Method for improving yield and purity of cyclic peptide compound produced by aspergillus fermentation and application

Abstract

The invention discloses a method for improving the yield and purity of cyclic peptide compounds produced by aspergillus fermentation and application, components of a fermentation culture medium for producing the cyclic peptide compounds by aspergillus fermentation are optimized through a single factor test and an orthogonal test, and it is found that the yield of JBIR-15 is effectively improved by adding a certain amount of amino acid, the yield reaches 984 mg / L, and the yield of the JBIR-15 reaches 984 mg / L. The yield of JBIR-15 (321mg / L) is increased by about 3.07 times compared with that of JBIR-15 (321mg / L) The purity is greater than or equal to 95%, the biological activity is effectively improved, and the compound can be used for preparing anti-tumor drugs and / or anti-inflammatory drugs.

Description

(I) Technical Field

[0002] This invention relates to a method and its application for improving the yield and purity of cyclic peptide compounds produced by Aspergillus fermentation. (II) Background Technology

[0004] Marine fungi, particularly *Aspergillus* species, are considered important resources for lead drug development due to their ability to produce specific secondary metabolites. In recent years, researchers have obtained various natural products from marine-derived *Aspergillus* species, which can produce secondary metabolites such as polyketides, alkaloids, peptides, and terpenes, exhibiting antibacterial, antiviral, and anticancer activities, all of which demonstrate the important role of *Aspergillus* in marine microbiology. Addressing the decreasing availability of novel secondary metabolites from *Aspergillus*, researchers have adopted the "One Fungus, Multiple Metabolites" (OSMAC) strategy to synthesize novel secondary metabolites. This strategy includes methods such as modifying culture medium composition, fermentation conditions, co-culturing, and adding chemical epigenetic modifiers. Furthermore, adding specific precursors for corresponding synthetic pathways is also an efficient strategy to increase the yield of target products, providing a basis for further improving the innovative capabilities of *Aspergillus* secondary metabolites. The energy, inorganic salts, and water required for microbial growth all come from the culture medium. Due to the influence of the type and concentration of nutrients, the types and yields of microbial metabolites vary greatly in different culture media. Therefore, optimizing the composition of the culture medium is one of the most important ways to increase the yield of microbial metabolites.

[0005] Zheng et al. previously isolated the cyclic peptide compound JBIR-15 from the fermentation broth of Aspergillus PT06-1. This compound exhibited selective antibacterial activity against Candida albicans. However, no studies have yet investigated its antitumor or anti-inflammatory activities, and there are few published reports on the fermentation medium composition for high-yield production of the cyclic peptide compound JBIR-15 from Aspergillus. Through preliminary screening, Czapek's medium was found to be effective in producing the target product JBIR-15. Therefore, Czapek's medium was selected as the basal medium, and its composition was optimized accordingly. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a method for improving the yield and purity of cyclic peptide compounds produced by Aspergillus fermentation and its application in the preparation of antitumor and / or anti-inflammatory drugs. Simultaneously, by screening and optimizing the composition of the fermentation medium, the yield of cyclic peptide compounds is effectively increased. Through optimized separation and extraction methods, the purity of the target product is ≥95%, and its biological activity is effectively improved.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides a method for improving the yield and purity of cyclic peptide compounds produced by Aspergillus fermentation, the method comprising the following steps:

[0010] (1) The activated Aspergillus seed culture was inoculated into the fermentation medium and fermented at 20-30°C and 150-200 rpm for 7-17 days to obtain a fermentation broth containing cyclic peptides. The fermentation medium consisted of: 10-50.0 g / L carbon source, 0.5 g / L potassium chloride, 0.5 g / L magnesium sulfate heptahydrate, 2.0-6.0 g / L nitrogen source, 1.0 g / L dipotassium hydrogen phosphate, 0.01 g / L ferrous sulfate, 10-50 mmol / L amino acids, natural pH, and water as the solvent.

[0011] (2) Filter the fermentation broth obtained in step (1) (preferably filter with eight layers of gauze) to separate the bacterial culture and cells. Extract the bacterial culture with an equal volume of ethyl acetate. Take the upper extract and repeat the extraction 1-3 times (preferably 2 times). Combine the upper extracts and concentrate under reduced pressure to dry state to obtain crude extract.

