Alterations of the Benzoxazinoid Profiles of Uninjured Maize

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Alterations of the Benzoxazinoid Profiles of Uninjured Maize Seedlings During Freezing, Storage, and Lyophilization Hans Albert Pedersen,* Kirsten Heinrichson, and Inge S. Fomsgaard Department of Agroecology, Aarhus University, Forsøgsvej 1, Flakkebjerg, DK-4200 Slagelse, Denmark ABSTRACT: Benzoxazinoids are highly studied compounds due to their biological activity and presence in several cereals. They include compound classes such as hydroxamic acids and lactams and usually occur as inactive glucosides in unstressed plants. Injury to the plant causes enzymatic hydrolysis of the inactive glucosides to the biologically active hydroxamic acid and lactam aglucones. The hydroxamic acids further undergo spontaneous hydrolysis to benzoxazolinones in aqueous solution. Extraction methods that do not cause immediate inactivation of enzymes result in accumulation of aglucones in samples. Using HPLC-MS to profile benzoxazinoids in maize seedlings subjected to several sample preparation techniques, we have found that hydroxamic acid aglucones and benzoxazolinones are present in uninjured maize seedlings, but that the benxozazinoid profile varies depending on sample treatment, potentially underrepresenting the glucoside content and overrepresenting the aglucone and benzoxazolinone content. KEYWORDS: benzoxazinoids, hydroxamic acids, DIMBOA, DIMBOA-glc, transformation, extraction, lyophilization, HPLC-MS

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INTRODUCTION The benzoxazinoids may be divided into the benzoxazolinones, hydroxamic acids, and lactams, the latter two existing either as glucosides or aglucones. As their systematic names are long, they are commonly abbreviated according to Figure 1. Their biosynthesis is well described in the literature,1,2 and they are present in young cereal plants at high concentrations.3 In uninjured plants, they exist primarily as hydroxamic acid glucosides and are stored in the vacuole.4 Upon mechanical damage to the plant, subcellular compartments are broken, thereby bringing the glucosides into contact with βglucosidases5 hitherto stored in plastids.4,6 This causes hydrolysis of the biologically inactive glucosides releasing biologically active aglucones. The hydroxamic acid aglucones are unstable in aqueous environments and decompose spontaneously into benzoxazolinones.7,8 The degradation processes are summarized in Figure 2 for DIMBOA-glc, 8. The discovery of the benzoxazolinones BOA, 11, and MBOA, 12, in 1957 raised questions because those substances could not be isolated from intact plants immersed in boiling water, whose enzymes had thus been inactivated.9 This prompted the search for precursors, leading to the discovery of the glucosidic and agluconic forms of the hydroxamic acid DIBOA, 3, in rye seedlings10 and DIMBOA, 4, in wheat and maize seedlings in 1959,11 and it became apparent that the benzoxazinoids occur in plants as inactive hydroxamic acid glucosides that are hydrolyzed by β-glucosidases5 when the plant is subjected to a challenge or damaged mechanically. The benzoxazinoids have since been found to occur in many cereals as well as several nongraminaceous plant species in two orders of dicots.12 Since their discovery the benzoxazinoids have been the subject of steadily increasing research efforts. Searching Web of Science results in 60 papers on the topic of “benzoxazinoids” that have been published since 2010, but since nomenclature usage has changed over time, this excludes a great number of papers. Eighteen papers on “benzoxazinone © 2017 American Chemical Society

derivatives” from the FATEALLCHEM project, in which our laboratory participated, were published in a dedicated section of the Journal of Agricultural and Food Chemistry in 2006 alone.13−30 Benzoxazinoids have primarily been of interest as defense compounds of agroecological relevance,2,4,9,31 but research into their dietary relevance has revealed that they occur in high concentrations in cereals and cereal-based foods32,33 and are absorbed and metabolized in animals and humans.34−41 Because of the degradation pathway in Figure 2, the measured concentrations of hydroxamic acid aglucones and benzoxazolinones in plant material were observed to depend heavily on sample treatment prior to extraction.42 The degradation process makes proper sample treatment during and after extraction essential, for both analytical and preparative purposes, and one should consider which reactions should be stopped or allowed to proceed, depending on whether the aim is to isolate glucosides, hydroxamic acid aglucones, or benzoxazolinones. When the aim of a study is quantitative, both reactions must be minimized or stopped to reveal a true picture of the benzoxazinoid profile at the moment of sampling. While immersion in boiling solvent is an effective way to inactivate enzymes, it may potentially lead to undesired chemical transformations. Freeze-drying is widely used as a gentle technique for removing water from frozen sample material, but questions have been raised about its appropriateness for benzoxazinoid analysis due to the potential for enzymatic reactions to occur during the freezing process42 or in samples that are not fully dry or frozen. We hypothesized that, provided that samples were fully and immediately frozen prior to lyophilization and then placed under low-pressure conditions Received: Revised: Accepted: Published: 4103

