Method for optimizing fermentation yield of doxylamine compound and preparing doxylamine compound
By optimizing the culture medium and fermentation process, and combining adsorption resin and liquid chromatography separation technology, the problems of low yield and high toxicity of doxamine compounds were solved, and high-yield and high-purity doxamine compounds with good antibacterial activity and selectivity were achieved.
Patent Information
- Application Number
- CN202511583819.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-31
- Publication Date
- 2026-03-13
AI Technical Summary
The low fermentation yield, high cytotoxicity and hemolytic toxicity, and poor selectivity of doxamine compounds limit their development and application.
By optimizing the composition and conditions of the culture medium in shake flasks and fermenters, adopting a fed-batch fermentation process, and combining non-ionic macroporous adsorption resins and liquid chromatography separation technology, the fermentation yield and purity of doxoride compounds were improved.
It significantly increased the fermentation yield of doxamine compounds, with a maximum increase of 1160 times, reduced cytotoxicity, enhanced antibacterial activity, and provided broad prospects for clinical application.
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Figure CN121653203A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a method for optimizing the fermentation yield of doxamine compounds and preparing doxamine compounds. Background Technology
[0002] The development and widespread use of antibiotics have greatly improved the clinical treatment of bacterial infections, effectively controlling a variety of infectious diseases. However, due to the overuse and irrational use of some antibiotics, pathogenic bacteria are constantly mutating under selective pressure, evolving drug resistance and exacerbating the problem of multidrug resistance. Currently, the availability of effective antibiotics in clinical practice is becoming increasingly scarce, and drug-resistant bacterial infections have become a serious global problem threatening human health. To address this challenge, there is an urgent need to develop novel antimicrobial compounds with new mechanisms of action.
[0003] Docetin antibiotics were first purified in 1986 by Takahashi's team at the National Institute of Health in Japan from the subspecies *Cryophilis* TS-1980 of *Nocardiopsis mutabilis*. They exhibit broad-spectrum inhibitory effects against a variety of Gram-positive and Gram-negative bacteria. However, due to limitations in spectroscopic techniques at the time, the precise chemical structure of docetin remained unidentified until 2023, when Olga Genilloud's research group in Spain isolated six docetin compounds from the genus *C. cryophile* CA-258035. They identified the structures of docetin compounds for the first time and discovered that they possess a pyrazine-alkylguanidine skeleton.
[0004] However, the initial fermentation yield of doxamine is very low. Previous studies have found that fermentation of psychrophilic C. cryophile ZJ4-3B using ISP2 liquid medium yields only 0.1–0.3 mg / mL of doxamine. Currently, there is a lack of technologies for the large-scale production and preparation of this type of antibiotic. At the same time, this type of compound also exhibits problems such as cytotoxicity, hemolytic toxicity, and poor selectivity, which greatly limits its subsequent development and application. Summary of the Invention
[0005] The purpose of this invention is to overcome the problems of low yield of existing doxamine compounds and the cytotoxicity, hemolytic toxicity, and poor selectivity of existing doxamine compounds. Therefore, there is an urgent need to provide a method for optimizing the fermentation yield of doxamine compounds and for preparing doxamine compounds.
[0006] The objective of this invention can be achieved through the following technical solutions:
[0007] One of the technical solutions of the present invention provides a method for optimizing the fermentation yield of doxorubicin compounds, comprising the following steps:
[0008] S1. Inoculate Klostridium psychrophile ZJ4-3B into shake flask seed culture medium and culture with shaking to obtain Klostridium psychrophile ZJ4-3B shake flask seed liquid;
[0009] S2. The psychrophilic Klostridium ZJ4-3B seed culture obtained in step S1 is inoculated into the seed culture medium of the fermenter, and cultured with aeration and shaking to obtain the psychrophilic Klostridium ZJ4-3B fermentation seed culture.
