Targeted Separation of Ziziphus jujuba Pulp Polyphenols: Adsorption Kinetics Characteristics of AB-8 Resin and Product Structure Analysis

Abstract

To address the challenge of purifying bioactive polyphenols from the complex matrix of Ziziphus jujuba Mill. var. spinosa pulp, this study established an integrated purification protocol combining Deep Eutectic Solvent (DES) extraction with macroporous adsorption resin (MAR) enrichment. Among five screened resins, AB-8 exhibited superior selectivity, achieving a maximum adsorption capacity of 62.48 mg polyphenols/g dry resin and a desorption ratio of 83.40%. Kinetic analysis revealed that the adsorption process strictly followed a pseudo-second-order model (R² = 0.999), indicating a mechanism dominated by chemisorption. Through dynamic optimization, optimal column parameters were determined as a loading concentration of 2.4 mg/mL, a flow rate of 1.0 mL/min, and elution with 70% (v/v) ethanol. Structural characterization via UV-Vis and FT-IR confirmed the effective removal of polysaccharide and protein impurities, while High-Performance Gel Permeation Chromatography (HPGPC) indicated a low-molecular-weight distribution (M_w approx. 1073 Da). Furthermore, HPLC-MS profiling definitively identified eight key constituents, including chlorogenic acid, catechin, rutin, and quercetin. Collectively, this work elucidates the adsorption mechanism and provides a scalable, efficient technical foundation for the high-purity preparation of jujube polyphenols.

1. Introduction

Ziziphus jujuba Mill. var. spinosa is a highly valued medicinal and edible plant resource in China [1]. Its pulp is recognized as a rich source of bioactive secondary metabolites, particularly phenolic acids (e.g., chlorogenic acid) and flavonoids (e.g., catechin), which exhibit potent antioxidant, anti-inflammatory, and neuroprotective activities [2,3]. These properties offer broad prospects for developing natural functional ingredients and health-promoting foods. However, the complex matrix of jujube pulp, characterized by high levels of polysaccharides and proteins, leads to significant co-extraction issues. This often results in crude extracts with low polyphenol purity, which compromises their stability and complicates subsequent structural characterization. Recent trends emphasize the use of green solvents like Deep Eutectic Solvents (DESs) for efficient extraction [4,5,6]; Deep Eutectic Solvents (DESs), typically composed of a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD), have emerged as a promising class of sustainable solvents. Due to their extensive hydrogen bond networks, DESs exhibit tunable physicochemical properties and exceptional solubilizing capacity for phenolic compounds, often surpassing conventional solvents like water or ethanol. For instance, DESs have demonstrated remarkable biodegradability and efficiency in extracting bioactive oleoresins from Ferula gummosa roots compared to conventional solvents [7]. Despite these advantages, the application of DESs is often hindered by their inherent high viscosity and low vapor pressure, which make the recovery of target compounds from the DES matrix challenging. However, despite these advantages, advanced extraction techniques often face hurdles related to operational complexity, product stability, and initial capital investment [8]. Consequently, developing a robust downstream separation strategy is crucial to recover polyphenols from the viscous DES matrix effectively. Therefore, to overcome the challenges posed by the high viscosity and complex background of DES, it is essential to develop robust post-extraction purification protocols.

To overcome the separation challenges posed by the complex DES matrix, Macroporous Adsorption Resins (MARs) offer a highly effective solution. Compared to traditional separation methods, macroporous adsorption resins (MARs) are highly effective for enriching plant polyphenols due to their excellent stability, reusability, and selective adsorption capacities [9,10,11]. MARs are durable polymers with a permanent pore structure and high specific surface area. They separate compounds based on differences in molecular weight and polarity through mechanisms such as van der Waals forces and hydrogen bonding. Crucially, MARs can selectively adsorb non-polar to weakly polar polyphenols while allowing highly polar impurities (such as polysaccharides) and the water-soluble DES components to be washed away, thus achieving extraction and purification in a streamlined process.

The purification efficiency of MARs is governed by the complementarity between the resin’s pore architecture and polarity and the target molecule’s structural attributes, such as its aromaticity and hydroxyl distribution [12].

While the purification of jujube polyphenols using resins like AB-8 or D-101 has been explored, a systematic evaluation comparing different polarity and pore size systems remains insufficient [13,14]. Most existing studies lack an in-depth analysis of the synergistic relationship between resin physicochemical properties and the adsorption thermodynamics of structurally diverse jujube polyphenols. Furthermore, comprehensive structural elucidation utilizing multiple analytical platforms is essential to validate the enrichment effects. Therefore, this study systematically screened five MARs and employed a multi-technology strategy—including UV-Vis, FT-IR, and HPLC–MS—to establish a reproducible and efficient purification process. This work clarifies the structure–activity relationship between resin properties and polyphenol adsorption, providing a technical foundation for the high-purity preparation of jujube-derived bioactive factors.

2. Materials and Methods

2.1. Materials and Instruments

The flesh of the Chinese Ziziphus jujuba Mill. var. spinosa: provided by Dongying Guangyuan Biotechnology Co., Ltd., (Dongying, China) (mature fruits in September 2024); macroporous adsorption resins: NKA-II (styrene type), AB-8 (styrene type), S-8 (styrene type), HPD-100 (styrene type), D-101 (styrene type), all purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China); reference substances: catechin (≥98%), epicatechin (≥98%), quercetin (≥98%), rutin (≥98%), chlorogenic acid (≥98%), caffeic acid (≥98%), ferulic acid (≥98%), esculetin (≥98%), naringenin (≥98%), all purchased from Shanghai Yuenye Biotechnology Co., Ltd. (Shanghai, China); chemical reagents: anhydrous ethanol, hydrochloric acid, sodium hydroxide, Choline chloride, Levulinic acid, all analytical grade, and all purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); ultrapure water.

