Soraphen a and its preparation method and application

Abstract

The present application relates to a kind of new aromatic heteroterpene compound solenopsin A, it has significant bacteriostatic effect on fusarium graminearum, fusarium oxysporum and fusarium sambucinum, can be used to prepare anti-agricultural plant pathogenic fungi drug, with good medicinal prospect.

Description

[0001] This invention is a divisional application of Chinese patent application filed on August 22, 2025, with application number 2025111821626, entitled "A type of *Follium schoenleinii* and its preparation of schoenlein A and its application". Technical Field

[0002] This invention relates to a serotonin A, its preparation method, and its application. Background Technology

[0003] Plant diseases are a significant factor hindering sustainable agricultural development, causing damage to crops and forestry worldwide and incalculable harm to humankind. Plant diseases are typically caused by plant pathogens such as fungi, nematodes, bacteria, and viruses, with fungi being the primary pathogens. Reports indicate that fungal diseases result in a 10% to 16% reduction in global crop yields annually.

[0004] Currently, chemical fungicides remain the primary means of controlling plant fungal diseases. However, long-term and excessive use of chemical fungicides can easily lead to drug resistance in pathogens and poses potential hazards to the environment and human health. Therefore, developing novel, highly efficient, low-toxicity, and environmentally friendly agents against plant pathogenic fungi has become a current research hotspot. Microbial metabolite pesticides, which use metabolites produced by microbial fermentation as active ingredients to control pests, diseases, weeds, rodents, and other harmful organisms or to promote plant growth and development, represent an important direction for future green pesticide research and development.

[0005] Macrofungi are a treasure trove of various bioactive natural products, producing a wide variety of chemical components, including alkaloids, steroids, nucleosides, terpenes, coumarins, and fatty acids. Many of these can effectively control plant diseases, providing abundant natural resources for the development of new biological pesticides and playing an important role in the development of new green fungicides. Summary of the Invention

[0006] The purpose of this invention is to provide a serotonin A for resisting plant fungal pathogens, its preparation method, and its application.

[0007] The present invention adopts the following technical solution: A type of clofenacin A, with the general formula: .

[0008] The application of the above-mentioned serotonin A in the preparation of drugs for the prevention and control of plant fungal diseases.

[0009] A bactericide comprising the aforementioned serotonin A.

[0010] A method for preparing the above-mentioned serotonin A, which utilizes *Fomitopsis serotonin* with accession number CCTCC NO: M20251808 (… Piptoporellus soloniensis )preparation.

[0011] Among them, the Solon micropore fungus ( Piptoporellus soloniensis The accession number is CCTCCNO: M20251808, and it was deposited at the China Center for Type Culture Collection on August 8, 2025, located in Wuhan, Hubei Province, China.

[0012] Furthermore, the preparation method specifically includes the following steps: (1) Take out the preserved Solon small peel tube mother culture test tube, pick out the mycelium block under aseptic conditions and inoculate it on sterilized PDA medium, set the constant temperature incubator at 25℃ and incubate for 7 days; (2) The activated strain was inoculated into sterile PDB liquid culture medium and cultured on a shaker at 25°C and 150 rpm for 7 days as the seed liquid for solid fermentation; (3) Mix rice and water evenly, autoclave at 121℃ for 20 min to obtain rice culture medium; inoculate the seed liquid into the rice culture medium; culture at room temperature for 60 days; (4) Add an equal volume of ethyl acetate to the obtained fermentation product, stir thoroughly, soak for 6 h, then remove the supernatant and evaporate to dryness by rotation. Repeat the extraction 3 times and combine to obtain the total extract. (5) Mix the total extract with ultrapure water in a volume ratio of 1:1 to 1:2, stir evenly, add ethyl acetate in an equal volume to the extract aqueous solution, stir evenly again, let stand for 30 min, take the upper layer solution, repeat the extraction with ethyl acetate three times, then distill the extract under reduced pressure until no ethyl acetate flows out, control the temperature not to exceed 55℃, collect the extract, dry at a temperature below 40℃ to obtain ethyl acetate extract; (6) Mix the ethyl acetate extract with 1.5 to 1.8 times the mass of silica gel of 100 to 200 mesh, stir thoroughly, dry, grind evenly into powder, so that the extract sample is fully adsorbed on the silica gel particles, and then perform silica gel column chromatography at normal pressure. The silica gel column is packed with silica gel of 200 to 300 mesh. Use a petroleum ether / ethyl acetate system for gradient elution. The components eluted in a 10:1 gradient are separated by semi-preparative liquid phase separation to obtain the product, which is serotonin A.

