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Article: Doheny-Adams, Timothy, Redeker, Kelly Robert orcid.org/0000-0002-1903-2286, Kittipol, Varanya et al. (2 more authors) (2017) Development of an efficient glucosinolate extraction method. Plant Methods. 17. ISSN 1746-4811 https://doi.org/10.1186/s13007-017-0164-8

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[email protected] https://eprints.whiterose.ac.uk/ Plant M ethods Development of an efficient glucosinolate extraction method --Manuscript Draft--

Manuscript Number: PLME-D-16-00100R1 Full Title: Development of an efficient glucosinolate extraction method Article Type: Methodology

Funding Information: Biotechnology and Biological Sciences Prof Ian Bancroft Research Council (BB/L002124/1) Biotechnology and Biological Sciences Prof Sue E Hartley Research Council (BB/K020463/1)

Abstract: Abstract

Background

Glucosinolates, anionic sulfur rich secondary metabolites, have been extensively studied because of their occurrence in the agriculturally important brassicacae and their impact on human and animal health. There is also increasing interest in the biofumigant properties of toxic glucosinolate hydrolysis products as a method to control agricultural pests. Evaluating biofumigation potential requires rapid and accurate quantification of , but current commonly used methods of extraction prior to analysis involve a number of time consuming and hazardous steps; this study aimed to develop an improved method for glucosinolate extraction.

Results

Three methods previously used to extract glucosinolates from brassicacae tissues, namely extraction in cold methanol, extraction in boiling methanol, and extraction in boiling water were compared across tissue type (root, stem leaf) and four brassicacae species (B. juncea, S. alba, R. sativus, and E. sativa). Cold methanol extraction was shown to perform as well or better than all other tested methods for extraction of glucosinolates with the exception of glucoraphasatin in R. sativus shoots. It was also demonstrated that lyophilisation methods, routinely used during extraction to allow tissue disruption, can reduce final glucosinolate concentrations and that extracting from frozen wet tissue samples in cold 80% methanol is more effective.

Conclusions

We present a simplified method for extracting glucosinolates from plant tissues which does not require the use of a freeze drier or boiling methanol, and is therefore less hazardous, and more time and cost effective. The presented method has been shown to have comparable or improved glucosinolate extraction efficiency relative to the commonly used ISO method for major glucosinolates in the species studied: and gluconasturtiin in B. juncea; sinalbin, , and gluconasturtiin in S. alba; glucoraphenin and glucoraphasatin in R. sativus; and glucosatavin, glucoerucin and glucoraphanin in E. sativa. Corresponding Author: Timothy William Doheny-Adams, Ph.D University of York UNITED KINGDOM Corresponding Author Secondary Information: Corresponding Author's Institution: University of York Corresponding Author's Secondary Institution: First Author: Timothy William Doheny-Adams, Ph.D

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation First Author Secondary Information: Order of Authors: Timothy William Doheny-Adams, Ph.D Kelly Redeker Varanya Kittipol Ian Bancroft Sue E Hartley Order of Authors Secondary Information: Response to Reviewers: We would like to thank the reviewers for their time and effort and their suggestions for improving the manuscript. We have addressed these suggestions to the best of our ability. Corrections have been highlighted in yellow in the document and the line numbers of those corrections are included below.

REVIEWER #1: 1.The authors should provide the some chromatograms of their real smaples analysis as a supporting materials:

Response: A supporting document with example chromatograms has been uploaded.

2.The size of the column they used should be provided. For example, what is the size of "C18 column" (line 233). Response: the size of the column has been added (line 237)

REVIEWER #2

LN 103-104: A description of the desulfation process/mechanism and or reference is needed. Why is this a necessary step?

Response: Now reads “Samples are subsequently desulfated by ion exchange chromatography on a DEAE sephadex column to remove impurities.” Whether it is a necessary step or not and the steps involved are explored later in the manuscript with a direct comparison between desulfated and non-desulfated extractions.

LN112: self-dimerisation via formation of a disulfide linkage during the extraction procedure. Response: Changed to: “In addition, prior to 2002 the major glucosinolate in leaves of E. sativa, 4-mercaptobutyl glucosinolate, was missed due to self-dimerisation via formation of disulfide linkages during extraction [22]”

LN113-115: How many glucosinolates possess free thiol groups and the ability to self- react? The main difficulties lie in inhibiting activity and degradation via heat or oxidation. Response:We appreciate the reviewers comment and agree that the vast majority of glucosinolates are not capable of self-dimerization. However, the previous sentence clearly demonstrates that at least one, important, glucosinolate has this potential. Therefore, we feel that this sentence remains valid as stated.

LN156: Table m2 could be converted to text to reduce space. Response: We are willing to perform this action if the journal is concerned about space, otherwise we are content to let the table remain.

LN160: What were average conditions for grinding prior to milling? Response: Lines 161-165 now read: “(i) Freeze dried plant tissue was homogenised to a roughly ground powder (approximately 0.1cm particle size) using a grinder (Lloytron, E5601BK) Homogenised ground samples were milled at a frequency of 20 Hz for 10 minutes (Retch, MM400) with 2 steel ball bearings to a fine powder (particle diameter <0.1mm). Samples were then sealed and stored at 20°C for up to 9 months.”

LN161: Frequency in Hz Response: corrected 2 instances (lines 161, 168) LN188: rev/min - rpm

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Response: corrected (line 191 and 375) LN190: micron symbol µ Response: corrected (line 193) LN192-197: replace incubation with heated (one presumes this was not conducted in an incubation chamber. Response: “incubation” has been changed to “heated” (lines 197, 198)

LN195: magnetic stirrer hotplate corrected (line 198)

LN200: krmp? corrected to “8000 rpm” (line 203)

LN215: small letter p to represent para-nitrocatechol corrected (line 218)

LN215: check consistency re: sulfate vs sulphate Response: changed all instances of “sulphate” to “sulfate” (line 218)

LN218: Is DAE meant to be DEAE? Response: Yes, corrected (line 221)

LN223: For reduction of disulfide linkages, from dimerized… please spell out TCEP. Response: changed and added. Now reads: “…. For the reduction of disulphide linkages, from dimerized desulfoglucosatavin in E. sativa extracts 3g TCEP (Tris(2- carboxyethyl)phosphine hydrochloride powder Sigma, C4706) was…” (line 226 and 227)

LN228: High performance liquid chromatography Response: amended (line 228)

LN 492: 'krpm' vs rpm? Response: Changed “8 krpm” to “8000 rpm” for lines 494 and 496.

LN246: Provide a bold title for Mass Spectrometry Response: added (line 250)

LN257: First mention of TBA and ACN please spell out in first instance Response: amended (line 262 and 263)

LN273: Capital T used for time, check consistency earlier t is used Response: all “T=” have been changed to “t=” (lines 278, 636)

Acknowledgements have been amended to recognise the time and effort contributed by the reviewers (line 520).

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Manuscript Click here to download Manuscript Development of an efficient glucosinolate extraction method.docx Click here to view linked References