March 14, 2017 April 27, 2017 April 30, 2017 April 30, 2017 DOI: 10.1021/acs.jafc.7b01158 J. Agric. Food Chem. 2017, 65, 4103−4110

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Journal of Agricultural and Food Chemistry

Figure 1. Structures of benzoxazinoids quantitated in the present study.

Figure 2. Degradation of 8 to 4 via enzymatic hydrolysis and further to 12 spontaneously in aqueous medium. into the stack. After cultivation for 5 d, the seedlings were harvested. Two batches of seedlings were produced in this way: one for each of experiments A and B. Harvesting and Extraction of Seedlings. Two experiments, A and B, were carried out. In the first, three methods (A1−A3) of sample handling and extraction were compared, and in the second, six methods (B1−B6) were compared to expand upon the first experiment. Seven replicates were used to test each sample-handling method (except A1 and A2, where only six replicates were used). Method A1. This method was adapted from that used by Cambier et al.42 Each single seedling was harvested individually by removal from the cultivation tray and immediate submersion in methanol boiling under reflux in a round-bottomed flask equipped with a heating mantle and a magnetic stirrer. One hundred milliliters of methanol was brought to a boil while stirring under reflux. The condenser was momentarily removed, and using tweezers the intact seedling was dropped or gently pushed though the neck of the flask before replacing the condenser. After 15 min the extract thus obtained was decanted. A further 75 mL of methanol was added to the flask and the seedling homogenized using an Ultra-Turrax blender before boiling was repeated for a further 15 min. The homogenate from the second boiling was filtered through filter paper into the extract previously obtained from that seedling. In this manner two combined extracts were obtained for each individual seedling. The extracts were stored briefly at −18 °C before analysis by HPLC-MS.

so that water loss from the sample occurred entirely by sublimation, lyophilization was then a fully adequate process for preparing samples of benzoxazinoid-containing plant material for analysis. To this end, we tested a range of sample preparation methods with the aim of determining which one would result in the most accurate reflection of the benzoxazinoid profile of whole maize seedlings.

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EXPERIMENTAL PROCEDURES

Cultivation of Seedlings. One hundred milliliters of commercial game cover “Jægermajs” maize seeds (Danish Agro, Aarhus, Denmark) measured in a 250 mL bottle were soaked in Milli-Q water, made up to the 200 mL mark, overnight before being placed between double layers of tissue paper soaked with deionized water, sealed in plastic bags, and placed in the dark for 2 d to allow pregermination to begin. Germinating seeds were then transferred selectively to cultivation trays where germination and cultivation were continued for another 5 d. The cultivation trays were stackable and measured 17 × 17 × 4 cm (width × length × height) and had narrow slits in the bottom through which water could drain. Twenty-five seedlings were arranged in a 5 × 5 array in each tray. The seedlings were watered twice daily by pouring 250 mL of water over the cultivation trays and allowing it to drain through the slits in the bottom of the tray. Trays were stacked six or seven on top of each other during the germination and cultivation, with each stack topped by a lid with slits distributing the water poured 4104

