Distribution of procyanidins and their metabolites in rat plasma and tissues after an acute intake of hazelnut extract

Abstract

Procyanidins are present in a wide range of dietary foods and their metabolism is well known. Nevertheless, the biological target and their distribution are topics lacking information. The purpose of the present work was to study the metabolism and distribution of procyanidins and their metabolites in rat plasma and different tissues, such as liver, brain, lung, kidney, intestine, testicle, spleen, heart and thymus, after 2 h of an acute intake of hazelnut extract rich in procyanidins (5 g kg⁻¹ of rat body weight). The interest of an acute intake of procyanidins instead of repeated low doses from daily ingestion of is to achieve a concentration of metabolites in the tissues that allows their detection and quantification. The results showed that catechin and epicatechin-glucuronide, methyl catechin and epicatechin-glucuronide and methyl catechin and epicatechin-sulphate were detected in plasma samples at the μmol level. On the other hand, catechin-glucuronide, methyl catechin-glucuronide and methyl catechin-sulphate were identified in some tissues, such as thymus, intestine, lung, kidney, spleen and testicle at the nmol level. Procyanidins with a low grade of polymerization (dimers and trimers) were detected in plasma samples and the intestine. Additionally, a wide range of simple aromatic acids from fermentation by the colonic microflora was detected in all tissues studied.

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1 Introduction

It is well known that polyphenols present in food are highly metabolized before their absorption. In recent years, attention has focused on the digestion and gastrointestinal metabolism of procyanidins.^1–6 Prior to absorption, procyanidins are hydrolyzed by digestive enzymes or colonic microflora⁷ and during the absorption step, procyanidins are conjugated in the small intestine, resulting in a wide range of conjugated metabolites, from the combination of sulphatation, glucuronidation and methylation.^8–10 Additionally, the colonic microflora also participates in the last step of the procyanidin metabolism, generating new small molecules by hydrolysis, mainly simple aromatic acids. Secondly, procyanidins are metabolized by the liver where they can be modified into a variety of metabolites, mainly glucuronide conjugates.

Throughout digestion, hydrolysis and metabolism change the molecular structure of procyanidins, leading to a large number of different molecules. These structural modifications may exert a negative influence on their biological activities, as occurs with the antioxidant activity, which decreases drastically when the hydroxyl group is modified.⁶ Different studies have shown the bioavailability of procyanidins by studying the concentration of their metabolites in plasma and urine.^4,11–13 Nevertheless, determination of the bioavailability of polyphenol metabolites in tissues may be much more important than knowledge of their plasma concentrations.⁷

There is a lack of knowledge about the specific target organs where the metabolites derived from ingested procyanidins accumulate. The existing studies related to the distribution of procyanidins in tissues focus on evaluating the behavior of a single molecule, such as epicatechin, by detecting this compound and its metabolites in some rat tissues.^6,14 However, it is well known that foods contain a complex mixture of phenolic compounds^6,14 making the study of metabolism, distribution and accumulation of procyanidins in the body more difficult.

These are the reasons why we report in this paper on a comprehensive study of the absorption, metabolism and distribution in plasma and body tissues (thymus, heart, brain, spleen, testicle, intestine, kidney, lung and liver) of (+)-catechin and (−)-epicatechin and procyanidins with a low degree of polymerization (dimers and trimers) following the oral intake of a high dose of hazelnut extract in rats (5 g kg⁻¹ of rat body weight). To observe and understand the future potential benefits of polyphenols, taking into account their short life in plasma, the studies should be carried out during the postprandial state, immediately after ingestion.^15,16 So, the aim of an acute intake of procyanidins instead of repeated low doses from daily ingestion of them is to achieve a concentration of procyanidin metabolites in the tissues that allow their detection and quantification. This fact may be very useful in future repeated low dose experiments, facilitating the understanding of future results.