[0012] (3) Dissolve the crude extract obtained in step (2) in petroleum ether and acetone in a volume ratio of 1:1, separate it on a silica gel column, use petroleum ether and acetone in a volume ratio of 1:1 for gravity elution, elute for 8-10 column volumes, use dichloromethane and methanol in a volume ratio of 15:1 as the developing solvent for thin-layer chromatography monitoring, collect the eluent with an Rf value of 0.2-0.3, concentrate it under reduced pressure to dryness, and obtain the crude product;

[0013] (4) The crude product obtained in step (3) was dissolved in methanol and filtered through a 0.22 μm polytetrafluoroethylene organic filter membrane. The filtrate was eluted isocratically using a semi-preparative high performance liquid chromatograph with a mobile phase of methanol and water at a volume ratio of 60:40 and a flow rate of 2 mL / min. The eluent was collected for 26.2 min and concentrated under reduced pressure to dryness to obtain the cyclic peptide compound JBIR-15.

[0014] Furthermore, the Aspergillus mentioned in step (1) is preferably Aspergillus (… Aspergillus sp . L14-OE::laeA2, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20232057, deposited on October 27, 2023, and published in patent application CN117778204.

[0015] Furthermore, the cyclic peptide compound is JBIR-15, with the following structural formula:

[0016]

[0017] Furthermore, the fermentation conditions for step (1) are: 30°C, 200 rpm, and fermentation for 15 days.

[0018] Furthermore, in step (1), the carbon source is one or more of sucrose, glucose, starch, maltose or mannitol, more preferably sucrose; the nitrogen source is one or more of sodium nitrate, yeast powder, beef extract or peptone, more preferably sodium nitrate; and the amino acid is one or more of ornithine, alanine, valine, tryptophan or lysine, more preferably lysine.

[0019] Furthermore, the preferred composition of the fermentation medium in step (1) is: 50 g / L sucrose, 6 g / L sodium nitrate, 30 mmol / L lysine, 0.5 g / L KCl, 0.5 g / L MgSO4·7H2O, 1.0 g / L K2HPO4, 0.01 g / L FeSO4, with natural pH and water as the solvent.

[0020] Furthermore, a semi-preparative high-performance liquid chromatograph was used: Aono LC-2000, and the chromatographic column was: Agilent ZORBAX SB-C18 (250 mm × 9.4 mm, 5 µm).

[0021] Furthermore, before inoculating the Aspergillus into the fermentation medium, it is first inoculated into PDA solid medium for activation, and then inoculated into PDB liquid medium to prepare a seed culture. The seed culture is inoculated into the fermentation medium at a volume concentration of 10%. The seed culture is prepared as follows: Aspergillus is streaked onto PDA solid medium and activated in a fungal incubator at 30-37°C for 3-5 days (preferably 30°C, 3 days), and then inoculated into PDB liquid medium and cultured at 30-37°C and 150-200 rpm for 3-5 days (preferably 30°C, 200 rpm, 3 days) to obtain the seed culture. The PDA solid medium consists of 200.0 g / L potato, 20.0 g / L glucose, and 15.0-20.0 g / L agar, with a natural pH and water as the solvent. The PDB liquid medium consists of 200.0 g / L potato and 20.0 g / L glucose, with a natural pH and water as the solvent.

[0022] Furthermore, the fermentation is carried out in the fermenter under the following conditions: the seed liquid is inoculated into the fermenter containing the fermentation medium at a volume concentration of 10%; the stirring speed of the fermenter is 150-260 rpm, the aeration rate is 15 vm, the culture temperature is 30℃, the culture time is 15 days, and the dissolved oxygen is controlled at 40-60% during the stable growth period of the strain.

[0023] The JBIR-15 prepared by the method of the present invention can be used to prepare anti-tumor drugs, wherein the tumor cells include neuroblastoma LN229.

[0024] The JBIR-15 prepared by the method of the present invention can be used to prepare anti-inflammatory drugs, including drugs with anti-macrophage activity, wherein the macrophages include mouse microglia BV2.

[0025] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0026] This invention optimized the fermentation medium composition for Aspergillus fermentation to produce cyclic peptides through single-factor and orthogonal experiments, and found that adding a certain amount of amino acids effectively increased the yield of JBIR-15, which reached 984 mg / L, about 3.07 times higher than the yield of JBIR-15 (321 mg / L) produced by fermentation in the initial medium.

