Article pubs.acs.org/JAFC
Total Myrosinase Activity Estimates in Brassica Vegetable Produce Edward B. Dosz,† Kang-Mo Ku,‡,§ John A. Juvik,‡ and Elizabeth H. Jeffery*,† †
Department of Food Science and Human Nutrition and ‡Department of Crop Sciences, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801-3838, United States S Supporting Information *
ABSTRACT: Isothiocyanates, generated from the hydrolysis of glucosinolates in plants of the Brassicaceae family, promote health, including anticancer bioactivity. Hydrolysis requires the plant enzyme myrosinase, giving myrosinase a key role in health promotion by brassica vegetables. Myrosinase measurement typically involves isolating crude protein, potentially underestimating activity in whole foods. Myrosinase activity was estimated using unextracted fresh tissues of five broccoli and three kale cultivars, measuring the formation of allyl isothiocyanate (AITC) and/or glucose from exogenous sinigrin. A correlation between AITC and glucose formation was found, although activity was substantially lower measured as glucose release. Using exogenous sinigrin or endogenous glucoraphanin, concentrations of the hydrolysis products AITC and sulforaphane correlated (r = 0.859; p = 0.006), suggesting that broccoli shows no myrosinase selectivity among sinigrin and glucoraphanin. Measurement of AITC formation provides a novel, reliable estimation of myrosinase-dependent isothiocyanate formation suitable for use with whole vegetable food samples. KEYWORDS: broccoli, kale, myrosinase activity, sulforaphane, isothiocyanates
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INTRODUCTION Brassica vegetable crops contain diverse health-promoting compounds including glucosinolates (GSL), flavonoids, carotenoids, and vitamins. Glucosinolates themselves are not bioactive but are converted into multiple hydrolysis products, including isothiocyanates and nitriles, through the action of the endogenous enzyme myrosinase.1 The health benefits of broccoli are strongly associated with isothiocyanates including sulforaphane (SF), phenethyl isothiocyanate (PEITC), and allyl isothiocyanate (AITC). The chemical structure of hydrolysis products depends on the structure of the GSL side chain and reaction conditions, including the presence of myrosinase cofactors such as epithiospecifier protein (ESP). Abundance of ESP favors formation of nitriles, which not only lack anticancer activity, but are alternative hydrolysis products to bioactive isothiocyanates, thus lowering isothiocyanate levels.1,2 Recent studies report that destruction of myrosinase during processing of commercially available frozen broccoli products was the probable cause for little to no formation of the anticancer compound SF upon crushing the material.3,4 We and others find that in the absence of myrosinase, only about 10% of the glucoraphanin present in a supplement or processed broccoli product appears as SF and its metabolites in plasma and urine.5,6 A larger study found that SF absorption from a broccoli product lacking myrosinase may vary between 93% inhibition (Figure 4A). To avoid this interference for the ABTS-glucose assay, after
leaf samples, measured by either glucose or AITC assays. In all, when myrosinase activity from all tissues was compared across the two assays, although it was not strong, there was a significant, positive correlation between glucose appearance and AITC formation (r = 0.488, n = 21, p = 0.0252). This suggests that the two assays measure the same activity, although not optimally. Therefore, we chose to determine if data were more similar between the two assays at earlier time points. In reviewing change in enzyme rates over time, Figure 2 shows that rates were initially linear, but no longer linear by 5 min, whether using appearance of glucose (Figure 2A) or formation of AITC (Figure 2B). Background glucose levels are low in semipurified myrosinse preparations but greatly confounded the glucose appearance assay in homogenates at short time points (Figure 2A). For example, at 5 min, there was insufficient glucose formed to differentiate glucose production from background glucose present in the whole tissue sample, causing great variability in repeat measures (Figure 2A). Thus, it was not until 30 min that leaf samples showed significantly greater activity than root samples, and not until 40 min that floret samples showed significantly greater activity than root samples (Figure 2A). Both assays identified leaf as having the greatest myrosinase activity per gram DW of tissue and root the least, even though under the conditions used, linearity was possibly only present for the first 3 min of incubation. In leaf tissue, neither glucose nor AITC appearance at 5 min was significantly different from their appearance at 30 min, showing that the reaction had almost stopped by 5 min under these conditions. In the literature the glucose release or sinigrin disappearance assays are often run for 5 min or more9,11,18,20,23 and assume linearity of glucose formation during the entire assay,18 which is likely true for these simplified systems using extracted myrosinase but could greatly underestimate myrosinase activity and not accurately reflect the real-time activity of myrosinase if the reaction slows as we observed in the unextracted tissue. We considered the possibility that substrate loss, when sinigrin is converted to hydrolysis products, could be responsible in part for the change seen in linearity of myrosinase activity. However, on the basis of product formation at 30 min, >40% substrate remained in samples from the AITC method. A previous study suggested that AITC can react with protein, resulting in
Figure 3. Scheme of ABTS-glucose assay showing site of potential risk of antioxidant interference. 8097
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Figure 4. Endogenous antioxidant interference of glucose measurement, when using the ABTS-glucose assay: (A) interference of gallic acid (1.17− 150 μM) on measurement of glucose (fixed at 5 μg); (B) percent interference by endogenous antioxidant compounds in ‘Marathon’ root, leaf, or floret broccoli tissues. Extracts generated following incubation were either undiluted or diluted 96-fold. Values are means ± SD (n = 3). (∗∗) Significantly different from no gallic acid, p < 0.01. Different letters indicate significantly different values based on LSD test (p < 0.05).
