KR101262257B1 - New Natural Herbicidal Compound 3-Hydroxy-9H-xanthen-9one from Burcucumber (Sicyos angulatus L.) and Preparation Method Thereof - Google Patents

Abstract

The present invention discloses a novel compound isolated from Sicyos angulatus L. and represented by the following structural formula and having herbicidal activity.

Description

New herbicidal active substance 3-hydroxy-9H-xanthene-9-one isolated from barbed gourd and its separation method (New Natural Herbicidal Compound 3-Hydroxy-9H-xanthen-9one from Burcucumber (Sicyos angulatus L.) and Preparation Method Thereof}

The present invention relates to a novel compound having herbicidal activity, specifically 3-hydroxy-9H-xanthene-9-one (3-hydroxy-9H-xanthene-9-source), which is a novel herbicidal substance isolated from Sicyos angulatus L. Hydroxy-9H-xanthen-9one) and a separation method thereof, and a herbicide composition comprising the same.

Spiny bark is a vine plant native to North America, grows over 4m, has ridges, dense hairs, and has 3-10 pieces of long oval fruit with thin thorns (Lee Chang-bok. 2003. 775).

It is a weed that is difficult to control once it occurs on river banks, riverbeds, railroads, wastelands, forests, etc., which has neglected its management due to its habitual habits. (Lim, SH, HY Kim, HJ Choi, KH Kim and S. Kim. 2005. Distribution pattern of Burcucumber ( Sicyos angulatus ) at chuncheon, Korea. Korean Society of Pesticide Science Autumn Conference 2005 Collection. p.65).

The growth characteristics of spiny gourd include (1) growing faster than other plants, (2) shading while growing other plants, and (3) releasing allergic inhibitors (allelopathy) into the environment. It can be said that it inhibits the germination or growth of seed. These botanical traits make prickly pear easily dominant in its surroundings.

Kim et al. (2004) reported to academia that methanol extracts of spiny gourd had the herbicidal activity while conducting a series of studies to search for herbicidal active substances from Korean native plants (Kim Mi-sung, Weo-sung, DB Khoa). , Hee-Yeon Kim, Hae-Jin Choi, Hyun-Hyun Choi, Soo-Jung Kwon, Soon-Bae Park, Dong-Sik Park, Sang-Sup Han, and Seong-Moon Kim 2004. Exploration of Korean Native Plants Containing Herbicides Ⅱ.

Huh et al. (2006) found that methanol extracts of spiny gourds (rapeseed) napus ) The DAB fraction (GR ₅₀ value, 43 ㎍ / g) with high herbicidal activity was isolated by fractionation-directed isolation method to detect the herbicidal active substances contained in the seedlings. The chemical structure of the compounds contained in the fractions has not been elucidated (Hoo Soo Jung, Kwon Soon Bae, Kum Hee Kim, Kyun Hyun Hyun, Choi Hae Choi, Hee Yeon Kim, Sung Moon Kim. 2006. Herbicide Activity of Prickly Pear.

Accordingly, the inventors of the present invention have completed the present invention as a result of diligent research to isolate novel herbicidal active substances from spiny foil and to reveal their chemical structures.

An object of the present invention is to isolate and identify novel compounds having herbicidal activity from spiny foil.

Moreover, an object of this invention is to reveal the chemical structure of the said novel compound.

The present invention also aims to assay the herbicidal activity of the novel compounds.

In order to achieve the above object, the present invention provides a novel compound which is separated from barley foil and represented by the following structural formula and has herbicidal activity.

The present invention also provides a herbicide composition containing a novel compound having the above structural formula as an active ingredient.

In addition, the present invention (A) extracting at least one of the leaves or stems of the spiny foil with methanol; (B) dissolving the methanol extract of the spiny gourd in dichloromethane to obtain a dichloromethane fraction; (C) eluting the dichloromethane fraction with a dichloromethane-ethyl acetate mixed solution (13: 1, v / v) in an adsorption column chromatography to obtain an MD fraction having an Rf value of 0.37 to 0.00; (D) The MD fraction was eluted with dichloromethane-ethyl acetate-hexane-methanol mixed solution (5: 4: 3: 1.5, v / v / v / v) by adsorption column chromatography, and the Rf value was 0.17 to 0.00. Obtaining a phosphorus MDC fraction; And (E) eluting and fractionating the MDC fraction with a dichloromethane-ethylacetate-methanol-hexane mixed solution (8: 3: 3: 2, v / v / v / v) by adsorption column chromatography. It provides a method for separating a novel compound having herbicidal activity and represented by the structural formula above from the bakbak, including obtaining an MDCC fraction.

