Stability of Dietary Polyphenols under the Cell Culture Conditions

Article pubs.acs.org/JAFC

Stability of Dietary Polyphenols under the Cell Culture Conditions: Avoiding Erroneous Conclusions Jianbo Xiao*,†,‡,§ and Petra Högger† †

Universität Würzburg, Institut für Pharmazie und Lebensmittelchemie, Am Hubland, 97074 Würzburg, Germany State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Taipa, Macau, China § Department of Nutrition and Food Hygiene, School of Public Health, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China ‡

ABSTRACT: Most data of bioactivity from dietary polyphenols have been derived from in vitro cell culture experiments. In this context, little attention is paid to potential artifacts due to chemical instability of these natural antioxidants. An early degradation time (CT10) and half-degradation time (CT50) were defined to characterize the stability of 53 natural antioxidants incubated in Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C. The degree of hydroxylation of flavones and flavonols significantly influenced the stability in order resorcinol-type > catechol-type > pyrogallol-type, with the pyrogallol-type being least stable. In contrast, any glycosylation of polyphenols obviously enhanced their stability. However, the glycosylation was less important compared to the substitution pattern of the nucleus rings. Methoxylation of flavonoids with more than three hydroxyl groups typically improved their stability as did the hydrogenation of the C2C3 double bond of flavonoids to corresponding flavanoids. There was no significant correlation between the antioxidant potential of polyphenols and their stability. Notably, the polyphenols were clearly more stable in human plasma than in DMEM, which may be caused by polyphenol−protein interactions. It is strongly suggested to carry out stability tests in parallel with cell culture experiments for dietary antioxidants with catechol or pyrogallol structures and pyrogallol-type glycosides in order to avoid artifacts. KEYWORDS: antioxidants, polyphenols, stability, structure−stability relationship, cell culture

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INTRODUCTION Dietary polyphenols are of great general interest due to their diverse bioactivity such as antioxidant, antidiabetes, antiinflammation, or anticancer activity.1−6 Their health effects depend on their bioavailability, which is influenced by the stability, formation of complexes with other components, interaction with food, uptake from the intestine, and both hepatic metabolism and metabolism by gut bacteria.7−13 Many dietary polyphenols have been explored to undergo considerable transformation and to show low bioavailability.14 It has been estimated that less than 5% of polyphenols are absorbed and reach the plasma unchanged. Most of the cellular effects ascribed to polyphenols have been deduced from in vitro cell culture experiments which allow detailed investigations of certain signaling pathways or the influence on gene transcription under controlled conditions. For example, in a typical cytotoxicity and proliferation assay for studying anticancer potential, the individual polyphenol or the extracts rich in polyphenols are typically incubated with various cell lines for up to a few days.15−17 For intestinal absorption and transportation experiments with human epithelial cells, polyphenols or their extracts are usually incubated for less than 1 h under the cell culture conditions.18−21 However, it is still not clear what happens with most polyphenols during this incubation period. Individual compounds might undergo local metabolism in the cells they encounter,21 or they might reveal chemical instability under the cell culture conditions.14 In this context, the influence of the cell culture medium on the stability and fate of polyphenols is not routinely taken into account, which might lead to imprecise conclusions. There are limited © 2015 American Chemical Society

investigations regarding the stability of polyphenols in cell culture. Herein, we investigated the structure−stability relationship of polyphenols (Table 1) incubated with Dulbecco’s modified Eagle’s medium (DMEM) under the cell culture conditions up to 180 min. We detected a significant relationship between the structure of natural polyphenols and their stability. It is strongly suggested to carry out stability tests in parallel with cell culture experiments for natural polyphenols with catechol or pyrogallol structures and pyrogallol-type glycosides in order to avoid artifacts.

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EXPERIMENTAL PROCEDURES

Chemicals. Daidzein (>95.0%), piceatannol (>98.0%), formononetin (>98.0%), myricetin (>97.0%), 7,8-dihydroxyflavone (>98.0%), pinostilbene (>97.0%), kaempferol hydrate (>97.0%), resveratrol (>98.0%), piceid (>95.0%), 4′-hydroxyflavanone (>98.0%), fisetin (>96.0%), naringenin, hesperetin (>97.0%), baicalein (>98.0%), isorhapontigenin (>95.0%), puerarin (>98.0%), apigenin (>98.0%), oxyresveratrol (>95.0%), epigallocatechin-3-gallate (EGCG) (>98.0%), 3-methoxyflavone (>98.0%), pterostilbene (>98.0%), 3,4′dihydroxyflavone (>97.0%), 3-hydroxy-4′-methoxyflavone (>98.0%), 3-hydroxyflavone (>98.0%), 6-methoxyflavone (>98.0%), genistein (>96.0%), rutin trihydrate (>98.0%), and chrysin (>97.0%) were purchased from Tokyo Chemical Industry Co., Ltd. (Shanghai, China). Baicalin (95.0%), aposide (>98.0%), vitexin (>98.0%), luteolin (>98.0%), luteolin 7-O-glucoside (>98.0%), hispidulin (>97.0%), Received: Revised: Accepted: Published: 1547

November 18, 2014 January 8, 2015 January 21, 2015 January 21, 2015 DOI: 10.1021/jf505514d J. Agric. Food Chem. 2015, 63, 1547−1557