2.2. Preparation of Crude Polyphenol Extract from Wild Jujube (Ziziphus jujuba Mill. var. spinosa) Pulp

First, the pretreatment of Ziziphus jujuba Mill. var. spinosa was carried out referring to the method described by Du et al. [15]. Ziziphus jujuba Mill. var. spinosa was sorted to remove rotten fruits and impurities, followed by separation of seeds. The processed pulp was dried at low temperature, ground, and sieved through an 80-mesh screen, and then stored in a sealed, light-proof container for subsequent use. Next, the deep eutectic solvent (DES) was prepared according to the synthesis protocol reported by Pinho et al. [16]. Choline chloride (as a hydrogen bond acceptor) and levulinic acid (as a hydrogen bond donor) were mixed at a molar ratio of 1:1.2, and heated with stirring at 90 °C to form a homogeneous, transparent deep eutectic solvent (DES-2, the optimal extraction solvent). Subsequently, based on the extraction strategies for bioactive compounds reviewed by Ling and Hadinoto [17], Ziziphus jujuba Mill. var. spinosa powder was mixed with DES-2 containing 20% water at a solid-to-liquid ratio of 1:30 g/mL, and subjected to constant-temperature extraction at 60 °C for 40 min. After extraction, the mixture was centrifuged at 8000 rpm/min for 10 min, and the supernatant was collected to obtain the crude polyphenol extract. The initial concentration of the extract was determined, and then it was stored at −20 °C for further use.

2.3. Pretreatment (Activation) of Macroporous Adsorption Resins (MARs)

The pretreatment and activation of macroporous adsorption resins (MARs) were performed following the procedure described by Hou et al. [9]. An appropriate amount of each type of macroporous adsorption resin (MAR) was weighed and immersed in anhydrous ethanol for 24 h, with stirring once every 8 h to ensure uniform swelling. The resin was then repeatedly rinsed with ultrapure water until no ethanol odor was detected in the effluent. Subsequently, the resin was soaked in 5% (v/v) HCl solution for 4 h to remove alkaline impurities, followed by rinsing with ultrapure water until the pH of the effluent reached 7. Next, the resin was immersed in 5% (w/v) NaOH solution for 4 h to eliminate acidic impurities, and again rinsed with ultrapure water until the effluent pH was neutral (pH = 7). The activated resin was soaked in ultrapure water for 2 h, and then subjected to suction filtration to remove surface moisture. It was then dried in a vacuum drying oven (40 °C, 0.09 MPa) until a constant weight was attained, cooled to room temperature, and stored in a sealed container for subsequent use.

2.4. Screening of Macroporous Adsorption Resins (Static Adsorption–Desorption Experiments)

2.4.1. Static Adsorption Experiments

We precisely weighed 1.0 g of each type of pre-treated MAR (NKA-II, AB-8, S-8, HPD-100, D-101) and placed it in a 250 mL conical flask. We added 50 mL of a crude polyphenol extract solution diluted to a polyphenol concentration of 1.0 mg/mL (determined by the Folin–Ciocalteu method) to each flask. The flasks were placed in a constant-temperature water bath shaker (Shanghai Yiheng Technology Co., Ltd., Shanghai, China) and shaken at 30 °C and 180 rpm for 24 h to ensure complete adsorption. Equilibrium was considered reached when the polyphenol concentration in the supernatant showed no significant difference between consecutive sampling intervals, as verified by the adsorption kinetic curves (Section 3.2). After filtration, we took the supernatant and determined the polyphenol concentration (C₂) after adsorption using the UV spectrophotometry method (wavelength 765 nm, Folin–Ciocalteu method) [18]. The adsorption capacity (Q) and adsorption rate were calculated according to formulas (1) and (2):

$$\mathrm{Adsorbing} \mathrm{capacity} (\mathrm{mg} \mathrm{polyphenols}/\mathrm{g} \mathrm{dry} \mathrm{resin}) ={\displaystyle \frac{({\mathrm{C}}_1-{\mathrm{C}}_2)}{\mathrm{W}}}\times \mathrm{V}$$

(1)

$$\mathrm{Adsorption} \mathrm{rate} (\mathrm{\%})={\displaystyle \frac{({\mathrm{C}}_1-{\mathrm{C}}_2)}{{\mathrm{C}}_2}}\times 100\mathrm{\%}$$

(2)

where C₁ is the mass concentration of the polyphenol solution before adsorption (mg/mL); C₂ is the mass concentration of polyphenols in the solution after adsorption (mg/mL); V is the volume of the crude sour jujube polyphenol extract added before adsorption (mL); W is the mass of the dry adsorption resin (g).

2.4.2. Static Desorption Experiment

MARs that had reached adsorption equilibrium were rinsed three times with ultrapure water (10 mL per rinse) to remove residual crude extract from the surface. After blotting surface moisture with filter paper, the resins were transferred to 250 mL conical flasks, and 50 mL of 70% (v/v) ethanol aqueous solution was added to each flask. The flasks were shaken at 30 °C and 180 rpm for 24 h. The desorption equilibrium was considered reached when the polyphenol concentration in the supernatant showed no significant difference between consecutive measurements. The supernatant was collected after filtration, and the polyphenol concentration after desorption (C₃) was determined [16]. The desorption rate was calculated using the following formula:

$$\mathrm{Desorption} \mathrm{rate} (\mathrm{\%})={\displaystyle \frac{{\mathrm{C}}_3\times {\mathrm{V}}_3}{\mathrm{W}}}\times \mathrm{Q}\times 100\mathrm{\%}$$

(3)

where C₃ is the mass concentration of polyphenols in the desorption solution (mg/mL); V₃ is the volume of the desorption solution (mL); W is the mass of the dry adsorption resin (g); Q is the adsorption capacity of the resin (mg/g).

