KR20070065942A - Herbicidal plant extracts from burcucumber sicyos angulatus l. and separation method thereof - Google Patents

Abstract

An extract of Sicyos angulatus L. is provided to have excellent herbicidal activity, thereby being used as a herbicidal composition. The method for separating a herbicidal plant extract from Sicyos angulatus L. comprises the steps of: (a) extracting Sicyos angulatus L. with an organic solvent such as methanol, ethanol, propanol, butanol, and pentanol and then drying it; (b) dissolving the product obtained from the step(a) in dichloromethane to obtain a dichloromethane fraction; (c) subjecting the dichloromethane fraction to column chromatography using a mixture solution of hexane, dichloromethane, ethylacetate and methanol, wherein the volumetric ratio thereof is 4:1:6:0.5, to obtain a DA fraction having the herbicidal activity; (d) subjecting the DA fraction to column chromatography using a mixture solution of ethylacetate, dichloromethane, hexane and methanol, wherein the volumetric ratio thereof is 4:1:3:0.5, to obtain a DAB fraction having the herbicidal activity; and (e) subjecting the DAB fraction to column chromatography using a mixture solution of ethylacetate, hexane and methanol, wherein the volumetric ratio thereof is 9:1:0.1 to obtain a DABB fraction having the herbicidal activity.

Description

Herbicidal Activity Extracts Isolated from Spiny Bak and Methods of Separation {Herbicidal Plant Extracts from Burcucumber Sicyos angulatus L. and Separation Method

1 is a photograph showing a visible foil as the main material of the present invention.

2 is a flowchart illustrating a process for separating the herbicidal active material from spiny gourds.

The present invention relates to a herbicide comprising a spiny gourd extract having herbicidal activity, a method for separating the herbicidal active substance from the spiny gourd, and a spiny gourd extract having herbicidal activity.

Spiny gourd ( Sicyos angulatus L.) is a vine plant native to North America, grows over 4m, has ridges, dense hairs, and has 3-10 pieces of ellipsoidal fruit. Illustration of Plants for Primary Colors, Hyangmun Temple (pp.775).

It is reported that thorn bark is a weed that is difficult to control once it occurs on riverbanks, riverbeds, railroads, wastelands, forests, etc., where management is neglected because of its habitual habits. Lim, SH, HY Kim, HJ Choi, KH Kim and S. Kim. 2005. Distribution Pattern of Burcucumber ( Sicyos angulatus ) at Chuncheon, Korea.

Since the availability of spiny gourd has been raised in Korea as a plant of park crops (Lee Won-Hyung, Kim Sam-Bo, Kwak Byung Hwa. 1991. Characteristics of Sicyos angulatus L. growing in Andong Province and its applicability as gourd and crop plant. The Korean Society for Horticultural Science 32 (3): 299-304), currently used as a substitute for gourd and pumpkin to overcome the damage caused by the cultivation of foliar crops (Lee, HW, SB Kim and BW Kwack. 1991. of Sicyos angulatus L. growing wild in Andong area and its potential as rootstock for cucurbitaceous crops.J. Korean Soc.Hort.Sci. 32 (3): 299-304).

In Korea, the use of prickly gourd has not been reported in other fields besides the use of large tree, but in China, the seed of prickly gourd is medicinal (鄭漢臣, 吳光 第, 1990. 中國 本 草圖 錄 卷 七).商務印書館 (香港) 有限公司 人民 衛生 出版社 合作 出版. P.159). Seeds of spiny gourd are known to have antipyretic and insecticidal effects to lower the heat of the stomach, to control vomiting from pregnancy, and to be used as an insect repellent for roundworms and duodenal insects. However, to date, it is not known which of the bioactive substances contained in the seeds of thorny seed shows such medicinal effects.

To date, there is no research on the activity of extracts isolated from spiny plants. In particular, as the present inventors attempt to extract the extract from the thorn gourd is not known to have a herbicidal activity, the research to use the thorny extract having such a herbicidal activity as a herbicide has not been reported yet.