[0027] The present invention also provides a method for improving the purity of cyclic peptide compounds, such that the purity is ≥95%, and effectively improves their biological activity, enabling them to be used in the preparation of antitumor drugs and / or anti-inflammatory drugs. (iv) Description of the attached drawings

[0029] Figure 1 This is the HPLC chromatogram of the fermentation broth from Example 1.

[0030] Figure 2 The image shows the UV absorption spectrum of the JBIR-15 standard solution in Example 1.

[0031] Figure 3 The regression curve is shown for the JBIR-15 standard in Example 1.

[0032] Figure 4 The HPLC chromatogram of JBIR-15 is shown in step 1 of Example 2.

[0033] Figure 5 For step 1 of Example 2, JBIR-15 1 H NMR spectrum.

[0034] Figure 6 For step 1 of Example 2, JBIR-15 13 C10 NMR spectrum.

[0035] Figure 7 This is the ESI-MS positive source image of JBIR-15 from step 1 of Example 2. (V) Detailed Implementation Methods

[0037] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0038] Example 1: Investigation of different components, concentrations, and fermentation times in fermentation culture medium

[0039] 1. Fermentation culture and product detection

[0040] (1) Activation culture: Take out Aspergillus CCTCC NO: M20232057 stored in glycerol tube from the -80℃ freezer, and inoculate it into PDA solid medium with an inoculation loop in a clean bench using the streak method. Activate the medium by inverting it in a fungal incubator at 30°C for 3 days. The composition of PDA solid medium is: 200.0 g / L potato, 20.0 g / L glucose, 15.0~20.0 g / L agar, natural pH, water as solvent, and autoclave at 121°C for 20 min.

[0041] (2) Seed culture: In a clean bench, pick up a single colony from the PDA solid medium with an inoculation loop and inoculate it into PDB liquid medium. Incubate at 30°C and 200 rpm for 3 days to obtain seed culture. The composition of PDB liquid medium is: 200.0 g / L potato, 20.0 g / L glucose, natural pH, and water as solvent. Autoclave at 121°C for 20 min and fill the container to 50% capacity.

[0042] (3) Fermentation culture: The seed culture was inoculated into a 150 mL Erlenmeyer flask containing 80 mL of initial fermentation medium at a volume concentration of 10%. The flask was shaken and cultured in a shaker at 30℃ and 200 rpm for 15 days to obtain the fermentation broth. Three parallel experiments were set up. The initial fermentation broth culture medium consisted of: 30.0 g / L sucrose, 3.0 g / L sodium nitrate, 0.5 g / L KCl, 0.5 g / L MgSO4·7H2O, 1.0 g / L K2HPO4, and 0.01 g / L FeSO4, with natural pH and water as the solvent. The broth was autoclaved at 121℃ for 20 min, and the volume of the broth was 50% of the container capacity.

[0043] (4) Detection of target product content

[0044] Take 1 mL of the fermentation broth obtained in step (3) and use HPLC to detect the peak area of ​​product JBIR-15. Based on the standard curve of JBIR-15 standard concentration versus peak area, the content of product JBIR-15 is calculated to be 321 mg / L. The chromatogram is shown in [reference needed]. Figure 1 As shown.

[0045] The HPLC detection conditions are as follows:

[0046] Chromatographic column: Agilent ZORBAX XDB-C18 (250 mm × 4.6 mm, 5µm); mobile phase A was methanol; mobile phase B was 0.1% formic acid aqueous solution; flow rate: 1.0 mL / min, injection volume: 10 μL, column temperature: 30℃, gradient elution, UV detection wavelength: 302 nm;

[0047] The gradient elution program was as follows: 0 min, 55% mobile phase A, 45% mobile phase B; 30 min, 65% mobile phase A, 35% mobile phase B; 40 min, 100% mobile phase A, 0% mobile phase B; 50 min, 55% mobile phase A, 45% mobile phase B.

[0048] (5) Construction of standard curve

[0049] Standard stock solution: Accurately weigh 8.1 mg JBIR-15 standard, dissolve it in methanol, and dilute to a 25 mL volumetric flask to prepare the standard stock solution with a concentration of 0.324 mg / mL.