Journal of Agricultural and Food Chemistry

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Table 1. Results of the Stability Experiments of Various Classes of Polyphenols (Initial Concentration 10 μM) in DMEM under the Cell Culture Condition (37 °C, 5% CO2)a

a

Results depict mean and SD of n = 3 samples. 1548

DOI: 10.1021/jf505514d J. Agric. Food Chem. 2015, 63, 1547−1557


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wogonoside (>98.0%), galangin (>97.0%), gallocatechin-3-gallate (GCG) (>98.0%), epicatechin (EC) (>98.0%), epigallocatechin (EGC) (>98.0%), kaempferide (>97.0%), quercetin (>97.0%), catechin (C) (96.0%), quercitrin (>98.0%), isorhametin (>98.0%), myricitrin (>98.0%), formononetin 7-O-glucoside (>98.0%), genistin (>98.0%), daidzin (>96.0%), biochanin A (>98.0%), naringin (>95.0%), narirutin (>97.0%), hesperidin (>97.0%), liquiritigenin (>98.0%), and liquiritin (>98.0%) were obtained from Aladdin Chemistry Co. Ltd. (Shanghai, China). Methanol and acetonitrile (HPLC grade) were purchased from Baker (J.T. Baker). Penicillin, streptomycin, L-glutamine, and fetal bovine serum (FBS) were all purchased from Biochrom AG (Berlin, Germany). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Sigma (St Louis, MO). MiliQ water was used throughout the experiments. Other organic solvents and reagents were of analytical reagent grade. Instrumentation and Chromatographic Conditions. The high performance liquid chromatography (HPLC) system consisted of a Waters 1525 Binary HPLC pump, a Waters 2487 Dual λ absorbance detector, and a Waters 717 autosampler. Separation was carried out on a YMC America C18 column (4.6 mm × 150 mm; 5 μm) (Pennsylvania). The polyphenols were classified into seven groups: flavones, multihydroxyflavonols, flavonols, flavanones, isoflavones, catechins, and stilbenoids (Table 1). The mobile phase consisted of 0.1% phosphoric acid (A) and acetonitrile (B) and was degassed prior to use (Table 2). The flow rate was 1.0 mL/min. Stability of Polyphenols in Cell Culture Medium. Polyphenols were dissolved in methanol or DMSO to obtain a 10−2 mol/L standard stock solutions. DMEM was supplemented with 10% FBS, 50 U/mL penicillin, 100 mg/L streptomycin, and L-glutamine. The standard stock solutions of polyphenols were diluted to 1 × 10−3 mol/L with DMEM before testing. 50 μL of the diluted polyphenol solution was added to 4950 μL DMEM (prewarmed to 37 °C in a 5% CO2 atmosphere 30 min prior to experiments); then, the mixtures were incubated at 37 °C in 5% CO2. At 1, 10, 30, 60, 90, 120, and 180 min, a sample of 500 μL was withdrawn and immediately mixed with 1000 μL cold methanol (−20 °C) and kept at −20 °C for 30 min to precipitate the proteins. After centrifugation at 12 000 × g for 10 min at 4 °C, a 50 μL aliquot was injected to the HPLC system. Stability of Polyphenols in Human Plasma. Human plasma from three volunteers was obtained from a local blood bank (Bayerisches Rotes Kreuz, München, Germany). The standard stock solutions of flavonols (myricetin, myricitrin, quercetin, and kaempferol) were diluted to 1 × 10−3 mol/L with MiliQ water before testing. A 50 μL portion of the diluted flavonol solution was added to 4950 μL of the pooled human plasma (prewarmed to 37 °C for 30 min prior to experiments); then, the mixtures were incubated at 37 °C. After 1, 10, 30, 60, 90, 120, and 180 min, a sample of 500 μL was withdrawn and immediately mixed with 1000 μL cold methanol (−20 °C) and kept at −20 °C for 30 min to precipitate the proteins. After centrifugation at 12 000 × g for 10 min at 4 °C, a 50 μL aliquot was injected to the HPLC system. Determination of Protein Concentrations in DMEM and Human Plasma. The protein concentrations in DMEM and human plasma were determined using the BCA protein assay (Thermo Scientific).22 For calibration curves BSA (125 μg/mL to 2000 μg/mL) was used as standard. DMEM and human plasma were diluted with PBS before testing. Data Analysis. An early degradation time (CT10) and a half degradation time (CT50) have been defined to characterize the stability of a compound (initial concentration of C μM) in a specific biological or biochemical system. CT10 and CT50 represent the time that is required for 10% and 50%, respectively, degradation of an individual compound in vitro. The CT10 and CT50 of each compound were derived from time-response curves. The time−concentration curve was fitted using linear, polynomial, or exponential data regression. CT10 and CT50 values were defined as the time when the concentration of polyphenol decreased to 10% or 50% of the initial concentration.

Table 2. Composition of Gradient Mobile Phases for HPLC Separations time/min 0 3 10 15 30 35 40 45 50 0 5 10 20 30 40 50 55 60 0 5 10 20 30 40 45 0 3 10 20 30 35 45 55 60 0 10 15 20 25 27 30 0 5 10 22 25 33 42 45

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0.1% phosphoric acid Flavones (280 nm) 90% 90% 85% 85% 75% 70% 65% 52% 90% Multihydroxyflavonols (360 nm) 90% 90% 85% 77% 75% 60% 50% 50% 90% Less-hydroxyflavonols (280 nm) 85% 85% 75% 60% 55% 52% 85% Flavanones (280 nm) Isoflavones (280 nm) 95% 90% 85% 85% 80% 75% 70% 60% 95% Catechins (270 nm) 95% 95% 90% 80% 75% 70% 95% Stilbenoids (280 nm) 92% 90% 80% 75% 75% 50% 50% 92%

acetonitrile 10% 10% 15% 15% 25% 30% 35% 48% 10% 10% 10% 15% 23% 25% 40% 50% 50% 10% 15% 15% 25% 40% 45% 48% 15% 5% 10% 15% 15% 20% 25% 30% 40% 5% 5% 5% 10% 20% 25% 30% 5% 8% 10% 20% 25% 25% 50% 50% 8%



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Statistical Analysis. All experiments were repeated three times. All values were expressed as means ± standard deviations.

