procyanidin fraction (55.28 ± 3.83 mg/g) was primarily composed of polymeric procyanidin B1, epicatechin, and some catechin. The procyanidin fraction accounted for 65.37% of total polyphenols, consistent with previous reports (Figure D). The anthocyanin fraction consisted of five anthocyanidins (cyanidin, pelargonidin, malvidin, peonidin, delphinidin) conjugated with galactose, glucose, arabinose, or xylose. Flavonoids were mainly present as glycosides; namely kaempferol, quercetin, and isorhamnetin conjugated with various sugars including galactose, glucose, arabinose, xylose and rhamnose. The phenolic acids were mainly derivatives of caffeoylquinic acid (chlorogenic acid, new chlorogenic acid, quinic acid, caffeic acid), derivatives of hydroxycinnamic acid (para-coumaric acid, ferulic acid, mustard acid), and derivatives of hydroxybenzoic acid (catechuic acid, gallic acid, gentiolic acid). Gallic tannins (such as gallic acetyl glucose) were also present, and possibly formed by hydrolysis of gallic acid followed by combination with polyols under acid, alkali, or enzyme catalysis.

1. Types and Contents of AMP among Three Extraction Methods .

				mass charge ratio (m/z)				polyphenol content (μg/g)
number	type	identified constituent	molecular formula	[M]+	[M–H]−	fragmentation (m/z)	RT (min)	DES extract	80% methanol extract	60% ethanol extract
1	anthocyanins	cyanidin-3-O-galactoside	C21H21O11	447.1042		287.0545	4.22	7322.67 ± 112.46	2107.81 ± 136.76	529.38 ± 26.85
2		cyanidin-3-O-glucoside	C21H21O11	447.1122		287.0545	5.94	3267.42 ± 242.43	1294.67 ± 67.86	275.46 ± 24.67
3		cyanidin-3-O-xyloside	C20H19O10	417.2971		287.0545	6.49	583.36 ± 174.76	210.53 ± 12.53
4		cyanidin-3-O-arabinoside	C20H19O10	417.2971		287.0545	6.60	1649.53 ± 64.60	125.35 ± 13.45
5		pelargonidin-3-O-arabinoside	C20H19O9	401.1065		271.0620	8.89	204.31 ± 23.46	95.42 ± 8.32
6		pelargonidin-3-O- xyloside	C20H19O9	401.1065		271.0620	8.02	180.46 ± 32.67	41.53 ± 3.42
7		pelargonidin-3-O-glucoside	C21H21O10	431.0672		271.0620	7.86	1649.56 ± 78.87	10.42 ± 5.22
8		pelargonidin-3-O-galactoside	C21H21O10	431.0672		271.0620	7.74	1233.78 ± 78.43	116.42 ± 10.62
9		peonidin-3-O-galactoside	C22H23O11	461.1526		301.0702	0.59	674.32 ± 55.63
10		peonidin-3-O-glucoside	C22H23O11	461.1526		301.0811	1.04	454.43 ± 36.74
11		delphinidin-3-O-arabinoside	C27H31O17	433.1236		303.0509	6.63	533.24 ± 24.84
12		delphinidin-3-O-xyloside	C27H31O17	433.1213		303.0509	6.67	284.38 ± 34.68
13		malvidin-3-O-xyloside	C23H25O12	461.1562		331.0811	6.56	124.64 ± 28.65	83.41 ± 6.73
14		malvidin-3-O-arabinoside	C23H25O12	461.1704		331.0811	6.50	1292.46 ± 98.90	37.25 ± 4.86
15		malvidin-3-O-galactoside	C22H23O11	491.1173		331.0811	5.84	1802.67 ± 136.47	144.50 ± 9.59
16		malvidin-3-O-glucoside	C22H23O11	491.1173		331.0811	6.60	1784.39 ± 131.98	164.52 ± 9.75
17	procyanidins	procyanidin B1/B2/B3/B4	C22H23O11		577.1672	289.0732	2.85	34125.59 ± 2655.67	5672.09 ± 352.62	3536.52 ± 169.38
18		epicatechin	C15H14O6		289.0145	179.6788,165.0773	12.81	19182.75 ± 978.48	2795.27 ± 187.23	1683.91 ± 96.91
19		catechin	C15H14O6		289.0145	179.6788, 165.0773	13.65	1966.75 ± 197.63