Hereinafter, a process of separating 3-hydroxy-9H-xanthene-9-one (3-Hydroxy-9H-xanthen-9one), which is a novel compound represented by the above structural formula, from Sicyos angulatus L. I will explain.

The novel compound 3-hydroxy-9H-xanthene-9-one according to the invention is characterized in that it is isolated from spinach. After extracting dried thorny sample with methanol and carrying out seed bioassay on rapeseed, the herbicidal force (GR ₅₀ value, about 50% of plant growth) was 1,597㎍ / g It was.

The methanol extract was re-fractionated with ethyl acetate, butanol, dichloromethane, hexane and water, and the GR ₅₀ value of the fractions was the highest. The GR ₅₀ value of the dichloromethane fraction was the highest at 954 µg / g and the recovery was 2.42. Was%.

The highly active dichloromethane fraction was eluted with dichloromethane-ethyl acetate mixed solution (13: 1, v / v) by adsorption column chromatography. The GR ₅₀ values of the MA fraction with an Rf value of 1.00, the MB fraction with 0.80, the MC fraction with 0.77 to 0.65, and the MD fraction with 0.37 to 0.00 were found to have the GR ₅₀ value of 960 µg / g. The recovery rate was the highest at 83.90%.

The highly active MD fractions were eluted with dichloromethane-ethylacetate-hexane-methanol mixed solution (5: 4: 3: 1.5, v / v / v / v) by adsorption column chromatography. The GR ₅₀ values of the MDA fractions with Rf values of 1.00 to 0.57, the MDB fractions with 0.50 to 0.40, and the MDC fractions with 0.17 to 0.00 were found to have the greatest GR ₅₀ values of 737 µg / g and the recovery rate. The highest was 34.98%.

The high activity MDC fraction was eluted with dichloromethane-ethylacetate-methanol-hexane mixed solution (8: 3: 3: 2, v / v / v / v) by adsorption column chromatography. The GR ₅₀ values of MDCA fractions with Rf values of 1.00, MDCB fractions with 0.90 to 0.82, MDCC fractions with 0.70, MDCD fractions with 0.60 and MDCE fractions with 0.17 to 0.00 were found to have a GR ₅₀ value of 146 µg / MD. The highest yield was g and the recovery was 5.65%.

GC-MS, ¹ H-NMR, ¹³ C-NMR analysis to determine the chemical structure of the isolated MDCC fractions, the MDCC fraction has a molecular weight of 212.0, the chemical structure of C ₁₃ H ₈ O ₃ It was found to be 3-hydroxy-9H-xanthene-9-one represented by the structural formula (3-Hydroxy-9H-xanthen-9one).

According to the present invention, it is possible to isolate and identify a novel compound 3-hydroxy-9H-xanthene-9-one (3-Hydroxy-9H-xanthen-9one) having herbicidal activity from Sicyos angulatus L. .

In addition, the novel compound 3-hydroxy-9H-xanthene-9-one having herbicidal activity isolated from the barley of the present invention is used to develop a new herbicide. mother compound).

In addition, the novel compound 3-hydroxy-9H-xanthene-9-one having herbicidal activity isolated from the prickly pear of the present invention is different from the conventional herbicide structure. It has a new structure and can be used as a parent compound in the development of herbicides that attack new modes of action in the future.

1 is a photograph of the herbicidal active plants used in Example 1 of the present invention ( Sicyos angulatus L.).
2 is a mass spectrum analysis result of the 3-hydroxy-9H-xanthene-9-one (3-Hydroxy-9H-xanthen-9one) isolated from the visible foil of the present invention.
Figure 3 shows the results of ¹ H-NMR spectrum analysis of 3-hydroxy-9H-xanthene-9-one (3-Hydroxy-9H-xanthen-9one) isolated from spiny foil of the present invention.
Figure 4 is a ¹³ C-NMR spectrum analysis of the 3-hydroxy-9H-xanthene-9-one herbicidal active material isolated from the visible foil of the present invention (3-Hydroxy-9H-xanthen-9one).
Figure 5 is a photograph showing the herbicidal activity of weed bark and herbicides of 3-hydroxy-9H-xantene-9-one (3-Hydroxy-9H-xanthen-9one) isolated from spines of the present invention. . It is a photograph which shows the herbicidal activity after 7 days of 0, 100, 200, 300, 400, 500 microgram / g treatment from left to right, respectively.
FIG. 6 is a photograph showing herbicidal activity against weed varieties and weed varieties of 3-hydroxy-9H-xanten-9-one herbicide active substance isolated from spiny foil of the present invention. . It is a photograph which shows the herbicidal activity after 7 days of 0, 100, 200, 300, 400, 500 microgram / g treatment from left to right, respectively.

