Glycosylated Benzoxazinoids Are Degraded during Fermentation of

Article pubs.acs.org/JAFC

Glycosylated Benzoxazinoids Are Degraded during Fermentation of Wheat Bran Otto Savolainen,*,†,§ Jenna Pekkinen,† Kati Katina,# Kaisa Poutanen,†,# and Kati Hanhineva† †

Institute of Public Health and Clinical Nutrition, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland Department of Biology and Biological Engineering, Division of Food and Nutrition Science, Chalmers University of Technology, Gothenburg, Sweden # VTT Technical Research Centre of Finland, P.O. Box 1000, 02044 VTT Espoo, Finland §

S Supporting Information *

ABSTRACT: Benzoxazinoids are plant secondary metabolites found in whole grain cereal foods including bread. They are bioavailable and metabolized in humans, and therefore their potential bioactivity is of interest. However, effects of food processing on their content and structure are not yet studied. This study reports effects of bioprocessing on wheat bran benzoxazinoid content. Benzoxazinoid glycosides were completely degraded during fermentation, whereas metabolites of benzoxazinoid aglycones were formed. Fermentation conditions did not affect the conversion process, as both yeast and yeast/ lactic acid bacteria mediated fermentations had generally similar impacts. Likewise, enzymatic treatment of the bioprocess samples did not affect the conversion, suggesting that these compounds most likely are freely bioavailable from the grain matrix and not linked to the cell wall polymers. Additionally, the results show that benzoxazinoids undergo structural conversion during the fermentation process, resulting in several unknown compounds that contribute to the phytochemical intake and necessitate further analysis. KEYWORDS: benzoxazinoids, bioprocessing, metabolomics, mass spectrometry

■

INTRODUCTION Benzoxazinoids are a group of allelochemical defense compounds found in some cereal crops including wheat, rye, and maize.1−4 They can be divided into three structural classes: (1) benzoxazolinones, for example, 1,3-benzoxazol-2-one (BOA) and 6-methoxy-1,3-benzoxazol-2-one (MBOA); (2) hydroxamic acids, for example, 2,4-dihydroxy-1,4-benzoxazin-3one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3one (DIMBOA); and (3) lactams, for example, 2-hydroxy-1,4benzoxazin-3-one (HBOA) and 2-hydroxy-7-methoxy-1,4benzoxazin-3-one (HMBOA) (Figure 1). In planta, they are stored as glycosides in the vacuoles and enzymatically released when needed to respond to microbial attack or other environmental stimuli.3,5 In cereal crops benzoxazinoids are mainly found in the vegetative partsroots and leaves.2 Only recently has it been shown that they are also present in the kernels of wheat and rye, therefore contributing to the dietary phytochemical intake.6,7 In wheat, benzoxazinoids are particularly abundant in the germ and bran, whereas in rye their main location is in the bran.8 The major benzoxazinoids in wheat and rye are DIBOA and HBOA diglycosides, although their concentrations are much higher in rye than in wheat (DIBOA diglycoside, 78 ng/g dry matrix in rye grain and 3 ng/g dry matrix in wheat grain; HBOA diglycoside, 6 ng/g dry matrix in rye grain and 0.4 ng/g dry matrix in wheat grain).8 Benzoxazinoids have also been found in conventionally baked breads7 and water-soaked or -boiled pearled rye and rye flakes.8 Owing to their high bioactive potential, there has been increasing interest in the bioavailability and metabolism of © 2015 American Chemical Society

dietary benzoxazinoids within the past few years. Adhikari et al. showed that benzoxazinoids are absorbed and metabolized in pigs as they found HBOA-hex to be the most dominant benzoxazinoid in blood and excreted in urine after feeding a rye-containing diet to pigs.9 Another study in rats fed a ryebased diet showed that certain benzoxazinoids were detected in plasma and urine as glucuronidated conjugates.10 In a recent human study consumption of whole grain sourdough rye bread was found to increase urinary content of a HBOA glucuronide.11 Interestingly, also a novel metabolite group, namely, hydroxylated phenylacetamides, was described to be excreted in urine as sulfate or glucuronide conjugate after rye consumption. Phenylacetamides, 2-hydroxy-N-(2hydroxyphenyl)acetamide (HHPAA) and N-(2hydroxyphenyl)acetamide (HPAA), are potentially derived from benzoxazinoids, because they have been described as conversion products of soil microbial degradation of benzoxazinoids.12,13 This was of special interest as the excretion of HPAA sulfate, HHPAA sulfate and glucuronide, and HBOA glucuronide differentiated between consumption of sourdough and nonsourdough fermented rye breads.11 Thereafter, phenylacetamides have been detected also in postprandial plasma samples after a meal enriched with rye bran, which shows their rapid bioavailability in circulation.14 Received: Revised: Accepted: Published: 5943