2 Materials and methods

2.1 Reagents

Internal standard (IS) catechol, and the standards of (−)-epicatechin, (+)-catechin, (−)-epigallocatechin, (−)-epigallocatechin-3-O-gallate, gallic acid, p-hydroxybenzoic acid, protocatechuic acid, phenylacetic acid and 3-(4-hydroxyphenyl)propionic acid were purchased from Sigma Aldrich (St. Louis, MO, USA) and procyanidin dimer B2 [epicatechin-(4β-8)-epicatechin], 2-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid and 3-(2,4-dihydroxyphenyl)propionic acid from Fluka Co. (Buchs, 125 Switzerland). The acetonitrile (HPLC-grade), methanol (HPLC-grade), acetone (HPLC-grade) and glacial acetic acid (≥99.8%) were of analytical grade (Scharlab, Barcelona, Spain). Ortho-phosphoric acid 85% was purchased from MontPlet& Esteban S. A. (Barcelona, Spain). Formic acid and L(+)-ascorbic acid (reagent grade) were all provided by ScharlauChemie (Barcelona, Spain). Ultrapure water was obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA, USA).

2.2 Hazelnut procyanidin extract

A hazelnut extract used as a source of procyanidins was kindly supplied by La Morella Nuts S.A. (Reus, Spain). The extract was produced from hazelnut skins by solid/liquid extraction using a mixture of water and acetone based on the previous work by Ortega et al.¹⁷ The resulting extract was rotary evaporated until all of the acetone was eliminated, and then freeze-dried and stored at −18 °C in N₂ atmosphere. The procyanidin composition of hazelnut skin extract was analysed according to the method in Ortega et al.¹⁸

2.3 Treatment of animals and plasma and tissues collection

Three-month-old male Wistar rats were obtained from Charles River Laboratories (Barcelona, Spain). The rats were housed in cages on a 12h light–12h dark schedule at controlled temperature (22 °C). They were subjected to a standard diet of a commercial chow, PanLab A04 (Panlab, Barcelona, Spain), and waterat libitum. The animals were then kept in fasting conditions for between 16 and 17 h with only access to tap water. Subsequently, a single acute dose of 5 g of hazelnut extract/kg of body weight dispersed in water was administered to the rats (n = 10) by intragastric gavage. Two hours later, the animals were anaesthetized with isoflurane (IsoFlo, VeterinariaEsteve, Bologna, Italy) and euthanized by exsanguinations. Blood samples were collected from the abdominal aorta with heparin-moistened syringes. The plasma samples were obtained by centrifugation (2000g, 30 min at 4 °C) and stored at −80 °C until the chromatographic analysis of procyanidin metabolites. Additionally, a control group of rats (n = 10) were maintained in fasting conditions with no intake of the extract and were similarly euthanized. The thymus, heart, liver, intestine, testicle, lung, kidney, spleen and brain of rats were excised, stored at −80 °C and freeze-dried for procyanidin extraction and chromatographic analysis. The study was approved by The Animal Ethics Committee of the University of Lleida (CEEA 03-02/09, 9th November 2009). All experiments with rats were performed in compliance with the relevant laws and University of Lleida guidelines.

2.4 Extraction of procyanidins from plasma and tissues

The method used to extract procyanidins and their metabolites from plasma and tissues was based on the methodologies described in our previous papers.^12,19 In order to clean-up the biological matrix and preconcentrate the phenolic compounds, the plasma samples were pretreated by microelution solid-phase extraction (μSPE), and the rat tissue samples were pretreated by a combination of a liquid-solid extraction (LSE) and μSPE. Briefly, the extraction was realized with 60 mg of freeze-dried tissue in which 50 μl of ascorbic acid 1%, 50 μl of catechol 20 mg l⁻¹ (dissolved in phosphoric acid 4%) as an internal standard and 100 μl of phosphoric acid 4% were added. The sample was extracted four times with 400 μl of water/methanol/phosphoric acid 4% (94/4/1, v/v/v). In each extraction, 400 μl of extraction solution was added. The sample was sonicated during 30 s maintaining it in a freeze water bath to avoid heating and it was then centrifuged for 15 min, at 14 000 rpm at 20 °C. The supernatants were collected, and then the extracts were treated with μSPE before the chromatographic analysis of the procyanidins and their metabolites.