[0050] Standard solutions: Accurately pipette 0.2, 1, 2, 4, 6, and 7 mL of the standard stock solution and dilute to 10 mL volumetric flasks with methanol, yielding concentrations of 0.00648, 0.0324, 0.0648, 0.1296, 0.1944, and 0.2268 mg / mL, respectively.

[0051] The standard solutions of various concentrations were analyzed under the HPLC chromatographic conditions described above. The peak area of ​​the characteristic peak at 16.513 min was recorded. A regression curve equation was calculated with the injection concentration of JBIR-15 standard solution (mg / mL) as the x-axis and the peak area as the y-axis. The UV absorption chromatogram of the standard solution (0.1296 mg / mL) is shown below. Figure 2 .

[0052] The results showed that JBIR-15 exhibited good linearity in the range of 0.00648 ~ 0.2268 mg / mL, with the linear equation being y = 22204x - 50.756 and r = 0.9994. The regression curve is shown below. Figure 3 .

[0053] Determination of the limit of detection (LOD) and the limit of quantitation (LOQ): A standard solution (0.1296 mg / mL) was injected at different volumes (2, 4, 6, 8, 10 μL) under the above HPLC chromatographic conditions. The injection volume at which the peak area was 3 times (S / N=3) and 10 times (S / N=10) the noise level was taken as the LOD and LOQ. The LOD of JBIR-15 was 0.2398 ng, and the LOQ was 0.7995 ng.

[0054] Precision experiment: A standard solution (0.1296 mg / mL) was injected six times consecutively, and the relative standard deviation (RSD) of the peak area was recorded to examine intra-day precision. Inter-day precision was determined by injecting six times a day for three consecutive days. The results showed that the intra-day precision RSD was 1.69%, and the inter-day precision RSD was 1.34%.

[0055] Recovery experiment: Nine portions of fermentation broth with a known JBIR-15 content (0.215 mg / mL) were divided into three groups. Reference solution was precisely added to each group at low (0.1672 mg / mL), medium (0.2090 mg / mL), and high (0.2510 mg / mL) concentrations, respectively. HPLC detection showed that the recoveries of JBIR-15 were 97.5%, 101.2%, and 95.8%, respectively, with an average recovery rate of 98.18% and an RSD of 2.80%.

[0056] 2. Single-factor condition optimization

[0057] (1) Selection of amino acid types and amounts

[0058] The following amino acids were added to the initial fermentation medium in step 1 at different concentrations: 30 mmol / L (ornithine, alanine, valine, tryptophan, lysine); 15 mmol / L each of alanine and ornithine; 15 mmol / L each of alanine and valine; 15 mmol / L each of ornithine and valine; and 10 mmol / L each of alanine, ornithine, and valine. Other operations and conditions remained unchanged, and each group was tested in triplicate, with the initial fermentation medium without added amino acids serving as a control. After 15 days of fermentation, 1 mL of the fermentation broth was analyzed by HPLC to determine the content of the target compound JBIR-15. The results are shown in Table 1. The results show that the content of JBIR-15 was highest when the amino acid was lysine.

[0059] Table 1. Effects of different amino acids on JBIR-15 content

[0060]

[0061] After determining lysine as the optimal amino acid through the above experiments, the amount of amino acid added was investigated. Lysine concentrations of 10, 20, 30, 40, and 50 mmol / L were added to determine the optimal concentration, with other conditions remaining constant. Each group was tested in triplicate. After 15 days of fermentation, 1 mL of fermentation broth was taken and the content of the target compound JBIR-15 was analyzed by HPLC. The results are shown in Table 2. The results show that the highest content of JBIR-15 was achieved when the lysine concentration was 30 mmol / L.

[0062] Table 2. Effect of lysine addition on JBIR-15 content

[0063]

[0064] (2) Product content at different fermentation times

[0065] Add 30 mmol / L lysine to the initial fermentation medium in step 1, and change the fermentation time to 7, 9, 11, 13, 15, 17, and 19 days, respectively, while keeping other operations and conditions the same. The content of the target compound JBIR-15 is shown in Table 3. The results show that the JBIR-15 content is highest when the fermentation time is 17 days, but considering the time and fermentation cost, the final fermentation time was determined to be 15 days.