November 7, 2014 June 2, 2015 June 4, 2015 June 4, 2015 DOI: 10.1021/acs.jafc.5b00879 J. Agric. Food Chem. 2015, 63, 5943−5949

Article

Journal of Agricultural and Food Chemistry

Figure 1. Molecular structures of the different benzoxazinoids exemplified with the most common compounds of each chemical class The asterisk (∗) indicates the compounds found in this study. In addition to aglycone forms, also the diglycoside and glucoside forms of DIBOA, DIMBOA, and HBOA were identified and analyzed in this study.

Figure 2. Schematic illustration of the study setup. Ground wheat bran was fermented in four different conditions (P1−P4). All conditions included equal amounts of yeast (Kazachstania exigua) at the start. In addition, conditions P2 and P4 included added lactic acid bacteria (Lactobacillus brevis), and conditions P3 and P4 had mixture of enzymes with cellulase, xylanase, β-glucanase, and α-amylase activity. Samples were collected at the start (0 h) and after 12 and 24 h of fermentation. LC-qTOF-MS analytics was used to study the effects of different fermentation conditions on the resulting benzoxazinoid metabolite profile.

grains or flakes and that they are released into water in notable amounts during such processing.8 Sourdough fermentation combined with baking has been shown to effectively degrade DIBOA diglycoside and increase the amount of BOA and benzoxazinoid-related metabolite HPAA,11,14 but whether the degradation was due to fermentation or baking is not known. In the current study, we used a nontargeted liquid chromatography quadrupole time-of-flight mass spectrometry (LC-qTOF-MS) metabolite profiling approach with the aim of examining the effects of different fermentation conditions on the benzoxazinoid profile of wheat bran sourdoughs. The effect of different microbial contents (yeast, and yeast with lactic acid bacteria) as well as impact of cell wall degrading enzymes was evaluated.

Benzoxazinoids are highly bioactive compounds that have been suggested to even have pharmaceutical potential and may therefore have implications on health as part of the diet, as has been reviewed recently.15 For example, benzoxazinoids have been reported to have antiallergenic and anti-inflammatory, as well as appetite suppressing effects in humans.3,4,16−18 Additionally, benzoxazinoids may reduce cellular glucose uptake.19 On the other hand, benzoxazinoids have also been found to be potent polyploidy-inducing agents in human-derived cell lines (HepG2 and HeLa), which has raised concern for their potential adverse health effects.20,21 For phenylacetamides, no reports on bioactivity and potential effects on health are found. Whereas it is unclear whether benzoxazinoids, or potentially their metabolized forms, for example, phenylacetamides, have an impact on human metabolism, there is a need to understand the effect of different food-processing technologies on their content. It is known that soaking and boiling can considerably change the content of benzoxazinoids and their glycosides in

■

MATERIALS AND METHODS

Materials. Commercial wheat bran (Fazer Mills, Lahti, Finland) was ground using a TurboRotor mill (Mahltechnik Görgens GmbH, 5944

DOI: 10.1021/acs.jafc.5b00879 J. Agric. Food Chem. 2015, 63, 5943−5949

Article


Table 1. Identified Benzoxazinoid Derivatives and Unknown Compounds with Molecular Formula, m/z of the Molecular Ion in ESI(−), Fragment Ions in ESI(−), Collision Energy (CE) Used for Fragmentation, Retention Time (rt), and References Used in Identification putative ID

mol formula

m/z

DIMBOA diglycoside DIBOA diglycoside DIBOA glycoside HBOA diglycoside HBOA glycoside HHPAA BOA unknown 1 unknown 2 unknown 3 unknown 4

