Alkaloid compounds derived from endophytic fungi of saffron and their preparation and application

Abstract

The invention discloses an alkaloid compound derived from saffron endophytic fungi, and preparation and application thereof. The invention extracts, purifies and separates two new alkaloid compounds 11-acetyl-pseurotin A2 (Ⅰ) and 20-demethyl-fumiquinazoline C (Ⅱ) from saffron endophytic fungi fermentation products for the first time. The alkaloid compounds can be used to resist plant pathogens, have the effect of biological control, and have broad application prospects in the field of agriculture.

Description

(I) Technical field

[0001] The invention relates to two alkaloid compounds in endophytic fungi of saffron and their preparation and application in agricultural production. (II) Background technology

[0002] Natural products have always been an important source for discovering innovative drugs with novel structures and new mechanisms of action. They mainly come from plants and their endophytic fungi, especially the secondary metabolites of endophytic fungi in plants. They have the characteristics of diverse skeletons, novel structures, multiple targets, and low toxicity and side effects. They are usually evolved through natural selection, can effectively combine with biological macromolecules, and exhibit good biological activity.

[0003] Endophytic fungi, also known as "asymptomatic fungi", may exist in all plants, and it is estimated that the number of their species may exceed 1.2 million. In 1993, Stierle A et al. isolated an endophytic fungus, Taxomycesandreanae, from the inner bark of Taxus brevifolia, which can produce paclitaxel (Taxol) with anticancer activity. Since then, the study of secondary metabolites of endophytic fungi in plants has attracted widespread attention. At present, secondary metabolites of endophytic fungi have been used as antioxidants, pesticides, insecticides, etc., and even used as antibiotics, anticancer drugs and immunosuppressants in clinical practice. Therefore, the study of endophytic fungi in plants has great economic value and far-reaching scientific significance.

[0004] Saffron is the dried stigma of the style of Crocus sativus L., a plant of the Iridaceae family. It is also known as saffron, gardenia, saffron, saffron, etc. It is a traditional precious Chinese medicine in my country and was included in the catalog of medicine and food homology in 2020. According to the "Chinese Pharmacopoeia", saffron tastes sweet, is neutral in nature, and is oriented to the heart and liver meridians. It can promote blood circulation and remove blood stasis, cool blood and detoxify, and relieve depression and calm the mind. It is often used for amenorrhea, postpartum blood stasis, fever and rashes, depression, palpitations and madness. At present, there are few studies on various aspects of endophytic fungi in saffron, especially the systematic separation of secondary metabolites of endophytic fungi in saffron. At present, the endophytic fungi isolated and identified from saffron plants mainly include Aspergillus, Alternaria, Penicillium, Trichoderma, Fusarium, Alternaria, Paecilomyces, Epicoccum, Mortierella, etc., among which Aspergillus fungi include Aspergillus fumigatus.

[0005] Aspergillus fungi are widely distributed in nature and are a common saprophytic fungus. Currently, there are more than 180 known species of Aspergillus fungi, which are widely used in food fermentation industries such as sauce making and wine making, as well as agricultural production fields such as biofertilizers and biological control. According to literature reports, the secondary metabolites currently isolated from this genus of fungi are rich in structural types, including alkaloids, terpenes, polyketides, steroids, benzene derivatives, etc., and have anti-inflammatory, antibacterial, anti-tumor, antiviral, anti-diabetic, antioxidant, neuroprotective and plant growth promotion biological activities. Therefore, screening and discovering new active ingredients against plant pathogens from Aspergillus fungi has important research value. (III) Summary of the invention

[0006] The invention provides an alkaloid compound derived from endophytic fungi of saffron, a preparation method thereof and application of the alkaloid compound in agricultural production, which can be used in agricultural production fields such as biological fertilizer and biological control.

[0007] The technical solution adopted by the present invention is:

[0008] The present invention provides an alkaloid compound derived from endophytic fungi of saffron, the structural formula of which is shown in formula (I) or formula (II):

[0009]

[0010] The compounds represented by formula (I) and formula (II) can be fermented, extracted and separated from saffron endophytic fungi such as Aspergillus fumigatus, or prepared by artificial synthesis.

[0011] The present application also provides a method for preparing the alkaloid compound, which is carried out according to the following steps:

[0012] (1) Fermentation: Inoculate saffron endophytic fungus Aspergillus fumigatus into a rice culture medium, seal the medium, and ferment it at 26° C. for 21 days. Soak the culture in ethyl acetate overnight, filter it, and concentrate the filtrate under reduced pressure to obtain a crude extract. The rice culture medium is prepared by mixing rice and ultrapure water, wherein the volume ratio of the ultrapure water is 1-6:1 (preferably 1.25:1) based on the mass of rice. After sealing the medium with a bacterial filter plug, sterilize it at 121° C. for 30 min.

