KR20030068634A - Antifungal composition for control of plant powdery mildew comprising chrysophanol or parietin as active ingredient - Google Patents

Abstract

PURPOSE: Provided is an antifungal composition for the control of plant powdery mildew comprising chrysophanol or parietin as an active ingredient. The parietin and chrysophanol have more excellent control effect on plant powdery mildew than fenarimol and polyoxin A, respectively. CONSTITUTION: An antifungal composition for the control of plant powdery mildew is characterized by comprising chrysophanol represented by the formula(1) or parietin represented by the formula(2), as an active ingredient, in the amount of 0.12-200μg/ml. The chrysophanol or parietin is obtained by extracting Rumex crispus and Rumex japonica.

Description

Plant powdery mildew control composition containing chrysopanol or parietin as an active ingredient {ANTIFUNGAL COMPOSITION FOR CONTROL OF PLANT POWDERY MILDEW COMPRISING CHRYSOPHANOL OR PARIETIN AS ACTIVE INGREDIENT}

Plant powdery mildew disease is a representative disease that occurs in plants such as cucumber, pumpkin and red bean powder caused by Sphaerotheca fuliginea , powdery powder of rose caused by Sphaerotheca pannosa , and grapefruit spp. Podosphaera apple powdery mildew caused by leucotricha ) and powdery mildew disease caused by Erysiphe graminis .

Fungicides used to control plant powdery mildew can be largely divided into azole type fungicides, morpholine (morpolines) and piperidines type fungicides. The azole fungicides are known to have antimicrobial activity by inhibiting the C14 dimethylation step (DMI) during sterol biosynthesis (Berg et al., Sterol biosynthesis inhibitors-pharmaceutical and agrochemical aspects , 168, 1988; Koller, Target sites of fungicide action , 139, 1992), and fungicides belonging to this group include fenarimol, cyproconazole, epoxyconazole and tebuconazole. Morpholines and piperidines disinfectants are known to inhibit Δ ¹⁴ -reductase and △ ⁸ → △ ⁷ -isomerase (Baloch et al., Phytochemistry , 26). , 663, 1987) Fungicides belonging to this group include penpropimorph, tridemorph, and fenpropidin.

Many countries, including Europe, have reported on the development of strains that are resistant to DMI compounds or cross-resistant to fungicides belonging to DMI (Gisi et al . , Proc. British Crop Prot. Cof.-Pests and Diseases , 3 , 857, 1986). In the case of morpholine fungicides, resistant bacteria have emerged, and the use of chemical agents for controlling plant diseases is increasing (Hardwick et al., Crop Protection , 13 , 93, 1994). In addition, strobilurins fungicides that inhibit respiratory processes and anilopyrimidines fungicides that inhibit amino acid biosynthesis processes have been developed (Godwin et al . , Proc. British Crop Prot. Cof.-Pests and Diseases , 2 , 435, 1992; Heye et al ., Proc.British Crop Prot.Cof.-Pests and Diseases , 2 , 501, 1994), commercially available and resistant to powdery mildew resistant bacteria against strobiliurin- based fungicides. It was almost impossible to do.

Currently, developed countries are conducting a lot of research for the development of new pesticides. However, as the development of new pesticides that meet the conditions of pesticide penetration, persistence and antimicrobial efficacy becomes more difficult, the efficiency of new pesticide development has recently been improved. In order to improve the situation there are many attempts to find new antimicrobial actives from natural products, microorganisms and the like.

