Food Phytochemicals for Cancer Prevention I - ACS Publications

Chapter 14

Downloaded by UNIV OF MARYLAND COLL PARK on March 28, 2014 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0546.ch014

High-Performance Liquid Chromatographic Determination of Glucosinolates in Brassica Vegetables J. M . Betz and W. D. Fox Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, DC 20204

Glucosinolates are naturally occurring constituents of Brassica vegetables. The term refers to a class of more than 100 sulfur-containing glycosides that yield thiocyanate, nitrile, or isothiocyanate derivatives upon enzymatic hydrolysis. These compounds are important because of their potential toxicity and because epidemiologic and other evidence indicates that some of them may inhibit some carcinogenic processes when consumed as part of the normal diet. Accordingly, quantitation of these compounds in processed and unprocessed foods has become important. Existing analytical methods are time consuming and labor intensive. Solid phase extraction (SPE) of broccoli extracts, followed by reverse phase ion pair high performance liquid chromatography (HPLC) of intact, nonderivatized glucosinolates, provides a rapid and simple method for evaluation of glucosinolate loss during food processing.

The varieties of the genus Brassica that are closely related to cabbage and are consumed as greens or vegetables were all derived from sea cabbage (Brassica oleracea var. sylvestris L.), a coastal species found wild in much of western Europe (1). According to Nieuwhof (2), cruciferous plants have been used medicinally and as food for millennia. Gout, diarrhea, coeliac trouble, stomach trouble, deafness, headache, wound healing, and mushroom poisoning are some of the conditions for which they were used (2). As food these plants were eaten raw, cooked, or pickled (3-5). Chemistry The major bioactive constituents of the Brassicaceae (Cruciferae) are a class of sulfur-containing glycosides called glucosinolates and their stable thiocyanate, nitrile, and isothiocyanate hydrolysis products. The glucosinolates generally occur in the form of a potassium salt, whose general structure is shown in Figure 1 (6). The R groups (aglycones) are unstable alkyl or aryl side chains which, upon being

This chapter not subject to U.S. copyright Published 1994 American Chemical Society

In Food Phytochemicals for Cancer Prevention I; Huang, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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FOOD PHYTOCHEMICALS I: FRUITS AND VEGETABLES

released by enzymatic hydrolysis, are responsible for the characteristic flavors and odors of the Brassicas (7-12). The glycone portion of nearly all glucosinolates consists of a single β-D-glucose moiety, although at least two exceptions have been noted (13,14). More than 100 glucosinolates have been reported (75). These compounds are distributed over eleven families of dicotyledonous angiosperms (76). The glucosinolates previously identified in broccoli are shown in Table I.


^S-CeHnOo N - O S O 3 -

R = aryl or alkyl s i d e chain

Figure 1. General glucosinolate structure. Table I. Glucosinolates in Broccoli R group

Trivial name

3-MethylsulfinylpropylAllyl2(7?)-Hydroxy-3-butenyl2(S)-Hydroxy-3-butenyl4-Methylthiobutyl4-Methylsulfinylbutyl2-Phenylethyl3-Indolylmethyl3(A^Methoxy)indolylmethyl-

Glucoiberin Sinigrin Progoitrin ' e/?/-Progoitrin ' Glucoerucin Glucoraphanin Gluconasturtiin Glucobrassicin Neoglucobrassicin

a

a

a b

a b

3

a

Standards available for this study. These isothiocyanates cyclize to form oxazolidinethiones.

b

The current systematic approach to glucosinolate nomenclature was proposed by Ettlinger et al. (17). In this system, the name of the side chain was prefixed to the term glucosinolate. Readers desiring more information on this subject should consult the works of VanEtten and Tookey (18), Challenger (79), Kjaer (20), and Fenwick et al. (21). Despite the number of glucosinolates which have been described, individual species generally produce only a few types in abundance (27). More important are the quantity and pattern of distribution of individual glucosinolates within species, which depend on factors such as plant part examined (22), stage of development, horticultural variety, cultivation conditions, climate, and agricultural practices (23-27). The volatile constituents of Brassica vegetables are generated by the hydro lysis of glucosinolates by myrosinase (thioglucoside glucohydrolase, E C 3:2:3:1) (19,28). This enzyme is located in specialized myrosin cells in Cruciferae (29). Details of the complex interrelationships between the glucosinolates, myrosinase and its isoenzymes, cofactors, and the individual hydrolysis products have emerged


