Description

ANTIBIOTIC AND INSECT COMPOSITION INCLUDING ANTIBIOTICS AS EFFECTIVE COMPONENTS EXTRACTED FROM MUSKRAT EXCREMENT

Technical Field

[1] The present invention relates to a method of preparing antibiotics extracted from muskrat excrement and an antibiotic and insecticidal composition comprising the same. More specifically, the present invention relates to a method of preparing antibiotics against food poisoning bacteria, plant pathogens and pests such as termite extracted from muskrat excrement and an anti-plant pathogenic, anti-food poisoning and insecticidal composition comprising the antibiotics as an active ingredient. Background Art

[2] The muskrat (Ondatra zibethicus) is a mammalian which belongs to order Rodentia and family Muridae, is oval-shaped, has a body length of about 35cm and a tail length of 25cm and weighs about lkg. Muskrats are common near marsh and lake which is full of weed. They act from spring to late autumn and have a limited action in winter. They are herbivorous, but they also eat fish or aquatic animals. They practice monogamy and have a fine breeding. Female muskrats breed from the 4 month to the 9' month and have 2 or 3 litters a year.

[3] Particularly, male muskrats have two sachets and produce 5 to 8g of muskrat must per year.

[4] Muskrat musk can be used as a raw material for perfumes, cosmetics and medicines, and thus muskrat has a high potency to be developed as an animal source for livestock industry. Since most of musk which is distributed domestically is an imitation of musk, a development of muskrat must as a replacement of musk is much needed. Pharmaceutical companies and herbal medicine industries completely depend on import, because they do not secure muskrats for obtaining muskrat musk.

[5] The state of research and development and its application in the outside are summarized in Table 1 :

[6] Table 1

[7] [8] The studies of muskrat were focused on muskrat musk. Up to now, any other utilities of muskrat except for muskrat must have not been studied. Disclosure of Invention Technical Problem

[9] The present inventors have investigated a method to utilize muskrat excrement and, as a result, have found that antibiotics extracted from the muskrat excrement by proper organic solvents can be used as an active ingredient for an anti-plant pathogenic composition, an anti-food poisoning composition or an insecticidal composition.

[10] Therefore, it is an object of the present invention to provide a method of preparing antibiotics extracted from muskrat excrement.

[H] It is another object of the present invention to provide an anti-plant pathogenic composition which comprises the antibiotics as an active ingredient. [12] It is still another object of the present invention to provide an antibiotic composition which comprises the antibiotics as an active ingredient.

[13] It is yet another object of the present invention to provide an anti-food poisoning composition which comprises the antibiotics as an active ingredient. [14] It is a further object of the present invention to provide an insecticidal composition which comprises the antibiotics as an active ingredient. Technical Solution

[15] In a first aspect, the present invention provides a method of preparing the 1^st antibiotic extract which comprises the steps of drying muskrat excrement in the shade, treating the dried excrement with ethyl acetate for a shaking extraction, filtrating the extract and concentrating the filtrate under vacuum.

[16] In a second aspect, the present invention provides a method of preparing the 2nd

antibiotic extract which comprises the steps of treating the residue left after the filtration in the 1^st embodiment with ethanol for a shaking extraction, filtrating the extract and concentrating the filtrate under vacuum.

[17] In a third aspect, the present invention provides a method of preparing the 3rd antibiotic extract which comprises the steps of treating the residue left after the filtration in the 2nd embodiment with chloroform for a shaking extracting, filtrating the extract and concentrating the filtrate under vacuum.

[18] In a fourth aspect, the present invention provides an antibiotic composition which comprises the 1^st antibiotic extract prepared by the method of the 1^st embodiment as an active ingredient.

[19] In a fifth aspect, the present invention provides an anti-plant pathogenic composition which comprises one or more extracts selected from the 1^st and 2° antibiotic extracts prepared by the methods of the 1^st and 2° embodiments as an active ingredient.

