Bioactive components and antioxidant activities of oak cup crude extract and its four partially purified fractions by HPD-100 macroporous resin chromatography

Peipei Yin; Lingguang Yang; Ke Li; Hang Fan; Qiang Xue; Xiang Li; Liwei Sun; Yujun Liu

doi:10.1016/j.arabjc.2016.09.018

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Original article

12 (

); 249-261

doi:

10.1016/j.arabjc.2016.09.018

Bioactive components and antioxidant activities of oak cup crude extract and its four partially purified fractions by HPD-100 macroporous resin chromatography

Peipei Yin, Lingguang Yang, Ke Li, Hang Fan, Qiang Xue, Xiang Li, Liwei Sun^⁎, Yujun Liu^⁎

National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China

⁎Corresponding authors at: National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University, Qinghuadonglu No. 35, Haidian District, Beijing 100083, China. lsun2013@bjfu.edu.cn (Liwei Sun), yjliubio@bjfu.edu.cn (Yujun Liu)

Received: 2016-7-26, Accepted: 2016-9-18, Published: 2016-02-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Documents have revealed that leaves, barks and woods from oak trees are rich in phenols, yet little is known about them in Mongolian oak (Quercus mongolica) cups, the by-product waste with large biomass. In the present work, composition analysis through total phenols, flavonoids and tannins, and condensed tannins, and HPLC profiles and antioxidant assessment by DPPH, ABTS, reducing power, ORAC and CAA were performed to evaluate oak cup crude extracts and the resulting four fractions (Fr. I, Fr. II, Fr. III and Fr. IV) prepared by HPD-100 macroporous resin chromatography. Among the optimal 50% ethanol crude extract (ECE) and its subsequent four fractions, Fr. II, on the basis of the oak cups, collected the majority of total phenols, flavonoids and tannins, and condensed tannins from ECE though lower contents of all compositions than those in Fr. III when based on the extract with an exception of condensed tannins. And the highlight of Fr. II is that it possessed the strongest antioxidant ability from the perspective of extract. These findings imply that composition of condensed tannins as well as its contents might take dominate role in antioxidant ability of oak cups. This was further corroborated through HPLC profiles as compositions in ECE and the four fractions are all distinguished from each other. HPLC also exhibited the great enrichment of ellagic acid in Fr. III. Moreover, the correlation analysis indicated that there is exclusive correlation existed between total flavonoids and antioxidant assays among all composition analysis including ellagic acid, which further confirmed the main influencing factor to antioxidant ability of oak cup may be composition of condensed tannins as well as its contents because the condensed tannins were constituted by flavonoid units, and ellagic acid may not be a strong antioxidant contributor in oak cups. Therefore, Fr. II should be chosen as the best fraction to be further explored. In addition, this established HPD-100 macroporous resin chromatography procedure could be developed as a simple and effective procedure for preparation of ellagic acid from oak cups.

Keywords

Mongolian oak (Quercus mongolica) cups

Flavonoids

Condensed tannins

Antioxidant activities

Macroporous resin

HPLC

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1 Introduction

For decades, natural antioxidant is received increasing attention due to its safety, functionality and last but not least the contribution to human health. Natural antioxidants such as simple phenolics and flavonoids are commonly found in fruits, vegetables and herbs, and their common function is to serve as antioxidant against oxidative stress formed by oxidizing agents and free radicals (Antolovich et al., 2000; Matkowski and Wołniak, 2005; Sarikurkcu et al., 2009 ). Owing to safe and favorable antioxidant function, a trend has been formed to take full advantage of natural antioxidants in various industries, including food, cosmetics and pharmaceutical industry (Araújo et al., 2015; Kusumawati and Indrayanto, 2013; Marino et al., 2015 ). However, abundant and low-cost natural antioxidant resources are still lacking. Therefore, seeking a novel and ideal natural antioxidant resource throughout plant kingdom is quite urgent.

Mongolian oak (Quercus mongolica) in the Castanea family is largely distributed in China, Russia, Mongolia, Japan and Korea. In China, they are mainly found in Heilongjiang, Jilin, Liaoning, Inner Mongol, Hebei and Shandong provinces, and were used as non-commercial or ecological public-welfare forests (He et al., 2016), or their timber was commonly utilized for making furniture or producing wine barrels (Omar et al., 2013). However, in both cases, oak produces a wide variety of by-products discarded as waste, including leaves or shells and oak cups, thus leading to huge resource waste and environment pollution due to their large biomass. Therefore, making full use of these by-products is of great significance as well as urgent.

Many studies on oak trees mainly concentrated on the leaves, acorns or shells. It had been reported for the first time that sawtooth oak (Quercus acutissima) shell waste could be used as materials for bio-ethanol production (Yang et al., 2015). Custódio et al. (2014) documented the in vitro antioxidant and inhibitory activities of hexane, methanol and water extracts of cork oak (Quercus suber). They found that compounds in the leaves and acorns of cork oak could alleviate symptoms associated with Alzheimer diseases and other neurodegenerative ailments as well as diabetes. Tahmouzi (2014) and Moreno-Jimenez et al. (2015) reported that the extracts of Zagros oak (Quercus brantii) and red oak (Quercus spp.) leaves possess antioxidant, anti-inflammatory, anticarcinogenic and antimicrobial activities. However, oak cups, the abundant outer layer around the acorn (diameter 1.5–1.8 cm in length and 0.8–1.5 cm in width) remain to be investigated. Therefore, it is of great interest that whether the oak cups could be used as a potential source of natural antioxidants or functional food ingredients.

The conventional separation methods of bioactive compounds from plants have been studied using ion exchange, solvent extraction, high-speed counter-current chromatography (Liu et al., 2010a–c ), preparative high-performance liquid chromatography (HPLC) (Wang et al., 2014), and solid-phase extraction (SPF) (Da Silva Santos et al., 2014), which exhibited certain disadvantages including organic solvent wastage, high cost, time-consuming procedure, low recoveries and environmental pollution (Li et al., 2015) as well as many advantages. Recently, macroporous resin attracted much more attention on isolating the bioactive constituents from natural resources (Jia and Lu, 2008; Liu et al., 2010a–c; Zhao et al., 2011 ). Macroporous resins, which possess different characteristics including specific surface area, polarity, particle size, pore diameter, are economic, environmental friendly and easy to be regenerated (Jia and Lu, 2008; Yang et al., 2016).

Despite many analytical methods being available for assessing antioxidant capacity, there are no approved standardized methods by far. Sometimes, different antioxidant determination methods have led to widely conflicting results that are extremely difficult to interpret (Frankel and Meyer, 2000). This is mainly attributable to the distinct chemical principles underlying these methods. For instance, DPPH, ABTS, reducing power and ORAC are methods used to evaluate antioxidant activity in vitro, while cellular antioxidant activity (CAA) is an approach used to evaluate the antioxidant based on cells. The first three assays are based on electron transfer reaction, while ORAC assay is based on hydrogen atom transfer reaction. Therefore, a precise or comprehensive evaluation of antioxidant capacity requires the use of various methods with different mechanisms for inhibiting oxidation (Frankel and Meyer, 2000).

In virtue of research gap for oak cup, the aim of present study was to, for the first time, separate phenols from oak cups by macroporous resins, and unveiling of its bioactive compounds and their antioxidant activity for the purpose of making thorough use of oak cup.

2 Materials and methods

2.1

2.1 Plant materials and chemicals

Mongolian oak (Quercus mongolica Fisch. ex Ledeb.) cups were collected from Grand Khingan in Heilongjiang province on September 2014, and authenticated by Dr. Zhonghua Liu, Beijing Forestry University. The oak cups were air-dried until equilibrium humidity, ground and screened through a 1-mm mesh, and then stored in darkness at room temperature for further use.

ABTS [2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulfonate)], DPPH (1,1-diphenyl-2-picrylhydrazyl), DCFH-DA (2′,7′-dichlorofluorescin diacetate), AAPH [2,2′-Azobis (2-amidinopropane) dihydrochloride], ellagic acid, Trolox, and Folin–Ciocalteu were purchased from Sigma–Aldrich Chemical (St. Louis, MO, USA). Gallic acid, catechin and rutin were obtained from National Institutes for Food and Drug Control (Beijing, China). All other chemicals and reagents were of analytical grade.

2.2

2.2 Solvent extraction

To select the optimal extraction solvent for preparation of bioactive constituents, 20 mL of various solvents, namely, 0%, 30%, 50%, 80% and 100% v/v ethanol: water solutions, was added to each of a 2-g ground powders of oak cups, respectively. Each mixture was extracted by heating reflux for 1 h at 80 °C. After cooling to room temperature, the resulting slurries were filtered through filter paper (Whatman No. 1) and the residue was re-extracted as described above with two extra 20 mL of respective corresponding solvent. Finally, filtrates of the three times were pooled and diluted to a volume of 60 mL with corresponding extraction solvents in order to obtain five crude extract solutions, which were analyzed for selecting the optimal extract solvent based on composition measurements of total phenols, flavonoids and tannins, and condensed tannins.

Subsequently, 50 g of ground powder was extracted for three times with the optimal extracting solvent (500 mL for each time) selected according to the procedures described above, and the combined supernatants (1500 mL) were rotarily evaporated at 60 °C till one third of the volume left. The resulting slurries were re-diluted to the volume of 1500 mL with distilled water and stood overnight to obtain the supernatant, and this 1500 mL of supernatant was used for further experiments.

