Ultrasound-Assisted Extraction, Macroporous Resin Purification, and Antioxidant Activity of Chlorogenic Acid from Eucommia ulmoides Leaves

Abstract

Chlorogenic acid from Eucommia ulmoides leaves is a promising natural antioxidant for food applications, yet its extraction and purification require optimization to improve yield and purity. This study employed ultrasound-assisted ethanol extraction on fresh leaves, evaluating the effects of ethanol concentration, solid-to-liquid ratio, extraction time, and temperature on CGA yield. Optimal extraction parameters were determined using response surface methodology. Purification with NKA-II macroporous resin increased CGA purity to 82.72%. SEM analysis revealed wrinkled and porous surface structures, while FTIR confirmed the presence of characteristic hydroxyl, carbonyl, and aromatic groups. Under optimized conditions (70% ethanol, solid-to-liquid ratio 1:20 g/mL, 58 °C, 61 min), the extraction yield reached 6.96%. In vitro assays demonstrated strong antioxidant activity, with scavenging rates of 96.01% for DPPH, 89.69% for hydroxyl, and 99.82% for ABTS radicals at 5 mg/mL. These findings provide an efficient method for obtaining chlorogenic acid from Eucommia ulmoides leaves and support its potential as a functional food ingredient.

1. Introduction

Eucommia ulmoides (E. ulmoides), the only species of the genus Eucommia (family Eucommiaceae), is an endemic tree species to China and has long been regarded as an important traditional medicinal resource [1,2,3,4,5]. Owing to its diverse bioactive constituents and associated health-promoting effects, E. ulmoides has been widely applied in health products, functional foods, and related food applications [4,6]. Phytochemical studies have shown that this species contains various bioactive compounds, including lignans, phenylpropanoids, flavonoids, iridoids, and triterpenoids, which are associated with multiple biological activities [7,8,9]. These constituents exhibit multiple physiological effects, among them antioxidant [9,10], anti-inflammatory [11], blood pressure lowering [12], hypolipidemic [13], hypoglycemia [14,15], and antiviral [16]. While the bark has historically been the primary medicinal part, recent evidence indicates that the leaves share a similar chemical profile [13,17,18]. Notably, E. ulmoides leaves have been officially approved in China as a medicinal and edible resource, highlighting their considerable potential for further research and development in the food and health-related fields.

Chlorogenic acid (CGA), a widespread phenolic compound in plants, occurs at elevated concentrations in honeysuckle and E. ulmoides [19,20]. It exhibits a range of beneficial properties, including anti-aging, antibacterial, antiviral, lipid-lowering, and hypoglycemic properties [19,21,22]. Previous studies have demonstrated that, within E. ulmoides, CGA is preferentially accumulated in the leaves. For example, Yan et al. [23] reported significantly higher CGA content in leaves compared to bark. Reports suggest that the concentration of this compound in the leaves can vary widely, generally falling between 1% and 5.5% [24]. These findings highlight E. ulmoides leaves as a promising source for CGA extraction. Given its high abundance and application potential, developing an efficient and reliable extraction method is essential for further utilization.

CGA is a polar compound that is sensitive to strong light and high temperatures, yet it shows good solubility and stability in ethanol-based systems [25]. Several methods have been used to extract CGA from E. ulmoides, including ultrasound-assisted extraction, organic solvent extraction, water extraction, and enzyme-assisted extraction [26,27,28,29]. However, conventional solvent extraction and enzymatic methods often involve drawbacks such as safety concerns, environmental impact, high costs, and stringent operational conditions [30,31]. In contrast, ultrasound-assisted extraction has attracted attention as an effective alternative due to its ability to enhance mass transfer and improve extraction efficiency through cavitation-induced disruption of plant tissues [26,31,32].

In addition to CGA, E. ulmoides leaf extracts contain polysaccharides and proteins, which necessitates an appropriate purification step [33]. Among the available separation techniques, macroporous resin adsorption is widely used for natural product enrichment due to its high capacity, environmental compatibility, and suitability for scale-up [34,35]. The adsorption performance of target compounds depends strongly on resin polarity, with strongly polar resins proving effective for CGA isolation [19,33,36].

Based on the physicochemical properties of CGA and the principles of solid–liquid extraction, four key variables were selected for optimization: ethanol concentration, solid–liquid ratio, extraction temperature, and extraction time. Ethanol concentration affects solvent polarity and CGA solubility, the solid–liquid ratio influences mass transfer driving force, temperature enhances solubility and diffusion while requiring control to prevent degradation of CGA’s ester bond, and extraction time governs equilibration kinetics. These parameters represent the primary controllable factors in ultrasound-assisted extraction of phenolic compounds, as consistently reported in previous optimization studies [25,31,37,38]. Ultrasound power and frequency were fixed at 300 W and 40 kHz, respectively, based on preliminary trials confirming stable cavitation without excessive heating or CGA degradation.