20	flavonoids	kaempferol	C15H10O6		285.0673	257.0341, 241.0310, 193.0213	0.52	3672.86 ± 192.58	2787.38 ± 148.91	1561.71 ± 88.11
21		kaempferol-3-O-galactoside	C21H20O11		447.0886	285.0367	6.49	1784.28 ± 97.92	52.81 ± 1.81
22		kaempferol- 3-O-glucoside	C21H20O11		447.0886	285.0367	6.57	1683.29 ± 65.12	53.27 ± 4.88
20		kaempferol-3-O-xyloside	C20H18O10		417.108	285.0367	6.79	1265.19 ± 72.60	24.69 ± 1.96
23		kaempferol-3-O-arabinoside	C20H18O10		417.0803	285.0367	6.69	1175.83 ± 42.97	45.82 ± 3.91
25		kaempferol-3-O-rhamnoside-rutinoside	C27H30O15		594.5643	285.0367	4.78	84.92 ± 9.82
26		kaempferol-3-O-β-d-glucosyl (1–2) rhamnoside	C27H30O15		593.1623	285.0367	4.24	43.74 ± 6.75
27		quercetin	C15H10O7		301.0012	273.0377, 257.0328, 193.0213	5.94	3984.82 ± 112.47	1923.91 ± 146.73	735.27 ± 38.74
28		quercetin-3-O-arabinoside	C20H18O11		433.1190	301.0012	6.54	1277.37 ± 84.59
29		quercetin-3-O-xyloside	C20H18O11		433.1190	301.0012	6.84	1283.78 ± 89.36
30		quercetin-3-O-galactoside	C21H20O12		463.2957	301.0012	5.68	1363.83 ± 122.75
31		quercetin-3-O-glucoside	C21H20O12		463.2973	301.0012	5.59	1443.72 ± 87.49	44.92 ± 6.82
32		quercetin-3-O-robinoside	C27H30O16		609.1463	463.2957, 301.0012	4.24	2212.83 ± 197.48	26.67 ± 1.76
33		quercetin-3–0-rutinoside	C27H30O16		609.1463	463.2957, 301.0012	4.02	2562.43 ± 198.27	623.41 ± 74.28	263.78 ± 82.56
34		quercetin-3–0-vicianoside	C28H33O18		626.1492	463.2973, 301.0012	6.45	57.62 ± 9.16	16.53 ± 1.61
35		quercetin-3-o-galactoside-7-o-glucoside	C27H34O19		626.1452	463.2973, 301.0012	4.05	38.25 ± 3.51
36		glucogallin	C21H21O10		332.0751	332.0751	0.95	2765.62 ± 86.28	1124.82 ± 83.46
37		1,2,3,6-tetra-O-galloyl-b-d-glucose	C34H28O22		788.1081	332.0751	5.46	1563.24 ± 98.42
38		1,2,3,4,6-penta-O-glucose	C41H32O30		940.1140	332.0751	5.97	964.82 ± 60.86
39		isorhamnetin-3-O-β-d-glucose-7-O-β-d-gentiobioside	C34H42O22		802.2132	316.2263, 477.4184, 639.4326	4.38	76.32 ± 8.15
40	organic acid	chlorogenic acid	C16H18O9		353.0857	191.0548, 179.0334, 135.0442	3.24	5783.39 ± 378.21	2984.72 ± 285.38	1643.71 ± 57.18
41		neochlorogenic acid	C16H18O9		353.0857	191.0548, 179.0334, 135.0442	3.62	258.56 ± 62.81	1823.71 ± 96.35	1232.35 ± 127.28
42		cryptochlorogenic acid	C16H18O9		353.0857	191.0548, 179.0334	3.35	1764.82 ± 81.37	454.39 ± 63.72	1674.78 ± 168.32
43		caffeic acid	C9H8O4		179.6478	135.0442, 161.2245, 135.0442	2.13	664.71 ± 78.23	54.67 ± 8.37	26.82 ± 1.72
44		ferulic acid	C10H10O4		193.1308	179.0334, 163.0286, 135.0442	1.24	394.53 ± 46.72	66.56 ± 7.72
45		p-coumaric acid	C9H8O3		163.0482	119.0297	1.04	264.67 ± 33.35	94.58 ± 8.81
46		quinic acid	C7H12O6		191.0422	163.0334	0.61	221.57 ± 53.62	112.43 ± 24.45
47		sinapinic acid	C11H12O5		223.0475	223.0460	0.86	126.25 ± 23.17
48		salvianolic acid C	C26H20O10		491.3692	293.2366	4.01	49.25 ± 6.72
49		protocatechuic acid	C7H6O4		153.0678	109.4241	0.56	198.23 ± 8.41	69.93 ± 5.27	7.01 ± 0.56
		total						84555.18 ± 3845.92	20991.9 ± 1642.1	16591.54 ± 974.82

^aRetention time of compounds was similar among the three extraction methods, but slightly different. In the table, retention time after DES extraction is taken as the standard.

