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Alkaloid that inhibits biosynthesis of mycotoxins and method for screening for mycotoxin inhibitors

a technology of mycotoxin and inhibitor, applied in the field of alkaloid compound, can solve the problems of crop and food commodity contamination by mycotoxin producing fungi, limited to a slight reduction in crop yield, and achieve the effect of inhibiting biosynthesis

Inactive Publication Date: 2005-06-02
BOARD OF TRUSTEES OPERATING MICHIGAN STATE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

[0012] 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 alkenyl piperidine amide, which can be isolated from Piper nigrum. The alkenyl 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 ...

Problems solved by technology

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.
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.
Also, products from animals that have been fed aflatoxin contaminated feed may also become contaminated.
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.
In areas of the world where regulations do not exist, aflatoxin is a serious health problem (CAST, 1989).
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.
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.

Method used

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  • Alkaloid that inhibits biosynthesis of mycotoxins and method for screening for mycotoxin inhibitors
  • Alkaloid that inhibits biosynthesis of mycotoxins and method for screening for mycotoxin inhibitors
  • Alkaloid that inhibits biosynthesis of mycotoxins and method for screening for mycotoxin inhibitors

Examples

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Effect test

example 1

[0069] The method for identifying compounds that inhibit mycotoxin biosynthesis is illustrated using ground black pepper (Piper nigrum) extracts and assaying for compounds that inhibit aflatoxin biosynthesis.

[0070] Ground black pepper (500 mg) was extracted in 2 L cyclohexanol overnight under a fume hood with constant stirring. After 24 hours, the mixture was filtered through Whatman paper and the extracted pepper filtrate was removed and in a similar manner to the above, extracted with chloroform followed by absolute ethanol. The filtrates from each extraction were concentrated by rotary evaporation and analyzed by thin layer chromatography (TLC).

[0071] The extracts were separated on TLC plates using a solvent system consisting of chloroform:acetone:toluene (25:40:35 v:v:v). Afterwards, the TLC plates were dried in a fume hood to evaporate any solvents left on the plates, and then placed silica-side down on a UV transilluminator and UV absorbent spots were traced on an acetate sh...

example 2

[0075] The following is a procedure for isolating Cp2 from ground pepper extracts.

[0076] Black ground pepper was suspended in cyclohexane (1:2 w:v) with constant stirring overnight. The mixture was filtered and then reextracted with cyclohexane for an additional four hours. The filtrates were combined and concentrated by rotary evaporation under vacuum at 40° C. After rotary evaporation, the cyclohexane extract was observed to have two phases. These phases were separated yielding a phase readily soluble in cyclohexane and a less dense phase readily soluble in ethanol. Both phases were tested to determine which phase contained the bioactive compound indicated in Example 1. An amount consisting of 10 μl of both phases were loaded onto separate TLC plates and the plates were developed using a solvent system consisting of chloroform:toluene:acetone (25-40:35 v:v:v). After development, the plates were dried overnight. Then the GUS bioassay was performed as shown in Example 1. The bioact...

example 3

[0079] Cp2 has a demonstrable effect on aflatoxin biosynthesis and nor-1 transcription. This example used Aspergillus parasiticus SU-1, a wild-type aflatoxin producing isolate, and consisted of analyzing aflatoxin biosynthesis by TLC and aflatoxin mRNA transcription by Northern analysis.

[0080] A nutritional shift protocol (Skory et al., Appl. Environ. Microbiol. 56: 3315-3320 (1990)) was used with the modifications below to determine the effect of Cp2 on expression of the nor-1 gene and aflatoxin biosynthesis. Six cultures of fungi were incubated in PMS (peptone mineral salts), a medium that does not induce biosynthesis of aflatoxin. After 48 hours, the mycelium from each culture were separately transferred to GMS (glucose mineral salts), a medium that induces biosynthesis of aflatoxin. At the same time Cp2 was added to five of the six cultures to provide cultures having a final concentration of 2.6 μg / ml, 26 μg / ml, 39 μg / ml, 52 μg / ml, and 78 μg / ml, respectively. Thirty-six hours l...

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Abstract

The present invention provides an alkaloid compound that is an inhibitor of mycotoxin biosynthesis. The alkaloid is an alkenyl piperidine amide wherein the alkenyl is a C18 alkenyl with one or more double bonds. The alkaloid inhibits transcription of the fungus genes nor-1, tri5, and ipnA. The present invention further provides a method for identifying compounds, which inhibit biosynthesis of aflatoxin in Aspergillus spp. and deoxynivalenol in Gibberella spp. without inhibiting growth of the fungus in vitro.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional application Ser. No. 60 / 156,381, which was filed Sep. 28, 1999.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] None. BACKGROUND OF THE INVENTION [0003] (1) Field of the Invention [0004] 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 alkenyl piperidine amide wherein the alkenyl is a C18 alkenyl 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 ...

Claims

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Application Information

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IPC IPC(8): A61K31/4453C07D295/185C12N15/80
CPCA61K31/4453C12N15/80C07D295/185
Inventor TRAIL, FRANCESHAMMERSCHMIDT, RAYMONDLINZ, JOHNXU, HAIXINVELASQUEZ, LUISANNIS, SEANNA
Owner BOARD OF TRUSTEES OPERATING MICHIGAN STATE UNIV
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