OASIS HLB μElution Plates 30 μm (Waters, Milford, MA, USA) were used. Briefly, these were conditioned sequentially with 250 μl of methanol and 250 μl of 0.2% acetic acid. 350 μL of phosphoric acid 4% was added to 350 μL of tissue extract, and then this mixture was loaded onto the plate. The loaded plates were washed with 200 μl of Milli-Q water and 200 μl of 0.2% acetic acid. Then, the retained molecules (procyanidins and their metabolites) were eluted with 2 × 50 μl of acetone/Milli-Q water/acetic acid solution (70/29.5/0.5, v/v/v). The eluted solution was directly injected into the chromatographic system, and the sample volume was 2.5 μl.

2.5 Analysis of procyanidins and their metabolites by UPLC-ESI-MS/MS

Procyanidins were analysed by Acquity Ultra-Performance™ liquid chromatography from Waters (Milford MA, USA) and tandem MS, as reported in our previous studies.^12,20 Briefly, the column was Acquity high strength silica (HSS) T3 (100 mm × 2.1 mm i.d., 1.8 μm particle size) with 100% silica particles, from Waters (Milford MA, USA). The mobile phase was 0.2% acetic acid as eluent A and acetonitrile as eluent B. The flow-rate was 0.4 ml min⁻¹ and the analysis time 12.5 min.

Tandem MS analyses were carried out on a triple quadrupole detector (TQD) mass spectrometer (Waters, Milford MA, USA) equipped with a Z-spray electrospray interface. The ionization technique was electrospray ionization (ESI). The procyanidins and their metabolites were analyzed in negative ion mode and the data was acquired through selected reaction monitoring (SRM). Two SRM transitions were studied for each analyte, the most sensitive transition being selected for quantification and a second one for confirmation purposes (Additional Information). The dwell time established for each transition was 30 ms. Data acquisition was carried out with the MassLynx v 4.1 software.

(+)-Catechin, (−)-epicatechin, dimer B2 [epicatechin-(4β-8)-epicatechin], gallic acid, p-hydroxybenzoic acid, protocatechuic acid, phenylacetic acid, 2-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid, 3-(4-hydroxyphenyl)propionic acid, 3-(2,4-dihydroxyphenyl)propionic acid were quantified using the calibration curves of the respective standards. Due to the lack of standards for some metabolites, 5-(hydroxyphenyl)-γ-valerolactone was quantified using the calibration curve of 3-(4-hydroxyphenyl)propionic acid, 5-(3,4-dihydroxyphenyl)- γ-valerolactone was quantified using the calibration curve of 3-(2,4-dihydroxyphenyl)propionic acid, and isoferulic acid was quantified using the calibration curve of ferulic acid.

2.6 Statistical analysis

The data on the procyanidin metabolite concentration are expressed as mean values ± standard error (n = 10). The data were analyzed by Student t test to assess the significant differences between the control group and the treated group two hours after the acute intake of the hazelnut procyanidin extract. All statistical analysis was carried out using STATGRAPHICS Plus 5.1.

3 Results

The composition of the hazelnut extract used as a source of procyanidins in this study is summarized in Table 1. Procyanidins with low grade of polymerization (dimers) were the most abundant with 15 ± 1.3 μmol g⁻¹ of extract, followed by catechin with 6.3 ± 0.54 μmol g⁻¹ of extract. Procyanidin trimers and tetramers were present in the extract with 4.9 ± 0.32 μmol and 0.20 ± 0.01 μmol g⁻¹ of extract, respectively. Two hours after the acute intake of the hazelnut extract, several metabolites were detected in the rat plasma samples (Table 2). An intense metabolism (methylation, sulfation and glucuronidation) of the monomers, catechin and epicatechin, was observed. The glucuronidated forms of catechin and epicatechin were quantified at 1763 ± 132 μmol l⁻¹ and 154 ± 12 μmol l⁻¹, respectively. The methyl-glucuronidated and methyl-sulphate forms of both monomers were also quantified, methyl-catechin-glucuronide being the main metabolite (1103 ± 98 μmol l⁻¹). However, procyanidin dimers and trimers were detected as unconjugated forms with 20 ± 1.3 μmol l⁻¹ and 1748 ± 145 μmol l⁻¹, respectively. None of these procyanidin metabolites were detected in plasma samples from the rat control group (data not shown).

