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Identification of microbial metabolites derived from in vitro fecal fermentation of different polyphenolic food sources
Nutrition 28 (2012) 197–203
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Basic nutritional investigation
Identification of microbial metabolites derived from in vitro fecal fermentation of different polyphenolic food sources Margherita Dall’Asta M.Sc., Luca Calani M.Sc., Marianna Tedeschi M.Sc., Lucia Jechiu M.Sc., Furio Brighenti Ph.D., Daniele Del Rio Ph.D. * The 42 Laboratory of Phytochemicals in Physiology, Human Nutrition Unit, Department of Public Health, University of Parma, Via Volturno, Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 March 2011 Accepted 7 June 2011
Objective: The biological effects of dietary polyphenols are linked to their bioavailability and catabolism in humans. The colon, with its symbiotic microbiota, is an active site where complex polyphenolic compounds are possibly modified to smaller and more absorbable molecules. The aim of this study was to identify the major metabolites derived from microbial colonic fermentation of some common polyphenol-rich foods. Methods: An in vitro fecal fermentation model was applied to 16 polyphenol-rich foods and polyphenolic precursors. Phenolic metabolites were identified by high-performance liquid chromatography coupled with tandem mass spectrometric detection. Results: Twenty-four phenolic fermentation metabolites were characterized. Some metabolites were common to several polyphenol-rich foods, whereas others were characteristic of specific sources. Conclusion: The metabolites identified in vitro likely are generated in the human colon after consumption of polyphenol-rich foods. Their occurrence in plasma and/or urine should be considered when evaluating the bioavailability of polyphenols from specific food groups in humans and in the definition of markers of exposure to specific foods or food groups in epidemiologic studies. However, the search for these and other microbial metabolites after a feeding study in vivo should consider their possible further conjugation at the level of the liver. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Polyphenols Colonic fermentation Microbial metabolites Tandem mass spectrometry
Introduction The increasing interest in polyphenols has been triggered by hundreds of studies that have associated polyphenol-rich diets to several health effects in humans [1–4]. This large family of secondary plant metabolites is widely distributed in several foods and beverages, in particular in red wine, coffee, tea, fruits, vegetables, and cereals [5]. Until recently, polyphenols principally have been recognized as health-promoting dietary components owing to their antioxidant activity, but currently they are thought to be involved in more complex physiologic mechanisms linked to the prevention of different pathologies [1,5]. Credibly, the bioactivity of these phytochemicals has been linked to their bioavailability and catabolism in humans [6] and, as a matter of fact, it is becoming clear that the biological effects should not be attributed to the native compounds present in foods, but rather to their * Corresponding author. Tel.: þ39-0521-903830; fax: þ39-0521-903832. E-mail address: [email protected] (D. Del Rio). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.06.005
metabolites of various origins gaining contact with the internal compartments [7]. Because of this increased awareness, in recent years, much effort has been devoted to understand polyphenol metabolism in humans, but a complete picture still has to be drawn. The small intestine was always considered a key tissue in polyphenol absorption and metabolism; however, although dietary polyphenols are indeed modified and absorbed through the small intestine before entering the systemic circulation, most are not efficiently absorbed, thus reaching the large intestine. Here, the human microbiome induces drastic transformations that are very different from those exerted by human intestinal and hepatic enzymes [8]. The human colon hosts a highly complex microbial ecosystem that interacts symbiotically with the host and operates as a bioreactor with a virtually unlimited metabolic potential [9]. The gut microbiota carries out chemical reactions to modify phenolic skeletons and allows the absorption of a range of lower-weight metabolites. Microbial enzymes can hydrolyze glycosides, glucuronides, sulfates, amides, esters, and lactones and operate ring-cleavage, reduction, decarboxylation, demethylation,
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and dehydroxylation reactions [10]. The bioavailability of polyphenols is starting to be reconsidered in light of these observations. As an example, chlorogenic acids and flavan-3-ols produce their unique spectra of colonic metabolites, which are excreted in urine in substantial amounts corresponding to 29% of intake after coffee and approximately 71% after green tea consumption [11]. Moreover, the biological effects of polyphenols are starting to be re-evaluated under the spotlight of microbial metabolism. As an example, some polyphenolic metabolites generated in vivo in the colon have been recently shown to counteract in vitro two key features of diabetic complications, namely protein glycation and neurodegeneration [12]. To gain more information on which molecules should be looked for in future bioavailability and exposure studies and investigated in experimental in vitro models of bioactivity, we planned this study to identify the major metabolites derived in vitro from microbial colonic fermentation of a range of different polyphenol rich-foods that contribute significantly to the intake of polyphenols in the common Western diet. Materials and methods Chemicals L-cysteine hydrochloride monohydrate and Fe(II)-sulfate heptahydrate were purchased from AppliChem (Darmstadt, Germany). Bile salts, calcium chloride, (þ)-arabinogalactan, tryptone, yeast extract, inulin, buffered peptone water, Dulbecco’s phosphate buffer saline, casein sodium salt from bovine milk, pectin from citrus fruits, mucin from porcine stomach-type III, xylan from Birchwood, sodium hydrogen carbonate, potassium hydrogen phosphate, magnesium sulfate monohydrate, guar gum, Tween 80, and resazurin redox indicator were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pure phenolic standards for high-performance liquid chromatographic (LC) and tandem mass spectrometric (MS/MS) analyses of phloroglucinol, pyrogallol, pyrocatechol, benzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2-hydroxybenzoic acid, ocoumaric acid, m-coumaric acid, p-coumaric acid, quinic acid, dihydrocaffeic acid, dihydroferulic acid, 3-hydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, syringic acid, gallic acid, and protocatechuic acid were purchased from Sigma-Aldrich. Anhydrous dipotassium hydrogen phosphate, soluble starch, 37% hydrochloric acid, methanol, 99% formic acid, and acetone were from Carlo Erba Reagents (Milan, Italy). Potassium chloride and sodium chloride were obtained from Merk (Darmstadt, Germany).
Samples Sixteen polyphenol rich foods were analyzed in this study: fresh strawberries, blueberries, raspberries, blackberries, Tarocco blood orange, onion, clarified apple juice, pomegranate juice, dark chocolate (90% cocoa), red wine (Merlot), oat bran, wheat bran, flaxseeds, black and green tea, and ground coffee. Sample pre-treatment For in vitro fermentation experiments, strawberry, blueberry, blackberry, raspberry, and Tarocco blood orange juices were obtained from fresh fruits. Each fruit sample was homogenized with a mixer (La Moulinette, Moulinex, Ecully Cedex, France) and centrifuged at 35 000 g (Sigma, 3K-30) for 10 min to separate the solid fraction. Raw onion was finely minced and oat bran, wheat bran, flaxseeds, and chocolate were milled with a grinder (La Moulinette, Moulinex) to a fine powder. Brewed black and green teas were prepared by adding boiled water to black and green tea bags and adding ascorbic acid and citric acid to tea brews to stabilize the polyphenols. Red wine was treated with a rotary evaporator (BÜCHI Rotavapor R-200, Flawil, Switzerland) to remove ethanol. Each sample was stored at 80 C until in vitro fermentation. For LC-MS/MS analysis of food samples, supernatants obtained from centrifugation of fresh fruits (strawberries, raspberries, blueberries, blackberries, and Tarocco blood oranges) were acidified with formic acid (1%, v/v). Polyphenol extraction from mashed onion was carried out with 1% (v/v) formic acid in methanol under sonication for 30 min and subsequent centrifugation. Oat bran, wheat bran, and flaxseed powders were hydrolyzed with 1.2 M HCl in a methanol/water (50%, v/v) solution at 90 C for 120 min. Black and green teas were acidified with formic acid (1%, v/v). Chocolate polyphenols were extracted with an acetone/water (70%, v/v) solution. Red wine and apple and pomegranate juices
were centrifuged at 10 000 g for 10 min (Sigma, 3K-30) and analyzed without additional treatment before injection. All samples were stored at 80 C until LCMS/MS analysis. In vitro fermentation growth medium preparation The composition for 1 L of growth medium was 5 g of soluble starch, 5 g of peptone, 5 g of tryptone, 4.5 g of yeast extract, 4.5 g of NaCl, 4.5 g of KCl, 2 g of pectin, 4 g of mucin, 3 g of casein, 2 g of arabinogalactan, 1.5 g of NaHCO3, 0.69 g of MgSO4$H2O, 1 g of guar, 0.8 g of L-cysteine HCl$H2O, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.4 g of bile salt, 0.08 g of CaCl2, 0.005 g of FeSO4$7H2O, 1 mL of Tween 80, and 4 mL of resazurin solution (0.025%, w/v) as an anaerobic indicator. The growth medium was sterilized at 121 C for 15 min in glass vessels (50 mL) before sample preparation. Fecal slurry Fresh fecal samples were collected from three healthy donors who did not have previous intestinal disease, were not treated with antibiotics for the previous 3 mo, and followed a polyphenol-free diet for 2 d before fecal collection. Samples were immediately stored in an anaerobic jar and then diluted with Dulbecco’s phosphate buffer saline at 10% (v/v) and homogenized to obtain a 20% (w/w) slurry to be used as the fermentation starter. Fermentation conditions The final fermentation volume was 22 mL: 45.5% growth medium, 45.5% fecal slurry, and 9% food sample extract. The fermentation starter was introduced in the vessel containing sterilized growth medium, sealed with a rubber seal, and flushed through a double needle with nitrogen to create an anaerobic condition. Samples were then introduced to the vessel through the needle and incubated for 24 h at 37 C at 200 strokes/min in a Dubnoff bath (ISCO, Milan, Italy). After 5 and 24 h of incubation, the fermentation samples were centrifuged, and 2 mL was filtered through a 0.2-mm nylon filter and stored at 80 C. All samples were fermented independently with each fermentation starter (derived from each donor) and all experiments were carried out in triplicate. This procedure generated nine chromatographic analyses for each food sample. High-performance LC/electrospray interface/MS/MS analyses Phenolic compounds originally derived phenolic metabolites were separation module equipped with a trometer fitted with an electrospray Separations were performed using reverse-phase column (Waters).