[0013] Furthermore, the semi-preparative liquid chromatography separation conditions are as follows: COSMOSIL C18 column, 250 mm × 10 mm, column temperature 25℃, wavelength 210 nm, MeCN-H2O, 80:20, 3 mL / min. t R =28.0 min.

[0014] The beneficial effects of this invention are as follows: The *Fotomyces solani* of this invention is fermented using rice culture medium, and the resulting fermentation product exhibits significant antibacterial activity against *Fusarium graminearum*, *Fusarium oxysporum*, and *Fusarium verticillatum*. Further purification of the fermentation product using chemical methods yields a novel aromatic heteroterpene compound, clarifying the antibacterial component of the fermentation product and indicating its promising medicinal potential for use in the preparation of drugs against pathogenic fungi of agricultural plants. The preparation process of this invention utilizes abundant and inexpensive raw materials and is simple. Attached Figure Description

[0015] Figure 1 Photograph of wild fruiting bodies.

[0016] Figure 2 This is a photograph of the purified mycelium after isolation and culture.

[0017] Figure 3 This is the high-resolution mass spectrum of the compound.

[0018] Figure 4 For compounds 1 H NMR spectrum.

[0019] Figure 5 For compounds 13 C NMR spectrum.

[0020] Figure 6 The image shows the HMBC spectrum of the compound.

[0021] Figure 7 The HSQC spectrum of the compound is shown.

[0022] Figure 8 For compounds 1 H- 1 H COSY spectrum.

[0023] Figure 9 The NOESY spectrum of the compound is shown. Detailed Implementation

[0024] The present invention can be further understood through the embodiments given below, but they are not intended to limit the invention.

[0025] Example 1: *Follium solani* (Sorrentosporum salina*) Piptoporellus soloniensis Screening and preservation of ) (1) Isolation of strains Fresh fruiting bodies (e.g., ...) were collected from Liaohe River Source National Forest Park, Pingquan City, Chengde City, Hebei Province (longitude E118.5214, latitude N41.3204, altitude 1110 meters). Figure 1(As shown). Rinse the collected fresh fruiting bodies with running water to remove surface dirt and dry them. Then, wipe the cap and stipe with 75% alcohol cotton balls for 1-2 minutes for disinfection. Finally, rinse with sterile water 1-2 times and blot dry with sterile filter paper. In a laminar flow hood, use a sterile scalpel to longitudinally cut the stipe and cap at the junction to expose the internal fleshy tissue. Cut into tissue blocks of about 0.3 cm. Inoculate the tissue blocks onto PDA medium (200 g potato extract, 20 g glucose, 18 g agar, 1000 mL distilled water) using a sterile inoculation needle or forceps and incubate at 25°C in the dark for 4-5 days. Finally, select colonies free of contaminants and with dense, pale yellow mycelium. Use a 0.5 cm diameter sterile punch to create mycelial cakes on the colonies around the tissue blocks. Transfer these to fresh PDA medium and repeat 1-2 times to obtain pure mycelium (e.g., ...). Figure 2 The culture was incubated in the dark at 25℃ for 6 days, and the strain was prepared for use. The strain number was QA-2-29.

[0026] (2) Identification of strains Molecular identification was performed using wild fruiting body tissues and pure cultured mycelia obtained from isolation as materials.

