Article pubs.acs.org/JAFC
Degradation of Curcuminoids by in Vitro Pure Culture Fermentation Suryani Tan,†,§,# Thusitha W. T. Rupasinghe,#,⊗ Dedreia L. Tull,#,⊗ Berin Boughton,⊗,△ Christine Oliver,† Chris McSweeny,□ Sally L. Gras,#,⊥ and Mary Ann Augustin*,† †
CSIRO Food and Nutrition Flagship, 671 Sneydes Road, Werribee, Victoria 3030, Australia Department of Chemical and Biomolecular Engineering, #Bio21 Molecular Science and Biotechnology Institute, ⊥The ARC Dairy Innovation Hub, Department of Chemical and Biomolecular Engineering, ⊗Metabolomics Australia, and △School of Botany, The University of Melbourne, Parkville, Victoria 3010, Australia □ CSIRO Agriculture Flagship, 306 Carmody Road, St. Lucia, Queensland 4067, Australia §
S Supporting Information *
ABSTRACT: Colonic bacteria may mediate the transformation of curcuminoids, but studies of this metabolism are limited. Here, the metabolism of curcuminoids by Escherichia fergusonii (ATCC 35469) and two Escherichia coli strains (ATCC 8739 and DH10B) was examined in modified medium for colon bacteria (mMCB) with or without pig cecal fluid. LC-MS analysis showed that 16−37% of curcumin, 6−16% of demethoxycurcumin (DMC) and 7−15% of bis-demethoxycurcumin (Bis-DMC), and 7− 15% of bis-demethoxycurcumin (Bis-DMC) were converted following 36 h of fermentation, with the amount of curcuminoids degraded varying depending on the bacterial strain and medium used. Three metabolites (dihydrocurcumin (DHC), tetrahydrocurcumin (THC), and ferulic acid (FA)) were found in fermentation cultures with all strains used. In addition, a compound with m/z [M − H]− 470 was found and identified to be a curcumin adduct (curcumin−L-cysteine), using accurate mass FT-ICR-MS. This study provides insights into the bacterial metabolism of curcuminoids. KEYWORDS: curcumin, curcuminoids, Escherichia coli, gut bacteria, metabolism
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curcuminoids by gut bacteria.12 Studies on rats showed that about 38% of curcumin ingested remains in the cecum and large intestine of rats.13 Furthermore, ∼75% of curcumin administered orally (1 g/kg) in rats was excreted in feces, but only traces appeared in urine and blood plasma, suggesting that ∼25% of curcumin could be metabolized in the gut.14 Despite the importance of such microbiota-mediated transformation of curcumin, studies on the microbial metabolism of curcuminoids are limited. The human large intestine hosts a highly complex microbial ecosystem containing ∼1014 bacterial cells comprising approximately 1000 different species.15,16 These gut microbiota are capable of transforming polyphenolic compounds by ring cleavage, reduction, decarboxylation, demethylation, and dehydroxylation, thereby allowing the absorption of lower weight metabolites. 17 Six biologically relevant bacterial strains (Bifidobacteria longum BB536, Bifidobacteria pseudocatenulatum G4, Escherichia coli K-12, Enterococcus faecalis JCM 5803, Lactobacillus acidophilus, and Lactobacillus casei) were capable of degrading curcumin, with at least 56% reduction, as judged by loss of the parent compound.18 A recent study reported that a bacterium isolated from human feces, later confirmed to be E. coli, has curcumin-converting ability.19 The enzyme responsible for this curcumin-converting ability was named CurA and once isolated from E. coli strain K-12, substrain DH10B was able to transform curcumin to DHC and
INTRODUCTION The curcuminoids curcumin, demethoxycurcumin (DMC), and bis-demethoxycurcumin (Bis-DMC) are naturally occurring yellow-orange polyphenolic compounds found in the rhizomes of Curcuma longa and other Curcuma species.1,2 The putative health benefits of curcuminoids are related to their potential antiinflammatory,3 antioxidant,4,5 and anticancer6,7 properties, which are influenced by the chemical changes that occur in these compounds and their absorption in vivo.8 Several studies have examined the conversion of curcuminoids in vivo.9−12 After oral (1 g/kg) and intraperitoneal (0.1 g/kg) administrations to mice, curcumin was first biotransformed to dihydrocurcumin (DHC) and tetrahydrocurcumin (THC), and these compounds were then converted to monoglucuronide conjugates.10 In addition, there was a reduction of curcumin through an endogenous reductase and subsequent glucuronidation by UDP-glucuronosyl transferases. 10 Incubation of curcuminoids with liver slices from rat resulted in the reduction of curcumin to DHC, THC, hexahydrocurcumin, and octahydrocurcumin, which were consequently glucuronidated, with tetrahydro, hexahydro, and octahydro forms being mainly present as glucuronides or conjugated with sulfate.11 It was also established that the metabolism of DMC and Bis-DMC follows the same pathways as curcumin with similar reductive products obtained.11 Studies have provided insight into the breakdown of encapsulated curcumin in bread that were found in the serum, urine, and feces of humans. Phenolic acids such as vanillic acid and ferulic acid (FA) were found to be major metabolites, and it was postulated that the presence of phenolic acids in these samples after 24 h was due to the metabolism of nonabsorbed © 2014 American Chemical Society
Received: Revised: Accepted: Published: 11005
July 1, 2014 September 3, 2014 October 15, 2014 October 15, 2014 dx.doi.org/10.1021/jf5031168 | J. Agric. Food Chem. 2014, 62, 11005−11015
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Table 1. Parametersa from LC-Neg-ESI-MS/MS Data for Curcuminoids Curcumin, DMC, Bis-DMC, Known Metabolites DHC, THC, and FA, and a New Compound Detected in the Fermentation Culture MRM parameters compound
Rt (min)
MRM transitions (m/z)
fragmentor voltage
collision energy (CE)
LOD (μM)
LOQ (μM)
curcumin
5.99
367−134b 367−149 367−173
100 100 100
20 20 10
0.01
0.025
DMC
5.97
337−217b 337−173
110 110
10 10
0.01
0.025
Bis-DMC
5.94
307−143b 307−119
110 110
10 10
0.01
0.025
DHCc
5.10
369−219b 369−134 369−149
110 110 110
10 10 25
THC
5.60−6.05
371−235b 371−99 371−135
120 120 120
10 30 30
0.025
0.05
ferulic acid (FA)
1.35
193−134b 193−178
100 100
10 10
0.01
0.025
curcumin adduct (curcumin−L-cysteine)d
5.46
470−167b 470−392 470−152
120 120 120
10 10 10
β-estradiol-17-acetate
6.34
313−185
230
35
a
Rt, retention time; LOD, limit of detection; LOQ, limit of quantification. bPeak area from this transition was used for quantification. cIdentification and confirmation based on molecular weight and MS/MS fragmentation patterns. dCompound confirmation based on accurate mass from FT-ICRMS and structural identification based on MS/MS fragmentation patterns. Response for DHC and curcumin adduct (curcumin−L-cysteine) was assumed to be the same as that for curcumin. β-estradiol-17-acetate, were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Acetonitrile was purchased from Burdick & Jackson (Gillman, SA, Australia), methanol and ethyl acetate were supplied by Merck Pty Ltd. (Kilsyth, Vic, Australia), and all solvents were of HPLC grade. Formic acid and ammonium acetate (analytical grade) were from Ajax FineChem (Waltham, MA, USA). Redistilled deionized water with a resistivity of 18.2 mΩ was used in all experiments. Pig cecal fluid (PCF) was sourced from an abattoir (Laverton, VIC, Australia). The PCF was centrifuged at 16000g for 40 min (Sorvall RC6, Thermo Scientific) to remove solid particles, and the supernatant was stored at −20 °C until further use. Bacterial Strains. Escherichia fergusonii ATCC 35469 (E469) and E. coli ATCC 8739 (E739) from human fecal isolates were obtained from American Type Culture Collection (Manassas, VA, USA). E. coli DH10B (E10B) was from Invitrogen (VIC, Australia). All strains were stored at −80 °C in de Man−Rogosa−Sharpe medium (MRS medium; Oxoid; SA, Australia) supplemented with 30% (v/v) glycerol as a cryoprotectant. Medium and Growth Conditions for Fermentation Experiments. Modified medium for colon bacteria (mMCB), which supports the growth of colonic bacteria,20 was selected for fermentation experiments. The medium consisted of 0.65% (all w/v) bacteriological peptone (Oxoid), 0.5% soy peptone (Oxoid), 0.3% yeast extract (Oxoid), 0.25% tryptone (Oxoid), 0.45% NaCl, 0.2% KCl, 0.05% MgSO4·7H2O, 0.045% CaCl2·2H2O, 0.02% NaHCO3, 0.02% MnSO4· H2O, 0.0005% FeSO4·7H2O, 0.0005% ZnSO4·7H2O, 0.04% L-cysteine HCl·H2O as a reducing agent, and 1.0 mL of resazurin solution (made from 0.01% w/v) as an anaerobic indicator. This growth medium was supplemented with 5% (v/v) PCF for selected experiments. This
then THC in the presence of nicotinamide adenine dinucleotide phosphate (NADPH).19 The mechanism of CurA is presumed to occur through the reduction of CC bonds only and not CO bonds on the heptadienone linkage of curcumin.19 To gain more information on the metabolism of curcuminoids by gut bacteria, we studied the in vitro interaction of curcuminoids with a pure culture of Escherichia fergusonii (ATCC 35469 (E469)) and two strains of E. coli (ATCC 8739 (E739) and DH10B (E10B)). These bacteria were chosen as they all contain the YncB gene that encodes for CurA. A basic local alignment search tool (BLAST) program revealed that the predicted amino acid sequence of Escherichia fergusonii ATCC 35469 (E469) and E. coli ATCC 8739 (E739) exhibited 90% and 97% similarity, respectively, to the deduced amino acid sequence of CurA from E. coli DH10B (E10B). The amount of curcuminoids degraded by the three Escherichia bacteria was quantified using liquid chromatography−mass spectrometry (LC-MS) and multiple reaction monitoring (MRM). The breakdown products produced were identified and characterized using tandem mass spectrometry (MS/MS) and high-resolution Fourier Transform-ion cyclotron resonance MS (FT-ICR-MS) and then quantified using MRM.