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RESULTS Stability of Polyphenols in Cell Culture Medium. After incubation with DMEM at 37 °C for 1, 10, 30, 60, 90, 120, and 180 min, the concentrations of polyphenols were determined to compile time−concentration response curves. The 10T10 and 10 T50 values of polyphenols (initial concentration 10 μM) in DMEM at 37 °C varied considerably (Table 1). Some polyphenols, such as myricetin, C, EGCG, GCG, and EGC, were significantly degraded within 10 min of incubation (10T10 < 1 min, 10T50 < 1 min). However, most polyphenols such as isoflavones flavanones and less-hydroxyflavonols were very stable even after 3 h incubation (10T10 > 180 min, 10T50 > 180 min). Flavonols. Hydroxylation. 1. Hydroxylation on Ring B. The hydroxylation at the ring B of flavonols significantly affected the stability (Figure 1). Myricetin (3′,4′,5′-OH) (10T10

Figure 2. Methoxylation on ring B of multihydroxyflavonols (initial concentration 10 μM) improved the stability in DMEM incubated at 37 °C in 5% CO2. These results are the mean of three independently performed experiments.

(10T10 < 1 min and 10T50 = 14.40 min versus 10T10 < 1 min and T50 = 14.40 min); the methoxylation at the C-4′ position of kaempferol (10T10 = 46.68 min and 10T50 = 119.47 min) significantly enhanced its stability (kaempferide, 10T10 = 150.02 min and 10T50 > 180 min). However, the methoxylation of less hydroxylated flavonols such as 3-hydroxyflavonol slightly compromised their stability (Figure 3). 3,4′-Dihydroxyflavone and 4′-hydroxyflavone showed a slightly higher stability than 3hydroxy-4′-methoxyflavone and 4′-methoxyflavone (Table 1). 10

Figure 1. Stability of multihydroxyflavonols (initial concentration 10 μM) in DMEM incubated at 37 °C in 5% CO2. These results are the mean of three independently performed experiments.

< 1 min and 10T50 < 1 min; Table 1) was highly instable, degraded to 26% within 1 min, and completely disappeared within 10 min. Quercetin (3′,4′-OH) (10T10 = 4.66 min and 10 T 50 = 17.96 min) decreased to 25% of the initial concentration within 30 min and almost disappeared after 120 min incubation. Kaempferol (4′-OH) (10T10 = 46.68 min and 10T50 = 119.47 min) was more stable than myricetin and quercetin. Moreover, kaempferol was found to degrade linearly while quercetin degraded exponentially. Galangin without any hydroxyl groups on ring B appeared to be most stable over a 3 h time course (10T10 > 180 min and 10T50 > 180 min). Likewise, 3,4′-dihydroxyflavone was more stable than 3-hydroxyflavone (Figure 3). 2. Hydroxylation on Ring A. Quercetin (3,5,7,3′,4′-OH) (10T10 = 4.66 min and 10T50 = 17.96 min) exhibited slightly higher stability as compared to fisetin (3,7,3′,4′-OH) (10T10 < 1 min and 10T50 = 14.40 min; Figure 1). Methoxylation. The methoxylation at ring B of multihydroxyflavonols apparently improved their stability (Figure 2). Isorhametin revealed a slightly higher stability than quercetin

Figure 3. Methoxylation of flavonols (initial concentration 10 μM) slightly weakened the stability in DMEM incubated at 37 °C in 5% CO2. These results are the mean of three independently performed experiments.

Glycosylation. In the present study, the glycosylation of flavonols significantly enhanced their stability. Compared with quercetin (10T10 = 4.66 min and 10T50 = 17.96 min; Figure 4), quercitrin and rutin were more stable during 3 h of incubation in DMEM medium with T10 > 180 min and T50 > 180 min. After 3 h of incubation, quercetin almost completely disappeared, but quercitrin and rutin barely degraded (Figure 4). Myricitrin (10T10 = 20.66 min and 10T50 = 63.95 min) also showed higher stability than myricetin (10T10 < 1 min and 10T50 1550



