Didylactam A, a novel lactam compound derived from marine mangrove fungi, and its preparation method and applications.

Abstract

The application provides a new lactam compound Didylactam A from a marine mangrove fungus, and a preparation method and application thereof. The structural formula of the Didylactam A is shown as formula (I). The lactam compound can effectively inhibit the proliferation of hepatoma cells, and the compound has significant anti-hepatoma activity, and can be used as a candidate drug for resisting hepatoma, and provides a new drug preparation method for treating hepatoma. Moreover, the compound is derived from a natural marine mangrove fungus, and the strain is a microorganism, and the compound can be prepared on a large scale without destroying an ecological system, and is easy to obtain. Therefore, the Didymella sp. CYSK-4 fungus, the lactam compound Didylactam A and the microbial source preparation method thereof have important application values.

Description

Technical Field

[0001] This invention belongs to the field of microbial pharmaceuticals. More specifically, it relates to a method for preparing Didylactam A, a lactam compound derived from Didymella sp. CYSK-4, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most common primary liver cancer in adults and the fourth leading cause of cancer-related deaths worldwide. HCC has been found to be more common in patients with chronic liver diseases such as liver fibrosis and cirrhosis. Current clinical treatment strategies for HCC include surgical intervention, image-guided ablation, radiotherapy, transarterial therapy, and newly established systemic therapies. Chemotherapy is one of the most effective treatments for tumors, especially those that easily spread throughout the body or have metastasized. However, most clinical anti-tumor drugs have significant side effects. Furthermore, there is a possibility of tumor resistance and recurrence. Therefore, given the rising global incidence of cancer, developing new and effective methods, including novel anti-tumor drugs, to prevent or treat liver cancer is of paramount importance.

[0003] Natural products are an important source for the discovery and development of drugs, especially anticancer drugs. Approximately 70% of anticancer drugs approved by the U.S. Food and Drug Administration (FDA) are derived from natural products or their derivatives. Lactam compounds are a class of polyketide / nonribosomal peptide hybrid alkaloids derived from fungi. Studies have shown that these compounds have significant antitumor activity. For example, patent CN107686817A discloses a lactam compound derived from the endophytic fungus *Chlorella vulgaris*, which exhibits activity against breast cancer, colon cancer, lung cancer, glioblastoma, and prostate cancer. Summary of the Invention

[0004] This invention addresses the shortcomings of existing anti-liver cancer drugs by providing a lactam compound, Didylactam A, derived from the fungus Didymella sp. CYSK-4, to exert a significant anti-liver cancer effect.

[0005] A second objective of this invention is to provide a Didymella sp. CYSK-4 strain.

[0006] A third objective of this invention is to provide a method for preparing the aforementioned lactam compound Didylactam A.

[0007] A fourth objective of this invention is to provide the application of Didylactam A in the preparation of anti-liver cancer drugs.

[0008] The fifth objective of this invention is to provide the application of the fermentation broth of the Didymella sp. CYSK-4 strain in the preparation of anti-liver cancer drugs.

[0009] The sixth objective of this invention is to provide an anti-liver cancer drug.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention provides a lactam compound, DidylactamA, derived from the fungus Didymella sp. CYSK-4, having the chemical structure shown in formula (Ⅰ):

[0012]

[0013] The lactam compound Didylactam A of this invention has a basic skeleton of 5 / 6 / 5 / 6 / 5 / 5 / 5 / 6 / 13. Although it is very different from traditional oncology drugs (such as cisplatin, ET-743, paclitaxel, etc.), it can effectively inhibit the growth of liver cancer cells Hepa1-6 and Huh7, indicating that the compound has significant anti-liver cancer activity and is suitable for preparation as an anti-liver cancer drug, providing a new source of drug material for the treatment of liver cancer.

[0014] The present invention also provides a Didymella sp. CYSK-4 strain, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 25, 2023, with accession number GDMCC No: 63750.

[0015] In addition, the present invention also provides a method for preparing the above-mentioned lactam compound Didylactam A, which is obtained by fermentation of the above-mentioned Didymella sp. CYSK-4 strain.

[0016] Preferably, the preparation method includes the following steps:

[0017] S1. The Didymella sp. CYSK-4 strain was inoculated into a seed culture medium and cultured to obtain a seed culture solution;

[0018] S2. The seed culture obtained in S1 is inoculated into a fermentation medium for culture to obtain the fermentation product;

[0019] S3. The fermentation product obtained in S2 is sequentially filtered, extracted, concentrated, subjected to silica gel column chromatography, gel column chromatography, and purified to obtain the lactam compound Didylactam A.

[0020] Preferably, in the seed culture medium described in S1, the mass-to-volume ratio of glucose to potato juice is 6-40 g: 150-1000 mL.

