Preparation method of chaetocidin d and application thereof in prevention and treatment of apple tree rot disease

Abstract

The application belongs to the technical field of biological control of plant diseases, and discloses a preparation method of chaetomassine D and application of the chaetomassine D in prevention and treatment of apple tree damping-off. 32 H 36 N2O5. The chaetomassine D has good prevention and treatment effects on the apple tree damping-off, and the protection effect is equivalent to the treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for plant diseases, and particularly relates to a method for preparing chalcogenin D and its application in the control of apple tree rot disease. Background Technology

[0002] Apple canker, caused by the ascomycete *Valsa mali*, is a major disease affecting apple trees, widely distributed across various fruit-growing regions in my country. It causes the bark to rot and decay, and in severe cases, can lead to tree death and orchard destruction, resulting in significant economic losses. Currently, disease control still relies primarily on chemical agents, but long-term, excessive use of chemical agents can lead to environmental pollution and increased pathogen resistance. Biological control, with its environmentally friendly characteristics, is increasingly contributing to the green development of the fruit industry. The foundation and key to biological control lies in the exploration of highly efficient biocontrol resources. Plants contain a large number of potential biocontrol fungi that can produce bioactive secondary metabolites. These metabolites can effectively inhibit pathogen growth and stimulate the host plant to acquire resistance. Therefore, utilizing the active substances produced by endophytic fungi for the biological control of apple canker has potential advantages.

[0003] Chaetoglobosin D is a type of chaetochalasin, an alkaloid belonging to the macrocyclic polyketide class and also the cytochalasin class, possessing good biological activity. However, reports on chaetoglobosin D in agricultural plant diseases are extremely limited, with the only reported effect being its control of grape white rot (Coniothyrium diplodiella). Furthermore, the isolation and purification process of this compound is difficult, and the standard product is not available from major reagent companies such as Aladdin. Therefore, exploring a simple preparation method for chaetoglobosin D and clarifying its role in the control of agricultural plant diseases, especially apple tree canker, is of great significance.

[0004] Based on the above analysis, the existing technology has the following problems and defects: there are very few reports on chamomile D in agricultural plant diseases, the separation and purification process of the compound is difficult, and the standard product is not available for sale in major reagent companies such as Aladdin. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for preparing chalcogenin D and its application in the prevention and control of apple tree canker.

[0006] This invention is achieved by separating the antibacterial substance using chromatographic methods and identifying it using spectroscopic methods. The preparation method of the chalcogenin D includes the following steps:

[0007] The first step is to filter the fermentation broth, take the filtrate and mix it with an equal volume of ethyl acetate, let it stand and separate into layers, and take the upper ethyl acetate phase; concentrate the ethyl acetate phase under reduced pressure, pack it into a column using a wet method, and then perform silica gel column chromatography using a dry method.

[0008] The second step involves passing the ethyl acetate petroleum ether fraction through a gel column, purifying the fraction using thin-layer chromatography, combining identical fractions, and preparing the product using a semi-preparative high-performance liquid chromatograph (LC-2030CD). The product is analyzed by high-performance liquid chromatography, and a single ultraviolet absorption peak is observed. The compound corresponding to this peak is labeled as compound 004A.

[0009] The third step involved mass spectrometry, nuclear magnetic resonance spectroscopy, and other analyses, which identified the compound as chalcogenin D.

[0010] Furthermore, the first step mobile phase is a gradient elution of petroleum ether:ethyl acetate = 5:1-1:1 and dichloromethane:methanol = 20:1-0:1, with each two column volumes constituting one fraction. The ratio of the mobile phase is adjusted once for each fraction, and the silica gel is 300-400 mesh.

[0011] Furthermore, the components that inhibit V. mali in the first step MG2 fermentation broth were found in the silica gel column chromatography fractions of ethyl acetate:petroleum ether = 2:1, 1:1, and dichloromethane:methanol = 20:1, 10:1.

[0012] Furthermore, the gel column filler in the second step is Sephadex LH-20 gel (25-100 pm) containing hydroxypropyl dextran.

[0013] Furthermore, the second step mobile phase was 55%-80% methanol aqueous solution, the detection wavelength was 254nm, the chromatographic column was YMC-PackODS-AC185μmI.D.1.0×250mm, and the flow rate was 3mL / min.