[20] In a sixth aspect, the present invention provides insecticidal composition which comprises one or more extracts selected from the 1^st, 2° and 3^r antibiotic extracts prepared by the methods of the 1^st, 2° and 3^r embodiments as an active ingredient.

[21] Hereinafter, the present invention is further described in detail with the reference of

FIG. 1.

[22] First, muskrat excrement was dried under the shade. The drying is carried out, but not limited to, at ambient temperature, which is preferred in the aspect of economics. Further, the dried excrement is preferably triturated to improve the effect of extraction.

[23] In order to extract most of antibiotics contained in the excrement, the excrement is treated with 5 to 10 parts by weight of organic solvent selected from ethyl acetate, chloroform, dichlorome thane, acetone and methanol based on 100 parts by weight of the dried excrement for a shaking extraction. After filtrating the extract, the filtrate is concentrated under vacuum to obtain the 1^st antibiotic extract. It is preferred to use ethyl acetate as an organic solvent for extraction.

[24] The residue left after the above filtration is treated with an organic solvent selected from ethanol, dichloromethane, acetone and methanol for a shaking extraction, the extract is filtrated and the filtrate is concentrated under vacuum to obtain the 2° antibiotic extract. It is preferred to use ethanol as an organic solvent for extraction. The amount of organic solvent used for extraction is preferably 5 to 10 parts by weight based on 100 parts by weight of the residue.

[25] The residue left after the above filtration is treated with an organic solvent selected from chloroform, dichloromethane, acetone and methanol for a shaking extraction, the extract is filtrated and the filtrate is concentrated under vacuum to obtain the 3rd antibiotic extract. It is preferred to use chloroform as an organic solvent for extraction.

The amount of organic solvent used for extraction is preferably 5 to 50 parts by weight based on 100 parts by weight of the residue.

[26] The shaking extractions for the preparation of the 1^st, 2° and 3^r extract are carried out, but not limited to, at 150 to 200rpm for 10 to 14 hours, which maximizes the extraction effect.

[27] The shaking extractions are repeated at least three times to collect the concentrate under vacuum.

[28] As shown in FIG. 2, the 1^st antibiotic extract has an antibiotic activity against

Bacillus subtilis, Salmonella typhimurium, Staphylococcus aureus, Vibrio parahemolyticus and Escherichia coli 0157:H7.

[29] Therefore, the present invention provides an antibiotic composition which comprises the 1^st antibiotic extract as an active ingredient. The composition can further comprise, but not limited to, glycerol ester, pyroligneous liquid, etc.

[30] Furthermore, as shown in FIG. 3, the 1^st and 2° antibiotic extracts have an anti- plant pathogenic activity against Rhizoctonia solani, Pythium ultimum, Sclerotinia sclerotiorum, Phytophthora capsici and Fusarium oxysporum.

[31] Therefore, the present invention provides an anti-plant pathogenic composition which comprises one or more extracts selected from the 1^st and 2° extracts as an active ingredient. In the aspect of effect, it is preferred to use the 1^st extract and the 2° extract in a weight ratio of 50 to 70:50 to 30. The composition can further comprise, but not limited to, glycerol ester, pyroligneous liquid, etc.

[32] As shown in FIG. 4, the 1^st, 2° and 3^r antibiotic extracts have an insecticidal activity against termite, acarid, mite, etc.

[33] Therefore, the present invention provides an insecticidal composition which comprised one or more extract selected from the 1^st, 2° and 3^r antibiotic extracts as an active ingredient. In the aspect of effect, it is preferred to use the 1^st and 2° extracts mixed in a weight ratio of 50 to 70:50 to 30, the 2° and 3^r extracts mixed in a weight ratio of 50 to 70:50 to 30, the 1^st and 3^r extracts mixed in a weight ratio of 50 to 70:50 to 30, and the 1^st, 2° and 3^r extracts mixed in a weight ratio of 25 to 35:40 to 55: 10 to 35. The composition can further comprise, but not limited to, natural insecticides derived from plants.

