Method for producing extracts containing strobilurin derivatives from the fungus Favolaschia sp.

Abstract

Page 1 of 10 Invention details A name that reflects an invention. Method for producing extracts containing strobilurin derivatives from the fungus Favolaschia sp. Branches of science related to invention. 5. Biotechnology and chemistry related to methods for producing extracts containing derivative compounds. Strobilurins from the fungus Favolaschia sp. (Method for the production of crude extracts) comprisingofstrobilurinderivativesfromthethefungiFavolaschiasp.) Background in the relevant art or science. Agricultural production is a key driver of the global economy; achieving good agricultural yields is crucial. Therefore, 10 is a key target for farmers, but a major obstacle causes losses in agricultural production. Due to problems caused by diseases and pests, controlling plant diseases that negatively affect crop yields is crucial. In particular, fungal plant diseases are another major cause affecting agricultural productivity. Currently, chemicals are used to prevent and control fungal plant diseases in order to increase yield and reduce losses. Losses from agricultural production in the global market have led to the development of a variety of synthetic chemicals to inhibit fungal growth. 15. There are many plant disease control products sold in the market, and it has been found that if these synthetic chemicals leave residues that can cause problems... The accumulation of these substances will have a significant negative impact on both living organisms and the environment; therefore, due to these factors... Therefore, research is necessary to find natural products that are safe for consumption. Prevent and control plant diseases to achieve good yields with minimal impact on living organisms and the environment. Research reports indicate that natural products derived from fungi possess good pharmacological activity and are potentially beneficial. 20. They are diverse and highly safe because they are biological substances created through biosynthesis. The survival of living organisms is ensured by strobilurin, another type of bioactive substance. In particular, its antifungal activity (Sauter,H.et.al.EvolutionofaNewClassofActiveSubstances). (Angew.Chem.,Int.Ed.1999,38,1328-1349) found that this substance was isolated from fungi in the Basidiomycetes group. (Basidiomycetes), which are important sources of the bioactive compound strobilurin, can be isolated from nature. 25. It was released as a single compound by chromatography, and its chemical structure was proven using chemical techniques. Spectroscopy, along with molecular formula analysis using high-resolution mass spectrometry and principal structure analysis. Chemically, it contains a beta-methoxyacrylate group that exhibits a relationship with... Biological activity (Balba,HJReviewofStrobilurinFungicideChemicals.Environ.Sci. However, some research has found that strobilurin in the form of certain single compounds... Thirty species, when left for a period of time, show decreased stability and reduced biological activity. too Page 2 of 10. Preliminary research by the research group has found that strobilurins become more stable when in the form of... Furthermore, there have never been any reports on the use of fungal extracts containing strobilurins. The components were previously used to inhibit plant pathogens; therefore, this invention developed a manufacturing process. An extract from the fungal strain Favolaschia calocera (BCC36684) containing derivative compounds. 5-Strobilurin has inhibitory activity against the leaf spot pathogen (Alternaria brassicicola, ABr), and its use is in the form of an extract. Reducing the separation steps to obtain unstable single compounds and increasing efficiency. Its good biological activity can be further developed for use as an inhibitor of plant pathogens. The nature and purpose of the invention. This invention relates to a method for producing extracts containing strobilurin derivatives from fungi. 10. Favolaschia sp., which is composed of Favolaschia sp. fungi cultured on a growth medium. The product is then filtered to separate the liquid first phase of the fungal product from the solid second phase of the fungal product. Solid and fractional extraction of first and / or second part fungal products using solvents. Organic methods are used to obtain the first and / or second extract products, respectively. In one form, methods for producing extracts containing strobilurin derivatives may be further developed. 15. The strobilurin derivative components of the extracted product were analyzed using the liquid analysis technique. High-performance liquid chromatography connected to a UV detector. This invention aims to produce an extract containing strobilurin derivatives from fungi. Favolaschia sp., particularly the fungal strain Favolaschia calocera (BCC36684), has inhibitory activity. The leaf spot pathogen caused by the fungus Alternaria brassicicola can be further developed for use as a substance. 