Article pubs.acs.org/JAFC
Tracking (Poly)phenol Components from Raspberries in Ileal Fluid Gordon J. McDougall,*,† Sean Conner,† Gema Pereira-Caro,‡ Rocio Gonzalez-Barrio,‡ Emma M. Brown,§ Susan Verrall,† Derek Stewart,†,∥ Tanya Moffet,§ Maria Ibars,§ Roger Lawther,⊥ Gloria O’Connor,⊥ Ian Rowland,# Alan Crozier,‡ and Chris I. R. Gill§ †
Enhancing Crop Productivity and Utilisation Theme, Environmental and Biochemical Sciences Group, The James Hutton Institute, Invergowrie, Dundee DD2 5DA, Scotland ‡ Plant Products and Human Nutrition Group, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Joseph Black Building, Glasgow G12 8QQ, Scotland § Northern Ireland Centre for Food and Health, Centre for Molecular Biosciences, University of Ulster, Cromore Road, Coleraine BT52 1SA, Northern Ireland ∥ School of Life Sciences, Heriot-Watt University, Edinburgh EH14 4AS, Scotland ⊥ Altnagelvin Area Hospital, Western Health and Social Care Trust, Glenshane Road, Londonderry BT47 6SB, Northern Ireland # Hugh Sinclair Unit of Human Nutrition, Department of Food and Nutritional Sciences, University of Reading, P.O. Box 226, Whiteknights, Reading RC6 6AP, England S Supporting Information *
ABSTRACT: The (poly)phenols in ileal fluid after ingestion of raspberries were analyzed by targeted and nontargeted LC−MSn approaches. Targeted approaches identified major anthocyanin and ellagitannin components at varying recoveries and with considerable interindividual variation. Nontargeted LC−MSn analysis using an orbitrap mass spectrometer gave exact mass MS data which were sifted using a software program to select peaks that changed significantly after supplementation. This method confirmed the recovery of the targeted components but also identified novel raspberry-specific metabolites. Some components (including ellagitannin and previously unidentified proanthocyanidin derivatives) may have arisen from raspberry seeds that survived intact in ileal samples. Other components include potential breakdown products of anthocyanins, unidentified components, and phenolic metabolites formed either in the gut epithelia or after absorption into the circulatory system and efflux back into the gut lumen. The possible physiological roles of the ileal metabolites in the large bowel are discussed. KEYWORDS: polyphenols, digestion, bioactivity, gut health, bioavailability, metabolism
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INTRODUCTION The importance of fruit consumption to health was highlighted recently by Ezzati and Riboli, who ranked a diet low in fruits third (∼4%) in terms of individual risk factors attributable to global disease burden, after high blood pressure (∼7%) and smoking (∼6%).1 A similar observation was reported for colorectal cancer where low fruit and low vegetable consumption was associated with a moderately increased risk.2 Among the constituents of fruit and vegetables, (poly)phenol secondary metabolites are of great interest due to their putative bioactive properties and their relatively high daily intake levels (∼800 mg/day).3 Commonly consumed fruits such as strawberries (Fragaria), blueberries (Vaccinium), or raspberries (Rubus), ingested as fresh or processed forms, contain relatively high levels of total (poly)phenols (100−300 mg/100 g of fresh mass). As a consequence, anthocyanin intakes in Finnish and French populations have been reported at approximately 40 mg/day.3,4 The large variations evident in consumption patterns for berry (poly)phenolics reflect both individual preferences in diet and the impact of horticulture. Indeed, the (poly)phenol compositions of berry fruits are known to be strongly influenced by genetic and environmental factors,5−7 including species,8 variety, weather, and cultivation method,9 ripeness at harvest,10 and storage conditions.11 © 2014 American Chemical Society
Raspberries (Rubus idaeus), a commonly consumed berry in Europe, are rich in (poly)phenols, including the anthocyanins cyanidin 3-O-sophoroside and cyanidin 3-O-glucoside and the ellagitannins sanguiin H-6 and sanguiin H-10.12−14 The importance of bioavailability in relation to putative bioactivity of (poly)phenolics has become a major focus of attention in recent years,15,16 with ileostomy studies providing information on events occurring in the proximal gastrointestinal tract and compounds which, in volunteers with an intact colon, would pass from the small to the large intestine. Raspberry feeding studies with ileostomists have shown that substantial amounts of (poly)phenolic compounds in the berries pass into the large intestine.14,17 Expanding on this earlier work, we investigated the fate of colon-available raspberry (poly)phenolic compounds following the acute ingestion of a 300 g raspberry puree by ileostomists. Ileal fluid was collected for up to 8 h after consumption of raspberries and analyzed by targeted LC−MSn against standard compounds and untargeted LC−MS2 using an orbitrap mass spectrometer to identify novel components that Received: Revised: Accepted: Published: 7631
