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
1999-06-30
2003-02-18
McElwain, Elizabeth F. (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
C800S260000, C800S265000, C800S270000, C800S276000, C800S278000, C800S279000, C800S298000, C800S301000, C800S302000
Reexamination Certificate
active
06521818
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to methods for developing novel, highly chemoprotectant crucifer germplasm. In particular, this invention relates to methods for developing novel, highly chemoprotectant broccoli and cauliflower germplasm. The invention relates to the development of germplasm which contains significant quantities of chemoprotective compounds that modulate mammalian enzymes which are involved in metabolism of carcinogens. These compounds induce the activity of Phase 2 enzymes, without inducing biologically significant activities of those Phase 1 enzymes that activate carcinogens. More specifically, the novel germplasm contains an enhanced alkyl/indole glucosinolate ratio.
This invention further provides a method of germinating cruciferous seeds and sprouts under conditions which increase antibiotic activity against a range of human pathogens, and which increase the chemoprotectant activity of the sprouts.
2. Description of the Related Art
It is widely recognized that diet plays a large role in controlling the risk of developing cancers and that increased consumption of fruits and vegetables reduces cancer incidence in humans. It is now believed that a major mechanism of protection depends on the presence of chemical components in plants that, when delivered to mammalian cells, elevate levels of Phase 2 enzymes that detoxify carcinogens.
Phase 2 enzymes are effective by detoxifying electrophilic forms of carcinogens which would otherwise damage DNA. Compounds which elevate the level of Phase 2 enzymes are termed “selective inducers.” Selective inducers of Phase 2 enzymes are designated monofunctional inducers. This means that they induce only Phase 2 enzymes, without significantly inducing Phase 1 enzyme activities. In contrast, compounds which induce both Phase 2 and Phase 1 enzymes are designated bifunctional inducers. (see Prochaska & Talalay (1988)
Cancer Res.
48: 4776-4782). The monofunctional inducers are nearly all electrophiles and belong to at least 9 distinct chemical classes. (see Prestera et al. (1993)
Proc. Natl. Acad. Sci. USA
90: 2963-2969; Khachick et al. (1999) in ANTIOXIDANT FOOD SUPPLEMENTS IN HUMAN HEALTH, Packer, L. et al. (eds) pp 203-229, Academic Press, San Diego). The only apparent common property, shared by almost all of these inducers is their ability to react with thiol groups.
Monofunctional inducers are thus chemoprotective agents which reduce the susceptibility of mammals to the toxic and neoplastic effects of carcinogens. Chemoprotectors can be of plant origin or synthetic compounds. Synthetic analogs of naturally occurring inducers have also been generated and shown to block chemical carcinogenesis in animals. (see Posner et al. (1994)
J. Med. Chem.
37: 170-176; Zhang et al. (1994)
Proc. Natl. Acad. Sci. USA
91: 3147-3150; Zhang et al. (1994)
Cancer Res
. (Suppl) 54: 1976s-1981s).
Highly efficient methods have been developed for measuring the potency of plant extracts to increase or induce the activities of Phase 2 enzymes (Prochaska & Santamaria (1988)
Anal. Biochem.
169: 328-336 and Prochaska et al. (1992)
Proc. Natl. Acad. Sci. USA
89: 2394-2398). In addition, these methods have been employed for isolating the compounds responsible for the inducer activities in plants and for evaluating the anticarcinogenic activities of these compounds and their synthetic analogs (Zhang et al. (1992)
Proc. Natl. Acad. Sci. USA
89: 2399-2403 and Posner et al. (1994)
J. Med. Chem.
17: 170-176).
These methods have identified crucifer plants as a possible source of inducer activity. However, the levels of inducer activity among individual plants are highly variable, depending on the variety and on the growth and harvesting conditions.
