KR100760444B1 - Red Pepper Anthrax Control Composition Using Anemarrhena asphodeloides Leaf Extract and Separation Compounds - Google Patents

Abstract

The present invention relates to an extract of Anemarrhena asphodeloides leaf having an activity against red pepper anthrax and a method for preparing an active fraction and a single compound from the extract. More specifically, the extract is extracted with methanol and concentrated under reduced pressure. Butanol fractions obtained by sequentially extracting and extracting various organic solvents and gitonin purely separated from these fractions can be effectively controlled by treating pepper anthrax.

Jimo, Chilli, Anthrax, Antifungal, Prevention, Control

Description

Composition for Control Anthracnose (Colletotrichum gloeosporioides) with extracts and isolated compound from leaves of Anemarrhena asphodeloides}

Figure 1 is a schematic of the methanol extract preparation method of the leaves.

Figure 2 is a schematic diagram of a method for producing the hexane, ethyl acetate, butanol, and water fractions extracted from methanol extract of the hair leaves.

Figure 3 is a schematic of a method for pure separation of gitonin (gitonin) by column chromatography from the butanol fraction of the leaves of the hair.

Figure 4 shows the antifungal activity against red pepper anthracnose fungi of hexane, ethyl acetate, butanol, and water fractions extracted from methanol extract of gimo leaves.

Figure 5 shows the antifungal activity of pepper anthrax fungi according to the concentration of the compound gitonin (gitonin) isolated from the leaves.

Figure 6 shows the antifungal activity of pepper anthrax fungi against red pepper pulp at different concentrations of the compound gitonin (gitonin) isolated from the leaves.

Figure 7 shows the nuclear magnetic resonance (NMR) analysis and fast atomic bombardment (FAB) mass spectrometry of the compound gitonin isolated from the leaves of the hair.

Figure 8 is a schematic of the chemical structure of the compound gitonin (kitonin) isolated from the leaves of the hair.

The present invention relates to a pepper anthracnose control agent mainly containing natural products, and more particularly, fractions obtained by extracting the leaves of Anemarrhena asphodeloides with methanol, and concentrated under reduced pressure, and the compounds separated from the fractions. It relates to a pepper anthrax control agent using.

Chemical synthetic pesticides are effective in controlling pests and germs in the short term, but in the long term, they cause resistance to pests and germs, causing more and more toxic pesticides to be used or controlled. In addition, chemical synthetic pesticides destroy natural food chains and exterminate microorganisms beneficial to natural organisms and plants.

Anthracnose is a disease that occurs mainly in crops such as apples, grapes, peppers, and watermelons. It occurs frequently in hot and humid climates. It is mainly in the genus Collectotrichum and in the genus Gloosporium. Mold is the cause. The disease results in spots on the leaves, stems, fruits, and flowers of the plant, which grow in size, shrinking tissue and causing the plant to wither and die.

Pepper anthrax begins to occur around mid-June, when the flowers begin to bloom. The fungus causes anthrax to multiply rapidly in a humid environment, so it must be sprayed with pesticides after rain. In general, pepper farmers spray several times a fungicide between June and August to control anthrax. Nevertheless, the causative fungus of anthrax is increasingly resistant to chemical synthetic fungicides and spreads easily from the areas where the pesticides are not sprayed to the entire field.

Therefore, the present inventors confirmed that the methanol extract obtained by extracting the gimo leaves with methanol and concentrated under reduced pressure has antifungal activity against pepper anthrax. The methanol extract is suspended in water and distributed sequentially with hexane, ethyl acetate, and butanol solvent. Among the fractions of the hexane layer, ethyl acetate layer, butanol layer and water layer obtained from the extract, it was confirmed that the butanol fraction has antifungal activity against pepper anthrax, and the butanol fraction has antifungal activity against pepper anthrax by column chromatography. After the material was purely separated, the chemical structure was identified to complete the present invention.

