In vitro evaluation of the antioxidant and anti-inflammatory activities of sulphated metabolites of catechins
Evaluación in vitro de las actividades antioxidante y antiinflamatoria de metabolitos sulfatados de catequinas

Abstract

Catechins are major polyphenols in many plant foods that have been related to health promotion. In the human organism, they are largely metabolised to different conjugated metabolites (i.e. glucuronide, sulphate and methylated derivatives), which are further found in plasma and would be thus able to reach the biological targets. Therefore, in vitro assays aiming to elucidate the biological effects of dietary catechins should also consider their metabolites and not only the original compounds. In this article, the in vitro antioxidant and anti-inflammatory activities of different catechin and epicatechin sulphates, one of the less studied catechin metabolites, have been evaluated. Since these compounds are not commercially available, they had to be first synthesised in the laboratory. The in vitro antioxidant activity was assessed using the ferric reducing power (FRAP) assay and two methods based on the ability to scavenge the ABTS^•+ radical cation at different pH values. Sulphation of (epi)catechin lead to a decrease in the antioxidant activity that was greater when the sulphate moiety was located in the catechin B-ring than in A-ring. Despite this, all the studied (epi)catechin sulphates still behave as better antioxidants than α-tocopherol in the radical scavenging assays carried out at pH 7.4, suggesting that they might act as efficient antioxidants in physiological conditions. The anti-inflammatory potential was assessed by evaluating the ability of the compounds to reduce the concentration of nitric oxide (NO) secreted by macrophages (RAW 264.7) after activation with a bacterial lipopolysaccharide (LPS). In the range of studied concentrations (1–300 μM), all the (epi)catechin sulphates caused a dose-dependent inhibition in NO production that even slight was statistically significant in most cases in relation to controls (LPS-activated cells without catechins), whereas the parent catechins did not show any effect in NO production in our experimental conditions. None of the assayed compounds showed any cytotoxic effect in macrophages up to the highest concentration used (300 μM). The obtained results suggested possible antioxidant and immuno-modulatory roles of the sulphated metabolites of catechins.

Las catequinas son polifenoles mayoritarios en muchos alimentos vegetales y han sido relacionadas con la promoción de la salud. En el organismo humano son biotransformadas a diferentes metabolitos conjugados (glucurónidos, sulfatos y derivados metilados), que aparecen luego en plasma y serían, por tanto, capaces de alcanzar los potenciales objetivos biológicos. Por ello, los ensayos in vitro que tratan de esclarecer los efectos biológicos de las catequinas de la dieta deben considerar también estos metabolitos y no sólo los compuestos originales. En este trabajo se han evaluado las actividades antioxidante y antiinflamatoria in vitro de diferentes sulfatos de catequina y epicatequina, un tipo de metabolitos apenas estudiados. Estos compuestos no están disponibles comercialmente, por lo que fueron sintetizados en el laboratorio. La actividad antioxidante se evaluó mediante el método FRAP y dos métodos basados en la capacidad para captar el radical ABTS^•+ a dos valores diferentes de pH (4,5 y 7,4). Se observó que la sulfatación produce una disminución en la actividad antioxidante de las catequinas, mayor cuando el grupo sulfato se encuentra en el anillo B que en el A. No obstante, todos los sulfatos estudiados se comportan como mejores antioxidantes que el α-tocoferol en el ensayo realizado a pH 7,4, sugiriendo que podrían actuar como antioxidantes eficaces en situación fisiológica. La actividad antiinflamatoria se evaluó a partir de la capacidad de los compuestos para reducir la concentración de óxido nítrico (NO) secretada por macrófagos (RAW 264,7) tras su activación con lipopolisacárido bacteriano (LPS). En el intervalo de concentraciones estudiadas (1–300 μM) todos los sulfatos de (epi)catequina producían una inhibición en la formación de NO dosis-dependiente pequeña pero estadísticamente significativa con relación a los controles (células activadas con LPS sin catequina), mientras que la catequinas originales no tenían ningún efecto en la producción de NO. Ninguno de los compuestos ensayados mostró efectos citotóxicos en los macrófagos hasta la concentración máxima ensayada (300 μM). Los resultados obtenidos sugieren un posible papel antioxidante e inmuno-modulador de los metabolitos sulfatados de catequinas.