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MATERIALS AND METHODS
Materials. Commercially available curcumin, which consists of a mixture of three naturally occurring curcuminoids, curcumin (80.1%), DMC (15.6%), and Bis-DMC (2.6%), pure standards of two possible breakdown products, THC and FA, and the internal standard (IS) used, 11006
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Figure 1. Growth of Escherichia fergusonii (E469) (A), E. coli 739 (E739) (B), and E. coli DH10B (E10B) (C) in mMCB with or without Tween 20 and PCF and supplemented with curcuminoids. Each data point is the average of three biological replicates ± SD. at 805g for 5 min at 0 °C (Eppendorf centrifuge 5810R) to remove cell debris. The supernatant was kept at −20 °C prior to extraction. Curcuminoids and Metabolite Extraction. The procedure for extraction of curcuminoids and transformed metabolites was adapted and optimized from that of Saradhi et al.21 Briefly, 0.5 mL of supernatant and 1 mL of ethyl acetate with 100 μg/mL IS (β-estradiol-17-acetate) were mixed. The sample was then vortexed and centrifuged at 11000g for 5 min (Eppendorf Centrifuge 5424). The upper organic layer (∼1 mL) was transferred to a clean microfuge tube. The same extraction procedure was repeated using the lower aqueous layer. Both upper organic layers were then combined and dried (SpeedVac, Christ RVC 233, John Morris Scientific) for about 2 h at 20 °C or until completely dry. The dried extracts were then resuspended in 100 μL of LC-MS running buffer containing 10 mM ammonium acetate in an acetonitrile/water (50%, v/v) solution, vortexed, and further diluted by combining 10 μL of resuspended extract and 90 μL of LC-MS running buffer. This solution was then vortexed and centrifuged at 11000g for 5 min, and 80 μL was transferred to vials, which were capped and ready for LC-MS analysis. Identification and Quantification of Extracted Curcuminoids and Metabolites. Chromatographic separation of curcuminoids and metabolites was performed with an LC 1200 series system (Agilent Technologies, Santa Clara, CA, USA), which consisted of a Binary Pump SL and Hi-P-Autosampler (with injection volume of 2 μL). Elution was achieved with an Agilent Poroshell 120 EC-C8 column (2.1 × 50 mm, with a particle size of 2.7 μm). The mobile phases consisted of 10 mM ammonium acetate and 0.1% (v/v) formic acid in water (A) or in acetonitrile (B). A linear gradient was applied as follows with a flow rate of 0.3 mL min−1: 0−5 min, 20% B; 5−8.1 min, 80% B; 8.1−10.0 min, 20% B; with a total run time of 10 min. Analysis was performed using a 6460 triple-quadrupole mass spectrometer (Agilent Technologies) equipped with an electrospray ionization (ESI) source operating in negative mode. For the identification and quantification of compounds,
medium was prepared anaerobically and transferred (10 mL) to glass tubes in an anaerobic culturing hood. For subculture preparations, Escherichia fergusonii and E. coli were cultured individually on lysogeny agar plates containing 1% (all w/v) tryptone, 0.5% yeast extract, 1% NaCl, and 2% agar (Oxoid). Under these conditions colonies were visible after 24 h of incubation at 37 °C. One colony of each culture was then grown in separate glass tubes containing 10 mL of mMCB, as above, under anaerobic conditions at 37 °C for 2 h. For fermentation assays, 100 μL of the subculture was inoculated into separate glass tubes containing 10 mL of mMCB and supplemented with curcuminoids at a concentration of 0.3 mM (solubilized in 3% (v/v) of Tween 20). Blank control samples containing only mMCB or mMCB + PCF but without curcuminoids were also inoculated. The initial pH of the fermentation medium was pH 6.27 ± 0.02. A set of abiotic samples with or without curcuminoids in mMCB or mMCB + PCF were maintained to account for the stability of curcuminoids at the start and end of fermentation. Fermentation temperature was kept constant at 37 °C and maintained for 36 h. Samples were acquired at 12 different time points for further analysis. The fermentation was performed on three separate occasions. Bacterial Growth. The growth curves of bacterial strains were obtained by measuring the optical density (OD) at 600 nm using a Nanodrop 2000C spectrophotometer with a UV−vis detector (Thermo-Fisher Scientific). Triplicate measurements were obtained, and data are presented as the mean ± SD. Quenching and Cell Lysis. Following incubation, bacterial metabolism was quenched by adding 2 mL of cold methanol/water (60%, v/v) solution to 1 mL of cell culture. To lyse the cell, samples were frozen in liquid nitrogen, thawed on dry ice, and then sonicated in a microfuge tube within a sonicator bath for ∼3 min. This lysis procedure was repeated 10 times. The samples were then vortexed and centrifuged 11007
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Figure 2. MS/MS spectra of DHC and curcumin from triple-quadrupole mass spectrometer operating in negative mode with a fragmentor voltage of 110 V and collision energy of 25 V (A) and 10 V (B) and their neutral chemical structures and fragmentation sites, DHC (top) and curcumin (bottom). The product ions shown on the spectra are the [M − H]− ions formed by loss of a proton from either of the phenol moieties. a MRM analysis was employed, tracking the transition indicative of parent and product ion specific for each compound. Previous direct infusion experiments were performed to optimize the fragmentor voltage and collision energy (CE). After infusion had been performed, the following parameters were fixed: drying N2 temperature, 300 °C; gas flow, 7 L min−1; nebulizer pressure, 15 psi; sheath gas temperature, 400 °C; sheath gas flow, 10 L min−1; capillary voltage, 2000 V; nozzle voltage, 1000 V; cell accelerator voltage, 7 V; dwell time, 20 ms. Detailed transitions of parent molecules and product ions, MS parameters (fragmentor voltage and CE), limits of detection (LODs), and limits of quantification (LOQs) are listed in Table 1. The concentrations of curcuminoids curcumin, DMC, and Bis-DMC were quantified on the basis of a calibration curve for curcumin. It was assumed that the response factors for DMC and Bis-DMC were the same as that for curcumin. The metabolites THC and FA were also quantified using standards. For the quantification of the metabolite with m/z [M − H]− 369 and the compound with m/z [M − H]− 470, it was assumed that these also had the same response factor as curcumin. The area under the curve from the LC-MS spectra of each compound was normalized using the IS and then converted to concentration using the standard calibration curves. Separate spiking experiments with known quantities of the curcuminoids, and the metabolites THC and FA in mMCB medium were carried out. Recoveries for all five compounds were ∼80%. All values given in the tables and figures were corrected for recovery (assuming 80% for all compounds). Identification of Compounds with m/z [M − H]− 369 and m/z [M − H] − 470. LC-MS/MS using a triple-quadrupole mass spectrometer and accurate mass using FT-ICR-MS were used to confirm the identity of compounds with m/z [M − H]− 369 and m/z [M − H]− 470. LC-MS/MS. Chromatographic separation conditions were as described above. Analysis was performed using a 6460 triple-quadrupole mass spectrometer (Agilent Technologies) equipped with an ESI ion
source operating in negative mode. A product ion scan was employed for confirmation of the metabolite. The acquisition parameters were as follows: CE, 10, 20, and 35 V; fragmentor voltage, 110 V; MS scan range from 50 m/z (min) to 500 m/z (max); drying N2 temperature, 300 °C; gas flow, 7 L min−1; nebulizer pressure, 15 psi; sheath gas temperature, 400 °C; sheath gas flow, 10 L min−1; capillary voltage, 2000 V; noozle voltage, 1000 V. FT-ICR-MS. A Bruker Daltonics (Bremen, Germany) SolariX 7 T Hybrid MALDI/ESI-FT-ICR-MS was used for isolation and high mass resolution analysis. The instrument was operated in ESI negative ionization mode with TD acquisition = 1M, MS detection (resolution 260000 at m/z 400) using an external calibration of sodium trifluoroacetate solution, and MS/MS detection (resolution 45000 at m/z 400). The sample was infused at a rate of 2 μL min−1 using the syringe pump, and a total of 20 scans were collected and averaged. The following general instrument conditions were set: capillary voltage, 4200 V; drying gas flow, 4.0 L min−1; drying gas temperature, 180 °C; nebulizer gas flow rate, 0.8 bar; source accumulation, 0.001 s; ion accumulation, 0.5 s; ion cooling time, 0.01 s; flight time, 0.001 s. Various collision voltages were used with a collision gas flow rate of 40%. Data were analyzed using Bruker Compass Data Analysis 4.2 (Build 383.1) software. Statistical Analysis. Statistical analysis was performed using the statistical package Minitab for Windows (version 17). Differences in the data were analyzed using one-way analysis of variance (ANOVA). The effect of two different treatments, (i) without PCF and (ii) with PCF, on the ability of the three bacterial strains to degrade curcuminoids at 36 h of fermentation was also tested. Tukey pairwise comparisons were used to identify the differences and statistical significance defined as p < 0.05.
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RESULTS AND DISCUSSION Growth of Escherichia fergusonii and Two E. coli Strains. The three Escherichia bacterial strains demonstrated a
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Figure 3. Full scan MS mass spectra of isolated curcumin adduct displaying isotopic profile (A) and the calculated isotopic profile of the predicted C24H25NO7S compound (B).
growth curve typical of batch culture in mMCB media, with a lag phase of around 2−3 h followed by exponential growth and a stationary phase beyond 8 h (Figure 1). The growth of all strains was not markedly affected by the addition of Tween 20 to solubilize the curcumin (3%, v/v) (Figure 1). Others have observed that Tween 20 (0.5%, w/w) in 0.1 M phosphate buffer disintegrated the cell wall and cytoplasmic membrane of Listeria monocytogenes and Salmonella typhimurium.22 It is likely that differences in the responses of bacteria to curcumin may vary with growth medium and microbial species. Indeed, the addition of medium was observed to counter the nonreversible damage caused to cells grown in buffer.23 The addition of PCF (5%, v/v) slightly increased bacterial growth and the OD600 nm increased by ∼0.02−0.05 units (Figure 1). PCF is known to assist the growth of facultative anaerobic bacteria. Similar findings were observed previously when media containing either ruminal fluid or pig cecal extract supported the growth of a larger number of gut bacteria compared to a medium rich in nutrients.24 The growth of E469, E739, and E10B in the first 8 h of fermentation was not affected by the supplementation of 0.3 mM curcuminoids, as shown by the profiles (Figure 1). However, a decline in OD600 nm was observed for E469 and E10B during the stationary phase, indicating a change in metabolism. A similar decline was observed for E469 in the presence of PCF and Tween 20. The pH values at the start and end of the fermentation were 6.27 ± 0.02 and 6.00 ± 0.05, respectively. Curcumin is stable at these pH values, but at alkaline pH >7.5, it degraded chemically within 30 min to ferulic acid, feruloylmethane, and vanillin.25,26
Therefore, changes in curcumin are likely to be due to bacterial metabolism. Identity of Compounds Formed in Fermentation Cultures with Curcumin. The upper organic and lower aqueous fractions for all extracted samples were examined by LCMS using a full MS scan operating in both negative and positive modes from m/z 50 to 800 (method S1, Supporting Information). Four compounds, in addition to the curcuminoids added, were identified from the full scan LC-MS spectra of the upper organic fraction of inoculated samples supplemented with curcuminoids. The full scan LC-MS spectra showing the total ion chromatogram of sample with 0.3 mM curcuminoids inoculated with E469 in mMCB and the extracted full mass spectra of one of the compounds are shown in Figure S1 (Supporting Information). Two of these additional compounds, THC and FA, were identified by comparison to standards. The other two compounds with m/z (all in [M − H]−) 369 and 470 were identified using LC-MS/MS and FT-ICR-MS, respectively. Compounds with m/z [M − H]− 369 and 470 were confirmed to be dihydrocurcumin (DHC) and a curcumin adduct (curcumin−L-cysteine), respectively, as discussed below. No metabolites could be identified in the lower aqueous fractions. Confirmation of Compounds with m/z [M − H]− 369. Full scan LC-MS of samples grown in the presence of curcuminoids gave rise to product ion with m/z [M − H]− 369 (data not shown), consistent with the [M − H]− ion of DHC (370 MW). To ensure that this compound putatively identified as DHC was not an isotope of curcumin, LC-MS/MS of DHC and curcumin in negative mode were performed and their spectra together with neutral chemical structures are shown in Figure 2. A fragmentor 11009
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Table 2. Calculation of Molecular Formulas from High-Resolution FT-ICR-MS Analysis of the Compound (m/z [M − H]− 470) and Most Abundant Fragment Ions Observed from the Analysis of Collision-Induced Decay (CID) MS/MS Data metabolite
formula
curcumin L-cysteine curcumin + cysteine curcumin−L-cysteine 392 fragment 167 fragment
C21H20O6 C3H7NO2S
calcd formula
C24H27NO8S C24H25NO7S C23H22NO5− C8H7O2S−
neutral mass
calcd m/z, [M − H]−
obsd m/z, [M − H]−
error (ppm)
368.12544
367.11871
367.11876
−0.25
489.14574 471.13462 393.15762 168.0245
470.12789 392.15035 167.01722
470.12809 392.15045 167.01725
−0.41 −0.27 −0.14
Figure 4. MS/MS analysis of curcumin adduct (curcumin−L-cysteine) from FT-ICR-MS (A) and triple-quadrupole mass spectrometer (B) and the proposed chemical structure of the adduct. MS/MS fragmentation patterns of curcumin adduct (curcumin−L-cysteine) from both instruments showed two intense fragment ions at nominal masses of 392 and 167.