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T50 > 180 min, Figure 5), which suggested that the hydroxylation in ring A of flavones decreased the stability of flavones. The influence of various substitutions in ring A on the stability of flavones was determined as resorcinol-type (chrysin) > catechol-type (7,8-dihydroxyflavone) > pyrogallol-type (baicalein). 3. Hydroxylation in Ring C (C-3 Position). Galangin (5,7dihydroxyflavonol) and chrysin (5,7-dihydroxyflavone) without any hydroxyl groups at ring B were all very stable in DMEM (10T10 > 180 min and T50 > 180 min). Compared to flavonols with hydroxyl groups on ring B [kaempferol (10T10 = 46.68 min and 10T50 =119.47 min), and quercetin (10T10 = 4.66 min and 10 T50 =17.96 min)], flavones [apigenin (10T10 > 180 min and 10 T50 > 180 min), and luteolin (10T10 = 56.35 and 10T50 > 180)] were found to be more stable during 3 h incubation in DMEM (Table 1 and Figures 1 and 5). Obviously, a hydroxylation in the ring C (C-3 position) of flavones with hydroxyl groups on ring B reduced their stability. Glycosylation. The sugar moieties are in C-7 or C-8 position of ring A (Table 1). Luteolin 7-O-glucoside obviously exhibited higher stability than luteolin. Baicalin (10T10 < 1 min and 10T50 = 27.31 min) also showed a higher stability than baicalein (10T10 < 1 min and 10T50= 3.04 min). Even a glycoside attached at the C-7 positon of baicalein was still instable and entirely decomposed after 3 h incubation in DMEM. Isoflavones. All the isoflavones tested were very stable during 3 h incubation in DMEM (10T10 > 180 min and 10T50 > 180 min), which might be due to their few hydroxyl groups in the rings A and B. The concentrations of isoflavones after 3 h incubated in DMEM (initial concentration 10 μM) (Table 1) were determined as biochanin A (10.81 ± 0.22 μM) > genistein (10.30 ± 0.26 μM) > daidzein (10.28 ± 0.16 μM) > puerarin (10.04 ± 0.13 μM) > formononetin (9.92 ± 0.14 μM) > genistin (9.67 ± 0.10 μM) > formononetin 7-O-glucoside (9.11 ± 0.13 μM) > daidzin (9.01 ± 0.02 μM). Hydrogenation of the C2C3 Double Bond. Naringenin, naringin, narirutin, hesperetin, hesperidin, liquiritigenin, and liquiritin were stable during 3 h incubation in DMEM (10T10 > 180 min and 10T50 > 180 min; Table 1). In contrast, dihydromyricetin was degraded during the incubation period (10T10 < 1.0 min and 10T50 = 1.91 min). Compared with the flavonoid apigenin, the flavanoid naringenin displayed a slightly higher stability (10T10 > 180 min and 10T50 > 180 min). However, dihydromyricetin was very instable (10T10 < 1 min and 10T50 = 1.91 min) with more than 90% being decomposed within 10 min incubation. However, dihydromyricetin was more stable than myricetin (10T10 < 1 min and 10T50 < 1 min). Catechins. Catechins are the major polyphenols in green tea leaves. The major catechins of green tea extract are C, EC, EGC, EGCG, EGC, and GCG. The order of stability of catechins was determined as ECG> EC > EGCG> GCG > EGC ≈ C (Figure 6 and Table 1). EGC and C were extremely instable and completely decomposed within 1 min. EGCG and GCG revealed significant instability within 10 min. The initial concentrations (10 μM) of catechins decreased within 10 min: ECG (9.8 ± 1.1 μM) > EC (9.6 ± 0.1 μM) > EGCG (2.1 ± 0.3 μM) > GCG (0.76 ± 0.8 μM) > EGC = C (0) (Figure 7). Apparently, the pyrogallol-type catechins showed lower stability than the catechol-type catechins. The galloylation of catechins obviously improved their stability. However, specific to various substitutions on ring B, the degree of influence was quite different. For the pyrogallol-type catechins, a galloylation

Figure 4. Glycosylation of flavonols (initial concentration 10 μM) improved the stability in DMEM incubated at 37 °C in 5% CO2. These results are the mean of three independently performed experiments.

< 1 min). Even with the glycoside attached at the C-3 positon of myricitrin, it was still instable and entirely vanished after 3 h incubation. Apparently, the pyrogallol moiety in myricetin and myricitrin were the key structures influencing the stability. Flavones. Hydroxylation. 1. Hydroxylation in Ring B. The hydroxylation at the ring B of flavones obviously influenced the stability (Figure 5). Chrysin (no hydroxyl group in the B-ring)

Figure 5. Stability of flavones (initial concentration 10 μM) in DMEM incubated at 37 °C in 5% CO2. These results are the mean of three independently performed experiments.

and apigenin (4′-OH in ring B) were very stable during 3 h incubation in DMEM (10T10 > 180 min and 10T50 > 180 min). Compared with chrysin and apigenin, luteolin (3′,4′-OH on ring B) showed a slightly lower stability (T10 = 56.35 min and T50 > 180 min). The general pattern of flavone stability was determined as chrysin > apigenin > luteolin. Apparently, the hydroxylation on ring B of flavones would slightly increase the stability. 2. Hydroxylation in Ring A. Baicalein (5,6,7-OH) (10T10 < 1 min and 10T50 = 3.04 min) showed significantly lower stability than chrysin (5,7-OH) (10T10 > 180 min and T50 > 180 min) and 7,8-dihydroxyflavone (7,8-OH) (10T10 = 104.1 min and 1551



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Figure 6. Stability of catechins (initial concentration 10 μM) in DMEM incubated at 37 °C in 5% CO2. These results are the mean of three independently performed experiments.

Figure 8. Stability of stilbenoids (initial concentration 10 μM) in DMEM incubated at 37 °C in 5% CO2. These results are the mean of three independently performed experiments.

glucoside) (10T10 > 180 min and 10T50 > 180 min) was the most stable compound among stilbenoids tested: it was more stable than resveratrol (3,5,4′-OH). The concentrations of stilbenoids after 3 h incubated in DMEM (initial concentration 10 μM) (Figure 9) were determined as piceid (10.8 ± 0.6 μM) >

Figure 7. Concentration of catechins after 10 min incubated in DMEM. These results are the mean of three independently performed experiments.

