Flavonoid metabolites transport across a human BBB model

Food Chemistry 149 (2014) 190–196

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Flavonoid metabolites transport across a human BBB model Ana Faria a,b,c,⇑, Manuela Meireles a, Iva Fernandes b, Celestino Santos-Buelga d, Susana Gonzalez-Manzano d, Montserrat Dueñas d, Victor de Freitas b, Nuno Mateus b, Conceição Calhau a,e a

Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal Chemistry Investigation Centre (CIQ), Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal Faculty of Nutrition and Food Sciences, University of Porto, 4200-465 Porto, Portugal d Grupo de Investigación Polifenoles (GIP-USAL), Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno s/n, E-37007 Salamanca, Spain e CINTESIS Center for Research in Health Technologies and Information Systems, Faculty of Medicine, Porto University, Rua Dr. Plácido da Costa, 4200-450 Porto, Portugal b c

a r t i c l e

i n f o

Article history: Received 11 June 2013 Received in revised form 5 September 2013 Accepted 22 October 2013 Available online 30 October 2013 Keywords: Anthocyanins Blood-brain barrier Flavanoids Flavonoids metabolites hCMEC/D3 cells Transport

a b s t r a c t This study aimed to evaluate the transmembrane transport of different flavonoids (flavan-3-ols, anthocyanins and flavonols) and some of their metabolites (methylated and conjugated with glucuronic acid) across hCMEC/D3 cells (a blood–brain barrier (BBB) model). Further metabolism of the tested compounds was assayed and their transport modulated in an attempt to elucidate the mechanisms behind this process. The transport across hCMEC/D3 cells was monitored in basolateral media at 1, 3 and 18 h by HPLC-DAD/MS. All the flavonoids and their metabolites were transported across hCMEC/D3 cells in a time-dependent manner. In general, the metabolites showed higher transport efficiency than the native flavonoid. No further biotransformation of the metabolites was found as consequence of cellular metabolism. Anthocyanins and their metabolites crossed this BBB cell model in a lipophilicity-dependent way. Quercetin transport was influenced by phosphatase modulators, suggesting a phosphorylation/dephosphorylation regulation mechanism. Overall, this work suggests that flavonoids are capable of crossing the BBB and reaching the central nervous system. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Numerous epidemiological studies have indicated that individuals with a high consumption of fruits and vegetables have a reduced incidence of age-associated illness and neurodegenerative diseases (Singh, Arseneault, Sanderson, Murthy, & Ramassamy, 2008; Spencer, 2009). Different classes of flavonoids have shown positive effects on Alzheimers disease (Marambaud, Zhao, & Davies, 2005) and in some parameters associated with Parkinsons disease (Guo et al., 2007). Flavonoids and flavonoid-rich food consumption have been associated with the amelioration of several neuronal pathological conditions, as well as memory improvement and ageing (Andrade & Assuncao, 2012; Andres-Lacueva et al., 2005).

Abbreviations: ALP, alkaline phosphatase; BBB, blood–brain barrier; CNS, central nervous system; RBE4, rat brain endothelial cell; hCMEC/D3, human brain endothelial cell. ⇑ Corresponding author at: Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, Al. Prof. Hernâni Monteiro, 4200-319 Porto, Portugal. Tel./fax: +351 22 551 36 24. E-mail address: [email protected] (A. Faria). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.10.095

The blood–brain barrier (BBB), besides fulfilling a protective role by severely limiting the movement of substances into the brain (Palmer, 2010), also has the crucial role of supplying essential nutrients, hormones and drugs, and eliminating metabolites from the brain. One of the main issues on flavonoid bioavailability that remains unclear concerns the mechanisms by which flavonoids (conjugated or unconjugated) are able to reach the central nervous system (CNS) and the molecules which are responsible for their biological effects. The appearance of flavonoids in plasma, and hence in the blood, is the main evidence for the assumption that these compounds can reach the brain. In fact, flavonoids have been detected in the plasma of human volunteers after ingestion of beverages or foods rich in these compounds (Mullen, Borges, Lean, Roberts, & Crozier, 2010). Among the different flavonoid classifications, flavan-3-ols are one of the most studied due to their abundance in the human diet. Different studies have detected several flavan-3-ols and their metabolites in the plasma of human healthy volunteers after consumption of green tea (Del Rio et al., 2010). The main metabolites found in the plasma were methylated, sulphated and glucuronidated conjugates (Del Rio et al., 2010; Stalmach et al.,

