Compound derived from reed endophytic fungus and preparation method and application thereof

Abstract

The application discloses a compound byssochlanone B, and a structure formula of the compound is shown in the following.The application also discloses a preparation method and application of the byssochlanone B, and the application separates and purifies endophytic fungi from collected sample reed plants, identifies the strains, and carries out in-vitro anti-tumor activity screening, then selects the best active strain for large-scale fermentation, and carries out systematic separation and purification on the fermentation product, obtains the monomer of the byssochlanone B, then carries out structure identification on the monomer through a spectrum method, and obtains activity data through in-vitro experiments, so that a foundation is laid for new microbial drug development, and it is found through in-vitro experiments that the byssochlanone B has certain anti-tumor activity.

Description

Technical Field

[0001] This invention belongs to the field of microbial pharmaceutical technology, specifically relating to two novel compounds in the fermentation products of endophytic fungi of reed (Phragmites australis), namely, byssochlanones A and byssochlanones B in the fermentation products of a strain of Byssochlamys sp. Furthermore, this invention also relates to methods for preparing these compounds, and the uses of byssochlanones A and byssochlanones B. This invention is a divisional application of application number 2023106865358. Background Technology

[0002] Over the course of its long evolutionary history, fungal species have achieved a biodiversity far exceeding that of any other organism except insects, spreading to every corner of the world. Besides forming different reproductive forms (hyphae, spores, and sclerotia, etc.) to adapt to diverse biotic and abiotic environments, fungi have, more importantly, evolved a unique set of chemical defense mechanisms during their life cycle: fungal secondary metabolites. Plant endophytic fungi refer to fungi living in different tissues or organs of the host plant. This also includes saprophytic fungi that parasitize the surface of the plant at a certain stage of their life cycle, as well as latent pathogenic fungi and mycorrhizal fungi that pose no immediate threat to the host. Due to their long-term co-evolution with the host, a mutually beneficial symbiotic relationship has formed between them. Research shows that plant endophytic fungi, due to their unique living environment, have evolved special survival strategies and have gradually become an important source of bioactive natural products.

[0003] Reed is a tall, perennial aquatic or wetland grass that grows along irrigation ditches, riverbanks, and marshes. It is found worldwide, and its leaves, flowers, stems, roots, and shoots are all used medicinally. Reed stems and roots can also be used in the paper industry and in biological agents. The reeds used in this invention were collected from the Zhalong Wetland in Heilongjiang Province. Endophytic fungi in the Zhalong Wetland represent a unique group of habitat fungi. The Zhalong Wetland region is characterized by year-round waterlogging, predominantly saline marsh soil, and significant temperature variations, fostering a unique ecological plant community. Endophytic fungi living in this special habitat, due to the uniqueness of their host environment (adapting to both biotic and abiotic factors), possess special secondary metabolic pathways. Reed endophytic fungi are an excellent source of active metabolites worthy of in-depth research.

[0004] There are currently no reports on the use of reed endophytic fungi to prepare new active compounds. Summary of the Invention

[0005] Purpose of the invention: To overcome the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide two new compounds in the fermentation products of a genus of endophytic fungi of reeds, namely Byssochlamys sp., and to provide the preparation method and uses of the two new compounds.

[0006] Technical solution: To solve the above technical problems, the present invention provides compound byssochlanone A, the structural formula of which is as follows:

[0007]

[0008] The molecular formula of byssochlanone A is C9H. 14 O3 has a molecular weight of 170.

[0009] The present invention also includes a method for preparing the compound byssochlanone A, comprising the following steps: subjecting Byssochlamys sp. PA-2003 to solid-state fermentation, solvent extraction, silica gel column chromatography, and semi-preparative liquid chromatography.

[0010] Among them, the genus Byssochlamys sp. PA-2003 is deposited at the China Center for Type Culture Collection (CCTCC) on March 31, 2023, with accession number CCTCC NO: M 2023449.