1 2 1 3 4 5 2 Development of an efficient glucosinolate extraction method 6 7 8 3 Doheny-Adams T., Redeker K., Kittipol V., Bancroft I., Hartley S. E. 9 10 4 11 12 13 5 14 15 16 17 18 6 Author information 19 20 7 All Authors are employed at the University of York, in the United Kingdom 21 22 23 8 Postal address: 24 25 9 Department of Biology 26 27 10 University of York 28 29 30 11 Wentworth Way, York 31 32 12 YO10 5DD 33 34 35 13 United Kingdom 36 37 38 14 39 40 41 15 Timothy Doheny-Adams: [email protected] 42 43 44 16 Kelly Redeker: [email protected] 45 46 47 48 17 Varanya Kittipol: [email protected] 49 50 51 18 Ian Bancroft: [email protected] 52 53 54 19 Sue Hartley: [email protected] 55 56 57 20 58 59 60 61 62 63 1 64 65 21 1 2 3 22 4 Abstract 5 6 7 23 Background 8 9 24 Glucosinolates, anionic sulfur rich secondary metabolites, have been extensively studied because of 10 11 12 25 their occurrence in the agriculturally important brassicacae and their impact on human and animal 13 14 26 health. There is also increasing interest in the biofumigant properties of toxic glucosinolate 15 16 27 hydrolysis products as a method to control agricultural pests. Evaluating biofumigation potential 17 18 19 28 requires rapid and accurate quantification of glucosinolates, but current commonly used methods of 20 21 29 extraction prior to analysis involve a number of time consuming and hazardous steps; this study 22 23 30 aimed to develop an improved method for glucosinolate extraction. 24 25 26 27 31 Results 28 29 32 Three methods previously used to extract glucosinolates from brassicacae tissues, namely extraction 30 31 33 in cold methanol, extraction in boiling methanol, and extraction in boiling water were compared 32 33 34 34 across tissue type (root, stem leaf) and four brassicacae species (B. juncea, S. alba, R. sativus, and E. 35 36 35 sativa). Cold methanol extraction was shown to perform as well or better than all other tested 37 38 36 methods for extraction of glucosinolates with the exception of glucoraphasatin in R. sativus shoots. 39 40 41 37 It was also demonstrated that lyophilisation methods, routinely used during extraction to allow 42 43 38 tissue disruption, can reduce final glucosinolate concentrations and that extracting from frozen wet 44 45 46 39 tissue samples in cold 80% methanol is more effective. 47 48 49 40 Conclusions 50 51 41 We present a simplified method for extracting glucosinolates from plant tissues which does not 52 53 42 require the use of a freeze drier or boiling methanol, and is therefore less hazardous, and more time 54 55 56 43 and cost effective. The presented method has been shown to have comparable or improved 57 58 44 glucosinolate extraction efficiency relative to the commonly used ISO method for major 59 60 61 62 63 2 64 65 45 glucosinolates in the Brassicaceae species studied: sinigrin and gluconasturtiin in B. juncea; sinalbin, 1 2 46 glucotropaeolin, and gluconasturtiin in S. alba; glucoraphenin and glucoraphasatin in R. sativus; and 3 4 5 47 glucosatavin, glucoerucin and glucoraphanin in E. sativa. 6 7 8 48 9 10 11 49 12 Background 13 14 50 15 16 17 51 Glucosinolates, B-thioglucoside N-hydroxysulfate derivatives, are secondary metabolites found in 18 19 20 52 brassicaceae and related families [1]. Over 120 glucosinolates, which differ in variable aglycone side 21 22 53 chains derived from an alpha-amino acid, have been identified and classified into aliphatic, aromatic 23 24 54 and indole glucosinolates [2, 3]. Due to their prevalence in cultivated vegetables, spices, oils and 25 26 27 55 animal feed, glucosinolates and their hydrolysis products have been much studied in the context of 28 29 56 their effects on human and animal nutrition [4, 5]. Glucosinolates and their breakdown products 30 31 57 have also been a focus of studies in dietary prevention of disorders linked to oxidative stress such as 32 33 34 58 cancer and gastric ulcers [2, 6, 7] and more recently, potential undesirable dietary effects such as 35 36 59 genotoxicity of glucosinolate breakdown products in broccoli [8] and Pak Choi [9]. The breakdown of 37 38 39 60 glucosinolates has also been studied because of their potential use as agricultural pesticides in a 40 41 61 technique known as biofumigation. In biofumigation a glucosinolate-rich crop is mulched into the 42 43 62 field, releasing toxic secondary glucosinolate by-products, in order to reduce the incidence of pests, 44 45 46 63 weeds and diseases in the following arable and horticultural crops [10, 11, 12, 13]. 47 48 49 64 Evaluating biofumigation potential requires rapid and accurate quantification of glucosinolates, but 50 51 65 current commonly used methods of extraction prior to analysis involve a number of time consuming 52 53 54 66 and potentially hazardous steps. These steps are (i) lyophilisation, or freeze drying, and tissue 55 56 67 disruption, (ii) extraction in water or methanol, (iii) purification of extract, typically by desulfation on 57 58 68 DEAE sephadex, and (iv) separation and analysis of (desulfo)glucosinolates. These steps are outlined 59 60 61 62 63 3 64 65 69 in figure 1 and discussed in more depth below. This study aimed to improve glucosinolate extraction 1 2 70 methods by finding alternatives to commonly used steps which are unnecessary or likely to 3 4 5 71 introduce variability. 6 7 72 8 9 10 73 INSERT FIGURE 1 HERE 11 12 74 13 14 15 75 Myrosinase, an enzyme found in brassicacae and compartmentalised in cells in close proximity to 16 17 76 glucosinolates, is responsible for hydrolysing glucosinolates upon plant tissue disruption. Accurate 18 19 20 77 analysis of glucosinolates therefore requires inactivation of myrosinase prior to tissue disruption. 21 22 78 This is achieved by first freezing then freeze drying the tissue which allows disruption by milling or 23 24 79 grinding to occur in the absence of water (fig 1). Lyophilisation, or freeze drying, is used to remove 25 26 27 80 water from glucosinolate-containing tissues while preventing myrosinase mediated glucosinolate 28 29 81 hydrolysis through thermal inhibition. Publications on freeze drying plant tissue have focussed 30 31 32 82 primarily on the production of heat or its implications in generating oxygen sensitive foodstuffs (e.g.- 33 34 83 space, military or extreme-sport foodstuffs and instant coffee) [14]. To our knowledge, no study has 35 36 84 yet examined the efficiency of freeze drying in maintaining glucosinolate concentrations. Freeze 37 38 39 85 drying functions on the principle of sublimation: pressure is reduced below the triple point of water 40 41 86 (6.12 mbar, 0.01°C) at which point sublimation of ice from the sample occurs. The cooling effect of 42 43 87 sublimation should be high enough to ensure the sample remains below 0°C for the initial stage of 44 45 46 88 freeze drying, thus minimizing enzyme-driven glucosinolate hydrolysis. Rapid sample loading and 47 48 89 rapid initial pressure drop are also required to avoid sample defrosting before pressure is reduced 49 50 90 below 6.12 mbar. Leaves have a high surface area to volume ratio and may defrost quickly, 51 52 53 91 activating myrosinase and reducing final glucosinolate concentration. Despite the importance of the 54 55 92 freeze drying process in glucosinolate extraction, many authors do not report details which are likely 56 57 58 93 to affect final concentrations of glucosinolates (e.g.- how samples are transported, temperature of 59 60 94 the room, whether a heating/cold plate is used and time taken for the pressure to drop). 61 62 63 4 64 65 95 The most commonly used methods for extraction of glucosinolates from plant material are based on 1 2 96 the ISO 9167-1 method [15; highlighted in grey in figure 1], which was designed for extraction of 3 4 5 97 glucosinolates from B. napus seed and has been adapted to suit the needs of researchers examining 6 7 98 glucosinolate profiles of other plant species and tissue types. Although freeze drying is not explicitly 8 9 10 99 detailed in the ISO 9167-1 method, it is an implicit requirement in order to avoid myrosinase 11 12 100 mediated glucosinolate hydrolysis during disruption of leaf, stem or root tissues. Once the plant 13 14 101 tissue is prepared, the ISO 9167-1 extraction is carried out at 75°C in 70% methanol for 10 minutes. 15 16 17 102 Heating the sample is thought to be an essential step to denature myrosinase, thus preventing 18 19 103 enzymatic hydrolysis of glucosinolates [16]. Samples are subsequently desulfated by ion exchange 20 21 104 chromatography on a DEAE sephadex column to remove impurities. Desulfoglucosinolates are then 22 23 24 105 separated and identified using HPLC with a reverse phase C18 column and a UV or MS detector. 25 26 106 Hazards associated with boiling methanol [17] and the time required for extractions using this 27 28 107 method have led researchers to seek alternatives. Replacing heated methanol with boiling water is 29 30 31 108 reported to have comparable [18, 19], and in some cases better [20], extraction efficiencies. 32 33 109 Although most glucosinolates are thermostable for the typical 10-30 minute heating period, indole 34 35 36 110 glucosinolates such as 4-hydroxy- and 4-methoxyglucobrassicin have been reported to 37 38 111 degrade quickly at temperatures below 100°C [21]. In addition, prior to 2002 the major glucosinolate 39 40 112 in leaves of E. sativa, 4-mercaptobutyl glucosinolate, was missed because it self-dimerises via 41 42 43 113 formation of disulphide linkages during extraction [22]. A major challenge therefore to ensuring 44 45 114 consistent and repeatable GSL analysis is to create extraction conditions in which myrosinase is 46 47 115 inactive, and glucosinolates do not self-react or degrade. A single study, conducted exclusively on 48 49 50 116 radish roots, has demonstrated that cold extraction in 80% methanol does not cause appreciable 51 52 117 reduction in glucosinolate concentrations compared to more conventional heated extraction 53 54 55 118 methods [23]. However, myrosinase activity can vary dramatically [24] and whether this method is 56 57 119 suitable for extraction of glucosinolates from other glucosinolate containing plants has not 58 59 120 previously been assessed. 