Journal of Food Composition and Analysis 64 (2017) 78–84 Contents lists available at ScienceDirect Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca Original research article ☆ ☆☆ Challenges of developing a valid dietary glucosinolate database , T ⁎ Xianli Wua, , Jianghao Sunb, David B. Haytowitza, James M. Harnlyb, Pei Chenb, Pamela R. Pehrssona a Nutrient Data Laboratory, USDA ARS Beltsville Human Nutrition Research Center, 10300 Baltimore Ave., Beltsville, MD 20705, USA b Food Composition and Methods Development Laboratory, USDA ARS Beltsville Human Nutrition Research Center, 10300 Baltimore Ave., Beltsville, MD 20705, USA ARTICLE INFO ABSTRACT Chemical compounds studied in this article: Glucosinolates are a group of important cancer chemo-preventive sulfur-containing compounds in cruciferous Glucoraphanin (PubChem CID: 656556) vegetables. To estimate their dietary intake, there is a great need to develop a valid glucosinolate database. The 4-Hydroxy glucobrassicin (PubChem CID: aim of this study was to investigate the key challenges in developing such database. First, three commonly used 9576741) enzyme deactivation methods (blanching, steaming and microwaving) were compared with samples of raw Hydroxy glucobrassicin (PubChem CID: untreated broccoli and kale. Steaming and microwaving were found to effectively deactivate myrosinase, both 123131529) led to significantly higher glucosinolate values that blanching (∼15–50% higher). Glucosinolates in untreated Glucobrassicin (PubChem CID: 656506) 4-Methoxy-glucobrassicin (PubChem CID: broccoli was similar to that of blanching broccoli, while glucosinolates were not detected in untreated kale. Heat 656563) treatment was also shown to alter the profiles of individual glucosinolate. Quantification of total glucosinolates Neoglucobrassicin (PubChem CID: 9576416) of four common vegetables was compared by the two most commonly used analytical methods (ISO 9167-1 Gluconapoleiferin (PubChem CID: 92024315) method and cyclocondensation method). Except for kale, the results from ISO 9167-1 were much higher (∼6–8 Sinigrin (PubChem CID: 23682211) fold) than that from cyclocondensation method in other three vegetables. In conclusion, the sample preparation Methoxy-glucobrassicin (PubChem CID: procedure, analytical method for quantification and the compounds to be measured must be considered and 6122984) validated in order to develop a valid dietary glucosinolate database. Keywords: Glucosinolate Isothiocyanate Cruciferous Myrosinase Database Enzyme Brassica Food composition Food analysis 1. Introduction variety of nutrients and phytonutrients including vitamins, carotenoids, folate, selenium, glucosinolates and flavonoids. Nonetheless, a large Cruciferous or Brassica vegetables refer to a large group of com- body of evidence accumulated in the past decades suggested that the monly consumed vegetables, including broccoli, kale, cabbages, cauli- cancer chemo-preventive effects of cruciferous vegetables may largely flower, Brussels sprouts, etc. Epidemiological evidence from pro- be attributed to glucosinolates and their breakdown products (Clarke, spective cohort studies and retrospective case–control studies have 2010). linked consumption of cruciferous vegetables to reduced risk of various Glucosinolates are sulfur rich, anionic secondary metabolites found cancers, including lung (Wu et al., 2015), gastric (Bosetti et al., 2012), principally in the plant order Brassicales. Over 350 genera and 3000 colorectal (Azeem et al., 2015), breast (Bosetti et al., 2012), bladder species in Brassicaceae (older name, Cruciferae), as well as a number of (Al-Zalabani et al., 2016) and prostate cancer (Chan et al., 2009). Like non-cruciferous plants, contain glucosinolates; the genus Brassica con- most other vegetables, cruciferous vegetables are good sources of a tain most of the commonly consumed vegetables (Fahey et al., 2001). ☆ This paper was originally submitted as an oral presentation at the 39th NNDC held from May 16–18, 2016 in Alexandria, VA, USA. ☆☆ Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. ⁎ Correspondence author. E-mail address: [email protected] (X. Wu). http://dx.doi.org/10.1016/j.jfca.2017.07.014 Received 29 October 2016; Received in revised form 19 January 2017; Accepted 7 July 2017 Available online 11 July 2017 0889-1575/ Published by Elsevier Inc. X. Wu et al. Journal of Food Composition and Analysis 64 (2017) 78–84 in plant foods. The results will be used to develop a data quality eva- luation system for dietary glucosinolates. 