[0011] The specific preparation method of the compound byssochlanone A includes: inoculating the *Byssochlamys* sp. PA-2003 onto PDA medium and culturing it; then inoculating the medium containing the inoculum into a medium containing rice and distilled water to obtain fermentation products; then using ethyl acetate for ultrasonic extraction to obtain an ethyl acetate extract; then performing column chromatography with silica gel using gradient elution to obtain five components A, B, C, D, and E; component B is prepared using a semi-preparative high-performance liquid chromatography (HPLC) method, wherein the preparation conditions of the semi-preparative HPLC method are: detection wavelength of 254 nm, chromatographic column of reversed C-18 column, mobile phase of methanol and water with a volume ratio of 65:35, and collecting the eluent after 25-26 minutes to obtain the monomeric compound byssochlanone A.

[0012] The mass-to-volume ratio of rice to distilled water is 80:100 g / mL.

[0013] The silicone is 200-300 mesh silicone.

[0014] The gradient elution conditions are as follows: gradient elution is performed using a petroleum ether-ethyl acetate system with volume ratios of 100:1, 50:1, 25:1, 5:1 and 1:1, respectively.

[0015] The present invention also includes the use of the compound byssochlanone A in the preparation of drugs for the prevention or treatment of tumors.

[0016] The tumors mentioned include, but are not limited to, cervical cancer or cecal adenocarcinoma.

[0017] This invention also provides the compound byssochlanone B, whose structural formula is as follows:

[0018]

[0019] The molecular formula of byssochlanone B is C9H. 14 O4 has a molecular weight of 186.

[0020] The present invention also includes a method for preparing the compound byssochlanone B, comprising the following steps: subjecting Byssochlamys sp. PA-2003 to solid-state fermentation, solvent extraction, silica gel column chromatography, and semi-preparative liquid chromatography.

[0021] The specific preparation method of the compound byssochlanone B includes: inoculating the *Byssochlamys* sp. PA-2003 onto PDA medium and culturing it; then inoculating the medium containing the inoculum into a medium containing rice and distilled water to obtain fermentation products; then using ethyl acetate for ultrasonic extraction to obtain an ethyl acetate extract; then performing column chromatography with silica gel using gradient elution to obtain five components A, B, C, D, and E; component B is prepared using a semi-preparative high-performance liquid chromatography (HPLC) method, wherein the preparation conditions of the semi-preparative HPLC method are: detection wavelength of 254 nm, chromatographic column of reversed C-18 column, mobile phase of methanol and water with a volume ratio of 65:35, and collecting the eluent after 19-20 minutes to obtain the monomeric compound byssochlanone B.

[0022] The mass-to-volume ratio of rice to distilled water is 80:100 g / mL.

[0023] The silicone is 200-300 mesh silicone.

[0024] The gradient elution conditions are as follows: gradient elution is performed using a petroleum ether-ethyl acetate system with volume ratios of 100:1, 50:1, 25:1, 5:1 and 1:1, respectively.

[0025] The present invention also includes the use of the compound byssochlanone B in the preparation of drugs for the prevention or treatment of tumors.

[0026] The tumors mentioned include, but are not limited to, cervical cancer or cecal adenocarcinoma.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention is the first to discover an endophytic fungus of the genus Byssochlamys sp. in the study of endophytic fungi of reeds. The new compounds byssochlanones A and byssochlanones B obtained from the ethyl acetate extract of this fungus have certain antitumor activity against cervical cancer or cecal adenocarcinoma. Compounds byssochlanone A and byssochlanones B can be used as lead compounds for antitumor drugs. Attached Figure Description

[0028] Figure 1 Structural diagrams of compounds byssochlanones A and byssochlanones B;

[0029] Figure 2 Experimental and calculated ECD plots of compound byssochlanone A;

[0030] Figure 3 Experimental and calculated ECD plot of compound byssochlanone B. Detailed Implementation

[0031] The embodiments of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0032] The method for isolating, purifying, and fermenting endophytic fungi of the present invention is a conventionally used method known in the art. When further confirmation is needed, all technical information, including technical details, can be easily found in relevant textbooks and related technical documents.

[0033] The method for separating and identifying the structure of byssochlanones A and byssochlanones B in this invention is a conventional method in the field.

[0034] The organic solvent used in the organic solvent separation and preparation method of the present invention is a conventional organic solvent in the art.

[0035] Example 1: Obtaining strain Byssochlamys sp. PA-2003

[0036] 1. Reed sample collection

[0037] Phragmites australis, collected in June 2020 from Zhalong Wetland, Qiqihar, Heilongjiang, China.

