Compound derived from endophytic fungi of reed and preparation method and application thereof

Abstract

The present invention discloses a compound byssochlanone A, and its structural formula is shown below. The present invention also discloses a preparation method and application of byssochlanone A. The present invention separates and purifies endophytic fungi from collected sample reed plants, identifies these strains and screens their anti-tumor activity in vitro, then selects strains with the best activity for large-scale fermentation, and systematically separates and purifies the fermentation products, obtains monomers of byssochlanones A, identifies their structure by spectral methods, and obtains activity data by in vitro experiments, laying a foundation for the development of new microbial drugs. In vitro experiments have also found that it has certain anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial medicine, and specifically relates to two new compounds in the fermentation products of endophytic fungi of reed (Phragmites australis), namely, new compounds byssochlanones A and byssochlanones B in the fermentation products of a strain of Byssochlamys sp.; in addition, the present invention also relates to a preparation method of the compound, and the use of byssochlanones A and byssochlanones B. Background Art

[0002] During the long process of evolution, the biodiversity of fungal species far exceeds that of other organisms except insects, and they are found in every corner of the world. In addition to forming different reproductive bodies (hyphae, spores, and sclerotia, etc.) to adapt to different biological and abiotic environments, fungi have also evolved a unique set of chemical defenses in the process of their life reproduction: fungal secondary metabolites. Endophytic fungi refer to fungi that live in different tissues or organs of host plants. They also include saprophytic fungi that parasitize on their surfaces at a certain stage in their life history, as well as latent pathogenic fungi and mycorrhizal fungi that do not harm the host temporarily. Due to their long-term co-evolution with the host, a mutualistic symbiotic relationship has been formed between the two. Studies have shown that due to their special living environment, endophytic fungi in plants have evolved special survival strategies and have gradually become an important source of active natural products.

[0003] Reed is a tall grass of perennial aquatic or wetland, grows beside irrigation ditches, river bank marshes, etc., and is grown all over the world, and reed leaves, reed flowers, reed stems, reed roots, and asparagus can all be used as medicine. Reed stems and reed roots can also be used in papermaking industry, and biological preparations. Reed in the present invention is collected from Zhalong Wetland, Heilongjiang Province, and Zhalong Wetland Plant Endophytic Fungi is a class of special habitat fungi group, and Zhalong Wetland area is perennially waterlogged, and soil is mainly based on salinized marsh soil, and unique ecological environments such as large temperature differences breed unique ecological plant communities, and the endophytic fungi living in such special habitat plants contain special secondary metabolic pathways due to the uniqueness of their host environment (adapting to the influence of its biological and abiotic factors). Endophytic fungi in reed are just one of them a good source of active metabolites that can be studied in depth.

[0004] There is no report on the use of endophytic fungi in reed to prepare new active compounds. Summary of the invention

[0005] Purpose of the invention: To address the deficiencies 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 reed endophytic fungus, Byssochlamys sp., and to provide a preparation method and use of the two new compounds.

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

[0007]

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

[0009] The present invention also includes a method for preparing the compound byssochlanone A, which comprises the following steps: subjecting Byssochlamys sp. PA-2003 to solid fermentation, solvent extraction, silica gel chromatography and semi-preparative liquid phase separation in sequence to obtain the byssochlanone A.

[0010] Among them, the Byssochlamys sp. PA-2003 is preserved in the China Center for Type Culture Collection, the preservation date is March 31, 2023, and the preservation number is: CCTCC NO: M 2023449.

[0011] The specific preparation method of the compound byssochlanone A includes: inoculating the Byssochlamys sp. PA-2003 onto a PDA culture medium for cultivation, then inoculating the culture medium containing the strain into a culture medium containing rice and distilled water to obtain a fermentation product, then using ethyl acetate for ultrasonic extraction to obtain an ethyl acetate extract, and then using silica gel to perform column chromatography using gradient elution to obtain five components A, B, C, D, and E. Component B is prepared using semi-preparative high performance liquid chromatography, and the preparation conditions of the semi-preparative high performance liquid chromatography are: the detection wavelength is 254 nm, the chromatographic column is a reverse C-18 column, the mobile phase is methanol and water in a volume ratio of 65:35, and the eluate from 25 to 26 minutes is collected to obtain the monomer compound byssochlanone A.

[0012] Wherein, the mass volume ratio of the rice to the distilled water is 80:100 g / mL.

[0013] Wherein, the silica gel is 200-300 mesh silica gel.