The inventors of the present invention, while conducting research to develop a herbicidal active material from domestic native plants, confirmed that the extract extracted from spinach gourd with an organic solvent has a herbicidal activity, the extract of spiny gourd, rape, drizzle, hair agar, eggs It has been found that it has high herbicidal activity against weed plants when applied to weed plants. In addition, fractions of thorny organic solvent extract dried and tested in the in vitro (herbicidal activity), as a result of the weeds, including the rapeseed, dongsae, hair amaranth, algae weed to show the herbicidal activity to complete the present invention Was done.

Therefore, an object of the present invention is to provide a spiny gourd extract having herbicidal activity from spiny gourd.

And another object of the present invention is to provide a method for separating the herbicidal active substance from spiny gourd.

The present invention can provide a herbicide comprising a thorn gourd extract having herbicidal activity.

The present invention for achieving the above-mentioned object is characterized by having a herbicidal activity as an extract obtained by extracting thorn gourd with an organic solvent.

In another aspect, the present invention includes a fraction extract having a herbicidal activity obtained by further comprising the step of extracting the thorn gourd with an organic solvent and fractionating with dichloromethane or ethyl acetate.

In the present invention, the organic solvent used to obtain the spiny gourd extract having the herbicidal activity can be used as long as the spiny gourd extract can have the herbicidal activity. As one example of such an organic solvent in the present invention, any one or more selected from methanol, ethanol, propanol, butanol and pentanol may be used.

The present invention includes a herbicide containing a spiny gourd extract having a herbicidal activity by extracting the spiny gourd with an organic solvent.

In the present invention, the barley gourd extract having the herbicidal activity was measured on the weeds including rapeseed, dracaena, hair amaranth, and egg shells. It can be seen that there is a herbicidal effect on these weeds.

In the present invention, in the herbicide comprising a spiny gourd extract having a herbicidal activity by extracting the spiny gourd with an organic solvent, using a spiny gourd extract alone or in addition to a carrier commonly used in herbicides for the formulation of herbicides. May contain.

The carrier may be any material as long as it can facilitate the application of the herbicidal active ingredient to the weeds, or can facilitate the storage, or to facilitate the transport or operation. The carrier may also be a liquid or a solid, usually a gas, including a substance compressed to form a liquid, and any carrier commonly used in the manufacture of herbicides may be used.

Natural and synthetic clays and silicates, such as natural silica, such as diatomaceous earth, as an example of the solid carrier in the above; Magnesium silicates such as talc; Magnesium aluminum silicates such as etherpulzite and vermiculite; Aluminum silicates such as kaolinite, montmorillonite and mica; Calcium carbonate; Calcium sulfate; Ammonium sulfate; Synthetic hydrated silicon oxides and synthetic calcium or aluminum silicates; Elements such as carbon and sulfur; Natural and synthetic resins such as coumarone resins, polyvinyl chloride and styrene polymers and copolymers; Solid polychlorophenols; bitumen; Waxes; And one or more selected from superphosphates.

And water as an example of a liquid carrier; Alcohols such as isopropanol and glycols; Ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone and cyclohexanone; Ethers; Aromatic or araliphatic hydrocarbons such as benzene, toluene and xylene; Pertrolium fractions such as kerosine and light mineral oils; Any one or more selected from chlorinated hydrocarbons such as carbon tetrachloride, perchloroethylene and trichloroethane can be used.

On the other hand, the present invention includes a method for separating the herbicidal active substance from spiny gourd.

Separation method of the herbicidal active substance from spiny gourd of the present invention,

(a) extracting and drying the spiny gourd with an organic solvent;

(b) dissolving the dried thorn organic solvent extract with dichloromethane to obtain a dichloromethane fraction;

(c) obtaining DA fraction of herbicidal activity from column chromatography using a mixed solution of hexane-dichloromethane-ethylacetate-methanol from the dichloromethane fraction obtained in step (b);

(d) obtaining DAB fractions of the herbicidal activity from column chromatography using a mixed solution of ethyl acetate-dichloromethane-hexane-methanol from the DA fraction obtained in step (c);

(e) obtaining a herbicidal DABB fraction from the DAB fraction obtained in step (d) using column mixture of ethyl acetate-hexane-methanol in the column chromatography (see FIG. 2).

