Insecticidal extract from legume plants and method of preparing the same

Abstract: The invention may be summarized as follows. The present invention provides insecticides comprised of naturally-occurring compounds, such as, for example, PA1b-related peptides and terpenoid saponins. The present invention also provides for identification and characterization of a synergistic insecticidal effect between peptides and saponins extracted from plants. Furthermore, simplified extraction procedures are provided that avoid the use of a chloroform defatting step or a column chromatography step. (end of abstract)

Agent: Ronald I. Eisenstein Nixon Peabody LLP - Boston, MA, US
Inventors: Wesley G. Taylor, Paul G. Fields, Daniel H. Sutherland
USPTO Applicaton #: #20060216367 - Class: 424757000 (USPTO)
Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Plant Material Or Plant Extract Of Undetermined Constitution As Active Ingredient (e.g., Herbal Remedy, Herbal Extract, Powder, Oil, Etc.), Containing Or Obtained From Leguminosae (e.g., Legumes Such As Soybean, Kidney Bean, Pea, Lentil, Licorice, Etc.)

Insecticidal extract from legume plants and method of preparing the same description/claims

The Patent Description & Claims data below is from USPTO Patent Application 20060216367, Insecticidal extract from legume plants and method of preparing the same.

Brief Patent Description - Full Patent Description - Patent Application Claims

FIELD OF INVENTION

[0001] The present invention relates to an insecticidal composition that comprises compounds that occur naturally in legumes. The present invention further relates to a method of isolating the compounds from their natural source or setting. More particularly, the present invention relates to a legume extract comprising an insecticidal peptide, saponin, or a combination thereof.

BACKGROUND OF THE INVENTION

[0002] Insects have been a problem for crops and stored food products since the dawn of agriculture. Since the 1950's synthetic insecticides have been the method of choice to control insect infestations in crop fields, stored grain, warehouses and food processing facilities. However, there are several reasons that alternatives to synthetic insecticides are required. Insects have developed resistance to several synthetic insecticides. Many consumers want no insecticide residues on their food. The application of synthetic insecticides has become more onerous. For example, the fumigant aluminum phosphide in the U.S.A. has a new label which requires more sampling of product and greater notification of the public.

[0003] Some insecticides are being phased out because they are harmful to the environment. Methyl bromide, widely used as a fumigant, is an ozone-depleting substance that will be banned after 2005 in most countries. Phosphine, the most widely used grain fumigant, is also being restricted in its use. New protectants are needed that are safe for humans and the environment.

[0004] To control insects there are a number alternatives to synthetic insecticides; physical, biological, or botanical. Botanical insecticides have a long history of use in agricultural product protection. Pyrethrums, usually synergized with piperonyl butoxide, are used extensively commercially to control stored-product insects. Neem has been used for centuries in India in stored grain. However, other than spices, food plants have not been widely used to control insects.

[0005] It has long been known that legume seeds contain a wide range of chemicals with toxic or deterrent effects against insect pests. For example, pea seeds (Pisum sativum L.) and their extracts are toxic to many insects, especially Sitophilus spp. (Bodnaryk et al, U.S. Pat. No. 5,955,082, issued Sep. 21, 1999; Delobel et al, PCT publication WO99/58695 published Nov. 18, 1999). A multigenic family of small linear, 37-amino acid peptides, was isolated from peas (Higgins et al., 1986) and variants of this cysteine-rich pea albumin (PA1b) were found to be toxic to stored-product insects and aphids (Delobel et al., WO99/58695). Delobel et al. isolated from peas and sequenced an insecticidal variant of PA1b with a mass of 3741 Da. Gressent et al., 2003) have studied the binding of this 3741 variant to microsomal fractions of wheat-feeding and pea feeding weevils. Other legume seeds have been studied genetically for the presence of insect-toxic seed albumins (Louis et al., 2004).