May 13, 2014 June 25, 2014 July 6, 2014 July 7, 2014 dx.doi.org/10.1021/jf502259j | J. Agric. Food Chem. 2014, 62, 7631−7641
Journal of Agricultural and Food Chemistry
Article
U.K.) maintained at 40 °C. The mobile phase, pumped at a flow rate of 1 mL/min, was 1% aqueous formic acid (A) and 99% methanol containing 1% formic acid (B), establishing a linear gradient from 10% to 40% B over 60 min. Chromatograms were recorded at 280, 325, 365, and 520 nm. After passing through the flow cell of the diode array detector, the column elute was split, and 0.3 mL/min was directed to an LCQ DecaXP ion trap mass spectrometer fitted with an electrospray interface (ESI) operating in either positive ionization mode for anthocyanins or negative ionization mode for ellagic acid and ellagitannins. Analyses were carried out using full-scan, data-dependent MS2 scanning from m/z 150 to m/z 2000. With the ESI in positive ionization mode, the capillary temperature was 300 °C, the amount of sheath gas was 50 units, the amount of auxiliary gas was 35 units, and the source voltage was 2 kV. For negative ionization, the capillary temperature was 325 °C, the amounts of the sheath and auxiliary gases were both 40 units, and the source voltage was 4 kV. Anthocyanins were quantified from their chromatographic peak areas recorded at 520 nm and expressed as cyanidin 3-O-glucoside equivalents, ellagitannins detected at 260 nm were expressed as punicalagin equivalents, and ellagic acid and ellagic acid conjugates monitored at 365 nm were quantified in ellagic acid equivalents. Untargeted Analysis of Raspberry Metabolites in Ileal Fluids. Ileal samples were extracted by the procedure described above with small differences. The frozen samples were thawed and vortexed, and duplicate 2.0 ± 0.1 g samples were weighed into 15 mL centrifuge tubes. These samples were extracted using 3 mL of UPW containing 1% formic acid and 20 mM DDC. The tubes were vortexmixed for 3 × 30 s and then sonicated in a water bath for 1 min. All procedures were carried out at 5 °C. After centrifugation (2500g, 10 min, 5 °C), the supernatants were transferred to new tubes. The pellets were extracted twice using 3 mL of 1% formic acid in methanol containing 20 mM DDC and the supernatants combined and vortexmixed. A subsample of 4 mL was removed and dried in a Speed-Vac. The dried samples were resuspended in 10% acetonitrile containing 0.2% formic acid and prepared in filter vials prior to analysis using the orbitrap LC−MS system. Untargeted analysis of the ileal fluids was performed on an HPLC system consisting of a quaternary pump (Thermo Fisher Scientific, Accella 600) and a PDA detector (Thermo Fisher Scientific, Accella) coupled to an LTQ orbitrap mass spectrometer (Thermo Fisher Scientific, Stafford House, Boundary Way, Hemel Hempstead, U.K.). Replicate 10 μL samples were injected onto a 2 × 150 mm (4 μm) Synergy Hydro-RP 80 fitted with a C18 4 × 2 mm Security Guard cartridge (Phenomenex Ltd.). Sample and column temperatures were maintained at 6 and 30 °C, respectively. The samples were analyzed at a flow rate of 0.3 mL/min using a binary mobile phase of (A) 0.1% aqueous formic acid and (B) 0.1% formic acid in acetonitrile/water (1:1, v/v) and the following gradient: 0−4 min, 5% B; 4−22 min, 5− 50% B; 22−32 min, 50−100% B. Mass detection was carried out using an LTQ orbitrap mass spectrometer in negative ESI mode. Two scan events were employed; full-scan analysis was followed by data-dependent MS/MS of the three most intense ions using collision energies of 45 eV source voltage (set at 3.4 kV) in wide-band activation mode. The instrument was optimized (tuned) against morin at a resolution of 30 000 (to maximize the number of scans across peaks but retain a reasonable resolution) in a range of 80−2000 mass units. For optimal electrospray ionization, the source conditions were set at a source temperature of 300 °C, a sheath gas amount of 60 arbitrary units, and an auxiliary gas amount of 5 arbitrary units. Prior to analysis, the mass accuracy of the instrument was assured by calibrating using a preparative ESI negative ion calibration tuning solution (containing 2.9 μg/mL sodium dodecyl sulfate, 5.4 μg/mL sodium taurocholate, and 0.001% Ultramark 1621) following the manufacturer’s protocols. SIEVE Analysis. Mass spectral data from the orbitrap analysis were applied to the SIEVE software program (Thermo Fisher Scientific, Hemel Hempstead, U.K; http://www.thermoscientific.com/en/ product/sieve-software-differential-expression.html). The SIEVE method compares and contrasts multiple samples on the basis of the nature of the experimental setup. In this case, the samples were labeled
could enter the colon with the potential to influence colonic health.16,18
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MATERIALS AND METHODS