[0013] (2) Extraction: The crude extract from step (1) is dispersed into a suspension with ultrapure water, extracted with petroleum ether, ethyl acetate, and n-butanol in sequence, the ethyl acetate extract is collected, and concentrated to dryness to obtain an ethyl acetate phase;

[0014] (3) Silica gel chromatography: The ethyl acetate phase in step (2) was dissolved with a mixed solution of methanol and dichloromethane, and silica gel was added to mix the sample. After drying, the sample was loaded onto a silica gel chromatography column (silica gel 100-200 mesh, column height 100 cm, inner diameter 16 cm, column packing height 46 cm), and eluted with dichloromethane / ethyl acetate in different volume ratios (V / V=1:0, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1), and then gradient eluted with dichloromethane / methanol in different volume ratios (V / V=5:1, 2:1, 1:1, 0:1), and the elution rate was 10. 0mL / min, the elution volume of different elution solvents is 2L, every 500mL of sample is collected and concentrated, and thin layer chromatography monitoring is performed using dichloromethane / ethyl acetate with a volume ratio of 2:1 as a developing solvent, and fractions with similar spots are combined to collect 11 components, which are respectively recorded as components Fr.1 to Fr.11 after drying, among which the Rf value of component Fr.6 is 0.053-0.868; the methanol and dichloromethane mixed solution is prepared by mixing methanol and dichloromethane with a volume ratio of 1:5, and the volume dosage of the mixed solution is 8-2mL / g based on the mass of the ethyl acetate phase;

[0015] (4) Silica gel chromatography: Component Fr.6 was dissolved in a 2:1 volume ratio petroleum ether / ethyl acetate solution and loaded onto a silica gel chromatography column (silica gel 200-300 mesh, column height 80 cm, inner diameter 8 cm, column packing height 42 cm). Petroleum ether / ethyl acetate in different volume ratios (V / V = 2:1, 1:1, 0:1) was used as the eluent. The elution rate was 20 mL / min and the elution volume of different elution solvents was 600 mL. Thin layer chromatography monitoring was performed using a 1:1 volume ratio petroleum ether / ethyl acetate as the developing solvent. The fractions of the spots were separated to obtain 13 components, which were dried and recorded as components Fr.6-1 to Fr.6-13, among which the Rf values ​​of component Fr.6-5 were 0.161, 0.232, 0.304, 0.393, 0.446, and 0.535; the Rf values ​​of component Fr.6-6 were 0.125, 0.179, 0.286, 0.321, 0.375, 0.429, and 0.536; the volume of the petroleum ether / ethyl acetate solution used for dissolution was 25-5 mL / g based on the mass of component Fr.6;

[0016] (5) Compounds of formula (I):

[0017] The component Fr.6-5 was dissolved in dichloromethane / methanol in a volume ratio of 1:1 and then loaded onto a hydroxypropyl dextran gel column Sephadex LH-20. The mobile phase was dichloromethane / methanol in a volume ratio of 1:1 for elution and purification. The elution rate was 10 mL / min and the elution volume was 3000 mL. The column liquid was monitored by thin layer chromatography using ethyl acetate / dichloromethane in a volume ratio of 1:2 as a developing solvent. The components with the same spots were combined and dried to obtain 4 fractions, which were respectively recorded as Fr.6-5-1 to Fr.6-5-4, among which the Rf value of the component Fr.6-5-3 was 0.098-0.745; the volume of the dichloromethane / methanol used for dissolution was 25-5 mL / g based on the mass of the component Fr.6-5;

[0018] Select component Fr.6-5-3, dissolve it in dichloromethane, and then load it onto a silica gel chromatography column (silica 200-300 mesh, column height 40 cm, inner diameter 6 cm, column height 28 cm), use dichloromethane / methanol with different volume ratios (V / V=1:0, 100:1, 50:1, 10:1) as eluent, the elution rate is 12 mL / min, the elution amount of different elution solvents is 500 mL, and dichloromethane / methanol with a volume ratio of 10:1 is used as a developing agent for thin layer chromatography monitoring, and fractions with similar spots are combined and dried to obtain 8 fractions, which are respectively recorded as Fr.6-5-3-1 to Fr.6-5-3-8, among which the Rf value of component Fr.6-5-3-5 is 0.059-0.784; the volume of dichloromethane used for dissolution is 30-5 mL / g based on the mass of component Fr.6-5-3;

[0019] Component Fr.6-5-3-5 was dissolved in methanol and then subjected to semi-preparative HPLC (C 18 The column was used with 49% acetonitrile-water system as the mobile phase, the flow rate was 1.5 mL / min, the detection wavelengths were 210 nm, 254 nm, and 365 nm, the column temperature was 35 °C, and t R =21.1min) separation, collecting the mobile phase with a retention time of 21.1min, and drying by rotary evaporation to obtain the alkaloid compound represented by formula (I); the volume amount of methanol used for dissolution is 40-10mL / g based on the mass of component Fr.6-5-3-5;

[0020] (6) Compound of formula (II):

[0021] The component Fr.6-6 was dissolved in a dichloromethane / methanol solution with a volume ratio of 1:1, and then loaded onto a hydroxypropyl dextran gel column Sephadex LH-20. The mobile phase was dichloromethane / methanol with a volume ratio of 1:1 for elution and purification. The elution rate was 10 mL / min, and the elution volume was 3000 mL. The column liquid was monitored by thin layer chromatography using ethyl acetate / dichloromethane with a volume ratio of 2:1 as a developing solvent. The components with the same spots were combined and dried to obtain 5 fractions, which were respectively recorded as Fr.6-6-1 to Fr.6-6-5, among which the Rf value of the component Fr.6-6-4 was 0.116-0.692; the volume of the dichloromethane / methanol solution used for dissolution was 25-5 mL / g based on the mass of the component Fr.6-6;