Advantageous Effects

[34] According to the present invention, the antibiotics extracted from muskrat excrement with ethyl acetate have a potent antibiotic effect against gram negative Salmonella among food poisoning bacteria and thus, they are effective in treating and preventing food poisoning. Further, they have a potent antibiotic effect against plant pathogens and, thus, provide a high utility as an agricultural fertilizer and a natural and

environmental-friendly agricultural material. Furthermore, they can replace chemical insecticides to control termites which vastly damage cultural assists. Brief Description of the Drawings

[35] FIG. 1 is a flow chart showing the process for obtaining the 1^st antibiotic extract

(ethyl acetate extract), the 2° antibiotic extract (ethanol extract) and the 3^r antibiotic extract (chloroform extract).

[36] FIG. 2 is a photograph showing the antibiotic activity of the organic extract

(EtOAc) obtained from muskrat excretion against B. sutillus (A) and S. typhimurium (B). Nos. 1 to 4 indicate plant extracts, No. 5 indicates the ethyl acetate extract obtained from muskrat musk, No. 6 indicates the ethanol extract obtained from muskrat musk, No. 7 indicates the 1^st antibiotic extract (ethyl acetate extract) and No. 8 indicates the 2° antibiotic extract (ethanol extract).

[37] FIG. 3 is a photograph showing the antibiotic activity of the organic extracts

(EtOAc, EtOH, CHCl ) obtained from muskrat excretion against R. solani (A), P. ultimum (B) and B. cinerea (C). No. 1 indicates the 1^st antibiotic extract (ethyl acetate extract), No. 2 indicates the 2° antibiotic extract (ethanol extract) and No. 3 indicates the 3^r antibiotic extract (chloroform extract).

[38] FIG. 4 is a photograph showing the insecticidal activity of the organic extracts

(EtOAc, EtOH, CHCl ) obtained from muskrat excretion against termite. (A) is a photograph immediately after the disk is treated with termite, (B) is a photograph 8 days after the disk is treated with termite and (C) is a photograph 28 days after the disk is treated with termite. The top disk is numbered as No. 1 and the remaining disks were clockwise numbered as Nos. 2 to 7. Nos. 1 and 5 indicate non-treated filter paper disk, No. 2 indicates the 1^st antibiotic extract (ethyl acetate extract) -treated filter paper disk, No. 3 is empty, No. 4 indicates the 2° antibiotic extract (ethanol extract)-treated filter paper disk, No. 6 indicates the 3^r antibiotic extract (chloroform extract)-treated filter paper disk and No. 7 indicates a center (where the start mites are placed). Best Mode for Carrying Out the Invention

[39] Material

[40] The muskrat excretion used in the present invention was obtained from the muskrat bred at Pusan University attached muskrat-run. Gram positive Bacillus subtillus ATCC 9372 and gram negative Samonella typhimurium were used and, as plant pathogens, Rhizoctonia solani, Pythium ultimum and Botrytis cinerea were used.

[41]

[42] Example 1 : Solvent extraction of muskrat excrement

[43] As shown in FIG. 1, the muskrat excretion obtained from the muskrat bred at Pusan

University attached muskrat-run was dried under the shade.

[44] 200ml of ethyl acetate was added to 2Og of the dried excrement and shaking extraction (150rpm, 12 hours) was repeated three times. The extracts were filtrated and the filtrates were concentrated under vacuum with a rotating concentrator to obtain the 1^st antibiotic extract (ethyl acetate extract).

[45] As for the residue left after the filtration of ethyl acetate extract, the above steps were carried out except for using ethanol in stead of ethyl acetate to thereby collect the 2° antibiotic extract (ethanol extract).

[46] As for the residue left after the filtration of ethanol extract, the above steps were carried out except for using chloroform in stead of ethanol to thereby collect the 3^r antibiotic extract (chloroform extract).

