Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-04-08
2002-01-22
Benzion, Gary (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S269000, C800S264000, C800S273000, C800S267000, C800S298000, C435S410000
Reexamination Certificate
active
06340784
ABSTRACT:
FIELD OF INVENTION
The present invention relates to methods for the selective increase of anticarcinogenic glucosinolate derivatives in Brassica species, and to Brassica species with enhanced levels of anticarcinogenic glucosinolate derivatives and in particular edible Brassica vegetables with elevated levels of the anticarcinogenic glucosinolate derivatives 4-methylsulfinylbutyl isothiocyanate and/or 3-methylsulfinylpropyl isothiocyanate. The present invention also provides methods for selection of genetic combinations of broccoli containing high levels of anticarcinogenic glucosinolate derivatives and methods to evaluate the anticarcinogenic properties of these genetic combinations. The invention further relates to compositions of matter comprising Brassica vegetables with concentrations of 4-methylsulfinylbutyl glucosinolate and/or 3-methylsulfinylpropyl glucosinolate between 10 and 100 &mgr;moles/g dry weight.
BACKGROUND ART
The present invention provides methods for the production of Brassica vegetables with elevated levels of specific glucosinolates and derivatives thereof. In particular the invention provides methods for the production and selection of Brassica vegetables with elevated levels of 3-methylsulfinylpropyl and/or 4-methylsulfinylbutyl glucosinolates. These glucosinolates are converted by the activity of the enzyme myrosinase into isothiocyanate derivatives which have been demonstrated to be potent inducers of phase II detoxification enzymes, elevated activity of which is associated with reduced susceptibility to the neoplastic effects of carcinogens. The invention provides genetic combinations which 1.) exhibit elevated levels of 4-methylsulfinylbutyl glucosinolate and/or 3-methylsulfinylpropyl glucosinolate and 2.) exhibit low activity of the GSL-ALK allele which encodes an activity capable of converting these glucosinolates into the alkenyl derivatives, which do not posses the anti-carcinogenic properties of the isothiocyanate derivatives of these glucosinolates and 3.) suitable myrosinase activity capable of producing isothiocyanate derivatives of said glucosinolates. Accordingly these genetic combinations provide elevated levels of specific glucosinolates, reduced production of alkenyl derivatives of these glucosinolates and favoured production of isothiocyanate derivatives of said glucosinolates. The invention further relates to the use of genetic markers to select the genetic combinations described above.
It is known that a diet high in vegetables is associated with a reduction in the risk of certain types of cancer and hence it is desirable to include a significant amount of vegetables in the human diet. The anticarcinogenic activity of vegetables has been associated with the presence of several classes of secondary metabolites. Evidence is growing that some of these secondary metabolites are involved in lowering the risk of certain types of cancer and hence are considered anticarcinogenic. Accordingly, enhancing the level of anticarcinogenic metabolites provides a useful strategy for the reduction of cancer risk, in complementation with dietary advice to increase the consumption of vegetables.
The precise mechanism by which vegetables provide a decreased risk of many types of cancer is not known with certainty, but there are many lines of evidence which support the involvement of vegetables in the prevention of cancer. In particular, the role of cruciferous vegetables in the prevention of cancer is widely supported through epidemiological studies and more recently biochemical studies. One class of secondary metabolites that is implicated in the beneficial effects of cruciferous vegetables is the isothiocyanate derivatives of certain glucosinolates. Four complementary pieces of evidence suggest that isothiocyanates derived from the hydrolysis of methylsulfinylalkyl glucosinolates found in crucifers may be important in the human diet in reducing the risk of cancer. (1.) Dietary provision of cruciferous vegetables protects rodents against chemically induced cancer (Wattenberg, L. W. (1985)
Cancer Res.