[0021] More preferably, the potato juice is prepared by adding water to potatoes, heating to boiling, and simmering to make potato juice; wherein the mass ratio of potatoes to water is (0.8 to 1.2):1, and most preferably 1:1.

[0022] Preferably, the culture in S1 is a shaker culture, specifically a shaker culture at 20–30°C and 100–280 rpm for 48–168 h. Most preferably, it is a culture at 28°C and 140 rpm for 168 h.

[0023] Preferably, the fermentation medium in S2 contains 25-75g of starch matrix and 0.5-3g of sea salt per 100mL-500mL of water. More preferably, it contains 50g of starch matrix and 3g of sea salt per 100mL of water.

[0024] More preferably, the starch matrix includes, but is not limited to, rice starch.

[0025] Preferably, the culture in S2 is a static culture, specifically a static culture at 20–30°C for 20–60 days. More preferably, a static culture at 25°C for 40 days.

[0026] Preferably, the extraction in S3 is performed using pure methanol.

[0027] Preferably, the silica gel column chromatography in step S3 involves gradient elution using petroleum ether-ethyl acetate as the mobile phase.

[0028] More preferably, in the mobile phase, the volume fraction of petroleum ether changes during elution as follows: 100% → 90% → 80% → 70% → 60% → 50% → 40% → 30%; and the volume fraction of ethyl acetate changes as follows: 0% → 10% → 20% → 30% → 40% → 50% → 60% → 70%. The volume of eluent used for each gradient is 25 to 35 times the sample volume, most preferably 30 times.

[0029] Preferably, the gel column chromatography described in S3 uses a gradient elution with methanol-dichloromethane as the mobile phase.

[0030] More preferably, the volume ratio of methanol to dichloromethane in the mobile phase is (1-2):1, and most preferably 1:1.

[0031] Preferably, the purification described in S3 is column chromatography purification or high-performance liquid chromatography purification.

[0032] The lactam compound Didylactam A described in this invention inhibits apoptosis and proliferation of liver cancer cells and can be used to prepare anti-liver cancer drugs, providing a new source of pharmaceutical materials for the treatment of liver cancer. Therefore, the application of the above-mentioned lactam compound Didylactam A in the preparation of anti-liver cancer drugs should be within the scope of protection of this invention. Furthermore, since the fermentation broth of the Didymella sp. CYSK-4 strain can be fermented to obtain the above-mentioned lactam compound Didylactam A, its application in the preparation of anti-liver cancer drugs, as well as anti-liver cancer drugs containing the above-mentioned lactam compound Didylactam A or the fermentation broth of the above-mentioned Didymella sp. CYSK-4 strain, should also be within the scope of protection of this invention.

[0033] Preferably, the liver cancer cells are Hepa1-6 and Huh7.

[0034] The application of Didylactam A in the preparation of anti-liver cancer drugs.

[0035] Application of the fermentation broth of Didymella sp. CYSK-4 strain in the preparation of anti-liver cancer drugs.

[0036] The present invention provides an anti-liver cancer drug containing the fermentation broth of the Didylactam A or Didymella sp. CYSK-4 strains.

[0037] The present invention has the following beneficial effects:

[0038] 1. Didylactam A, a lactam compound of the present invention, can effectively inhibit the proliferation of liver cancer cells Hepa1-6 and Huh7, and has significant cytotoxic activity. It is suitable for preparation as an anti-liver cancer drug, providing a new source of drug material for the treatment of liver cancer.

[0039] 2. The Didymella sp. CYSK-4 strain used in this invention is a microorganism that can be prepared on a large scale without disrupting the ecosystem and is easy to obtain. Attached Figure Description

[0040] Figure 1 Morphological diagram of Didymella sp. CYSK-4 strain in PDA medium.

[0041] Figure 2 It is the single crystal structure of Didylactam A, a lactam compound.

[0042] Figure 3This is a comparison chart of the measured ECD and calculated ECD of the lactam compound Didylactam A. Expt.ECDof 1 represents the measured ECD comparison result for Didylactam lactam compounds, and Calcd.ECD of 1 represents the calculated ECD comparison result for Didylactam lactam compounds.

[0043] Figure 4 The effects of the lactam compound Didylactam A on liver cancer cells Hepa1-6 and Huh7 were investigated. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0045] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0046] Example 1: Obtaining Didymella sp. CYSK-4 strain

[0047] 1. Isolation and screening of bacterial strains:

[0048] Three single colonies were isolated from branches of *Chrysanthemum indicum*, a mangrove plant collected from the Shankou Mangrove Reserve in Guangxi Zhuang Autonomous Region, China. These three colonies were screened using commercially available Bengal Red agar, and the fastest-growing hyphae were selected to obtain a dark green strain, named CYSK-4.