60 61 62 63 5 64 65 121 A desulfation step is often carried out post extraction to purify desulfoglucosinolates and improve 1 2 122 accuracy and identification from HPLC. However, the desulfation reaction of glucosinolates can be 3 4 5 123 affected by feedback inhibition of the enzyme which causes incomplete desulfation of glucosinolates 6 7 124 [25]. In addition, rhamnopyranosyloxy-benzyl glucosinolates extracted from M. oleifera have been 8 9 10 125 shown to be completely converted and degraded by the desulfation purification step [26]. Due to 11 12 126 these drawbacks, and the additional time and potential error extra steps can introduce, some 13 14 127 authors have skipped the purification and desulfation steps entirely [19, 26, 27] (fig 1). 15 16 17 18 128 We have tested each stage of glucosinolate analysis from the roots, stems and leaves of B. juncea, S. 19 20 129 alba, R. sativus, E. sativa and B. napus and suggest a number of adjustments/improvements which 21 22 130 can be made to reduce the costs, time and variability associated with glucosinolate analysis. 23 24 25 131 Specifically, this study aims to address the following questions: 26 27 28 132 1) How do lyophilisation conditions affect glucosinolate concentrations? 29 30 133 2) Is lyophilisation a necessary step for glucosinolate extraction from green tissues? 31 32 33 134 3) Do extractions in hot methanol, cold methanol and boiling water yield comparable 34 35 135 glucosinolate concentrations across a range of brassicacae species and tissue types? 36 37 136 4) How do desulfation time and enzyme concentration affect final glucosinolate 38 39 40 137 concentrations? 41 42 138 5) Is desulfation a necessary step for glucosinolate extraction from green tissue? 43 44 45 139 46 47 48 49 140 50 51 52 141 53 54 55 142 Materials and methods 56 57 58 143 59 60 61 62 63 6 64 65 Experiment Fig Species Tissue Freeze Extraction Desulfation HPLC 1 2 drying/tis 3 4 5 sue 6 7 disruption 8 9 10 1-Effect of freeze drier 2 B. napus Leaves FD-A or Cold 0.3 U/ml ISO 9167-1 11 12 on GSL concentration FD-B methanol for 24 H method 13 14 /mill 15 16 17 2-Comparison of GSL 3 B. napus Leaves FD-A or Cold 0.3 U/ml ISO 9167-1 18 19 extraction from freeze -20°C methanol for 24 H method 20 21 22 dried tissue with methanol 23 24 extraction from wet 25 26 tissue 27 28 29 3-Comparison of 6, 7 R. sativus Leaves, FD-A Hot 0.3 U/ml ISO 9167-1 30 31 extraction methods B. juncea Stems, methanol, for 24 H method 32 33 S. alba Roots Cold 34 35 36 E. sativa methanol, 37 38 Boiling 39 40 41 water 42 43 4-Comparison of 8,9 R. sativus Leaves, FD-A Cold 0.3 U/ml ISO 9167-1 44 45 desulfation/purification B. juncea Stems, methanol for 12, 24, Method for 46 47 48 methods S. alba Roots 48 H, and desulfoGSL, 49 50 E. sativa 5U/ml for Herzallah 51 52 16 H or and Holly 53 54 55 filtration method for 56 57 intact GSLs. 58 59 60 144 Table m1: summary of methods used 61 62 63 7 64 65 145 Plant material 1 2 146 B. napus used in the freeze drying tests were grown in 1 L pots filled with Terra-green in a controlled 3 4 5 147 temperature glasshouse (regulated from 17.6°C to 27.7°C). At 3-4 weeks post germination, leaves 6 7 148 were removed and halved down the limits of the midrib, excluding the midrib from the final sample. 8 9 149 Leaf halves were immediately frozen in liquid nitrogen and stored at -80°C for a maximum of 1 week. 10 11 12 13 150 B. juncea (cv. ISCI99), R. sativus (cv. Bento), S. alba (cv. Ida Gold) and E. sativa (cv. Nemat) plants 14 15 151 were grown by Barworth agriculture ltd. in a sandy loam soil dominated fields (coordinates: 16 17 152 53.000371, -0.290404) from 31/07/2014 to 25/09/2014. Total stem and total leaves were cut from 18 19 20 153 flowering plants and immediately frozen in liquid nitrogen; root samples were gently washed and 21 22 154 dried before freezing in liquid nitrogen. Samples were stored at -80°C for a maximum of 2 months. 23 24 25 155 Freeze drying 26 27 28 156 Samples wrapped loosely in aluminium foil were transported on dry ice and loaded into one of two 29 30 157 freeze driers (table m2). Maximum loading time was 30 seconds. 31 32 33 Freeze drier Room temp Cooling Time to Lowest Freezer Model 34 35 36 (°C) plate 5 mbar pressure temperature 37 38 (s) (mbar) (°C) 39 40 41 A 22 Yes 90 0.12 -45 Lyotrap, LTE 42 43 scientific ltd. 44 45 1 chamber. 46 47 48 B 28 No 65 0.16 -53 Thermo, Heto 49 50 Powerdry LL3000. 51 52 4-6 chambers. 53 54 55 158 Table m2: Freeze drier characteristics 56 57 58 159 59 60 61 62 63 8 64 65 160 Tissue disruption 1 2 161 (i) Freeze dried plant tissue was homogenised to a roughly ground powder (approximately 3 4 5 162 0.1cm particle size) using a grinder (Lloytron, E5601BK) Homogenised ground samples were 6 7 8 163 milled at a frequency of 20 Hz for 10 minutes (Retch, MM400) with 2 steel ball bearings to a 9 10 164 fine powder (particle diameter <0.1mm). Samples were then sealed and stored at 20°C for 11 12 13 165 up to 9 months. 14 15 16 166 (ii) Frozen fresh B. napus leaf halves (experiment 2, table m1) were placed in 2ml eppendorf vials 17 18 19 167 and stored at -20°C. 1.755ml of 80% methanol precooled at -20°C, 25µl of 5mM sinigrin and 2 small 20 21 168 ball bearings were added. Samples were milled for 10 minutes at frequency 20 Hz (TissueLyser II, 22 23 169 Qiagen). Final concentrations of methanol were estimated by incorporating average leaf moisture 24 25 26 170 content of fresh B. napus leaves according to equation (1). Final concentration of methanol ranged 27 28 171 from 79.3% to 79.9% and leaf moisture content accounted for less than 1% of final liquid volume. 29 30 31 32 172 33 푀푒푂퐻푖 푀푒푂퐻푖 푀푒푂퐻푓 푐 × 푉 34 (1) C = 푎푣 푑푙 푀푒푂퐻푖 35 푚 × 푚 + 푉 36 173 Where cMeOHf is final methanol concentration (%) 37 38 174 cMeOHi is initial methanol concentration (90%) 39 40 175 V is initial methanol volume (1.755 ml) 41 MeOHi 42 43 176 mav is the average moisture content per dry weight (in this case 0.22 ml/g) 44 45 177 mdl dry mass of leaf sample (g) 46 47 48 178 Glucosinolate extraction 49 50 51 179 Extractions were carried out in one of three ways (fig 1m). In each case 50µl of a 5mM 52 53 180 gluctropaeolin (for B. juncea samples) or 20 mM sinigrin (for all other samples) internal standard was 54 55 181 added: 56 57 58 59 60 61 62 63 9 64 65 182 Hot methanol extraction (based on the ISO 9167-1 method): 1 2 183 0.1g of plant material was preheated at 75°C for 3 minutes in a 20ml falcon tube. 4.95ml of 70:30 3 4 5 184 methanol:water, preheated to 75°C and the internal standard was added. The sample was 6 7 185 incubated at 75°C for 10 minutes, and manually shaken every 2 minutes. The sample was then 8 9 186 centrifuged at 4000 rpm (Jouan, model:B 3.11) for 10 minutes. Supernatent was stored at -20°C or 10 11 12 187 desulfated directly. 13 14 15 188 Cold methanol extraction (Ishida et al. 2011, [23]): 16 17 189 5 ml of 80:20 methanol:water at 20°C was added to 0.1g plant tissue and the internal standard was 18 19 20 190 added. The sample was shaken and left to stand for 30 minutes at room temperature. The sample 21 22 191 was then mixed at 70 rpm with a platform rocker for a further 30 minutes (Bibby, STR6) before 23 24 192 centrifugation at 4000 rpm (Jouan, model:B 3.11) for 10 minutes. Supernatent was then filtered 25 26 27 193 through a 0.22 µm syringe filter (Millex GP) for direct injection on HPLC, or unfiltered if applied to 28 29 194 sephadex column in a purification step. 30 31 32 195 Boiling water extraction (adapted from Herzallah and Holley, 2012, [19]): 33 34 35 196 25 ml of boiling water was added to 0.1g of freeze dried and milled plant tissue in a 150ml 36 37 197 erlenmeyer flask and the internal standard was added. Sample was heated at 100°C and stirred with 38 39 198 a magnetic stirrer hot plate for 10 minutes. Sample was heated for a further 4 H at 70°C before 40 41 42 199 centrifugation at 4000 rpm (Jouan, model:B 3.11) for 10 minutes. Sample was topped up to 20ml 43 44 200 with deionised water. 45 46 47 201 48 Purification and determination of activity of sulfatase: 49 50 202 Sulfatase from Helix pomatia type H-1 (Sigma, S9626) was purified according to Wathalet et al. 51 52 203 (1999) [25]. 25 mg of sulfatase was added to 1ml 40% ethanol and centrifuged at 8000 rmp for 1 53 54 204 minute (eppendorf centrifuge, 54151). The supernatant was transferred to a fresh 2ml eppendorf 55 56 57 205 tube, 1ml of pure ethanol was added to precipitate the sulfatase before being centrifuged at 8krmp 58 59 60 61 62 63 10 64 65 206 for 1 minute. The supernatant was discarded and the sulfatase pellet air dried and redissolved in 2ml 1 2 207 of water. 3 4 5 6 208 Activity of sulfatase was determined based on the ISO 9167-1 method. 1 ml of buffered 0.15mM 7 8 209 sinigrin solution (3ml of 5mM sinigrin, adjusted to 100ml with a solution containing 40ml 0.2% 9 10 210 ethylene diamine, 73ml 0.2% acetic acid; adjusted to pH 5.8 ) in a quartz cuvette was placed in a UV 11 12 13 211 spectrometer set to 229nm. At t=0, 25µl of diluted and undiluted purified sulfatase was added to the 14 15 212 cuvette and measurements taken over the course of 4 hours. The tangent to t=0 was plotted and its 16 17 18 213 gradient (ΔA/Δt) measured. Activity was calculated using equation (2): 19 20 21 214 22 23 훥퐴 × 5.7 (2) Activity (U/ml) = 24 훥푡퐴푒 25 215 Where ΔA/Δt is the gradient at t=0 and Ae is the difference between absorbance at equilibrium and 26 27 28 216 absorbance at t=0. 