Macroporous adsorbent resin-based wheat bran polyphenol extracts inhibition effects on H₂O₂-induced oxidative damage in HEK293 cells

Abstract

In the present study, polyphenol-rich extracts of wheat bran (PEWB) were prepared via adsorption on macroporous resins and desorption with ethanol. Extraction was performed using aqueous ethanol and four different types of macroporous adsorbent resins for isolation. Specifically, the properties of the macroporous resins were investigated by adsorption and desorption tests. Total polyphenolic content of PEWB was determined using the Folin–Ciocalteu method, and its resistance effects against hydrogen peroxide (H₂O₂)-induced oxidation on HEK293 cells were assessed by cell viability and the reactive oxygen species (ROS) assay. The results indicate that resin NKA-9 displayed excellent adsorption and separation ability, as well as provided insight into the generation of PEWB from wheat bran extracts. In addition, these results suggest that pretreating HEK293 cells with PEWB prior to H₂O₂ exposure exhibited a significantly increased survival ratio and reduced the ROS levels. Further investigation involving the phenolic content of PEWB identification and quantification demonstrated that ferulic acid was the most abundant phenolic compound in a number of extracts, which was also confirmed with MTT and ROS assays. Our study revealed that PEWB can prevent HEK293 cells from H₂O₂-induced oxidative damage.

---

1. Introduction

Cereals are an important part of human nutrition, which provide a wide range of nutrients and biologically active compounds. Cereal bran, separated in the milling process during the production of refined flours¹ are rich in phenolic acids² and have been widely used for providing health benefits to consumers in addition to general nutrition. Specifically, phenolic acids and flavonoids are present in cereals in both free and conjugated forms, reaching the highest concentrations in the aleurone layer of cereal grains.³ They have excellent antioxidant activity,⁴ such as displaying anti-inflammatory responses, preventing low-density lipoprotein oxidation and possessing antithrombotic, antihypertensive, and carcinostatic behavior.⁵ Wheat bran, which is produced in enormous quantities worldwide as an important byproduct of the cereal industry, serves as a good source of dietary fibre and antioxidants.^6,7 Previous studies reported that wheat bran extracts (WBEs) possess various physiological activities, such as anticancer capacity,⁸ indicating that the antioxidant ability of wheat in the diet is associated with its antitumor activity.⁹

Several approaches of enriching and separating active constituents from the fermentation broth have been reported, such as liquid–liquid extraction,^10,11 solid–liquid extraction,^12–15 solid phase extraction,¹⁶ membrane filtration,¹⁷ ion exchange, and adsorption.¹⁸ Among all these methods, adsorption has attracted the most attention, due to its low cost, high efficiency, and simple operating procedure.¹ Macroporous resins are durable polar, nonpolar, or slightly hydrophilic polymers that display a high adsorption capacity and high recovery, with a low cost.¹⁹ Recently, macroporous resin adsorption technology has gained popularity in the pharmaceutical field, and it has also been used for polyphenol and flavonoids purification.^20–22 The effects of different macroporous adsorbent resins (DA201-C, NKA-II, NKA-9, and H1020) on the purification of WBEs were evaluated. Static, dynamic adsorption, and desorption tests were applied in our experiments. Further investigation on the phenolic content of polyphenol-rich extracts of wheat bran (PEWB) was also performed. The resistance effect of WBEs against H₂O₂-induced oxidative damage has been reported previously. However, to the best of our knowledge, there are very few studies involving PEWB resistance against oxidative damage on cells. Therefore, to have a deep understanding about the ability of PEWB to prevent H₂O₂-induced cell damage is of significance.

2. Materials and methods

2.1. Chemicals and reagents

Raw wheat bran was provided from the Yihai Kerry Food Industry Co., Ltd. (Kunshan, China). HEK 293 cells were obtained from the American Type Culture Collection. Ferulic acid, o-coumaric acid, p-coumaric acid, and gallic acid (Sigma-Aldrich, St. Louis, MO, USA), HG-DMEM (Gibco BRL, Life Technologies, USA), trypsin–EDTA solution (Beyotime, Jiangsu, China), fetal bovine serum (FBS; Sijiqing, Zhejiang, China) and MTT (Sigma, St. Louis, MO, USA) were purchased from commercial suppliers. ROS assay kit was purchased from the Beyotime Institute of Biotechnology (Haimen, China). An SH-1000 Lab microplate reader (Corona Electric Co. Ltd., Ibaragi, Japan), SpectraMax M5 Multifunctional microplate reader (Molecular Devices, USA) and Acquity UPLC-TQD system (Waters, Milford, MA) were used in the experiments.