[0014] Furthermore, in the second step, the product was analyzed by high performance liquid chromatography, and a single ultraviolet absorption peak was observed at 17.552 min.

[0015] Furthermore, the third step uses a 55%-80% methanol-water solution as the mobile phase, a detection wavelength of 254 nm, and a ThermoScientific column. TM Hypersil TM ODSC 185μm 4.6×250mm, flow rate 1mL / min.

[0016] Furthermore, the tested endophytic Chaetomium globosum strain MG2 was identified as Chaetomium globosum and has been deposited at the China General Microbiological Culture Collection Center (CGMCC) No. 40070.

[0017] Another object of the present invention is to provide a chalcogenin D prepared by the method of the preparation of chalcogenin D, wherein the molecular formula of chalcogenin D is C 32 H 36 N2O5.

[0018] Another object of the present invention is to provide an application of the aforementioned chalcogenin D in the prevention and control of apple tree rot disease.

[0019] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0020] First, addressing the technical problems existing in the prior art and the difficulty in solving them, this paper closely analyzes, in conjunction with the technical solution to be protected by this invention and the results and data obtained during the research and development process, how the technical solution of this invention solves the technical problems, and the inventive technical effects brought about by solving these problems. The specific description is as follows:

[0021] This invention, through the isolation of secondary metabolites of Chaetomium MG2 and the application of activity tracking technology, obtained an active substance with a significant inhibitory effect on V. mali, providing a material basis for the development of highly efficient and low-toxicity microbial pesticides.

[0022] Second, considering the technical solution as a whole or from the perspective of the product, the technical effects and advantages of the technical solution to be protected by this invention are specifically described as follows: The efficacy of the substance chalcogenin D against apple tree rot was determined. The results showed that chalcogenin D has a good control effect on apple tree rot, and exhibits significant antibacterial activity against mycelial growth and spore germination. Its protective and therapeutic effects on detached branches are comparable.

[0023] Third, as supplementary evidence of the inventive step of the claims of this invention, it is also reflected in the following important aspects:

[0024] (1) The technical solution of this invention fills a technical gap in the industry both domestically and internationally:

[0025] Control of V. mali using chlamydospermum erythrorhizon D.

[0026] (2) Whether the technical solution of the present invention solves the technical problem that people have long wanted to solve but have never been able to solve successfully:

[0027] It provides valuable material resources for the prevention and control of apple tree rot disease. Attached Figure Description

[0028] Figure 1 This is a flowchart of the preparation method of chalcogenin D provided in the embodiments of the present invention;

[0029] Figure 2 This is a schematic diagram of the MG2 fermentation broth material separation method provided in an embodiment of the present invention;

[0030] Figure 3 This is a schematic diagram of the preliminary separation and bioassay results provided in an embodiment of the present invention;

[0031] Figure 4 This is a schematic diagram of the semi-preparative high-performance liquid chromatography purification of compound 004A provided in the embodiments of the present invention;

[0032] Figure 5 This is a schematic diagram of the HPLC analysis of compound 004A after purification, provided in an embodiment of the present invention.

[0033] Figure 6 It is compound 004A provided in the embodiments of the present invention. 13 Schematic diagram of C NMR analysis;

[0034] Figure 7 It is compound 004A provided in the embodiments of the present invention. 1 Schematic diagram of H NMR analysis;

[0035] Figure 8 This is a positive and negative ion mass spectrometry analysis chromatogram of compound 004A provided in the embodiments of the present invention;

[0036] Figure 9 This is a schematic diagram illustrating the identification of compound 004A provided in an embodiment of the present invention;

[0037] Figure 10 This invention provides an embodiment of the inhibitory effect of chalcogenin D on the mycelial growth of apple tree rot fungus; (a) Inhibition effect diagram of the isolated substance; (b) Statistical analysis diagram of the inhibition effect of the isolated substance;

[0038] Figure 11 This invention relates to the effect of chamomilein D on the hyphal morphology of apple tree rot pathogens; A: Hyphae morphology of normally growing hyphae; B: Hyphae morphology after treatment with chamomilein D.

[0039] Figure 12 The present invention provides the inhibitory effect of chamomilein D on the germination of apple tree rot spores; (a) the effect of chamomilein D on the germination rate of conidia; (b) the effect of chamomilein D on the inhibition rate of conidia germination.

