KR20090048934A - Separation method of herbicide composition containing methyl para hydroxybenzoate as active ingredient and separation method of methyl para hydroxy benzoate - Google Patents

Abstract

The present invention is a herbicide composition containing methyl para p-hydroxy benzoate, a novel herbicidally active substance isolated from trichophyllum as an active ingredient, a method of using the compound for herbicidal use, and trichophytic herbicides A method for separating the compound from the present invention, wherein the compound isolated by the present invention is a natural herbicide active material, has a unique structure different from the structure of herbicides developed so far, a new mode of action It can be used as a lead compound in the development of new herbicides that attack.

Methyl para hydroxybenzoate, trigeminal vinegar, herbicide, herbicidal activity,

Description

Herbicide composition comprising methyl-p-hydroxybenzoate as an efficient content and method for Purifying the same from Epimedium koreanum Nakai }

The present invention relates to a herbicide composition containing methyl para hydroxybenzoate as an active ingredient and to a method for separating methyl para hydroxy benzoate from trichophytium, more specifically isolated from trichophyte. A herbicide composition comprising methyl para p-hydroxy benzoate, a novel herbicidal active substance, as an active ingredient, a method for using the compound for herbicidal use, and a method for separating the compound from trifoliar vinegar. It is about.

Trilobite leaf koreanum Nakai) is a perennial plant of the Berberidaceae. Its height is 40 ~ 80cm, and the rhizome is connected to the base of the old stalk sideways and grows upright at the end (Park Jong-hee, Lee Jung-gyu. 2000 .. Commercial medicinal plants (Illustration Book, Shinil Corp. Seoul, pp.204-206).

Trilobite lobule is ovate with leaf tip sharp and base bottom. 1-2 leaves in the main stem rebirth and has the characteristic of dividing twice by three (Fig. 1), the flower is yellowish-white (Lee Chang-bok. 2003. primary plant picture book. Hyangmunsa. Seoul. Pp.393).

The outpost of trichophyte vinegar is called yinyangkuk, and its extract has pharmacological effects such as expanding the pupil of the frog, increasing the voluntary movement of the rat, and promoting the semen secretion of the dog. It is known to have been used as a tonic for the treatment of sonic (Park Jong-hee, Lee Jung-gyu. 2000. Illustrated commercial medicinal plants. Shinil Corp. Seoul. Pp.204-206).

Many studies have been conducted on the bioactive natural substances contained in trifoliate vinegar. -α-rhamnoside, ikarisoside AF, maltol, n-alkanes, phytosterols, phytosteryl, quercetin, salidroside (Itaeseong. 2005. Epimedium koreanum Nakai) extract. Master's Thesis, Kangwon National University), β-sitosterol, β-sitosterol 3-O-β-D-glucopyranoside (Kang Sam-sik, Kim Joo-sun, Kang Yun-jung, Han Hye-kyung. Yin Yang Guk, Korean Journal of Pharmacognosy 21 (1): 56 -59), caohuoside-B, epimedokoreanoside-I (Li, WK, PG Xiao, GZ Tu, LB Ma, and RY Zhang. 1995. Flavonol glycosides from Epimedium koreanum . Phytochemistry 38 (1): 263-265), epimedokoreanin A (Li, WK, PG Xiao, and RY Zhang. 1995. A difuranoflavone from Epimedium koreanum . Phytochemistry 38 (3): 807-808), baohuoside I (Li, WK, JQ Pan, MJ Lu, RY Zhang and PG Xiao. 1995. A 9,10-dihydrophenanthrene derivative from Epimedium koreanum . Phytochemistry 39 (1): 231-233) and the like have been reported to exist.

In addition, research on manufacturing methods for the development of various industrial products using trilobite vinegar has been actively conducted, and the situation is mainly concentrated in the field of health drinks and medical products.