In the study, ultrasound-assisted extraction was used to recover CGA from E. ulmoides leaves, with parameters optimized via response surface methodology following single-factor experiments. The resulting extract was subsequently purified using NKA-II macroporous resin, and the antioxidant activity of the purified CGA was evaluated. This integrated approach provides a practical basis for developing CGA-enriched extracts from E. ulmoides leaves as natural antioxidants and functional ingredients in food applications. Unlike earlier work that primarily focused on extraction optimization, the present study combines food-grade ultrasound extraction, resin purification, structural characterization, and functional assessment to offer a complete, application-oriented processing route.

2. Materials and Methods

2.1. Materials

The fresh E. ulmoides leaves are picked from Henan Jin Duzhong Agricultural Science and Technology Co., Ltd., in Xuchang of Henan Province, China. Fresh leaves were selected to minimize potential thermal degradation or chemical alteration of CGA and other labile phenolic compounds that might occur during conventional high-temperature drying [25]. Put the E. ulmoides leaves into a constant-temperature electric hot air blower box (model DH6-924385-l, Shanghai Xinmiao Medical Treatment Apparatus Manufacturing Co., Ltd., Shanghai, China) at 40 °C until a constant weight was achieved. This standardized mild drying step eliminates moisture variability, enabling extraction yields to be expressed on a dry weight basis and ensuring reliable comparison with literature data from dried materials. After drying, use a crusher (model FTT-2500G, Dongguan Fangtai Electric Appliance Co., Ltd., Dongguan, China) to crush the E. ulmoides leaves and sieve them through a 60-mesh screen. Store them in a refrigerator (model MR-540WSPZE, Midea Group Co., Ltd., Foshan, China) at 4 °C for future use.

2.2. HPLC Analysis of CGA

2.2.1. Preparation of CGA Standard Stock Solution

A CGA standard (10 mg; Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China) was accurately weighed and dissolved in an acetonitrile (HPLC grade, Shanghai Yien Chemical Technology Co., Ltd., Shanghai, China)–water solution to obtain a stock solution with a concentration of 10 mg/mL. The stock solution was stored at −20 °C until further use.

2.2.2. CGA Standard Curve Plotting

CGA standard stock solutions were respectively absorbed to prepare a standard series with concentrations of 0.5, 1, 2, 3, 4, 5 mg/mL, and determined by HPLC (model LC-20, Shimadzu Corporation, Kyoto, Japan). Chromatographic conditions are: ZORBAX Stable Bond C18 chromatographic column (4.6250 mm, 5 m, 2.5 mL) (Agilent Technologies, Inc., Santa Clara, CA, USA); Mobile phase: acetonitrile-water (13:87, v/v), wavelength: 327 nm, injection volume: 10 μL, flow rate: 1 mL/min, column temperature 30 °C. Repeat the measurement three times for each concentration and record the peak area. The linear regression equation of CGA obtained is y = (7 × 10⁶)x − 85,109 (R² = 0.9997) (Figure 1). The results indicated that CGA had a good linear relationship within the range of 0.5 to 5 mg/mL.

Figure 1.

CGA standard curve.

2.3. Extraction of CGA

Uniformly ground E. ulmoides leaf powder (1.0 g) was mixed with a specified volume of ethanol (analytical grade, Tianjin Tianli Chemical Reagent Co., Ltd., Tianjin, China) and subjected to ultrasound-assisted extraction using an ultrasonic cleaner (JP-060ST, Shenzhen Jiemeng Cleaning Equipment Co., Ltd., Shenzhen, China) equipped with a temperature-controlled water-bath system. The apparatus operates at a fixed frequency of 40 kHz with a nominal maximum output power of 300 W, which was kept constant throughout all experiments. Given the constant sample mass, this setting corresponds to a power density of 300 W/g, calculated based on the nominal output power. The selected power level was determined according to preliminary trials and was maintained unchanged to ensure experimental reproducibility and to isolate the effects of extraction temperature, time, and solvent concentration [26,31]. Extraction was conducted for the designated duration at the preset temperature, which was controlled by a circulating water bath to within ±1 °C of the target value. The extract was subsequently centrifuged at 4000 r/min for 15 min (TDL-5, Shanghai Anting Scientific Instrument Factory, Shanghai, China). The supernatant was collected, and the CGA content was determined by HPLC. The extraction yield of CGA was calculated according to Equation (1):

X = C × V × N/M × 100% (1)

where X represents the extraction yield of CGA (%), C is the CGA concentration determined from the calibration curve (mg/mL), V is the total volume of the extract (mL), N is the dilution factor, and M is the mass of E. ulmoides leaf powder (g).