[0010] S3. The psychrophilic Klostridium ZJ4-3B fermentation seed liquid obtained in step S2 was inoculated into the fermenter culture medium, aerated and shaken, and the fermentation liquid containing the increased production of dopsisamines AD was collected.
[0011] In some specific embodiments, in steps S1 and S2, both the shake flask seed culture medium and the fermenter seed culture medium consist of the following components by mass percentage: 1% malt extract, 0.4% yeast extract, and 0.4% glucose.
[0012] In step S3, the fermentation medium consists of the following components: 0.1% MgCl·6H2O, 0.2% NH4Cl, 0.2% CaCO3, 0.1% K2HPO4, 0.1% NaCl, 0.5% sucrose, 0.2% sodium sulfate, 2% soluble starch, and 0.1% trace element mixture; wherein, the trace element mixture per 100 mL consists of: 100 mg MnCl2·4H2O, 100 mg ZnCl2, and 100 mg FeCl2·4H2O, and the pH is adjusted to 7.0–7.1.
[0013] In some specific embodiments, in step S1, the process conditions for obtaining the psychrophilic Klosella ZJ4-3B shake flask seed culture are: cultured at 30°C and 220 rpm for 2-3 days.
[0014] In step S2, the inoculation volume ratio of the psychrophilic Klostridium ZJ4-3B seed culture to the fermentation seed medium was 2%. The process conditions for obtaining the psychrophilic Klostridium ZJ4-3B fermentation seed culture by aeration and shaking were as follows: aeration rate of 0.3 vvm, tank pressure of 0.01-0.03 MPa, rotation speed of 100-150 rpm, and culture for 1-2 days.
[0015] In step S3, the inoculation volume ratio of the psychrophilic Klostridium ZJ4-3B fermentation seed liquid to the fermentation medium is 15%–17%. The aeration and shaking culture conditions are as follows: aeration rate of 0.5 vvm, tank pressure of 0.01–0.03 MPa, rotation speed of 100–150 rpm from 0 to 36 h, rotation speed of 200–300 rpm after 36 h, and culture for 5–7 days.
[0016] The second technical solution of the present invention is to provide a doxylamine compound, characterized in that it is selected from any one or more of the structures shown in Formula I:
[0017] In the formula, R1 is H or OH; R2 is H or OH; R3 is H or R4 is H or R5 is H or
[0018] In some specific embodiments, the doxylamine compound is selected from any one or more of the following group:
[0019]
[0020] Understandably, in Dopsisamine E(1), R1 = R3 = H, R2 = OH.
[0021] In Dopsisamine F(2), R1=R2=OH,
[0022] In Dopsisamine G(3), R1 = H, R2 = OH,
[0023] In Dopsisamine H(4), R1=R2=R3=H,
[0024] In Dopsisamine I (5), R1=R2=R3=R4=R5=H.
[0025] The third technical solution of the present invention provides a method for preparing doxylamine compounds, which further includes the following steps:
[0026] S401. The fermentation broth as described in one of the technical solutions is adsorbed by a non-ionic macroporous adsorption resin. The resin adsorbed with the fermentation broth is eluted sequentially by pure water, 20% methanol aqueous solution, and 2‰ TFA-acetone. The crude extract is obtained by concentrating the 2‰ TFA-acetone eluent.
[0027] S402. The crude extract obtained in step S401 is dissolved in methanol and purified by liquid chromatography. Mobile phase A is 2‰ trifluoroacetic acid aqueous solution and mobile phase B is acetonitrile. Gradient elution is performed at a flow rate of 16.0 mL / min to obtain doxamine compounds with the structure shown in Formula I.
[0028] In some specific embodiments, in step S402, the gradient elution process conditions are as follows: 0-15 min gradient elution, with mobile phase B increasing from 20% to 45%; 15-20 min isocratic elution, with mobile phase B kept constant at 99%; 20-25 min isocratic elution, with mobile phase B kept constant at 20%.
[0029] The fourth technical solution of the present invention is to provide an antibacterial drug composition, the antibacterial drug composition comprising:
[0030] (1) a doxamine compound as described in the second technical solution above, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; and (2) a pharmaceutically acceptable carrier.