Chemicals. Cyanidin 3-O-glucoside was purchased from Apin Chemicals (Abingdon, Oxford, U.K.), cyanidin 3-O-sambubioside 5-Oglucoside was supplied by Polyphenols (Sandnes, Norway), morin and ellagic acid were obtained from Sigma-Aldrich (Poole, Dorset, U.K.), and punicalagin was obtained from Chromadex (United States). HPLC-grade solvents were obtained from Rathburn Chemicals (Walkerburn, U.K.). Formic acid and sodium diethyldiothiocarbamate were purchased from Sigma-Aldrich, and acetic acid was purchased from BDH (Poole, U.K.). Plant Material and Processing. Raspberries (30 kg, R. idaeus variety Glen Ample) were purchased from Peter Marshall & Co., Alyth, Perthshire, U.K. The raspberries were picked as commercially ripe and transported to the James Hutton Institute on the day of picking, where they were washed in distilled water, left to drain on sieves, and then pureed in 10 kg batches using a catering homogenizer (Fimar MX40S fixed speed stick blender). The procedure did not homogenize the seeds. The purees were combined and continually stirred using an overhead paddle stirrer at 75 rpm (to prevent settlement) and ladled into food-grade bags in 300 ± 1 g amounts. The purees were frozen, transported to the University of Ulster, and stored at −20 °C prior to use in the ileostomy feeding studies. Ileostomy Feeding Study. The study (11/NI/0112) was conducted with the prior approval of the Office for Research Ethics Committees Northern Ireland (ORECNI) and the University of Ulster Ethical Committee and with the informed consent of participants who were recruited from clinics at Altnagelvin Area Hospital, with the assistance of a colorectal consultant (Dr. R. Lawther) and nurse specialist (Dr. G. O’Connor). Eleven ileostomists (six males, five females, mean age 44 ± 12 yr) who had undergone terminal ileostomies and were at least 1.5 years postoperative prior to the study and were nonsmokers undertook the study. Participants were required to follow a diet low in polyphenolic compounds as described previously,14 avoiding fruits and vegetables, nuts, high-fiber products, and beverages such as tea, coffee, and fruit juices, as well as abstaining from consumption of alcohol for 48 h prior to the beginning of the study. Following an overnight fast, the participants provided an ileal fluid sample (T 0 h), then consumed 300 g of homogenized raspberries within 10 min, and continued the low polyphenolic diet, with provision of a standard lunch, for a further 8 h until provision of the second ileal fluid sample (T 8 h). The ileal fluid samples were collected and processed within 30 min. The volumes and pH values of the ileal fluid were recorded, before dilution with ice-cold distilled water as required, dependent on the viscosity, and before the fluid was homogenized in a chilled Waring blender for 30 s and storage of aliquots at −80 °C in preparation for subsequent analysis. Targeted Analysis of Raspberry Phenolics in Ileal Fluids. Duplicate samples of ileal fluid (2 ± 0.1 g) were spiked with 10 μg of cyanidin 3-O-sambubioside 5-O-glucoside as an internal standard and homogenized in 3 mL of 1% aqueous formic acid (FA) containing 20 mM sodium diethyldiothiocarbamate (DDC) for 1 min at 24 000 rpm using an Utra-Turrax homogeneizer. The samples were centrifuged for 20 min at 4000g at 4 °C, and the supernatants were collected. The pellet was re-extracted twice with 1% formic acid in methanol containing 20 mM DDC, and the three supernatants were pooled. Portions (4 mL) of this extract were reduced to dryness in vacuo using a SpeedVac concentrator. The residues were resuspended in 50 μL of 1% formic acid in methanol and 450 μL of 1% formic acid, and 100 μL aliquots were analyzed by HPLC-PDA−MS2 (PDA = photodiode array). The samples were analyzed on a Surveyor HPLC system comprised of an HPLC pump, a PDA detector, scanning from 200 to 700 nm, and an autosampler cooled to 4 °C (Thermo Finnigan, San Jose, CA). Separations of ellagic acid derivatives, ellagitannins, and anthocyanins from raspberry were performed using a Synergi 4 μm RP-POLAR 80 Å 250 × 4.6 mm i.d. reversed-phase column (Phenomenex, Macclesfield, 7632
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and the group mean ileal fluid sample mass at baseline (T 0 h) was 187.8 ± 73 g (range 107.4−318.0 g) with a group mean pH value of 5.6 ± 0.4 (range 5.0−6.3). The group mean ileal fluid mass for the T 8 h sample (post berry consumption) was 268. ± 108 g (range 62−470 g) with a group mean pH value of 6.6 ± 0.6 (range 5.3−7.5). In both cases the group mean ileal fluid masses and pH values were significantly (p < 0.05) increased compared to those of the baseline fasted samples. Eight anthocyanins were detected in Glen Ample raspberries (Table 1). Although the composition was comparable to that