A. Faria et al. / Food Chemistry 149 (2014) 190–196

2010). These metabolites, as well as some native forms, were also found in the plasma of human volunteers after ingestion of chocolate and cocoa (Baba et al., 2000). Anthocyanins are another class of flavonoids present in the human diet (e.g., red wine, and red fruits) that naturally occur as glycosides of the flavylium cation. Supplementation of the human diet with rich sources of anthocyanins resulted in their appearance in the plasma in their intact forms (Milbury, Vita, & Blumberg, 2010). Studies with quercetin (a major flavonol in the human diet) also showed that this compound, in its native form, as well as its metabolites, are available in human plasma after food intake (Mullen, Edwards, & Crozier, 2006). Some of the biologic effects of these compounds may be due not only to the unconjugated compounds but also to their metabolites (Del Rio et al., 2010; Mullen et al., 2010). Nevertheless, whether flavonoids and/or their metabolites can pass through the BBB and reach the central nervous system is another issue, as the information available is still scarce. The aim of this study was to evaluate the transmembrane transport of flavonoids and some of their metabolites (methylated and conjugated with glucuronic acid) across a human BBB cell line, hCMEC/D3 cells, to identify any further metabolisation and to modulate the transport in an attempt to elucidate mechanisms behind this process. hCMEC/D3 cells are an immortalised cell line of human capillary cerebral endothelial cells, herein used as a BBB model. These cells, often applied in research to model the BBB, remain phenotypically nontransformed, maintain many characteristics of human brain endothelial cells (Mkrtchyan et al., 2009) and retain protein transporters, receptors and junctions expressed at the human BBB (Ohtsuki et al., 2013). Thus, hCMEC/D3 appears to constitute an interesting model to study the transmembrane flavonoid transport across the BBB, without interference from other tissue elements. The actual challenge regards the knowledge about dietary (or other) factors that may interfere with this transport and modulate their access to the CNS, which could be the first step for future dietary recommendations. 2. Materials and methods 2.1. Reagents (+)-catechin, ()-epicatechin, quercetin dehydrate, Minimum Essential Medium, Ham’s F10, neomycine, penicillin G, amphotericin B, streptomycin, HEPES, trypsin–EDTA and collagen type I from Rat tail, (Sigma–AldrichÒ, Madrid, Spain); foetal bovine serum (FBS), basic fibroblast growth factor and Hanks’ Balance Salt Solution (HBSS) (Gibco, Barcelona, Spain), Endothelial Basal Medium-2 (EBM-2), VEGF, IGF-1, EGF, basic FGF, hydrocortisone, ascorbate and gentamycin were from Clonetics (Cambrex BioScience, Wokingham, UK). Delphinidin-3-O-glucoside, cyanidin-3-O-glucoside and malvidin-3-O-glucoside were purchased from Extrasynthese SA (Genay, France). Catechin, epicatechin, quercetin and anthocyanin metabolites were synthesised and purified in the laboratory according to the literature (Fernandes, Marques, de Freitas, & Mateus, 2013; Fernandes et al., 2009; Gonzalez-Manzano, Gonzalez-Paramas, Santos-Buelga, & Duenas, 2009). 2.2. Cell and culture conditions The hCMEC/D3 cell line was kindly supplied by Dr. Pierre-Olivier Couraud (INSERM U. 567, Université René Descartes, Paris, France). Cells were maintained in a humidified atmosphere of 5% CO2-95% air at 37 °C, between passages 26 and 30. Cells were grown in EBM-2 medium supplemented with VEGF, IGF-1, EGF, basic FGF, hydrocortisone, ascorbate, gentamycin and 2.5% foetal bo-

