Lactones compound, preparation method and application

Abstract

The invention belongs to the field of biological medicine, discloses a lactone compound, a preparation method and application, and particularly relates to a separation and purification method of the lactone compound and the inhibitory activity of the lactone compound on fusarium graminearum and valsa mali Miyabe.The separation and purification method comprises the steps of Cladosporium tenuissimum large-scale fermentation, methanol ultrasonic extraction, vacuum concentration and ethyl acetate extraction. According to the method, the lactone compounds in the Cladosporidium tenuissimum are separated and purified through the steps of normal-phase column chromatography, high-performance liquid chromatography separation and purification and the like, and the lactone compounds are obtained from the Cladosporidium tenuissimum solid fermentation product in combination with multiple modern spectrum analysis technologies such as nuclear magnetic resonance spectrum and high-resolution mass spectrum. The compound is obtained by being separated from a solid rice fermentation product of Cladosporium tenuissimum, and the Cladosporium tenuissimum is separated from kadsura longepedunculata. The lactone compound disclosed by the invention has relatively good inhibitory activity on fusarium graminearum and valsa mali Miyabe, and can be used for preparing an antifungal drug.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a method for separating and purifying lactone compounds in Cladosporium tenuissimum and application of the method in preparing antifungal drugs. Background Art

[0002] In humanity's long battle against disease, drug-resistant fungi have become a growing challenge. With the increasing number of immunocompromised patients, the emergence of fungal resistance, and the overuse of antifungal drugs in agriculture, animal husbandry, and clinical settings, the risk of fatal fungal infections in humans is increasing. Lactones are a class of organic compounds containing an internal ester structure, typically formed through esterification. As part of the polyketide family, lactones have attracted widespread attention due to their diverse structures and bioactivities, including antifungal, cytotoxic, antibacterial, anti-inflammatory, and immunomodulatory properties. They possess important physiological and pharmacological properties in many organisms, such as antifungals, anticancer drugs, immunosuppressants, and hormones. In recent years, an increasing number of researchers have investigated lactones to explore their antifungal activity, providing important insights into the development and production of new antifungal drugs. Currently, the practical application of existing lactone antifungal drugs is limited by toxicity, drug interactions, and drug resistance, leading to the increasing morbidity and mortality of fungal infections. In addition, traditional activity-based screening methods face many challenges. Not only are they prone to repeated discovery of existing lactone antifungal drugs, but they also lack new antifungal targets, which makes the research and development of new antifungal drugs difficult. Therefore, the search for new lactone antifungal drugs is urgent. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a lactone compound from Cladosporium tenuissimum, a preparation method and an application thereof. The compound is isolated from a solid rice fermentation product of a strain of Cladosporium tenuissimum, which is isolated from Schisandra chinensis and has strong inhibitory activity against Fusarium culmorum and Valsa maliMiyabe.

[0004] It should be noted that Schisandra chinensis (Schisandra chinensis), a member of the Magnoliaceae family, is primarily used to treat chronic cough, nocturnal emission, spermatorrhea, bedwetting, frequent urination, chronic diarrhea, spontaneous night sweats, thirst due to yin deficiency, internal heat, palpitations, and insomnia. Its chemical components primarily include triterpenes, lignans, and steroids. Modern pharmacological and clinical studies have demonstrated that extracts and chemical components from this genus possess anti-rheumatic, antioxidant, and anti-inflammatory activities, and can also be used to treat rheumatism and liver diseases. The symbiotic bacteria of Schisandra chinensis form a complex and intriguing microbial community, interacting closely with the plant. Research has shown that these bacteria can produce a variety of plant-derived natural products with unique chemical structures and biological activities. These bacteria are closely associated with plant secondary metabolism, participating in the synthesis and regulation of secondary metabolites such as terpenes, thereby influencing the medicinal properties and quality of the plant.

