Screening of Key Antioxidant Compounds of Longan (Dimocarpus

Sep 13, 2014 - Seed Extract by Combining Online Fishing/Knockout, Activity Evaluation, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, an...
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Screening of Key Antioxidant Compounds of Longan (Dimocarpus longan Lour.) Seed Extract by Combining Online Fishing/Knockout, Activity Evaluation, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, and High-Performance Liquid Chromatography Electrospray Ionization Mass Spectrometry Methods Jinyu Chen,† Zhen-zhen Ge,† Wei Zhu,† Ze Xu,† and Chun-mei Li*,†,‡ †

College of Food Science and Technology, and ‡Key Laboratory of Environment Correlative Food Science, Ministry of Education, Huazhong Agricultural University, Wuhan, Hubei 430070, People’s Republic of China ABSTRACT: To figure out the key phenolic compounds accounting for the antioxidant effects of longan (Dimocarpus longan Lour.) seed extract, online fishing/knockout method, activity evaluation assays, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR−MS), and high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC− ESI−MS) analysis were used jointly for the first time. p-Coumaric acid−glycoside, (S)-flavogallonic acid, ellagic acid derivative, and methyl-ellagic acid glucopyranoside were first identified in longan seeds. In addition, our study revealed that ellagic acid as well as its derivative and p-coumaric acid−glycoside had important contribution to the potent antioxidant activity of longan seed extract, while gallic acid, corilagin, (S)-flavogallonic acid, methyl-ellagic acid glucopyranoside, and ethyl gallate showed very little contribution to the total antioxidant activity of longan seed extract. The combining use of the online fishing/knockout method, activity evaluation assays, FT-ICR−MS, and HPLC−ESI−MS analysis is a useful and simple strategy for screening of key bioactive compounds from complex extracts. KEYWORDS: longan seed, phenolic compounds, online knockout, FT-ICR−MS, HPLC−ESI−MS

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INTRODUCTION Longan (Dimocarpus longan Lour.) is cultivated widely in southern China and southeast Asia.1 The fruit of longan has become one of the most favored subtropical fruits in China because of its delicate flavor and sweet taste. It is often consumed as fresh or in processed forms, such as dried flesh and canned products. During processing, about 17% of the fresh weight of whole fruits remains as waste in the form of seeds,2 which are abundant in antioxidant phenolic compounds3,4 and can be served as a good source of functional ingredients. Extracts of longan seed were reported to exert potent antioxidant activities on scavenging free radicals.2,5,6 However, there is limited information on the analysis of antioxidant compounds in longan seeds. Therefore, phenolic compounds accounting for the potent antioxidant effects of longan seeds are not clear. In addition, the current data on key antioxidant compounds of longan seeds are inconsistent or contrary. For example, Soong et al.6 suggested that gallic acid, corilagin, and ellagic acid had great contribution to the total antioxidant activity of longan seed extract, while Rangkadilok et al.1 revealed that the strong antioxidant activity of polyphenols in longan seeds may be derived from other unknown phenols beyond gallic and ellagic acids. Thus, further investigation on the structure of the phenolic constituents as well as their antioxidant contributions is needed. Screening and evaluation of bioactive compounds from crude extracts is not easy because of the complex components and the possible synergistic and/or suppressive actions of the constituents with each other.7 A conventional way is to isolate © 2014 American Chemical Society

chemical compounds and evaluate their activities one by one. Because a single active constituent can hardly reproduce the entire efficacy of the whole extract, this method ignores the importance of interrelationships of the multiple components. Other methods, including mass-spectrometry- and functionbased screening strategies,8,9 are highly available. However, contribution of individual components on the efficacy of the extract is not taken into account in most strategies. Recently, Liu et al.7 established the fishing and knockout strategy of a chemical marker, which was proven to be a useful approach for screening of the bioactive group that represented the efficacy of a complex extract. This strategy was performed according to the following steps: (1) screening out the target fractions/peaks in an extract fingerprint, (2) knocking out target fractions/peaks, and (3) evaluating the bioactivities of the knockout components. After comparison of the bioactivities of samples with target fractions/peaks missing, the bioactivity contribution of each component can be evaluated and the bioactive group in an extract that played significant role can be figured out. When this method is applied, we could obtain all types of components from an extract easily, such as main components, bioactive components, characteristic components, or a combination of them. An additional advantage of this method is the Received: Revised: Accepted: Published: 9744

