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

Abstract

Induction of phase 2 detoxication enzymes [e.g., glutathione transferases, epoxide hydrolase, NAD(P)H: quinone reductase, and glucuronosyltransferases] is a powerful strategy for achieving protection against carcinogenesis, mutagenesis, and other forms of toxicity of electrophiles and reactive forms of oxygen. Since consumption of large quantities of fruit and vegetables is associated with a striking reduction in the risk of developing a variety of malignancies, it is of interest that a number of edible plants contain substantial quantities of compounds that regulate mammalian enzymes of xenobiotic metabolism. Thus, edible plants belonging to the family Cruciferae and genus Brassica (e.g., broccoli and cauliflower) contain substantial quantities of isothiocyanates (mostly in the form of their glucosinolate precursors) some of which (e.g., sulforaphane or 4-methylsulfinylbutyl isothiocyanate) are very potent inducers of phase 2 enzymes. Unexpectedly, 3-day-old sprouts of cultivars of certain crucifers including broccoli and cauliflower contain 10-100 times higher levels of glucoraphanin (the glucosinolate of sulforaphane) than do the corresponding mature plants. Glucosinolates and isothiocyanates can be efficiently extracted from plants, without hydrolysis of glucosinolates by myrosinase, by homogenization in a mixture of equal volumes of dimethyl sulfoxide, dimethylformamide, and acetonitrile at -50 degrees C. Extracts of 3-day-old broccoli sprouts (containing either glucoraphanin or sulforaphane as the principal enzyme inducer) were highly effective in reducing the incidence, multiplicity, and rate of development of mammary tumors in dimethylbenz(a)anthracene-treated rats. Notably, sprouts of many broccoli cultivars contain negligible quantities of indole glucosinolates, which predominate in the mature vegetable and may give rise to degradation products (e.g., indole-3-carbinol) that can enhance tumorigenesis. Hence, small quantities of crucifer sprouts may protect against the risk of cancer as effectively as much larger quantities of mature vegetables of the same variety.

Figure 1

Induction of quinone reductase in Hepa 1c1c7 murine hepatoma cells as a function of concentration of glucosinolates (Left) and of an extract of mature fresh broccoli (Right). (Left) Five glucosinolates (GS). Curves: 1, glucoraphanin (the glucosinolate of sulforaphane); 2, gluconapin (3-butenyl-GS); 3, sinigrin (allyl-GS); 4, progoitrin (2-hydroxy-3-butenyl-GS); 5, glucobrassicin (indol-3-ylmethyl-GS). Solid lines, myrosinase treated; dashed lines, untreated. (Right) A triple solvent extract of mature broccoli obtained from a local supermarket. Induction potencies were measured, in the absence and presence of purified myrosinase, in 96-well microtiter plates containing serial dilutions of aqueous solutions of the pure glucosinolates (expressed as micromolar concentrations) or of broccoli extracts (expressed as fr. wt. equivalents in μg per well). Note the logarithmic concentration scales. From the CD value of 30 μg fr. wt. equivalent per well, one can calculate that this sample of mature broccoli contained 33,300 units/g fr. wt. of latent inducer activity (compare with Fig. 2).

Figure 2

Quinone reductase inducer activities of randomly selected fresh and frozen broccoli samples purchased at Baltimore area supermarkets. Florets of seven frozen broccoli lots representing five national brands (Hanover, America’s Choice, Green Giant, BirdsEye, and BelAir) were extracted with cold triple solvent. Each bar represents the analysis of a portion of a pooled 3- to 5-lb. sample from a single lot (1 lb. = 0.45 kg). Mean inducer potencies of the frozen broccoli samples were 12,000 units/g fr. wt. Twenty-two fresh bunches (one to four heads each) of conventionally and organically grown broccoli were obtained from local supermarkets. The florets were homogenized in cold triple solvent and assayed for inducer activity in the presence of added purified myrosinase. Mean potency is 35,000 units/g fr. wt. Negligible quantities of inducer activity were detected in most samples before addition of myrosinase (see text).

Figure 3

Effect of age on the inducer potency of broccoli sprout extracts. Broccoli sprouts were harvested daily for 9 days. Extracts were prepared by homogenization in cold triple solvent and assayed for inducer activity with myrosinase and ascorbate added to the microtiter well plates. Inducer potencies of the sprouts are expressed both “per mg fresh plant weight” (Upper) and “per plant” (Lower). Mean fresh weights were determined on samples of at least 20 sprouts per time point. The initial seed weight is about 3.5 mg, and sprout weight increases by about 6.6 mg/day. Inducer specific activities of myrosinase-treated sprout extracts are compared with those of seeds. Without myrosinase treatment, extracts contained <1,000 units/g fr. wt.

Figure 4

Comparison of glucosinolate HPLC profiles of 2-day-old broccoli sprouts with those of florets from heads of mature broccoli grown from the same seeds of cultivar SAGA. All samples were extracted with triple solvent and the component glucosinolates were resolved by paired-ion chromatography (18). Extracts equivalent to 62.4 mg fr. wt. of mature broccoli and 9.45 mg fr. wt of sprouts were analyzed. Glucosinolates were identified by reanalysis after treatment with myrosinase; the shaded black peaks disappeared upon such treatment. The glucosinolates are as follows. Peaks: A, glucoraphanin; B, 4-hydroxyglucobrassicin; C, glucoerucin; D, glucobrassicin; E, neoglucobrassicin. The chemical identities of peaks A, C, D, and E were confirmed by mass spectrometry and NMR (18). In the sprout extract, the major peaks are glucoraphanin (16.6 μmol/g fr. wt.) and glucoerucin (5.41 μmol/g fr. wt.), and only 0.71 μmol/g fr. wt. of 4-hydroxyglucobrassicin. The mature broccoli extract contained glucoraphanin (1.08 μmol/g fr. wt.) and the indoles glucobrassicin (1.67 μmol/g fr. wt) and neoglucobrassicin (0.62 μmol/g fr. wt.).

Figure 5

Effects of hot-water extracts of 3-day-old broccoli sprouts on mammary tumor development in DMBA-treated female Sprague–Dawley rats. The analytical composition of the glucosinolate and isothiocyanate extracts is given in Table 1. The animals were examined for tumors at weekly intervals. (Upper) Tumor incidence (percent of rats with tumors). (Lower) Tumor multiplicity (no. of tumors/no. of rats at risk). Progression of tumor development in the single control group is shown in all panels. The animals received daily intragastric doses of 25 or 100 μmol of glucosinolates (Left) and 25, 50, or 100 μmol of isothiocyanates (Right) at age 47–51 days. The 25-μmol doses corresponded to 81 and 106 mg of lyophilized extract for the glucosinolate and isothiocyanate preparations, respectively. Statistical significance of the retardation of tumor development (incidence) compared with controls was as follows. For 25 and 100 μmol doses of glucosinolates, P = 0.0079 and 0.138, respectively. For 25, 50, and 100 μmol doses of isothiocyanates, P = 0.214, 0.0422, and 0.0058, respectively.