[0005] Cladosporium fungi can produce a wide variety of secondary metabolites, many of which have antiviral, antimalarial, and antibacterial activities. These substances also have a good effect in protecting plants from other biotic and abiotic stresses. For example, a new cyclohexene derivative, cladscyclitols B, and a new ribofuranose phenol derivative, 4-O-α-D-ribofuranose-2-pentyl-3-benzyl alcohol, were obtained from the extract of the mangrove-derived fungus Cladosporium sp. JJM22. They showed effective inhibitory activity against α-glucosidase, IC 50 The values were 2.95 and 2.05 μM, respectively. An extract of Cladosporium sp., a symbiotic fungus found in the leaves of European olive trees, inhibited the growth of 27 different Candida species, including Candida albicans, C. lypolitica, C. tropicalis, C. sphaerica, C. krusei, C. guilliermondii, C. parapsilosis, C. norvegicus, C. glabrata, and C. kefyr. The inhibitory effect increased with increasing concentration, reaching its highest level at 30 mg / mL, effectively inhibiting fungal growth. This demonstrates that Cladosporium sp. symbiotic fungi have great potential for future anti-α-glucosidase and antifungal activities.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first technical purpose of the present invention is to provide a lactone compound having the chemical structural formula:

[0008] A.

[0009]

[0010] The compound is named: pandangolide 1, and its molecular formula is C 12 H 20 O4, white powder;

[0011] HR-ESI-MS:m / z 245.1377[M+H] + (calcd.for C 12 H 21 O4,245.1384);

[0012] B.

[0013]

[0014] The compound is named: pandangolide 1a, and its molecular formula is C 12 H 20 O5, white powder;

[0015] HR-ESI-MS: m / z 267.1208[M+Na] + (calcd.for C 12 H 20 O5Na,267.1206);

[0016] C.

[0017]

[0018] The compound is named: de-o-methyldiaporthin, and its molecular formula is C 12 H 12 O5, white powder;

[0019] HR-ESI-MS:m / z 237.0749[M+H] + (calcd.for C 12 H 13 O5,237.0757);

[0020] D.

[0021]

[0022] The compound is named: (4S,5S,11S)-iso-cladospolide B, and its molecular formula is C 12 H 20 O4, white powder;

[0023] HR-ESI-MS:m / z 229.1425[M+H] + (calcd.for C 12 H 21 O4,229.1434).

[0024] It should be noted that the lactone compounds of the present invention 1 H-NMR spectrum, 13 C-NMR spectrum and mass spectrum data are attached Figure 1-12 .

[0025] The second technical object of the present invention is to provide a method for preparing the lactone compounds as described above, wherein the lactone compounds pandangolide 1, pandangolide 1a, de-o-methyldiaporthin and (4S,5S,11S)-iso-cladospolide B are isolated from the solid fermentation product of Cladosporium tenuissimum.

[0026] Furthermore, the solid fermentation product of Cladosporium tenuissimum is prepared by culturing the seed liquid of Cladosporium tenuissimum on a solid rice culture medium.

[0027] Furthermore, the solid rice culture medium is used to carry out large-scale fermentation culture of Cladosporium tenuissimum, and the specific steps are as follows:

[0028] S1. Solid rice culture medium preparation: Add 40 g of rice and 60 mL of deionized water to each of 220 500 mL Erlenmeyer flasks. Seal with a breathable membrane and sterilize in an autoclave (121°C, 30 min). Cool and set aside.

[0029] S2. PDB medium preparation: Add 1.05 g potato dextrose broth and 30 mL deionized water to each of six 50 mL Erlenmeyer flasks. Seal the flasks with a breathable membrane and sterilize them in an autoclave (121°C, 30 min). Then, sterilize all flasks in a clean bench under UV light for 15 min.

[0030] S3. Seed solution preparation: Inoculate the bacterial cake of Cladosporium tenuissimum into PDB medium, with an inoculation area of about 1cm per bottle. 2 The bacterial cake was labeled and cultured in a constant temperature shaker for 5 days (28°C, 120 rpm);

[0031] S4. Under sterile conditions, 800 μL of seed solution was inoculated into 220 500 mL conical flasks of solid rice culture medium and statically cultured at a constant temperature of 28°C for 23 days.