2.2. Adsorbents

Three types of macroporous resins—NKA-II, NKA-9 and H1020—were purchased from Nankai Hecheng S & T Co., Ltd. (Tianjing, China), while the DA201-C resin was purchased from the Jiangsu Suqing Company (Jiangsu, China). All of the resins were pretreated with 100% ethanol for 24 h, and then washed with 5% HCl and 5% NaOH solution followed by distilled water. Other chemicals used in this study were of analytical grade.

2.3. Preparation of PEWB

The wheat bran was washed three times to remove starch, and then heated at 50 °C for 12 h. The dried wheat bran was ground into a powder by a hammer mill and passed through a 100-mesh filter. The bran was extracted twice with 80% ethanol at an 8 : 1 ratio (v/w) for 15 h at room temperature. The mixture was centrifuged for 20 min at 9000 × g and evaporated at 40 °C. The ethanol extract was further freeze-dried and stored in the dark in a sealed container at 4 °C for further analysis.

WBEs were then added to the column (2.6 cm × 80 cm) containing the macroporous resin and the outflow liquid was collected and added to the column containing four macroporous resins. The WBEs were subjected to DA201-C, NKA-II, NKA-9 and H1020 macroporous resins via column chromatography elution with a gradient of ethanol–H₂O (20 : 80, 40 : 60, 60 : 40, and then 80 : 20) to acquire four major fractions. The fractions were detected using a UV detector at a wavelength of 310 nm. The products were collected, concentrated, and lyophilized for the subsequent experiments. PEWB was obtained after the freeze-drying process. Various types of eluotropic fractions were dissolved in sterile 1% ethanol, filtered with a sterile 0.2 μm filter, attenuated with a DMEM-based culture medium, and then added to the cell culture.

2.4. Total phenolic content

The total phenolic content of the WBEs was determined using the Folin–Ciocalteu reagent.²³ The mixture contained 100 μL of WBEs in dimethyl sulfoxide (DMSO), 500 μL of the Folin–Ciocalteu reagent, and 1.5 mL of 20% sodium carbonate. The final volume was made up to 10 mL with pure water. After 2 h of reaction at ambient temperature, the phenolic content was calculated using a standard curve prepared with gallic acid at a wavelength of 765 nm. The reactions were repeated in triplicate.

2.5. Static adsorption and desorption tests

2.5.1 Adsorptive properties of resins. All the four macroporous resins were screened through static adsorption tests. First, the crude extract (30 mL) was added into the pretreated resin (1 g dry weight basis each) in an Erlenmeyer flasks and shaken at 200 rpm for 24 h at a temperature of 25 °C to reach the adsorption equilibrium.²⁴ Then, the resins were washed with 30 mL of double-distilled water, and then desorbed with 30 mL 95% ethanol v/v and shaken at 200 rpm for 4 h at 25 °C. These experiments were repeated in triplicate. The following equations were used to quantify the adsorption capacity, as well as the ratios of adsorption and desorption.

Adsorption capacity:

Adsorption ratio:

where Q_e is the adsorption capacity, representing the mass of adsorbate adsorbed on 1 g of dry resin at the adsorption equilibrium; E is the adsorption ratio, representing the percentage of total adsorbate being adsorbed at the adsorption equilibrium; C₀ and C_e are the initial and equilibrium concentrations of the sample solutions, respectively; V₀ is the initial volume added into the flask, and is the mass of the dry resin.

Desorption ratio:

where D is the desorption ratio in (%); C_d is the concentration of the desorption solution (mg mL⁻¹); V_d is the volume of the desorption solution; C₀, C_e, and V₀ are the same as defined above.