[0022] Component Fr.6-6-4 was selected and dissolved in dichloromethane and then loaded onto a silica gel chromatography column (silica 200-300 mesh, column height 40 cm, inner diameter 6 cm, column packing height 24 cm). The eluent was dichloromethane / methanol (V / V=1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1). The elution rate was 12 mL / min, and the elution volume of different elution solvents was 400 mL. Thin layer chromatography monitoring was performed using dichloromethane / methanol in a volume ratio of 10:1 as a developing solvent, and fractions with similar spots were combined and dried to obtain 9 parts, recorded as Fr.6-6-4-1 to Fr.6-6-4-9, wherein the Rf value of component Fr.6-6-4-2 was 0.100-0.633; the volume of dichloromethane used for dissolution was 25-10 mL / g based on the mass of component Fr.6-6-4;

[0023] Component Fr.6-6-4-2 was dissolved in methanol and purified by semi-preparative HPLC (C 18 The column was used with 45% acetonitrile-water system as the mobile phase, the flow rate was 1.5 mL / min, the detection wavelengths were 210 nm, 254 nm, and 365 nm, the column temperature was 35 °C, and t R =18.8min), collecting the mobile phase with a retention time of 18.8min, and drying by rotary evaporation to prepare the alkaloid compound represented by formula (II); the volume of methanol used for dissolution is 40-10mL / g based on the mass of component Fr.6-6-4-2.

[0024] Preferably, the saffron endophytic fungus in step (1) is Aspergillus fumigatus Y0107, which is deposited in the General Microbiological Center of China Microorganism Culture Collection Committee, with a deposit date of May 17, 2023, a deposit number of CGMCC No.40629, and an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0025] Preferably, in step (1), slant activation and seed expansion culture are first performed before the Aspergillus fumigatus is inoculated into the rice culture medium, and the seed liquid is inoculated into the rice culture medium with an inoculum amount of 1-5% by volume, and the slant activation and seed culture method is as follows: inoculating the Aspergillus fumigatus into the PDA culture medium, culturing at 26° C. for 48 hours, and obtaining the slant thalli; the slant culture medium is composed of: 120 g potato, 12 g glucose, 10 g agar, and 600 mL water; then inoculating the slant thalli into the seed culture medium, culturing at 26° C. and 150 rpm for 24 hours, and obtaining the seed liquid; the seed culture medium is composed of: 80 g potato, 8 g glucose, and 400 mL water.

[0026] Preferably, the ethyl acetate extraction in step (1) is performed 1-5 times, the amount of ethyl acetate used in the first extraction is 40-10 mL / g based on the weight of the culture, and the amount of ethyl acetate used in the second to fifth extractions is 20-5 mL / g based on the weight of the culture.

[0027] Preferably, the amount of ultrapure water used in step (2) is 25-10 mL / g based on the weight of the crude extract; the amounts of petroleum ether, ethyl acetate and n-butanol used are all 25-8 mL / g based on the weight of the crude extract.

[0028] The compounds of the present invention can be extracted and separated after fermentation by endophytic fungi of plants, and after the structures of the compounds are determined, they can also be prepared by artificial synthesis and other methods.

[0029] The present invention also provides an application of the alkaloid compound in preparing a plant pathogen inhibitor, wherein the plant pathogens include Agrobacterium tumefaciens, Pantoea agglomerans, Ralstonia solanacearum, Erwinia sp. and Xanthomonas oryzae.

[0030] The present invention also provides an Aspergillus fumigatus Y0107 for preparing the alkaloid compound, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit date of May 17, 2023, a deposit number of CGMCC No.40629, and an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0031] Compared with the prior art, the beneficial effects of this application are mainly reflected in:

[0032] (1) The present invention extracts, purifies and separates two new alkaloid compounds 11-acetyl-pseurotin A2 (Ⅰ) and 20-demethyl-fumiquinazoline C (Ⅱ) from the fermentation products of endophytic fungi of saffron for the first time. The compounds can be used to resist plant pathogens and have the effect of biological control. They have broad application prospects in the field of agriculture.

[0033] (2) The compounds of the present invention have certain activity against plant pathogens. (IV) Description of the drawings

[0034] Figure 1 11-acetyl-pseurotin A2(Ⅰ) 1 H NMR spectrum.

[0035] Figure 2 11-acetyl-pseurotin A2(Ⅰ) 13 C NMR spectrum.

[0036] Figure 3 20-demethyl-fumiquinazoline C(Ⅱ) 1 H NMR spectrum.