[0006] Bodnaryk et al. (U.S. Pat. No. 5,955,082) developed an extraction procedure comprising treatment steps using chloroform and hot 80% methanol to obtain crude insecticidal pea extracts. The aqueous methanol extracts from defatted, protein-rich flour were partially purified with reversed phase C8 silica. Activity was found in fractions obtained by elution of the C8 silica column with methanol. These C8 extracts displayed insecticidal and antifeedant activity against rice weevil [Sitophilus oryzae (L.)] and other stored-product insects but the active ingredients of C8 extracts were not identified. Furthermore, the use and disposal of chloroform is expensive, and insecticides containing residual chloroform may be of limited use, particulary with regard to food-grade crops or products. The use of column chromatography is also expensive and may limit industrial scale-up.

[0007] Sitophilus spp., S. granarius (L.), S. oryzae (L.) and S. zeamais Motschulsky, are examples of serious, cosmopolitan pests of stored cereals. Previous work has shown that mixing pea seed with wheat seed at a ratio of 1:1 reduced S. oryzae populations by 70%. However, mixing equal weights of whole peas and wheat is not a practical means for controlling pests.

[0008] None of the previous approaches has been adapted for industrial use in controlling insects.

[0009] Thus, there is a need for alternative natural extracts and methods of preparing the same, for controlling insect spoilage of agricultural products.

SUMMARY OF THE INVENTION

[0010] The present invention relates to an insecticidal composition that comprises compounds that occur naturally in legumes. The present invention further relates to a method of isolating the compounds from their natural source or setting. More particularly, the present invention relates to a legume extract comprising an insecticidal peptide, saponin, or a combination thereof.

[0011] It is an object of the invention to provide an insecticidal composition and a process for preparing the same.

[0012] According to the present invention there is provided an insecticidal, alcohol-soluble extract of a non-defatted legume seed material, the extract comprising an insecticidal peptide to saponin ratio ranging from 10:1 to 1:10. As an example, the extract may comprise at least one PA1b-related peptide comprising a sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:9. In another example, the saponin may be a triterpenoid saponin and may further, be selected from the group consisting of: soyasaponin I, soyasaponin II, soyasaponin III, soyasaponin VI, dehydrosoyasaponin I, echinocystic acid 3-glucoside, glycyrrhizic acid, hederacoside C, beta-escin, and alpha-hederin.

[0013] According to the present invention there is provided a method of preparing an insecticidal alcohol soluble legume extract comprising: extracting a non-defatted legume seed material with a lower molecular weight alcohol, for example C(1-4) alcohol. Methods of the present invention may further comprise precipitating the alcohol soluble extract with an organic acid.

[0014] In an example of the method of the present invention the organic acid may be selected from the group consisting of: benzoic, acetic, trichloroacetic, trifluoroacetic, sorbic, citric, formic, and propionic acids.

[0015] According to the present invention there is provided an insecticidal composition comprising an isolated PA1b-related peptide and an isolated saponin, the peptide to saponin ratio ranging from 10:1 to 1:10. In an example of the insecticidal composition of the present invention the PA1b-related peptide comprises a sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:9. In another example, the saponin is a triterpenoid saponin, and may optionally be selected from the group consisting of: soyasaponin I, soyasaponin II, soyasaponin III, soyasaponin VI, dehydrosoyasaponin I, echinocystic acid 3-glucoside, glycyrrhizic acid, hederacoside C, beta-escin, and alpha-hederin.

[0016] According to the present invention there is provided a method of controlling insects comprising exposing the insects to an effective amount of an extract or composition of the present invention. In an example, the composition or extract may comprise dehydrosoyasaponin I.

[0017] According to the present invention there is provided an insecticidal alcohol soluble plant extract comprising a PA1b-related peptide. By "PA1b-related peptide" is meant any peptide that comprises a sequence that is at least 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78% or 80% identical to any one of the sequences represented in FIGS. 18-21 and 23 (SEQ ID Nos:1-60), and exhibits insecticidal activity alone or in combination with a saponin.

