Article pubs.acs.org/JAFC
Bioavailability of Hydroxycinnamic Acids from Crepidiastrum denticulatum Using Simulated Digestion and Caco‑2 Intestinal Cells Hee Ju Lee,† Kwang Hyun Cha,† Chul Young Kim,‡ Chu Won Nho,†,§ and Cheol-Ho Pan*,†,§ †
Functional Food Center, Korea Institute of Science and Technology (KIST), Gangneung 210-340, Republic of Korea College of Pharmacy, Hanyang University, Ansan 426-791, Republic of Korea § Department of Biological Chemistry, Korea University of Science and Technology (UST), Youseong-gu, Dajeon 305-350, Republic of Korea ‡
ABSTRACT: Hydroxycinnamic acids have antioxidant properties and potentially beneficial effects on human health. This study investigated the digestive stability, bioaccessibility, and permeability of hydroxycinnamic acids from Crepidiastrum denticulatum using simulated digestion and Caco-2 intestinal cells. The major compounds of C. denticulatum were determined to be four hydroxycinnamic acids [caftaric acid, chlorogenic acid, chicoric acid, and 3,5-di-O-caffeoylquinic acid (3,5-DCQA)] and one flavonoid (luteolin-7-O-glucuronide) by high-performance liquid chromatography and electrospray ionization mass spectrometry. Hydroxycinnamic acids from C. denticulatum were rapidly released in the stomach and duodenum phase, maximizing the possibility of absorption in the intestinal Caco-2 cells. The digestive stability and bioaccessibility of hydroxycinnamic acids from C. denticulatum were markedly low after simulated digestion and remained minimal in the soluble fraction of the ileum phase. Unlike the four hydroxycinnamic acids, luteolin-7-O-glucuronide was stable in terms of digestive stability and bioaccessibility during simulated digestion. The cell permeabilities (Papp A to B/Papp B to A) of caftaric acid (0.054) and chlorogenic acid (0.055) were higher than those of chicoric acid (0.011) and 3,5-DCQA (0.006) in general. That of luteolin-7-O-glucuronide was not detectable, showing its low absorption in Caco-2 cells. These results indicate that the rapid release of hydroxycinnamic acids in the stomach and duodenum phase may increase the potential for absorption in Caco-2 cells, and that luteolin-7-O-glucuronide, which was stable in terms of digestive stability and bioaccessibility, has relatively low absorption compared with hydroxycinnamic acids. KEYWORDS: hydroxycinnamic acid, Crepidiastrum denticulatum, bioavailability, simulated digestion, Caco-2 intestinal cells
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INTRODUCTION The hydroxycinnamic acids, a major class of phenolic compounds, are found in plant-derived foods.1,2 Natural derivatives of hydroxycinnamic acids are represented by their quinic acids or glucose esters, which are present in fruits and vegetables.3 Hydroxycinnamic acids play an important role in total polyphenol intake. The daily consumption of hydroxycinnamic acids is usually 211 mg/day, although differences exist depending on the studied group.4 Chlorogenic acid, the ester of caffeic acid and quinic acid, is found in many types of fruit (apples, cherries, berries, and pears) and is also a major component of coffee.5 Caftaric acid (caffeoyltartaric acid) is found as the major phenolic component of grapes and wines. The caftaric acid content depends on the species, cultivar, ripeness, and winemaking process.6 Chicoric acid is a 2,3dicaffeoyltartaric acid, which was isolated from Cichorium intybus for the first time, and has been reported to be present in significant amounts in Echinacea purpurea.7,8 Additionally, 3,5di-O-caffeoylquinic acid (3.5-DCQA), which is the ester of two caffeic acids and one quinic acid, is reported as a characteristic compound in Asteraceae plants.9 Hydroxycinnamic acids have antioxidant properties and potentially beneficial effects on human health as protective agents against cancer, cardiovascular and inflammatory disease, and aging.10−12 Crepidiastrum denticulatum (the common name is e-go-deulppae-gi in Korean), an edible vegetable, grows in East Asia in countries such as Korea, Japan, and China.13 Young roots and © 2014 American Chemical Society