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gradient were the same as HPLC analysis described above. For mass spectroscopy (MS), the heated capillary temperature and spray needle voltage were 350 °C and 4 kV, respectively. The drying gas (N2) flow rate was set at 10 L/min, and mass spectra scale ranged from m/z 100 to 2000. Online Knockout. The strategy included two main steps: (1) screening out the target fractions/peaks in an extract fingerprint and (2) knocking out target fractions/peaks. In this strategy, any component in an extract can be fished or knocked out. On the basis of the method reported by Liu et al.,7 the online knockout experiments of LSPs were conducted on a SHIMADZU liquid chromatograph system (Shimadzu Co., Kyoto, Japan) with a semi-preparative HPLC column (ZORBAX SB-C18, 9.4 × 250 mm, 5 μm, Agilent). Samples were dissolved in methanol at 50 mg/mL before injecting 20 μL into the system. The mobile phase and gradient were the same as described above for analytical HPLC, except that the flow rate was 4 mL/min. Different ingredients of the extract were separated by the liquid chromatography (LC) column and eluted out from the detector cell. We collected different fractions/peaks manually. To collect accurately, we connected the detector cell with a short PEEK tubing with a narrow inner diameter (Agilent, inner diameter of 0.13 mm). When the elution began, the eluate was collected from the PEEK tube in a brown container to keep away from light. As soon as the target fraction/peak appeared, the target fraction/peak was collected in another flask separately. Once the collection of the target fraction/ peak was finished, the residual components were collected in the former container continuously. All of the target fractions or peaks were completely fished from the total extract by repeating this step. Different kinds of samples collected by the online fishing or knockout method were using a rotary evaporator under vacuum to remove solvent and stored at 4 °C for HPLC analysis and antioxidant activity assays. Assays for Antioxidant Activity. Determination of the Reducing Power. The ferric-reducing antioxidant power (FRAP) assay was according to the methods by Dudonne et al. and Ozgen et al.,11,12 with some modification. LSPs were dissolved in methanol to give various concentrations (20, 40, 60, 80, and 100 μg/mL). A total of 1 mL of various LSP solutions was mixed with 0.2 M phosphate buffer (0.2 mL, pH 6.6) and 0.3% potassium ferricyanide (1.5 mL) in a test tube. The mixture was kept in a water bath at 50 °C for 20 min. Then, 10% trichloroacetic acid (1 mL), 0.3% ferric chloride (0.5 mL), and distilled water (3 mL) were added to the mixture before the absorbance was measured at 700 nm. Determination of the DPPH-Scavenging Activity. The DPPHscavenging activity of LSPs was evaluated according to the methods by Cervato et al.13 and Goupy et al.14 A total of 2 mL of sample (10 μg/ mL) dissolved in methanol was mixed with 2 mL of 0.2 mM DPPH solution. The mixture was incubated in the dark at 25 °C for 30 min before the absorbance was measured at 517 nm. The reaction mixture without a test sample was used as the control. The scavenging capacity of LSPs was calculated as follows: scavenging rate (%) = (1 − absorbance of sample/absorbance of control) × 100. The activity loss of each sample with target fraction/peak missing was calculated as follows: activity loss (%) = (1 − scavenging rate of sample with one fraction or peak missing/scavenging rate of sample before knockout) × 100. Determination of the Oxygen Radical Absorbance Capacity (ORAC). The measurement of the ORAC assay was based on the method by Huang et al.15 A total of 25 μL of sample solution (0.1 mg/ mL, dissolved in methanol) was transferred to a 96-well polystyrene plate. Then, 150 μL of 8.16 × 10−5 mM fluorescein solution diluted in 75 mM phosphate buffer (pH 7.4) was added. The plate was covered with a lid to maintain a constant temperature and preincubated (37 °C) for 10 min. The reaction was initiated by adding 25 μL of 153 mM AAPH. The result was recorded on a fluorescent reader every 5 min for 80 min, with the excitation wavelength at 485 nm and emission wavelength at 530 nm. The standard curve was obtained using Trolox (a water-soluble derivative of vitamin E with antioxidant property) as a standard. The Trolox standard was prepared as follows: 0.250 g of Trolox was dissolved in 50 mL of 75 mM phosphate buffer (pH 7.4)

determination of the bioactivity contribution ratio of certain compounds from a complex extract (7). Therefore, the present study aimed to figure out the key phenolic compounds accounting for the antioxidant effects of longan seed extract using a combination of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR−MS), high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC−ESI−MS) analysis, online fishing or knockout, and antioxidant-based screening strategies. Detailed information on the chemical structure−antioxidant activity relationship of longan seed extract will allow us to exploit it as a potentially beneficial phytonutrient.