The present invention is to provide a pepper anthracnose control agent which replaces the chemical synthetic pesticides and has a natural product as a main component. In other words, the mycelial growth inhibitory activity of pepper extracts of methanol extract of methanol extract extracted with methanol was investigated, and the hexane layer obtained by sequentially dispensing hexane, ethyl acetate and butane as a solvent by suspending methanol extract in water, Among the fractions of ethyl acetate layer, butanol layer, and water layer, butanol fractions having excellent antifungal activity against pepper anthracnose disease were separated by column chromatography. To identify the chemical structure.

The present invention is an antifungal activity assay for the pepper anthrax of methanol extract obtained by extracting Anemarrhena asphodeloides with methanol, and the hexane layer, ethyl acetate obtained by sequentially extracting the extract from the methanol extract using hexane, ethyl acetate, butanol solvent. Antifungal Activity Test of Red Pepper Anthrax Disease and Fractions of Butanol and Water Layers, and Butanol Fractions with High Antifungal Activity by Column Chromatography Antifungal activity assay for pepper anthrax by concentration was performed.

Example 1 Pathogen Spore Harvesting

The strain used in the experiment was pepper anthrax (Colletotrichum gloeosporioides, KACC strain number 40003), which was distributed by the Korea Agricultural Microbiological Resource Center (KACC), RDA, and used as an experimental pathogen. Spreading the pepper anthrax pathogens were inoculated in the medium (PDA, potato dextrose agar) medium and incubated in the incubator (Vision Science, VS-1203P4N) for 26 days in the dark state for 7 days to form spores. The spores formed were harvested by the addition of sterile distilled water, and the spore concentration was measured with a hemocytometer under an optical microscope to make a spore suspension of 1 × 10 ⁷ spores / ml and stored in a refrigerator (4 ° C). Used.

Example 2 Room Test of Pepper Anthrax Antifungal Activity of Gemini Methanol Extract

The hairs collected in late July were divided into leaves and roots, cut into 3 ~ 4 cm, and shaded. 10 g of each of the dried hair leaves and roots were ground with a mill (Hanil, FM680T), and 100 ml of methanol (100%) was added thereto, followed by extraction at room temperature for 24 hours, followed by filtration with a filter paper (Whatman No. 3). Methanol extract, which was the filtrate obtained by repeating the above extraction filtration operation twice, was concentrated under reduced pressure at 40 ° C. using a vacuum concentrator to obtain 1.5 g and 1.2 g of the dried methanol extract, respectively. Accurately weigh each of these extracts by 0.1g, dissolve in 10ml of methanol and chloroform 1: 1 (v: v) solution to make 10,000ppm, and then absorb 50µl of circular paper (Advantec, 8mm) and dry it. Soured.

Preparation of Potato dextrose agar (PDA) medium was dissolved 39 g of PDA (Difco co.) In 1 liter of distilled water, sterilized at 121 ° C for 15 minutes (Autoclave) and cooled to 50 ° C. The concentration was adjusted to 1 × 10 ⁴ spores / ml, and the culture medium was uniformly dispensed in a disposable petri dish (green cross, 9 cm) before the medium solidified, and used as an experimental medium. A circular paper (Advantec, 8mm) absorbed from methanol extract samples of the leaves and roots was placed on the above experimental medium and cultured in an incubator at 26 ° C. for 3 days, and then visually observed to measure the diameter of the inhibitory ring (clear zone). One result is shown in Table 1.

Table 1. Antifungal Activity Assays of Methanol Extracts from Leaves and Roots of Red Pepper Anthrax Against Pepper Anthrax

As a result of assaying antifungal activity of methanol extract of root and root against red pepper anthrax, it was determined that methanol extract of leaf showed strong activity, and there was a substance that inhibited growth and spore germination of pepper anthrax on leaf.