2.5. Plotting of Adsorption–Desorption Kinetic Curves for Macroporous Adsorption Resins

2.5.1. Adsorption Kinetic Curve

Precisely weighed 1.0 g of pre-treated MAR was transferred to a 250 mL conical flask, followed by the addition of 50 mL of crude polyphenol extract with a concentration of 1.0 mg/mL. The flask was placed in a constant-temperature water bath shaker and agitated at 30 °C and 180 rpm. Aliquots of 50 μL were sampled at 0, 1, 2, 4, 6, 8, 10, 12, and 24 h—after each sampling, 50 μL of ultrapure water was supplemented to maintain a constant system volume [19]. The polyphenol concentration was determined, and the adsorption rate was calculated. A static adsorption kinetic curve was plotted, with time as the abscissa and adsorption rate as the ordinate.

2.5.2. Desorption Kinetic Curve

MARs that had reached adsorption equilibrium were rinsed with ultrapure water until no polyphenols were detected in the effluent (UV detection showed no absorbance). After suction filtration, the resins were transferred to a 50 mL conical flask, and 25 mL of 50% (v/v) ethanol aqueous solution was added. The flask was placed in a constant-temperature water bath shaker and continuously agitated for desorption at 25 °C and 180 rpm for 24 h. During this period, 50 μL of supernatant was sampled every 1 h—50 μL of ultrapure water was supplemented after each sampling—and the polyphenol concentration was determined [19]. The desorption rate was calculated, and a static desorption kinetic curve was plotted.

2.6. Optimization of Dynamic Adsorption–Desorption Process Parameters for Macroporous Adsorption Resins

2.6.1. Effect of Sample Loading Concentration on Adsorption Performance of Macroporous Resins

A 10.0 g amount of pre-treated macroporous resin was wet-packed into a glass chromatography column (0.6 cm × 50 cm). The sample solution was loaded under fixed conditions: pH = 3 (adjusted with 1.0 mol/L HCl), loading volume = 100 mL, and loading flow rate = 1.0 mL/min. The loading concentrations were set at 0.4, 0.8, 1.6, 2.4, 3.2, and 4.0 mg/mL, respectively. Effluent fractions were collected every 10 mL, and the concentration of Ziziphus jujuba Mill. var. spinosa polyphenols in each fraction was determined. The breakthrough point was defined as the point when the polyphenol concentration in the effluent reached 1/10 of the initial loading concentration [20]. This point was recorded to determine the optimal loading concentration.

2.6.2. Effect of Sample Loading Flow Rate on Adsorption Performance of Macroporous Resins

A 10.0 g amount of pre-treated macroporous resin was wet-packed into a glass chromatography column (0.6 cm × 50 cm). The aqueous sample solution was loaded under fixed conditions: pH = 3, loading volume = 100 mL, and loading concentration = 2.0 mg/mL. The loading flow rates were set at 0.5, 1.0, 1.5, 2.0, and 2.5 mL/min, respectively. Effluent fractions were collected every 10 mL, and the concentration of Ziziphus jujuba Mill. var. spinosa polyphenols in each fraction was determined. The breakthrough point was defined as the point when the polyphenol concentration in the effluent reached 1/10 of the initial loading concentration. This point was recorded to determine the optimal loading flow rate.

2.6.3. Determination of Optimal Ethanol Concentration in Eluent

A 10.0 g amount of pre-treated macroporous resin was wet-packed into a glass chromatography column (0.6 cm × 50 cm). Adsorption was performed under the following conditions: sample solution pH = 3, loading concentration = 2.0 mg/mL, loading volume = 100 mL, and loading flow rate = 1.0 mL/min. After the resin reached adsorption saturation, impurities were rinsed off with a sufficient volume of deionized water. Elution was then carried out using 40%, 50%, 60%, 70% and 95% (v/v) ethanol aqueous solutions at a flow rate of 1.0 mL/min. Effluent fractions were collected every 10 mL, and the concentration of Ziziphus jujuba Mill. var. spinosa polyphenols in each fraction was determined to investigate the effect of ethanol concentration on the desorption performance of the macroporous resin.

2.6.4. Plotting Dynamic Elution Curve

After the resin was fully saturated by loading at a flow rate of 2 mL/min, elution was carried out using an eluent with 70% ethanol volume fraction at a flow rate of 2 mL/min. The effluent was collected every 5 mL, and the polyphenol mass concentration in the even-numbered tubes was determined.

2.6.5. Optimization of Sample Loading Volume

To determine the maximum processing capacity of the resin column, dynamic adsorption tests were conducted. Under fixed conditions (2.4 mg/mL concentration, 1.0 mL/min flow rate), the sample solution was continuously loaded onto the column. The effluent was collected in fractions, and the polyphenol concentration was monitored. The dynamic breakthrough curve was plotted to determine the saturation point and the optimal loading volume before significant leakage (defined as C/C₀ = 10%) occurred [9].

2.6.6. Determination of Polyphenol Concentration in Jujube Fruit Pulp

The purified polyphenols from jujube fruit pulp were freeze-dried in the dark and weighed [21]. The calculation formula is as follows:

$$\mathrm{P}\mathrm{u}\mathrm{r}\mathrm{i}\mathrm{t}\mathrm{y} \mathrm{o}\mathrm{f} \mathrm{p}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{p}\mathrm{h}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{l}\mathrm{s} \mathrm{i}\mathrm{n} \mathrm{j}\mathrm{u}\mathrm{j}\mathrm{u}\mathrm{b}\mathrm{e} \mathrm{f}\mathrm{r}\mathrm{u}\mathrm{i}\mathrm{t} \mathrm{p}\mathrm{u}\mathrm{l}\mathrm{p} \left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{C}\mathrm{V}}{\mathrm{m}}}\times 100\mathrm{\%}$$

(4)

where C is the mass concentration of the purified and freeze-dried jujube polyphenol extract in mg/mL; V is the volume of the solution in mL; m is the mass of the purified and freeze-dried jujube fruit pulp polyphenol powder in mg.