[0031] The fifth technical solution of the present invention provides the use of the doxylamine compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, as described in the second technical solution, for the preparation of a formulation or drug, wherein the formulation or drug is used for:
[0032] (1) To inhibit or kill Gram-negative bacteria and / or Gram-positive bacteria and / or mycobacteria; and / or (2) To prevent and / or treat infectious diseases caused by Gram-negative bacteria, Gram-positive bacteria or mycobacteria.
[0033] In some specific embodiments, the mycobacterium is Mycobacterium tuberculosis, the Gram-negative bacterium is Escherichia coli, and the Gram-positive bacterium is methicillin-resistant Staphylococcus aureus.
[0034] In some specific embodiments, the infectious disease caused by the mycobacterium is pulmonary tuberculosis, the infectious disease caused by the Escherichia coli is extraintestinal infection (exemplary, such as urinary tract infection, abdominal infection, etc.), and the infectious disease caused by methicillin-resistant Staphylococcus aureus is pneumonia.
[0035] Compared with the prior art, the present invention has the following beneficial effects:
[0036] (1) This invention significantly improved the fermentation yield of dopsisamine AD under fed-batch fermentation conditions by optimizing the culture medium at the shake-flask level and scaling up the fermenter stepwise. The yield can be increased by up to 1160 times, providing a solid yield foundation for the further development and drug research of dopsisamine AD. Moreover, the extraction method of this invention is mature, the process is simple, and the yield of the product is high.
[0037] (2) This invention isolated and identified five novel compounds, Dopsisamines EI, with trace amounts of dopsisamine structures. Compared with previously reported dopsisamine compounds, these compounds exhibit different numbers and positions of isopentenyl and hydroxyl substitutions. All products were confirmed to have correct structures by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Due to the increased number and different positions of isopentenyl and hydroxyl substitutions, the activity test results of Dopsisamines EI in this invention show enhanced antibacterial activity and significantly reduced cytotoxicity. It exhibits good antibacterial activity and selectivity against Escherichia coli, methicillin-resistant Staphylococcus aureus, and Mycobacterium tuberculosis, and is expected to be developed into an antibacterial lead compound with broad clinical application prospects. Attached Figure Description
[0038] Figure 1 This is a comparison graph of liquid chromatography-mass spectrometry analysis of dopsisamines AD in crude extract before and after fermentation optimization. Figure 2 The fermentation optimization yield improvement curve for the dopsisamine compound AD. Figure 3 The images show the ultraviolet absorption spectra of compounds 1-5 of this invention. Figures 4 to 10 The HR-ESI-MS spectrum of compound 1 of the present invention is shown below. 1 H NMR spectrum, 13 C NMR spectrum, 1 H- 1 H COSY spectrum, HMBC spectrum, HSQC spectrum, NOESY spectrum. Figures 11-17 The HR-ESI-MS spectrum of compound 2 of the present invention, 1 H NMR spectrum, 13 C NMR spectrum, 1 H- 1 H COSY spectrum, HMBC spectrum, HSQC spectrum, NOESY spectrum. Figures 18-24 The HR-ESI-MS spectrum of compound 3 of the present invention is shown below. 1 H NMR spectrum, 13 C NMR spectrum, 1 H- 1 HCOSY spectrum, HMBC spectrum, HSQC spectrum, NOESY spectrum. Figures 25-31 The HR-ESI-MS spectrum of compound 4 of this invention is shown below. 1 H NMR spectrum, 13 C NMR spectrum, 1 H- 1 H COSY spectrum, HMBC spectrum, HSQC spectrum, NOESY spectrum. Figures 32-38 The HR-ESI-MS spectrum of compound 5 of the present invention, 1H NMR spectrum, 13 C NMR spectrum, 1 H- 1 H COSY spectrum, HMBC spectrum, HSQC spectrum, NOESY spectrum. Detailed Implementation
[0039] The psychrophilic Krossella ZJ4-3B involved in this invention ( Crossiella cryophile ZJ4-3B, with accession number CGMCC No. 33991, was deposited on March 26, 2025, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China.