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MATERIALS AND METHODS

Plant Materials. Fresh and fully mature longan fruits (cv. Shixia), which were harvested from south China, were transported to the laboratory for subsequent processing. The longan seeds from nondisease fruits were collected and freeze-dried at −60 °C for 48 h. All of the samples were homogenized to a powder using a stainless-steel grinder and then stored at −20 °C prior to analysis. Chemicals. Analytical or high-performance liquid chromatography (HPLC)-grade solvents and reagents were used. Methanol, acetonitrile, and formic acid were purchased from Fisher Scientific (Waltham, MA). Trolox, fluorescein disodium, and 2,2-diphenylpicrylhydrazyl (DPPH) were obtained from Sigma-Aldrich (St. Louis, MO). 2,2′Azobis(2-amidinopropane) dihydrochloride (AAPH) was purchased from Wako Chemicals USA (Richmond, VA). D3520 macroporous resin was purchased from Nankai Chemical Plant (Tianjin, China). Preparation of Longan Seed Polyphenols (LSPs). LSPs were extracted according to the modified method by Soong et al.6 A total of 5 g of dried longan seed powder was refluxed in 100 mL of aqueous ethanol (90%, v/v) at 70 °C for 1.5 h, using a heating mantle to control the temperature and a water-jacketed condenser to return the vapor to the pot. The extract was filtered with a 30 μm Whatman filter paper under vacuum, and the residue was extracted 2 more times. The extract was evaporated under reduced pressure at 35 °C to remove the solvent before lyophilization. The crude polyphenol extract was purified on D3520 macroporous resin.10 Approximately 10 g of the crude extract was dissolved in 50 mL of 40% ethanol and loaded on a glass column (4.2 × 60 cm) packed with D3520 macroporous resin. Then, 1000 mL of distilled water was used to remove the sugar, and 1000 mL of 40% ethanol was used to elute the phenols at a flow rate of 1.5 mL/min. After elution, the solvent was removed using a rotary evaporator under vacuum, the residue was lyophilized, and LSPs were obtained. Samples were dissolved in methanol to 1 mg/mL prior to HPLC analysis. FT-ICR−MS Analysis. FT-ICR mass spectra were collected on a Bruker Apex-Qe-FT-ICR mass spectrometer equipped with a 9.4 T superconducting magnet (Bruker Co., Chicago, IL). The LSP extract was dissolved in methanol at 0.1 μg/μL. Then, 100 μL of the solution was imported into an electrospray ionization (ESI) technique at a flow rate of 100 μL/h, with the temperature at 180 °C. Mass spectra were obtained in negative-ion mode, and the mass scale ranged from m/z 100 to 2000. HPLC−ESI−MS Analysis. The separation of LSPs by HPLC was performed on an Agilent 1100 liquid chromatograph system (Santa Clara, CA) with a ZORBAX TC-C18 (4.6 × 250 mm, 5 μm, Agilent) column. Separation was accomplished at 30 °C with 0.4% formic acid in water (solvent A) and methanol (solvent B) at 1.0 mL/min using a gradient as follows: 0−10 min, 5−25% B; 10−20 min, 25−55% B; 20− 30 min, 55% B; 30−35 min, 55−5% B; and re-equilibration with 5% B for 5 min. Samples were filtered through a 0.45 μm cellulose acetate spin filter before injecting 10 μL, and the eluate was monitored at 280 nm. Electrospray ionization mass spectroscopy (ESI−MS) analysis was conducted on an Agilent 1100 LC−MS spectrometer (Santa Clara, CA) equipped with a diode array detector and an Agilent 6300 ion trap (Santa Clara, CA) in negative-ion mode. The mobile phase and 9745

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to give a 0.02 M stock solution. The stock solution was diluted with the same phosphate buffer to 50, 25, 12.5, 6.25, and 3.125 μM working solutions. The net area under the curve (AUC) of the standard and samples was calculated as