14. BETZ AND FOX

Glucosinolates and Brassica Vegetables

183

slowly (29-31). When cells of Brassica are ruptured in the presence of water, endo genous myrosinase catalyzes the hydrolysis of glucosinolate into glucose and an unstable product which undergoes a spontaneous Lossen-type rearrangement. The rearrangement yields bisulfate ion and the characteristic thiocyanate, nitrile, or isothiocyanate product, the exact nature of which depends on several additional factors, although the nitrile is usually favored (32-34). Glucosinolates treated with exogenous myrosinase at neutral pH yield aglycones which undergo rearrangement to produce the volatile isothiocyanates (35). Glucosinolates treated with the enzyme under acidic conditions (pH 3) yield nitriles rather than isothiocyanates, as do those hydrolyzed in the presence of F e and those which possess a β- or γ-hydroxyl group in the parent side chain (15,21,33,36). These latter products (hydroxyisothiocyanates) are unstable and spontaneously cyclize to form the oxazolidine-2-thiones (OZTs). Indole glucosinolates yield either the 3-carbinol (I3C) (via an unstable iso thiocyanate), upon hydrolysis at pH 7, or the 3-nitrile [indole-3-acetonitrile (I3A)], and subsequently indole-3-acetic acid, under acidic conditions (36-38). The 3carbinol may condense to the diindolyl methane or react with ascorbic acid to form ascorbigenin (39). Epithionitrile and thiocyanate products may also be formed from gluco sinolate precursors. The former requires the action of an epithiospecifier protein and F e on the unstable 2-hydroxy-3-butenyl intermediate (33,34,36-42). The mechanism of thiocyanate formation is presently unknown (75), but it has been suggested that a labile thiocyanate-forming factor similar to epithiospecifier protein may be present in the limited number of genera from which the thiocyanates have been isolated (27).


+ +

+ +

Biological Activity Glucosinolates and their hydrolysis products have been shown to exhibit both acute and chronic toxic effects in mammalian systems. Early studies were focused on goi trogenic effects of the Cruciferae (43,44). Thiocyanates (45) and L-5-vinyl-2-thiooxazolidone (goitrin) (46) produced by glucosinolate hydrolysis (47) were iden tified as the goitrogenic factors. Goitrin was determined to be the more important compound since its activity could not be reversed by added dietary iodine (45,48). In vitro experiments demonstrated that progoitrin and e/?/-progoitrin were hydro lyzed to R- and 5-goitrin by native gut flora and were thus toxic even in the absence of thioglucosidase activity (49-52). Embryotoxicity has been reported for a number of hydrolysis products (53). Acute rodent L D 5 0 values for glucosinolate hydrolysis products have been determined (53,54), and hepatic, renal, and pancreatic lesions have been reported following ingestion of OZTs (50) and nitriles (53,55-58). Longer-term adverse effects of glucosinolates and their hydrolysis products are more difficult to identify. Syrian hamsters fed a dried cabbage diet had increased pancreatic carcinoma formation (59), and cabbage fed to 1,2-dimethylhydrazine-treated mice produced a higher incidence of adenocarcinomas (60). Indole-3-carbinol fed to 1,2-dimethylhydrazine-treated rats during initiation caused an increase in carcinogenesis (61). Diets containing 10 and 20% cabbage enhanced tumorigenicity of 1,2-dimethylhydrazine, whereas diets of 40% cabbage provided a protective effect in mice (62). Sauerkraut incubated with simulated gastric fluid displayed alkylating activity which was enhanced when nitrite was added to the test solution (63). Liithy et al. (64,65) reported that goitrin is nitrosated by treatment



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with nitrite under stomach conditions to form N-nitroso-5-vinyl-2-oxazolidone, a mutagen in the Ames Salmonella assay. Albert-Puleo (65) provides a review of some of the medicinal uses of Cruciferae, with special emphasis on the anticancer activity of the family. Several epidemiological studies on diet and cancer have been performed which suggest a decrease in colon cancer risk associated with frequent ingestion of vegetables, especially cabbage and other cruciferous vegetables (67-71). Hepatic tumorigenicity of aflatoxin B i in rats can be reduced by dietary cabbage and cauliflower (72,73). Likewise, the incidence of dimethylbenz[a]anthra cene-induced mammary tumors in rats can be reduced by oral administration of cabbage and cauliflower (74), and I3C and 3,3'-diindolylmethane (75). Both of these compounds and I3A also inhibit benzo[

Food Phytochemicals for Cancer Prevention I - ACS Publications

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