Elsevier

Phytochemistry

Volume 65, Issue 9, May 2004, Pages 1273-1281
Phytochemistry

Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli

https://doi.org/10.1016/j.phytochem.2004.04.013Get rights and content

Abstract

Sulforaphane, an isothiocyanate from broccoli, is one of the most potent food-derived anticarcinogens. This compound is not present in the intact vegetable, rather it is formed from its glucosinolate precursor, glucoraphanin, by the action of myrosinase, a thioglucosidase enzyme, when broccoli tissue is crushed or chewed. However, a number of studies have demonstrated that sulforaphane yield from glucoraphanin is low, and that a non-bioactive nitrile analog, sulforaphane nitrile, is the primary hydrolysis product when plant tissue is crushed at room temperature. Recent evidence suggests that in Arabidopsis, nitrile formation from glucosinolates is controlled by a heat-sensitive protein, epithiospecifier protein (ESP), a non-catalytic cofactor of myrosinase. Our objectives were to examine the effects of heating broccoli florets and sprouts on sulforaphane and sulforaphane nitrile formation, to determine if broccoli contains ESP activity, then to correlate heat-dependent changes in ESP activity, sulforaphane content and bioactivity, as measured by induction of the phase II detoxification enzyme quinone reductase (QR) in cell culture. Heating fresh broccoli florets or broccoli sprouts to 60 °C prior to homogenization simultaneously increased sulforaphane formation and decreased sulforaphane nitrile formation. A significant loss of ESP activity paralleled the decrease in sulforaphane nitrile formation. Heating to 70 °C and above decreased the formation of both products in broccoli florets, but not in broccoli sprouts. The induction of QR in cultured mouse hepatoma Hepa lclc7 cells paralleled increases in sulforaphane formation.

Pre-heating broccoli florets and sprouts to 60 °C significantly increased the myrosinase-catalyzed formation of sulforaphane (SF) in vegetable tissue extracts after crushing. This was associated with decreases in sulforaphane nitrile (SF Nitrile) formation and epithiospecifier protein (ESP) activity.

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Introduction

Cruciferous plants such as broccoli (Brassica oleracea var. italica) contain glucosinolates, secondary metabolites that are part of an elegant herbivory defense mechanism (reviewed in Bones and Rossiter, 1996). When the tissue of these plants is damaged, glucosinolates are released from the vacuoles of myrosin cells and are hydrolyzed by cytosolic myrosinase, a thioglucoside glucohydrolase enzyme (EC 3.2.3.1). The spontaneous products of this reaction at neutral pH have been shown to be isothiocyanates (Gil and MacLeod, 1980b), and these compounds are the principal products of glucosinolate hydrolysis in a number of crucifers (Cole, 1976). However, other cruciferous plants, such as rape (Brassica napus) form predominantly nitriles from these glucosinolates (Gil and MacLeod, 1980c; Gil and MacLeod, 1980a; Lambrix et al., 2001), indicating that different products may be formed from the same glucosinolate, depending upon the plant species.

Sulforaphane (1) [4-(methylsulfinyl)butyl isothiocyanate], an isothiocyanate from broccoli, was initially identified as a potential anticarcinogen by its capacity to induce quinone reductase (QR), a phase II detoxification enzyme, in Hepa lclc7 cell culture (Zhang et al., 1992). The phase II detoxification enzyme system is an inducible set of xenobiotic defense enzymes that can intercept bioactivated carcinogens, protecting against DNA damage in the mammal (Talalay et al., 1995). Since its identification, experimental evidence has accumulated regarding the efficacy of sulforaphane as a cancer preventative agent in a number of animal models of carcinogenesis. Sulforaphane has been shown to inhibit chemically-induced cancer in rats and mice (Zhang et al., 1994; Fahey et al., 2002). Although there are no clinical studies showing prevention of cancer with sulforaphane, a recent clinical study evaluated absorption of broccoli extracts perfused into the small intestine and found that approximately 75% of the sulforaphane was absorbed (Petri et al., 2003). In that study levels of two detoxification enzymes, glutathione-S-transferase and UDP-glucuronosyl transferase, were induced more than 2-fold in exfoliated enterocytes released into the perfusate, indicating the potential for anticarcinogenic action of sulforaphane in these human subjects. Sulforaphane has also been observed to inhibit growth and stimulate apoptosis in several cancer cell lines in culture (Gamet-Payrastre et al., 2000; Chiao et al., 2002), indicating potential antiproliferative activity.

Although investigations of the effects of storage and cooking on the glucosinolate content of broccoli have been performed (Goodrich et al., 1989; Vallejo et al., 2003), surprisingly little research has examined the effects of storage or cooking on formation of sulforaphane in dietary broccoli. In current literature examining the chemoprotective effects of sulforaphane, the assumption is made that sulforaphane is the sole product of the hydrolysis of its precursor glucosinolate, glucoraphanin (2) (Fahey et al., 1997; Shapiro et al., 1998). However, a nitrile analog to sulforaphane, sulforaphane nitrile (3) [5-(methylsulfinyl)pentane nitrile] may actually be the predominant hydrolysis product of glucoraphanin (Matusheski et al., 2001; Mithen et al., 2003). Sulforaphane nitrile has recently been shown not to possess the anticarcinogenic properties of sulforaphane (Matusheski and Jeffery, 2001; Basten et al., 2002). Thus the potential health benefit of broccoli as a result of sulforaphane formation is compromised by the alternative formation of an inactive nitrile when broccoli is crushed.