Keywords:

• catechin
• epicatechin
• sulphated metabolites
• antioxidant
• anti-inflammatory
• flavonoids
• RAW 264.7
• NO

Palabras clave:

• catequina
• epicatequina
• metabolitos sulfatados
• antioxidante
• anti-inflamatori
• flavonoides
• RAW 264,7
• NO

Introduction

Flavonoids are plant secondary metabolites that are present in a broad range of commonly consumed fruits and vegetables and plant-derived products and have been related to health promotion. Flavan-3-ols (catechins and proanthocyanidins) are among the more widespread flavonoids in the human diet and several epidemiological studies have shown that the intake of flavan-3-ol-rich products (i.e. tea, red wine, apples or chocolate) is inversely associated with the risk of coronary heart disease, cancer and immuno-disfunctions (Cook & Samman, 1996; Cooper, Donovan, Waterhouse, & Williamson, 2008; Nakachi, Suemasu, & Suga, 1998; Williamson & Manach, 2005). These compounds possess a broad set of pharmacological activities demonstrated in different in vitro, ex vivo and animal assays. Catechins are acknowledged antioxidants with free radical scavenging (Mira, Silva, Rocha, & Manso, 1999; Rice-Evans, Miller, & Paganga, 1996) and metal ion chelating properties (Mira et al., 2002), they act as powerful inhibitors of low density lipoproteins (LDL) oxidation in vitro (Steinberg, Bearden, & Keen, 2003), and are able to modulate inflammatory processes (Saito, Hosoyama, Ariga, Kataoka, & Yamaji, 1998), reduce platelet aggregation, inhibit the growth of human cancer cell lines (Kashiwada, Nonaka, Nishioka, & Chang Lee, 1992), decrease DNA damage, and delay tumour promotion in mouse (Bomser, Singletary, Wallig, & Smith, 1999; Simonetti, Ciappellano, Gardana, Bramati, & Pietta, 2002).

Health effects of these compounds depend on their bioavailability. It has been shown that catechins are absorbed from the human intestinal tract, largely metabolised and distributed as conjugated derivatives in bloodstream (Baba et al., 2000). Methyl, sulphate and glucuronide conjugates have been described as circulating metabolites of catechins (Baba, Osakabe, & Natsume, 2001; Donovan et al., 1999, 2001; Natsume et al., 2003). Circulating metabolites would be those able to reach the biological targets and, therefore, they should be main actors to explain the health effects associated to the intake of catechins and flavonoids in general. Conjugated metabolites are likely to possess different biological properties than do parent compounds, and therefore, in vitro studies should also consider metabolites rather those only commercially available compounds as found in foodstuffs (Kroon et al., 2004). The nature and position of the conjugation can be expected to have an effect on their biological activities. Previous works have shown that the nature and position of the conjugating residues have a large effect on the bioactivity of flavonoids (Cao, Sofic, & Prior, 1997; Day, Bao, Morgan, & Williamson, 2000; Williamson, Barron, Shimoi, & Terao, 2005). Therefore, in order to better understand the in vivo effects of dietary catechins, standards of the conjugated metabolites are required, so that their biological activity can be assessed in in vitro assays. However, most catechin metabolites are not commercially available and must, therefore, be prepared in the laboratory by synthetic approaches or isolated from suitable sources. In previous studies of our group methods, the hemisynthesis of conjugated metabolites of catechins, including different sulphated derivatives, was optimised (Gonzalez-Manzano, Gonzalez-Paramas, Santos-Buelga, & Dueñas, 2009). Some data on the in vitro antioxidant activity of glucuronide and methylether derivatives have also been obtained (Dueñas, Gonzalez-Manzano, Gonzalez-Paramas, & Santos-Buelga, 2010). However, data are hardly available regarding the activity of sulphates due, among others, to their low stability that makes difficult their preparation and identification.