20 biological products that inhibit plant pathogens, are environmentally friendly, and help reduce the use of chemical pesticides. Brief description of the painting. Figure 1 shows the chemical structure of the standard substance strobilurin. Figure 2 shows the chromatogram of the strobilurin standard substance using high-performance liquid chromatography. With a UV detector. Figure 3 shows the chromatogram of the broth extract from the fungus Favolaschia calocera. (BCC36684) cultured in PYGM medium under static conditions for 40 days using the technique... High-performance liquid chromatography connected to a UV detector was used to compare the results with the standard substance strobilurin. Figure 4 shows the chromatogram of the cell extract from the fungus Favolaschia calocera (BCC). 36684) cultured in PYGM medium under static conditions for 40 days using the liquid technique. 30-stage high-performance chromatography connected to a UV detector, compared with the standard substance strobilurin. Page 3 of 10. Full disclosure of the invention. This invention relates to a method for producing an extract from the fungal species Favolaschia sp. containing... Strobillurin derivatives exhibiting inhibitory activity against the leaf spot pathogen (Alternaria brassicicola, ABr) through culture. Fungi under various conditions and the identification of strobilurin derivatives by comparison with standard substances. 5-Strobilurin (Figure 1) using high-performance liquid chromatography technique connected to a UV detector under the following conditions: appropriate Method for producing extracts containing strobilurin derivatives from the fungus Favolaschia sp. Methods for producing extracts containing strobilurin derivatives from the fungus Favolaschia sp. The methods include... The steps involved include: 10a. The cultured Favolaschia sp. fungus is filtered to separate the product. The first part of the fungus, which is liquid, is separated from the second part, which is solid, and b. Extraction of the first and / or second fungal products using organic solvents. To obtain the first extract product and / or the second extract product, respectively. In one form, methods for producing extracts containing strobilurin derivatives may be further developed. 15. By analyzing the strobilurin derivative components of the extracted product. The fungus Favolaschia sp. was selected from Favolaschia calocera. Each step has the following additional details: The process of cultivating the fungus Favolaschia sp. This invention involved experiments to find the optimal conditions for obtaining an extract with antimicrobial activity. The 20 best plant diseases for which suitable fungal culture methods are used to produce strobilurin derivatives include: The fungal hyphae or spores are transplanted into a liquid culture medium and incubated under aerobic conditions. As follows: Transplanting the mycelium or spores of the fungus Favolaschia sp. into liquid medium or inoculum medium. Nutritional supplements such as liquid minimum salt media may be repeated 2 times. 25 times to provide optimal fungal growth as a starting point for secondary metabolite production. Incubation in an aerobic environment by shaking the transplanted fungus at a speed of 200 to 220 revolutions per minute. Minutes (rpm) at a temperature of 20 to 30 degrees Celsius, with the optimal temperature being between 25 and 28 degrees Celsius. For 2 to 15 days, as an option, this inoculum culture can be repeated a second time in a larger culture container. Larger size or a greater number of culture flasks. Page 4 of 10. The obtained inoculum is transferred to a specific culture medium, which is a suitable medium for inoculum formation. In general, the volume of the starting inoculum is approximately 1 / 10 of the volume of the specific culture medium. With the generation of secondary metabolites using selective culture media from PDB, MEB, and PY. One or more combinations of PYGM (polygonal globulin) can be used for fermentation, and the fermentation can be done under static conditions. 5 or shake for 10 to 80 days at a temperature of 20 to 30 degrees Celsius, with the optimal temperature being 25 to 28 degrees. After the appropriate fermentation period, the fermentation product is extracted by filtration. To separate the culture medium and mycelial fractions for further preparation of extracts. The extraction process for preparing extract samples. Extraction of primary and / or secondary fungal products using organic solvents. 10. To obtain the first and / or second extract products, respectively, based on the amount of substance. Strobillurin derivatives can be extracted from both the culture medium and the mycelium through a process... Filter to separate the culture medium (liquid) and fibrous portion (solid), then extract as follows: Extraction of the first part of the fungal product, or the extraction of strobilurin derivatives, from the culture medium. (Liquid) Made by extracting the first part of the fungal product or the (liquid) culture medium using a solvent. 