[0049] 2. Identification of the strain:

[0050] Morphological identification: The morphological characteristics of strain CYSK-4 on commercially available PDA medium are dark green, forming radiating filaments, such as... Figure 1 As shown.

[0051] Molecular biological identification: Didymella sp. CYSK-4 strain was identified as belonging to the genus Didymella sp. by molecular biological ITS sequencing and comparison.

[0052] In summary, strain CYSK-4 was identified as belonging to the genus Didymella sp. and was deposited on August 25, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC No: 63750. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0053] Example 2: Obtaining Didylactam A, a lactam compound derived from Didymella sp. CYSK-4 strain

[0054] S1. Seed culture

[0055] S1-1. Preparation of seed culture medium: Mix 2400mL of potato juice (potatoes and water in a mass ratio of 1:1 are heated to boiling and boiled to make potato juice) and 48g of glucose, and evenly distribute them into 8 500mL Erlenmeyer flasks. Sterilize at 121℃ for 30min.

[0056] S1-2. Didymella sp. CYSK-4 strain was inoculated into seed culture medium and cultured in a shaker at 28℃ and 140rpm for 168h to obtain seed culture solution;

[0057] S2. Fermentation culture

[0058] S2-1. Preparation of fermentation culture medium: Dissolve 50g of rice starch and 3g of sea salt in 100mL of tap water and sterilize at 121℃ for 15min.

[0059] S2-2. Inoculate 10 mL of the seed culture obtained in S1 into an Erlenmeyer flask containing fermentation medium, and incubate at 25°C for 40 days to obtain the fermentation product;

[0060] S3. Product purification

[0061] The fermentation product obtained from S2 was filtered to obtain bacterial cells. The bacterial cells were soaked in methanol three times (24 h each time), and then concentrated under reduced pressure at 50 °C to obtain a crude methanol extract. The crude methanol extract was subjected to silica gel column chromatography (using petroleum ether-ethyl acetate as the mobile phase for gradient elution, wherein the volume fraction of petroleum ether during elution changed as follows: 100% → 90% → 80% → 70% → 60% → 50% → 40% → 30%; and the volume fraction of ethyl acetate during elution changed as follows: 0% → 10% → 20% → 30% → 40% → 50% → 60% → 70%; wherein the volume of eluent used for each gradient was 30 times the sample volume). The eluent was collected and divided into 8 fractions. The 5th fraction (i.e., the 60% petroleum ether-40% ethyl acetate fraction) was subjected to Sephadex gel chromatography. LH-20 column chromatography (eluting with methanol and dichloromethane at a volume ratio of 1:1) yielded three subfractions 5-1 to 5-3. These three subfractions were then subjected to vanillin sulfate color development; subfraction 5-1, which showed a blue color, was the target fraction. Subfraction 5-1 was then further eluted using a silica gel column (dichloromethane:methanol = 200:1) to obtain subfractions 5-1-1 to 5-1-3. These three subfractions were then subjected to vanillin sulfate color development; subfraction 5-1-3, which showed a blue color, was the target fraction. Subfraction 5-1-3 was then purified using Ultimate XB-C18 reversed-phase high-performance liquid chromatography (T...). R =20.4min)(The solvent for high performance liquid chromatography was methanol:water =9:1), thus obtaining Didylactam lactam compound, named Didylactam A.

[0062] Example 3: Structural determination of Didylactam A

[0063] Based on the combined analysis of nuclear magnetic resonance spectroscopy, mass spectrometry, single-crystal testing, and ECD comparison, the A-structure of Didylactam was analyzed, and the results are as follows:

[0064] (1) Didylactam A:C 42 H 51 NO9, HRESI-MS: m / z 736.3434 [M+Na]+ (theoretical value 736.3462).

[0065] The NMR data of Didylactam A are shown in Table 1, and the single crystal structure is as follows: Figure 2 As shown.

[0066] Table 1. NMR data of Didylactam A (100MHz / 400MHz, TMS, ppm)

[0067]

[0068]

[0069] Comparison of measured and calculated ECD of Didylactam A as follows: Figure 3 As shown.

[0070] Therefore, based on the results of the structural test, the structural formula of Didylactam A can be determined as shown in equation (Ⅰ):

[0071]

[0072] Example 4: Anti-hepatocellular carcinoma activity of the lactam compound Didylactam A

[0073] The Huh7 and Hepa1-6 cell lines were purchased from the cell bank of the Shanghai Institutes for Biological Sciences (Shanghai, China). Huh7 and Hepa1-6 cells were cultured in DMEM medium (Invitroen, Carlsbad, CA, USA) supplemented with 10% FBS (Hyclone, Logan, UT, USA), 1% penicillin / streptomycin (Invitrogen), and 2 mM L-glutamine, as recommended by the supplier.