2.7. Structural Analysis of Polyphenolic Active Components in Jujube Pulp

2.7.1. UV Spectroscopy Analysis

The freeze-dried polyphenol powder was dissolved in 50% (v/v) ethanol to prepare a solution with a concentration of 0.1 mg/mL, followed by UV scanning over the wavelength range of 200–800 nm to identify characteristic absorption maxima. Flavonoids typically exhibit distinct absorption bands at 250–270 nm and 300–330 nm, whereas phenolic acids show a prominent peak near 280 nm [22].

2.7.2. FT-IR Spectroscopy Analysis

Fourier Transform Infrared Spectroscopy (FT-IR) spectra were acquired with a TENSOR 27 Fourier transform mid-infrared spectrometer (Bruker Technology Co., Ltd., Karlsruhe, Germany) using the KBr pellet method with a sample-to-KBr mass ratio of 1:100. Scans were performed in the wavenumber range of 400–4000 cm⁻¹ to identify key functional groups, including 3200–3600 cm⁻¹ (O–H stretching vibration), 1600–1650 cm⁻¹ (aromatic ring skeletal vibrations), and 1200–1300 cm⁻¹ (C–O stretching vibration) [23].

2.7.3. Determination of Molecular Weight Distribution

The molecular weight distribution of the purified Ziziphus jujuba pulp fraction was determined using High-Performance Gel Permeation Chromatography (HPGPC) (Agilent Technologies, Santa Clara, CA, USA). The chromatographic system was equipped with an Alltech 6000 Evaporative Light Scattering Detector (ELSD) (Agilent Technologies, Santa Clara, CA, USA). The detector operating conditions were optimized with a drift tube temperature of 110 °C, a carrier gas flow rate of 2.0 L/min, and a gain setting of 2. Separation was achieved on a TSKgel G3000PWxl (Agilent Technologies, Santa Clara, CA, USA) column maintained at 35 °C. The mobile phase consisted of distilled water eluted at a flow rate of 0.6 mL/min. The molecular weight was calculated by comparing the retention times of the sample with a calibration curve constructed using a standard of known molecular weights. Data acquisition and processing were performed using the Origin 2024 (OriginLab Corporation, Northampton, MA, USA).

2.7.4. HPLC-MS Analysis

(1) Chromatographic conditions: Separation was achieved on an Agilent ZORBAX SB-C18 (Agilent Technologies, Santa Clara, CA, USA) column (250 mm × 4.6 mm, 5 μm) with a mobile phase consisting of 0.1% (v/v) formic acid in water (A) and acetonitrile (B). A gradient elution program was applied as follows: 0–5 min, 5% B; 5–20 min, 5–25% B; 20–30 min, 25–40% B; 30–35 min, 40–95% B. The flow rate was maintained at 1.0 mL/min, column temperature at 30 °C, and injection volume at 10 μL. (2) Mass spectrometry conditions: Detection was performed using electrospray ionization in positive ion mode (ESI⁺) with a scan range of m/z 100–1000 and collision energy ramped between 10 and 30 eV. (3) Component identification: Active constituents were tentatively identified by comparing retention times and MS fragmentation patterns with reference standards (e.g., catechin, chlorogenic acid) and by matching observed molecular ions with literature-reported data (e.g., [M+H]⁺ at m/z 291.08 for catechin and m/z 355.10 for chlorogenic acid) [24].

2.8. Statistical Analysis

All values are presented as means ± standard deviations. Data were analysed using Origin 2024 (OriginLab Corporation, Northampton, MA, USA) and SPSS 26.0 (IBM Corporation, Armonk, NY, USA) statistical software. Statistical significance was set at p < 0.05 and determined using Duncan’s multiple range test.

3. Results

3.1. Screening Results of Macroporous Adsorption Resins

The physical and chemical characteristics of the five screened macroporous adsorption resins (D101, HPD-100, AB-8, NKA-2, and S-8) are summarized in

Table 1. Physicochemical properties of the five candidate macroporous adsorption resins.

Resin	Polarity	Appearance	Specific Surface Area (m2/g)	Particle Size Range (mm)	Average Pore Diameter (nm)
D101	Nonpolar	Milky white	500–550	0.3–1.25	9–11
HPD-100	Nonpolar	Milky white	500–550	0.3–1.25	8.5–9.5
AB-8	Weakly polar	Milky white	480–520	0.3–1.25	13–14
NKA-2	polar	Reddish-brown	250–290	0.3–1.25	14.5–15.5
S-8	Strong polar	Light yellow	100–120	0.3–1.25	28–30

Note: Data were provided by the manufacturer (Tianjin Guangfu Fine Chemical Research Institute). All resins consist of a polystyrene backbone except where specified. Specific surface area and average pore diameter were determined using the BET and BJH methods, respectively.

. These resins encompass a wide range of polarities, from nonpolar to strongly polar, and specific surface areas ranging from 100 to 550 m²/g [25]. Notably, AB-8 is a weakly polar resin with a moderate average pore diameter (13–14 nm) and a substantial specific surface area (480–520 m²/g), which may provide an ideal structural environment for the adsorption of polyphenolic molecules through hydrophobic interactions and π-π stacking interactions [13,25]. In contrast, the strongly polar S-8 resin exhibits the smallest specific surface area (100–120 m²/g), potentially limiting its total adsorption capacity for the target compounds.