2. Materials and methods 2.1. Chemicals and reagent Methanol-HPLC grade, Sodium Acetate, DEAE Sepharose CL–6B suspension, Imidazole formate and Sinigrin hydrate, Benzene-1,2-di- Fig. 1. Chemical structure of glucosinolate. thiol, sulfatase (from Helix pomatia) and myrosinase were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1,3-benzodithiole-2-thione Chemically, glucosinolates are β-thioglucoside N-hydroxysulfates (also was synthesized in the Food Composition and Method Development Lab known as (Z)-N-hydroxysulfates or S-glucopyranosyl thiohydroximates) and purified with thin layer chromatography (TLC). The purity (96.2%) (Fahey et al., 2001)(Fig. 1). The number of reported glucosinolates is was examined with nuclear magnetic resonance spectroscopy (NMR) now approaching 200, which differ from each other by the side chain R (Bruker AVIII–600 MHz, Rheinstetten, Germany). (Clarke, 2010). The glucosinolates are grouped by the structures of their side chain R as three major groups: aliphatic, aromatic and heterocylic 2.2. Plant materials and processing methods (indole) glucosinolates (Fahey et al., 2001). Types of glucosinolates and their concentration vary enormously Selected cruciferous vegetables of the Brassica oleracea species among different cruciferous vegetables, both qualitatively and quanti- (broccoli – Italica Group; kale – Acephala Group; collard greens – tatively, according to species and cultivar, tissue type, growing stage, Acephala Group; and red cabbage – Capitata Group) were purchased environmental factors, insect attack and microorganism intrusion from local grocery stores in Beltsville, MD. The vegetables were cleaned (Holst and Williamson, 2004), which complicates the interpretation of by rinsing with tap water for approximately 60–90 s, followed by rin- epidemiological studies. The total glucosinolate intake in German po- sing with distilled water. Inedible parts (leaves/scales, hard stems) were pulation was estimated as 14.2 ± 1.1 mg/day for men and cut off and the vegetables were cut into 5 cm × 3 cm pieces. Vegetable 14.8 ± 1.3 mg/day for women (Steinbrecher and Linseisen, 2009); pieces were mixed thoroughly prior to further processing. Samples 6.5 mg/day, among which 35% were of indole type in a Spanish adult without further processing (control) were freeze-dried immediately population (Agudo et al., 2008) and the national mean daily intake is after cutting. calculated to be 46.1 mg in fresh material, and 29.4 mg in cooked in UK Three common enzyme deactivation methods are blanching, (Sones et al., 1984). However, these crude dietary intake estimates were steaming and microwaving. The vegetables were cut just before cooking made based on very limited data or dietary exposure to cruciferous to prevent enzyme reaction and oxidation. For blanching, tap water was vegetables. There is no dietary intake of glucosinolates estimation in added into a stainless steel pot and was heated to boiling. Vegetable US, due to lack of adequate dietary glucosinolate composition data. To pieces were put into the pot for 85 s, then quickly removed from the establish the link between the intake of Brassica vegetables and the risk boiling water and spread on a plate to cool. The cooked vegetables were of cancer and other chronic diseases, to estimate the dietary intake of freeze-dried before analysis. For steaming, the steamer basket was in- glucosinolates and to precisely understand the mechanisms of their serted to a stainless steel pot containing approximately 2.5 cm (1 in) of health benefits, there is a great need of for a valid database of dietary water, with the bottom of steamer basket approximately 5 cm (2 in) glucosinolates. above the water surface. The water in the pot was brought to a boil over An extensive literature search found only one published study at- high heat and the vegetables pieces were scattered over the steamer tempted to develop a dietary glucosinolate database (McNaughton and basket and steamed in medium heat for 5 min. When the vegetables Marks, 2003). This paper was published in 2003 and has since been were done, they were quickly removed from the steamer basket and widely cited in epidemiological studies and used to estimate dietary were spread on a plate to cool. For microwaving, vegetable pieces was intake of dietary glucosinolates. However, only 18 references were in- put in a microwave safe bowl with a teaspoon of water. The bowl was cluded in developing this database, and among them 10 studies used then covered with microwave-safe plastic wrap and microwaved for the glucose-release methods, 5 used high performance liquid chroma- 1 min at full power (1200W, 2450 MHz) in a microwave oven tography (HPLC) and 3 used gas chromatography (GC) based methods. (Panasonic Model NE-12521, Newark, NJ, USA). The samples were then Because