Hereinafter, the content of the present invention will be described in more detail in the following examples, but the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

Example  One : Thorny night  Preparation of Methanol Extracts

The prickly gourd sample used in this example was collected in Yasan, Toegye-dong, Chuncheon-si, Gangwon-do, 2009, as shown in FIG. The thorny ground parts (leaves and stems) were collected and vulgarized and completely ground using a grinder. 1Kg of dry sample was taken and placed in a 5L Erlenmeyer flask containing 2L of methanol, shaken at 100rpm for 24 hours, and extracted twice. The extracted methanol was placed in a 250 mL round bottom flask, completely concentrated using a rotary evaporator, and 50 mL of distilled water (hereinafter referred to as 'dH ₂ 0') was added thereto. The dried product in the flask was dissolved well using dH ₂ 0 and then dried in a lyophilizer. Lyophilized samples were stored in a refrigerator at -20 ° C.

Example  2 : Thorny night  Herbicide Activity of Methanol Extract

In this example, the seed assay (seed bioassay) was performed to assay the herbicidal activity of the methanol extract of the thorn gourd obtained in Example 1.

The methanol extract of Example 1 was used to prepare a stock solution to be 10,000 μg / g using dH ₂ 0, and a treated solution was prepared using the stock solution. 5 liters of rapeseed ( Brassica) on 1 g of sand napus L.) seeds were treated with a seeded 24-well tissue culture test plate, and the test plate was placed on a plant growth at a temperature of 25 ° C., a humidity of 50%, and a brightness of 250 μmol / m ² / s. Growing for days. After 7 days of treatment, the rapeseed biomass was measured to obtain a GR ₅₀ value (a dose capable of inhibiting 50% of plant growth) for each sample, and the experiment was repeated three times.

Methanol extract was obtained in order to assay herbicidal activity of thorn gourd, and the extract was subjected to seed assay on rape, as shown in Table 1 below.

Solvent fractionation layer Herbicide activity (GR ₅₀ value, ㎍ / g) Methanol 1,597

Example  3: Thorny night  Derived dichloromethane Fraction  Herbicide activity

100 ml of ethyl acetate, butanol, dichloromethane, hexane, and water were added to a separatory funnel containing 10 g of methanol extract dried from the thorny gourd of Example 1 each time, and the solvent polarity was gradually increased. Fractionated by soluble fraction. Each fraction layer was subjected to a concentration step of completely removing the solvent by a rotary pressure reducer and freeze-dried at -50 ° C for 3 days. Treatment solutions of varying concentrations were prepared by adding water from each of the lyophilized fractions, and seed assay was performed as described in Example 2 above to assay herbicidal activity for each fraction.

The methanol extract of spiny gourd was partitioned into ethyl acetate, butanol, dichloromethane, hexane and water, and each fraction was concentrated using a rotary evaporator, lyophilized and tested for herbicide activity on rapeseed, ethyl acetate, butanol. GR ₅₀ values and recovery rates of, dichloromethane, hexane and water are shown in Table 2.

Solvent fractionation layer Herbicide activity (GR ₅₀ value, ㎍ / g) Recovery rate (%) Hexane 6,103 1.42 Dichloromethane 954 2.42 Ethyl acetate 980 0.33 Butanol 3,853 1.49 water 9,655 26.89

According to Table 2, the herbicidal activity of the dichloromethane fraction was the highest (954 ㎍ / g).

Example  4 : Thorny night  Derived from dichloromethane MD Fraction  Herbicide activity

In Example 3, the dichloromethane fraction having the highest herbicidal activity among the fractions of the visible thin methanol extract dried product using a solvent was separated by column chromatography. First, 200 g of silica gel (Silica gel 60 F ₂₅₄ , 0.040 ~ 0.063 mm, Merck) was filled in a glass column (5 cm in diameter, 100 cm in length) under reduced pressure, and a dichloromethane fraction sample was placed thereon. After addition of the sample, eluted with dichloromethane-ethyl acetate mixed solution (13: 1, v / v), and then Ve / Vt (elution volume / column bed volume) 1.00 (MA), 0.80 (MB), 0.77-0.65 ( MC), 0.37-0.00 (MD) fractions were obtained.