present in foods and the fermentationanalyzed using a Waters 2695 Alliance Micromass Quattro Micro Api mass specinterface (ESI; Waters, Milford, MA, USA). an Atlantis dC18-3 mm (2.1 150 mm)
Food samples An initial survey of the polyphenol content was carried out operating the MS in scan mode from 100 to 1000, 1500, or 2000 mass-to-charge ratio (m/z), depending on the food sample. The ESI source worked in negative ionization mode. The source temperature was 120 C, the desolvation temperature was 350 C, the capillary voltage was 2.8 kV, and the cone voltage was 35 V. Moreover, a second survey was carried out in positive ionization mode with the MS operating in scan mode from 100 to 1000 m/z to tentatively identify anthocyanins. The source temperature and desolvation temperature were always set to 120 C and 350 C, respectively, whereas capillary voltage was 3.5 kV and cone voltage was 38 V. The mobile phase, pumped at a flow rate of 0.17 mL/min, was a 40-min linear gradient of 5% to 40% acetonitrile in 1% aqueous formic acid. A specific multiple reaction monitoring (MRM) analysis was then developed for each sample based on the scan survey observations, with the ESI source working in negative mode. The collision energy used for MS/MS identifications was set at 25 eV. For anthocyanin-rich samples, a specific MRM analysis was developed with the source operating in positive ionization mode. In this case, the mobile phase, pumped at a flow rate of 0.17 mL/min, was a 30-min linear gradient of 5% to 35% acetonitrile in 1% aqueous formic acid and the collision energy used for MS/MS identifications was set at 20 eV. Fermented samples Fermented samples were initially analyzed using the mass spectrometer operating in scan mode, in negative ionization from 100 to 800 m/z. The mobile phase, pumped at a flow rate of 0.17 mL/min, was a 35-min linear gradient of 0% to 40% acetonitrile in 1% aqueous formic acid. After a first rough identification of the putative metabolites, a specific MRM method was developed for each sample and run with a 10-min linear gradient of 0% to 20% acetonitrile in 1% aqueous formic acid. The ESI source tuning was the same as described earlier, but the fragmentation was reached applying a collision energy equal to 25 or 30 eV,
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Table 1 Polyphenolic precursors and metabolites derived from in vitro colonic fermentation after 5 and 24 h of 16 food samples Food sample
Phenolic precursors*
Metabolitesy
5h
24 h
Raspberries
pelargonidin hexoside; cyanidin hexoside; cyanidin rutinoside; cyanidin sophoroside; cyanidin sambubioside rhamnoside; coumaroyl hexosides; caffeoylhexosides; salicylic acid. cyanidin pentoside; delphinidin pentoside; cyanidin hexoside; delphinidin hexoside; petunidin hexoside; malvidin hexoside; peonidin hexoside; catechin; epicatechin; coumaroylhexosides; coumaroylquinic acids; caffeoylhexosides; caffeylquinic acids; feruloyl hexosides; feruloylquinic acids; sinapic acid hexosides; procyanidin dimers b-type; salicylic acid
protocatechuic acid benzoic acid
D D
D D
gallic acid coumaric acid protocatechuic acid quinic acid dihydrocaffeic acid hydroxybenzoic acid protocatechuic acid
ND D D D D D D
D D D D D D D
gallic acid quinic acid (3,4-dihydroxyphenyl)acetic acid phloroglucinol dihydroferulic acid dihydrosinapic acid dihydroferulic acid dihydrosinapic acid protocatechuic acid dihydroferulic acid homovanillic acid enterodiol enterolactone dihydrocaffeic acid 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone (3,4-dihydroxyphenyl)acetic acid protocatechuic acid hydroxybenzoic acid salicylic acid dihydroferulic acid sinapic acid protocatechuic acid
D D D D D D D D D D ND ND D D D D D D D D D D
D D D D D D D D D D D D D D D D D D ND D D D
quinic acid 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone dihydrocaffeic acid protocatechuic acid gallic acid pyrogallol phlorogucinol syringic acid protocatechuic acid phlorogucinol pyrogallol coumaric acid gallic acid 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone 5-(30 -hydroxyphenyl)-g-valerolactone protocatechuic acid dihydrocaffeic acid homovanillic acid quinic acid caffeic acid dihydroferulic acid quinic acid dihydrocaffeic acid ferulic acid protocatechuic acid hydroxybenzoic acid phloroglucinol pyrogallol gallic acid 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone protocatechuic acid dihydrocaffeic acid 5-(30 -hydroxyphenyl)-g-valerolactone quinic acid
D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D ND D
D D ND D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D
Blueberries
Blackberries
Strawberries Onion Oat bran Wheat bran
pelargonidin hexoside; cyanidin hexoside; cyanidin malonylhexoside; cyanidin dioxaloylhexoside; cyanidin rutinoside; coumaroylhexoside; caffeoylhexoside; quercetin hexoside; quercetin glucuronide; isorhamnetin glucuronide; salicylic acid. pelargonidin hexoside; cyanidin hexoside; pelargonidin rutinoside; coumaroyl hexosides; caffeoyl hexosides; hydroxybenzoic acid; salicylic acid. quercetin; quercetin hexosides; isorhamnetin hexoside; quercetin dihexosides; isorhamnetin dihexoside ferulic acid; ferulic acid derivative; sinapic acid; avenanthramide A; avenanthramide B; salicylic acid coumaric acid; ferulic acid; ferulic acid derivative; sinapic acid; salicylic acid
Flaxseed
coumaric acids; ferulic acid; coumaroyl hexosides; caffeoyl hexosides; ferulic hexosides; secoisolariciresinol; secoisolariciresinol hexosides; secoisolariciresinol dihexosides; salicylic acid
Dark chocolate
epicatechin; catechin; procyanidin dimers b-type; procyanidin trimers b-type; procyanidin tetramer b-type
Orange juice
delphinidin hexoside; cyanidin malonyl hexoside; cyanidin dioxaloylhexoside; cyanidin hexoside; cyanidin sophoroside; delphinidin rutinoside; coumaroyl hexoside; feruloyl hexoside; sinapic acid hexoside; kaempferol hexoside; narirutin; didymin; eriocitrin; diosmin; hesperidin caffeic acid; catechin; epicatechin; coumaroylquinic acids; caffeoylquinic acids; phloretin hexoside; quercetin rhamnoside; quercetin hexosides; phloretin xylosyl hexoside; procyanidin dimers b-type; procyanidin trimers b-type; procyanidin tetramers b-type pelargonidin hexoside; cyanidin hexoside; delphinidin hexoside; cyanidin dihexoside; delphinidin dihexoside; gallic acid; ellagic acid; ellagic acid pentoside; ellagic acid rhamnoside; ellagic acid acetylpentoside; galloylhexahydroxydiphenoyl-hexose; punicalin; punicalagin a; punicalagin b
Apple juice
Pomegranate juice
Black tea
gallic acid; catechin; epicatechin; gallocatechin; epigallocatechin; coumaroylquinic acids; galloylquinic acid; caffeoylquinic acids; catechin gallate; epicatechin gallate; kaempferol hexosides; gallocatechin gallate; epigallocatechin gallate; quercetin hexosides; theaflavin; procyanidin dimers btype; kaempferol rutinoside; prodelphinidin dimers b-type; rutin; theaflavin gallate; theaflavin digallate; quercetin-rhamnose-hexose-rhamnose
Coffee
ferulic acid; coumaroylquinic acids; caffeoylquinic acids; feruloylquinic acids; dicaffeoylquinic acids; salicylic acid
Green tea
gallic acid; catechin; epicatechin; gallocatechin; epigallocatechin; coumaroylquinic acids; galloylquinic acid; caffeoylquinic acids; catechin gallate; epicatechin gallate; quercetin hexosides; kaempferol rutinoside; rutin
(Continued)
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Table 1 (Continued ) Food sample
Phenolic precursors*
Metabolitesy
5h
24 h
Red wine