[0027] Wild-collected fruiting bodies were sampled to obtain their mycelial tissue, which was dried at low temperature (45℃). The pure culture was cultured in PDA medium to obtain fresh mycelium, which was then ground with liquid nitrogen. Genomic DNA was extracted using the Ezup column-type super plant genomic DNA extraction kit (Sangon Biotech (Shanghai) Co., Ltd.). The obtained DNA solution (DNA template) was stored at -20℃ for later use.

[0028] ITS-PCR experiments were performed using universal primers ITS1 / ITS4 (ITS1: TCCGTAGGTGAACCTGCGG, ITS4: TCCTCGCTTATTGATATGC) synthesized by Sangon Biotech (Shanghai) Co., Ltd. Amplification was performed on a Biometra PCR instrument. The PCR reaction mixture (total 25 µL) consisted of 12.5 µL 2×Es Taq MasterMix, 1 µL DNA template, 1 µL each of forward and reverse primers (10 µM), and dd H2O to a final volume of 25 µL. The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min → 35 cycles (94℃ denaturation for 45 s, 55℃ annealing for 45 s, 72℃ extension for 1 min) → 72℃ extension for 10 min → storage at 4℃.

[0029] PCR products were directly sent for bidirectional sequencing, which was performed by Sangon Biotech (Shanghai) Co., Ltd. The ITS sequences of the fruiting bodies and mycelia obtained from the sequencing were assembled and submitted to GenBank to obtain the accession number PV842231. Online BLAST homology comparison was performed in the NCBI database, and the ITS sequence of this strain showed a sequence similarity of 99.84% with that of *Fomitopsis lanceolata* (KR605803).

[0030] Based on the morphological identification of the collected samples according to the "Illustrated Handbook of Macrofungi Resources in China", the characteristics of *Fotomyces solonensis* are as follows: The fruiting body is a brown-rot fungus, mainly growing on broad-leaved trees, solitary, annual, with short lateral stalks or sessile, imbricate, leathery when fresh, and corky when dry. The cap is semi-circular or circular, up to 28 cm in diameter and up to 30 mm thick in the center; the surface is milky white when fresh, turning ochre when dry; the edge is sharp, wavy when fresh, and inrolled when dry. The pore surface is milky white when fresh, turning ochre when dry, without refractive reaction; nearly circular, 4-5 per millimeter; the edge is thin or slightly thick, entire. The flesh is creamy white when fresh, fleshy, and pale yellow or pale pinkish-yellow when dry, spongy or corky, up to 20 mm thick. The tubes are the same color as the pore surface and can be up to 10 mm long. The stipe is creamy when fresh, turning light ochre when dry, covered with fine hairs or smooth, reaching up to 2 cm in length and diameter. Basidiospores are 4.8–6 × 2.8–3.8 μm, elliptical, colorless, thin-walled, smooth, non-starchy, and not blue-loving. The mycelial system is dimorphic, with clamp connections in the reproductive hyphae. Both the fruiting body and mycelium have a strong tropical fruit aroma.

[0031] Molecular biological identification revealed that its ITS molecular sequence determination and analysis results were similar to those of *Follium schoensis*. Combining morphological and microscopic characteristics with molecular identification results, strain QA-2-29 was identified as *Follium schoensis*. Piptoporellus soloniensis ).

[0032] The above-mentioned Solon microporous fungus ( Piptoporellus soloniensis It was deposited on August 8, 2025 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20251808, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. Example 2: Preparation method of clopyralid A

[0033] (1) Take out the mother culture tube of *Follium sarmentosum* that has been stored at 4℃ and is covered with hyphae, and pick out a 1cm section under aseptic conditions. 2 The bacterial blocks of different sizes were inoculated onto sterilized PDA medium and incubated at 25°C for 7 days.

[0034] (2) The activated strain was inoculated into four 500 mL Erlenmeyer flasks containing 250 mL of sterile PDB liquid culture medium (200 g potato extract, 20 g glucose, and 1000 mL distilled water), and cultured on a shaker at 25 °C and 150 rpm / min for 7 days as the seed culture for solid fermentation.