191

vine serum (FBS), 100 U/ml penicillin G, 0.25 mg/ml amphotericin B and 100 mg/ml streptomycin, as recommended by the manufacturer (Lonza Walkersville, Inc). The cell medium was changed every 48 h, and the cells reached confluence after 5–6 days incubation. For subculturing, the cells were dissociated with 0.25% trypsin–EDTA, diluted 1:5 and subcultured in petri dishes collagen-coated with a 21 cm2 growth area (Corning CostarÒ, Badhoevedorp, The Netherlands). For the experiments, cells were seeded on transwell inserts (collagen-coated polytetrafluoroethylene membrane, 0.4 lm pore size, 12 mm diameter, Corning CostarÒ). Inserts were placed in 12 well plates. All experiments were performed 9–10 days after initial seeding. 2.3. Transport studies Transepithelial electrical resistance (TEER) of cells grown in the transwell was measured using an epithelial voltohmmeter, fitted with planar electrodes (EVOM; World Precision Instruments, Stevenage, UK). Experiments were conducted only in cell monolayers that showed a TEER > 100 O.cm2. The medium was removed and cells were washed with HBSS’ medium with 1.0 mM MgCl2 and 0.25 mM CaCl2, pH 7.4. The flavonoid solution in HBSS with 0.1% of FBS was added to the apical side of the cells and the same medium free polyphenols were added to the basolateral compartment. The transepithelial transport was followed, as a function of time, at 37 °C. Samples were taken from the basolateral side and replaced by fresh medium. The samples were frozen at 20 °C until HPLC analysis. 2.4. HPLC analysis Catechin, epicatechin and their metabolites were analysed by HPLC (Elite Lachrom system (L-2130)) on a 150  4.6 mm i.d. reversed-phase C18 column (Merck, Darmstadt); detection was carried out using a diode array detector (L-2455). The solvents were A: H2O/HCOOH (9.9:0.1), and B: CH3CN. The program initiated with 93% A and 7% B for 4 min and a gradient of 7–25% B over 46 min, at a flow rate of 0.5 ml/min. The column was washed with 100% B for 10 min and then stabilised at the initial conditions for another 10 min. For quercetin and its metabolites, the same apparatus and solvents were used. The program initiated with 95% A and 5% B for 15 min and a gradient of 5–70% B over 20 min, at a flow rate of 0.5 ml/min. The column was washed with 100% B for 10 min and then stabilised at the initial conditions for another 10 min. HPLC analysis of the anthocyanins and their methylated products was performed on the same apparatus equipped with a 250  4.6 mm i.d. reversed phase C18 column (Merck, Darmstadt); detection was carried out at 520 nm (for 40 -Me-Dp3gluc the detection was carried out at 503 nm) using a diode array detector (L-2455). The solvents were A: H2O/HCOOH (9:1), and B: H2O/CH3CN/HCOOH (6:3:1). The gradient consisted of 26–45% B for 50 min, 45–85% B for 25 min and 85–0% B for 10 min, at a flow rate of 1.0 ml/min. The column was washed with 100% B for 20 min and then stabilised at the initial conditions for another 20 min. Detected peaks were scanned between 200 and 700 nm. For LC–MS analyses, a liquid chromatograph (Hewlett–Packard 1100 series) equipped with a Thermo Finnigan (Hypersil GoldÒ) reversed-phase column (150 mm  4.6 mm, 5 lm, C18) thermostated at 25 °C was used. The samples were analysed using the same solvents, gradients, injection volume and flow rate referred above for HPLC analysis. The mass detector was a Finnigan LCQ DECA XP MAX (Finnigan Corp., San Jose, CA) quadrupole ion trap equipped with an atmospheric pressure ionisation (API) source, using an electrospray ionisation (ESI) interface. The vaporiser and