functionality. For DHC, which possesses a reduced alkene with two hydrogen atoms, the corresponding mass shifted product ions 175 and 219 were observed (Figure 2). The product ion 173 was also observed for DHC, where the presence of a single alkene functionality and corresponding fragmentation at the ketone produce an identical product ion to that of curcumin. Confirmation of Compound with m/z [M − H]− 470. To putatively identify the compound with m/z [M − H]− 470, high mass accuracy FT-ICR-MS mass spectra of the compound were collected. Direct infusion of the crude culture media provided a complex signal; further isolation of a narrow mass range (10 m/ z) centered around m/z of 470 found a single peak at m/z [M − H]− 470.12809 as shown in Figure 3. Calculation of the molecular formula from the accurate mass gave only a single possible formula, C24H25NO7S (calculated m/z [M − H]− 470.12789), with a very low mass error of −0.41 ppm, as shown in Table 2. The isotopic pattern of the compound with m/
voltage of 110 V and CE of 25 and 10 V were applied to both compounds, producing product ions of m/z 134, 149, 173, 175, and 219 for DHC and m/z 134, 149, 173, and 217 for curcumin (Figure 2). Differences between the product ions of DHC and curcumin were attributed to their chemical structures. Curcumin possesses two alkenyl CC bonds within the heptadienone backbone,1 whereas DHC is the reduced form of curcumin in which one of the alkenes has been reduced.19 DHC has been previously reported as the intermediate breakdown product of curcumin by the enzyme CurA, through the reduction of an alkene present on the heptadienone chain.19 The common product ions of both curcumin and DHC can be rationalized through the presence of at least one CC bond, where the observed [M − H]− product ions m/z 134 and 149 correspond to fragmentation across the enone functionality (149) with a further loss of a single methyl group (134) (Figure 2). The other product ions for curcumin, 173 and 217, correspond to fragmentation at the ketone 11010
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Figure 5. Amount of curcuminoids: curcumin (A, B), DMC (C, D), and Bis-DMC (E, F) measured during fermentation with Escherichia fergusonii (E469), E. coli 739 (E739), and E. coli DH10B (E10B) in mMCB without PCF (A, C, E) or with PCF (5%, v/v) (B, D, F). The amount of curcuminoids in the upper organic extracts was quantified using LC-MS/MS and expressed as total micromoles in the original experimental broth of 10.1 mL. The amounts of curcuminoids in the control (abiotic) samples were measured at 0 and 36 h. Each data point is the average of three biological replicates ± SD.
z [M − H]− 470 from FT-ICR-MS is similar to the calculated isotopic peak spacing of the compound, as shown in Figure 3. L-Cysteine was present in the growth medium (mMCB) to reduce the oxido/reduction potential. The amount added far exceeds that required for the formation of the curcumin−Lcysteine adduct. The predicted formula of the metabolite matches an addition product of curcumin (C21H20O6) and Lcysteine (C3H7NO2S) with a loss of water. We propose an initial 1,4-addition product, curcumin adduct (curcumin−L-cysteine), with a possible chemical structure as shown in Figure 4, which undergoes dehydration either in vitro or within the MS source. In support of our putative assignment, curcumin possesses two α,βunsaturated ketone moieties that can act as Michael acceptors to nucleophilic attack through a Michael-type addition reaction mechanism.27 Furthermore, curcumin has previously been found to bind covalently to nucleophilic sulfhydryls and the selenol moiety of selenocysteine.28,29 Thioredoxin reductase, a selenocysteine-containing enzyme, was irreversibly inhibited by curcumin through a Michael-type addition reaction only in the presence of NADPH, with inhibition persisting after the removal of curcumin.28 To explore the mechanism of formation of the putatively identified curcumin adduct, we performed a separate experiment by incubating mMCB with and without 0.3 mM curcuminoids at 37 °C for 24 h and removed the bacterial cells to produce spent media. Both spent media samples were further supplemented with 0.3 mM curcuminoids and incubated for another 24 h. The curcumin adduct was observed only in spent media where curcuminoids were co-incubated with mMCB (data not shown). This suggests that the curcumin adduct is formed only in the presence of bacteria. We propose that this curcumin adduct may be formed through a Michael-type addition, possibly in the
presence of NADPH. It is probable that NADPH was produced in the fermentation cultures in this study through bacterial synthesis or another biological reductant may be employed. Additional MS/MS analysis by FT-ICR-MS and triplequadrupole MS showed two intense fragment ions at nominal masses of 392 and 167, as demonstrated in Figure 4. Accurate mass analysis gave masses of 392.15045 and 167.01725 with calculation of the molecular formula indicating empirical formulas of C23H22NO5− (calculated, 392.15035; mass error, −0.26 ppm) and C8H7O2S− (calculated mass, 167.01722; mass error, −0.14 ppm) as probable fragmentation products of a curcumin−L-cysteine adduct, as shown in Table 2. Although isolation and elucidation of the exact chemical structure is beyond the scope of the current study, data presented here can be used to assist in future determination of the exact chemical structure of the compound with m/z [M − H]− 470. Quantification of Compounds Formed in Fermentation Cultures with Curcumin. Change in Curcuminoid Concentrations during Fermentation. The extent to which the three curcuminoids (curcumin, DMC, and Bis-DMC) were degraded by the bacteria E469, E739, and E10B during batch fermentation for 36 h was determined using LC-MS. The effect of supplementing the media with PCF (5%, v/v) on the ability of all three strains to degrade curcuminoids was examined. Blank control samples containing only mMCB or mMCB with PCF without curcuminoids showed no or negligible signal interference in the LC-MS spectra of curcuminoids and metabolites. The amounts of curcuminoids (curcumin, DMC, and BisDMC) present in abiotic control samples in mMCB (Figure 5A,C,E) and in mMCB with PCF (Figure 5B,D,F) did not change at the start and end of fermentation. This may be expected as curcumin is stable at the pH of the fermentation 11011
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Table 3. Measured Curcuminoids Curcumin, DMC, Bis-DMC, Metabolite FA, and Curcumin Adduct Curcumin−L-Cysteine Detected in the Fermentation Culturea fermentation medium: mMCB
fermentation medium: mMCB with 5% (v/v) PCF
E. fergusonii E469
E. coli E739
E. coli E10B
E. fergusonii E469
E. coli E739
E. coli E10B
curcumin initial t = 0 h (μmol) final t = 36 h (μmol) % converted
2.20 ± 0.09 1.38 ± 0.03 37.3 ± 3.79a
2.13 ± 0.06 1.71 ± 0.06 19.9 ± 1.65bc
2.16 ± 0.02 1.69 ± 0.01 20.8 ± 0.42bc
2.19 ± 0.04 1.68 ± 0.03 23.2 ± 1.24b
2.13 ± 0.07 1.80 ± 0.04 15.8 ± 1.45c
2.17 ± 0.01 1.82 ± 0.05 16.1 ± 2.09c
DMC initial t = 0 h (μmol) final t = 36 h (μmol) % change
0.43 ± 0.004 0.40 ± 0.003 7.09 ± 1.42ab
0.42 ± 0.005 0.37 ± 0.002 16.3 ± 5.57a
0.42 ± 0.002 0.39 ± 0.010 8.10 ± 2.11ab
0.43 ± 0.007 0.39 ± 0.011 9.12 ± 2.21ab
0.43 ± 0.015 0.40 ± 0.008 5.69 ± 1.69b
0.43 ± 0.006 0.40 ± 0.016 7.78 ± 4.91ab
Bis-DMC initial t = 0 h (μmol) final t = 36 h (μmol) % change
0.08 ± 0.001 0.07 ± 0.001 7.22 ± 0.76b
0.08 ± 0.001 0.07 ± 0.001 10.8 ± 1.33ab
0.08 ± 0.001 0.07 ± 0.001 14.8 ± 2.40a
0.08 ± 0.001 0.07 ± 0.002 7.21 ± 2.04b
0.07 ± 0.002 0.07 ± 0.001 6.58 ± 1.65b
0.08 ± 0.002 0.07 ± 0.002 9.60 ± 2.79b
ferulic acid (FA) final t = 36 h (μmol) curcuminoid equivb (μmol)
0.13 ± 0.005 0.07 ± 0.003b
0.06 ± 0.002 0.03 ± 0.001c
0.05 ± 0.005 0.03 ± 0.003c
0.14 ± 0.003 0.08 ± 0.002a
0.04 ± 0.003 0.02 ± 0.002d
0.03 ± 0.003 0.02 ± 0.002d
curcumin−L-cysteine final t = 36 h (μmol) curcuminoid equivc (μmol)
0.43 ± 0.02 0.43 ± 0.02a
0.23 ± 0.01 0.23 ± 0.01c
0.21 ± 0.01 0.21 ± 0.01cd
0.28 ± 0.02 0.28 ± 0.02b
0.17 ± 0.01 0.17 ± 0.01d
0.22 ± 0.01 0.22 ± 0.01c
% curcuminoids accountedd
86.8 ± 2.96b
90.8 ± 2.57ab
90.4 ± 0.91ab
92.6 ± 1.32a
93.5 ± 1.06a
94.4 ± 2.22a
compound
a
The values for the amounts have been corrected for the recoveries of compounds and represent the amount present in 10.1 mL of fermentation medium. This was 80% for curcumin, DMC, Bis-DMC, and FA, as determined from separate spiking experiments. As curcumin−L-cysteine was not commercially available, spiking experiments could not be carried out, and the values are those as determined without correction for recovery. Means with the same letters within the same row are not significantly different. bCurcuminoid equivalents (equiv) were calculated assuming the following: (i) 1 mol of curcumin gives 2 mol of FA and 1 mol of DMC gives 1 mol of FA. (ii) The initial curcuminoid mixture contained 80.1:15.6 (curcumin/ DMC); therefore, FA obtained from this mixture on a molar basis would be 1.837. cCurcuminoid equivalents (equiv) were calculated assuming 1 mol of curcumin gives 1 mol of curcumin−L-cysteine. dPercent curcuminoids accounted for {[μmol of remaining curcuminoids + μmol FA/1.837) + μmol curcumin−L-cysteine)]/(initial μmol curcumin + initial μmol DMC + initial μmol Bis-DMC)} × 100.