DOI: 10.1021/acs.jafc.7b01158 J. Agric. Food Chem. 2017, 65, 4103−4110

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Journal of Agricultural and Food Chemistry Method A2. This method introduced a frozen storage step between the flash-freezing and extraction included in method 2. Each seedling was removed from the cultivation tray and submerged immediately in liquid nitrogen for 30 s. The liquid nitrogen was subsequently replenished to keep the seedlings submerged while they were transported to the freezer, where the nitrogen was allowed to evaporate, and stored at −18 °C for 2 weeks. Following storage, the seedlings were removed from the freezer one at a time and immediately extracted following the procedure described for method A1. Method A3. This method was identical to method A2, except that the extraction step was performed using a model 350 accelerated solvent extractor (ASE) (Dionex, Sunnyvale, CA). For each sample, 0.1 g homogenized, lyophilized seedling material was placed in a 33 mL ASE cell as follows: A cellulose filter was placed in the bottom of the cell, followed by 5 g of Ottawa sand. The sample material was then added, followed by another 5 g of Ottawa sand and another cellulose filter. The filter was depressed slightly into the cell and the cell agitated to mix the sample material and the sand. The filter was then gently depressed as fully as allowed by the sand and the sample, and the remainder of the cell filled with glass beads. The samples were then extracted using the following ASE parameters: preheat, 5 min; heat, 5 min; static, 3 min; flush, 60% volume; purge, 60 s; 4 cycles; pressure, 107 Pa; temperature, 80 °C. The extraction solvent consisted of methanol/water/acetic acid (80:19:1, v/v/v). Method B1. This method was identical to method A1, differing only in that it was applied to the second batch of seedlings and that minor aspects of the HPLC-MS analysis were changed. Method B2. This method introduced a flash-freezing step prior to the procedure outlined for methods A1 and B1. Each single seedling was removed from the cultivation tray and submerged immediately in liquid nitrogen for 30 s before being transferred to boiling methanol and extracted as described for methods A1 and B1. Method B3. This method introduced a frozen storage step between the flash-freezing and extraction included in method B2. Each seedling was removed from the cultivation tray and submerged immediately in liquid nitrogen for 30 s. The liquid nitrogen was subsequently replenished to keep the seedlings submerged while they were transported to the freezer, where the nitrogen was allowed to evaporate, and stored at −18 °C for 2 weeks. Following storage, the seedlings were removed from the freezer one at a time and immediately extracted following the procedure described for method A1. Method B4. This method added a lyophilization step after the flashfreezing and frozen storage steps in method B3 and before the extraction used in method A1. After storage for 2 weeks the seedlings were placed in the freeze-dryer for a further 2 weeks, after which they were removed and extracted in boiling methanol. Methods B5 and B6. These methods were identical to methods B3 and B4, respectively, but seedlings were stored at −80 °C instead of −18 °C. Analysis of the Benzoxazinoid Content of Seedlings. HPLCMS Analysis. For experiment A, the methanolic extracts were evaporated and redissolved in 40 mL of methanol, which was then diluted by a factor of 5 to a final solvent composition of methanol/ water/acetic acid (40:59.5:0.5, v/v/v). ASE extracts were diluted by a factor of 4 to the same solvent composition. For experiment B, aliquots of methanolic maize seedling extract were diluted to either 2.5, 10, or 20 times the original volume and a solvent composition of methanol/water/acetic acid (40:59.5:0.5 v/v/v). After dilution, 1 mL aliquots of extracts were filtered through a 0.22 μm PTFE filter and analyzed by chromatography on a 1200 HPLC (Agilent, Santa Clara, CA) interfaced with a 3200 QTrap mass spectrometer (Sciex, Framingham, MA). An injection volume of 10 μL was used. The column used for chromatography was a 250 mm × 2 mm i.d., 4 μm, Synergi Polar RP-80A, with a 4 mm × 2 mm i.d. guard column of the same material (Phenomenex, Torrance, CA). The column temperature was maintained at 30 °C. The gradient used eluent A, consisting of 7% acetonitrile in water, and eluent B, consisting of 78% acetonitrile in water. Both eluents contained 20 mM acetic acid modifier. The

gradient began at 0% B, ramping to 8% B at 1 min, 10% B at 3 min, 70% B at 13 min, and finally 90% B at 14 min, which was held until 16 min before returning to 0% B at 17 min and reequilibrating until 23 min. Compounds were detected by negative-mode ESI-MS/MS using a multiple reaction monitoring (MRM) method. The instrumentdependent parameters were as follows: curtain gas, 30 psi; CAD gas, −2; temperature, 550 °C; gas 1, 60 psi; gas 2, 50 psi; interface heater, on; ion spray voltage, −4200 V. Compound-dependent parameters and retention times are given in Table 1. Quantitation was carried out