[0066] Table 3. Effects of different fermentation times on JBIR-15 content

[0067]

[0068] (3) Selection of carbon source type and amount

[0069] Add 30 mmol / L lysine to the initial fermentation medium in step 1, and change the carbon source sucrose to glucose, starch, maltose, and mannitol, respectively, while keeping other conditions and operations unchanged. Each group was tested in triplicate. After 15 days of fermentation, 1 mL of fermentation broth was taken and the content of the target compound JBIR-15 was determined by HPLC. The results are shown in Table 4. The results show that the JBIR-15 content was highest when sucrose was used as the carbon source.

[0070] Table 4. Effects of different carbon sources on JBIR-15 content

[0071]

[0072] After determining sucrose as the optimal carbon source through the above experiments, the sucrose concentration was investigated. Optimal concentrations were determined by adding 10, 20, 30, 40, and 50 g / L of sucrose, with other conditions remaining constant. Each group was tested in triplicate. After 15 days of fermentation, 1 mL of the fermentation broth was analyzed by HPLC to determine the content of the target compound JBIR-15. The results are shown in Table 5. The results indicate that the JBIR-15 content was highest when the sucrose concentration was 50 g / L.

[0073] Table 5. Effects of different sucrose concentrations on JBIR-15 content

[0074]

[0075] (4) Selection of nitrogen source type and amount

[0076] Add 30 mmol / L lysine to the initial fermentation medium in step 1, change the sucrose concentration to 50 g / L, and change the nitrogen source sodium nitrate to yeast extract, beef extract, peptone, and ammonium sulfate, respectively, while keeping other conditions and operations unchanged. Each group was tested in triplicate. After 15 days of fermentation, 1 mL of fermentation broth was taken and the content of the target compound JBIR-15 was determined by HPLC. The results are shown in Table 6. The results show that the JBIR-15 content was highest when sodium nitrate was used as the nitrogen source.

[0077] Table 6. Effects of different nitrogen sources on JBIR-15 content

[0078]

[0079] After determining sodium nitrate as the optimal nitrogen source through the above experiments, the concentration of sodium nitrate was investigated. The optimal concentration was determined by adding 2, 3, 4, 5, and 6 g / L, with other conditions remaining constant. Each group was tested in triplicate. After 15 days of fermentation, 1 mL of the fermentation broth was analyzed by HPLC to detect the content of the target compound JBIR-15. The results are shown in Table 7. The results show that the JBIR-15 content was highest when the sodium nitrate concentration was 5 g / L.

[0080] Table 7. Effects of different nitrogen source concentrations on JBIR-15 content

[0081]

[0082] Through the above single-factor experiments, the optimal carbon source was determined to be 50 g / L sucrose, the optimal nitrogen source to be 5 g / L sodium nitrate, the optimal amino acid to be 30 mmol / L lysine, and the optimal fermentation time to be 15 days.

[0083] 3. Orthogonal experiment

[0084] Based on the aforementioned experiment, four factors were selected: sucrose concentration (A), sodium nitrate concentration (B), lysine concentration (C), and fermentation time (D). A four-factor, three-level orthogonal experiment was designed (Table 8). Each group was repeated three times to determine the interaction between the factors. After fermentation, 1 mL of fermentation broth was taken and the content of the target compound JBIR-15 was detected by HPLC. The results are shown in Table 9.

[0085] Table 8. Factor Level Table for Orthogonal Experiments

[0086]

[0087] Table 9. Effects of interactions among various factors on JBIR-15 content.

[0088]

[0089] Based on the initial fermentation medium, through single-factor experiments and orthogonal experiments, the optimal fermentation medium composition for Aspergillus fermentation to produce JBIR-15 was determined to be: 50 g / L sucrose, 6 g / L sodium nitrate, 30 mmol / L lysine, 0.5 g / L KCl, 0.5 g / L MgSO4·7H2O, 1.0 g / L K2HPO4, 0.01 g / L FeSO4, natural pH, and water as the solvent.

[0090] 4. Shake flask culture in optimal fermentation medium

[0091] Using the optimal fermentation medium from step 3 and the conditions from step 1, the yield of JBIR-15 in the fermentation broth reached 984 mg / L, which is about 3.07 times higher than the yield of JBIR-15 (321 mg / L) from the initial medium.