Inflammatory processes are characterised by an increase in the synthesis and release of a variety of pro-inflammatory mediators, such as reactive oxygen and nitrogen species (ROS and RNS), influencing the integrity of the tissue. Macrophages are involved in the inflammatory response. These cells have important roles in the immune system. But under some circumstances, septic shock, rheumatoid arthritis and atherosclerosis, macrophages have been described to have harmful effects probably due to non-regulation and excess in the secretion of inflammatory modulators like nitric oxide (NO). The synthesis of NO is catalysed by the enzyme nitric oxide synthase (NOS). There are three isoforms of NOS, two of them named constituent and calcium-dependent (cNOS), which synthesised NO in normal conditions, and one inducible and calcium-independent (iNOS) that is weakly active in physiological conditions, but it is induced in response to various inflammatory stimuli. iNOS is present in cells involved in inflammatory processes as macrophages and induced by different pro-inflammatory cytokines and bacterial endotoxins products (i.e. LPS). The production of NO excess accelerates the formation of ROS, damages cellular macromolecules, such as proteins, DNA and lipids, and triggers many detrimental cellular responses (Radi, Beckman, Bush, & Freeman, 1991a, 1991b; Yermilov, Rubio, & Ohshima, 1995). Therefore, inhibition of NO and radical scavenging activity are properties that could improve the pathogenesis of several diseases including sepsis, cancer, diabetes, renal disease and atherosclerosis, which are characterised by abnormal iNOS expression and high NO production (Beckman & Koppenol, 1996; Cooke & Dzau, 1997). Flavanols from various natural sources have been shown to be able to inhibit NOS activity and/or NO production in cultured cells (Ho, Hwang, Shen, & Lin, 2007; Tsai, Tsai, Yu, & Ho, 2007).

The aim of the present study was to assess the antioxidant and anti-inflammatory activities of catechin and epicatechin (Supplementary Figure 1) and some of their sulphated metabolites. Antioxidant and antiradical activities were assessed using the FRAP and ABTS methods, and the anti-inflammatory activity was assessed by evaluating the ability of catechin and epicatechin to reduce the concentration of nitric oxide (NO) secreted by macrophages (RAW 264.7) after activation with a bacterial lipopolysaccharide.

Materials and methods

Standards and reagents

HPLC-grade methanol was purchased from Carlo Erba (Rodano, Italy). Pyridine and HPLC-grade acetonitrile were obtained from Merck KGaA (Darmstadt, Germany). Trifluoroacetic acid was purchased from Riedel-de-Haën (Seeize, Germany). Analytical grade acetic acid glacial was obtained from Panreac (Barcelona, Spain). (−)-Epicatechin, trioxide-N-triethylamine complex, Sephadex LH-20, dioxane, Neutral red, Griess reagent, LPS (lipopolysaccharide), PBS, were purchased from Sigma-Aldrich (Madrid, Spain). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, trypsin and L-glutamine were purchased from Lonza (Barcelona, Spain). All reagents used were of analytical grade.

Preparation of (epi)catechin sulphates

Catechin and epicatechin sulphates were synthesised by the method described by Gonzalez-Manzano et al. (2009) based on the procedure reported by Jones, Jukes-Jones, Verschoyle, and Farmer (2005) for the preparation of quercetin sulphates. Briefly, (epi)catechin (500 mg) was dried by adding dry pyridine until dissolved, the solvent was rotary evaporated and the residue recovered in dioxane (50 mL). Ten-fold molar excess of sulphur trioxide-N-triethylamine complex was then added to the solution under a nitrogen atmosphere and allowed to react at 40°C in a water bath for 90 min during which the products of sulphation precipitated out and stuck to the glass. Dioxane was decanted, the precipitate redissolved in methanol:water (10:90, v/v) and the mixture containing epicatechin sulphates fractioned on a Sephadex LH-20 column (350 × 30 mm). Elution was carried out with 10% aqueous ethanol (500 mL) and 20% aqueous ethanol (500 mL) to separate monosulphates from diversely substituted sulphates and the remaining catechin. The fraction containing a mixture of monosulphates was evaporated to dryness under vacuum, redissolved in ultra-pure water and further submitted to semipreparative HPLC for the isolation of individual catechin sulphates using a Waters 600 chromatograph. Separation was performed in a Ultracarb C18 ODS20 5-μm (10 × 250 mm) column from Phenomenex (Supelco Ascentis, Bellefonte, PA, USA). The solvents were (A) 5% acetic acid, and (B) methanol, establishing a gradient from 0 to 10% B in 40 min, and 10 to 12% B in 20 min, at a flow rate of 3 mL/min. Detection was carried out at 280 nm in an UV-VIS model 486 detector coupled to the chromatographic system. The peaks were collected in a fraction collector and their identity checked by HPLC-DAD-ESI/MS. The compounds were freeze-dried and kept in a dry cold place. Previous to their use, the purity of the sulphates was checked again by HPLC-DAD-ESI/MS so as to verify that they were not degraded during storage.