either before or after, and the program was set to discern which MS signals were consistently present and increased in the aftersupplementation samples. As the SIEVE software automatically detects and integrates peaks, it can also provide lists of peaks that are consistently decreased after feeding. SIEVE parameters were chosen which have been shown in our laboratory to be effective for this pairwise comparison of samples. These parameters define the optimum and maximum numbers of peaks, peak threshold values, a delayed retention start of 5 min to dismiss unbound material, and suitable peak width parameters to allow for drifts in retention time between runs and samples. These settings have been used to accurately align and “bin” signals from mass spectral data from multiple chromatograms. The output, as peak areas for the detected peaks, can be accessed in Excel file format and used to develop principal component analysis (PCA) plots and other models. Data Analysis and Conditions. Statistical analysis was carried out on all the variables collected from the LC−MS analysis of the samples. Data were obtained as peak areas from the SIEVE automatic integration software and consisted of 862 variables or potential peaks. Three approaches were taken: First, PCA, using univariate scaling, was applied to all the samples, and components 1−4 described 42% of the variation in the data set, with component 1 describing almost 17%. Second, a discriminant analysis (optimized partial least squares, OPLS) was performed with two classifications (before feeding and after feeding), resulting in a model that described 33% of the variation, 14% of which described the after feeding classification. The Q2 score for this model was 0.974. These analyses were performed using SIMCA-P 12.0.1.0 software. The third analysis performed was pairwise metabolite−metabolite correlations (using Genstat for Windows, 16th ed., VSN International Ltd., Hemel Hempstead, U.K.), determined by Pearson’s correlation coefficient (r) test using two sample sets, first all samples and then a subset consisting of the “after feeding” samples. This technique was used to define components whose patterns of abundance matched those of components identified by the targeted analysis and provide a subset which was likely to have arisen from the berry material. Extraction and Analysis of Extracts from Raspberry Seeds. Raspberry seeds were isolated from the purees by repeated sieving and washing using ice-cold distilled water. The seeds, which were free of attached material, were dried using paper tissues and stored at −18 °C. The extraction procedure was essentially the same as noted above for the untargeted study, but only 200 mg of seeds was extracted and the volumes were reduced to 1 mL. After the 1 mL extracts were dried in the Speed-Vac, they were resuspended in 5% acetonitrile containing 0.1% FA and prepared in filter vials. Samples of resolubilized extracts were analyzed using an LCQDECA system, comprising a Surveyor autosampler, pump, and PDA detector, and a Thermo-Finnigan ion trap mass spectrometer controlled by Xcalibur software. A 2 mm × 150 mm C18 column (Synergi Hydro C18 with polar end-capping, Phenomenex Ltd.) was eluted with a binary solvent of (A) 0.1% aqueous formic acid and (B) 0.1% formic acid in acetonitrile at a flow rate of 200 μL/min using a 30 min gradient of 5−40% B. The PDA detector scanned three discrete channels at 280, 365, and 520 nm. The mass data were collected in negative or positive mode scanning over the mass range m/z 80−2000. The MS detector was tuned against morin in negative mode and cyanidin 3-O-glucoside in positive mode. Two scan events were employed; full-scan analysis was followed by data-dependent MS/MS of the most intense ions using collision energies of 45% of the source voltage (set at 3 kV) in wide-band activation mode. The capillary temperature was set at 250 °C, with the sheath gas pressure at 60 psi and auxiliary gas pressure at 15 psi.
Table 1. Identification and Quantification of Phenolic Compounds Detected in Raspberries peak
compd
1
cyanidin 3,5-Odiglucoside cyanidin 3-Osophoroside cyanidin 3-O(2″-Oglucosyl) rutinoside pelargonidin 3O-sophoroside cyanidin 3-Oglucoside cyanidin 3-O(2″-O-xylosyl) rutinoside cyanidin 3-Orutinoside pelargonidin 3O-glucoside
2 3
4 5 6 7 8
[m/z]+
MS2 m/z
concna (μmol/300 g)
18.1
611
449, 287
0.6 ± 0.1
18.9
611
449, 287
22 ± 1
19.9
757
611, 287
4.2 ± 0.3
23.5
595
271
0.25 ± 0.01
24.2
449
287
9±1
25.2
727
581, 287
0.3 ± 0.1
27.1
595
287
2.2 ± 0.2
28.9
433
271
0.1 ± 0.1
total anthocyanins
38.2 ± 3.2
34.6
433b
257, 229
1.2 ± 0.1
10
ellagic acid Opentoside ellagic acid
35.6
301b
257, 229 total ellagic acid
4.8 ± 0.1 6.0 ± 0.2
11
sanguiin H-10
18.9
783b
43 ± 3
12
sanguiin H-6
26.0
1869b
13
lambertianin C
29.2
1401b
1265, 1103, 933, 631 1567, 1265, 933, 631 1869, 1567, 1265, 933, 631 total ellagitannins
9
a
tR (min)
61 ± 3 20 ± 2 124 ± 8
Data expressed as mean values ± SD (n = 2). b[m/z]−.
detected previously in this variety of raspberries,14,19,20 the amounts of anthocyanins and the ellagic acid derivatives were lower than in the berries used in a previous feeding study.14 The overall levels of ellagitannins were similar, but the profile was different, with lower levels of sanguiin H-6 and higher amounts of sanguiin H-10 and lambertianin C. These differences may reflect differences in growing location or year-to-year variation, but the lower content of anthocyanins could indicate that the raspberries were not as ripe as those used in the previous study, which were picked directly from the field as opposed to being part of a shipment of raspberries to be delivered to a supermarket.