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the capillary voltages were 5 kV and 4 V, respectively. The capillary temperature was set to 325 °C. Nitrogen was used as both the sheath and auxiliary gas at flow rates of 80 and 30, respectively (in arbitrary units). Spectra were recorded in positive ion mode between m/z 120 and 1500. The mass spectrometer was programmed to do a series of three scans: a full mass, a zoom scan of the most intense ion in the first scan, and a MS–MS of the most intense ion using relative collision energy of 30 and 60. 2.5. Statistical analysis Values are expressed as the arithmetic mean ± SEM. Statistical significance of the difference between various groups was evaluated by two-way analysis of variance (ANOVA) followed by the Bonferroni test. Differences were considered significant when p < 0.05. 3. Results and discussion 3.1. General The hCMEC/D3 cell line is an immortalised human brain endothelial cell line, which retains most of the morphological and functional characteristics of brain endothelial cells, even without coculture with glial cells, constituting a reliable in vitro model of the human BBB (Ohtsuki et al., 2013; Weksler et al., 2005). Recent work analysed and validated this cell line as a model of the human BBB, useful for examining mechanisms of drug transport across human BBB (Ohtsuki et al., 2013). hCMEC/D3 cells were cultured on semi-permeable supports and the transepithelial electrical resistance (TEER) of cells was measured at the beginning of experiment and only inserts with a TEER > 100 O.cm2 were used. After 18 h the TEER was measured again and only inserts which retained a TEER > 100 O.cm2 were considered. With this model, the transcellular transport of some of the flavonoid’s most relevant metabolites were studied. Data regarding flavonoid metabolites transport are scarce and diffuse. It is well known that flavonoids undergo biotransformation either by methylation, conjugation with glucuronic acid, sulphates or glutathione (Crozier, Jaganath, & Clifford, 2009; Del Rio et al., 2010; Stalmach et al., 2010). Hence, these forms gained biological relevance in the health effects attributed to flavonoids. The transport of some metabolites from different flavonoid classes (flavan3-ols, anthocyanins and flavonols) (please see Supplementary material) was studied in this BBB model and compared to the transport of their unconjugated forms. 3.2. Flavan-3-ols The transport of epicatechin and catechin has already been studied in this cell model in previous work (Faria et al., 2011). The transport of two methylated metabolites of epicatechin (40 -O-methylepicatechin and 30 -O-methylepicatechin) was studied and compared with epicatechin. Both metabolites crossed the hCMEC/D3 cells in a time-dependent manner with a tendency to do so more efficiently than epicatechin. Indeed, after 3 h incubation, 40 -O-methylepicatechin transport was significantly higher than epicatechin, and after 18 h incubation transport of both methylated metabolites was significantly higher than the parent compound (Fig. 1a). The same experiments were performed at 4 °C at 1 and 3 h (18 h was not included because cell viability could be compromised) in order to inhibit transporter-mediated processes. The transport of the flavanol metabolites under these conditions could indicate a passive diffusion process for these molecules as a mechanism to