AUC = 0.5 + f1 /f0 + ... + fi /f0 + ... + f15 /f0 + 0.5(f16 /f0 ) where f 0 is the initial fluorescence reading at 0 min and f i is the fluorescence reading at time i. Final ORAC values were expressed as micromoles of Trolox equivalents per liter of samples. Data Analysis. All of the determinations were performed in triplicate. Data were expressed as the mean ± standard deviation (SD) and evaluated by one-way analysis of variation (ANOVA) of SPSS 19.0 (SPSS, Inc., Chicago, IL) with Duncan’s multiple range test. p values of fraction 3 > fraction 1. The same results were also obtained in the FRAP assay (Figure 3). Our results suggested that fraction 1 had very little contribution to the antioxidant activity of LSPs, which was in line with the results by Rangkadilok et al.1 Therefore, we ignored it in the following study. As described above, fraction 3 was mainly composed of ellagic acid, which was reported to

Table 2. Scavenging Rate and Activity Loss of Fraction 2 and Different Samples against the DPPH Radicala sample by online knockout fraction 2 sample 4 (fraction 2-1 missing) sample 5 (fraction 2-2 missing) sample 6 (fraction 2-3 missing)

scavenging rate (%) 41.13 34.89 24.68 34.11

± ± ± ±

0.004 0.012 0.025 0.006

a b c b

activity loss (%) 15.16 ± 0.028 b 40.00 ± 0.06 a 17.05 ± 0.016 b

The data are expressed as the mean ± SD (n = 3). Mean values in the same column with unlike letters are significantly different (p < 0.05).

a

scavenging effect of each sample with target fraction knockout was in the following order: sample 4 (fraction 2-1 missing) > sample 6 (fraction 2-3 missing) > sample 5 (fraction 2-2 missing), which meant that the order of the contribution of each fraction was fraction 2-2 > fraction 2-3 > fraction 2-1. When fraction 2-2 was knocked out, the scavenging effect of sample 5 (fraction 2-2 missing) reduced significantly, demonstrating that fraction 2-2 had vital contribution to the total radical scavenging activity of fraction 2, while the missing of fractions 2-1 and 2-3 did not affect the scavenging rate notably. The FRAP assay (Figure 5) provided further support for the above conclusion. These findings have led to a conclusion that fraction 2-2 was the target bioactive group in longan seed extract. However, a further online fishing or knockout program was needed to screen the target bioactive compounds.

Figure 3. Reducing power of total polyphenols, sample 1 (fraction 1 missing), sample 2 (fraction 2 missing), and sample 3 (fraction 3 missing). Bars with different letters at the same concentration differ significantly (p < 0.05). 9747

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Table 3. Scavenging Rate and Activity Loss of Fraction 2-2 and Different Collections against the DPPH Radicala collection by online knockout fraction 2-2 collection 1 collection 2 collection 3 collection 4 collection 5 collection 6 collection 7 collection 8

(peak (peak (peak (peak (peak (peak (peak (peak

1 2 3 4 5 6 7 8

missing) missing) missing) missing) missing) missing) missing) missing)

scavenging rate (%) 81.54 28.37 54.55 41.72 34.23 40.19 35.16 57.27 69.31

± ± ± ± ± ± ± ± ±

0.008 0.007 0.008 0.007 0.008 0.005 0.011 0.008 0.007

a h d e g f g c b

activity loss (%) 65.20 33.10 48.83 58.02 50.71 56.88 29.77 15.00

± ± ± ± ± ± ± ±

0.009 a 0.01 e 0.009 d 0.01 b 0.006 c 0.013 b 0.01 f 0.009 g

The data are expressed as the mean ± SD (n = 3). Mean values in the same column with different letters are significantly different (p < 0.05).

a

Figure 5. Reducing power of fraction 2, sample 4 (fraction 2-1 missing), sample 5 (fraction 2-2 missing), and sample 6 (fraction 2-3 missing). Bars with unlike letters at the same concentration are significantly different (p < 0.05).