[0040] Figure 13The embodiments of this invention provide the inhibitory effect of chalcogenin D on the germination of apple tree rot spores; A. Conidia germinating after normal culture for 24 hours; B. Conidia germinating after treatment with 2 μg / mL 004A for 24 hours; C. Conidia germinating after normal culture for 48 hours; D. Conidia germinating after treatment with 2 μg / mL 004A for 48 hours;

[0041] Figure 14 The embodiments of the present invention provide the following effects on the control of apple tree rot disease on detached branches: (a) protective effect; (b) statistical analysis of protective effect; (c) therapeutic effect; and (d) statistical analysis of therapeutic effect. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0043] like Figure 1 As shown, the preparation method of spherical chitin D provided in this embodiment of the invention includes the following steps:

[0044] S101: After filtering the fermentation broth, take the filtrate and mix it with an equal volume of ethyl acetate. After standing and separating the layers, take the upper ethyl acetate phase. After concentrating the ethyl acetate phase under reduced pressure, pack it into a column using a wet method and then perform silica gel column chromatography using a dry method.

[0045] S102: The ethyl acetate:petroleum ether = 1:1 fraction was further purified by gel column chromatography and then prepared by semi-preparative high performance liquid chromatography. The product was analyzed by high performance liquid chromatography, and a single ultraviolet absorption peak was observed at 17.552 min. The compound corresponding to this peak was labeled as compound 004A.

[0046] S103: Mass spectrometry, nuclear magnetic resonance spectroscopy, and other analyses identified compound 004A as chalcogenin D, with the molecular formula C. 32 H 36 N2O5.

[0047] To demonstrate the inventiveness and technical value of the technical solution of this invention, this section provides specific product or related technology application examples of the technical solution claimed.

[0048] The chalcogenin D provided in this embodiment of the invention will be used to prepare pesticide compounds for the prevention and control of apple tree rot. It is expected that by applying chalcogenin D externally, the mycelial growth and spore germination of apple tree rot pathogens can be inhibited, thereby achieving the effect of antibacterial and bactericidal action.

[0049] I) Experimental Materials

[0050] (1) Test culture medium

[0051] PDA medium: Peel 200g of potatoes, cut them into pieces, boil them in double-distilled water for 20 minutes, then filter them through four layers of gauze into a 1L measuring cup, add 20g of glucose and 15g of agar, stir well, bring the volume to 1L with double-distilled water, and autoclave at 121℃ for 20 minutes.

[0052] PDB medium: Peel 200g of potatoes, cut them into pieces, boil them in double-distilled water for 20 minutes, then filter them through four layers of gauze into a 1L measuring cup, add 20g of glucose and stir well, bring the volume to 1L with double-distilled water, and autoclave at 121℃ for 20 minutes.

[0053] (2) Test strains

[0054] The tested endophytic *C. globosum* strain MG2 was isolated from healthy *Chaenomeles speciosa* in our laboratory and identified as *C. globosum*. It was preserved on small slant agar at 4℃. This strain is currently deposited at the China General Microbiological Culture Collection Center (CGMCC No. 40070). The CGMCC is located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. Telephone: 010-64807355, Fax: 010-64807288, Email: cgmcc@im.ac.cn. http: / / www.cgmcc.net cGMCC No. 40070; Biological material (strain) used for reference: MG2, suggested classification name: Chaetomium globosum; This biological material (strain) was received by this depository on February 14, 2022, and registered. At your request, it will be preserved for thirty years from that date, with an extension of five years if a request for biological material samples is received before the expiration date; The viability of this biological material (strain) was tested by this depository on February 14, 2022, and the result was: viable. Test pathogen: *V. mali*, *V. globosum*, isolated and preserved by the Fruit Tree Disease Pathogen Biology and Integrated Management Research Team, College of Plant Protection, Northwest A&F University;

[0055] II) Specific Experimental Procedure

[0056] (1) Activation culture of strain

[0057] In a clean bench, a small amount of mycelium to be activated is picked and inoculated onto PDA medium. After 2-3 days of dark incubation at 25°C, activation is complete.