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RESULTS AND DISCUSSION This study confirms and extends previous research that examined the fate of anthocyanins, the major ellagitannins, and ellagic acid derivatives from raspberries excreted in ileal fluid by subjects with an ileostomy.14 The 11 ileostomists participating in the current study had a mean age of 44 ± 12 yr, 7633
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Table 2. Recovery of Anthocyanins, Ellagic Acid, and Ellagitannins in Ileal Fluid Collected 0−8 h after Consumption of 300 g of Raspberriesa compd
S1
S2
S3
S4
S5
S6
cyanidin 3-Osophoroside cyanidin 3-O-(2″O-glucosyl) rutinoside pelargonidin 3-Osophoroside cyanidin 3-Oglucoside cyanidin 3-Orutinoside total anthocyanins
8.0 ± 0.7
36
4.2 ± 0.4
19
7±2
33
6.6 ± 0.8
30
0.41 ± 0.06
2
6±2
27
1.7 ± 0.5
40
1.6 ± 0.1
38
1.7 ± 0.7
39
1.7 ± 0.3
40
0.11 ± 0.01
3
1.6 ± 0.4
38
0.1 ± 0.1
40
0.10 ± 0.01
40
0.10 ± 0.02
40
0.08 ± 0.01
32
nd
0
0.08 ± 0.02
32
0.1 ± 0.1
1
0.4 ± 0.1
4
0.06 ± 0.01
1
0.12 ± 0.03
1
nd
0
0.09 ± 0.01
1
0.3 ± 0.1
14
0.10 ± 0.01
4
0.04 ± 0.01
2
0.03 ± 0.01
1
0.03 ± 0.02
1
0.2 ± 0.03
9
10.2 ± 1.5
27
6.4 ± 0.6
17
8.9 ± 2.7
23
8.5 ± 1.1
22
0.55 ± 0.09
1
7.9 ± 2.5
21
ellagic acid Opentoside ellagic acid total ellagic acid
0.3 ± 0.1
25
0.5 ± 0.1
42
0.50 ± 0.03
42
0.6 ± 0.3
50
0.05 ± 0.01
42
0.5 ± 0.2
42
7.2 ± 0.6 7.5 ± 0.7
150 125
10 ± 3 10.5 ± 3.1
208 175
14.6 ± 0.1 15.1 ± 0.1
304 252
12 ± 6 12.6 ± 6.3
250 210
0.8 ± 0.1 0.85 ± 0.11
17 14
12 ± 3 12.5 ± 3.2
250 208
31 14 0 18
6±1 17 ± 7 0.7 ± 0.6 23.7 ± 8.6 S8
14 28 4 19
8±4 32 ± 2 2.4 ± 0.7 42.4 ± 8.7 S9
19 52 12 34
6.5 ± 0.2 29 ± 19 4±2 39.5 ± 21.2 S10
15 47 20 32
0.6 ± 0.04 0.4 ± 0.06 nd 1.0 ± 0.1 S11
cyanidin 3-O-sophoroside cyanidin 3-O-(2″-O-glucosyl)rutinoside pelargonidin 3-O-sophoroside cyanidin 3-O-glucoside cyanidin 3-O-rutinoside total anthocyanins
4±1 1.0 ± 0.2 0.05 ± 0.01 0.3 ± 0.1 0.4 ± 0.1 5.7 ± 1.4
ellagic acid O-pentoside ellagic acid total ellagic acid sanguiin H-10 sanguiin H-6 lambertianin C total ellagitannins
sanguiin H-10 13.5 ± 0.6 sanguiin H-6 8.5 ± 1.7 lambertianin C nd total ellagitannins 22.0 ± 2.3 compd
18 24 20 3 18 15
6.8 ± 0.2 1.66 ± 0.01 0.10 ± 0.01 0.07 ± 0.01 0.11 ± 0.02 8.7 ± 0.2
31 39 40 1 5 23
0.5 ± 0.1 8.6 ± 0.5 9.1 ± 0.6
40 179 152
0.4 ± 0.1 13 ± 1 13.4 ± 1.1
33 271 223
4±2 23 ± 4 0.4 ± 0.2 27.4 ± 6.2
9 38 2 22
16 64 35 43
7±2 39 ± 5 7±5 53 ± 9
6.3 ± 0.5 1.51 ± 0.03 0.07 ± 0.01 0.06 ± 0.01 0.9 ± 0.1 8.8 ± 0.6
1 1 0 0.8
8±2 19 ± 3 nd 27 ± 5
19 31 0 22 S12
29 36 28 1 41 23
3.5 ± 0.8 0.9 ± 0.4 0.04 ± 0.01 0.11 ± 0.08 0.02 ± 0.01 4.6 ± 1.3
16 21 16 1 1 12
0.5 ± 0.1 9±1 9.5 ± 1.1
40 187 158
0.13 ± 0.01 7.3 ± 0.7 7.4 ± 0.7
11 152 124
0.8 ± 0.3 7±2 8 ± 2.3
33 83 134
7.4 ± 0.3 19 ± 7 2.3 ± 0.6 28.7 ± 7.9
17 31 12 23
14 43 5 27
5±3 12 ± 8 1.1 ± 0.8 27 ± 5
13 20 6 15
6±1 26 ± 2 1±1 33 ± 4
4.03 ± 2.1 1 ± 0.55 0.02 ± 0.01 0.09 ± 0.05 0.57 ± 0.3 5.7 ± 1.7
19 24 8 0.3 23 15
In the first column for each subject, the data are expressed as mean values in micromoles ± standard error (n = 2). The numbers in the second column for each subject represent the amounts recovered as a percentage of the quantity ingested. nt = not detected. Anthocyanins, ellagic acid, and ellagitannins were not detected in ileal fluid samples collected at time 0 h before raspberry intake. S1 = subject 1, S2 = subject 2, and so on. Subject 7 did not complete the study, and therefore 11 subjects are described.