C21H29NO15 C20H27NO14 C14H17NO9 C20H27NO13 C14H17NO8 C8H9NO3 C7H5NO2

534.1457 504.1362 342.0828 488.1407 326.0883 166.0524 134.0254 150.0550 150.0552 188.0917 224.0553

MS/MS m/z 192.0281, 164.0324, 162.0207, 180.0303, 162.0179, 134.0213, 164.0334 164.0356 118.0453, 108.0422 78.047 108.041, 107.035 108.042, 107.032 153.3879, 139.0272, 178.0526, 162.0529,

CE (eV)

rt

ref

149.0164 134.0226, 108.0422 108.0419

20 20 20 10 10 20 20

6 6 6 6 6 11 6

126.0841, 118.03662, 107.1137, 108.0512, 93.0216 144.0437, 134.0236, 118.0424, 108.05473, 78.0903

20 20

3.47 3.07 3.42 3.01 3.39 2.89 3.52 3.03 3.22 3.67 2.75

Dormagen, Germany) to a median particle size of 160 μm. Enzymes used in fermentations 3 and 4 were Depol 740l (Biocatalyst Ltd., UK) and Grindamyl A1000 (Danisco, Denmark). Their enzyme activities are presented in Supporting Information Table 1. The yeast used in all fermentations was Kazachstania exigua (VTT C-81116), and the lactic acid bacteria (LAB) starter used in fermentations 2 and 4 was Lactobacillus brevis (VTT E-95612). Bran Fermentations. Four different liquid fermentations (P1, yeast; P2, yeast + LAB; P3, yeast + enzymes; and P4, yeast + LAB + enzymes), containing bran and water (10:90 w/w) with an initial inoculum of ca. 106−107 cfu/mL for both starter cultures employed, were made as duplicates. The fermentations were performed in 1 L BIOSTAT QPlus bioreactors (Sartorius Stedim Biotech GmbH, Germany) in an anaerobic environment, which was created by a flow of nitrogen into the bottom of the fermentor. Preparation of the sourdoughs and process parameters can be found in the Supporting Information (Tables 2 and 3). Samples (285 g) were taken at 0, 12, and 24 h, freeze-dried (Lyovac GT 2, Steris GmbH, Germany; or Epsilon 2-25DS, Christ, Germany), and milled (Bamix, Switzerland and Retsch ZM 200, Retsch GmbH, Germany) to pass a 0.5 mm sieve. The process scheme for fermentations and sampling is illustrated in Figure 2. Acidification. Acidity (pH and total titratable acids (TTA)) of the bioprocessed bran was determined from a suspension containing 5 g of bran sample and 50 mL of distilled water in an automatic titrator (Mettler Toledo titrator T50). After pH measurement, the suspension was titrated with 0.1 M NaOH to a final pH of 8.5. TTA value was expressed as the amount of NaOH used in milliliters per 10 g of sample. Sample Preparation and LC-qTOF-MS Analyses. For LCqTOF-MS analyses 100 mg of each freeze-dried and ground fermentation sample was weighed. The samples were extracted by vigorous shaking with 80% methanol for 30 min at room temperature using a Multi Reax shaker (Heidolph Instruments GmbH, Germany), centrifuged for 10 min at 6708g (7 °C), and filtered through a 0.2 μm PTFE membrane (PALL Corp., USA). Prior to analysis, samples were stored for 12 h at 6 °C. Metabolite profiling was carried out on an LCqTOF-MS system consisting of a 1290 ultrahigh-performance liquid chromatograph (UHPLC) with a 6540 ultrahigh-definition (UHD) accurate-mass qTOF mass spectrometer (Agilent Technologies, USA). Separation of metabolites was performed using a 2.1 × 100 mm 1.8 μm particle size Agilent ZORBAX Eclipse XDB-C18 column (Agilent Technologies) thermostated at 50 °C. The mobile phase consisted of 0.1% of formic acid in water (phase A) and 0.1% of formic acid in methanol (phase B). The gradient program was as follows: 2−100% of B in 10 min, held at 100% of B for 4.5 min, 100−2% of B in 0.01 min, and held at 2% of B for 1.99 min. The mobile phase flow rate was 0.4 mL/min, and the sample injection volume was 1 μL. The instrument was equipped with an Agilent Jet Stream electrospray source and was operated in negative ionization mode with the following source parameters: drying gas flow rate, 11 L/min; gas temperature, 325 °C;