29 30 31 217 The activity for Sulfatase from Helix pomatia type H-1 (Sigma, S9626) given by the supplier is 32 33 218 determined by desulfation of p-nitrocatechol sulfate and is an order of magnitude higher than the 34 35 36 219 activity measured for desulfation of sinigrin using this method. 37 38 39 220 Desulfation of glucosinolates 40 41 221 As per the ISO 9067-1 method, columns were prepared with 0.5ml sephadex slurry (2g DEAE 42 43 222 sephadex beads in 30 ml 2M acetic acid.) and activated with 2ml imizadole formate (6M). Columns 44 45 46 223 were washed twice with 1ml water. The column was washed twice with 1ml 20mM sodium acetate 47 48 224 (pH 4.0) and 75µl of purified sulfatase was added (5U/ml or 0.3U/ml). Columns were incubated at 49 50 51 225 room temperature for either 12, 24 or 48 hours before elution of desulfoglucosinolates with two 1ml 52 53 226 volumes of water. For the reduction of disulphide linkages, from dimerized desulfoglucosatavin in E. 54 55 227 sativa extracts 3g TCEP (Tris(2-carboxyethyl)phosphine hydrochloride powder Sigma, C4706) was 56 57 58 228 added to 1 ml of desulfated extract. Desulfoglucosinolates were stored at -20°C before high 59 60 229 performance liquid chromatography analysis. 61 62 63 11 64 65 230 For the high sulfatase treatment, between 0.5 and 1 ml of sample was added due to insufficient 1 2 231 sample volume remaining. 3 4 5 6 232 HPLC 7 8 233 A Waters 600E system controller attached to a Waters 717 autosampler, Waters 996 photodiode 9 10 234 array detector and SphereClone 5µ ODS(2) column (Phenomonex) were used for separation and 11 12 13 235 detection of desulfo and intact glucosinolates. 14 15 16 236 HPLC analysis of desulfoglucosinolates – adapted from ISO 9167-1 17 18 237 A reverse phase C18 column (Phenomonex, SphereClone 5µ ODS(2), 150mm x 4.6mm) was 19 20 21 238 equilibrated for 30 min with a mobile phase which consisted of 100% diH2O. Flow rate was set to 22 23 239 1ml/min and samples separated according to programme for desulfoglucosinolates detailed in table 24 25 240 m3. Mobile phase solutions were degassed for 30 minutes in a sonicator (Decon, Sussex England). 26 27 28 29 241 Solution A: 100% diH2O 30 31 242 Solution B: 70:30, diH2O:acetonitrile 32 33 34 Time % solution A % solution B Transition 35 36 37 0 100 0 38 39 30 0 100 Linear gradient 40 41 35 0 100 42 43 44 40 100 0 Linear gradient 45 46 50 100 0 47 48 49 243 Table m3: mobile phase conditions for separation of desulfoglucosinolates. 50 51 52 244 Desulfoglucosinolates were quantified using 229nm wavelength within the UV spectrum. The HPLC 53 54 245 PDA detector allowed a full spectrum analysis from 180 to 800nm, allowing comparative UV-Visible 55 56 57 246 spectra analysis, which aided in identifying unknown glucosinolates. Through standard injections and 58 59 247 HPLC-MS identification we were able to confirm the id’s of these reported glucosinolates. Desulfated 60 61 62 63 12 64 65 248 purified standards: sinigrin (sigma aldrich), glucotropaeolin, glucoraphenin, glucoraphanin, 1 2 249 glucerucin, glucobrassicin, gluconasturtiin, sinalbin, and glucoiberin (phytoplan). 3 4 5 6 250 Mass spectrometry 7 8 251 Major glucosinolates for which no commercial standard is available were identified using an MS 9 10 252 detector (Bruker maXis UHR-TOF) with the following settings: 11 12 13 253 Source: Standard electrospray (flow split 1/10 from LC) 14 15 254 Nebulizer: 2.0 bar 16 17 255 Dry gas: 6.0 L/min 18 19 20 256 Dry gas heater: 250C 21 22 257 Capillary voltage: 3500 V 23 24 25 258 Ion polarity: positive 26 27 259 Spectra rate: 1Hz 28 29 30 260 HPLC analysis of intact glucosinolates – adapted from Herzallah and Holly, 2012 [19] 31 32 261 A C18 column (Phenomonex, SphereClone 5u ODS(2)) was equilibrated for 3 h with a mobile phase 33 34 35 262 which consisted of 80 mL (0.02 M) TBA (tetrabutylammonium bromide) and 20 mL ACN 36 37 263 (acetonitrile) with detection at 229 nm. The flow rate was set at 1.0 ml/min and separated according 38 39 40 264 to programme for desulfoglucosinolates detailed in table m3. 41 42 43 265 Solution A: 100% TBA (0.02M) 44 45 266 Solution B: 70:30, TBA (0.02M):acetonitrile 46 47 48 267 Glucosinolates were quantified using the chromatogram from 229nm and standard curves were 49 50 51 268 constructed using pure sinigrin (sigma aldrich), glucotropaeolin, glucoraphenin, glucoraphanin, 52 53 269 glucerucin, glucobrassicin, gluconasturtiin, sinalbin, progoitrin and glucoiberin (phytoplan). 54 55 56 270 In the case of glucoraphasatin in R. sativus leaves and glucotropaeolin in B. juncea minor alterations 57 58 59 271 were made to avoid peaks co-eluting. The mobile phase programme for R. sativus leaves was 100% A 60 61 62 63 13 64 65 272 for 5 minutes, followed by a 35 minute linear gradient to 66% B followed by a 5 minute linear 1 2 273 gradient to 100% B followed by a 5 minute linear gradient to 100% A . For B. juncea leaves, an 3 4 5 274 isocratic 85:15, TBA (0.02M):acetonitrile mobile phase for 70 minutes was used. 6 7 8 275 Determination of myrosinase activity 9 10 276 Activity of pure myrosinase was tested in water and 80% methanol solutions containing 0.25mM 11 12 13 277 sinigrin and 0.1 mM ascorbic acid, a myrosinase cofactor (Burmeister et al. 2000). Myrosinase was 14 15 278 added at t=0 and absorbance of sinigrin at 229nm was measured over the course of an hour. Activity 16 17 279 was measured at room temperature (25°C). 18 19 20 21 280 Determination of glucosinolate thermostability 22 23 281 A 50µl of 10mM sinigrin, 10mM glucotropaeolin, 10mM glucobrassicin solution was added to 0.95ml 24 25 282 water or 70% methanol preheated to 100°C or 75°C respectively and sealed in 1.5ml eppendorf 26 27 28 283 tubes. Samples were maintained at either 100°C or 75°C for 5, 10, 30 and 60 minutes and intact 29 30 284 glucosinolate concentrations analysed with HPLC following the adapted Herzallah and Holly (2012) 31 32 285 method [19]. 33 34 35 36 286 Calculation of glucosinolate content 37 38 287 Glucosinolate content, expressed in µmol/g were calculated according to the ISO 9067-1 method 39 40 288 (equation (3)): 41 42 43 44 289 (3) 45 퐴푔 푛 100 46 퐺푙푢푐표푠푖푛표푙푎푡푒 푐표푛푡푒푛푡 = 퐴푠 × 푚 × 퐾푔 × 100−푤 47 48 290 Where Ag is the peak area corresponding to desulfoglucosinolate; 49 50 291 As is the peak area corresponding to internal standard; 51 52 292 n is the quantity, in micromoles, of the internal standard; 53 54 55 293 m is the mass of the test portion; 56 57 294 Kg is the response factor of the desulfoglucosinolate relative to the internal standard; 58 59 295 w is the moisture and volatile matter content, expressed as a percentage by mass of the test sample. 60 61 62 63 14 64 65 296 Statistical analysis 1 2 297 Paired two tailed t-test analysis were carried out on total B. napus glucosinolate content per leaf half 3 4 5 298 in experiments 1 and 2 with Microsoft excel (table m1). For determination of significance of effect of 6 7 299 method on final glucosinolate content estimates in experiments 3 and 4 (table m1), repeat measure 8 9 300 ANOVA analyses were carried out for each glucosinolate with R statistical software package (version 10 11 12 301 3.3.1). 13 14 15 302 16 17 18 303 19 Results and discussion 20 21 22 304 1 Lyophilisation 23 24 305 Modifications to the ISO9167-1 method (specifically created for the extraction and analysis of 25 26 27 306 glucosinolates from oil rape seed samples) are required for analysis of plant green tissues (leaves, 28 29 307 stems and roots). A number of prior-to-analysis steps, such as sampling in the field, cleaning (if 30 31 308 required), freezing, crushing, storage or/and shipping and reduction of sample amount have been 32 33 34 309 discussed by Wathelet et al. (2004) and are not revisited here [28]. These preliminary steps are 35 36 310 followed by lyophilisation, or freeze drying, to remove water from glucosinolate containing tissues 37 38 311 while preventing myrosinase mediated glucosinolate hydrolysis through thermal inhibition. This 39 40 41 312 process allows subsequent tissue disruption without risking glucosinolate degradation. 42 43 44 313 We tested reproducibility of glucosinolate concentrations extracted after lyophilisation in separate 45 46 314 freeze driers (table 1). Fresh B. napus leaves were halved, loosely wrapped in foil, flash frozen in 47 48 49 315 liquid nitrogen and transported in dry ice to be dried in separate freeze driers (table 1). Total 50 51 316 glucosinolate concentrations were significantly higher in samples dried in freeze drier A than freeze 52 53 317 drier B (fig 2a). In addition, samples dried in freeze drier B developed a darker hue and deformed 54 55 56 318 more than samples in dried in freeze drier A (fig 2b). Plant tissue samples have been shown to 57 58 319 deform during the freeze drying process when temperatures exceed the glass transition state and 59 60 61 62 63 15 64 65 320 melting point of water [29]. It is likely that samples placed into freeze drier B may have defrosted 1 2 321 before the pressure had reduced below the 6.12 mbar required for sublimation due to higher 3 4 5 322 temperatures and the lack of cooling plate. As a result, enzyme mediated hydrolysis of 6 7 323 glucosinolates may have occurred at the initial stage. Additionally, as sublimation slows over time 8 9 10 324 due to the remaining water vapour passing through a dry layer of increasing thickness and because 11 12 325 water is increasingly more tissue bound, the sample temperature may have increased to above 0°C 13 14 326 in freeze drier B, causing defrosting. 