[0037] Figure 4 20-demethyl-fumiquinazoline C(Ⅱ) 13 C NMR spectrum. (V) Specific implementation methods

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0040] In the following preparation examples, the specific rotation test was completed by a JASCO P-1020 polarimeter; NMR was measured using a Bruker Avance III 400 instrument; HR-ESI-MS was measured using a Waters Synapt G2 instrument; the silica gel used was produced by Qingdao Ocean Chemical Company of China; Sephadex LH-20 dextran gel was produced by Pharmacia of Sweden; the semi-preparative HPLC was a Waters 2535 model, and a YMC-Pack ODS-A (5 μm, 10 mm×250 mm) semi-preparative chromatographic column; all reagents were produced by Shanghai Zhanyun Chemical Co., Ltd. and Shanghai McLean Biochemical Technology Co., Ltd.

[0041] Example 1: Isolation, purification and identification of endophytic fungi in saffron

[0042] Normal and intact saffron plants, including bulbs, lateral buds and buds, were collected from Sandu Saffron Cooperative in Jiande City, Zhejiang Province. They were washed with running water to remove surface soil, dust and other impurities, and then dried. The following steps were performed in sequence in a clean workbench: soaking in 75% ethanol for 30 seconds, rinsing with sterile water, soaking in 5% sodium hypochlorite solution for 3 minutes, rinsing with sterile water, repeating the whole process 3 times, and drying. Take the above surface-sterilized samples and cut the lateral buds of saffron into about 2mm thick pieces with sterile scissors. 3 Place the block in the shape of an equilateral triangle on a PDA medium plate, repeat 3 plates, and culture at 26°C in the dark. At the same time, apply the last rinse sterile water to the PDA medium to ensure that the isolated strain is an endophytic fungus rather than a miscellaneous fungus. Observe at any time, and after the colonies grow on the edge of the tissue, pick out colonies with different colors or obvious morphological differences and inoculate them on the plate and continue to culture. Use the gradient dilution streaking method to perform 3-5 rounds of colony purification until the growth rate of the single colony picked out is similar and the edges are clear. It can be preliminarily judged that it has been completely purified and strain Y0107 is obtained.

[0043] Through observation, it was found that the strain Y0107 grew fast on the PDA medium, with a colony diameter of about 60 mm, abundant aerial hyphae, white at first, and then turned dark blue from the center, showing a fluffy appearance. An appropriate amount of saffron endophytic fungus hyphae was scraped under a sterile environment and sent to Ningbo Testo Biotechnology Co., Ltd., and genomic DNA was extracted from the fungal tissue using the silicon matrix adsorption column method of the kit, and 2Taq Master Mix was used to perform PCR amplification on all the extracted saffron endophytic fungus DNAs, and the amplified products were sequenced (bidirectional sequencing) for the first generation, and the ITS sequencing result was shown in SEQ ID NO.1.

[0044] The ITS sequencing results were searched for the DNA sequence with the highest similarity in GeneBank using BLSAT in the NCBI website. Together with the sequencing result sequences, the neighbor-joining method was used to draw an evolutionary tree, and the strain Y0107 was identified as Aspergillus fumigatus and named Aspergillus fumigatus Y0107 (GenBank accession no. MZ854147, which is currently deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit date of May 17, 2023, and a deposit number of CGMCC No. 40629.

[0045] ITS sequencing

[0046] .

[0047] Example 2: Preparation of alkaloid compounds

[0048] 1. Fermentation:

[0049] Aspergillus fumigatus Y0107 was inoculated into a PDA medium and cultured at 26°C for 48 hours to obtain a slant culture medium; the composition of the slant culture medium was: 120 g potato, 12 g glucose, 10 g agar, and 600 mL water; the slant culture medium was then inoculated into a seed culture medium, cultured at 26°C and 150 rpm for 24 hours to obtain a seed solution; the composition of the seed culture medium was: 80 g potato, 8 g glucose, and 400 mL water.

[0050] The seed liquid was inoculated into a conical flask containing rice culture medium (120 g of rice was placed in a 1 L conical flask, 150 mL of ultrapure water was added, and the flask was sealed with a filter plug and sterilized at 121° C. for 30 min) at a volume concentration of 3%, and 140 flasks were inoculated respectively, and each flask was sealed and then allowed to stand for fermentation at 26° C. for 21 days. After the fermentation of the strain was completed, the 140 flasks of culture were combined and soaked in 18 L of ethyl acetate overnight. After suction filtration, the filter cake was repeatedly soaked in ethyl acetate overnight for 4 times, each time with 10 L of ethyl acetate, and the filtrates were combined and concentrated under reduced pressure to obtain a total crude extract (182.1 g).

[0051] 2. Extraction: Disperse 182.1 g of the total crude extract into a suspension with 2.5 L of ultrapure water, extract three times with 2.5 L of petroleum ether, 2 L of ethyl acetate, and 2 L of n-butanol, collect the ethyl acetate phase extract, concentrate it, transfer it to a beaker, and dry it to obtain the ethyl acetate phase (49.3 g).