[0058] (2) Preparation of MG2 fermentation broth

[0059] Under aseptic conditions, collect MG2 colonies that have been cultured for 7 days, and use a punch to create 5mm diameter mycelial cakes along the outermost edge of the colony. Place 10 mycelial cakes into each Erlenmeyer flask containing 400ml of LPDB medium and incubate at 25℃ and 160 rpm. -1 After 12 days of cultivation (shaking), the mycelium was separated from the fermentation filtrate by filtering with two layers of sterile filter paper using a circulating water multi-purpose vacuum pump (SHB-Ⅲ) to separate the substances as soon as possible.

[0060] (3) Preliminary separation of active substances from MG2

[0061] At least 50 LMG2 fermentation broth was collected and filtered. The solid (mycelium) was extracted with methanol and tested to find no activity, indicating the presence of active substances in the fermentation filtrate. Next, an equal volume of ethyl acetate was added to the supernatant of the MG2 fermentation broth for three extractions. The liquid was then subjected to silica gel column chromatography (specific conditions: petroleum ether:ethyl acetate = 5:1-1:1, dichloromethane:methanol = 20:1-0:1). For each fraction (mixture) obtained in each step, 10 μL was dropped onto a small filter paper disc and cultured against the pathogen on PDA medium to determine if each fraction possessed antibacterial biological activity.

[0062] (4) Further separation of active substances in MG2

[0063] The active component was further purified using gel column chromatography, followed by thin-layer chromatography. The relatively pure component was then subjected to semi-preparative high-performance liquid chromatography (HPLC) with multiple purifications using 55%-80% methanol-water solution to finally obtain a single pure compound with biological activity, named 004A. Its peak conditions were determined using a liquid chromatography column. Its chemical structure was identified using nuclear magnetic resonance analysis (C-NMR, 1H NMR, and mass spectrometry) (procedure as follows). Figure 2 ).

[0064] (5) Determination of the antibacterial activity of the isolated substance

[0065] The inhibitory effect of MG2 fermentation broth on V. mali was determined using the mycelial growth rate method. Substance 004A was mixed with melted PDA medium to prepare culture plates with concentrations of 10, 5, 2.5, 1.25, and 0.625 μg / mL. After solidification, a V. mali mycelial cake (5 mm in diameter) cultured for 2-3 days was inoculated in the center of each plate. PDA medium without the substance served as the control group. Three biological replicates were performed for each treatment, and the plates were incubated at 25°C until the control completely covered the entire culture dish.

[0066] Colony measurement: Colony diameter was determined using the cross-hatching method. After the control group colonies had almost completely covered the entire PDA medium plate, the colony diameters of the control group and the treatment group were measured separately. The inhibition rate was calculated using the following formula:

[0067] Mycelial growth inhibition rate (%) = [(Control group colony diameter - Treatment group colony diameter) / Control group colony diameter] × 100

[0068] (6) Effects of isolated substances on the hyphal morphology of apple tree rot fungus

[0069] This experiment was conducted using a scanning electron microscope. Referring to (4), a dish containing the drug was prepared. After culturing for 3 days, the edges of normally growing *V. mali* hyphae were cut off with a blade as the control group, and the edges of *V. mali* hyphae after antagonism by the isolated substance were cut off as the treatment group. Both were fixed in 4% (v / v) glutaraldehyde phosphate buffer (100mM, pH=6.8) at 4℃ for 24h, then rinsed 4 times with 0.1M PBS buffer (pH=6.8), dehydrated (30%, 50%, 70%, 80%, and 90% ethanol for 20min each; finally, anhydrous ethanol for 30min, repeated 3 times), dried near the CO2 spot, mounted on a stage, sputter-coated with gold, and observed (samples were placed under 10.0-kV voltage on an S-4800 field emission scanning electron microscope) and photographed.

[0070] (7) Determination of the inhibitory effect of the isolated substance on the germination of apple tree rot fungus spores

[0071] Take a 5mm diameter mycelium cake of the rot-causing fungus and inoculate it upside down onto a blank PDA medium. Incubate at 25℃ in the dark until the entire medium is covered. Then, spread the medium out and incubate at room temperature. When yellow conidial horns appear in the medium, use an inoculation needle to pick up the conidial horns and suspend them in sterile water under sterile conditions to prepare a concentration of 1×10⁻⁶. 5 —1×10 6 A spore suspension of 1 conidia / mL.