2.3

2.3 Fractionation of the oak cup crude extracts

For the 1500 mL of supernatant prepared above, 300 mL was rotarily evaporated and dried with water bath at 60 °C to prepare the crude extract. The remaining 1200 mL was subjected to a chromatography column (600 mm × 60 mm i.d.) packed with HPD 100 macroporous resin for partial purification (Cangzhou Bonchem Co., Ltd, Cangzhou, China). After full absorption, the macroporous resins column was sequentially eluted with 2000 mL of distilled water, 2000 mL of 30% aqueous ethanol, 1500 mL of 50% aqueous ethanol and 1500 mL of 80% aqueous ethanol in order to obtain collections I, II, III and IV, respectively. Subsequently, these four collections were evaporated and dried in the water bath at 60 °C to obtain Fractions I, II, III and IV. The total eluting yield of the four fractions accounted for 66.83% of the ECE, the unabsorbed ECE by macroporous resin was around 19.40%, and the remaining was those kept by the macroporous resins within the column. Finally, these four fractions, together with the crude extract, were stored at −20 °C for further analysis.

2.4

2.4 Determination of total phenols and total tannins

Total phenols of the crude extract and four fractions were evaluated using Folin–Ciocalteu’s method reported by Sumczynski et al. (2015) with modifications. In brief, 20 μL of appropriately diluted solution of the crude extract or each of the fractions was mixed with 40 μL of 25% Folin–Ciocalteu reagent in corresponding well of a 96-well microplate. After 5 min standing at room temperature, 140 μL of sodium carbonate solution (700 mM) was added to each well and the plate was shaken for 30 s at 500 rpm in a microplate shaker. The microplate was then covered and incubated in the dark at 40 °C for 30 min, followed by reading at 765 nm using a microplate reader (Tecan infinite 200, Swiss). The results were expressed as gallic acid equivalents from the calibration curve of gallic acid standard solutions (0–400 mg/L) and expressed as mg of gallic acid equivalents per gram of a sample (mg GAE/g; the word ‘sample’ here and after refers to the crude extract or four fractions by HPD 100 macroporous column chromatography). All assays were performed in triplicate.

Total tannins in the crude extract and four fractions were determined based on phosphomolybdium tungstic acid-casein reaction described by Zhao et al. (2011) with modifications. Briefly, 25 mL of appropriately diluted solution of the crude extract or each of the four fractions was mixed with 100 mg of casein and incubated at room temperature for 3 h with shaking at 200 rpm. After incubation, this mixture was filtered through a 0.45-μm filter to collect the supernatant, named as the sample after CASEIN-Precipitating Reaction. The rest steps were the same as the method for determination of total phenols. Total tannins equal the difference of total phenols between samples before and after CASEIN-Precipitating Reaction. All determinations were conducted in triplicate and the results were expressed as equivalent of μmol gallic acid (GAE) per gram of a sample.

2.5

2.5 Determination of total flavonoid

Total flavonoids of the crude extract and four fractions were examined by an aluminum chloride colorimetric assay described by Sumczynski et al. (2015) with modifications. Briefly, 120 μL of appropriately diluted solution of the crude extract or each of the four fractions was mixed with 8 μL of 50 mg/mL sodium nitrite in the designated well of a 96-well microplate, and then the microplate was stood for 6 min before addition of 8 μL of 100 mg/mL aluminum chloride into each well. After a 5-min incubation at room temperature, 100 μL of 40 mg/mL sodium hydroxide was added to each well. Subsequently, the mixture was mixed thoroughly by pipetting up and down for 10 times. The microplate was then covered and incubated in the dark at room temperature for 30 min, and then absorbance measurement was read at a wavelength of 410 nm by using the microplate reader. All determinations were performed in triplicate and results were expressed as rutin equivalents from the calibration curve of rutin standard solution (0–100 mg/L) and expressed as mg of rutin equivalents per gram of a sample (mg RE/g).

2.6

2.6 Determination of condensed tannins

Condensed tannins was measured by a vanillin method described by Saad et al. (2014) with some modifications. In brief, 20 μL of appropriately diluted solution of the crude extract or each of the fractions was mixed with 120 μL of 4% vanillin-methanol solution in the designated well of a 96-well microplate, followed by addition of 60 μL of HCl (original solution) to each well, and then the 96-well plate was shaken for 5 min at 200 rpm by using an orbital shaker. After incubation in the dark for 15 min at room temperature, absorbance determination was performed at 500 nm by using the microplate reader. All examinations were performed in triplicate and results were expressed as catechin equivalents from the calibration curve of catechin standard solution (0–400 mg/L) and expressed as mg of catechin equivalents per gram of a sample (mg CE/g).

2.7

2.7 Antioxidant activities of the crude extract and its four fractions

2.7.1

2.7.1 DPPH^• scavenging activity

DPPH (1,1-diphenyl-2-picryl hydrazyl) free radical scavenging activity of the crude exact or each of the four fractions was determined by using the method of Alañón et al. (2011) with slight modification. Briefly, 10 μL of the standard (Trolox 10–400 mg/L, final concentration), samples or blank (distilled water) was added to corresponding well, followed by addition of 40 μL of 1 mM freshly prepared DPPH solution and 190 μL of methanol to each well. The 96-well plate was then shaken at 200 rpm for 1 min by using an orbital shaker. After incubated for 30 min at room temperature in the dark, the microplate reader was used to examine absorbance at a wavelength of 517 nm for assessing the value. The radical scavenging activity (RSA) for DPPH^• was calculated as RSA (%) = (A₀ − As)/A₀ × 100, where As is the absorbance of the sample solution and A₀ is the absorbance of the blank solution.

2.7.2

2.7.2 ABTS radical scavenging activity

ABTS radical cation (ABTS^+•) scavenging capacity of the crude exact or each of the four fractions was analyzed by the method reported by Alañón et al. (2011) with minor modifications. ABTS^+• was generated by the reaction of a 7-mM aqueous solution of ABTS with a 2.4-mM aqueous solution of potassium persulfate (K₂S₂O₈) in equivalents. Then, the mixture was kept in the dark at room temperature for 12–16 h. The ABTS^+• solution was then diluted with methanol at a ratio of 1:48 to an absorbance of 0.70 ± 0.02 at a wavelength of 734 nm to produce an ABTS^+• working solution. After the ABTS^+• working solution was prepared, 5 μL of standard (20–100 mg/L Trolox, final concentration), samples or blank (distilled water) was quickly added to corresponding wells in a 96-well microplate, followed by 200 μL of ABTS^+• working solution to each well. After incubation for 5 min at 30 °C in the dark, the absorbance was read at 734 nm by using the 96-well plate reader. The RSA for ABTS was also calculated as RSA (%) = (A₀ − As)/A₀ × 100, where As is the absorbance of the sample solution and A₀ is the absorbance of the blank solution.

2.7.3

2.7.3 Reducing power capacity

The reducing power was determined according to the methods of Zhao et al. (2011) with minor modifications. Briefly, 0.4 mL of methanol solutions (0.05–0.2 mg/mL) of the crude exact or each of the four fractions was mixed with 1 mL of phosphate buffer (0.2 M, pH = 6.6) and 1 mL of 1% (w/v) potassium ferricyanide (K₃[Fe(CN)₆]), and the mixture was incubated in a water bath at 50 °C for 20 min. After addition of 0.5 mL trichloroacetic acid (10%, w/v), the mixture was incubated at room temperature for 10 min. Subsequently, 1 mL of this mixture was mixed with 1 mL of distilled water and 0.2 mL of ferric chloride solution (0.1%, w/v). Finally, the absorbance was measured at 700 nm using an ultraviolet spectrophotometer (Shimadzu, Kyoto, Japan). A calibration curve was prepared using Trolox (0–600 mg/L). Results were expressed as the concentration of the crude extract or each of the four fractions versus absorbance at 700 nm.

2.7.4

2.7.4 Oxygen radical absorbance capacity (ORAC)

The ORAC assay was conducted according to Sun et al. (2012). The reaction was carried out in 75 mM phosphate buffer (pH = 7.4) and all of the reagents in the ORAC assay were prepared with this phosphate buffer. The experiment was protected from direct light because of light-sensitivity of fluorescein. In brief, 75 μL of fluorescein solution (0.2 μM) was added to each well of the 96-well plate, and then 25 μL of Trolox standards (5–50 μM, final concentration), samples or 75 mM phosphate buffer (reagent blank) was added to corresponding well. After shaking at 250 rpm for 5 min, the plate was kept in the dark and incubated in a 37 °C-prewarmed oven for 15 min. Once the incubation is completed, 100 μL of 37 °C-prewarmed AAPH was quickly added to each well by a 12-channel multipipet. The microplate was then immediately placed in the 96-well microplate reader and fluorescence was recorded every 1.5 min for 75 min with an excitation at 530 nm and emission at 485 nm. The net Area under Curve (AUC) of samples and standards was calculated by subtracting the AUC of the blank. Results were calculated by comparing the net AUC of the sample with those of the standard. All examinations were performed in triplicate and the results were expressed as μmol of Trolox equivalent (TE)/g of a sample.

2.7.5

2.7.5 CAA

CAA is a cell-based assay to detect foods, phytochemicals and dietary supplements for potential antioxidant activity. The CAA assay was measured using the method reported by Wolfe and Liu (2007) with slight modification. Briefly, human hepatocellular carcinoma (HepG2) cells were seeded in wells of a 96-well microplate at a density of 5 × 10⁴/well with 100 μL of culture medium supplemented with 10% FBS, 4 mM l-glutamine and 1% penicillin–streptomycin. After twenty-four hours culture at 37 °C with 5% CO₂, the culture medium was removed, and cells were washed cleanly by PBS. Subsequently, HepG2 cells in the wells were subjected to a 1-h incubation with 100 μL of the crude extract or each of the four fractions at different concentrations (10, 25 and 50 μg/mL) plus 25 μM DCFH-DA dissolved in antioxidant treatment medium (DMEM with 4 mM l-glutamine and 10 mM Hepes). The medium was then removed and 100 μL of AAPH (600 μM) dissolved in oxidant treatment medium (HBSS with 10 mM Hepes) was added to each well. The microplate was immediately placed in the 96-well microplate reader and fluorescence was recorded every 5 min for 60 min with an excitation at 538 nm and emission at 485 nm. The control wells contained the cells treated with DCFH-DA and AAPH, and the blank wells contained the cells treated with DCFH-DA and HBSS in the absence of AAPH. Quercetin (0.1–10 μM) was used as a standard and the standard curve was set up based on the Area under Curve of fluorescence versus time. CAA values were calculated as μmol of quercetin equivalents per gram of a sample.