[0052] 3. Silica gel chromatography: 49.3 g of the ethyl acetate phase was dissolved in a mixed solution of 30 mL of methanol and 180 mL of dichloromethane, and 75 g of silica gel was added to mix the sample. After drying, the sample was loaded on a silica gel chromatography column (silica gel 100-200 mesh, column height 100 cm, inner diameter 16 cm, column height 46 cm), and eluted with dichloromethane / ethyl acetate in different volume ratios (V / V=1:0, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1), and then eluted with dichloromethane / methanol in different volume ratios (V / V=5:1, 2:1, 1:1, 0:1) for gradient elution. The elution rate was 100 mL / min, the elution volume of different elution solvents was 2 L, and the sample was collected every 500 mL. Thin layer chromatography monitoring was carried out using dichloromethane / ethyl acetate with a volume ratio of 2:1 as the developing solvent. The fractions with similar spots were combined and 11 components were collected. After drying, they were recorded as component Fr.1 to component Fr.11, among which the yield of component Fr.6 was 2.16 g, and the Rf values ​​of component Fr.6 were 0.053, 0.263, 0.395, 0.447, 0.526, 0.684, 0.763, and 0.868, respectively.

[0053] 4. Silica gel chromatography: 2.16 g of component Fr.6 was dissolved in 20 mL of a 2:1 petroleum ether / ethyl acetate solution, and then loaded onto a silica gel chromatography column (silica gel 200-300 mesh, column height 80 cm, inner diameter 8 cm, column packing height 42 cm). Petroleum ether / ethyl acetate in different volume ratios (V / V=2:1, 1:1, 0:1) was used as the eluent. The elution rate was 20 mL / min, and the elution volume of different elution solvents was 600 mL. Petroleum ether / ethyl acetate in a volume ratio of 1:1 was used as the developing solvent for thin layer chromatography monitoring. The fractions with similar spots were combined and 13 components were separated and dried, and recorded as components Fr.6-1 to Fr.6-13; among them, the yields of component Fr.6-5 and component Fr.6-6 were 253 mg and 575.6 mg respectively, and the Rf values ​​of component Fr.6-5 were 0.161, 0.232, 0.304, 0.393, 0.446, and 0.535 respectively; the Rf values ​​of component Fr.6-6 were 0.125, 0.179, 0.286, 0.321, 0.375, 0.429, and 0.536 respectively.

[0054] 5. Compounds of formula (I):

[0055] 253 mg of component Fr.6-5 was dissolved in 5 mL of 1:1 dichloromethane / methanol by volume and loaded onto a hydroxypropyl dextran gel column Sephadex LH-20. The product was eluted and purified using dichloromethane / methanol by volume ratio of 1:1 as the mobile phase. The elution rate was 10 mL / min and the elution volume was 3000 mL. The column liquid was monitored by thin layer chromatography using ethyl acetate / dichloromethane by volume ratio of 1:2 as the developing solvent. The components with the same spots were combined and dried to obtain 4 fractions, which were respectively recorded as components Fr.6-5-1 to Fr.6-5-4; the yield of component Fr.6-5-3 was 162.9 mg, and the Rf values ​​of component Fr.6-5-3 were 0.098, 0.196, 0.235, 0.392, 0.471, 0.549, and 0.745, respectively.

[0056] Fr.6-5-3 (162.9 mg) was selected and dissolved in 4 mL of dichloromethane and then loaded on a silica gel chromatography column (silica 200-300 mesh, column height 40 cm, inner diameter 6 cm, column height 28 cm), and dichloromethane / methanol with different volume ratios (V / V=1:0, 100:1, 50:1, 10:1) was used as the eluent. The elution rate was 12 mL / min, and the elution volume of different elution solvents was 500 mL. The dichloromethane / methanol with a volume ratio of 10:1 was used as the eluent. The developing agent was used for thin layer chromatography monitoring, and the fractions with similar spots were combined and dried to obtain 8 fractions, which were recorded as Fr.6-5-3-1 to Fr.6-5-3-8; among which the yield of component Fr.6-5-3-5 was 37.6 mg, and the Rf values ​​of component Fr.6-5-3-5 were 0.059, 0.157, 0.314, 0.392, 0.431, 0.471, 0.529, 0.569, 0.588, 0.667, and 0.784, respectively.

[0057] Fr.6-5-3-5 (37.6 mg) was dissolved in 1 mL of methanol and purified by semi-preparative HPLC (C 18 The mobile phase was 49% acetonitrile-water system, the flow rate was 1.5 mL / min, the detection wavelengths were 210 nm, 254 nm, 365 nm, and the column temperature was 35 °C. The mobile phase with a retention time of 21.1 min was collected and dried by rotary evaporation to obtain 11-acetyl-pseurotin A2(Ⅰ) (1.7 mg, t R =21.1min). 1 H NMR spectrum is shown in Figure 1 , 13 C NMR spectrum is shown in Figure 2 .

[0058] 6. Compound of formula (II)

[0059] Dissolve 575.6 mg of component Fr.6-6 in 10 mL of a 1:1 dichloromethane / methanol solution and load onto a hydroxypropyl dextran gel column Sephadex. LH-20 was purified by elution with dichloromethane / methanol in a volume ratio of 1:1 as the mobile phase. The elution rate was 10 mL / min and the elution volume was 3000 mL. The lower column liquid was monitored by thin layer chromatography using ethyl acetate / dichloromethane in a volume ratio of 2:1 as the developing solvent. The components with the same spots were combined and dried to obtain 5 fractions, which were recorded as components Fr.6-6-1 to Fr.6-6-5. The yield of component Fr.6-6-4 was 264.3 mg, and the Rf values ​​of component Fr.6-6-4 were 0.116, 0.192, 0.256, 0.372, 0.385, 0.488, 0.538, 0.651, and 0.692, respectively.