[0040] Example 1 This example provides a method for optimizing the fermentation yield of doxorubicin compounds, including the following steps:
[0041] (1) Solid Petri dish culture: The solid culture medium can be prepared using conventional techniques. The composition (by mass percentage) is: 1% malt extract, 0.4% yeast extract, 0.4% glucose, and 15-20% agar. Sterilize the solid culture medium at 115℃ for 30 minutes to prepare solid petri dishes. Streak the psychrophilic Cryophile ZJ4-3B strain onto the solid petri dish, invert it at 30℃, and incubate at a constant temperature for 4-5 days until a large number of white spores are produced. When there is no contamination, it can be harvested and used to obtain the petri dish cultured strain.
[0042] (2) Shake-flask seed culture: The shake-flask seed culture medium can be prepared using conventional techniques. Its components (by weight percentage) are: 1% malt extract, 0.4% yeast extract, and 0.4% glucose. The above components are added to a 250mL Erlenmeyer flask, and deionized water is added to bring the volume to 50mL. After sterilization, the mixture is cooled to room temperature to obtain the shake-flask seed culture medium. Approximately 1cm of the fermentation strain *C. cryophile* ZJ4-3B is scraped off. 2 The spores were transferred to a shake flask seed culture medium and cultured at 30°C using conventional methods for 2-3 days at 220 rpm to obtain the shake flask seed solution.
[0043] (3) Fermentation seed culture: The fermentation seed culture medium can be prepared using conventional techniques. The composition (by mass percentage) is: 1% malt extract, 0.4% yeast extract, and 0.4% glucose. The above components are placed in a 10L fermenter, and deionized water is added to make up to 6L. After sterilization, the mixture is cooled to room temperature to obtain the fermentation seed culture medium. The shake flask seed liquid is inoculated into the fermentation seed culture medium at an inoculation ratio of 2%. The culture conditions are: aeration rate of 0.3 vvm, tank pressure of 0.015-0.025 MPa, rotation speed of 100 rpm, and fermentation for 1-2 days to obtain the fermentation seed liquid.
[0044] (4) Fermentation in a fermenter: The fermentation medium can be prepared using conventional techniques, with the following composition (mass percentage): MgCl·6H2O 0.1%, NH4Cl 0.2%, CaCO3 0.2%, K2HPO4 0.1%, NaCl 0.1%, sucrose 0.5%, sodium sulfate 0.2%, soluble starch 2%, and a micronutrient mixture 0.1%; wherein, the micronutrient mixture per 100mL consists of: MnCl2·4H2O 100mg, ZnCl2 100mg, and FeCl2·4H2O 100mg. The pH is adjusted to 7.0-7.1. 5L of the above-mentioned fermenter seed culture is inoculated into 30L of fermentation medium, with an inoculation ratio of approximately 16.7%, for liquid fermentation. The culture conditions were: aeration rate of 0.5 vvm, tank pressure of 0.015-0.025 MPa, rotation speed of 150 rpm for 0-36 h, rotation speed of 250 rpm after 36 h, and fermentation for 5-7 days.
[0045] (5) Separation and purification of doxamine compounds: The fermentation broth obtained from the above fermenter was collected, and the upper fermentation broth was collected by high-speed centrifugation. 10-20% of Amberlite XAD16 non-ionic macroporous adsorption resin was added, and the mixture was rotated and adsorbed on a shaker at 220 rpm for 12 h. The resin was collected by filtration and eluted three times with pure water, 20% methanol-water, and 2‰ TFA-acetone. The crude extract was obtained by vacuum compression of the 2‰ TFA-acetone solution. Liquid chromatography-mass spectrometry analysis showed that the metabolic richness was significantly improved compared with fermentation in the same volume of ISP2 medium (i.e., the seed culture in the shake flask was inoculated into 30 L of ISP2 medium at a volume ratio of 16.7% and cultured at 30℃ and 220 rpm for 5-7 days). Figure 1 As shown.