(peaks 1 > 4 > 6 > 5 > 3 > 2 > 7 > 8). It was evident that peaks 1, 4, and 6 performed a great contribution to the total antioxidant activity of fraction 2-2. Besides, the contribution rates of peaks 5 and 3 were 50.71 and 48.83%, respectively, which were a little lower than those of peaks 1, 4, and 6. However, very limited decreases on the DPPH-scavenging effects of collections with peaks 2, 7, and 8 knockout were observed, revealing that the three peaks had a small contribution to the bioactivity of fraction 2-2 in comparison to other peaks. The ORAC assay was used to further confirm the contribution of each peak to the antioxidant activity of fraction 2-2. As illustrated in Figure 7, the ORAC value of each

We obtained good separation of fraction 2-2 by adjusting the HPLC elution program as follows: the mobile phase consisted of 0.4% formic acid mixed with double distilled water (solvent A) and methanol/acetonitrile (50:50, v/v) (solvent B); the gradient program started from 0 min (5% B) to 10 min (25% B) and 20 min (32% B) and then was re-equilibrated for 10 min with 5% B for the next analysis. The HPLC fingerprint of fraction 2-2 was finally obtained with favorable separation. Likewise, peaks 1−8 in fraction 2-2 were knocked out one by one. By comparison of the obtained chromatograms (Figure 6),

Figure 7. ORAC values of fraction 2-2 and different collections with target peak knockout. All of the results are expressed as micromoles of Trolox equivalents per liter.

Figure 6. Chromatograms of peak knockout under the same HPLC conditions: (a) chromatogram with fraction 2-2, (b) chromatogram with peak 1 knockout, (c) chromatogram with peak 2 knockout, (d) chromatogram with peak 3 knockout, (e) chromatogram with peak 4 knockout, (f) chromatogram with peak 5 knockout, (g) chromatogram with peak 6 knockout, (h) chromatogram with peak 7 knockout, and (i) chromatogram with peak 8 knockout.

collection was in the following order: collections 8 (peak 8 missing) > 7 (peak 7 missing) > 5 (peak 5 missing) > 2 (peak 2 missing) > 3 (peak 3 missing) > 1 (peak 1 missing) > 6 (peak 6 missing) > 4 (peak 4 missing), demonstrating that the contribution of each peak to the antioxidant activity of fraction 2-2 declined as follows: peaks 4 > 6 > 1 > 3 > 2 > 5 > 7 > 8. The minor discrepancy between the results of DPPH and ORAC assays may be due to the different systems.2,24 Taken together, DPPH and ORAC analyses suggested that peaks 1, 4, and 6 could be the main contributors to the total antioxidant activity of fraction 2-2. These specific bioactive compounds may represent the remarkable antioxidant effect of polyphenols in longan seeds.

it was obvious that the target peaks in fraction 2-2 were knocked out completely. Similarly, DPPH and ORAC assays were used to screen and evaluate the possible target bioactive compounds in fraction 2-2. The scavenging rates of all of the collections with target peaks missing were summarized in Table 3. The DPPH scavenging property of each collection declined as follows: collections 8 (peak 8 missing) > 7 (peak 7 missing) > 2 (peak 2 missing) > 3 (peak 3 missing) > 5 (peak 5 missing) > 6 (peak 6 missing) > 4 (peak 4 missing) > 1 (peak 1 missing), consistent with the results of contribution rates 9748

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Table 4. HPLC−ESI−MS Analysis for Individual Phenolic Compounds of Fraction 2-2 of Longan Seed Extract peak number

retention time (min)

[M − H]− (m/z)

1 2 3 4 5 6 7 8

13.41 13.63 14.01 14.65 14.91 15.77 16.61 17.97

291 291 633 387 469 611 477 197

MS2 ions (m/z) 247, 247, 463, 369, 425, 397, 462, 169,

205, 205, 301, 207, 301 301, 301, 125

161 161 275 163, 119 229 169

compound unknown unknown corilagin p-coumaric acid glycoside (S)-flavogallonic acid ellagic acid derivative methyl-ellagic acid glucopyranoside ethyl gallate