[0072] Refer to (4) to prepare a series of gradient-grade culture dishes. Add 20 μL of the prepared *V. mali* conidial suspension and gently spread evenly using a sterile spreader. Use a culture medium without any substance as a control. Perform three biological replicates for each treatment, and repeat the experiment three times. Incubate at 25℃ in the dark for approximately 24 hours. Detect spore germination under an optical microscope (40×10). Germination is defined as the length of the germ tube exceeding half the diameter of the conidium. Calculate the conidial germination rate and germination inhibition rate.

[0073] Germination inhibition rate (%) = [(Germination rate of control conidia - Germination rate of treated conidia) / Germination rate of control conidia] × 100

[0074] (8) Determination of the effect of separated substances on the prevention and control of apple tree rot disease

[0075] Branch treatment: Collect Fuji apple branches (1-2 years old), cut them into 10cm lengths, select branches with uniform thickness and growth, rinse them with clean water, disinfect them with 0.6% sodium hypochlorite solution for 15-20 minutes, rinse them with sterile water 3-4 times, let them dry, seal both ends of the branches with melted paraffin, and let them dry.

[0076] Protective effect: Holes (5mm diameter) were punched in the treated branches. The treatment group was then treated with 10μL of substance 004A (added in two separate drops, concentration = 500μg / mL). After air drying, the branches were inoculated with a fungal cake containing the rot pathogen. A sterile water treatment served as the control group. After 5 days of incubation at room temperature with humidity, the occurrence of apple tree rot was observed, the length of lesions was measured, and the control effect was calculated. Six branches were used for each treatment, with one inoculation point per branch, and the experiment was repeated three times.

[0077] Treatment effect: Holes (5mm diameter) were punched in the treated branches, and then inoculated with a fungal cake of rot pathogens. After 1 day, the fungal cake was scraped off. In the treatment group, 10 μL of substance 004A was added to the hole (added in two drops, concentration = 500 μg / mL). A sterile water treatment served as the control group. After culturing in a room temperature humidified incubator for 5 days, the occurrence of apple tree rot was observed, the length of lesions was measured, and the control effect was calculated. Six branches were used for each treatment, with one inoculation point per branch, and the treatment was repeated three times.

[0078] III) Test Results

[0079] (1) Preparation and acquisition of spherical chitin D compound

[0080] The active compound was isolated and purified from the fermentation filtrate of *Chaetomium MG2* using extraction, vacuum concentration, and multi-stage chromatographic chromatography (silica gel column chromatography, gel column chromatography, and semi-preparative high-performance liquid chromatography) with "bioactivity tracking" as the indicator. The specific steps are as follows: 1. After filtering the fermentation broth, the filtrate was mixed with an equal volume of ethyl acetate, allowed to stand for separation, and the upper ethyl acetate phase was collected. The ethyl acetate phase was concentrated under reduced pressure, packed into a column using a wet method, and then subjected to silica gel column chromatography using a dry method. The mobile phase was a gradient elution of petroleum ether:ethyl acetate = 5:1-1:1 and dichloromethane:methanol = 20:1-0:1, with each two column volumes constituting one fraction. The mobile phase ratio was adjusted once for each fraction (all reagents were analytical grade). The silica gel was 300-400 mesh (produced by Qingdao Haiyang Chemical). The presence of components inhibiting *V. mali* in the MG2 fermentation broth was found in the ethyl acetate:petroleum ether = 2:1, 1:1, and dichloromethane:methanol = 20:1 and 10:1 fractions obtained by silica gel column chromatography. Figure 3The ethyl acetate:petroleum ether = 1:1 fraction was prepared by semi-preparative high performance liquid chromatography (mobile phase: 55%-80% methanol aqueous solution, detection wavelength: 254nm, column: YMC-PackODS-AC185μmI.D.1.0×250mm, flow rate: 3mL / min). Figure 4 The product was analyzed by analytical high-performance liquid chromatography (HPLC), and a single ultraviolet absorption peak was observed at 17.552 min. Figure 5 The compound corresponding to this peak was labeled as compound 004A. (Mobile phase: 55%-80% methanol aqueous solution, detection wavelength: 254nm, column: ThermoScientific) TM Hypersil TM ODSC (185 μm, 4.6 × 250 mm, flow rate 1 mL / min) was analyzed by mass spectrometry, nuclear magnetic resonance spectroscopy, etc. Figures 6-8 Compound 004A was identified as chaetoglobosin D, with the molecular formula C004. 32 H 36 N2O5( Figure 9 ).