2.8

2.8 HPLC analysis

HPLC was applied to analyze the difference of the chromatographic profile and to determine ellagic acid contents in the crude extract obtained with the optimal concentrations of aqueous ethanol and each of the four fractions. A Shimadzu HPLC system (Shimadzu, Kyoto, Japan) equipped with a SPDM20A ultraviolet detector and Eclipse XDB-C18 column (Agilent, 250 mm × 4.6 mm i.d., 5 μm), and a SIL-20AC TH autosampler controlled by an analytical software (LC Solution-Release 1.23SP1) were applied. Detections for the chromatographic profile of the crude extract and the four fractions were carried out using the method reported by Fernandes et al. (2011) with slight modification. Two solvents were applied for elution: A-water/acetic acid (99: 1; v/v) and B-water/acetonitrile/acetic acid (79: 20: 1; v/v/v). The gradient program was as follows: 0–30 min, 5–30% B; 30–70 min, 30–80% B; 70–85 min, 80–90% B; 85–105 min, 90–100% B; 105–135 min, 100% B, and the monitor wavelength was at 280 nm with a flow rate at 0.3 mL/min. The injection volume was 20 μL and column thermostat was set at 25 °C. The chromatographic column was washed with 100% B for 20 min and stabilized with the initial conditions for another 20 min before running next sample.

Determination of ellagic acid was conducted in conditions as follows: the mobile phase was 0.2% (v/v) phosphoric acid in water (A) and acetonitrile (B) (A: B = 80: 20). The monitor wavelength was set at 254 nm and flow rate was 1 mL/min, and the column thermostat was set at 30 °C. The optimum crude extract and the four fractions were diluted at a concentration of 0.2 mg/mL and then filtered through a 0.22 μm nylon filter in triple times. A standard curve was set up based on the peak areas of different concentrations of ellagic acid.

2.9

2.9 Statistical analysis

Data were presented as mean ± standard deviation (SD) of three parallel measurements. The statistical significance (t-test: two-sample equal variance, using two-tailed distribution) was determined by using Microsoft Excel statistical software (Microsoft Office Excel 2016, Microsoft Corp. Redmond, WA, USA). P values (<0.05) were set to be significant. The correlations were analyzed using SPSS for Windows (Version 17.0, SPSS Inc., Chicago, IL, USA) and were calculated using the correlation coefficient statistical option in the Pearson test.

3 Results and discussion

3.1

3.1 Effect of ethanol concentrations on extraction efficiency

To determine an optimal solvent for preparation of phenols from oak cups, five different solvents (0%, 30%, 50%, 80% and 100% v/v ethanol: water) were compared. As shown in Table 1, preparative effects by the five different solvents on total phenols, flavonoids and tannins, and condensed tannins all exhibited a similar increasing tendency with a maximum at 50% aqueous ethanol and decreased to the lowest at 100% ethanol. Total phenols, flavonoids and tannins, and condensed tannins in the five extracts all exhibited significant differences (P < 0.05), except that condensed tannins between pure water and 80% aqueous ethanol extracts. Our findings that the 50% aqueous ethanol extract contained the highest total phenols, flavonoids and tannins, and condensed tannins is in agreement with previous studies (Turkmen et al., 2006; Ćujić et al., 2016). Based on its satisfactory extraction, together with the low toxicity and high safety at a trace level after evaporation to dryness, 50% ethanol was finally selected as the optimal extraction solvent for oak cups, and its extract was named as ECE (50% ethanol crude extract) hereafter.

Table 1 Total phenols, flavonoids and tannins, and condensed tannins contents in oak cup extracts.

Extraction method	Content (mg/g cup)
Extraction method	Total phenols^a	Total flavonoid^b	Total tannins^a	Condensed tannins^c
Water	59.81 ± 1.43^A	88.63 ± 2.13^A	40.27 ± 0.54^A	6.72 ± 0.37^A
30% ethanol	70.93 ± 5.03^B	95.32 ± 1.19^B	50.39 ± 2.12^B	10.87 ± 0.42^B
50% ethanol	99.82 ± 2.86^C	102.27 ± 3.19^C	77.01 ± 0.67^C	11.71 ± 0.46^C
80% ethanol	34.86 ± 1.51^D	46.15 ± 2.19^D	26.87 ± 0.69^D	7.06 ± 0.56^A
100% ethanol	18.31 ± 1.27^E	25.52 ± 0.49^E	12.05 ± 0.68^E	4.44 ± 0.13^D

^A,B,C,D,EIn each column different letters mean significant differences between two groups (P < 0.05).

a Data expressed in mg equivalent of gallic acid (GAE) to 1 g of cup.

b Data expressed in mg equivalent of rutin (RE) to 1 g of cup.

c Data expressed in mg equivalent of catechin (CE) to 1 g of cup.

In general, tannins can be classified into two groups, the hydrolyzable tannins and the condensed tannins. Based on the data in Table 1, condensed tannins accounted for 36.85%, 26.27%, 21.57%, 16.69%, and 15.21% of the total tannins (CE/GAE) in the five extracts obtained by 100%, 80%, 30%, 50%, and 0% ethanol: water (v/v) solutions, respectively. These results unveiled that total tannins in oak cups may be constituted mainly by hydrolyzable tannins (accounting for 63.15–84.79%), or aqueous ethanol was suitable for extracting hydrolyzable tannins but not condensed tannins. Onem et al. (2014) reported that tannins from oak cups and beards of acorns were primarily hydrolyzable tannins (ellagitannins). Our previous report (Zhao et al., 2011) also showed that the predominant tannins extracted with aqueous ethanol from chestnut (Castanea mollissima Blume) burs, also in Castanea family, were hydrolyzable tannins. Based on all these results, it may be implied that the tannins in oak cups were mainly hydrolyzable rather than condensed tannins. Furthermore, we found that total tannins accounted for 65.81–77.15% of the total phenols in the five extracts, suggesting that total tannins might be the major constituent in the oak cup phenols, and thus hydrolyzable tannins (e.g., ellagitannins) might be its major components.

3.2

3.2 Total phenols, flavonoids and tannins, and condensed tannins of the ECE and the four fractions

For the purpose of use of non-toxic organic solvents in conventional separation techniques, macroporous resin was utilized for further isolation of bioactive components from the ECE, and the ECE was fractionated into four fractions, with Fraction I obtained with distilled water (labeled as Fr. I hereafter), Fraction II with 30% aqueous ethanol (Fr. II), Fraction III with 50% aqueous ethanol (Fr. III), and Fraction IV with 80% aqueous ethanol (Fr. IV), respectively.

The total phenols, flavonoids and tannins, and condensed tannins of Frs II and III were all significantly higher than those of the ECE (Table 2), suggesting that the four bioactive components had been largely enriched in these two fractions. The total phenols and tannins and the condensed tannins of Fr. IV were relatively higher than those of the ECE, but the total flavonoids were lower than those of the ECE. In contrast, the total phenols, flavonoids and tannins in Fr. I were significantly lower than those in the ECE, and the condensed tannins showed no significant difference when compared to that of the ECE.

Table 2 Total phenols, flavonoids and tannins, and condensed tannins in the ECE and the four fractions per gram of extract.

	ECE	Fr. I	Fr. II	Fr. III	Fr. IV
TP mg GAE/g extract	519.50 ± 11.29^A	255.74 ± 7.29^B	626.67 ± 8.87^C	796.77 ± 28.99^D	621.83 ± 44.31^C
TF mg RE/g extract	598.30 ± 9.39^A	383.48 ± 7.63^B	754.80 ± 11.28^C	797.40 ± 17.22^D	463.38 ± 16.24^E
TT mg GAE/g extract	388.23 ± 13.01^A	120.52 ± 12.31^B	513.10 ± 11.83^C	608.95 ± 24.57^D	448.76 ± 31.75^E
CT mg CE/g extract	63.97 ± 6.5^A	68.44 ± 5.33^A	101.17 ± 7.92^B	94.73 ± 10.45^B	84.29 ± 9.40^C

^A,B,C,D,EIn each line different letters mean significant differences between two groups (P < 0.05).

TP: total phenols; TF: total flavonoids; TT: total tannins; CT: condensed tannins.

The condensed tannins in Frs I–IV was all higher than that in the ECE (Table 2), implying that the condensed tannins has been well enriched by different concentrations of aqueous ethanol through macroporous resin. Based on the data in Table 2, the condensed tannins in Frs II, III, IV and the ECE all accounted for less than 20% of the total tannins, further indicating that condensed tannins was not the predominant tannins in the oak cups. In contrast, the condensed tannins in Fr. I made up 56.79% of the total tannins, significantly higher than those from the other three fractions, which exhibited to be significantly different from that in the Frs II (only 19.72%), III (15.56%), IV (18.78%) and the ECE (16.48%), implying that water, which possesses the highest polarity in the four eluting solvents (water and 30%, 50% and 80% aqueous ethanol), was optimal in enriching condensed tannins from the oak cups.