[0014] Wherein, the gradient elution conditions are: gradient elution is performed using a petroleum ether-ethyl acetate system, and the volume ratios are 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 preparing drugs for preventing or treating tumors.

[0016] The tumor includes but is not limited to cervical cancer or cecal adenocarcinoma.

[0017] The present invention also provides a compound byssochlanone B, whose structural formula is as follows:

[0018]

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

[0020] The present invention also includes a method for preparing the compound byssochlanone B, which comprises the following steps: subjecting Byssochlamys sp. PA-2003 to solid fermentation, solvent extraction, silica gel chromatography and semi-preparative liquid phase separation in sequence to obtain the compound.

[0021] The specific preparation method of the compound byssochlanone B includes: inoculating the Byssochlamys sp. PA-2003 onto a PDA culture medium for cultivation, then inoculating the culture medium containing the strain into a culture medium containing rice and distilled water to obtain a fermentation product, then using ethyl acetate for ultrasonic extraction to obtain an ethyl acetate extract, then using silica gel to perform column chromatography using gradient elution to obtain five components A, B, C, D, and E, and component B is prepared using semi-preparative high performance liquid chromatography. The preparation conditions of the semi-preparative high performance liquid chromatography are: the detection wavelength is 254 nm, the chromatographic column is a reverse C-18 column, the mobile phase is methanol and water in a volume ratio of 65:35, and the eluent from 19 to 20 minutes is collected to obtain the monomer compound byssochlanone B.

[0022] Wherein, the mass volume ratio of the rice to the distilled water is 80:100 g / mL.

[0023] Wherein, the silica gel is 200-300 mesh silica gel.

[0024] Wherein, the gradient elution conditions are: gradient elution is performed using a petroleum ether-ethyl acetate system, and the volume ratios are 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 preparing drugs for preventing or treating tumors.

[0026] The tumor includes but is 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 in reeds. The new compounds byssochlanones A and byssochlanones B obtained from the ethyl acetate extraction part of the fungus have certain anti-tumor activity against cervical cancer or cecal adenocarcinoma. The compounds byssochlanone A and byssochlanones B can be used as lead compounds of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The structures of the compounds byssochlanones A and byssochlanones B;

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

[0030] Figure 3 Experimental and calculated ECD diagrams of compound byssochlanone B. DETAILED DESCRIPTION

[0031] The embodiments of the present invention will be described in detail below, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0032] The separation, purification and fermentation methods of the endophytic fungi of the present invention are commonly used methods known in the art. When further confirmation is needed, all technical information including technical details can be easily consulted from relevant teaching materials and related technical literature.

[0033] The method for separating and identifying the structures of byssochlanones A and byssochlanones B of the present invention is a conventional method in the art.

[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 the strain Byssochlamys sp. PA-2003

[0036] 1. Reed sample collection

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

[0038] 2. The preparation method of the strain comprises the following steps:

[0039] 1) Reed roots are washed, dried, and sterilized; the sterilization method comprises the following steps: rinse with 75% ethanol for 30 seconds in a sterile operating table; rinse with sterile water three times; rinse with 5% sodium hypochlorite solution for 3 minutes; rinse with sterile water three times; rinse with 75% ethanol for 30 seconds; rinse with sterile water three times.

[0040] 2) After drying, the pieces are inoculated on the culture medium and cultured in a constant temperature incubator;

[0041] 3) When mycelium grows out, transfer the fungus to a new culture medium for further cultivation and transfer;

[0042] 4) After purification, the fungus strain was transferred to a PDA medium slant test tube to obtain the fungal strain, which was numbered PA-2003 and kept for future use.

[0043] Identification of fungus: After culturing on PDA medium for 3 days, mycelium was scraped and DNA was extracted by CTAB method. Then, the DNA was amplified by PCR and molecularly identified using ITS sequence, and the identification result was Byssochlamys sp.

[0044] The strain Byssochlamys sp. PA-2003 is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan, China, the deposit date is March 31, 2023, and the deposit number is: CCTCC NO: M2023449.

[0045] Example 2 Preparation of fermentation products

[0046] Byssochlamys sp. PA-2003 was used as the fermentation strain, and solid fermentation, solvent extraction, silica gel chromatography and semi-preparative liquid phase separation methods were successively used to separate the new compounds byssochlanones A and byssochlanones B of the present invention. The structures of the above new compounds were determined by mass spectrometry, nuclear magnetic resonance and other methods.