a
Five of the major anthocyanins in the raspberries were consistently detected in the ileal fluids collected from the 11 donors after supplementation (Table 2). The most abundant anthocyanin in the raspberries, cyanidin 3-O-sophoroside, was also the main anthocyanin in the ileal fluids, but the recovery varied greatly between the subjects (from 2% to 36% of intake, mean 21.7%, Table 3). Other anthocyanins that were less abundant in the raspberries, such as cyanidin 3-O-(2″glucosyl)rutinoside and pelargonidin 3-O-sophoroside, often had higher precentage recoveries than cyanidin 3-O-sophoroside. The order of recovery was cyanidin 3-O-(2″-glucosyl)rutinoside > pelargonidin 3-O-sophoroside > cyanidin 3-Osophoroside > cyanidin 3-O-rutinoside > cyanidin 3-Oglucoside (mean recoveries 28.6%, 24.0%, 21.7%, 9.2%, and 1.3%, respectively), but again with substantial interindividual variation. These recoveries were lower than but showed the same order as those observed previously.14
The major ellagitannins detected in raspberries were also detected in the ileal samples after supplementation (Tables 2 and 3). Sanguiin H-6 was the most abundant in the ileal samples, but as with the anthocyanins, there was a large interindividual variation in recoveries (1−64%, mean 30.5%). Sanguiin H-10 was generally recovered at higher levels (1− 31%, mean 13.8%) than lambertianin C (0−35%, mean 8.2%), but again with large interindividual variation. The recovery figures may reflect their innate relative stability to the conditions of the upper gastrointestinal tract, but it also possible that some components are formed through degradation of larger ellagitannins. For example, sanguiin H-6 is a potential degradation product of lambertianin C, and sanguiin H-10 could be formed during the degradation of sanguiin H-6. Indeed, in vitro digestion studies indicated an increased recovery of sanguiin H-10 isomers which was suggested to be due to breakdown of the larger ellagitannins.21,22 7634
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Table 3. Mean Recovery of Anthocyanins, Ellagic Acid, and Ellagitannins in Ileal Fluid Collected from 11 Volunteers 0− 8 h after Consumption of 300 g of Raspberries recovery compd
μmol (mean ± SE)
% of intake
cyanidin 3-O-sophoroside cyanidin 3-O-(2″-O-glucosyl)rutinoside pelargonidin 3-O-sophoroside cyanidin 3-O-glucoside cyanidin 3-O-rutinoside total anthocyanins
4.7 ± 0.8 1.2 ± 0.3 0.06 ± 0.02 0.11 ± 0.04 0.23 ± 0.06 6.30 ± 1.22
21.7 28.6 24.0 1.3 9.2 16.3
ellagic acid O-pentoside ellagic acid total ellagic acid
0.4 ± 0.1 8.4 ± 1.5 8.8 ± 1.6
33.0 175.7 146.6
sanguiin H-10 sanguiin H-6 lambertianin C total ellagitannins
6.0 ± 1.3 18.7 ± 4.9 1.6 ± 0.9 26.3 ± 7.1
13.8 30.5 8.2 21.2
Although the levels of ellagic acid pentoside were generally reduced (recoveries 11−50%, mean 33%, Tables 2 and 3), ellagic acid was detected in the ileal fluids from all subjects at levels generally higher than in the raspberries (17−304%, mean 175.7%). This increase in ellagic acid most probably is a result of the degradation of ellagitannins and/or ellagic acid pentosides and release of ellagic acid as noted in model studies.23 However, in the previous study14 only sanguiin H-6 and ellagic acid were detected. It is very noticeable that the recoveries of (poly)phenols in certain subjects were relatively high (e.g., subject 4) whereas those in other subjects (e.g., subject 5) were very low. This interindividual variation may be related to the differing physiology of the subjects. In particular, subject 5 had the lowest output of ileal fluid, which may indicate slow passage of the berries through the intestine or lower levels of intestinal secretions. Subject 1 had the highest ileal output and had higher than average recovery of anthocyanins but poorer recovery of sanguiin H-6 (Table 2). Overall, the targeted analysis provided evidence for the differential stability of the anthocyanin, ellagitannin, and ellagic acid derivatives following consumption of raspberries and largely confirmed previous findings.14 For the nontargeted approach, the ileal samples were examined by LC−MS/MS using an orbitrap mass spectrometer capable of exact mass determination. This confirmed the presence of the major anthocyanins, ellagitannins, and ellagic acid derivatives detected by the targeted analysis as can be seen in a characteristic absorbance profile of the “before”- and “after”-supplementation samples (Figure 1a). The presence of anthocyanins could be detected by examining the elution profiles at 520 nm (Figure 1b). There was a good association between the targeted and nontargeted analyses as indicated by a comparison of the levels of anthocyanins and the levels of the major ellagitannin peak, sanguiin H-6 (see the Supporting Information, Figure S1). Indeed, it was apparent that the interindividual variabilities in recoveries were similar between the targeted and nontargeted analyses. However, it also became clear that certain subjects had very different profiles. In particular, subjects 1 and 5, and to a lesser extent subject 10, gave a different PDA profile in the after-supplementation samples (Supporting Information, Figure S2). Their profiles
Figure 1. (a) Characteristic UV traces for ileal samples before and after supplementation. Traces from subject 6 are shown before and after (red) supplementation with raspberries. The black arrows denote the positions of the sanguiin H-6, ellagic acid pentose, and ellagic acid peaks. The dashed arrow denotes the position of the main anthocyanin peak. The figure in the top right corner is the full-scale deflection of the PDA detector at 280 nm. (b) Traces at 520 nm of ileal samples before and after supplementation. Traces from subject 6 are shown before and after (red) supplementation with raspberries. The dashed arrow denotes the position of the main anthocyanin peak. The figure in the top right corner is the full-scale deflection of the PDA detector at 520 nm.