The extracting or eluting yield, and the total phenols, flavonoids and tannins, and the condensed tannins of the ECE and the four fractions per gram of oak cups are shown in Table 3. Among the four fractions, the eluting yield of Fr. II was the highest, accounting for 45.23% of that of ECE, followed by significant decreases in Frs I, III and IV (14.49%, 3.87%, and 3.25%, respectively). Besides the highest eluting yield, total phenols, flavonoids, tannins and condensed tannins in Fr. II were the highest and the percentage of total phenols, flavonoids and tannins, and the condensed tannins in Fr. II alone accounted for 76.30%, 77.10%, 80.71% and 73.77%, respectively, of the total yields of Frs I–IV, suggesting that most of the bioactive compounds existed in Fr. II. In addition, total phenols, flavonoids and tannins, and condensed tannins in all these four fractions accounted for 71.51%, 74.00%, 74.06% and 97.01% of those in the ECE, and all of which were higher than those of the total eluting yield (66.83%), suggesting that most of bioactive constituents, especially the condensed tannins (97%), were further enriched from macroporous resins by different concentrations of aqueous ethanol. This further indicated that macroporous resin was a superior isolation method for oak cup extract.

Table 3 Extracting or eluting yield and total phenols, flavonoids and tannins, and condensed tannins in the ECE and the four fractions per gram of oak cup.

	ECE	Fr. I	Fr. II	Fr. III	Fr. IV
Yield mg/g cup	185.43 ± 4.49^A	26.86 ± 1.75^B	83.87 ± 1.27^C	7.17 ± 0.19^D	6.02 ± 0.22^E
TP mg GAE/g cup	99.82 ± 2.86^A	7.12 ± 0.58^B	54.46 ± 2.23^C	5.92 ± 0.62^D	3.88 ± 0.80^E
TF mg RE/g cup	102.27 ± 3.19^A	9.49 ± 0.61^B	58.35 ± 0.94^C	5.27 ± 0.37^D	2.57 ± 0.29^E
TT mg GAE/g cup	77.01 ± 0.67^A	3.46 ± 0.35^B	46.03 ± 1.06^C	4.66 ± 0.19^D	2.88 ± 0.20^E
CT mg CE/g cup	11.71 ± 0.46^A	1.81 ± 0.20^B	8.38 ± 0.66^C	0.67 ± 0.05^D	0.50 ± 0.045^E

^A,B,C,D,EIn each line different letters mean significant differences between two groups (P < 0.05).

In a previous study, Ogawa et al. (2008) used Diaion HP-20, Chromatorex ODS 1024T, and Sephadex LH-20 chromatography to fractionate the seed shells of Aesculus turbinate, and they obtained the highly purified fractions of polyphenols. However, their purification method was involved in three different chromatography, including macroporous resin and normal-phase and reverse-phase chromatography. In addition, too much toxic methanol was used in their method. In this sense, the one-step HPD-100 macroporous resin chromatography procedure in our study was a simple, effective and environment-friendly method for isolation and enrichment of phenols from oak cups.

3.3

3.3 Antioxidant capacities of the ECE and the four fractions

Many analytical methods are used to measure the antioxidant activity of substances, yet little is known about the comparability of the tested results between different methods. Previous researches have shown that one single method can hardly reflect comprehensive antioxidant capacity generated by a series of complex compounds in the plant, because different antioxidant compounds may act through distinct mechanisms against oxidizing agents (Marazza et al., 2012; Xiao et al., 2014). For this reason, five different antioxidant assays were employed to detect the antioxidant capacities of the ECE and four fractions. Among these methods, DPPH, ABTS, reducing power and ORAC were all chemical methods, while CAA was a cell-based biological antioxidant assay.

3.3.1

3.3.1 DPPH and ABTS antioxidant capacities

Free radical scavenging ability (RSA) of the ECE and four fractions were firstly evaluated with the changes of absorbances caused by the reductions in DPPH^• and ABTS free radicals (Fig. 1), and the more rapidly the absorbance changes, the more potent the antioxidant activity of the extract presents in terms of its hydrogen atom-donating capacity (Wang et al., 2013). As shown in Fig. 1A, DPPH^• scavenging activity of the ECE increased in a dose-dependent manner, and it increased rapidly up to a concentration at 0.05 mg/mL, then relatively slowly at concentrations between 0.05 and 0.2 mg/mL. The antioxidant activities of Frs II and III were higher and those of Frs I and IV were lower than those of the ECE at all concentrations, among which Fr II exhibited the highest, while Fr IV displayed the lowest capacities of scavenging free radicals, respectively. These suggest that the bioactive compounds might be more effectively enriched by the Frs II and III than those by Frs I and IV. Similar tendency was found in ABTS assay (Fig. 1B). The ABTS free radical scavenging activity of the ECE showed an increase in a concentration-dependent manner, and it increased slowly at first, then quickly, and finally increased slowly again. The same antioxidant order to that found in DPPH assay (Fig. 1A) was observed at all the measured concentrations as follows: Fr. II > Fr. III > ECE > Fr. I > Fr. IV. The structure of phenolic compounds is a key determinant of their radical scavenging activity, and the antioxidant activity usually depends on the numbers and positions of the hydroxyl groups in relation to the carboxyl functional group (Hayes et al., 2011). As the four fractions were subsequently eluted through macroporous resins by different polar solvents, the compositions as well as the contents were all different. Hence, we speculated that the high antioxidant activity of Frs II and III might be attributed to their distinct chemical structure. Additionally, it had been reported that the high molecular weight phenols (tannins) had stronger ability than the other specific functional groups to quench ABTS free radical and their effectiveness depended on its molecular weight, number of aromatic rings, and nature of hydroxyl group’s substitution (Cai et al., 2014; Hagerman et al., 1998). Therefore, it can be implied that the high capacities of ABTS free radical scavenging activity of Frs II and III might be due to the presence of a larger amount of high molecular weight phenols than those of the ECE and the other two fractions.

Antioxidant capacity of the ECE and four fractions by DPPH and ABTS colorization assays. Four concentrations of ECE and the four fractions were chosen to be subjected to DPPH and ABTS to examine their corresponding DPPH (A) and ABTS (B) radical scavenging activity. The absorbance was determined at 517 nm for DPPH, and at 734 nm for ABTS. Trolox was used as the positive control to ensure that the results were reliable. The results were presented as mean ± SD of three independent experiments (n = 3) and expressed as the concentrations of the ECE or each of the four fractions versus DPPH or ABTS radical scavenging activity (RSA) (%).

Defined as concentration value of the ECE or each of the four fractions when eliminating 50% of DPPH^• and ABTS radicals, EC₅₀ for DPPH and ABTS exhibited a same order: Fr. II < Fr. III < ECE < Fr. I < Fr. IV (Table 4; A lower EC50 value indicated a stronger antioxidant ability). The results showed that levels of total phenols, flavonoids and tannins in Fr. II are lower than those of Fr. III as shown in Table 2; however, antioxidant ability of Fr. II is contradictorily higher than that of Fr. III (Table 4), indicating that those three components may take minor responsible for the fact of higher antioxidant capacity of Fr. II than that of Fr. III, or Fr. II may contain certain phenols, flavonoids and/or tannins with much higher antioxidant activity that were lower in concentration or not included in Fr. III, provided that the abilities of the components to react with different reagents (e.g., Folin–Ciocalteu reagent and aluminum chloride) in different assays were negligible. On the other hand, condensed tannins in Fr. II is higher than that in Fr. III, yet no significant difference in condensed tannins existed between these two fractions (Table 2), and those may imply that the fact of superiority of antioxidant ability of Fr. II to Fr. III was more attributed to the composition of condensed tannins rather than its contents. Furthermore, constituents contained in Fr. II should have greater polarity than those in Fr. III as they were eluted by 30% and 50% ethanol aqueous, respectively, and thus it could be concluded that the condensed tannins with high polarity might made more contribution to the antioxidant activity.

Table 4 Antiradical activities in the ECE and four fractions.

EC₅₀ (μg/mL)
	ECE	Fr. I	Fr. II	Fr. III	Fr. IV
DPPH	140.0 ± 8.3^A	344.8 ± 48.7^B	101.3 ± 17.1^C	141.3 ± 6.4^D	561.8 ± 62.2^E
ABTS	122.0 ± 7.3^A	214.3 ± 32.2^B	82.3 ± 7.2^C	123.2 ± 6.4^D	337.0 ± 28.1^E

^A,B,C,D,EIn each line different letters indicate significant differences between two groups (P < 0.05).

ECE: 50% ethanol crude extract.

In our study, the EC₅₀ values of the ECE and four fractions (101.3–561.8 μg/mL) were significantly lower than those of ascorbic acid (2210 μg/mL) (Harzallah et al., 2016), indicating the high potentiality of oak cups as a source for natural antioxidants. Moreover, Frs II and III were eluted by 30% and 50% aqueous ethanol, respectively, indicating that the polarity of bioactive compounds might be similar to 30% and 50% aqueous ethanol. According to Table 3, the yield of Fr. II was much higher than that of the other three fractions, suggesting that 30% ethanol was the best elution solvent for isolating components with the highest antioxidant activities. All these results indicated that Fr. II was the best fraction with the highest yield and antioxidant activity when compared to those of the ECE and the other three fractions.