[0032] In the above-mentioned method for preparing lactone compounds, the solid fermentation product of Cladosporium tenuissimum is ultrasonically extracted five times with an equal volume of methanol, the five extracts are combined, and after concentration under reduced pressure, it is extracted five times with an equal volume of ethyl acetate, and the five extracts are combined to obtain an ethyl acetate phase extract;

[0033] The ethyl acetate extract was subjected to silica gel column chromatography with a gradient elution of petroleum ether-ethyl acetate (volume ratio, 100:0, 100:1, 40:1, 20:1, 18:1, 14:1) to obtain fractions A to E; fraction D was subjected to gradient elution by medium- and low-pressure liquid chromatography (methanol-water, 30%-100%, 20 mL / min, detection wavelengths set at 210, 254 and 365 nm) to obtain fractions D1 to D6; fraction D2 was sequentially subjected to isocratic elution by Sephadex LH-20 (solvent: methanol) and preparative high performance liquid chromatography (methanol-water, 65%, 2 mL / min, detection wavelengths set at 210 and 254 nm) to obtain compound pandangolide 1 (t R =20.5min,10.2mg) and pandangolide 1a(t R =23.8min,9.4mg); Fraction D5 was gradient eluted with RP-18 chromatography column under the conditions of methanol:water (10%→100%) to obtain fractions D5.1 to D5.3. Fraction D5.2 was sequentially purified by Sephadex LH-20 column (volume ratio, solvent: chloroform:methanol=1:1) and silica gel column chromatography with petroleum ether-ethyl acetate (volume ratio, 1:1, 1:2, 1:3, 1:4, 1:5) gradient elution to obtain compounds de-o-methyldiaporthin (9.2mg) and (4S,5S,11S)-iso-cladospolide B (10.3mg).

[0034] The third technical purpose of the present invention is to provide a use of the lactone compound as described above in the preparation of antifungal drugs.

[0035] It should be noted that the lactone compounds of the present invention have good inhibitory activity against Fusarium culmorum and Valsa maliMiyabe, and can be used to prepare antifungal drugs.

[0036] Specifically, the lactone compounds pandangolide 1, pandangolide 1a and (4S,5S,11S)-iso-cladospolide B exhibited strong inhibitory activity against Fusarium culmorum, with MIC values of 12.5, 12.5 and 50 μM, respectively. The compounds pandangolide 1, pandangolide 1a and de-o-methyldiaporthin exhibited strong inhibitory activity against Valsa maliMiyabe, with MIC values of 12.5, 50 and 25 μM, respectively. The compound (4S,5S,11S)-iso-cladospolide B exhibited moderate inhibitory activity against Valsa maliMiyabe, with an MIC value of 100 μM. The MIC values of the positive control carbendazim against Fusarium culmorum and Valsa maliMiyabe were both 50 μM.

[0037] The fourth technical purpose of the present invention is to provide an antifungal drug, which contains the lactone compound described in the present invention.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1) The present invention obtains a lactone compound from Cladosporium tenuissimum for the first time, and the lactone compound from Cladosporium tenuissimum of the present invention is tested for its inhibitory activity against Fusarium culmorum and Valsa maliMiyabe in vitro. The results show that the lactone compounds pandangolide 1, pandangolide 1a and (4S,5S,11S)-iso-cladospolide B from Cladosporium tenuissimum exhibit strong inhibitory activity against Fusarium culmorum, with MIC values of 12.5, 12.5 and 50 μM, respectively. The compounds pandangolide 1, pandangolide 1a and de-o-methyldiaporthin exhibit strong inhibitory activity against Valsa maliMiyabe. maliMiyabe) showed strong inhibitory activity with MIC values of 12.5, 50 and 25 μM, respectively. The compound (4S,5S,11S)-iso-cladospolide B showed moderate inhibitory activity against Valsa maliMiyabe with a MIC value of 100 μM. The MIC value of the positive control carbendazim against Fusarium culmorum and Valsa maliMiyabe was 50 μM.

[0040] 2) The lactone compounds of the present invention have strong inhibitory activity against Fusarium culmorum and Valsa mali Miyabe, can be used as antifungal drugs, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0042] Figure 1 The lactone compound pandangolide 1 of the present invention 1 H-NMR spectrum;

[0043] Figure 2The lactone compound pandangolide 1 of the present invention 13 C-NMR spectrum;

[0044] Figure 3 is the HRESIMS spectrum of the lactone compound pandangolide 1 of the present invention;

[0045] Figure 4 The lactone compound pandangolide 1a of the present invention 1 H-NMR spectrum;

[0046] Figure 5 The lactone compound pandangolide 1a of the present invention 13 C-NMR spectrum;

[0047] Figure 6 is the HRESIMS spectrum of the lactone compound pandangolide 1a of the present invention;

[0048] Figure 7 The lactone compound de-o-methyldiaporthin of the present invention 1 H-NMR spectrum;