[0038] 2. The method for preparing the strain includes the following steps:

[0039] 1) Wash and dry the reed roots, then disinfect them. The disinfection method includes the following steps: rinse with 75% ethanol for 30 seconds in a sterile operating table; rinse 3 times with sterile water; rinse with 5% sodium hypochlorite solution for 3 minutes; rinse 3 times with sterile water; rinse with 75% ethanol for 30 seconds; rinse 3 times with sterile water.

[0040] 2) After drying, cut the pieces and inoculate them onto the culture medium, then incubate in a constant temperature incubator;

[0041] 3) Once the mycelium has grown, transfer the fungus to a new culture medium for further cultivation and inoculation;

[0042] 4) After purification, transfer the sample to a slant tube of PDA medium to obtain the fungal strain, which is numbered PA-2003 and kept for later use.

[0043] Strain identification: After culturing on PDA medium for 3 days, mycelia were scraped off and DNA was extracted using the CTAB method. The DNA was then amplified by PCR and molecularly identified using ITS sequencing, and the strain was identified as *Byssochlamys* sp.

[0044] This strain, Byssochlamys sp. PA-2003, is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan, China, on March 31, 2023, with accession number CCTCC NO: M 2023449.

[0045] Example 2 Preparation of fermentation products

[0046] Using *Byssochlamys* sp. PA-2003 as the fermentation strain, the novel compounds byssochlanones A and byssochlanones B of this invention were isolated by a series of methods including solid-state fermentation, solvent extraction, silica gel column chromatography, and semi-preparative liquid chromatography. The structures of these novel compounds were determined using mass spectrometry and nuclear magnetic resonance.

[0047] The strain *Byssochlamys* sp. PA-2003 was cultured on potato dextrose agar (PDA) for 5 days. Fifty 500 mL Erlenmeyer flasks (approximately 1 cm × 1 cm) containing the strain were then inoculated with the culture medium into each flask containing 80 g of rice and 100 mL of water. Each flask was pre-sterilized using an autoclave. All flasks were incubated at 25°C for 5 weeks to collect the fermentation product. The fermentation product was then extracted three times at room temperature with twice its volume of ethyl acetate using ultrasonic extraction for 20 minutes each time, followed by vacuum evaporation of the solvent. The ethyl acetate extract (14.5 g) was subjected to column chromatography (CC) on silica gel (200-300 mesh) (purchased from Qingdao Haiyang Chemical Co., Ltd.) using five gradients (petroleum ether-ethyl acetate, successively 100:1, 50:1, 25:1, 5:1, and 1:1, v / v), with a solvent elution volume of 3 column volumes (280 cm³) for each gradient. 3 Five crude components were obtained, designated as components A, B, C, D, and E respectively. Component B (243 mg) was prepared using a semi-preparative high-performance liquid chromatography (HPLC) method. The preparation conditions were: detection wavelength 254 nm, reverse-phase C-18 column, and methanol and water as the mobile phase (65:35, v / v), yielding compound 1 (4.5 mg, t...). R =25.4min) and 2(3.8mg,t R =19.8min).

[0048] Structural identification: This invention comprehensively utilizes mass spectrometry (MS) and proton nuclear magnetic resonance (NMR) spectroscopy. 1 H-NMR), carbon nuclear magnetic resonance (NMR) 13 Analytical techniques such as C-NMR and two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) are used to identify the structures of monomeric compounds in fermentation products.

[0049] The structural formula of compound 1 is as follows:

[0050]