significantly enhanced the stability. For catechol-type catechins, a galloylation only slightly increased the stability. Moreover, the catechins with 2,3-cis structure exhibited higher stability than the catechins with 2,3-trans structure. Stilbenoids. A hydroxylation, glycosylation, and methylation of resveratrol significantly influenced the stability of its derivatives isorhapontigenin, oxyresveratrol, pinostilbene, pterostilbene, and piceid (Table 1, Figure 8). Compared with resveratrol (3,5,4′-OH) (10T10 = 165.92 min and 10T50 > 180 min), piceatannol (3,5,4′,5′-OH) (10T10 = 5.68 min and 10T50= 22.51 min) and oxyresveratrol (3,5,4′,6′−OH) (10T10 = 11.57 min and 10T50= 59.12 min) with an additional hydroxyl group on ring B were instable during the 3 h incubation. The stability of resorcinol-type stilbenoids was higher than that of catecholtype stilbenoids. The 5′-methoxylation of piceatannol to isorhapontigenin (3,5,4′-OH,5′-OCH3) (10T10 = 81.14 min and 10T50 > 180 min) clearly improved the stability. The methoxylation on ring A of resveratrol to pinostilbene (3,4′OH,5-OCH3) (10T10 > 180 min and T50 > 180 min) and pterostilbene (4′-OH,3,5-OCH3) (10T10 > 180 min and 10T50 > 180 min) slightly enhanced the stability. Piceid (3, 4′-OH,5-O-

Figure 9. Concentration of stilbenoids after 3 h incubated in DMEM at 37 °C in 5% CO2. These results are the mean of three independently performed experiments.

pterostilbene (10.7 ± 0.9 μM) > pinostilbene (10.4 ± 0.04 μM) > resveratrol (8.7 ± 0.09 μM) > isorhapontigenin (7.9 ± 0.2 μM) > oxyresveratrol (1.8 ± 0.22 μM) > piceatannol (0). It was concluded that the glycosylation and methoxylation of OH moiety on stilbenoids would enhance the stability and the hydroxylation of stilbenoids decreases the stability. Correlation of the Stability with the Antioxidant Potential of the Compounds. The correlation between the stability and the antioxidant properties of polyphenols was 1552



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analyzed using published data of the antioxidant potential.23 There was a weak linear relationship between the stability and antioxidant potential (Figure 10, R2(10T10)=0.5399, R2(10T50)=

Figure 11. Stability of flavonols (initial concentration 10 μM) in human plasma incubated at 37 °C in 5% CO2. These results are the mean of three independently performed experiments.

min. The 10T50 values of myricitrin, quercetin, and kaempferol (initial concentration 10 μM) in DMEM at 37 °C were determined as 119.47, 17.96, and 63.95 min (Table 1). Protein Concentrations in DMEM and Human Plasma. The protein concentration in DMEM supplemented with 10% FBS was determined as 0.41 g/L; the total protein concentration in human plasma was 61.7 g/L.

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DISCUSSION Numerous researchers in biological and medicinal areas use cell cultures for their experiments, which provide information on cell proliferation and apoptosis, cell signaling, and regulation of gene expression and protein synthesis under controlled and reproducible conditions.24 However, when working with polyphenols it must be taken into consideration that these compounds might not be stable under the cell culture conditions and that artifacts might arise.24 The results of our study revealed that the factors influencing the stability of polyphenol in cell culture medium include the initial concentration of polyphenol, temperature, the presence and concentration of proteins, and the pH value. In contrast, the polyphenols’ stability in organic solvents depends mainly on their initial concentration and the temperature. Thus, the stability of polyphenols in cell culture medium differs from that in organic solvents. Several groups have investigated the stability of selected tea catchins under the cell culture conditions.15,25 However, the structure−stability relationship of dietary polyphenols has not been systematically and comprehensively investigated for the main groups of polyphenols and their major representatives. The present study revealed a systematic influence of the chemical stucture of polyphenols on their stability under the cell culture conditions (Figure 12). We defined and used for the first time an early degradation (CT10) and a half degradation time (CT50) to characterize and compare the stability of the individual compounds. It was concluded that polyphenols with divergent structure significantly differed regarding their stability in cell culture medium. The stability of flavonoids was mainly correlated with structural motifs of the molecules, which also frequently affect their reactivity and bioactivity in a variety of physiological processes.26 Important structural motifs were

Figure 10. Correlation between the stability of polyphenols in DMEM and their antioxidant properties. The data of the antioxidant potential of polyphenols are expressed as TEAC values (mM) based on the ABTS assay and DPPH assay.23

0.6336). For example, the TEAC values (ABTS) of flavonols were determined as quercetin > kaempferol > myricetin > galangin,23 which is different from the following order of stability: galangin > kaempferol > quercetin > myricetin. Rutin (TEACABTS = 2.02 mM; TEACDPPH = 2.33 mM) showed much higher antioxidant potential than baicalin (TEACABTS = 1.55 mM; TEACDPPH = 1.79 mM).23 However, rutin (10T10 > 180 min and 10T50 > 180 min) was much more stable than baicalin under the cell culture conditions. Stability of Flavonols in Human Plasma. The stablility of flavonols such as myricetin, myricitrin, quercetin, and kaempferol, which were particularly instable under the cell culture conditions, was further investigated. It was found that myricetin was still highly instable in human plasma; however, myricetin revealed increased stability in human plasma as compared to DMEM. After incubation with DMEM at 37 °C for 10 min, there was no more myricetin detectable (Figure 1), but when incubated with human plasma at 37 °C for 180 min, myricetin was still present (Figure 11). The stability of myricitrin, quercetin, and kaempferol (initial concentration 10 μM) in human plasma at 37 °C was determined as 10T50 > 180 1553



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Figure 12. Structural and molecular properties of polyphenols that influence the stability of polyphenols.