cyanidin hexoside; peonidin hexoside; delphinidin hexoside; petunidin hexoside; malvidin hexoside; peonidin acetyl hexoside; malvidin hexoside acetaldehyde; petunidin acetyl hexosides; malvidin acetyl hexosides; malvidin hexoside pyruvate; cyanidin coumaroyl hexoside; delphinidin coumaroyl hexoside; petunidin coumaroyl hexoside; malvidin coumaroyl hexosides; malvidin caffeoyl hexosides; protocatechuic acid; coumaric acid; gallic acid; caffeic acid; cis–resveratrol; trans–resveratrol; epicatechin; catechin; trans– cutaric acid; quercetin; gallocatechin; epigallocatechin; trans–caftaric acid; trans–fertaric acid; cis–resveratrol hexoside; trans–resveratrol hexoside; astilbin; quercetin glucuronide; procyanidin dimers b-type; prodelphinidin dimers b-type; procyanidin trimers b-type
5-(30 ,40 -dihydroxyphenyl)-g-valerolactone 3-(3-hydroxyphenyl)propionic acid protocatechuic acid gallic acid pyrogallol phloroglucinol coumaric acid quinic acid
D D D D D D D D
D D D D D D D D
D, detectable; ND, not detectable * Native phenolic compounds were identified with high-performance liquid chromatographic/electrospray interface/tandem mass spectrometric analyses. y Metabolites detected or not detected with high-performance liquid chromatographic/electrospray interface/tandem mass spectrometric analyses in at least one of the three different fecal fermentations after 5 and 24 h. depending on the target metabolites. Additional Daughters scan analyses were performed on fermentation samples to confirm ambiguous identifications together with the injection of pure standards, when available. For all analyses, desolvation and cone gas (N2) were 750 and 50 L/h, respectively. For all MRM methods, the collision gas used was argon.
Table 2 Mass spectrometric characteristics of native phenolic compounds and their microbial metabolites identified in the study (negative ionization) Compound
Results Polyphenolic precursors and metabolites derived from in vitro microbial colonic fermentation of the 16 foods are summarized in Table 1. Each reported metabolite was detected in at least one of the three different fecal fermentations after 5 or 24 h of fermentation. Identification of the native phenolic compounds and metabolites was based on high-performance LC/ ESI/MS/MS analyses, and a summary of mass spectrometric specific molecular patterns in negative and positive ionization is presented in Tables 2 and 3, respectively. Among the compounds generated during in vitro fermentation, protocatechuic acid was the most relevant metabolite, because it was identified in several fermented samples (i.e., raspberries, blueberries, blackberries, flaxseeds, chocolate, orange juice, pomegranate juice, apple juice, coffee, red wine, black and green tea; Fig. 1). Quinic and gallic acids were also identified in several fermented samples and in food samples as native compounds. Dihydrocaffeic acid was mainly found in coffee (Fig. 2) but was also present in fermented black tea, green tea, and apple juice samples. Other interesting compounds generated after flaxseed microbial fermentation were enterolactone (Fig. 3) and enterodiol. Valerolactones, such as 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone, 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone (Fig. 4) and 5-(30 hydroxyphenyl)-g-valerolactone, were detected in chocolate, apple juice, black and green teas, and red wine fermented samples. Dihydrosinapic and dihydroferulic acids were principally detected in oat bran and wheat bran fermented samples, but dihydroferulic acid was also generated after fermentation of coffee and flaxseeds. Phloroglucinol was identified after onion, red wine, and green tea microbial colonic fermentation. Discussion This study was performed to identify the main molecules generated by the effect of human microbiota acting on different common classes of polyphenols. The polyphenolic profile of the analyzed food samples agrees with previous works [13–17]. As previously reported in the literature, protocatechuic acid was widely distributed as a fermentation metabolite and its abundant presence leads to several hypotheses. Vitaglione et al. [18] described this hydroxybenzoic acid as the major metabolite
[M-H] (m/z) Fragment ions (m/z)
Astilbin 449 Avenanthramide A 298 Avenanthramide B 328 Benzoic acid 121 Caffeic acid 179 Caffeoyl-hexosides 341 Caffeoylquinic acids 353 Trans–caftaric acid 311 Catechin 289 Catechin gallate 441 Coumaric acid 163 Coumaroyl hexosides 325 Coumaroylquinic acids 337 Trans-cutaric acid 295 Dicaffeoylquinic acids 515 Didymin 593 Dihydrocaffeic acid 181 (3,4-dihydroxyphenyl)acetic acid 167 0 0 207 5-(3 ,4 -dihydroxyphenyl)-gvalerolactone Dihydroferulic acid 195 Dihydrosinapic acid 225 Diosmin 607 Ellagic acid 301 Ellagic acid pentoside 433 Ellagic acid rhamnoside 447 Ellagic acid acetylpentoside 475 Enterodiol 301 Enterolactone 297 Epicatechin 289 Epicatechin gallate 441 Epigallocatechin 305 Epigallocatechin gallate 457 Eriocitrin 595 Trans–fertaric acid 325 Ferulic acid 193 Ferulic acid derivative d Feruloylquinic acids 367 Feruloyl hexosides 355 Gallic acid 169 Gallocatechin 305 Gallocatechin gallate 457 Galloylquinic acid 343 Galloyl-hexahydroxydiphenoyl-hexose 633 Hesperidin 609 Hydroxybenzoic acid 137 191 5-(30 -hydroxyphenyl)-g-valerolactone 3-(3-hydroxyphenyl)propionic acid 165 Isorhamnetin dihexoside 639 Isorhamnetin hexoside 477 Isorhamnetin glucuronide 491
179; 173; 179; 245;
163; 163; 173; 191; 59;
153; 229;
245;
193; 173; 191; 193;
315;
303 254 284 77 135 135 191 179 137 169 119 119 191 163 353 285 137 123 163 136 151 299 257 301 301 301 253 253 137 169 179 169 287 193 134 134 193 134 125 179 169 169 301 301 93 147 121 477 315 315
M. Dall’Asta et al. / Nutrition 28 (2012) 197–203 Table 2 (Continued ) Compound
[M-H] (m/z) Fragment ions (m/z)
Kaempferol hexoside 447 Kaempferol rutinoside 593 Homovanillic acid 181 Narirutin 579 Phloretin hexoside 435 Phloretin xylosyl hexoside 567 Phloroglucinol 125 Pyrogallol 125 Procyanidin dimers B-type 577 Procyanidin trimers B-type 865 Procyanidin tetramers B-type 1153 Punicalin 781 Prodelphinidin dimers B-type 593 Procyanidin trimers B-type 865 Protocatechuic acid 153 Punicalagin A 1083 Punicalagin B 1083 Quercetin 301 Quercetin dihexosides 625 Quercetin hexosides 463 Quercetin glucuronide 477 Quercetin rhamnoside 447 Quercetin-rhamnose-hexose-rhamnose 755 Quinic acid 191 Cis-resveratrol 227 Trans-resveratrol 227 Cis–resveratrol hexoside 389 Trans–resveratrol hexoside 389 Rutin 609 Salicylic acid 137 Sinapic acid 223 Sinapic acid hexosides 385 Syringic acid 197 Secoisolariciresinol 361 Secoisolariciresinol dihexosides 685 Secoisolariciresinol hexosides 523 Theaflavin 563 Theaflavin digallate 867 Theaflavin gallate 715 223 5-(30 ,40 ,50 -trihydroxyphenyl)-gvalerolactone
285 285 137 271 273 273 51 51; 41 289; 407 125; 289; 577 577; 865 601 289; 407 577 109 541; 781 541; 781 151 301; 463 301; 151 301 301 301; 609 85 159; 185 159; 185 227 227 301 93 149; 208 223 153; 182 165 361; 523 361; 165 137 715; 137 137 179
m/z, mass-to-charge ratio; [M-H], Negatively charged molecular ion