pressure of nebulizer gas, 45 psi; Vcap, 3500 V; fragmentor, 100 V; skimmer, 45 V. The mass scan range was m/z 50−1600. For tandem mass spectrometry (MS/MS) analyses three different collision energies (10, 20, and 40 V) were used. The mass-axis was calibrated online during the analyses with two reference mass ions. Data acquisition was conducted with MassHunter Acquisition B.04.00 (Agilent Technologies). Data Processing. Data were extracted using MassHunter Qualitative analysis B.05.00 (Agilent Technologies) using the “Find Compounds by Molecular Feature” function. Peaks were extracted by combining isotopes, sodium adducts, and dimers for specific “compounds”. For compound alignment data were output into the Mass Profiler Professional software (version 2.2, Agilent Technologies, Santa Clara, CA, USA). Main emphasis on data analysis was laid on analyzing the benzoxazinoids, which typically elute at 2−4 min in our chromatographic conditions; therefore, the collection of signals for the data analysis was focused on this region. Compounds were identified on the basis of their accurate mass, molecular formula, and MS/MS fragmentation pattern. A heatmap chart was generated with Multiexperiment Viewer (MeV) software.22,23

■

RESULTS AND DISCUSSION Identification of the Benzoxazinoid Compounds. Tentatively identified benzoxazinoids in wheat bran ferments included DIBOA diglycoside, DIBOA glycoside, HBOA diglycoside, HBOA glycoside, DIMBOA diglycoside, and BOA aglycone. All reported compounds were detected in negative electrospray ionization as molecular ions. The recorded MS/MS fragments of detected benzoxazinoid glycosides and metabolites are presented in Table 1, as well as references used for confirmation of identification. MS/MS fragmentation spectra for DIBOA and HBOA diglycoside and HHPAA with extracted ion chromatograms are presented in Figure 3. The fragmentation pattern of benzoxazinoid glycosides was analogical to that found earlier (Table 1; Figure 3).6 Identification of the phenylacetamide HHPAA, a potential microbial conversion product of benzoxazinoids, was based on the fragmentation pattern showing major fragments at m/z 118.0453 and 108.0422 (Figure 3), which is in accordance with that reported earlier.11 There were two signals (m/z 150.055 with retention time (rt) of 3.0 and 3.2 min) exhibiting the molecular mass and MS/MS fragmentation corresponding to phenylacetamide HPAA as reported earlier.11 However, the structural difference between these two compounds could not be resolved, and therefore they are reported as unknowns (Table 1). Additionally, the data were searched for other earlier reported conversion products of benzoxazinoids,24 but none were found. However, there were two metabolites formed 5945


Article


Figure 3. Extracted ion chromatograms in ESI(−) LC-qTOF-MS analysis for DIBOA diglycoside (A), HBOA diglycoside (B), and HHPAA (C) with MS/MS fragmentation spectra for each of the compounds.

during the fermentation (m/z 189.092 at 3.67 min and m/z 224.055 at 2.75 min), which had fragmentation pattern and retention time referring potentially to benzoxazinoid derived metabolites (Table 1). The ion with m/z 189.092 gave several fragment ions including m/z 118.037, 107.114, and 108.051,

which were observed also for the benzoxazinoid compounds eluting in this same chromatographic region. Additionally, the unknown compound with m/z 224.055 resulted in MS/MS fragments of m/z 162.053, 134.024, 118.042, and 108.055, which are very close in mass to the ions observed for other 5946


Article


Figure 4. Heatmap representation of the tentatively identified benzoxazinoids and their conversion products in the different bioprocesses (P1−P4) during fermentation (0, 12, and 24 h). The signal abundances were row-wise normalized. The color coding indicates the abundance of each metabolite: yellow, high abundance; blue, low abundance; gray, no value.