15. Dissolve the organic ethyl acetate at a ratio ranging from 1 to 1-5 by volume; this process can be repeated as an alternative method. The ethyl acetate fraction was distilled twice and then evaporated dry using a vacuum distillation apparatus to obtain the product. Extraction of the first phase, or the extract of the liquid portion of the broth. The extraction of secondary fungal products, or the extraction of strobilurin derivatives, from the mycelium. (Solid) By soaking the fiber section in the organic solvent methanol and incubating at room temperature for 2 days. 20. Next, filter it through filter paper to obtain the methanol fraction. You may choose to use filter paper number 1. Then, take this fraction... Methanol is further concentrated using vacuum distillation to obtain an extract with a specific characteristic. The concentrated liquid was then mixed with distilled water and extracted with ethyl acetate in a ratio of distilled water to ethyl acetate. The acetate ratio is in the range of 1:1-5 by volume, and then the ethyl acetate (top layer) is further concentrated. Using a vacuum distillation apparatus to obtain the secondary extract or cell extract. Page 5 of 10. The procedure for analyzing the strobilurin derivative components of the extract product is as follows: Comparison was made with a standard substance, strobilurin, using high-performance liquid chromatography (HPLC) connected to a sample. UV measurement The measurement of strobilurin derivatives, which are substances that absorb UV light, is possible. 5. High-performance liquid chromatography (HPLC) technique can be used in conjunction with a UV detector under various conditions and phase ratios. Optimal movement (Table 1) is determined using data obtained from recording chemical profiles in the wavelength range of 190-600 Hz. Nanometers were used to compare the chromatograms between the standard substance strobilurin (Figure 2) and the extract. The liquid culture (broth) and fibrous extract (cell) were analyzed using the operating conditions of a liquid reagent. High-performance liquid chromatography is connected to a UV detector as follows: 10-Type Column: C18 type fixed phase packed column, particle size 5 micrometers, diameter... Inner diameter 4.6 mm, length 150 mm. -Column temperature: 35 degrees Celsius -Mobile phase flow rate: 0.6 ml / min -Volume of substance for injection: 2-20 microliters (extract concentration 10 milligrams per milliliter) 15. Wavelengths recorded: 190-600 nanometers. -Wavelength analyzed: 210 nanometers - Mobile phase type A: Water containing 0.05 percent by volume of formic acid. - Mobile phase type B: Acetonitrile with formic acid as an ingredient at 0.05 percent by volume. Table 1 shows the ratio of the type A mobile phase to the type B mobile phase. 20 Time (minutes) Phase movement type A (percentage) Phase movement type B (percentage) 08020 181585 201585 250100 25 300100 20. Testing the inhibitory activity against the leaf spot pathogen (Alternaria brassicicola, ABr). The extract was tested for its inhibitory activity against the leaf spot pathogen (Alternaria brassicicola, ABr) using the inhibitory method. Spore germination inhibition according to the method of Chutrakul et al. (2013). (Chutrakul,C.et.al.Thepotentialofafluorescent-basedapproachforbioassayofantifungal 30 agentsagainstchilianthracnosediseaseinThailand.BiosciBiotechnolBiochem,2013,77,259– 25265.) to find the percentage of fungal inhibition (%inhibition) which can be calculated from equation (1). Page 6 of 10. Inhibition percentage = [1-(FU / FU)] x 100(1) Test sample, control sample. Where FU and FU are the average fluorescence values ​​measured from the sample wells. Test sample, control sample. Test and control samples (dimethylsulfoxide (DMSO) solution, concentration...) 2.5 percent), respectively. 5 examples of producing extracts using the manufacturing method described in this invention. Example 1: Production of an extract from the fungal strain Favolaschia sp. containing derivative compounds. Strobillurin with inhibitory activity against the leaf spot pathogen (Alternaria brassicicola, ABr). 1. The fungal strain Favolaschia sp. was cultured on PDA agar at 25 degrees Celsius. The jelly portion is cut into pieces approximately 1 square centimeter in size and placed into 250-milliliter containers. 