Table 1. Compound-Dependent HPLC-MS Parameters for the MRM Method Used To Quantitate Benzoxazinoids in Maize Seedlingsa compound

MRM transition

tR (min)

DP (V)

EP (V)

CE (V)

CXP (V)

1 2 3 4 5 6 7 8 9 10 11 12

164/108 194/138 180/134 164/149 326/164 356/194 342/134 372/164 488/164 504/134 134/42 164/149

10.2 11.8 9.6 11.5 7.1 9.2 7.1 9.3 4.5 4.6 13.1 13.8

−28 −28 −12 −24 −34 −30 −22 −22 −45 −36 −45 −24

−3 −3 −1.5 −4 −4 −4 −4 −4 −9 −4 −11 −4

−23 −19 −10 −20 −20 −22 −24 −18 −30 −38 −40 −20

−1.5 −2.5 −2.5 −1 −4.9 −11 −4 −5 −4 −2 −1 −1

a

Abbreviations: retention time (tR), declustering potential (DP), entrance potential (EP), collision energy (CE), and cell exit potential (CXP).

against external standards using calibration curves ranging from 0 to 800 ng/mL. Analytical standards were either synthesized or obtained as gifts during prior projects43 or as part of an ongoing patenting process.44 Statistical Analysis. For experiment B, Levene’s test for equality of variances was applied for each compound measured across all treatments. The full data set was then analyzed by multifactor ANOVA to detect which factors (flash-freezing, frozen storage, lyophilization, and dilution) were significant for specific compounds. At each dilution level, the relative standard deviation (RSD) was calculated within each combination of compound and treatment. The average RSD across treatments for each compound was then calculated and compared to the other dilution levels to decide which data set (one for each dilution level) to analyze. Analyses were performed using Statgraphics 5.1 software.

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RESULTS AND DISCUSSION The major benzoxazinoids known from maize are the 7methoxylated compounds shown in Figure 2, and these compounds are therefore often central to quantitative studies of benzoxazinoids in maize. To achieve a result reflective of the in planta concentrations of these compounds at the time of analysis, it is imperative that enzymatic degradation processes be arrested and that subsequent spontaneous degradation of hydroxamic acid aglucones be limited as much as possible. It has been suggested that hydroxamic acid aglucones are only found in maize seedlings when these are improperly handled prior to analysis.42 Meihls et al.31 have used cold extraction after homogenization under liquid nitrogen to extract benzoxazinoids while avoiding transformations. Cambier et al.3,42 extracted seedling tissue in boiling methanol to avoid transformations and reported that glucosides were degraded to aglucones when samples were frozen and thawed. 4105


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Figure 3. Average concentrations of benzoxazinoids (A) derived from glucoside 8, (B) derived from glucoside 6, (C) derived from glucoside 7, and (D) derived from glucoside 5, in maize seedlings subjected to three sample preparation and extraction methods in experiment A. Error bars show the standard error of the mean (n = 6 for methods A1 and A2, n = 7 for method A3).

Figure 4. Average concentrations of benzoxazinoids (A) derived from glucoside 8, (B) derived from glucoside 6, (C) derived from glucoside 7, and (D) derived from glucoside 5, in maize seedlings subjected to varying sample preparation methods in experiment B. Error bars show the standard error of the mean (n = 7).

To test whether flash-freezing followed by frozen storage and freeze-drying could provide an acceptable alternative to extraction with boiling methanol, we compared several methods in two experiments. In experiment A we first compared

immediate extraction of fresh maize seedlings with boiling methanol to seedlings that had been flash-frozen, stored at −18 °C, lyophilized, and homogenized before extraction with either boiling methanol or ASE. In experiment B we subjected 4106