In other words, it relates to a health beverage, a method of manufacturing a health beverage containing trichophytic vinegar (Korean Patent No. 10-0463098), a method of producing folk liquor using Yin Yang Kwak (Korean Patent Application No. 2001-0068828) The manufacturing method (Korean Patent No. 10-0484789) and the like are disclosed.

In addition, the pharmaceutical relates to a pharmaceutical composition (Korean Patent Application No. 2005-0084420), a method for producing an extract from trichophytic herbaceous plants (Korea Patent Publication No. 84-001510) And the like are disclosed.

To date, the herbicidal activity of trilobite vinegar has been disclosed in Korean Patent Laid-Open Publication No. 2007-0065943 for a method for separating herbicidal active substances from trilobite vinegar. According to the method, the GR ₅₀ values isolated from the ethyl acetate layer are 241, EADBA, EADBB and EADBC fractions with very high herbicidal activity of 399, 253 μg g ^{- 1 have} been disclosed, but the identity of the EADBA, EADBB and EADBC fractions has not been revealed.

The present invention has been made to solve the problems of the prior art as described above, an object of the present invention is to provide a herbicide composition containing a herbicidal active compound derived from natural products isolated from trigeminal leaf vinegar as an active ingredient.

It is another object of the present invention to provide a method of using the above-mentioned compound derived from natural products isolated from trilobite vinegar for herbicidal use.

Another object of the present invention is to provide a method for separating the above compounds from trilobite vinegar.

The present invention provides a herbicide composition containing a methyl para p-hydroxybenzoate compound represented by the following formula as an active ingredient in order to achieve the above object.

<Formula>

The present invention provides a method of using the above compound for herbicidal use.

The present invention also provides a method for separating the above compounds from trilobite vinegar.

Methyl para-hydroxybenzoate, a compound isolated by the present invention, is isolated from trichloromethane dichloromethane extract with very high herbicidal activity, and can be used as a new herbicide as a herbicidal substance whose structure is determined. In addition, it has a unique structure that is different from the structure of herbicides developed so far, and may be used as a lead compound in the development of new herbicides that attack a new mode of action in the future.

The first aspect of the present invention relates to a herbicide composition containing a methyl para hydroxybenzoate compound represented by the following formula as an active ingredient.

<Formula>

The herbicide composition according to the present invention is characterized in that the compound is separated from the trilobite vinegar, and the content of the compound is not particularly limited, and may be appropriately selected according to need.

The herbicide composition according to the present invention contains the compound as an active ingredient of herbicidal activity, and is also known in the art as pigments, thickeners, diarrhoeareagents, surfactants or mixtures thereof. And the like, but are not limited thereto.

Rapeseed ( Brassica) as a plant to be controlled by the compound which is an active ingredient of the herbicide composition according to the present invention Cruciferaceae, such as napus L.) is preferred, but is not limited thereto.

The second aspect of the present invention relates to a method of using a methyl-p-hydroxybenzoate compound represented by the following formula for herbicidal use.

<Formula>

In the method according to the present invention, the compound is characterized in that it is separated from trilobite.

The method according to the invention can be varied in many ways and depends on the nature of the soil, the method of application, the crops, the weeds to be controlled, the main climatic conditions, and the method of application, the time of application and other factors governed by the target crop. Can be influenced.

The third aspect of the present invention, (a) extracting the dried sample of trigeminal vinegar with methanol; (b) obtaining a dichloromethane fraction from the methanol extract; (c) obtaining a highly active fraction (DA) having herbicidal activity having a GR ₅₀ value of <500 μg g ⁻¹ in a vacuum liquid chromatography using a hexane-ethyl acetate solution from the dichloromethane fraction. step; And (d) obtaining a fraction (DAB) having herbicidal activity having a GR ₅₀ value of 132 μg g ⁻¹ in vacuum liquid chromatography from the DA fraction; It relates to a separation method of methyl para hydroxy benzoate (methyl-p-hydroxybenzoate) compound represented by the following formula from the trigeminal leaf.