[0049] Figure 8 The lactone compound de-o-methyldiaporthin of the present invention 13 C-NMR spectrum;

[0050] Figure 9 is the HRESIMS spectrum of the lactone compound de-o-methyldiaporthin of the present invention;

[0051] Figure 10 The lactone compound (4S, 5S, 11S)-iso-cladospolide B of the present invention 1 H-NMR spectrum;

[0052] Figure 11 The lactone compound (4S, 5S, 11S)-iso-cladospolide B of the present invention 13 C-NMR spectrum;

[0053] Figure 12 This is the HRESIMS spectrum of the lactone compound (4S,5S,11S)-iso-cladospolide B of the present invention. DETAILED DESCRIPTION

[0054] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0055] It should be noted that the experimental materials are as follows:

[0056] Culture medium:

[0057] Solid rice medium: 40.00 g rice, 60.00 mL water, pH natural; medium was autoclaved at 121°C for 30 min before use.

[0058] Experimental reagents:

[0059] Methanol, acetone, and chloroform are all industrial-grade reagents and are used after redistillation; ethyl acetate and petroleum ether are analytical-grade reagents; organic solvents such as chromatographic acetonitrile, DMSO, and chromatographic methanol are of analytical or chromatographic grade depending on the actual situation.

[0060] Preparation of thin layer chromatography (TLC) vanillin sulfuric acid color developer: 80 mL of concentrated sulfuric acid was slowly added to 200 g of ice while stirring. Then 5 g of vanillin (vanillin) was placed in 100 mL of anhydrous ethanol and stirred until dissolved. The dissolved vanillin anhydrous ethanol solution was added to ice water.

[0061] Experimental instruments:

[0062] Nuclear magnetic resonance: Agilent DD2400-MR (TMS internal standard); High performance liquid chromatography-high resolution mass spectrometry: ABSciex Triple 4600; polarimeter Rudolph Autopol III MCP5300; high performance liquid chromatographs: Waters 1525, Agilent 1260 and Arc HPLC analytical type; infrared spectrometer: PerkinElmer Frontier (potassium bromide tablet method); ultraviolet spectrometer: Shimadzu UV-2550; circular dichroism spectrometer: Chirascan CD; medium and low pressure liquid chromatograph: Büchi Flash Systems X-10; Microplate Reader: Synergy neo2; Fungal Incubator: BSD-TX345; Rotary Evaporator: Büchi Rotavapor R-100; Circulating Water Multi-Purpose Vacuum Pump: SHB-Ⅲ (Zhengzhou Great Wall Science & Technology Industry & Trade Co., Ltd.); Low-Temperature Coolant Circulating Pump: DLSB-10 / 20 (Zhengzhou Great Wall Science & Technology Industry & Trade Co., Ltd.); Clean Bench: SW-OJ-2F (Suzhou Antai Air Technology Co., Ltd., Sujing Group); Vertical Steam Sterilizer: BKQ-B12011 (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory). Liquid chromatography columns: Waters XBridge C18 column (19 mm × 250 mm, 5 μm), Waters XBridge C18 column (10 × 250 mm, 5 μm), XBridge C18 column (4.6 × 250 mm, 5 μm); column chromatography silica gel (100–200 mesh, 200–300 mesh, and 300–400 mesh) and thin-layer chromatography silica gel (silica gel H) were all produced by Qingdao Ocean Chemical Plant; hydroxypropyl dextran gel Sephadex LH-20 was produced by GE Healthcare Life Sciences, and RP-C18 reverse-phase silica gel was produced by YMC.

[0063] Example 1: A lactone compound from Cladosporium tenuissimum and a method for its separation and purification

[0064] The lactone compounds pandangolide 1, pandangolide 1a, de-o-methyldiaporthin, and (4S,5S,11S)-iso-cladospolide B of the present invention are isolated from the solid fermentation product of a strain of Cladosporium tenuissimum. The strain is isolated from Schisandra chinensis and identified as Cladosporium tenuissimum by fungal ITS rDNA strain identification. The ITS sequence is:

[0065] The preservation and activation culture conditions for Cladosporium tenuissimum are as follows: The strain is preserved using PDA slant culture medium. To activate the strain, the strain is inoculated onto a PDA plate and incubated at 28°C for 7 days until the plate is covered with mycelium. The PDA plate medium contains 200g of potato, 20g of glucose, and 16g of agar powder per liter. 1. Large-Scale Fermentation Culture of Cladosporium tenuissimum:

[0066] The strain was fermented on a large scale using solid rice culture medium. The specific steps are as follows:

[0067] S1. Solid rice culture medium preparation: Add 40 g of rice and 60 mL of deionized water to each of 220 500 mL Erlenmeyer flasks. Seal with a breathable membrane and sterilize in an autoclave (121°C, 30 min). Cool and set aside.