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Journal of Agricultural and Food Chemistry otherwise unharmed maize seedlings to flash-freezing, storage for 2 weeks at −18 °C or −80 °C, and optional freeze-drying afterward and compared this to direct extraction of fresh seedlings with boiling methanol. Figure 3 shows the concentrations of benzoxazinoids detected during experiment A. For all glucoside−aglucone pairs included in the experiment, extraction with boiling methanol produced an excess of glucoside over aglucone by a factor of 2 or more, but when the seedlings were subjected to flash-freezing, storage, and lyophilization, this picture changed dramatically, regardless of whether they were extracted by boiling in methanol or ASE using methanol/water/acetic acid (80:19:1, v/v/v). In fact, the two extraction methods produced remarkably similar profiles. This indicated that dramatic alterations in the benzoxazinoid profile were possible during sample preparation, and we therefore decided to investigate each of these steps in more detail in experiment B, but using only one extraction method for simplicity. In experiment B we tested whether flash-freezing followed by frozen storage and freeze-drying could provide an acceptable alternative to extraction with boiling methanol by subjecting otherwise unharmed maize seedlings to flash-freezing, storage for 2 wk at −18 °C or −80 °C, and optional freeze-drying afterward. Several benzoxazinoids were included in the analysis as both aglucones, 1−4, and glucosides, 5−8, and when the seedlings were extracted directly with boiling methanol, the glucoside concentrations of DIMBOA-glc, 8, and HBOA-glc, 5, were 13 and 35 times higher, respectively, than those of their aglucones, DIMBOA, 4, and HBOA, 1 (Figure 4). Subjecting seedlings to flash-freezing immediately prior to extraction in boiling methanol altered the aglucone:glucoside ratio for 4 and 8 so that the aglucone concentration was nearly 1.5 times the glucoside concentration. For HMBOA, 2 and HMBOA-glc, 6, the excess of glucoside dropped from 15:1 to 2:1, but for DIBOA, 3, and DIBOA-glc, 7, as well as 1 and 5 the glucosides still outweighed the aglucones by factors of 12 and 14 respectively. In absolute terms the concentration of all aglucones was far higher after flash-freezing, but the only glucoside which decreased was 8 (Figure 4A-D). The treatments applied after flash-freezing did not appear to matter nearly as much as the flash-freezing itself. Levene’s test showed significant inequality of variance for 1− 4 and 11 across treatments. With the exception of BOA, 11, those compounds are all aglucones produced by enzymatic hydrolysis of their corresponding glucosides, 5−8, upon injury to the seedling, with 11 being a spontaneous degradation product of 3, and thus also indirectly influenced by enzymatic hydrolysis. The variance of the concentrations of those compounds was considerably larger in samples subjected to treatments B2−B6 than in those subjected to treatment B1 (Figure 4). Multifactor ANOVA (Table 2) revealed that dilution of the extract prior to analysis was significant only for the dihexose 9, the benzoxazolinone 11, and the lactam 2. The former two were low-abundance compounds where coverage became problematic when samples were diluted, while the p value for 2 fell between 0.01 and 0.05, so that the significance may have been due to chance. On the other hand, all compounds except for 7 were affected by one or more of the remaining factors. Visual inspection of residual plots did not suggest deviations from normality although the variances were unequal in some cases. Surprisingly, for all compounds, the RSD was lowest in the least diluted extract (Table 3). Additionally, the data set from the least diluted extracts

Table 2. Significance of Sample Treatment Effects in Methods B1−B6 for Measured Concentrations of Benzoxazinoids in Maize Seedlings As Determined by Multifactor ANOVAa

a

compound

flash-freezing

storage

lyophilization

dilution

1 2 3 4 5 6 7 8 9 10 11 12

* *** − *** * − − *** − − − ***

** * *** * *** * − − − * * −

*** *** *** * − − − * − − * −

− * − − − − − − ** − ** −

*p < 0.05; ** p < 0.01; *** p < 0.001; − p > 0.05.

Table 3. Relative Standard Deviation (%) Averages across Methods B1−B6 for Each Compound and Dilution Levela dilution factor compound

2.5×

10×

20×

1 2 3 4 5 6 7 8 9 10 11 12

29 23 64 30 33 20 47 23 28 38 80 26

37 26 76 34 35 25 49 27 43 42 − 28

36 29 66 37 36 26 53 28 57 39 − 31

a

Values of zero, where compounds were not detected, were excluded from the calculations. BOA was not detected in the 10× and 20× dilutions.