Chrysophanol and parietin belonging to the anthraquinone compounds are extracted from various plants such as Rumex crispus and Rumex japonica , or Aspergillus spp. It has been reported to be produced by Penicillium spp. And Alternaria spp., And these natural compounds are anticancer activity (Lin et al . , J. Natural Products , 64 , 674, 2001), Antioxidant activity (Bezabih et al., Planta Medica , 67 , 340, 2001; Yen et al., Food Chemistry , 70 , 437, 2000), antimutagenic activity (Choi et al., PlantaMedica , 63 , 11, 1997), topoisomerase II ( topoisomerase Ⅱ) inhibitory activity (US Patent Application No. 544203), toxicity to mice upon intraperitoneal injection (Bachmann et al. , J. Agric. Food Chem. , 27 , 1342, 1979), in vitro against poliovirus Antiviral activity (Semple et al., Antiviral Research , 49 , 169, 2001) and athlete's foot Antimicrobial activity against Trichophyton mentagrophytes (Agarwal et al . , J. Ethnopharmacol. , 72 , 43, 2000) has been reported. However, no in vitro and in vivo antimicrobial activity of anthraquinone compounds against plant pathogens has been reported, especially Sphaerotheca spp. And Staphylococcus spp. ( Podosphaera spp.) And Erysiphe spp. Have not been reported antimicrobial activity.

Accordingly, the present inventors have completed the present invention by confirming that the compound obtained by separating and purifying the anthraquinone-based compounds chrysophanol and parietin from the root of Rinja has excellent control against barley and cucumber powdery mildew.

An object of the present invention is to provide a plant powdery mildew control composition having excellent control activity.

1 shows the electron collision mass spectrum of chrysopanol,

Figure 2 shows the electron collision mass spectrum of parietin,

Figure 3 shows the hydrogen nuclear magnetic resonance spectrum of chrysopanol,

Figure 4 shows the hydrogen nuclear magnetic resonance spectrum of parietin,

5 is a photograph showing the antimicrobial effect of parietin on cucumber powdery mildew.

In order to achieve the above object, the present invention provides a plant powdery mildew control composition containing an anthraquinone-based compound of formula 1 or 2 chrysophanol or parietin as an active ingredient.

Formula 1

Formula 2

The chrysopanol or parietin-containing composition of the present invention can control powdery mildew, which occurs in plants such as cucumbers, pumpkins, red beans, roses, apples, varicose, etc., and is particularly effective in controlling barley and cucumber powdery mildew.

Chrysophanol and parietin used as an active ingredient in the composition according to the present invention can be obtained by a conventional solvent extraction method from Sokjak as follows. Specifically, for example, Rumex crispus or Rumex japonica is chopped and extracted with methanol, and the methanol extract obtained by concentration under reduced pressure is dissolved in distilled water and then hexane or dichloromethane Extract with an organic solvent, and the extract thus obtained is concentrated under reduced pressure. The concentrate was chromatographed (developing solvent: hexane: acetone: methanol mixture) to obtain an active fraction, which was then chromatographed using hexane: ethyl acetate (about 3: 1, v / v) as a developing solvent to form chrysopanol. And parietin can be purified purely.

Chrysophanol or parietin of the present invention exhibits excellent control against powdery mildew when foliar sprayed on plants at a concentration of 0.12 μg / ml to 200 μg / ml.

Hereinafter, the specific configuration and operation of the present invention will be described with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1 Isolation and Purification of Antimicrobial Active Materials

5 kg of the sample obtained by drying 10 kg of Rumex crispus in vivo was crushed with a blender, and then 10 L of methanol was added thereto and stored at room temperature for 7 days. The methanol extract was filtered using filter paper, and the filtrate was concentrated under reduced pressure.

Methanol extract of Rumex crispus was dissolved in 3 L of distilled water, and then extracted three times with 3 L of hexane and dichloromethane, respectively. The extracts were combined and concentrated under reduced pressure to obtain 33.44 g of a sample. Got it. The obtained sample was added to silica gel column chromatography (5.0 × 60 cm, Kaigel gel 60, 70-230 mesh, 500 g: Merck), followed by hexane: acetone: methanol (70: 9: 1, 50: 9: 1, v / v / v) was eluted with developing solvent. The eluate was separated and analyzed by thin layer chromatography (Kaygel gel 60 F254, 0.25 mm; Merck) using hexane: acetone: methanol (50: 9: 1, v / v / v) as the developing solvent. According to the pattern of TLC divided into five fractions, such as F1, F2, F3, F4 and F5, each fraction was treated in an amount of 100 ㎍ / ㎖ as in Example 1 to assay the antimicrobial activity in vivo against plant powdery mildew F1 sample (3.77 g) and F2 sample (5.53 g) showing high antimicrobial activity were obtained.