[0047] The strain Byssochlamys sp. PA-2003 was cultured on potato dextrose agar (PDA) medium for 5 days. The culture medium containing the strain of about 1 cm×1 cm was inoculated into 50 500 mL Erlenmeyer flasks, each containing 80 g of rice and 100 mL of water, and each flask was sterilized by autoclave in advance. All flasks were incubated at 25°C for 5 weeks to collect the fermentation products. The fermentation products were then ultrasonically extracted with 2 volumes of ethyl acetate at room temperature for 3 times, each for 20 minutes, and then the solvent was evaporated in vacuo. The ethyl acetate extract (14.5 g) was subjected to column chromatography (CC) using silica gel (200-300 mesh) (purchased from Qingdao Ocean Chemical Co., Ltd.) using 5 gradients (petroleum ether-ethyl acetate, 100:1, 50:1, 25:1, 5:1 and 1:1, v / v), and the solvent elution volume of each gradient was 3 column volumes (column volume was 280 cm 3 ), a total of 5 crude fractions were obtained, which were recorded as A, B, C, D, and E. Component B (243 mg) was prepared by semi-preparative high performance liquid chromatography. The preparation conditions were: the detection wavelength was 254 nm, the chromatographic column was a reverse C-18 column, and methanol and water were used as the mobile phase (65:35, v / v), to obtain compound 1 (4.5 mg, t R =25.4min) and 2 (3.8mg, t R =19.8min).

[0048] Structural identification: The present invention comprehensively uses mass spectrometry (MS), nuclear magnetic resonance spectroscopy ( 1 H-NMR), carbon nuclear magnetic resonance spectroscopy ( 13 C-NMR) and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy were 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 was an amorphous powder. HR-ESI-MS gave a quasi-molecular ion peak of m / z 193.0824 [M+Na] + (C9H 14 O3Na theoretical value: 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 olefin proton signals; the compound also contains three hydrogen signals connected 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). 13 C NMR suggests that the compound contains 9 carbon atoms, including 1 carbonyl carbon (δ C 177.7), 2 olefinic carbons (δ C 127.1 and 131.4), 2 oxygen-linked carbons (δ C 64.8 and 70.1), 2 methine carbons (δ C 38.0 and 51.5) and 2 methyl carbons (δ C 17.8 and 21.8), with 1 The information provided by H NMR is completely consistent. 1 H- 1 H COSY results indicate that the compound contains structural fragments of propenyl and hydroxyethyl. 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, as well as H-5 and δ in HMBC C 177.7 (C-2) related, H-4 and δ C 177.7 (C-2), H-3 and C-2 (δ C 177.7) and C-5 (δ C 70.1), indicating that there is a furanone structural fragment in the structure. H 4.13 (1H, qd, J = 6.6, 2.4 Hz, H-6) and δ C 51.5(C-3) related, δ H 5.47 (1H, dd, J = 15.0, 8.4 Hz, H-8) and δ C38.0(C-4) correlations respectively indicate that the hydroxyethyl group is located at the C-3 position and the propenyl group is located at the C-4 position. Therefore, the planar structure of the compound was determined to be a furanone derivative. In NOESY, H-3 is correlated with CH3-7, and H-3 is correlated with H-4, indicating that these protons are on the same side of the ring. At the same time, the coupling constant between H-3 and H-6 is larger at 9.0Hz, and the coupling constant between H-3 and H-4 is smaller at 2.4Hz, which also supports the above conclusion. In order to further identify the absolute configuration of the compound, the calculated ECD curve and the measured ECD curve were compared. The results show that the calculated ECD spectrum of (3S, 4S, 6S)-1 is consistent with the measured ECD spectrum. Therefore, the structure of the compound was determined and named byssochlanone A.

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

[0053]

[0054] Compound 2 is an amorphous powder. HR-ESI-MS gives a quasi-molecular ion peak of m / z 209.0769 [M+Na] + (C9H 14 O4Na theoretical value: 209.0790). A detailed comparison of the NMR data of compound 2 and 1 shows that compound 2 is an analog of 1. The only difference is that compound 2 has an additional oxygen-linked quaternary carbon (δ C 77.9), one less methyl carbon, and also supported by 2D NMR. In HMBC, H-5 and H-4 are similar to δ C 77.9 (C-3), indicating that H-3 in compound 1 was replaced by the hydroxyl group in 2. Considering the consistency of the biological sources of the two compounds, we proposed that the hydroxyl group was in the β orientation. By comparing the measured ECD curve with the calculated ECD curve, the absolute configuration of C-3 in compound 2 was determined to be the R configuration. Therefore, the structure of compound 2 was determined and named byssochlanone B.