Table 1 Procyanidin composition of hazelnut skin extract^a

Compound	Concentration (μmol g^−1)
a Data expressed as mean values±standard error (n = 5). b Quantified as dimer B2.
(+)-Catechin	6.3 ± 0.54
(−)-Epicatechin	2.4 ± 0.13
(−)Epigallocatechin	2.6 ± 0.21
(−)-Epigallocatechin-3-O- gallate	0.09 ± 0.00
Procyanidin dimers^b	15 ± 1.3
Procyanidin trimers^b	4.9 ± 0.32
Procyanidin tetramers^b	0.20 ± 0.01

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Table 2 Plasma concentration of metabolites after an acute intake of 5g kg⁻¹ of body weight of hazelnut skin extract^a

Compound	Concentration (μmol l^−1)
a Data expressed as mean values ± standard error (n = 10).
Catechin-glucuronide	1763 ± 132
Epicatechin-glucuronide	154 ± 12
Methyl-catechin-glucuronide	1103 ± 98
Methyl-epicatechin-glucuronide	59 ± 3.4
Methyl-catechin-sulphate	18 ± 1.5
Methyl-epicatechin-sulphate	21 ± 2.0
Procyanidin dimers	20 ± 1.3
Procyanidin trimers	1748 ± 145

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In relation to the distribution and accumulation of procyanidin metabolites in the rat tissues, a wide range of metabolites resulting from small intestine or liver metabolism (conjugated derivatives) or from colonic microflora fermentation (simple aromatic acids) were investigated by HPLC-MS/MS (Table 2, ESI†). Differences in the concentration of metabolites between samples from the control group and the rats after the acute intake of hazelnut extract were analyzed with the Student t test to assess the significant differences. Table 3 lists the metabolites that showed statistically significant differences (p < 0.01 and p < 0.05) in their concentration between tissues from the control group and the group treated with the procyanidin extract. The analysis of the intestines showed high concentrations of conjugated derivatives of catechin, such as catechin-glucuronide and methyl-catechin-glucuronide, the latter being the main metabolite quantified (218 ± 20 nmol g⁻¹ tissue). Besides, the free form of procyanidin dimers and trimers were detected exclusively in this organ. Related to simple aromatic acids, protocatechuic acid, protocatechuicsulphate acid and gallic acid were only detected in the intestines after the acute intake of the extract. In contrast to the intestines, only protocatechuic acid was detected in the livers after the acute intake of hazelnut extract (15 ± 1.3 nmols g⁻¹ tissue).

Table 3 Quantities of metabolites in different tissues from control rats and from rats after an acute intake of nuts skin extract^ab