E739 (19.9%) and E10B (20.8%), there were no significant differences (p > 0.05) between degradation by E739 and E10B in the absence of PCF. Similar results were obtained in the presence of PCF, where curcumin conversion was significantly higher (p < 0.05) with E469 (23.2%) compared to E739 (15.8%) and E10B (16.1%). The effects of PCF addition on curcumin degradation were strain dependent. In the presence of PCF, the degradation of curcumin by E469 was significantly reduced (p < 0.05). However, the addition of PCF had no significant effect (p > 0.05) on the ability of E739 and E10B to degrade curcumin. The amounts of DMC altered in the fermented cultures for all strains in mMCB were not significantly different (p > 0.05). This was also the case for all strains in mMCB with added PCF (Table 3). The change in the amount of DMC in the presence of E739 was significantly higher (p < 0.05) in mMCB (16.3%) than in mMCB with added PCF (5.7%), but changes in DMC amounts were not significantly affected by the addition of PCF (p > 0.05) when E469 or E10B was used. The amount of Bis-DMC in mMCB changed by E10B (14.8%) was significantly higher (p < 0.05) than that changed by E469 (7.2%), but there were no significant differences (p > 0.05) between E739 and E469. The ability of all three strains to convert curcumin in mMCB was in the order E10B > E739 ∼ E469. The changes in the amounts of Bis-DMC in the presence of all three strains were not significantly different (p > 0.05) in mMCB with
media (pH 6.00−6.27 over the fermentation course of 36 h). It is only at pH >7.5 that curcumin is unstable.25,26 The absence of an observable change in abiotic samples shows that there is no chemical degradation of curcuminoids over this time and confirms that any change in curcuminoids in the presence of the cultures is the result of bacterial metabolism. The degradation profiles of curcuminoids (curcumin, DMC, and Bis-DMC) in the presence of bacteria over the 36 h fermentation period are also shown in Figure 5. In the absence of PCF, a decrease in curcumin was observed until about 8−10 h, coincident with the onset of the stationary phase (Figure 1); the level of curcumin then stayed relatively constant until 36 h (Figure 5A), whereas with PCF, the level of curcumin decreased until around 12 h and then stayed relatively constant until 36 h (Figure 5B). A slight decrease was also observed in the amounts of DMC and Bis-DMC following 36 h of fermentation, as demonstrated in Figure 5C,D and E,F, respectively. The initial measured amounts of curcuminoids in fermentation cultures at 0 and 36 h are given in Table 3. The significant differences between the ability of the three bacterial strains to degrade curcuminoids in mMCB with or without PCF at 36 h were compared. The ability of all three strains to convert curcumin, in mMCB media with or without PCF, was in the order E469 > E10B ∼ E739. Whereas E469 degraded 37.3% of curcumin, which is significantly higher (p < 0.05) than that with 11012
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Figure 6. Measured amounts of FA (A, B) and curcumin adduct (curcumin−L-cysteine) (C, D) during fermentation with Escherichia fergusonii (E469), E. coli 739 (E739), and E. coli DH10B (E10B) in mMCB without PCF (A, C) or with PCF (5%, v/v) (B, D). The amount of FA and curcumin adduct (curcumin−L-cysteine) in the upper organic extracts were quantified using LC-MS/MS and expressed as total micromoles in experimental broth of 10.1 mL. Each data point is the average of three biological replicates ± SD.
Figure 7. Proposed degradation of the three curcuminoids (curcumin, DMC, and Bis-DMC) and breakdown products formed by Escherichia fergusonii (E 469), E. coli 739 (E739), and E. coli DH10B (E10B). Numbers in parentheses represent the theoretical conversion of curcuminoids to metabolites. D, detected; N.D, not detected in this study.
curcumin−L-cysteine during fermentation is shown in Figure 6, and the amounts quantified after 36 h are given in Table 3. In mMCB without PCF, the formation of FA by E469 (0.07 μmol) was significantly higher (p < 0.05) than the amounts formed by E739 and E10B (0.03 μmol), but there were no significant differences (p > 0.05) between E739 and E10B. In mMCB with PCF, the formation of FA by E469 (0.08 μmol) was also significantly higher (p < 0.05) than that by E739 and E10B (0.02 μmol), but there were no significant differences (p > 0.05)
added PCF. The ability of E469 and E739 to convert Bis-DMC was not significantly affected by the addition of PCF (p > 0.05). However, a significant increase (p < 0.05) was observed for changes in Bis-DMC when PCF was not added to mMCB in the presence of E10B (14.8% compared from 9.6%). Quantification of Compounds Formed in Fermentation Cultures with Curcumin. DHC and THC were minor metabolites of curcumin degradation, and they constitute 0.05) between E739 and E10B. In mMCB with PCF, the formation of curcumin−Lcysteine by E469 (0.28 μmol) was significantly higher (p < 0.05) than those by E10B (0.22 μmol) and E739 (0.17 μmol). In the same medium, the formation of curcumin−L-cysteine by E10B was also significantly higher (p < 0.05) than that by E739. The formation of curcumin−L-cysteine by E469 and E739 in the absence of PCF (0.43 and 0.23 μmol, respectively) was significantly higher (p < 0.05) than in the presence of PCF (0.28 and 0.17 μmol, respectively). The formation of curcumin− L-cysteine by E10B was not significantly different (p > 0.05) in the absence (0.21 μmol) or presence of PCF (0.22 μmol). Accountability for Curcuminoids. The amount of curcumin accounted for was also calculated and found to be 87−94%. The amount of curcumin accounted for was not significantly different (p > 0.05) between the three Escherichia bacteria in mMCB or mMCB with PCF (Table 3). However, the amount of curcumin accounted for E469 in the presence of PCF (92.6%) was significantly higher (p < 0.05) than in the absence of PCF (86.8%), but there were no significant differences (p > 0.05) for E739 and E10B in the presence or absence of PCF. The level of accountability, which represents remaining curcuminoids (curcumin, DMC, and Bis-BMC) and curcuminoid equivalents for conversion into ferulic acid and curcumin−L-cysteine, is high. In a different system, when curcuminoids were incubated with rat liver slices, there was ∼50% of curcumin and DMC and ∼75% for Bis-DMC accounted for.11 These authors attributed the lack of accountability for the total amount of curcuminoids to possible spontaneous chemical degradation in aqueous solution and conversion to other breakdown products that were not detected by the methods used.11 Proposed Pathways of Degradation of Curcuminoids by Bacterial Strains Examined. The proposed pathways of degradation of the three curcuminoids are demonstrated in Figure 7. Curcumin is converted to DHC and then THC by the enzyme CurA, through the reduction of CC bonds on the heptadienone chain.19 In studies involving ingestion of curcumin in humans and animals it was found that this pathway was rapid and that curcumin and metabolites were subsequently glucuronidated or sulfated by enzymes present in the liver.10,11 Both curcumin and DHC can also be converted to FA, as shown in Figure 7. FA was found to be one of the major metabolites in the serum, urine, and feces of humans after consumption of encapsulated curcumin in bread, and the authors suggested that the presence of FA was probably as a result of gut bacteria metabolism.12 Our data are consistent with this hypothesis. In our in vitro system, the presence of a curcumin adduct, curcumin−L-cysteine, was also found. This adduct was observed only in fermented samples in the presence of curcuminoids and bacteria and not present in abiotic samples. The adduct was also observed only in spent media where curcuminoids were coincubated with mMCB, confirming that the presence of bacteria was necessary for the formation of the curcumin adduct. However, from these studies alone it is not possible to assign
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ASSOCIATED CONTENT
S Supporting Information *
Method S1, profiling of breakdown products using full scan LCMS; Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(M.A.A.) E-mail: [email protected]. Funding
S.T. is supported by a CSIRO OCE Postgraduate Scholarship and a University of Melbourne MIFRS Scholarship. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank A/Prof Stuart Dashper of University of Melbourne for the use of the anaerobic culturing facilities. We also thank Patrick Daniel of Department of Primary Industries for supplying the pig cecal fluid.
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ABBREVIATIONS USED DMC, demethoxycurcumin; Bis-DMC, bis-demethoxycurcumin; DHC, dihydrocurcumin; THC, tetrahydrocurcumin; FA, ferulic acid; E. coli, Escherichia coli; NADPH, nicotinamide 11014
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(18) Jazayeri, S. D.; Mustafa, S.; Manap, M. Y.; Ali, A. M.; Ismail, A.; Faujan, N. H.; Shaari, M. Y. Survival of Bifidobacteria and other selected intestinal bacteria in TPY medium supplemented with curcumin as assessed in vitro. Int. J. Probiotics Prebiotics 2009, 4, 15−22. (19) Hassaninasab, A.; Hashimoto, Y.; Tomita-Yokotani, K.; Kobayashi, M. Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism. Proc. Natl. Acad. Sci. U.S.A., Early Ed. 2011, 1−6. (20) Van der Meulen, R.; Makras, L.; Verbrugghe, K.; Adriany, T.; De Vuyst, L. In vitro kinetic analysis of oligofructose consumption by Bacteroides and Bifidobacterium spp. indicates different degradation mechanisms. Appl. Environ. Microbiol. 2006, 72, 1006−1012. (21) Saradhi, U.; Ling, Y. H.; Wang, J. A.; Chiu, M.; Schwartz, E. B.; Fuchs, J. R.; Chan, K. K.; Liu, Z. F. A liquid chromatography-tandem mass spectrometric method for quantification of curcuminoids in cell medium and mouse plasma. J. Chromatogr., B 2010, 878, 3045−3051. (22) Cherepova, N.; Veljanov, D. Effect of sorbitan monolaurate polyoxyalkylene (Tween 20) on the ultrastructure of some bacteria. Cytobios 1994, 80, 179−185. (23) Ingram, L. O. Ethanol tolerance in bacteria. Crit. Rev. Biotechnol. 1990, 9, 305−319. (24) Salanitro, J. P.; Muirhead, P. A. Quantitative method for gaschromatographic analysis of short chain monocarboxylic and dicarboxylic-acids in fermentation media. Appl. Microbiol. 1975, 29, 374−381. (25) Tonnesen, H. H.; Karlsen, J. Studies on curcumin and curcuminoids. 5. Alkaline-degradation of curcumin. Z. Lebensm.-Unters. -Forsch. 1985, 180, 132−134. (26) Tonnesen, H. H.; Karlsen, J. Studies on curcumin and curcuminoids. 6. Kinetics of curcumin degradation in aqueous-solution. Z. Lebensm.-Unters. -Forsch. 1985, 180, 402−404. (27) Gupta, S. C.; Prasad, S.; Kim, J. H.; Patchva, S.; Webb, L. J.; Priyadarsini, I. K.; Aggarwal, B. B. Multitargeting by curcumin as revealed by molecular interaction studies. Nat. Prod. Rep. 2011, 28, 1937−1955. (28) Fang, J. G.; Lu, J.; Holmgren, A. Thioredoxin reductase is irreversibly modified by curcumin − a novel molecular mechanism for its anticancer activity. J. Biol. Chem. 2005, 280, 25284−25290. (29) Jung, Y. J.; Xu, W. P.; Kim, H.; Ha, N.; Neckers, L. Curcumininduced degradation of ErbB2: a role for the E3 ubiquitin ligase CHIP and the Michael reaction acceptor activity of curcumin. Biochim. Biophys. Acta−Mol. Cell Res. 2007, 1773, 383−390.