Example 3 Anti-fungal Activity of Red Pepper Anthrax Disease of Fractions Extracted from Methanol Extracts of Hairy Leaves

As in Example 2, 360 g of dried leaves of Anemarrhena asphodeloides were pulverized, and 6 L of methanol (100%) was extracted for 24 hours at room temperature, followed by filtration with a filter paper (Whatman No. 3). The methanol extract obtained by repeating the above extraction filtration operation twice was concentrated under reduced pressure at 40 ° C. using a vacuum concentrator to obtain 58 g of dried methanol extract. 51 g of this methanol extract was weighed and suspended in 1 L of water, and then placed in a 3 L separatory funnel, and 1 L of hexane was added thereto. After 24 hours, the hexane layer was recovered and concentrated at 80 ° C. 4.46 g of dried hexane fractions were obtained. After the extraction of the nucleic acid, the remaining water suspension was placed in a separatory funnel with 1 L of ethyl acetate solvent, and after 24 hours, the ethyl acetate layer was recovered and concentrated under reduced pressure at 38 ° C. The ethyl acetate fraction was dried twice. Got. After the extraction of ethyl acetate, the remaining water suspension was added 1 L of butanol solvent to the separatory funnel, and after 24 hours, the butanol layer was recovered and concentrated under reduced pressure at 60 ° C. The above procedure was repeated twice to obtain 20.07 g of butanol fraction. The water suspension remaining after the butanol partition extraction was concentrated under reduced pressure at 50 ° C. to obtain 24.57 g of dried water fractions. Hexane, ethyl acetate, butanol, and water fractions obtained by partition extraction in this manner were prepared at a concentration of 10,000 ppm to assay antifungal activity of pepper anthrax, showing strong antifungal activity in the butanol fraction as shown in Table 2.

In view of the above results, it was determined that the butanol fraction contained the substance showing the antifungal activity in the butanol fraction among the fractions of the hexane layer, the ethyl acetate layer, the butanol layer, and the water layer obtained by partitioning and extracting the methanol extract of the gelatin leaf with the solvent.

Table 2. Antifungal Activity Assay of Red Pepper Anthrax Against Fractions of Methanol Extracts from Gemini Leaf

Example 4 Isolation, Structure Identification, and Antifungal Activity Assay of Red Pepper Anthrax Antifungal Activity

The separation of pure compounds showing pepper antithrax antifungal activity in butanol fraction is as follows. The butanol fraction of Example 3 (20 g) was mixed with silica gel (Merck, 7734) to prepare a fine powder from induction, and then placed on top of a silica column-filled glass column (5 cm in diameter) and subjected to column chromatography. Separation was carried out by the method. The solvent was first flowed into the ratio of chloroform and methanol at a ratio of 10 to 1, and then chloroform and methanol were sequentially flowed in a ratio of 5 to 1, 2 to 1, and 1 to 1, and 400 ml of each was collected in each test tube. The obtained solution was assayed by the antifungal activity indoor assay method of Example 2, and the isolates showing the activity were collected and concentrated, and again separated by column chromatography using ODS gel (ODS gel, Yamazen 50 µm). It was. The solvent was poured into each test tube with 100 ml of water and methanol in a ratio of 4 to 1, then in a ratio of 2 to 1 and 1 to 1, and finally to 100% methanol. The obtained solution was assayed again by the antifungal activity assay of Example 2, and the isolates showing antifungal activity were collected and concentrated under reduced pressure (40 占 폚) to obtain 60 mg of pure compound.

This pure compound was found in UV light (254, 365 nm) when mobile phase was developed with chloroform: methanol (3: 1, v / v) on thin layer chromatograph (silica gel 60F ₂₅₄ , Merck 0.2mm). When the solution was developed with 15% sulfuric acid (H ₂ SO ₄ : water = 15:85) solution, purple material was identified and the Rf value was 0.1.