Among the three extraction methods, the DES approach achieved the highest AMP extraction yield (84.56 ± 3.85 mg/g) (P < 0.05). This yield was 3.91-fold and 6.11-fold higher than that obtained using the conventional 80% methanol extraction method (25.36 mg/g) and the 60% ethanol method (16.27 mg/g), respectively. The significantly richer and more diverse polyphenolic profile obtained by the DES method can be attributed to its unique physicochemical properties. The DES system, composed of choline chloride and lactic acid, possesses an extensive and tunable hydrogen-bonding network. This network likely facilitates stronger and more selective interactions (e.g., hydrogen bonding, ionic, and dipole–dipole interactions) with a wider range of polyphenolic compounds, including those with higher polarity or molecular weight that are poorly solubilized by conventional organic solvents. Furthermore, water in DES acts a glue-like effect, acting as a bridge between DES components. The polarity of the DES can be finely adjusted by varying the water content, potentially enabling a more comprehensive solvation of polyphenols with diverse chemical structures and polarities present in the complex plant matrix. This synergistic effect of enhanced interactions and tunable solvation capacity likely contributes to the superior extraction efficiency and compositional richness observed for the DES extract. These results clearly demonstrate that ultrasound-assisted DES extraction outperforms traditional organic solvent methods in terms of AMP extraction efficiency and the compositional diversity of the extract.

3.4. In Vitro Hypoglycemic and Hypolipidemic Activity Assay

Blueberry fruit contain natural antioxidants such as proanthocyanidins and anthocyanins. They are popular with domestic and international consumers in the food, pharmaceutical, and cosmetic industries because of their pleasant taste, potent antioxidant activity, and hypoglycemic and hypolipidemic effects. Blueberry is generally regarded as the most valuable berry for development. However, the polyphenol content in blueberry is lower than that in AMP. Therefore, the hypoglycemic and hypolipidemic activities of AMP were compared with those of a similar extract from blueberry (BP) to provide a reference for evaluating the developmental potential of AMP.

3.4.1. α-Glucosidase Inhibition Activity of BP and AMP

The AMP and blueberry polyphenols (BP) were subjected to an in vitro simulated digestion treatment, and then the effect of the undigested and digested AMP and BP on α-glucosidase activity were determined. As shown in Figure , AMP and BP in their digested and undigested forms inhibited α-glucosidase inhibition activity. The undigested AMP demonstrated significant inhibitory activity (76.4 ± 1.2%), which decreased to 15.1 ± 0.4% after gastric digestion and then increased to 30.4 ± 1.2% following intestinal digestion (P < 0.05). Similarly, the α-glucosidase inhibitory activity of undigested BP was 68.2 ± 0.9%, decreasing to 5.9 ± 0.2% after gastric digestion and then slightly increasing to 9.1 ± 0.2% after intestinal digestion (P < 0.05). Statistically significant differences (indicated by different lowercase letters in Figure ) were determined via one-way ANOVA followed by Tukey’s post hoc test, comparing the mean inhibition rates among the three experimental groups (Undigested AMP, Gastric-digested AMP, Intestinal-digested AMP, and Undigested BP, Gastric-digested BP, Intestinal-digested BP) at each independent time point. This analysis aimed to identify significant differences in inhibitory potency attributable to the digestion process (for the same extract) and between the two extract sources (AMP vs BP) at equivalent digestion stages. Notably, compared with gastric-digested BP, gastric-digested AMP exhibited 2.56-fold higher α-glucosidase inhibitory activity; and intestinal-digested AMP showed 3.34-fold higher α-glucosidase inhibitory activity than that of intestinal-digested BP.

4.

Inhibition rate of AMP and BP on α-glucosidase. Values are mean ± SD (n = 3). Different lowercase letters above the bars indicate statistically significant differences (P < 0.05) among the groups at each digestion stage, as determined by one-way ANOVA followed by Tukey’s post hoc test.