adenine dinucleotide phosphate; LC-MS, liquid chromatography−mass spectrometry; IS, internal standard; PCF, pig cecal fluid; E469, Escherichia fergusonii ATCC 35469; E739, Escherichia coli ATCC 8739; E10B, Escherichia coli DH10B; mMCB, modified medium for colon bacteria; OD, optical density; ESI, electrospray ionization; MRM, multiple reaction monitoring; CE, collision energy; Rt, retention time; LOD, limit of detection; LOQ, limit of quantification; AUC, area under the curve; FTICR-MS, Fourier transform-ion cyclotron resonance-mass spectrometer
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REFERENCES
(1) Sharma, R. A.; Gescher, A. J.; Steward, W. P. Curcumin: the story so far. Eur. J. Cancer 2005, 41, 1955−1968. (2) Strimpakos, A. S.; Sharma, R. A. Curcumin: preventive and therapeutic properties in laboratory studies and clinical trials. Antioxid. Redox Signal. 2008, 10, 511−545. (3) Begum, A. N.; Jones, M. R.; Lim, G. P.; Morihara, T.; Kim, P.; Heath, D. D.; Rock, C. L.; Pruitt, M. A.; Yang, F. S.; Hudspeth, B.; Hu, S. X.; Faull, K. F.; Teter, B.; Cole, G. M.; Frautschy, S. A. Curcumin structure-function, bioavailability, and efficacy in models of neuroinflammation and Alzheimer’s disease. J. Pharmacol. Exp. Ther. 2008, 326, 196−208. (4) Aggarwal, B. B.; Sundaram, C.; Malani, N.; Ichikawa, H. Curcumin: the Indian solid gold. In Molecular Targets and Therapeutic Uses of Curcumin in Health and Disease; Springer: New York, 2007; Vol. 595, pp 1−75. (5) Joe, B.; Vijaykumar, M.; Lokesh, B. R. Biological properties of curcumin-cellular and molecular mechanisms of action. Crit. Rev. Food Sci. Nutr. 2004, 44, 97−111. (6) Ireson, C.; Orr, S.; Jones, D. J. L.; Verschoyle, R.; Lim, C. K.; Luo, J. L.; Howells, L.; Plummer, S.; Jukes, R.; Williams, M.; Steward, W. P.; Gescher, A. Characterization of metabolites of the chemopreventive agent curcumin in human and rat hepatocytes and in the rat in vivo, and evaluation of their ability to inhibit phorbol ester-induced prostaglandin E-2 production. Cancer Res. 2001, 61, 1058−1064. (7) Ireson, C. R.; Jones, D. J. L.; Orr, S.; Coughtrie, M. W. H.; Boocock, D. J.; Williams, M. L.; Farmer, P. B.; Steward, W. P.; Gescher, A. J. Metabolism of the cancer chemopreventive agent curcumin in human and rat intestine. Cancer Epidemiol. Biomarkers Prev. 2002, 11, 105−111. (8) Williamson, G.; Clifford, M. N. Colonic metabolites of berry polyphenols: the missing link to biological activity? Br. J. Nutr. 2010, 104, S48−S66. (9) Holder, G. M.; Plummer, J. L.; Ryan, A. J. Metabolism and excretion of curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) in the rat. Xenobiotica 1978, 8, 761−768. (10) Pan, M. H.; Huang, T. M.; Lin, J. K. Biotransformation of curcumin through reduction and glucuronidation in mice. Drug Metab. Dispos. 1999, 27, 486−494. (11) Hoehle, S. I.; Pfeiffer, E.; Solyom, A. M.; Metzler, M. Metabolism of curcuminoids in tissue slices and subcellular fractions from rat liver. J. Agric. Food Chem. 2006, 54, 756−764. (12) Vitaglione, P.; Lumaga, R. B.; Ferracane, R.; Radetsky, I.; Mennella, I.; Schettino, R.; Koder, S.; Shimoni, E.; Fogliano, V. Curcumin bioavailability from enriched bread: the effect of microencapsulated ingredients. J. Agric. Food Chem. 2012, 60, 3357−3366. (13) Ravindranath, V.; Chandrasekhara, N. In vitro studies on the intestinal absorption of curcumin in rats. Toxicology 1981, 20, 251−257. (14) Wahlstrom, B.; Blennow, G. A study on the fate of curcumin in the rat. J. Acta Pharmacol. Toxicol. 1978, 43, 86−92. (15) Fava, F.; Danese, S. Intestinal microbiota in inflammatory bowel disease: friend of foe? World J. Gastroenterol. 2011, 17, 557−566. (16) Tuohy, K. M.; Conterno, L.; Gasperotti, M.; Viola, R. Upregulating the human intestinal microbiome using whole plant foods, polyphenols, and/or fiber. J. Agric. Food Chem. 2012, 60, 8776−8782. (17) Aura, A. M. Microbial metabolism of dietary phenolic compounds in the colon. Phytochem. Rev. 2008, 7, 407−429. 11015
dx.doi.org/10.1021/jf5031168 | J. Agric. Food Chem. 2014, 62, 11005−11015
Degradation of Curcuminoids by in Vitro Pure Culture Fermentation Suryani Tan,†,§,# Thusitha W. T. Rupasinghe,#,⊗ Dedreia L. Tull,#,⊗ Berin Boughton,⊗,△ Christine Oliver,† Chris McSweeny,□ Sally L. Gras,#,⊥ and Mary Ann Augustin*,† †
CSIRO Food and Nutrition Flagship, 671 Sneydes Road, Werribee, Victoria 3030, Australia Department of Chemical and Biomolecular Engineering, #Bio21 Molecular Science and Biotechnology Institute, ⊥The ARC Dairy Innovation Hub, Department of Chemical and Biomolecular Engineering, ⊗Metabolomics Australia, and △School of Botany, The University of Melbourne, Parkville, Victoria 3010, Australia □ CSIRO Agriculture Flagship, 306 Carmody Road, St. Lucia, Queensland 4067, Australia §
S Supporting Information *
ABSTRACT: Colonic bacteria may mediate the transformation of curcuminoids, but studies of this metabolism are limited. Here, the metabolism of curcuminoids by Escherichia fergusonii (ATCC 35469) and two Escherichia coli strains (ATCC 8739 and DH10B) was examined in modified medium for colon bacteria (mMCB) with or without pig cecal fluid. LC-MS analysis showed that 16−37% of curcumin, 6−16% of demethoxycurcumin (DMC) and 7−15% of bis-demethoxycurcumin (Bis-DMC), and 7− 15% of bis-demethoxycurcumin (Bis-DMC) were converted following 36 h of fermentation, with the amount of curcuminoids degraded varying depending on the bacterial strain and medium used. Three metabolites (dihydrocurcumin (DHC), tetrahydrocurcumin (THC), and ferulic acid (FA)) were found in fermentation cultures with all strains used. In addition, a compound with m/z [M − H]− 470 was found and identified to be a curcumin adduct (curcumin−L-cysteine), using accurate mass FT-ICR-MS. This study provides insights into the bacterial metabolism of curcuminoids. KEYWORDS: curcumin, curcuminoids, Escherichia coli, gut bacteria, metabolism
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curcuminoids by gut bacteria.12 Studies on rats showed that about 38% of curcumin ingested remains in the cecum and large intestine of rats.13 Furthermore, ∼75% of curcumin administered orally (1 g/kg) in rats was excreted in feces, but only traces appeared in urine and blood plasma, suggesting that ∼25% of curcumin could be metabolized in the gut.14 Despite the importance of such microbiota-mediated transformation of curcumin, studies on the microbial metabolism of curcuminoids are limited. The human large intestine hosts a highly complex microbial ecosystem containing ∼1014 bacterial cells comprising approximately 1000 different species.15,16 These gut microbiota are capable of transforming polyphenolic compounds by ring cleavage, reduction, decarboxylation, demethylation, and dehydroxylation, thereby allowing the absorption of lower weight metabolites. 17 Six biologically relevant bacterial strains (Bifidobacteria longum BB536, Bifidobacteria pseudocatenulatum G4, Escherichia coli K-12, Enterococcus faecalis JCM 5803, Lactobacillus acidophilus, and Lactobacillus casei) were capable of degrading curcumin, with at least 56% reduction, as judged by loss of the parent compound.18 A recent study reported that a bacterium isolated from human feces, later confirmed to be E. coli, has curcumin-converting ability.19 The enzyme responsible for this curcumin-converting ability was named CurA and once isolated from E. coli strain K-12, substrain DH10B was able to transform curcumin to DHC and
INTRODUCTION The curcuminoids curcumin, demethoxycurcumin (DMC), and bis-demethoxycurcumin (Bis-DMC) are naturally occurring yellow-orange polyphenolic compounds found in the rhizomes of Curcuma longa and other Curcuma species.1,2 The putative health benefits of curcuminoids are related to their potential antiinflammatory,3 antioxidant,4,5 and anticancer6,7 properties, which are influenced by the chemical changes that occur in these compounds and their absorption in vivo.8 Several studies have examined the conversion of curcuminoids in vivo.9−12 After oral (1 g/kg) and intraperitoneal (0.1 g/kg) administrations to mice, curcumin was first biotransformed to dihydrocurcumin (DHC) and tetrahydrocurcumin (THC), and these compounds were then converted to monoglucuronide conjugates.10 In addition, there was a reduction of curcumin through an endogenous reductase and subsequent glucuronidation by UDP-glucuronosyl transferases. 10 Incubation of curcuminoids with liver slices from rat resulted in the reduction of curcumin to DHC, THC, hexahydrocurcumin, and octahydrocurcumin, which were consequently glucuronidated, with tetrahydro, hexahydro, and octahydro forms being mainly present as glucuronides or conjugated with sulfate.11 It was also established that the metabolism of DMC and Bis-DMC follows the same pathways as curcumin with similar reductive products obtained.11 Studies have provided insight into the breakdown of encapsulated curcumin in bread that were found in the serum, urine, and feces of humans. Phenolic acids such as vanillic acid and ferulic acid (FA) were found to be major metabolites, and it was postulated that the presence of phenolic acids in these samples after 24 h was due to the metabolism of nonabsorbed © 2014 American Chemical Society
Received: Revised: Accepted: Published: 11005
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Table 1. Parametersa from LC-Neg-ESI-MS/MS Data for Curcuminoids Curcumin, DMC, Bis-DMC, Known Metabolites DHC, THC, and FA, and a New Compound Detected in the Fermentation Culture MRM parameters compound
Rt (min)
MRM transitions (m/z)
fragmentor voltage
collision energy (CE)
LOD (μM)
LOQ (μM)
curcumin
5.99
367−134b 367−149 367−173
100 100 100
20 20 10
0.01
0.025
DMC
5.97
337−217b 337−173
110 110
10 10
0.01
0.025
Bis-DMC
5.94
307−143b 307−119
110 110
10 10
0.01
0.025
DHCc
5.10
369−219b 369−134 369−149
110 110 110
10 10 25
THC
5.60−6.05
371−235b 371−99 371−135
120 120 120
10 30 30
0.025
0.05
ferulic acid (FA)
1.35
193−134b 193−178
100 100
10 10
0.01
0.025
curcumin adduct (curcumin−L-cysteine)d
5.46
470−167b 470−392 470−152
120 120 120
10 10 10
β-estradiol-17-acetate
6.34
313−185
230
35
a
Rt, retention time; LOD, limit of detection; LOQ, limit of quantification. bPeak area from this transition was used for quantification. cIdentification and confirmation based on molecular weight and MS/MS fragmentation patterns. dCompound confirmation based on accurate mass from FT-ICRMS and structural identification based on MS/MS fragmentation patterns. Response for DHC and curcumin adduct (curcumin−L-cysteine) was assumed to be the same as that for curcumin. β-estradiol-17-acetate, were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Acetonitrile was purchased from Burdick & Jackson (Gillman, SA, Australia), methanol and ethyl acetate were supplied by Merck Pty Ltd. (Kilsyth, Vic, Australia), and all solvents were of HPLC grade. Formic acid and ammonium acetate (analytical grade) were from Ajax FineChem (Waltham, MA, USA). Redistilled deionized water with a resistivity of 18.2 mΩ was used in all experiments. Pig cecal fluid (PCF) was sourced from an abattoir (Laverton, VIC, Australia). The PCF was centrifuged at 16000g for 40 min (Sorvall RC6, Thermo Scientific) to remove solid particles, and the supernatant was stored at −20 °C until further use. Bacterial Strains. Escherichia fergusonii ATCC 35469 (E469) and E. coli ATCC 8739 (E739) from human fecal isolates were obtained from American Type Culture Collection (Manassas, VA, USA). E. coli DH10B (E10B) was from Invitrogen (VIC, Australia). All strains were stored at −80 °C in de Man−Rogosa−Sharpe medium (MRS medium; Oxoid; SA, Australia) supplemented with 30% (v/v) glycerol as a cryoprotectant. Medium and Growth Conditions for Fermentation Experiments. Modified medium for colon bacteria (mMCB), which supports the growth of colonic bacteria,20 was selected for fermentation experiments. The medium consisted of 0.65% (all w/v) bacteriological peptone (Oxoid), 0.5% soy peptone (Oxoid), 0.3% yeast extract (Oxoid), 0.25% tryptone (Oxoid), 0.45% NaCl, 0.2% KCl, 0.05% MgSO4·7H2O, 0.045% CaCl2·2H2O, 0.02% NaHCO3, 0.02% MnSO4· H2O, 0.0005% FeSO4·7H2O, 0.0005% ZnSO4·7H2O, 0.04% L-cysteine HCl·H2O as a reducing agent, and 1.0 mL of resazurin solution (made from 0.01% w/v) as an anaerobic indicator. This growth medium was supplemented with 5% (v/v) PCF for selected experiments. This
then THC in the presence of nicotinamide adenine dinucleotide phosphate (NADPH).19 The mechanism of CurA is presumed to occur through the reduction of CC bonds only and not CO bonds on the heptadienone linkage of curcumin.19 To gain more information on the metabolism of curcuminoids by gut bacteria, we studied the in vitro interaction of curcuminoids with a pure culture of Escherichia fergusonii (ATCC 35469 (E469)) and two strains of E. coli (ATCC 8739 (E739) and DH10B (E10B)). These bacteria were chosen as they all contain the YncB gene that encodes for CurA. A basic local alignment search tool (BLAST) program revealed that the predicted amino acid sequence of Escherichia fergusonii ATCC 35469 (E469) and E. coli ATCC 8739 (E739) exhibited 90% and 97% similarity, respectively, to the deduced amino acid sequence of CurA from E. coli DH10B (E10B). The amount of curcuminoids degraded by the three Escherichia bacteria was quantified using liquid chromatography−mass spectrometry (LC-MS) and multiple reaction monitoring (MRM). The breakdown products produced were identified and characterized using tandem mass spectrometry (MS/MS) and high-resolution Fourier Transform-ion cyclotron resonance MS (FT-ICR-MS) and then quantified using MRM.