leaves of C. denticulatum are consumed as kimchi (fermented food), as a vegetable, and as a powder in Korea. C. denticulatum has been reported to have cancer chemopreventive activity and to prevent cell death induced by oxidative stress.14,15 In addition, Crepidiastrum lanceolatum, which is used as a vegetable in Japan, has been reported to show antioxidative activity due to chicoric acid, chlorogenic acid, and caffeic acid.16 Most studies related to the bioavailability of hydroxycinnamic acid have focused on caffeic acid and chlorogenic acid. Chlorogenic acid has markedly reduced absorption in humans and rats.17,18 In fact, the absorption of chlorogenic acid occurs mainly in the colon after hydrolysis by intestinal microbes. The absorption of chlorogenic acid in the small intestine represented ∼8% using the in situ perfusion model, reflecting its the poor intestinal bioavailability.19 The bioavailability of other hydroxycinnamic acids has not yet been reported. In this study, we identified various hydroxycinnamic acids from C. denticulatum by high-performance liquid chromatography (HPLC) and mass spectrometry (MS) and evaluated the digestive stability and bioaccessibility of hydroxycinnamic acids using an in vitro simulated digestion model. Furthermore, we Received: Revised: Accepted: Published: 5290
January 19, 2014 May 12, 2014 May 19, 2014 May 19, 2014 dx.doi.org/10.1021/jf500319h | J. Agric. Food Chem. 2014, 62, 5290−5295
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cultures were grown for 21−29 days to obtain a differentiated intestinal cell monolayer. Transepithelial electrical resistance (TEER) measurements were taken twice weekly using a Millipore Millicell-ERS system (Millipore Corp., Bedford, MA) to ensure the monolayer was intact. Once the TEER values reached 600−800 Ω/cm2, the monolayer was used for transport study. To initiate the transport experiments, the culture media in the donor and receiving compartments were carefully aspirated, and the cells were rinsed with prewarmed transport medium [HBSS supplemented with 25 mM HEPES (pH 7.4)]. Following a 30 min equilibration with the transport medium at 37 °C, the cells were incubated with each hydroxycinnamic acid for 2 h for transport studies. The transport studies were performed in both directions as follows. For apical-to-basolateral (a-to-b) transport, 1.2 mL of transport medium was added in the basolateral part, 0.45 mL of each hydroxycinnamic acid (2 mM) dissolved in the transport medium was added in the apical part, and 0.05 mL of the sample (C0) was immediately withdrawn into a collection tube. After 2 h, 0.6 mL of basolateral transport medium was withdrawn for the analysis and replenished with an equal volume of fresh transport medium. For basolateral-to-apical (b-to-a) transport, 1.25 mL of each hydroxycinnamic acid dissolved in the transport solution was added in the basolateral part and a 0.05 mL sample was immediately withdrawn, followed by 0.4 mL of transport medium being added in the apical part; 0.2 mL of apical transport medium was withdrawn after 2 h and replenished with the same volume of fresh transport medium. The TEER values were measured after the experiment to confirm the cell integrity status. All of the collected samples were added to the same volume of 0.6% formic acid in acetonitrile to increase the stability of hydroxycinnamic acids prior to liquid chromatography−MS analysis. The apparent permeability coefficient (Papp) was calculated as
investigated the intestinal cell permeability of hydroxycinnamic acids in a Caco-2 cell transport model.