It is now known that most of the inducer activity of crucifer plants is due to the presence and amounts of isothiocyanates and their biogenic precursors, glucosinolates. Glucosinolates are converted to isothiocyanates by the enzyme myrosinase, which is a thioglucoside glucohydrolase. Normally, myrosinase and glucosinolates are separated in the cell. If the cell is damaged, resulting in disruption of cellular compartmentalization, myrosinase comes into contact with glucosinolates, and converts them to isothiocyanates.
Although glucosinolates are not themselves inducers of mammalian Phase 2 enzymes, their conversion products, by virtue of myrosinase activity, are. Thus, it is the isothiocyanate products which are potent monofunctional inducers of Phase 2 enzymes in the murine hepatoma cell bioassay of QR activity.
However, not all glucosinolates produce isothiocyanates which are inducers of Phase 2 enzymes. Certain glucosinolates (e.g. alkylthioalkyl glucosinolates) produce isothiocyanates that are potent chemoprotective agents. Other glucosinolates (e.g. indole glucosinolates), produce compounds such as indole-3-carbinol and indole-3-acetonitrile, that are problematic for several reasons. First, such compounds are bifunctional inducers; that is, they induce both Phase 2 and Phase 1 enzymes. Phase 1 enzymes can activate xenobiotics thereby creating carcinogens. (Prochaska & Talalay (1988)
Cancer Res.
48: 4776-4782). Second, the indole glucosinolates are only weak inducers of Phase 2 enzymes (Fahey J W et al. (1998) Chapter 2 in FUNCTIONAL FOODS FOR DISEASE PREVENTION I. Shibamoto T. et al. (eds), pp 16-22, ACS Symposium Series 701, Am Chem Soc, Washington D.C.). Third, these compounds themselves function as tumor promoters (Kim D J et al. (1997)
Carcinogenesis
18(2):377-381). Finally, these compounds can form condensation products under the acid conditions encountered in the stomach, which are potent carcinogens very similar to dioxin (TCDD) (Bjeldanes L F et al. (1991)
Proc Nat Acad Sci USA
88:9543-9547).
Thus, the amounts of inducer activity depends upon both the quality and quantity of glucosinolates present in crucifer plants. Market stage broccoli and cauliflower, for example, contain among the highest levels of the alkylthioalkyl glucosinolates, 4-methylsulfinylbutyl and 3-methylsulfinylpropyl glucosinolate, thus far identified in vegetables. They also contain, however, similar levels of the indole glucosinolates, glucobrassicin (indolyl-3-methyl glucosinolate), neoglucobrassicin, and 4-hydroxyglucobrassicin. Broccoli and cauliflower germinated seeds, sprouts, and young plantlets, on the other hand, contain higher concentrations of glucosinolates than do the market stage vegetable. The amount of glucosinolates present in the sprouts may depend to some extent upon the leakage of glucosinolates from the seeds upon imbibition and germination.
The processes of seed imbibition and germination, as well as priming, osmoconditioning, matri-conditioning and the like, though primarily associated with a net influx of water to the seed and seedling, also typically involve the leaking or leaching of chemicals from the germinating seed. The amount of chemicals leaking from the seed can be regulated by the milieu in which the seed is placed, although some leakage is inevitable. Furthermore, the amount of leakage may also be related to the quality of the seed lot, and to the type of seed.
The leachates of cruciferous seeds can exhibit potent antibiotic activity. This activity is effective not only against a range of human pathogens, but also against other microbes which commonly thrive or co-exist in commercial green sprout (such as bean sprout or green leafy sprout) production systems, which thus effectively contaminate these systems. The antibiotic activity of germinating crucifer seeds, in both the seeds and seedlings and the leachate resulting therefrom, is related to the glucosinolate content of the seed. While the leachates of alfalfa seed, the primary raw material of the green sprouts industry, actually stimulate the growth of
Escherichia coli
, leachates of cruciferous seeds (e.g., broccoli, cauliflower, kale, daikon, cabbage, arugula) contain glucosinolates and their isothiocyanate congeners (e.g., glucoraphanin and sulforaphane), which inhibit the gro
Ibrahim Medina A.
John Hopkins School of Medicine
McElwain Elizabeth F.
LandOfFree
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