By extracting the spiny gourd with the organic solvent in the above it is possible to obtain a spiny gourd extract having a herbicidal activity, but in order to obtain a more active herbicidal component material, the above-mentioned steps (b) to (e) may be further performed. .

Hereinafter, a method of separating the herbicidal active material from the thorn gourd using steps (a) to (e) will be described in detail.

In the step (a), the organic solvent may be obtained an organic solvent extract from the spiny foil using at least one selected from methanol, ethanol, propanol, butanol, pentanol.

The thorn extract using the organic solvent can be dried by any one or more methods selected from natural drying, hot air drying, far-infrared drying, micro drying, lyophilization to obtain an organic solvent extract dried from the thorn foil. In the present invention, such a drying method is sufficient if those skilled in the art can be selectively carried out selectively, detailed description of the drying method will be omitted below.

When methanol is used as the solvent, the visible bioassay is obtained from thorny methanol extract, which is obtained by extracting spiny gourd with methanol, freeze-driing spiny gourd methanol extract, and targeting rape ( Brassica napus L.). ) And the herbicidal activity was measured, and the GR ₅₀ value (amount of drug that inhibits plant growth by 50%) was 328 µg · g ⁻¹ .

Step (b) is a step of obtaining a dichloromethane fraction by dissolving the dried extract of thorn organic solvent with dichloromethane. In addition to dichloromethane, the extracted organic solvent extract was extracted with ethyl acetate, butanol, hexane, and water, and the killing power of each fraction was analyzed by seed bioassay. The GR ₅₀ value of the dichloromethane fraction was 214 µg. It was confirmed that there is an excellent herbicidal activity of g ^-1 .

Step (c) is a step of obtaining DA fraction, DB fraction of the herbicidal activity in column chromatography using a mixed solution of hexane-dichloromethane-ethylacetate-methanol from dichloromethane fraction having excellent herbicidal activity. In this case, hexane-dichloromethane-ethyl acetate-methanol may use a mixed solution composed of a mixing ratio of 2 to 6: 0.5 to 3: 4 to 8: 0.1 to 1 (v / v / v / v), more preferably A mixed solution of hexane-dichloromethane-ethylacetate-methanol (4: 1: 6: 0.5, v / v / v / v) can be used.

Step (d) is to obtain the herbicidal DAA fraction, DAB fraction, DAC fraction in the column chromatography using a mixed solution of ethyl acetate-dichloromethane-hexane-methanol from the DA fraction of the excellent herbicidal activity from step (c) Step. In this case, ethyl acetate-dichloromethane-hexane-methanol may be used a mixed solution consisting of a mixing ratio of 2 to 6: 0.5 to 3: 1 to 5: 0.1 to 1.0 (v / v / v / v), more preferably A mixed solution of ethyl acetate-dichloromethane-hexane-methanol (4: 1: 3: 0.5, v / v / v / v) can be used.

Step (e) is a step of obtaining the DABB fraction of the herbicidal activity in column chromatography using a mixed solution of ethyl acetate- hexane- methanol from the DAB fraction of the excellent herbicidal activity obtained in step (d). In this case, ethyl acetate-hexane-methanol may be a mixed solution having a mixing ratio of 7 to 9: 0.5 to 2: 0.05 to 0.3 (v / v / v), and more preferably ethyl acetate-hexane-methanol (9: 1: 0.1, v / v / v) mixed solution can be used.

As a result of assaying the herbicidal activity of each fraction of step (c) to (e) in vitro, the fraction having excellent herbicidal activity with GR ₅₀ value of 142 µg · g ⁻¹ was called 'DABB'. Can be obtained).

The present invention refers to a herbicide comprising a herbicidal active substance separated from spiny foil by steps (a) to (e) above.

