Calbistrin compound with antifungal activity as well as preparation method and application of calbistrin compound

Abstract

The invention belongs to the technical field of phytochemistry, and particularly discloses a calbistrin compound with antifungal activity as well as a preparation method and application of the calbistrin compound. The compound disclosed by the invention is prepared from a fermentation crude extract of a strain of tobacco endophytic fungus Aspergillus japonicus TE-739D, and the compound disclosed by the invention is prepared from the fermentation crude extract of the tobacco endophytic fungus Aspergillus japonicus TE-739D. The compounds have been subjected to detailed structural identification by modern spectroscopy techniques, such as high resolution mass spectrometry HRESIMS and superconductive nuclear magnetic resonance spectroscopy NMR. Through SciFindern database retrieval, the compound is found to be a compound with a new structure, and the compound is named as Japonidiene G. The Calbistrin compound Japonidiene G has strong inhibitory activity on various plant pathogenic fungi, the activity of the Calbistrin compound Japonidiene G on alternaria alternata and phytophthora capsici is superior to that of a positive drug carbendazim, and the activity of the Calbistrin compound Japonidiene G on botrytis cinerea is equivalent to that of the positive drug carbendazim.

Description

Technical Field

[0001] This invention belongs to the field of phytochemistry technology, specifically relating to a calbistrin-like compound with antifungal activity, its preparation method, and its application. Background Technology

[0002] Plant diseases, as key biological stressors restricting agricultural production, not only significantly inhibit normal plant growth and development but also directly lead to sharp reductions in crop yields and deterioration in quality, making them one of the major threats to agricultural production. Among these, fungal diseases comprise over 30,000 species, accounting for more than 70% of all plant diseases, becoming the most significant biological factor in field disease occurrence. While currently widely used chemical control methods can quickly control diseases in a short period, their long-term negative effects are becoming increasingly prominent. For example, long-term use can easily lead to pathogen resistance, pesticide residues causing environmental pollution, and potentially jeopardizing food security. Against this backdrop, biological control strategies based on microbial natural products, with their significant advantages such as high environmental friendliness, strong targeting, and good non-target biological safety, are playing an increasingly important role in agricultural disease control. In February 2025, the Ministry of Agriculture and Rural Affairs released the "Key Areas of National Agricultural Science and Technology Innovation (2024-2028)," proposing to strengthen support for the research and registration of green pesticides such as biopesticides, and listing the creation of green pesticides and microbial pesticides as key directions. Discovering novel, efficient, and safe biopesticides based on microbial natural products has become a new trend in agricultural biopharmaceutical research and development.

[0003] Calbistrin compounds are primarily secondary metabolites synthesized by fungi of the genera *Penicillium* and *Aspergillus*. These compounds have been proven to be a rare class of fungal antibiotics, exhibiting highly selective inhibitory activity against fungi, particularly *Candida*. Furthermore, these compounds have been reported to possess various medicinal values, such as antitumor activity, cholesterol reduction, and as promoters of nerve growth factor production. Chinese invention patent "Application of Calbistrins in Plant Disease Control" (CN101473821B) discloses two Calbistrins compounds, Calbistrin A and Calbistrin B. Calbistrins compounds exhibit extremely strong antibiotic activity against pathogenic fungi of the genus *Phytophthora*, with Calbistrin B having a MIC (minimum inhibitory concentration) of only 1.25 μg / mL against *Phytophthora capsici*.

[0004] However, there are currently few reported Calbistrin compounds with antifungal activity, and they target a limited number of fungi. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a calbistrin-like compound with antifungal activity, its preparation method, and its application. The calbistrin-like compound involved in this invention is a novel compound that has not been previously reported, and its inhibitory activity against a variety of plant pathogenic fungi has been discovered for the first time. This compound has the potential to be developed into a novel microbial natural product pesticide.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A calbistrin-like compound with antifungal activity, the compound having the molecular formula C 17 H 26 O4, the molecular structure is as follows:

[0008]

[0009] The present invention discloses a method for preparing the above-mentioned compound containing a polyhydronaphthalene skeleton, wherein Aspergillus japonicus TE-739D mycelium is selected for fermentation culture, and the fermentation broth is separated and purified.