2.5.2 Static adsorption experiment. The adsorption kinetics of the NKA-II, NKA-9, H1020 and DA201-C resins were evaluated by adding crude extracts (30 mL each) into the pretreated resin (on a 1 g dry weight basis each) in Erlenmeyer flasks and shaking at 200 RPM for 10 h at 25 °C using a thermal shaker. 100 μL of the solutions was taken out for the analysis of the total phenol concentrations in the liquid phase at different time intervals using the Folin–Ciocalteu reagent method.

2.6. Dynamic adsorption and desorption tests

The dynamic adsorption and desorption experiments were performed using a column (26 × 800 mm, Shanghai Qite Analytical Instrumental, Shanghai, China) packed with the four hydrated macroporous resins. The bed volume (BV) was 350 mL and the flow rate was 2 mL min⁻¹. For the dynamic desorption experiment, both gradient and isocratic elution were carried out, with a total loading of 400 mL of crude extract on the NKA-9 column mentioned above. For gradient elution, the column was successively eluted with 3 to 6 BV of each of the ethanol aqueous solutions (20%, 40%, 60%, and 80%). The eluting solvent was changed when the absorbance of the eluate at 310 nm showed little alteration. For isocratic elution, after the column was washed with 3 BV of double-distilled water, all the ethanol eluents were collected and concentrated to dryness under reduced pressure at 40 °C. The resin-refined sample was stored and frozen for further experiment.

2.7. Cell viability assay

Cell viability was determined using the MTT assay, which was performed as previously described^25,26 with minor modifications. Briefly, HEK 293 cells were seeded at a density of 3 × 10⁴ cells per well in 96-well plate (Costar 3599; Corning, NY) for 12 h of attachment. The PEWB and ferulic acid (positive control) were dissolved in ethanol and diluted with HG-DMEM. The cells were treated with PEWB or ferulic acid for 1 h, and then exposed to 1 mM H₂O₂ for 2 h. After replacing the medium with fresh medium, 20 μL of MTT solution (5 mg mL⁻¹ in PBS) was added and the cells were incubated at 37 °C for 4 h with 5% CO₂. The medium was then carefully removed, and 150 μL of colored formazan dissolved in DMSO was added in its place. The plate was shaken for 10 min, and the absorbance was measured at 570 nm using a microplate reader (SH-1000 Lab microplate reader). The cell viability was expressed as % = [MTT OD value of treated cells/MTT OD value of control cells]. All assays were performed with at least three individual experiments with at least six replicate measurements.

2.8. ROS assay

HEK293 cells were seeded at 3 × 10⁴ cells per well in clear-bottomed, black-walled, 96-well plates (Costar 3606; Corning, NY) for 12 h of attachment. Then, the cells were incubated with a carboxy-2′,7′-dichloro-dihydro-fluorescein diacetate probe for 20 min and washed twice with PBS. Then, the cells were exposed to WBEs, PEWB and ferulic acid for 1 h and H₂O₂ for 2 h. Fluorescence was recorded at 488 nm (excitation) and 525 nm (emission) wavelengths using a SpectraMax M5 microplate reader. All assays were performed in at least three individual experiments, each with six replicate measurements.

2.9. Chromatographic system and conditions

All the samples were analyzed using the Acquity UPLC-TQD system (Waters, Milford, MA), including an autosampler, photodiode array detector and an MS pump equipped with an electrospray ionization (ESI) probe as the interface. The samples were separated by ultra-high performance liquid chromatography using an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) with a mobile phase consisting of acetonitrile solution (A) and 0.1% (v/v) formic acid water solution (B) at a flow rate of 0.3 mL min⁻¹ and an injection volume of 1 μL.