Tissue	Metabolite (nmol g^−1 tissue)	Control	Acute intake
a Letters in bold represent the phase II metabolites of procyanidins. b Data expressed as mean values ± standard error (n = 10). c Mean values within a column with unlike superscript letter were significantly different. Signification level (p < 0.01) between control tissues and tissues obtained after an acute intake of nuts skin extract. d Mean values within a column with unlike superscript letter were significantly different. Signification level (p < 0.05) between control tissues and tissues obtained after an acute intake of nuts skin extract.
Intestine	Catechin - glucuronide	n.d. ^b	42 ± 3.2^d
	Methyl-catechin - glucuronide	n.d. ^b	218 ± 20^d
	Dimer	n.d. ^b	27 ± 1.9^d
	Trimer	n.d. ^b	7 ± 0.4^d
	Protocatechuic acid	n.d.^b	32 ± 1.8^d
	Protocatechuic-sulphate acid	n.d.^b	18 ± 1.7^d
	Gallic acid	n.d.^b	24 ± 1.6^d
Liver	Protocatechuic acid	n.d.^b	15 ± 1.3 ^b
Thymus	Methyl-catechin - glucuronide	n.d. ^b	2.7 ± 0.13^d
	Vanillic acid	67 ± 6.0^b	76 ± 3.7^c
Spleen	Methyl-catechin - glucuronide	n.d. ^b	1.5 ± 0.13^c
	Vanillic acid	17 ± 0.9^b	20 ± 1.8^c
Testicle	Catechin - glucuronide	n.d. ^b	2.2 ± 0.32^d
	Methyl-catechin - glucuronide	n.d. ^b	2.3 ± 0.15^d
	p-Hydroxyphenylacetic acid	n.d.^b	19 ± 1.1^d
	o-Hydroxyphenylacetic acid	n.d.^b	19 ± 1.2^d
Lung	Epicatechin	n.d. ^b	59 ± 5.1^d
	Catechin - glucuronide	n.d. ^b	19 ± 1.9^d
	Methyl-catechin - glucuronide	n.d. ^b	23 ± 2.5^d
	p-Hydroxybenzoic acid	46 ± 2.9^b	65 ± 7.0^c
	Protocatechuic-sulphate acid	n.d.^b	18 ± 1.0^d
Heart	Vanillic acid	165 ± 15.5^b	203 ± 15^c
	Protocatechuic acid	n.d.^b	110 ± 5.5^d
	3-Hydroxyphenylvaleric acid	85 ± 8.1^b	98 ± 7.6^d
	5-Dihydroxyphenyl-γ-valerolactone	n.d.^b	91 ± 9.0^d
	Trimethyluric acid	92 ± 9.2^b	124 ± 11^c
Brain	Vanillic acid	21 ± 2.1^b	18 ± 1.6^c
	p-Hydroxyphenylpropionic acid	n.d.^b	15 ± 1.1^c
	m-Hydroxyphenylpropionic acid	21 ± 1.9^b	18 ± 1.0^c
Kidney	Methyl-catechin - sulphate	n.d. ^b	1.8 ± 0.12^d
	Catechin - glucuronide	n.d. ^b	5.1 ± 0.45^d
	Methyl-catechin - glucuronide	4.0 ± 0.23 ^b	17 ± 1.4^d
	p-Hydroxybenzoic acid	31 ± 3.0^b	36 ± 2.3^c
	p-Hydroxyphenylacetic acid	20 ± 1.9^b	29 ± 2.1 ^d
	m-Hydroxyphenylpropionic acid	17 ± 1.1^b	24 ± 2.6^c
	Protocatechuic acid	18 ± 1.8^b	39 ± 4.0^d
	Protocatechuic-sulphate acid	12 ± 1.1^b	22 ± 6.8^d
	Methyl gallate	13 ± 1.1^b	16 ± 1.0^d

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Methyl-catechin-glucuronide was only quantified (2.7 ± 0.13 nmol g⁻¹ tissue) in the thymus from the treated rats. A wide range of simple aromatic acids were quantified in this tissue (Table 2, ESI†), but only the concentration of vanillic acid showed significant differences (p < 0.05) between the control and treated groups with 76 ± 37 nmol g⁻¹ tissue after the acute intake of the hazelnut extract. As in the thymus, methyl-catechin-glucuronide was only detected in the spleens after an acute intake of extract (1.5 ± 0.13 nmol g⁻¹ tissue), and the concentration of vanillic acid in the spleens increased significantly (p < 0.05) after the intake of the extract.

Two catechin-conjugated metabolites were detected in the testicles after the acute intake of the hazelnut extract, these being catechin-glucuronide with 2.2 ± 0.32 nmol g⁻¹ tissue and methyl-catechin-glucuronide with 2.3 ± 0.15 nmol g⁻¹ tissue. Additionally, two hydroxylated forms of phenylacetic acid, p- and o-hydroxyphenylacetic acids were only quantified after the intake of the hazelnut extract with a similar concentration.