[0081] (2) Inhibitory effect of chamomile D on the growth of apple tree rot pathogens

[0082] In vitro antibacterial activity assays showed that, compared to the control group, chalcogenin D significantly inhibited the mycelial growth of apple tree rot pathogens. Figure 10 ).

[0083] (3) Effects of chalcogenin D on the morphology of apple tree rot pathogens

[0084] Scanning electron microscopy revealed that the hyphae of the apple tree rot pathogen in the control group grew normally, with slender, smooth, and relatively uniform thickness, and the tips of the hyphae were quite pointed. After antagonism by the substance chalcogenin D, the protoplasm of the hyphae severely leaked out, resulting in an uneven, wrinkled, and sunken surface; the leaked protoplasm even caused the hyphae to clump together. This demonstrates that chalcogenin D has a significant impact on the hyphal morphology of the apple tree rot pathogen. Figure 11 ).

[0085] (4) Inhibitory effect of chamomile D on spore germination of apple tree rot fungus

[0086] Experiments showed that the substance chalcogenin D could significantly inhibit the germination of spores. A concentration of 2 μg / mL of chalcogenin D could achieve a 100% inhibition rate against the spore germination of apple tree rot pathogens. Figure 12Meanwhile, spore morphology and germination rate were severely affected. Compared with the control group, the germination of spores of the putrefactive fungus treated with chalcogenin D was inhibited, and protoplasmic exudation occurred after 48 hours. Figure 13 ).

[0087] (5) Effect of chamomile D on the control of apple tree rot on detached branches

[0088] The efficacy of chalcanthogenetic acid D against apple tree canker on detached branches was determined. The results showed that chalcanthogenetic acid D had a good control effect on apple tree canker, and its protective and curative effects were comparable. Figure 14 This lays the foundation for the further research and development of chalcogenin D and the study of its prevention and control mechanisms.

[0089] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing sphaeropsidin D, characterized by, The preparation method of the spherical chitin D Includes the following steps: The first step involved filtering the MG2 fermentation broth and mixing the filtrate with an equal volume of ethyl acetate. After standing and separating the layers, the upper ethyl acetate phase was collected. The ethyl acetate phase was concentrated under reduced pressure and then packed into a column using a wet method. The sample was then loaded onto a silica gel column for dry chromatography. The mobile phase consisted of a gradient elution of petroleum ether:ethyl acetate = 5:1-1:1 and dichloromethane:methanol = 20:1-0:

1. Each two column volumes constituted one fraction, and the ratio of the mobile phase was adjusted for each fraction. The second step involves purifying the ethyl acetate:petroleum ether = 1:1 fraction and then preparing it using a semi-preparative high performance liquid chromatograph. The product was analyzed by high performance liquid chromatography (HPLC), and a single ultraviolet absorption peak was observed. The compound corresponding to this peak was labeled as compound 004A. The mobile phase of the semi-preparative HPLC was 55%-80% methanol aqueous solution, the detection wavelength was 254 nm, the chromatographic column was YMC-PackODS-AC185μmI.D.1.0×250 mm, and the flow rate was 3 mL / min. The third step involved mass spectrometry, nuclear magnetic resonance spectroscopy, and other analyses, which identified the compound as chalcogenide D. The tested endophytic Chaetomium strain MG2 was identified as Chaetomium globosum C.globosum , and has been deposited with the China General Microbiological Culture Collection Center (CGMCC) under No. 40070.

2. The method for preparing chalcogenin D as described in claim 1, characterized in that, The first step uses a gradient elution of petroleum ether:ethyl acetate = 5:1-1:1 and dichloromethane:methanol = 20:1-0:1, with each two column volumes constituting one fraction. The ratio of the mobile phase is adjusted once for each fraction, and the silica gel is 300-400 mesh.

3. The method for preparing chalcogenin D as described in claim 1, characterized in that, The second step involved analyzing the product using an analytical high-performance liquid chromatograph, which revealed a single ultraviolet absorption peak at 17.552 min.

4. The method for preparing chalcogenin D as described in claim 1, characterized in that, The mobile phase of the high-performance liquid chromatograph is 55%-80% methanol-water solution, the detection wavelength is 254nm, the chromatographic column is ThermoScientific™ Hypersil™ ODSC185μm 4.6×250mm, and the flow rate is 1mL / min.

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