HPLC-DAD-ESI/MS analyses

The analyses were performed in a 1200 Series Agilent Technologies chromatograph (Agilent, Technologies, Waldbronn, Germany) which was equipped with a diode array detector (model G1315C), degasser (G1379B), binary gradient pump (G1367C), thermoautosampler (G1312B) and column oven (G1316B), and coupled to a Chemstation Software (Agilent Technologies). An Ascentis Express C18 (2.1 × 100 mm; 2.7 μm particle size) column thermostatted at 30°C was used. Solvents were (A) 0.1% formic acid in water, and (B) acetonitrile; the elution gradient was from 0% to 5% B over 5 min, 5–10% B over 5 min, isocratic elution for 10 min, 10–20% B over 10 min, 20–50% B over 3 min using a flow rate of 0.5 mL/min. Double online detection was carried out in the DAD using 280 nm as preferred wavelength and in a mass spectrometer (MS) connected to HPLC system via the DAD cell outlet.

MS detection was performed in an API 3200 Qtrap (Applied Biosystems, Darmstadt, Germany) equipped with an ESI source and a triple quadrupole-ion trap mass analyser that was controlled by the Analyst 5.1 software. Zero grade air served as the nebuliser gas (30 psi) and turbo gas for solvent drying (400°C, 40 psi). Nitrogen served as the curtain (20 psi) and collision gas (medium). The quadrupols were set at unit resolution. The ion spray voltage was set at −4000 V in the negative mode. Detection was performed in enhanced MS (EMS) mode using the following parameters: DP −40 V, EP −7 V, CE −20. Enhanced product ion (EPI) mode was used to obtain the fragmentation pattern of the parent ion(s) using parameters of DP −40 V, EP −10 V, CE −25 and CES 0.

In vitro evaluation of the antioxidant activity

ABTS/peroxidase assay. The assay was carried out according to Villaño, Fernández-Pachón, Troncoso, and García-Parrilla (2004). Free radicals were generated by an enzymatic system consisting of horseradish peroxidase enzyme, its oxidant substrate (hydrogen peroxide) and the ABTS chromophore. The radical was generated by a reaction between 1.5 mM ABTS, 15 μM hydrogen peroxide and 0.25 μM peroxidase in 50 mM glycine–HCl buffer (pH 4.5). The final volume was 60 mL, yielding a final concentration of 30 μM of the ABTS^*+ radical cation. The blank reference cuvette contained glycine–HCl buffer. Once the radical was formed, the sample was added and the decrease in absorbance was monitored. The assay was carried out at room temperature. The reaction started by adding 100 μL of test sample to 2 mL of ABTS^•+ solution, the samples were vortexed for 10 s, and the absorbance at 414 nm was measured after 2 min of reaction using a Hewlett Packard UV–Visible HP 8453 spectrophotometer (Palo Alto, CA, USA). Two independent experiments in triplicate were performed for each of the assayed compounds. In each case, six different dilutions were prepared in 50% aqueous methanol and submitted to the reaction. The results were expressed as Trolox-equivalent antioxidant capacity (TEAC) values, defined as the concentration of Trolox (mM) giving the same percentage change of absorbance of the ABTS as that of 1 mM of the assayed antioxidant. TEAC values were obtained by interpolating the decrease in absorbance on the calibration curve obtained using Trolox solutions from 30 to 1000 μM.

ABTS/persulphate assay. The ABTS^•+ radical was produced by the oxidation of 7 mM ABTS with potassium persulphate (2.45 mM final concentration) in water. The mixture was allowed to stand in the dark at room temperature for 12–16 h before use, and then the ABTS^•+ solution was diluted with phosphate buffered saline (PBS) at pH 7.4 and equilibrated at 30°C to give an absorbance of 0.7 ± 0.02 at 734 nm. A 50 μL of 50% aqueous methanol solution of the test compounds was mixed with 2 mL of the ABTS^•+ preparation, vortexed for 10 s, and the absorbance measured at 734 nm after 4 min of reaction at 30°C. Different dilutions of each of the test compounds were assayed and the results were obtained by interpolating the absorbance on a calibration curve obtained with Trolox (30–1000 μM). The results were expressed as TEAC values. Two independent experiments were performed in triplicate for each of the assayed compounds.