3.3.2

3.3.2 Reducing power

Reducing power is a method to examine the antioxidant ability of plant extracts based on a single electron transfer reaction. As presented in Fig. 2, all the absorbances were highly correlated with the concentrations of extracts (R² > 0.98), indicating that the reducing power assay was reliable for estimating the antioxidant capacity of oak cup extracts. Based on this assay, the reducing power is positively correlated with the slope. Accordingly, the reducing power of the ECE exhibited a linear dose-dependent increase. The reducing power of Frs II (218.4 ± 3.1 mg Trolox/g) and III (165.7 ± 1.6) was both higher, while those of Frs I (95.4 ± 1.5) and IV (45.2 ± 0.4) were both lower than that of ECE (154.4 ± 3.1) at all measured concentrations, and these four fractions all displayed an linear and dose-dependent increase in the reducing power. The results showed that the sequence in the reducing power was consistent with those of DPPH and ABTS radical scavenging assays with respect to antioxidant activity, namely, Fr. II > Fr. III > ECE > Fr. I > Fr. IV. The antioxidant activity of Fr. II was 1.41- and 4.83- times higher than those of ECE and Fr. IV, respectively (Table 5). In addition, it had been reported that the reducing properties in foods were associated with reductones (Harzallah et al., 2016). The phenolic compounds present in the ECE and four fractions may act in a similar fashion as reductones by donating electrons and quenching free radicals.

Reducing power of the ECE and four fractions. Four concentrations (0.05, 0.1, 0.15 and 0.2 mg/mL) were examined to evaluate the reducing power for ECE and the four fractions, and the absorbance was read at 700 nm. Trolox was used as the positive control to ensure that the results are rigorous. Results were expressed as mean ± SD of three independent experiments (n = 3) and as the concentration of the ECE or each of the four fractions versus absorbance at 700 nm. The high slope of the line indicated strong reducing power.

Table 5 Correlation analyses.

RP: reducing power; EA: ellagic acid.

^* Significant correlation at P < 0.05.

^** Positive significant correlation at P < 0.01.

3.3.3

3.3.3 ORAC and CAA

The ORAC assay, which could closely reflect the antioxidant capacity in biological systems (Prior et al., 2005), is a method for assessing total antioxidant activity. The assay is based on a hydrogen atom transfer reaction in which peroxyl radical ROO^• abstracts a hydrogen atom from the antioxidant compounds. The ORAC values of ECE and four fractions are shown in Fig. 3A. The ORAC value of Fr. II was the highest, followed by those of the ECE, Fr. III, Fr. I and Fr. IV. This tendency was a little different from DPPH, ABTS and reducing power, with the ORAC value of the ECE is higher than that of the Fr. III (but no significant difference), while the antioxidant activity of Fr. III was significantly higher than that of the ECE in DPPH, ABTS and reducing power assays. This difference might be due to the different chemical properties of bioactive compound and the distinct mechanisms of these four different antioxidant methods.

Antioxidant activity of the ECE and four fractions by ORAC and CAA. Samples of the ECE and four fractions (n = 3) were analyzed for ORAC (A) and values were expressed as Trolox Equivalents (TE)/g. The same samples (n = 4) were also analyzed by CAA (B) and the obtained values were expressed as Quercetin Equivalents/g.

Moreover, comparing the antioxidant activity of Rhodiola, a widely used Chinese herbal medicine with recognized strong antioxidant capacity, the values of ORAC in our study were much higher than those of Rhodiola extract (229.70 ± 17.02 mg Trolox/g) (Sun et al., 2012). Sharpe et al. (2016) had reported that the ORAC value of green tea was approximately 525.63 mg Trolox/g, which was still much lower than the ORAC value of the ECE and Frs I–III in our study. This also implied the superior antioxidant activity of oak cups.

The CAA assay based on HepG2 cells is a highly biologically relevant approach which reflects the absorption, metabolism and distribution aspects of antioxidants at the cellular level (Wolfe and Liu, 2007). The principle of CAA is as follows: DCFH-DA diffuses into the cell to form polar DCFH, which is trapped within the cell. AAPH is also able to diffuse into cells and spontaneously decomposed to form peroxyl radicals. These peroxyl radicals attack the cell membrane to produce more radicals and convert (oxidize) the intracellular DCFH to the fluorescent DCF.

The level of oak cup extracts used in our experiments, including the ECE and four fractions, was under the non-toxic level to HepG2 cells (data not shown). Quercetin and kaempferol were used as positive controls to test the reliabilities of CAA. Our results showed that the EC₅₀ values of quercetin and kaempferol were 5.92 ± 0.07 μM and 7.85 ± 0.51 μM, respectively, both lower than those reported by Wolfe and Liu (2007). After calculation, kaempferol was 75.37 ± 11.22 μmol of QE/100 μmol in our study, consistent with that reported by Wolfe and Liu (2007) (75.3 ± 4.7 μmol of QE/100 μmol), indicating that CAA values in the present experiments are reliable. The slight difference in EC₅₀ value between our results and that of Wolfe and Liu (2007) may be due to the different free radicals used (ABAP or AAPH) and the condition of HepG2 cells (Liu and Huang, 2014).

As shown in Fig. 3B, CAA values of the ECE, and Frs II and III were all significantly higher than those of the Frs I and IV, with Fr. II being the highest, followed by the ECE, Fr. III, Fr. I and Fr. IV. These results were in agreement with those of ORAC, thus also a little different from data in the DPPH, ABTS and reducing power assays.

Furthermore, the antioxidant activities of Fr. II and the ECE were even higher than those of black tea extract (16.4 μmol QE/100 mg) (Liu and Huang, 2014), suggesting that oak cup extract might play a superior antioxidant role against the AAPH-generated peroxyl radicals by protection against oxidant attack to cell membrane, thus preventing from the oxidation of DCFH to DCF in HepG2 cells.

The CAA and ORAC assays, together with DPPH, ABTS and reducing power methods, suggested that the antioxidant activity of Fr. II was the highest, suggesting the profound significance of Fr. II for further study.

3.4

3.4 Chromatographic profiles and ellagic acid contents in the ECE and the four fractions

To probe into the relationship between antioxidant ability and constituents in the ECE and its four fractions, HPLC was applied. As shown in Fig. 4, ECE contained relatively complete but low concentration constituents of oak cups, and ellagic acid, one of the main compounds, was observed at about 120 min which is validated by its standard labeled as EA. After fractionation of ECE with macroporous resins, most of the constituents were observed to be enriched in Frs I and II though their chromatographic profiles were notably different. The bioactive constituents in Fr. I were mainly washed out from 10 min to 60 min and from 70 min to 100 min, and ellagic acid is observed to be run out at 120 min. In contrast, those of Fr. II were primarily washed out from 30 min to 120 min, indicating the differences of components between Fr. I and Fr. II. The chromatographic profile of Fr. III is shown in Fig. 4 in two formats labeled as Fr. III-1 and Fr. III-2, and the difference existed in the scale of y-axis. Fr. III-1 showed that the main bioactive constituents in Fr. III were washed out at 60 min with one single peak and from 75 min to 125 min with several peaks, suggesting that constituents enriched in Fr. III including ellagic acid were far less polar than those in Fr. I and Fr. II. Moreover, Fr. III-2 clearly demonstrated that Fr. III enriched the largest amount of ellagic acid in all these fractions, which was calculated to be much higher than the ECE and the other three fractions (contents of EA in Fr. III is 4.09 folds of that in ECE, and 7.10 folds of that in Fr. I, 6.68 folds of that in Fr. II, 4.70 folds of that in Fr. IV; also see Fig. 5). In addition, Fr. IV was displayed to contain none but a comparative level of ellagic acid to Frs I and II, indicating the complete enrichment of Frs I, II and III for the bioactive compositions from the ECE.

HPLC chromatograms of the ECE, four fractions and ellagic acid. The ECE and each of the four fractions were applied to a C18 RP-HPLC column and elution was achieved by a gradient at a flow rate of 0.3 mL/min and was monitored at 280 nm. ECE or each of the four fractions (2 mg/mL) and ellagic acid standard (8 μg/mL) was subjected to HPLC to perform composition analysis. ECE indicates 50% ethanol crude extract of oak cups, Frs I–IV mean fractions obtained from HPD-100 macroporous resin chromatography, fraction III was exhibited in two formats labeled as Fr. III-1 and Fr III-2, and EA designates ellagic acid standard.

Ellagic acid contents of the ECE and four fractions. HPLC was used to determine the ellagic acid contents in ECE and the four fractions. Results were expressed as mg ellagic acid/g and presented as mean ± SD of three independent experiments (n = 3). The different letters mean significant difference (P < 0.05).

Ellagic acid contents of the ECE and the four fractions are shown in Fig. 5. Fr. III presented the highest content of ellagic acid (58.76 ± 0.41 mg/g extract), followed by the ECE (14.35 ± 0.15 mg/g extract), Fr. IV (12.50 ± 0.21 mg/g extract), Fr. II (8.80 ± 0.25 mg/g extract) and Fr. I (8.28 ± 0.24 mg/g extract). Our results indicated that this HPD-100 macroporous resin chromatography procedure can succeed in partially separation of complete components in ECE, and the established procedure provided an effective, low cost and simple method for enrichment of bioactive constituents and purification of ellagic acid from oak cups.

3.5

3.5 Correlation analysis among total phenols, flavonoids, tannins, condensed tannins and antioxidant capacities

There was no correlation among total phenols, flavonoids and tannins, and condensed tannins, except that total phenols were significantly positively correlated with total tannins (R² = 0.988, P < 0.01), as shown in Table 5 (data above the horizontal dash line). The correlations among the antioxidant assays were highly positive (0.879 < R² < 0.993, P < 0.05), indicating that these five assays, at both chemical and biological levels, provided comparable values when they were used for estimating the antioxidant capacity of oak cup extracts (Table 5; data on the right side of the vertical dash line).