The observed variations in inhibitory activity across digestion stages are intrinsically linked to the stability, transformation, and bioavailability of the key polyphenolic constituents in AMP and BP. The sharp decline in α-glucosidase inhibition after gastric digestion can be attributed to the instability of many polyphenols under highly acidic conditions (pH 2.0). Anthocyanins, particularly the abundant cyanidin glycosides in AMP, are known to undergo structural transformation (e.g., conversion to chalcones or degradation) at low pH, which can significantly diminish their enzyme–inhibitory potency. Similarly, certain flavonoid glycosides and phenolic acids may experience acid hydrolysis or conformational changes affecting their ability to interact with the enzyme’s active site. The partial recovery of inhibitory activity in the intestinal phase, although not to the level of the undigested extract, suggests that some polyphenols remain stable or are converted into bioactive forms in the neutral-to-weakly alkaline environment. Moreover, the intestinal phase involves bile salts and pancreatin, which can form mixed micelles, potentially improving the solubility and accessibility of less polar polyphenolic aglycones to the enzyme.

Anthocyanins are known to inhibit α-glucosidase, with cyanidin-3-O-galactoside and cyanidin-3-O-glucoside showing the strongest inhibitory activity. These two compounds represent the most abundant anthocyanins in AMP. In contrast, the main anthocyanin compounds in blueberry are delphinidin and malvidin chloride. Quercetin and its derivatives, widely recognized as blood glucose modulators, have been extensively studied in clinical trials and evaluated for their antidiabetic potential. They are known to act as mixed-type or noncompetitive inhibitors of α-glucosidase, with their inhibitory efficacy often preserved or modified postdigestion depending on their glycosylation pattern and the digestive environment. Chlorogenic acid and caffeic acid also have significant α-glucosidase inhibitory activity. Whereas gallic acid itself also inhibits α-glucosidase, our study identified two more gallic acid derivatives: 1,2,3,6-tetra-O-galloyl-β-d-glucose and 1,2,3,4,6-penta-O-galloyl-β-d-glucose, which are currently being investigated as targeted hypoglycemic agents. These chemicals present complex molecular structures with their multiple phenolic rings and ester bonds. This could make them probably resist digestive degradation due to their structures, contributing to the sustained inhibitory activity observed. All these bioactive compounds with glucose-lowering properties are present in both AMP and BP, although their concentrations are substantially higher in AMP, which directly correlates with the consistently stronger inhibitory activity of AMP across all digestion stages.

α-Glucosidase is mainly located on the membrane of intestinal epithelial cells, and is a key hydrolytic enzyme in carbohydrate metabolism. Its activity affects blood glucose levels, so it is often used as a target molecule for the detection of hypoglycemic active ingredients. That is, compounds that inhibit α-glucosidase ultimately result in lower blood glucose levels. Because many active compounds are pH sensitive, their activity can be fully or partially lost in the acidic gastric environment. Upon reaching the intestinal tract, however, the active components can be directly absorbed by small intestinal epithelial cells, where they can exert inhibitory effects on α-glucosidase. The results demonstrated that, compared with the gastric environment (strongly acidic), the intestinal environment (weakly alkaline) is more conducive to the release of hypoglycemic active substances from A. melanocarpa, as also noted in a study on blueberry. Furthermore, the results show that the hypoglycemic activity of AMP is stronger than that of BP, consistent with the results of Čakar’s comparative study on the α-glucosidase inhibitory capacity of these two berries, although the DES extracts demonstrated even greater inhibitory potential.

3.4.2. Inhibition of Pancreatic Lipase

Figure illustrates the inhibitory effects of AMP and BP, before and after simulated digestion, on pancreatic lipase activity. The pancreatic lipase inhibition rate of undigested AMP was 100 ± 2.2%, that of AMP after gastric digestion was 100 ± 1.9%, and that of AMP after digestion with intestinal juice was 92.7 ± 2.9% (P < 0.05). A similar trend was observed for BP. The pancreatic lipase inhibition rate of undigested BP was 100 ± 1.5%, that of BP after gastric digestion was 95.5 ± 1.1%, and that of BP after digestion with intestinal juice was 89.3 ± 1.7% (P < 0.05). Thus, undigested AMP and BP completely inhibited pancreatic lipase activity; gastric-digested AMP exhibited 1.05-fold higher inhibition than that of gastric-digested BP; and intestinal-digested AMP exhibited 1.04-fold higher inhibition than that of intestinal-digested BP. The statistical significance indicated by different lowercase letters in Figure follows the same comparative logic as in Figure . Comparisons are made among the experimental groups at each independent digestion stage to evaluate the impact of digestion and the difference between the two extracts.