10. Each bottle contains three types of liquid culture media: PDB, MEB, and PYGM. 25 ml of starter culture after incubating at 25°C for 7 days while shaking (200 rpm). It will be transferred to a 1000 ml bottle containing 250 ml of the three liquid culture media mentioned above. The liquid formula, experimental conditions, and incubation time are as follows: -Three liquid formula recipes: PDB, MEB, and PYGM (Table 2) 15. Shaking conditions during incubation: 10 and 20 days. -Still condition, incubation time: 20 and 40 days. Table 2 shows the formulas of three liquid formulations: PDB, MEB, and PYGM. Weight by volume Liquid food recipe ingredients (grams per liter) 20 PDB Cassava Starch 4 (PDB) Dextrose 20 Malt extract 6 MEB Maltose 1.8 (MEB) Dextrose 6 25 Yeast extract 1.2 Bactopeptone 5 Yeast extract 20 PYGM Glucose sugar 10 (PYGM) Potassium dihydrogen phosphate (KHPO)1 24 30 Magnesium sulfate heptahydrate (MgSO₄⁻⁷HO) 0.5 42 Page 7 of 10. 2. Extraction to prepare two components: a liquid portion (broth) and a fibrous portion (cell). 2.1. Extraction of strobilurin derivatives from the culture medium (liquid) by taking the portion... The culture medium (liquid) was extracted with the organic solvent ethyl acetate at the following ratio: The 1:1 ratio by volume was repeated twice. The ethyl acetate fraction was then evaporated dry using a distillation apparatus. 5. Vacuum evaporation yields an extract of the liquid medium (broth). 2.2. Extraction of strobilurin derivatives from the fibrous (solid) portion by soaking the fiber portion. Incubate at room temperature for 2 minutes using 100 ml of methanol, an organic solvent. After that, it is filtered through filter paper to obtain the methanol portion. The methanol portion is then used... Concentration can be increased using vacuum distillation to obtain an extract with specific characteristics. 10. It is a concentrated liquid form, and 50 ml of distilled water is added to it. It is then extracted using ethyl acetate. 100 milliliters, then the ethyl acetate portion (the top layer) is further concentrated. Vacuum distillation yields extracts from the fibrous (cell) portion. Example 2: Testing the inhibitory activity of fungal extracts against leaf spot disease (Alternaria brassicicola, ABr). Favolaschia sp. 15. The extract from the fungal strain Favolaschia sp. was tested for its inhibitory activity against the leaf spot pathogen (Alternaria). Follow these steps: 1. Preparation of the inoculum for leaf spot disease (Alternaria brassicicola, ABr). This test used a 25 µl suspension of Alternaria cell spores. Brassicicola DOAC0436(BCC42724) in 12.5 percent minimum salt medium (Minimum -5 20 Salt Medium (MM) was added to a 384-well microplate test well at a concentration of 1x10⁻⁶. Place one milliliter of spores in the container and leave it at room temperature for 3 hours to allow the spores to grow. 2. The inhibitory activity against plant pathogens was tested using the fluorescence emission measurement method of substance 5, (6)Carboxyfluorescenediacetate(CFDA)fluorometric (detection) by preparing the tested sample and diluting it with 2.5 percent dimethyl sulfoxide. Add 25% (dimethylsulfoxide; DMSO) to each prepared test well and incubate for 16 minutes. After 18 hours, add a solution mixed with 40 percent glycerol and 0.05 ml. Molar of 5,(6)carboxyfluorescenediacetate(CFDA) In 25 microliters of 70 percent dimethylsulfoxide (DMSO). Leave it in the dark for 5 to 10 minutes, then rinse the plate with water and add 25 microliters of distilled water. 30. Fluorescence was measured using a SpectraMax M5 microplate reader. (Multi-detection microplate reader, SpectraMax M5) at wavelengths of 485 and 535 nanometers. meters (nm) and then calculate the percentage of fungal inhibition (%inhibition) using equation (1). Page 8 of 10. Inhibition percentage = [1-(FU / FU)] x 100(1) Test sample, control sample. Where FU and FU are the average fluorescence values ​​measured from the sample wells. Test sample, control sample. Test and control samples (dimethylsulfoxide (DMSO) solution) (2.5 percent concentration) respectively. Table 3 shows the results of the inhibitory activity of fungal extracts on leaf spot disease (Alternaria brassicicola, ABr). Varieties of Favolaschia sp. in PYGM medium under different conditions. MIC level 80 Sample sequence 10 (micrograms per milliliter) 1Favolaschiasp._Shake_10 days_PYGM_broth6.25 2Favolaschia sp._Shake_10 days_PYGM_cell50.0 3Favolaschiasp._Shake_20 days_PYGM_broth6.25 4Favolaschiasp._Shake_20 days_PYGM_cell12.50 15 5 Favolaschia sp._Settled_20 days_PYGM_broth12.50 6Favolaschia sp._settled_20 days_PYGM_cell12.50 7Favolaschiasp._Settled_40 days_PYGM_broth6.25 8Favolaschiasp._settled_40 days_PYGM_cell0.781 Standard substances Amphotericin B (0.781) 20 positive Note: MIC is the minimum concentration of a substance that can inhibit the growth of microorganisms. inhibitory concentration) Example 3: Cytotoxicity testing of an active fungal extract from the Favolaschia sp. strain. 10. Inhibits leaf spot disease (Alternaria brassicicola, ABr). 