FRAP assay. Ferric reducing ability was evaluated according to Benzie and Strain (1996) with minor modifications. The FRAP reagent contained 10 mM of 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) solution in 40 mM HCl, 20 mM FeCl₃.6H₂O and acetate buffer (300 mM, pH 3.6) (1:1:10, v/v). A 100 μL of 50% aqueous methanol solution of the test compounds was added to 3 mL of the FRAP reagent, and the absorbance was measured at 593 nm after incubation at room temperature for 6 min, using the FRAP reagent as a blank. Different dilutions of each of the test compounds were assayed and the results were obtained by interpolating the absorbance on a calibration curve obtained with Trolox (30–1000 μM). The results were expressed as TEAC values. Two independent experiments were performed in triplicate for each of the assayed compounds.

Determination of NO-suppressing activity

The murine monocyte/macrophage cell line RAW 246.7 was used. The medium consisted of DMEM with 4.5 g/L glucose and L-glutamine supplemented with 10% FBS and 1% penicillin/streptomycin (5000 U/mL). For experiments, cells were seeded in 6-well plates with a density of 5.10⁵ cells in 2.5 mL of medium, and cultured for 48 h until the cells reached 80% confluence. Cells were incubated with different concentrations of the assayed compounds in the range 1–300 μM for 3 h, and after that time they were washed twice with PBS in order to eliminate the compounds that were not absorbed by the cells. Subsequently, the cells were activated with 500 ng/mL LPS for 20 h, with the exception of the non-activated controls. Uptake of the neutral red dye was used as a measure of cell viability in response to the different compounds tested, as described by Park, Rimbach, Saliou, Valacchi, and Packer (2000). The NO concentration was determined in the culture medium by the Griess reaction; the absorbance was measured at 540 nm. Nitrite production was normalised to the protein content measured by Bradford method. All assays were performed in triplicate.

Statistical analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) using the PC software package, SPSS (version 13.0; SPSS Inc., Chicago). Significant differences were assessed with an LSD test (p < 0.01).

Results and discussion

Preparation of (epi)catechin sulphates

Sulphated metabolites were prepared by chemical hemisynthesis as indicated above and published elsewhere (Gonzalez-Manzano et al., 2009). A mixture of (epi)catechin monosulphates was first obtained (yield 10–20%) from which individual compounds were separated by semipreparative HPLC. Four epicatechin and two catechin monosulphates could be obtained (yields ranging 1–4% depending on the compound) in sufficient amount and with sufficient purity to be used in the in vitro activity assays.

These compounds were identified based on their chromatographic behaviour and ESI/MS fragmentation patterns, as described by Gonzalez-Manzano et al. (2009), as the 5-, 7-, 3′- and 4′-O-sulphates of epicatechin (Supplementary Figure 2), and 3′- and 4′-O-sulphates of catechin. No catechin derivatives substituted on A-ring could be isolated as individual pure stable compounds. Supplementary Table 1 shows the retention time and UV and mass spectral data of the obtained monosulphates compared to their original flavan-3-ol. As it can be observed, the different compounds showed absorption spectra with maximum wavelengths around 280 nm and the sulphation produced a small hypsochromic shift in the maximum wavelength with regard to the corresponding catechin that was especially noticeable when the substitution was produced at position 4′ (−5 nm; Supplementary Figure 3). Obviously, all monosulphates showed the same pseudomolecular [M-H]⁻ ion at m/z 369, although their fragmentation patterns differed depending on the ring of substitution, either A or B. The precise position of the sulphate residue was first tentatively established from their chromatographic elution (Gonzalez-Manzano et al., 2009), although the actual identity of the derivatives substituted in the B-ring (i.e. the 3′- and 4′-O-sulphates) could be further confirmed by NMR (Dueñas, Gonzalez-Manzano, Gonzalez-Paramas, & Santos-Buelga, submitted for publication).