[0068] S2. PDB medium preparation: Add 1.05 g potato dextrose broth and 30 mL deionized water to each of six 50 mL Erlenmeyer flasks. Seal the flasks with a breathable membrane and sterilize them in an autoclave (121°C, 30 min). Then, sterilize all flasks in a clean bench under UV light for 15 min.

[0069] S3. Seed solution preparation: Inoculate the bacterial cake of Cladosporium tenuissimum into PDB medium, with an inoculation area of about 1cm per bottle. 2 The bacterial cake was labeled and cultured in a constant temperature shaker for 5 days (28°C, 120 rpm);

[0070] S4. Under sterile conditions, 800 μL of seed solution was inoculated into 220 500 mL conical flasks of solid rice culture medium and statically cultured at a constant temperature of 28°C for 23 days.

[0071] 2. The specific extraction and separation methods are as follows:

[0072] After large-scale fermentation finishes, in all 500mL Erlenmeyer flasks, add equal-volume methanol, stop fermentation, solid rice culture medium-methanol solution is moved in the 30L plastic bucket, use Buchner funnel suction filtration after using large-scale ultrasonic instrument ultrasonic extraction 30min, filter residue is transferred back in the plastic bucket and adds methanol to repeat top step and extract 4 times again, merge whole extracts, extracting solution steams with large-scale rotary evaporator concentrating under reduced pressure until without methanol, measure and spin out methanol alcohol content, if alcohol content is greater than 80% (v / v), methyl alcohol is reusable, after concentrating, obtains crude extract.The crude extract that is dissolved in water is extracted 5 times with equal-volume ethyl acetate again, gets supernatant and concentrates through rotary evaporation and has obtained ethyl acetate phase extractum. The ethyl acetate extract was subjected to silica gel column chromatography with a gradient elution of petroleum ether-ethyl acetate (volume ratio, 100:0, 100:1, 40:1, 20:1, 18:1, 14:1) to obtain fractions A to E; fraction D was subjected to gradient elution by medium- and low-pressure liquid chromatography (methanol-water, 30%-100%, 20 mL / min, detection wavelengths set at 210, 254 and 365 nm) to obtain fractions D1 to D6; fraction D2 was sequentially subjected to isocratic elution by Sephadex LH-20 (solvent: methanol) and preparative high performance liquid chromatography (methanol-water, 65%, 2 mL / min, detection wavelengths set at 210 and 254 nm) to obtain compound pandangolide 1 (t R =20.5min,10.2mg) and pandangolide 1a(t R =23.8min,9.4mg); Fraction D5 was gradient eluted with RP-18 chromatography column under the conditions of methanol:water (10%→100%) to obtain fractions D5.1 to D5.3. Fraction D5.2 was sequentially purified by Sephadex LH-20 column (volume ratio, solvent: chloroform:methanol=1:1) and silica gel column chromatography with petroleum ether-ethyl acetate (volume ratio, 1:1, 1:2, 1:3, 1:4, 1:5) gradient elution to obtain compounds de-o-methyldiaporthin (9.2mg) and (4S,5S,11S)-iso-cladospolide B (10.3mg).

[0073] The chemical structures of the lactone compounds pandangolide 1, pandangolide 1a, de-o-methyldiaporthin and (4S,5S,11S)-iso-cladospolide B are as follows:

[0074] A.

[0075]

[0076] The compound is named: pandangolide 1, and its molecular formula is C 12 H 20 O4, white powder;

[0077] HR-ESI-MS:m / z 245.1377[M+H] + (calcd.for C 12 H 21 O4,245.1384);

[0078] B.

[0079]

[0080] The compound is named: pandangolide 1a, and its molecular formula is C 12 H 20 O5, white powder;

[0081] HR-ESI-MS: m / z 267.1208[M+Na] + (calcd.for C 12 H 20 O5Na,267.1206);

[0082] C.