[0010] Aspergillus japonicus TE-739D is deposited by the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40901, classified as Aspergillus japonicus, on October 25, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] Preferably, the specific steps include:

[0012] (1) Select Aspergillus japonicus TE-739D mycelium for fermentation culture to obtain fermentation broth;

[0013] (2) The fermentation broth was extracted with ethyl acetate and concentrated to obtain a crude extract.

[0014] (3) After mixing the crude extract with silica gel, gradient elution was performed by vacuum silica gel column chromatography.

[0015] (4) Collect the fraction Fr.5 eluted under the elution conditions of petroleum ether to ethyl acetate volume ratio of (0:10)-(3:7) and elute it through a reversed-phase silica gel column;

[0016] (5) The subfraction Fr.5.3 eluted under 70% methanol-water conditions was purified by gel column chromatography and semi-preparative high performance liquid chromatography to obtain the compound described above.

[0017] Preferably, the specific operation of step (1) is as follows: the mycelium of the activated strain Aspergillus japonicus TE-739D is picked and inoculated onto a PDA plate medium, cultured at 28°C for 5 days, the mycelium block on the PDA plate is cut and transferred to potato glucose water medium, cultured at room temperature for 30 days to obtain fermentation broth.

[0018] Preferably, the specific operation of step (2) is as follows: the fermentation broth in step (1) is extracted with ethyl acetate by ultrasonic extraction and concentrated under vacuum to obtain crude extract. The conditions for vacuum concentration are: vacuum degree 0.09 MPa, and concentration to a non-fluid state.

[0019] Preferably, the specific operation of step (3) is as follows: after dissolving the crude extract in methanol, 100-200 mesh silica gel is added and mixed, and then the components are separated by vacuum silica gel column chromatography at a vacuum degree of 0.05 MPa. The components are eluted by petroleum ether-ethyl acetate mixtures with volume ratios of 100:0, 95:5, 90:10, 80:20, 70:30, 50:50, 30:70, and 0:100, and methanol-ethyl acetate mixtures with volume ratios of 10:90, 20:80, and 30:70, respectively. Similar components are combined by thin-layer chromatography to obtain 7 components Fr.1-Fr.7 with polarity from small to large.

[0020] Preferably, the specific operation of step (4) is as follows: the fraction Fr.5 eluted from the petroleum ether-ethyl acetate 30:70 mixed system and the 0:100 mixed system is eluted by Lobar LiChroprep RP-18 reverse silica gel column chromatography gradient elution, the elution system is methanol-water volume ratio of 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, and the fractions are separated and combined to obtain 6 subfractions Fr.5.1-5.6.

[0021] Preferably, the subfraction Fr.5.3 obtained by elution system of 70% methanol-water is first separated by Sephadex LH-20 gel chromatography column and then purified by semi-preparative high performance liquid chromatography with mobile phase of 24% acetonitrile-water to obtain the compound.

[0022] The present invention also claims protection for the use of calbistrin-like compounds with antifungal activity in the treatment of plant pathogenic fungi.

[0023] Furthermore, the plant pathogenic fungus is *Tobacco Star*, *Phytophthora capsici*, or *Botrytis cinerea*.