15 16 17 327 18 19 20 Freeze drier Room temp Cooling plate Time to 5 Lowest Freezer 21 22 (°C) mbar (s) pressure temperature 23 24 25 (mbar) (°C) 26 27 A 22 Yes 90 0.12 -45 28 29 B 28 No 65 0.16 -53 30 31 32 328 Table 1: Freeze drier characteristics 33 34 35 329 36 37 38 330 INSERT FIGURE 2 HERE 39 40 41 331 42 43 44 332 These results underline the need for a more substantive study to assess optimal conditions for 45 46 333 freeze drying plant tissues for glucosinolate analysis. It is clear that differences in freeze drying can 47 48 49 334 introduce significant variability in retained glucosinolate concentrations (fig 2a). 50 51 335 A cold methanol extraction method may be sufficient to 1) inactivate myrosinase and 2) efficiently 52 53 54 336 extract glucosinolates, precluding the need for the lyophilisation step altogether. We tested this by 55 56 337 comparing glucosinolates extracted from one half of a B. napus leaf in 80% methanol without freeze 57 58 59 60 61 62 63 16 64 65 338 drying against glucosinolates extracted from the other half, first dried in freeze drier A and then 1 2 339 extracted using the cold methanol extraction method. 3 4 5 340 No significant difference in final glucosinolate concentration was found between the two methods 6 7 341 (fig. 3). Freeze drying is an energy intensive and costly process requiring long drying times under 8 9 10 342 continuous vacuum and the significant effect of freeze drier parameters on final glucosinolate 11 12 343 concentrations (fig. 2a) highlights a potential source of variation between studies. If long term 13 14 344 storage of plant tissue samples is not required, skipping the freeze drying step and extracting 15 16 17 345 glucosinolates directly into cold methanol (-20°C) is cheaper, quicker and less hazardous. 18 19 346 20 21 347 INSERT FIGURE 3 HERE 22 23 24 348 25 26 27 349 2 Extraction 28 29 350 Some authors have highlighted that glucosinolates, specifically indole glucosinolates, are heat 30 31 32 351 sensitive and are significantly degraded in temperatures ≥75°C in less than 10 minutes [21]. This has 33 34 352 serious implications for accuracy and reliability of the ISO 9167-1 extraction method, which 35 36 353 recommends extractions occur in boiling 70% methanol (75° C) for 10 minutes, as well as the less 37 38 39 354 commonly used boiling water extraction (100° C). In order to first test whether thermal degradation 40 41 355 of glucosinolates was likely to occur with these methods we measured the glucosinolate 42 43 44 356 concentrations of pure sinigrin (aliphatic), glucotropaeolin (aromatic) and glucobrassicin (indole) in 45 46 357 boiling water (fig 4) and boiling 70% methanol (data not shown). Sinigrin and glucotropaeolin did not 47 48 358 significantly decrease over 60 minutes suggesting that extraction in boiling water or methanol is 49 50 51 359 unlikely to affect the concentrations of these glucosinolates. However, glucobrassicin was thermally 52 53 360 degraded at 100°C and data from extractions carried out at these temperatures or above (such as 54 55 361 with microwave based methods) may underestimate the concentration of glucobrassicin and other 56 57 58 362 indole glucosinolates. Boiling an extract in water for 10 minutes degrades glucobrassicin by an 59 60 363 estimated 7%. 61 62 63 17 64 65 364 INSERT FIGURE 4 HERE 1 2 365 3 4 5 366 6 7 8 367 Activity of pure myrosinase was tested at 25° C in water and 80% methanol solutions containing 9 10 368 0.25mM sinigrin and 0.1 mM ascorbic acid, a myrosinase cofactor [30]. Absorbance of sinigrin at 11 12 13 369 229nm, at room temperature (25°C), was measured over the course of an hour after myrosinase 14 15 370 addition. Myrosinase was inactive in 80% methanol (fig 5) suggesting that heating methanol at 75°C 16 17 18 371 for 10 minutes in order to inactivate myrosinase may be an unnecessary step for extracting 19 20 372 glucosinolates from plant tissue. 21 22 23 373 INSERT FIGURE 5 HERE 24 25 26 374 Glucosinolates from B. juncea, S. alba, R. sativus and E. sativa leaves, stems and roots were 27 28 29 375 extracted (i) in boiling water for 10 minutes followed by a 4h incubation at 70°C, (ii) in 70% methanol 30 31 376 at 75°C, or (iii) in 80% methanol at room temperature (~20°C) for 30 minutes standing followed by 32 33 34 377 30 minutes shaking at 70 rpm. All extracts were centrifuged and desulfated with sulfatase according 35 36 378 to the ISO 9167-1 method. Major glucosinolates from these species can be found in table 2. 37 38 39 Common name Chemical name Structure Species, tissue type 40 41 42 43 Sinigrin 2-propenyl Aliphatic B. juncea L, S, R 44 45 46 Glucoraphenin 4-methylsulfinyl-3-butenyl Aliphatic R. sativus L, S, R 47 48 Glucoraphanin 4-methylsulfinylbutyl Aliphatic E. sativa L, S, R 49 50 Glucosatavin mercaptobutyl Aliphatic E. sativa L, S, R 51 52 Glucoraphasatin 4-Methylthio-3-butenyl Aliphatic R. sativus L, S, R 53 54 or hydroxyglucoerucin 55 56 57 Glucoerucin Methylthiobutyl Aliphatic E. sativa S, R 58 59 S. alba, R 60 61 62 63 18 64 65 Sinalbin 4-Hydroxybenzyl Aromatic S. alba L, S, R 1 2 Glucotropaeolin Benzyl Aromatic S. alba L, S, R 3 4 Gluconasturtiin phenylethyl Aromatic B. juncea R 5 6 7 S. alba R 8 9 Methoxyglucobrassicin 4-methoxy-3-indolylmethyl Indole S. alba R 10 11 379 Table 2: Glucosinolates examined in this study. L, S and R correspond to leaf, stem and root respectively. 12 13 380 Letters in bold represent major glucosinolates of those tissues (>10 µmol/g dry weight). 14 15 16 381 17 18 19 382 Figure 6 compares glucosinolate concentrations obtained using the cold methanol method and 20 21 22 383 boiling water method normalised against the ISO 9167-1 boiling methanol method. For most 23 24 384 glucosinolates, across most tissue types and species, the three extraction methods yield similar 25 26 27 385 results. We found that extraction with cold methanol produced a significantly higher estimated 28 29 386 concentration of sinalbin in S. alba and sinigrin in B. juncea than the hot methanol extraction (fig 6). 30 31 387 Surprisingly, given the sensitivity of glucobrassicin to thermal degradation (fig 4), extraction in 32 33 34 388 boiling water did not significantly reduce the concentration of the indole glucosinolate: 35 36 389 methoxyglucobrassicin relative to the other two methods. However, glucosatavin was extracted with 37 38 390 lower efficiency from leaves of E. sativa using the boiling water method (fig 6). It seems unlikely that 39 40 41 391 this glucosinolate is less thermostable than other glucosinolates and was therefore degraded by the 42 43 392 extraction method since reduced extraction efficiencies are not observed for stem and root samples. 44 45 46 393 There are no published explanations or hypotheses that might help to explain the observed lower 47 48 394 extraction efficiencies for glucosatavin using the boiling water method. Glucoraphasatin extraction 49 50 395 using cold methanol appears to be significantly less effective than the standard ISO method (fig 6), 51 52 53 396 however this was driven by poor extraction efficiencies from R. sativus stems (fig 7). Ishida et al. 54 55 397 reported a significant 5% increase in glucoraphasatin concentrations extracted from R. sativus roots 56 57 398 using the cold methanol method [23]. In this study, extraction efficiencies of glucoraphenin in R. 58 59 60 61 62 63 19 64 65 399 sativus roots with a cold methanol method were comparable to extraction efficiencies using the 1 2 400 boiling methanol method (fig 7). 3 4 5 6 401 INSERT FIGURE 6 HERE 7 8 9 402 INSERT FIGURE 7 HERE 10 11 12 403 No glucosinolates were detected in a subset of samples extracted in cold water indicating the 13 14 15 404 presence of active myrosinase leading to their degradation (data not shown). However, the cold 16 17 405 methanol extraction did not significantly affect the concentration of the internal standard relative to 18 19 406 the boiling methanol method (data not shown), providing additional evidence that myrosinase is 20 21 22 407 inactivated in 80% methanol without heating (fig 5). 23 24 25 408 These data demonstrate that 80% cold methanol can be used instead of boiling methanol to extract 26 27 409 glucosinolates across a broad spectrum of brassicacae species and tissue types. With the exception 28 29 30 410 of glucoraphasatin in R. sativus shoots, replacing hot 70% methanol with cold 80% methanol did not 31 32 411 significantly reduce glucosinolate concentrations, yet marginally increased recovery of sinalbin in S. 33 34 412 alba and sinigrin in B. juncea. It is advised, due to reduction in steps and hazard as well as improved 35 36 37 413 or comparable glucosinolate recovery, that a cold methanol extraction is used instead of a boiling 38 39 414 methanol extraction for most glucosinolate containing green tissues. 40 41 42 415 3 Purification 43 44 45 416 Purification of extract according to the ISO 9167-1 method is carried out by introducing 1ml of 46 47 417 extract to a column containing 0.5ml of sephadex solution. The column is rinsed with a 20mM 48 49 418 acetate buffer at pH 4.0 to avoid possible reduction of indole glucosinolates recovery [28]. 75µl of 50 51 52 419 sulfatase solution with an activity above 0.05 U/ml is applied and left to act overnight. We tested the 53 54 420 extraction efficiency of the ISO 9167-1 purification step at the described pH 4.0, at 20°C for 12, 24 55 56 57 421 and 48 hours. Complete desulfation of glucosinolates in rapeseed extract required a minimum of 11 58 59 422 hours in operating conditions of 30°C and pH 5.8 [25] so it was expected that an overnight 12 hour 60 61 62 63 20 64 65 423 desulfation period may be insufficient for complete desulfation of samples at room temperature. 1 2 424 Figure 8 shows absorbance values for representative desulfoglucosinolate solutions from B. juncea, 3 4 5 425 S. alba, R. sativus and E. sativa extracts treated with sulfatase solution for 12, 24 or 48 hours. In 6 7 426 most cases, 12 and 24 hour incubation periods were insufficient for complete desulfation of 8 9 10 427 glucosinolates. Glucoraphenin decreased in all R. sativus leaf samples tested, from 24 to 48 hours, 11 12 428 while recovery of the internal standard increased, suggesting that specifically this 13 14 429 desulfoglucosinolate is degraded during the purification process (fig 8). 