2.10. Identification of phenolic compounds by UPLC-MS

The analysis performed was used to identify phenolic compounds in the different extracts, which may have antioxidant activity. Specifically, extracts were dissolved in methanol (1 mg mL⁻¹), diluted with 50% acetonitrile, centrifuged at 10 000 rpm and syringe filtered using 0.22 μm polyvinylidene difluoride filters. The starting condition for each experiment was 5 : 95 mobile phase A (acetonitrile): mobile phase B (water + 0.1% (v/v) formic acid) held for 0.8 min, with a ramp-up to 10 : 90 (A : B) at 1.2 min, then to 15 : 85 for 2.4 min, and held for 1.3 min. Further gradient increases were carried out to 21 : 79 by 4.0 min and to 50 : 50 by 7.8 min, with a change to 100 : 0 (A : B) by 8.8 min, and then held for 0.5 min before finally being reconditioned to the initial starting conditions. The mass spectrometer was operated in negative mode ESI. The detection of the four phenolics was conducted in multiple reaction monitoring (MRM) mode. Individual compounds were identified using MRM with a specific precursor–production transition: m/z 163.08 > 119.64 for o-coumaric acid; m/z 163.08 > 119.15 for p-coumaric acid; m/z 193.00 > 134.10 for ferulic acid; m/z 169.05 > 125.00 for gallic acid. Detected phenolic compounds were quantified against standard curves generated with commercial phenolic standards. Results were expressed as milligrams of compound per gram of extract (mg compound per g extract).

2.11. Statistical analysis

All the experiments were performed in triplicate, and data are expressed as mean ± SD based on three separate experiments. Statistical analysis²⁷ was performed using the Student's t-test and one-way SPSS (16.0 software) analysis. Probability values of p < 0.05 were considered to be significant.

3. Results and discussion

3.1. Macroporous resin purification of PEWB

Macroporous adsorbent resins have been applied successfully in the separation and isolation of effective components from many natural products, offering an efficient approach with a high absorption capacity, low operating costs, low solvent consumption, a moderate purification effect, and easy regeneration.^18,28,29 Macroporous adsorbent resins have typically been selected based on the chemical nature of the phenolic acid and physical properties such as polarity, surface areas and average pore diameter. The adsorption capacity of a polymeric adsorbent is proportional to its specific surface area; a higher specific surface area promotes enhanced adsorption capacity. In the present study, four types of macroporous resins were analyzed and their physical properties are summarized in Table 1. Generally, resins with a weak polar structure have a strong affinity for weakly polar phenolic acids, and resins with higher polarity exhibit stronger adsorption abilities toward polar substances. Among the four resins tested, H1020 and DA201-C showed significantly higher adsorption capacities compared to the others. Adsorption capacity is usually determined by the degree of compatibility and similarity between adsorbent and adsorbate. Specifically, the NKA-II displayed a higher adsorption–desorption ratio than the other resins (p < 0.05) (Table 2). The non-polar resins (DA201-C and H1020) and polar resin (NKA-II) all exhibited high adsorption capabilities. However, NKA-II exhibited a relatively lower desorption capability for PEWB. This could be attributed to NKA-II possessing a strong affinity for the solute. Similar results were also observed for glycyrrhetinic acid mono-glucuronide separation from the crude extracts of fermentation broths by macroporous resins.¹

Table 1 Physical properties of the macroporous resins used

Resins	Polarity	Moisture content (%)	Particle diameter	Surface area (m^2 g^−1)	Average pore diameter (nm)
DA201-C	Non-polar	50–60	0.40–1.25	1000–1300	3.0–4.0
H1020	Non-polar	50–60	0.30–1.25	700–1000	12.0–17.0
NKA-II	Polar	42–52	0.30–1.25	160–200	14.5–15.5
NKA-9	Polar	67–73	0.30–1.25	250–290	15.5–16.5

---

Table 2 Adsorption capacity, adsorption, and desorption ratios of phenol acid on different macroporous resins^a

Resin	Adsorption capacity (mg g^−1)	Adsorption ratio (%)	Desorption ratio (%)
a Results are mean ± SD (n = 6). Numbers followed by different letters are significantly different at the level of p < 0.05 according to the Duncan test.
DA201-C	1.25 ± 0.03^b	80.6 ± 0.9^b	68.8 ± 6.9^b
H1020	1.27 ± 0.06^b	81.7 ± 3.4^b	63.7 ± 7.5^b
NKA-II	1.20 ± 0.05^b	77.9 ± 3.2^b	47.3 ± 2.0^a
NKA-9	1.03 ± 0.01^a	67.1 ± 0.9^a	87.2 ± 3.0^c

