Improving the health benefit of broccoli by maximizing sulforaphane formation: A role for epithiospecifier protein

The isothiocyanate sulforaphane is a potent anticarcinogen derived from broccoli. For sulforaphane to be formed, fresh broccoli tissue must be crushed, allowing the myrosinase-catalyzed hydrolysis of the glucosinolate precursor, glucoraphanin. A common assumption has been that sulforaphane is the sole product of glucoraphanin hydrolysis, but several studies have demonstrated that a nitrite analog to sulforaphane is also formed. However, little research has examined the relative bioactivity of this sulforaphane nitrite compared to sulforaphane, or the mechanism underlying the fractional formation of the nitrite or isothiocyanate products from glucoraphanin hydrolysis in broccoli. Our objectives were to develop an efficient means to purify sulforaphane and sulforaphane nitrite from broccoli seed, to test the relative bioactivity of sulforaphane and sulforaphane nitrite, to examine the effects of processing treatments on the formation of sulforaphane and sulforaphane nitrite from broccoli, and to identify specific physiological factors responsible for the formation of the nitrite. In this thesis, four sets of experiments, each corresponding to a specific objective, are presented. A preparative HPLC method has been developed to purify sulforaphane and sulforaphane nitrite in gram quantities from broccoli seed. The purified compounds were tested for their bioactivity in cell culture and in the rat, and sulforaphane nitrite was found to be far less potent than sulforaphane as an inducer of the phase II detoxification enzymes quinone reductase (QR) and glutathione S-transferase (GST). Mild heat treatment was found to dramatically increase the formation of sulforaphane from broccoli while decreasing the formation of the alternative nitrite product. A heat sensitive epithiospecifier protein (ESP) was identified as the primary nitrile-forming factor, and this protein was cloned from a commercial broccoli cultivar for analysis. Recombinant ESP was found to direct the formation of sulforaphane nitrite from purified glucoraphanin when low concentrations of iron were present (0.1 mM). The activity of ESP in 20 commercial broccoli genotypes was significantly correlated with sulforaphane nitrite formation. The studies presented in this dissertation have better defined the formation of sulforaphane in fresh broccoli as it is eaten in the diet. By developing processing methods to lower ESP activity, or by breeding to reduce expression of this protein, broccoli products may be developed that convert maximal amounts of glucoraphanin to the anticarcinogen sulforaphane.