[0092] 5. Optimal fermentation medium for fermenter cultivation

[0093] 4L of the optimized fermentation medium from step 3 was added to a 10L fermenter, and the seed culture prepared in step 1 was inoculated at a volume concentration of 10% via flame inoculation. The fermenter was stirred at 150-260 rpm, with an aeration rate of 15 v / m, a culture temperature of 30℃, and a culture time of 15 days. Dissolved oxygen was controlled at 40-60% during the stationary phase of the strain's growth. The yield of JBIR-15 in the fermentation broth was measured using the method in step 1, reaching 893 mg / L, which is approximately 2.69 times higher than the yield of JBIR-15 (321 mg / L) from the initial culture medium fermentation. Therefore, the optimized fermentation conditions can be further used for industrial production.

[0094] Example 2: Purification, identification, and content determination of the target compound JBIR-15

[0095] 1. Preparation of target compound JBIR-15

[0096] (1) Activation culture: Take out Aspergillus CCTCC NO: M20232057 stored in glycerol tube from the -80℃ freezer, and inoculate it into PDA solid medium (same as in Example 1) using the streak method in a clean bench. Activate the culture by inverting at 30°C for 3 days in a fungal incubator.

[0097] (2) Seed culture: In a clean bench, pick up a single colony from the PDA solid medium with an inoculation loop and inoculate it into PDB liquid medium (same as in Example 1). Culture at 30°C and 200 rpm for 3 days to obtain seed culture;

[0098] (3) Fermentation culture: The seed culture was inoculated into a 150 mL Erlenmeyer flask containing 80 mL of the fermentation medium optimized in Example 1 at a volume concentration of 10%. The flask was shaken and cultured in a shaker at 30℃ and 200 rpm for 15 days to obtain the fermentation broth. The fermentation broth culture medium consisted of 50.0 g / L sucrose, 6.0 g / L sodium nitrate, 30 mmol / L lysine, 0.5 g / L KCl, 0.5 g / L MgSO4·7H2O, 1.0 g / L K2HPO4, and 0.01 g / L FeSO4, with a natural pH and water as the solvent. The flask was autoclaved at 121℃ for 20 min, and the volume of the flask was 50% of the container capacity.

[0099] 2. Purification and identification of the product

[0100] (1) Filter 2400 mL of the fermentation broth obtained in step 1 through eight layers of gauze to separate the bacterial culture and the bacterial cells. Extract the bacterial culture with an equal volume of ethyl acetate, take the upper extract, repeat the extraction twice, combine the upper extracts, concentrate under reduced pressure to dry state, and obtain 7.36 g of crude extract.

[0101] (2) Take 2.59 g of the crude extract from step (1), dissolve it in 10 mL of petroleum ether:acetone = 1:1 (volume ratio), and separate it on a silica gel column (silica gel particle size 300-400 mesh, silica gel packing amount is 10 times the sample mass). Use petroleum ether and acetone at a volume ratio of 1:1 for gravity elution, elute for 10 column volumes, use methanol and dichloromethane at a volume ratio of 1:15 as the developing solvent for thin-layer chromatography monitoring, collect the eluent with an Rf value of 0.2-0.3, concentrate under reduced pressure to dry state, and obtain 457 mg of crude product of the target compound. Take a sample and use HPLC as described in Example 1 for detection. The purity is determined to be 75% by the peak area of ​​liquid chromatography.

[0102] (3) Dissolve all the crude product obtained in step (2) in 5 mL of methanol. The results are shown in the figure. Figure 4 As shown. Then, it was filtered through a 0.22 μm polytetrafluoroethylene organic filter membrane, and isocratic eluted using a semi-preparative high-performance liquid chromatography (HPLC) system with a mobile phase of methanol and water at a volume ratio of 60:40 and a flow rate of 2 mL / min. The eluent was collected after 26.2 min, concentrated under reduced pressure to a dry state, and then sampled using nuclear magnetic resonance (NMR) spectroscopy. 1 H NMR, 13 The structures of the compounds were identified by 12C NMR and ESI-MS. 1 H NMR see Figure 5 , 13 C NMR (see) Figure 6 See ESI-MS positive source plot. Figure 7225 mg of the target compound JBIR-15 (purity ≥95%) was obtained.