[0083]

[0084] The compound is named: de-o-methyldiaporthin, and its molecular formula is C 12 H 12 O5, white powder;

[0085] HR-ESI-MS:m / z 237.0749[M+H] + (calcd.for C 12 H 13 O5,237.0757);

[0086] D.

[0087]

[0088] The compound is named: (4S,5S,11S)-iso-cladospolide B, and its molecular formula is C 12 H 20 O4, white powder;

[0089] HR-ESI-MS:m / z 229.1425[M+H] + (calcd.for C 12H 21 O4,229.1434).

[0090] The NMR spectra and mass spectra of the above lactone compounds are shown in Figure 1-12 .

[0091] Example 2: Inhibitory activity test of lactone compounds from Cladosporium tenuissimum against Fusarium culmorum and Valsa maliMiyabe:

[0092] The lactone compounds pandangolide 1, pandangolide 1a, de-o-methyldiaporthin and (4S,5S,11S)-iso-cladospolide B were tested for their inhibitory activity against Fusarium culmorum and Valsa mali Miyabe using the microdilution method.

[0093] (1) Preparation of YPD liquid culture medium: Add 5 g yeast extract, 10 g peptone, and 450 mL deionized water to two 500 mL conical flasks. Prepare 100 mL of 20% glucose solution in another 250 mL conical flask. Seal the three conical flasks with air-permeable membranes and sterilize them in an autoclave (121°C, 20 min). After sterilization, when the temperature drops to about 50°C, add 50 mL of 20% glucose solution to each of the two 500 mL conical flasks, mix well, place in a clean bench, sterilize with UV light for 15 min, and cool for later use.

[0094] (2) Preparation of YPD solid culture medium: 5 g yeast extract, 10 g peptone, 450 mL deionized water, and 10 g agar were added to two 500 mL conical flasks. 100 mL of 20% glucose solution was prepared in another 250 mL conical flask. The three conical flasks were sealed with air-permeable membranes and sterilized in an autoclave (121°C, 20 min). After sterilization, when the temperature dropped to about 50°C, 50 mL of 20% glucose solution was added to each of the two 500 mL conical flasks. The mixture was then transferred to a culture dish, placed in a clean bench, and sterilized under UV light for 15 min. The mixture was cooled and set aside.

[0095] (3) Preparation of bacterial solution: Under sterile conditions, the mycelia of Gibberella tatarica and Black rot fungus of apple were inoculated into YPD solid medium respectively, marked and placed in a constant temperature incubator for 3 days (28°C); after the two plant pathogenic fungi were cultured in YPD solid medium, about 1 cm 2The bacterial cake was inoculated into YPD liquid culture medium and cultured in a constant temperature incubator at 28°C and 120 rpm for 24 h to obtain bacterial liquids of Gibberella graminearum and Black rot fungus of apples;

[0096] (4) Determination of initial bacterial concentration: The bacterial solution was added to a 96-well plate and the absorbance was measured at 600 nm using a microplate reader. The initial bacterial concentrations of Gibberella graminearum and Psoralea truncatula were 0.6-0.8 OD.

[0097] (5) Determination of minimum inhibitory concentration: The powders of the lactone compounds pandangolide 1, pandangolide 1a, de-o-methyldiaporthin and (4S,5S,11S)-iso-cladospolide B and the positive control carbendazim were dissolved in dimethyl sulfoxide (DMSO) to prepare sample solutions, which were added to the 96-well plates in (4) and cultured in a constant temperature incubator at 28°C for 2 days. The minimum inhibitory concentration (MIC) of the lactone compounds and the positive control was determined by observing the growth of Gibberella zeae and Black rot Fungus.

[0098] The inhibitory activities of the lactone compounds pandangolide 1, pandangolide 1a, de-o-methyldiaporthin and (4S,5S,11S)-iso-cladospolide B against Fusarium culmorum and Valsa mali Miyabe were analyzed by in vitro bioassay. The results showed that the lactone compounds pandangolide 1, pandangolide 1a and (4S,5S,11S)-iso-cladospolide B from Cladosporium tenuissimum showed strong inhibitory activities against Fusarium culmorum, with MIC values of 12.5, 12.5 and 50 μM, respectively. The compounds pandangolide 1, pandangolide 1a and de-o-methyldiaporthin had strong inhibitory activities against Valsa mali Miyabe. maliMiyabe) showed strong inhibitory activity with MIC values of 12.5, 50 and 25 μM, respectively. The compound (4S,5S,11S)-iso-cladospolide B showed moderate inhibitory activity against Valsa maliMiyabe with a MIC value of 100 μM. The MIC value of the positive control carbendazim against Fusarium culmorum and Valsa maliMiyabe was 50 μM. The detailed data of MIC values against Fusarium culmorum and Valsa maliMiyabe are shown in Table 1.