As shown in Table 5 (data below the horizontal and on the left side of the vertical dash lines), there was no significant correlation among total phenols, tannins, condensed tannins, ellagic acid, and DPPH, ABTS, reducing power, ORAC and CAA, while total flavonoids was significantly positively correlated with DPPH, ABTS and reducing power, indicating that total flavonoids is the most important contributor to antioxidant capacity in oak cup extracts.

It is generally accepted that flavonoids can be enriched by using 30% ethanol in the macroporous resin chromatography. In our study, Fr. II were prepared by 30% ethanol through HPD-100 macroporous resin chromatography and our data indeed showed high contents of flavonoids in the Fr. II, being consistent with the common view. Fr. III-2 displayed that Fr. III contained extremely high level of ellagic acid. And our experiment demonstrated that ellagic acid can be inaccurately colorized as flavonoid in the total flavonoid colorization assays though it in fact belongs to non-flavonoid polyphenol, which can explain why Fr. III showed high contents of flavonoids assay and even higher than that of Fr. II (Table 2). Therefore, the facts that flavonoids alone were positively correlated with antioxidant capacity and antioxidant capacity of Fr. II were stronger than that of Fr. III might be related to composition as well as contents of flavonoids except to ellagic acid. It is defined that condensed tannins are polymers of 2–50 (or more) flavonoid units that are joined by carbon–carbon bonds. In this sense, therefore, we hypothesized that compositions of condensed tannins as well as contents might make a considerable contribution to antioxidant capacities of Fr. II. In addition, Fr. II was indeed determined to have different constituents with Fr. III as demonstrated by HPLC profile, which provided solid evidence for above hypothesis, and condensed tannins with high polarity might take major responsible for highest antioxidant activity of Fr. II.

Collectively, the bioactive components in these four fractions were eluted subsequently by different solvents, thus the compositions of these four fractions were both different with each other and this was indeed exhibited by HPLC profile. Therefore, the fact that positive correlation of total flavonoids to DPPH, ABTS and reducing power might not only depend upon the amount but the composition of bioactive components as well.

Previous studies reported that the total antioxidant capacity of plant extracts is not only affected by the content of antioxidants, but their chemical composition as well (Ávila-Reyes et al., 2010). This explains that different components in plant extracts contribute unequally to their total antioxidant ability (Zou et al., 2016). In this study, we observed that those antioxidant activities were not clearly associated with the contents of total phenols and total tannins. This result was consistent with the report of Ávila-Reyes et al. (2010), who found that the antioxidant activities were not clearly associated with the contents of total phenol. Therefore, our finding indicated that flavonoids may be more important for antioxidant activity than any particular phenolic concentration, and this was further confirmed by enrichment of flavonoids in Fr. II in our work.

4 Conclusion

The current study investigated the impact of different concentrations of aqueous ethanol on the extraction and isolation of bioactive compounds by HPD-100 macroporous resin chromatography from the Mongolian oak cups, as well as its antioxidant capacity. Overall, oak cups contained considerable phenols, flavonoids and tannins, and exhibited high antioxidant capacity. Our data demonstrated that 50% aqueous ethanol was the most effective extraction solvent and Fr. II eluted with 30% aqueous ethanol from HPD-100 macroporous resin exhibited the highest extract yield. Fr. II also collected the majority of total phenols, flavonoids, tannins and condensed tannins from the ECE. In the sense of antioxidant ability, Fr. II displayed consistent highest antioxidant activity in the antioxidant assays. Therefore, Fr. II should be chosen as the best fraction to be further explored. In addition, enrichment of major amounts of ellagic acid in Fr. III indicated that the one-step HPD-100 macroporous resin chromatography procedure could be applied as the simple and effective preparative procedure for preparation of ellagic acid from oak cups. Therefore, oak cups, an abundant and low-cost resource, possessed great potential to be explored as a source of bioactive compounds and natural antioxidants. In general, our study extends the knowledge on the bioactive composition and antioxidant ability and applications of oak cups in pharmaceutical, cosmetic or food industries. Nevertheless, identification of specific compounds from the ECE and the four fractions, and confirmation of the key components contributing to the antioxidant activity should require to be further studied to evaluate the effects of fractionation of macroporous resin on the variation of oak cups’ phenol constituents and their bioactivities as well as the composition-activity relationship.

Author contributions

Peipei Yin, Lingguang Yang, Liwei Sun and Yujun Liu designed the study, carried out the research and drafted the manuscript. Ke Li, Hang Fan, Qiang Xue and Xiang Li participated in the experiments. Yujun Liu provided facilities and reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgment

This work was financially supported by the Fundamental Research Funds for the Central Universities (No. BLYJ201413) and Ph.D. Programs Foundation of Ministry of Education (20120014110006), China.