[0024] The beneficial effects of this invention are:

[0025] The Calbistrin compounds described in this invention were prepared from the crude fermentation extract of the tobacco endophytic fungus *Aspergillus japonicus* TE-739D. They were then processed using modern spectroscopic techniques, such as high-resolution mass spectrometry (HRESIMS). Figure 2 ) and superconducting nuclear magnetic resonance (NMR) spectroscopy (with Figure 3 and 4 The novel Calbistrin-like compounds underwent detailed structural identification using SciFinder. n A database search revealed that this compound was a previously unreported novel structure, and it was named Japonidiene G (see appendix). Figure 1 The extraction and preparation method provided in this invention can accurately and rapidly separate and purify the target compound Japonidiene G. The Calbistrin-like compound Japonidiene G can inhibit the growth of various plant pathogenic fungi. Specifically, this compound exhibits superior activity against *Tobacco Star Fever* and *Phytophthora capsici* compared to the positive control fungicide carbendazim; its activity against *Botrytis cinerea* is comparable to that of carbendazim. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 This is a molecular structure diagram of Japonidiene G, a Calbistrin-like compound prepared in Example 1 of the present invention.

[0028] Figure 2 This is a high-resolution mass spectrum of Japonidiene G, a Calbistrin-like compound prepared in Example 1 of this invention.

[0029] Figure 3 The 1H NMR spectrum (500MHz, DMSO-d6) of Japonidiene G, a Calbistrin-like compound prepared in Example 1 of this invention.

[0030] Figure 4The carbon spectrum (125MHz, DMSO-d6) of Japonidiene G, a Calbistrin-like compound prepared in Example 1 of this invention. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1

[0033] A method for preparing a calbistrin-like compound with antifungal activity (named Japonidiene G) is as follows:

[0034] (1) In a sterile operating table, the mycelia of the activated strain Aspergillus japonicus TE-739D were picked and inoculated onto PDA agar plates and cultured in a constant temperature incubator for 5 days at 28℃. After the culture was completed, the mycelial blocks (1cm×1cm) on the PDA plates were cut and transferred to 1L Erlenmeyer flasks (300mL per flask) containing Potato Dextrose Water (brand: Haibo Biotechnology; serial number: HB0233-4). The culture medium had been sterilized at high temperature (121℃, 20min).

[0035] (2) After inoculation, the Erlenmeyer flask was allowed to ferment at room temperature for 30 days, with light and dark treatments for 12 hours each. After fermentation, the culture medium was extracted with ethyl acetate (300 mL / bottle) using ultrasonic extraction (ultrasonic power 800 W, ultrasonic working time 30 min) and concentrated under vacuum (vacuum degree 0.09 MPa) to obtain crude extract.

[0036] (3) After dissolving the crude extract in methanol, 100-200 mesh silica gel was added and mixed. Then, the components were separated by silica gel column chromatography under reduced pressure (vacuum degree 0.05Mpa). Petroleum ether-ethyl acetate mixtures with volume ratios of 100:0, 95:5, 90:10, 80:20, 70:30, 50:50, 30:70, and 0:100, and methanol-ethyl acetate mixtures with volume ratios of 10:90, 20:80, and 30:70 were used for elution. Similar components were combined by thin-layer chromatography to obtain 7 components Fr.1-Fr.7 with increasing polarity.

[0037] (4) The fraction Fr.5 eluted from the petroleum ether-ethyl acetate mixture of 30:70 and 0:100 was further eluted by gradient chromatography on a Lobar LiChroprep RP-18 reverse silica gel column with methanol-water (volume ratios of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%) to obtain six subfractions Fr.5.1-5.6.

[0038] (5) The subfraction Fr.5.3 (elution system of 70% methanol-water) was first separated by Sephadex LH-20 gel chromatography column and then purified by semi-preparative high performance liquid chromatography (24% acetonitrile-water) to finally obtain the compound Japonidiene G.