---

3.2. Adsorption kinetics of macroporous resins

In general, the selection of proper resins should be in accordance with their physical and chemical properties. Basically, polarity, surface area and average pore diameter are important factors for selecting a proper resin. The adsorption kinetics curves of the four resins over time (600 min) for PEWB are shown in Fig. 1. The adsorption capacities dramatically increased in the first 60 min, then slowly in the following time and eventually reached equilibrium at approximately 420 min. The fast initial rate may be due to the occurrence of adsorption into the easily accessible mesopores of the particles. The later slower uptake can be attributed to high mass transfer resistance inside the particle during the process. Similar results were also observed in the flavonoids purification from Houttuynia cordata Thunb by macroporous resins.²⁰ The results indicate that the adsorption capacity of NKA-9 was lower compared with the others. Based on the adsorption kinetics curves, 60 min was sufficient to achieve adsorption capacities of PWBE for these four resins.

Fig. 1 Static adsorption tests of the four macroporous resins.

3.3. Dynamic adsorption and desorption experiments

Dynamic adsorption and desorption results are affected by several factors, such as the feed and flow rates of the eluting solvent, feed pH value, temperature, initial concentration and the ratio of column height to the diameter. The desorbing solvent for the macroporous resin was usually ethanol because it can be recycled easily and it has a low cost and little toxicity to the samples. Different concentrations of ethanol solutions were used to perform desorption tests in order to find the most suitable desorption solution. In this study, WBEs were subjected to DA201-C, NKA-II, NKA-9 and H1020 macroporous resins via column chromatography and were eluted with a gradient of ethanol–H₂O (20 : 80, 40 : 60, 60 : 40, and then 80 : 20) to generate fractions. In the experiments, the feed rate of 3 mL min⁻¹ and eluting solvent flow rate of 2 mL min⁻¹ were fixed. Sixteen samples were obtained after using the four macroporous resins: PEWB (D-2, D-4, D-6, D-8), PEWB (H-2, H-4, H-6, H-8), PEWB (NII-2, NII-4, NII-6, NII-8) and PEWB (N9-2, N9-4, N9-6, N9-8) (Fig. 2).

Fig. 2 Dynamic adsorption and desorption tests of the four macroporous resins. (a) DA201-C, (b) H1020, (c) NKA-II and (d) NKA-9 (D-2, H-2, NII-2, N9-2, 20% ethanol eluents; D-4, H-4, NII-4, N9-4, 40% ethanol eluents; D-6, H-6, NII-6, N9-6, 60% ethanol eluents; D-8, H-8, NII-8, N9-8, 80% ethanol eluents).

Fig. 3 TIC chromatograms from a standard solution of gallic acid (A), p-coumaric acid (B), ferulic acid (C), o-coumaric acid and (D) methanol, TIC (total ion concentration) chromatogram.

3.4. Effects of PEWB on H₂O₂ injured cell growth and viability in HEK293 cells

MTT and related assays are widely used for cell viability evaluation after exposure to toxic chemicals or materials.^30,31 In our experiment, cell viability, demonstrated by the MTT assay, showed time- and dose-dependent decreases in cell viability. Cell viability significantly decreased when HEK293 cells were incubated for 2 h, suggesting that HEK293 cells were damaged in the presence of H₂O₂, and the IC₅₀ value of H₂O₂ was 0.98 ± 0.02 mM. Therefore, 1 mM H₂O₂ was used to induce injury for further experiments. In addition, the cytotoxic and resistance effects of PEWB on HEK293 cells were performed before the subsequent assay. The samples did not show significant cytotoxicity in cell proliferation over 12 h and 24 h (data not shown).

The resistance effects of various PEWB prepared using the different macroporous resins were further investigated and there was considerable variation in cell viability corresponding with increased ethanol concentration used to collect the fractions (Fig. 4). The highest cell viability was obtained with PEWB (N9-4), which was separated using the NKA-9 macroporous adsorbent resin at a 40 : 60 ethanol–water ratio. HEK293 cells pretreated with PEWB prior to exposure to H₂O₂ exhibited increased cell viability. However, the other macroporous adsorbent resins showed better cell viability at an ethanol–water ratio of 40 : 60. N9-4 displayed the greatest protection effect on HEK293 cells against oxidative damage and was selected for use in further investigations.