References

Alañón M.E., Castro-Vázquez L., Díaz-Maroto M.C., Gordon M.H., Pérez-Coello M.S., . A study of the antioxidant capacity of oak wood used in wine ageing and the correlation with polyphenol composition. Food Chem.. 2011;128:997-1002.
[Google Scholar]
Antolovich M., Prenzler P., Robards K., Ryan D., . Sample preparation in the determination of phenolic compounds in fruits. The Analyst. 2000;125:989-1009.
[Google Scholar]
Araújo M., Pimentel F.B., Alves R.C., Oliveira M.B.P.P., . Phenolic compounds from olive mill wastes: health effects, analytical approach and application as food antioxidants. Trends Food Sci. Technol.. 2015;45:200-211.
[Google Scholar]
Ávila-Reyes J.A., Almaraz-Abarca N., Delgado-Alvarado E.A., González-Valdez L.S., del Toro G.V., Durán Páramo E., . Phenol profile and antioxidant capacity of mescal aged in oak wood barrels. Food Res. Int.. 2010;43:296-300.
[Google Scholar]
Cai H., Xie Z., Liu G., Sun X., Peng G., Lin B., Liao Q., . Isolation, identification and activities of natural antioxidants from Callicarpa kwangtungensis Chun. PLoS ONE. 2014;9:e93000.
[Google Scholar]
Ćujić N., Šavikin K., Janković T., Pljevljakušić D., Zdunić G., Ibrić S., . Optimization of polyphenols extraction from dried chokeberry using maceration as traditional technique. Food Chem.. 2016;194:135-142.
[Google Scholar]
Custódio L., Patarra J., Alberício F., Neng N.D.R., Nogueira J.M.F., Romano A., . Phenolic composition, antioxidant potential and in vitro inhibitory activity of leaves and acorns of Quercus suber on key enzymes relevant for hyperglycemia and Alzheimer’s disease. Ind. Crop. Prod.. 2014;64:45-51.
[Google Scholar]
Da Silva Santos V., Bisen-Hersh E., Yu Y., Cabral I.S., Nardini V., Culbreth M., Teixeira da Rocha J.B., Barbosa F. Jr., Aschner M., . Anthocyanin-rich acai (Euterpe oleracea Mart.) extract attenuates manganese-induced oxidative stress in rat primary astrocyte cultures. J. Toxicol. Environ. Health Part A. 2014;77:390-404.
[Google Scholar]
Fernandes A., Sousa A., Mateus N., Cabral M., de Freitas V., . Analysis of phenolic compounds in cork from Quercus suber L by HPLC-DAD/ESI-MS. Food Chem.. 2011;125:1398-1405.
[Google Scholar]
Frankel E.N., Meyer A.S., . The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. J. Sci. Food Agric.. 2000;80:1925-1941.
[Google Scholar]
Hagerman A.E., Riedl K.M., Jones G.A., Sovik K.N., Ritchard N.T., And P.W.H., Riechel T.L., . High molecular weight plant polyphenolics (tannins) as biological antioxidants. J. Agric. Food. Chem.. 1998;46:1887-1892.
[Google Scholar]
Harzallah A., Bhouri A.M., Amri Z., Soltana H., Hammami M., . Phytochemical content and antioxidant activity of different fruit parts juices of three figs (Ficus carica L.) varieties grown in Tunisia. Ind. Crop. Prod.. 2016;83:255-267.
[Google Scholar]
Hayes J.E., Allen P., Brunton N., O’Grady M.N., Kerry J.P., . Phenolic composition and in vitro antioxidant capacity of four commercial phytochemical products: Olive leaf extract (Olea europaea L.), lutein, sesamol and ellagic acid. Food Chem.. 2011;126:948-955.
[Google Scholar]
He F., Yang B., Wang H., Yan Q., Cao Y., He X., . Changes in composition and diversity of fungal communities along Quercus mongolica forests developments in Northeast China. Appl. Soil Ecol.. 2016;100:162-171.
[Google Scholar]
Jia G., Lu X., . Enrichment and purification of madecassoside and asiaticoside from Centella asiatica extracts with macroporous resins. J. Chromatogr. A. 2008;1193:136-141.
[Google Scholar]
Kusumawati I., Indrayanto G., . Chapter 15 – Natural Antioxidants in Cosmetics. In: Atta ur R., ed. Studies in Natural Products Chemistry. Elsevier; 2013. p. :485-505.
[Google Scholar]
Li H., Liu Y., Jin H., Liu S., Fang S., Wang C., Xia C., . Separation of vitexin-4“-O-glucoside and vitexin-2”-O-rhamnoside from hawthorn leaves extracts using macroporous resins. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci.. 2015;1007:23-29.
[Google Scholar]
Liu P., Du Y., Zhang X., Sheng X., Shi X., Zhao C., Zhu H., Wang N., Wang Q., Zhang L., . Rapid analysis of 27 components of Isodon serra by LC-ESI-MS-MS. Chromatographia. 2010;72:265-273.
[Google Scholar]
Liu S., Huang H., . Assessments of antioxidant effect of black tea extract and its rationals by erythrocyte haemolysis assay, plasma oxidation assay and cellular antioxidant activity (CAA) assay. J. Funct. Foods. 2014;18:1095-1105.
[Google Scholar]
Liu W., Zhang S., Zu Y.-G., Fu Y.-J., Ma W., Zhang D.-Y., Kong Y., Li X.-J., . Preliminary enrichment and separation of genistein and apigenin from extracts of pigeon pea roots by macroporous resins. Bioresource Technol.. 2010;101:4667-4675.
[Google Scholar]
Liu Y., Liu J., Chen X., Liu Y., Di D., . Preparative separation and purification of lycopene from tomato skins extracts by macroporous adsorption resins. Food Chem.. 2010;123:1027-1034.
[Google Scholar]
Marazza J.A., Nazareno M.A., de Giori G.S., Garro M.S., . Enhancement of the antioxidant capacity of soymilk by fermentation with Lactobacillus rhamnosus. J. Funct. Foods. 2012;4:594-601.
[Google Scholar]
Marino A., Paterniti I., Cordaro M., Morabito R., Campolo M., Navarra M., Esposito E., Cuzzocrea S., . Role of natural antioxidants and potential use of bergamot in treating rheumatoid arthritis. PharmaNutrition. 2015;3:53-59.
[Google Scholar]
Matkowski A., Wołniak D., . Plant phenolic metabolites as the free radical scavengers and mutagenesis inhibitors. BMC Plant Biol.. 2005;5:1-2.
[Google Scholar]
Moreno-Jimenez M.R., Trujillo-Esquivel F., Gallegos-Corona M.A., Reynoso-Camacho R., Gonzalez-Laredo R.F., Gallegos-Infante J.A., Rocha-Guzman N.E., Ramos-Gomez M., . Antioxidant, anti-inflammatory and anticarcinogenic activities of edible red oak (Quercus spp.) infusions in rat colon carcinogenesis induced by 1,2-dimethylhydrazine. Food Chem. Toxicol.. 2015;80:144-153.
[Google Scholar]
Ogawa S., Kimura H., Ai N., Katsube T., Jisaka M., Yokota K., . Fractionation and structural characterization of polyphenolic antioxidants from seed shells of Japanese horse chestnut (Aesculus turbinata BLUME) J. Agric. Food. Chem.. 2008;56:12046-12051.
[Google Scholar]
Omar M., Matsuo Y., Maeda H., Saito Y., Tanaka T., . New ellagitannin and galloyl esters of phenolic glycosides from sapwood of Quercus mongolica var. crispula (Japanese oak) Phytochem. Lett.. 2013;6:486-490.
[Google Scholar]
Onem E., Gulumser G., Akay S., Yesil-Celiktas O., . Optimization of tannin isolation from acorn and application in leather processing. Ind. Crop. Prod.. 2014;53:16-22.
[Google Scholar]
Prior R.L., Wu X., Schaich K., . Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food. Chem.. 2005;53:4290-4302.
[Google Scholar]
Saad H., Khoukh A., Ayed N., Charrier B., Bouhtoury F.C.-E., . Characterization of Tunisian Aleppo pine tannins for a potential use in wood adhesive formulation. Ind. Crop. Prod.. 2014;61:517-525.
[Google Scholar]
Sarikurkcu C., Arisoy K., Tepe B., Cakir A., Abali G., Mete E., . Studies on the antioxidant activity of essential oil and different solvent extracts of Vitex agnus castus L. fruits from Turkey. Food Chem. Toxicol.. 2009;47:2479-2483.
[Google Scholar]
Sharpe E., Hua F., Schuckers S., Andreescu S., Bradley R., . Effects of brewing conditions on the antioxidant capacity of twenty-four commercial green tea varieties. Food Chem.. 2016;192:380-387.
[Google Scholar]
Sumczynski D., Bubelova Z., Sneyd J., Erb-Weber S., Mlcek J., . Total phenolics, flavonoids, antioxidant activity, crude fibre and digestibility in non-traditional wheat flakes and muesli. Food Chem.. 2015;174:319-325.
[Google Scholar]
Sun L., Isaak C.K., Zhou Y., Petkau J.C., O K., Liu Y., Siow Y.L., . Salidroside and tyrosol from Rhodiola protect H9c2 cells from ischemia/reperfusion-induced apoptosis. Life Sci.. 2012;91:151-158.
[Google Scholar]
Tahmouzi S., . Optimization of polysaccharides from Zagros oak leaf using RSM: antioxidant and antimicrobial activities. Carbohyd. Polym.. 2014;106:238-246.
[Google Scholar]
Turkmen N., Sari F., Velioglu Y.S., . Effects of extraction solvents on concentration and antioxidant activity of black and black mate tea polyphenols determined by ferrous tartrate and Folin-Ciocalteu methods. Food Chem.. 2006;99:835-841.
[Google Scholar]
Wang C., Shi L., Fan L., Ding Y., Zhao S., Liu Y., Ma C., . Optimization of extraction and enrichment of phenolics from pomegranate (Punica granatum L.) leaves. Ind. Crop. Prod.. 2013;42:587-594.
[Google Scholar]
Wang E., Yin Y., Xu C., Liu J., . Isolation of high-purity anthocyanin mixtures and monomers from blueberries using combined chromatographic techniques. J. Chromatogr. A. 2014;1327:39-48.
[Google Scholar]
Wolfe K.L., Liu R.H., . Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. J. Agric. Food. Chem.. 2007;55:8896-8907.
[Google Scholar]
Xiao Y., Xing G., Rui X., Li W., Chen X., Jiang M., Dong M., . Enhancement of the antioxidant capacity of chickpeas by solid state fermentation with Cordyceps militaris SN-18. J. Funct. Foods. 2014;10:210-222.
[Google Scholar]
Yang J., Jiang J., Zhang N., Miao C., Wei M., Zhao J., . Enhanced enzyme saccharification of Sawtooth Oak shell using dilute alkali pretreatment. Fuel. 2015;139:102-106.
[Google Scholar]
Yang Q., Zhao M., Lin L., . Adsorption and desorption characteristics of adlay bran free phenolics on macroporous resins. Food Chem.. 2016;194:900-907.
[Google Scholar]
Zhao S., Liu J.Y., Chen S.Y., Shi L.L., Liu Y.J., Ma C., . Antioxidant potential of polyphenols and tannins from burs of Castanea mollissima Blume. Molecules. 2011;16:8590-8600.
[Google Scholar]
Zou Z., Xi W., Hu Y., Nie C., Zhou Z., . Antioxidant activity of Citrus fruits. Food Chem.. 2016;196:885-896.
[Google Scholar]

Introduction

Materials and methods

Plant materials and chemicals
Solvent extraction
Fractionation of the oak cup crude extracts
Determination of total phenols and total tannins
Determination of total flavonoid
Determination of condensed tannins
Antioxidant activities of the crude extract and its four fractions
HPLC analysis
Statistical analysis

Results and discussion

Effect of ethanol concentrations on extraction efficiency
Total phenols, flavonoids and tannins, and condensed tannins of the ECE and the four fractions
Antioxidant capacities of the ECE and the four fractions
Chromatographic profiles and ellagic acid contents in the ECE and the four fractions
Correlation analysis among total phenols, flavonoids, tannins, condensed tannins and antioxidant capacities

Conclusion

Author contributions

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[1] Alañón M.E., Castro-Vázquez L., Díaz-Maroto M.C., Gordon M.H., Pérez-Coello M.S., . A study of the antioxidant capacity of oak wood used in wine ageing and the correlation with polyphenol composition. Food Chem.. 2011;128:997-1002.
[Google Scholar]

[2] Antolovich M., Prenzler P., Robards K., Ryan D., . Sample preparation in the determination of phenolic compounds in fruits. The Analyst. 2000;125:989-1009.
[Google Scholar]

[3] Araújo M., Pimentel F.B., Alves R.C., Oliveira M.B.P.P., . Phenolic compounds from olive mill wastes: health effects, analytical approach and application as food antioxidants. Trends Food Sci. Technol.. 2015;45:200-211.
[Google Scholar]

[4] Ávila-Reyes J.A., Almaraz-Abarca N., Delgado-Alvarado E.A., González-Valdez L.S., del Toro G.V., Durán Páramo E., . Phenol profile and antioxidant capacity of mescal aged in oak wood barrels. Food Res. Int.. 2010;43:296-300.
[Google Scholar]

[5] Cai H., Xie Z., Liu G., Sun X., Peng G., Lin B., Liao Q., . Isolation, identification and activities of natural antioxidants from Callicarpa kwangtungensis Chun. PLoS ONE. 2014;9:e93000.
[Google Scholar]

[6] Ćujić N., Šavikin K., Janković T., Pljevljakušić D., Zdunić G., Ibrić S., . Optimization of polyphenols extraction from dried chokeberry using maceration as traditional technique. Food Chem.. 2016;194:135-142.
[Google Scholar]

[7] Custódio L., Patarra J., Alberício F., Neng N.D.R., Nogueira J.M.F., Romano A., . Phenolic composition, antioxidant potential and in vitro inhibitory activity of leaves and acorns of Quercus suber on key enzymes relevant for hyperglycemia and Alzheimer’s disease. Ind. Crop. Prod.. 2014;64:45-51.
[Google Scholar]

[8] Da Silva Santos V., Bisen-Hersh E., Yu Y., Cabral I.S., Nardini V., Culbreth M., Teixeira da Rocha J.B., Barbosa F. Jr., Aschner M., . Anthocyanin-rich acai (Euterpe oleracea Mart.) extract attenuates manganese-induced oxidative stress in rat primary astrocyte cultures. J. Toxicol. Environ. Health Part A. 2014;77:390-404.
[Google Scholar]