[0046] Using the external standard method, a standard curve was established with pure doxoride to calculate the doxoride content in the fermentation broth. The results showed a significant increase in the yield of doxoride metabolites, such as... Figures 1-2As shown, the highest yield of Dopsisamine A (DopA) is approximately 290 mg / L, the highest yield of Dopsisamine B (DopB) is approximately 155 mg / L, the highest yield of Dopsisamine C (DopC) is approximately 394 mg / L, and the highest yield of Dopsisamine D (DopD) is approximately 10 mg / L. Compared to fermentation in the same volume on ISP2 medium, the optimized method in this embodiment increased the yield of Dopsisamine A (DopA) from approximately 0.25 mg / L to a maximum of 290 mg / L, an increase of nearly 1160 times; the yield of Dopsisamine B (DopB) from approximately 0.16 mg / L to a maximum of 155 mg / L, an increase of nearly 969 times; the yield of Dopsisamine C (DopC) from approximately 0.28 mg / L to a maximum of 394 mg / L, an increase of nearly 1407 times; and the yield of Dopsisamine D (DopD) from approximately 0.21 mg / L to a maximum of 10 mg / L, an increase of nearly 48 times.
[0047] Example 2 This example provides a doxoride compound and its preparation method. Based on the crude extract obtained in Example 1, the method further includes the following steps:
[0048] 1.8 g of crude extract was dissolved in methanol and purified by preparative high-performance liquid chromatography (HPLC). A Reprosil Basic C18 column (21.2 × 250 mm, 5 μm) was used for gradient elution at a flow rate of 16.0 mL / min. Mobile phase A was 2‰ trifluoroacetic acid aqueous solution, and mobile phase B was acetonitrile. The elution program was as follows: gradient elution from 0 to 15 min, with mobile phase B increasing from 20% to 45%; isocratic elution from 15 to 20 min, with mobile phase B remaining constant at 99%; and isocratic elution from 20 to 25 min, with mobile phase B remaining constant at 20%.
[0049] Compound 1 (9.5 mg, t) was finally obtained from the fermentation broth in the fermenter. R =14.6min), compound 2 (10.5mg, t R =16.8min), compound 3 (7.8mg, t R =17.9min), compound 4 (6.3mg, t R =17.3min), compound 5 (5.2mg, t R =10.8 min). The new compounds 1-5 were named Dopsisamine E, Dopsisamine F, Dopsisamine G, Dopsisamine H, and Dopsisamine I, respectively, and identified as follows:
[0050] Compound Dopsisamine E(1): a yellowish-brown oily substance; readily soluble in methanol, DMSO, and pyridine; exhibits UV absorption peaks at 200 nm, 224 nm, and 304 nm, such as... Figure 3 As shown. Figure 4 The HR-ESI-MS spectrum of compound 1 shows its [M+H] content. + The peak at m / z 686.5566 indicates that its molecular formula is C. 36 H 67 O2N 11 HR-ESI-MS spectra were obtained using Thermo QExactive orbital trap high-resolution mass spectrometry with methanol as the solvent. Figure 5 Compound 1 is dissolved in Pyridine-d5 1 H-NMR spectrum, measured at 600 MHz. Figure 6 Compound 1 is dissolved in Pyridine-d5 13 C10-NMR spectra, measured at 150 MHz. Figures 7-10 Compound 1 dissolved in Pyridine-d5 1 H- 1 The NMR spectrum of compound 1 was studied using HCOSY, HMBC, HSQC, and NOESY spectra, and further analyzed. 1 H and 13 The C signals were assigned, as shown in Table 1. The final determined structure of compound 1 is shown in [Table 1]. Figure 4 .