were detected only from 12 h onward, showing highest accumulation at 24 h (Figures 4 and 5). One potential mediator for the compound conversion in the fermentation is the microbial metabolism in the bioprocess. Of the four fermentation methods used, P1 and P3 did not contain added lactic acid bacteria (LAB) but only the yeast, whereas in fermentations P2 and P4 a LAB starter was included. However, despite the differential bacterial regime, the fermentation method did not affect the degradation of glycosides and formation of other compounds including BOA and HHPAA (Figure 4). The pH decreased in all fermentations from 6.7 to an average of 4.8 during 24 h, and the use of LAB starter (P2 and P4) had only a slight additional 0.5 unit decrease in pH (Supporting Information (Table 4)). This slight difference in pH had no effect on the metabolic conversions. The lowered pH in fermentations without added LAB is most likely due to the growth of naturally occurring bacteria in the bran as has been demonstrated earlier.26 Thus, the degradation of benzoxazinoid glycosides or formation of BOA and HHPAA was quite similar after two different types of fermentation irrespective of the microbial content. There were two unknown metabolites, which were produced mainly in either the fermentation containing yeast solely (P1; unknown 3, m/z 188.092) or after the fermentation with yeast, LAB, and enzymes (P4; unknown 1, m/z 150.055) referring to differential impact of the fermentation regime to the metabolic conversion (Figure 4). However, these two compounds remained unknown, similarly as two compounds that were produced in all of the fermentation conditions, which highlights the necessity of further structural analysis to understand the metabolic conversions of benzoxazinoid compounds during food processing. In fermentations P3 and P4 enzyme preparations with cellulase, xylanase, β-glucanase, and α-amylase activities (Supporting Information (Table 1)) were used to modify the cereal cell wall structure and to release phytochemicals into the fermentation matrix.27 With the application of enzymes in fermentation, the amount of phytochemicals in the dough can be modified; for example, the phenolic acids can be released from arabinoxylan.27 This can further improve the bioaccessibility and bioavailability of these compounds.28,29 Even when the sampling procedure was maintained as similar as possible for the four different fermentation conditions, the metabolic conversions seem to start rapidly after mixing the constituents of the fermentation mixture, as was suggested by the differences in the metabolite levels at the onset of the fermentation (0 h).

benzoxazinoid compounds, including DIMBOA and HHPAA. We have recently observed this same unknown compound to be excreted in urine of mice after feeding with rye bran and, intriguingly, this compound was excreted in significantly higher levels when rye bran was fermented with yeast.25 Effects of Fermentation on the Benzoxazinoid Compounds. The relative intensities of the identified compounds had major differences during all four bioprocesses as visualized in a heatmap chart (Figure 4). Prior to fermentation (0 h), both the mono- and diglycoside derivatives of DIBOA, DIMBOA, and HBOA were found in all four fermentation conditions. Although our analytical method was not quantitative, it is worth mentioning that at the onset of fermentation we detected the largest peak areas for DIBOA and HBOA diglycosides (Figure 5), which have been shown to be

Figure 5. Average abundance of benzoxanoid derivatives and metabolites as relative peak areas plotted against fermentation time. The abundances from the different bioprocesses were combined.

the most abundant benzoxazinoids in wheat bran.8 There were no benzoxazinoid aglycones or any of the potential degradation products detected prior to fermentation in any of the samples, except for a minor signal for BOA. Between 0 and 12 h of fermentation all di- and monoglycoside derivatives of benzoxazinoids were degraded in all of the tested fermentation conditions. In contrast, the breakdown products of DIBOA and HBOA, namely, BOA and HHPAA, as well as the several unknown compounds potentially derived from benzoxazinoids, 5947


Article


fermentation to modify the composition of phytochemicals embedded in the cereal matrix and the need to examine the effects of food processing on the phytochemical composition to be able to evaluate dietary effects at the molecular level.