Peak identification relied on retention time matching with the CGA standard and UV detection at 327 nm. Method selectivity was verified by the standard addition method: known amounts of CGA were spiked into leaf extract samples, yielding linear peak area increases (recovery 95–104%, R² > 0.997) with no deviation or additional peaks, confirming the absence of co-eluting interference under the optimized conditions.

2.4. Single-Factor Experiments

Single-factor experiments were conducted to evaluate the effects of individual extraction parameters on CGA yield. E. ulmoides leaf powder (1.0000 g) was used in each experiment. The investigated parameters included ethanol concentration (40%, 50%, 60%, 70%, and 80%), solid–liquid ratio (1:10, 1:15, 1:20, 1:25, and 1:30 g/mL), extraction temperature (40, 50, 60, 70, and 80 °C), and extraction time (15, 30, 45, 60, and 75 min).

2.5. Response Surface Methodology Design

Based on a single factor, a four-factor three-level experimental Design was conducted using Box-Behnken in the Design-Expert software (version 13, SAS Institute Inc., Cary, NC, USA). A total of 29 experimental runs were performed. The experimental data were statistically analyzed, and the optimal extraction conditions were determined by evaluating the regression model, three-dimensional response surface plots. The predicted optimal conditions were subsequently validated through confirmatory experiments.

2.6. Purification of CGA

NKA-II macroporous resin (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China), a polar polystyrene-based resin, was employed for the purification of CGA. The selection of this resin was based on preliminary adsorption tests and literature reports demonstrating its strong affinity for phenolic acids such as CGA [19,33,36]. The polar nature of NKA-II facilitates hydrogen bonding and dipole interactions with the hydroxyl and carboxyl groups of CGA, enhancing its selective adsorption from the aqueous ethanolic crude extract. The purification procedure was as follows: the crude extract was loaded onto the resin column at a flow rate of 2 bed volumes (BV). Impurities were removed by sequential washing with 4 BV of distilled water followed by 3 BV of 20% ethanol. Elution of CGA was then performed using 6 BV of 60% ethanol. The eluate was collected, concentrated under vacuum, and freeze-dried to yield the purified CGA. Purity of the final product was assessed by HPLC.

2.7. Analysis of the Microstructure of CGA

The microstructure of CGA was tested using a scanning electron microscope (SEM) (model SEM 5000X, Guoyi Quantum Technology (Hefei) Co., Ltd., Hefei, Anhui Province, China) at magnifications of 1000×, 5000×, 10,000× and 50,000× and at a working voltage of 5 kv.

2.8. Fourier Transform Infrared (FTIR) Analysis

FTIR spectroscopy was applied to analyze the functional groups of the purified CGA isolated from E. ulmoides. Prior to spectral acquisition, the sample was thoroughly dried at room temperature to reduce potential interference from residual moisture. FTIR spectra were collected in the wavenumber range of 4000–400 cm⁻¹ at a spectral resolution of 4 cm⁻¹, with 32 scans averaged for each measurement. Air was used as the background reference, and the obtained spectra were subsequently analyzed to identify the characteristic absorption bands of the sample. FTIR spectra were recorded to confirm the presence of characteristic functional groups in the purified fraction. Spectra were compared with literature data for CGA [39,40,41]. While FTIR identifies key groups (hydroxyl, carbonyl, aromatic rings), definitive compound identification relied on HPLC retention time matching with the authentic standard.

2.9. Antioxidant Activity of CGA

2.9.1. DPPH Radical Scavenging Assay

The DPPH radical scavenging assay was performed according to the method described by Wang et al. [42]. with minor adjustments. Briefly, 2.0 mL of CGA solution at varying concentrations (1, 2, 3, 4, and 5 mg/mL) was mixed with 2.0 mL of DPPH methanolic solution (Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China). The mixture was incubated at 25 °C for 30 min in the dark, after which absorbance was measured at 517 nm (A₁). Absorbance of the sample with anhydrous ethanol in place of DPPH solution was recorded as A₂. Blank absorbance (A₀) was obtained by replacing the sample with distilled water. Vitamin C (VC) served as positive control, following the same procedure.

2.9.2. ABTS Radical Scavenging Assay

The ABTS radical scavenging assay was adapted from the procedure reported by Ramesh et al. [43]. with minor modifications. In brief, 100 µL of CGA solution at concentrations of 1, 2, 3, 4, and 5 mg/mL was added to separate test tubes containing 1.8 mL of ABTS working solution (Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China). After thorough mixing, the mixtures were incubated in the dark for 6 min. Absorbance was then measured at 734 nm (A₁). Sample absorbance without ABTS (A₂) was obtained by substituting distilled water for the ABTS solution, while blank absorbance (A₀) was recorded by replacing the sample with distilled water. VC was included as a positive control, following the identical protocol.