[0051] Table 1. NMR data of compound 1
[0052]
[0053]
[0054] Compound Dopsisamine F(2): a yellowish-brown oily substance; readily soluble in methanol, DMSO, and pyridine; exhibits UV absorption peaks at 200 nm, 224 nm, and 304 nm, such as... Figure 3 As shown. Figure 11 The HR-ESI-MS spectrum of compound 2 shows its [M+H] content. + The peak at m / z 770.6144 indicates that its molecular formula is C. 41 H 75 O3N 11 HR-ESI-MS spectra were obtained using Thermo QExactive orbital trap high-resolution mass spectrometry with methanol as the solvent. Figure 12Compound 2 is soluble in Pyridine-d5 1 H-NMR spectra were measured at 5℃ and 600MHz. Figure 13 Compound 2 is soluble in Pyridine-d5 13 C-NMR spectra were measured at 5℃ and 150MHz. Figures 14-17 These are compounds 2 dissolved in Pyridine-d5, respectively. 1 H- 1 The NMR spectrum of compound 2 was studied using HCOSY, HMBC, HSQC, and NOESY spectra, and its analysis was performed. 1 H and 13 The C signal was assigned, as shown in Table 2. The final determined structure of compound 2 is shown in [Table 2]. Figure 11 .
[0055] Table 2 NMR data for compound 2
[0056]
[0057]
[0058] Compound Dopsisamine G(3): a yellowish-brown oily substance; readily soluble in methanol, DMSO, and pyridine; exhibits UV absorption peaks at 200 nm, 224 nm, and 304 nm, such as... Figure 3 As shown. Figure 18 The HR-ESI-MS spectrum of compound 3 shows its [M+H]+ peak at m / z 754.6196, indicating its molecular formula is C. 41 H 75 O2N 11 HR-ESI-MS spectra were obtained using Thermo QExactive orbital trap high-resolution mass spectrometry with methanol as the solvent. Figure 19 Compound 3 is soluble in Pyridine-d5 1 H-NMR spectrum, measured at 600 MHz. Figure 20 Compound 3 is soluble in Pyridine-d5 13 C-NMR spectrum, measured at 150 MHz. Figures 21-24 Compound 3 dissolved in Pyridine-d5, respectively. 1 H- 1 The NMR spectrum of compound 3 was studied using H COSY, HMBC, HSQC, and NOESY spectra, and its analysis was performed. 1 H and 13 The C signal was assigned, as shown in Table 3. The final determined structure of compound 3 is shown in [Table 3]. Figure 18 .
[0059] Table 3 NMR data for compound 3
[0060]
[0061]
[0062] Compound Dopsisamine H(4): a yellowish-brown oily substance; readily soluble in methanol, DMSO, and pyridine; exhibits UV absorption peaks at 200 nm, 224 nm, and 304 nm, such as... Figure 3 As shown. Figure 25 The HR-ESI-MS spectrum of compound 4 shows its [M+H]+ peak at m / z 670.5620, indicating that its molecular formula is C. 36 H 67 ON 11 HR-ESI-MS spectra were obtained using Thermo QExactive orbital trap high-resolution mass spectrometry with methanol as the solvent. Figure 26 Compound 4 is soluble in Pyridine-d5 1 H-NMR spectrum, measured at 600 MHz. Figure 27 Compound 4 is soluble in Pyridine-d5 13 C-NMR spectrum, measured at 150 MHz. Figures 28-31 Compound 4 dissolved in Pyridine-d5 1 H- 1 The NMR spectrum of compound 4 was studied using H COSY, HMBC, HSQC, and NOESY spectra, and its analysis was performed. 1 H and 13 The C signal was assigned, as shown in Table 4. The final determined structure of compound 4 is shown in [Table 4]. Figure 25 .