Surprisingly, the analysis of the lungs from the treated group of rats revealed the presence of high concentrations of the free form of epicatechin with 59 ± 5.1 nmol g⁻¹ tissue (Table 3). Besides the two conjugate forms of catechin quantified in other tissues, catechin-glucuronide with 19 ± 1.9 nmol g⁻¹ tissue and methyl-catechin-glucuronide with 23 ± 2.5 nmol g⁻¹ tissue. Additionally, protocatechuic-sulphate acid was only detected after the acute intake of hazelnut extract.

Finally, as an essential pathway of excretion, the kidneys were analyzed and a high number of metabolites were detected. Methyl-catechin-sulphate and catechin-glucuronide were only detected after the acute intake of the extract, with 1.8 ± 0.12 nmol g⁻¹ tissue and 5.1 ± 0.45 nmol g⁻¹ tissue, respectively. Additionally, methyl-catechin-glucuronide was also detected in the control group, but its concentration was significant lower (p < 0.05) than in the kidneys from the treated rats (Table 3). Besides, the concentration of different simple aromatic acids increased significantly (p < 0.05 and p < 0.01) after the acute intake of hazelnut extract. Methyl gallate was only detected in the kidneys. This metabolite was also detected in the control group, but its level increased significantly (p < 0.05) after the acute intake of the extract.

4 Discussion

The main objective of this work was to evaluate the metabolism and distribution of procyanidin metabolites in rat bodies, including their distribution in plasma and tissues. As far as we know, the present study shows for the first time the distribution of procyanidin metabolites in a wide range of rat tissues after an acute intake of a complex mixture of procyanidins contained in a food matrix, this being a hazelnut skin extract. The extract used was rich in procyanidin dimers, and the main monomer present in the extract was catechin (Table 1).

After the ingestion of the hazelnut skin extract, the monomers catechin and epicatechin were absorbed, appearing at high concentrations in the plasma as conjugated forms (Table 2). The main metabolites detected in the plasma in our study were glucuronidated and methyl-glucuronidated conjugates and this agrees with the studies by El Mohsen et al.²¹ and Harada et al.²² The presence of the methylated forms of the glucuronide and sulphate conjugates of catechin and epicatechin could be explained by the ingestion of a large amount of catechin contained in the hazelnut extract.⁶ This high absorption and conjugation of procyanidins in glucuronidated, sulphated and methylated forms observed in the present study is in agreement with previous studies, which also analyzed biological fluids after the ingestion of such rich sources of procyanidins as chocolate,^23–25 tea²⁶ or grape seed extract.^1,27 Nevertheless, not only were the conjugated forms of catechin and epicatechin able to reach the bloodstream; the low grade of polymerization of procyanidins, such as dimers and trimers, were detected in plasma two hours after the extract intake, similar to that observed by other authors.^1,28–30 However, the free forms of catechin and epicatechin were not detected.

The absorption of procyanidins initially takes place during transfer through the small intestine and subsequently, by the liver,²¹ resulting in a wide range of metabolites. These metabolites may reach other organs through the bloodstream. Fig. 1 shows the distribution and accumulation of procyanidin metabolites in the rat tissues observed in the present study, resulting from metabolism in the small intestine or liver (conjugated derivatives) or from the fermentation of colonic microflora (simple aromatic acids). In order to assess the major metabolites accumulated in different tissues as a result of the ingestion of a procyanidin-rich extract, the difference between the amount quantified in tissues obtained from the treated group and the quantities found in the tissues from the control group for each compound was calculated. The characteristic procyanidin metabolism conducted by the intestine and liver may explain the presence of catechin-glucuronide, methyl-catechin-glucuronide and methyl-catechin-sulphate in some organs, like the thymus, lung, kidney, spleen or testicles, two hours after the acute intake of the hazelnut extract.

Fig. 1 The increase of concentration of phenolic acids and procyanidin metabolites quantified in different tissues. The increase is expressed as nanomoles, obtained by the difference between the amount quantified in the tissues obtained after an acute intake of the nuts skin extract (treated group) and the amount quantified in the control tissues.