contained the smallest amount of missing values due to undetected compounds. The missing values were almost entirely due to 11 not being detected at the factor 10 and 20 dilution levels. In the absence of a significant dilution effect on the majority of compounds, and even a slightly lower variability in more concentrated samples, the lowest dilution factor was chosen for statistical analysis of differences owing to sample handling. Table 3 lists the relative standard deviations averaged across sample treatments for each compound, at all three dilution levels. The compounds most relevant to maize, 4, 8, and 12, are part of the same degradation pathway (Figure 2) and account for over 90% of the benzoxazinoids measured in this study. Figure 4A shows this subset of the data. The profiles are remarkably similar for all sample treatments except the immediate extraction in boiling methanol. This suggests that flash-freezing was ineffective at negating the effect of βglucosidases on 8 in the whole seedlings, but that subsequent treatment mattered little. Interestingly, the total molar quantities increased from approximately 9 μmol/g with immediate boiling extraction to 16−17 μmol/g for all the other treatments. This suggests that 4107


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Journal of Agricultural and Food Chemistry there was either a rapid biosynthesis occurring during the flashfreezing process or a rupture of cells making the extraction especially more effective for the aglucones. Improved extraction efficiency upon flash-freezing seems unlikely, however, since benzoxazinoids are primarily thought to be stored as inactive glucosides in subcellular compartments,1 and only roughly twothirds of those glucosides were still extracted (Figure 4A). Depending on tissue localization and age, however, aglucones may be present to varying degrees, even in uninjured plants.2,27,30,42,45 Another possibility is that interaction of boiling methanol with fresh tissue somehow caused the glucosides to be less extractable. If the total increase is due to an improvement in extraction efficiency, there is also enzymatic hydrolysis of glucosides occurring at the same time. A similar pattern was seen for the 7-methoxylated lactams 2 and 6, with a very modest decrease in the concentration of the glucoside accompanied by at least a fivefold increase in the concentration of the aglucone. The elevated total amount of compounds derived from 8 (4, 8, and 12) found in seedlings treated with methods B2−B6 may also have been due, at least partly, to an undetected reservoir of related diglycosylated metabolites such as the hypothetical hexose derivative (DIMBOA-glc-hex) of 8, analogous to the dihexose, 10, we detected, or to unknown transformation pathways. It should be noted, however, that the concentrations of the dihexoses 9 and 10 were quite low compared to their corresponding monoglucosides, 5 and 7, so it is not obvious that DIMBOA-glc-hex could play such a role. For the non-7-methoxylated lactams and hydroxamic acids, 1 and 3, there appeared to be no loss of glucoside following flashfreezing, frozen storage, or lyophilization, although again, the aglucone concentration appeared to increase. Finally, as expected from the tendency of hydroxamic acid aglucones to spontaneously degrade in aqueous media, the concentrations of the benzoxazolinones 11 and 12 resulting from degradation of 3 and 4, respectively, were higher in the samples that also had elevated concentrations of hydroxamic acids. It is tempting, however, to speculate on the existence of an alternative biosynthetic route to the benozoxazolinones, perhaps by conversion of hydroxamic acids prior to the glucosylation step. Irrespective of whether the observed changes were due to enzymatic liberation of aglucones in combination with increased extraction efficiency or biosynthesis, it is clear that immediate enzymatic inactivation was not achieved with any method except, perhaps, method B1. Increased extraction efficiency along with inactivation of enzymes should have produced an elevated quantitation result for the glucosides in methods B2−B6, and this did not happen. It is possible that the thickness of the kernels, which remained attached to the seedlings, prevented them from being flash-frozen, and that the majority of the enzymatic transformations took place there, as both the roots and the shoots were very thin, and both cold extraction and hot extraction techniques have previously been employed in quantitation of benzoxazinoids.31,42 Interestingly, for the compounds derived from 8, there was relatively little difference among methods B2−B6, suggesting that after flashfreezing, further treatment did not greatly influence the quantitation result. This suggests that flash-freezing did result in enzymatic inactivation, but that it did not happen fast enough to reflect the original glucoside concentration in the undisturbed seedling upon quantitation. The question remains, however, as to whether the excess equivalents of aglucones are best explained by increased extraction efficiency in methods B2−B6 or some other factor.