[0063] Table 4 Compound 4 1 H NMR and 13 C NMR data
[0064]
[0065]
[0066] Compound Dopsisamine I(5): a yellowish-brown oily substance; readily soluble in methanol, DMSO, and pyridine; exhibits UV absorption peaks at 200 nm, 224 nm, and 304 nm, such as... Figure 3 As shown. Figure 32The HR-ESI-MS spectrum of compound 5 shows a [M+H]+ peak at m / z 670.5620, indicating that its molecular formula is C. 26 H 51 ON 11 HR-ESI-MS spectra were obtained using Thermo QExactive orbital trap high-resolution mass spectrometry with methanol as the solvent. Figure 33 Compound 5 is soluble in Pyridine-d5 1 H-NMR spectrum, measured at 600 MHz. Figure 34 Compound 5 is soluble in Pyridine-d5 13 C-NMR spectrum, measured at 150 MHz. Figures 35-38 These are compounds 5 dissolved in Pyridine-d5, respectively. 1 H- 1 The NMR spectrum of compound 5 was studied using HCOSY, HMBC, HSQC, and NOESY spectra, and its analysis was performed. 1 H and 13 The C signal was assigned, as shown in Table 5. The final determined structure of compound 5 is shown in [Table 5]. Figure 32 .
[0067] Table 5 Compound 5 1 H NMR and 13 C NMR data
[0068]
[0069] Example 3: Evaluation of the antibacterial activity and selectivity of the novel dopsisamine compound, Dopsisamine EI.
[0070] The minimum inhibitory concentration (MIC) of the compound was determined using the standard dilution method. The test was conducted at 37°C, and the MIC was measured after 16-20 hours of incubation. Ciprofloxacin was used as a control. The MTT assay was used to test the toxicity of the compound against the Vero cell line.
[0071] Tables 6 to 8 show the measured MICs of compounds 1-5 against Mycobacterium tuberculosis H37Ra, Escherichia coli, and methicillin-resistant Staphylococcus aureus, and their CC values for toxicity to Vero cell lines. 50The calculated selectivity index (SI) was also used. Dopsisamine EI showed superior antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium H37Ra compared to Dopsisamine AD. Against Escherichia coli, Dopsisamine E, F, and G exhibited superior antibacterial activity compared to Dopsisamine AD. Dopsisamine G, a triisopentenyl-substituted product, showed the best antibacterial activity against Escherichia coli and MRSA. Furthermore, Dopsisamine G exhibited lower cytotoxicity than Dopsisamine AD, demonstrating the highest selectivity index and high selectivity.
[0072] Table 6. Minimum inhibitory concentrations and selectivity indices of compounds 1-5 against Mycobacterium tuberculosis.
[0073]
[0074] Table 7. Minimum inhibitory concentrations and selectivity indices of compounds 1-5 against Escherichia coli.
[0075]
[0076] Table 8. Minimum inhibitory concentrations and selectivity indices of compounds 1-5 against methicillin-resistant Staphylococcus aureus.
[0077]
Claims
1. A method for optimizing the fermentation yield of doxylamine compounds, characterized in that, Includes the following steps: S1. Inoculate Klostridium psychrophile ZJ4-3B into shake flask seed culture medium and culture with shaking to obtain Klostridium psychrophile ZJ4-3B shake flask seed liquid; S2. The psychrophilic Klostridium ZJ4-3B seed culture obtained in step S1 is inoculated into the seed culture medium of the fermenter, and cultured with aeration and shaking to obtain the psychrophilic Klostridium ZJ4-3B fermentation seed culture. S3. The psychrophilic Klostridium ZJ4-3B fermentation seed liquid obtained in step S2 was inoculated into the fermenter culture medium, aerated and shaken, and the fermentation liquid containing the increased production of dopsisamines AD was collected.
2. The method for optimizing the fermentation yield of doxylamine compounds according to claim 1, characterized in that, In steps S1 and S2, both the shake flask seed medium and the fermenter seed medium consist of the following components: By weight percentage, malt extract 1%, yeast extract 0.4%, glucose 0.4%; In step S3, the fermentation medium consists of the following components: 0.1% MgCl·6H2O, 0.2% NH4Cl, 0.2% CaCO3, 0.1% K2HPO4, 0.1% NaCl, 0.5% sucrose, 0.2% sodium sulfate, 2% soluble starch, and 0.1% trace element mixture; wherein, the trace element mixture per 100 mL consists of: 100 mg MnCl2·4H2O, 100 mg ZnCl2, and 100 mg FeCl2·4H2O, and the pH is adjusted to 7.0–7.