Figure 13. Potential sites of polyphenols affecting the stability are schematically illustrated. The up arrows represent increasing the stability; the down arrows represent decreasing the stability.

the stability of flavonoids has been discussed previously,32 there is limited information on the stability of flavonoid O-glycosides in DMEM. Herein, the influence of glycosylation of dietary flavonols and flavones on the stability in DMEM medium was investigated. C or O-Glycosylation of flavonoids obviously enhanced the stability. It was reported that C-glycosylated flavonoids are more stable in comparison with aglycones or Oglycosylated flavonoids.33 However, we found that apigenin Cand O-glycosides were very stable during 3 h incubation in DMEM (T10 > 180 min and T50 > 180 min), which were similar to the aglycone. It has been reported that the glycosylation of flavonoids decreases antioxidant activity,34 and this negative effect may result from the properties of sugar itself, rather than from the loss of a free hydroxyl group.35 Plumb et al. have also reported that antioxidant capacities of flavonol glycosides present in tea decrease as the number of glycosidic moieties increased.36 Moreover, the glycosylation is not the crucial factor deciding the stability of flavones and flavonols, which depends on the substitutions on the nucleus rings of flavonoids. In the present study it was found that the hydrogenation of the C2C3 double bond of flavonoids to corresponding flavanoids increased the stability. However, along with disparate substitutions on the nucleus rings, the degree of influence is not identical. Planarity of the C ring in flavonoids maybe important for the stability, as the molecules with saturated C2C3 bonds (flavanones and certain others) permit more twisting of ring B with reference to ring C. A C2C3 double bond increases the delocalization of π-electrons across those conjugated systems and the p-conjugation of the bond linking the rings B and C, which favors near-planarity of the two rings.37 Stilbenoids are phytoalexins become activated when plants are stressed and are important polyphenols with the C6−C2−C6 structure.38 The typical natural stilbenoids are resveratrol and its 3-glucoside, piceid.39 We found that the glycosylation and methoxylation of stilbenoids enhanced the stability, and the

determined as follows (1) for flavonoids: (a) the number and position of the hydroxyl groups on the rings A, B, and C; (b) the methoxyl or methyl groups in the rings A and B; (c) the class and position of the glycosides; (d) the degree of saturation of the ring C (the presence or absence of a C2C3 double bond). Motifs follow (2) for silbenoids: (a) the number and position of the hydroxyl and methoxyl moieties on the rings A and B; (b) the class and position of the glycosides. Motifs follow (3) for catechins: pyrogallol-type or catechol-type for ring B; (b) galloylation on the ring C; (c) 2,3-cis or 2,3-trans structure. The structure−stability relationships of polyphenols determined under the cell culture conditions were summarized (Figure 13). Hydroxylation on flavonoids usually weakened the stability. The hydroxyl substitutions on the nucleus rings of flavonoids influencing the stability were determined as resorcinol-type > catechol-type > pyrogallol-type. Maini et al. investigated the UVA-light and aqueous stability of galangin, kaempferol, quercetin, and myricetin and analyzed the stability of these flavonols in DMEM at room temperature (25 °C).27 They also observed a lower stability of these compounds under the cell culture conditions compared to in aqueous solution. As expected, the temperature is also an important factor affecting the stability. The lower stability of catechol-type and pyrogalloltype flavonols is likely due to more hydrogen-bond donating hydroxyl groups and the formation of an ortho-quinone on ring B, which favors the formation of a critical carbocation intermediate on ring C during the oxidative decomposition.28,29 The hydroxyl group at C-5 position can form a hydrogen bond with the carbonyl group at C-5 position, which may enhance the stability. However, hydroxylation on ring A of flavonols is not the key factor influencing the stability. Natural dietary flavonols are found mainly as 3- and 7-Oglycosides, and the plant flavones are mainly found as 4′- and 7O-glycosides.5,30,31 Even though the effect of glycosylation on 1554



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of polyphenols follow these requirements: (a) the o-dihydroxyl (3′,4′-diOH, i.e., catechol) structure in the B-ring, (b) the C2− C3 double bond (in conjugation with the 4-oxo group), (c) the presence of both 3-OH and 5-OH groups for the maximal radical scavenging capacity and the strongest radical absorption. (d) In the absence of o-dihydroxyl structure in the B ring, the hydroxyl substituents in a catechol structure on the A ring are able to compensate and become a larger determinant of flavonoid antiradical activity. These summarized structural features are partially similar to the partial structures that make the molecule more prone to instability under the cell culture conditions. It was obvious to speculate that the polyphenols’ degree of stability in cell culture medium merely reflected their antioxidant properties. However, in the present study it was found that there was no strong correlation between the antioxidant potential of polyphenols and their stability. Consequently, it can be assumed that oxidation processes are not the only factor governing the stability of polyphenols in biological systems. Other influences such as polyphenol− protein interactions or complex formation might play an important role, too. It has been reported that binding to human plasma proteins improved the polyphenols’ stability and protected against oxidation.43 Our results are consistent with that observation. We found that polyphenols revealed a higher stability in human plasma as compared to DMEM. The protein concentration determined in human plasma was 150-fold higher than in DMEM which suggested that the adsorption of polyphenols to plasma proteins contributed to their enhanced stability in this matrix. The degree of plasma protein binding has been shown to be highly variable among individual polyphenolic compounds,44 and it needs to be investigated how strong the extent of protein binding correlates with the compounds’ stability in plasma or cell culture medium. At this point, it is not clear whether similar degraded or oxidized polyphenol products can be found in human plasma and cell culture medium. The compounds may exhibit different degradation or oxidation patterns, which might also help explaining the different results from cell experiments in vitro and animal/human experiments in vivo.53 It also has to be considered that the degraded or metabolized polyphenol might exhibit a bioactivity which might be different from the parent compound.21 In 2010, D’Archivio et al. criticized that a large number of in vitro studies tested polyphenols rather than their active metabolites.54 So, we might be looking at the wrong compounds when investigating the mechanisms contributing to the health benefits of polyphenols. Recently, it was shown that metabolites of resveratrol (resveratrol 3-O-sulfate, resveratrol 3O-glucuronide, and resveratrol 4′-O-glucuronide) inhibited human colon carcinoma and metastatic cell proliferation through an accumulation of cells in DNA replication phase and by inducing DNA damage.55 Further investigations should clarify the precise mechanisms of degradation of polyphenols in cell culture medium and plasma and take the bioactivity of the resulting derivatives into consideration. In the case that an individual polyphenol displays pronounced instability in cell culture medium while exerting significant cellular effects, it should be considered that not the mother compound but a degradation product or metabolite is the actual bioactive principle.56,57 Further isolation and identification of the final degraded products is necessary to explore the mechanism, as well as to investigate the bioactivity of the derivatives and metabolites of polyphenols. To summarize, it is essential to