[0018] In an example of the present invention, a PA1B-related peptide comprises an amino acid sequence of Cys-(Xaa)n-(Xaa)n-Cys-(Xaa)n-Pro-(Xaa)n-(Xaa)n-Pro-Cys-(Xaa)n-(Xaa)n-Cys-- (Xaa)n-Cys-(Xaa)n-Pro-(Xaa)n-(Xaa)n-(Xaa)n-Cys (SEQ ID NO:63), where each Xaa is independently selected from any amino acid and each n independently equals 1, 2, 3, 4, or 5. In another example of the present invention, a PA1B-related peptide comprises an amino acid sequence of Cys-(Xaa)n-(Xaa)n-Cys-(Xaa)n-Pro-(Xaa)n-(Xaa)n-Pro-Pro-Cys-(Xaa)n-(Xaa)n-- Cys-(Xaa)n-Cys-(Xaa)n-Pro-(Xaa)n-(Xaa)n-(Xaa)n-Cys-(Xaa)n-Pro (SEQ ID NO:64), where each Xaa is independently selected from any amino acid and each n independently equals 1, 2, 3, 4, or 5. In again another example of the present invention, a PA1B-related peptide comprises an amino acid sequence of Cys-Xaa-Xaa-(Xaa)n-Cys-Xaa-Pro-Xaa-(Xaa)n-Xaa-Xaa-Pro-Cys-Xaa-Xaa-Xaa-(Xa- a)n-Cys-Xaa-Cys-Xaa-Pro-Xaa-(Xaa)n-Xaa-Xaa-Xaa-Xaa-(Xaa)n-Cys (SEQ ID NO:65), where each Xaa is independently selected from any amino acid and each n independently equals 0, 1, 2, 3, or 4. In still another example of the present invention, a PA1B-related peptide comprises an amino acid sequence of Cys-Xaa-Xaa-(Xaa)n-Cys-Xaa-Pro-Xaa-(Xaa)n-Xaa-Pro-Pro-Cys-Xaa-Xaa-Xaa-(Xa- a)n-Cys-Xaa-Cys Xaa-Pro-Xaa-(Xaa)n-Xaa-Xaa-Xaa-Xaa-(Xaa)n-Cys-(Xaa)n-Xaa-Pro (SEQ ID NO:66), where each Xaa is independently selected from any amino acid and each n independently equals 0, 1, 2, 3, or 4. In yet another example of the present invention, a PA1B-related peptide comprises an amino acid sequence of Cys-Xaa-Xaa-(Xaa)n-Cys-Ser-Pro-Phe-(Xaa)n-Xaa-Pro-Pro-Cys-Xaa-Xaa-Xaa-(Xa- a)n-Cys-Xaa-Cys-Xaa-Pro-Xaa-(Xaa)n-Leu-Xaa-(Xaa)n-Gly-Xaa-Cys-(Xaa)n-Xaa-P- ro (SEQ ID NO:67), where each Xaa is independently selected from any amino acid, and each n independently equals 0, 1, 2, 3, or 4. It will be understood that each n independently indicates multiples of an Xaa residue. For example, where n equals zero an Xaa residue is removed, where n equals one a single residue is represented, where n equals two a double Xaa sequence is represented with each Xaa being independently selected from any amino acid, where n equals three a triple Xaa sequence is represented with each Xaa being independently selected from any amino acid, etc. In a further example, a PA1B-related peptide comprises an amino acid sequence having the general formula X1-C--X2-C--X3-C--X4-C--X5-C--X6-C--X7 as previously defined in WO99/58695 (Delobel et al.).

[0019] Alcohols for use with the present invention, may be empirically determined by persons skilled in the art. As an example lower molecular weight alcohols may be used. In further examples the alcohol may be selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, tert-butanol, 1-isobutanol, and 2-buten-1-ol. Use of ethanol may be advantageous in regards to insecticidal protection of food-grade crops and products. In still another example, the alcohol is an about 50-98% aqueous solution of the alcohol, and may optionally be an about 60-95% aqueous solution of the alcohol. The temperature of the alcohol is not critical to the present invention.

[0020] There are potential commercial applications of this invention for control of insects attacking stored grains in home, farm or elevator locations and on ships. Worldwide, there is a great deal of interest in developing botanical or natural insect control products, especially from food grade materials. The combination of field peas as a raw material source and aqueous alcohol as extraction solvent should be attractive to industry.

[0021] The methods, extracts, or compositions of the present invention might find considerable utility in, or when exporting grain to, warmer areas of the world inhabited by major storage pests, for example the rice weevil.

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