1.
3. The method for optimizing the fermentation yield of doxylamine compounds according to claim 1, characterized in that, In step S1, the process conditions for obtaining the psychrophilic Klosella ZJ4-3B shake flask seed culture are: cultured at 30℃ and 220rpm for 2-3 days. In step S2, the inoculation volume ratio of the psychrophilic Klostridium ZJ4-3B seed culture to the fermentation seed medium was 2%. The process conditions for obtaining the psychrophilic Klostridium ZJ4-3B fermentation seed culture by aeration and shaking were as follows: aeration rate of 0.3 vvm, tank pressure of 0.01-0.03 MPa, rotation speed of 100-150 rpm, and culture for 1-2 days. In step S3, the inoculation volume ratio of the psychrophilic Klostridium ZJ4-3B fermentation seed liquid to the fermentation medium is 15%–17%. The aeration and shaking culture conditions are as follows: aeration rate of 0.5 vvm, tank pressure of 0.01–0.03 MPa, rotation speed of 100–150 rpm from 0 to 36 h, rotation speed of 200–300 rpm after 36 h, and culture for 5–7 days.
4. A doxylamine compound, characterized in that, Selected from any one or more of the structures shown in Formula I below: In the formula, R1 is H or OH; R2 is H or OH; R3 is H or R4 is H or R5 is H or 5. The doxylamine compound according to claim 4, characterized in that, The doxylamine compounds are selected from any one or more of the following group:
6. A method for preparing a doxylamine compound, characterized in that, It also includes the following steps: S401. The fermentation broth as described in any one of claims 1 to 3 is adsorbed by a non-ionic macroporous adsorption resin. The resin adsorbed with the fermentation broth is eluted sequentially by pure water, 20% methanol aqueous solution, and 2‰ TFA-acetone. The crude extract is obtained by concentrating the 2‰ TFA-acetone eluent. S402. The crude extract obtained in step S401 is dissolved in methanol and purified by liquid chromatography. Mobile phase A is 2‰ trifluoroacetic acid aqueous solution and mobile phase B is acetonitrile. Gradient elution is performed at a flow rate of 16.0 mL / min to obtain doxamine compounds with the structure shown in Formula I.
7. The method for preparing doxylamine compounds according to claim 6, characterized in that, In step S402, the gradient elution process conditions are as follows: 0-15 min gradient elution, with mobile phase B increasing from 20% to 45%; 15-20 min isocratic elution, with mobile phase B kept constant at 99%; 20-25 min isocratic elution, with mobile phase B kept constant at 20%.
8. An antibacterial drug composition, characterized in that, The antibacterial drug composition includes: (1) The doxylamine compound as described in claim 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; and (2) Pharmaceutically acceptable carrier.
9. The use of the doxylamine compound as claimed in claim 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that, For use in the preparation of a formulation or drug, said formulation or drug being used for: (1) Inhibits or kills Gram-negative bacteria and / or Gram-positive bacteria and / or mycobacteria; and / or (2) Prevention and / or treatment of infectious diseases caused by Gram-negative bacteria, Gram-positive bacteria or mycobacteria.
10. The use according to claim 9, characterized in that, The mycobacterium is Mycobacterium tuberculosis, the Gram-negative bacteria is Escherichia coli, and the Gram-positive bacteria is methicillin-resistant Staphylococcus aureus. The infectious disease caused by the mycobacterium is pulmonary tuberculosis, the infectious disease caused by the Escherichia coli is extraintestinal infection, and the infectious disease caused by methicillin-resistant Staphylococcus aureus is pneumonia.