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

[0056]

[0057] Example 3 Study on the anti-tumor activity of compound byssochlanone A

[0058] The MTT method 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 with DMEM medium containing 10% fetal bovine serum. The cell concentration was adjusted according to different cell growth rates and 6×10 cells were added to each well. 4 The cells were added to a 96-well plate at a density of , and cultured in a 5% CO2 incubator at 37°C for 24 hours. Subsequently, the compound byssochlanone A dissolved in dimethyl sulfoxide (DMSO) at five different concentrations (50, 10, 2, 0.4, 0.08 μM) was added thereto, with three parallel wells for each concentration and a blank control group, and cultured for 24 hours. Then, the cells were incubated with 10 μL MTT (5 mg / mL) for 4 hours. The supernatant was carefully discarded, and 200 μL DMSO was added. The crystals were gently shaken to dissolve, and the optical density (OD) value of each well was measured on a microplate reader at a wavelength of 570 nm. The average value of 3 wells in each group was taken as the average OD value of each well. The growth inhibition rate (GIR) of byssochlanone A on each tumor cell was calculated according to the formula GIR = (1-experimental group OD / control group OD) × 100%, and the half inhibitory concentration (IC) was calculated by Origin software. 50 ). The anti-tumor experimental screening results are shown in Table 2.

[0059] Table 2 Antitumor experimental screening results of compound byssochlanone A (IC 50 μM)

[0060] sample Cervical cancer cells (HeLa) Human cecal adenocarcinoma cells (HCT-8) byssochlanone A ＞50 45.4 Paclitaxel (positive control) 1.40 3.14

[0061] The experimental results in Table 2 show that the compound byssochlanone A has certain cytotoxic activity against human cecal adenocarcinoma cells, and its IC 50 It is 45.4 μM. Therefore, it can be seen that the compound byssochlanone A described in the present invention can be used as a lead compound of anti-tumor drugs.

[0062] Example 4 Study on the anti-tumor activity of compound byssochlanone B

[0063] The anti-tumor activity of the compound byssochlanone B was determined by the same method as in Example 3. The anti-tumor experimental screening results are shown in Table 3.

[0064] Table 3 Antitumor experimental screening results of compound byssochlanone B (IC a0 μM)

[0065] sample Cervical cancer cells (HeLa) Human cecal adenocarcinoma cells (HCT-8) byssochlanone B 38.1 21.3 Paclitaxel (positive control) 1.40 3.14

[0066] The experimental results in Table 3 show that the compound byssochlanone B has an IC a0 The IC value of human cecal adenocarcinoma cells (HCT-8) was 38.1 μM. 50 It is 21.3 μM. Therefore, the compound byssochlanones B described in the present invention can also be used as a lead compound of anti-tumor drugs.

Claims

1. The compound byssochlanone A has the following structural formula: .

2. The method for preparing the compound byssochlanone A according to claim 1, characterized in that: The following steps are involved: The genus Mycorrhiza ( Byssochlamys sp. )PA-2003 is sequentially subjected to solid fermentation, solvent extraction, silica gel chromatography and semi-preparative liquid phase separation, wherein the Mycelial Fungus genus ( Byssochlamys sp. )PA-2003 was deposited in China Center for Type Culture Collection on March 31, 2023, with a deposit number of CCTCC NO: M 2023449. The specific preparation method of the compound byssochlanone A comprises: Byssochlamys sp. ) PA-2003 is inoculated into a PDA medium for culture, and then the culture medium containing the strain is inoculated into a culture medium containing rice and distilled water for culture to obtain a fermentation product, and then ethyl acetate is used for ultrasonic extraction to obtain an ethyl acetate extract, and then the ethyl acetate extract is subjected to column chromatography using silica gel using gradient elution to obtain five components A, B, C, D, and E, and component B is prepared using semi-preparative high performance liquid chromatography, and the preparation conditions of the semi-preparative high performance liquid chromatography are as follows: the detection wavelength is 254 nm, the chromatographic column is a reverse C-18 column, and the mobile phase is methanol and water in a volume ratio of 65:35, and the eluate from 25 to 26 minutes is collected to obtain a monomer compound byssochlanone A, the mass volume ratio of rice and distilled water is 80:100 g / mL, the silica gel is 200-300 mesh silica gel, and the gradient elution conditions are as follows: gradient elution is performed using a petroleum ether-ethyl acetate system, and the volume ratios are 100:1, 50:1, 25:1, 5:1 and 1:1, respectively.

3. Use of the compound byssochlanone A according to claim 1 in the preparation of a drug for preventing or treating a tumor, wherein the tumor is cecal adenocarcinoma.

Images