Fig. 4 Effects of PEWB on cell viability in H₂O₂-injured HEK293 cells. HEK293 cells were pre-incubated with extracts for 2 h prior to treatment with 1 mM H₂O₂ for 2 h. After the treatment, cell viability was determined by an MTT analysis (n = 6). Data are shown as means ± SD.

3.5. Effect of PEWB on intracellular ROS accumulation

It is well demonstrated that the treatment of HEK293 cells with H₂O₂ resulted in nuclear damage, loss of mitochondrial membrane potential, and elevated ROS levels. Specially, ROS generation is regarded as an indicator for cells being under abnormal physiological conditions.³² As shown in Fig. 5a, the ROS levels greatly increased when the HEK293 cells were exposed to 1 mM H₂O₂. However, when the HEK293 cells were pretreated with ferulic acid and PEWB, the ROS levels were significantly lower. Thus, it is reasonable to assume that PEWB plays important role in preventing oxidative damage in HEK293 cells upon exposure to H₂O₂.

Fig. 5 Effect of WBEs and PEWB on H₂O₂ induced intracellular ROS. (a) WBEs (1 mg mL⁻¹) and ferulic acid, (b) DA201-C, (c) H1020, (d) NKA-II and (e) NKA-9 (D-2, H-2, NII-2, N9-2, 20% ethanol eluents; D-4, H-4, NII-4, N9-4, 40% ethanol eluents; D-6, H-6, NII-6, N9-6, 60% ethanol eluents; D-8, H-8, NII-8, N9-8, 80% ethanol eluents.

The formulations that were separated at an ethanol–water ratio of 40 : 60 through macroporous adsorbent resins showed decreased ROS levels (Fig. 5b–e). N9-4 has proven to be the best formulation of PEWB that could reduce the ROS levels of cells to the same extent as ferulic acid. These results indicate that PEWB (N9-4) had the strongest ability to decrease the ROS levels in cells (Fig. 5e).

3.6. UPLC-TDQ analysis

UPLC-TDQ analysis of each extract was performed and total ion chromatograms of the standard solutions of the four phenolic compounds are shown in Fig. 3. The results are shown in Table 3. The phenolic compounds identified in each sample are as follows: PWEB (D-2, D-4, D-6, D-8; H-2, H-4, H-6, H-8; NII-2, NII-4, NII-6, NII-8; N9-2, N9-4, N9-6, N9-8). For each sample, the total number of phenolics quantified in the 40% ethanol extract was greater than that in the corresponding 60% ethanol extract. This result further is confirmed with the results observed in the MTT and ROS assays. Ferulic acid was the most abundant phenolic compound in a number of extracts, including N9-4 (35.49 ± 2.7 mg g⁻¹), D-4 (27.46 ± 1.03 mg g⁻¹), H-4 (24.71 ± 2.17 mg g⁻¹), NII-4 (24.07 ± 1.76 mg g⁻¹). Similar results were also observed for phenolic acid concentrations in spring and winter wheat.³³ p-Coumaric acid was identified in a number of extracts except for the 60% and 80% ethanol extracts. These extracts also showed increased antioxidant activity. Ferulic acid is an antioxidant chemical³⁴ and it may play an important role in the antioxidant activities of these extracts.

Table 3 Quantification of phenolic compounds in PWBE using UPLC-TDQ. Values are expressed as mg of compound per gram of extract (mg g⁻¹), where the annotation ‘n/d’ represents compounds not detected. Results followed by different letters (a–h) are significantly different at the level of p < 0.05 according to Duncan test. Results are mean ± SD (n = 6).