[0051] Compound 1 is an amorphous powder. HR-ESI-MS yielded a quasi-molecular ion peak at m / z 193.0824 [M+Na]. + (C9H 14 Theoretical value of O3Na: 193.0841). Analysis 1 H NMR spectrum, δ H 1.11 (3H, d, J = 6.6 Hz) and 1.63 (3H, d, J = 6.6 Hz) are two methyl signals; δ H 5.47 (1H, dd, J = 15.0, 8.4 Hz) and 5.60 (1H, m) are two alkene proton signals; the compound also contains three hydrogen signals bonded to oxygen atoms: δH 3.80 (1H, t, J = 9.0 Hz), 4.26 (1H, t, J = 9.0 Hz), 4.26 (1H, t, J = 9.0 Hz); and two hydrogen signals located in the high-field region: δ H 2.46 (1H, dd, J = 9.0, 2.4 Hz) and 3.30 (1H, m). Analysis 13 12C NMR indicates that the compound contains 9 carbons, including 1 carbonyl carbon (δ¹⁸). C 177.7), 2 olefinic carbons (δ) C 127.1 and 131.4), 2 oxygen-containing carbons (δ C 64.8 and 70.1), 2 methylene carbons (δ C 38.0 and 51.5) and 2 methyl carbons (δ C 17.8 and 21.8), with 1 The information from H NMR is completely consistent. From 1 H- 1 H COSY deduced that the compound contains structural fragments of propenyl and hydroxyethyl groups. The compound contains C-2(δ) C 177.7), C-3(δ) C 51.5), C-4 (δ) C 38.0) and C-5 (δ C 70.1) These four carbon signals, and H-5 and δ in HMBC C 177.7 (C-2) correlation, H-4 and δ C 177.7 (C-2) is related, H-3 is related to C-2 (δ) C 177.7) and C-5 (δ C The correlation of 70.1) indicates the presence of a furanone structural fragment in this structure. In HMBC, δ H 4.13 (1H, qd, J = 6.6, 2.4 Hz, H⁻⁶) and δ C 51.5(C-3) correlation, δ H 5.47 (1H, dd, J = 15.0, 8.4 Hz, H-8) and δ CThe correlation at 38.0 (C-4) indicates that the hydroxyethyl group is located at C-3 and the propenyl group at C-4. Therefore, the planar structure of the compound was determined to be a furanone derivative. In NOESY, the correlations between H-3 and CH3-7, and between H-3 and H-4, indicate that these protons are on the same side of the ring. Furthermore, the larger coupling constant between H-3 and H-6 (9.0 Hz) and the smaller coupling constant between H-3 and H-4 (2.4 Hz) also support this conclusion. To further determine the absolute configuration of the compound, the calculated and measured ECD curves were compared. The results showed that the calculated ECD spectrum of (3S, 4S, 6S)-1 was consistent with the measured ECD spectrum. Therefore, the structure of the compound was determined and it was named byssochlanone A.

[0052] The structural formula of compound 2 is as follows:

[0053]

[0054] Compound 2 is an amorphous powder, and HR-ESI-MS yielded a quasi-molecular ion peak at m / z 209.0769 [M+Na]. + (C9H 14 Theoretical value of O4Na: 209.0790. Detailed comparison of NMR data between compounds 2 and 1 indicates that compound 2 is an analogue of 1. The only difference is that compound 2 has an additional oxygen-bound quaternary carbon (δ¹⁰). C 77.9), missing one methylene carbon, and also supported by two-dimensional NMR, in HMBC, H-5 and H-4 with δ C The correlation at 77.9 (C-3) indicates that the H-3 in compound 1 is replaced by the hydroxyl group in compound 2. Considering the consistency of the biological origin of the two compounds, we propose that the hydroxyl group is β-oriented. By comparing the measured ECD curves and the calculated ECD curves, the absolute configuration of C-3 in compound 2 is determined to be R configuration. Therefore, the structure of compound 2 is determined and named byssochlanone B.

[0055] Table 1. Compounds 1-2 1 H NMR (600MHz, DMSO-d6) and 13 C NMR (150MHz, DMSO-d6) data

[0056]