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MATERIALS AND METHODS
Samples and Chemicals. α-Amylase, pepsin, pancreatin, pancreatic lipase, and bile extract were purchased from Sigma-Aldrich for simulated digestion. All cell culture reagents, including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, 1 M 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), phosphatebuffereds saline (PBS), 100× penicillin and streptomysin, and trypsin/ EDTA, were obtained from Gibco (Carlsbad, CA). Hank’s balanced salt solution (HBSS) and 100× nonessential amino acids were purchased from Sigma-Aldrich. Transwell permeable polycarbonate inserts (0.4 μm) and 12-well cell culture plates were obtained from Corning Inc. Solvents used for HPLC were purchased from Fisher Scientific (Springfield, NJ). Unless indicated, all other chemicals were supplied by Sigma-Aldrich. The standard for quantitative analysis, chlorogenic acid, was purchased from Aldrich Chemical Co. (St. Louis, MO). Caftaric acid, chicoric acid, luteolin-7-O-glucuronide, and 3,5DCQA were purified from C. denticulatum by open column chromatography and semipreparative HPLC. Chemical structures were confirmed by 1H and 13C nuclear magnetic resonance data by comparison with the references.16,20,21 Sample Preparation. The whole plants of C. denticulatum were collected at the Wild Vegetable Experiment Station, Pyeongchang, Korea, in June 2012. For simulated digestion, the whole plants of C. denticulatum were dried at room temperature and ground to a powder before being used. All extracts were filtered (0.45 μm, 13 mm, syringe filter GHP, Smartpor) and analyzed in the HPLC system. All experiments were conducted in triplicate. In Vitro Simulated Digestion. Simulated digestion was performed according to the method described by Garrett et al.22 with minor modifications. To simulate in vitro digestion, digestive steps were conducted sequentially. Briefly, C. denticulatum powder (1 g) was homogenized in 10 mL of saline solution containing 120 mM NaCl, 5 mM KCl, and 6 mM CaCl2 (pH 5.5). Then, 1000 units of αamylase was added, and the pH was adjusted to 6.5. After the saline solution had been added to a volume of 12.5 mL, the samples were incubated at 37 °C for 5 min in a shaking water bath (Lab companion, Jeio Tech, Korea) at 95 rpm for the simulated oral phase of digestion. To mimic the gastric phase of human digestion, the pH of the sample was acidified to 2.2 with HCl and 0.5 mL of a porcine pepsin solution (0.075 g/mL in 0.1 N HCl) was added. The samples were suspended in a saline solution to a volume of 15 mL and incubated at 37 °C for 2 h in a shaking water bath at 95 rpm. To simulate the intestinal phase more accurately and specifically, that phase was separated into three intestinal parts (duodenum, jejunum, and ileum) and simulated sequentially. In the duodenum part, 250 mg of bile extract, 0.5 mL of pancreatic lipase (0.01 g/mL of 0.1 M sodium bicarbonate), and 0.5 mL of pancreatin (0.08 g/mL saline) were added and the pH was increased to 4.0 by adding 1 M sodium bicarbonate. Samples were incubated for 30 min at 37 °C (final volume of 20 mL). In the jejunum part, the pH was adjusted to 5.5 and the sample incubated for 90 min at 37 °C (final volume of 22.5 mL). Finally, the pH was adjusted to 7.0 and the sample incubated for 300 min at 37 °C (final volume of 25 mL) to mimic the ileum part. At the end of each digestion, digestates (100 μL) were collected and added to aqueous ethanol [1:1 (v/v)] to determine the bioaccessibility of hydroxycinnamic acids. To measure the digestive stability of hydroxycinnamic acids, digestates of each intestinal phase were added to ethanol (25 mL) and extracted by sonication for 1 h. After the mixtures had been centrifuged at 12300g for 10 min, their supernatants were collected and filtered through a 0.45 μm GHP filter prior to HPLC analysis. Transport of Hydroxycinnamic Acids via a Caco-2 Cell Monolayer. Caco-2 cells (HTB-37, American Type Culture Collection, Rockville, MD) were maintained for cellular transport experiments, according to the method of Hubatsch et al.23 For the experiments, cells were seeded at a density of 3 × 105 cells/cm2 on the tissue culture-treated polycarbonate filter in 12-transwell plates. The
Papp =
∂Q AC0 ∂t
where ∂Q/∂t is the permeability rate, A is the surface area of the membrane filter, and C0 is the initial concentration in the donor compartment (the apical compartment in the a-to-b transport study and the basolateral compartment in the b-to-a transport study). HPLC Analysis. HPLC was used for the quantitative analysis of hydroxycinnamic acids in C. denticulatum during simulated digestion. The HPLC system consisted of an Agilent Series 1200 liquid chromatography system with a G1379B vacuum degasser, a G1312A binary pump, a G1329A autosampler, a G1316A column oven, a G1315B diode array detector (DAD), and ChemStation (version B.02.01, Agilent, Waldbronn, Germany). The separation was undertaken with a Shiseido Capcell pak MG II C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase consisted of 0.3% formic acid in water (solvent A) and 0.3% formic acid in acetonitrile (solvent B) with the following conditions: 82:18 [A:B (v/v)] isocratic mode for 30 min. The flow rate was 1.0 mL/min, and the oven temperature was set at 35 °C. Detection was conducted at 330 nm, and the injection volume of the standards and extracts was 10 μL. HPLC−PDA−MS Analysis. HPLC−PDA−MS analysis was used for the identification of major compounds in C. denticulatum and the quantitative analysis of hydroxycinnamic acids during the intestinal Caco-2 cell permeability test. All analyses were performed using a Thermo HPLC−PDA−MS system (Thermo Fisher Scientific Inc., San Jose, CA) equipped with an ACCELA photodiode array detector, an autosampler, a quarternary pump, and an LCQ FLEET ion trap mass spectrometer with an electrospray ionization source. Thermo Xcalibur (version 2.1) was used for data acquisition and processing. The mobile phase was the same as that previously described in the HPLC analysis section. A Waters Acquity BEH C18 column (3.0 mm × 100 mm, 1.7 μm) was used. The gradient mode was as follows: from 0 to 10 min, initial mobile phase solvent A/B [90:10 (v/v)]; from 10 to 45 min, 70:30 linear gradient and reconditioning steps to initial condition for 15 min. The flow rate was 150 μL/min, and detection was conducted at 330 nm. The injection volume of samples was 10 μL. The mass spectrometer conditions were as follows: negative ion mode; mass 5291
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Figure 1. (A) HPLC chromatogram of C. denticulatum extracts and structures of four hydroxycinnamic acids and one flavonoid and (B) ESI-MS spectra of five compounds.
Table 1. Calibration Curve, Precision, LOD, and LOQ of Five Compounds in C. denticulatum calibration curve compound caftaric acid chlorogenic acid chicoric acid luteolin-7-O-glucuronide 3,5-DCQA
regressive equation y y y y y
= = = = =
25.399x 24.963x 32.962x 16.006x 29.750x
− 9.299 − 12.613 − 42.775 + 7.051 + 73.041
precision, RSD (%) r2
linear range (μg/mL)
intraday (n = 3)
interday (n = 3)
LOD (μg/mL)
LOQ (μg/mL)
0.9997 0.9999 1.0000 0.9998 0.9999
3.90625−125 7.8125−62.5 3.90625−1000 3.90625−62.5 3.90625−500
0.09 0.55 2.39 0.36 0.44
0.12 0.56 1.73 0.97 0.11
0.82 0.90 0.93 0.13 0.22
2.48 2.74 2.83 0.39 0.66
range, m/z 100−1000; capillary voltage, −7.0 V; tube lens, −45 V; sheath gas flow rate (N2), 50 arbitrary units; auxiliary gas flow rate (N2), 12 arbitrary units; capillary temperature, 350 °C. Data Analysis. A minimum of three independent observations were made in each experiment. All data are expressed as means ± the standard error of the mean (SEM). Statistical comparisons between the experimental groups and the control group were performed using the Student’s t test. The Student−Newman−Keuls test was performed to separate the means of experimental groups when significant differences (P < 0.05) occurred. Statistical analyses were performed using GraphPad Prism version 5.
ions at m/z 311, and fragmentation in the MS experiment produced a caffeic acid (m/z 179) as a prominent fragment. Peak 1 appeared to be caftaric acid. Peak 2 was identified as chlorogenic acid by mass spectra. Fragmentation of the [M − H]− ion at m/z 353 was characterized by the loss of a caffeic acid moiety, resulting in a product ion at m/z 191. Peak 3 showed [M − H]− ions at m/z 473. The fragmentation of peak 3 produced a product ion at m/z 311 (loss of a caffeoyl moiety) and m/z 293 (loss of the caffeic acid and an additional loss of water). Peak 3 was assigned to chicoric acid. Peak 4 showed [M − H]− ions at m/z 461 and another characteristic fragment at m/z 285 of the aglycone, corresponding to the loss of a glucuronide moiety. Therefore, peak 4 was assigned to luteolin7-O-glucuronide following a comparison against the reference compound. Peak 5 showed [M − H]− ions at m/z 515 and a product ion at m/z 353 after the loss of one caffeic acid. This fragment is typical of dicaffeoylquinic acid. Peak 5 was identified as 3,5-DCQA through comparison with an authentic compound. Five compounds were identified as caftaric acid, chlorogenic acid, chicoric acid, luteolin-7-O-glucuronide, and 3,5-DCQA by comparing their MS data with references24,25 and
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RESULTS AND DISCUSSION Identification and Quantitative Analysis of Hydroxycinnamic Acids in C. denticulatum. The aqueous ethanol extracts of C. denticulatum were injected into the HPLC system. Following separation, five major compounds were detected in their HPLC profiles with a DAD chromatogram (Figure 1A). HPLC−ESI-MS was used to identify the four hydroxycinnamic acids and one flavonoid, and the mass spectra of the five compounds are shown in Figure 1B. Peak 1 showed [M − H]− 5292
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Figure 2. Digestive stability (◆, solid line) and release (■, dotted line) of four hydroxycinnamic acids and one flavonoid during simulated digestion: (A) caftaric acid, (B) chlorogenic acid, (C) chicoric acid, (D) luteolin-7-O-glucuronide, and (E) 3,5-DCQA. A different letter for each digestive stage indicates significant differences (P < 0.05) between the treatment means of each compound’s recovery or release.