The herbicidal activity of the MA, MB, MC, and MD fractions isolated from the dichloromethane fractions as shown in Example 2 above is shown in Table 3 below.

Solvent fractionation layer Herbicide activity (GR ₅₀ value, ㎍ / g) Recovery rate (%) MA > 2,000 5.12 MB > 2,000 1.38 MC > 2,000 4.71 MD 960 83.90

As shown in Table 3, the herbicidal activity of the MD fraction of the four fractions separated from the dichloromethane fraction was the highest (960 ㎍ / g).

Example  5: Thorny night  Derived from dichloromethane MDC Fraction  Herbicide activity

The MD extract, which had the most herbicidal effect among the extracts (MA, MB, MC, MD) separated from the dichloromethane extract of spiny gourd in Example 4, was separated by column chromatography. First, 200 g of silica gel (Silica gel 60 F ₂₅₄ , 0.040 ~ 0.063 mm, Merck) was filled in a glass column (5 cm in diameter, 100 cm in length) under reduced pressure, and the MD fraction sample was placed thereon. After addition of the sample, the mixture was eluted with dichloromethane-ethyl acetate-hexane-methanol mixed solution (5: 4: 3: 1.5, v / v / v / v), and then Ve / Vt (elution volume / column bed volume) 1.00- Fractions of .057 (MDA), 0.50-0.40 (MDB), and 0.17-0.00 (MDC) were obtained.

The herbicidal activity of the MDA, MDB, and MDC fractions as described in Example 2 above is shown in Table 4, and the results obtained by performing the seed assay.

Solvent fractionation layer Herbicide activity (GR ₅₀ value, ㎍ / g) Recovery rate (%) MDA > 2,000 5.4 MDB > 2,000 0.6 MDC 737 34.98

As shown in Table 4, the herbicidal activity of the MDC fraction of the three fractions (MDA, MDB, MDC) separated from the MD fraction was the highest (737 ㎍ / g).

Example  6: Thorny night  Derived from dichloromethane MDCC Fraction  Herbicide activity

The MDC extract, which had the most herbicidal effect among the extracts (MDA, MDB, MDC) separated from the spiny dichloromethane extract in Example 5, was separated by column chromatography. First, 200 g of silica gel (Silica gel 60 F ₂₅₄ , 0.040 ~ 0.063 mm, Merck) was filled into a glass column (5 cm in diameter, 100 cm in length) under reduced pressure, and the MDC fraction sample was placed thereon. After sample addition, the mixture was eluted with a dichloromethane-ethyl acetate-methanol-hexane mixed solution (8: 3: 3: 2, v / v / v / v), and then Ve / Vt (elution volume / column bed volume) 1.00 ( MDCA), 0.90-0.82 (MDCB), 0.70 (MDCC), 0.60 (MDCD), 0.17-0.00 (MDCE) fractions were obtained.

The herbicidal activity of five fractions (MDCA, MDCB, MDCC, MDCD, MDCE) isolated from the MDC extracts is shown in Table 5 by performing the seed assay as described in Example 2 above.

Solvent fractionation layer Herbicide activity (GR ₅₀ value, ㎍ / g) Recovery rate (%) MDCA 772 1.74 MDCB > 1,000 3.04 MDCC 146 5.65 MDCD 455 5.65 MDCE > 1,000 21.96

As shown in Table 5, among the five fractions separated from the MDC fraction, the herbicidal activity of the MDCC fraction was the highest at 146 µg / g, and the recovery was 6.65%.

Example  7: Thorny night  Derived herbicidal substances MDCC Fraction  Chemical analysis

Compound of the MDCC fraction obtained in Example 6 is a yellow amorphous powder, the molecular ion peak in m / z 212 [M] ⁺ in the EI mass spectrum (Fig. 2).

^{In the 1} H-NMR spectrum (FIG. 3), signals corresponding to seven characteristic aromatic protons were observed, of which three were hydrogen, δ 8.23 (1H, dd, J = 2.0, 8.0 Hz), 6.99 (1H, dd, J = 2.0, 8.0 Hz), 6.94 (1H, d, J = 2.0 Hz) correspond to the ABX system, and the remaining four hydrogens are δ 7.55 (1H, d, J = 8.4 Hz), 7.81 (1H, dt, J = 1.7, 8.0 Hz), 7.44 (1H, dt, J = 1.0, 7.0 Hz), 8.14 (1H, d, J = 8.6 Hz), respectively. In addition, a phenolic hydroxy proton signal was observed at δ 9.75.