were dominated by two peaks (tR = 9.72 and 13.76 min) with PDA and m/z data characteristic of caffeoylquinic acids (PDA maximum ∼320 nm; [M − H], m/z 353; MS2, m/z 191), arguably indicative of coffee intake by the subjects during the supplementation period.24 The chromatographic profiles were examined visually to discern peaks that consistently increased after supplementation, but the human element was supplemented by a nontargeted method. The MS data from all of the samples including blanks and the quality control samples were examined using the SIEVE software program, which selects peaks that increase or decrease significantly after supplementation. The SIEVE approach is completely unbiased and provides data to produce principal component analysis plots illustrating the differences and similarities between the samples (see Figure 2). The before and after samples were separated mainly on the t[1] axis (which explains 17% of the variation). Certain before and after samples 7635
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variation between replicate extractions as illustrated in the PCA plot (Figure 2) could have been caused by different numbers of seeds being present in replicate samples. To examine which components could be extracted from the raspberry seeds, the seeds were separated from some of the original raspberry puree, then extracted under identical conditions, and examined using similar LC conditions but using an ion-trap MS detector (Figure 3). A number of components were identified in the seed extracts that had been identified in the ileal samples. The major component was the ellagitannin sanguiin H-6, but there were also other ellagitannins and some proanthocyanidin (PAC) components (see Table 5). In particular, the seeds contained a peak with PDA and MS properties consistent with a proanthocyanidin dimer composed of (epi)afzelchin and (epi)catechin (peak 12, Table 5; PDA maximum 275 nm; [M − H], m/z 561; MS2, m/z 543, 435, 289) and a proanthocyanidin dimer composed of (epi)catechin units (peak 8, Table 5; PDA maximum 275 nm; [M − H], m/z 577; MS2, m/z 559, 533, 289). There was also evidence for larger PACs containing (epi)afzelchin groups which was supported by further analyses of different extracts of the seeds (results not shown). These findings highlight the possible contribution of raspberry seeds to the availability of phenolic components in ileal fluids. Raspberry seeds can make a considerable contribution to the total berry mass. Seed/flesh ratios of 20% have been reported, although values of ∼5% are more common for cultivated varieties, such as Glen Ample (Dr. Nikki Jennings, The James Hutton Institute, personal communication). Of course, the components extractable from the seeds (or the pulp) in the ileal fluid may not be equivalent to those extractable during digestion in the gastrointestinal tract. Such components may only become accessible during colonic digestion of the raspberry material and would be subject to degradation by fermentation. Raspberry seeds have evolved to survive gastrointestinal digestion. and it would be interesting to compare the components extractable from seeds after ileal digestion with those that are expelled in feces. In essence, the components found by this nontargeted approach to be increased after raspberry supplementation can be split into four classes: (1) those that were also identified by targeted analysis, (2) those which are related to the targeted components, but may have been extracted from the seeds (Table 5), (3) components present in the raspberries which have become more abundant due to their stability in the gastrointestinal tract, and (4) unknown components which may also encompass some potential breakdown products or metabolites of the original berry polyphenols (Table 4). Components suggestive of breakdown of ellagitannins include ellagic acid, which was identified in the targeted approach (Table 1). The presence of (epi)catechin and derivatives in the ileal fluid after supplementation (see Table 4) suggests breakdown of the proanthocyanidins, perhaps from the raspberry seeds (Table 5). Indeed, it is notable that epicatechin and (epi)afzelchin were not detected in the raspberry seed extracts. Components such as dihydroxybenzoic acid and hydroxybenzoic acid could appear due to anthocyanin degradation as the B-ring fragments from cyanidin and pelargonidin aglycons, respectively.25,26 The putatively identified hydroxybenzoic acid glucoside and benzoic acid components could also arise from anthocyanin degradation. The hydroxycinnamic acids p-coumaric and caffeic acids have also been reported to be formed through the degradation of anthocyanins.26 However, these hydroxycinnamic acids were
Figure 2. Principal component analysis of the samples from the ileostomy study using the SIEVE process. Black markers represent the before samples, and the red markers represent the after samples. The blue markers are the quality control samples, which were prefeeding samples (subject 10) given blind before extraction. Axis t[1] explains 17% of the variation and t[2] 9%. The double-headed arrow shows the separation of replicate samples along axis t[1]. The circles annotate samples discussed in the text.