[9] Fernandes A., Sousa A., Mateus N., Cabral M., de Freitas V., . Analysis of phenolic compounds in cork from Quercus suber L by HPLC-DAD/ESI-MS. Food Chem.. 2011;125:1398-1405.
[Google Scholar]

[10] Frankel E.N., Meyer A.S., . The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. J. Sci. Food Agric.. 2000;80:1925-1941.
[Google Scholar]

[11] Hagerman A.E., Riedl K.M., Jones G.A., Sovik K.N., Ritchard N.T., And P.W.H., Riechel T.L., . High molecular weight plant polyphenolics (tannins) as biological antioxidants. J. Agric. Food. Chem.. 1998;46:1887-1892.
[Google Scholar]

[12] Harzallah A., Bhouri A.M., Amri Z., Soltana H., Hammami M., . Phytochemical content and antioxidant activity of different fruit parts juices of three figs (Ficus carica L.) varieties grown in Tunisia. Ind. Crop. Prod.. 2016;83:255-267.
[Google Scholar]

[13] Hayes J.E., Allen P., Brunton N., O’Grady M.N., Kerry J.P., . Phenolic composition and in vitro antioxidant capacity of four commercial phytochemical products: Olive leaf extract (Olea europaea L.), lutein, sesamol and ellagic acid. Food Chem.. 2011;126:948-955.
[Google Scholar]

[14] He F., Yang B., Wang H., Yan Q., Cao Y., He X., . Changes in composition and diversity of fungal communities along Quercus mongolica forests developments in Northeast China. Appl. Soil Ecol.. 2016;100:162-171.
[Google Scholar]

[15] Jia G., Lu X., . Enrichment and purification of madecassoside and asiaticoside from Centella asiatica extracts with macroporous resins. J. Chromatogr. A. 2008;1193:136-141.
[Google Scholar]

[16] Kusumawati I., Indrayanto G., . Chapter 15 – Natural Antioxidants in Cosmetics. In: Atta ur R., ed. Studies in Natural Products Chemistry. Elsevier; 2013. p. :485-505.
[Google Scholar]

[17] Li H., Liu Y., Jin H., Liu S., Fang S., Wang C., Xia C., . Separation of vitexin-4“-O-glucoside and vitexin-2”-O-rhamnoside from hawthorn leaves extracts using macroporous resins. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci.. 2015;1007:23-29.
[Google Scholar]

[18] Liu P., Du Y., Zhang X., Sheng X., Shi X., Zhao C., Zhu H., Wang N., Wang Q., Zhang L., . Rapid analysis of 27 components of Isodon serra by LC-ESI-MS-MS. Chromatographia. 2010;72:265-273.
[Google Scholar]

[19] Liu S., Huang H., . Assessments of antioxidant effect of black tea extract and its rationals by erythrocyte haemolysis assay, plasma oxidation assay and cellular antioxidant activity (CAA) assay. J. Funct. Foods. 2014;18:1095-1105.
[Google Scholar]

[20] Liu W., Zhang S., Zu Y.-G., Fu Y.-J., Ma W., Zhang D.-Y., Kong Y., Li X.-J., . Preliminary enrichment and separation of genistein and apigenin from extracts of pigeon pea roots by macroporous resins. Bioresource Technol.. 2010;101:4667-4675.
[Google Scholar]

[21] Liu Y., Liu J., Chen X., Liu Y., Di D., . Preparative separation and purification of lycopene from tomato skins extracts by macroporous adsorption resins. Food Chem.. 2010;123:1027-1034.
[Google Scholar]

[22] Marazza J.A., Nazareno M.A., de Giori G.S., Garro M.S., . Enhancement of the antioxidant capacity of soymilk by fermentation with Lactobacillus rhamnosus. J. Funct. Foods. 2012;4:594-601.
[Google Scholar]

[23] Marino A., Paterniti I., Cordaro M., Morabito R., Campolo M., Navarra M., Esposito E., Cuzzocrea S., . Role of natural antioxidants and potential use of bergamot in treating rheumatoid arthritis. PharmaNutrition. 2015;3:53-59.
[Google Scholar]

[24] Matkowski A., Wołniak D., . Plant phenolic metabolites as the free radical scavengers and mutagenesis inhibitors. BMC Plant Biol.. 2005;5:1-2.
[Google Scholar]

[25] Moreno-Jimenez M.R., Trujillo-Esquivel F., Gallegos-Corona M.A., Reynoso-Camacho R., Gonzalez-Laredo R.F., Gallegos-Infante J.A., Rocha-Guzman N.E., Ramos-Gomez M., . Antioxidant, anti-inflammatory and anticarcinogenic activities of edible red oak (Quercus spp.) infusions in rat colon carcinogenesis induced by 1,2-dimethylhydrazine. Food Chem. Toxicol.. 2015;80:144-153.
[Google Scholar]

[26] Ogawa S., Kimura H., Ai N., Katsube T., Jisaka M., Yokota K., . Fractionation and structural characterization of polyphenolic antioxidants from seed shells of Japanese horse chestnut (Aesculus turbinata BLUME) J. Agric. Food. Chem.. 2008;56:12046-12051.
[Google Scholar]

[27] Omar M., Matsuo Y., Maeda H., Saito Y., Tanaka T., . New ellagitannin and galloyl esters of phenolic glycosides from sapwood of Quercus mongolica var. crispula (Japanese oak) Phytochem. Lett.. 2013;6:486-490.
[Google Scholar]

[28] Onem E., Gulumser G., Akay S., Yesil-Celiktas O., . Optimization of tannin isolation from acorn and application in leather processing. Ind. Crop. Prod.. 2014;53:16-22.
[Google Scholar]

[29] Prior R.L., Wu X., Schaich K., . Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food. Chem.. 2005;53:4290-4302.
[Google Scholar]

[30] Saad H., Khoukh A., Ayed N., Charrier B., Bouhtoury F.C.-E., . Characterization of Tunisian Aleppo pine tannins for a potential use in wood adhesive formulation. Ind. Crop. Prod.. 2014;61:517-525.
[Google Scholar]

[31] Sarikurkcu C., Arisoy K., Tepe B., Cakir A., Abali G., Mete E., . Studies on the antioxidant activity of essential oil and different solvent extracts of Vitex agnus castus L. fruits from Turkey. Food Chem. Toxicol.. 2009;47:2479-2483.
[Google Scholar]

[32] Sharpe E., Hua F., Schuckers S., Andreescu S., Bradley R., . Effects of brewing conditions on the antioxidant capacity of twenty-four commercial green tea varieties. Food Chem.. 2016;192:380-387.
[Google Scholar]

[33] Sumczynski D., Bubelova Z., Sneyd J., Erb-Weber S., Mlcek J., . Total phenolics, flavonoids, antioxidant activity, crude fibre and digestibility in non-traditional wheat flakes and muesli. Food Chem.. 2015;174:319-325.
[Google Scholar]

[34] Sun L., Isaak C.K., Zhou Y., Petkau J.C., O K., Liu Y., Siow Y.L., . Salidroside and tyrosol from Rhodiola protect H9c2 cells from ischemia/reperfusion-induced apoptosis. Life Sci.. 2012;91:151-158.
[Google Scholar]

[35] Tahmouzi S., . Optimization of polysaccharides from Zagros oak leaf using RSM: antioxidant and antimicrobial activities. Carbohyd. Polym.. 2014;106:238-246.
[Google Scholar]

[36] Turkmen N., Sari F., Velioglu Y.S., . Effects of extraction solvents on concentration and antioxidant activity of black and black mate tea polyphenols determined by ferrous tartrate and Folin-Ciocalteu methods. Food Chem.. 2006;99:835-841.
[Google Scholar]

[37] Wang C., Shi L., Fan L., Ding Y., Zhao S., Liu Y., Ma C., . Optimization of extraction and enrichment of phenolics from pomegranate (Punica granatum L.) leaves. Ind. Crop. Prod.. 2013;42:587-594.
[Google Scholar]

[38] Wang E., Yin Y., Xu C., Liu J., . Isolation of high-purity anthocyanin mixtures and monomers from blueberries using combined chromatographic techniques. J. Chromatogr. A. 2014;1327:39-48.
[Google Scholar]

[39] Wolfe K.L., Liu R.H., . Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. J. Agric. Food. Chem.. 2007;55:8896-8907.
[Google Scholar]

[40] Xiao Y., Xing G., Rui X., Li W., Chen X., Jiang M., Dong M., . Enhancement of the antioxidant capacity of chickpeas by solid state fermentation with Cordyceps militaris SN-18. J. Funct. Foods. 2014;10:210-222.
[Google Scholar]

[41] Yang J., Jiang J., Zhang N., Miao C., Wei M., Zhao J., . Enhanced enzyme saccharification of Sawtooth Oak shell using dilute alkali pretreatment. Fuel. 2015;139:102-106.
[Google Scholar]

[42] Yang Q., Zhao M., Lin L., . Adsorption and desorption characteristics of adlay bran free phenolics on macroporous resins. Food Chem.. 2016;194:900-907.
[Google Scholar]

[43] Zhao S., Liu J.Y., Chen S.Y., Shi L.L., Liu Y.J., Ma C., . Antioxidant potential of polyphenols and tannins from burs of Castanea mollissima Blume. Molecules. 2011;16:8590-8600.
[Google Scholar]

[44] Zou Z., Xi W., Hu Y., Nie C., Zhou Z., . Antioxidant activity of Citrus fruits. Food Chem.. 2016;196:885-896.
[Google Scholar]

Bioactive components and antioxidant activities of oak cup crude extract and its four partially purified fractions by HPD-100 macroporous resin chromatography