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MATERIALS AND METHODS
Materials. Commercially available curcumin, which consists of a mixture of three naturally occurring curcuminoids, curcumin (80.1%), DMC (15.6%), and Bis-DMC (2.6%), pure standards of two possible breakdown products, THC and FA, and the internal standard (IS) used, 11006
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Figure 1. Growth of Escherichia fergusonii (E469) (A), E. coli 739 (E739) (B), and E. coli DH10B (E10B) (C) in mMCB with or without Tween 20 and PCF and supplemented with curcuminoids. Each data point is the average of three biological replicates ± SD. at 805g for 5 min at 0 °C (Eppendorf centrifuge 5810R) to remove cell debris. The supernatant was kept at −20 °C prior to extraction. Curcuminoids and Metabolite Extraction. The procedure for extraction of curcuminoids and transformed metabolites was adapted and optimized from that of Saradhi et al.21 Briefly, 0.5 mL of supernatant and 1 mL of ethyl acetate with 100 μg/mL IS (β-estradiol-17-acetate) were mixed. The sample was then vortexed and centrifuged at 11000g for 5 min (Eppendorf Centrifuge 5424). The upper organic layer (∼1 mL) was transferred to a clean microfuge tube. The same extraction procedure was repeated using the lower aqueous layer. Both upper organic layers were then combined and dried (SpeedVac, Christ RVC 233, John Morris Scientific) for about 2 h at 20 °C or until completely dry. The dried extracts were then resuspended in 100 μL of LC-MS running buffer containing 10 mM ammonium acetate in an acetonitrile/water (50%, v/v) solution, vortexed, and further diluted by combining 10 μL of resuspended extract and 90 μL of LC-MS running buffer. This solution was then vortexed and centrifuged at 11000g for 5 min, and 80 μL was transferred to vials, which were capped and ready for LC-MS analysis. Identification and Quantification of Extracted Curcuminoids and Metabolites. Chromatographic separation of curcuminoids and metabolites was performed with an LC 1200 series system (Agilent Technologies, Santa Clara, CA, USA), which consisted of a Binary Pump SL and Hi-P-Autosampler (with injection volume of 2 μL). Elution was achieved with an Agilent Poroshell 120 EC-C8 column (2.1 × 50 mm, with a particle size of 2.7 μm). The mobile phases consisted of 10 mM ammonium acetate and 0.1% (v/v) formic acid in water (A) or in acetonitrile (B). A linear gradient was applied as follows with a flow rate of 0.3 mL min−1: 0−5 min, 20% B; 5−8.1 min, 80% B; 8.1−10.0 min, 20% B; with a total run time of 10 min. Analysis was performed using a 6460 triple-quadrupole mass spectrometer (Agilent Technologies) equipped with an electrospray ionization (ESI) source operating in negative mode. For the identification and quantification of compounds,
medium was prepared anaerobically and transferred (10 mL) to glass tubes in an anaerobic culturing hood. For subculture preparations, Escherichia fergusonii and E. coli were cultured individually on lysogeny agar plates containing 1% (all w/v) tryptone, 0.5% yeast extract, 1% NaCl, and 2% agar (Oxoid). Under these conditions colonies were visible after 24 h of incubation at 37 °C. One colony of each culture was then grown in separate glass tubes containing 10 mL of mMCB, as above, under anaerobic conditions at 37 °C for 2 h. For fermentation assays, 100 μL of the subculture was inoculated into separate glass tubes containing 10 mL of mMCB and supplemented with curcuminoids at a concentration of 0.3 mM (solubilized in 3% (v/v) of Tween 20). Blank control samples containing only mMCB or mMCB + PCF but without curcuminoids were also inoculated. The initial pH of the fermentation medium was pH 6.27 ± 0.02. A set of abiotic samples with or without curcuminoids in mMCB or mMCB + PCF were maintained to account for the stability of curcuminoids at the start and end of fermentation. Fermentation temperature was kept constant at 37 °C and maintained for 36 h. Samples were acquired at 12 different time points for further analysis. The fermentation was performed on three separate occasions. Bacterial Growth. The growth curves of bacterial strains were obtained by measuring the optical density (OD) at 600 nm using a Nanodrop 2000C spectrophotometer with a UV−vis detector (Thermo-Fisher Scientific). Triplicate measurements were obtained, and data are presented as the mean ± SD. Quenching and Cell Lysis. Following incubation, bacterial metabolism was quenched by adding 2 mL of cold methanol/water (60%, v/v) solution to 1 mL of cell culture. To lyse the cell, samples were frozen in liquid nitrogen, thawed on dry ice, and then sonicated in a microfuge tube within a sonicator bath for ∼3 min. This lysis procedure was repeated 10 times. The samples were then vortexed and centrifuged 11007
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Figure 2. MS/MS spectra of DHC and curcumin from triple-quadrupole mass spectrometer operating in negative mode with a fragmentor voltage of 110 V and collision energy of 25 V (A) and 10 V (B) and their neutral chemical structures and fragmentation sites, DHC (top) and curcumin (bottom). The product ions shown on the spectra are the [M − H]− ions formed by loss of a proton from either of the phenol moieties. a MRM analysis was employed, tracking the transition indicative of parent and product ion specific for each compound. Previous direct infusion experiments were performed to optimize the fragmentor voltage and collision energy (CE). After infusion had been performed, the following parameters were fixed: drying N2 temperature, 300 °C; gas flow, 7 L min−1; nebulizer pressure, 15 psi; sheath gas temperature, 400 °C; sheath gas flow, 10 L min−1; capillary voltage, 2000 V; nozzle voltage, 1000 V; cell accelerator voltage, 7 V; dwell time, 20 ms. Detailed transitions of parent molecules and product ions, MS parameters (fragmentor voltage and CE), limits of detection (LODs), and limits of quantification (LOQs) are listed in Table 1. The concentrations of curcuminoids curcumin, DMC, and Bis-DMC were quantified on the basis of a calibration curve for curcumin. It was assumed that the response factors for DMC and Bis-DMC were the same as that for curcumin. The metabolites THC and FA were also quantified using standards. For the quantification of the metabolite with m/z [M − H]− 369 and the compound with m/z [M − H]− 470, it was assumed that these also had the same response factor as curcumin. The area under the curve from the LC-MS spectra of each compound was normalized using the IS and then converted to concentration using the standard calibration curves. Separate spiking experiments with known quantities of the curcuminoids, and the metabolites THC and FA in mMCB medium were carried out. Recoveries for all five compounds were ∼80%. All values given in the tables and figures were corrected for recovery (assuming 80% for all compounds). Identification of Compounds with m/z [M − H]− 369 and m/z [M − H] − 470. LC-MS/MS using a triple-quadrupole mass spectrometer and accurate mass using FT-ICR-MS were used to confirm the identity of compounds with m/z [M − H]− 369 and m/z [M − H]− 470. LC-MS/MS. Chromatographic separation conditions were as described above. Analysis was performed using a 6460 triple-quadrupole mass spectrometer (Agilent Technologies) equipped with an ESI ion
source operating in negative mode. A product ion scan was employed for confirmation of the metabolite. The acquisition parameters were as follows: CE, 10, 20, and 35 V; fragmentor voltage, 110 V; MS scan range from 50 m/z (min) to 500 m/z (max); drying N2 temperature, 300 °C; gas flow, 7 L min−1; nebulizer pressure, 15 psi; sheath gas temperature, 400 °C; sheath gas flow, 10 L min−1; capillary voltage, 2000 V; noozle voltage, 1000 V. FT-ICR-MS. A Bruker Daltonics (Bremen, Germany) SolariX 7 T Hybrid MALDI/ESI-FT-ICR-MS was used for isolation and high mass resolution analysis. The instrument was operated in ESI negative ionization mode with TD acquisition = 1M, MS detection (resolution 260000 at m/z 400) using an external calibration of sodium trifluoroacetate solution, and MS/MS detection (resolution 45000 at m/z 400). The sample was infused at a rate of 2 μL min−1 using the syringe pump, and a total of 20 scans were collected and averaged. The following general instrument conditions were set: capillary voltage, 4200 V; drying gas flow, 4.0 L min−1; drying gas temperature, 180 °C; nebulizer gas flow rate, 0.8 bar; source accumulation, 0.001 s; ion accumulation, 0.5 s; ion cooling time, 0.01 s; flight time, 0.001 s. Various collision voltages were used with a collision gas flow rate of 40%. Data were analyzed using Bruker Compass Data Analysis 4.2 (Build 383.1) software. Statistical Analysis. Statistical analysis was performed using the statistical package Minitab for Windows (version 17). Differences in the data were analyzed using one-way analysis of variance (ANOVA). The effect of two different treatments, (i) without PCF and (ii) with PCF, on the ability of the three bacterial strains to degrade curcuminoids at 36 h of fermentation was also tested. Tukey pairwise comparisons were used to identify the differences and statistical significance defined as p < 0.05.