F1 sample and F2 sample were added to silica gel column chromatography (5.0 × 60 cm, Kaigel gel 60, 70-230 mesh, 400 g), and then hexane: ethyl acetate (3: 1) was separated with a developing solvent. 0.96 g of the orange substance, 1.37 g of the orange substance and 0.36 g of the yellow substance were separated purely from the F2 sample.

Example 2 Structure Determination of Chrysophanol and Parietin

The two purely separated actives were subjected to mass spectrometry using a mass spectrometer (JEOL JMS-DX303; JEOL Ltd., Tokyo, Japan). The electron impact mass spectrum revealed that the two active substances had molecular weights of 254 (identified as chrysopanol) and 284 (identified as parietin), and the molecular formulas were C ₁₅ H ₁₀ O ₄ and C _{16, respectively.} Estimated to H ₁₂ O ₅ ( FIGS. 1 and 2 ).

In addition, to determine the complete molecular structure of the two active substances, 20 mg of each active substance was completely dried, dissolved in CDCl _{3, and placed} in a 5 mm nuclear magnetic resonance (NMR) tube, followed by NMR analysis (Bruker-AMX 500 (500 MHz). ). ¹ H-NMR was measured at 500 MHz and ¹³ C-NMR at 125 MHz. The two active substances were determined to be chrysophanol and parietin, respectively, via hydrogen nuclear magnetic resonance spectra ( FIGS. 3 and 4 ).

Example 3: Control Effect of Chrysopanol and Parietin on Plant Disease

Example 1 I of Cree sofa play and Paris ethynyl away from the living body (in vivo) rice blast, rice sheath pattern in order to investigate the antimicrobial activity in blight, tomato gray mold, tomato late blight, wheat leaf rust, and wheat powdery mildew, etc. Experiments were conducted on six plant diseases. Chrysophanol or parietin was dissolved in acetone and then added to an aqueous solution containing Tween 20 at a concentration of 250 μg / ml to control the concentration of chrysophanol or parietin to 50 to 200 μg / ml. The final acetone concentration of the sample was adjusted to 10%. As a control, a solution containing 10% acetone and 250 μg / ml Tween 20 was used. Two pots were used for each plant disease, and medicinal sprays of foliar were air-dried for 24 hours and then inoculated with each plant pathogen.