Peak 7, with a m/z of 477, was identified as methyl-ellagic acid glucopyranoside on the basis of a previous study29 and the observed mass spectra. The fragment ions at m/z 462 ([M − H − 15]−) and m/z 301 ([M − H − 15 − 161]−) corresponded to the loss of a methyl unit and a glucosyl unit, respectively. Ellagic acid at m/z 301 was also identified. Peak 8, with a m/z of 197, was determined to be ethyl gallate, which yielded the characteristic fragment ion at m/z 169 (gallic acid) by losing an ethyl unit. The fragment ion at m/z 125 resulted from the continuing loss of CO2, further confirming the proposed structure.30,31 This compound was previously identified in longan seeds by Zheng et al.2 In summary, the combination of FT-ICR−MS analysis, HPLC−ESI−MS analysis, online fishing or knockout, and antioxidant-based screening strategies was used to screen the key phenolic compounds accounting for the potent antioxidant effects of longan seeds for the first time. Ellagic acid and its derivative were proven to have important contribution to the antioxidant activity of LSPs. A new compound, probably pcoumaric acid-glycoside, had the greatest contribution to the antioxidant capacity of LSPs, while gallic acid, corilagin, (S)flavogallonic acid, methyl-ellagic acid glucopyranoside, and ethyl gallate did not affect the potent antioxidant activity of LSPs, showing a small contribution to the total bioactivity. Further work on the detailed structures of the newly found phenolic compounds of p-coumaric acid-glycoside and ellagic acid derivative with high antioxidant activity and their structure−activity relationships is needed.

Therefore, we used HPLC−ESI−MS to explore the structures of peaks 1−8 in fraction 2-2. The results are summarized in Table 4. HPLC−MS analysis in negative-ion mode revealed that peaks 1 and 2 had a common molecular mass and fragmentation pattern, suggesting a pair of isomers. The molecular ion [M − H]− was observed at m/z 291. Fragment ions were found at m/z 247, 205, and 161 in the MS2 spectrum. The fragment at m/z 247 corresponded to a loss of 44 amu resulting from the elimination of a CO2 molecule from the molecular ion. The fragment ion at m/z 205 was derived from the loss of two ethenone, and the fragment ion at m/z 161 came from the further loss of a CO2 molecule. The ESI−MS fragmentation patterns of peaks 1 and 2 were very similar to that of catechin but with dominant peaks Δ2 amu larger than that of catechin. It was reported to exist in strawberry fruits in a previous study,25 but the detailed structure was unknown. Peak 3 showed a distinct molecular ion at m/z 633, and its three characteristic MS2 fragments ions were observed at m/z 463, 301, and 275, indicating that it was galloyl-HHDPglucopyranose (corilagin).21 The fragment at m/z 463 was attributed to the loss of a galloyl unit from the parent molecule, and the ion at m/z 301 was defined as ellagic acid.26 It was also reported to be one of the major polyphenol components of longan seeds by Sudjaroen et al.18 The molecular ion of peak 4 was at m/z 387. Four characteristic fragment ions were found at m/z 369, 207, 163, and 119, in the MS2 spectrum of m/z 387. The first fragment ion ([M − H − 18]−) at m/z 369 resulted from the elimination of a water molecule from the parent molecule. The fragment ions at m/z 207 ([M − H − 18 − 162]−) and m/z 163 ([M − H − 18 − 162 − 44]−) corresponded to the further loss of a glucosyl moiety and the continuing loss of a CO2 molecule. The fragment ion at m/z 163, with its typical production ion at m/z 119, was identified as p-coumaric acid.27,28 Thus, it was deduced that peak 4 was probably a p-coumaric acid−glucose conjugate linked by a carboxyl group, but further work is needed to verify the structure of this compound. Peak 5 presented a molecular ion [M − H]− at m/z 469 with two characteristic MS2 fragment ions at m/z 425 and 301, respectively. The former came from the neutral loss of CO2, and the latter was identified as ellagic acid at m/z 301. On the basis of the mass spectra data,23 peak 5 was identified as (S)flavogallonic acid. Peak 6 showed a molecular ion at m/z 611 and two strong fragment ions at m/z 397 and 301. Besides, the fragment ion at m/z 229 was observed, which is indicative of the presence of ellagic acid (m/z 301). Because no more structural information was available, we deduced that this compound could be an ellagic acid derivative.

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AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: 86-27-87282966. E-mail: [email protected]. edu.cn. Funding

This study was supported by the National Science and Technology Pillar Program during the 12th Five-Year Plan Period (2012BAD31B03), the Chinese Ministry Program for New Century Excellent Talents in University (NCET-120865), and the Fundamental Research Funds for the Central Universities (2013PY022). Notes

The authors declare no competing financial interest.

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dx.doi.org/10.1021/jf502995z | J. Agric. Food Chem. 2014, 62, 9744−9750