[0039] Example 2

[0040] In this embodiment, the molecular structure of the compound Japonidiene G prepared in Example 1 was determined by high-resolution mass spectrometry (HRESIMS) and one-dimensional / two-dimensional nuclear magnetic resonance spectroscopy (1D, 2DNMR). The physicochemical properties of compound Japonidiene G are as follows:

[0041] Properties: Pale yellow oily substance; Solubility: Easily soluble in methanol and DMSO; Molecular formula: C 17 H 26 O4; Optical rotation: [α] 25 D +97.5 (c=0.08, MeOH); UV absorption spectrum λ max 239 nm; High-resolution mass spectrometry (HRESIMS, with appendix) Figure 2 ): m / z277.1799[M-H2O+H] + (Theoretical value C) 17 H 25 O3 + ,277.1798); 1H NMR spectral data (attached) Figure 3 ) and carbon spectrum data (attached) Figure 4 See Table 1.

[0042] Table 1: 1H and 1C NMR spectra of the Calbistrin compound Japonidiene G (solvent: DMSO-d6)

[0043]

[0044] Example 3

[0045] This embodiment tests the growth-inhibiting activity of the Calbistrin-like compound Japonidiene G prepared in Example 1 against seven plant pathogenic fungi. The specific process is as follows:

[0046] The seven plant pathogenic fungi involved in this invention are: *Fusarium oxysporum* (Schl.) F.sp. cucumerinum Owen (fusarium wilt of cucumber), *Alternaria alternate* (Fries) Keisslar (tobacco red spot causal agent), *Fusarium graminearum* Schw. (wheat sclerosis), *Alternaria malirob.* (apple leaf spot causal agent), *Phytophthora capsici* Leonian (pepper phytosis), *Colletotrichum orbiculare* Arx. (cucumber anthracnose causal agent), and *Botrytis cinerea* Pers. (grape gray mold causal agent). These plant pathogenic fungi were provided by the College of Chemistry and Pharmacy, Qingdao Agricultural University.

[0047] The tested plant pathogenic fungi were inoculated into PDB medium and cultured at 28°C for 3–5 days. The bacterial suspension was then diluted with PDB medium to a concentration of 10. 6 CFU / mL, used for MIC determination. Dissolve approximately 1 mg of the compound Japonidiene G in approximately 100 μL of DMSO, mix thoroughly, and dilute to a final concentration of 2560 μg / mL as the sample solution. Carbendazim is used as the positive control. Add 10 μL of the compound Japonidiene G (or positive control carbendazim) and 190 μL of bacterial suspension to well 1 of a 96-well microplate. Add 100 μL of bacterial suspension to the remaining 10 wells. After thoroughly mixing well 1, transfer 100 μL to well 2, halving the sample concentration each time. The final sample concentrations in each well are 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL, respectively. In 96-well microplates, different concentrations of the drug were added to the bacterial suspension of the test bacteria. After incubation, the growth of indicator bacteria in a well was observed. If the indicator bacteria grew in a well, it indicated that the drug concentration in that well was insufficient to inhibit the growth of the bacteria; the liquid in that well would be turbid, and the transmittance would be significantly reduced. Conversely, if the liquid in that well was clear, the transmittance would not decrease significantly. The lowest sample concentration at which the growth of indicator bacteria was completely inhibited in a well was defined as the MIC of the compound.

[0048] The results, as shown in Table 2, revealed that the compound Japonidiene G exhibited strong inhibitory activity against *Aureobasidium aizoon*, *Phytophthora capsici*, and *Botrytis cinerea*. Specifically, Japonidiene G showed superior activity against *Aureobasidium aizoon* (MIC = 4 μg / mL) compared to the positive control agent carbendazim (MIC = 16 μg / mL); its activity against *Phytophthora capsici* (MIC = 4 μg / mL) was superior to that of carbendazim (MIC = 8 μg / mL); and its activity against *Botrytis cinerea* (MIC = 4 μg / mL) was comparable to that of carbendazim (MIC = 4 μg / mL).

[0049] Table 2: Growth inhibitory activity (MIC, μg / mL) of the Calbistrin compound Japonidiene G against seven plant pathogenic fungi.