[47] For the comparison, the ethyl acetate extract and the ethanol extract obtained from the muskrat musk of male 24 month-aged muskrat bred at Pusan University attached muskrat-run were prepared according to the above method. Mode for the Invention

[48] Example 2: Antibiotic activity of the 1 ^"antibiotic extract against bacteria and food poisoning pathogens

[49] First, to determine the antibiotic activity of the 1^st antibiotic extract prepared in

Example 1, B. subtillus and S. typhimurium were used as test bacteria. One platinum loop of bacteria which were grown in a slant culture was inoculated into 5ml of nutrient broth medium and the bacteria were cultured through three passages at 3O⁰C for 18 to 24 hours each time.

[50] The plate medium to test the antibiotic effect was prepared as follows: after adding the sterilized basal medium for culturing bacteria (agar 1.5%) to petridishes in an amount of 15ml/dish and the medium was solidified. The medium (agar 0.75%) to form a medial layer was added to test tubes in an amount of 2.5ml/tube and sterilized. While keeping the tubes at 45⁰C in a water bath, 0.1ml of test specimens was aseptically added to the tubes. After mixing it, the mixture was evenly layered on the basal medium and the medium was solidified to prepare two-layered plate medium for the inoculation of bacteria.

[51] To determine the antibiotic ability to said bacteria and food poisoning pathogens, agar disk plate method was used. Each fractions were dissolved in ethyl acetate and ethanol in a concentration of 50mg/ml, the solutions were filtrated through 0.45D membrane (Millipore Inc., USA) to sterilize bacteria, and the filtrates were absorbed into sterilized filter paper disks (Toyo, 8mm, Japan) in an amount of 2OD (lmg)/paper.

[52] Next, the extraction solvents were evaporated from the papers and the papers were put on the plate medium and closely attached. After standing the dishes in a 4⁰C refrigerator for 1 hour and culturing the microorganisms in a 3O⁰C incubator for 24 to 48

hours, the diameter of clear zone around disks was measured.

[53] The result is shown in FIG. 2. FIG. 2(A) is a photograph to B. sutillus and 2(B) to S. typhimurium.

[54] In FIG. 2, Nos. 1 to 4 indicate plant extracts, No. 5 indicates the ethyl acetate extract obtained from muskrat musk, No. 6 indicates the ethanol extract obtained from muskrat musk, No. 7 indicates the 1^st antibiotic extract (ethyl acetate extract) and No. 8 indicates the 2° antibiotic extract (ethanol extract).

[55] As shown in FIG. 2, the 1^st antibiotic extract (ethyl acetate extract, No. 7) showed a remarkably superior antibiotic activity against B. sutillus and S. typhimurium compared with the 2° antibiotic extract (ethanol extract, No. 8), the ethyl acetate extract obtained from muskrat musk (No. 5) and the ethanol extract obtained from muskrat musk (No. 6).

[56]

[57] Example 3: Antibiotic activity of the 1 ^~and 2 ^~antibiotic extracts against plant pathogens

[58] Using the 1^st antibiotic extract (ethyl acetate extract), the 2° antibiotic extract

(ethanol extract) and the 3^r antibiotic extract (chloroform extract) prepared in Example 1, antibiotic test against plant pathogens of R. solani (A), P. ultimum (B) and B. cinerea were carried out.

[59] Each pathogens were inoculated into potato dextrose agar medium (PDA), each extracts was dissolved in extraction solvent in a concentration of 50mg/ml, the solution was filtrated through 0.45D membrane (Millipore Inc., USA) to sterilize bacteria, and the filtrates were absorbed into sterilized filter paper disks (Toyo, 8mm, Japan) in an amount of 2OD (lmg)/paper.

[60] Next, the extraction solvents were completely evaporated from the papers and the papers were put on the agar media which were inoculated with plant pathogens and closely attached. While culturing the plant pathogens in a 25⁰C incubator, their growth was observed.

[61] The result is shown in FIG. 3. FIG. 3(A) is a photograph to R. solani, 3(B) is a photograph to P. ultimum and 3(C) is a photograph to B. cinerea. In FIG. 3, No. 1 indicates the 1^st antibiotic extract (ethyl acetate extract), No. 2 indicates the 2° antibiotic extract (ethanol extract) and No. 3 indicates the 3^r antibiotic extract (chloroform extract).