45, 1-8.). (2.) Methylaulfinylalkyl isothiocyanates are known to be potent inducers of phase II detoxification enzymes in murine hepatoma Hepa 1c1c7 cells in culture (Zhang, Y., Talalay, P., Cho, C.-G., & Posner, G. H. (1992)
Proc. Natl. Acad. Sci. USA
89, 2399-2403 and Tawfiq, N., Heaney, R. K., Plumb, J. A., Fenwick, G. R., Musk, S. R. R., & Williamson, G. (1995)
Carcinogenesis
16, 1191-1194.), which are associated with reduced susceptibility of mammals and mammalian cell cultures to the toxic and neoplastic effects of carcinogens. (3.) Sulforaphane (4-methylsulfinylbutyl isothiocyanate) blocks the formation of mammary tumors in Sprague-Dawley rats treated with 9,10-dimethyl-1,2-benzanthracene (Zhang, Y., Kensler, T. W., Cho, C.-G., Posner, G. H., & Talalay, P. (1994)
Proc. Natl. Acad. Sci. USA
91, 3147-3150.). (4.) Epidemiological studies show that people with high levels of vegetables in their diet are less susceptible to cancer (Block, G., Patterson, B., & Suber, A. (1992)
Nutr. and Cancer
18, 1-19.). Thus the beneficial effects of a diet high in certain glucosinolates may included a reduction in the risk of cancer. However, it appears that only certain glucosinolates and more accurately, certain derivatives of specific glucosinolates may be primarily responsible for the beneficial effect.
There are numerous individual glucosinolates in cruciferous plants. Glucosinolates have a common glycone moiety and a variable aglycone side chain. The structure of the glucosinolate side chain varies in length and chemical composition.
Glucosinolates are formed by the action of a number of enzymes, encoded by a small number of glucosinolate biosynthetic alleles (GSL alleles). In the glucosinolate pathway, methionine is converted to homo-methionine and dihomo-methionine by the activity of the GSL-ELONG allele. Homo-methionine is eventually converted to 3-methylthiopropyl glucosinolate followed by conversion to 3-methylaulfinylpropyl glucosinolate by the activity of GSL-OXID allele and finally 2-propenyl glucosinolate by the activity of GSL-ALK allele. Dihomo-methionine is converted to 4-methylthiobutyl glucosinolate, then to 4 methylsulfinylbutyl glucosinolate by the activity of GSL-OXID allele, then to 3-butenyl glucosinolate by the activity of GSL-ALK allele and finally converted to 2-hydroxy-3-butenyl glucosinolate by the activity of GSL-OH allele.
In general, the 3-methylsulfinylpropyl glucosinolates and 4-methylthiobutyl glucosinolates produce non-volatile isothiocyanates and hence these particular glucosinolates contribute little to flavour. In contrast, the volatile alkenyl derivatives can contribute to flavour, both positively and negatively, dependant on the plant species and particular glucosinolate derivative.
In
B. oleracea
vegetables, glucosinolates have either a three or four carbon side chain. Glucosinolates can be hydrolysed by the action of myrosinase which is often induced upon tissue damage. Many vegetables have alkenyl (2-propenyl and 3-butenyl) glucosinolates which result in the production of volatile products upon hydrolysis through the action of myrosinase. Some vegetables contain a 2-hydroxy-3-butenyl glucosinolate called progoitrin. This glucosinolate produces an unstable isothiocyanate that spontaneously cyclizes to produce oxazolidone-2-thiones, which are undesirable in diets due to their goitrogenic properties. Isothiocyanates derived from alkenyl and hydroxyalkenyl glucosinolates can have both positive and negative effects on flavour.
Broccoli accumulates low levels of glucosinolates with 4-metylsulfinylbutyl and 3-methylsulfinylpropyl side chains since broccoli has a greatly reduced activity of the GSL-ALK allele, responsible for the conversion of glucosinolates into alkenyl derivatives. It is believed that the popularity of broccoli as a vegetable is due in part to the relatively modest contribution to taste made by 4-metylsultinylbutyl and 3-methylsulfinylpropyl glucosinolate derivatives in contrast to the strong flavour
Faulkner Kathy
Mithen Richard
Williamson Gary
Benzion Gary
Kruse David H
Lorusso & Loud
Plant Bioscience Limited
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