cross this barrier. This seems plausible since the methylated molecules are more lipophilic than the parent molecule. However, when the transport experiment was performed at 4 °C, where passive diffusion can still occur, no increased transport of the methylated metabolites through the cells in relation to epicatechin was observed at 1 h (Fig. 1b), but some transport was observed at 3 h, although significantly lower than at 37 °C. This suggested that passive diffusion was not the only pathway by which epicatechin metabolites cross the BBB, and a membrane transporter could be involved. After 18 h of incubation with epicatechin metabolites, the basolateral media was analysed by HPLC–MS. From this analysis, it was possible to identify and confirm the peak of epicatechin ([M+H]+ at m/z 291) and the peak of the metabolite ([M+H]+ at m/z 305). No other identifiable peaks were detected (data not shown). In previous work, it was possible to detect epicatechin conjugated with glucuronic acid in this model (Faria et al., 2011). Another flavanol metabolite, 40 -methylcatechin, was tested and the results obtained presented the same trend: the methylated metabolite was transported through the cells in a time-dependent manner and this transport was significantly higher than that observed for catechin after 1, 3 and 18 h of incubation at 37 °C (Fig. 1c). Experiments were also performed at 4 °C and no transport of the compounds was observed after 1 h incubation. However, after 3 h incubation, both catechin and 40 -O-methylcatechin transport was significantly reduced compared with that at 37 °C, although for the metabolite, the transport efficiency was still considerable (Fig. 1d). This showed that methylation could be a key factor in increasing the bioavailability of the compounds, probably due to an increase in lipophilicity. Nevertheless, the results obtained in the experiments at 4 °C suggested that these compounds could use two different routes to cross the cell: one involving a transporter and another by passive diffusion. Also, similarly to what was observed for epicatechin and its metabolites, no further conjugation of catechin metabolites could be observed. This could be of interest since molecules are usually conjugated with glucuronic acid to facilitate excretion; therefore, the inhibition of this reaction may prevent the exit of the molecules, leaving them to exert their biological effects. 3.3. Anthocyanins Anthocyanins are common flavonoids present in food with the particularity of occurring with glycoside moieties. They are one of the few flavonoids that have been reported to be absorbed in their glycoside form (Crozier et al., 2009; Milbury et al., 2010), resulting in different biokinetics than the other flavonoids. Because they appear in the blood stream in a glycosidic form it is possible that they reach the BBB in their native form. From this perspective, three anthocyanins with different polarities were assayed: delphinidin-3-O-glucoside (Dp-3-gl), cyanidin-3-O-glucoside (Cy-3-gl) and malvidin-3-O-glucoside (Mv-3-gl). All the tested anthocyanins were able to cross the hCMEC/D3 cells in a timedependent manner but with different efficiencies. Dp-3-gl showed the lowest transport efficiency, followed by Cy-3-gl and Mv-3-gl (Table 1). After 18 h of incubation, Cy-3-gl (16.0 ± 0.6%) and Mv-3-gl transport (20.0 ± 3.3%) was significantly higher than that of Dp-3-gl (11.6 ± 0.6%). The differences in the ability to pass through the cells seem to be related with their hydrophobicity: Dp-3-gl < Cy-3-gl < Mv-3-gl, suggesting the influence of polarity of anthocyanins in this transport, although the structural features of the anthocyanins might also play a role. A 40 -methylated metabolite of Dp-3-gl and a mixture of 0 3 /40 -O-methylcyanidin-3-O-glucoside were also tested. Both metabolites were able to cross BBB monolayer in a time dependent manner. Similarly to what was observed with the flavan-3-ols, the

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(a)

*

*

Transport efficiency at 3h (%)

Transport efficiency (%)

40

*

30 20 10 0

40

(b)

Epicatechin 3'-MeEpi 4'-MeEpi

*

30

*

20

* 10

*

37ºC

18

3

1

0 4ºC

Transport efficiency (%)

40

(c) *

*

30

*

20 10 0

Transport efficiency at 3h (%)

Time (h)

40

*

30

*

20 10

*

18

3

0

1

Catechin 4'-MeCat

(d)

37ºC

4ºC

Time (h) Fig. 1. Transport efficiency of 30 lM epicatechin, 30 -O-methylepicatechin, 40 -O-methylepicatechin (a) and of 30 lM catechin and 40 -O-methylcatechin (c) in hCMEC/D3 cells (Apical ? Basolateral). Transport efficiency after 3 h of incubation of 30 lM epicatechin, 30 -O-methylepicatechin and 40 -O-methylepicatechin (b) and of 30 lM catechin and 40 -O-methylepicatechin (d) in hCMEC/D3 cells at 37 °C and 4 °C. Transport efficiency percentages were calculated based on (compound concentrations at the basolateral side overtime)/(compound concentrations at the apical side at the zero hours)*100. ⁄Significantly different from epicatechin or catechin (p < 0.05).