[0099] Table 1 MIC values of lactone compounds against F. culmorum and Valsamali Miyabe

[0100]

[0101] a Positive control; b NA: no activity

[0102] In summary, the lactone compounds pandangolide 1, pandangolide 1a, de-o-methyldiaporthin and (4S,5S,11S)-iso-cladospolide B can be developed as lead compounds for antifungal drugs.

[0103] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A lactone compound, characterized in that The structural formula of the compound is: A、 The molecular formula of the compound is C 12 H 20 O4, named: pandangolide 1; B、 The molecular formula of the compound is C 12 H 20 O5, named: pandangolide 1a; C、 The molecular formula of the compound is C 12 H 12 O5, designated as: de-o-methyldiaporthin; D、 The molecular formula of the compound is C 12 H 20 O4, named: (4S,5S,11S)-iso-cladospolide B.

2. A method for preparing a lactone compound according to claim 1, characterized in that: The lactone compounds pandangolide 1, pandangolide 1a, de-o-methyldiaporthin and (4S, 5S, 11S)-iso-cladospolide B are isolated from the solid fermentation product of Cladosporium tenuissimum.

3. The method for preparing a lactone compound according to claim 2, wherein: The solid fermentation product of Cladosporium tenuissimum is prepared by culturing the seed liquid of Cladosporium tenuissimum on a solid rice culture medium.

4. The method for preparing a lactone compound according to claim 3, wherein The large-scale fermentation culture of Cladosporium tenuissimum using solid rice culture medium is carried out in the following specific steps: S1, solid rice culture medium preparation: add 40g rice and 60mL deionized water to 220 500mL Erlenmeyer flasks, seal with breathable membrane, sterilize in autoclave, and cool for use; S2. PDB medium preparation: Add 1.05 g potato dextrose broth and 30 mL deionized water to each of six 50 mL Erlenmeyer flasks. Seal the flasks with a breathable membrane and sterilize them in an autoclave. Then, place all flasks in a clean bench and sterilize them under UV light for 15 min. S3. Seed solution preparation: Inoculate the bacterial cake of Cladosporium tenuissimum into PDB medium, with an inoculation area of about 1cm per bottle. 2 The bacterial cake was labeled and cultured in a constant temperature shaker at 28°C and 120 rpm for 5 days; S4. Under sterile conditions, 800 μL of seed solution was inoculated into 220 500 mL conical flasks of solid rice culture medium and statically cultured at a constant temperature of 28°C for 23 days.

5. The method for preparing a lactone compound according to claim 4, wherein The solid fermentation product of Cladosporium tenuissimum was ultrasonically extracted five times with an equal volume of methanol, the five extracts were combined, concentrated under reduced pressure, and then extracted five times with an equal volume of ethyl acetate, the five extracts were combined to obtain an ethyl acetate phase extract; The ethyl acetate extract was eluted by silica gel column chromatography with a gradient elution of petroleum ether and ethyl acetate in a volume ratio of 100:0, 100:1, 40:1, 20:1, 18:1, and 14:1 to obtain fractions A to E. Fraction D was subjected to gradient elution by medium and low pressure liquid chromatography to obtain fractions D1 to D6. Fraction D2 was sequentially eluted by Sephadex LH-20 column and preparative high performance liquid chromatography by isocratic elution to obtain compounds pandangolide 1 and pandangolide 1a. Fraction D5 was gradient eluted by RP-18 column chromatography to obtain fractions D5.1 to D5.

3. Fraction D5.2 was sequentially eluted by Sephadex LH-20 column and silica gel column chromatography with a gradient elution of petroleum ether and ethyl acetate to obtain compounds de-o-methyldiaporthin and (4S,5S,11S)-iso-cladospolide B.

6. Use of the lactone compound according to claim 1 in the preparation of antifungal drugs.

7. An antifungal drug, characterized in that: The medicine contains the lactone compound as claimed in claim 1.

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