The herbicide comprising the herbicidal active material separated from prickly pear by step (a) to (e) of the present invention comprises the herbicidal active material separated from prickly pear by step (a) to (e). It may further comprise a carrier used alone or commonly used in herbicides for the formulation of herbicides.

In the herbicide comprising a herbicidal active material separated from prickly pear by step (a) to (e) of the present invention, the herbicidal active material separated from prickly pear by step (a) to (e) When formulated to include a carrier, 10 to 99.9% by weight of the herbicidal active material separated from the visible foil in the step (a) to (e), 0.1 to 90% by weight of the carrier can be used.

Hereinafter, the content of the present invention will be described in more detail through examples. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited thereto.

<Example 1>

Kwangbak samples were collected from Jinae-ri, Chuncheon-si, Gangwon-do from July to September 2005 (see Fig. 1).

The leaf area of the spiny gourd was washed with water, dried, and ground to 0.6 mm using a grinder. 1 kg of the pulverized spinach dried sample was placed in a 5L Erlenmeyer flask containing 2 liters of methanol (L), and extracted twice with a 100 rpm shaker for 24 hours.

The methanol extract obtained above was passed through a Buchner funnel with filter paper to remove the residue, followed by complete concentration to completely remove methanol using a rotary pressure reducer (EYELA NE-1101), and then a flask. 50 mL of water was added after the addition.

The dried product in the flask was dissolved well with water, and then dried at -50 ° C for 3 days using a lyophilizer (ILSHIN LAB CO.) To obtain a methanol extract. Lyophilized samples were used for herbicidal activity assays while stored in refrigerators at 4 ° C.

As a herbicidal activity assay for the barley methanol extract dried obtained above, a seed bioassay was used. Methanol extract dried product was diluted with water to prepare a stock solution (to 10,000 g · g ⁻¹ ), and then a treatment solution having a different concentration was prepared from the stock solution. Concentration ^{0㎍ · g -1, 2,000㎍ · g} -1, 4,000㎍ · g -1, 6,000㎍ · g -1, 8,000㎍ · g -1 and 10,000㎍ · g ^-1 of the processing solution on sand 1g Five rapeseed ( Brassica napus L.) seeds were treated on a 24-well tissue culture test plate with teeth. Test plates were placed on plant growth at a temperature of 25 ° C., a humidity of 70%, and a light intensity of 250 μmol · m ⁻² · s ⁻¹ and grown for 7 days. After 7 days of treatment, the live weight of the rapeseed seedlings was measured to calculate the GR ₅₀ value (amount that can inhibit the growth of the plant by 50%) for each sample. All experiments were repeated three times.

To test the herbicidal activity of prickly gourd, seed bioassay was carried out on the rapeseed methanol extract, and the GR ₅₀ value was 328 µg · g ⁻¹ .

<Example 2>

Using the thorn methanol extract dried in Example 1, weeds ( Leptochloa chinensi s), Amaranthus retroflexus , Cyperus diformis , representative of weeds, broadleaf, and quadruple, respectively, in vitro Seeds were subjected to herbicidal activity using seed bioassay.

Methanol extract dried product was diluted with water to prepare a stock solution at 2,000 µg · g ⁻¹ , and then a treatment solution having a different concentration was prepared from the stock solution. Concentration ^{0㎍ · g -1,, 250㎍ ·} g -1,, 500㎍ · g -1,, 1,000㎍ · g -1,, 1,500㎍ · g -1,, 2,000㎍ · g -1, the The treated solution was treated in a culture tube (12 × 75 mm) in which 65 mg of sand and weed seeds were dented. Growth was placed on a constant temperature growth (25 ° C., 14 hours photoperiod, 250 μmol · m ⁻² · s ⁻¹ condition) in which humidity of 70% was maintained. All experiments were repeated three times, and the herbicidal activity was assessed by the weeding power for each weed in units of 0-100 (0, invalid; 100, complete test) after 1 week.