[0060] Fr.6-6-4 (264.3 mg) was selected and dissolved in 5 mL of dichloromethane and then loaded onto a silica gel chromatography column (silica 200-300 mesh, column height 40 cm, inner diameter 6 cm, column packing height 24 cm). The eluent was dichloromethane / methanol (V / V = 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 2:1). The elution rate was 12 mL / min, and the elution volume of different elution solvents was 400 mL. The volume ratio was 10 Thin layer chromatography monitoring was carried out using 1:1 dichloromethane / methanol as the developing solvent. The fractions with similar spots were combined and dried to obtain 9 parts, which were recorded as Fr.6-6-4-1 to Fr.6-6-4-9; the yield of component Fr.6-6-4-2 was 102.3 mg, and the Rf values ​​of component Fr.6-6-4-2 were 0.100, 0.233, 0.300, 0.367, 0.433, 0.484, 0.533, and 0.633, respectively.

[0061] Fr.6-6-4-2 (102.3 mg) was dissolved in 2.5 mL of methanol and the mixture was purified by semi-preparative HPLC (C 18 The mobile phase was 45% acetonitrile-water system, the flow rate was 1.5 mL / min, the detection wavelengths were 210 nm, 254 nm, 365 nm, and the column temperature was 35 °C. The mobile phase with a retention time of 18.8 min was collected and dried by rotary evaporation to obtain 20-demethyl-fumiquinazoline C (Ⅱ) (2.1 mg, t R =18.8min). 1 H NMR spectrum is shown in Figure 3 , 13 C NMR spectrum is shown in Figure 4 .

[0062] The compounds 11-acetyl-pseurotin A2(Ⅰ) and 20-demethyl-fumiquinazoline C(Ⅱ) were identified as new alkaloid compounds. The spectral and physicochemical data are as follows:

[0063] 11-Acetyl-pseurotin A(Ⅰ): white solid, molecular formula is C 24 H 27 NO9. 1 H NMR (400MHz, TMS, CDCl3, δ H(in ppm): 8.28 (2H, d, J = 7.2 Hz, H-23, H-19), 7.64 (1H, t, J = 7.6 Hz, H-21), 7.49 (2H, d, J = 8 Hz, H-22, H-20), 7.38 (1H, s, NH), 5.80 (1H, q, J = 4 Hz, H-11), 5.75 (1H, m, H-13), 5.38 (1H, t, J = 4 Hz, 10 Hz, H-12), 4.77 (1H, d, J = 5.6 Hz, H-10), 4.62 (1H, d, J = 11.2 Hz, H-9), 4.03 (1H, d, J = 12.4 Hz, 9-OH), 3.38 (3H, s, OMe), 2.26 (1H, m, H a -14), 2.15 (1H, m, H b -14), 2.04 (3H, s, H-25), 1.80 (3H, s, H-16), 0.97 (3H, t, J = 7.6 Hz, H-15).

[0064] 13 C NMR (100 MHz, TMS, CDCl3, δc in ppm): 197.5 (C-4), 194.5 (C-17), 185.1 (C-2), 170.5 (C-24), 165.2 (C-6), 140.2 (C-13), 134.8 (C-21), 132.5 (C-18), 130.7 (C-19, C-23), 128.9 (C-20, C-22), 122.3 (C-12), 113.6 (C-3), 91.9 (C-5), 89.7 (C-8), 74.0 (C-9), 71.7 (C-11), 70.7 (C-10), 51.8 (OMe), 21.6 (C-14), 21.2 (C-25), 14.1 (C-15), 5.8 (C-16); HR-ESI-MS [M+Na] + m / z 496.1582 (calcd for 496.1564, Δ = 3.6 ppm).

[0065] 20-Demethyl-fumiquinazoline C (Ⅱ): Pale yellow solid, molecular formula C 23 H 19 N5O4. 1 H NMR (400 MHz, TMS, CDCl3, δ Hin ppm): 8.36(1H,dd,J=1.6Hz,7.6Hz,H-10),7.84(1H,dd,J=1.6Hz,7.2Hz,H-8),7.79(1H, dd,J=1.6Hz,8Hz,H-7),7.61(1H,td,J=1.2Hz,8Hz,H-9),7.46(1H,d,J=7.6Hz,H-27),7.3 6(1H,m,H-26),7.33(1H,dd,J=1.2Hz,8Hz,H-25),7.23(1H,d,J=8Hz,H-24),6.92(1H,br. S,NH-2),5.73(1H,d,J=7.2Hz,H-14),5.42(1H,d,J=7.6Hz,H-18),3.57(1H,d,J=8.8Hz,H a -20),3.47(1H,m,H b -20),2.94(1H,dd,J=7.2Hz,15.2Hz,H a -15),2.19(1H,d,J=15.2Hz,H b -15),2.05(3H,s,H-16).