Interestingly, the fermentation together with yeast and the mixture of enzymes had the highest amount of the diglycoside conjugates of DIBOA, DIMBOA, and HBOA. This could be due to the enzymatic processing of the bran structure resulting in increased release of diglycoside conjugates to the fermentation media. In fermentation mixture P4 the mixture of enzymes was also added, but together with the LAB starter, which could boost the subsequent conversion of the digycosides and hence result in lower abundances of diglycosides when compared to the fermentation without the addition of LAB. However, the exact location of benzoxazinoids in the bran matrix is not yet studied, and thus the potential to increase their release by enzymatic treatment is only speculative. Detailed kinetic study would require quantitative targeted methods, which is currently not possible as not all of the detected compounds are available as commercial standards, and at the end of the fermentation process the levels of the main end products were relatively similar without striking differences between the different fermentation conditions. In previous studies it has been demonstrated that boiling and soaking affect both the profile and the apparent concentration of benzoxazinoids in wheat.8 Whereas it has been speculated that sourdough fermentation may be involved in altering the benzoxazinoid concentration in whole grain bread,11 it was still unclear if this was due to the fermentation or the baking process. Our results clearly show that extensive structural conversions occur on benzoxazinoids during fermentation without baking, including degradation of benzoxazinoid glycosides and formation of HHPAA. The earlier finding of HHPAA glucuronide and sulfate as one of the most discriminatory signals for whole grain sourdough rye bread intake11 suggests that HHPAA is bioavailable, as has also been shown in a postprandial study involving rye bran enriched diets. 14 Conversion of HHPAA was suggested to be postabsorptive metabolism,11 although on the basis of our results this could equally be due to direct absorption of the metabolite present in sourdough bread. However, focused quantitative analyses are required to resolve the kinetics and dose−response relationship of this metabolite. In our analysis, two signals potentially corresponding to HPAA were detected in the fermented products, but detailed identification could not be accomplished. HPAA has been previously detected in bread and was suggested to be a result from degradation of benzoxazinoid glycosides.11 However, an alternative way of formation could be due to thermal processing during baking from HHPAA via dehydroxylation. Additionally, in our analysis, we observed unknown metabolites emerging in the sourdough process, which could potentially be of benzoxazinoid origin based on similarity in MS/MS fragments when compared with the identified metabolites. Therefore, it is possible that other, yet unknown, metabolized forms of benzoxazinoids are present in products involving sourdough fermentation and interestingly, as we recently observed, some of these seem to be also bioavailable and excreted in urine of mice after feeding with rye bran.25 In conclusion, we showed for the first time in a controlled fermentation that benzoxazinoid glycosides are degraded during fermentation and that HHPAA and BOA, conversion products of benzoxazinoids, alongside other potential metabolites of benzoxazinoid origin, are formed in sourdough. We also showed that the conversion was not remarkably affected by using lactic acid bacteria or enxymes in the fermentation in addition to yeast starter. This result emphasizes the potential of

■

ASSOCIATED CONTENT

S Supporting Information *

Tables 1−4. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jafc.5b00879.

■

AUTHOR INFORMATION

Corresponding Author

*(O.S.) Department of Biology and Biological Engineering, Food Science, Chalmers University of Technology, Kemivägen 10, Gothenburg, Sweden. E-mail: [email protected]. Phone: +46(0)317723813. Funding

Funding from the Academy of Finland and Biocenter Finland is gratefully acknowledged. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Ville Koistinen is acknowledged for assistance in the preparation of the figures and Miia Reponen for assistance in the LC-qTOF-MS analyses.