Antioxidant activity

The antioxidant activity of epicatechin and catechin and their sulphate conjugates was assessed using FRAP and ABTS scavenging assays. The FRAP assay evaluates the ability of a substance to reduce Fe³⁺ to Fe²⁺; since the antioxidant activity of a substance is usually correlated to its reducing capacity this assay provides a reliable method to evaluate the antioxidant activity (Benzie & Strain, 1996). The ABTS assay measures the ability of an antioxidant to scavenge the ABTS^•+ radical cation. In the original method developed by Miller et al. (1993), metmyoglobin and H₂O₂ were used to generate ferrylmyoglobin, which then reacted with ABTS to form the ABTS^•+ radical. Different strategies have been further implemented for ABTS^•+ generation, using either chemical or enzyme reactions; chemical generation usually requires longer times, whereas enzymatic generation is faster and the reaction conditions are milder. In this study, two different assays have been employed differing in the way of generation of the ABTS^•+ radical and the pH value used: an enzymatic protocol using horseradish peroxidase at pH 4.5, and a chemical assay using persulphate at pH value of 7.4, close to physiological conditions. Even though the results obtained in the FRAP and the ABTS/peroxidase assays do not reflect the physiological situation in plasma, they were included because they are widely used in the bibliography, which allow comparing our results with those obtained by other authors. On the other hand, by using the three assays a range of pH values was covered, which could be of interest when considering not only plasma but also other physiological environments (e.g. stomach or colon).

Supplementary Table 2 shows the values of antioxidant activity obtained in the three assays for the different (epi)catechin monosulphates compared with those obtained for the parent catechins and α-tocopherol as a reference antioxidant. As already reported (Dueñas et al., 2010), the two catechins showed significantly higher TEAC values than α-tocopherol in the three in vitro assays. Epicatechin revealed as a better antioxidant than catechin in the ABTS scavenging assays, whereas no significant differences between them were found regarding their reducing capacity as evaluated by the FRAP assay. A possible explanation of this distinct behaviour might be found in the larger charge area of epicatechin compared to catechin (Saint-Cricq de Gaulejac, Vivas, De Freitas, & Bourgeois, 1999), which could provide a more effective electron delocalisation, since no relevant differences exist between both compounds regarding the acidity of their phenolic hydroxyl groups nor their ability to act as hydrogen and electron donors (Cren-Olivé & Rolando, 2003).

As expected, the sulphated derivatives showed less antioxidant activity than their parent catechins, which can be explained by the decrease in the number of free hydroxyls, a structural feature important for the antioxidant properties of flavonoids (Rice-Evans et al., 1996). The presence of free o-dihydroxyl groups in the B-ring is also known as an important feature for the antioxidant activity of the flavonoids (Rice-Evans et al., 1996; Silva et al., 2002), and therefore substitution of one of these hydroxyls could be expected to affect more the antioxidant properties than the substitution of hydroxyls at A-ring. This presumption was confirmed by comparison of the results obtained in the ABTS/persulphate assay for the different studied epicatechin sulphates for which lower antioxidant capacity was found when sulphation was produced in B-ring than in A-ring.

As it can also be observed in Supplementary Table 2, greater antioxidant activity for catechins and their monosulphates was obtained at pH 7.4 (ABTS/persulphate assay) than at acidic pH (i.e. ABTS/peroxidase and FRAP assays). An increase in the radical scavenging capacity of catechins with the increase in the pH of the medium was also observed by other authors (Lemanska et al., 2001; Muzolf, Szymusiak, Gliszczynska-Swiglo, Rietjens, & Tyrakowska, 2008). This might be explained by an increase in the electron donating ability upon deprotonation of catechins at increasing pH values. Interestingly, catechin 3′-O-sulphate was the sulphated metabolite with higher antioxidant activity in this study, superior to all the assayed epicatechin sulphates, in contrast with what happened for the parent catechins. Similar result was found in the case of methylated derivatives, among which 3′-O-methyl-catechin showed greater antioxidant activity than other catechin and epicatechin counterparts (Dueñas et al., 2010).