[0074] Drug administration group: Before drug treatment, 1×10 4 Cells were seeded into 96-well plates and cultured overnight. Didylactam A, a lactam compound, was dissolved in DMSO (10 mM) and diluted with culture medium to various concentrations (0 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM). Cells were then treated with each concentration for 48 hours. A working solution of 5 mg / ml MTT (Genview, Houston, TX, USA) (10 μl / 100 μl DMEM medium) was added to each well for 4 hours to form purple formazan crystals. The purple formazan crystals were then dissolved in 150 μl dimethyl sulfoxide (Sangon Biotech, Shanghai, China). The absorbance of the samples was measured at 490 nm using a microplate spectrophotometer. Data are presented as mean ± standard deviation, with each experiment performed in triplicate.

[0075] According to the formula The inhibition rate was calculated, and then the IC50 of the compound was fitted using GraphPad software. 50 Curves were used to determine the anti-hepatocellular carcinoma activity of the compounds, and the results are shown in Table 2 and... Figure 4 As shown.

[0076] Negative control group: Didylactam A, a lactam compound, was replaced with DMSO in the treatment group.

[0077] Blank group: Didylactam A, a lactam compound in the drug administration group, was replaced with DMEM medium.

[0078] Three parallel samples were set up for each of the drug administration group, negative control group, and blank group.

[0079] Table 2 Results of anti-hepatocellular carcinoma activity

[0080]

[0081] Drug administration groups (Hepa1-6) 5.17±0.06 The treatment group (Huh7) 10.57±1.15 Negative control group (Hepa1-6) - Negative control group (Huh7) - Blank group (Hepa1-6) - Blank group (Huh7) -

[0082] From Table 2 and Figure 4 It was found that the negative control group and the blank group could not inhibit the proliferation of liver cancer cells Huh7 and Hepa1-6. The IC50 of the lactam compound Didylactam A on liver cancer cells Hepa1-6 and Huh7 was [not specified]. 50 The concentrations of 5.17±0.06 μM and 10.57±1.15 μM, respectively, effectively inhibited the proliferation of liver cancer cells, indicating that Didylactam A of the present invention has significant anti-liver cancer activity and is suitable for preparation as an anti-liver cancer drug, providing a new source of drug material for the treatment of liver cancer.

[0083] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A kind Didymella Didylactam A, a lactam compound derived from sp., is characterized by, The lactam compound Didylactam A has the chemical structure shown in formula (Ⅰ): Equation (Ⅰ).

2. A single plant Didymella sp. CYSK-4 strain, characterized by, The Didymella sp. CYSK-4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 25, 2023, with accession number GDMCC No: 63750.

3. The claim 2 Didymella The use of sp. CYSK-4 in the preparation of Didylactam A, the lactam compound of claim 1.

4. The method for preparing Didylactam A, a lactam compound according to claim 1, characterized in that, Includes the following steps: S1. As described in claim 2 Didymella sp. CYSK-4 strain was inoculated into seed culture medium and cultured to obtain seed culture solution; S2. The seed culture medium obtained in S1 is inoculated into a fermentation medium and cultured to obtain the fermentation product; S3. The fermentation product obtained in S2 is subjected to soaking, extraction, filtration, concentration, silica gel column chromatography, gel column chromatography, and purification in sequence to obtain Didylactam A; S3 describes silica gel column chromatography using petroleum ether and ethyl acetate as mobile phases for gradient elution. The volume fraction of petroleum ether changes during elution as follows: 100%→90%→80%→70%→60%→50%→40%→30%; the volume fraction of ethyl acetate changes during elution as follows: 0%→10%→20%→30%→40%→50%→60%→70%. S3 describes gel column chromatography: gradient elution is performed using dichloromethane and methanol as the mobile phase, and the packing material is Sephadex LH-20. The volume ratio of methanol to dichloromethane in the mobile phase is (1~2):

1.

5. The preparation method according to claim 4, characterized in that, In the seed culture medium described in S1, the mass-to-volume ratio of glucose to potato juice is 6–40 g: 150–1000 mL.

6. The preparation method according to claim 4, characterized in that, The fermentation medium described in S2 contains 25-75 g of starch matrix and 0.5-3 g of sea salt per 100-500 mL of fermentation medium.

7. The use of Didylactam A, the lactam compound of claim 1, in the preparation of an anti-hepatocellular carcinoma drug.

8. An anti-liver cancer drug, characterized in that, It contains Didylactam A, a lactam compound as described in claim 1.

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