Figure 3. (A) Content of soluble chicoric acid at various pH values in the absence of gastric enzymes and (B) solvent partition of the digestate at various pH values to evaluate insoluble chicoric acid under each condition (upper phase, ethyl acetate; lower phase, aqueous fraction).
co-injections of authentic standards. Five concentrations of each standard compound were injected in triplicate to establish the calibration curves. As shown in Table 1, the limits of detection (LOD) and quantification (LOQ) for each standard were determined at the standard deviation (SD) of the response and the slope with values of ∼3.3 and ∼10, respectively. Intra- and interday variability was determined three times each day and on three separate days. Variations are indicated by the relative standard deviations (RSDs). Digestive Stability and Bioaccessibility of Hydroxycinnamic Acids in C. denticulatum during Simulated Digestion. The contents of hydroxycinnamic acids in C. denticulatum were measured by HPLC to evaluate the digestive stability and bioaccessibility of the major compounds (Figure 2). The stability of each compound was checked by measuring the amount of compound remaining after each digestion step. Values for chlorogenic acid, chicoric acid, and 3,5-DCQA remained at 66.6, 65.1, and 57.0%, respectively, during the initial digestion phase from food to the mouth, while the value for caftaric acid decreased only slightly throughout the
digestion phase from the mouth to the jejunum, indicating that caftaric acid was stable under general digestive conditions compared with chlorogenic acid, chicoric acid, and 3,5-DCQA. Values for caftaric acid, chlorogenic acid, and chicoric acid remained at 38.2, 20.4, and 23.1%, respectively, after harsh digestion in the ileum at pH 7.0 for 5 h. The value for 3,5DCQA was also markedly degraded after simulated digestion and remained at only 5.1% in the soluble fraction of the ileum phase. Unlike the four hydroxycinnamic acids, luteolin-7-Oglucuronide was stable and was preserved after total simulated digestion (residual percentage, 65.3%). The degradation of caftaric acid, chlorogenic acid, chicoric acid, and 3,5-DCQA might be dependent on the pH, and the stability of these compounds tends to be decreased under higher-pH conditions (pH >6.0).26 Bioaccessibility is defined as the relative amount of phytochemicals “released” from a complex food matrix during digestion, which could potentially also be available for absorption.27 Five compounds were released from their food matrices during simulated digestion, and the digestates of the 5293
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Table 2. Apparent Permeability Coefficients (Papp) of Hydroxycinnamic Acids in the Caco-2 Model compound caftaric acid chlorogenic acid chicoric acid luteolin-7-O-glucuronide 3,5-DCQA
Papp A to B (×10−6 cm/s)a 0.119 0.046 0.038 NDd 0.025
Papp B to A (×10−6 cm/s)b
Papp A to B/Papp B to Ac
± ± ± ± ±
0.054 0.055 0.011 NDd 0.006
± 0.010 ± 0.006 ± 0.004
2.197 0.839 3.502 7.850 4.337
± 0.003
0.075 0.174 0.188 0.265 0.211
a
Papp A to B, transport of the compounds from the apical to basolateral compartment. bPapp B to A, transport from the basolateral to apical compartment. c The Papp A to B/Papp B to A ratio is the ratio of Papp A to B to Papp B to A. Data are means ± SEM (n = 3−6). dNo detection.
