Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2000-09-26
2004-11-30
Desai, Rita (Department: 1626)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C546S245000
Reexamination Certificate
active
06825216
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
None.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to an alkaloid compound that inhibits biosynthesis of particular products of secondary metabolism. In particular, the present invention relates to an alkenylene piperidine amide wherein the alkenylene alkenylene with one or more double bonds, which can be isolated from
Piper nigrum
, that inhibits transcription of fungus genes nor-1, tri5, ver-1, verA, fas-1a, omt-1, alfR and ipnA. The present invention further relates to a method for identifying compounds that inhibit the biosynthesis of mycotoxins in fungi. In particular, a method for identifying compounds that inhibit biosynthesis of aflatoxin in
Aspergillus
spp. and deoxynivalenol in
Gibberella
spp.
(2) Description of Related Art
Mycotoxins are a group of structurally heterogeneous secondary metabolites produced by a diverse group of fungal plants pathogens. Infestation of crops and food commodities by mycotoxin producing fungi is a serious problem in view of the immunosuppressive, carcinogenic, cytotoxic, and teratogenic effects of the compounds in humans and animals. One of the most economically important mycotoxins worldwide is aflatoxin, a polyketide produced by several
Aspergillus
spp. Aflatoxin is the best studied of the mycotoxins and much of the molecular biology of the biosynthetic pathway has been determined in
Aspergillus flavus, Aspergillus parasiticus
, and
Aspergillus nidulans. Aspergillus flavus
produces aflatoxin B1 and aflatoxin B2 whereas
Aspergillus parasiticus
produces in addition aflatoxin G1 and G2.
Aspergillus nidulans
, which is not considered to be an agricultural threat, has been used as a model genetic system for studies of aflatoxin biosynthesis because it produces sterigmatocystin, an aflatoxin precursor. The genes for aflatoxin biosynthesis are clustered in all three species. The molecular biology of aflatoxin biosynthesis is reviewed by Trail et al., in
Microbiol.
141: 755-765 (1995).
Aspergillus flavus
and
Aspergillus parasiticus
are weak pathogens of corn, cotton, peanut, and nut crops: their effect is limited to a slight reduction in crop yield. However, the significant consequence of crops infected with either of these fungi is contamination by aflatoxin, which is produced under certain conditions during the infection. Traditional control strategies such as breeding crops for resistance to the fungi or chemical treatments of crops to prevent infection by the fungi have not been effective.
Aflatoxin is a secondary metabolite that appears to be the most potent naturally occurring carcinogen known (Council for Agricultural Science and technology (CAST), 1989). It is suspected of being responsible for the high incidence of human liver cancer in many areas of the world (Eaton and Gallagher,
Ann. Rev. Pharmacol. Toxicol.
34: 135-139 (1994)). Aflatoxin is introduced into the food chain by preharvest and postharvest contamination of foods and feeds. Also, products from animals that have been fed aflatoxin contaminated feed may also become contaminated. Currently, the U.S. Food and Drug Administration limits the allowable amount of aflatoxin in food to 20 ppb, with slightly higher levels allowed in feeds. Because the level of aflatoxin in products destined for human consumption is strictly regulated in the U.S., aflatoxin contamination is primarily of economic importance. However, even though aflatoxin levels in foods is limited to 20 ppb, the effect of chronic exposure to low levels of aflatoxin on human health is unknown. Thus, some European countries require the presence of aflatoxin in foods intended for human consumption to be 0 ppb. In areas of the world where regulations do not exist, aflatoxin is a serious health problem (CAST, 1989).
Approaches to control of aflatoxin have been broadly grouped into preharvest and postharvest strategies. Proper grain storage can greatly reduce contamination postharvest, and some decontamination methods, while costly, are used, e.g., ammoniation. However, most research efforts at control of aflatoxin has been directed at the preharvest elimination of infection and contamination, since the ability to control preharvest contamination would reduce the need for postharvest elimination. Preharvest methods have included agricultural practices such as irrigation strategies designed to eliminate stress to crops associated with drought, which appears to increase production of aflatoxin by the fungus. Other methods include using regionally adapted varieties of crop plants. However, these methods have been expensive to implement and have not been completely effective. Chemical control methods have also been ineffective at controlling infection by these fungi.
The development of host plants that are resistant to
Aspergillus
infection and aflatoxin contamination has not been as successful as have programs for breeding resistance to other pathogens. In general, the resistant varieties that have been made are unstable from growing season to growing season and from region to region. Also, screening plants for resistance to colonization by
Aspergillus
spp. and aflatoxin contamination has been difficult. In corn, and frequently in cotton, inoculation methods have been difficult, often requiring wounding the plant to introduce the fungus, which may overwhelm the plants natural resistance reactions making it difficult to evaluate the plants resistance mechanisms (
Cotty, Plant Dis.
73: 489-492 (1989)).
Methods have been developed for inhibiting mycotoxin production in crops. For example, U.S. Pat. No. 5,942,661 to Keller discloses a method of inhibiting mycotoxin production by introducing into the plant a gene encoding a lipoxygenase pathway enzyme of the mycotoxin. The method may produce transgenic plants that are substantially resistant to mycotoxin contamination. Mycotoxin resistance is further increased by introducing into the plant antisense genes for the 9-hyperoxide fatty acid producing lipoxygenases. However, reducing aflatoxin contamination by making transgenic plants resistant to aflatoxin production is expensive and time consuming, and since transformation efficiencies varies from plant species to plant species, the method may not be successful for all plant species. Furthermore, the long-term effect of introducing transgenic plants into the environment is unknown.
Since traditional methods for controlling fungal infection and/or production of aflatoxin by breeding, chemicals, or transgenic plants have not been completely effective, there is a need for an inexpensive and effective method for either controlling infection of crops by fungi such as
Aspergillus
spp. or
Gibberella
spp., or controlling the biosynthesis and accumulation of mycotoxins such as aflatoxin or deoxynivalenol in plants infected with fungi such as
Aspergillus
spp or
Gibberella
spp., respectively. There is also a need for a rapid and inexpensive method for identification of chemicals or compounds in natural extracts that inhibit production of mycotoxins such as aflatoxin and deoxynivalenol.
SUMMARY OF THE INVENTION
The present invention provides a substantially pure alkaloid compound that inhibits the biosynthesis of particular products of secondary metabolism. In particular, the present invention provides an alkenylene piperidine amide, which can be isolated from
Piper nigrum
. The alkenylene piperidine amide inhibits transcription from the nor-1 promoter, the tri5 promoter, ver-1 promoter, the verA promoter, the omt-1 promoter, the fas-1a promoter, alfR promoter, the ipna promoter, and mutant thereof. In a preferred embodiment the alkenylene piperidine amide inhibits at least transcription of the nor-1 promoter of
Aspergillus parasiticus
, the tri5 promoter of
Gibberella pulicaris
or the ver-1 promoter of
Aspergillus nidulans
without killing the fungus in vitro. In a preferred embodiment, the alkenylene is a C18 alkenylene with one or more double bonds. Preferably, the C
18
alkenylene has two to four do
Hammerschmidt Raymond
Linz John E.
Trail Frances
Board of Trustees of Michigan State University
Coppins Janet L
Desai Rita
McLeod Ian C.
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