15 16 17 18 430 INSERT FIGURE 8 HERE 19 20 21 431 Not all glucosinolates are desulfated on the column at the same rate [31], meaning that incomplete 22 23 432 desulfation of extractions is likely to yield imprecise results: overestimating or underestimating the 24 25 26 433 final concentration of glucosinolates which are desulfated quicker or slower respectively than the 27 28 434 internal standard. In addition, relative and total concentrations of glucosinolates and degradation or 29 30 435 rearrangement of glucosinolates during this process can also affect final concentrations [26, 31]. Use 31 32 33 436 of higher sulfatase concentrations than outlined in the ISO method has been suggested for 34 35 437 glucosinolate analysis in B. napus and B. oleracea [25, 31]. Figure 9 compares relative glucosinolate 36 37 438 concentrations from B. juncea, S. alba, R. sativus and E. sativa purified with a low activity sulfatase 38 39 40 439 solution (0.3U/ml) for 12H, 24H and 48H, a high activity sulfatase solution (5U/ml) and intact 41 42 440 glucosinolates. All concentrations have been normalised to the intact glucosinolate values. 43 44 441 Desulfated glucosinolates concentrations obtained with high concentration sulfatase compared well 45 46 47 442 with intact glucosinolates (fig 9). However, both high sulfatase as well as low sulfatase treatments 48 49 443 yielded lower glucoraphenin content estimates. Coupled with the reduction of the recovery of 50 51 52 444 desulfoglucoraphenin from 24H to 48H (fig 8), these data suggest that glucoraphenin is degraded or 53 54 445 transformed during the desulfation process. 55 56 57 446 INSERT FIGURE 9 HERE 58 59 60 61 62 63 21 64 65 447 Shorter desulfation times and lower sulfatase concentrations resulted in underestimation of the 1 2 448 concentrations of glucoraphenin from R. sativus, glucoraphanin and glucosatavin from E. sativa, 3 4 5 449 sinigrin from B. juncea, and sinalbin from S. alba and an overestimation of the concentrations of 6 7 450 glucoraphasatin in R. sativus roots (fig 9). The overnight (12H-24H) incubation with 0.3 U/ml 8 9 10 451 sulfatase solution yields inaccurate results for most major glucosinolates examined in this study. The 11 12 452 ISO9167-1 method suggests that a diluted purified sulfatase solution with an activity exceeding 13 14 453 0.05U/ml should be used, which is shown to be insufficient for glucosinolate analysis from plant 15 16 17 454 samples and conditions examined in this study (fig 9). Instead, if a desulfation step is carried out, use 18 19 455 of a higher concentration of purified sulfatase (in this case, 5U/ml) is advised. 20 21 22 456 In all E. sativa leaf samples tested, recovery of monomeric desulfo-glucosatavin decreased and 23 24 25 457 recovery of dimeric desulfo-glucosatavin increased between 24 and 48 hours. Bennet et al. (2002) 26 27 458 previously hypothesised that dimeric glucosatavin is unlikely to be found in vivo and is probably an 28 29 459 artefact of the extraction process [22]. We can confirm that glucosatavin forms dimers as a result of 30 31 32 460 the desulfation step of the extraction and that without carrying this step out and instead quantifying 33 34 461 intact glucosinolates, no dimeric glucosatavin was detected in these samples. 35 36 37 462 Given that glucoraphenin concentration estimates are lower from methods employing a desulfation 38 39 40 463 step, and that this step is also responsible for the dimerization of glucosatavin, analysis of intact 41 42 464 glucosinolates is preferable in most instances. It is out of the scope of this study to compare or 43 44 465 improve separation and detection methods but it should be noted that major glucosinolates in this 45 46 47 466 study were accurately measured by a HPLC-UV method adapted from Herzallah and Holley (2012) 48 49 467 [19]. For examination of low abundance glucosinolates, and to avoid any potential inaccuracies due 50 51 52 468 to contamination it is advised that an alternative HPLC method such as those suggested in Lee et al. 53 54 469 (2013) or Forster et al. (2015) be used instead [26, 32]. 55 56 57 470 58 59 60 471 Suggested method for glucosinolate extraction: 61 62 63 22 64 65 472 Tissue disruption 1 2 473 Depending on whether freeze drying is required: 3 4 5 6 474 1a – Freeze samples loosely wrapped in foil in liquid nitrogen and store at -80°C. Transport samples 7 8 475 to freeze drier in dry ice. Rapidly load samples onto a cool plate in freeze drier and ensure the 9 10 476 pressure drops to below 5 mbar in under 2 minutes. Mill samples once dried and store in airtight 11 12 13 477 containers in the dark. 14 15 478 16 17 479 or 18 19 20 21 480 1b – Freeze 50mg samples in liquid nitrogen in 2ml eppendorf tubes and store at -80°C (for larger 22 23 481 samples use larger tubes). Add a volume of 80% methanol precooled to -20°C ensuring that final 24 25 482 methanol concentration remains above 78% according to equation (1) in materials and methods. 26 27 28 483 Add an appropriate volume of internal standard sinigrin or glucotropaeolin (e.g. 100µM final 29 30 484 concentration). Disrupt tissue by adding 2 small ball bearings and agitating with a tissue lyser (e.g. 31 32 33 485 tissuelyserII, Qiagen) for 10 minutes at 20 rev/s. Alternatively use a plastic pestle to thoroughly grind 34 35 486 the sample taking care that to keep the media below 0°C. Continue directly to 2b. 36 37 38 487 Extraction 39 40 488 2a For freeze dried tissue (1a). To 0.1g tissue, add 5ml of 80% methanol and 50µL of 20mM sinigrin 41 – 42 43 489 solution. Then 44 45 46 490 2b – shake sample once and leave to stand for 30 minutes. Shake sample for a further 30 minutes 47 48 491 (70 rev/s). Centrifuge at 4000 rpm and transfer supernatant to a fresh tube. 49 50 51 52 492 Desulfation 53 54 493 If desulfation is required, a high concentration sulfatase solution should be prepared by dissolving 55 56 494 15-25mg sulfatase in 1ml 40% ethanol and centrifuge at 8000 rmp for 1 minute. Transfer 57 58 59 495 supernatant to a fresh 2ml eppendorf tube and add 1ml of pure ethanol to precipitate the sulfatase 60 61 62 63 23 64 65 496 and centrifuge at 8000 rpm for 1 minute. Discard the supernatant and air dry the pellet before re- 1 2 497 dissolving in 2ml of water. Proceed with desulfation according to ISO9167-1 method. 3 4 5 6 498 7 8 9 499 Conclusions 10 11 12 500 In this study we compared different methods for extracting and purifying glucosinolates from B. 13 14 501 napus, B. junea, S. alba, E. sativa and R. sativus green tissues to highlight unnecessary or hazardous 15 16 502 steps. We have presented a simplified method for extracting glucosinolates from plant tissues which 17 18 19 503 does not require the use of a freeze drier or boiling methanol, and is therefore less hazardous, and 20 21 504 more time and cost effective. The presented method has been shown to have comparable or 22 23 505 improved glucosinolate extraction efficiency relative to the commonly used ISO method for major 24 25 26 506 glucosinolates in the Brassicaceae species studied: sinigrin and gluconasturtiin in B. juncea; sinalbin, 27 28 507 glucotropaeolin, and gluconasturtiin in S. alba; glucoraphenin and glucoraphasatin (roots but not 29 30 31 508 shoots) in R. sativus; and glucosatavin, glucoerucin and glucoraphanin in E. sativa. 32 33 34 509 Declarations 35 36 37 510 38 Authors’ contributions 39 40 41 511 TDA, KR, and SEH organized the project. VK carried out sample preparation and glucosinolate 42 43 512 extractions on B. napus leaves. TDA performed all other experiments, analyzed the data, and wrote 44 45 46 513 the paper; SEH, IB and KR reviewed and edited the manuscript. All authors read and approved the 47 48 514 final manuscript. 49 50 51 52 515 Acknowledgements 53 54 516 We are grateful to Catherine Lilley, (University of Leeds), Peter Urwin (University of Leeds), Andy 55 56 517 Barker (Barworth Agriculture ltd.) and Helen Barker (Barworth Agriculture ltd.) for collection of leaf, 57 58 59 518 stem and root samples used in this study. We would also like to thank Thomas Hartley (University of 60 61 62 63 24 64 65 519 York) for statistical advice. We would also like to express our gratitude to the BBSRC for funding this 1 2 520 study. Finally, we would like to thank the reviewers for their time and effort in reviewing this 3 4 5 521 manuscript. 6 7 8 522 Competing interests 9 10 523 The authors declare that they have no competing interests. 11 12 13 14 524 Funding 15 16 525 This work was supported by UK Biotechnology and Biology Sciences Research Council 17 18 526 (BB/L002124/1) and (BB/K020463/1). 19 20 21 22 527 Ethics approval (NA) 23 24 25 528 Consent for publication (NA) 26 27 28 529 Availability of data and Material 29 30 31 530 Material used in this study is stored at the University of York and is available on request. Datasets 32 33 531 analysed in this study are available from the corresponding author on request. 34 35 36 532 37 38 39 40 533 References 41 42 534 [1] Clarke DB. Glucosinolates, structures and analysis in food. Analytical Methods. 2010;2(4):310-25. 43 44 535 [2] Fahey JW, Zalcmann AT, Talalay P. The chemical diversity and distribution of glucosinolates and 45 46 47 536 among plants. 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Chemoprotective glucosinolates and 11 12 547 isothiocyanates of broccoli sprouts metabolism and excretion in humans. Cancer Epidemiology 13 14 548 Biomarkers & Prevention. 2001 May 1;10(5):501-8. 15 16 17 549 [8] Latté KP, Appel KE, Lampen A. Health benefits and possible risks of broccoli–an overview. Food 18 19 550 and Chemical Toxicology. 