Figure 5. Correlation between myrosinase measurements using exogenous (singirin) or endogenous glucosinolate in 21 broccoli and kale tissue samples: (A) sinigrin (10 mM) or no glucosinolate addition, glucose assay; (B) sinigrin (2 mM) or no glucosinolate addition, AITC and SF assay. Pearson’s correlation was calculated on the basis of the mean activity for each assay; n = 3.
appearance of glucose in 21 test samples. The tissue total GSL concentrations ranged from 1.86 to 70.9 μmol/g (concentration during the assay, 0.093−3.54 mM, Supporting Information Table S1). There was a highly significant correlation between myrosinase activity using endogenous GSL and exogenous GSL (10 mM sinigrin) measured as glucose appearance (r = 0.744, n = 21, p < 0.0001; Figure 5A). This shows that myrosinase activity can be measured using endogenous GSL over this naturally occurring GSL concentration range. The GSL concentration varied significantly from tissue to tissue; averaged values of total broccoli floret and root GSL concentration from five cultivars were 16.2 and 14.8 μmol/g DW, respectively which were considerably higher concentrations than those observed in leaf tissue (4.0 μmol/g DW, Supporting Information). Averaged values of kale root and leaf tissue GSL concentrations were 44.3 and 10.0 μmol/g DW, respectively (Supporting Information). However, this method is only feasible to use for short time measurements and for screening plant tissues that contain sufficient GSL. Because glucoraphanin is usually the predominant GSL found in broccoli, we hypothesized that its hydrolysis would correlate to the myrosinase activity determined previously. We found myrosinase activity using the formation of either AITC or SF to
myrosinase-dependent glucose formation was complete, we evaluated the use of 96-fold diluted sample extracts to estimate glucose. At this dilution, plant endogenous antioxidant compounds interfered with glucose generation less than 1, 6, and 3% for root, leaf, and floret tissues, respectively (Figure 4B). This dilution not only allows us to minimize the potential interference from antioxidant compounds but also to decrease the concentration of endogenous glucose within the capacity range of the ABTS-glucose assay. Published myrosinase activity assays typically use the addition of excess exogenous GSL; in most cases sinigrin is used and activity is reported in terms of sinigrin hydrolysis.23 Often, however, myrosinase activity is being measured in samples in which other GSL or isothiocyanates are the compounds of interest. The use of a GSL not endogenous or not found in high concentrations in a cruciferous vegetable may skew results, particularly if substrate specificity exists between myrosinase and endogenous GSL, as previously shown for Crambe abyssinica seeds.28 In this respect we hypothesized that using endogenous GSL to measure myrosinase activity should also be possible if the sample contains sufficient endogenous substrate to ensure that myrosinase (and not substrate) is rate limiting. Hydrolysis of endogenous GSL was measured as the 8098
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be significantly correlated (r = 0.860, n = 8, p = 0.0062; Figure 5B). This suggests that the ITC formation-based myrosinase assay described here can be utilized to measure myrosinase activity using endogenous, as well as exogenous, GSL. For example, myrosinase activity in broccoli, watercress, and horseradish might be measured by SF, PEITC, and AITC formation, respectively. However, it is necessary to remember that whereas this may provide an accurate measure for ingested foods, the presence of ESP, and possibly other myrosinase protein cofactors, will cause underestimation of absolute myrosinase activity. Because in vivo formation and bioavailability of ITC from GSL in broccoli is affected by processing conditions,29 more study should be conducted using foods after the cooking process. In conclusion, evaluating myrosinase activity from multiple broccoli varieties, we find that leaf and floret tissues have relatively greater activity than root tissue, on a per gram DW basis. Kale varieties exhibited the same trend, with leaf tissues possessing greater myrosinase activity than root. Myrosinase activity based on the formation of AITC can be useful in determining short-term enzymatic rates of myrosinase, with incubation times limited to 1−3 min. Samples can be run and hydrolysis products extracted rapidly, allowing a quick turnaround time for a few activity estimations. When using brassica vegetable tissues, the glucose release assay cannot be run for such short times, due to high baseline interference by endogenous glucose: on average, 30 min was required to reliably measure glucose formation. For limited sampling, the glucose assay also takes longer to perform, with multiple steps and incubations. However, if there are a large number of samples, performance time per sample is reduced radically, making glucose release a better high-throughput method, as long as samples are prepared in such a way that background glucose does not interfere, such as the use of a crude protein extract in place of tissue samples. However, we found that up to 36% of myrosinase activity was discarded in the pellet during generation of the crude protein extract. Whether soluble and insoluble myrosinases are similar with regard to activity or even substrate specificity is not known, although we found that changing substrates from sinigrin to the endogenous substrate glucoraphanin did not alter myrosinase measurements using the AITC method. The proposed AITC formation assay can measure activity during the short-term linear phase of total myrosinase activity (∼3 min) when used for whole brassica vegetable samples. It also has the capacity to evaluate hydrolysis of endogenous glucoraphanin. Whereas this assay appears most relevant for estimating myrosinase activity in terms of food consumption, further studies are needed to understand the impact of cooking on myrosinase activity in whole homogenates, particularly to determine how soluble and insoluble myrosinase enzymes are affected by postharvest processing.
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Goodwin Ave., Room 467, Urbana, IL 61801-3838, USA. Phone: (217) 333-3820. E-mail: ejeff
[email protected]. Author Contributions §
K.-M. K. contributed equally to this work.
Funding
Our thanks to Sakata Seed Co. for their support of E.J. and E.D. and for USDA/NIFA 2010-65200-20398 to E.J. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED ABTS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); AITC, allyl isothiocyanate; ESP, epithiospecifier protein; GSL, glucosinolate; PEITC, phenethyl isothiocyanate; SF, sulforaphane
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ASSOCIATED CONTENT
S Supporting Information *
Table S1 (glucosinolate concentrations of broccoli and kale cultivars). This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*(E.H.J.) Mail: Department of Food Science and Human Nutrition, University of Illinois at Urbana−Champaign, 905 S. 8099
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