5.

Inhibition rate of AMP and BP on pancreatic lipase. Values are mean ± SD (n = 3). Different lowercase letters above the bars indicate statistically significant differences (P < 0.05) among the groups at each digestion stage, as determined by one-way ANOVA followed by Tukey’s post hoc test.

The robust inhibition of pancreatic lipase by both AMP and BP, even after digestion, underscores the remarkable stability and bioaccessibility of the responsible polyphenolic compounds, particularly proanthocyanidins. Proanthocyanidins, especially the highly polymerized forms abundant in AMP, are known to resist digestive degradation due to their complex condensed structures and multiple interflavan bonds. They can inhibit pancreatic lipase through a multimechanistic approach: forming noncovalent complexes with the enzyme via hydrogen bonding and hydrophobic interactions, thereby blocking the active site or causing conformational changes; (2) binding to the substrate (triglycerides) or the lipid–water interface, reducing substrate availability; and (3) precipitating proteins including the enzyme itself. , The slightly lower inhibition in the intestinal phase compared to the gastric phase might be attributed to partial hydrolysis of some esterified phenolic acids (e.g., chlorogenic acid derivatives) or interactions with bile salts and phospholipids, which could sequester some polyphenols into micelles, slightly reducing their direct access to the enzyme. The fact that AMP maintained near-complete inhibition postgastrically and only a slight decrease intestinally, compared to BP, aligns with its significantly higher content of these stable, high-molecular-weight proanthocyanidins (as shown in Table ).

Pancreatic lipase functions in the small intestine, and plays a central catalytic role in human lipid metabolism. It breaks down fats into free fatty acids, glycerol, and monoglycerides, enabling their absorption. In this way, it controls the blood lipid content. Inhibition of this enzyme can reduce the absorption rate of fat in the intestine. Lachowicz-Wiśniewska et al. demonstrated that AMP, especially highly polymerized proanthocyanidins, could effectively inhibit the activity of pancreatic lipase as measured by an in vitro method. Notably, AMP contains the highest known concentration of proanthocyanidins among plant materials. Based on the results of our study and other studies, we speculate that abundant proanthocyanidins are responsible for the strong hypolipidemic capacity of AMP.

The results demonstrate that gastrointestinal digestive solution of both AMP and BP exhibit potent hypolipidemic ability. The gastric environment (acidic) proved more favorable than the intestinal environment (weakly alkaline) for the release of hypolipidemic active compounds from both AMP and BP. Furthermore, AMP demonstrated superior hypolipidemic activity compared with that of BP, and it should be noted that DES-extracted AMP showed stronger hypolipidemic ability.

4. Conclusion

The study demonstrates the clear superiority of ultrasound-assisted deep eutectic solvent (DES) extraction for obtaining Aronia melanocarpa polyphenols (AMP). The DES-extracted AMP exhibited not only a richer diversity and higher content of polyphenolic compounds but also significantly stronger in vitro antioxidant capacity (scavenging ABTS⁺, DPPH, and hydroxyl radicals). Following simulated digestion, DES-AMP also showed markedly greater inhibitory effects on α-glucosidase and pancreatic lipase compared to blueberry polyphenols. The DES-extracted AMP holds distinct potential for application in the functional food and nutraceutical sectors. Specifically, its intended forms of application can be delineated into two primary pathways: as a core active ingredient in dietary supplements (e.g., capsules, tablets, or powdered formulations targeting glycemic and lipid control), and as a natural bioactive food additive. For the latter, its potent antioxidant and enzyme–inhibitory properties make it suitable for fortifying a range of food products such as functional beverages, dairy items, health bars, and baked goods, where it can enhance shelf life and impart metabolic health benefits. This research provides a theoretical and experimental foundation for the development of value-added AMP-based ingredients, clearly outlining these viable application routes for industry consideration.

Y.S.: Writingoriginal draft, formal analysis, data curation. H.Z.: Writingreview and editing, investigation. D.G.: Investigation, formal analysis, data curation. P.W.: Formal analysis, review and editing. C.L.: Review and editing, supervision.

The authors declare no competing financial interest.