^A total of 13 carbons were identified in the ¹³ C-NMR spectrum (FIG. 4), and carbon corresponding to 12 aromatic rings was δ 164.9, 159.3, 157.4, 135.8, 129.5, 127.4, 125.2, 123.1, 119.1, 116.3, 115.0, Each was observed at 103.6 and the δ 176.2 signal corresponding to one carbonyl group was also identified.

Based on the spectroscopic analysis data as above, the compound was named 3-hydroxy-9H-xanthene-9-one (3-Hydroxy-9H-xanthen-9one).

The analysis results of the MDCC compound are as follows. EI-MS m / z 212.0 ( Calcd for C ₁₃ H ₈ O ₃ : 212.00). ¹ H-NMR spectrum (400 MHZ, CDCl ₃ ) δ: 9.75 (OH), 8.23 (1H, dd, J = 2.0, 8.0 Hz, H-1), 8.14 (1H, d, J = 8.6 Hz, H- 8), 7.81 (1H, dt, J = 1.7, 8.0 Hz, H-6), 7.55 (1H, d, J = 8.4 Hz, H-5), 7.44 (1H, dt, J = 1.0, 7.0 Hz, H-7), 6.99 (1H, doublet of doublets, J = 2.0, 8.0 Hz, H-2), 6.94 (1H, d, J = 2.0 Hz, H-4). ¹³ C-NMR spectrum (100 MHz, CDCl ₃ ) δ: 176.2 (C-9), 164.9 (C-3), 159.3 (C-4a), 157.4 (C-4b), 135.8 (C-6), 129.5 (C-1), 127.4 (C-8), 125.2 (C-7), 123.1 (C-8a), 119.1 (C-5), 116.3 (C-2), 115.0 (C-9a), 103.6 ( C-4)

Example  8 : Thorny night  Derived herbicidal active substance 3-hydroxy-9H- Xanthene -9-membered (3- Hydroxy -9H- xanthen -9 one Herbicidal activity of

In this embodiment, the hydroxyapatite dichloromethane extract in Example 6 and the chemical structure of the 3-hydroxy-9H-xanthene-9-one in Example 7 (3-Hydroxy-9H-xanthen-9one) The herbicidal activity of the MDCC fractions identified by Echinochloa crus-galli var.caudata Kitagawa and Digitaria cilaris (Retz. Koel).

As in Example 2, the MDCC fraction of Example 6 was added to 0, 100, 200, 300, 400, 500 μg / in a 24-well tissue culture test plate on which 1 bar of sand and 5 seeds of seeds were dented on 1 g of sand. g and the test plate was placed on a plant growth at a temperature of 25 ° C., a humidity of 50% and a luminous intensity of 250 μmol / m ² / s for 7 days, and grown 7 days after treatment. Was measured to obtain a GR ₅₀ value for each sample.

As a result, the herbicidal activity of the bark and the leek of the MDCC extract was 237.0 μg / g and 175 μg / g, respectively. The results for the bark are shown in FIG. 5, and the results for the cold water are shown in FIG. 6.

In summary, the MDCC fractions of which the chemical structure of the present invention is identified as 3-hydroxy-9H-xanthene-9-one are not only rape, which is broadleaf weed, but also bark and weed, It was found that there was an excellent herbicidal effect on the cranberries.

Claims (3)

delete Herbicide composition characterized in that it contains a compound represented by the following structural formula and isolated from Sicyos angulatus L. as an active ingredient.

(A) extracting at least one of the leaves or stems of Sicyos angulatus L. with methanol;
(B) dissolving the methanol extract of the spiny gourd in dichloromethane to obtain a dichloromethane fraction;
(C) eluting the dichloromethane fraction with a dichloromethane-ethyl acetate mixed solution (13: 1, v / v) by adsorption column chromatography to obtain a fraction having an Rf value of 0.37 to 0.00;
(D) The fraction having the Rf value of 0.37 to 0.00 was eluted with a dichloromethane-ethyl acetate-hexane-methanol mixed solution (5: 4: 3: 1.5, v / v / v / v) by adsorption column chromatography. Obtaining a fraction having an Rf value of 0.17 to 0.00; And
(E) eluting fractions having an Rf value of 0.17 to 0.00 by dichloromethane-ethylacetate-methanol-hexane mixed solution (8: 3: 3: 2, v / v / v / v) by adsorption column chromatography Obtaining a fraction having an Rf value of 0.70,
A method for separating a compound having herbicidal activity from the thorn foil and represented by the following structural formula.

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