[0103] Semi-preparative high-performance liquid chromatograph: Aono LC-2000, chromatographic column: Agilent ZORBAX SB-C18 (250mm × 9.4mm, 5µm).

[0104] Example 3: Activity determination of target compound JBIR-15

[0105] 1. In vitro antitumor activity

[0106] Initial screening: Tumor cells in Table 10 (all purchased from the Cell Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured in DMEM medium containing 10% domestic fetal bovine serum at 37°C and 5% CO2. The cells were passaged every 1-2 days until they adhered to the culture vessel.

[0107] According to 1 10 4 Cells were seeded at a density of 1:1 in 96-well plates and incubated overnight at 37°C with 5% CO2. After cell attachment, the old culture medium was discarded, and the plates were divided into experimental wells, control wells, and blank wells. 10 μL of fresh serum-free DMEM medium containing 20 μM JBIR-15 (prepared in Example 2) was added to the experimental wells. 10 μL of serum-free DMEM medium containing 20 μM doxorubicin (antibiotic) was added to the control wells. 10 μL of DMEM medium containing 10% domestic fetal bovine serum was added to the blank wells. Three replicates were set for each well. After incubation for 48 h, 10 μL of CCK-8 solution was added to each well, and the OD value was measured at 450 nm using a microplate reader to detect the inhibition rate. If the inhibition rate was greater than 50%, a second screening was performed. The results are shown in Table 10.

[0108] Inhibition rate = [(OD control wells - OD experimental wells) / (OD control wells - OD blank wells)] × 100%

[0109] JBIR-15 prepared in Example 2 was diluted with serum-free DMEM medium to set five concentrations (0.363, 0.725, 1.250, 2.50, and 5.00 μM). Cell culture and seeding procedures were the same as in the initial screening. After 48 h of drug treatment, 10 μL of CCK-8 solution was added, the inhibition rate was detected, and the IC50 value was calculated.

[0110] The results show that JBIR-15 has a good inhibitory rate on neuroblastoma LN229 cells and good anti-neuroblastoma LN229 cell activity.

[0111] Table 10 Evaluation of the antitumor activity of JBIR-15

[0112]

[0113] 2. Anti-inflammatory activity

[0114] Initial screening: Mouse microglia (BV2) and mouse mononuclear macrophages (RAW264.7) in Table 11 were cultured in DMEM medium containing 10% domestic fetal bovine serum at 37°C and 5% CO2. They were passaged every 1-2 days.

[0115] According to 1 10 4 Cells were seeded at a density of 96-well plates and incubated overnight at 37°C with 5% CO2. After cell attachment, the old culture medium was discarded, and the plates were divided into experimental wells, control wells, and blank wells. 10 μL of fresh serum-free DMEM medium containing 20 μM JBIR-15 (prepared in Example 2) was added to the experimental wells. 10 μL of serum-free DMEM medium containing 20 μM dexamethasone was added to the control wells. 10 μL of DMEM medium containing 10% domestic fetal bovine serum was added to the blank wells. Three replicates were set for each well. LPS (lipopolysaccharide) was added after 1 hour, and incubation continued for 24 hours. The supernatant was collected, and the NO level was measured using the Griess method. The OD value was measured at 562 nm using a microplate reader to detect the inhibition rate. If the inhibition rate was greater than 50%, a second screening was performed. The results are shown in Table 11.

[0116] JBIR-15 prepared in Example 2 was diluted with serum-free DMEM medium to set five concentrations (0.725, 1.250, 2.50, 5.00, and 10 μM). After 24 h of drug treatment, 10 μL of 1 mg / mL LPS aqueous solution was added, and the IC50 value was calculated. The results are shown in Table 11.

[0117] The results show that JBIR-15 has a good inhibitory rate on BV2 cells and good anti-inflammatory activity.