The free forms of catechin and epicatechin were not detected in either the plasma or tissues, except in the lungs where the free form of epicatechin was quantified at an even higher level than the conjugated forms of catechin (Fig. 1). On the contrary, the free forms of procyanidin dimers and trimers were only quantified in the plasma but not in the tissues. So, this may confirm the absorption of low grade of polymerization procyanidins but not the disposition in tissues, probably because molecular weight made the interaction of dimers and trimers with tissue proteins difficult, as occurs with similar molecules, such as tannins. This interaction is fundamental to allow the fixation between the bioactive compounds in the tissues or to exert their biological activities.³¹Procyanidin dimers and trimers were only detected in the intestine, probably as a result of the hydrolysis of the most highly polymerized procyanidins in the hazelnut extract that cannot be absorbed in their native form, or as a result of an incomplete hydrolysis into the monomeric forms, catechin and epicatechin, that occurs during digestion and the first 1–4 h of colonic fermentation.³² However, the accumulation of these in specific target organs has not been demonstrated at least two hours after the ingestion of the extract. Differences in the nature of the tissue metabolites and blood metabolites may be related to the specific uptake or elimination of some of the tissue metabolites or the intracellular metabolism.⁷

In relation to the balance between the stereoisomers catechin and epicatechin, the main metabolites quantified in plasma were the conjugated forms of catechin. However, conjugated forms of epicatechin were also quantified, but at lower concentrations. Despite their presence in plasma, epicatechin conjugate forms were not detected in any tissue except the lungs, where the free form of epicatechin was found. This major accumulation of the conjugated forms of catechin in the tissues compared with epicatechin conjugates may be related to the higher levels of catechin in the hazelnut extract. Another possible explanation could be related to the influence of the stereochemical configuration of flavanols in the level and metabolism of flavanols in humans, recently reported by Ottaviani et al.³³ The results of this study showed a major oral absorbability of epicatechin after the oral intake of a low-flavanol cocoa-based dairy-containing drink matrix, enriched with catechin and epicatechin isolated from cocoa powder preparations. In contrast, our results demonstrate a greater absorbability of catechin, probably as a consequence of the higher concentration of its conjugated metabolites measured in the plasma, and the exclusive accumulation of these metabolites in some tissues.

The analysis of the metabolism and tissue distribution of procyanidins showed important differences in the nature and accumulation of metabolites two hours after the intake of the hazelnut extract (Table 3 and Fig. 1). The concentrations of the catechin metabolites resulting from metabolism in the small intestine or liver (conjugated derivatives) ranged from 1.5 to 23 nmol aglycone equivalents g⁻¹ tissue, and the concentration of the potential metabolites formed from colonic microflora fermentation (simple aromatic acids) ranged from 2.6 to 110 nmol g⁻¹ tissue (Fig. 1). Additionally, flavonoids are also rapidly excreted in the bile and urine.^34,35 Both excretion pathways were observed in the results obtained. An example of recirculation in bile, corresponding to the phase II biotransformation in the liver, could be the presence of catechin-glucuronide and methyl catechin-glucuronide in the intestine (Fig. 1); and the quantification of some catechin metabolites (methyl catechin-sulphate, catechin-glucuronide and methyl catechin-glucuronide) in the kidney may indicate the excretion of procyanidin metabolites through the urine.

The nature of the intake and the time of tissue sampling may be of great importance, depending on the kinetics of the accumulation and elimination of procyanidins in the tissues. In this study, it was chosen to sample the extract two hours after ingestion so that this time corresponded with the maximum concentration of procyanidin metabolites in the plasma observed in previous studies.^19,36 With regard to the nature of the ingestion, a single and acute intake of procyanidins was done in the present work to carry out a pharmacokinetic study. However, long treatments with procyanidins may provide different kinds and numbers of metabolites in the tissues, as occurred in the study performed by Urpi-Sarda, et al.,¹⁴ in which catechin and epicatechin metabolites were found in the brain after three weeks of cocoa diet. Thus, the ability of procyanidin metabolites to cross the blood–brain barrier and target the brain could be affected by the dose and duration of the treatment with procyanidins. Additionally, the presence of catechin metabolites in the tissues two hours after the ingestion of hazelnut extract, together with the results of Urpi-Sarda, et al.,¹⁴ may indicate that, with an adequate combination of time and doses, procyanidin metabolites could accumulate in tissues.