2.9.3. Hydroxyl Radical Scavenging Assay

The hydroxyl radical scavenging assay was adapted from the method of Kramberger et al. [44]. with minor modifications. To each reaction tube, 100 µL of CGA solution at concentrations of 1, 2, 3, 4, and 5 mg/mL was added, followed by 1 mL of 0.15 mmol/L FeSO₄, 0.4 mL of 2 mmol/L salicylic acid, 1.0 mL of 4.0 mmol/L H₂O₂, and 0.4 mL of distilled water. The mixtures were incubated at 37 °C for 1 h. Absorbance was measured at 510 nm (A₁). Sample absorbance without salicylic acid (A₂) was determined by substituting distilled water for the salicylic acid solution, while blank absorbance (A₀) was obtained by replacing the sample with distilled water. VC served as the positive control under the same conditions.

The calculation formula for DPPH radical scavenging effect, ABTS radical scavenging ability and hydroxyl radical scavenging effect is as follows:

Scavenged hydroxy (%) = 1 − (A₁ − A₂)/A₀ × 100% (2)

where A₁ is the absorbance of the sample, A₂ is the absorbance of the blank, and A₀ is the absorbance of the control without sample. This formula was applied uniformly to DPPH, ABTS, and hydroxyl radical assays, as it is the standard method for comparing radical scavenging capacity across different mechanisms [45,46]. Note that DPPH and ABTS primarily involve single electron transfer or hydrogen atom transfer, while hydroxyl radicals are highly reactive and non-selective.

2.10. Statistical Analysis

All extraction and antioxidant assays were performed with three independent biological replicates (n = 3), with each replicate representing separately prepared samples from distinct batches or independent extraction runs. For HPLC analysis, each biological sample was injected in triplicate (technical replicates) to ensure analytical precision. Data are expressed as mean ± standard error. One-way analysis of variance was performed using IBM SPSS Statistics (v.27.0, IBM Corp., Armonk, NY, USA), and figures were generated using GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, USA). Different letters denote significant differences (p < 0.05), while identical letters indicate no significant differences (p > 0.05)

3. Results

3.1. Single-Factor Experiment

By investigating four factors—ethanol concentration (Figure 2a), solid-to-liquid ratio (Figure 2b), extraction temperature (Figure 2c), and extraction time (Figure 2d)—it was found that each factor significantly influenced the extraction yield of eucommia CGA, exhibiting a trend of initial increase followed by decrease. Peak values were observed at 70% ethanol (yield 6.47%), solid-liquid ratio of 1:20 g/mL (6.66%), temperature of 60 °C (6.83%), and ultrasonic duration of 60 min (6.71%). These results indicate that appropriate ethanol concentration, solid-to-liquid ratio, extraction temperature, and ultrasonication duration play crucial roles in enhancing the extraction efficiency of CGA.

Figure 2.

Effect of ethanol concentration (a), solid-liquid ratio (b), extraction temperature (c), and extraction time (d) on extraction rate of CGA. The same lowercase letter above the bars indicates no significant difference (p > 0.05), while different letters indicate significant differences (p < 0.05).

3.2. Response Surface Optimization of the Extraction Process of E. ulmoides CGA

3.2.1. Analysis of Variance and Regression Model

Four factors, namely ethanol concentration (A), solid-liquid ratio (B), extraction temperature (C), and extraction time (D), were selected to optimize the extraction rate of CGA. The experimental design scheme is shown in Table 1.

Table 1.

Test factor level table of the response surface.

Level	Factors
	A (%)	B (g/mL)	C (°C)	D (min)
−1	60	1:15	50	45
0	70	1:20	60	60
1	80	1:25	70	75

In order to optimize the process conditions for ultrasound-assisted extraction of CGA leaves, the four-factor three-level Box-Behnken test was adopted. The test was carried out according to the design scheme of the response surface test, and the results are shown in Table 2.

Table 2.

Response surface design and results.