2011 Dec 31;49(12):3287-309. 20 21 551 [9] Wiesner M, Schreiner M, Glatt H. High mutagenic activity of juice from pak choi (Brassica rapa 22 23 24 552 ssp. chinensis) sprouts due to its content of 1-methoxy-3-indolylmethyl glucosinolate, and its 25 26 553 enhancement by elicitation with methyl jasmonate. Food and Chemical Toxicology. 2014 May 27 28 554 31;67:10-6. 29 30 31 555 [10] Ngala BM, Haydock PP, Woods S, Back MA. Biofumigation with Brassica juncea, Raphanus 32 33 556 sativus and Eruca sativa for the management of field populations of the potato cyst nematode 34 35 36 557 Globodera pallida. Pest management science. 2015 May 1;71(5):759-69. 37 38 558 [11] Lord JS, Lazzeri L, Atkinson HJ, Urwin PE. Biofumigation for control of pale potato cyst 39 40 559 nematodes: activity of Brassica leaf extracts and green manures on Globodera pallida in vitro and in 41 42 43 560 soil. Journal of agricultural and food chemistry. 2011 Jun 30;59(14):7882-90. 44 45 561 [12] Mattner SW, Porter IJ, Gounder RK, Shanks AL, Wren DJ, Allen D. Factors that impact on the 46 47 562 ability of biofumigants to suppress fungal pathogens and weeds of strawberry. Crop Protection. 2008 48 49 50 563 Aug 31;27(8):1165-73. 51 52 564 [13] Bellostas N, Kachlicki P, Sørensen JC, Sørensen H. Glucosinolate profiling of seeds and sprouts of 53 54 55 565 B. oleracea varieties used for food. Scientia Horticulturae. 2007 Nov 20;114(4):234-42. 56 57 566 [14] Ratti C. Hot air and freeze-drying of high-value foods: a review. Journal of food engineering. 58 59 567 2001 Sep 30;49(4):311-9. 60 61 62 63 26 64 65 568 [15] ISO 9167-1, 1992 (NA 057-05-05 AA – Joint committee of DIN and DGF for the analysis of fats, 1 2 569 oils and products thereof, related and primary products. (2012): Rapeseed Determination of 3 – 4 5 570 glucosinolate content – Part 1: Method using high-performance liquid chromatography (ISO 9167– 6 7 571 1:1992/DAM 1:2012), German version EN ISO 9167-1:1995/prA1:2012.) 8 9 10 572 [16] Ares AM, Nozal MJ, Bernal JL, Bernal J. Optimized extraction, separation and quantification of 11 12 573 twelve intact glucosinolates in broccoli leaves. Food chemistry. 2014 Jun 1;152:66-74. 13 14 574 [17] Church AS, Witting MD. Laboratory testing in ethanol, methanol, ethylene glycol, and 15 16 17 575 isopropanol toxicities. The Journal of emergency medicine. 1997 Oct 31;15(5):687-92. 18 19 576 [18] Rangkadilok N, Nicolas ME, Bennett RN, Premier RR, Eagling DR, Taylor PW. Determination of 20 21 577 sinigrin and glucoraphanin in Brassica species using a simple extraction method combined with ion- 22 23 24 578 pair HPLC analysis. Scientia Horticulturae. 2002 Dec 6;96(1):27-41. 25 26 579 [19] Herzallah S, Holley R. Determination of sinigrin, sinalbin, allyl-and benzyl isothiocyanates by RP- 27 28 580 HPLC in powder extracts. LWT-Food Science and Technology. 2012 Jul 31;47(2):293-9. 29 30 31 581 [20] D. F. Stoin and R. D. Dogaru, Bull. USAMV-CN, 2007, 63, 77–82 32 33 582 [21] Oerlemans K, Barrett DM, Suades CB, Verkerk R, Dekker M. Thermal degradation of 34 35 36 583 glucosinolates in red cabbage. Food Chemistry. 2006 Mar 31;95(1):19-29. 37 38 584 [22] Bennett RN, Mellon FA, Botting NP, Eagles J, Rosa EA, Williamson G. Identification of the major 39 40 585 glucosinolate (4-mercaptobutyl glucosinolate) in leaves of Eruca sativa L.(salad rocket). 41 42 43 586 Phytochemistry. 2002 Sep 30;61(1):25-30. 44 45 587 [23] Ishida M, Kakizaki T, Ohara T, Morimitsu Y. Development of a simple and rapid extraction 46 47 588 method of glucosinolates from radish roots. Breeding science. 2011;61(2):208-11. 48 49 50 589 [24] Piekarska A, Kusznierewicz B, Meller M, Dziedziul K, Namieśnik J, Bartoszek A. Myrosinase 51 52 590 activity in different plant samples; optimisation of measurement conditions for spectrophotometric 53 54 55 591 and pH-stat methods. Industrial Crops and Products. 2013 Oct 31;50:58-67. 56 57 592 [25] Wathelet JP, Mabon N, Marlier M. Determination of glucosinolates in rapeseed improvement of 58 59 593 the official HPLC ISO method (precision and speed). In Proceedings of the 10th International 60 61 62 63 27 64 65 594 Rapeseed Congress 1999 (p. 185). Gosford, NSW, Australia: The Regional Institute Ltd.. 1 2 595 [26] Förster N, Ulrichs C, Schreiner M, Müller CT, Mewis I. Development of a reliable extraction and 3 4 5 596 quantification method for glucosinolates in Moringa oleifera. Food chemistry. 2015 Jan 1;166:456- 6 7 597 64. 8 9 10 598 [27] Troyer JK, Stephenson KK, Fahey JW. Analysis of glucosinolates from broccoli and other 11 12 599 by hydrophilic interaction liquid chromatography. Journal of Chromatography 13 14 600 A. 2001 Jun 15;919(2):299-304. 15 16 17 601 [28] Wathelet JP, Iori R, Leoni O, Rollin P, Quinsac A, Palmieri S. Guidelines for glucosinolate analysis 18 19 602 in green tissues used for biofumigation. Agroindustria. 2004;3(3):257-66. 20 21 603 [29] Karathanos VT, Anglea SA, Karel M. Structural collapse of plant materials during freeze-drying. 22 23 24 604 Journal of Thermal Analysis and Calorimetry. 1996 Nov 1;47(5):1451-61. 25 26 605 [30] Burmeister WP, Cottaz S, Rollin P, Vasella A, Henrissat B. High resolution X-ray crystallography 27 28 606 shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic 29 30 31 607 base. Journal of Biological Chemistry. 2000 Dec 15;275(50):39385-93. 32 33 608 [31] Hennig K, Verkerk R, Bonnema G, Dekker M. Pitfalls in the desulphation of glucosinolates in a 34 35 36 609 high-throughput assay. Food chemistry. 2012 Oct 15;134(4):2355-61. 37 38 610 [32] Lee JG, Bonnema G, Zhang N, Kwak JH, de Vos RC, Beekwilder J. Evaluation of glucosinolate 39 40 611 variation in a collection of turnip (Brassica rapa) germplasm by the analysis of intact and desulfo 41 42 43 612 glucosinolates. Journal of agricultural and food chemistry. 2013 Apr 10;61(16):3984-93. 44 45 46 613 47 48 49 614 50 51 52 615 53 54 55 56 616 57 58 59 617 60 61 62 63 28 64 65 618 1 2 3 619 4 5 6 7 8 620 Figure legends 9 10 11 621 12 13 622 Fig 1: A broad outline of common extraction methods used for glucosinolate analysis. Highlighted in grey is the 14 15 623 ISO 9167-1 method which was originally intended for glucosinolate extraction from B. napus seed but is 16 17 624 commonly used for glucosinolate extraction and analysis in all glucosinolate containing plant tissues. 18 19 20 625 21 22 23 626 Fig 2: (a) total glucosinolate concentration of B. napus leaf halves dried in freeze drier B are significantly lower 24 25 (paired t-test, p=0.009) than leaf halves dried in freeze drier A; (b) B. napus leaf tissue dried with freeze drier B 26 627 27 28 628 is deformed and darker Error bars represent standard error. 29 30 31 629 32 33 34 630 Fig 3: There is no difference in final glucosinolate concentrations between freeze drying or direct extraction in - 35 36 631 20°C methanol. B. napus leaves were cut in half and frozen. One half was freeze dried prior to glucosinolate 37 38 632 extraction, the other half was extracted directly into -20°C methanol (n=12; paired t-test, p=0.15; R squared = 39 40 633 0.96). The dashed line represents equivalence of x and y. 41 42 43 634 44 45 46 635 Fig 4: Concentrations of representative aliphatic (sinigrin) and aromatic (glucotropaeolin) glucosinolates were 47 48 636 not reduced over the course of an hour at 100°C. The representative indole glucosinolate (glucobrassicin) is 49 50 51 637 degraded at 100°C. Asterisks represent significant difference from concentration at t=0 (paired t-test, p<0.05). 52 53 54 638 55 56 57 639 Fig 5: Spectrophotometric analysis of sinigrin hydrolysis kinetics in water and 80% methanol (n=3) by purified 58 59 640 myrosinase (0.05mg/ml) at room temperature (25°C). 60 61 62 63 29 64 65 641 1 2 3 642 Fig 6: Extraction of glucosinolates (≥1 µmol/g) in plant tissues across the three extraction methods. 4 5 643 Glucosinolate concentrations from the cold methanol and boiling water extraction methods are normalised to 6 7 644 the glucosinolate concentrations obtained from the ISO9167-1 (75°C methanol) method (n=4-12). Error bars 8 9 645 represent standard error. Asterisks represent a significant effect of extraction method on glucosinolate 10 11 12 646 concentration (repeat measure ANOVA, p<0.05). 13 14 15 647 16 17 18 648 Fig 7: The cold extraction method yields less glucoraphasatin in R. sativus stems relative to the ISO 9167-1 19 20 649 (boiling methanol) extraction method (n=4). Values normalised to the ISO method results. Error bars represent 21 22 650 standard error. Asterisks represent significant difference from the ISO 9167-1 (boiling methanol) method 23 24 651 (paired t test, p<0.05). 25 26 27 652 28 29 30 653 Fig 8: Absorbance values for representative desulfoglucosinolate extracts from B.juncea, S. alba, R. sativus and 31 32 33 654 E. sativa extracts treated with sulfatase solution for 12, 24 or 48 hours. These values are reflective of 34 35 655 desulfoglucosinolate recovery and not the initial glucosinolate concentration. 36 37 38 656 39 40 41 657 Fig 9: Desulfoglucosinolate content extracted from B. juncea, E. sativa and R. sativus tissue incubated with 75µl 42 43 658 low concentration sulfatase (0.3 U/ml) over 12H, 24H or 48H, and with a high concentration sulfatase (5U/ml) 44 45 659 over 24H, normalised to glucosinolate content of the same samples prior to sulfatase treatment. E. sativa leaf 46 47 48 660 samples were treated with TCEP post desulfation to undimerise didesulfoglucosatavin. Asterisks indicate a 49 50 661 significant effect of purification method on glucosinolate concentration (repeat measure ANOVA, p<0.05). ‘d’ 51 52 662 indicates the purification method yields a significantly different glucosinolate concentration relative to the 53 54 663 intact glucosinolates (paired t-test, p<0.05). 55 56 57 664 58 59 60 61 62 63 30 64 65 Figure 1 Click here to download Figure Figure 1.pptx