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RESULTS AND DISCUSSION Growth of Escherichia fergusonii and Two E. coli Strains. The three Escherichia bacterial strains demonstrated a
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Figure 3. Full scan MS mass spectra of isolated curcumin adduct displaying isotopic profile (A) and the calculated isotopic profile of the predicted C24H25NO7S compound (B).
growth curve typical of batch culture in mMCB media, with a lag phase of around 2−3 h followed by exponential growth and a stationary phase beyond 8 h (Figure 1). The growth of all strains was not markedly affected by the addition of Tween 20 to solubilize the curcumin (3%, v/v) (Figure 1). Others have observed that Tween 20 (0.5%, w/w) in 0.1 M phosphate buffer disintegrated the cell wall and cytoplasmic membrane of Listeria monocytogenes and Salmonella typhimurium.22 It is likely that differences in the responses of bacteria to curcumin may vary with growth medium and microbial species. Indeed, the addition of medium was observed to counter the nonreversible damage caused to cells grown in buffer.23 The addition of PCF (5%, v/v) slightly increased bacterial growth and the OD600 nm increased by ∼0.02−0.05 units (Figure 1). PCF is known to assist the growth of facultative anaerobic bacteria. Similar findings were observed previously when media containing either ruminal fluid or pig cecal extract supported the growth of a larger number of gut bacteria compared to a medium rich in nutrients.24 The growth of E469, E739, and E10B in the first 8 h of fermentation was not affected by the supplementation of 0.3 mM curcuminoids, as shown by the profiles (Figure 1). However, a decline in OD600 nm was observed for E469 and E10B during the stationary phase, indicating a change in metabolism. A similar decline was observed for E469 in the presence of PCF and Tween 20. The pH values at the start and end of the fermentation were 6.27 ± 0.02 and 6.00 ± 0.05, respectively. Curcumin is stable at these pH values, but at alkaline pH >7.5, it degraded chemically within 30 min to ferulic acid, feruloylmethane, and vanillin.25,26
Therefore, changes in curcumin are likely to be due to bacterial metabolism. Identity of Compounds Formed in Fermentation Cultures with Curcumin. The upper organic and lower aqueous fractions for all extracted samples were examined by LCMS using a full MS scan operating in both negative and positive modes from m/z 50 to 800 (method S1, Supporting Information). Four compounds, in addition to the curcuminoids added, were identified from the full scan LC-MS spectra of the upper organic fraction of inoculated samples supplemented with curcuminoids. The full scan LC-MS spectra showing the total ion chromatogram of sample with 0.3 mM curcuminoids inoculated with E469 in mMCB and the extracted full mass spectra of one of the compounds are shown in Figure S1 (Supporting Information). Two of these additional compounds, THC and FA, were identified by comparison to standards. The other two compounds with m/z (all in [M − H]−) 369 and 470 were identified using LC-MS/MS and FT-ICR-MS, respectively. Compounds with m/z [M − H]− 369 and 470 were confirmed to be dihydrocurcumin (DHC) and a curcumin adduct (curcumin−L-cysteine), respectively, as discussed below. No metabolites could be identified in the lower aqueous fractions. Confirmation of Compounds with m/z [M − H]− 369. Full scan LC-MS of samples grown in the presence of curcuminoids gave rise to product ion with m/z [M − H]− 369 (data not shown), consistent with the [M − H]− ion of DHC (370 MW). To ensure that this compound putatively identified as DHC was not an isotope of curcumin, LC-MS/MS of DHC and curcumin in negative mode were performed and their spectra together with neutral chemical structures are shown in Figure 2. A fragmentor 11009
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Table 2. Calculation of Molecular Formulas from High-Resolution FT-ICR-MS Analysis of the Compound (m/z [M − H]− 470) and Most Abundant Fragment Ions Observed from the Analysis of Collision-Induced Decay (CID) MS/MS Data metabolite
formula
curcumin L-cysteine curcumin + cysteine curcumin−L-cysteine 392 fragment 167 fragment
C21H20O6 C3H7NO2S
calcd formula
C24H27NO8S C24H25NO7S C23H22NO5− C8H7O2S−
neutral mass
calcd m/z, [M − H]−
obsd m/z, [M − H]−
error (ppm)
368.12544
367.11871
367.11876
−0.25
489.14574 471.13462 393.15762 168.0245
470.12789 392.15035 167.01722
470.12809 392.15045 167.01725
−0.41 −0.27 −0.14
Figure 4. MS/MS analysis of curcumin adduct (curcumin−L-cysteine) from FT-ICR-MS (A) and triple-quadrupole mass spectrometer (B) and the proposed chemical structure of the adduct. MS/MS fragmentation patterns of curcumin adduct (curcumin−L-cysteine) from both instruments showed two intense fragment ions at nominal masses of 392 and 167.
functionality. For DHC, which possesses a reduced alkene with two hydrogen atoms, the corresponding mass shifted product ions 175 and 219 were observed (Figure 2). The product ion 173 was also observed for DHC, where the presence of a single alkene functionality and corresponding fragmentation at the ketone produce an identical product ion to that of curcumin. Confirmation of Compound with m/z [M − H]− 470. To putatively identify the compound with m/z [M − H]− 470, high mass accuracy FT-ICR-MS mass spectra of the compound were collected. Direct infusion of the crude culture media provided a complex signal; further isolation of a narrow mass range (10 m/ z) centered around m/z of 470 found a single peak at m/z [M − H]− 470.12809 as shown in Figure 3. Calculation of the molecular formula from the accurate mass gave only a single possible formula, C24H25NO7S (calculated m/z [M − H]− 470.12789), with a very low mass error of −0.41 ppm, as shown in Table 2. The isotopic pattern of the compound with m/
voltage of 110 V and CE of 25 and 10 V were applied to both compounds, producing product ions of m/z 134, 149, 173, 175, and 219 for DHC and m/z 134, 149, 173, and 217 for curcumin (Figure 2). Differences between the product ions of DHC and curcumin were attributed to their chemical structures. Curcumin possesses two alkenyl CC bonds within the heptadienone backbone,1 whereas DHC is the reduced form of curcumin in which one of the alkenes has been reduced.19 DHC has been previously reported as the intermediate breakdown product of curcumin by the enzyme CurA, through the reduction of an alkene present on the heptadienone chain.19 The common product ions of both curcumin and DHC can be rationalized through the presence of at least one CC bond, where the observed [M − H]− product ions m/z 134 and 149 correspond to fragmentation across the enone functionality (149) with a further loss of a single methyl group (134) (Figure 2). The other product ions for curcumin, 173 and 217, correspond to fragmentation at the ketone 11010
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Figure 5. Amount of curcuminoids: curcumin (A, B), DMC (C, D), and Bis-DMC (E, F) measured during fermentation with Escherichia fergusonii (E469), E. coli 739 (E739), and E. coli DH10B (E10B) in mMCB without PCF (A, C, E) or with PCF (5%, v/v) (B, D, F). The amount of curcuminoids in the upper organic extracts was quantified using LC-MS/MS and expressed as total micromoles in the original experimental broth of 10.1 mL. The amounts of curcuminoids in the control (abiotic) samples were measured at 0 and 36 h. Each data point is the average of three biological replicates ± SD.
z [M − H]− 470 from FT-ICR-MS is similar to the calculated isotopic peak spacing of the compound, as shown in Figure 3. L-Cysteine was present in the growth medium (mMCB) to reduce the oxido/reduction potential. The amount added far exceeds that required for the formation of the curcumin−Lcysteine adduct. The predicted formula of the metabolite matches an addition product of curcumin (C21H20O6) and Lcysteine (C3H7NO2S) with a loss of water. We propose an initial 1,4-addition product, curcumin adduct (curcumin−L-cysteine), with a possible chemical structure as shown in Figure 4, which undergoes dehydration either in vitro or within the MS source. In support of our putative assignment, curcumin possesses two α,βunsaturated ketone moieties that can act as Michael acceptors to nucleophilic attack through a Michael-type addition reaction mechanism.27 Furthermore, curcumin has previously been found to bind covalently to nucleophilic sulfhydryls and the selenol moiety of selenocysteine.28,29 Thioredoxin reductase, a selenocysteine-containing enzyme, was irreversibly inhibited by curcumin through a Michael-type addition reaction only in the presence of NADPH, with inhibition persisting after the removal of curcumin.28 To explore the mechanism of formation of the putatively identified curcumin adduct, we performed a separate experiment by incubating mMCB with and without 0.3 mM curcuminoids at 37 °C for 24 h and removed the bacterial cells to produce spent media. Both spent media samples were further supplemented with 0.3 mM curcuminoids and incubated for another 24 h. The curcumin adduct was observed only in spent media where curcuminoids were co-incubated with mMCB (data not shown). This suggests that the curcumin adduct is formed only in the presence of bacteria. We propose that this curcumin adduct may be formed through a Michael-type addition, possibly in the
presence of NADPH. It is probable that NADPH was produced in the fermentation cultures in this study through bacterial synthesis or another biological reductant may be employed. Additional MS/MS analysis by FT-ICR-MS and triplequadrupole MS showed two intense fragment ions at nominal masses of 392 and 167, as demonstrated in Figure 4. Accurate mass analysis gave masses of 392.15045 and 167.01725 with calculation of the molecular formula indicating empirical formulas of C23H22NO5− (calculated, 392.15035; mass error, −0.26 ppm) and C8H7O2S− (calculated mass, 167.01722; mass error, −0.14 ppm) as probable fragmentation products of a curcumin−L-cysteine adduct, as shown in Table 2. Although isolation and elucidation of the exact chemical structure is beyond the scope of the current study, data presented here can be used to assist in future determination of the exact chemical structure of the compound with m/z [M − H]− 470. Quantification of Compounds Formed in Fermentation Cultures with Curcumin. Change in Curcuminoid Concentrations during Fermentation. The extent to which the three curcuminoids (curcumin, DMC, and Bis-DMC) were degraded by the bacteria E469, E739, and E10B during batch fermentation for 36 h was determined using LC-MS. The effect of supplementing the media with PCF (5%, v/v) on the ability of all three strains to degrade curcuminoids was examined. Blank control samples containing only mMCB or mMCB with PCF without curcuminoids showed no or negligible signal interference in the LC-MS spectra of curcuminoids and metabolites. The amounts of curcuminoids (curcumin, DMC, and BisDMC) present in abiotic control samples in mMCB (Figure 5A,C,E) and in mMCB with PCF (Figure 5B,D,F) did not change at the start and end of fermentation. This may be expected as curcumin is stable at the pH of the fermentation 11011
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Table 3. Measured Curcuminoids Curcumin, DMC, Bis-DMC, Metabolite FA, and Curcumin Adduct Curcumin−L-Cysteine Detected in the Fermentation Culturea fermentation medium: mMCB
fermentation medium: mMCB with 5% (v/v) PCF
E. fergusonii E469
E. coli E739
E. coli E10B
E. fergusonii E469
E. coli E739
E. coli E10B
curcumin initial t = 0 h (μmol) final t = 36 h (μmol) % converted
2.20 ± 0.09 1.38 ± 0.03 37.3 ± 3.79a
2.13 ± 0.06 1.71 ± 0.06 19.9 ± 1.65bc
2.16 ± 0.02 1.69 ± 0.01 20.8 ± 0.42bc
2.19 ± 0.04 1.68 ± 0.03 23.2 ± 1.24b
2.13 ± 0.07 1.80 ± 0.04 15.8 ± 1.45c
2.17 ± 0.01 1.82 ± 0.05 16.1 ± 2.09c
DMC initial t = 0 h (μmol) final t = 36 h (μmol) % change
0.43 ± 0.004 0.40 ± 0.003 7.09 ± 1.42ab
0.42 ± 0.005 0.37 ± 0.002 16.3 ± 5.57a
0.42 ± 0.002 0.39 ± 0.010 8.10 ± 2.11ab
0.43 ± 0.007 0.39 ± 0.011 9.12 ± 2.21ab
0.43 ± 0.015 0.40 ± 0.008 5.69 ± 1.69b
0.43 ± 0.006 0.40 ± 0.016 7.78 ± 4.91ab
Bis-DMC initial t = 0 h (μmol) final t = 36 h (μmol) % change
0.08 ± 0.001 0.07 ± 0.001 7.22 ± 0.76b
0.08 ± 0.001 0.07 ± 0.001 10.8 ± 1.33ab
0.08 ± 0.001 0.07 ± 0.001 14.8 ± 2.40a
0.08 ± 0.001 0.07 ± 0.002 7.21 ± 2.04b
0.07 ± 0.002 0.07 ± 0.001 6.58 ± 1.65b
0.08 ± 0.002 0.07 ± 0.002 9.60 ± 2.79b
ferulic acid (FA) final t = 36 h (μmol) curcuminoid equivb (μmol)
0.13 ± 0.005 0.07 ± 0.003b
0.06 ± 0.002 0.03 ± 0.001c
0.05 ± 0.005 0.03 ± 0.003c
0.14 ± 0.003 0.08 ± 0.002a
0.04 ± 0.003 0.02 ± 0.002d
0.03 ± 0.003 0.02 ± 0.002d
curcumin−L-cysteine final t = 36 h (μmol) curcuminoid equivc (μmol)
0.43 ± 0.02 0.43 ± 0.02a
0.23 ± 0.01 0.23 ± 0.01c
0.21 ± 0.01 0.21 ± 0.01cd
0.28 ± 0.02 0.28 ± 0.02b
0.17 ± 0.01 0.17 ± 0.01d
0.22 ± 0.01 0.22 ± 0.01c
% curcuminoids accountedd
86.8 ± 2.96b
90.8 ± 2.57ab
90.4 ± 0.91ab
92.6 ± 1.32a
93.5 ± 1.06a
94.4 ± 2.22a
compound
a
The values for the amounts have been corrected for the recoveries of compounds and represent the amount present in 10.1 mL of fermentation medium. This was 80% for curcumin, DMC, Bis-DMC, and FA, as determined from separate spiking experiments. As curcumin−L-cysteine was not commercially available, spiking experiments could not be carried out, and the values are those as determined without correction for recovery. Means with the same letters within the same row are not significantly different. bCurcuminoid equivalents (equiv) were calculated assuming the following: (i) 1 mol of curcumin gives 2 mol of FA and 1 mol of DMC gives 1 mol of FA. (ii) The initial curcuminoid mixture contained 80.1:15.6 (curcumin/ DMC); therefore, FA obtained from this mixture on a molar basis would be 1.837. cCurcuminoid equivalents (equiv) were calculated assuming 1 mol of curcumin gives 1 mol of curcumin−L-cysteine. dPercent curcuminoids accounted for {[μmol of remaining curcuminoids + μmol FA/1.837) + μmol curcumin−L-cysteine)]/(initial μmol curcumin + initial μmol DMC + initial μmol Bis-DMC)} × 100.