<Formula>

The separation method according to the invention may further comprise the step of recrystallizing the DAB fraction obtained in step (d).

Hereinafter, a process of separating the methyl parahydroxybenzoate compound represented by the above formula from the trigeminal leaf vinegar will be described in detail.

Methyl parahydroxy benzoate compound according to the present invention is trigeminal leaf ( Epimedium koreanum Nakai) isolated from the outpost, extracts dried trigeminal vinegar with methanol, and the extract was subjected to seed bioassay on rapeseed ( Brassica napus L.) as a result of GR ₅₀ value (50% inhibition of plant growth). Dose) was 381 μg g ⁻¹ .

The methanol extract was repartitioned with ethyl acetate, butanol, dichloromethane, hexane and water, and the bactericidal power of the fraction was assayed. As a result, the GR ₅₀ value of the dichloromethane extract was 214 μg g ⁻¹ .

The highly active dichloromethane extract was fractionated by hexane-ethyl acetate solution using vacuum liquid chromatography, and the herbicidal activity of each fraction was assayed, and the GR ₅₀ value was <500 μg g ^-1 . An active fraction (hereinafter referred to as "DA"') was obtained.

The highly active DA fractions obtained above were fractionated by hexane-ethyl acetate solution using vacuum solution chromatography, and the herbicidal activity of each fraction was assayed, and then the highly active fraction having a GR ₅₀ value of 132 μg g ^-1 (hereinafter, Is called "DAB".

In order to elucidate the chemical structure of the isolated DAB fraction, EI-MS, ¹ H-NMR, ¹³ C-NMR analysis was performed, and the result was methyl parahydroxybenzoate having a molecular weight of 152 and a chemical formula of C ₈ H ₈ O ₃ . Turned out to be.

Hereinafter, the content of the present invention will be described in detail through examples and test 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 (Preparation of Methanol Extracts of Trifolium Leaf)

Samji-gu vinegar samples used for the experiment were collected in July 2005 at the Northern Experimental Center, Gangwon-do Agricultural Research and Development Center, Cheongyang 6-ri, Kimhwa-eup, Cheorwon-gun, Gangwon-do.

The leaves and stems of the Trifolium vinegar were shaded and crushed to 0.6 mm using a grinder. 200 g of the pulverized dry sample was taken and placed in a 5 L Erlenmeyer flask containing 2 L of methanol, and extracted twice with a 24 rpm shaker for 24 hours.

The methanol extract obtained above was passed through a Buchner funnel with filter paper to remove the residue, and then concentrated by using a rotary vacuum condenser (EYELA NE-1101) to remove methanol completely. 50 mL was added.

The dried product in the flask was dissolved well with distilled water, and then dried at −50 ^° C. for 3 days using a freeze dryer (ILSHIN LAB CO.). Lyophilized samples were stored in 4 ^° C. refrigerators for use in herbicidal assay and structural analysis.

The herbicidal activity assay was obtained by extracting the trigeminal vinegar methanol extract from rape ( Brassica). Seed bioassay was used to investigate the effects on the germination and growth of napus L. plants.

Distilled methanol extract was diluted with distilled water to prepare a stock solution (to 10,000 μg g ⁻¹ ), and then a treatment solution having a different concentration was prepared from the stock solution.

The treatment solution was treated with a 24-well tissue culture test plate in which 5 rapeseed seeds were dented on 1 g of sand, and the assay plate was subjected to a temperature of 25 ^° C., a humidity of 70% and a brightness of 250 μmol. The plants were grown on m ⁻² s ⁻¹ plants and grown for 7 days.

After 7 days of treatment, the live weight of rapeseed seedlings was measured to calculate the GR ₅₀ value (amount that can inhibit 50% of plant growth) for each sample, and the herbicidal force was measured. All experiments were repeated three times.

Methanol extract was obtained in order to assay the herbicidal activity of trilobite vinegar, and the extract was subjected to seed bioassay on rapeseed. As shown in FIG. 2, the GR ₅₀ value was 381 μg g ⁻¹ .