obtained from the biodegradation of cyanidine glycosides, the main anthocyanins identified in red wine, orange juice, and berries, with the only exception of strawberries. In fact, strawberries are rich in pelargonidin, which, according to the chemical structure with only one hydroxy group on the B ring, is unlikely to produce this phenolic acid. Conversely, the protocatechuic acid identified in chocolate, apple juice, and black and green tea fermented samples could derive from the decomposition of flavan-3-ols, as shown by Urpi-Sarda et al. [19] who demonstrated that this acid is the main metabolite found in rat urine after consumption of a flavan-3-ol–rich chocolate. Another hypothesis on protocatechuic acid formation is based on the conversion of chlorogenic, caffeic, and ferulic acids, which were detected in coffee and flaxseed samples [20]. Other widely distributed compounds identified after fermentation were quinic and gallic acids. Probably these two molecules were the product of microbial esterases: quinic acid likely was released through the hydrolysis of chlorogenic acids and galloylquinic acid, whereas gallic acid could derive from degalloylation of flavan-3-ols in green tea, galloylquinic acid and theaflavin gallates in black tea, or the release from gallotannins in pomegranate. Dihydrocaffeic acid was detected after coffee, black tea, and green tea and apple juice fermentation and likely originated
201
Table 3 Mass spectrometric characteristics of anthocyanins identified in the study (positive ionization) Compound
[M]þ (m/z)
Cyanidin coumaroyl hexoside Cyanidin dihexoside Cyanidin dioxaloylhexoside Cyanidin hexoside Cyanidin malonyl hexoside Cyanidin pentoside Cyanidin rutinoside Cyanidin sambubioside rhamnoside Cyanidin sophoroside Delphinidin coumaroyl hexoside Delphinidin dihexoside Delphinidin hexoside Delphinidin pentoside Delphinidin rutinoside Malvidin acetyl hexosides Malvidin hexoside Malvidin hexoside acetaldehyde Malvidin hexoside pyruvate Pelargonidin hexoside Pelargonidin rutinoside Peonidin acetyl hexoside Peonidin hexoside Petunidin acetyl hexosides Petunidin hexoside Petunidin coumaroyl hexoside
595 611 593 449 535 419 595 727 611 611 627 465 435 611 535 493 517 561 433 579 505 463 521 479 625
Fragment ions (m/z)
287; 287; 287; 433;
449;
287;
287 287 287 287 449 287 449 581 287 303 303 303 303 303 331 331 355 399 271 433 301 301 317 317 317
m/z, mass-to-charge ratio; [M]þ, Positively charged molecular ion
from caffeoyl- and dicaffeoyl-quinic acid precursors [21]. Enterolactone and enterodiol identified from flaxseed microbial fermentation were previously described by Wang [22] as the major metabolites of lignans, which are distinctive polyphenols in flaxseeds. Valerolactones such as 5-(30 ,40 -dihydroxyphenyl)g-valerolactone, 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone, and 5-(30 -hydroxyphenyl)-g-valerolactone were previously studied as typical products of monomeric and oligomeric flavan-3-ols microbial metabolism [23,24]. Two valerolactones were identified after 5 and 24 h of fermentation of black and green tea samples, whereas 5-(30 -hydroxyphenyl)-g-valerolactone was present after 24 h only, indicating that dehydroxylation could be a late metabolic step of flavan-3-ols degradation [24]. Dihydrosinapic and dihydroferulic acids were the characteristic metabolites of oat and wheat bran fermentation and likely derived from the dehydrogenation of native phenolic acids. Phloroglucinol was previously identified as a transient intermediate in the breakdown of quercetin [8], a molecule largely present in onion, red wine, and black and green teas. Interestingly, urolithins were previously identified as biomarkers of pomegranate juice consumption, being microbial products of ellagitannins and ellagic acid [25]. However, these molecules were not identified in the present study. This could be
Fig. 1. Multiple reaction monitoring chromatogram of protocatechuic acid detected in fermented raspberry sample after 5 h.
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Fig. 4. Multiple reaction monitoring chromatograms of 5-(3,40 -dihydroxyphenyl)g-valerolactone (M6) in (A) 5-h and (B) 24-h fermented chocolate samples. Fig. 2. Mass spectrum comparison between dihydrocaffeic acid in (A) fermented coffee sample and (B) pure standard.
explained by the extreme individual variability in urolithin production [25] or by the fact that their precursor polyphenols need to undergo specific modifications in the small intestine to interact subsequently with the colonic microbiota. According to the interindividual variability hypothesis, a recent study on the in vitro colonic bioconversion of polyphenols from black tea, red wine, and grape juice showed remarkable differences in the production of metabolites owing to microbial (and therefore enzymatic) differences in different gut microbiota [26]. The metabolites identified in the present in vitro study also are likely to be generated in the human colon after consumption of the foods analyzed, being formed by the action of bacteria of human fecal origin. It has recently been reported that, in a fermentation experiment similar to the one applied in our study, polyphenols from apple juice manufactured products able to inhibit histone deacetylase [27]. These products acted in an additive or a synergistic manner together with butyrate in exerting this action, which can induce differentiation and lead to apoptosis of precancerous and tumor cells. Because polyphenols added to fermentable dietary fiber did not eliminate in vivo the positive effects of the latter, increased the cecal butyrate pool, and increased the ring fission of rutin, hyperoside (quercetin 3O-galactoside), and quercitrin (quercetin 3-O-rhamnoside) in the colon [28], this led to the hypothesis that phenolic metabolites also can act to prevent colon cancer. The search for these microbial metabolites in vivo after a proper feeding study should consider their possible further conjugation at the level of the liver. In fact, when absorbed in the colon, microbial metabolites are transported by the portal circulation to the liver, where they may undergo phase II enzyme action to generate a wide pattern of possible circulating metabolites. These microbial metabolites are generated hours after
Fig. 3. Multiple reaction monitoring chromatogram of enterolactone from flaxseed microbial colonic fermentation.
food intake and are, therefore, good candidates for exposure studies, which usually evaluate biological fluids (urine in particular) in fasting conditions. A limitation of the study that should be considered is that, in vivo, the selected food samples would have undergone several digestive steps before entering the large intestine and being fermented, and these steps were not reproduced in our in vitro system. In fact, if the action of digestive enzymes per se could not change the structure of the investigated phenolic compounds, it is not known from the literature if their action on the food matrix could influence their physical accessibility for microbial fermentation. Moreover, a relevant fraction of the ingested polyphenols interacts in vivo with conjugating phase II enzymes at the small intestinal level [7] and, therefore, most phenolics reach the colon as methyl, glucuronide, or sulfate derivatives. This aspect could modify the fermentation process to an extent that was not measurable in the present work.
Conclusion Having targeted systematically the entire set of putative metabolites derived from the polyphenol fermentation of a range of common foods, this work brings new insights to better evaluate the bioavailability and putative preventive activity of polyphenols from specific food groups in humans. Moreover, based on these observations, some of the identified metabolites could also be used to evaluate exposure to specific foods or food groups in epidemiologic studies.