Other cruciferous plants, including crambe seed (Crambe abyssinica), garden cress (Lepidium sativum) and other members of the Brassica species such as rapeseed (Brassica napus) and white cabbage (Brassica oleracea) have all been shown to form nitriles (Daxenbichler et al., 1977). A protein, called epithiospecifier protein (ESP), has been identified in some crucifers that appears responsible for the formation of epithionitriles (Tookey, 1973; Petroski and Tookey, 1982). This protein does not catalyze glucosinolate hydrolysis by itself, but instead directs the products of glucosinolate hydrolysis toward epithionitriles, rather than isothiocyanates. Epithiospecifier protein requires iron for its activity, and heat treatment has been shown to decrease the formation of epithionitriles in seeds of turnip rape (Brassica campestris; Kirk and Macdonald, 1974). A recent study in the experimental plant Arabidopsis thaliana suggests that ESP may regulate nitrile formation in addition to epithionitrile formation (Lambrix et al., 2001). Here, we test the hypothesis that ESP is present in broccoli, and supports the formation of inactive sulforaphane nitrile at the expense of formation of the potent anticarcinogen sulforaphane (Fig. 1). Our objectives were to examine the formation of sulforaphane and sulforaphane nitrile from glucoraphanin in fresh broccoli from several commercial cultivars, and to examine the effects of heating on sulforaphane and sulforaphane nitrile formation, ESP activity, and potential bioactivity of extracts prepared from fresh broccoli florets and broccoli sprouts.

Section snippets

Glucoraphanin content of commercial broccoli cultivars

Glucoraphanin content determined on lyophilized powders of 7 commercial broccoli cultivars varied significantly, ranging from 4.4±0.4 to 16.4±0.9μmol/g dry weight. Several of the cultivars examined were also represented in a previous study, where 50 broccoli accessions and a number of other cruciferous vegetable crops were examined for glucosinolate content (Kushad et al., 1999). Our results were in agreement with the earlier study, cv. Brigadier containing the highest glucoraphanin content and

Materials

Broccoli seeds for the production of sprouts and mature broccoli were gifts from Asgrow Seed Co. (cv. Majestic, Everest, Packman and Baccus), from Peto Seed Co. (Seminis Seeds; cv. Brigadier, Peto 7) and from Sakata Seed Co. (cv. Saga). Organic solvents (HPLC grade) were purchased from Fisher Scientific (Fairlawn, NJ). Purified epi-progoitrin and benzyl glucosinolate were purchased from Dr. Jens Sørensen at the Bioraf Denmark Foundation (Copenhagen, Denmark). Isothiocyanates, nitriles and

Acknowledgements

This research was supported by grants from the USDA (National Research Initiative 99-35503-7010). The authors thank William R. Bagby Jr. of Flatland Hydroponics, Thomasboro, IL for providing equipment and guidance for broccoli seed sprouting. Also thanks to Asgrow Seed Co., Peto Seed Co. and Sakata Seed Co. for donating seed stock utilized for broccoli production in this work.

References (48)

  • J.T.O Kirk et al.

    1-Cyano-3,4-epithiobutane: a major product of glucosinolate hydrolysis in seeds from certain varieties of Brassica campestris

    Phytochemistry

    (1974)
  • A.J MacLeod et al.

    The occurrence and activity of epithiospecifier protein in some cruciferae seeds

    Phytochemistry

    (1985)
  • R.J Petroski et al.

    Interactions of thioglucoside glucohydrolase and epithiospecifier protein of cruciferous plants to form 1-cyanoepithioalkanes

    Phytochemistry

    (1982)
  • H.J Prochaska et al.

    Direct measurement of NAD(P)H:quinone reductase from cells cultured in microtiter wells: a screening assay for anticarcinogenic enzyme inducers

    Anal. Biochem.

    (1988)
  • P Talalay et al.

    Chemoprotection against cancer by phase 2 enzyme induction

    Toxicol. Lett.

    (1995)
  • G.P Basten et al.

    Sulforaphane and its glutathione conjugate but not sulforaphane nitrile induce UDP-glucuronosyl transferase (UGT1A1) and glutathione transferase (GSTA1) in cultured cells

    Carcinogenesis

    (2002)
  • A.M Bones et al.

    The myrosinase–glucosinolate system, its organisation and biochemistry

    Physiol. Plant.

    (1996)
  • A.F Brown et al.

    Glucosinolate profiles in broccoli (Brassica oleracea L.): stability over environments and implications for cancer chemoprotection

    J. Am. Soc. Hort. Sci.

    (2002)
  • J.W Chiao et al.

    Sulforaphane and its metabolite mediate growth arrest and apoptosis in human prostate cancer cells

    Int. J. Oncol.

    (2002)
  • R.A Cole

    Volatile components produced during ontogeny of some cultivated crucifers

    J. Sci. Food Agric.

    (1980)
  • M.E Daxenbichler et al.

    Glucosinolates and derived products in cruciferous vegetables. Identification of organic nitriles from cabbage

    J. Agric. Food Chem.

    (1977)
  • S Eriksson et al.

    Identification and characterization of soluble and insoluble myrosinase isoenzymes in different organs of Sinapis alba

    Physiol. Plant.

    (2001)
  • J.W Fahey et al.

    Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors

    Proc. Natl. Acad. Sci. USA

    (2002)
  • J.W Fahey et al.

    Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens

    Proc. Natl. Acad. Sci. USA

    (1997)
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