Static Screening and Selection of Macroporous Adsorption Resins (MARs) The static adsorption and desorption experiments revealed significant variations in the enrichment efficacy among the five tested MARs for Ziziphus jujuba Mill. var. spinosa polyphenols (Figure 1). Overall, AB-8 resin was identified as the most suitable medium for the purification process, demonstrating a superior comprehensive performance that significantly outperformed the other candidates (p < 0.05) [13,26]. Quantitative Performance Comparison: Among the tested resins, AB-8 exhibited the highest efficiency, achieving a maximum adsorption capacity (AC) of 62.48 mg polyphenols/g dry resin, an adsorption ratio (A) of 71.01 ± 1.09%, and a desorption ratio (D) of 83.40 ± 0.67% [26]. In sharp contrast, S-8 exhibited the poorest performance, with an AC of only 31.59 mg polyphenols/g dry resin and an adsorption ratio of 35.90 ± 0.98%, which were approximately 50% lower than those of AB-8. The intermediate resins, including D101, HPD-100, and NKA-2, showed moderate capacities but failed to match the balanced adsorption–desorption profile of AB-8. Mechanistic Analysis of Resin–Polyphenol Affinity: The exceptional performance of AB-8 is primarily attributed to the synergistic effect of its physicochemical attributes and the structural characteristics of the jujube polyphenols. As a weakly polar polystyrene-based resin, AB-8 possesses an aromatic backbone that facilitates strong stacking and hydrophobic interactions with the aromatic rings of phenolic acids and flavonoids. Furthermore, its mesoporous structure and high specific surface area provide optimal accessibility and minimize steric hindrance for the diffusion of target molecules into the inner pores [27]. Discussion in the Context of Recent Research Trends: Recent studies (2020–2025) emphasize that the purification efficiency of MARs is highly sensitive to the initial extraction solvent. For polyphenols extracted using deep eutectic solvents (DESs), as performed in this study, the choice of resin is critical because the high viscosity and hydrogen-bonding potential of the DES matrix can interfere with traditional analysis. The high desorption ratio (>83%) of AB-8 indicates excellent reversibility, which is consistent with recent findings that weak-polar resins are more effective for recovering medium-polarity polyphenols compared to highly non-polar resins like D101 [28]. This reversibility is essential for maintaining the bioactive integrity of the enriched fractions for downstream functional applications.

3.2. Adsorption–Desorption Kinetic Characteristics

The adsorption kinetics of sour jujube polyphenols on AB-8 resin exhibited an initial rapid adsorption phase followed by a gradual approach to equilibrium (Figure 2A). The adsorption capacity (qₜ) increased sharply during the first 90 min, reaching approximately 40–50 mg/g, and thereafter remained nearly constant. This behavior is typical for macroporous resins and is primarily driven by the large initial concentration gradient between the polyphenol solution and the resin surface, which promotes rapid external mass transfer and surface adsorption. As adsorption sites become progressively occupied, both intraparticle diffusion and overall adsorption rates slow down, ultimately leading to equilibrium.

Similar kinetic patterns have been reported for plant polyphenols on weak-polar polystyrene resins. Yang et al. (2024) observed a comparable “fast-then-stable” kinetic profile when purifying white tea flavonoids using AB-8, attributing the rapid phase to concentration-driven diffusion and the plateau phase to site saturation [29]. Likewise, Cao et al. (2024) reported that phenolics from Plantago depressa exhibited rapid initial uptake followed by a diffusion-controlled equilibrium stage on AB-8 resin [30]. These results confirm that AB-8 provides efficient mass-transfer kinetics and favorable adsorption dynamics for sour jujube polyphenols, supporting its suitability for subsequent column-scale dynamic adsorption optimization.

The adsorption kinetics of sour jujube polyphenols on AB-8 were well fitted by the pseudo-second-order model (Figure 2B), with an excellent correlation coefficient (R² = 0.999). The close agreement between the calculated and experimental equilibrium adsorption capacities indicates that the adsorption process is primarily governed by chemisorption, involving specific interactions between phenolic hydroxyl groups and the aromatic domains of the polystyrene resin.

Pseudo-second-order kinetics have been widely reported for plant polyphenols on macroporous resins. Wang and Wang (2023) demonstrated that flavonoids from Salicornia europaea followed pseudo-second-order adsorption on macroporous resin due to affinity-driven interactions [31]. Yang et al. (2024) also found that white-tea flavonoids exhibited pseudo-second-order behavior during AB-8 adsorption [29]. These findings collectively confirm the suitability of AB-8 for efficient adsorption and provide theoretical support for its use in subsequent dynamic purification.

As shown in (Figure 2C), the desorption kinetics of the polyphenols from the jujube pulp by AB-8 resin exhibited a typical “rapid desorption–gradual equilibrium” pattern: within the first 120 min, the desorption amount rapidly increased, and then stabilized at approximately 40 mg/g. This indicates that the major reversibly bound polyphenol molecules immediately migrated from the resin pores to the external solution in the initial stage, and the desorption process was mainly driven by the initial concentration gradient; as the concentration difference between the eluent and the resin decreased, the diffusion rate slowed down, eventually reaching desorption equilibrium.

3.3. Optimization of Dynamic Adsorption–Desorption Process Parameters of Macroporous Resin

3.3.1. Influence of Sample Concentration on Adsorption Effect of Macroporous Resin

From the curve of the effect of the feed concentration on the adsorption rate (Figure 3A), it can be seen that the adsorption rate first increases and then decreases with the increase in the feed concentration. The adsorption rate reaches the maximum (about 81%) at a concentration of 2.4 mg/mL. At the low concentration stage (≤2.4 mg/mL), as the concentration increases, the mass transfer driving force between the polyphenol solution and the resin strengthens, and there are sufficient adsorption sites on the resin, which is conducive to the adsorption of polyphenol molecules, so the adsorption rate continues to rise. When the concentration exceeds 2.4 mg/mL, the adsorption sites on the resin gradually become saturated, and at the same time, the excessively high concentration reduces the concentration gradient in the mass transfer process, resulting in insufficient adsorption driving force, and the adsorption rate decreases accordingly.

3.3.2. The Influence of Sample Volume on the Adsorption Effect of Macroporous Resin

From the curve depicting the relationship between sample loading volume and polyphenol concentration in the effluent (Figure 3B), it is observed that the polyphenol concentration in the effluent exhibits a trend of slow initial increase followed by rapid elevation as the sample loading volume increases. Specifically, at low sample volumes (≤800 mL), the polyphenol concentration in the effluent remains at a low level with a gentle upward slope. However, once the volume exceeds 800 mL, the concentration rises sharply.