■

REFERENCES

(1) Sicker, D.; Frey, M.; Schulz, M.; Gierl, A. Role of natural benzoxazinones in the survival strategy of plants. Int. Rev. Cytol. 2000, 198, 319−346. (2) Villagrasa, M.; Guillamon, M.; Labandeira, A.; Taberner, A.; Eljarrat, E.; Barcelo, D. Benzoxazinoid allelochemicals in wheat: distribution among foliage, roots, and seeds. J. Agric. Food Chem. 2006, 54, 1009−1015. (3) Niemeyer, H. M. Hydroxamic acids derived from 2-hydroxy-2H1,4-benzoxazin-3(4H)-one: key defense chemicals of cereals. J. Agric. Food Chem. 2009, 57, 1677−1696. (4) Macias, F. A.; Marin, D.; Oliveros-Bastidas, A.; Molinillo, J. M. Rediscovering the bioactivity and ecological role of 1,4-benzoxazinones. Nat. Prod. Rep. 2009, 26, 478−489. (5) Cambier, V.; Hance, T.; de Hoffmann, E. Non-injured maize contains several 1,4-benzoxazin-3-one related compounds but only as glucoconjugates. Phytochem. Anal. 1999, 10, 119−126. (6) Hanhineva, K.; Rogachev, I.; Aura, A. M.; Aharoni, A.; Poutanen, K.; Mykkanen, H. Qualitative characterization of benzoxazinoid derivatives in whole grain rye and wheat by LC-MS metabolite profiling. J. Agric. Food Chem. 2011, 59, 921−927. (7) Pedersen, H. A.; Laursen, B.; Mortensen, A.; Fomsgaard, I. S. Bread from common cereal cultivars contains an important array of neglected bioactive benzoxazinoids. Food Chem. 2011, 127, 1814− 1820. (8) Tanwir, F.; Fredholm, M.; Gregersen, P. L.; Fomsgaard, I. S. Comparison of the levels of bioactive benzoxazinoids in different wheat and rye fractions and the transformation of these compounds in homemade foods. Food Chem. 2013, 141, 444−450. (9) Adhikari, K. B.; Laursen, B. B.; Lãrke, H. N.; Fomsgaard, I. S. Bioactive benzoxazinoids in rye bread are absorbed and metabolized in pigs. J. Agric. Food Chem. 2012, 60, 2497−2506. (10) Adhikari, K. B.; Lãrke, H. N.; Mortensen, A. G.; Fomsgaard, I. S. Plasma and urine concentrations of bioactive dietary benzoxazinoids and their glucuronidated conjugates in rats fed a rye bread-based diet. J. Agric. Food Chem. 2012, 60, 11518−11524.