As a result of seed bioassay of the edible bird, hairy amaranth, and sorghum, the methanol extract (2,000㎍ · g ⁻¹ ) showed higher than 80% high herbicidal effect on all weed species. (See Table 1). However, the treatment of 1,000 μg · g ⁻¹ showed a high herbicidal effect of 80% or more for drunkards and eggs, while no herbicidal effects were found for hairy manure. From these results, it was confirmed that thorny methanol extract inhibited the growth of drunkard, hairy stalk, and eggplant swan when the dose was 2,000 g · g ⁻¹ or more.

Table 1. Herbicidal Effects of Methanol Extracts of Rhizome amaranth on Drunken, Hairy Amaranth and Egg Bran

Treatment concentration (㎍ · g ⁻¹ ) Drake Bird ( Leptochloa chinensi s) Hairy amaranth ( Amaranthus retroflexus ) Eggshells ( Cyperus diformis ) 2,000 97 85 100 1,000 83 0 98 500 10 0 33 250 0 0 0 0 0 0 0

<Example 3>

For each 10 g of the methanol extract dried from the thorns of Example 1 using methanol, 100 mL of hexane, dichloromethane, ethyl acetate, butanol, and water were each fractionated by soluble fraction of each solvent, increasing the polarity of the solvent step by step. Each fraction layer was subjected to complete concentration to completely remove each solvent with a rotary pressure reducer and freeze-dried at -50 ° C for 3 days.

Concentration to different concentrations using water from a lyophilized each fraction is ^{0㎍ · g -1, 250㎍ · g} -1, 500㎍ · g -1, 1,000㎍ · g -1, 1,500㎍ · g - ¹ , 2,000 μg · g ⁻¹ was prepared and carried out using the method mentioned in Example 1 above for the herbicidal assay for each fraction.

Methanol extract of spiny gourd was partitioned into hexane, dichloromethane, ethyl acetate, butanol and water, each fraction was completely concentrated by rotary pressure concentrator and lyophilized, and the rapeseed was tested by hexane, dichloro The GR ₅₀ values for the methane, ethyl acetate, butanol and water fractions were> 2,000 μg g- ¹ (recovery 28.8%), 214 μg g- ¹ (recovery 0.8%) and 129 μg g- ¹ (recovery 0.5%) ), 288 µg · g ⁻¹ (7.3% recovery) and 1,117 µg · g ⁻¹ (43.1% recovery) (see Table 2).

Table 2. Herbicidal Activity of Solvent Fraction Layer Isolated from Prickly Methanol Extract

Solvent fractionation layer Herbicidal power (GR ₅₀ , ㎍ · g ^-1 ) Hexane > 2,000 Dichloromethane 214 Ethyl acetate 129 Butanol 288 water 1,117

<Example 4>

The dichloromethane fraction showing high killing power in the fraction obtained by distilling the dried prickly methanol extract in Example 3 using a solvent was added to a hexane-dichloromethane-ethylacetate-methanol solution (4: 1: 6: 0.5, v / v / v / v), eluting with 10 mL of adsorption (Silica gel 60, 0.040 to 0.063 mm, Merck) column chromatography to obtain DA fraction (Rf, 0.05 to 1.00) and DB fraction (Rf, 0.00 to 0.02). .

Each fraction was completely concentrated with a rotary decompression thickener, and the herbicidal assay of the DA and DB fractions was carried out using the method of Example 1, and the GR ₅₀ values of these fractions were 57 μg g ⁻¹ (recovery rate, respectively). 44.1%) and 97 μg · g ⁻¹ (recovery rate 45.0%), the DA killing power was higher than that of DB (see Table 3).

Table 3. Herbicidal Activity of DA, DB Fractions Fractionated from Prickly Dichloromethane Fractionation

Solvent fractionation layer Herbicidal power (GR ₅₀ , ㎍ · g ^-1 ) DA 57 DB 97

Example 5

The DA fraction, which had high herbicidal activity in Example 4, was adsorbed by ethyl acetate-dichloromethane-hexane-methanol solution (4: 1: 3: 0.5, v / v / v / v) (Silica gel 60, 0.040 to 0.063 mm). , Merck) Elution fractions of 10 mL in column chromatography gave DAA fractions (Rf, 0.50 to 1.00), DAB fractions (Rf, 0.05 to 0.41), and DAC fractions (Rf, 0.00 to 0.05).