Samples	o-Coumaric acid	p-Coumaric acid	Ferulic acid	Gallic acid
D-2	0.30 ± 0.03^b	13.19 ± 0.02^e	20.34 ± 1.53^de	1.83 ± 0.06^e
D-4	0.92 ± 0.03^e	12.33 ± 0.21^d	27.46 ± 1.03^g	1.80 ± 0.08^e
D-6	n/d	0.74 ± 0.06^a	0.31 ± 0.02^a	n/d
D-8	n/d	1.32 ± 0.05^a	0.62 ± 0.03^a	0.21 ± 0.02^b
H-2	n/d	11.83 ± 0.14^d	18.93 ± 0.53 ^cd	2.00 ± 0.04^f
H-4	1.51 ± 0.02^f	13.01 ± 0.41^e	24.71 ± 2.17^f	2.44 ± 0.05^h
H-6	n/d	0.76 ± 0.11^a	0.21 ± 0.02^a	0.04 ± 0.01^a
H-8	0.41 ± 0.02^c	9.05 ± 0.19^b	21.23 ± 1.78^e	1.49 ± 0.02^c
NII-2	n/d	10.27 ± 0.87^c	17.11 ± 1.51^bc	1.63 ± 0.03^d
NII-4	0.85 ± 0.06^d	12.34 ± 0.22^d	24.07 ± 1.76^f	2.10 ± 0.06^g
NII-6	n/d	n/d	0.13 ± 0.06^a	n/d
NII-8	n/d	0.91 ± 0.03^a	0.84 ± 0.07^a	0.20 ± 0.01^b
N9-2	n/d	10.33 ± 0.51^c	16.20 ± 1.01^b	1.70 ± 0.03^d
N9-4	0.92 ± 0.05^e	16.79 ± 0.68^f	35.49 ± 2.7^h	2.08 ± 0.09 ^fg
N9-6	n/d	1.10 ± 0.20^a	0.18 ± 0.01^a	n/d
N9-8	0.02 ± 0.01^a	0.86 ± 0.09^a	0.55 ± 0.07^a	0.18 ± 0.01^b

---

4. Conclusions

Preparative separation of PEWB with macroporous resins was successfully achieved. Among the four resins investigated, NKA-9 resin exhibited the best separation performance for PEWB on the oxidative ability (ROS assay) compared to the others. The adsorption–desorption method was shown to be more efficient than other conventional methods due to higher efficiency, lower cost and procedural simplicity. Our work also demonstrates that the pretreatment of PEWB increased the ability to inhibit H₂O₂-induced oxidative damage in HEK293 cells. The resistance effects can be attributed to the ability of phenolic acid components to neutralize radicals and other ROS. Further investigations on the presence of phenolics using UPLC-TQD analysis revealed that some 40% ethanol extracts with high antioxidant activity also contained large amounts of ferulic acid, compared with the 60% ethanol extracts. The results indicate that NKA-9 had good adsorption and separation ability, which can be used for the preparation of PEWB from the WBEs.

Acknowledgements

This work was sponsored by the Qing Lan Project, China Postdoctoral Science Foundation (Grant no. 2014M560396), Jiangsu Planned Projects for Postdoctoral Research Funds (Grant no. 1402072C), and the National Key Technology R&D Program (Grant no. 2013AA102201).