[0057] Example 3: Study on the antitumor activity of compound byssochlanone A

[0058] The MTT assay was used to determine the growth inhibitory effect of compound byssochlanone A on human cervical cancer cells (HeLa) and human cecal adenocarcinoma cells (HCT-8). Cervical cancer cells (HeLa) and human cecal adenocarcinoma cells (HCT-8) in the logarithmic growth phase were digested with trypsin and diluted into single-cell suspensions in DMEM medium containing 10% fetal bovine serum. Cell concentrations were adjusted according to different cell growth rates, and cells were stored at 6 × 10⁶ cells per well. 4 Cells were added at a density of 100 μL to 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. Subsequently, five different concentrations (50, 10, 2, 0.4, 0.08 μM) of byssochlanone A dissolved in dimethyl sulfoxide (DMSO) were added to each well, with three parallel wells for each concentration. A blank control group was also included. Cells were incubated for 24 hours. Then, cells were incubated with 10 μL MTT (5 mg / mL) for 4 hours. The supernatant was carefully discarded, and 200 μL LDMSO was added. The mixture was gently shaken to dissolve the crystals, and the optical density (OD) value of each well was measured at 570 nm using a microplate reader. The average OD value of each well was taken as the mean OD value. The growth inhibition rate (GIR) of byssochlanone A on each tumor cell type was calculated using the formula GIR = (1 - experimental group OD / control group OD) × 100%. The half-maximal inhibitory concentration (IC50) was then calculated using Origin software. 50 The results of the antitumor experimental screening are shown in Table 2.

[0059] Table 2. Screening results of antitumor assays for compound byssochlanone A (IC50, 1000 mg / L). 50 μM)

[0060] byssochlanone A >50 45.4 Paclitaxel (positive control) 1.40 3.14

[0061] Table 2 shows that compound byssochlanone A has certain cytotoxic activity against human cecal adenocarcinoma cells, with an IC50 value of [missing information]. 50 The concentration was 45.4 μM. Therefore, the compound byssochlanone A described in this invention can serve as a lead compound for antitumor drugs.

[0062] Example 4: Study on the antitumor activity of compound byssochlanone B

[0063] The antitumor activity of compound byssochlanone B was determined using the same method as in Example 3, and the results of the antitumor screening experiment are shown in Table 3.

[0064] Table 3. Screening results of the antitumor assay for compound byssochlanone B (IC50, 1000 mg / kg). 50 μM)

[0065] byssochlanone B 38.1 21.3 Paclitaxel (positive control) 1.40 3.14

[0066] Table 3 shows the experimental results, indicating that compound byssochlanone B is effective against human cervical cancer cells (HeLa) IC50. 50 The concentration was 38.1 μM, and the IC50 value for human cecal adenocarcinoma cells (HCT-8) was [missing value]. 50 The concentration was 21.3 μM. Therefore, the compound byssochlanones B described in this invention can also serve as a lead compound for antitumor drugs.

Claims

1. The compound byssochlanone B has the following structural formula: 。 2. The method for preparing the compound byssochlanone B according to claim 1, characterized in that, Includes the following steps: The genus *Cymbidium* ( Byssochlamys sp. PA-2003 was obtained by sequentially performing solid-state fermentation, solvent extraction, silica gel column chromatography, and semi-preparative liquid chromatography. The fungus *Hymenopterus* genus (…) Byssochlamys sp. PA-2003 is deposited at the China Center for Type Culture Collection (CCTCC) on March 31, 2023, with accession number CCTCC NO: M 2023449. The depositary address is Wuhan University, Wuhan, China. The specific preparation method of the compound byssochlanone B includes: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Byssochlamys sp. PA-2003 was inoculated onto PDA medium and cultured. Then, the medium containing the inoculum was inoculated into a medium containing rice and distilled water to obtain fermentation products. Ethyl acetate was then extracted using ultrasonic extraction with ethyl acetate to obtain an ethyl acetate extract. The ethyl acetate extract was then subjected to gradient elution with silica gel to obtain five fractions: A, B, C, D, and E. Fraction B was prepared using a semi-preparative high-performance liquid chromatography (HPLC) method. The preparation conditions for this semi-preparative HPLC method were: detection wavelength of 254 nm, a reverse-phase C-18 column, and a mobile phase of methanol and water at a volume ratio of 65:

35. The eluent was collected at 19–20 minutes to obtain the monomer compound byssochlanone B. The mass-to-volume ratio of rice to distilled water was 80:100 g / mL. The silica gel was 200–300 mesh. The gradient elution conditions were: a petroleum ether-ethyl acetate system with volume ratios of 100:1, 50:1, 25:1, 5:1, and 1:

1.

3. The use of the compound byssochlanone B of claim 1 in the preparation of a medicament for the prevention or treatment of tumors, wherein the tumor is cervical cancer or cecal adenocarcinoma.

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