hydroxylation of stilbenoids decreased the stability. Long et al. found that pyrogallol-type compounds decompose in cell culture media to generate significant amounts of H2O2.40 3,4,5,4′-Tetrahydroxystilbene and 3,3′,4,4′,5,5′-trihydroxystilbene, but not resveratrol, generate measurable amounts of H2O2 when incubated in DMEM at a concentration of 50 μM.41 Some polyphenols displayed instability in cell culture medium. It has been reported that reactive oxygen species are formed under most cell culture conditions.14 For example, EGCG has been shown to be instable and subject to oxidation and polymerization, resulting in the formation of reactive oxygen species.14,15 It appears that also different cell culture media might have a different influence on the stability of polyphenols. In the present study, it was found that EGCG (initial concentration 10 μM) was very instable in DMEM without cells (10T10 < 1 min and 10T50 < 1 min). It was reported that in McCoy’s 5A culture media, the 25T50 of EGCG (initial concentration 10 μM) was less than 30 min; surprisingly, the stability was prolonged to 130 min in the presence of HT-29 cells.15 In contrast, it was reported that quercetin (initial concentration 25 μM) was very instable when incubated with DMEM in the presence of HT29 cells (25T50 = 30 min).42 In the present study, the 10T50 of quercetin (initial concentration 10 μM) was 17.96 min with DMEM in the absence of cells, which is not consistent with the notion that cells enhance the stability of quercetin. It needs further investigations to clarify the stability of polyphenols in the presence of cells. Generally, it seems possible that various cellular enzymes compromise the compounds’ stability via degradation or metabolism. On the other side, adsorption of polyphenols to cellular proteins might enhance the stability of the compounds. Such a stabilizing effect has been described for polpyphenols attached to plasma proteins.43,44 Several factors, such as pH, proteins, antioxidant levels, and the presence of metal ions, might influence the stability of polyphenols, and pH is possibly the most critical determinant.15,45 In DMEM, quercetin was shown to be very stable at pH of 6 and decomposed under pH of 7 and 8.45 Other constituents in DMEM obviously also influenced its stability because quercetin was reported to be more stable in H2O than in DMEM. However, most supplements for cell culture media, such as HEPES, glucose, sodium pyruvate, antibiotics, and FBS, had a neglible influence on the stability of quercetin.45 Yet, it is not clear for how these factors influence other polyphenols. Kosińska et al. investigated the stability of polyphenol extracts from cocoa, green tea, and strawberries in HBSS buffer.46 They found that most of the polyphenols decomposed during 2 h of incubation. EGCG and EGC were very vulnerable in HBSS both at pH 6.5 and 7.4; their specific molecular structure was discussed to be responsible for this phenomenon.46 The flavan-3-ols from cocoa were more stable at pH 6.5 than at pH 7.4. The major oxidative products of EGCG in McCoy’s 5A culture media were theasinensin and its dimer.15 However, the mechanism should be further elucidated. Autooxidation was observed for EGCG in air-saturated Tris buffer, which may generate •O2− and quinones to form H2O2.47 It was reported that the stability of EGCG was significantly enhanced in the presence of SOD in the cell culture medium, which prolonged the T50 of EGCG to 24 h. The presence of 5% serum in the medium also prolonged the T50 of EGCG to 1 h.47 On the basis of many previous and recent findings,48−52 it appears that favorable structures required for radical scavenging 1555