The comparison of the results here presented with those obtained for methylated catechins studied in a previous work (Dueñas et al., 2010) showed that sulphation produces a greater decrease in the antioxidant activity than methylation at the same position of the flavanol. As an example, a TEAC value of 3.59 ± 0.37 was determined for 3′-O-methyl-catechin in the ABTS/persulphate assay (Dueñas et al., 2010), which is 50% higher than the one obtained here for catechin 3′-O-sulphate (2.35 ± 0.04; Supplementary Table 2). Similar observations were made for quercetin sulphates and methylated derivatives (Dueñas, Surco-Laos, Gonzalez-Manzano, Gonzalez-Paramas, & Santos-Buelga, 2011). The differences in the antioxidant capacity between both types of metabolites (i.e. sulphated and methylated derivatives) might be because the methoxyl groups still retain some electron-donating properties which could provide a more effective electron delocalisation, thus conferring some stability to the phenoxyl radicals, as suggested by Rice-Evans et al. (1996). Nevertheless, despite their lower antioxidant activity compared with catechins and methylated metabolites, all the assayed monosulphates still behave as better antioxidants than α-tocopherol in the radical scavenging assays carried out at pH 7.4, suggesting that they might act as efficient antioxidants in physiological conditions.

Anti-inflammatory activity

The ability of catechin and epicatechin and their sulphated derivatives in the B-ring to inhibit the production of nitric oxide (NO) secreted by macrophages (RAW 264.7) after activation with a bacterial lipopolysaccharide (LPS) was evaluated as a measure of their anti-inflammatory activity. Furthermore, the cytotoxicity of the tested compounds at the concentrations used in the assays was also checked.

Non-stimulated control cells produced a negligible amount of NO, whilst a substantial production of NO existed in LPS-stimulated macrophages. As it is shown in Supplementary Figure 4, the pre-treatment of the stimulated cells with catechin or epicatechin did not show any effect in NO production compared with controls (LPS-stimulated macrophages cultivated in the absence of the assayed compounds). However, the studied (epi)catechin sulphates caused a dose-dependent inhibition in NO production that even slight was statistically significant in most cases in relation to controls. In the cytotoxicity assay, the viability of the cells treated with the compounds tested up to concentrations of 300 μM was not significantly modified in relation to the active control. As for the antioxidant activity, it was catechin 3′-O-sulphate metabolite that showed the highest NO inhibition activity.

Controversial results have been previously published about the inhibition of NO production by flavan-3-ols. Monomers (catechins) and dimeric flavanols have been reported to repress NO production (Park et al., 2000), whereas no such ability of procyanidin dimers and oligomers to inhibit the NO production in LPS-activated RAW cells was found by Stevens et al. (2002). More recently, Terra et al. (2007) observed that procyanidin oligomers were able to inhibit iNOS expression in RAW 264.7 macrophages stimulated with LPS plus interferon-γ, but not the monomeric forms (catechin and epicatechin). The results obtained here support the lack of NO inhibiting activity by catechins in a monocyte–macrophage cell model, whereas they point to some activity in the case of their sulphated metabolites, suggesting a possible immuno-modulatory role in the inflammatory process of these latter.

Concluding remarks

Sulphation of (epi)catechin results in a decrease of the antioxidant activity in relation with the parent catechins. However, sulphated metabolites still retain significant radical scavenging activity at pH 7.4, significantly better than the well recognised antioxidant α-tocopherol. Furthermore, (epi)catechin sulphates produce a slight dose-dependent inhibition in NO production in the cell culture that was not observed for their precursor catechins, without inducing significant cytotoxicity.

Sulphated metabolites have been less reported than methylated and glucuronidated conjugates, although they should also be relevant circulating forms (Williamson et al., 2005). Their less frequent identification may be due to their easy acid-catalysed cleavage that makes difficult their extraction in intact form. Despite this, Morand, Manach, Donovan, and Remesy (2001), using differential hydrolyses, concluded that catechin was present in rat plasma mostly as glucurono-sulpho conjugates (68%) and sulphates (22%). In general, the circulating levels of both native and metabolic forms of flavonoids in human plasma are in the nanomolar to low micromolar range (Loke et al., 2009; Spencer, El Mohsen, Minihane, & Mathers, 2008), which are lower than those concentrations assayed in this study. Nevertheless, it must be taken into account that an accumulation of metabolites may happen in some target sites in the organism, especially in lipid–water surfaces (e.g. cell membranes), thus overcoming their isotropic dilution and reaching sufficiently high concentrations to afford an efficient activity (Laranjinha, 2010). All in all, the observations made suggest that sulphated metabolites could act as potential antioxidants in physiological conditions as well as exert an immuno-modulatory activity, thus contributing to the beneficial effects associated to the consumption of flavonoid-rich food.