Each fraction was completely concentrated with a rotary decompressor, and the herbicidal assay was performed using the method of Example 1, and the GR ₅₀ value of the DAB fraction was 43 µg · g ⁻¹ (41.6% recovery). 4).

Table 4. Herbicidal Activity of DAA, DAB, and DAC Fractions Fractionated from Spiny DA Fractions

Solvent fractionation layer Herbicidal power (GR ₅₀ , ㎍ · g ^-1 ) DAA 381 DAB 43 DAC 146

<Example 6>

DAB fraction having high biocidal activity in Example 5 was adsorbed with ethyl acetate-hexane-methanol solution (9: 1: 0.1, v / v / v) (Silica gel 60, 0.040 ~ 0.063mm, Merck) in column chromatography Elution fractions of 10 mL gave DABA (Rf, 0.94 to 1.00), DABB (Rf, 0.50 to 0.58), DABC (Rf, 0.36 to 0.44), and DABD (Rf, 0.00 to 0.36) fractions.

Each fraction was completely concentrated in a rotary pressure reducer, and the herbicidal assay was performed using the method of Example 1, and the GR ₅₀ value of each fraction was> 1,000 µg · g ⁻¹ (yield 8.5%), 142 µg. The herbicidal activity of DABB was the highest with g ^-1 (6.3% recovery), 443 µg-g ^-1 (3.3% recovery), and> 1,000 µg-g ^-1 (66.4% recovery) (see Table 5).

Table 5. Herbicidal Activity of DABA, DABB, DABC, DABD Fractions Fractionated from Spiny DAB Fractions

Solvent fractionation layer Herbicidal power (GR ₅₀ , ㎍ · g ^-1 ) DABA > 1,000 DABB 142 DABC 443 DABD > 1,000

Through the present invention, the extract fraction of Korean native plant Sicyos angulatus L. has excellent herbicidal activity and can be used as a composition of a new herbicide.

In the case of identifying and identifying the herbicidal active substances contained in the thorn extract fraction, it can be used directly as a new herbicide or as a parent compound for the development of new herbicides.

As described above, although described with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and modified within the scope of the present invention without departing from the spirit and scope of the invention described in the claims below. It will be appreciated that it can be changed.

Claims (6)

Spiny gourd extract with herbicidal activity obtained by extracting spiny gourd with organic solvent. The spiny gourd extract according to claim 1, further comprising the step of extracting the spiny gourd with an organic solvent and then fractionating it with dichloromethane. The spiny gourd extract of claim 1 or 2, wherein the organic solvent is any one or more selected from methanol, ethanol, propanol, butanol, and pentanol. (a) extracting and drying the spiny gourd with an organic solvent; (b) dissolving the dried thorn organic solvent extract with dichloromethane to obtain a dichloromethane fraction; (c) Sicily in column chromatography using a mixed solution of hexane-dichloromethane-ethylacetate-methanol (4: 1: 6: 0.5, v / v / v / v) from the dichloromethane fraction obtained in step (b) Obtaining an active DA fraction; (d) Herbicidal activity in column chromatography using a mixed solution of ethyl acetate-dichloromethane-hexane-methanol (4: 1: 3: 0.5, v / v / v / v) from the DA fraction obtained in step (c) Obtaining a DAB fraction of the; (e) obtaining the herbicidal DABB fraction from the DAB fraction obtained in step (d) using column mixture of ethyl acetate-hexane-methanol (9: 1: 0.1, v / v / v) in column chromatography Separation method of the herbicidal active material from spiny gourd comprising a. The method of claim 4, wherein the organic solvent is any one or more selected from methanol, ethanol, propanol, butanol, and pentanol. A herbicide comprising a thorny leaf extract having the herbicidal activity of any one of claims 1 to 3.

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