[0050]

[0051] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0052] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A calbistrin-like compound with antifungal activity, characterized in that, The compound has the molecular formula C. 17 H 26 O4, the molecular structure is as follows:

2. The method for preparing a calbistrin-like compound with antifungal activity according to claim 1, characterized in that, Select mycelia of Aspergillus japonicus TE-739D for fermentation culture. The fermentation broth is then separated and purified. Aspergillus japonicus TE-739D is deposited by the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40901, classified as Aspergillus japonicus, on October 25, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

3. The method for preparing a calbistrin-like compound with antifungal activity according to claim 2, characterized in that, Specifically, the steps include the following: (1) Select Aspergillus japonicus TE-739D mycelium for fermentation culture to obtain fermentation broth; (2) The fermentation broth was extracted with ethyl acetate and concentrated to obtain a crude extract. (3) After mixing the crude extract with silica gel, gradient elution was performed by vacuum silica gel column chromatography. (4) Collect the fraction Fr.5 eluted under the elution conditions of petroleum ether to ethyl acetate volume ratio of (0:10)-(3:7) and elute it through a reversed-phase silica gel column; (5) The subfraction Fr.5.3 eluted under 70% methanol-water conditions was purified by gel column chromatography and semi-preparative high performance liquid chromatography to obtain the compound as described in claim 1.

4. The method for preparing a calbistrin-like compound with antifungal activity according to claim 3, characterized in that, The specific operation of step (1) is as follows: the mycelium of the activated strain Aspergillus japonicus TE-739D is picked and inoculated onto PDA plate medium, cultured at 28℃ for 5 days, the mycelium block on the PDA plate is cut and transferred to potato glucose water medium, cultured at room temperature for 30 days to obtain fermentation broth.

5. The method for preparing a calbistrin-like compound with antifungal activity according to claim 3, characterized in that, The specific operation of step (2) is as follows: the fermentation broth in step (1) is extracted with ethyl acetate using ultrasonic extraction, with an ultrasonic power of 800W and an ultrasonic working time of 10s. The crude extract is obtained by vacuum decompression concentration. The conditions for vacuum decompression concentration are: vacuum degree of 0.09Mpa and concentration to a non-fluid state.

6. The method for preparing a calbistrin-like compound with antifungal activity according to claim 3, characterized in that, The specific operation of step (3) is as follows: after dissolving the crude extract in methanol, add 100-200 mesh silica gel and mix. Then, separate the components by vacuum silica gel column chromatography under reduced pressure of 0.05 MPa. Use petroleum ether-ethyl acetate mixtures with volume ratios of 100:0, 95:5, 90:10, 80:20, 70:30, 50:50, 30:70, and 0:100, and methanol-ethyl acetate mixtures with volume ratios of 10:90, 20:80, and 30:70 for elution. Use thin-layer chromatography to combine similar components to obtain 7 components Fr.1-Fr.7 with polarity from small to large.

7. The method for preparing a calbistrin-like compound with antifungal activity according to claim 6, characterized in that, The specific operation of step (4) is as follows: the fraction Fr.5 eluted from the petroleum ether-ethyl acetate 30:70 mixed system and the 0:100 mixed system is eluted by LobarLiChroprep RP-18 reverse silica gel column chromatography gradient elution. The elution system is methanol-water volume ratio of 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, and the fractions are separated and combined to obtain 6 subfractions Fr.5.1-5.

6.

8. The method for preparing a calbistrin-like compound with antifungal activity according to claim 7, characterized in that, The subfraction Fr.5.3 obtained by elution with a 70% methanol-water system was first separated by Sephadex LH-20 gel chromatography column and then purified by semi-preparative high performance liquid chromatography with a mobile phase of 24% acetonitrile-water to obtain the compound as described in claim 1.

9. The application of the calbistrin-like compounds with antifungal activity as described in claim 1 in the fight against plant pathogenic fungi.

10. The application as described in claim 9, characterized in that, The plant pathogenic fungi are *Tobacco Star*, *Phytophthora capsici*, or *Botrytis cinerea*.

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