Sequence Number	A (%)	B (g/mL)	C (°C)	D (min)	R (%)
1	60	1:15	60	60	6.53
2	80	1:15	60	60	6.15
3	60	1:25	60	60	6.21
4	80	1:25	60	60	5.99
5	70	1:20	50	45	6.12
6	70	1:20	70	45	6.06
7	70	1:20	50	75	6.10
8	70	1:20	70	75	5.95
9	60	1:20	60	45	6.03
10	80	1:20	60	45	6.21
11	60	1:20	60	75	5.98
12	80	1:20	60	75	6.04
13	70	1:15	50	60	6.35
14	70	1:25	50	60	6.06
15	70	1:15	70	60	6.03
16	70	1:25	70	60	6.19
17	60	1:20	50	60	6.35
18	80	1:20	50	60	6.42
19	60	1:20	70	60	6.00
20	80	1:20	70	60	6.01
21	70	1:15	60	45	6.07
22	70	1:25	60	45	6.02
23	70	1:15	60	75	6.24
24	70	1:25	60	75	6.01
25	70	1:20	60	60	6.95
26	70	1:20	60	60	6.97
27	70	1:20	60	60	6.93
28	70	1:20	60	60	6.98
29	70	1:20	60	60	6.89

3.2.2. Regression Model Analysis

Regression analysis was conducted on the data in Table 2 using Design-Expert software. Table 3 presents the results of variance analysis of the model and the significance test results of each coefficient.

Table 3.

Variance analysis of regression model.

Source	Sum of Squares	df	Mean Square	F-Value	p-Value
Model	0.2670	14	0.0191	81.45	<0.0001
A	8.33 × 10−6	1	8.33 × 10−6	35.46	0.0231
B	0.0016	1	0.0016	6.95	0.0694
C	0.0040	1	0.0040	17.22	0.0010
D	0.0007	1	0.0007	2.88	0.1117
AB	0.0000	1	0.0000	0.1068	0.7487
AC	0.0000	1	0.0000	0.0000	0.0300
AD	0.0016	1	0.0016	6.83	0.0204
BC	0.0006	1	0.0006	2.67	0.1246
BD	0.0001	1	0.0001	0.4270	0.5240
CD	0.0090	1	0.0090	38.54	<0.0001
A2	0.1250	1	0.1250	533.91	<0.0001
B2	0.0897	1	0.0897	382.98	<0.0001
C2	0.0897	1	0.0897	382.98	<0.0001
D2	0.0841	1	0.0841	358.94	<0.0001
Residual	0.0033	14	0.0002
Lack of Fit	0.0028	10	0.0003	2.12	0.2439
Pure Error	0.0005	4	0.0001
Cor Tptal	0.2703	28

As presented in Table 3, the response surface model yielded an F-value of 81.45 (p < 0.0001). Significant linear effects were observed for ethanol concentration (A) and extraction temperature (C), along with notable interaction effects for AC, AD, and CD, as well as quadratic terms A², B², C², and D² (p < 0.05). The fitted multiple linear regression equation was Y = 6.27 + 0.0008A − 0.0117B − 0.0183C − 0.0075D + 0.0025AB + 0.02AD − 0.0125BC + 0.005BD − 0.0475CD − 0.1388A² − 0.1176B² − 0.1176C² − 0.1138D², with R² = 0.9879, adjusted R² = 0.9757, and predicted R² = 0.9382. These coefficients confirm strong model fit and predictive performance. Based on F-values, the factors ranked in decreasing order of influence on CGA yield were ethanol concentration, extraction temperature, solid-liquid ratio, and extraction time.

Three-dimensional response surfaces (Figure 3) illustrate the interactions among factors affecting CGA yield. Each surface displayed a clear parabolic curvature, reflecting effective quadratic fitting and the presence of an optimum. Yields increased to a maximum before declining with further changes in parameters, a pattern consistent across interactions. Moderate levels of each factor favored higher extraction rates, supporting the reliability and predictive accuracy of the regression model.

Figure 3.

Response surface plots showing the interactive effects of extraction parameters on the extraction rate of CGA. (a) Ethanol concentration and solid–liquid ratio; (b) Ethanol concentration and extraction temperature; (c) Ethanol concentration and extraction time; (d) Solid–liquid ratio and extraction temperature; (e) Solid–liquid ratio and extraction time; (f) Extraction temperature and extraction time. The color scale represents the extraction rate of CGA (%), with warmer colors (red/yellow) indicating higher rates and cooler colors (blue/green) indicating lower rates.

The regression model predicted optimal extraction conditions of 70.43% ethanol, solid-liquid ratio of 1:19.84 g/mL, temperature of 58.38 °C, and ultrasonic time of 61.33 min, corresponding to a projected yield of 6.98%. For practical applicability, the parameters were adjusted to 70% ethanol, 1:20 g/mL solid-liquid ratio, 58 °C, and 61 min. Under these conditions, triplicate experiments gave an average yield of 6.96%.

3.3. Results of CGA Purification

The CGA samples obtained after purification with NKA-II macroporous resin and subsequent freeze-drying were analyzed by HPLC, and the results are presented in Figure 4. As shown in Figure 4a, the standard CGA exhibited a single sharp peak at approximately 2.0 min with high purity. In contrast, the crude extract before purification (Figure 4b) displayed the CGA peak along with several impurity peaks, indicating lower purity. After purification (Figure 4c), the CGA peak became predominant, with significantly reduced impurity peaks, confirming the effective removal of impurities. The purity of CGA in the final purified sample was calculated to be 82.72% based on peak area normalization. These results demonstrate that the ultrasound-assisted extraction combined with NKA-II macroporous resin purification is an efficient process for obtaining relatively high-purity CGA from Eucommia ulmoides leaves.