Acknowledgements

This work has been financially supported by Spanish Ministerio de Ciencia e Innovación through the projects AGL2007-66108-C04-02 and AGL2006-05453 and the Consolider-Ingenio 2010 Programme (FUN-C-FOOD, CSD2007-00063).

Supplementary material

Supplementary Figure 1. Structure of catechins.

Figura 1. Estructura de catequinas.

Supplementary Figure 2. Chromatogram recorded at 280 nm showing the profile of the epicatechin monosulphates obtained upon synthesis. Peaks are identified as (1) epicatechin-5-O-sulphate, (2) epicatechin-3′-O-sulphate, (3) epicatechin-7-O-sulphate, (4) epicatechin-4′-O- sulphate, and (5) epicatechin.

Figura 2. Cromatograma registrado a 280 nm donde se muestra el perfil de monosulfatos de epicatequina obtenidos en la reacción de síntesis. Los picos corresponden a (1) epicatequina-5-O-sulfato, (2) epicatequina-3′-O-sulfato, (3) epicatequina-7-O-sulfato, (4) epicatequina-4′-O-sulfato, y (5) epicatequina.

Supplementary Figure 3. UV spectra of epicatechin-4′-O-sulphate (dotted line) and epicatechin (continuous line).

Figura 3. Espectros UV de epicatequina-4′-O-sulfato (línea de puntos) y de epicatequina (línea continua).

Supplementary Figure 4. Percentage of inhibition in NO production secreted by different catechins and their sulphated derivatives. The data are expressed as the average±standard deviation of three independent experiments. Values with an asterisk (*) are significantly different compared to controls (cells treated in the same way without catechins) (p <0.05).

Figura 4. Porcentaje de inhibición en la producción de NO secretado inducido por las catequinas y sus derivados sulfatados. Los resultados están expresados como media±desviación estándar de tres experimentos independentes. Los valores señalados con un asterisco (*) muestran diferencias significativas con respecto a los controles (células cultivadas en condiciones idénticas pero sin catequinas) (p <0.05).

Supplementary Table 1. Chromatographic, UV and mass spectral data obtained for epicatechin and their monosulphated metabolites. Peak numbers refer to Figure 2.
Tabla 1. Tiempo de retención en HPLC, longitud de onda de máxima absorción en el UV y datos de espectrometría de masas obtenidos para epicatequina y sus derivados sulfatados. La numeración de los picos hace referencia a la Figura 2.

Compounds	Peak number	Retention time (min)	λmax (nm)	Pseudo-molecular ion [M-H]^− (m/z)	MS^2 spectra
Epicatechin 5-O-sulphate	1	2.6	278	369	217, 245, 289
Epicatechin 3′-O-sulphate	2	2.8	277	369	231, 245, 289
Epicatechin 7-O-sulphate	3	2.2	275	369	217, 245, 289
Epicatechin 4′-O-sulphate	4	3.6	274	369	231, 245, 289
Epicatechin	5	5.7	279	289	123, 139, 165

Supplementary Table 2. TEAC values of antioxidant activity of the assayed compounds.
Tabla 2. Valores TEAC de actividad antioxidante de los compuestos estudiados.

Compounds	ABTS/peroxidase assay	ABTS/persulphate assay	FRAP
Catechin	1.34 ± 0.02^d	3.84 ± 0.01^f	1.53  ± 0.22^c
Epicatechin	2.07 ± 0.09^e	4.26 ± 0.25^f	1.34 ± 0.01^b,c
Catechin 3′-O-sulphate	2.00 ± 0.12^e	2.35 ± 0.04^e	1.30 ± 0.09^b,c
Epicatechin 4′-O-sulphate	0.71 ± 0.03^b	1.30 ± 0.14^b	0.57 ± 0.00^a
Epicatechin 3′-O-sulphate	0.40 ± 0.01^a	1.46 ± 0.04^b,c	0.43 ± 0.00^a
Epicatechin 5-O-sulphate	0.43 ± 0.00^e	1.75 ± 0.09^c,d	0.58 ± 0.06^a
Epicatechin 7-O-sulphate	0.56 ± 0.01^a,b	1.99 ± 0.01^d	0.36 ± 0.02^a
α-Tocopherol	1.01 ± 0.02^c	0.98 ± 0.03^a	1.18 ±  0.01^b
Note: ^a,b,c,d,e,f Values in the same column with different letters are significantly different: LSD (p <0.05).