[0035] (3) Add 100 g of rice and 90 mL of water to a conical flask (500 mL × 150 mm) and mix well. Autoclave at 121 °C for 20 min and let cool. Inoculate 40 flasks of rice culture medium with each flask of seed liquid. Incubate at room temperature for 60 days.

[0036] (4) The obtained fermentation product was collected in a 50 L iron drum, an equal volume of ethyl acetate was added, and the mixture was stirred thoroughly using a portable mixer. After soaking for 6 h, the supernatant was extracted and evaporated to dryness. The extraction was repeated 3 times and the extracts were combined to obtain the total extract.

[0037] (5) Mix the extract with ultrapure water in a volume ratio of 1:1 to 1:2, stir evenly, add ethyl acetate in an equal volume to the extract aqueous solution, stir evenly again, let stand for 30 min, take the upper layer solution, repeat this process three times with ethyl acetate, then distill the extract under reduced pressure on a rotary evaporator until no ethyl acetate flows out, control the temperature not to exceed 55℃, collect the extract, and dry the obtained product in a vacuum drying oven at a temperature below 40℃ to obtain ethyl acetate extract.

[0038] (6) Mix ethyl acetate extract with 1.5-1.8 times its mass of 100-200 mesh silica gel, stir thoroughly, dry, and grind evenly into powder so that the extract sample is fully adsorbed onto the silica gel particles. Then perform atmospheric pressure silica gel column chromatography. The silica gel column is packed with 200-300 mesh silica gel. Use a petroleum ether / ethyl acetate gradient elution system. The gradient elution process is 100:0, 25:1, 10:1, 5:1, 3:1, 1:1, 0:100. Elute three column volumes for each gradient and collect the eluent for each gradient. Distill under reduced pressure at 45-50℃. The fraction eluted in the above 10:1 gradient is passed through a semi-preparative liquid chromatography system (COSMOSIL column C18, 250 mm × 10 mm, column temperature 25℃, wavelength 210 nm, MeCN-H2O, 80:20, 3 mL / min). t R Separate the product (28.0 min) to obtain serotonin A. Example 3: Structural determination of clopyralid A

[0039] The structure of clofibrin A was determined using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy data (e.g. Figures 3-9 High-resolution mass spectrometry data: HREISMS [M-H]- m / z 431.2438, calcd for C 25 H 36 O6, 431.2434. The nuclear magnetic resonance spectral data are shown in Table 1.

[0040] Table 1. Nuclear magnetic resonance spectral data of serotonin A .

[0041] Based on the above results, the general structural formula of clopyralid A is determined to be: .

[0042] The chemical name of serotonin A is 6-(dimethoxymethyl)-2,4-dihydroxy-3-(((3E,5E)-3,7,11-trimethyldodecano-3,5,10-trien-1-yl)oxy)benzaldehyde, with a molecular weight of 432.2512 and a molecular formula of C2. 25 H 36 O6 is a white powder that is soluble in organic solvents such as methanol, acetonitrile, and chloroform.

[0043] Example 4: Inhibitory effect of serotonin A on plant pathogenic fungi The compound fuscin A was dissolved in DMSO to prepare a stock solution with a concentration of 10,000 μg / mL. This stock solution was then diluted 2 / 3 times with DMSO to create six series of concentration gradients. The different concentrations of fuscin A solutions were added to PDA medium that had been heated to thaw and then cooled to 55°C. After mixing, the mixture was poured into petri dishes to prepare drug-containing plates, which were then allowed to cool and stand.

[0044] PDA plates containing an equal volume of DMSO were used as a solvent control, and osthol was used as a control reagent. Fusarium graminearum (Fusarium graminearum) was incubated at 25°C. Fusarium graminearum Fusarium oxysporum ( Fusarium oxysporum ) and Fusarium verticillata ( Fusarium verticillioides Pre-culture was carried out by punching a 5 mm diameter hole at the edge of the pre-cultured colony, then inoculating the colony into the center of the drug-containing plate, and incubating at 25°C until the control was almost fully colonized.