This phenomenon stems from the variation in the adsorption capacity of the resin. During the low-volume stage, AB-8 resin possesses abundant available adsorption sites, enabling sufficient adsorption of jujube pulp polyphenols, thus resulting in minimal polyphenol residues in the effluent. As the sample volume increases, the adsorption sites of the resin gradually approach saturation. When the volume exceeds the resin’s saturated adsorption capacity, excess polyphenols can no longer be effectively bound and are consequently discharged with the effluent, leading to a drastic surge in polyphenol concentration in the effluent.

3.3.3. The Influence of Ethanol Concentration on the Desorption Effect of Macroporous Resin

From the curve illustrating the relationship between ethanol volume fraction and polyphenol concentration in the eluate (Figure 3C), it is observed that the polyphenol concentration in the eluate exhibits a trend of rapid initial increase followed by a plateau as the ethanol concentration rises. Specifically, within the ethanol concentration range of 40% to 70%, the polyphenol concentration in the eluate surges rapidly from approximately 9 mg/g to 25 mg/g. However, when the ethanol concentration exceeds 70%, the rate of increase in polyphenol concentration slows down significantly and stabilizes.

This phenomenon is directly correlated with the elution capacity of ethanol. At low ethanol concentrations, as an organic solvent, increasing ethanol concentration enhances its ability to disrupt the binding forces (hydrophobic interactions and hydrogen bonds) between polyphenols and the resin matrix. This disruption facilitates the desorption of more resin-adsorbed polyphenols into the eluate, leading to the rapid rise in polyphenol concentration. When the ethanol concentration reaches 70%, its elution capacity is sufficiently strong to achieve nearly complete desorption of the jujube pulp polyphenols from the resin. Further increases in ethanol concentration beyond this point yield marginal improvements in elution efficiency, resulting in the plateau of the curve.

3.3.4. Drawing Dynamic Elution Curve

The dynamic elution curve of this experiment (Figure 3D) shows a characteristic of slow rise–rapid peak–gradual decline: during the initial elution period (0–20 min), the polyphenol concentration in the effluent rose slowly (remaining below 5 mg/mL); around 50 min of elution, the concentration reached its peak (approximately 21 mg/mL); then, the concentration dropped rapidly and gradually stabilized.

This trend is directly related to the mass transfer law during the elution process: at the beginning of elution, the ethanol eluent first fills the voids of the chromatography column and initially contacts the resin surface, with only a small amount of polyphenols being desorbed, so the concentration is relatively low; as elution proceeds, the eluent penetrates deeper into the resin pores, and a large amount of polyphenols bound to the resin are desorbed, causing the concentration to rise rapidly to its peak; after the elution time exceeds 50 min, most of the polyphenols on the resin have been desorbed, and the remaining polyphenols with strong binding forces are very few, so the concentration of the effluent gradually decreases and remains at a low level, indicating that the elution process is basically completed.

3.4. Physicochemical Characterization and Structural Identification of Polyphenolic Active Components in Jujube Pulp

3.4.1. Ultraviolet and Infrared Spectral Analysis

As shown in Figure 4A, the infrared spectra of the acid jujube pulp polyphenols before and after purification by macroporous resin show significant differences. The O-H stretching vibration peak in the range of 3300–3400 cm⁻¹ of the purified sample is sharper, indicating that matrix impurities such as polysaccharides and proteins have been effectively removed, thus making the phenolic hydroxyl signal purer. The intensity of the aromatic C-H vibration peak near 3005 cm⁻¹ is enhanced, reflecting the high selective adsorption capacity of the resin for aromatic structures. Meanwhile, the characteristic C=O stretching vibration of phenolic acids was mainly located at approximately 1710–1730 cm⁻¹, which became more distinguishable after resin purification. Additionally, in the 1500–1600 cm⁻¹ range, the C=C skeleton vibration peaks (1564, 1547, 1492 cm⁻¹) are significantly enhanced [32], which is in line with the typical characteristics of flavonoids and phenolic acids; simultaneously, the C-O stretching vibration peaks in the 1000–1200 cm⁻¹ range (such as 1066, 1154 cm⁻¹) are also significantly enhanced, suggesting that the phenolic hydroxyl and aromatic ether bond structures are more prominent, preliminarily confirming their phenolic nature [33]. The out-of-plane C-H bending vibration at approximately 895 cm⁻¹ is significantly enhanced in the purified sample, indicating an enrichment of ortho- or para-substituted aromatic ring structures. Overall, the AB-8 macroporous resin can effectively remove non-target components such as polysaccharides, significantly improving the clarity of characteristic functional group peaks (such as hydroxyl, carbonyl, aromatic rings, and C-O bonds) of polyphenols, verifying its excellent enrichment effect on acid jujube polyphenols and laying a reliable foundation for subsequent structural characterization and biological activity research.

As shown in Figure 4B, the purified jujube pulp polyphenols with AB-8 resin exhibit a shoulder peak near 360 nm, which is attributed to the conjugated carbonyl (C=O) groups in phenolic acids and flavonoids, indicating that the purified product contains a relatively complete conjugated structure system [34]. The UV spectrum confirms that AB-8 can effectively retain and enrich jujube pulp polyphenols with aromatic rings and conjugated structures, providing basic information for subsequent structural identification.

3.4.2. Molecular Weight Profile and Comparative Analysis

The HPGPC chromatogram (Figure 4C) of the sample exhibited a single, symmetrical peak with a retention time centered at approximately 9.86 min, indicating a relatively homogeneous distribution of the components. The calculated molecular weight distribution (Figure 4D) revealed that the weight-average molecular weight of the sample was approximately 1073 Da, with the distribution ranging primarily from 100 to 4000 Da.