were separated from the others; e.g., the S1-2 (after) replicates were displaced from the other after samples, and the S12-1 (before) samples were also separated from the other before samples. In most cases, the replicate extractions were tightly bunched, but some replicates (e.g., S3-2) were slightly but definitely separated (see the arrows, Figure 2). The SIEVE output data were also plotted as bar charts to illustrate peaks/ metabolites that show similar patterns of increased or decreased levels across the subjects. For example, the first approach taken was to select peaks that not only increased after supplementation but also had a pattern of recovery similar to that of the main ellagitannin peak, sanguiin H-6, across the subjects (see Figure S3a, Supporting Information). This subjective approach should focus on components that have arisen from the supplementation. However, when we analyzed the SIEVE data using an unbiased approach, a near-identical list of peaks was generated (results not shown). It is also possible to select for peaks that systematically decreased across the subjects after raspberry intake, but although this may be very interesting, this was not a priority in this study. Using this approach, we identified peaks such as ellagic acid pentose (Figure S3b) and an unidentified component with m/z = 355 (Figure S3c). Once this subset of peaks had been identified and the quality of the peaks was checked to ensure that they were not artifacts, this subset of peaks was assessed against previous reports and the m/z, MS2, and exact mass data were employed to putatively identify the components (Table 4). It was noted that the ileal samples contained intact raspberry seeds that had survived the digestion process. Although the ileal samples were well mixed before extraction, they were not milled in a fashion that would homogenize the seeds, as this does not occur in vivo. Therefore, it is possible that some of the 7636
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Table 4. Putative Identification of Elevated MS Signals in Ileal Samples after Raspberry Supplementationa
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Table 4. continued
Shading indicates the compounds were also found in the extracts of raspberry seeds. Asterisks denote doubly charged species, [M − 2H]2−. Underlined masses are the main MS2 fragments. Formulas in italics are predicted for MS2 fragments.
a
also detected in ileal fluid after intake of a mixed fruit juice,27 where they may have arisen from intake of hydroxycinnamic acid derivatives. Some other components, such as pcoumaroylquinic acid, indole 3-acetylaspartic acid, and the putative gibberellin-like derivatives, were present in raspberry in relatively low amounts, but became more apparent due to their stability in the small intestine. The stringency of the SIEVE approach is illustrated in the case of the putative identification of a methylepicatechin sulfate derivative. Although three signals with m/z 383 can be discerned in most ileal samples after supplementation, only one peak was consistently and significantly increased. In addition, a peak putatively attributable to dihydrocaffeic acid
sulfate (tR = 5.1 min; m/z 261; MS2, m/z 181 and 143) could be discerned in many after samples but was not selected by the SIEVE method. However, a set of three peaks with m/z 355 were identified by the SIEVE approach. These peaks all gave an exact mass that yielded a predicted formula of C15H15O10 with a low mass deviation (∼1 ppm). This formula matches that of a caffeic acid glucuronide, which has been identified in urine and plasma after coffee intake.28 Indeed, the METLIN database provides this compound as a potential hit (http://metlin. scripps.edu/metabo_info.php?molid=96063). However, the MS2 data do not fit, and in particular, there is no fragment at m/z 179 as expected for caffeic acid. Therefore, this component remains unidentified. Recent work by Sun et al. identified m/z 7638
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ferulic acid. Sulfated derivatives of phenolic components of fruit juices have been identified in ileal fluid after juice intake.27 However, further studies are required to confirm the identity of these derivatives. Such phase II metabolites could be produced in the wall of the small intestine, efflux back into the lumen, and as a consequence appear in ileal fluid. An alternative route could involve their absorption into the bloodstream and return to the small intestine via enterohepatic recirculation in the bile. However, recent studies with (−)-epicatechin metabolites indicate that recycling by the bile is at best a minor event.30,31 In conclusion, targeted and nontargeted LC−MSn techniques have been used to identify components in ileal fluid that increase after raspberry intake. These components have survived digestion in the upper gastrointestinal tract and in healthy subjects with an intact functioning colon would enter the large intestine where they may influence gut function and health.19 Understanding which components enter the colon is of benefit to estimate the possible health effects that may occur. Previous work has shown that components that enter the colon are subject to degradation by resident microbiota, usually to a simpler set of metabolites which may have their own potential bioactive effects.22,32 This study confirmed previous work14 that illustrated that considerable amounts of ellagitannins, ellagic acid derivatives, and anthocyanins survive and may enter the colon. However, the untargeted approach has extended the list of “known” components and identified new compounds that may arise from the seeds present in the raspberry purees as breakdown products of the original berry anthocyanins (e.g., hydroxybenzoic acid derivatives), a set of components that are enriched due to their stability, and unknown components yet to
Figure 3. UV trace of components extracted from isolated raspberry seeds. The peaks denoted are described in Table 5. The figure in the top right corner is the full-scale deflection of the PDA detector at 280 nm.
signals at 191.0193 in strawberry extracts as citric acid,29 and the main MS2 fragment of the m/z 355 component is a good match for this structural formula (C6H7O7, mass deviation