Example 2 (Separation of Solvent Fractionation Layer from Methanol Extract of Trifolium Leaf)

100 ml of hexane, dichloromethane, ethyl acetate, butanol, and distilled water were added to a separatory funnel containing 10 g of methanol extract dried from the trilobite vinegar of Example 1, and the solubility of the solvent was gradually increased. Fractions were fractionated.

Each fraction layer was concentrated to remove the solvent completely with a rotary vacuum concentrator, and lyophilized at −50 ^° C. for 3 days. Treatment solutions of varying concentrations were prepared from each of the lyophilized fractions using water, and the seed bioassay mentioned in Example 1 above was performed for the biocidal assay for each fraction.

The methanol extracts of the trifolium vinegar were partitioned into hexane, dichloromethane, ethyl acetate, butanol, and distilled water, and each fraction was concentrated with a rotary vacuum condenser, lyophilized, and the results of assaying the herbicidal power of rapeseed are shown in Table 1 below. GR ₅₀ values of hexane, dichloromethane, ethyl acetate, butanol and distilled water fractions were> 5,000 μg g ^-1 (10.5% recovery), 231 μg g ^-1 (4.8% recovery), 969 μg g ^-1 (recovery rate) 13.0%), 1,311 μg g ^-1 (17.3% recovery), 1,458 μg g ^-1 (53.8 recovery).

Table 1 Herbicidal Activity of Solvent Fractions Isolated from Methanol Extracts

Solvent fractionation layer Killing power (GR ₅₀ , μg g ^-1 ) Hexane > 5,000 Dichloromethane 231 Ethyl Acetate 969 Butanol 1,311 Water 1,458

Example 3 (Separation of Dichloromethane Fraction (DA))

In Example 2, the trichloromethane methanol extract was dried using a solvent, and the dichloromethane fraction and silica gel, which showed high killing power, were coated and dried in a ratio of 1 to 2, and then crushed and dried in a glass column (5 cm in diameter). , 500 cm was charged in a length of 100 cm).

Eleven fractions (DA, DB, DC, DE, DF, DG, DH, DI, DJ, DK) were obtained by sequentially eluting 1 L at a solvent ratio as shown in Table 2 below.

TABLE 2 Dichloromethane Fraction Separation Solvent Ratio

Solvent ratio Elution Fraction Hexane-ethyl acetate solution (85:15) 1L DA Hexane-ethyl acetate solution (80:20) 1L Hexane-ethyl acetate solution (70:30) 1L Hexane-ethyl acetate solution (60:40) 1L Hexane-ethyl acetate solution (50:50) 1L DB Hexane-ethyl acetate solution (40:60) 1L DC Hexane-ethyl acetate solution (30:70) 1L DD Hexane-ethyl acetate solution (20:80) 1L Hexane-ethyl acetate solution (10:90) 1L DE Ethyl Acetate Solution (100%) 1L Ethyl Acetate Solution-Methanol (90:10) 1L DF Ethyl Acetate Solution-Methanol (80:20) 1L DG Ethyl Acetate Solution-Methanol (70:30) 1L Ethyl Acetate Solution-Methanol (60:40) 1L DH Ethyl Acetate Solution-Methanol (50:50) 1L Ethyl Acetate Solution-Methanol (40:60) 1L DI Ethyl Acetate Solution-Methanol (30:70) 1L Ethyl Acetate Solution-Methanol (20:80) 1L DJ Ethyl Acetate Solution-Methanol (10:90) 1L Methanol (100%) 1L DK Acetone (100%) 1L

Each fraction was completely concentrated in a rotary vacuum concentrator, and the results of obtaining GR ₅₀ values by assaying 11 herbicidal activities using the same seed bioassay method as in Example 1 are shown in Table 3 below.