This molecular weight profile differs significantly from many crude Ziziphus jujuba polysaccharides reported in the recent literature. Typically, bioactive Ziziphus jujuba polysaccharides are characterized as high-molecular-weight macromolecules. For instance, Li et al. (2023) reported that polysaccharides extracted from jujube fruits across different regions exhibited values ranging within 114–1730 kDa [35]. Similarly, Ji et al. (2022) isolated a high-molecular-weight fraction with an average Ziziphus jujuba polysaccharide weight of 241 kDa [36].

In contrast, the notably lower molecular weight observed in this study suggests that the purified fraction is likely composed of oligosaccharides or low-molecular-weight phenolic conjugates, rather than long-chain polysaccharides. This aligns with findings by Jia et al. (2025), who noted that degradation treatments (such as ultrasonic or enzymatic hydrolysis) can reduce the molecular weight of jujube polysaccharides to the range of 3–9 kDa, thereby enhancing their solubility and bioavailability [37]. The low polydispersity index (PDI) observed in our sample further confirms that the purification process effectively removed high-molecular-weight aggregates, yielding a refined fraction with potential for high absorption efficiency in biological systems [38].

3.4.3. Liquid Chromatography–Mass Spectrometry (LC-MS) Result Analysis

The total ion chromatogram (TIC) of the AB-8 purified polyphenol fraction from Ziziphus jujuba Mill. var. spinosa pulp is presented in Figure 5. The chromatogram exhibited a complex multi-peak profile within the retention time range of 0–30 min, indicating that the purified sample still contained a variety of phenolic acids, flavonoids, and their derivatives. Several small and sharp peaks observed in the 0–5 min region are characteristic of low-molecular-weight and highly polar phenolic acids, such as gallic acid and protocatechuic acid, which generally exhibit short retention times due to their strong hydrophilicity. Similar early-eluting phenolic acids have also been reported in jujube and other fruit extracts purified by macroporous resins [39]. The most intensive and concentrated peak cluster appeared in the retention time window of 8–12 min. In particular, the peak groups observed at 8.18, 8.32, 8.37, and 8.46 min are highly consistent with the reported elution behavior of chlorogenic acid and its positional isomers (neochlorogenic acid and cryptochlorogenic acid), while peaks detected around 11.20–11.45 min may correspond to catechin and epicatechin, which are typical flavan-3-ols commonly found in jujube fruits [40]. In addition, several medium-intensity peaks distributed in the range of 12–20 min suggest the presence of more complex flavonoids and phenolic esters, whose longer retention times reflect increased hydrophobicity and molecular weight. The relatively weak but continuous signals observed beyond 20 min further indicate the presence of trace amounts of high-hydrophobicity phenolic derivatives or aglycones.

To further confirm the identities of the major phenolic constituents, eight representative phenolic standards were selected for targeted LC–MS comparison analysis (Figure 6,

Table 2. Identification of the major polyphenolic compounds from Ziziphus jujuba Mill. var. spinosa pulp by HPLC–MS.

Peak no.	Compound Name	RT (min)	Precursor Ion (m/z)	Adduct	Fragment Ions (m/z)	Compound Category
A	Ferulic acid	3.85	195.06	[M+H]+	177, 145	Hydroxycinnamic acid
B	p-Coumaric acid	4.2	165.04	[M+H]+	119	Hydroxycinnamic acid
C	Catechin	4.15	291.08	[M+H]+	139, 123	Flavan-3-ol
D	Quercetin	10.79	303.05	[M+H]+	153, 137	Flavonol
E	Rutin	4.73	611.16	[M+H]+	465.10, 303.05	Flavonoid glycoside
F	Chlorogenic acid	10.72	355.1	[M+H]+	163.04	Phenolic acid ester
G	Gallic acid	4.2	171.03	[M+H]+	127	Hydroxybenzoic acid
H	Naringenin	7.87	273.08	[M+H]+	153	Flavanone

Note: Compounds were identified by comparing their retention times (RT), precursor ions, and MS/MS fragmentation patterns with authentic standards and literature data. All mass-to-charge ratios (m/z) were acquired in the positive electrospray ionization (ESI⁺) mode.

). As illustrated in Figure 6A–H, ferulic acid, p-coumaric acid, catechin, quercetin, rutin, chlorogenic acid, gallic acid, and naringenin were unambiguously identified based on the consistency of retention times and characteristic molecular ion peaks with those of authentic standards. Specifically, ferulic acid and p-coumaric acid exhibited protonated molecular ions at m/z 195.06 and 165.04, respectively, along with diagnostic fragment ions resulting from the loss of methoxy and carboxyl functional groups. Catechin showed a typical [M+H]⁺ ion at m/z 291.08, accompanied by fragment ions at m/z 139 and 123 generated via retro-Diels–Alder cleavage of the flavan-3-ol ring system. Quercetin and rutin were identified by their characteristic precursor ions at m/z 303.05 and 611.16, respectively, with rutin further exhibiting fragment ions at m/z 465.10 and 303.05 corresponding to the stepwise loss of glycosidic moieties. Chlorogenic acid was confirmed based on its [M+H]⁺ ion at m/z 355.10 and the diagnostic fragment ion at m/z 163.04 originating from the caffeic acid moiety.

Similarly, gallic acid and naringenin were identified by their molecular ions at m/z 171.03 and 273.08, respectively, together with their typical fragmentation behaviors. The identification of these representative phenolic acids and flavonoids demonstrates that AB-8 resin purification effectively enriched structurally diverse and bioactive polyphenols from jujube pulp, including hydroxybenzoic acids, hydroxycinnamic acids, flavan-3-ols, and flavonoid aglycones/glycosides. Consistent with previous studies on macroporous resin-purified fruit polyphenols [39], the concentration of major antioxidant compounds such as chlorogenic acid and catechin in the AB-8-treated fraction was significantly increased.

3.5. Discussion

In the present study, a robust and efficient strategy for the separation and purification of polyphenols from Ziziphus jujuba Mill. var. spinosa pulp was successfully developed based on macroporous adsorption resin technology. Among the five tested resins, AB-8 exhibited superior comprehensive performance in terms of adsorption rate, desorption efficiency, and adsorption capacity, highlighting its strong affinity for jujube polyphenols. Kinetic analysis demonstrated that the adsorption process followed a pseudo-second-order model, indicating that chemisorption dominated the interaction between polyphenolic compounds and the resin matrix.