Figure 4.

HPLC chromatograms of CGA standard (a), crude extract (b), and purified sample (c).

3.4. Results of Microstructure Analysis of CGA

SEM images reveal that CGA forms flocculent aggregates composed of irregular polygonal and short rod-shaped particles (Figure 5). The particles are tightly packed yet exhibit a porous architecture. At higher magnification, the particle surfaces show distinct wrinkles and pores. While SEM alone cannot confirm chemical identity, the observed microstructure is attributed to a CGA-rich material, consistent with the HPLC purity of 82.72% (Figure 4). This type of porous and roughened surface morphology typically corresponds to an increased specific surface area, which may enhance contact with solvents and facilitate dissolution. the observed dense yet porous microstructure suggests that the obtained product possesses morphological characteristics often associated with favorable dissolution behavior, supporting further studies on its functional performance.

Figure 5.

SEM images of purified CGA from Eucommia ulmoides leaves at different magnifications. (a) 1000×; (b) 5000×; (c) 10,000×; (d) 50,000×.

3.5. FTIR Analysis

As illustrated in Figure 6, the FTIR spectrum of the purified CGA displayed a broad absorption band centered at 3355 cm⁻¹, corresponding to the O–H stretching vibrations of phenolic and carboxylic hydroxyl groups. A prominent peak at 1687 cm⁻¹ was assigned to the stretching vibration of the ester carbonyl (C=O) group. The bands observed at 1605 cm⁻¹ and 1521 cm⁻¹ are characteristic of aromatic C=C skeletal vibrations. Furthermore, absorption bands appearing at 1288, 1192, and 1115 cm⁻¹ were mainly associated with C–O stretching vibrations. Taken together, these spectral features are in good agreement with the characteristic FTIR absorption patterns of CGA reported in previous studies.

Figure 6.

FTIR spectrum of CGA isolated from E. ulmoides.

3.6. Results of Antioxidant Activity Assays

Figure 7 illustrates the concentration-dependent antioxidant capacity of purified CGA. Scavenging activity against DPPH, hydroxyl, and ABTS radicals increased markedly with rising mass concentration. At 5 mg/mL, clearance rates reached 96.01% for DPPH radicals, 89.69% for hydroxyl radicals, and 99.82% for ABTS radicals—values approaching saturation and indicative of robust performance. Based on the concentration–response curves, the IC₅₀ values of CGA were calculated by linear interpolation to be 0.84 mg/mL for DPPH radicals, 0.93 mg/mL for ABTS radicals, and 1.28 mg/mL for hydroxyl radicals, respectively. These results demonstrate that CGA possesses effective and broad-spectrum radical scavenging capacity in vitro, with comparatively higher activity toward DPPH and ABTS radicals than hydroxyl radicals.

Figure 7.

Scavenging rates of CGA. (a) DPPH radical scavenging rate; (b) Hydroxyl radical scavenging rate; (c) ABTS radical scavenging rate.

4. Discussion

CGA is a key active component in E. ulmoides, formed by the condensation of hydrophilic quinic acid and hydrophobic caffeic acid to create a condensed phenolic acid [22]. The molecule features oxidizable hydroxyl groups and an ester linkage prone to hydrolysis at elevated temperatures [35]. Consequently, CGA exhibits favorable solubility in solvents of moderate polarity while showing thermal instability [9]. The yield of CGA in this work proved sensitive to ethanol concentration, solid-liquid ratio, extraction temperature, and ultrasonic duration. Each parameter followed a similar pattern: yields rose to a peak before declining at higher values. Maximum extraction occurred at 70% ethanol, beyond which further increases led to gradual reductions. Higher ethanol concentrations, while promoting CGA dissolution, may also extract liposoluble impurities, thereby lowering solvent selectivity and adversely affecting CGA recovery [47]. Solvent volume directly influences CGA yield through the solid-liquid ratio. Within the tested range, maximum extraction occurred at 1:20 g/mL, followed by a decline and subsequent stabilization at higher ratios. Temperature displayed a comparable pattern: elevated levels accelerate molecular diffusion and component dissolution [37,38], yet CGA—as a heat-sensitive compound—degrades beyond 70 °C due to ester bond hydrolysis, thereby lowering yields [48]. Short ultrasonic times limit cell wall disruption and CGA release, while prolonged exposure risks degrading the extracted CGA via mechanical shear and promotes impurity solubilization, compromising efficiency [38]. Liu et al. [49] reported a CGA yield of 4.27 mg/g from dandelin using ultrasound-assisted extraction. Li Jichang et al. [38]. applied response surface methodology to optimize CGA extraction, attaining 0.013 mg/mL. Response surface results here align with Qin Rulan et al. [50]., confirming ethanol concentration as the dominant factor affecting CGA yield. The decline in CGA yield at temperatures above 58 °C and extraction times beyond 61 min (Figure 2c,d) aligns with the known thermal and oxidative instability of CGA, which contains an ester bond and catechol moiety prone to hydrolysis and oxidation [25,38]. Ultrasound cavitation can induce localized high temperatures and free radicals that could exacerbate these effects under harsh conditions [45]. The optimized parameters (58 °C, 61 min) were selected to maximize extraction efficiency while operating below reported thresholds for significant degradation (>70 °C or prolonged exposure) [25,48]. Mild pre-drying at 40 °C further minimized potential loss during sample preparation. Although direct quantification of degradation products was not performed in this study, the observed yield trends and high final purity (82.72%) indicate that degradation was effectively controlled under the chosen conditions.