[0045] The colony diameter of each treatment was measured using the cross-sectional method, and the inhibition rate was calculated based on the colony diameter.

[0046] Inhibition rate (%) = [(control colony diameter - drug-treated colony diameter) / control colony diameter] × 100.

[0047] Colony diameter (mm) = Average diameter measured by the cross-cross method - 5.

[0048] The toxicity regression equation was calculated using DPS v7.05 based on the inhibition rate and compound concentration, and the half-inhibitory concentration (EC5) was calculated. 50 The values ​​were calculated. Each treatment was set up with three technical replicates, and the experiment was repeated three times. The measurement results are shown in Table 2 below.

[0049] Table 2. Inhibitory effect of clopyralid A on plant pathogenic fungi (EC50) 50 (μg / mL) .

[0050] Table 2 shows that sorghumin A exhibits inhibitory effects against all pathogenic fungi of the genus *Fusarium*, and its antibacterial effect is comparable to that of the positive control osthol. Specifically, the EC50 of sorghumin A against *Fusarium graminearum*, *Fusarium oxysporum*, and *Fusarium verticillatum* is [not specified in the original text]. 50 The values ​​were 24.75±0.21 μg / mL, 27.63±0.66 μg / mL, and 33.62±0.45 μg / mL, respectively, among which the inhibitory activity against Fusarium graminearum was the strongest.

[0051] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A clopyralid A, characterized in that, The general formula is: 。 2. The use of serotonin A as described in claim 1 in the preparation of a drug for preventing and controlling plant fungal diseases.

3. A bactericide, characterized in that, It contains serotonin A as described in claim 1.

4. A method for preparing serotonin A as described in claim 1, characterized in that, It utilizes *Follium sarmentosum* with accession number CCTCC NO: M20251808 (…). Piptoporellus soloniensis )preparation.

5. The preparation method according to claim 4, characterized in that, It includes the following steps: (1) Take out the preserved Solon small peel tube mother culture test tube, pick out the mycelium block under aseptic conditions and inoculate it on sterilized PDA medium, set the constant temperature incubator at 25℃ and incubate for 7 days; (2) The activated strain was inoculated into sterile PDB liquid culture medium and cultured on a shaker at 25°C and 150 rpm for 7 days as the seed liquid for solid fermentation; (3) Mix rice and water evenly, autoclave at 121℃ for 20 min to obtain rice culture medium; inoculate the seed liquid into the rice culture medium; culture at room temperature for 60 days; (4) Add an equal volume of ethyl acetate to the obtained fermentation product, stir thoroughly, soak for 6 h, then remove the supernatant and evaporate to dryness by rotation. Repeat the extraction 3 times and combine to obtain the total extract. (5) Mix the total extract with ultrapure water in a volume ratio of 1:1 to 1:2, stir evenly, add ethyl acetate in an equal volume to the extract aqueous solution, stir evenly again, let stand for 30 min, take the upper layer solution, repeat the extraction with ethyl acetate three times, then distill the extract under reduced pressure until no ethyl acetate flows out, control the temperature not to exceed 55℃, collect the extract, dry at a temperature below 40℃ to obtain ethyl acetate extract; (6) Mix the ethyl acetate extract with 1.5 to 1.8 times the mass of silica gel of 100 to 200 mesh, stir thoroughly, dry, grind evenly into powder, so that the extract sample is fully adsorbed on the silica gel particles, and then perform silica gel column chromatography at normal pressure. The silica gel column is packed with silica gel of 200 to 300 mesh. Use a petroleum ether / ethyl acetate system for gradient elution. The components eluted in a 10:1 gradient are separated by semi-preparative liquid phase separation to obtain the product, which is serotonin A.

6. The preparation method according to claim 5, characterized in that, The semi-preparative liquid chromatography separation conditions were as follows: COSMOSIL C18 column, 250 mm × 10 mm, column temperature 25℃, wavelength 210 nm, MeCN-H2O, 80:20, 3 mL / min. t R =28.0 min.

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