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Basic nutritional investigation
Identification of microbial metabolites derived from in vitro fecal fermentation of different polyphenolic food sources Margherita Dall’Asta M.Sc., Luca Calani M.Sc., Marianna Tedeschi M.Sc., Lucia Jechiu M.Sc., Furio Brighenti Ph.D., Daniele Del Rio Ph.D. * The 42 Laboratory of Phytochemicals in Physiology, Human Nutrition Unit, Department of Public Health, University of Parma, Via Volturno, Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 March 2011 Accepted 7 June 2011
Objective: The biological effects of dietary polyphenols are linked to their bioavailability and catabolism in humans. The colon, with its symbiotic microbiota, is an active site where complex polyphenolic compounds are possibly modified to smaller and more absorbable molecules. The aim of this study was to identify the major metabolites derived from microbial colonic fermentation of some common polyphenol-rich foods. Methods: An in vitro fecal fermentation model was applied to 16 polyphenol-rich foods and polyphenolic precursors. Phenolic metabolites were identified by high-performance liquid chromatography coupled with tandem mass spectrometric detection. Results: Twenty-four phenolic fermentation metabolites were characterized. Some metabolites were common to several polyphenol-rich foods, whereas others were characteristic of specific sources. Conclusion: The metabolites identified in vitro likely are generated in the human colon after consumption of polyphenol-rich foods. Their occurrence in plasma and/or urine should be considered when evaluating the bioavailability of polyphenols from specific food groups in humans and in the definition of markers of exposure to specific foods or food groups in epidemiologic studies. However, the search for these and other microbial metabolites after a feeding study in vivo should consider their possible further conjugation at the level of the liver. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Polyphenols Colonic fermentation Microbial metabolites Tandem mass spectrometry
Introduction The increasing interest in polyphenols has been triggered by hundreds of studies that have associated polyphenol-rich diets to several health effects in humans [1–4]. This large family of secondary plant metabolites is widely distributed in several foods and beverages, in particular in red wine, coffee, tea, fruits, vegetables, and cereals [5]. Until recently, polyphenols principally have been recognized as health-promoting dietary components owing to their antioxidant activity, but currently they are thought to be involved in more complex physiologic mechanisms linked to the prevention of different pathologies [1,5]. Credibly, the bioactivity of these phytochemicals has been linked to their bioavailability and catabolism in humans [6] and, as a matter of fact, it is becoming clear that the biological effects should not be attributed to the native compounds present in foods, but rather to their * Corresponding author. Tel.: þ39-0521-903830; fax: þ39-0521-903832. E-mail address: [email protected] (D. Del Rio). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.06.005
metabolites of various origins gaining contact with the internal compartments [7]. Because of this increased awareness, in recent years, much effort has been devoted to understand polyphenol metabolism in humans, but a complete picture still has to be drawn. The small intestine was always considered a key tissue in polyphenol absorption and metabolism; however, although dietary polyphenols are indeed modified and absorbed through the small intestine before entering the systemic circulation, most are not efficiently absorbed, thus reaching the large intestine. Here, the human microbiome induces drastic transformations that are very different from those exerted by human intestinal and hepatic enzymes [8]. The human colon hosts a highly complex microbial ecosystem that interacts symbiotically with the host and operates as a bioreactor with a virtually unlimited metabolic potential [9]. The gut microbiota carries out chemical reactions to modify phenolic skeletons and allows the absorption of a range of lower-weight metabolites. Microbial enzymes can hydrolyze glycosides, glucuronides, sulfates, amides, esters, and lactones and operate ring-cleavage, reduction, decarboxylation, demethylation,
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and dehydroxylation reactions [10]. The bioavailability of polyphenols is starting to be reconsidered in light of these observations. As an example, chlorogenic acids and flavan-3-ols produce their unique spectra of colonic metabolites, which are excreted in urine in substantial amounts corresponding to 29% of intake after coffee and approximately 71% after green tea consumption [11]. Moreover, the biological effects of polyphenols are starting to be re-evaluated under the spotlight of microbial metabolism. As an example, some polyphenolic metabolites generated in vivo in the colon have been recently shown to counteract in vitro two key features of diabetic complications, namely protein glycation and neurodegeneration [12]. To gain more information on which molecules should be looked for in future bioavailability and exposure studies and investigated in experimental in vitro models of bioactivity, we planned this study to identify the major metabolites derived in vitro from microbial colonic fermentation of a range of different polyphenol rich-foods that contribute significantly to the intake of polyphenols in the common Western diet. Materials and methods Chemicals L-cysteine hydrochloride monohydrate and Fe(II)-sulfate heptahydrate were purchased from AppliChem (Darmstadt, Germany). Bile salts, calcium chloride, (þ)-arabinogalactan, tryptone, yeast extract, inulin, buffered peptone water, Dulbecco’s phosphate buffer saline, casein sodium salt from bovine milk, pectin from citrus fruits, mucin from porcine stomach-type III, xylan from Birchwood, sodium hydrogen carbonate, potassium hydrogen phosphate, magnesium sulfate monohydrate, guar gum, Tween 80, and resazurin redox indicator were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pure phenolic standards for high-performance liquid chromatographic (LC) and tandem mass spectrometric (MS/MS) analyses of phloroglucinol, pyrogallol, pyrocatechol, benzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2-hydroxybenzoic acid, ocoumaric acid, m-coumaric acid, p-coumaric acid, quinic acid, dihydrocaffeic acid, dihydroferulic acid, 3-hydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, syringic acid, gallic acid, and protocatechuic acid were purchased from Sigma-Aldrich. Anhydrous dipotassium hydrogen phosphate, soluble starch, 37% hydrochloric acid, methanol, 99% formic acid, and acetone were from Carlo Erba Reagents (Milan, Italy). Potassium chloride and sodium chloride were obtained from Merk (Darmstadt, Germany).
Samples Sixteen polyphenol rich foods were analyzed in this study: fresh strawberries, blueberries, raspberries, blackberries, Tarocco blood orange, onion, clarified apple juice, pomegranate juice, dark chocolate (90% cocoa), red wine (Merlot), oat bran, wheat bran, flaxseeds, black and green tea, and ground coffee. Sample pre-treatment For in vitro fermentation experiments, strawberry, blueberry, blackberry, raspberry, and Tarocco blood orange juices were obtained from fresh fruits. Each fruit sample was homogenized with a mixer (La Moulinette, Moulinex, Ecully Cedex, France) and centrifuged at 35 000 g (Sigma, 3K-30) for 10 min to separate the solid fraction. Raw onion was finely minced and oat bran, wheat bran, flaxseeds, and chocolate were milled with a grinder (La Moulinette, Moulinex) to a fine powder. Brewed black and green teas were prepared by adding boiled water to black and green tea bags and adding ascorbic acid and citric acid to tea brews to stabilize the polyphenols. Red wine was treated with a rotary evaporator (BÜCHI Rotavapor R-200, Flawil, Switzerland) to remove ethanol. Each sample was stored at 80 C until in vitro fermentation. For LC-MS/MS analysis of food samples, supernatants obtained from centrifugation of fresh fruits (strawberries, raspberries, blueberries, blackberries, and Tarocco blood oranges) were acidified with formic acid (1%, v/v). Polyphenol extraction from mashed onion was carried out with 1% (v/v) formic acid in methanol under sonication for 30 min and subsequent centrifugation. Oat bran, wheat bran, and flaxseed powders were hydrolyzed with 1.2 M HCl in a methanol/water (50%, v/v) solution at 90 C for 120 min. Black and green teas were acidified with formic acid (1%, v/v). Chocolate polyphenols were extracted with an acetone/water (70%, v/v) solution. Red wine and apple and pomegranate juices
were centrifuged at 10 000 g for 10 min (Sigma, 3K-30) and analyzed without additional treatment before injection. All samples were stored at 80 C until LCMS/MS analysis. In vitro fermentation growth medium preparation The composition for 1 L of growth medium was 5 g of soluble starch, 5 g of peptone, 5 g of tryptone, 4.5 g of yeast extract, 4.5 g of NaCl, 4.5 g of KCl, 2 g of pectin, 4 g of mucin, 3 g of casein, 2 g of arabinogalactan, 1.5 g of NaHCO3, 0.69 g of MgSO4$H2O, 1 g of guar, 0.8 g of L-cysteine HCl$H2O, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.4 g of bile salt, 0.08 g of CaCl2, 0.005 g of FeSO4$7H2O, 1 mL of Tween 80, and 4 mL of resazurin solution (0.025%, w/v) as an anaerobic indicator. The growth medium was sterilized at 121 C for 15 min in glass vessels (50 mL) before sample preparation. Fecal slurry Fresh fecal samples were collected from three healthy donors who did not have previous intestinal disease, were not treated with antibiotics for the previous 3 mo, and followed a polyphenol-free diet for 2 d before fecal collection. Samples were immediately stored in an anaerobic jar and then diluted with Dulbecco’s phosphate buffer saline at 10% (v/v) and homogenized to obtain a 20% (w/w) slurry to be used as the fermentation starter. Fermentation conditions The final fermentation volume was 22 mL: 45.5% growth medium, 45.5% fecal slurry, and 9% food sample extract. The fermentation starter was introduced in the vessel containing sterilized growth medium, sealed with a rubber seal, and flushed through a double needle with nitrogen to create an anaerobic condition. Samples were then introduced to the vessel through the needle and incubated for 24 h at 37 C at 200 strokes/min in a Dubnoff bath (ISCO, Milan, Italy). After 5 and 24 h of incubation, the fermentation samples were centrifuged, and 2 mL was filtered through a 0.2-mm nylon filter and stored at 80 C. All samples were fermented independently with each fermentation starter (derived from each donor) and all experiments were carried out in triplicate. This procedure generated nine chromatographic analyses for each food sample. High-performance LC/electrospray interface/MS/MS analyses Phenolic compounds originally derived phenolic metabolites were separation module equipped with a trometer fitted with an electrospray Separations were performed using reverse-phase column (Waters).