E739 (19.9%) and E10B (20.8%), there were no significant differences (p > 0.05) between degradation by E739 and E10B in the absence of PCF. Similar results were obtained in the presence of PCF, where curcumin conversion was significantly higher (p < 0.05) with E469 (23.2%) compared to E739 (15.8%) and E10B (16.1%). The effects of PCF addition on curcumin degradation were strain dependent. In the presence of PCF, the degradation of curcumin by E469 was significantly reduced (p < 0.05). However, the addition of PCF had no significant effect (p > 0.05) on the ability of E739 and E10B to degrade curcumin. The amounts of DMC altered in the fermented cultures for all strains in mMCB were not significantly different (p > 0.05). This was also the case for all strains in mMCB with added PCF (Table 3). The change in the amount of DMC in the presence of E739 was significantly higher (p < 0.05) in mMCB (16.3%) than in mMCB with added PCF (5.7%), but changes in DMC amounts were not significantly affected by the addition of PCF (p > 0.05) when E469 or E10B was used. The amount of Bis-DMC in mMCB changed by E10B (14.8%) was significantly higher (p < 0.05) than that changed by E469 (7.2%), but there were no significant differences (p > 0.05) between E739 and E469. The ability of all three strains to convert curcumin in mMCB was in the order E10B > E739 ∼ E469. The changes in the amounts of Bis-DMC in the presence of all three strains were not significantly different (p > 0.05) in mMCB with
media (pH 6.00−6.27 over the fermentation course of 36 h). It is only at pH >7.5 that curcumin is unstable.25,26 The absence of an observable change in abiotic samples shows that there is no chemical degradation of curcuminoids over this time and confirms that any change in curcuminoids in the presence of the cultures is the result of bacterial metabolism. The degradation profiles of curcuminoids (curcumin, DMC, and Bis-DMC) in the presence of bacteria over the 36 h fermentation period are also shown in Figure 5. In the absence of PCF, a decrease in curcumin was observed until about 8−10 h, coincident with the onset of the stationary phase (Figure 1); the level of curcumin then stayed relatively constant until 36 h (Figure 5A), whereas with PCF, the level of curcumin decreased until around 12 h and then stayed relatively constant until 36 h (Figure 5B). A slight decrease was also observed in the amounts of DMC and Bis-DMC following 36 h of fermentation, as demonstrated in Figure 5C,D and E,F, respectively. The initial measured amounts of curcuminoids in fermentation cultures at 0 and 36 h are given in Table 3. The significant differences between the ability of the three bacterial strains to degrade curcuminoids in mMCB with or without PCF at 36 h were compared. The ability of all three strains to convert curcumin, in mMCB media with or without PCF, was in the order E469 > E10B ∼ E739. Whereas E469 degraded 37.3% of curcumin, which is significantly higher (p < 0.05) than that with 11012
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Figure 6. Measured amounts of FA (A, B) and curcumin adduct (curcumin−L-cysteine) (C, D) during fermentation with Escherichia fergusonii (E469), E. coli 739 (E739), and E. coli DH10B (E10B) in mMCB without PCF (A, C) or with PCF (5%, v/v) (B, D). The amount of FA and curcumin adduct (curcumin−L-cysteine) in the upper organic extracts were quantified using LC-MS/MS and expressed as total micromoles in experimental broth of 10.1 mL. Each data point is the average of three biological replicates ± SD.
Figure 7. Proposed degradation of the three curcuminoids (curcumin, DMC, and Bis-DMC) and breakdown products formed by Escherichia fergusonii (E 469), E. coli 739 (E739), and E. coli DH10B (E10B). Numbers in parentheses represent the theoretical conversion of curcuminoids to metabolites. D, detected; N.D, not detected in this study.
curcumin−L-cysteine during fermentation is shown in Figure 6, and the amounts quantified after 36 h are given in Table 3. In mMCB without PCF, the formation of FA by E469 (0.07 μmol) was significantly higher (p < 0.05) than the amounts formed by E739 and E10B (0.03 μmol), but there were no significant differences (p > 0.05) between E739 and E10B. In mMCB with PCF, the formation of FA by E469 (0.08 μmol) was also significantly higher (p < 0.05) than that by E739 and E10B (0.02 μmol), but there were no significant differences (p > 0.05)
added PCF. The ability of E469 and E739 to convert Bis-DMC was not significantly affected by the addition of PCF (p > 0.05). However, a significant increase (p < 0.05) was observed for changes in Bis-DMC when PCF was not added to mMCB in the presence of E10B (14.8% compared from 9.6%). Quantification of Compounds Formed in Fermentation Cultures with Curcumin. DHC and THC were minor metabolites of curcumin degradation, and they constitute 0.05) between E739 and E10B. In mMCB with PCF, the formation of curcumin−Lcysteine by E469 (0.28 μmol) was significantly higher (p < 0.05) than those by E10B (0.22 μmol) and E739 (0.17 μmol). In the same medium, the formation of curcumin−L-cysteine by E10B was also significantly higher (p < 0.05) than that by E739. The formation of curcumin−L-cysteine by E469 and E739 in the absence of PCF (0.43 and 0.23 μmol, respectively) was significantly higher (p < 0.05) than in the presence of PCF (0.28 and 0.17 μmol, respectively). The formation of curcumin− L-cysteine by E10B was not significantly different (p > 0.05) in the absence (0.21 μmol) or presence of PCF (0.22 μmol). Accountability for Curcuminoids. The amount of curcumin accounted for was also calculated and found to be 87−94%. The amount of curcumin accounted for was not significantly different (p > 0.05) between the three Escherichia bacteria in mMCB or mMCB with PCF (Table 3). However, the amount of curcumin accounted for E469 in the presence of PCF (92.6%) was significantly higher (p < 0.05) than in the absence of PCF (86.8%), but there were no significant differences (p > 0.05) for E739 and E10B in the presence or absence of PCF. The level of accountability, which represents remaining curcuminoids (curcumin, DMC, and Bis-BMC) and curcuminoid equivalents for conversion into ferulic acid and curcumin−L-cysteine, is high. In a different system, when curcuminoids were incubated with rat liver slices, there was ∼50% of curcumin and DMC and ∼75% for Bis-DMC accounted for.11 These authors attributed the lack of accountability for the total amount of curcuminoids to possible spontaneous chemical degradation in aqueous solution and conversion to other breakdown products that were not detected by the methods used.11 Proposed Pathways of Degradation of Curcuminoids by Bacterial Strains Examined. The proposed pathways of degradation of the three curcuminoids are demonstrated in Figure 7. Curcumin is converted to DHC and then THC by the enzyme CurA, through the reduction of CC bonds on the heptadienone chain.19 In studies involving ingestion of curcumin in humans and animals it was found that this pathway was rapid and that curcumin and metabolites were subsequently glucuronidated or sulfated by enzymes present in the liver.10,11 Both curcumin and DHC can also be converted to FA, as shown in Figure 7. FA was found to be one of the major metabolites in the serum, urine, and feces of humans after consumption of encapsulated curcumin in bread, and the authors suggested that the presence of FA was probably as a result of gut bacteria metabolism.12 Our data are consistent with this hypothesis. In our in vitro system, the presence of a curcumin adduct, curcumin−L-cysteine, was also found. This adduct was observed only in fermented samples in the presence of curcuminoids and bacteria and not present in abiotic samples. The adduct was also observed only in spent media where curcuminoids were coincubated with mMCB, confirming that the presence of bacteria was necessary for the formation of the curcumin adduct. However, from these studies alone it is not possible to assign
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ASSOCIATED CONTENT
S Supporting Information *
Method S1, profiling of breakdown products using full scan LCMS; Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(M.A.A.) E-mail: [email protected]. Funding
S.T. is supported by a CSIRO OCE Postgraduate Scholarship and a University of Melbourne MIFRS Scholarship. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank A/Prof Stuart Dashper of University of Melbourne for the use of the anaerobic culturing facilities. We also thank Patrick Daniel of Department of Primary Industries for supplying the pig cecal fluid.
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ABBREVIATIONS USED DMC, demethoxycurcumin; Bis-DMC, bis-demethoxycurcumin; DHC, dihydrocurcumin; THC, tetrahydrocurcumin; FA, ferulic acid; E. coli, Escherichia coli; NADPH, nicotinamide 11014
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adenine dinucleotide phosphate; LC-MS, liquid chromatography−mass spectrometry; IS, internal standard; PCF, pig cecal fluid; E469, Escherichia fergusonii ATCC 35469; E739, Escherichia coli ATCC 8739; E10B, Escherichia coli DH10B; mMCB, modified medium for colon bacteria; OD, optical density; ESI, electrospray ionization; MRM, multiple reaction monitoring; CE, collision energy; Rt, retention time; LOD, limit of detection; LOQ, limit of quantification; AUC, area under the curve; FTICR-MS, Fourier transform-ion cyclotron resonance-mass spectrometer
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