Nuclear magnetic resonance (NMR) analysis and high-speed atomic bomb (FAB) mass spectrometry were used to identify chemical structures of pure compounds. The nuclear magnetic resonance spectrometer was a Bruker Advance Digital 400 spectrometer. (400MHz, USA) was used, and pure compounds were analyzed by dissolving in pyridine-d ₅ substituted with deuterium. The high-speed atomic bomb (FAB) mass spectrometer used a JEOL JMS 700 mass spectrometer (Japan). In the proton ( ¹ H) nuclear magnetic resonance (NMR) spectrum, two singlet signals due to methyl groups in the high magnetic field were observed at 0.494 and 0.612 ppm. In the carbon ( ¹³ C) nuclear magnetic resonance (NMR) spectrum, a total of 50 carbon signals were observed, of which carbon peaks attributable to ketal carbon were observed at 110.06 ppm. ) It was confirmed that the steroid saponin (steroid saponin) system. Fast atom bombardment (FAB) mass spectrometry showed that [M.Na] ⁺ was found at m / z 1,073 and published in Wang Y., O. Kazuhiro, K. Ryoji and Y. Kazuo. 1996. In a comprehensive comparison with Steroidal Saponins from Fruits of Tribulus Terrestris.Phytochemistry 42 (5): 1417-1422), this compound is a combination of gitonin (gitonin, (25R, S) -5α-spirostan-2α, 3β-diol-3 -O-β-D-galactopyranosyl (1-2)-[β-D-xylopyranosyl (1-3)]-β-D-glucopyranosyl (1-4) -β-D-galactopyranoside)).

The results of the indoor assay of antifungal activity according to the treatment concentrations for pepper anthrax cultivated in PDA medium of Gitonin compounds, which were identified by pure separation from the above, are shown in Table 3.

The antifungal activity assay of gitonin treated with red pepper anthrax showed a strong activity even at 50 ppm and at lower concentrations than that of Propineb, which is registered as an anthrax pesticide.

Table 3. Antifungal Activity of Red Pepper Anthrax by Treatment Concentrations of Gitonin Isolated from Leaves

Example 6 Anti-fungal Activity Assay of Red Pepper Anthrax by Treatment of Red Chili Pepper from Gitonin Compound Isolated from Hair

20 mg of Gitonin isolated from the hair was dissolved in 200 ml of water, and 5 µl of Tween 20 solution was added thereto to adjust the concentration of the base material to 1,000 ppm, and the solution of this concentration was 50 ppm and 100 ppm. Diluted to 250 ppm, 500 ppm.

Pepper anthracnose pathogen of Example 1 was inoculated in a medium (PDA) medium and cultured in a 26 ℃ thermostat (cancer) for 7 days to form spores. The spores formed were harvested by the addition of sterile distilled water, and spore suspensions were irradiated with a hemocytometer under a light microscope to obtain 1 × 10 ⁷ spores / ml. Pathogens were inoculated with a spore suspension on the red pepper flesh with a needle, and then spores were inoculated. The spores were inoculated at a concentration of 50, 100, 250, 500 ppm on the inoculation part.

The peppers treated in this way were incubated at 26 ° C. for 5 days, and then the lesion area ratio was investigated. The effect of Gitonin on pepper anthrax was calculated by the control value according to the following equation.

Control value (%) = (lesion area ratio of 1-treatment zone / lesion area ratio of no treatment zone) × 100

As shown in Table 4, the control value of Gitonin from red pepper leaves was 95% at 250ppm of Gitonin compound and 98% at 50ppm of dithianon, a conventional pesticide. The response was lower than that of Propineb.

As a result, the butanol fraction obtained by extracting the gimo leaves with methanol, concentrated under reduced pressure, and then sequentially partitioned with various organic solvents, and Gitonin purely separated from these fractions were treated effectively with pepper anthrax. I can control it.

Table 4. Antifungal Activity Test of Gitonin from Pepper Leaves on Pepper Anthrax

As described above, in the present invention, the methanol extract extracted from the hair leaves, butanol fraction obtained from the methanol extract, and the single compound, Gitonin, which is purely separated from the butanol fraction, inhibit the mycelial growth of fungal anthracnose fungus. Can be used as a preventive and control agent for pepper anthrax using fractions or gitonin substances and is a very useful invention for the natural pesticide industry.

Claims (3)

Chilli anthracnose control composition comprising methanol extract obtained by extracting Anemarrhena asphodeloides leaves with methanol and concentrated under reduced pressure The composition of claim 1, wherein the methanol extract is suspended in water, partitioned and extracted with hexane and ethyl acetate, and the remaining water suspension is partitioned and extracted with butanol, followed by concentration under reduced pressure. The method of claim 2, wherein the butanol fraction is a pepper anthracnose control composition comprising a steroid saponin-based gitonin compound obtained by separating the column chromatography method

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