Through systematic optimization of dynamic column parameters, the optimal operational conditions were established at a loading concentration of 2.4 mg/mL, a flow rate of 1.0 mL/min, and an ethanol elution concentration of 70% (v/v). Under these conditions, efficient enrichment and effective recovery of polyphenols were achieved, with stable performance and good reproducibility. Furthermore, the combined characterization using UV, FT-IR, and HPLC–MS confirmed that the purified fraction was significantly enriched in major bioactive polyphenols, particularly chlorogenic acid and catechin.

Overall, this work provides a practical and scalable purification protocol for obtaining high-purity polyphenols from Ziziphus jujuba Mill. var. spinosa pulp. The established process not only enhances the utilization efficiency of jujube resources but also offers a reliable technical foundation for the development of functional food ingredients, natural antioxidants, and value-added nutraceutical products derived from Ziziphus jujuba Mill. var. spinosa.

The systematic screening and optimization results highlight the high efficacy of macroporous adsorption resin technology for the purification of polyphenols from Ziziphus jujuba Mill. var. spinosa pulp. The superior performance of AB-8 resin over the other four candidates is primarily governed by its physicochemical compatibility with jujube polyphenols. As a weakly polar polystyrene-based resin, AB-8 provides a favorable microenvironment for the adsorption of medium-polarity phenolic compounds, such as chlorogenic acid and catechin, through hydrophobic interactions and stacking between the resin’s aromatic matrix and the phenolic rings.

The adsorption kinetics on AB-8 were exceptionally well-fitted by the pseudo-second-order model (R² = 0.999), which strongly indicates that the rate-limiting step is a chemisorption process rather than simple physical occlusion. This involves the formation of specific chemical bonds or strong interactions, likely hydrogen bonding between the phenolic hydroxyl groups and the active sites on the resin surface. During dynamic optimization, the observed peak in adsorption rate at a loading concentration of 2.4 mg/mL suggests that this concentration provides a sufficient mass transfer driving force while avoiding premature site saturation or increased viscosity that would hinder diffusion. Furthermore, the transition to 70% ethanol for elution efficiently disrupted the binding forces, achieving nearly complete recovery of the target components.

The multi-platform characterization further validated the purification efficiency. FT-IR spectra indicated that matrix interferences, specifically polysaccharides and proteins, were effectively removed, as evidenced by the sharpened phenolic hydroxyl signals and enhanced aromatic skeletal vibration peaks. The identification of eight major phenolic constituents by HPLC–MS, including phenolic acids (e.g., ferulic and gallic acids) and flavonoids (e.g., rutin and naringenin), confirms that AB-8 resin maintains a broad-spectrum enrichment capacity for structurally diverse bioactive molecules; these findings emphasize that the established purification protocol successfully preserves the chemical integrity and diversity of the jujube polyphenolic fraction.

3.6. Comparative Analysis and Future Perspectives

While this study established an efficient purification protocol using AB-8 resin, it is valuable to compare this approach with other emerging stabilization and characterization strategies. For example, nanoparticle fabrication has been successfully employed to enhance the antioxidative stability of Chondrus ocellatus polyphenols [41]. Although nano-encapsulation offers superior stability, macroporous resin adsorption provides a more cost-effective and scalable solution for initial bulk purification. Furthermore, the rigorous structural characterization performed in our study aligns with current high-standard quality evaluation protocols, similar to the microstructural and chemical assessments used for specialty crops like Coffea arabica with designation of origin [42] and the comprehensive phytochemical profiling of Brassica oleracea extracts [43]. These comparisons highlight that while different matrices require tailored strategies, the combination of DES extraction and resin purification balances purity, cost, and scalability effectively.

3.7. Economic Feasibility and Environmental Sustainability

The economic viability of the proposed process relies on the reusability of the AB-8 resin and the low cost of the DES components (choline chloride and levulinic acid). Unlike single-use solid-phase extraction cartridges, AB-8 resin can be regenerated multiple times with ethanol, significantly reducing operational costs in industrial applications.

Regarding environmental impact, although DESs are widely recognized for their low toxicity and biodegradability [7], their large-scale application must consider solvent recovery. As noted in recent reviews on hydrolyzed collagen production, while advanced extraction methods offer functional benefits, optimizing the balance between energy consumption (e.g., for removing viscous DES) and yield is crucial for sustainability [8]. In our protocol, the water-soluble nature of DES allows it to be effectively separated from the hydrophobic polyphenols during the resin adsorption phase, minimizing solvent waste and preventing contamination of the final product.

4. Conclusions

This study successfully established a scalable and efficient purification protocol for polyphenols from Ziziphus jujuba Mill. var. spinosa pulp using AB-8 macroporous resin, which exhibited superior selectivity with an adsorption capacity of 62.48 mg/g and a high desorption ratio of 83.40%. Kinetic analysis elucidated that the adsorption process follows a pseudo-second-order model (R² = 0.999), driven primarily by chemisorption mechanisms. Through dynamic process optimization, maximum efficiency was achieved with a loading concentration of 2.4 mg/mL and 70% (v/v) ethanol elution. Crucially, multi-spectral characterization validated the quality of the purified product: FT-IR analysis confirmed the effective removal of polysaccharide and protein impurities, while HPGPC revealed a homogenous low-molecular-weight distribution (M_w approx. 1073 Da) favorable for bioavailability. Furthermore, LC-MS profiling definitively identified eight key bioactive constituents, including chlorogenic acid, catechin, rutin, and quercetin, confirming the resin’s broad-spectrum enrichment capability. Collectively, this work provides a reproducible, cost-effective technical foundation for the industrial value-added utilization of jujube resources in functional foods and nutraceuticals.