The NKA-II resin purification increased CGA purity to 82.72%, a level sufficient for many functional food applications. Phenolic-rich extracts in the 70–90% purity range are widely accepted as natural antioxidant ingredients in food matrices, where co-extracted compounds may provide synergistic benefits while maintaining cost-effectiveness and scalability [45,51]. Although higher purity (>95%) could be achieved through additional techniques such as preparative chromatography, these would significantly increase cost, complexity, and yield loss, making them less practical for food-grade ingredient development. The present one-step resin method was therefore optimized to balance purity, yield, and industrial feasibility. Chromatographic comparison showed a marked reduction in both the number and intensity of impurity peaks after purification, with the CGA peak predominating, consistent with the resin’s selective adsorption for phenolic acids. NKA-II resin achieves adsorption primarily via hydrophobic interactions and hydrogen bonding while repelling hydrophilic impurities [52]. This is supported by recent work. Liu et al. [49] employed the analogous NKA-II resin in an ultrasonic-assisted deep eutectic solvent system for CGA from E. ulmoides leaves, with adsorption following pseudo-second-order kinetics and the Freundlich model. Jiang et al. [48]. reported enhanced CGA recovery and purity using deep eutectic solvents combined with macroporous resins. The purity obtained here is slightly lower than that achieved with integrated optimization approaches, likely due to the absence of auxiliary techniques. Nonetheless, the NKA-II resin method is simple, cost-effective, and scalable, producing a fraction suitable for bioactivity assessment and functional food applications.

In comparison with previously reported methods employing ultrasound-assisted extraction combined with macroporous resin purification for CGA from E. ulmoides leaves [33,34,48], the workflow proposed in this study demonstrates several practical advantages. The optimized extraction conditions (58 °C for 61 min) are milder in both temperature and duration than those reported in some earlier studies that applied higher temperatures or prolonged extraction times [25,37], which may contribute to reduced energy consumption and improved stability of CGA. In addition, the use of aqueous ethanol as the sole solvent throughout both extraction and purification simplifies the overall operation, avoids intermediate solvent exchange, and is consistent with food-grade and environmentally friendly processing principles. The optimized solid–liquid ratio of 1:20 g/mL represents a balanced compromise between extraction efficiency and solvent consumption, preventing the excessive solvent usage observed in protocols employing higher ratios. Furthermore, the direct compatibility between the ethanolic extract and subsequent NKA-II macroporous resin purification enhances the continuity of the process, thereby improving its practical feasibility and potential scalability.

Previous studies have also indicated that CGA–related compounds in the solid state often form aggregated structures with distinct microstructural features, and such morphology is generally associated with their dissolution properties and transport behaviors [39,53]. In line with this, the microstructural characteristics observed in this study reveal that the purified CGA exhibits a consolidated morphology with a certain degree of porosity. This type of porous and high-surface-area morphology is consistent with the potential for favorable dissolution performance [51]. Overall, from a microstructural perspective, the SEM results suggests the possibility of suitable dissolution behavior, which would be valuable for its further application. A limitation of this study is the lack of dissolution or release kinetics data, which prevents quantitative assessment of how the observed porous microstructure influences functional properties such as solubility or bioavailability. While SEM suggests favorable characteristics, future work should include these measurements to confirm microstructure-performance relationships.