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(7) Manach, C.; Scalbert, A.; Morand, C.; Rémésy, C.; Jiménez, L. Polyphenols: Food sources and bioavailability. Am. J. Clin. Nutr. 2004, 79, 727−747. (8) Clifford, M. N. Diet-derived phenols in plasma and tissues and their implications for health. Planta Med. 2004, 70, 1103−1114. (9) Xiao, J. B.; Hö gger, P. Influence of diabetes on the pharmacokinetic behavior of natural polyphenols. Curr. Drug Metab. 2014, 15, 23−29. (10) Xiao, J. B.; Kai, G. Y. A review of dietary polyphenol-plasma protein interactions: Characterization, influence on the bioactivity, and structure-affinity relationship. Crit. Rev. Food Sci. Nutr. 2012, 52, 85− 101. (11) Xiao, J. B.; Högger, P. Advance in pharmacokinetics of bioactive polyphenols. Curr. Drug Metab. 2014, 15, 1−2. (12) Kanakis, C. D.; Hasni, I.; Bourassa, P.; Tarantilis, P. A.; Polissiou, M. G.; Tajmir-Riahi, H. A. Milkbeta-lactoglobulin complexes with tea polyphenols. Food Chem. 2011, 127, 1046−1055. (13) Dubeau, S.; Samson, G.; Tajmir-Riahi, H. A. Dual effect of milk on the antioxidant capacity of green, Darjeeling, and English breakfast teas. Food Chem. 2010, 122, 539−545. (14) Sang, S.; Lambert, J. D.; Yang, C. S. Bioavailability and stability issues in understanding the cancer preventive effects of tea polyphenols. J. Sci. Food Agric. 2006, 86, 2256−2265. (15) Hong, J.; Lu, H.; Meng, X.; Ryu, J. H.; Hara, Y.; Yang, C. S. Stability, cellular uptake, biotransformation, and efflux of tea polyphenol (−)-epigallocatechin-3-gallate in HT-29 human colon adenocarcinoma cells. Cancer Res. 2002, 62, 7241−7246. (16) Yuan, L.; Wei, S. P.; Wang, J.; Liu, X. B. Isoorientin induces apoptosis and autophagy simultaneously by reactive oxygen species (ROS)-related p53, PI3K/Akt, JNK, and p38 signaling pathways in HepG2 cancer cells. J. Agric. Food Chem. 2014, 62, 5390−5400. (17) Yuan, L.; Wang, J.; Xiao, H. F.; Wu, W. Q.; Wang, Y. T.; Liu, X. B. MAPK signaling pathways regulate mitochondrial-mediated apoptosis induced by isoorientin in human hepatoblastoma cancer cells. Food Chem. Toxicol. 2013, 53, 62−68. (18) Lee, W. C.; Peng, C. C.; Chang, C. H.; Huang, S. H.; Chyau, C. C. Extraction of antioxidant components from Bidens pilosa flowers and their uptake by human intestinal Caco-2 cells. Molecules 2013, 18, 1582−1601. (19) Boyer, J.; Brown, D.; Liu, R. H. Uptake of quercetin and 1uercetin 3-glucoside from whole onion and apple peel extracts by Caco-2 cell monolayers. J. Agric. Food Chem. 2004, 52, 7172−7179. (20) Uhlenhut, K.; Högger, P. Facilitated cellular uptake and suppression of inducible nitric oxide synthase (iNOS) by a metabolite of maritime pine bark extract (Pycnogenol). Free Radic. Biol. Med. 2012, 53, 305−313. (21) Kurlbaum, M.; Mülek, M.; Högger, P. Facilitated uptake of a bioactive metabolite of maritime pine bark extract (Pycnogenol) into human erythrocytes. PLoS One 2013, 8, e63197. (22) Walker, J. M. The Bicinchoninic Acid (BCA) Assay for Protein Quantitation. The Protein Protocols Handbook; Humana Press: Totowa, NJ, 1996. (23) Cai, Y. Z.; Sun, M.; Xing, J.; Luo, Q.; Corke, H. Structure− radical scavenging activity relationships of phenolic compounds from traditional Chinese medicinal plants. Life Sci. 2006, 78, 2872−2888. (24) Halliwell, B. Cell culture, oxidative stress, and antioxidants, avoiding pitfalls. Biomed. J. 2014, 37, 99−105. (25) Sang, S.; Lee, M. J.; Hou, Z.; Ho, C. T.; Yang, C. S. Stability of tea polyphenol (−)-epigallocatechin-3-gallate and formation of dimers and epimers under common experimental conditions. J. Agric. Food Chem. 2005, 53, 9478−9484. (26) Kumar, S.; Pandey, A. K. Chemistry and biological activities of flavonoids, an overview. Sci. World J. 2013, 162750. (27) Maini, S.; Hodgson, H. L.; Krol, E. S. The UVA and aqueous stability of flavonoids is dependent on B-ring substitution. J. Agric. Food Chem. 2012, 60, 6966−6976. (28) Krishnamachari, V.; Levine, L. H.; Zhou, C.; Pare, P. W. In vitro flavon-3-ol oxidation mediated by a B ring hydroxylation pattern. Chem. Res. Toxicol. 2004, 17, 795−804.

account for polyphenol stability in cell culture experiments to avoid erroneous conclusions. In conclusion, the influence of cell culture conditions on the stability and fate of polyphenols is rarely considered, which might result in misleading conclusions about the compound’s bioactivity or the active principle. In the current investigation, the structure−stability relationship of polyphenols incubated with the cell culture medium DMEM was comprehensively investigated. Some polyphenols, especially aglycones with catechol or pyrogallol structures and pyrogallol-type glycosides, were evidently instable. The additional hydroxyl moieties attached to ring B of flavonols obviously weakened the compound’s stability under the cell culture conditions. The glycosylation of a hydroxyl group of the pyrogallol moiety in flavones slightly enhanced the stability. It appears that the glycosylation of isoflavones decreased and the methoxylation of isoflavones increased their stability. The C2C3 double bond in conjugation with a 4-oxo group is also considered as an important role for the stability. The hydrogenation of the C2 C3 double bond of flavanoids to corresponding flavonoids increased the stability. We suggest that the stability of polyphenols in a biological system is not solely governed by the oxidation processes, because the degradation tendency of these polyphenols was not highly correlated with their antioxidant potential. Polyphenols were considerably more stable in human plasma as compared to DMEM, which may be based on polyphenol−protein interactions enhancing the compounds’ stability.10 Our results might be used to further investigate the mechanism of degradation or metabolism of polyphenols in cell culture and human plasma, which might stimulate the search for the actual bioactive compounds derived from polyphenols.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 (0) 17681574937. E-mail: jianboxiao@yahoo. com. Funding

The authors are grateful for financial support by the Alexander von Humboldt Foundation (Germany). Notes

The authors declare that they have no conflict of interests. The authors declare no competing financial interest.

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REFERENCES

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Stability of Dietary Polyphenols under the Cell Culture Conditions

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