Quantification of benzoxazinoids and their metabolites in Nordic breads
Introduction
Whole grains contain all the parts of the cereal grain, i.e. bran, germ and endosperm, and have a higher content of vitamins, minerals and fiber, along with phytochemicals, such as lignans, alkylresorcinols, sterols and phenolic acids, as compared to their refined grain counterparts (Fardet, 2010). Epidemiological studies have shown that higher whole grain consumption is associated with a reduced risk of lifestyle-related illnesses, such as cardiovascular diseases, cancer, metabolic syndrome and diabetes (de Munter et al., 2007, Fardet, 2010, Mellen et al., 2008, Murtaugh et al., 2003, Sahyoun et al., 2006) although the mechanisms behind the protective effects are not fully understood. Benzoxazinoids (Bx) are a group of phytochemicals which were previously believed to be mainly present in young rye and wheat plants as part of their natural defense system (Sicker & Schulz, 2002). Later, these compounds were reported in commonly consumed breads and flours (Adhikari et al., 2015a). As Bx are proposed to be bioactive and are absorbed by humans and animals it has been suggested that they could be a contributing factor to the health benefits of whole grains (Adhikari, Laerke, Mortensen, & Fomsgaard, 2012a; Adhikari, Laursen, Laerke, & Fomsgaard, 2012b). In previous studies individual Bx have been shown to have antimicrobial (Yalcin et al., 2003), anti-inflammatory (Otsuka, Hirai, Nagao, & Yamasaki, 1988), anti-cancer (Roberts et al., 1998, Wu et al., 2012) and antidepressant properties (Adhikari et al., 2015a; Rosenfeld, Berger, & Negus, 2003). Currently, however, there are no conclusive data on the adverse or beneficial health effects of these compounds in humans. Bx are commonly divided into three groups according to their structures (Table 1) (Niemeyer, 2009): benzoxazolinones such as benzoxazolin-2-one (BOA); lactams, such as 2-hydroxy-1,4-benzoxazin-3-one (HBOA) and its glycoside 2-β-d-glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-Glc); hydroxamic acids, such as 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its glycoside 2-β-d-glucopyranosyloxy-4-hydroxy-1,4-bezoxazin-3-one (DIBOA-Glc). Diglycosides of HBOA and DIBOA (HBOA-di-Glc and DIBOA-di-Glc, respectively) have also been reported but their structures have not been confirmed (Hanhineva et al., 2011). The composition and concentration of Bx in foods are dependent on a range of factors, such as the cultivar, growing conditions of the plants and the food processing methods (Adhikari et al., 2015b; Fomsgaard, Mortensen, & Carlsen, 2004). During food processing the composition and concentrations of Bx can change again via enzymatic, microbial or spontaneous chemical reactions. Different routes have been suggested for the metabolism of Bx by humans, though it is not yet fully understood (Adhikari et al., 2012b; 2013; Beckmann et al., 2013).
To improve understanding of the population level exposure to Bx, broader and better estimation of the Bx content in breads and flours is needed. However, quantification of Bx is hampered by the lack of commercial standards for quantification, especially for the glycoside derivatives. Additionally, the large variations of Bx concentrations within cultivars, as well as due to baking and fermentation processes, are not well understood (Beckmann et al., 2013), and understanding the effect of these processes on Bx content will aid in determining human exposure. Thus, the quantification of Bx in a range of commercially available foods is necessary, in order to obtain a better understanding of the amounts consumed. In this paper a method, including the synthesis of isotopically labeled HBOA-d4 and HHPAA-d4 internal standards, is reported, using UHPLC-MS/MS for the quantification of nine Bx (HBOA, DIBOA, HHPAA, HPAA, BOA, HBOA-Glc, DIBOA-Glc, HBOA-di-Glc, DIBOA-di-Glc) in 30 commercially available breads and flours from the Nordic countries.
Section snippets
Synthesis of standards (HBOA, HHPAA, HBOA-d4 and HHPAA-d4)
The synthesis of HBOA (4) and HHPAA (5) started with the amidation of 2-aminophenol (1) with dichloroacetyl chloride (2) (5 equiv.) providing dichloroacetyl-2-aminophenol (3) in a pure form after recrystallization in methanol. Compound 3 was then treated with saturated sodium bicarbonate solution to generate HBOA (4) (Fig. 1 a) (Yalcin et al., 2003). HHPAA (5) was obtained by reduction of (4) using sodium borohydride (14 equiv.) in methanol, giving 5 (Chatterjee, Sharma, Banerji, & Basa, 1990).
Synthesis of standards (HBOA, HHPAA, HBOA-d4 and HHPAA-d4)
The amidation of 2-aminophenol with dichloroacetyl chloride provided pure dichloroacetyl-2-aminophenol after recrystallization in 48–76% yield and finally HBOA in 32% yield. Reduction of HBOA into HHPAA was obtained in 19% yield. The deuterium-labeled HBOA-d4 and HHPAA-d4 were synthesized following the same synthesis route, starting with reduction of d4-2-nitrophenol in 97% yield. Yields obtained from the synthesis of HBOA and HHPAA were similar to previously reported results (Chatterjee et
Conclusions
The present study reported Bx concentrations of a wide variety of different flour and breads and illustrated how the Bx profile was affected by the bread type, raw material and fermentation. These data are currently lacking in the literature and are needed for assessment of population level exposure. The developed method was suitable and stable for the analysis of Bx in breads. These data can be used in future for estimating daily dietary intake of Bx and related metabolites, which is crucial
Conflicts of interest
The study was partly funded by Bertobos foundation. The PhD work of MV Lind is partly supported by an unrestricted grant from Cereal Partners Worldwide, a joint venture between Nestlé SA and General Mills Ltd. Any of the study sponsors played no role in the design, methods, data management and analysis, or in the decision to publish.
Acknowledgements
Nils-Gunnar Carlsson and Annette Almgren are acknowledged for their assistance in practical work of this study. Dr. Dieter Sicker is acknowledged for providing standard compound DIBOA. Bertebos foundation is acknowledged for partially funding the study.
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