[0118] Table 11 Evaluation of the anti-inflammatory activity of JBIR-15

[0119]

Claims

1. A method for improving the yield and purity of cyclic peptide compounds produced by fermentation of engineered Aspergillus, characterized in that, The method includes the following steps: (1) The activated Aspergillus seed culture was inoculated into the fermentation medium and fermented at 20-30°C and 150-200 rpm for 7-17 days to obtain a fermentation broth containing cyclic peptides. The fermentation medium consisted of: 10-50.0 g / L carbon source, 0.5 g / L potassium chloride, 0.5 g / L magnesium sulfate heptahydrate, 2.0-6.0 g / L nitrogen source, 1.0 g / L dipotassium hydrogen phosphate, 0.01 g / L ferrous sulfate, 10-50 mmol / L amino acids, natural pH, and water as the solvent. (2) Filter the fermentation broth obtained in step (1) to separate the bacterial culture and the bacterial cells. Extract the bacterial culture with an equal volume of ethyl acetate, take the upper extract, repeat the extraction 1-3 times, combine the upper extracts, concentrate under reduced pressure to dry state, and obtain crude extract. (3) Dissolve the crude extract obtained in step (2) in petroleum ether and acetone in a volume ratio of 1:1, separate it on a silica gel column, use petroleum ether and acetone in a volume ratio of 1:1 for gravity elution, elute for 8-10 column volumes, use dichloromethane and methanol in a volume ratio of 15:1 as the developing solvent for thin-layer chromatography monitoring, collect the eluent with an Rf value of 0.2-0.3, concentrate it under reduced pressure to dryness, and obtain the crude product; (4) The crude product obtained in step (3) was dissolved in methanol and filtered through a 0.22 μm polytetrafluoroethylene organic filter membrane. The filtrate was eluted isocratically using a semi-preparative high performance liquid chromatograph with a mobile phase of methanol and water at a volume ratio of 60:40 and a flow rate of 2 mL / min. The eluent was collected for 26.2 min and concentrated under reduced pressure to dryness to obtain cyclic peptide compounds.

2. The method as described in claim 1, characterized in that, The Aspergillus is Aspergillus ( Aspergillus sp . CCTCCNO: M20232057.

3. The method as described in claim 1, characterized in that, The cyclic peptide compound is JBIR-15, with the following structural formula: 。 4. The method as described in claim 1, characterized in that, In step (1), the carbon source is one or more of sucrose, glucose, starch, maltose or mannitol; the nitrogen source is one or more of sodium nitrate, yeast powder, beef extract or peptone.

5. The method as described in claim 1, characterized in that, In step (1), the amino acid is one or more of the following: ornithine, alanine, valine, tryptophan, and lysine.

6. The method as described in claim 1, characterized in that, The fermentation medium consisted of: 50.0 g / L sucrose, 0.5 g / L potassium chloride, 0.5 g / L magnesium sulfate heptahydrate, 6.0 g / L sodium nitrate, 1.0 g / L dipotassium hydrogen phosphate, 0.01 g / L ferrous sulfate, 30 mmol / L lysine, with a natural pH and water as the solvent.

7. The method as described in claim 1, characterized in that, Before inoculating the Aspergillus into the fermentation medium, it is first activated by inoculating it into PDA medium, and then inoculated into PDB medium to prepare a seed culture. The seed culture is inoculated into the fermentation medium at a volume concentration of 1-10%. The seed culture is prepared as follows: Aspergillus is streaked onto PDA solid medium and activated in a fungal incubator at 30-37°C for 3-5 days, and then inoculated into PDB liquid medium and cultured at 30-37°C and 150-200 rpm for 3-5 days to obtain the seed culture. The composition of PDA solid medium is: 200.0 g / L potato, 20.0 g / L glucose, 15.0-20.0 g / L agar, natural pH, and water as the solvent. The composition of PDB liquid medium is: 200.0 g / L potato, 20.0 g / L glucose, natural pH, and water as the solvent.

8. The method as described in claim 1, characterized in that, The fermentation was carried out in a fermenter under the following conditions: the seed culture was inoculated into the fermenter containing the fermentation medium at a volume concentration of 10%; the stirring speed of the fermenter was 150-260 rpm; the aeration rate was 15 vm; the culture temperature was 30℃; the culture time was 15 days; and the dissolved oxygen was controlled at 40-60% during the stable growth period of the strain.

9. The use of a cyclic peptide compound prepared by the method of claim 1 in the preparation of an antitumor drug.

10. The use of a cyclic peptide compound prepared by the method of claim 1 in the preparation of an anti-inflammatory drug.

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