[62] As shown in FIG. 3, the 1^st antibiotic extract (ethyl acetate extract) had the strongest antibiotic activity against P. ultimum, and also showed a superior antibiotic activity against R. solani, but did not have any activity against B. cinerea.

[63] The 2° antibiotic extract (ethanol extract) had the strongest antibiotic activity against R. solani, and also showed a superior antibiotic activity against P. ultimum, but

did not have any activity against B. cinerea.

[64] The 3^r antibiotic extract (chloroform extract) showed a weak antibiotic activity against all of plant pathogens tested.

[65]

[66] Example 4: Insecticidal activity of the 1 ^~. 2 ^~and 3 "^antibiotic extracts

[67] Using the 1^st antibiotic extract (ethyl acetate extract), the 2° antibiotic extract

(ethanol extract) and the 3^r antibiotic extract (chloroform extract) prepared in Example 1, insecticidal effect against termites which vastly damage wood structures were tested.

[68] Termites were collected from rotten wood material in a hill near Milyang. Since termites can easily digest cellulose, filter papers which was treated with the extract or not were supplied and the number of survival termites was counted.

[69] Termites were placed on the tray which was constructed to test the effect of killing and damaging insects with a fixed amount of sterilized soil. Termites could freely move without restriction to the treated and control groups. Each extracts was dissolved in the extraction solvent in a concentration of 50mg/ml, the solution was filtrated through 0.45D membrane (Millipore Inc., USA) to sterilize bacteria, and the filtrates were absorbed into sterilized filter paper disks (Toyo, 8mm, Japan) in an amount of 2OD (lmg)/paper. Then, the extraction solvents were completely evaporated from the papers.

[70] The filter papers treated or not with each extract were randomly placed on six compartments located in edges of the tray constructed in a laboratory. 100 termites were put on the center compartment, and the consumption of filter papers treated or not with the extracts and the number of survivals was checked.

[71] The result is shown in FIG. 4. FIG. 4(A) is a photograph immediately after the disk is treated with termite, 4(B) is a photograph 8 days after the disk is treated with termite and 4(C) is a photograph 28 days after the disk is treated with termite. The top disk in A, B and C is numbered as No. 1 and the remaining disks were clockwise numbered as Nos. 2 to 7. Nos. 1 and 5 indicate non-treated filter paper disk, No. 2 indicates the 1^st antibiotic extract (ethyl acetate extract)-treated filter paper disk, No. 3 is empty, No. 4 indicates the 2° antibiotic extract (ethanol extract)-treated filter paper disk, No. 6 indicates the 3^r antibiotic extract (chloroform extract)-treated filter paper disk and No. 7 indicates a center (where the start mites are placed).

[72] As a result, most of the non-treated filter papers (Nos. 1 and 5) were ingested after 1 week. Around 28 days tested, most of the filter paper treated with chloroform extract was ingested, some of the filter papers treated with the 2° antibiotic extract (No. 4) were ingested, and the remarkable reduction of the number of termites started.

[73] After 1 month, about 20 termites were survived and the filter paper treated with the

1^st antibiotic extract (No. 2) was not ingested at all, which shows the strongest

antibiotic activity. Industrial Applicability

[74] The present invention will be useful for the biomedicine industry to treat and prevent food poisoning bacteria, as shown from the result that the ethyl acetate extract obtained from muskrat excrement has a potent antibiotic effect against gram negative Salmonella bacteria among food poisoning bacteria with a potential that the extract may also be effective against gram positive food poisoning bacteria. Further, since the extracts from muskrat excrement have a potent antibiotic effect against plant pathogens, when the muskrat excretion is used in agriculture, it would provide a high utility as an agricultural fertilizer and a natural and environmental-friendly agricultural material. Furthermore, the extracts from muskrat excrement have an insecticidal effect, and thus can replace chemical insecticides to control termites which vastly damage cultural assists.