[0066] 13 C NMR (100MHz, TMS, CDCl3, δc in ppm): 170.3(C-1),170.0(C-21),159.8(C-12),150.3(C-4),146.5(C-6),138.1(C- 28),136.2(C-23),135.1(C-8),130.5(C-25),128.7(C-9),128.5(C-7),127.4(C-10 ),126.6(C-24),125.0(C-26),121.4(C-11),115.9(C-27),90.2(C-17),87.4(C-18 ),84.2(C-3),53.0(C-20),51.6(C-14),32.0(C-15),24.8(C-16); HR-ESI-MS[M+Na] + m / z 452.1331 (calcd for 452.1335, Δ=-0.88ppm).

[0067] Example 3: Effects of alkaloid compounds on plant pathogens

[0068] Plant pathogens: Agrobacterium tumefaciens (No. TS343653), Pseudomonas alcaligenes (No. TS275617), Ralstonia solanacearum (No. TS341223), Erwinia sp. (No. TS326150) and Xanthomonas oryzae (No. TS337700) were purchased from Ningbo Testo Biotechnology Co., Ltd.

[0069] Cultivation of plant pathogens: The five plant pathogens (Agrobacteriumtumefaciens, Pantoeaagglomerans, Ralstonia solanacearum, Erwinia sp. and Xanthomonasoryzae) tested in Table 1 were inoculated into a new solid Sabouraud medium by the plate streak method and cultured at 30°C for 12 hours for recovery. Use sterile distilled water to pick an appropriate amount of bacteria and inoculate it into 40mL Sabouraud medium, parallel twice, and culture it in a constant temperature shaking shaker at 24°C for 12 hours. Use sterile Sabouraud medium to prepare the test strain into an appropriate bacterial suspension, detect it by UV-visible spectrophotometer, and dilute it to OD600 of 0.10-0.11 as a bacterial suspension of plant pathogens.

[0070] Solid Sabouraud medium composition: beef extract 3.0g, peptone 10.0g, NaCl 5.0g, distilled water 1L, natural pH, agar 9-12g.

[0071] Sabouraud medium composition: beef extract 3.0 g, peptone 10.0 g, NaCl 5.0 g, distilled water 1 L, pH natural.

[0072] Activity test: The anti-plant pathogenic bacteria activity of 11-acetyl-pseurotin A2 (Ⅰ), 20-demethyl-fumiquinazoline C (Ⅱ), streptomycin (positive control group) and the ethyl acetate phase (EAL) prepared in step 2 of Example 1 was detected by two-fold dilution method. The specific operation was as follows:

[0073] Add 100 μL of Sabouraud culture solution to each well around the 96-well plate, and then add the bacterial suspension of plant pathogens to the remaining 60 wells, 50 μL per well. The initial concentration of compounds (I) and (II) is 200 μg / mL, and the initial concentration of the ethyl acetate phase is 400 μg / mL. Add 50 μL to the second well, mix well, take 50 μL from the second well and add it to the third well according to the two-fold dilution method, mix well, and then take 50 μL from the third well to the fourth well, and repeat this process to the eleventh well. Use streptomycin as a positive control, repeat three times, and culture at 30°C for 24 hours. By observing the infection of plant pathogens, it was found that 100μg / mL 11-acetyl-pseurotin A2 (Ⅰ) and 50μg / mL 20-demethyl-fumiquinazoline C (Ⅱ) could inhibit the growth of plant pathogens and had a certain antibacterial effect, but the antibacterial effect was slightly weaker than that of the positive control group. The antibacterial effect can be further improved by structural modification in the later stage. This shows that the compounds (Ⅰ) and (Ⅱ) described in the present invention have certain agricultural production significance.

[0074] Table 1 Data on the anti-plant pathogenic activity of compounds (I) and (II)

[0075]

[0076] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. An alkaloid compound derived from endophytic fungi of saffron, characterized in that: The structural formula of the alkaloid compound is shown in formula (II):

2. A method for preparing the alkaloid compound according to claim 1, characterized in that: The method is carried out according to the following steps: (1) Fermentation: Inoculate the saffron endophytic fungus Aspergillus fumigatus into a rice culture medium, seal it, and ferment it at 26° C. for 21 days, soak the culture in ethyl acetate overnight, filter it, and concentrate the filtrate under reduced pressure to obtain a crude extract; the rice culture medium is prepared by mixing rice and ultrapure water, and the volume ratio of the ultrapure water is 1-6:1 based on the mass of rice; (2) Extraction: The crude extract from step (1) is dispersed into a suspension with ultrapure water, extracted with petroleum ether, ethyl acetate, and n-butanol in sequence, the ethyl acetate extract is collected, and concentrated to dryness to obtain an ethyl acetate phase; (3) Silica gel chromatography: The ethyl acetate phase in step (2) was dissolved with a mixed solution of methanol and dichloromethane, and silica gel was added to mix the sample. After drying, the sample was loaded onto a silica gel chromatography column and eluted with dichloromethane / ethyl acetate in a volume ratio of 1:0, 50:1, 20:1, 10:1, 5:1, 2:1, and 1:1, and then gradient eluted with dichloromethane / methanol in a volume ratio of 5:1, 2:1, 1:1, and 0:

1. The elution rate was 100 mL / min. The elution volume of the reagent is 2L, and every 500mL of sample is collected and concentrated. Thin layer chromatography monitoring is performed using dichloromethane / ethyl acetate with a volume ratio of 2:1 as a developing solvent, and fractions with similar spots are combined to collect 11 components. After drying, they are respectively recorded as components Fr.1 to Fr.11, among which the Rf value of component Fr.6 is 0.053-0.868; the methanol and dichloromethane mixed solution is prepared by mixing methanol and dichloromethane with a volume ratio of 1:5; (4) Silica gel chromatography: Component Fr.6 was dissolved in a petroleum ether / ethyl acetate solution with a volume ratio of 2:1 and then loaded onto a silica gel chromatography column. The petroleum ether / ethyl acetate with a volume ratio of 2:1, 1:1, and 0:1 was used as the eluent. The elution rate was 20 mL / min and the elution volume of different elution solvents was 600 mL. The petroleum ether / ethyl acetate with a volume ratio of 1:1 was used as the developing solvent for thin layer chromatography monitoring. Fractions with similar spots were combined to separate 13 components, which were dried and recorded as components Fr.6-1 to Fr.6-13. Among them, the Rf values ​​of component Fr.6-5 were 0.161, 0.232, 0.304, 0.393, 0.446, and 0.535; the Rf values ​​of component Fr.6-6 were 0.125, 0.179, 0.286, 0.321, 0.375, 0.429, and 0.

536. (5) Compound of formula (II): The component Fr.6-6 was dissolved in a dichloromethane / methanol solution with a volume ratio of 1:1 and then loaded onto a hydroxypropyl dextran gel column Sephadex LH-20. The mobile phase was dichloromethane / methanol with a volume ratio of 1:

1. The elution rate was 10 mL / min and the elution volume was 3000 mL. The column liquid was monitored by thin layer chromatography using ethyl acetate / dichloromethane with a volume ratio of 2:1 as a developing solvent. The components with the same spots were combined and dried to obtain 5 fractions, which were respectively recorded as Fr.6-6-1 to Fr.6-6-5, among which the Rf value of component Fr.6-6-4 was 0.116-0.692; Select component Fr.6-6-4, dissolve it in dichloromethane, and then load it onto a silica gel chromatography column. Use dichloromethane / methanol with a volume ratio of 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, and 2:1 as elution solvents. The elution rate is 12 mL / min, and the elution volume of different elution solvents is 400 mL. Use dichloromethane / methanol with a volume ratio of 10:1 as a developing solvent for thin layer chromatography monitoring. Combine fractions with similar spots and dry them to obtain 9 parts, which are recorded as Fr.6-6-4-1 to Fr.6-6-4-9, among which the Rf value of component Fr.6-6-4-2 is 0.100-0.633; Component Fr.6-6-4-2 was dissolved in methanol, and semi-preparative HPLC was used to collect the mobile phase with a retention time of 18.8 min, and after rotary evaporation and drying, an alkaloid compound represented by formula (II) was prepared; the semi-preparative HPLC was carried out using C 18 The column was used with 45% acetonitrile-water system as the mobile phase, the flow rate was 1.5 mL / min, the detection wavelengths were 210 nm, 254 nm, and 365 nm, the column temperature was 35 °C, and t R =18.8min.

3. The method according to claim 2, characterized in that The saffron endophytic fungus Aspergillus fumigatus is Aspergillus fumigatus Y0107, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of May 17, 2023, a deposit number of CGMCC No. 40629, and an address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

4. The method according to claim 2, characterized in that Step (1) The Aspergillus fumigatus is first activated by slant and expanded by seed culture before being inoculated into the rice culture medium. The seed liquid is inoculated into the rice culture medium with an inoculum amount of 1-5% by volume. The slant activation and seed culture method is as follows: the Aspergillus fumigatus is inoculated into the PDA culture medium, and cultured at 26° C. for 48 hours to obtain the slant thalli; the slant culture medium consists of: 120 g potato, 12 g glucose, 10 g agar, and 600 mL water; the slant thalli is then inoculated into the seed culture medium, and cultured at 26° C. and 150 rpm for 24 hours to obtain the seed liquid; the seed culture medium consists of: 80 g potato, 8 g glucose, and 400 mL water.

5. The method according to claim 2, characterized in that The ethyl acetate extraction in step (1) is performed 1-5 times, with the amount of ethyl acetate used in the first extraction being 40-10 mL / g based on the weight of the culture, and the amount of ethyl acetate used in the second to fifth extractions being 20-5 mL / g based on the weight of the culture.

6. The method according to claim 2, characterized in that The amount of ultrapure water used in step (2) is 25-10 mL / g based on the weight of the crude extract; the amount of petroleum ether, ethyl acetate and n-butanol used is 25-8 mL / g based on the weight of the crude extract.

7. Use of the alkaloid compound according to claim 1 in the preparation of a plant pathogen inhibitor, characterized in that: The plant pathogenic bacteria are Agrobacterium tumefaciens, Pantoea agglomerans, Ralstonia solanacearum, Erwinia sp. or Xanthomonas oryzae.

8. An Aspergillus fumigatus Y0107 for preparing the alkaloid compound described in claim 1, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit date of May 17, 2023, a deposit number of CGMCC No.40629, and an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

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