The FTIR spectrum of CGA extracted from E. ulmoides leaves displayed several characteristic absorption bands indicative of its functional groups. The broad peak observed at 3355 cm⁻¹ corresponds to the stretching vibrations of hydroxyl (-OH) groups, reflecting the polyphenolic nature of CGA, consistent with previous reports on phenolic acids [39,40,54,55]. The strong absorption at 1687 cm⁻¹ is assigned to the C=O stretching vibration of the carboxylic group, confirming the presence of ester and carboxyl functionalities in the molecule, which are crucial for its antioxidant activity [40,45]. The bands at 1605, 1521, and 1443 cm⁻¹ are attributed to the aromatic C=C stretching vibrations, indicating the benzene ring structure typical of CGA. These peaks are consistent with those reported in the analysis of plant-derived CGA and related polyphenols [41,56]. Furthermore, the absorption at 1288 cm⁻¹ corresponds to the C–O stretching of the ester linkage, while the peaks at 1192 and 1115 cm⁻¹ can be assigned to C–O–C vibrations and phenolic C–O stretching, respectively, which are indicative of glycosidic and ester bonds in CGA [32,41,45,57]. Collectively, these spectral features match those reported for CGA and confirm the presence of key functional groups—hydroxyl, carbonyl, and aromatic rings—in the purified material [39,40,41]. It should be noted that while FTIR supports the presence of these groups, it cannot exclusively distinguish CGA from other phenolic acids with similar functional moieties. The observed profile is consistent with a molecule possessing the structural basis for antioxidant activity, aligning with the radical scavenging results presented [41,54].

The in vitro antioxidant activity of purified CGA showed concentration-dependent scavenging of DPPH, ABTS, and hydroxyl radicals. IC₅₀ values were 0.84, 0.93, and 1.28 mg/mL, respectively, which are comparable to those reported for CGA from other plant sources under similar assay conditions [45]. For example, CGA from E. ulmoides leaves extracted with natural deep eutectic solvents exhibited lower IC₅₀ values of 0.075 mg/mL (DPPH), 0.020 mg/mL (ABTS), and 0.314 mg/mL (hydroxyl radical) [8], while CGA from coffee beans showed DPPH IC₅₀ values in the range of 0.5–1.0 mg/mL [58].The slightly higher values in this study may reflect differences in assay protocols and extract purity. While assays were conducted up to 5 mg/mL to establish full dose–response curves, substantial activity was already evident at 1–2 mg/mL, aligning with typical usage levels of phenolic antioxidants in food systems (0.05–0.5%, w/v) [46,51]. Vitamin C served as a standard positive control due to its established efficacy and widespread use in food-related assays [59]. Structurally similar phenolics were not included, as the focus was on evaluating CGA itself rather than comparative ranking. Crude extracts were not tested in parallel, but the marked increase in CGA purity (to 82.72%) after purification strongly suggests that the enhanced antioxidant performance is primarily attributable to CGA enrichment. These results support the potential of CGA from E. ulmoides leaves as a natural antioxidant ingredient in functional foods, although in-matrix stability and bioaccessibility studies are needed for practical application.

A limitation of this study is the use of leaves from a single harvest and location, which restricts evaluation of batch-to-batch or seasonal variability in phytochemical composition. This approach was chosen to provide a standardized baseline during method optimization [25]. Future studies should validate the optimized extraction and purification parameters using leaves from multiple harvests and growing regions to ensure broader applicability and robustness.

5. Conclusions

This study established an optimized ultrasound-assisted extraction and NKA-II resin purification protocol for CGA from E. ulmoides leaves, achieving a maximum extraction yield of 6.96% (70% ethanol, 1:20 solid–liquid ratio, 58 °C, 61 min) and a final purity of 82.72%. Structural characterization by SEM and FTIR confirmed the the retention of typical morphology and functional groups, and in vitro assays demonstrated strong antioxidant activity. These results provide a simple, food-grade method for producing CGA-enriched extracts with purity suitable for functional food applications. The protocol offers operational simplicity, scalability potential, and cost-effectiveness compared to more complex multi-step approaches. However, further validation across multiple raw material batches, pilot-scale trials, technoeconomic evaluation, and life-cycle assessment are needed to confirm industrial feasibility and environmental sustainability.

Author Contributions

Conceptualization, Q.W. and X.L.; methodology, Q.W.; investigation, Q.W.; resources, Y.Z.; data curation, Q.W.; writing—original draft preparation, Q.W.; writing—review and editing, X.L.; visualization, Q.W.; formal analysis, Q.W.; supervision, X.L. and K.L.; project administration, Y.Z., Y.Y. and K.L.; funding acquisition, Y.Z., X.X. and Y.Y. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Conflicts of Interest

Author Xiaoxiao Liang is employed by Henan Guang’an Biotechnology Co., Ltd., and Author Keke Li is employed by Henan Golden Lily Biotechnology Co., Ltd., These companies provided technical support and resources as part of a university-industry cooperation project. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding Statement

This work was funded by the National Natural Science Foundation of China (32130099). The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.