present in foods and the fermentationanalyzed using a Waters 2695 Alliance Micromass Quattro Micro Api mass specinterface (ESI; Waters, Milford, MA, USA). an Atlantis dC18-3 mm (2.1 150 mm)
Food samples An initial survey of the polyphenol content was carried out operating the MS in scan mode from 100 to 1000, 1500, or 2000 mass-to-charge ratio (m/z), depending on the food sample. The ESI source worked in negative ionization mode. The source temperature was 120 C, the desolvation temperature was 350 C, the capillary voltage was 2.8 kV, and the cone voltage was 35 V. Moreover, a second survey was carried out in positive ionization mode with the MS operating in scan mode from 100 to 1000 m/z to tentatively identify anthocyanins. The source temperature and desolvation temperature were always set to 120 C and 350 C, respectively, whereas capillary voltage was 3.5 kV and cone voltage was 38 V. The mobile phase, pumped at a flow rate of 0.17 mL/min, was a 40-min linear gradient of 5% to 40% acetonitrile in 1% aqueous formic acid. A specific multiple reaction monitoring (MRM) analysis was then developed for each sample based on the scan survey observations, with the ESI source working in negative mode. The collision energy used for MS/MS identifications was set at 25 eV. For anthocyanin-rich samples, a specific MRM analysis was developed with the source operating in positive ionization mode. In this case, the mobile phase, pumped at a flow rate of 0.17 mL/min, was a 30-min linear gradient of 5% to 35% acetonitrile in 1% aqueous formic acid and the collision energy used for MS/MS identifications was set at 20 eV. Fermented samples Fermented samples were initially analyzed using the mass spectrometer operating in scan mode, in negative ionization from 100 to 800 m/z. The mobile phase, pumped at a flow rate of 0.17 mL/min, was a 35-min linear gradient of 0% to 40% acetonitrile in 1% aqueous formic acid. After a first rough identification of the putative metabolites, a specific MRM method was developed for each sample and run with a 10-min linear gradient of 0% to 20% acetonitrile in 1% aqueous formic acid. The ESI source tuning was the same as described earlier, but the fragmentation was reached applying a collision energy equal to 25 or 30 eV,
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Table 1 Polyphenolic precursors and metabolites derived from in vitro colonic fermentation after 5 and 24 h of 16 food samples Food sample
Phenolic precursors*
Metabolitesy
5h
24 h
Raspberries
pelargonidin hexoside; cyanidin hexoside; cyanidin rutinoside; cyanidin sophoroside; cyanidin sambubioside rhamnoside; coumaroyl hexosides; caffeoylhexosides; salicylic acid. cyanidin pentoside; delphinidin pentoside; cyanidin hexoside; delphinidin hexoside; petunidin hexoside; malvidin hexoside; peonidin hexoside; catechin; epicatechin; coumaroylhexosides; coumaroylquinic acids; caffeoylhexosides; caffeylquinic acids; feruloyl hexosides; feruloylquinic acids; sinapic acid hexosides; procyanidin dimers b-type; salicylic acid
protocatechuic acid benzoic acid
D D
D D
gallic acid coumaric acid protocatechuic acid quinic acid dihydrocaffeic acid hydroxybenzoic acid protocatechuic acid
ND D D D D D D
D D D D D D D
gallic acid quinic acid (3,4-dihydroxyphenyl)acetic acid phloroglucinol dihydroferulic acid dihydrosinapic acid dihydroferulic acid dihydrosinapic acid protocatechuic acid dihydroferulic acid homovanillic acid enterodiol enterolactone dihydrocaffeic acid 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone (3,4-dihydroxyphenyl)acetic acid protocatechuic acid hydroxybenzoic acid salicylic acid dihydroferulic acid sinapic acid protocatechuic acid
D D D D D D D D D D ND ND D D D D D D D D D D
D D D D D D D D D D D D D D D D D D ND D D D
quinic acid 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone dihydrocaffeic acid protocatechuic acid gallic acid pyrogallol phlorogucinol syringic acid protocatechuic acid phlorogucinol pyrogallol coumaric acid gallic acid 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone 5-(30 -hydroxyphenyl)-g-valerolactone protocatechuic acid dihydrocaffeic acid homovanillic acid quinic acid caffeic acid dihydroferulic acid quinic acid dihydrocaffeic acid ferulic acid protocatechuic acid hydroxybenzoic acid phloroglucinol pyrogallol gallic acid 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone protocatechuic acid dihydrocaffeic acid 5-(30 -hydroxyphenyl)-g-valerolactone quinic acid
D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D ND D
D D ND D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D
Blueberries
Blackberries
Strawberries Onion Oat bran Wheat bran
pelargonidin hexoside; cyanidin hexoside; cyanidin malonylhexoside; cyanidin dioxaloylhexoside; cyanidin rutinoside; coumaroylhexoside; caffeoylhexoside; quercetin hexoside; quercetin glucuronide; isorhamnetin glucuronide; salicylic acid. pelargonidin hexoside; cyanidin hexoside; pelargonidin rutinoside; coumaroyl hexosides; caffeoyl hexosides; hydroxybenzoic acid; salicylic acid. quercetin; quercetin hexosides; isorhamnetin hexoside; quercetin dihexosides; isorhamnetin dihexoside ferulic acid; ferulic acid derivative; sinapic acid; avenanthramide A; avenanthramide B; salicylic acid coumaric acid; ferulic acid; ferulic acid derivative; sinapic acid; salicylic acid
Flaxseed
coumaric acids; ferulic acid; coumaroyl hexosides; caffeoyl hexosides; ferulic hexosides; secoisolariciresinol; secoisolariciresinol hexosides; secoisolariciresinol dihexosides; salicylic acid
Dark chocolate
epicatechin; catechin; procyanidin dimers b-type; procyanidin trimers b-type; procyanidin tetramer b-type
Orange juice
delphinidin hexoside; cyanidin malonyl hexoside; cyanidin dioxaloylhexoside; cyanidin hexoside; cyanidin sophoroside; delphinidin rutinoside; coumaroyl hexoside; feruloyl hexoside; sinapic acid hexoside; kaempferol hexoside; narirutin; didymin; eriocitrin; diosmin; hesperidin caffeic acid; catechin; epicatechin; coumaroylquinic acids; caffeoylquinic acids; phloretin hexoside; quercetin rhamnoside; quercetin hexosides; phloretin xylosyl hexoside; procyanidin dimers b-type; procyanidin trimers b-type; procyanidin tetramers b-type pelargonidin hexoside; cyanidin hexoside; delphinidin hexoside; cyanidin dihexoside; delphinidin dihexoside; gallic acid; ellagic acid; ellagic acid pentoside; ellagic acid rhamnoside; ellagic acid acetylpentoside; galloylhexahydroxydiphenoyl-hexose; punicalin; punicalagin a; punicalagin b
Apple juice
Pomegranate juice
Black tea
gallic acid; catechin; epicatechin; gallocatechin; epigallocatechin; coumaroylquinic acids; galloylquinic acid; caffeoylquinic acids; catechin gallate; epicatechin gallate; kaempferol hexosides; gallocatechin gallate; epigallocatechin gallate; quercetin hexosides; theaflavin; procyanidin dimers btype; kaempferol rutinoside; prodelphinidin dimers b-type; rutin; theaflavin gallate; theaflavin digallate; quercetin-rhamnose-hexose-rhamnose
Coffee
ferulic acid; coumaroylquinic acids; caffeoylquinic acids; feruloylquinic acids; dicaffeoylquinic acids; salicylic acid
Green tea
gallic acid; catechin; epicatechin; gallocatechin; epigallocatechin; coumaroylquinic acids; galloylquinic acid; caffeoylquinic acids; catechin gallate; epicatechin gallate; quercetin hexosides; kaempferol rutinoside; rutin
(Continued)
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Table 1 (Continued ) Food sample
Phenolic precursors*
Metabolitesy
5h
24 h
Red wine