It is clear that the highest concentration of glucosides and the lowest concentration of expected degradation products was achieved using method B1. Furthermore, both hydroxamic acids and benzoxazolinones were detected using this method, contrary to reports in the literature.3,42 This difference may be due to advances in instrumentation and lowering of detection limits over the last 10−15 years or simply to the sensitivity of maize seedlings to injury, though great care was taken when manipulating them. Immediate extraction of maize seedlings in boiling methanol produced the highest quantitation result for benzoxazinoid glucosides compared to hydroxamic acid aglucones and benzoxazolinones. This suggests that, while impractical for field studies, it was the method producing the results closest to the undisturbed benzoxazinoid profile, at least in the case of glucosides. The question remains, however, whether this was also the case for the aglucones. When comparing experiments A and B, the benzoxazinoid profiles in the fresh seedlings extracted directly with boiling methanol where somewhat different, although the same protocol was applied (methods A1 and B1 were equivalent), indicating a batch effect. Concentrations of glucosides were generally lower in experiment A and higher in experiment B, while the opposite was true for aglucones. Notably also the accumulation of 4 when seedlings were not extracted directly was higher in experiment A than in experiment B. The underlying tendencies, however, of higher glucoside concentration with direct extraction in boiling methanol, and accumulation of aglucones when this was not done, were consistent across the two experiments, and another four experiments we carried out (not shown, data available on request) yielded similar results, resembling mostly experiment B. Although the current conclusions can be applied with certainty only to maize seedlings, they have potentially important implications for field studies. It may be impractical to immediately perform a boiling extraction in the field, and an apparent solution to this problem may be to apply a model to calculate original metabolite concentrations from those actually measured. Such a model would, however, suffer from the large variability seen for the abundant compounds in methods B2− B6 and should therefore rely on a sample size large enough to reflect this variability. Flash-freezing intact seedlings prior to further treatment including frozen storage and lyophilization altered the benzoxazinoid profile considerably compared to the fresh seedlings subjected to immediate extraction with boiling methanol, but no great differences in the concentration of major compounds were found across subsequent treatments after flash-freezing. Hydroxamic acid aglucones and benzoxazolinones were detected even in intact seedlings extracted immediately with boiling methanol, suggesting that these compounds are present at low concentrations in uninjured seedlings The results suggest that immediate extraction in boiling methanol is most appropriate for enzymatic inactivation and that while flash-freezing with liquid nitrogen does inactivate enzymes, it does not do so fast enough in cases where parts of the seedlings, such as the remaining kernel, cannot be immediately frozen through. The lack of difference between treatments after flash-freezing suggests that the altered benzoxazinoid profile results neither from frozen storage nor from lyophilization, but this requires further study and is only of consequence if a successful method of freezing is applied. 4108