Rice pots, tomatoes, barley, and wheat were sown in a plastic pot of 4.5 cm in diameter with about 70% of water or gardening soil, and grown in a greenhouse at 25 ± 5 ° C for 1 to 3 weeks. Rice blasts are seedlings of the 2nd to 3rd leaves and Magnaporte gricia, the causative agent of blasts.Magnaporthe grisea,Spore Suspension (5 × 10)⁵Spores / ml) were spray inoculated, treated for one day on a 25 ° C. humidifier, followed by incubation for 5 days in a constant temperature chamber at 25 ° C. to induce onset. Rice leaf herbaceous blight is the three leaf seedlings, which is the causative agent of leaf herbaceous blight, which is tannate porous cucurumeris (Thanatephorus cucumeris,Korea Research Institute of Chemical Technology) was inoculated with a medium obtained by incubating for 7 days (90 g of wheat bran, 15 g of chaff, 100 ml of distilled water), and treated for 4 days in a humidified environment at 25 ° C. for 4 days in a constant temperature room at 25 ° C. Incubation was induced by culturing. Tomato blight is a phytophthora infestans, a causative agent of late blight on tomato seedlings.Phytophthora infestans,Kangnung National University Yuzu sleeping bag (10⁵The influx of influenza suspension extracted from the saccharin sac / ml) was inoculated and treated for 4 days in a humidified environment at 25 ° C. to induce the onset. Tomato gray mold disease is caused by Botrytis disease, a causative agent of gray mold disease in tomato seedlings.Botrytis cinerea,Spore Suspension of Korea Research Institute of Chemical Technology⁶Spores / ml), and onset for 3 days on 20 ° C. Wheat rust is a fungus that is known to be a parasitic bacterium in leafy seedlings.Puccinia recondita,Incheon University) spores were sprayed with 250 μg / ml Tween 20 solution in an amount of 0.67 g spores / l, sprayed and induced for one day in a 20 ° C. wet room, and transferred to a constant temperature room at 20 ° C. for 6 days. . Barley powdery mildew disease is the etiology of E. coli, which is a causative agent of powdery mildew, passaged from host plants to seedlings of barley.Erysiphe graminisf. sp.hordei,The inoculation was carried out by shaking the spores of the Korea Research Institute of Chemical Technology) and induced the onset in a room temperature of 20 ℃.

After 7 days of rice blast, wheat red rust, and wheat flour, 8 days after rice leaf blight, and 3 days and 4 days after tomato gray mold and tomato late blight, the disease area ratios were examined.

The results of the control effect of chrysopanol and parietin on each plant disease are shown in Table 1 .

Plant diseases Concentration (µg / ml) Rice fever Rice leaf blight Tomato gray mold Tomato plague Wheat Red Rust Wheat flour Chrysopanol 50 65 0 21 25 0 98 100 75 0 21 16 20 98 200 80 0 14 25 7 100 Parietin 50 40 0 8 0 0 100 100 50 0 0 0 0 100 200 20 0 0 0 0 100

As shown in the above results, chrysopanol and parietin showed high antimicrobial activity against barley powdery mildew, especially parietin showed 100% control effect at all tested concentrations. In addition to barley flour, chrysopanol has shown some control effects against rice blasts.

Example 4 50% Control Concentration for Barley Powder Disease (EC ₅₀ )

In order to determine the 50% control concentrations of chrysopanol and parietin against barley powdery mildew, experiments were carried out at various concentrations on barley powdery mildew as in Example 3 as follows. Chrysophanol was treated with 3.12 μg / ml at the highest concentration with 100 μg / ml at the highest concentration, and parietin at 0.12 μg with 30 μg / ml at the highest concentration. / Ml was treated to the lowest concentration. As a control drug, fenarimol, known as fenarimol (manufactured by Syngenta, USA), was used, and the treatment concentration was the same as that of parietin. The control group was treated with Tween 20 solution containing 10% acetone at a concentration of 250 μg / ml. The drug was treated one day before the inoculation, incubated in a constant temperature room at 20 ° C. for 7 days, and then examined for incidence. All treatments were repeated five times, and after obtaining the control value for each experiment, 50% control agent concentration was obtained, and the results are shown in Table 2 .

Active substance EC ₅₀ (μg / ml) Chrysopanol 4.7 Parietin 0.48 Fenanri Mall (Control) 0.15

As in the above results, parietin showed lower antibacterial activity than feminarimol, but the 50% control drug concentration was 1 μg / ml or less, indicating a very strong antimicrobial activity.

Example 5 Persistence and Therapeutic Effect of Chrysopanol and Pariethins on Barley Powder Disease

In order to investigate the persistence and therapeutic effect of chrysopanol and parietin on barley powdery mildew, the same experiment as in Example 3 was carried out by foliar spray treatment of the drug 7 days, 4 days or 1 day before strain inoculation of barley seedlings. Was carried out.