Table 1 Transport efficiency of 100 lM delphinidin-3-O-glucoside (Dp-3-gl), 40 -O-methyldelphinidin-3-O-glucoside (40 -Me-Dp-3-gl), cyanidin-3-O-glucoside (Cy-3-gl), 40 -O-methyl/30 -O-methylcyanidin-3-O-glucoside (40 /30 Me-Cy-3-gl) and malvidin-3O-glucoside (Mv-3-gl) in hCMEC/D3 cells (Apical ? Basolateral). Results are presented as transport efficiency (%) (mean ± SEM). Transport efficiency percentages were calculated based on (compound concentrations at the basolateral side overtime)/(compound concentrations at the apical side at the zero hours)*100. Compound

Dp-3-gl 40 Me-Dp-3-gl Cy-3-gl 40 /30 Me-Cy-3-gl Mv-3-gl *

Transport efficiency (%) 1h

3h

18 h

5.0 ± 0.7 5.5 ± 1.3 8.0 ± 1.1 9.2 ± 2.0 5.3 ± 0.1

8.8 ± 1.1 11.5 ± 1.0 12.6 ± 0.9 13.4 ± 1.2 13.3 ± 2.4

11.6 ± 0.6 17.6 ± 1.7* 16.0 ± 0.6* 19.0 ± 1.4# 20.0 ± 3.3*

Significantly different from delphinidin-3-O-glucoside (p < 0.05). Significantly different from cyanidin-3-O-glucoside (p < 0.05).

#

methylated forms were able to cross BBB barrier more efficiently than the unconjugated forms, with significantly differences after 18 h of incubation (Table 1). In the LC–MS analysis of the basolateral media after 18 h of incubation, the tested compounds were detected but no additional metabolites were observed. Again, the hypothesis that anthocyanins transport could be influenced by their lipophilicity, suggesting the involvement of passive diffusion, is strengthened by these results. The insertion of a methyl group in the anthocyanin structure affected its transport in a positive way. 3.4. Flavonols Quercetin, a major flavonol present in the human diet, has been extensively studied for its biological properties. Similarly to other flavonoids, quercetin is subjected to metabolisation in the organism, with conjugation with glucuronic acid being one of the most

common pathways. Therefore, 3-O-glucuronyl-quercetin (3-GlucQ) transport was tested in the hCMEC/D3 cell line. This metabolite was found to be transported across the hCMEC/D3 cells in a time-dependent manner (Fig. 2a). Interestingly, the transport efficiency was significantly higher at all incubation times when compared to the non-metabolised compound. Again, transport was completely inhibited at 4 °C after 1 h of incubation, and after 3 h of incubation, only a small increase in the transport efficiency of the metabolite was observed (Fig. 2b). LC–MS analysis of the 18 h of incubation samples revealed the presence of both quercetin and 3-GlucQ, yet no other metabolites were identified. Efficiency of quercetin transport was very low (around 8% after 18 h incubation, Fig. 2a), agreeing with previous results obtained with a rat BBB cell model, RBE4 (Faria et al., 2010). This low transport efficiency could be explained by (i) a low uptake at the apical membrane, (ii) a low transport through the basolateral membrane, (iii) a high metabolisation/degradation rate or (iv) an increased efflux. In order to understand and possibly reverse the low transport efficiency of quercetin, additional experiments were performed. An interaction between flavonoids and efflux transporters is documented but this relationship is not yet clear. Efflux transporters are essential components of the BBB, controlling the entrance of xenobiotics to the brain, limiting the bioavailability and distribution of these compounds. P-glycoprotein, in particular, has been detected at the apical membranes of epithelial cells in the excretory organs, such as the intestine, liver, kidney and endothelial cells in the BBB. Nevertheless, besides flavonoid interaction with efflux transporters, some reports have suggested them as substrates of these transporters (Vaidyanathan & Walle, 2001). Since Breast Cancer Resistance Protein (BCRP) and P-glycoprotein are both expressed in the BBB and in the present cell line (Youdim, Shukitt-Hale, & Joseph, 2004), cells were pretreated for 48 h with specific inhibitors: rhodamine 123 and cyclosporine A, respectively. The aim was to investigate whether the transport across BBB of quercetin

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(a) *

15 10

Transport efficiency at 3h (%)

Transport efficiency (%)

20

*

*

5 0

(b)

20

Quercetin 3-GlucQ

*

15 10 5

*

18

3

1

0 4ºC

37ºC

Time (h) Fig. 2. Transport efficiency of 30 lM quercetin and 3-glucuronyl-quercetin in hCMEC/D3 cells (Apical ? Basolateral) (a). Transport efficiency after 3 h of incubation, with 30 lM quercetin and 3-glucuronyl-quercetin in hCMEC/D3 cells at 37 °C and 4 °C (b). Transport efficiency percentages were calculated based on (compound concentrations at the basolateral side overtime)/(compound concentrations at the apical side at the zero hours)*100. ⁄Significantly different from quercetin (p < 0.05).

was increased by inhibiting those efflux transporters. An increase in quercetin transport efficiency after 1 h of incubation either with cyclosporine and rhodamine 123 was observed (Table 2), which suggested some involvement of efflux transporters in the low efficiency of quercetin uptake.