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(14) Etzerodt, T.; Nielsen, S. T.; Mortensen, A. G.; Christophersen, C.; Fomsgaard, I. S. Elucidating the transformation pattern of the cereal allelochemical 6-methoxy-2-benzoxazolinone (MBOA) and the trideuteriomethoxy analogue [D3]-MBOA in soil. J. Agric. Food Chem. 2006, 54, 1075−1085. (15) Fomsgaard, I. S. Chemical ecology in wheat plant-pest interactions. How the use of modern techniques and a multidisciplinary approach can throw new light on a well-known phenomenon: allelopathy. J. Agric. Food Chem. 2006, 54, 987−990. (16) Fomsgaard, I. S.; Mortensen, A. G.; Idinger, J.; Coja, T.; Blümel, S. Transformation of benzoxazinones and derivatives and microbial activity in the test environment of soil ecotoxicological tests on Poecilus cupreus and Folsomia candida. J. Agric. Food Chem. 2006, 54, 1086− 1092. (17) Fritz, J. I.; Braun, R. Ecotoxicological effects of benzoxazinone allelochemicals and their metabolites on aquatic nontarget organisms. J. Agric. Food Chem. 2006, 54, 1105−1110. (18) Hansen, L. M. Effect of 6-methoxybenzoxazolin-2-one (MBOA) on the reproduction rate of the grain aphid (Sitobion avenae F.). J. Agric. Food Chem. 2006, 54, 1031−1035. (19) Jia, C.; Kudsk, P.; Mathiassen, S. K. Joint action of benzoxazinone derivatives and phenolic acids. J. Agric. Food Chem. 2006, 54, 1049−1057. (20) Krogh, S. S.; Mensz, S. J. M.; Nielsen, S. T.; Mortensen, A. G.; Christophersen, C.; Fomsgaard, I. S. Fate of benzoxazinone allelochemicals in soil after incorporation of wheat and rye sprouts. J. Agric. Food Chem. 2006, 54, 1064−1074. (21) Lo Piparo, E.; Fratev, F.; Lemke, F.; Mazzatorta, P.; Smiesko, M.; Fritz, J. I.; Benfenati, E. QSAR models for Daphnia magna toxicity prediction of benzoxazinone allelochemicals and their transformation products. J. Agric. Food Chem. 2006, 54, 1111−1115. (22) Lo Piparo, E.; Smiesko, M.; Mazzatorta, P.; Benfenati, E.; Idinger, J.; Blümel, S. Preliminary analysis of toxicity of benzoxazinones and their metabolites for Folsomia candida. J. Agric. Food Chem. 2006, 54, 1099−1104. (23) Macías, F. A.; Marín, D.; Oliveros-Bastidas, A.; Castellano, D.; Simonet, A. M.; Molinillo, J. M. G. Structure-activity relationship (SAR) studies of benzoxazinones, their degradation products, and analogues. Phytotoxicity on problematic weeds Avena fatua L. and Lolium rigidum Gaud. J. Agric. Food Chem. 2006, 54, 1040−1048. (24) Macías, F. A.; Marín, D.; Oliveros-Bastidas, A.; Chinchilla, D.; Simonet, A. M.; Molinillo, J. M. G. Isolation and synthesis of allelochemicals from Gramineae: benzoxazinones and related compounds. J. Agric. Food Chem. 2006, 54, 991−1000. (25) Martyniuk, S.; Stochmal, A.; Macías, F. A.; Marín, D.; Oleszek, W. Effects of some benzoxazinoids on in vitro growth of Cephalosporium gramineum and other fungi pathogenic to cereals and on Cephalosporium stripe of winter wheat. J. Agric. Food Chem. 2006, 54, 1036−1039. (26) Mathiassen, S. K.; Kudsk, P.; Mogensen, B. B. Herbicidal effects of soil-incorporated wheat. J. Agric. Food Chem. 2006, 54, 1058−1063. (27) Mogensen, B. B.; Krongaard, T.; Mathiassen, S. K.; Kudsk, P. Quantification of benzoxazinone derivatives in wheat (Triticum aestivum) varieties grown under contrasting conditions in Denmark. J. Agric. Food Chem. 2006, 54, 1023−1030. (28) Stochmal, A.; Kus, J.; Martyniuk, S.; Oleszek, W. Concentration of benzoxazinoids in roots of field-grown wheat (Triticum aestivum L.) varieties. J. Agric. Food Chem. 2006, 54, 1016−1022. (29) Villagrasa, M.; Guillamón, M.; Eljarrat, E.; Barceló, D. Determination of benzoxazinone derivatives in plants by combining pressurized liquid extraction-solid-phase extraction followed by liquid chromatography-electrospray mass spectrometry. J. Agric. Food Chem. 2006, 54, 1001−1008. (30) Villagrasa, M.; Guillamón, M.; Labandeira, A.; Taberner, A.; Eljarrat, E.; Barceló, D. Benzoxazinoid allelochemicals in wheat: distribution among foliage, roots, and seeds. J. Agric. Food Chem. 2006, 54, 1009−1015. (31) Meihls, L. N.; Handrick, V.; Glauser, G.; Barbier, H.; Kaur, H.; Haribal, M. M.; Lipka, A. E.; Gershenzon, J.; Buckler, E. S.; Erb, M.;

Taken overall, the results suggest that close attention to enzymatic inactivation must be paid in laboratory studies and in field studies. Where immediate extraction with boiling methanol may be impractical or impossible, it is necessary to be aware of the effect of the treatment, potentially even including a comparative laboratory vs field study as part of the experimental work. A further question remains as to whether the effect of treatment, so important for compounds derived from 8 in the present study on maize seedlings, would be equally important for compounds derived from 7 in species, such as rye, where those compounds dominate, and this will require further study.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +45 87158178. E-mail: [email protected]. ORCID

Hans Albert Pedersen: 0000-0003-2289-7384 Funding

This work was supported financially by the Danish Council for Strategic Research (grant no. B4084-00002) as part of the ERACAPS project BENZEX. Notes

The authors declare no competing financial interest.

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REFERENCES

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Alterations of the Benzoxazinoid Profiles of Uninjured Maize

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