In addition, in order to investigate the treatment effect of plant powdery mildew by chrysopanol and parietin, barley powdery mildew was inoculated first, and then, after 1 or 2 days, the medicated leaf was sprayed. Seedlings of barley used in this experiment were sown on the same day, and each experiment was repeated five times. The control group was treated with a 250 μg / ml Tween 20 solution containing 10% acetone, and feminarimol was used as a control. The results are shown in Table 3 .

Active substance Concentration (µg / ml) Drug treatment time and control effect (%) (± standard deviation) Pre-inoculation treatment Post Inoculation Treatment 7 days 4 days 1 day 1 day 2 days Chrysopanol 50 18 ± 21 72 ± 7.5 97 ± 2.1 78 ± 5.4 44 ± 12 Parietin 5 53 ± 5.3 100 ± 0 100 ± 0 47 ± 5.3 33 ± 0 Fenanri Mall (Control) 5 60 ± 8.0 100 ± 0 100 ± 0 100 ± 0 100 ± 0

As shown in Table 3, chrysopanol and parietin according to the present invention have some lasting and therapeutic effects. Particularly, parietin has a slightly lower therapeutic effect compared to feminarimol, but the sustaining effect is similar. .

Example 6: Control Effects of Chrysophanol and Parietin on Cucumber Powdery mildew

In order to investigate the control activities of chrysopanol and parietin on cucumber powdery mildew disease, experiments were conducted using adult cucumbers. Inoculation of Sphaerotheca fuliginea , a causative agent of powdery mildew, was caused by natural infection, and in the case of pharmaceutical treatment, chrysophanol was used at concentrations of 100 μg / ml or 33.3 μg / ml, parietin and feminarimol. Polyoxin B was sprayed onto the foliar surface of infected adult cucumber at a concentration of 30 μg / ml or 10 μg / ml and polyoxin B at a concentration of 100 μg / ml or 33.3 μg / ml. After treatment with the drug, the disease was induced in the greenhouse at 25 ± 5 ° C for 1 week, followed by a second treatment to induce the disease for another week. Subcutaneous aging of cucumbers excluded one and two leaves, and the lesions of three to fifteen leaves were examined. The results are shown in Table 4 below.

Active substance Concentration (µg / ml) Control price (%) Chrysopanol 100 78 ± 9.6 33.3 47 ± 9.1 Parietin 30 64 ± 8.3 10 66 ± 6.3 Reference drug Feminari mall 30 62 ± 5.8 10 28 ± 11 Polyoxine ^* B 100 26 ± 8.7 33.3 6.9 ± 9.9 *: Dongbu Hannong Chemical Co., Ltd.

As in the above results, chrysopanol and parietin showed excellent control activity against cucumber powdery mildew. In particular, parietin showed better control activity than feminarimol used as a control agent, and chrysophanol also showed better control activity than polyoxine B.

Plant powdery mildew control composition containing chrysophanol and parietin according to the present invention as an active ingredient is very effective in controlling plant powdery mildew.

Claims (4)

Plant powdery mildew control composition containing chrysophanol of formula (1) or parietin (parietin) of formula (2) as an active ingredient. Formula 1 Formula 2 The method of claim 1, The plant powdery mildew is a composition characterized in that the powdery mildew or cucumber powdery mildew. The method of claim 1, A composition comprising chrysopanol or parietin at a concentration of 0.12 μg / ml to 200 μg / ml. The method of claim 1, Chrysophanol or parietin, (1) Rumex crispus and Rumex japonica roots were chopped, extracted with methanol, concentrated under reduced pressure, (2) Methanol extract was dissolved in distilled water, extracted with hexane or dichloromethane and concentrated under reduced pressure, (3) The obtained extract was subjected to column chromatography using a hexane: acetone: methanol mixed solvent to obtain an antimicrobial fraction, (4) fractionation of the obtained antimicrobial active fraction by chromatography using a hexane: ethyl acetate mixed solvent Composition obtained by a process comprising a.