References

1. S. P. Zou, J. J. Zhou, I. Kaleem, L. P. Xie, G. Y. Liu and C. Li, Sep. Sci. Technol., 2012, 47, 1055–1062.
2. S. Senter, R. Horvat and W. Forbus, J. Food Sci., 1983, 48, 798–799.
3. M. Naczk and F. Shahidi, J. Pharm. Biomed. Anal., 2006, 41, 1523–1542.
4. Y. P. Zou, Y. H. Lu and D. Z. Wei, J. Agric. Food Chem., 2004, 52, 5032–5039.
5. S. Kaviarasan, K. Vijayalakshmi and C. Anuradha, Plant Foods Hum. Nutr., 2004, 59, 143–147.
6. X. P. Yuan, J. Wang and H. Y. Yao, Food Chem., 2005, 90, 759–764.
7. L. Yu, Food Chem., 2005, 90, 311–316.
8. L. Liu, K. M. Winter, L. Stevenson, C. Morris and D. N. Leach, Food Chem., 2012, 130, 156–164.
9. J. W. Carter, R. Madl and F. Padula, Nutr. Res., 2006, 26, 33–38.
10. J. Liu, X. X. Wang and Z. Zhao, J. Sci. Food Agric., 2014, 94, 126–130.
11. H. Wu, G. Li, S. Liu, Z. Ji, Q. Zhang, N. Hu, Y. Suo and J. You, Food Anal. Methods, 2015, 8, 685–695.
12. Q. Xu, M. Li, L. Zhang, J. Niu and Z. Xia, Langmuir, 2014, 30, 11103–11109.
13. Q. Xu, M. Li, J. Niu and Z. Xia, Langmuir, 2013, 29, 13743–13749.
14. G. Li, S. Liu, Z. Sun, L. Xia, G. Chen and J. You, Food Chem., 2015, 170, 123–130.
15. C. Gu and C. Shannon, J. Mol. Catal. A: Chem., 2007, 262, 185–189.
16. H. Guo, P. Zhang, J. Wang and J. Zheng, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2014, 951–952, 89–95.
17. X. Gong, Phys. Chem. Chem. Phys., 2013, 15, 10459–10465.
18. Y. Zhao, B. Chen and S. Yao, Sep. Purif. Technol., 2007, 52, 533–538.
19. J. K. Zhang, X. Y. Zhu, F. L. Luo, C. D. Sun, J. Z. Huang, X. Li and K. S. Chen, J. Sep. Sci., 2012, 35, 128–136.
20. Y. Zhang, S. F. Li, X. W. Wu and X. Zhao, Chin. J. Chem. Eng., 2007, 15, 872–876.
21. F. J. Yang, L. Yang, W. J. Wang, Y. Liu, C. J. Zhao and Y. G. Zu, Int. J. Mol. Sci., 2012, 13, 8970–8986.
22. M. Murzakhmetova, S. Moldakarimov, L. Tancheva, S. Abarova and J. Serkedjieva, Phytother. Res., 2008, 22, 746–751.
23. L. L. Yu, S. Haley, J. Perret, M. Harris, J. Wilson and M. Qian, J. Agric. Food Chem., 2002, 50, 1619–1624.
24. R. Yang, D. M. Meng, Y. Song, J. Li, Y. Y. Zhang, X. S. Hu, Y. Y. Ni and Q. H. Li, J. Agric. Food Chem., 2012, 60, 8450–8456.
25. Y. Liu, J. Fang, Y. J. Kim, M. K. Wong and P. Wang, Mol. Pharm., 2014, 11, 1651–1661.
26. B. R. Schroeder, M. I. Ghare, C. Bhattacharya, R. Paul, Z. Yu, P. A. Zaleski, T. C. Bozeman, M. J. Rishel and S. M. Hecht, J. Am. Chem. Soc., 2014, 136, 13641–13656.
27. Z. Qiao, C. Xia, S. Shen, F. D. Corwin, M. Liu, R. Guan, J. R. Grider and L. Y. Qiao, PLoS One, 2014, 9, e114536.
28. S. Zhao, J. Y. Liu, S. Y. Chen, L. L. Shi, Y. J. Liu and C. Ma, Molecules, 2011, 16, 8590–8600.
29. E. Silva, D. Pompeu, Y. Larondelle and H. Rogez, Sep. Purif. Technol., 2007, 53, 274–280.
30. Y. C. Wang, Y. Y. Ma, Y. Zheng, J. Song, X. Yang, C. Bi, D. R. Zhang and Q. Zhang, Int. J. Pharm., 2013, 441, 728–735.
31. R. E. Chenbo Dong, L. Sargent, M. L. Kashon, D. Lowry, Y. Rojanasakul and C. Zoica Dinu, Environ. Sci.: Nano, 2014, 6, 595–603.
32. A. Manke, S. Luanpitpong, C. B. Dong, L. Y. Wang, X. Q. He, L. Battelli, R. Derk, T. A. Stueckle, D. W. Porter, T. Sager, H. L. Gou, C. Z. Dinu, N. Q. Wu, R. R. Mercer and Y. Rojanasakul, Int. J. Mol. Sci., 2014, 15, 7444–7461.
33. J. Zuchowski, K. Jonczyk, L. Pecio and W. Oleszek, J. Sci. Food Agric., 2011, 91, 1089–1095.
34. M. Srinivasan, A. R. Sudheer and V. P. Menon, J. Clin. Biochem. Nutr., 2007, 40, 92.

---