As regards the colonic metabolism, a variety of simple aromatic acids were detected in the intestine and tissues, probably as products of the colonic fermentation of procyanidins. Some of these simple aromatic acids have been quantified in a previous study after the colonic fermentation of a cocoa cream by in vitro and in vivo models.³² For example, protocatechuic acid, found in all tissues except the testicles, or hydroxyphenylacetic acid, could be intermediate fermentation products of phenylacetic and 3-(4-hydroxyphenyl)-propionic acids.³²p-Hydroxybenzoic acid quantified in the lungs and kidneys has been described as the final fermentation product of catechin.³²

The presence of protocatechuic sulphate acid in the lungs may indicate enzymatic metabolism after the colonic fermentation, possibly due to a trans-membrane intestine metabolism. Protocatechuic acid has been described as a fermentation product of catechin and dimer B2,^32,35 from the decarboxylation of the 3,4-dihydroxyphenylpropionic acid and its subsequent dehydroxylation to p-hydroxybenzoic acid, this being a common compound quantified in tissues in this study. Additionally, some compounds, such as 3-hydroxypenylpropionic acid and p-hydroxybenzoic acid, usually found in urine^34,35 were detected in the kidneys. In fact, 3-hydroxyphenylpropionic acid has been described as the main urinary metabolite after an ingestion of dimer B3.³⁵

Only protocatechuic acid was detected in the liver, despite its role in the procyanidin metabolism. A previous study by Urpi-Sarda, et al.¹⁴ showed the accumulation of some procyanidin metabolites in the liver was probably related to a continuous intake of procyanidins over weeks. On the other hand, the presence of a wide range of simple aromatic acids in heart tissue with significant differences in their concentration compared with the control group, may be related to the potential health benefits of procyanidins, especially in the context of cardiovascular health.³⁷

To sum up, after an acute intake of a procyanidin-rich extract, the procyanidins were absorbed, metabolized and distributed around the body. As a consequence, some conjugated derivatives and simple aromatic acids, such as procyanidin metabolites, were detected in the plasma and tissues. The main accumulation of the conjugated metabolites (mainly glucuronide conjugates) of procyanidins was observed in the lung. This disposition may indicate a temporary accumulation of procyanidin metabolites in tissues, probably related to the dose and duration of the treatment. The main accumulation of simple aromatic acids, probably resulting from the hydrolytic metabolism of procyanidins, was observed in the heart. Based on the results of this study, it would be important to consider the possible role of these simple aromatic acids accumulated in the tissues as a result of the intake of procyanidins. So, studying the distribution of procyanidin metabolites in these tissues should be the starting point for knowing the metabolic target and the first step towards understanding how procyanidin acts at a cellular level.

Acknowledgements

The present study was supported by the CENIT program from the Spanish Minister of Industry and by a consortium of companies led by La Morella Nuts S.A. (Reus, Catalonia, Spain) with the following: Shirota Functional Foods, S.L., KRAFT; BTSA, Biotecnologías Aplicadas, S.L., Selección Batallé, S.A., Industrial Técnica Pecuaria, S.A., Neuron BioPharma, S.A., Grupo Leche Pascual, S.A.U., Innaves, S.A. This study was also supported by the Catalan Government (Interdepartmental Commission for Research and Technological Innovation) through the A. Serra grant.

We also wish to thank Carme Piñol (in charge) and Roser Pane (technician) from the Animal Facility Service of University of Lleida for their technical support. We are indebted to Sergi Traver for his help with the development of the experimental.

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Footnote

† Electronic supplementary information (ESI) available: Optimized SRM conditions and concentration of metabolites in different tissues of rat control group and rat group after an acute intake of the nuts skin extract. See DOI: 10.1039/c1fo10083a

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