Table 2 Transport efficiency of 30 lM quercetin (Q) and after 48 pretreatment with 1 lM cyclosporine A and 25 lM rhodamine 123 in hCMEC/D3 cells (Apical ? Basolateral). Results are presented as transport efficiency (%) (mean ± SEM). Transport efficiency percentages were calculated based on (compound concentrations at the basolateral side overtime)/(compound concentrations at the apical side at the zero hours)*100. Compound

3.5. Modulation of flavonoids transport

Transport efficiency (%)

Quercetin (Q) Q + cyclosporin A Q + rhodamine 123

In order for flavonoids to reach their targets, in this particular case the neurons, the BBB needs to be crossed. In this work, different classes of flavonoids were shown to be able to cross a human BBB model. Thus, in addition, several different compounds were tested to evaluate their impact on flavonoid transport efficacy. Previous work in an intestinal cell line, the continuous exposure to flavonoids was able to influence their own transport by changing the expression of transporters (Faria et al., 2009). Bearing this in mind, cells were pretreated with 30 lM of epicatechin, catechin and quercetin in cell medium for 48 h. The transport of these flavonoids (30 lM) was studied for 18 h after this treatment. No significant changes were observed in the transport of catechin and epicatechin. However, quercetin transport was significantly increased after 18 h of incubation (Fig. 3). This result suggested that quercetin transport was influenced by the continue exposure of the BBB to this flavonoid, but that was not the case concerning catechins. The effect of the pretreatment with other compounds was also tested. First, a 48 h pretreatment with 100 lM progesterone and 100 lM b-estradiol did not affect epicatechin nor catechin

*

1h

3h

18 h

2.9 ± 0.6 7.9 ± 2.6* 7.2 ± 2.1*

5.2 ± 1.3 8.7 ± 2.2 7.4 ± 3.0

6.5 ± 1.2 9.8 ± 2.7 7.0 ± 2.0

Significantly different from Q (p < 0.05).

transport but had a significant effect on quercetin transport (Table 3), which was significantly increased. Both progesterone and b-estradiol are alkaline phosphatase activators. Since only quercetin’s transport was affected by these hormones, different mechanisms may be suggested for different flavonoid classes. Quercetin transport probably involves a mechanism dependent on phosphorylation/dephosphorylation. This observation is relevant since quercetin has multiple cellular targets with important repercussions in cell biology such as regulation of transcription factors, signal transduction pathways, and mediators of cell death (Dajas, 2012). Also, it has been described as a broad kinase modulator, acting on several signalling cascades (Williams, Spencer, & Rice-Evans, 2004) and an inducer of alkaline phosphatase (Lea, Ibeh, Deutsch, Hamid, & desBordes, 2010).

Without pre-treatm ent

Transport efficiency (%)

30

With pre-treatm ent

Epicatechin

Catechin

Quercetin *

20

10

1 3 18

1 3 18

1 3 18

1 3 18

1 3 18

1 3 18

0 Time (h) Fig. 3. Transport efficiency of 30 lM epicatechin, catechin and quercetin after 48 h treatment in hCMEC/D3 cells (Apical ? Basolateral). Results are presented as transport efficiency (%) (mean ± SEM). Transport efficiency percentages were calculated based on (compound concentrations at the basolateral side overtime)/(compound concentrations at the apical side at the zero hours)*100. ⁄Significantly different from quercetin (p < 0.05).