5948


Article

Journal of Agricultural and Food Chemistry (11) Beckmann, M.; Lloyd, A. J.; Haldar, S.; Seal, C.; Brandt, K.; Draper, J. Hydroxylated phenylacetamides derived from bioactive benzoxazinoids are bioavailable in humans after habitual consumption of whole grain sourdough rye bread. Mol. Nutr. Food Res. 2013, n/a. (12) Zikmundova, M.; Drandarov, K.; Bigler, L.; Hesse, M.; Werner, C. Biotransformation of 2-benzoxazolinone and 2-hydroxy-1,4benzoxazin-3-one by endophytic fungi isolated from Aphelandra tetragona. Appl. Environ. Microbiol. 2002, 68, 4863−4870. (13) Zikmundova, M.; Drandarov, K.; Hesse, M.; Werner, C. Hydroxylated 2-amino-3H-phenoxazin-3-one derivatives as products of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) biotransformation by Chaetosphaeria sp., an endophytic fungus from Aphelandra tetragona. Z. Naturforsch., C: J. Biosci. 2002, 57, 660−665. (14) Hanhineva, K.; Keski-Rahkonen, P.; Lappi, J.; Katina, K.; Pekkinen, J.; Savolainen, O.; Timonen, O.; Paananen, J.; Mykkanen, H.; Poutanen, K. The postprandial plasma rye fingerprint includes benzoxazinoid-derived phenylacetamide sulfates. J. Nutr. 2014, 144, 1016−22. (15) Adhikari, K. B.; Tanwir, F.; Gregersen, P. L.; Steffensen, S. K.; Jensen, B. M.; Poulsen, L. K.; Nielsen, C. H.; Høyer, S.; Borre, M.; Fomsgaard, I. S. Benzoxazinoids: cereal phytochemicals with putative therapeutic and health-protecting properties. Mol. Nutr. Food Res. 2015, n/a−n/a. (16) Rosenfeld, M. J.; Forsberg, S. R. Compounds for use in weight loss and appetite seppression in humans. U.S. Patent 7,507,731 B2, 2009. (17) Poupaert, J.; Carato, P.; Colacino, E.; Yous, S. 2(3H)benzoxazolone and bioisosters as “privileged scaffold” in the design of pharmacological probes. Curr. Med. Chem. 2005, 12, 877−885. (18) Otsuka, H.; Hirai, Y.; Nagao, T.; Yamasaki, K. Antiinflammatory activity of benzoxazinoids from roots of Coix lachryma-jobi var. ma-yuen. J. Nat. Prod. 1988, 51, 74−79. (19) Bharathkumar, H.; Sundaram, M. S.; Jagadish, S.; Paricharak, S.; Hemshekhar, M.; Mason, D.; Kemparaju, K.; Girish, K. S.; Basappa; Bender, A.; Rangappa, K. S. Novel benzoxazine-based aglycones block glucose uptake in vivo by inhibiting glycosidases. PLoS One 2014, 9, No. e102759. (20) Buchmann, C. A.; Nersesyan, A.; Kopp, B.; Schauberger, D.; Darroudi, F.; Grummt, T.; Krupitza, G.; Kundi, M.; Schulte-Hermann, R.; Knasmueller, S. Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), two naturally occurring benzoxazinones contained in sprouts of Gramineae are potent aneugens in human-derived liver cells (HepG2). Cancer Lett. 2007, 246, 290−299. (21) Arroyo, E.; Chinchilla, N.; Molinillo, J. M. G.; Macias, F. A.; Astola, A.; Ortiz, M.; Valdivia, M. M. Aneugenic effects of benzoxazinones in cultured human cells. Mutat. Res., Genet. Toxicol. Environ. Mutagen. 2010, 695, 81−86. (22) Saeed, A. I.; Bhagabati, N. K.; Braisted, J. C.; Liang, W.; Sharov, V.; Howe, E. A.; Li, J.; Thiagarajan, M.; White, J. A.; Quackenbush, J. TM4 microarray software suite. Methods Enzymol. 2006, 411, 134− 193. (23) Saeed, A. I.; Sharov, V.; White, J.; Li, J.; Liang, W.; Bhagabati, N.; Braisted, J.; Klapa, M.; Currier, T.; Thiagarajan, M.; Sturn, A.; Snuffin, M.; Rezantsev, A.; Popov, D.; Ryltsov, A.; Kostukovich, E.; Borisovsky, I.; Liu, Z.; Vinsavich, A.; Trush, V.; Quackenbush, J. TM4: a free, open-source system for microarray data management and analysis. BioTechniques 2003, 34, 374−378. (24) Fomsgaard, I. S.; Mortensen, A. G.; Carlsen, S. C. Microbial transformation products of benzoxazolinone and benzoxazinone allelochemicals − a review. Chemosphere 2004, 54, 1025−1038. (25) Pekkinen, J.; Rosa-Sibakov, N.; Micard, V.; Keski-Rahkonen, P.; Lehtonen, M.; Poutanen, K.; Mikkänen, H.; Hanhineva, K. Amino acid-derived betaines dominate as urinary markers for rye bran intake in mice fed high-fat diet − a non-targeted metabolomics study. Mol. Nutr. Food Res. 2015, n/a−n/a. (26) Savolainen, O. I.; Coda, R.; Suomi, K.; Katina, K.; Juvonen, R.; Hanhineva, K.; Poutanen, K. The role of oxygen in the liquid fermentation of wheat bran. Food Chem. 2014, 153, 424−431.

(27) Nordlund, E.; Katina, K.; Aura, A.-M.; Poutanen, K. Changes in bran structure by bioprocessing with enzymes and yeast modifies the in vitro digestibility and fermentability of bran protein and dietary fibre complex. J. Cereal Sci. 2013, 58, 200−208. (28) Anson, N. M.; Selinheimo, E.; Havenaar, R.; Aura, A. M.; Mattila, I.; Lehtinen, P.; Bast, A.; Poutanen, K.; Haenen, G. R. Bioprocessing of wheat bran improves in vitro bioaccessibility and colonic metabolism of phenolic compounds. J. Agric. Food Chem. 2009, 57, 6148−6155. (29) Mateo Anson, N.; Aura, A. M.; Selinheimo, E.; Mattila, I.; Poutanen, K.; van den Berg, R.; Havenaar, R.; Bast, A.; Haenen, G. R. Bioprocessing of wheat bran in whole wheat bread increases the bioavailability of phenolic acids in men and exerts antiinflammatory effects ex vivo. J. Nutr. 2011, 141, 137−143.

5949


Glycosylated Benzoxazinoids Are Degraded during Fermentation of

Recommend Documents