Tissue Plant tissue freeze dried and milled disruption Frozen plant tissue disrupted in -20°C Glucosinolates Glucosinolates Glucosinolates 80% Extraction extracted in 70% extracted in extracted 80% methanol methanol at 75°C water at 100°C methanol at RT

Extract treated with Extract treated with Filtered without Desulfation low (0.05-0.3U/ml) high ( 0.5-1 U/ml) sulfatase treatment sulfatase solution sulfatase solution

Seperation and HPLC analysis identification Figure 2 Click here to download Figure Figure 2.pptx (a) (b) 14

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2 Meantotal glucosinolate content (µmol/g)

0 AB Figure 1003 Click here to download Figure Figure 3.pptx

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20 Total glucosinolate content from freeze dried extraction (µmol/g) extraction dried freeze from content glucosinolate Total

0 0 20 40 60 80 100 Total glucosinolate content from wet extraction (µmol/g) Figure 4 Click here to download Figure Figure 4.pptx 1.2 Sinigrin Glucotropaeolin Glucobrassicin

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0 5 10 30 60 Time (min) Figure 5 Click here to download Figure Figure 5.pptx 1.4

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0.2 Water 80% Methanol 0 0 10 20 30 40 50 60 70 Time (min) Figure 6 Normalised glucosinolate content 1.2 1.4 0.2 0.4 0.6 0.8 1 0 * Boiling methanol (ISO) * * Cold methanol Boiling water Click here to download Figure Figure 6.pptx * * Figure 71.2 Click here to download Figure Figure 7.pptx Boiling methanol Cold methanol

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0 Root Stem Figure 8 Click here to download Figure Figure 8.pptx 1200000 Glucoraphanin Glucoerucin sinigrin gluconasturtiin 1000000 sinalbin glucoraphenin glucotropaeolin glucosatavin 800000 dimeric glucosatavin

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0 0 10 20 30 40 50 60 Time (h) Figure 91.6 Click here to download Figure Figure 9.pptx Intact glucosinolates High sulfatase 12H Low sulfatase c 12H Low sulfatase c 24H Low sulfatase c 48H 1.4 d * d d * 1.2 * * * * 1 d d dd d d 0.8 dd d d

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0 Example chromatograms

Click here to access/download Supplementary Material Example Chromatograms.docx