significant differences between the P app B to A of each hydroxycinnamic acid, and the ratio of Papp A to B to Papp B to A was approximately 5−9 times higher in chlorogenic acid and caftaric acid than in chicoric acid and 3,5-DCQA. According to previous sudies,29,30 chlorogenic acid was not well absorbed in the Caco-2 cell transport model, rat chronic absorption study, or human clinical study. The ratio of Papp A to B to Papp B to A of caftaric acid, chicoric acid, and 3,5-DCQA was even lower than that of chlorogenic acid. This result indicates that some efflux mechanisms may exist with hydroxycinnamic acids in their transport across the Caco-2 cell monolayer. Luteolin-7-Oglucuronide, which was the most stable compound among the five main components from C. denticulatum during simulated digestion, has a Papp B to A far higher than those of four hydroxycinnamic acids. This agreed well with other studies that demonstrated that flavonoid glycosides such as luteolin-7-Oglucuronide were poorly absorbed in Caco-2 cells because of the active extrusion of P-glycoprotein.31−33 Interestingly, chicoric acid is most abundant in C. denticulatum, followed by caftaric acid, 3,5-DCQA, and chlorogenic acid. However, caftaric acid and chlorogenic acid might be more important hydroxycinnamic acids in humans when considering their entire bioavailability such as their digestive stability, their efficiency of release from the food matrix, and their intestinal absorption. Further research using in vivo rat models and clinical studies will be required to clarify the bioavailability of these hydroxycinnamic acids. In conclusion, this study shows that hydroxycinnamic acids from C. denticulatum were rapidly accessible in the stomach and duodenum phase, maximizing the possibility of absorption in intestinal Caco-2 cells. However, the intestinal cell permeability of hydroxycinnamic acids indicated that some efflux mechanisms might exist with hydroxycinnamic acids in their transport across the Caco-2 cell monolayer. The digestive stabilities of hydroxycinnamic acids from C. denticulatum were markedly low after simulated digestion and remained minimal in the soluble fraction of the ileum phase. Further, in vivo rat models and human clinical studies are needed to clarify the bioavailability of hydroxycinnamic acids.
soluble fraction were available for uptake by absorptive epithelial cells.28 Because of their hydrophilic properties, 1.12, 0.28, and 2.52 mg of caftaric acid, chlorogenic acid, and chicoric acid, respectively, were released from the food matrix. A significant increase appeared in the bioaccessibility of caftaric acid (87.8%) and chlorogenic acid (61.3%) during simulated digestion (Figure 2A,B). More than 60% of caftaric and chlorogenic acid was released in the stomach and duodenum phase. However, the bioaccessibility of chicoric acid was very low through the mouth and stomach step, which suddenly underwent a change in pH from 6.5 to 2.2 (Figure 2C). Because of the pH change, chicoric acid (stomach phase, 1.36 mg) was aggregated during digestion and was insoluble in the digestate. The solubility of chicoric acid was shown at various pH values in the absence of gastric enzymes (Figure 3A) and was low at pH 2 and 3 compared to that at pH 4 and 5. This result may explain why the result in Figure 2C was obtained. The additional experiment was conducted using the solvent partition of chicoric acid from the digestate to evaluate the insoluble chicoric acid at various pH values (Figure 3B). The lower phase (aqueous fraction) and upper phase (ethyl acetate fraction) were analyzed by HPLC to evaluate the chicoric acid content. Chicoric acid, which was insoluble at pH 10−6 cm/s, except for that of chlorogenic acid (0.839 × 10−6 cm/s). In particular, there were
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +82-33-650-3652. Fax: +82-33-650-3679. E-mail:
[email protected]. Funding
This work was supported by a KIST intramural research grant (2Z04220). Notes
The authors declare no competing financial interest. 5294
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ACKNOWLEDGMENTS We thank the Wild Vegetable Experiment Station (Pyeongchang, Korea) for supplying the cultivated C. denticulatum.
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ABBREVIATIONS USED
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REFERENCES
3,5-DCQA, 3,5-dicaffeoylquinic acid; TEER, transepithelial electrical resistance; DAD, diode array detector; SEM, standard error of the mean; LOD, limit of detection; LOQ, limit of quantification; RSD, relative standard deviation
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dx.doi.org/10.1021/jf500319h | J. Agric. Food Chem. 2014, 62, 5290−5295