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Table 3 Transport efficiency of 30 lM epicatechin, catechin and quercetin through hCMEC/D3 cells alone, or in the presence of progesterone (100 lM), b-estradiol (100 lM) and caffeine (10 lM) (Apical ? Basolateral). Results are presented as transport efficiency (%) (mean ± SEM). Transport efficiency percentages were calculated based on (compound concentrations at the basolateral side overtime)/(compound concentrations at the apical side at the zero hours)*100. ()-Epicatechin Time (h)

Progesterone 100 lM b-Estradiol 100 lM Caffeine 10 lM *

(+)-Catechin

Quercetin

1

3

18

1

3

18

1

16.3 ± 1.5

23.0 ± 1.0

24.9 ± 1.0

12.4 ± 1.5

15.6 ± 1.5

18.8 ± 1.1

2.9 ± 0.6

3 5.2 ± 1.3

18 6.5 ± 1.2

17.3 ± 1.8 21.8 ± 4.1 13.3 ± 2.2

24.2 ± 1.5 27.7 ± 5.1 17.9 ± 2.8

23.3 ± 1.4 29.6 ± 3.6 19.3 ± 2.9

12.7 ± 2.5 13.5 ± 1.8 6.0 ± 1.2*

18.5 ± 3.6 18.4 ± 2.9 12.1 ± 1.7

20.3 ± 2.1 21.5 ± 2.7 19.3 ± 2.3

13.6 ± 1.2* 6.9 ± 1.2 2.0 ± 0.4

12.2 ± 1.4 10.9 ± 1.4* 1.9 ± 0.2*

15.4 ± 5.0* 14.3 ± 2.5* 2.0 ± 0.3*

Significantly different from epicatechin, catechin or quercetin alone (p < 0.05).

Moreover, the effect of caffeine upon epicatechin, catechin and quercetin transport was also studied. Caffeine is consumed worldwide and has the potential to affect health, behaviour, memory and other physiopathological processes (Cunha & Agostinho, 2010). Caffeine has been described to have an enhancer effect on memory performance in human and animal studies (Cunha & Agostinho, 2010). Furthermore, caffeine is present in tea, an important catechins source. In this study, cells were treated with caffeine (10 lM) for 48 h before the transport assays of epicatechin, catechin and quercetin. Epicatechin and catechin transport was found to decrease after treatment. On the other hand, quercetin transport, which was already very low, was further significantly reduced after treatment for 18 h with caffeine (10 lM) (Table 3). Caffeine is a well-known alkaline phosphatase inhibitor (Casiglia, Spolaore, Ginocchio, & Ambrosio, 1993; Tsuang, Sun, Chen, Sun, & Chen, 2006), and the inhibitory effect of caffeine on quercetin transport could be related with this fact, since the ALP activators tested increased quercetin transport, again suggesting the involvement of a phosphorylation/dephosphorylation regulation mechanism of quercetin transport across these cells. 4. Conclusions Overall, flavonoid metabolites were capable of crossing the BBB more efficiently than the unconjugated compounds. Anthocyanins and their metabolites were able of crossing this BBB cell model in a lipophilicity-dependent way. Flavonol (i.e., quercetin) transport is thought to be modulated by phosphatase modulators suggesting a possible phosphorylation/dephosphorylation regulation mechanism. The results obtained in this work suggested that flavonoids are capable of reaching CNS, possibly correlating with some in vivo neuroprotective effects already described in the literature for these compounds. Acknowledgements Financial support from Fundação para a Ciência e Tecnologia (FCT) – Fundo Social Europeu, Programa Operacional Potencial Humano da EU (POPH), PTDC/AGR-TEC/2227/2012, SFRH/BPD/ 75294/2010, SFRH/BD/78367/2011 and SFRH/BPD/86173/2012 is gratefully acknowledged. The GIP-USAL is financially supported by the Spanish Government through the Project BFU2012-35228 and the Consolider-Ingenio 2010 Programme (grant CSD200700063). The authors thank Dr. Pierre-Olivier Couraud (INSERM U. 567, Université René Descartes, Paris, France) for hCMEC/D3 cell line. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.foodchem.2013.10.095.

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