25. Investigation of the cytotoxicity of fungal extract from the strain Favolaschia sp. against Vero cells. (Vero-African green monkey kidney fibroblast) uses a method to measure the amount of green fluorescent protein. Fluorescent protein (GFP) in microplates (Hunt, L. et al. GFP-expressing mammalian cells for fast,sensitive,noninvasivecellgrowthassessmentinakineticmode.Biotechnol.Bioeng.1999, 1565,201-205) Methods for preparing cells. Page 9 of 10. This experiment used cells that produce green fluorescent protein (GFP), which was obtained by transfecting the pEGFP-N1 plasmid. (Clontech) was introduced into Vero cells (ATCCCCL-81) by culturing the cells in a liquid medium containing minimall. 10% Minimalsential Medium (MEM) serum of inactivated calf embryos. Heat-inactivated fetal bovine serum 1 millimolar sodium pyruvate (sodium 5-pyruvate, 2.2 millimolar sodium bicarbonate, and 0.8 milligrams per milliliter. Geneticin was incubated at 37 degrees Celsius in an incubator with controlled humidity and gas concentration. The 5% carbon dioxide-containing cells to be used for cell cytotoxicity testing will be cells in the mature stage. Cells undergoing logphase growth are prepared as suspensions in liquid medium without geniticin. 4 Concentration 3.3 ± 10 cells per milliliter. 10. Vero cytotoxicity test using GFP quantification in microplates. This test method uses a 384-well microplate with the sample to be tested and the reagent prepared. The positive standard ellipticine is provided as a solution at various concentrations by dilution to 5. Add 5% dimethylsulfoxide (DMSO) in three increments, then add... These sample solutions, or 5 percent dimethyl sulfoxide, which is a negative control sample, are added to the microscope. 15 plates, each well containing 5 microliters, were followed by 45 cells prepared in liquid medium per well, as described above. Microliters were then used to incubate the cells for 4 days at 37 degrees Celsius in an incubator with maintained humidity and... A carbon dioxide concentration of 5 percent was measured in fluorescence units after a specified time. (Fluorescence Unit, FU) with a SpectraMax M5 microplate reader at a wavelength... 485 and 535 nanometers, using the average FU measured on the start date of the test as the subtractive value. 20 was taken from the FU value measured on day 4, and the resulting value was used to calculate the percentage of cytotoxicity (%). cytotoxicity) with equation (2) Percentage of cytotoxicity = [1-(FU / FU)] x 100(2) Test sample, control sample. Where FU and FU are the average fluorescence values ​​measured from the sample wells. Test sample, control sample. Test wells and negative control sample wells were used respectively to determine the concentration of the growth-inhibiting substance. 25% of the cells obtained 50% (IC50%) from the graph showing the relationship between substance concentration and percentage values. 50 Cytotoxicity (% cytotoxicity) was determined using SOFTMaxPro software (Moleculard device). Page 10 of 10 pages. Table 4. Results of cytotoxicity testing of the fungal extract of the strain Favolaschia sp. with inhibitory activity. Leaf spot disease (Alternaria brassicicola, ABr) IC level 50 Sample sequence (micrograms per milliliter) 5 1Favolaschiasp._Settled_40 days_PYGM_broth greater than 50 2Favolaschiasp._settled_40 days_PYGM_cell17.74 Positive standard substance Ellipticine 1.26 Note: IC50 is the concentration of a substance that can inhibit cell growth by 50 percent (inhibitory). 50 concentration50) 105. Example 4: Measurement of strobilurin derivative types by comparison with strobilurin standard substances. Bilurin was detected using high-performance liquid chromatography connected to a UV detector derived from fungal extracts. Favolaschia sp. exhibits inhibitory effect on leaf spot disease (Alternaria brassicicola, ABr). Use the data obtained from recording the chemical profile in the wavelength range of 190-600 nanometers using the liquid filtration device. High-performance liquid chromatography (HPLC) connected to a UV detector under optimal conditions was used for comparison. 15 10. Chromatogram comparing the standard substance strobilurin (Figure 2) with the extract of the liquid medium (broth) and other substances. Extraction of the hyphae (cell) from the fungus Favolaschia sp. cultured in PYGM medium under static conditions. After 40 days, it was found that... -The broth extract can produce at least one strobilurin derivative. The three compounds are 9-methoxystrobilurin A (9-MSA), audemancin, and 3-methoxystrobilurin. 20 15A (oudemansinA;OA), and oudemansinI (oI) (Figure 3). -The extract from the fibrous (cell) portion can produce derivatives in the strobilurin group as a component. The main components include at least seven substances, namely 9-methoxystrobilurin A (9-MSA). 9-methoxystrobilurinE (9-MSE), 9-methoxystrobilurinG (9- methoxystrobilurinG;9-MSG),strobilurinG(strobilurinG;SG),audemancinA 2520(oudemansinA;OA), oudemansinG(oudemansinG;OG), and oudemansinI (oudemansinI;OI)(Figure 4) The best way to invent something. As previously stated in the section on full disclosure of the invention.

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