cyanidin hexoside; peonidin hexoside; delphinidin hexoside; petunidin hexoside; malvidin hexoside; peonidin acetyl hexoside; malvidin hexoside acetaldehyde; petunidin acetyl hexosides; malvidin acetyl hexosides; malvidin hexoside pyruvate; cyanidin coumaroyl hexoside; delphinidin coumaroyl hexoside; petunidin coumaroyl hexoside; malvidin coumaroyl hexosides; malvidin caffeoyl hexosides; protocatechuic acid; coumaric acid; gallic acid; caffeic acid; cis–resveratrol; trans–resveratrol; epicatechin; catechin; trans– cutaric acid; quercetin; gallocatechin; epigallocatechin; trans–caftaric acid; trans–fertaric acid; cis–resveratrol hexoside; trans–resveratrol hexoside; astilbin; quercetin glucuronide; procyanidin dimers b-type; prodelphinidin dimers b-type; procyanidin trimers b-type
5-(30 ,40 -dihydroxyphenyl)-g-valerolactone 3-(3-hydroxyphenyl)propionic acid protocatechuic acid gallic acid pyrogallol phloroglucinol coumaric acid quinic acid
D D D D D D D D
D D D D D D D D
D, detectable; ND, not detectable * Native phenolic compounds were identified with high-performance liquid chromatographic/electrospray interface/tandem mass spectrometric analyses. y Metabolites detected or not detected with high-performance liquid chromatographic/electrospray interface/tandem mass spectrometric analyses in at least one of the three different fecal fermentations after 5 and 24 h. depending on the target metabolites. Additional Daughters scan analyses were performed on fermentation samples to confirm ambiguous identifications together with the injection of pure standards, when available. For all analyses, desolvation and cone gas (N2) were 750 and 50 L/h, respectively. For all MRM methods, the collision gas used was argon.
Table 2 Mass spectrometric characteristics of native phenolic compounds and their microbial metabolites identified in the study (negative ionization) Compound
Results Polyphenolic precursors and metabolites derived from in vitro microbial colonic fermentation of the 16 foods are summarized in Table 1. Each reported metabolite was detected in at least one of the three different fecal fermentations after 5 or 24 h of fermentation. Identification of the native phenolic compounds and metabolites was based on high-performance LC/ ESI/MS/MS analyses, and a summary of mass spectrometric specific molecular patterns in negative and positive ionization is presented in Tables 2 and 3, respectively. Among the compounds generated during in vitro fermentation, protocatechuic acid was the most relevant metabolite, because it was identified in several fermented samples (i.e., raspberries, blueberries, blackberries, flaxseeds, chocolate, orange juice, pomegranate juice, apple juice, coffee, red wine, black and green tea; Fig. 1). Quinic and gallic acids were also identified in several fermented samples and in food samples as native compounds. Dihydrocaffeic acid was mainly found in coffee (Fig. 2) but was also present in fermented black tea, green tea, and apple juice samples. Other interesting compounds generated after flaxseed microbial fermentation were enterolactone (Fig. 3) and enterodiol. Valerolactones, such as 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone, 5-(30 ,40 -dihydroxyphenyl)-g-valerolactone (Fig. 4) and 5-(30 hydroxyphenyl)-g-valerolactone, were detected in chocolate, apple juice, black and green teas, and red wine fermented samples. Dihydrosinapic and dihydroferulic acids were principally detected in oat bran and wheat bran fermented samples, but dihydroferulic acid was also generated after fermentation of coffee and flaxseeds. Phloroglucinol was identified after onion, red wine, and green tea microbial colonic fermentation. Discussion This study was performed to identify the main molecules generated by the effect of human microbiota acting on different common classes of polyphenols. The polyphenolic profile of the analyzed food samples agrees with previous works [13–17]. As previously reported in the literature, protocatechuic acid was widely distributed as a fermentation metabolite and its abundant presence leads to several hypotheses. Vitaglione et al. [18] described this hydroxybenzoic acid as the major metabolite
[M-H] (m/z) Fragment ions (m/z)
Astilbin 449 Avenanthramide A 298 Avenanthramide B 328 Benzoic acid 121 Caffeic acid 179 Caffeoyl-hexosides 341 Caffeoylquinic acids 353 Trans–caftaric acid 311 Catechin 289 Catechin gallate 441 Coumaric acid 163 Coumaroyl hexosides 325 Coumaroylquinic acids 337 Trans-cutaric acid 295 Dicaffeoylquinic acids 515 Didymin 593 Dihydrocaffeic acid 181 (3,4-dihydroxyphenyl)acetic acid 167 0 0 207 5-(3 ,4 -dihydroxyphenyl)-gvalerolactone Dihydroferulic acid 195 Dihydrosinapic acid 225 Diosmin 607 Ellagic acid 301 Ellagic acid pentoside 433 Ellagic acid rhamnoside 447 Ellagic acid acetylpentoside 475 Enterodiol 301 Enterolactone 297 Epicatechin 289 Epicatechin gallate 441 Epigallocatechin 305 Epigallocatechin gallate 457 Eriocitrin 595 Trans–fertaric acid 325 Ferulic acid 193 Ferulic acid derivative d Feruloylquinic acids 367 Feruloyl hexosides 355 Gallic acid 169 Gallocatechin 305 Gallocatechin gallate 457 Galloylquinic acid 343 Galloyl-hexahydroxydiphenoyl-hexose 633 Hesperidin 609 Hydroxybenzoic acid 137 191 5-(30 -hydroxyphenyl)-g-valerolactone 3-(3-hydroxyphenyl)propionic acid 165 Isorhamnetin dihexoside 639 Isorhamnetin hexoside 477 Isorhamnetin glucuronide 491
179; 173; 179; 245;
163; 163; 173; 191; 59;
153; 229;
245;
193; 173; 191; 193;
315;
303 254 284 77 135 135 191 179 137 169 119 119 191 163 353 285 137 123 163 136 151 299 257 301 301 301 253 253 137 169 179 169 287 193 134 134 193 134 125 179 169 169 301 301 93 147 121 477 315 315
M. Dall’Asta et al. / Nutrition 28 (2012) 197–203 Table 2 (Continued ) Compound
[M-H] (m/z) Fragment ions (m/z)
Kaempferol hexoside 447 Kaempferol rutinoside 593 Homovanillic acid 181 Narirutin 579 Phloretin hexoside 435 Phloretin xylosyl hexoside 567 Phloroglucinol 125 Pyrogallol 125 Procyanidin dimers B-type 577 Procyanidin trimers B-type 865 Procyanidin tetramers B-type 1153 Punicalin 781 Prodelphinidin dimers B-type 593 Procyanidin trimers B-type 865 Protocatechuic acid 153 Punicalagin A 1083 Punicalagin B 1083 Quercetin 301 Quercetin dihexosides 625 Quercetin hexosides 463 Quercetin glucuronide 477 Quercetin rhamnoside 447 Quercetin-rhamnose-hexose-rhamnose 755 Quinic acid 191 Cis-resveratrol 227 Trans-resveratrol 227 Cis–resveratrol hexoside 389 Trans–resveratrol hexoside 389 Rutin 609 Salicylic acid 137 Sinapic acid 223 Sinapic acid hexosides 385 Syringic acid 197 Secoisolariciresinol 361 Secoisolariciresinol dihexosides 685 Secoisolariciresinol hexosides 523 Theaflavin 563 Theaflavin digallate 867 Theaflavin gallate 715 223 5-(30 ,40 ,50 -trihydroxyphenyl)-gvalerolactone
285 285 137 271 273 273 51 51; 41 289; 407 125; 289; 577 577; 865 601 289; 407 577 109 541; 781 541; 781 151 301; 463 301; 151 301 301 301; 609 85 159; 185 159; 185 227 227 301 93 149; 208 223 153; 182 165 361; 523 361; 165 137 715; 137 137 179
m/z, mass-to-charge ratio; [M-H], Negatively charged molecular ion