Referring to Table 3 below, the GR ₅₀ values of the fractions DA, DB, DC, DE, DF, DG, DH, DI, DJ, DK were <500 μg g ⁻¹ (9.5% recovery) and 971 μg g ^{−1, respectively.} (Recovery rate 3.5%), 445 μg g ^-1 (recovery rate 5.67%), 725 μg g ^-1 (recovery rate 8.0%), 1,033 μg g ^-1 (recovery rate 7.56%), 768 μg g ^-1 (recovery rate 3.78%), 575 μg g ^-1 (recovery rate 17.0%), 543 μg g ^-1 (recovery rate 3.83%),> 5,000 μg g ^-1 (recovery rate 4.0%), 3,187 μg g ^-1 (recovery rate 3.0%), 4,700 μg g ^-1 As a recovery rate of 3.5%, the killing power of DA is the highest among 11 fractions.

TABLE 3 Herbicidal Activity of DA-DK Fractions Fractionated from the Trichloromium Dichloromethane Fractions

Solvent fractionation layer Killing power (GR ₅₀ , μg g ^-1 ) DA <500 DB 971 DC 445 DD 725 DE 1,033 DF 768 DG 575 DH 543 DI > 5,000 DJ 3,187 DK 4,700

Example 4 (Separation of DAB Fraction from DA Fraction)

The DA fraction, which had high herbicidal activity in Example 3, was separated using VLC (vacuum liquid chromatography). First, 200 g of silica gel was filled in a glass column under reduced pressure (5 cm in diameter and 15 cm in length), and a DA fraction coating sample (silica gel: DA fraction = 1: 2, w / w) was placed thereon.

After addition of the sample, 1L of hexane-ethyl acetate mixed solution was prepared at the same ratio as in Table 4 below, and eluted sequentially, and five fractions (DAA, DAB, DAC, DAD, and DAE) were obtained.

TABLE 4 DA fraction separation solvent ratio

Solvent ratio Elution Fraction Hexane-ethyl acetate solution (98: 2) 1L DAA Hexane-ethyl acetate solution (95: 5) 1L DAB Hexane-ethyl acetate solution (92: 8) 1L DAC Hexane-ethyl acetate solution (89:11) 1L DAD Hexane-ethyl acetate solution (86:14) 1L DAE

Each fraction was completely concentrated with a rotary vacuum concentrator, and then subjected to a herbicidal assay using the seed bioassay method as in Example 1 to obtain GR ₅₀ values.

Referring to Table 5, the GR ₅₀ values of fractions DA, DB, DC, DE, DF, DG, DH, DI, DJ, DK are 403 μg g ^-1 (recovery rate 10.11%) and 132 μg g ^-1 (recovery rate) 14.61%), 355 μg g ^-1 (13.48% recovery), 620 μg g ^-1 (19.10% recovery), 938 μg g ^{- 1} (42.70% recovery) with the lowest GR ₅₀ values of the DAB fraction. have.

TABLE 5 Herbicidal Activity of DAA, DAB, DAC, DAD, DAE Fractions Fractionated from Trichophyte DA Fraction

Solvent fractionation layer Killing power (GR ₅₀ , μg g ^-1 ) DAA 403 DAB 132 DAC 355 DAD 620 DAE 938

Example 5 (Recrystallization of DAB Fractions)

The DAB fraction, which had high herbicidal activity in Example 4, was completely concentrated, dissolved in 3 mL of ethyl acetate, and then sealed when the crystallization step was reached by adding 5 mL of hexane, and recrystallized in a refrigerator at -20 ^o C for 14 days. .

Example 6 (Structure Analysis of Recrystallized Fraction)

The compound recrystallized in Example 5 was analyzed by MS spectrometry (Ion source, EI, 70 eV; ion source temperature, 250 ° C.) using a mass spectrometer (Micromass, M363 series, UK). As can be seen, molecular ions (M +) were found at m / z 152 and characteristic fraction ions were found at 121, 93 and 65 (FIG. 4).