obtained from the biodegradation of cyanidine glycosides, the main anthocyanins identified in red wine, orange juice, and berries, with the only exception of strawberries. In fact, strawberries are rich in pelargonidin, which, according to the chemical structure with only one hydroxy group on the B ring, is unlikely to produce this phenolic acid. Conversely, the protocatechuic acid identified in chocolate, apple juice, and black and green tea fermented samples could derive from the decomposition of flavan-3-ols, as shown by Urpi-Sarda et al. [19] who demonstrated that this acid is the main metabolite found in rat urine after consumption of a flavan-3-ol–rich chocolate. Another hypothesis on protocatechuic acid formation is based on the conversion of chlorogenic, caffeic, and ferulic acids, which were detected in coffee and flaxseed samples [20]. Other widely distributed compounds identified after fermentation were quinic and gallic acids. Probably these two molecules were the product of microbial esterases: quinic acid likely was released through the hydrolysis of chlorogenic acids and galloylquinic acid, whereas gallic acid could derive from degalloylation of flavan-3-ols in green tea, galloylquinic acid and theaflavin gallates in black tea, or the release from gallotannins in pomegranate. Dihydrocaffeic acid was detected after coffee, black tea, and green tea and apple juice fermentation and likely originated
201
Table 3 Mass spectrometric characteristics of anthocyanins identified in the study (positive ionization) Compound
[M]þ (m/z)
Cyanidin coumaroyl hexoside Cyanidin dihexoside Cyanidin dioxaloylhexoside Cyanidin hexoside Cyanidin malonyl hexoside Cyanidin pentoside Cyanidin rutinoside Cyanidin sambubioside rhamnoside Cyanidin sophoroside Delphinidin coumaroyl hexoside Delphinidin dihexoside Delphinidin hexoside Delphinidin pentoside Delphinidin rutinoside Malvidin acetyl hexosides Malvidin hexoside Malvidin hexoside acetaldehyde Malvidin hexoside pyruvate Pelargonidin hexoside Pelargonidin rutinoside Peonidin acetyl hexoside Peonidin hexoside Petunidin acetyl hexosides Petunidin hexoside Petunidin coumaroyl hexoside
595 611 593 449 535 419 595 727 611 611 627 465 435 611 535 493 517 561 433 579 505 463 521 479 625
Fragment ions (m/z)
287; 287; 287; 433;
449;
287;
287 287 287 287 449 287 449 581 287 303 303 303 303 303 331 331 355 399 271 433 301 301 317 317 317
m/z, mass-to-charge ratio; [M]þ, Positively charged molecular ion
from caffeoyl- and dicaffeoyl-quinic acid precursors [21]. Enterolactone and enterodiol identified from flaxseed microbial fermentation were previously described by Wang [22] as the major metabolites of lignans, which are distinctive polyphenols in flaxseeds. Valerolactones such as 5-(30 ,40 -dihydroxyphenyl)g-valerolactone, 5-(30 ,40 ,50 -trihydroxyphenyl)-g-valerolactone, and 5-(30 -hydroxyphenyl)-g-valerolactone were previously studied as typical products of monomeric and oligomeric flavan-3-ols microbial metabolism [23,24]. Two valerolactones were identified after 5 and 24 h of fermentation of black and green tea samples, whereas 5-(30 -hydroxyphenyl)-g-valerolactone was present after 24 h only, indicating that dehydroxylation could be a late metabolic step of flavan-3-ols degradation [24]. Dihydrosinapic and dihydroferulic acids were the characteristic metabolites of oat and wheat bran fermentation and likely derived from the dehydrogenation of native phenolic acids. Phloroglucinol was previously identified as a transient intermediate in the breakdown of quercetin [8], a molecule largely present in onion, red wine, and black and green teas. Interestingly, urolithins were previously identified as biomarkers of pomegranate juice consumption, being microbial products of ellagitannins and ellagic acid [25]. However, these molecules were not identified in the present study. This could be
Fig. 1. Multiple reaction monitoring chromatogram of protocatechuic acid detected in fermented raspberry sample after 5 h.
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Fig. 4. Multiple reaction monitoring chromatograms of 5-(3,40 -dihydroxyphenyl)g-valerolactone (M6) in (A) 5-h and (B) 24-h fermented chocolate samples. Fig. 2. Mass spectrum comparison between dihydrocaffeic acid in (A) fermented coffee sample and (B) pure standard.
explained by the extreme individual variability in urolithin production [25] or by the fact that their precursor polyphenols need to undergo specific modifications in the small intestine to interact subsequently with the colonic microbiota. According to the interindividual variability hypothesis, a recent study on the in vitro colonic bioconversion of polyphenols from black tea, red wine, and grape juice showed remarkable differences in the production of metabolites owing to microbial (and therefore enzymatic) differences in different gut microbiota [26]. The metabolites identified in the present in vitro study also are likely to be generated in the human colon after consumption of the foods analyzed, being formed by the action of bacteria of human fecal origin. It has recently been reported that, in a fermentation experiment similar to the one applied in our study, polyphenols from apple juice manufactured products able to inhibit histone deacetylase [27]. These products acted in an additive or a synergistic manner together with butyrate in exerting this action, which can induce differentiation and lead to apoptosis of precancerous and tumor cells. Because polyphenols added to fermentable dietary fiber did not eliminate in vivo the positive effects of the latter, increased the cecal butyrate pool, and increased the ring fission of rutin, hyperoside (quercetin 3O-galactoside), and quercitrin (quercetin 3-O-rhamnoside) in the colon [28], this led to the hypothesis that phenolic metabolites also can act to prevent colon cancer. The search for these microbial metabolites in vivo after a proper feeding study should consider their possible further conjugation at the level of the liver. In fact, when absorbed in the colon, microbial metabolites are transported by the portal circulation to the liver, where they may undergo phase II enzyme action to generate a wide pattern of possible circulating metabolites. These microbial metabolites are generated hours after
Fig. 3. Multiple reaction monitoring chromatogram of enterolactone from flaxseed microbial colonic fermentation.
food intake and are, therefore, good candidates for exposure studies, which usually evaluate biological fluids (urine in particular) in fasting conditions. A limitation of the study that should be considered is that, in vivo, the selected food samples would have undergone several digestive steps before entering the large intestine and being fermented, and these steps were not reproduced in our in vitro system. In fact, if the action of digestive enzymes per se could not change the structure of the investigated phenolic compounds, it is not known from the literature if their action on the food matrix could influence their physical accessibility for microbial fermentation. Moreover, a relevant fraction of the ingested polyphenols interacts in vivo with conjugating phase II enzymes at the small intestinal level [7] and, therefore, most phenolics reach the colon as methyl, glucuronide, or sulfate derivatives. This aspect could modify the fermentation process to an extent that was not measurable in the present work.
Conclusion Having targeted systematically the entire set of putative metabolites derived from the polyphenol fermentation of a range of common foods, this work brings new insights to better evaluate the bioavailability and putative preventive activity of polyphenols from specific food groups in humans. Moreover, based on these observations, some of the identified metabolites could also be used to evaluate exposure to specific foods or food groups in epidemiologic studies.
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