¹ H-NMR and ¹³ C-NMR analyzes were performed using a Bruker DPX 400 MHz (9.4T) nuclear magnetic resonance apparatus (FT-NMR Spectrometer), CDCl ₃ as solvent, and tetramethyl as internal standard. Silane (tetramethylsilane, TMS) was used.

^{As a result of 1} H-NMR (400 MHz), as shown in FIG. 5, δ (ppm) 3.9 (3H, t, -CH ₃ ), 5.65 (1H, s, OH), 6.85 (2H, d, H -1, H-4), 7.95 (2H, d, H-2, H-3) was confirmed the hydrogen signal. ^As a result of performing ¹³ C-NMR (400 MHz), as shown in Figure 6, it was able to confirm the carbon signal at δ (ppm) 52, 115, 122, 132, 160, 167.

The active substance fraction isolated from trilobite herbicide by trichophyllium by EI-MS, ¹ H-NMR, and ¹³ C-NMR analysis has a molecular weight of 152 and a chemical formula of C ₈ H ₈ O ₃ , aka methyl paraoxybenzoate. It was determined with methyl para hydroxybenzoate, which is well known as (methylparaben).

Example 7 (Preparation of Manufacturing Composition)

A herbicide composition containing 20% by weight of methylparahydroxybenzoate compound, 30% by weight of surfactant, and 50% by weight of liquid carrier, prepared in Examples 5 and 6 above was prepared.

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.

Since the herbicidally active material separated from the trilobite vinegar by the present invention is a herbicidally active material derived from natural products, unlike the herbicidally active material produced by the chemical synthesis method, it is expected to be spotlighted in terms of eco-friendly agriculture. It can be usefully used in domestic and foreign pesticide related fields.

According to the present invention, methyl para p-hydroxybenzoate, a herbicidal active substance isolated from trichloromethane dichloromethane extract having a very high herbicidal activity and having a determined structure, can be used as a new herbicide and has been developed to date. It is expected to be used as a lead compound in the development of new herbicides that attack a new mode of action in the future because of its unique structure.

Figure 1 is a trichophyll outpost photo.

Figure 2 is the herbicidal activity assay results of methanol extract of Trichophyte herb using a 24-well tissue culture test plate.

Figure 3 is a simplified illustration of the separation process of the herbicide active material DAB from trilobite vinegar.

Figure 4 is the result of performing the EI-MS analysis to determine the structure of the herbicidal active material DAB contained in trichophytic dichloromethane extract.

Figure 5 is the result of performing the ¹ H-NMR analysis to determine the structure of the herbicidal active material DAB contained in trifoliate dichloromethane extract.

Figure 6 is the result of performing ¹³ C-NMR analysis to determine the structure of the herbicidal active material DAB contained in trichlorophyll dichloromethane extract.

Claims (6)

Herbicide composition containing a methyl para hydroxybenzoate compound represented by the following formula as an active ingredient. <Formula>

The herbicide composition of claim 1, wherein said compound is isolated from trilobite vinegar. A method of using a methyl para hydroxybenzoate compound represented by the following formula for herbicidal use. <Formula>

4. The method of claim 3, wherein said compound is isolated from trilobite vinegar. (a) extracting a dry sample of trifoliar vinegar with methanol; (b) obtaining a dichloromethane fraction from the methanol extract; (c) obtaining a highly active fraction (DA) having herbicidal activity having a GR ₅₀ value of <500 μg g ⁻¹ in a vacuum liquid chromatography using a hexane-ethyl acetate solution from the dichloromethane fraction. step; And (d) obtaining a fraction (DAB) having herbicidal activity having a GR ₅₀ value of 132 μg g ⁻¹ in vacuum liquid chromatography from the DA fraction; Separation method of methyl para hydroxybenzoate (methyl-p-hydroxybenzoate) compound represented by the following formula from the trigeminal foliar comprising. <Formula>

6. The method of claim 5, further comprising recrystallizing the DAB fraction obtained in step (d).

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